JPH0269497A - Clavamine, production thereof and insecticide composition using the same compound - Google Patents

Abstract

NEW MATERIAL:A compound represented by the formula or salts thereof. USE:An insecticide having no inhibition activity against glutamic acid receptors. PREPARATION:A poison gland of a joro spider (Nephila clavata) is ground as necessary and then subjected to extraction, isolation and purification to obtain clavamine(salt). In addition clavamine may be obtained by bonding 2,4- dihydroxyphenylacetyl group or the peptide fragment having a terminal 2,4- dihydroxyphenylacetyl group of clavamine with a peptide fragment corresponding to the residual part of clavamine and removing protecting groups as necessary.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to lavamine, which is useful as an insecticide. The compound is found in Joro 5pider.

It is a component contained in the poisonous glands of Nephila clavata).

BACKGROUND OF THE INVENTION A compound contained in the venom glands of the Negro spider, which has been purified and isolated from these venom glands and is in a substantially pure form, is described in JP-A-60-184021. Various properties of these pure compounds have been confirmed, and it has also been confirmed that they have a glutamate receptor inhibitory effect. These inhibitory activities have been confirmed by measuring the excitatory nonadhesive postpotential (EPSP) at the epineurium neuromuscular junction.The compound has a molecular m500
±200, warping force gel thin IV! Chromatography [Silica gel Merck 760. n-propyl alcohol
Water mixed solvent 7:3 (v/v)) and Rf value is 0.20.

The patent application No. 62-23147 also describes 41. Some compounds have been disclosed that have a glutamate receptor inhibitory effect. The compound is 2.4-
Hydroquine has a structure with a phenylacetyl group at one end, a polyamine followed by an arginyl group at the other end.

In Japanese Patent Application No. 63-105080, a new chemically synthesized amide compound which exhibits a glutamate receptor inhibitory effect has been synthesized.

Problems to be Solved by the Invention Although the active components in the venom glands of the spider spider have been partially elucidated as mentioned above, they are still isolated, and therefore there are many unknowns such as physical properties, structure, and chemical synthesis methods. There are ingredients. The objects of the present invention have been described above. Confirm the existence of a new active substance different from known compounds obtained from the venom glands of the Japanese spider spider, establish isolation and purification methods and chemical synthesis methods, confirm its biological activity, and discover its uses. That's true.

Means for Solving the Problems The present inventors conducted various studies to achieve the above object, and unexpectedly found that the invention was disclosed in the well-known Japanese Patent Application Laid-Open No. 60184021 and the earlier patent application No. 62-23147. While extracts from the venom glands of the spider spider had high glutamate receptor inhibitory activity, we discovered a fraction that did not exhibit such high glutamate receptor inhibitory activity, but had strong insecticidal activity against insects such as Chikaie chikara. The present invention was completed after further intensive studies regarding the purification and isolation methods, structures, chemical synthesis methods, etc.

That is, the present invention provides: (1) Clavamine or a salt thereof represented by the following formula [hereinafter referred to as formula (1)].

(Left below) i'JI++ C=NH 011Ni1 4g\,


CI-1.

/1 Moxibustion/ C0NII+

CII+tlo
r CI-t, CIl+


CH3CILCON
ll CI ON HCHyCH+CII pcI
I tc Il+N tl G OCI-1+CI(z
N 11 CIItCHtCH*C112N II C
OCHN 11 COCI-1+N +-I COCT
-IN Hz formula (+) Structure of clavamine (
2) Joro 5pider (N)
1. A method for producing Clapamine or a salt thereof, which comprises crushing the venom glands of S. ephilaclavata as desired, and then extracting, isolating and purifying the venom glands.

(3) 2. -1-dihydroxyphenylacetyl group or 2.・Clavamine or a salt thereof, which is characterized in that a peptide fragment of clavamine having a 1-dihydroxyphenylacetyl group at the end is bonded to a peptide fragment corresponding to the remaining portion of clavamine, and the protective group is removed if necessary. manufacturing method.

(4) An insecticide composition comprising clavamine or a salt thereof and an agriculturally acceptable carrier.

Examples of the salt of compound (1) include salts with inorganic acids or organic acids. Examples of inorganic acid salts include hydrochloride, sulfate, carbonate, and nitrate. Examples of organic acid salts include formate,
Examples include acetate, propionate, sulfate, succinate, benzoate, valatoluene 7, sulfonate, and the like. Compound (1) may also be a complex salt with metals such as calcium, zinc, magnesium,
Examples include cobalt, copper, and iron.

In addition, the amino acid constituting clavamine represented by formula (+) may be any of the L-form, D-form, and DL-form, and the L-form is more preferable.

The compound of the present invention represented by the above formula (1) has a molecule of 792, and has a 2,4-dihydroxyphenylacetyl group followed by an asparaginyl group at one end, similar to other known substances obtained from the venom gland of the spider spider. , the other end is the N-terminus of arginine, followed by a glycyl group, an alanyl group, and an amino acid, which has a special structure in which they are connected via peptide bonds. In these respects, the clavamine of the present invention is disclosed in the above-mentioned Japanese Patent Application Laid-Open No. 60-18402.
It is clearly distinguished from the substance obtained from the venom glands of the spider spider disclosed in No. 1 and Japanese Patent Application No. 62-23147. It also differs in structure from the synthetic amide compound specifically disclosed in Japanese Patent Application No. 63-105080.

In addition, the clavamine of the present invention has the physical and chemical properties detailed below, and is largely distinguished from the above-mentioned known substances in that it does not exhibit a very high glutamate receptor inhibitory effect, but it has a strong It is a compound that has a unique biological activity of exhibiting insecticidal activity.

