Biotin-targeted pd-l1 inhibitor and preparation method and application thereof

Abstract

The application provides a biotin-targeted PD-L1 inhibitor and a preparation method and application thereof, and belongs to the technical field of organic compound synthesis. In the application, 2,6-dibromotoluene and 3-aminobenzoic acid pinacol ester are used as starting materials, and through Suzuki coupling reaction, amide condensation, Suzuki coupling reaction and reductive amination reaction, a compound SW-1 is synthesized, and the molecular structure of the compound SW-1 is confirmed. The compound SW-1 can significantly inhibit the activity of PD-L1, has a significant antitumor effect, and is an effective PD-L1 inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of organic compound synthesis technology, and particularly relates to the field of chemical pharmaceutical raw material preparation technology, specifically to a biotin-targeted PD-L1 inhibitor and its preparation method and application. Background Technology

[0002] Tumor immunotherapy inhibits and eliminates tumors by restarting and maintaining the body's tumor-immune cycle and reshaping the endogenous anti-tumor immune response. With its controllable adverse reactions, it has become a mainstream technology in the clinical treatment of malignant tumors. Among existing immunotherapy products, immune checkpoint monoclonal antibodies, represented by PD-1 / PD-L1 (programmed death receptor 1 / programmed death ligand 1) inhibitors, are the most widely used in clinical practice, demonstrating definite therapeutic effects against many types of malignant tumors and representing a core and popular target in current anti-tumor drug development.

[0003] Currently, mainstream PD-1 / PD-L1 inhibitors in clinical practice are all large-molecule antibody drugs. However, due to limitations in their molecular physicochemical properties, these drugs have inherent technical drawbacks: Firstly, large molecules have difficulty penetrating the dense matrix barrier of solid tumors. The high expression of extracellular matrix components such as collagen and hyaluronic acid in the tumor microenvironment further hinders drug penetration into deeper tumor layers. The drugs tend to accumulate in the perivascular region of the tumor, making it difficult to target deep tumor cells, ultimately resulting in a low overall objective response rate for these antibody drugs against solid tumors. Secondly, systemic infusion of PD-L1 antibodies easily leads to off-target effects, non-specifically activating the immune system in normal tissues and inducing various immune-related adverse events (irAEs). irAEs can affect multiple systems throughout the body, with diverse clinical manifestations ranging in severity from mild flu-like symptoms to life-threatening toxicity. Therefore, developing small-molecule PD-L1 inhibitors with advantages in oral absorption, strong tissue penetration, and tumor-targeting action to compensate for the shortcomings of antibody drugs in the treatment of solid tumors has significant clinical application value and industrialization prospects.

[0004] In the prior art, Example 202 of patent application CN105705489A discloses a class of resorcinol diphenyl methyl ether derivatives. This compound can block the protein interaction between PD-1 and PD-L1, possessing PD-L1 inhibitory activity and anti-tumor development potential. However, as verified by HTRF in vitro binding assay, the IC50 of this known compound against the PD-1 / PD-L1 interaction is only 61 nM, indicating weak target inhibitory activity, which is insufficient to meet the development needs of highly effective clinical drugs. Based on the above-mentioned deficiencies in the prior art, it is necessary to provide a novel small molecule PD-L1 inhibitor that significantly enhances target inhibitory activity. Summary of the Invention

[0005] This invention provides a biotin-targeted PD-L1 inhibitor and its preparation method. By designing a novel biotin-targeted PD-L1 inhibitor, the invention effectively overcomes the shortcomings of existing technologies, provides a key solution for improving anti-tumor efficacy, and has significant clinical value and broad application prospects.

[0006] Specifically, the first objective of this invention is to provide a biotin-targeted PD-L1 inhibitor, wherein the biotin-targeted PD-L1 inhibitor is a compound represented by formula (I), or a stereoisomer of the compound represented by formula (I), or a pharmaceutically acceptable salt of the compound represented by formula (I), or a pharmaceutically acceptable salt of a stereoisomer of the compound represented by formula (I). The structural formula of the compound represented by formula (I) is as follows: (I).

[0007] A second objective of this invention is to provide a method for preparing the biotin-targeted PD-L1 inhibitor, comprising the following steps: S1, Compound A1 and Compound A2 undergo a Suzuki coupling reaction to generate intermediate A3; S2, intermediate A3 and compound A4 undergo an amide condensation reaction to generate intermediate A5; S3, intermediate A5 and compound A6 undergo a Suzuki coupling reaction to generate intermediate A7; S4, intermediate A7 and compound A8 undergo a reductive amination reaction to generate compound SW-1, which is the biotin-targeted PD-L1 inhibitor; The synthetic route for the biotin-targeted PD-L1 inhibitor is as follows: .

[0008] In a preferred embodiment, the molar ratio of compound A1 to compound A2 in step S1 is 1:(1~2).

[0009] In the preferred embodiment, the reaction conditions in step S1 are as follows: under inert gas protection, in the presence of palladium catalyst and acid-binding agent, compound A1 and compound A2 react at 70℃~90℃ for 8~12 hours.

[0010] In a further preferred embodiment, the palladium catalyst in step S1 is at least one of tetra(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, and bis(tritert-butylphosphine)palladium (0).

[0011] In a further preferred embodiment, the acid-binding agent in step S1 is at least one of potassium carbonate, sodium carbonate, and potassium acetate.

[0012] In a preferred embodiment, in step S1, compound A1 and compound A2 react in a mixed solvent consisting of an ether solvent and water in a volume ratio of 5:1.

[0013] In a further preferred embodiment, the ether solvent in step S1 is 1,4-dioxane.

[0014] In the preferred embodiment, the molar ratio of intermediate A3 to compound A4 in step S2 is 1:(1~2).

[0015] In the preferred embodiment, the reaction conditions in step S2 are as follows: in the presence of a condensing agent and an acid-binding agent, intermediate A3 reacts with compound A4 at 20°C to 30°C for 1 to 5 hours.

[0016] In a further preferred embodiment, the condensing agent in step S2 is at least one of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole.

[0017] In a further preferred embodiment, the acid-binding agent in step S2 is at least one of N,N-diisopropylethylamine and triethylamine.

[0018] In the preferred embodiment, in step S2, intermediate A3 reacts with compound A4 in a polar aprotic organic solvent.

[0019] In a further preferred embodiment, the polar aprotic organic solvent in step S2 is N,N-dimethylformamide.

[0020] In the preferred embodiment, the molar ratio of intermediate A5 to compound A6 in step S3 is 1:(1~2).

