Volume 12, Issue 2, Pages 237-247 (February 2005) A Neoceptor Approach to Unraveling Microscopic Interactions between the Human A2A Adenosine Receptor and Its Agonists  Kenneth A. Jacobson, Michihiro Ohno, Heng T. Duong, Soo-Kyung Kim, Susanna Tchilibon, Michal Cˇesnek, Antonín Holý, Zhan-Guo Gao  Chemistry & Biology  Volume 12, Issue 2, Pages 237-247 (February 2005) DOI: 10.1016/j.chembiol.2004.12.010 Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 1 Structures of Nonselective Adenosine Receptor Ligands (3, 4) and Several Amine Derivatives (1, 2) Previously Studied at A2AAR Neoceptors Chemistry & Biology 2005 12, 237-247DOI: (10.1016/j.chembiol.2004.12.010) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 2 Synthesis of N6-Derivatized Adenosine Analogs 5 and 6 Reagents: (A) (i) ethylendiamine, rt., 24 hr; (ii) 1 N HCl, MeOH-H2O (1:1), 55°C, 36 hr, 65%. (B). (i) 2,2-dimethoxypropane, p-TsOH, acetone, 4 hr, 85%; (ii) TBDMS-Cl, imidazole, pyridine, 1 hr, 83%; (iii) guanidine solution, DABCO, 3 hr, 70%; (iv) Bu4N+F-, THF, 1hr, 95%; (v) KMnO4, KOH, H2O, 5 hr, 50%; (vi) H2SO4, 3 hr, 60°C, 84%; (vii) (a) ClCOOEt, N,N- i-Pr2NEt, DMF, 0°C; (b) EtNH2 in MeOH, 0°C 1 hr, RT 24 hr, 50%. Chemistry & Biology 2005 12, 237-247DOI: (10.1016/j.chembiol.2004.12.010) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 3 Synthetic Routes to Various Mono- and Disubstituted Adenosine-5′-Uronamide Derivatives (A) Synthesis of adenosine-5′-uronamide analogs 1 and 8–10. (B) Synthesis of N6-(2-methylbenzyl)adenosine-5′-uronamide analogs 12–15. Reagents: (i) CrO3, AcOH, 18%; (ii) TMSCHN2, MeOH, 88%; (iii) RNH2, DMF, 60°C; (iv) 1N HCl MeOH-H2O (1:1) 55°C, 2 d; (v) RP-SiO2 C18, for 7, Amberlite CG-50, for 11 and 12–15; (vi) Boc-NH(CH2)nNH2, iPr2NEt, DMF, 60°C, 54%–97%; (vii) Amberlite CG-50, 13%–67%; (viii) 2-MeBnBr; (ix) ethylamine or ethanolamine, MeOH, heat, Dimroth rearrangement and aminolysis of ester. Chemistry & Biology 2005 12, 237-247DOI: (10.1016/j.chembiol.2004.12.010) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 4 Pharmacological Characterization of Two Neoagonists Indicates Selective Interaction with Neoceptors Derived from the A2AAR Binding (A and B) and functional (C and D) effects of two adenosine derivatives, the hydrazide derivative 7 (A) and the N6-(2-methylbenzyl)-5′-aminoethyluronamide derivative 14 (B) at WT (■) and mutant A2AARs (T88D [▴], N181D [♦], and Q89D [▾]) transiently expressed in COS-7 cells. In the binding experiments, cell membranes (10–20 μg protein) were incubated with the radiolabeled antagonist [3H]ZM241385 (2.0 nM) in duplicate, together with increasing concentrations of the competing nucleoside, in a final volume of 0.4 ml Tris-HCl buffer (50 mM, pH 7.4) at 25°C for 120 min. Results were from a representative experiment performed in duplicate. The Ki values listed in Table 1 were from at least three separate experiments. In the functional experiments, cells expressing WT or mutant receptors were then treated with agonist 7 (C) or 14 (D) in the presence of rolipram (10 μM) and adenosine deaminase (3 units/ml) and incubated at 37°C for 1 hr. cAMP accumulation was determined using a competitive protein binding method [29]. The EC50 values (n = 3) determined for stimulation of cAMP formation were (nM, mean ± SEM): WT, 826 ± 138; T88D, 2970 ± 980; Q89D, 5.1 ± 0.8; N181D, 120 ± 22 for 7; and WT, 5800 ± 1230; T88D, 12,600 ± 3200; Q89D, 58 ± 12; N181D, 52 ± 6 for 14. Chemistry & Biology 2005 12, 237-247DOI: (10.1016/j.chembiol.2004.12.010) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 5 Proposed Modes of Docking of Two Neoagonists in Rhodopsin-Based Homology Models of Neoceptors Derived from the A2AAR The binding site of (A) the N181D-A2AAR/1 and (B) the Q89D-A2AAR/7 complex. All ligands are displayed as ball-and-stick models in the atom-by-atom color, and the side chains of human A2AAR are shown as stick models. The H bonding between ligand and the mutant receptor is displayed in yellow. The A2AAR is represented by a tube model with a different color for each TM domain (TM3 in yellow, TM5 in green, TM6 in cyan, and TM7 in purple). Chemistry & Biology 2005 12, 237-247DOI: (10.1016/j.chembiol.2004.12.010) Copyright © 2005 Elsevier Ltd Terms and Conditions