JPH08119983A - New compound wf14865, its production and its use - Google Patents

Abstract

PURPOSE: To obtain a new compound for treating and preventing muscular dystrophy, Alzheimer's dementia, etc., having inhibiting action on cathepsin B and L by culturing WF14865-producing yeast belonging to the genus Anixiopsis in a nutrient medium. CONSTITUTION: A WF14865 producing yeast belonging to the genus Anixiopsis [e.g. Anixiopsis stercoraria No. 14865 (FERM BP 4689), etc.] is cultured in a nutrient medium and a product is collected from the resultant cultured products to provide a new compound WF 14865 consisting of a compound (A) which is colorless powder, has the molecular formula, C16 H25 N5 O5 , is soluble in methanol and water, slightly soluble and insoluble in chloroform and positive to ferric chloride reaction and negative to Ehrlich's reaction and has a specific rotation, [α]<25> D=30.0 deg. (C=1.0, CH3 OH) and contains isoleucine or the compound (B) which has a molecular formula and solubility similar to the compound A and is white powder and is negative to ferric chloride reaction and positive to Ehrlich's reaction and has a specific rotation, [α]<23> D=+35 deg. (C=0.7, CH3 OH) and contains leucine.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel compound WF148
65A and WF14865B. More specifically, the present invention relates to novel compounds WF14865A and W that play a role in inhibiting cathepsin B and / or L activity.
F14865B, WF14865 belonging to Ascomycete genus Anixiopsis Hansen 1897 in nutrient medium
WF14865A and W
Method for producing F14865B, and WF14865
It relates to a pharmaceutical composition containing A or WF14865B and a pharmaceutically acceptable carrier.

[0002]

2. Description of the Related Art Recently, cathepsin B which is a lysosomal cysteine protease
It was suggested that L and / or L are involved in joint destruction caused by inflammatory joint diseases such as rheumatoid arthritis and osteoarthritis [Biochemical Pharmacolog
y, 44 , 1201 (1992)]. In addition, cathepsin B and / or L is associated with muscular dystrophy and vacuolar peripheral myopathy.
It is thought to cause intractable muscle collapse diseases such as lar distal myopathy. Cathepsins B and L are also enzymes involved in the formation of senile plaques in the brain of patients with Alzheimer's disease.

Therefore, cathepsin B and / or L
Inhibitors are expected to be useful against these diseases. Traditionally, for example, the microbial products antipain and leupeptin are effective but reversible inhibitors of cysteine proteases [McConnell et a
l., 33 J. Med. Chem. 86-93 (1990); Sutherland eta
l. 110 Biochem. Biophys. Res. Commun. 332-38 (198
2)], they also inhibit serine proteases [Um
ezawa, 45 Meth. Enzymol. 678-95 (1976)]. Compound E
64 and its synthetic analogues are more selective inhibitors [Barret et al., 201 Biochem. J. 189-98 (1982); Gr.
inde, 701 Biochem. Biophys. Acta. 328-33 (1982)
], It disappears from the blood circulation too quickly when used in vivo [Hashida et al., 91 J. Bi.
ochem. 1373-80 (1982)].

[0004]

DISCLOSURE OF THE INVENTION The present inventors have screened novel compounds having cathepsin B and / or L inhibitory activity from microbial products. As a result of the screening, the present inventors isolated the novel compounds WF14865A and WF14865B from the culture of a strain belonging to the ascomycete genus Anixopsis.

The present invention relates to cathepsin B and / or L
Compounds WF14865A and WF, which have an inhibitory action against chlorophyll and have physicochemical properties as described below
14865B is provided. The present invention also cultivates a WF14865-producing strain belonging to the genus Ascomycete Anixiopsis in a nutrient medium, and obtains the compound WF14865 from the obtained culture.
A method for producing a compound WF14865A or WF14865B, which comprises collecting A or WF14865B. Furthermore, the present invention is useful as a pharmaceutical composition for mammals for treating and preventing the above-mentioned various diseases caused by cathepsin B and / or L activity,
Compound WF14865A or WF14 as an active ingredient
The present invention provides a pharmaceutical composition containing 865B and a pharmaceutically acceptable carrier. Furthermore, the present invention provides a drug that exhibits cathepsin B and / or L inhibitory activity, and an anixiopsis stercoraria.
a) No. 14865 to provide a biologically pure culture.

The novel compound WF14865A has the following physicochemical properties. a) Appearance: colorless powder b) Molecular formula: C ₁₆ H ₂₅ N ₅ O ₅ c) FAB-MS: m / z 368 (M + H) HRFAB-MS: m / z Measured value: 368.192
4 Theoretical value C ₁₆ H ₂₅ N ₅ O ₅ + H: 368.1934 d) Infrared absorption spectrum: 3290, 2970, 1680, 1640, 1560, 1460, 1430, 1390, 12 shown in FIG.
00, 1140 900, 800cm ^-1 e) Solubility: Soluble: Methanol, Water Poorly soluble: Acetone Insoluble: Chloroform f) Color reaction Positive: Ninhydrin reaction Ferric chloride reaction Cerium sulfate reaction Reaction with iodine vapor Negative: Ehrlich reaction Maurish reaction Diacetyl reaction g) ¹ H-NMR spectrum (400 MHz, CD ₃ OD)
(See Fig. 2) δ ppm: 6.52 (1H, s), 4.17 (1H, d, J = 8Hz), 3.69 (1
H, d, J = 2Hz), 3.51 (1H, d, J = 2Hz), 3.27 (1H, m), 3.22
(1H, m), 2.50 (2H, m), 1.90-1.73 (3H, m), 1.54 (1
H, m), 1.20 (1H, m), 0.94 (3H, d, J = 7Hz), 0.92 (3H,
t, J = 7Hz) h) ¹³ C-NMR spectrum (100 MHz, CD ₃ OD)
(Shown in Fig. 3) δ ppm: 173.4 (s), 170.6 (s), 168.7 (s), 148.6 (s), 1
28.3 (s), 110.0 (d), 59.7 (d), 54.2 (d), 53.3 (d), 39.3
(t), 37.8 (d), 29.0 (t), 26.1 (t), 22.6 (t), 15.9 (q),
11.3 (q) i) Specific rotation: [α] _D ²⁵ = + 30.0 ° (c = 0.1, CH ₃ OH) j) Amino acid analysis: isoleucine

