JPH11106399A - Peptide derivative and antifungal agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new derivative having excellent antifungal activity and high safety, effective for treating profound mycosis such as serious invasive candidiases as an antifungal agent, comprising a peptide derivative having a specific amino acid sequence of D isomer or L-isomer. SOLUTION: This new peptide derivative (salt) has an amino acid sequence of D-isomer or L-isomer of formula I [Xaa is an amino acid residue of formula II (R is H, a lower alkyl, a lower alkoxy or a halogen)]}, is excellent in antifungal activity, safe and useful for treating serious profound mycosis such as invasive candidiases, etc. A peptide derivative containing specific 6 amino acid residues and an amidated C-terminal group among an amino acid sequence constituting bovine lactoferrin is found to have strong antifungal actions. The incorporation of a unnatural type amino acid residue into the part of the sequence of the peptide derivative is found to show excellent antifungal activity. The peptide derivative is obtained by synthesizing the amino acid sequence by using a solidified-phase synthesis method, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to a novel peptide derivative or a pharmaceutically acceptable salt thereof, and a use thereof. More specifically, the invention of this application relates to safe peptide derivatives that can be used as pharmaceuticals, especially as antifungal agents.

[0002]

2. Description of the Related Art Severe deep mycosis such as invasive candidiasis is often a fatal disease, and its incidence is increasing worldwide. Originally, it is thought that the main defense mechanism of the host organism against fungi such as Candida is nonspecific immunity by neutrophils, and if this defense mechanism functions normally, there is a risk of infection with fungi. Is less. However, in recent years, the number of patients with malignant tumors (in particular, hematopoietic malignant tumors such as acute leukemia and malignant lymphoma) and AIDS and other underlying diseases that cause a decrease in the immune function of the living body has been increasing. It has been attributed to an increase in the incidence of mycosis. It is also considered that the frequent use of medical treatments such as anticancer agents and immunosuppressants is one of the factors that facilitate fungal infection. In addition, antimicrobial antibiotics, heavy use of steroid hormones, long-term central parenteral nutrition, use of intravenous catheters, etc. are also considered as risk factors for developing deep mycosis (see, for example, Clinical and Microbial Studies, Vol. 17, 265). Page, 1990).

[0003] The most effective means for treating such deep mycosis is chemotherapy with an antifungal agent. Currently, drugs that have been put into practical use as therapeutic agents for deep mycosis include antibacterial agents. Much less compared to just six of amphotericin, flucytosine, miconazole, fluconazole, itraconazole, and ketoconazole. on the other hand,
Numerous reports have been made on peptides or derivatives thereof that have antibacterial and antifungal activities against various microorganisms [for example, Biochimica et Biophysica Acta, No. 1197].
Vol. 109, 1994], one of which is lactoferrin.

[0004] Lactoferrin is a natural iron-binding protein contained in tears, saliva, peripheral blood, milk and the like, and has an antibacterial and antifungal action against harmful microorganisms such as Escherichia coli, Candida and Clostridium. It is known to show [e.g., Journal of Pediatrics, Vol. 94, page 1, 19
1979; Archives of Disease in Childhood,
67, 657, 1992].

It is also known that lactoferrin degradation products, lactoferrin-derived peptides, and peptides having homology to these have antibacterial and antifungal activities. For example, an antibacterial agent containing a lactoferrin hydrolyzate as an active ingredient (JP-A-5-32068),
Antimicrobial peptide consisting of at least 20 amino acid residues (Japanese Patent Application Laid-Open No. 5-92994), antimicrobial peptide consisting of 5 amino acid residues (Japanese Patent Application Laid-Open No. 5-148296), and 3 to 6 amino acid residues An amino acid sequence identical to that of an antimicrobial peptide comprising a base (JP-A-5-148297), a lactoferrin degradation product, a peptide isolated from a lactoferrin degradation product, or a peptide isolated from a lactoferrin degradation product An antioxidant containing a synthesized peptide as an active ingredient (JP-A-6-199687)
Patent Publication), peptide derivatives and their uses (Japanese Unexamined Patent Application Publication No.
No. 76190) is known.

[0006]

As described above, only six antifungal agents are used for the treatment of deep mycosis, and in order to effectively cope with various types of fungal infections. There has been a long-awaited demand for new antifungal agents based on a new mechanism. The present invention has been made in view of the above-mentioned situation, has excellent antifungal activity, is safe, and has a novel antifungal peptide derivative based on a completely new mechanism, and an antibacterial peptide derivative. It is intended to provide a novel antifungal agent as an active ingredient.

