WO2023165165A1 - Benzoimidazopyrazine-3-carboxamide compound targeting a2a and anti-tumor immunological function thereof - Google Patents

Abstract

The present invention relates to the technical field of pharmaceutics, and particularly relates to a benzoimidazopyrazine-3-carboxamide compound targeting A2A and an anti-tumor immunological function thereof. The present invention provides a subtype-selective adenosine A2A receptor antagonist, i.e., a micromolecular compound 1-amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide, having a structure represented by formula (I). According to the present invention, the micromolecular compound can specifically target A2AR, inhibit cAMP accumulation, promote cytokine release in immune cells, enhance the killing of tumor cells by immune cells in co-culture, and enhance the efficacy of tumor immunotherapies. The present invention exhibits a significant anti-tumor immunological enhancement effect at the molecular level and the cellular level and in a mouse cancer model, and is prospective in application to tumor immunotherapies.

Description

target A 2A benzimidazolopyrazine-3-carboxamide and its tumor immune function technical field

The invention belongs to the technical field of medicine, and in particular relates to benzimidazolopyrazine-3-carboxamide targeting _A2A and its tumor immune function.

Background technique

Cancer immunotherapy based on immune checkpoint inhibition or adoptive cell therapy has revolutionized the way cancer is treated, yet a large proportion of patients do not benefit from such treatments. Multiple redundant and non-redundant immunosuppressive pathways active in the tumor microenvironment (TME) may partly explain the low response rate to current immune checkpoint therapies. A key mechanism of cancer immune evasion is the production of high levels of immunosuppressive adenosine within the TME. A large amount of adenosine in the tumor microenvironment can inhibit the proliferation and maturation of immune cells such as T cells and NK cells and the production of immune cytokines, thereby causing immune damage to the body and promoting the growth of tumor cells. Not only that, adenosine also has a direct proliferation effect on tumor cells. Moreover, targeted blockade of the main effectors of this class of adenosine receptors has now been shown to promote anti-tumor immunity in various clinical cancer models, thereby enhancing the efficacy of standard cancer therapy and other immune checkpoint blockade therapies.

Adenosine receptors belong to G protein-coupled receptors (GPCR), which can be subdivided into four subtypes, A _2A , A _2B , A ₁ and A ₃ . Under physiological conditions, the levels of extracellular ATP (eATP) and eADO are both stay in the nanomolar range. However, after cell death or cell stress caused by hypoxia, nutrient starvation or inflammation, ATP will be rapidly released into the extracellular space to reach a micromolar concentration. Under the regulation of the factor HIF-1, the two enzymes CD39 and CD73 will convert extracellular adenosine triphosphate ATP into AMP and then into adenosine, resulting in a large amount of extracellular adenosine accumulated in the TEM. However, the high concentration of extracellular adenosine mainly interacts with A _2AR on the surface of immune cell membrane, and A _2AR promotes the increase of adenylate cyclase (AC) activity and increases intracellular cyclic phosphate by binding to Gs/Golf protein. The content of adenosine (cAMP), which in turn activates many ion channels and receptors by activating protein kinase A (PKA), transmits immunosuppressive signals to immune cells, thereby inhibiting T cells, NK cells, macrophages and dendrites The proliferation, maturation, infiltration of immune cells such as DC cells and the production of immune cytokines eventually lead to immune injury of the body and immune escape of tumor cells. Therefore, blocking the signal transmission mediated by adenosine _A2A receptors by designing _A2A receptor antagonist drug molecules, thereby preventing the immune loss of the body and inhibiting the growth of tumor cells, is currently in the clinical research stage. Novel tumor immunotherapy strategies.

technical problem

So far, there is no adenosine _A2A receptor antagonist drug molecule that has been officially approved for marketing, and only seven small molecule _A2A receptor antagonists are in the clinical research stage. They are AZD4635 from AstraZeneca and AZD4635 from Corvus CPI-444, PBF-509 from Novartis, SHR5126 from Hengrui Medicine, CS3005 from CStone, EXS21546 from Exscientia, and AB928 developed by Arcus Biosciences that simultaneously target _A2A and _A2B . These 7 candidate drug molecules are being clinically tested for common tumor diseases such as melanoma, lung squamous cell carcinoma, colon cancer, pancreatic cancer, kidney cancer, bladder cancer, ovarian cancer, and prostate cancer. Their effects on human specific tumors The specific curative effect and toxicity data have not yet been published, and it is unknown whether they will be successfully marketed in the future. Therefore, the development of new adenosine _A2A receptor antagonist drugs has important scientific research significance and clinical medical application value.

technical solution

In order to overcome the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a subtype-selective adenosine A _2A receptor antagonist, which is 1-amino-N-(pyridin-2-ylmethyl)benzene [4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecular compound, the present invention finds 1-amino-N-(pyridin-2-ylmethyl)benzo[ The 4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecular compound has a good effect of enhancing tumor immunity and is widely used in the preparation of tumor immunotherapy drugs.

