JP3500187B2 - Novel spider venom derivative, method for producing the same, and glutamate receptor blocker containing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel spider venom derivative containing a D-amino acid, a process for producing the same, and a glutamate receptor blocker containing the same, and more particularly, a novel spider neurocyte-mediated neurotransmitter action. The present invention relates to a spider venom derivative, a method for producing the same, and a glutamate receptor blocker containing the same.

[0002]

BACKGROUND OF THE INVENTION Glutamic acid functions as an excitatory transmitter in the neuromuscular junction of arthropods and the cranial nervous system of mammals. Further, it has been suggested that glutamate is involved in higher brain functions such as memory and learning, and elucidating the function of glutamate receptors is an important medical and physiological issue. Therefore, compounds that block the neurotransmission of glutamate can be an extremely powerful research tool in achieving the above-mentioned object. On the other hand, when glutamic acid is excessively present, it is also known to cause abnormal excitation of nerve cells and promote death of nerve cells. The excitotoxicity of glutamate is
The effects on the individual of the death of nerve cells that are never regenerated, such as those seen during cerebral ischemia, are significant. To reduce such glutamate excitotoxicity, it is therapeutically beneficial to temporarily block glutamate-mediated neurotransmission.

In recent years, a component that blocks neurotransmission by glutamate has been found in biotoxins. For example, during poisoning of Nephila spp., JSTX, nephilatoxin (Nep
hilatoxin) (Y. Aramaki et al., Pro
c. Acad. Jpn., Ser. B, 62,359 (1986), T. Toki et a
l., Biomed. Res., 9, 421 (1988), and T. Toki et a.
l., Jpn. J. Sanit. Zool., 41, 9 (1990)]. The present inventors have applied for patents by discovering natural toxic components of the Japanese Nephila spp., Efficiently synthesizing these compounds, synthesizing various derivatives, and developing an activity evaluation method. Kaihei 1-294734, JP-A-4-54142, JP-A-4-91067, JP-A-4-198162, JP-A-5-1005, JP-A-5-17348, and JP-A-4-17455]. These compounds have asparagine, ornithine,
It is a compound containing amino acids such as arginine and polyamines such as cadaverine, putrescine and spermine. The amino acids contained in the molecules of these compounds are all L-forms, but the present inventors have changed the L-amino acids found in natural products to D-amino acids in the course of conducting a structure-activity relationship study of spider venom components. It was found that the activity showing the original spider venom is retained even if it is replaced. The present invention is a further development of the studies conducted by the present inventors up to now, and an object thereof is to provide a novel spider venom derivative containing a D-amino acid or a salt thereof, which has a high blocking activity against glutamate receptors. Especially.

[0004]

The present inventors have completed the present invention as a result of intensive research to solve the above problems. That is, the present invention provides a compound represented by the general formula (I) R ¹ (CH ₂ ) _m CO-D-Asn-NH (CH ₂ ) _n NH-R ² ... (I) (wherein R ¹ is an alkyl group or cycloalkyl). Group, an aryl group, an aralkyl group or a heterocyclic group, R ² represents an aminoalkylcarbonyl group, R ¹ and R ² may have a substituent, D-Asn represents a D-asparagine residue. Shows, m
Represents 0 or 1, and n represents an integer of 2 to 5. The present invention provides a spider venom derivative or a salt thereof and a method for producing the same. The present invention also provides a glutamate receptor blocker containing the spider venom derivative represented by the general formula (I) or a salt thereof as an active ingredient.

The present invention will be described in detail below. In the general formula (I), the alkyl group represented by R ¹ is
For example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl,
Heptyl, octyl, decyl, dodecyl, tridecyl,
Examples thereof include linear or branched alkyl groups having 1 to 20 carbon atoms such as tetradecyl, hexadecyl, octadecyl and eicosyl. As the cycloalkyl group, for example,
Examples thereof include cycloalkyl groups having 5 to 8 carbon atoms such as cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. Examples of the aryl group include phenyl,
Groups such as 1-naphthyl, 2-naphthyl, anthryl, phenanthryl and the like can be mentioned. Examples of the aralkyl group include groups such as benzyl, phenethyl, benzhydryl, trityl and the like. Examples of the heterocyclic group include groups such as indolyl, furyl, thienyl, pyranyl, chromenyl, pyrrolyl, imidazolyl, pyrazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidinyl, piperidino, piperidyl, piperazinyl, morpholino and morpholinyl. These are, for example:
A halogen atom, a lower alkyl group having about 1 to 6 carbon atoms, a hydroxyl group, an alkoxy group, a carbonyl group, a dialkylamino such as 4-chlorophenyl, 4-methylphenyl, and 5- (dimethylamino) -1-naphthyl group. It may have a substituent such as a group, an amino group and a nitro group.

