JPH0827143A - Ucf76 compound - Google Patents

Abstract

PURPOSE:To obtain UCF76 compound having excellent antibacterial and antitumor activities by culturing a microorganism belonging to Streptomyces genus. CONSTITUTION:This UCF76 compound of the formula (when X<1> and X<2> in combination represent a single bond, R<1> is H and R<2> is -OCH3; when X<1> and X<2> in combination represent -O-, R<1> and R<2> in combination represent a single bond) is obtained by culturing Streptomyces sp. UCF76 strain in a medium using glucose, starch, etc., as carbon sources and ammonium chloride, a meat extract, etc., as nitrogen sources, and further adding inorganic salts such as potassium chloride, magnesium sulfate, etc., at need, preferably by using a culturing method with agitation in depth at 16-37 deg.C (preferably 25-37 deg.C) and pH4-10 (preferably 6-8) for usually 1-7 days. Further, an aqueous ammonium or ammonium carbonate solutions is used for adjusting pH of the medium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a UCF76 compound having antibacterial and antitumor activity and useful as an antibacterial and antitumor agent.

[0002]

[Prior art]

 Formula (II)

[0003]

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UCF13 compound represented by the formula (III), which has antibacterial and antitumor activity (JP-A-4-154778):

[0005]

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Frenolysin represented by the formula [J.Am.Chem.Soc., 90 , 1325 (1968)] having the antibacterial action, formula (IV)

[0007]

[Chemical 4]

Frenolysin B represented by [Journal
J. Antibiotics, 31 , 959 (1
978)] and formula (V)

[0009]

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Deoxyfrenolicin represented by [Journal of Antibiotics, 3
1 , 959 (1978)] is known.

[0011]

The object of the present invention is to provide a compound having excellent antibacterial and antitumor activity.

[0012]

According to the invention, the formula (I)

[0013]

[Chemical 6]

(In the formula, when X ¹ and X ² together represent a single bond, R ¹ represents hydrogen, R ² represents —OCH ₃ , and X 1
^{When 1} and X ² together represent -O-, R ¹ and R ²
Taken together represent a single bond) to provide UCF76 compounds having antibacterial and antitumor activity. The compound is obtained by culturing a microorganism belonging to the genus Streptomyces.

The present invention will be described in detail below. UCF76
In the compound, formula (Ia)

[0016]

[Chemical 7]

The compound represented by the formula UCF76-B, also represented by the formula (Ib)

[0018]

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The compound represented by the formula (1) is named UCF76-C. The physicochemical properties of the UCF76 compound are as shown below. (i) UCF76-B Property: Yellow powder Specific rotation: [α] _D ²³ = + 196 ° (c = 0.18, CH ₃ OH) Mass spectrum: positive ion FAB-MS m / z 361.1284 [M + H] ⁺ (Theoretical value 361.1287 C ₁₉ H ₂₁ O ₇ ) Molecular formula: C ₁₉ H ₂₀ O ₇

UV absorption spectrum: λmax nm (ε) (CH
₃ OH): 421 (4300), 269 (9900), 250 (9500), 211 (34700). Infrared absorption spectrum: νmax cm ^-1 (KBr): 3471,1749,
1639,1614,1458,1284,1257,1167,783,710.

¹ H-NMR spectrum (400 MHz, CDCl ₃ ): δ pp
m (multiplicity, coupling constant, integral): 11.90 (brs, 1H), 7.65 (dd, J
= 2.2,7.6Hz, 1H), 7.62 (t, J = 7.6Hz, 1H), 7.26 (dd, J = 2.2,7.
6Hz, 1H), 4.84 (dd, J = 2.8,10.6Hz, 1H), 4.66 (d, J = 2.4Hz, 1
H), 4.30 (dt, J = 2.4,6.7Hz, 1H), 3.74 (s, 3H), 2.84 (d, J = 6.7
Hz, 2H), 1.80 (m, 1H), 1.72 (m, 1H), 1.64 (m, 1H), 1.55 (m, 1
H), 1.00 (t, J = 7.2Hz, 3H).