For realizing the above-mentioned purpose of the invention, the technical scheme that the invention adopts is:

A subtype-selective adenosine _A2A receptor antagonist which is 1-amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1,2-a] Pyrazine-3-carboxamide small molecular compound (its molecular formula is C ₁₇ H ₁₄ N ₆ O, molecular weight is 318.34), the structure of the antagonist is shown in formula (I):

The present invention also provides the use of the subtype-selective adenosine _A2A receptor antagonist in the preparation of drugs targeting adenosine _A2A receptors.

It is found through research that the 1-amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecular compound of the present invention can be Specifically targeting A _2A R.

The present invention also provides the application of the subtype-selective adenosine _A2A receptor antagonist in the preparation of tumor immunotherapy drugs.

The research of the present invention found that the 1-amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecular compound of specific structure The invention has a good effect of enhancing tumor immunity, and has wide application in preparing tumor immunotherapy drugs.

Preferably, the tumor includes but not limited to breast cancer.

Preferably, the immunotherapy is to inhibit the accumulation of cAMP.

Preferably, the immunotherapy is to promote immune cells to secrete cytokines. Further, the cytokine is IL-2.

Preferably, the immunotherapy is to enhance the killing effect of immune cells on tumors.

It is found through research that the 1-amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecular compound of the present invention can be Specifically targeting _A2A R, inhibiting the accumulation of cAMP, promoting the release of immune cell cytokines, enhancing the killing of immune cells on tumor cells in co-culture, and enhancing the effect of tumor immunotherapy. At present, the response rate of tumor immunotherapy is low, and immune escape occurs frequently. The present invention provides an important reference for the development of new drugs targeting _A2AR enhanced immunotherapy, and provides a certain treatment strategy for reducing immune escape, and has a good application prospect.

The invention also provides the use of the subtype-selective adenosine _A2A receptor antagonist in the preparation of drugs for inhibiting tumor cell growth.

Preferably, the tumor cells include but not limited to breast cancer cells.

Through the experiment of Balb/c mouse subcutaneous cell transplantation tumor model, it was found that 1-amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3- Formamide small molecule compounds can inhibit the growth of breast cancer cells.

The present invention also provides a drug targeting adenosine A _2A receptors or a drug for tumor immunotherapy or a drug for inhibiting tumor cell growth, the drug uses the subtype-selective adenosine A _2A receptor Antagonist as the main active ingredient.

Preferably, in the above application and drug regimen, the subtype-selective adenosine _A2A receptor antagonist also includes 1-amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazole A pharmaceutically acceptable salt or solvate of a[1,2-a]pyrazine-3-carboxamide small molecule compound.

The term "acceptable salt" refers to the acidic and/or basic salts formed by the above-mentioned compounds or their stereoisomers with inorganic and/or organic acids and bases, including zwitterionic salts (inner salts), and quaternary Ammonium salts, such as alkylammonium salts. These salts may be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the above-mentioned compound, or its stereoisomer, with a certain amount of acid or base as appropriate (for example, equivalent). These salts may form precipitates in solution and be collected by filtration, or may be recovered after evaporation of the solvent, or may be obtained by freeze-drying after reaction in an aqueous medium.

More preferably, the pharmaceutically acceptable salt is a pharmaceutically acceptable inorganic or organic salt.

Further, pharmaceutically acceptable salts include but are not limited to: sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid Phosphate, Isonicotinate, Lactate, Salicylate, Acid Citrate, Tartrate, Oleate, Tannate, Pantothenate, Bitartrate, Ascorbate, Succinate, Horse Tonate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate (methanesulfonate salt), ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate; or ammonium salts (such as primary amine salts, secondary amine salts, tertiary amine salts, quaternary ammonium salts), Metal salts (eg sodium salts, potassium salts, calcium salts, magnesium salts, manganese salts, iron salts, zinc salts, copper salts, lithium salts, aluminum salts).

Preferably, in the above pharmaceutical regimen, one or more pharmaceutically acceptable carriers, diluents or excipients are also included.

The term "pharmaceutically acceptable" means that a certain carrier, diluent or excipient, and/or the salt formed is usually chemically or physically compatible with other ingredients constituting a pharmaceutical dosage form, and physiologically compatible with compatible with the receptor.

Preferably, in the above drug regimen, the dosage forms of the drug include but are not limited to injections, capsules, tablets, pills, and granules.

