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With the recent market approval of Pitolisant (Wakix®), the interest in clinical application for novel multifunctional histamine H3 receptor antagonists has clearly increased. Several combinations of different H3R pharmacophores with pharmacophoric elements of other G-protein coupled receptors, transporters, or enzymes have been synthesized by numerous pharmaceutical companies and academic institutions. Since central nervous system disorders are characterized by diverse physiological dysfunctions and deregulations of a complex network of signaling pathways, optimal multipotent drugs should simultaneously and peculiarly modulate selected groups of biological targets. Interestingly, very recent studies have shown that some clinically evaluated histamine H3 receptor antagonists possess a nanomolar affinity for sigma-1 receptor binding sites, suggesting that this property might play a role in their overall efficacy. The sigma-1 receptor, unusual and yet obscure protein, is supposed to be involved in numerous CNS pathologies through neuroprotection and neuroplasticity. These two different biological structures, histamine H3 and sigma-1 receptors, combined, can represent a potential fruitful target for therapeutic developments in tackling numerous human diseases.

Aims One of the therapeutic approaches in the treatment of obesity is the use of histamine H3 receptor ligands. Histamine plays a significant role in eating behavior because it causes a loss of appetite and is considered to be a satiety signal released during food intake. Material and methods Histamine ligands were selected based on the preliminary studies which included determination of intrinsic activity and selected pharmacokinetic parameters. Female Wistar rats were fed palatable feed for 28 days and simultaneously the tested compounds were administered intraperitoneally at a dose of 10 mg/kg b.w./day. Rats’ weight was evaluated daily and calories intake was evaluated once per week. At the end of experiment insulin and glucose tolerance tests was performed. Plasma levels of cholesterol, triglycerides, leptin, insulin, glucose, C-peptide and CRP were also determined. In order to rule out false-positive results the influence of tested compounds on spontaneous activity of rats was monitored. Results Animals fed palatable feed and treated with KSK-61 or KSK-63 compounds showed the slowest weight gain which was comparable to the one observed in control animals. Both compounds with the highest pharmacological activity have also similar pharmacokinetic properties with quite long half-life and high volume of distribution indicating that they can freely cross most biological barriers. Some compounds, especially KSK-63, compensated for metabolic disorders. Conclusion The presented study proves that search among the active histamine H3 receptor ligands for the new therapeutic agents to treat obesity is justified. Compounds KSK-61 and KSK-63 can be considered as the leading structures.

N9-Benzyl-substituted imidazo-, pyrimido- and 1,3-diazepino[2,1-f]purinediones were designed as dual-target-directed ligands combining A2A adenosine receptor (AR) antagonistic activity with blockade of monoamine oxidase B (MAO-B). A library of 37 novel compounds was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and for their ability to inhibit MAO-B. A systematic modification of the tricyclic structures based on a xanthine core by enlargement of the third heterocyclic ring or attachment of various substituted benzyl moieties resulted in the development of 9-(2-chloro-6-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione (9u; Ki human A2AAR: 189 nM and IC50 human MAO-B: 570 nM) as the most potent dual acting ligand of the series displaying high selectivity versus related targets. Moreover, some potent, selective MAO-B inhibitors were identified in the group of pyrimido- and 1,3-diazepino[2,1-f]purinediones. Compound 10d (10-(3,4-dichlorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-2,4(3H,6H)-dione) displayed an IC50 value at human MAO-B of 83 nM. Analysis of structure–activity relationships was complemented by molecular docking studies based on previously published X-ray structures of the protein targets. An extended biological profile was determined for selected compounds including in vitro evaluation of potential hepatotoxicity calculated in silico and antioxidant properties as an additional desirable activity. The new molecules acting as dual target drugs may provide symptomatic relief as well as disease-modifying effects for neurodegenerative diseases, in particular Parkinson’s disease.

In an attempt to find new dual acting histamine H3 receptor (H3R) ligands, we designed a series of compounds, structurally based on previously described in our group, a highly active and selective human histamine H3 receptor (hH3R) ligand KSK63. As a result, 15 obtained compounds show moderate hH3R affinity, the best being the compound 17 (hH3R Ki = 518 nM). Docking to the histamine H3R homology model revealed two possible binding modes, with key interactions retained in both cases. In an attempt to find possible dual acting ligands, selected compounds were tested for antioxidant properties. Compound 16 (hH3R Ki = 592 nM) showed the strongest antioxidant properties at the concentration of 10−4 mol/L. It significantly reduced the amount of free radicals presenting 50–60% of ascorbic acid activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, as well as showed antioxidative properties in the ferric reducing antioxidant power (FRAP) assay. Despite the yet unknown antioxidation mechanism and moderate hH3R affinity, 16 (QD13) constitutes a starting point for the search of potential dual acting H3R ligands-promising tools for the treatment of neurological disorders associated with increased neuronal oxidative stress.

