JP2593495B2 - Compound TAN-999, production method and use thereof - Google Patents

Description

The present invention relates to a novel compound TAN-999 (hereinafter "TAN-999") useful as a therapeutic agent for infectious diseases such as bacteria and fungi and malignant tumors.
And may be abbreviated as ").

BACKGROUND OF THE INVENTION Staurosporine (AM-2282) -Journal.
Journal of Antibiotic
s), 30, 275, 1977 and JCS Chemical Communication (JCSChem.Comm.), 800, 1978
Year], this compound differs from TAN-999 in the presence or absence of a methoxy group, and TAN-999 is a novel compound. In terms of biological activity, this class of alkaloid compounds inhibit protein kinases [Journal of
Journal of Antibiotics, 34
Volume, 1059, 1986; 35, 706, 1987] and platelet aggregation inhibitory activity [Agricultural and Biological chemistry (Agricultural and Biologica)
l, Chemistry), 50, 2723, 1986].

Problems to be Solved by the Invention Phagocytic cells such as macrophages usually not only phagocytose, digest, and detoxify foreign foreign substances and waste products in vivo, but also suppress growth of tumor cells, It has extremely diverse capabilities such as controlling ecological functions by producing active substances, establishing and expressing immunity, and plays an important role in host defense. In recent years, infection with bacteria, fungi, viruses, etc., malignant tumors and their treatment, administration of immunosuppressants in organ transplantation, etc., have reduced the function of host phagocytes and lymphocytes, etc., resulting in resistance to infection and other diseases. The deterioration of the disease, the susceptibility to various diseases, and the seriousness of the disease have become major problems in the medical field. In order to overcome these problems, there is a need in the art for novel compounds having an activating effect on phagocyte functions such as macrophages or intermediate materials for synthesizing those compounds.

Means for Solving the Problems In view of the present situation, the present inventors have repeatedly studied a macrophage activator from a new viewpoint. As a result, among a large number of microorganisms isolated from soil, a certain microorganism is cultured in an appropriate medium, thereby activating a mouse-derived cultured macrophage, and a compound having an action of significantly changing the morphology is added to the medium. Knowing that it could accumulate, the compound was isolated and its physicochemical and biological properties confirmed that it was a novel compound, which was named TAN-999. It was also found that the microorganism belongs to the genus Nocardiopsis. The present inventors have further studied based on these findings and completed the present invention.

That is, the present invention provides a method of culturing (1) TAN-999 or a salt thereof, and (2) a TAN-999-producing bacterium belonging to the genus Nocardiopsis in a medium, producing and accumulating TAN-999 in the culture. It is intended to provide a method for producing TAN-999, which is characterized in that it is collected.

The bacterium that produces TAN-999 used in the method of the present invention may be any microorganism that belongs to the genus Nocardiopsis and has the ability to produce TAN-999. An example thereof is the C-71425 strain isolated from soil, and the bacterial properties of the strain are as follows. Properties on various media are at 28 ° C unless otherwise indicated.
The cells were cultured for 14 days and observed according to a conventional method. The color tone was measured using a color harmony manual (Color Harmony Ma
nual), 4th edition [Container Corporation of America, 19
Published in 1958].

1. Form The formation of aerial mycelium and spores is moderately observed on a medium such as yeast extract / malt extract agar, starch inorganic salt agar, glycerol / asparagine agar. The branch of the aerial hyphae is a simple branch, no axle branch is observed, and a chain of spores is observed at the tip of the branch. No special structures such as fungal hypha, sclerotium, spore sac, etc. are observed. Observation with an electron microscope shows that the surface of the spores is smooth, the spores are ovoid or cylindrical, and their size is 0.3 to 0.6 × 0.7 to 1.2 microns, usually 10 or more. Also, these spores do not show motility.

