Pharmacological properties and procognitive effects of ABT-288, a potent and selective histamine H3 receptor antagonist.

Blockade of the histamine H(3) receptor (H(3)R) enhances central neurotransmitter release, making it an attractive target for the treatment of cognitive disorders. Here, we present in vitro and in vivo pharmacological profiles for the H(3)R antagonist 2-[4'-((3aR,6aR)-5-methyl-hexahydro-pyrrolo[3,4-b]pyrrol-1-yl)-biphenyl-4-yl]-2H-pyridazin-3-one (ABT-288). ABT-288 is a competitive antagonist with high affinity and selectivity for human and rat H(3)Rs (K(i) = 1.9 and 8.2 nM, respectively) that enhances the release of acetylcholine and dopamine in rat prefrontal cortex. In rat behavioral tests, ABT-288 improved acquisition of a five-trial inhibitory avoidance test in rat pups (0.001-0.03 mg/kg), social recognition memory in adult rats (0.03-0.1 mg/kg), and spatial learning and reference memory in a rat water maze test (0.1-1.0 mg/kg). ABT-288 attenuated methamphetamine-induced hyperactivity in mice. In vivo rat brain H(3)R occupancy of ABT-288 was assessed in relation to rodent doses and exposure levels in behavioral tests. ABT-288 demonstrated a number of other favorable attributes, including good pharmacokinetics and oral bioavailability of 37 to 66%, with a wide central nervous system and cardiovascular safety margin. Thus, ABT-288 is a selective H(3)R antagonist with broad procognitive efficacy in rodents and excellent drug-like properties that support its advancement to the clinical area.

H4 receptor antagonism exhibits anti-nociceptive effects in inflammatory and neuropathic pain models in rats.

The histamine H(4) receptor (H(4)R) is expressed primarily on cells involved in inflammation and immune responses. To determine the potential role of H(4)R in pain transmission, the effects of JNJ7777120, a potent and selective H(4) antagonist, were characterized in preclinical pain models. Administration of JNJ7777120 fully blocked neutrophil influx observed in a mouse zymosan-induced peritonitis model (ED(50)=17 mg/kg s.c., 95% CI=8.5-26) in a mast cell-dependent manner. JNJ7777120 potently reversed thermal hyperalgesia observed following intraplantar carrageenan injection of acute inflammatory pain (ED(50)=22 mg/kg i.p., 95% CI=10-35) in rats and significantly decreased the myeloperoxide activity in the carrageenan-injected paw. In contrast, no effects were produced by either H(1)R antagonist diphenhydramine, H(2)R antagonists ranitidine, or H(3)R antagonist ABT-239. JNJ7777120 also exhibited robust anti-nociceptive activity in persistent inflammatory (CFA) pain with an ED(50) of 29 mg/kg i.p. (95% CI=19-40) and effectively reversed monoiodoacetate (MIA)-induced osteoarthritic joint pain. This compound also produced dose-dependent anti-allodynic effects in the spinal nerve ligation (ED(50)=60 mg/kg) and sciatic nerve constriction injury (ED(50)=88 mg/kg) models of chronic neuropathic pain, as well as in a skin-incision model of acute post-operative pain (ED(50)=68 mg/kg). In addition, the analgesic effects of JNJ7777120 were maintained following repeated administration and were evident at the doses that did not cause neurologic deficits in rotarod test. Our results demonstrate that selective blockade of H(4) receptors in vivo produces significant anti-nociception in animal models of inflammatory and neuropathic pain.

Design of a new histamine H3 receptor antagonist chemotype: (3aR,6aR)-5-alkyl-1-aryl-octahydropyrrolo[3,4-b]pyrroles, synthesis, and structure-activity relationships.