The novel compound WF14865B has the following physicochemical properties. a) Appearance: White powder b) Molecular formula: C ₁₆ H ₂₅ N ₅ O ₅ c) FAB-MS: m / z 368 (M + H) HRFAB-MS: m / z Measured value: 368.192
4 Theoretical value C ₁₆ H ₂₅ N ₅ O ₅ + H: 368.1934 d) Infrared absorption spectrum: 3300, 2960, 1680, 1650, 1620, 1550, 1470, 1440, 13 shown in FIG.
80, 1250 1130, 900 cm ^-1 e) Solubility: Soluble: Methanol, water Poorly soluble: Acetone Insoluble: Chloroform f) Color reaction Positive: Ninhydrin reaction Cerium sulfate reaction Ferric chloride-potassium ferricyanide reaction Iodine vapor and Reaction Ehrlich reaction Negative: ferric chloride reaction Maurish reaction g) ¹ H-NMR spectrum (300 MHz, CD ₃ OD)
(Shown in Fig. 5) δ ppm: 6.50 (1H, s), 4.36 (1H, dd, J = 9Hz, J = 6Hz),
3.53 (1H, d, J = 2Hz), 3.36 (1H, d, J = 2Hz), 3.30-3.14
(2H, m), 2.49 (2H, m), 1.78 (2H, m), 1.70-1.54 (3H,
m), 0.97 (3H, d, J = 6Hz), 0.92 (3H, d, J = 6Hz) h) ¹³ C-NMR spectrum (75 MHz, CD ₃ OD)
(See Fig. 6) δ ppm: 174.6 (s), 174.0 (s), 170.2 (s), 148.7 (s), 1
28.4 (s), 109.9 (d), 55.6 (d), 54.1 (d), 53.6 (d), 41.7
(t), 39.3 (t), 29.0 (t), 26.0 (d), 23.4 (q), 22.6 (t),
21.9 (q) i) Specific rotation: [α] _D ²³ = + 35 ° (c = 0.7, CH ₃ OH) j) Amino acid analysis: Leucine

WF14865A (5- [3- [N-
[[(2R, 3R) -3-trans- (oxycarbonyl) oxiran-2-yl] carbonyl] -L-isoleucyl] aminopropanyl] -1H-imidazole-2
The structural formula of -ylamine) is as follows.

[0009]

Embedded image

WF14865B (5- [3- [N-
The structural formula of [[(2R, 3R) -3-trans- (oxycarbonyl) oxiran-2-yl] carbonyl] -L-leucyl] aminopropanyl] -1H-imidazol-2-ylamine) is as follows: is there.

[0011]

Embedded image

In the present specification, the above compound WF1486
5A and WF14865B are also collectively referred to as WF14865.

WF14865 is an anixiopsis stercoraria no . 1486
WF belonging to Ascomycetes Anixopsis such as 5
It is produced by culturing a 14865-producing bacterium in a nutrient medium and collecting WF14865 from the obtained culture.
Among the WF14865 producing bacteria belonging to the Ascomycetes Aniki shea Opsys genus, Aniki Shi Opsys Suterukoraria (Ani
xiopsis stercoraria) No. 14865 was newly isolated from the soil sample collected by the present inventors in Kanoya City, Kagoshima Prefecture. Anixiopsis stercoraria No. The freeze-dried sample of 14865 is an international depository institute under the Budapest Treaty, Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology (1-3, Higashi, Tsukuba, Ibaraki, Japan (postal code 30
5)) on June 6, 1994, with deposit number FERM BP
Deposited as -4689.

The methods utilizing the specific microorganisms mentioned in the present specification are merely for the purpose of explanation, and the production of the novel compound WF14865 is not limited thereto. The present invention also includes all variants capable of producing WF14865, including naturally occurring variants as well as X-rays, UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, or Also included are artificial variants produced from the aforementioned microorganisms by conventional methods such as treatment with 2-aminopurine and the like.

Anixiopsis stelaria (Anixi
opsis stercoraria) No. 14865 has the following morphological, culture and physiological properties.

WF14865 producing strain No. 14865
Properties of strain No. 14865 was isolated from a soil sample collected in Kanoya City, Kagoshima Prefecture. This microorganism spread rapidly in various media and formed yellowish white colonies. This strain No. 14865 is anamorph in some of the agar medium (chrysosporium - morphs (Chrysos
porium- morph)) and teleomorphs (asci). Ascidium is an ascidian shell, in which the asci are irregularly arranged. The asci have a unilamellar membrane and morphology are subglobular to ovoid with a fugitive wall. Ascospores are lenticular and covered with an irregular reticulum. Based on the morphological characteristics, this strain is considered to belong to the ascomycete genus Anixopsis (Y. Otani: Seiya Ito's Mycological Flora of Jap.
an.Vol.III, No.2, 310p., Yokendo LTD., Tokyo, 198
8).

The culture properties of various agar media are shown in Table 1.
Shown in On YpSs agar medium, colonies grow extensively and extensively, and their diameter is 6.5-7.0c at 25 ° C. for 2 weeks.
reached m. The surface of the colony was flat and light, and the color was pale red to white. The color of the back surface was reddish gray to yellowish white. Ascidian and conidial structures were observed. The colonies on the corn meal agar medium grew extensively and expanded, and the diameter reached 5.0 to 6.5 cm under the same conditions. The surface of the colony was flat, thin, powdery, and yellowish white in color. The color of the back surface was also yellowish white. Conidial structure was observed.

Morphological properties were determined on the basis of cultivation on YpSs agar medium. The obturator shell is thin, smooth, and spherical to subspherical in size, with a size of 130-29.
At 0 μm, it becomes grayish when matured. Eight spores are contained inside the ascus, and the morphology is subspherical to ovoid with a disappearing wall, and the size is 7.5-12.0μ in diameter.
It was m. Ascospores are lenticular and have a diameter of 3.5
-5.5 μm, covered with irregular reticulum. Anamorphs consist of Aleuro-type conidia and segmental-type conidia.
The terminal and lateral Aleurotic conidia are colorless, have no septum, are smooth, and have a pear-shaped to club-shaped form.
It has a cut-out base and has a size of 4.5-10.0 × 4.
It was 0-6.5 μm. The mid-segmented conidia in the middle are colorless, have no septum, are smooth, have a cylindrical to barrel form, and are cut at both ends and have a size of 5.0-1.
It was 2.5 × 2.5-5.0 μm. Vegetative hyphae were smooth, septate, colorless and branched. The mycelium had a cylindrical shape and the width thereof was 1.0 to 4.5 μm.

