1, 2, 4-oxadiazole biphenyl compound as well as preparation method and application thereof

Abstract

The invention provides a 1, 2, 4-oxadiazole biphenyl compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides a 1, 2, 4-oxadiazole biphenyl compound which has a structure as shown in a formula h. By introducing dioxin, amide bridges and chlorine atoms, the water solubility of the compound can be improved. In addition, potassium trimethylsilanolate is used as an alkali reagent of an SZUKI coupling reaction, methylbenzene is used for replacing trichloroacetic anhydride to serve as a solvent of a cyclization reaction, and a mixed solvent of dichloromethane and methanol is used as a reaction solvent of reductive amination to optimize the reaction. When the 1, 2, 4-oxadiazole biphenyl compound provided by the invention is used as a PD-L1 small-molecule inhibitor, the 1, 2, 4-oxadiazole biphenyl compound has good targeting property and anti-proliferative activity, and a basis is provided for further enriching a small-molecule inhibitor compound library and researching the structure-function relationship of the 1, 2, 4-oxadiazole inhibitor.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a 1,2,4-oxadiazole biphenyl compound, its preparation method, and its application. Background Technology

[0002] In most countries around the world, cancer is the first or second leading cause of death. At the same time, population aging is accelerating the incidence of cancer.

[0003] Traditional cancer treatments mainly include surgery, radiotherapy, and chemotherapy. However, current anti-tumor drugs often have many common side effects, such as ulcers caused by the similarity or similarity between normal skin and mucous membranes and tumor cell targets; gastrointestinal reactions; liver and kidney toxicity due to difficulties in drug metabolism; and hair loss, neurotoxicity, and bone marrow suppression due to cell damage. In recent years, tumor immunotherapy drugs have gradually gained favor among researchers. These drugs eliminate tumor cells by enhancing the body's natural immune defense against tumors. They are mainly classified as immune checkpoint inhibitors, therapeutic antibodies, and cancer vaccines. This new cancer therapy that inhibits negative immune regulation has greatly improved the overall survival rate of patients with advanced metastatic cancer. Among them, immune checkpoint inhibitors have the most mature clinical research and the most widespread application.

[0004] Immune checkpoints are a class of immunosuppressive molecules expressed on immune cells that regulate the level of immune activation. They play a crucial role in preventing autoimmune responses; simply put, they are small protein molecules produced by immune cells that regulate autoimmune function. PD-1 / PD-L1 is one such very important immune checkpoint. Inhibitors of PD-1 / PD-L1 restore T cell activity and activate the immune system to kill tumor cells by preventing PD-1 from binding to PD-L1. Currently, the FDA has approved several PD-L1 antibodies for marketing. Among them, pembrolizumab and atezolizumab are used to treat various types of tumors. However, the application of antibody drugs is limited due to their poor oral bioavailability and immune-related adverse events (irAEs). Small molecule inhibitors of the PD-1 / PD-L1 immune checkpoint are still in preclinical research, and no small molecule inhibitors have been successfully marketed. Therefore, expanding the types of small molecule inhibitors of the PD-1 / PD-L1 immune checkpoint is of great significance for cancer treatment.

[0005] Patent application CN 111718310 B discloses a series of small molecule inhibitors mainly composed of 1,3,4-oxadiazole compounds, including the structure and synthetic route of a 1,2,4-oxadiazole compound named 2-[((3-{[(3-(2-methyl-[1,1'-biphenyl]-3-yl)-1,2,4-oxadiazole-5-yl)amino]benzyl)amino)ethanol], with the following structural formula:

[0006]

[0007] The aforementioned PCT patent application discloses that it can inhibit the binding of programmed cell death receptor 1 / programmed cell death ligand 1 (PD-1 / PD-LI) to a certain extent. However, repeating the experiment along its route results in many byproducts and low yield, which is not conducive to further expanding the library of similar 1,2,4-oxadiazole compounds and further studying the structure-activity relationship. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a 1,2,4-oxadiazole biphenyl compound, its preparation method, and its applications. The 1,2,4-oxadiazole biphenyl compound provided by this invention exhibits good targeting and anti-proliferative activity when used as a small molecule inhibitor of PD-L1. Furthermore, the synthetic route provided by this invention has a short reaction time and high yield, enabling efficient and economical preparation of 1,2,4-oxadiazole biphenyl compounds.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a method for preparing 1,2,4-oxadiazole biphenyl compounds, comprising the following steps:

[0011] In the presence of a palladium catalyst and a basic reagent, a compound having the structure shown in formula a undergoes a Suzuki coupling reaction with a compound having the structure shown in formula b to obtain a compound having the structure shown in formula c.

[0012]

[0013] A compound having the structure shown in formula c undergoes a nucleophilic addition reaction with an aqueous hydroxylamine solution to give a compound having the structure shown in formula d.

[0014]

[0015] A compound having the structure shown in formula d undergoes a cyclization reaction with trichloroacetic anhydride to give a compound having the structure shown in formula e.

[0016]

[0017] A compound having the structure shown in formula e undergoes a condensation reaction with 3-aminobenzyl alcohol to give a compound having the structure shown in formula f.

