JPH08109182A - Compound rp-1551s - Google Patents

Abstract

PURPOSE: To obtain the subject new bioactive substance produced from a microorganism belonging to genus Penicillium and having antimicrobial activity. CONSTITUTION: These compounds RP-1551s are expressed by formula I [R<1> and R<2> are united together to form a group of formula II (2'-positioned and 5'-positioned H is respectively anti-arrangement to 8-positioned H), or R<1> is H and R<2> is a group of formula III, etc.]. In the compound of the formula I, a compound in which R<1> and R<2> are united to form a group of the formula II has the following physicocheminal properties: appearance: yellow powder; melting point: 124.0-128.0 deg.C; specific rotatory power: [α]<25> D=-135 deg. (C=0.12, CH3 OH); molecular formula: C35 H29 O6 Cl; high resolution FAB mass spectrum: m/z=461.1738(M+H)<+> ; FAB mass spectrum: m/z=461(M+H)<+> , etc. The compound of the formula I is obtained by culturing a microorganism belonging to genus Penicillium in a medium and picking from the cultured substance.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to penicillium (Pen
icillium ), which is produced by a microorganism belonging to the genus icillium and has an antibacterial action.

[0002]

2. Description of the Related Art Substances having a structure similar to those of RP-1551s described later are represented by the formula (VI) or the formula (VI).
I)

[0003]

[Chemical 6]

Isochromophyllon I represented by
(Isochromophilone I) or Isochromophilone II
(Isochromophilone II) [Journal of Antibiotics], 46 , 1908
-1911 (1993)], human immunodeficiency virus (HIV)
It has been reported as a substance that inhibits the binding between the surface antigen CD4 and the envelope protein gp120.

Formula (VIII) or Formula (IX)

[0006]

[Chemical 7]

[0007] Ketoviridin B (Chae
toviridin B) or ketoviridin C (Chaetoviridin
C) refers to Chemical Pharmaceutical Bulletin [(Chemical & Pharmaceutical Bulletin), 38 , 625-62
8 (1990)] as a dye. Formula (X)

[0008]

Embedded image

TL-3 and TL-4 represented by
Annual Meeting of Natural Organic Compounds (October 11-13, 1993, Kyoto;
It is reported as a pigment in the abstracts of the lectures pp290-297). Of the above compounds, TL-3 and TL-4 have the same planar structure as the compound (RP-1551-1) in which R ¹ and R ² are united to represent the formula (II),
The two are different with respect to the relative configuration at the 2'and 5'positions with respect to the 8th position.

[0010]

An object of the present invention is to provide a novel bioactive substance having antibacterial activity.

[0011]

The present invention provides a compound of formula (I)

[0012]

[Chemical 9]

{In the formula, R ¹ and R ² are (1) integrated into formula (II)

[0014]

[Chemical 10]

(In the formula, 2'-position hydrogen and 5'-position hydrogen are 8
Position hydrogen and each have an anti-configuration) or (2) R ¹ represents hydrogen and R ² is of the formula (III)

[0016]

[Chemical 11]

Or (3) together form the formula (IV)

[0018]

[Chemical 12]

Or (4) together represents the formula (V)

[0020]

[Chemical 13]

Compound RP-1551
Regarding kind. The compound can be obtained by culturing a microorganism belonging to the genus Penicillium.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION Among the compounds represented by the formula (I), compounds in which R ¹ and R ² are united to represent the formula (II) are RP-1551-1, R ¹ represents hydrogen, Compounds in which R ² represents formula (III) are RP-1551-2, R ¹
And R ² together form a compound of formula (IV)
The compound in which P-1551-3, R ¹ and R ² are united and represents the formula (V) is referred to as RP-1551-4. In addition, these compounds are collectively referred to as RP-1551s.

RP-1551s are all equilibrium mixtures of two geometric isomers (E-form and Z-form) with respect to the configuration of side-chain trisubstituted olefins. The equilibrium is rapid and both isomers cannot be isolated in pure form. Similar equilibria have been reported for compounds isochromophylones having similar structures as described above. RP-1551
In any of the compounds of 4, the E form is usually the main component and the Z form is the minor component.
It varies depending on the isolation and purification method and storage conditions, and is not constant.

The physicochemical properties of RP-1551s are shown below, and all of them relate to the E and Z isomer mixture samples obtained by the methods described in the examples below. In addition, only for ¹³ C and ¹ H-NMR spectra,
Since it was possible to observe and attribute the signals of the E-form and the Z-form separately, they are described separately. Compound R
The physicochemical properties of P-1551s are shown below.

