Selective Adenosine Receptor (AR) ligands modulate AR activity showing promise as clinical candidate

Synthesis and evaluation of adenosine derivatives as A1, A2A, A2B, and A3 adenosine receptor ligands containing boron clusters as phenyl isosteres and selective A3 agonists

Bednarska-Szczepaniak et al., 2021

Adenosine is an organic compound that is naturally present in the human body. It helps build RNA and many small molecules and supplies chemical energy for cells in the form of ATP. Adenosine interacts with the extracellular domain of G-protein coupled receptors (A1AR, A2AAR, A2BAR, and A3AR) and regulates nervous, circulatory, endocrine, and immunological functions.

The levels of adenosine increase in certain conditions like hypoxia, inflammation, or ischemia. This molecule acts as a protectant under these conditions and could be explored for therapeutic purposes. Already the A2A AR agonist regadenoson is being evaluated as an agent that reduces COVID-19-induced lung injury through inhibition of hyperinflammation ( Identifier: NCT04606069).

Researchers are testing potent and selective AR ligands. But they face many challenges, such as low-tissue specificity of ARs in the body and cross-activation of nontarget AR receptors by highly potent selective ligands.

One of the ways of solving this problem is to develop ligands that don’t activate 100% of the receptors but only those essential for achieving the desired clinical outcomes. One example is nucleoside-boron cluster conjugates, including adenosine derivatives that could represent potent and more selective AR ligands.

Researchers at the Institute of Medical Biology at PAS (Lodz, Poland); the Institute of Biochemistry and Biophysics (Warsaw, Poland); the Medical University of Lodz (Poland) and the National Institute of Diabetes & Digestive & Kidney Diseases (NIH, USA) synthesized 30 adenosine derivatives with either inorganic boron clusters or organic phenyl groups to test this hypothesis. They evaluated the effects of organic and inorganic modifications on the affinity of ligands for the A1, A2A, A2B, and A3 ARs.

Study Overview

Patients and Biological Materials

Fifty-seven healthy volunteers donated blood for platelet aggregation and thrombus formation assays.

Inhibition of human blood platelets aggregation

The researchers centrifuged the blood for 12 min at 190xg to obtain platelet-rich plasma (PRP) at a 2 x 108 platelets/mL final concentration. They added a tested compound dissolved in DMSO. Then, they induced platelet aggregation by adding ADP to each sample to a final concentration of 10 mM. They monitored this process for 10 min using an optical aggregometer.

Formation of thrombus in flow conditions

The researchers tested the effects of the compounds on the formation of thrombus. For that, they used Cellix’s VenaFlux platform.

First, the researchers pre-coated the microchannels of Vena8 Fluoro+ biochips with Type 1 collagen. They incubated citrated blood with anti-CD61 PE-conjugated antibodies to label blood platelets. They added tested compounds or 0.1 % DMSO 5 minutes before measurements. The researchers recalcified the samples with 1mM CaCl2 shortly before running in the system. Then, they perfused the samples with a shear force of 20 dyn/cm2 for 5 min. After that, they used a microscope equipped with a camera to visualize the thrombi in the channels. They estimated the individual thrombi using a dedicated ZEN software.


The main findings of these experiments were:

Of all the compounds tested, compounds 18 and 40, modified with a phenyl group, significantly decreased ADP-induced platelet activation by approximately 50%, as highlighted in Table 4. Other compounds did not inhibit activation by less than 50%. The compound 24 inhibited platelet aggregation by 20%.

Table 4 from Bednarska-Szczepaniak et al. ADP-induced platelet aggregation in platelet-rich plasma in the presence of the compounds 7, 8 , 15-18, 23, 24, 35, 37, 39 and 40.

Collagen induced thrombi formation in whole blood under shear forces as illustrated in Fig. 3 from Bednarska-Szczepaniak et al. The thrombi in samples treated with compound 40 were smaller than the thrombi in the untreated samples. Notably, compound 18 did not exert an inhibitory effect under these conditions.

Fig. 3 from Bednarska-Szczepaniak et al. Effects of compound 40 on the formation of thrombi deposited on collagen under flow conditions. A) Representative images of fluorescently labelled thrombi formed in blood treated with a vehicle (left panel) or 50 mM compound 40 (right panel). B) Comparison of the sizes of thrombi. The sizes of thrombi visualized in the channels were quantified using dedicated software. Each dot represents the summarized area of thrombi larger than 50 mm2 formed in the blood of a particular donor treated either with vehicle or compound 40. Significant differences were determined using a paired Student's t-test, P < 0.01, n = 9.

Other experiments in this study

In addition to the experiments mentioned above, the researchers of the study conducted several analyses to verify the functionality of the synthesized compounds. The main findings were:

  • Most tested ligands tended to bind A3 AR over other ARs preferentially.

  • Compounds 18, 40 and 24 showed nanomolar A3 affinity

  • Among the boron cluster-containing compounds, the highest A3 affinity was for adenosine derivative 41 modified at C2.

  • Analogs bearing boron clusters showed a lower binding affinity for adenosine receptors than the corresponding phenyl analogs.

  • Several boron cluster modified adenosine ligands showed significantly higher A3 receptor selectivity than the corresponding phenyl analogs.

Investigating the chemistry and biology of innovative adenosine derivatives containing boron clusters can expand the possibilities of AR ligand types available for testing as potential drug candidates.

What do I need to get started?

Would you like to run similar experiments in your lab and don’t know how to start? This is what you´ll need:

  • Vena8 Fluoro+ biochip – to mimic human blood vessels and model blood clots, see further details below.

  • Mirus Evo pump – to control shear rates (flow rates) in the biochip; this enables you to set the shear rate at a setting which models flow rates for thrombosis in micropillaries or other vessels.

  • Microenvironmental chamber – this is a temperature-controlled frame, the biochip sits in this and it keeps everything at 370C. The microenvironmental chamber sits on the microscope stage.

  • Inverted microscope – we supply the Zeiss AxioVert A1 with the VenaFlux Pro option or the Zeiss AxioObserver7 with the VenaFlux Elite option.

  • Digital camera – to capture images and video recordings. We supply the Prime BSI Express with both the VenaFlux Pro and Elite options. This is an excellent camera with a high frame rate suitable for thrombosis studies.

  • Image Pro Cell Analysis software – to analyse the images and videos from your experiments.

If you already have some of these items (such as the inverted microscope, camera, or cell analysis software), we recommend the VenaFlux Starter kit. Our options suit all budgets. Take a look at them on our eShop.


Katarzyna Bednarska-Szczepaniak, Adam Mieczkowski, Aleksandra Kierozalska, Dijana Pavlovi Safti, Konrad Głabała, Tomasz Przygodzki, Lidia Stanczyk, Kamil Karolczak, Cezary Watała, Harsha Rao, Zhan-Guo Gao, Kenneth A. Jacobson, Zbigniew J. Lesnikowski.

Synthesis and evaluation of adenosine derivatives as A1, A2A, A2B and A3 adenosine receptor ligands containing boron clusters as phenyl isosteres and selective A3 agonists. European Journal of Medicinal Chemistry (2021), 223, 113607. doi:

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