[0018]

[0019] Under the action of the Des Martin reagent, the compound having the structure shown in formula f is oxidized to the compound having the structure shown in formula g;

[0020]

[0021] Under the action of a reducing agent, a compound having the structure shown in formula g undergoes a reductive amination reaction with ethanolamine to obtain a 1,2,4-oxadiazole compound having the structure shown in formula h.

[0022]

[0023] Preferably, the alkaline reagent is potassium trimethylsilanolate; the molar ratio of the compound having the structure shown in formula a to the alkaline reagent is 1:1 to 5;

[0024] The molar ratio of the compound having the structure shown in Formula a to the compound having the structure shown in Formula b is 1:1 to 5.

[0025] The Suzuki coupling reaction was carried out at a temperature of 100–105 °C for 30–60 min.

[0026] Preferably, the mass concentration of the hydroxylamine aqueous solution is 40-60%;

[0027] The nucleophilic addition reaction is carried out at a temperature of 90–100 °C for 6–10 h.

[0028] Preferably, the molar ratio of the compound having the structure shown in formula d to trichloroacetic anhydride is 1:1 to 5;

[0029] The reaction solvent for the cyclization reaction is toluene; the mass ratio of the compound having the structure shown in formula d to the volume of toluene is 100 mg: 2-20 mL;

[0030] The cyclization reaction is carried out at a temperature of 100–110°C for 2–3 hours.

[0031] Preferably, the molar ratio of the compound having the structure shown in formula e to 3-aminobenzyl alcohol is 1:4 to 6;

[0032] The condensation reaction is carried out at a temperature of 130–150°C for 8–12 hours.

[0033] Preferably, the molar ratio of the compound having the structure shown in formula f to the Dess-Martin reagent is 1:1 to 5;

[0034] The oxidation temperature is 0–20°C, and the time is 20–30 min.

[0035] Preferably, the reducing agent is sodium cyanoborohydride, and the molar ratio of the compound having the structure shown in formula g to the reducing agent is 1:1 to 5;

[0036] The molar ratio of the compound having the structure shown in formula g to ethanolamine is 1:1 to 5;

[0037] The reaction solvent for the reductive amination reaction is a mixture of dichloromethane and methanol in a volume ratio of 1:1, and the mixture contains 6 wt% acetic acid.

[0038] The reductive amination reaction is carried out at a temperature of 20–50°C for a time of 4–8 hours.

[0039] This invention provides a 1,2,4-oxadiazole biphenyl compound having the structure shown in formula h:

[0040]

[0041] This invention provides the application of the above-mentioned 1,2,4-oxadiazole biphenyl compounds in the preparation of PD-L1 small molecule inhibitors.

[0042] This invention provides the application of the above-mentioned 1,2,4-oxadiazole biphenyl compounds in the preparation of antitumor drugs.

[0043] This invention provides a 1,2,4-oxadiazole biphenyl compound having the structure shown in formula h. By introducing dioxins, amide bridges, and chlorine atoms, this invention enhances the compound's water solubility and its ability to promote PD-L1 protein dimerization, effectively inhibiting the binding of PD-1 / PD-L1 proteins. When used as a small molecule inhibitor of PD-L1, the 1,2,4-oxadiazole biphenyl compound provided by this invention exhibits good targeting and anti-proliferative activity, providing a foundation for further enriching the small molecule inhibitor compound library and studying the structure-activity relationship of 1,2,4-oxadiazole inhibitors. The results of the examples show that the 1,2,4-oxadiazole compound provided by this invention is effective against three normal cell lines: human lung cell line BEAS-2b, human kidney cell line HEK-293T, and human liver cell line L02, IC 50 All concentrations were above 25 μmol / L, indicating that the 1,2,4-oxadiazole compounds provided in this invention have good in vitro safety. For the murine breast cancer cell line 4T-1 and the murine colon cancer cell line MC38, the IC50 of the 1,2,4-oxadiazole compounds provided in this invention... 50 All concentrations below 20 μmol / L showed some anti-proliferative activity. In in vitro PD-1 / PD-L1 binding inhibition experiments, their IC50 values ​​were... 50Less than 1 μmol / L. In vivo antitumor activity experiments showed that, compared with the untreated group, the 1,2,4-oxadiazole compounds provided by this invention could significantly inhibit tumor growth (administered at a dose of 30 mg / Kg, intraperitoneal injection), and did not cause weight loss or death during treatment. There was no obvious damage to the conventional tissues of the group treated with the compound, indicating that the effective dose of the compound of this invention is well tolerated.

