JPH01294734A - Basic amide compound - Google Patents

Abstract

NEW MATERIAL:A compound of the formula (wherein m, p and q are each 0 or 1, n is 0-4, R1 is H or an acyl group, and R2 is a hydroxy or amino group). USE:A new basic amide compound composed of a basic amino acid or polyamine having a histamine releasing activity. PREPARATION:Poison glands of silk spiders are extracted with a mixed solvent of a dilute aqueous acid solution (e.g., aqueous trifluoroacetic acid solution) with an organic solvent (e.g., acetonitrile), and the extract is purified with a high-performance chromatography of excellent resolution.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to a novel basic amide compound composed of a basic amino acid and a polyamine having histamine-releasing activity.

(b) Prior Art Spider venom is known to exhibit glutamate receptor inhibiting action. The inventor has focused on the glutamate receptor inhibitory effect of the spider venom, and has conducted research such as isolation of its active substance, elucidation of its chemical structure, and structure-activity relationship. As a result, the glutamate receptor inhibitor JSTX.

N5TX was isolated and its chemical structure determined [Y.

Aramaki et at, Proc, Jpn
, Acad,, Set, B, 62°359 (198
6), Y, Aramaki et at,, Bio
med, Res, 8°167 (1987), Y, A
ramaki et at,, 1 bid,, 8,2
41° (1987).

(c) Problems to be Solved by the Invention However, biological activities other than glutamate receptor inhibitory action have not been investigated, and no other physiologically active substances have been found.

(d) Means for Solving the Problems As a result of examining other biological activities of Negro spider venom, the present inventor found that the crude extract of venom glands had remarkable histamine-releasing activity and neurotransmission-blocking activity, and isolated this active substance. , succeeded in determining its chemical structure.

Thus, according to the present invention, there is provided a basic amide compound represented by the general formula % (where % represents a hydrogen atom or an acyl group, and Rt represents a hydroxy or amino group).

As is clear from the above general formula, the compound of this invention has (ornithine) Ill-aspartic acid or asparagine-kataverine-(pretoanine) n-(ornithine) p-(arginine) q as a constituent component. Preferred compounds include, when m=1, 1) n=3. p=0. q=0 or i
ii) n = 2, p = 0, q =
0 and m=0, then 1) n=0. p=l, q=
1; 1i) n-1, p=l, q=l, 1ii) n=1
．． p=0. q=0; iv) n engineering 3. p=0.
q=o, and v) n=2. An example is a combination of p=0 and q=0.

R1 is a hydrogen atom or indole-3-acetyl,
Acyl groups such as 6-hydroxyindole-3-acetyl and 2,4-dihydroxyphenylacetyl are preferred.

Next, a method for producing the compound of this invention will be explained. The compound of this invention is basically a Nephi spider.
It can be obtained from the extraction and purification steps from the venom glands of S. la clavata.

Extraction is performed using a mixed solvent of a dilute aqueous acid solution and an organic solvent. At this time, the poison glands may be left standing in the mixed solvent for a long time, or may be carried out in a short time using a stirrer or a centrifuge. As the dilute acid aqueous solution, 0.5% or less trifluoroacetic acid, trichloroacetic acid, acetic acid, 0.05% or less hydrochloric acid, etc. can be used. As the organic solvent, alcohols such as methanol and ethanol, and nitriles such as acetonitrile can be used. The mixing ratio of organic solvent to dilute acid aqueous solution is 70
% (V8) or less.

Since the extract contains multiple active ingredients, high performance liquid chromatography, which has excellent separation ability, is used to purify the extract. As the column, a reverse phase type column or a cation exchange type column can be used. It is also possible to purify by using these columns in combination. The solvent used for purification with a reversed-phase column may be the solvent used for extracting the poisonous component, but acetonitrile is the most desirable organic solvent. When using a cation exchange column, pi (=6.5 to 7.5
The components are adsorbed using a sodium phosphate buffer, and each component is separated and eluted using a concentration gradient of a salt such as sodium chloride. The poisonous components in the eluent were detected using an ultraviolet detector at 210n.
It is obtained by measuring the absorbance at m to 28 Or+m and fractionating the eluted portion corresponding to each peak.

