JPH06340670A - Novel physiologically active substance and the production process therefor - Google Patents

Abstract

PURPOSE:To obtain a new physiologically active substance which is obtained from a culture mixture of a specific microorganism blonging to the genus Verticilium and useful as an antitumor agent with reduced side-effect, specifically inhibiting the proliferation of human-originating cancer cells without inhibiting proliferation of non-tumorigenic cells. CONSTITUTION:A microorganism belonging to the genus Verticillium, producing a novel physiologically active substance of formula I [R<1> is H, OH; R<2> is H, formula: CH2CH=C(CH3)2; R<1>, R<2> combine to together form group of formula II or III; R<3> is H, OH; R<4>-R<6> are H, group of formula IV], for example, Verticillium balanoides MCI 2825 strain, is inoculated to a culture medium and cultured under shaking at 27 deg.C for 11 days. Acetone is added to the culture mixture to effect extraction and the extract is filtered with Celite, then the acetone is distilled off under reduced pressure, ethyl acetate is added to the residue to effect extraction and the extract is concentrated under reduced pressure. The oily residue is subjected to reverse-phase partition chromatography to effect purification, whereby the objective new physiologically active substance such as antitumor agent represented by formula I is obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel physiologically active substance and a method for producing the same, and more particularly to a novel physiologically active substance having a cancer cell growth inhibitory action and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION In the field of chemotherapy for cancer, many chemical substances have been put to practical use as pharmaceuticals, but in many cases, not only are the drug effects insufficient, but The problem of tumor cell resistance to drugs also complicates clinical use [see 47th Annual Meeting of the Japanese Cancer Society, pages 12 to 25 (1988)]. Under such circumstances, the development of new antitumor substances is always required in the field of cancer treatment.

In addition, since the mechanism of action of conventional antitumor substances is based on the inhibitory action on the basic mechanism of cell division and proliferation, the action is not limited to cancer cells but to normal cells. Also gives a non-specific cytotoxic effect,
As a result, side effects during drug administration have become a major clinical problem, which is not always a satisfactory situation. Therefore, the appearance of antitumor agents that act specifically on cancer cells and have no side effects is desired.

The present invention, which is always required,
The present invention aims to solve these problems by providing a novel antitumor compound that can be a more effective drug than a known antitumor substance and establishing a method for producing the same.

[0005]

[Means for Solving the Problems] The present inventors focused on the fact that microorganisms produce various physiologically active substances such as antibiotics, collected a large number of samples from nature, and separated a large number of them from various species. As a result of repeated studies on microbial cultures, Verticillium
It was found that a substance having an antitumor property was produced in a culture of a microorganism belonging to the genus. Isolate this material,
The structure was clarified, and it was found that this substance has a good cancer cell growth inhibitory action and shows almost no suppressive effect on the growth of cells having no tumorigenicity, and the present invention was completed. That is, the gist of the present invention is the following general formula (I)

[0006]

[Chemical 2]

The novel bioactive substance represented by and a method for producing the same exist. The present invention will be described in detail below. The compounds of the present invention include the following.

[0008]

[Chemical 3]

[0009]

[Chemical 4]

[0010]

[Chemical 5]

The compound of the present invention can be obtained, for example, by culturing a compound-producing bacterium of the present invention belonging to the genus Verticillium and isolating it from the culture. Such a compound-producing bacterium of the present invention may be any microorganism as long as it is a microorganism belonging to the genus Verticillium and is capable of producing the compound of the present invention in an amount sufficient to be collected in the culture solution. Good. An example of such a strain is Verticillium rosenous ( Ve) belonging to an incomplete bacterium newly isolated from litter by the present inventors.
rticillium balanoides ) MCI
2825 strain (hereinafter referred to as “this strain” or “MCI28
25). MCI2825
The microbiological properties of No. bacterium are as follows.

1. Morphological characteristics a) On potato-dextrose agar medium (PDA), 2
Properties at 0 ° C. Colonies are 3 to 4 cm in diameter in 3 weeks and grow slowly. The color tone of the colony is a yellowish orange color. Occasionally fan-shaped sectors are formed in part of the colony. The sector has a white color. The back has a light brown color. Basal hyphae are colorless, have septa, and branch. Width 2.5-3.4 μm. Aerobic hyphae are well developed. Conidia stalks are more upright or irregular than basal hyphae or aerial hyphae, colorless, have septa,
Two to four phialides form a ring for each verse. The phialide is slightly swollen at the base, the tip is tapered, the length is 6-28 μm, the width is 2-2.8 μm at the base, and 1 at the tip.
-1.5 μm, conidia exhibit fiaro-type conidia, acorn-shaped balanoid, have an adhesive substance on the head, size: 2.5-3.7 μm × 1.5-2.3 μm, colorless, Agglomerates and agglomerates at the tip of the fear ride.

(B) Potato carrot agar medium (PC
A) Properties at 20 ° C on top Colonies reach a diameter of 4 cm in 3 weeks. Growth retardation. The color of the colony is light yellowish brown. The back side is a pale yellowish brown ~
The other morphological properties, which show a glazed yellow-orange color, show the same characteristics as those on the potato dextrose agar medium.

[0014]

[Table 1] 2. Physiological properties Growth temperature (cultivated on PDA for 2 weeks): 15 to 30 ° C Optimum temperature: 27 ° C Growth pH (cultured on LCA liquid medium at 27 ° C for 2 weeks):
3-9 Optimum pH: 6-8

3. Taxonomic device The present strain (MCI 2825) shows 1) a fiaro-type conidial formation mode, 2) a phialide is from a conidia peduncle 2-
Deuterium (Deute)
romycotina) -Incomplete filamentous fungal mesh (Hypho)
mycestes) to the genus Verticillium.
W. Gams & A. Van Zaayen (198
2), Neth. J. Pl. Path. 88: 57-7
8, and W. Gams (1988) Neth. J. P
l. Path. According to the monograph of the genus Verticillium of 94: 123-148, 18 species are recognized in this genus. These species groups are nematophagous species that are parasitic on nematodes.
s) and saprophytes.

The present strain (MCI 2825) was searched according to the above-mentioned reference such as Gams et al., And it was found that this strain had acorn-shaped conidia and contained an adhesive substance in the head of the conidia. Therefore, it was in agreement with the morphological characteristics of Verticillium rose noise. V. bala
Since noides are known as nematode parasites, this strain (MCI 2825) is also a soil-active Caenorhabditis elegans.
I tried an infection experiment. However, the infectivity was not confirmed. Presence or absence of nematode infectivity has a problem of host specificity, and it is difficult to consider it as a main trait for species identification. Therefore, due to the morphological characteristics of this strain, Verticillium rose noise MC
It was identified as No. I2825.

[0017] MCI 2825 is a product of the Institute of Biotechnology, Institute of Biotechnology, Institute of Biotechnology, No. 13554 (FER).
It has been deposited as MP-13554). In general, the bacterium of the genus Verticillium tends to change its properties as seen in other fungi. For example, even if the strain is MCI2825 or a mutant strain (spontaneous or inducible) derived from this strain, a zygote or a genetic recombinant, any one capable of producing the compound of the present invention can be used in the method of the present invention. Can be used for

In the present invention, the above-mentioned bacterium is cultivated in a medium containing nutrients that can be utilized by ordinary microorganisms. As the nutrient source, for example, glucose, starch syrup, dextrin, sucrose, starch, molasses, animal / vegetable oil, etc. can be used. As the nitrogen source, for example, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate,
Urea or the like can be used. In addition, if necessary, sodium, potassium, calcium, magnesium, cobalt,
It is effective to add inorganic salts capable of producing chlorine, phosphoric acid, sulfuric acid and other ions. In addition, organic and inorganic substances that help the growth of bacteria and promote the production of the compound of the present invention can be appropriately added.

As a culture method, a culture method under aerobic conditions is suitable. A suitable temperature for culturing is 20 to 30 ° C, but in most cases, the culturing is performed at around 26 to 30 ° C. The production of the compound of the present invention varies depending on the medium and culture conditions, but the accumulation generally reaches an optimum within 3 to 20 days. When the amount of the compound of the present invention accumulated in the culture becomes maximum, the culture is stopped and the target substance is isolated from the culture medium. Among the compounds of the present invention, the above-mentioned compounds 1 to 5 are fat-soluble substances,
In isolating and purifying compounds 1 to 5 from the culture,
It can be performed by utilizing the characteristic. That is, for example, a solvent extraction method using ethyl acetate, chloroform, etc .; silica gel, alumina, ODS, DIAION HP-20.
Column chromatography or high performance liquid chromatography using synthetic adsorbents such as (manufactured by Mitsubishi Kasei) or gel filtration agents such as Sephadex LH-20 (manufactured by Pharmacia); preparative thin layers using silica gel as a carrier. Chromatography and the like are effective.