Noting the worldwide rapid increase in the prevalence of overweight and obesity new effective drugs are now being sought to combat these diseases. Histamine H3 receptor antagonists may represent an effective therapy as they have been shown to modulate histamine synthesis and release and affect a number of other neurotransmitters (norepinephrine, acetylcholine, γ-aminobutyric acid, serotonin, substance P) thus influencing the food intake. Based on the preliminary studies determining affinity, intrinsic activity, and selected pharmacokinetic parameters, two histamine H3 receptor ligands were selected. Female rats were fed palatable food for 28 days and simultaneously administered the tested compounds intraperitoneally (i.p.) at a dose of 10 or 1 mg/kg b.w./day. Weight was evaluated daily and calorie intake was evaluated once per week. The plasma levels of cholesterol, triglycerides, leptin, adiponectin, ghrelin, corticosterone, CRP and IL-6 were determined at the end of experiment. The glucose tolerance test was also performed. To exclude false positives, the effect of tested compounds on spontaneous activity was monitored during the treatment, as well as the amount of consumed kaolin clay was studied as a reflection of possible gastrointestinal disturbances comparable to nausea. The histamine H3 receptor antagonists KSK-59 and KSK-73 administered i.p. at a dose of 10 mg/kg b.w. prevented weight gain in a rat model of excessive eating. They reduced adipose tissue deposits and improved glucose tolerance. Both compounds showed satisfying ability to penetrate through biological membranes determined in in vitro studies. Compound KSK-73 also reduced the caloric intake of the experimental animals what indicates its anorectic effect. These results show the pharmacological properties of histamine H3 receptor antagonists, (4-pyridyl)piperazine derivatives, as the compounds causing not only slower weight gain but also ameliorating some metabolic disorders in rats having the opportunity to overeat.

A library of 34 novel compounds based on a xanthine scaffold was explored in biological studies for interaction with adenosine receptors (ARs). Structural modifications of the xanthine core were introduced in the 8-position (benzylamino and benzyloxy substitution) as well as at N1, N3, and N7 (small alkyl residues), thereby improving affinity and selectivity for the A2A AR. The compounds were characterized by radioligand binding assays, and our study resulted in the development of the potent A2A AR ligands including 8-((6-chloro-2-fluoro-3-methoxybenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12d; Ki human A2AAR: 68.5 nM) and 8-((2-chlorobenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12h; Ki human A2AAR: 71.1 nM). Moreover, dual A1/A2AAR ligands were identified in the group of 1,3-diethyl-7-methylxanthine derivatives. Compound 14b displayed Ki values of 52.2 nM for the A1AR and 167 nM for the A2AAR. Selected A2AAR ligands were further evaluated as inactive for inhibition of monoamine oxidase A, B and isoforms of phosphodiesterase-4B1, -10A, which represent classical targets for xanthine derivatives. Therefore, the developed 8-benzylaminoxanthine scaffold seems to be highly selective for AR activity and relevant for potent and selective A2A ligands. Compound 12d with high selectivity for ARs, especially for the A2AAR subtype, evaluated in animal models of inflammation has shown anti-inflammatory activity. Investigated compounds were found to display high selectivity and may therefore be of high interest for further development as drugs for treating cancer or neurodegenerative diseases.

Taking into account that multidrug resistance (MDR) is the main cause for chemotherapeutic failure in cancer treatment, the ability of novel histamine H3 receptor ligands to reverse the cancer MDR was evaluated, using the ABCB1 efflux pump inhibition assay in mouse MDR T-lymphoma cells. The most active compounds displayed significant cytotoxic and antiproliferative effects as well as a very potent MDR efflux pump inhibitory action, 3-5-fold stronger than that of reference inhibitor verapamil. Although these compounds possess weak antagonistic properties against histamine H3 receptors, they are valuable pharmacological tools in the search for novel anticancer molecules. Furthermore, for the most active compounds, an insight into mechanisms of action using either, the luminescent Pgp-Glo™ Assay in vitro or docking studies to human Pgp, was performed.

Among still comparatively few G protein-coupled receptors, the adenosine A2A receptor has been co-crystallized with several ligands, agonists as well as antagonists. It can thus serve as a template with a well-described orthosteric ligand binding region for adenosine receptors. As not all subtypes have been crystallized yet, and in order to investigate the usability of homology models in this context, multiple adenosine A1 receptor (A1AR) homology models had been previously obtained and a library of lead-like compounds had been docked. As a result, a number of potent and one selective ligand toward the intended target have been identified. However, in in vitro experimental verification studies, many ligands also bound to the A2AAR and the A3AR subtypes. In this work we asked the question whether a classification of the ligands according to their selectivity was possible based on docking scores. Therefore, we built an A3AR homology model and docked all previously found ligands to all three receptor subtypes. As a metric, we employed an in vitro/in silico selectivity ranking system based on taxicab geometry and obtained a classification model with reasonable separation. In the next step, the method was validated with an external library of, selective ligands with similarly good performance. This classification system might also be useful in further screens.

Multi-target-directed ligands seem to be an interesting approach to the treatment of complex disorders such as Alzheimer’s disease. The aim of the present study was to find novel multifunctional compounds in a non-imidazole histamine H3 receptor ligand library. Docking-based virtual screening was applied for selection of twenty-six hits which were subsequently evaluated in Ellman’s assay for the inhibitory potency toward acetyl- (AChE) and butyrylcholinesterase (BuChE). The virtual screening with high success ratio enabled to choose multi-target-directed ligands. Based on docking results, all selected ligands were able to bind both catalytic and peripheral sites of AChE and BuChE. The most promising derivatives combined the flavone moiety via a six carbon atom linker with a heterocyclic moiety, such as azepane, piperidine or 3-methylpiperidine. They showed the highest inhibitory activities toward cholinesterases as well as well-balanced potencies against H3R and both enzymes. Two derivatives were chosen - 5 (IC50 = 0.46 μM (AChE); 0.44 μM (BuChE); Ki = 159.8 nM (H3R)) and 17 (IC50 = 0.50 μM (AChE); 0.76 μM (BuChE); Ki = 228.2 nM (H3R)), and their inhibition mechanism was evaluated in kinetic studies. Both compounds displayed non-competitive mode of AChE and BuChE inhibition. Compounds 5 and 17 might serve as good lead structures for further optimization and development of novel multi-target anti-Alzheimer’s agents.