In the general formula (I), examples of the aminoalkylcarbonyl group represented by R ² include glycine residue, beta-alanine residue, gamma-aminobutanoic acid residue and the like. Further, histidine residue, lysine residue, ornithine residue, arginine residue, putreanine residue, N
-(Aminopropyl) putreanine residue, 8-amino-
4-azaoctanoic acid residue, may have a substituent such as 12-amino-4,9-diazadodecanoic acid residue,
Further, for example, a combination of plural kinds of groups such as an arginyl ornithine residue and an arginyl putreanine residue may be used.

The compound represented by the general formula (I) is a pharmacologically acceptable salt, for example, an inorganic acid such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, formic acid or triacetic acid. It may be an organic acid such as fluoroacetic acid, propionic acid, oxalic acid, glycolic acid, maleic acid, succinic acid and malic acid.

Among the spider venom derivatives represented by the general formula (I) or salts thereof, particularly representative compounds include a nephilatoxin derivative represented by the following general formula (I-1).

[0009]

[Chemical 3]

(In the formula, R ³ represents -NHCO (CH ₂ ) ₂ NH (CH ₂ ) ₄ NH ₂ or -NHCO (CH ₂ ) ₂ NH (CH ₂ ) ₄ NH (CH ₂ ) ₃ NH ₂ . The compound represented by the general formula (I) is a compound represented by the following formula (III) obtained by de-Boc reaction of an azide compound represented by the following formula (II) in the presence of an acid as shown in the following reaction formula (1). The compound represented by the following formula (IV) is reacted in the organic solvent with the D-asparagine derivative represented by the following formula (IV) as shown in the reaction formula (2), and the obtained compound represented by the following formula (V) is present in the presence of an acid. A compound represented by the following formula (VI) obtained by de-Boc reaction as shown in the reaction formula (3) in an organic solvent, as shown in reaction formula (4), an acid represented by the following formula (VII) or an ester thereof. The resulting compound represented by the following formula (VIII) is reduced to an azide group of the compound represented by the following formula (VIII) in an organic solvent as shown in the reaction formula (5) to obtain a compound represented by the following formula (IX). , A protective group is attached as shown in reaction formula (6). It can be synthesized by introducing an aminoalkylcarbonyl group and reducing the resulting compound represented by the following formula (X) as shown in reaction formula (7) to remove the protecting group.

[0011]

[Chemical 4]

However, in the above series of formulas, R ¹ , R ² , D-Asn, m
And n have the same meanings as described above, Boc is a t-butyloxycarbonyl group, and R ⁴ is R ² in which a protecting group is introduced into the amino group.
Indicates a group.

The removal of the Boc group shown in the above reaction formula (1) is carried out by using the starting material, an azide compound represented by the formula (II), with hydrochloric acid,
It is carried out by treating with an inorganic acid such as hydrobromic acid, an organic acid such as acetic acid or trifluoroacetic acid, or a mixture thereof. The reaction is usually carried out in a solvent system such as dimethylformamide, dimethylsulfoxide and methylene chloride, which does not adversely influence the reaction. The reaction is completed in about 30 minutes to several hours by stirring at about 0 to 50 ° C. still,
The azide compound represented by the formula (II) which is a starting material can be produced by the method described in JP-A-4-91067. That is, a compound represented by the formula (i) H ₂ N (CH ₂ ) _n OH (i) (in the formula, n has the same meaning as described above) and a t-type compound such as di-t-butyl-dicarbonate. A butyloxycarbonylating reagent and a formula (ii) R ⁵ X (ii) (wherein, R ⁵ represents an alkanesulfonyl group, an arylsulfonyl group or an aralkylsulfonyl group, and X represents a halogen atom). The compound is reacted to form the compound of formula (ii
i) A compound represented by BOC-NH (CH ₂ ) _n OR ⁵ (iii) (in the formula, BOC, n and R ⁵ have the above-mentioned meanings), and the compound and azide such as sodium azide are obtained. The azide compound represented by the formula (II) can be obtained by reacting with a compound.

