JPH0987265A - Amino acid derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new amino acid derivative useful for treating osteoporosis, etc., as a cathepsin L inhibitor from culture products of a specific microorganism belonging to the genus Arthrobotrys. SOLUTION: This compound is represented by the formula. Furthermore, the compound is preferably obtained by culturing a microorganism (FERM P-15138) belonging to the genus Arthrobotrys and having producing ability of the compound and collecting from the cultured products, and inperfect filamentous fungus separated from stem of gramineous plant collected and cut down in Ogawa town, Hiki district in Saitama prefecture is used as the microorganism and the cultivation is preferably carried out under aerobic conditions at about 20-25 deg.C and about pH5-7.5 for 4-7 days.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an amino acid derivative and a salt thereof, which are useful as a drug, particularly as a cathepsin L inhibitor.
The present invention relates to a method for producing them using a microorganism and a medicine containing them as an active ingredient.

[0002]

2. Description of the Related Art In recent years, with the rapid increase of the elderly population, the prolongation of the menopausal age of women, and the increase in patients with renal dialysis osteoporosis or malignant hypercalcemia, the content of osteogenesis imperfections or promotion of bone breakdown has been described. It is becoming increasingly important to prevent or treat bone diseases. Among them, osteoporosis frequently causes fractures,
Since it causes bedridden elderly people, development of an effective preventive or therapeutic drug therefor is desired.

[0003] Once formed, bone is not completely unchanged. Bone is always made and broken by osteoblasts and osteoclasts, and it is based on the balance between bone formation and bone resorption. The supporting tissue of bone is mainly composed of collagen fibers which are organic and calcium salts which are inorganic, and these are combined to form light and strong bone. Although there are many different causes of bone breakdown, recent studies have revealed that there are two molecular factors that cause bone breakdown. The first concerns calcium absorption and impaired deposition,
The second is due to the enhanced degradation of collagen fibers, which are bone supporting tissues.

[0004] The former involves the supply, transfer, absorption and deposition of calcium, and mainly involves vitamin D derivatives, female hormones and the like. For the prevention or treatment of such diseases, a therapy for supplementing or maintaining calcium is employed, and active vitamin D ₃ preparations, calcium preparations and the like are used. Hormonal agents such as estrogen preparations and calcitonin preparations have been used for the purpose of suppressing demineralization from bone.

[0005] On the other hand, the latter is a core component of the bone supporting tissue.
This is due to the decomposition of the lage fiber.
Collagenase, a protease that breaks down proteins
I have been. However, recent studies indicate that these collages
Fiber is not degraded by normal collagenase,
Cathepsin L, a type of protease in the zyme
And decomposed very well.
Psin L plays a leading role in collagen fiber degradation in bone
(BIOmedica629,7
(6), (1992)).

[0006] Accordingly, compounds that effectively inhibit cathepsin L are expected to be effective as preventive or therapeutic agents for bone diseases such as osteoporosis, malignant hypercalcemia, and Paget's disease of bone.

On the other hand, as cysteine protease inhibitors having an epoxysuccinic acid structure isolated from natural products, estatins and catestatins, including E64, have been reported [Agri
c. Biol. Chem. 42 , 523 (1978), J. Antibiot. 42 , 136.
9 (1989), JP-A-7-70098] and JP-A-7-70098.
No. 70098 discloses that all of these compounds have cathepsin L inhibitory activity. However, the compound YM-57409 of the present invention is a novel compound not described in the literature, and its structure is clearly different from those of known cathepsin L inhibitors.

[0008]

SUMMARY OF THE INVENTION The present invention relates to osteoporosis,
An object of the present invention is to provide a novel compound useful for bone diseases such as malignant hypercalcemia and Paget's disease.

More specifically, it is an object of the present invention to provide a novel compound that inhibits cathepsin L in place of the conventional therapy for supplementing or maintaining calcium in resorbable bone disease. This is because cathepsin L promotes the decomposition of collagen fibers which are the main component of the bone supporting tissue.

