Polypeptide-drug conjugates having novel structures and uses thereof

Abstract

Novel polypeptide drug conjugates and uses thereof are disclosed, specifically, compounds of formula (V): [Formula 1] JPEG2024531309000060.jpg72170

Description

[Technical Field]

[0001] The present invention relates to a polypeptide-drug conjugate having a novel structure and uses thereof, specifically to a compound represented by formula (V) or a pharmaceutically acceptable salt thereof:

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This invention claims priority to CN2021109452856, filed August 17, 2021, CN2021109767108, filed August 24, 2021, CN2021112144653, filed October 19, 2021, CN2021112236320, filed October 20, 2021, CN2021114658736, filed December 3, 2021, and CN2022100034604, filed January 4, 2022. [Background technology]

[0003] In 2020, there were approximately 19.3 million new cases of cancer worldwide, with nearly 10 million deaths. Currently, standardized treatments are available for most early-stage cancer patients. However, due to factors such as disease progression and drug resistance or insensitivity to conventional treatment modalities, some cancer patients, especially those with advanced cancer, have limited treatment options and less than ideal therapeutic effects. Therefore, the research into tumor treatment drugs with new targets, new mechanisms, and new structures has always been an urgent challenge in the field of tumor therapy.

[0004] EphA2 is a novel tumor-associated target that has attracted much attention in recent years, reported to regulate processes related to carcinogenesis and tumor progression. Unlike most Eph kinases, EphA2 is primarily confined to rapidly proliferating epithelial cells in adults. Studies have shown that EphA2 is overexpressed in various cancers, including prostate, lung, esophageal, colorectal, cervical, ovarian, and skin cancers. High EphA2 expression in tumors is directly associated with clinical symptoms such as poor prognosis, increased risk of metastasis, and shortened survival in cancer patients. The signaling pathway comprised by EphA2 and its ligand, ephrin A1, induces and suppresses various downstream kinases, including ERK and AKT, thereby controlling the migration, activity, and proliferation of malignant cells. Therefore, EphA2 can serve as a target for drug delivery and tumor therapy in tumor tissues. Summary of the Invention

[0005] The present invention provides a compound of formula (V) or a pharmaceutically acceptable salt thereof: [ka] Selected from Xi, Xii and Xiii are each independently selected from Cys, hCys, βCys, Pen, Dap and N-methyl-Dap, and Xi, Xii and Xiii are not simultaneously Cys; Or, [ka] Selected from and Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, Pen, Dap, and N-methyl-Dap.

[0006] The present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, Pen, Dap, and N-methyl-Dap.

[0007] The present invention also provides a compound represented by formula (I') or a pharmaceutically acceptable salt thereof: [ka] During the ceremony, Xi, Xii, and Xiii are each independently selected from Cys, hCys, βCys, Pen, Dap, and N-methyl-Dap; The condition is that Xi, Xii and Xiii are not simultaneously Cys.

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

[0009] The present invention also provides a compound represented by the formula: or a pharmaceutically acceptable salt thereof. [ka] JPEG0007734891000006.jpg217170JPEG0007734891000007.jpg74170

[0010] The present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a solid tumor in which EphA2 is overexpressed.

[0011] The present invention also provides a method for treating a solid tumor in which EphA2 is overexpressed in a subject in need thereof, comprising providing the subject with a therapeutically effective amount of the above-described compound or a pharmaceutically acceptable salt thereof.

[0012] The present invention refers to the following manufacturing method. [ka] JPEG0007734891000009.jpg102170

[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 EphA2 protein 1. Purpose of the test The affinity of a test substance for the target protein EphA2 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) EphA2 (Cat. No. 13926-H08HD, Sino Biological)

[0015] 3. Test Plan In this study, the amino coupling method was used: the target protein EphA2 was directly immobilized on a CM5 chip using a Biacore 8K. The test substance was then diluted to a predetermined concentration gradient in a buffer solution (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 EphA2. The final data was 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 EphA2 and have significant growth inhibitory effects on cells cultured in vitro in tumor cell lines NCI-H1975 and SK-OV-3. They also exhibit significant tumor growth inhibitory effects in human ovarian cancer SK-OV-3 cell subcutaneous xenograft tumor models and human prostate cancer PC-3 subcutaneous xenograft tumor models, with clear dose-response relationships. The compounds of the present invention exhibit good pharmacokinetic properties, rapid clearance in plasma, low accumulation, and low MMAE release. They also have good stability in liver microsomes, plasma, and whole blood in vitro, demonstrating good potential for drug discovery. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows tumor growth curves of the compounds of the present invention in a human prostate cancer cell subcutaneous xenograft tumor model. [Figure 2] FIG. 2 shows the change curve of animal body weight in a human prostate cancer cell subcutaneous xenograft tumor model in response to the compound of the present invention. [Figure 3] FIG. 3 shows the tumor growth curve of the compound of the present invention in a BALB / c nude mouse model of human ovarian cancer SK-OV-3 cell subcutaneous xenograft tumors. [Figure 4] FIG. 4 shows the weight change rate of animals in a BALB / c nude mouse model of human ovarian cancer SK-OV-3 cell subcutaneous xenograft tumors in response to the compound of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[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 can 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] "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.