The introduction of the D-asparagine residue shown in the above reaction formula (2) is carried out by a conventional peptide synthesis method, that is, an active ester method in which p-nitrophenyl ester, N-hydroxysuccinimide, etc. are introduced, and a condensing agent. As the DCC method using dicyclohexylcarbodiimide, and the DCC-additive method which is a combination of these methods. Removal of the Boc group shown in the above reaction formula (3) is performed in the same manner as in the case of reaction formula (1).

The introduction of the acyl group represented by the above reaction formula (4) is
Ordinary peptide synthesis methods, that is, active ester method in which p-nitrophenyl ester, N-hydroxysuccinimide, etc. are introduced, DCC method using dicyclohexylcarbodiimide as a condensing agent, and DCC which is a combination of these methods.
-Additive method is used. The reduction of the azide group represented by the above reaction formula (5) is usually carried out by catalytic reduction in the presence of a reduction catalyst. As the reduction catalyst, for example,
Platinum, platinum oxide, palladium black, palladium carbon, Raney nickel, etc. can be used. The amount of the reducing catalyst used is usually about 0.001 to 1 times the weight of the compound represented by the formula (VIII). The catalytic reduction reaction is carried out, for example, in a solvent such as water, methanol, ethanol, isopropanol, diethyl ether, dioxane, tetrahydrofuran or the like, 1
It is completed in 1 to 24 hours by stirring in a hydrogen gas atmosphere of about -10 kg / cm ^{2 at} about -30 ° C to about the boiling point of the solvent.

The introduction of the aminoalkylcarbonyl group represented by the above reaction formula (6) is carried out in the same manner as the introduction of the acyl group represented by the reaction formula (4). The amino group in the aminoalkylcarbonyl group can be protected with a protecting group such as Boc, p-toluenesulfonyl, methylsulfonyl, benzyloxycarbonyl and the like which is commonly used in peptide synthesis. Removal of the protecting group shown in the above reaction formula (7) is
The treatment is carried out with the same acid as in the case of (1), or the same catalytic reduction as in the case of reaction formula (5).

The target compound of the present invention can be isolated and purified by, for example, a solvent extraction method, a recrystallization method, a thin layer chromatography method, a column chromatography method, a high performance liquid chromatography method and the like. The obtained compound can be identified by known methods such as melting point, elemental analysis, NMR spectrum, mass spectrum and IR spectrum.

Evaluation of the activity of the compounds of the present invention is described in JP-A-5-
The method described in Japanese Patent No. 17348, that is, a method of evaluating the activity by administering the compound of the present invention to insects such as flies and cockroaches, and examining how the insects recover from paralysis, or crustaceans such as lobster and It can be carried out by a method of assaying by generating an excitatory postsynaptic potential in a neuromuscular preparation of an insect and examining the influence of the compound administered to the nerve fiber on the potential. The compound of the present invention shows remarkable biological activity in the above assay method and is useful as a glutamate receptor blocker.

[0019]

As described above, according to the present invention, it was possible to provide a novel spider venom derivative having glutamate receptor blocking properties.

[0020]

EXAMPLES The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 D represented by the formula (XVI) according to the following synthetic scheme 1
-Nefiratoxin-12 was synthesized.

