JP3063941B2 - Didemethyl allosamidine and its production - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to didemethylarosamidine useful in the fields of medicine, agricultural chemicals, veterinary medicine and the like, and a method for producing the same.

[0002]

2. Description of the Related Art Chitin is a main component of the cell wall of fungi, and in the process of repeated division and growth of fungi, consistent synthesis and decomposition of chitin are performed. On the other hand, chitin is a main component of the epidermis of insects, and it is known that synthesis and decomposition of chitin are also skillfully controlled in the process of molting and growing of insects.

[0003] Chitinase is a major enzyme involved in the metabolic system of chitin, and it is therefore expected that an inhibitor of this enzyme could be a new type of antifungal agent or insect growth regulator.

Hitherto, as a chitinase inhibitor, allosamidin (Allosamidin: S. Sakuda et al., JA)
ntibiotics. 40, 296-300, 1987), Methylallosamidin: Shohei Sakuta, Proceedings of the 28th Symposium on Natural Organic Compounds, p.70, 1986, Demethylallosamidin: S .Sakuda et al.,
Agric. Biol. Chem., 54, 1333-1335, 1990, JP-A-3-58792), glucoarosamidines A and B,
Methyl-N-demethylarosamidine (Glucoallosamidi
n.A and B, Methyl-N-demethylallosamidin: Y.
Nishimoto et al., J. Antibiotics, 44, 716-722, 1
991) is known.

[0005]

However, Candida albicans (Candida albicans) of these compounds described above.
The inhibitory activity of pathogenic fungi such as da Albicans) on chitinase was not sufficient, and it was necessary to develop a more potent chitinase inhibitor. In addition, since all of these compounds have low lipophilicity and insufficient permeability of the insect epidermal layer and the fungal membrane, it is necessary to develop a compound having higher lipophilicity or a compound which can be easily modified to increase lipophilicity. was there.

In view of this situation, the present inventors have developed an allosamidine compound which has a stronger inhibitory activity against fungal chitinase and has high lipophilicity, or is easy to synthesize with a derivative for enhancing lipophilicity. I thought it was important to explore.

[0007]

Accordingly, the present inventors have conducted a wide search from the natural world using the inhibitory activity against Candida albicans-derived chitinase as an index and found that a microorganism of the genus Streptomyces has the following chemical structural formula [I]: It was found that the represented didemethylallosamidine was produced, and based on this, the present invention was completed. That is, the present invention relates to didemethyl allosamidine represented by the following chemical structural formula.

[0008]

Embedded image

In order to produce didemethylallosamidine, the compound of the present invention, a bacterium having the ability to produce the compound, for example, Streptomyces sp.
s sp. ) AJ9463 (FERM BP-2801) is cultured in an appropriate medium, and the compound is collected from the culture.

[0010] Streptomyces sp.
ces sp. ) The mycological properties of AJ9463 (FERM BP-2801) are as follows. (1) Morphological properties Under microscopic observation, basal hyphae diverge and grow well on various nutrient agar media to form well-extended aerial hyphae. No ring branch is observed. Many spore chains are not straight or have a gentle curve. The mature spores are elliptical and have a size of about 1 × (1.5-2) μm, the surface is smooth but wrinkled, the spore chain is 10 to several tens, and the boundaries between spores are somewhat blurred. is there. In addition, spores, sclerotia, motile spores, etc. are not observed.

(2) Growth condition in various media The color tone in each of the following media is indicated by the Japan Color Institute,
Color standards were used. Sucrose / nitrate agar Medium It grows moderately, forms a slightly gray aerial mycelium on almost colorless growth, and has no remarkable color or soluble pigment on the back surface. Glucose-asparagine agar medium It grows well, and almost no aerial hyphae are observed on light brown growth, and no remarkable color or soluble pigment on the reverse side is observed. Glycerin-asparagine agar medium (ISP5 medium) Medium growth or poor growth, almost white and gray aerial hyphae are formed on almost colorless growth, and no remarkable color or soluble pigment on the back side is observed. Starch agar medium (ISP4 medium) It grows well, forms sparsely white to gray to black aerial hyphae on grayish reddish brown growth, and no remarkable color or soluble pigment on the back side is observed. Tyrosine agar medium (ISP7 medium) Medium growth or poor growth, white-grey aerial mycelia slightly formed on almost colorless growth, and no remarkable soluble pigment on the back side is observed. Nutrient agar medium It grows well, forms a light gray-brown abundant gray aerial mycelium, and has no noticeable color or soluble pigment on the back. Yeast-malt agar medium (ISP2 medium) Growing well, forming a light gray brown mycelia on light brown growth, no remarkable color on the back,
No soluble pigment is observed, but only slightly grayish yellowish brown. Oatmeal agar medium (ISP3 medium) Grew well, formed a gray aerial mycelium abundantly on grayish brown growth, no noticeable color on the back side, no soluble pigment, but slight ash It is brownish.

