JPH0832723B2 - Antifungal peptide - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel antifungal peptide useful as a therapeutic agent for fungal infections.

[Conventional technology]

Conventionally, there are many other fungal infectious agents such as amphotericin B, flucytosine and miconazole, but there are problems in efficacy and toxicity. In particular, there are very few low-toxic drugs effective for systemic infections, which have been increasing in recent years.

[Problems to be Solved by the Invention]

An object of the present invention is to provide a highly active and low toxic agent as a therapeutic agent for fungal infections.

[Means for solving the problem]

The present inventors have conducted research aiming at the search for new antibiotics, and have investigated Aureobasium pullulans (Aureobas).
idium pullulans) No.R106 [Micromachinery Research Article 1938 (FERM
BP-1938)] and other antibiotics represented by the following general formula (II) produced by microorganisms belonging to the genus Aureobasidium were found (Japanese Patent Application No. 1-37363, “Novel antibiotics R106 and The manufacturing method and use "and Heisei 6
"New antibiotic R106" filed for patent on March 19) (In the formula, R is a methyl group or an ethyl group, X ₁ is MePhe or β-HOMePhe or Phe, X ₂ is allo-I1e or Val or Leu, X ₃ is MeVal or Val, X ₄ is β-HOMeVal or γ-. HOMeVal or MeVal or V
al or N, β-MeAsp or MePhe or β-HOMePhe or MeDH _{2, 3} Val or MeDH _3, a ₄ Val. Furthermore, the inventors of the present invention have conducted extensive studies on derivatives of antibiotic R106, and as a result, have shown that antibiotic R106 represented by the general formula (II) above.
The present invention was completed by finding that a novel linear peptide represented by the following general formula (I), obtained by hydrolyzing lactic acid under alkaline conditions, has antifungal activity and low toxicity.

(In the formula, R is a methyl group or an ethyl group, Y ₁ is MePhe or β-HOMePhe or Phe or Sar, Y ₂ is allo-I1e or Val or Leu, Y ₃ is MeVal or Val, Y ₄ is β-HOMeVal or γ-HOMeVal or MeVal or V
al or N, β-MeAsp or MePhe or β-HOMePhe or MeDH ₂ , ₃ Val or MeDH ₃ , ₄ Val or Sar) used in the present specification including the above general formulas (I) and (II) The amino acid abbreviations are as shown below.

Val: valine MeVal: N-methylvaline β-HOMeVal: β-hydroxy-N-methylvaline γ-HOMeVal: γ-hydroxy-N-methylvaline MeDH ₂ , ₃ Val: N-methyl-2,3-didehydrovaline MeDH ₃ , ₄ Val: N-methyl-3,4-didehydrovaline Phe: phenylalanine MePhe: N-methyl-phenylalanine β-HOMePhe: β-hydroxy-N-methylphenylalanine allo-Ile: alloisoleucine Leu: Leucine Pro: Proline N, β-MeAsp: N, β-Dimethylaspartate Sar: Sarcosin As specific examples of the peptide represented by the general formula (I) of the present invention, the compounds shown in Table 1 can be mentioned.

Specific examples of the antibiotic R106 represented by the general formula (II) include the compounds shown in Table 2.

The compound represented by the general formula (I) of the present invention can be produced by hydrolyzing the ester bond contained in the antibiotic R106 of the general formula (II) under alkaline conditions. Usually, the compound of the general formula (II) is dissolved in a hydrophilic solvent such as methanol, ethanol or dimethylsulfoxide, and an alkaline aqueous solution is added to the solution to react. The alkali is not particularly limited, but sodium hydroxide and potassium hydroxide are preferably used. Alkali concentration is 0.01 ~
2N is preferable, and the reaction is carried out at a temperature of 0 ° C to the boiling point, but usually room temperature is sufficient. The reaction time varies depending on the compound, but is several hours to several days.

In the compound of general formula (II), β-HOMePhe is used as X _1.
And a compound containing β-HOMeVal as X ₄ are β-HOMePhe and β-HOMeV under the alkaline conditions described above.
A compound in which the amino acid side chain of al undergoes a reverse aldol reaction and is converted to Sar is obtained as the main product. Also, compounds containing MeDH _{2, 3} Val as X ₄ is under normal conditions is hardly go reaction, compounds of the present invention is obtained by refluxing.

The physicochemical and biological properties of the representative compounds in the present invention are as follows.

