Structure-activity relationships of 9-alkyladenine and ribose-modified adenosine derivatives at rat A3 adenosine receptors

Abstract

9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5'- (N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3-6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(beta-D-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 microM. 2-Chloro-9-[2-amino-2,3-dideoxy-beta-D-5-(methylcarbamoyl)- arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3'-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3'-OH and 4'-CH2OH groups of adenosine are not required for activation at A3 receptors. A number of 2',3'-dideoxyadenosines and 9-acyclic-substituted adenines appear to inhibit adenylyl cyclase at the allosteric "P" site.

Figure 1

Structures of adenosine (1 and 2) and adenine (3 and 4) derivatives studied as adenosine receptor A₃ agonists and A₁/A₂ antagonists, respectively.

Figure 2

Agonist-elicited inhibition of adenylyl cyclase via rat A₃ receptors in transfected CHO cells: circles, NECA; squares, Cl-IB-MECA triangles, compound 35.

Scheme 1^a

^aReagents: (a) 3-iodobenzylamine-HCl, triethylamine, EtOH, rt; (b) CH₃I, K₂CO₃, DMF; (c) NH₂NH₂; (d) NH₂CH₃/THF; (e) DMF, triethylamine, CH₃O₂CCH₂NH₂-HCl; (f) CH₃(CH₂)₂NH₂; (g) CH₃(CH₂)₅NH₂; (h) NaOCH₃, MeOH; (i) NaSCH₃, DMF–DME; (j) NaSH, pyridine.

Scheme 2^a

^a Reagents: (a) R’I (9, 12), R’Br (13), or (2,2-dimethyl-1,3-dioxolan-4-yl)methyl p-toluenesulfonate, R (10, 47) or S (ll), and K₂CO₃, DMF; (b) 1 N HCl, 90 °C, 1 h; (c) NaSCH₃, DMF.

Scheme 3^a

^a Reaction conditions: (a) Ac₂O, pyridine, rt, 24 h; (b) SnCl₄, MeCN, N⁶-(3-iodobenyl)-2-chloroadenine,50 °C; (c) conc NH₄OH, reflux.

Scheme 4^a

^a Reagents: (a) i. CS₂, NaH, MeI, THF, ii. Bu₃SnH, Et₃B, benzene; (b) NH₃/MeOH; (c) RuO₂, NaIO₄, CHCl₃:CH₃CN:H₂O (2:2:3); (d) MeOH, EDAC, DMAP; (e) CH₃NH₂/THF; (f) H₂SO₄, Ac₂O, AcOH.

Scheme 5^a

^a Reagents: (a) TMSOTf, Cl(CH)₂Cl; (b) NH₃MeOH; (c) PhOC(S)Cl, DMAP, AcCN; (d) n-Bu₃SnH, Et₃B, benzene.

Scheme 6^a

^a Reagents: (a) methanesulfonyl chloride, pyridine, CH₂Cl₂; (b) NaN₃, DMF, 100 °C; (c) PPh₃, NH₄OH, THF–MeOH; (d) DAST.