¹³ C-NMR spectrum (100 MHz, CDCl ₃ ): δ
ppm (Multiplicity): 189.1 (s), 183.4 (s), 171.6 (s) 161.8 (s), 1
46.6 (s), 141.1 (s), 136.7 (d), 131.7 (s), 124.9 (d), 119.4
(d), 114.9 (s), 70.8 (d), 67.4 (d), 60.3 (d), 51.9 (q), 35.7
(t), 32.9 (t), 19.6 (t), 13.6 (q).

Solubility: dimethyl sulfoxide (DMSO),
Soluble in chloroform, acetone and ethyl acetate, water,
Insoluble in hexane Color reaction: Positive for iodine reagent Thin layer chromatography: Rf value 0.3 thin layer; silica gel thin layer (HPTLC plate Art.5715, Merck) Developing solvent: hexane: ethyl acetate (2: 1 v / v The spot of UCF76-B after development can be detected by an iodine reagent.

(Ii) UCF76-C Property: Light yellow solid Specific rotation: [α] _D ²⁶ = + 44 ° (c = 0.17, CH ₃ OH) Mass spectrum: positive ion FAB-MASS m / s 345.0972 [M + H]
⁺ (Theoretical value 345.0974 C ₁₈ H ₁₇ O ₇₎ Molecular Formula: C ₁₈ H ₁₆ O ₇

UV absorption spectrum: λmax nm (ε) (CH
₃ OH): 365 (4900), 277 (3300), 236 (14800). Infrared absorption spectrum: νmax cm ^-1 (KBr): 1788,1699,
1651,1456,1284,1246,1161,1001,928,733.

¹ H-NMR spectrum (400 MHz, CDCl ₃ ): δ pp
m (multiplicity, coupling constant, integral): 11.31 (s, 1H), 7.69 (t, J =
7.7Hz, 1H), 7.62 (dd, J = 1.0,7.7Hz, 1H), 7.31 (dd, J = 1.0,8.
4Hz, 1H), 5.46 (d, J = 4.9Hz, 1H), 4.67 (dd, J = 3.1,11.6Hz, 1
H), 4.48 (dd, J = 4.9,6.5Hz, 1H), 2.88 (dd, J = 6.5,18.3Hz, 1
H), 2.63 (d, J = 18.3Hz, 1H), 1.91 (m, 1H), 1.68 (m, 1H), 1.57
(m, 2H), 1.03 (t, J = 7.3Hz, 3H).

¹³ C-NMR spectrum (100 MHz, CDCl ₃ ): δ
ppm (Multiplicity): 194.1 (s), 188.4 (s), 173.7 (s), 162.3 (s),
137.8 (d), 131.5 (s), 125.2 (d), 120.3 (d), 114.4 (s), 69.3
(d), 67.4 (d), 64.5 (d), 64.4 (s), 60.0 (s), 35.3 (t), 29.3
(t), 18.7 (t), 13.6 (q).

Solubility: Soluble in dimethyl sulfoxide (DMSO), chloroform, acetone and ethyl acetate, insoluble in water and hexane Color reaction: Positive for iodine reagent Thin layer chromatography: Rf value 0.24 thin layer; silica gel thin layer ( HPTLC plate Art.5715, manufactured by Merck) Development solvent; hexane: ethyl acetate (2: 1 v / v) After development, the UCF76-C spot can be detected by an iodine reagent.

The above data was measured by the following equipment. Mass analysis: JEOL JMS-HX110 / 110A Mass spectrometer UV absorption spectrum: Shimadzu UV-2200 spectrophotometer Infrared absorption spectrum: JEOL JIR-RFX3001 Infrared spectrophotometer Nuclear magnetic resonance spectrum: JEOL JNM-α400 Nuclear magnetic resonance device Optical rotation: JASCO DIP-370 type digital polarimeter UCF76 The biological activity of the compound is as shown below. (A) Antibacterial action against various bacteria The minimum growth inhibitory concentration (M
IC) is shown in Table 1.

The antibacterial activity is bactotryptone (manufactured by Difco) 3 g / l, meat extract 3 g / l, yeast extract 1 g / l,
The measurement was carried out by an agar dilution method using a medium (pH 7) consisting of glucose 1 g / l and agar 16 g / l.

