Delayed administration of a potent cyclin dependent kinase and glycogen synthase kinase 3 beta inhibitor produces long-term neuroprotection in a hypoxia-ischemia model of brain injury.

We compared the neuroprotective efficacy of a potent and CNS-penetrant cyclin dependent kinase (CDK) and glycogen synthase kinase 3 beta (GSK3beta) inhibitor (Compound 1) in juvenile (postnatal day 21; P21) and adult C57Bl/6 mice (postnatal day 60; P60) using a model of hypoxic-ischemic brain injury (HI). Neuronal cell counts and density measures from brain sections stained with Cresyl Violet revealed that exposure of P21 mice to 60 min of HI resulted in extensive damage to the ipsilateral cornu ammonis 1 (CA1) region of the hippocampus (40% cell loss) and striatum (30% cell loss) 7 days later. Exposure of P60 mice to 40 min of HI produced a similar pattern of cell loss. Intraperitoneal administration of Compound 1 (3 mg/kg) 1, 5 and 9 h after 60 min of HI did not reduce brain injury in P21 mice relative to vehicle controls. By contrast, in P60 mice, this treatment significantly decreased cell loss in the ipsilateral hippocampus (10% cell loss) and striatum (15% loss) relative to vehicle controls. Terminal uridine deoxynucleotidyl transferase (TUNNEL) positive cell counts and infarct volume were also substantially reduced in P60 mice treated with Compound 1. A motor coordination test performed twice weekly until 5 weeks post-HI confirmed that Compound 1 produced long lasting functional recovery. Our results indicate that Compound 1 produced long lasting neuroprotective effects in adult but not juvenile mice suggesting that inhibition of the CDKs and GSK3beta plays a distinct neuroprotective role in the juvenile and adult brain.

1192U90 in animal tests that predict antipsychotic efficacy, anxiolysis, and extrapyramidal side effects.

1192U90 was developed on the assumption that antagonism of 5-HT2 receptors efficacy yields more potently than D2 receptors against positive and negative symptoms of schizophrenia with minimal liability for extrapyramidal side effects (EPSs), and that 5-HT1A agonism further reduces EPSs and provides anxiolytic and antidepressant activity. 1192U90 was submitted to four tests that predict antipsychotic efficacy (antagonism of apomorphine-induced climbing in mouse, antagonism of apomorphine-induced circling in rats with unilateral 6-OHDA lesions, antagonism of amphetamine-induced hyperlocomotion in rat, and inhibition of conditioned avoidance in rat), two tests of 5-HT2 function (antagonism of 5-MeODMT-induced head twitches in mouse and antagonism of 5-HTP-induced wet dog shakes in rat), and three tests that predict EPS liability (antagonism of apomorphine-induced stereotypy in mouse and rat and induction of catalepsy in mouse). ED50s (mg/kg PO) were as follows: climbing 10.1, circling 7.9, hyperlocomotion 6.6, and avoidance 5.7; head twitches 5 and wet dog shakes 4.6; stereotypy in mouse 91.1, stereotypy in rat 133.4, and catalepsy 192.4. The ratio of ED50 for stereotypy antagonism to ED50 for climbing antagonism was 9 (compared to 4, 3, and 4 for clozapine, risperidone, and haloperidol). The ratio of ED50 for catalepsy induction to ED50 for climbing antagonism was 19 (compared to 7, 2, and 17 for clozapine, risperidone, and haloperidol). 1192U90 was also submitted to three tests that predict anxiolysis: It produced only a small increase in punished lever pressing for food in rat (Geller-Seifter conflict test), which is specific for rapid-onset efficacy, but produced large increases in punished key pecking for food in pigeon and cork gnawing in rat, which identify the delayed onset 5-HT1A agonists such as buspirone. The results suggest that 1192U90 would be effective for positive and negative symptoms of schizophrenia, with minimal liability for EPSs, and may also have anxiolytic properties.

Synthesis and evaluation of heterocyclic carboxamides as potential antipsychotic agents.

