JP2003012687A - Antibiotic substance caprazamycin d, g, d1, and g1 and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new antibiotic substance having new molecular skeleton and exhibiting excellent antibiotic action on bacteria. SOLUTION: The antibiotic substance is caprazamycin D and G expressed by general formula (I) [R is 10-methyl-undecyl in caprazamycin D and 9-methyl- undecyl in caprazamycin G] and caprazamycin D1 and G1 expressed by general formula (II) [R is 10-methyl-undecyl in caprazamycin D1 and 9-methyl-undecyl in caprazamycin G1]. The antibiotic substance is produced by the culture of Streptomyces sp. MK730-62F2 (FERM BP-7218). The caprazamycin D, G, D1 and G1 have excellent antibiotic activity against various acid-fast fungi and bacteria and their drug-resistant strains.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to caprazamycin (capramicin) which is a novel antibiotic having excellent antibacterial activity.
azamycin) D, caprazamycin G, caprazamycin D1 and caprazamycin G1 or pharmaceutically acceptable salts thereof. The present invention also includes caprazamycin D, caprazamycin G, caprazamycin D1.
And a method for producing caprazamycin G1. Further, the present invention relates to a pharmaceutical composition, particularly an antibacterial composition, which contains at least one of caprazamycin D, G, D1 and G1 or a salt thereof as an active ingredient. Furthermore, the present invention relates to the novel antibiotics caprazamycin D, G, D1 and G1 and the known antibiotics caprazamycin A,
A method for producing caprazamycin A, B, C, D, E, F, G, D1, G1 which comprises culturing Streptomyces sp. MK730-62F2 which has the property of producing B, C, E, F Includes.

[0002]

2. Description of the Related Art Antibiotics such as rifampicin, kanamycin, streptomycin, viomycin, capreomycin, and cycloserine are used as antibacterial agents in chemotherapeutics for bacterial infectious diseases, particularly in the case of chemotherapeutic diseases for acid-fast bacteria. .

In chemotherapy for bacterial infections, it is a serious problem that the bacteria causing the infection become drug resistant. Particularly, in the chemotherapy of infections with acid-fast bacilli, acid-fast bacilli having resistance to rifampicin, kanamycin, streptomycin, viomycin, capreomycin, cycloserine and the like have emerged, which has become a social problem.
There is a strong demand for new chemotherapeutic agents effective in the treatment of drug-resistant acid-fast bacterium infections. There is also a strong demand for new chemotherapeutic agents that are effective in treating infections of atypical acid-fast bacilli for which chemotherapy has not been established. Therefore, unlike known antibiotics that have been used conventionally, it is strongly desired to discover or create novel synthetic compounds and novel antibiotics having novel chemical structures and excellent properties such as high antibacterial activity. Is desired. The present inventors have conducted various studies for the purpose of providing a novel antibiotic having an excellent antibacterial activity that can meet the above demands.

First, the present inventors have found that Streptomyces
We provide caprazamycins A, B, C, E and F that show strong antibacterial activity against acid-fast bacterium, which is produced by SPK MK730-62F2 strain (deposited with the deposit number of FERM BP-7218). (International publication of PCT application PCT / JP00 / 05415 WO
01 / 12643A1 publication, reference, issued February 22, 2001).

Caprazamycin A, B, C, E and F
Is the following general formula (A) [In the formula, R is a tridecyl group in caprazamycin A, 11-methyl-dodecyl group in caprazamycin B, dodecyl group in caprazamycin C, and undecyl group in caprazamycin E, And caprazamycin F is a 9-methyl-decyl group].

The above-mentioned caprazamycin A, B,
As a substance having a structural skeleton related to the chemical structures of C, E, and F, Streptomyces sp SN-1061M strain (FERM BP-580
0) Fermentation products of known antibiotics liposidomycin A, B, C (JP-A-61-282088) and liposidomycins G, H (JP-A-2-306992), and other liposides Mycins [The Journal of Antibiotics 51 Vol. 7
Issue 647-654 (1998); The Journal of Antibiotics, 5
Volume 2, Issue 3, Pages 281-287 (1999); Journal of the American
Chemical Society, Vol. 110, No. 13, 4416-4417 (1988); Th
e Journal of Organic Chemistry, Volume 57 Issue 24 6392-6403
Page (1992); PCT International Publication WO 97/41248].

[0007] In the above-mentioned PCT International Publication WO 97/41248, as concrete new compounds actually isolated and obtained, lipocidomycin Z, L, M, K, N, A- (III )
, C- (III), X- (III), Y- (III), Z- (III), C- (III), V-
(III), A- (III), G- (III), M- (III), K- (III), N- (II
A total of 17 compounds of I), A- (IV) and C- (IV) are listed (see Table 1 of WO 97/41248).

[0008]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention There is still a strong demand for new antibiotics having excellent antibacterial activity, which are effective for the treatment of bacterial infections, particularly infectious diseases of resistant bacteria. Is being continued by many researchers.

[0009]

DISCLOSURE OF THE INVENTION Following the discovery of caprazamycins A, B, C, E and F described above, the present inventors conducted research to discover new and useful antibiotics. As a result, this time, Streptomyces sp. MK730-62
It was found that the F2 strain produced four new antibiotics separately from the above-mentioned caprazamycins A, B, C, E and F. We have succeeded in isolating and obtaining these four new antibiotics. It was discovered that these four new antibiotics have excellent antibacterial activity against acid-fast bacteria and drug-resistant bacteria. The inventors further succeeded in determining their chemical structure by analyzing these four new antibiotics. Then, it was confirmed that these four new antibiotics are novel compounds having a common skeleton with caprazamycin A, B, C, E, and F, and caprazamycin D, caprazamycin G, and caprazamycin G, respectively. They were named plazamycin D1 and caprazamycin G1. It was discovered that these new compounds can be represented by the following formula (Ia), formula (Ib), formula (IIa) and formula (IIb), respectively.

Moreover, a new antibiotic, caprazamycin D, discovered and isolated by the present inventors this time,
It was found that G, D1 and G1 all differ from the known ones in any of the liposidomycins in terms of chemical structure. Furthermore, it was also confirmed that none of the known chemically synthesized compounds is similar to caprazamicins. Therefore, it was confirmed by the present inventors that caprazamycin D, G, D1 and G1 obtained this time are novel compounds.

