An inverse agonist selective for alpha5 subunit-containing GABAA receptors enhances cognition.

Alpha5IA is a compound that binds with equivalent subnanomolar affinity to the benzodiazepine (BZ) site of GABA(A) receptors containing an alpha1, alpha2, alpha3, or alpha5 subunit but has inverse agonist efficacy selective for the alpha5 subtype. As a consequence, the in vitro and in vivo effects of this compound are mediated primarily via GABA(A) receptors containing an alpha5 subunit. In a mouse hippocampal slice model, alpha5IA significantly enhanced the burst-induced long-term potentiation of the excitatory postsynaptic potential in the CA1 region but did not cause an increase in the paroxysmal burst discharges that are characteristic of convulsant and proconvulsant drugs. These in vitro data suggesting that alpha5IA may enhance cognition without being proconvulsant were confirmed in in vivo rodent models. Hence, alpha5IA significantly enhanced performance in a rat hippocampal-dependent test of learning and memory, the delayed-matching-to-position version of the Morris water maze, with a minimum effective oral dose of 0.3 mg/kg, which corresponded to a BZ site occupancy of 25%. However, in mice alpha5IA was not convulsant in its own right nor did it potentiate the effects of pentylenetetrazole acutely or produce kindling upon chronic dosing even at doses producing greater than 90% occupancy. Finally, alpha5IA was not anxiogenic-like in the rat elevated plus maze nor did it impair performance in the mouse rotarod assay. Together, these data suggest that the GABA(A) alpha5-subtype provides a novel target for the development of selective inverse agonists with utility in the treatment of disorders associated with a cognitive deficit.

An orally bioavailable, functionally selective inverse agonist at the benzodiazepine site of GABAA alpha5 receptors with cognition enhancing properties.

(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (13) has been identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors. 13 is orally bioavailable, readily penetrates the CNS, and enhances performance in animal models of cognition. It does not exhibit the convulsant, proconvulsant, or anxiogenic activity associated with nonselective GABA(A) inverse agonists.

An orally active, water-soluble neurokinin-1 receptor antagonist suitable for both intravenous and oral clinical administration.

1-(5-[[(2R,3S)-2-([(1R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethyl]oxy)-3-(4-fluorophenyl)morpholin-4-yl]methyl]-2H-1,2,3-triazol-4-yl)-N,N-dimethylmethanamine hydrochloride 3 is a high affinity, orally active, h-NK(1) receptor antagonist with a long central duration of action and a solubility in water of >100 mg/mL. The construction of the 5-dimethylaminomethyl 1,2,3-triazol-4-yl unit, which incorporates the solubilizing group of 3, was accomplished by thermal rearrangement of a propargylic azide in the presence of dimethylamine. Compound 3 is highly effective in pre-clinical tests that are relevant to clinical efficacy in emesis and depression.

2-Aryl indole NK1 receptor antagonists: optimisation of the 2-aryl ring and the indole nitrogen substituent.

Novel 2-aryl indole hNK1 receptor ligands were prepared utilising palladium cross-coupling chemistry of a late intermediate as a key step. Compounds with high hNK1 receptor binding affinity and good brain penetration (e.g., 9d) were synthesised.

The novel NK1 receptor antagonist MK-0869 (L-754,030) and its water soluble phosphoryl prodrug, L-758,298, inhibit acute and delayed cisplatin-induced emesis in ferrets.

The anti-emetic profile of the novel brain penetrant tachykinin NK1 receptor antagonist MK-0869 (L-754,030) 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluor o)phenyl-4-(3-oxo-1,2,4-triazol-5-yl)methylmorpholine and its water soluble prodrug, L-758,298, has been examined against emesis induced by cisplatin in ferrets. In a 4 h observation period, MK-0869 and L-758,298 (3 mg/kg i.v. or p.o.) inhibited the emetic response to cisplatin (10 mg/kg i.v.). The anti-emetic protection afforded by MK-0869 (0.1 mg/kg i.v.) was enhanced by combined treatment with either dexamethasone (20 mg/kg i.v.) or the 5-HT3 receptor antagonist ondansetron (0.1 mg/kg i.v.). In a model of acute and delayed emesis, ferrets were dosed with cisplatin (5 mg/kg i.p.) and the retching and vomiting response recorded for 72 h. Pretreatment with MK-0869 (4-16 mg/kg p.o.) dose-dependently inhibited the emetic response to cisplatin. Once daily treatment with MK-0869 (2 and 4 mg/kg p.o.) completely prevented retching and vomiting in all ferrets tested. Further when daily dosing began at 24 h after cisplatin injection, when the acute phase of emesis had already become established, MK-0869 (4 mg/kg p.o. at 24 and 48 h after cisplatin) prevented retching and vomiting in three out of four ferrets. These data show that MK-0869 and its prodrug, L-758,298, have good activity against cisplatin-induced emesis in ferrets and provided a basis for the clinical testing of these agents for the treatment of emesis associated with cancer chemotherapy.

