JPH06339395A - Novel bioactive substance and method for producing the same - Google Patents

Abstract

(57) [Summary] [Structure] A novel physiologically active substance represented by the following formula (I) was obtained by degrading a culture of a bacterium belonging to the genus Streptomyces. The substance has a proline hydroxylase inhibitory activity. [Chemical 1] [Effect] Since the novel physiologically active substance of the present invention has a proline hydroxylase inhibitory activity at a low concentration, it is useful as a therapeutic drug for organ fibrosis such as liver cirrhosis and pulmonary fibrosis.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel physiologically active substance having a tetracycline skeleton and a method for producing the same,
Specifically, it relates to a novel physiologically active substance having proline hydroxylase inhibitory activity and a method for producing the same.

[0002]

PRIOR ART AND PROBLEMS TO BE SOLVED BY THE INVENTION It is said that organ fibrosis such as liver cirrhosis and pulmonary fibrosis is caused by an abnormal increase in collagen. As one of the fibrosis inhibitors, An inhibitor of proline hydroxylase, which is one of the enzymes involved in collagen biosynthesis, is considered. Inhibition of proline hydroxylation by inhibitors results in low hydroxylated collagen molecules, which are non-functional and can only be released from the cells in small amounts into the extracellular space.
Furthermore, low-hydroxylated collagen cannot be introduced into the collagen matrix, and degradation by proteolysis proceeds very easily. These effects result in an overall decrease in the amount of extracellularly deposited collagen. Therefore, an inhibitor of proline hydroxylase is an effective means for treating a disease in which the deposition of collagen has a significant influence on the clinical picture.

Conventionally, pyridine-2,4-dicarboxylic acid derivatives and pyridine- have been used as inhibitors of proline hydroxylase.
2,5-dicarboxylic acid derivative (Eur. J. Bioch
em. , 138 , 239-245 (1984); JP-A 61-60655; 63-216870; 63.
No. 216871; No. 63-216873; No. 63-
238059) and various peptides (JP-B-52-29740; JP-A-53-27273; JP-A-2-42098, etc.), but newer types of drugs are emerging. Was desired.

[0004]

[Means for Solving the Problems] The present inventors have focused on that microorganisms produce various physiologically active substances such as antibiotics, and have taken many samples in nature and separated from them. a result of studying on culture of a microorganism, found that the culture degradation products in certain strains belonging to Streptomyces (Streptomyces) genus include substances having a prolyl hydroxylase inhibiting activity, its structure The present invention has been completed by clarifying the above.

That is, the gist of the present invention lies in a physiologically active substance represented by the following formula (I) and a method for producing the same.

[0006]

[Chemical 6] [In the above formula, R ¹ is a hydrogen atom,

[0007]

[Chemical 7] Or

[0008]

[Chemical 8] R ² is a hydrogen atom,

[0009]

[Chemical 9] Or

[0010]

[Chemical 10] (R ³ and R ⁴ each independently represent a hydrogen atom or a methyl group)]

The present invention will be described in detail below. Examples of the novel physiologically active substance of the present invention include the following compounds. In the following, the novel physiologically active substance of the present invention may be collectively referred to as "compound group A2121".

A2121-1

[Chemical 11]

[Chemical 12]

A2121-2

[Chemical 13]

[Chemical 14]

A2121-3

[Chemical 15]

[Chemical 16]

A2121-4

[Chemical Formula 17] R ¹ = H

[Chemical 18]

A2121-5

Embedded image R ¹ = H

[Chemical 20]

A2121-6

Embedded image R ¹ = H

[Chemical formula 22]

A2121-7

[Chemical formula 23]

[Chemical formula 24]

A2121-8

[Chemical 25]

[Chemical formula 26]

A2121-9

Embedded image R ¹ = H

[Chemical 28]

A2121-10

Embedded image R ¹ = H

Embedded image R ² = H

The compound group A2121 of the present invention is obtained by, for example, culturing a compound group A2121-producing bacterium belonging to the genus Streptomyces and isolating the compound from the culture solution, or if necessary, hydrolyzing or adding a solvent to the culture. Can be disassembled. In the present invention, the compound group A2121-producing bacterium is a microorganism belonging to the genus Streptomyces, and the compound group A in a sufficient amount to be collected in the culture solution.
Any material having the ability to produce 2121 may be used. Examples of such strains include Streptomyces sp. ( Streptomyces s) newly isolated from soil by the present inventors.
p. ) MCI 2836 (hereinafter abbreviated as "this strain" or "MCI 2836 bacterium"). MCI
The microbiological properties of No. 2836 are as follows.

A) Morphological characteristics As spore-forming medium, glycerin / asparagine agar medium, starch / inorganic salt agar medium, yeast extract / malt extract agar were used and observed after culturing at 27 ° C. for 14 days. The color tone of the colony is yellowish white to yellow to light brown at the initial stage of culture, and greenish brown gray on the 14th day of culture. The basal hyphae are branched and elongated and no fragmentation is observed. It forms abundant aerial hyphae and forms spiral segmental spores with 1 to 5 turns on the aerial hyphae. The surface structure is smooth.

B) Properties on each medium ISP [International Streptomyces Project (International Strep
[Tomices Project)] The properties after culturing on various specified media at 27 ° C. for 14 days are as follows.

[0025]

[Table 1] Type of medium Item Properties of MCI 2836 bacterium ──────────────────────────────────── Loin-nitrate G Good, dark yellow to grayish olive Agar medium RC Grayish olive AM Very poor SP Red brown S No formation ────────────────────── ─────────────── Glucose / Asparagine G Good, Gray brown to brown Agar RC Dark brown AM Rich SP SP Brown S Poor, 1 to 5 revolutions of spiral ────── ────────────────────────────── Glycerin / asparagine G good, light brown gray agar RC dark brown (ISP5) AM rich, Velvety SP Brown S Medium, 1 to 3 turns of spiral ────────── ───────────────────────── Starch / inorganic salt G Good, dark brown to greenish agar medium Light brown gray (ISP4) RC Dark brown AM Abundant, velvety SP Red brown S Medium, 1 to 5 turns of spiral ───────────────────────────────── ───

[0026]

[Table 2] Type of medium Item Properties of MCI 2836 bacterium ───────────────────────────────────── Tyrosine Agar medium G Medium brown gray (modified by Okanishi) RC Brown gray AM Not formed SP Dark brown S Not formed ─────────────────────── ─────────────── Normal agar medium G Poor, Brown gray RC Brown gray AM No formation SP SP Dark brown S No formation ─────────────── ────────────────────── Yeast extract G Good, white-brown gray Malt extract agar medium RC brown gray (ISP2) AM rich, velvety SP Dark brown S Poor, 1 to 4 revolutions of helix ──────────────────────────────────── Automy G Medium, Brown Gray Agar RC Brown Gray (ISP8) AM Rich, Velvety SP Red Brown S S Poor, 1 to 3 turns of spiral ─────────────────── ─────────────────

[0027]

