New 2,N6-Disubstituted adenosines: Potent and selective A1 adenosine receptor agonists

A number of adenosine analogues substituted in the 2- and N⁶-positions were synthesized and evaluated for affinity, functional potency and intrinsic activity at the A₁ and A_2A adenosine receptors (AR). Three classes of N⁶-substituents were tested; norbornen-2-yl (series 1), norborn-2-yl (series 2) and 5,6-epoxynorborn-2-yl (series 3). The halogens; fluoro, bromo, and iodo were evaluated as C-2 substituents. All compounds showed relatively high affinity (nanomolar) for the A₁AR and high potency for inhibiting (−)isoproterenol-stimulated cAMP accumulation in hamster smooth muscle DDT1 MF-2 cells with the 2-fluoro derivatives from each series having the highest affinity. All of the derivatives showed the same intrinsic activity as CPA. At the A2AAR, all of the derivatives showed relatively low affinity and potency (micromolar) for stimulating cAMP accumulation in rat pheochromocytoma PC-12 cells. The intrinsic activity of the derivatives compared to CGS 21680 was dependent upon the halogen substituent in the C-2 position with most showing partial agonist activity. Of particular interest is 2-iodo-N⁶-(2S-endo-norborn-2-yl)adenosine (5e), which is over 100-fold selective for the A₁AR, is a full agonist at this receptor subtype and has no detectable agonist activity at the A_2AAR.

2-Amino-3-aroyl-4,5-alkylthiophenes: Agonist allosteric enhancers at human A1 adenosine receptors

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A₁ adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A₁AR (hA₁AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a-i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a-h), three 3-aroyl-2-carboxy-4,5- dimethylthiophenes (15a-c), 10 2-amino-3-benzoyl-5,6-dihydro 4H-cyclopenta[b]thiophenes (17a-j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a-n), and 15 2-amino- 3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a-o). An in vitro assay employing the A₁AR agonist [¹²⁵I]ABA and membranes from CHO-K1 cells stably expressing the hA¹AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist- A₁AR-G protein ternary complex. Compounds 3a-i had little or no AE activity, and compounds 12a-h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds 17a-c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds 17a-j, 18a-n, and 19a-o. AE activity increased with the size of the -(CH₂)_n- bridge, n ) 3 < n ) 4 < n ) 5. The 3-carbethoxy substituents of 17a, 18a, and 19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA₁AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.

Potential antiarrhythmic and cardioprotective agents based on adenosine

N-Ethylcarboxamidoadenosine (12) was synthesised from adenosine (1) and the 6-chloro-2’,3’-O-isopropylidene-AT-ethylcarboxamidoadenosine (25) was synthesised from inosine (19). Employing molecular modelling techniques and the results from previous structure activity relationships it was possible to design and synthesise a N6-substituted N-ethylcarboxamidoadenosines which possessed an oxygen in the N6-substituent either in the form of an epoxide (which was obtained by cpoxidising an alkene with m-CPBA or dimethyldioxirane) or in the form of a cyclic ether as was the case for N6-((tetrahydro-2H--pyran--2-yl)methyl-N-ethylcarboxamidoadenosine (78). These compounds were tested for their biological activity at the A1 adenosine receptor by their ability to inhibit cAMP accumulation in DDT, MF2 cells. The EC50 values obtained indicated that the N6-(norborn-5-en-2-yl)-N-ethylcarboxamidoadenosines were the most potent. Of theseN6-(S-endo-norbrn-5-en-2-yI)-N-ethylcarboxaniidoadenosine (56) was the most potent (0.2 nM). N6-(exo-norborn-5-en-2-yl)-2-iodo-N-ethylcarboxamidoadenosine (79) was synthesised from guanosine (22) and was also evaluated for its potency at the A, receptor (24.8 ± 1.5 nM). At present 79 is being evaluated for its selectivity for the A1 receptor compared to the other three receptor subtypes (A2a, A2b, A3). A series of N6-(benzyl)-N-ethylcarboxamidoadenosines were synthesised with substitutions at the 4-position of the phenyl ring. Another series of compounds were synthesised which replaced the methylene spacer between the N6H and the N6-aromatic or lipophilic substituent The replacement groups -were carbonyl and trans-2- cyclopropyl moieties. The N6-acyl compounds were obtained by reacting 2’,3’-O- di(tert-butyldimethylsilyl)-AT-ethylcarboxamidoadenosinc (59) with the appropriate acid chloride and then deprotecting with lelrabutylammonium fluoride in tetrahydrofuran. The compound N6-(4-(1,2-dihydroxy)ethyl)benzyl-N- ethylcarboxamidoadenosine (125) was synthesised by the reaction of 4-(1,2-0- isopropylidene-ethyl)benzyl aminc (123) with 6-chloro-2,3-0-isopropylidene-N- ethylcarboxamidoadenosine (25). Compound 123 was synthesised from an epoxidation of vinylbenzyl phthalimide (118) followed by an acidic ring opening to yield the diol which was isopropylidenated to yield 4-(l,2-O-isopropylidene- elhyl)benzyl phlhalimide (122), It was hoped that the presence of the diol functionality in 125 would increase water solubility whilst maintaining potency at the A3 receptor.