No. The 14865 strain can grow in the range of 6 ° C to 36 ° C, and the optimum growth temperature is 26 ° C to 31 ° C.
° C. These temperature data were measured on a potato-glucose agar medium (manufactured by Nissui Co., Ltd.).

According to the classification criteria of the genus Anixopsis (E. Gueho and C. De Vroey: Canadian Journal of Bo
tany, 64, pp.2207-2210, 1986), No. The 14865 strain is an Anixiopsis stella coral
ercoraria) strain, deposit number FERM BP at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry
Deposited as -4689 (deposit date: June 6, 1994).

[0021]

[Table 1]

^* : The composition of malt extract agar medium, Czapek agar medium, and MY20 agar medium is JCM Catalogu.
e of Strains (Nakase, T., 5th ed., 503p., Japan Co
llection of Microorganisms and Life Science Resear
ch Information Section of the Institute of Physical
and Chemical Research, Saitama, 1992).

These characteristics were observed after culturing at 25 ° C. for 14 days. Metune Handbook of Color (Kornerup, A. and JH Wanscher, Methuen Handboo
k ofColor, 3rd ed., 525p., Methuen, London, 1978)
Based on the above, the color tone is described.

Generally, WF14865 is WF1486
A nutrient medium containing a carbon source and a nitrogen source capable of assimilating 5 producing bacteria, preferably under aerobic conditions (eg, shaking culture,
It can be produced by culturing in submerged culture or the like).

The preferred carbon source in the nutrient medium is glucose,
Carbohydrates such as fructose, glycerin and starch. Other carbon sources that can be contained include lactose, arabinose, xylose, dextrin, molasses and the like.

Suitable nitrogen sources include yeast extract, peptone, gluten meal, cottonseed flour, soybean flour, corn steep liquor, dried yeast, etc., as well as ammonium salts (for example, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids. Inorganic and organic nitrogen compounds such as.

Although it is advantageous to use a combination of a carbon source and a nitrogen source, if the raw material has a low purity and contains a small amount of growth factors and a considerable amount of inorganic nutrients, it is suitable for use. Need not be used.
If necessary, an inorganic salt such as calcium carbonate, sodium phosphate, potassium phosphate, sodium iodide, potassium iodide, magnesium salt, or cobalt chloride may be added to the medium. If necessary, an antifoaming agent such as liquid paraffin, higher alcohols, vegetable oils, mineral oils or silicones may be added, especially when the culture solution foams significantly.

As a condition for mass production, aerobic submerged culture is preferred for production of WF14865. For small production,
Shake or surface culture in flasks or bottles. Furthermore, when culturing in a large tank, WF148
In order to prevent the growth delay in the production process of No. 65, it is preferable to inoculate the production tank with the growth-type microorganism. Therefore, first of all, a relatively small amount of medium is inoculated with spores or mycelia of microorganisms to produce a microbial vegetative inoculum by culturing the inoculated medium, and the cultivated vegetative inoculum is aseptically stored in a large tank. Move to. As the medium for producing the growth-type inoculum, a medium that is substantially the same as or slightly different from the medium used in the main culture of WF14865 can be used.

The stirring and aeration of the culture mixture can be carried out in various ways. Agitation can be accomplished with a propeller or similar mechanical agitation device, by rotating or shaking the fermentor, by various pumping devices, or by passing sterile air through the culture. Aeration can be accomplished by passing sterile air through the fermentation mixture.

Culturing is usually carried out at about 4 ° C. to about 40 ° C., preferably about 20 ° C. to about 30 ° C., for 50 hours to 100 hours. This can vary depending on fermentation conditions and scale.

The WF14865 produced in this way is
Harvested from the culture by known methods commonly used to recover other fermentation products such as antibiotics.

In general, the majority of WF14865 produced is found in the culture mycelium cake. Therefore, WF1
4865 is a fungal cake extract obtained by filtering or centrifuging a culture solution, extraction using a conventional solvent, concentration under reduced pressure, lyophilization, pH adjustment, a conventional resin (for example, anion or Treatment with cation exchange resin, nonionic adsorption resin), conventional adsorbent (activated carbon,
It is isolated by conventional methods such as treatment with silicic acid, silica gel, cellulose, alumina), crystallization, recrystallization and the like.

WF14865 can also be synthesized by a synthesis method as shown in Examples below. WF14865
Since it has a carboxyl group, it can be converted into an ester form by a conventional method. Examples of ester form are:
Examples thereof include alkyl esters (esters with alkyl such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl) and aralkyl esters (benzyl ester).

The following tests detail some of the biological properties of WF14865. Test Example 1 (Inhibition of cathepsin B activity in vitro by WF14865A or WF14865B.) Cathepsin B activity is a fluorescence-producing peptide N-.
Carbobenzoxy-L-Arg-L-Arg-AMC
7-amino-4 from (Z-Arg-Arg-AMC)
-Methylcoumarin (AMC) emission was measured by detection with excitation at 360 nm and emission at 460 nm. All reactions were performed on microtiter plates. 50 μl of inhibitor was added to 10 μM Z-Arg-
Arg-AMC, 5 mM dithiothreitol and 1
Serial dilutions were made with 100 mM sodium phosphate buffer solution (pH 6.0) containing mM EDTA. Reaction is 75
ng / ml human liver cathepsin B (Athens Research
& Technology, USA). The mixture was incubated at 37 ° C. for 60 minutes, and the reaction was terminated by adding 100 μl of 100 mM sodium monochloroacetate dissolved in a buffer solution (pH 4.3) containing 30 mM sodium acetate and 70 mM acetic acid. A Titertech Fluorocan II spectrophotometer (flow Laboratories, Australia) was used for fluorescence measurements. The fluorescence of the reagent blank (without enzyme) was subtracted from the test measurements. The calibration was performed with free AMC having different concentrations.