Annelated purinedione derivatives have been shown to act as possible multiple-target ligands, addressing adenosine receptors and monoaminooxidases. In this study, based on our previous results, novel annelated pyrimido- and diazepino[2,1-f]purinedione derivatives were designed as dual-target-directed ligands combining A2A adenosine receptor (AR) antagonistic activity with blocking monoamine oxidase B. A library of 19 novel compounds was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and for their ability to inhibit MAO-B. This allowed 9-(2-chloro-6-fluorobenzyl)-3-ethyl-1-methyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione (13 e; Ki human A2A AR: 264 nM and IC50 human MAO-B: 243 nM) to be identified as the most potent dual-acting ligand from this series. ADMET parameters were estimated in vitro, and analysis of the structure-activity relationships was complemented by molecular-docking studies based on previously published X-ray structures of the protein targets. Such dual-acting ligands, by selectively blocking A2A AR, accompanied by the inhibition of dopamine metabolizing enzyme MAO-B, might provide symptomatic and neuroprotective effects in, among others, the treatment of Parkinson disease.

The pharmacological profile of pitolisant, a histamine H3 receptor antagonist/inverse agonist, indicates that this compound might reduce body weight and metabolic disturbances. Therefore, we studied the influence of pitolisant on body weight, water and sucrose intake as well as metabolic disturbances in the high-fat and high-sugar diet-induced obesity model in mice. To induce obesity, male CD-1 mice were fed a high-fat diet consisting of 40% fat blend for 14 weeks, water and 30% sucrose solution available ad libitum. Glucose tolerance test was performed at the beginning of week 15. Insulin tolerance was tested the day after. At the end of study, plasma levels of triglycerides and cholesterol were determined. Pitolisant at dose of 10 mg/kg bw (ip) was administrated during 14 days, starting from the beginning of week 13. Metformin at dose of 100 mg/kg bw (ip) was used as reference drug. Mice fed with high-fat diet and sucrose solution showed more weight gain throughout the 12-week period of inducing obesity. Animals fed with high-fat diet and treated with pitolisant (for the next 14 days) showed significantly less weight gain than mice from the control group consuming a high-fat feed. In the group treated with pitolisant, glucose levels were significantly lower than glucose levels of control obese mice after glucose load. The plasma triglyceride levels in pitolisant-treated mice were significantly lower compared with those in control obese group. In conclusion, pitolisant has a favorable influence of body weight and improves glucose tolerance and the lipid profile in obese mice.

This study focuses on the design, synthesis, molecular modeling and biological evaluation of a novel group of alkyl-1,3,5-triazinyl-methylpiperazines. New compounds were synthesized and their affinities for human histamine H4 receptor (hH4R) were evaluated. Among them, 4-(cyclohexylmethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (14) exhibited hH4R affinity with a Ki of 160 nM and behaved as antagonist in functional assays: the cellular aequorin-based assay (IC50 = 32 nM) and [35S]GTPγS binding assay (pKb = 6.67). In addition, antinociceptive activity of 14 in vivo was observed in Formalin test (in mice) and in Carrageenan-induced acute inflammation test (in rats).

Background: : One of the best known to date GPCR class A (Rhodopsin) includes more than 100 orphan receptors for which the endogenous ligand is not known or is unclear. One of them is N-arachidonyl glycine receptor, named GPR18, a receptor that has been reported to be activated by Δ9-THC, endogenous cannabinoid receptors agonist anandamide and other cannabinoid receptor ligands suggesting it could be considered as third cannabinoid receptor. GPR18 activity, as well as its distribution might suggest usage of GPR18 ligands in treatment of endometriosis, cancer, and neurodegenerative disorders. Yet, so far only few GPR18 antagonists have been described, thus only ligand-based design approaches appear to be most useful to identify new ligands for this orphan receptor. Methods: : Main goal of this study, GPR18 inactive form homology model was built on the basis of the evolutionary closest homologous template: Human P2Y1 Receptor crystal structure. Results: : Obtained model was further evaluated and showed active/nonactive ligands differentiating properties with acceptable confidence. Moreover, it allowed for preliminary assessment of proteinligand interactions for a set of previously described ligands. Conclusion:: Thus collected data might serve as a starting point for a discovery of novel, active GPR18 blocking ligands.