Physicochemical data of RP-1551-1
(E form: Z form = approx. 3.5: 1) Property: Yellow powder Melting point: 124.0 to 128.0 ° C Specific rotation: [α] _D ²⁵ = -135 ° (c = 0.12, CH ₃ OH) Molecular formula: C ₂₅ H ₂₉ O ₆ Cl FAB Mass spectrum: m / z 461 (M + H) ⁺ High resolution FAB mass spectrum: m / z 461.1738 (M + H) ⁺ UV absorption spectrum: λ _max (CH ₃ OH) nm (ε) 458
(sh 8,700), 430 (22,300), 409 (28,300), 391 (sh 2
4,900), 355 (22,300), 341 (sh 20,400), 251 (18,90)
0) Red external absorption spectrum: ν _max (KBr) cm ^-1 3425, 173
4, 1628, 1558, 1522, 1236, 1111, 843 ¹³ C-NMR spectrum (100MHz, CDCl ₃ ): δ ppm (multiplicity) 1) E isomer 189.17 (s), 172.44 (s), 158.42 (s) , 147.83 (d), 147.02
(d), 142.21 (d), 140.83 (s), 131.93 (s), 116.43 (d), 1
15.35 (s), 109.78 (s), 105.38 (d), 102.53 (s), 84.12
(s), 73.49 (d), 57.21 (d), 47.41 (d), 44.88 (t), 35.05
(d), 30.10 (t), 24.11 (q), 20.59 (q), 20.24 (q), 12.38
(q), 11.92 (q) 2) Z isomer 189.25 (s), 172.44 (s), 158.15 (s), 147.02 (d), 145.36
(d), 140.68 (s), 133.86 (d), 129.92 (s), 118.81 (d), 1
15.35 (s), 110.1 (s), 106.06 (d), 102.53 (s), 84.12
(s), 73.49 (d), 57.15 (d), 47.47 (d), 44.88 (t), 34.09
(d), 30.29 (t), 24.06 (q), 20.97 (q), 20.59 (q), 20.07
(q), 11.97 (q) ¹ H-NMR spectrum (400MHz, CDCl ₃ ): δ ppm (integral,
Multiplicity, coupling constant J (Hz)) 1) E isomer 7.71 (1H, s), 7.06 (1H, d, 15.8), 6.58 (1H, s), 6.07 (1H,
d, 15.8), 5.66 (1H, d, 10.0), 4.62 (1H, m), 3.36 (1H, d, 9.
0), 2.99 (1H, d, 9.0), 2.47 (1H, m), 2.39 (1H, d, 14.3),
1.87 (1H, dd, 14.3,11.5), 1.84 (3H, d, 1.0), 1.45 (1H, m),
1.42 (3H, d, 6.4), 1.41 (3H, s), 1.34 (1H, m), 1.01 (3H,
d, 6.6), 0.86 (3H, t, 7.4) 2) Z isomer 7.75 (1H, s), 7.45 (1H, d, 15.6), 6.60 (1H, s), 6.16 (1H,
d, 15.6), 5.51 (1H, d, 10.0), 4.62 (1H, m), 3.37 (1H, d, 9.
0), 3.00 (1H, d, 9.0), 2.64 (1H, m), 2.39 (1H, d, 14.3),
1.91 (3H, d, 1.2), 1.87 (1H, dd, 14.3,11.5), 1.45 (1H, m),
1.42 (3H, d, 6.4), 1.41 (3H, s), 1.34 (1H, m), 1.01 (3H,
d, 6.6), 0.86 (3H, t, 7.4)

