RU2837445C2 - Conjugated drug based on tricyclic polypeptide and its use - Google Patents

Abstract

FIELD: chemistry; pharmaceutics.

SUBSTANCE: invention relates to a conjugated drug based on a tricyclic polypeptide, particularly a compound of formula (III), or a pharmaceutically acceptable salt thereof, and use thereof in treating solid tumours in which nectin-4 is overexpressed.

EFFECT: what is presented is a conjugated drug based on a tricyclic polypeptide for treating solid tumours in which nectin-4 is overexpressed.

10 cl, 4 dwg, 16 tbl, 9 ex

Description

Field of technology

The present invention relates to a conjugated drug based on a tricyclic polypeptide and its use, in particular to a compound represented by formula (III) or a pharmaceutically acceptable salt thereof.

State of the art

Nectin-4 (poliovirus receptor-like protein 4, PVRL4) is a newly emerging tumor-associated target in recent years and belongs to the nectin protein family, which includes four major subtypes, i.e. nectins 1-4. Together with nectin-like molecule (Necl), nectin-4 is an immunoglobulin-like cell adhesion molecule, playing an essential role in the formation and maintenance of intercellular adhesion and tight junctions. Nectins-1, -2 and -3 are widely expressed in normal human tissues, and nectin-4 is mainly expressed at high levels in embryos and placenta, but its expression level is significantly reduced in adults. Studies show that nectin-4 is overexpressed in various tumors such as bladder cancer, urothelial cancer, breast cancer, triple-negative breast cancer, lung cancer, gastric cancer, esophageal cancer, etc., making it a potential target for the treatment of these cancers. Currently, the biological antibody-drug conjugate enfortumab vedotin developed for this target was approved for sale in the United States in 2019. Its primary indication as the only marketed drug for this target is metastatic urothelial cancer, and clinical efficacy studies are also underway for several indications. Therefore, the development of chemotherapeutic drugs targeting nectin-4 has broad application prospects.

The essence of the invention

The present invention provides a compound represented by formula (III) or a pharmaceutically acceptable salt thereof,

,

where R ₁ is selected from H and C _1-3 alkyl;

R ₂ is selected from H, C _1-4 alkyl and ;

n is selected from 1, 2, 3, and 4; and

each of Xi, Xii and Xiii is independently selected from Cys, hCys, βCys and Pen;

MMAE - monomethyl auristatin E;

PABC - .

In some embodiments of the present invention, the above compound is selected from the structures represented by formulas (III-1) and (III-2),

, ,

where R ₁ , R ₂ , Xi, Xii and Xiii are defined herein.

The present invention further provides a compound or a pharmaceutically acceptable salt thereof selected from

, , .

The present invention further provides a pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a compound or a pharmaceutically acceptable salt thereof according to the present invention, for use in the treatment of solid tumors in which nectin-4 is overexpressed.

The present invention further provides the use of a compound or a pharmaceutically acceptable salt thereof according to the present invention, as well as a pharmaceutical composition according to the present invention, in the preparation of a medicament for the treatment of solid tumors in which nectin-4 is overexpressed.

The present invention further provides a method of treating solid tumors in which nectin-4 is overexpressed, comprising administering to a subject a compound or a pharmaceutically acceptable salt thereof of the present invention.

The present invention further provides a method for treating solid tumors in which nectin-4 is overexpressed, comprising administering to a subject a pharmaceutical composition of the present invention.

The present invention further provides a product comprising a compound or a pharmaceutically acceptable salt thereof of the present invention for use in the treatment of solid tumors in which nectin-4 is overexpressed.

The present invention further provides a product comprising the pharmaceutical composition of the present invention for use in the treatment of solid tumors in which nectin-4 is overexpressed.

The present invention further provides the following method for producing:

The present invention further provides the following testing method.

Test Method 1: Testing the Binding Ability of the Compounds of the Present Invention to Nectin-4 Protein

1. The purpose of the experiment

To test the affinity of the test compound for the target protein nectin-4 using the SPR method.

2. Materials and devices

Biacore 8K (GE Healthcare)

96-well plate (cat. no. 650101, Greiner Bio-One)

CM5 Chip (Cat. No. BR-1005-30, GE Healthcare)

Amine Immobilization Kit (Cat. No. BR-1000-50, GE Healthcare)

EDC

NHS

1 M ethanolamine

10 mM sodium acetate, pH 4.5 (Cat. No. BR-1003-50, GE Healthcare)

DMSO (cat. no. D4540, Sigma)

P20 (Cat. No. BR-1000-54, GE Healthcare)

PBS (Cat. No. BR-1006-72, GE Healthcare)

Nectin-4 (Cat. No. 1006-72, GE Healthcare)

3. Experimental protocol

In this experiment, the amino group immobilization method is adopted. The target protein nectin-4 is directly immobilized on the CM5 chip using Biacore 8K. The compound to be tested as an analyte is then diluted with a buffer solution (10 mM PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5% DMSO and 0.05% P20) to the required concentration gradient for multi-cycle kinetic detection, in which each cycle consists of sample injection for 180 seconds and dissociation for 180 seconds, and then the next cycle is performed, to obtain the affinity kinetic analysis data of the target protein nectin-4 and the compound to be tested. Using Biacore Insight evaluation software (version 2.0.15.12933), the final data were subjected to kinetic fit analysis based on a 1:1 model.

4. Methodology and procedure of the experiment

1) Preparation of buffer solution: 10 mM PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5% DMSO and 0.05% P20.

2) Activation of CM5 chip: CM5 chip was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL/min for 420 seconds.

3) Target protein binding: The target protein was diluted to 10 μg/mL with 10 mM sodium acetate (pH 4.5) and bound at a flow rate of 10 μL/min for 284 s. Channels #1, #2, and #3 on the chip were used in the experiment, and the binding results were 1639.9 RU, 1747.8 RU, and 1702.2 RU, respectively.

4) Blocking of CM5 chip: The CM5 chip is blocked with 1 M ethanolamine at a flow rate of 10 μl/min for 420 seconds.

5) Preparation of analyte concentrations: The compound to be tested is diluted with a buffer solution. The compound to be tested is diluted from 100 nM to 0.78 nM with a 2-fold gradient.

6) Injection sample analysis: Each concentration of the working solution of the compound to be tested corresponds to one cycle at a flow rate of 30 µL/min for binding for 180 seconds and dissociation for 180 seconds. The last cycle is a 5% DMSO solvent correction cycle.

7) All results are subjected to kinetic correspondence analysis based on a 1:1 model.

TECHNICAL EFFECT

The compounds of the present invention have a strong binding effect to nectin-4 and a pronounced anti-tumor cell proliferation activity in vitro; exhibit a strong anti-tumor effect in a subcutaneous tumor model in mice in vivo, and have excellent metabolic stability and PK properties in vitro.

Brief description of graphic materials

Fig. 1 shows a tumor growth curve with a compound of the present invention in a subcutaneous tumor xenograft model of human lung cancer cells NCI-H292.

Fig. 2 shows the weight change curve of animals with a compound of the present invention in a xenograft model of subcutaneous tumor from human lung cancer cells NCI-H292.