A new histamine H3 receptor (H3R) antagonist chemotype 1 was designed by combining key pharmacophoric elements from two different precursor structural series and then simplifying and optimizing the resulting combined structural features. First, analogues were made based on a previously identified conessine-based H3R antagonist series. While the first analogues 11 and 15 showed no antagonistic activity to H3R, the mere addition of a key moiety found in the reference compound 7 (ABT-239) elevated the series to high potency at H3R. The hybrid structure (16b) was judged too synthetically demanding to enable an extensive SAR study, thus forcing a strategy to simplify the chemical structure. The resulting (3aR,6aR)-5-alkyl-1-aryl-octahydropyrrolo[3,4-b]pyrrole series proved to be highly potent, as exemplified by 17a having a human H3 K(i) of 0.54 nM, rat H3 K(i) of 4.57 nM, and excellent pharmacokinetics (PK) profile in rats (oral bioavailability of 39% and t(1/2) of 2.4 h).

Structure-activity studies on a series of a 2-aminopyrimidine-containing histamine H4 receptor ligands.

A series of 2-aminopyrimidines was synthesized as ligands of the histamine H4 receptor (H4R). Working in part from a pyrimidine hit that was identified in an HTS campaign, SAR studies were carried out to optimize the potency, which led to compound 3, 4- tert-butyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-ylamine. We further studied this compound by systematically modifying the core pyrimidine moiety, the methylpiperazine at position 4, the NH2 at position 2, and positions 5 and 6 of the pyrimidine ring. The pyrimidine 6 position benefited the most from this optimization, especially in analogs in which the 6- tert-butyl was replaced with aromatic and secondary amine moieties. The highlight of the optimization campaign was compound 4, 4-[2-amino-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl]benzonitrile, which was potent in vitro and was active as an anti-inflammatory agent in an animal model and had antinociceptive activity in a pain model, which supports the potential of H 4R antagonists in pain.

The alkaloid conessine and analogues as potent histamine H3 receptor antagonists.

The naturally occurring alkaloid, conessine (6), was discovered to bind to histamine H3 receptors in a radioligand-based high-throughput screen. Conessine displayed high affinity at both rat and human H3 receptors (pKi = 7.61 and 8.27) and generally high selectivity against other sites, including histamine receptors H1, H2, and H4. Conessine was found to efficiently penetrate the CNS and reach very high brain concentrations. Although the very slow CNS clearance and strong binding to adrenergic receptors discouraged focus on conessine itself for further development, its potency and novel steroid-based skeleton motivated further chemical investigation. Modification based on introducing diversity at the 3-nitrogen position generated a new series of H3 antagonists with higher in vitro potency, improved target selectivity, and more favorable drug-like properties. One optimized analogue (13c) was examined in detail and was found to be efficacious in animal behavioral model of cognition.

In vitro SAR of pyrrolidine-containing histamine H3 receptor antagonists: trends across multiple chemical series.

Structure-activity relationships (SAR) were analyzed within a library of diverse yet simple compounds prepared as histamine H3 antagonists. The libraries were constructed with a variety of low molecular weight pyrrolidines, selected from (R)-2-methylpyrrolidine, (S)-2-methylpyrrolidine, and pyrrolidine.

Synthesis, potency, and in vivo profiles of quinoline containing histamine H3 receptor inverse agonists.

A new structural series of histamine H3 receptor antagonist was developed. The new compounds are based on a quinoline core, appended with a required basic aminoethyl moiety, and with potency- and property-modulating heterocyclic substituents. The analogs have nanomolar and subnanomolar potency for the rat and human H3R in various in vitro assays, including radioligand competition binding as well as functional tests of H3 receptor-mediated calcium mobilization and GTPgammaS binding. The compounds possessed favorable drug-like properties, such as good PK, CNS penetration, and moderate protein binding across species. Several compounds were found to be efficacious in animal behavioral models of cognition and attention. Further studies on the pharmaceutic properties of this series of quinolines discovered a potential problem with photochemical instability, an issue which contributed to the discontinuation of this series from further development.

An 80-amino acid deletion in the third intracellular loop of a naturally occurring human histamine H3 isoform confers pharmacological differences and constitutive activity.

