Characterization of the histamine H4 receptor binding site. Part 1. Synthesis and pharmacological evaluation of dibenzodiazepine derivatives

A series of dibenzodiazepine derivatives was synthesized to probe the binding site of the recently discovered histamine H4 receptor (H4R). Optimization of the lead structure clozapine (2) resulted in (E)-7-chloro-11-(4-methylpiperazin-1-yl)dibenzo[b,f][1,4]oxazepine (7j), a potent H4R agonist (H4R, pKi = 7.6). Pharmacological data suggests that the series of nonimidazole compounds can be used to describe the orthosteric binding site of the H4R because both 2 and 7j displace [3H]histamine in a competitive manner. Furthermore, it is demonstrated that the effects of 7j are competitively antagonized by the selective H4R antagonist JNJ 7777120 (1), indicating considerable overlap of their binding sites. On the basis of the derived structure-activity relationships and additional pharmacological results, a pharmacophore model was constructed, which will be the premise for the design of novel H4R ligands.     

Studies on histaminergic compounds, III. Synthesis and histamine H2-activity of a series of corresponding histamine and dimaprit analogues

Structure-activity relationships of a series of histamine analogues were found to be identical, except for one minor discrepancy, to those of a series of corresponding dimaprit analogues. It was concluded that histamine and dimaprit most probably interact with the same binding sites of the histamine H₂-receptor.     

H3 receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450.

1. Thioperamide (TP), an imidazole and a highly potent, specific antagonist of the histamine H3 receptor, inhibited the secretion of cortisol from bovine isolated adrenocortical cells (IC50 0.20 microM) and in the rat (5 mg kg-1) prevented both basal and stress-induced secretion of corticosterone. 2. In adrenocortical microsomes, low affinity binding of [3H]-histamine (KD 27.7 microM) was potently inhibited by TP (Ki 0.33 microM). 3. In adrenocortical microsomal membranes, both histamine and TP yielded type II difference absorption spectra, characteristic of the interaction between imidazole and cytochrome P450 enzymes. Dissociation constants for binding to P450, calculated from spectral data, were 15.9 microM and 1.5 mM for histamine, and 0.3 microM and 3.7 microM for TP. 4. In view of previously reported evidence for an intracellular mediator role of histamine in platelets, the present findings suggest a physiological role for histamine in the modulation of adrenal P450 monooxygenases that generate adrenocortical steroids. 5. The results suggest that direct adrenocortical inhibition by thioperamide at a non-H3 intracellular site must be taken into account in studies designed to elucidate functional roles of H3 receptors.     

Characterization of histamine H2-receptors in human neutrophils with a series of guanidine analogues of impromidine

Summary Human neutrophils possess an NADPH oxidase which catalyzes superoxide (O _inf2 ^sup– ) formation and is activated by chemotactic peptides. Histamine inhibits O _inf2 ^sup–1 formation via H₂-receptors (Burde et al. 1989). We characterized the neutrophil H₂-receptor with a series of new guanidine-type H₂-agonists structurally derived from impromidine. Histamine inhibited O _inf2 ^sup– formation with an IC₅₀ value of 6.7 ± 1.2 M. Five aryloxy- and arylthioalkylguanidines were less potent and effective than histamine. Several arpromidine-like phenyl(pyridylalkyl)guanidines were either full or partial H₂-agonists. Some guanidines possess a three-membered carbon chain connecting the aromatic rings and the guanidine group; they were similarly potent and effective as histamine. Shortening or elongation of the carbon chain substantially decreased the potency and intrinsic activity of the guanidines. Halogenation of the phenyl ring did not substantially affect the potency and intrinsic activity of the compounds in comparison to the non-substituted parent compound. The H₂-antagonist, famotidine, competitively antagonized inhibition of O _inf2 ^sup– formation caused by the guanidine, arpromidine, with a pA₂ value of 6.84. The H₂-antagonist, cimetidine, differentially counteracted inhibition caused by partial and full H₂-agonists. Partial H₂-agonists antagonized the effects of histamine. The inhibitor of phosphodiesterases, 3-isobutyl-lmethylxanthine, additively enhanced the inhibitory effects of histamine and guanidines. The properties of the neutrophil H₂-receptor were compared with literature data concerning properties of the H₂-receptor of the guinea pig atrium. In the latter system, guanidines are full H₂-agonists with potencies of up to 125-fold of that of histamine. Our data indicate that guanidines inhibit O _inf2 ^sup– formation in human neutrophils via H₂-receptors. The structure/activity relationship for the neutrophil H₂-receptor substantially differs from the one for the H₂-receptor in the guinea pig atrium, suggesting that the neutrophil H₂-receptor has cell type-specific properties. Other possibilities to explain the differences between H₂-receptors in these systems are discussed. Send offprint requests to R. Seifert at the above address     

Structure-activity relationships in histamine H2 receptor antagonists: construction of a binding site model

Structure-Activity Relationships in Histamine H₂-Receptor Antagonists: Construction of a Binding Site Model For a series of compounds with H₂-antihistaminic activity and of amino acids as models of receptor binding sites, interaction energies were calculated using the Monopole Bond Polarizability method. Because of a significant correlation between calculated binding energies and antagonistic activities, glutamic acid is predicted to be a potential binding site for H₂-antagonists of the investigated type.     

