Tricyclic polypeptide conjugate drugs and uses thereof

Abstract

Tricyclic polypeptide conjugate drugs and uses thereof are provided, specifically compounds of formula (III) and pharma- ceutically acceptable salts thereof: embedded image JPEG2024535472000058.jpg54170

Description

[Technical Field]

[0001] The present invention relates to tricyclic polypeptide conjugate drugs and uses thereof, and in particular to compounds of formula (III) and pharmaceutically acceptable salts thereof:

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This invention claims priority to CN2021111505175, filed September 29, 2021, and CN2021112166281, filed October 19, 2021. [Background technology]

[0003] Nectin-4 (poliovirus receptor-like 4, PVRL4, or nectin-4) has emerged as a novel tumor-associated target in recent years. It belongs to the nectin protein family, which mainly consists of four subtypes, nectin-1 to -4. Together with nectin-like molecules (Necl), it constitutes an immunoglobulin-like cell adhesion molecule and plays an important role in cell-cell adhesion and the formation and maintenance of tight junctions. Nectin-1, -2, and -3 are widely distributed in normal human tissues, while nectin-4 is highly expressed mainly in the embryo and placenta, with its in vivo expression level significantly reduced in adults. Nectin-4 is overexpressed in various tumors, including bladder cancer, urothelial cancer, breast cancer, triple-negative breast cancer, lung cancer, gastric cancer, and esophageal cancer, making it a potential target for the treatment of related cancers. Currently, a biological antibody conjugate drug, enfortumab vedotin, developed against this target was approved for sale in the United States in 2019. It is the only commercially available drug targeting this target, and its primary indication is metastatic urothelial carcinoma. Clinical efficacy studies are also underway for many other indications. Therefore, the development of chemotherapy drugs targeting Nectin-4 holds promise for widespread use. Summary of the Invention

[0004] The present invention provides compounds of formula (III) and pharmaceutically acceptable salts thereof: [ka] During the ceremony, R1 is H and C 1~3 selected from alkyl groups, R2 is H, C 1~4 Alkyl groups and [ka] Selected from n is selected from 1, 2, 3 and 4; Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, and Pen.

[0005] In some embodiments of the present invention, the compound is selected from the structures of formula (III-1) and formula (III-2): [ka] wherein R1, R2, Xi, Xii and Xiii are as defined in the present invention.

[0006] The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, R1 is H or C 1~3 selected from alkyl groups, R2 is H, C 1~4 Alkyl groups and [ka] Selected from n is selected from 1, 2, 3 and 4; Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, Pen, Dap, and N-methyl-Dap.

[0007] In some embodiments of the present invention, the compound is selected from the structures of formula (I-1) and formula (I-2): [ka] wherein R1, R2, Xi, Xii and Xiii are as defined in the present invention.

[0008] Further embodiments of the present invention are any combination of the above variables.

[0009] The present invention also provides a compound represented by the formula: [ka] JPEG0007774349000008.jpg213170

[0010] The present invention also provides pharmaceutical compositions containing a therapeutically or prophylactically effective amount of a compound according to the present invention or a pharmaceutically acceptable salt thereof.

[0011] The present invention also provides use of the compound of the present invention, a pharmaceutically acceptable salt, and the above-mentioned pharmaceutical composition in the manufacture of a medicament for treating a solid tumor in which Nectin-4 is overexpressed.

[0012] The compounds of the present invention also provide the following methods for preparing them. [ka] JPEG0007774349000010.jpg191170

[0013] The present invention also provides the following test method. Test method 1: Test of the binding ability of the compound of the present invention to Nectin-4 protein 1. Purpose of the test The affinity of a test substance for the target protein Nectin-4 is detected using the SPR method.

[0014] 2. Materials and Equipment Biacore 8K (GE Healthcare) 96-well plate (catalog number 650101, Greiner Bio-One) CM5 chip (catalog number BR-1005-30, GE Healthcare) Amine Coupling Kit (Cat. No. BR-1000-50, GE Healthcare) EDC NHS 1M ethanolamine 10mM Sodium Acetate pH 4.5 (Cat. No. BR-1003-50, GE Healthcare) DMSO (Cat. No. D4540, Sigma) P20 (Catalog No. BR-1000-54, GE Healthcare) PBS (Cat. No. BR-1006-72, GE Healthcare) Nectin-4 (Cat. No. 1006-72, GE Healthcare)

[0015] 3. Test Plan In this study, the amino coupling method was used: the target protein, nectin-4, was directly immobilized on a CM5 chip using a Biacore 8K. The test substance was then diluted to a predetermined concentration gradient in buffer (10 mM PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5% DMSO, 0.05% P20) and subjected to multi-cycle kinetic measurements. Each cycle consisted of 180 seconds of loading and 180 seconds of dissociation, followed by the next cycle to obtain affinity kinetic data for the target protein, nectin-4. The final data were subjected to kinetic fitting analysis using a 1:1 model using Biacore Insight Evaluation Software (v2.0.15.12933).

[0016] 4. Test methods and procedures 1) Prepare buffer: 10 mM PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5% DMSO, 0.05% P20.

[0017] 2) Activate the CM5 chip with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 seconds.

[0018] 3) Coupling of the target protein. Dilute the target protein to 10 μg / mL with 10 mM sodium acetate (pH 4.5) and couple for 284 seconds at a flow rate of 10 μL / min. In the test, channels 1, 2, and 3 of the chip were used, and the coupling results were 1639.9 RU, 1747.8 RU, and 1702.2 RU, respectively.

[0019] 4) Block the CM5 chip with 1 M ethanolamine at a flow rate of 10 μL / min for 420 seconds.

[0020] 5) To obtain the analyte concentration, the test substance is diluted using a buffer solution. The test substance is diluted from 100 nM to 0.78 nM in a 2-fold gradient.

[0021] 6) Load and analyze. Each concentration of the test substance working solution was counted as one cycle, with binding time of 180 seconds and dissociation time of 180 seconds at a flow rate of 30 μL / min. The final cycle was a calibration cycle with 5% DMSO solvent.