The present inventors isolated and purified Compounds 1 to 15 by using the means and methods as described above, and examined their physicochemical properties. As a result of another study, Compounds 1 to 15 had the above structures. It was clarified that the compound is a compound represented by the formula, and that this compound is a novel compound. Compound 16 can be obtained by the same method as described above, but desirably,
It is obtained by decomposing compound 1 or compound 2. Decomposition of compound 1 or compound 2 is usually carried out by reacting in a suitable solvent containing water under acidic conditions. As the acid used here, commonly used hydrochloric acid, acetic acid, sulfuric acid, malonic acid, tartaric acid, oxalic acid or the like can be used. Compounds 1 to 16 can be produced in this manner, and the physical properties of these compounds are as follows.

A) Compound 1 1) Appearance: white crystals 2) Melting point: 185-187 ° C. 3) EI mass spectrum: m / e 535 (M ⁺ ), 5
20,182 4) High resolution mass spectrum: (C ₃₂ H ₄₁ NO ₆ ) calculated value: 535.2932 actual measurement value: 535.2939 5) ultraviolet absorption spectrum: methanol solution λ _MAX : 230 nm (ε = 3.5 ×) 10 ⁴ ) 281 nm (ε = 7.9 × 10 ³ ) 6) Infrared absorption spectrum: KBr method ν _MAX : 3424, 2980, 2934, 1454, 1
375, 1304, 1090, 1049, 1009, 9
24,743 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: deuterated acetone solution δ _ppm : 1.15 (3H, s); 1.22 (3H,
s); 1.23 (3H, s); 1.24 (3H, s);
1.26 (3H, s); 1.31 (3H, s); 2.7
1 (1H, d, J = 6.5 Hz); 3.52 (1H,
s); 3.55 (1H, d, J = 9.5 Hz); 3.6
0 (1H, s); 3.99 (1H, d, J = 9.5H
z); 4.34 (1H, dd, J = 9.0, 9.0H)
z); 4.67 (1H, d, J = 6.5Hz); 6.9.
3 (2H, m); 7.28 (1H, m); 7.32 (1
H, m); 9.91 (1H, brs)

8) Carbon nuclear nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 153.01 (s); 140.96 (s); 1
25.75 (s); 120.09 (d); 119.22
(D); 118.55 (d); 116.59 (s); 1
12.15 (d); 95.94 (d); 77.97.
(S); 75.16 (s); 71.98 (d); 71.
77 (d); 71.40 (d); 67.82 (s); 6
3.04 (d); 60.24 (d); 57.09
(S); 51.21 (s); 50.58 (d); 43.
00 (s); 28.20 (q); 27.41 (t); 2
6.72 (t); 24.22 (q); 21.11.
(T); 18.99 (q); 18.44 (q); 16.
54 (q); 16.17 (q) 9) Specific rotation: acetone solution

[0024]

[Chemical 6]

B) Compound 2 1) Appearance: white crystal 2) Melting point: 157-159 ° C. 3) EI mass spectrum: m / e 519 (M ⁺ ), 5
04,182 4) High resolution mass spectrum: (C ₃₂ H ₄₁ NO ₅ ) calculated value: 519.2983 measured value: 519.2965 5) ultraviolet absorption spectrum: methanol solution λ _MAX : 230 nm (ε = 3.3 ×) 10 ⁴ ) 282 nm (ε = 7.5 × 10 ³ ) 6) Infrared absorption spectrum: KBr method ν _MAX : 3422, 2934, 1453, 1375, 1
304, 1119, 1098, 980, 924, 741
cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: deuterated acetone solution δ _ppm : 1.14 (3H, s); 1.18 (3H,
s); 1.27 (3H, s); 1.30 (3H, s);
1.68 (6H, s); 3.44 (1H, s); 3.5
1 (1H, d, J = 9.0 Hz); 3.53 (1H,
s); 4.02 (1H, d, J = 9.5 Hz); 4.3
3 (1H, dd, J = 9.0, 9.0Hz); 5.18
(1H, m); 5.58 (1H, d, J = 6.5H
z); 6.95 (2H, m); 7.30 (1H, m);
7.33 (1H, m); 9.90 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 152.86 (s); 140.77 (s); 1
37.63 (s); 125.56 (s); 123.38.
(D); 119.89 (d); 119.03 (d); 1
18.15 (d); 116.36 (s); 111.98.
(D); 92.80 (d); 77.77 (s); 74.
43 (s); 71.75 (d); 71.69 (d); 7
1.23 (d); 67.60 (s); 60.35
(D); 51.01 (s); 50.40 (d); 42.
78 (s); 28.25 (q); 27.24 (t); 2
6.54 (t); 24.95 (q); 20.93
(T); 18.27 (q); 18.04 (q); 16.
51 (q); 15.99 (q) 9) Specific rotation: acetone solution

[0028]

[Chemical 7]

C) Compound 3 1) Appearance: white crystals 2) Melting point: 280-290 ° C. (decomposition) 3) EI mass spectrum: m / e 521 (M ⁺ ), 5
06,182 4) High resolution mass spectrum: (C ₃₂ H ₄₃ NO ₅ ) Calculated value: 521.3139 Measured value: 521.3133 5) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 230 nm (ε = 3.6 ×) 10 ⁴ ) 282 nm (ε = 8.3 × 10 ³ ) 6) Infrared absorption spectrum: KBr method ν _MAX : 3407, 2973, 2938, 1451, 1
366, 1302, 1256, 1111, 1044, 9
26,750 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: deuterated chloroform solution δ _ppm : 1.10 (3H, s); 1.21 (3H,
s); 1.26 (3H, s); 1.27 (3H, s);
1.64 (3H, s); 1.69 (3H, s); 3.4
1 (1H, d, J = 9.0 Hz); 3.59 (1H,
s); 3.94 (2H, d, J = 6.5Hz); 3.9
7 (1H, d, J = 9.0 Hz); 4.18 (1H, d
d, J = 9.0, 9.0 Hz); 4.61 (1H,
s); 5.24 (1H, t, J = 6.5Hz); 7.0
6 (2H, m); 7.28 (1H, m); 7.42 (1
H, m); 7.72 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: deuterated chloroform solution δ _ppm : 151.88 (s); 139.69 (s); 1
37.22 (s); 125.18 (s); 120.65
(D); 120.45 (d); 119.64 (d); 1
18.49 (d); 117.65 (s); 111.43.
(D); 79.06 (s); 77.89 (s); 74.
74 (d); 71.41 (d); 68.83 (s); 6
7.22 (d); 64.33 (d); 58.05
(T); 50.69 (s); 50.09 (d); 42.
30 (s); 30.36 (t); 27.77 (t); 2
7.42 (t); 27.20 (t); 25.68
(Q); 23.54 (q); 20.62 (t); 19.
17 (q); 18.78 (q); 17.94 (q); 1
6.00 (q) 9) Specific rotation: chloroform solution

[0032]

[Chemical 8]

D) Compound 4 1) Appearance: white crystals 2) Melting point: 184-187 ° C. 3) EI mass spectrum: m / e 619 (M ⁺ ), 6
04,548,2664) High resolution mass spectrum: C
₃₇ H ₄₉ NO ₇ Calculated value: 619.3507 Measured value: 619.3351 1 5) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 234 nm (ε = 3.8 × 10 ⁴ ) 283 nm (ε = 7.3 × 10 ³⁾ ) 6) Infrared absorption spectrum: KBr method ν _MAX : 3434, 2980, 2934, 1456, 1
379, 1117, 1090, 1049, 1009, 9
24,831,804 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 1.16 (3H, s); 1.20-1.26
(15H, m); 1.31 (3H, s); 1.36 (3
H, s); 2.71 (1H, d, J = 6.5 Hz);
3.45 (1H, s); 3.55 (1H, d, J = 9.
5Hz); 3.60 (1H, s); 3.99 (1H,
d, J = 9.5 Hz); 4.35 (1H, dd, J =
9.0, 9.0 Hz); 4.67 (1H, d, J = 6.
5Hz); 6.88 (1H, d, J = 8.0Hz);
7.20 (1H, s); 7.27 (1H, d, J = 8.
0Hz); 9.77 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 153.09 (s); 141.63 (s); 1
30.47 (s); 124.66 (s); 120.75
(D); 118.62 (d); 116.73 (s); 1
12.42 (d); 96.21 (d); 78.23.
(S); 75.39 (s); 72.21 (d); 72.
03 (d); 71.65 (d); 68.04 (s); 6
5.26 (d); 63.29 (d); 60.50
(D); 58.24 (s); 57.29 (s); 51.
47 (s); 50.81 (d); 43.26 (s); 3
6.19 (t); 28.46 (q); 27.71
(T); 27.02 (t); 24.96 (q); 24.
48 (q); 21.36 (t); 19.26 (q); 1
9.12 (q); 18.71 (q); 16.79
(Q); 16.40 (q) 9) Specific rotation: acetone solution

