JPS63190864A - Glutamic acid receptor-inhibiting substance - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (R is H or formula II; m and n are 3 or 4; p is 0 or 1) and salt thereof. USE:A glutamic acid receptor-inhibiting substance capable of utilizing also to mechanism of memory and explication of cerebral nerve disease, etc. PREPARATION:An insect such as silk spiders, preferably only poison gland thereof is taken out, crushed in an extract solvent (preferably acetonitrile-water mixed solvent) by an atomizer, homogenizer, ultrasonic wave, etc., extracted and separated by a centrifugal separator, etc., to provide the compound expressed by formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel compound or a salt thereof having a glutamate receptor inhibitory effect.

BACKGROUND OF THE INVENTION When arthropods such as insects are bitten by Nephila spiders, such as Nephila clavata and Nephila maculata, they become paralyzed and then become prey to the spiders.

Therefore, it was thought that a chemical substance that paralyzed the nerves of insects and other arthropods was present in the bodies of spiders.

Problems to be Solved by the Invention However, the chemical substances that paralyze the nerves of insects and other arthropods that exist in the bodies of spiders are extremely difficult to isolate. The chemical structure was unknown.

Means for Solving the Problems The present inventors elucidated the chemical structure of a compound that paralyzes the nerves of arthropods obtained from the venom glands of spiders, and conducted further studies to complete the present invention.

The compound of the present invention is a compound represented by the following formula [1] or a salt thereof.

C0NH.

[In the formula, R is a hydrogen atom or a group is abbreviated as Arg], m and n are each 3 or 4
, p represents 0 or 1] Examples of the salt of compound [I] include salts with inorganic acids or organic acids. Examples of inorganic acid salts include hydrochloride, disulfate, carbonate, and nitrate. Organic acid salts include formate,
Examples include acetate, propionate, sulfate, succinate, benzoate, and paratoluenesulfonate. The α-amino acid constituting compound [I] is L-form or D-amino acid.
Although it is an L-form, the L-form is more preferable.

First, the method for producing the compound [+] of the present invention will be explained.

Manufacturing consists of extraction and purification steps. Extraction is carried out using an extraction solvent after removing the body of the spider, preferably only the poisonous glands. It is better to crush the insect body (preferably the poison gland) in advance, but it is more preferable to crush it in an extraction solvent. For crushing, an atomizer, a homogenizer, etc. are used, but crushing by ultrasonic waves is also used. As the extraction solvent, an aqueous solvent such as water, an aqueous electrolyte solution, or a mixed solvent of water and an organic solvent that can be uniformly mixed with water is used. As the electrolyte aqueous solution, saline, ammonium chloride aqueous solution, sodium sulfate aqueous solution, etc. are used. The electrolyte aqueous solution used has a concentration range of 0 to 1.5% (W/V), preferably 0.3 to 0.5% (W/V). Examples of organic solvents that can be uniformly mixed with water include alcohols such as methanol, ethanol, and propatool, nitrites such as acetonitrile, and ketones such as acetone. The most preferred extraction solvent is a mixed solvent of acetonitrile and water. Extraction can be carried out by allowing it to stand for a long time, but it can also be carried out in a short time using a conventional stirrer or centrifuge. It is advantageous to use a centrifuge because it is easy to separate the extract and the residue after extraction. The speed of centrifugation is 1.0
00-100°000 revolutions/min, centrifugation time 5 minutes to 3
Good time.

The active ingredient can be conveniently tracked by measuring the excitatory postpotential (EPSP) of the neuromuscular junction of the walking leg of the spiny lobster. The measurement method will be described later. The measurement results show that the active ingredients have migrated to the extract. Even when an attempt is made to dissolve the active ingredient in a common organic solvent such as ethyl acetate or methylene chloride, it remains in the aqueous layer without being substantially dissolved.

Next, the extract is purified, but it is desirable to remove the extraction solvent before purification. The extraction solvent can be removed using a conventional solvent removal method, such as vacuum distillation or freeze drying. As a purification operation, gel filtration, purification using an ion exchange resin, silica gel preparative thin layer chromatography, high performance liquid chromatography, etc. may be used, or separation and purification by high performance liquid chromatography is preferred since it contains a large number of active ingredients. Examples of the solvent used for separation and purification include the aqueous solvent used for extraction. The separated and purified active ingredient, ie, the compound [+] of the present invention, can be stored as a lyophilized product. Multiple Compounds Obtained [1F can be confirmed to be a single compound by silica gel thin layer chromatography, gel filtration, and electrophoresis.