[Table 3] Type of medium Item Properties of MCI 2836 No. bacterium ──────────────────────────────────── Bennett Agar medium G Medium, brown ash to gray RC Brown Gray AM Medium, powdery SP Dark brown S No formation ───────────────────────── ──────────── Calcium malate G Medium, light yellow agar medium RC yellow AM No formation of SP No formation of S No formation of calcium Malate assimilating capacity ───── ──────────────────────────────── G: Growth, RC: Backside color tone, AM: Aerial mycelium, SP: Soluble pigment , S: Sporulation

[0028]

[Table 4] c) Physiological properties 1) Growth temperature range 9 to 40 ° C 2) Growth pH range pH 4 to 8 3) Gelatin liquefaction positive 4) Starch hydrolysis positive 5) Skim milk coagulation positive peptone positive 6 ) Positive melanin-like pigment formation (on peptone / yeast extract / iron agar, tryptone / yeast extract agar and tyrosine agar medium) 7) Nitrate reduction positive 8) Resistance to NaCl Viable at 5%, non-viable at 7% 9) Assimilation of carbon source D-glucose + L-arabinose + D-xylose + D-fructose + sucrose + L-rhamnose + raffinose + inositol + D-mannitol, which showed some growth even on a carbon source-free medium. ++: Use, ±: Doubt to use, −: Do not use

D) Cell component The cell wall amino acid type of this strain MCI2836 is:
Since L, L-diaminopimelic acid was detected by analysis of the whole cell hydrolyzate, it was confirmed to be cell wall type I type. Regarding the cell wall sugar composition, glucose and ribose were detected in the whole cell hydrolyzate, but no characteristic pattern was observed.

E) Taxonomic consideration From the above results, it was revealed that the present strain MCI2836 belongs to the genus Streptomyces . When the strain described in ISP was searched for the identification of the strain, this strain was found to be E. B. Shirring
& D. Gottlieb, Int. J. Syst. B
act. Vol. 18, NO. 2: p165 (198
6), Vol. 22, NO. 4: p323-325 (1
972), Streptomyces
resistomycificus and S. neya
It was suggested to be a species similar to Gawaensis .

However, as shown in the table below, some differences such as peptonization of litmus milk and reduction of nitrate were observed in the culture properties and various physiological properties. Therefore, this strain MCI2836 was treated with Strepto
myces sp . Was identified.

[0032]

[Table 5] S. resistomycificus S. neyagawaensis MCI 2836 (IFO12814) (IFO13477) Growth pH range pH5-11 pH5-11 pH4-8 Litmus milk peptone- ± + coagulation- + + nitrate reduction --- + melanin-like Pigmentation PYFe medium + + + Try-Y medium + + + Tyrosine medium + + + Starch hydrolysis + + + NaCl resistance 5% + + + 7% − − − 10% − − − Carbon source utilization D -Glucose + + + L-arabinose + + + D-xylose + w + w + D-fructose +-+ sucrose + + + L-rhamnose + + + raffinose + + + inositol + + + + D-mannitol + + +

PTFe medium: peptone / yeast extract / iron agar medium Try-Y medium: tryptone, yeast extract agar medium +: positive (strong), + w: positive (weak), −: negative, ±: suspicious MCI 2836 , FERM P-13555 deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology.

In general, Streptomyces spp. Tend to change their properties as seen with other fungi. For example, even if the strain is MCI2836 or a mutant strain (spontaneous or inducible) derived from this strain, a zygote or a gene recombinant, those having the ability to produce the compound group A2121 are all the methods of the present invention. Can be used for

In the present invention, the above-mentioned bacteria are cultivated in a medium containing nutrients that can be utilized by ordinary microorganisms. As the nutrient source, glucose, starch syrup, dextrin, sucrose, starch, sugar concentrate, animal / vegetable oil and the like can be used.
As a nitrogen source, soybean flour, wheat germ, malt powder, corn steep liquor, cottonseed meal, meat extract, peptone,
Yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used. In addition, it is effective to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions, if necessary. In addition, an organic substance and / or an inorganic substance capable of supporting the growth of the bacterium and promoting the production of the compound group A2121 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 group A2121 varies depending on the medium and culture conditions,
Usually, the accumulation reaches the optimum level within 3 to 10 days, and thus the culture is stopped when the accumulated amount of the compound group A2121 in the culture becomes maximum.

Since the compound group A2121 is a fat-soluble substance, when the compound group A2121 is purified from the culture,
It can be performed by utilizing the characteristic. That is, a solvent extraction method using ethyl acetate, chloroform, etc .; silica gel, alumina, okdadecyl silica gel, synthetic adsorbents such as Diaion HP-20 (manufactured by Mitsubishi Kasei), and Sephadex LH-20 (manufactured by Pharmacia). Column chromatography with a gel filtration agent or the like; preparative thin layer chromatography with silica gel or the like as a carrier, and recrystallization with water or various organic solvents in some cases are effective.

The above-mentioned A2121-4 to 10 are A2.
It can also be obtained by hydrolyzing or solvolyzing 121-1 to 123-1. Specifically, the decomposition of A2121-1 to 3 is usually carried out by reacting under acidic conditions in a solvent such as water, ethanol or methanol.
In order to increase the solubility of A2121-1 to A2121-1, it is effective to add a solvent such as dimethyl sulfoxide or acetonitrile to the reaction solution. As the acid used here, commonly used hydrochloric acid, acetic acid, sulfuric acid, trifluoroacetic acid or the like can be used. A2121 used for disassembly
-1 to 3 may be a culture solution as it is, a crudely purified product of the culture solution, a mixture with A2121-1 to 10, or isolated and purified A2121-1 to A212-1 to 3. A
Since 2121-4 to 10 are fat-soluble substances, when isolating and purifying them from the reaction product, their characteristics can be utilized and the same method as described above can be used.

The present inventors produced the compound group A2121 by using the means and method described above, and investigated the physicochemical properties thereof. As a result, the compound group A2121 is represented by the structural formula (I). It was clarified that this compound is a new compound and that this compound is a novel compound. Compound group A2121 can be produced in this manner, and its physical properties are as follows.

