Design, synthesis, and biological evaluation of triazole-pyrimidine-methylbenzonitrile derivatives as dual A2A/A2B adenosine receptor antagonists

Abstract A series of novel dual A2A/A2B AR antagonists based on the triazole-pyrimidine-methylbenzonitrile core were designed and synthesised. The A2A AR antagonist cAMP functional assay results were encouraging for most target compounds containing quinoline or its open-ring bioisosteres. In addition, compound 7i displayed better inhibitory activity on A2B AR (IC50 14.12 nM) and higher potency in IL-2 production than AB928. Moreover, molecular docking studies were carried out to explain the rationality of molecular design and the activity of compound 7i. Further studies on 7f and 7i revealed good liver microsomes stabilities and acceptable in vivo PK profiles. This study provides insight into the future development of dual A2A/A2B AR antagonists for cancer immunotherapy.


General Information
All chemicals were purchased from commercial suppliers and used without further purification. Air or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Reactions were monitored by thin layer chromatography (TLC), and spots were visualized with iodine vapor or by irradiation with UV light. Flash column chromatography was performed using the Qingdao Haiyang flash silica gel (200-300 mesh). All yields were reported as isolated yields. 1 H NMR and 13 C NMR spectra were recorded on the Bruker ( 1 H, 400 or 600MHz) spectrometer with tetramethylsilane as the internal standard. Chemical shifts (δ) are reported in parts per million (ppm) relative to the reference solvents used. The following abbreviations were used to report the multiplicities of the peaks: br, broad; m, multiplet; s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets.
High resolution mass spectrometry results were recorded on Thermo Q-Exactive timeof-flight LC/MS system.

The Ramachandran plot for the homology model of hA2B AR
The primary sequence of hA2B AR was obtained from the NCBI/UNIPROT online database (www.ncbi.nlm.nih.gov/protein/P29275.1) as follows: The sequence identity of homology model and the template was 61.92%. And the homology model has been checked using the Ramachandran plot application in Discovery Studio 2017 R2 ( Figure S1). The data showed that 98.2% (326/332) of the residues are in favorable regions and 1.2% (4/332) in permitted regions. These data indicate that the model has high stereochemical quality, since models with more than 90% of residues in favorable and allowed regions are considered excellent and therefore, suitable for molecular docking studies.

Method
Metabolic stabilities of compounds 7f, 7i and AB928 in liver microsomes were studied with pooled human (male) liver microsomes solution (20 mg/mL) and male Sprague-  (Table S1).

Samples collection and quantitative analysis
Approximately 200 μL of blood was collected at each time point. All blood samples were put into plastic microcentrifuge tubes containing Heparin-Na as anticoagulant.
microcentrifuge tubes with blood samples and anticoagulant were inverted several times for proper mixing of the tube contents to centrifugation for plasma. Plasma samples will be centrifuged at 12000 rpm for 8 min at 4 ℃ to obtain the supernatant.
The serum sample (25 μL) was treated with acetonitrile (100 μL), after which the mixture was vortex-mixed for 8 min and centrifuged at 10000 rpm for 10 min at 4 ℃.
The supernatant layer was collected and then 50 μL of supernatant was injected for the UPLC-MS/MS analysis.

Chromatography and mass spectrometry conditions
UPLC was performed using an ACQUITY TM Table S1.

Standard solutions and work curve
Stock solutions of 1.00 mg/mL 7f, 7i, and AB928 were prepared in acetonitrile and S44 stored at -20 ℃. Calibration solutions of 7f, 7i, and AB928 from 2.5 to 1000 ng/mL were diluted with mice blank plasma. A one-step protein precipitation was adapted as follows: a 20 μL plasma sample was transferred into a 1.5 mL centrifuge tube, and 100 μL of acetonitrile (including 375 ng/mL IS) was added to precipitate the protein. The samples were vortexed for 8 min, followed by centrifugation at 10000 rpm for 5 min.
Then, the supernatants were transferred to an autosampler bottle, and 50 μL of sample was immediately injected into the UPLC-MS/MS system for analysis.