A tricyclic compound, and a preparation method and application thereof

Abstract

The application belongs to the technical field of compounds, and particularly relates to a tricyclic compound and a preparation method and application thereof. The tricyclic compound provided by the application has a molecular structure shown in formula (I), can significantly degrade PD-L1 protein, has good oral bioavailability (F>60%), and the synthesis route of the tricyclic compound avoids dangerous oxidation-reduction reagents such as lithium aluminum hydride and DMP, has high product yield, is not prone to produce by-products, has high atomic utilization rate, produces less waste, and is beneficial to environmental protection and industrial production requirements.

Description

Technical Field

[0001] This invention relates to the field of compound technology, and more specifically, to a tricyclic compound, its preparation method, and its application. Background Technology

[0002] Immune checkpoint blockade (ICB) is an innovative therapy that uses the immune system to recognize and eliminate tumor cells. First-generation immune checkpoint inhibitors, represented by programmed cell death receptor 1 / programmed cell death ligand 1 (PD-1 / PD-L1), have gradually become the cornerstone of immuno-oncology (IO) therapy. PD-1 / PD-L1 antibodies have seen the most rapid progress in the field of immuno-oncology, with Opdivo (nivolumab) and Keytruda (pembrolizumab) demonstrating good efficacy in the treatment of melanoma and non-small cell lung cancer. However, clinical studies have found that the overall response rate (ORR) of PD-L1 antibodies is low (<30%). Meanwhile, the disadvantages of large molecule antibody drugs are also very obvious. First, they have poor penetration into relevant tissues and tumor cells, long metabolic half-lives, and can only be administered by injection. Second, antibody drugs are immunogenic, which can lead to serious adverse reactions, such as immune cytokine storm syndrome and fatal myocarditis. Moreover, the manufacturing and purification process of antibody drugs is very complex, resulting in very high production costs (O drug, annual out-of-pocket treatment cost for patients is 140,000 RMB; Keytruda, annual out-of-pocket treatment cost for patients is 140,000 RMB). Compared with large molecule antibody drugs, small molecule compounds have many advantages in pharmacodynamics. For example, small molecule compounds have better oral bioavailability, high penetration into relevant tissues and tumor cells, low manufacturing costs, and reasonable half-lives. Therefore, the development of small molecule PD-L1 modulators is expected to enable oral administration, reduce the economic burden of medication for patients, and at the same time compensate for the shortcomings of antibodies, which has very good clinical application prospects and practical significance. Most reported small molecule compounds exert their anticancer effects by blocking the PD-1 / PD-L1 interaction. However, the oral bioavailability of reported PD-L1 small molecule inhibitors is low, and further improvements in bioavailability are needed (Reference: J Med Chem . 2021, 64(11):7390-7403.doi: 10.1021 / acs.jmedchem.1c00010.), thereby achieving oral administration.

[0003] PD L1 protein is an important immunosuppressive molecule that contributes to tumor immune escape and tumor progression. Its overexpression can transmit negative regulatory signals, leading to T cell exhaustion. Therefore, the design and synthesis of PD-L1 degraders are aimed at reducing or downregulating PD. Theoretically, increasing PD-L1 protein levels could further enhance anti-cancer efficacy. This is because PD-L1 degraders can promote PD-L1 protein degradation, meaning they can more thoroughly relieve the inhibitory effect of PD-L1 on T cells, thereby enhancing the immune system's ability to attack tumors and more completely activating the immune system. In contrast, PD-L1 inhibitors primarily alleviate inhibition by preventing the binding of PD-L1 to PD-1, rather than necessarily eliminating the PD-L1 molecule itself. Furthermore, research has found that PD-L1 overexpression is associated with immune escape; reducing PD-L1 protein levels can counteract this upregulation mechanism, helping to prevent tumor cells from escaping immune surveillance. For some tumors insensitive to traditional treatments, PD-L1 degraders can provide a new treatment strategy, improving treatment efficacy by enhancing the immune system's attack capabilities. Additionally, PD-L1 degraders have a more durable therapeutic effect because they can degrade PD-L1 molecules, reducing their expression on the surface of tumor cells. This helps prevent tumor cells from escaping immune attack by re-expressing PD-L1, thus making the treatment effect more lasting. Currently, foreign scientists have reported on lysosomal targeted chimeras (LYTACs) technology, which can effectively degrade PD-L1 protein (literature: Nature 2020, 584(7820):291-297. doi: 10.1038 / s41586-020-2545-9.), but LYTACs have too large a molecular weight to be administered orally. Further scientific research has found that biphenyl-based PD-L1 small molecule inhibitors can dimerize PD-L1 protein, followed by endocytosis, and then lysosomal degradation of PD-L1 protein (Reference: J Med Chem 2022, 65(5):3879-3893. doi: 10.1021 / acs.jmedchem.1c01682.), Small changes in the structure of biphenyl-based PD-L1 small molecule inhibitors can lead to huge differences in degradation efficiency. This information suggests that by optimizing the structure of biphenyl-based PD-L1 small molecule inhibitors, it is possible to obtain orally available inhibitors that can effectively degrade PD. L1, a small molecule degrader, meets unmet clinical needs and benefits a large number of cancer patients.

