CN116903595A - Pyrimidine diketone compound and preparation method and application thereof - Google Patents

Pyrimidine diketone compound and preparation method and application thereof Download PDF

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CN116903595A
CN116903595A CN202310640474.1A CN202310640474A CN116903595A CN 116903595 A CN116903595 A CN 116903595A CN 202310640474 A CN202310640474 A CN 202310640474A CN 116903595 A CN116903595 A CN 116903595A
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丁宗保
程斌斌
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Zhuhai Campus Of Zunyi Medical University
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Abstract

The invention belongs to the technical field of compounds, and provides a pyrimidinedione compound, a preparation method and application thereof. The pyrimidinedione compound provided by the invention can obviously inhibit the mutual combination of PD-1/PD-L1, has high inhibition activity on CD73, is a PD-L1/CD73 double-target compound, has an obvious effect, and can easily predict the pharmacokinetics PK of a single molecule. Therefore, the method has very good prospect in potential double immunotherapy and is beneficial to improving the effect of tumor immunotherapy.

Description

Pyrimidine diketone compound and preparation method and application thereof
Technical Field
The invention relates to the technical field of compounds, in particular to a pyrimidinedione compound, a preparation method and application thereof.
Background
The first generation of immune checkpoint inhibitors, represented by apoptosis receptor 1/apoptosis ligand 1 (PD-1/PD-L1), have gradually become the cornerstone of oncology-oncology (IO) treatment. However, clinical studies have found that the overall response rate of PD-L1 inhibitors is low (ORR < 30%), and finding a new generation of tumor immune targets that are safe and effective and that produce synergistic effects with PD-1/PD-L1 is the focus of current phase attention. At present, researchers have attempted to combine PD-L1 inhibitors with other anticancer drugs for the treatment of cancer.
For solid Tumor treatment, to overcome drug resistance and improve curative effect, an important aspect is to relieve the inhibition of Tumor micro-environment (TME) on immune effector cells. TME is a very complex system, consisting of a variety of cells, cytosol, enzymes, cytokines, metabolites, etc., and has the characteristics of significant hypoxia, low pH and high pressure, and is greatly different from normal tissues. One of the important immunosuppressive mechanisms is mediated by the CD 73-Adenosine (Adenosine receptor type 2 a) metabolic signaling pathway. Adenosine can inhibit the immune killing effect of T cells through adenosine receptor (A2 AR) to make tumors realize immune escape, and CD73 is a key enzyme for catalyzing adenosine production. In addition, CD73 exerts immunosuppressive effects by modulating tumor immune microenvironment and reduces T cell infiltration, complementing the PD-1/PD-L1 axis. Thus, the CD73 target axis is considered a new generation of tumor immunotherapeutic targets.
However, there are drawbacks to the combination therapy of anti-PD-1/PD-L1 antibodies and CD73 inhibitors, including unpredictable Pharmacokinetics (PK)/Pharmacodynamics (PD), overlapping of toxic side effects caused by the combination of two or more drugs, and interactions of the drugs to affect therapeutic effects, etc. In practical clinical applications, combination therapy still presents a certain safety risk. Therefore, development of a novel dual-target inhibitor is needed, which not only has excellent anti-tumor efficacy, but also has small toxic and side effects, and the therapeutic effect is not affected by the interaction of the drugs.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a pyrimidine diketone compound, and a preparation method and application thereof. The pyrimidinedione compound provided by the invention has a molecular structure shown in the following formula (I), can be used as a double-target inhibitor, has better anti-tumor efficacy than monotherapy, has small toxic and side effects, and can not interact with medicines.
In a first aspect, the invention provides a pyrimidinedione compound.
Specifically, a pyrimidinedione compound has a molecular structure as shown in the following formula (I):
wherein R is 1 Selected from CH 3 And/or halogen; r is R 2 Selected from CH 3 And/or halogen; ring a is selected from 6-10 membered heteroaryl; ring B is selected from 6-10 membered heteroaryl.
Preferably, the pyrimidinedione compound has any one of the following structural formulas la, lb or ic:
wherein R is 1 Selected from CH 3 And/or halogen; r is R 2 Selected from CH 3 And/or halogen; x is X 1 -X 24 Each independently selected from N or CH.
Further preferably, the pyrimidinedione compound has any one of the following structural formulas 1 to 8:
the second aspect of the invention provides a method for preparing a pyrimidinedione compound.
