Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.

A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred ß-arrestin2 over ß-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

Piperidine-containing histamine H3 receptor antagonists of the carbamate series: the alkyl derivatives.

A series of N-alkyl urethanes, potential histamine H3 receptor antagonists, was prepared. Carbamate derivatives were synthesized from appropriate isocyanates and N-piperidinoalkan-1-ols. The novel compounds were evaluated for histamine H3 receptor activity in vitro on the guinea pig ileum. Some selected compounds were tested in vivo after p.o. application to mice and in vitro for selectivity towards other histamine receptors (H1, H2) in functional assays in the guinea pig. The most potent H3 receptor antagonist in vitro was compound 14 (pA2 = 7.2). Compound 14 was equipotent at M3 receptors and lacked H3 receptor activity in vivo. Predictions of octanol-water partition coefficient (Pallas) and metabolic fate (MetabolExpert, METEOR) were used to explore potential reasons for this absence of in vivo activity.

Synthesis and histamine H1 receptor agonist activity of a series of 2-phenylhistamines, 2-heteroarylhistamines, and analogues.

New histamine derivatives characterized by a (substituted) aryl, heteroaryl, benzyl, or heteroarylmethyl substituent in the C2 position of the imidazole ring have been prepared from appropriate imidates or amidines, respectively, and 2-oxo-4-phthalimido-1-butyl acetate (1). The compounds were screened as potential H1 receptor agonists on the isolated guinea pig ileum. The 3-halogenated 2-phenylhistamines (halogen = Br (35) and I (36)) were equipotent with histamine, while 2-(3-(trifluoromethyl)phenyl)histamine (2-[2-(3-(trifluoromethyl)phenyl)-1H-imidazol-4-yl]ethanamine (39)) was significantly more potent than histamine (39: pD2 = 6.81, relative activity = 128%). The 2-substituted histamine analogues were partial H1 receptor agonists on the endothelium-denuded isolated guinea pig aorta with pEC50 values generally smaller than observed on the guinea pig ileum, but the rank order of potency was found to be similar. The contractile effects on guinea pig ileum and aorta, respectively, could be blocked concentration-dependently by the H1 receptor antagonist mepyramine, yielding KB values for mepyramine in the nanomolar range. In vitro compounds 35 and 39 bound to [3H]mepyramine-labeled guinea pig cerebellar membranes with a pKi of 6.1 and 5.9, respectively. However, upon iv administration, 35 (3-100 mg/kg) and 39 (3-300 mg/kg) failed to inhibit the binding of [3H]mepyramine to mouse cerebral cortex in vivo, thereby indicating that these histamine derivatives are not able to penetrate the blood-brain barrier. In functional in vitro studies on histamine H2, H3, and other neurotransmitter receptors the selectivity of 39 was found to be 2138 (H1:H2), > 64 (H1:H3), 1000 (H1:M3), 105 (H1:alpha 1), 708 (H1:beta 1), and 71 (H1:5HT2A). Thus compound 39 is the most potent and selective H1 receptor agonist reported so far. These results make meta-substituted 2-phenylhistamines, especially 2-(3-(trifluoromethyl)phenyl)- and 2-(3-bromophenyl)histamine (39 and 35, respectively) valuable experimental tools for the selective stimulation of histamine H1 receptors and the study of H1 receptor-mediated functions.

Iodoaminopotentidine and related compounds: a new class of ligands with high affinity and selectivity for the histamine H2 receptor.