[0022] 7) All results will be subjected to kinetic fitting analysis using a 1:1 model. [Effects of the Invention]

[0023] The compounds of the present invention have very strong binding activity to Nectin-4, exhibit significant antitumor cell proliferation activity in vitro, and exhibit strong tumor-suppressing effects in an in vivo mouse subcutaneous tumor model. They also have excellent in vitro metabolic stability and PK properties. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows the tumor growth curve of the compound of the present invention in a human lung cancer NCI-H292 cell subcutaneous xenograft tumor model. [Figure 2] FIG. 2 shows the change curve of animal body weight in a human lung cancer NCI-H292 cell subcutaneous xenograft tumor model in response to the compound of the present invention. [Figure 3] Figure 3 shows the tumor growth curves in which the administration of the compounds of the present invention was initiated in groups when the average tumor volume reached approximately 500-600 mm3 in a BALB / c nude mouse model of subcutaneous xenograft tumors of human breast cancer MDA-MB-468 cells. [Figure 4] Figure 4 shows the rate of change in animal body weight when the administration of the compound of the present invention was initiated in groups when the average tumor volume in a BALB / c nude mouse model of subcutaneous xenograft tumors of human breast cancer MDA-MB-468 cells reached approximately 500-600 mm3.

[0025] Definitions and Explanations: Unless otherwise specified, the following terms and expressions used herein have the following meanings: Unless specifically defined, a particular term or expression is to be understood in its ordinary, undefined or ambiguous sense. When a trade name is mentioned herein, it refers to the corresponding product or its active ingredient.

[0026] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are medically determined to be suitable for use in contact with human or animal tissue, are not toxic or irritating, and are not likely to cause an allergic reaction or other problem or complication, and for which the benefit-risk ratio is reasonable.

[0027] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention prepared from a compound having certain substituents of the present invention and a relatively non-toxic acid or base. When a compound of the present invention contains a relatively acidic functional group, a base addition salt may be obtained by contacting the compound with a sufficient amount of base in a pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, magnesium salts, or similar salts. When a compound of the present invention contains a relatively basic functional group, an acid addition salt may be obtained by contacting the compound with a sufficient amount of acid in a pure solution or in a suitable inert solvent. Some of the compounds of the present invention contain basic or acidic functional groups and can therefore be converted into any base or acid addition salt.

[0028] The pharmaceutically acceptable salts of the present invention may be synthesized from parent compounds that contain an acid or base group by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or free base form of these compounds with the stoichiometrically appropriate base or acid in water, an organic solvent, or a mixture of both.

[0029] The term "pharmaceutically acceptable excipient" refers to an inert substance administered with an active ingredient to facilitate administration of the active ingredient, and includes, but is not limited to, any glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, disintegrant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved for use in humans or animals (e.g., livestock) by the China Food and Drug Administration. Non-limiting examples of such excipients include calcium carbonate, calcium phosphate, various sugars and various starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0030] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or salts thereof with pharmaceutically acceptable excipients. The pharmaceutical composition is intended to facilitate administration of the compounds of the present invention to a living body.

[0031] The pharmaceutical compositions of the present invention may be prepared by combining the compounds of the present invention with suitable pharmaceutically acceptable additives, and can be prepared, for example, as solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols and the like.

[0032] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that perform functions similar to those of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (e.g., an α-carbon bonded to a hydrogen, a carboxy group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds whose structure differs from that of a typical amino acid but which perform a similar function to a naturally occurring amino acid.

[0033] The amino acid sequences of the present invention include the standard one-letter or three-letter abbreviations for the 20 naturally occurring amino acids.

[0034] The term "treatment" includes inhibiting, alleviating, arresting or reversing the progression or severity of an existing condition or disease.

[0035] The term "therapeutically effective amount" or "effective amount" refers to the amount of a compound of the present invention that achieves the following effects: (i) treating or preventing a particular disease, condition, or disorder; (ii) alleviating, ameliorating, or eliminating one or more symptoms of a particular disease, condition, or disorder; or (iii) preventing or delaying the onset of one or more symptoms of a particular disease, condition, or disorder described herein. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells, shrink tumor size, inhibit (i.e., reduce to some extent, and preferably stop) cancer cell invasion of surrounding organs, inhibit (i.e., reduce to some extent, and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate one or more symptoms associated with cancer to some extent. The extent to which a drug prevents the growth and / or kills existing cancer cells may be cytostatic and / or cytotoxic.

[0036] Unless otherwise specified, the term "isomer" is intended to include geometric isomers, cis / trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

[0037] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. Such compounds contemplated by the present invention include cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, as well as racemic and other mixtures, e.g., enantiomer- or diastereomer-enriched mixtures, all of which are within the scope of the present invention. Substituents such as alkyl groups may have additional asymmetric carbon atoms. All of these isomers and mixtures thereof are within the scope of the present invention.

[0038] Unless otherwise specified, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of one another.

[0039] Unless otherwise stated, the terms "cis / trans isomers" or "geometric isomers" result from the inability to freely rotate about a double bond or a single bond of a ring-forming carbon atom.

[0040] Unless otherwise specified, the term "diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another.

[0041] Unless otherwise specified, "(+)" represents dextrorotatory, "(-)" represents levorotatory, and "(±)" represents racemic.

[0042] Unless otherwise specified, JPEG0007774349000011.jpg36170

[0043] Unless otherwise specified, the terms "enriched in one isomer," "enriched in an isomer," "enriched in one enantiomer," or "enantiomer-enriched" refer to an isomer or enantiomer that is present in an amount of less than 100% and that is at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.6%, or at least 99.7%, or at least 99.8%, or at least 99.9%.

[0044] Unless otherwise specified, the terms "isomeric excess" or "enantiomeric excess" refer to the difference in relative percentage abundance of two isomers or two enantiomers. For example, if one isomer or enantiomer is present at 90% and the other isomer or enantiomer is present at 10%, the isomeric or enantiomeric excess (ee) is 80%.

