Use of an inverse agonist radioligand [3H]A-317920 reveals distinct pharmacological profiles of the rat histamine H3 receptor

Selective radioligands for histamine H(3) receptors have been used to characterize H(3) receptor pharmacology by radioligand binding assays and to determine H(3) receptor distribution by tissue autoradiography. Here we report the synthesis and receptor binding characterization of [(3)H]A-317920 (furan-2-carboxylic acid(2-[4-[3-([3,5-(3)H]4-cyclopropanecarbonyl-phenoxy)-propyl]-piperazin-1-yl]-1-methyl-2-oxo-ethyl)-amide), a high affinity inverse agonist radioligand for the rat H(3) receptor. The binding of [(3)H]A-317920 to rat cortical and cloned H(3) receptors revealed fast on- and slower off-rate kinetics with calculated K(d) values in agreement with those determined in saturation binding assays (0.2 nM for both receptors). Further, we compared [(3)H]A-317920 with the agonist [(3)H](N)-alpha-methylhistamine ([(3)H]NalphaMH) as radioligand tools to study receptor pharmacology. Agonists and antagonists displaced [(3)H]NalphaMH with one-site binding characteristics and Hill slopes approached unity. In contrast, although antagonists exhibited one-site binding, [(3)H]A-317920 displacement by agonists was best fit by two-site binding models, and the potencies of the high affinity, GDP-sensitive sites correlated with the potencies defined in [(3)H]NalphaMH binding. Unlike [(125)I]iodoproxyfan, [(3)H]A-317920 exhibits potent and selective binding to rat H(3) receptors with low binding to non-H(3) sites, including cytochrome P450. These findings show that [(3)H]A-317920 is a potent rat H(3) receptor antagonist radioligand and has utility for studying H(3) receptor pharmacology.     

Search for histamine H(3) receptor ligands with combined inhibitory potency at histamine N-methyltransferase: omega-piperidinoalkanamine derivatives

In an effort to design new hybrid compounds with dual properties, i.e. binding affinity at histamine H(3) receptors and inhibitory potency at the catabolic enzyme histamine N(tau)-methyltransferase (HMT), a novel series of 1-substituted piperidine derivatives was synthesized. This alicyclic heterocycle is structurally linked via aminoalkyl spacers of variable lengths to additional aromatic carbo- or hetero-cycles. These new hybrid drugs were pharmacologically evaluated regarding their binding affinities at recombinant human H(3) receptors, stably expressed in CHO cells, and in a functional assay for their inhibitory potencies at rat kidney HMT. All compounds investigated proved to be H(3) receptor ligands with binding affinities in the micro- to nanomolar concentration range despite significant differences in the type of the aromatic moiety introduced. The most potent compound in this series was the quinoline derivative 20 (K(i) = 5.6 nM). Likewise, all new ligands studied showed impressive HMT inhibitory activities. Here, compounds 5, 10, 14 and 18-20 exhibited submicromolar potencies (IC(50) = 0.061-0.56 microM). The aminomethylated quinoline 19 showed almost the same, well balanced nanomolar activities on both targets. In this study, new hybrid compounds with a dual mode biological action were developed. These pharmacological agents are valuable leads for further development and candidates for treatment of histamine-dependent disorders.     

Recent advances in histamine H3/H4 receptor ligands

Advances in molecular biology have brought fresh impetus to the field of research into histamine receptor ligands with the recent cloning of human histamine H₃ and H₄ receptors. Numerous promising potential therapeutic targets have been suggested for H₃ receptor ligands based on various pharmacological investigations, e.g., Alzheimer’s disease, schizophrenia, obesity, epilepsy, inflammatory diseases, sleep disorders and attention-deficit hyperactivity disorder. Concurrent to the availability of the receptors, the number of leads has also largely increased in the last few years, with some hybrid compounds acting simultaneously at two targets. In particular, the large number of different non-imidazole H₃ receptor antagonists possessing high affinities and selectivities and the first compound in clinical testing raise the hope for having a drug available in the market in the near future. The first studies with H₄ receptor ligands show promising anti-inflammatory and immunomodulatory properties.     