[0044] This invention provides a method for preparing the aforementioned 1,2,4-oxadiazole biphenyl compounds. Using 1,4-benzodioxane-6-borate pinacol ester and 3-bromo-2-chlorobenzonitrile as starting materials, the invention yields 1,2,4-oxadiazole biphenyl compounds with the structure shown in formula h through a series of Suzuki coupling reactions, nucleophilic addition reactions, cyclization reactions, condensation reactions, oxidation reactions, and reductive amination reactions. Furthermore, this invention optimizes the reaction by using potassium trimethylsilanolate as the base reagent for the Suzuki coupling reaction, toluene as the solvent for the cyclization reaction, and a mixed solvent of dichloromethane and methanol as the reaction solvent for the reductive amination. Compared to similar compounds in the prior art, the synthetic route of this invention is greatly optimized, with shorter reaction times, higher product yields (most steps have yields of not less than 90%), and enables the efficient and economical preparation of 1,2,4-oxadiazole biphenyl compounds. Attached Figure Description

[0045] Figure 1 The synthetic route for 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide;

[0046] Figure 2 The HTRF test results are for 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide;

[0047] Figure 3 The results of the antiproliferative activity tests of 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide against different cells;

[0048] Figure 4 The results of tumor growth inhibition in different groups at different days after treatment. Detailed Implementation

[0049] This invention provides a method for preparing 1,2,4-oxadiazole biphenyl compounds, comprising the following steps:

[0050] In the presence of a palladium catalyst and a basic reagent, a compound having the structure shown in formula a undergoes a Suzuki coupling reaction with a compound having the structure shown in formula b to obtain a compound having the structure shown in formula c.

[0051]

[0052] A compound having the structure shown in formula c undergoes a nucleophilic addition reaction with an aqueous hydroxylamine solution to give a compound having the structure shown in formula d.

[0053]

[0054] A compound having the structure shown in formula d undergoes a cyclization reaction with trichloroacetic anhydride to give a compound having the structure shown in formula e.

[0055]

[0056] A compound having the structure shown in formula e undergoes a condensation reaction with 3-aminobenzyl alcohol to give a compound having the structure shown in formula f.

[0057]

[0058] Under the action of the Des Martin reagent, the compound having the structure shown in formula f is oxidized to the compound having the structure shown in formula g;

[0059]

[0060] Under the action of a reducing agent, a compound having the structure shown in formula g undergoes a reductive amination reaction with ethanolamine to obtain a 1,2,4-oxadiazole compound having the structure shown in formula h.

[0061] In this invention, under the action of a palladium catalyst and a basic reagent, a compound having the structure shown in formula a undergoes a Suzuki coupling reaction with a compound having the structure shown in formula b to obtain a compound having the structure shown in formula c. In this invention, the palladium catalyst includes all catalysts containing metallic palladium, preferably a divalent palladium catalyst, specifically preferably a [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex. In this invention, the molar ratio of the compound having the structure shown in formula a to the palladium catalyst is preferably 1:0.01 to 1, more preferably 1:0.05 to 0.5, and even more preferably 1:0.05 to 0.1.

[0062] In this invention, the alkaline reagent is preferably potassium trimethylsilanolate (TMSOK). TMSOK exhibits good organic solubility and is inexpensive. Experiments show that compared to traditional coupling alkaline reagents such as potassium carbonate and potassium acetate, the reaction time is shortened from several hours to 15-60 minutes, and fewer byproducts are produced. In this invention, the molar ratio of the compound having the structure shown in formula a to the alkaline reagent is preferably 1:1-5, more preferably 1:2-4.

[0063] In this invention, the molar ratio of the compound having the structure shown in Formula a to the compound having the structure shown in Formula b is preferably 1:1 to 5, more preferably 1:1 to 3, and even more preferably 1:1 to 1.5.

[0064] In this invention, the reaction solvent for the suzuki coupling reaction is preferably dioxane. The suzuki coupling reaction is preferably carried out under nitrogen protection, with a preferred temperature of 100–105°C and a preferred time of 30–60 min, more preferably 40–50 min. Compared to existing technologies, the preparation method provided by this invention shortens the reaction time from 12 h to less than 1 h, requires only 0.05 equivalents of catalyst, significantly improving synthesis efficiency and reducing costs.

[0065] In this invention, after the Suzuki coupling reaction, the obtained Suzuki coupling reaction solution is preferably post-treated, and the post-treatment preferably includes the following steps:

[0066] The reaction solvent in the Suzuki coupling reaction solution is removed, and the resulting residue is extracted, the organic phases are combined, dried, and separated by column chromatography. In this invention, the solvent used for extraction is preferably a mixture of ethyl acetate and water, and the volume ratio of ethyl acetate to water is preferably 1:1 to 2.

[0067] In this invention, the process of the Suzuki coupling reaction is shown in formula m:

[0068]

[0069] After obtaining the compound having the structure shown in formula c, the compound having the structure shown in formula c undergoes a nucleophilic addition reaction with an aqueous hydroxylamine solution to obtain the compound having the structure shown in formula d. In this invention, the mass concentration of the aqueous hydroxylamine solution is preferably 40-60 wt%, more preferably 50 wt%. In this invention, the mass ratio of the compound having the structure shown in formula c to the volume ratio of the hydroxylamine solution is preferably 1 g: 10-100 mL, more preferably 1: 20-50 mL.

[0070] In this invention, the aqueous hydroxylamine solution serves as both a reactant and a solvent for the nucleophilic addition reaction.