Aims Histamine H3 receptors ligands act anorectic by blocking the H3 autoreceptors in the CNS, that results in increased synthesis and disinhibition of histamine release. Histamine further influencing H1 receptors participates in the leptin-dependent inhibition of food intake. It also affects the peripheral metabolism by increasing white adipose tissue lipolysis. Thus, ligands such as KSK19 with significant antagonistic properties at the H3 receptor might serve as an useful treatment for obesity. Materials and methods To induce obesity, female CD-1 mice were fed a high-fat diet for 14 weeks. The test compound at the doses of 10 or 15 mg/kg, i.p. was administrated for 21 days. Glucose and insulin tolerance tests was performed at the beginning of week 15. At the end of study, amount of intraperitoneal fat pads, AlAT, IL-6 and TNF-α plasma levels were determined. Results Animals fed with high-fat diet and treated with test compound at the dose of 15 mg/kg showed significantly less weight gain, than mice from the control group. The use of KSK19 for 21 days in obese mice also significantly improved glucose tolerance and insulin resistance. In the tested doses KSK19 did not affect locomotor activity neither in lean nor in obese mice after single i.p. administration, but spontaneous activity increased during three hour after twentieth administrations. Conclusion KSK19 is a strong, selective histamine H3 receptor antagonist with a favorable influence on body weight after multiple administration at the dose of 15 mg/kg. Moreover it significantly improves glucose tolerance.

A series of 4-pyridylpiperazine derivatives with varying regulatory region substituents proved to be potent histamine H3 receptor (H3R) ligands in the nanomolar concentration range. The most influential modification that affected the affinity toward the H3R appeared by introducing electron-withdrawing moieties into the distal aromatic ring. In order to finally discuss the influence of the characteristic 4-pyridylpiperazine moiety on H3R affinity, two Ciproxifan analogues 2 and 3 with a slight modification in their basic part were obtained. The replacement of piperazine in 3 with piperidine in compound 2, led to slightly reduced affinity towards the H3R (Ki = 3.17 and 7.70 nM, respectively). In fact, 3 showed the highest antagonistic properties among all compounds in this series, hence affirming our previous assumptions, that the 4-pyridylpiperazine moiety is the key element for suitable interaction with the human histamine H3 receptor. While its structural replacement to piperidine is also tolerated for H3R binding, the heteroaromatic 4-pyridyl moiety seems to be essential for proper ligand-receptor interaction. The putative protein-ligand interactions responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at the H3R, as well as drug-like properties of ligands were evaluated using in vitro methods. Moreover, pharmacological in vivo test results of compound 9 (structural analogue of Abbott’s A-331440) clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound.

Design and development of multitarget-directed ligands (MTDLs) has become a very important approach in the search of new therapies for Alzheimer’s disease (AD). In our present research, a number of xanthone derivatives were first designed using a pharmacophore model for histamine H3 receptor (H3R) antagonists/inverse agonists, and virtual docking was then performed for the enzyme acetylcholinesterase. Next, 23 compounds were synthesised and evaluated in vitro for human H3R (hH3R) affinity and inhibitory activity on cholinesterases. Most of the target compounds showed hH3R affinities in nanomolar range and exhibited cholinesterase inhibitory activity with IC50 values in submicromolar range. Furthermore, the inhibitory effects of monoamine oxidases (MAO) A and B were investigated. The results showed low micromolar and selective human MAO B (hMAO B) inhibition. Two azepane derivatives, namely 23 (2-(5-(azepan-1-yl)pentyloxy)-9H-xanthen-9-one) and 25 (2-(5-(azepan-1-yl)pentyloxy)-7-chloro-9H-xanthen-9-one), were especially very promising and showed high affinity for hH3R (Ki = 170 nM and 100 nM respectively) and high inhibitory activity for acetylcholinesterase (IC50 = 180 nM and 136 nM respectively). Moreover, these compounds showed moderate inhibitory activity for butyrylcholinesterase (IC50 = 880 nM and 394 nM respectively) and hMAO B (IC50 = 775 nM and 897 nM respectively). Furthermore, molecular docking studies were performed for hH3R, human cholinesterases and hMAO B to describe the mode of interactions with these biological targets. Next, the two most promising compounds 23 and 25 were selected for in vivo studies. The results showed significant memory-enhancing effect of compound 23 in dizocilpine-induced amnesia in rats in two tests: step-through inhibitory avoidance paradigm (SIAP) and transfer latency paradigm time (TLPT). In addition, favourable analgesic effects of compound 23 were observed in neuropathic pain models. Therefore, compound 23 is a particularly promising structure for further design of new MTDLs for AD.

A series of twenty new chlorophenoxyalkylamine derivatives (9-28) was synthesized and evaluated on their binding properties at the human histamine H3 receptor (hH3R). The spacer alkyl chain contained five to seven carbon atoms. The highest affinities have shown the 4-chloro substituted derivatives 10 and 25 (Ki=133 and 128 nM, respectively) classified as antagonists in cAMP accumulation assay (EC50=72 and 75 nM, respectively). Synthesized compounds were also evaluated for anticonvulsant activity in Antiepileptic Screening Program (ASP) at National Institute of Neurological Disorders and Stroke (USA). Two compounds (4-chloro substituted derivatives: 20 and 26) were the most promising and showed in the MES seizure model in rats (after ip administration) ED50 values of 14 mg/kg and 13.18 mg/kg, respectively. Protective indexes (PI=TD50/ED50) were 3.2 for 20 and 3.8 for 26. Moreover, molecular modeling and docking studies were undertaken to explain affinity at hH3R of target compounds, and the experimentally and in silico estimation of properties like lipophilicity and metabolism was performed. Antiproliferative effects have been also investigated in vitro for selected compounds (10 and 25). These compounds neither possessed significant antiproliferative and antitumor activity, nor modulated CYP3A4 activity up to concentration of 10 μM.

