This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.

This Letter describes the discovery of GSK189254 and GSK239512 that were progressed as clinical candidates to explore the potential of H3 receptor antagonists as novel therapies for the treatment of Alzheimer's disease and other dementias. By carefully controlling the physicochemical properties of the benzazepine series and through the implementation of an aggressive and innovative screening strategy that employed high throughput in vivo assays to efficiently triage compounds, the medicinal chemistry effort was able to rapidly progress the benzazepine class of H3 antagonists through to the identification of clinical candidates with robust in vivo efficacy and excellent developability properties.

5-hydroxytryptamine (5-HT) is found in many areas of the CNS, and has been implicated in the modulation of learning and memory processes. The 5-HT6 receptor is a member of the seven transmembrane G protein coupled family of receptors and is positively coupled to adenylate cyclase. 5-HT6 receptor antagonists can increase the release of several neurotransmitters in the brain and enhance cognitive processes in rodent models. Several 5-HT6 antagonists are being developed as symptomatic agents for the treatment of memory deficits in Alzheimer's disease and other dementias, including SB-742457. SB-742457 was profiled in standard in vitro binding and functional assays. SB-742457 had high affinity for native 5-HT6 receptors expressed in rat striatum (pKi = 9.56) consistent with its binding affinity and functional potency (cAMP assay) at the recombinant human receptor (pKi = 9.68; pA2 = 9.2). SB-742457 is > 100 fold selective for the human 5-HT6 receptor over all other 5-HT receptors, ion channels and transporters examined apart from 5-HT2a. (pKi 8.0). Following oral administration in rats, SB-742457 exhibited high 5-HT6 receptor occupancy in the striatum (ED50 = 0.3mg/kg from ex vivo binding studies) indicative of good brain penetration. In vivo oral activity of SB-742457 and the cholinesterase inhibitor Donepezil was assessed in a range of neurochemical and animal models of cognition including aged Morris Water Maze and Passive Avoidance. In these studies a sub-chronic administration of SB-742457 or Donepezil (0.03 and 0.3mg/kg) was able to significantly improve the spatial learning ability of aged animals, with SB-742457 (1.5mg/kg) promoting an apparently more prolonged beneficial effect on memory retention. Acute administration of both compounds reversed scopolamine induced amnesia in a passive avoidance paradigm, though with different dose-response profiles. Given its beneficial effects, on learning and memory in preclinical species the 5-HT6 antagonist SB-742457 has the potential to be a new therapeutic agent for the symptomatic treatment of Alzheimer's disease and other dementias.

Starting from a benzazepine sulfonamide 5-HT(6) receptor antagonist lead with limited brain penetration, application of a strategy of conformational constraint and reduction of hydrogen bond donor count led to a novel series of tricyclic derivatives with high 5-HT(6) receptor affinity and excellent brain:blood ratios.

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H 3 receptor antagonist with high affinity for human (pK(i) = 9.59-9.90) and rat (pK(i) = 8.51-9.17) H-3 receptors. GSK189254 is >10,000-fold selective for human H-3 receptors versus other targets tested, and it exhibited potent functional antagonism (pA(2) = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC(50) = 8.20 versus basal guanosine 5'-O-(3-[S-35]thio)triphosphate binding] at the human recombinant H-3 receptor. In vitro autoradiography demonstrated specific [H-3]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H 3 binding was detected in medial temporal cortex samples from severe cases of Alzheimer's disease, suggesting for the first time that H-3 receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(-)-alpha-methyl[imidazole-2,5(n)-H-3]histamine dihydrochloride ([H-3]R-alpha-methylhistamine) binding (ED50 = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3-3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H-3 receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID50 = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimer's disease and other cognitive disorders.