Fig. 3 shows the tumor growth curve with the compound of the present invention administered in groups when the average tumor volume reached about 500-600 mm ³ in a subcutaneous tumor xenograft model of human breast cancer cells MDA-MB-468 in BALB/c nude mice.

Fig. 4 shows the rate of change in weight of animals with the compound of the present invention administered in groups when the average tumor volume reached about 500-600 mm ³ in a xenograft model of subcutaneous tumor from human breast cancer cells MDA-MB-468 in nude BALB/c mice.

Definitions and description

Unless otherwise stated, the following terms and phrases used in this document are intended to have the following meanings. Unless otherwise stated, a particular term or phrase is not to be considered vague or unclear, but is to be interpreted in accordance with its generally accepted meaning. Where trade names appear in this document, they are intended to refer to their respective products or their active ingredients.

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic reaction or other problems or complications in accordance with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention prepared from the compound using the specific substituents disclosed herein and a relatively non-toxic acid or base. If the compounds of the present invention contain relatively acidic functional groups, base addition salts can be prepared by contacting such compounds with a sufficient amount of base in neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or the like. If the compounds of the present invention contain relatively basic functional groups, acid addition salts can be prepared by contacting such compounds with a sufficient amount of acid in neat solution or a suitable inert solvent. Certain specific compounds of the present invention contain basic and acidic functional groups and thus can be converted into any base or acid addition salt.

The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from the parent compound containing an acid radical or a basic group. Typically, such salts are prepared by reacting these compounds in the form of a free acid or base with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.

The term "pharmaceutically acceptable excipient" refers to an inert material that is administered with the active ingredient and facilitates the administration of the active ingredient, including, but not limited to, any glidant, sweetener, diluent, preservative, dye/coloring agent, flavor enhancer, surfactant, wetting agent, dispersing agent, disintegrating agent, suspending agent, stabilizer, isotonic agent, solubilizer, or emulsifier approved by the State Food and Drug Administration for use in humans or animals (e.g., pets). Non-limiting examples of an excipient include calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oil, and polyethylene glycol.

The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or their salts and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds of the present invention to the body.

The pharmaceutical composition of the present invention can be prepared by combining the compound of the present invention with suitable pharmaceutically acceptable excipients, for example, in the form of solid, semi-solid, liquid or gaseous compositions such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres, aerosols, and the like.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code as well as those amino acids that have been subsequently modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (e.g., an alpha carbon atom bonded to a hydrogen atom, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methylmethionine sulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics are chemical compounds whose structure differs from the general chemical structure of amino acids, but which function similarly to naturally occurring amino acids.

The amino acid sequences of the present invention comprise standard one-letter or three-letter codes for twenty natural amino acids.

The term "treatment" includes inhibiting, slowing, stopping, or reversing the progression or severity of an existing symptom or condition.

The term "therapeutically effective amount" or "effective amount" means an amount of a compound of the present invention that provides 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 may reduce the number of cancer cells; reduce the size of a tumor; inhibit (i.e., slow down and preferably stop to some extent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with cancer to some extent. To the extent that a drug can prevent the growth of existing cancer cells and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.

Unless otherwise specified, the term "isomers" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention encompasses all such compounds, including cis and trans isomers, (-) and (+) enantiomers, ( R ) and ( S ) enantiomers, diastereomers, ( D ) isomers, ( L ) isomers, as well as racemic mixtures and other mixtures, such as enantiomerically enriched or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as an alkyl group. All such isomers and mixtures thereof are included within the scope of the present invention.

Unless otherwise specified, the term "enantiomers" or "optical isomers" refers to stereoisomers that are mirror images of each other.

Unless otherwise noted, the term "cis-trans isomers" or "geometric isomers" derives from the inability of the double bond or single bond at the carbon atom forming the ring to rotate freely.

Unless otherwise specified, the term "diastereomer" refers to stereoisomers in which the molecules have two or more chiral centers and are not mirror images of each other.

Unless otherwise noted, "(+)" means dextrorotatory, "(-)" means levorotatory, and "(±)" means racemic mixture.

Unless otherwise stated, the absolute configuration of the stereocenter is represented by bonds in the form of wedge-shaped solid lines ( ) and connections in the form of wedge-shaped dotted lines ( ), and the relative configuration of the stereocenter is represented by bonds in the form of straight solid lines ( ) and connections in the form of straight dotted lines ( ); connections in the form of wedge-shaped solid lines ( ) or connections in the form of wedge-shaped dotted lines ( ) are represented by wavy lines ( ), or connections in the form of straight solid lines ( ) or connections in the form of straight dotted lines ( ) are represented by wavy lines ( ).

Unless otherwise specified, the term "enriched in one isomer," "isomerically enriched," "enriched in one enantiomer," or "enantiomerically enriched" means that one isomer or enantiomer is present in a content that is less than 100% and greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise stated, the term "isomeric excess" or "enantiomeric excess" refers to the difference in relative percentage values 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%, then the isomeric or enantiomeric excess (ee value) is 80%.

Optically active ( R )- and ( S )-isomers, as well as D- and L -isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. The desired enantiomer of a compound of the present invention can be prepared by asymmetric synthesis or derivatization using chiral auxiliaries, whereby the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to yield the pure desired enantiomer. Alternatively, if the molecule contains a basic functionality such as an amino group or an acidic functionality such as a carboxyl group, a diastereomeric salt is formed with the appropriate optically active acid or base, followed by separation of the diastereomers by conventional methods well known in the art, followed by recovery to yield the pure enantiomer. In addition, separation of enantiomers and diastereomers is usually accomplished by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (e.g. formation of carbamates from an amine).

The compounds of the present invention may contain non-naturally occurring isotopic fractions of atoms at one or more of the atoms comprising the compounds. For example, the compounds may be labeled with radioactive isotopes such as tritium ( ^3H ), iodine-125 ( ^125I ), or C-14 ( ^14C ). Also, for example, a deuterated drug may be formed by substituting deuterium for hydrogen, wherein the bond formed between a deuterium atom and a carbon atom is stronger than the bond formed between an ordinary hydrogen atom and a carbon atom, and the deuterated drug has advantages such as reduced toxic side effects, increased drug stability, increased therapeutic efficacy, and a long biological half-life, compared to a non-deuterated drug. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

If the listed joining groups do not specify the direction of their joining, then the direction of joining is arbitrary. For example, in , if the attachment group L is -MW-, -MW- can be attached to ring A and ring B in the same direction as the left-to-right reading order, forming , or it can be attached to ring A and ring B in the opposite direction from left to right reading order, forming Combinations of attached groups, substituents and/or their variants are permissible only if such combinations result in the production of stable compounds.

