JPH0238600B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a cyclic peptide represented by the following general formula and an acid addition salt thereof. These cyclic peptides and their salts are useful as intermediates in the production of semi-synthetic antifungal agents. These cyclic peptides are produced by deacylating a cyclic peptide antibiotic represented by the general formula below. In the formula, R is a saturated or unsaturated fatty acid side chain, and the definitions of R ¹ , R ² , R ³ and R ⁴ will be described later. In this specification, for convenience, the deacylated cyclic peptide represented by the above formula and the cyclic peptide skeleton excluding the acyl group R or COR ⁵ of the acylated cyclic peptide represented by the above formula or the following formula are used. It is sometimes referred to as a "cyclic peptide core" or simply "nucleus." We have discovered a method for producing a cyclic peptide core by enzymatically removing R from the fatty acid side chain. This manufacturing method uses antibiotics in an aqueous solvent.
The deacylation reaction is brought into contact with an enzyme produced from a microorganism belonging to the family Actinoplanaceae until the deacylation reaction is substantially completed. The preferred production method of the present invention uses microorganisms belonging to the genus Actinoplanes, particularly
It consists of cleaving fatty acid side chains using an enzyme produced from Actinoplanes utahensis NRRL12052. Deacylation usually consists of adding a suitable antibiotic to the A.utahensis medium and culturing the medium until deacylation is substantially complete. The nuclei thereby obtained are separated from the fermentation broth by methods well known to those skilled in the art. These nuclei are useful in that they can be reacylated into new antibiotics. In the present invention, each definition of the cyclic peptide core represented by the formula is as follows. R ¹ represents H or OH. And when R ¹ is H, R ² is H, and R ³ and R ⁴ are both H or both OH. Also, when R ¹ is OH, R ² is H, and R ³ is OH or C ₁ to C ₆
is alkoxy and R ⁴ is OH, or
R ² is CONH ₂ and R ³ and R ⁴ are both OH. One of the core embodiments of the present invention includes:
Examples include those in which R ¹ , R ³ and R ⁴ are OH and R ² is H, and this nucleus is known as the A-30912A nucleus. The A-30912A nucleus has the formula: R
is linoleoyl, stearoyl, or palmitoyl, and R ¹ , R ² , R ³ and R ⁴ are A-30912A
It is produced by deacylating a cyclic peptide antibiotic that has the same meaning as nuclear. Cyclic peptide antibiotics where R is linoleoyl are known as A-30912 Factor A, and antibiotics where R is stearoyl are known as tetrahydro-A-30912 Factor A,
The antibiotic in which R is palmitoyl is also known as acreacin A. A-30912 Factor A A-30912 Factor A is Factor B, C, D, E, F
It is one of the factors in the A-30912 complex that also contains G and G. For the A-30912 complex and individual factors A through G, see Marvin
M. Hoehn and Karl H. Michel, U.S. Patent No.
It is described in No. 4024245. A-30912 factor A is
Antibiotic A as described in U.S. Pat. No. 4,024,246 by Calvin. E. Higgens and Karl H. Michel
Same as −22082. A-30912 factor A is also
Antibiotic echinocandin B
[F.Benz et al., Helv.Chim.Acta57, 2459~
2477 (1974) and Swiss Patent No. 568386]
and antibiotic SL7810/F [C.Keller-Juslen
et., Tetrahedron Letters (46), 4147-4150
(1976) and Belgian Patent No. 834289]
It is also known that it is the same as Keller-Juslen et al., in the formula, R is linoleoyl, R ¹ , R ³ and R ⁴ are OH,
It was proposed that the structure in which R ² is H is designated as A-30912 factor A. Tetrahydro-A-30912 Factor A Tetrahydro-A-30912 Factor A (Tetrahydro-SL7810/F; Tetrahydroechinocandin B)
In the formula, R is stearoyl, and R ¹ ,
It has a structure in which R ³ and R ⁴ are OH and R ² is H, and Belgian Patent No. 834289 and F.Benz et al.
Helv.Chim.Acta. 57 , 2459–2477 (1974) by al.
It is described in. For convenience, this material is hereinafter referred to as Tetrahydro-A-30912A. Acleacin A Accreacin A is one component of the acreacin complex. The acreasin complex consists of one major component (acreasin A) and six minor components (acreasins B, C, D, E, F, and G), and these components of acreasin were disclosed in the US patent by K. Mizuno et al. No. 3978210. As discussed in Belgian Patent No. 859067,
Acreacin A is tetrahydro-A- except that the stearoyl side chain is replaced by palmitoyl.
It is a cyclic peptide with the same structure as 30912A. A-30912A nucleus Novel cyclic peptide nucleus of the present invention, namely A-
30912 factor A (echinocandin B, SL7810/F),
Tetrahydro-A-30912 Factor A, as well as the core of acreacin A, have a structure in which R ¹ , R ³ and R ⁴ are OH and R ² is H. A-30912A core is a white amorphous substance, water,
dimethylformamide, dimethyl sulfoxide,
It is soluble in solvents such as and methanol, and insoluble in solvents such as chloroform, toluene, and diethyl ether. The experimental formula of the A-30912A nucleus is C ₃₄ H ₅₁ N ₇ O ₁₅ ,
The molecular weight is 797.83. Figure 1 shows the infrared absorption spectrum of the A-30912A nucleus measured with a KBr disk. The following absorption maximum is observed. 3340 (wide, OH, hydrogen bond),
2970, 2930 and 2890 (CH stretching, aliphatic CH ₃ ,
CH ₂ , CH), 1660 and 1625 (multiple carbonyl C=O), 1510-1550, 1430-1450 (CH bending),
1310~1340, 1230~1260, 1080, 835, 650 (width), and 550 (width) cm ^-1 . A- in 66% aqueous dimethylformamide
Electrotitration of 30912A nuclei yields a pka value of approximately 7.35 (initial pH
7.32) indicates the existence of one applicable group. A-30912A nuclei can be separated by high performance liquid chromatography (HPLC), and the approximate retention time (k') when separated using the conditions described below is 11.52 minutes. Column: 4 x 300mm Packing material: Silica gel/ _C18 solvent: Ammonium acetate-acetonitrile-
Water (1:2:97) Flow rate: 3 ml/min Pressure: 2500 psi Detector: Tunable wavelength UV (230 nm) Sensitivity: 0 to 0.4 AUFS Production of substrate for A-30912A nucleus A-30912A nucleus in the present invention is A- 30912 Factor A, Tetrahydro-A-30912 Factor A, or acreasin A. These substrates are
Although it may be used as a purified substance, it is not necessarily necessary to purify it. Therefore, for example, A-
A-30912 complex in which 30912 factor A is the main component is A
-30912A may be used as a substrate for producing nuclei. A-30912 Factor A A-30912 Factor A is produced by performing deep aeration fermentation using any of the following microorganisms. 1) Aspergillus rugulosus NRRL8113;
2) Aspergillus nidulans NRRL 8112; 3)
Aspergillus nidulans var.echinulatus A-
32204, NRRL3860; 4) Aspergillus rugulosus
NRRL8039; or 5) Aspergillus nidulans
var.roseus, NRRL11440. A.nidulans var.roseus NRRL11440 strain A-
When used to produce 30912 Factor A, a conjugate of factors is obtained, which is conveniently referred to as the A-42355 antibiotic conjugate. A-30912 factor A is the main factor contained in the A-42355 antibiotic complex, and in addition,
Factors B, D and H are present in small amounts. Tetrahydro-A-30912A Tetrahydro-A-30912A is prepared by reducing A-30912 Factor A by standard hydrogenation procedures until the two double bonds in the linoleoyl side chain have been reduced. Acleacin A Acleacin A is Aspergillus aculeatus
Produced by fermentation of NRRL8075, which means
It is described in US Pat. No. 3,978,210, which is hereby incorporated by reference. Another embodiment of the core of the invention is that R ¹ and
Both R ² are H and R ³ and R ⁴ are both OH, which is known as the A-30912B nucleus. A
-30912B core is produced by deacylating a cyclic peptide antibiotic in which R is linoleoyl or stearoyl and R ¹ , R ² , R ³ and R ⁴ have the same meanings as A-30912B core. Ru. Cyclic peptide antibiotics where R is linoleoyl are known as A-30912 Factor B, and antibiotics where R is stearoyl are known as tetrahydro-A-30912 Factor B. A-30912 factor B A-30912 factor B is a factor A, C, D, E, F
It is one of the factors in the A-30912 complex that also contains G and G. For the A-30912 complex, see Marvin M. Hoehn and Karl H.
Michel and U.S. Pat. No. 4,024,245. A-30912 factor B is the antibiotic echinocandin C [R. Traber et al., Helv.
Chim. Acta. 62 , 1252-1267 (1979)] and the antibiotic SL7810/F- (Belgian patent no.
834289)). Traber et al. proposed that echinocandin C has a structure in which R ¹ and R ⁵ are both H, R ³ and R ⁴ are both OH, and R is linoleoyl. Further, Traber et al., in the formula, R ¹ and R ² are both H, and R ³ and R ⁴
are both OH and R is stearoyl, it was proposed that the structure be tetrahydro-echinocandin C. Tetrahydro-A-30912 Factor B Tetrahydro-A-30912 Factor B (Tetrahydro-SL7810/F-; Tetrahydroechinocandin C) is a compound in which R ¹ and R ² are both H
and has a structure in which R ³ and R ⁴ are both OH and R is stearoyl, and has a structure in which R 3 and R 4 are both OH and R is stearoyl, and it is
No. 834289 and Helv.Chim. by R.Traber et al.
Acta 62 , 1252-1267 (1979). For convenience, this substance is hereinafter referred to as tetrahydro-A-
It is written as 30912B. A-30912B nucleus Novel cyclic peptide nucleus of the present invention, namely A-
30912 Factor B (Echinocandin C, SL7810/F-
) and the nucleus of tetrahydro-A-30912B, in the formula, R ¹ and R ² are both H,
It has a structure in which both R ³ and R ⁴ are OH. The experimental formula for the A-30912B nucleus is C ₃₄ H ₅₁ N ₇ O ₁₄ ,
The molecular weight is 781.83. Production of A-30912B Cores Production of Substrates A-30912B nuclei are produced from A-30912 Factor B or Tetrahydro-A-30912B. A-
30912 factor B is not a major component of the antibiotic complex that produces it, so before it is used as a substrate,
It must be purified to the extent that other co-produced factors are removed. A-30912 Factor B A-30912 Factor B is produced by performing deep aeration enzymes with any of the microorganisms listed below. 1) Aspergillus rugulosus NRRL8113;
2) Aspergillus nidulans var.echinulatus A-
32204, NRRL3860; 3) Aspergillus rugulosus
NRRL8039; or 4) Aspergillus nidulans
var.roseus, NRRL11440. A.nidulans var.roseus NRRL11440 strain A-
When used to produce 30912 factor B, a conjugate of factors is obtained, which is conveniently referred to as the A-42355 antibiotic conjugate. A-30912 factor A is the main factor contained in the A-42355 antibiotic complex, and in addition,
A-30912 Factors B, D and H are present in small amounts. Tetrahydro-A-30912B Tetrahydro-A-30912B is prepared by reducing A-30912 Factor B by standard hydrogenation procedures until the two double bonds in the linoleoyl side chain have been reduced. Another embodiment of the core of the invention is that R ¹ , R ² ,
Both R ³ and R ⁴ are H, which means A-
Known as the 30912D nucleus. The A-30912D nucleus has the formula in which R is linoleoyl or stearoyl, and R ¹ , R ² , R ³ and R ⁴ are A-
Produced by deacylating a cyclic peptide antibiotic synonymous with 30912D. A cyclic peptide antibiotic in which R is linoleoyl is known as A-30912 Factor D, and an antibiotic where R is stearoyl is known as tetrahydro-A-30912 Factor D. A-30912 factor D A-30912 factor D is a factor A, B, C, E, F
It is one of the factors in the A-30912 complex that also contains G and G. For the A-30912 complex, see Marvin M. Hoehn and Karl. H.
Michel and U.S. Pat. No. 4,024,245. A-30912 factor D is the antibiotic echinocandin D [R. Traber et al., Helv.
Chim. Acta. 62 , 1252-1267 (1979)] and the antibiotic SL7810/F- (Belgian patent no.
834289)). ^{Traber et} al ^.
We proposed that a structure in which R ⁴ is all H and R is linoleoyl be used as echinocandin D. In addition, Traber ^{et al} .
