Electrophilic derivatives of purines as irreversible inhibitors of A1 adenosine receptors

Abstract

Functionalized congeners derived from 1,3-dipropyl-8-phenylxanthine and from N 6 -phenyladenosine were derivatized to contain electrophilic groups (isothiocyanate, Nhydroxysuccinimide ester, maleimide, sulfonyl chloride, or α-haloacyl group) capable of reaction with nucleophiles on biopolymers. The goal was to inhibit chemically the A 1 adenosine receptor by using reactive agonist and antagonist ligands. Some of the electrophilic derivatives were synthesized through acylation of amine-functionalized congeners using hetero-or homobifunctional reagents available for protein cross-linking. The affinity for A 1 adenosine receptors was evaluated in competitive binding assays by using rat and bovine brain membranes. Several xanthine and adenosine thiourea derivatives prepared from 1,3-and l,4-phenylene diisothiocyanate (DITC) were potent irreversible inhibitors of adenosine receptors. Derivatives of m-DITC, at concentrations between 10 and 500 nM, irreversibly eliminated binding at 90% of the A 1 -receptor sites. Receptor affinity of both xanthine and adenosine derivatives containing distal phenylthiourea substituents was diminished by electron-donating groups on the ring.Adenosine modulates a variety of physiological functions. 1 It acts as an inhibitor of neuronal firing and the release of neurotransmitters, 1b an inhibitor of platelet aggregation, 1c a cardiac depressant and a vasodilator, 1d a vasoconstrictor 1e (e.g., in the renal afferent arterioles and in the skin), and an immunosuppressant. 1f Most of the physiological effects of adenosine result from binding to discrete membrane-bound adenosine receptors of the A 1 or A 2 subtypes. 1g The xanthine drugs caffeine and theophylline, and many synthetic analogues, 2 act as competitive antagonists at adenosine receptors.Alkylating or acylating ligands that form a stable covalent bond with a receptor have been synthesized for a number of receptors, including opiate, 3,4 phencyclidine, 5 adrenergic, 6 and benzodiazepine receptors. 7 The utility of such chemically irreversibly bound ligands for characterizing receptors in membranes and in physiological systems, 4,5 and in receptor identification, 8 has been demonstrated. For adenosine receptors, only photoaffinity labels 1g,9 have hitherto been described. We now report a set of reactive adenosine receptor ligands, derived from agonist 10 and antagonist 11 functionalized congeners. These ligands contain electrophilic acylating and alkylating groups capable of reaction with nucleophilic residues in proximity to the adenosine receptor binding site.

Molecular probes for muscarinic receptors: derivatives of the M1-antagonist telenzepine

“…Compounds 10-13, which contain the isothiocyanate group for chemical affinity labeling, were prepared from the corresponding phenylenediisothio-cyanate (DITC) derivatives (7,8), present in large stoichiometric excess to avoid formation of bis-adducts. Compound 15, which is a fluorescent eosin derivative intended for the technique of photosensitized labeling of proteins in proximity to the receptor (19), was also prepared from the amine congener 4 and the appropriate isothiocyanate.…”

“…Under irreversible conditions, at a concentration 83 times its K i value, 13 resulted in a 62% loss of Compounds 10 and 13 showed some selectivity for the forebrain versus cardiac receptors in irreversible binding. There is considerable potential for using these affinity labels and related derivatives as pharmacological probes (7,8,22), particularly if selectivity is demonstrated in further testing and structural modification.…”

“…Such molecular probes include irreversible affinity labels containing chemically-and photochemically-reactive groups (5,(7)(8)(9), fluorescent labels and other spectroscopic reporter groups (10)(11), biotinylated probes (10,12). We have developed a functionalized congener approach to the design of muscarinic ligands (13,14), which in this study is extended to sites for the attachment of reporter groups to high-affinity antagonists.…”

[3H]XAC (xanthine amine congener) is a radioligand for A2-adenosine receptors in rabbit striatum

“…Derivatives of XAC have been used as spectroscopic and chemical affinity probes at A 1 -adenosine receptors (Stiles and Jacobson, 1988;Jacobson et al, 1987Jacobson et al, , 1989b). An agarose affinity column containing an immobilized form of XAC was used successfully to concentrate A 1 -adenosine receptors from bovine brain (Olah et al, 1989) and purify to homogeneity (Nakata, 1989) the rat brain A 1 -adenosine receptor.…”

“…Materials XAC, (8-cyclopentyl-1,3-dipropyl-2-thioxanthine), N,N α -Me 2 -XAC (8- [2-dimethylaminoethyl[amino[carbonyl[methyl[oxy-phenyl]]]]]-1,3-dipropyl-xanthine), and the corresponding monomethyl XAC analog were synthesized by methods reported (Jacobsen et al, 1989a). m-and p-DITC-XAC isomers (1,3-dipropyl-8-[4-[[[[[2-[[[(isothiocyanatophenyl) (Jacobson et al, 1989b) -ethylcarboxamidoadenosine) and its N α -p-aminophenylacetyl derivative, PAPA-APEC, were prepared as described (Jacobson et al, 1990). PD115,]]]-1,3-dipropylxanthine) was the generous gift of Dr J. Bristol.…”