JPH07123995A - Tripeptide compound - Google Patents

Abstract

PURPOSE: To obtain a new tripeptide compound having cathepsin L-inhibiting action and useful for prevention and treatment of bone diseases such as osteoporosis, malignant tumor hypercalcemia and bone Paget's disease.

CONSTITUTION: This compound is represented by the formula (R is an aldehyde or hydroxymethyl), e.g. isovaleryl-L-tyrosyl-L-valyl-L-phenylalaninal. Furthermore, the compound of the formula is obtained by culturing a microorganism of Streptomyces sp.-Q-21705 (FERM P-13917) belonging to the genus Streptomyces and having ability to produce the compound of the formula in a culture medium, producing and accumulating the tripeptide compound in the culture product and collecting tripeptide from the culture product.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to novel compounds Q-21705-A and Q-21705-B which are useful for bone diseases such as osteoporosis, malignant neoplastic hypercalcemia and Paget's disease of bone, and a method for producing them. It is about.

[0002]

2. Description of the Related Art In recent years, with the rapid increase in the elderly population, the extension of the menopausal age of women, and the increase in the number of patients with renal dialysis osteoporosis or malignant neoplastic hypercalcemia, the promotion of bone dysplasia or bone collapse has been considered. The prevention or treatment of such bone diseases is becoming increasingly important. Among them, osteoporosis frequently causes fractures.
Since it causes a bedridden old man, the development of an effective preventive or therapeutic drug is desired.

Bone, once formed, is not at all unchanged. Bone is always created and destroyed by osteoblasts and osteoclasts, which is a balance between bone formation and bone resorption. The supporting tissue of bone is mainly collagen fibers, which are organic, and calcium salts, which are inorganic, and these two combine to form a light and strong bone. There are various causes of bone destruction, but recent studies have revealed that there are two molecular factors that cause bone destruction. The first
Relates to defective absorption and deposition of calcium, and the second is due to enhanced degradation of collagen fibers, which are bone supporting tissues.

The former is concerned with calcium supply, transfer, absorption, and deposition, and vitamin D derivatives, female hormones, etc. are mainly involved. For the prevention or treatment of such diseases, a therapy to supplement or maintain calcium is adopted, and active vitamin D ₃ preparations, calcium preparations and the like are used. Furthermore, hormone agents such as estrogen preparations and calcitonin preparations are used for the purpose of suppressing demineralization from bone.

On the other hand, the latter is a core component of bone supporting tissue.
Due to the decomposition of Lagen fiber, it has been
Collagenase, a protease that decomposes
It has come. But from recent research, these collagens
Fiber is not degraded by normal collagenase and
Cathepsin L, a kind of protease in zome
It was revealed that the
Pushin L plays a leading role in degrading collagen fibers in bone
Have been considered to be the same (BIOmedica 629,7
(6), (1992)).

Therefore, a compound that effectively inhibits cathepsin L is expected to be effective as a preventive or therapeutic drug for bone diseases such as osteoporosis, malignant neoplastic hypercalcemia and Paget's disease of bone.

Under these circumstances, a preventive and / or therapeutic agent for bone diseases containing a certain dipeptide as an active ingredient is disclosed in Japanese Patent Laid-Open Publication No. Hei 5-
No. 155764. In addition, Agric. Biol.
Chem., 799 49 (3), (1989) discloses naturally occurring strepin P-1 which is a tripeptide and describes the inhibitory action of cysteine proteases papain and calpain. However, Strepin P-1
Has a structure different from that of the compound of the present invention, and neither suggests nor discloses inhibition of cathepsin L.

[0008]

The object of the present invention is to provide novel Q-21705-A and Q-2 useful for bone diseases such as osteoporosis, malignant neoplastic hypercalcemia and Paget's disease of bone.
1705-B compound.

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

Another object of the present invention is to provide a method for producing novel Q-21705-A and Q-21705-B compounds from the novel Q-21705 strain and a method for obtaining both compounds by chemical synthesis.

[0011]

That is, the present invention is a tripeptide compound represented by the following general formula or a salt thereof.