Heterocyclic analogues of 1192U90, 2-amino-N-(4-(4-(1,2-benzisothiazol-3-yl)-1-piperazinyl)-butyl) benzamide hydrochloride (1), were prepared and evaluated as potential antipsychotic agents. These analogues were evaluated in vitro for their binding to the dopamine D2, serotonin 5-HT2, and serotonin 5-HT1a receptors and in vivo for their ability to antagonize the apomorphine-induced climbing response in mice. Nine different types of heterocyclic carboxamides were studied in this investigation (i.e., pyridine-, thiophene-, benzothiophene-, quinoline-, 1,2,3,4-tetrahydroquinoline-, 2,3-dihydroindole-, indole-, benzimidazole-, and indazolecarboxamides). Two derivatives exhibited potent in vivo activities comparable to 1: 3-amino-N-(4-(4-(1,2-benzisothiazol-3-yl)-1-piperazinyl)butyl)-2 -pyridinecarboxamide (16) and 3-amino-N-(4-(4-(1,2-benzisothiazol-3-yl)-1-piperazinyl)butyl) -2-thiophenecarboxamide (29). Furthermore, these derivatives were found to be much less active in behavioral models predictive of extrapyramidal side effects than in the mouse climbing assay, which predicts antipsychotic activity. Carboxamides 16 and 29 were selected for further evaluation as potential backup compounds to 1.

Effect of linking bridge modifications on the antipsychotic profile of some phthalimide and isoindolinone derivatives.

A series of phthalimide and isoindolinone derivatives bridged to 4-(1,2-benzisothiazol-3-yl)-1-piperazinyl was prepared. The compounds were evaluated in vitro at dopamine D2 and serotonin 5-HT1a and 5-HT2 receptors and in vivo for their ability to antagonize the apomorphine-induced climbing response in mice. The effects of bridge length and conformation on the biological activity of these potential antipsychotic agents are discussed. A four-carbon spacer provided optimal activity within the two homologous series. Conformational investigations of the lead compound, isoindolinone 2, were conducted in an attempt to account for the superior activity observed for the butyl derivatives. On the basis of NMR and molecular modeling studies, two types of folded structures were proposed and several conformationally restrained analogues were synthesized. In general, restrictions incorporated within the linking bridge were detrimental to activity.

Cyclic benzamides as mixed dopamine D2/serotonin 5-HT2 receptor antagonists: potential atypical antipsychotic agents.

A series of novel 4-(4-(1,2-benzisothiazol-3-yl)-1-piperazinyl)butyl) cyclic amides was prepared and evaluated as potential antipsychotic agents. The target compounds were examined in vitro for their binding affinities to the dopamine D2, serotonin 5-HT2, and serotonin 5-HT1a receptors and in vivo for their ability to antagonize the apomorphine-induced climbing response in mice. Derivatives that exhibited good D2/5-HT2 selectivity in vitro and good potency in vivo were selected for further evaluation in tests designed to assess their potential extrapyramidal side effect liability. Structural modifications discussed herein focus on the bicyclic amide subunit leading to the preparation of a variety of heterocyclic ring systems (i.e., phthalimide, isoindolinone, isoquinolinone, benzazepinone, indazolone, phthalazinone, 4-methyl phthalazinone, benzisothiazolone 1,1-dioxide, benzotriazinone, homophthalimide, benzisothiazolone, phthalazinedione, quinazoline, and saturated phthalazinones). The potency and selectivity within this series was found to be dependent on ring size, nature of the covalent linking unit, relative position of the functional groups, degree of unsaturation, and relative stereochemistry. In general, the cyclic benzamides examined in this investigation exhibited receptor binding activities indicative of potential atypical antipsychotic agents. Several of these derivatives possessed in vivo activities that suggest they would be useful in the treatment of schizophrenia and would have a low propensity to induce extrapyramidal side effects. Two potent analogues were identified and selected for further evaluation: 2-(4-(4-(1,2-benzisothiazol-3-yl)-1-piperazinyl)butyl)-1-isoind olinone (31) and (+-)-cis-2-(4-(4-(1,2-benzisothiazol-3-yl)-1-piperazinyl)-butyl)- 4a,5,6,7,8,8a-hexahydro-1(2H)-phthalazinone hydrochloride (52).