(1) Physicochemical properties Table 3 shows the physicochemical properties of the representative compounds of the present invention shown in Table 1.

(2) Biological properties i) Antifungal activity Table 4 shows the minimum growth inhibitory concentrations (MIC, μg / ml) of typical compounds of the present invention against fungi.

Kashiton medium (glucose 2.0%, bacto-casitone 0.9%, yeast extract 1.0%, KH ₂ PO ₄ 0.1%, Na ₂ HPO ₄ was used for measurement.
0.1%, sodium citrate 1.0%, agar 2.0%, the above concentrations were all performed by the agar dilution method using w / v).

ii) Toxicity The representative compounds 1 , 5 and 10 of the present invention were intraperitoneally administered to mice at 200 mg / kg and once, respectively, and no abnormality was observed in the mice with any of the compounds.

Next, the present invention will be described more specifically with reference to reference examples and examples, but the present invention is not limited to these examples.

Reference Example Production of Antibiotics R106-I to XVI Aureobasidium pullulans No.R106 (FERM BP-1938)
The liquid medium [Difco yeast nitrogen base 0.67%
(W / v), glucose 2% (w / v)] 500 ml containing 100 ml
The contents were shaken in an Erlenmeyer flask at 27 ° C. for 2 days to obtain a seed culture solution. 1000 ml of this seed culture was added to 100 l of liquid medium [glucose 2%, (NH ₄ ) ₂ SO ₄ 0.5%, KH ₂ PO ₄ 0.15%, MgSO ₄ .7H
_{2 O0.05%, CaCl 2 0.01%} , NaCl0.01% ( all concentrations expressed in terms of _{w / v), FeCl 3 0.5μg} / ml, in 200l volumes changer over Sulfur Mentor containing the ZuSO ₄ 0.5μg / ml] The cells were inoculated and aerated with stirring at 25 ° C. for 90 hours (aeration rate 100 l / min, stirring 150 rpm).

The obtained culture broth was centrifuged to collect the bacterial cells, and 10 l of acetone was added to the bacterial cells to perform an extraction operation. The acetone extract was concentrated under reduced pressure, and the acetone was distilled off, followed by ethyl acetate 1
It was extracted twice with. The extract was concentrated under reduced pressure and evaporated to dryness, then the residue was dissolved in 100 ml of acetonitrile and divided into 30 times. High-performance liquid chromatography for preparative filtration [Column: Soken Pack C
₁₈ (manufactured by Soken Chemical Industry Co., Ltd.) 5 cmφ × 50 cm, mobile phase: 70% (v / v) acetonitrile water detection: UV _{230 nm} ], and 18 eluated activities were peaked at the retention time shown in Table 5. The fractions were collected and concentrated under reduced pressure to obtain white powders of R106-I to XVI in the amounts shown in Table 5.

Example 1 Production of compounds 1 and 2 R106-I (105 mg) was dissolved in methanol (9 ml), 1N aqueous sodium hydroxide solution (3 ml) was added, and the mixture was stirred at room temperature overnight.
The reaction solution was neutralized with a 1N aqueous hydrochloric acid solution and then concentrated. Water was added to the concentrate, acidified with hydrochloric acid, and extracted with ethyl acetate. The extract was washed with water, dried, and concentrated under reduced pressure to dryness, leaving a residue of 103 m.
got g. High-performance liquid chromatography for preparative separation (column: Nucleosyl-C ₁₈ (M _O Nagel), 10 m
mφ × 250 mm, mobile phase: 70% (v / v) aqueous acetonitrile, detection: UV _{230 nm} ], and the peaks of Compounds 1 and 2 were collected. Minute toga fraction was concentrated to dryness under reduced pressure, Compound 1 55 mg, 2
3 mg was obtained.

Compound 1 molecular weight: FAB-MS m / z 1061 (M + H), 1083 (M + Na) Amino acid analysis: sarcosine, proline, alloisoleucine, leucine, phenylalanine Specific rotation ▲ [α] ²⁰ _D ▼ -178.8 (C1.0, methanol) ) Compound 2 molecular weight: FAB-MS m / z 1119 (M + H), 1141 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine Example 2 Preparation of compound 3 20 mg of R106-IIa was dissolved in 6 ml of methanol, and 1N water was added. 2 ml of an aqueous sodium oxide solution was added, and the mixture was stirred at room temperature overnight. The same operation as in Example 1 was carried out to obtain 5 mg of compound 3 .