Therefore, in the first aspect of the present invention, the following general formula (I) [In the formula, R is 10-methyl-undecyl group- (CH ₂ ) ₉ CH (CH ₃ ) ₂ in caprazamycin D, and caprazamycin G is
In R is 9-methyl - undecyl group _{- (CH 2) 8 CH (} CH 3) CH 2 C
H ₃ ] and the antibiotics caprazamycin D and caprazamycin G, or a pharmaceutically acceptable salt thereof.

Caprazamycins D and G represented by the general formula (I) according to the first aspect of the present invention include caprazamycin D of the following formula (Ia) and caprazamycin G of the following formula (Ib). To do. (1) The following formula (Ia) Caprazamycin D represented by [in the general formula (I), R is 10
-Compound when it is a methyl-undecyl group]. (2) The following formula (Ib) Caprazamycin G represented by [R in the general formula (I) is 9
-Compound when it is a methyl-undecyl group].

Further, in the second invention, the following general formula (II) [In the formula, R is 10-methyl-undecyl group- (CH ₂ ) ₉ CH (CH ₃ ) ₂ in caprazamycin D 1, and R is 9-methyl-undecyl group- (C
H ₂ ) ₈ CH (CH ₃ ) CH ₂ CH ₃ ].
There is provided caprazamycin D1 and caprazamycin G1, or pharmaceutically acceptable salts thereof.

The caprazamycins D1 and G1 represented by the general formula (II) according to the second aspect of the present invention include caprazamycin D1 of the following formula (IIa) and caprazamycin G1 of the following formula (IIb). To do. (1) The following formula (IIa) Caprazamycin D1 [in the general formula (II), R is 1
A compound having a 0-methyl-undecyl group]. (2) The following formula (IIb) Caprazamycin G1 represented by [R in the general formula (II) is 9
-Compound when it is a methyl-undecyl group].

The caprazamycin D of the formula (Ia) according to the first aspect of the present invention has the following physicochemical properties. (1) Appearance: colorless powder (2) Molecular formula: C ₅₂ H ₈₅ N ₅ O ₂₂ (3) High resolution mass spectrometry (HRFABMS: positive ion mode) Experimental value: 1132.5776 (M + H) ⁺ calculated value: 1132.5764 (4 ) Specific rotation: [α] _D ²³ -3.0 ° (c 1, methanol) (5) Ultraviolet absorption spectrum (in methanol) λmax nm (ε): 262 (9,200) Shown in FIG. 1 of the accompanying drawings. (6) Infrared absorption spectrum: shown in FIG. 2 of the accompanying drawings. (7) Proton nuclear magnetic resonance spectrum A proton NMR spectrum measured at room temperature in deuterated methanol at 500 MHz is shown in FIG. 3 of the accompanying drawings. (8) Carbon 13 Nuclear magnetic resonance spectrum Carbon measured at 125MHz in deuterated methanol at room temperature
The 13 NMR spectrum is shown in Figure 4 of the accompanying drawings.

(9) Solubility Soluble in methanol, dimethyl sulfoxide (DMSO) and water, but insoluble in acetone and ethyl acetate. (10) The Rf value when developed with a solvent of butanol-methanol-water (4: 1: 2) on thin layer chromatography of TLC silica gel 60F ₂₅₄ (manufactured by Merck) is 0.41.

The caprazamycin D of the first invention is an amphoteric substance, and its pharmaceutically acceptable salt is a salt with an organic base such as a quaternary ammonium salt, or a salt with various metals, Examples thereof include salts with alkali metals such as sodium salts, addition salts with organic acids such as acetic acid, and addition salts with inorganic acids such as hydrochloric acid.

The caprazamycin G of the formula (Ib) according to the present invention has the following physicochemical properties.

(1) Appearance: colorless powder (2) Molecular formula: C ₅₂ H ₈₅ N ₅ O ₂₂ (3) High resolution mass spectrometry (HRFABMS: positive ion mode) Experimental value 1132.5769 (M + H) ⁺ calculated value 1132.5764 ( 4) Specific rotation: [α] _D ²³ -4.2 ° (c 0.61, methanol) (5) Ultraviolet absorption spectrum (in methanol) λmax nm (ε): 262 (9,000) Shown in Figure 9 of the accompanying drawings. (6) Infrared absorption spectrum: shown in FIG. 10 of the accompanying drawings. (7) Proton Nuclear Magnetic Resonance Spectrum A proton NMR spectrum measured at 500 MHz in deuterated methanol at room temperature is shown in FIG. 11 of the accompanying drawings. (8) Carbon 13 Nuclear magnetic resonance spectrum Carbon measured at 125MHz in deuterated methanol at room temperature
The 13 NMR spectrum is shown in Figure 12 of the accompanying drawings.

(9) Solubility Soluble in methanol, DMSO and water, but insoluble in acetone and ethyl acetate. (10) The Rf value when developed with a solvent of butanol-methanol-water (4: 1: 2) on thin layer chromatography of TLC silica gel 60F ₂₅₄ (manufactured by Merck) is 0.41.

The caprazamycin G of the first present invention is an amphoteric substance, and its pharmaceutically acceptable salt is a salt with an organic base such as a quaternary ammonium salt, or a salt with various metals, Examples thereof include salts with alkali metals such as sodium salts, addition salts with organic acids such as acetic acid, and addition salts with various inorganic acids such as hydrochloric acid.

The caprazamicin D1 of the formula (IIa) according to the second aspect of the present invention has the following physicochemical properties. (1) Appearance: colorless powder (2) Molecular formula: C ₄₃ H ₆₉ N ₅ O ₁₈ (3) High resolution mass spectrometry (HRFABMS: positive ion mode) Experimental value: 944.4686 (M + H) ⁺ calculated value: 944.4716 (4 ) Specific rotation: [α] _D ²⁵ + 13.9 ° (c 0.91, methanol) (5) Ultraviolet absorption spectrum (in methanol) λ max nm (ε): 262 (8,300) Shown in FIG. 5 of the accompanying drawings. (6) Infrared absorption spectrum: shown in FIG. 6 of the accompanying drawings. (7) Proton nuclear magnetic resonance spectrum A proton NMR spectrum measured at 500 MHz in deuterated methanol at room temperature is shown in FIG. 7 of the accompanying drawings. (8) Carbon-13 nuclear magnetic resonance spectrum The carbon-13 NMR spectrum measured at room temperature in a mixed solvent of deuterated methanol / heavy water (= 10: 1) at 125 MHz is shown in FIG. 8 of the accompanying drawings.