[0030] The therapeutic dose of a compound of the invention may be determined, for example, by the particular therapeutic application, the manner of administration of the compound, the patient's condition, the judgment of the prescribing physician, etc. The proportion or concentration of a compound of the invention in a pharmaceutical composition is not necessarily constant and will depend on various factors, including dosage, chemical characteristics (e.g., hydrophobicity), and route of administration.

[0031] The term "treatment" means administering a compound or formulation according to the present invention to improve or eliminate a disease or one or more symptoms associated with said disease, and includes the following: (i) arresting the disease or disease state, i.e., arresting its progression; (ii) alleviating the disease or disease state, i.e., causing the disease or disease state to disappear.

[0032] The term "therapeutically effective amount" refers to an amount of a compound of the present invention that (i) treats a particular disease, condition, or disorder, (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The "therapeutically effective amount" of a compound of the present invention will vary depending on the compound, the condition and its severity, the mode of administration, and the age of the mammal being treated, but can be determined by one of ordinary skill in the art based on their knowledge and the present disclosure.

[0033] In the present invention, unless otherwise specified, throughout the specification and the appended claims, the term "comprise" and its English equivalents, such as "comprises" and "comprising," are to be understood in an open and non-exclusive sense, meaning "including, but not limited to."

[0034] As used throughout the specification, "one embodiment" or "an embodiment" or "another embodiment" or "in some embodiments" means that at least one embodiment includes the associated particular referenced element, structure, or feature described in that embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in some embodiments" in different places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, particular elements, structures, or features may be combined in any suitable manner in one or more embodiments.

[0035] Unless otherwise specified, the term "isomer" is intended to include geometric isomers, cis / trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

[0036] 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.

[0037] Unless otherwise specified, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of one another.

[0038] 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.

[0039] 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.

[0040] Unless otherwise specified, "(+)" represents dextrorotatory, "(-)" represents levorotatory, and "(±)" represents racemic.

[0041] Unless otherwise specified, [ka]

[0042] 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%.

[0043] 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%.

[0044] 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).

[0045] The compounds of the present invention may contain unnatural proportions of isotopes of one or more atoms that constitute the compounds. For example, tritium ( 3H), iodine-125( 125 I) or carbon-14 ( 14 The compounds can be labeled with radioactive isotopes such as CI, ...

[0046] 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] Combinations of the above connecting groups, substituents and / or variants thereof are permissible only if such combinations result in stable compounds.

[0047] 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: [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.

[0048] Unless otherwise specified, the term "C 1~4 The term "alkyl group" refers to a linear or branched saturated hydrocarbon group consisting of 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.

[0049] Unless otherwise specified, in the present invention, the amino acid X and TATA are connected by a sulfhydryl group on the amino acid residue, for example, when Xi is Pen, [ka] Represents.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] The solvent used in the present invention may be a commercially available product.

[0054] 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, MMAE represents monomethylauristatin E, and the specific structure is [ka] In the formula, 1Nal represents 1-naphthylalanine, hArg or 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 or D-Asp represents D-aspartic acid, Fmoc represents a 9-fluorenylmethyloxycarbonyl group, Boc represents a tert-butoxycarbonyl group, Trt represents a trityl group, Pbf represents a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group, and PBS represents phosphate buffered saline.

[0055] 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.

[0056] Example 1 [ka]

[0057] Synthetic Route: [ka]

[0058] Step 1: Synthesis of the TFA salt of Peptide 1 The polypeptides are synthesized using standard stepwise synthesis methods.

[0059] 1) DMF (20 mL) was added to a vessel containing Rink amide MBHA resin (0.5 mmol, 1.85 g, substrate: 0.27 mmol / g) and the resin was allowed to swell for 2 hours.

[0060] 2) Suction filter, then rinse with DMF three times, bubbling with nitrogen for 30 seconds each time.

[0061] 3) Add 20% piperidine / DMF, then react for 30 minutes.