A preparation method of a pyrimidinedione compound comprises the following steps:
(1) Firstly, performing coupling reaction on a halogenated compound of a formula (II) and 2, 4-dimethoxy-pyrimidine-5-boric acid to generate an intermediate 1;
(2) The intermediate 1 undergoes demethylation reaction under acidic conditions to produce intermediate 2:
(3) The intermediate 2 undergoes hydrolysis reaction under the action of a catalyst to generate an intermediate 3;
(4) The intermediate 3 and the biphenyl diamino compound are subjected to condensation reaction under the action of a condensing agent to generate an intermediate 4;
(5) The intermediate 4 further undergoes condensation reaction under the action of a condensing agent to generate the pyrimidinedione compound;
the molecular structure of the halogenide of formula (II) is as follows:
wherein ring A is selected from 6-10 membered heteroaryl.
Preferably, in step (1), the coupling reaction is carried out at a temperature of 80-100℃for a period of 1-10 hours.
Preferably, in the step (2), the demethylation reaction is carried out at a temperature of 70-90 ℃ for a time of 1-10 hours.
Preferably, in step (2), the reagent used in the demethylation reaction comprises at least one of hydrochloric acid, trifluoroacetic acid, and boron tribromide.
Preferably, in the step (3), the temperature of the hydrolysis reaction is 20-30 ℃ and the time is 10-15 hours.
Preferably, in the step (3), the hydrolysis reaction adopts a hydrolysis reagent, and the hydrolysis reagent comprises at least one of lithium hydroxide, lithium bromide and sodium hydroxide.
Preferably, in step (4) and step (5), the condensation reaction employs a condensing agent comprising at least one of 2- (7-azabenzotriazol) -N, N' -tetramethylurea hexafluorophosphate, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, and N, N "-carbonyldiimidazole.
Preferably, in the step (4), the temperature of the condensation reaction is 20-30 ℃ and the time is 0.5-3 hours; in the step (5), the temperature of the condensation reaction is 20-30 ℃ and the time is 0.5-3 hours.
The preparation reaction formula of the pyrimidinedione compound is shown as follows:
wherein R is 1 Selected from CH 3 Halogen; r is R 2 Selected from CH 3 Halogen; ring a is selected from 6-10 membered heteroaryl; ring B is selected from 6-10 membered heteroaryl groups
In a third aspect, the invention provides the use of a pyrimidinedione compound.
An application of pyrimidinedione compounds in preparing medicines for treating cancer.
Use of pyrimidinediones in the manufacture of a medicament for the treatment of a disease associated with PD-1/PD-L1 and/or CD 73.
Preferably, the medicament further comprises a pharmaceutically acceptable carrier and an auxiliary material.
A dual-target inhibitor of CD73 and PD-L1, comprising the pyrimidinedione compound.
Compared with the prior art, the invention has the following beneficial effects:
the pyrimidinedione compound provided by the invention has a molecular structure shown in a formula (I), can obviously inhibit the mutual combination of PD-1/PD-L1, has high inhibition activity on CD73, is a PD-L1/CD73 double-target compound, has an obvious effect, has double targeting capability for a single pyrimidinedione compound molecule, and is easy to predict the Pharmacokinetics (PK) of a single molecule. Therefore, the pyrimidine diketone compound provided by the invention has very good prospect in dual immunotherapy, and is beneficial to improving the effect of tumor immunotherapy.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
The progress of the reaction according to the invention is monitored by conventional monitoring methods, such as TLC (thin layer chromatography), LCMS (liquid chromatography) or NMR (nuclear magnetic resonance), generally by taking the reaction substrate as the end point when it disappears.
In the following specific examples, the liquid chromatography conditions used for detection and identification of the prepared compounds were: island LCMS2020, G1322A degasser, G1312 binary high pressure pump, G1329A autosampler, G1316A column oven, G4212B diode array detector. The column was Xbridge C18 (50 mm. Times.4.6 mm,5.0 μm) and was eluted with a gradient of deionized water as mobile phase A and acetonitrile containing 0.1% trifluoroacetic acid as mobile phase B, with the following procedure:
time (min) Mobile phase a (volume%) Mobile phase B (volume%)
0.01 95 5
1.50 95 5
3.00 5 95
3.50 5 95
4.00 95 5
5.00 95 5
The flow rate was 1.5mL/min, the column temperature was 40℃and the detection wavelength was 254nm.