A structure in which R ³ and R ⁴ are both H and R is stearoyl was proposed to be the antibiotic tetrahydroechinocandin D. Tetrahydro-A-30912 Factor D (Tetrahydro-SL7810/F-; Tetrahydroechinocandin D) is hereinafter referred to as Tetrahydro-A-
It is called 30912D. A-30912D nucleus Novel cyclic peptide nucleus of the present invention, namely A-
30912 Factor D (Echinocandin D, SL7810/F-
) and the nucleus of tetrahydro-A-30912D are
In the formula, R ¹ , R ² , R ³ and R ⁴ are all H. The experimental formula for the A-30912D nucleus is C ₃₄ H ₅₁ N ₇ O ₁₂ ,
The molecular weight is 749.83. Preparation of A-30912D Nuclei Substrate Preparation A-30912D nuclei may be prepared from A-30912 Factor D or Tetrahydro-A-30912D. A-30912
Since Factor D is not a major component of the antibiotic complex that produces it, it must be purified to the extent that other co-produced factors are removed before it can be used as a substrate. A-30912 Factor D A-30912 Factor D is produced by performing deep aeration enzymes with any of the microorganisms listed below. 1) Aspergillus rugulosus NRRL8113;
2) Aspergillus nidulans var.echinulatusA−
32204, NRRL3860; 3) Aspergillus rugulosus
NRRL8039; or 4) Aspergillus nidulans
var.roseus.NRRL11440. A.nidulans var.roseus NRRL11440 strain A-
When used to produce 30912 Factor D, a conjugate of factors is obtained, which is conveniently referred to as the A-42355 antibiotic conjugate. A-30912 factor A is the main factor contained in the A-42355 antibiotic complex, and in addition,
A-30912 Factors B, D and H are present in small amounts. Tetrahydro-A-30912D Tetrahydro-A-30912D is prepared by reducing A-30912 Factor D by standard hydrogenation procedures until the two double bonds in the linoleoyl side chain have been reduced. Yet another embodiment of the invention is that R ¹ and R ⁴ are both OH, R ² is H, and R ³ is C ₁ -C ₆ alkoxy, which is known as an A-30912H type nucleus. ing. The A-30912H type nucleus deacylates a cyclic peptide antibiotic in which R is linoleoyl or stearoyl, and R ¹ , R ² , R ³ and R ⁴ have the same meanings as the A-30912H type nucleus. Manufactured by. In the formula, R is linoleoyl and R ³
A cyclic peptide antibiotic in which is methoxy is A-
30912 The antibiotic known as factor H, where R is stearoyl and R ³ is methoxy, is
Tetrahydro-A-30912 is known as Factor H. In the formula, R is linoleoyl and R ³
Cyclic peptide antibiotics in which R is _C2 - _C6 alkoxy are known as lower alkoxy congeners of A-30912 Factor H, R is stearoyl,
Antibiotics in which R ³ is C ₂ -C ₆ alkoxy are known as lower alkoxy congeners of Tetrahydro-A-30912 Factor H. A-30912 Factor H A-30912 Factor H is a factor A, C, D, E, F.
It is one of the factors in the A-30912 complex that also contains G and G. For the A-30912 complex, see Marvin M. Hoehn and Karl H.
Michel and U.S. Pat. No. 4,024,245. A-30912 factor H is a later discovered A-30912 factor, and is disclosed in Karl H. Michel's U.S. Patent Application No. 117739 (filed February 1, 1980).
It has been described as antibiotic A-30912 factor H. This US application is a continuation-in-part of the now dismissed US Patent Application No. 46,875, filed June 8, 1970. Homolog of A-30912 Factor H Following the discovery of the structure of A-30912 Factor H, A
The lower alkoxy congeners of factor H-30912 have become appreciated as useful products. Until now, alkoxy derivatives were not recognized to have useful effects;
It was simply manufactured for use as a tool for determining structure. A-30912 factor H lower C ₂
~ _C6 alkoxy congeners are prepared from A-30912 Factor A. A-30912 Factor A is produced by performing deep aeration enzymes with any of the microorganisms listed below. 1) Aspergillus rugulosus NRRL8113;
2) Aspergillus nidulans NRRL8112; 3)
Aspergillus nidulans var.echinulatus A-
32204, NRRL3860; 4) Aspergillus rugulosus
NRRL8039 (described in Belgian patent no. 834289);
or 5) Aspergillus nidulans var.roseus,
NRRL11440. Since the A-30912H core contains an amino moiety, it may exist in the form of a salt. This kind of salt is
Useful as an intermediate and used for purification purposes. Pharmaceutically acceptable salts of the A-30912H core are particularly useful because they shorten the purification steps of the final product. Pharmaceutically acceptable salts generally include:
Salts are meant that do not increase the toxicity of the product to warm-blooded animals. Acid addition salts of A-30912H nuclei are formed when reacted with inorganic or organic acids through conventional reaction operations. Typical inorganic or organic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid,
Acetic acid, benzoic acid, sulfamic acid; tartaric acid, citric acid, maleic acid, succinic acid, ascorbic acid,
Glycolic acid, lactic acid, fumaric acid, palmitic acid,
Cholic acid, pamoic acid, mucic acid, D-glutamic acid, d-camphoric acid, glutaric acid, phthalic acid,
Lauric acid, stearic acid, salicylic acid, methanesulfonic acid, benzenesulfonic acid, sorbic acid,
Picric acid, cinnamic acid, and other suitable acids may be exemplified. Production of the A-30912H nucleus Production of the substrate The A-30912H nucleus, i.e., a compound in which R ¹ and R ⁴ are both OH, R ² is H, and R ³ is methoxy, is the A-30912 factor H. or from tetrahydro-A-30912H.
Since A-30912 Factor H is not a major component of the antibiotic complex that produces it, it must be purified to the extent that other co-produced factors are removed before it can be used as a substrate. In the formula, R ¹ and R ⁴ are both OH,
A where ^R2 is H and ^R3 is _C2 - _C6 alkoxy
The substrate for producing the -30912H nucleus (A-30912H homologue) is the reaction of A-30912 Factor A or tetrahydro-A-30912A with a suitable alcohol to give the corresponding _C2 - _C6 alkoxy derivative. It is obtained by Since A-30912 Factor A is a major component of the antibiotic complex that produces it, this process is the preferred method for producing A-30912 Factor H and tetrahydro-A-30912H. A-30912 Factor H A-30912 Factor H is produced by performing deep aeration fermentation using any of the following microorganisms. 1) Aspergillus rugulosus NRRL8113 or 2) Aspergillus nidulans var.roseus
NRRL11440. A.nidulans var.roseus NRRL11440
When used to produce 30912 Factor H, a conjugate of factors is obtained, which is conveniently referred to as the A-42355 antibiotic conjugate. A-30912 factor A is the main factor contained in the A-42355 antibiotic complex, and in addition,
A-30912 Factors B, D and H are present in small amounts. A-30912H Homologues A-30912H Homologues are prepared by reacting A-30912 Factor A or Tetrahydro-A-30912A with the appropriate corresponding alcohol, where R ¹ and R ⁴ are both OH, and R ² is H and R ³ is C ₂
It is produced by converting the desired alkoxy derivative which is ~ _C6 alkoxy. This is A-
30912 Factor H and Tetrahydro-A-30912H are the preferred methods of making them. In the formula, R
A-30912H congeners in which is stearoyl and R ³ is C ₂ -C ₆ alkoxy can be prepared by a) preparing tetrahydro-A-30912A and reacting it with a suitable alcohol to give an alkoxy derivative, or b )
A-30912 Factor A is reacted with a suitable alcohol to form an alkoxy derivative, which is obtained by reducing the double bond in the linoleoyl side chain. Tetrahydro derivative Tetrahydro-A-30912A, Tetrahydro-
A-30912H, and in the formula, R ³ is C ₂ to C ₆
Compounds in which R is alkoxy and R is stearoyl refer to A-30912 factors A and H, and compounds in which R ³ is C ₂ -C ₆ alkoxy and R is linoleoyl, as well as compounds in which R is It is produced by reduction by conventional hydrogenation operations until the bond is reduced. Another embodiment of the core of the invention is that R ¹ , R ³ and
R ⁴ is OH and R ² is carboxamide, which is known as the S-31794/F-1 nucleus.
The S-31794/F-1 nucleus has the formula in which R is myristoyl and R ¹ , R ² , R ³ and R ⁴ are S-
It is obtained by deacylating a cyclic peptide antibiotic having the same meaning as 31794/F-1 nucleus. S31794/F-1 The S31794/F-1 core of the present invention is obtained by deacylating the cyclic peptide antibiotic S-31794/F-1. Antibiotic S31794/F-1 is a West German published patent no.
No. 2,628,965 and US Pat. No. 4,173,629, Acrophialophora limonispora nov.
Spec.Dreyfuss et Muller NRRL8075 is an antibiotic with antifungal action and has the following properties. Melting point 178-180℃ (dec.) (amorphous form) or 181-
183°C (dec.) (crystal); [α] ²⁰ _D −24° (c=0.5,
CH ₃ OH) or +37° (c=0.5, methanol)
(Crystal); UV maximum absorption (in methanol) 194nm
(E1% 1cm=807), 225nm (shoulder) (E1% 1cm=132),
276nm (E1% 1cm=12.8), 284nm (shoulder) (E1% 1cm=
10.5); ^13C -NMR (heavy methanol, 190mg/1.5
ml, internal standard tetramethylsilane) 176.2,
175.0, 173.7, 172.6, 172.0, 171.8, 171.7,
168.6, 157.7, 132.5, 129.0, 115.9, 76.6, 75.5,
74.0, 71.0, 70.5, 69.7, 68.0, 62.2, 58.3, 57.0,
56.2, 55.4, 52.9, 51.2, 39.7, 38.8, 36.6, 34.8,
32.8, 30.6, 26.7, 23.5, 19.7, 14.3, 11.1
(PPM): Elemental analysis values (dried at 100℃ for 2 hours under high vacuum), carbon 55.5-56.5%, hydrogen 7.5-7.7%, nitrogen
10.5−10.8%, enzyme 25.5−26.0%; methanol,
Soluble in ethanol, pyridine, dimethyl sulfoxide, slightly soluble in water, chloroform, ethyl acetate, diethyl ether, benzene, hexane. Has antifungal effects (especially against Candida albicans). Antibiotic S31794/F-1 has the formula:
R is myristoyl, R ¹ , R ³ and R ⁴ are
It is said to have a structure in which it is OH and R ² is carboxamide. S31794/F-1 nucleus The novel cyclic peptide nucleus of the present invention, namely the antibiotic S31794/F-1 nucleus, has a structure in which R ¹ , R ³ and R ⁴ are OH and R ² is carboxamide. ing. The empirical formula of _the S31794/F-1 nucleus is _C35H52N8O16 and _the molecular weight is _840.87 . Production of S31794/F-1 nucleus Production of substrate S31794/F-1 nucleus is an antibiotic S31794/F-1
Manufactured from. Antibiotic S31794/F-1 is Acrophialophora
Produced by deep aeration culture of limonispora NRRL8095. This microorganism is NRRL (Northern
Regional Research Laboratory, US
Department of Agriculture, Agriculture
Research Culture Collection, North Central
Region, Peoria, Illinois 61604), and is publicly available under the assigned NRRL number. Since these nuclei each have an amino moiety, they may exist in the form of salts, which are useful as intermediates and are used for purification purposes. Pharmaceutically acceptable salts of the core are particularly useful as they shorten the purification steps of the final product. By pharmaceutically acceptable salts is meant salts that do not increase the overall toxicity of the product to warm-blooded animals. Nucleic acid addition salts are formed when reacted with inorganic or organic acids by conventional reaction procedures. Typical inorganic or organic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, acetic acid, benzoic acid, sulfamic acid, tartaric acid, citric acid, maleic acid, succinic acid, ascorbic acid, Glycolic acid, lactic acid, fumaric acid, palmitic acid, cholic acid,
Pamoic acid, mucic acid, D-glutamic acid, d-camphoric acid, glutaric acid, phthalic acid, lauric acid,
stearic acid, salicylic acid, methanesulfonic acid,
Benzene sulfonic acid, sorbic acid, picric acid,
Cinnamic acid and other suitable acids may be mentioned. Final purification of individual antibiotics is preferably carried out using reverse phase high performance low pressure liquid chromatography (HPLPLC) using silica gel/C ₁₈ as adsorbent. In this case, the crude antibiotic complex is dissolved in a solvent and adsorbed onto a column equilibrated with the same solvent. The column is eluted with solvent and the collected fractions are analyzed using bioautography using Candida albicans or UV (based on relative retention times).
monitored by. Combine fractions containing the same antibiotic factor. In order to extract each factor in its pure form, it may be necessary to further separate it by chromatographic operation. The crude antibiotic conjugate of A-30912 or A-42355 can be prepared, for example, by dissolving whole broth or mycelium in methanol.
Obtained by extraction with chloroform. When chromatographically treating these antibiotics, methanol-water-acetonitrile (7:2:
1) is preferably used as the solvent system. Crude S31794/F-1 antibiotic is obtained by extracting the whole broth with ethyl acetate-isopropanol (4:1) and chromatographing the extract. Each factor is identified by thin layer chromatography (TLC). It is preferable to use silica gel as the adsorbent. High carbon content silica gel TLC, benzene-methanol (7:3) solvent system, and Candida
A- using albicans bioautography
The R _f values of 30912 factors A to G are shown in the table.

[Table] High carbon content silica gel TLC and Candida
The approximate R _f values for A-30912 factors A, B, C, D and H using different solvent systems using C. albicans bioautography are shown in the table.

[Table] Solvent system a: Ethyl acetate-methanol (3:2) b: Ethyl acetate-methanol (7:3) c: Acetonitrile-water (95:5) d: Ethyl acetate-ethanol-acetic acid (40:60: 0.25) A-30912 factors A, B, C, D and H are identified by HPLPLC using the following conditions. Column: Glass (0.8 x 15.0cm) Packing material: Nucleosil 10−C ₁₈ (Machery−
(Nagel and Company), using the slurry filling method of Example 7. Solvent: Methanol-water-acetonitrile (7:2:1) Sample amount: 8 mcl Sample size: 8 mcg Column temperature: Room temperature Flow rate: 1.8 ml/min Pressure: Approximately 200 psi Detector: UV at 222 nm (ISCO Model 1800
Variable Wavelength UV−Visible
Absorbance Monitor) Pump: LDC Duplex Minipump Injection: Loop injection A-30912 factors A, B, D, under these conditions
The approximate retention times for and H are summarized in the table.