The method for producing clavamine or a salt thereof of the present invention will be described below.

The clavamine of the present invention can be obtained by extracting and purifying the venom glands of the spider spider.

In order to enhance the effect of extraction, it is preferable to add a crushing step prior to extraction.

Crushing can be carried out according to conventional methods. Examples include crushing with an atomizer, homogenizer, and ultrasonic waves. Usually, it is convenient to homogenize in a suitable solvent such as water, physiological saline, or a phosphate buffer solution with a pH of about 7.4.

For extraction, normal extraction methods can be applied. - For boat fishing, it is efficient to carry out homogenization in a solvent such as those mentioned above using a stirrer, centrifuge, etc. If a crushing treatment such as homogenization in a solvent is performed in advance, the extraction operation can be carried out directly, which is convenient. When using a centrifuge, the centrifugation speed is 1,000 to 100,000 revolutions/minute, and the centrifugation time is about 5 minutes to 3 hours.

Purification was performed using gel filtration (molecular sieves), purification using ion exchange resins, and silica gel thin layer chromatography (hereinafter referred to as T
This can be carried out using purification means such as LC (sometimes abbreviated as LC) and high performance liquid chromatography (hereinafter sometimes abbreviated as HPLC). Particularly preferred is molecular sieve and HPLC. Furthermore, since clavamine exists as a salt with metal ions, it is desirable to pass it through column chromatography using an SP-Sephadex column or the like to remove such metal ions. More specifically, for example, molecular sieves using Sephadex G-10 columns, MPG-ODS columns)
A combination of lPLc and sP-Sephadex column chromatography can be mentioned as a particularly suitable purification method.

The target product purified in this way can be analyzed by TLC or HPLC.
can confirm that it is a single substance. To give a specific method of such TLC, for example, the plate may be made of microcrystalline cellulose, silica gel, etc.
Solvents include propyl alcohol/water, phenol/
This can be done by using a mixed solvent such as water and detecting with fluorescamine or the like. By taking both components detected as a single spot on TLC, an isolated and purified substantially pure component can be obtained.

Clavamine, isolated and purified in this way and confirmed as a single substance, was analyzed by gas chromatography (hereinafter referred to as GC).
), Edman decomposition, Dancil (hereinafter referred to as D
When amino acid analysis is carried out by conversion (abbreviated as NS), etc., 'H-NMR and 13C-NMR (spectrometry, mass spectrometry (hereinafter abbreviated as MS)) are used.
The structure can be determined by analytical means such as

Specifically, the structure of clavamine was determined by first analyzing the hydrolyzate of the purified product by gas chromatography (GC).
．． 4-nohydroxyphenylacetic acid (DMA), alanine (Ala), aspartic acid (Asp), arginine (
The presence of putreanine (Arg) and putreanine (P at) was confirmed.

Next, by Edman degradation and dansyl (DNS) conversion, the amino acid sequence was converted to Arg-Gly-A
It was determined that la. Furthermore, it was estimated that Put-Cad-asparagine (Asn)-DHA was bound to this, and this assumption was supported by tH- and C-NMR spectrometry and mass spectrometry (MS).

It was thus revealed that clavamine has a structure with a molecular weight of 792 as shown in FIG.

Furthermore, various methods for chemically synthesizing clavamine having such a structure were also investigated.

Clavamine or its salt represented by formula (1) is usually
2,4-dihydroxyphenylacetyl group or 2,4
It is produced by linking a peptide fragment of clavamine having a -dihydroxyphenylacetyl group with a peptide fragment corresponding to the remaining portion of clavamine, and removing the protecting group if necessary.

That is, since the compound of the present invention has five peptide groups -CONH- in the molecule, focusing on any one of the peptide groups, the compound of the present invention has R-C
It can be represented by ON H-11', and the compound of the present invention may be prepared by combining RCO- and -N I-1-R'.

Here, R and R' respectively represent groups on both sides of the clavamine that sandwich the peptide bond of the clavamine of interest.

For example, by combining Gly-Arg-If or by combining Gly-Arg-If
Combine with Arg -If or Gly 4-A
Combine with rg-11 or Gly 4-Arg-I
I or Gly 4-Arg-H
It is manufactured by a method such as combining.

More specifically, the target compound of the present invention is RCOOH,
It is produced by condensing a reactive derivative thereof or a salt thereof with HzNR'.

(R and R' are as defined above.) Asn-4Cad 4-PLIL ←Ala 4-Gl
r-Arg-tl.

Ala-Gly　←＾rg-tl.

110-Put 4-Ala-Gly4-Arg-II
.

(B) 110-Ala4-Gly4-Arg-H,
Usually, clavamine is synthesized by reacting (A) 2,4-dihydroxyphenylacetic acid or
2. A compound having a 4-dihydroxyphenylacetyl group followed by a corresponding fragment of clavamine (hereinafter referred to as compound (A)) or a derivative thereof or a salt thereof and (B) the remaining portion of clavamine. A compound having a corresponding fragment (hereinafter referred to as compound (B)).

) or a derivative thereof or a salt thereof.

Compounds (A) and (I3) may have functional groups that do not participate in the condensation reaction for extending the peptide chain protected, if necessary.

When the above compounds (A) and (1113) have a protecting group, clavamine or a salt thereof can be obtained by removing the protecting group after the condensation reaction of compounds (A) and (B).

As such protecting groups, the protecting groups commonly used in the field of peptide synthesis can be applied here.

