Muneharu Miyake

Synthesis of β-Lactams by Convenient Annelation of Imines

Abstract 2-Acyl-3-oxo-4,5-benzo-1,2-thiazoline 1,1-dioxide (3) provides good yield routes to monocyclic 3-lactams (6) and tricyclic β-lactams (7) from a variety of imines (4) and 1-substituted-3,4-dihydroisoquinolines (5).

Cyclophane and acyclic cyclophane: Novel channel blockers of N-methyl-d-aspartate receptor

The effects of cyclophanes (CPCn, CPPy and TGDMAP) and acyclic cyclophane (ATGDMAP) on various glutamate receptors were studied with these receptors expressed in Xenopus oocytes using voltage-clamp recording. CPCn, CPPy, TGDMAP and ATGDMAP were found to inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2), but not Ca(2+)-permeable AMPA receptors (GluR1), Ca(2+)-nonpermeable AMPA receptors (GluR1/GluR2) and metabotropic glutamate receptors (mGluR1alpha). The inhibition of NR1/NR2A receptors by these compounds was more potent than those of the other NMDA receptor subtypes. At a resting potential (-70 mV), the IC(50) values of CPCn, CPPy, TGDMAP and ATGDMAP for NR1/NR2A receptors were 0.5+/-0.1, 1.0+/-0.2, 8.0+/-0.8 and 4.9+/-0.5 microM, respectively. The inhibition by these compounds was voltage-dependent, that is, the degree of inhibition was in the order of negative holding potentials, -100 mV>-70 mV>-20 mV. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by CPCn. The inhibition by CPCn was not altered significantly in the mutants at the critical asparagines in the M2 loop, NR1 N616, NR2B N615 and NR2B N616, these residues are known to form the narrowest region of the channel and the binding site of Mg(2+). However, mutations at NR1 N650, located in the vestibule of channel pore, and NR1 D669, located in the extracellular region, reduced the inhibition by CPCn, suggesting that these amino acid residues interact with CPCn. These results suggest that CPCn interacts directly with the mouth or vestibule of the ion channel, like a lid.

Cleft-type cyclophanes confer neuroprotection against excitatory neurotoxicity in vitro and in vivo through inhibition of NMDA receptors.

The cleft-type cyclophanes (ACCn, DNCn and TsDCn) were found to strongly inhibit macroscopic currents at heteromeric NMDA receptors (NR1/NR2) but not AMPA receptors expressed in Xenopus oocytes at voltage-clamp recording. The inhibition by cleft-type cyclophanes was voltage-dependent, because the inhibition was larger at -100 mV than at -20 mV. Mutations at NR1 N650, located in the vestibule of the channel pore, reduced the inhibition by DNCn and TsDCn, suggesting that the residue (N650) interacts with these cleft-type cyclophanes. Cell toxicity of TsDCn on SH-SY5Y cells was slightly weaker than that of memantine. The neuroprotective effects of cleft-type cyclophanes against cell damage caused by NMDA were investigated in cultured rat hippocampal neurons. Addition of 10 microM DNCn or TsDCn into the medium ablated the neurotoxicity induced by NMDA, and a similar effect was also observed with memantine. The neuroprotective effects of cleft-type cyclophanes were then assayed on NMDA-induced seizures in mice. Intracerebroventricular injection of TsDCn (5 mg/mouse) decreased the seizure induced by intraperitoneal injection of NMDA (115 mg/kg) in mice. The results demonstrate that these cleft-type cyclophanes interact directly with the extracellular mouth of the NMDA channel pore and exhibit neuroprotective effects on NMDA-induced excitatory toxicity in primary cultured neurons and mice.

Anionic cyclophanes as hosts for cationic aromatic guests

Abstract Negatively-charged cyclophanes ( 2a,2b ) having eight carboxylate groups on the aromatic rings were designed and synthesized. It is shown that they work as hosts that form inclusion complexes selectively with positively-charged aromatic compounds as guests in alkaline water.

Antagonism of NMDA receptors by butanesulfonyl-homospermine guanidine and neuroprotective effects in in vitro and in vivo

The polyamine derivative BsHSPMG (butanesulfonyl-homospermine with guanidine group) was found to inhibit macroscopic currents strongly at heteromeric N-methyl-D-aspartate (NMDA) receptors (NR1/NR2A and NR1/NR2B) and Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (homomeric glutamate receptor 1) receptors expressed in Xenopus laevis oocytes on voltage-clamp recording. The IC(50) values of BsHSPMG for NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors were 0.016, 0.021, 5.4, and 9.0 μM, respectively. BsHSPMG inhibited the activity of NR1/NR2A and NR1/NR2B receptors more strongly and did it for those of NR1/NR2C and NR1/NR2D receptors more weakly than a therapeutic drug of Alzheimers disease, memantine. The inhibition by BsHSPMG was voltage-dependent, since it was prominent at -100 mV compared to that at -20 mV. Mutations including NR1 N616Q, E621Q, N650A, L655A, T807C, NR2B W559L, M562S, W607L, N616Q, and V620E, among others, reduced the inhibition by BsHSPMG, suggesting that BsHSPMG penetrates the channel pore of NMDA receptors deeply. The toxicity of BsHSPMG in neuroblastoma SH-SY5Y cells was much weaker than that of memantine. The effect of BsHSPMG was measured on the focal cerebral ischemia induced by occlusion (1 h) of the middle cerebral artery in mice. BsHSPMG applied before or after occlusion greatly reduced the volume of infarct in mice. These findings demonstrate that BsHSPMG penetrates the NMDA channel pore and exhibits neuroprotective effects against excitatory toxicity in mice.