More preferably, preferably, in the above drug regimen, the dosage form of the drug is an injection.

Beneficial effect

Compared with the prior art, the beneficial effects of the present invention are: the present invention discloses a subtype-selective adenosine _A2A receptor antagonist, which is 1-amino-N-(pyridin-2-ylmethyl base) benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecule compound, the molecular formula is C ₁₇ H ₁₄ N ₆ O, and the molecular weight is 318.34. The present invention has found through research that the 1-amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecule The compound can specifically target _A2AR , inhibit the accumulation of cAMP, promote the release of immune cell cytokines, enhance the killing of tumor cells by immune cells in co-culture, and enhance the effect of tumor immunotherapy. It shows obvious tumor immune enhancing effect at the molecular level, cellular level and cancer mouse model, is expected to be applied to tumor immunotherapy, and has a wide range of applications in the preparation of tumor immunotherapy drugs.

Description of drawings

Figure 1 shows the targeting properties of LDH-E-4 to A _2A R and the results of cell function tests (A is the IC ₅₀ curve of LDH-E-4 binding to A _2A and A ₁ receptors; B is LDH-E-4 IC ₅₀ curve for inhibiting cAMP accumulation; C is the EC ₅₀ curve for LDH-E-4 to promote IL-2 release);

Figure 2 is the effect of LDH-E-4 on enhancing immune cells to kill tumor cells in vivo and in vitro (A is that LDH-E-4 enhances the killing ability of PBMCs to MDA-MB-231 breast cancer cells; B is the effect on mice subcutaneous breast cancer cells Tumor volume changes during administration in the xenograft tumor model; C is the quality map of the exfoliated breast cancer subcutaneous tumor after 26 days of administration; D is the appearance map of the exfoliated breast cancer subcutaneous tumor after 26 days of administration).

Embodiments of the present invention

Specific embodiments of the present invention will be further described below. It should be noted here that the descriptions of these embodiments are used to help understand the present invention, but are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not constitute a conflict with each other.

The experimental methods in the following examples, if no special instructions, are conventional methods, and the test materials used in the following examples, if no special instructions, all can be purchased through conventional commercial channels.

Example 1 1-Amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecule compound (LDH-E- 4) Preparation

The structure of LDH-E-4 is shown below:

The preparation of this compound comprises the steps:

(1) Preparation of compound shown in formula 3:

According to the above reaction formula, the compound shown in Formula 1 (1,2-phenylenediamine, 3.2 g, 30 mmol) and 50 mL of acetic acid (AcOH) were added to the reaction flask at room temperature, and the starting materials were slowly added after cooling in an ice bath. That is, the compound shown in Formula 2 (methyl 2,2,2-trichloroimidoacetate, 4 mL, 30 mmol) was stirred at room temperature for 2 hours after the addition, and TLC showed that the reaction was complete. After the reaction was completed, the reactant was filtered, and the obtained filter cake was washed with water (3×25 mL) and vacuum-dried to obtain the compound represented by formula 3 (2-trichloromethyl-benzopyrimidine) (7.1 g, 93%).

(2) Preparation of compound shown in formula 4:

According to the above reaction formula, the compound shown in Formula 3 (2-trichloromethylbenzopyrimidine, 5.9g, 25mmol) was cooled to 0°C, and then ammonia in 1,4-dioxane solution was added at 0°C ( 0.40M, 125mL) and sealed, stirred at room temperature for 2 hours, concentrated under reduced pressure after TLC showed that the reaction was complete, and passed through the column to obtain the compound shown in Formula 4 (2-cyanobenzopyrimidine, 2.7g, 76%).

(3) Preparation of compound shown in formula 6:

According to the above reaction formula, acetonitrile MeCN (40mL), N,N-diisopropylethylamine DIPEA (4.4g, 34mmol), the compound shown in formula 4 (2-cyanobenzopyrimidine, 2.6 g, 17mmol) and the compound shown in Formula 5 (3-bromo-2-oxopropyl acetate, 3.3g, 17mmol) were added to the reaction flask, stirred at -15°C for 2 hours, after the reaction was shown by TLC Diluted with water, then extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and passed through the column to obtain the compound (1-(2-(4-methoxyphenyl)-2-oxaethyl )-1 Hydrogen-benzopyrimidine-2-cyano) (3.5 g, 80%). The spectrum information of the product is as follows:

¹ HNMR (400MHz, Chloroform-d) δ7.89–7.86(m,1H),7.50–7.40(m,2H),7.30–7.28(m,1H),5.28(s,2H),4.81(s,2H ), 2.22(s,3H).