Another object of the present invention is to provide a method for producing the above-mentioned novel compound using a microorganism belonging to the genus Arthrobotris.

[0011]

Means for Solving the Problems Under the state of the art, the present inventors have conducted extensive research and research on cathepsin L inhibitors produced by microorganisms. As a result, the incomplete filamentous fungus belonging to the genus Arthrobotris has the following formula ( The present invention has been accomplished by producing a novel amino acid derivative represented by I) or a salt thereof and finding that the compound has an excellent cathepsin L inhibitory activity.

That is, the present invention provides a compound of the formula (I)

[0013]

Embedded image

And a salt thereof. Further, according to the present invention, it is preferable that the microorganism having the ability to produce the amino acid derivative (I) or a salt thereof belonging to the genus Arthrobotris is cultured, and the amino acid derivative (I) or a salt thereof is collected from the culture. A method for producing the amino acid derivative (I) or a salt thereof is provided.

Further, according to the present invention, a medicament containing the amino acid derivative (I) or a salt thereof as an active ingredient,
In particular, a cathepsin L inhibitor, more specifically, a prophylactic and / or therapeutic agent for a bone disease such as osteoporosis, malignant neoplastic calcium or bone paget disease is provided.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION

(Compound) The compound (I) of the present invention forms an acid addition salt.
Compound (I) is isolated as a free base under ordinary culture conditions and isolation conditions, but may be produced or isolated as an acid addition salt depending on the conditions. Further, the free base can be subjected to a usual salt-forming reaction to produce an acid addition salt. Such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and pivalic acid. Acid addition salts with organic acids such as acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, glycolic acid, lactic acid, malic acid, tartaric acid, glutamic acid, aspartic acid, carbonic acid, methanesulfonic acid and ethanesulfonic acid Is mentioned.

The compound (I) of the present invention or a salt thereof has an asymmetric carbon atom and has a diastereoisomers (racemic, enantiomeric and acid addition salts with an optically active acid). Ma) exists. As the active ingredient of the compound of the present invention and the medicament, natural optical isomers are suitable, and the present invention includes all of the separated optical isomers and mixtures thereof.

The compound of the present invention may be isolated as a hydrate or various solvates or may have a polymorph depending on isolation conditions. Also included are solvates, various solvates, and compounds in all crystal forms.

(Microorganism) The microorganism used in the present invention is:
An amino acid derivative (I) belonging to the genus Arturobotris
Alternatively, it is characterized by its ability to produce salts. Specific examples of the microorganism include Arthrobotrys sp. Q29331.

This Q29331 strain is an incomplete filamentous fungus isolated from the stem of a cut grass plant collected in Ogawa-machi, Hiki-gun, Saitama Prefecture, and has the following mycological properties. 1. Properties in Various Media (1) Potato / Glucose Agar Medium The growth is very fast, and the colony becomes 61 mm in diameter when cultured at 24 ° C. for 4 days. The aerial hyphae are in the form of felt and initially take on a yellowish brown color from white. Conidia are formed very well. (2) Malt extract agar medium The growth is very fast, and the colony becomes 59 mm in diameter when cultured at 24 ° C. for 4 days. Aerial hyphae are wool-like and yellowish-brown. Conidia are well formed. (3) Tzapek agar medium Growth is worse than potato / glucose agar medium.
Culturing for 4 days at 4 ° C. gives a colony of 34 mm in diameter. No aerial mycelium is formed, and only the underlying mycelium spreads thinly and turns white. Conidia are hardly formed. (4) Sabouraud agar medium The growth is fast, and the colony has a diameter of 4 when cultured at 24 ° C. for 4 days.
6 mm. The aerial mycelium is wool-like and initially takes on a yellowish brown color from white. The back has a yellow-brown color. Conidia are well formed.

2. Physiological properties Growth temperature: grows in the range of 10-30 ° C, optimal growth temperature is 20-25 ° C.