[0062] 4) Filter by suction, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0063] 5) Add a solution of Fmoc-protected amino acid, bubble with nitrogen for 30 seconds, then add a condensing agent, bubble with N2 and react for about 1 hour.

[0064] 6) Repeat steps 2 to 5 to condense the next amino acid. [Table 1]

[0065] Polypeptide cleavage and purification: 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.

[0066] 2) The polypeptide is precipitated with ice-cold isopropyl ether and centrifuged (3000 rpm, 3 minutes).

[0067] 3) Wash twice more with isopropyl ether.

[0068] 4) Dry to obtain the TFA salt of compound Peptide 1.

[0069] Step 2: Synthesis of Peptide 2 acetate salt Crude Peptide 1 TFA salt (1.40 g, 470 μmol) was dissolved in 50% MeCN / HO (500 mL). TATA (140 mg, 560 μmol) was slowly added to the stirring solution at room temperature. The reaction mixture was stirred at room temperature for 30 min. The pH was then adjusted to 8 with NH4HCO3, and the reaction mixture was continued to stir at room temperature for 12 h. When LCMS showed complete reaction, stirring was stopped and the acetate salt of Peptide 2 was purified by reverse-phase preparative chromatography (A: 0.075% TFA in HO, B: CH3CN). [Table 2]

[0070] The synthesis of the acetate salts of the other two polypeptides, Peptides 3 and 4, is similar to that of Peptide 2. [Table 3]

[0071] Step 3: Synthesis of the TFA salt of intermediate INT_1 Compound 1-1 (200.0 mg, 178.0 μmol) is dissolved in DMF (5 mL), and DIEA (31.0 μL, 178.0 μmol) is added at 0°C and stirred for 10 minutes. At the same time, compound 1-2 (290.4 mg, 890.1 μmol) is 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 is added dropwise to the reaction solution of compound 1-2 while stirring. This reaction solution is stirred at 0°C for 30 minutes. The reaction solution is filtered to remove insoluble residues, and the filtrate is directly purified by reverse phase preparative chromatography (TFA system) to obtain the TFA salt of compound INT_1.

[0072] Step 4: Synthesis of acetate salt of PDC_1 The TFA salt of the polypeptide compound peptide 2 (87.0 mg, 27.1 μmol) was dissolved in DMF (0.5 mL), and then DIEA (12.9 μL, 74.4 μmol) was added and stirred at room temperature for 10 minutes. The TFA salt of the compound INT_1 (36.2 mg, 27.1 μmol) was then dissolved in DMF (0.3 mL) and added dropwise to the above TFA salt of the polypeptide compound peptide 2, followed by stirring at room temperature for 2 hours. When LC-MS showed the reaction was complete, the reaction was stopped, the reaction solution was filtered to remove insoluble residues, and the filtrate was directly purified by reverse-phase preparative chromatography (TFA system), lyophilized, and then converted to the AcOH salt via preparative chromatography to obtain the acetate salt of PDC_1.

[0073] Example 2 [ka]

[0074] Example 3 [ka] The preparation methods for the acetate salts of PDC_2 and PDC_3 are the same as those for the acetate salt of PDC_1. [Table 4]

[0075] Example 4 [ka]

[0076] Synthetic Route: [ka] JPEG0007734891000025.jpg200170

[0077] 1. Synthesis of intermediate 3: Polypeptide synthesis: The polypeptides are synthesized using standard stepwise synthesis methods.

[0078] 1) Add DCM to a vessel containing CTC Resin (10.0 mmol, 10.0 g) and Fmoc-Asp(OAll)-OH.

[0079] 2) Add DIEA (4.00 eq) and then stir for 2 hours.

[0080] 3) Add MeOH (10.0 mL) and stir for 30 minutes.

[0081] 4) Filter by suction, then rinse with DMF three times, bubbling with nitrogen for 30 seconds each time.

[0082] 5) Add 20% piperidine / DMF and then react for 30 minutes.

[0083] 6) Filter by suction, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0084] 7) Add a solution of Fmoc-protected amino acid, bubble with nitrogen for 30 seconds, then add a condensing agent, bubble with N2 and react for about 1 hour.

[0085] 8) Repeat steps 4-7 to condense the next amino acid until the Fmoc-Lys(Alloc)-OH connection is complete.

[0086] 9) Suction filter, then rinse with DMF three times and DCM three times, bubbling with nitrogen for 30 seconds each time.

[0087] 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.

[0088] 11) Suction filter, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0089] 12) Ring closure: Add the condensation agent solution HATU (2.85 eq.) and DIEA (6.0 eq.), bubble N2, and react for about an hour.