[0071] In this invention, the temperature of the nucleophilic addition reaction is preferably 90-100°C, more preferably 95°C; the time is preferably 6-10 hours, more preferably 7-8 hours.

[0072] In this invention, after the nucleophilic addition reaction, the resulting nucleophilic addition reaction solution is preferably post-treated, and the post-treatment preferably includes the following steps:

[0073] The nucleophilic addition reaction solution was mixed with ice water to terminate the reaction, and solid-liquid separation was performed. The resulting solid was then dried.

[0074] In this invention, the solid-liquid separation is preferably achieved through filtration; this invention does not have any special requirements for the drying method, and any drying method well known to those skilled in the art can be used.

[0075] In this invention, the nucleophilic addition reaction process is shown in formula n:

[0076]

[0077] After obtaining the compound having the structure shown in formula d, the compound having the structure shown in formula d undergoes a cyclization reaction with trichloroacetic anhydride to obtain the compound having the structure shown in formula e. In this invention, the molar ratio of the compound having the structure shown in formula d to trichloroacetic anhydride is preferably 1:1 to 5, more preferably 1:1.2 to 4, and even more preferably 1:1.2 to 3.

[0078] In this invention, the reaction solvent for the cyclization reaction is preferably toluene. In this invention, the mass-to-volume ratio of the compound having the structure shown in formula d to toluene is preferably 100 mg: 2–20 mL, more preferably 100 mg: 5–10 mL. Compared with the prior art, this invention uses toluene instead of acid anhydrides as the reaction solvent, reducing the generation of byproducts and significantly saving solvent costs, which is beneficial to improving product yield.

[0079] In this invention, the temperature of the cyclization reaction is preferably 100-110°C, more preferably 105°C, and the time is preferably 2-3 hours, more preferably 2.5 hours.

[0080] In this invention, after the cyclization reaction, the resulting cyclization reaction solution is preferably post-treated, and the post-treatment preferably includes the following steps:

[0081] The organic solvent in the cyclization reaction solution is removed, and the resulting residue is extracted, the organic phases are combined, and separated by column chromatography. In this invention, the solvent used for extraction is preferably a mixture of dichloromethane and water, and the volume ratio of dichloromethane to water is preferably 1:2 to 3.

[0082] In this invention, the reaction process of the cyclization reaction is shown in formula o:

[0083]

[0084] After obtaining the compound having the structure shown in Formula e, the compound having the structure shown in Formula e undergoes a condensation reaction with 3-aminobenzyl alcohol to obtain the compound having the structure shown in Formula f. In this invention, the molar ratio of the compound having the structure shown in Formula e to 3-aminobenzyl alcohol is preferably 1:4 to 6, more preferably 1:5.

[0085] In this invention, the reaction solvent for the condensation reaction is preferably N,N-dimethylformamide (DMF). In this invention, the condensation reaction is preferably carried out under nitrogen protection, the temperature of the condensation reaction is preferably 130–150°C, more preferably 140°C, and the time is preferably 8–12 h, more preferably 9–10 h.

[0086] Following the condensation reaction, the present invention preferably performs post-treatment on the resulting condensation reaction solution, the post-treatment preferably including the following steps:

[0087] The condensation reaction solution was extracted with ethyl acetate, the organic phases were combined, and the organic phases were separated by column chromatography.

[0088] In this invention, the reaction process of the condensation reaction is shown in formula p:

[0089]

[0090] After obtaining the compound having the structure shown in formula f, it is oxidized to the compound having the structure shown in formula g under the action of a Dysmart reagent (DMP). In this invention, the molar ratio of the compound having the structure shown in formula f to the Dysmart reagent is preferably 1:1 to 5, more preferably 1:1.2 to 2.2, and even more preferably 1:1.5 to 2. In this invention, the preferred structural formula of the Dysmart reagent is:

[0091] In this invention, the reaction solvent during oxidation is preferably one or more of dichloromethane (DCM), acetonitrile, and N,N-dimethylformamide. In this invention, the oxidation temperature is preferably 0–30°C, more preferably 5–10°C, and the oxidation time is preferably 10–120 min, more preferably 20–60 min, and even more preferably 20–30 min.

[0092] After oxidation, the present invention preferably performs post-treatment on the resulting oxidation reaction solution, the post-treatment preferably including the following steps:

[0093] The oxidation reaction solution is washed, subjected to vacuum distillation, and extracted. The extracted organic phases are combined and separated by column chromatography. In this invention, the solvent used for extraction is preferably a mixture of ethyl acetate and water, and the volume ratio of ethyl acetate to water is preferably 1:1 to 3.

[0094] In this invention, the oxidation reaction process is shown in formula q:

[0095]

[0096] After obtaining the compound having the structure shown in formula g, the compound having the structure shown in formula g undergoes a reductive amination reaction with ethanolamine under the action of a reducing agent to obtain a 1,2,4-oxadiazole compound having the structure shown in formula h. In this invention, the reducing agent is preferably sodium cyanoborohydride, and the molar ratio of the compound having the structure shown in formula g to the reducing agent is preferably 1:1 to 5, more preferably 1:2 to 4. In this invention, the molar ratio of the compound having the structure shown in formula g to ethanolamine is preferably 1:1 to 5, more preferably 1:1 to 3, and even more preferably 1:1.2.

