JP2003503416A - New compound - Google Patents

Abstract

  (57) [Summary] The present invention provides a novel antimicrobial compound FR21956 substance, and further provides a method for producing the FR21956 substance by culturing a microorganism producing the FR21956 substance in a nutrient medium and collecting the FR21956 substance from the cultured medium. Furthermore, an antimicrobial agent containing the FR21956 substance and a carrier, a pharmaceutical composition comprising an effective amount of the FR21956 substance and a pharmaceutically acceptable carrier, a method of applying the FR21956 substance to a microorganism to kill the microorganism, and a FR219556 substance, Provide use for the treatment of infectious diseases caused by pathogenic microorganisms.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a novel antibacterial compound or a salt thereof which is useful as a medicine and is hereinafter referred to as FR231956.

[0002]

DISCLOSURE OF THE INVENTION

  The present invention relates to a novel antibacterial compound FR231956 or a salt thereof.

More specifically, a novel antibacterial compound FR231956 or a salt thereof having an antibacterial activity against pathogenic microorganisms, particularly pathogenic fungi, a method for producing the same, a pharmaceutical composition containing them and useful as an antibacterial agent, and pharmaceuticals thereof. Regarding the use of.

Therefore, one object of the present invention is to provide a novel compound FR231956 or a salt thereof, which is useful for treating infectious diseases caused by pathogenic microorganisms.

Another object of the invention is to order Sordaria araneosa A in a nutrient medium.
Fermentation of FR231956 producing strains such as TCC36386 results in FR2319
It is to provide a method for producing 56 substances or salts thereof.

Still another object of the present invention is to provide a pharmaceutical composition containing FR231956 substance or a salt thereof as an active ingredient.

Another object of the present invention is to provide use of FR231956 substance or its salt for treating infectious diseases caused by pathogenic microorganisms.

The structure of the novel compound FR231956 substance is the antifungal antibiotic (Helveti
See ca Chimica Acta, 1971, 54 , 1178-1190).
It is composed of the sordarin skeleton. The FR231956 substance is a strain ATCC36 of the fungal species Sordaria araneosa, known as a sordarin-producing strain.
Manufactured by fermenting 386.

British Patent Specification No. 1,162,027 describes strain Sordaria arane
It describes the production of the antibiotic SL2266, which was later named sordaline, carried out by culturing osaNRRL3196 (ATCC36386). Japanese Patent Publication J62040292 discloses a compound zofimarin having a sordarin skeleton and reports that it has antifungal activity. Semi-synthetic sordarin derivatives have been reported in PCT applications WO 96/14326, WO 96/14327 and WO 98/15178.

A novel compound FR231 with excellent antifungal activity and broad spectrum of action
956 will be described below.

[0011]   The novel compound FR231956 substance is represented by the following formula.

[0012]

[Chemical 2]

The FR231956 substance has the following physicochemical properties. Molecular formula: C ₃₆ H ₅₀ O ₁₁ Elemental analysis: C ₃₆ H ₅₀ O ₁₁・ 1 / 2H ₂ O Calculated value: C 64.75, H 7.70 Measured value: C 64.64, H 7.93 Molecular weight: ESI-MS (+) m / z 681 (M + Na), ESI-MS (-) m / z 657 (MH) Melting point: 98-110 ℃ (decomposition) Specific rotation: [α] D (23 ℃) + 21 ° (c = 0.5, methanol) ) Ultraviolet absorption spectrum: λmax (methanol): 222 nm (ε = 13000) Solubility: Soluble: Methanol, ethyl acetate, chloroform, dimethylsulfoxide Insoluble: Water, n-hexane Color reaction: Positive: Iodine vapor reaction, sulfuric acid Cerium reaction negative: Maurish reaction, ninhydrin reaction, Ehrlich reaction, Dragendorff reaction, iron chloride reaction thin layer chromatography (TLC): * E. Made by Merck