Structure-based discovery and binding site analysis of histamine receptor ligands

ABSTRACT

Introduction: The application of structure-based drug discovery in histamine receptor projects was previously hampered by the lack of experimental structures. The publication of the first X-ray structure of the histamine H1 receptor has been followed by several successful virtual screens and binding site analysis studies of H1-antihistamines. This structure together with several other recently solved aminergic G-protein coupled receptors (GPCRs) enabled the development of more realistic homology models for H2, H3 and H4 receptors.

Areas covered: In this paper, the authors review the development of histamine receptor models and their application in drug discovery.

Expert opinion: In the authors’ opinion, the application of atomistic histamine receptor models has played a significant role in understanding key ligand-receptor interactions as well as in the discovery of novel chemical starting points. The recently solved H1 receptor structure is a major milestone in structure-based drug discovery; however, our analysis also demonstrates that for building H3 and H4 receptor homology models, other GPCRs may be more suitable as templates. For these receptors, the authors envisage that the development of higher quality homology models will significantly contribute to the discovery and optimization of novel H3 and H4 ligands.     

Effects of histamine H3 receptor ligands in experimental models of anxiety and depression

Histamine H₃ receptor ligands have been proposed to be of potential therapeutic interest for the treatment of different central nervous system disorders; however, the psychopharmacological properties of these drugs have not been studied extensively. In this work, we investigated the possible involvement of histamine H₃ receptor function in experimental models of anxiety (elevated plus-maze) and depression (forced swimming test). Male Sprague-Dawley rats were treated IP with the histamine H₃ receptor agonist R-α-methylhistamine (10 mg/kg) or the histamine H₃ receptor antagonist thioperamide (0.2, 2 and 10 mg/kg) and 30 min afterwards the time spent in the open arms of an elevated plus-maze was registered for 5 min. The immobility time of male OF1 mice in the forced swimming test was recorded for 6 min, 1 h after the IP administration of R-α-methylhistamine (10 and 20 mg/kg), thioperamide (0.2, 2, 10 and 20 mg/kg) or another histamine H₃ receptor antagonist, clobenpropit (5 mg/kg). The locomotor activity of mice was checked in parallel by means of an activity meter. Both saline controls and active drug controls were used in all the paradigms. Neither thioperamide nor R-α-methylhistamine significantly changed animal behaviour in the elevated plus-maze. R-α-methylhistamine and the higher dose of thioperamide assayed (20 mg/kg) were also inactive in the forced swimming test. By contrast, thioperamide (0.2–10 mg/kg) dose-dependently decreased immobility, the effect being significant at 10 mg/kg (33% reduction of immobility); clobenpropit produced an effect qualitatively similar (24% reduction of immobility). None of these histamine H₃ receptor antagonists affected locomotor activity. These preliminary results suggest that the histamine H₃ receptor blockade could be devoid of anxiolytic potential but have antidepressant effects. Besides, the stimulation of these receptors does not seem to be followed by changes in the behavioural parameters studied. Received: 1 July 1998/Final version: 8 September 1998     

Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist

The human Neuropeptide Y (NPY) receptors 1 (hY1), 2 (hY2), 4 (hY4), and the mouse type 5 (mY5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPgammaS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gqo5, Gqi5 and Gqi9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY1, hY2 and hY4 receptors coupled to Gqo5, Gqi5 and Gqi9, the mY5 receptor stimulated transient calcium release only when co-expressed with Gqi9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY5 receptor with high affinity (Ki=32.1+/-1.8 nM), competitively antagonized NPY-mediated GTPgammaS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.     