[Table] Production of enzyme 1 Producing microorganism Enzymes useful for deacylation of antibiotics in the present invention include microorganisms belonging to the family Actinoplanaceae,
In particular, it is produced from a microorganism belonging to the genus Actinoplanes, preferably Actinoplanes utahensis NRRL12052. The enzyme is the enzyme used to deacylate penicillins (Walter J. Kleinschmidt, Walter E.
Wright, Frederick W. Kavanagh and
U.S. Patent No. 3150059 by William M. Stark
may be the same as the A.
A preferred method for culturing S. utahensis NRRL12052 to produce this enzyme is described in Preparation Example 1, but it is well known to those skilled in the art that other methods may be used. Actinoplanaceae is a relatively recent family of the order Actinomycetales, first discovered by Dr. John N.
Described by J. Elisha Mitchell Sci. Sco. 71 , 148, the family was established in 1955.
~155 (1955)]. The nature of this family and many individual genera is described in Bergey's Manual of Determinative
Bacteriology, 8th ed., RE Buchanan and
NEGibbons, Eds., The Williams & Wilkins
Co., Brltimore, Md., 1974, pp. 706-723, and the following 10 genera have been distinguished so far. 1Actinolanes (standard genus and most universal genus): . Spirillospora;. Streptosporangium; ． Amorphosporangium;.
Ampullariella; ． Pilimelia;. Planomonospora;.
Planobispora;. Dactylosporangius; and. Kitasatoa. The following species have been isolated so far and their properties have been shown: Actinoplanes
phillippinensis, Actinoplanes armeniacus,
Actinoplanes utahensis, and Actinoplanes
missouriensis；Spirillospora albida;
Streptosporangium roseum,
Streptosporangium vulgare,
Streptosporangium roseumvar,
hollandensis，Streptosporangium album，
Streptosporangium vilidialbum,
Amorphosporangium auranticolor,
Ampullariella regularis，Ampullariella
campanulata，Ampullariella lobata，
Ampullariella digitata，Pilimeria terevasa，
Pilimelia anulata, Planomonospora
parontospora, Planospora venezuelensis,
Planobispora longispora, Planobispora
rosea, Dactylosporangium aurantiacum, and Dactylosporangium thailandense. The genus Actinoplanes is a preferred source of enzymes useful in the present invention. Within the genus Actinoplanes, the species Actinoplanes utahensis is a particularly preferred source of enzymes. Representative strains are designated by the numbers below.
It is available from (see above). Actinoplanes utahensisA8316.2.6 NRRL12052 Actinoplanes missouriensisA1307
NRRL12053 Actinoplanes sp.A7413.3 NRRL8122 Streptosporangium roseumsubsp.
hollandensis A3663 NRRL12064 Actinoplanes sp.A7411 NRRL12065 Above strain Actinoplanes utahensis
A8316.2.6, Actinoplanes missouriensis
A1307, Streptosporangium roseum subsp.
hollandensis A3693, and Actinoplanes sp.
Both A7411s were transferred to international deposits under the Budapest Treaty on December 6, 1989, and are given the same international deposit numbers as above. A.utahensis NRRL12052 is ATCC
(American Type Culture Collection; 12301
Parklawn Drive, Rockville, Md. 20852)
It was derived from the parent strain deposited as A.utahensis ATCC14539. A.utahensis
ATCC14539 strain may be used as an enzyme source. A. missouriensis NRRL12053 was given to ATCC as A.
missouriensis ATCC14538, and is derived from a strain that serves as another enzyme source. When attempting to assay the activity of an arbitrarily selected strain of the Actinoplanaceae family in carrying out the deacetylation of the present invention, it can be carried out as follows. A suitable growth medium is inoculated with microorganisms and cultured at about 28°C for 2-3 days using a rotary shaker. Add antibiotic substrate to this. Keep the pH of the fermentation medium around 6.5 and
monitor using S. albicans. This is described in Procedure E, and the fact that the microorganism has the necessary deacylation activity is demonstrated by the disappearance of antibiotic activity. However, this method requires that 1) the existence of the nucleus itself be detected by HPLC;
This must be demonstrated either by detection by analysis or 2) by confirming restoration of activity by reacylation with an appropriate side chain. 2 Conditions for Enzyme Production Enzyme production is performed under conditions suitable for the growth of Actinoplanaceae, ie, at 25-30°C and pH 5.0-8.0 with aeration and stirring. The culture medium must contain the following: a) Assimilable carbon sources such as sucrose, glucose, glycerol;
b) nitrogen sources such as peptone, urea, ammonium sulfate; c) phosphate sources such as soluble phosphate; and d) inorganic salts that are generally effective in promoting the growth of microorganisms. Effective amounts of enzyme are generally obtained about 40 to 60 hours after growth ring formation, but the culture is continued for some time thereafter.
The amount of enzyme produced depends on the species of microorganism.
It also changes depending on growth conditions. Although the produced deacylating enzyme can be separated and purified from the culture medium and used, usually a culture medium containing bacterial cells is used as a crude enzyme solution to deacylate the substrate. As is clear in this field,
Bacteria that produce enzymes, such as Actinoplanes utahensis NRRL12052, can be mutated. For example, artificial mutations and mutations in these strains can be obtained by treatment with various known mutagens, such as ultraviolet light, X-rays, high frequency waves, radiation, and chemicals.
Natural mutants, artificial mutants, and mutant strains obtained from Actinoplanaceae that produce enzymes are all used in the present invention. C. Deacylation Conditions The substrate is preferably added after the Actinoplanaceae medium has been incubated for at least 48 hours. The concentration of substrate in the medium conversion can vary widely. However, to maximize enzyme utilization and substantially complete deacylation within 24 hours, the concentration of substrate will generally be about 0.5-1.0 mg/ml. Lower concentrations can be used, but the enzyme is not used to its full potential. High concentrations can also be used, but unless the fermentation time is extended, the substrate will not be completely deacylated. In order to convert the substrate antibiotic into the corresponding nuclei of the invention, the fermentation medium is best kept at a pH of about 6.0-7.0. At pH below 6, deacylation progresses slowly, and when pH exceeds 6, the substrate also
The nuclei that are formed also gradually lose their stability. For maximum stability, PH 6.0 is preferred, although deacylation does not proceed as quickly at PH 6.0 (approximately 30-36 hours). A pH of about 6.5 is preferred for very fast deacylation (about 24 hours) without significant losses. In fermenters equipped with a stirrer, the PH value can be measured using an automatic PH sensing type.
Can be controlled by a PH controller. In some cases, such as fermentation in flasks, 0.1
The PH value is controlled by adding molar phosphate buffer. Continue culturing the medium for approximately 24 hours after adding the substrate. Substrate purification affects the rate of deacylation. For example, a substrate with a purity of 50% or higher will undergo deacylation at a rate of approximately 0.8 to 1.2 mg in 24 hours, whereas with a substrate of lower purity, deacylation will proceed more slowly. do. The substrate may be added multiple times. For example, in a small tank, add 0.3-0.5 mg/ml of antibiotic.
May be added at least 5 times every 12 hours or 0.7 mg/ml twice in a large tank. The deacylation reaction can be accomplished over a wide temperature range, for example at about 20-45°C, but preferably at about 25-30°C, particularly preferably at about 26°C.
Let's do it. This is the optimal condition for deacylation and substrate and nuclear stability. D Substrate It is preferable to use a purified antibiotic as the substrate. The antibiotic substrate has antifungal activity, but not antibacterial activity. Thus, the substrate material may include bacterial cells or their spores that can grow in the deacylation fermentation medium. Such contaminants can affect the deacylation reaction or the stability of the raw antibiotic or product core. Therefore, it is important to keep the substrate sterile. Since autoclaving destroys most of the antibiotic substrate, it is preferable to sterilize by treating with ethylene oxide while maintaining a constant atmospheric pressure. E Monitoring of Deacylation The raw material is an antibiotic with antifungal properties, particularly active against Candida albicans. For this reason, it is preferred to use C. albicans to determine the amount of substrate present. The resulting core is soluble in water but biologically inert. Therefore, it is a presumptive test for deacylation with reduced biological activity. Since the substrate is slightly soluble in the broth, a sample of the broth and an alcoholic extract of the fermentation solids are assayed together. F. Use of Resting Cells An alternative method of deacylation is accomplished by removing the Actinoplanaceae cells from the culture medium, resuspending them in buffer, and carrying out the deacylation as described in Procedure C. When using this operation,
Enzymatically active mycelia can be used again. For example, A.utahensis NRRL12052 mycelium retains deacylation activity even after being stored in the refrigerator (4-8°C) or frozen (-20°C) for one month or more. A preferred buffer is 0.1 molar phosphate buffer. G. Immobilized Enzymes An alternative to the deacylation method is to make the enzymes immobilized by procedures known to those skilled in the art (Biomedical Applications of Immobilized Enzymes).
Enzymes and Proteins, Thomas Ming Swi
Chang, Ed., Plenum Press, New York,
1977; see volume 1). The motility-inhibiting enzyme is used within the column (and other suitable forms of reactants) to effect the deacylation. Furthermore, the movement of microorganisms themselves can be inhibited and the microorganisms themselves can be used as catalysts for deacylation reactions. Utility of Nuclei Nuclei and their acid addition salts are useful intermediates in the production of synthetic antifungal agents. Useful antifungal agents made from these cores are currently under application.
Applications by Bernard J. Abbott and David S. Fukuda and two currently pending applications by Manuel Debono, both entitled “Derivatives of Cyclic Peptide Cores,” were filed on the same date. An application has been filed. In the above definitions, the term "alkyl" refers to a monovalently saturated, straight or branched hydrocarbon group. The term "alkenyl" refers to a monounsaturated straight or branched hydrocarbon group containing up to three double bonds. This unsaturated hydrocarbon chain may have either a cis or trans configuration. The designation " _C6 - _C24 " means that the straight-chain or branched hydrocarbon has from 6 to 24 carbon atoms. The compound of formula is α on ornithine of the appropriate nucleus.
They are prepared by acylating the amino group with a suitable acyl side chain by conventional methods to form an amide bond. Generally, this acylation is carried out by reacting the core with an activated derivative of the acid corresponding to the desired acyl side chain. The term "activated derivative" as used herein refers to a derivative that has been activated so that the acyl group of the acylating agent can be coupled with a primary amine group to form an amide bond linking the acyl side chain and the nucleus. . Suitable activated derivatives, their preparation and their use as acylating agents for primary amines are known to those skilled in the art. Preferred activated derivatives include (a) acid halides (e.g. acid chlorides), (b) acid anhydrides (e.g. alkoxide acid anhydrides, aryloxide acid anhydrides), (c) activated esters (e.g. 2,
4,5-trichlorophenyl ester).
Another method of activating the carboxy function is to react the carboxylic acid with a carbodiimide (eg N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide). In this case, the generated reactive intermediate is not isolated because it is unstable, but is directly reacted with the primary amine. If the amino acid contains a functional group that can be acylated in addition to its amino group, the reagent used to introduce the N-alkanoyl group or N-alkenoyl group should be protected in advance before reacting with the amino acid. This is well known to those skilled in the art. As the protecting group, it is appropriate to use a protecting group for a side chain functional group, which is well known to those skilled in the art in peptide synthesis. Such groups are known,
The choice of protecting groups and their use is well known to those skilled in the art (e.g., “Protective
Croups In Organic Chemistry”, M.Mc Omie,
Editor, Plenum Press, NY, 1973). The compounds of the formula inhibit the growth of pathogenic fungi and are therefore useful in controlling the growth of fungi (as preservatives) on environmental surfaces, and are also useful in controlling fungi growth on environmental surfaces. It is useful in the treatment of infectious diseases. The compounds are particularly active against Candida albicans and are therefore particularly useful in the treatment of candidosis. Compound activity can be determined using standard microbiological test methods such as in vitro agar plate disk diffusion or agar dilution, or in vitro using mice infected with C.albicans.
Proven by in vivo tests. The compound is
Trichophyton mentagrophytes (dermatophytes),
Saccharomyces pastorianus and Neurospora
It also shows activity against crassa. Certain compounds (eg, Reference Example 19, shown in the table) exhibit significant blood levels when administered orally to mice. When a compound of the formula in which R ⁵ is p-(n-octyloxy)benzoyl was administered intravenously to dogs at 100 mg/Kg per day for 5 days, no outward signs of toxicity were observed; An increase in SGPT levels was observed. When used systemically, the dose of a compound of formula
It depends on the specific compound used, the type and severity of the infection, and the physical condition of the person being treated. It is advisable to begin treatment with a low dose and increase the dose until the desired antifungal effect is achieved. The compounds may be administered by injection intravenously or intramuscularly in the form of a sterile aqueous solution or suspension. If necessary, known pharmaceutically acceptable preservatives, buffers, solubilizing agents, suspending agents, etc. may be added to this preparation. Additionally, other additives such as common salt or glucose may be added to make the solution isotonic. The proportions and types of such additives are well known to those skilled in the art. Certain compounds of the formula can be administered orally systemically, as they provide significant blood concentrations upon oral administration (see Reference Example 19, Table). For oral use, these compounds can be placed in capsules, capsules, etc. with pharmaceutically acceptable carriers or excipients.