Structure-activity relationships of a series of substituted benzamides: potent D2/5-HT2 antagonists and 5-HT1a agonists as neuroleptic agents.

A series of substituted (4-(4-(1,2-benzisothiazol-3-yl)-1-piperazinyl)butyl)benzamide derivatives was prepared and evaluated as potential atypical antipsychotic agents. The target compounds were readily prepared from their benzoyl chloride, benzoic acid, or isatoic anhydride precursors, and they were evaluated in vitro for their ability to bind to dopamine D2, serotonin 5-HT2, and serotonin 5-HT1a receptors. To assess the potential antipsychotic activity of these compounds, we investigated their ability to inhibit the apomorphine-induced climbing response in mice. Selected compounds were evaluated further to determine their side-effect potentials. Structure-activity relationships of both mono- and polysubstituted benzamides are discussed herein. While several analogues had potent in vitro and in vivo activities indicative of potential atypical antipsychotic activity, anthranilamide 77 (1192U90) ddemonstrated a superior pharmacological profile. As a result of this investigation, 1192U90 (2-amino-N-(4-(4-(1,2-benzisothiazol-3-yl)-1-piperazinyl)butyl)ben zamide hydrochloride) was selected for further evaluation and is currently in phase I clinical trials as a potential atypical antipsychotic agent.

Conformationally restricted analogues of remoxipride as potential antipsychotic agents.

Several conformationally restricted derivatives of (S)-3-bromo-N-((1-ethyl-2-pyrrolidinyl)methyl)-2,6-dimethoxybenzamide (remoxipride) were synthesized and evaluated in vitro for their ability to inhibit [3H]raclopride binding at the dopamine D-2 receptor. The cyclic benzamides designed to mimic the intramolecular hydrogen bonding of desmethylremoxipride (4, FLA-797) included 2,3-dihydro-4H-1,3-benzoxazin-4-ones, 2,3-dihydro-4H-1,3-benzthiazin-4-ones, phthalimides, 1-isoindolinones, 1,2-benzisothiazol-3(2H)-ones, and 1,2-benzisothiazol-3(2H)-one 1,1-dioxides. In this series, enhanced affinities to the dopamine D-2 receptor were not observed. The phthalimidine analogue 24b ((S)-6-chloro-2-(1-ethylpyrrolidinyl)-1-isoindolinone) exhibited the highest affinity to the dopamine D-2 receptor with an IC50 of 1.3 microM, which was equipotent to remoxipride.

Synthesis and structure-activity relationships of trisubstituted phenyl urea derivatives as neuropeptide Y5 receptor antagonists.

1-((1R,2R)-2-Hydroxy-1-methyl-2-phenylethyl)-1-methyl-3-(4-phenoxyphenyl)urea (1) was identified as a hit from the screening of the neuropeptide Y5 (NPY5) receptor. This lead was optimized for in vitro potency by changing the stereochemistry, the phenylethyl segment, the urea portion, and the 4-phenoxyphenyl group on the molecule. Over 40 analogues of 1 were prepared to study the structure-activity relationship for this series. The most potent compounds in this class have IC(50)s less than 0.1 nM at the NPY5 receptor (e.g., 40f, 44a, and 47). To determine the functional activity for this series of compounds, selected analogues were tested in a cellular assay measuring forskolin-induced cyclic AMP accumulation in 293 cells transfected with the human NPY5 receptor. All urea analogues tested in the functional assay acted as antagonists (e.g., 1, 32, 40a, and 44e).

4-(Heteroarylthio)-2-biphenylyltetrazoles as nonpeptide angiotensin II antagonists.

