Discovery of novel 2-(4-aryl-2-methylpiperazin-1-yl)-pyrimidin-4-ones as glycogen synthase kinase-3ß inhibitors.

We herein describe the results of further evolution of glycogen synthase kinase (GSK)-3ß inhibitors from our promising compounds containing a 3-methylmorpholine moiety. Transformation of the morpholine moiety into a piperazine moiety resulted in potent GSK-3ß inhibitors. SAR studies focused on the nitrogen atom of the piperazine moiety revealed that a phenyl group afforded potent inhibitory activity toward GSK-3ß. Docking studies indicated that the phenyl group on the piperazine nitrogen atom and the methyl group on the piperazine make cation-π and CH-π interactions with GSK-3ß respectively. 4-Methoxyphenyl analogue 29 showed most potent inhibitory activity toward GSK-3ß with good in vitro and in vivo pharmacokinetic profiles, and 29 demonstrated a significant decrease in tau phosphorylation after oral administration in mice.

MKC-231, a choline uptake enhancer: (3) Mode of action of MKC-231 in the enhancement of high-affinity choline uptake.

MKC-231, a putative cholinergic activity, is reported to improve learning and memory impaired in AF64A-treated animals. MKC-231 enhances high-affinity choline uptake (HACU) known as the rate-limiting step of acetylcholine (ACh) synthesis. We investigated the mode of action (MOA) of HACU enhancement by MKC-231. Intracerebroventricular (i.c.v.) injections of AF64A (3 nmol/brain) resulted in significant HACU reduction in hippocampal synaptosomes. Treatment with MKC-231 increased Vmax of HACU and Bmax of [3H]-HC-3 binding 1.6 and 1.7-fold, respectively. In studies of [3H]-MKC-231 binding and Biacore analysis, MKC-231 showed noticeable affinity for cloned high-affinity choline transporters (CHT1). The present study suggests that MKC-231 directly affects trafficking of CHT1 and increases the numbers of transporter, working for HACU, at the synaptic membrane.

MKC-231, a choline-uptake enhancer: (1) long-lasting cognitive improvement after repeated administration in AF64A-treated rats.

MKC-231 is reported to increase high-affinity choline uptake (HACU) in vitro and improve learning impairment on a single oral administration in AF64A-treated rats. In this study, we investigated the effects of repeated administration of MKC-231 (1 and 3 mg/kg, p.o., 8 days) on learning impairment in the water-maze task in AF64A-treated rats 1, 24, 48, and 72 h after the last dose. Significant cognitive improvement was observed for 24 h, however, concentration measurement studies indicated MKC-231 was not detected in the brain by this time. We also studied the effects of 8-days repeated administration of MKC-231 on HACU 1, 24, 48, and 72 h after the last dose and observed an increase of HACU similar in time course with cognitive improvement. From these results, we discussed the possibility that MKC-231 could induce long-lasting procognitive effects by changing the choline transporter regulation system.

MKC-231, a choline uptake enhancer: (2) Effect on synthesis and release of acetylcholine in AF64A-treated rats.

The effect of MKC-231 on acetylcholine (ACh) synthesis and release was studied in the hippocampus of normal and AF64A-treated rats. AF64A (3 nmol/brain, i.c.v.) produced significant reduction of high-affinity choline uptake (HACU) and high K+-induced ACh release in hippocampal synaptosomes. Treatments with MKC-231 (10(-8) and 10(-7) M) showed significant reverse of the decrease in both HACU and ACh release. In hippocampal slices superfused with choline-containing artificial cerebro-spinal fluid (ACSF), high K+-induced ACh release was gradually decreased by repeated alteration of resting and high K+ stimulations in AF64A-treated rats. However, addition of MKC-231 (10(-8) to 10(-7) M) in the superfusate reduces this decrease. In vivo microdialysis studies indicate MKC-231 (10 mg/kg, p.o.) significantly reversed reduction of basal ACh concentrations in AF64A-treated rats, measured by radioimmunoassay without a cholinesterase inhibitor in the perfusate. These results indicate MKC-231 improves AF64A-induced cholinergic hypofunction by enhancing HACU, subsequently facilitating ACh synthesis and release in vitro and in vivo.

Onset of competence to respond to activin A in isolated eight-cell stage Xenopus animal blastomeres.

Single animal hemisphere blastomeres isolated from the eight-cell stage Xenopus embryos differentiate into mesoderm when treated with activin A, whereas when cultured without activin they form atypical epidermis. The mesoderm tissue induced by activin is different between dorsal and ventral blastomeres. In the present study, the duration and timing of activin treatment was varied, in order to identify the critical stage when animal blastomeres acquire competence to respond to activin A. It was shown that the critical time was 45 min after blastomere isolation, which corresponds approximately to NF stage 6 (32-cell stage) of normal development. The competence gradually increased during the morula stages.