[0022]

[Chemical 5]

(1) 5-azido-1-represented by the formula (XII)
Synthesis of (N-Boc-D-asparaginyl) aminopentane

[0024]

[Chemical 6]

5-Azido-1-N-Boc-aminopentane (XI) (1.39 g) was dissolved in anhydrous dichloromethane (4.7 ml), trifluoroacetic acid (4.7 ml) was added to this solution, and the mixture was stirred at room temperature for 2 hours. did. The solvent and trifluoroacetic acid were distilled off under reduced pressure, dichloromethane (5 ml) was added to the residue to dissolve it, and the solvent was distilled off again. This operation was repeated 3 times to remove trifluoroacetic acid. The amine obtained was dissolved in dimethylformamide (30 ml) and N-methylmorpholine (NMM, 6.7 ml) was added, followed by N-Boc-D-asparagine-p.
-Nitrophenol ester (2.50 g) was added and the mixture was stirred at room temperature for 12 hours. The reaction solution was diluted with ethyl acetate (1.2 liter), and this was washed successively with 18% aqueous sodium chloride solution (50 ml) 4 times, water (50 ml) once, and saturated aqueous sodium chloride solution (50 ml) 3 times. The organic layer was dried over magnesium sulfate and then filtered, and the solvent was distilled off under reduced pressure.
The residue was purified by silica gel column chromatography (silica gel 90 g, dichloromethane / ethanol = 30/1 to 20/1 volume ratio) and 5-azido-1- (N-Boc-D-asparaginyl) aminopentane (XII) 1.60 g. (Yield 77%) was obtained as white crystals. [Α] _D = -16.3 ° (c = 0.69, CHCl ₃ ) (2) Synthesis of 5-azido-1- (indole-3-acetyl-D-asparaginyl) aminopentane represented by formula (XIII)

[0026]

[Chemical 7]

1.60 g of 5-azido-1- (N-Boc-D-asparaginyl) aminopentane (XII) was dissolved in anhydrous dichloromethane (3.6 ml), trifluoroacetic acid (3.6 ml) was added, and the mixture was stirred at room temperature for 3 hours. It was stirred. After distilling off the solvent and trifluoroacetic acid under reduced pressure, dichloromethane (5 ml) was added to the residue.
Was added and dissolved, and the solvent was distilled off again. This operation was repeated 3 times to remove trifluoroacetic acid. The obtained amine was dissolved in anhydrous dimethylformamide (20 ml), and N-
Methylmorpholine (5.15 ml), then indoleacetic acid-
N-Hydroxysuccinimide ester (2.50 g) was added and the mixture was stirred at room temperature for 12 hours. The reaction solution was diluted with ethyl acetate (1 liter), and this was washed successively with 18% aqueous sodium chloride solution (50 ml) 4 times, water (50 ml) once, and saturated aqueous sodium chloride solution (50 ml) 3 times. The organic layer was dried over magnesium sulfate and then filtered, and the solvent was distilled off under reduced pressure. The residue is silica gel column chromatography (silica gel 90
g, dichloromethane / acetone / ethanol = 10/1 /
0-3 / 1 / 0-2 / 1 / 0.5 (volume ratio) to obtain white crystals of 5-azido-1- (indole-3-acetyl-
1.13 g (yield 60%) of D-asparaginyl) aminopentane (XIII) was obtained. [Α] _D = + 1.6 ° (c = 0.19, MeOH) (3) Synthesis of protected form of D-nefilatoxin-12 represented by formula (XV)

[0028]

[Chemical 8]

5-azido-1- (indole-3-acetyl-D-asparaginyl) aminopentane (XIII) 300 m
g in ethanol (64 ml), 10 wt% Pd-C 50 mg
Was added and the mixture was stirred at room temperature under a hydrogen gas atmosphere for 2 hours. The catalyst was filtered off with a membrane filter, and the obtained filtrate was concentrated under reduced pressure. The residue (compound (XIV)) was dissolved in anhydrous dimethylformamide (14 ml), to which triethylamine (0.5 ml) was added, followed by 8-azido-N-Boc-4-azaoctanoic acid-p-nitrophenol ester (450 mg) in dimethyl. Formamide solution (10 ml) was added, and the mixture was stirred at room temperature for 12 hours. The reaction solution was diluted with ethyl acetate (1.2 liters), and this was sequentially added with an 18% aqueous sodium chloride solution (50 ml) 4 times.
Once, with water (50 ml) once, saturated aqueous sodium chloride solution (50
ml) and washed 3 times. The organic layer was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained crude crystals were dissolved in methanol (5 ml), distilled water (7.5 ml) was added while cooling this to 0 ° C. in an ice bath, and the precipitated crystals were collected by filtration with a membrane filter. After drying under reduced pressure, 392 mg (yield) of the protected form of D-nefilatoxin-12 (XV) was obtained as white crystals.
81%). [Α] _D = -3.7 ° (c = 0.35, MeOH) (4) Synthesis of _D -nefilatoxin-12 represented by formula (XVI)

