JPH10330364A - New substance egs-1300-1 and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a new compound, having activities for inhibiting sialic acid transferases and effective as an inhibitor, etc., of cancer metastasis. SOLUTION: This compound is represented by the formula (R is linoleoyl) or its pharmacologically acceptable salt. The physicochemical properties thereof manifest properties such as a red glassy solid. The compound is preferably produced by culturing a microorganism, belonging to the genus Penicillium and having the ability to produce the above compound (FERM BP-5869) on a culture medium at 16-37 deg.C temperature and pH 4-10 for 1-7 days according to a liquid culture method, especially a submerged spinner culture method, etc., and isolating the resultant compound from a cultured product. The daily dose of the compound for an adult is usually preferably about 0.01-2 mg/kg.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel EGS-1300-1 compound having sialyltransferase inhibitory activity, a pharmacologically acceptable salt thereof, and a method for producing the same.

[0002]

2. Description of the Related Art It is known that sialic acid (for example, N-acetylneuraminic acid) exists on cell surfaces as glycoproteins, glycolipids, polysaccharide sugar chains, and the like. This sialic acid is used for nervous function, cancer, inflammation, cell differentiation, viral infection,
It is regarded as important in immunity and the like, and is attracting medical and pharmacological attention as a specific active molecule localized on the cell surface.

Since it is known that sialyltransferase transfers sialic acid to a sugar chain in a living body, compounds that inhibit the action of sialyltransferase include nervous functions, cancer, and the like. It is expected to be useful in elucidating inflammation, cell differentiation, virus infection, immunity, etc. Particularly in some cancer cells, it has recently been reported that the content of sialic acid occupying the non-reducing end of the sugar chain on the membrane surface and the metastatic ability of cancer cells show a positive correlation [Shimono et al., Am. J.
Gastroenterol., 89, 101-105 (1994); Nakayama et a
l., Cancer, 75, 2051-2056 (1995)]. Therefore, a compound that inhibits the action of sialyltransferase is expected to be effective as a cancer metastasis inhibitor.

[0004] Low molecular weight compounds having an inhibitory effect on sialyltransferase include N-acetyllactosamine derivatives [Kajihara et al., Carbohydrate Research, 247, 1].
79-193 (1993)] and substrate derivatives such as sialic acid derivatives (JP-A-8-198891 and JP-A-198892).

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide compounds having inhibitory activity on sialyltransferase and a method for producing them.

[0006]

According to the present invention, there is provided a compound of formula (I)

[0007]

Embedded image

A compound having a sialyltransferase inhibitory activity represented by the formula (wherein R represents linoleoyl):
-1 or a pharmaceutically acceptable salt thereof is provided. Further, according to the present invention, belonging to the genus Penicillium, cultivating a microorganism having the ability to produce EGS-1300-1 in a medium, EGS-1300-1 characterized by collecting EGS-1300-1 from the culture, A manufacturing method is provided.

[0009]

DETAILED DESCRIPTION OF THE INVENTION The present invention will be described in detail. Less than,
Among the compounds represented by the formula (I),

[0010]

Embedded image

The compound represented by the following formula is designated as compound EGS-1300-1a. The physicochemical properties of compound EGS-1300-1a are as shown below. The following data was measured with the following equipment. Melting point: Yanagimoto Seisakusho Micro melting point apparatus Specific rotation: JASCO DIP-370 Digital polarimeter Ultraviolet absorption spectrum: Shimadzu Corporation UV-2200 spectrometer Red outside absorption spectrum: JEOL JIR-RFX3001 Spectrometer FAB Mass spectrum and high resolution FAB mass spectrum: JEOL JMS-HX110 / 110A mass spectrometer ¹³ C and ¹ H NMR spectrum: JEOL JNM-α400 nuclear magnetic resonance apparatus