High Performance Liquid Chromatography (HPLC): Conditions: Column: YMC-Pack ODS-AM-303 ** (4.6 mmφ x 250 mmL) Mobile phase: 60% acetonitrile containing 0.5% NaH ₂ PO ₄ .2H ₂ O Aqueous solution flow rate: 1 ml / min Detection: UV, 210 nm Retention time: 12.8 minutes **: YMC Corporation infrared absorption spectrum: νmax (KBr): 3420, 2960, 2870, 1720, 1460, 1380, 1220, 1160, 1100, 1070,
1000, 900 cm ^{-1 1} H Nuclear magnetic resonance spectrum: (500 MHz, CD ₃ OD) δ (ppm): 9.78 (1H, s), 6.02 (1H, dd, J = 3 and 1 Hz), 5.65 (1H , m), 5.47 (1H, dd
, J = 4 and 3 Hz), 4.50 (1H, d, J = 1 Hz), 4.24 (1H, dd, J = 6 and 2 Hz)
, 3.97 (1H, d, J = 10 Hz), 3.78 (1H, dd, J = 4 and 1 Hz), 3.75 (1H, d, J =
10 Hz), 3.70 (1H, m), 3.34 (3H, s), 3.31 (1H, dd, J = 9 and 3 Hz), 3.15
(1H, m), 2.93 (1H, dd, J = 6 and 2 Hz), 2.75-2.70 (2H, m), 2.34 (1H, m
), 2.16 (1H, m), 2.08-2.02 (3H, m), 1.98 (3H, d, J = 2 Hz), 1.88-1.69 (4H,
m), 1.36 (3H, d, J = 6 Hz), 1.27 (3H, d, J = 6 Hz), 1.26-1.20 (2H, m), 1.00
(3H, d, J = 7 Hz), 0.96 (3H, d, J = 7 Hz), 0.96 (1H, m), 0.82 (3H, d, J = 7 H
z) ¹³ C nuclear magnetic resonance spectrum: (125 MHz, CD ₃ OD) δ (ppm): 207.1 (d), 176.0 (s), 167.3 (s), 150.5 (s), 140.2 (d), 133.9 (s ), 131.3
(d), 100.6 (d), 79.6 (d), 76.5 (t), 74.8 (s), 71.1 (d), 70.4 (d), 69.9 (
d), 66.9 (s), 59.6 (s), 57.9 (q), 57.8 (d), 57.3 (d), 53.4 (d), 53.0 (d)
, 47.3 (d), 42.8 (d), 42.6 (d), 33.2 (t), 32.5 (d), 30.6 (t), 29.9 (t),
28.8 (d), 27.3 (t), 23.2 (q), 21.6 (q), 20.5 (q), 18.4 (q), 17.9 (q), 14
.2 (q) Material properties: acidic materials

FR231956 substance is Sordaria araneosa in nutrient medium
It can be produced by fermentation of a FR231956-producing strain such as ATCC36386. Sordaria araneosa ATCC36386 is an American
n Type Culture Collection, 12301 Park
Lawn Drive, Rockville, Maryland 20852,
Saved by USA and distributable. It is to be understood that the production of FR231956 material is not limited to the use of the particular organisms described herein and is merely exemplary. Natural mutants and artificial mutants that can be produced from the above-mentioned organisms by conventional methods such as X-ray irradiation, ultraviolet irradiation, N-methyl-N′-nitro-N-nitrosoguanidine, and treatment with 2-aminopurine. The present invention encompasses the use of any mutant strain capable of producing the FR231956 substance.

Manufacture of FR231956 substance FR231956 substance is Sordaria araneosa in nutrient medium.
FR231956-producing strains such as ATCC36386 are produced when grown in a nutrient medium containing assimilable carbon and nitrogen sources under aerobic conditions (eg shake culture, submerged culture, etc.).

Preferred carbon sources in the nutrient medium are carbohydrates such as glucose, sucrose, starch, fructose, glycerin and the like.

[0018] Preferable nitrogen sources include peanut flour, yeast extract, peptone, gluten meal, cottonseed flour, soybean meal, soybean meal, corn steep liquor, dry yeast, wheat germ, and the like, and inorganic or organic nitrogen compounds, For example, ammonium salts (for example, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids and the like can be mentioned.

The carbon source and the nitrogen source are advantageously used in combination, but low-purity raw materials containing trace amounts of growth factors and significant amounts of mineral nutrients are also suitable for use, and are therefore used in pure form. No need. If desired, the medium is supplemented with mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, zinc salts, iron salts or cobalt salts. Good.

[0020] If necessary, an antifoaming agent such as liquid paraffin, fatty oil, vegetable oil, mineral oil, or silicone may be added, especially when the medium is significantly foamed.

Agitation and aeration of the culture mixture may be carried out in a variety of ways, eg, using a propeller or similar mechanical agitation device, or by rotating or shaking the fermentor. May be.

Fermentation may vary depending on fermentation conditions and scale, but is usually about 10 ° C.
It is carried out at a temperature of -40 ° C, preferably 20 ° C to 35 ° C for about 24 hours to 120 hours.