[0012]

[Chemical 2]

(In the formula, R means an aldehyde group or a hydroxymethyl group.) Further, according to the present invention, a microorganism belonging to the genus Streptomyces and having the ability to produce the above tripeptide compound. Is provided in the medium, the tripeptide compound is produced and accumulated in the culture, and the tripeptide is collected from the culture.

Furthermore, according to the present invention, there is provided a cathepsin L inhibitor comprising the above tripeptide compound or a salt thereof as an active ingredient. Further, according to the present invention, there is provided a prophylactic or therapeutic agent for osteoporosis, malignant neoplastic calciumemia or Paget's disease of bone, which comprises the above tripeptide compound or a salt thereof as an active ingredient.

The present invention will be described in more detail below. The compound of the present invention has an asymmetric carbon atom and has various isomers. The present invention also includes separated forms of these isomers and mixtures thereof. Further, the compound of the present invention may form a salt. Specific examples of the salt include salts of inorganic bases such as sodium and potassium, and salts of organic bases such as triethylamine, diethylamine and pyridinium ion.

The strain producing the compound of the present invention is, for example, a microorganism Streptomyces sp. Q isolated from soil collected in Kalimantan Island, Indonesia.
-21705 strain can be mentioned.

The mycological properties of this strain will be described below. 1. Morphological characteristics This strain grows relatively well on oatmeal agar medium and tyrosine agar medium, and the color of basal hyphae is yellowish gray to yellowish brown. Aerial mycelia are well formed on Bennett's agar and appear white. On the spore stalk branched from the aerial mycelium, a few spores are linked to a few spores, and the spores are curved and coiled as they mature. In liquid culture, no fragmentation of basal hyphae is observed. Observation by an electron microscope shows that the spores have a cylindrical shape,
The size is 0.3-0.6 μm × 0.8-1.2 μm,
The surface is smooth. No special organs such as sporangia and motile spores are observed.

2. Properties on various agar media Properties on various agar media are as follows. Unless otherwise specified, the cells were cultured at 27 ° C. for 21 days and observed according to a conventional method. The description of the color tone was based on the color standard (Japan Color Research Institute).

[0019]

[Table 1]

(Note) G: Growth temperature A: Settlement of aerial mycelium and its hue R: Hue of the back surface S: Soluble pigment 3. Physiological properties

[0021]

[Table 2]

(Note) The growth temperature range and the optimum growth temperature are 7-, at each temperature (5, 10, 15, 20, 24, 27, 32, 37, 40, 45, 50 ° C).
It is an observation result until 21st. Unless otherwise indicated, the observation results after 2 weeks at 27 ° C. are shown. 4. Utilization of carbon source

[0023]

[Table 3]

(Note) +: Grows ±: Suspicious growth −: Does not grow 5. Chemical analysis of bacterial components LECHVALIER et al. (LECHVALIER, MP et al; pp 277-2
38 in DIETZ, A. et al ed., Actinomycete Taxonomy,
According to the analysis of the acid hydrolyzate of this strain according to SIM Special Publication No. 6, 1980), LL-diaminopimelic acid was detected (cell wall type I). Major bacterial menaquinone was MK-9 (H _8). From the above Q
In summary, the properties of the -21705 strain are yellow gray to yellow brown in the basic mycelia and white in the aerial mycelia. A few, three to twenty or more spore chains are formed on the aerial hyphae, and the basal hyphae are not fragmented. Cell wall type is I, major menaquinone is MK-
9 (H ₈ ).

Bacterial species having the above-mentioned various properties are described in various documents (Berg
ey's Manual of Systematic Bacteriology vol.4,1989.
, Etc., this strain is found to be Streptomyces ( Str
It is considered to be a strain belonging to the genus eptomyces ). Therefore, this strain was designated as Streptomyces sp.
It was named 21705.

This strain has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, under the deposit number FERM P-13917.

It should be noted that although microorganisms are prone to mutate artificially or naturally, the Streptomyces sp. Q-21705 strain of the present invention can be used in addition to the strains isolated from nature, ultraviolet rays, X-rays, It also includes those artificially mutated by chemical agents and their natural mutants.

(Production Method) The Q-21705 compound is produced by culturing Streptomyces sp. Strain Q-21705 in a medium and collecting it from the culture. The culturing method is carried out according to the culturing method of general microorganisms, but the submerged culturing method is generally advantageous in a liquid medium.

