(N)-methanocarba 2,N6-disubstituted adenine nucleosides as highly potent and selective A3 adenosine receptor agonists

Abstract

A series of ring-constrained (N)-methanocarba-5'-uronamide 2,N(6)-disubstituted adenine nucleosides have been synthesized via Mitsunobu condensation of the nucleobase precursor with a pseudosugar ring containing a 5'-ester functionality. Following appropriate functionalization of the adenine ring, the ester group was converted to the 5'-N-methylamide. The compounds, mainly 2-chloro-substituted derivatives, were tested in both binding and functional assays at human adenosine receptors (ARs), and many were found to be highly potent and selective A(3)AR agonists. Selected compounds were compared in binding to the rat A(3)AR to assess their viability for testing in rat disease models. The N(6)-(3-chlorobenzyl) and N(6)-(3-bromobenzyl) analogues displayed K(i) values at the human A(3)AR of 0.29 and 0.38 nM, respectively. Other subnanomolar affinities were observed for the following N(6) derivatives: 2,5-dichlorobenzyl, 5-iodo-2-methoxybenzyl, trans-2-phenyl-1-cyclopropyl, and 2,2-diphenylethyl. Selectivity for the human A(3)AR in comparison to the A(1)AR was the following (fold): the N(6)-(2,2-diphenylethyl) analogue 34 (1900), the N(6)-(2,5-dimethoxybenzyl) analogue 26 (1200), the N(6)-(2,5-dichlorobenzyl) and N(6)-(2-phenyl-1-cyclopropyl) analogues 20 and 33 (1000), and the N(6)-(3-substituted benzyl) analogues 17, 18, 28, and 29 (700-900). Typically, even greater selectivity ratios were obtained in comparison with the A(2A) and A(2B)ARs. The (N)-methanocarba-5'-uronamide analogues were full agonists at the A(3)AR, as indicated by the inhibition of forskolin-stimluated adenylate cyclase at a concentration of 10 microM. The N(6)-(2,2-diphenylethyl) derivative was an A(3)AR agonist in the (N)-methanocarba-5'-uronamide series, although it was an antagonist in the ribose series. Thus, many of the previously known groups that enhance A(3)AR affinity in the 9-riboside series, including those that reduce intrinsic efficacy, may be adapted to the (N)-methanocarba nucleoside series of full agonists.

Figure 1

The complex of the hA₃AR with (A) 18, (B) 24, and (C) 29 in the putative binding site. The nucleosides are displayed as ball-and-stick models, and the side chains of hA₃AR are shown as stick models. The H-bonding between ligand and hA₃AR is displayed in yellow. The A₃AR is represented by a tube model with a different color for each TM domain (TM3 in yellow, TM4 in green, TM5 in cyan, TM6 in blue, TM7 in purple). Residues that interacted with ligand in the putative binding site were; T94 (3.36), N150 (EL2), Q167 (EL2), F168 (EL2), S181 (5.42), M177 (5.38), V178 (5.39), N250 (6.55), I268 (7.39), S271 (7.42), and H272 (7.43).

Scheme 1

Reagents and conditions: i) 2,6-dichloropurine, DIAD, TPP, THF, r.t.; ii) RNH₂, TEA, MeOH; iii) 40% aq. MeNH₂, MeOH; iv) 10% CF₃COOH in MeOH, H₂O, 70°C; v) 6-chloro-2-iodopurine or 6-chloro-2-methylthiopurine or 6-chloro-2-aminopurine, DIAD, TPP, THF, r.t.; vi) 3-chlorobenzylamine, TEA, MeOH.

Scheme 2

Reagents and conditions: i) NBS, benzoyl peroxide in dry CCl₄, reflux, 3h; ii) potassium phthalimide, in dry DMF at 80°C, 3h; iii) NH₂NH₂, in EtOH; reflux, 24h; iv) CH₃I, K₂CO₃ in DMF at r.t. overnight; v) AcONH₄, NaCNBH₃ in dry MeOH, 48h; vi) CuI, (PPh₃)₂PdCl₂, in dry Et₂NH; vii) benzyl bromide, K₂CO₃ in DMF at r.t. overnight; viii) LiAlH₄, in dry THF, reflux, 3h; ix) (Boc)₂O, 10% Et₃N in MeOH, 45°C, 1 h; x) 2-bromoacetamide, K₂CO₃ in DMF at r.t. overnight; xi) 15% TFA in CH₂Cl₂, 45 min.

Scheme 3

Reagents and conditions: i) tert-butyl nitrite, methyl disulphide, acetonitrile, r.t. ii) aq. NaOH, i-PrOH,THF.

Chart 1

Chemical structures of adenosine derivatives used in the present study and containing a 9-ribose moiety (1), a 9-ribose-5′-N-methyluronamide moiety (2), or an (N)-methanocarba moiety (3 - 5).