The purified component can be confirmed to be a single compound by means such as high performance liquid chromatography, H-layer chromatography, and electrophoresis. The chemical structure of each component is determined by fragmentation due to acid partial decomposition, amino acid and polyamine analysis, and measurement of NMR spectrum, mass spectrum, IR spectrum, and U* spectrum. Compound activity is assayed at each stage of purification by examining histamine release from rat intraperitoneal mast cells.

The assay method will be explained below. Rat is Wistar, 30
Use 0-350g. After decapitating and exsanguinating the rat under ether anesthesia, mast cell medium (
Abbreviated as MCM; 150mM NaCl.

3.7d KCl, 3. OmM NatHPO,,
3.5mM Kl, PO.

0.9mM esclg, 0.1% glucose, 0,
2% bovine serum albumin) 20! ! Inject Q and massage for 1-2 minutes.

Then, the MCM suspension of mast cells was removed from the peritoneal cavity.
Centrifuge at 1100 rpm for 5 minutes at 4°C.

The pellet is further washed 2-3 times with 4x (l) of MCM and used for activity assay.
This 20μρ was suspended in MCM to a concentration of 0.9%
Add to 20 μρ of the sample dissolved in NaCl aqueous solution,
℃ for 5 minutes. After that, water cooled and 4000 rpm.
55% trichloroacetic acid is added to 30 μg of the supernatant obtained by centrifugation at 5000 rpm for 5 minutes, stirred, and centrifuged at 110,000 rpm for 5 minutes to quantify histamine in the supernatant. Histamine quantification is performed by high performance liquid chromatography using the 0PA-post-column detection method.

The activity of each sample was determined by 0.1% Triton X-10
The amount of histamine when treated as described above is 100 and is expressed as a percentage. The compounds of this invention exhibit significant histamine-releasing activity by this assay.

Similarly, mastoparan, a peptide in bee venom that exhibits histamine-releasing activity, is rich in basic amino acids, and the amino group in this side chain is thought to play an important role in expressing the activity ( Y, Hirai et a
l,, CheIIp, Phara+, Bull,
, 27°1942 (1979), K, Takam
Atsu et al., FEBS, Lett.

Month 4.123 (1983)).

The compound of this invention is also composed of a basic amino acid and a polyamine, and is considered to exhibit strong basicity. Spider venom is obtained with a hydrophobic substituent attached to this basic moiety, but this substituent can be cleaved by hydrolysis with an acid to obtain only the hydrophilic and basic moiety. This moiety also exhibits histamine-releasing activity like the other compounds, and the histamine-releasing activity of multiple compounds with the same substituent group is determined by the strength of basicity, which can be judged by the strength of adsorption to the cation exchange resin. Since this is almost parallel to the above, it is thought that the part consisting of basic amino acids and polyamines is important for the histamine-releasing activity of the present compound.

In addition, in the state purified from spider venom, the substituent (R8)
However, since it has a partial structure that is unstable to oxidation, such as an indole skeleton, it is possible to obtain a more stable compound without losing activity by cutting this and then introducing a more stable acyl group. be.

As an acyl group, an alkanoyl such as acetyl, propionyl, butyryl, hexanoyl, heptanoyl;
Arylcarbonyl such as benzoyl, naphthalenecarbonyl; methoxycarbonyl, ethoxycarbonyl,
Examples include acyl groups derived from organic carboxylic acids such as alkoxycarbonyl such as butoxycarbonyl; aryloxycarbonyl such as phenoxycarbonyl; aralkylcarbonyl such as benzylcarbonyl and phenethylcarbonyl. Introduction of these acyl groups can be carried out using active derivatives of organic carboxylic acids (acid halides, acid anhydrides, active imide derivatives, active ester derivatives, etc.) according to conventional methods.

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

Example 1 The venom glands removed from 10 Joro spiders were 0.1 jIQ.
Ultrasonication was performed for 5 minutes in a 60% acetonitrile aqueous solution containing 1% trifluoroacetic acid to form a uniform suspension. This was centrifuged at 1200 rpm for 5 minutes, and the supernatant was directly separated by high performance liquid chromatography. The column is manufactured by Tosoh Corporation, 0DS-80TM (4,6X250
Elution was carried out using a linear concentration gradient method from 5% acetonitrile containing 0.1% trifluoroacetic acid to 60% acetonitrile aqueous solution containing 001% trifluoroacetic acid at a flow rate of 1.0 x Q 1 min.

