JPH0624970A - Metabolism regulating type l-glutamic acid receptor agonist - Google Patents

Abstract

PURPOSE:To obtain a metabolism regulating type L-glutamic acid receptor agonist useful as a biochemical reagent. CONSTITUTION:This agonist contains (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl) glycine of formula I or (2S,1'S,2'S,3'S)-2-(2,3-dicarboxycyclopropyl)glycine of formula II as an active ingredient. These compounds are agonists specific to a metabolism regulating type receptor of L-glutamic acid and useful as a study on higher grade nervous function such as memory or learning. Further, these compounds can be expected for development to therapeutic agents for various kinds of neurodegenerative diseases, e.g. therapeutic agents for epilepsy, Huntington's chorea, Parkinson's disease, etc., as blocking agents of L-glutamic acid recpetor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a new use of 2- (2,3-dicarboxycyclopropyl) glycine, which is an L-glutamic acid receptor agonist.
More specifically, it relates to a metabotropic L-glutamic acid receptor agonist that plays a major role in the study of the L-glutamic acid receptor.

The development of metabolically regulated L-glutamic acid receptor agonists provides clues for the elucidation of the physiological function of L-glutamic acid receptors, in particular the molecular mechanism of memory and learning formation, and the development of blockers. Therefore, it can be expected to be applied to the treatment of neurological disorders such as epilepsy, Huntingson's disease, Parkinson's disease, and neuromutation.

[0003]

BACKGROUND OF THE INVENTION L-Glutamic acid is used as a transmitter of excitatory nerve stimulation in the central nervous system of mammals, and as a neuroexcitotoxin that destroys nerve cells and causes various brain and nerve diseases. It is attracting attention as a substance that is important in the formation of learning.

The L-glutamic acid receptor is linked to such various physiological functions, and by the introduction of an exogenous agonist group, (a) NMDA (N-methyl) is introduced. -D-aspartic acid) type (b) KA (kainic acid) type (c) AMPA (ampa) type are classified into three subtypes. AMP here
A is α-amino-3-hydroxy-5-methyl-4-
Isoxazole propionic acid. Further, the KA (kainic acid) type and the AMPA (ampa) type may be collectively referred to as a non-NMDA type.

Conventionally, NMDA type receptors are considered to be the center of neuroexcitotoxins, and it is presumed that nerve cells are destroyed by excessive activation of the receptors, which in turn trigger various neurological diseases. ing.

Regarding this NMDA type receptor, (2S, 1'R, 2'S) -2- (2-carboxycyclopropyl) glycine is a potent NMDA type agonist superior to NMDA by Ofuna et al. Discloses that the folded conformation of glutamic acid activates the NMDA receptor (JP-A-1-09356).
3).

Regarding non-NMDA type receptors, Ofuna et al. (2S, 1'R, 2'R, 3'R) -2
-(2-Carboxy-3-methoxymethylcyclopropyl) glycine and (2S, 1'R, 2'R, 3'R) -2-
It has been disclosed that (2-carboxy-3-benzyloxymethylcyclopropyl) glycine is a non-NMDA type agonist (Tetrahedron L.
etters 31: 4049-4052 1990
Year; Brain Res. , 550, 152-156
P. 1991).

In these NMDA and non-NMDA type receptors, the receptor and the ion channel regulated by the receptor are present as a receptor-channel complex, and a transmitter is bound to the receptor. It is a type that is classified as an "ion channel type receptor" in which ion channels are directly opened and closed (Metabolism 26
Vol. 6, pp. 535-542, 1989).

On the other hand, Sugiyama et al. Have a certain species in the L-glutamic acid receptor, in which the receptor and the effector regulated by the receptor exist as separate molecular species, and a transmitter binds to the receptor. , The effector is regulated via the second messenger system activated by this G protein, and the receptive reaction is triggered.
It has been found that there are types of receptors classified as “metabolite-type receptors” (Nature 325, 5
31 pages 1987).