3. Morphological characteristics Strains grown on potato-glucose agar medium were observed under a microscope. Conidiophores: Stand up from the basal hypha, have a length of 300 μm or less, and a diameter of 3 to 6 μm. It hardly branches and is separated by a septum, and conidia are formed only at the tips of conidiophores. The tip where the conidia are formed is in the shape of a small tooth protrusion, producing 5 to 15 conidia. Conidia: formed in a symposioform. The size is 12-4
0 × 3-14 μm, colorless, smooth, pear-shaped or oval. The cells are bicellular with septum formed transversely, and the tip cells are slightly larger. Rarely, three-cell and four-cell types are observed.

Based on the above mycological properties, the taxonomic position of strain Q29331 was determined by The Genera of Funjay.
Spol Rating in Pure Culture 3rd Edition (Arx. JA von, 1981, The Genera of Fungi Sporul
ating in Pure Culture, 3rded., Cramer, Vaduz). As a result, this strain was determined to belong to the incomplete fungus genus Arthrobotrys . In addition, the Orshot reference (Oorschot, CAN
van, 1985, Taxonomy of the Dactylaria complex, V.
A review of Arthrobotrys and allied genera.Stud
ies in Mycology 26, p61-96), etc., and the properties of this strain were not completely identical to those of the known strain. Therefore, this strain was transferred to Arthrobotrys sp.
29331. This strain was obtained from the National Institute of Advanced Industrial Science and Technology under the accession number FERM P-1513.
Deposited as No. 8.

Microorganisms are liable to be mutated artificially or spontaneously. Therefore, the microorganisms used in the present invention include, in addition to the above-mentioned Arthrobotris sp. Q29331 strain isolated from nature, ultraviolet rays, X-rays,
Those artificially mutated by a chemical agent or the like and natural mutants thereof are also included. In addition, Q2 used in the present invention
The 9331 strain has been isolated and obtained from nature, and can be easily obtained by restoring the strain deposited with the Biotechnology Research Institute.

(Production Method) The compound of the present invention belongs to the genus Arthrobotris, and is produced by culturing a microorganism having the ability to produce the compound of the present invention, and collecting the compound of the present invention from the culture. The cultivation is carried out according to a general microorganism culturing method, but it is usually advantageous to carry out a submerged culture method using a liquid medium.

The medium used for the culture may be any medium containing a nutrient used by Arthrobotris sp. Q29331, and a synthetic medium, a semi-synthetic medium or a natural medium is used. The composition of the medium may be, for example, L-arabinose, D-xylose, D-glucose, D-fructose, sucrose, inositol, L-rhamnose, raffinose, D-mannitol, mannose, melibiose, lactose, D-galactose as a carbon source. , Maltose, trehalose, salicin, xanthine, chitin, starch, glucose, dextrin,
Glycerin, vegetable oil, etc., as a nitrogen source, meat extract, peptone, gluten meal, cottonseed meal, soybean flour, peanut flour,
Fish meal, corn steep liquor, dried yeast, yeast extract,
Ammonium chloride, ammonium sulfate, ammonium nitrate, urea and other organic and inorganic nitrogen sources are used. Further, as metal salts, Na, K, Mg, Ca, Zn, F
e, Co and other sulfates, nitrates, chlorides, carbonates, phosphates and the like are added as necessary.

Further, if necessary, a substance for promoting the production of the compound of the present invention such as methionine, cysteine, cystine, thiosulfate, methyl oleate, lard oil, silicone oil, and a surfactant, or an antifoaming agent can be added. .

As the culturing conditions, it is generally advantageous to culture under aerobic conditions, and the culturing temperature is in the range of about 10 to 30 ° C, preferably about 20 to 25 ° C. Good results can be obtained by adjusting the pH of the medium to a range of about 4.5 to 8.5, preferably about 5 to 7.5. The cultivation period is appropriately set depending on the composition of the medium and the temperature conditions.
Days, preferably about 4 to 7 days.