[0045] Optically active (R)- and (S)-isomers, and D- and L-isomers, can be prepared using asymmetric synthesis, chiral reagents, or other conventional techniques. Enantiomers of certain compounds of the present invention may be prepared by asymmetric synthesis or derivatization with chiral auxiliaries. The diastereomeric mixture of products can be separated and the auxiliary groups removed to yield the desired pure enantiomers. Alternatively, if the molecule contains basic (e.g., amino) or acidic (e.g., carboxy) functional groups, the diastereomeric salts can be formed with an appropriate optically active acid or base, followed by separation and subsequent recovery to yield the pure enantiomers using conventional methods known in the art. Separation of enantiomers and diastereomers is also commonly accomplished by chromatography, which utilizes chiral stationary phases, optionally combined with chemical derivatization (e.g., carbamate formation from amines).

[0046] The compounds of the present invention may contain unnatural proportions of isotopes of one or more atoms that constitute the compounds. For example, tritium ( 3 H), iodine-125( 125 I) or carbon-14( 14 The compounds can be labeled with radioactive isotopes such as CI, ...

[0047] When the direction of connection is not specified for a listed connecting group, there is no restriction on the direction of connection, for example, [ka] The connecting group L is -MW-, and in this case, -MW- connects ring A and ring B in the same direction as reading from left to right. [ka] or by connecting ring A and ring B in the opposite direction from left to right, [ka] Combinations of the above connecting groups, substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0048] Unless otherwise specified, if a group has one or more connectable sites, any one or more of the sites on the group can be connected to other groups via a chemical bond. If the destination of the chemical bond is not specified and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at that site is reduced by the number of connecting chemical bonds to form a group of the corresponding valence. The chemical bond connecting the site to other groups is: For example, in -OCH3, the linear solid bond indicates that the functional group is connected to another functional group via the oxygen atom of the functional group. [ka] In the formula, a linear dashed bond represents a bond between the nitrogen atoms of the group and another functional group, [ka] In the figure, the wavy lines indicate that the phenyl group is connected to other functional groups via the 1st and 2nd carbon atoms.

[0049] Unless otherwise specified, the term "C 1~4 The term "alkyl group" refers to a linear or branched saturated hydrocarbon group having 1 to 4 carbon atoms. 1~4 The alkyl group is C 1~2 , C 1~3 , C 2~3It may contain an alkyl group, and may be monovalent (e.g., a methyl group), divalent (e.g., a methylene group), or polyvalent (e.g., a methine group). 1~4 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl, and t-butyl) groups.

[0050] Unless otherwise specified, the term "C 1~3 The term "alkyl group" refers to a linear or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. 1~3 Alkyl groups are C 1~2 , C 2~3 It may contain an alkyl group, and may be monovalent (e.g., a methyl group), divalent (e.g., a methylene group), or polyvalent (e.g., a methine group). 1~3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl) groups, and the like.

[0051] Unless otherwise specified, in the present invention, amino acids Xi, Xii and Xiii are connected to TATA by the sulfhydryl groups on the residue, e.g., when Xi is Pen, [ka]

[0052] The structure of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. When the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional technical means in this field. For example, when using single crystal X-ray diffraction (SXRD), diffraction intensity data is collected from a cultivated single crystal using a Bruker D8 venture diffractometer, with a CuKα radiation source and a φ / ω scanning method. After collecting the relevant data, the crystal structure can be analyzed by a direct method (Shelxs97) to confirm the absolute configuration.

[0053] The compounds of the present invention include the specific embodiments described below, embodiments made in combination with other chemical synthetic methods, and equivalent alternatives familiar to those skilled in the art, and preferred embodiments can be prepared by a variety of synthetic methods familiar to those skilled in the art, including, but not limited to, the examples of the present invention.

[0054] Compounds are named according to conventional naming conventions in the art or using the software ChemDraw®; commercially available compounds are named as given in the manufacturer's catalogue.

[0055] The solvent used in the present invention may be a commercially available product, and the ratio of the mixed solvent described in the present invention is a volume ratio, for example, 20% MeCN / HO means that the volume of MeCN in the mixed solvent accounts for 20%.

[0056] The following abbreviations are used in the present invention: eq. represents equivalent, SPPS represents solid phase peptide synthesis, TFA represents trifluoroacetic acid, DIEA represents diisopropylethylamine, DMF represents N,N-dimethylformamide, HATU represents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, EDC represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, NHS represents N-hydroxysuccinimide, TIS represents triisopropylsilane, DTT represents DL-1,4-dithiothreitol, and TATA represents [ka] MMAE represents monomethyl auristatin E, the structure of which is [ka] and PABC is [ka] Cit represents L-citrulline, Val represents L-valine, and Glutaryl represents [ka] and β-Ala represents: [ka] and Sar represents [ka] and Sar10 represents [ka] Cys represents L-cysteine, and hCys represents [ka] and βCys represents [ka] and Pen represents [ka] and N-methyl-Dap represents [ka] In the formula, 1Nal represents 1-naphthylalanine, hArg represents L-homoarginine, Hyp represents L-hydroxyproline, Trp represents L-tryptophan, Pro represents L-proline, Thr represents L-threonine, Ser represents L-serine, Asp represents L-aspartic acid, dAsp represents D-aspartic acid, Fmoc represents a 9-fluorenylmethyloxycarbonyl group, Boc represents a tert-butoxycarbonyl group (Boc), Trt represents a trityl group, Pbf represents a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group, and PBS represents phosphate buffered saline. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention will now be described in detail using examples, which are not intended to limit the present invention in any way. Detailed descriptions of the present invention, including specific embodiments, are provided herein. It will be apparent to those skilled in the art that various modifications and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0058] Example 1 [ka]

[0059] Synthetic Route: [ka] JPEG0007774349000032.jpg222170

[0060] 1. Synthesis of Compound 3 Polypeptide synthesis: The polypeptides are synthesized using standard stepwise synthesis methods.

[0061] 1) Add DCM to a vessel containing CTC Resin (10.0 mmol, 10 g, substrate: 1.0 mmol / g) and Fmoc-Asp(OAll)-OH (3.95 g, 10.0 mmol, 1.0 eq).

[0062] 2) Add DIEA (4.0 eq) and then stir for 2 hours.