GSK207040 (5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-2-pyrazinecarboxamide) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1H-1,4-diazepine) are novel and selective non-imidazole histamine H3 receptor antagonists from distinct chemical series with high affinity for human (pKi=9.67±0.06 and 9.49±0.09, respectively) and rat (pKi=9.08±0.16 and 9.12±0.14, respectively) H3 receptors expressed in cerebral cortex. At the human recombinant H3 receptor, GSK207040 and GSK334429 were potent functional antagonists (pA2=9.26±0.04 and 8.84±0.04, respectively versus H3 agonist-induced changes in cAMP) and exhibited inverse agonist properties (pIC50=9.20±0.36 and 8.59±0.04 versus basal GTPγS binding). Following oral administration, GSK207040 and GSK334429 potently inhibited cortical ex vivo [3H]-R-α-methylhistamine binding (ED50=0.03 and 0.35mg/kg, respectively). Functional antagonism of central H3 receptors was demonstrated by blockade of R-α-methylhistamine-induced dipsogenia in rats (ID50=0.02 and 0.11mg/kg p.o. for GSK207040 and GSK334429, respectively). In more pathophysiologically relevant pharmacodynamic models, GSK207040 (0.1, 0.3, 1 and 3mg/kg p.o.) and GSK334429 (0.3, 1 and 3mg/kg p.o.) significantly reversed amnesia induced by the cholinergic antagonist scopolamine in a passive avoidance paradigm. In addition, GSK207040 (0.1, 0.3 and 1mg/kg p.o.) and GSK334429 (3 and 10mg/kg p.o.) significantly reversed capsaicin-induced reductions in paw withdrawal threshold, suggesting for the first time that blockade of H3 receptors may be able to reduce tactile allodynia. Novel H3 receptor antagonists such as GSK207040 and GSK334429 may therefore have therapeutic potential not only in dementia but also in neuropathic pain.

Histamine H3 receptor antagonists can increase the release of several neurotransmitters in the brain and enhance cognition in rodent models. Several H3 antagonists are being developed as symptomatic agents for the treatment of cognitive deficits in Alzheimer's Disease and other dementias. To investigate the in vitro and in vivo properties of GSK189254, a novel non–imidazole histamine H3 receptor antagonist. GSK189254 was profiled in standard in vitro binding and functional assays. In vivo oral activity of GSK189254 was assessed in several assays including ex vivo binding, cognition models and EEG. GSK189254 had high affinity for native H3 receptors expressed in human and rat cerebral cortex (pKi = 9.59 and 8.58 respectively) and for human and rat recombinant H3 receptors (pKi = 9.90 and 9.17 respectively). GSK189254 was highly selective for H3 receptors (>10000–fold vs other receptors, ion channels and transporters) and exhibited high functional antagonist potency at the human recombinant H3 receptor in vitro (pA2 = 9.1 vs imetit–induced inhibition of forskolin–stimulated cAMP accumulation). In vitro autoradiography studies with [3H]–GSK189254 in rat brain slices demonstrated a specific binding pattern consistent with H3 receptor distribution in cortex, hippocampus, striatum and hypothalamus. Specific [3H]–GSK189254 labelling was also observed in post–mortem cortex and hippocampus from human control and Alzheimer's disease brains. Following oral administration in rats, GSK189254 exhibited high H3 receptor occupancy in the cerebral cortex (ED50 = 0.17mg/kg from ex vivo binding studies) indicative of good brain penetration, and potently reversed drinking induced by the H3 selective agonist (R)–alpha–methylhistamine (ID50 = 0.05mg/kg), supporting functional blockade of H3 receptors in vivo. GSK189254 (0.1–3mg/kg) exhibited efficacy in a number of rodent models of cognitive function including object recognition (48h delay), passive avoidance (scopolamine–induced amnesia) and water maze (aged animals). In addition, GSK189254 induced a transient increase in wakefulness in the rat during the light phase, and a corresponding decrease in slow wave sleep 2. Given these effects on cognition and alertness in preclinical species, the H3 antagonist GSK189254 has the potential to be a new therapeutic agent for the symptomatic treatment of Alzheimer's Disease and other dementias.

Bisaryl cyclic ureas have been identified as high affinity 5-HT2C receptor antagonists with selectivity over 5-HT2A and 5-HT2B. Compounds such as 8 and 22 have shown oral activity in a centrally mediated pharmacodynamic model of 5-HT2C function in rodents.

The novel 8-piperazinyl-2,3-dihydropyrroloisoquinoline template was synthesized in nine steps. The template was N-substituted to give a series of compounds showing binding to human cloned 5-HT1A, 5-HT1B and 5-HT1D receptors with pKi's greater than 9 and selectivities up to 1000-fold against other serotonin, dopamine and adrenergic receptors. Several compounds were shown to possess weak partial agonist activity in cloned receptors, which translated to antagonism in in vitro studies.

A newly developed apparatus for automated behavioural analysis, Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS), has been further validated with respect to the ability of the system to detect the pharmacodynamic effects of standard pharmacological tools. Data were obtained from rats administered with mCPP (reversal with SB242084), 8-OH-DPAT (reversal with WAY100635), amphetamine (reversal with haloperidol) and angiotensin, with the focus on locomotor activity, feeding and drinking behaviours. The data captured and analysed by LABORAS, suggests that the automated system is able to detect pharmacologically induced changes in behaviour, reliably and efficiently, with a significant reduction in the number of animals required, and reduced operator input.