In order to isolate and purify the desired compound of the present invention from a culture, a method for isolating and purifying a physiologically active substance from a culture of a usual microorganism is applied. Since the target substance is contained in the culture solution and in the cells, the cells are separated by centrifugation or filtration, and then the active substance is isolated and purified from the filtrate and the cells. That is, using cathepsin L inhibitory activity as an index,
Separation by means used in the production of general physiologically active substances utilizing differences in solubility and solubility in an appropriate solvent, differences in adsorption affinity to various adsorbents, and differences in distribution between two liquid phases. , Collected and purified. These methods may be used alone as needed or combined in any order and applied repeatedly.

(Pharmaceutical) Since the compound of the present invention has a cathepsin L inhibitory action, it can be used as a medicament for treating and preventing bone diseases involving cathepsin L.

The cathepsin L inhibitory activity of the compound of the present invention was confirmed by the following method.

Cathepsin L Inhibitory Activity Assay Using Cathepsin L derived from human kidney,
Cathepsin L inhibitory activity was measured by partially modifying the method described in in Enzymology Vol. 80 p. 540-541. In a buffer (pH 5.5) containing sodium acetate (340 mM), acetic acid (60 mM) and disodium EDTA (4 mM),
Add human kidney-derived cathepsin L (protogen),
A 20 ng / ml enzyme solution was prepared. On the other hand, dithiothreitol was added to the above buffer to a concentration of 8 mM, and then carbobenzoxy-L-phenylalanine-L-arginine-4-methylcoumalyl-7-amide (manufactured by Peptide Laboratories) was added to a concentration of 1 μM. In addition, a substrate solution was prepared. Enzyme solution 100 μl, substrate solution 100 μl
And 2 μl of a methanol solution of the test sample were mixed at 37 ° C.
For 30 minutes. Sodium monochloroacetate (100 mM), sodium acetate (30 m
M) and a reaction stop solution (pH:
4.3) After adding 50 μl to stop the reaction, the fluorescence of the reaction solution containing the released 4-methylcoumalyl-7-amide was measured at an excitation wavelength of 355 nm and an observation wavelength of 460 nm.
The inhibition rate was determined by the following equation. In addition, 250 μl of distilled water was added to 2 μl of a methanol solution of the measurement sample, and the mixture was incubated.

Inhibition rate (%) = [1− (sample value−blank value) / (control value−blank value)] × 100

To use the compound of the present invention for the above purpose,
Usually, it is administered orally or parenterally. The dosage is age,
Depending on body weight, symptoms, therapeutic effect, administration method, treatment time, etc., usually 0.1 mg per adult per day
It is orally administered once a day in a range of 100 mg, preferably 1 mg to 10 mg, or divided into two to several times, or 0.1 mg to 100 mg per adult per day.
The parenteral dose may be administered once or several times a day in the range of mg, or may be continuously administered intravenously in the range of 1 hour to 24 hours a day. Since the dose varies under various conditions, a dose smaller than the above dose range may be sufficient.

As the solid composition for oral administration according to the present invention, tablets, powders, granules and the like are used. In such solid compositions, the one or more active substances comprise at least one inert diluent, such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, metasilicate. It is mixed with magnesium aluminate and the like. The composition may contain, in a conventional manner, additives other than an inert diluent, for example, a lubricant such as magnesium stearate, a disintegrant such as calcium cellulose glycolate, a stabilizer such as lactose, glutamic acid or It may contain a solubilizing or solubilizing agent such as aspartic acid. Tablets or pills can be sucrose, gelatin,
It may be coated with a sugar coating such as hydroxypropylcellulose or hydroxypropylmethylcellulose phthalate, or a film of a gastric or enteric substance.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, commonly used inert diluents, For example, it contains purified water and ethanol. The composition may contain, in addition to the inert diluent, adjuvants such as wetting agents and suspending agents, sweetening agents, flavoring agents, fragrances, and preservatives.
Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Aqueous solutions and suspensions include, for example, distilled water for injections and physiological saline. Examples of the water-insoluble solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and surfactants such as polysorbate 80 (trade name). Such compositions may also contain adjuvants such as preserving, wetting, emulsifying, dispersing, stabilizing (eg, lactose) and solubilizing or dissolving aids (eg, glutamic acid, aspartic acid). .
These are, for example, filtration through a bacteria retaining filter,
It is sterilized by blending of a bactericide or irradiation. They can also be used to produce sterile solid compositions which are dissolved in sterile water or sterile injectable solvents before use.