[0004] Regarding the chemical synthesis of biphenyl-based PD-L1 small molecule inhibitors, patent (CN115417870A) reports the following synthetic route:

[0005] When the applicant replicated this synthetic route, the intermediate J5 was synthesized according to the method reported in the patent (as shown below, see reference). J Med Chem 2021 64(11) ,7390 The synthesis of compound 23a in 7403 has a low yield. In addition, this route uses dangerous redox reagents such as lithium aluminum hydride and DMP, and the post-processing is complicated, generating a lot of waste liquid, which is not conducive to environmental protection and industrial production requirements.

[0006]

[0007] Referring to the patented synthesis method, the applicant discovered that the second step, J3 to J4, using TFA to remove the protecting group, easily generates the byproduct BP1. Meanwhile, under acidic conditions, the terminal amino group of intermediate J4 readily forms a salt, reducing the nucleophilicity of the amino group. This makes the reductive amination reaction from J5 to J6 in step 3 difficult, resulting in a low yield of the target product J6 and the easy formation of the byproduct BP2. Therefore, in addition to discovering entirely new PD-L1 degrading agents, it is also necessary to design a novel tricyclic PD-L1 small molecule inhibitor to compensate for the shortcomings of existing technologies. Summary of the Invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art, and proposes a tricyclic compound, its preparation method and application.

[0009] The tricyclic compound provided by this invention has the molecular structure shown in formula (Ⅰ), which can effectively reduce PD-L1 protein and has high oral bioavailability, showing great promise for clinical application.

[0010] A first aspect of the present invention provides a tricyclic compound.

[0011] A tricyclic compound, characterized by having the molecular structure shown in formula (Ⅰ):

[0012] Equation (I); Where A is selected from , , , , , .

[0013] A second aspect of the present invention provides a method for preparing a tricyclic compound.

[0014] A method for preparing a tricyclic compound includes the following steps:

[0015] Where A is selected from , , , , , ; (1) First, T1 and T2 undergo a Suzuki coupling reaction to generate intermediate T3; (2) Intermediates T3 and T4 undergo a Miyaura coupling reaction to generate intermediate T5; (3) Intermediates T5 and T6 undergo a Suzuki coupling reaction to generate intermediate T7; (4) Intermediate T7 and T8 undergo a Borge reduction reaction to generate intermediate T9; (5) Intermediate T9 further undergoes an Fmoc protection reaction with T10 to generate intermediate T11; (6) Intermediate T11 loses its Boc protecting group under the action of 4M HCl to generate intermediate T12; (7) Intermediates T12 and T13 undergo a condensation reaction to generate intermediate T14; (8) Intermediate T14 was deprotected by Fmoc protecting group under the action of 20% piperidine to generate target product 1-6.

[0016] Preferably, in step (1), the temperature of the Suzuki coupling reaction is 50-80 °C and the time is 1-10 hours.

[0017] Preferably, in step (2), the temperature of the Miyaura coupling reaction is 70-90 °C and the time is 1-10 hours.

[0018] Preferably, in step (3), the temperature of the Suzuki coupling reaction is 80-100 °C and the time is 1-10 hours.

[0019] Preferably, in step (4), the Boch reduction reaction uses a reducing agent, which includes at least one of sodium cyanoborohydride, sodium borohydride acetate, and sodium borohydride.

[0020] Preferably, in step (5), the Fmoc protecting group is added, and the reaction conditions require the addition of an organic base, which includes at least one of triethylamine and DIPEA.

[0021] Preferably, in step (6), the Boc protecting group is removed, and the reaction needs to be carried out under acidic conditions, wherein the acidic conditions protect at least one of 4M HCl / ethyl acetate, TFA, and 4M HCl / dioxane.

[0022] Preferably, in step (7), the condensation reaction is carried out using a condensation agent, which includes at least one of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N''-carbonyldiimidazole.

[0023] Preferably, in step (7), the temperature of the condensation reaction is 20-30 °C and the time is 0.5-3 hours.

[0024] Preferably, in step (8), the Fmoc protecting group is removed, the reaction solvent is DMF, and the reaction time is 0.5-3 hours.

