Neli Melman

N6-Substituted adenosine derivatives: selectivity, efficacy, and species differences at A3 adenosine receptors

The activation of the human A(3) adenosine receptor (AR) by a wide range of N(6)-substituted adenosine derivatives was studied in intact CHO cells stably expressing this receptor. Selectivity of binding at rat and human ARs was also determined. Among N(6)-alkyl substitutions, small N(6)-alkyl groups were associated with selectivity for human A(3)ARs vs. rat A(3)ARs, and multiple points of branching were associated with decreased hA(3)AR efficacy. N(6)-Cycloalkyl-substituted adenosines were full (</=5 carbons) or partial (>/=6 carbons) hA(3)AR agonists. N(6)-(endo-Norbornyl)adenosine 13 was the most selective for both rat and human A(1)ARs. Numerous N(6)-arylmethyl analogues, including substituted benzyl, tended to be more potent in binding to A(1) and A(3) vs. A(2A)ARs (with variable degrees of partial to full A(3)AR agonisms). A chloro substituent decreased the efficacy depending on its position on the benzyl ring. The A(3)AR affinity and efficacy of N(6)-arylethyl adenosines depended highly on stereochemistry, steric bulk, and ring constraints. Stereoselectivity of binding was demonstrated for N(6)-(R-1-phenylethyl)adenosine vs. N(6)-(S-1-phenylethyl)adenosine, as well as for the N(6)-(1-phenyl-2-pentyl)adenosine, at the rat, but not human A(3)AR. Interestingly, DPMA, a potent agonist for the A(2A)AR (K(i)=4nM), was demonstrated to be a moderately potent antagonist for the human A(3)AR (K(i)=106nM). N(6)-[(1S,2R)-2-Phenyl-1-cyclopropyl]adenosine 48 was 1100-fold more potent in binding to human (K(i)=0.63nM) than rat A(3)ARs. Dual acting A(1)/A(3) agonists (N(6)-3-chlorobenzyl- 29, N(6)-(S-1-phenylethyl)- 39, and 2-chloro-N(6)-(R-phenylisopropyl)adenosine 53) might be useful for cardioprotection.

Chronic administration of selective adenosine A1 receptor agonist or antagonist in cerebral ischemia

The effect of chronic administration of selective adenosine A1 receptor agonists and antagonists on the outcome of cerebral ischemia is entirely unknown. Therefore, we have investigated the impact of such regimens on the hippocampal adenosine A1 receptor density, and on the recovery from 10 min forebrain ischemia in gerbils. While acutely administered N6-cyclopentyladenosine (CPA) given at 0.02 mg/kg resulted only in a significant reduction of mortality, at 1 mg/kg it improved both survival and neuronal preservation in the hippocampal CA1 region. Acute treatment with 1,3-dipropyl-8-cyclopentylxanthine (CPX) significantly worsened the outcome and enhanced neuronal destruction. The effects of chronic administration of these drugs (15 days followed by 1 drug-free day) were opposite. Thus, although chronic CPA at 0.02 mg/kg did not have any effect at all, at 1 mg/kg both survival and neuronal preservation were significantly poorer than in controls, while chronic CPX resulted in a significant improvement of both measures. These results were not accompanied by adenosine A1 receptor up- or downregulation. Our study indicates that highly selective adenosine analogues may have therapeutic potential in treatment of cerebral ischemia/stroke and possibly other neurodegenerative disorders as well.

Acyl‐hydrazide derivatives of a xanthine carboxylic congener (XCC) as selective antagonists at human A2B adenosine receptors