[0097] In this invention, the reaction solvent for the reductive amination is preferably a mixture of dichloromethane and methanol in a volume ratio of 1:1, and the mixture preferably contains 6 wt% acetic acid. In this invention, the mass ratio of the compound having the structure shown in formula g to the volume ratio of the reaction solvent is preferably 100 mg: 1–6 mL, more preferably 1: 2–4 mL.

[0098] In this invention, the temperature of the reductive amination reaction is preferably 20-50°C, more preferably 30-40°C, and the time is preferably 4-8 hours, more preferably 5-6 hours.

[0099] Following the reductive amination reaction, the present invention preferably performs post-treatment on the obtained reductive amination reaction solution, the post-treatment preferably including the following steps:

[0100] The reaction solvent in the reducing amination reaction solution is removed, and the resulting residue is extracted, the organic phases are combined, and separated by column chromatography. In this invention, the solvent used for extraction is preferably a mixture of ethyl acetate and water, and the volume ratio of ethyl acetate to water is preferably 1:1 to 3.

[0101] In this invention, the process of the reductive amination reaction is shown in formula r:

[0102]

[0103] This invention provides a 1,2,4-oxadiazole biphenyl compound, chemically named 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide. In this invention, the 1,2,4-oxadiazole biphenyl compound has the structure shown in formula h:

[0104]

[0105] This invention provides the application of the above-mentioned 1,2,4-oxadiazole biphenyl compounds in the preparation of PD-L1 small molecule inhibitors.

[0106] This invention provides the application of the above-mentioned 1,2,4-oxadiazole biphenyl compounds in the preparation of antitumor drugs. In this invention, the tumor is preferably triple-negative breast cancer.

[0107] The following examples illustrate the 1,2,4-oxadiazole biphenyl compounds, their preparation methods, and applications provided by this invention, but these should not be construed as limiting the scope of protection of this invention.

[0108] Example 1: Preparation of 1,2,4-oxadiazole biphenyl compounds

[0109] (1) Preparation of 2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)benzonitrile

[0110] 1.5 g (5.73 mmol, 1 eq.) of 1,4-benzodioxane-6-borate pinacol ester was dissolved in 30 mL of dioxane. 1.2366 g (5.73 mmol, 1 eq.) of 3-bromo-2-chlorobenzonitrile and 1.03 g (8.022 mmol, 1.4 eq.) of potassium trimethylsilanolate were added. The system was protected with nitrogen and mixed thoroughly. Heating was carried out at 100 °C. 236.64 mg of Pd(II) (0.29 mmol, 0.05 eq.) catalyst was added, and the reaction was stopped after 30 min. The solvent was removed, and the mixture was extracted. The combined organic phases were dried. Column chromatography was used to purify and obtain compound c 2.8658 g, with a yield of 92%. Compared with the yield reported in the literature, this synthetic method shortens the reaction time from 12 h to less than 1 h, and requires only 0.05 equivalents of catalyst, greatly improving the synthetic efficiency and reducing the cost.

[0111]

[0112] 1 H NMR (300MHz, CDCl3) δ7.64(dd,J=7.7,1.7Hz,1H),7.54(dd,J=7.8,1.7Hz,1H),7.39(t,J=7.7Hz,1H),6.97-6.91(m,2H),6.90-6.85(m,1H),4.31(s,4H).

[0113] (2) Synthesis of (Z)-2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N'-hydroxybenzamide

[0114] 1.5 g of compound c (7.77 mmol, 1 eq.) was weighed and added to 30 mL of hydroxylamine aqueous solution (50% by mass). The reaction system was heated and stirred in an oil bath at 100 °C for 8 h. After the reaction was completed, ice water was added to terminate the reaction, and a white solid precipitated. After filtration and drying, 1.67 g of white solid, which was compound d, was obtained, with a yield of 95%. Compared with similar literature, the yield of this synthetic method is doubled, and the post-processing is simple.

[0115]

[0116] 1 H NMR (300MHz, DMSO) δ9.41(s,1H),7.46-7.28(m,3H),6.99-6.81(m,3H),5.84(s,2H),4.29(s,4H). 13C NMR (201MHz, DMSO) δ150.36,142.53,142.26,139.39,134.13,131.44,130.97,130.18,129.46,126.11,121.70,117.34,116.22,63.54,63.50.

[0117] (3) Synthesis of 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-5-(trichloromethyl)-1,2,4-oxadiazole

[0118] 650 mg of compound d (2.87 mmol, 1 eq.) was weighed and dissolved in 32.5 mL of toluene. 630 μL of trichloroacetic anhydride (3.45 mmol, 1.2 eq.) was added, and the mixture was heated to 110 °C for 2 h before stopping the reaction. The solvent was removed, and the organic phases were combined. Column chromatography was used to purify compound e to obtain 997.5 mg, with a yield of 94%. Compared with similar literature, this synthetic method improves the yield by more than 60%, and the use of toluene instead of anhydride as the solvent significantly reduces costs.