It is administered in the form of tablets or powder. The types and proportions of these carriers and excipients can be easily determined by those skilled in the art. For the treatment of vaginal candidiasis, compounds of the formula can be administered in combination with pharmaceutically acceptable excipients suitable for intravaginal use. Formulations for vaginal administration are well within the skill of those skilled in the art. Examples are provided below to further illustrate the operation of the invention. Production Example 1 Fermentation of Actinoplanes utahensis A preserved strain of Actinoplanes utahensis NRRL12052 is prepared and stored on an agar slant. This slant medium is selected from the following: Medium Amount of A component used Plektsk Oatmill 60.0g Yeast 2.5g K ₂ HPO ₄ 1.0g Plektsk Mineral Stock* 5.0ml Agar 25.0g Deionized water Appropriate amount (total amount 1) PH before sterilization, approx. 5.9; Water PH after adjustment by adding sodium oxide, 7.2; PH after sterilization, approximately 6.7. *Composition of Tuapetsk Mineral Stock: Ingredient usage amount FeSO ₄ 7H ₂ O (dissolved in 2 ml of concentrated hydrochloric acid)
2g KCl 100g MgSO ₄・7H ₂ O 100g Deionized water appropriate amount (total amount 1) Medium Amount of B component used Potato dextrin 5.0g Yeast extract 0.5g Casein enzyme hydrolyzate ^* 3.0g Beef extract 0.5g Glucose 12.5g Corn starch 5.0g Meat peptone 5.0g Blackstrap 2.5g MgSO ₄・7H ₂ O 0.25g CaCO ₃ 1.0g Tuapetsk mineral stock 2.0ml Agar 20.0g Deionized water Appropriate amount (total amount 1) ^* N -Z-Amine A, Humko Sheffield
Chemical, Lyndhurst, NJ Actinoplanes utaensis NRRL12052
is inoculated onto a slant and cultured at 30°C for about 8 to 10 days.
Approximately half of this slant culture is used to inoculate 50 ml of a growth medium with the following composition. Ingredients Usage: Plektsk Oatmeal 20.0g Sucrose 20.0g Yeast 2.5g Detailer's Dry Grain ^* 5.0g K ₂ HPO ₄ 1.0g Plektsk Mineral Stock 5.0ml Deionized water Appropriate amount (total amount 1) PH by sodium hydroxide Adjustment of, 7.4; pH after sterilization, approximately 6.8. ^* National Disfillers Products Co., 99Park
Ave., New York, NY. The inoculated medium is shaken (2 inch diameter arc, 250 rpm) at 30°C for approximately 72 hours in a 250 ml wide-mouth Erlenmeyer flask. This cultured medium may be used directly to inoculate a secondary growth medium. It can also be stored, preferably under a stream of nitrogen, for later use. In order to store this cultured bacteria,
Separate it into small vials as shown below. Add 2 ml of culture medium to glycerin-lactose solution [WADailey and CE Higgens, “Preservation
and Storage of Microorganisms in the Gas
Phase of Liquid Nitrogen; Cryobiol 10 , 364
~367 (1973) for details] into each vial along with 2 ml. The resulting suspension is stored in a nitrogen atmosphere. Using 1 ml of the bacterial cell suspension thus preserved,
Next, inoculate 50 ml of growth medium (composition as above). Cultivation is performed in the same manner as described above. To obtain a large amount of inoculating culture, 10 ml of the primary culture should be mixed with a secondary growth medium of the same composition.
Inoculate 400ml. Secondary cultures are performed in two wide-mouth Erlenmeyer flasks at 30° C. for approximately 48 hours with shaking (250 rpm on a 2-inch diameter arc). Using the secondary culture (800ml) obtained in this way,
Inoculate one of the following sterile manufacturing media 100. Amount of medium components used (g/) Peanut organisms 10.0 Soluble meat peptone 5.0 Sucrose 20.0 KH ₂ PO ₄ 0.5 K ₂ HPO ₄ 1.2 MgSO ₄・7H ₂ O 0.25 Tap water Appropriate amount (total volume 1) 121℃, 16-18psi After sterilization for 45 minutes at
6.9. Amount of medium components used (g/) Sucrose 30.0 Peptone 5.0 K ₂ HPO ₄ 1.0 KCl 0.5 MgSO ₄・7H ₂ O 0.5 FeSO ₄・7H ₂ O 0.002 Deionized water Appropriate amount (total amount 1) Adjust to PH7.0 with hydrochloric acid ;PH after sterilization, approximately 7.0. Medium component usage (g/) Glucose 20.0 NH ₄ Cl 3.0 Na ₂ SO ₄ 2.0 ZnCl ₂ 0.019 MgCl ₂・6H ₂ O 0.304 FeCl ₃・6H ₂ O 0.062 MnCl ₂・4H ₂ O 0.035 CuCl ₂・2H ₂ O 0.005 CaCO ₃ 6.0 KH ₂ PO ₄ ^* 0.67 Tap water Appropriate amount (total amount 1) ^* Sterilize separately and add aseptically. Final pH approximately 6.6. The inoculated medium is fermented for 42 hours at approximately 30°C using 165 fermentation tanks. The medium is stirred at approximately 200 rpm with a conventional stirrer and aerated with sterile air such that the amount of dissolved oxygen exceeds 30% of air saturation at atmospheric pressure. Production Example 2 Production of A-42355 antibiotic complex A Shake flask fermentation Aspergillus nidulans var.roseus
NRRL11440 is cultured and stored on an agar slant with the following composition. Ingredients usage Glucose 5g Yeast extract 2g CaCO ₃ 3g Vegetable juice ^* 200ml Agar ^** 20g Deionized water Appropriate amount (total amount 1) (Initial pH 6.1) ^* V-8Juice, Campbell Soup Co.,
Camden, NJ ^** Meer Corp. Aspergillus nidulans var.roseus
NRRL11440 is inoculated onto the above slant medium and cultured at 25°C for about 7 days. Cover mature slopes with water;
Scrape off the spores with a sterile loop. The resulting suspension was further suspended in 10 ml of sterile deionized water and 1 ml was used to prepare growth medium 55 in a 250 ml flask.
Inoculate ml. The composition of this growth medium is as follows. Ingredients usage Shuucrose 25g Blackstrap 36g Corn steep liquor 6g Malt extract 10g K ₂ HPO ₄ 2g Casein enzyme hydrolyzate ^* 10g Tap water 1100ml (Initial pH 6.5-6.7) ^* N-Z-Case, Humko Sheffield
Chemical, Lyndhurst, NJ After inoculation, incubate at 25°C for 48 hours with shaking at 250 rpm (rotary shaker). in the middle,
After 24 hours, transfer the medium to a blender (Waring
Homogenize for 1 minute at low speed (type) for 1 minute, then continue culturing for the remaining 24 hours. Alternatively, the medium may be cultured for 48 hours and then homogenized at low speed for 15 seconds. This culture may be used to inoculate a medium for shaking culture or a medium for secondary growth culture. Alternatively, it may be stored in a nitrogen atmosphere for later use. For this purpose, the culture is stored in a number of small vials as follows. The culture is mixed with an equal volume of suspension solution having the following composition: Ingredients used: Glycerol 20ml Lactose 10g Deionized water Appropriate amount (total volume 100ml) Pour 4ml of this suspension into small sterile vials with screw caps and store in a nitrogen atmosphere. The stock suspension thus obtained is used for inoculating agar slants or liquid media. If using a slant, incubate in a bright place at 25°C for 7 days. B Tank Culture To obtain a large inoculating culture, 10 ml of the primary culture was used to inoculate 400 ml of secondary growth medium of the same composition and incubated at 25°C in two wide-mouthed Erlenmeyer flasks for 24 hours. Incubate with shaking for an hour (shaking on a 2 inch diameter arc at 250 rpm). The secondary culture thus obtained (800 ml) is used to inoculate one of the following sterile manufacturing media 100. Media component usage ZnSO ₄・7H ₂ O 0.00455g / Soluble meat peptone ^* 30.5g / Soy flour 15.5g / Tapioca dextrin ^** 2.0g / Blackstrap 10.5g / Casein enzyme hydrolyzate ^*** 8.5g / Na ₂ HPO ₄ 4.5g / MgSO ₄・7H ₂ O 5.5g / FeSO ₄・7H ₂ O 0.1g / Cottonseed oil 40.0ml (Defoaming agent) ^**** 1.0ml Tap water 1000.0ml (Initial pH 6. 8~7.0) ^* OMPeptone, Amber Laboratories,
Juneaw, Wisc. ^** Stadex11, AEStaley Cr., Decatur,
I11. ^*** N-Z-Amine A.Humko Sheffield
Chemical, Lyndhurst, NJ ^**** P2000, Dow Corning, Midland,
Michigan Medium component usage Glucose 2.5% Starch 1.0% Soluble meat peptone ^* 1.0% Blackstrap 1.0% CaCO ₃ 0.2% MgSO ₄・7H ₂ O 0.05% Casein enzyme hydrolyzate ^** 0.4% (defoaming) ^*** 0.02% Tap Water Required ^* OM Peptone ^** NZ-Amine A ^*** Antifoam “A”, Dow Corning Inoculated medium was incubated at 25°C in a 165 fermentation tank.
Let it ferment for about 7 days. The medium is aerated with sterile air so that the amount of dissolved enzyme is greater than 30% of air saturation. C. Tertiary Growth Medium When performing large-scale fermentations of more than 100 cells, it is always advisable to use a tertiary culture to inoculate large tanks. A preferred composition of the tertiary growth medium is as follows. Ingredients usage Shuucrose 25g Blackstrap 25g Corn steep liquor 6g Casein enzyme hydrolyzate ^* 10g Malt extract 10g K ₂ HPO ₄ 2g Tap water 1000ml (Initial pH 6.1) ^* N-Z-Case Production example 3 Isolation of A-42355 antibiotic complex Fermentation was performed using the production medium according to the method described in Production Example 2, and methanol (4280) was added to the obtained whole culture (4127) and stirred well for 1 hour. Superaid (Hyflo Super-cel, diatomaceous earth,
Johns-Manville Products Corp.).
Add 5N hydrochloric acid to the solution to adjust the pH to 4.0, and extract twice with equal volumes of chloroform. Combine the chloroform extracts and concentrate under reduced pressure to ca. Add this concentrate to about 200 g of diethyl ether and
Precipitate the 42355 complex. This was taken to obtain 2775 g of A-42355 composite as an off-white powder. Preparation Example 4 Isolation of A-30912 Factor A 1 g of the A-42355 antibiotic conjugate prepared as described in Preparation Example 3 is dissolved in 7 ml of methanol-water-acetonitrile (7:2:1). After filtering this solution, it is passed through a loop by valve operation to a glass column (3.7 cm inner diameter x 35 cm, Michel-Miller high performance low pressure liquid chromatography (HPLPLC) column, Ace Glass Incorporated, Vineland,
NJ08360). LP-1/C ₁₈ for the column
Silica gel reverse phase resin (10-20 microns) (manufacturing example)
10) using methanol-water-acetonitrile (7:2:1) according to the slurry filling method described in Example 11. A valveless piston type FMI pump (maximum flow rate 19.5 ml/min) is used to move the solvent at a flow rate of 9 ml/min under a pressure of approximately 100 psi, collecting fractions every minute. Antibiotic elution was performed using UV light with an optical unit (ISCO Type 6).
Monitor (ISCO Model UA-5, Instrument
Specialist Co., 4700 Superior Ave., Lincoln,
Nebraska68504) and monitor at 280 nm. The fractions (approximately 112-140) are combined, 20 ml of water is added, the pH is adjusted to 4.0 with 1N hydrochloric acid, and the mixture is extracted twice with an equal volume of chloroform. The chloroform layers are combined and concentrated in vacuo to give an oil. When this is dissolved in t-butanol and freeze-dried, A-
42355 Factor A (A-30912 Factor A; A-22082) 524
Get mg. Preparation Example 5 Isolation of A-30912 Factor B The A-42355 complex is isolated as described in Preparation Example 3. However, the concentrated chloroform extract (285) can be used on a silica gel column (Crace
Chromatograph on silica gel 150, grade 62) at a flow rate of 2/min. The column is washed with chloroform (200) and eluted with acetonitrile (500) and then acetonitrile-water (98:2) at a flow rate of 1/min. Approximately 200 fractions are collected and analyzed individually for biological activity. Bioassay Candida albicans
The paper disk assay method is used on agar plates inoculated with . Fractions 77 to 103 (1365) are combined and concentrated under reduced pressure. When the concentrated solution (4.5) is passed through, the A-42355 complex (119
g) is obtained. The liquid is evaporated to dryness and the resulting residue is redissolved in an appropriate amount of methanol. Addition of this methanol solution to 10 volumes of diethyl ether precipitates the antibiotic complex containing Factor B. When this is taken and dried, 24 g of A-42355 complex containing a large amount of factor B is obtained as an off-white powder. A- containing a large amount of factor B obtained in this way
Dissolve the 42355 complex in 8 ml of methanol-water-acetonitrile (7:2:1). After this solution has passed, it is passed through the loop through a glass column (3.7 cm inner diameter x 35 cm, Michel-Miller column).
to be introduced. The column is LP-1/C ₁₈ silica gel reverse phase resin (10-20 microns) (according to Production Example 10)
methanol-water-acetonitrile (7:2:
1) is used to fill through the loop with valve operation. The slurry filling method described in Production Example 11 is used. A valveless piston type FMI pump is used to move the solvent at a flow rate of 10 ml/min under a pressure of approximately 100 psi, collecting fractions every minute. Antibiotic elution was carried out in the same manner as in Example 1, using a UV monitor.