[0021] In the preferred embodiment, the reaction conditions in step S3 are as follows: under inert gas protection, in the presence of palladium catalyst and acid-binding agent, intermediate A5 reacts with compound A6 at 70℃~90℃ for 8~12 hours.

[0022] In a further preferred embodiment, the palladium catalyst in step S3 is at least one of tetra(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, and bis(tritert-butylphosphine)palladium (0).

[0023] In a further preferred embodiment, the acid-binding agent in step S3 is at least one of potassium acetate, sodium carbonate, and potassium carbonate.

[0024] In a preferred embodiment, in step S3, intermediate A5 and compound A6 react in a mixed solvent consisting of an ether solvent and water in a volume ratio of 5:1.

[0025] In a further preferred embodiment, the ether solvent in step S3 is 1,4-dioxane.

[0026] In a preferred embodiment, the molar ratio of intermediate A7 to compound A8 in step S4 is 1:(1~2).

[0027] In the preferred embodiment, the reaction conditions in step S4 are as follows: in the presence of a reducing agent and a weak organic acid, intermediate A7 reacts with compound A8 at 20°C to 30°C for 1 to 5 hours.

[0028] In a further preferred embodiment, the reducing agent in step S4 is at least one of NaBH3CN, NaBH(OAc)3, and NaBH4.

[0029] In a further preferred embodiment, the organic weak acid in step S4 is at least one of acetic acid and formic acid.

[0030] In a preferred embodiment, in step S4, intermediate A7 and compound A8 react in a mixed solvent consisting of an aprotic polar solvent and a protic polar solvent in a volume ratio of 10:1.

[0031] In a further preferred embodiment, the aprotic polar solvent in step S4 is dichloromethane.

[0032] In a further preferred embodiment, the proton polar solvent in step S4 is methanol.

[0033] A third objective of this invention is to provide the use of the biotin-targeted PD-L1 inhibitor in the preparation of antitumor drugs related to the PD-L1 target.

[0034] In a preferred embodiment, the antitumor drug targets liver cancer and / or melanoma.

[0035] A fourth object of the present invention is to provide a pharmaceutical composition comprising the biotin-targeted PD-L1 inhibitor.

[0036] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) The compound SW-1 provided by this invention is a small molecule inhibitor of PD-L1, which has excellent in vivo anti-tumor pharmacological effects and can effectively exert in vivo anti-tumor therapeutic effects (IC500 on PD-1 / PD-L1 activity inhibition). 50 The concentration was 5.6 nM, and the IC50 value for B16-F10 and HepG2 cells was 5.6 nM. 50 The concentrations were 0.5 µM and 2.4 µM, respectively, for LO2 normal hepatocytes (IC50). 50(>20μM) showed no cytotoxicity, and all experimental mice in the SW-1 administration group survived in the toxicity test. It has good drug development potential and clinical application transformation value, and has no obvious systemic toxic side effects.

[0037] (2) The method of preparing compound SW-1 in this invention has high atom utilization (overall yield up to 84.55%) and generates less waste, which is conducive to environmental protection and industrial production requirements. Attached Figure Description

[0038] Figure 1 The 1H NMR spectrum of compound SW-1 prepared in Example 1 of this invention; Figure 2 The image shows the carbon NMR spectrum of compound SW-1 prepared in Example 1 of this invention. Figure 3 The HPLC chromatogram of compound SW-1 in Example 1 of the present invention is shown below. Figure 4 This is a graph showing the therapeutic effect of compound SW-1 in a tumor-bearing mouse model in Example 4 of the present invention. Detailed Implementation

[0039] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0040] The reaction process of the present invention is monitored using conventional monitoring methods (such as TLC (thin-layer chromatography) or NMR (nuclear magnetic resonance)), and the reaction endpoint is generally defined as the disappearance of the reaction substrate.

[0041] In the following specific embodiments, the high-performance liquid chromatography (HPLC) conditions used for purity determination of compound SW-1 were as follows: Shimadzu LC-20AD / T, DGU-20A5R degasser, CTO-20A column oven, and SPD-20A UV-Vis detector. The chromatographic column was an Xbridge C18 (50 mm × 4.6 mm, 5.0 μm), with deionized water as mobile phase A and acetonitrile containing 0.1% (v / v) trifluoroacetic acid as mobile phase B, using gradient elution at a flow rate of 0.65 mL / min. The gradient elution program was as follows: 0–1 min, 30% (v / v) mobile phase B; 1–16 min, mobile phase B linearly increased to 60% (v / v); 16–17 min, maintained at 60% mobile phase B; 17–25 min, mobile phase B linearly decreased to 30% (v / v); 25–30 min, maintained at 30% (v / v) mobile phase B; column temperature was 30 °C, injection volume was 5 μL, and detection wavelength was 254 nm.

[0042] The compounds disclosed in this invention also relate to their available forms, such as metabolites, hydrates, solvates, prodrugs, salts, especially pharmaceutically acceptable salts, and coprecipitates.

[0043] Furthermore, the compounds of the present invention exist in a free form, for example, as a free base or free acid or zwitterion, or in the form of a salt. The salt can be any pharmaceutically acceptable salt, organic or inorganic addition salt, especially any pharmaceutically acceptable organic or inorganic addition salt.

[0044] Pharmaceutically acceptable salts of the compounds of this invention may be acid addition salts of the compounds of this invention carrying nitrogen atoms in the chain or ring, acid addition salts of sufficiently alkalized compounds of this invention, acid addition salts formed with inorganic acids, or acid addition salts formed with organic acids. For example, the acids that add to the compounds of this invention are selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanoic acid, digluconic acid, 3-hydroxy-2-naphthic acid, nicotinic acid, dihydroxynaphthic acid, pectinic acid, persulfate, 3-phenylpropionic acid, bitter acid, etc. Acids, neopentanoic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheponic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, hemisulfonic acid, or thiocyanate.

[0045] The compounds of the present invention may contain non-natural proportions of isotopes on one or more atoms constituting the compound, for example, by replacing hydrogen with deuterium to form deuterated drugs.

[0046] In this invention, the term "stereoisomer" refers to compounds having the same chemical composition but differing in the spatial arrangement of atoms or groups. Stereoisomers include enantiomers, diastereomers, and conformational isomers. The term "enantiomer" refers to two stereoisomers of a compound that are non-overlapping mirror images of each other. The term "diastereomer" refers to stereoisomers having two or more chiral centers whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, or biological activity. Mixtures of diastereomers can be separated using chiral HPLC.

[0047] The Suzuki coupling reaction described in this invention is a common organic named reaction in the art.