Physicochemical data of RP-1551-2
(E form: Z form = approx. 3.3: 1) Property: Yellow powder Melting point: 54.0-55.0 ° C Specific rotation: [α] _D ²⁹ = + 24.5 ° (c = 0.50, CH ₃ OH) Molecular formula: C ₂₄ H ₃₁ O ₅ Cl FAB Mass spectrum: m / z 435 (M + H) ⁺ High resolution FAB mass spectrum: m / z 435.1909 (M + H) ⁺ UV absorption spectrum: λ _max (CH ₃ OH) nm (ε) 460
(sh 10,400), 432 (sh26,500), 409 (33,100), 391 (s
h 28,600), 356 (25,500), 340 (sh 22,600), 319 (sh
15,600), 250 (22,300) Red External absorption spectrum: ν _max (KBr) cm ^-1 3431, 170
9, 1624, 1558, 1520, 1213, 964, 850 ¹³ C-NMR spectrum (100MHz, CDCl ₃ ): δ ppm (multiplicity) 1) E isomer 209.84 (s), 191.45 (s), 158.25 (s) , 147.89 (d), 145.30
(d), 142.28 (d), 141.58 (s), 131.96 (s), 119.22 (s), 1
16.40 (d), 106.57 (s), 105.02 (d), 74.11 (s), 63.82
(d), 51.20 (t), 41.64 (t), 40.26 (d), 35.06 (d), 30.12
(t), 26.75 (q), 22.43 (q), 20.26 (q), 12.39 (q), 11.93
(q) 2) Z isomer 209.87 (s), 191.45 (s), 157.97 (s), 145.41 (d), 145.34
(d), 141.43 (s), 133.86 (d), 129.89 (s), 118.83 (d), 1
10.47 (s), 106.88 (s), 105.71 (d), 74.11 (s), 63.82
(d), 51.17 (t), 41.58 (t), 40.26 (d), 34.11 (d), 30.30
(t), 26.73 (q), 22.43 (q), 20.98 (q), 20.10 (q), 11.99
(q) ¹ H-NMR spectrum (400MHz, CDCl ₃ ): δ ppm (integral,
Multiplicity, coupling constant J (Hz)) 1) E isomer 7.41 (1H, s), 7.03 (1H, d, 15.6), 6.50 (1H, s), 6.04 (1H,
d, 15.6), 5.65 (1H, d, 9.5), 4.17 (1H, m), 3.43 (1H, dd, 9.
5,3.5), 3.09 (1H, dd, 18.1,3.5), 2.48 (1H, m), 2.46 (1H,
m), 2.45 (1H, m), 2.34 (1H, dd, 18.1,9.5), 1.82 (3H, d, 1.
2), 1.42 (1H, m), 1.31 (3H, s), 1.26 (1H, m), 1.15 (3H, d,
6.4), 1.00 (3H, d, 6.5), 0.85 (3H, t, 7.5) 2) Z isomer 7.45 (1H, s), 7.42 (1H, d, 15.6), 6.52 (1H, s), 6.13 ( 1H,
d, 15.6), 5.50 (1H, d, 10.7), 4.17 (1H, m), 3.44 (1H, dd,
9.5,3.4), 3.10 (1H, dd, 18.1,3.4), 2.63 (1H, m), 2.48 (1
H, m), 2.46 (1H, m), 2.35 (1H, dd, 18.1,9.5), 1.90 (3H, d,
1.2), 1.42 (1H, m), 1.31 (3H, s), 1.26 (1H, m), 1.16 (3H,
d, 6.3), 1.01 (3H, d, 6.8), 0.86 (3H, t, 7.5)

Physicochemical data of RP-1551-3
(E form: Z form = about 2.8: 1) Property: Yellow powder Melting point: 91.0-93.0 ℃ Specific rotation: [α] _D ³⁰ = -335 ° (c = 0.066, CH ₃ OH) Molecular formula: C ₂₅ H ₂₇ O ₅ Cl FAB Mass spectrum: m / z 443 (M + H) ⁺ High resolution FAB mass spectrum: m / z 443.1612 (M + H) ⁺ UV absorption spectrum: λ _max (CH ₃ OH) nm (ε) 462
(sh 11,100), 433 (27,100), 412 (31,800), 391 (sh
25,800), 360 (21,700), 342 (sh 19,400), 241 (22,30
0) Red external absorption spectrum: ν _max (KBr) cm ^-1 3444, 178
4, 1693, 1668, 1631, 1558, 1520, 1431, 1242, 1090,
962, 847 ¹³ C-NMR spectrum (100MHz, CDCl ₃ ): δ ppm (multiplicity) 1) E isomer 90.06 (s), 184.31 (s), 168.38 (s), 158.79 (s), 148.44
(d), 147.89 (d), 146.22 (d), 142.78 (d), 140.65 (s), 1
31.94 (s), 130.44 (d), 116.14 (d), 113.51 (s), 109.31
(s), 105.11 (d), 83.58 (s), 54.56 (d), 42.71 (d), 35.1
0 (d), 30.09 (t), 23.38 (q), 20.22 (q), 18.73 (q), 12.3
9 (q), 11.92 (q) 2) Z isomer 190.01 (s), 184.31 (s), 168.35 (s), 158.51 (s), 147.89
(d), 146.22 (d), 145.89 (d), 140.51 (s), 134.34 (d), 1
30.40 (t), 129.89 (s), 118.54 (d), 113.51 (s), 109.60
(s), 105.79 (d), 83.55 (s), 54.56 (d), 42.71 (d), 35.1
0 (d), 30.27 (t), 23.38 (q), 20.95 (q), 20.05 (q), 18.7
3 (q), 11.99 (q) ¹ H-NMR spectrum (400MHz, CDCl ₃ ): δ ppm (integral,
Multiplicity, coupling constant J (Hz)) 1) E isomer 7.41 (1H, s), 7.03 (1H, d, 15.6), 7.01 (1H, dq, 15.7,6.9),
6.55 (1H, s), 6.43 (1H, dq, 15.7,1.7), 6.05 (1H, d, 15.
6), 5.66 (1H, d, 9.8), 4.01 (1H, s), 4.00 (1H, s), 2.47 (1
H, m), 1.97 (3H, dd, 6.9,1.7), 1.83 (3H, d, 1.2), 1.63 (3
H, s), 1.42 (1H, m), 1.3 (1H, m), 1.01 (3H, d, 6.6), 0.86
(3H, t, 7.5) 2) Z isomer 7.46 (1H, s), 7.43 (1H, d, 15.6), 7.01 (1H, dq, 15.7,6.9),
6.52 (1H, s), 6.43 (1H, dq, 15.7,1.7), 6.14 (1H, d, 15.
6), 5.52 (1H, d, 10.0), 4.01 (1H, s), 4.01 (1H, s), 2.62
(1H, m), 1.97 (3H, dd, 6.9,1.7), 1.90 (3H, d, 1.2), 1.63
(3H, s), 1.42 (1H, m), 1.30 (1H, m), 1.00 (3H, d, 6.6), 0.
85 (3H, t, 7.5)