N-substituted piperidinyl alkyl imidazoles: discovery of methimepip as a potent and selective histamine H3 receptor agonist

In this study, we continue our efforts toward the development of potent and highly selective histamine H(3) receptor agonists. We introduced various alkyl or aryl alkyl groups on the piperidine nitrogen of the known H(3)/H(4) agonist immepip and its analogues (1-3a). We observed that N-methyl-substituted immepip (methimepip) exhibits high affinity and agonist activity at the human histamine H(3) receptor (pK(i) = 9.0 and pEC(50) = 9.5) with a 2000-fold selectivity at the human H(3) receptor over the human H(4) receptor and more than a 10000-fold selectivity over the human histamine H(1) and H(2) receptors. Methimepip was also very effective as an H(3) receptor agonist at the guinea pig ileum (pD(2) = 8.26). Moreover, in vivo microdialysis (in rat brain) showed that methimepip reduces the basal level of brain histamine to about 25% after a 5 mg/kg intraperitoneal administration.     

The effect of a novel, dual function histamine H1 receptor antagonist/5-lipoxygenase enzyme inhibitor on in vivo dermal inflammation and extravasation

Leukotrienes and histamine are thought to play important roles in the development of dermatitis. This study evaluated the in vivo efficacy of 5-{4-[(aminocarbonyl)(hydroxy)amino]but-1-ynyl}-2-(2-{4-[(R)-(4-chlorophenyl)(phenyl)methyl]piperazin-1-yl}ethoxy)benzamide (ucb 35440), a dual function histamine H1 receptor antagonist/5-lipoxygenase enzyme inhibitor, in mouse skin. A single application of phorbol 12-myristate 13-acetate (PMA) was used to induce an acute inflammatory response over a 6-h period. PMA was applied on days 0, 2, 4, 7 and 9 to generate a chronic inflammatory response measured on day 10. ucb 35440 was applied topically at 1 h pre-PMA challenge and 3 h post-PMA challenge in the acute model. In the chronic PMA model, ucb 35440 was applied topically twice a day (AM and PM) on days 7, 8 and 9. Dose-response studies revealed that ucb 35440 inhibited PMA-induced ear weight gain with a 57% inhibition measured using a 3% w/v topical solution in the acute model. The compound appeared less potent in the chronic model with 43% inhibition measured using a 3% w/v topical solution of ucb 35440. Qualitative histologic assessment in PMA challenged ears showed that ucb 35440 produced a moderate reduction of polymorphonuclear cell infiltration in the acute model whereas, a more substantial reduction in polymorphonuclear infiltration was noted in the chronic model. In addition, the oral efficacy of ucb 35440 was evaluated in vivo against histamine-induced extravasation in guinea pig skin. Single oral doses of ucb 35440 (10 mg/kg in 0.5% methylcellulose suspension) at 1, 2, 6 or 24 h pre-histamine challenge produced minimal inhibition of histamine-induced extravasation in the dermis. However, when ucb 35440 (10 mg/kg in a 0.5% methylcellulose suspension) was orally administered 24 and 2 h prior to dermal histamine challenge, significant inhibition of extravasation was observed. Similar inhibition of histamine-induced extravasation was observed when animals were orally dosed twice a day (AM and PM 10 mg/kg in a 0.5% methylcellulose suspension) for 5.5 days prior to dermal histamine challenge. Collectively, these results suggest that ucb 35440 may represent an important therapeutic class for the treatment of dermatologic inflammatory conditions.     

Synthesis and histamine H(3) and H(4) receptor activity of conformationally restricted cyanoguanidines related to UR-PI376