Example 1
A pyrimidinedione compound, named N, N '- (2, 2' -dimethyl- [1,1 '-diphenyl ] -3,3' -diyl) bis (6- (2, 4-dioxo-1, 2,3, 4-tetrahydropyrimidin-5-yl) pyridazin-3-amide) (abbreviated as compound 1), having the molecular structure shown below:
the preparation method of the compound 1 comprises the following steps:
(1) Into a reaction flask were charged the compound of formula (II) (1 g,5.79 mmol), 2, 4-dimethoxy-pyrimidine-5-boronic acid (1.85 g,6.95 mmol) and [1,1' -bis (diphenylphosphine) ferrocene]Palladium dichloride dichloromethane complex (423.43 mg,579.48 mu mol), cesium carbonate (1.58 g,11.59 mmol), 1, 4-dioxane/water=5/1 (30 mL), vacuum nitrogen substitution 3 times, stirring and heating to 90 ℃ under nitrogen protection, reacting for 5 hours, monitoring by TLC, cooling to 25 ℃ after the raw materials are reacted completely, passing through column chromatography silica gel column, using petroleum ether: the ethyl acetate system was eluted (PE: ea=2:1) to give intermediate 1 (1.2 g, yield 74.96%) as a white solid. LCMS: (MS-ESI, M/z) [ M+H ]] + ＝277.1。
(2) To the reaction flask were added intermediate 1 (1.2 g,4.34 mmol) and 1M aqueous hydrochloric acid (30 mL), stirred and warmed to 80℃for 2 hours, LCMS was monitored to complete the reaction, cooled to 0℃and a large amount of white solid precipitated, filtered to give intermediate 2 (900.00 mg, yield 83.48%) as a white powdery solid. LCMS: (MS-ESI, M/z) [ M+H ]] + ＝249.1。
(3) Into a reaction flask were charged intermediate 2 (900.00 mg,3.63 mmol) and lithium hydroxide (130.25 mg,5.44mmo at room temperaturel) methanol/water=5/1 ratio 20ml,25 ℃,12 hours, LCMS monitoring, after the reaction of the starting materials is complete, 1M hydrochloric acid hydrolysate is added to adjust ph=6-7, a large amount of white solid is precipitated, filtered to obtain white powdery solid intermediate 3 (800.00 mg, yield 94.21%). LCMS: (MS-ESI, M/z) [ M+H ]] + ＝235.1。
(4) To a reaction flask was added intermediate 3 (100.00 mg, 427.04. Mu. Mol), 2 '-dimethyl- [1,1' -diphenyl at room temperature]-3,3 '-diamine (90.66 mg,427.04 μmol), 2- (7-azabenzotriazol) -N, N' -tetramethylurea hexafluorophosphate (162.38 mg,427.04 μmol), N-diisopropylethylamine (66.23 mg,512.45 μmol), N-dimethylformamide (5 mL), reaction for 1 hour at 25 ℃, TLC monitoring, after completion of the starting reaction, 20mL water quench reaction was added, separated, ethyl acetate extraction 3 times (30 mL x 3), merging the ethyl acetate phases, concentrating under reduced pressure, adding silica gel mix, column chromatography silica gel column chromatography, column chromatography with dichloromethane: the methanol system eluted (DCM: meoh=40:1) to give intermediate 4 (150.00 mg, 81.98%) as a white powder. LCMS: (MS-ESI, M/z) [ M+H ]] + ＝429.2。
(5) To a reaction flask was added intermediate 4 (100.00 mg, 233.40. Mu. Mol), 6- (2, 4-dioxo-1, 2,3, 4-tetrahydropyrimidin-5-yl) pyridazine-3-carboxylic acid (54.66 mg, 233.40. Mu. Mol), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (88.75 mg, 233.40. Mu. Mol), N, N-diisopropylethylamine (36.20 mg, 280.08. Mu. Mol), N, N-dimethylformamide (5 mL), 25℃for 3 hours, TLC monitored, after the completion of the reaction of the starting material, 20mL of water quenched reaction, split, ethyl acetate extraction 3 times (30 mL. Times 3), merging the ethyl acetate phases, concentrating under reduced pressure, adding silica gel for column chromatography, column chromatography of silica gel using methylene chloride: the methanol system eluted (DCM: meoh=10:1) to give compound 1 (85.00 mg, 53.17%) as a white powder.
The reaction formula of the compound 1 is as follows:
results of liquid chromatography-mass spectrometry combination test of compound 1: LCMS:(MS-ESI,m/z):[M+H] + nuclear magnetic hydrogen spectrum test results= 645.2: 1 H NMR(400MHz,DMSO)δ11.71(s,2H),11.62(s,2H),10.61(s,2H),8.63(d,J＝9.0Hz,2H),8.57(d,J＝4.7Hz,2H),8.32(d,J＝9.0Hz,2H),7.78(d,J＝7.9Hz,2H),7.35(t,J＝7.8Hz,2H),7.05(d,J＝7.4Hz,2H),2.05(s,6H).