Molecular weight: FAB-MS m / z 1047 (M + H), 1069 (M + Na) Amino acid analysis: sarcosine, proline, alloisoleucine, leucine, phenylalanine Example 3 Preparation of compound 4 20 mg of R106-IIb was dissolved in 6 ml of methanol and 1N hydroxylated. 2 ml of sodium aqueous solution was added, and the mixture was stirred at room temperature overnight. The same operation as in Example 1 was carried out to obtain 5 mg of compound 4 .

Molecular weight: FAB-MS m / z 1047 (M + H), 1069 (M + Na) Amino acid analysis: sarcosine, proline, valine, leucine, phenylalanine Example 4 Preparation of compound 5 48 mg of R106-III was dissolved in 12 ml of methanol and 1N sodium hydroxide was added. 4 ml of an aqueous solution was added, and the mixture was stirred at room temperature overnight. The same operation as in Example 1 was carried out to obtain 12 mg of compound 5 .

Molecular weight: FAB-MS m / z 1119 (M + H), 1141 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine Example 5 Preparation of compounds 6 , 7 and 8 R106-IV 60 mg was dissolved in methanol 9 ml to give 1N. 3 ml of aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature overnight. In the same manner as in Example 1, 18 mg of compound 6 , 7 mg of 7 and 8 were added.
5 mg was obtained.

Compound 6 molecular weight: FAB-MS m / z 971 (M + H), 993 (M + Na) amino acid analysis: sarcosine, proline, alloisoleucine, leucine, phenylalanine Compound 7 molecular weight: FAB-MS m / z 1077 (M + H), 1099 (M + Na) amino acid Analysis: sarcosine, proline, alloisoleucine, leucine, phenylalanine Compound 8 Molecular weight: FAB-MS m / z 1029 (M + H), 1051 (M + Na) Amino acid analysis: sarcosine, proline, alloisoleucine, leucine, phenylalanine Example 6 Compound Preparation of 9 R106-V (70 mg) was dissolved in methanol (6 ml), 1N aqueous sodium hydroxide solution (2 ml) was added, and the mixture was stirred at room temperature overnight. Example 1
13 mg of compound 9 was obtained by performing the same operation as above.

Molecular weight: FAB-MS m / z 1047 (M + H), 1069 (M + Na) Amino acid analysis: sarcosine, proline, valine, alloisoleucine, leucine, phenylalanine Example 7 Preparation of compound 10 32 mg of R106-VI was dissolved in 15 ml of methanol to give 8N. 2.5 ml of an aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature overnight. The same operation as in Example 1 was carried out to obtain 14 mg of compound 10 .

Molecular weight: FAB-MS m / z 1103 (M + H), 1125 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine Specific rotation ▲ [α] ²⁰ _D ▼ -153.2 (C1.0, methanol) Example 8 Preparation of Compound 11 20 mg of R106-VII was dissolved in 6 ml of methanol, 2 ml of 2N sodium hydroxide aqueous solution was added, and the mixture was stirred overnight at room temperature. The same operation as in Example 1 was carried out to obtain 10 mg of compound 11 .

Molecular weight: FAB-MS m / z 1089 (M + H), 1111 (M + Na) Amino acid analysis: proline, valine, alloisoleucine,
Leucine, phenylalanine Example 9 Preparation of compound 12 R106-VIII (30 mg) was dissolved in methanol (3 ml), 1N sodium hydroxide (1 ml) was added, and the mixture was stirred at room temperature overnight. Example 1
6 mg of compound 12 was obtained by performing the same operation as above.

Molecular weight: FAB-MS m / z 1061 (M + H), 1083 (M + Na) Amino acid analysis: sarcosine, proline, leucine, phenylalanine Example 10 Preparation of compound 13 R106-IX 13 mg was dissolved in methanol 3 ml, 2N sodium hydroxide aqueous solution 1 ml. Was added, and the mixture was stirred at room temperature for 13 days. The same operation as in Example 1 was carried out to obtain 6 mg of compound 13 .

Molecular weight: FAB-MS m / z 1133 (M + H), 1155 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine Example 11 Preparation of compound 14 R106-Xa (30 mg) was dissolved in methanol (6 ml), 2N aqueous sodium hydroxide solution was added. 2 ml was added, and the mixture was stirred overnight at room temperature. The same operation as in Example 1 was carried out to obtain 11 mg of compound 14.