WF14865A and WF14865B
The results obtained when is used as an inhibitor are shown in Tables 2 and 3, respectively. In the above measurement, WF14865
Cathepsin B (Lot. CB9302) is used for A, and cathepsin B (Lot. CB) is used for WF14865B.
9402) was used.

[0036]

[Table 2]

[0037]

[Table 3]

Test Example 2 (WF14865A or WF1
Inhibition of cathepsin L activity in vitro by 4865B. ) Cathepsin L activity was measured similarly to cathepsin B activity using human liver cathepsin L (final concentration 50 ng / ml, Calbiochem, USA) and Z-Phe-Arg-AMC as substrate. WF14865A and WF14
The results obtained when using 865B as the inhibitor are shown in Tables 4 and 5, respectively. In the above measurement, WF1
Cathepsin L (Lot. 793993) is present in 4865A.
And WF14865B has cathepsin L (Lo
t. B10573) was used.

[0039]

[Table 4]

[0040]

[Table 5]

Test Example 3 (acute toxicity) WF14865A or WF14865B (dose: 3)
(00 mg / kg) was intraperitoneally administered to 5 ICR mice (female, 5 weeks old). Observed for one week, no abnormal symptoms were observed.

WF14865A and W in the present invention
F14865B is a strong cathepsin B and / or L
Because of its inhibitory effect, rheumatoid arthritis, osteoarthritis, malaria, gingivitis, metabolic bone disease (osteoporosis), muscular dystrophy, vacuolar peripheral myopathy (vacuolar distal myopathy)
myopathy) and mammals (eg, humans, mice, rats, cats, dogs, rabbits, cows, pigs) for treating and preventing diseases caused by increased cathepsin B and / or L activity such as Alzheimer's disease Used as a medicine.

WF14865A or W in the present invention
F14865B can be administered to a mammal (eg, human, mouse, rat, cat, as a mixture with a pharmaceutically acceptable carrier).
It can be orally or parenterally administered to dogs, rabbits, cows, pigs) in the form of pharmaceutical compositions such as capsules, tablets, granules, powders, buccal agents, sublingual agents and solutions.

The pharmaceutically acceptable carrier includes various organic or inorganic carrier materials conventionally used for pharmaceutical use, such as excipients (eg, sucrose, starch, mannitol, etc.).
Sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, etc.), binders (eg, cellulose, methyl cellulose, hydroxypropyl cellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose,
Starch, etc.), disintegrating agent (eg, starch, carboxymethyl cellulose, calcium salt of carboxymethyl cellulose, hydroxypropyl-starch, sodium salt of glycol-starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, etc.), lubricant (eg, Magnesium stearate, aerosil, talc,
Sodium lauryl sulfate, etc.), flavoring agents (eg, citric acid, menthol, glycine, orange powder, etc.), preservatives (eg, sodium benzoate, sodium bisulfite, methylparaben, propylparaben, etc.), stabilizers (eg, quenching agent). Acid, sodium citrate, acetic acid, etc.),
Suspending agents (eg, methylcellulose, polyvinylpyrrolidone, aluminum stearate, etc.), dispersants (eg, surfactants, etc.), aqueous diluents (eg, water), oils (eg, sesame oil, etc.), and bases Wax (for example,
Cocoa butter, polyethylene glycol, white petrolatum, etc.)
Is exemplified.

WF14865A and WF14865B
The dose of can be varied depending on various factors such as the type of disease, the weight and / or age of the patient, and the route of administration. WF14865A or WF14865B is about 0.1-100 mg / kg / day, preferably 1-50 m
g / kg is administered parenterally, especially subcutaneously, intravenously, muscularly or rectally. When used as an oral agent, WF14865A or WF14865B is preferably administered at about 0.1-500 mg / kg, more preferably 10-300 mg / kg, per day.

[0046]

EXAMPLES Examples will be given below to explain the present invention. Example 1: Isolation of WF14865A (1) No. Cultivation of 14865 strain Sucrose (4%), cottonseed meal (2%), dry yeast (1%),
Polypeptone (1%), potassium dihydrogen phosphate (0.2
%), Calcium carbonate (0.2%), and polysorbate 80 (0.1%) in an aqueous seed medium (12
0 ml) was poured into each of three 500 ml Erlenmeyer flasks and sterilized at 120 ° C. for 30 minutes. 2 weeks at 25 ℃,
From the mature slope culture grown on YpSs agar, one platinum loop No. Each seed flask was inoculated with 14865 strain. The inoculated flask was placed on a rotary shaker (2
Shaking was performed at 25 rpm at 20 rpm and an amplitude of 5.1 cm for 4 days. The resulting seed culture was inoculated into 20 liters of sterile culture medium. Corn starch (2%), glycerin (1%), peanut powder (1%), corn steep liquor (0.5%) and calcium carbonate (0.
Aqueous medium containing 2%) was poured into a 30 liter stainless steel jar fermentor. PH of medium
Was adjusted to 6.0 with aqueous NaOH before sterilization at 120 ° C. for 30 minutes. The culture was carried out at 25 ° C. for 5 days while aerating at 20 liter / min and stirring at 180 rpm.

No. in culture 14865 (WF1486
5A) production by reverse phase column (YMCAM303 ODS 250x4.6mm
(Inner diameter) HPLC (H, YMC, Japan)
ighPerformance Liquid Chromatography: high performance liquid chromatography). The solvent system is 0.
It was 25% aqueous methanol containing 05% trifluoroacetic acid (TFA), and its detection wavelength was 210 nm.