The medium used for the culture may be any medium containing a nutrient source used by the Streptomyces sp. Q-21705 strain, and a synthetic medium, a semi-synthetic medium or a natural medium may be used. The composition of the medium is, for example, glucose, fructose, starch, vegetable oil or the like as the carbon source, meat extract, peptone, gluten meal, cottonseed meal, soybean flour, peanut flour, fish meal, corn steep liquor, dry yeast as the nitrogen source. , Yeast extract, ammonium sulfate, ammonium nitrate, urea and other organic and inorganic nitrogen sources are used. Further, as the metal salt, Na, K, M
g, Ca, Zn, Fe and other sulfates, nitrates, chlorides,
Carbonate, phosphate, etc. are added as needed.

Furthermore, if necessary, a compound-accelerating substance of the present invention such as methionine, cystine, cystine, thiosulfate, methyl oleate, lard oil, silicone oil, or a surfactant, or an antifoaming agent can be added. .

As the culture conditions, it is generally advantageous to culture under aerobic conditions, and the culture temperature is in the range of about 15 to 45 ° C, preferably about 24 to 37 ° C. Good results are obtained by adjusting the pH of the medium to a range of about 5-10, preferably about 6-8. The culture period is appropriately set according to the composition of the medium and the temperature conditions.

In order to isolate and collect the desired Q-21705 compound from the culture, a usual method for isolating a physiologically active substance from a culture of a microorganism is applied. Since the target substance is contained in the culture solution and in the microbial cells, the microbial cells are separated by centrifugation or filtration, and then the active substance is extracted from the filtrate and the microbial cells. That is, by the means used for the production of general physiologically active substances, which utilizes the difference in solubility and solubility in an appropriate solvent, the difference in adsorption affinity for various adsorbents, the difference in distribution between two liquid phases, and the like. , Separated, collected and purified. These methods can be used alone, or can be combined and repeated in any order, if desired. Next, a method for chemically synthesizing the compound of the present invention and its salt will be described.

[0033]

[Chemical 3]

(In the formula, Pa is an amino-terminal protecting group, and P is
c is a protecting group for a carboxyl group, X is a leaving group such as a halogen atom or a hydroxy group, THF is tetrahydrafuran, and WSCD is 1-ethyl-3- (3- (N, N-dimethylamino) propyl. ) Carbodiimide hydrochloride was added to HO
BT is 1-hydroxybenzotriazole, AcO
Et means ethyl acetate and DIBAL-H means diisobutylaluminum hydride. ) For example, valyl-phenylalanine derivative (II) is used as a starting material, condensed with a tyrosine derivative to give tyrosyl-valyl-phenylalanine derivative (III), and then 2-methylpropyl is added to the amino terminal and tyrosyl group of tripeptide (III). A carbonyl group is introduced by a condensation reaction to give a tripeptide (IV), and finally, the terminal carboxyl group is reduced simultaneously with the cleavage of the ester of the tyrosyl group,
The compound (I) of the present invention is obtained. However, in order to obtain the compound of the present invention, the order of condensing amino groups is arbitrary. For example, using a tyrosyl-valyl derivative as a starting material,
Phenylalanine may be condensed. Moreover, the order of the steps of introducing the 2-methylpropylcarbonyl group does not matter.

Peptide (II) and peptide (III)
The amino terminal of is preferably protected with a protecting group (Pa). This protecting group is deprotected in reaction A before the condensation reaction.

Pa and Pc are preferably protecting groups usually used in the peptide field. Specific examples of the amino group-protecting group (Pa) include a benzyloxycarbonyl group,
p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, p-chlorobenzyloxycarbonyl group, 1,1-dimethylethoxycarbonyl group, isobornyloxycarbonyl group, p-biphenylisopropyloxycarbonyl group, 3, 5-dimethoxy-α, α-dimethylbenzyloxycarbonyl group, 9-
Examples thereof include a fluorenylmethyloxycarbonyl group and a methylsulfonylethoxycarbonyl group.

Examples of the protecting group (Pc) for the carboxyl group include, for example, benzyl group, p-nitrobenzyl group, p-methoxybenzyl group, diphenylmethyl group, substituted benzyl groups such as benzhydryl group, tert.
-Butyl group, methyl group, ethyl group, phenacyl group, trichloroethyl group and the like can be mentioned.