Monitor at 280nm. Combine fractions 102-110,
Concentration under reduced pressure gives an oil. Add a small amount of this
-When dissolved in butanol and lyophilized, A-
Obtain 22 mg of 30912 factor B. Production Example 6 Isolation of A-30912 Factor D Concentrated chloroform extracts (3800 and 3800 and
4007) and chromatographed on a silica gel column (Grace, grade 62). Wash the column with chloroform and elute with acetonitrile and acetonitrile-water (98:2). about
Two hundred fractions are collected and tested for biological activity by the paper disc method on agar inoculated with Candida albicans. When the active fractions (850) are combined and the solution (0.7) concentrated under reduced pressure is added to 10 volumes of diethyl ether, the A-43255 complex containing a large amount of factor D is precipitated. When this is taken and dried, an A-42355 complex containing a large amount of factor D is obtained as a gray powder. The A-42355 complex (1.0 g) containing a large amount of factor D thus obtained was dissolved in 5 ml of methanol-water-acetonitrile (7:2:1). After this solution has passed, it is introduced into a silica gel column (3.7 cm internal diameter x 30 cm, Michel-Miller column) through a loop using a valve operation. The column is packed with LP-1/C ₁₈ silica gel reverse phase resin (10-20 microns) (per Preparation Example 10). Filling is carried out by the slurry filling method described in Production Example 11 using methanol-water-azetonitrile (7:2:1). Using a valveless piston type FMI pump, under a pressure of approximately 45psi,
Move the solvent at a flow rate of 8 ml/min. Collect fractions every 2 minutes. Antibiotic elution was performed using a UV monitor (ISCO Type 6) equipped with an optical unit (ISCO Type 6).
Monitor at 280nm using Model UA-5). Fractions 96-108 are combined and concentrated under reduced pressure to give an oil. This is dissolved in a small amount of t-butanol and lyophilized to obtain 89 mg of A-30912 factor D. Production Example 7 Isolation of A-30912 Factor H Dissolve A-42355 antibiotic body (5.0 g) obtained by the method described in Production Example 3 in 35 ml of methanol-water-acetonitrile (7:2:1). After passing through the solution, the glass column (3.7 cm inner diameter x 42 cm, Michel-
Miller column). LP in the column
1/C ₁₈ silica gel reverse phase resin (10-20 microns)
, methanol-water-acetonitrile (7:2:
1) according to the description in Preparation Example 11.
Using a valveless piston type FMI pump, approx.
The solvent is transferred at a flow rate of 13 ml/min under a pressure of 120 psi, collecting fractions every 2 minutes. Antibiotic elution is
Monitor with 280nm UV. Fractions 112-132,
It is combined with fractions 106 to 117 that were purified separately and concentrated under reduced pressure to obtain an oily substance. Dissolve this in a small amount of t-butanol and freeze-dry to obtain crude A-30912.
Obtain 173 mg of factor H. Crude A-30912 factor H (150 mg) in methanol
Dissolve in 8 ml of water-acetritrile (7:2:1), filter the resulting solution, and introduce into a glass column (2.0 cm inner diameter, 32 cm) in the same manner as above.
The solvent is transferred at a flow rate of 8 ml/min under a pressure of approximately 80 psi, collecting fractions every 3 minutes. Antibiotic elution is
Monitor at 280nm. Fractions 17 and 18 are combined and concentrated under reduced pressure to obtain an oil. This is dissolved in a small amount of t-butanol and lyophilized to obtain 29 mg of A-30912 factor H. Production Example 8 Production of Antibiotic S31794/F-1 Antibiotic S31794/F-1 is obtained by culturing Acrophialophore limonispora NRL8095 in deep water under shaking and/or aeration. Culture at pH 3 to 8 under shaking and/or aeration.
and preferably at a pH of 5 to 7, at a temperature of 15 to 30°C, preferably at a temperature of 18 to 27°C for 48 to 360 hours, preferably at a temperature of 120 to 360 hours.
Do it for 288 hours. Treat the culture broth (90) with ethyl acetate-isopropanol (4:1, 90) and homogenize for 30 minutes at room temperature. Separate the organic layer and concentrate under reduced pressure at about 40°C. The obtained residue was poured onto 10 times the amount of silica gel and chloroform-methanol (from 95:5 to
60:40). The fractions having antifungal activity are combined and chromatographed on a 100-fold volume of Sephadex LH-20 using methanol. Of the fractions eluted from the Sephadex column, the fractions with antifungal activity are combined to 100%
Chromatograph on double volume of silica gel (0.05-0.2 nm) using a solvent system of chloroform-methanol-water (71:25:4). The eluted fractions having antifungal activity are combined and concentrated under reduced pressure to obtain crude antibiotic S31794/F-1. This is dissolved in a small amount of methanol, diethyl ether is added to precipitate it, and the precipitate is dried at 25-30°C under high vacuum to obtain S31794/F-1 as a white amorphous powder with a melting point of 178-180°C. Add this to 10 times the amount of ethyl acetate.
Crystallized from methanol-water (80:12:8) and dried at 20°C under high vacuum, melting point 181-183°C.
(decomposition) to obtain crystals of S31794/F-1. Production Example 9 Isolation of Antibiotic S31794/F-1 The crude antibiotic S31794/F-1 produced as described in Production Example 8 was chromatographed on a Sephadex column and then passed through a loop through a valve operation to a silica gel column. (Michel-Miller
Column). LP-1/C ₁₈ silica gel reverse phase resin (10-20 microns) obtained by the method described in Production Example 10 was added to the column through a valve-operated loop, and chloroform-methanol-water (71:28:4) was added to the column. ). For filling, slurry filling described in Production Example 11 is used. A valveless piston type FMI pump is used to move the solvent. Antibiotic elution is monitored using a UV monitor at 280 nm. Fractions with antifungal activity are collected and concentrated under reduced pressure to produce antibiotics.
Obtain S31794/F-1. Production example 10 Silica gel/C ₁₈ reverse phase resin production process 1 Hydrolyzable LP-1 silica gel (1000g, QuantumCorp.,
Whatman) was added to a mixture of concentrated sulfuric acid (1650 ml) and concentrated nitric acid (1650 ml) in a round-bottomed flask.
Shake to ensure thorough suspension. Heat the mixture overnight (16 hours) on a steam bath with a water jacket condenser attached to the flask. The mixture is cooled in an ice bath and carefully filtered using a melt glass filter. After washing this silica gel with deionized water until the pH becomes neutral, it is placed under reduced pressure.
Dry at 100℃ for 2 days. Step 2: First Allylation The dry silica gel obtained in Step 1 is transferred to a round bottom flask and suspended in toluene (3.5). This is heated on a steam bath for 2 hours to remove some residual water by azeotropy. Octadecyltrichlorosilane (321ml,
Aldrich Chemical Company) and the reaction mixture was refluxed overnight (16 hours) at about 60° C. with gentle stirring. During this time, be careful not to bend the stirrer near the bottom of the flask. This is to prevent the silica gel particles from being crushed. The mixture is cooled, the silanized silica gel is removed, washed with toluene (3) and acetone (3), and air-dried overnight (16-20 hours). Add this to 3.5 flasks of acetonitrile-water (1:1).
and carefully stirred at room temperature for 2 hours.
After filtering and washing with acetone (3), air dry overnight. Step 3: Second silylation Repeat the first silylation using octadecyltrichlorosilane (200 ml). The suspension is refluxed at 60° C. for 2 hours with careful stirring. The final product was taken, washed with toluene (3) and methanol (6), and kept under vacuum at 50°C overnight (16-20°C).
time) Stir. Production example 11 Michael Miller column (Michel)
Miller Column) Slurry Packing Method General A Column can be packed by this operation. B For filling silica gel and silica gel reverse phase resin, for example, Quantum LP-1, particle size
10-20 microns and LiChroprep RP-8 and RP-18, particle size 25-40 microns are preferred. However, other silica gels, e.g.
Shandons ODS Hypersil, 5 micron particle size, was successfully loaded by this procedure as well as other types of resin. C This slurry filling generally requires pressures below 200 psi and flow rates of 5 to 40 ml/min, but
This depends on the volume and size of the column. The pressure for filling is 30~30% higher than the pressure used for actual separation.
Preferably 50 psi higher. By doing so, it is possible to prevent the adsorbent from being further compressed during separation. Through this operation, a column packed with reversed-phase silica gel can be used for several years without loss of efficiency. D A sudden pressure drop causes cracks and channels in the packing material, significantly reducing column efficiency. Therefore, it is important to gradually reduce the pressure to zero whenever the pump is turned off. E Volume of column (Ace Glass Cat.No.,
unpacked): 5795-04, 12ml; 5795-10, 110
ml; 5795-16, 300ml; 5795-24, 635ml; and 5796-34, 34ml. The time required to fill a F glass column ranges from several minutes to several hours, depending on the column size and the skill level of the operator. Details 1 The glass column and the storage column are connected by a coupling (the volume of the storage column is twice the volume of the main column). Stand both columns vertically (storage column on top). 2 Weigh the packing material (approx. for 200 ml of column).
100g). 3. Add about 5 times the volume of solvent to the filler.
As a solvent, 70-80% methanol and 20-30%
Using a mixture of % water. 4. Shake well until all particles are wet and leave overnight to ensure complete penetration of the solvent into the particles. Remove the supernatant by tilting. 5. Slurry the resin with enough solvent to load into the storage column and quickly pour into the storage column. Fill the column with the same solvent in advance,
The storage column is filled with solvent before injecting the slurry. Larger slurry volumes give better results, but they either (a) require large volume storage columns, or (b) require multiple refills of the storage columns during the packing operation. 6. Close the storage column using a Teflon plug at the bottom of the column (see Figure 1 of U.S. Pat. No. 4,131,547,
(See plug No. 3). Connect to the pump and immediately begin pumping the solvent. Ace Cat No.
When using a 13265-25 pump or similar solvent delivery system, use maximum flow rate (approximately 20 ml/min). 7 Continue until the column is completely filled with adsorbent. During this time, the pressure must not exceed the maximum capacity of the column (approximately 200 psi for large columns and 300 psi for analytical columns). Often the pressure is 200psi
Less than that is sufficient. 8. When the pressure exceeds the maximum value, reduce the flow rate. The pressure is thereby reduced. 9 Once the column is filled with adsorbent, turn off the pump and reduce the pressure to zero. Remove the storage column and install a press column in its place. Fill the precolumn with solvent and a small amount of adsorbent and pump at maximum pressure until the column is completely filled. For other points, please refer to general information. Whenever the pump is stopped, the pressure should be gradually reduced to prevent the formation of cracks or channels in the fill material. 10 Release the pressure and carefully remove the precolumn. Remove a small amount (2-4 mm) of adsorbent from the top of the column with a small spatula and place one or two filters on top of the column. Gently press down on the top of the adsorbent and push in the Teflon plug until it is firmly attached. Connect to a pump, apply pressure (usually less than 200 psi) and observe through the glass wall to see if the resin is further compressed. If the adsorbent moves (which tends to happen in large columns), this will create a dead space or flow path, so repeat step 9 above. Production Example 12 Production of Tetrahydro-A-30912A Dissolve Factor A of A-30912 in ethanol. on the other hand,
Platinum oxide is reduced in absolute ethanol to lead to platinum, which is then used to catalytically reduce A-30912 Factor A. Reduction is carried out using positive pressure hydrogen until the reaction is complete (approximately 2-3 hours). The reaction mixture is filtered and concentrated under reduced pressure. The residue was dissolved in a small amount of t-butanol and lyophilized to obtain tetrahydro-A-
Obtain 30912A. Production example 13 Production method of Tetrahydro-A-301912B Dissolve A-30912 factor B in ethanol. on the other hand,
Platinum oxide is reduced in absolute ethanol to lead to platinum, which is then used to catalytically reduce A-30912 factor B. Reduction is carried out using positive pressure hydrogen until the reaction is complete (approximately 2-3 hours). The reaction mixture is filtered and concentrated under reduced pressure. The residue was dissolved in a small amount of t-butanol and lyophilized to give tetrahydro-A
- Obtain 30912B. Production Example 14 Process for producing Tetrahydro-A-30912D Dissolve A-30912 Factor D in ethanol. on the other hand,
Platinum oxide is reduced in absolute ethanol to lead to platinum, which is then used to catalytically reduce A-30912 Factor D. Reduction is carried out using positive pressure hydrogen until the reaction is complete (approximately 2-3 hours). The reaction mixture is filtered and concentrated under reduced pressure. The residue was dissolved in a small amount of t-butanol and lyophilized to obtain tetrahydro-A-
Get 30912D. Production example 15 Process for producing tetrahydro-A-30912H Soak A-30912 factor H in ethanol. on the other hand,
Platinum oxide is reduced in absolute ethanol to lead to platinum, which is then used to catalytically reduce A-30912 Factor H. Reduction is carried out using positive pressure hydrogen until the reaction is complete (approximately 2-3 hours). The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in a small amount of t-butanol and lyophilized to obtain tetrahydro-A-
Get 30912H. Example 1 A Deacylation of A-30912 factor A Using slant medium A and production medium,
Fermentation of A.utahensis is carried out as described in Production Example 1. Culture in production medium is approximately 42 hours. Crude -30912 factor A (340g, this product is A-
Contains approximately 19.7g of 30912 factor A) in ethanol 1.5
Dissolve and add to the fermentation medium. Deacylation of A-30912 factor A is
Monitored by assay using Candida albicans. Fermentation continues until completion of deacylation is indicated by loss of activity against C. albicans. B Isolation of A-30912A nucleus Contains the nucleus generated from about 20g of A-30912 factor A,
The fermentation process (100) obtained as described in step A is filtered and the bacterial cake is discarded. The resulting clear liquid (approx.