N3-substituted xanthines as irreversible adenosine receptor antagonists

8-Cyclopentyl-3-(3-(4-fluorosulfonylbenzoyl)oxy)propyl-propylxanthine (44, FSCPX) has been reported to exhibit potent and selective irreversible antagonism of the A1 adenosine receptor when using in vitro biological preparations. However, FSCPX (44) suffers from cleavage of the ester linkage separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine pharmacophore when used in in vivo biological preparations or preparations containing significant enzyme activity, presumably by esterases. Cleavage of the ester linkage renders FSCPX (44) inactive in terms of irreversible receptor binding. In order to obtain an irreversible A1 adenosine receptor antagonist with improved stability, and to further elucidate the effects of linker structure on pharmacological characteristics, several FSCPX (44) analogues incorporating the chemoreactive 4-(fluorosulfonyl)phenyl moiety were targeted, where the labile ester linkage has been replaced by more stable functionalites. In particular, ether, alkyl, amide and ketone linkers were targeted, where the length of the alkyl chain was varied from between one to five atoms. Synthesis of the target compounds was achieved via direct attachment of the N-3 substituent to the xanthine. These compounds were then tested for their biological activity at the A1 adenosine receptor via their ability to irreversibly antagonise the binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, ( 9) to the A1 adenosine receptor of DDT1 MF-2 cells. For comparison, the xanthines were also tested for their ability to inhibit the binding of [3H]-4-(2-[7-amino-2-{furyl} {1,2,4}- triazolo{2,3-a} {1,3,5}triazin-5-ylamino-ethyl)]phenol ([3H]ZM241385, 36) to the A2A adenosine receptor of PC-12 cells. The results suggest that the length and chemical composition of the linker separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine ring contribute to the potency and efficacy of the irreversible A1 adenosine receptor ligands. Like FSCPX (44, IC50 A1 = 11.8 nM), all derivatives possessed IC50 values in the low nM range under in vitro conditions. Compounds 94 (IC50 A1 = 165 nM), 95 (IC50 A1 = 112 nM) and 96 (IC50 A1 = 101 nM) possessing one, three and five methylene spacers within the linkage respectively, exhibited potent and selective binding to the A1 adenosine receptor versus the A2A adenosine receptor. Compound 94 did not exhibit any irreversible binding at A1 adenosine receptors, while 95 and 96 exhibit only weak irreversible binding at A1 adenosine receptors. Those compounds containing a benzylic carbonyl separating the 4-(fluorosulfonyl)phenyl moiety from the xanthine ring in the form of an amide (119, IC50 A1 = 24.9 nM, and 120, IC50 A1 = 21 nM) or ketone (151, IC50 A1 = 14 nM) proved to be the most potent, with compound 120 exhibiting the highest selectivity of 132-fold for the A receptor over the A2A receptor. compounds 119, 120 and 151 also strongly inhibited the binding of [3H]DPCPX irreversibly (82%, 83% and 78% loss of [3H]DPCPX binding at 100 nM respectively). compounds 120 and 151 are currently being evaluated for use in in vivo studies. Structure-activity studies suggest that altering the 8-cycloalkyl group of A1 selective xanthines for a 3-substituted or 2,3-disubstituted styryl, combined with N-7 methyl substitution will produce a compound with high affinity and selectivity for the A2A adenosine receptor over the A1 adenosine receptor. Compound 167 (IC50 A2A = 264 nM) possessing 8-(m-chloro)styryl substitution and the reactive 4-(fluorosulfonyl)phenyl moiety separated from the xanthine ring via an amide linker in the 3-position (as for 119 and 120), exhibited relatively potent binding to the A2A adenosine receptor of PC-12 cells, with a 16-fold selectivity for that receptor over the A1 adenosine receptor. However, compound 167 exhibited only very weak irreversible binding at A2A adenosine receptors. Overall, at this stage of biological testing, compound 120 appears to possess the most advantageous characteristics as an irreversible antagonist for the A1 adenosine receptor. This can be attributed to its high selectivity for the A1 adenosine receptor as compared to the A2A adenosine receptor. It also has relatively high potency for the A1 adenosine receptor, a concentration-dependent and selective inactivation of A1 adenosine receptors, and unbound ligand is easily removed (washed out) from biological membranes. These characteristics mean compound 151 has the potential to be a useful tool for the further study of the structure and function of the A1 adenosine receptor.