[0031]

[Table 1]

(B) Inhibition of growth on BALB3T3 / H-ras cells Using 96-well microtiter plate with MEM medium containing 10% fetal bovine serum (manufactured by Nissui; hereinafter referred to as medium A)
BALB3T3 / H-ras cells adjusted to 3.0 x 10 ⁴ cells / ml (ATC
0.1 ml of C HTB22) was dispensed into each well of the plate.
The plate was incubated in a carbon dioxide gas incubator at 37 ° C. for 20 hours, and then the test compound was appropriately diluted with the medium A.
Add 0.1 ml each and place in a carbon dioxide incubator 3
It was cultured at 7 ° C for 72 hours. After removing the culture supernatant, the residue was washed once with physiological saline, treated with 0.1 ml methanol for 10 minutes to fix the cells, and then 0.1 ml of Giemsa stain solution [Gimza stain stock solution Merck Art 9204 (Merck)]. : Saline = 1: 1
The cells were stained with 0] for 5 minutes. After removing the staining solution, remove the residue
It was washed once with 0.2 ml of water. Then 0.1N hydrochloric acid 0.2ml
After extracting the dye with, use a microplate reader to
The absorbance at 0 nm was measured.

Cell proliferation was determined by comparing the absorbance of untreated cells and cells treated with known concentrations of test compound.
The concentration of test compound that inhibits 0% (IC ₅₀ ) was calculated. The results are shown in Table 2.

[0034]

[Table 2]

Next, a method for producing the UCF76 compound will be described. UCF76 belongs to the genus Streptomyces, UC
It is produced by culturing a microorganism capable of producing F76 in a medium, producing and accumulating UCF76 in the culture, and collecting UCF76 from the culture. Microorganisms capable of producing UCF76 belong to the genus Streptomyces, UCF
Any strain can be used as long as it is a strain capable of producing 76. In addition, if any of these strains are mutated by an artificial mutagenesis method, for example, ultraviolet irradiation, X-ray irradiation, treatment with a mutagenic agent, etc. Can be used. A specifically preferred example is the Streptomyces sp. UCF76 strain.

Streptomyces species ( Stre
The mycological properties of the ptomyces sp.) UCF76 strain are as follows. It should be noted that the determination of the said properties, international Streptomyces how Streptomyces (Streptomyces) project (ISP) is recommended for the Streptomyces species characterization [EB Schelling (EB Shirling) and D. Gottlieb (D.Gottli
eb), International Journal Systematic Bacteriology (Int. J. Syst. Bacteriol.), 1
6 , 313-340 (1966)].

The isomer of diaminopimelic acid in the hydrolyzate of whole cells is determined by the method of B. Becker et al. [Appl. Microbiol., 1
2 , 421-423 (1964)]. The morphological examination was carried out using an optical microscope, and especially the morphology of the spore surface was carried out using a scanning electron microscope.

To assign color names, refer to the Color Harmony Manual [Container Corporation of America].
rporation of America), 4th edition (1985)]. 1. Morphological properties 1) Hyphae aerial hyphae formation; aerial hyphae fragmentation and motility;

2) Spores Presence / absence of spore formation and position of formation; Formation in aerial hyphae Presence / absence of formation of sporangia and position of spores: Number of spores linked on asporeless stalk; 10 or more numerous spores linked Shape when straightening; characteristic of straight or curved spores surface structure; smooth shape and size; short rod shape, about 0.5-0.6 μm x 0.8-
1.0 μm Motility and presence of flagella; none

3) Others Chlamydospores; unfocused hyphae; non-pseudospores; non-hyphae division pattern; simple branching

2. Culture Properties UCF76 strain shows normal or vigorous growth in commonly used synthetic and natural media, and the basal hyphae show a yellow to light brown system. A brownish soluble pigment may be produced by the medium. 28 ℃, 14 days, U on various media
The characteristics of growth and color when the CF76 strain is cultured are shown below.

