JPH01233275A - Bioactive substance - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula (R1 and R2: one is H and another is OH; named as bioactive substance A and B). USE:A medicine having growth inhibiting action against cancer culturing cell and some Gram-positive bacteria. PREPARATION:On the whole applying correspondingly to the case of producing general fermentation products, Pseudeurotium sp. F-1771 (FERM P-9798) is cultivated under an aerobic condition, in a culture medium containing various nutriments. Next, the compound expressed by the formula produced in a culture solution is isolated applying correspondingly general method for isolating fermentation products to afford the compound expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to novel physiologically active substances 1771A and B.

[Background Art] As a compound structurally similar to general formula (1) of the present invention, 0valicin [He1vetika Chem
ica Acta.

Volume 51, page 1395 (1968) is known. There are also reports on its biological activity [Journa
l ofMedical Plant Re5erch
5erch p. 2131, (1978)].

[Disclosure of the Invention] As a result of repeated exploratory research in order to obtain a new substance with antitumor activity, the present inventors discovered that a specific microorganism inhibits the proliferation of cultured cancer cells. The present inventors have discovered that a novel physiologically active substance having the following properties can be produced, and have completed the present invention.

We have identified this new active substance as '1771A, (-
At maximum (I) R'=H, R"=OH) J and'
1771B (-R'=OH, R"= at maximum (1)
H) Named J.

The strain producing the novel physiologically active substances 1771A and 1771B of the present invention is a strain newly isolated by the present inventors from soil collected in Yoshimi-cho, Hiki-gun, Saitama Prefecture, and the name of the microorganism is 'P
seudeurotium sp, F-1771J and Microorganism Deposit No.
RM P-9798) J, which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

The mycological properties of this strain are shown below.

■ Morphology This strain grows well on wort agar, potato/glucose agar, oatmeal agar, YpSS agar, etc., and forms conidia well. When a colony grown on an oatmeal agar medium is observed under a microscope, the hyphae are transparent, have septa, and are highly branched.
The diameter is 1.5 to 2.5μ, and the shape is straight or cylindrical with a slight bend at the top, and gradually tapers toward the tip. The conidia are symbosio-shaped, and a single conidium initially attaches to the tip of the conidiophore, and as time passes in culture, new growth points are extended and formed. Conidia are oval and the size is 4.5-6.0μ x 3.5
~5μ, and its surface exhibits a smooth surface.

(2) Properties on media The following Table 1 shows the results of macroscopic observation when cultured on various media at 28°C for 14 days.

■ Physiological and ecological properties 1) Optimal growth conditions The optimal growth conditions for this strain are YpSs medium at pH 5.
, 0-7.6, and temperature 20-35°C.

2) Growth range The growth range of this strain is in YpSs medium between pH 3.3 and
9.8, temperature 16-38°C.

3) Distinction between aerobic and anaerobic; from the morphological characteristics and culture characteristics that are more than aerobic, this strain is
It was suggested that it may belong to the genus Pseudeurotium. Based on the above-mentioned properties, we have compiled the following books, including Yotoshi Uda-1 Keisuke Tsubaki-1 Illustrated Encyclopedia of Fungi (1978) and C. Booth's '5
studies of Pyrenomycetes N
Th1elavia.

with notes on some allied
genera (C, M, I Mycol, paper
s, 83.11 (1961)) J and A, C, 5
'Thegenera Anixiops' by talk
is hansen and pseudoeurot
Antonie van
Leeuwenhoek, Vol, 21.65 (195
5) As a result of a comparative study with many known strains reported in J.A., the conidiophores are straight, or the upper part is slightly bent and the symbosio-type conidia are attached to the tapered tip. Based on the characteristics, this strain is Pseudeurot.
It was revealed that the strain belonged to the genus Mium. However, ascus formation, which is considered to be the most important factor in determining the species, was not observed in this strain during the culture period, and the species could not be determined.
It was named tium sp, F-1771.

Next, the production of novel physiologically active substances 1771A and 1771B by this strain is carried out roughly in the same manner as in the production of general fermentation products.

That is, Pseude is grown in a medium containing various nutritional substances.
urotium sp. F-1771 is cultured under aerobic conditions.