[0119]

[0120] 1 H NMR (800MHz, CDCl3) δ7.85 (dd, J=7.7, 1.7Hz, 1H), 7.50 (dd, J=7.6, 1.7Hz, 1H), 7.43 (t, J=7.7Hz, 1H), 6.98-6.90 (m, 3H), 4.33-4.30 (m, 4H).

[0121] (4) Synthesis of 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(hydroxymethyl)phenyl)-1,2,4-oxadiazole-5-carboxamide

[0122] 1 g (2.3 mmol, 1 eq.) of compound e and 1.43 g (11.6 mmol, 5 eq.) of 3-aminobenzyl alcohol were weighed into a dry two-necked flask. The system was evacuated and protected with nitrogen. 20 mL of DMF was added, and the mixture was heated in an oil bath at 130 °C for 8 h. The mixture was extracted with ethyl acetate, and the combined organic phases were purified by column chromatography to obtain compound f 910 mg, with a yield of 42.5%.

[0123]

[0124] 1H NMR (300MHz, DMSO) δ11.31(s,1H),7.96-7.81(m,1H),7.83(d,J=1.9Hz,1H),7.72-7.56(m,3H),7.36(t,J=7.8Hz ,1H),7.16(dt,J=7.8,1.2Hz,1H),7.02-6.87(m,3H),5.26(t,J=5.7Hz,1H),4.52(d,J=5.7Hz,2H),4.30(s,4H).

[0125] (5) Synthesis of 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-formylphenyl)-1,2,4-oxadiazole-5-carboxamide

[0126] 130.6 mg (0.28 mmol, 1 eq.) of compound f was weighed and dissolved in 2 mL of DCM. 254.6 mg (0.60 mmol, 2 eq.) of DMP was added in an ice bath. The reaction progress was monitored by TLC. After the starting material spot disappeared, the reaction mixture was washed with saturated sodium thiosulfate solution to remove residual DMP, DCM was removed under reduced pressure, and the mixture was extracted with ethyl acetate. The combined organic phases were purified by column chromatography to obtain compound g 117.5 mg, with a yield of 90.0%.

[0127]

[0128] 1 H NMR (300MHz, DMSO) δ11.62(s,1H),10.04(s,1H),8.43(t,J=1.8Hz,1H),8.11(ddd,J=8.1,2.3,1.2Hz,1H),7.90(dd,J =6.0,3.4Hz,1H),7.78(dt,J=7.6,1.3Hz,1H),7.73-7.57(m,3H),7.02-6.94(m,2H),6.97-6.87(m,1H),4.30(s,4H).

[0129] (6) Synthesis of 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide

[0130] 619.4 mg (1.43 mmol, 1 eq.) of compound g was weighed and dissolved in 12 mL of a 1:1 DCM:MeOH mixture. 0.8 mL of glacial acetic acid and 103.2 μL (1.71 mmol, 1.2 eq.) of ethanolamine were added to the mixture, and the mixture was stirred. Finally, 448.58 mg (7.14 mmol, 5 eq.) of sodium cyanoborohydride was added. After 5 h, the solvent in the reaction system was removed under reduced pressure. The organic phases were extracted, combined, and dried. Column chromatography was used to purify and obtain 586.7 mg of compound h, namely 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-((((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide), with a yield of 65.6%. Compared with similar literature, this synthetic method significantly improves the synthetic efficiency.

[0131]

[0132] 1 H NMR (300MHz, DMSO) δ11.39(s,1H),7.95-7.84(m,2H),7.72(d,J=8.1Hz,1H),7.63(d,J=5.9Hz,2H),7.39(t,J=7.8Hz,1H),7.28(d, J=7.7Hz,1H),7.03-6.87(m,3H),4.78(s,1H),4.30(s,4H),3.87(s,2H),3.55(t,J=5.6Hz,2H),2.71(t,J=5.7Hz,2H).MS(ESI)for C 26 H 23 ClN4O5[M+H] + :calcd.508.14; found.507.32.

[0133] In this embodiment, the synthetic route for 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide is as follows: Figure 1 As shown.

[0134] Comparative Example 1

[0135] The difference from step (1) of Example 1 is that 1,4 eq of trimethylsilanopotassium was replaced with 4 eq of potassium acetate. All other operations were the same, the yield was 85.6%, the reaction time was increased from 0.5 h to 12 h, and thin-layer chromatography showed more byproducts.

[0136] Comparative Example 2

[0137] The solvent toluene in step (3) of Example 1 was replaced with trichloroacetic anhydride, and the rest of the operation was the same. The yield of compound e was 16%. Thin-layer chromatography showed that there were many byproducts and a large amount of trifluoroacetic anhydride was consumed, which was expensive.

[0138] Comparative Example 3

[0139] The volume of toluene solvent in step (3) of Example 1 was replaced with 2 mL per 100 mg of compound d instead of 5 mL, and all other operations were the same. The yield of the obtained compound was 46.5%, and thin-layer chromatography showed a large number of byproducts.