[0036]

[Chemical 9]

E) Compound 5 1) Appearance: white crystal 2) Melting point: 170-172 ° C. 3) EI mass spectrum: m / e 603 (M ⁺ ), 5
88,250 4) High resolution mass spectrum: C ₃₇ H ₄₉ NO ₆ calculated value: 603.3557 actual value: 603.3549 5) ultraviolet absorption spectrum: methanol solution λ _MAX : 233 nm (ε = 3.4 × 10 ^4). ) 286 nm (ε = 8.6 × 10 ³ ) 6) Infrared absorption spectrum: KBr method ν _MAX : 3422, 2980, 2934, 1454, 1
375, 1117, 1090, 1049, 1009, 9
24,831,802 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 1.15 (3H, s); 1.24 (3H,
s); 1.25 (3H, s); 1.25 (3H, s);
1.28 (3H, s); 1.32 (3H, s); 1.7
2 (3H, s); 1.74 (3H, s); 2.73 (1
H, d, J = 6.5 Hz); 3.37 (2H, d, J =
7.5 Hz); 3.57 (1H, d, J = 9.5H)
z); 3.60 (1H, s); 4.00 (1H, d, J
= 9.5 Hz); 4.36 (1H, dd, J = 9.0,
9.0 Hz); 4.69 (1H, d, J = 6.5H)
z); 5.36 (1H, m); 6.82 (1H, d, J
= 8.0 Hz); 7.14 (1 H, s); 7.19 (1
H, d, J = 8.0 Hz); 9.78 (1H, brs)

8) Carbon nuclear nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 153.26 (s); 139.58 (s); 1
32.43 (s); 130.89 (s); 126.05
(S); 121.07 (d); 117.69 (d); 1
16.34 (s); 111.96 (d); 96.02.
(D); 78.02 (s); 75.22 (s); 72.
05 (d); 71.83 (d); 71.47 (d); 6
7.87 (s); 63.11 (d); 60.30
(D); 57.14 (s); 51.22 (s); 50.
57 (d); 43.04 (s); 34.87 (t); 2
8.27 (q); 27.50 (t); 26.77
(T); 25.57 (q); 24.30 (q); 21.
18 (t); 19.07 (q); 18.47 (q); 1
7.51 (q); 16.60 (q); 16.22 (q) 9) Specific rotation: acetone solution

[0040]

[Chemical 10]

F) Compound 6 1) Appearance: white crystals 2) Melting point: 138-142 ° C. 3) EI mass spectrum: m / e 603 (M ⁺ ), 5
88,521,506,250 4) High resolution mass spectrum: (C ₃₇ H ₄₉ NO ₆ ) Calculated value: 603.3558 Measured value: 603.35325) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 232, 284nm 6 ) Infrared absorption spectrum: KBr method ν _MAX : 3424, 2934, 1456, 1385, 1
309,1124,1046cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 1.20 (3H, s); 1.20 (3H,
s); 1.29 (3H, s); 1.33 (3H, s);
1.73 (3H, s); 1.76 (3H, s); 1.7
8 (3H, s); 3.52 (1H, d, J = 9.5H
z); 3.58 (3H, m); 4.06 (1H, d, J
= 9.5 Hz); 4.12 (2H, m); 4.35 (1
H, dd, J = 9.0, 9.0 Hz); 5.24 (1
H, m); 5.39 (1H, m); 5.71 (1H,
d, J = 6.0 Hz); 6.74 (1H, d, J = 7.
0 Hz); 6.88 (1H, dd, J = 8.0, 7.0)
7.13 (1H, d, J = 8.0 Hz);
90 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 152.37 (s); 142.17 (s); 1
40.96 (s); 132.81 (s); 131.38
(S); 125.25 (s); 125.02 (d); 1
24.58 (d); 120.69 (d); 118.84
(D); 116.33 (s); 110.20 (d); 9
2.29 (d); 78.06 (s); 74.96
(S); 72.13 (d); 72.01 (d); 71.
58 (d); 67.98 (s); 61.36 (t); 6
0.66 (d); 51.09 (s); 50.93
(D); 43.16 (s); 32.60 (t); 28.
59 (q); 26.91 (t); 25.68 (q); 2
1.28 (t); 20.66 (q); 18.67
(Q); 17.81 (q); 16.87 (q); 16.
33 (q)

G) Compound 7 1) Appearance: white crystal 2) Melting point: 202-204 ° C. 3) EI mass spectrum: m / e 605 (M ⁺ ), 5
90, 534, 266 4) High resolution mass spectrum: (C ₃₇ H ₅₁ NO ₆ ) Calculated value: 605.3714 Actual value: 605.3730 5) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 234, 282 nm 6) Red External absorption spectrum: KBr method ν _MAX : 3422, 2932, 1636, 1458, 1
375, 1115, 1046, 928, 810cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: deuterated acetone solution δ _ppm : 1.17 (3H, s); 1.26 (3H,
s); 1.26 (3H, s); 1.30 (3H, s);
1.34 (3H, s); 1.38 (3H, s); 1.6
8 (3H, s); 1.72 (3H, s); 2.38 (1
H, dd, J = 13.0, 11.0 Hz); 3.30
(1H, s); 3.41 (1H, d, J = 9.0H
z); 3.52 (1H, s); 3.97 (1H, dd,
J = 9.0, 3.0 Hz); 4.03 (2H, m);
4.21 (1H, dd, J = 9.0, 9.0 Hz);
4.31 (1H, s); 5.27 (1H, m); 6.9
0 (1H, d, J = 8.0 Hz); 7.22 (1H,
s); 7.29 (1H, d, J = 8.0 Hz); 9.7
9 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 153.20 (s); 141.67 (s); 1
36.84 (s); 130.47 (s); 124.72.
(S); 122.13 (d); 120.76 (d); 1
18.64 (d); 116.75 (s); 112.44.
(D); 79.43 (s); 77.98 (s); 75.
75 (d); 72.16 (d); 68.97 (s); 6
7.94 (d); 65.29 (d); 64.21
(D); 58.71 (t); 58.25 (s); 51.
46 (s); 50.91 (d); 43.14 (s); 3
6.22 (t); 30.32 (t); 28.98
(T); 27.76 (t); 26.99 (t); 25.
65 (q); 24.98 (q); 23.84 (q); 2
1.43 (t); 19.63 (q); 19.15
(Q); 18.68 (q); 17.86 (q); 16.
43 (q)

H) Compound 8 1) Appearance: white crystals 2) Melting point: 152-155 ° C. 3) EI mass spectrum: m / e 603 (M ⁺ ), 5
88,532,250 4) High resolution mass spectrum: (C ₃₇ H ₄₉ NO ₆ ) Calculated value: 603.3558 Measured value: 603.3570 5) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 232, 284 nm 6) Red External absorption spectrum: KBr method ν _MAX : 3434, 2932, 1636, 1456, 1
375, 1306, 1117, 1049, 1009cm
^-1

7) Hydrogen nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 1.19 (3H, s); 1.24 (3H,
s); 1.26 (3H, s); 1.26 (3H, s);
1.28 (3H, s); 1.33 (3H, s); 1.7
0 (3H, s); 1.73 (3H, s); 2.73 (1
H, d, J = 6.0 Hz); 3.63 (4H, m);
4.02 (1H, d, J = 9.5 Hz); 4.37 (1
H, dd, J = 9.0, 9.0 Hz); 4.69 (1
H, d, J = 6.0 Hz); 5.39 (1 H, m);
6.74 (1 H, d, J = 7.0 Hz); 6.88 (1
H, dd, J = 7.5, 7.5 Hz); 7.12 (1
H, d, J = 8.0 Hz); 9.84 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 152.37 (s); 140.99 (s); 1
32.85 (s); 131.39 (s); 125.28.
(S); 125.05 (d); 120.72 (d); 1
18.86 (d); 116.32 (s); 110.16.
(D); 96.17 (d); 78.10 (s); 75.
37 (s); 72.19 (d); 72.00 (d); 7
1.61 (d); 68.01 (s); 63.25
(D); 60.44 (d); 57.25 (s); 51.
12 (s); 50.93 (d); 43.20 (s); 3
2.63 (t); 28.42 (q); 26.92
(T); 25.71 (q); 24.43 (q); 21.
30 (t); 19.21 (q); 18.66 (q); 1
7.84 (q); 16.75 (q); 16.36 (q)