(4) Preparation of compound shown in formula 7:

According to the above reaction formula, the compound described in formula 6 (3-(2-cyano-1H-benzo[d]imidazol-1-yl)-2-oxopropyl acetate, 3.3g, 13mmol), Ammonium acetate NH ₄ OAc (5.1g, 66mmol) and acetic acid HOAc (10mL) were added to a sealed tube, stirred at 95°C for 1 hour, after TLC showed that the reaction was complete, a saturated sodium bicarbonate solution was added to neutralize the reaction until no more bubbles were generated, and used Dichloromethane extraction (3 × 100mL), the organic phase was collected, the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and passed through the column to obtain the compound (1-aminobenzo[4,5]imidazo[ 1,2-a]pyrazin-3-yl)methyl acetate) (2.8 g, 85%). The spectrum information of the product is as follows:

¹ H NMR (500MHz, Chloroform-d) δ7.96–7.86(m,3H),7.56–7.46(m,3H),6.06(s,2H),5.10(s,2H),2.16(s,3H) .

(5) Preparation of compound shown in formula 8:

According to the above reaction formula, the compound shown in formula 7 (1-aminobenzo[4,5]imidazo[1,2-a]pyrazin-3-yl)methyl acetate, 2.8, 11mmol), 4-di Methylaminopyridine DMAP (134mg, 1.1mmol) and triethylamine TEA (4.5g, 44mmol) were dissolved in 50mL tetrahydrofuran THF, and cooled to 0°C, then di-tert-butyl dicarbonate (Boc ) ₂ O (6.0g, 27.5mmol), stirred at room temperature for 4 hours, added water 100mL after TLC showed that the reaction was complete, extracted with dichloromethane (3×100mL), combined the organic phase, washed the organic phase with 100mL saturated brine, sulfuric acid Drying over sodium, concentrating under reduced pressure, and passing through the column, the compound ((1-(di(tert-butoxycarbonyl)amino)benzo[4,5]imidazo[1,2-a]pyrazine- 3-yl) methyl acetate, 4.2 g, 84%). The spectrum information of the product is as follows:

¹ HNMR (400MHz, Chloroform-d) 8.46(s, 1H), 8.07(d, J=8.3Hz, 1H), 7.97(d, J=8.3Hz, 1H), 7.64(t, J=7.7Hz, 1H ), 7.53 (t, J=7.7Hz, 1H), 5.29 (s, 2H), 2.16 (3H, s), 1.41 (s, 18H).

(6) Preparation of compound shown in formula 9:

According to the above reaction formula, the compound shown in formula 8 ((1-(di(tert-butoxycarbonyl)amino)benzo[4,5]imidazo[1,2-a]pyrazin-3-yl)acetic acid Methyl ester, 4.1g, 9mmol) was dissolved in 50mL methanol MeOH. After cooling to 0°C, potassium carbonate (3.7g, 27mmol) was slowly added, stirred at room temperature for 4h, and the reaction was completed by TLC. After the reaction, 100mL of water was added, and dichloro Methane extraction (3 × 100mL), combined organic phase, organic phase was washed with saturated brine (100mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and passed through the column to obtain the compound shown in formula 9 ((3-(hydroxymethyl ) tert-butyl benzo[4,5]imidazo[1,2-a]pyrazin-1-yl)carbamate, 67%, 1.9 g). The spectrum information of the product is as follows:

¹ H NMR (400MHz, Chloroform-d) δ8.56(s, 1H), 8.12(s, 1H), 7.96(d, J=8.3Hz, 1H), 7.89(d, J=8.3Hz, 1H), 7.61–7.57(m,1H), 7.50–7.60(m,1H), 4.85(s,2H), 1.56(s,9H).

(7) Preparation of compound shown in formula 10:

According to the above reaction formula, the compound shown in formula 9 ((3-(hydroxymethyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-yl) tert-butyl carbamate, 1.9 g, 6mmol) was dissolved in 50mL of dichloromethane DCM, and after cooling to 0°C, slowly added Dess-Martin oxidant——Dess-Martin periodinane DMP (3.6g, 8.4mmol), stirred at room temperature for 4h, and the reaction was shown by TLC After completion, filter, add saturated aqueous sodium bicarbonate until no bubbles are generated, then extract with dichloromethane (3×100mL), combine the organic phases, wash the organic phases with saturated brine (100mL), dry over anhydrous sodium sulfate, reduce Concentrate under reduced pressure and pass through the column to obtain the compound shown in formula 10 ((3-formylbenzo[4,5]imidazo[1,2-a]pyrazin-1-yl) tert-butyl carbamate, 1.4g, 75%). The spectrum information of the product is as follows:

¹ H NMR (500MHz, Chloroform-d) δ10.17(s,1H),8.82(s,1H),8.03–8.00(m,2H),7.70–7.67(m,1H),7.61–7.57(m, 1H), 1.61(s, 9H).

