WO1998043955A1

WO1998043955A1 - Tetramic acid compound

Info

Publication number: WO1998043955A1
Application number: PCT/JP1998/001485
Authority: WO
Inventors: Takako Sugawara; Kayo Sanada; Hisao Ikeya; Tsutomu Ando; Masabumi Shibuya; Tadayasu Okazaki
Original assignee: Taisho Pharmaceutical Co., Ltd.
Priority date: 1997-04-02
Filing date: 1998-03-31
Publication date: 1998-10-08
Also published as: AU6521098A

Abstract

A tetramic acid compound is represented by formula (1). This compound is useful as a drug having an antitumor effect.

Description

Description Tetranic acid compound

Technical field

The present invention relates to a novel tetonic acid compound having an antitumor effect

Background branch art

Several compounds such as Tenuazonic acid have been reported as compounds having tetramic acid in its structure, but no compound having an antitumor effect is known.

An object of the present invention is to provide a novel bioactive substance having an antitumor effect.

Disclosure of the invention

The present inventors have isolated a large number of strains from soil and plants in order to achieve the above-mentioned object, and have conducted various studies on metabolites of the strains. As a result, a novel physiological activity in which a certain strain has an antitumor activity It found that producing substance, _c ie have completed the present invention, the present invention provides a compound of formula (1)

(Hereinafter abbreviated as TELOOIO).

The strain that produces TELOOIO is a strain newly isolated from the natural world by the present inventors, and has the name of microorganism rstreptomvces sp. (Address: 1-35-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan, Deposit date: February 26, 1998). The bacteriological properties of this strain are shown below.

A. Morphological properties

The basal hypha grows well and branches, but no division is observed. The formation of aerial mycelium and the formation of vesicles are good on oatmeal agar medium and 3 other mediums. No sporulation in the agar medium is observed. It forms a dense spiral spore chain (350 spores) of 600 turns, which is simply branched from aerial hyphae. The spores are 1.1.13 fi m in length, 0.70.9 m in width, cylindrical in shape, and smooth in surface. No sporangia sclerotium formation and spore motility were observed.

B. Cultural properties

Table 1 below shows the results of macroscopic observations when cultured for 28 ^ 14 days on various media. In addition, the color display quoted the system color name of the Japan Standards Association and JIS color name book (1985).

Aerial bacterium Thread 3 Ronichi Agar Agar medium

Culture and shaping color production sucrose · moderate medium bright medium moderate very light yellow none nitric acid (5-9 / 3)

(5Y7. 5/1)

Crucos · Moderate Very light yellow None. Raki'n slightly to light grayish yellow

(10YR9 / 3 10

(YR7 / 3)

K'lyserin · Good Good Yellowish color Good Dark grayish yellow ~ none None;, ° Rak. (5Y6 / 1) Dark yellow

(5Y4. 5/3

5Y2. 5/2. 5)

Star i-T. 良好 Good Good Grayish yellow Good Blueish yellow None

(7.5Y6.5.3 / 3) (7.5Y4.5.5 / 3)

Tyrosine good good bright yellow good: dark dark red brown

(5Y8 / 3) (10YR2 / 2)

Ordinary nutrition Weak None None Medium Medium Medium Grayish yellow Good Dull red None

of i (7.5.Y6. 5/3)

(10YR7 / 7)

Automated Good Good Yellowish gray Good Grayish yellow None

(5Y6 / 1) (10YR5 / 3)

What. H. Ton · Medium None Gray Yellow Blackish brown Iron · Iron (10YR5 / 3) Physiological properties

The growth temperature test was observed for 1 week, the carbon source utilization test was observed for 2 weeks, and the others were observed after 3 weeks of culture.

① Growth temperature range: 12 ~ 38, optimal growth temperature: 26 ~ 3 1

② Liquefaction of gelatin: positive.

③ Coagulation of skim milk: negative

④ Peptone conversion of skim milk: positive

⑤Melanin-like pigment production: positive

Hydrolysis of starch: positive

⑦Utilization of carbon source (on Pridham Gottlieb agar medium)

L-arabinose: + L—Ramulose

D—Xylose: + Raffinose +

D—glucose: + inositol +

D—fracture: + D—mannitol +

Sucrose: +

(+: Use: Use slightly)

D. Chemical taxonomic properties

(1) Presence / absence of diaminopimelic acid and optical isomer: L L-type diaminopimelic acid was detected.

② menaquinone: MK- 9 (H ₆₎ and MK- 9 (H ₈₎ was detected as a main component. MK-9 (H ₄ ) was also detected in small amounts.

From the above properties, the TA-0358 strain can be classified as an actinomycete belonging to the genus Streptomvces, and this strain was named Streptomvces sp. TA-0358.