A liquid medium is mainly used as the medium, and glucose, molasses, starch, etc. can be used alone or in combination as secondary sources. As the nitrogen source, polypeptone, soybean flour, yeast extract, etc. can be used alone or in combination. In addition, 177 physiologically active substances help the growth of bacterial strains.
Organic substances and inorganic salts that promote the production of 1A and B can be added as necessary. As the antifoaming agent, adecanol, silicone, etc. can be used.

For the culture method, aerobic culture such as shaking culture or aerated agitation culture is suitable, and the pH is 3-7.25-37°C for 2-5 days, preferably pH 6-7.28-30°C for 3-4 days. Incubate for days.

The physiologically active substances 1771A and 1771B produced in this culture solution can be isolated according to a general method for collecting fermentation products. That is, after the completion of culture, the culture filtrate separated by centrifugation or filtration is treated with Diaion H.
Lower alcohols are adsorbed onto P-20 (product name: manufactured by Mitsubishi Kasei), etc.

Elute with acetone etc. Eluted physiologically active substance 17
After concentrating the fractions 71A and B, they are transferred to a nonaqueous solvent such as benzene, ethyl acetate, or chloroform, and concentrated to form a syrup. This syrup was dissolved again in an organic solvent such as benzene, acetic acid ethyl ester, or chloroform, and then subjected to column chromatography using silica gel, Sephadex LH-20 (trade name; manufactured by Pharmacia).
subjected to gel filtration and reversed phase chromatography using
By collecting active ingredients, physiologically active substance 1771A
and B can be purified and isolated.

Physiologically active substances 'Jj1771A and B of the present invention exhibit a significant growth-inhibitory effect on cultured cancer cells and also exhibit a growth-inhibitory effect on certain types of Durham-positive bacteria, and are therefore useful as medicines [Example ] Hereinafter, the present invention will be specifically explained with reference to Examples and Test Examples.

Example 1 (1) Pseudeuroti was added to a sterile liquid medium containing 4 g of glucose and 1 g of polypeptone per 100 t+tQ.
Inoculated with um sp, F-1771 strain, and incubated at 30°C19
After 6 hours of shaking culture, sterile medium 3 with the same composition as the seed culture was prepared using 50P of di-Fγ-mentor.
0P was inoculated with 0.61 of the above seed culture solution, and 30″C196
Incubate with aeration and agitation for an hour.

(After the completion of culture, the culture solution 852 for 3 groups was filtered, the obtained filtrate 801 was adsorbed on 5ffi of Diaion HP-20 (product name; manufactured by Mitsubishi Kasei), and 80% acetone solution 1
After elution with 01, the eluate was concentrated to 1.52. This concentrate was extracted twice with equal volumes of acetic acid ethyl ester. The acetic acid ethyl ester layer was dehydrated with anhydrous sodium sulfate and then concentrated to obtain 2.5 g of a brown syrup-like substance.

■) Dissolve 2.5 g of syrup-like substance in 5 mQ of chloroform and fill with 200 ml of silica gel (Kiesel Gel 60. trade name, manufactured by Merck & Co., Ltd.) prepared with chloroform.
Adsorb onto a column of chloroform 300+T11!
, chloroform-methanol (99,8: 0.2)
Eluted with 300ynll of mixed solvent of
Collect both parts containing 71A and obtain 0.3 of the brown syrupy substance.
I got g.

(4) Dissolve 0.3 g of the brown syrupy substance from the previous section in 5 ml of methanol, and gel it with methanol using a 500 mQ column packed with Sephadex LH-20 (trade name, manufactured by Pharmacia) prepared with methanol. Filtration was performed. The active fractions were collected and concentrated to give a brown powder 1
I got 20+v.

(5) 120 ml of the brown powder obtained in the previous section was dissolved in 5 ml of 70% methanol. This solution was subjected to reverse phase high performance liquid chromatography (Deverosil, diameter 10 I x length 25
cm, manufactured by Senshu Kagaku Co., Ltd.) at a column temperature of 50
Elution was repeated with 70% methanol at a flow rate of 4 mL/min at a temperature of 4 mL, and both fractions with a retention time of around 5.7 minutes were collected. Both fractions were concentrated to dryness to obtain 25 quarts of white powder. 25 ml of this white powder was dissolved in 5 ml of methanol, and gel filtration was performed using a Sephadex LH-20 (trade name, manufactured by Pharmacia) column. The obtained active fractions were collected and concentrated to dryness to obtain 21■ as a white powder. This powder 21cm was dissolved in a mixed solvent of n-hexane-acetone (10:1) at 50 DEG C. and allowed to stand at room temperature to obtain 131Tg of the physiologically active substance 1771A in the form of colorless plate-like crystals.

