Liquid chromatography-mass spectrometric method for determination of the non-imidazole H3-receptor antagonist UPR1056 in rat plasma.

The non-imidazole H3 receptor antagonist UPR1056 was dosed in plasma samples from rats individually administered with a single i.p. dose of 1.25 mg/kg by means of a newly validated HPLC-MS method. UPR1056 was extracted from rat plasma by protein precipitation with acetonitrile and was separated by linear gradient elution, employing water and methanol both additioned with 0.05% trifluoroacetic acid as mobile phases. UPR1056 was detected in MS using an electrospray ion source operating in positive ion mode. Acquisition was performed in single ion monitoring mode at m/z=349.3. The method was validated over the concentration range of 17.43-1743 ng/mL (50-5000 pmol/mL). Within- and between-run precision for the low, mid and high quality controls (QC) levels were 6.75% or less and accuracy ranged from 95.8 to 107.6%. The lower limit of quantification was 17.43 ng/mL. The analysis of the time course of UPR1056 concentrations over the 24-h period revealed a C(max) of 1147 ng/mL after 2 h from peripheral administration of the non-imidazole H(3)-receptor antagonist, with a prolonged elimination half-life of over 9 h.

Dual-acting drugs: an in vitro study of nonimidazole histamine H3 receptor antagonists combining anticholinesterase activity.

Dual-acting compounds that combine H(3) antagonism with anticholinesterase properties are currently emerging as a novel and promising therapeutic approach in the treatment of multifactorial disorders primarily characterized by cholinergic deficits such as Alzheimer's disease. A series of novel nonimidazole H(3) ligands was developed from the chemical manipulation of 1,1'-octa-, -nona-, and -decamethylene-bis-piperidines--H(3) antagonists that had been the subject of previous investigations. These compounds were evaluated for in vitro binding affinity, antagonistic potency, and selectivity at rodent and human histamine H(3) receptors, inhibitory activity at rat brain cholinesterase, and in vivo CNS access and cholinomimetic effects. Within the present series, the tetrahydroaminoacridine hybrid 18 stands out as one of the most attractive molecules, synergistically combining nanomolar and selective H(3) antagonism with remarkable anticholinesterase activity. From this original starting point, it is hoped that future investigations will lead to dual-acting compounds that can selectively enhance central cholinergic neurotransmission and thus facilitate the treatment of cognitive disorders.

Stereospecific effects of benzo[d]isothiazolyloxypropanolamine derivatives at beta-adrenoceptors: synthesis, chiral resolution, and biological activity in vitro.

We performed the asymmetric synthesis of four enantiopure benzo[d] isothiazo-3-or 5-yloxypropanolamine derivatives, previously described as competitive antagonists at beta-adrenoceptors. The chemical characterization of each enantiomer was accomplished by (1)H NMR and HPLC/DAD/CD. The direct chromatographic separation of the enantiomers via chiral HPLC was investigated. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). The enantiomers obtained had enantiomeric purities suitable for biological assays. Tested in isolated rat cardiac and intestinal tissues to evaluate their effects at beta(1)- and beta(3)-adrenoceptors, the (S)-enantiomers revealed a higher degree of antagonism than (R)-enantiomers at both subtypes, even though their activity was greater at the cardiac beta(1)-subtype. The potent and cardiospecific antagonistic effect exerted by the compounds tested suggests that the benzisothiazole moiety could be an interesting scaffold for discovering new chiral beta-blocking drugs.

Synthesis and structure-activity relationships for biphenyl H3 receptor antagonists with moderate anti-cholinesterase activity.

The combination of antagonism at histamine H(3) receptors and inhibition of acetylcholinesterase has been recently proposed as an approach to devise putative new therapeutic agents for cognitive diseases. The 4,4'-biphenyl fragment has been reported by us as a rigid scaffold leading to potent and selective non-imidazole H(3)-antagonists. Starting from these premises, the current work presents an expanded series of histamine H(3) receptor antagonists, characterized by a central 4,4'-biphenyl scaffold, where the structure-activity profile of both mono-basic and di-basic compounds is further explored and their ability to inhibit rat brain cholinesterase activity is determined. The steric properties and basicity of the terminal groups were modulated in symmetrical compounds, carrying identical substituents, and in asymmetrical compounds, having a piperidine ring at one end and different groups at the other. The length of the linker connecting the biphenyl scaffold to the terminal groups was also modulated. Binding studies at rat and human H(3) receptors evidenced the highest binding affinities for di-basic compounds, in the order of nM concentrations, and that the steric requirements for the two terminal groups are different. Many potent compounds showed good selectivity profiles over the other histamine receptors. Interestingly, some derivatives displayed a moderate ability to inhibit rat brain cholinesterase, for example compound 12 (1-[2-(4'-piperidinomethyl-biphenyl-4-yl)ethyl]piperidine) has a pIC(50)=5.96 for cholinesterase inhibition and high H(3) receptor binding affinity and antagonist potency (pK(i)=8.70; pK(B)=9.28). These compounds can be considered as rigid analogs of a recently reported class of dual-acting compounds and as a promising starting point for the design of new H(3)-antagonists with anti-cholinesterase activity.