The synthesis and biological evaluation of a new class of histamine H2 antagonists with N-cyano-N'-[omega-[3-(1-piperidinylmethyl)phenoxy] alkyl]guanidine partial structure are described as part of an extensive research program to find model compounds for the development of new radioligands with high H2 affinity and specific activity. High receptor affinity is achieved by an additional (substituted) aromatic ring, which is connected with the third guanidine N by a carbon chain spacer and an amine, carboxamide, ester, or sulfonamide link ("polar group"). In functional studies for H2 antagonistic activity and other pharmacological actions [e.g. H1 antihistaminic, antimuscarinic, antiadrenergic (alpha 1, beta 1), 5-HT2 blocking activity] in the isolated guinea pig atrium and ileum and rat aorta and tail artery, the compounds proved to be highly potent and selective histamine H2 receptor antagonists. The H2 antagonistic activity is mainly depending on the length of both the N'-alkyl chain (chain A) and the N"-spacer (chain B). Compounds with a C3 chain A and a C2 chain B are most potent in the preferred group of substances, i.e., the carboxamide series. A wide variety of substituents at the aromatic ring is tolerated, among them iodine, amino, and azido groups. These compounds are up to 32 times more potent than cimetidine in the isolated guinea pig right atrium. The replacement of the carboxamide by an ester group (44c) is well tolerated, while replacement of the cyanoguanidine by an urea group results in nearly 100-fold decrease in activity (46c,e). The iodinated benzamides are among the most potent H2 antagonists known so far. The [125I]-labeled form of 31f ([125I]iodoaminopotentidine, [125I]-N-[2-(4-amino-3-iodobenzamido) ethyl]-N'-cyano-N"-[3-[3-(1-piperidinylmethyl) phenoxy]propyl]guanidine) and its photolabile analogue 31h ([125I]iodoazidopotentidine, [125I]-N-[2-(4-azido-3- iodobenzamido)ethyl]-N'-cyano-N"-[3-[3-(1-piperidinyl-methyl)pheno xy] propyl]guanidine) proved to be useful probes for reversible and irreversible labeling of the histamine H2 receptor. Radioligand binding studies in guinea pig cerebral membranes revealed considerably higher H2 receptor affinity for 31f (pKi = 9.15), 31h (pKi = 8.58), and some analogues than functional experiments (guinea pig atrium), presumably reflecting an easier access to the H2 receptors in membranes.

Histaprodifens: synthesis, pharmacological in vitro evaluation, and molecular modeling of a new class of highly active and selective histamine H(1)-receptor agonists.

A new class of histamine analogues characterized by a 3, 3-diphenylpropyl substituent at the 2-position of the imidazole nucleus has been prepared outgoing from 4,4-diphenylbutyronitrile (4b) via cyclization of the corresponding methyl imidate 5b with 2-oxo-4-phthalimido-1-butyl acetate or 2-oxo-1,4-butandiol in liquid ammonia, followed by standard reactions. The title compounds displayed partial agonism on contractile H(1) receptors of the guinea-pig ileum and endothelium-denuded aorta, respectively, except 10 (histaprodifen; 2-[2-(3, 3-diphenylpropyl)-1H-imidazol-4-yl]ethanamine) which was a full agonist in the ileum assay. While 10 was equipotent with histamine (1), methylhistaprodifen (13) and dimethylhistaprodifen (14) exceeded the functional potency of 1 by a factor of 3-5 (13) and 2-3 (14). Compounds 10 and 13-17 relaxed precontracted rat aortic rings (intact endothelium) with relative potencies of 3.3- up to 28-fold (compared with 1), displaying partial agonism as well. Agonist effects were sensitive to blockade by the selective H(1)-receptor antagonist mepyramine (pA(2) approximately 9 (guinea-pig) and pA(2) approximately 8 (rat aorta)). The affinity of 10 and 13-17 for guinea-pig H(1) receptors increased 20- to 100-fold compared with 1. Two lower homologues of 10 were weak partial H(1)-receptor agonists while two higher homologues of 10 were silent antagonists endowed with micromolar affinity for rat and guinea-pig H(1) receptors. In functional selectivity experiments, 10, 13, and 14 did not stimulate H(2), H(3), and several other neurotransmitter receptors. They displayed only low to moderate affinity for these sites (pA(2) < 6). For a better understanding of structure-activity relationships, the interaction of 1 and 10, 13 and 14 within the transmembrane (TM) domains of the human histamine H(1) receptor were studied using molecular dynamics simulations. Remarkable differences were found between the binding modes of 10, 13, and 14 and that of 1. The imidazole ring of 10, 13, and 14 was placed 'upside down' compared with 1, making the interaction of the N(pi)-atom with Tyr431 possible. This new orientation was mainly caused by the space filling substitution at the 2-position of the imidazole ring and influenced the location of the protonated N(alpha)-atom which was positioned more between TM III and TM VI. This orientation can explain both the increased relative potency and the maximum effect of 10, 13, and 14 compared with 1. Compound 13 (methylhistaprodifen; N(alpha)-methyl-2-[2-(3, 3-diphenylpropyl)-1H-imidazol-4-yl]ethanamine) is the most potent histamine H(1)-receptor agonist reported so far in the literature and may become a valuable tool for the study of physiological and pathophysiological H(1)-receptor-mediated effects.