Physicochemical data of compound EGS-1300-1a : red glassy solid FABMS spectrum: m / z positive mode: 1134.1136 (M + Na) ⁺ , 1112.1114
(M ⁺ H) + negative mode; 1110 · 1112 (MH) ^- High resolution FABMS spectrum: m / z positive ^{mode; 1112.6357 (M + H) +} , Δ = -0.1 mmu (C
Negative mode; 1110.6216 (MH) ^- , Δ = + 1.4 mmu (as ₆₂ H ₉₆ O ₁₂ N ³⁵ ClP)
₆₂ H ₉₄ O ₁₂ N ³⁵ ClP) UV spectrum (CH ₃ OH): λ _max nm (ε) 478 (6,200),
370 (37,100), 345 sh (35,000), 232 (28,900) IR spectrum (CHCl ₃ ): ν _max cm ^-1 3400, 2927, 173
4, 1699, 1591, 1506, 1456, 1371, 1086 ¹ H NMR spectrum (500 MHz, CDCl ₃ , internal standard TMS):
δppm (integral, multiplicity, coupling constant) 8.23 (1H, br s), 6.
96 (1H, s), 6.92 (1H, d, J = 15.1 Hz), 6.18 (1H, d, J
= 15.1 Hz), 5.68 (1H, d, J = 9.9 Hz), 5.40-5.29 (8H,
m), 5.15 (1H, m), 4.32 (1H, dd, J = 11.5, 2.1 Hz), 4.
19 (1H, m), 4.11 (1H, m), 4.09 (1H, m), 4.04 (2H,
m), 3.85 (1H, m), 3.77 (1H, m), 2.76 (4H, t, J = 6.5
Hz), 2.48 (1H, m), 2.25 (4H, m), 2.13 (3H, s), 2.0
4 (8H, m), 1.80 (3H, s), 1.55 (4H, m), 1.52 (3H,
s), 1.43 (2H, m), 1.38-1.20 (28H, m), 1.01 (3H, d,
J = 6.6 Hz), 0.89 (6H, t, J = 7.4 Hz), 0.88 (3H, t, J =
6.9 Hz) ¹³ C NMR spectrum (125 MHz, CDCl ₃ , internal standard TMS),
The number of signals and the number of carbon atoms do not match because some signals overlap: δppm (multiplicity)
195.3 (s), 183.9 (s), 173.7 (s), 173.5 (s), 170.7
(s), 148.5 (s), 148.0 (d), 145.6 (d), 144.6 (s), 1
44.0 (d), 132.2 (s), 130.3 (d), 130.0 (d), 128.2
(d), 127.9 (d), 114.7 (d), 113.6 (s), 111.4 (d), 1
01.8 (s), 85.4 (s), 70.5 (d), 63.5 (t), 63.2 (t),
62.7 (t), 53.7 (t), 35.0 (d), 34.3 (t), 34.1 (t), 3
2.0 (t), 31.6 (t), 30.1 (t), 29.76 (t), 29.73 (t),
29.63 (t), 29.42 (t), 29.39 (t), 29.38 (t), 29.34
(t), 29.27 (t), 29.22 (t), 29.18 (t), 27.26 (t), 2
7.24 (t), 25.7 (t), 25.0 (t), 24.9 (t), 23.4 (q), 2
2.6 (t), 20.4 (q), 20.2 (q), 14.13 (q), 14.09 (q),
12.6 (q), 12.0 (q) ³¹ P NMR spectrum (161.7 MHz, CDCl ₃ , 85% external standard)
Phosphoric acid): δppm 0.70

The pharmacologically acceptable salts of the compound EGS-1300-1 include pharmacologically acceptable acid addition salts, metal salts,
Includes ammonium salts, organic amine addition salts, amino acid addition salts and the like. Acid addition salts include inorganic acid salts such as hydrochloride, sulfate and phosphate, acetate, maleate, fumarate,
Organic salts such as tartrate, citrate and lactate can be mentioned. Examples of metal salts include alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, and aluminum. Salts, zinc salts, etc .; ammonium salts, such as ammonium and tetramethylammonium; organic amine addition salts, such as morpholine and piperidine; and amino acid addition salts, such as glycine and phenylalanine. , Aspartic acid, lysine and the like.

Next, a method for producing the compound EGS-1300-1 will be described. Compound EGS-1300-1 belongs to the genus Penicillium, and cultured in a medium a microorganism having EGS-1300-1 producing ability,
EGS-1300-1 was produced and accumulated in the culture, and EG was extracted from the culture.
Obtained by collecting S-1300-1. As a microorganism capable of producing EGS-1300-1, belongs to the genus Penicillium,
Any strain having EGS-1300-1 producing ability can be used. In addition, artificial strains of these strains, for example, mutant strains mutated by ultraviolet irradiation, X-ray irradiation, mutagen treatment, etc. or naturally mutated strains, can also produce EGS-1300-1. If present, it can be used in the present invention.

A particularly favorable example is Penicillium sp. G-1300 strain. The present inventors have found that a substance having a sialyltransferase inhibitory activity (EGS-1300-1) is produced in a culture solution obtained by culturing a G-1300 strain isolated from soil in a medium.

The bacteriological properties of the representative strain Penicillium sp. G-1300 producing the compound of the present invention are described below.

