JP4395579B2 - Antifungal compound - Google Patents

Description

  The present invention relates to novel compounds having antifungal activity.

  The incidence of deep mycosis tends to increase as the number of susceptible patients increases, and an effective therapeutic drug is desired. Currently, there are only 6 drugs for the treatment of deep mycosis that are marketed in Japan, and 3 of these are azole drugs (miconazole, fluconazole, itraconazole). In addition to its bacteriostatic action, fluconazole, a representative example, is concerned about the emergence of resistant bacteria as the amount used increases. On the other hand, amphotericin B, which is a polyene antibiotic having a high bactericidal effect, is highly toxic and is not necessarily a safe drug. For these reasons, medical satisfaction with deep fungal infections is generally low, and there is still a great demand for new drugs that are bactericidal and fungal selective.

  The subject of this invention is providing the antifungal compound useful as an active ingredient of the pharmaceutical for the prevention and / or treatment of fungal infection.

  As a result of intensive studies to solve the above problems, the inventors of the present invention include a novel zinc chelate compound represented by the following formula in a culture of a microorganism belonging to the genus Nocardia, It was found to have strong antibacterial activity against fungi. Conventionally, no zinc chelate compound having an antifungal action has been known, and this compound has a very specific structure and action. The present invention has been completed based on the above findings.

  That is, according to the present invention, the following formula (I):

で表される化合物又はその塩が提供される。好ましい態様によれば、下記の式（II）：

Or a salt thereof is provided. According to a preferred embodiment, the following formula (II):

で表される化合物又はその塩が提供される（式中の立体標記は絶対配置である）。

Or a salt thereof is provided (in which the three-dimensional designation is an absolute configuration).

  In another aspect, a medicament for the prevention and / or treatment of fungal infection, comprising a compound represented by the above formula (I) or formula (II) or a salt thereof as an active ingredient Is provided by the present invention. And a method for preventing and / or treating a fungal infection, comprising the use of a compound represented by the above formula (I) or formula (II) or a salt thereof for the manufacture of the above medicament; A method comprising the step of administering a prophylactic and / or therapeutically effective amount of a compound represented by (I) or formula (II) or a salt thereof to a mammal, including a human, is provided by the present invention.

  From still another aspect, the method for producing a compound represented by the above formula (I) was obtained by culturing a bacterium producing the compound represented by the above formula (I) belonging to the genus Nocardia. A method comprising the step of separating and collecting the compound from the culture is provided. Nocardia transvalensis can be used as a producer of the compound represented by the above formula (I) belonging to the genus Nocardia.

  According to the present invention, a novel compound having an antifungal action or a salt thereof is provided, and a medicament containing this compound or a salt thereof as an active ingredient is useful as a medicament for the prevention and / or treatment of fungal infections.

  The compound of the present invention is a compound represented by the above formula (I). Stereoisomers such as any optically active or diastereoisomer of this compound are also included within the scope of the present invention. In addition to pure forms of stereoisomers, any mixture of stereoisomers and racemates are also included within the scope of the present invention. The compounds of the present invention may exist in the form of a salt, and the free form of the compound or salt thereof may exist as a hydrate or solvate, any of which are within the scope of the present invention. Is done. Although the kind of salt is not specifically limited, For example, the acid addition salt of mineral acids, such as hydrochloride, may be formed. Of the compounds of the present invention, a compound represented by the formula (II) is preferable.

  The compound of the present invention can be easily produced by culturing a bacterium producing the compound of the present invention belonging to the genus Nocardia. Further, the compound of the present invention can be produced synthetically or semi-synthetically by using a compound obtained from a culture of microorganisms as a production intermediate. The microorganism belonging to the genus Nocardia is not particularly limited as long as it is a microorganism capable of producing the compound of the present invention. For example, Nocardia transvalensis can be preferably used. As Nocardia transvalensis, for example, Nocardia transvalensis strain IMF10065 can be used.

  Nocardia transvalensis strain IMF10065 is a strain isolated from clinical materials. As of November 4, 2003, RIKEN Microbial System Preservation Facility (2-1 Hirosawa, Wako-shi, Saitama, Japan) JCM12242 and the applicant guarantees the preservation and distribution of the strain. This strain corresponds to a microorganism that cannot be deposited in the Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology) (1-1 Higashi 1-chome, Tsukuba City, Ibaraki, Japan).

  Nocardia transvalensis strain IMF10065 has the following morphological characteristics. When this strain is cultured on Mueller Hinton agar medium containing 0.2% glucose, it has long branched mycelia and aerial mycelium as seen in a kind of Actinomycetes. By lengthening the culture, several spores were observed at the tip of the aerial hyphae, and further rupture of the vegetative mycelium was observed. From the fact that vegetative mycelia was observed to be broken, it was presumed to belong to the genus Nocardia.