Recently, we identified highly potent agonists of the human histamine H(4) receptor (hH(4) R) among a series of imidazolylbutylcyanoguanidines. Aiming at improved selectivity for the hH(4) R relative to the H(3) receptor (hH(3) R), the flexible tetramethylene linker connecting imidazole ring and cyanoguanidine group was replaced by conformationally restricted carbocycles. Introduction of a para- or a meta-phenylene spacer yielded only very weakly active compounds at both hH(3) R and hH(4) R (investigated in [(35) S]GTPγS binding assays using Sf9 insect cell membranes expressing hH(x) R subtypes). By contrast, the incorporation of a more flexible cyclohexane-1,4-diyl linker resulted in EC(50) or K(B) values ≥110 nM at hH(4) R and hH(3) R. Quality of action, potency and receptor subtype selectivity of the investigated compounds depend on the stereochemistry: Cis-configured diastereomers prefer the hH(4) R and are partial agonists, whereas trans-isomers are antagonists at the hH(4) R. At the hH(3) R the trans-diastereomers are superior to the cis-isomers by a factor of 10. The results on imidazolylcycloalkylcyanoguanidines suggest that variation of ring size and optimization of the stereochemistry may be useful to increase the potency and selectivity of hH(4) R agonists relative to the hH(3) R.     

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.     

组胺H3受体拮抗剂／反向激动剂pitolisant的合成

目的对组胺H3受体拮抗剂／反向激动剂pitolisant进行合成工艺研究。方法以对氯氯苄、丙二酸二乙酯为起始原料,经过缩合、水解、脱羧、还原、酯化、取代反应制得pitolisant。结果与结论经过6步反应合成目标化合物pitolisant,其结构经1H—NMR及MS确证。对其中多步反应条件进行了工艺考察及优化,总收率为31．4％（以对氯氯苄计）。     

Constitutive activity of H3 autoreceptors modulates histamine synthesis in rat brain through the cAMP/PKA pathway

We previously described that agonist-activated histamine H3 autoreceptors inhibit the stimulation of histamine synthesis mediated by calcium/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA respectively) in histaminergic nerve endings. In the absence of an agonist H3 receptors show partial constitutive activity, so we hypothesized that suppression of constitutive activity by an inverse agonist could stimulate these transduction pathways. We show here that the H3 inverse agonist thioperamide increases histamine synthesis in rat brain cortical slices independently from the amounts of extracellular histamine. Thioperamide effects were mimicked by the inverse agonists clobenpropit and A-331440, but not by the neutral antagonist VUF-5681. In contrast, coincubation with VUF-5681 suppressed thioperamide effects. The effects of thioperamide were completely blocked by the PKA inhibitor peptide myristoyl-PKI14-22, a peptide that did not block depolarization stimulation of histamine synthesis. In addition, thioperamide effects required depolarization and were impaired by blockade of N-type calcium channels (mediating depolarization), but not by CaMKII inhibition. These results indicate that constitutive activity of H3 receptors in rat brain cortex inhibits the adenylate cyclase/PKA pathway, and perhaps also the opening of N-type voltage sensitive calcium channels, but apparently not CaMKII.     

The challenge of drug discovery of a GPCR target: analysis of preclinical pharmacology of histamine H3 antagonists/inverse agonists

Although the histamine H(3) receptor was identified pharmacologically in 1983, and despite widespread pharmaceutical interest in the target, no compound interacting specifically with this site has undergone successful clinical examination to develop the necessary proof-of-concept data. Therefore, clinical knowledge of the therapeutic potential of H(3) receptor antagonists in neuropsychiatric diseases, in metabolic diseases or in sleep disorders has yet to determine if the preclinical data that show broad efficacy in animal models of the aforementioned states are relevant to current unmet medical needs. H(3) receptors are complex, with species-related sequence differences that impact pharmacological responses. The receptors have a complex gene organization that provides opportunity for multiple slice isoforms, most of which remain poorly characterized even within a species. H(3) receptors are constitutively active, although the extent of this could vary either between species and/or receptor splice isoforms, both of which may provide opportunity for preferential coupling to different G-proteins. Thus, it is not surprising that the pharmacological effects of known H(3) ligands are complex and diverse, since these agents may act both as agonists and antagonists in different systems. Moreover, other compounds show inverse agonism in some models but neutral antagonist activity in others. Some of this diversity may be related to different ligand-dependent receptor activation states or to the effects of key amino acids important for ligand recognition. This commentary provides an overview of these complexities as applied to the H(3) receptor and the challenges these intricacies create for drug discovery.     