Unless otherwise stated, when a group has one or more attachment sites, any one or more sites of that group may be attached to other groups by chemical bonds. If the chemical bonds are non-positionally attached and H atoms are present at the attachment sites, the number of H atoms at the site will be reduced accordingly with the number of chemical bonds attached to form a group with the corresponding valence when the chemical bonds are added. Chemical bonds attaching sites to other groups may be represented by bonds in the form of straight solid lines ( ), connections in the form of straight dotted lines ( ) or wavy lines ( ). For example, the bond as a straight solid line in -OCH ₃ represents attachment to another group via an oxygen atom in the group; the bonds as straight dotted lines in represent attachment to other groups through both ends of the nitrogen atom in the group; wavy lines in represent attachment to other groups via carbon atoms in the phenyl group at positions 1 and 2.

Unless otherwise specified, the term "C _1-4 alkyl" represents a straight or branched saturated hydrocarbon group consisting of 1 to 4 carbon atoms. C _1-4 alkyl includes C _1-2 alkyl, C _1-3 alkyl, C _2-3 alkyl, and the like; which may be monovalent (e.g. methyl), divalent (e.g. methylene), or polyvalent (e.g. methine). Examples of C _1-4 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, sec-butyl, and tert-butyl), and the like.

Unless otherwise specified, the term "C _1-3 alkyl" represents a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. C _1-3 alkyl includes C _1-2 alkyl, C _2-3 alkyl, and the like; which may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of C _1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise stated, amino acids Xi, Xii and Xiii in the present invention are attached to TATA via a thiol group in the residue. For example, when Xi is Pen, both of And represent ; and when Xi is Cys, then both of And represent .

The structure of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional technical means in the art. For example, in the case of single crystal X-ray diffraction (SXRD), the diffraction intensity data of the cultured single crystal is collected using a Bruker D8 Venture diffractometer, wherein the light source is CuKα radiation and the scanning mode is φ/ω, and after collecting the corresponding data, the absolute configuration can be confirmed by further resolving the crystal structure using a direct method (Shelxs97).

The compounds of the present invention can be prepared by various synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combination with other chemical synthetic methods, and equivalents thereof well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention.

Compounds are named according to conventional nomenclature in the art or using ChemDraw® software, and for commercially available compounds, supplier catalog names are used.

The solvent used in the present invention is commercially available. The ratio of the mixed solvents provided in the present invention is a volume ratio. For example, 20% MeCN/ _H2O means that the volume of MeCN in the mixed solvent is 20%.

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

Detailed description of the invention

The following examples describe the present invention in detail, but they are not intended to impose any unfavorable limitation on the present invention. The present invention has been described in detail herein and specific embodiments thereof have also been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments without departing from the spirit and scope of the present invention.

Example 1

Synthesis path:

1. Synthesis of compound 3

Polypeptide synthesis

The polypeptide was synthesized using a standard stepwise synthesis method.

1) Add DCM to a container containing CTC resin (10.0 mmol, 10 g, with 1.0 mmol/g substrate) and Fmoc-Asp(OAll)-OH (3.95 g, 10.0 mmol, 1.0 equiv).

2) Add DIEA (4.0 equiv.) and stir for 2 hours.

3) Add MeOH (0.3 ml) and stir for 30 min.

4) The resulting mixture is dried and then rinsed three times with DMF, each time bubbling with nitrogen for 30 seconds.

5) Add 20% piperidine/DMF and then allow the system to react for 30 min.

6) The resulting mixture is dried and then rinsed five times with DMF, bubbling with nitrogen for 30 seconds each time.

7) Add the solution of the next amino acid with the Fmoc protecting group for 30 seconds, then add the condensing agent, and allow the system to react under _N2 bubbling for about 1 hour.

8) Repeat steps 4-7 to condense the next amino acid until the addition of Fmoc-Lys(Alloc)-OH is complete. The order of addition of amino acids and condensing agents used to synthesize compound 3 is shown in Table 1.

9) The resulting mixture is dried and then rinsed three times with DMF and three times with DCM, bubbling with nitrogen for 30 seconds each time.

10) De-OAll and Alloc: Pd( _PPh3 ) ₄ (0.1 equiv.) and _PhSiH3 (10.0 equiv.) were added to the resin solution in DCM, and the system was reacted under _N2 bubbling for about 15 min and dried. This step was repeated three times.

11) The resulting mixture is dried and then rinsed five times with DMF, bubbling with nitrogen for 30 seconds each time.

12) Cyclization: A solution of condensing agent HATU (22.85 equiv.) and DIEA (6.0 equiv.) are added and the system is allowed to react under _N2 bubbling for about 1 hour.

13) The resulting mixture is dried and then rinsed three times with DMF and three times with methanol, each time bubbling with nitrogen for 30 seconds, and dried.

Table 1. Order of addition

No. Source material Condensing agent 1 Fmoc-Asp(OAll)-OH (1.0 eq.) DIEA (4.0 equiv.) 2 Fmoc-hArg(Pbf)-OH (3.0 eq.) HBTU (2.85 equiv.) and DIEA (6.0 equiv.) 3 Fmoc-Lys(Alloc)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.)

Splitting and cleaning

The digestion buffer (20% HFIP/DCM) was added to the flask containing the side chain protected polypeptide and stirred at room temperature for 30 min x 2 times. The solution was collected and dried in a spin dryer, purified by reverse phase process (NH ₄ HCO ₃ system) and lyophilized to obtain intermediate 3.

Table 2. Cleaning conditions

Separation conditions Dissolution conditions Dissolution in DMF Equipment model AUNO DAC-100 Mobile phase A: H ₂ O (10.0 mM NH ₄ HCO ₃ /H ₂ O) B: _CH3CN Gradient of separation 30-70% - 40 min, holding time: 20 min Column type Welch Xtimate®C18, 250*50 mm, 10 µm, 120Å Flow rate 250 ml/min Wavelength 214/254 nm Temperature 25°C

2. Synthesis of TFA salt of compound 4

The polypeptide was synthesized using a standard stepwise synthesis method.

1) Add DMF to a container containing Rink amide MBHA resin (0.5 mmol, 1.56 g, with 0.32 mmol/g substrate) and allow the resin to swell for 2 hours.

2) The resulting mixture is dried and then rinsed three times with DMF, each time bubbling with nitrogen for 30 seconds.

3) Add 20% piperidine/DMF and then allow the system to react for 30 min.

4) The resulting mixture is dried and then rinsed five times with DMF, bubbling with nitrogen for 30 seconds each time.

5) The amino acid solution with the Fmoc protecting group is added for 30 seconds, followed by the condensing agent, and the system is allowed to react under _N2 bubbling conditions for about 1 hour.

6) Repeat steps 2-5 to condense the next amino acid.

The order of addition of amino acids and condensing agents used to synthesize compound 4 is shown in Table 3.