Neuroprotection by Tosyl-Polyamine Derivatives through the Inhibition of Ionotropic Glutamate Receptors

Tosyl-polyamine derivatives such as N-{4-[4-(guanidinobutylamino)-butylamino]butyl}-4-methylbenzenesulfonamide trihydrochroride (TsHSPMG) have been found to strongly inhibit macroscopic currents through heteromeric N-methyl-d-aspartate (NMDA) receptors (NR1/NR2A, NR1/NR2B) and Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (homomeric glutamate receptor 1) receptors expressed in Xenopus laevis oocytes on voltage-clamp recording. In the present study, it was found that the inhibition of NMDA receptor activity induced by tosyl-polyamine derivatives was voltage-dependent. Some mutations located in the intracellular region of the channel pore, such as NR1 E621Q and NR2B W607L, reduced the inhibition by tosyl-polyamine derivatives, suggesting that tosyl-polyamine derivatives penetrate deeply into the channel pore of NMDA receptors. The neuroprotective effects of tosyl-polyamine derivatives against cell injury caused by NMDA were investigated in cultured rat hippocampal neurons. Addition of 1 μM TsHSPMG to medium ablated the neurotoxicity induced by NMDA, and a similar effect was observed with 30 μM memantine. The neuroprotective effects of tosyl-polyamine derivatives on NMDA-induced seizures in mice were also assayed. Intracerebroventricular or intravenous injection of TsHSPMG (0.1 or 0.5 mg/kg) decreased the seizures induced by intraperitoneal injection of NMDA in mice. These findings indicate that tosyl-polyamine derivatives exhibit neuroprotective effects not only in primary cultured neurons but also in mice.

Differential effects of linear and cyclic polyamines on NMDA receptor activities.

The linear polyamine spermine enhances N-methyl-d-aspartate (NMDA) receptors activity at depolarized membrane potential and shows a voltage-dependent block. Spermine potentiates NMDA receptor currents in the presence of saturating concentrations of glutamate and glycine, but cyclic polyamines such as CP2323 do not. CP2323 inhibited the currents most potently amongst 10 kinds of cyclic polyamines tested. The inhibition was prominent at heteromeric NR1/NR2A and NR1/NR2B receptors but not at NR1/NR2C and NR1/NR2D receptors expressed in Xenopus oocytes. Inhibition by CP2323 was voltage-dependent, because the degree of inhibition was in the order -100mV>-70mV>-20mV. It was 10-100 times more prominent than inhibition by spermine. The inhibitory potency of both CP2323 and spermine was attenuated by the mutations around the vestibule of the channel pore at NR1 W563, N650, T807, and NR2B Y646. Inhibition by CP2323 was hardly affected by the mutations of NR1 N616 and E621, whereas inhibition by spermine was reduced by these mutations. The results suggest that CP2323 interacts with the vestibule region of the NMDA receptor and does not enter deep into the channel. Mutations of NR2B W607 greatly reduced the inhibition by CP2323 and spermine, suggesting that the mutation of this residue may cause the change of the channel structure. Neuroprotective effects of cyclic polyamines against cell damage caused by NMDA were compared with those of spermine in cultured rat hippocampal neurons. Addition of CP2323, but not spermine, into the medium attenuated the neurotoxicity induced by NMDA. These results indicate that CP2323 functions as a channel blocker of the NMDA receptor.

Synthesis of a novel water-soluble cyclophane having eight carboxymethylthiomethyl groups on the aromatic rings

A novel cyclophane (9) having eight carboxyl groups that are attached on the aromatic rings via a spacer (-CH 2 SCH 2 -) was designed and synthesized as a host for cationic, anionic, and neutral aromatic guests in alkaline water. Macrocyclization of 7 with 6 was carried out under high dilution condition to give the objective 8 in 17% yield

Inclusion Complex Formation between a Novel Anionic Cyclophane and Cationic Aromatic Guests in Aqueous Solution

1 H-Nmr spectral studies have shown that a negatively charged cyclophane (2) having eight carboxylate groups that are bonded directly on the aromatic rings forms 1:1 inclusion complexes selectively with positively charged aromatic comounds as guests in alkaline water