[0007]

Means for Solving the Problems The inventors of the present application have:
As a result of a detailed study of the antifungal activity of the lactoferrin-derived peptides and their derivatives disclosed in the above-mentioned prior art invention, a specific 6 amino acid residue (Arg) of the amino acid sequence constituting bovine lactoferrin was determined.
-Arg-Trp-Gln-Trp-Arg), and found that a derivative having the same amino acid sequence and amidated C-terminus has strong antifungal activity.

Further, based on this fact, the inventors of the present application have pursued a peptide derivative which expresses a stronger antifungal activity, and as a result, a part of the sequence has a non-natural amino acid residue which has not existed before. By incorporating the groups, a series of peptide derivatives with significant antifungal activity have been successfully created. That is, the present invention provides a first invention which solves the above-mentioned problem, as follows:

[0009]

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Wherein Xaa is the following equation (2)

[0011]

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(Wherein, R represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom)
Represents an amino acid residue represented by. And a pharmacologically acceptable salt thereof having a D-form or L-form amino acid sequence represented by the formula: The present invention also provides a second invention as follows:

[0013]

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[Where Xaa is the following formula (2)

[0015]

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(Wherein, R represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom)
Represents an amino acid residue represented by. As an active ingredient, a peptide derivative having a D-form or L-form amino acid sequence represented by the formula: or a mixture of two or more compounds selected from the group consisting of pharmacologically acceptable salts thereof. Provide an antifungal agent.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide derivative according to the first invention of this application can be synthesized by a known method such as a solid phase method and a liquid phase method. In the case of the solid phase method, each amino acid block is sequentially condensed using a solid phase resin for peptide synthesis to extend the peptide chain. For each amino acid block, use the amino acid derivative in which the N-terminal and side chain reactive groups are protected with an appropriate protecting group, and repeat the condensation reaction and the N-terminal deprotection reaction from the C-terminal side to construct the desired peptide chain. I do.
Next, the peptide in which the N-terminal and side chain reactive groups are protected is cleaved from the resin, taken out, amidated at the C-terminal, and further deprotected to obtain the desired peptide derivative. be able to.

Since the peptide derivatives of the present invention are all derivatives in which the C-terminal is amidated, a solid-phase resin for synthesizing a C-terminal amide peptide can be used instead of a usual solid-phase resin. In this case, it is convenient because the process can be simplified and the resin removal and the deprotection can be performed simultaneously depending on how the protecting group is selected. A so-called automatic peptide synthesizer in which the above operations are mechanized or automated can also be used.

In the case of the liquid phase method, an amino acid derivative in which the reactive group at the N-terminal or C-terminal and the side chain is protected by an appropriate protecting group is basically converted into each amino acid block by a commonly known peptide synthesis method. Using this, the desired peptide chain can be constructed by repeating the condensation reaction and the N-terminal or C-terminal deprotection reaction. In the liquid phase method, in addition to the sequential chain lengthening method of sequentially extending the peptide chain from the C-terminal side or the N-terminal side, the target peptide chain is divided into appropriate fragments, and each fragment chain is synthesized. A so-called fragment condensation method in which each is condensed to construct a final peptide chain can also be applied.

The peptide derivative of the present invention is characterized by containing a non-natural amino acid residue, and the amino acid corresponding to this amino acid residue portion or a protected derivative thereof can be prepared by any known method. Can be synthesized by Some examples are as follows. As shown in the following reaction formula, first, only the carboxyl group at the α-position is selectively tert-butylesterified, and glutamic acid mono-tert-butyl ester to which a 9-fluorenylmethoxycarbonyl group (hereinafter abbreviated as Fmoc) is bound ( A) is reacted with a desired aniline derivative (B) in the presence of a suitable condensing agent such as a carbodiimide, and
The moc glutamic acid mono-tertbutyl ester γ-anilide derivative (C) is obtained. At this time, a condensation additive such as 1-hydroxybenzotriazole (HOBt) and N-hydroxysuccinimide (HOSu) can be used in combination.

Next, Fmoc glutamic acid mono-tert
Butyl ester γ-anilide derivative (C) is subjected to tert-butyl esterification in a strong acid such as trifluoroacetic acid to give Fmoc glutamic acid γ-aniline represented by the general formula (D).
Anilide derivatives can be synthesized.

[0022]

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(Wherein, R represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom.) The peptide derivative of the present invention can be prepared by a known method such as liquid chromatography, column chromatography, recrystallization and the like. It can be purified. Further, synthetic intermediates, synthetic raw materials, and the like used in the production of the peptide derivative of the present invention can be purified by the same method.

Each amino acid residue constituting the peptide derivative of the present invention includes not only L-form but also D-form. That is, the antifungal activity of the peptide derivative of the present invention is not affected by whether the amino acid residue constituting the compound of the present invention is in L-form or D-form. In addition,
In the examples described later, the peptide derivative is synthesized using a peptide synthesizer and an amino acid to which Fmoc is bound as a raw material for synthesis. However, each Fmoc amino acid may be used in an L-form or a D-form according to the purpose of synthesis. Therefore, when it is necessary to clarify these distinctions, the symbol L or D is described before the symbol representing the amino acid.