[0063] 3) Add MeOH (0.3 mL) and stir for 30 minutes.

[0064] 4) Suction filter, then rinse with DMF three times, bubbling with nitrogen for 30 seconds each time.

[0065] 5) Add 20% piperidine / DMF and then react for 30 minutes.

[0066] 6) Filter by suction, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0067] 7) Add the next solution of Fmoc-protected amino acid, and after 30 seconds, add the condensing agent. Bubble N2 and react for about an hour.

[0068] 8) Repeat steps 4 to 7 to condense the next amino acid. The order of adding the amino acids and condensation reagents used in the synthesis of compound 3 is as shown in Table 1. Continue this process until the connection of Fmoc-Lys(Alloc)-OH is completed.

[0069] 9) Suction filter, then rinse with DMF three times and DCM three times, bubbling with nitrogen for 30 seconds each time.

[0070] 10) Release OAll and Alloc. Add Pd(PPh3)4 (0.1 eq) and PhSiH3 (10.0 eq) to the DCM resin solution, bubble with N2, react for about 15 minutes, suction filter, and repeat this step three times.

[0071] 11) Suction filter, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0072] 12) Ring closure: Add the condensation agent solution HATU (2.85 eq.) and DIEA (6.0 eq.), bubble with N2, and react for about an hour.

[0073] 13) Filter with suction, then rinse with DMF three times and wash with methanol three times, bubbling nitrogen for 30 seconds each time, filter with suction, and dry. [Table 1]

[0074] Cutting and Purification: Cleavage buffer solution (20% HFIP / DCM) was added to the flask containing the side-chain protected polypeptide and stirred at room temperature for 30 min twice. The solution was collected, spun dry, purified by reverse-phase preparative chromatography (NHHCO system), and lyophilized to give intermediate 3. [Table 2]

[0075] 2. Synthesis of TFA salt of compound 4 The polypeptides are synthesized using standard stepwise synthesis methods.

[0076] 1) DMF is added to a vessel containing Rink amide MBHA resin (0.5 mmol, 1.56 g, substrate: 0.32 mmol / g) and the resin is allowed to swell for 2 hours.

[0077] 2) Suction filter, then rinse with DMF three times, bubbling with nitrogen for 30 seconds each time.

[0078] 3) Add 20% piperidine / DMF, then react for 30 minutes.

[0079] 4) Filter by suction, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0080] 5) Add the Fmoc-protected amino acid solution, and after 30 seconds, add the condensing agent. Bubble N2 and react for approximately 1 hour.

[0081] 6) Repeat steps 2 to 5 to condense the next amino acid.

[0082] The order of adding the amino acids and condensation reagents used in the synthesis of compound 4 is as shown in Table 3. [Table 3]

[0083] Cleavage and purification of polypeptides: 1) Add cleavage buffer solution (90% TFA / 2.5% TIS / 2.5% H2O / 5.0% DTT) to the flask containing the side-chain protected polypeptide and stir at room temperature for 2 hours.

[0084] 2) The polypeptide is precipitated with ice-cold isopropyl ether and centrifuged (3 minutes, 3000 rpm).

[0085] 3) Wash twice more with isopropyl ether.

[0086] 4) The crude polypeptide is dried to obtain the TFA salt of intermediate 4.

[0087] 2. Synthesis of acetate salt of compound Peptide_1 The crude TFA salt of intermediate 4 (2.4 g) was dissolved in 50% MeCN / HO (1 L). TATA (0.5 mmol) was slowly added to the stirred solution at room temperature. The reaction mixture was stirred at room temperature for 30 minutes, and then the pH was adjusted to 8 with NH4HCO3. The reaction mixture was stirred at room temperature for 12 hours. When LCMS showed the reaction was complete, the stirring was stopped and the compound Peptide_1 acetate salt was obtained by purification using reverse phase preparative chromatography. [Table 4]

[0088] The synthesis of Peptide_2 and Peptide_3 may be performed with reference to the synthesis of Peptide_1, and their structures are shown in Table 5. [Table 5]

[0089] 4. Synthesis of TFA salt of compound INT_1 Compound 1-1 (200.0 mg, 178.0 μmol) was dissolved in DMF (5 mL) and added with DIEA (31.0 μL, 178.0 μmol) at 0 °C and stirred for 10 minutes. Meanwhile, compound 1-2 (290.4 mg, 890.1 μmol) was also dissolved in DMF (5 mL) in a separate reaction flask and stirred at 0 °C for 10 minutes. Then, at 0 °C, the reaction solution of compound 1-1 was added dropwise to the reaction solution of compound 1-2 while stirring. This reaction solution was stirred at 0 °C for 30 minutes. The reaction solution was filtered to remove insoluble residues, and the filtrate was directly purified by reverse-phase preparative chromatography (mobile phase: A: 0.075% TFA in H2O, B: CH3CN, gradient: 10%-40% (B), 42 minutes) to obtain the TFA salt of compound INT_1.

[0090] 5. Synthesis of acetate salt of compound PDC_1 The acetate salt of compound Peptide_1 (34.0 mg, 10.1 μmol) was dissolved in DMF (0.3 mL), and then DIEA (7.00 μL, 40.4 μmol) was added and stirred at room temperature for 10 minutes. Subsequently, the TFA salt of compound INT_1 (13.4 mg, 10.1 μmol) was dissolved in DMF (0.2 mL) and added dropwise to the above reaction solution, followed by stirring at room temperature for 2 hours. The reaction solution was filtered to remove insoluble residues, and the filtrate was directly purified by reverse-phase preparative chromatography (mobile phase: A: 0.075% TFA in H2O, B: CH3CN, gradient: 10%-40% (B), 42 min), lyophilized, and then preparatively converted to the acetate salt to obtain the acetate salt of compound PDC_1. MS m / z: 1533.4 (M+3H) + ) / 3.

[0091] Example 2 [ka]

[0092] Synthetic Route 1: The acetate salt of PDC_2 was prepared by following the synthetic route for the acetate salt of PDC_1 (Peptide_2 replaces Peptide_1 in the reaction). MS m / z: 1500.5 (M+3H + ) / 3.