Phosphorylated morpholine acetal human neurokinin-1 receptor antagonists as water-soluble prodrugs.

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.

Structural optimization affording 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4- (3-oxo-1,2,4-triazol-5-yl)methylmorpholine, a potent, orally active, long-acting morpholine acetal human NK-1 receptor antagonist.

Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.

In vitro and in vivo predictors of the anti-emetic activity of tachykinin NK1 receptor antagonists.

The ability of tachykinin NK1 receptor antagonists to inhibit GR73632 (D-Ala-[L-Pro9,Me-Leu8]substance P-(7-11))-induced foot tapping in gerbils was employed as an indirect measure of brain penetration and this was compared with their ability to prevent acute emesis induced by cisplatin in ferrets. (+)-GR203040 ((2S,3S and 2R,3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin- 3-yl)-amine), CP-99,994 ((2S,3S)-cis-3-(2-methoxybenzylamino)-2-phenyl piperidine) dihydrochloride), and L-742,694 (2-(S)-(3,5-bis(trifluoromethyl)benzyloxy)-3-(S)-phenyl-4-(5-(3-oxo-1,2, 4-triazolo)methylmorpholine) potently inhibited GR73632-induced foot tapping (ID50 < or = 0.85 mg/kg), and acute retching induced by cisplatin (ID50 < or = 0.18 mg/kg). RPR100893 ((3aS,4S,7aS)-7,7-diphenyl-4-(2-methoxyphenyl)-2-[(S)-2-(2-m ethoxyphenyl)proprionyl] perhydroisoindol-4-ol) was not a potent antagonist of retching (ID50 4.1 mg/kg) or foot tapping (ID50 > 10 mg/kg). High doses (3-10 mg/kg) of CGP49823 ((2R,4S)-2-benzyl-1-(3,5-dimethylbenzoyl)-N-[(4-quinolinyl)methyl] -4-piperineamine) dihydrochloride), FK888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-propyl]-N-methy l-N-phenylmethyl-L-3-(2-naphthyl)-alaninamide), and LY303870 ((R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(pi peridinyl)piperidin-1-yl)acetyl)amino]propane) were required to inhibit foot tapping; these agents were not anti-emetic in this dose range. SR140333 ((S)-1-[2-[3-(3,4-dichlorphenyl)-1 (3-isopropoxyphenylacetyl)piperidin-3-yl] ethyl]-4-phenyl-1 azaniabicyclo [2.2.2]octane; 3-10 mg/kg) failed to inhibit foot tapping or emesis. Affinities for the human and ferret tachykinin NK1 receptor were highly correlated (r = 0.93, P = 0.0008). Inhibition of foot tapping in gerbils, but not NK1 receptor binding affinity, predicted anti-emetic activity in ferrets (r = 0.75, P < 0.01). These findings confirm that the anti-emetic activity of tachykinin NK1 receptor antagonists is dependent on brain penetration.

N-heteroaryl-2-phenyl-3-(benzyloxy)piperidines: a novel class of potent orally active human NK1 antagonists.

The preparation of a series of N-heteroarylpiperidine ether-based human NK1 antagonists is described. Two of the compounds 3-[-(2S,3S)-3-(((3,5-bis(trifluoromethyl)phenyl)methyl)oxy)- 2-phenylpiperidino}methyl]-1,2,4-triazole (11) and 5-[¿(2S,3S)-3-(((3,5-bis(trifluoromethyl)-phenyl)methyl)oxy)-2- phenylpiperidino}methyl]-3-oxo-1,2,4-triazolone (12)), in particular, are orally bioavailable and exhibited significant improvements in potency, both in vitro and in vivo, over the lead (carboxamidomethyl)piperidine ether 1. Rat liver microsome studies on a selected number of compounds from this series show the triazolone heterocycle to be considerably more stable than the others. Furthermore, both 11 and 12 have been profiled in a number of assays that may be predictive of the clinical utility of substance P antagonists.