(8) Preparation of compound shown in formula 11:

According to the above reaction formula, the compound (3-formylbenzo[4,5]imidazo[1,2-a]pyrazin-1-yl) tert-butyl carbamate shown in formula 10, 1.4g, 4.5mmol ), sodium dihydrogen phosphate (2.2g, 18mmol), and isopentene 2-methylbut-2-ene (1.6g, 22mmol) were dissolved in a mixed solution of 40mL tetrahydrofuran THF and 20mL water, and slowly added sub Sodium chlorate (1.6g, 18mmol), stirred at 0°C for 3h, after the reaction was shown by TLC, an aqueous solution of sodium thiosulfate (3.6g, 22mmol) was added at 0°C to quench the reaction, followed by extraction with dichloromethane (3× 100mL), the organic phases were combined, the organic phase was washed with saturated brine (100mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Carbonyl)amino)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxylic acid, 886 mg, 60%). The spectrum information of the product is as follows:

¹ H NMR (400MHz,DMSO-d ₆ )δ9.46(s,1H),8.56(s,1H),7.98-7.96(m,1H),7.65–7.54(m,2H),1.50(s,9H ).

(9) Preparation of compound shown in formula 13:

According to the above reaction formula, the compound shown in Formula 11 (1-((tert-butoxycarbonyl)amino)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxylic acid, 67mg, 0.2mmol), compound shown in formula 12 (2-aminomethylpyridine, 32mg, 0.3mmol), 1-hydroxybenzotriazole HOBt (41mg, 0.3mmol) were dissolved in 2mL dichloromethane DCM, cooled to 0 After ℃, dicyclohexylcarbodiimide DCC (62mg, 0.3mmol) was slowly added, stirred at room temperature for 16 hours, after TLC showed that the reaction was complete, it was filtered with silica gel, and washed with a mixture of dichloromethane:methanol=50:1 to obtain The filtrate was concentrated under reduced pressure to obtain the compound shown in formula 13 ((3-((pyridin-2-ylmethyl)carbamoyl)benzo[4,5]imidazo[1,2-a]pyrazine-1 -yl) tert-butyl carbamate), and used directly in the next reaction.

(10) Preparation of target compound 14a (LDH-E-4):

According to the above reaction formula, the compound shown in formula 13 (3-((pyridin-2-ylmethyl)carbamoyl)benzo[4,5]imidazo[1,2-a]pyrazin-1-yl ) tert-butyl carbamate) was dissolved in 2 mL of dichloromethane DCM, and after cooling to 0°C, 0.2 mL of trifluoroacetic acid TFA was slowly added, stirred at room temperature for 4 hours, and after the reaction was shown by TLC, saturated aqueous sodium bicarbonate solution was added until no longer Bubbles were generated, and extracted with dichloromethane (3×50mL), the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and passed through the column to obtain the target compound LDH-E- 4 (1-Amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide, 45 mg, 70%). The spectrum information of the product is as follows:

¹ H NMR (500MHz, DMSO-d ₆ ) δ8.95(s, 1H), 8.81(t, J=6.0Hz, 1H), 8.56(d, J=4.8Hz, 1H), 8.45(d, J= 8.2Hz, 1H), 7.92(d, J=8.2Hz, 1H), 7.78(t, J=7.7Hz, 1H), 7.56–7.46(m, 4H), 7.39(d, J=7.8Hz, 1H) , 7.30 (t, J=6.2Hz, 1H), 4.68 (d, J=5.8Hz, 2H). ¹³ C NMR (126MHz, DMSO-d ₆ ) δ164.2, 158.2, 149.4, 143.6, 137.3, 136.3, 130.6, 130.2, 126.4, 123.6, 122.8, 121.9, 120.7, 113.6, 111.5, 44.6.

Example 2 Study on the characteristics of LDH-E-4 specifically targeting A _2A R

(1) Cell culture

The A2A-HEK293 and A1-HEK293 (HEK293 cells used in this experiment were purchased from ATCC, and the construction of the overexpression cell line refers to "Borodovsky, A., et al., Small molecule AZD4635 inhibitor of A2AR signaling rescues immune cell function including CD103+ dendritic cells enhancing anti-tumor immunity.Journal for ImmunoTherapy of Cancer, 2020.8(2):p.e000417."), MBA-MD-231 (purchased from ATCC) cells were cultured in 10% fetal bovine serum and 1% double antibody In DMEM medium (penicillin and streptomycin), 4T1 and PBMC cells were cultured in RPIM-1640 medium containing 10% fetal bovine serum and 1% double antibody (penicillin and streptomycin). Culture in a cell incubator at 37°C, 5% CO ₂ .