Table 3. Order of addition

No. Source material Condensing agent 1 Fmoc-Cys(Trt)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 2 Fmoc-Trp(Boc)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 3 Fmoc-Hyp(tBu)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 4 Fmoc-Pro-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 5 Fmoc-Thr(tBu)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 6 Fmoc-Ser(tBu)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 7 Fmoc-Trp(Boc)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 8 Fmoc-Asp(OtBu)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 9 Fmoc-hArg(Pbf)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 10 Fmoc-Met-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 11 Fmoc-Pen(Trt)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 12 Fmoc-dAsp(OtBu)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 13 Fmoc-1Nal-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 14 Fmoc-Pro-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 15 Fmoc-Cys(Trt)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 16 Compound 3 (2.0 equiv.) HATU (1.90 equiv.) and DIEA (4.0 equiv.) 17 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 18 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 19 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 20 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 21 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 22 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 23 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 24 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 25 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 26 Fmoc-Sar-OH (5.0 equiv.) HATU (4.75 equiv.) and DIEA (10.0 equiv.) 27 Fmoc-β-Ala-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.)

Cleavage and purification of the polypeptide

1) Add digestion buffer (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.

2) The polypeptide is precipitated with ice-cold isopropyl ether and centrifuged using a centrifuge (3 min, 3000 rpm).

3) The resulting polypeptide is washed with isopropyl ether two more times.

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

2. Synthesis of acetate salt of compound peptide_1

The crude TFA salt of intermediate 4 described above (2.4 g) was dissolved in 50% MeCN/H ₂ O (1 L), and TATA (0.5 mmol) was slowly added to the stirred solution at room temperature. The reaction mixture was stirred at room temperature for 30 min, and then the pH was adjusted to 8 with NH ₄ HCO ₃ . The reaction mixture was stirred at room temperature for an additional 12 h. When LC-MS showed the reaction to be complete, stirring was stopped. The mixture was purified by a reversed-phase process to afford the acetate salt of compound peptide_1.

Table 4. Cleaning conditions

Conditions for the first cleaning and separation Conditions for the second cleaning and separation Dissolution conditions Dissolution in 20% MeCN/ _H2O Dissolution in 20% MeCN/ _H2O Equipment model Welch Sail-1000 Gilson GX-281 Mobile phase A: _H2O (0.075% TFA/ _H2O ) A: _H2O (0.5% AcOH/ _H2O ) B: _CH3CN B: _CH3CN Gradient of separation 25-44% - 40 min, holding time: 30 min 14-70% - 40 min, holding time: 24 min Column type Welch Xtimate® C18, 250*50 mm, 10 µm, 120Å YMC-Actus Triart C18, 250*30 mm, 5 µm, 120Å Flow rate 80 ml/min 20 ml/min Wavelength 214/254 nm 214/254 nm Temperature 25°C 25°C

Peptide_2 and peptide_3 were synthesized according to the synthesis of peptide_1, and their structures are shown in Table 5.

Table 5. Structural sequences of compounds peptide_1~3

Peptide_1 Peptide_2 Peptide_3

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 DIEA (31.0 μL, 178.0 μmol) was added at 0 °C and stirred for 10 min. Compound 1-2 (290.4 mg, 890.1 μmol) was dissolved in DMF (5 mL) in another reaction flask and stirred at 0 °C for 10 min. Then, the reaction solution of compound 1-1 at 0 °C was added dropwise to the stirred reaction solution of compound 1-2 at 0 °C and stirred at 0 °C for 30 min. The reaction solution was filtered to remove insoluble residues. The filtrate is directly purified by a reversed-phase process (mobile phase: A: _H2O containing 0.075% TFA, B: _CH3CN , gradient: 10%-40% (B), 42 min) to obtain the TFA salt of compound INT_1.

5. Synthesis of acetate salt of compound PDC_1

The acetate salt of the 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 thereto and stirred at room temperature for 10 minutes. Then, the TFA salt of the compound INT_1 (13.4 mg, 10.1 μmol) was dissolved in DMF (0.2 mL), added dropwise to the above reaction solution, and then stirred at room temperature for 2 hours. The reaction solution was filtered to remove insoluble residues. The filtrate was directly purified by a reversed-phase process (mobile phase: A: _H2O containing 0.075% TFA, B: _CH3CN , gradient: 10%-40% (B), 42 min), lyophilized, and then converted to the acetate salt by work-up to give the acetate salt of compound PDC_1. MS m/z: 1533.4 (M+3H ⁺ )/3.

Example 2

Synthesis path 1:

Acetate salt of PDC_2 is prepared according to the synthesis route of acetate salt of PDC_1 (replacing peptide_1 with peptide_2 in the reaction). MS m/z: 1500.5 (M+3H ⁺ )/3.

Synthesis path 2:

Step 1. Synthesis of intermediate product 5

The polypeptide was synthesized using a standard stepwise synthesis method.

1) Add DMF to a container containing Rink amide MBHA resin (5.0 mmol, 8.33 g, with 0.60 mmol/g substrate) and allow the resin to swell for 2 hours.

2) The resulting mixture is dried and then rinsed three times with DMF, each time bubbling with nitrogen for 30 seconds.

3) Add 20% piperidine/DMF and then allow the system to react for 30 min.

4) The resulting mixture is dried and then rinsed five times with DMF, bubbling with nitrogen for 30 seconds each time.

5) The amino acid solution with the Fmoc protecting group is added for 30 seconds, followed by the condensing agent, and the system is allowed to react under _N2 bubbling conditions for about 1 hour.

6) Repeat steps 2-5 to condense the next amino acid.

The order of addition of amino acids and condensing agents used to synthesize intermediate 5 is shown in Table 6.

Table 6. Order of addition

No. Source material Condensing agent 1 Fmoc-Cys(Trt)-OH (3.0 eq.) HBTU (2.85 equiv.) and DIEA (6.0 equiv.) 2 Fmoc-Trp(Boc)-OH (3.0 eq.) HBTU (2.85 equiv.) and DIEA (6.0 equiv.) 3 Fmoc-Hyp(tBu)-OH (3.0 eq.) HBTU (2.85 equiv.) and DIEA (6.0 equiv.) 4 Fmoc-Pro-OH (3.0 equiv.) HBTU (2.85 equiv.) and DIEA (6.0 equiv.) 5 Fmoc-Thr(tBu)-OH (3.0 eq.) HBTU (2.85 equiv.) and DIEA (6.0 equiv.) 6 Fmoc-Ser(tBu)-OH (3.0 eq.) HBTU (2.85 equiv.) and DIEA (6.0 equiv.) 7 Fmoc-Trp(Boc)-OH (3.0 eq.) HBTU (2.85 equiv.) and DIEA (6.0 equiv.) 8 Fmoc-Asp(OtBu)-OH (3.0 eq.) HBTU (2.85 equiv.) and DIEA (6.0 equiv.) 9 Fmoc-HArg(Pbf)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 10 Fmoc-Met-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 11 Fmoc-Pen(Trt)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 12 Fmoc-dAsp(OtBu)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 13 Fmoc-1Nal-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 14 Fmoc-Pro-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 15 Fmoc-Cys(Trt)-OH (3.0 eq.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 16 Fmoc-Asp(OAll)-OH (2.0 eq.) HATU (1.90 equiv.) and DIEA (4.0 equiv.) 17 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 18 Fmoc-Lys(Alloc)-OH (2.0 eq.) HATU (1.90 equiv.) and DIEA (4.0 equiv.) 19 Removing OAll and Alloc Pd( _PPh3 ) ₄ (0.1 equiv.) and _PhSiH3 (10.0 equiv.) 20 Cyclization HATU (2.85 equiv.) and DIEA (6.0 equiv.) 21 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 22 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 23 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 24 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 25 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 26 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 27 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 28 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 29 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 30 Fmoc-Sar-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.) 31 Fmoc-β-Ala-OH (3.0 equiv.) HATU (2.85 equiv.) and DIEA (6.0 equiv.)