Among compounds (A) and (B), examples of the salt of the compound having a carboxyl group include inorganic base salts and organic base salts. Examples of inorganic base salts include alkali metal salts (eg, sodium salts, potassium salts, etc.), alkaline earth metal salts (eg, calcium salts, etc.). Compound (A).

Examples of the base salt of 115 in (I3) include trimedelamine salt, trimedelamine salt, tert-butyldimedylamine salt, cyclohexylamine salt, dibenzylmedylamine salt, hennoldimedylamine salt NN -Dimethylaniline salt, pyridine salt, 1-quinoline salt, etc. are exemplified.

On the other hand, examples of the salt of the compound containing an amino group among compounds (A) and (B) include salts with inorganic acids or organic acids. Inorganic acid salts include hydrochloride, hydrobromide, sulfate, nitrate, and phosphate, and organic acid salts include formate and acetate.

Examples include trifluoroacetate, methanesulfonate, and p-toluenesulfonate.

Among compounds (A) and (13), the carboxyl group of the compound that provides the carboxyl group that participates in the condensation reaction that extends the peptide chain may remain free, but by leading it to a reactive derivative thereof, C The terminal may be activated and subjected to the reaction.

The derivatives of the above compounds (A) and (B) mean compounds with protected functional groups and/or reactive derivatives of the carboxyl groups. Examples of reactive derivatives of compounds (A) and (B) include acid halides, acid at, acid anhydrides, mixed acid anhydrides, active amides, active esters, and active thioesters.

The acid halides of compounds (Δ) and ([3) include, for example, acid chlorides and acid bromides, and the mixed acid anhydrides include monoalkyl carbonic acid mixed acid anhydrides (for example, (A),
(B) and monomethyl carbonate, monoethyl carbonate, monoisopropyl carbonate, and monoisobutyl carbonate.

Mono-tert-butyl carbonate, monobenzyl carbonate, mono(
(mixed acid anhydrides with p-nitrobenol) carbonic acid, monoallyl carbonic acid, etc.), mixed acid anhydrides with aliphatic carboxylic acids (e.g. (A), (B) with acetic acid, trichloroacetic acid, anoacetic acid, propionic acid, butyric acid, isoacetic acid) Butyric acid.

mixed acid anhydrides with valeric acid, isovaleric acid, bivaric acid, trifluoroacetic acid, trichloroacetic acid, acetoacetic acid, etc.), mixed acid anhydrides of aromatic carboxylic acids (e.g. (A), (B)
and mixed acid anhydrides of benzoic acid, p-toluic acid, p-chlorobenzoic acid, etc.), mixed acid anhydrides of organic sulfonic acids (for example, (A), (B) and methanesulfonic acid, ethanesulfonic acid, benzenesulfone) Examples of active amides include amides with nitrogen-containing heterocyclic compounds (e.g. (A), (B)).
These nitrogen-containing heterocyclic compounds include alkyl groups, alkoxy groups, halogen atoms, and oxo groups.

(which may have a substituent such as a thioxo group or an alkylthio group) are exemplified. As active esters (A) and (B), all those used for this purpose in the field of peptide synthesis can be used, and organic phosphates (e.g., jetoxyphosphate, diphenoxyphosphate, etc.) are preferred. Phenyl ester, 2.4-
Dinitrophenyl ester, cyanomethyl ester, pentachlorophenyl ester, N-hydroxyphthalimide ester, N-hydroxyphthalimide ester,
l-hydroxybenzotriazole ester, 6-chloro-1-hydroxybenzotriazole ester, 1-
Examples include hydroxy-IH-2 to pyridone ester. The active thioesters (A) and (B) include esters with aromatic heterocyclic thiol compounds (for example, 2-pyridylthiol ester, 2-benzothiazolylthiol ester, etc., where these heterocycles are alkyl groups, alkoxy groups, etc.). , which may have a substituent such as a halogen atom or an alkylthio group).

2g of hydroxyl groups on the benzene ring of the starting compound (A) may be protected. Protecting groups include substituted or unsubstituted alkanoyl groups (e.g. acetyl, propionyl,
trifluoroacetyl), substituted oxycarbonyl groups (e.g. methoxycarbonyl, ethoxycarbonyl,
isopropoxycarbonyl, tert-butoxycarbonyl, fenoquinecarbonyl, benzyloxycarbonyl, p-methylbenzyloxycarbonyl, benzhydryloxycarbonyl, etc.), 1ert-butyl group, aralkyl JJ (e.g. benzyl, p-methylbenzyl, p-methoxybenzyl, p-chlorobenzyl.

Examples include benzhydryl, trityl, etc.), substituted silyl groups (eg, trimethylsilyl, tert-butyldimethylsilyl, etc.).

The imino group of the starting compound (B) is protected.

good. Protecting groups for imino groups include substituted or unsubstituted alkanoyl groups (eg, pormylacetyl, monochloroacetyl, trichloroacetyl).

monoiodoacetyl, 3-oxobutyryl, p-chlorophenylacetyl, p-chlorophenoxyacetyl, etc.), aromatic carbonyl groups (such as benzoyl, p-t
ert-butylbenzoyl, etc.), substituted oxycarbonyl groups (methoxycarbonyl, ethquinocarbonyl, t
ert-butoxycarbonyl, benzyloxycarbonyl, p-chlorobenoloxycarbonyl, p-methylbenzyloxycarbonyl, penzhydryloxycarbonyl, etc.).