Pharmacological characterization of the new histamine H4 receptor agonist VUF 8430

Background and purpose:

We compare the pharmacological profiles of a new histamine H₄ receptor agonist 2-(2-guanidinoethyl)isothiourea (VUF 8430) with that of a previously described H₄ receptor agonist, 4-methylhistamine.

Experimental approach:

Radioligand binding and functional assays were performed using histamine H₄ receptors expressed in mammalian cell lines. Compounds were also evaluated ex vivo in monocyte-derived dendritic cells endogenously expressing H₄ receptors and in vivo in anaesthetized rats for gastric acid secretion activity.

Key results:

Both VUF 8430 and 4-methylhistamine were full agonists at human H₄ receptors with lower affinity at rat and mouse H₄ receptors. Both compounds induced chemotaxis of monocyte-derived dendritic cells. VUF 8430 also showed reasonable affinity and was a full agonist at the H₃ receptor. Agmatine is a metabolite of arginine, structurally related to VUF 8430, and was a H₄ receptor agonist with micromolar affinity. At histamine H₃ receptors, agmatine was a full agonist, whereas 4-methylhistamine was an agonist only at high concentrations. Both VUF 8430 and agmatine were inactive at H₁ and H₂ receptors, whereas 4-methylhistamine is as active as histamine at H₂ receptors. In vivo, VUF 8430 only caused a weak secretion of gastric acid mediated by H₂ receptors, whereas 4-methylhistamine, dimaprit, histamine and amthamine, at equimolar doses, induced 2.5- to 6-fold higher output than VUF 8430.

Conclusions and implications:

Our results suggest complementary use of 4-methylhistamine and VUF 8430 as H₄ receptor agonists. Along with H₄ receptor antagonists, both agonists can serve as useful pharmacological tools in studies of histamine H₄ receptors.     

Molecular modeling and pharmacological analysis of species-related histamine H(3) receptor heterogeneity

Presynaptic histamine H(3) receptors (H(3)R) regulate neurotransmitter release in the central nervous system, suggesting an important role for H(3) ligands in human diseases such as cognitive disorders, sleep disturbances, epilepsy, or obesity. Drug development for many of these human diseases relies upon rodent-based models. Although there is significant sequence homology between the human and rat H(3)Rs, some compounds show distinct affinity profiles. To identify the amino acids responsible for these species disparities, various mutant receptors were generated and their pharmacology studied. The N-terminal portion was shown to determine the species differences in ligand binding since a chimeric H(3)R containing N-terminal human and C-terminal rat receptor sequences exhibited similar pharmacology to the human receptor. Sequence analysis and molecular modeling studies suggested key amino acids at positions 119 and 122 in transmembrane region 3 play important roles in ligand recognition. Mutant receptors changing amino acids 119 or 122 of the human receptor to those in the rat improved ligand binding affinities and functional potencies of antagonist ligands, confirming the significant role that these amino acids play in species-related pharmacological differences. A model has been developed to elucidate the ligand receptor interactions for H(3)Rs, and pharmacological aspects of this model are described.     

Characteristics of recombinantly expressed rat and human histamine H3 receptors

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor.     

Histamine H4 receptor-RGS fusion proteins expressed in Sf9 insect cells: A sensitive and reliable approach for the functional characterization of histamine H4 receptor ligands