Further, as a means for specifying and displaying the peptide derivative of the example obtained corresponding to each Fmoc amino acid used as a raw material, for example, it is displayed as shown in the following formula.

[0026]

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In this example, the Xaa portion is L
This shows that the Fmoc amino acid in the form is a peptide derivative synthesized using the Fmoc amino acid in the D form for the other 5 amino acid residue equivalent parts. The peptide derivative of the present invention is characterized in that it contains non-natural amino acid residues, and these amino acid residues are formally a side chain terminal amide of glutamine which is one of natural amino acid residues. Since it can be considered that the hydrogen atom on the nitrogen atom of the group is substituted by a phenyl group (which may have a substituent), the hydrogen atom is represented by a shorthand notation as shown in the following formula.

[0028]

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(Wherein, R represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom.) The specific structural formula thereof is as shown in the following formula.

[0030]

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(Wherein, R represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom.) The pharmacologically acceptable salts of the peptide derivative of the present invention are further described below. Examples thereof include a metal complex and an acid addition salt. The metal complex is, for example, a complex of zinc, iron, calcium, magnesium or aluminum. As acid addition salts,
Hydrochloride, hydrobromide, sulfate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, acetate, trifluoroacetate,
Citrate, benzoate, succinate, malate, ascorbate, tartrate and the like can be exemplified.
Further, a carboxylate, for example, a sodium salt or a potassium salt with an alkali metal, or an ammonium salt such as a calcium salt or a magnesium salt with an alkaline earth metal may be used.

Next, the antifungal agent according to the second invention of this application will be described. The antifungal agent of the present invention contains, as an active ingredient, any one compound selected from the group consisting of the peptide derivatives and their pharmacologically acceptable salts or a mixture of two or more thereof. . Further, the antifungal agent of the present invention may contain a known antifungal agent in addition to the above-mentioned active ingredient.

The antifungal agent of the present invention can be prepared in a conventional manner, for example, as an ointment, gel, paste, cream, spray, suspension, emulsion, syrup, tablet, sugar, capsule, granule, powder, etc. It can also be processed. The dosage of the antifungal agent of the present invention varies depending on the subject to be treated, the condition, the age, and the like, but preferably about 0.1 to 50 mg per administration is usually administered about 1 to 3 times a day. In the case of superficial mycosis, an ointment containing 0.01 to 5% (weight; hereinafter the same unless otherwise specified) may be topically applied about 1 to 3 times a day.

In the specification of this application, the meanings of antibacterial activity (antibacterial agent) and antifungal activity (antifungal agent) are distinguished from each other. Next, test examples will be shown, and the action and effect of the antifungal peptide derivative of the present invention will be described in more detail. Test Example 1 This test was performed to examine the sensitivity of the peptide derivative of the present invention to fungi. (1) Test strains The following four strains were used as test strains.

Candida albicans ATCC 90028 Candida tropicalis ATCC 750 Candida parapsilosis ATCC 90018 Candida glabrata ATCC 90030 These strains are all of the American type.
It is readily available from the Culture Collection (ATCC). (2) Test method 1) Preparation of medium 2 g of Bacto Peptone (manufactured by Difco) and 4 g of D-glucose (manufactured by Wako Pure Chemical Industries, Ltd.) were added to distilled water for injection (manufactured by Otsuka Pharmaceutical Factory Co., Ltd., hereinafter referred to as distilled water). ), Adjusted to a total volume of 200 ml, sterilized in an autoclave at 121 ° C for 15 minutes, and stored at 4 ° C until use. 2) Preparation of test substance Peptide derivative of the present invention prepared by the same method as in Examples 1 to 3, Example 6, and Example 7, and compound prepared by the same method as Comparative Examples 1 and 2 Was dissolved in distilled water to prepare a 1 mg / ml solution, which was used as a reference solution. This reference solution was stored frozen at -20 ° C or lower until use. 3) Preparation of Positive Control Drug Amphotericin B (manufactured by Sigma), miconazole (manufactured by Sigma), and fluconazole (manufactured by Pfizer) used as the positive control drug. The contents of the diflucan capsule were extracted with ethanol, and the residue under reduced pressure was concentrated. Recrystallized from ethyl acetate-hexane.) Was dissolved in dimethyl sulfoxide (Sigma; cell culture) and 10 mg / m 2
A 1-concentration solution was prepared and used as a stock solution. This stock solution was stored frozen at -20 ° C or lower until use, and was diluted 10-fold (1 mg / ml) with distilled water at the time of use, and this was used as a standard solution. 4) Preparation of inoculum bacterial solution Calculation of the number of bacteria in the culture solution One streak from the slant was inoculated into the medium (2 ml),
Incubate at 35 ° C. for 19 hours, and use a spectrophotometer at a wavelength of 6
The OD value at 60 nm was measured. On the other hand, part of the medium (50
μl) in a fresh medium (5 ml), and transferred to 35 ° C.
For 6 hours, and the OD value of the bacterial solution and the viable cell count were measured. The viable cell count was measured by diluting the bacterial solution 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ -fold, smearing 0.1 ml of each on an SDA agar medium, culturing, and counting the number of colonies that appeared the next day. A calibration curve of OD value / number of bacteria was obtained, and the number of bacteria per 1 ml of bacterial solution was calculated.