[0090] 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 5]

[0091] Cutting and Purification: 5) Add cleavage buffer solution (20% HFIP / DCM) to the flask containing the side-chain protected polypeptide and stir at room temperature twice for 30 minutes. The solution is collected, spun dry, and separated by reverse-phase preparative chromatography to give intermediate 3. [Table 6]

[0092] 2. Synthesis of intermediate 4 Polypeptide synthesis: The polypeptides are synthesized using standard stepwise synthesis methods.

[0093] 1) DMF (20 mL) is added to a vessel containing Rink amide MBHA resin (0.5 mmol, 1.85 g) and the resin is allowed to swell for 2 hours.

[0094] 2) Suction filter, then rinse with DMF three times, bubbling with nitrogen for 30 seconds each time.

[0095] 3) Add 20% piperidine / DMF, then react for 30 minutes.

[0096] 4) Filter by suction, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0097] 5) Add the Fmoc-amino acid solution and bubble with nitrogen for 30 seconds, then add the condensing agent and bubble with N2 to react for about an hour.

[0098] 6) Repeat steps 2 to 5 to condense the next amino acid. [Table 7]

[0099] Polypeptide cleavage and purification: 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.

[0100] 2) The polypeptide is precipitated with ice-cold isopropyl ether and centrifuged (3000 rpm, 3 minutes).

[0101] 3) Wash twice more with isopropyl ether.

[0102] 4) The crude polypeptide is dried to obtain the TFA salt of intermediate 4 (crude).

[0103] 3. Synthesis of TFA salt of intermediate 5 The crude TFA salt of intermediate 4 (1.40 g, 470 μmol) was dissolved in 50% MeCN / HO (500 mL). TATA (140 mg, 560 μmol) was slowly added to the stirring solution at room temperature. The reaction mixture was stirred at room temperature for 30 min. The pH was then adjusted to 8 with NH4HCO3, and the reaction mixture was continued to stir at room temperature for 12 h. When LCMS showed the reaction was complete, the stirring was stopped and the crude TFA salt of intermediate 5 was purified by reverse-phase preparative chromatography (A: 0.075% TFA / HO, B: CH3CN). [Table 8]

[0104] 4. Synthesis of acetate salt of compound PDC_4 Dissolve the TFA salt of intermediate 5 (60.0 mg, 18.6 μmol) in DMF (0.3 mL), then add DIEA (12.9 μL, 74.4 μmol) and stir at room temperature for 10 minutes. Then, dissolve the TFA salt of compound INT_1 (24.8 mg, 18.6 μmol) in DMF (0.2 mL) and add it dropwise to the reaction solution of intermediate 5 above, then stir at room temperature for 2 hours, filter the reaction solution to remove insoluble residues, and directly purify the filtrate by reverse phase preparative chromatography (TFA system), lyophilize, and then convert it into the acetate salt by preparative chromatography to obtain the acetate salt of compound PDC_4.

[0105] Example 5 [ka]

[0106] Synthetic Route: [ka]

[0107] Synthesis of intermediate 6: The polypeptides are synthesized using standard stepwise synthesis methods.

[0108] 1) DMF (30 mL) was added to a vessel containing Rink amide MBHA resin (0.5 mmol, 1.8 g, substrate: 0.28 mmol / g) and the resin was allowed to swell for 2 hours.

[0109] 2) Suction filter, then rinse with DMF three times, bubbling with nitrogen for 30 seconds each time.

[0110] 3) Add 20% piperidine / DMF, then react for 30 minutes.

[0111] 4) Filter by suction, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0112] 5) Add the Fmoc-protected amino acid solution, and after 30 seconds, add the condensing agent. Bubble N2 and react for approximately 1 hour.

[0113] 6) Repeat steps 2 to 5 to condense the next amino acid. [Table 9]

[0114] The Fmoc protecting group was removed with 20% piperidine / DMF for 30 minutes. The condensation reaction was detected by a color reaction, and the resin was washed with DMF 3-5 times after each reaction.

[0115] Polypeptide cleavage and purification: 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.

[0116] 2) The polypeptide is precipitated with ice-cold isopropyl ether and centrifuged (3000 rpm, 3 minutes).

[0117] 3) Wash twice more with isopropyl ether.

[0118] 4) The crude product is dried to obtain intermediate 6.

