A pd-l1 / ezh2 bifunctional inhibitor and preparation method and application thereof

Abstract

The application provides a PDL1 / EZH2 bifunctional inhibitor and a preparation method and application thereof, and belongs to the technical field of organic compound synthesis. In the application, phenylboronic acid and 2,6-dibromotoluene are used as starting materials, and through Suzuki coupling reaction, Miyaura coupling reaction, Suzuki coupling reaction, Birch reduction reaction, Fmoc protection reaction, Boc protection group removal reaction, condensation reaction and Fmoc protection group removal reaction, a compound PE-1 is synthesized, and the molecular structure of the compound PE-1 is confirmed. The compound PE-1 can simultaneously significantly inhibit the activities of PD-L1 and EZH2, has a significant antitumor effect, and has good oral bioavailability, and is an orally administrable PD-L1 / EZH2 bifunctional inhibitor.

Description

Technical Field

[0001] This invention relates to the field of organic compound synthesis technology, specifically to a PD L1 / EZH2 bifunctional inhibitors, their preparation methods, and applications. Background Technology

[0002] Immune checkpoint blockade therapy has become an important means of cancer treatment, among which PD 1 / PD L1 (programmed death receptor 1 / programmed death ligand 1) immune checkpoint inhibitors have shown significant efficacy in various malignant tumors by reactivating T-cell immune responses. However, clinical practice has shown that PD... 1 / PD L1 monotherapy has significant limitations: low objective response rate (only 15%–25% in most solid tumors), widespread primary and secondary resistance, making it difficult to meet clinical needs. The core reason is that the tumor microenvironment contains multiple immunosuppressive mechanisms, and simply blocking PD... 1 / PD The L1 pathway is insufficient to reverse the highly immunosuppressed tumor microenvironment and is therefore insufficient to fully activate the anti-tumor immune system.

[0003] EZH2 (enhancer homolog 2), as the core catalytic subunit of PRC2 (multicomb repressor complex 2), is a key epigenetic immune regulatory target. EZH2 inhibits the expression of immune-related genes, reduces CD8 (Cluster of Differentiation 8) positive T cell infiltration and activation, and promotes the accumulation of immunosuppressive cells by catalyzing H3K27me3 (trimethylation of histone H3 at lysine 27), thereby mediating immune escape and disease progression (PD). 1 / PD L1 resistance leads to a "cold tumor" phenotype in tumors. Studies have confirmed that PD... L1 and EZH2 are co-expressed and synergistic in tumors, forming an immunosuppressive network. High expression of both is closely associated with tumor progression and poor prognosis. Therefore, simultaneously blocking PD... 1 / PD The L1 and EZH2 pathways can synergistically activate immune mechanisms at both the immune checkpoint and epigenetic regulation levels, significantly improving efficacy and overcoming drug resistance.

[0004] Currently, relevant clinical trials are exploring PD. 1 / PD Combining L1 inhibitors with EZH2 inhibitors has significant drawbacks: high risk of drug-drug interactions, pharmacokinetic mismatch, uneven in vivo exposure, poor adherence, and increased toxicity, making it difficult to achieve optimal synergistic benefits. In contrast, PD... L1 / EZH2 bifunctional small molecule inhibitors can act on two targets simultaneously with a single molecule, achieving stronger synergistic effects, more stable PK / PD (pharmacokinetic / pharmacodynamic) characteristics, better oral drugability, and avoiding many drawbacks of combination therapy. They are a new generation of drugs with greater clinical advantages.

[0005] Although dual-target strategies have become an important research and development direction, existing drugs still suffer from problems such as insufficient oral bioavailability, poor target selectivity, and limited structural types. In particular, there is a lack of orally available, highly effective, and low-toxicity drug-drug combinations. The lack of bifunctional small molecule inhibitors of L1 / EZH2 cannot meet clinical needs. Therefore, it is necessary to develop novel structures, high-activity inhibitors, and those with both PD and other therapeutic effects. 1 / PD Bifunctional small molecule inhibitors that block L1 and inhibit EZH2 activity and have good oral bioavailability have become urgently needed. Summary of the Invention

[0006] In view of the above problems, the present invention provides a PD L1 / EZH2 bifunctional inhibitors and their preparation methods. A novel PD inhibitor was designed. L1 / EZH2 dual-target inhibitor compounds effectively compensate for the shortcomings of existing technologies, providing key solutions for overcoming immunotherapy resistance, improving anti-tumor efficacy, and achieving convenient oral administration, and have important clinical value and broad application prospects.

[0007] Specifically, in order to achieve the above objectives, the PD provided by the present invention The L1 / EZH2 bifunctional 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 of formula (I) is as follows: (I).

[0008] The method for preparing the PD-L1 / EZH2 bifunctional inhibitor provided by the present invention 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 a Miyaura coupling reaction to generate intermediate A5; S3, intermediate A5 and compound A6 undergo a Suzuki coupling reaction to generate intermediate A7; S4. Intermediate A7 reacts with compound A8 in a Birch reduction reaction to generate intermediate A9. S5, intermediate A9 reacts with compound A10 via an Fmoc protection reaction to generate intermediate A11; S6. Intermediate A11 loses its Boc protecting group under the action of acid to generate intermediate A12; S7. Intermediate A12 undergoes a condensation reaction with compound A13 to generate intermediate A14; S8 and intermediate A14 lose the Fmoc protecting group under the action of piperidine to generate compound PE-1, which is the PD-L1 / EZH2 bifunctional inhibitor. The synthetic route for the PD-L1 / EZH2 bifunctional inhibitor is as follows: .

[0009] In the preferred embodiment, the molar ratio of compound A1 to compound A2 in step S1 is 1:(1~2).

[0010] In the preferred embodiment, the reaction conditions in step S1 are: under inert gas protection, in the presence of a palladium catalyst, and at 50°C to 80°C for 1 to 10 hours.

[0011] In a preferred embodiment, the molar ratio of intermediate A3 to compound A4 in step S2 is 1:(1~2).

[0012] In the preferred embodiment, the reaction conditions in step S2 are: under inert gas protection, in the presence of a palladium catalyst, and at 70°C to 90°C for 1 to 10 hours.

