Table 5. Kinetics of Binding for Synthetic Adenosine and NECA Phenoxycyclopentyl Derivatives to the Orthosteric Binding Site at Human and Rat A₁R.

compd	R1	R2	hA1R				rA1R
			kon (k3) ×105 (M–1 min–1)a	koff (k4) (min–1)b	pKdc	RT (min)d	kon (k3) ×105 (M–1 min–1)a	koff (k4) (min–1)b	pKdc	RT (min)d
24	–CH2OH	H	4.78 ± 0.93	0.048 ± 0.004	6.97 ± 0.06	21.56 ± 2.13	2.70 ± 0.34	0.053 ± 0.001	6.70 ± 0.06	18.91 ± 0.30
25	–CH2OH	p-t-Bu	1.48 ± 0.04	0.054 ± 0.005	6.44 ± 0.03	19.12 ± 2.01	4.84 ± 0.37	0.484 ± 0.173	6.17 ± 0.30	5.53 ± 3.31
26	–CH2OH	m-OMe	2.97 ± 0.27	0.037 ± 0.003	6.90 ± 0.01	27.61 ± 2.09	3.32 ± 0.52	0.085 ± 0.007	6.58 ± 0.03	12.06 ± 1.03
27	–CH2OH	m-Br	10.12 ± 1.29	0.038 ± 0.012	7.47 ± 0.09	33.13 ± 10.40	13.30 ± 1.21	0.050 ± 0.005	7.42 ± 0.03	20.40 ± 1.86
28	–CH2OH	o-Cl	11.61 ± 1.31	0.052 ± 0.004	7.34 ± 0.04	19.55 ± 1.48	17.92 ± 1.30	0.048 ± 0.005	7.58 ± 0.01	21.46 ± 2.08
29	–CH2OH	m-Cl	25.90 ± 2.14	0.040 ± 0.003	7.81 ± 0.02	25.37 ± 1.70	30.07 ± 4.02	0.071 ± 0.010	7.63 ± 0.12	15.20 ± 2.50
30	–CH2OH	p-Cl	1.01 ± 0.31	0.056 ± 0.009	6.22 ± 0.13	19.46 ± 3.34	2.51 ± 0.90	0.056 ± 0.001	6.58 ± 0.15	17.90 ± 0.25
49	–CONHEt	H	16.43 ± 2.81	0.028 ± 0.002	7.63 ± 0.01	32.82 ± 1.02	25.12 ± 2.08	0.037 ± 0.002	7.83 ± 0.05	27.61 ± 1.66
50	–CONHEt	p-t-Bu	1.53 ± 0.11	0.091 ± 0.010	6.23 ± 0.06	11.47 ± 1.48	6.29 ± 0.62	0.070 ± 0.007	6.95 ± 0.01	14.76 ± 1.64
51	–CONHEt	m-OMe	4.27 ± 0.03	0.027 ± 0.007	7.23 ± 0.11	42.68 ± 10.88	11.91 ± 1.33	0.030 ± 0.006	7.62 ± 0.11	38.05 ± 8.74
52	–CONHEt	m-Br	5.57 ± 2.56	0.075 ± 0.038	6.91 ± 0.04	27.87 ± 14.23	4.46 ± 1.28	0.056 ± 0.008	6.85 ± 0.13	19.17 ± 2.68
53	–CONHEt	o-Cl	17.27 ± 1.12	0.041 ± 0.010	7.66 ± 0.11	29.44 ± 6.91	37.59 ± 1.91	0.048 ± 0.003	7.90 ± 0.00	21.23 ± 1.22
54	–CONHEt	m-Cl	20.28 ± 1.11	0.043 ± 0.013	7.75 ± 0.16	34.70 ± 13.43	35.85 ± 3.95	0.062 ± 0.007	7.76 ± 0.10	16.78 ± 2.22
55	–CONHEt	p-Cl	3.75 ± 1.04	0.076 ± 0.024	6.71 ± 0.03	17.48 ± 4.79	11.62 ± 0.26	0.064 ± 0.003	7.26 ± 0.01	15.65 ± 0.78

^ak_on (k₃) for ligands as determined using NanoBRET binding assays using either human or rat Nluc-A₁R expressing HEK 293 cells and determined through fitting with the ″kinetics of competitive binding″ model.³⁵

^bk_off (k₄) for ligands determined as in footnote a.

^cKinetic dissociation constant (pK_d) for each ligand as determined from k_off/k_on.

^dResidence time of each ligand as determined by the reciprocal of the k_off. All data are the mean ± SEM of at least three independent repeats conducted in duplicate.