Physicochemical data of RP-1551-4
(E form: Z form = about 2.4: 1) Property: Yellow powder Melting point: 82.0 ~ 84.0 ° C Specific rotation: [α] _D ²⁴ = -306 ° (c = 0.20, CH ₃ OH) Molecular formula: C ₂₅ H ₂₉ O ₆ Cl FAB Mass spectrum: m / z 461 (M + H) ⁺ High resolution FAB mass spectrum: m / z 461.1717 (M + H) ⁺ UV absorption spectrum: λ _max (CH ₃ OH) nm (ε) 461
(sh 26,100), 433 (59,300), 413 (70,700), 392 (sh
57,900), 359 (48,700), 342 (sh 42,000), 253 (28,00
0) Red external absorption spectrum: ν _max (KBr) cm ^-1 3444, 177
8, 1716, 1626, 1556, 1516, 1433, 1244, 1093, 849,
681 ¹³ C-NMR spectrum (100MHz, CDCl ₃ ): δ ppm (multiplicity) 1) E isomer 202.19 (s), 183.97 (s), 168.22 (s), 158.79 (s), 148.58
(d), 146.45 (d), 142.93 (d), 140.66 (s), 131.95 (s), 1
16.10 (d), 113.28 (s), 109.30 (s), 105.14 (d), 83.62
(s), 64.13 (d), 56.95 (d), 50.81 (t), 42.46 (d), 35.13
(d), 30.11 (t), 23.40 (q), 22.96 (q), 20.23 (q), 12.39
(q), 11.93 (q) 2) Z isomer 202.19 (s), 184.07 (s), 168.20 (s), 158.51 (s), 146.48
(d), 146.01 (d), 140.51 (s), 134.47 (d), 129.89 (s), 1
18.51 (d), 113.28 (s), 109.58 (s), 105.82 (d), 83.62
(s), 64.13 (d), 56.91 (d), 50.76 (t), 42.46 (d), 34.20
(d), 30.28 (t), 23.40 (q), 22.96 (q), 20.97 (q), 20.07
(q), 12.00 (q) ¹ H-NMR spectrum (400MHz, CDCl ₃ ): δ ppm (integral,
Multiplicity, coupling constant J (Hz)) 1) E isomer 7.44 (1H, s), 7.05 (1H, d, 15.7), 6.55 (1H, s), 6.06 (1H,
d, 15.7), 5.68 (1H, d, 9.8), 4.28 (1H, m), 3.888 (2H, s),
3.115 (1H, dd, 17.4,8.6), 2.80 (1H, dd, 17.4,3.2), 2.47
(1H, m), 1.83 (3H, d, 1.0), 1.62 (3H, s), 1.42 (1H, m), 1.
30 (1H, m), 1.24 (3H, d, 6.1), 1.01 (3H, d, 6.6), 0.855 (3
H, t, 7.5) 2) Z isomer 7.480 (1H, s), 7.45 (1H, d, 15.6), 6.570 (1H, s), 6.15 (1
H, d, 15.6), 5.53 (1H, d, 9.8), 4.28 (1H, m), 3.892 (2H,
s), 3.121 (1H, dd, 17.4,8.6), 2.81 (1H, dd, 17.4,3.2),
2.62 (1H, m), 1.91 (3H, d, 1.2), 1.62 (3H, s), 1.42 (1H,
m), 1.30 (1H, m), 1.25 (3H, d, 6.4), 1.01 (3H, d, 6.6), 0.
860 (3H, t, 7.5) Of the above compounds, RP-1551-1 has the same planar structure as TL-3 and TL-4, but for the 8-position
Both differ in the relative configuration at the 2'and 5'positions. Regarding TL-3 and TL-4, the 7-position methyl group and 8-position hydrogen, the 8-position hydrogen and 2'-position hydrogen, and the 2'-position hydrogen and 5'-position hydrogen are all in syn configuration. On the other hand, RP-1
In 551-1, the 7-position methyl group and 8-position hydrogen, and the 2'-position hydrogen and the 5'-position hydrogen all have the syn configuration, but the 8-position hydrogen and the 2'-position hydrogen have the anti configuration. This is due to the NOESY experiment and the difference N in the NMR spectrum.
In the OE experiment, the 7-position methyl group and the 8-position hydrogen, and the 2'-position hydrogen
It was determined that NOE was observed between the 5'-position hydrogen and NOE was not observed between the 8-position hydrogen and the 2'-position hydrogen. Therefore, RP-1551-1 is TL
-3 or TL-4 is clearly a different substance.

