JPH0643322B2 - Antifungal agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to an antifungal agent containing heptaene V-28-3M as an active ingredient.

[Conventional technology]

In recent years, bacteriopathies have been easily and effectively treated by the use of many chemotherapeutic agents, mainly antibiotics. However, by using these chemotherapeutic agents, deep-seated mycosis caused by infection with so-called fungi such as mold and yeast has emerged as one problem. The present applicant has already discovered a novel heptaene antibiotic having antifungal activity and applied for a patent (see Japanese Patent Laid-Open No. 61-189224).
Although this substance has strong antifungal activity, it is highly toxic to animals and there is room for improvement.

[Problems to be Solved by the Invention]

The problem to be solved by the present invention is to obtain a new antifungal agent having stronger antibacterial activity against fungi and low toxicity.

[Means for Solving the Problems]

In view of the above circumstances, the present inventors have a heptaene antibiotic V-28-3 (hereinafter V-28-3) for the purpose of searching for a new antifungal agent having a stronger antibacterial activity against fungi and having a lower toxicity. -3
Abbreviated) was synthesized. As a result, methyl-esterified V-28-3 (hereinafter abbreviated as V-28-3M) was detected as V-2 against fungi.
8-3 It was found that it is a novel heptaene having stronger antibacterial activity and completed the antifungal agent of the present invention.

V-28-3M, which is the active ingredient of the present invention, can be produced by subjecting V-28-3 to a conventional methyl esterification reaction. An example is as follows. V-28-3M can be produced by adding a diazomethane solution prepared according to a conventional method to a suspension of V-28-3 in methyl alcohol at ice temperature, and stirring and reacting the mixture at room temperature for about 30 minutes. The starting compound V-28-3 can be prepared according to the method described in JP-A No. 61-189224.

V-28-3M produced by the reaction can be separated and purified, for example, from the residue concentrated under reduced pressure from the reaction solution by means generally used for isolation of antibiotics.

The V-28-3M thus obtained is Novapack C18 as a carrier.
(Manufactured by Waters), 10 mM ammonium acetate buffer (pH 8.5) containing 65% acetonitrile as a mobile phase,
In high performance liquid chromatography (hereinafter, abbreviated as HPLC, flow rate 1.0 ml / min) using a UV (405 nm and 254 nm) detector as a detector, a single peak is given at 7.5 minutes, and the following physicochemical properties are shown.

1) Molecular weight: 1126 [FABMS, (M + H) ⁺ ; 1127] 2) Molecular formula: C ₅₉ H ₈₆ N ₂ O ₁₉ 3) Optical rotation ▲ [α] ²⁰ _D ▼: + 296 ° (c = 0.1%, DMSO) ) 4) Melting point: Browning at 135 ° C 5) UV spectrum: As shown in Fig. 6) Solubility in solvents: Insoluble in water, diethyl ether, hexane, chloroform Slightly soluble in methanol, ethanol, n-butanol Dimethyl Soluble in sulfoxide and dimethylformamide 7) Color reaction: phenol sulfate: positive, ferric chloride:
Negative, ninhydrin: positive, concentrated sulfuric acid: positive, iodine: positive 8) ¹ H-NMR spectrum: as shown in FIG. 2 9) ¹³ C-NMR spectrum: as shown in FIG. 3: 10) The V-28-3M thus obtained has a strong antibacterial activity and is useful as an active ingredient of a novel fungal agent having low toxicity. For example, the antibacterial activity (MIC) of V-28-3M against Candida albicans ATCC 10231 is 0.39.
μg / ml, which shows stronger antibacterial activity than amphotericin B and V-28-3. In addition, 5 of V-28-3M against L1210 cells
The 0% growth inhibitory concentration is 0.23 μg / ml, which is about 6 times that of V-28-3. Furthermore, the LD ₅₀ is 5 for acute toxicity tests.
It is 0 mg / kg or more, which is extremely lower than V-28-3, pertricin B, pertricin B methyl ester, amphotericin B and amphotericin B methyl ester. Furthermore, in a subacute toxicity test using male Fisher rats (5 weeks old, 6 animals per group), no death was observed when intravenously administered at a dose of 20.0 mg / kg or less for 5 consecutive days. It can be confirmed that the toxicity is low.

The antifungal agent containing V-28-3M as an active ingredient is useful as a therapeutic agent for fungal diseases involving humans including fungi, fungi such as yeast, and fungal diseases for treating mammals. Inhibiting or killing fungal growth in the body by formulating tablets, capsules or elixirs for oral administration and sterile solutions or suspensions for parenteral administration You can The active ingredient used in the present invention can be administered to patients (animals and humans) in need of such treatment in a dose range of 10 to 500 mg per patient. The dose may vary depending on the severity of the illness, the weight of the patient and other factors recognized by those of ordinary skill in the art.