(9) Solubility Soluble in methanol, DMSO and water, but insoluble in acetone and ethyl acetate. (10) The Rf value when developed with a solvent of butanol-methanol-water (4: 1: 2) on thin layer chromatography of TLC silica gel 60F ₂₅₄ (manufactured by Merck) is 0.41.

The caprazamicin D1 of the second present invention is an amphoteric substance, and its pharmaceutically acceptable salt is:
Salts with organic bases such as quaternary ammonium salts, salts with various metals, salts with alkali metals such as sodium salts, addition salts with organic acids such as acetic acid, or various inorganics such as hydrochloric acid. Examples thereof include addition salts with acids.

The caprazamicin G1 of the formula (IIb) according to the second aspect of the present invention has the following physicochemical properties. (1) Appearance: colorless powder (2) Molecular formula: C ₄₃ H ₆₉ N ₅ O ₁₈ (3) High resolution mass spectrometry (HRFABMS: positive ion mode) Experimental value 944.4720 (M + H) ⁺ calculated value 944.4716 (4) Ratio Optical rotation: [α] _D ²⁵ + 14.6 ° (c 0.91, methanol) (5) Ultraviolet absorption spectrum (in methanol) λmax nm (ε): 262 (8,300) Shown in FIG. 13 of the accompanying drawings. (6) Infrared absorption spectrum: shown in FIG. 14 of the accompanying drawings. (7) Proton nuclear magnetic resonance spectrum A proton NMR spectrum measured at room temperature in deuterated methanol at 500 MHz is shown in FIG. 15 of the accompanying drawings. (8) Carbon 13 Nuclear magnetic resonance spectrum Carbon measured at 125MHz in deuterated methanol at room temperature
The 13 NMR spectrum is shown in Figure 16 of the accompanying drawings.

(9) Solubility Soluble in methanol, DMSO and water, but insoluble in acetone and ethyl acetate. (10) The Rf value when developed with a solvent of butanol-methanol-water (4: 1: 2) on thin layer chromatography of TLC silica gel 60F ₂₅₄ (manufactured by Merck) is 0.41.

The caprazamycin G1 of the second present invention is an amphoteric substance, and its pharmaceutically acceptable salt is:
Salts with organic bases such as quaternary ammonium salts, salts with various metals, salts with alkali metals such as sodium salts, addition salts with organic acids such as acetic acid, or various inorganics such as hydrochloric acid. Examples thereof include addition salts with acids.

The liposidomycins Z, L, M, K and N shown in the above-mentioned WO 97/41248 are represented by the following general formula (B): [Wherein R ₁ has the meaning shown in Table 1 below according to Table ₁ of WO 97/41248], and the molecular formula and molecular weight shown in Table 1 It is a compound with.

Further, liposidomycin A- (II) and C- (II) shown in WO 97/41248 are represented by the following general formula (C): [Wherein R ₁ is according to Table ₁ of WO 97/41248,
It has the meaning shown in Table 2 below], and is a compound having the molecular formula and molecular weight shown in Table 2.

Furthermore, liposidomycin X- (III), Y- (II
I), Z- (III), C- (III), V- (III), A- (III), G- (III), M
-(III), K- (III) and N- (III) are represented by the following general formula (D) [Wherein R ₁ is according to Table ₁ of WO 97/41248,
It has the meaning shown in Table 3 below] and has the molecular formula and molecular weight shown in Table 3.

Furthermore, liposidomycin A- (IV) and C- (IV) are represented by the following general formula (E): [Wherein R ₁ is according to Table ₁ of WO 97/41248,
It has the meaning shown in Table 4 below] and has the molecular formula and molecular weight shown in Table 4.

[0032]

Caprazamycin D, G, D according to the invention
It is clearly seen that 1 and G1 do not match any of the known liposidomycins shown above.

Caprazamycins D and G represented by the general formula (I) according to the first aspect of the present invention and caprazamycin D1 represented by the general formula (II) according to the second aspect of the present invention.
And G1 have the biological properties described below. That is, caprazamycin D, caprazamycin G, caprazamycin D1 and caprazamycin G1 have antibacterial activity against gram-positive bacteria including drug-resistant bacteria and acid-fast bacteria, and drug-resistant bacteria. Indicates. The antibacterial activity of caprazamycin D, G, D1 and G1 against these bacteria was tested as follows.

Test Example 1 The antibacterial spectrum of caprazamycin D against various microorganisms was measured by the multiple dilution method on a normal agar medium containing 1% glycerin based on the standard method of the Japanese Society of Chemotherapy. The results are shown in Table 5 below.

Test Example 2 The antibacterial spectrum of caprazamycin D against various microorganisms other than those shown in Table 5 was measured by the multiple dilution method on the Mueller Hinton agar medium based on the standard method of the Japanese Society of Chemotherapy. . The results are shown in Table 6.

Test Example 3 The antibacterial spectrum of caprazamycin G against various microorganisms was measured by a multiple dilution method on a normal agar medium containing 1% glycerin based on the standard method of the Japanese Society of Chemotherapy. The results are shown in Table 7.

Test Example 4 The antibacterial spectrum of caprazamycin G against various microorganisms other than those shown in Table 7 was measured by the multiple dilution method on the Mueller Hinton agar medium based on the standard method of the Japanese Society of Chemotherapy. . The results are shown in Table 8.

Test Example 5 The antibacterial spectrum of caprazamycin D1 against various microorganisms was measured by a multiple dilution method on a normal agar medium containing 1% glycerin based on the standard method of the Japanese Society of Chemotherapy. The results are shown in Table 9.

Test Example 6 The antibacterial spectrum of caprazamycin D1 against various microorganisms other than those shown in Table 9 was measured by the multiple dilution method on the Mueller Hinton agar medium based on the standard method of the Japanese Society of Chemotherapy. . The results are shown in Table 10.