Preparation of Inoculated Bacterial Solution A scrape from the slant was inoculated into the above medium (2 ml),
After culturing at 35 ° C. for 19 hours, a part thereof was aseptically collected, and the OD value at a wavelength of 660 nm was measured with a spectrophotometer (the OD value was about 1.0, and the number of bacteria was about 1 × 10 ^7. / Ml).

As a result of inoculating a 1/100 amount of this bacterial solution into a fresh medium (10 to 15 ml), culturing at 35 ° C. for 6 hours, and measuring the OD value at a wavelength of 660 nm,
At 0.1-0.2, the bacterial count was calculated to be about 1 × 10 ⁶ cells / ml. The culture was first diluted 50-fold and then 10-fold with the above-mentioned medium, and diluted with an inoculum (2 × 10 ³ cells / m 2).
l) was prepared. If it could not be used within 15 minutes after preparation, it was stored at 4 ° C and used within 2 hours. 5) Preparation of Drug Diluent The medium was dispensed into a 96-well flat-bottom microplate using a multipipette, 160 μl in the first row and 100 μl in the second to tenth rows. In the 11th row, 100 μl was dispensed into the upper four rows (growth control), and 200 μl into the lower four rows (negative control).

[0038] Diluted solution (concentration: 1 mg / ml) of test substance and control drug prepared in 2) and 3) above, 40 µm
1 was added to the first row and mixed, of which 100 μl was added to 2
Added to row. Repeat this operation up to the 10th column to
Serial dilutions were performed. In the last ten rows, 100 μl was collected and discarded. 6) Bacterial inoculation and culturing The inoculated bacterial solution prepared in 4) was dispensed in 100 μl portions into all four holes in the first to tenth and eleventh rows and cultured at 35 ° C. for 2 days. 7) Judgment The end point is defined as the concentration at which growth is completely blocked visually with a microplate viewer, as the minimum growth inhibitory concentration (hereinafter referred to as M
Described as IC value. ). (3) Test results The results of this test are as shown in Table 1. Samples produced by the same method as in each of the examples in the table are peptide derivatives of the present invention, and samples produced by the same method as in the comparative example are those of the specific amino acid sequence constituting bovine lactoferrin. 6 amino acid residues (Arg-Arg
-Trp-Gln-Trp-Arg) is a compound having the same amino acid sequence as above, a derivative in which the C-terminus is amidated, and known as a peptide derivative having an antibacterial action or an antifungal action.

The compound of Comparative Example 1 is a compound represented by LArg-LArg-LTrp-LGln-LTrp-LArg-NH2 in which all six amino acid residues are in the L form. The compound of Comparative Example 2 is an enantiomer of Comparative Example 1 described above, in which all six amino acid residues are in the D-form, and are represented by DArg-DArg-DTrp-DGln-DTrp-DArg-NH2. .

No difference in the antifungal activity between the compound of Comparative Example 1 and the compound of Comparative Example 2, that is, the difference between the L-form and the D-form was observed for any fungi. Candid
All the compounds of the comparative examples had an MIC value of 12.5 μg / ml against a albicans ATCC 90028.
On the other hand, all of the peptide derivatives of the present invention had MIC values of 6.25 to 12.5 μg / ml, and an antifungal activity equivalent to or twice that of the compound of Comparative Example was observed.

With respect to Candida tropicalis ATCC 750, all of the compounds of Comparative Examples had MIC values of 3.13 μm.
g / ml. In contrast, the peptide derivatives of the present invention all have MIC values of 1.56 to 3.13 μg.
/ Ml, and an antifungal effect equivalent to or twice that of the compound of Comparative Example was observed. Candida parapsilosis ATC
For C 90018, the compounds of Comparative Examples were all MI
The C value was 50 μg / ml. In contrast, the peptide derivatives of the present invention all have MIC values of 12.5 to 12.5.
It was 25 μg / ml, and the antifungal activity was 2 to 4 times that of the compound of Comparative Example.