As shown in the examples, Nephilac
lavata) as compound location] I a, I
b, Ic.

Four types of compounds of Id are found in Neph spiders.
ila maculata) or: ;Ie, [f, and Ig, three types of compounds can be obtained as a single product.

The chemical structure of compound [+] is determined by amino acid analysis, I) (-N
MR,,” CN MR, mass spectrum, IR.

This is done using U■ etc.

The structures of compounds Ib, Ic, Id and Ig are as follows.

C0NI+.

-NH[(C1l,)mNH]p(C1l,)nNll
R[I b n=H, m= 3, n= 3
, p=i or n=H, m=3. n=4. p=II
c n=H, m=4. n=3. p=11d
R=Arg, m=3, n=4, p=1
or n=Arg, m=4, n=3, p=
11g R=Arg, n=4, p=0
The active ingredient is assayed using the neuromuscular synapse of the isepi walking leg. The extensor muscles of the ise-epi walking leg are exposed, and the potential is measured using a glass tube microelectrode filled with 3M potassium chloride aqueous solution. The activity of the inhibitor is determined by either (1) or (2).

(1) The nerves controlling the extensor muscles are separated into single fibers and electrically stimulated using a suction electrode. When excitatory nerve stimulation causes glutamate to be released from the nerve terminal and binds to a receptor, an excitatory postsynaptic potential (EPSP) of 3 to 5 mV is generated in the postsynaptic membrane.

Glutamate receptor inhibitors reduce this EPSP by 50
% or more or disappear.

(2) Fill a glass tube microelectrode with glutamic acid (IM, pH 8), bring it close to the neuromuscular nonabs, apply an inward current of about 10"9A, and electrophoretically flow 1M5 glutamic acid. The membrane usually has a length of 3 to 5 m.
A depolarizing potential of V (glutamate potential) is created, which is proportional to the strength of the applied current. Glutamate receptor inhibitors reduce or eliminate this glutamate potential by 50% or more.

Action/Effect The glutamate receptor inhibiting action of compound [1] is an irreversible action that does not disappear even after washing with water.

This indicates that the binding of the compound [1] of the present invention to the glutamate receptor is much stronger than that of known glutamate receptors. Therefore, it is believed that labeling the compound of the present invention will be useful in research such as isolation, structural analysis, and local analysis of glutamate receptors. It is also thought to be useful in elucidating the mechanisms of memory and cranial nerve disorders, even though glutamic acid is not involved.
The value for personal use is high.

Example 1 Nephila clava from Japan
La) 5 venom glands obtained from 2.5 animals were placed in a glass homogenizer, 60% aqueous acetonitrile (acetonitrile/water - 60:40 V/V) ld was added, and homogenized for 5 minutes. The homogenized mushrooms were centrifuged at 5,000 rpm for 10 minutes using a centrifuge. After lyophilizing the centrifuged supernatant using a freeze dryer,
It was dissolved in 0 μg of water, and a chromatogram was created using high performance liquid chromatography (hereinafter abbreviated as HPLC). For HPLC, a reverse phase distribution column (T
SKgel 0DS-120T) was used. The size of the column was 250 mm in length and 4.6 mm in inner diameter, and the resin used was 5 μm in diameter. Column temperature is 40°C
1. A mixed solvent of 0.02% hydrochloric acid and acetonitrile was used as the elution solvent, and a linear gradient method was used. i.e. 0.02%
Elution was started with only hydrochloric acid, and the acetonitrile content was increased by 1% (V/V) every minute, and ended when the acetonitrile content was 60% (V/V). The flow rate was 500 μm2/min. The obtained chromatogram is as shown in FIG. Four types of single products are changed, and the order of flow is Ia,
Ib, 1. c, Id.

Example 2 Nephi spider from Babua New Guinea
la maculata), a chromatogram as shown in FIG. 2 was obtained in exactly the same manner as in Example 1.

Three types of single products were obtained, and the order of outflow was Ie, [.

It is Ig.

Example 3 Nephila clava from Japan
ta) 0.5 g of venom glands obtained from 200 animals was placed in a glass homogenizer, and 60% aqueous acetonitrile (acetonitrile; water = 60:40 V/V) IO- was added and homogenized for 5 minutes. The homogenized product was centrifuged in a centrifuge at 10,000 rpm for 30 minutes.