[0025] A third aspect of the present invention provides an application of a tricyclic compound.

[0026] Application of a tricyclic compound in the preparation of cancer treatment drugs.

[0027] The use of a tricyclic compound in the preparation of drugs for treating diseases related to PD-1 / PD-L1.

[0028] Preferably, the drug further includes pharmaceutically acceptable salts, carriers, and excipients.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a tricyclic compound with the molecular structure shown in formula (I), which can significantly degrade PD-L1 protein and has high oral bioavailability. It is an orally administered PD-L1 degrading agent with significant effects. Furthermore, the synthetic route of the above tricyclic compound avoids dangerous redox reagents such as lithium aluminum hydride and DMP, and also avoids steps in existing technologies that result in low yields of the target product and easy generation of byproducts. It has high atom utilization and generates less waste, which is beneficial to environmental protection and industrial production requirements. Attached Figure Description

[0030] Figure 1 The proton NMR spectrum of compound 1 was prepared for Example 1.

[0031] Figure 2 The proton NMR spectrum of compound 2 prepared in Example 2.

[0032] Figure 3 The proton NMR spectrum of compound 3 prepared in Example 3 is shown.

[0033] Figure 4 The proton NMR spectrum of compound 4 was prepared for Example 4.

[0034] Figure 5 The proton NMR spectrum of compound 5 was prepared for Example 5.

[0035] Figure 6 The proton NMR spectrum of compound 6 prepared in Example 6 is shown. Detailed Implementation

[0036] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0037] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0038] 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.

[0039] In the following specific embodiments, the liquid chromatography conditions used for the detection and identification of the prepared compounds were: Shimadzu LCMS 2020, 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, using gradient elution. The specific gradient elution program is as follows:

[0040] The flow rate was 1.5 mL / min, the column temperature was 40℃, and the detection wavelength was 254 nm.

[0041] the term 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.

[0042] Furthermore, the compounds of the present invention can 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.

[0043] 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, for example, acid addition salts of sufficiently basic compounds of the present invention, for example, acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, disulfuric acid, phosphoric acid, or nitric acid, or acid addition salts formed with organic acids, such as 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, etc. Acids, including digluconic acid, 3-hydroxy-2-naphthic acid, nicotinic acid, dihydroxynaphthic acid, pectinic acid, persulfate, 3-phenylpropionic acid, picric acid, 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.

[0044] 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.

[0045] As used herein, the term "stereoisomer" refers to compounds that have the same chemical composition but differ in the spatial arrangement of atoms or groups. Stereoisomers include enantiomers, diastereomers, and conformational isomers.

[0046] As used in this article, the term "enantiomer" refers to two stereoisomers of a compound that are non-overlapping mirror images of each other.

[0047] As used herein, the term "diastereomer" refers to a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers possess different physical properties, such as melting point, boiling point, spectral properties, or biological activity. Mixtures of diastereomers can be separated using chiral HPLC.

[0048] The degradation effect of the compound on PD-L1 was detected by Western blot (WB) assay. The results showed that the compound could efficiently degrade PD-L1 protein.

[0049] Those skilled in the art will understand that some 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 exist as a single stereoisomer (e.g., enantiomer, diastereomer) and mixtures thereof in any proportion, such as racemates, and, where appropriate, as tautomers and geometric isomers.

[0050] Example 1 A tricyclic compound, named 6-(2,4-diketone-1,2,3,4-tetrahydropyrimidin-5-yl)-nitrogen-(2-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethyl)pyridazine-3-carboxamide (referred to as Compound 1), has the following molecular structure:

[0051] The preparation method of compound 1 includes the following steps: (1) Compound (T1) (5 g, 41.01 mmol), T2 (10.25 g, 41.01 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloromethane complex (334.47 mg, 410.07 µmol), TMSOK (7.89 g, 61.51 mmol), and 1,4-dioxane / water = 5 / 1 (50 mL / 10 mL) were added to the reaction flask. The mixture was purged with nitrogen three times. Under nitrogen protection, the mixture was stirred and heated to 60 °C for 2 hours, monitored by TLC. The mixture was then cooled to 25 °C, and the reaction solution was evaporated to dryness. The solution was purified by column chromatography using silica gel and eluted with a petroleum ether:ethyl acetate system (PE:EA = 200:1) to obtain a colorless oily intermediate T3 (9.5 g, yield 93.74%). LCMS: (MS-ESI, m / z ): [M+H] + =247.1.