Novel partial agonists for the histamine H(3) receptor with high in vitro and in vivo activity.

Novel branched N-alkylcarbamates and aliphatic ethers derived from 3-(1H-imidazol-4-yl)propanol were prepared. The compounds were investigated on two functional histamine H(3)-receptor assays. Some compounds displayed partial agonist activity on synaptosomes of rat brain cortex but behaved as pure competitive antagonists on the guinea pig ileum. Under in vivo conditions after po application to mice, some of the compounds showed partial or full agonist activity. Highest in vivo potency was found for the 3,3-dimethylbutyl ether 10 (ED(50) = 0.29 mg/kg, full intrinsic activity). These novel agonists are structurally diverse from classical aminergic histamine H(3)-receptor agonists (e.g., (R)-alpha-methylhistamine, imetit) as they lack a basic moiety in the side chain, which is supposed to be important for the activation of the receptor protein. The selectivity for the histamine H(3) receptor was proven by determination of H(1)- and H(2)-receptor activity on functional assays of the guinea pig.

New histamine H(3)-receptor ligands of the proxifan series: imoproxifan and other selective antagonists with high oral in vivo potency.

Histamine H(3)-receptor antagonists of the proxifan series are described. The novel compounds possess a 4-(3-(phenoxy)propyl)-1H-imidazole structure and various functional groups, e.g., an oxime moiety, on the phenyl ring. Synthesis of the novel compounds and X-ray crystallography of one highly potent oxime derivative, named imoproxifan (4-(3-(1H-imidazol-4-yl)propyloxy)phenylethanone oxime), are described. Most of the title compounds possess high antagonist potency in histamine H(3)-receptor assays in vitro as well as in vivo in mouse CNS following po administration. Structure-activity relationships are discussed. Imoproxifan displays subnanomolar potency on a functional assay on synaptosomes of rat cerebral cortex (K(i) = 0.26 nM). In vivo, imoproxifan increases the central N(tau)-methylhistamine level with an ED(50) of 0.034 mg/kg po. A receptor profile on several functional in vitro assays was determined for imoproxifan, demonstrating high selectivity toward the histamine H(3) receptor for this promising candidate for further development.

The selective histamine H1-receptor agonist 2-(3-trifluoromethylphenyl)histamine increases waking in the rat.

The effects of the selective histamine H1-receptor agonist, 2-(3-trifluoromethylphenyl)histamine, were studied in rats implanted with electrodes for chronic sleep recordings. 2-(3-Trifluoromethylphenyl)histamine (80-120 micrograms) injected into the left lateral ventricle increased wakefulness, whereas slow wave sleep was reduced. Pretreatment with pyrilamine (2.0 mg/kg) prevented the effects of the H1-receptor agonist on wakefulness and slow wave sleep. Our results further support the involvement of histamine in the modulation of the waking state.

H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain.