2. Properties on various classification media (cultured at 28 ° C for 14 days) 1) Sucrose nitrate agar medium Growth: good Aerial mycelia: medium, white Back color: light ivory (2ca) to light tan (3gc) soluble Pigment: None 2) Yeast extract / malt extract agar medium Growth / good Aerial mycelia: Medium, white Back color: Light wheat (2ea)-Light amber (3ic) Soluble pigment: None 3) Oat-meal agar medium Growth: good Aerial mycelium: medium, white Back color: light ivory (2ca)-light wheat (2ea) Soluble pigment: none 4) Starch inorganic salt agar medium Growth: good aerial mycelium: medium, white Color: Light ivory (2ca) to maize (2ga) Soluble pigment: None 5) Glycerol / asparagine agar medium Growth: Good Aerial mycelium: Medium, white Back color: Light ivory (2ca) to medium (2ga) Soluble pigment: None 6) Glucose / asparagine agar medium Growth: Good Aerial mycelium: Medium, white Back color: Light ivory (2ca) to maize (2ga) Soluble pigment: None 7) Peptone / Yeast extract・ Iron agar medium growth / medium aerial mycelium: not settled Back color: honey gold (2ic) soluble pigment: none 8) Tyrosine agar medium growth: good aerial mycelium: medium, white back color: light ・Wheat (2ea) to light gold (2ic) Soluble pigment: None 9) Calcium malate agar medium Growth: Medium Aerial mycelium: poor, white to pearl (2ba) Back color: light ivory (2ca) Soluble pigment: None 3. Physiological properties 1) Growth temperature: 10 ° C to 34 ° C Optimal temperature: 17 ° C to 30 ° C 2) Gelatin liquefaction: positive 3) Starch hydrolysis: positive 4) Milk peptone: positive 5) Mi Coagulation: negative 6) Nitrate reduction: positive 7) Melanin-like pigment formation: negative 8) Utilization of carbon source: D-sorbitol + sucrose + i-inositol + lactose + D-mannose + raffinose + D- Xylose + trehalose + L-arabinose + mannose + D-galactose + soluble starch + D-glucose + glycerol + D-fructose + melibiose + rhamnose + maltose + cellulose-ribose + no addition-basal medium: Pleedham and Gotley Analysis of Diaminopimelic Acid and Sugar during Hydrolysis of Whole Cells This bacterium contains meso-diaminopimelic acid in the hydrolyzate of all cells and does not contain arabinose and majulose.

To summarize the above taxonomic properties, the C-71425 strain
The aerial mycelium tip is bent, the spore surface is smooth,
It does not form mycelium and does not produce motile spores, its development color is pale yellow or yellowish brown, the aerial mycelium is white, meso-diaminopimelic acid is detected from the cells, but arabinose and majulose The bacterium belongs to cell wall type III and the sugar type belongs to type C by not being detected. Based on the above properties, edited by R.E.Buffanan and N.E.Gibbons, the Barges Manual of Deterministic Bacteriology (Berge
y's Manual of Determinative Bacteriology)
The strain C-71425 was found to belong to the genus Nocardiopsis as a result of a search for bacterial species according to the No. ed., 1974 and ninth edition, 1986. It was designated as P (Nocardiopsis sp.) C-71425.

The C-71425 strain was obtained from the Fermentation Research Institute (IFO) on November 11, 1987 under the accession number IFO-14673, and the bacterium was obtained on November 18, 1987 from the Ministry of International Trade and Industry, It has been deposited with the Research Institute (FRI) under the accession number FERM P-9713.

Bacteria producing TAN-999 belonging to the genus Nocardiopsis,
As in the case of other actinomycetes, for example, irradiation with ultraviolet rays, X-rays, radiation, etc., isolation of single spores, various mutagenesis treatments, and mutation by other means can be carried out. Even mutant strains which are substantially different from the taxonomic properties described above and have the property of producing the compound can be used in the method of the present invention.

