JPH0368519A - Glutamate antagonist - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a glutamate antagonist that inhibits glutamate receptor function in the central nervous system.

[Conventional technology]

Glutamate plays a major role as an excitatory neurotransmitter in the central nervous system, but on the other hand, excess glutamate is known to destroy nerve cells. That is, glutamate is thought to cause excessive excitement in nerve cells, leading to cell death, and this phenomenon is called excitotoxicity. Such excitotoxicity is a cause of neurodegenerative disorders, and drugs having glutamate antagonistic effects are useful as therapeutic agents for neurodegenerative disorders.

As a glutamate antagonist, for example, 6-nitro-tucyanoquinoxaline-2,3-dione is known (
Eur, J., Pharn+acology, 156
．． 177-180 (1988) and 5science
, 241.70HI988)).

[Purpose of the invention]

The present invention provides a glutamate antagonist,
In particular, it is an object of the present invention to provide a medicament that selectively acts on the glycine-regulated site of the glutamate receptor in the central nervous system and has fewer side effects.

[Structure of the invention]

The glutamate antagonist of the present invention contains 6,7-cycloquinoxaline-23-dione as an active ingredient. This compound is known as a synthetic intermediate for photographic desensitizers (Japanese Patent Publication No. 1300/1983), and is described in, for example, the above publication or Liebigs Ann, Chem.
, 745-76N, 1982).

Glutamate is one of the excitatory neurotransmitters in the mammalian central nervous system, and glutamatergic nerves have been identified particularly in the pathway from the cerebral cortex to the thalamus and striatum, as well as in the cerebellum and hippocampus. Currently, quisqualic acid (Q) is the glutamate receptor in glutamatergic nerves.
A) receptors, kainic acid (KA) receptors and N
It has been recognized that at least three subtypes called -methyl-0-aspartate (NMDA) receptors exist, and among these, NMDA receptors are known to exist only in mammals.

There are three main antagonists that act on NMD A receptors.
Two types are known. One is a competitive antagonist to the receptor itself, such as CPP (3(2-carboxypiperazin-4-yl)propane-1-phosphate),
AP-5 (2-amino-5 phosphonopentanoic acid), AP
-7 (2-amino 7-phosphonohebutanoic acid) and the like are known.

The other is a blocker of ion channels associated with receptors, such as MK-801 (D-5-methyl-10,11-dihydro-58-dibenzoCa, d]
cyclohepten-5,10 imine) and the like are known. Another type is an inhibitor of the glycine regulatory site associated with the receptor; for example, 7-chlorokynurenic acid is known.

On the other hand, recent rapid progress in basic research has led to the elucidation of the physiological role of glutamate as an excitatory transmitter, as well as the relationship with various pathological conditions. As a result, neuronal excitotoxicity is associated with neurodegenerative disorders such as Alzheimer's disease, chorea, medullocerebellar degeneration, stroke, cerebral palsy, epilepsy, hypoglycemic neuropathy, cerebral ischemia, and carbon oxide poisoning. It has been suggested that this may be the cause.

Under these circumstances, the present inventors have discovered that the quinoxaline derivatives are NMDA receptor antagonists, particularly selective inhibitors of the glycine regulatory site.

The quinoxaline derivative represented by the above formula, which is the active ingredient of the present invention, exhibits strong glutamate antagonism and is therefore effective as a therapeutic agent for the above-mentioned neurodegenerative disorders. It is expected that this product will be an excellent drug with few side effects.

The glutamate antagonist of the present invention can be administered orally or parenterally. That is, it can be administered orally in commonly used dosage forms such as tablets, capsules, syrups, suspensions, etc., or it can be administered in the form of liquids such as solutions, emulsions, and suspensions by injection. It can be administered as a drug. It can also be administered rectally in a form of execution. Such dosage forms include conventional carriers, excipients, binders,
It can be manufactured according to a general method by blending the active ingredient with a stabilizer and the like. When used in the form of an injection, a buffer, solubilizing agent, isotonic agent, etc. may be added.

The dosage and frequency of administration vary depending on the symptoms, age, body weight, administration form, etc., but when administered orally, the dose for adults is usually 1 to 100 mg per day, and when administered parenterally, it is 0.00 mg per day. 1 to 10 mg can be administered once or in divided doses.

The present invention will be explained in detail with reference to Examples below.

Example 1 The fact that the quinoxaline derivative, which is the active ingredient of the glutamate antagonist of the present invention, is an antagonist that acts on the NMDA receptor is due to the fact that [3H]-801 bound to the NMDA ion channel in a glutamate-dependent manner is excluded from its binding site. It can be demonstrated by measuring the ability to

Its properties as a competitive antagonist to the receptor itself are
['H]Gl bound to the NMDA receptor itself
U(glutamic acid) or [:'Ily(glycine) bound to the glycine regulatory site can be shown to be inhibitors of the glycine regulatory site by measuring their ability to eliminate HICPP. The ability to exclude can be shown by specifying fill.

These activities are ICs. This value can be expressed as C3HI MK-801, ['ll]Glu, respectively.
(:'tt)CPP and ['lf)Gry's 50
It represents the concentration (μM) of the test compound required to eliminate %.