The effect of histamine and related drugs on the tritium overflow evoked electrically (0.3 Hz) or by introduction of Ca2+ ions into Ca2+-free K+-rich (25 mmol/l) medium containing tetrodotoxin was studied in superfused guinea-pig brain cortex, cerebellum, hippocampus or hypothalamus slices and in mouse brain cortex slices preincubated with 3H-noradrenaline. The electrically evoked tritium overflow in guinea-pig cortex slices was inhibited by histamine; the H3 receptor antagonist clobenpropit reversed the effect of histamine to a slight facilitation. The facilitatory effect of histamine (obtained in the presence of clobenpropit) was not affected by the H1 receptor antagonist mepyramine but abolished by the H2 receptor antagonist ranitidine. In the absence of clobenpropit, ranitidine augmented the inhibitory effect of histamine. In slices superfused in the presence of ranitidine, the evoked overflow was inhibited by histamine and, more potently, by the H3 receptor agonist R-alpha-methylhistamine in a concentration-dependent manner (maximum inhibitory effect obtained for both agonists 30-35%). The concentration-response curve of histamine was shifted to the right by the H3 receptor antagonist thioperamide. R-alpha-methylhistamine inhibited the electrically evoked tritium overflow also in guinea-pig cerebellar, hippocampal and hypothalamic slices. In cortex slices superfused in the presence of clobenpropit, the H2 receptor agonists impromidine and, less potently, R-sopromidine facilitated the evoked overflow in a concentration-dependent manner. S-Sopromidine only tended to increase the evoked overflow. The effect of impromidine was counteracted by the H2 receptor antagonists ranitidine and cimetidine. The extent of the maximum facilitatory effect of impromidine (by 15-20%) was about the same when (i) the Ca2+ concentration in the medium was reduced from 1.3 to 0.98 mmol/l, (ii) the time of exposure to impromidine was reduced from 28 to 8 min or (iii) cerebellar, hippocampal or hypothalamic slices were used instead of cortical slices. The Ca2+-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca2+-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H3 receptors and facilitated via H2 receptors not located presynaptically. In the mouse brain cortex, only inhibitory H3 receptors occur. The extent of the H3 receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex.

Histaprodifen, methylhistaprodifen, and dimethylhistaprodifen are potent H1-receptor agonists in the pithed and in the anaesthetized rat.

Selective H2- and H3-receptor agonists, exhibiting an at least tenfold higher potency than histamine itself at the respective receptors, have been known for several years. Selective H1-receptor agonists with a potency exceeding that of histamine have become available only recently; the most potent are methylhistaprodifen and dimethylhistaprodifen [Nalpha-methyl- and Nalpha,Nalpha-dimethyl-2(3,3-diphenylpropyl)histamine, respectively] with 3.4- and 2.4-fold higher potencies than histamine in vitro (in the guinea-pig ileum). The aim of the present study was to examine whether these compounds and the parent compound histaprodifen are potent H1-receptor agonists in the pithed and in the anaesthetized rat. In pithed, vagotomized rats diastolic blood pressure was decreased by 2-(2-thiazolyl)ethanamine i.v. (which was used as a reference H1-receptor agonist) and by histaprodifen, methylhistaprodifen, and dimethylhistaprodifen; the maximum decrease was about 45 mmHg for each compound, and the potencies, expressed as pED50, the negative logarithm of the dose (in mole per kilogram body weight) eliciting a half-maximal response, were 7.23, 7.55, 8.43 and 8.12, respectively. The dose/response curves of the four compounds were shifted to the right to about the same extent by the H1-receptor antagonist dimetindene (1 micromol/kg i.v.). The vasodepressor response was not affected by combined i.v. administration of the H2- and H3-receptor antagonists ranitidine and thioperamide, by combined i.v. administration of the alpha1- and alpha2-adrenoceptor antagonists prazosin and rauwolscine, and by the beta-adrenoceptor antagonist propranolol i.v. but was attenuated by the inhibitor of NO synthase, N(omega)-nitro-L-arginine methyl ester i.v. In anaesthetized rats 2-(2-thiazolyl)ethanamine, histaprodifen, methylhistaprodifen and dimethylhistaprodifen i.v. also decreased diastolic blood pressure in a manner sensitive to dimetindene i.v. Our data show that histaprodifen and, in particular, methyl and dimethylhistaprodifen are highly potent H1-receptor agonists in vivo.