When producing a salt or a reactive derivative thereof from (A) or (B) or introducing a protecting group therein, it can be easily carried out using a known method or a method analogous thereto. In the reaction between compound (A) and compound (B), for example, a reactive derivative of starting compound (A) may be reacted with compound (I3) as a substance isolated from the reaction mixture, or as a substance isolated from the reaction mixture. The reaction mixture containing the reactive derivative of the starting material compound (A) may be directly reacted with the compound (B). When the carboxyl group-providing compound of compounds (A) and (B) is reacted with the other compound in the form of a free acid or salt, a suitable condensing agent is used.

Examples of condensing agents include N,N'-disubstituted carbodiimides such as N,N'-dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole, N,
Azolides such as N'-thiocarbonyldiimidazole, e.g. N-ethoxycarbonyl-2-ethoxy-
Dehydrating agents such as 1,2-dihydroquinoline, phosphorus oxychloride, and alkoxyacetylene, and 2-halogenopyridinium salts such as 2-chloropyridinium methyl iodide and 2-fluoropyridinium methyl iodide are used. When these condensing agents are used, the reaction is thought to proceed via a reactive derivative of compound (A) or (B).

The reaction between compound (A) and compound (B) is generally carried out in a solvent, and a solvent that does not inhibit the reaction is appropriately selected.

Examples of such solvents include dioxane, tetrahydrofuran, and diethyl ether.

Ethers such as tert-butyl methyl ether, diisopropyl ether, ethylene glycol dimethyl ether, esters such as ethyl formate, ethyl acetate, butyl acetate, halogens such as dichloromethane, chloroform, carbon tetrachloride, trichlene, l,2-dichloroethane, etc. Hydrocarbons such as hexane, benzene, toluene, etc., such as formamide, N.

Amides such as N-dimethylformamide, N,N-dimethylacetamide, e.g. acetone.

Methyl ethyl ketone. In addition to ketones such as methyl isobutyl ketone, nitriles such as acetonitrile and propionitrile, dimethyl sulfoxide,
Sulfolane, hexamethylphosphoramide, water, etc. are used alone or as a mixed solvent. Compound (A), (
Among B), the amount of the compound in which an amino group participates in the condensation reaction is usually + to 5 mol per 1 mol of the other compound.

Preferably it is 1 to 3 moles. Reaction is -80 to 80°C
1 Preferably -40 to 50°C, most preferably -30 to
It is carried out at a temperature of 30°C. The reaction time is compound (A)
The reaction time is usually 1 minute to 72 hours, preferably 15 minutes to 24 hours, depending on the type of compound (B), the type of solvent (and the mixing ratio in the case of a mixed solvent), and the reaction temperature. When the acid halide of compound (A) or (B) is used, the reaction can be carried out in the presence of a deoxidizing agent for the purpose of removing released hydrogen halide from the reaction system. Examples of such deoxidizers include inorganic bases such as sodium carbonate, potassium carbonate, calcium carbonate, and sodium bicarbonate, such as triethylamine and tripropylamine.

Tributylamine, diisopropylethylamine.

cyclohexyltmethylamine, pyridine, lutidine,
γ-Corinone, N,N-dimethylaniline.

N-methylpipedilline, N-methylpyrrolinone.

Examples of N-methylmorpholine include alkylene oxides such as propylene oxide and epichlorohydrin.

Compound (A) and compound (B) are reacted as described above.
It was also. The target compound (1) of the present invention can be obtained by removing the protecting group and purifying if necessary.

For example, methods commonly used in the field of peptide synthesis can be used as is for removing the protecting group. For example, methoxycarbonyl, ethoxycarbonyl, tert-butquine carbonyl, phenoxycarbonyl, etc. can be converted to benzyloxycarbonyl by acid (eg, hydrochloric acid or trifluoroacetic acid).

p-Methylbenzyloxycarbonyl, benzhydryloxycarbonyl, etc. can be converted to benzyl, p-methylbenzyloxycarbonyl, etc. by catalytic reduction.
-Methylbenzyl, p-methoxybenzyl, p-chlorobenzyl, benzhydryl, tridel, etc. by acid (e.g. trifluoroacetic acid) or catalytic reduction; trimethylsilyl, ter'j-butyldimethylsilyl, etc. by water alone or in the presence of acetic acid. Can be detached.

When removing the above-mentioned protecting group, after isolating compound (1) with protected hydroxyl group, amino group, (mino group) from the reaction mixture obtained from the reaction of compound (A) and compound (B), The protecting group may be removed or the reaction mixture may be used as it is.

Hydroxyl group. Amino group. The imino group-protected compound (A) and the target compound (A) of the present invention are purified by an extraction method.

Gel filtration. ion exchange resin column chromatography,
This can be carried out using known purification methods such as silica gel preparative thin layer chromatography, high performance liquid chromatography, and recrystallization. Compound (A) and compound (B) can be prepared manually by a known method or a method analogous thereto.

For example, starting from a known compound (hereinafter referred to as compound (2)) represented by the formula (in the formula, -Bzσ represents a benzyl group, Z- represents a benzyloxycarbonyl group, and other symbols are as defined above). Preferred specific methods for the substance include a production method that extends a peptide chain.

Compound (2) used as a starting material in the above method is Tetrahedron Letters 28. 3509-3510
(1987).

However, although starting material (2) has a benzyl group and a benzyloxocarbonyl group protecting the hydroxyl group and the amino group, it is not limited to these protecting groups, and other protecting groups commonly used in peptide synthesis may be used. Needless to say, they can be replaced.