In vitro and in vivo pharmacological analysis of imidazole-free histamine H3 receptor antagonists: promising results for a brain-penetrating H3 blocker with weak anticholinesterase activity.

The pharmacological profiling of potent histamine H(3)-ligands initiated in a previous study is completed here. In vitro functional and binding studies revealed that several derivatives were selective H(3)-antagonists with nanomolar potency at human and guinea-pig histamine receptors, able to inhibit rat brain cholinesterase at micromolar concentrations and devoid of any cytotoxicity on cultured cells. Ex vivo binding experiments in rats showed that the most potent H(3)-antagonist, compound 5, had a prompt and long-lasting presence in the central nervous system (CNS), inhibiting [(3)H](R)-alpha-methylhistamine cortical binding [ED(50) (dose that elicits a 50% response) = 0.63 mg/kg intraperitoneally (i.p.)]. In the passive-avoidance test, compound 5, at 1.25 mg/kg i.p., was as effective as the anti-Alzheimer drug donepezil in attenuating scopolamine-induced amnesia in rats. These results suggest that a good CNS penetration and multitarget activity could account for the antiamnesic effect of this weak cholinesterase inhibitor and potent H(3)-antagonist (compound 5). This result represents a potential benchmark for the development of non-imidazole H(3)-antagonists/cholinesterase inhibitors with therapeutic potential in cognitive disorders.

In vitro pharmacology at human histamine H3 receptors and brain access of non-imidazole alkylpiperidine derivatives.

In this report, we describe the pharmacological profile of alkylpiperidine derivatives at human histamine H(3) receptors and their ability to access central histamine H(3) receptors in rodents. The three most attractive compounds exhibit high affinity and antagonistic potency (pK(i) ranging from 8.56 to 8.35) towards human histamine H(3) receptors stably expressed in SK-N-MC cells and, in contrast to thioperamide, they show slightly greater affinity for human than for rodent H(3) receptors. In ex vivo binding tests, they displayed a limited brain penetration since they displace [(3)H](R)-alpha-methylhistamine from rat cerebral cortex after intraperitoneal administration at doses four times higher than thioperamide. Among these compounds, derivative 5 tends to counteract partially scopolamine-induced amnesia in passive avoidance task. On the whole, these findings contribute to the identification of the requirements of increasingly drug-like non-imidazole H(3) antagonists.

Dibasic non-imidazole histamine H3 receptor antagonists with a rigid biphenyl scaffold.

A class of rigid, dibasic, non-imidazole H3 antagonists was developed, starting from a series of previously described flexible compounds. The original polymethylene chain between two tertiary amine groups was replaced by a rigid scaffold, composed by a phenyl ring or a biphenyl fragment. Modulation of the distance between the two amine groups, and of their alkyl substituents, was driven by superposition of molecular models and docking into a receptor model, resulting in the identification of 1,1'-[biphenyl-4,4'-diylbis(methylene)]bis-piperidine (5) as a subtype-selective H3 antagonist with high binding affinity (pKi=9.47) at human H3 histamine receptor.

Synthesis and biological evaluation of new non-imidazole H3-receptor antagonists of the 2-aminobenzimidazole series.

A novel series of non-imidazole H(3)-receptor antagonists was developed, by chemical modification of a potent lead H(3)-antagonist composed by an imidazole ring connected through an alkyl spacer to a 2-aminobenzimidazole moiety (e.g., 2-[[3-[4(5)-imidazolyl]propyl]amino]benzimidazole), previously reported by our research group. We investigated whether the removal of the imidazole ring could allow retaining high affinity for the H(3)-receptor, thanks to the interactions undertaken by the 2-aminobenzimidazole moiety at the binding site. The imidazole ring of the lead was replaced by a basic piperidine or by a lipophilic p-chlorophenoxy substituent, modulating the spacer length from three to eight methylene groups; moreover, the substituents were moved to the 5(6) position of the benzimidazole nucleus. Within both the 2-alkylaminobenzimidazole series and the 5(6)-alkoxy-2-aminobenzimidazole one, the greatest H(3)-receptor affinity was obtained for the piperidine-substituted compounds, while the presence of the p-chlorophenoxy group resulted in a drop in affinity. The optimal chain length was different in the two series. Even if the new compounds did not reach the high receptor affinity shown by the imidazole-containing lead compound, it was possible to get good H(3)-antagonist potencies with 2-aminobenzimidazoles having a tertiary amino group at appropriate distance.