Macroscopic observation When cultivated on a malt extract agar medium at 25 ° C., the diameter of the colonies was 28 to 30 mm on the 7th day of culture and 48 to 5 mm on the 14th day of culture.
Reaches 0 mm. The surface of the settlement is grayish green-blue to dark gray, and the surrounding area is orange. In addition, the backside of the colony turns orange to brown. A brownish pigment appears in the medium.

Using a potato-glucose agar medium,
When cultured at 25 ° C, the diameter of the colonies is 32-34 on day 7 of culture.
mm, reaching 55-57 mm on day 14 of culture. The surface of the settlement is grayish green-blue to dark gray, and the surrounding area is orange. In addition, the backside of the colony turns orange to brown. A brownish pigment appears in the medium. The optimal growth temperature of this bacterium is 11-32 ° C, and it grows best around 26 ° C. It can grow at pH 2-11, and grows best around pH 6.

[0019] Using an optical microscope observation malt extract agar, observation by an optical microscope of this bacteria when cultured for 2 weeks at 25 ° C. is as follows. Hyphae have septum and are smooth and well-branched. The conidiophores do not form a bundle, but stand singly from mycelium and stand up. Conidiophores are smooth, with septum, width 2.0-3.2μ
m, length up to 180 μm. 7 to 11 phialides are formed at the tip of the conidiophore, and conidium is formed from the phialide tip. The phialide has a stocky shape, is colorless and smooth, has a length of 8.0 to 12.5 μm, a width of 3.0 to 5.5 μm at the widest part, and a width of 1.0 to 1.5 μm at the tip. The ontogeny mode of the conidium is the endogenous budding type, and its formation mode is the phialo type. Phialo-conidia are unicellular, spherical to subspherical, smooth, 2.0-3.0 μm in diameter. Conidia alone are colorless, but when assembled they appear golden.
In this strain, only the anamorph described above is observed, and no teleomorph is observed.

Based on the mycological properties described above, the taxonomic position of the fungus was referred to as "The Generala of Funjay Sporrating in Pure Culture 2nd Edition [The G
enera of Fungi Sporulating in Pure Culture, 2nd e
d., Cramer, Vaduz, JA von Arx, (1974)].
( Penicillium) . The present inventors referred to this strain as "Penicilium sp.
00 (Penicillium s p. G-1300) ", and the name is FERM BP-5869, which is a microfabrication laboratory of the Ministry of Industrial Science and Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan). Was deposited on March 13, 1997.

In culturing the EGS-1300-1 producing bacterium of the present invention, a usual culturing method used for culturing fungi is applied. As the medium to be used, any of a synthetic medium and a natural medium can be used as long as the medium appropriately contains a carbon source, a nitrogen source, an inorganic substance, etc. which can be used by microorganisms. As a carbon source, glucose, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, starch, dextrin, carbohydrates such as molasses, citric acid, organic acids such as malic acid, acetic acid, fumaric acid, methanol, ethanol, etc. And hydrocarbons such as methane, ethane, propane and n-paraffin, etc. are used alone or in combination.

Examples of the nitrogen source include ammonium salts such as ammonium chloride, ammonium nitrate, ammonium sulfate and ammonium phosphate, aspartic acid, glutamine,
Amino acids such as cystine and alanine, urea, peptone,
Meat extract, yeast extract, dried yeast, corn steep,
Liquor, soy flour, casamino acid and the like are used alone or in combination.

As inorganic substances, potassium monohydrogen phosphate,
Potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, nickel sulfate, zinc sulfate, copper sulfate, sodium chloride, potassium chloride, calcium chloride, cobalt chloride, calcium carbonate And barium carbonate.
In addition, if necessary, a substance that promotes the growth of bacterial cells or the production of EGS-1300-1 such as vitamins, for example, thiamine, is added to the medium. When the microorganism used requires a specific substance, the substance is added.

The cultivation is carried out by a liquid culture method, particularly a submerged stirring culture method, at a temperature of 16 to 37 ° C., preferably 25 to 32 ° C., at pH 4 to 10, preferably pH 6 to 8. Usually 1
By culturing for 77 days, the target compound EGS-1300-1 is produced and accumulated in the culture and in the cells. Ammonia water or ammonium carbonate solution is used for adjusting the pH of the medium. The culture is stopped when the amount of EGS-1300-1 produced in the culture reaches a maximum.