The synthesis and biological activity of 4-tert-pentylphenoxypropyl derivatives are described in this manuscript. All compounds (except one) showed human histamine H3 receptor affinity with Ki values below 760 nM. The inhibitory activity toward human monoamine oxidase B (hMAO B) was evaluated using a fluorometric Amplex-Red assay, and most of the compounds were effective in the submicromolar range. Among them, 1-(3-(4-tert-pPentylphenoxy)propyl)pyrrolidine (5) exhibited hMAO B inhibitory activity with an IC50 value of 4.5 nM. In addition, hMAO B inhibition by 5 was shown to be non-competitive and reversible. Further, recently described potent histamine H3 receptor ligands – 4-tert-pentylphenoxyalkyl derivatives (with a 4–8 carbon spacer) – were evaluated for hMAO B inhibitory activity, and some of them displayed activity in the submicromolar range. Selected compounds were also tested for human MAO A (hMAO A) inhibitory potencies and exhibited no activity. Moreover, molecular modeling studies were carried out for tested compounds to explain their molecular mechanism of hMAO B inhibition and the selectivity of compounds for hMAO B over hMAO A.

As a continuation of our search for novel histamine H3 receptor ligands, a series of new acetyl and propionyl phenoxyalkylamine derivatives (2–25) was synthesized. Compounds with three to four carbon atoms alkyl chain spacer, composed of six various 4N-substituted piperazine moieties were evaluated for their binding properties at human histamine H3 receptors (hH3R). In vitro test results proved the 4-pyridylpiperazine moiety as crucial element for high hH3R affinity (hH3R Ki = 5.2–115 nM). Moreover introduction of carbonyl group containing residues in the lipophilic part of molecules instead of branched alkyl substituents resulted in increased affinity in correlation to previously described series, whereas propionyl derivatives showed slightly higher affinities than those of acetyl (16 and 22 vs. 4 and 10; hH3R Ki = 5.2 and 15.4 nM vs. 10.2 and 115 nM, respectively). These findings were confirmed by molecular modelling studies, demonstrating multiple ligand-receptor interactions. Furthermore, pharmacological in vivo test results of compound 4 clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound. Likewise, its protective action against hyperglycemia and the development of overweight has been shown. In order to estimate drug-likeness of compound 4, in silico and experimental evaluation of metabolic stability in human liver microsomes was performed.

As a continuation of our search for novel histamine H3 receptor ligands, a series of twenty four new tert-butyl and tert-pentyl phenoxyalkylamine derivatives (2−25) was synthesized. Compounds with three to four carbon atoms alkyl chain spacer were evaluated for their binding properties at human histamine H3 receptor (hH3R). The highest affinities were observed for 4-pyridyl derivatives 4, 10, 16 and 22 (Ki = 16.0–120 nM). As it has been shown in docking studies, those specific heteroaromatic 4-N piperazine substituents might interact with one of the key receptor interacting amino acids. Moreover, the most promising compounds exhibited anticonvulsant activity in the maximal electroshock-induced seizure (MES) model in mice. Furthermore, the blood-brain barrier penetration, the functional H3R antagonist potency as well as the pro-cognitive properties in the passive avoidance test were demonstrated for compound 10. In order to estimate drug-likeness of compound 10, in silico and experimental evaluation of metabolic stability in human liver microsomes was performed. In addition, paying attention to the results obtained within this study, the 4-pyridyl-piperazino moiety has been established as a new bioisosteric piperidine replacement in H3R ligands.

Since its discovery in 1983, followed by gene cloning in 1999, the histamine H3 receptor served as an outstanding target for drug discovery. The wide spectrum of possible therapeutic implications makes H3R's one of the most researched areas in the vast GPCR ligands field - started from imidazole containing ligands, through various successful imidazole replacements, with recent introduction of Wakix® to pharmaceutical market. One such replacement is piperazine moiety, a significant versatile scaffold in rational drug design for most of the GPCR ligands. Therefore, herein, we review ligands built on piperazine, as well as its seven membered analogue azepine, that target H3R’s and their potential therapeutical applications, in order to elucidate the current state of the art in this vast field. Due to a high level of structural divergence among compounds described herein, we decided to divide them into groups, where the key division element was the position of nitrogen basicity decreasing moieties in (homo)piperazine ring. Paying attention to a number of published structures and their overall high biological activity, one can realize that the (homo)piperazine scaffold bids a versatile template also for histamine H3 receptor ligands. With two possible substitution sites and therefore a number of possible structural combinations, piperazine derivatives stand as one of the largest group of high importance among H3R ligands.

As a continuation of our search for novel histamine H3 receptor ligands a series of twenty new tert-amyl phenoxyalkylamine derivatives (2-21) was synthesized. Compounds of four to eight carbon atoms spacer alkyl chain were evaluated on their binding properties at human histamine H3 receptor (hH3R). The highest affinities were observed for pentyl derivatives 6-8 (Ki=8.8-23.4nM range) and among them piperidine derivative 6 with Ki=8.8nM. Structures 6, 7 were also classified as antagonists in cAMP accumulation assay (with EC50=157 and 164nM, respectively). Moreover, new compounds were also evaluated for anticonvulsant activity in Antiepileptic Screening Program (ASP) at National Institute of Neurological Disorders and Stroke (USA). Seven compounds (2-4, 9, 11, 12 and 20) showed anticonvulsant activity at maximal electroshock (MES) test in the dose of 30mg/kg at 0.5h. In the subcutaneous pentetrazole (scMET) test compound 4 showed protection at 100 and 300mg/kg dose at mice, however compounds showed high neurotoxicity in rotarod test at used doses. Also, molecular modeling studies were undertaken, to explain affinity of compounds at hH3R (taking into the consideration X-ray analysis of compound 18). In order to estimate "drug-likeness" of selected compounds in silico and experimental evaluation of lipophilicity, metabolic stability and cytotoxicity was performed.