Nakanishi, who is one of the present inventors, succeeded in isolating this metabotropic L-glutamic acid receptor (hereinafter abbreviated as mGluR) and analyzing its structure by the method of molecular biology (Nature 349). Vol. 760, page 1991), and from the search for homologues thereof, three more mGluRs were isolated and classified into types 1 to 4 (hereinafter abbreviated as mGluRs 1 to 4, respectively).

[0011]

[Problems to be Solved by the Invention] Of these, mGluR1
Promotes inositol triphosphate turnover, and is often found in Purkinje cells in the cerebellum, dentate gyrus / CA2-3 area in the hippocampus, thalamus and the like. Further, mGluR2 suppresses the activation of adenylate cyclase and is present in Golgi cells of the cerebellum, dentate gyrus of the hippocampus, cerebral cortex and the like. To clarify the role of these mGluRs in vivo, drugs that specifically activate each are required. However, there are some compounds known to date that are suitable for this use. There wasn't.

[0012]

[Means for Solving the Problems] Nakagawa et al., Ishida et al., Among carboxycyclopropylglycines, extende
Electrophysiological studies show that (2S, 1 ′S, 2 ′S) -2- (2-carboxycyclopropyl) glycine, which fixes the conformation of d-type glutamic acid, is an agonist that significantly activates mGluR. Was also confirmed biochemically (Nakagawa et al .: European J. Phar
macol. 84, 205, 1990. Ishida et al .: Brain Research, Volume 537 311
P. 1990).

Further, the inventors of the present invention conducted extensive studies on agonists of mGluR and found that they were of extended type and f type.
A carboxycyclopropylglycine derivative having a structure in which an old-type conformation is fixed in the same molecule is represented by the formula (1) (2S, 1'R, 2'R, 3'R)-
2- (2,3-dicarboxycyclopropyl) glycine (hereinafter abbreviated as DCG-I)

[Chemical 3] And expressed by equation (2) (2S, 1'S, 2'S, 3'S)
-2- (2,3-dicarboxycyclopropyl) glycine (hereinafter abbreviated as DCG-II)

[Chemical 4] The activity of these compounds on mGluR1 and 2 was examined.

That is, Nakanishi et al. (Nature 349)
Vol. 760, page 1991), the cloned receptor genes are constitutively expressed in mammalian cells, and mGluR1 activity is inositol triphosphate turnover, and mGluR2 activity is phosphocholine-stimulated cells. Suppression of increase in internal cyclic adenosine 3 ′, 5′-monophosphate (hereinafter abbreviated as cAMP) concentration was measured as an index. As a result, DCG-I and D
It was found that CG-II is a specific agonist of mGluR2, and completed the present invention.

DCG-I, one of the compounds of the present invention
Can be synthesized, for example, as shown in Scheme I below.

[Chemical 5] (In the formula, TBS represents a t-butyldimethylsilyl group, and B
oc represents a t-butoxycarbonyl group. )

In the above scheme, first, (1R, 7S, 8R, 9R) -3-aza-9-t-butyldimethylsilyloxymethyl-4,4-dimethyl-5 represented by the formula (3).
Oxatricyclo [6.1.0.0 ^{3 '7]} nonane -2
-On (Tetrahedron Letters 3
1 volume 4049-4052 described in 1990) is de-t-TBS-ized by a known method to obtain an alcohol derivative represented by the formula (4).

The obtained alcohol compound was dissolved in water and ethanol without purification, alkali-hydrolyzed with 3 equivalents of barium hydroxide or the like, neutralized with sulfuric acid and the insoluble matter was filtered off, and the filtrate was triethylamine. PH was adjusted to 9 with and treated with BOC by di-t-butyl dicarbonate treatment,
(2S, 1'R, 2'R, 3'R) -N- represented by the formula (5)
tert-butoxycarbonyl-2- (2-carboxy-3
-Hydroxymethylcyclopropyl) glycinol.