Test Example 7 The antibacterial spectrum of caprazamycin G1 against various microorganisms was measured by a multiple dilution method on a common agar medium containing 1% glycerin based on the standard method of the Japanese Society of Chemotherapy. The results are shown in Table 11.

Test Example 8 The antibacterial spectrum of caprazamycin D1 against various microorganisms other than those shown in Table 11 was measured by the multiple dilution method on the Mueller Hinton agar medium based on the standard method of the Japanese Society of Chemotherapy. . The results are shown in Table 12.

Furthermore, according to the third aspect of the present invention, which belongs to the genus Streptomyces and is represented by the above-mentioned general formula (I), caprazamycin D and caprazamycin G, and general formula (II). Of at least one of caprazamycin D1 and caprazamycin G1 are cultivated, and caprazamycin D, G, D1 is obtained from the culture.
And at least one of G1 is provided, which provides a process for the production of the antibiotics caprazamycin D, G, D1 and / or G1.

The bacteria producing the antibiotics caprazamycin D, G, D1 and G1 used in the third method of the present invention are caprazamycins D, G having the above-mentioned physicochemical and biological properties. As long as it has the ability to produce D1 and G1, it can be used regardless of its species and can be selected from a wide range of microorganisms. Among such microorganisms, a specific preferable example of a bacterium producing the antibiotic caprazamycin is isolated from soil of Hawaii and Oahu Island at the Institute for Microbial Chemistry in March 1997 by the present inventors. There is also an actinomycete strain with the strain number MK730-62F2.

The mycological properties of the MK730-62F2 strain are described below. 1. Morphology MK730-62F2 strain extends aerial hyphae that are relatively longer than the branched basal hyphae and forms a helix of 5-10 turns at the tip. The mature spore chain links 10 to 50 oval spores, and the spore size is about 0.5-0.6 x 0.8-1.0 micron. The surface of the spores is smooth. Rotation branch, fungal filament,
There are no sporangia or motile spores.

2. Regarding the description of the growth state color in various media The standard of the color shown in [] is the Color Harmony Manual (Container Corporation of America) of the Continer Corporation of America.
Color harmony manual). (1) Sucrose / nitrate agar medium (27 ° C culture) White aerial hyphae grew slightly on the development of light yellow [2ea, Lt Wheat], and no soluble pigment was observed. (2) Glycerin / asparagine agar medium (ISP-medium 5,
27 ℃ culture) Light yellow [2ea, Lt Wheat] ~ Light yellow tea [2ng, Dull Gol
On the development of [d], aerial hyphae of gray [3dc, Natural] to light ash [d] are settled. No soluble dye is found.

(3) Starch / inorganic salt agar medium (ISP-medium 4, 27 ° C. culture) light yellow [2ea, Lt Wheat] to light yellow tea [2lg, Mustard Ta
n] grows, and aerial hyphae of white to bright ash [d] are settled. No soluble dye is found. (4) Tyrosine agar medium (ISP-medium 7, 27 ℃ culture) On the growth of light yellow tea [2le, Mustard ~ 2ng, Dull Gold], attach aerial mycelia of gray [b, Oyster White ~ 3dc, Natural]. It produces a soluble dye in dark tea. (5) Yeast / malt agar medium (ISP-medium 2, 27 ° C culture) For the development of light yellow tea [2ie, Lt Mustard Tan ~ 3ic, Lt Amber], gray [b, OysterWhite] ~ bright ash [d] Settle aerial mycelia. No soluble dye is found. (6) Oatmeal agar medium (ISP-medium 3, 27 ° C culture) On the development of light yellow [2ea, Lt Wheat], gray white [3dc, Natu
Ral] to bright ash [d] aerial hyphae are settled and no soluble pigment is observed.

3. Physiological properties (1) Growth temperature range Glucose / asparagine agar medium (glucose 1.0
%, Asparagine 0.05%, dipotassium phosphate 0.05%, string agar 2.5%, pH 7.0), 10 ℃, 20 ℃, 24 ℃, 27
As a result of testing at each temperature of ℃, 30 ℃, 37 ℃, 45 ℃ and 50 ℃, this strain, except 10 ℃, 45 ℃ and 50 ℃, from 20 ℃ to 37 ℃
It was grown in the range of ° C. The optimum temperature for growth is around 30 to 37 ° C.

(2) Starch hydrolysis (starch / inorganic salt agar medium, ISP-medium 4, 27 ° C. culture) Starch hydrolysis was observed from about 3 days after the culture, and its action is moderate. (3) Generation of melanin-like pigment (tryptone, yeast,
Broth, ISP-medium 1; peptone-yeast-iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; both cultures at 27 ° C) Both media are positive. (4) Utilization of carbon source (Pridham-Gotrieve agar medium, ISP-medium 9, 27 ° C. culture) D-glucose, L-arabinose, D-fructose, sucrose, inositol, rhamnose, raffinose and D-mannitol are used And grow up, D-
Probably also uses xylose.

(5) Reduction reaction of nitrate (peptone water containing 0.1% potassium nitrate, ISP-medium 8, 27 ° C. culture) Negative. (6) Liquefaction of gelatin (simple gelatin, 20 ° C culture; glucose / peptone gelatin, 27 ° C culture) No liquefaction was observed in the simple gelatin after 40 days of culture. In case of glucose, peptone and gelatin, after culturing
About 40 days, it showed weak liquefaction. (7) Coagulation and peptone conversion of skim milk (10% skim milk, 37 ° C culture) Without coagulation, peptone conversion started about 7 days after cultivation and was completed on the 14th day.

In summary of the above properties, the MK730-62F2 strain extends aerial hyphae having helix formation from branched basal hyphae. The surface of spores is smooth. In various media,
On the development of light yellow to light yellow tea, aerial hyphae of gray to light ash are grown. Soluble pigments were not found except for melanin-like pigments. The optimum growth temperature is around 30-37 ℃. The formation of melanin-like pigment is positive, and the water-degradability of starch is moderate. The 2,6-diaminopimelic acid contained in the cell wall is LL-type, and the major menaquinone in the bacterial cells is MK-9 (H8).
And MK-9 (H6).