In addition, this strain is cultured in a brain heart infusion liquid medium containing 2% glucose after shaking culture at 250 rpm, 30 ° C. for 72 hours. The grown cells are sterilized with formalin and centrifuged (3000 rpm × 10 minutes). Collected, washed twice with distilled water, then dried by vacuum freeze-drying, and cell wall components (diaminopimelic acid, cell wall composition, lipid composition) based on Bergey's manual of Determinative Bacteriology 9th ed, Williams & Willkins, Baltimore, 1993 As a result of analysis, meso-diaminopimelic acid (meso-DAP) was detected from the amino acid analysis results, and arabinose and galactose were detected from the sugar analysis results. The lipid analysis results confirmed the presence of mycolic acid as a cell wall lipid component, and the type was Nocardia Type (including Rhodococcus). It was revealed that isoprenoid quinone, which is a component of the electron transfer system, is MK-8 (H ₄ , cycle) as a main component, and this strain was confirmed to be a microorganism belonging to the genus Nocardia. In addition, by extracting genomic DNA from this strain and determining the sequence of 16S rDNA, and further comparing the homology, phylogenetic analysis based on the 16S rDNA nucleotide sequence (Methods for identifying actinomycetes, edited by Japan Actinomycetes Study Group, 1985), it was confirmed to belong to Nocardia transvalensis.

  Further, this strain had the physiological biochemical properties shown in Table 1 below. Judging from these results, this strain was identified as Nocardia transvalensis.

ノカルジア属に属する本発明の化合物の産生菌を培養することにより、培養物中に本発明の化合物が蓄積されるので、培養物から容易に本発明の化合物を分離・採取することができる。上記の培養に用いる培地の種類は特に限定されず、炭素源、窒素源、及び無機塩などを適宜の割合で含有するものであれば合成培地又は天然培地のいずれでも好適に用いることができる。必要に応じて、ビタミン類又はその他栄養物質を適宜加えてもよい。

By culturing the bacterium producing the compound of the present invention belonging to the genus Nocardia, the compound of the present invention is accumulated in the culture, so that the compound of the present invention can be easily separated and collected from the culture. The kind of the medium used for the culture is not particularly limited, and any of a synthetic medium and a natural medium can be suitably used as long as it contains a carbon source, a nitrogen source, an inorganic salt, and the like in appropriate proportions. If necessary, vitamins or other nutrient substances may be added as appropriate.

  Examples of the carbon source include sugars such as glucose, maltose, fructose, sucrose and starch, alcohols such as glycerol and mannitol, amino acids such as glycine, alanine and asparagine, and fats and oils such as soybean oil and olive oil. One or more general carbon sources can be used. Examples of the nitrogen source include one or more organic nitrogen-containing compounds such as soybean powder, corn steep liquor, beef extract, peptone, yeast extract, amino acid mixture, or fish meal, or inorganic nitrogen compounds such as ammonium salts and nitrates. Can be used. As the inorganic salt, for example, calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, copper sulfate, manganese chloride, zinc sulfate, cobalt chloride, and various phosphates may be added as necessary. Further, an antifoaming agent such as vegetable fat, polypropylene alcohol and the like can be added as necessary.

  The culture temperature can be appropriately changed as long as the microorganism grows and the compound of the present invention is efficiently produced, but is preferably about 20 to 37 ° C, more preferably about 27 ° C. The pH at the start of the culture is preferably near neutral. The culture time is not particularly limited, but is usually about 5 to 8 days. In general, the cultivation may be terminated when the production amount of the compound of the present invention reaches an amount that can be collected, and preferably reaches the maximum. The culture method is not particularly limited, and any method may be employed as long as it is a commonly used method such as solid layer culture or normal stirring culture.

  The method for separating and collecting the compound of the present invention from the culture is not particularly limited, and one or a combination of two or more methods usually used for separating and collecting a low molecular weight compound from a microorganism culture can be used. it can. For example, various ion exchange resins, nonionic adsorption resins, gel filtration chromatography, chromatography using an adsorbent such as activated carbon, alumina or silica gel, or a separation method using high performance liquid chromatography, or crystallization, vacuum concentration, or Freeze-drying means can be used alone or in appropriate combination.

  The compound of the present invention has an antibacterial action against various fungi and is useful as an active ingredient of a medicament for the prevention and / or treatment of fungal infections. As the medicament of the present invention, a substance selected from the group consisting of the compound represented by the above formula (I) and a salt thereof, and a hydrate and a solvate thereof can be used. Two or more kinds of substances may be used in combination. As the medicament of the present invention, the above-mentioned substance as an active ingredient may be administered as it is, but it is preferable to prepare and administer a pharmaceutical composition containing the above-mentioned substance as an active ingredient and a pharmaceutical additive. .