Then, by treatment with diazomethane, the formula (6) is obtained.
A methyl ester compound represented by (2S, 1′R, 2′R, 3 ′
R) -N-tert-butoxycarbonyl-2- (2-methoxycarbonyl-3-hydroxymethylcyclopropyl) glycinol, which is further treated with Jones reagent and then with diazomethane to give a trimethyl ester represented by the formula (7): (2S, 1'R, 2'R, 3'R)-
N-tert-butoxycarbonyl-2- (2,3-dimethoxycarbonylcyclopropyl) glycine methyl ester can be hydrolyzed to obtain the compound of formula (1).

DCG-II, one of the compounds of the present invention
Can be synthesized, for example, as in the following scheme II in the same manner as DCG-I described above.

[Chemical 6] (In the formula, TBS represents a t-butyldimethylsilyl group, and B
oc represents a t-butoxycarbonyl group. ) In the above scheme II, (1S, 7S, 8S,
9S)-3-aza -9-t-butyldimethylsilyloxy-4,4-dimethyl-5-oxatricyclo [6.1.0.0 ^{3 '7]} nonane-2-one (Tetr
ahedron Letters Volume 31 4049-
4052, described in 1990) as a starting material.

[Action]

DCG-I, one of the compounds of the present invention
Shows mGluR in the inositol phosphate turnover assay in mGluR1 expressed in Chinese Hamster Ovary cells (hereinafter abbreviated as CHO cells).
MGl that suppresses the production of cAMP in mGluR2 expressed in CHO cells without exhibiting agonistic activity at 1
It was found to be a specific agonist of uR2.

It was also found that DCG-II has no agonist activity on mGluR1, but is a specific agonist showing mGluR2 activity similar to that of L-glutamic acid.

[0022]

The present invention will now be described in more detail by way of examples, which should not be construed as limiting the scope of the present invention.

Reference Example 1. (2S, 1'R, 2'R, 3'R)-
2- (2,3-dicarboxycyclopropyl) glycine
Synthesis of (DCG-I) (1) Synthesis of DCG-I was carried out according to Scheme I above.

Step 1. (2S, 1'R, 2'R, 3'R)
-N-tert-butoxycarbonyl-2- (2-methoxy
Carbonyl-3-hydroxymethylcyclopropyl) g
Synthesis of (6) of Ricinol (1R, 7S, 8R, 9R) -3-Aza-9-t-butyldimethylsilyloxymethyl-4,4-dimethyl-5-
Oxatricyclo [6.1.0.0 ^{3 '7]} nonane -2
200 mg (0.64 mmol) of -one (3) was dissolved in 2 ml of tetrahydrofuran (hereinafter abbreviated as THF), and tetra-n-butylammonium fluoride (1M was added under ice cooling.
(/ THF solution) (1 ml) and the mixture was stirred for 10 minutes to obtain an alcohol compound (4).

The obtained alcohol compound (4) was dissolved in 2 ml of water and 2 ml of ethanol without purification, 606 mg (1.92 mmol) of barium hydroxide was added, and the mixture was heated to 80 ° C.
And stirred for 3 hours. After neutralization with diluted sulfuric acid, the insoluble material was filtered off, and the filtrate was adjusted to pH 9 with triethylamine.

Di-t-butyl dicarbonate 14
6 μl (0.64 mmol) and 2 ml of dioxane were added, and the mixture was stirred at room temperature for 16 hours. After washing the reaction solution with ether, the aqueous layer was adjusted to pH 1 with 1N hydrochloric acid and extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over magnesium sulfate, and the solvent was evaporated under reduced pressure to give amorphous (5). An ether solution of diazomethane was added to this to give a methyl ester, and the title compound (6) was quantitatively obtained.

The physical properties of the obtained compound (6) are shown below.