From the above data, it was found that the compounds RP-1551s are novel compounds. The biological activity of compounds RP-1551s against various bacteria is shown below.

Test Example 1 Antibacterial activity against various bacteria Antibacterial activity was 3 g / L bactotryptone (manufactured by Difco), 3 g / L meat extract, 1 g / L yeast extract, 1 g / L glucose, agar
It was measured by an agar dilution method using a medium (pH 7.0) having a composition of 16 g / L. Minimum growth inhibitory concentration (M
IC) is shown in Table 1.

[0031]

[Table 1]

The compound RP-1551s are compounds belonging to the genus Penicillium and having the ability to produce the compound RP-1551s are cultured in a medium to produce and accumulate the compounds RP-1551s in the culture. It is produced by collecting RP-1551s. Compound RP-15
As the microorganism capable of producing 51s, any strain can be used as long as it is a strain belonging to the genus Penicillium and capable of producing the compound RP-1551s. In addition, these strains can be artificially mutagenized, for example, by ultraviolet irradiation, X
The compound RP-can also be a mutant strain mutated by irradiation with radiation, treatment with a mutagen or the like, or a mutant strain naturally mutated.
It can be used in the present invention as long as it has the ability to produce 1551s.

A particularly preferred example is Penicillium sp. SPC-21609 strain. Penicillium s
p.) SPC-21609 strain has the following mycological properties. Macroscopic observation When cultured at 24 ° C using malt extract agar medium,
The diameter of the colony reaches 23 to 24 mm on the 7th day of culture and reaches about 43 mm on the 14th day of culture. On the 7th day of culture, the central part of the colony is orange, the periphery is white, the back side is dark orange, and yellow soluble pigment is put out in the medium.

Using potato-glucose agar medium,
When cultivated at 25 ° C, the diameter of the colony was about 2 on the 7th day of culturing.
4 mm, reaching about 40 mm on day 14 of culture. The appearance of the colony on the 7th day of culture is the same as that on the malt extract agar medium.
The optimum growth temperature of this strain is 15 to 36 ° C, and it grows best at around 28 ° C. The pH that can grow is 2 to 8, and the optimum growth pH is 5 to 6. Observation with an optical microscope The following are the results of observation with an optical microscope of this strain when cultured on a malt extract agar medium.