[0040]

Table 6 A2121-1 1) Appearance: yellow powder 2) Melting point: 158-159.5 ° C 3) Molecular weight: 1199 (SIMS negative)
M ^-) 4) Molecular _{formula: C 59 H 77 NO 25 5} ) Ultraviolet absorption spectrum: acetonitrile solution _{λ MAX: 243nm (ε = 3.6} × 10 4) 433nm (ε = 5.1 × 10 3) 6) CD spectrum: in acetonitrile solution λ _EXT : 245 nm (Δε = 6.7) 259 nm (Δε = 0.0) 260 nm (Δε = −1.0) 264 nm (Δε = 0.0) 285 nm (Δε = 27.4) 314 nm (Δε = 0.0) 355 nm (Δε = −3.7) 7) Infrared absorption spectrum: KBr method ν (cm ⁻¹ ): 3414, 2936, 1732, 169
0,1628,1595,1115,1067,100
1 8) hydrogen jittery magnetic resonance spectrum: dimethylsulfoxide -d ₆ solution _{δ ppm: 0.63 (3H, brs} ); 0.99 (9H, d, J = 6.5Hz); 1.10 (3H, d,
J = 6.0Hz); 1.11 (3H, d, J = 5.5Hz); 2.91 (1H, m); 3.03 (3H, m);
3.12 (2H, m); 3.79 (1H, q, J = 7.0Hz); 3.83 (1H, q, J = 6.5Hz);
3.89 (3H, s); 4.02 (1H, q, J = 6.5Hz) 4.13 (1H, brs); 4.42 (1H, d, J = 7.0Hz); 4.61 (1H, d, J = 5.5H
z); 4.64 (1H, d, J = 6.5Hz); 4.74 (3H, m); 4.80 (1H, brs); 6.40
(1H, s); 7.35 (1H, s); 9.21 (1H, brs); 9.46 (1H, brs,); 12.76
(1H, brs,); 13.42 (1H, s); 18.03 (1H, brs)

[0041]

9) Carbon nuclear magnetic resonance spectrum: δ _{ppm in} dimethyl sulfoxide-d ₆ solution: 190.26 (s); 188.81 (s); 178.43 (s); 173.11 (s); 16
8.09 (s); 166.63 (s); 161.75 (s); 160.76 (s); 152.26 (s); 13
3.45 (s); 124.26 (s); 119.16 (d); 112.20 (s); 110.20 (d); 10
2.44 (d); 101.79 (d); 99.68 (s); 98.51 (d); 98.39 (d); 98.28
(d); 98.21 (d); 84.91 (s); 78.77 (d); 78.17 (d); 75.42 (d); 7
4.17 (d); 74.01 (d); 73.81 (d); 73.67 (d); 69.99 (d); 69.92
(d); 66.34 (d); 66.15 (d); 64.85 (d); 57.03 (q); 41.73 (t); 4
1.52 (d); 30.97 (t); 30.76 (t); 30.56 (t); 29.95 (t); 28.79
(t); 27.38 (t); 24.30 (t); 24.10 (t); 23.93 (t); 23.75 (t); 2
2.43 (t); 18.32 (q); 18.04 (q); 17.58 (q); 16.94 (q); 16.92
(q); 16.76 (q)

[0042]

Table 8 A2121-2 1) Appearance: yellow powder 2) Melting point: 162-164 ° C 3) Molecular weight: 1213 (SIMS negative)
M ^-) 4) Molecular _{formula: C 60 H 79 NO 25 5} ) Ultraviolet absorption spectrum: acetonitrile solution _{λ MAX: 243nm (ε = 3.7} × 10 4) 433nm (ε = 5.6 × 10 3)

[0043]

6) CD spectrum: λ _{EXT in} acetonitrile solution: 248 nm (Δε = 7.4) 258 nm (Δε = 0.0) 262 nm (Δε = −3.3) 267 nm (Δε = 0.0) 285 nm ( Δε = 28.7) 314 nm (Δε = 0.0) 355 nm (Δε = −3.9) 7) Infrared absorption spectrum: KBr method ν (cm ⁻¹ ): 3412, 2936, 1732, 169
0,1630,1595,1115,1069,100
3 8) hydrogen jittery magnetic resonance spectrum: dimethylsulfoxide -d ₆ solution _{δ ppm: 0.63 (3H, brs} ); 0.99 (9H, d, J = 6.0Hz); 1.10 (3H, d,
J = 5.5Hz); 1.11 (3H, d, J = 5.5Hz); 1.26 (3H, d, J = 7.5Hz); 2.9
2 (1H, m); 3.03 (3H, m); 3.11 (2H, m); 3.79 (1H, q, J = 6.5Hz);
3.83 (1H, q, J = 6.5Hz); 3.89 (3H, s); 4.02 (1H, q, J = 6.5Hz) 4.
13 (1H, brs); 4.42 (1H, d, J = 7.5Hz); 4.60 (1H, d, J = 8.0Hz);
4.64 (1H, d, J = 6.0Hz); 4.73 (3H, m); 4.80 (1H, brs); 6.40 (1
H, s); 7.35 (1H, s); 9.20 (1H, brs); 9.46 (1H, brs,); 12.79 (1
H, brs,); 13.42 (1H, s); 18.02 (1H, brs)

[0044]

TABLE 10 9) carbon jittery magnetic resonance spectrum: dimethylsulfoxide -d ₆ solution _{δ ppm: 190.27 (s);} 188.83 (s); 178.44 (s); 173.13 (s); 17
1.23 (s); 171.20 (s); 169.89 (s); 169.84 (s); 161.77 (s); 16
0.76 (s); 152.27 (s); 133.46 (s); 124.27 (s); 119.17 (d); 11
2.20 (s); 110.21 (d); 102.44 (d); 101.80 (d); 99.68 (s); 98.
52 (d); 98.40 (d); 98.23 (d); 84.92 (s); 78.78 (d); 78.18
(d); 75.43 (d); 74.14 (d); 74.02 (d); 73.82 (d); 73.69 (d); 7
0.00 (d); 69.86 (d); 66.35 (d); 66.16 (d); 64.97 (d); 64.85
(d); 56.62 (q); 45.78 (q); 45.65 (q); 41.54 (d); 30.97 (t); 3
0.76 (t); 30.56 (t); 29.89 (t); 28.97 (t); 28.89 (t); 27.39
(t); 24.29 (t); 24.10 (t); 23.93 (t); 23.76 (t); 22.43 (t); 2
2.35 (t); 18.31 (q); 18.03 (q); 17.57 (q); 16.93 (q); 16.91
(q); 16.76 (q); 16.68 (q); 13.53 (q); 13.51 (q)

[0045]

[Table 11] A2121-3 1) Appearance: Yellow powder 2) Melting point: 165-167 ° C 3) Molecular weight: 1227 (SIMS negative)
M ^-) 4) Molecular _{formula: C 61 H 81 NO 25 5} ) Ultraviolet absorption spectrum: acetonitrile solution _{λ MAX: 242nm (ε = 3.7} × 10 4) 434nm (ε = 5.4 × 10 3)

[0046]

6) CD spectrum: λ _{EXT in} acetonitrile solution: 245 nm (Δε = 7.6) 257 nm (Δε = 0.0) 260 nm (Δε = −3.5) 267 nm (Δε = 0.0) 285 nm ( Δε = 29.0) 314 nm (Δε = 0.0) 357 nm (Δε = −3.6) 7) Infrared absorption spectrum: KBr method ν (cm ⁻¹ ): 3395, 2936, 1732, 169
0,1630,1595,1115,1069,100
3 8) hydrogen jittery magnetic resonance spectrum: dimethylsulfoxide -d ₆ solution _{δ ppm: 0.63 (3H, brs} ); 0.97 (3H, d, J = 6.5Hz); 0.99 (6H, d,
J = 6.5Hz); 1.10 (3H, d, J = 6.0Hz); 1.11 (3H, d, J = 6.0Hz); 1.3
2 (3H, s); 1.33 (3H, s); 2.92 (1H, m); 3.03 (3H, m); 3.11 (2H,
m); 3.79 (1H, q, J = 6.5Hz); 3.83 (1H, q, J = 6.5Hz); 3.89 (3H,
s); 4.02 (1H, q, J = 6.5Hz); 4.12 (1H, brs); 4.41 (1H, d, J = 7.5
Hz); 4.60 (1H, d, J = 8.0Hz); 4.64 (1H, d, J = 6.5Hz); 4.72 (3H,
m); 4.80 (1H, brs); 6.39 (1H, s); 7.35 (1H, s); 9.20 (1H, br
s); 9.46 (1H, brs,); 12.80 (1H, brs,); 13.42 (1H, s); 18.03
(1H, brs)