The human histamine H₄ receptor (hH₄R), co-expressed with Gα_i2 and Gβ₁γ₂ in Sf9 cells, is highly constitutively active. In the steady-state GTPase assay, the full agonist histamine (HA) induces only a relatively small signal (∼20-30%), resulting in a low signal-to background ratio. In order to improve this system for ligand screening purposes, the effects of the regulators of G-protein signaling (RGS) RGS4 and RGS19 (GAIP) were investigated. RGS4 and GAIP were fused to the C-terminus of hH₄R or co-expressed with non-fused hH₄R, always combined with Gα_i2 and Gβ₁γ₂. The non-fused RGS proteins did not significantly increase the relative effect of HA. With the hH₄R-RGS4 fusion protein the absolute GTPase activities, but not the relative HA-induced signal were increased. Fusion of hH₄R with GAIP caused a selective increase of the HA signal, resulting in an enhanced signal-to-noise ratio. A detailed characterization of the hH₄R-GAIP fusion protein (co-expressed with Gα_i2 and Gβ₁γ₂) and a comparison with the data obtained for the non-fused hH₄R (co-expressed with Gα_i2 and Gβ₁γ₂) led to the following results: (i) the relative agonist- and inverse agonist-induced signals at hH₄R-GAIP are markedly increased. (ii) Compared to the wild-type hH₄R, standard ligands show unaltered potencies and efficacies at hH₄R-GAIP. (iii) Like hH₄R, hH₄R-GAIP shows high and NaCl-resistant constitutive activity. (iv) hH₄R-GAIP shows the same G-protein selectivity profile as the non-fused hH₄R. Collectively, hH₄R-GAIP provides a sensitive test system for the characterization of hH₄R ligands and can replace the non-fused hH₄R in steady-state GTPase assays.     

3-Methoxylphenylpropyl amides as novel receptor subtype-selective melatoninergic ligands: characterization of physicochemical and pharmacokinetic properties

1. Developing subtype-selective melatoninergic ligands has been a subject of considerable interest in drug discovery. A series of 3-methoxyphenylpropyl amide derivatives showing selective binding capacity to type 2 melatonin receptor with subnanomolar range of affinities has been identified recently by our laboratory. In the present study, their physicochemical properties, Caco-2 cell and mdr1-MDCK cell permeability, plasma protein binding, and metabolic stability were investigated.

2. The selected compounds are lipophilic in nature, exhibiting aqueous solubility ranging from 40 to 200 µg/mL. Cell permeability studies on Caco-2 and mdr1-MDCK model revealed that they were readily transported through intestinal epithelium and possessed high penetration potential through blood–brain barrier, implying good oral absorption and central nervous system (CNS) distribution potential. They also showed substantial binding to human plasma protein ranging from 78.5% to 92.3%.

3. These compounds were, however, subjected to rapid cytochrome P450-mediated degradation in rat and human liver microsomes with in vitro half-life of 9.5–31.9?min in rat and 5.5–66.7?min in human, which were much shorter than that of melatonin (～73?min).

4. Metabolite profiling unveiled that C6-ether linkage and methoxy substituents were likely the major metabolic soft spots in their structures, which provided important information for further improvement of their structural stability.

     

4-(omega-(alkyloxy)alkyl)-1H-imidazole derivatives as histamine H(3) receptor antagonists/agonists

In an effort to develop new histamine H(3) receptor antagonists usable as pharmacological tools we present here novel unsymmetrical ether derivatives. Etherification of different omega-(1H-imidazol-4-yl)alkyl scaffolds led to compounds containing alkyl chains of increasing lengths either with or without unsaturated termini, cycloalkyl or arylalkyl moieties, or additional heteroatoms. When investigated in an in vitro assay on rat synaptosomes, the majority of compounds displayed potencies in the low nanomolar concentration range at the H(3) receptor, e.g., 4-(3-(3-cyclopentylpropyloxy)propyl)-1H-imidazole (27, K(i) = 7 nM). FUB 465, 4-(3-(ethoxy)propyl)-1H-imidazole (14), a useful tool for the characterization of constitutive activity of H(3) receptors in vivo in rodents, proved to be of high oral in vivo potency in mice (ED(50) = 0.26 mg/kg). Further, the influence of chosen compounds on specific [(35)S]GTPgammaS binding was assayed on HEK293 cell membranes expressing the human histamine H(3) receptor revealing partial agonism of the compounds in this particular model. These distinct responses are further hints for "protean agonism" in this class of compounds. Additionally, selected compounds were functionally investigated in vitro on isolated organs of the guinea-pig at H(3), H(1), and H(2) receptors.