Tachykinin NK1 receptor antagonists act centrally to inhibit emesis induced by the chemotherapeutic agent cisplatin in ferrets.

These studies have compared the pharmacological profile of two non-peptide human type neurokinin1 (hNK1) receptor selective antagonists, L-741,671 and a quaternised compound L-743,310. In radioligand binding studies L-741,671 and L-743,310 had high affinity for ferret and cloned hNK1 receptors [Ki (nM) ferret 0.7 and 0.1; human 0.03 and 0.06, respectively] but low affinity for rodent NK1 receptors [Ki (nM) 64 and 17, respectively] suggesting that ferret receptors have hNK1-like binding pharmacology. Studies in vivo showed that L-741,671 and L-743,310 had equivalent functional activity in the periphery (ID50s of 1.6 and 2 micrograms/kg i.v., respectively) as measured by inhibition of plasma protein extravasation evoked in the oesophagus of guinea pigs by resiniferatoxin (7 nmol/kg i.v.). Using an in situ brain perfusion technique in anaesthetised rats, L-741,671 was shown to be much more brain penetrant than the quaternary compound L-743,310 which had an entry rate similar to the poorly brain penetrant plasma marker inulin. These compounds thus provided an opportunity to compare the anti-emetic effects of equi-active hNK1 receptor antagonists with and without brain penetration to central NK1 receptor sites. When tested against cisplatin-induced emesis in ferrets, L-741,671 (0.3, 1 and 3 mg/kg i.v.) produced marked dose-dependent inhibition of retching and vomiting but L-743,310 was inactive at 3 and 10 micrograms/kg i.v. In contrast, direct central injection of L-741,671 and L-743,310 (30 micrograms) into the vicinity of the nucleus tractus solitarius or L-743,310 (200 micrograms) intracisternally was shown to inhibit retching and vomiting induced by i.v. cisplatin. L-741,671 and L-743,310 had equivalent functional activity, at the same dose, against cisplatin-induced emesis when injected centrally. These observations indicated that had L-743,310 penetrated into the brain after systemic administration it would have been active in the cisplatin-induced emesis assay and so show that brain penetration is essential for the anti-emetic action of systemically administered NK1 receptor antagonists.

Enantiospecific inhibition of emesis induced by nicotine in the house musk shrew (Suncus murinus) by the neurokinin1 (NK1) receptor antagonist CP-99,994.

Effects of the NK1 receptor antagonist CP-99,994 on nicotine-induced emesis were examined in Suncus murinus. CP-99,994 (3 and 10 mg/kg i.p.) attenuated emesis to (-)nicotine (4 mg/kg s.c.). CP-100,263 (3 and 10 mg/kg i.p.), the enantiomer of CP-99,994 with 1000 fold lower affinity for the NK1 receptor was without effect and RP67580 reduced emesis only at a dose of 30 mg/kg i.p. Responses to NK1 antagonists were ranked according to their affinities for the Suncus murinus NK1 receptor.

An investigation into the discriminative stimulus and reinforcing properties of the CCKB-receptor antagonist, L-365,260 in rats.

The discriminative stimulus properties of the selective CCKB-receptor antagonist, L-365,260 were evaluated in rats trained to discriminate diazepam (2 mg/kg) or morphine (5 mg/kg) from vehicle, using a two-lever food reinforced technique. In the diazepam drug discrimination, the benzodiazepine-receptor agonist FG8205 (0.063-2 mg/kg) produced dose-related drug associated responding, whereas L-365,260 (0.125-4 mg/kg) treated animals showed vehicle appropriate behaviour. In rats trained to discriminate morphine from saline, L-365,260 (0.063-4 mg/kg) produced saline lever responding. When a dose of 1 mg/kg L-365,260 was administered in combination with morphine, the dose response curve for drug lever responding was not significantly affected. This was in contrast to the effect produced by the opiate antagonist naloxone (0.3 mg/kg) which shifted the dose-response curve to the right. Another group of rats underwent training to discriminate a dose of 6 mg/kg L-365,260 from vehicle. None of the animals learned the discrimination within 50 daily training sessions. In addition, unlike morphine (3 mg/kg), or changing the training dose of cocaine, intravenous administration of L-365,260 (0.3-10 mg/kg) did not modify lever pressing or the number of injections received by rats trained to self administer cocaine (0.25 mg/injection). L-365,260 (0.1-3 mg/kg) produced a dose-related inhibition of pentagastrin-stimulated gastric acid secretion in vivo. When administered dissolved in a mixture of ethanol/propylene glycol/saline, the ID50 was 0.83 mg/kg, and when suspended in an ethanol/carboxymethylcellulose vehicle, it was 0.7 mg/kg. It was concluded: 1) that L-365,260 does not produce discriminative stimuli similar to either diazepam or morphine; 2) that the potentiation of morphine-induced behaviour by L-365,260 does not extend to the discriminative stimulus properties of morphine; 3) that L-365,260 itself does not produce readily discriminable interoceptive stimuli in rats; and 4) that L-365,260 does not substitute for the reinforcing drug cocaine.