(3) Physiological properties It grows in a growth temperature range of 10 to 42 ° C. Gelatin liquefaction Glucose-peptone gelatin medium at 20 ° C and 27 ° C
C., and liquefaction of gelatin was observed in each case. Starch hydrolysis Positive on starch agar medium (ISP4 medium). Coagulation / peptone formation of skim milk Strong coagulation was observed at 37 ° C, and no peptone formation was observed. At 27 ° C, no coagulation was observed and strong peptone formation was observed. Formation of melanin-like pigment Negative on tyrosine agar medium (ISP7 medium).

Utilization of carbon source (Pridham Gottlieb medium) Use: D-glucose, D-xylose, L-rhamnose, raffinose Some use: D-fructose, sucrose Not use: L-arabinose, inositol, D -Mannit The 2,6-diaminopimelic acid contained in the cell wall was of the LL-type.

From the above properties, it was found that this strain AJ9463 was Streptomyces sp.

The culturing method basically follows the culturing method of general microorganisms, but usually, submerged culture in a liquid medium is advantageous. The medium used for the culture may be any medium containing a nutrient source that can be used by the producing bacteria. That is, as a carbon source, glucose, fructose, starch, dextrin, etc. are used, and as a nitrogen source, meat extract, casein, gluten, yeast extract, soybean powder, corn stave liquor, urea, ammonium sulfate, ammonium phosphate, etc. Used. In addition, inorganic salts such as sodium hydrogen phosphate, magnesium sulfate, and calcium carbonate can be used as needed. In the culture, when foaming is severe, a small amount of an antifoaming agent such as a silicon compound, a higher alcohol, or a vegetable oil may be added.

The culturing temperature is preferably from 20 to 38 ° C, and most preferably around 27 ° C. The culture time is preferably about 1 to 10 days,
It can be appropriately changed depending on the culture conditions.

Didemethyl allosamidine produced by culturing is mainly accumulated in the cells, and is generally separated from the cells separated by centrifugation, filtration or the like, for example, by means used for isolating antibiotics. And purified. Specifically, a solvent extraction method using a lower alcohol such as methanol or n-butanol, adsorption column chromatography using silica gel, diatomaceous earth, Avicel, alumina, or the like, Toyopearl HW40 (a carrier for gel filtration manufactured by Tosoh Corporation) Filtration, various ion-exchange chromatography, reversed-phase partition column chromatography using octadecylated silica gel as a carrier, and HPL
C. Further, isolation and purification can be carried out by sequentially or appropriately combining purification means such as a countercurrent distribution method, crystallization, and recrystallization.

The thus isolated and purified didemethylallosamidine has the following physicochemical properties. a) Appearance; white powder b) Molecular weight; 594 (FABMS; m / z 595 (M + H) ⁺ , glycerol matrix) c) Molecular formula; C ₂₃ H ₃₈ N ₄ O ₁₄ d) Ultraviolet absorption spectrum; 0.1 N in acetic acid E) ¹ H-nuclear magnetic resonance spectrum; FIG. 1 (600 MHz, 0.3%
(In heavy water containing biacetic acid). f) ¹³ C-nuclear magnetic resonance spectrum; the following signal is shown in heavy water containing 150 MHz and 0.3% biacetic acid (δ) (however, it is considered that this compound takes a plurality of conformations in the present solvent and this is due to this). And several small signals other than the following. 171.0 (Q), 170.8 (Q), 159.3 (Q), 97.6 (CH),
96.3 (CH), 84.0 (CH), 81.7 (CH), 77.2 (C
H), 77.2 (CH), 73.9 (CH), 70.5 (CH), 6
9.6 (CH), 67.1 (CH), 66.0 (CH), 63.2 (C
H), 61.2 (CH), 57.9 (CH 2), 56.5 (CH 2),
49.9 (CH), 49.5 (CH), 48.4 (CH), 17.9 (C
_{H 3), 17.9 (CH 3} )

The structural formula of this didemethyl allosamidine is as described above. Unlike the known allosamidines, it is a novel compound in which the amino group at the 7-position of allosamizoline is demethylated, and is a novel compound of Candida albicans,
It was found to show strong inhibitory activity against chitinase from Saccharomyces cerevisiae and Trichoderma sp.