[0047]

9) Carbon nuclear magnetic resonance spectrum: δ _{ppm in} dimethylsulfoxide-d ₆ solution: 190.26 (s); 188.83 (s); 178.42 (s); 173.72 (s); 17
3.10 (s); 172.12 (s); 161.74 (s); 160.75 (s); 152.25 (s); 13
3.44 (s); 124.25 (s); 119.15 (d); 112.19 (s); 110.20 (d); 10
2.43 (d); 101.77 (d); 99.67 (s); 98.49 (d); 98.37 (d); 98.19
(d); 98.11 (d); 84.91 (s); 78.77 (d); 78.16 (d); 75.41 (d); 7
4.05 (d); 73.99 (d); 73.78 (d); 73.66 (d); 69.98 (d); 69.80
(d); 66.34 (d); 66.14 (d); 64.95 (d); 57.03 (q); 49.30 (s); 4
1.51 (d); 30.97 (t); 30.75 (t); 30.56 (t); 29.94 (t); 29.88
(t); 28.95 (t); 27.37 (t); 24.29 (t); 24.09 (t); 23.92 (t); 2
3.78 (t); 22.53 (q); 22.49 (q); 22.37 (t); 22.30 (t); 18.32
(q); 18.04 (q); 17.58 (q); 16.94 (q); 16.92 (q); 16.75 (q)

[0048]

[Table 14] A2121-4 1) Appearance: Yellow powder 2) Melting point: 157-159 ° C 3) Molecular weight: 971 (SIMS negative)
M ^-) 4) Molecular _{formula: C 47 H 57 NO 21 5} ) Ultraviolet absorption spectrum: acetonitrile solution _{λ MAX: 242nm (ε = 3.1} × 10 4) 433nm (ε = 4.0 × 10 3) 6) CD spectrum: in acetonitrile solution λ _EXT : 250 nm (Δε = 7.1) 286 nm (Δε = 20.5) 314 nm (Δε = 0.0) 351 nm (Δε = -3.3)

[0049]

Table 15 7) Infrared absorption spectrum: KBr method ν (cm ⁻¹ ): 3403, 2936, 1734, 169
0, 1628, 1595, 1248, 1224, 111
5,1071,10018 8) Hydrogen nuclear magnetic resonance spectrum: δ _{ppm in} dimethylsulfoxide-d ₆ solution: 0.64 (3H, d, J = 5.0Hz); 0.99 (9H, d, J = 6.5Hz); 2.68
(1H, dd, J = 12.0,4.0Hz); 3.03 (2H, m); 3.40 (2H, s); 3.46 (1
H, brs); 3.79 (1H, q, J = 7.0Hz); 3.83 (1H, q, J = 6.5Hz); 3.89
(3H, s) 4.02 (1H, q, J = 6.5Hz); 4.13 (1H, brs); 4.62 (1H, d, J = 8.0H
z); 4.74 (3H, m); 4.79 (1H, brs); 6.39 (1H, s); 6.93 (1H, br
s); 7.37 (1H, s); 9.17 (1H, brs); 9.40 (1H, brs,); 12.77 (1H,
brs,); 13.38 (1H, s); 17.99 (1H, brs) 9) Carbon nuclear magnetic resonance spectrum: dimethylsulfoxide-d _{6 in} solution δ _ppm : 192.31 (s); 190.04 (s); 178.51 (s) ; 173.01 (s); 16
8.07 (s); 166.63 (s); 161.80 (s); 160.63 (s); 151.21 (s); 13
3.34 (s); 124.04 (s); 119.26 (d); 112.50 (s); 110.30 (d); 10
1.74 (d); 98.35 (d); 98.24 (d); 98.17 (d); 98.00 (s); 78.15
(d); 77.90 (s); 74.12 (d); 73.77 (d); 73.63 (d); 69.90 (d); 6
6.32 (d); 66.12 (d); 64.82 (d); 57.01 (q); 43.50 (d); 41.73
(t); 29.97 (t); 28.93 (t); 27.11 (t); 24.27 (t); 24.07 (t); 2
3.90 (t); 23.73 (t); 22.40 (t); 17.56 (q); 16.95 (q); 16.92
(q); 16.76 (q)

[0050]

[Table 16] A2121-5 1) Appearance: Yellow powder 2) Melting point: 161-162.5 ° C 3) Molecular weight: 985 (SIMS negative)
M ^-) 4) Molecular _{formula: C 48 H 59 NO 21 5} ) Ultraviolet absorption spectrum: acetonitrile solution _{λ MAX: 242nm (ε = 3.7} × 10 4) 433nm (ε = 5.0 × 10 3) 6) CD spectrum: in acetonitrile solution λ _EXT : 250 nm (Δε = 10.4) 285 nm (Δε = 24.2) 314 nm (Δε = 0.0) 357 nm (Δε = −4.5) 7) Infrared absorption spectrum: KBr Method ν (cm ^-1 ): 3401, 2938, 1732, 169
0, 1628, 1595, 1248, 1223, 111
3,1071,1003 8) Hydrogen nuclear magnetic resonance spectrum: δ _{ppm in} dimethyl sulfoxide-d ₆ solution: 0.63 (3H, d, J = 5.5Hz); 0.99 (9H, m); 1.26 (3H, d, J =
7.5Hz) 2.68 (1H, dd, J = 12.5,4.5Hz); 3.03 (2H, m); 3.49 (H, q, J = 7.5
Hz) 3.79 (1H, q, J = 6.5Hz); 3.83 (1H, q, J = 6.5Hz); 3.89 (3H, s) 4.02 (1H, q, J = 6.0Hz); 4.13 (1H, brs) 4.62 (1H, d, J = 8.5H
z); 4.74 (3H, m); 4.80 (1H, brs); 6.39 (1H, s); 6.95 (1H, br
s); 7.37 (1H, s); 9.18 (1H, brs); 9.40 (1H, brs,); 12.78 (1H,
brs,); 13.38 (1H, s); 17.99 (1H, brs)

[0051]