[0037]

EXAMPLES The compounds of the present invention and the method for producing the same have been described below, but will be described in more detail with reference to the following examples.
However, the compound of the present invention is not limited at all by these Examples.

Example 108 Medium (potato starch 2%, glucose 1%, polypeptone 0.5%, yeast extract 0.5%, NaC1
0.4%, pH 7.0).
l Dispense 100ml into an Erlenmeyer flask with a navel
Sterilized at 21 ° C. for 20 minutes. To this medium, scrape and inoculate the mycelia of the Q29331 strain grown on the slope agar medium,
Shaking culture at 200 rpm was performed at 24 ° C. for 3 days to obtain a seed culture solution. Next, P10 medium (2% glucose, glycerol 3%, corn steep liquor 2%, polypeptone 0.5%, 0.5% yeast extract, MgSO ₄ · 7H ₂ O
0.1%, 0.2% CaCO ₃ , pH 6.5)
(Liter), 100 ml of this medium was dispensed into a 500 ml Erlenmeyer flask, sterilized at 121 ° C. for 20 minutes, and inoculated with a seed culture solution at a rate of 2%.
Zero rotation shaking culture was performed at 24 ° C. for 5 days. 20 l of the thus obtained culture solution was filtered to obtain a culture filtrate 15.
1 was obtained. The culture filtrate was diluted with 4 DIAION HP-20.
The column was adsorbed to a packed column, washed with about 10 l of water, and eluted with 20 l of 80% acetone aqueous solution. The eluate was concentrated under reduced pressure to about 4 liters, and 400 ml of the strongly acidic cation exchange resin Dowex 50xl (H ⁺ type) was added.
Adsorbed to the packed column. After washing with about 1.5 l of water, elution was carried out with 2 l of 0.5N aqueous ammonia. After extracting the eluate with an equal amount of n-butanol, the aqueous layer was dried under reduced pressure to obtain 12.4 g of a crude active fraction. The crude active fraction was subjected to ODS column chromatography using SSC-ODS-7515-12 (manufactured by Senshu Kagaku) (2 id × 45 cm). When 10% acetonitrile-0.1% trifluoroacetic acid was used for the mobile phase, the active fraction eluted in a fraction of 275-625 ml. The active fraction was concentrated under reduced pressure, and then purified with Sephadex LH-20 (2 id × 90 cm) using methanol.
1.24 g of an active fraction containing M-57409 was obtained.
This was dissolved in a small amount of methanol and subjected to preparative high performance liquid chromatography [column: STR-ODS-H15
i. d. x250mm (manufactured by Shimadzu Corporation), detection: UV
210 nm, mobile phase: 8% acetonitrile-0.1% trifluoroacetic acid, flow rate: 8 ml / min. ]. The fraction showing a single peak at a retention time of 16 to 19 minutes was collected and concentrated to dryness to give Y as a colorless oily substance.
80 mg of M-57409 was obtained.

Cathepsin L inhibitory activity of YM-57409 IC _{50 of} YM-57409 determined by the above pharmacological test method
The value was 8.7 × 10 ^-9 M.