The eluate was divided into 20 portions of 2×Q, and each portion was examined for histamine release activity. For the activity assay, each fraction was once dried and dissolved again in a 0.9% NaCl aqueous solution of 2ytQ, and 20 μg of each fraction was used for the assay.

The results of the activity assay are shown in Figure 1. Fraction 7 showing activity in Figure 1 was further separated using the same column using an 18% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid as an eluent, and each peak showing absorption at 220 nm was separated. Six types of peaks were obtained. The components in each peak were subjected to high performance liquid chromatography analysis under the same conditions and confirmed to be a single component. As a result of measuring the UV spectrum using a photodiode array detector (JASCO Corporation, MULTI 320) during analysis, all components were found to have a wavelength of 220 nm, 2g0r+.
It showed an absorption maximum at 29Or+m. A portion of these components was taken and hydrolyzed in 5°7N-HCI at 130°C for 9 hours.
, Ox 5D, w), the amino acid and polyamine compositions were determined.

The results are shown in Table 1. Next, add these ingredients to 5.7N-
The components were fragmented by hydrolysis in HCI at 1°C for 20 minutes. After hydrolysis, 0DS (manufactured by Tosoh Corporation)
Each fragment was separated by high performance liquid chromatography using a -80TM column, and each peak having an absorption at 210 nm was fractionated. The amino acid and polyamine compositions of each fragment were determined by the method described above, and the amino terminal residues of each fragment were examined by dansylation. That is, each fragment was mixed with IOμg of dansyl chloride (100μ9/lit in acetone solution) in 10μQ of 0.1M triethylamine-carbonate buffer (pH=8.2).
was added and reacted at 37°C for 1 hour.

After the reaction, the solvent was distilled off and the residue was dissolved in 5.7N-HCI at 130%
After hydrolysis for 9 hours at °C, dansylated amino acids and polyamines in the decomposition product were identified by high performance liquid chromatography using an octadecyl-4PW column (4,0x150zx) manufactured by Tosoh Corporation. Table 2-1 shows the fragments of each component and the sequence of each component found through the above study.
It is shown in 2-2. Discrimination between aspartic acid and asparagine in this sequence was performed using pitrifluoroacetoxy-iodobenzene (BTI). That is, 100 p mole to 1 n mole of each spider venom component was dissolved in 20 μQ of 50% acetonitrile aqueous solution,
Add 20 μg of BTI acetonitrile solution (5 mM), and react at 60° C. for 8 to 10 minutes. Acetone 3 after reaction
Add 0 μg and heat at 60° C. for 10 minutes to decompose excess BTI.

BTI is used in reactions that partially convert terminal amide groups into amines. In this reaction, asparagine becomes a basic amino acid (β-amino-alanine), and after hydrolysis,
It can be distinguished from aspartic acid. That is, if aspartic acid is detected after hydrolysis after BTI treatment, it means that aspartic acid is contained in the sequence, and if β-amino-alanine is detected, asparagine is contained in the sequence. As a result of examining each toxic component, it was found that component 1.2 contained aspartic acid, and components 3, 4, and 5.6 contained asparagine. Note that R1 shown in Table 2-2 does not contain amino acids or polyamines, and the UV spectrum measured by a photodiode array detector has absorption maximums of 220 nm, 280 nm,
290 ns, which was the same as the original component. When the amino terminals of each of the original components were examined by dansylation, we found that
Since dansyl-aspartic acid and α-dansyl-ornithine were not detected, it was revealed that this R was bonded to the α-amino group of the amino terminal residue of the original component. Therefore, R8 was fractionated by liquid chromatography and its NMR was measured. Measurement was performed using JEOL GX- in DtO.
400, the signal was 3.83 (s),
7.12(t).

7.20(t), 7.26(s), 7.46(d), 7
．． 57(d) respectively. Further, by comparing the elution position in reverse phase high performance liquid chromatography and the UV spectrum, R3 was found to be indole-3-acetic acid.

Table 3 shows the histamine release activity of each of these components.