4.5 (Mitsubishi Chemical Industries, Ltd.) series at a flow rate of 200 ml/min, and discard the passed liquid. The column is then washed with 8 column volumes of deionized water (PH 6.5-7.5) to remove residual broth. This washing liquid is discarded. The column is eluted using a water-methanol (7:3) mixture (85) at a flow rate of 200-300 ml/min. Elution is monitored by the following procedure. A sample sampled from each elution fraction is divided into two halves, one portion is concentrated to a small volume and treated with an acid chloride (eg, myristoyl chloride). This operation uses the method described in Reference Example 1. This product and the other untreated sample are tested for activity against Candida albicans. Untreated samples show no activity;
If the acylated sample shows activity, the fraction A
- Contains 30912A nucleus. The fraction containing A-30912A nuclei is concentrated under reduced pressure to a small amount and freeze-dried to obtain approximately 97 g of crude nuclei. C A- by reversed phase liquid chromatography
Purification of 30912A Nuclei Crude A- obtained as described in Step B
Dissolve 30912A nuclei (25 g) in water: acetonitrile: acetic acid: pyridine (96:2:1:1) (300 ml) and dissolve Lichroprep RP-18 (particle size 25-40 microns) (MC/BManufacturingChemisis, Inc.).
4 stainless steel column packed with E/M, Cincinnati, OH). This column is a high-performance liquid chromatography instrument (Chromatospac Prep100unit;
JobinYvon，16～18Rue du
Canal 191160 Longjumeau, France). The column is operated at a pressure of 90-100 psi and a flow rate of approximately 60 ml/min using the same solvent as above. In the separated state, the optical unit (ISCO
Type 6) UV monitor (ISCO
Absorption Monitor Model UA-5,
Instrumentation SpecialtiesCo.
4700 Superior Ave., Lincoln, Nebraska
68504) at 280 nm. Per minute
Collect 500 ml fractions. Based on absorption at 280nm, A-30912A
The fractions containing the nucleus are combined, concentrated under reduced pressure, and lyophilized to obtain 2.6 g of the nucleus. The amount of solvent required to carry out this chromatographic operation is 7-8. Characteristics of D A-30912A nucleus (a) Laboratory: C ₃₄ H ₅₁ N ₇ O ₁₅ (b) Molecular weight: 797.83 (c) White amorphous solid. Soluble in water, dimethylformamide, dimethyl sulfoxide and methanol, insoluble in chloroform, toluene and diethyl ether. (d) Infrared absorption spectrum (smelling potassium tablets):
3340 (wide, OH, hydrogen bond), 2970, 2930 and 2890 (CH stretching, aliphatic CH ₃ , CH ₂ CH);
1660 and 1625 (multiple carbonyl C=0);
1510~1550; 1430~1450 (CH angle); 1310~
1340; 1230-1260; 1080; 835, 650 (width)
and 550 (width) cm ^-1 . (e) Electrotitration in 66% aqueous dimethylformamide shows the presence of one titratable group with a pka value of approximately 7.35 (initial pH 7.32). (f) HPLC retention time (K′): 11.52 min. Conditions: Column: 4 x 300 mm Packing material: Silica gel/ _C18 Solvent system: Ammonium acetate: Acenanitrile:
Water (1:2:97) Flow rate: 3ml/partial pressure Pressure: 2500psi Detector: Tunable wavelength UV (230nm) Sensitivity: 0-0.4AUFS Example 2 Example 1 using Tetrahydro-A-30912A as a substrate A-30912A nucleus was produced by the method of
And refine. Example 3 A-30912A nuclei are prepared and purified by the method of Example 1 using acreacin A as a substrate. Example 4 A Deacylation of A-30912 Factor B Fermentation of A. Utahhensis is carried out as described in Production Example 1 using a production medium. After culturing for about 48 hours, A-30912 factor B is dissolved in a small amount of methanol and added to the fermentation medium. Deacylation of A-30912 factor B is
Monitor by paper disc method for Candida albicans or Neurospora crassa. Fermentation is continued until completion of deacylation is confirmed by disappearance of biological activity. B Isolation of A-30912B Nuclei Pass the fermentation process obtained as described in Step A and discard the cell cake. The resulting transparent solution (diaion, highly porous polymer,
Pass through a column packed with HP-series (manufactured by Mitsubishi Chemical Industries, Ltd.) and discard the passing liquid. The column is washed with 8 column volumes of deionized water at pH 6.5-7.5 to remove remaining fermentation liquid, and the washing liquid is discarded. The column is eluted with water-methanol (7:3) and the eluent is monitored as follows.
A sample of each eluted fraction is taken, divided into two parts, one part is concentrated to a small volume, and then treated with an acid chloride (eg, myristoyl chloride) using the method described in Preparation Example 11. For this product and the other untreated sample,
Assay activity against Candida albicans.
If the untreated sample shows no activity and the acylated sample shows activity, this fraction is classified as A-30912B.
This means that it contains a nucleus. The fraction containing the A-30912B nucleus was concentrated under reduced pressure to a small amount,
Lyophilize to obtain crude cores. C Purification of A-30912B nuclei by reverse phase liquid chromatography The crude A-30912B nuclei obtained in step B above were purified with water.
Acetonitrile-acetic acid-pyridine (96:2:
1:1) and add this solution to Lichroprep.
RP-18, particle size 25-40 microns (MC/B
ManufacturingChemists, Inc.E/M,
Chromatograph on a column packed with (Cincinnati, OH). This column is Chromatospac
Prep100unit (Jobin Yvon, 16〜18Rue du
Canal 91160 Longjumeau, France). The column is operated with the same solvent at a flow rate of approximately 60 ml/min under a pressure of 90-100 psi. The separation status was monitored using a UV monitor (ISCO AbsorptionMonitor Model) equipped with an optical unit (ISCO Type 6).
UA-5, Instrumentation Specialties Co.,
4700 Superion Ave., Lincoln,
Nebraska68504) at 280nm. Based on absorption at 280nm, A-30912B
The fractions containing the nuclei are combined, concentrated under reduced pressure, and lyophilized to obtain purified A-30912B nuclei. Example 5 A-30912B cores are prepared and purified by the method of Example 4 using tetrahydrofuran A-30912B as a substrate. Example 6 A Deacylation of A-30912 Factor D Fermentation of A.utahensis is carried out as described in Production Example 1 using a production medium. After culturing for about 48 hours, A-30912 Factor D is dissolved in a small amount of methanol and added to the fermentation medium. Deacylation of A-30912 factor D is
Monitor with paper disc method for Candida albicans or Neurospora crassa.
Fermentation is continued until completion of dealylation is confirmed by loss of biological activity. B Isolation of A-30912D Nuclei Strain the fermentation broth obtained as described in Step A and discard the cell cake. The resulting transparent liquid was added to HP-20 resin (Diaion, highly porous polymer, HP-Series, manufactured by Mitsubishi Chemical Industries, Ltd.)
Pass through a column packed with . The column was then soaked in 88 mL of deionized water with a pH of 6.5 to 7.5.
Wash the column volume to remove the remaining fermentation liquid and discard the washing liquid. The column is eluted with water-methanol (7:3) and the eluent is monitored as follows. A sample is taken from each eluted fraction, divided into two parts, one part is concentrated to a small volume, and then treated with an acid chloride (eg, myristoyl chloride) using the method described in Reference Example 1. This product and the other untreated sample are assayed for activity against Candida albicans. If the untreated sample shows no activity and the acylated sample shows activity, this fraction contains A-30912D nuclei. The fraction containing the A-30912D core is concentrated under reduced pressure to a small amount and lyophilized to obtain the crude core. C Purification of A-30912D Nuclei by Reversed Phase Liquid Chromatography The crude A-30912D nuclei obtained in Step B are purified according to Step C of Example 4. Based on absorption at 280nm, A-30912D
Fractions containing nuclei are combined, concentrated under reduced pressure, and lyophilized to obtain purified A-30912D nuclei. Example 7 An A-30912D nucleus was produced by the method of Example 6 using tetrahydro-A-30912D as a substrate,
and refine it. Example 8 A Deacylation of A-30912 Factor H Fermentation of A.utahensis is carried out as described in Production Example 1 using a production medium. After culturing for about 48 hours, A-30912 Factor H is dissolved in a small amount of methanol and added to the fermentation medium. Deacylation of A-30912 factor H is performed by Candida
Monitor by paper disc method for C. albicans or Neurospora crassa. Fermentation is continued until completion of deacylation is confirmed by disappearance of biological activity. B Isolation of A-30912H Nuclei Strain the fermentation broth obtained as described in Step A and discard the cell cake. The resulting transparent liquid was added to HP-20 resin (Diaion, highly porous polymer, HP-Series, manufactured by Mitsubishi Chemical Industries, Ltd.)
Pass through a column packed with . This column was then soaked in deionized water with a pH of 6.5 to 7.5,
Wash with 8 column volumes to remove remaining fermentation liquid,
Discard the washing liquid. The column is eluted with water-methanol (7:3) and the eluent is monitored as follows. A sample is taken from each elution fraction, divided into two parts, one part is concentrated to a small amount, and then an acid chloride (for example, myristoyl) is added using the method described in Reference Example 1.
chloride). This product and other untreated samples are assayed for activity against Candida albicans. If the untreated sample shows no activity and the acylated sample shows activity, this fraction contains A-30912H nuclei. The fraction containing the A-30912H nucleus is concentrated under reduced pressure to a small volume and lyophilized to obtain the crude nucleus. C Purification of A-30912H nucleus by reverse phase liquid chromatography Crude A- obtained as described in step B
The 30912H nuclei are purified according to the procedure of Example 4, Step C. Based on absorption at 280nm, A-30912H
Fractions containing nuclei are combined, concentrated under reduced pressure, frozen, and lyophilized to obtain purified A-30912 nuclei. Example 9 A-30912H core is prepared and purified according to the method of Example 8 using tetrahydro-A-30912H as a substrate. Example 10 An A-30912H nucleus was produced according to the method of Example 2,
refine. However, A-30912 factor H, which serves as a substrate, can be obtained from A-30912 factor A by the following procedure. Antibiotic A-30912 Factor A (19.6 mg) was dissolved in dimethylformamide (1 ml), acidic methanol (3% HCl, 0.06 ml) was added thereto, stirred overnight at room temperature, and then concentrated under reduced pressure. The obtained residue was purified using reverse phase silica gel (LP-1/C ₁₈ , manufactured by the method described in Production Example 10) and methanol-water-acetonitrile (7:2:1) as the elution solvent.
When treated with HPLPLC as described in Production Example 7, A-
1.4 mg of 30912 Factor H (a compound in which R ¹ and R ⁴ are both hydroxy, R ² is hydrogen, R ³ is methoxy, and R is linolesyl) is obtained. Example 11 Using a compound in which R ³ is ethoxy and R is linoleoyl as a substrate, a core of type A-30912H in which R ³ is ethoxy in the general formula was prepared by the method of Example 8, and purified. do.
The substrate is prepared similarly to the method described in Example 10. Example 12 Using a compound in the general formula in which R ³ is n-propoxy and R is linoleoyl as a substrate,
By the method of Example 8, a nucleus of type A-30912H in the general formula in which R ³ is n-poxy is obtained.
The substrate is purified similarly to the method described in Example 10. Example 13 Using a compound in which R ³ is isobutoxy and R is linoleoyl as a substrate, a nucleus of type A-30912H in which R ³ is isoptoxy in the general formula is prepared by the method of Example 8, and refine. Example 14 Using a compound in which R ³ is n-pentyloxy and R is linoleoyl as a substrate, by the method of Example 8, the compound in the general formula
A core of type A-39012H, where R ³ is n-pentyloxy, is prepared and purified. Example 15 A compound in which R ³ is n-hexyloxy and R is linoleoyl in the general formula is used as a substrate, and by the method of Example 8, in the general formula
A nucleus of type A-30912H, in which R ³ is n-hexyloxy, is prepared and purified. Example 16 Using a compound in which R ³ is ethoxy and R is stearoyl in the general formula as a substrate, Example 8
A nucleus of the A-30912H type in the general formula in which R ³ is ethoxy is prepared and purified by the method described above. Example 17 Using a compound in which R ³ is 2-ethyl-1-butoxy and R is linoleoyl in the general formula as a substrate, a compound in which R ³ in the general formula is 2-ethyl-1-butoxy is prepared by the method of Example 8. A-
Produce and purify 30912H type nuclei. Example 18 A compound in which R ³ is 3-methyl-1-butoxy and R is linoleoyl in the general formula is used as a substrate, and a compound in which R ³ is 3-methyl-1-butoxy in the general formula is prepared by the method of Example 8. A-
Produce and purify 30912H type nuclei. Example 19 A. Deacylation of antibiotic S31794/F-1 Fermentation of A.utahensis is carried out as described in Production Example 1 using a production medium. After culturing for about 48 hours, antibiotic S31794/F-1 is dissolved in a small amount of methanol and added to the fermentation medium. Deacylation of S31794/F-1 is
Monitor with paper disk method for Candidaalbicans. Fermentation is continued until completion of deacylation is confirmed by disappearance of biological activity. B Isolation of S31794/F-1 Nuclei Strain the fermentation broth obtained as described in Step A and discard the cell cake. The resulting clear liquid is passed through a column containing HP-20 resin (Diaion, Highly Porous Polymer, HP-series, Mitsubishi Chemical Industries, Ltd. product), and the passed liquid is discarded. The column was then soaked in 88 mL of deionized water with a pH of 6.5 to 7.5.