For Candida glabrata ATCC 90030, all of the compounds of the comparative examples had MIC values of 50 μg /
ml. On the other hand, the peptide derivatives of the present invention all had MIC values of 12.5 to 50 μg / ml, and showed an antifungal activity equivalent to or four times that of the compound of Comparative Example. As is evident from these test results, all of the peptide derivatives of the present invention generally exhibited an antifungal action superior to known peptides. In addition,
Similar tests were performed for other peptide derivatives,
Almost equivalent results were obtained.

[0043]

[Table 1]

Test Example 2 This test was conducted to examine the acute toxicity of the peptide derivative of the present invention. (1) Test animals Four-week-old male ddY mice (purchased from Japan SLC) were acclimated for at least one week, and then divided into four groups (five animals per group). (2) Test method 100 mg or 500 mg of the peptide derivative of the present invention produced by the same method as in Example 1 per kg of body weight
Was dissolved in distilled water at a ratio of 1 and administered once by oral gavage to test acute toxicity. (3) Test results No fatal cases were found in the groups administered at the rates of 100 mg / kg body weight and 500 mg / kg body weight. Therefore, the LD ₅₀ value of the peptide derivative of the present invention is 500 mg
/ Kg body weight or more, and the toxicity was found to be extremely low. The same test was conducted for other peptide derivatives, but almost the same results were obtained.

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

[0046]

【Example】

Example 1 Peptide synthesizer (Applied Perkin Elmer)
Made by Biosystems Division. Type 433A. Hereinafter, it is abbreviated as "manufactured by PE Co." ), And a solid-phase peptide synthesis method using the same equipment's instruction manual and Shepard [Journal
Of Chemical Society Parkin I (Journa
l of Chemical Society Perkin I), page 538, 1
981], the peptide was synthesized as follows.

First, 397 mg of Fmoc amide resin (manufactured by PE) which is a solid-phase resin for synthesizing a C-terminal amide peptide.
(0.25 mmol), the deprotection group reaction and the condensation reaction were repeated by the synthesis program of the peptide synthesizer to extend the peptide chain. That is, first 20%
Piperidine-containing N-methylpyrrolidone (manufactured by PE; hereinafter, N-methylpyrrolidone is abbreviated as NMP)
Fmoc, which is an amino-protecting group of the solid phase resin, was cleaved off and washed with NMP. Then, Fmoc amino acid [Fmoc-D-Arg (Pmc) -OH (manufactured by Watanabe Chemical Industry Co., Ltd.)] was added to FastMo.
c Condensed using (registered trademark) Regent Kit (manufactured by PE) and washed with NMP. Hereinafter, the operations from the cleavage of the Fmoc group to the condensation washing of the Fmoc amino acid were repeated.

However, after the next step, the Fmoc amino acid was added in the order of the steps, Fmoc-D-Trp (Boc) -OH (manufactured by Watanabe Chemical Industry Co., Ltd.), Fmoc-D-Gln (4Cl-Ph) -OH (see Reference Example below). Fmoc-D-Trp (Boc) -OH (manufactured by Watanabe Chemical Industry) and Fmoc-D-Arg (Pmc) -OH (manufactured by Watanabe Chemical Industry)
Was used. 1.0 mmol of each Fmoc amino acid
The amount corresponding to was used in a special cartridge. Conditions for the condensation reaction and deprotection conditions were automatically controlled by a feedback monitoring system attached to the apparatus. After the completion of the peptide chain extension reaction, 20
The N-terminal Fmoc group is cleaved with NMP containing 5% piperidine, washed with NMP and dichloromethane (manufactured by PE), dried under vacuum, and protected with a protected peptide resin 764 mg (yield 9).
5%).

322 mg of the above protected peptide resin (0.
1 mmol) to ethanedithiol (Watanabe Chemical Industry Co., Ltd.)
1.2 ml, meta-cresol (manufactured by Watanabe Chemical Industry Co., Ltd.)
4 ml and thioanisole (manufactured by Watanabe Chemical Industry Co., Ltd.)
4 ml of the mixture was stirred at room temperature under a stream of argon for 15 minutes, cooled with ice, and further stirred for 10 minutes. Then, trifluoroacetic acid (manufactured by Watanabe Chemical Industry Co., Ltd .; hereinafter abbreviated as TFA) 15
Then, 2.7 ml of trimethylsilyl bromide (manufactured by Watanabe Chemical Industry Co., Ltd.) was added, and the mixture was stirred for 50 minutes. Thereafter, the resin was filtered off with a glass filter, and the filtrate was quickly concentrated under reduced pressure. Preliminarily cooled anhydrous diethyl ether (manufactured by Kokusan Chemical) was added to the residue to convert the peptide into a white powder.

