Synthesis and pharmacological investigation of novel 2-aminothiazole-privileged aporphines

A series of apomorphine ((-)-1, APO)-derived analogues ((+/-)-3, (-)-4-(-)-6) were designed and synthesized by hybridizing APO with a privileged 2-aminothiazole functionality which was lent from the orally available anti-parkinsonian drug, pramipexole (2). Among these hybridized compounds, catecholic aporphine (-)-6 shows good affinity at the D(2) receptor with K(i) of 328nM, slightly less potent (3-fold), but more selective against the D(1) receptor than that of the parent compound, APO. Although possessing reduced affinity at the D(2) receptor, aporphines 15 and 18 show significant potency at both the D(1) and 5-HT(1A) receptors. The former compound is equipotent at both receptors (K(i): 116 and 151nM, respectively), while the latter is 8-fold more potent at the D(1) (K(i): 78nM) than at the 5-HT(1A) receptors (K(i): 640nM). These results indicate that the catechol fragment is critical for the D(2) receptor binding of the anti-parkinsonian drug, APO ((-)-1), but not necessary for binding at the D(1) and 5-HT(1A) receptors.     

Design, synthesis, and pharmacological evaluation of novel tetrahydroprotoberberine derivatives: selective inhibitors of dopamine D₁ receptor

A series of new tetrahydroprotoberberine (THPB) derivatives were designed, synthesized, and tested for their binding affinity towards dopamine (D(1) and D(2)) and serotonin (5-HT(1A) and 5-HT(2A)) receptors. Many of the THPB compounds exhibited high binding affinity and activity at the dopamine D(1) receptor, as well as high selectivity for the D(1) receptor over the D(2), 5-HT(1A), and 5-HT(2A) receptors. Among these, compound 19c exhibited a promising D(1) receptor binding affinity (K(i)=2.53nM) and remarkable selectivity versus D(2)R (inhibition=81.87%), 5-HT(1A)R (inhibition=61.70%), and 5-HT(2A)R (inhibition=24.96%). Compared with l-(S)-stepholidine (l-SPD) (D(1)K(i)=6.23nM, D(2)K(i)=56.17nM), compound 19c showed better binding affinity for the D(1) receptor (2.5-fold higher) and excellent D(2)/D(1) selectivity. Functional assays found compounds 18j, 18k, and 19c are pure D(1) receptor antagonists. These results indicate that removing the C10 hydroxy group and introducing a methoxy group at C11 of the pharmacophore of l-SPD can reverse the function of THPB compounds at the D(1) receptor. These results are in accord with molecular docking studies.     

Synthesis and in vitro binding of N-phenyl piperazine analogs as potential dopamine D3 receptor ligands

A series of N-(2-methoxyphenyl)piperazine and N-(2,3-dichlorophenyl)piperazine analogs were prepared and their affinities for dopamine D(2), D(3), and D(4) receptors were measured in vitro. Binding studies were also conducted to determine if the compounds bound to sigma (sigma(1) and sigma(2)) and serotonin (5-HT(1A), 5-HT(2A), 5-HT(2B), 5-HT(2C), 5-HT(3), 5-HT(4), 5-HT(5), 5-HT(6), and 5-HT(7)) receptors. The results of the current study revealed a number of compounds (12b, 12c, 12e, and 12g) having a high affinity for D(3) (K(i) at D(3) receptors ranging from 0.3 to 0.9 nM) versus D(2) (K(i) at D(2) receptors ranging from 40 to 53 nM) receptors and a log P value indicating that they should readily cross the blood brain barrier (log P = 2.6-3.5). All of the compounds evaluated in this study had a high affinity for serotonin 5-HT(1A) receptors. These compounds may be useful as probes for studying the behavioral pharmacology of the dopamine D(3) receptor, as well as lead compounds for the development of radiotracers for studying D(3) receptor regulation in vivo with the functional imaging technique, positron emission tomography.     