[0030]

[Chemical 9]

D-nephilatoxin-12 protected form (XV) 12
8 mg was dissolved in anhydrous dichloromethane (1.3 ml), trifluoroacetic acid (0.6 ml) was added, and the mixture was stirred at room temperature for 3 hours. After distilling off the solvent and trifluoroacetic acid under reduced pressure, 1,2-
Dichloroethane (2 ml) was added and dissolved, and the solvent was distilled off again. This operation was repeated to remove trifluoroacetic acid. Dissolve the residue in 15% acetonitrile aqueous solution,
After passing through a -pak, ethanol (5 ml) was added to the filtrate, and the solvent was evaporated under reduced pressure. Ethanol (5 ml) was added to the residue to dissolve it, and the solvent was distilled off again. This operation was repeated 3 times to completely remove water. The residue was dried under reduced pressure with a vacuum pump to obtain a reddish purple oil (175 mg). The obtained reddish purple oil (175 mg) was dissolved in ethanol (20 ml) to give 10 wt% Pd-
17 mg of C was added, and the mixture was stirred at room temperature under a hydrogen gas atmosphere for 3.5 hours. The catalyst was filtered off with a membrane filter, and the obtained filtrate was concentrated under reduced pressure. The residue was passed through a pretreatment column (Chemco Sep ODS-A, 250 mg) using a 30% aqueous acetonitrile solution containing 0.1% trifluoroacetic acid. The obtained eluate was applied to a reverse phase HPLC column (TOSOH, TSK-Gel ODS-120.
T, 4.6 x 250 mm), 10% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid was used as a mobile phase, and D
-85 mg of nephilatoxin-12 (XVI) was obtained.

Example 2 D-nefilatoxin-8 represented by the formula (XVIII) was synthesized according to the following synthesis scheme 2.

[0033]

[Chemical 10]

(1) Synthesis of protected form of D-nefilatoxin-8 represented by the formula (XVII)

[0035]

[Chemical 11]

5-azido-1- (indole-3-acetyl-D-asparaginyl) aminopentane (XIII) 160 m
g dissolved in methanol (30 ml), 10 wt% Pd-C 80 mg
Was added and the mixture was stirred at room temperature under a hydrogen gas atmosphere for 2 hours. The catalyst was filtered off with a membrane filter, and the filtrate was concentrated under reduced pressure. The residue was dissolved in anhydrous dimethylformamide (10 ml), to which N-methylmorpholine (0.55 ml) was added, followed by 12
-Azido-bis-N-Boc-4,9-diazadodecanoic acid-N
A solution of hydroxysuccinimide ester (250 mg) in dimethylformamide (4 ml) was added, and the mixture was stirred at room temperature for 12 hours. The reaction solution was diluted with ethyl acetate (1 liter), and this was washed successively with 18% aqueous sodium chloride solution (50 ml) 4 times, water (50 ml) once, and saturated aqueous sodium chloride solution (50 ml) 3 times. The organic layer was dried over magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel 90 g, chloroform / acetone / ethanol = 4/1 / 0.6 volume ratio), and the protected form of D-nefilatoxin-8 (XVII) was made amorphous.
0 mg (77% yield) was obtained. [Α] _D = -3.0 ° (c = 0.54, MeOH) (2) Synthesis of D-nefilatoxin-8 represented by formula (XVIII)

[0037]

[Chemical 12]