[0093] Synthetic Route 2: [ka]

[0094] Step 1: Synthesis of Intermediate 5 The polypeptides are synthesized using standard stepwise synthesis methods.

[0095] 1) DMF was added to a vessel containing Rink amide MBHA resin (5.0 mmol, 8.33 g, substrate: 0.60 mmol / g) and the resin was allowed to swell for 2 hours.

[0096] 2) Suction filter, then rinse with DMF three times, bubbling with nitrogen for 30 seconds each time.

[0097] 3) Add 20% piperidine / DMF, then react for 30 minutes.

[0098] 4) Filter by suction, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0099] 5) Add the Fmoc-protected amino acid solution, and after 30 seconds, add the condensing agent. Bubble N2 and react for approximately 1 hour.

[0100] 6) Repeat steps 2 to 5 to condense the next amino acid.

[0101] The order of adding the amino acids and condensation reagents used in the synthesis of intermediate 5 is as shown in Table 6. [Table 6]

[0102] 7) Add cleavage buffer solution (90% TFA / 2.5% TIS / 2.5% H2O / 5.0% DTT) to the flask containing the side-chain protected polypeptide and stir at room temperature for 2 hours.

[0103] 8) The polypeptide is precipitated with ice-cold isopropyl ether and centrifuged (3 minutes, 3000 rpm).

[0104] 9) Wash twice more with isopropyl ether.

[0105] 10) The crude polypeptide is dried to obtain crude intermediate 5.

[0106] Step 2: Synthesis of acetate salt of Peptide_2 Crude intermediate 5 (15.0 g) was dissolved in 50% MeCN / HO (5 L). TATA (7.5 mmol) was slowly added to the stirred solution at room temperature. The reaction mixture was stirred at room temperature for 30 minutes, and then the pH was adjusted to 8 with NH4HCO3. The reaction mixture was continued to stir at room temperature for 12 hours. When LCMS showed the reaction was complete, the stirring was stopped and the mixture was purified by reverse-phase preparative chromatography (primary purification: mobile phase A: H2O containing 0.075% TFA, B: CH3CN, gradient: 10%-40% B phase, 42 min, retention time: 29 min; secondary purification: mobile phase A: H2O containing 0.5% AcOH, B: CH3CN, gradient: 20%-40% B phase, 40 min, retention time: 21 min; tertiary purification: mobile phase A: H2O containing 0.5% AcOH, B: CH3CN, gradient: 16%-36% B phase, 40 min, retention time: 26 min) to obtain the acetate salt of intermediate Peptide_2.

[0107] Step 3: Synthesis of acetate salt of PDC_2 The acetate salt of intermediate Peptide_2 (369 mg) and INT_1 (150 mg) were dissolved in DMF (6.00 mL) and DIEA (58.0 mg) was added. The mixture was stirred at room temperature for 5 hours, and when LC-MS showed the reaction was complete, the reaction was stopped. The reaction mixture was filtered to remove insoluble residues, and the filtrate was directly purified by reverse-phase preparative chromatography (mobile phase: A: H2O containing 0.075% TFA, B: CH3CN, gradient: 10%-40% (B), 42 min), lyophilized, and then converted to the AcOH salt via preparative chromatography to obtain the acetate salt of PDC_2. MS m / z: 1500.3 (M+3H) + ) / 3.

[0108] Example 3 [ka] The acetate salt of PDC_3 was prepared by following the synthesis method of the acetate salt of PDC_1 (Peptide_3 was substituted for Peptide_1 in the reaction). MS m / z: 1504.8 (M+3H + ) / 3.

[0109] Biological Test Data: Test Example 1: Test of the binding ability of the compound of the present invention to Nectin-4 protein 1. Purpose of the test The affinity of a test substance for the target protein Nectin-4 is detected using the SPR method.

[0110] 2. Materials and Equipment Biacore 8K (GE Healthcare) 96-well plate (catalog number 650101, Greiner Bio-One) CM5 chip (catalog number BR-1005-30, GE Healthcare) Amine Coupling Kit (Cat. No. BR-1000-50, GE Healthcare) EDC NHS 1M ethanolamine 10mM Sodium Acetate pH 4.5 (Cat. No. BR-1003-50, GE Healthcare) DMSO (Cat. No. D4540, Sigma) P20 (Catalog No. BR-1000-54, GE Healthcare) PBS (Cat. No. BR-1006-72, GE Healthcare) Nectin-4 (Cat. No. 1006-72, GE Healthcare)

[0111] 3. Test Plan In this study, the amino coupling method was used: the target protein, nectin-4, was directly immobilized on a CM5 chip using a Biacore 8K. The test substance was then diluted to a predetermined concentration gradient in buffer (10 mM PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5% DMSO, 0.05% P20) and subjected to multi-cycle kinetic measurements. Each cycle consisted of 180 seconds of loading and 180 seconds of dissociation, followed by the next cycle to obtain affinity kinetic data for the target protein, nectin-4. The final data were subjected to kinetic fitting analysis using a 1:1 model using Biacore Insight Evaluation Software (v2.0.15.12933).

[0112] 4. Test methods and procedures 1) Prepare buffer: 10 mM PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5% DMSO, 0.05% P20.

[0113] 2) Activate the CM5 chip with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 seconds.

[0114] 3) Coupling of the target protein. Dilute the target protein to 10 μg / mL with 10 mM sodium acetate (pH 4.5) and couple for 284 seconds at a flow rate of 10 μL / min. In the test, channels 1, 2, and 3 of the chip were used, and the coupling results were 1639.9 RU, 1747.8 RU, and 1702.2 RU, respectively.

[0115] 4) Block the CM5 chip with 1 M ethanolamine at a flow rate of 10 μL / min for 420 seconds.

[0116] 5) To obtain the analyte concentration, the test substance is diluted using a buffer solution. The test substance is diluted from 100 nM to 0.78 nM in a 2-fold gradient.