7-Arylpiperazinylalkyl and 7-tetrahydroisoquinolinylalkyl derivatives of 8-alkoxy-purine-2,6-dione and some of their purine-2,6,8-trione analogs as 5-HT(1A), 5-HT(2A), and 5-HT(7) serotonin receptor ligands

On the basis of our earlier studies with the serotonin receptor ligands in the group of 1,3-dimethyl-3,7-dihydropurine-2,6-dione derivatives, a series of new arylpiperazinylalkyl and tetrahydroisoquinolinylalkyl analogs of 8-alkoxy-1,3-dimethyl-3,7-dihydropurine-2,6-dione (10-25) and 1,3-dimethyl-7,9-dihydro-3H-purine-2,6,8-trione (26-30) were synthesized and their 5-HT(1A), 5-HT(2A), and 5-HT(7) receptor affinities were determined. The new compounds 17, 18, 20, and 21 were found to be highly active 5-HT(1A) receptor ligands (K(i)=11-19nM) with diversified affinity for 5-HT(2A) receptors (K(i)=15-253nM). Compounds 12, 13, 15, and 19 were moderately potent 5-HT(2A) ligands (K(i)=23-57nM), whereas 17, 18, 24, and 25 showed distinct affinity for 5-HT(7) receptors (K(i)=51-83nM). Purine-2,6,8-triones showed weak affinities for 5-HT(1A) and 5-HT(7) receptors; among them, 27 and 29 were classified as 5-HT(2A) receptor ligands. The selected compounds 17 and 21 were pharmacologically evaluated to determine their functional activities at pre-(hypothermia in mice) and post-(lower lip retraction in rats) synaptic 5-HT(1A) receptors. Compound 17 showed features of a potential agonist of pre- and post-synaptic 5-HT(1A) receptors, whereas 21 was classified as a potential, weak partial agonist of postsynaptic sites. Last of all, the most interesting compound 17 tested in behavioral models showed potential anxiolytic and antidepressant activities.     

Benzimidazole derivatives. Part 5: design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new mixed benzimidazole-arylpiperazine derivatives were designed by incorporating in general structure III the pharmacophoric elements of 5-HT(1A) and 5-HT(3) receptors. Compounds 1-11 were synthesized and evaluated for binding affinity at both serotoninergic receptors, all of them exhibiting high 5-HT(3)R affinity (K(i)=10-62nM), and derivatives with an o-alkoxy group in the arylpiperazine ring showing nanomolar affinity for the 5-HT(1A)R (K(i)=18-150nM). Additionally, all the synthesized compounds were selective over alpha(1)-adrenergic and dopamine D(2) receptors (K(i)>1000-10,000nM). Compound 3 was selected for further pharmacological characterization due to its interesting binding profile as mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both receptors (5-HT(1A): K(i)=18.0nM, 5-HT(3): K(i)=27.2nM). In vitro and in vivo findings suggest that this compound acts as a partial agonist at 5-HT(1A)Rs and as a 5-HT(3)R antagonist. This novel mixed 5-HT(1A)/5-HT(3) ligand was also effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test, suggesting a potential interest in the treatment of cognitive dysfunction.     

The influence of substitution at aromatic part of 1,2,3,4-tetrahydroisoquinoline on in vitro and in vivo 5-HT(1A)/5-HT(2A) receptor activities of its 1-adamantoyloaminoalkyl derivatives.

Further structure-activity relationship (SAR) studies with the 1,2,3,4-tetrahydroisoquinoline (THIQ) class of 5-HT(1A) ligands led to the synthesis of new 1-adamantoyloaminoalkyl derivatives. The impact of substituent variations in the aromatic part of THIQ moiety on 5-HT(1A) and 5-HT(2A) receptor affinities, as well as in vivo functional properties of the investigated compounds were discussed. It was found that those modifications reduced the binding affinity for 5-HT(1A) receptors (in comparison with unsubstituted THIQ derivatives); however, the majority of new compounds still remained potent 5-HT(1A) ligands (K(i)=4.9-46 nM) and most of them showed features of partial agonists of postsynaptic 5-HT(1A) receptors. At the same time, their 5-HT(2A) receptor affinity was slightly increased (K(i)=40-1475 nM), which resulted in a loss of 5-HT(2A)/5-HT(1A) selectivity. 5-Br,8-OCH3 derivative-the most potent, mixed 5-HT(1A)/5-HT(2A) ligand-produced activation of presynaptic 5-HT(1A) receptors and showed properties of a 5-HT(2A) receptor antagonist.     