7) Add digestion buffer (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.

8) The polypeptide is precipitated with ice-cold isopropyl ether and centrifuged using a centrifuge (3 min, 3000 rpm).

9) The resulting polypeptide is washed with isopropyl ether two more times.

10) The crude polypeptide is dried to obtain the crude intermediate product 5.

Step 2. Synthesis of acetate salt of compound peptide_2

The crude intermediate 5 (15.0 g) was dissolved in 50% MeCN/H ₂ O (5 L), and TATA (7.5 mmol) was slowly added to the stirred solution at room temperature. The reaction mixture was stirred at room temperature for 30 min, and then the pH was adjusted to 8 with NH ₄ HCO ₃ . The reaction mixture was stirred at room temperature for an additional 12 h. When LC-MS showed the reaction to be complete, stirring was stopped. The mixture was purified by a reversed phase process (first purification: mobile phase: A: _H2O containing 0.075% TFA, B: _CH3CN , gradient: 10%-40% phase B, 42 min, retention time: 29 min; second purification: mobile phase: A: _H2O containing 0.5% AcOH, B: _CH3CN , gradient: 20%-40%, 40 min, retention time: 21 min; third purification: mobile phase: A: _H2O containing 0.5% AcOH, B: _CH3CN , gradient: 16%-36% (B); 40 min, retention time: 26 min) to give the acetate salt of the intermediate product peptide_2.

Step 3. Synthesis of PDC_2 acetate salt

The acetate salt of the 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 h. When LC-MS showed that the reaction was complete, the reaction was stopped. The reaction solution was filtered to remove insoluble residues. The filtrate was directly purified by a reversed-phase process (mobile phase: A: _H2O containing 0.075% TFA, B: _CH3CN , gradient: 10%-40% (B), 42 min), lyophilized, and then converted to AcOH salt by work-up to give the acetate salt of PDC_2. MS m/z: 1500.3 (M+3H ⁺ )/3.

Example 3

The acetate salt of PDC_3 was prepared according to the synthesis of the acetate salt of PDC_1 (replacing peptide_1 with peptide_3 in the reaction). MS m/z: 1504.8 (M+3H ⁺ )/3.

Biological analysis data

Test Example 1. Testing the Binding Ability of the Compounds of the Present Invention to Nectin-4 Protein

1. The purpose of the experiment

To determine the affinity of the compound to be tested for the target protein nectin-4 using the SPR method.

2. Materials and devices

Biacore 8K (GE Healthcare)

96-well plate (cat. no. 650101, Greiner Bio-One)

CM5 Chip (Cat. No. BR-1005-30, GE Healthcare)

Amine Immobilization Kit (Cat. No. BR-1000-50, GE Healthcare)

EDC

NHS

1 M ethanolamine

10 mM sodium acetate, pH 4.5 (Cat. No. BR-1003-50, GE Healthcare)

DMSO (cat. no. D4540, Sigma)

P20 (Cat. No. BR-1000-54, GE Healthcare)

PBS (Cat. No. BR-1006-72, GE Healthcare)

Nectin-4 (Cat. No. 1006-72, GE Healthcare)

3. Experimental protocol

In this experiment, the amino group immobilization method is adopted. The target protein nectin-4 is directly immobilized on the CM5 chip using Biacore 8K. The compound to be tested as an analyte is then diluted with a buffer solution (10 mM PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5% DMSO and 0.05% P20) to the required concentration gradient for multi-cycle kinetic detection, in which each cycle consists of sample injection for 180 seconds and dissociation for 180 seconds, and then the next cycle is performed, to obtain the affinity kinetic analysis data of the target protein nectin-4 and the compound to be tested. Using Biacore Insight evaluation software (version 2.0.15.12933), the final data were subjected to kinetic fit analysis based on a 1:1 model.

4. Experimental methodology and procedure

1) Preparation of buffer solution: 10 mM PBS, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5% DMSO and 0.05% P20.

2) Activation of CM5 chip: CM5 chip was activated with 400 mM EDC and 100 mM NHS at a flow rate of 10 μL/min for 420 seconds.

3) Target protein binding: The target protein was diluted to 10 μg/mL with 10 mM sodium acetate (pH 4.5) and bound at a flow rate of 10 μL/min for 284 s. Channels #1, #2, and #3 on the chip were used in the experiment, and the binding results were 1639.9 RU, 1747.8 RU, and 1702.2 RU, respectively.

4) Blocking of CM5 chip: The CM5 chip is blocked with 1 M ethanolamine at a flow rate of 10 μl/min for 420 seconds.

5) Preparation of analyte concentrations: The compound to be tested is diluted with a buffer solution. The compound to be tested is diluted from 100 nM to 0.78 nM with a 2-fold gradient.

6) Injection sample analysis: Each concentration of the working solution of the compound to be tested corresponds to one cycle at a flow rate of 30 µL/min for binding for 180 seconds and dissociation for 180 seconds. The last cycle is a 5% DMSO solvent correction cycle.

7) All results are subjected to kinetic correspondence analysis based on a 1:1 model.

5. Experimental results

The experimental data of five effective concentrations were selected for kinetic fit analysis based on the 1:1 model using Biacore Insight evaluation software (version 2.0.15.12933). The results are shown in Table 7.

Table 7. Results of the study of binding of the compounds of the present invention to the human nectin-4 protein by the SPR method

Compound k _D of human nectin-4 protein according to SPR (nM) Acetate salt PDC_1 1.4 Acetate salt PDC_2 0.43 ^a Acetate salt PDC_3 4.13

^a Average of two tests.

Conclusion: The compounds of the present invention have a strong binding effect to nectin-4.

Test Example 2. Antiproliferative Activity of the Compounds of the Present Invention in Vitro against NCI-H292 and MDA-MB-468 Cells

1. The purpose of the experiment

To investigate the inhibitory effect of the compounds of the present invention on cell proliferation by detecting the effect of the compounds of the present invention on cellular activity in vitro in the tumor cell lines MDA-MB-468 and NCI-H292.

2. Experimental plan

Cell culturing

Tumor cell lines were cultured in a 37°C incubator without _CO2 and in a 37°C incubator with 5% _CO2 according to the culture conditions shown in Table 8, respectively. Cells were passaged periodically, and cells in the logarithmic growth phase were selected for seeding.