(2) Isolation and purification of WF14865A After completion of the culture, the culture solution (40 liters) was filtered, and 40 liters of methanol were added to the mycelium cake while stirring. The mixture was left overnight and filtered. The extract was concentrated under reduced pressure until it became an aqueous solution, and the polymer adsorbent HP-20
(Mitsubishi Kasei Co., Ltd., 1.5 liters). After washing the column with water, the desired material was eluted from the column with 50% aqueous methanol (6 liters). The eluate was concentrated to dryness (about 4.5 g), dissolved in a small amount of water,
It was subjected to C-ODS-AM (YMC, Japan, 2 liters) column chromatography. Set the column to 0.
Elution was performed with a 10% aqueous acetonitrile solution containing 2% TFA. Active fractions were collected and concentrated to dryness to give crude residue 83
1 mg was obtained. 100 mg crude residue with a small amount of 0.1%
It was dissolved in a 12.5% acetonitrile aqueous solution containing TFA and packed in a reverse phase column YMC-ODS-AM (YMC packed column, SH-343-5AM, YMC, Japan). The column was eluted with the same solvent as above, and the active fractions were collected. The collected fractions were dried and redissolved in 25% methanol in water containing 0.1% TFA, reverse phase column YM.
C-ODS-AM (YMC packed column, SH-343-5AM, YMC, Japan) was packed. Column 0.1% T
Elution was performed with 25% aqueous methanol containing FA. The active fraction was concentrated under reduced pressure and freeze-dried to give 11 mg of WF14865A as a pure powder.

Example 2: Isolation of WF14865B (1) No. Cultivation of strain 14865 Sucrose (4%), cottonseed flour (2%), dried yeast (1%),
Polypeptone (1%), potassium dihydrogen phosphate (0.2
%), Calcium carbonate (0.2%), and polysorbate 80 (0.1%) in an aqueous seed medium (12
0 ml) was poured into each of three 500 ml Erlenmeyer flasks and sterilized at 120 ° C. for 30 minutes. 2 weeks at 25 ℃,
From the mature slope culture grown on YpSs agar, one platinum loop No. Each seed flask was inoculated with 14865 strain. The inoculated flask was placed on a rotary shaker (2
Shaking was performed at 20 rpm and an amplitude of 5.1 cm) at 25 ° C. for 3 days.

Corn starch (2%), glycerin (1
%), Corn steep liquor (0.5%), peanut powder (1%), ammonium sulfate (0.5%),
An aqueous medium containing calcium carbonate (1%) and β-cyclodextrin (1%) was poured into a 30-liter stainless steel jar fermentor. The pH of the medium should be 6N NaO before sterilization at 120 ° C for 30 minutes.
It was adjusted to 6.0 with an aqueous H solution.

The seed culture obtained was inoculated into 20 liters of sterile culture medium. The culture was carried out at 25 ° C. for 4 days while aerating at 20 liter / min and stirring at 200 rpm.

No. in culture 14865 (WF1486
5B) is produced by reverse phase column (YMCAM303 ODS 250x4.6mm
HPLC using (inner diameter), YMC, Japan)
Monitored by. The solvent system is acetonitrile-tetrahydrofuran (THF) -water (12.5: 2.0: 85.5) containing 0.1% trifluoroacetic acid (TFA).
The detection wavelength was 210 nm.

(2) Isolation and purification of WF14865B The culture solution (320 liters) thus obtained was filtered using a layer of diatomaceous earth. Add 320 liters of tap water to the mycelial cake while stirring and adjust the pH to 2.0 with concentrated sulfuric acid.
Adjusted to. The mixture was left at room temperature for 5 hours and filtered. The extract was adjusted to 6.5 with 6N aqueous NaOH,
The polymer was adsorbed on Diaion HP-20 (Mitsubishi Kasei Co., Ltd., 21 liters). The column was washed with water and then eluted with 50% aqueous methanol. The eluate (8
4 liters) was concentrated under reduced pressure to 40 liters. The aqueous solution was passed through Diaion HP-20 (10 liters). Water, 10% methanol water, and 15
After washing with% methanol water, the desired material was eluted with 40% methanol water. The eluate was concentrated under reduced pressure to 10 liters and 10 ml TFA was added. The concentrated solution was mixed with YMC-gel (ODS-AM, 120-S50, YMC,
2 liters) column chromatography. Acetonitrile-THF containing 0.1% TFA
-Eluted with water (5: 2: 93). Collect the active fractions,
It was concentrated under reduced pressure to an aqueous solution and freeze-dried. After dissolving with a small amount of water, the crude yellowish powder was resubmitted to YMC-gel (2 liters) column chromatography eluting with a similar solvent system. The active fraction was freeze-dried to obtain a pale yellowish powder (389.9 mg). 4 ml
This crude product, dissolved in water, was loaded onto a YMC-gel (2 liter) column and eluted with acetonitrile-THF-water (10: 2: 88) containing 0.1% TFA.
The active fraction was concentrated under reduced pressure and freeze-dried. 2.5
White powder (243.8 mg) dissolved in ml of water was added to Y
MC packed column (D-ODS-15B, S-15, 250x30mm (inner diameter))
Using acetonitrile-THF-water (10: 2: 8).
8) was used as a mobile phase and was subjected to preparative HPLC eluting at a flow rate of 18 ml / min. Active fractions containing the compound of interest were pooled and concentrated to dryness under reduced pressure. The dried material is dissolved in a small amount of water and a YMC packed column (ODS-AM, SH-343-5AM, S-5, 250x) with the same solvent system as above at a flow rate of 10 ml / min.
It was isolated by preparative HPLC using 20 mm (inside diameter). After concentrating to remove the organic solvent, the aqueous solution was diluted with Di.
Passed through aion HP-20 (30 ml). The column was washed with water and then eluted with methanol. The eluate (12
0 ml) was evaporated to dryness, suspended in ethanol and then dried, 64.3 mg WF148 as a pure powder.
65B was obtained.

Production Example 1 (2R, 3R) -trans-2,3-epoxysuccinic acid diethyl ester ( Chem. Pharm. Bull, 35, 1098 (198
Synthesized according to 7)) (1.20 g) in ethanol (5 m
To l), an ethanol solution (11 ml) of potassium hydroxide (422 mg) was added dropwise under ice cooling. After stirring at the same temperature for 1.5 hours, the solvent was concentrated under reduced pressure. 5% of the obtained residue
After dissolving in an aqueous solution of potassium hydrogen sulfate (100 ml),
Sodium chloride was added to saturation and ethyl acetate (10
It was extracted with 0 ml × 3). The ethyl acetate solution was washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was concentrated under reduced pressure to give (2R, 3R) -trans-2,3-epoxysuccinic acid ethyl ester (1.02 g) as an oil. Got as. ¹ H-NMR (300MHz, CDCl ₃ ): δ 1.32 (t, 3H, J = 7.2Hz),
3.72 (d, 1H, J = 2.0Hz), 3.73 (d, 1H, J = 2.0Hz), 4.29
(q, 2H, J = 7.2Hz).