(2) Affinity test of compounds for A _2A R and A ₁ R

1) Preparation of NECA

① Preparation of NECA mother solution: Weigh 0.00157g NECA, dissolve in 250μL sterile water, aliquot into 30μL/tube, and store at -20°C.

②Preparation of NECA working solution: Take 10 μL NECA mother solution and add 990 μL ddH ₂ O to make 1 mL.

2) Configuration of [ ³ H]-ZM 241385 working fluid and [ ³ H]-DPCPX working fluid

①[ ³ H]-ZM 241385 and [ ³ H]-DPCPX (purchased from China Tongfu Co., Ltd.) mother solution: Take the original solution and aliquot it, 5 μL each (the concentration of the original solution is 20 μM), and store at -20°C.

②[ ³ H]-ZM 241385 and [ ³ H]-DPCPX working solution: 3nM, [ ³ H]-ZM 241385 and [ ³ H]-DPCPX final concentration: 1nM.

3) Cell collection, lysis, membrane protein extraction

① Passage A _2A -HEK293 cells into a 10×10 cm culture dish, culture them at 37°C and 5% CO ₂ to a density of 90%, and use them for experiments. Take out the Petri dish, discard the culture medium, and rinse twice with 3 mL of PBS.

②Add 1mL LPBS to the Petri dish and let it stand for 3min.

③ Rinse the cells, then transfer to a 1.5mL centrifuge tube, and centrifuge at 3500rpm, 4°C for 8min.

④ Pour off the supernatant, add 1mL lysis buffer and PMSF (Lysis:PMSF=100:1), and incubate at 4°C for 30min.

⑤ Pass the needle (1mL needle) under ice bath 15 times.

⑥ Add two-thirds of the reaction solution lysis buffer (about 3mL) to the centrifuge tube, and centrifuge at high speed (15000rpm, 4°C) for 20min.

⑦ Pour off the supernatant, add reaction buffer 1mL, and pass the needle (1mL needle) under ice bath for 15 times. Add reaction buffer (about 3mL) to two thirds of the volume in the centrifuge tube, and centrifuge at high speed (15000rpm, 4°C) for 20min.

⑧ Pour off the supernatant, dissolve the extracted protein in 500 μL reaction buffer, pass through the needle about 10 times to get the membrane protein solution. The protein concentration was determined using BCA reagent, and stored in a -80°C refrigerator after aliquoting for radioligand binding experiments.

4) Radioligand binding experiment

① The sample addition system is shown in Table 1:

Table 1 Sample loading system for radioligand binding experiments

② Membrane protein solution (60 μg/tube, containing ADA 10 μg/mL), radioligand 1nM [ ³ H]-ZM241385, 1nM [ ³ H]-DPCPX were added to each tube, NECA with a final concentration of 10 Uμ was added to the non-specific tube, and the compound (Example 1) 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 M7 different concentrations of compounds were added to the tubes, shaken and mixed, and incubated at 37°C After 30 min, the reaction was terminated in a water bath, and the free ligand and the bound ligand were separated by vacuum filtration using GF/C glass fiber filter paper, and washed three times with pre-cooled 50 mM Tris-HCl, about 1 mL each time. Remove the film, place it upside down on a tray, and bake for 3 minutes until dry. Put the dried small disc filter membranes into scintillation tubes in sequence, and add 2mL of scintillation fluid. [ ³ H] counts were performed in a liquid scintillation counter. Three replicate tubes were made for each junction point, and the average value was taken.

5) Data processing and statistical methods

Compound inhibition rate (I%)=(total binding tube cpm-compound cpm)/(total binding tube cpm-non-specific binding tube cpm)×100%. GraphPad Prism software was used to conduct statistical analysis of the experimental results and make graphs. And Ki=IC ₅₀ /(1+(L/Kd)), all the data are expressed as mean±standard error, one-way ANOVA test is used for comparison between groups, p<0.05 is considered statistically significant. where Ki is the affinity constant of the competing ligand, _IC50 is the concentration of compound that displaces 50% of radioligand binding, [L] is the free concentration of radioligand, and Kd is the dissociation constant of radioligand.

(3) Experimental results

Table 2 Affinity of compounds with A _2A R and A ₁ R

The Ki of the test compound for A _2AR can test its binding ability to A _2AR , and then the selectivity can be tested by testing the Ki of the compound and the A1 receptor. As shown in Table 2 and the experimental results in Figure 1(A), the Ki of this compound for A _2AR is 155.4±1.1pM, and it has extremely high targeting for A _2AR , and the Ki for A _1R is 10.58±1pM. 0.7nM, and the affinity to A _2AR is 68 times that of A _1R , with extremely high selectivity.