The search for novel lead from the group of various substituted N-piperazine ether derivatives was performed. Acyl- and pyridylpiperazine ethyl/propyl ethers were obtained via three different synthetic pathways. Affinity to histamine H3 receptor was established, as well as, for selected compounds, selectivity towards histamine H4R. Docking studies to the histamine H3R homology model strengthened the position of (4-(3-(4-(3-chlorobenzoyl)piperazin-1- yl)propoxy)phenyl)(cyclopropyl)methanone (compound 26) as a novel lead for further studies on histamine H3 receptor antagonist/inverse agonist.

A novel series of 4-pyridylpiperazine derivatives with varying alkyl linker length and eastern part substituents proved to be potent histamine H 3 receptor (hH 3 R) ligands in the nanomolar concentration range. While paying attention to their alkyl linker length, derivatives with a six methylene linker tend to be more potent than their five methylene homologues. Moreover, in the case of both phenoxyacetyl- and phenoxypropionyl- derivatives, an eight methylene linkers possess lower activity than their seven methylene homologues. However, in global analysis of collected data on the influence of alkyl linker length, a three methylene homologues appeared to be of highest hH 3 R affinity among all described 4-pyridylpiperazine derivatives from our group up to date. In the case of biphenyl and benzophenone derivatives, compounds with para- substituted second aromatic ring were of higher affinity than their meta analogues. Interestingly, benzophenone derivative 18 showed the highest affinity among all tested compounds (hH 3 R K i = 3.12 nM). The likely protein-ligand interactions, responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at H 3 R, as well as drug-like properties of selected ligands were evaluated using in vitro methods.

The use of atypical antipsychotic drugs like olanzapine is associated with side effects such as sedation and depression-like symptoms, especially during the initial period of the use. It is believed that the occurrence of these undesirable effectsis mainly the result of the histamine H1receptors blockade by olanzapine. In addition, use of olanzapine increases the level of triglycerides in the blood, which correlates with growing obesity. The aim of this study was to investigate the influence of pitolisant – H3 histamine antagonist - on subchronic olanzapine-induced depresion-like symptoms, sedation and hypertriglicerydemia. Forced swim test was conducted to determinate depressive-like effect of olanzapine and antidepressive-like activity during the co-administered pitolisant. The test was performed after the first and fifteenth day of the treatment of the mice. The spontaneous activity of the mice was measured on the fourteenth day of the treatment with a special, innovative RFID-system (Radio-frequency identification system) – TraffiCage (TSE-Systems, Germany). Triglyceride levels were determined on the sixteenth day of the experiment after 15 cycles of drug administration. Daily olanzapine treatment (4 mg/kg b.w., i.p., d.p.d) for 15 days significantly induces sedation (p

In recent years, multitarget-directed ligands have become an interesting strategy in a search for a new treatment of Alzheimer's disease. Combination of both: a histamine H3 receptor antagonist/inverse agonist and a cholinesterases inhibitor in one molecule could provide a new therapeutic opportunity. Here, we present biological evaluation of histamine H3 receptor ligands-chlorophenoxyalkylamine derivatives against cholinesterases: acetyl- and butyrylcholinesterase. The target compounds showed cholinesterase inhibitory activity in a low micromolar range. The most potent in this group was 1-(7-(4-chlorophenoxy)heptyl)homopiperidine (18) inhibiting the both enzymes (EeAChE IC50=1.93μM and EqBuChE IC50=1.64μM). Molecular modeling studies were performed to explain the binding mode of 18 with histamine H3 receptor as well as with cholinesterases.

Bassem Sadek,1 Ali Saad,1 Gniewomir Latacz,2 Kamil Kuder,2 Agnieszka Olejarz,2 Tadeusz Karcz,2 Holger Stark,3 Katarzyna Kieć-Kononowicz2 1Department of Pharmacology and Therapeutics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates; 2Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, Poland; 3Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Düsseldorf, Germany Abstract: A series of twelve novel non-imidazole-based ligands (3–14) was developed and evaluated for its in vitro binding properties at the human histamine H3 receptor (hH3R). The novel ligands were investigated for their in vivo protective effects in different seizure models in male adult rats. Among the H3R ligands (3–14) tested, ligand 14 showed significant and dose-dependent reduction in the duration of tonic hind limb extension in maximal electroshock (MES)-induced seizure model subsequent to acute systemic administration (5, 10, and 20mg/kg, intraperitoneally), whereas ligands 4, 6, and 7 without appreciable protection in MES model were most promising in pentylenetetrazole (PTZ) model. Moreover, the protective effect observed for ligand 14 in MES model was lower than that observed for the reference drug phenytoin and was entirely abrogated when rats were co-administered with the brain-penetrant H1R antagonist pyrilamine (PYR) but not the brain-penetrant H2R antagonist zolantidine (ZOL), demonstrating that histaminergic neurotransmission by activation of postsynaptically located H1Rs seems to be involved in the protective action. On the contrary, PYR and ZOL failed to abrogate the full protection provided by 4 in PTZ model and the moderate protective effect by 14 in strychnine (STR) model. Moreover, the experimental and in silico estimation of properties such as metabolism was performed for five selected test compounds. Also, lipophilicity using planar reversed-phase thin-layer chromatography method was included for better understanding of the molecular properties of the tested compounds. Additionally, the absorption, distribution, metabolism, and elimination and toxicity parameters were evaluated for the most promising compounds 2, 4, 6, 7, and 14 utilizing in vitro methods. These interesting results highlight the potential of H3R ligands as new antiepileptic drugs or as adjuvants to available epilepsy medications. Keywords: histamine H3 receptors, antagonists, anticonvulsant, R-(α)-methyl-histamine, pyrilamine, zolantidine