The hypha has a partition wall and exhibits colorless to yellowish brown or reddish brown, and is smooth and well branched. The conidia peduncle does not form a bundle, but stands alone from the hyphae. Conidia peduncle is smooth, colorless to pale yellowish brown, and has a length of about 200.
The width is about 3 μm. 3 to the tip of the conidia handle
Five metres are formed, and 3-5 phialides are formed at the tip of the metres. In addition, a ramie may be formed between the conidia peduncle and the metre. The metre is cylindrical and has a length of 6 to 14 μm and a width of 1.5 to 3.5 μm. Fear ride has a tongue-like shape and a length of 9-12μ
m, the width is 2 to 3.5 μm in the widest part. The ramie is cylindrical and has a length of about 20 μm and a width of about 2 μm. The ontogeny of conidia is of the endophytic type, which is the fiaro type. Conidia are formed from the tip of the phialide and form a chain. Phialoconidia are unicellular, colorless, elliptical or subspherical, with a length of 2-3 μm and a width of 1.5-2 μm.
m, the surface is smooth. In this strain, only the above-mentioned anamorph was observed, and there was a teleomorph.
ph) is not observed.

From the above-mentioned mycological properties, the taxonomic position of this bacterium is determined as "The Genera of Fanjayi Poring in Pure Culture 2nd Edition (The Ge
neraof Fungi Sporulating in Pure Culture, 2nd ed.
Cramer, Vaduz, JA vonArx, 1974) ”, the strain was identified as belonging to the genus Penicillium .

This strain is Penicillium species
It is named ( Penicillium sp.) SPC-21609, and has been deposited as FERM BP-4768 at the Institute of Biotechnology, Institute of Industrial Science, Agency of Industrial Science and Technology, on August 4, 1994, based on the Budapest Treaty. In culturing the compound RP-1551-1 producing bacterium of the present invention, a usual culturing method used for culturing a filamentous fungus is applied. The medium to be used may be either a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, an inorganic substance and the like that can be assimilated by the bacterium.

As carbon sources, glucose, sucrose, starch, dextrin, mannose, maltose,
Molasses, mashed potatoes and other carbohydrates, citric acid,
Organic acids such as malic acid, acetic acid and fumaric acid, alcohols such as methanol and ethanol, hydrocarbons such as methane, ethane, propane and n-paraffin, amino acids such as glutamic acid and glycerol are used.

As the nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium phosphate, aspartic acid, glutamine,
Amino acids such as cystine and alanine, urea, peptone,
Meat extract, yeast extract, dry yeast, malt extract, corn steep liquor, soybean powder, soluble vegetable
Protein, cottonseed meal, soybean casein, casamino acid, pharmaceutical media, vegetable juice, etc. are used.

As the inorganic substance, potassium monohydrogen phosphate,
Potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate, cobalt sulfate, zinc sulfate, calcium pantothenate, ammonium molybdate, potassium aluminum sulfate, barium carbonate. , Calcium carbonate, cobalt chloride, salt and the like are used.

In addition, if necessary, the growth of bacterial cells such as vitamins and thiamine or the compound RP-1551 is added to the medium.
Add substances that promote the production of varieties. When the microorganism used requires a specific substance, the substance is added. Culture is performed by shaking culture, aeration stirring culture, or the like for 15 to 35
It is carried out at a temperature of ° C and a pH near neutral. Usually, after 3 to 15 days of culture, the maximum accumulation of compound RP-1551s is reached.

Compound RP-1551 accumulated in the culture medium
When isolating and collecting the species from the culture medium, a usual method for collecting a physiologically active substance from the culture medium is applied. That is, extraction of cell components with an organic solvent such as acetone or methanol, filtration, cell removal by centrifugation, adsorption resin, silica gel, silanized silica gel, reverse phase silica gel, aluminum oxide, cellulose, diatomaceous earth, silicic acid. Compounds RP-1551s are isolated by adsorption / desorption treatment of the active substance by column chromatography or thin layer chromatography using magnesium, gel filtration agent, ion exchange resin or the like, or partitioning with an appropriate solvent system.

The compounds RP-1551s in the above purification step were detected by a fluorescent agent-containing silica gel (Kieselgel F254).
, Manufactured by Merck & Co., Inc.) and subjected to iodine reaction or 253.7 nm ultraviolet irradiation method. Examples of the present invention will be shown below.

[0044]

【Example】

[0045] As Example 1 seed, Penicillium sp. (Penicillium s
p.) SPC-21609 strain (FERM BP-4768) was used, and 3 g of mashed potato broth (Snow Brand Milk Products Co., Ltd.) was used as the first type medium.
/ DL, glucose 10g / dL, yeast extract 0.5g / dL
A medium having a composition of (pH 6.5) was used. The inoculum 1 platinum loop was inoculated into 10 mL of the above-mentioned type 1 medium contained in a thick test tube, and cultured with shaking at 25 ° C for 5 days (type 1 culture).