First, the catalytic hydrogenation reaction A for deprotection will be described. Palladium / carbon is preferably used as the hydrogenation catalyst, and palladium black,
You may use palladium hydroxide etc. As a hydrogen source,
Hydrogen gas is preferably used, but instead of hydrogen gas,
Cyclohexene, formic acid, 1,4-cyclohexadiene and the like may be used. As the solvent, THF, methanol,
Any of those commonly used in catalytic hydrogenation reactions such as ethanol and ethyl acetate may be used. Further, depending on the protecting group used, not only hydrogenation but also a commonly used deprotection reaction is applied.

Next, the condensation reaction B of a peptide with an amino acid such as tyrosine will be described. In this reaction, it is preferable to use a condensing agent, and as the condensing agent, 1-ethyl-
3- (3- (N, N-dimethylamino) propyl) carbodiimide (WSCD) can be preferably used.
In addition, N, N-dicyclohexylcarbodiimide (DCC), diphenylphosphoryl azide (DDP)
A), isobutyl chloroformate, carbonyldiimidazole benzotriazolyl-N-hydroxytrisdimethylaminophosphonium hexafluorophosphide salt (Bop reagent), and other condensing agents generally used for peptide bond formation can be used.

As an additive which may be used together with a condensing agent such as WSCD and DCC, there is HOBT, and N-
Examples thereof include hydroxysuccinimide (HONSu) and 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOOBt).

Instead of the carboxylic acid derivative, an active ester may be used for condensation. An active ester obtained by reacting with a phenol-based compound such as p-nitrophenol, an N-hydroxysuccinimide, an N-hydroxylamine-based compound such as 1-hydroxybenzotriazole; a carbonic acid monoalkyl ester, or an organic acid The resulting mixed acid anhydride, diphenylphosphoryl chloride, or a phosphoric acid-based mixed acid anhydride obtained by reacting with N-methylmorpholine; an acid azide obtained by reacting an ester with hirorazine or an alkyl nitrite; an acid chloride or an acid It can be produced by applying a C-terminal activation method using an acid halide such as bromide; a symmetric acid anhydride and the like.

Depending on the method applied, it may be preferable to carry out the reaction in the presence of a base such as triethylamine, pyridine, N-methylmorpholine or the like, for the reaction to proceed smoothly. The reaction is usually performed in a solvent under cooling to room temperature. As the solvent used, a commonly used solvent such as chloroform, carbon tetrachloride, cycloloethane, tetrahydrofuran, dioxane, dimethoxymethane, dimethoxyethane, ethyl acetate, benzene, dimethylformamide, dimethylsulfoxide, or any mixed solvent thereof is used. To be

Reaction C is a condensation reaction of isovaleryl chloride. You may use the condensation method similar to reaction B using isovaleryl acid.

Reaction D reduces the ester group at the terminal of the carboxyl group to an aldehyde. Diisobutylaluminum hydride (DIBAL-H) is preferably used for this reaction. In addition to this, bis (2-methoxyethoxy) aluminum hydride, aluminum sodium hydride bis (N-methylpiperazino) aluminum hydride,
Diisobutylaluminum sodium hydride and the like are also used. In addition to toluene, the solvent may be any organic solvent commonly used for reduction of metal hydrides such as THF, ether and hexane.

The compound according to the invention thus produced is isolated and purified in free form or as its salt. Isolation and purification are carried out by appropriately applying usual chemical operations such as extraction, crystallization, recrystallization and various kinds of chromatography.

The racemic compound can be optically resolved by using an appropriate starting compound or by a general racemic resolution method (for example, a diastereomeric salt with a general optically active acid (tartaric acid etc.) is introduced. Etc.) can lead to stereochemically pure isomers. The diastereomeric mixture can be separated by a conventional method, for example, fractional crystallization or chromatography.

[0047]

EXAMPLES The compounds of the present invention and the method for producing the same have been described above, but the details will be described below with reference to Examples.
However, the compound of the present invention is not limited to these examples.