  The administration route of the medicament of the present invention is not particularly limited and can be administered orally or parenterally, but it is desirable to appropriately select the most effective route for prevention and / or treatment of fungal infection. Examples of the pharmaceutical composition suitable for oral administration include capsules, powders, tablets, granules, fine granules, emulsions, syrups, solutions, suspensions, etc., and pharmaceuticals suitable for parenteral administration. Examples of the composition include inhalants, sprays, rectal agents, injections, drops, ointments, creams, transdermal absorption agents, transmucosal absorption agents, eye drops, nasal drops, ear drops, Examples thereof include a tape and a patch, but the form of the medicament of the present invention is not limited thereto.

  Among pharmaceutical compositions suitable for oral administration, for example, liquid preparations such as emulsions and syrups include water; sugars such as sucrose, sorbit and fructose; glycols such as polyethylene glycol and propylene glycol; sesame oil, olive oil, soybean oil and the like Oils; Preservatives such as p-hydroxybenzoates; and other additives for pharmaceutical use such as strawberry flavors and flavors such as peppermint. Solid preparations such as capsules, tablets, powders, and granules include excipients such as lactose, glucose, sucrose, and mannitol; disintegrants such as starch and sodium alginate; lubricants such as magnesium stearate and talc; It can be produced using a binder such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin; a surfactant such as a fatty acid ester; a plasticizer such as glycerin.

  Among pharmaceutical compositions suitable for parenteral administration, liquid preparations in the form of injections, drops, eye drops and the like can be prepared preferably as sterilized isotonic liquid preparations. For example, injections can be prepared using an aqueous medium consisting of a salt solution, a glucose solution, or a mixture of saline and glucose solution. Rectal administration agents can be prepared usually in the form of suppositories, using a carrier such as cacao butter, hydrogenated fat or hydrogenated carboxylic acid. For the preparation of the propellant, a non-irritating carrier that facilitates absorption by dispersing the above-mentioned substance as an active ingredient as fine particles can be used. Examples of such a carrier include lactose and glycerin, and the form of the preparation can be selected from forms such as aerosol and dry powder. However, the pharmaceutical additives used in the production of the medicament of the present invention are not limited to those described above, and any additives that can be used by those skilled in the art may be used.

  The dose and frequency of administration of the medicament of the present invention are not particularly limited, and an appropriate dose is selected according to conditions such as the type and severity of fungal infection, the presence or absence of the underlying disease, the patient's age, weight, etc. Is possible.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by the following examples.

Example 1
Dispense 25 ml of basic medium consisting of 1% glycerol, 1% glucose and brain heart infusion broth into a 50 ml Erlenmeyer flask, autoclaved at 121 ° C for 15 minutes in an autoclave, and after cooling, nocardia transvalensis IMF10065 strain Vaccinated. This was precultured by shaking culture (250 rpm) for 5 days at 30 ° C.
100 ml main culture medium consisting of glucose 3%, Pharmamedia 0.6%, Nissin Soya Flour FT 0.75%, K ₂ HPO ₄ 0.1%, CaCO ₃ 0.55%, (NH ₄ ) ₂ SO ₄ 0.5% in a 500 ml Erlenmeyer flask Were sterilized in the same manner, and 20 of the preculture solution was inoculated at 1% v / v. The culture was carried out by shaking culture (250 rpm) at 31 ° C. for 7 days, and this operation was repeated 4 times.

The obtained culture 8L was centrifuged to recover the cells. Methanol was added to the obtained bacterial cells for extraction, and the bacterial cells were removed with a filter paper. This extract was concentrated to dryness using an evaporator. Water and ethyl acetate were added to the concentrate at a ratio of 1: 1, and the ethyl acetate layer was recovered. The ethyl acetate extract was concentrated to dryness with an evaporator and subjected to silica gel chromatography. Silica gel FL100D (Fuji Silysia) was used as the silica gel, and the column size was 3.5 × 20 cm. Elution was performed with 300 ml each of chloroform: methanol = 1: 0, 9: 1, 3: 1, 2: 1, 1: 1, 0: 1. The 1: 0 eluted fraction was concentrated with an evaporator. The concentrate was again subjected to silica gel chromatography. The gel used was silica gel FL100D. Elution was performed with toluene: ethyl acetate = 3: 1, and 5.0 ml was collected in fractions, and the active fraction was collected using growth inhibitory activity against Trichophyton mentagrophytes as an index. The collected active fraction was fractionated by thin layer chromatography. As thin layer chromatography, a PLC plate 20 × 20 cm silica gel 60F ₂₅₄ , 0.5 mm (Merck) was used, and toluene: ethyl acetate = 3: 1 was used as a developing solvent. The portion having UV254 absorption was recovered, and the recovered material was subjected to gel filtration chromatography. Sephadex LH-20 (Amersham) was used as the carrier, and the column size was 1.5 × 40 cm. Elution was performed with a solvent of chloroform: methanol = 1: 1. 3 ml was collected as one fraction, and the active fraction was collected and concentrated by the above assay method.