The production of TEL0010 is carried out in a manner similar to the production of general fermentation products by culturing Streptomvces sp. TA-0358 strain under aerobic conditions in a medium containing various nutrients. The medium is mainly a liquid medium and consists of a carbon source, a nitrogen source and inorganic salts. If necessary, vitamins, precursors and defoamers can be added, and the pH is adjusted to around 7. Examples of carbon sources include glucose, sucrose, dextrin, Lyserin, starch, etc. may be used alone or as a mixture. As the nitrogen source, for example, meat extract, oatmeal, yeast extract, soybean powder, polypeptone, corn 'Stip', urea, ammonium salt or the like is used alone or in combination. As the inorganic salt, for example, monopotassium phosphate, magnesium sulfate, sodium chloride, carbonated carbonate or the like is used alone or in combination. Adecanol, a silicon compound, or the like can be used as an antifoaming agent.

Aerobic cultivation such as shaking cultivation and aeration and stirring culturing is suitable for the culturing method. Incubate for days.

In order to isolate TEL0010 produced by this culture, a general method for collecting fermentation products may be used. For example, the following method is effective. That is, after completion of the culture, a culture filtrate is obtained by centrifugation or filtration, and Diaion HP-20

(Mitsubishi Kasei Co., Ltd.), etc., and then eluted with an organic solvent such as lower alcohol or acetone. The cells are extracted with an organic solvent such as lower alcohol or acetone. Then, the bacterial cell extract and the eluate from the adsorption resin are combined and concentrated under reduced pressure to remove the organic solvent. This is concentrated to a syrup. This syrup is dissolved again in an organic solvent such as benzene, ethyl acetate, acetone, methanol, and chromatographies. TEL0010 can be purified and isolated by subjecting it to HPLC and high performance liquid chromatography.

The structural formula of TEL0010 obtained by the above-described purification method was determined as shown in Formula (1) by analyzing the molecular weight, ultraviolet absorption spectrum, 'Η-NMR spectrum, ¹³ C-NMR spectrum, and the like.

The physicochemical properties of TEL0010 are shown below. .

(a) Appearance: pale yellow powder

(b) Melting point: 287-291 ° C (decomposition)

(c) Molecular weight: 39 1 (d) Molecular formula: C ₂₂ H ₃₃ N0 ₅

(e) HR E I mass spectrum

Measured value: 39 1.23 8 1

Calculated as Theoretical: 3 9 1.23 59 (C _S N0 ₅ )

(f) E I mass spectrum

/ z 39 1 (M) +

(g) Specific rotation:

[a] _D ²⁵ : + 69.7 ° (c = 0.2, Me OH)

(h) UV absorption spectrum:

λ max n m (ε)

Me OH: 243 (1700), 284 (16000)

Me OH + HC 1: 225 (5900), 286 (1500) MeOH + NaOH: 244 (12600), 283 (1900) (i) Infrared absorption spectrum:

Figure 1 shows the results measured by the KBr method.

(j) — NMR spectrum:

Figure 2 shows the measurement results at 500 MHz in heavy pyridine.

(k) ^13C— NMR spectrum:

FIG. 3 shows the results of measurement at 125 MHz in heavy pyridine.

(1) Solubility in solvents:

Soluble in methanol, ethanol, dimethyl sulfoxide

Black mouth form, poorly soluble in ethyl acetate

Insoluble in water, hexane, benzene, ether

(m) Color reaction:

Positives: I ₂ , H ₂ S〇 ₄ , ammonium sulfate molybdate

Negative: ninhydrin, anthrone sulfate-(n) Basic, acidic, neutral: weakly acidic. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the infrared absorption spectrum of TEL0010 measured by the KBr method.

FIG. 2 shows the 'H-NMR spectrum of TEL0010 measured at 500 MHz in heavy pyridine.

FIG. 3 shows a ¹³ C-NMR spectrum of TEL0010 measured at 125 MHz in heavy pyridine. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to Examples and Test Examples.

A solution containing 2.0% soluble starch, 0.5% glucose, 0.3% NZ case, 0.2% yeast extract, 0.5% fish mill, and 0.2% calcium carbonate (ΡΗ7.0) 100 ml of the body medium was placed in a 500 ml Erlenmeyer flask, and sterilized at 120 at 2 atm for 20 minutes. Next, Streptomvces sp. TA-0358 strain was inoculated into this sterile medium, and the cells were subjected to rotary shaking culture at 28 rpm at 200 rpm for 2 days to obtain seed culture. Next, using a 50-volume Jap-Amen group, glycerol 2.0%, dextrin 2.0%, soybean flour 1.0%, Pharmamedia 0.5%, polypeptone 0.1%, Mg S 〇 ₄ · 7 Eta ₂ 〇 0. 0 5%, C o C l 2 '6 H 2 〇 0.000 5%, carbonate Karushiu arm 0.3% sterile production medium 3 0 consisting of (pH 7. 0) 1 was inoculated with 400 ml of the seed culture solution, and cultured at 28 rpm at 150 rpm for 4 days with aeration and stirring.