[0013] In a preferred embodiment, the molar ratio of intermediate A5 to compound A6 in step S3 is 1:(1~2).

[0014] In the preferred embodiment, the reaction conditions in step S3 are: under inert gas protection, in the presence of a palladium catalyst, and at 80°C to 100°C for 1 to 12 hours.

[0015] In a preferred embodiment, the molar ratio of intermediate A7 to compound A8 in step S4 is 1:(1~2).

[0016] In a preferred embodiment, the reducing agent in step S4 is at least one of NaBH3CN, NaBH(OAc)3, and NaBH4.

[0017] In the preferred embodiment, the reaction conditions in step S4 are: reaction at 20℃~30℃ for 1~5 hours.

[0018] In a preferred embodiment, the molar ratio of intermediate A9 to compound A10 in step S5 is 1:(1~2).

[0019] In the preferred embodiment, the reaction conditions in step S5 are: reacting at 20°C to 30°C for 1 to 5 hours in the presence of an acid-binding agent.

[0020] In the preferred embodiment, in step S6, the Boc protecting group in intermediate A11 reacts with the H group in the acid. + The molar ratio is 1:(1~50).

[0021] In a preferred embodiment, the acid in step S6 is at least one of trifluoroacetic acid and hydrochloric acid.

[0022] In the preferred embodiment, the reaction conditions in step S6 are: reaction at 20℃~30℃ for 1~5 hours.

[0023] In a preferred embodiment, the molar ratio of intermediate A12 to compound A13 in step S7 is 1:(1~2).

[0024] In a preferred embodiment, in step S7, intermediate A12 and compound A13 undergo a condensation reaction in the presence of a condensing agent, wherein the condensing agent is at least one of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole.

[0025] In the preferred embodiment, the reaction conditions in step S7 are: reacting at 20°C to 30°C for 1 to 5 hours in the presence of an acid-binding agent.

[0026] In a preferred embodiment, the amount of piperidine used in step S8 is 10-100 mL of piperidine per gram of intermediate A14.

[0027] In the preferred embodiment, the reaction conditions in step S8 are: reaction at 20℃~30℃ for 1~5 hours.

[0028] The present invention also provides the PD Application of L1 / EZH2 bifunctional inhibitors in the preparation of antitumor drugs targeting PD-L1 and EZH2.

[0029] In a preferred embodiment, the antitumor drug targets colon cancer and / or melanoma.

[0030] The present invention also provides a pharmaceutical composition comprising the PD. L1 / EZH2 bifunctional inhibitor.

[0031] In a preferred embodiment, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipients.

[0032] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) The compound PE-1 provided by the present invention is a PD-L1 / EZH2 bifunctional small molecule inhibitor compound with excellent in vivo anti-tumor pharmacological effects. It can effectively exert the in vivo anti-tumor therapeutic effect and has good drug development potential and clinical application transformation value. Compared with existing single-target drugs and traditional combination drug regimens, it has good in vivo drug safety and no obvious systemic toxic side effects.

[0033] (2) The method for preparing compound PE-1 in this invention has high atom utilization and generates less waste, which is conducive to environmental protection and industrial production requirements. Attached Figure Description

[0034] Figure 1 The 1H NMR spectrum of compound PE-1 prepared in Example 1 of this invention; Figure 2 The image shows the carbon NMR spectrum of compound PE-1 prepared in Example 1 of this invention. Figure 3 This is a comparison chart showing the therapeutic effects of compound PE-1 and the control compound against colon cancer in tumor-bearing mice in Example 2 of the present invention. Figure 4 This is a comparison chart showing the therapeutic effects of compound PE-1 and the control compound against melanoma cancer in tumor-bearing mice in Example 2 of the present invention. Detailed Implementation

[0035] 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.

[0036] The reaction process of this invention is monitored using conventional monitoring methods (such as TLC (thin-layer chromatography), LCMS (liquid chromatography-mass spectrometry) or NMR (nuclear magnetic resonance)). The reaction endpoint is generally defined as the disappearance of the reaction substrate.

[0037] In the following specific embodiments, the high-performance liquid chromatography (HPLC) conditions used for the detection and identification of the prepared compounds were as follows: Shimadzu LCMS2020, G1322A degasser, G1312 binary high-pressure pump, G1329A autosampler, G1316A column oven, and G4212B diode array 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% trifluoroacetic acid as mobile phase B, and gradient elution was performed. The specific gradient elution program is shown in Table 1.

[0038] Table 1. Gradient elution program for mobile phase

[0039] The flow rate was 1.5 mL / min, the column temperature was 40 °C, and the detection wavelength was 254 nm.

[0040] Unless otherwise stated, the following terms appearing in this specification and claims have the following meanings: The present invention also relates to available forms of the compounds disclosed herein, such as metabolites, hydrates, solvates, prodrugs, salts, especially pharmaceutically acceptable salts, and coprecipitates.

[0041] 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.

[0042] Pharmaceutically acceptable salts of the compounds of the present invention may be, for example, acid addition salts of the compounds of the present invention carrying nitrogen atoms in the chain or ring, or acid addition salts of sufficiently basic compounds of the present invention, or 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 the present 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.

[0043] The compounds of the present invention may contain non-natural proportions of isotopes on one or more atoms constituting the compound, for example, replacing hydrogen with deuterium to form deuterated drugs.

[0044] 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.

[0045] In this invention, Fmoc refers to 9-fluorenyloxycarbonyl, and Boc refers to tert-butyloxycarbonyl.

[0046] The Suzuki coupling reaction, Miyaura coupling reaction, Suzuki coupling reaction, and Birch reduction reaction described in this invention are common named organic reactions in the field.

[0047] 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.