[Table 17] 9) carbon jittery magnetic resonance spectrum: dimethylsulfoxide -d ₆ solution _{δ ppm: 192.31 (s);} 190.03 (s); 178.51 (s); 173.02 (s); 17
1.20 (s); 171.15 (s); 169.87 (s); 169.81 (s); 161.79 (s); 16
0.63 (s); 151.20 (s); 133.33 (s); 124.03 (s); 119.25 (d); 11
2.49 (s); 110.30 (d); 101.73 (d); 98.34 (s); 98.17 (d); 97.9
8 (s); 78.14 (d); 77.89 (s); 74.07 (d); 73.75 (d); 73.62 (d);
69.82 (d); 66.31 (d); 66.11 (d); 64.92 (d); 64.81 (d); 57.01
(q); 45.73 (d); 45.60 (d); 43.50 (d); 29.96 (t); 28.93 (t); 2
7.11 (t); 24.26 (t); 24.06 (t); 23.90 (t); 23.74 (t); 22.39
(t); 22.31 (t); 17.55 (q); 16.95 (q); 16.92 (q); 16.77 (q); 1
6.69 (q); 13.54 (q); 13.52 (q)

[0052]

[Table 18] A2121-6 1) Appearance: Yellow powder 2) Melting point: 167-168.5 ° C 3) Molecular weight: 999 (SIMS negative)
M ^-) 4) Molecular _{formula: C 49 H 61 NO 21 5} ) Ultraviolet absorption spectrum: acetonitrile solution _{λ MAX: 242nm (ε = 3.7} × 10 4) 431nm (ε = 5.1 × 10 3) 6) CD spectrum: in acetonitrile solution λ _EXT : 248 nm (Δε = 10.1) 286 nm (Δε = 25.2) 313 nm (Δε = 0.0) 357 nm (Δε = −4.6)

[0053]

Table 19 7) Infrared absorption spectrum: KBr method ν (cm ⁻¹ ): 3393, 2938, 1732, 169
0, 1628, 1595, 1248, 1223, 111
5,1071,1003 8) Hydrogen nuclear magnetic resonance spectrum: δ _{ppm in} dimethylsulfoxide-d ₆ solution: 0.63 (3H, d, J = 5.0Hz); 0.98 (3H, d, J = 6.5Hz); 0.99
(3H, d, J = 6.5Hz); 1.33 (3H, s); 1.32 (3H, s) 2.68 (1H, dd, J = 12.5,5.0Hz); 3.03 (2H, m); 3.46 (1H, brs ) 3.79 (1H, q, J = 6.5Hz); 3.83 (1H, q, J = 6.5Hz); 3.89 (3H, s) 4.03 (1H, q, J = 6.5Hz); 4.13 (1H, brs); 4.62 (1H, d, J = 8.5H
z); 9) Carbon nuclear magnetic resonance spectrum: δ _{ppm in} dimethyl sulfoxide-d ₆ solution: 192.30 (s); 190.03 (s); 178.50 (s); 173.69 (s); 17
3.02 (s); 172.10 (s); 161.79 (s); 160.63 (s); 151.20 (s); 13
3.33 (s); 124.04 (s); 119.25 (d); 112.49 (s); 110.30 (d); 10
1.73 (d); 98.34 (d); 98.16 (d); 98.08 (d); 97.98 (s); 78.14
(d); 77.89 (s); 74.01 (d); 73.75 (d); 73.62 (d); 69.78 (d); 6
6.31 (d); 66.11 (d); 64.91 (d); 57.00 (q); 49.28 (s); 43.50
(d); 29.96 (t); 28.92 (t); 27.10 (t); 24.26 (t); 24.06 (t); 2
3.90 (t); 23.75 (t); 22.52 (q); 22.47 (q); 22.27 (q); 17.55
(q); 16.94 (q); 16.92 (q); 16.74 (q);

[0054]

Table 20 A2121-7 1) Appearance: yellow powder 2) Melting point: 178-181 ° C 3) Molecular weight: 771 (SIMS negative)
M ^-) 4) Molecular formula: C ₃₈ H ₄₅ NO ₁₆

[0055]

Table 21 5) Ultraviolet absorption spectrum: in acetonitrile solution λ _MAX : 242 nm (ε = 3.5 × 10 ⁴ ) 435 nm (ε = 5.1 × 10 ³ ) 6) CD spectrum: in acetonitrile solution λ _EXT : 246 nm (Δε = 6.8) 257 nm (Δε = 0.0) 263 nm (Δε = −4.2) 268 nm (Δε = 0.0) 285 nm (Δε = 27.8) 314 nm (Δε = 0.0) 356 nm (Δε = −3.7) 7) Infrared absorption spectrum: KBr method ν (cm ⁻¹ ): 3424, 2934, 2872, 169
0, 1628, 1595, 1248, 1227, 116
5, 1113, 1067, 988 8) Hydrogen nuclear magnetic resonance spectrum: δ _{ppm in} dimethyl sulfoxide-d ₆ solution: 0.65 (3H, d, J = 4.5Hz); 1.10 (3H, d, J = 5.5Hz); 1.11
(3H, d, J = 5.0Hz); 2.90 (3H, m); 3.03 (1H, m); 3.12 (2H, m); 3.
37 (1H, dd, J = 11.5,6.0Hz); 3.42 (1H, d, J = 18.5Hz); 3.74 (1
H, dd, J = 18.5,5.5Hz); 3.89 (3H, s); 4.12 (1H, brs); 4.42 (1
H, d, J = 7.5Hz); 4.57 (1H, d, J = 7.0Hz); 4.64 (1H, d, J = 7.0H
z); 6.40 (1H, s); 7.34 (1H, s); 9.20 (1H, brs); 9.45 (1H, br
s); 13.42 (1H, s); 18.04 (1H, brs)

[0056]

9) Carbon nuclear magnetic resonance spectrum: δ _{ppm in} dimethyl sulfoxide-d ₆ solution: 190.24 (s); 188.81 (s); 178.41 (s); 173.09 (s); 16
1.71 (s); 160.73 (s); 152.28 (s); 133.42 (s); 124.23 (s); 11
9.15 (s); 112.17 (s); 110.20 (d); 102.42 (d); 101.98 (d); 9
9.71 (s); 98.41 (d); 78.78 (d); 75.40 (d); 75.32 (d); 73.98
(d); 69.97 (d); 57.03 (q); 41.50 (d); 30.96 (t); 30.75 (t); 3
0.54 (t); 30.13 (t); 29.88 (t); 27.38 (t); 18.32 (q); 18.04
(q); 17.71 (q);

[0057]

[Table 23] A2121-8 1) Appearance: Yellow powder 2) Melting point: 190.5-192.5 ° C 3) Molecular weight: 657 (SIMS negative)
M ^-) 4) Molecular _{formula: C 32 H 35 NO 14 5} ) Ultraviolet absorption spectrum: acetonitrile solution _{λ MAX: 242nm (ε = 3.7} × 10 4) 434nm (ε = 5.4 × 10 3) 6) CD spectrum: in acetonitrile solution λ _EXT : 247 nm (Δε = 7.2) 257 nm (Δε = 0.0) 263 nm (Δε = −3.8) 268 nm (Δε = 0.0) 286 nm (Δε = 29.1) 314 nm (Δε = 0.0) 354 nm (Δε = −4.1)

[0058]