I) Compound 9 1) Appearance: white crystal 2) Melting point: 191-195 ° C. 3) EI mass spectrum: m / e 503 (M ⁺ ), 4
88,182 4) High-resolution mass spectrum: (C ₃₂ H ₄₁ NO ₄ ) calculated value: 503.3034 measured value: 503.302 5) ultraviolet absorption spectrum: methanol solution λ _MAX : 229, 280 nm 6) infrared absorption Spectrum: KBr method ν _MAX : 3422, 2936, 1684, 1653, 1
456, 1377, 1304, 1167, 1098, 1
069,980,743 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 1.05 (3H, s); 1.13 (3H,
s); 1.20 (3H, s); 1.30 (3H, s);
1.70 (3H, s); 1.71 (3H, s); 2.3
8 (1H, dd, J = 13.0, 11.0 Hz); 2.
68 (1H, dd, J = 13.0, 6.5Hz); 3.
47 (1H, s); 3.65 (1H, d, J = 10.0
Hz); 3.93 (1H, dd, J = 10.5, 7.0)
4.0) (1 H, d, J = 9.5 Hz);
18 (1H, m); 5.59 (1H, d, J = 6.0H
z); 6.97 (2H, m); 7.31 (1H, dd,
J = 7.5, 2.0 Hz); 7.35 (1H, dd, J
= 7.5, 2.0 Hz); 9.89 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 150.99 (s); 141.44 (s); 1
37.91 (s); 125.90 (s); 123.96.
(D); 120.60 (d); 119.58 (d); 1
18.69 (d); 117.81 (s); 112.47.
(D); 93.22 (d); 74.91 (s); 73.
76 (d); 72.43 (d); 71.87 (d); 6
4.56 (s); 56.83 (d); 51.03
(S); 50.35 (d); 41.65 (d); 40.
48 (s); 32.29 (t); 30.31 (t); 2
8.66 (q); 27.69 (t); 25.39
(Q); 24.70 (t); 22.48 (t); 18.
47 (q); 17.00 (q); 16.13 (q); 1
4.76 (q)

J) Compound 10 1) Appearance: white crystals 2) Melting point: 120-123 ° C. 3) EI mass spectrum: m / e 505 (M ⁺ ), 4
90,421,182 4) High resolution mass spectrum: (C ₃₂ H ₄₃ NO ₄ ) Calculated value: 505.3190 Measured value: 503.3318 5) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 230, 281 nm 6) Red External absorption spectrum: KBr method ν _MAX : 3424, 2934, 1451, 1377, 1
304,1152,1146,1111,741cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: deuterated acetone solution δ _ppm : 1.05 (3H, s); 1.11 (3H,
s); 1.26 (3H, s); 1.30 (3H, s);
1.68 (3H, s); 1.73 (3H, s); 2.3
8 (1H, dd, J = 13.0, 11.0 Hz); 2.
68 (1H, dd, J = 13.0, 6.5Hz); 3.
41 (1H, s); 3.52 (1H, d, J = 9.0H
z); 3.84 (1H, dd, J = 10.0, 7.5H
z); 3.99 (1H, d, J = 9.0 Hz); 4.0
4 (2H, m); 4.33 (1H, s); 5.28 (1
H, m); 6.98 (2H, m); 7.32 (1H,
d, J = 7.0 Hz); 7.36 (1H, d, J = 7.
0Hz); 9.89 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 151.06 (s); 141.42 (s); 1
36.84 (s); 125.91 (s); 122.11.
(D); 120.57 (d); 119.57 (d); 1
18.68 (d); 117.78 (s); 112.46.
(D); 79.50 (s); 75.88 (d); 73.
45 (d); 68.09 (d); 65.20 (s); 6
0.04 (d); 58.69 (t); 51.01
(S); 50.41 (d); 41.50 (d); 40.
21 (s); 32.26 (t); 27.71 (t); 2
5.66 (q); 24.82 (t); 23.78
(Q); 22.58 (t); 19.62 (q); 17.
86 (q); 16.20 (q); 14.75 (q)

K) Compound 11 1) Appearance: white crystal 2) Melting point: 113-115 ° C. 3) EI mass spectrum: m / e 587 (M ⁺ ), 5
72,250 4) High resolution mass spectrum: (C ₃₇ H ₄₉ NO ₅ ) Calculated value: 587.3609 Measured value: 587.3593 5) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 232, 284 nm 6) Infrared absorption Spectrum: KBr method ν _MAX : 3424, 2934, 1715, 1653, 1
454, 1375, 1306, 1119, 1100, 1
047,982 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 1.19 (3H, s); 1.20 (3H,
s); 1.30 (3H, s); 1.33 (3H, s);
1.70 (3H, s); 1.71 (3H, s); 1.7
3 (3H, s); 1.76 (3H, s); 2.66 (1
H, ddd, J = 13.5, 13.5, 5.0 Hz);
3.45 (1H, s); 3.52 (1H, d, J = 9.
5Hz); 3.59 (3H, m); 4.06 (1H,
d, J = 9.0 Hz); 4.35 (1H, dd, J =
9.0, 9.0 Hz); 5.18 (1 H, m); 5.3
9 (1H, td, J = 6.5, 2.5 Hz); 5.59
(1H, d, J = 6.5 Hz); 6.74 (1H, d,
J = 7.5 Hz); 6.88 (1H, dd, J = 8.
0, 7.5 Hz); 7.12 (1H, d, J = 8.0H
z); 9.84 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 152.62 (s); 141.20 (s); 1
37.97 (s); 132.92 (s); 131.47.
(S); 125.39 (s); 125.12 (d); 1
23.94 (d); 120.79 (d); 118.95.
(D); 116.44 (s); 110.29 (d); 9
3.26 (d); 78.28 (s); 74.87
(S); 72.21 (d); 72.19 (d); 71.
70 (d); 68.04 (s); 60.84 (d); 5
1.18 (s); 51.02 (d); 43.28
(S); 32.69 (t); 30.21 (t); 29.
30 (t); 28.72 (q); 27.03 (t); 2
5.78 (q); 25.39 (q); 21.38
(T); 18.77 (q); 18.48 (q); 17.
91 (q); 16.97 (q); 16.43 (q)

L) Compound 12 1) Appearance: white crystals 2) Melting point: 260-264 ° C. (decomposition) 3) EI mass spectrum: m / e 589 (M ⁺ ), 5
74,505,250 4) High resolution mass spectrum: (C ₃₇ H ₅₁ NO ₅ ) Calculated value: 589.3765 Measured value: 589.337745) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 232, 284 nm 6) Red External absorption spectrum: KBr method ν _MAX : 3399, 2928, 1734, 1456, 1
373, 1275, 1113, 1047 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: deuterated acetone solution δ _ppm : 1.18 (3H, s); 1.26 (3H,
s); 1.29 (3H, s); 1.33 (3H, s);
1.67 (3H, s); 1.72 (3H, s); 1.7
3 (3H, s); 1.76 (3H, s); 2.56 (1
H, dd, J = 12.0,10.5Hz); 2.67
(1H, ddd, J = 13.5, 13.5, 5.5H
z); 3.29 (1H, s); 3.40 (1H, d, J
= 9.0 Hz); 3.51 (1H, s); 3.59 (2
H, m); 3.97 (1H, d, J = 9.0 Hz);
4.03 (2H, m); 4.20 (1H, dd, J =
9.0, 9.0 Hz); 4.30 (1H, s); 5.2
7 (1H, m); 5.39 (1H, m); 6.74 (1
H, d, J = 7.0 Hz); 6.87 (1H, dd, J
= 7.5, 7.5 Hz); 7.12 (1H, d, J =
8.0 Hz); 9.80 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 152.64 (s); 141.17 (s); 1
36.81 (s); 132.87 (s); 131.43
(S); 125.37 (s); 125.10 (d); 1
22.11 (d); 120.73 (d); 118.91
(D); 116.40 (s); 110.25 (d); 7
9.41 (s); 77.97 (s); 75.72
(D); 72.13 (d); 68.97 (s); 67.
92 (d); 64.19 (d); 58.69 (t); 5
1.11 (s); 51.03 (d); 43.10
(S); 32.67 (t); 30.28 (t); 28.
95 (t); 26.91 (t); 25.74 (q); 2
5.62 (q); 23.81 (q); 21.37
(T); 19.62 (q); 18.66 (q); 17.
87 (q); 17.83 (q); 16.41 (q)