The structure–activity relationships (SAR) of 8‐phenyl‐1,3‐dipropylxanthine derivatives in binding to recombinant human A2B adenosine receptors were explored, in order to identify selective antagonists. Based on the finding of receptor selectivity in MRS 1204, containing an N‐hydroxysuccinimide ester attached through the p‐position of the 8‐phenyl substituent [Jacobson et al. (1999): Drug Dev. Res., 47:45–53], a hydrazide and its more stable imide derivatives were synthesized. The hydrazide of XCC (8‐[4‐[[[carboxy]methyl]oxy]phenyl]‐1,3‐dipropylxanthine) was acylated with a variety of mono‐ and dicarboxylic acids. Ki values were determined in the adenosine receptor binding assays. At recombinant human A2B receptors expressed in membranes of HEK‐293 cells, antagonist radioligands used were the xanthine 125I‐ABOPX (125I‐3‐(4‐amino‐3‐iodobenzyl)‐8‐oxyacetate‐1‐propyl‐xanthine) and the nonxanthine antagonist [3H]ZM 241385 ([3H]4‐(2‐[7‐amino‐2‐{furyl}{1,2,4}triazolo{2,3‐a}{1,3,5}triazin‐5‐ylamino‐ethyl)phenol). The initial screening utilized rat A1/A2A receptors and human A3 receptors, and selected compounds were examined at the human A1/A2A subtypes. A 1,2‐dimethylmaleimide derivative, 14 (MRS 1595), bound to human A2B receptors with a Ki of 19 nM and proved to be selective vs. human A1/A2A/A3 receptors by 160‐, and 35‐fold, respectively. Enprofylline (3‐propylxanthine) is slightly selective for A2B receptors, suggesting removal of the 1‐propyl group; however, combination of the 1‐H‐3‐Pr and 8‐phenyl substituents eliminated the selectivity. Other potent and moderately selective A2B antagonists were a tetrahydrophthaloyl derivative 18b (MRS 1614, Ki value 10 nM) and amino acid conjugates of the XCC‐hydrazide, i.e., the glutarimide 24b (MRS 1626, Ki value 13 nM), and protected dipeptide 27 (MRS 1615, Ki value 11 nM). Drug Dev. Res. 47:178–188, 1999. Published 1999 Wiley‐Liss, Inc.

A SURVEY OF NONXANTHINE DERIVATIVES AS ADENOSINE RECEPTOR LIGANDS

The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [3H]PIA or [3H]CGS 21680 in rat brain membranes or [125I]AB-MECA in CHO cells stably transfected with rat A3 receptors. Several new classes of adenosine antagonists (e.g. 5-oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11-hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidinone, folic acid, and cytochalasin H and J bound to A3 receptors selectively. Moreover, cytochalasin A, which bound to A1 adenosine receptors with Ki value of 1.9 μM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A1 receptor binding.

Pyran Template Approach to the Design of Novel A3 Adenosine Receptor Antagonists.

A3 adenosine receptor antagonists have potential as anti‐inflammatory, anti‐asthmatic, and anti‐ischemic agents. We previously reported the preparation of chemical libraries of 1,4‐dihydropyridine (DHP) and pyridine derivatives and identification of members having high affinity at A3 adenosine receptors. These derivatives were synthesized through standard three‐component condensation/oxidation reactions, which permitted versatile ring substitution at five positions, i.e., the central ring served as a molecular scaffold for structurally diverse substituents. We extended this template approach from the DHP series to chemically stable pyran derivatives, in which the ring NH is replaced by O and which is similarly derived from a stepwise reaction of three components. Since the orientation of substituent groups may be conformationally similar to the 1,4‐DHPs, a direct comparison between the structure activity relationships of key derivatives in binding to adenosine receptors was carried out. Affinity at human A3 receptors expressed in CHO cells was determined vs. binding of [125I]AB‐MECA (N6‐(4‐amino‐3‐iodobenzyl)‐5′‐N‐methylcarbamoyladenosine). There was no potency‐enhancing effect, as was observed for DHPs, of 4‐styryl, 4‐phenylethynyl, or 6‐phenyl substitutions. The most potent ligands in this group in binding to human A3 receptors were 6‐methyl and 6‐phenyl analogs, 3a (MRS 1704) and 4a (MRS 1705), respectively, of 3,5‐diethyl 2‐methyl‐4‐phenyl‐4H‐pyran‐3,5‐dicarboxylate, which had Ki values of 381 and 583 nM, respectively. These two derivatives were selective for human A3 receptors vs. rat brain A1 receptors by 57‐fold and 24‐fold, respectively. These derivatives were inactive in binding at rat brain A2A receptors, and at recombinant human A2B receptors displayed Ki values of 17.3 and 23.2 μM, respectively. The selectivity, but not affinity, of the pyran derivatives in binding to the A3 receptor subtype was generally enhanced vs. the corresponding DHP derivatives. Drug Dev. Res. 48:171–177, 1999. Published 1999 Wiley‐Liss, Inc.

New base-altered adenosine analogues: Synthesis and affinity at adenosine A1 and A2A receptors

N6-Substituted adenosine analogues containing cyclic hydrazines or chiral hydroxy (ar)alkyl groups, designed to interact with the S2 and S3 receptor subregions, have been synthesized and their binding to the adenosine A1 and A2A receptors have been investigated. Examples of both types of compounds were found to exhibit highly selective binding (Ki in low nM range) to the rat A1 receptor.