Divalent cations increase the binding capacity of the [3H]mepyramine binding site, a possible histamine H1 receptor, in rat liver membranes

The [3H]mepyramine binding to rat liver membranes was about 2-fold higher in the presence of 2 mM CaCl2 than in the absence. However, the [3H]mepyramine binding to rat brain membranes was not affected by the presence of 2 mM CaCl2. Scatchard analysis showed that 2 mM CaCl2 did not change the affinity (KD) of liver membranes to [3H]mepyramine, but increased the binding capacity (Bmax). CaCl2 had a half maximal effect (ED50) at 3 microM and a maximal effect at concentrations of more than 10 microM. Other divalent cations, Mg++, Mn++, Sr++ and Ba++, also increased the [3H]mepyramine binding, whereas monovalent cations, Na+, K+, Li+ and Rb+, had no effect.     

Synthesis and in vitro pharmacology of a series of new chiral histamine H3-receptor ligands: 2-(R and S)-Amino-3-(1H-imidazol-4(5)-yl)propyl ether derivatives

To investigate stereospecificity and the mechanism of activation of the histamine H3-receptor, a series of 2-(R and S)-amino-3-(1H-imidazol-4(5)-yl)propyl ether derivatives were synthesized. In these compounds, the structures of the well-known antagonist iodoproxyfan and the full agonists R- or S-(alpha)-methylhistamine were combined in one molecule. The obtained "hybrid" molecules were tested for H3-receptor affinity on rat cerebral cortex. Some selected compounds were further screened for H3-receptor functional activity with GTPgamma[35S] autoradiography studies using rat brain tissue sections. The affinity of all the synthesized compounds (-log Ki = 5.9-7.9) was lower than that found for iodoproxyfan or two of its analogues; however, the compounds showed stereospecificity. The S-configuration of the series of 2-amino-3-(1H-imidazol-4(5)-yl)propyl ether derivatives, which resembles the stereochemistry of R-(alpha)-methylhistamine, was more favorable. Incorporation of an amino group in the propyl chain of iodoproxyfan and analogues did not alter the antagonistic behavior for compounds with an aromatic side chain. However, when also the aromatic moiety was replaced by a cyclohexyl group, the compounds behaved as agonists. This indicates that an interaction between the side chain amino group and the H3-receptor protein is involved in H3-receptor activation. The 2-(S)-amino-3-(1H-imidazol-4(5)-yl)propyl cyclohexylmethyl ether (23) has H3-receptor agonistic properties with high affinity for the histamine H3-receptor (-log Ki = 7.9 +/- 0.2) and might serve as a useful tool for further studies concerning drug design and receptor-ligand interactions.     

Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor

The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinct receptor isoforms, of which isoforms 1, 2, and 4, encode functional proteins. Detailed pharmacology on isoforms 1 (unspliced receptor), and 2 (which has an 80 amino acid deletion within the third intracellular loop of the protein) revealed that both isoforms displayed robust responses to a series of known H(3) agonists, while all agonists tested displayed increased potency at isoform 2 relative to isoform 1. Histamine, N(alpha)-methylhistamine, and R(-) and S(+)-alpha-methylhistamine are 16-23-fold more potent, while immepip and imetit are three to fivefold more potent. Antagonist experiments revealed a rank order of potency at both isoforms of clobenpropit>iodophenpropit>thioperamide, and these drugs are fivefold less potent at isoform 2 than isoform 1. To further explore the pharmacology of H(3) receptor function, we screened 150 clinically relevant neuropsychiatric drugs for H(3) receptor activity, and identified a small number of antipsychotics that possess significant antagonist activity.     

Synthesis and histamine H2-receptor activity of heterocyclic impromidine analogues.