example 2
A pyrimidinedione compound, named N, N '- (2, 2' -dimethyl- [1,1 '-diphenyl ] -3,3' -diyl) bis (5- (2, 4-dioxo-1, 2,3, 4-tetrahydropyrimidin-5-yl) pyrazin-2-amide) (abbreviated as compound 2), having the molecular structure shown below:
the preparation method of the compound 2 is the same as in example 1, except that the step (1)Replaced by
Compound 2 (41 mg, 40.22% yield). LCMS: (MS-ESI, M/z) [ M+H ]] + ＝645.2 1 H NMR(400MHz,DMSO)δ11.68(s,2H),11.60(s,2H),10.61(s,2H),9.33(s,2H),9.12(s,2H),8.32(d,J＝7.4Hz,2H),7.72(d,J＝7.8Hz,2H),7.34(t,J＝7.8Hz,2H),7.05(d,J＝7.4Hz,2H),2.01(s,6H).
Example 3
A pyrimidinedione compound, named N, N ' - (2, 2' -dimethyl- [1,1' -diphenyl ] -3,3' -diyl) bis (2 ',4' -dioxo-1 ',2',3',4' -tetrahydro- [5,5' -bipyrimidine ] -2-amide) (abbreviated as compound 3), having the molecular structure shown below:
the preparation method of the compound 3 is the same as in example 1, except that the step (1)Replaced by
Compound 3 (39 mg, 40.2% yield). LCMS: (MS-ESI, M/z) [ M+H ]] + ＝645.2 1 H NMR(400MHz,DMSO)δ11.73(s,2H),11.62(s,2H),10.61(s,2H),8.53(s,2H),8.27(s,2H),8.12(d,J＝7.5Hz,2H),7.79(d,J＝7.9Hz,2H),7.34(t,J＝7.9Hz,2H),7.05(d,J＝7.5Hz,2H),2.06(s,6H).
Example 4
A pyrimidinedione compound, named N, N ' - (2, 2' -dimethyl- [1,1' -diphenyl ] -3,3' -diyl) bis (2 ',4' -dioxo-1 ',2',3',4' -tetrahydro- [2,5' -bipyrimidine ] -5-amide) (abbreviated as compound 4), having the molecular structure shown below:
the preparation method of the compound 4 is the same as in example 1, except that the step (1)Replaced by
Compound 4 (41 mg, yield 50.6%). LCMS: (MS-ESI, M/z) [ M+H ]] + ＝645.2 1 H NMR(400MHz,DMSO)δ11.74(s,2H),11.62(s,2H),10.55(s,2H),8.83(s,2H),8.67(s,2H),8.12(d,J＝7.4Hz,2H),7.74(d,J＝7.9Hz,2H),7.34(t,J＝7.9Hz,2H),7.05(d,J＝7.4Hz,2H),2.01(s,6H).
Example 5
A pyrimidinedione compound, named N, N '- (2, 2' -dimethyl- [1,1 '-diphenyl ] -3,3' -diyl) bis (6- (2, 4-dioxo-1, 2,3, 4-tetrahydropyrimidin-5-yl) pyridazin-3-amide) (abbreviated as compound 5), having the molecular structure shown below:
the preparation method of the compound 5 is the same as in example 1, except that the step (4)Replaced by->
Compound 5 (37 mg, 39.9% yield). LCMS: (MS-ESI, M/z) [ M+H ]] + ＝685.1 1 H NMR(400MHz,DMSO)δ11.70(s,2H),11.61(s,2H),10.62(s,2H),8.64(d,J＝9.0Hz,2H),8.55(d,J＝4.7Hz,2H),8.32(d,J＝9.0Hz,2H),7.99(d,J＝7.9Hz,2H),7.55(t,J＝7.8Hz,2H),7.24(d,J＝7.4Hz,2H).
Example 6
A pyrimidinedione compound, named 6- (2, 4-dioxo-tetrahydropyrimidin-5-yl) -N- (3 ' - (6- (2, 4-dioxo-1, 2,3, 4-tetrahydropyrimidin-5-yl) pyridazin-4-amide) -2,2' -dimethyl- [1,1' -diphenyl ] -3-yl) pyridazin-3-amide (abbreviated as compound 6), having the molecular structure shown below:
the preparation method of the compound 6 is the same as in example 1, except that the step (5)Replaced by->
Compound 6 (37 mg, yield 37.6%). LCMS: (MS-ESI, M/z) [ M+H ]] + ＝645.2.. 1 H NMR(400MHz,DMSO)δ11.73(s,2H),11.64(s,2H),10.62(s,2H),8.63(m,2H),8.57(m,2H),8.35(m,2H),7.78(d,J＝7.9Hz,2H),7.35(t,J＝7.8Hz,2H),7.05(d,J＝7.4Hz,2H),2.05(s,6H).