Wash the column volume to remove the remaining fermentation liquid and discard the washing liquid. The column is eluted with water-methanol (7:3) and the eluent is monitored as follows. A sample is taken from each eluted fraction, divided into two parts, one part is concentrated to a small volume, and then treated with an acid chloride (eg, myristoyl chloride) using the method described in Reference Example 1. This product and other treated samples are assayed for activity against Candida albicans. If the untreated sample shows no activity and the acylated sample shows activity, this fraction contains S-31794/F-1 nuclei. The fraction containing the S-31794/F-1 nucleus is concentrated under reduced pressure to a small amount and freeze-dried to obtain a crude nucleus. Purification of C S31794/F-1 nucleus by reverse phase liquid chromatography Crude product obtained as described in Step B
S31794/F-1 nuclei are purified according to the procedure of Step C of Example 4. Based on absorption at 280nm, S31794/F
- Combine the fractions containing 1 nucleus, concentrate under reduced pressure,
After freeze-drying, purified S31794/F-1 nuclei are obtained. Preparation of Abbott and Fukuda Derivatives The following procedures illustrate the preparation of compounds of general formula by the active ester method. The special compounds obtained by this method have the general formula
This is a compound in which R ⁵ is CH ₃ (CH ₂ ) ₁₁ — and R ¹ , R ² , R ₃ , and R ⁴ have the same meaning as each nucleus. Reference production example 1 2,4,5-trichlorophenyl tritecanoate n-tridecanoic acid (Sigma Chemical co.)
12.5g, 2,4,5-trichlorophenol
11.5 g of N,N'-dicyclohexylcarbodiimide and 12.0 g of N,N'-dicyclohexylcarbodiimide were dissolved in 650 ml of methylene chloride and stirred at room temperature for 16 hours. The reaction mixture was filtered and dried under reduced pressure to obtain 22 g of 2,4,5-trichlorophenyl tridecanoate. This is purified by column chromatography on silica gel (Woelm) using toluene as eluent. Fractions are monitored by TLC with detection with short wavelength UV light. The fractions containing the purified product are combined and evaporated to dryness under reduced pressure. Reference Example 1 Acylation of A-30912A nucleus with 2,4,5-trichlorophenyl tridecanoate Dissolve 6.0 g of 2,4,5-trichlorophenyl tridecanoate and 4.5 g of A-30912A nucleus in 600 ml of dimethylformamide. , stir at room temperature for 16 hours. The solvent is distilled off under reduced pressure, and the resulting residue (12 g) is slurried with 500 ml of methylene chloride for 45 minutes and filtered. Discard the liquid and remove the remaining solid with methanol.
Extract with 500ml. Filter the methanol extract,
Concentrate under reduced pressure to obtain 5.0 g of crude product. This crude product is purified by reverse phase HPLC as follows. A sample of 1 g of the crude product was dissolved in 5 ml of methanol and injected into a stainless steel column (1 x 32 inches) packed with LP-1/C ₁₈ resin (see Preparation Examples 10 and 11) in a water-methanol-acetonitrile solution. Elute with a solvent system consisting of (3:3:4). Elution was performed using an LDC duplex pump (Milton-
Roy), under pressure of 1000-1500psi, 11-12
Perform at a flow rate of ml/min. The effluent is monitored at 280 nm using an ultraviolet detector (ISCO-UA-5). Collect fractions (21-24 ml) every 2 minutes. Fractions containing the target product are collected and dried under reduced pressure. Product yield is 550 mg. Repeating the above chromatography four times yields samples of 620 mg, 520 mg, 670 mg and 490 mg of further purified product. Total yield is 2.8g. According to the above procedure, add 40g of A-30912A nucleus to 2,
Reaction with 4,5-trichlorophenyl n-tridecanoate gives 2.6 g of the purified title compound. Combine this with the product obtained earlier (5.4 g). Mass ions (M ⁺ +Na ⁺ ) by FDMS:
1016 (theoretical value: M ⁺ + Na ⁺ = 1016). HPLC using water-methanol-acetonitrile (2:1:2) as a solvent system (C ₁₈ Micro Bondapak, WastersCo.)
shows only one peak. Reference Example 2 Acylation of A-30912B nucleus with 2,4,5-trichlorophenyl n-tridecanoate 2,4,5-trichlorophenyl n-tridecanoate (3. mmol) and A-30912B nucleus (1 mmol) in dimethylformamide (DMF)
(200 ml), stirred at room temperature for 16 hours, and the solvent was distilled off under reduced pressure. Pour the residue into methylene chloride (300ml)
Slurry for 45 minutes and filter the resulting mixture. Discard the liquid and extract the solid with methanol (300ml). The methanol extract is filtered and concentrated under reduced pressure to obtain the crude product. This is purified by reverse phase HPLC as follows. A sample (1 g) of the crude product was dissolved in methanol (5 g).
ml) and injected into a stainless steel column (1 x 32 inches) packed with LP-1/C ₁₈ resin (see Preparation Examples 10 and 11) in water-methanol-acetonitrile (3:3:4). is eluted as a solvent system. Elution was performed using an LDC duplex pump (Milton-
Roy), under pressure of 1000-1500psi, 11-12
Perform at a flow rate of ml/min. The eluent is monitored at 280 nm using a UV detector (ISCO-UA-5). Collect fractions (21-24 ml) every 2 minutes. Fractions containing the desired product are combined and dried under reduced pressure to give the final product. The purified product is subjected to reverse phase
Analysis is performed by thin layer chromatography on a C ₁₈ plate (What-man KC ₁₈ ) using water-methanol-acetonitrile (1:2:2 v/v) as the solvent system. After development, observe the plate under UV light to detect the product. Reference Example 3 Acylation of A-30912D nucleus with 2,4,5-trichlorophenyl n-tridecanoate 2,4,5-trichlorophenyl n-tridecanoate (3.3 mmol) and A-30912D nucleus (1 mmol) ) in dimethylformamide (200ml)
and stir at room temperature for 16 hours. The solvent was distilled off under reduced pressure, and the resulting residue was dissolved in methylene chloride (300 ml).
Slurry and strain for 45 min. Discard the liquid;
Extract the solid with methanol (300ml). The methanol extract is filtered and concentrated under reduced pressure to obtain the crude product. The crude product was subjected to reverse phase treatment in the same manner as in Reference Example 2.
Purify by HPLC. Reference Example 4 Acylation of A-30912H nucleus with 2,4,5-trichlorophenyl n-tridecanoate 2,4,5-trichlorophenyl n-tridecanoate (3.3 mmol) and A-30912H nucleus (1 mmol) ) in dimethylformamide (200ml)
After stirring at room temperature for 16 hours, the solvent was distilled off under reduced pressure. Slurry the residue with methylene chloride (300 ml) for 45 minutes and filter the mixture. Discard the liquid;
Extract the residue with methanol (300ml). The methanol extract is filtered and concentrated under reduced pressure to obtain a crude product. The crude product was subjected to reverse phase treatment in the same manner as in Reference Example 2.
Purify by HPLC treatment. Reference Example 5 Using a compound in which R ³ in the general formula is n-propoxy (manufactured by the method of Example 12) as a starting material and following the procedure in Reference Example 4, R ³ in the general formula is n-propoxy. R ⁵ is CH ³ -
(CH ₂ ) ₁₁ - is obtained. Reference Example 6 Acylation of S31794/F-1 nucleus with 2,5,6-trichlorophenyl/n-tridecanoate 2,4,5-trichlorophenyl/n-tridecanoate (3.3 mmol) and S31794/F -1
Nuclei (1 mmol in dimethylformamide (200 ml)
After stirring at room temperature for 16 hours, the solvent was distilled off under reduced pressure. The residue was dissolved in methylene chloride (300ml).
Slurry for 45 minutes, discard the liquid, and extract the residue with methanol (300 ml). The methanol extract is filtered and concentrated under reduced pressure to obtain the crude product. The crude product was subjected to reverse phase treatment in the same manner as in Reference Example 2.
Purify by HPLC. Production of debono group derivative In the general formula, R ⁵ is

Compounds of the formula, ie debono compounds of the general formula, can be prepared. Reference Example 2 Production of N-(n-dodecanoyl)-p-aminobenzoic acid p-Aminobenzoic acid (5.5 g; 40 mmol) was dissolved in pyridine (100 ml), and n-dodecanoyl chloride (8.74 g; 40 mmol) dropwise. Stir the mixture for 3 hours and pour into water (3). The generated precipitate was collected and dried under reduced pressure to give N-(n
-dodecanoyl)-P-aminobenzoic acid (11.01 g) is obtained. Reference production example 3 Production of N-(n-dodecanoyl)-p-aminobenzoic acid/2,4,5-trichlorophenyl ester N-(n-dodecanoyl)-p-aminobenzoic acid (11.01g; 34.5 mmol) , 2,4,5-trichlorophenol (7.5 g; 38 mmol) and N,N'-dicyclohexylcarbodiimide (6.94 g; 34.5 mmol) were dissolved in methylene chloride (250 ml).
Stir for 3.5 hours at room temperature and filter.
The liquid was concentrated under reduced pressure, and the residue was crystallized from acetonitrile-water to give N-(n-dodecanoyl)-P-
Obtain aminobenzoic acid trichlorophenyl ester (12.84g). Reference Example 7 Acylation of A-30912A nucleus A-39012A nucleus (8.16 g; 10.2 mmol) and N-(n-doridecanoyl)-p-aminobenzoic acid/2,4,5-trichlorophenyl ester were dissolved in dimethylformamide. (100 ml) and stir at room temperature for 15 hours. The solvent is removed under reduced pressure and the residue is washed twice with diethyl ether. Discard the washing liquid;
The washed residue was dissolved in methanol (50 ml), and a Prep Pak-500/C ₁₈ column (Waters
PrepLC/
Purify by reverse phase HPLC using System500unit. The column is constantly eluted with water-methanol-acetonitrile (25:65:10 v/v) at a pressure of 500 psi. The fractions are analyzed by thin layer chromatography on a silica gel plate using water-methanol-acetonitrile (25:65:10 volume ratio) as the solvent.
Combine the fractions containing the target product and lyophilize to obtain A.
-30912A nuclear N- (n-dodecanoyl) -p-
Obtain 3.5 g of aminobenzoyl derivative. Reference Example 8 Acylation of A-30912B nucleus A-30912B nucleus (10.2 mmol) and N-
(n-dodecanoyl)-P-aminobenzoic acid 2,
4,5-Trichlorophenyl ester (10.2 mmol) is dissolved in dimethylformamide (100 ml) and stirred at room temperature for 15 hours. Remove the solvent under reduced pressure,
The resulting residue is washed twice with dimethyl ether. The washing solution was discarded, the washed residue was dissolved in methanol (50 ml), and the mixture was subjected to reverse phase analysis using PrepLC/System500unit using a Prep Pak-500/C ₁₈ column as the stationary phase.
Purify by HPLC. The column was filled with water-methanol-acetonyl (25:65:
10v/v). The fractions are analyzed by thin layer chromatography on a silica gel plate using water-methanol-acetonitrile (25:65:10 v/v) as the solvent system. The fractions containing the desired product were combined and lyophilized to extract the N-
A (n-dodecanoyl)-P-aminobenzoyl derivative is obtained. Reference Example 9 Acylation of A-30912D nucleus A-30912D nucleus (10.2 mmol) and N-
(n-dodecanoyl)-p-aminobenzoic acid 2,
4,5-Trichlorophenyl ester (10.2 mmol) is dissolved in dimethylformamide (100 ml) and stirred at room temperature for 15 hours. Remove the solvent under reduced pressure,
The resulting residue is washed twice with diethyl ether. The washing solution was discarded, the washed residue was dissolved in methanol (50 ml), and PrepLC/ _{System500unit} (Waters
Purified by reverse-phase HPLC by S.A. Associates, Inc., Milford, Massachusetts). The column is
Elute constantly with water-methanol-acetonitrile (25:65:10 v/v) at a pressure of 500 psi. The fractions are analyzed by thin layer chromatography on a silica gel plate using water-methanol-acetonitrile (25:65:10 v/v) as the solvent system. Fractions containing the desired product were combined and lyophilized to yield A-
An N-(n-dodecanoyl)-p-aminobenzoyl derivative with a 39012D core is obtained. Reference Example 10 Acylation of A-30912H nucleus A-30912H nucleus (10.2 mmol) and N-
(n-dodecanoyl)-p-aminobenzoic acid 2,
4,5-Trichlorophenyl ester (10.2 mmol) is dissolved in dimethylformamide (100 ml) and stirred at room temperature for 15 hours. Remove the solvent under reduced pressure,
The resulting residue is washed twice with diethyl ether.
Discard the washing liquid and add the washed residue to methanol (50ml).
A Prep Pek-500/C ₁₈ column (Water Asso-ciates, Inc.) was used as the stationary phase.
PrepLC/System500unit (Water Assocciaties,
Inc., Milford, Massachusetts)
Purify by HPLC. Galum was prepared using water-methanol-acetonitrile (25:65:
10v/v). The fractions are analyzed by thin layer chromatography on a silica gel plate using water-methanol-acetonitrile (25:65:10 v/v) as the solvent system. The fractions containing the desired product were combined and lyophilized to remove the N of the A-30912H nucleus.
-(n-dodecanoyl)-p-aminobenzoyl derivative is obtained. Reference Example 11 Using a compound in which R ³ is isobutoxy in the general formula (produced by the method of Example 13) and following the procedure in Reference Example 9, in the general formula R ³ is isobutoxy and R ⁵ is

A compound of the formula is obtained. Reference Example 12 Acylation of A-31794/F-1 nucleus S31794/F-1 nucleus (10.2 mmol) and N-
(n-dodecanoyl)-p-aminobenzoic acid 2,
4,5-Trichlorophenyl ester (10.2 mmol) is dissolved in dimethylformamide (100 ml) and stirred at room temperature for 15 hours. Remove the solvent under reduced pressure,
The resulting residue is washed twice with dimethyl ether. The washing solution was discarded, the washed residue was dissolved in methanol (50 ml), and a Prep Pak-500/C ₁₈ column (WaterAssociates, Inc.) was used as the stationary phase.