Novel histaprodifen analogues as potent histamine H1-receptor agonists in the pithed and in the anaesthetized rat.

We have shown previously that histaprodifen and its Nalpha-substituted analogues methylhistaprodifen and dimethylhistaprodifen are highly potent H1-receptor agonists in vivo. The aim of the present study was to examine the influence of four newly synthesized histaprodifen analogues, 3-fluoro-methylhistaprodifen (1), Nalpha-imidazolylethylhistaprodifen (2), bis-histaprodifen (3) and Nalpha-methyl-bis-histaprodifen (4), on the cardiovascular system in the pithed and in the anaesthetized rat. In pithed and vagotomized rats, diastolic blood pressure (which was increased to 80-85 mmHg by vasopressin infusion) was decreased dose dependently by methylhistaprodifen (the reference compound) and by compounds 1-4. The maximum decrease was about 47-50 mmHg for methylhistaprodifen and compounds 1, 2 and 3. Their potencies, expressed as pED50 (the negative logarithm of the dose in mole per kilogram body weight that decreased diastolic blood pressure by 25 mmHg), were 8.31, 8.23, 8.26 and 7.84, respectively. With compound 4 the maximal effect was not achieved at doses up to 1 micromol/kg (the latter dose decreased blood pressure by about 30 mmHg; pED50 approximately 6.5). The vasodepressor effect of the five compounds was attenuated by the H1-receptor antagonist dimetindene (1 micromol/kg) but was not changed by combined administration of the H2- and H3-receptor antagonists ranitidine and thioperamide (1 micromol/kg each), by combined administration of the alpha1- and alpha2-adrenoceptor antagonists prazosin and rauwolscine (1 micromol/kg each) or by the beta-adrenoceptor antagonist propranolol (3 micromol/kg). In anaesthetized rats methylhistaprodifen and compounds 1-4 induced almost the same fall in blood pressure as in pithed and vagotomized animals; the effects were sensitive to blockade by dimetindene (1 micromol/kg). Higher doses of compounds 1 and 2 (1 micromol/kg) increased heart rate in pithed and vagotomized rats in a manner sensitive to propranolol (3 micromol/kg) but insensitive to dimetindene (3 micromol/kg). The same dose of methylhistaprodifen and of compounds 3 and 4 failed to affect heart rate. We conclude that the agonistic potency of compounds 1 and 2 at H1-receptors in the cardiovascular system of the rat equals that of methylhistaprodifen, the most potent histamine H1-receptor agonist available until recently. Compounds 1 and 2 exhibit sympathomimetic activity at high doses.

Synthesis and autoradiographic evaluation of a novel high-affinity Tc-99m ligand for the 5-HT2A receptor.

The synthesis and in vitro autoradiography of a novel Tc-99m ligand with subnanomolar affinity to the 5-HT2A receptor is reported. The complex combines the 4-(4-fluoro)-benzoyl piperidine portion derived from the 5-HT2A receptor antagonist ketanserin with a neutral oxotechnetium(V) chelate in form of a mixed ligand "3 + 1" unit containing the SNS/S donor set. The analogous rhenium compound has been synthesized as a surrogate for the Tc-99m complex for use in receptor binding assays and for complete structural characterization. In competition experiments the Tc-99 complex as well as its Re analogue display subnanomolar affinity toward the 5-HT2A receptor (Ki 0.44 nM for Tc, 0.25 nM for Re). The subnanomolar 5-HT2A receptor binding of the Re complex was confirmed by functional in vitro antagonism of contractile effects evoked by 5-HT in rat arterial tissue. Re 1 inhibited 5-HT-induced, 5-HT2A receptor-mediated contractions of isolated rat tail artery in a competitive fashion and possessed nanomolar affinity (pA2 = 9.08, pA2 representing the negative decadic logarithm of the Re 1/5-HT2A-receptor dissociation constant [mol/L]). Like ketanserin, Re 1 displayed moderate affinity for adrenergic alpha1D (pA2 = 8.23) and histamine H1 receptors (pA2 = 8.00), and was >600-fold up to 10,700-fold less active at several neurotransmitter receptor subtypes. In vitro autoradiographic studies clearly indicate the accumulation of the Tc-99m compound in 5-HT2A-receptor-rich areas of the brain. This enrichment can be blocked by 5-HT2A receptor antagonists such as mianserin and ketanserin and is therefore specific.