1) Sucrose / nitrate agar medium Growth condition: normal Color of basal mycelium; white (a) Growth condition of aerial mycelium and its color; vigorous, white (a) Soluble pigment; None

2) Glucose / asparagine agar medium Growth state; normal Color of basal hyphae; bamboo (2gc) to camel (3ie) Growth status of aerial mycelium and its color tone; poor, white (a) soluble pigment; none

3) Glycerin / asparagine agar medium Growth condition; normal color of basal mycelium; light tan (3gc) to cork tan (4ie) aerial mycelium growth condition and its color; normal, shell pink (5b
a) Soluble pigment; production (brown)

4) Starch / inorganic salt agar medium Growth state: vigorous color of basal mycelium; light tan (3 gc) aerial mycelium growth state and its color tone: vigorous, white (a) soluble pigment; production (brown)

5) Tyrosine agar growth condition; vigorous color of basal hyphae; light brown (4 ng) aerial hyphae growth and its color tone; usually, white (a) to Dusk (10fe) soluble pigment; production (brown) )

6) Nutrient agar medium Growth condition: Vigorous color of basic hyphae: Colonial yellow (2ga) to bamboo
(2gc) Aerial hyphae growth status and its color tone; poor, white (a) soluble pigment; none

7) Yeast-malt agar medium Growth state: swelling Color of basal mycelium; cinnamon (3le) Settling state of aerial mycelium and its color; Poor, white (a) Soluble pigment; Production (brown)

8) Oatmeal agar medium Growth condition; normal color of basal hyphae; light wheat (2ea) aerial mycelium growth and its color tone; poor, white (a) soluble pigment; none

3. Physiological Properties The physiological properties of the UCF76 strain are shown below. The results for the growth temperature range after 10 days and for the other temperature after 28 to 28 ° C for 2 to 3 weeks are shown. 1) Growth temperature range; 6.0 to 43.0 ℃ 2) Gelatin liquefaction; No 3) Starch hydrolysis; No 4) Skim milk powder coagulation and peptonization; Peptone formation 5) Melanin-like pigment formation (i) Peptone Yeast / iron agar; no (ii) tyrosine agar; yes

6) Utilization of carbon source (basic medium: Pleedham-Gotlieve agar medium) In the following, + means that the material is used, and-is that it is not used. L-arabinose; -D-xylose; + D-glucose; + sucrose; -raffinose; -D-fructose; + L-rhamnose; + inositol; -D-mannitol; +

4. Chemotaxonomic properties Optical isomers of diaminopimelic acid in bacterial cells; LL type and above. Chemotaxonomically, the classification of actinomycetes by MP Lechevalier and HA Lechevalier [International Journal Systematic Bacteriology (Int.J.Syst.Bacterio
l.), 20 , 435-443 (1970)], it is classified into cell wall type I (LL-diaminopimelic acid, glycine). Further, morphologically, it is appropriate to assign this strain to the genus Streptomyces by combining morphological characteristics of the strain such as formation of spore chains in aerial hyphae.

As a result of the above, this strain was designated as Streptomyces sp. UCF76.
The strain is 21st June 1994 based on the Budapest Treaty.
FERM BP- at the Institute of Biotechnology, Institute of Biotechnology
Deposited as 4702. In culturing the UCF76 compound-producing bacterium of the present invention, a usual actinomycete culture method is applied. As the medium, either synthetic medium or natural medium can be used as long as it is a medium containing carbon source, nitrogen source, inorganic substances and necessary growth and production promoting substances which can be assimilated by microorganisms.

Carbon sources include glucose, starch, dextrin, mannose, fructose, sucrose,
Carbohydrates such as lactose, xylose, arabinose, mannitol, molasses are used alone or in combination. Further, depending on the assimilation capacity of the bacterium, hydrocarbons, alcohols, organic acids, glycerol and the like are also used.
As the nitrogen source, ammonium chloride, ammonium nitrate, ammonium sulfate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder, casamino acid, etc. may be used alone or in combination.

In addition, if necessary, sodium chloride,
Inorganic salts such as potassium chloride, magnesium sulfate, magnesium phosphate, calcium carbonate, potassium dihydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate and copper sulfate are added. Furthermore, the growth of used bacteria and UC
A trace component that promotes the production of F76 can be appropriately added.

As a culture method, a liquid culture method, particularly a deep agitation culture method is suitable. The culture is 16 to 37 ° C, preferably
It is carried out at a temperature of 25 to 32 ° C. and a pH of 4 to 10, preferably 6 to 8, and is usually completed in 1 to 7 days, and UCF76 is produced and accumulated in the culture solution and cells. Ammonia water or ammonium carbonate solution is used to adjust the pH of the medium. The culture is stopped when the maximum production in the culture is reached.