Example 1 Glucose 1%, potato starch 2%, yeast extract (DIFCO LABORATORI)
ES) 0.5%, polypeptone (DIFCO LABORATORIES
A medium (pH 7.0) containing 0.5% and CaCO ₃ 0.4% was prepared, and this medium was placed in a 500 ml Erlenmeyer flask for 100 each.
It was dispensed by ml and sterilized at 121 ° C. for 20 minutes. This medium was inoculated by scraping the hyphae of the Q-21705 strain from the original slant and shaking culture at 210 rpm for 3 days at 27 ° C. to obtain a seed culture solution. Next, 5 L of the above medium was prepared, and this medium was placed in a 500 ml Erlenmeyer flask for 100 m each.
The seed culture solution was inoculated at a rate of 4% into one that had been aliquoted by 1 and sterilized at 121 ° C for 20 minutes, and shake culture at 210 rpm was performed at 27 ° C for 3 days. The culture broth thus obtained was filtered together with diatomaceous earth, and 4.5 L of the culture broth thus obtained was adjusted to pH 10 with a 1N aqueous sodium hydroxide solution and extracted with the same amount of ethyl acetate. The ethyl acetate layer was washed with a pH 3 aqueous hydrochloric acid solution and then concentrated to dryness to obtain 213 mg of a crude active fraction. This crude active fraction was used as Wako Gel C
200 (manufactured by Wako Pure Chemical Industries, Ltd.) (inside diameter 2 cm, length 19 c
m. ) Was used for silica gel column chromatography. The mobile phase was chloroform-methanol / 98: 2.
(300 mL) and 97: 3 (1 L), the active fraction was chloroform-methanol / 9.
Elution at 7: 3. This active fraction was purified by eluting with methanol by column chromatography of LH-20 (Pharmacia) (inner diameter 2 cm, length 90 cm),
Active fraction 33.8 containing Q-21705-A and B
mg was obtained. This is dissolved in a small amount of methanol and preparative high performance liquid chromatography (manufactured by Shimadzu, trade name, ST
R-ODS-H. Inner diameter is 10 mm and length is 250 mm
Column was used. UV detection at 205 nm. Mobile phase: 57% methanol, flow rate: 3 mL / min. ). Fractions showing a single peak at a retention time of 20 to 21 minutes were collected, concentrated, extracted with ethyl acetate, and the obtained ethyl acetate layer was concentrated to dryness to give Q-2170.
0.5 mg of 5-A was obtained as a white powder. Similarly, fractions showing a single peak with a retention time of 18 to 20 minutes were collected, concentrated, extracted with ethyl acetate, and the obtained ethyl acetate layer was concentrated to dryness to give Q-21705-B a white color. 18.7 mg was obtained as a powder. Q-21705-A and Q-2
1 shows a structural formula of 1705-B.

[0049]

[Chemical 4]

Example 2 First Step 7 g of carbobenzoxy-L-valyl-L-phenylalanine methyl ester was added to 100 ml of methanol and THF.
40 ml and 10% palladium-carbon 350 mg were added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 2 hours. The reaction solution was filtered and the solvent was distilled off to obtain a crude product of L-valyl-L-phenylalanine methyl ester. This L-Varyl-L
-2.67 g of phenylalanine methyl ester, 100 ml of dichloromethane, 2.5 g of carbobenzoxy-L-tyrosine, 1.69 g of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride and 1-hydroxybenzotriazole 1 0.39 g was added, and the mixture was stirred at room temperature for 2 days. The reaction solution was washed with brine, the precipitate formed in the oil layer was collected by filtration, and carbobenzoxy-L-tyrosyl-L-
4.80 g of crude product of valyl-L-phenylalanine methyl ester was obtained.

To 3.49 g of the obtained crude carbobenzoxy-L-tyrosyl-L-valyl-L-phenylalanine methyl ester, 200 ml of methanol, 50 ml of ethyl acetate and 750 mg of 10% palladium-carbon were added, and the mixture was placed in a hydrogen atmosphere. , Stirred at room temperature for 1.5 hours. The reaction solution was filtered, the solvent was distilled off, and a crude product of L-tyrosyl-L-valyl-L-phenylalanine methyl ester 2.61 was obtained.
g was obtained. To 2.61 g of this crude L-tyrosyl-L-valyl-L-phenylalanine methyl ester was added 100 ml of dichloromethane and 0.88 of isovaleryl chloride.
g and 0.74 g of triethylamine were added, and the mixture was allowed to stand at room temperature for 13
Stir for hours. 100 ml of dichloromethane, 0.73 g of isovaleryl chloride and 0.68 g of triethylamine were added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was washed successively with saturated aqueous sodium hydrogen carbonate solution and 0.1N aqueous hydrochloric acid solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was recrystallized from diethyl ether-methanol to obtain 1.62 g of N, O-diisovaleryl-L-tyrosyl-L-valyl-L-phenylalanine methyl ester.