[0048] A specific embodiment of the present invention provides a PD The preparation method of the L1 / EZH2 bifunctional inhibitor includes the following steps: S1. Compound A1 and compound A2 undergo a Suzuki coupling reaction to generate intermediate A3: In a mixed solvent consisting of an ether organic solvent and water, in the presence of a palladium catalyst and an acid-binding agent, and under the protection of an inert gas, compound A1 and compound A2 react at a molar ratio of 1:(1~2) at 50℃~80℃ for 1~10 hours to obtain intermediate A3. S2. Intermediate A3 and compound A4 undergo a Miyaura coupling reaction to generate intermediate A5: In an ether-based organic solvent, in the presence of a palladium catalyst and an acid-binding agent, and under inert gas protection, intermediate A3 and compound A4 react at a molar ratio of 1:(1~2) at 70℃~90℃ for 1~10 hours to obtain intermediate A5. S3. Intermediate A5 and compound A6 undergo a Suzuki coupling reaction to generate intermediate A7: In a mixed solvent consisting of an ether organic solvent and water, in the presence of a palladium catalyst and an acid-binding agent, and under the protection of an inert gas, intermediate A5 and compound A6 react at a molar ratio of 1:(1~2) at 80℃~100℃ for 1~12 hours to obtain intermediate A7. S4. Intermediate A7 and compound A8 undergo Birch reduction reaction to generate intermediate A9: In an organic solvent, in the presence of a reducing agent, intermediate A7 and compound A8 react at a molar ratio of 1:(1~2) at 20℃~30℃ for 1~5h to obtain intermediate A9. S5. Intermediate A9 reacts with compound A10 via an Fmoc protection reaction to generate intermediate A11: In an organic solvent, in the presence of an acid-binding agent, intermediate A9 and compound A10 react at a molar ratio of 1:(1~2) at 20℃~30℃ for 1~5h to obtain intermediate A11. S6. Intermediate A11 undergoes a Boc protecting group removal process under acidic conditions to generate intermediate A12: In an organic solvent, intermediate A11 reacts with acid at 20℃~30℃ for 1~5 hours. The reaction solution is then concentrated under reduced pressure to obtain intermediate A12. The Boc protecting group in intermediate A11 reacts with the H+ in the acid... + The molar ratio is 1:(1~50); S7. Intermediate A12 and compound A13 undergo a condensation reaction to generate intermediate A14: In an organic solvent, in the presence of a condensing agent and an acid-binding agent, intermediate A12 and compound A13 react at 20℃~30℃ for 1~5h to obtain intermediate A14; the molar ratio of intermediate A12 to compound A13 is 1:(1~2); S8. Intermediate A14 is deprotected by Fmoc protecting group under the action of piperidine to generate compound PE-1: Intermediate A14 is added to an organic solvent containing 24% (v / v) to 100% (v / v) piperidine and reacted at 20℃ to 30℃ for 1 to 5 h to obtain compound PE-1; wherein, the amount of piperidine used is 10 to 100 mL of piperidine per gram of intermediate A14.

[0049] The main reaction route of the above preparation method is as follows: .

[0050] 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.

[0051] As an example, the reaction temperature in step S1 is 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, but is not limited to this.

[0052] As an example, the reaction time in step S1 can be 1h, 2h, 2.5h, 3h, 5h, 6h, 8h, or 10h, but is not limited to this.

[0053] 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.

[0054] As an example, the reaction temperature in step S2 is 70°C, 75°C, 80°C, 85°C, or 90°C, but is not limited to this.

[0055] As an example, the reaction time in step S2 can be 1h, 2h, 2.5h, 3h, 4h, 4.5h, 5h, 6h, 8h, or 10h, but is not limited to this.

[0056] 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.

[0057] As an example, the reaction temperature in step S3 is 80°C, 85°C, 90°C, 95°C, or 100°C, but is not limited to this.

[0058] As an example, the reaction time in step S3 is 1h, 2h, 2.5h, 3h, 4h, 4.5h, 5h, 5.5h, 6h, 8h, 9h, 10h or 12h, and is not limited thereto.

[0059] 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.

[0060] As an example, the reaction temperature in step S4 is 20°C, 25°C, or 30°C, but is not limited to this.

[0061] 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.

[0062] As an example, the molar ratio of intermediate A9 to compound A10 in step S5 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.

[0063] As an example, the reaction temperature in step S5 is 20°C, 25°C, or 30°C, but is not limited to this.

[0064] As an example, the reaction time in step S5 is 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, or 5h, but is not limited to this.

[0065] As an example, in step S6, the Boc protecting group in intermediate A11 reacts with the H group in the acid. + The molar ratio is 1:1, 1:2, 1:3, 1:4, 1:5, 1:8, 1:10, 1:12, 1:15, 1:16, 1:18, 1:20, 1:25, 1:30, 1:40 or 1:50, and is not limited to these.

[0066] As an example, the reaction temperature in step S6 is 20°C, 25°C, or 30°C, but is not limited to this.

[0067] As an example, the reaction time in step S6 is 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, or 5h, but is not limited to this.

[0068] As an example, the molar ratio of intermediate A12 to compound A13 in step S7 is 1:1, 1:1.02, 1:1.05, 1:1.06, 1:08, 1:1.1, 1:1.11, 1:1.15, 1:1.2, 1:1.3, 1:1.5, 1:1.6, 1:1.8, or 1:2, and is not limited thereto.

[0069] As an example, the reaction temperature in step S7 is 20°C, 25°C, or 30°C, but is not limited to this.

[0070] As an example, the reaction time in step S7 is 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, or 5h, but is not limited to this.

[0071] As an example, the volume-to-mass ratio of piperidine to intermediate A14 in step S8 is 10 mL: 1 g, 15 mL: 1 g, 20 mL: 1 g, 22 mL: 1 g, 24 mL: 1 g, 25 mL: 1 g, 30 mL: 1 g, 35 mL: 1 g, 40 mL: 1 g, 50 mL: 1 g, 50 mL: 1 g, 60 mL: 1 g, 65 mL: 1 g, 70 mL: 1 g, 75 mL: 1 g, 80 mL: 1 g, 85 mL: 1 g, 90 mL: 1 g, or 100 mL: 1 g, but is not limited to this.

[0072] As an example, the reaction temperature in step S8 is 20°C, 25°C, or 30°C, but is not limited to this.

[0073] As an example, the reaction time in step S8 is 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, or 5h, but is not limited to this.

[0074] In some preferred embodiments, the ether organic solvent in step S1 is 1,4-dioxane, and the volume ratio of 1,4-dioxane to water is 5:1.