After completion of the culture, the obtained culture solution 301 was separated into bacterial cells and supernatant by centrifugation. The cells were extracted with acetone 61 and methanol 61, and the two extracted fractions were combined and concentrated. After the concentrated solution was extracted with 4 L of ethyl acetate to remove the fat-soluble fraction, it was further extracted with 6 L of n-butanol, and the n-butanol fraction was concentrated. The culture supernatant was added with Diaion HP-20 (trade name: manufactured by Mitsubishi Kasei Corporation) 21 and stirred to adsorb the active substance, and then eluted with acetone 61 and then methanol 61. The two eluates were combined, concentrated under reduced pressure, and further combined with a concentrated solution from the cells. The concentrated solution was extracted with ethyl acetate to remove the fat-soluble fraction, and then extracted with n-butanol 41 to concentrate the n-butanol layer. An active substance was precipitated by adding black hole form to the concentrated solution to obtain 3.58 g of a white powder. The obtained powder was dissolved in methanol and subjected to silica gel column chromatography (capacity: 600 ml) using a form-monoethanol methanol system. The active fraction eluted with 20% methanol (2.1 L) and 50% methanol (2.0 L) was collected and concentrated under reduced pressure. This was dissolved in methanol, divided into two portions, and subjected to gel filtration using a column of Sephadex LH-20 (trade name, manufactured by Pharmacia Co., Ltd.) (prepared by Pharmacia) with a gel (volume: 500 ml). The residue was concentrated to dryness under reduced pressure to obtain 1.71 g of TEL0010.

The purity was confirmed by high performance liquid chromatography using the following conditions. Column size 4.6 (f> X 1 50mm

Support 〇DS silica gel (YMC)

Solvent composition 60% acetonitrile, 40% water (PH 3.5)

Flow velocity 1.0 ml / in

Temperature 50

Detection wavelength 2 15 nm

Equipment Waters M-6 00

Retention time 7.2 minutes Test example 1 HL-60 cell growth inhibition test

(Sample)

TEL0010 was dissolved in DMSO so as to have a concentration of 1 OmgZm 1 and diluted with sterile water to a target concentration.

(Test cell)

Human promyelocytic leukemia cells HL—60

(Test method)

The the HL- 6 0 cells cultured with RPM I- 1 640 medium, 1 2-well flat bottom pre - was added to a ^{1 X 1 0 5 cells / m} 1 bets, then the Yo are the object density tone The prepared sample was added. The test cells were cultured at 37 for 48 hours in a 5% carbon dioxide incubator, and then the live cells were measured, and the 50% inhibitory concentration (IC _so value) was calculated from the sample concentration and the inhibition rate. Adriamycin was used as a control. The results are shown in Table 2. Test example 2 KB cell growth inhibition test

(Sample)

TELOOIO was dissolved in DMSO so as to have a concentration of 1 OmgZm 1 and diluted with sterile water to the desired concentration.

(Test cell)

Human nasopharyngeal carcinoma cells KB

(Test method)

A cell suspension of KB cells (10 ³ cells / 100 ul / well) was added to a flat-bottomed 96-well microplate, and cultured for 24 hours. After adding a sample of a desired concentration, the cells were further cultured for 72 hours. After completion of the culture, MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] reagent was added and reacted for 4 hours. After completion of the reaction, the absorbance was measured to obtain the ratio of the absorbance of the sample processing unit to the absorbance of the controls opening one Le was determined 50% inhibitory concentration (IC _5. Value). Adriamycin was used as a control. The results are shown in Table 2. Table 2

Industrial moments available

As shown in Table 2, the compound of the present invention has a growth inhibitory effect on HL-60 cells and KB cells, and is therefore useful as an antitumor agent. -To this end, the compounds of the present invention are added with conventional bulking agents, binders, disintegrants, pH regulators, solubilizers, etc., and tablets, pills, capsules, granules are produced by conventional formulation techniques , Powders, solutions, emulsions, suspensions, injections and the like. The compound of the present invention can be administered to an adult patient orally or parenterally in 0.1 to 50 OmgZ days once or several times a day. This dose can be appropriately adjusted according to the type of disease, age, weight, and symptoms of the patient.

Claims

The scope of the claims

formula

Tetramic acid compound represented by <

2. An antitumor agent comprising the tetramic acid compound according to claim 1 as an active ingredient <