[Table 1]

Step 2. (2S, 1'R, 2'R, 3'R)
-N-tert-butoxycarbonyl-2- (2,3-dime
Toxycarbonylcyclopropyl) glycine methyl s
Synthesis of ter (7) 70 mg (0.24 mm ) of the obtained methyl ester (6)
ol) is dissolved in 2 ml of acetone and Jones is cooled under ice.
The reagent was added, and the mixture was stirred under ice cooling for 1 hour and at room temperature for further 3 hours. Isopropyl alcohol was added under ice cooling to decompose excess reagents, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over magnesium sulfate, the solvent was distilled off under reduced pressure, the resulting residue was added with an ether solution of diazomethane for methyl esterification, and subjected to silica gel column chromatography (methanol / chloroform = 3/9
Purification in 7) yielded 75 mg of the title compound (7) (yield 90%).

The physical properties of the obtained compound (7) are shown below.

[Table 2]

Step 3. (2S, 1'R, 2'R, 3'R)
-2- (2,3-dicarboxycyclopropyl) glycy
(DCG-I) (1) synthetic trimethyl ester (7) 58 mg (0.17 mmo
l) was dissolved in 1 ml of THF, 1 ml of a 1M aqueous sodium hydroxide solution was added, and the mixture was cooled under ice-cooling for 5 hours and further at room temperature for 24 hours.
Stir for hours. To this, 1 ml of 2N hydrochloric acid was added and the mixture was allowed to stand at room temperature for 18
Stir for hours. The residue after concentration under reduced pressure was diluted with water, subjected to Dowex 50W × 4 column chromatography, washed with water, and eluted with 1N ammonia water. Aqueous ammonia was distilled off under reduced pressure, pH was adjusted to 2 with 1N hydrochloric acid, and crystallized from water-ethanol to obtain 22 mg of the title compound (yield 65%).

The physical properties of the obtained compound (1) are shown below.

[Table 3]

Reference Example 2. (2S, 1'S, 2'S, 3'S)-
2- (2,3-dicarboxycyclopropyl) glycine
Synthesis of (DCG-II) (2) Synthesis of DCG-II was carried out according to the above scheme II in Reference Example 1
It carried out by the method similar to.

(1S, 7S, 8S, 9S) -3-Aza-9
-T- butyldimethylsilyloxy-4,4-dimethyl-5-oxatricyclo [6.1.0.0 ³ '
⁷ ] Nonan-2-one (8) 300 mg (0.96 mm
ol) as a starting material and in the same manner as in Example 1 to obtain 82 mg of the trimethyl ester compound (12) (yield 25%).
The obtained trimethyl ester form (12) 65 mg (0.
Similarly, 10 mg of the title compound was obtained from (19 mmol) (yield 26%).

The physical properties of the obtained compound (2) are shown below.

[Table 4]

A synthetic intermediate (2S, 1'S, 2'S, 3 '
The physical properties of S) -N-tert-butoxycarbonyl-2- (2-methoxycarbonyl-3-hydroxymethylcyclopropyl) glycinol (11) are shown below.

[Table 5]

Intermediate (12) (2S, 1'S, 2 '
S, 3'S) -N-tert-butoxycarbonyl-2- (2
The physical properties of 3,3-dimethoxycarbonylcyclopropyl) glycine methyl ester are shown below.

[Table 6]

Example 1. Metabotropic L-glutamic acid receptor
Step of measuring the agonist activity of the body 1. CHO-mGluR1 cells and CHO-
CHO-mGluR1 cells and CH used in assay for making mGluR2 cells
O-mGluR2 cells were prepared by Nakanishi et al. (Recent Pr).
ogress in Horn Research
46, 59-84, 1990). Ie cloned mGluR1 or mG
The gene for luR2 was incorporated into pdKCR, which is an expression vector for mammalian cells, and introduced into CHO cells lacking dehydrofolate reductase by the calcium phosphate method to obtain CHO-mGluR1 cells and CHO-mGlu.
R2 cells were created. These cells were passaged by adding penicillin and streptomycin to Dulbecco's modified Eagle medium (hereinafter abbreviated as DMEM medium) containing 10% dialyzed fetal bovine serum, 1% proline, and 2 mM glutamine.
The culture was carried out at 37 ° C. under 5% CO ₂ . The cells used in the following assay are not limited to CHO-mGluR1 cells and CHO-mGluR2 cells, and cells in which these receptors are expressed on the cell surface by a known method can be used.