In this article, we pharmacologically characterized two naturally occurring human histamine H3 receptor (hH3R) isoforms, hH3R(445) and hH3R(365). These abundantly expressed splice variants differ by a deletion of 80 amino acids in the intracellular loop 3. In this report, we show that the hH3R(365) is differentially expressed compared with the hH3R(445) and has a higher affinity and potency for H3R agonists and conversely a lower potency and affinity for H3R inverse agonists. Furthermore, we show a higher constitutive signaling of the hH3R(365) compared with the hH3R(445) in both guanosine-5'-O-(3-[35S]thio) triphosphate binding and cAMP assays, likely explaining the observed differences in hH3R pharmacology of the two isoforms. Because H3R ligands are beneficial in animal models of obesity, epilepsy, and cognitive diseases such as Alzheimer's disease and attention deficit hyperactivity disorder and currently entered clinical trails, these differences in H3R pharmacology of these two isoforms are of great importance for a detailed understanding of the action of H3R ligands.

Novel heterocyclic-substituted benzofuran histamine H3 receptor antagonists: in vitro properties, drug-likeness, and behavioral activity.

Three novel heterocyclic benzofurans A-688057 (1), A-687136 (2), and A-698418 (3) were profiled for their in vitro and in vivo properties as a new series of histamine H(3) receptor antagonists. The compounds were all found to have nanomolar potency in vitro at histamine H(3) receptors, and when profiled in vivo for CNS activity, all were found active in an animal behavioral model of attention. The compound with the most benign profile versus CNS side effects was selected for greater scrutiny of its in vitro properties and overall drug-likeness. This compound, A-688057, in addition to its potent and robust efficacy in two rodent behavioral models at blood levels ranging 0.2-19 nM, possessed other favorable features, including high selectivity for H(3) receptors (H(3), K(i)=1.5 nM) versus off-target receptors and channels (including the hERG K(+) channel, K(i)>9000 nM), low molecular weight (295), high solubility, moderate lipophilicity (logD(pH7.4)=2.05), and good CNS penetration (blood/brain 3.4x). In vitro toxicological tests indicated low potential for phospholipidosis, genotoxicity, and CYP(450) inhibition. Even though pharmacokinetic testing uncovered only moderate to poor oral bioavailability in rat (26%), dog (30%), and monkey (8%), and only moderate blood half-lives after i.v. administration (t(1/2) in rat of 2.9h, 1.7h in dog, 1.8h in monkey), suggesting poor human pharmacokinetics, the data overall indicated that A-688057 has an excellent profile for use as a pharmacological tool compound.

4-[6-(2-Aminoethyl)naphthalen-2-yl]benzonitriles are potent histamine H3 receptor antagonists with high CNS penetration.

4-[6-(2-Tertiaryaminoethyl)naphthalen-2-yl]benzonitriles are conformationally constrained histamine H3 receptor antagonists with high potency and selectivity. The analogs were designed around a naphthalene core, with the goal of enhancing lipophilicity and CNS penetration, as compared to a previously reported benzofuran series. The SAR of the tertiary amine moiety is similar to that reported for the benzofuran series, with analogs bearing a 2-methylpyrrolidine substituent possessing the greatest rat and human H3 receptor binding affinities.

Lack of efficacy of melanin-concentrating hormone-1 receptor antagonists in models of depression and anxiety.

The aim of this study was to validate melanin-concentrating hormone (MCH)-1 receptor antagonism as a potential treatment of mood disorders. We attempted to replicate the effects previously reported with SNAP-7941 and expanded the investigation to three other orally bioavailable MCH-1 receptor antagonists with good brain penetration. SNAP-7941 (3-30 mg/kg, i.p.) and T-226296 (5-60 mg/kg, p.o.) (+/- racemate), were evaluated in the rat forced swim and mouse tail suspension tests. (+)SNAP-7941 (3-10 mg/kg, p.o.) was also tested in a modified 5-min rat forced swim protocol as previously reported. A-665798 (3-30 mg/kg, p.o.) and A-777903 (3-30 mg/kg, p.o.) were tested in mouse tail suspension and rat Vogel tests. None of the compounds showed meaningful efficacy in the paradigms tested. The lack of efficacy with four structurally different MCH-1 receptor antagonists does not support a role for therapeutic treatment of depression/anxiety via this mechanism of action.