For example, in the case of a production method using the above compound (2) as a starting material, a compound having a peptide fragment of the optionally protected amino acid sequence A la - G Iy - Arg in compound (2) or a salt thereof, for example. Alternatively, compound (2) or a salt thereof may be condensed with alanine or a derivative thereof or a salt thereof, and this product may be condensed with glycine or a derivative thereof or a salt thereof. Furthermore, clavamine can also be produced by condensing the product with arginine, a derivative thereof, or a salt thereof. In this case, functional groups of alanine, glycine, arginine, or peptide fragments thereof that do not participate in peptide chain extension are usually protected as described above before being subjected to the reaction. The protecting group is
Again, a variety of protecting groups commonly used in the field of peptide synthesis may be selected. In particular, a tert-butquine carbonyl group or a benzyloxycarbonyl group is generally preferred for an amino group, and a benzyl group is generally preferred for a hydroxyl group.

Similar to the protecting group of compound (2), the protecting group of the above amino acid and peptide fragment can be removed by the method described above whenever the reaction is completed. In particular, trifluoroacetic acid is used for the protecting group of the tert-butoxycarbonyl group, and C ss O *H C
It is usually a preferred embodiment to remove each protecting group by using a F, C O O H system or the like.

Generally, when trifluoroacetic acid or the like is used, this can be carried out by dissolving or suspending the solution in the trifluoroacetic acid and leaving it to stand or stirring.

On the other hand, in such a condensation reaction that extends the peptide chain, the carboxyl group can be left free as described above or reacted by leading it to a reactive derivative as described above. Among the above-mentioned reactive derivatives at the C-terminus, examples include mixed acid anhydrides with carbonic acid monoalkyl esters, active esters such as p-nitrophenyl ester, N-phthalimide ester, N-oxysuccinimide ester, etc. is mentioned as a preferable example.

A specific method for producing clavamine using compound (2) as a starting material includes, for example, a route as shown in the following formula.

In the formula, Cat, Put, Asn, Ala, Gly, Ar
g, Bz12゜Z is 0 meaning as written, Boc is,
tert-butoquinocarbonyl group, O9u represents succinimidoxy group, and TFA represents trifluoroacetic acid.

Taking the synthesis route shown in FIG. 2 as an example, the manufacturing method will be specifically described below.

(a) Step The reaction from compound (2) to compound (3) can be carried out according to the conditions for carrying out condensation reactions in the field of peptide synthesis, as described above. Specifically, the condensation reaction of compounds (A) and (B) can be carried out under the conditions described above.

For example, compound (2) is dissolved in a reaction-inert solvent such as DMF or DMSO, and an alanine derivative (usually the N-terminus is protected with an amino protecting group and the C-terminus is protected as described above) together with a base such as triethylamine. Those derived from reactive derivatives are preferred.

For example, Boc-Ala-OS u) can be added and stirred. The reaction temperature is approximately -4
The temperature is 0 to 50°C, preferably about 00C to 30°C, and the reaction time is about 15 minutes to 24 hours.

The ratio of compound (2) and alanine derivative used is approximately 1:1.
The molar ratio is ~1:3, preferably about 1:1-1:2.

(b) Step The reaction from compound (3) to compound (4) involves selectively removing the N-terminal protecting group of compound (3) using the method described above, and combining the resulting product with a glycine derivative (usually It is preferable that the N-terminus is protected with an amino protecting group, and the C-terminus is derived from a reactive derivative as described in "-". For example, Boc-
This step is carried out by condensing G ly -OS u) under the same conditions as in step (a).

(c) Step The N-terminal protecting group is removed from compound (4) by the method described above, and the resulting product and arginine or its derivative (usually, the amino group and imino group are protected with the respective protecting group, Furthermore, it is preferable that the C-terminus is guided to a reactive derivative. For example, Z-Arg(Z2)-08+(CH3)t
cHcH,0COCI2 reaction mixture) from -40 to 5
Approximately 30 minutes to 50 minutes at 0°C, preferably -30 to 30°C
Compound (5) is usually obtained by reacting for about a period of time.

(d) Step Clavamine, which is the object compound of the present invention, can be obtained by removing the protecting group of compound (5) according to the protecting group removal method as described above.

The method for producing clavamine has been specifically explained using the synthetic route shown in Figure 2 as an example. By combining, in short, a peptide chain of 2゜4-dihydroquinphenylacetic acid or a peptide fragment having a 2゜4-dihydrogynephenylacetyl group at the terminal and then an amino acid sequence of clavamine, a derivative thereof, or a salt thereof. Clavamine or its salt can be produced by extending the process.

The in vivo bioassay of clavamine or its salts was carried out by releasing young larvae of C. chinensis into an aqueous solution containing both components of each purification step and observing the mortality rate after 3 hours.

For more information, please refer to Culex mosqu
10 heads of 1st to 2nd instar larvae of Ito were placed in an ice cream cup (50d). The mice were released into a sample solution, and the mortality rate was measured after 3 hours. Regarding clavamine, a sample solution containing an amount equivalent to five poison glands showed a 100% mortality rate. Hereinafter, the activity equivalent to one poison gland was determined as 1 unit (U).

Clavamine or a salt thereof of the present invention has insecticidal activity and can be used as an insecticide.

When using the clavamine or its salt of the present invention as an insecticide, the form that can be taken by general agricultural chemicals, that is, clavamine or its salt is dissolved or dispersed in a suitable liquid carrier depending on the purpose of use, and a suitable solid carrier is used. Mixed with a carrier or adsorbed, emulsion.