Put; Butre1nin, Arg; Arginine Ca
dH Cadaverine 2 RI-0rn-Asp-Cad-Put
-Put3 R, -^sx-Cad-Orn
-Arg4 R, -Asx-Cad-Put
-Arg5 R, -^5x-Cad-Orn
ASX; 7 people Parakin Orn; Ornitino Put;
Putrenine Arg; Full X Con Cad; Cadaverine 2 Asp-Cad-Put-Put,
0rn-Asx-Cad, RI-3Asx-Cad, C
ad-Orn-Arg, Asx-Cad-Orn-Ar
g, RI-4Asx-Cad, Asx-Cad-Put
-Arg, RI-5Asx-Cad, R, Example 2 Both parts 5.6 shown in FIG.
Separation was performed using cation exchange chromatography. The analytical conditions and chromatogram are shown in Figure 2.

The beaks 5', 7', and 8' in Fig. 2 are further connected to the above-mentioned ODS.
Purification was performed using a -80TM column to obtain five components ranging from 1'' to 5''.

The chromatogram and conditions during purification are shown in FIG. During purification, the UV spectrum of each component was measured using a photodiode array detector, and the results were 220 nm, 255 nm.
nll, absorption maximum was observed at 280+v. A portion of each component was taken and hydrolyzed in 5.7N-HCI at 130° C. for 9 hours, followed by amino acid polyamine decomposition, and the compositions shown in Table 4 were obtained. Next, these components were fragmented by hydrolysis in 5.7N-HCI at 100° C. for 20 minutes, and the fragments were separated using the aforementioned ODS-80TM column, and the amino acid polyamine composition of the fragments was investigated. Furthermore, the amino terminal residues of each fragment were investigated by dansylation. Table 5-11 shows the amino acid polyamine sequences of each component found through the above studies. Table 5-2 shows the fragments obtained from each component.
It was shown to.

R1- in Table 5-2 is the same as the original component, 220 nm.

255r++s, has an absorption maximum at 2g0nII+,
Furthermore, since the α-amino group of the amino terminal residue of the original components was not dansylated, R,- was found to be a substituent bonded to the α-amino group of the amino terminal residue of each component.

This RI- was fractionated by liquid chromatography and FD-M
When the S spectrum was measured, the molecular weight was found to be 191.

Furthermore, after methylating RI- with diazomethane, GC-
Cleavage pattern and UV when measuring mass spectra
From the spectrum and elution position in high-performance liquid chromatography, R1- is 6
- It turned out to be hydroxyindole-3-acetic acid.

When 'H-NMR was measured in heavy water, it was 3.90p.
pm(2H,s), 7.05ppa+(IH,s), 7
．． 10 ppm (IH, s) was observed, but this could not be detected because there is a hydroxyl group at the 6-position of the indole ring, and the protons at the 5- and 7-positions were replaced with deuterium due to substitution in heavy water. It is thought that only the protons at the 2nd and 4th positions of the indole ring appear. Furthermore, when aspartic acid and asparagine in the sequence were distinguished by the method described in Example 1, all of them were found to be asparagine. Furthermore, when these components were examined for their histamine release activity from rat intraperitoneal mast cells, they all showed significant activity.

The results are shown in Table 6.

2″ R+-0rn-Asx-Cad-Put-P
ut3″R,-0rn-Asx-Cad-Put
-Put-Put4' R+-Asx-Cad
-Put-Put-Put2″ Asx-Cad-P
ut-Put, 0rn-Asx-Cad, R+-
3″ Asx-Cad-Put-Put-Put,
0rn-Asx-Cad, R+ -4' Asx
-Cad, Asx-Cad-Put-Put-Put,
R+1 Example 3 The venom glands of 20 spider spiders were extracted with 1 m5 of 0.1% trifluoroacetic acid aqueous solution, and the extract was extracted with Tosoh Co., Ltd.
After adding to DS-PAK and washing with 2J112 0.1% trifluoroacetic acid aqueous solution, washing with 30% acetonitrile aqueous solution 2xQ containing 0.1% trifluoroacetic acid, collecting the eluted components, and further washing as described above. ODS-80T
Separation was performed by reverse phase high performance liquid chromatography using an M column. The chromatogram and separation conditions are shown in FIG. Peak 3 in FIG. 4 was separated and purified using the same column using a 5% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid as an eluent to obtain two components, 1"' and 2"'. As a result of measuring the UV spectrum with a photodiode array detector, 21On showed an absorption maximum at 11.280 nm. After hydrolyzing these components in the same manner as Fu, the amino acid and polyamine compositions were determined. The results are shown in Table 7. In addition, each component was dissolved in 5.7N-HCI at iooo℃.
, after 20 minutes of hydrolysis and fragmentation, each fragment was separated,
The amino acids, polyamine composition, and amino terminal residues of each fragment were investigated using the methods described above. As a result, the arrangement shown in Table 8-■ was obtained. Further, each fragment is shown in Table 8-2.