Mass spectrum (m / z): 610 (M +
H) ⁺ nuclear magnetic resonance spectrum (400 MHz, CDCL ₃ , T
MS internal standard) δ: 0.75 to 0.9 (12H, m), 1.03 (3
H, d), 1.95 to 2.1 (4H, m), 2.21.
(1H, septet, J = 6.8Hz), 2.39
(2H, d, J = 7.3 Hz), 3.0 to 3.5 (2
H, m), 3.5-3.15 (2H, m), 3.70.
(3H, s), 4.24 (1H, dd, J = 6.6,
8.5 Hz), 4.72 (1H, q, J = 7.3H)
z), 4.85 (1H, q, J = 7.0 Hz), 6.1
5 (1H, d, J = 7.8Hz), 6.51 (1H,
d, J = 7.8 Hz), 6.63 (1H, d, J = 8.
3Hz), 6.97 (2H, d, J = 8.3Hz),
7.12 (2H, d, J = 6.3Hz), 7.15 ~
7.3 (5H, m).

Second Step 6 ml of dry toluene was added to 400 mg of N, O-diisovaleryl-L-tyrosyl-L-valyl-L-phenylalanine methyl ester obtained in the first step. To the toluene solution, 3.97 ml of a 0.93 M diisobutylaluminum hydride hexane solution was added dropwise at -78 ° C, and then the mixture was stirred for 5 minutes. After adding 5 ml of water and returning to room temperature, 1N hydrochloric acid was added and then extracted with ethyl acetate. After washing with a saturated aqueous solution of sodium hydrogen carbonate, it was dried over anhydrous sodium sulfate, and then the solvent was distilled off. The residue was purified by silica gel column chromatography. From the fraction eluted with chloroform-methanol (95: 5),
72 mg of a mixture of isovaleryl-L-tyrosyl-L-valyl-L-phenylalaninal (Q-21705-A) and isovaleryl-L-tyrosyl-L-valyl-L-phenylalaninol (Q-21705-B) was obtained. It was

50 mg of the mixture of Q-21705-A and Q-21705-B obtained in the second step was purified by preparative high performance liquid chromatography under the same conditions as in Example 1 to obtain Q-21705-A (19 mg). And Q-2170
5-B (8 mg) was obtained.

The cathepsin L inhibitory activity of the compound of the present invention is
It was confirmed by the following method. Cathepsin L Inhibitory Activity Assay Method Using Cathepsin L derived from human kidney,
The method described in in Enzymology Vol. 80 p. 540-541 was partially modified to measure the cathepsin L inhibitory activity.

Sodium acetate (340 nM), acetic acid (60 m
M), disodium EDTA (4 mM) and dithiothreitol (8 mM) in 70 μl of a buffer (pH 5.5), 150 μl of human kidney-derived cathepsin L (manufactured by Protogen; 20 ng / ml of the above buffer) and a measurement sample 5 μl of methanol solution
Was added and incubated at 30 ° C. for 1 minute. Then, 75 μl of carbobenzoxy-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide (manufactured by Peptide Lab) (20 nM buffer solution) was added to give 3
This was made up to 00 μl and further incubated for 10 minutes. A reaction stop solution (pH 4.3) containing sodium monochloroacetate (100 mM), sodium acetate (30 mM), and acetic acid (70 mM) in the reaction solution was measured at an excitation wavelength of 370 nm and an observation wavelength of 460 nm. The inhibition rate was calculated by the following formula. However, the blank value was the fluorescence of the reaction solution having the above composition, which was prepared by adding the reaction stop solution in advance and incubating.