Example 3 Study on the characteristics of LDH-E-4 inhibiting cAMP accumulation

(1) Compound and buffer configuration

Assay buffer containing 1 x HBSS (Sigma), 0.1% BSA (Perkin Elmer), 20 mM HEPES (Gibco) and 100 nM IBMX (Sigma) was prepared. Use Simulation buffer to configure 8× test compound stock solution (10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 M) and 8× CGS21680 stock solution (50 nM). Prepare 20× cAMP-d2 and 20× anti-cAMP-Eu3+ detection reagent solutions using Lysis & Detection Buffer.

(2) HTRF method to test intracellular cAMP content

HEK293-A _2A cells were seeded in 384-well plates containing 38,000 cells per well in 15 μL of assay buffer. 2.5 μL of the test compound solution was added to the designated wells of the above-mentioned 384-well plate and incubated at 37°C for 10 minutes. Then, 2.5 μL of CGS21680 stock solution was added to the 384-well plate, and incubated at 37° C. for another 30 minutes (the final volume of the reaction system was 20 μL). Finally, 10 μL of cAMP-d2 and 10 μL of anti-cAMP-Eu3+ detection reagent were added to each well of the well plate, and incubated at room temperature for 1 h. Data were collected on a microplate reader at wavelengths of 665 nm and 620 nm.

(3) Experimental results

Table 3 IC ₅₀ of Compounds Inhibiting Accumulation of cAMP

The combination of adenosine and _A2AR can cause the accumulation of intracellular cAMP, which in turn activates a series of downstream pathways, and finally leads to immunosuppression. As shown in the experimental results in Table 3 and Figure 1(B), the IC ₅₀ of this compound for inhibiting cAMP accumulation induced by CGS21680 (50nM) was 97.2±4.4nM, indicating that it has a better effect of inhibiting cAMP accumulation.

Example 4 LDH-E-4 promotes PBMC to secrete cytokines

(1) PBMC separation

1) Pour 20mL of human peripheral blood (from volunteers of the School of Pharmacy, Sun Yat-Sen University, and the blood was drawn by the East Campus Hospital of Sun Yat-Sen University) into 40mL of sterile PBS buffer for dilution;

2) Take a 50mL centrifuge tube, add 10mL Ficoll solution, and carefully add 20Ml of diluted peripheral blood to the upper layer with a dropper;

3) Place the centrifuge tube in a horizontal centrifuge, and centrifuge at 300g density gradient for 20min at 4°C;

4) After centrifugation, the tube is divided into four layers, the upper layer is plasma and PBS, the lower layer is red blood cells, and the floating floc layer in the middle is the target mononuclear cells (PBMC).

5) Use a dropper to absorb the flocculent mononuclear cells floating in the middle, place them in a new 50mL centrifuge tube, add 3 times the volume of RPMI-1640, centrifuge at 1500rpm for 10min, and discard the supernatant to obtain the PBMC cell pellet.

(2) The compound promotes the release of IL-2 from PBMC

1) Inoculate the isolated PBMC cells into a 96-well plate with 20,000 cells per well, and incubate overnight at 37°C and 5% CO ₂ ;

2) Add LDH-E-4 (10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 , 10 ^-8 , 10 ^-9 , 10 ^-10 M) to the 1640 medium in the well plate for pre-incubation for 1 h;

3) After 1 hour, add NECA (1 μM) and incubate for 1 hour;

4) After 1 hour, add CD3 and CD28 (400ng/mL) to activate for 24 hours;

5) After 24 hours, the supernatant was collected, and the IL-2 content in the culture medium was tested by Elisa.

(4) Experimental results

Table 4 EC ₅₀ of compounds promoting IL-2 release

The combination of adenosine and A _2A R can cause immunosuppression, leading to a decrease in the secretion of cytokines by immune cells and weakening the function of immune cells. As shown in Table 4 and the experimental results in Figure 1(C), the EC ₅₀ of this compound against the reduction of IL-2 release caused by NECA (1 μM) is 342.8±3.6nM, indicating that it has a good effect on promoting immune cells to secrete cytokines Effect.

Example 5 The effect of LDH-E-4 on increasing tumor killing effect on immune cells in co-culture

(1) The separation and activation of PBMCs are the same as in Example 4;

(2) Co-culture of PBMC and breast cancer cells

1) Spread MDA-MB-231 breast cancer cells into a 96-well plate (20,000/well) and culture overnight;

2) Add overnight activated PBMCs (100,000/well) to the wells pre-laid with MDA-MB-231;

3) Add LDH-E-4 (1 μM) to the 1640 medium for pre-incubation for 1 h;

4) After 1 hour, add NECA (1 μM) for co-culture and incubate for 24 hours;

5) Detect the content of lactate dehydrogenase (LDH) in the medium. The destruction of the cell membrane structure caused by apoptosis or necrosis will cause the LDH in the cytoplasm to be released into the medium, and the relative amount of LDH in the medium is detected. It can represent the relative death of MDA-MB-231 cells caused by PBMC.