The medium used for cultivation of the compound-producing bacterium may be liquid or solid as long as it contains a nutrient that can be used by the bacterium, but it is more appropriate to use a liquid medium for large-scale treatment. The medium is appropriately mixed with a carbon source, a nitrogen source, an inorganic substance, and a micronutrient which can be assimilated by the compound-producing bacterium. Examples of the carbon source include glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, fats and oils (eg, soybean oil, lard oil, chicken oil, etc.), n-paraffin, and the like as a nitrogen source. Is, for example, meat extract, yeast extract, dried yeast,
Soy flour, corn steep liquor, peptone, cottonseed flour, molasses, urea, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.) and the like are used. Further, salts containing sodium, potassium, calcium, magnesium and the like, metal salts such as iron, manganese, zinc, cobalt and nickel, salts such as phosphoric acid and boric acid, and salts of organic acids such as acetic acid and propionic acid are appropriately used. . Other amino acids (eg, glutamic acid, aspartic acid, alanine,
Lysine, methionine, proline, etc.), peptides (eg,
Dipeptides, tripeptides, etc.), vitamins (eg,
B ₁ , B ₂ , nicotinic acid, B ₁₂ , C, etc.) and nucleic acids (eg, purines, pyrimidines, derivatives thereof, etc.) may be contained. Of course, an inorganic or organic acid, alkali, buffer or the like may be added for the purpose of adjusting the pH of the medium, or an appropriate amount of an oil or fat, a surfactant or the like may be added for the purpose of defoaming. During liquid culture, the pH of the medium should be around neutral, especially
A pH of about 6-8 is preferred. The culture temperature is preferably about 24 ° C. to 30 ° C., and the culture time is preferably about 48 hours to 120 hours.

The titer of TAN-999 produced over the course of the culture is determined by a liquid dilution method using mouse-derived cultured macrophages Mm1 as test cells. Usually, the production of TAN-999 reaches its maximum in about 2-3 days of culture.

In order to collect the target compound TAN-999 from the culture, TAN-999 is a compound that is basic and shows lipophilicity, and can be collected by a general means utilizing this property.

Since TAN-999 in the culture is contained in the cells and the filtrate, the culture is adjusted to pH 6 to 11, preferably pH 8 to 10, and then water-immiscible organic solvent such as dichloromethane, ethyl acetate, methyl isobutyl ketone or A method of extracting an active substance by adding isobutanol or the like is used. In addition, an organic solvent miscible with water, for example, acetone or methanol, is added to the culture solution, and the mixture is stirred and extracted. After insolubles are filtered off, the organic solvent in the extract is distilled off under reduced pressure to obtain an aqueous solution. Or an adsorptive resin such as Amberlite XAD-II (manufactured by Rohm and Haas, USA), DIAION HP-20 (manufactured by Mitsubishi Kasei, Japan) or Amberlite SP-207 (Mitsubishi). A method of performing a chromatography method using, for example, Kasei Co., Japan) is advantageously used. In order to elute the target active substance from the column using the adsorptive resin, water or a water-containing solvent such as water-containing methanol or water-containing acetone is advantageously used. The extract or eluate thus obtained is concentrated under reduced pressure to obtain a crude substance containing TAN-999.

Various chromatographic methods are advantageously used to further purify the crude material to obtain pure TAN-999. As a carrier, silica gel, cellulose, Sephadex LH-20
(Pharmacia, Sweden) etc. are used,
These are usually performed by column chromatography.
To elute the active substance from the column, a suitable organic solvent such as ethyl acetate, dichloroethane, acetone, methanol or the like, alone or in a mixed solvent is used. When the eluate is concentrated and left in a cool place, or the concentrated residue is dissolved in a suitable crystallization solvent such as diethyl ether, ethyl acetate, methanol or a mixture thereof, and left in a cool place,
A crystal of TAN-999 is obtained.

Since TAN-999 is a basic substance, a salt of TAN-999 can be obtained by treating with a suitable mineral acid. These are prepared according to a method known per se. Examples of the type of salt include hydrochloride, sulfate, phosphate and the like.

Thus, the physicochemical properties of TAN-999 obtained in Example 2 described below are as follows.

1) Appearance: colorless crystal 2) Melting point: mp 221 ° C (decomposition point) 3) Specific rotation: ▲ [α] ²¹ _D ▼ + 42 ゜ (in C0.5, DMF) 4) Mass spectrum measured value: m / Z 496 (M ⁺ , EI-MS) 5) Elemental analysis: (%) Found; C, 68.59; H, 5.64; N, 10.86 Calculated; C, 68.90; H, 5.78; N, 11.08; O, 14.24 ^* (* Calculated as including 0.5 mole of water) 6) Molecular formula: C ₂₉ H ₂₈ N ₄ O ₄ 7) Ultraviolet absorption (UV) spectrum: in methanol, FIG. 296 (1215), 341 (394), 352 (shoulder), 368 (222) See FIG.