Further, the fact that the amino group at the 7-position of allosamizoline is not methylated in didemethylarosamidine is advantageous when introducing a highly lipophilic substituent into it, and it does not affect insect epidermis and fungal membranes. It was considered advantageous for synthesizing a highly lipid-soluble chitinase inhibitor with increased permeability.

As described above, didemethylarosamidine represented by the chemical structural formula [I] can be obtained from Candida albicans,
Shows inhibitory activity against chitinase derived from Saccharomyces cerevisiae and Trichoderma sp.

On the other hand, all of these compounds had low cytotoxicity, and showed no growth inhibition on mouse ascites breast cancer cells or human leukemia cells even when added at a concentration of 1 mg / ml.

Therefore, by containing any of these compounds, it can be used, for example, as an antifungal agent. Antifungal agents containing these as active ingredients are useful as therapeutic agents for mycosis involving fungi such as molds and yeasts which are included in humans, and for treating mammals. Inhibit or kill fungal growth in living organisms by formulating as a tablet, capsule or elixir for oral administration, or aseptic solution or suspension for parenteral administration Can be. When the compound of the present invention is used as the active ingredient, it can be administered to patients (animals and humans) in need of such treatment in a dose range of 10 to 1000 mg per patient. Dosages may vary depending on the severity of the disease, the weight of the patient and other factors recognized by those skilled in the art.

The compounds can be used as physiologically acceptable salt compounds or admixtures, and include physiologically acceptable vehicles, carriers, excipients, binders, preservatives,
It is admixed with stabilizers, flavoring agents and the like in unit dosage form as required by generally accepted pharmaceutical practice. The amount of active substance in these compositions or preparations is such that a suitable dosage in the range indicated is obtained.

Specific agents that can be mixed, such as tablets and capsules, are as follows. Binders such as arabic gum, corn starch or gelatin, excipients such as microcrystalline cellulose, leavening agents such as alginic acid,
Lubricants such as magnesium stearate, solubilizing agents such as sodium desoxycholate, sweeteners such as sucrose and lactose, flavoring agents such as peppermint, and the above-mentioned type of material when the unit dosage form is a capsule A liquid simple substance such as oil and fat can be contained. Various other materials may be present as coatings or to otherwise alter the physical form of the dosage unit. For example, tablets may be coated with shellac, sucrose or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparapen as preservatives, a dye and flavoring such as cherry or orange flavor.

Sterile compositions for injection include active substances in vehicles such as water for injection, sesame oil, coconut oil, peanut oil,
It can be formulated according to the usual pharmaceutical practice of dissolving or suspending naturally occurring vegetable oils such as cottonseed oil and the like or synthetic fat vehicles such as ethyl oleate and the like. Buffers, preservatives, antioxidants and the like can be combined as needed.

[0027]

【Example】

(1) Production of didemethylallosamidine A pH 7.2 culture medium comprising 1 g of glucose, 0.1 g of meat extract, 0.2 g of peptone, 0.1 g of yeast extract and 100 ml of water was prepared and injected into a 500 ml Sakaguchi flask. After sterilizing at 120 ° C for 20 minutes, Streptomyces sp. AJ9463
One loopful of a slant culture of (FERM BP-2801) was inoculated into a platinum loop and cultivated by rotary stirring at 28 ° C. and 150 rpm for 2 days to obtain a primary seed mother liquor. On the other hand, 1 liter of a medium having the same composition as described above was prepared, and 100 ml of the medium was dispensed into 10 500 ml Sakaguchi flasks.
Sterilized at ℃ for 30 minutes. 2 ml of the primary seed mother liquor was added thereto, and the mixture was cultivated by rotating and stirring at 28 ° C. and 150 rpm for 5 days to obtain a culture solution. The cells were collected from the culture solution thus obtained by suction filtration.

To the obtained cells, 600 ml of 80% methanol was added and extracted overnight. The extract is filtered using Nutsche,
The cells and the extract were separated. The obtained extract was concentrated under reduced pressure to remove methanol, and then distilled water was added to adjust the total liquid volume to 80 ml. The whole amount was adsorbed on an activated carbon column of φ2.7 × 14 cm, washed with distilled water (80 ml), and 10% ethanol 16
Elution was performed sequentially with 0 ml and 400 ml of 50% ethanol. The fraction eluted with 50% ethanol was concentrated under reduced pressure to remove ethanol. Distilled water is added to this to bring the total liquid volume to 20 ml,
The pH was adjusted to 5.0 with acetic acid.