Detection of multiple H3 receptor affinity states utilizing [3H]A-349821, a novel, selective, non-imidazole histamine H3 receptor inverse agonist radioligand.

1. A-349821 is a selective histamine H3 receptor antagonist/inverse agonist. Herein, binding of the novel non-imidazole H3 receptor radioligand [3H]A-349821 to membranes expressing native or recombinant H3 receptors from rat or human sources was characterized and compared with the binding of the agonist [3H]N--methylhistamine ([3H]NMH). 2. [3H]A-349821 bound with high affinity and specificity to an apparent single class of saturable sites and recognized human H3 receptors with 10-fold higher affinity compared to rat H3 receptors. [3H]A-349821 detected larger populations of receptors compared to [3H]NMH. 3. Displacement of [3H]A-349821 binding by H3 receptor antagonists/inverse agonists was monophasic, suggesting recognition of a single binding site, while that of H3 receptor agonists was biphasic, suggesting recognition of both high- and low-affinity H3 receptor sites. 4. pKi values of high-affinity binding sites for H3 receptor competitors utilizing [3H]A-349821 were highly correlated with pKi values obtained with [3H]NalphaMH, consistent with labelling of H3 receptors by [3H]A-349821. 5. Unlike assays utilizing [3H]NMH, addition of GDP had no effect on saturation parameters measured with [3H]A-349821, while displacement of [3H]A-349821 binding by the H3 receptor agonist histamine was sensitive to GDP. 6. In conclusion, [3H]A-349821 labels interconvertible high- and low-affinity states of the H3 receptor, and displays improved selectivity over imidazole-containing H3 receptor antagonist radioligands. [3H]A-349821 competition studies showed significant differences in the proportions and potencies of high- and low-affinity sites across species, providing new information about the fundamental pharmacological nature of H3 receptors.

Evidence for tolerance following repeated dosing in rats with ciproxifan, but not with A-304121.

Blockade of presynaptic histamine H(3) receptors with potent and selective ligands improves cognitive function in rodents and there is significant interest in developing such drugs for long-term symptomatic treatment of CNS disorders such as attention deficit hyperactivity disorder (ADHD). Unfortunately, little is known about the effects of repeated exposure to H(3) receptor antagonists/inverse agonists. We therefore investigated the effects of acute and repeated daily administration of two potent, brain penetrating H(3) receptor antagonists/inverse agonists, ciproxifan and A-304121, on rat body weight, food and water intake, core temperature and locomotor activity, as well as H(3) receptor density and gene expression levels. Methylphenidate, used clinically for the treatment of ADHD, was included as an additional comparator. Ciproxifan, an imidazole-based compound, decreased food intake over the first 10 days and locomotor activity acutely, but these effects were lost after further repeated administration. The ex vivo binding studies revealed increased H(3) receptor density in rats following repeated administration of ciproxifan for 10 or 15 days; however, H(3) receptor gene expression was not changed. In contrast, rats treated with the non-imidazole, A-304121, did not differ from controls on any measure during the observation period, while rats treated with methylphenidate exhibited hyperthermia and hyperactivity. The implications for potential long-term treatment with H(3) receptor antagonists in CNS disorders such as ADHD are discussed.

Synthesis and SAR of 5-amino- and 5-(aminomethyl)benzofuran histamine H3 receptor antagonists with improved potency.

A new series of H3 receptor antagonists was discovered with nanomolar and subnanomolar affinities at human and rat H3 receptors. Starting from an earlier, more structurally limited series of benzofurans, the present series of compounds demonstrated increased structural variety and flexibility with greater in vitro potency. One compound in particular, [2-[2-(2-(R)-methylpyrrolidin-1-yl)ethyl]benzofuran-5-yl](5-nitropyridin-2-yl)amine (7h), gave the best binding potency (human K(i) of 0.05 nM, rat K(i) of 0.11 nM), which represented a 9-fold (in human) and an 11-fold (in rat) improvement over ABT-239 (compound 5), a compound previously reported to have excellent in vitro potency and in vivo efficacy. The synthesis, SAR of the H3 binding affinities, in vitro assay for phospholipidosis, and pharmacokinetic properties of the new compounds are described.