8) Infrared absorption (IR) spectrum: in KBr, FIG. 2, νma
x cm ^-1 , main absorption 3430, 2950, 1680, 1590, 1460, 1420, 1360, 1320, 12
90, 1260, 1210, 1120, 1040, 840, 800, 760, 640, 60
0 See attached FIG.

9) ^-1H nuclear magnetic resonance (NMR) spectrum: 300MHz, CDCl
_3. Among them, δ ppm J (Hz) 9.40 (d, J = 7.8), 7.75 (d, J = 8.5), 7.46 (t), 7.
46 (d), 7.35 (t), 7.27 (d, J = 8.0), 6.96 (dd, J
= 2.0), 6.53 (d, J = 5.6), 6.28 (s), 4.96 (2H, q,
J = 17), 3.95 (3H, s), 3.87 (d, J = 3.5), 3.44 (3H,
s), 3.35 (m), 2.75 (dd, J = 14.7, 4.0), 2.38
(M), 2.33 (3H, s), 1.57 (3H, s).

10) ¹³ CNMR spectrum; 75 MHz, in _{CDCl 3, δppm 173.8ls, 157.49s,} 141.04s, 136.67s, 131.48s, 130.6
3s, 127.09s, 126.50d, 124.88d, 123.55s, 120.94d, 1
19.65d, 118.92s, 118.58s, 114.88s, 114.24s, 107.94
d, 106.89d, 100.72d, 91.06s, 84.26d, 80.12d, 57.24
q, 55.78q, 50.36d, 45.92t, 33.38q, 30.29t, 29.69
q. (However, s: singlet (singlet), d: doublet (doublet), t: triplet (triplet), q: quartet (quartet)) 11) Thin layer chromatography (TLC): Carrier: silica gel 60F ₂₅₄ (manufactured by Merck Development solvent: ethyl acetate: methanol (96: 4) Rf = 0.11 12) High performance liquid chromatography (HPLC): carrier: ODS column (YMC-Pack A312, manufactured by Yamamura Chemical Laboratory) Mobile phase: 55% acetonitrile /0.01M phosphate buffer (p
H6.3) Flow rate: 2ml / min Detection: 254nm Rt = 6.0 (min) (Staurosporine: Rt = 6.3min) 13) Color reaction: Positive: Ninhydrin, Ehrlich, Dragendorff, Burton and Greek, Leeback reaction 14) the material classification: various analyzes of the above basic fat-soluble substances mentioned physicochemical properties and NMR spectra (COZY method, ¹ H- ¹³ C correlation (Correla
The chemical structure of TAN-999 was determined by the following formulas using the following methods: COLOC method, NOESY method).

The biological activity of TAN-999 can be demonstrated by the following experiment.

First, Table 1 shows the effect of the mouse culture macrophage on Mml cells.

Activating action: ability to induce activated macrophage-like morphology +: positive, ±: positive but weak,-: 37 in a Git medium (Nippon Pharmaceutical, Japan) containing a negative drug under 5% CO ₂
After culturing at 3 ° C. for 3 days, the cell morphology was observed.

Moreover, the poor phagocytic ability of the latex particles of the mouse culture macrophages Mnl and J774A-1 cultured for 2 days in the presence of TAN-999 5 ng / ml was enhanced about 9 times and 12 times, respectively, as compared with the case where no drug was added. Was. Next, a 10% proteose peptone (Difco, USA) solution was intraperitoneally administered to C57BL / 6 (10-week-old, female) mice, and peritoneal cells were collected on the fourth day, and plastic adherent cells were extracted from the peritoneal cells. Gather
Using this as a macrophage, TAN-999 mouse,
The effect on macrophages was examined. As a result, TAN
-999 Culture in the presence of 0.1 ng / ml for 2 days [RPMI 1640 medium containing 10% (v / v) fetal calf serum (Wittacar M. A. Bio Products, USA) (Whitaka M. A. Bio Products) Macrophages to zymosan (Zicuma, USA) have a phagocytic ability-dependent active oxygen release capacity (measured by a luminol-dependent chemiluminescence method) that is about twice that of the case without drug addition. Had been strengthened.