[0075] 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).

[0076] In some preferred embodiments, the acid-binding agent in step S1 is at least one of potassium carbonate, sodium carbonate, and potassium acetate.

[0077] 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 200:1.

[0078] In some preferred embodiments, the ether solvent in step S2 is 1,4-dioxane.

[0079] In some preferred embodiments, the palladium catalyst in step S2 is at least one of tetra(triphenylphosphine)palladium, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, and bis(tritert-butylphosphine)palladium(0).

[0080] In some preferred embodiments, the acid-binding agent in step S2 is at least one of potassium acetate, sodium carbonate, and potassium carbonate.

[0081] In some preferred embodiments, after the reaction in step S2 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 100:1.

[0082] In some preferred embodiments, the ether-based organic solvent in step S3 is 1,4-dioxane, and the volume ratio of 1,4-dioxane to water is 8:1.

[0083] 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).

[0084] In some preferred embodiments, the acid-binding agent in step S3 is at least one of potassium carbonate, potassium acetate, and sodium carbonate.

[0085] In some preferred embodiments, after the reaction in step S3 is completed, the reaction solution is extracted with an organic solvent that is poorly soluble in water, and the organic phase is subjected to silica gel column chromatography. The eluent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 50:1. As an example, the organic solvent that is poorly soluble in water is ethyl acetate.

[0086] In some preferred embodiments, the organic solvent in step S4 is a mixture of dichloromethane and methanol in a volume ratio of 10:1.

[0087] In some preferred embodiments, the reducing agent in step S4 is at least one of NaBH3CN, NaBH(OAc)3, and NaBH4.

[0088] 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 50:1.

[0089] In some preferred embodiments, the organic solvent in step S5 is dichloromethane.

[0090] In some preferred embodiments, the acid-binding agent in step S5 is at least one of N,N-diisopropylethylamine and triethylamine.

[0091] In some preferred embodiments, after the reaction in step S5 is completed, water is added to the reaction solution, the organic phase is separated and extracted with a water-insoluble organic solvent, 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 1:1 volume ratio. As an example, the water-insoluble organic solvent is dichloromethane.

[0092] In some preferred embodiments, the organic solvent in step S6 is at least one of dichloromethane, ethyl acetate, and 1,4-dioxane.

[0093] In some preferred embodiments, the acid in step S6 is at least one of trifluoroacetic acid and hydrochloric acid.

[0094] In a further preferred embodiment, when the organic solvent in step S6 is dichloromethane, the acid is trifluoroacetic acid; or in step S6, intermediate A11 is added to ethyl acetate containing 4M HCl and reacted at 20℃~30℃ for 1~5h; or in step S6, intermediate A11 is added to 1,4-dioxane containing 4M HCl and reacted at 20℃~30℃ for 1~5h.

[0095] In some preferred embodiments, the condensing agent in step S7 is at least one of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole.

[0096] In a further preferred embodiment, the condensing agent in step S7 is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole; the molar ratio of intermediate A12, compound A13, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole is 1:(1~2):(1~3):(1~3). As an example, the molar ratio of intermediate A12, compound A13, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole in step S7 is 1:1:1:1, 1:1.05:1.1:1.1, 1:1.1:1.2:1.2, 1:1.1:1.3:1.3, 1:1.1:1.5:1.5, 1:1.3:1.5:1.5, 1:1.5:1.8:1.8, 1:1.5:2:2, or 1:2:3:3, but is not limited to these.

[0097] In some preferred embodiments, the organic solvent in step S7 is N,N-dimethylformamide (DMF).

[0098] In some preferred embodiments, the acid-binding agent in step S7 is at least one of N,N-diisopropylethylamine and triethylamine.

[0099] In some preferred embodiments, after the reaction in step S7 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 dichloromethane and methanol in a volume ratio of 50:1. As an example, the organic solvent that is sparingly soluble in water is ethyl acetate.

[0100] In some preferred embodiments, in step S8, intermediate A14 is added to DMF containing 24% (v / v) piperidine; or intermediate A14 is added to piperidine (i.e., the organic solvent in step S8 is piperidine).

[0101] In some preferred embodiments, after the reaction in step S8 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, 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. As an example, the organic solvent that is sparingly soluble in water is ethyl acetate.

[0102] Example 1 This embodiment provides a PD The L1 / EZH2 bifunctional inhibitor has the following chemical name: N -(2-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethyl)-4,6-dimethyl-2-oxo-1,2-dihydropyridine-3-carboxamide (hereinafter referred to as compound PE-1), has the following chemical structural formula: .

[0103] The preparation method of compound PE-1 includes the following steps: S1. Add compound A1 (5g, 41.01mmol), compound A2 (10.25g, 41.01mmol), tetrakis(triphenylphosphine)palladium (473.87mg, 410.07µmol), potassium carbonate (8.50g, 61.51mmol), 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 50°C for 4 hours, monitored by TLC (developing solvent: a mixed solvent of PE and EA in a volume ratio of 200:1). 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 200:1) to obtain a colorless oily intermediate A3 (9.6 g, yield 94.73%). LCMS: (MS-ESI, m / z): [M+H]+=247.1.

[0104] S2, take the 3 obtained in step S1 bromine 2 methyl 1,1' Biphenyl (intermediate A3, 9.5 g, 38.85 mmol) was dissolved in 1,4-dioxane, and pinacol diboronic acid ester (compound A4, 14.80 g, 58.27 mmol), 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (284.23 mg, 388.45 µmol) and potassium acetate (5.72 g, 58.27 mmol) were added. The reaction was carried out at 80 °C for 6 hours under nitrogen atmosphere. The reaction was monitored by thin-layer chromatography (developing solvent: a mixed solvent of PE and EA in a volume ratio of 100:1). After the reaction was complete, the reaction solution was evaporated to dryness and column chromatography was performed using silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.). The solution was eluted with a petroleum ether:ethyl acetate system (PE and EA in a volume ratio of 100:1) to give a colorless oily intermediate A5 (11.2 g, yield 98.00%). LCMS: (MS-ESI, m / z): [M+H]+=295.2.