Then, the mixture was transferred to a centrifuge tube and centrifuged (2500
rpm, 10 minutes), the supernatant was discarded, cold diethyl ether was newly added, and the operation of sufficiently stirring and centrifuging again was repeated four times. Thereafter, the peptide precipitate was vacuum-dried, dissolved in water and freeze-dried to obtain about 42 mg of the desired crude peptide derivative. The whole amount of the crude peptide derivative was dissolved in water, centrifuged (15000 rpm, 5 minutes), the supernatant was filtered with a 0.45 μm filter, and purified by high performance liquid chromatography under the following conditions. The column is a reversed-phase Lichrospher 100 RP-18 (e) 2
50 × 10 mm (manufactured by Merck) was used. The eluent is 0.
Using 1% TFA / water as solution A and 80% acetonitrile / A solution as solution B, elution was performed with a linear concentration gradient from solution A to solution B. Lyophilize the main peak fraction again, and

[0051]

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As a result, about 17.4 mg (yield: about 16%) of the peptide derivative of the present invention was obtained as a white powder. Example 2 Instead of Fmoc-D-Gln (4Cl-Ph) -OH, Fmoc-D-Gln (4Br-Ph) -OH 523 produced by the same method as in Reference Example 2 described later.
Example 1 except that mg (1.0 mmol) was used.
By the same method as

[0053]

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As a result, about 23.0 mg (yield: about 20%) of the peptide derivative of the present invention as a white powder was obtained. Example 3 Instead of Fmoc-D-Gln (4Cl-Ph) -OH, Fmoc-D-Gln (4F-Ph) -OH462m produced by the same method as in Reference Example 3 described later.
g (1.0 mmol), except that the following formula was used in the same manner as in Example 1.

[0055]

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As a result, about 35.7 mg (yield: about 33%) of the peptide derivative of the present invention as a white powder was obtained. Example 4 Instead of Fmoc-D-Gln (4Cl-Ph) -OH, 444 mg of Fmoc-D-Gln (Ph) -OH produced by the same method as Reference Example 4 described later.
(1.0 mmol) by the same method as in Example 1 except that

[0057]

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As a result, about 21.9 mg (yield: about 20%) of the peptide derivative of the present invention in the form of a white powder was obtained. Example 5 Instead of Fmoc-D-Gln (4Cl-Ph) -OH, Fmoc-D-Gln (4Me-Ph) -OH 458 produced by the same method as Reference Example 5 described later.
Example 1 except that mg (1.0 mmol) was used.
By the same method as

[0059]

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As a result, about 26.0 mg (yield: about 23%) of the peptide derivative of the present invention was obtained as a white powder. Example 6 Instead of Fmoc-D-Gln (4Cl-Ph) -OH, Fmoc-D-Gln (3Me-Ph) -OH 458 produced by the same method as in Reference Example 6 described later.
Example 1 except that mg (1.0 mmol) was used.
By the same method as

[0061]

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As a result, about 23.8 mg (yield: about 21%) of the peptide derivative of the present invention was obtained in the form of a white powder. Example 7 Instead of Fmoc-D-Gln (4Cl-Ph) -OH, Fmoc-D-Gln (4OMe-Ph) -OH475 produced by the same method as Reference Example 7 described later.
Example 1 except that mg (1.0 mmol) was used.
By the same method as

[0063]

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As a result, about 11.6 mg (yield: about 11%) of the peptide derivative of the present invention in the form of a white powder was obtained. Example 8 About 10 peptide derivatives obtained by the same method as in Example 1
mg was dissolved in 5 ml of 0.02 mol / l hydrochloric acid, further diluted to 20 ml with water, and freeze-dried to obtain about 10 mg of a hydrochloride of the compound of Example 1 in the form of a white powder. Example 9 A tablet having the following composition was produced by a conventional method.

Lactose (manufactured by Wako Pure Chemical Industries) 78.2 (%) Peptide derivative obtained by the same method as in Example 1 1.2 Magnesium stearate (manufactured by Wako Pure Chemical Industries) 20.0 Example 10 An injection having the following composition was produced by a conventional method. The peptide derivative obtained by the same method as in Example 1 was dissolved in physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) at a rate of 6.0 mg / ml,
The solution was passed through a sterile filtration filter to produce an injection. Example 11 An ointment having the following composition was produced by a conventional method. Except for peptide derivatives, all commercial products were used.

Vaseline 26.3 (%) Paraffin 5.3 Cetostearyl alcohol 2.1 Propylene glycol 10.5 Polyoxyethylene / polyoxypropylene glycol ether 3.2 Peptide derivative obtained by the same method as in Example 1 0 5.5 Purified water 52.1 Example 15 A skin external preparation having the following composition was produced by a conventional method. In addition,
All commercial products were used except for peptide derivatives.