At the end of the fermentation, for the recovery of the FR231956 substance, the cultivated medium is subjected to various methods commonly used for the recovery and purification of physiologically active substances, for example suitable solvents or solvents using mixtures of several solvents. It is subjected to extraction, chromatography, or recrystallisation from a suitable solvent or mixture of solvents.

The free form FR231956 substance is treated with an inorganic or organic base such as sodium or potassium hydroxide, ammonium hydroxide or ethanolamine, and further treated with an amino acid such as glycine, lysine or glutamic acid to form a salt thereof. It can be converted.

Biological Properties of FR231956 Substances FR231956 substances are pathogenic microorganisms, especially pathogenic yeasts (eg Cand).
It has a strong antibacterial activity against pathogenic fungi such as ida albicans). Therefore, the FR231956 substance or a pharmaceutically acceptable salt thereof is useful as an antibacterial agent, especially as an antifungal agent used for the treatment of infectious diseases in humans or animals. The above infections include surface, skin, subcutaneous and systemic fungal infections such as respiratory infections, gastrointestinal tract infections, cardiovascular infections, ureteral infections, candidiasis and chronic mucosal candidiasis (eg oral cavity infections. And vaginal candidiasis), skin infections caused by fungi, cutaneous and mucocutaneous candidiasis, dermatophytosis (eg ringworm and ringworm, tinea pedis, paronychia, pityriasis, intertritis, candida). Vulva, candida glans, and otitis externa). They can also be used as prophylactic agents to prevent systemic and topical fungal infections in immunocompromised patients (eg AIDS patients, patients undergoing cancer treatment or organ transplant patients).

As an example showing the pharmacological action of FR231956, some biological data are explained below. Test 1 (antibacterial activity) The antibacterial activity of the FR231956 substance was measured by the continuous medium dilution method using a 96-well microtiter plate in 100 μl of yeast nitrogen basic dextrose medium. The inoculum was adjusted to 1 × 10 ⁵ colony forming units / ml. Candida albicans was cultured at 37 ° C. for 24 hours and Cryptococcus neoformans was cultured at 37 ° C. for 48 hours in a 5% CO ₂ incubator. After incubation, the growth inhibition of microorganisms in each well was determined by microscopic observation. The results are shown as MEC (minimum effective concentration: μg / ml) values (Table 1).

[0027]

[Table 1]

From the test results, it was found that the FR231956 substance of the present invention has antibacterial activity (particularly antifungal activity). The pharmaceutical composition of the present invention is in the form of a pharmaceutical preparation containing FR231956 substance or a pharmaceutically acceptable salt thereof as an active ingredient together with an organic or inorganic carrier or excipient suitable for external use, enteral or parenteral administration. , For example in solid, semi-solid or liquid form. The active ingredient is suitable for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, injections, ointments, liniments, eye drops, lotions, gels, creams and uses. Alternatively, it may be formulated with a conventional non-toxic pharmaceutically acceptable carrier.

Carriers that can be used include water, glucose, lactose, acacia gum, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, and during production. Other carriers suitable for use may be mentioned, in solid, semi-solid or liquid form, as well as auxiliaries, stabilizers, thickeners, solubilizers, colorants and fragrances. be able to.

The target compound or a pharmaceutically acceptable salt thereof is contained in the pharmaceutical preparation in an amount sufficient to produce a desired antibacterial action depending on the course or symptoms of the disease.

For application of the composition to humans, it is preferably applied by intravenous, intramuscular, topical, transdermal, pulmonary or oral administration or inhalation. The dose or therapeutically effective amount of the FR231956 substance or a pharmaceutically acceptable salt thereof varies depending on the age and symptoms of the individual patient to be treated, but in general, when administered intravenously, it is 1 per kg of human body weight. In the case of intramuscular administration of an amount ranging from 0.01 to 20 mg of FR231956 substance per day, FR231956 per kg body weight of human per day
An amount of the substance in the range of 0.1 to 20 mg is used for the treatment or prophylaxis of infectious diseases in the range of 0.5 to 50 mg of FR231956 substance per kg of human body weight per day for oral administration.