Next, Table 2 shows the growth inhibitory effect of TAN-999 on mouse tumor cells.

As is clear from the above physicochemical properties and biological properties, TAN-999 is a novel compound, which activates macrophages and shows a strong growth inhibitory effect on tumor cells. It is.

When compound (I) is used as an antitumor agent, a diluent,
It is mixed with excipients, carriers and the like to form a pharmaceutically acceptable formulation, and is orally or parenterally administered.

For example, when used as an injection, the salt of compound (I) may be used as a water-soluble injection in an amount of 0.1 mg / kg to 10 mg / day for an adult.
mg / kg is administered intravenously.

Examples Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto. The percentages (%) in the medium represent weight / volume percentages unless otherwise specified.

Example 1 Yeast extract and malt extract Nocardiopsis sp. C-71425 (IFO-14673, F
ERMP-9713) with glucose 2 in a 200 ml Erlenmeyer flask
%, Soluble starch 3%, raw soy flour 1%, corn steep liquor 1%, peptone 0.5%, NaCl 0.3%, CaCO ₃ 0.5
% Of the seed medium (pH 7.0), and cultured on a rotary shaker at 28 ° C. for 48 hours to obtain a pre-culture liquid. 5 ml of the obtained preculture was transferred to 500 ml of a seed medium in a 2000 ml Sakaguchi flask and cultured at 28 ° C. for 48 hours on a reciprocating shaker to obtain a seed culture. Transfer 500 ml of this seed culture into a 50-volume stainless steel tank containing 30 seed medium (same composition as the above seed medium), aeration 30 / min, stirring 280 rpm, internal pressure 1
The culture was performed under the condition of kg / cm ² . 5 of the obtained culture solution was 200
Glucose in stainless steel tank 0.5
%, Dextrin 5%, defatted soy flour 3.5%, CaCO ₃ 0.7%
Transplanted into 120 main media (pH 7.0) containing
The cells were cultured for 96 hours under the conditions of 0 / min, 180 rpm / min, and an internal pressure of 1 kg / cm ² .

Example 2 After adjusting the culture solution 110 obtained in Example 1 to pH 9 with a dilute sodium hydroxide solution, ethyl acetate 110 was added thereto.
After stirring for 60 minutes, 4.5 kg of Hyflo Super Cell was added to the solution, followed by filtration. The obtained extracted ethyl acetate layer 80 is washed with water 25
After washing with water, the mixture was concentrated, and the oily concentrate was subjected to silica gel column chromatography (silica gel: 50 g). The eluate of ethyl acetate: methanol (90:10) was concentrated and kept cool to obtain 1.1 g of TAN-999 as pale yellow crystals. Also,
The crystal mother liquor and the recovered fractions in the silica gel chromatography were collected, concentrated, and the concentrate was subjected to silica gel chromatography again, and treated in the same manner as described above to obtain TA.
588 mg of N-999 crystals were obtained.

Example 3 100 mg of the TAN-999 crystals obtained in Example 2 were dissolved in 30 ml of chloroform, and a precipitate formed in a solution into which hydrochloric acid gas had been introduced. This was collected by filtration to obtain 87 mg of TAN-999 monohydrochloride. Was done.

▲ [α] ²⁰ _D ▼ -12.8 ゜ (C 0.5 in MeOH) Elemental analysis (as C ₂₉ H ₂₈ N ₄・ 1.5HCl ・ 2.5H ₂ O) Found: C, 59.11; H, 6.00; N, 9.54 ; Cl, 9.00 Calculated: C, 58.41; H, 5.83; N, 9.40; Cl, 8.92

[Brief description of the drawings]

1 and 2 show the UV spectrum and IR spectrum of compound TAN-999, respectively.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication C12R 1:01)

Claims (3)

(57) [Claims] 1. A compound TAN-999 represented by the following formula (I) or a salt thereof. 2. A compound TAN- characterized by culturing a compound TAN-999-producing bacterium belonging to the genus Nocardiopsis in a medium, producing and accumulating TAN-999 in the culture, and collecting the TAN-999. 999 manufacturing method. 3. A macrophage activator comprising the compound TAN-999 or a salt thereof.