Structure-activity relationships of arylbenzofuran H3 receptor antagonists.

An SAR study of histamine H3 receptor antagonists based on substituted (R)-2-methyl-1-[2-(5-phenyl-benzofuran-2-yl)-ethyl]-pyrrolidines is presented.

Effects of histamine H3 receptor antagonists in two models of spatial learning.

Despite the well-described attention and short-term memory enhancing effects of H3 receptor antagonists, and evidence to suggest a close relationship between central histaminergic and cholinergic systems, there is a paucity of evidence for a role for H3 receptor blockade in spatial learning. To address this, we investigated two H3 receptor antagonists in a visual discrimination water maze in rats, and in a Barnes circular maze in mice. Thioperamide and ciproxifan significantly attenuated a scopolamine-induced deficit in the water maze task, while only ciproxifan showed a modest attenuation in the Barnes maze. Taken together, these data suggest a role for H3 receptors in spatial learning that appears to be task-dependent.

G protein-dependent pharmacology of histamine H3 receptor ligands: evidence for heterogeneous active state receptor conformations.

Previously reported pharmacological studies using the imidazole-containing histamine H3 receptor ligands GT-2331 (Cipralisant) and proxyfan resulted in a range of classifications (antagonist, agonist, and protean) for these compounds. We examined the role that the signaling system, with particular emphasis on the type of G protein, had on the pharmacology observed for H3 ligands. Ligands were assessed using assays measuring neurotransmitter release, cAMP, and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding. Whereas clobenpropit and ciproxifan were consistently antagonists, GT-2331, proxyfan, and imetit exhibited differential activity. Although GT-2331 and proxyfan exhibited little agonist activity in neurotransmitter release assays, both demonstrated full agonism relative to (R)-alpha-methylhistamine in cAMP assays. In [35S]GTPgammaS binding assays, GT-2331 and proxyfan demonstrated partial agonism. Imetit showed full agonism in most assays, but it was slightly less efficacious in a neurotransmitter release assay and in [35S]GTPgammaS binding at the human H3 receptor. To further examine these ligands, we coexpressed G alpha16 or chimeric G alpha q/i5 in human embryonic kidney cells expressing the human H3 receptor and assayed intracellular calcium and cAMP levels. GT-2331, proxyfan, and imetit demonstrated full agonism in all assays of cAMP activity. However, in cells expressing G alpha16, they exhibited minimal agonism in calcium mobilization assays, whereas imetit showed partial agonism. When G alpha q/i5 was used, the activity of both GT-2331 and proxyfan increased, whereas imetit became a full agonist. These results demonstrate that GT-2331 and proxyfan's differential pharmacology at the H3 receptor depends on the type of G protein used and provide indirect evidence for differential ligand-bound active states that mediate signaling by the H3 receptor.

Assessment of pharmacology and potential anti-obesity properties of H3 receptor antagonists/inverse agonists.

Histamine is a key neurotransmitter that alters central nervous system functions in both behavioural and homeostatic contexts through its actions on the histamine (H) subreceptors H(1), H(2) and H(3) G-protein-coupled receptors. H(3)receptors have a diverse central nervous system distribution where they function as both homo- and hetero-receptors to modulate the synthesis and/or release of several neurotransmitters. H(3) receptors are constitutively active, which implies that antagonists of H(3) receptors may also function as inverse agonists to alter the basal state of the receptor and uncouple constitutive receptor-G-protein interactions. Reference H(3) antagonists such as thioperamide and ciproxifan, administered either centrally or systemically, have been shown to cause changes in food consumption and/or body weight in proof-of-concept studies. More recently, several non-imidazole-based H(3) antagonists/inverse agonists have also been described with efficacy in at least one animal model of human obesity. Considerable preclinical effort remains necessary before such compounds achieve therapeutic success or failure. Moreover, ongoing research in a number of laboratories has shed new insights into the effects of H(3) ligands in the control of feeding, appetite and body weight, which offer different results and conclusions. The goal of this review is to appraise these findings and forecast whether any H(3) antagonists/inverse agonists will provide clinical utility to treat human obesity.