[0119] Synthesis of TFA salt of intermediate 7: Crude polypeptide intermediate 6 (1.20 g, 418 μmol) was dissolved in 50% MeCN / HO (500 mL). TATA (150 mg, 600 μmol) was slowly added to the stirring solution at room temperature. The reaction mixture was stirred at room temperature for 30 min. The pH was then adjusted to 8 with NHHCO, and the reaction mixture was continued to stir at room temperature for 12 h. When LCMS showed complete reaction, the stirring was stopped and the mixture was purified by reverse-phase preparative chromatography (TFA system) to give the TFA salt of intermediate 7. [Table 10]

[0120] Synthesis of acetate salt of PDC_5: The TFA salt of polypeptide intermediate 7 (60.6 mg) and the TFA salt of compound INT_1 (25.0 mg) were dissolved in DMF (0.7 mL) and DIEA (9.68 mg, 74.9 μmol, 13.0 μL) was added. After stirring at room temperature for 2 hours, LC-MS showed the reaction was complete. The reaction was stopped and the reaction solution was filtered to remove insoluble residues. The filtrate was directly purified by reverse-phase preparative chromatography (TFA system), lyophilized, and then converted to the acetate salt via preparative chromatography to obtain the acetate salt of PDC_5.

[0121] Example 6 [ka] The acetate salt of PDC_6 was prepared by following the synthesis method of Example 5, except that in the order of ingredients in Table 9 of Example 5, raw material 1 was replaced with Fmoc-Cys(Trt)-OH (2.0 eq.) and raw material 15 was replaced with Fmoc-hCys(Trt)-OH (3.0 eq.).

[0122] Example 7 [ka]

[0123] Synthetic Route: [ka]

[0124] Synthesis of intermediate 8: The polypeptides are synthesized using standard stepwise synthesis methods.

[0125] 1) DMF (30 mL) was added to a vessel containing Rink amide MBHA resin (0.5 mmol, 1.5 g, substrate: 0.33 mmol / g) and the resin was allowed to swell for 2 hours.

[0126] 2) Suction filter, then rinse with DMF three times, bubbling with nitrogen for 30 seconds each time.

[0127] 3) Add 20% piperidine / DMF, then react for 30 minutes.

[0128] 4) Filter by suction, then rinse with DMF five times, bubbling with nitrogen for 30 seconds each time.

[0129] 5) Add the Fmoc-protected amino acid solution, and after 30 seconds, add the condensing agent. Bubble N2 and react for approximately 1 hour.

[0130] 6) Repeat steps 2 to 5 to condense the next amino acid. [Table 11]

[0131] The Fmoc protecting group was removed with 20% piperidine / DMF for 30 minutes. The condensation reaction was detected by a color reaction, and the resin was washed with DMF 3-5 times after each reaction.

[0132] Polypeptide cleavage and purification: 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.

[0133] 2) The polypeptide is precipitated with ice-cold isopropyl ether and centrifuged (3000 rpm, 3 minutes).

[0134] 3) Wash twice more with isopropyl ether.

[0135] 4) The crude product is dried to obtain intermediate 8.

[0136] Synthesis of TFA salt of intermediate 9: Crude polypeptide intermediate 8 (1.50 g, 500 μmol) was dissolved in 50% MeCN / HO (500 mL). TATA (186 mg, 750 μmol) was slowly added to the stirring solution at room temperature. The reaction mixture was stirred at room temperature for 30 min. The pH was then adjusted to 8 with NHHCO, and the reaction mixture was continued to stir at room temperature for 12 h. When LCMS showed complete reaction, the stirring was stopped and the mixture was purified by reverse-phase preparative chromatography (TFA system) to give the TFA salt of intermediate 9. [Table 12]

[0137] Synthesis of acetate salt of PDC_7: The TFA salt of polypeptide intermediate 9 (40.0 mg) and the TFA salt of compound INT_1 (17.2 mg) were dissolved in DMF (0.5 mL) and DIEA (6.36 mg, 49.2 μmol) was added. After stirring at room temperature for 5 hours, LC-MS showed the reaction was complete. The reaction was stopped and the reaction solution was filtered to remove insoluble residues. The filtrate was directly purified by reversed-phase preparative chromatography (TFA system), lyophilized, and then converted to the acetate salt by preparative chromatography to obtain the acetate salt of PDC_7.

[0138] Example 8 [ka] The acetate salt of PDC_8 was prepared by following the synthesis method of Example 7, except that in the order of ingredients in Table 11 of Example 7, raw material 1 was replaced with Fmoc-Pen(Trt)-OH (2.85 eq.) and raw material 15 was replaced with Fmoc-Cys(Trt)-OH (2.85 eq.). [Table 13]

[0139] Biological Testing Test Example 1: Test of the binding ability of the compound of the present invention to EphA2 protein 1. Purpose of the test The affinity of a test substance for the target protein EphA2 is detected using the SPR method.