This solution was treated with 50 mM ammonium acetate (pH
5.0) SP-Sephadex C-25 (Pharmacia)
), And subsequently eluted with 50 mM ammonium acetate (pH 5.0). Fractions containing didemethyl allosamidine were collected and lyophilized.

From this freeze-dried product, capsule pack ODS
Column (Capcell Pak _C18 SG120 column; φ4.6 × 2
Didemethyl allosamidine was purified by high performance liquid chromatography using 50 mm, filler particle size 5 μm, manufactured by Shiseido Co., Ltd.). The flow rate was 1 ml / min, and elution was performed by a linear gradient method in which the concentration of acetonitrile in an aqueous solution of 10 mM ammonium acetate (pH 8.9) was increased from 0% to 50% in 30 minutes. The components were detected by ultraviolet absorption at 220 nm.

Didemethyl allosamidine was eluted at 10.9 minutes, and after fractionation, concentration under reduced pressure, and lyophilization, 0.3 mg of a white powder was obtained. The elution times of allosamidine and demethyl allosamidine at this time were 15.0 minutes and 12.2 minutes, respectively.

(2) Measurement of Chitinase Inhibitory Activity of Didemethylallosamidine (i) Preparation of Chitinase Enzyme Solution Candida albicans (C. albicans) chitinase was prepared by the following method. First, glucose 4g,
Sabouraud's medium (p) containing 1 g of polypeptone and 100 ml of water
H5.6) was prepared and poured into a 500 ml Erlenmeyer flask. 1
After sterilization at 20 ° C for 20 minutes, Candida albicans AT
One loopful of CC 10231 slant culture was inoculated into a loop and incubated at 37 ° C for 2 hours.
After culturing with shaking for one day, a mother liquid of the first seed was obtained. On the other hand, 1 liter of a medium having the above composition was prepared, and 100 ml of a 500-ml Erlenmeyer flask was prepared.
Each was dispensed and sterilized at 120 ° C. for 20 minutes. 2 ml of the primary seed mother liquor was added thereto, followed by shaking culture at 37 ° C. for 2 days.
A second seed mother liquor was obtained. Further, 50 l of a medium having the above composition was prepared, poured into a 100 l culture tank, and sterilized at 120 ° C. for 30 minutes. To this, add 1 liter of the above-mentioned secondary seed mother liquor,
Cultured for hours. The stirring speed at this time is 140 rpm, and the aeration speed is
It was 100 l / min. The culture solution thus obtained was centrifuged in a Sharpless centrifuge to recover the cells. The wet weight of the obtained cells was 250 g.

The obtained cells were suspended in 800 ml of a cell disruption buffer (50 mM Bis-Tris, 0.25 M saccharose, 1 mM disodium ethylenediaminetetraacetate, pH 6.5),
Dyno-mill (MK-2GX, 0.25-0.5)
mm) twice to disrupt the cells. The obtained cell lysate was centrifuged at 9000 g for 30 minutes, and the supernatant was recovered. The supernatant was ultracentrifuged at 152,000 g for 1 hour, and the supernatant was recovered. The supernatant was concentrated about 5-fold with a limiting filtration membrane (Toyo Ultrafilter UP-20) having an exclusion limit molecular weight of 20,000, and this was used as a crude chitinase enzyme solution of Candida albicans in the following assay.

The baker's yeast (S. cerevisiae) chitinase was prepared by the following method. First, 400 g of baker's yeast (Kanebuchi Chemical Industry Co., Ltd.) was added to an enzyme extraction buffer (0.1% digitonin (Wako Pure Chemical Industries, Ltd.), 0.1% β-mercaptoethanol, 25 mM female (MES: Hanoi Chemical Co., Ltd.). Pharmaceutical Co., Ltd.
The suspension was suspended in 2 l and shaken at 30 ° C. and 120 spm for 2 hours to extract the enzyme. This extract was centrifuged at 12000 g for 10 minutes, and the supernatant was recovered. The supernatant is passed through an ultrafiltration membrane (UP
-20) and concentrated about 5-fold. To the concentrate, 800 ml of a citrate buffer (pH adjusted to 3.0 by adding 0.15 M aqueous solution of sodium citrate to 0.15 M aqueous solution of citric acid) was added, and the resulting precipitate was removed by centrifugation (0 ° C., 12000 g, 10 min). After that, the supernatant was again passed through an ultrafiltration membrane (UP-20) for about 80 hours.
It was concentrated to ml. This concentrated liquid was used as baker's yeast chitinase in the following assay.

