Table 1.

Binding assays of fluorescent adenosine derivatives at three ARs (human ARs, and mouse ARs for compound 2). Structures are shown in Figure 1. When a reference is given, the K_i values listed appear in that source.

Compound	Fluorophore	Ki (nM) or % inhibitiona			Efficacyb	Ref.
		A1AR	A2AAR	A3AR	A3AR
1, MRS5218	Cy5	h: 36%	4730	17.2	94.4%	[22]
		m: 143±10c	717±19c	261±29c	NDc	N/A
2, MRS5704	4-pyrene	8%	3110	68.3	77.8%	[21]
3	1-pyrene	11%	4%	660	97.1%	[21]
4	AlexaFluor 488	0%	23%	416	37.8%	[19]
5	Squaraine- Rotaxane	0%	2%	239	111%	[19]
6	IR700DX	ND	ND	1320±110	ND	N/A
7	Alexa Fluor 488	12±4%	7±2%	400±210	ND	N/A
8	Alexa Fluor 488	1860±440	46%	290±50	ND	N/A

ND, not determined. N/A, not applicable.

^aBinding in membranes of CHO or HEK293 (A_2A only) cells stably expressing one of three hAR subtypes. The binding affinity for A₁, A_2A and A₃ARs was expressed as K_i values using agonist radioligands [³H]N⁶-R-phenylisopropyladenosine (R-PIA), [³H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamido-adenosine (CGS21680), or [¹²⁵I]N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyl-uronamide (I-AB-MECA), respectively. A percent in parentheses refers to inhibition of binding at 10 µM.

^bIn inhibition of forskolin-stimulated cyclic AMP production in hA₃AR-transfected CHO cells. At 10 µM, in comparison to the maximal effect of 10 µM 5′-N-ethylcarboxamidoadenosine (=100%). Data are expressed as mean±standard error (n = 3, unless noted). Data from Tosh et al. [19,21,22].

^cCompetition radioligand binding assays using [¹²⁵I]I-AB-MECA (A₁ and A₃ARs) and [³H]CGS21680 (A_2AAR) were conducted with membranes prepared from HEK293 cells expressing recombinant mA₁, A_2A, or A₃ARs. The data (n = 3–4) are expressed as K_i values. A percent in parentheses refers to inhibition of radioligand binding at 10 µM. ND, not determined. N/A, not applicable.