[0048] Those skilled in the art will understand that compounds of formula (I) may contain one or more chiral centers, and thus have two or more stereoisomers. Therefore, the compounds of the present invention may be a single stereoisomer (e.g., an enantiomer, a diastereomer) or a mixture of multiple stereoisomers in any proportion. For example, they may exist in racemic form, and, where appropriate, in the form of their tautomers or geometric isomers.

[0049] The present invention provides a biotin-targeted PD-L1 inhibitor, wherein the biotin-targeted PD-L1 inhibitor is a compound represented by formula (I), or a stereoisomer of the compound represented by formula (I), or a pharmaceutically acceptable salt of the compound represented by formula (I), or a pharmaceutically acceptable salt of a stereoisomer of the compound represented by formula (I). The structural formula of the compound represented by formula (I) is as follows: (I).

[0050] Specific embodiments of the present invention also provide a method for preparing the biotin-targeted PD-L1 inhibitor, comprising the following steps: S1. Compound A1 and compound A2 undergo a Suzuki coupling reaction to generate intermediate A3: In a mixed solvent consisting of ether solvent and water, in the presence of palladium catalyst and acid-binding agent, under inert gas protection, compound A1 and compound A2 react at a molar ratio of 1:(1~2) at 70℃~90℃ for 8~12 hours to obtain intermediate A3. S2. Intermediate A3 and compound A4 undergo an amide condensation reaction to generate intermediate A5: In a polar aprotic organic solvent, in the presence of a condensing agent and an acid-binding agent, intermediate A3 and compound A4 react at 20℃~30℃ for 1~5h to obtain intermediate A5; the molar ratio of intermediate A3 to compound A4 is 1:(1~2). S3. Intermediate A5 and compound A6 undergo a Suzuki coupling reaction to generate intermediate A7: In a mixed solvent consisting of ether solvent and water, in the presence of palladium catalyst and acid-binding agent, under inert gas protection, intermediate A5 and compound A6 react at a molar ratio of 1:(1~2) at 70℃~90℃ for 8~12 hours to obtain intermediate A7. S4. Intermediate A7 and compound A8 undergo a reductive amination reaction to generate compound SW-1: In a mixed solvent composed of aprotic polar solvent and protic polar solvent, in the presence of a reducing agent and a weak organic acid, intermediate A7 and compound A8 react at a molar ratio of 1:(1~2) at 20℃~30℃ for 1~5h to obtain compound SW-1.

[0051] The main reaction route of the above preparation method is as follows: .

[0052] As an example, the molar ratio of compound A1 to compound A2 in step S1 is 1:1, 1:1.05, 1:1.08, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.8 or 1:2, and is not limited thereto.

[0053] As an example, the reaction temperature in step S1 is 70°C, 75°C, 80°C, 85°C, or 90°C, but is not limited to this.

[0054] As an example, the reaction time in step S1 is 8h, 9h, 10h, 11h or 12h, but is not limited to this.

[0055] As an example, the molar ratio of intermediate A3 to compound A4 in step S2 is 1:1, 1:1.05, 1:1.08, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.8 or 1:2, and is not limited thereto.

[0056] As an example, the reaction temperature in step S2 is 20°C, 25°C, or 30°C, but is not limited to this.

[0057] As an example, the reaction time in step S2 is 1h, 2h, 2.5h, 3h, 4h, 4.5h or 5h, but is not limited to this.

[0058] As an example, the molar ratio of intermediate A5 to compound A6 in step S3 is 1:1, 1:1.05, 1:1.08, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.8 or 1:2, and is not limited thereto.

[0059] As an example, the reaction temperature in step S3 is 70°C, 75°C, 80°C, 85°C, or 90°C, but is not limited to this.

[0060] As an example, the reaction time in step S3 is 8h, 9h, 10h, 11h or 12h, but is not limited to this.

[0061] As an example, the molar ratio of intermediate A7 to compound A8 in step S4 is 1:1, 1:1.05, 1:1.08, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.8, or 1:2, and is not limited thereto.

[0062] As an example, the reaction temperature in step S4 is 20°C, 25°C, or 30°C, but is not limited to this.

[0063] As an example, the reaction time in step S4 is 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, or 5h, but is not limited to this.

[0064] In some preferred embodiments, the volume ratio of the ether solvent to water in step S1 is 5:1. As an example, the ether solvent is 1,4-dioxane.

[0065] In some preferred embodiments, the palladium catalyst in step S1 is at least one of tetra(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, and bis(tritert-butylphosphine)palladium (0).

[0066] In some preferred embodiments, the acid-binding agent in step S1 is at least one of potassium carbonate, sodium carbonate, and potassium acetate.

[0067] In some preferred embodiments, after the reaction in step S1 is completed, the temperature is lowered to room temperature, the solvent is removed by rotary evaporation, and the residue is subjected to silica gel column chromatography with the eluent being a mixture of petroleum ether and ethyl acetate in a ratio of 20:1.

[0068] In some preferred embodiments, the condensing agent in step S2 is at least one of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole.

[0069] In a further preferred embodiment, the condensing agent in step S2 is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; the molar ratio of intermediate A3, compound A4, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1:(1~2):(1~2). As an example, the molar ratio of intermediate A3, compound A4, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate in step S2 is 1:1:1, 1:1.05:1.1, 1:1.1:1.2, 1:1.1:1.3, 1:1.1:1.5, 1:1.3:1.5, 1:1.5:1.8, 1:1.5:2, or 1:2:2, and is not limited thereto.

[0070] In some preferred embodiments, the polar aprotic organic solvent in step S2 is N,N-dimethylformamide (DMF).

[0071] In some preferred embodiments, the acid-binding agent in step S2 is at least one of N,N-diisopropylethylamine and triethylamine.

[0072] In some preferred embodiments, after the reaction in step S2 is completed, water is added to the reaction solution, the organic phase is separated and extracted with an organic solvent that is sparingly soluble in water, the organic phase is concentrated under reduced pressure, the residue is added to silica gel for concentration, and then subjected to silica gel column chromatography. The eluent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 15:1. As an example, the organic solvent that is sparingly soluble in water is ethyl acetate.

[0073] In some preferred embodiments, the volume ratio of the ether solvent to water in step S3 is 5:1. As an example, the ether solvent is 1,4-dioxane.

[0074] In some preferred embodiments, the palladium catalyst in step S3 is at least one of tetra(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, and bis(tritert-butylphosphine)palladium (0).

[0075] In some preferred embodiments, the acid-binding agent in step S3 is at least one of potassium acetate, sodium carbonate, and potassium carbonate.

