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. 2022 Oct 21;65(21):14864–14890. doi: 10.1021/acs.jmedchem.2c01414

Table 4. Kinetics of Binding for Synthetic Adenosine and NECA Benzyloxycyclopentyl Derivatives to the Orthosteric Binding Site at Human and Rat A1R.

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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
adenosine     1.65 ± 0.24 0.048 ± 0.002 6.53 ± 0.07 21.15 ± 0.84 0.52 ± 0.22 0.079 ± 0.007 5.71 ± 0.17 12.86 ± 1.10
BnOCPA –CH2OH H 1.47 ± 0.33 0.068 ± 0.002 6.30 ± 0.09 14.75 ± 0.56 1.57 ± 0.54 0.054 ± 0.007 6.40 ± 0.11 19.42 ± 2.21
NECA     2.04 ± 0.07 0.049 ± 0.005 6.63 ± 0.06 20.97 ± 1.98 2.29 ± 0.39 0.066 ± 0.008 6.53 ± 0.04 15.84 ± 2.05
19 –CH2OH m-OMe 2.51 ± 0.36 0.041 ± 0.004 6.77 ± 0.02 24.64 ± 1.98 2.52 ± 0.31 0.083 ± 0.016 6.49 ± 0.03 13.22 ± 2.10
20 –CH2OH m-Br 0.56 ± 0.12 0.080 ± 0.016 5.84 ± 0.02 14.84 ± 3.83 1.50 ± 0.29 0.097 ± 0.008 6.17 ± 0.09 10.56 ± 0.81
21 –CH2OH p-Br 0.66 ± 0.02 0.065 ± 0.010 6.02 ± 0.06 16.72 ± 3.01 1.13 ± 0.09 0.066 ± 0.010 6.24 ± 0.03 16.05 ± 2.09
22 –CH2OH o-Cl 2.75 ± 0.14 0.055 ± 0.012 6.74 ± 0.10 22.22 ± 6.21 5.07 ± 0.26 0.090 ± 0.015 6.77 ± 0.06 12.10 ± 2.06
23 –CH2OH m-Cl 1.40 ± 0.08 0.051 ± 0.015 6.48 ± 0.09 24.12 ± 5.29 2.85 ± 0.22 0.060 ± 0.006 6.68 ± 0.06 17.13 ± 1.54
44 –CONHEt m-OMe 1.12 ± 0.09 0.110 ± 0.024 6.04 ± 0.10 10.59 ± 2.34 1.96 ± 0.13 0.141 ± 0.018 6.15 ± 0.06 7.41 ± 0.82
45 –CONHEt m-Br 0.83 ± 0.06 0.095 ± 0.011 5.94 ± 0.02 10.78 ± 1.28 2.95 ± 0.41 0.121 ± 0.012 6.38 ± 0.06 8.51 ± 0.82
46 –CONHEt p-Br 1.01 ± 0.06 0.076 ± 0.007 6.13 ± 0.03 13.55 ± 1.36 2.14 ± 0.14 0.058 ± 0.003 6.56 ± 0.01 17.30 ± 1.02
47 –CONHEt o-Cl 3.64 ± 0.33 0.044 ± 0.009 6.94 ± 0.08 25.70 ± 4.63 7.31 ± 0.61 0.046 ± 0.002 7.20 ± 0.03 21.98 ± 1.13
48 –CONHEt m-Cl 1.80 ± 0.17 0.040 ± 0.006 6.67 ± 0.05 27.96 ± 5.57 7.54 ± 0.30 0.073 ± 0.004 7.02 ± 0.03 13.90 ± 0.72
a

kon (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

b

koff (k4) for ligands determined as in footnote a.

c

Kinetic dissociation constant (pKd) for each ligand as determined from koff/kon.

d

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