Table 8. Cell lines and methods of their cultivation

Cell line Cell type Source Cat. No. Growth characteristics Cultivation method NCI-
H292 Human lung cancer ATCC CRL-1848 Adhesive RPMI 1640 + 10% FBS MDA-
MB-468 Human breast cancer ATCC HTB-132 Adhesive Leibovitz medium L-15 + 10% FBS

Cell seeding

The cells in the logarithmic growth phase were collected and centrifuged at 1000 rpm for 3 min at room temperature. The supernatant was removed and the cells were resuspended in 5 ml of the culture medium. Then, 20 μl of the cell suspension was pipetted and mixed with trypan blue at a ratio of 1:1 for staining for 3 min to determine cell viability and count viable cells. The cell density was adjusted to 3000 cells/well. Then, 90 μl of the cell suspension was added to each well of the culture plate, and cell-free culture medium was added to the blank control wells. The culture plate was incubated overnight in an incubator at 37°C without _CO2 and with 100% relative humidity and in an incubator at 37°C with 5% _CO2 and 100% relative humidity, respectively.

Preparing a tablet with a stock solution of the compound

Preparation of a 400X compound stock plate. The compound to be tested is gradient diluted with DMSO from the highest to the lowest concentration.

Table 9. Dilution concentrations in a 400X stock plate

1 2 3 4 5 6 7 8 9 Concentration (µM) 400 133.33 44,444 14,815 4,938 1,646 0.549 0.183 0.061

Preparation of 10X working solution of compound and treatment of cells with compound

Preparation of 10X compound working solution: 78 μL of cell culture medium is added to a 96-well V-bottom plate, and 2 μL of each concentration of compound solution is pipetted from the 400X compound stock plate into the cell culture medium in the 96-well plate. Then, 2 μL of DMSO is added to the vehicle medium control and the blank control. After adding the compound or DMSO, the mixture is uniformly purged with a multichannel pipette. Drug addition: 10 μL of 10X compound working solution is added to the cell culture plate. Then, 10 μL of the cell culture medium and DMSO mixture is added to the vehicle medium control and the blank control. The 96-well cell culture plate is placed back in the incubator for 72 hours.

CellTiter-Glo Luminescent Cell Viability Assay

Detection is performed according to the instructions of the Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega-G7573). The luminescent signal is detected using the EnVision® Multi-Mode Plate Reader (EnVision 2104-10).

Data Analysis

The inhibition rate (IR) of the compound to be tested is calculated according to the following formula: IR (%) = (1- (RLU of compound - RLU of blank) / (RLU of vehicle control - RLU of blank) * 100%. The inhibition rates of the compounds at different concentrations are calculated in Excel. The inhibition curves are then plotted using GraphPad Prism 6.02 software and the relevant parameters including minimum inhibition rate, maximum inhibition rate and IC ₅₀ are calculated.

The results of the experiment are shown in Table 10.

Table 10. Results of the tumor cell proliferation inhibition assay by the compound of the present invention

Tumor cell line Compound IC ₅₀ (µM) Minimum inhibition level (%) Maximum inhibition rate (%) NCI-H292 Acetate salt PDC_2 0,0421 1.55 81.32 MDA-MB-468 Acetate salt PDC_2 0.0323 -10.28 86.28

The difference in IC ₅₀ between the two runs is within 2-fold.

Conclusion: The compound of the present invention has a significant inhibitory effect on the proliferation of cells cultured in vitro in the tumor cell lines MDA-MB-468 and NCI-H292.

Test Example 3. In vivo efficacy of the compounds of the present invention in the NCI-H292 human lung cancer cell subcutaneous tumor xenograft model

1. Objective of the Experiment: To investigate the efficacy of the compounds of the present invention in vivo in a xenograft mouse model of subcutaneous tumor from human lung cancer cells NCI-H292.

2. Experimental plan

Cell culture. NCI-H292 human lung cancer cells (ATCC, Manassas, VA, Cat. #CRL-1848) are grown as monolayers in vitro in RPMI 1640 medium supplemented with 10% fetal calf serum in an incubator at 37°C with 5% CO ₂ . Cells are routinely digested twice weekly with pancreatic enzyme/EDTA for passaging. When cell densities reach 80-90% and cell numbers are adequate, cells are harvested, counted, and inoculated.

Animals. "Nude" BALB/c mice, females, 6-8 weeks old, weighing 17-21 grams.

Tumor inoculation: Inoculate 0.2 ml (1×10 ⁷ NCI-H292 subcutaneously into the right back of each mouse.

Efficacy study: When the average tumor volume reached approximately 100-200 ^mm3 , the compound was administered intravenously to groups of 6 animals per group. The dose was 1.5 mg/kg, and the frequency of administration was QW×3.

Observations. The preparation and any modifications of this experimental protocol will be implemented only after evaluation and approval by the Institutional Animal Care and Utilization Committee (IACUC) of WuXi AppTec Shanghai. The use and welfare of laboratory animals will comply with the guidelines of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Daily monitoring of animal health and mortality will be performed as a routine examination, including observation of tumor growth and drug treatment effects on the daily behavior of animals, such as behavioral activity, food and water consumption, weight changes (weight is measured twice a week), signs of appearance or other abnormal conditions. Animal mortality and adverse events within a group will be recorded based on the number of animals in each group.

Termination of the experiment. Animals should be euthanized if their health continues to deteriorate, if the tumor volume exceeds 2000 mm ³ , or if they are severely ill or in pain.

Data Analysis: For comparisons between two groups, the T-test is used. For comparisons between three or more groups, a one-way ANOVA is used. If the F-values differ significantly, multiple comparisons should be performed after the ANOVA analysis. All data are analyzed using SPSS 17.0. p < 0.05 indicates a significant difference.

Composing a medicinal product

Frequency of preparation of the medicinal product. Before the first administration, a homogeneous initial solution is prepared, divided into portions and cryopreserved in a refrigerator at -80°C.

Volume of administration. The volume of administration is adjusted according to the weight of the animal (volume of administration = 10 µl/g)

Table 11. Detailed protocol for connection formation

Compound Package Method of compilation Concentration (mg/ml) Storage conditions Carrier medium -- 25 mM L-histidine (pH = 7), 10% sucrose
Weigh out 5 g of sucrose and add 50 ml of sterile water to make a 10% sucrose solution.
Weigh out 193.94 mg of L-histidine, add 10% sucrose, mix, adjust pH to 7 and dilute to 50 ml to obtain a homogeneous solution. -- 4°C Acetate salt PDC_2 2.0 mg/flask Add 2 mg PDC_2 to 1.8 ml carrier medium, mix by vortexing and dissolve to form 1.0 mg/ml homogeneous transparent solution. Distribute 0.3 ml/flask into 3 test tubes (for efficiency analysis) and store at -80°C. 1.0 -80°C Take 0.3 ml of 1.0 mg/ml PDC_2 solution, add 1.7 ml of solvent, mix by vortexing and dissolve to form a homogeneous transparent solution. 0.15 Ready to use

Experimental results. See Fig. 1 and Fig. 2.

Conclusion of the experimental results. The compound of the present invention exhibits a significant tumor growth inhibition effect in the NCI-H292 human lung cancer cell subcutaneous tumor xenograft model.

Test Example 4. Study of the pharmacodynamic characteristics of the compounds of the present invention in vivo in a xenograft model of a subcutaneous tumor from human breast cancer cells MDA-MB-468 in “nude” BALB/c mice

The aim of the experiment was to evaluate the efficacy of the compounds of the present invention in vivo in a xenograft model of a subcutaneous tumor from human breast cancer cells MDA-MB-468.