[0052] (2) Take the 3 obtained in step 1). bromine 2 methyl 1,1' Biphenyl (T3, 9.5 g, 38.44 mmol) was dissolved in 1,4-dioxane, and pinacol diboronate (14.64 g, 57.66 mmol), tetraphenylphosphine palladium (444.99 mg, 384.41 µmol), and potassium acetate (5.66 g, 57.66 mmol) were added. The reaction was carried out at 85 °C for 10 hours under nitrogen atmosphere. After the reaction was monitored by thin-layer chromatography, the reaction solution was evaporated to dryness, and column chromatography was performed using silica gel. The solution was eluted with a petroleum ether:ethyl acetate system (PE:EA = 100:1) to give a colorless oily intermediate T5 (11 g, yield 97.27%). LCMS: (MS-ESI, m / z ): [M+H] + =295.2.

[0053] (3) Take the intermediate T5 (11 g, 37.39 mmol) obtained in step 2), add 2-methyltetrahydrofuran / water (V / V = 10 / 1, 50 mL / 5 mL) as a solvent, and add 6 chlorine 2 methoxy Pyridine 3 Formaldehyde (6.42 g, 37.39 mmol), tetraphenylphosphine palladium (432.82 mg, 373.89 µmol), and cesium carbonate (18.27 g, 56.08 mmol) were reacted at 100 °C for 10 h under nitrogen atmosphere. After the reaction was complete as monitored by thin-layer chromatography, 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, dissolved, and separated by column chromatography. Elution was performed with a petroleum ether:ethyl acetate system (PE:EA = 50:1) to give intermediate T7 (10.8 g, 95.22%). LCMS: (MS-ESI, m / z ): [M+H] + =304.2.

[0054] (4) Take intermediate T7 (10.8 g, 35.60 mmol) obtained in step 3), Boc ethylenediamine (8.56 g, 53.40 mmol), and NaBH3CN (3.36 g, 53.40 mmol) and add them to a 100 mL round-bottom flask. The reaction solvent is dichloromethane / methanol (10 / 1, 40 mL / 4 mL). React at 0 °C for 2 h. After the reaction of the starting materials is complete as detected by TLC, the solvent is removed by vacuum distillation, and the mixture is separated by column chromatography. The intermediate T9 (15 g, 94.14%) is obtained by elution with a dichloromethane:methanol system (DCM:MeOH=50:1). LCMS: (MS-ESI, m / z): [M+H] + = 448.3.

[0055] (5) At room temperature, intermediate T9 (15 g, 33.51 mmol), Fmoc-OSu (11.31 g, 33.51 mmol), DIPEA (6.5 g, 50.27 mmol), and dichloromethane (50 mL) were added to the reaction flask. The reaction was carried out at 25°C for 1 hour under TLC monitoring. After the starting material was completely reacted, 20 mL of water was added to quench the reaction. The mixture was separated, extracted three times with dichloromethane (30 mL × 3), concentrated under reduced pressure, and concentrated again with silica gel. The mixture was then passed through a silica gel column for column chromatography and eluted with a petroleum ether:ethyl acetate system (PE:EA = 1:1) to obtain intermediate T11 (22 g, yield 98%). LCMS: (MS-ESI, m / z ): [M+H] + = 670.3.

[0056] (6) At room temperature, T11 (22 g, 32.84 mmol), 4M HCl, ethyl acetate (5 mL), and dichloromethane (40 mL) were added to the reaction flask. The reaction was carried out for 1 hour under TLC monitoring. After the starting material was completely reacted, the mixture was concentrated under reduced pressure to obtain intermediate T12 (18.5 g, yield 98.87%). LCMS: (MS-ESI, m / z ): [M+H] + = 570.3.

[0057] (7) At room temperature, add intermediates T12 (500.00 mg, 877.65 µmol), T13 (205.52 mg, 877.65 µmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (400.45 mg, 1.05 mmol), N,N-diisopropylethylamine (226.87 mg, 1.76 mmol), and N,N-dimethylformamide (5 mL) to the reaction flask. React at 25°C for 1 hour, monitored by TLC. After the starting materials have reacted completely, add 20 mL of water to quench the reaction. Separate the mixture and extract three times with ethyl acetate (30 mL each time). 3) Combine the ethyl acetate phases, concentrate under reduced pressure, add silica gel and concentrate again, then perform column chromatography on silica gel, eluting with a dichloromethane:methanol system (DCM:MeOH = 40:1) to give intermediate T14 (650.00 mg, yield 94.24%). LCMS: (MS-ESI, m / z ):[M+H] + = 786.3.