The test method is as follows.

[3H] ■-801 binding test Crude synaptic membrane preparation was obtained from Wistar male rat computer J
'! lii [then 50mM] in lis acetate buffer (p
I+ 7.4), 50.000g, 30
A washing operation by centrifugation for 3 minutes was performed three times. Sediment is 0
．． It was stored frozen in a 32 M sucrose aqueous solution at 1 tffi at -80°C. Freeze when in use! ! 1! After melting the suspension at room temperature, it was diluted with 0.08% Triton x-ioo at 2°C for 10
Pretreatment was performed for 1 minute. After pretreatment, the preparations were subjected to the above-mentioned centrifugal separation twice and subjected to the binding experiment. For binding experiments, membrane preparation (approximately 250 μglt white) was mixed with 5 nM 1:')
1] MK-801 (29,4Ci/mmol) and 2°C
Alternatively, the reaction was performed at 30°C for 30 minutes. The reaction was stopped by suction filtration using a Watt 7 GF/B glass filter. Radioactivity on the filter was measured by liquid scintillation method (measurement efficiency 40-42%).

Non-specific binding was calculated from radioactivity in the presence of 0.1mM -801.

C') l) [; Preparation of brain synaptic membrane preparation for Il+ binding test! ! ill: After preparing a crude synaptic membrane preparation from the brain of Wistar male rough) (200-250 g), it was added to 50 sM) Lis acetate buffer (pH 7).
, 4) and centrifuged at 50,000xg for 30 minutes.

After repeating this washing operation three times, the sample was washed with 0.32M
The suspension was frozen and stored at -80°C in a sucrose aqueous solution.

Before use, the frozen suspension was thawed at room temperature and pretreated with 0.08% Triton X-10.0 at 2.degree. C. for 10 minutes. After the treatment, a washing operation by centrifugation was performed twice.

C'HE Glu binding experiment: Membrane preparation (approximately 100 μg) was mixed with 10 nM ['H] Glu (30 ci/mmol)
and 50mM) in lis acetate buffer (pl+ 7.4) at 2°C for 10 minutes. Reaction stop is Watt 7nG
This was carried out by a suction filtration method using an F/B glass filter. liMDA sensitive binding is Q, lmM NMD^
was calculated by subtracting the binding in the presence of (NMOA-insensitive binding) from the total binding.

['ll) CP P binding test m'AI, crude synaptic membrane preparation from rat computer, 5 (
1mM) using lis acetate buffer (pH 7,4),
．． The cells were washed three times by centrifugation at 000g for 30 minutes. The obtained precipitate was suspended in a 0.32 M sucrose aqueous solution and stored frozen at -80°C. When using, this suspension was melted at room temperature and then heated with 0.08% Triton X-100 at 2°C.
Pretreatment was performed for 10 minutes. The pretreated sample was subjected to the above-mentioned washing operation twice and then used for the experiment. Binding experiments were performed using this suspension (
Approximately 250 μg protein) was mixed with 10 nM [”
H:l CPP (30.7Ci/mmol) at 2°C, 1
The reaction was carried out for 0 minutes. The reaction was stopped by suction filtration using a Whatman 6F/B glass filter. Non-specific binding was calculated from radioactivity in the presence of 1mM Glu.

[:')1'1Gay binding test Preparation of brain synapse M specimen: After preparing a rough synaptic membrane mass from rat brain, prepare a wooden specimen 5011114)! 50,000g using Is acetate buffer (pH 7,4), 3
A washing operation by centrifugation for 0 minutes was performed three times. The precipitate was suspended in a 0.32M sucrose aqueous solution and stored frozen at -80°C. Before use, after melting at room temperature, the sample was pretreated with O, Oa% Triton X-100 at 2°C for 10 minutes.

After pretreatment, the specimens were subjected to the above-mentioned washing operation twice and were used for experiments.

['H) Gly binding experiment: Synapse membrane preparation (150
−200 μg protein) to 10 nM (″)I]Gly(
40Ci/mmol) in the same IT solution at 2°C for 10 minutes. The reaction was stopped by suction filtration using a Watt 7 GF/B glass filter.

Nonspecific binding was calculated from the radioactivity in the presence of I mu GIy#.

Test Results (1) The active ingredient of the present invention inhibited [3H]-801 binding in the presence of 10 μM glutamic acid. I at that time
C. The value was 1.12±0.08 μM.

(2) C381Glu binding of the active ingredient of the present invention, [3H]
Table 1 shows the results of comparing the effects on CPP binding and ['ll]Gry binding with those of structurally similar compounds.

Table 1 CNQX 73J 16.4
5.31Q X >100
98.6 44.1CNOX:6-=)o-
7-C7/Quinoxaline-2°3-dione X: Quinoxaline-2,3-dione As is clear from Table 1, the active ingredient of the present invention selectively acts on the G1y regulatory site at extremely low concentrations.

Claims (2)

[Claims] (1) A glutamate antagonist containing 6,7-dichloroquinoxaline-2,3-dione as an active ingredient. (2) The glutamate antagonist according to claim 1, which is a therapeutic agent for neurodegenerative disorders.