M) Compound 13 1) Appearance: white crystals 2) Melting point: 102-105 ° C. 3) EI mass spectrum: m / e 587 (M ⁺ ), 5
72,503,250 4) High resolution mass spectrum: (C ₃₇ H ₄₉ NO ₅ ) Calculated value: 587.3609 Measured value: 587.3617 5) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 233, 283 nm 6) Red External absorption spectrum: KBr method ν _MAX : 3424, 2932, 1719, 1636, 1
454, 1375, 1306, 1098, 1046, 9
80 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: deuterated acetone solution δ _ppm : 1.17 (3H, s); 1.20 (3H,
s); 1.30 (3H, s); 1.33 (3H, s);
1.70 (3H, s); 1.71 (3H, s); 1.7
3 (3H, s); 1.75 (3H, s); 2.37 (1
H, dd, J = 13.0, 11.0 Hz); 3.38
(2H, d, J = 7.3 Hz); 3.44 (1H,
s); 3.52 (1H, d, J = 9.5 Hz); 3.5
7 (1H, s); 4.05 (1H, d, J = 9.5H
z); 4.35 (1H, dd, J = 9.0, 9.0H)
z); 5.18 (1H, m); 5.37 (1H, m);
5.59 (1 H, d, J = 6.0 Hz); 6.82 (1
H, dd, J = 8.5, 1.5 Hz); 7.14 (1
H, s); 7.19 (1H, d, J = 8.0 Hz);
9.75 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 153.51 (s); 139.81 (s); 1
37.98 (s); 132.70 (s); 131.13.
(S); 126.31 (s); 125.90 (d); 1
23.96 (d); 121.30 (d); 117.94.
(D); 116.59 (s); 112.19 (d); 9
3.27 (d); 78.28 (s); 74.87
(S); 72.23 (d); 72.20 (d); 71.
72 (d); 68.05 (s); 60.86 (d); 5
1.47 (s); 50.86 (d); 43.30
(S); 35.11 (t); 30.24 (t); 29.
31 (t); 28.73 (q); 27.75 (t); 2
7.05 (t); 25.79 (q); 25.40.
(Q); 21.44 (t); 18.75 (q);
50 (q); 17.73 (q); 16.98 (q); 1
6.45 (q)

N) Compound 14 1) Appearance: white crystal 2) Melting point: 231-235 ° C. (decomposition) 3) EI mass spectrum: m / e 589 (M ⁺ ), 5
74,505,250 4) High resolution mass spectrum: (C ₃₇ H ₅₁ NO ₅ ) Calculated value: 589.3765 Measured value: 589.33788 5) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 233, 283 nm 6) Red External absorption spectrum: KBr method ν _MAX : 3430, 2930, 1725, 1454, 1
377, 1277, 1103, 1044 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: deuterated acetone solution δ _ppm : 1.15 (3H, s); 1.25 (3H,
s); 1.29 (3H, s); 1.32 (3H, s);
1.67 (3H, s); 1.72 (6H, s); 1.7
4 (3H, s); 2.37 (1H, dd, J = 13.
0, 11.0 Hz); 3.28 (1H, s); 3.38
(2H, d, J = 7.5 Hz); 3.40 (1H, d,
J = 9.0 Hz); 3.51 (1H, s); 3.96
(1H, d, J = 9.0 Hz); 4.03 (2H,
m); 4.20 (1H, dd, J = 9.0, 9.0H)
z); 4.31 (1H, s); 5.27 (1H, m);
5.36 (1H, m); 6.81 (1H, d, J = 7.
5 Hz); 7.14 (1H, s); 7.19 (1H,
d, J = 8.5 Hz); 9.74 (1H, brs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 153.52 (s); 139.77 (s); 1
36.82 (s); 132.63 (s); 131.08
(S); 126.28 (s); 125.87 (d); 1
22.11 (d); 121.24 (d); 117.89
(D); 116.55 (s); 112.15 (d); 7
9.42 (s); 77.96 (s); 75.72
(D); 72.15 (d); 68.97 (s); 67.
92 (d); 64.20 (d); 58.71 (t); 5
1.39 (s); 50.85 (d); 43.10
(S); 35.08 (t); 30.29 (t); 28.
96 (t); 27.73 (t); 26.93 (t); 2
5.77 (q); 25.64 (q); 23.82
(Q); 21.43 (t); 19.62 (q); 18.
64 (q); 17.85 (q); 17.70 (q); 1
6.43 (q)

O) Compound 15 1) Appearance: white crystal 2) Melting point: 135-138 ° C. 3) EI mass spectrum: m / e 489 (M ⁺ ), 4
74,405,390,182 4) High resolution mass spectrum: (C ₃₂ H ₄₃ NO ₃ ) Calculated value: 489.3241 Measured value: 489.3327 15) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 229,281 nm 6 ) Infrared absorption spectrum: KBr method ν _MAX : 3401, 2932, 2859, 1726, 1
454, 1375, 1275, 1107, 748, 71
0 cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 1.06 (3H, s); 1.11 (3H,
s); 1.12 (3H, s); 1.16 (3H, s);
1.64 (3H, s); 1.69 (3H, s); 2.3
7 (1H, dd, J = 13.0, 11.0 Hz); 2.
67 (1H, dd, J = 13.0, 6.5Hz);
47 (1H, d, J = 2.5 Hz); 3.67 (1H,
dd, J = 11.5, 2.0 Hz); 3.87 (1H,
dd, J = 8.5, 8.5 Hz); 3.93 (1H, d
d, J = 11.0, 6.5 Hz); 4.00 (1H, d
d, J = 11.5, 6.5 Hz); 6.97 (2H,
m); 7.30 (1H, d, J = 8.0 Hz); 7.3
4 (1H, d, J = 7.5 Hz); 9.91 (1H, b
rs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 151.19 (s); 141.43 (s); 1
34.03 (s); 125.93 (s); 124.06.
(D); 120.56 (d); 119.57 (d); 1
18.67 (d); 117.76 (s); 112.48.
(D); 76.04 (s); 74.60 (d); 74.
42 (d); 61.90 (s); 59.34 (t); 5
6.14 (d); 51.05 (s); 50.51
(D); 41.83 (d); 40.15 (s); 32.
37 (t); 30.25 (t); 27.74 (t); 2
6.79 (t); 25.64 (q); 24.92
(T); 23.69 (q); 22.63 (t); 20.
97 (q); 17.91 (q); 16.46 (q); 1
4.82 (q)

P) Compound 16 1) Appearance: white crystals 2) Melting point: 176-179 ° C. (decomposition) 3) EI mass spectrum: m / e 453 (M ⁺ ), 4
38, 182 4) High resolution mass spectrum: (C ₂₇ H ₃₅ NO ₅ ) Calculated value: 453.2514 Actual value: 453.2537 5) Ultraviolet absorption spectrum: Methanol solution λ _MAX : 230 (ε = 3.1 ×) 10 ⁴ ) 282 (ε = 7.6 × 10 ³ ) 6) Infrared absorption spectrum: KBr method ν _MAX : 3407, 2936, 1456, 1373, 1
302, 1100, 1046, 930, 747cm ^-1

7) Hydrogen nuclear magnetic resonance spectrum: deuterated acetone solution δ _ppm : 1.17 (3H, s); 1.20 (3H,
s); 1.22 (3H, s); 1.33 (1H, s);
3.32 (1H, s); 3.36 (1H, d, J = 9.
0 Hz); 3.54 (1H, s); 3.99 (1H,
d, J = 9.0 Hz); 4.21 (1H, dd, J =
9.0, 9.0 Hz); 4.64 (1H, s); 6.9
5 (2H, m); 7.29 (1H, m); 7.34 (1
H, m); 8.02 (1H, s); 9.90 (1H, b
rs)

8) Carbon nuclear magnetic resonance spectrum: heavy acetone solution δ _ppm : 153.12 (s); 141.02 (s); 1
25.95 (s); 120.29 (d); 119.42
(D); 118.55 (d); 116.77 (s); 1
12.30 (d); 77.82 (s); 76.85.
(D); 72.86 (s); 71.99 (d); 69.
29 (s); 68.04 (d); 64.27 (d); 5
1.40 (s); 50.87 (d); 43.03
(S); 30.18 (t); 28.89 (t); 28.
25 (q); 27.64 (t); 26.88 (t); 2
4.05 (q); 21.37 (t); 18.61
(Q); 16.38 (q) 9) Specific rotation: acetone solution

[0074]

[Chemical 11]

[0075]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist. Example 1 Corn starch 3.0%, soybean powder 3.5%, soybean oil 0.
2%, wheat germ 1.5%, dipotassium hydrogen phosphate 0.
Medium containing 2% and 0.5% calcium carbonate (pH
40 ml of 6.0) in 20 200 ml Erlenmeyer flasks
Each was dispensed and autoclaved at 121 ° C. for 20 minutes.