Example 7
A pyrimidinedione compound, named N- (2, 2' -dichloro-3 ' - (6- (2, 4-dioxo-1, 2,3, 4-tetrahydropyrimidin-5-yl) pyridazin-4-amide) - [1,1' -diphenyl ] -3-yl) -6- (2, 4-dioxo-1, 2,3, 4-tetrahydropyrimidin-5-yl) pyridazin-3-amide (abbreviated as compound 7), having the molecular structure shown below:
the preparation method of the compound 7 is the same as in example 1, except that the step (4)Replaced by->Step (5)/(x)>Replaced by->
Compound 7 (40 mg, yield 42.3%). LCMS: (MS-ESI, M/z) [ M+H ]] + ＝685.1 1 H NMR(400MHz,DMSO)δ11.73(s,2H),11.64(s,2H),10.62(s,2H),8.63(m,2H),8.57(m,2H),8.35(m,2H),7.99(d,J＝7.9Hz,2H),7.55(t,J＝7.8Hz,2H),7.24(d,J＝7.4Hz,2H).
Example 8
A pyrimidinedione compound, named N, N '- (2, 2' -dimethyl- [1,1 '-diphenyl ] -3,3' -diyl) bis (6- (2, 4-dioxo-1, 2,3, 4-tetrahydropyrimidin-5-yl) pyridazin-4-amide) (abbreviated as compound 8), having the molecular structure shown below:
the preparation method of the compound 8 is the same as in example 1, except that the step (1)Replaced byStep (5)/(x)>Replaced by->
Compound 8 (31 mg, yield 32.4%). LCMS: (MS-ESI, M/z) [ M+H ]] + ＝645.2 1 H NMR(400MHz,DMSO)δ11.72(s,2H),11.62(s,2H),10.61(s,2H),8.81(s,2H),8.66(s,2H),8.37(s,2H),7.78(d,J＝7.9Hz,2H),7.35(t,J＝7.8Hz,2H),7.05(d,J＝7.4Hz,2H),2.07(s,6H).
Product effect test
1. Inhibition effect test on PD-1/PD-L1
The detection method of the PD-1/PD-L1 protein-protein interaction inhibition activity is a biochemical level-based homogeneous time-resolved fluorescence method (homogeneous time resolved fluorescence, HTRF) and utilizes HTRF (homogeneous time-resolved fluorescence) technology, and the detection method simply and rapidly characterizes the compounds and antibody blockers in a high-throughput form. The interaction between PD-L1 and PD-1 (HTRF donor) and anti-Tag 2 labeled with XL665 (HTRF acceptor) were detected using europium-labeled anti-Tag 1. When the donor and acceptor antibodies are in close proximity due to PD-L1 and PD1 binding, excitation of the donor antibody triggers Fluorescence Resonance Energy Transfer (FRET) to the acceptor antibody, which in turn emits specifically at 665 nm. The above specific signal is proportional to the extent of PD1/PD-L1 interaction. Blocking the PD1/PD-L1 interaction by a compound or antibody results in a decrease in HTRF signal. Thus, the intensity of fluorescence generated was measured by a microplate reader and the amount of activity of the compound blocking PD-1/PD-L1 was reflected in a ratio of 665nm/620 nm.
The main reagents used in the above test were as follows:
1. preparation of experiments
(1) Dilution of compound mother liquor: each test compound was diluted to 20mmol/L of compound mother liquor. The amount of DMSO required to dilute each compound can be calculated according to formula c=n/v=m/M/V. After the compound mother solution is prepared, the mother solution is diluted into 10 mu M, 3.333 mu M, 1.111 mu M, 0.370 mu M, 0.123 mu M, 0.041 mu M, 0.013 mu M and 0.004 mu M of diluents carried by the kit according to a gradient dilution method, and the diluted solutions are uniformly mixed for later use.
(2) Dilution of PD-L1 protein mixture: 7mL of PD-L1 protein in the kit is taken, and 510mL of diluent is added to dilute the protein, and the protein is uniformly mixed for standby.
(3) Dilution of PD-1 protein mixture: taking 5mL of PD-1 protein in the kit, adding 550mL of diluent to dilute the protein, and uniformly mixing for later use.
(4) Preparing a mixed test solution: respectively taking 5 mu L of Anti-Tag-Eu in the kit 3+ And 20. Mu.L of Anti-Tag-XL665, then 975. Mu.L of Detection buffer (Detection buffer) was added to dilute it, and mixed well for use.