Protected form of D-nefilatoxin 8 (XVII) 6
6.3 mg was dissolved in anhydrous dichloromethane (0.14 ml), trifluoroacetic acid (0.13 ml) was added, and the mixture was stirred at room temperature for 3 hours. After the solvent and trifluoroacetic acid were distilled off under reduced pressure, methanol (1 ml) was added to the residue to dissolve it, and the solvent was distilled off again. This operation was repeated to remove trifluoroacetic acid. The residue was dissolved in acetic acid (4 ml) and 10 wt% Pd-C 40 mg
Was added and the mixture was stirred at room temperature under a hydrogen gas atmosphere for 4 hours. The catalyst was filtered off with a membrane filter, and the filtrate was concentrated under reduced pressure. The residue was pretreated with a 30% aqueous acetonitrile solution containing 0.1% trifluoroacetic acid (Chemco Sep O
DS-A, 250 mg). The eluate obtained was analyzed using a reverse-phase HPLC column (TOSOH, TSK-Gel ODS-120T) to give 0.1%
A 10% aqueous solution of acetonitrile containing trifluoroacetic acid was used as a mobile phase to collect D-nefilatoxin-8 (XVIII).
18 mg was obtained.

Example 3 With respect to D-nefilatoxin-12 obtained in Example 1 and D-nephylatoxin-8 obtained in Example 2,
The paralytic effect on insects was examined by the method described in JP-A-5-17348. That is, a German cockroach (female adult) anesthetized with ether was used with a microsyringe on the chest of 10 rats per group, and the D-nephilatoxin-12 obtained in Example 1 and the D-nephila obtained in Example 2 were used. An aqueous solution of toxin-8 was administered. Water was similarly administered as a control and compared with the sample administration group. Table 1 shows the recovery time from paralysis.

[0040]

[Table 1]

Note) *: Water was administered as a control.

As is clear from Table 1, it is clear that the compounds obtained in Examples 1 and 2 of the present invention have a greater paralyzing effect on German cockroaches than the control group.

Front page continuation (51) Int.Cl. ⁷ identification code FI C07D 209/18 C07D 209/18 (58) Fields investigated (Int.Cl. ⁷ , DB name) CA (STN) CAOLD (STN) REGISTRY (STN )

Claims (4)

(57) [Claims] 1. General formula (I) R ¹ (CH ₂ ) _m CO-D-Asn-NH (CH ₂ ) _n NH-R ² ... (I) (wherein R ¹ is an alkyl group or a cycloalkyl group. , An aryl group, an aralkyl group or a heterocyclic group, R ² represents an aminoalkylcarbonyl group, R ¹ and R ² may have a substituent, D-Asn represents a D-asparagine residue , M
Represents 0 or 1, and n represents an integer of 2 to 5. ) A spider venom derivative represented by or a salt thereof. 2. A spider venom derivative or a salt thereof according to claim 1, wherein the spider venom derivative represented by the general formula (I) or a salt thereof is a nephilatoxin derivative represented by the general formula (I-1). . [Chemical 1] (In the formula, R ³ is -NHCO (CH ₂ ) ₂ NH (CH ₂ ) ₄ NH ₂ or -NHCO (C
_{H 2) 2 NH (CH 2} ) shows a _{4 NH (CH 2) 3 NH} 2. ) 3. A compound represented by the following formula (III) obtained by de-Boc conversion of an azide compound represented by the following formula (II) in the presence of an acid is represented by the following formula (IV) in an organic solvent. The compound represented by the following formula (V) obtained by reacting with a D-asparagine derivative is de-Boc-derivatized in the presence of an acid, and the obtained compound represented by the following formula (VI) is added in an organic solvent to the following formula ( VII)
In an acylation by reacting with an acid or an ester thereof, the azido group of the compound represented by the following formula (VIII) is reduced in an organic solvent to obtain the compound represented by the following formula (IX) The spider venom derivative or its derivative according to claim 1, wherein the compound represented by the following formula (X) is introduced to reduce the protecting group by introducing an aminoalkylcarbonyl group having a protecting group attached thereto. Salt production method. [Chemical 2] (In the above series of formulas, R ¹ , R ² , D-Asn, m and n have the same meanings as described above, Boc is a t-butyloxycarbonyl group, and R ⁴ is a protecting group introduced into an amino group. Indicates the R ² group.) 4. A glutamate receptor blocker containing the spider venom derivative or its salt according to claim 1 as an active ingredient.