[0076] In some preferred embodiments, after the reaction in step S3 is completed, the solvent is removed by rotary evaporation of the reaction solution, and the residue is subjected to silica gel column chromatography with the eluent being a mixture of petroleum ether and ethyl acetate in a ratio of 10:1.

[0077] In some preferred embodiments, the reducing agent in step S4 is at least one of NaBH3CN, NaBH(OAc)3, and NaBH4.

[0078] In some preferred embodiments, after the reaction in step S4 is completed, the solvent is removed by vacuum distillation, and the residue is subjected to silica gel column chromatography. The eluent is a mixture of dichloromethane and methanol in a volume ratio of 10:1.

[0079] In some preferred embodiments, the volume ratio of the aprotic polar solvent to the protic polar solvent in step S4 is 10:1. As an example, the aprotic polar solvent is dichloromethane, and the protic polar solvent is methanol.

[0080] In some preferred embodiments, the organic weak acid in step S4 is at least one of glacial acetic acid and formic acid.

[0081] Example 1 This embodiment provides a biotin-targeted PD-L1 inhibitor, chemically named N-(4''-(((3-amino-2,2-dimethyl-3-oxopropyl)amino)methyl)-3''-methoxy-2'-methyl-[1,1':3',1''-triphenyl]-3-yl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thiopheno[3,4- d Imidazol-4-yl)pentanamide (hereinafter referred to as compound SW-1), has the following chemical structural formula: .

[0082] The synthetic route for compound SW-1 is as follows: .

[0083] The synthesis method of compound SW-1 specifically includes the following steps: S1. Add compound A1 (2g, 8.00mmol), compound A2 (1.75g, 8.00mmol), tetrakis(triphenylphosphine)palladium (92.47mg, 80.02µmol), potassium carbonate (1.66g, 12.00mmol), and a mixed solvent of 1,4-dioxane and water in a 5:1 ratio (50mL / 10mL) to the reaction flask. The mixture was purged with nitrogen three times under vacuum. Under nitrogen protection, the mixture was stirred and heated to 80°C for 10 hours, monitored by TLC (evolving solvent: a mixed solvent of PE and EA in a volume ratio of 20:1). The mixture was then cooled to 25°C, and the solvent was evaporated by rotary evaporation. The solution was then passed through a column chromatography column using silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.), eluted with a petroleum ether (PE):ethyl acetate (EA) system (PE to EA volume ratio of 20:1), yielding a colorless oily intermediate A3 (2.0 g, yield 95.34%). LCMS: (MS-ESI, m / z): [M+H]+ = 262.1.

[0084] S2. At room temperature, intermediate A3 (1 g, 3.81 mmol), compound A4 (1.03 g, 4.20 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.5 g, 3.94 mmol), N,N-diisopropylethylamine (986.05 mg, 7.63 mmol), and N,N-dimethylformamide (DMF, 20 mL) were added to the reaction flask. The reaction was carried out at 25 °C for 1 hour and monitored by TLC (developing solvent: PE to EA volume ratio was...). The reaction was completed using a 15:1 mixed solvent. After the reactants reacted completely, 20 mL of water was added to quench the reaction. The mixture was separated, extracted three times with ethyl acetate (30 mL x 3), and the ethyl acetate phases were combined. The mixture was concentrated under reduced pressure, and silica gel (silica gel mass to concentrated residue mass ratio 30:1) was added for further concentration. The mixture was then filtered through a column using silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.) and eluted with a petroleum ether:ethyl acetate system (PE to EA volume ratio 15:1) to obtain intermediate A5 (1.8 g, yield 96.61%). LCMS: (MS-ESI, m / z): [M+H]+ = 488.2.

[0085] S3. Take intermediate A5 (500 mg, 1.02 mmol) obtained in step S2, add a mixed solvent of 1,4-dioxane and water at a volume ratio of 5:1 (25 mL / 5 mL), add compound A6 (268.31 mg, 1.02 mmol), tetrakis(triphenylphosphine)palladium (11.83 mg, 10.24 µmol), and potassium carbonate (212.21 mg, 1.54 mmol), and react at 80 °C for 10 hours under nitrogen atmosphere. Monitor the reaction using a thin-layer chromatography plate. (The developing solvent was a mixed solvent of PE and EA in a volume ratio of 10:1.) After the reaction was complete, the reaction solution was evaporated to dryness, water was added, and then extracted with ethyl acetate. The organic phase was dried over saturated brine and anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation. The mixture was separated by column chromatography using silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.). Elution was performed with a petroleum ether:ethyl acetate system (PE and EA in a volume ratio of 10:1) to give intermediate A7 (550 mg, yield 98.82%). LCMS: (MS-ESI, m / z): [M+H]+ = 544.2.

[0086] S4. Take intermediate A7 (100 mg, 183.93 µmol), compound A8 (32.05 mg, 275.90 µmol), NaBH3CN (17.34 mg, 275.90 µmol), and glacial acetic acid (1.10 mg, 18.39 µmol) obtained in step S3 and add them into a 100 mL round-bottom flask. The reaction solvent is a mixed solvent of dichloromethane and methanol in a volume ratio of 10:1 (40 mL / 4 mL). React at 25 °C for 2 h. After the reaction of the reactants was complete, as determined by TLC (using a mixed solvent of DCM and MeOH in a volume ratio of 10:1), the solvent was removed by vacuum distillation. Separation was then performed by column chromatography using silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.). Elution was carried out with a dichloromethane (DCM):methanol (MeOH) system (DCM to MeOH volume ratio of 10:1) to obtain compound SW-1 (110 mg, yield 92.89%, HPLC purity 98.36%). See [link to HPLC]. Figure 3 ).

[0087] The proton and carbon NMR spectra of compound SW-1 are shown below. Figure 1 and Figure 2 The detection and characterization results are as follows: 1H NMR (400 MHz, d6-DMSO) δ 10.04 (s, 1H), 8.71 (s, 1H), 7.70 (s, 1H), 7.63-7.53 (m, 2H), 7.45 (d, J = 7.5 Hz, 1H), 7.35 (dd, J = 18.7, 8.3 Hz, 3H), 7.28-7.19 (m, 2H), 7.13-6.95 (m, 3H), 6.42 (d, J = 29.6 Hz, 2H), 4.31 (s, 1H), 4.12 (d, J = 26.4 Hz, 3H), 3.90 (s, 3H), 3.13 (s, 1H), 2.93 (s, 2H), 2.87-2.79 (m, 1H), 2.60 (s, 1H), 2.34 (t, J = 6.5 Hz, 2H), 2.09 (s, 3H), 1.70-1.37 (m, 6H), 1.21 (s, 6H).