Ring-substituted histaprodifen analogues as partial agonists for histamine H(1) receptors: synthesis and structure-activity relationships.

Thirteen racemic benzene ring-substituted analogues of histaprodifen (8a; 2-[2-(3,3-diphenylpropyl)-1H-imidazol-4-yl]ethanamine), a novel lead for potent and selective histamine H(1)-receptor agonists, have been prepared from substituted 4,4-diphenylbutyronitriles 5 via cyclization of the corresponding methyl butyrimidates 6 with 2-oxo-4-phthalimido-1-butyl acetate in liquid ammonia, followed by deprotection. Nitriles 5 were accessible by alkylation of either substituted diphenylmethanes with 3-bromopropionitrile or diethyl malonate with substituted 1-chloro-diphenylmethanes and subsequent standard reactions. The title compounds 8 displayed partial agonism on contractile H(1) receptors of the guinea-pig ileum (E(max) = 2-98% relative to histamine) and, compared with the endogenous agonist, were endowed with agonist potencies of 4-92%. The meta fluorinated (8c) and meta chlorinated (8f) analogues showed the highest relative potency in this series (95% confidence limits 85-99% and 78-102%), but did not exceed the value of the lead 8a (99-124%). Compound 8c (2-[2-[3-(3-fluorophenyl)-3-phenylpropyl]-1H-imidazol-4-yl]ethanamine ) was a partial agonist at contractile H(1) receptors of the guinea-pig aorta (relative potency 154% vs. 100% for histamine) and at relaxation-mediating endothelial H(1) receptors of the rat aorta (relative potency 556% vs. 100% for histamine) and matched with the functional behaviour of 8a. Agonism observed for each compound was sensitive to blockade by the selective H(1)-receptor antagonist mepyramine (pA(2) approximately 9 (guinea-pig) and pA(2) approximately 8 (rat aorta)). All histaprodifen analogues 8 stimulated neither histaminergic H(2)/H(3) nor cholinergic M(3) receptors. They displayed only low to moderate affinity for these sites (H(2): pD'(2) < 5; H(3)/M(3): pA(2) < 6). With regard to the substitution pattern on the benzene ring, there was no correlation between the histaprodifen series and the corresponding derivatives of another selective H(1)-receptor agonist, viz. 2-phenylhistamine.

Influence of imidazole replacement in different structural classes of histamine H(3)-receptor antagonists.

The reference compounds for histamine H(3)-receptor antagonists carry as a common feature an imidazole moiety substituted in the 4-position. Very recently novel ligands lacking an imidazole ring have been described possessing a N-containing non-aromatic heterocycle instead. In this study we investigated whether imidazole replacement, favourably by a piperidine moiety, is generally applicable to different structural classes of reference compounds, e.g., thioperamide, carboperamide, clobenpropit, FUB 181, ciproxifan, etc. While replacement led to a loss of affinity for many of the compounds, it was successfully applied to some ether derivatives. The piperidine analogues of FUB 181 and ciproxifan, 3-(4-chlorophenyl)propyl 3-piperidinopropyl ether hydrogen oxalate (6) and cyclopropyl 4-(3-piperidinopropyloxy)phenyl methanone hydrogen maleate (7), almost maintained in vitro affinities, pK(i) values of 7.8 and 8.4, respectively, and showed high potency in vivo after p.o. administration (ED(50) values of 1.6 and 0.18 mg/kg, respectively).