Trichoderma sp.
The chitinase of p.) was prepared by adding commercially available Chitinase T-1 (manufactured by Asahi Industry Co., Ltd.) to a Mcl Lvanine buffer solution (a 0.1 M aqueous citric acid solution and a 0.2 M aqueous disodium hydrogen phosphate solution) to adjust the pH.
(Adjusted to 5.2) to a concentration of 50 μg / ml and subjected to the following assay.

(Ii) Assay for Chitinase Inhibitory Activity In a vial (20 ml), Candida
Albicans chitinase enzyme solution 50 μl, 100 μg / ml
4-methylumbelliferyl β-DN, N ', N "triac
etyl-chitotrioside (hereinafter abbreviated as 4MBTC. SIG)
MA Co.) 50 μl aqueous solution, 50 mM Bis-Tris buffer (pH
6.5) 75 μl and 25 μl of 0.1N acetic acid were added and reacted at 37 ° C. for 30 minutes. At this time, 50 μl of enzyme solution was used as a blank.
Was replaced with 50 mM Bis-Tris buffer (pH 6.5). After the reaction, a 0.5 M glycine-sodium hydroxide buffer solution (pH 10.4) was added to make a total volume of 5 ml.
Fluorometer (MPF-Made by Hitachi, Ltd.)
The fluorescence intensity was measured in 4). The excitation wavelength at this time is 350n
m, the fluorescence wavelength was 440 nm. The difference between the reading of the fluorescence intensity upon addition of the enzyme and the reading of the blank was taken as chitinase activity.

When measuring the inhibitory activity of didemethylallosamidine, these were dissolved in 0.1 N acetic acid,
μl was added instead of 25 μl of 0.1N acetic acid in the above reaction system. When measuring the inhibitory activity of baker's yeast and Trichoderma sp. On chitinase, the addition buffer in the above reaction system was changed from a 50 mM bis-Tris buffer (pH 6.5) to a 0.1 M citrate buffer (pH 3.0). )
And MclLvanine buffer (pH 5.2).

(Iii) Measurement Results The enzyme inhibitory activities of didemethylarosamidine against chitinase of Candida albicans, Saccharomyces cerevisiae and Trichoderma sp. Are as shown in Table 1 below.

[0039]

[Table 1]

Didemethylallosamidine cytotoxicity Mouse ascites breast cancer cells FM3A were inoculated at a concentration of 1 × 10 ⁵ cells / ml in Dulbecco's modified MEM medium supplemented with 10% fetal bovine serum. Didemethyl allosamidine
mg / ml, and cultured at 37 ° C. for 3 days. The cultured mouse ascites breast cancer cell FM3A was observed under a microscope, but no growth inhibition was observed.

Human leukemia cells K562 were inoculated at a concentration of 1 × 10 ⁵ cells / ml in a medium obtained by adding 10% fetal bovine serum to a FRMI1640 medium, and didemethyl allosamidine was added to the medium.
mg / ml, and cultured at 37 ° C for 3 days. The cultured human leukemia cells K562 were observed under a microscope, but no growth inhibition was observed.

[0042]

Industrial Applicability The didemethylallosamidine of the present invention has an inhibitory effect on chitinases derived from Candida albicans, Saccharomyces cerevisiae and Trichoderma sp. Accordingly, an antifungal agent and a chitinase inhibitor can be provided. Further, this compound can be easily mass-produced from the place where it is produced by fermentation.
Furthermore, didemethyl allosamidine has a primary amine in the molecule, which is considered to be advantageous for introducing a highly liposoluble substituent and synthesizing a more effective derivative.

[Brief description of the drawings]

FIG. 1 is a diagram showing a proton nuclear magnetic resonance spectrum of didemethylallosamidine.

Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12R 1: 465) (58) Investigated field (Int.Cl. ⁷ , DB name) C07H 1/00-23/00 C12P 19/00-19 / 64 A61K 31/00-49/00 A61P 1/00-43/00 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. Didemethyl allosamidine represented by the following chemical structural formula: 2. A method for culturing a microorganism belonging to the genus Streptomyces and capable of producing didemethylallosamidine according to claim 1, and collecting didemethylallosamidine from the culture. Of the described didemethyl allosamidine