In addition, Compound 1-15 producing strain, Vert
icillium balanoids MCI282
1 platinum loop was inoculated for 5 days at 27 ° C for 21 days.
Shaking culture was performed at 0 revolutions. Separately, a medium having the same composition as the above was prepared, and 80 ml of the medium was dispensed into 100 500 ml Erlenmeyer flasks, respectively, and the medium was kept at 121 ° C.
It was autoclaved for 0 minutes. The main fermentation medium was inoculated with 4 ml each of the seed culture solution and cultured at 27 ° C. for 11 days at 210 rotations. Acetone (7 liters) was added to the obtained culture for extraction, the mixture was filtered through Celite, and acetone was distilled off under reduced pressure to obtain an extract (7.1 liter).

The extract thus obtained was extracted with an equal amount of ethyl acetate and concentrated under reduced pressure to obtain 4.55 g of an oily substance. The oily substance obtained was subjected to reverse phase partition chromatography using octadecyl silica gel. M
CI GEL ODS 1MY (manufactured by Mitsubishi Kasei) 10g
After dusting and drying under reduced pressure, MCI GEL ODS 1M
90 g of Y was placed on a column packed with an acetonitrile-water mixture (2: 3), and an acetonitrile-water mixture (2:
3) Washed with 500 ml. Next, the mixture was washed with 500 ml of an acetonitrile-water mixed liquid (3: 2) and 200 ml of an acetonitrile-water mixed liquid (4: 1). Then, it was eluted with 300 ml of an acetonitrile-water mixture (4: 1), and then with 100 ml of acetonitrile. The eluates were collected and concentrated to dryness under reduced pressure to obtain 0.92 g of an oily substance. This oily substance was dissolved in 17 ml of acetonitrile, and 1.5 ml of this was dissolved in CAPCELL PAK C18 (30
mm × 250 mm) (manufactured by Shiseido Co., Ltd.) was used for preparative high performance liquid chromatography. After washing with 162 ml of an acetonitrile-water mixed solution (4: 1), elution was carried out with an acetonitrile-water mixed solution (4: 1), and 9 ml each was collected to obtain a fraction containing Compound 1 (fractions 14 to 1).
5) and a fraction containing compound 4 (fraction 18-
19), a fraction containing compound 2 (fractions 24 to
26), a fraction containing compound 3 (fractions 28-
30), the fraction containing compound 5 (fraction 48-
50) was obtained. Fractionation was similarly repeated for the remaining oily substance solution, and the obtained fractions were combined and concentrated to dryness under reduced pressure to give Compound 1 crude powder 89.0 mg, Compound 4
Coarse powder 39.5 mg, compound 2 coarse powder 110.0 mg,
Compound 3 crude powder 47.2 mg, Compound 5 crude powder 33.6
mg was obtained. The crude powder of compound 1 was recrystallized from n-hexane to obtain 69.9 mg of crystals of compound 1. In addition, Compound 2 crude powder was recrystallized from n-hexane to obtain 66.2 mg of Compound 2 crystals. Further, the crude powder of compound 3 was recrystallized from acetonitrile to obtain 24.6 mg of crystals of compound 3. Silica gel 60H (product of Merck)
Separation was performed by column chromatography packed with 5 g.
After developing with 25 ml of dichloromethane, developing with 25 ml of dichloromethane-methanol mixture (199: 1), and further with dichloromethane-methanol mixture (99: 1) 15 m
Unfolded with l. Dichloromethane-methanol mixture (9
The elution was carried out with 10 ml of 9: 1) and then with 15 ml of a dichloromethane-methanol mixture (197: 3). The eluate was concentrated to dryness under reduced pressure to obtain 32.1 mg of white powder.
Further, the white powder was separated by column chromatography packed with 5 g of silica gel.

Develop with 25 ml of n-hexane, then n
-Hexane-ethyl acetate mixed solution (9: 1) 25 ml, n-
Developing with 25 ml of hexane-ethyl acetate mixed solution (4: 1) and 15 ml of n-hexane-ethyl acetate mixed solution (7: 3),
It was eluted with 10 ml of n-hexane-ethyl acetate mixed solution (7: 3). Collect the eluate, concentrate under reduced pressure to dryness, 19.8m
g of white powder was obtained. This was recrystallized from n-hexane to obtain 15.4 mg of compound 4 crystals. The crude powder of compound 5 was subjected to column chromatography packed with 5 g of silica gel. After developing with 25 ml of dichloromethane,
Dichloromethane-methanol mixture (199: 1) 25 m
It was developed with 1, and a dichloromethane-methanol mixture (9
It was developed with 25 ml of 9: 1) and eluted with 10 ml of a dichloromethane-methanol mixture (197: 3). The eluate was concentrated to dryness under reduced pressure to obtain 21.7 mg of white powder. This was recrystallized from n-hexane to give a crystal of compound 5 12.5
mg was obtained. The physical properties of these compounds are as described above.

Example 2 In the same manner as in Example 1, Verticillium b
Alanoids MCI2825 was cultured in 200 500 ml Erlenmeyer flasks, extracted with acetone, and then extracted with ethyl acetate to obtain 10.68 g of an oily substance.
This oily substance was subjected to reverse phase partition chromatography using octadecyl silica gel. MCI GEL O
DS 1MY 20g dust, dried under reduced pressure, MCI
180 g of GELODS 1MY was placed on a column packed with an acetonitrile-water mixture (1: 1) and washed with 1 liter of an acetonitrile-water mixture (2: 3). Next, the mixture was washed with 1 liter of an acetonitrile-water mixed liquid (3: 2) and 600 ml of an acetonitrile-water mixed liquid (4: 1). Then, acetonitrile-water mixture (4: 1) 200
It was eluted with ml, and the eluate was concentrated to dryness under reduced pressure to obtain 0.37 g of fraction 1-A. Further, it was eluted with 200 ml of an acetonitrile-water mixture (4: 1), and the eluate was concentrated to dryness under reduced pressure to obtain 0.33 g of fraction 1-B. Further, it was eluted with 400 ml of an acetonitrile-water mixture (9: 1), and the eluate was concentrated to dryness under reduced pressure to obtain 2.71 g of fraction 1-C. Further, it was eluted with 600 ml of an acetonitrile-water mixture (9: 1), and the eluate was concentrated to dryness under reduced pressure to give 1.99 g of fraction 1-.
I got D.

Next, Fraction 1-A was dissolved in 4 ml of acetonitrile, and 2 ml of it was dissolved in Inertsil P
It was subjected to preparative high performance liquid chromatography equipped with REP-ODS (50 mm × 250 mm) (manufactured by GL Science). Acetonitrile-water mixture (13: 7) 315
After washing with 0 ml, an acetonitrile-water mixed solution (13:
7) Elution was performed using 175 ml. The remaining Fraction 1-A was similarly separated, and the eluate was collected and concentrated to dryness under reduced pressure to give Fraction 2-A 9.7 mg. Further, Fraction 2-A was dissolved in 0.5 ml of acetonitrile, and the mixture was mixed with CAPCELL.
It was subjected to preparative high performance liquid chromatography equipped with PAK C18 (30 mm × 250 mm). After washing with 666 ml of an acetonitrile-water mixed liquid (7: 3), elution was performed using 36 ml of an acetonitrile-water mixed liquid (7: 3).
The eluate was concentrated to dryness under reduced pressure to obtain 3.7 mg of compound 6.

Fraction 1-B was dissolved in a mixed solution of ethyl acetate-acetonitrile, the insoluble matter was filtered off, the filtrate was concentrated to dryness under reduced pressure, and 0.30 g of the obtained oily substance was dissolved in 2 ml of acetonitrile. , CAPCELL PAK C18 (3
It was subjected to preparative high performance liquid chromatography equipped with 0 mm × 250 mm). Acetonitrile-water mixture (13:
7) After washing with 4 liters, it was eluted with 150 ml of an acetonitrile-water mixed solution (13: 7). The eluate was concentrated to dryness under reduced pressure to obtain Fraction 3-A15.6 mg. Further, it was eluted with 225 ml of an acetonitrile-water mixture (13: 7), and the eluate was concentrated to dryness under reduced pressure to give Fraction 3-B19.7 m.
g was obtained. This fraction 3-B was dissolved in 0.7 ml of a mixed solution of ethyl acetate-acetonitrile, and CAPCELL P
It was subjected to preparative high performance liquid chromatography equipped with AK C18 (30 mm × 250 mm). After washing with 648 ml of an acetonitrile-water mixed solution (4: 1), elution was performed with 36 ml of an acetonitrile-water mixed solution (4: 1), and the eluate was concentrated to dryness under reduced pressure to obtain 7.1 mg of a fraction 4-A. .