When the EGS-1300-1 accumulated in the culture is isolated and collected from the culture, a general method for collecting a normal physiologically active substance from the culture is applied. For example, the culture is separated by filtration into a culture filtrate and cells, and the cells are extracted with methanol or the like. Next, the extract is diluted and passed through a polystyrene-based adsorbent, for example, Diaion HP 20 (manufactured by Mitsubishi Chemical Corporation) to adsorb the active component, and then eluted with a mixed solvent of acetone and methanol. Concentrate the eluate, and by high performance liquid chromatography,
The target compound EGS-1300-1 is obtained. In addition, the detection of EGS-1300-1 during the culture and purification operations can be traced using the ultraviolet absorption of EGS-1300-1 by silica gel thin-layer chromatography or high-performance liquid chromatography (HPLC) as a guide. .

The present compound EGS-1300-1 may exist in stereoisomers, and the present invention includes those isomers or a mixture of these isomers in any ratio. Compound EGS-13
When obtaining the salt of 00-1, when the compound EGS-1300-1 is obtained in the form of a salt, it may be purified as it is.
When it is obtained in a free form, it may be dissolved or suspended in a suitable solvent and an acid may be added to form a salt.

The compound EGS-1300-1 or a pharmacologically acceptable salt thereof may be present in the form of an adduct with water or various solvents, and these adducts are also included in the present invention. Is done. Next, the biological activity of the compound EGS-1300-1 will be described in Test Examples.

Test Example Sialyltransferase Inhibition Test The sialyltransferase inhibitory effect was measured by the following method. (1) Sample The sialyltransferase was fused with protein A.
Gal β 1,4 GlcNAc α2,3 sialyltransferase Namalba K
What was secreted into the culture supernatant of JM-1 cells was used.
5-336963, JP-A-6-277052). (2) Measurement method Compound EGS-1300-1 and 0.1 mM tetrasaccharide Galβ 1,4 GlcNAc β 1,3 Gal b 1,4 fluorescently labeled with aminopyridine
Sialyltransferase activity was measured using Glc (lacto-N-neotetraose) as an acceptor substrate and 5 mM CMP-NeuAc as a donor substrate. Add 5 ml enzyme solution and
Add 0.1 M sodium cacodylate (pH 6.8), 0.01 M manganese chloride, 0.45% Triton X-100 to a final volume of 30 ml
And kept at 37 ° C. for 2 hours. Thereafter, the sample was boiled for 5 minutes to stop the reaction. After centrifuging the sample at 12000xg,
The supernatant is subjected to high performance liquid chromatography (HP
LC). HPLC reaction product (α2,3 sialyl lacto-N-neotetraose) and unreacted substrate (lacto-
(N-neotetraose). HPLC analysis conditions _{Column: TSK-gel ODS-80T M} (Tosoh Corp.) eluent: 20 mM ammonium acetate (pH 4.0) flow rate: 1.0 ml / min Detection: Fluorescence intensity (excitation wavelength 320 nm, detection wavelength 400 nm) tests The results are shown in FIG.

According to FIG. 1, compound EGS-1300-1 showed a clear sialyltransferase inhibitory action. From the above test results, it was confirmed that the compound EGS-1300-1 has an excellent sialyltransferase inhibitory activity and is useful as a therapeutic agent for cancer and the like. Compound EGS-1300-1 can be administered as it is or orally or parenterally as various pharmaceutical compositions. Examples of the dosage form of such a pharmaceutical composition include tablets, pills, powders, granules, capsules,
Suppositories, injections, drops and the like can be mentioned.

For the preparation of the above dosage forms, generally known methods are applied, for example, various excipients, lubricants, binders, disintegrants, suspending agents, isotonic agents, emulsifiers, It may contain an absorption promoter or the like. The carrier used in the pharmaceutical composition includes, for example, water, distilled water for injection, physiological saline,
Glucose, fructose, white sugar, mannitol, lactose, starch, corn starch, potato starch, cellulose, methyl cellulose, carboxymethyl cellulose calcium, hydroxypropyl cellulose, alginic acid, talc, sodium citrate, calcium carbonate, calcium hydrogen phosphate, stearin. Examples thereof include magnesium acid, urea, silicone resin, sorbitan fatty acid ester, and glycerin fatty acid ester, and these are appropriately selected according to the type of preparation.

The dose of the compound EGS-1300-1 is determined depending on the desired therapeutic effect, the administration method, the treatment period, the age and weight of the patient, etc.
Injection, infusion, rectal administration with suppository or skin patch), usually 0.01 to 2 mg / k per adult per day
g. Hereinafter, embodiments of the present invention will be described with reference to examples.