Synthesis and biological evaluation of the novel histamine H3 receptor ligands is described. Two series of ethers (aliphatic and aromatic) have been prepared by four different methods. Compounds were evaluated for their affinities at recombinant human H3 receptor stably expressed in CHO cells. The ethers show from low to moderate in vitro affinities in nanomolar concentration range. The most potent compound was the 1-[3-(4-tert-butylphenoxy)propyl]-4-piperidino-piperidine 16 (hH3R Ki = 100 nM). Several members of the new series investigated under in vivo conditions, proved to be inactive.

The expansion of fluorescent techniques for studying the ligand-receptor interaction resulted in a burst of the novel fluorescent ligands development. The discovery of the ligand, that is of high affinity to the receptor and whose localization could be easily visualized, even on the single cell level, gave the researchers a strong impulse to investigate that field of GPCR ligands. Moreover, paying attention to the "non pharmacological" advantages of these ligands, as well as the techniques to be used, fluorescent ligands are becoming treated more seriously, as the ligands themselves, and as novel, useful tools for studying GPCRs. Herein, we review results described in the literature, starting from the year 2000, in the field of the fluorescent GPCR small, non-peptide ligands according to the affinity to the selected receptors (histamine, adenosine, adrenergic, cannabinoid, muscarinie, neuropeptide Y and serotonine) as well as the fluorophores that have been used to tag the molecules.

Within the constantly growing number of histamine H4 (H4R) receptor ligands there is a large group of azine derivatives. A series of novel compounds in the group of 4-methylpiperazine-1,3,5-triazine-2-amines were designed and obtained. Considered structures were modified at the triazine 6-position by introduction of variously substituted arylethenyl moieties. Their affinities to histamine H4 receptors were evaluated in radioligand binding assays with use of Sf9 cells, transiently expressing human H4R. Pharmacological studies results allowed to identify 4-[(E)-2-(3-chlorophenyl)ethenyl]-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (Ki = 253 nM) as the most potent compound in the present series.

An artificial polypeptide receptor (APR) library was created by using the self-organization of N-lipidated peptides attached to cellulose via m-aminophenylamino-1,3,5-triazine. The response of the library was probed using a series of novel H3 receptor ligands. Since no guidelines on how to design an APRs selective vs certain receptor types exist, a diverse set of amino acids (Ala, Trp, Pro, Glu, His, Lys and Ser) were used and coupled with one of three gating fatty acids (palmitic, ricinoleic or capric). A competitive adsorption-desorption of an appropriate reporter dye was used for the indirect visualization of the interactions of guests with particular receptors. The resulted library response to individual inhibitors was then arranged in a matrix, preprocessed and analyzed using the principal component analysis (PCA) and partial least squares (PLS) method. The most important conclusion obtained from the PCA analysis is that the library differentiates the probed compounds according to the lipophilicity of the gating unit. The PC3 with a dominant absolute contribution of the receptors containing Glu allowed for the best separation of the ligands with respect to their activity. This conclusion is in agreement with the fact that Glu 206 is a genuine ligand counterpart in the natural histamine receptor.

To determine the potential of histamine H3 receptor (H3R) ligands as new antiepileptic drugs (AEDs), aromatic ether, and diether derivatives (1-12) belonging to the nonimidazole class of ligands, with high in-vitro binding affinity at human H3R, were tested for their in-vivo anticonvulsive activity in the maximal electroshock (MES)-induced and pentylenetetrazole (PTZ)-kindled seizure models in rats. The anticonvulsive effects of a systemic injection of 1-12 on MES-induced and PTZ-kindled seizures were evaluated against the reference AED phenytoin (PHT) and the structurally related H3R antagonist/inverse agonist pitolisant (PIT). Among the most promising ligands 2, 4, 5, and 11, there was a significant and dose-dependent reduction in the duration of tonic hind limb extension (THLE) in MES-induced seizure subsequent to administration of 4 and 5 [(5, 10, and 15 mg/kg, intraperitoneally (i.p.)]. The protective effects observed for the 1-(3-(3-(4-chlorophenyl)propoxy)propyl)-3-methylpiperidine derivative 11 at 10 mg/kg, i.p. were significantly greater than those of PIT, and were reversed by pretreatment with the central nervous system penetrant H1R antagonist pyrilamine (PYR) (10 mg/kg). Moreover, the protective action of the reference AED PHT, at a dose of 5 mg/kg (without considerable protection in the MES model), was significantly augmented when coadministered with derivative 11 (5 mg/kg, i.p.). Surprisingly, pretreatment with derivative 7 (10 mg/kg, i.p.), an ethylphenoxyhexyl-piperidine derivative without considerable protection in the MES model, potently altered PTZ-kindled seizure, significantly prolonged myoclonic latency time, and clearly shortened the total seizure time when compared with control, PHT, and PIT. These interesting results highlight the potential of H3R ligands as new AEDs or as adjuvants to available AED therapeutics.