Table 24 7) Infrared absorption spectrum: KBr method ν (cm ^-1 ): 3412, 2934, 2872, 168
8, 1628, 1595, 1248, 1227, 116
3,1113,1057,990 8) hydrogen jittery magnetic resonance spectrum: dimethylsulfoxide -d ₆ solution _{δ ppm: 0.64 (3H, d} , J = 4.5Hz); 1.12 (3H, d, J = 6.0Hz); 2.90
(3H, m); 3.10 (1H, dd, J = 12.5,4.5Hz); 3.26 (1H, dq, J = 11.5,
6.0Hz); 3.42 (1H, d, J = 18.0Hz); 3.75 (1H, dd, J = 18.5,5.5H
z); 3.89 (3H, s); 4.12 (1H, brs); 4.57 (1H, d, J = 9.5Hz); 4.62
(1H, d, J = 8.5Hz); 4.67 (1H, d, J = 5.0Hz); 4.73 (1H, d, J = 5.5H)
z); 6.40 (1H, s); 7.34 (1H, s); 9.19 (1H, brs); 9.46 (1H, br
s); 13.42 (1H, brs); 18.05 (1H, brs) 9) Carbon nuclear magnetic resonance spectrum: dimethylsulfoxide-d _{6 in} solution δ _ppm : 190.25 (s); 188.92 (s); 178.42 (s); 173.07 (s); 16
1.63 (s); 160.74 (s); 152.27 (s); 133.42 (s); 124.26 (s); 11
9.08 (d); 112.15 (s); 110.20 (d); 101.97 (d); 99.71 (s); 98.
64 (d); 75.73 (d); 75.28 (d); 69.95 (d); 69.68 (d); 57.03
(q); 41.53 (d); 31.09 (t); 30.75 (t); 30.72 (t); 30.11 (t); 2
7.36 (t); 18.22 (q); 17.69 (q);

[0059]

Table 25 A2121-9 1) Appearance: Yellow powder 2) Melting point: 205-206 ° C (decomposition) 3) Molecular weight: 543 (SIMS negative)
M ^-) 4) Molecular formula: C ₂₆ H ₂₅ NO ₁₂

[0060]

Table 26 5) Ultraviolet absorption spectrum: in acetonitrile solution λ _MAX : 241 nm (ε = 3.5 × 10 ⁴ ) 433 nm (ε = 5.0 × 10 ³ ) 6) CD spectrum: in acetonitrile solution λ _EXT : 250 nm (Δε = 10.3) 286 nm (Δε = 25.3) 313 nm (Δε = 0.0) 355 nm (Δε = −4.6) 7) Infrared absorption spectrum: KBr method ν (cm ⁻¹ ): 3409 , 2932, 1686, 165
3,1626,1595,1250,1225,111
1,1069,10538 8) Hydrogen nuclear magnetic resonance spectrum: δ _{ppm in} dimethyl sulfoxide-d ₆ solution: 0.65 (3H, d, J = 4.0Hz); 1.26 (1H, m); 1.41 (1H, m);
1.85 (2H, m); 2.67 (1H, dd, J = 12.0,4.0Hz); 2.90 (2H, m); 3.3
2 (1H, dd, J = 18.0,5.0Hz); 3.40 (1H, d, J = 18.0Hz); 3.89 (3H,
s); 4.14 (1H, brs); 4.59 (1H, d, J = 9.0Hz); 4.67 (1H, d, J = 4.0
Hz); 6.38 (1H, s); 6.93 (1H, brs); 7.36 (1H, s); 9.17 (1H, br
s); 9.40 (1H, brs ); 13.37 (1H, s); 18.01 (1H, brs) 9) carbon jittery magnetic resonance spectrum: dimethylsulfoxide -d ₆ solution _{δ ppm: 192.28 (s);} 190.02 (s ); 178.54 (s); 173.03 (s); 16
1.80 (s); 160.63 (s); 151.24 (s); 133.34 (s); 124.04 (s); 11
9.27 (d); 112.50 (s); 110.31 (d); 101.97 (d); 98.64 (s); 77.
89 (s); 75.31 (d); 72.81 (d); 69.98 (d); 57.01 (q); 43.52
(d); 30.76 (t); 30.16 (t); 27.13 (t); 17.70 (q);

[0061]

[Table 27] A2121-10 1) Appearance: Red-yellow powder 2) Melting point: 198-202 ° C 3) Molecular weight: 429 (FD-MS M ⁺ ) 4) Molecular formula: C ₂₀ H ₁₅ NO ₁₀ 5) Ultraviolet absorption spectrum : Acetonitrile solution λ _MAX : 241 nm (ε = 3.4 × 10 ⁴ ) 431 nm (ε = 4.9 × 10 ³ ) 6) CD spectrum: Acetonitrile solution λ _EXT : 248 nm (Δε = 7.9) 285 nm ( Δε = 34.7) 312 nm (Δε = 0.0) 354 nm (Δε = −4.13) 7) Infrared absorption spectrum: KBr method ν (cm ⁻¹ ): 3380,1672,1622,159
3,1250,1223,1113 8) hydrogen jittery magnetic resonance spectrum: dimethylsulfoxide -d ₆ solution _{δ ppm: 2.63 (1H, dd} , J = 12.0,4.0Hz); 3.34 (1H, d, J = 18.5H
z); 3.40 (1H, dd, J = 18.5,4.0Hz); 3.89 (4H, m); 5.92 (1H, br
s); 6.37 (1H, s); 6.89 (1H, brs); 7.46 (1H, s); 9.11 (1H, br
s); 9.38 (1H, brs,); 13.36 (1H, s); 18.16 (1H, brs) 9) Carbon nuclear magnetic resonance spectrum: dimethyl sulfoxide-d ₆ solution at 70 ° C. δ _ppm : 26.81 (t); 44.82 (d); 56.62 (q); 68.04 (d); 77.71
(s); 97.72 (s); 109.92 (d); 112.72 (s); 118,23 (d); 124.01
(s); 133.52 (s); 150.07 (s); 160.53 (s); 161.51 (s); 172.91
(s); 178.22 (s); 189.49 (s); 191.95 (s); 193.33 (s); 195.07
(s)

[0062]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples unless it exceeds the gist. Example 1 Malt powder 3.5%, corn starch 3.0%, corn steep liquor 1.5%, Pharmamedia 1.5%,
A medium (pH 7.0) containing 0.5% of sun grains and 0.2% of calcium carbonate is dispensed into 40 200 ml Erlenmeyer flasks by 40 ml, and sterilized under high pressure at 121 ° C. for 20 minutes.

To this, one platinum loop of MCI2836 was inoculated and shake-cultured at 27 rpm at 210 rpm for 3 days. Separately, a medium having the same composition as above is prepared, 80 ml of the medium is dispensed into 100 500 ml Erlenmeyer flasks, and autoclaved at 121 ° C. for 20 minutes. 4 ml each of the seed culture solution was inoculated into this main fermentation medium,
Incubate with shaking at 210 rpm for 5 days at 0 ° C. The obtained culture is centrifuged to obtain a culture supernatant of 2.0.
1 and 3.2 l of cultured cells.