Fraction 1-C was further subjected to reverse layer partition chromatography using octadecyl silica gel. Fraction 1-C was mixed with acetonitrile-water (4: 1) 5
It is dissolved in 0 ml and 100 g of MCI GEL ODS 1MB (manufactured by Mitsubishi Kasei Co.) is mixed with acetonitrile-water (4:
Place on a column packed with 1), acetonitrile-
It was washed with 500 ml of a water mixture (4: 1). Next, after washing with 50 ml of an acetonitrile-water mixed solution (17: 3),
Elution was performed with 200 ml of an acetonitrile-water mixture (17: 3), and the eluate was concentrated to dryness under reduced pressure to give Fraction 5-A 0.97 g.
Got Acetonitrile-water mixture (17: 3) 1
00 ml was eluted and concentrated to dryness under reduced pressure to give fraction 5-B0.6.
1 g was obtained.

Fraction 5-B was dissolved in 50 ml of n-hexane and loaded on a column of 50 g of silica gel 60H filled with hexane, 250 ml of n-hexane, 250 ml of n-hexane-ethyl acetate mixed solution (9: 1), and further n-hexane. It was washed with 100 ml of a hexane-ethyl acetate mixed solution (4: 1). Next, 150 ml of n-hexane-ethyl acetate mixed solution (4: 1)
And eluted with n-hexane-ethyl acetate mixed solution (7:
3) Elution was carried out with 50 ml, and the eluates were combined and concentrated to dryness under reduced pressure to obtain 0.32 g of fraction 6-A.

Fraction 5-A and Fraction 6-A are combined for 11 m
After dissolving in 1 ethyl acetate-acetonitrile mixed solution, 3 ml of it was dissolved in Inertsil PREP-ODS (5
It was subjected to preparative high performance liquid chromatography equipped with 0 mm × 250 mm). Elution was performed while changing the composition of the acetonitrile-water mixed solution from 4: 1 to 1500 ml linearly to 17: 3, and 25 ml each was collected. Fractions 55-60, 68-71, 73-81, 82-8
5, 86-102 and 103-114 were collected and concentrated to dryness under reduced pressure. Fractionation was repeated in the same manner for the remaining solution, and the obtained fractions were combined and concentrated to dryness under reduced pressure to obtain fraction 7-A23.7 mg, fraction 7-B61.
4 mg, fraction 7-C344.1 mg, fraction 7-D58
9.6 mg, fraction 7-E328.4 mg, fraction 7-F2
8.4 mg was obtained.

Fraction 3-A, Fraction 4-A, and Fraction 7-A
Were dissolved in 1.4 ml of ethyl acetate, and YMC-Pa
ck Polymer C18 (30mm × 300m
m) was attached to preparative high performance liquid chromatography. After washing with 423 ml of acetonitrile-water mixture (4: 1), 72 ml of acetonitrile-water mixture (4: 1)
Was eluted with. The eluate was concentrated to dryness under reduced pressure, and fraction 8
-A14.6 mg was obtained. Further, this fraction 8-A was dissolved in 1 ml of a mixed solution of ethyl acetate-acetonitrile,
ack Polymer C18 (30mm × 300m
m) was attached to preparative high performance liquid chromatography. After washing with 648 ml of acetonitrile-water mixture (3: 1), 36 ml of acetonitrile-water mixture (3: 1)
Was eluted with. The eluate was concentrated to dryness under reduced pressure to obtain 5.26 mg of compound 7.

Fraction 7-B was dissolved in 2 ml of acetonitrile, 1 ml of which was dissolved in YMC-Pack Polymer.
It was subjected to preparative high performance liquid chromatography equipped with r C18 (30 mm × 300 mm). Acetonitrile-
After washing with 414 ml of a water mixture (17: 3), elution was performed with 36 ml of an acetonitrile-water mixture (17: 3). In the same way, perform fractionation for the remaining lysate,
The eluates were combined and concentrated to dryness under reduced pressure to give compound 8
mg was obtained.

Fraction 7-C was added to acetonitrile-acetone-
Dissolve in 6 ml of ethyl acetate mixture, 3 ml of which is dissolved in YM
C-Pack Polymer C18 (30mm × 3
It was subjected to preparative high performance liquid chromatography equipped with (00 mm). Acetonitrile-water mixture (4: 1) 360 m
After washing with l, 9 ml of each fraction was collected, and fractions 21 to 25 and fractions 32 to 37 were collected and concentrated to dryness under reduced pressure. Similarly, the remaining lysate was fractionated, combined with each eluate and concentrated to dryness under reduced pressure to obtain fraction 9-A62.6 mg and fraction 9-B50.8 mg.
Got

Fraction 9-A was dissolved in 2 ml of acetonitrile, and CAPCELL PAK C18 (30 mm × 25
It was subjected to preparative high performance liquid chromatography equipped with 0 mm). 522 ml of acetonitrile-water mixture (4: 1)
After washing with water, acetonitrile-water mixture (4: 1) 45
Elute with ml. The eluate was concentrated to dryness under reduced pressure and the fraction 10
-A27.6 mg was obtained.

Fraction 9-B was dissolved in 1.6 ml acetonitrile, 0.8 ml of which was dissolved in CAPCELL PAK.
It was subjected to preparative high performance liquid chromatography equipped with C18 (30 mm × 250 mm). After washing with 360 ml of an acetonitrile-water mixture (4: 1), 9 ml fractions were collected and fractions 21 to 25 and 28 were collected.
~ 32 were collected and concentrated to dryness under reduced pressure. In the same manner, the remaining lysate was fractionated, combined with each eluate and concentrated to dryness under reduced pressure to obtain fraction 11-A16.5m.
g, fraction 11-B19.1 mg was obtained.

Fractions 10-A and 11-A were combined, dissolved in 5 ml of dichloromethane, placed on a column of 5 g of silica gel 60H filled with dichloromethane, washed with 25 ml of dichloromethane, and further with 5 ml of dichloromethane-methanol mixture (99: 1). did. Then dichloromethane-
Elution was performed with 15 ml of a mixed solution of methanol (99: 1), and the eluates were combined and concentrated to dryness under reduced pressure to obtain 7.4 mg of compound 9.

Fraction 11-B was dissolved in 1 ml of acetonitrile, and CAPCELL PAK C18 (30 mm × 2)
It was subjected to preparative high performance liquid chromatography equipped with 50 mm). Acetonitrile-water mixture (4: 1) 585 m
After washing with l, acetonitrile-water mixture (4: 1) 3
Eluted with 6 ml. The eluate was concentrated to dryness under reduced pressure to obtain 14.4 mg of compound 10.

Fraction 7-D was dissolved in 4 ml of an ethyl acetate-acetonitrile mixed solution, and 1.5 ml of the solution was dissolved in YMC-P.
ack Polymer C18 (30mm × 300m
m) was attached to preparative high performance liquid chromatography. After washing with 441 ml of acetonitrile-water mixture (17: 3), acetonitrile-water mixture (17: 3) 72
Elute with ml. Similarly, with respect to the remaining solution, fractionation was repeated twice, and the eluates were combined and concentrated to dryness under reduced pressure to obtain fraction 12-A (8.9 mg).

Fraction 12-A was dissolved in 1 ml of methanol, and CAPCELL PAK C18 (30 mm × 25
It was subjected to preparative high performance liquid chromatography equipped with 0 mm). After washing with 378 ml of a methanol-water mixture (9: 1), elution was carried out with 27 ml of a methanol-water mixture (9: 1). The eluate was concentrated to dryness under reduced pressure to give compound 11 5.
2 mg was obtained.

Fraction 7-E was dissolved in 4 ml of ethyl acetate-acetonitrile mixture, and 2 ml of it was dissolved in YMC-Pac.
k Polymer C18 (30mm x 300mm)
It was subjected to preparative high performance liquid chromatography equipped with.
After washing with 540 ml of acetonitrile-water mixture (17: 3), 63 ml of acetonitrile-water mixture (17: 3)
Eluted at. Similarly, the remaining lysate is fractionated, and the eluates are combined and concentrated to dryness under reduced pressure.
A24.6 mg was obtained. This fraction 13-A was dissolved in 5 ml of n-hexane-ethyl acetate mixed solution, and loaded on a column of 5 g of silica gel 60H filled with hexane, and 25 ml of n-hexane and n-hexane-ethyl acetate mixed solution (19:
1) 25 ml, n-hexane-ethyl acetate mixed solution (9:
1) 25 ml, and n-hexane-ethyl acetate mixed solution (1
7: 3) Washed with 15 ml. Next, it was eluted with 10 ml of a mixed solution of n-hexane-ethyl acetate (17: 3), and the eluate was concentrated to dryness under reduced pressure to obtain 11.6 mg of fraction 14-A. Further, n-hexane-ethyl acetate mixed solution (17: 3) 15 ml
And eluate was concentrated to dryness under reduced pressure to give fraction 14-B.
7.3 mg was obtained.