Adenosine receptor agonists as potential therapeutic agents

Adenosine is a naturally occurring compound within the human body. It is known to exert a wide range of physiological effects. A range of compounds which elict a similar response to adenosine in vivo have been synthesised and evaluated as cardioprotective agents.

Agonist allosteric enhancers at A1 adenosine receptors

Pharmaceutical agents that may be protective to the heart during ischemia were targeted in this project. Analogs to a lead compound containing potential cardioprotective properties were prepared. In the pharmacological evaluation five compounds were found to be more potent than the lead compound.

Comonitoring of adenosine and dopamine using the wireless instantaneous neurotransmitter concentration system : proof of principle : laboratory investigation

Object

The authors of previous studies have demonstrated that local adenosine efflux may contribute to the therapeutic mechanism of action of thalamic deep brain stimulation (DBS) for essential tremor. Real-time monitoring of the neurochemical output of DBS-targeted regions may thus advance functional neurosurgical procedures by identifying candidate neurotransmitters and neuromodulators involved in the physiological effects of DBS. This would in turn permit the development of a method of chemically guided placement of DBS electrodes in vivo. Designed in compliance with FDA-recognized standards for medical electrical device safety, the authors report on the utility of the Wireless Instantaneous Neurotransmitter Concentration System (WINCS) for real-time comonitoring of electrical stimulation–evoked adenosine and dopamine efflux in vivo, utilizing fast-scan cyclic voltammetry (FSCV) at a polyacrylonitrile-based (T-650) carbon fiber microelectrode (CFM).
Methods

The WINCS was used for FSCV, which consisted of a triangle wave scanned between −0.4 and +1.5 V at a rate of 400 V/second and applied at 10 Hz. All voltages applied to the CFM were with respect to an Ag/AgCl reference electrode. The CFM was constructed by aspirating a single T-650 carbon fiber (r = 2.5 μm) into a glass capillary and pulling to a microscopic tip using a pipette puller. The exposed carbon fiber (the sensing region) extended beyond the glass insulation by ~ 50 μm. Proof of principle tests included in vitro measurements of adenosine and dopamine, as well as in vivo measurements in urethane-anesthetized rats by monitoring adenosine and dopamine efflux in the dorsomedial caudate putamen evoked by high-frequency electrical stimulation of the ventral tegmental area and substantia nigra.
Results

The WINCS provided reliable, high-fidelity measurements of adenosine efflux. Peak oxidative currents appeared at +1.5 V and at +1.0 V for adenosine, separate from the peak oxidative current at +0.6 V for dopamine. The WINCS detected subsecond adenosine and dopamine efflux in the caudate putamen at an implanted CFM during high-frequency stimulation of the ventral tegmental area and substantia nigra. Both in vitro and in vivo testing demonstrated that WINCS can detect adenosine in the presence of other easily oxidizable neurochemicals such as dopamine comparable to the detection abilities of a conventional hardwired electrochemical system for FSCV.
Conclusions

Altogether, these results demonstrate that WINCS is well suited for wireless monitoring of high-frequency stimulation-evoked changes in brain extracellular concentrations of adenosine. Clinical applications of selective adenosine measurements may prove important to the future development of DBS technology.

Wireless instantaneous neurotransmitter concentration system-based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring - laboratory investigation

Object  In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time.

Methods  The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme–linked electrode to measure glutamate; and 3) a multiple enzyme–linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig.

Results   The WINCS, which is designed in compliance with FDA-recognized consensus standards for medical electrical device safety, successfully measured dopamine, glutamate, and adenosine, both in vitro and in vivo. The WINCS detected striatal dopamine release at the implanted CFM during DBS of the MFB. The DBS-evoked adenosine release in the rat thalamus and MCS-evoked glutamate release in the pig cortex were also successfully measured. Overall, in vitro and in vivo testing demonstrated signals comparable to a commercial hardwired electrochemical system for FPA.