[0105] S3. Take intermediate A5 (11.2 g, 38.07 mmol) obtained in step S2, add a mixed solvent of 1,4-dioxane and water at a volume ratio of 8:1 (80 mL / 10 mL), and add 6... chlorine 2 methoxy Pyridine 3 Formaldehyde (compound A6, 6.53 g, 38.07 mmol), tetrakis(triphenylphosphine)palladium (439.92 mg, 380.69 µmol), and potassium carbonate (7.89 g, 57.10 mmol) were reacted at 90 °C for 12 hours under nitrogen atmosphere. The reaction was monitored by thin-layer chromatography (TLC) using a mixed solvent of PE and EA (50:1 v / v). After complete reaction, the reaction solution was 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. Separation was performed 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:EA v / v, 50:1 v / v) to give intermediate A7 (11 g, yield 95.25%). LCMS: (MS-ESI, m / z ): [M+H] + =304.2.

[0106] S4. Take intermediate A7 (11 g, 36.26 mmol) obtained in step S3, N-tert-butoxycarbonylethylenediamine (compound A8, 8.71 g, 54.39 mmol), and NaBH3CN (3.42 g, 54.39 mmol) and add them to a 200 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). React at 25 °C for 2 h. After the reaction is complete, the solvent is removed by vacuum distillation, and the mixture is separated by column chromatography with silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.). The mixture is eluted with a dichloromethane (DCM):methanol (MeOH) system (DCM:MeOH volume ratio of 50:1) to obtain intermediate A9 (16 g, yield 98.59%). LCMS: (MS-ESI, m / z): [M+H]+=448.3.

[0107] S5. At room temperature, add intermediate A9 (16 g, 35.75 mmol), 9-fluorenylmethylsuccinimino carbonate (Fmoc-OSu, compound A10, 12.06 g, 35.75 mmol), N,N-diisopropylethylamine (DIPEA, 9.24 g, 71.50 mmol) and dichloromethane (80 mL) to the reaction flask, and react at 25 °C for 1 hour. TLC monitoring (developing solvent: a mixed solvent of PE and EA in a 1:1 volume ratio), after the reactants reacted completely, 20 mL of water was added to quench the reaction. The mixture was separated, extracted three times with dichloromethane (40 mL × 3), and the organic phases were combined. The mixture was concentrated under reduced pressure, and silica gel (silica gel mass to concentrated residue mass ratio of 30:1) was added for further concentration. The sample was then passed through a column chromatography column with silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.), and eluted with a petroleum ether:ethyl acetate system (PE and EA in a 1:1 volume ratio) to obtain intermediate A11 (23.8 g, yield 99.40%). LCMS: (MS-ESI, m / z): [M+H]+ = 670.3.

[0108] S6. At room temperature, intermediate A11 (23.8 g, 35.53 mmol), trifluoroacetic acid (TFA, 40 mL), and dichloromethane (40 mL) were added to a reaction flask. The reaction was carried out at 25 °C for 2 hours, monitored by TLC (developing solvent: a mixed solvent of DCM and MeOH in a volume ratio of 10:1). After the starting material was completely reacted, the mixture was concentrated under reduced pressure to obtain intermediate A12 (20.1 g, yield 99.30%). LCMS: (MS-ESI, m / z): [M+H]+ = 570.3.

[0109] S7. At room temperature, add intermediate A12 (1 g, 1.76 mmol), compound A13 (322.76 mg, 1.93 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (300.77 mg, 2.63 mmol), N,N-diisopropylethylamine (453.73 mg, 3.51 mmol), 1-hydroxybenzotriazole (355.78 mg, 2.63 mmol), and N,N-dimethylformamide (DMF, 20 mL) to the reaction flask. React at 25°C for 1 hour, and monitor by TLC (developing solvent: [missing information - likely a specific solvent]). The reaction mixture (a 20:1 volume ratio of DCM to MeOH) was used as a solvent. After the reactants reacted completely, 20 mL of water was added to quench the reaction. The mixture was separated and extracted three times with ethyl acetate (30 mL × 3). The ethyl acetate phases were combined, concentrated under reduced pressure, and concentrated by adding silica gel (silica gel mass to concentrated residue mass ratio of 30:1). The solution was then purified by column chromatography using silica gel (200-300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.). Elution was performed with a dichloromethane:methanol system (DCM to MeOH volume ratio of 50:1) to give intermediate A14 (1.2 g, yield 95.10%). LCMS: (MS-ESI, m / z): [M+H]+ = 719.5.

[0110] S8. At room temperature, intermediate A14 (1.2 g, 1.67 mmol) and DMF solution containing 24% (v / v) piperidine (50 mL, containing 12 mL piperidine) were added to the reaction flask. The reaction was carried out at 25 °C for 2 hours and monitored by TLC (the developing solvent was a mixed solvent of DCM and MeOH in a volume ratio of 10:1). After the starting material was completely reacted, 50 mL of water was added to quench the reaction. The mixture was separated and extracted three times with ethyl acetate (40 mL × 3). The mixture was concentrated under reduced pressure, and the solvent was evaporated. The residue was subjected to column chromatography on silica gel (200~300 mesh, Huanghai Chemical Industry Research Institute (Tianjin) Co., Ltd.) and eluted with a mixture of dichloromethane and methanol (dichloromethane:methanol = 10:1, volume ratio) to give compound PE-1 (800 mg, yield 96.50%).

[0111] The main reaction route of the above preparation method is as follows: .

[0112] The proton and carbon NMR spectra of compound PE-1 are shown below. Figure 1 and Figure 2 The detection and characterization results are as follows: 1 H NMR (400 MHz, d 6-DMSO) δ 9.24 (s, 1H), 7.84 (d, J= 7.1 Hz, 1H), 7.46 (m, 2H), 7.36 (m, 5H), 7.24 (d, J = 7.2 Hz, 1H), 7.11 (d, J = 7.3 Hz, 1H), 6.01 (s, 1H), 3.90 (s, 3H), 3.77 (s, 2H), 3.18 (s, 2H), 2.74 (d, J = 5.9 Hz, 2H), 2.33 (s, 3H), 2.17 (s, 6H). 13 C NMR (101 MHz, d 6-DMSO) δ 166.30, 162.68, 160.62, 155.67, 154.27, 146.24, 142.92, 142.04, 141.22, 138.12, 133.20, 129.57, 129 .16, 128.63, 127.37, 125.93, 119.44, 117.39, 109.61, 53.53, 48.57, 46.73, 38.70, 21.67, 18.77, 18.62. MS m / z: calcd forC 30 H 32 N4O3496.24, found 497.35 [M+H] + HPLC t R 3.177 min, purity 98.51%.