Fragment R6- shown in Table 8-2 has the same 21O
It showed absorption at nI11, 280 nm, and since the α-amino groups of aspartic acid and ornithine at the amino terminals were not dansylated, R8- was found to be a substituent bonded to the α-amino groups of these amino-terminal residues. Further, this substituent was found to be 2,4-dihydroxyphenylacetic acid from the elution position in high performance liquid chromatography and the UV spectrum. When these components were examined for their histamine release activity from rat intraperitoneal mast cells, they all showed significant activity. The results are shown in Table 9.

1'' R+-Asx-Cad-Orn-Arg2
″' R+-0rn-Asx-Cad-Put-P
utR, ;2,4-ditFa4 diphenylacetyl group Table 8-2 1” Asx-Cad-Orn-＾rg, Asx
-Cad, R+-Example 4 Component 3 obtained in Example P11 in 5.7N 1ICI
00°C for 15 minutes to fragment the ODS-
Each fragment was purified using an 80TM column to obtain only the portion from which the substituents were removed. In addition, the components l″ and 2″ obtained in Example 2
, 3″ was fragmented by hydrolysis in 5.7N-HCI at 100°C for 20 minutes, and purified using an ODS-8OTM column.
The part excluding the substituents is similarly thin. When the histamine release activity of these basic moieties from rat intraperitoneal mast cells was measured, all showed activity. Table 1 shows the results and the amino acid and polyamine sequences of the basic part obtained.
0.

Neurotransmission-blocking spider venom has the effect of inhibiting neurotransmission by blocking glutamate receptors in arthropods.This time, we investigated the effects of the histamine-releasing active ingredient on neuromuscular synapses of the spiny polypod, and on neurotransmission. I looked into it. By inserting a recording electrode into the extensor muscle of the Isepi multipod, separating the innervating nerve into single fibers and stimulating them, an excitatory postsynaptic potential (EPSP) is generated, and each component is administered to the nerve fiber. , investigated the effects of toxic components on EPSP.

As a result, 3 and 4 in Example 1, 2'', 3'', 4'' in Example 2, and 0' in Example 3 were EPS.
The amplitude of P was significantly decreased. That is, it has been found that these toxic components not only release histamine, but also exhibit the activity of blocking nerve transmission in arthropods.

Table 11 shows the amino acid and polyamine sequences of each component and the decrease in EPSP.

Table 11

[Brief explanation of the drawing]

FIG. 1 is a graph showing the histamine release activity of the elution fraction according to Example 1, FIG. 2 is a chromatogram of elution fraction 5.6 according to Example 1, and FIG. 3 is the peak according to Example 2. Chromatograms for 5', 7', and 8' and FIG. 4 are chromatograms of the elution fraction according to Example 3. Figure 1, Order No. 1) Figure 2, Figure 3 (minutes)

Claims (10)

[Claims] (1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (m, p, q are 0 or 1, n is 0 to 4, R_1 represents a hydrogen atom or an acyl group, R_2 represents a hydroxy or amino group) A basic amide compound represented by (2) R_1 is an indole-3-acetyl group, m
2. The compound according to claim 1, wherein: =1, n=3, p=0, q=0. (3) R_1 is an indole-3-acetyl group, m
2. The compound according to claim 1, wherein: =1, n=2, p=0, q=0. (4) R_1 is an indole-3-acetyl group, m
The compound according to claim 1, wherein: =0, n=0, p=1, q=1. (5) R_1 is an indole-3-acetyl group, m
The compound according to claim 1, wherein: =0, n=1, p=1, q=1. (6) R_1 is an indole-3-acetyl group, m
The compound according to claim 1, wherein: =0, n=1, p=0, q=0. (7) R_1 is a hydrogen atom, m=0, n=3, p=
0, q=0. (8) R_1 is a hydrogen atom, m=0, n=2, p=
0, q=0. (9) R_1 is a hydrogen atom, m=0, n=1, p=
1. The compound according to claim 1, wherein q=1. (10) R_1 is a hydrogen atom, m=0, n=0, p
The compound according to claim 1, wherein q=1.