[0117] 6) Load and analyze. Each concentration of the test substance working solution was counted as one cycle, with binding time of 180 seconds and dissociation time of 180 seconds at a flow rate of 30 μL / min. The final cycle was a calibration cycle with 5% DMSO solvent.

[0118] 7) All results will be subjected to kinetic fitting analysis using a 1:1 model.

[0119] 5. Test Results The test data of five effective concentrations were selected and subjected to kinetics fitting analysis using the Biacore Insight Evaluation Software (V2.0.15.12933) with a 1:1 model. The results are shown in Table 7. [Table 7]

[0120] In conclusion, the compounds of the present invention have very strong binding activity to Nectin-4.

[0121] Test Example 2: In vitro antiproliferative activity of compounds of the present invention against NCI-H292 and MDA-MB-468 cells 1. Purpose of the test The cytostatic effect of the compounds of the present invention is studied by detecting the influence on the in vitro cellular activity of the compounds in tumor cell lines MDA-MB-468 and NCI-H292.

[0122] 2. Study Design: Cell culture: The tumor cell lines are cultured under the culture conditions shown in Table 8 in an incubator at 37°C without CO2 and at 37°C with 5% CO2, respectively. They are passaged periodically to obtain cells in the logarithmic growth phase for plating. [Table 8]

[0123] Cell plating: Cells in the logarithmic growth phase are harvested and centrifuged at 1000 rpm for 3 minutes at room temperature. The supernatant is aspirated, and the cells are resuspended in 5 mL of culture medium. 20 μL of cell suspension is aspirated and mixed 1:1 with trypan blue for 3 minutes to detect cell viability and count live cells. The cell density is adjusted to 3000 cells / well. 90 μL of cell suspension is added to each well of the culture plate, and culture medium without cells is added to blank control wells. The culture plates are incubated overnight in an incubator at 37°C without CO2 or at 37°C with 5% CO2 and 100% relative humidity, respectively.

[0124] Compound storage plate preparation: Prepare a 400x compound stock plate. Dilute test compounds in a gradient from highest to lowest concentration in DMSO. [Table 9]

[0125] Preparation of 10x compound working solutions and cell treatment with compounds: Prepare a 10x compound working solution. Add 78 μL of cell culture medium to a V-bottom 96-well plate, and aspirate 2 μL of each compound solution from the 400x compound storage plate and add it to the cell culture medium in the 96-well plate. Add 2 μL of DMSO to the solvent control and blank control. After adding the compound or DMSO, mix thoroughly by pipetting with a multichannel pipette. For dosing, add 10 μL of the 10x compound working solution to the cell culture plate. Add 10 μL of DMSO-cell culture medium mixture to the solvent control and blank control. Return the 96-well cell plate to the incubator and culture for 72 hours.

[0126] CellTiter-Glo Luminescent Assay for Cell Vitality Detection: The procedure was carried out according to the instructions for the Promega CellTiter-Glo Luminescent Cell Activity Detection Kit (Promega-G7573). Luminescent signals were detected using an EnVision® Multi-mode Plate Reader (EnVision 2104-10) microplate reader.

[0127] Data Analysis: Formula: IR(%)=(1-(RLU) 化合物 -RLU ブランク対照 ) / (RLU 溶媒対照 -RLU ブランク対照 The inhibition rate (IR) of the detected compound was calculated by multiplying the inhibition rate by 100%. The inhibition rates of the compounds at different concentrations were calculated using Excel, and then an inhibition curve was created using the software GraphPad Prism 6.02, showing the minimum inhibition rate, maximum inhibition rate, and IC 50 Calculate relevant parameters including

[0128] The test results are shown in Table 10. [Table 10]

[0129] In conclusion, the compounds of the present invention have obvious antiproliferative effects on the tumor cell lines MDA-MB-468 and NCI-H292 cultured in vitro.

[0130] Test Example 3: In vivo pharmacodynamics of compounds of the present invention in a human lung cancer NCI-H292 cell subcutaneous xenograft tumor model 1. The purpose of the study is to investigate the in vivo pharmacodynamics of the compounds of the present invention in a mouse model of human lung cancer NCI-H292 cell subcutaneous xenograft tumors. 2. Study Design: ■ Cell culture. Human lung cancer NCI-H292 cells (ATCC, Manassas, VA, catalog number CRL-1848) were cultured in vitro in monolayers in RPMI 1640 medium supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Cells were passaged twice weekly using a standard trypsin-EDTA digestion method. When the cell confluency reached 80%-90% and the desired number of cells was reached, the cells were harvested, counted, and inoculated.

[0131] ■ The animals used were female BALB / c nude mice, 6 to 8 weeks old and weighing 17 to 21 g.

[0132] ■ Tumor inoculation: 0.2 mL (1 × 10 7 100 (individuals) of NCI-H292 cells were subcutaneously inoculated into the right back of each mouse.

[0133] For pharmacodynamic studies, the mean tumor volume was approximately 100-200 mm 3 When the rats reached the maximum, intravenous administration was started in groups of 6 rats per group at a dose of 1.5 mg / kg, administered QW×3 times.

[0134] ■ Observation. The formulation of the study protocol and any amendments will be evaluated and approved by the Shanghai Pharmacopoeia Kangde Animal Ethics Committee (IACUC). The use and welfare of test animals will be carried out in accordance with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animal health and mortality will be monitored daily, and routine inspections will include observation of tumor growth and the effects of drug treatment on the animals' daily activities, such as behavior, food and water intake, weight changes (weighed twice a week), appearance and signs, or other abnormal conditions. The number of animal deaths and side effects within each group will be recorded based on the number of animals in each group.

[0135] The study was terminated when the animal's health condition continued to deteriorate or the tumor volume exceeded 2000mm. 3 If the animal exceeds the age limit, or is in severe illness or pain, euthanasia will be performed.

[0136] ■For data analysis, a t-test is used to compare two groups. One-way ANOVA is used to compare three or more groups. If there is a significant difference in the F value, multiple comparisons are performed after the ANOVA analysis. All data is analyzed using SPSS 17.0. A value of p<0.05 is considered to be a significant difference.

