5'-N-substituted carboxamidoadenosines as agonists for adenosine receptors.

Novel as well as known 5'-N-substituted carboxamidoadenosines were prepared via new routes that provided shorter reaction times and good yields. Binding affinities were determined for rat A1 and A2A receptors and human A3 receptors. EC50 values were determined for cyclic AMP production in CHO cells expressing human A2B receptors. On all receptor subtypes relatively small substituents on the carboxamido moiety were optimal. Selectivity for the A3 receptor was found for several analogues (1a, 1d, 1h, and 1k). On A1 receptors a number of compounds, but not 5'-N-ethylcarboxamidoadenosine (NECA, 1b), showed small GTP shifts, which could be indicative of lower intrinsic activities at the A1 receptor. At the A2B receptor, derivatives 1i-k with modified ethyl substituents had reduced activities compared to the A2B reference agonist NECA (1b). Thiocarboxamido derivatives (8b and 8c) displayed considerable although decreased A2B receptor activity. The X-ray structure determination of compound 8b was carried out. Due to intramolecular hydrogen bonding between the carboxamido NH and the purine N3 in the crystal structure, the ribose moiety of this compound is in a syn conformation. However, theoretical calculations support that NECA (1b), and less so 8b, can readily adopt both the syn and the anti conformation, therefore not excluding the proposed anti mode of binding to the receptor.

A functional screening of adenosine analogues at the adenosine A2B receptor: a search for potent agonists.

Various adenosine analogues were tested at the adenosine A2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A2B receptors. 5'-N-Substituted carboxamidoadenosines were most potent. 5'-N-Ethylcarboxamidoadenosine (NECA) was most active with an EC50 value of 3.1 microM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N6) of the purine ring system. The most active N6-substituted derivative N6-methyl-NECA was 5 fold less potent than NECA. C8- and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3- and 7-deazaanalogues were not active.

Derivatives of the triazoloquinazoline adenosine antagonist (CGS 15943) having high potency at the human A2B and A3 receptor subtypes.

The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1, 5-c]quinazolin-5-amine (CGS 15943) binds nonselectively to human A1, A2A, and A3 receptors with high affinity. Acylated derivatives and one alkyl derivative of the 5-amino group and other modifications were prepared in an effort to enhance A2B or A3 subtype potency. In general, distal modifications of the N5-substituent were highly modulatory to potency and selectivity at adenosine receptors, as determined in radioligand binding assays at rat brain A1 and A2A receptors and at recombinant human A3 receptors. In Chinese hamster ovary cells stably transfected with human A2B receptor cDNA, inhibition of agonist-induced cyclic AMP production was measured. An N5-(2-iodophenyl)acetyl derivative was highly selective for A2A receptors. An (R)-N5-alpha-methyl(phenylacetyl) derivative was the most potent derivative at A3 receptors, with a Ki value of 0.36 nM. A bulky N5-diphenylacetyl derivative, 13, displayed a Ki value of 0. 59 nM at human A3 receptors and was moderately selective for that subtype. Thus, a large, nondiscriminating hydrophobic region occurs in the A3 receptor in proximity to the N5-substituent. A series of straight-chain N5-aminoalkylacyl derivatives demonstrated that for A2B receptors the optimal chain length occurs with three methylene groups, i.e., the N5-gamma-aminobutyryl derivative 27 which had a pA2 value of 8.0 but was not selective for A2B receptors. At A1, A2A, and A3 receptors however the optimum occurs with four methylene groups. An N5-pivaloyl derivative, which was less potent than 27 at A1, A2A, and A3 receptors, retained moderate potency at A2B receptors. A molecular model of the 27-A2B receptor complex based on the structure of rhodopsin utilizing a "cross-docking" procedure was developed in order to visualize the environment of the ligand binding site.

Synthesis and copper-dependent antimycoplasmal activity of amides and amidines derived from 2-amino-1,10-phenanthroline.

A series of both aliphatic and aromatic amides and aromatic amidines derived from 2-amino-1,10-phenanthroline (3) according to the Topliss scheme were synthesized and subsequently tested for antimycoplasmal potency. Although the compounds themselves showed no activity, in the presence of a nontoxic copper concentration of 40 microM all compounds appeared to be very active against Mycoplasma gallisepticum K154. The most active compounds were found in the amide series and show growth inhibition in the nanomolar range. These compounds are 4 times more active than tylosin, a macrolide antibiotic, which is used therapeutically in veterinary practice. In the presence of copper, amides derived from 3 are more active than corresponding amidines. Increased activity following derivatization of 3 may be due to the presence of a third coordination site for copper in the title compounds. Evaluation of biological data revealed that antimycoplasmal activity of amides derived from 3 is dependent on lipophilicity. For these amides a good linear correlation was found between antimycoplasmal activity and hydrophobic fragmental values for substituents considered. This quantitative structure-activity relationship study indicated that antimycoplasmal activity was increased upon a decrease of these hydrophobic fragmental values.

Synthesis and copper-dependent antimycoplasmal activity of 1-amino-3-(2-pyridyl)isoquinoline derivatives. 2. Amidines.

In our search for new compounds with antimycoplasmal activity, a series of aromatic amidines derived from 1-amino-3-(2-pyridyl)isoquinoline (1) was synthesized. In the presence of 40 microM copper the most active compounds show growth inhibition of Mycoplasma gallisepticum in the nanomolar range. These compounds are 3 times as active as tylosin, an antimycoplasmal therapeutic agent that is used in veterinary practice. In the presence of copper, amidines derived from 1 are 2-3 times more active than the corresponding amides. Furthermore it was established that for these compounds too, the presence of a 2,2'-bipyridyl moiety is a necessary prerequisite for antimycoplasmal activity. As for the amides, antimycoplasmal activity of amidines derived from 1 is dependent on the hydrophobic fragmental value of the aromatic nucleus of the amidine moiety. A quantitative structure-activity relationship established the optimal hydrophobic fragmental value of this part of the molecule to be zero.