Novel aryloxy-8-azabicyclo[3.2.1]oct-3-enes with 5-HT transporter and 5-HT1A affinity

Joining aryl 8-azabicyclo[3.2.1]oct-3-enes with aryloxyethanes and aryloxypropanes produces novel series of compounds 11 and 12 with potent 5-HT-T affinity and moderately potent 5-HT(1A) affinity. Moreover, several of these compounds possess functional 5-HT(1A) antagonism. Optimal compounds are, 4-indolyloxyethane 21, 4-indolyloxypropanes 25, and 27, which possess potent 5-HT-T affinity (5-HT-T K(i): 21: 1.2nM, 25: 0.54nM, 27: 0.38nM) and good 5-HT(1A) affinity/antagonism (5-HT(1A)K(i), [(35)S]GTPgammaS: E(max) (%): 21: 111.1nM, 0%; 25: 173.2nM, 0%; 27: 107nM, 0%).     

The influence of modifications in imide fragment structure on 5-HT(1A) and 5-HT(7) receptor affinity and in vivo pharmacological properties of some new 1-(m-trifluoromethylphenyl)piperazines

New, flexible (7, 9, 11 and 13) and rigid (8, 10, 12 and 14) imides with a 1-(m-trifluorophenyl)piperazine fragment and a tetramethylene or a 1e,4e-cyclohexylene spacer, respectively, showed very high affinity (K(i)=0.3-34 nM) and agonistic in vivo activity for 5-HT(1A) receptors. Flexible new compounds and the previously described 5 also bound to 5-HT(7) receptors (K(i)=21-134 nM). Selected glutarimide derivatives, that is, the most potent postsynaptic 5-HT(1A) receptor agonist rigid compound 8 and its flexible analogue 7, as well as the previously described full agonist-rigid compound 6 and the partial agonist-its flexible counterpart 5 exhibited moderate affinity for alpha(1)-adrenoceptors (K(i)=85 - 268 nM), but were practically devoid of any affinity for dopamine D(2) sites. Those glutarimides demonstrated anxiolytic- (5 and 7) and antidepressant-like (5, 6 and 8) activity in the four-plate and the swim tests in mice, respectively; at the same time, however, they inhibited the locomotor activity of mice. The antidepressant-like effect of 8 was significantly stronger than that induced by imipramine used as a reference antidepressant.     

1-Aryl-4-(4-succinimidobutyl)piperazines and their conformationally constrained analogues: synthesis, binding to serotonin (5-HT1A, 5-HT2A, 5-HT7), alpha1-adrenergic, and dopaminergic D2 receptors, and in vivo 5-HT1A functional characteristics

Starting with the structure of potent 5-HT(1A) ligands, that is, MM77 [1-(2-methoxyphenyl)-4-(4-succinimidobutyl)piperazine, 4] and its constrained version 5 (MP349), previously obtained in our laboratory, a series of their direct analogues with differently substituted aromatic ring (R=H, m-Cl, m-CF(3), m-OCH(3), p-OCH(3)) were synthesized. The flexible and the corresponding 1e,4e-disubstituted cyclohexane derivatives were designed in order to investigate the influence of rigidification on 5-HT(1A) affinity, selectivity for 5-HT(2A), 5-HT(7), D(1), and D(2) binding sites and functional profile at pre- and postsynaptic 5-HT(1A) receptors. The new compounds 19-25 were found to be highly active 5-HT(1A) receptor ligands (K(i)=4-44 nM) whereas their affinity for other receptors was: either significantly decreased after rigidification (5-HT(7)), or controlled by substituents in the aromatic ring (alpha(1)), or influenced by both those structural modifications (5-HT(2A)), or very low (D(2), K(i)=5.3-31 microM). Since a distinct disfavor towards rigid compounds was observed for 5-HT(7) receptors only, it seems that the bioactive conformation of chain derivatives at those sites should differ from the extended one. Several in vivo models were used to asses functional activity of 19-25 at pre- (hypothermia in mice) and postsynaptic 5-HT(1A) receptors (lower lip retraction in rats and serotonin syndrome in reserpinized rats). Unlike the parent antagonists 4 and 5, all the new derivatives tested were classified as partial agonists with different potency, however, similar effects were observed within pairs (flexible and rigid) of the analogues. The obtained results indicated that substitution in the aromatic ring, but not spacer rigidification, controls the 5-HT(1A) functional activity of the investigated compounds. Moreover, an o-methoxy substituent in the structure of 5 seems to be necessary for its full antagonistic properties. Of all the new compounds studied, trans-4-(4-succinimidocyclohexyl)-1-(3-trifluoromethylphenyl)piperazine 24 was the most potent 5-HT(1A) receptor ligand in vitro (K(i)=4 nM) and in vivo, with at least 100-fold selectivity for the other receptors tested.     