Molecular weight: FAB-MS m / z 1089 (M + H), 1111 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine Example 12 Preparation of compound 15 28 mg of R106-Xb was dissolved in 6 ml of methanol, and a 2N aqueous sodium hydroxide solution was used. 2 ml was added, and the mixture was stirred overnight at room temperature. The same operation as in Example 1 was carried out to obtain 8 mg of compound 15 .

Molecular weight: FAB-MS m / z 1089 (M + H), 1111 (M + Na) Amino acid analysis: proline, valine, leucine, phenylalanine Example 13 Preparation of compound 16 Dissolve 15 mg of R106-XI in 2 ml of 2N sodium hydroxide aqueous solution. Was added, and the mixture was stirred at room temperature for 4 days. The same operation as in Example 1 was carried out to obtain 7 mg of compound 16 .

Molecular weight: FAB-MS m / z 1089 (M + H), 1111 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine Example 14 Preparation of compound 17 Dissolve 27 mg of R106-XII in 6 ml of methanol, and add 1N aqueous sodium hydroxide solution. 2 ml was added, and the mixture was stirred overnight at room temperature. The same operation as in Example 1 was carried out to obtain 13 mg of compound 17 .

Molecular weight: FAB-MS m / z 1101 (M + H), 1123 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine Example 15 Preparation of compound 18 R106-XIII 32 mg was dissolved in methanol 6 ml, 1N aqueous sodium hydroxide solution. 2 ml was added, and the mixture was stirred overnight at room temperature. The same operation as in Example 1 was carried out to obtain 13 mg of compound 1 and compound 18
Was obtained.

Molecular weight: FAB-MS m / z 1167 (M + H), 1189 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine Example 16 Preparation of compound 19 21 mg of R106-XIV was dissolved in 6 ml of methanol, and a 2N aqueous sodium hydroxide solution was used. 2 ml was added, and the mixture was stirred overnight at room temperature. By the same procedure as in Example 1, 8 mg of compound 19 was obtained.

Molecular weight: FAB-MS m / z 1089 (M + H), 1111 (M + Na) Amino acid analysis: proline, valine, alloisoleucine,
Leucine, Phenylalanine Example 17 Preparation of Compound 20 28 mg of R106-XV was dissolved in 6 ml of methanol, 2 ml of 1N sodium hydroxide was added, and the mixture was stirred overnight at room temperature. The same operation as in Example 1 was carried out to obtain 16 mg of compound 20 .

Molecular weight: FAB-MS m / z 1151 (M + H), 1173 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine Example 18 Preparation of Compound 21 19 mg of R106-XVI was dissolved in 6 ml of methanol, and a 2N aqueous sodium hydroxide solution was used. 2 ml was added and refluxed for 4 hours. Example 1
The same operation as the above was performed to obtain 5 mg of Compound 21 .

Molecular weight: FAB-MS m / z 1101 (M + H), 1123 (M + Na) Amino acid analysis: proline, alloisoleucine, leucine, phenylalanine [Effect of the invention] The antifungal peptide of the present invention has low toxicity and candida albicans, etc. It has an antibacterial activity against the pathogenic fungus of (1) and is useful as a therapeutic agent for mycosis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1: 645) (72) Inventor Junko Yamamoto 3-4-1 Seta, Otsu City, Shiga Prefecture Central Research Institute Co., Ltd. (72) Inventor Fumiyo Haruna 34-1 Seta, Otsu City, Shiga Prefecture, Saka Brewery Co., Ltd. Central Research Institute (72) Teruya Nakamura 34-1 Seta, Otsu City, Shiga Prefecture (72) Inventor Hideyo Yamaguchi 2-15-5 Kuriya, Tama-ku, Kawasaki City, Kanagawa Prefecture (72) Inventor Katsuhisa Uchida 3-163-3 Nakamura, Nerima-ku, Tokyo (56) References 3-22995 (JP, A) JP-A-2-138296 (JP, A)

Claims (1)

[Claims] 1. An antifungal peptide represented by the following general formula (I). (In the formula, R is a methyl group or an ethyl group, Y ₁ is MePhe or β-HOMePhe or Phe or Sar, Y ₂ is allo-I1e or Val or Leu, Y ₃ is MeVal or Val, Y ₄ is β-HOMeVal or γ-HOMeVal or MeVal or V
al or N, β-MeAsp or MePhe or β-HOMePhe or MeDH _{2, 3} Val or MeDH _3, a ₄ Val or Sar. )