From these properties, the MK730-62F2 strain is considered to belong to the genus Streptomyces .
Therefore, as a result of searching for closely related known strains, Streptomyces diasta
tochromogenes , Literature, International Journal of
Systematic Bacteriology, Vol. 22, p. 290, 1972), Streptomyce
s resistomycificus , Literature, International Journal of
Systematic Bacteriology, 18, pp. 165 pp., 1968), Streptomyces Korinusu (Streptomyces collinus, literature, International Journal of Systematic Bacteriolo
gy, 18: 100, 1968) and Streptomyces aurantiogriseu
s , Literature, International Journal of Systematic Bacte
rio logy, vol. 18, p. 297, 1968). Next, the above-mentioned 4 strains preserved by the present institute and the MK730-62F2 strain were compared and examined in practice. The results are shown in Table 13 below.

[0053]

[0054]

As is clear from Table 13 above, MK730-62
The F2 strain showed similar characteristics to any of the species compared in Table 13. However, Streptomyces registomysix exhibits a light yellow to brown-brown color in the development, the soluble pigment takes on a brown color, and it does not peptone defatted milk.
It was different from the 0-62F2 strain. In addition, Streptomyces corinus exhibits aerial hyphae in the form of straight to loop, and does not peptate defatted milk, and Streptomyces aurantioglyceus has a soluble dye tinge brown and liquefies simple gelatin. In terms of reducing nitrates, MK730-62F2
Distinguished from strains. On the other hand, Streptomyces diastatochromogenes was very similar to the MK730-62F2 strain except that the liquefaction of simple gelatin was positive. However, at present, the MK730-62F2 strain cannot be identified as a strain of Streptomyces diastatochromogenes. So MK
The 730-62F2 strain was designated Streptomyc
es sp.) MK730-62F2.

The MK730-62F2 strain was purchased from the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology located at Central No. 1-1, 1-1, Tsukuba, Ibaraki, Japan (Independent administration on April 1, 2001). A deposit application was made to the Patent Biological Depository Center of the National Institute of Advanced Industrial Science and Technology, and it was accepted as FERM P-17067 on November 27, 1998. In addition, on the transfer acceptance date of July 12, 2000, the MK730-62F2 strain was deposited at the above depository with the deposit number of FERM BP-7218 under the terms of the Budapest Treaty.

In the third method of the present invention, the antibiotics caprazamycin D, G, D1 and G1 are produced as follows. That is, the antibiotics caprazamycin D, G,
D1 and G1 are produced by caprazamycin D, G, D
1. Producing bacteria producing at least one of G1 and G1 (simply called caprazamycin D, G, D1, G1 producing bacteria) is inoculated into a nutrient medium, and antibiotics caprazamycin D, G, D1,
It is carried out by culturing at a favorable temperature for the production of G1. By culturing caprazamycin D, G, D1, G1 producing bacteria, antibiotics caprazamycin D, G, D1 and G1
A culture containing is obtained.

As the nutrient medium used for such a purpose, a nutrient medium that can be used for culturing actinomycetes is used. As a nutrient source, for example, commercially available soybean powder, peptone, yeast extract, meat extract, corn steep liquor, nitrogen sources such as ammonium sulfate can be used. Further, a carbon source such as a carbohydrate such as tomato paste, glycerin, starch, glucose, galactose, dextrin or fat can be used. Further, inorganic salts such as salt and calcium carbonate can be added and used. In addition, a trace amount of metal salt can be added if necessary. Any of these may be used as long as it is used by caprazamycin D, G, D1, G1 producing bacteria and is useful for the production of antibiotics caprazamycin D, G, D1, G1. Known actinomycete culture materials Can all be used.

Antibiotics Caprazamycin D, G, D1,
For the production of G1, a microorganism belonging to the genus Streptomyces and capable of producing the antibiotics caprazamycin D, G, D1 and G1 is used. Specifically, it was revealed by the present inventors that the isolated Streptomyces sp. MK730-62F2 (FERM BP-7218) produced by the present inventors produced the antibiotics caprazamycin D, G, D1 and G1. However, other strains capable of producing caprazamycin D, G, D1 and G1 can be isolated from nature by a conventional method for isolation of antibiotic-producing bacteria. In addition, including Streptomyces sp. MK730-62F2, the caprazamycin D, G, D1, and G1 producing bacteria are subjected to radiation irradiation and other mutation treatments.
There is still room for increasing G, D1, and G1 productivity. Furthermore, the antibiotics caprazamycin D, G, D1, and G1 can be produced by genetic engineering techniques.

To obtain seed mothers for the production of caprazamycin D, G, D1 and G1, MK73 was used on agar medium.
The growth product obtained from the slant culture of the 0-62F2 strain is used. In producing the antibiotics caprazamycin D, G, D1 and G1, it is preferable to aerobically culture caprazamycin D, G, D1 and G1 producing bacteria belonging to the genus Streptomyces in an appropriate medium. Conventional means can be used to collect the desired caprazamycins D, G, D1 and G1 from the culture. The culture temperature is caprazamycin D,
There is no particular restriction as long as the growth of G, D1 and G1 producing bacteria is not substantially inhibited and these antibiotics can be produced. The culture temperature can be appropriately selected according to the caprazamycin D, G, D1 and G1 producing bacteria to be used,
Preferably, the temperature within the range of 25 to 30 ° C. can be mentioned.

The production of caprazamycin D, G, D1 and G1 by this MK730-62F2 strain usually reaches its maximum in 3 to 9 days, but generally continues until sufficient antibacterial activity is imparted to the medium. The time course of the titer of caprazamycin D, G, D1, and G1 in this culture solution was determined by the HPLC method, or by the cylindrical plate method using Mycobacterium smegmatis or Mycobacterium bake as the test bacteria. Can be measured.

In the third method of the present invention, caprazamycin D, G, from the culture obtained as described above,
Collect at least one of D1 and G1. As a method for collecting caprazamycin D, G, D1, and G1, means used for collecting metabolites produced by microorganisms can be appropriately used. For example, by using a means of extraction with an organic solvent immiscible with water, a means of utilizing a difference in adsorption affinity for various adsorbents, a gel filtration method, chromatography using countercurrent distribution, etc., alone or in combination. From the culture, caprazamycin D, G, D1 and G1 can be collected individually or as a mixture of any two or more thereof.