Cell culture. MDA-MB-468 human breast cancer cells (ATCC, Manassas, VA, Cat# HTB-132) are grown as monolayers in vitro in L-15 culture medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in a 37°C incubator with 0% _CO2 . Cells are routinely digested twice weekly with pancreatic enzyme/EDTA for passaging. When 80-90% cell saturated and cell numbers are adequate, cells are harvested, counted, and inoculated.

Animals. BALB/c nude mice, female, 6-8 weeks old, weighing 18-22 grams, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.

Tumor inoculation: 0.2 ml (1×10 ⁷ ) of MDA-MB-468 cells (with Matrigel, in a 1:1 volume ratio) were inoculated subcutaneously into the right back of each mouse.

Volume of administration. The volume of administration is adjusted according to the weight of the animal (volume of administration = 10 µl/g)

Formulation of drug product. The compound to be tested was formulated as a 1.5 mg/mL homogeneous solution using 25 mM L-histidine (pH = 7) in 10% sucrose as the carrier medium and stored in a refrigerator at -80°C. On the day of administration, the homogeneous solution was diluted to the appropriate concentration for IV administration (intravenous injection) to the group.

Grouping. When the average tumor volume reaches approximately 500-600 mm ³ , the compound is administered in groups. The dose is 5 mg/kg, and the frequency of administration is QW×4.

The results of the experiment are shown in Fig. 3 and Fig. 4.

Conclusion of the experimental results. In the MDA-MB-468 human breast cancer cell subcutaneous tumor xenograft model, the compound of the present invention in large-volume groups still exhibits a significant tumor growth inhibition effect, correlation with the dose level, no adverse reactions, average weight loss not reaching 5% or more, and satisfactory safety.

Test Example 5. Pharmacokinetic Analysis of the Compounds of the Present Invention in Rat Blood Plasma

A. Purpose of the experiment

Testing the pharmacokinetic characteristics of the compounds of the present invention in SD rats.

B. Experimental procedures

Pharmacokinetic characteristics of the compound after intravenous injection are tested in rats according to a standard protocol. The compound to be tested is formulated as a clear solution using 25 mM L-histidine (pH = 7) in 10% sucrose as a carrier medium. Two rats are given a single intravenous injection of 3 mg/kg of the compound to be tested. Whole blood is collected at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration to obtain blood plasma. The concentration of the compound of the present invention and the concentration of its potential metabolite MMAE are analyzed by LC-MS/MS, and the pharmacokinetic parameters are calculated using Phoenix WinNonlin software.

C. Experimental results

The results of the experiment are shown in Table 12.

Table 12. Results of pharmacokinetic testing in rats

Connection No. Parameter Meaning Acetate salt PDC_2 Dose (mg/kg) 3 C ₀ (μM) 6.9 T _1/2 (h) 0.7 Vd _ss (l/kg) 0,1 CL (ml/min/kg) 2.7 AUC _0-last (μM h) 4.4 MMA C _max (µM) 0.07 AUC _0-last (μM h) 0.11

Conclusion: The compound of the present invention has a short half-life and rapid clearance in the blood of rats, and the exposure to the MMAE metabolite is only about 1/40 of that of the compound of the present invention, indicating its satisfactory safety.

Test Case 6. Pharmacokinetic Analysis in Cynomolgus Macaque Plasma

A. Purpose of the experiment

Testing the pharmacokinetic characteristics of the compounds of the present invention in cynomolgus monkeys.

B. Experimental procedures

The pharmacokinetic characteristics of the compound after intravenous injection are tested in cynomolgus monkeys according to a standard protocol. The compound to be tested is formulated as a clear solution using 25 mM L-histidine (pH = 7) in 10% sucrose as the carrier medium. Cynomolgus monkeys are given a single intravenous injection of 1 mg/kg of the compound to be tested. Whole blood is collected at 0.083, 0.25, 0.5, 1, 2, 4, 8 and 24 hours after administration to obtain plasma. The concentration of the compound to be tested and the concentration of its potential metabolite MMAE are analyzed by LC-MS/MS and the pharmacokinetic parameters are calculated using Phoenix WinNonlin software.

C. Experimental results

The results of the experiment are shown in Table 13.

Table 13. Results of pharmacokinetic testing in cynomolgus monkeys

Connection No. Parameter Meaning Acetate salt PDC_2 Dose (mg/kg) 1 C ₀ (μM) 3.1 T _1/2 (h) 1.5 Vd _ss (l/kg) 0.2 CL (ml/min/kg) 3.6 AUC _0-last (μM h) 1.0 MMA C _max (µM) 0,00 AUC _0-last (μM h) 0,00

Conclusion: The compound of the present invention is characterized by a short half-life and rapid clearance in the blood of cynomolgus macaque, the release of MMAE into the blood below the detection limit and satisfactory safety.

Test Example 7. Metabolic stability of the compounds of the present invention in hepatocytes of mice, rats, cynomolgus monkeys and humans

A. Purpose of the experiment

To investigate the metabolic stability of the compounds of the present invention in hepatocytes of mice, rats, cynomolgus monkeys and humans.

B. Experimental procedures

Incubation is performed in 96-well plates using the external extraction method. Several 96-well plates for sample precipitation, designated as T0, T15, T30, T60, T90, T0-MC, T90-MC, and a blank matrix are respectively prepared. The recovery culture medium and the incubation culture medium are collected in advance and placed in a water bath at 37°C for preheating. Frozen hepatocytes of CD-1 mice, SD rats, cynomolgus monkeys, and humans are removed from the liquid nitrogen reservoir, recovered, and diluted to 0.51× ¹⁰⁶ cells/mL with the incubation culture medium. Then, 198 μl of hepatocyte suspension (0.5× ¹⁰⁶ cells/ml) were added to the pre-warmed incubation plates, and 198 μl of hepatocyte-free incubation culture medium were added to the T0-MC and T90-MC incubation plates as the culture medium control groups. All the incubation plates were pre-incubated in an incubator at 37°C for 10 minutes. Then, 2 μl of the working solutions of the test sample and the control compound were added and mixed evenly. The incubation plates were placed on a shaker inside the incubator for incubation. Three replicates were prepared for each time point. The incubation conditions were 37°C, saturation humidity, and 5% _CO2 . In the test system, the final concentration of the test sample is 1 μM, the final concentration of the control sample is 3 μM, the final concentration of hepatocytes is 0.5×10 ⁶ cells/mL, and the total final concentration of the organic solvent is 1.0%, while the final concentration of DMSO is 0.1%.

After the incubation was completed, the incubation plates were removed at the appropriate time, 25 μl of the compound-cell mixture and the control compound-cell mixture were removed from the incubation plates and added to the sample plates containing 125 μl of the stop buffer solution, respectively. 25 μl of the hepatocyte-free incubation culture medium was directly added to the blank sample plate. All sample plates were sealed with film, shaken at 600 rpm on a plate shaker for 10 minutes, and then centrifuged at 3220×g for 20 minutes. The supernatant of the test sample and the control compound was diluted with purified water at a ratio of 1:3. All samples were uniformly mixed and then analyzed by LC-MS/MS.