50 mL of a culture solution (first culture solution) obtained by the first culture was placed in a 300 mL Erlenmeyer flask.
Was inoculated into the second type medium. The composition of the second type medium is the same as that of the first type medium. The second seed culture was performed at 28 ° C. for 2 days. 10 mL of the obtained second seed culture was inoculated into 50 mL of the main fermentation medium contained in a 300 mL Erlenmeyer flask. Main fermentation medium: sucrose 3g / dL, soluble starch 2g / d
L, dry yeast (Asahi Breweries) 0.5 g / dL, malt extract 1 g / dL, corn steep liquor 0.5 g /
A medium having a composition of dL, vegetable juice (Campbell) 20 mL / dL, and calcium carbonate 1.5 g / dL (pH 6.5) was used. The main fermentation culture was performed by shaking culture at 25 ° C. for 7 days.

800 mL of the obtained fermentation culture solution was filtered, 800 mL of methanol was added to the separated cells, and the mixture was stirred and filtered. 800 mL of deionized water was added to the filtrate, and 80 mL of Diaion HP-20ss filled with deionized water was added.
It was passed through a column (registered trademark manufactured by Mitsubishi Kasei). After washing the column with 300 mL of 50% aqueous methanol, 300 mL
Was eluted with a mixed solvent of methanol / acetone (7: 3). The eluate was concentrated under reduced pressure to give 794 mg of a yellow oil. This substance was dissolved in a chloroform solution containing a small amount of 10% 2-butanone, and 100 mL of silica gel (Merck, Art.773 manufactured by Merck Co., Ltd.) was filled with a solvent having the same composition.
4) It was placed on the top of the column and the active substance was eluted using the solvent of the same composition. When 20 mL of the eluate was collected, compound RP-1551-1 was eluted in fraction numbers 16 to 32. These fractions were collected and concentrated to dryness under reduced pressure to 1
34 mg of yellow solid was obtained. This material was dissolved in a small amount of 70% acetonitrile aqueous solution, and 150 mL of reverse-phase silica gel (YMC, ODS) filled with a solvent having the same composition was used.
-AQS50) was placed on the upper end of the column and eluted with a solvent of the same composition. If 20 mL of the eluate is collected, the compound RP
-1551-1 was eluted in fraction numbers 29-32. After collecting these fractions, they were concentrated to dryness under reduced pressure to give compound R
52 mg of P-1551-1 was obtained.

[0048] As Example 2 seed, Penicillium sp. (Penicillium s
p.) SPC-21609 strain (FERM BP-4768) was used, and 3 g of mashed potato broth (Snow Brand Milk Products Co., Ltd.) was used as the first type medium.
/ DL, glucose 10g / dL, yeast extract 0.5g / dL
A medium having a composition of (pH 6.5) was used. The inoculum 1 platinum loop was inoculated into 10 mL of the above-mentioned type 1 medium contained in a thick test tube, and cultured by shaking at 25 ° C for 7 days (type 1 culture).

5 mL of the culture solution (first culture solution) obtained by the first culture was inoculated into 50 mL of the second culture medium contained in a 300 mL Erlenmeyer flask. The composition of the second type medium is the same as that of the first type medium. The second seed culture was carried out at 25 ° C. for 3 days. 50 mL of the obtained second seed culture was inoculated into 400 mL of the main fermentation medium contained in a 2 L Erlenmeyer flask. As main fermentation medium, sucrose 3 g / dL, soluble starch 2 g / dL,
Dry yeast (Asahi Breweries) 0.5 g / dL, malt extract 1 g / dL, corn steep liquor 0.5 g / dL,
A medium having a composition of 20 mL / dL of vegetable juice (Campbell) and 1.5 g / dL of calcium carbonate (pH 6.5) was used. The main fermentation culture was performed by shaking culture at 25 ° C. for 7 days.