[0140] 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) EphA2 (Cat. No. 13926-H08HD, Sino Biological)

[0141] 3. Test Plan In this study, the amino coupling method was used: the target protein EphA2 was directly immobilized on a CM5 chip using a Biacore 8K. The test substance was then diluted to a predetermined concentration gradient in a buffer solution (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 EphA2. The final data was subjected to kinetic fitting analysis using a 1:1 model using Biacore Insight Evaluation Software (v2.0.15.12933).

[0142] 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.

[0143] 2) Activate the CM5 chip with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL / min for 420 seconds.

[0144] 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.

[0145] 4) Block the CM5 chip with 1 M ethanolamine at a flow rate of 10 μL / min for 420 seconds.

[0146] 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.

[0147] 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.

[0148] 7) All results will be subjected to kinetic fitting analysis using a 1:1 model.

[0149] 5. Test Results The test data of five effective concentrations were selected, and the kinetics fitting analysis was performed on the compound of the present invention using Biacore Insight Evaluation Software (V2.0.15.12933) with a 1:1 model. The results are shown in Table 14. [Table 14]

[0150] In conclusion, the compounds of the present invention have very strong binding activity to EphA2.

[0151] Test Example 2: In vitro antiproliferative activity of compounds of the present invention against NCI-H1975 and SK-OV-3 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 NCI-H1975 and SK-OV-3.

[0152] 2. Study Design: Cell culture: The tumor cell lines are cultured under the culture conditions shown in Table 15 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 15]

[0153] Cell plating: Cells in the logarithmic growth phase were harvested and centrifuged at 1000 rpm for 3 minutes at room temperature. The supernatant was aspirated, and the cells were resuspended in 5 mL of medium. 20 μL of cell suspension was aspirated and mixed 1:1 with trypan blue for 3 minutes to detect cell viability and count live cells. The cell density was adjusted to 4000 cells / well for NCI-H1975 or 5000 cells / well for SK-OV-3. 90 μL of cell suspension was added to each well of the culture plate, and culture medium without cells was added to blank control wells. The culture plates were then incubated overnight in an incubator at 37°C without CO2 or at 37°C with 5% CO2 and 100% relative humidity, respectively.

[0154] Compound storage plate preparation: Prepare a 400x compound stock plate. Dilute test compounds in a gradient from highest to lowest concentration in DMSO. [Table 16]

[0155] 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 compound 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 as shown in Table 1. Add 10 μL of DMSO-cell culture medium mixture to the solvent control and blank control. The final DMSO concentration is 0.25%. Return the 96-well cell plate to the incubator and culture for 72 hours.

[0156] 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.

[0157] 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

[0158] The test results are shown in Table 17. [Table 17]

[0159] In conclusion, the compounds of the present invention have obvious antiproliferative effects on the tumor cell lines NCI-H1975 and SK-OV-3 cultured cells in vitro.

[0160] Test Example 3: In vivo pharmacodynamic study of the compounds of the present invention in a BALB / c nude mouse model of human prostate cancer PC-3 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 prostate cancer PC-3 cell subcutaneous xenograft tumor model.

[0161] Cell culture. Human prostate cancer PC-3 cells (ATCC-CRL-1435) were cultured in vitro by wall adhesion. The culture conditions were F-12K medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured at 37°C in a 5% CO2 incubator. Cells were passaged twice a week 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.

[0162] The animals were male BALB / c nude mice, 6-8 weeks old, weighing 22-27g. A total of 70 mice were inoculated for the pharmacodynamics test, and 40 pharmacodynamic mice were included. They were provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

[0163] Tumors were inoculated in 0.1 mL (10 × 10 6 PC-3 cells (1000 cells / 10 ... 3 Group dosing was initiated when the animals reached 100 mg / kg, with each group consisting of 8 animals (n=8).

[0164] The dose volume is adjusted based on the animal's weight (dose volume = 10 μL / g).

[0165] Drug Preparation: Compounds are prepared as 1 mg / mL homogeneous solutions in 50 mM acetate buffer (pH=5), 10% sucrose, and stored in a refrigerator at -80°C. On the day of administration, they are diluted to the corresponding concentration for IV administration.

[0166] For pharmacodynamic studies, tumors with a mean volume of 150–200 mm 3 When the dose reaches 100mg / kg, the group-specific administration will start. The doses will be 0.25mg / kg, 0.5mg / kg, and 1.5mg / kg. The administration frequency will be QW x 3 weeks.

[0167] The test results are shown in Figures 1 and 2.