Discovery of histamine H3 receptor antagonistic property of simple imidazole-free derivatives: Preliminary pharmacological investigation.

The histamine H3 receptor subtype negatively modulates the release of various neurotransmitters such as histamine, glutamate, norepinephrine, acetylcholine and many others mainly in the CNS and H3 antagonists have been developed to treat central diseases characterized by neurotransmission disturbance such as schizophrenia, memory/learning and sleep disorders. In search for non-imidazole histamine H3 receptor antagonists, currently indicated as a promising class of H3 blockers, a series of simple alkylpiperidine derivatives has been studied to attain a preliminary pharmacological profile. The compounds were characterized in vitro in terms of binding affinity, antagonistic potency and selectivity at rodent H3 receptors. The imidazole-free derivatives possessed moderate to pronounced antagonistic potency at guinea-pig ileal H3 receptor consistent with binding affinity at rat brain H3 receptors and showed a favourable receptor selectivity profile. For the compound 5, with the highest affinity at rat H3 receptors, comparable values were calculated in binding (pKi = 8.35) and functional (pA2 = 8.22) assays in SK-N-MC cells stably expressing human H3 receptors. These findings indicate to extend the investigation to pharmacokinetic property and central effects to gain deeper knowledge on the pharmacological potential of this compound.

Synthesis of 1,2-benzisothiazolyloxypropanolamine derivatives and investigation of their activity at beta-adrenoceptors.

The synthesis of 3-methoxy-1,2-benzisothiazole derivatives, substituted in position 5- (compounds 1-7) or 7- (compounds 8-14), with oxypropanolaminic side chains and the pharmacological investigation on their activity at beta-adrenoceptors are described. Compounds were prepared in an attempt to explore the ability of the benzisothiazole ring to interact with the beta-adrenoceptor site and to establish whether oxypropanolaminic derivatives recognise the beta3-adrenoceptor subtype. All the products were tested on rat atria, bladder and small intestine, which preferentially (but not exclusively) express beta1-, beta2- and beta3-adrenoceptors, respectively. When compared with the reference, non-specific, beta-adrenoceptor agonist isoprenaline, the products tested did not show any consistent beta-adrenoceptor agonistic activity in the different models. Most compounds relaxed smooth muscle preparations, but such effect was resistant to the blockade by propranolol (1 micromol/l), ICI 118,551 (1 micromol/l) or bupranolol (1-10 micromol/l), thus excluding that the spasmolytic effect involves any beta-adrenoceptors. When tested as antagonists, some of these products showed a concentration-dependent attenuation of the isoprenaline-induced effects in rat atria, without affecting beta-adrenoceptor-mediated relaxation in smooth muscle. These data confirm the ability of the benzisothiazole ring to interact with beta-adrenoceptors, but the substitution in 5- or 7-positions with oxypropanolaminic groups does not generate compounds endowed with specific activity at beta3-adrenoceptors. Conversely, most of these compounds behave as (specific) antagonists at beta1- (cardiac) adrenoceptors. At the maximum concentrations tested (1-100 micromol/l), these compounds also exert direct spasmolytic and negative chronotropic effects, which could be related to a blockade of Ca2+-dependent mechanisms at an intracellular level and/or an anaesthetic-like activity at plasma membranes.

Validation of a histamine H3 receptor model through structure-activity relationships for classical H3 antagonists.

Histamine H(3) receptor is a G protein-coupled receptor whose activation inhibits the synthesis and release of histamine and other neurotransmitters from nerve endings and is involved in the modulation of different central nervous system functions. H(3) antagonists have been proposed for their potential usefulness in diseases characterized by impaired neurotransmission and they have demonstrated beneficial effects on learning and food intake in animal models. In the present work, a 3D model of the rat histamine H(3) receptor, built by comparative modeling from the crystallographic coordinates of bovine rhodopsin, is presented with the discussion of its ability to predict the potency of known and new H(3) antagonists. A putative binding site for classical, imidazole-derived H(3) antagonists was identified by molecular docking. Comparison with a known pharmacophore model and the binding affinity of a new rigid H(3) antagonist (compound 1, pK(i)=8.02) allowed the characterization of a binding scheme which could also account for the different affinities observed in a recently reported series of potent H(3) antagonists, characterized by a 2-aminobenzimidazole moiety. Molecular dynamics simulations were employed to assess the stability and reliability of the proposed binding mode. Two new conformationally constrained benzimidazole derivatives were prepared and their binding affinity was tested on rat brain membranes; compound 9, designed to reproduce the conformation of a known potent H(3) antagonist, showed higher potency than compound 8, as expected from the binding scheme hypothesized.