[0057]

[Equation 1] Inhibition rate (%) = [1- (sample value-blank value)
/ (Control value-blank value)] x 100 Q-21705-A and Q-2 calculated by the above method
The IC ₅₀ values (M) of the inhibitory activity of 1705-B are summarized in Table 4.

[0058]

[Table 4]

Table 5 shows the physical property data of Q-21705-A and B.

[0060]

[Table 5]

[0061]

INDUSTRIAL APPLICABILITY Since the compound of the present invention has a cathepsin L inhibitory action, it is useful for treating and preventing bone diseases associated with cathepsin L, such as osteoporosis, malignant hypercalcemia, Paget's disease of bone and the like. .

When the compound of the present invention, its non-toxic salt, or its hydrate is used for the above purpose, it is usually administered orally or parenterally. The dose varies depending on the age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually 0.1 mg to 100 mg per adult per day, preferably 1 mg to 10 mg, once a day. Oral administration in divided doses or 1 per adult
It is administered in the range of 0.1 mg to 100 mg per day by parenteral administration once to several times a day, or from 1 hour a day.
Intravenous continuous administration within a range of 24 hours. Since the dose varies depending on 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 comprises at least one inert diluent such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, metasilicic acid. It is mixed with magnesium aluminate. According to a conventional method, the composition comprises additives other than an inert diluent, for example, a lubricant such as magnesium stearate, a disintegrant such as calcium fibrin glycolate, a stabilizer such as lactose, glutamic acid or asparagine. It may contain a solubilizing agent such as an acid. If necessary, the tablets or pills may be coated with a film of a gastric or enteric substance such as sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate.

Liquid compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like, and generally used inert diluents. For example, it contains purified water and ethanol. This composition may contain, in addition to an inert diluent, auxiliary agents such as a wetting agent and a suspending agent, a sweetening agent, a flavoring agent, an aromatic agent and a preservative.
Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of the aqueous solution and suspension include distilled water for injection and physiological saline. Examples of the non-water-soluble 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 further contain adjuvants such as preservatives, wetting agents, emulsifiers, dispersants, stabilizers (eg lactose), solubilizers (eg glutamic acid, aspartic acid). These are sterilized by, for example, filtration through a bacteria-retaining filter, addition of a germicide, or irradiation. These can also be used by preparing a sterile solid composition and dissolving it in sterile water or a sterile solvent for injection before use.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location A61K 38/55 ADU C07K 5/087 8318-4H // (C12P 21/02 C12R 1: 465) A61K 37/64 ADU (72) Inventor Kyoko Teramura 4-11-11 Shozuzawa, Itabashi-ku, Tokyo Joyfast No. 205 (72) Inventor Yasuyo Shimizu 2-1-30 Fujimidai, Kunitachi, Tokyo National House 2-102 (72) Inventor Shiba saki Filled 3-18-13 Hasune, Itabashi-ku, Tokyo Rumi Hasune 409 (72) Inventor Yuuo Takahashi 500-5 Minamitajima, Kawagoe City, Saitama Prefecture (72) Inventor Hirayama Rev. Tsukuba, Ibaraki Prefecture Ichi-Ninomiya 2-5-9 Rumi Tsukuba No. 319 (72) Inventor Benjamin Steawan Indonesia Jakarta 10310 Jalan Lengbang No. 11 (72) Inventor Ratna Mulni Rantia Tomojo Indonesia Jakarta 11510 Artie 008 Arvie 009 Comple Box Greenville block Wow number 12

Claims (4)

[Claims] 1. A tripeptide compound represented by the following general formula or a salt thereof. [Chemical 1] (In the formula, R means an aldehyde group or a hydroxymethyl group.) 2. A microorganism belonging to the genus Streptomyces and having the ability to produce the tripeptide compound according to claim 1, is cultured in a medium, and the tripeptide compound is produced and accumulated in the culture, The method for producing a tripeptide compound according to claim 1, wherein the tripeptide is collected from the culture. 3. A cathepsin L inhibitor comprising the tripeptide compound or the salt thereof according to claim 1 as an active ingredient. 4. A prophylactic or therapeutic agent for osteoporosis, malignant neoplastic calcemia or Paget's disease of bone, which comprises the tripeptide compound or a salt thereof according to claim 1 as an active ingredient.