A series of 4-(heteroarylthio)-2-biphenylyltetrazoles was prepared, and the compounds were examined for their ability to displace [3H]AII from angiotensin II receptors. Analogues that exhibited significant receptor binding affinities at less than 10 microM were investigated further for potential antagonism of angiotensin II-mediated contraction of rabbit isolated aortic rings. Three 4-(heteroarylthio)-2-biphenylyltetrazoles were identified that exhibited sub-micromolar angiotensin II receptor binding affinities. These compounds and two reference agents, saralasin and losartan (DUP-753), exhibited concentration-dependent reversal of angiotensin II contraction in isolated aortic rings parallel to their receptor binding affinities. Molecular modeling studies were conducted to examine the conformational effects of the novel sulfide bridging unit contained in these 4-(heteroarylthio)-2-biphenylyltetrazoles. The biological effects of the sulfide bridge as well as alterations in the heteroaromatic moiety were investigated, and the resulting structure--activity relationships are discussed.

Structure-activity relationships of a series of pyrrolo[3,2-d]pyrimidine derivatives and related compounds as neuropeptide Y5 receptor antagonists.

Neuropeptide Y (NPY) has been shown to play an important role in the regulation of food intake and energy balance. Pharmacological data suggests that the Y5 receptor subtype contributes to the effects of NPY on appetite, and therefore a Y5 antagonist might be a useful therapeutic agent for the treatment of obesity. In attempts to identify potential Y5 antagonists, a series of pyrrolo[3, 2-d]pyrimidine derivatives was prepared and evaluated for their ability to bind to Y5 receptors in vitro. We report here the synthesis and initial structure-activity relationship investigations for this class of compounds. The target compounds were prepared by a variety of synthetic routes designed to modify both the substitution and the heterocyclic core of the pyrrolo[3,2-d]pyrimidine lead 1. In addition to identifying several potent Y5 antagonists for evaluation as potential antiobesity agents, a pharmacophore model for the human Y5 receptor is presented.

Behavioral characterization of neuropeptide Y knockout mice.

An extensive behavioral characterization was conducted with mice lacking the gene for neuropeptide Y (NPY) including response to 24 and 48 h fast and challenge with small molecule antagonists of NPY receptors implicated in mediating the feeding effects of NPY (i.e., Y1 and Y5). In addition, wildtype (WT) and NPY knockout (KO) mice were tested in locomotor monitors, elevated plus maze, inhibitory avoidance, acoustic startle, prepulse inhibition, and hot plate assays. One of the major findings was that the NPY KO mice have a reduced food intake relative to WT controls in response to fasting. Also, based on data from the behavioral models, the NPY KO mice may have an anxiogenic-like phenotype, and appear to be hypoalgesic in the hot plate paradigm. The data from these studies provide further evidence of involvement of NPY in energy balance, anxiety, and possibly nociception.

Is clozapine selective for the dopamine D4 receptor?

Binding of 3H-spiperone and 3H-raclopride to membranes of cells stably-transfected with a human dopamine D2 receptor clone was investigated, as was that of 3H-spiperone to those stably-transfected with a human D4 receptor clone. 3H-spiperone and 3H-raclopride labeled the same number of sites in the D2 receptor preparation. The inhibition of binding by clozapine, spiperone, (-) eticlopride, haloperidol and the novel substituted benzamide 1192U90 was also investigated. Clozapine and 1192U90 showed greater inhibition of 3H-raclopride binding than 3H-spiperone binding to the D2 receptor. Comparison with inhibition of 3H-spiperone binding to the D4 receptor revealed that clozapine and 1192U90 displayed apparent selectivity (as assessed by Ki ratios) for the D4 receptor when compared with binding of 3H-spiperone, but not 3H-raclopride, to the D2 receptor.

Analogues of the potential antipsychotic agent 1192U90: amide modifications.

Analogues of 2-amino-N-(4-(4-(1,2-benzisothiazol-3-yl)-l-piperazinyl) -butyl)benzamide hydrochloride (1192U90) were prepared and evaluated in receptor binding assays for the dopamine D2, serotonin 5-HT1a, and serotonin 5-HT2 receptors. Eight compounds have been synthesized in which the amide group of 1192U90 has been replaced with a variety of functional groups (i.e. ester, ketone, thioamide, butyramide, butyranilide, sulfonamide, alkoxyamide and hydrazide). These compounds exhibited moderate to potent affinities (0.55-200 nM) for all three receptors. Several analogues exhibited improved selectivity for the 5-HT2 receptor with D2/5-HT2 binding ratios greater than 1192U90.