It is used in the following dosage forms: oil, wettable powder, powder (1 granule, 3 tablets), spray, ointment, etc. These preparations may contain, if necessary, emulsifiers, suspending agents, spreading agents, penetrating agents, wetting agents, stabilizers, etc., and can be prepared by methods known per se.

The content ratio of active ingredients in insecticides varies depending on the purpose of use, but for emulsions, wettable powders, etc., the appropriate content is about 10 to 90% by weight, and for oils, powders, etc., it is about 0.1% to 10% by weight. The appropriate concentration for granules is about 1% to 20% by weight, but these concentrations may be changed as appropriate depending on the purpose of use. When using emulsions, hydrating agents, etc.
Dilute with water, etc. to increase the amount (for example, 100 to 100,000)
(double) and disperse.

Examples of liquid carriers (solvents) used include water, alcohols (e.g., methyl alcohol, ethyl alcohol, n-propyl alcohol, isopropyl alcohol, ethylene glycol, etc.), ketones (e.g., acetone, methyl ethyl ketone, etc.), and ethers. (e.g., dioxane, tetrahydrofuran, ethylene glycol monomethyl ether, diethylene glycol monomethyl ether, propylene glycol monomethyl ether, etc.), aliphatic hydrocarbons (e.g., Keroane,
Toura, fuel oil 1 machine oil, etc.), aromatic hydrocarbons (for example, henzhen, toluene, kinshin, solvent naphtha, methylnaphthalene, etc.), halogenated hydrocarbons (
(e.g., methylene chloride, chloroform, carbon tetrachloride, etc.), acid amides (e.g., dimethylformamide, dimethylacetamide, etc.), esters (e.g., ethyl acetate, butyl acetate, etc.).

Solvents such as fatty acid glycerol ester, etc.) and nitriles (for example, acetonitrile, propionitrile, etc.) are suitable, and these solvents may be used alone or in combination of two or more in an appropriate ratio.

Solid carriers (diluents and bulking agents) include vegetable powders (eg, soybean flour, tobacco flour, wheat flour, wood flour, etc.), mineral powders (eg, kaolin, bentonite).

Clays such as acid clay, talcs such as talc and waxite powder, silicas such as diatom powder and mica powder, etc.), alumina, sulfur powder, activated carbon, etc. are used, and one or more of these may be used as appropriate. Mix and use in appropriate proportions.

Examples of ointment bases include polyethylene glycol, pectin, polyhydric alcohol esters of higher fatty acids such as monostearic acid glycerol ester, cellulose derivatives such as methyl cellulose, sodium alginate, bentonite, higher alcohols such as glycerin, etc. Alcohol, petrolatum, white petrolatum, liquid! (One or more of rough-in, lard, various vegetable oils, lanolin, dehydrated lanolin hardened oil, resins, etc., or a mixture of these with various surfactants shown below may be used.)

Emulsifier, exhibition? Surfactants used as surfactants, penetrants, dispersants, etc. may include soaps, polyoxene ethylene alkyl aryl ethers [e.g., Neugen, E.N. 142 (E-AI4'2)] as necessary. Nonal ■ manufactured by Daiichi Kogyo Seiyaku Co., Ltd., manufactured by Toho Chemical Co., Ltd.], alkyl sulfates [eg. -, +, 4
0. Manufactured by Kao (K)], 7'tL4>L=X2. acid salts [e.g., Neogea ■, Neoge:/T o, manufactured by Daiichi Kogyo Seiyaku (Maji) 2 Neopelle Sox ■ manufactured by Kao Corporation''.

Polyethylene glycol ethers [e.g., Nonibo->L
=85”, Tsuyubo/L=lOOo,/Yus!-/L.

160 ■, manufactured by Sanyo Chemical Co., Ltd.], polyvalent A7 record S-/u JF! [(@I1. Nonionic and anionic surfactants such as Wee, 2oO, and Weeer 800 manufactured by Kao Corporation are used.

Furthermore, the compounds of the present invention may be used with other types of insecticides (pyrethrin insecticides, phosphorus insecticides, carbamate insecticides, natural insecticides, etc.), acaricides, nematicides, herbicides, and plant hormones. , ltibiotic growth regulators, disinfectants (e.g., copper-based disinfectants, organochlorine-based disinfectants, organic acid-based disinfectants, phenol-based disinfectants, etc.), synergists.

It is also possible to mix and use attractants, repellents, pigments, fertilizers, etc.

Effects of the Invention The clavamine of the present invention can be extracted and purified from the venom glands of the spider spider or synthesized, and can be obtained by the following i.
It has insecticidal activity as shown by the results of n vivo bioassay. Furthermore, the insecticidal activity of lavamine is further enhanced by metal ions. .

Accordingly, clavamine or its salts can provide insecticidal compositions.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited to these examples.

Example 1 (1) Crushing and water extraction of poison glands 4280 N. clavata were collected from the mountains and fields of the Kinki region, and two pairs of poison glands were taken out from each. Poison gland wet weight 4
．． 18 M1 of water was added to 5 g and heat treated at 100° C. for 2 minutes. It was homogenized by crushing it with a glass rod. This solution was centrifuged at 1000 Orpm for 10 minutes. 1Bml of supernatant was obtained. This supernatant has a dry weight of 300 m
There was g. The specific activity was 9.60/mg. By repeating this operation, an extract with a total dry weight of 1 g was obtained.

(2) Molecular sieve using Sephadex G-10 (
Transfer an amount of supernatant equivalent to one batch of step 1) to Sephadex G.
-10 (1.7 cm x 50 cm) column.