N-Propylnoraporphin-11-O-yl carboxylic esters as potent dopamine D(2) and serotonin 5-HT(1A) receptor dual ligands

A small series of N-propylnoraporphin-11-O-yl carboxylic esters with variant ester lengths were synthesized and their binding potencies at dopamine receptors (D(1), D(2)) and serotonin receptors (5-HT(1A), 5-HT(2A)) were evaluated. Monoesters 3a-f showed binding potency of 100 nM or less for the D(2) receptor, and potency of 10-30 nM for the 5-HT(1A) receptor. Butyryl ester 3d was found to be the best compound possessing the highest potency for both receptors, with K(i) values of 55 and 12 nM for D(2) and 5-HT(1A) receptors, respectively. There is no correlation between the binding potency and the length of the monoesters, but the diesters 9 and 10 were inactive for the D(2) receptor. The dual binding profile of these monoesters for the D(2) and 5-HT(1A) receptors may be useful for the treatment of neuropsychiatric disorders.     

Synthesis, receptor binding and functional studies of mesoridazine stereoisomers

The four stereoisomers of mesoridazine were synthesized and evaluated in D2, 5-HT1A, 5-HT2A, 5-HT2C, D1, and D3 receptor binding and functional assays. Two isomers demonstrated potent D2 receptor binding (Ki < 3 nM) and functional antagonism (IC50 < or = 10 nM) activities. These two isomers also showed moderate affinity for the 5-HT2A and D3 receptors. A third isomer was devoid of significant D2 receptor binding, but did have moderate affinity for the 5-HT2A and D3 receptors. The fourth isomer demonstrated poor affinity for all the receptors tested. Most significantly, the stereochemistry of the sulfoxide moiety played a dominant role in the observed structure-activity relationship (SAR).     

Discovery of 3-aryl-3-methyl-1H-quinoline-2,4-diones as a new class of selective 5-HT6 receptor antagonists

A 5,7-dichloro-3-phenyl-3-methyl-quinoline-2,4-dione (11a) has been identified in a random screen as a lead for 5-HT(6) antagonist. During the lead optimization process, several analogs were synthesized and their biological activities were investigated. Within this series, several compounds display high binding affinity and selectivity for the 5-HT(6) receptor. In particular, 3-(4-hydroxyphenyl)-3-methyl-quinoline-2,4-dione (12f) exhibits high affinity (K(i)=12.3 nM) for 5-HT(6) receptor with good selectivity over other serotonin and dopamine (D(1)-D(4)) receptor subtypes. In a functional adenylyl cyclase stimulation assay, this compound exhibited considerable antagonistic activity (IC(50)=0.61 microM).     

Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine derivatives as 5-HT1A serotonin receptor ligands

A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT1A receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and alpha1-, alpha2-adrenergic receptors. A o-OCH3 derivative (2e) bound at 5-HT1A sites with subnanomolar affinity (IC50 = 0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents.     

Binding of antipsychotic drugs to human brain receptors focus on newer generation compounds

Using radioligand binding assays and post-mortem normal human brain tissue, we obtained equilibrium dissociation constants (K(d)s) for nine new antipsychotic drugs (iloperidone, melperone, olanzapine, ORG 5222, quetiapine, risperidone, sertindole, ziprasidone, and zotepine), one metabolite of a new drug (9-OH-risperidone), and three older antipsychotics (clozapine, haloperidol, and pimozide) at nine different receptors (alpha1-adrenergic, alpha2-adrenergic, dopamine D2, histamine H1, muscarinic, and serotonin 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2C receptors). Iloperidone was the most potent drug at the two adrenergic receptors. ORG 5222 was the most potent drug at dopamine D2 and 5-HT2c receptors, while ziprasidone was the most potent compound at three serotonergic receptors (5-HT1A, 5-HT1D, and 5-HT2A). At the remaining two receptors, olanzapine was the most potent drug at the histamine H1 receptor (Kd=0.087 nM); clozapine at the muscarinic receptor (Kd=9 nM). Certain therapeutic and adverse effects, as well as certain drug interactions can be predicted from a drug's potency for blocking a specific receptor. These data can provide guidelines for the clinician in the choice of antipsychotic drug.     