Production Example 2 Phthalimidoacetaldehyde diethyl acetal (1
8.0 g) was dissolved in trifluoroacetic acid (200 ml), stirred at room temperature for 30 minutes, and trifluoroacetic acid was concentrated under reduced pressure. The obtained phthalimidoacetaldehyde was dissolved in benzene (300 ml), (triphenylphosphoranylidene) -2-propanone (21.8 g) was added, and the mixture was heated under reflux for 5.5 hr. After the solvent was concentrated under reduced pressure, the obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate, 3: 1), and 2- (4-oxopent-2-enyl) isoindole-1,3-dione (10 .2g) was obtained. References: J. Org. Chem ., 59,
7299 (1994) mp 84-85 ℃ ¹ H-NMR (300MHz, DMSO-d ₆ ): δ 2.19 (s, 3H), 4.39 (d,
2H, J = 4.6Hz), 6.04 (dt, 1H, J = 1.3, 16.2Hz), 6.87
(dt, 1H, J = 4.6, 16.2Hz), 7.82-7.92 (m, 4H) ESI-M
S [M + H] ⁺ : 230.

Production Example 3 2- (4-Oxopento-2-enyl) isoindole-1,3-dione (8.20 g) and triethylamine (5.73 g) were dissolved in anhydrous benzene (200 ml), and the mixture was stirred under a nitrogen stream. At room temperature, trimethylsilyl triflate (10.0 g) was added with stirring. After stirring at the same temperature overnight, saturated aqueous sodium hydrogen carbonate solution (100 m
1) was added, and the mixture was extracted with ether (600 ml). After washing with water and drying over anhydrous magnesium sulfate, the solvent was concentrated under reduced pressure, and the resulting residue was tetrahydrofuran (200 ml).
Dissolved in. Sodium hydrogen carbonate (4.76) was added to this solution.
g) and N-bromosuccinimide (7.38 g) were added at room temperature, the mixture was stirred for 1 hr, saturated aqueous sodium hydrogen carbonate solution (100 ml) was added, and the mixture was extracted with ether (300 ml). The extract was washed with saturated brine, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate, 4: 1).
Purified by and crystallized from ether-hexane to give 2-
(5-Bromo-4-oxopent-2-enyl) isoindole-1,3-dione (5.62 g) was obtained. Reference: J. Org. Chem ., 59, 7299 (1994) mp 119-120 ℃ ¹ H-NMR (300MHz, DMSO-d ₆ ): δ 4.39-4.44 (m, 4H),
6.28 (dt, 1H, J = 1.3, 16.1Hz), 7.00 (dt, 1H, J = 4.5,
J = 16.1Hz), 7.83-7.95 (m, 4H) MS [M + H] ⁺ : 308.

Production Example 4 A solution of 2- (5-bromo-4-oxopent-2-enyl) isoindole-1,3-dione (3.00 g) in dimethylformamide (40 ml) was stirred at room temperature under a nitrogen stream at room temperature. Add acetylguanidine (3.07g),
The mixture was stirred at the same temperature for 96 hours. The solvent was concentrated under reduced pressure, water (300 ml) was added to the obtained residue, and the precipitated crystals were collected by filtration, washed with water and dried to give N- [5- [3- (1,
3-dioxo-1,3-dihydroisoindole-2-
Yl) propenyl] -1H-imidazol-2-yl]
Acetamide (1.85 g) was obtained. References: J. Or
g. Chem ., 59, 7299 (1994) mp 237-240 ℃ ¹ H-NMR (300MHz, DMSO-d ₆ ): δ 2.01 (s, 3H), 4.28 (d,
2H, J = 5.8Hz), 6.06 (dt, 1H, J = 5.8, 15.6Hz), 6.35
(d, 1H, J = 15.6Hz), 6.78 (s, 1H), 7.80-7.91 (m, 4
H) ESI-MS [M + H] ⁺ : 311.

Production Example 5 N- [5- [3- (1,3-dioxo-1,3-dihydroisoindol-2-yl) propenyl] -1H-imidazol-2-yl] acetamide (630 mg) was added to acetic acid. (40 ml) and methanol (40 ml)
10% Palladium carbon (200 mg) was added, and catalytic reduction was performed for 5 hours under a hydrogen atmosphere at 3 atm. After removing the catalyst by filtration, the solvent was concentrated under reduced pressure, and the residue was washed with ethyl acetate (50 m
It was dissolved in 1). The solution was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was concentrated under reduced pressure and crystallized from ether-hexane to give N- [5- [3- (1,3-dioxo-1,
3-Dihydroisoindol-2-yl) propanyl]
-1H-imidazol-2-yl] acetamide (55
0 mg) was obtained. mp 226-228 ℃ ¹ H-NMR (300MHz, DMSO-d ₆ ): δ 1.80-1.91 (m, 2H),
2.39-2.50 (m, 2H), 3.60 (t, 2H, J = 7.0Hz), 6.44
(s, 1H), 7.78-7.91 (m, 4H) ESI-MS [M + H] ⁺ : 313.

Production Example 6 N- [5- [3- (1,3-dioxo-1,3-dihydroisoindol-2-yl) propanyl] -1H-imidazol-2-yl] acetamide (620 mg) in ethanol Add hydrazine monohydrate (5 ml) to the solution (30 ml).
(00 ml) was added and the mixture was heated under reflux for 1 hr. The solvent was concentrated under reduced pressure, 1M hydrochloric acid (20 ml) was added, and the mixture was stirred at 70 ° C. for 30 min. The precipitated insoluble material was filtered off, the solvent was concentrated under reduced pressure, 6N hydrochloric acid (20 ml) was added to the residue, and the mixture was heated under reflux for 24 hours. By concentrating the solvent under reduced pressure, 5- (3-
Aminopropanyl) -1H-imidazol-2-ylamine dihydrochloride (424 mg) was obtained as a powder. ¹ H-NMR (300MHz, DMSO-d ₆ ): δ 1.79-1.90 (m, 2H),
2.46-2.58 (m, 2H), 2.68-2.80 (m, 2H), 6.63
(s, 1H) ESI-MS [M + H] ⁺ : 141.