When the melting point of this colorless plate-like crystal was measured, it was found to be 126-1
The temperature was 28°C.

The physicochemical properties of the physiologically active substance 1771A are shown below.

(1) Appearance: colorless plate-like crystals (2) Mass spectrometry value m/z 266 (M”) (3) Elemental analysis value; C1, H! as ff1O4 Calculated values: C; 67, 67X, H; 8.27%.

Actual value: C; 67.05X, l (; 9.12%.

(4) [α Ko: -108,2”(c-0,1
(methanol solution) (5) UV absorption spectrum: Measurement with ethanol solution shows terminal absorption.

(6) IR absorption spectrum: The spectrum measured in chloroform is shown in FIG.

(7)'H-NMR spectrum: The spectrum measured at 400 MH2L in deuterated chloroform (CDC!) is shown in FIG.

(8) "C-NMR spectrum: The spectrum measured at 100 MH! in deuterated chloroform (CDCI!s) is shown in FIG.

Example 2 (1) Pseudeurotium was added to a sterile liquid medium containing 4 g of glucose and 1 g of polypeptone per 100 mQ.
sp.

F-1771 strain was inoculated and cultured with shaking at 30°C for 96 hours. Next, using a jar fermenter with a content of 4250j2, 0.62% of the seed culture solution was inoculated into a sterile medium 302 having the same composition as the seed culture, and cultured at 30''C with aeration for 96 hours.

(After completing the culture, filter the culture solution 851! for the three groups, and use the obtained filtrate 802 as Diaion HP-20 (product name;
(manufactured by Mitsubishi Kasei) 51, 80% acetone solution 10
After elution with j2, the eluate was concentrated to 1.52. This concentrate was extracted twice with equal volumes of acetic acid ethyl ester. The acetic acid ethyl ester layer was dehydrated with anhydrous sodium sulfate and then concentrated to obtain 2.5 g of a brown syrup-like substance.

a]) 2.5 g of syrup-like substance was dissolved in 5 ml of chloroform, and 200 mQ filled with silica gel (Keymol Gel 60. trade name, manufactured by Merck & Co., Ltd.) prepared with chloroform was prepared.
Allow 300ml of chloroform to be adsorbed onto the column. Mixed solvent 3 of chloroform-methanol (99,8:0.2)
It was eluted at 00 mQ, and both fractions containing the physiologically active substance 1771B were collected to obtain 0.4 g of a yellow, white-coloured substance.

(Dissolve 0.4 g of the yellow syrup-like substance in the front section in 20 ml of methanol, and gel filtrate with methanol using a 500 mQ column packed with Sephadex LH-20 (trade name, manufactured by Pharmacia) prepared with methanol. The active fractions were collected and concentrated to give 200 ITg of a yellow oil.

(5') Dissolve the yellow oily substance obtained in the previous section in 10 ml of 70% methanol, and perform reverse phase high performance liquid chromatography (Deverosil, diameter iom) prepared with a solvent of the same composition.
m x length 25 cm, manufactured by Senshu Kagakusha),
Column temperature: 50°C2 flow rate: 4ml/Zmin.

Repeated elution with 70% methanol, retention time 6.2
I collected both minutes before and after. Both fractions were concentrated to dryness to obtain 1 and 70 square meters of a white oily substance. This oily substance was dissolved in 2 ml of methanol, and Cefadeclas LH-20 (trade name,
Gel filtration was performed using a column (manufactured by Pharmacia). The obtained active fractions were collected and concentrated to dryness to obtain 62+yg of the physiologically active substance 1771B as a colorless and transparent oily substance.

The physicochemical properties of the physiologically active substance 1771B are shown below.

(1) Appearance: Colorless oil (2) Mass spectrometry value: FABMS m/z 283 (M+1)'' (3) High-resolution mass spectrometry value: Calculated value as C+sHi*0*; 282.147 Actual value, 282.151 ( 4) [α], knee 25.3° (c-0-1+methanol solution) (5) UV absorption spectrum: Measurement with ethanol solution shows terminal absorption.