The robust of fluorescent techniques to study the ligand-receptor interaction followed by rapidly developing fluorescence imaging techniques resulted in a burst of the novel fluorescent ligands development. Taking into consideration not only ligand's high affinity to the receptor, but also their fluorescent properties to visualize the interaction even in the single cell level, gives the researchers a strong impulse to investigate that field of GPCR ligands. Moreover, paying attention to the "non pharmacological" advantages of these ligands, as well as the techniques to be used, fluorescent ligands become commonly used pharmacological tools to study GPCRs. Herein we report on the results described in the literature since late 2007 in the field of the fluorescent GPCR small, non-peptide ligands according the receptor affinity, fluorophores that has been used to tag the molecules and their fluorescent properties as well as their application in GPCR research.

A series of novel 2-amino-4-(4-methylpiperazin-1-yl)-1,3,5-triazine derivatives with different aryl substituents in the 6-position was designed, synthesized and evaluated for histamine H4 receptor (H4R) affinity in Sf9 cells expressing human H4R co-expressed with G-protein subunits. Triazine derivative 8 with a 6-(p-chlorophenyl) substituent showed the highest affinity with hH4R Ki value of 203 nM and was classified as an antagonist in cAMP accumulation assay. This compound, identified as a new lead structure, demonstrated also anti-inflammatory properties in preliminary studies in mice (carrageenan-induced edema test) and neither possessed significant antiproliferative activity, nor modulated CYP3A4 activity up to concentration of 25 μM. In order to discuss structure–activity relationships molecular modeling and docking studies were undertaken.

The study presents novel biological properties of diether derivatives of homo- or substituted piperidine ligands of the histamine H(3) receptor. The compounds were evaluated for their inhibitory potency against acetylcholinesterase (AChE) from the electric eel and butyrylcholinesterase (BuChE) from horse serum. The most interesting multifunctional compound 13 displayed high affinity for the cloned hH(3) R (K(i) = 3.48 nM) and moderate inhibitory potency against both enzymes (IC(50) AChE = 7.91 µM and BuChE = 4.97 µM). Molecular modeling studies revealed interactions with key amino acid residues in the homology model of histamine H(3) receptor ligands, as well as the binding model for AChE and BuChE in the catalytic and peripheral active sites.

Syntheses and physicochemical properties of N-cycloalkyl-substituted imidazo-, pyrimido- and 1,3-diazepino[2,1-f]purinediones are described. These derivatives were synthesized by cyclization of 7-halogenoalkyl-8-bromo-1,3-dimethylxanthine derivatives with aminocycloalkanes. The obtained compounds (1-33) were evaluated for their affinity to rat adenosine A(1) and A(2A) receptors. Selected compounds were additionally investigated for affinity to the human A(1), A(2A), A(2B) and A(3) receptor subtypes. The results of the radioligand binding assays at adenosine A(1) and A(2A) receptors showed that most of the compounds exhibited adenosine A(2A) receptor affinity at micromolar or submicromolar concentrations; an annelated pyrimidine ring was beneficial for A(2A) affinity. The most potent A(2A) ligands of the present series were compounds 6 (K(i) 0.33 μM rat A(2A), 0.31 μM human A(2A)), 8 (K(i) 0.98 μM rat A(2A), 0.42 μM human A(2A)) and 15 (K(i) 0.24 μM rat A(2A), 0.61 μM human A(2A)) with the latter one showing high A(2A) selectivity. In NaCl shift assay, 15 was shown to be an antagonist at A(2A) receptors. This result was confirmed for the best compounds 6, 8, 15 in cAMP accumulation studies. A 3D-QSAR equation with a good predicting power (q(2) = 0.88) for A(2A) AR affinity was obtained. The compounds were evaluated in vivo as anticonvulsants in MES and ScMet tests and examined for neurotoxicity in mice (i.p.). Most of them showed anticonvulsant activity in chemically induced seizures; among them the diazepinopurinediones were the best (e.g. 31) showing protection in both tests on short time symptoms, without signs of neurotoxicity. Five compounds, 8, 17, 20, 29, and 31, exhibited anticonvulsant activity after peroral application in rats. Structure-activity relationships are discussed including the analysis of lipophilic and spatial properties. The new compounds, which contain a basic nitrogen atom and can therefore be protonated, may be good starting points for obtaining A(2A) antagonists with good water-solubility.

Synthesis and biological activities of a series of homo- or substituted piperidine unsymmetrical diethers are described. The novel compounds were evaluated for histamine H3 receptor binding affinities at recombinant human H3 receptor stably expressed in HEK-293 cells. All diethers showed in vitro affinities in nanomolar concentration range. The most potent compounds are 1-[3-(3-(4-chlorophenoxy)propoxy)propyl]-3-methylpiperidine 11 (Ki = 3.2 nM) and 1-[3-(3-(4-chlorophenoxy)propoxy)propyl]azepane 13 (Ki = 3.5 nM).