From the separated bacterial cells, caprazamycins are extracted by a solvent extraction method using an appropriate organic solvent or an elution method by disruption of the bacterial cells. As well as caprazamycin D,
G, D1 and G1 can be isolated and collected. Thus, the aforementioned antibiotics caprazamycin D, G, D
1 and G1 are obtained separately or as a mixture of two or more. In addition, caprazamycin D from caprazamycin A, B, C, E, F produced at the same time,
The separation of G, D1 and G1 and the separation of caprazamycin D, G, D1 and G1 from each other can be carried out by high performance liquid chromatography (HPLC) using a suitable developing solvent, as exemplified in the examples below. It can be carried out.

The above-mentioned Streptomyces sp. MK7
The 30-62F2 strain (FERM BP-7218) not only produces caprazamycin D, G, D1 and G1 of the present invention when cultured, but also known caprazamycins A, B, C and E.
And F can also be produced.

Therefore, in the fourth aspect of the present invention, the known antibiotics caprazamycin A, B, C, E and F and the antibiotics caprazamycin D and caprazamycin represented by the above general formula (I) are used. G and Streptomyces sp. MK730-62F2 (patented by the National Institute of Advanced Industrial Science and Technology, which has the property of producing the antibiotics caprazamycin D1 and caprazamycin G1 represented by the general formula (II) according to claim 4) The strain deposited at the Biological Depository Center under the terms of the Budapest Treaty under the deposit number of FERM BP-7218) was cultivated under aerobic conditions, and the antibiotics caprazamycin A, B, C, E and F and the antibiotics caprazamycin D, G, D1 and G1 respectively, separately collected,
Methods of making the antibiotics caprazamycin A, B, C, D, E, F and G and caprazamycin D1 and G1 are provided. The fourth method of the present invention can be carried out in the same manner as the third method of the present invention.

Further, in the fifth aspect of the present invention, the general formula (I)
And the general formula
(II) at least one of caprazamycin D1 and G1 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier,
There is provided a pharmaceutical composition containing the active ingredient in admixture.
In the pharmaceutical composition according to the fifth aspect of the present invention, the compound of caprazamycin D or G of general formula (I) or the compound of caprazamycin D1 or G1 of general formula (II) as an active ingredient is pharmaceutically It may be in the form of a composition containing a conventional liquid carrier such as ethanol, hydrous ethanol, water, physiological saline, or a solid carrier such as crystalline cellulose, starch and the like.

Caprazamicin D or G of the general formula (I), which is the active ingredient used in the pharmaceutical composition of the fifth invention,
Alternatively, caprazamicin D1 or G1 of the general formula (II) or a salt thereof can be administered orally. Alternatively, they can be administered parenterally, such as by nasal drop, intravenous or intramuscular or subcutaneous injection, or intraperitoneal administration. For oral administration, in the fifth pharmaceutical composition of the present invention, caprazamycin D or G of the general formula (I) or caprazamycin D1 or G1 of the general formula (II) as an active ingredient, Alternatively, the salt is mixed with a pharmaceutically acceptable conventional solid or liquid carrier, and the resulting mixture is formulated into a powder, tablet, capsule, suspension, syrup, etc. You can

Caprazamycin D, G, D1 or G1 as an active ingredient to be incorporated in the pharmaceutical composition of the fifth invention.
The ratio of the amount of the drug may vary depending on the dosage form.
It should be in the range of 90%. When the composition of the fifth aspect of the present invention is formulated for injection, a desirable formulation form is a sterile aqueous solution or sterile lyophilizer containing the above-mentioned caprazamycin as an active ingredient. The liquid carrier used here is preferably water, physiological saline, ethanol, hydrous ethanol, glycerol, propylene glycol, vegetable oil, or the like.

Caprazamycin D, G, D1 or G1, used as an active ingredient in the pharmaceutical composition of the present invention,
Alternatively, the dose of the salt varies depending on the type of bacterial infection to be treated, the purpose of treatment and the degree of symptoms.
The optimal dose of caprazamycin D, G, D1 or G1 can be determined by an expert in an appropriate preliminary test.

[0070]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below with reference to Examples. Example 1 Production of the antibiotics caprazamycin D, G, D1 and G1 Streptomyces sp. MK cultivated on an agar slant medium
730-62F2 (deposited with accession number FERM BP-7218), galactose 2%, dextrin 2%, glycerin 1%, Bactosoyton (manufactured by Difco) 1%, corn steep liquor 0.5%, ammonium sulfate 0.2%, calcium carbonate 0.2
Liquid medium (adjusted to pH 7.4) containing 5% Erlenmeyer flask (5
The solution was dispensed in an amount of 110 ml each in a volume of 00 ml) and inoculated into a medium sterilized at 120 ° C. for 20 minutes by a conventional method. After inoculation, this was cultivated at 30 ° C. for 2 days with rotary shaking to obtain a seed culture medium.

Tomato paste (Kagome) 2.4%, dextrin 2.4%, yeast extract (Oriental) 1.2
% Of cobalt chloride and 0.0006% of cobalt chloride (adjusted to pH 7.4) 15 liters of a medium was prepared in a tank culture tank (30 liters volume) and used as a production medium after sterilization. In this production medium,
An amount of 2% of the above seed culture medium was inoculated, and tank culture was carried out at 27 ° C. for 6 days under the culture conditions using an aeration rate of 15 liters per minute and a stirring rate of 200 rpm.

The culture broth thus obtained was centrifuged to separate 12 liters of the culture filtrate from the cells. Continuing,
6 liters of methanol was added to the cells and well stirred, and the antibiotics known as caprazamycin A, B, C, E from the cells were added.
And F and the new antibiotic caprazamycin D,
G, D1 and G1 were extracted with methanol (hereinafter, these antibiotics caprazamycin A, B, C, D,
E, F, G, D1 and G1 may be collectively referred to as caprazamycins). Combine this culture filtrate with the bacterial cell extract (methanol extract), and add 18 liters of the mixture obtained from this merger to the aromatic synthetic adsorbent Diaion HP.
-20 ml (manufactured by Mitsubishi Chemical Co., Ltd. of Japan) was passed through a column of 750 ml to adsorb caprazamycins.