Step 2. Assay of mGluR1 The activity of DCG-I and DCG-II against mGluR1 was determined by Nakanishi et al. (Nature 344: 760 1).
991). That is, first, the cells are treated as follows. 1) Day 1: 3 × 10 ⁵ pieces / hole CHO in a 6-hole plate
-Seeding the mGluR1 cells and culturing by the method of Nakanishi et al. 2) Day 2: Replacement with DMEM medium containing ³ H-inositol and culturing in the same manner. 3) Day 3: a) Phosphate buffered saline (Phospha)
te buffered saline (hereinafter abbreviated as PBS), and incubated for 20 minutes. b) Incubation for 20 minutes after changing to LiCl / PBS. c) After changing to LiCl / PBS containing the test compound, 20
Minute culture. d) Quenched the reaction with trichloroacetic acid and released ³ H
-Extract inositol phosphate. Then, using the ion-exchange resin from the obtained extract, ³
H-inositol phosphate is isolated and radioactivity is measured in a liquid scintillation counter to assay agonist activity on mGluR1.

As a result, DCG-I and DCG-II
Was found to have no agonistic activity on mGluR1.

Step 3. Assay of mGluR2 The activity of DCG-I and DCG-II on mGluR2 was determined by Sugama et al. (Biochem. Biophys. A).
cta 1011, 75, 1989) cAMP
It was measured by applying a concentration measuring method. That is, first, the cells are treated as follows. 1) Day 1: 2 × 10 ⁵ pieces / hole on a 12-hole plate
Seeding O-mGluR2 cells and culturing in the same manner as on day 1 of step 2. 2) Day 3: a) PBS (IBMX / PB) containing 1 mM isobutylmethylxanthine (hereinafter abbreviated as IBMX).
After changing to S), incubate for 20 minutes. b) IB containing 10 μM phosphocholine and a test compound
After exchanging with MX / PBS, incubate for 10 minutes. d) cAM released by stopping the reaction with trichloroacetic acid
Elute P. Then, the eluted cAMP was added to Amersham's cAMP.
Quantify using a measurement kit.

As a result, DCG-I was 3 × 10 ^-7 M, D
CG-II showed 50% inhibition of maximum inhibition at a concentration of 8 × 10 ^-6 M. When this was compared with glutamic acid itself, DCG-I showed about 30 times the activity, and DCG-II showed almost the same activity as glutamic acid.

From these results, DCG-I and DC
G-II was found to be an agonist specific to mGluR2.

[0046]

INDUSTRIAL APPLICABILITY According to the present invention, (2S, 1′R, 2′R, 3′R) -2 is an agonist specific to mGluR2 which is one of the L-glutamic acid metabotropic receptors. −
(2,3-dicarboxycyclopropyl) glycine (D
CG-I) (1) and (2S, 1'S, 2'S, 3'S)-
2- (2,3-Dicarboxycyclopropyl) glycine (DCG-II) (2) is provided.

The DCG-I and DCG-II are mG
Since it is an agonist specific to luR2, it is a useful probe for studying higher nerve functions such as memory and learning as a biochemical reagent, and at the same time, through the development of L-glutamic acid receptor blockers, various nerve mutations It will lead to the development of remedies for diseases.

Claims (1)

[Claims] 1. Formula (1): (2S, 1'R, 2'R, 3'R) -2- (2,3
-Dicarboxycyclopropyl) glycine or a compound of formula (2) (2S, 1'S, 2'S, 3'S) -2- (2,3
-Dicarboxycyclopropyl) glycine as an active ingredient, a metabotropic L-glutamic acid receptor agonist.