2. Experimental procedure
(1) mu.L of each of the compound 1-8 dilutions was added to each well of the 96-well plate, and each compound was added from top to bottom in order of concentration from top to bottom, and each compound was tested for 8 concentrations (10. Mu. Mol/L, 3.333. Mu. Mol/L, 1.111. Mu. Mol/L, 0.370. Mu. Mol/L, 0.123. Mu. Mol/L, 0.041. Mu. Mol/L, 0.013. Mu. Mol/L, 0.004. Mu. Mol/L), three sub-wells per concentration. Centrifuge at 1000rpm for 1min.
(2) mu.L (2.5X) of PD-L1 mixture was added to each well and centrifuged at 1000rpm for 1min.
(3) To each well, 4. Mu.L (2.5X) of PD-1 mixture was added, centrifuged at 1000rpm for 1min and incubated at room temperature for 15min.
(4) To each well 10. Mu.L (2X) of the test mixture was added and centrifuged at 1000rpm for 1min.
(5) The incubation was carried out at room temperature for 120min, and fluorescence values (excitation wavelength Ex:320nm, emission wavelengths Em:620nm and 665 nm) were read using a Tecan microplate reader.
(6) The inhibition ratio (inhibition)% = (1- (signal value 665nm/620nm per well-low control group average)/(high control group average-low control group average)) ×100 was calculated as follows. Wherein, the high control group: no compound was added to the reaction system group, and only an equivalent amount of DMSO solution was added thereto. Low control group: the PD-1 mixed solution is not contained, and only the equal amount of the detection mixed solution is added. In this assay system, the final DMSO concentration was 0.5%.
(7) Fitting dose-response curve: the log value of the concentration is taken as the X axis, the inhibition rate is taken as the Y axis, and a log (inhibitor) vs. response-Variable slope fit quantitative response curve of analysis software GraphPad Prism 5 is adopted, so that the IC of each compound on the enzyme activity is obtained 50 Values.
3. Experimental results
Table 1 test results for compounds 1-8
According to the in vitro experimental results of Table 1, the compounds 1-8 of the invention can obviously inhibit the mutual combination of PD-1/PD-L1, and the effect is obvious.
2. Inhibition effect test on CD73
1. Experimental method
The test was performed in the presence of 25mM Tris and 25mM MgCl 2 Is (B) Tris-MgCl 2 In a buffer. The specific operation steps are as follows:
(1) With Tris-MgCl 2 Buffer Human CD73 (Sino Biological; 10904-H08H) was formulated as a 3 Xstock solution and added to 96 Kong Baiban at 20. Mu.L/well to a final concentration of 0.1. Mu.g/mL;
(2) With Tris-MgCl 2 The final concentration of the compound to be tested was diluted to a 3 Xmother liquor with a proper concentration gradient, and compounds 1 to 8 (concentration: 60. Mu. Mol/L, 30. Mu. Mol/L, 10. Mu. Mol/L, 3.333. Mu. Mol/L, 1.111. Mu. Mol/L, 0.370. Mu. Mol/L, 0.123. Mu. Mol/L, 0.041. Mu. Mol/L) were added to the 96-well test white plate as described above at 20. Mu.L/well, respectively,after mixing, incubating for 30min at normal temperature, and simultaneously setting a positive control group (without adding compounds 1-8) and a negative control group (without adding CD 73);
(3) With Tris-MgCl 2 Preparing AMP (Sigma; A1752-5G) into 3 Xmother solution, adding into the above 96 Kong Baiban according to 20 μl/well, mixing to obtain final concentration of 100 μM, and incubating at 37deg.C for 60min;
(4) With Tris-MgCl 2 ATP (Sigma; A7699-1G) was prepared as 7 Xstock solution and added to 96 Kong Baiban as described above at a final concentration of 100. Mu.L/well, mixed well, incubated for a further 5min and detected using the ATP-GLO kit (Promega; G7573).
The inhibition ratio of the compound on the Human-CD73 is calculated according to the following formula, then the compound concentration is taken as an X axis, the inhibition ratio is taken as a Y axis, and IC of the compound on the Human-CD73 inhibition is calculated by Prism software 50 Values. Wherein the inhibition ratio (inhibition)% = ((positive control signal value-compound signal value of different concentrations)/(positive control signal value-negative control signal value)) ×100.
2. Experimental results
TABLE 2 inhibition of CD73 by Compounds 1-8
According to the in vitro experimental results of the table 2, the compounds 1 to 8 provided by the invention can obviously inhibit the activity of a CD73 target.