[0088] 13C NMR (101 MHz, d6-DMSO) δ 179.54, 171.76, 163.14, 157.72, 142.80,142.47, 142.40, 139.66, 132.49, 131.25, 129.22, 129.09, 128.91, 126.02,124.16, 121.57, 120.16, 118.03, 112.40, 61.46, 59.61, 56.09, 55.81, 48.01,36.65, 28.65, 28.51, 25.51, 24.16, 18.92. HRMS m / z: calcd for C 36 H 46 N5O4S [M+H]+ 644.3271, found 644.3281.

[0089] Example 2 This embodiment provides the same biotin-targeted PD-L1 inhibitor (compound SW-1) as in Example 1. The preparation method of compound SW-1 includes the following steps: S1. Add compound A1 (2.50 g, 10.00 mmol), compound A2 (2.63 g, 12.00 mmol), 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (73.19 mg, 0.10 mmol), sodium carbonate (1.59 g, 15.00 mmol), and a mixed solvent of 1,4-dioxane and water in a 5:1 ratio (60 mL / 12 mL) to the reaction flask. The mixture was purged with nitrogen three times and then stirred and heated to 85°C for 8 hours under nitrogen protection. The reaction was monitored by TLC (the developing solvent was a mixed solvent of PE and EA in a volume ratio of 20:1). The mixture was then cooled to 25°C and the reaction solution was rotary evaporated until the solvent was completely evaporated. The solution was then passed through a column chromatography silica gel (200-300 mesh) and eluted with a petroleum ether (PE): ethyl acetate (EA) system (PE to EA in a volume ratio of 20:1) to give a colorless oily intermediate A3 (2.45 g, yield 93.43%).

[0090] S2. At room temperature, intermediate A3 (1.20 g, 4.58 mmol), compound A4 (1.34 g, 5.49 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.61 g, 6.87 mmol), triethylamine (926.43 mg, 9.16 mmol), and N,N-dimethylformamide (DMF, 25 mL) were added to the reaction flask. The reaction was carried out at 20 °C for 3 hours and monitored by TLC (developing solvent: PE and...). The reaction was completed using a mixed solvent of EA (volume ratio 15:1). After the reaction was complete, 25 mL of water was added to quench the reaction. The mixture was separated and extracted three times with ethyl acetate (35 mL × 3). The ethyl acetate phases were combined and concentrated under reduced pressure. Silica gel (silica gel mass to concentrated residue mass ratio 30:1) was added and the mixture was concentrated. The sample was then passed through a column chromatography silica gel (200~300 mesh) and eluted with a petroleum ether:ethyl acetate system (PE to EA volume ratio 15:1) to obtain intermediate A5 (2.1 g, yield 93.92%).

[0091] S3. Take intermediate A5 (600 mg, 1.23 mmol) obtained in step S2, add a mixed solvent of 1,4-dioxane and water at a volume ratio of 5:1 (25 mL / 5 mL), add compound A6 (386.37 mg, 1.47 mmol), 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (8.99 mg, 12.28 μmol), and potassium acetate (180.84 mg, 1.84 mmol), and incubate at 85 °C under nitrogen atmosphere. After 8 hours of reaction, the reaction was monitored by thin-layer chromatography (developing solvent: a mixed solvent of PE and EA in a volume ratio of 10:1). Once the reaction was complete, the reaction solution was evaporated to dryness, water was added, and then extracted with ethyl acetate. The organic phase was dried over saturated brine and anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation. The mixture was separated by column chromatography on silica gel (200-300 mesh) and eluted with a petroleum ether:ethyl acetate system (PE and EA in a volume ratio of 10:1) to obtain intermediate A7 (660 mg, yield 98.82%).

[0092] S4. Take intermediate A7 (120 mg, 220.87 μmol), compound A8 (46.15 mg, 397.29 μmol), NaBH(OAc)3 (84.20 mg, 397.29 μmol), and glacial acetic acid (1.33 mg, 22.07 μmol) obtained in step S3 and add them to a 100 mL round-bottom flask. The reaction solvent is a mixture of dichloromethane and methanol in a volume ratio of 10:1 (50 mL / 5 mL). React at 20 °C for 3 h. After the reaction is complete, the solvent is removed by vacuum distillation, and the mixture is separated by column chromatography on silica gel (200-300 mesh). The mixture is eluted with a dichloromethane (DCM):methanol (MeOH) system (DCM:MeOH volume ratio of 10:1) to obtain compound SW-1 (130 mg, yield 91.48%).

[0093] Example 3 This embodiment provides the same biotin-targeted PD-L1 inhibitor (compound SW-1) as in Example 1. The preparation method of compound SW-1 includes the following steps: S1. Add compound A1 (1.50 g, 6.00 mmol), compound A2 (1.97 g, 9.00 mmol), bis(tri-tert-butylphosphine)palladium(0) (30.67 mg, 60.00 μmol), potassium acetate (883.52 mg, 9.00 mmol), and a mixed solvent of 1,4-dioxane and water in a ratio of 5:1 (40 mL / 8 mL) to the reaction flask. The mixture was purged with nitrogen three times and stirred and heated to 75°C for 12 hours under nitrogen protection. The reaction was monitored by TLC (the developing solvent was a mixed solvent of PE and EA in a volume ratio of 20:1). The mixture was then cooled to 25°C and the reaction solution was rotary evaporated until the solvent was completely evaporated. The solution was then passed through a column chromatography silica gel (200-300 mesh) and eluted with a petroleum ether (PE): ethyl acetate (EA) system (PE to EA in a volume ratio of 20:1) to give a colorless oily intermediate A3 (1.46 g, yield 92.8%).

[0094] S2. At room temperature, intermediate A3 (1.00 g, 3.81 mmol), compound A4 (1.40 g, 5.72 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (2.18 g, 5.72 mmol), N,N-diisopropylethylamine (986.05 mg, 7.63 mmol), and N,N-dimethylformamide (DMF, 20 mL) were added to the reaction flask. The reaction was carried out at 30 °C for 2 hours and monitored by TLC (developing solvent: [missing information]). The reaction was completed using a mixed solvent of PE and EA in a volume ratio of 15:1. 20 mL of water was added to quench the reaction, and the mixture was separated. The mixture was extracted three times with ethyl acetate (30 mL × 3). The ethyl acetate phases were combined, concentrated under reduced pressure, and concentrated with silica gel (the mass ratio of silica gel to concentrated residue was 30:1). The mixture was then passed through a column chromatography silica gel (200-300 mesh) and eluted with a petroleum ether:ethyl acetate system (PE and EA in a volume ratio of 15:1) to obtain intermediate A5 (1.8 g, yield 96.61%).