Ethyl paraoxybenzoate 0.1 (%) Butyl paraoxybenzoate 0.1 Lauromacrogol 0.5 Cetanol 20.0 White petrolatum 40.0 Purified water 34.3 Obtained by the same method as in Example 1. Peptide Derivative 5.0 Next, a production example of a compound for comparison with the peptide derivative of the present invention will be shown as a comparative example. Comparative Example 1 Instead of Fmoc-D-Gln (4Cl-Ph) -OH, Fmoc-L-Gln (Trt) -OH
OH (manufactured by PE) was used in the same manner as in Example 1 except that 597 mg (1.0 mmol) was used and the L-form (all manufactured by PE) was used instead of the D-form for all other amino acid blocks. Depending on the method,

[0068]

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The compound represented by the following formula (4) in the form of a white powder:
3.2 mg (about 44% yield) were obtained. Comparative Example 2 Instead of Fmoc-D-Gln (4Cl-Ph) -OH, Fmoc-D-Gln (Trt) -OH
597 mg (1.0 mmol) of OH (manufactured by Watanabe Chemical Industry Co., Ltd.)
By the same method as in Example 1 except that

[0070]

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The compound represented by the following formula (4) in the form of a white powder:
9.0 mg (about 50% yield) was obtained. Next, a production example of an intermediate for producing the peptide derivative of the present invention will be shown as a reference example. Reference Example 1 [Production of Fmoc-D-Gln (4Cl-Ph) -OtBu] 1276 mg of Fmoc-D-Gln-OtBu (manufactured by Watanabe Chemical Industry Co., Ltd.)
(3.0 mmol), 551 mg (3.6 mmol) of 1-hydroxybenzotriazole monohydrate (manufactured by Shimadzu Corporation)
l) and 20 ml of methylene chloride (manufactured by Kokusan Chemical Co., Ltd.) were cooled on ice, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Kokusan Chemical Co., Ltd.) 6
90 mg (3.6 mmol) were added in small portions. After completion of the addition, the mixture was stirred under ice cooling for a while and then at room temperature for 2 hours. The reaction mixture was ice-cooled again, and a methylene chloride solution of 383 mg (3.0 mmol) of 4-chloroaniline (manufactured by Tokyo Chemical Industry Co., Ltd.) was added dropwise. After the completion of dropping, under ice cooling for a while,
The mixture was further stirred at room temperature overnight. The solvent was distilled off, and ethyl acetate and 1N-hydrochloric acid were added to the residue to separate the organic layer.
The extract was washed once with an aqueous sodium bicarbonate solution and then once with a saturated saline solution, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel-column chromatography (ethyl acetate: hexane = 1: 50 to 1: 2 as a solvent).
use. ),

[0072]

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The white crystals of Fmoc-D-Gln (4
About 1051 mg (about 65.5% yield) of Cl-Ph) -OtBu were obtained. Reference Example 2 [Production of Fmoc-D-Gln (4Cl-Ph) -OH] 803 mg of Fmoc-D-Gln (4Cl-Ph) -OtBu of Reference Example 1 (1.
5 mmol) was added and dissolved under ice-cooling with 6 ml of trifluoroacetic acid (manufactured by Watanabe Chemical Industry Co., Ltd.), and the mixture was stirred at room temperature for 4 hours under protection of a calcium chloride tube. Excess trifluoroacetic acid was distilled off under reduced pressure at room temperature, and the residue was treated with ether and crystallized. The crystals are filtered off, washed several times with ether, further washed with hexane, dried and

[0074]

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The white crystals of Fmoc-D-Gln (4
About 598 mg (yield about 83.3%) of Cl-Ph) -OH was obtained. Reference Example 3 [Production of Fmoc-D-Gln (4Br-Ph) -OtBu] Except that 516 mg (3.0 mmol) of 4-bromoaniline (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of 4-chloroaniline. By the same method as in Reference Example 1,

[0076]

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The white crystals of Fmoc-D-Gln (4
About 1179 mg of Br-Ph) -OtBu (yield about 67.8%) was obtained. Reference Example 4 [Production of Fmoc-D-Gln (4Br-Ph) -OH] Fmoc-D-Gln produced by the same method as Reference Example 3 instead of Fmoc-D-Gln (4Cl-Ph) -OtBu (4Br-Ph) -OtBu 869
Example 2 except that mg (1.5 mmol) was used.
By the same method as

[0078]

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The white crystals of Fmoc-D-Gln (4
About 683 mg (yield about 87.0%) of Br-Ph) -OH was obtained. Reference Example 5 [Production of Fmoc-D-Gln (4F-Ph) -OtBu] Except that 333 mg (3.0 mmol) of 4-fluoroaniline (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of 4-chloroaniline. By the same method as in Reference Example 1,

[0080]

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The white crystals of Fmoc-D-Gln (4
About 1123 mg (yield about 72.2%) of F-Ph) -OtBu was obtained. Reference Example 6 [Production of Fmoc-D-Gln (4F-Ph) -OH] Fmoc-D-Gln produced by the same method as Reference Example 5 instead of Fmoc-D-Gln (4Cl-Ph) -OtBu (4F-Ph) -OtBu778m
g (1.5 mmol) by the same method as in Reference Example 2 except that g (1.5 mmol) was used.