3. Inhibition effect test on Jurkat/PD-1:CHO/PD-L1 assay
1. Principle of experiment
Overexpression of human PD-L1 receptor and TCR activator (activator) on CHO cell lines Jurkat cells express the NFAT fluorescent reporter gene and the human PD-L gene and the in vitro potency of PD-L1 inhibitors is examined in vitro. When PD-1/NFAT/Jurkat cells and CHO/PD-L1/TCR cells are co-cultured, TCR and Jurkat cell binding initiates NFAT fluorescent reporter expression. Whereas binding of PD-1 and PD-L1 on the surface of two cells inhibits TCR-induced NFAT reporter gene expression. When the test compound blocks the binding of the PD-1 and PD-L1 molecules on the surface of two cells, the TCR causes NAFT expression, and the NFAT expression level is in a dose-dependent relationship with the inhibition degree of the PD-1 and the PD-L1.
2. Experimental method
(1) CHO/PD-L1/TCR cells were cultured according to 35X 10 4 cells/mL were seeded into 96-well cell culture plates with 100. Mu.L of cell suspension per well, 37℃and 5% CO 2 Incubator, incubate overnight.
(2) On the day of the experiment, cell culture plate supernatants were discarded and tested in groups (divided into compound 1-8 groups, PD-L1 positive drug group, CD73 positive drug group, combination drug group) in which the administration concentrations were respectively 1-8 groups (concentration 60. Mu. Mol/L, 30. Mu. Mol/L, 10. Mu. Mol/L, 3.333. Mu. Mol/L, 1.111. Mu. Mol/L, 0.370. Mu. Mol/L, 0.123. Mu. Mol/L, 0.041. Mu. Mol/L), PD-L1 positive drug group (BMS-8 concentration 60. Mu. Mol/L, 30. Mu. Mol/L, 10. Mu. Mol/L, 3.333. Mu. Mol/L, 1.111. Mu. Mol/L, 0.370. Mu. Mol/L), 0.123. Mu. Mol/L, 0.041. Mu. Mol/L), CD73 positive drug group (LY-3475070 concentration 60. Mu. Mol/L, 30. Mu. Mol/L, 10. Mu. Mol/L, 3.333. Mu. Mol/L, 1.111. Mu. Mol/L, 0.370. Mu. Mol/L, 0.123. Mu. Mol/L, 0.041. Mu. Mol/L), combination drug group 1 (60. Mu. Mol/L BMS-8+1. Mu. M LY-3475070, 30. Mu. Mol/L BMS-8+1. Mu. MLY-3475070, 10. Mu. Mol/L BMS-8+1. Mu. M LY-3475070,3.333. Mu. Mol/L BMS-8+1. Mu. M-3475070,0.370. Mu. Mol/L BMS-8+1. Mu. M-3475070,0.123. Mu. Mol/LBMS-8+1. Mu. M-3475070,0.041. Mu. Mol/L BMS-8+1. Mu. M-3475070), combination group 2 (60. Mu. Mol/LLY-3475070+1. Mu.M BMS-8, 30. Mu. Mol/L LY-3475070+1. Mu.M BMS-8, 10. Mu. Mol/L LY-3475070+1. Mu. MBMS-8,3.333. Mu. Mol/L LY-3475070+1. Mu. M BMS-8,1.111. Mu. Mol/L LY-3475070+1. Mu. M BMS-8,0.370. Mu. Mol/L LY-3475070+1. Mu. M BMS-8,0.123. Mu. Mol/L LY-3475070+1. Mu. M BMS-8), and 50. Mu.L of compound working fluid was added to each well. Cell culture plates were incubated at 37℃with 5% CO 2 Incubation in the incubator was continued for 0.5 hours.
(3) Jurkat cells were then grown at 40X 10 4 cells/mL were seeded into 96-well cell culture plates, 50. Mu.L of cell suspension was seeded per well, 37℃and 5% CO 2 Incubators were incubated for 5 hours.
(4) After the incubation, the cell supernatant was removed and 100. Mu.L of Bright Glo detection reagent was added to each well of the cell culture plate for luminescence signal detection.
The PD-L1 positive drug used above was BMS-8, and the CD73 positive drug was LY-3475070.
3. Experimental results
TABLE 3 inhibitory Effect of Compounds on Jurkat/PD-1:CHO/PD-L1
According to the results shown in Table 3, the compounds 1-8 provided by the invention show remarkable inhibition effect in Jurkat/PD-1:CHO/PD-L1 assay test model, and are superior to PD-L1 positive drugs BMS-8, CD73 positive drug LY-3475070 and a combined administration group of two positive drugs.