Concentrated hydrochloric acid was added to the culture supernatant obtained here,
After adjusting the pH to 2, extraction was performed with an equal amount of ethyl acetate, the extract was concentrated under reduced pressure, and 5.74 g of oily substance A was extracted.
Got On the other hand, 4 liters of acetone was added to the cultured cells for extraction, filtered off with Celite, and the acetone was distilled off under reduced pressure. Concentrated hydrochloric acid was added to this residue to adjust the pH to 2, and the mixture was extracted with an equal amount of ethyl acetate. The extract was concentrated under reduced pressure to 1
2.11 g of oily substance B was obtained. The oily substance A and the oily substance B were combined and subjected to reverse layer partition chromatography using octadecyl silica gel. This oily substance was
GEL ODS1MY (manufactured by Mitsubishi Kasei Co., Ltd.) was loaded onto a column packed with 500 g and equilibrated with an acetonitrile-water mixed solution (3: 7), and then acetonitrile-water mixed solution (3: 7) 0.75.
Wash with l. Then, it was eluted with 1.25 l of acetonitrile, and the eluate was concentrated to dryness under reduced pressure to obtain 12.87 g of an oily substance.

The oily substance was again subjected to reverse layer partition chromatography using octadecyl silica gel. Oily substance MCI GEL ODS 1MY
Sprinkle on 20 g, dry under reduced pressure, then MCI GEL ODS
The column was loaded with 480 g of 1MY and equilibrated with an acetonitrile-water mixture (3: 7). The mixture was washed with 1.25 l of an acetonitrile-water mixed liquid (3: 7), washed with 1.25 l of an acetonitrile-water mixed liquid (2: 3), and further washed with 1.25 l of an acetonitrile-water mixed liquid (1: 1), and then acetonitrile-water. Elution was performed with 0.75 l of the mixed liquid (3: 2), and acetonitrile was distilled off under reduced pressure. The residue was extracted with ethyl acetate, dried over magnesium sulfate, and then concentrated under reduced pressure,
9.32 g of oily substance was obtained. 0.84 g of this oily substance was dissolved in 3 ml of acetonitrile and
rtsil PREP-ODS (50 mm x 250 m
m) (manufactured by GL Science Co.) was subjected to preparative high performance liquid chromatography. Acetonitrile-water mixed solution containing 0.002% trifluoroacetic acid (11: 9) 325 m
After washing with 1, the mixture was eluted with an acetonitrile-water mixture (11: 9) containing 0.002% trifluoroacetic acid, and the eluate was collected in 25 ml fractions.
4 to 26 is C-2, 28 to 31 is C-3, 34 to 37 is C-4, 38 to 43 is C-5, 49 to 54 is C-6, 5
7-62 were collected as C-7. In the same manner, 5 more times were collected, the obtained eluates C-1 to 7 were collected, acetonitrile was distilled off under reduced pressure, extraction was performed twice with an equal amount of ethyl acetate, and the extract was dried over magnesium sulfate. , Concentrated to dryness under reduced pressure to C-1 to D-1 (0.61 g), C
-2 to D-2 (0.19g), C-3 to D-3
(0.26 g), C-4 to D-4 (0.19 g), C
-5 to D-5 (0.27 g), C-6 to D-6
(0.17 g) and D-7 (0.22 g) was obtained from C-7.

102.8 mg of the fraction D-3 was dissolved in 3 ml of acetonitrile, and Inertsil PREP was added.
-Prepared high performance liquid chromatography equipped with ODS (50 mm x 250 mm). [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (11:
9) After washing with 800 ml, [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (11: 9) 1
Elute with 75 ml and concentrate the eluate to dryness under reduced pressure to give A2.
72.9 mg of a fraction containing 121-1 was obtained. Recrystallization was performed from a mixed solution of ethyl acetate and n-hexane to give A2121-
156.1 mg was obtained.

100.6 mg of fraction D-5 was dissolved in 3 ml of acetonitrile, and Inertsil PREP was added.
-Prepared high performance liquid chromatography equipped with ODS (50 mm x 250 mm). [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (11:
9) After washing with 1075 ml, [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (11: 9)
Elute with 150 ml, concentrate the eluate to dryness under reduced pressure, and
76.2 mg of a fraction containing 2121-2 was obtained. Recrystallization was performed from a mixed solution of ethyl acetate and n-hexane to give A2121.
-2 49.0 mg was obtained.

101.0 mg of the fraction D-7 was dissolved in 3 ml of acetonitrile, and Inertsil PREP was added.
-Prepared high performance liquid chromatography equipped with ODS (50 mm x 250 mm). [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (11:
9) After washing with 1500 ml, [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (11: 9)
Elute with 200 ml and concentrate the eluate to dryness under reduced pressure to give A
67.7 mg of a fraction containing 2121-3 was obtained. Recrystallization was performed from a mixed solution of ethyl acetate and n-hexane to give A2121.
-3 59.4 mg was obtained.

99.7 mg of the fraction D-2 was dissolved in 3 ml of acetonitrile, and Inertsil PREP-
It was subjected to preparative high performance liquid chromatography equipped with ODS (50 mm × 250 mm). [Acetonitrile containing 0.002% trifluoroacetic acid] -water mixture (11: 9)
After washing with 700 ml, [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (11: 9) 200
Elute with mL and concentrate the eluate to dryness under reduced pressure to give A212.
67.1 mg of a fraction containing 1-4 was obtained. Ethyl acetate and n
-Recrystallization from a mixed solution of hexane and A2121-4
51.2 mg was obtained.

Fraction D-4 (0.19 g) was dissolved in acetonitrile (3 ml), 2 ml of which was added to Inertsil.
It was subjected to preparative high performance liquid chromatography equipped with PREP-ODS (50 mm × 250 mm). [0.00
After washing with 1000 ml of 2% trifluoroacetic acid-containing acetonitrile] -water mixture (11: 9), [0.002
% Acetonitrile containing trifluoroacetic acid] -water mixture (1
1: 9) Eluted with 100 ml. The remaining acetonitrile solution was similarly fractionated, and the obtained eluates were combined and concentrated to dryness under reduced pressure to obtain 104.43 mg of a fraction containing A2121-5. Of this, 70.0 mg was recrystallized from a mixed solution of ethyl acetate and n-hexane,
A2121-5 of 61.7 mg was obtained.

100.1 mg of the fraction D-6 was dissolved in 3 ml of acetonitrile, and Inertsil PREP was added.
-Prepared high performance liquid chromatography equipped with ODS (50 mm x 250 mm). [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (11:
9) After washing with 1325 ml, [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (11: 9)
Elute with 175 ml and concentrate the eluate to dryness under reduced pressure to give A
61.9 mg of the fraction containing 2121-6 was obtained. Recrystallization was performed from a mixed solution of ethyl acetate and n-hexane to give A2121.
-6 45.0 mg was obtained.