[0140] Comparative Example 4

[0141] The solvent in step (6) of Example 1 was replaced with a simple dichloromethane solution instead of a mixed solution of DCM:MeOH = 1:1. All other operations were the same. The yield of the compound was 19.6%. The system changed from clear and transparent to turbid. Thin-layer chromatography showed that there were many byproducts.

[0142] Test Example 1

[0143] The PD-1 / PD-L1 homogeneous time-resolved fluorescence (HRF) binding assay of 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-((((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide obtained in Example 1 was performed. Specifically, the ability of the compound to inhibit PD-1 / PD-L1 interaction was investigated using a Cisbio PD-1 / PD-L1 HTRF binding assay kit (64ICP01PEG and 64ICP01-PEH). In the HTRFPD-1 / PD-L1 binding assay, the Anti-Tag1 antibody labeled with Europium (HTRF donor) bound to the Tag1-PD-L1 protein, and the Anti-Tag2 antibody labeled with XL665 (HTRF receptor) bound to the Tag-PD-1 protein. When PD-L1 and PD-1 bind, the HTRF donor and HTRF receptor move closer together. Excitation of the HTRF donor triggers FRET (fluorescence resonance energy transfer) to the HTRF receptor, which then emits light at 665 nm. A stronger emission signal indicates a stronger PD-1 / PD-L1 interaction. Therefore, adding compound h to an ELISA plate blocks the PD-1 / PD-L1 interaction, leading to a weakened HTRF signal. Measuring the HTRF signal can characterize the compound's ability to inhibit PD-1 / PD-L1.

[0144] The HTRF test results of 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-((((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide obtained in Example 1 are as follows: Figure 2 As shown. The IC50 of the target compound of this invention, 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide. 50 With a value of 565 nM, it can be concluded that the compound described in this invention can inhibit the binding of programmed cell death receptor 1 / programmed cell death ligand (PD-1 / PD-L1).

[0145] Test Example 2

[0146] 1.39 mg of compound 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-((((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide was weighed and dissolved in 100 μL DMSO. The solution was diluted with 1.2 mL of culture medium to obtain a stock solution of 21.9 μL, resulting in a concentration of 100 μmol / L. Further serial dilutions were performed using culture medium. Mouse breast cancer cell line 4T-1, mouse colon cancer cell line MC38, human lung normal cell line BEAS-2b, human kidney normal cell line HEK-293T, and human liver normal cell line LO2 were selected and prepared into a complete culture medium using DMEM + 10% embryonic bovine serum + 1% penicillin-dextrose antibody at 25 cm⁻¹. 2 Cultured in culture flasks, the cells are passaged when the coverage reaches 70%. The cells are digested with trypsin for about 3 minutes. Based on the characteristics of tumor cells, they are passaged to the third generation before use.

[0147] After passage to the third generation, cells were digested and prepared into a cell suspension, which was then uniformly pipetted and counted. The cell suspension was diluted to 3 × 10⁻⁶. 4 Tumor cells (cells / mL, with the outermost ring sealed with PBS) were added to each well. 100 μL of cell suspension was added to each well and incubated at 37°C with 5% CO2 for 8 hours. Then, 25 μL of drug solution was added to each well. After drug addition, the 96-well plate was incubated for another 40 hours. After incubation, the 96-well plate was removed, and 10 μL of CCK8 was added to each well. The plate was incubated for 1 hour. After incubation, the absorbance at 450 nm was measured using a microplate reader. The data were processed to obtain cell viability at different concentrations.

[0148] Figure 3 The results show the antiproliferative activity of 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide against different cells. Figure 3 In the table, A shows the antiproliferative activity test results against the human lung normal cell line BEAS-2b, the human kidney normal cell line HEK-293T, and the human liver normal cell line LO2; B shows the antiproliferative activity test results against the mouse breast cancer cell line 4T-1 and the mouse colon cancer cell line MC38; and C shows the results of further testing against the mouse breast cancer cell line 4T-1, with further precise measurement of IC50. 50 curve.

[0149] As can be seen, for the murine breast cancer cell line 4T-1, the IC50 of the 1,2,4-oxadiazole compounds provided in this invention is [high / low]. 50The concentration is around 19.68 μmol / L; for the murine colon cancer cell line MC38, the IC50 of the 1,2,4-oxadiazole compound provided in this invention is approximately 19.68 μmol / L. 50 The concentration range is between 12.5 and 25 μmol / L; the IC50 of the 1,2,4-oxadiazole compounds provided in this invention is [not specified] for human lung normal cell line BEAS-2b, human kidney normal cell line HEK-293T, and human liver normal cell line LO2. 50 The concentration range of 25–50 μmol / L indicates that the 1,2,4-oxadiazole compounds provided by this invention have good safety and anti-proliferative activity.