Uncoupling protein-3 (UCP-3) is a recently identified member of the mitochondrial transporter superfamily(1,2) that is expressed predominantly in skeletal muscle(1,2). However, its close relative UCP-1 is expressed exclusively in brown adipose tissue, a tissue whose main function is fat combustion and thermogenesis. Studies on the expression of UCP-3 in animals and humans in different physiological situations support a role for UCP-3 in energy balance and lipid metabolism(3,4). However, direct evidence for these roles is lacking. Here we describe the creation of transgenic mice that overexpress human UCP-3 in skeletal muscle. These mice are hyperphagic but weigh less than their wild-type littermates. Magnetic resonance imaging shows a striking reduction in adipose tissue mass. The mice also exhibit lower fasting plasma glucose and insulin levels and an increased glucose clearance rate. This provides evidence that skeletal muscle UCP-3 has the potential to influence metabolic rate and glucose homeostasis in the whole animal.

There is some controversy as to whether 5-HT2C receptor agonists are anxiogenic or anxiolytic. The effects of the novel 5-HT2C receptor agonist, ( S)-2-chloro-5-fluoro-indol-1-yl)-1-methyl ethylamine fumarate (RO 60 0175), in three models of anxiety were therefore tested. RO 60 0175 was found to induce hypolocomotion in rats at doses greater than 0.5 mg/kg s.c,, an effect reversed by the selective 5-HT2C receptor antagonist, SB-242084. RO 60 0175 did not elicit anxiolytic-like responses in the social interaction test under high light unfamiliar conditions, but suppressed both time spent in social interaction and locomotion at doses of 1 and 3 mg/kg s.c., suggesting a sedative response. In the Vogel conflict test, RO 60 0175 had no significant action on the number of shocks taken. In the Geller-Seifter test, RO 60 0175 (0.3 and 1 mg/kg s.c.) simultaneously reduced both unpunished and punished lever pressing, a profile consistent with sedation. Finally, RO 60 0175 was tested in a rat social interaction test under low light familiar conditions optimal for the detection of anxiogenic-like responses. At 1 and 3 mg/kg s.c., RO 60 0175 reduced both time spent in social interaction and concurrent locomotion, a profile more consistent with sedation than anxiogenesis. In conclusion, RO 60 0175 induced sedative-like responses via 5-HT2C receptor activation, but was neither anxiolytic, nor dearly anxiogenic at the doses tested. (C) 2000 Elsevier Science B.V. All rights reserved.

The effects of the K+ channel activators, levcromakalim, pinacidil and diazoxide, at comparable antihypertensive doses, on acute glucose tolerance and glibenclamide-induced hypoglycaemia were examined in conscious spontaneously hypertensive rats (SHR). Levcromakalim (0.15 mg·kg−1 p.o.) and pinacidil (1.0 mg·kg−1 p.o.) caused a slight, but short-lived, impairment of glucose tolerance following oral or s.c. administration of glucose (2.0 g.kg−1). This effect, although small, was abolished by the β-adrenoceptor blocker, propranolol (2.0 mg·kg−1 p.o.). Diazoxide (30.0 mg·kg−1 p.o.) caused a marked and sustained impairment of oral glucose tolerance and s.c. glucose tolerance, the profile of which was quantitatively and qualitatively different from levcromakalim or pinacidil and was not significantly affected by propranolol. Glibenclamide (1.0–10.0 mg·kg−1 p.o.) elicited a dose-related hypoglycaemic response. Levcromakalim or pinacidil had little or no significant effect on the hypoglycaemic response elicited by glibenclamide (3.0 mg·kg−1). Conversely, diazoxide both abolished and reversed glibenclamide-induced hypoglycaemia. We conclude that levcromakalim and pinacidil have only marginal and transient effects on glycaemic control in conscious SHR and that these disturbances are probably mediated indirectly via reflex activation of the sympathetic nervous system in response to blood pressure lowering. In addition, at active antihypertensive doses neither levcromakalim nor pinacidil significantly interfered with the ability of glibenclamide to reduce blood glucose concentration. Diazoxide's impairment of oral glucose tolerance, s.c. glucose tolerance and glibenclamide response confirms this drug's well known ability to activate pancreatic KATP channels.