[0058] (8) At room temperature, add T14 (100 mg, 127.25 µmol) and 20% piperidine DMF solution (5 mL) to the reaction flask. After reacting for 1 hour, monitor the reaction by TLC. After the starting material has reacted completely, add 20 mL of water to quench the reaction. Separate the mixture and extract it three times with ethyl acetate (30 mL × 3). Concentrate under reduced pressure and evaporate the solvent. Then, perform column chromatography (dichloromethane:methanol = 15:1) to obtain compound 1 (68 mg, yield 94.81%).

[0059] The reaction formula for compound 1 above is as follows: Characterization results of compound 1: 1 H NMR (400 MHz, DMSO) δ 11.58 (s, 1H), 9.43 (t, J = 5.9Hz, 1H), 8.56 (d, J = 9.0 Hz, 1H), 8.49 (d, J = 4.7 Hz, 1H), 8.21 (d, J = 8.9 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.47 (t, J = 7.3 Hz, 2H), 7.41 – 7.35 (m, 5H), 7.27 (d, J = 7.2 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 4.18 (s, 2H), 3.94 (s, 3H), 3.76 (d, J = 5.7 Hz, 2H), 3.21 (s, 2H), 2.17 (s, 3H). MS m / z : calcd forC 31 H 29 N7O4563.23, found 564.30 [M + H] + . Example 2 A tricyclic compound, named nitrogen-(2-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethyl)isonicotinamide (abbreviated as compound 2), has the following molecular structure: .

[0060] The preparation method of compound 2 is the same as that in Example 1, except that T13 in step (7) is used instead of the above method. Replace with The detection and characterization results of compound 2 are as follows: 1 H NMR (400 MHz, DMSO) δ 8.90 (t, J = 5.2 Hz, 1H), 8.72 (d, J = 5.5 Hz, 2H), 7.85 (d, J = 7.4 Hz, 1H), 7.79 (d, J = 5.8 Hz, 2H), 7.46 (t, J = 7.3 Hz, 2H), .37 (dd, J = 15.6, 7.6 Hz, 5H), 7.24 (d, J = 6.4 Hz, 1H), 7.13 (d, J = 7.4 Hz, 1H), 3.89 (s, 3H), 3.83 (s, 2H), 3.47 (d, J = 6.0 Hz, 2H), 2.84 (t, J = 6.3 Hz, 2H), 2.17 (s, 3H). MS m / z : calcd for C 28 H 28 N4O2452.22, found453.30 [M + H] + . Example 3 A tricyclic compound, named nitrogen-(2-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethyl)nicotinamide (abbreviated as compound 3), has the following molecular structure: .

[0061] The preparation method of compound 3 is the same as in Example 1, except that T13 from step (7) is used instead of the above method. Replace with .

[0062] Characterization results of compound 3: 1 H NMR (400 MHz, DMSO) δ 9.05 (s, 1H), 8.87 (s,1H), 8.75 – 8.66 (m, 1H), 8.28 – 8.20 (m, 1H), 8.10 (d, J = 7.3 Hz, 1H), 7.88(t, J= 7.0 Hz, 1H), 7.60 – 7.35 (m, 7H), 7.26 – 7.14 (m, 1H), 3.99 (s, 1H), 3.90 (s, 5H), 3.55 – 3.50 (m, 2H), 2.90 (dd, J = 14.8, 6.3 Hz, 2H), 2.17 (s,3H). MS m / z : calcd for C 28 H 28 N4O2452.22, found 453.30 [M + H] + . Example 4 A tricyclic compound, named nitrogen-(2-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethyl)pyridineamide (abbreviated as compound 4), has the following molecular structure: .

[0063] The preparation method of compound 4 is the same as in Example 1, except that T13 in step (7) is used instead of the above method. Replace with The detection and characterization results of compound 4: 1 H NMR (400 MHz, DMSO) δ 8.85 (t, J = 5.6 Hz, 1H), 8.65 (d, J = 4.5 Hz, 1H), 8.05 (t, J = 6.0 Hz, 1H), 7.98 (dd, J = 10.9, 4.4Hz, 1H), 7.82 (d, J = 7.4 Hz, 1H), 7.59 (dd, J = 6.6, 5.6 Hz, 1H), 7.46 (t, J = 7.3Hz, 2H), 7.40 – 7.31 (m, 4H), 7.23 (d, J = 6.2 Hz, 1H), 7.10 (d, J = 7.4 Hz, 1H), 3.88 (s, 3H), 3.77 (s, 2H), 3.48 (d, J = 6.1 Hz, 2H), 2.80 (t, J= 6.2 Hz, 2H),2.17 (s, 3H). MS m / z : calcd for C 28 H 28 N4O2452.22, found 453.30 [M + H] + . Example 5 A tricyclic compound, named nitrogen-(2-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethyl)imidazo[1,2-a]pyridin-6-carboxamide (abbreviated as compound 5), has the following molecular structure: .