Deionized water, 50% to this Diaion HP-20
2.25 liters of methanol water, 80% methanol water, 80% acetone water, and acetone were sequentially passed. A large amount of caprazamycin was eluted in the elution fraction with 80% acetone water. Also, since 50% methanol water elution fraction and 80% methanol water elution fraction also contained caprazamycins, both were combined again and the DIAION HP-20 column (7
50 ml), whereby the caprazamycins were adsorbed on the adsorbent of the column, and then 2.25 liter of 80% methanol water was passed through the column. Then from the column 80
Elution was performed with 2.25 liters of% acetone water. This eluate with 80% acetone water was combined with the above 80% acetone water elution fraction and concentrated to dryness under reduced pressure to obtain 10.1 g of a crude purified product containing caprazamycins.

A crude product containing this caprazamycin
10.1 g was dissolved in 50 ml of a mixed solvent of chloroform-methanol (= 1: 2), 50 ml of Kieselguhl (Merck, Art.10601) was added to the solution, and the solvent was concentrated to dryness under reduced pressure. The solid residue thus obtained by adsorbing caprazamycins to Kieselguhl was placed on a silica gel column (internal diameter 54 mm × length 200 mm) and subjected to chromatography. At this time, chloroform-methanol-water (= 4: 1: 0.1), chloroform-methanol-water (= 2: 1: 0.2), and chloroform-methanol-water (= 1: 1: 0.2) were used as developing solvents. Using 1.35 liters of each mixed solution, development was carried out sequentially.

The eluate from the silica gel column was fractionated by 20 g in fractions Nos. 1 to 53 and 19 g in fractions No. 54 to 117 by a fraction collector. The active fractions containing caprazamycin A, B, C, D, E, F and G were eluted in fractions No. 84 to 144, and the active fractions containing caprazamycin D1 and G1 in fractions No. 84 to 144 were eluted. Fraction Nos. 66 to 83 containing the caprazamycins A, B, C, D, E, F and G are collected and concentrated to dryness under reduced pressure.
625.3 mg of a crude product containing caprazamycin A, B, C, D, E, F and G was obtained. Also, Fraction No. 54 ~
117 were collected, concentrated to dryness under reduced pressure, and caprazamycin D1
1.28 g of a crude purified product containing and G1 was obtained.

First, caprazamycin A, B, C,
The crude purified products containing D, E, F and G were isolated and purified. When 62 ml of the above crude spermatozoa was dissolved by adding 5 ml of methanol, and the resulting solution was allowed to stand in the dark at 5 ° C., caprazamycin A, B, C, D, E,
537.3 mg of a precipitated precipitate fraction containing F and G was obtained. Next, this caprazamycin A, B, C, D, E, F, G
Precipitated precipitate fraction containing HPLC (CAPCELLPAK C18, φ20 × 25
0 mm, manufactured by Shiseido). In this HPLC, 50% acetonitrile water-0.05% formic acid (flow rate: 12.0 m
1 / min), caprazamycin A was eluted after 61 to 68 minutes, caprazamycin B was eluted after 52 to 60 minutes, and caprazamycin C was eluted after 39 to 41 minutes. After a minute, a mixture containing caprazamycin D and caprazamycin G, after 25 to 28 minutes, caprazamycin E was eluted, and after 22 to 25 minutes, caprazamycin F was eluted. Each active fraction was collected and concentrated to dryness under reduced pressure to obtain caprazamycin A (56.9
mg), caprazamycin B (90.3 mg), caprazamycin C (19.7 mg), a mixture containing caprazamycin D and caprazamycin G (162.9 mg), caprazamycin E (30.3 mg) and caprazamycin F (25.5 mg) was obtained.

Furthermore, 162.9 mg of the mixture containing caprazamycin D and caprazamycin G obtained above
It was purified using a PLC (CAPCELLPAK C18, φ20 × 250 mm, manufactured by Shiseido Co., Ltd.). In this HPLC, as a developing solvent, 50% acetonitrile water-0.025% trifluoroacetic acid (flow rate: 9.0 ml / mi
When developed by n), caprazamycin D was eluted after 55 to 69 minutes and caprazamycin G was eluted after 48 to 53 minutes.
The respective active fractions were collected and concentrated to dryness under reduced pressure to give caprazamycin D (69.7 mg) and caprazamycin G (39.0 mg).

Furthermore, 1.28 g of the crude purified product containing caprazamycin D1 and G1 obtained above was subjected to HPLC (CAPCE
Each component was isolated and purified using LLPAK C18, φ20 × 250 mm, manufactured by Shiseido. 45% acetonitrile water-0.05% trifluoroacetic acid as a developing solvent in this HPLC (flow rate: 12.0 ml
/ capillary), 36 to 49 minutes later, caprazamycin G1 and D1 were eluted, and these were collected and concentrated to dryness under reduced pressure to obtain 187 mg of a mixture of caprazamycin D1 and caprazamycin G1. It was In addition, HPLC
(CAPCELLPAK C18, φ20 × 250mm, manufactured by Shiseido Co., Ltd.) and developed with 44% acetonitrile water-0.025% trifluoroacetic acid (flow rate: 9.0 ml / min) as a developing solvent, caprazamycin D1 elutes after 46 to 52 minutes And after 41-44 minutes caprazamycin G1 was eluted. These elution fractions were collected and separately concentrated to dryness under reduced pressure to obtain caprazamycin D1 (54.1 mg) and caprazamycin G1 (57.6 mg).

[0079]

As described above, according to the present invention,
Caprazamycins D and G of the general formula (I) and caprazamycins D1 and G1 of the general formula (II) obtained as novel antibiotics are respectively various anti-acid bacteria,
It has excellent antibacterial activity against bacteria and their drug-resistant strains. Therefore, caprazamycin D of the present invention,
G, D1, G1 are effective and useful in treating infections with acid-fast bacteria and bacteria.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum of caprazamycin D in a methanol solution.

FIG. 2 is an infrared absorption spectrum of caprazamycin D measured by a KBr tablet method.

FIG. 3 is a proton nuclear magnetic resonance spectrum at 500 MHz measured in a deuterated methanol solution of caprazamycin D at room temperature.