Substituted 3-amino and/or 3-aminomethyl-3,4-dihydro-2H-1-benzopyrans: synthesis and biological activity

A series of new 3-amino, 3-aminomethyl-5-alkoxy-3,4-dihydro-2H-1-benzopyran and 5'-alkoxy-3',4'-dihydrospiro-[piperazine-2.3'(2'H)-benzopyran] derivatives was prepared and evaluated for affinity at 5-HT1A, 5-HT2A and D2 receptors. Two of the compounds (1f and 2b) can be considered as potent and selective 5-HT2A ligands. One compound (1g) demonstrated high affinity for 5-HT1A and D2 receptor binding sites and one compound (1d) proved to be a mixed 5-HT1A/5-HT2A ligand.     

Structure-intrinsic activity relationship studies in the group of 1-imido/amido substituted 4-(4-arylpiperazin-1-yl)cyclohexane derivatives; new, potent 5-HT1A receptor agents with anxiolytic- like activity

Introduction of 1,4-disubstituted cyclohexane ring in the structure of flexible long chain arylpiperazines resulted in linearly constrained, potent serotonin (5-HT)(1A) ligands. In order to trace structure-intrinsic activity relationships in this group, a new series of 1-substituted 4-(4-arylpiperazin-1-yl)cyclohexane derivatives with different cyclic imide/amide termini, and their flexible, tetramethylene analogues were synthesized and pharmacologically evaluated for 5-HT(1A) receptors. In vitro binding experiments revealed that all the compounds were potent 5-HT(1A) receptor agents (K(i) = 1.9-74 nM). Some derivatives tested additionally showed also high affinity for alpha(1)-adrenergic receptors (K(i) = 2.9-101 nM) and for 5-HT(7) receptors. Functional in vivo examination revealed that rigid ligands with o-OCH(3) group in the aryl moiety and cyclic imide system in the opposite terminal behaved like postsynaptic 5-HT(1A) receptor antagonists. On the other hand, unsubstituted, m-Cl, or m-CF(3) substituted derivatives as well as those with cyclic amide group in the terminal fragment exhibited agonistic or partial agonistic activity. Three out of four derivatives tested, that is, postsynaptic 5-HT(1A) antagonists 9 and 10, and partial agonist 16, showed anxiolytic-like activity in the conflict drinking (Vogel) test in rats.     

Affinity of (+/-)-pindolol, (-)-penbutolol, and (-)-tertatolol for pre- and postsynaptic serotonin 5-HT(1A) receptors in human and rat brain

There is considerable interest in the use of drugs that selectively block presynaptic (somatodendritic) serotonin 5-HT(1A) receptors for the adjunctive treatment of major depressive disorder. The 5-HT(1A)/beta-adrenoceptor ligands (+/-)-pindolol, (-)-tertatolol, and (-)-penbutolol are currently under clinical investigation, and knowledge of their affinity at different populations of central 5-HT(1A) receptors is needed. Here we have determined the affinity of these drugs for presynaptic and postsynaptic 5-HT(1A) receptors in postmortem human and rat brain using receptor autoradiography and the selective 5-HT(1A) radioligand [(3)H]WAY-100635. The binding of [(3)H]WAY-100635 was specific and saturable and showed high affinity in the rat dorsal raphe nucleus and hippocampus (K(D) = 1.5-1.7 nM). In competition studies, the three compounds had nanomolar affinity and produced monophasic displacement of [(3)H]WAY-100635 binding in all regions of both species. (-)-Penbutolol and (-)-tertatolol had similar affinity for pre-and postsynaptic 5-HT(1A) receptors in both rat and human brain. However, in the human, but not the rat, the affinity of (+/-)-pindolol in dorsal raphe nucleus (K(i) = 8.9 +/- 1. 1 nM) was slightly but significantly higher than that in hippocampus (K(i) = 14.4 +/- 1.5 nM in CA1). In summary, our data show that (+/-)-pindolol, (-)-tertatolol, and (-)-penbutolol are all high-affinity ligands at native human and rat 5-HT(1A) receptors. (-)-Penbutolol and (-)-tertatolol do not discriminate between the pre- and postsynaptic 5-HT(1A) sites tested in either species, but (+/-)-pindolol showed a slightly higher affinity for the presynaptic site in human brain. Further work is needed to establish whether the latter difference is clinically relevant.     