Water was flowed at a flow rate of 0.7 J/min. The eluent was 25
The absorbance at 4 nm was monitored to obtain a chromatogram as shown in FIG. It was found that the main active ingredient was present in the second peak. This component represented 76% of the total activity. This molecular sieve was operated again to obtain a total amount of 46 mg. This specific activity was I 63 U/mg.

(3) Using an MPG-ODS column [(PLC) Dissolve 10 mg of the active fraction obtained in the above procedure (2) in 1001 tQ of water, and apply it to an MPG ODS column (21.5 mm x 30
cm). 0.02% I]CQ-acetonitrile (4:I, v/v) mixture was flowed at a rate of 5.5 d/min. The eluate was monitored by absorbance at 214 nm. The active ingredient was present in both fractions corresponding to the first peak appearing on the chromatogram in FIG. This operation was repeated four times. The recovery rate from the aqueous extract was 69%, and the activity per dry weight was 574 U/mg. A dry weight of 12 mg was obtained.

(4) SP-Sephadex Column Chromatography - The aqueous solution of 12 mg of the purified product obtained in (3) above was converted into a metal salt, so 10 mg of it was added to an SP-Sephadex 7 deck. It was passed through a (Type II) column (1.5 cm x 2 cm) and concentrated to dryness. This was used as the final purified product. Approximately 9 mg of worms were obtained.

2 t + mol of the final purified product of (4) above was spotted on TLC microcrystalline cellulose (Tokyo Kasei Kogyo) of Cneyvvv and developed with n-propanol-water (7:3, v/v). When fluorescamine was sprayed and detected using a fluorescent spot, there was only a single spot as shown in Figure 3, confirming the presence of an amino group.

(6) HPLC of purified product 25 pmol of the final purified product from (4) above was added to MPG at 25°C.
-ODS column (4.6 mm x 150 cm) and eluted with an eluent containing 30 mM pentanesulfonic acid and 5% acetonitrile at 0.7+J/min. This solution contains 0
．． The ortho-phthalaldehyde solution was reacted in a conventional manner at 7+J/min and the fluorescence intensity was monitored. A single peak was shown as shown in FIG.

(7) GC of hydrolyzate of purified product 25 nmol calculated based on the molecular absorption coefficient of 3500 at 278 nm of the final purified product of (4) above is 105
Hydrolyzed with GNHCC for 0.16 hours.

After drying the hydrochloric acid, P, 0. It was further dried in a desiccator.

To this, 100 μQ of a mixed solution of isobutanol-3NHCQ (Gas Kuro Kogyo) was added, replaced with N, and heated at 110°C.
Heated for 30 minutes. After cooling and drying at room temperature, 25 μQ of ethyl acetate containing 5 μg of ethoxyformic anhydride and hebutafluorobutyric acid were added, replaced with N, and heated at +10° C. for 10 minutes. The reaction solution was dried and dissolved in 50 μσ ethyl acetate.
．． Gas chromato graph of 5μQ under the following conditions
Injected into dzu GC-8A. The temperature of the 0V-101 column (0.25 mm x 50 cm) was 220°C. The temperature of the vaporization chamber and detector was 270°C. The pressure of the carrier gas N was 2 kg/am''. Detection was performed using a flame ionization detector. The split ratio for injection into the column was 1=80. The chromatogram is shown in Figure 5. From the chromatogram, glycine (Gly)
.

The presence of cadaverine (Cad), 2,4-dihydroxyphenylacetic acid (DI-(Δ), alanine (Ala), aspartic acid (Asp), arginine (Arg), and butreanine (Put)) was confirmed.

Using 35 nmol of the purified product from (4) above, Edman degradation and DNS conversion were carried out in a conventional manner, and spots appearing in each degradation step were observed by TLC on 6N-HCQ polyamide. If Edman decomposition is not performed, the N end is Ar.
g1 In the first Edman degradation, cty appeared, and in the second time, Ala appeared, and then DHA appeared sequentially.

Put, Cad and Asp appeared. From this, the structure of spider venom, whose structure was previously determined, is A rg −G ly
The structure of formula (1) in which -A la is bonded was estimated.

(9) 'H-NMR-spectrometry of purified product The purified product from (4) above was dissolved in 0.5 dD20, and then two-dimensional 'H-N! The VIR spectrum was measured with a NM (1-Spectometer JNM-GX400 (JEOL Ltd.). The two-dimensional spectrum is shown in FIG. 6.

(10) Mass spectrometry (MS) of purified product Mass spectrometry (MS) of the purified product in (4) above
, 3000 (JEOL Ltd.) using triethanolamine as a matrix, the spectrum of high-speed atom collision FAB mass spectrometry is shown in FIG. In this way, (M++ +-t = 793) was observed.