FIG. 4 is a carbon-13 nuclear magnetic resonance spectrum at 125 MHz measured in a solution of caprazamycin D in deuterated methanol at room temperature.

FIG. 5 is an ultraviolet absorption spectrum of caprazamycin G in a methanol solution.

FIG. 6 is an infrared absorption spectrum of caprazamycin G measured by the KBr tablet method.

FIG. 7 is a proton nuclear magnetic resonance spectrum at 500 MHz measured in a solution of caprazamycin G in deuterated methanol at room temperature.

FIG. 8 is a carbon-13 nuclear magnetic resonance spectrum at 125 MHz measured in a deuterated methanol solution of caprazamycin G at room temperature.

FIG. 9 is an ultraviolet absorption spectrum of caprazamycin D1 in a methanol solution.

FIG. 10 is an infrared absorption spectrum of caprazamycin D1 measured by the KBr tablet method.

FIG. 11 is a proton nuclear magnetic resonance spectrum at 500 MHz measured at room temperature in a heavy methanol solution of Kapala mystery D1.

FIG. 12 is a carbon-13 nuclear magnetic resonance spectrum at 125 MHz measured in a deuterated methanol solution of caprazamycin D1 at room temperature.

FIG. 13 is an ultraviolet absorption spectrum of caprazamycin G1 in a methanol solution.

FIG. 14 is an infrared absorption spectrum of caprazamycin G1 measured by the KBr tablet method.

FIG. 15 is a proton nuclear magnetic resonance spectrum at 500 MHz measured in a deuterated methanol solution of caprazamycin G1 at room temperature.

FIG. 16 is a carbon-13 nuclear magnetic resonance spectrum at 125 MHz measured at room temperature in a deuterated methanol solution of caprazamycin G1.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // (C12P 17/16 C12R 1: 465 C12R 1: 465) (72) Inventor Masayuki Igarashi Atsugi City, Kanagawa Prefecture Nakamachi 4-10-4 Atsugi Green Corp. 802 (72) Inventor Hiroshi Naganawa 3-17, Denenchofuhonmachi, Ota-ku, Tokyo (72) Inventor Masa Hamada 17-2 Honshiocho, Shinjuku-ku, Tokyo F-term (reference) ) 4B064 AE54 CA04 CC06 CC12 CD02 CD19 CD22 CD30 CE03 CE08 CE09 CE10 DA02 4C057 AA01 BB03 CC03 CC04 DD03 LL10 LL41 4C086 AA01 AA02 AA03 AA04 EA11 MA01 MA04 MA05 NA14 ZB35

Claims (11)

[Claims] 1. The following general formula (I): [In the formula, R is 10-methyl-undecyl group- (CH ₂ ) ₉ CH (CH ₃ ) ₂ in caprazamycin D, and caprazamycin G is
In R is 9-methyl - undecyl group _{- (CH 2) 8 CH (} CH 3) CH 2 C
H ₃ ] and the antibiotics caprazamycin D and caprazamycin G, or a pharmaceutically acceptable salt thereof. 2. The following formula (Ia) 10. The antibiotic according to claim 1, which is caprazamycin D represented by the formula [a compound of the general formula (I) represented by claim 1 in which R is a 10-methyl-undecyl group]. 3. The following formula (Ib) The antibiotic according to claim 1, which is caprazamycin G represented by the formula [a compound of the general formula (I) represented by claim 1 in which R is a 9-methyl-undecyl group]. 4. The following general formula (II): [In the formula, R is 10-methyl-undecyl group- (CH ₂ ) ₉ CH (CH ₃ ) ₂ in caprazamycin D 1, and R is 9-methyl-undecyl group- (CH ₂ ) in caprazamycin G 1. ₈ CH (CH ₃ ) C
H ₂ CH ₃ ], which is a compound represented by the formula: caprazamycin D 1 and caprazamycin G 1, or a pharmaceutically acceptable salt thereof. 5. The following formula (IIa) 5. The antibiotic according to claim 4, which is caprazamycin D1 represented by the formula [a compound of the general formula (II) according to claim 4 in which R is a 10-methyl-undecyl group]. 6. The following formula (IIb) 5. The antibiotic according to claim 4, which is caprazamycin G1 (a compound of the general formula (II) according to claim 4 in which R is a 9-methyl-undecyl group). 7. The method according to claim 1, which belongs to the genus Streptomyces.
A small amount of the antibiotics caprazamycin D and caprazamycin G represented by the general formula (I) described in claim 1 and the antibiotics caprazamycin D1 and caprazamycin G1 represented by the general formula (II) according to claim 4 The antibiotics caprazamycin D, capraza, characterized in that at least one of caprazamycin D, G, D1 and G1 is collected from the culture, which produces a bacterium that produces both Process for producing mycin G, caprazamycin D1 and / or caprazamycin G1. 8. Caprazamycin D, G, D1 and G1
Streptomyces sp. MK730-62F2, which has been deposited with the deposit number of FERM BP-7218 under the terms of the Budapest Treaty at the Patent Biological Depository Center, National Institute of Advanced Industrial Science and Technology, as a bacterium producing at least one use,
The method according to claim 7. 9. A known antibiotic, caprazamycin A,
B, C, E and F and the antibiotics caprazamycin D and caprazamycin G represented by the general formula (I) described in claim 1 and the general formula (I
Streptomyces sp. MK730-62F2 (Independent Administrative Institution, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, FERM under the terms of the Budapest Treaty under the Budapest Treaty) The strain deposited under the accession number of BP-7218) is cultivated under aerobic conditions, and from the culture the antibiotics caprazamycin A, B, C, E and F and the antibiotic caprazamycin D, A method for producing the antibiotics caprazamycins A, B, C, D, E, F and G and caprazamycins D1 and G1 characterized in that G, D1 and G1 are separately collected. 10. The antibiotics caprazamycin D and caprazamycin G represented by the general formula (I) according to claim 1 and the antibiotics caprazamycin represented by the general formula (II) according to claim 4. A pharmaceutical composition comprising at least one of D1 and caprazamycin G1 or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier in admixture with the active ingredient. 11. The composition according to claim 10, which is an antibacterial composition.