[0082]

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Fmoc-D-Gln of white crystals represented by
About 638 mg of (4F-Ph) -OH was obtained (yield: about 91.9%). Reference Example 7 [Production of Fmoc-D-Gln (Ph) -OtBu] The same as Reference Example 1 except that 279 mg (3.0 mmol) of aniline (manufactured by Aldrich) was used instead of 4-chloroaniline. Depending on the method,

[0084]

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The white crystals of Fmoc-D-Gln (P
h) -OtBu About 1138 mg (yield about 75.8%) was obtained. Reference Example 8 [Production of Fmoc-D-Gln (Ph) -OH] Instead of Fmoc-D-Gln (4Cl-Ph) -OtBu, Fmoc-D-Gln (Ph) produced by the same method as Reference Example 7 ) -OtBu 751mg
(1.5 mmol), except that the following formula was used in the same manner as in Reference Example 2.

[0086]

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A white crystal of Fmoc-D-Gln (P
h) -OH (about 601 mg, about 90.2% yield). Reference Example 9 [Production of Fmoc-D-Gln (4Me-Ph) -OtBu] Except that 321 mg (3.0 mmol) of p-toluidine (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of 4-chloroaniline. By the same method as in Reference Example 1,

[0088]

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The white crystals of Fmoc-D-Gln (4
About 1090 mg (yield about 70.6%) of Me-Ph) -OtBu was obtained. Reference Example 10 [Production of Fmoc-D-Gln (4Me-Ph) -OH] Fmoc-D-Gln produced by the same method as Reference Example 9 instead of Fmoc-D-Gln (4Cl-Ph) -OtBu (4Me-Ph) -OtBu 772
Example 2 except that mg (1.5 mmol) was used.
By the same method as

[0090]

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The white crystals of Fmoc-D-Gln (4
About 649 mg (yield about 94.4%) of Me-Ph) -OH was obtained. Reference Example 11 [Production of Fmoc-D-Gln (3Me-Ph) -OtBu] Except that 321 mg (3.0 mmol) of m-toluidine (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of 4-chloroaniline. By the same method as in Reference Example 1,

[0092]

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A white crystal of Fmoc-D-Gln (3
About 983 mg (yield about 63.7%) of Me-Ph) -OtBu was obtained. Reference Example 12 [Production of Fmoc-D-Gln (3Me-Ph) -OH] Fmoc-D-Gln produced by the same method as Reference Example 3 instead of Fmoc-D-Gln (4Cl-Ph) -OtBu (3Me-Ph) -OtBu 772
Example 2 except that mg (1.5 mmol) was used.
By the same method as

[0094]

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The white crystals of Fmoc-D-Gln (3
About 642 mg (yield about 93.4%) of Me-Ph) -OH was obtained. Reference Example 13 [Production of Fmoc-D-Gln (4OMe-Ph) -OtBu] Except that 369 mg (3.0 mmol) of p-anisidine (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of 4-chloroaniline. By the same method as in Reference Example 1,

[0096]

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The white crystals of Fmoc-D-Gln (4
OMe-Ph) -OtBu (about 1437 mg, yield: about 90.3%) was obtained. Reference Example 14 [Production of Fmoc-D-Gln (4OMe-Ph) -OH] Fmoc-D-Gln produced by the same method as Reference Example 13 instead of Fmoc-D-Gln (4Cl-Ph) -OtBu (4OMe-Ph) -OtBu79
Except that 6 mg (1.5 mmol) was used, the following formula was obtained in the same manner as in Reference Example 2.

[0098]

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The white crystals of Fmoc-D-Gln (4
About 676 mg (yield about 95.0%) of OMe-Ph) -OH was obtained.

[0100]

As described above in detail, the invention of the present application provides a novel peptide derivative having excellent antifungal activity and being safe, and an antifungal agent containing the same as an active ingredient.

Claims (2)

[Claims] 1. The following formula (1) [Where Xaa is the following formula (2): (Wherein, R represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom). Or a pharmacologically acceptable salt thereof having a D-form or L-form amino acid sequence represented by the formula: 2. The following formula (1): [Where Xaa is the following formula (2): (Wherein, R represents a hydrogen atom, a lower alkyl group, a lower alkoxy group or a halogen atom). As an active ingredient, a peptide derivative having a D-form or L-form amino acid sequence represented by the formula: Antifungal agent.