Analogues of the H2-agonist impromidine with unsubstituted or substituted aromatic or non-aromatic heterocycles instead of the 5-methylimidazole group were prepared via the benzoylguanidine method and tested for H2-agonistic and H1-antagonistic activity in the guinea-pig isolated right atrium and ileum, respectively. Compounds with unsubstituted 2- or 3-pyridyl, 2-pyrazinyl, or 5-pyrimidyl thioether portion proved to be about equipotent in the atrium (about 2-4x histamine). Highest potency was found in the 5-chloro-3-pyridylthioethylguanidine 8h, that is about 20 times more potent as an H2-agonist than histamine, corresponding to about 8 times the activity of the unsubstituted parent compound 8g. It is demonstrated by the piperidinyl- and morpholinylalkyl guanidines 8m-o that an aromatic ring is not required in the affinity-conferring moiety of H2-agonists (8n: 1.3x histamine). Replacement of the (5-methylimidazol-4-yl)methylthio group in the H2-antagonist cimetidine by halogenated pyridyl, pyrazinyl and pyrimidinyl thioethers resulted in compounds inactive as H2-antagonists, giving further evidence for differences in the structural requirements of the affinity-conferring portions of H2-agonists and antagonists and their mode of interaction with the receptor protein.     

Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity

OBJECTIVE: This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. DESIGN AND METHODS: In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. RESULTS: Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. CONCLUSIONS: These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.     

Synthesis and structure-activity relationships of conformationally constrained histamine H(3) receptor agonists

Immepip, a conformationally constrained analogue of the histamine congener imbutamine, shows high affinity and functional activity on the human H(3) receptor. Using histamine and its homologues as prototypes, other rigid analogues containing either a piperidine or pyrrolidine ring in the side chain were synthesized and tested for their activities at the human H(3) receptor and the closely related H(4) receptor. In the series of piperidine containing analogues, immepip was found to be the most potent H(3) receptor agonist, whereas its propylene analogue 13a was identified as a high-affinity neutral antagonist for the human H(3) receptor. Moreover, replacement of the piperidine ring of immepip by a pyrrolidine ring led to a pair of enantiomers that show a distinct stereoselectivity at the human H(3) and H(4) receptor.     

Synthesis, monoamine transporter binding properties, and behavioral pharmacology of a series of 3beta-(substituted phenyl)-2beta-(3'-substituted isoxazol-5-yl)tropanes

Several 3beta-(substituted phenyl)-2beta-(3-substituted isoxazol-5-yl)tropanes (3a-t) were evaluated for their ability to inhibit radioligand binding at the DAT, 5-HTT, and NET as well as in gross observation and locomotor activity in mice and in rats trained to discriminate cocaine. All compounds showed high affinity for the DAT. The IC(50) values ranged from 0.5 to 26 nM. With the exception of 3e and 3f, which have no substituent on the 2beta-(1,2-isoxazole) ring, all compounds were selective for the DAT relative to the 5-HTT and NET. No compound showed death when dosed at 100 mg/kg; however, most compounds did show signs typical of dopamine activity. The ED(50) values for 2beta-(1,2-isoxazoles) that caused locomotor stimulation ranged from 0.2 to 12.8 mg/kg. Most compounds showed slower on-set and longer duration of action relative to cocaine. Surprisingly, 3beta-(4-methylphenyl)-2beta-[3-(4'-chlorophenyl)isoxazol-5-yl]tropane (3p) and 3beta-(4-methylphenyl)-2beta-[3-(4'-methylphenyl)isoxazol-5-yl]tropane (3r) did not produce significant increases in locomotor activity. In the cocaine discrimination test, all analogues showed full or at least 50% generalization with the exception of 3p, which did not show generalization. Importantly, both the locomotor activity and the cocaine discrimination ED(50)values were correlated with the DAT binding but not 5-HTT and NET binding. This provides further support for the dopamine hypothesis of cocaine abuse. High DAT affinity and selectivity, increased locomotor activity with slow onset and long duration of action, and generalization to cocaine shown by the 3beta-(substituted phenyl)-2beta-(3-substituted isoxazol-5-yl)tropanes are properties thought necessary for a pharmacotherapy for treating cocaine abuse.     

Characterization of the receptor binding profile of (3H)-dermorphin in the rat brain

Amphibian skin synthesizes a variety of biologically active peptides. Of these, dermorphin (Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is an extraordinarily potent opioid peptide up to 1000 times more active than morphine in inducing analgesia after intracerebroventricular administration. Dermorphin has little in common with the sequence of all hitherto known mammalian opioid peptides and is unique in having a d -amino acid residue in position 2. Specific binding properties of tritium labeled dermorphin were characterized in the rat brain. Scatchard or Hill analysis of equilibrium measurements performed over a large range of concentrations revealed a single population of dermorphin binding sites with a Kd value of 0.46 nM. Dermorphin and the selective μ-receptor ligand (d -Ala², MePhe⁴, Gly⁵-ol)- enkephalin (DAGO) had similar high potencies in competing with (³H)-dermorphin binding, whereas the inverse holds for the prototypical delta receptor ligand (d -Pen²,d -Pen⁵)-enkephalin (DPDPE), which exhibited a potency three orders of magnitude lower. Dermorphin was tested for its relative affinity to μ and delta binding sites by determining its potency in displacing (³H)-DAGO and (³H)-DPDPE from rat brain membrane preparations. Based on these comparisons, dermorphin exhibited a selectivity ratio Ki(DPDPE)/Ki(DA-GO) = 100, a value almost identical to that of DAGO, this ligand being considered as the protypical μ-receptor probe. The high affinity and selectivity of (³H)-dermorphin together with its very low nonspecific binding make this peptide a useful tool for dissecting the role(s) of the μ-receptor(s).     