[0168] In conclusion, the present study showed that the three dose groups of the compound of the present invention showed significant tumor growth inhibitory effects in a human prostate cancer PC-3 cell subcutaneous xenograft tumor model, and showed dose-relationships in the 0.25 mg / kg, 0.5 mg / kg, and 1.5 mg / kg groups.

[0169] Test Example 4: In vivo pharmacodynamic study of the compounds of the present invention in a BALB / c nude mouse model of human ovarian cancer SK-OV-3 cell subcutaneous xenograft tumors The purpose of the study was to evaluate the in vivo pharmacodynamics of the investigational drug in a human ovarian cancer SK-OV-3 cell subcutaneously xenograft tumor model.

[0170] Cell culture. Human ovarian cancer SK-OV-3 cells (ECACC-91091004) were cultured in vitro in monolayers in McCoy's 5a medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 incubator. Cells were passaged twice weekly using trypsin-EDTA digestion. When the cell confluency reached 80%-90% and the desired number of cells was reached, the cells were harvested, counted, and inoculated.

[0171] The animals were female BALB / c nude mice, 6-8 weeks old, weighing 18-21 g, provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

[0172] Tumors were inoculated with 0.2 mL (10 × 10 6 SK-OV-3 cells (1000 cells / mL) were subcutaneously inoculated into the right back of each mouse.

[0173] The dose volume is adjusted based on the animal's weight (dose volume = 10 μL / g).

[0174] Drug Preparation: Test compounds are prepared as 1 mg / mL homogeneous solutions in 50 mM acetate buffer (pH=5) with 10% sucrose and stored in a refrigerator at -80°C. On the day of administration, the compounds are diluted to the corresponding concentration for IV administration.

[0175] For pharmacodynamic studies, tumors with a mean volume of 150–200 mm 3 When the animals reached the age of 18, group administration was initiated, with each group consisting of 8 animals (n=8). The administration doses were 1 mg / kg and 3 mg / kg. The administration frequency was QW x 6 weeks.

[0176] The test results are shown in Figures 3 and 4.

[0177] In conclusion, the present study showed that the two dose groups of the compound of the present invention showed significant tumor growth inhibitory effects in a human ovarian cancer SK-OV-3 cell subcutaneous xenograft tumor model, and showed dose-related effects in the 1 mg / kg and 3 mg / kg groups.

[0178] Test Example 5: In vivo pharmacokinetic analysis of the compound of the present invention in mice A. Purpose of the test The in vivo pharmacokinetics of compounds of the invention are tested in CD-1 mice.

[0179] B. Test Procedure The pharmacokinetic properties of the compounds were tested in CD-1 mice after intravenous and oral administration using a standard protocol. The compounds of the present invention were prepared as clear solutions in 50 mM acetate buffer (pH = 5) and 10% sucrose. Two mice were administered a single intravenous injection of the test compound at 2 mg / kg. Whole blood was collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration, and plasma was prepared. The concentrations of the test compound and its potential metabolite MMAE were analyzed by LC-MS / MS. The peak blood concentration (C) was calculated using the software Phoenix WinNonlin. max ), clearance (Cl), half-life (T 1 / 2 ), volume of distribution (Vdss), area under the drug blood concentration-time curve (AUC 0~last ) and other pharmacokinetic parameters are calculated.

[0180] C. Test Results The test results are shown in Table 18. [Table 18]

[0181] In conclusion, the compounds of the present invention have a short half-life in the blood of CD-1 mice, are rapidly cleared, and release of MMAE into the plasma of CD-1 mice is very small.

[0182] Test Example 6: Pharmacokinetic analysis of the compound of the present invention in rat plasma The purpose of the study is to test the in vivo pharmacokinetics of the compounds of the present invention in SD rats. 6.1 Test the pharmacokinetic properties of compounds in rats after intravenous injection and oral administration using standard protocols. Prepare the test compound as a clear solution in 50 mM acetate buffer (pH=5) with 10% sucrose. Administer a single intravenous injection of the test compound at 2 mg / kg to two SD rats. At 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration, collect whole blood, prepare plasma, and analyze the concentrations of the test compound and its potential metabolite MMAE using LC-MS / MS. Calculate the pharmacokinetic parameters using Phoenix WinNonlin software.

[0183] The test results are shown in Table 19. [Table 19]

[0184] 6.2 The pharmacokinetic properties of the compound in rats after intravenous injection and oral administration were tested using a standard protocol. The test compound was prepared as a clear solution in 50 mM acetate buffer (pH=5) with 10% sucrose. Three male SD rats were administered a single intravenous injection of the test compound at 2.5 mg / kg, 3.75 mg / kg, or 5 mg / kg. Whole blood was collected at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 4 hours, and 24 hours after 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.