(6) IR absorption spectrum: The spectrum measured in chloroform is shown in FIG.

(7)'H-NMR spectrum: 400MH in deuterated chloroform (CDCIm)! Figure 5 shows the spectrum measured.

(8) "C-NMR spectrum: The spectrum measured at 100 MH in deuterated chloroform (CDCIm) is shown in FIG.

Test Example 1 (Proliferation inhibitory effect of physiologically active substance 1771A on various cancer cultured cells) (Sample) Physiologically active substance 1771A5■ obtained in Example 1 was dissolved in methanol and added to sterile physiological saline to the desired concentration. and diluted.

(Test cells) ■HL-60 Human leukemia ■P388 Mouse leukemia (Culture solution used) RPMI-1640 medium (Test method) Using the above culture solution, each cancer cell was grown at 2X10' to 1X10
'/+nQ, 2 ml each was dispensed into a 6-hole shear dish with a diameter of 35 m. Next, 50 μ of the sample pre-diluted to the desired concentration was added at the same time as the start of culture, and cultured in a 5% hypooxylic acid gas incubator for 3 to 4 days. After continuing, living cells were measured, and the IC value (concentration for 50% inhibition) was determined from the sample concentration and inhibition rate.

(result) The results are shown in Table 2.

Table 2 Test Example 2 (Proliferation inhibitory effect of physiologically active substance 1771A on various fungi) (Sample) Physiologically active substance 1771A1+m obtained in Example 1 was dissolved in methanol and diluted with a medium to reach the target concentration.

(Medium) Heart infusion agar medium was used as the bacterial medium, and Sabouraud agar medium was used as the fungal medium.

(Test method) The antibacterial activity of Inoculum size 10"CF U/mQ against various bacteria was investigated using the paper desk method.

(result) The results are shown in Table 3.

Table 3 Test Example 3 (Proliferation inhibitory effect of physiologically active substance 1771B on various cancer cultured cells) (Sample) 10 mg of physiologically active substance 1771B obtained in Example 2 was dissolved in methanol, and dissolved in sterile physiological saline to reach the target concentration. It was diluted with.

(Test cells) ■HL-60 Human leukemia ■P388 Mouse leukemia (Culture solution used) RPMI-1640 medium (Test method) Using the above culture solution, grow various cancer cells at 2X10' to 1X1
0'/mu, 2tt in a 6-hole shear tray with a diameter of 35mm.
It was dispensed in tll portions. Next, 5oPi of the sample diluted in advance to the desired concentration was added at the same time as the start of culture, and
After continuing to culture in a 5% hypooxylic acid gas incubator for days, living cells were measured, and the IC5s value (concentration for 50% inhibition) was determined from the sample concentration and inhibition rate.

(Results) The results are shown in Table 4 Test Example 4 (Proliferation inhibitory effect of physiologically active substance 1771B on various fungi) (Sample) 1 mg of physiologically active substance 1771B obtained in Example 2 was dissolved in methanol, and the target concentration was adjusted. It was diluted with medium so that

(Medium) Heart infusion agar medium was used as the bacterial medium, and Sabouraud agar medium was used as the fungal medium.

(Test method) The antibacterial activity of Inoculum size 10'CFU/mu against various bacteria was investigated using the paper desk method.

(result) The results are shown in Table 5.

Table 5

[Brief explanation of the drawing]

Figure 1 shows physiologically active substance 1 measured in chloroform.
The IR spectrum of 771A is shown. Figure 2 shows the 'H-NMR spectrum of physiologically active substance 1771A measured at 400 MHz in deuterated chlorophor A (CDCJs). Figure 3 shows 100%
“C-NM” of physiologically active substance 1771A measured with MH2
The R spectrum is shown. Figure 4 shows physiologically active substance 1 measured in chloroform.
The IR spectrum of 771B is shown. Figure 5 shows the 'H-NMR spectrum of physiologically active substance 1771B measured at 400 MHz in deuterated chloroform (CDG!).
“C-NM” of physiologically active substance 1771B measured at Hz
The R spectrum is shown.

Claims (1)

[Claims] 1) General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) (In the formula, R_1 and R_2 represent a hydrogen atom or a hydroxyl group. However, when R_1 is a hydrogen atom, R_2 is a hydroxyl group. (When R_1 is a hydroxyl group, R_2 is a hydrogen atom.)
A compound represented by