Benzisothiazoles and beta-adrenoceptors: synthesis and pharmacological investigation of novel propanolamine and oxypropanolamine derivatives in isolated rat tissues.

In an attempt to examine the ability of benzisothiazole-based drugs to interact with beta-adrenoceptors, a series of 1,2-benzisothiazole derivatives, which were substituted with various propanolamine or oxypropanolamine side chains in the 2 or 3 position, were synthesised and tested. The pharmacological activity of these compounds at the beta-adrenoceptors was examined using isolated rat atria and small intestinal segments, which preferentially express the beta1- and beta3-adrenoceptor-mediated responses, respectively. None of these products showed any beta-adrenoceptor agonistic activity. In contrast, the 2- and 3-substituted isopropyl, tert-butyl, benzyl, and piperonyl derivatives 2a-d and 3a-d elicited surmountable inhibition of the isoprenaline-induced chronotropic effects in the atria, suggesting competitive antagonism at the beta1-recognition site. The pA2 values revealed tert-butyl 3b and the isopropyl substituted piperonyl derivatives 3a to be the most effective. Remarkably, many of the 2-substituted propanolamines were less active than the corresponding 3-substituted oxypropanolamines. With the exception of compound 3b, none of these drugs antagonised the muscle relaxant activity of isoprenaline in the intestine, suggesting no effect on the beta3-adrenoceptors. These results confirm the ability of the benzisothiazole ring to interact with the beta-adrenoceptors, and demonstrate that 2-substitution with propanolamine or 3-substitution with oxypropanolamine groups yields compounds with preferential antagonistic activity at the cardiac beta1-adrenoceptors. The degree of antagonism depends strongly on both the nature of the substituent and its position on the benzisothiazole ring.

Synthesis and three-dimensional quantitative structure-activity relationship analysis of H3 receptor antagonists containing a neutral heterocyclic polar group.

Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis was applied to a series of H(3) receptor antagonists characterized by an imidazole ring, an alkyl spacer, and a heterocyclic polar moiety containing an imidazole or a thiazole ring, with a view to investigate the requirements for H(3) receptor affinity on rat cortex membranes. The compounds were aligned based on the hypothesis that the presence of a H-bond donor group in the polar portion of the molecule can increase H(3) receptor affinity. The 3D-QSAR analysis, which was performed using both the CoMFA and CoMSIA protocols, revealed that the presence of a H-bond donor group is not statistically relevant for H(3) receptor affinity. Based on this result, another alignment was adopted that took into consideration the structural features common to all compounds, namely the imidazole ring and the N atom with a free lone pair in the polar portion. The 3D-QSAR models thus obtained showed that H(3) receptor affinity is modulated by the position and direction of the intermolecular interaction elicited by the polar group in the ligands.

The role of HB-donor groups in the heterocyclic polar fragment of H3-antagonists. I. Synthesis and biological assays.

It has been recently reported that compounds composed of an imidazole connected through an alkyl spacer to a 2-aminobenzimidazole showed high affinity towards the H(3)-receptor. The guanidine fragment of the 2-aminobenzimidazole is probably involved in hydrogen bond interactions at the binding site, and is referred to as the 'polar fragment'. In the present work, starting from 2-aminobenzimidazole derivatives with a di-methylene spacer 1 (pK(i)=7.25) or a tri-methylene one 2 (pK(i)=8.90), we investigated the importance of the hydrogen bond (HB) donor groups at the polar fragment in the interaction with the H(3)-receptor. The replacement of 2-aminobenzimidazoles with different moieties [2-aminobenzothiazole, 3, 4; 2-thiobenzimidazole, 5, 6; 2-thiobenzothiazole, 7, 8; 2-thio-4-phenyl- or 2-thio-5-phenyl-N-methylimidazoles, 9-12] highlighted the effect of the polar group basicity on the optimal length of the alkyl chain: longer spacers were preferred with polar groups of moderate basicity whereas, in the presence of neutral polar groups, the best affinity values were obtained with di-methylene chains. Moreover, N-methylation at the 2-aminobenzimidazole moiety 13-16 revealed different behaviour for compounds having different spacer lengths. In fact, methylation of the exocyclic NH group maintained high affinity for the tri-methylene 2-aminobenzimidazole derivative, while a drop in affinity was observed for the annular N-methylation. An opposite trend characterised di-methylene derivatives. These observed SAR suggest that, within this class of compounds, the number of HB-donor groups can be lowered while maintaining high receptor affinity. Since the presence of HB-donor groups strongly affects brain access, this observation could be useful to design and prepare new H(3)-antagonists.