The concentration of the compound to be tested and the concentration of the control compound in the sample are determined semi-quantitatively by liquid chromatography with tandem mass spectrometry (LC-MS/MS) without standard curves and a quality control sample. The ratio of the peak area of the analyte to the peak area of the internal standard is used to express the concentration in the sample. Retention times, chromatogram acquisition, and integration of the analyte and internal standard chromatograms are processed by Analyst software (Sciex, Framingham, MA, USA).

C. Experimental results

The results of the experiment are shown in Table 14.

Table 14. Results of the study of metabolic stability of the compound of the present invention in hepatocytes of CD-1 mice, SD rats, cynomolgus monkeys and humans

Connection No. View T _1/2 (min) CL _{int (liver)}
(ml/min/kg) Acetate salt PDC_2 Mice CD-1 > 216.8 < 75.9 SD Rats > 216.8 < 29.9 Javan macaques > 216.8 < 23 Human > 216.8 < 17.8

Conclusion: The compound of the present invention has excellent metabolic stability in liver microsomes of 4 kinds.

Test Example 8. Metabolic stability of the compounds of the present invention in the S9 fraction of the kidneys of mice, rats, cynomolgus monkeys and humans

A. Purpose of the experiment

To investigate the metabolic stability of the compounds of the present invention in the S9 fraction of kidneys of mice, rats, cynomolgus monkeys and humans.

B. Experimental procedures

Materials: S9 fraction from CD-1 mouse, SD rat, cynomolgus monkey and human kidneys was purchased from BioIVT or XenoTech LLC and stored in a refrigerator at -80°C.

Experimental procedure. Eight 96-well plates, designated T0, T5, T15, T30, T45, T60, blank, and NCF60, were prepared. The first six incubation plates corresponded to reaction times of 0, 5, 15, 30, 45, and 60 minutes, respectively. No test sample or control compound was added to the blank plate.

To the T0, T5, T15, T30, T45, T60 plates, 2 μl of the working solution of the test sample (10 mM solution of the compound to be tested in DMSO diluted with 100% acetonitrile to 100 μM) and 100 μl of the working solution S9 (the concentration of kidney protein S9 is 1.0 mg/ml) are added, and to the “blank” plate, only the working solution S9 is added. The above incubation plates are then placed in a water bath at 37°C for pre-incubation for approximately 10 minutes.

After the pre-incubation, 98 μL of auxiliary enzyme working solution was added to each sample well in each plate except T0 to initiate the reaction. After incubation for the appropriate time (i.e., 5, 15, 30, 45, and 60 minutes), 600 μL of stop buffer (acetonitrile solution containing 100 ng/mL tolbutamide and 100 ng/mL labetalol) was added to each sample well to stop the reaction. Preparation of T0 plate: First, 600 μL of stop buffer was added to T0 plate, followed by 98 μL of auxiliary enzyme working solution. All sample plates were shaken uniformly and centrifuged at 3220×g for 20 minutes. Then 100 µl of supernatant from each well was removed and diluted in 300 µl of pure water for analysis by liquid chromatography-tandem mass spectrometry.

C. Experimental results

The results of the experiment are shown in Table 15.

Table 15. Results of the study of metabolic stability of the compound of the present invention in the S9 fraction of kidneys of CD-1 mice, SD rats, cynomolgus monkeys and humans

Connection No. View T _1/2 (min) CL _{int (S9)}
(µl/min/mg) Acetate salt PDC_2 Mice CD-1 > 145 < 9.6 SD Rats > 145 < 9.6 Javan macaques > 145 < 9.6 Human > 145 < 9.6

Conclusion: The compound of the present invention has excellent metabolic stability in the S9 fraction of kidneys from 4 species.

Test Example 9. Analysis of the stability of the compounds of the present invention in blood plasma of various species

A. Purpose of the experiment

To test the stability of the compounds of the present invention in the blood plasma of SD rats, cynomolgus monkeys and humans.

B. Experimental procedures

2 μL of the working solution of the compound to be tested (100 μM) are added to the respective incubation plates, including the T0, T10, T30, T60, T120, and T240 incubation plates. Three parallel wells are prepared for each sample. Then 98 μL of blank plasma from SD rats, cynomolgus monkeys, and humans are added to the respective incubation plates to which the working solution has been added. All samples are incubated in a water bath at 37 °C. The final incubation concentration of the compound to be tested is 2 μM. At the end of each incubation time point, the respective incubation plate is removed, stop buffer is added, pelleted, and centrifuged for 20 min. Then 150 μL of the supernatant is analyzed by LC-MS/MS. The concentration of the compound to be tested in the sample is determined semi-quantitatively by liquid chromatography with tandem mass spectrometry (LC-MS/MS).

C. Experimental results

The results of the experiment are shown in Table 16.

Table 16. Results of stability testing of the compound of the present invention in blood plasma of SD rats, cynomolgus monkeys and humans

Connection No. View T _1/2 (min) Acetate salt PDC_2 SD Rats > 578.1 Javan macaques > 578.1 Human > 578.1

Conclusion: The compound of the present invention has excellent metabolic stability, with a half-life of more than 578.1 min in all three species.

Claims (22)

1. A compound represented by formula (III) or a pharmaceutically acceptable salt thereof MMAE-RABC-Cit-Val-glutaryl-β-Ala-Sar10 where R ₁ is selected from H and C _1-3 alkyl; R ₂ is selected from H, C _1-4 alkyl and n is selected from 1, 2, 3, and 4; and each of Xi, Xii and Xiii is independently selected from Cys, hCys, βCys and Pen; MMAE - monomethyl auristatin E; RAVS - 2. A compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the structures represented by formulas (III-1) and (III-2), MMAE-RABC-Cit-Val-glutaryl-β-Ala-Sar10 MMAE-RABC-Cit-Val-glutaryl-β-Ala-Sar10 where R ₁ , R ₂ , Xi, Xii and Xiii are defined in paragraph 1. 3. A compound or a pharmaceutically acceptable salt thereof selected from 4. A pharmaceutical composition comprising a therapeutically or prophylactically effective amount of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, for use in the treatment of solid tumors in which nectin-4 is overexpressed. 5. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a medicinal product for the treatment of solid tumors in which nectin-4 is overexpressed. 6. Use of the pharmaceutical composition according to claim 4 in the preparation of a medicinal product for the treatment of solid tumors in which nectin-4 is overexpressed. 7. A method for treating solid tumors that overexpress nectin-4, comprising administering to a subject a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3. 8. A method for treating solid tumors in which nectin-4 is overexpressed, comprising administering to a subject the pharmaceutical composition of claim 4. 9. A product containing a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, for use in the treatment of solid tumors in which nectin-4 is overexpressed. 10. A product containing the pharmaceutical composition according to claim 4, for use in the treatment of solid tumors in which nectin-4 is overexpressed.