  The obtained concentrate was subjected to high performance liquid chromatography twice for purification. The first purification uses ODS 10mm x 250mm (JASCO) as the column, acetonitrile: water mixture as the solvent, and concentration gradient elution of 35-65% acetonitrile (flow rate: 5ml / min, 30 minutes). The active fraction was obtained by the above assay method. For the second purification, the above active fraction was used, ODS 10 mm x 250 mm (JASCO) as a column, methanol as a solvent, elution at a flow rate of 5 ml / min, and the active fraction was obtained by the above assay method. Obtained. The active fraction was concentrated and dried with an evaporator to obtain 7.2 mg of the compound of the present invention (compound represented by the above formula (II)) as a purified product.

The physicochemical data of the obtained compound are shown below. The internal standard of ¹ H-NMR spectrum used the peak of chloroform as δ7.24, and the internal standard of ¹³ C-NMR spectrum used the center peak of deuterated chloroform as δ77.0.
UV (methanol) λ _max (logε) 225 (4.47), 251 (4.05), 358 (3.65) nm.
FAB-MS m / z 655 [M ( ³⁷ Cl, ⁶⁶ Zn) + H] ⁺ , 653 [M ( ³⁷ Cl, ⁶⁴ Zn / ³⁵ Cl, ⁶⁶ Zn) + H] ⁺ , 651 [M ( ³⁵ Cl, ⁶⁴ Zn) + H] ⁺ , 637, 635, 633 (M-17), 307, 289, 176, 154, 136.
FAB-MS (+ KI) m / z 693 [M ( ³⁷ Cl, ⁶⁶ Zn) + K] ⁺ , 691 [M ( ³⁷ Cl, ⁶⁴ Zn / ³⁵ Cl, ⁶⁶ Zn) + K] ⁺ , 689 [M ( ³⁵ Cl, ⁶⁴ Zn) + K] ⁺ , 637, 635, 633 (M-17), 580, 578, 576, 344.5, 306, 288, 191, 154, 136.
ESI-MS (CH ₃ OH-CH ₃ CN-H ₂ O = 70: 20: 5) m / z 718 [M ( ³⁷ Cl, ⁶⁶ Zn) + Na + CH ₃ CN] ⁺ , 716 [M ( ³⁷ Cl , ⁶⁴ Zn / ³⁵ Cl, ⁶⁶ Zn) + Na + CH ₃ CN] ⁺ , 714 [M ( ³⁵ Cl, ⁶⁴ Zn) + Na + CH ₃ CN] ⁺ .
IR ν _max (KBr) cm- ¹ 3400 (sh), 3190, 2835, 1660, 1625, 1595 (infl.), 1520, 1460, 1365, 1340, 1235, 1150, 1085, 980, 965, 595.
^{CD (c 1.2 × 10- 4 mol} / L, methanol) [θ] (nm) + 24000 (217), + 9900 (238), 0 (244), - 11000 (254), - 16000 (276), 0 (317), + 1900 (347), 0 (387).

例２
例１で得られた本発明の化合物の抗真菌作用を調べた。抗真菌作用は医真菌学会の標準化方法（日本医真菌学会雑誌, 43, pp.107-123, 2002）に従って測定した。培地としてはBHI(Difco, U.S.A.）を用い、Trichophyton mentagrophytesについては72時間で判定し、それ以外の菌株については48時間で判定した。結果を最小発育阻止濃度（μg/ml)として以下の表３に示す。

Example 2
The antifungal activity of the compound of the present invention obtained in Example 1 was examined. The antifungal activity was measured according to a standardized method of the Japanese Society for Medical Mycology (Journal of the Japanese Society for Medical Mycology, 43, pp.107-123, 2002). BHI (Difco, USA) was used as the medium, and Trichophyton mentagrophytes were determined at 72 hours, and other strains were determined at 48 hours. The results are shown in Table 3 below as the minimum growth inhibitory concentration (μg / ml).

Claims (5)

Formula (I) below:

Or a salt thereof. Formula (II) below:

Or a salt thereof. A medicament for preventing and / or treating a fungal infection, comprising the compound or a salt thereof according to claim 1 or 2 as an active ingredient. A method for producing the compound or salt thereof according to claim 1 or 2, comprising culturing a bacterium producing the compound belonging to the genus Nocardia, and separating and collecting the compound or salt thereof from the obtained culture. Including methods. The method according to claim 4, wherein the producer is Nocardia transvalensis.