3. Rat pharmacokinetic testing
1. Test method
SD rats were administered by tail vein injection (compound 1, administration concentration 2 mg/kg). Pre-and post-dose 0, 5, 10, 15, 30 minutes, and 1,2,3,4, 6, 8, 12, 24 hours blood was withdrawn from the orbit by approximately 0.1mL, heparin was placed for anticoagulation. The blood was placed in a test tube, centrifuged for 10 minutes, serum was separated, 150. Mu.L of IS mixed methanol solution (concentration: 100 ng/mL) was added, and after centrifugation for 10 minutes at 16000g, 100. Mu.L of the supernatant was aspirated for UPLC-MS analysis to determine the compound concentration. And analyzing and processing pharmacokinetic data by utilizing a non-atrioventricular model method of WINNONLIN to obtain the pharmacokinetic parameters of the rat. The analysis conditions for UPLC-MS were as follows, chromatographic system: waters ACQUITY UPLC I-Class/Xex TQD; chromatographic column: CORTECS UPLC C18 Column (2.1 mm. Times.100 mm,1.6 μm); column temperature: 40 ℃; acetonitrile as mobile phase A,0.1% formic acid aqueous solution as B, flow rate set 0.4mL/min, separation of compound 1 from internal standard (Midazolam) using C18 chromatographic column, sample injection capacity 5 μl, and run time 3min. Mass spectrometry conditions: using positive ion mode electricitySpraying ESI source, multi-reaction monitoring (Multiple Reaction Monitoring, MRM) to generate ion fragments, wherein the capillary voltage is 1kV, the desolventizing gas temperature is 500 ℃, the source temperature is 150 ℃, the sheath gas flow rate is 50L/H, and the desolventizing gas flow rate is 1000L/H. Wherein AUC (0-∞) Representing the area under the blood concentration-time curve; t (T) max Time points representing maximum plasma concentrations; t is t 1/2 Representing half-life; c (C) max Indicating maximum blood concentration.
2. Experimental results
Table 4 pharmacokinetic data of compound 1 in rats
As can be seen from the results in table 4, the compound 1 provided in the present invention shows a suitable half-life, maximum plasma concentration, and area under the plasma concentration-time curve in the rat pharmacokinetic PK test model.

Claims (10)

1. A pyrimidinedione compound, which is characterized by having a molecular structure represented by the following formula (i):
wherein R is 1 Selected from CH 3 And/or halogen; r is R 2 Selected from CH 3 And/or halogen; ring a is selected from 6-10 membered heteroaryl; ring B is selected from 6-10 membered heteroaryl.
2. The pyrimidinedione compound of claim 1, having any one of the following formulas la, ib or ic:
wherein R is 1 Selected from CH 3 And/or halogen; r is R 2 Selected from CH 3 And/or halogen; x is X 1 -X 24 Each independently selected from N or CH.
3. The pyrimidinedione compound according to claim 2, which has any one of the following structural formulas 1 to 8:
4. a process for the preparation of a pyrimidinedione compound according to any one of claims 1-3, comprising the steps of:
(1) Firstly, performing coupling reaction on a halogenated compound of a formula (II) and 2, 4-dimethoxy-pyrimidine-5-boric acid to generate an intermediate 1;
(2) The intermediate 1 undergoes demethylation reaction under acidic conditions to produce intermediate 2:
(3) The intermediate 2 undergoes hydrolysis reaction to generate an intermediate 3;
(4) The intermediate 3 and the biphenyl diamino compound undergo condensation reaction to generate an intermediate 4;
(5) The intermediate 4 further undergoes condensation reaction to generate the pyrimidinedione compound;
the molecular structure of the halogenide of formula (II) is as follows:
wherein ring A is selected from 6-10 membered heteroaryl.
5. The process according to claim 4, wherein in the step (1), the coupling reaction is carried out at a temperature of 80 to 100℃for a period of 1 to 10 hours.
6. The process according to claim 4, wherein in the step (2), the demethylation reaction is carried out at a temperature of 70 to 90℃for a period of 1 to 3 hours.
7. The process according to claim 4, wherein in the step (3), the hydrolysis is carried out at a temperature of 20 to 30℃for a period of 10 to 15 hours.
8. The process according to claim 4, wherein in the step (4), the condensation reaction is carried out at a temperature of 20 to 30℃for a period of 0.5 to 3 hours; in the step (5), the temperature of the condensation reaction is 20-30 ℃ and the time is 0.5-3 hours.
9. Use of a pyrimidinedione compound according to any one of claims 1-3 in the manufacture of a medicament for the treatment of cancer.
10. A dual target inhibitor of CD73 and PD-L1 comprising a pyrimidinedione compound according to any one of claims 1-3.
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