Synthesis and copper-dependent antimycoplasmal activity of 1-amino-3-(2-pyridyl)isoquinoline derivatives. 1. Amides.

In order to investigate the antimycoplasmal activity of compounds structurally related to 2,2'-bipyridyl, a series of both aliphatic and aromatic amides derived from 1-amino-3-(2-pyridyl)isoquinoline were synthesized. The most active compounds appeared to be as active as Tylosin, an antimycoplasmal therapeutic that is used in veterinary practice, in the presence of a small nontoxic amount of copper. Furthermore, it was found that antimycoplasmal activity depends on the hydrophobic fragmental value of amide residue. A quantitative structure-activity relationship established the optimal hydrophobic fragmental value of the amide residue to be 0.30.

Substituent effects during the rat liver aldehyde dehydrogenase catalyzed oxidation of aromatic aldehydes.

The influence of the steric hindrance of halogen substituents was investigated in vitro by measuring the activity of yeast aldehyde dehydrogenase (aldehyde: NAD(P)+ oxidoreductase, EC 1.2.1.5) and of aldehyde dehydrogenases in subcellular rat liver fractions with a series of ortho- and para-halo-substituted benzaldehydes as substrates. Upon an increase in the size of the halogen substituent (F, Cl, Br), the reactivity of yeast aldehyde dehydrogenase to ortho-substituted benzaldehydes decreased drastically. The same phenomenon was observed with the unspecific aldehyde dehydrogenases in three rat liver fractions; cytoplasm, mitochondria and microsomes. The corresponding para-halobenzaldehydes (F, Cl, Br, I) did not reveal large differences in reactivity to the various rat liver aldehyde dehydrogenases. The aldehyde dehydrogenases in the rat liver microsomal fraction exhibited most clearly the regiospecificity. Enzymatic oxidation of 4-bromobenzaldehyde was more than 30-times faster then the ortho-isomer. The findings in this investigation confirm the suggestion that the steric hindrance of bulky ortho-substituents of benzaldehydes account for the slowing down of the aldehyde dehydrogenase-catalyzed oxidation of benzaldehydes to corresponding benzoic acids. The enzymatic oxidation of microsomal aldehyde dehydrogenase is strongly influenced by steric effects of benzaldehydes, bearing a halogen in ortho-position. We think that the microsomal aldehyde dehydrogenase might be the principal enzyme responsible for oxidation of halobenzaldehydes in rat liver.

Endocardial catheter mapping: wire skeleton technique for representation of computed arrhythmogenic sites compared with intraoperative mapping.

Guiding surgical therapy of ventricular tachycardia by preoperative endocardial catheter mapping necessitates improvement of the accuracy of localization of the arrhythmogenic site. We therefore used a new mathematical cineradiographic method during catheter mapping to compute the position of left ventricular arrhythmogenic sites relative to three anatomic reference points: the centers of aortic and mitral valve ostia and the left ventricular apex. To enable the surgeon to identify the position of the computed sites, a wire skeleton (one for each patient) representing a single or multiple arrhythmogenic site(s) relative to the anatomic reference points was constructed. This wire skeleton was inserted into the left ventricular cavity during surgery. Side branches of the device indicated preoperatively localized arrhythmogenic sites. Results in eight consecutive patients were compared with those of intraoperative simultaneous mapping of 64 endocardial sites. Sixteen morphologically distinct monomorphic ventricular tachycardias were mapped by catheter and 15 by intraoperative mapping. In 12 ventricular tachycardias an identical morphology was recorded during both techniques. The distance between arrhythmogenic sites localized with both methods was 1 cm or less in 11 of these 12 ventricular tachycardias and 2 cm in one ventricular tachycardia. These results indicate that endocardial catheter mapping combined with wire skeleton representation of computed positions of arrhythmogenic sites is reliable for guiding surgical therapy of ventricular tachycardia and since some of the ventricular tachycardias were inducible only during either preoperative or intraoperative mapping, both techniques have an additive value. In addition, the wire skeleton proved convenient during surgery by identifying the arrhythmogenic sites.

Endocardial catheter mapping: validation of a cineradiographic method for accurate localization of left ventricular sites.

To guide surgical therapy for ventricular tachycardia by preoperative endocardial catheter mapping, accurate anatomic localization of arrhythmogenic sites is mandatory. For this reason we developed a mathematical cineradiographic method to compute left ventricular sites relative to three anatomic reference points: the centers of aortic and mitral valve ostia and the left ventricular apex. To validate the method 14 epicardial left ventricular markers were implanted in four dogs to simulate arrhythmogenic sites. Distances between markers and the anatomic references were calculated and the results were compared with postmortem measurements. The difference between calculated and measured distances was 0.5 +/- 3.1 mm (mean +/- SD), confirming accurate localization of anatomic marker sites. However, in surgery the results have to be displayed in a practically applicable, unambiguous way. Therefore, wire skeletons were constructed to represent calculated endocardial marker sites relative to the anatomic reference points. To validate this approach, 14 markers were implanted in the left ventricular subendocardium in four dogs. Wire skeletons were constructed, one for each marker site, and inserted postmortem into the left ventricular cavity via a 2 cm incision. In all cases the correct indication of a marker site by the corresponding wire skeleton was confirmed by fluoroscopic inspection in multiple projections. This wire skeleton technique may enhance the practical usefulness of preoperative endocardial catheter mapping.