[0064] The preparation method of compound 5 is the same as in Example 1, except that T13 in step (7) is used instead of the above method. Replace with Characterization results of compound 5: 1 H NMR (400 MHz, DMSO) δ 9.17 (s, 1H), 8.76 (s, 1H), 8.17 – 7.97 (m, 2H), 7.88 (t, J = 7.0 Hz, 1H), 7.70 (dd, J = 9.5,1.7 Hz, 1H), 7.66 – 7.60 (m, 2H), 7.47 (t, J = 7.3 Hz, 2H), 7.40 – 7.34 (m,4H), 7.25 (dd, J = 7.3, 1.4 Hz, 1H), 7.15 (d, J = 7.4 Hz, 1H), 4.00 (s, 1H), 3.90(d, J = 3.6 Hz, 5H), 3.52 (d, J = 5.6 Hz, 2H), 2.90 (t, J = 6.2 Hz, 2H), 2.16 (s,3H). MS m / z : calcd for C 30 H 29 N5O2491.23, found 492.30 [M + H] + . Example 6 A tricyclic compound, named (E)-nitrogen-(2-(((2-methoxy-6-(2-methyl-[1,1'-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethyl)-3-(pyridin-3-yl)acrylamide (abbreviated as compound 6), has the following molecular structure: .

[0065] The preparation method of compound 6 is the same as in Example 1, except that T13 in step (7) is used instead of the above method. Replace with The detection and characterization results of compound 6 are as follows: 1 H NMR (400 MHz, DMSO) δ 8.76 (d, J = 1.6Hz, 1H), 8.55 (d, J = 3.8 Hz, 1H), 8.46 (d, J = 5.3 Hz, 1H), 8.10 (d, J = 7.3 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.89 (t, J = 7.1 Hz, 1H), 7.47 (dd, J = 16.1, 8.2Hz, 5H), 7.36 (dd, J = 12.2, 4.4 Hz, 3H), 7.24 (d, J = 6.4 Hz, 1H), 7.16 (d, J =7.4 Hz, 1H), 6.80 (dd, J = 15.9, 2.1 Hz, 1H), 4.01 (s, 1H), 3.94 – 3.86 (m,5H), 3.42 (d, J = 3.5 Hz, 2H), 2.88 – 2.82 (m, 2H), 2.17 (s, 3H). MS m / z : calcdfor C 30 H 30 N4O2478.24, found 479.30 [M + H] + . Comparative Example 1 paper J Med Chem.2021 Jun 10;64(11):7390-7403. doi: 10.1021 / acs.jmedchem.1c00010. Title: Novel Biphenyl Pyridines as Potent Small-Molecule Inhibitors Targeting the Programmed Cell Death-1 / Programmed Cell Death-Ligand1 Interaction. A series of tricyclic PD-L1 inhibitors were reported, with a reported yield of 16.99%. The structural formulas of representative compounds are as follows: .

[0066] Comparative Example 2 Patent CN115417870A. Invention title: PD-L1 & NAMPT dual-target inhibitor and its use. A series of tricyclic PD-L1 inhibitors have been reported, with representative compounds having the following structural formulas. Literature reports yields of 12%–14%, while the synthetic route of this application yields 71%–73%, with the following structural formulas (see Example 9 of Patent CN115417870A): .

[0067] Comparative Example 3 Patent CN115417870A. Invention title: PD-L1 & NAMPT dual-target inhibitor and its uses. A series of tricyclic PD-L1 inhibitors have been reported, with yields of 12%–14% reported in the literature. The yield using the synthetic route of this patent is 71%–73%. The structural formulas of representative compounds are as follows (see Example 15 of Patent CN115417870A):

[0068] Comparative Example 4 Patent CN115417870A. Invention title: PD-L1 & NAMPT dual-target inhibitors and their uses. A series of tricyclic PD-L1 inhibitors have been reported, with a reported yield of 13.56%. The structural formulas of representative compounds are as follows (see Example 25 of Patent CN115417870A): .

[0069] Product effectiveness test I. Degradation effect test of PD-L1 protein (Western blot method) 1. Cell Culture: MDA-MB-231 cells were cultured in Leibovitz's L-15 medium + 10% FBS + 1% penicillin. Streptomycin solution was used, and cells were cultured at 37°C and 5% CO2.