The centrifuged supernatant was freeze-dried using a freeze dryer, then dissolved again in Id water, and then added to a reverse phase distribution column (TSKgel 0DS-12 manufactured by Toyo Soda Co., Ltd.) as in Example 1.
0T) was used for separation and purification by HPLC.

The above aqueous solution was separated and purified a total of 10 times in 1/10 increments, and the separated identical compounds were collected together with N
This was used as an MR sample. The column size is 300 mm in length.
The inner diameter was 7 or 8 mm, and the resin diameter was 10 μm. Column temperature is 40°C5 Elution solvent is 0.02%
The same linear gradient method as in Example I was carried out using a mixed solvent of hydrochloric acid and acetonitrile. The flow rate was 1.5 d/min. After fractionation, the solvent was removed using a centrifugal evaporator similar to a freeze dryer, and NMR was measured in heavy water (D, O) or methyl sulfoquine (do DMSO).

Example 4 Nephi spider from Papua New Guinea
Samples for NMR were prepared in exactly the same manner as in Example 3 for N. la maculata), and NMR was prepared in exactly the same manner as in Example 3.
MR was measured.

The proton NMR spectra of Ib, Ic, Id, and Ig in Dto are shown in FIG.

Ig glue, proton NM in O and d,-DMSO
A comparison of the R spectra is as shown in FIG.

Proton-cosY-NMR in 1 g da-DMSO
The spectrum is as shown in FIG.

Note that NMR is JEOL GX270 or GX40.
0, BROKER WH270 (equipped with
th ASPECT2000) or AM400(e
equipped with ASPECT3000
).

In the heavy atom collision mass spectrum (FAB-MASS) of JEOL DX-303, Ig is M+H": m
Since a peak is observed at /z665, the molecular weight of 1g is 6.
It is 64.

When the UV spectrum of Ig was measured in a 0.02% hydrochloric acid-acetonitrile mixture (50:50 v/v), 21
It has an absorption maximum below 0 nm and at 280 nm. Furthermore, natural fluorescence with an excitation wavelength of 280 nm and a fluorescence wavelength of 315 nm is observed.

[Brief explanation of the drawing]

The chromatogram of the Joro spider is shown in Figure 1. The chromatogram of the giant recluse spider is shown in Figure 2. Proton NMR spectra in DtO of compounds 1b, Ic, Id and [g are shown in FIG. FIG. 4 shows a comparison of proton NMR spectra of 1 g of compound in glue, O, and da-DMso. d, -DM of 1 g of compound
The P-C7):/-COSY-NMR spectrum in SO is shown in FIG. ○ Procedural amendment [Format] Patent Application No. 23147 of 1988 2, Name of invention glutamate receptor inhibitor 3, Relationship with the person making the amendment Case Patent applicant address 2-27 Doshomachi, Higashi-ku, Osaka Name ( 2
93) Takeda Pharmaceutical Co., Ltd. Representative Junmasa Umemoto (and 1 other person) 1 Agent Tokyo contact information (Patent Regulations Division) Telephone 278-221.8.2
2195, Date of amendment order Drawing and document certifying power of attorney 7. Contents of amendment (1) As shown in the attached sheet, we will submit the entire drawing clearly engraved. (2) As shown in the attached document, we will submit a document evidencing the power of representation. 8. List of attached documents (1) Drawings (Figures 1 to 5)
1 copy (2) Power of attorney
2 or more

Claims (7)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R is a hydrogen atom or group ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼, m and n are each 3 or 4, p is 0 or 1) or a salt thereof. (2) The substance according to claim (1), wherein R is a group ▲ has a mathematical formula, chemical formula, table, etc. ▼, n is 4, and p is 0. (3) The substance according to claim (1), wherein R is a group ▲ has a mathematical formula, chemical formula, table, etc. ▼, m is 3, n is 4, and p is 1. (4) The substance according to claim (1), wherein R is a group ▲There are mathematical formulas, chemical formulas, tables, etc.▼, m is 4, n is 3, and p is 1. (5) The substance according to claim (1), wherein R is a hydrogen atom, m is 3, n is 3, and p is 1. (6) The substance according to claim (1), wherein R is a hydrogen atom, m is 3, n is 4, and p is 1. (7) The substance according to claim (1), wherein R is a hydrogen atom, m is 4, n is 3, and p is 1.