Production Example 7 5- (3-aminopropanyl) -1H-imidazole-
2-ylamine dihydrochloride (424 mg) and triethylamine (605 mg) in dimethylformamide (20
ml) at room temperature for t-butoxycarbonyl-L-isoleucine N-hydrosuccinimide ester (653 m
g) was added. After stirring at the same temperature overnight, the solvent was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate (60 ml). The solution was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous magnesium sulfate, concentrated under reduced pressure and crystallized from ether to give 5- [3- (t-butoxycarbonyl-L-isoleucyl). Aminopropanyl) -1H-imidazol-2-ylamine (348m
g) was obtained. mp 145-147 ℃ ¹ H-NMR (300MHz, CDCl ₃ ): δ 0.89 (t, 3H, J = 7Hz), 0.9
2 (d, 3H, J = 7Hz), 1.05-1.24 (m, 2H), 1.42 (s, 9
H), 1.45-1.68 (m, 1H), 1.69-1.92 (m, 2H), 2.39
-2.55 (m, 2H), 3.08-3.31 (m, 2H), 3.96-4.05
(m, 1H), 6.29 (s, 1H) ESI-MS [M + H] ⁺ : 354.

Production Example 8 5- [3- (t-butoxycarbonyl-L-isoleucyl) aminopropanyl] -1H-imidazol-2-ylamine (300 mg) in dioxane (10 ml)
And 4N hydrogen chloride in dioxane at room temperature (10 ml)
Was added. After stirring at the same temperature for 30 minutes, the solvent was concentrated under reduced pressure and pulverized from ether to give 5- [3- (L-isoleucyl) aminopropanyl] -1H-imidazole-2-
Ilamine dihydrochloride (449 mg) was obtained and used in the next reaction in total.

Production Example 9 (2R, 3R) -trans-2,3-epoxysuccinic acid diethyl ester (150 mg) and pentafluorophenol (172 mg) in dimethylformamide (5
ml) at room temperature with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (179 mg). After stirring at the same temperature for 3 hours, this solution was cooled with ice for 5 hours.
-[3- (L-isoleucyl) aminopropanyl) -1
H-imidazol-2-ylamine dihydrochloride (449m
g) and a dimethylformamide solution (10 ml) of N-methylmorpholine (172 mg) were added, and the mixture was stirred at room temperature overnight. The solvent was concentrated under reduced pressure, and the residue was washed with ethyl acetate (20 m
It was dissolved in 1), washed with saturated aqueous sodium hydrogen carbonate solution and saturated saline, and dried over anhydrous magnesium sulfate. The crude product obtained by concentrating the solvent under reduced pressure was subjected to reverse phase HPLC (1
After purification by linear gradient (150 minutes) of 0.1% trifluoroacetic acid aqueous solution of 0-30% acetonitrile, lyophilization was performed, and 5- [3- [N-[[(2R, 3
R) -3-trans- (Ethoxycarbonyl) oxiran-2-yl] carbonyl] -L-isoleucyl] aminopropanyl] -1H-imidazol-2-ylamine (125 mg) was obtained. ¹ H-NMR (300MHz, CD ₃ OD): δ 0.92 (t, 3H, J = 7Hz),
0.94 (d, 3H, J = 7Hz), 1.12-1.27 (m, 1H), 1.30
(t, 3H, J = 7Hz), 1.48-1.62 (m, 1H), 1.72-1.92
(m, 3H), 2.51 (m, 2H), 3.15-3.33 (m, 2H), 3.58
(d, 1H, J = 2Hz), 3.73 (d, 1H, J = 2Hz), 4.18 (d, 1H, J
= 8Hz), 4.25 (q, 2H, J = 7Hz), 6.52 (s, 1H) ESI-MS [M + H] ⁺ : 396, FAB-MS [M + H] ⁺ : 396.

[0063]Production Example 10 5- [3- [N-[[(2R, 3R) -3-trans-
(Ethoxycarbonyl) oxiran-2-yl] carbo
Nyl] -L-isoleucyl] aminopropanyl] -1H
-Imidazol-2-ylamine (80.3 mg)
Add to the tanol solution (2 ml) with potassium hydroxide (2 ml) under ice cooling.
0.5mg) ethanol solution (1ml) was added and the same temperature
And stirred for 2 hours. Concentrate the solvent to 1 ml under reduced pressure and
Ether (20 ml) was added and the precipitate was collected by filtration. Got
The crude product was purified by reverse phase HPLC (2-30% acetonitrile
Linear gradient of 0.1% trifluoroacetic acid aqueous solution
(150 minutes)) and then freeze-dried to give 5- [3
-[N-[[(2R, 3R) -3-trans- (oxy
Carbonyl) oxiran-2-yl] carbonyl] -L
-Isoleucyl] aminopropanyl] -1H-imidazo
Obtained ol-2-ylamine (7.5 mg).¹ H-NMR (300MHz, C D₃OD): δ 0.92 (t, 3H, J = 7Hz),
0.94 (d, 3H, J = 7Hz), 1.20 (m, 1H), 1.54 (m, 1H),
1.73-1.90 (m, 3H), 2.50 (m, 2H), 3.14-3.30 (m,
2H), 3.51 (d, 1H, J = 2Hz), 3.69 (d, 1H, J = 2Hz), 4.1
6 (d, 1H, J = 8Hz), 6.52 (s, 1H) FAB-MS [M + H]⁺ : 368 [α]_D ^{twenty three}= + 27 ° (c = 0.24, C H₃OH) IR (ν_max, KBr) 3280, 2970, 1680, 1640, 1560, 144
0, 1390, 1200, 1140 cm ^-1.

[0064]

EFFECT OF THE INVENTION WF14865A and WF14865
B has a strong inhibitory activity against cathepsin B and / or L activity, and is capable of treating and preventing various diseases caused by enhanced cathepsin B and / or L activity by oral or parenteral administration to mammals. Useful for.