The UCF76 accumulated in the culture is isolated and purified from the culture according to a conventional method for isolating and purifying ordinary microbial metabolites from the culture. For example, the culture is separated by filtration into a culture filtrate and cells, and the cells are extracted with chloroform, acetone or the like.
Then, the extract and the culture filtrate are combined and passed through a polystyrene-based adsorbent such as Diaion HP20 (manufactured by Mitsubishi Kasei Co.) to adsorb the active ingredient, and then ethyl acetate,
Elute with acetone, etc. The eluate is concentrated, and UCF76 is obtained by column chromatography with silica gel, high performance liquid chromatography and the like. The detection of UCF76 during the culture and isolation / purification operations can be carried out by using silica gel thin layer chromatography and then using an iodine reagent.

Examples of the present invention will be shown below.

[0059]

【Example】

Example 1. Streptomyces sp. U as inoculum
The CF76 strain (FERM BP-4702) was used. 5 g / g of bactotryptone (Difco) in a 2 L Erlenmeyer flask.
l, yeast extract 5g / l, meat extract 3g / l, soluble starch 10
g / l, glucose 10g / l, magnesium phosphate 0.5g / l
The cells were inoculated into 300 ml of a seed medium (pH 7.2 before sterilization) having the above composition, and cultured by shaking at 30 ° C. for 48 hours (rotation speed 200 rpm). Transfer the obtained seed culture to 18 L of fermentation medium having the following composition in a 30 L jar fermenter at a rate of 5% (volume),
Culture with aeration and stirring at 28 ° C (rotation speed 300 rpm, aeration rate 18 L /
Minutes).

Fermentation medium composition: Glycerol 25 g / l, glucose 25 g / l, dry yeast 15 g / l, potassium dihydrogen phosphate
0.5g / l, magnesium sulfate ・ 7H ₂ O 0.5g / l, magnesium phosphate 0.5g / l, sodium chloride 10g / l (before sterilization p
(H 7.0, adjusted with caustic soda) During the culture, the pH of the medium was not particularly controlled, and the culture was performed for 72 hours. The culture was separated into a culture filtrate and cells by filtration, and the cells were extracted with acetone. After concentrating the extract, a silica gel column (BW-300 manufactured by Fuji Devison Chemical Co., Ltd.) was used, and chloroform-methanol (250:
1 v / v) mixed solution was developed and fractionated. The active fraction eluted was concentrated, and the silica gel column (Lichroprep Si60 Merck
(Manufactured by the company) was developed with a mixed solution of hexane-ethyl acetate (2: 1 v / v) and fractionated. Concentrate the eluted active fraction,
High performance liquid chromatography (HPLC) was performed under the following conditions. The mixture was developed with an acetonitrile-water (3: 2 v / v) mixed solution, and the fraction containing UCF76-B (retention time: 44 to 46 min) and the fraction containing UCF76-C (retention time: 47 to 51 mi).
n) was separately collected. Each fraction was concentrated, water was added to the residue, and the mixture was extracted with ethyl acetate. The extract was dehydrated with anhydrous sodium sulfate and then concentrated to give U
66 mg of CF76-B yellow powder and 21.9 mg of UCF76-C light yellow solid were obtained, respectively.

HPLC preparative condition column: ODS 120A S-5 (SH363-5) (manufactured by YMC) Eluent: 60% acetonitrile Flow rate: 10 ml / min Detection: 238 nm Retention time: UCF76-B; 45 min, UCF76-C 49mi
n

[0062]

INDUSTRIAL APPLICABILITY According to the present invention, a UCF76 compound having antibacterial and antitumor activity can be provided.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keiko Ochiai 4-21-17 Kamiimazumi, Ebina City, Kanagawa Prefecture (72) Inventor Shiro Akinaga 1188 Shimochikari, Nagaizumi-cho, Sunto-gun, Shizuoka Prefecture

Claims (1)

[Claims] 1. Formula (I): (In the formula, when X ¹ and X ² together represent a single bond,
R ¹ represents hydrogen, R ² represents —OCH _3, and when X ¹ and X ² together represent —O—, R ¹ and R ² together represent a single bond). UCF76 compound.