[0095] S3. Take intermediate A5 (500 mg, 1.03 mmol) obtained in step S2, add a mixed solvent of 1,4-dioxane and water at a volume ratio of 5:1 (25 mL / 5 mL), add compound A6 (402.47 mg, 1.54 mmol), bis(tri-tert-butylphosphine)palladium(0) (5.23 mg, 10.24 μmol), and sodium carbonate (162.74 mg, 1.54 mmol), and react at 70 °C under nitrogen atmosphere for 1 minute. After 2 hours, the reaction was monitored on a thin-layer chromatography plate (the developing solvent was a mixed solvent of PE and EA in a volume ratio of 10:1). The reaction solution was then evaporated to dryness, water was added, and the mixture was extracted with ethyl acetate. The organic phase was dried over saturated brine and anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation. The mixture was separated by column chromatography on silica gel (200-300 mesh) and eluted with a petroleum ether:ethyl acetate system (PE and EA in a volume ratio of 10:1) to give intermediate A7 (530 mg, yield 95.23%).

[0096] S4. Take intermediate A7 (100 mg, 183.93 μmol), compound A8 (42.73 mg, 367.86 μmol), NaBH4 (13.92 mg, 367.86 μmol), and glacial acetic acid (1.10 mg, 18.39 μmol) obtained in step S3 and add them to a 100 mL round-bottom flask. The reaction solvent is a mixture of dichloromethane and methanol in a volume ratio of 10:1 (40 mL / 4 mL). The reaction is carried out at 30 °C for 1.5 h. After the reaction is complete, the solvent is removed by vacuum distillation and column chromatography is performed using silica gel (200-300 mesh). The mixture is eluted with a dichloromethane (DCM):methanol (MeOH) system (DCM:MeOH volume ratio of 10:1) to obtain compound SW-1 (108 mg, yield 91.20%).

[0097] Example 4 This embodiment further investigates the inhibitory effect of compound SW-1 prepared in Example 1 on PD-1 / PD-L1 interaction, its cytotoxicity to three cell types (B16-F10, HepG2, LO2), and its efficacy in treating melanoma in mice, as detailed below: 1. PD-1 / PD-L1 target testing 1.1 Experimental Objective and Principle The detection method for the inhibitory activity of PD-1 / PD-L1 protein-protein interaction is a biochemically based homogeneous time-resolved fluorescence (HTRF) assay. HTRF technology offers a simple, rapid, and high-throughput method for characterizing compounds and antibody blockers. PD-L1 carries Tag1, and PD-1 carries Tag2. Europium-labeled anti-Tag1 antibodies bind to Tag1-labeled PD-L1, while XL665-labeled anti-Tag2 antibodies bind to Tag2-labeled PD-1. When Tag1-labeled PD-L1 and Tag2-labeled PD-1 bind, the HTRF donor and HTRF receptor also come into close proximity (typically <10 nm). At this point, the HTRF donor is photoexcited and transfers fluorescence resonance energy (FRET) to the HTRF receptor, which emits characteristic fluorescence at 665 nm. The intensity of the fluorescence signal is directly proportional to the degree of PD-1 / PD-L1 interaction; that is, the stronger the fluorescence signal, the more PD-L1 with Tag1 and PD-1 with Tag2 bind. Therefore, blocking PD-1 / PD-L1 interaction with compounds or antibodies leads to a reduction in HTRF signal. If the HTRF donor and HTRF receptor do not bind, there is no energy transfer, and only the fluorescence emitted by the HTRF donor at 620 nm is detected. The fluorescence intensity is detected by a microplate reader, and the ratio of 665 nm to 620 nm reflects the extent to which the compound blocks PD-1 / PD-L1 activity.

[0098] 1.2 Reagent Information Table 1. Source information of reagents required for testing

[0099] 1.3. Dilution of the compound and preparation of the test solution (1) Dilution of the compound stock solution: In order to accurately measure the inhibitory effect of each compound on the binding ability of PD-1 / PD-L1, each compound needs to be diluted to a stock solution of 20 mmol / L. According to the formula C=n / V=m / M / V (C is the molar concentration, in mol / L; n is the amount of substance, in mol; V is the volume, in L; m is the mass of the compound, in g; M is the molar mass of the compound, in g / mol), the amount of DMSO required to dilute each compound can be calculated. After preparing the stock solution of the compound, dilute it sequentially with Diluent reagent (provided in the kit in Table 1) to obtain diluents of 10 μmol / L, 3.333 μmol / L, 1.111 μmol / L, 0.370 μmol / L, 0.123 μmol / L, 0.041 μmol / L, 0.013 μmol / L, 0.004 μmol / L, 0.001 μmol / L, and 0.0005 μmol / L, using a serial dilution method. Mix well and set aside.

[0100] (2) Dilution of PD-L1 protein mixture: Take 7 mL of PD-L1 protein from the kit, follow the instructions in the kit, add 510 mL of diluent to dilute it, mix well and set aside.

[0101] (3) Dilution of PD-1 protein mixture: Take 5 mL of PD-1 protein from the kit, follow the instructions in the kit, add 550 mL of diluent to dilute it, mix well and set aside.

[0102] (4) Preparation of mixed test solution: Take Anti-Tag-Eu3 from the kit separately + Mix 5 μL of Anti-Tag-XL665 with 5 μL of Anti-Tag-XL665 according to the kit instructions, then add 975 μL of Detection Buffer (the kit is included in Table 1) to dilute it and mix well.

[0103] 1.4 Experimental Testing Procedure (1) Add 2 μL of the compound stock solution dilution to each well of the 96-well plate and centrifuge at 1000 rpm for 1 min.

[0104] (2) Add 4 μL of PD-L1 protein mixture dilution to each well and centrifuge at 1000 rpm for 1 min.

[0105] (3) Add 4 μL of PD-1 protein mixture dilution to each well, centrifuge at 1000 rpm for 1 min, and incubate at room temperature for 15 min.

[0106] (4) Add 10 μL of mixed test solution to each well and centrifuge at 1000 rpm for 1 min.

[0107] (5) Incubate at room temperature for 120 min and read the fluorescence value using a Tecan microplate reader (excitation wavelength Ex: 320 nm; emission wavelength Em: 620 nm or 665 nm).

[0108] (6) Calculate the inhibition rate according to the following formula: Inhibition % = [1 - (signal value at 665nm / 620nm in each well - average value of low control group) / (average value of high control group - average value of low control group)] × 100%. The high control group is the group without compound treatment, and only the reaction system is added with an equal concentration of DMSO; the low control group is the group without PD-1 protein mixture dilution, and only the detection buffer is added.