Conclusions  By incorporating FPA, the chemical repertoire of WINCS-measurable neurotransmitters is expanded to include glutamate and other nonelectroactive species for which the evolving field of enzyme-linked biosensors exists. Because many neurotransmitters are not electrochemically active, FPA in combination with enzyme-linked microelectrodes represents a powerful intraoperative tool for rapid and selective neurochemical sampling in important anatomical targets during functional neurosurgery.

Blunted adenosine A2a receptor function in platelets in patients with major depression.

There is provisional evidence of involvement of adenosine in depression. In this study, the second messenger intracellular calcium response in platelets was measured in patients with major depression and controls using spectrofluorometry. The primary result of this study was a statistically significantly blunted second messenger response to agonist stimulation in the depressed group compared to the control group at the 50 and 100 nM and 1 microM dosage levels. This suggests that dysregulation of the adenosine A2a receptor may be present in depression.

Phylogeny of the Australian freshwater crayfish Cherax destructor-complex (Decapoda:Parastacidae) inferred from four mitochondrial gene regions

The phylogenetic relationships among 32 individuals of Australian freshwater crayfish belonging to the Cherax destructor-complex were investigated using a dataset comprising sequences from four mitochondrial gene regions: the large subunit rRNA (16S rRNA), cytochrome oxidase I (COI), adenosine triphosphatase 6 (ATPase 6), and cytochrome oxidase III (COIII). A total of 1602 bp was obtained, and a combined analysis of the data produced a tree with strong support (bootstrap values 94–100%) for three divergent lineages, verifying the phylogenetic hypotheses of relationships within the C. destructor species-complex suggested in previous studies. Overall, sequences from the 16S rRNA gene showed the least variation compared to those generated from protein coding genes, which presented considerably greater levels of divergence. The level of divergence within C. destructor was found to be greater than that observed in other species of freshwater crayfish, but interspecific variation among species examined in the present study was similar to that reported previously.

Modifying the bitterness of selected oral pharmaceuticals with cation and anion series of salts

Purpose. NaCl has proven to be an effective bitterness inhibitor, but the reason remains unclear. The purpose of this study was to examine the influence of a variety of cations and anions on the bitterness of selected oral pharmaceuticals and bitter taste stimuli: pseudoephedrine, ranitidine, acetaminophen, quinine, and urea.
Method. Human psychophysical taste evaluation using a whole mouth exposure procedure was used.
Results. The cations (all associated with the acetate anion) inhibited bitterness when mixed with pharmaceutical solutions to varying degrees. The sodium cation significantly (P < 0.003) inhibited bitterness of the pharmaceuticals more than the other cations. The anions (all associated with the sodium cation) also inhibited bitterness to varying degrees. With the exception of salicylate, the glutamate and adenosine monophosphate anions significantly (P < 0.001) inhibited bitterness of the pharmaceuticals more than the other anions. Also, there were several specific inhibitory interactions between ammonium, sodium and salicylate and certain pharmaceuticals.
Conclusions. We conclude that sodium was the most successful cation and glutamate and AMP were the most successful anions at inhibiting bitterness. Structure forming and breaking properties of ions, as predicted by the Hofmeister series, and other physical-chemical ion properties failed to significantly predict bitterness inhibition.

The influence of sodium salts on binary mixtures of bitter-tasting compounds

In order to study potential mixture interactions among bitter compounds, selected sodium salts were added to five compounds presented either alone or as binary bitter- ompound mixtures. Each compound was tested at a concentration that elicited ‘weak’ perceived bitterness. The bitter compounds were mixed at these concentrations to form a subset of possible binary mixtures. For comparison, the concentration of each solitary compound was doubled to measure bitterness inhibition at the higher intensity level elicited by the mixtures. The following sodium salts were tested for bitterness inhibition: 100 mM sodium chloride (salty), 100 mM sodium gluconate (salty), 100 and 20 mM monosodium glutamate (umami), and 50 mM adenosine monophosphate disodium salt (umami). Sucrose (sweet) was also employed as a bitterness suppressor. The sodium salts differentially suppressed the bitterness of compounds and their binary combinations. Although most bitter compounds were suppressed, the bitterness of tetralone was not suppressed, nor was the bitterness of the binary mixtures that contained it. In general, the percent suppression of binary mixtures of compounds was predicted by the average percent suppression of its two components. Within the constraints of the present study, the bitterness of mixtures was suppressed by sodium salts and sucrose independently, with few bitter interactions. This is consistent with observations that the bitter taste system integrates the bitterness of multi-compound solutions linearly.