Fraction D-1 was dissolved in 4 ml of acetonitrile, impurities were filtered off, and 2 ml of Inertsil was added.
It was subjected to preparative high performance liquid chromatography equipped with PREP-ODS (50 mm × 250 mm). [0.00
2% trifluoroacetic acid-containing acetonitrile] -water mixture (3: 7) was used for elution, 25 ml of the eluate was collected, and fractions 24 to 27 were E-1, 30 to 34 were E-2, and
83-93 were collected as E-3. Furthermore, the remaining acetonitrile solution is similarly fractionated, the obtained eluates are respectively collected, and the acetonitrile is distilled off under reduced pressure.
The mixture was extracted twice with an equal amount of ethyl acetate, dried over magnesium sulfate, and then concentrated to dryness under reduced pressure to remove A21 from E-1.
21-9 (97.6 mg), from E-2 to F-1 (2
2.1 mg) and E-3 gave F-2 (43.1 mg).

Fraction F-2 was dissolved in 1 ml of acetonitrile, and CAPCELL PAK C18 (30 mm × 25
0 mm) (manufactured by Shiseido Co., Ltd.) was attached. [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (2: 3) 369 ml
After washing with, elution with 36 ml of [0.002% trifluoroacetic acid-containing acetonitrile] -water mixture (2: 3),
The mixture was concentrated to dryness under reduced pressure to give A2121-7 (6.3 mg).

Fraction F-1 was dissolved in 1 ml of acetonitrile and the mixture was mixed with CAPCELL PAK C18 (30 mm × 25).
0 mm) was attached to the preparative high performance liquid chromatography. After washing with 225 ml of [0.002% acetonitrile containing trifluoroacetic acid] -water mixture (3: 7),
[Acetonitrile containing 0.002% trifluoroacetic acid]
-Elution with 36 ml of a water mixture (3: 7) and concentration to dryness under reduced pressure gave A2121-8 (8.8 mg).

Example 2 MCI2836 was cultured for 100 Erlenmeyer flasks in the same manner as in Example 1, and the obtained culture was centrifuged.
The culture supernatant was separated into 3.5 l and 3 l of cultured cells. Concentrated hydrochloric acid was added to the culture supernatant obtained here to adjust the pH to 3, and extraction was performed with an equal amount of ethyl acetate, and the extract was concentrated under reduced pressure to obtain 3.70 g of oily substance A. It was On the other hand, 4 l of acetone was added to the cultured cells for extraction, the cells were filtered off with Celite, and the acetone was distilled off under reduced pressure. Concentrated hydrochloric acid was added to this extract to adjust the pH to 3, and the mixture was extracted with an equal amount of ethyl acetate. The extract was concentrated under reduced pressure to obtain 8.45 g of oily substance B. The oily substance A and the oily substance B were combined and subjected to reverse layer partition chromatography using octadecyl silica gel. This oily substance was converted into MCI GEL ODS
1MY (made by Mitsubishi Kasei) is sprinkled with 20g, dried under reduced pressure,
The column was charged with 480 g of MCI GEL ODS1MY and equilibrated with an acetonitrile-water mixture (3: 7), and the column was washed with 2.5 l of an acetonitrile-water mixture (3: 7). Then acetonitrile-water mixture (2: 3) 2.5
1, further 0.5 l of acetonitrile-water mixture (1: 1)
After washing with 1., mixed solution of acetonitrile-water (1: 1) 2.
Elute with 0 l, then acetonitrile-water mixture (3:
2) Eluted with 2.5 l. The eluates were collected and concentrated to dryness under reduced pressure to obtain a residue of 4.82 g. 2.26 of this residue
g was dissolved in 50 ml of acetonitrile, 50 ml of 0.3 N sulfuric acid was added, and the mixture was reacted at room temperature for 5 hours. 250 ml of water was added to the reaction solution, and the mixture was extracted twice with 500 ml of ethyl acetate. The organic layers were collected, dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue (1.57 g) was crystallized twice from acetonitrile to give A2121-10 (495 mg). The physical properties of this substance are as described above.

Example 3 To 20.4 mg of A2121-2, 1 ml of a 0.3N trifluoroacetic acid-acetonitrile solution and 0.5 ml of water were added and dissolved, and the mixture was decomposed at room temperature for 5 hours. The precipitated crystals were collected by filtration to give A2121-10 5.0 mg. Test Example 1 Next, the action of inhibiting the activity of proline hydroxylase by A2121-10 is shown.

Measurement of the inhibitory activity of proline hydroxylase by A2121 is described in K. I. Kiviriko et al. [Eur. J. Biochemistry, 52 , 9-
16 (1975)] and using a partially purified enzyme prepared from a chicken embryo, (Pro-Pro-Gly) ₁₀ · 9H ₂
Using O as a substrate, R. E. Methods such as Rhoad [Met
hods in Enzymology, XVIIIB, 3
06 (1971)]. That is, reaction liquid 2
0.05M Tris-HCl buffer (pH 7.
8), the substrate 40 μg, ferrous sulfate 0.05 mM, ascorbic acid 2 mM, dithiothreitol 0.1 mM,
0.4 mg of bovine serum albumin, 0.1 mM α-ketoglutarate containing ¹⁴ C-labeled radioactive α-ketoglutarate, 50 μg of catalase and proline hydroxylase.
4 ml was contained and the reaction was performed at 37 ° C. for 20 minutes. Then, the reaction was stopped with the same amount of 25% trichloroacetic acid as the reaction solution, and the released carbon dioxide was collected and the radioactivity was measured. Similarly, perform the reaction with the reaction solution containing no substrate, subtract the radioactivity measurement value in the reaction not containing the substrate from the radioactivity measurement value in the reaction containing the substrate, and use it as the index of proline hydroxylase activity. The concentration of the inhibitory substance required to inhibit its activity by 50% was defined as 50% inhibitory concentration. as a result,
The 50% inhibitory concentration of A2121-10 was 0.9 μM.

[0078]

EFFECTS OF THE INVENTION The novel physiologically active substance compound group A2 of the present invention
121 has a proline hydroxylase inhibitory activity at a low concentration, and thus is useful as a therapeutic drug for organ fibrosis such as liver cirrhosis and pulmonary fibrosis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area A61K 31/65 ACS 9454-4C AED 9454-4C C12N 9/99 9359-4B (C12P 29/00 C12R (465) (72) Inventor Noriko Chiba 1000 Kamoshida-cho, Midori-ku, Yokohama, Kanagawa Sanryo Kasei Co., Ltd. (72) Inventor Reiko Sashida 1000, Kamoshida-cho, Midori-ku, Yokohama, Kanagawa Company Research Institute

Claims (2)

[Claims] 1. A novel physiologically active substance represented by the following formula (I). [Chemical 1] [In the above formula, R ¹ is a hydrogen atom, Or, R ² is a hydrogen atom, Or, (R ³ and R ⁴ each independently represent a hydrogen atom or a methyl group)] 2. The method according to claim 1, which belongs to the genus Streptomyces.
The method for producing a novel physiologically active substance according to claim 1, wherein the bacterium producing the novel physiologically active substance described above is cultured and separated.