We studied the stereoselective interaction between aryloxypropanolamines and the human 5-hydroxytryptamine1A (5-HT1A) receptor. R- and S-enantiomers of propranolol, penbutolol, and alprenolol were investigated for their ability to bind to human 5-HT1A wild-type and Asn386Val mutant receptors. Asn386 seemed to act as a chiral discriminator. Although both aryloxypropanol enantiomers displayed lower affinity for the mutant receptors, the affinities for the S-enantiomers were more affected. Receptor affinities of other structurally unrelated 5-HT1A ligands were not decreased by the mutation of Asn386 to valine. In addition, a series of analogues of propranolol with structural variation in the oxypropanolamine moiety was synthesized, and affinities for wild-type and Asn386Val mutant 5-HT1A receptors were determined. Both the hydroxyl and the ether oxygen atoms of the oxypropanol moiety seem to be required for binding at wild-type 5-HT1A receptors. The hydroxyl group of propranolol probably directly interacts with Asn386. The ether oxygen atom may be important for steric reasons but can also be involved in a direct interaction with Asn386. These findings are in agreement with the interactions of aryloxypropanolamines with Asn386 in rat 5-HT1A receptors that we previously proposed. The loss of affinity for propranolol by the Asn386Val mutation could be regained by replacement of the hydroxyl group of the ligand by a methoxy group. This modification of the propranolol structure has no effect on the affinity of both enantiomers for the wild-type 5-HT1A receptor, which provides an alternative hypothesis for the interaction of Asn386 with the oxypropanol oxygen atoms. According to this novel hypothesis, the oxypropanol oxygen atoms may both act as hydrogen bond acceptors from the NH2 group of Asn386.

We investigated the structural requirements for high 5-HT1A affinity of the agonist flesinoxan and its selectivity versus D2 receptors. For this purpose a series of arylpiperazine congeners of flesinoxan were synthesized and evaluated for their ability to displace [3H]-8-OH-DPAT and [3H]spiperone from their specific binding sites in rat frontal cortex homogenates and rat striatum, respectively. Variations were made in the N4-substituent and the arylpiperazine region. Effects of N4-substitution in the investigated compounds appeared to be quite similar for 5-HT1A- and D2-receptor affinity. Lipophilicity at a distance of four carbon atoms from the piperazine N4 atom seems to be the main contributing factor to affinity for both receptors. Our data show that the amide group in the flesinoxan N4-substituent is unlikely to interact with the 5-HT1A receptor but, instead, acts as a spacer. In contrast to the structure-affinity relationships (SARs) of the N4-substituents, selectivity for 5-HT1A versus D2 receptors was gained by the arylpiperazine substitution pattern of flesinoxan. Restriction of flexibility of the N4-(benzoylamino)ethyl substituent and its effect on 5-HT1A-receptor affinity and activity were also studied. Our data show that in the bioactive conformation, the N4-[(p-fluorobenzoyl)amino]ethyl substituent is probably directed anti-periplanar relative to the HN4 atom. These results were used to dock flesinoxan (1) and two of its congeners (27 and 33) into a model of the 5-HT1A receptor that we previously reported. Amino acid residues surrounding the N4-[(p-fluorobenzoyl)amino]ethyl substituent of flesinoxan and its congeners are also present in D2 receptors. In contrast, several residues that contact the benzodioxane moiety differ from those in D2 receptors. These observations from the 3D model agree with the 5-HT1A SAR data and probably account for the selectivity of flesinoxan versus D2 receptors.

In order to explore the structural requirements for high 5-HT1A affinity, a series of aryl-substituted N1-phenylpiperazines were synthesized and evaluated for their ability to displace [3H]-8-OH-DPAT from its specific binding sites in rat frontal cortex homogenates. We found 2-methoxy substitution to be favorable, while 4-methoxy substitution was detrimental for 5-HT1A affinity. Substitution with annelated rings at the 2,3-positions was highly favorable for all investigated compounds, with the exception of a pyrrole ring. All other substitutions, except fluoro, in this class of heterobicyclic phenylpiperazines decreased affinity in the order: ortho > para > meta. The loss of affinity in the ortho and para positions is probably due to steric factors: the substituents either cause steric hindrance with the receptor or prevent the compound from adopting the appropriate conformation for binding to the 5-HT1A receptor. Conformational analysis combined with structure-affinity relationships (SAR) indicates that our arylpiperazines may bind at the 5-HT1A receptor in a nearly coplanar conformation. Observed interactions of the compounds in our 5-HT1A receptor model appeared to be in agreement with SAR data. The aromatic part of the arylpiperazine moiety has pi-pi interactions with the aromatic residues Trp161 and Phe362 in helices IV and VI, respectively. The positively charged protonated basic nitrogen forms a hydrogen bond with the negatively charged Asp116 in helix III. The ammonium-aspartate complex is surrounded by aromatic residues Trp358 and Phe361 in helix VI. A lipophilic pocket is formed by Phe362, Leu366 (both helix VI), and the methyl group of Thr200 (helix V). In agreement with the model, addition of a methyl substituent to the structure of the benzodioxine analogue 12 in this region, yielding 13, is favorable for 5-HT1A receptor affinity. Unfavorable positions for substitution with bulky groups, like the 3- and 4-positions in the benzofuran compound 14, are explained by steric hindrance with the backbone atoms of helix V. Thus, we were able to rationalize the 5-HT1A SAR of existing N1-phenylpiperazines, as well as a series of newly synthesized bicyclic heteroarylpiperazines, in terms of receptor-ligand interactions. Several of these N4-unsubstituted compounds had affinities in the low-nanomolar range.

SummaryWe have developed a two-step enzyme immunoassay (EIA) that allows the quantitation of degradation products derived from fibrinogen (FbgDP) and that does not detect degradation products derived from cross-finked (XDP) or noncrosslinked fibrin (fdp).The EIA is based on two monoclonal antibodies (FDP-14 and Y-18), developed in our institute. FDP-14 is used as catching antibody. It complexes exclusively with degradation products, irrespective whether these are derived from fibrinogen or from fibrin. It does not complex with intact fibrinogen or fibrin. Y-18 is reactive with fibrinogen and fibrinopeptide A-comprising fibrinogen fragments. It is used, conjugated with horse-radish peroxy-dase, as tagging antibody.The FbgDP-EIA is highly specific, accurate and sensitive. The coefficient of variation is between 3 and 8%; the lower detection limit is less than 0.025 μg/ml.The assay has been applied to plasma from patients with suspected disseminated intravascular coagulation (DIC), to plasma from patients undergoing streptokinase (SK) therapy for acute myocardial infarction and to plasma from newborn babies.DIC patients had no or very low levels of FbgDP, but high levels of other degradation products, SK-treated patients showed high levels of degradation products two hours after termination of the SK infusion. A considerable fraction of these degradation products was shown to be FbgDP. Plasma from newborn babies contained elevated levels of FbgDP associated with prolonged prothrombin times.