In-vitro pharmacology of sarpogrelate and the enantiomers of its major metabolite: 5-HT2A receptor specificity, stereoselectivity and modulation of ritanserin-induced depression of 5-HT contractions in rat tail artery.

The new antiplatelet agent sarpogrelate (MCI-9042), its major metabolite (R,S)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2- propanol ((R,S)-M-1) and the enantiomers of (R,S)-M-1 were studied as antagonists at 5-HT2A receptors, 5-HT1-like receptors, 5-HT3 receptors, alpha 1-adrenoceptors, beta-adrenoceptors, histamine H1 receptors, histamine H2 receptors and muscarinic M3 receptors in various functional in-vitro assays. Sarpogrelate, (R,S)-M-1, (R)-M-1 and (S)-M-1, respectively, were competitive antagonists of 5-hydroxytryptamine (5-HT) at 5-HT2A receptors of rat tail artery with calculated pA2 values of 8.53, 9.04, 9.00 and 8.81, respectively. Sarpogrelate lacked prominent 5-HT1-like, 5-HT3, beta, H1, H2 and M3 antagonist activity and weakly blocked alpha 1-adrenoceptors (pKB = 6.30). (S)-M-1 showed weak affinity for 5-HT1-like receptors (pKB = 6.30), alpha 1- (pKB = 6.80) and beta- (pKB = 6.54) adrenoceptors, while (R)-M-1 was a weak antagonist at histamine H1 receptors (pKB = 6.49). Stereoselectivity of M-1 enantiomers was low. (R)-M-1 showed 1.6-fold, 2,3-fold and 2.5-fold higher antagonist activity than (S)-M-1 for 5-HT2A, H1 and M3 receptor, respectively. Affinity at beta-adrenoceptors and 5-HT1-like receptors was 5-fold and 3-fold higher for (S)-M-1 than for (R)-M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine analogues. 36th communication. Basically substituted histamine derivatives with H1-agonistic activity.

Piperidinoalkyl-, morpholinoalkyl- or [(pyrid-2-yl)methylthio]alkyl-substituents were introduced into position 2 of the essential histamine structure. These 2-substituted histamine derivatives were prepared via reaction of imidate hydrochlorides in liquid ammonia under pressure with 1,3-dihydroxypropanone followed by a stepwise build-up of the side chain in position 4 of the imidazole nucleus (route I) or by cyclization with 2-oxo-4-phthalimido-1-butyl acetate (route II) followed by deprotection of the primary amine function. The novel compounds were screened for H1-activity on the isolated guinea-pig ileum and for H2-activity on the isolated guinea-pig right atrium. While 10a, c-e proved to be weak partial H1-agonists, 10b, f were very weak H1-antagonists.

Piperidine-containing histamine H3 receptor antagonists of the carbamate series: the influence of the additional ether functionality.

Recently novel leads for histamine H3 receptor antagonists of the non-imidazole type have been described. As a continuation of this research eleven new carbamate derivatives possessing an additional ether functionality were prepared. The compounds were evaluated in vitro for their antagonist activity on isolated organs of guinea-pig (GP) H3 as well as H2, H1, and M3 receptors, respectively. All compounds investigated possessed moderate antagonist affinities at guinea-pig histamine H3 receptors (pA2 6.11-6.76). An ether functionality introduced in different places of the lipophilic part of carbamates differently influenced activity and selectivity toward H3, M3, and other histamine receptors tested.

Piperidine-containing histamine H3-receptor antagonists of the carbamate series: variation of the spacer length.

Ten carbamate derivatives have been prepared from appropriate isocyanates and omega-piperidino-1-alkanols. All compounds belong to the new generation of non-imidazole histamine H3-receptor ligands which may have beneficial pharmacokinetic properties compared with the classical imidazole-containing H3-receptor antagonists. The carbamates were evaluated in vitro for antagonist activity at guinea-pig (gp) H3, H2, H1, and M3 receptors, respectively. They displayed moderate affinity for H3 receptors (pA2 5.8-7.0 in the gp ileum assay) as well as low to moderate selectivities vis-à-vis H2 (gp atrium), H1 (gp ileum), and M3 (gp ileum) receptors. A typical member of this series is 7-piperidino-1-heptyl N-(4-phenyl-1-butyl)carbamate (17) with pA2 values of 7.02 (H3), 5.92 (H1), and 6.38 (M3), respectively, and a pD'2 value of 5.46 (H2).