Enantioselective inhibition of apomorphine-induced emesis in the ferret by the neurokinin1 receptor antagonist CP-99,994.

These studies have examined the effects of the selective neurokinin1 (NK1) receptor antagonist CP-99,994 on the retching and vomiting response to apomorphine. CP-99,994 (1-3 mg/kg i.p.) attenuated retching and vomiting induced by apomorphine (0.25 mg/kg s.c.) with complete inhibition of retching and vomiting at the 3 mg/kg dose. In contrast CP-100,263 (3 mg/kg i.p.), the enantiomer of CP-99,994 with 1000-fold lower affinity for the NK1 receptor, was without effect.

The tachykinin NK1 receptor antagonist CP-99,994 attenuates cisplatin induced emesis in the ferret.

The tachykinin NK1 receptor antagonist CP-99,994 ((+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine) (0.3-3 mg/kg i.v.), but not its inactive enantiomer CP-100,263, attenuated the retching and vomiting induced by cisplatin (10 mg/kg i.v.) in the ferret. CP-99,994, 3 mg/kg i.v., prevented vomiting in all ferrets tested. Since substance P is the preferred ligand at the NK1 receptor subtype these data support a role for the release of this peptide during the emetic response induced by cytotoxic chemotherapeutic agents.

The behavioral effects of heptyl physostigmine, a new cholinesterase inhibitor, in tests of long-term and working memory in rodents.

We assessed the effects of heptyl physostigmine, a new cholinesterase inhibitor, in a mouse tail-flick (TF) test, a mouse and rat passive avoidance test, a rat conditioned suppression-of-drinking (CSD) test, a rat Random Interval (RI) response rate test and a rat delayed matching-to-position (DMTP) test. In the TF test, a dose of 8.0 mg/kg of heptyl induced a significant antinociceptive effect that was in excess of 75% of the maximum possible effect 300 minutes after administration. In the mouse passive avoidance test, a dose of 3.0 mg/kg of heptyl fully reversed, and a dose of 1.0 mg/kg partially reversed, a scopolamine-induced (0.2 mg/kg) deficit. In the rat passive avoidance test, a dose of 8.0 mg/kg fully reversed a scopolamine-induced (0.2 mg/kg) deficit, while a dose of 4.0 mg/kg of heptyl was without effect. In the same experiment, a dose of 0.6 mg/kg of physostigmine partially reversed the scopolamine-induced deficit. In the CSD test, a dose of 8.0 mg/kg of heptyl fully reversed, and doses of 1.0 and 4.0 mg/kg of heptyl partially reversed, the deficit induced by scopolamine (0.4 mg/kg). In the RI response rate test, doses of 8.0 mg/kg and 0.6 mg/kg of physostigmine fully suppressed lever pressing for food rewards. Doses of 4.0 mg/kg of heptyl and below had no effect on lever-pressing rates. In the working memory test (DMTP), 4.0 mg/kg heptyl partially reversed the scopolamine-induced deficit (0.2 mg/kg) in the number of correct choices made, but did not affect the scopolamine-induced deficit in the number of trials completed.(ABSTRACT TRUNCATED AT 250 WORDS)

Postponement of satiety by blockade of brain cholecystokinin (CCK-B) receptors.

Exogenous cholecystokinin (CCK) decreases food intake and causes satiety in animals and man. However, it has not been established that endogenous CCK causes satiety or whether the response is mediated by peripheral-type (CCK-A) or brain-type (CCK-B) receptors. The development of potent and selective antagonists for CCK-A (MK-329) and CCK-B (L-365,260) receptors now allows these issues to be addressed. The CCK-A antagonist MK-329 and the CCK-B antagonist L-365,260 increased food intake in partially satiated rats and postponed the onset of satiety; however, L-365,260 was 100 times more potent than MK-329 in increasing feeding and preventing satiety. These results suggest that endogenous CCK causes satiety by an agonist action on CCK-B receptors in the brain.