[0032]

Example 1 Method for producing compound EGS-1300-1 Penicillium sp . G-1300 was used as a seed bacterium . The strain was placed in a 300 ml Erlenmeyer flask in a seed medium consisting of 30 g / L of mashed potato (snow mark), 5 g / L of yeast extract, and 100 g / L of glucose (pH before sterilization: 6.
5) Inoculated into 50 ml, and cultured with shaking at 25 ° C for 72 hours. The obtained seed culture solution was transferred at a rate of 10% (volume) to 500 ml of a fermentation medium having the following composition in a 2 L Erlenmeyer flask at 25 ° C
And shaking (200 rpm).

Fermentation medium composition: 20 g / L glucose, 20 g / L mashed potato (snow mark), 5 g / peptone (Far East)
L, potassium dihydrogen phosphate 5 g / L, magnesium phosphate
0.5 g / L (pH 6.0 before sterilization, adjusted with NaOH) During the culture, the culture was cultured for 96 hours without any special control of the pH of the medium. The cells were separated from the culture solution, methanol was added to the cells in an amount equal to that of the culture solution, and the mixture was stirred to obtain 2.5 L of filtrate. Dilute the filtrate with water and use polystyrene adsorbent, Diaion
The active substance was adsorbed by passing through a 400 ml HP 20 (manufactured by Mitsubishi Chemical Corporation) column. The active substance was eluted with a mixed solution of methanol, acetone and deionized water. The active fraction is diluted with water and the polystyrene-based adsorption resin agent, Diaion HP 20ss
The column was passed through a 300 ml column (manufactured by Mitsubishi Chemical Corporation) to adsorb the active substance. The column was eluted with a mixed solution of methanol, acetone and deionized water. The eluted active fractions are concentrated and subjected to high performance liquid chromatography (HPLC) under the following conditions.
The active fraction was fractionated by. The fractionated active fraction was further eluted with chloroform, methanol and water (800: 100: 5) by HPLC using a normal phase silica gel column to fractionate. The obtained active fraction was subjected to gel filtration with methanol using LH-20, whereby 1% of the active substance EGS-1300-1 was obtained.
4.2 mg were obtained. HPLC fractionation condition column: YMC-Pack SH-343-5AQ (manufactured by YMC) Eluent: solvent A: 95% methanol, solvent B: methanol / acetone = 7: 3 100% A → 100% B linear concentration gradient Elution flow rate: 2 ml / min

Formulation Example 1 Tablet EGS-1300-1 100 g, lactose 40 g, corn starch
18 g and carboxymethylcellulose calcium 10
g are mixed, and a 10% hydroxypropylcellulose solution is added and kneaded. The kneaded liquid was granulated by an extrusion granulator equipped with a 1.0 mm basket, and magnesium stearate was added to the granulated granules to obtain granules for tableting. 100 mg of EGS-1300-1
To obtain tablets of 8 mm diameter.

Formulation Example 2 A 10% hydroxypropylcellulose solution was added to a mixture consisting of 50 g of capsule EGS-1300-1, 80 g of lactose and 38 g of potato starch, and kneaded. After granulation, magnesium stearate is added, and the mixture is filled into hard capsules by a capsule filling machine to obtain a capsule containing 50 mg of EGS-1300-1 in 1 capsule (170 mg) by a conventional method.

Formulation Example 3 Soft capsules 10 g of EGS-1300-1 was dissolved in 100 g of soybean oil, and the resulting solution was injected into capsules by a conventional method to give 10 mg of EGS-1300-1 per capsule. To obtain a soft capsule.

[0037]

Industrial Applicability According to the present invention, there is provided a novel EGS-1300-1 compound having sialyltransferase inhibitory activity, a pharmacologically acceptable salt thereof, and a method for producing the same.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of Examples of the present invention in which the inhibitory activity on sialyltransferase was measured. Horizontal axis shows EGS-1300-1 concentration (μg / ml), vertical axis shows inhibition rate (%)
Represents

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:80) (C12P 17/12 C12R 1:80) (72) Inventor Yuzuru Matsuda 1-22-Nukii Minamimachi, Koganei-shi, Tokyo 7

Claims (2)

[Claims] 1. Formula (I): (Wherein, R represents linoleoyl)
S-1300-1 or a pharmacologically acceptable salt thereof. 2. A microorganism belonging to the genus Penicillium and having the ability to produce EGS-1300-1 is cultured in a medium, and the microorganism is cultured.
2. The method according to claim 1, wherein EGS-1300-1 is collected.
Manufacturing method of EGS-1300-1.