Importance of the lipophilic group in carbamates having histamine H3-receptor antagonist activity.

In order to evaluate changes in the lipophilic part of designed carbamates concerning their potential histamine H3-receptor antagonist properties a new series of O-[3-(1H-imidazol-4-yl)propanol]carbamates was derived containing N-mono- or di-alkenyl, alkynyl, cycloalkyl, or double-branched alkyl substituents. The compounds were tested in vitro for their H3-receptor antagonist activity on synaptosomes of rat cerebral cortex and shared moderate to high antagonist activity in vitro. In this series 3-(1H-imidazol-4-yl)propyl N-(4-pentenyl)carbamate (4) was the most potent compound in vitro (Ki = 6.3 nM). H3-receptor antagonist activity in the central nervous system (CNS) was detected for most compounds in the in vivo H3-receptor assay based upon measurement of brain N tau-methylhistamine levels after p.o. administration to mice. The most effective carbamate in vivo, 3-(1H-imidazol-4-yl)propyl N-(allyl)carbamate (3), showed higher CNS potency (ED50 = 0.48 mg/kg p.o.) than the reference antagonist thioperamide. For some novel carbamates their histamine H1- and H2-receptor activities were determined on isolated organs of guinea-pig thereby demonstrating their high H3-receptor selectivity.

Preparation and in vitro pharmacology of 5-HT4 receptor ligands. Partial agonism and antagonism of metoclopramide analogous benzoic esters.

Alicyclic ester analogues of the gastroprokinetic benzamide metoclopramide (1) and its ester congener SDZ 205557 (2), a 5-HT4 receptor antagonist, were prepared by O-alkylation of 4-amino-5-chloro-2-methoxybenzoate with N-(2-chloroethyl) substituted alicyclic amines. The bromo and iodo analogue of compound 13b (2-(1-piperidinyl)ethyl 4-amino-5-chloro-2-methoxybenzoate) were obtained by halogenation of dechloro-13b with N-halogenated succinimides. The series was evaluated in functional in vitro assays with regard to affinity for serotoninergic 5-HT4, 5-HT3 and muscarinic M3 receptors. The affinities for 5-HT3 and M3 receptors were below 6.0 (pKB or pA2). On 5-HT4 receptors in guinea-pig ileal longitudinal muscle and rat oesophagus, the majority of compounds revealed partial 5-HT4 receptor agonism susceptible to blockade by SDZ 205557, a reference 5-HT4 receptor antagonist (pKB = 7.25-7.73 (guinea-pig ileum) and 7.09-7.43 (rat oesophagus)). The relative agonist potency was in the range of 5-303% (5-HT: 100%). Compound 13b and its bromo analogue 17 were the most potent esters of the series. The enantiomers of 13g ((R)- and (S)-2-(2-methyl-1-piperidinyl)ethyl 4-amino-5-chloro-2-methoxybenzoate) interacted stereoselectively with 5-HT4 receptors and displayed enantiomeric potency ratios (R)/(S) of 4.3-8.7. There was an excellent correlation between (a) antagonist affinity on guinea-pig ileum and rat oesophagus, (b) relative agonist potency on guinea-pig ileum and rat oesophagus, and (c) between antagonist affinity and relative agonist potency within each assay (r2 > 0.91). The new compounds may serve as academic tools in evaluating the functional role of 5-HT4 receptors. The selective partial 5-HT4 receptor agonists presented in this paper may be useful to restore physiological motility and secretion in the gut with reduced or absent propensity to elicit tachycardia and desensitization of the intestinal target receptor.