About 10-500 mg of a compound or admixture of a physiologically acceptable salt of the above compound is generally accepted with physiologically acceptable vehicles, carriers, excipients, binders, preservatives, stabilizers, flavors and the like. Admixed in the unit dosage form required for pharmaceutical implementation. The amount of active substance in these compositions or preparations is such that a suitable dosage in the indicated range is obtained.

Specific miscible agents such as tablets, capsules and the like are the following: binders such as arabic gum, corn starch or gelatin; excipients such as microcrystalline cellulose; alginic acid and the like. Bulking agents; lubricants such as magnesium stearate; solubilizing agents such as sodium desoxycholate; sweetening agents such as sucrose and lactose; flavoring agents such as peppermint; the above-mentioned types when the dosage unit form is capsules. The material may further contain a liquid simple substance such as oil and fat. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, 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 propylparapenes as preservatives, a dye and flavoring such as cherry or orange flavor.

Sterile compositions for injection may be prepared by dissolving or suspending the active substance in a vehicle such as water for injection, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil or the like or a synthetic fat vehicle such as ethyl oleate or the like. It can be formulated according to conventional pharmaceutical practice. Buffer, preservative,
An antioxidant or the like can be bound if necessary.

〔The invention's effect〕

V-28-3M has a strong antibacterial activity against Candida alkans, has low toxicity, and can be used as a new antifungal agent. Hereinafter, the present invention will be described in detail with reference to Examples.

Example <Production Example> 100 ml of the medium having the composition shown in Table 1 was dispensed into a 500 ml flask and sterilized by heating at 120 ° C for 10 minutes. to this,
One platinum loop of Streptomyces arene V-28-3 (FERM P-8099, FERM BP-1537) grown on an agar slant of the same composition was inoculated and shake culture was carried out at 27 ° C for 3 days.

On the other hand, medium 20 of the same composition (defoamer, silicone KM-75
10 ml was added to a 30-volume jar fermenter, and the mixture was sterilized by heating at 120 ° C. for 10 minutes, and the seed culture solution was inoculated into the jar fermenter, followed by aeration-agitation culture at 27 ° C. for 24 hours. (Ventilation volume 1/2 V.VM; agitation number 20)
0 rpm). The culture solution was centrifuged (4000 rpm for 10 minutes), and 1.
6 kg of bacterial cell cake was collected. 80% methanol 1.5 was added to 500 g of the bacterial cell cake, and ammonia water was added to adjust the pH to 9.5.
A time extraction operation was performed. Lower the pH of the extract to 7.0 and add 1.5
After adding DIAION HP-20 (manufactured by Mitsubishi Kasei Co., Ltd.,
Adsorption carrier) column (10 × 30 cm). The column was washed with a small amount of 50% methanol and then eluted with 80% methanol containing 0.45% ammonia. The sections having antibacterial activity against Candida albicans HUT7501 were collected, concentrated under reduced pressure, the resulting yellowish brown precipitate was collected by centrifugation, washed with cold acetone and dried under reduced pressure to obtain 600 mg of yellowish green powder. Obtained. Suspend this powder in 100 ml of chloroform, and add High Flow Super Celcum (5 x
20 cm). Column with 100 ml chloroform,
After sequentially washing with 100 ml of a mixed solution of chloroform: methanol = 1: 1, elution was carried out with an eluent of acetonitrile: 0.05M ammonium acetate (pH 9.5) = 1: 1. The active fractions were collected, the solvent was removed under reduced pressure, the resulting precipitate was collected by centrifugation, washed with acetone, and then dried under reduced pressure.
20 mg of yellow powder was obtained. Of this, 60 mg was taken and dissolved in 5.0 ml of 66% hydrous tetrahydrofuran solution. This is L
It was loaded on an RP-1 column (C-18 reverse phase chromatography carrier manufactured by Whatman) and eluted with an eluent of acetonitrile: 0.05M ammonium acetate (pH 9.0) = 4.5: 5.5. The antibacterial active substance which is eluted after the antibacterial active substance is separated into two and eluted (the active substance which is eluted first is identified as the same substance as partricin B from its ¹ H-NMR spectrum). Were collected, the solvent was gradually removed under reduced pressure until a slight white turbidity occurred, and the mixture was stored at 5 ° C. overnight to precipitate yellow crystals. The yellow crystals were collected by centrifugation, washed with cold water and cold acetone and dried under reduced pressure to give 30 mg of
I got V-28-3.

The thus obtained V-28-3 (20 mg) was suspended in 4 ml of methyl alcohol, and a tetrahydrofuran solution of diazomethane was added dropwise to the suspension under ice cooling. After stirring the reaction solution at room temperature for 30 minutes, nitrogen gas was blown to the reaction solution to remove the solvent and obtain 21 mg of a dry solid. This dried solid was dissolved in a lower layer solution of a mixed solution of chloroform-methyl alcohol-water (2: 2: 1), and subjected to droplet AC distribution using the lower layer solution of the above mixed solution as a stationary phase and the upper phase solution as a mobile phase. . Evaluate the eluate by the above-mentioned HPLC,
Fractions containing only V-28-3M were combined and concentrated to dryness to isolate V-28-3M (15 mg) as a yellow powder.