[0113] Example 2 This example investigated the inhibitory effect of compound PE-1 in Example 1 on PD-1 / PD-L1 interaction, its inhibition rate on EZH2 enzyme activity, its pharmacokinetic effects in rats, and its therapeutic effects on melanoma and colon cancer in mice.

[0114] I. 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) method. HTRF is a simple, rapid, and high-throughput method for characterizing compounds and antibody blockers. PD-L1 carries Tag1, and PD-1 carries Tag2. Europium-labeled (HTRF donor) anti-Tag1 antibody binds to Tag1-labeled PD-L1, while XL665-labeled (HTRF receptor) anti-Tag2 antibody binds 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.

[0115] 1.2 Reagent Information Table 2. Source information of reagents required for testing

[0116] 1.3. Dilution of the compound and preparation of the test solution: (1) Dilution of the mother liquor of the compounds: In order to accurately measure the effect of each compound on PD 1 / PD To inhibit L1 binding, each compound needs to be diluted to a 20 mmol / L stock solution. The required amount of DMSO for each compound can be calculated using the formula C = n / V = m / M / V (where 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; and M is the molar mass of the compound in g / mol). After preparing the stock solutions, use Diluent reagent (included in the kit in Table 2) to sequentially dilute the stock solutions to 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, and 0.004 μmol / L, and mix well before use.

[0117] (2) PD Dilution of L1 protein mixture: Take 7 mL of PD-L1 protein from the kit, follow the instructions in the kit manual, add 510 mL of diluent to dilute it, mix well and set aside.

[0118] (3) PD 1. Dilution of the protein mixture: Take 5 mL of PD-1 protein from the kit, follow the instructions in the kit manual, add 550 mL of diluent to dilute it, and mix well for later use.

[0119] (4) Preparation of mixed test solution: Take Anti-Tag-Eu3 from the kit respectively + Mix 5 μL of Anti-Tag-XL665 and 20 μL of Anti-Tag-XL665 according to the kit instructions, then add 975 μL of Detection Buffer (the kit is included in Table 2) to dilute it, mix well and set aside.

[0120] 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.

[0121] (2) Add 4 μL of PD-L1 protein mixture dilution to each well and centrifuge at 1000 rpm for 1 min.

[0122] (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.

[0123] (4) Add 10 μL of mixed test solution to each well and centrifuge at 1000 rpm for 1 min.

[0124] (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).

[0125] (6) Calculate the inhibition rate according to the following formula: Inhibition rate % = [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, with only an equal amount of DMSO added to the reaction system; the low control group is the group without PD-1 protein mixture dilution, with only an equal amount of detection buffer added.

[0126] (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.

[0127] 1.5 Experimental Results Table 3. Test results of the compounds' inhibitory effect on PD-1 / PD-L1 activity.

[0128] In Table 3, 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 3 show that the compound PE-1 prepared in this invention can inhibit the binding of PD-1 / PD-L1.

[0129] II. Detection of EZH2 enzyme inhibitory activity The inhibition rate of the test compound on the EZH2 enzymatic reaction was determined using the commercial MTase-Glo™ methyltransferase assay kit (purchased from Promega, catalog number: V7601). All operations were performed strictly in accordance with the kit instructions.

[0130] 2.1 Experimental Preparation Using a white, opaque 384-well microplate, the following reagents were added sequentially: 1× detection buffer, recombinant human EZH2 holoenzyme complex (PRC2 complex), and methyl donor S. S-adenosylmethionine (SAM) and histone H3-derived substrate peptides were gently mixed and then briefly centrifuged to allow the reagents to settle to the bottom of the well.

[0131] 2.2 Sample addition and enzymatic reaction A gradient concentration (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM, DMSO final concentration ≤1% (v / v)) of the test compound was added to each well. Blank control wells (without test compound), negative control wells (without EZH2), and positive control wells were also provided. After sealing, the plates were incubated at 30°C in the dark for 60 minutes to initiate the EZH2-mediated methyl transfer reaction, catalyzing the H3K27 trimethylation of the substrate peptide and generating the byproduct S. Adenosine homocysteine ​​(SAH).

[0132] 2.3 Signal Conversion and Detection After incubation, add MTase-Glo to each well. TM The reagent converts the SAH generated in the reaction into ADP. After the reaction is carried out at room temperature in the dark, MTase-Glo™ detection solution is added to further convert ADP into ATP. ATP participates in the luciferase-catalyzed reaction and generates a chemiluminescent signal.

[0133] 2.4 Signal Reading and Calculation The luminescence values ​​of each well were detected using a multifunctional chemiluminescent microplate reader. The luminescence intensity was positively correlated with the amount of SAH generated, i.e., positively correlated with the EZH2 enzyme activity. The enzyme inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 - luminescence value of experimental group / luminescence value of negative control group) × 100%. The IC50 of the analyte against EZH2 was calculated by fitting a dose-response curve using GraphPad Prism. 50 value.

[0134] 2.5 Experimental Results Table 4. Results of the test compounds' inhibition rate on EZH2 enzyme activity.

[0135] As shown in Table 4, the compound PE-1 prepared in this invention can inhibit the enzyme activity of EZH2, and its effect is superior to that of compound 15 prepared in Example 15 of patent application CN115417870A and compound HP6 prepared in Example 6 of patent application CN115010658A. In the field of medicinal chemistry, there is a strict structure-activity relationship between compound structure and pharmacological action. Small changes in compound structure can lead to significant differences in pharmacological activity. Although the compound PE-1 of this invention has a similar structure to the compounds in patent applications CN115417870A and CN115010658A, its effect of inhibiting EZH2 enzyme activity is significantly more prominent than the latter two. Tazesta, a small molecule EZH2 inhibitor, was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., and used as a positive control to verify the stability and reliability of the test system.