[0070] 2. Plate preparation: One day in advance, add 2 ml of 1×10⁻⁶ solution to each well of a 6-well plate. 6 Cells were cultured overnight at a density of 2 × 10⁶ cells / ml, and the cell density reached 2 × 10⁶ cells / ml the next day. 6 Add a 10 μM solution of the test compound to each well at a concentration of 10 μM / ml and incubate at 37°C in a 5% CO2 incubator for 24 h.

[0071] 3. Extraction of cell proteins (1) Mix the RIPA lysis buffer and protease inhibitor.

[0072] (2) Centrifuge the cells and discard the culture medium. Resuspend the cells in PBS, centrifuge again, and retain the precipitate.

[0073] (3) Add RIPA mixed lysis buffer to each well according to the cell count, and transfer it to a 1.5ml EP tube after pipetting.

[0074] (4) Place the 1.5ml EP tube on ice for 10min, centrifuge at 12000g at 4℃ for 10min, and transfer the protein supernatant to a new 1.5ml EP tube.

[0075] 4. Bradford assay for total protein concentration (1) Dilute the supernatant protein 20 times, and take 3 μl of diluent + 100 μl of Bradford into a new tube.

[0076] (2) After mixing, take 90 μl into a 96-well plate and let stand for 5 min.

[0077] (3) The absorbance was measured by an ultraviolet spectrophotometer at a wavelength of 595 nm.

[0078] 5. SDS-polyacrylamide gel electrophoresis (1) Add SDS loading buffer to the protein sample and denature at 95℃ for 5 min.

[0079] (2) Carefully remove the comb from the gel, install the glass plate filled with SDS-PAGE gel on the electrophoresis rack with the short plate facing inward, and then place it in the electrophoresis tank. Pour 1× Tris-Gly electrophoresis buffer that has been pre-cooled overnight at 4°C into the electrophoresis tank.

[0080] (3) Add protein sample to each lane according to the principle of equal mass and volume, add 3 μl of protein marker to both sides, and add an equal volume of 1× loading buffer to the lane without sample. The sample loading volume for each well is 15-20 μl.

[0081] (4) Cover the electrophoresis tank, turn on the power, and perform electrophoresis at a constant voltage of 80V for 20-30 minutes. After the protein sample is pressed into a line, increase the voltage to 120V for about 40-60 minutes and continue electrophoresis until the bromophenol blue reaches the bottom of the separating gel.

[0082] 6. Transfer membrane (1) Prepare sponge, filter paper and PVDF membrane. Pour 1× transfer buffer and methanol, which have been pre-cooled overnight at 4℃, into enamel pans respectively. Activate the PVDF membrane by soaking it in methanol for 10 min. Soak the sponge and filter paper in the transfer solution.

[0083] (2) Remove the glass plates from the electrophoresis tank, separate the two glass plates, cut off the stacking gel, cut off the excess separating gel according to the size of the target protein, and place it in the transfer buffer.

[0084] (3) Place the PVDF membrane in the transfer fixer in the order of black gel and white film, gently removing air bubbles after each layer. Mark the PVDF membrane. Clamp the membrane and place it in the transfer electrophoresis tank with the black sides facing each other. Pour in the transfer buffer and close the lid.

[0085] (4) Place the transfer tank into a foam box filled with crushed ice. Turn on the power and transfer at a constant current of 260-280 mA for 60 minutes.

[0086] 7. Immunostaining (1) Closed 1) Prepare 5% skim milk powder, mix thoroughly and pour into the incubation box.

[0087] 2) After the transfer is complete, remove the PVDF membrane, rinse it with TBST to remove salt, and place it in an incubation box containing 5% skim milk powder. Shake it slowly on a shaker for 1 hour to seal it.

[0088] (2) Target protein and primary antibody reaction (PD-L1, abcam): 1) Prepare primary antibody reaction solution: Prepare a 15ml centrifuge tube, add 3ml of primary antibody diluent, add the primary antibody according to the dilution ratio indicated in the antibody instructions, invert and mix well, and place on ice.

[0089] 2) Transfer the PVDF membrane from the blocking solution to TBST and cut the bands according to the molecular weight of the target protein.

[0090] 3) Place the strip into a centrifuge tube containing the primary antibody reaction solution and incubate overnight at 4°C with shaking.

[0091] (3) Reaction of primary antibody with HRP-labeled secondary antibody: 1) Wash with TBST 3 times, 10 minutes each time.

[0092] 2) Preparation of secondary antibody reaction solution: Prepare a 15ml centrifuge tube, add 3ml of 5% skim milk powder, then add goat anti-rabbit IgG-HRP (1:5000) or goat anti-mouse IgG-HRP (1:5000), and mix by inverting.

[0093] 3) Place the strip into a plastic box containing the secondary antibody reaction solution and incubate at room temperature with shaking for 1 hour.