The physicochemical properties of this yellow powder were as follows.

1) Molecular weight: 1126 [FABMS, (M + H) ⁺ ; 1127] 2) Molecular formula: C ₅₉ H ₈₆ N ₂ O ₁₉ 3) Optical rotation ▲ [α] ²⁰ _D ▼: + 296 ° (c = 0.1%, DMSO) ) 4) Melting point: Browning at 135 ° C 5) UV spectrum: As shown in Fig. 6) Solubility in solvents: Insoluble in water, diethyl ether, hexane, chloroform Slightly soluble in methanol, ethanol, n-butanol Dimethyl Soluble in sulfoxide and dimethylformamide 7) Color reaction: phenol sulfate: positive, ferric chloride:
Negative, ninhydrin: positive, concentrated sulfuric acid: positive, iodine: positive 8) ¹ H-NMR spectrum: as shown in FIG. 2 9) ¹³ C-NMR spectrum: as shown in FIG. 3: 10) <Working> The antibacterial activity (MIC) of V-28-3M is shown in Table 2, and M against Candida albicans ATCC10231.
IC was lower than amphotericin B and V-28-3, and showed strong antibacterial activity.

The experiments shown in Table 2 were carried out using Sabraud agar medium in the conventional MI.
The measurement was carried out according to the C measurement method, and the judgment was made after 48 hours.

Next, the cytotoxicity of V-28-3M (50% growth inhibitory concentration)
As shown in the table, the 50% growth inhibitory concentration on L1210 cells was about 6 times that of V-28-3.

The experiments shown in Table 3 were carried out using RPMI 1640 medium according to a conventional method for measuring cell growth inhibition, and the evaluation was made 48 hours later.

Furthermore, the results of acute toxicity (LD ₅₀ ) of V-28-3M according to the conventional acute toxicity measurement method are shown in Table 4.

In addition, V-28- was added to male Fischer rats (5 weeks old, 1 group of 6 rats).
As a result of intravenous administration of 3M at doses of 1.25, 5.0 and 20.0 mg / kg for 5 consecutive days, no deaths were observed. On the other hand, amphotericin B 0.12, 0.6, 3.0 mg / k
When administered intravenously at a dose of g for 5 consecutive days, 3.0 mg /
Immediately after the first administration, 2 patients died in the kg administration group. 0.12
There were no deaths in the 0.6 mg / kg group.

V-28-3M was intravenously administered to male Fisher rats (5 weeks old, 6 rats in one group) at a dose of 0.8, 4.0, 20.0 mg / kg for 30 consecutive days, and a subacute toxicity test was conducted. . On day 15 of administration, in the 20 mg / kg administration group, coarse hair was observed in all cases, thinning in 4 cases, and decreased body temperature in 2 cases, but all recovered within 4 days, and abnormal transition on day 19 of administration was observed. I couldn't do it.
No abnormalities were observed in the other groups throughout the test period.

[Brief description of drawings]

Fig. 1 is a UV spectrum of V-28-3M measured in methyl alcohol, where the vertical axis shows the absorption intensity and the horizontal axis shows the wavelength (n
m). 2 and 3 are respectively 400 MHz,
It is a < ¹ > H-NMR and a < ¹⁵ > C-NMR spectrum figure measured at 100 megahertz. In the figure, the vertical axis indicates the absorption intensity and the horizontal axis indicates TM.
It is a chemical shift expressed in ppm based on S.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI technical display location C12R 1: 465) 7804-4B (72) Inventor Ryosaku Nomi 4-4 Ando, Asanan-ku, Hiroshima-shi, Hiroshima Prefecture 12-8 Examiner Naoko Tsukanaka

Claims (1)

[Claims] 1. A heptaene V- having the following physicochemical properties:
Antifungal agent containing 28-3M as an active ingredient (a) Property: Yellow powder (b) Molecular weight: 1126 [FABMS (M + H) ⁺ ; 1127] (c) Molecular formula: C ₅₉ H ₈₆ N ₂ O ₁₉ (d) Optical rotation ▲ [α] ²⁰ _D ▼: + 296 ° (0.1%, DMSO) (e) Melting point: Browning at 135 ° C (f) UV spectrum: As shown in Fig. 1 (g) Solubility in solvent: Insoluble in water, diethyl ether, hexane, chloroform Slightly soluble in methanol, ethanol, n-butanol Soluble in dimethyl sulfoxide, dimethylformamide (h) Color reaction: phenol sulfate: positive Ferric chloride:
Negative ninhydrin: Positive Concentrated sulfuric acid: Positive Iodine: Positive (i) ¹ H-NMR spectrum: As shown in Fig. 2 (j) ¹³ C-NMR spectrum: As shown in Fig. 3