[Brief description of drawings]

FIG. 1 is a diagram of an infrared absorption spectrum of WF14865A measured by a potassium bromide method.

FIG. 2 is a ¹ H-NMR (nuclear magnetic resonance) spectrum of WF14865A.

FIG. 3 is a diagram of a ¹³ C-NMR (nuclear magnetic resonance) spectrum of WF14865A.

FIG. 4 is a diagram of an infrared absorption spectrum of WF14865B measured by a potassium bromide method.

FIG. 5 is a ¹ H-NMR (nuclear magnetic resonance) spectrum of WF14865B.

FIG. 6 is a diagram of a ¹³ C-NMR (nuclear magnetic resonance) spectrum of WF14865B.

FIG. 7 is a reaction pathway diagram for synthesizing a novel compound WF14865A.

FIG. 8 is a reaction pathway diagram for synthesizing a novel compound WF14865A.

FIG. 9 is a reaction pathway diagram for synthesizing a novel compound WF14865A.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1: 645) (C12P 17/16 C12R 1: 645) (72) Inventor Sumio Kiyoen Tsuchiura, Ibaraki Prefecture 1132-1, Nagakuni-Higashi-cho, Ichi, Japan (72) Masahiro Kaoru, 4016-25 Tsuneura, Tsuchiura, Ibaraki Prefecture (72) Keiji Hemi, 668-37 Shimohirooka, Tsukuba, Ibaraki Prefecture

Claims (6)

[Claims] 1. A compound having the following properties (A) or (B), WF14865. Properties (A); a) Appearance: colorless powder b) Molecular formula: C ₁₆ H ₂₅ N ₅ O ₅ c) FAB-MS: m / z 368 (M + H) HRFAB-MS: m / z Measured value: 368.192
4 Theoretical value C ₁₆ H ₂₅ N ₅ O ₅ + H: 368.1934 d) Infrared absorption spectrum: 3290, 2970, 1680, 1640, 1560, 1460, 1430, 1390, 12 shown in FIG.
00, 1140 900, 800cm ^-1 e) Solubility: Soluble: Methanol, Water Poorly soluble: Acetone Insoluble: Chloroform f) Color reaction Positive: Ninhydrin reaction Ferric chloride reaction Cerium sulfate reaction Reaction with iodine vapor Negative: Ehrlich reaction Maurish reaction Diacetyl reaction g) ¹ H-NMR spectrum (400 MHz, CD ₃ OD)
(See Fig. 2) δ ppm: 6.52 (1H, s), 4.17 (1H, d, J = 8Hz), 3.69 (1
H, d, J = 2Hz), 3.51 (1H, d, J = 2Hz), 3.27 (1H, m), 3.22
(1H, m), 2.50 (2H, m), 1.90-1.73 (3H, m), 1.54 (1
H, m), 1.20 (1H, m), 0.94 (3H, d, J = 7Hz), 0.92 (3H,
t, J = 7Hz) h) ¹³ C-NMR spectrum (100 MHz, CD ₃ OD)
(Shown in Fig. 3) δ ppm: 173.4 (s), 170.6 (s), 168.7 (s), 148.6 (s), 1
28.3 (s), 110.0 (d), 59.7 (d), 54.2 (d), 53.3 (d), 39.3
(t), 37.8 (d), 29.0 (t), 26.1 (t), 22.6 (t), 15.9 (q),
11.3 (q) i) Specific optical rotation: [α] _D ²⁵ = + 30.0 ° (c = 1.0, CH ₃ OH) j) Amino acid analysis: Isoleucine Properties (B); a) Appearance: White powder b ) Molecular formula: C ₁₆ H ₂₅ N ₅ O ₅ c) FAB-MS: m / z 368 (M + H) HRFAB-MS: m / z Measured value: 368.192
4 Theoretical value C ₁₆ H ₂₅ N ₅ O ₅ + H: 368.1934 d) Infrared absorption spectrum: 3300, 2960, 1680, 1650, 1620, 1550, 1470, 1440, 13 shown in FIG.
80, 1250 1130, 900 cm ^-1 e) Solubility: Soluble: Methanol, water Poorly soluble: Acetone Insoluble: Chloroform f) Color reaction Positive: Ninhydrin reaction Cerium sulfate reaction Ferric chloride-potassium ferricyanide reaction Iodine vapor and Reaction Ehrlich reaction Negative: ferric chloride reaction Maurish reaction g) ¹ H-NMR spectrum (300 MHz, CD ₃ OD)
(Shown in Fig. 5) δ ppm: 6.50 (1H, s), 4.36 (1H, dd, J = 9Hz, J = 6Hz),
3.53 (1H, d, J = 2Hz), 3.36 (1H, d, J = 2Hz), 3.30-3.14
(2H, m), 2.49 (2H, m), 1.78 (2H, m), 1.70-1.54 (3H,
m), 0.97 (3H, d, J = 6Hz), 0.92 (3H, d, J = 6Hz) h) ¹³ C-NMR spectrum (75 MHz, CD ₃ OD)
(See Fig. 6) δ ppm: 174.6 (s), 174.0 (s), 170.2 (s), 148.7 (s), 1
28.4 (s), 109.9 (d), 55.6 (d), 54.1 (d), 53.6 (d), 41.7
(t), 39.3 (t), 29.0 (t), 26.0 (d), 23.4 (q), 22.6 (t),
21.9 (q) i) Specific rotation: [α] _D ²³ = + 35 ° (c = 0.7, CH ₃ OH) j) Amino acid analysis: Leucine 2. A WF14865-producing bacterium belonging to the genus Anixopsis is cultivated in a nutrient medium, and W is extracted from the obtained culture.
The method for producing the compound WF14865 according to claim 1, wherein F14865 is collected. 3. The WF14 according to claim 1 as an active ingredient.
A pharmaceutical composition comprising 865 and a pharmaceutically acceptable carrier. 4. The pharmaceutical composition according to claim 3, which is a drug for treating and / or preventing a disease caused by increased cathepsin B and / or L activity. 5. A cathepsin B and / or L inhibitor containing WF14865 or a salt thereof according to claim 1. 6. Anixopsis stelaria N
o. 14865 biologically pure culture.