[0185] The test results are shown in Table 20. [Table 20]

[0186] In conclusion, the compounds of the present invention have a short blood half-life in rats, are rapidly cleared, the PDC exposure increases with increasing dose, have good metabolic stability, and release a small amount of MMAE into rat plasma, which shows a certain degree of dose correlation.

[0187] Test Example 7: Pharmacokinetic analysis of the compound of the present invention in cynomolgus monkey plasma A. Purpose of the test The in vivo pharmacokinetics of compounds of the invention are tested in cynomolgus monkeys.

[0188] B. Test Procedure The pharmacokinetic properties of the compounds were tested in cynomolgus monkeys after intravenous injection and oral administration using standard protocols. The compounds of the present invention were prepared as clear solutions in 50 mM acetate buffer (pH=5) and 10% sucrose. Two cynomolgus monkeys were given a single intravenous injection of the test compound at 1 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 test compound and its potential metabolite MMAE were analyzed using LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software.

[0189] C. The test results are shown in Table 20. [Table 21]

[0190] In conclusion, when intravenously injected, the compounds of the present invention have a short half-life in the blood of cynomolgus monkeys, are rapidly cleared, and release of MMAE into the plasma of cynomolgus monkeys is very low.

[0191] Test Example 8: 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 rat, cynomolgus monkey and human plasma.

[0192] B. Test Procedure 2 μL of the test compound (100 μM) working solution 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 SD rat, cynomolgus monkey, and human blank plasma 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, 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 semi-quantitatively using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0193] C. Test Results The test results are shown in Table 21. [Table 22]

[0194] Using the above method, the incubation time of the test compound with human plasma was extended to 72 hours, and the test results are shown in Table 22. [Table 23]

[0195] In conclusion, the compounds of the present invention have excellent stability in the plasma of three species: SD rats, cynomolgus monkeys and humans.

[0196] Test Example 9: Stability analysis of the compounds of the present invention in whole blood of different species A. Purpose of the test The stability of the compounds of the present invention in whole blood of SD rats and cynomolgus monkeys is tested.

[0197] B. Test Procedure 2 μL of the test compound (100 μM) working solution 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 whole blood from SD rats and cynomolgus monkeys 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, and a stop solution was added to precipitate proteins. The plates were centrifuged for 20 minutes. 100 μL of the supernatant was removed and diluted with 300 μL of ultrapure water. After uniform mixing, the supernatant was analyzed by LC-MS / MS. The concentration of the test compound in the samples was measured semi-quantitatively using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0198] C. Test Results The test results are shown in Table 23. [Table 24]

[0199] In conclusion, the compounds of the present invention have excellent stability in the plasma of SD rats and cynomolgus monkeys.

[0200] Test Example 10: Metabolic stability of the compound of the present invention in cynomolgus monkey and human hepatocytes A. Purpose of the test The metabolic stability of the compounds of the present invention in cynomolgus monkey and human hepatocytes is investigated.

[0201] 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 cynomolgus monkey and human hepatocytes 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 the test sample 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%.

[0202] At the end of the incubation period, the incubation plates were removed, and 25 μL of the compound-cell mixture was added to each sample plate containing 125 μL of stop solution. 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 for 10 minutes, then centrifuged at 3220 g for 20 minutes. The supernatants of the test samples were diluted 1:3 with pure water. All samples were mixed uniformly and then analyzed by LC-MS / MS.

[0203] The concentration of the compounds of the present invention in samples was measured semiquantitatively using liquid chromatography-tandem mass spectrometry (LC-MS / MS), without the need for a standard curve 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 analyte and internal standard, chromatogram acquisition, and chromatogram integration were processed using Analyst software (Sciex, Framingham, MA, USA).

[0204] C. Test Results The test results are shown in Table 24. [Table 25]

[0205] In conclusion, the compounds of the present invention have excellent metabolic stability in liver microsomes of cynomolgus monkeys and humans.

Claims

1. A compound of formula (V) or a pharmaceutically acceptable salt thereof, 【Chemical 1】 During the ceremony, 【Chemistry 2】 Selected from Xi, Xii, and Xiii are each independently a compound selected from Cys, hCys, βCys, and Pen, or a pharmaceutically acceptable salt thereof.

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

3. A medicament for treating solid tumors in which EphA2 is overexpressed, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.

4. A drug for treating solid tumors in which EphA2 is overexpressed, comprising the compound of claim 2 or a pharmaceutically acceptable salt thereof.

5. A compound represented by the following formula: 【Chemistry 4】 【change】 【change】 【change】 Or, 【change】

Images