[0109] (7) Fitting the dose-response curve: Using the log value of concentration as the X-axis and the inhibition rate as the Y-axis, the dose-response curve was fitted using the log (inhibitor) vs. response variable slope function of the analysis software GraphPadPrism 5 to obtain the IC50 of the compound's inhibition of PD-1 / PD-L1 activity. 50 value.

[0110] 1.5 Experimental Results Table 2. Test results of the compounds' inhibitory effect on PD-1 / PD-L1 activity.

[0111] In Table 2, BMS-202 is a small molecule PD-1 / PD-L1 protein-protein interaction inhibitor developed by Bristol-Myers Squibb and purchased from Shanghai Biede Pharmaceutical Technology Co., Ltd. BMS-202 was used as a control compound to inhibit PD-1 / PD-L1 activity, and the control experiment was conducted according to step 1.4. The experimental results in Table 2 show that the compound SW-1 prepared in this invention can inhibit the binding of PD-1 / PD-L1, and its activity is superior to BMS-202.

[0112] 2. Cytotoxicity test of three cell types (B16-F10, HepG2, LO2) 2.1 Experimental Procedure The in vitro proliferative inhibitory activity of the target compound against human hepatocellular carcinoma HepG2 cells, mouse melanoma B16-F10 cells, and normal human hepatocytes LO2 was evaluated using the CCK-8 assay. B16-F10, HepG2, and LO2 cells were purchased from Wuhan Pronosai Life Sciences Co., Ltd. The CCK-8 assay kit was purchased from Abbkine, Inc. (catalog number BMU106-CN). The test cells were seeded at a density of 5000 cells per well in 96-well cell culture plates (purchased from Wuhan Pronosai Life Sciences Co., Ltd.) and incubated overnight at 37°C in a 5% CO2 incubator. Discard the original culture medium and replace each well with 100 μL of serum-free basal culture medium (DMEM, KGI Biotechnology, catalog number KGL1213) containing gradient concentrations of the target compound (20 μM, 10 μM, 3.3 μM, 1.1 μM, 0.37 μM, 0.12 μM, 0.04 μM, 0.01 μM). Continue incubation at a constant temperature for 48 h. Then add 10 μL of CCK to each well. 8. Incubate with the chromogenic working solution (included in the CCK-8 kit) in the dark for 1 hour. Measure the absorbance of each well using a microplate reader at a detection wavelength of 490 nm. Calculate the half-maximal inhibitory concentration (IC50) of the compound using a nonlinear regression model fitted with GraphPad Prism software. 50 The cell proliferation inhibitory activity of the compounds was characterized in this way. All the above in vitro cell experiments were independently repeated in triplicate.

[0113] 2.2 Experimental Results Table 3. Cytotoxicity test results of the test compounds against three cell types (B16-F10, HepG2, LO2).

[0114] Table 3 shows that SWS1 is a small molecule PD-1 / PD-L1 protein-protein interaction inhibitor disclosed in patent application CN115925717A (paragraph 0050 of the specification). The data in Table 3 show that the compound SW-1 prepared in this invention can inhibit the activity of HepG2 and B16F10 cells, with better effects than the prior art compounds SWS1 and BMS-202. Furthermore, compound SW-1 also shows efficacy against LO2 normal hepatocytes (IC50, 1000-1000-1000). 50(>20μM) SW-1 exhibits no cytotoxicity and demonstrates excellent selectivity for both cancer cells and normal tissues. Specifically, SW-1 maintains a therapeutic window with over 40-fold selectivity for B16-F10 cells compared to LO2 cells. SW-1's extremely high tumor selectivity significantly reduces the toxic side effects on normal tissues, providing a prominent safety advantage in the clinical application of anti-tumor drugs. It also possesses significant technological advantages compared to existing PD-1 / PD-L1 small molecule inhibitors.

[0115] 3. Testing the anti-tumor therapeutic effects of compounds 3.1 Experimental Materials The tumor cell line used in this experiment was the mouse melanoma cell line B16F10 (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.). The B16F10 cells were adapted for in vivo modeling in C57BL / 6 mice and are currently recognized as a classic tumor model cell for anti-tumor immunopharmacological evaluation. The model has high stability and good reproducibility, and can objectively evaluate the in vivo immunotherapeutic effects of the test compounds.

[0116] Healthy male C57BL / 6 mice aged 6-7 weeks were used as experimental animals. All experimental mice were provided by Liaoning Changsheng Biotechnology Co., Ltd., and the experimental animals had complete quality certificates. The animal housing environment met the SPF (specific pathogen-free) level barrier system housing standards. The temperature, humidity, and diurnal light rhythm of the housing environment were kept constant. The mice had free access to food and water. After one week of acclimatization, formal animal modeling and drug administration experiments were carried out to ensure the stability of the animals' physiological state and to eliminate the interference of environmental and physiological stress on the drug efficacy evaluation results.

[0117] 3.2 Experimental Methods Construction of a mouse subcutaneous allogeneic tumor transplantation model: B16F10 melanoma cells in good logarithmic growth phase and with normal activity were collected and resuspended in sterile PBS buffer (0.01 mol / L, pH 7.2–7.4) to prepare homogeneous single-cell suspensions. Cell viability (>95%) and cell suspension concentration (2 × 10⁻⁶) were strictly controlled. 6 Cells / mL). Under aseptic conditions, cells were seeded subcutaneously in the axilla of one forelimb of each mouse, with 2 × 10⁶ cells seeded per mouse. 5 Establish a mouse subcutaneous solid tumor transplantation model using one mouse per animal.

[0118] Following inoculation, mice were routinely observed daily for their mental state, eating habits, activity levels, and subcutaneous tumor growth. The long and short diameters of the tumors were measured periodically using calipers, and the tumor volume was recorded in real-time according to the tumor volume calculation formula. The mice were allowed to complete the process once the subcutaneous tumors had grown uniformly to 50 mm. 3 ~100mm 3When the modeling of the standard interval is successful, the tumor-bearing mice are randomly divided into 3 groups (6 mice in each group) to ensure that there is no statistically significant difference in the average tumor volume and body weight of the mice in each group, and to exclude the influence of baseline differences between groups on the efficacy evaluation.

[0119] After grouping, each group of tumor-bearing mice was administered the corresponding test compound and control drug for intervention treatment, strictly adhering to the pre-set dosage, route of administration, and dosing cycle throughout the entire treatment period. Throughout the entire drug treatment experiment, changes in body weight, mental state, diet, and activity levels of the mice in each group were continuously and dynamically monitored and recorded. Simultaneously, changes in tumor volume growth in each group were measured and recorded periodically. The inhibitory effect of the test compound on tumor growth and drug safety in mice were dynamically observed. The entire experiment strictly followed animal experimental ethical guidelines and standard operating procedures for antitumor pharmacodynamic evaluation.