Novel class of arylpiperazines containing N-acylated amino acids: their synthesis, 5-HT1A, 5-HT2A receptor affinity, and in vivo pharmacological evaluation

Novel arylpiperazines with N-acylated amino acids, selected on the basis of a preliminary screening of two libraries previously synthesized on SynPhase Lanterns, were prepared in solution and their affinity for 5-HT(1A), 5-HT(2A), and D(2) receptors was evaluated. The compounds bearing (3-acylamino)pyrrolidine-2,5-dione (19-26) and N-acylprolinamide (29-34) moieties showed high affinity for 5-HT(1A) (K(i)=3-47 nM), high-to-low for 5-HT(2A) (K(i)=4.2-990 nM), and low for D(2) receptors (K(i)=0.77-21.19 microM). All the new o-methoxy derivatives of (3-acylamino)pyrrolidine-2,5-diones tested in vivo revealed agonistic activity at postsynaptic 5-HT(1A) receptors, while m-chloro derivatives were classified as antagonists of these sites; similar relations were observed for o-methoxy (29) and m-chlorophenylpiperazine derivatives of N-acylprolinamides. The reported results show that the amino acid-derived terminal fragment modified the in vivo functional profile. Finally, the selected compounds 19 and 20, a 5-HT(1A) partial agonist and a full agonist, respectively, and 26, a mixed 5-HT(1A)/5-HT(2A) antagonist, were evaluated in preclinical animal models of depression and anxiety. The project allowed selecting the lead compound 20 which exhibited an anxiolytic-like effect in the four-plate test in mice and revealed distinct antidepressant-like effects in the forced swimming and tail suspension tests in mice.     

Synthesis and structure-affinity relationship investigations of 5-aminomethyl and 5-carbamoyl analogues of the antipsychotic sertindole. A new class of selective alpha1 adrenoceptor antagonists

A new class of selective alpha(1) adrenoceptor antagonists derived from the antipsychotic drug sertindole is described. The most potent and selective compound 1-(2-(4-[5-aminomethyl-1-(4-fluorophenyl)-1H-indol-3-yl]-1-piperidinyl)ethyl)-2-imidazolidinone (11) binds with 0.50 nM affinity for alpha(1) adrenergic receptors and with more than 44 times lower affinity for dopamine D(2),D(3), D(4) and serotonin 5-HT(1A), 5-HT(1B), 5-HT(2A) and 5-HT(2C) receptors. The molecular features providing high affinity for adrenergic alpha(1) receptors and high selectivity towards dopamine D(2) and serotonin 5-HT(2A) and 5-HT(2C) receptors are discussed.     

Benzimidazole derivatives. Part 1: Synthesis and structure-activity relationships of new benzimidazole-4-carboxamides and carboxylates as potent and selective 5-HT4 receptor antagonists

New benzimidazole-4-carboxamides 1-16 and -carboxylates 17-26 were synthesized and evaluated for binding affinity at serotonergic 5-HT4 and 5-HT3 receptors in the CNS. Most of the synthesized compounds exhibited moderate-to-very high affinity (in many cases subnanomolar) for the 5-HT4 binding site and no significant affinity for the 5-HT3 receptor. SAR observations and structural analyses (molecular modeling, INSIGHT II) indicated that the presence of a voluminous substituent in the basic nitrogen atom of the amino moiety and a distance of ca. 8.0 A from this nitrogen to the aromatic ring are of great importance for high affinity and selectivity for 5-HT4 receptors. These results confirm our recently proposed model for recognition by the 5-HT4 binding site. Amides 12-15 and esters 24 and 25 bound at central 5-HT4 sites with very high affinity (Ki = 0.11-2.9 nM) and excellent selectivity over serotonin 5-HT3, 5-HT2A, and 5-HT1A receptors (Ki > 1000-10,000 nM). Analogues 12 (Ki(5-HT4) = 0.32 nM), 13 (Ki(5-HT4) = 0.11 nM), 14 (Ki(5-HT4) = 0.29 nM) and 15 (Ki(5-HT4) = 0.54 nM) were pharmacologically characterized as selective 5-HT4 antagonists in the isolated guinea pig ileum (pA2 = 7.6, 7.9, 8.2 and 7.9, respectively), with a potency comparable to the 5-HT4 receptor antagonist RS 39604 (pA2 = 8.2). The benzimidazole-4-carboxylic acid derivatives described in this paper represent a novel class of potent and selective 5-HT4 receptor antagonists. In particular, compounds 12-15 could be interesting pharmacological tools for the understanding of the role of 5-HT4 receptors.