[0136] III. Rat Pharmacokinetic Test 3.1 Test Method Three male SD rats (6-8 weeks old, weighing 180-220g, n=3 per group) were administered compound PE-1 orally via gavage, intravenously, or intraperitoneally to test its oral bioavailability. Approximately 0.1 mL of blood was collected from the orbital sinus before administration and at 0 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration, and anticoagulated with heparin. The blood was collected in test tubes, centrifuged for 10 min, and serum was separated. 150 μL of a methanol solution containing an internal standard (internal standard compound concentration: 100 ng / mL) was added, and the solution was centrifuged at 16000g for 10 min. 100 μL of the supernatant was analyzed by UPLC-MS (ultra-high performance liquid chromatography-mass spectrometry) to determine the compound concentration. Pharmacokinetic data were processed using the WINNONLIN non-compartmental model method to obtain the rat pharmacokinetic parameters. The analytical conditions for UPLC-MS were as follows: Chromatographic system: Waters ACQUITY UPLC I-Class / Xevo TQD; Column: CORTECS UPLC C18 Column (2.1 mm × 100 mm, 1.6 μm); Column temperature: 40 °C; Mobile phase: A mixture of 0.1% formic acid (mobile phase A) and methanol (mobile phase B). The elution gradient was set as follows: Initially, mobile phase B was 30% (v / v) and mobile phase A was 70% (v / v); within 1–2 minutes, mobile phase B linearly increased to 90% (v / v) and maintained at this level for 1.5 minutes; subsequently, within 3.5–3.6 minutes, mobile phase B linearly decreased to 5% (v / v). The separation of compound PE-1 and the internal standard (Midazolam) was performed using a C18 column with an injection volume of 5 μL and a run time of 3 min. Mass spectrometry conditions: positive ion mode electrospray ionization (ESI) source, multiple reaction monitoring (MRM) generated ion fragments, capillary voltage 1 kV, desolvation gas temperature 500 °C, source temperature 150 °C, sheath gas flow rate 50 L / H, and desolvation gas flow rate 1000 L / H.

[0137] 3.2 Experimental Results Table 5. Pharmacokinetic data of compound PE-1 in rats.

[0138] According to the results in Table 5, the compound PE-1 provided by this invention showed a suitable half-life, maximum plasma concentration, and excellent area under the plasma concentration-time curve in the rat pharmacokinetic test model. The calculated oral bioavailability of compound PE-1 is 58.4%, which shows that it has the potential for oral administration.

[0139] IV. Testing the Anti-tumor Therapeutic Effects of Compounds 4.1 Experimental Materials The tumor cell lines used in this experiment were mouse melanoma cell line B16F10 (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) and mouse colon cancer cell line CT26 (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.). B16F10 cells were adapted for in vivo modeling in C57BL / 6 mice, and CT26 cells were adapted for in vivo modeling in BALB / c mice. Both cell lines are currently recognized as classic tumor model cells for evaluating antitumor immunopharmacological efficacy, exhibiting high model stability and reproducibility, and enabling objective evaluation of the in vivo immunopharmacological and antitumor effects of the test compounds.

[0140] The experimental animals were healthy male C57BL / 6 mice and BALB / c mice aged 6-7 weeks. 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, the 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.

[0141] 4.2 Experimental Methods Construction of a mouse subcutaneous allogeneic tumor model: B16F10 melanoma cells and CT26 colon cancer 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, with strict control over cell viability and suspension concentration. Under aseptic conditions, cells were subcutaneously seeded in the axilla of one forelimb of each mouse, with a cell count of 2 × 10⁶ cells per mouse. 5 Establish a mouse subcutaneous solid tumor transplantation model using one mouse per animal.

[0142] 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 3 When the modeling of the standard interval is successful, the tumor-bearing mice are randomly divided into groups to ensure that there is no statistically significant difference in the average tumor volume and body weight among the groups, thus excluding the influence of baseline differences between groups on the efficacy evaluation.

[0143] 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.

[0144] 4.3 Experimental Results The therapeutic effects of compound PE-1 provided by this invention on tumor-bearing mice in an in vivo anti-tumor model are as follows: Figure 3 , Figure 4 As shown. Among them, the EZH2 inhibitor positive control was performed using Tazemetostat (abbreviated as Taz); the mouse PD-L1 antibody was abbreviated as PD-L1 or anti-mouse PD-L1. Figure 3 and Figure 4 In this context, ns indicates that there is no statistically significant difference between groups (P≥0.05). This indicates that P < 0.01. This indicates that P < 0.001. This means P < 0.0001.

[0145] like Figure 3 As shown in Figure A, compound PE-1, administered orally at a dose of 50 mg / kg once daily (days 1-17, single daily dose), significantly reduced terminal tumor weight in mice compared to the control group (no drug intervention), achieving a tumor growth inhibition rate (TGI) of 69.9%. This in vivo antitumor activity was significantly superior to the selective EZH2 inhibitor tazestat monotherapy group (50 mg / kg, single daily dose, tumor growth inhibition rate of only 24.6%), and also significantly superior to the combination of tazestat (50 mg / kg) and the PD-L1 small molecule inhibitor BMS-202 (50 mg / kg, single daily dose, tumor growth inhibition rate of 54.4%). The results of continuous monitoring of tumor volume dynamics during the experimental period are shown below. Figure 3As shown in Figure B, the results are consistent with the aforementioned endpoint of tumor weight measurement. Throughout the 17-day overall experiment, the PE-1 group showed sustained inhibition of tumor progression. Results on day 17 showed that the PE-1 group achieved a tumor growth inhibition rate of 62.1%, significantly superior to the tazestat monotherapy group (tumor growth inhibition rate 22.6%) and the tazestat combined with BMS-202 group (tumor growth inhibition rate 46.1%). Furthermore, the tumor growth curve in the PE-1 group flattened after day 7 of administration, indicating that the compound achieves sustained and stable target binding in vivo. Safety monitoring results showed that no mice in any of the experimental groups experienced drug-related weight loss exceeding 10% (see Figure B). Figure 3 (See Figure C). The drug was well tolerated. Mouse survival analysis further confirmed that compound PE-1 possesses excellent anti-tumor therapeutic effects (see Figure C). Figure 3 (Figure D) Throughout the 17-day observation period, all mice in the PE-1 administration group survived. These results confirm that compound PE-1 exhibits good tolerability at effective antitumor doses, with a wide therapeutic window and high safety profile.