[0137] Drug preparation: Regarding the frequency of drug preparation, a uniform stock solution will be prepared before the first administration, and the solution will be dispensed and frozen and stored in a refrigerator at -80°C.

[0138] The dose volume is adjusted based on the animal's weight (dose volume = 10 μL / g). [Table 11]

[0139] The test results are shown in Figures 1 and 2.

[0140] In conclusion, the compounds of the present invention show obvious tumor growth inhibitory effects in human lung cancer NCI-H292 cell subcutaneous xenograft tumor model.

[0141] Test Example 4: In vivo pharmacodynamic study of the compounds of the present invention in a BALB / c nude mouse model of human breast cancer MDA-MB-468 cell subcutaneous xenograft tumors The purpose of the study is to evaluate the in vivo pharmacodynamics of compounds of the present invention in a human breast cancer MDA-MB-468 cell subcutaneous xenograft tumor model.

[0142] Cell culture. Human breast cancer MDA-MB-468 cells (ATCC, Manassas, VA, catalog number HTB-132) were cultured in vitro in monolayers in L-15 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C CO2-free incubator. Cells were passaged twice weekly using trypsin-EDTA digestion. When the cells reached a confluency of 80%-90% and the desired number of cells was reached, they were harvested, counted, and inoculated.

[0143] The animals were female BALB / c nude mice, 6-8 weeks old, weighing 18-22 g, provided by Beijing Weitong Lihua Co., Ltd.

[0144] Tumors were inoculated in 0.2 mL (1 × 10 7 1000 cells / ml) of MDA-MB-468 cells (supplemented with Matrigel, volume ratio 1:1) were subcutaneously inoculated into the right back of each mouse.

[0145] The dose volume is adjusted based on the animal's weight (dose volume = 10 μL / g).

[0146] Drug Preparation: Test compounds were prepared as 1.5 mg / mL homogeneous solutions in 25 mM L-histidine (pH 7) and 10% sucrose and stored in a refrigerator at -80°C. On the day of administration, the compounds were diluted to the corresponding concentrations for administration in the IV (intravenous) group.

[0147] For group administration, the mean tumor volume was approximately 500-600 mm 3 When the dose reaches 5 mg / kg, group-specific administration will be initiated, with the administration frequency being QW×4.

[0148] The test results are shown in Figures 3 and 4.

[0149] The study concluded that the compound of the present invention still showed significant tumor growth inhibitory effects in a human breast cancer cell MDA-MB-468 subcutaneous xenograft tumor model, even in large groups, and showed dose-related effects. No adverse reactions were observed and there was no case of an average body weight loss of 5% or more, indicating good safety.

[0150] Test Example 5: Pharmacokinetic analysis of the compound of the present invention in rat plasma A. Purpose of the test Test the in vivo pharmacokinetics of the compounds of the present invention in SD rats

[0151] B. Test Procedure The pharmacokinetic properties of the compound were tested in rats after intravenous injection using a standard protocol. The test compound was prepared as a clear solution in 25 mM L-histidine (pH=7) and 10% sucrose. Two rats were administered a single intravenous injection of the test compound at 3 mg / kg. After administration, whole blood was collected at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours, and plasma was prepared. The concentrations of the compound of the present invention and its potential metabolite MMAE were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[0152] C. Test Results The test results are shown in Table 12. [Table 12]

[0153] In conclusion, the compound of the present invention has a short blood half-life in rats, is rapidly cleared, and the exposure level of the metabolite MMAE is only about 1 / 40 of that of the compound of the present invention, indicating good safety.

[0154] Test Example 6: Pharmacokinetic analysis in cynomolgus monkey plasma A. Purpose of the test The in vivo pharmacokinetics of compounds of the invention are tested in cynomolgus monkeys.

[0155] B. Test Procedure The pharmacokinetic properties of compounds were tested in cynomolgus monkeys after intravenous injection using a standard protocol. The test compound was prepared as a clear solution in 25 mM L-histidine (pH 7) and 10% sucrose. Cynomolgus monkeys were administered a single intravenous injection of the test compound at 1 mg / kg. Whole blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours post-administration, and plasma was prepared. The concentrations of the test compound and its potential metabolite MMAE were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[0156] C. Test Results The test results are shown in Table 13. [Table 13]

[0157] In conclusion, the compounds of the present invention have a short half-life in the blood of cynomolgus monkeys, are rapidly cleared, and release of MMAE in the blood is below the detection limit, which indicates good safety.

[0158] Test Example 7: Metabolic stability of the compound of the present invention in mouse, rat, cynomolgus monkey and human hepatocytes A. Purpose of the test The metabolic stability of the compounds of the present invention is investigated in mouse, rat, cynomolgus monkey and human hepatocytes.

[0159] B. Test Procedure Incubation is performed in a 96-well plate using an external collection method. Several 96-well sample precipitation plates are prepared and named T0, T15, T30, T60, T90, T0-MC, T90-MC, and blank matrix, respectively. The resuscitation medium and incubation medium are removed in advance and placed in a 37°C water bath for pre-warming. Cryopreserved hepatocytes from CD-1 mice, SD rats, cynomolgus monkeys, and humans are removed from the liquid nitrogen tank and resuscitated. The cells are then cultured in the incubation medium at a concentration of 0.51 x 10 cells. 6 Dilute 198 μL of hepatocyte suspension (0.5 × 10) to 100 cells / mL. 6 198 μL of incubation medium without hepatocytes was added to the T0-MC and T90-MC incubation plates for the culture medium control group. All incubation plates were pre-incubated in a 37°C incubator for 10 minutes. 2 μL of test sample and control compound working solution was then added, mixed evenly, and incubated on a shaker in the incubator. Three parallel samples were prepared for each time point. The incubation conditions were 37°C, saturated humidity, and 5% CO2. In the test system, the final concentration of the test sample was 1 μM, the final concentration of the control sample was 3 μM, and the final concentration of hepatocytes was 0.5 × 10 6 The final concentration of the organic solvents was 1.0%, of which the final concentration of DMSO was 0.1%.