4-(2-[2-(2(R)-methylpyrrolidin-1-yl)ethyl]benzofuran-5-yl)benzonitrile and related 2-aminoethylbenzofuran H3 receptor antagonists potently enhance cognition and attention.

H(3) receptor antagonists based on a 2-aminoethylbenzofuran skeleton have been discovered, which are potent in vitro at human and rat H(3) receptors, with K(i) values of 0.1-5.8 nM. Analogues were discovered with potent (0.01-1 mg/kg) cognition and attention enhancing properties in animal models. One compound in particular, 4-(2-[2-(2(R)-methylpyrrolidin-1-yl)ethyl]benzofuran-5-yl)benzonitrile (ABT-239), combined potent and selective H(3) receptor antagonism and excellent pharmacokinetic and metabolic properties across species, with full efficacy in two behavioral models: a five-trial inhibitory avoidance acquisition model in rat pups at 0.1 mg/kg and a social recognition memory model in adult rats at 0.01 mg/kg. Furthermore, this compound did not stimulate locomotor activity and showed high selectivity for the induction of behavioral efficacy versus central nervous system based side effects. The potency and selectivity of this compound and of analogues from this class support the potential of H(3) receptor antagonists for the treatment of cognitive dysfunction.

Pharmacological properties of ABT-239 [4-(2-{2-[(2R)-2-Methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile]: II. Neurophysiological characterization and broad preclinical efficacy in cognition and schizophrenia of a potent and selective histamine H3 receptor antagonist.

Acute pharmacological blockade of central histamine H3 receptors (H3Rs) enhances arousal/attention in rodents. However, there is little information available for other behavioral domains or for repeated administration using selective compounds. ABT-239 [4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile] exemplifies such a selective, nonimidazole H3R antagonist with high affinity for rat (pK(i) = 8.9) and human (pK(i) = 9.5) H3Rs. Acute functional blockade of central H3Rs was demonstrated by blocking the dipsogenia response to the selective H3R agonist (R)-alpha-methylhistamine in mice. In cognition studies, acquisition of a five-trial, inhibitory avoidance test in rat pups was improved with ABT-239 (0.1-1.0 mg/kg), a 10- to 150-fold gain in potency, with similar efficacy, over previous antagonists such as thioperamide, ciproxifan, A-304121 [(4-(3-(4-((2R)-2-aminopropanoyl)-1-piperazinyl)propoxy)phenyl)(cyclopropyl) methanone], A-317920 [N-((1R)-2-(4-(3-(4-(cyclopropylcarbonyl) phenoxy)propyl)-1-piperazinyl)-1-methyl-2-oxoethyl)-2-furamide], and A-349821 [(4'-(3-((R,R)2,5-dimethyl-pyrrolidin-1-yl)-propoxy)-biphenyl-4-yl)-morpholin-4-yl-methanone]. Efficacy in this model was maintained for 3 to 6 h and following repeated dosing with ABT-239. Social memory was also improved in adult (0.01-0.3 mg/kg) and aged (0.3-1.0 mg/kg) rats. In schizophrenia models, ABT-239 improved gating deficits in DBA/2 mice using prepulse inhibition of startle (1.0-3.0 mg/kg) and N40 (1.0-10.0 mg/kg). Furthermore, ABT-239 (1.0 mg/kg) attenuated methamphetamine-induced hyperactivity in mice. In freely moving rat microdialysis studies, ABT-239 enhanced acetylcholine release (0.1-3.0 mg/kg) in adult rat frontal cortex and hippocampus and enhanced dopamine release in frontal cortex (3.0 mg/kg), but not striatum. In summary, broad efficacy was observed with ABT-239 across animal models such that potential clinical efficacy may extend beyond disorders such as ADHD to include Alzheimer's disease and schizophrenia.