[0120] 3.3 Experimental Results The therapeutic effects of compound SW-1 provided by this invention in an in vivo tumor-bearing mouse model are as follows: Figure 4 As shown. The mouse PD-L1 antibody (purchased from BioXcell, catalog number BE0101) is abbreviated as PD-L1 or anti-mouse PD-L1. This means P < 0.0001.

[0121] like Figure 4 As shown in Figure A: Compound SW-1, administered once daily via intraperitoneal injection at a dose of 10 mg / kg (solvent composed of 30% (v / v) PEG-300, 5% (v / v) DMSO, and 65% (v / v) saline), significantly reduced terminal tumor weight in mice (days 1-15) compared to the control group (administered the same volume of solvent composed of 30% (v / v) PEG-300, 5% (v / v) DMSO, and 65% (v / v) saline), achieving a tumor growth inhibition rate (TGI) of 64.9%. This in vivo antitumor activity was superior to the selective PD-L1 antibody group (10 mg / kg, tumor growth inhibition rate 44.2%). Safety monitoring results showed that no mice in any of the experimental treatment groups experienced drug-related weight loss exceeding 10% (see Figure A). Figure 4 (Figure B in the middle). The mouse survival analysis results further confirm that compound SW-1 possesses excellent anti-tumor therapeutic effects (see Figure B in the middle). Figure 4(See Figure C). Throughout the 15-day observation period, all mice in the SW-1 administration group survived. These results confirm that compound SW-1 exhibits good tolerability at effective antitumor doses, with a wide therapeutic safety window and high safety profile.

[0122] In summary, this invention provides a biotin-targeted PD-L1 inhibitor (compound SW-1) that possesses excellent in vivo antitumor pharmacological effects, can effectively exert in vivo antitumor therapeutic effects, and has good drug development potential and clinical application translation value.

[0123] The embodiments described above are merely preferred embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by anyone skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection claimed in this application and the content of the specification should be included within the scope of protection of this application.

Claims

1. A biotin-targeted PD-L1 inhibitor, characterized in that, The biotin-targeted PD-L1 inhibitor is a compound of formula (I), or a stereoisomer of the compound of formula (I), or a pharmaceutically acceptable salt of the compound of formula (I), or a pharmaceutically acceptable salt of a stereoisomer of the compound of formula (I). The structural formula of the compound represented by formula (I) is as follows: （I）。 2. The method for preparing the biotin-targeted PD-L1 inhibitor according to claim 1, characterized in that, Includes the following steps: S1, Compound A1 and Compound A2 undergo a Suzuki coupling reaction to generate intermediate A3; S2, intermediate A3 and compound A4 undergo an amide condensation reaction to generate intermediate A5; S3, intermediate A5 and compound A6 undergo a Suzuki coupling reaction to generate intermediate A7; S4, intermediate A7 and compound A8 undergo a reductive amination reaction to generate compound SW-1, which is the biotin-targeted PD-L1 inhibitor; The synthetic route for the biotin-targeted PD-L1 inhibitor is as follows: 。 3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of compound A1 to compound A2 is 1:(1~2). Or / and, in step S2, the molar ratio of intermediate A3 to compound A4 is 1:(1~2); Or / and, in step S3, the molar ratio of intermediate A5 to compound A6 is 1:(1~2); Or / and, in step S4, the molar ratio of intermediate A7 to compound A8 is 1:(1~2).

4. The preparation method according to claim 2, characterized in that, The reaction conditions in step S1 are as follows: under inert gas protection, in the presence of palladium catalyst and acid-binding agent, compound A1 and compound A2 react at 70℃~90℃ for 8~12 hours. Or / and, the reaction conditions in step S2 are: in the presence of a condensing agent and an acid-binding agent, intermediate A3 reacts with compound A4 at 20℃~30℃ for 1~5h; Or / and, the reaction conditions in step S3 are: under inert gas protection, in the presence of palladium catalyst and acid-binding agent, intermediate A5 reacts with compound A6 at 70℃~90℃ for 8~12 hours; Or / and, the reaction conditions in step S4 are: in the presence of a reducing agent and a weak organic acid, intermediate A7 reacts with compound A8 at 20℃~30℃ for 1~5h.

5. The preparation method according to claim 4, characterized in that, The palladium catalyst in step S1 is at least one of tetra(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, and bis(tritert-butylphosphine)palladium (0); Or / and, the acid-binding agent in step S1 is at least one of potassium carbonate, sodium carbonate, and potassium acetate; Or / and, the condensing agent in step S2 is at least one of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole; Or / and, the acid-binding agent in step S2 is at least one of N,N-diisopropylethylamine and triethylamine.

6. The preparation method according to claim 4, characterized in that, The palladium catalyst in step S3 is at least one of tetra(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, and bis(tritert-butylphosphine)palladium (0); Or / and, the acid-binding agent in step S3 is at least one of potassium acetate, sodium carbonate, and potassium carbonate; Or / and, the reducing agent in step S4 is at least one of NaBH3CN, NaBH(OAc)3, and NaBH4; Or / and, the organic weak acid in step S4 is at least one of acetic acid and formic acid.

7. The preparation method according to claim 2, characterized in that, In step S1, compound A1 and compound A2 react in a mixed solvent consisting of an ether solvent and water in a volume ratio of 5:1; Or / and, in step S2, intermediate A3 reacts with compound A4 in a polar aprotic organic solvent; Or / and, in step S3, intermediate A5 reacts with compound A6 in a mixed solvent consisting of an ether solvent and water in a volume ratio of 5:1; Or / and, in step S4, intermediate A7 reacts with compound A8 in a mixed solvent consisting of an aprotic polar solvent and a protic polar solvent in a volume ratio of 10:

1.

8. The preparation method according to claim 7, characterized in that, The ether solvent in step S1 is 1,4-dioxane; Or / and, the polar aprotic organic solvent in step S2 is N,N-dimethylformamide; Or / and, the ether solvent in step S3 is 1,4-dioxane; Or / and, in step S4, the aprotic polar solvent is dichloromethane; Or / and, in step S4, the proton polar solvent is methanol.

9. The use of the biotin-targeted PD-L1 inhibitor of claim 1 in the preparation of antitumor drugs related to the PD-L1 target.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the biotin-targeted PD-L1 inhibitor of claim 1.

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