Characterization of the binding site of the histamine H3 receptor. 1. Various approaches to the synthesis of 2-(1H-imidazol-4-yl)cyclopropylamine and histaminergic activity of (1R,2R)- and (1S,2S)-2-(1H-imidazol-4-yl)-cyclopropylamine

Various approaches to the synthesis of all four stereoisomers of 2-(1H-imidazol-4-yl)cyclopropylamine (cyclopropylhistamine) are described. The rapid and convenient synthesis and resolution of trans-cyclopropylhistamine is reported. The absolute configuration of its enantiomers was determined by single-crystal X-ray crystallographic analysis. The distinct trans-cyclopropylhistamine enantiomers were tested for their activity and affinity on the histamine H3 receptor. (1S,2S)-Cyclopropylhistamine (VUF 5297) acts as an agonist both on the rat cortex (pD2 = 7.1; alpha = 0.75) and on guinea pig jejunum (pD2 = 6.6; alpha = 0.75). Its enantiomer, (1R, 2R)-cyclopropylhistamine (VUF 5296), is about 1 order of magnitude less active. Both enantiomers show weak activity on H1 and H2 receptors. All synthetic attempts to cis-cyclopropylhistamine were unsuccessful. Nevertheless, the results of this study provide an ideal template for molecular modeling studies of histamine H3 receptor ligands.     

Novel histamine H3 receptor antagonists: affinities in an H3 receptor binding assay and potencies in two functional H3 receptor models.

1. We determined the affinities of ten novel H3 receptor antagonists in an H3 receptor binding assay and their potencies in two functional H3 receptor models. The novel compounds differ from histamine in that the aminoethyl side chain is replaced by a propyl or butyl chain linked to a polar group (amide, thioamide, ester, guanidine, guanidine ester or urea) which, in turn, is connected to a hexocyclic ring or to an alicyclic ring-containing alkyl residue [corrected]. 2. The specific binding of [3H]-N alpha-methylhistamine to rat brain cortex membranes was monophasically displaced by each of the ten compounds at pKi values ranging from 7.56 to 8.68. 3. Inhibition by histamine of the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]-noradrenaline was antagonized by the ten compounds and the concentration-response curve was shifted to the right with apparent pA2 values ranging from 7.07 to 9.20. 4. The electrically induced contraction in guinea-pig ileum strips (which was abolished by atropine) was inhibited by the H3 receptor agonists R-(-)-alpha-methylhistamine (pEC50 7.76), N alpha-methylhistamine (7.90) and imetit (8.18). The concentration-response curve of R-(-)-alpha-methylhistamine was shifted to the right by thioperamide (apparent pA2 8.79) and by the ten novel compounds (range of pA2 values 6.64-8.81). 5. The affinities and potencies were compared by linear regression analysis. This analysis was extended to thioperamide, the standard H3 receptor antagonist, which is also capable of differentiating between H3A and H3B sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Novel nonimidazole histamine H3 receptor antagonists: 1-(4-(phenoxymethyl)benzyl)piperidines and related compounds

In an extension of very recently published studies on successful imidazole replacements in some series of histamine H(3) receptor antagonists, we report on a new class of lipophilic nonimidazole antagonist having an aliphatic tertiary amino moiety connected to a benzyl template substituted in the 4-position by a phenoxymethyl group. The structural modifications were performed with the intention to avoid possible negative side effects reported for other series of antagonists. The novel compounds combine different characteristics of recently developed histamine H(3) receptor antagonists. The compounds were screened for their affinity in a binding assay for the human histamine H(3) receptor stably expressed in CHO-K1 cells and tested for their in vivo potency in the central nervous system of mice after oral administration. Different substitution patterns on the phenoxy group were used to optimize in vitro and/or in vivo potency leading to some compounds with low nanomolar affinity and high oral in vivo potency. Modifications of the basic piperidino moiety were performed by ring expansion, contraction, and opening. Selected compounds exhibited selectivity in functional assays on isolated organs of guinea-pig for H(3) vs H(1) and H(2) receptors. Unexpectedly, some of the novel antagonists also showed a slight preference for the human histamine H(3) receptor compared to their affinities for the guinea-pig H(3) receptor.