(3) Experimental results

A _2A R agonist NECA can inhibit the killing effect of PBMC on MDA-MB-231 breast cancer cells, as shown in the experimental results in Figure 2(A), LDH-E-4 can restore the killing effect of PBMC on MDA-MB-231 breast cancer cells cell killing ability.

Example 6 Study on Inhibition of Tumor Growth by LDH-E-4 in Vivo Model

(1) Effect of LDH-E-4 on mouse subcutaneous breast cancer cell xenograft model

1) Take breast cancer cells in the logarithmic growth phase (MDA-MB-231 breast cancer cells), digest and count, mix pre-cooled PBS and Matrigel at a ratio of 1:1, and resuspend the cells to obtain a concentration of 3×10 ⁵ cells/100 μL of cell suspension, placed on ice. Inject 100 μL of cell suspension into the subcutaneous sites on both sides of the abdomen and back of 4-5 week-old Balb/c mice;

2) When the subcutaneous tumor volume is about 50mm ³ , they are randomly divided into 4 groups. Groups and administration settings are as follows: control group, placebo every day (solvent used for instant medicine: 15% castor oil+85% sterilized PBS); LDH-E-4 (ip) group, administration every day, intraperitoneal injection, The dosage is 15mg/kg; the LDH-E-4(po) group is administered every day, orally, and the dosage is 15mg/kg;

3) Within 26 days of continuous administration, the body weight and tumor size of the mice were measured every day, and the body weight growth curve of the mice was drawn;

4) After 26 days of administration, the mice were sacrificed and dissected, and the subcutaneous tumors were peeled off and weighed.

(2) Experimental results

The experimental results are shown in Figure 2 (B-D), and Figure (2B) is a diagram of the tumor volume change during administration in the mouse subcutaneous breast cancer cell xenograft model, LDH-E-4 oral (po) and intraperitoneal injection group ( ip) can inhibit the tumor growth of normal immunized mice; LDH-E-4 intraperitoneal injection group can more significantly inhibit the tumor growth of normal immunized mice than LDH-E-4 oral group, shows that LDH-E-4 intraperitoneal injection It has a better effect; Figure (2C) shows that LDH-E-4 oral and intraperitoneal injection can significantly reduce the weight of breast cancer tumors, wherein the tumor inhibition rate of the LDH-E-4 oral group is about 43%, LDH-E The tumor inhibition rate of -4 intraperitoneal injection group is about 66%; Figure (2D) is the appearance of breast cancer subcutaneous tumor peeled off after 26 days of administration, indicating that LDH-E-4 oral (po) and intraperitoneal injection group (ip) All can inhibit the tumor growth of normal immunized mice, and the inhibitory effect of the intraperitoneal injection group (ip) is better.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and modifications to these embodiments still fall within the protection scope of the present invention.

Claims (10)

1. A subtype selective adenosine _A2A receptor antagonist, characterized in that the antagonist is 1-amino-N-(pyridin-2-ylmethyl)benzo[4,5]imidazo[1, 2-a] pyrazine-3-carboxamide small molecular compound, the structure of the antagonist is shown in formula (I):

   
2. Use of the subtype-selective adenosine _A2A receptor antagonist of claim 1 in the preparation of drugs targeting adenosine _A2A receptors.
3. The use of the subtype-selective adenosine _A2A receptor antagonist described in claim 1 in the preparation of tumor immunotherapy drugs.
4. The use according to claim 2, wherein the tumor includes but not limited to breast cancer.
5. The use according to claim 2, characterized in that the immunotherapy is to inhibit the accumulation of cAMP.
6. The application according to claim 2, characterized in that the immunotherapy is to promote immune cells to secrete cytokines.
7. The application according to claim 2, characterized in that the immunotherapy is to enhance the killing effect of immune cells on tumors.
8. The use of the subtype-selective adenosine _A2A receptor antagonist of claim 1 in the preparation of drugs for inhibiting tumor cell growth.
9. The use according to claim 8, wherein the tumor cells include but not limited to breast cancer cells.
10. A drug targeting adenosine _A2A receptors or a tumor immunotherapy drug or a drug for inhibiting tumor cell growth, characterized in that it is antagonized with the subtype-selective adenosine _A2A receptors described in claim 1 agent as the main active ingredient.

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