Pharmacological properties of YM-57409 Molecular formula C ₁₇ H ₃₂ N ₄ O ₅ Molecular weight 372 Mass spectrometry (m / z): ESI-MS 373 (M +
H) ⁺ UV spectrum: terminal absorption (in MeOH) IR spectrum (KBr) 3417, 2962, 1
680, 1635, 1205, 1138, 10
26 cm ^-1 (FIG. 1) Specific light intensity [α] ²⁵ _D = + 10.8 ° (c = 0.5 in M)
eOH) ¹ H-NMR (500 MHz, CD ₃ OD): δ (ppm): 0.97 (d, 3H), 0.98 (d, 3H), 1.44 (m, 1H),
1.63 (br, 1H), 1.72 (m, 4H), 1.78 (m, 1H), 1.93 (m, 2H), 2.
88 (br, 1H), 2.99 (br, 3H), 3.41 (m, 1H), 3.44 (t, 2H), 3.5
0 (brs, 1H), 3.54 (m, 1H), 3.64 (brs, 1H), 4.80 (1H) (FIG. 2) ¹³ C-NMR (125 MHz, CD ₃ OD): δ (ppm): 21.7, 23.7, 25.9, 26.1, 26.9, 27.1,
38.1, 40.4, 41.8, 44.0, 48.5, 49.9, 53.8, 54.1, 16
9.0, 171.2, 174.9 (Fig. 3)

Due to the above physicochemical properties, the compound of the present invention Y
M-57409 was identified as a substance having the following structure.

[0042]

Embedded image

The compound YM-57409 of the present invention is a cathepsin L inhibitor having the above-mentioned known epoxy succinic acid structure in that the C-terminal of leucine is an amide tertiary with a 4-aminobutyl group and a 3-aminopropyl group. The structure is obviously different from the substance.

[0044]

The amino acid derivative (I) and the salt thereof of the present invention have a cathepsin L inhibitory action, and
Is useful as a medicament for preventing and / or treating bone diseases such as osteoporosis, malignant neoplastic calcium, bone Paget disease and the like. Further, the production method of the present invention is useful as providing a method for advantageously producing a novel and useful compound of the present invention by a fermentation method.

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of YM-57409.

FIG. 2 is a ¹ H-nuclear magnetic resonance spectrum of YM-57409.

FIG. 3 is a ¹³ C-nuclear magnetic resonance spectrum of YM-57409.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location // (C12P 17/02 C12R 1: 645) (72) Inventor Osamu Nogi6 Nagayama 6, Ryugasaki-city, Ibaraki Pref. -15-9 (72) Inventor Tsutomu Sato 3-32-7-3 Akatsunashinmachi, Itabashi-ku, Tokyo 7-103 (72) Inventor Koji Nagai 3-24-16, Akabanekita, Kita-ku, Tokyo 206 Auberge Kirigaoka 206 (72) Invention Person Mitsuru Shibasaki 3-18-13 Hasune, Itabashi-ku, Tokyo 409 Hasumi, Rumane 409 (72) Inventor Kenichi Koyama 3-13-1, Ninomiya, Tsukuba-shi, Ibaraki Ruminomiya 321 (72) Inventor Hajime Okada Saitama 3-11-12 Midorigaoka, Ogawa-machi, Hiki-gun

Claims (6)

[Claims] 1. A compound of the formula (I) Or a salt thereof. 2. A microorganism belonging to the genus Arthrobotris and having the ability to produce the amino acid derivative (I) or a salt thereof according to claim 1, is cultured, and the amino acid derivative (I) or a salt thereof is collected from the culture. A process for producing said amino acid derivative (I) or a salt thereof. 3. The method according to claim 1, wherein the microorganism is Arthrobotris sp.
The production method according to claim 2, which is Q29331 (FERM P-15138). 4. A pharmaceutical comprising the amino acid derivative (I) according to claim 1 or a salt thereof as an active ingredient. 5. The medicament according to claim 4, which is a cathepsin L inhibitor. 6. The medicament according to claim 4, which is an agent for preventing and / or treating a bone disease.