[0146] To further verify the in vivo application potential of compound PE-1 as an immunotherapy candidate drug, and the synergistic effect of this compound with anti-PD-L1 immunotherapy drugs, this invention systematically evaluated the in vivo antitumor efficacy of compound PE-1 in a B16-F10 mouse melanoma model. Tumor-bearing C57BL / 6 mice with stable tumor growth were selected and randomly divided into four groups (n=5 per group) and treated with the drug for 14 consecutive days. The specific groupings were as follows: (1) control group; (2) anti-PD-L1 monoclonal antibody monotherapy group (10 mg / kg, intraperitoneal injection, once every 3 days); (3) compound PE-1 monotherapy group (50 mg / kg, oral administration, once daily); (4) compound PE-1 combined with anti-PD-L1 antibody therapy group.

[0147] like Figure 4 As shown in Figures A and B, PE-1 monotherapy significantly inhibited tumor growth, achieving a tumor growth inhibition rate of 48.7%, which was superior to the anti-PD-L1 antibody monotherapy group (tumor growth inhibition rate of 39.17%). Crucially, the combination therapy of PE-1 and the anti-PD-L1 antibody exhibited the strongest in vivo anti-tumor effect, with a tumor growth inhibition rate as high as 59.02%, clearly demonstrating that the combination of the two drugs has a significant synergistic anti-tumor effect. Figure 4The dynamic monitoring data of tumor volume in the C-plot further verified the above experimental conclusions: compared with the control group, the tumor volume reduction rate in the PE-1 monotherapy group reached 59.8%, and the tumor volume reduction rate in the anti-PD-L1 antibody monotherapy group was 44.9%. Throughout the entire treatment period, no significant weight loss was observed in any of the treatment groups (see Figure 1). Figure 4 Figure D in the figure confirms that compound PE-1, whether administered alone or in combination with anti-PD-L1 antibody, exhibits good in vivo safety with no significant systemic toxic side effects.

[0148] In summary, the compound PE-1 provided by this invention is a PD-L1 / EZH2 bifunctional small molecule inhibitor compound with excellent in vivo antitumor pharmacological effects. It can effectively exert in vivo antitumor therapeutic effects, has good drug development potential and clinical application transformation value, and has good in vivo drug safety compared with existing single-target drugs and traditional combination drug regimens, with no obvious systemic toxic side effects.

[0149] 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 PD-L1 / EZH2 bifunctional inhibitor, characterized in that, The PD-L1 / EZH2 bifunctional 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）。 2. The method for preparing the PD-L1 / EZH2 bifunctional 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 a Miyaura coupling reaction to generate intermediate A5; S3, intermediate A5 and compound A6 undergo a Suzuki coupling reaction to generate intermediate A7; S4. Intermediate A7 reacts with compound A8 in a Birch reduction reaction to generate intermediate A9. S5, intermediate A9 reacts with compound A10 via an Fmoc protection reaction to generate intermediate A11; S6. Intermediate A11 loses its Boc protecting group under the action of acid to generate intermediate A12; S7. Intermediate A12 undergoes a condensation reaction with compound A13 to generate intermediate A14; S8 and intermediate A14 lose the Fmoc protecting group under the action of piperidine to generate compound PE-1, which is the PD-L1 / EZH2 bifunctional inhibitor. The synthetic route for the PD-L1 / EZH2 bifunctional 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, In step S5, the molar ratio of intermediate A9 to compound A10 is 1:(1~2); Or / and, in step S6, the Boc protecting group in intermediate A11 reacts with the H in the acid. + The molar ratio is 1:(1~50); Or / and, in step S7, the molar ratio of intermediate A12 to compound A13 is 1:(1~2); Or / and, in step S8, the amount of piperidine used is 10-100 mL of piperidine per gram of intermediate A14.

5. The preparation method according to claim 2, characterized in that, In step S4, the reducing agent is at least one of NaBH3CN, NaBH(OAc)3, and NaBH4; Or / and, the acid mentioned in step S6 is at least one of trifluoroacetic acid and hydrochloric acid; Or / and, in step S7, intermediate A12 and compound A13 undergo a condensation reaction in the presence of a condensing agent, said condensing agent being at least one of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-hydroxybenzotriazole.

6. The preparation method according to claim 2, characterized in that, The reaction conditions in step S1 are: under inert gas protection, in the presence of palladium catalyst, at 50℃~80℃ for 1~10 hours; Or / and, the reaction conditions in step S2 are: under inert gas protection, in the presence of palladium catalyst, at 70℃~90℃ for 1~10 hours; Or / and, the reaction conditions in step S3 are: under inert gas protection, in the presence of a palladium catalyst, at 80℃~100℃ for 1~12 hours.

7. The preparation method according to claim 2, characterized in that, The reaction conditions in step S4 are: reaction at 20℃~30℃ for 1~5 hours; Or / and, the reaction conditions in step S5 are: in the presence of an acid-binding agent, the reaction is carried out at 20°C to 30°C for 1 to 5 hours; Or / and, the reaction conditions in step S6 are: react at 20℃~30℃ for 1~5 hours.

8. The preparation method according to claim 2, characterized in that, The reaction conditions in step S7 are: in the presence of an acid-binding agent, the reaction is carried out at 20℃~30℃ for 1~5 hours; Or / and, the reaction conditions in step S8 are: react at 20℃~30℃ for 1~5 hours.

9. The PD according to claim 1 Application of L1 / EZH2 bifunctional inhibitors in the preparation of antitumor drugs targeting PD-L1 and EZH2.

10. A pharmaceutical composition, characterized in that, Including the PD as described in claim 1 L1 / EZH2 bifunctional inhibitor.

Images