Fraction 14-A was dissolved in 1 ml of methanol, and CAPCELL PAK C18 (30 mm × 25
It was subjected to preparative high performance liquid chromatography equipped with 0 mm). After washing with 360 ml of a methanol-water mixture (9: 1), it was eluted with 45 ml of a methanol-water mixture (9: 1), the eluate was concentrated to dryness under reduced pressure, and fraction 15-A9.6.
mg was obtained. Further, methanol-water mixture (9: 1) 54 m
The eluate was concentrated to dryness under reduced pressure, and fraction 15-B was eluted.
9.2 mg was obtained.

Fraction 14-B was dissolved in 1 ml of methanol, and CAPCELL PAK C18 (30 mm × 25
It was subjected to preparative high performance liquid chromatography equipped with 0 mm). After washing with 360 ml of a methanol-water mixture (9: 1), the mixture was eluted with 45 ml of a methanol-water mixture (9: 1), the eluate was concentrated to dryness under reduced pressure, and fraction 16-A2.8.
mg was obtained. Methanol-water mixture (9: 1) 36m
The eluate was concentrated to dryness under reduced pressure, and fraction 16-B was eluted.
Obtained 1.5 mg.

Fractions 15-A and 16-A were combined and dissolved in 1 ml of methanol, and CAPCELLPAK C18 (3
It was subjected to preparative high performance liquid chromatography equipped with 0 mm × 250 mm). Methanol-water mixture (9: 1) 3
After washing with 60 ml, methanol-water mixture (9: 1)
Elution was performed with 36 ml, and the eluate was concentrated to dryness under reduced pressure to obtain 2.4 mg of compound 12.

Fractions 15-B and 16-B were combined and dissolved in 1 ml of methanol to obtain CAPCELLPAK C18 (3
It was subjected to preparative high performance liquid chromatography equipped with 0 mm × 250 mm). Methanol-water mixture (9: 1) 4
After washing with 14 ml, methanol-water mixture (9: 1)
Elution was performed with 45 ml, and the eluate was concentrated to dryness under reduced pressure to obtain 7.6 mg of compound 13.

Fraction 7-F was dissolved in 0.6 ml of a mixed solution of chloroform-acetonitrile, and CAPCELL PAK was dissolved.
It was subjected to preparative high performance liquid chromatography equipped with C18 (30 mm × 250 mm). After washing with 630 ml of an acetonitrile-water mixture (17: 3), elution was performed with 36 ml of an acetonitrile-water mixture (17: 3). The eluate was concentrated to dryness under reduced pressure to obtain Fraction 17-A 9.0 mg.

Fraction 1-D was dissolved in 50 ml of an acetonitrile-water mixture (7: 3) and subjected to reverse layer partition chromatography using octadecyl silica gel. MCI
Fraction 1-D solution was placed on a column packed with 100 g of GELODS 1MB using an acetonitrile-water mixed solution (7: 3), and the acetonitrile-water mixed solution (7: 3) 500 m.
1, then acetonitrile-water mixture (3: 1) 500 m
Wash with l. Next, an acetonitrile-water mixed solution (4:
1) After washing with 250 ml, it was eluted with 250 ml of an acetonitrile-water mixture (4: 1), and further eluted with 250 ml of an acetonitrile-water mixture (17: 3). The eluates were combined, concentrated to dryness under reduced pressure, and fractionated. 18-A1.55g was obtained.

Fraction 18-A was dissolved in 10 ml of an ethyl acetate-acetonitrile mixture, and 5 ml of it was dissolved in Ine.
rtsil PREP-ODS (50 mm x 250 m
m) was attached to preparative high performance liquid chromatography. Elution was performed while changing the composition of the acetonitrile-water mixed solution from 4: 1 to 1500 ml linearly to 17: 3, and 25 ml each was collected. Fraction 92-10
7, 108 to 112 were collected and concentrated to dryness under reduced pressure. The remaining lysate was similarly fractionated, and the obtained fractions were combined and concentrated to dryness under reduced pressure to obtain fraction 19-A.
82.3 mg and the fraction 19-B9.1 mg were obtained.

Fraction 19-A was dissolved in 1.5 ml of a mixed solution of ethyl acetate-acetonitrile, and CAPCELL PAK was added.
It was subjected to preparative high performance liquid chromatography equipped with C18 (30 mm × 250 mm). After washing with 648 ml of an acetonitrile-water mixed liquid (17: 3), elution was performed with 45 ml of an acetonitrile-water mixed liquid (17: 3). The eluate was concentrated to dryness under reduced pressure to obtain fraction 20-A22.3 mg.
This fraction 20-A was dissolved in n-hexane and the insoluble material was collected by filtration to give an insoluble fraction 20-AC 12.9 mg.

Fractions 17-A and 20-AC were combined to 1 ml.
Dissolve in acetonitrile, YMC-Pack Poly
It was subjected to preparative high performance liquid chromatography equipped with mer C18 (30 mm × 300 mm). After washing with 423 ml of acetonitrile-water mixture (9: 1), the mixture was eluted with 36 ml of acetonitrile-water mixture (9: 1), and the eluate was concentrated to dryness under reduced pressure to obtain 9.1 mg of compound 14.

Fraction 19-B was dissolved in 0.8 ml of a mixed solution of ethyl acetate-acetonitrile, and CAPCELL PAK was added.
It was subjected to preparative high performance liquid chromatography equipped with C18 (30 mm × 250 mm). After washing with 774 ml of an acetonitrile-water mixture (17: 3), elution was carried out with 27 ml of an acetonitrile-water mixture (17: 3). The eluate was concentrated to dryness under reduced pressure to obtain 1.0 mg of compound 15. The physical properties of these compounds are as described above.

Example 3 30.6 mg of compound 2 was dissolved in 30 ml of ethanol, and 0.6 ml of concentrated hydrochloric acid was added. The reaction solution was stirred at room temperature and after 30 minutes, 80 ml of saturated sodium hydrogen carbonate
Was added. Extraction was performed twice with 100 ml of dichloromethane, the extracts were collected, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained oily substance was crystallized from n-hexane to obtain 16.1 mg of crystals of compound 16. The physical properties of this compound are as described above.

Test Example 1 (Cell Growth Inhibitory Action) NIH3T3 cells are often used as a model of normal cells in that they have a contact inhibition phenomenon in proliferation and have no tumorigenicity when inoculated into animals. Is a stock. Using this cell line and a human-derived cancer cell line, the action of the present active substance on cell proliferation was examined by the following method.

NIH3T3 cells and human-derived cancer cell lines HeLaS3 cells, MCF7 cells, SW480 cells and MDA231 cells were seeded using a 96-well multititer plate, and fetal bovine serum-containing modified Darpecco's Eagle medium or fetal calf was prepared. After culturing in serum-containing L15 medium at 37 ° C. under 5% carbon dioxide gas for 4 hours, the active substance was added and the cells were further cultured for 4 days. The number of viable cells after 4 days was measured by Dominic A. X of Scudiero et al.
TT method (Cancer Research 48, 48
27 (1988)) as the absorbance at 490 nm. The absorbance was defined as C, and the absorbance in the system in which the test substance was not added at the same time was defined as D, and the cell growth inhibition rate by the test substance was calculated by the following formula.

[0108]

## EQU00001 ## Cell growth inhibition rate (%) = (D−C) / D × 100 Book required to inhibit growth of various cells by 50%
Concentration of active substance (IC ₅₀Values) are shown in Table 1.

[0109]

[Table 2]

[0110]

EFFECTS OF THE INVENTION The active substance of the present invention hardly inhibits the growth of cells having no tumorigenicity as shown in the above-mentioned Test Example 1, and has the action of specifically inhibiting the growth of human-derived cancer cells. Have. Therefore, it is useful as an antitumor agent with low side effects based on a novel action that has never been seen.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number in the agency FI technical display location C12R 1: 645) (72) Inventor Noriko Chiba Sanshikasei 1000, Kamoshidacho, Midori-ku, Yokohama Inside Research Institute, Inc. (72) Inventor Takashi Mikawa 1000 Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa Sanryo Kasei Co., Ltd.

Claims (2)

[Claims] 1. The following general formula (I): A novel physiologically active substance represented by. 2. Verticilium
The method for producing a novel physiologically active substance according to claim 1, wherein the bacterium that produces the novel physiologically active substance according to claim 1 belonging to the genus Lium) is cultured and the substance is collected from the culture.