The following examples are given by way of illustration of the invention and are not meant to be limiting. Example (1) Sordaria araneo for producing FR231956 substance
sa ATCC36386 fermented corn starch 2%, sucrose 1%, glycerol 1%, Pharmamedia 1%,
Liquid seed medium containing 1% gluten meal and 0.2% Tween 80 (160
ml) into each of three 500 ml Erlenmeyer flasks and
Sterilized at 30 ° C for 30 minutes. Sordaria araneosa ATCC363
One platinum loop of 86 slant cultures was inoculated into each seed flask. The inoculated flask was shaken with a rotary shaker (220 rpm, 5.1 cm stroke) at 25 ° C. for 4 days, and 480 ml (3 flasks) of the seed culture was denatured starch in a 30 liter jar fermenter. 3%, Pharmamedia 2%, Oatmeal 0
． 5%, (NH ₄ ) ₂ SO ₄ 0.3%, KH ₂ PO ₄ 2%, Na ₂ HPO ₄ · 12
H ₂ O 1.5%, ADECANOL LG-109 0.05% and Silicone K
20 liters of sterile production medium containing 0.05% M-70 were inoculated. The fermentation was carried out at 25 ° C. for 4 days under aeration of 20 l / min and stirring at 200 rpm.

The production of FR231956 material in the fermented medium was monitored by the HPLC analysis shown below. # HPLC analysis condition column: YMC Pack ODS-AM 303, S-5 120A (250 x 4.6 mm inner diameter, YMC Corporation)
Mobile phase: 60% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid Flow rate: 1 ml / min Detection: UV, 210 nm Retention time: FR231956 11.8 minutes (2) Separation and purification of FR231956 substance Culturing at room temperature for 2 hours with stirring The prepared medium (100 liters) was extracted with an equal volume of acetone. The mixture was filtered through diatomaceous earth. The filtrate was diluted with an equal volume of water and passed through a column (7 liters) of Diaion HP-20 (Mitsubishi Chemical Co., Ltd.) filled with 25% aqueous acetone solution. The column was washed with 50% aqueous methanol solution (20 liters) and eluted with methanol (40 liters). The active fraction (0 to 30 liters) was diluted with water to 75 liters, and 40% methanol aqueous solution filled YMC-GEL (O
DS-AM120-S50, YMC Corporation) column (2 liters). The column was eluted with 50% aqueous methanol (9.3 liters). Active fraction (
1.3-5.3 liters) is diluted with an equal volume of water, and a 25% methanol aqueous solution is charged Y
It was passed through a column (2 liters) of MC-GEL (ODS-AM120-S50, YMC Corporation). The column was washed with 40% aqueous acetonitrile containing _{0.5% NaH 2 PO 4 · 2H} 2 O (9 _{liters), 0.5% NaH 2 PO 4} · 2H 2 O
Elution was carried out with a 50% aqueous acetonitrile solution (9.95 liters) containing. Active fraction (6.15 to 9.45 liters) was diluted with water equal volume of 25% aqueous acetonitrile filled YMC-GEL containing _{0.25% NaH 2 PO 4 · 2H} 2 O (ODS-A
(M120-S50, YMC Corporation) column (2 liters). The column was washed with 50% aqueous methanol solution (4 liters) and washed with 97% aqueous methanol solution (
(2.6 liters). The active fraction (1.3-2.1 liter) was concentrated in vacuo to give a yellow residue. The residue was dissolved in a small amount of ethyl acetate, a large amount of n-hexane was added, and then dried to obtain 1044 mg of FR231956 as a pale yellow powder.

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Claims (10)

[Claims] 1. The following formula: FR231956 substance represented by. 2. The FR231956 substance or a salt thereof according to claim 1, which comprises culturing a FR231956 substance-producing microorganism in a nutrient medium and recovering the FR231956 substance or a salt thereof according to claim 1 from the cultured medium. Manufacturing method. 3. An antibacterial agent comprising the FR231956 substance or a salt thereof according to claim 1 and a carrier. 4. FR23195 according to claim 1 for use as a medicament.
6 substances or pharmaceutically acceptable salts thereof. 5. Use of the FR231956 substance according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament. 6. A pharmaceutical composition comprising the FR231956 substance according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, together with a pharmaceutically acceptable carrier or excipient. 7. A method for killing a microorganism by applying the FR231956 substance or a salt thereof according to claim 1 to the microorganism. 8. A method for killing a fungus by applying the FR231956 substance or a salt thereof according to claim 1 to the fungus. 9. A method for preventing and / or treating an infectious disease caused by a pathogenic microorganism, which comprises administering the FR231956 substance according to claim 1 or a pharmaceutically acceptable salt thereof to a human or an animal. 10. Use of the FR231956 substance according to claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating infectious diseases caused by pathogenic microorganisms.