[0160] At the end of the corresponding incubation time, the incubation plate was removed, and 25 μL of the compound and control compound / cell mixture was added to a sample plate containing 125 μL of stop solution, respectively. 25 μL of incubation medium without hepatocytes was added directly to the blank sample plate. All sample plates were sealed and shaken at 600 rpm on a shaker for 10 minutes, then centrifuged at 3220 g for 20 minutes. The supernatants of the test samples and control samples were diluted 1:3 with pure water. All samples were mixed uniformly and then analyzed by LC-MS / MS.

[0161] The concentrations of test compounds and controls in samples were measured semiquantitatively using liquid chromatography-tandem mass spectrometry (LC-MS / MS), without the use of standard curves or quality control samples. The concentration in the sample was expressed as the ratio of the analyte peak area to the internal standard peak area. The retention times of the analytes and internal standards, chromatogram acquisition, and chromatogram integration were processed using Analyst software (Sciex, Framingham, MA, USA).

[0162] C. Test Results The test results are shown in Table 14. [Table 14]

[0163] In conclusion, the compounds of the present invention have excellent metabolic stability in liver microsomes of four species.

[0164] Test Example 8: Metabolic stability of the compound of the present invention in kidney S9 of mice, rats, cynomolgus monkeys, and humans A. Purpose of the test The metabolic stability of the compounds of the present invention in kidney S9 of mice, rats, cynomolgus monkeys and humans is investigated.

[0165] B. Test Procedure As materials, CD-1 mice, SD rats, cynomolgus monkeys, and human kidney S9 are purchased from BioIVT or XenoTech LLC and stored in a refrigerator at -80°C.

[0166] For the test step, eight 96-well incubation plates were prepared and named T0, T5, T15, T30, T45, T60, Blank, and NCF60, respectively. The reaction time points corresponding to the first six incubation plates were 0 hours, 5 hours, 15 hours, 30 hours, 45 hours, and 60 minutes, respectively. No test sample or control compound was added to the blank plate.

[0167] To each of the T0, T5, T15, T30, T45, and T60 plates, add 2 μL of the test sample working solution (a 10 mM DMSO solution of the test compound diluted to 100 μM with 100% acetonitrile) and 100 μL of the S9 working solution (the kidney S9 protein concentration is 1.0 mg / mL). To a blank plate, add only the S9 working solution. Then, place the incubation plates in a 37°C water bath and preincubate for approximately 10 minutes.

[0168] After preincubation, 98 μL of coenzyme working solution was added to each sample well to initiate the reaction, except for the TO plate. After incubation for the appropriate time (e.g., 5, 15, 30, 45, or 60 minutes), 600 μL of stop solution (100 ng / mL tolbutamide and 100 ng / mL labetalol in acetonitrile) was added to each sample well to terminate the reaction. To prepare the TO plate, 600 μL of stop solution was first added to the TO plate, followed by 98 μL of coenzyme working solution. All sample plates were shaken to homogenize and centrifuged at 3220 g for 20 minutes. Then, 100 μL of supernatant was removed from each well and diluted with 300 μL of purified water for liquid chromatography-tandem mass spectrometry analysis.

[0169] C. Test Results The test results are shown in Table 15. [Table 15]

[0170] In conclusion, the compounds of the present invention have excellent metabolic stability in kidney S9 of the four species.

[0171] Test Example 9: Plasma stability analysis of compounds of the present invention in different species A. Purpose of the test The stability of the compounds of the present invention is tested in the plasma of SD rats, cynomolgus monkeys and humans.

[0172] B. Test Procedure Two microliters of a working solution of the test compound (100 μM) was added to the corresponding incubation plates, including T0, T10, T30, T60, T120, and T240 incubation plates, with each sample prepared in triplicate. Next, 98 μL of blank plasma from SD rats, monkeys, and humans was added to the incubation plates containing the working solution. All samples were incubated in a 37°C water bath. The final incubation concentration of the test compound was 2 μM. At the end of each incubation time point, the corresponding incubation plate was removed, a stop solution was added to precipitate proteins, and the plates were centrifuged for 20 minutes. 150 μL of the supernatant was removed and analyzed by LC-MS / MS. The concentration of the test compound in the samples was measured semiquantitatively using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0173] C. Test Results The test results are shown in Table 16. [Table 16]

[0174] In conclusion, the compounds of the present invention have excellent stability, since their half-lives in all three species are longer than 578.1 minutes.

Claims

1. A compound of formula (III) or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 During the ceremony, R １ is H and C １～３ selected from alkyl groups, R ２ is H, C １～４ Alkyl groups and 【Chemistry 2】 Selected from n is selected from 1, 2, 3 and 4; Xi, Xii, and Xiii are each independently a compound selected from Cys, hCys, βCys, and Pen, or a pharmaceutically acceptable salt thereof.

2. The compound is selected from the structures represented by formula (III-1) and formula (III-2), 【Transformation 3】 In the formula, R １ , R ２ 2. The compound of claim 1, wherein Xi, Xii and Xiii are as defined in claim 1, or a pharmaceutically acceptable salt thereof.

3. A compound represented by the following formula or a pharmaceutically acceptable salt thereof: 【Chemistry 4】 【change】

4. 10. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof.

5. 10. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the compound of claim 2 or a pharmaceutically acceptable salt thereof.

6. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of the compound of claim 3 or a pharmaceutically acceptable salt thereof.

7. 10. A product comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof for use in treating Nectin-4 overexpressing solid tumors.

8. 10. A product comprising the compound of claim 2 or a pharmaceutically acceptable salt thereof for use in treating Nectin-4 overexpressing solid tumors.

9. A product comprising the compound of claim 3 or a pharmaceutically acceptable salt thereof for use in treating Nectin-4 overexpressing solid tumors.

10. A product comprising the pharmaceutical composition of claim 4 for use in treating Nectin-4 overexpressing solid tumors.

11. A product comprising the pharmaceutical composition of claim 5 for use in treating Nectin-4 overexpressing solid tumors.

12. A product comprising the pharmaceutical composition of claim 6 for use in treating Nectin-4 overexpressing solid tumors.

13. A compound represented by the following formula or a pharmaceutically acceptable salt thereof: 【Transformation 5】

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