[0150] Test Example 3

[0151] To evaluate the antitumor activity of compound 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide, its in vivo antitumor efficacy was assessed using the C57BL / 6j mouse MC38 model. After the tumor volume reached approximately 50 cubic millimeters, mice were treated with either the drug BMS202 (control) or intraperitoneal injection of compound 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide (20 mg / kg) once daily for 12 consecutive days. At the end of the experiment, mice were euthanized by cervical dislocation, and the tumors were removed and weighed.

[0152] Tumor growth inhibition results for different groups at different days after treatment are shown in the figure. Figure 4 ( Figure 4 J30 represents the compound 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide). Figure 4 In the figures, A shows images of tumors removed in each group after treatment; B shows changes in mouse body weight during treatment (no statistically significant differences were found between groups); C shows changes in tumor volume during treatment (no statistically significant difference was found, p < 0.0001, n = 6); D shows the weight of tumors removed in each group (no statistically significant differences were found, p < 0.01, *** p < 0.001, n = 6); E shows the weight of different organs in each group (no statistically significant differences were found between groups). All data are expressed as mean ± standard deviation.

[0153] It can be seen that the compound 3-(2-chloro-3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)phenyl)-N-(3-(((2-hydroxyethyl)amino)methyl)phenyl)-1,2,4-oxadiazole-5-carboxamide significantly inhibited tumor growth without causing weight loss or death during treatment. Compared with the untreated group (NC), the treated group showed no significant damage to conventional tissues. These results indicate that the effective dose of this compound is well-tolerated.

[0154] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a 1,2,4-oxadiazole biphenyl compound, characterized in that, Includes the following steps: In the presence of a palladium catalyst and a basic reagent, a compound having the structure shown in formula a undergoes a Suzuki coupling reaction with a compound having the structure shown in formula b to obtain a compound having the structure shown in formula c. A compound having the structure shown in formula c undergoes a nucleophilic addition reaction with an aqueous hydroxylamine solution to give a compound having the structure shown in formula d. A compound having the structure shown in formula d undergoes a cyclization reaction with trichloroacetic anhydride to give a compound having the structure shown in formula e. A compound having the structure shown in formula e undergoes a condensation reaction with 3-aminobenzyl alcohol to give a compound having the structure shown in formula f. Under the action of the Des Martin reagent, the compound having the structure shown in formula f is oxidized to the compound having the structure shown in formula g; Under the action of a reducing agent, a compound having the structure shown in formula g undergoes a reductive amination reaction with ethanolamine to obtain a 1,2,4-oxadiazole compound having the structure shown in formula h.

2. The preparation method according to claim 1, characterized in that, The alkaline reagent is potassium trimethylsilanolate; the molar ratio of the compound having the structure shown in formula a to the alkaline reagent is 1:1 to 5; The molar ratio of the compound having the structure shown in Formula a to the compound having the structure shown in Formula b is 1:1 to 5. The Suzuki coupling reaction was carried out at a temperature of 100–105 °C for 30–60 min.

3. The preparation method according to claim 1 or 2, characterized in that, The mass concentration of the hydroxylamine aqueous solution is 40-60%; The nucleophilic addition reaction is carried out at a temperature of 90–100 °C for 6–10 h.

4. The preparation method according to claim 1, characterized in that, The molar ratio of the compound having the structure shown in formula d to trichloroacetic anhydride is 1:1 to 5; The reaction solvent for the cyclization reaction is toluene; the mass ratio of the compound having the structure shown in formula d to the volume of toluene is 100 mg: 2-20 mL; The cyclization reaction is carried out at a temperature of 100–110°C for 2–3 hours.

5. The preparation method according to claim 1 or 4, characterized in that, The molar ratio of the compound having the structure shown in Formula e to 3-aminobenzyl alcohol is 1:4 to 6; The condensation reaction is carried out at a temperature of 130–150°C for 8–12 hours.

6. The preparation method according to claim 1, characterized in that, The molar ratio of the compound having the structure shown in Formula f to the Des Martin reagent is 1:1 to 5; The oxidation temperature is 0–20°C, and the time is 10–120 min.

7. The preparation method according to claim 1, characterized in that, The reducing agent is sodium cyanoborohydride, and the molar ratio of the compound having the structure shown in formula g to the reducing agent is 1:1 to 5; The molar ratio of the compound having the structure shown in formula g to ethanolamine is 1:1 to 5; The reaction solvent for the reductive amination reaction is a mixture of dichloromethane and methanol in a volume ratio of 1:1, and the mixture contains 6 wt% acetic acid. The reductive amination reaction is carried out at a temperature of 20–50°C for a time of 4–8 hours.

8. A 1,2,4-oxadiazole biphenyl compound, characterized in that, It has the structure shown in equation h:

9. The use of the 1,2,4-oxadiazole biphenyl compound prepared by the preparation method according to any one of claims 1 to 7 or the 1,2,4-oxadiazole biphenyl compound according to claim 8 in the preparation of PD-L1 small molecule inhibitors.

10. The use of the 1,2,4-oxadiazole biphenyl compound prepared by the preparation method according to any one of claims 1 to 7 or the 1,2,4-oxadiazole biphenyl compound according to claim 8 in the preparation of antitumor drugs.

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