8 L of the obtained fermentation culture broth was filtered, 8 L of methanol was added to the separated cells, and the mixture was stirred and filtered. 8 L of deionized water was added to the filtrate, and 3 L of Diaion HP-20ss (manufactured by Mitsubishi Kasei: filled with deionized water:
It was passed through a (registered trademark) column. The column was washed with 9 L of 50% methanol aqueous solution and then eluted with 9 L of a methanol / acetone mixed solvent (7: 3). The eluate was concentrated under reduced pressure to obtain 8.3 g of a yellow oily substance. This substance was dissolved in a small amount of chloroform solution and placed on the top of a 1 L silica gel (Merck, Art.7734) column packed with a solvent of the same composition, 3 L chloroform, 3 L 10% 2-butanone. Chloroform containing 9 L of 20% 2-
Chloroform containing butanone and 3 L of methanol were sequentially eluted, and 1 L of the eluate was collected. Fraction No. 4 and Fraction Nos. 7 to 15 were dried under reduced pressure to obtain 382 mg and 1.8 g of yellow oily substances, respectively. The oily substance obtained from Fraction No. 4 was dissolved in a small amount of chloroform, placed on the upper end of a silica gel (Lichroprep Si60 manufactured by Merck & Co.) column, and eluted with chloroform. RP-1551-3 was eluted between the elution volumes of 180 mL and 370 mL. The eluate was dried under reduced pressure to obtain 66 mg of a crudely purified sample of RP-1551-3. 85% of the obtained crude purified sample
A column dissolved in an aqueous solution of methanol and equilibrated with a solvent having the same composition (Y-MC, D-ODS-5AQ, 2 cmφ).
RP-1551-3 by placing it on the upper end of (25 cm) and flowing an 85% aqueous methanol solution at a flow rate of 10 mL / min.
Was eluted. RP-1551-3 was eluted at a retention time of 23 minutes, and the eluate was collected and dried under reduced pressure to give R
22 mg of powder of P-1551-3 was obtained.

The oily substance obtained by concentrating and drying the above-mentioned fractions Nos. 7 to 15 was dissolved in a small amount of 85% aqueous methanol solution and equilibrated with a solvent having the same composition as reversed phase silica gel (YM.
C, ODS AQ-S50) column (150 mL) was placed on the upper end of the column and eluted with a solvent having the same composition. Eluate 1
Fractionation by 4 mL yields RP-1551-4 with fraction number 19
~ 22, RP-1551-2 is fraction number 23-27
In addition, RP-1551-1 was eluted in fraction numbers 29 to 34. Concentrate each fraction together under reduced pressure to dryness,
111 mg of the crude purified sample of RP-1551-4, RP-1
169 mg of the crude purified sample of 551-2, RP-1551-
787 mg of 1 was obtained. A crude purified sample of RP-1551-4 was dissolved in a small amount of 85% methanol aqueous solution, and an HPLC column (YMC, D-ODS-5AQ, 2 cmφ × 25) was used.
cm). The column was eluted with 85% aqueous methanol solution at a flow rate of 10 mL / min to give RP-155.
1-4 were eluted at a retention time of 16-18 minutes. The eluates were combined, concentrated to dryness under reduced pressure, and RP-1551-4 was added to 37%.
mg was obtained.

A crudely purified sample of RP-1551-2 was dissolved in a small amount of 85% aqueous methanol solution to prepare RP-1554-1.
Fractionation was performed under the same conditions as in the purification process of. RP-1551
-2 was eluted at a retention time of 18.5 to 20.5 minutes, and the eluate was collected and concentrated to dryness under reduced pressure to give RP-1.
47 mg of 551-2 was obtained.

[0053]

INDUSTRIAL APPLICABILITY According to the present invention, compounds RP-1551s having antibacterial activity can be provided.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 17/06 7432-4B 17/18 D 7432-4B (C12P 17/06 C12R 1:80) ( (C12P 17/18 C12R 1:80) (72) Inventor Katsuhiko Ando 1-17-8 Asahi-cho, Machida-shi, Tokyo (72) Inventor Yuzuru Matsuda 1-22-7 Nunui-cho, Koganei-shi, Tokyo (72) Inventor Katsunori Kita 255-7 Takehara, Nagaizumi-cho, Sunto-gun, Shizuoka (72) Inventor Yasuhiro Suzuki 2978-18 Shimokanuki Hamada, Numazu-shi, Shizuoka (72) Inventor Akira Mihara 1079-22 Kiso-cho, Machida-shi, Tokyo

Claims (5)

[Claims] 1. Formula (I): {In the formula, R ¹ and R ² are integrated by (1) into the formula (I
I) [Chemical formula 2] (Wherein the 2'-position hydrogen and the 5'-position hydrogen have an anti-configuration relationship with the 8-position hydrogen, respectively) or (2) R ¹
Represents hydrogen and R ² is of the formula (III): Or (3) taken together as a formula (IV) Or (4) together form the formula (V) The compound RP-1551s represented by. 2. R ¹ and R ² together form the formula (I
A compound according to claim 1 which represents I). 3. R ¹ represents hydrogen and R ² is of the formula (III)
The compound according to claim 1, which represents 4. R ¹ and R ² together form the formula (I
A compound according to claim 1 which represents V). 5. R ¹ and R ² together form the formula (V)
The compound according to claim 1, which represents