[0094] (4) Exposure: 1) Wash with TBST 3 times, 10 minutes each time.

[0095] 2) Mix equal volumes of solution A and solution B to prepare the colorimetric solution, and protect it from light.

[0096] 3) Place the strip in the black film plate, wipe off the excess water, add the color developer to fully cover the strip, and expose for color development.

[0097] Note: The development results were analyzed for grayscale using Image J, and the degradation ratio was obtained through normalization. The degradation effects of the compounds in this application at concentrations between 10 μM are shown in Table 1. For the degradation effect, "++++" indicates a degradation effect between 100% and 70%; "+++" indicates a degradation effect between 70% and 50%; "++" indicates a degradation effect between 50% and 30%; and "+" indicates a degradation effect <30%.

[0098] Table 1. Degradation test results of each compound on PD-L1

[0099] As shown in Table 1, compounds 1-6 effectively degraded PD-L1 protein in MDA-MB-231 cells, with better results than comparative compounds 1-4. 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 compounds of this invention have similar structures to comparative compounds 1-4, they exhibit better PD-L1 protein degradation effects than the comparative compounds.

[0100] II. Rat Pharmacokinetic Test 1. Testing Method SD rats were administered compound 3 (20 mg / kg) orally via gavage. Approximately 0.1 mL of blood was collected from the orbital cavity before administration and at 0, 5, 10, 15, 30 minutes, 1, 2, 3, 4, 6, 8, 12, and 24 hours after administration. The blood was anticoagulated with heparin. The blood was collected in test tubes, centrifuged for 10 minutes, and the serum was separated. 150 μL of a 100 ng / mL methanol solution (16000 g / mL) was added, and the mixture was centrifuged at 16000 g for 10 minutes. 100 μL of the supernatant was then analyzed by UPLC-MS 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 ℃; Acetonitrile was used as mobile phase A, 0.1% formic acid aqueous solution was used as mobile phase B, flow rate was set to 0.4 mL / min, and the separation of compound 1 and 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 was used, with ion fragments generated by multiple reaction monitoring (MRM), capillary voltage of 1 kV, desolvation gas temperature of 500 ℃, source temperature of 150 ℃, sheath gas flow rate of 50 L / H, and desolvation gas flow rate of 1000 L / H.

[0101] AUC （0-∞） This represents the area under the blood drug concentration-time curve; T max Indicates the time point of maximum plasma concentration; t 1 / 2 Indicates half-life; C max This indicates the maximum blood drug concentration.

[0102] 2. Experimental Results Table 2. Pharmacokinetic data of compound 3 in rats.

[0103] According to the results in Table 2, compound 3 provided by this invention showed a suitable half-life, maximum plasma concentration, and a very high area under the plasma concentration-time curve in the rat pharmacokinetic PK test model. Calculations showed that the oral bioavailability of compound 3 was greater than 60%, while the oral bioavailability of compound 1 reported in the literature was 22%, which was significantly lower than that of the compound of this invention. The comparison showed that the oral bioavailability of the compound of this invention was very high and it has the prospect of oral administration.

Claims

1. A tricyclic compound, characterized in that, It has any one of the following molecular structures: 、 、 。 2. The method for preparing the tricyclic compound according to claim 1, characterized in that, Includes the following steps: Where A is selected from , , ; (1) First, T1 and T2 undergo a Suzuki coupling reaction to generate intermediate T3; the reaction to generate intermediate T3 is carried out in a mixed solvent consisting of 1,4-dioxane and water in a volume ratio of 5:

1. (2) Intermediates T3 and T4 undergo a Miyaura coupling reaction to generate intermediate T5; the reaction to generate intermediate T5 uses 1,4-dioxane as a solvent. (3) Intermediates T5 and T6 undergo a Suzuki coupling reaction to generate intermediate T7; the reaction to generate intermediate T7 is carried out in a mixed solvent of 2-methyltetrahydrofuran and water in a volume ratio of 10:1; (4) Intermediate T7 and T8 undergo a Borge reduction reaction to generate intermediate T9; (5) Intermediate T9 further undergoes an Fmoc protection reaction with T10 to generate intermediate T11; (6) Intermediate T11 loses its Boc protecting group under the action of 4M HCl to generate intermediate T12; (7) Intermediates T12 and T13 undergo a condensation reaction to generate intermediate T14; (8) Intermediate T14 is deprotected by Fmoc protecting group under the action of 20% piperidine to generate the target product.

3. A pharmaceutical composition, characterized in that, It comprises the tricyclic compound of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. The use of the tricyclic compound of claim 1 in the preparation of drugs for treating PD-L1-targeted cancers.

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