Example 2 (100 mg, O, I l 3 mmol) was dissolved in DMF (l
Triethylamine (13.7 mg,
0.136 mmol) and Boc-^1a-O3u
(64.8 mg, 0.226 mmol) was added and stirred at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure, and ethyl acetate and water were added to the resulting oil to solidify it. Filter,
Washed with ethyl acetate and then water. Accommodation ff189.6m
g (JO, 0%) (69.6 mg, 70.0 μmol)
was dissolved in TF'A (21n1) and stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure, and ether was added to the obtained curved product to solidify it. Yield: 61.6 mg This solid was dissolved in DMF (8d) and triethylamine (
7.42 mg, 73.3 μmol) and Boc-Gly
-O3u (30.8 mg, 122 μmol) was added and stirred at room temperature for 20 hours. After concentrating the reaction solution under reduced pressure,
It solidified when ethyl acetate and water were added. It was filtered and washed with ethyl acetate and then with water. Yield 34.2 mg (46.9%
) Arg(Zz) -Z (5) (4) (30.0 mg, 28.5 μmol) in TFA
(Id) and stirred at room temperature for 35 minutes. The reaction solution was concentrated under reduced pressure, and ether was added to the obtained curved material to solidify it. The yield was 21.9+ng.
triethylamine (1.8 mg, 18 μm) dissolved in
ol) was added and cooled to -20°C (solution A). Separately ZA
rg(Z2)-OH (21.2 mg, 36.8 μmol) was dissolved in anhydrous tetrahydrofuran (LYE), and triethylamine (3.7 mg, 37 μmol) and isobutyl chloroformate (5.0 mg, 37 μmol) were dissolved at -20°C.
and stirred for 10 minutes. This solution was added to the previously prepared solution A at -20°C, stirred for 1 hour, and then stirred at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure, and ethyl acetate and water were added to the obtained curved product to solidify it. It was filtered and washed with ethyl acetate and then with water. Yield: 18.4 mg (48.7%) 11.3TFA (+) Dissolved in fluoromethanesulfonic acid-TFA-m-talesol-dioanisole (5:20:6:60285μσ) and stirred at 0°C for 1 hour. did. When ether was added to the reaction solution, a precipitate was obtained. This was dissolved in water and washed three times with ether, and the aqueous layer was purified by high performance liquid chromatography. (Cosmoseal 5 G,,, 8 x 25
0mm, 0.1% TFA-acetonitrile, 0-30%
(15 minutes), 30-60% (5 minutes) retention time 15,0.
Detection wavelength: 220 nm) Yield: 4.6 mg (41%) Amino acid analysis: Asp: Q, 94. Gly: 1
．． Q2. Ala:1.02. Put: 1.02.
Arg: 1.0ONMR: 1.1g (m, 2H
), 1.36 (d, 3B), 1.44 (m, 411).

1.56 (m, 211), 1.67 (m, 4H
), 1.93(m, 211), 2.63(t
, 2H), 2.73(ddd, 211), 3
．． 05(t, 2H), 3.10(+n, 4H
), 3.21 (m.

411), 3.26ct, 2H), 3.52(dd,
2+1), 4.01 (d, 2+1), 4.0
6 (t, LH), 4.24 (q, 1tl), 4.60 (
m, 1it), 6.44 (m, 211) 7.08 (d,
(Chemical shift with IHXIIOD as δ 6.70)
All of the amino acid fragments of clavamine synthesized in this example are five in number.

TLCI' (f value) of the composite product thus obtained.

The HPLC retention time, MS, and NMR spectra were completely consistent with those of the purified product, and furthermore, the GC of the hydrolyzate of the synthetic product
The results were completely consistent with those of the purified hydrolyzate.

Insecticidal Effect Test Example The insecticidal effect of purified and synthesized lavamine and the effect of metal ions were investigated.

Assuming that 1 X 1 O-'M of clavamine is present in one venom gland, that is, IU, 0.5 each of 3 X 1 O-'M purified and synthesized lavamine liquid or venom gland aqueous extract was prepared. Furthermore, an equimolar amount of metal ion 3 is added to these liquids.
A solution containing x10°0M was prepared. The pH of the 6th solution was adjusted to 7.0 with 1.6M sodium citrate. This solution was further diluted to the same extent and the insecticidal effect against the mosquito mosquito was measured.

The results are shown in Table 1.

As shown in Table 1, purified and synthesized lavamine exhibited insecticidal activity. Ca'“ is a metal ion contained in poison glands.
It was found that Pb2+ has an enhancing effect. Also, Fe
A weak enhancing effect was observed in 3+ chives.

Table 1 Purification and synthesis of Chikayeka Mortality rate for Rabamine Synthetic product Mg” 0 0

[Brief explanation of the drawing]

Fig. 1 shows a chromatogram obtained when an extract from the venom gland of the spider spider containing clavamine was passed through a Sephadex G-10 molecular sieve. Figure 2 shows the active fraction passed through the above molecular sieve, ~IPG
- Shows a chromatogram when applied to HPLC using an ODS column. In addition, 0.02% lICO,-acetonitrile (421v
/v) was changed to 0.02% HCσ-acetonitrile (1:l, v/v) at the point of the arrow ■. Also arrow■
At this point, a solution of 0.02% I-I CQ in acetonitrile was used. Figure 3 shows TLC of the final purified product of clavamine.
The chromatogram is shown below. FIG. 4 shows the HPLC chromatogram of the final purified clavamine. FIG. 5 shows a GC chromatogram of the HC & hydrolyzate derivatives of the final purified product of clavamine. Figure 6 shows the two-dimensional 'H-CO3Y-N of the final purified product of clavamine.
An MR spectrum is shown. FIG. 7 shows the FAB-mass spectrum of the final purified product of clavamine. 254nml plastic (da 6 friends)

Claims (4)

[Claims] (1) Formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ Clavamine or its salt represented by (2) Joro spider (Nep)
2. The method for producing clavamine or a salt thereof according to claim 1, wherein the venom glands of C. hilaclavata are crushed if desired, and then extracted from the venom glands and isolated and purified. (3) 2,4-dihydroxyphenylacetyl group or 2
, 4-dihydroxyphenylacetyl group at the end, and a peptide fragment corresponding to the remaining part of the clavamine, and if necessary, the protective group is removed. . (4) An insecticide composition comprising the compound according to claim 1 and an agriculturally acceptable carrier.