PrepLC／System500unit―(Water
Purify by reverse-phase HPLC by HPLC, Inc.). The column is constantly eluted with water-methanol-acetritrile (25:65:10 v/v) at a pressure of 500 psi. The fractions were separated on a silica gel plate in water-methanol-acetonitrile (25:65:10v/v).
Analyze by thin layer chromatography using a solvent system. The fractions containing the desired product are combined and lyophilized to obtain the N-(n-dodecanoyl)-p-aminobenzoyl derivative of S31794/F-1 core. Production of debono group derivatives In the general formula, R ⁶ is

Compounds having the formula, ie debono compounds of the general formula, may be prepared. Reference production example 4 Production of P-(n-octyloxy)benzoic acid p-Hydoxybenzoic acid (19.2g; 150 mmol)
Dissolve in 10% aqueous sodium oxide (120ml),
Add this to dimethyl sulfoxide (DMSO) (480 ml) preheated to 80°C. to this,
Further n-octyl bromide (28.95 g, 150 mmol) is added dropwise and the mixture is stirred at room temperature for 4 hours and then poured into 1200 ml of ice water. Add concentrated hydrochloric acid (30 ml) and leave to stand until precipitation is complete. Collect the precipitate,
After drying, crystallize from acetonitrile-water. Melting point 97-99℃ Elemental analysis (as C ₁₅ H ₂₂ O ₃ ) Calculated value: C, 71.97; H, 8.86. Actual value: C, 71.72; H, 9.10. Reference production example 5 p-(n-octyloxy) Benzoic acid 2,
Preparation of 4,5-trichlorophenyl ester p-(n-octyloxy)benzoic acid (6.18 g; 24.7 mmol), 2,4,5-trichlorophenol (5.39 g; 27.2 mmol), and N,N'- Dicyclohexylcarbodiimide (4.94 g, 24.7 mmol) was dissolved in methylene chloride (200 ml).
The solution was stirred at room temperature for 18 hours and filtered. The liquid is concentrated, and the resulting oil is crystallized from acetonitrile-water to obtain p-(n-octyloxy)benzoic acid 2,4,5-trichlorophenyl ester. NMR: δ4.02 (2H, t, J=
3Hz), 7.0 (1H, d, J=4Hz), 7.23 (s, 1H),
7.3 (s, 1H), 8.08 (d, 1H, J=4Hz). Reference Example 13 Acylation of A-30912A nucleus A-30912A nucleus (14.2 g, 17.8 mmol) and p-(n-octyloxy)benzoic acid 2,4,
5-Trichlorophenyl ester (15.32g, 35.7
mmol) in dimethylformamide (150 ml) and stir this solution at room temperature for 16-20 hours.
The solvent was distilled off under reduced pressure, and the residue was diluted with diethyl ether.
Wash twice with methine chloride. The washing liquid was discarded, and the washed residue was diluted with ethyl acetate:methanol (1:3) (80%
ml), using silica gel as the stationary phase,
Purify by high-speed HPLC using PrepLC/System500unit. The column is eluted stepwise with methanol-ethyl acetate (1:4 to 2:3). Fractions are analyzed by thin layer chromatography on silica gel (Merck) using ethyl acetate-methanol (3:2 v/v) as the solvent system. -30912A Combine the nuclear-free fractions and freeze-dry to obtain A-30912A.
A nuclear p-(n-octyloxy)benzoyl derivative is obtained. Yield 7.13g. M ⁺ +23:1052 (by FDMS) Reference Example 14 Acylation of A-30912B nucleus A-30912B nucleus (17.8 mmol) and p-
(n-octyloxy)benzoic acid (35.7 mmol)
Dissolve in dimethylformamide (150 ml) and stir at room temperature for 16-20 hours. The solvent was distilled off under reduced pressure, and the resulting residue was washed twice with diethyl ether and twice with methylene chloride. Discard the washing liquid and add the washed residue to ethyl acetate-methanol (1:3) (80ml).
using silica gel as the stationary phase.
Purify by HPLC using PrepLC/System500-unit. The column is eluted stepwise with a methanol-ethyl acetate (1:4 to 2:3) solvent system. Fractions are analyzed by thin layer chromatography on silica gel (Merck) using ethyl acetate-methanol (3:2 v/v) as the solvent system. A-
Fractions that do not contain the 30912B nucleus are collected and lyophilized to obtain the p-(n-octyloxy)benzoyl derivative of the A-30912B nucleus. Example 15 Acylation of A-30912D nucleus A-30912D nucleus (17.8 mmol) and p-
(n-octyloxy)benzoic acid 2,4,5-
Trichlorophenyl ester (35.7 mmol) is dissolved in dimethylformamide (150 ml) and stirred at room temperature for 16-20 hours. The solvent was removed under reduced pressure and the residue was washed twice with diethyl ether and then twice with methylene chloride. The washing liquid was discarded, and the washed residue was dissolved in ethyl acetate-methanol (1:3) (80 ml).
PrepLC using silica gel as the stationary phase/
Purify by HPLC using System500unit.
The column was made of methanol-ethyl acetate (1:4~
2:3). Fractions are analyzed by thin layer chromatography on silica gel using ethyl acetate-methanol (3:2 v/v) as the solvent system.
Fractions containing no A-30912B nucleus were collected and lyophilized to extract p-(n-octyloxy) of the A-30912D nucleus.
gives benzoyl derivatives. Reference Example 16 Acylation of A-30912H nucleus A-30912H nucleus (17.8 mmol) and p-
(n-ogtyloxy)benzoic acid 2,4,5-
Trichlorophenyl ester (35.7 mmol) is dissolved in dimethylformamide (150 ml) and the solution is stirred at room temperature for 16-20 hours. The solvent is removed under reduced pressure and the residue is washed twice with diethyl ether and then twice with methylene chloride. Discard the washing liquid and add the washed residue to ethyl acetate-methanol (1:3) (80ml).
using silica gel as the stationary phase.
Purify by PLC using PrepLC/System500unit. The column is eluted stepwise with a solvent system of methanol-ethyl acetate (1:4 to 2:3). Fractions are analyzed by thin layer chromatography on silica gel (Merck) using ethyl acetate-methanol (3:2 v/v) as the solvent system.
Fractions that do not contain the -30912H nucleus are collected and lyophilized to obtain p-(n-octyloxy) of the A-30912H nucleus.
A benzoyl derivative is obtained. Reference Example 17 Using a compound in which R ³ in the general formula is n-hexyloxy (produced by the method of Example 15) as a starting material and following the procedure in Example 16, R ³ in the general formula is n-hexyl oxide. So R ⁵ is

A compound of the formula is obtained. Reference Example 18 Acylation of S31794/F-1 nucleus S31794/F-1 nucleus (17.8 mmol) and p-
(n-octyloxy)benzoic acid 2,4,5-
Trichlorophenyl ester (35.7 mmol) is dissolved in dimethylformamide (150 ml) and this is stirred at room temperature for 16-20 hours. The solvent was removed under reduced pressure and the residue was washed twice with diethyl ether and then twice with methylene chloride. The washing solution was discarded, the washed residue was dissolved in ethyl acetate-methanol (1:3) (80 ml), and silica gel was used as the stationary phase.
Purify by HPLC using PrepLC/System500unit. The column is eluted stepwise with a methanol-ethyl acetate (1:4 to 2:3) solvent system.
Fractions are analyzed by thin layer chromatography on silica gel (Merck) using ethyl acetate-methanol (3:2 v/v) as the solvent system. S31794/F
-1 Collect the fractions that do not contain nuclei, freeze-dry them and make S-
A P-(n-octyloxy)benzoyl derivative of 31794/F-1 nucleus is obtained. Reference Example 19 The antifungal activity of the compound of formula 19 was determined by evaluating its in vitro activity using standard disk diffusion and agar dilution methods, and its in vivo activity against systemic fungal infections using standard methods using mice. It is proved by testing each of the following. The table shows the results of antifungal tests for representative compounds of the formula. Table 1 shows the in vitro test results of the compounds of Reference Examples 1, 7 and 13 by the agar plate disk diffusion method. The table shows the size of the inhibition zone (diameter in mm) for microorganisms produced by the test compound.
activity (measured by ). The table shows the test substance (μg/
The activity is indicated by the minimum inhibitory concentration (MIC) of the drug. The table shows the results of an in vitro test using the agar dilution method of p-(n-octyloxy)benzoyl derivatives of A-30912A nuclei against 5 strains of Candida albicans. The table shows the minimum inhibitory concentration (MIC) of the test substance required to inhibit the test bacteria.
mg/ml). The table shows the results of an in vivo test evaluating the effect of the derivative on Candida albicans A-26 infection in mice. In the same table, the activity is determined by the ED ₅₀ value (the dose required to cure 50% of the test mice).
(value expressed in mg/ml).
For those for which ED ₅₀ values were not obtained, activity was shown at the lowest dose that produced a significant antifungal effect.
In this study, male mice (al-
binomice, specific pathogen free) group,
Candida albicans A-26 is infected intravenously. Animals are exposed to X-rays for 8 minutes at a rate of approximately 50 roentgens per minute (total dose of 400 roentgens) 24 hours prior to infection in order to suppress the immune response to the infecting microorganism. At 0, 4 and 24 hours after infection,
Each group of mice is administered the test compound subcutaneously in several doses suspended in 33% polyethylene glycol (PEG)-water and the date of death of each animal is recorded. At each dose, the mean date of death was statistically compared (Student's ttest) between the infected-treated group and the infected-untreated group of 10 animals to determine whether treatment significantly prolonged survival. do. The table shows the results of testing the compounds of Examples 1, 7 and 13 for absorption after oral administration. In this study, 416 mg/Kg of test compound was administered at 33%
PEG400 - Suspended in water and forced ingestion into mice. Blood is collected from the orbital sinus after a certain period of time, and antibacterial activity is assayed as follows.
A 7mm disk impregnated with 20μ of whole blood,
The inhibition zone obtained by placing the test compound on agar inoculated with Aspergillus montevidensis A35137 and incubating it at 30°C for 40 hours is compared with the standard value obtained for the test compound to calculate the amount in the blood sample.

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【table】 [Brief explanation of drawings]

Figure 1 shows the infrared absorption spectrum of the A-30912A nucleus measured with a KBr disk.

Claims (1)

[Claims] 1. A cyclic peptide represented by the following formula and its acid addition salt: (In the formula, R ¹ represents H ^or OH.
is H, R ² is H, and R ³ and R ⁴ are both H or both OH. Also, R ¹ is
When OH, ^R2 is H, ^R3 is OH or _C1 - _C6 alkoxy, ^R4 is OH,
Or R ² is CONH ₂ and R ³ and R ⁴ are both
It's OH. ). 2 In the formula, R ¹ , R ³ and R ⁴ are all
2. The cyclic peptide and acid addition salt thereof according to claim 1, wherein R ² is OH and R 2 is H. 3 In the formula, R ¹ and R ² are both H,
Claim 1 in which R ³ and R ⁴ are both OH
Cyclic peptides and acid addition salts thereof as described in Section 1. 4. The cyclic peptide and acid addition salt thereof according to claim 1, wherein in the formula, R ¹ , R ² , R ³ and R ⁴ are all H. 5. The cyclic peptide and acid addition salt thereof according to claim 1, wherein in the formula, R ¹ and R ⁴ are both OH, R ² is H, and R ³ is CH ₃ O-. 6. The cyclic peptide and acid addition salt thereof according to claim 1, wherein in the formula, R ¹ and R ⁴ are both OH, R ² is H, and R ³ is C ₂ to _{C 6} alkoxy. 7 ^R3 is ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, n-pentyloxy, n-hexyloxy, 2-ethyl-1-butoxy or 3-methyl-1-butoxy Cyclic peptide according to claim 6 and acid addition salt thereof. 8 In the formula, R ¹ , R ³ and R ⁴ are all
The cyclic peptide and acid addition salt thereof according to claim 1, wherein R ² is OH and R 2 is [Formula]. 9 The following formula: (In the formula, R ¹ represents H ^or OH.
is H, R ² is H, R ³ and R ⁴ are both H or OH, and R is stearoyl or linoleoyl. Also, when R ¹ is OH, R ² is H, and R ⁴ is OH,
R ³ is OH and R is stearoyl, linoleoyl, or palmitoyl, or
^R3 is _C1 - _C6 alkoxy and R is stearoyl or linoleoyl. Further, when R ¹ , R ³ and R ⁴ are all OH and R ² is [Formula], R is myristoyl. ) is brought into contact with a crude enzyme solution obtained by culturing a deacylation enzyme-producing bacterium belonging to the genus Actinoplanes in an aqueous solvent: (In the formula, R ¹ represents H ^or OH.
is H, R ² is H, and R ³ and R ⁴ are both H or both OH. Also, R ¹ is
When OH, ^R2 is H, ^R3 is OH or _C1 - _C6 alkoxy, ^R4 is OH,
Or R ² is CONH ₂ and R ³ and R ⁴ are both
It's OH. ) A method for producing a cyclic peptide represented by and an acid addition salt thereof. 10 In the formula, R ¹ , R ³ and R ⁴ are OH, R ² is H, and R is linoleoyl, stearoyl or palmitoyl. The antibiotic is brought into contact with the crude enzyme solution. The manufacturing method according to scope item 9. 11 In the formula, R ¹ and R ² are both H, R ³ and R ⁴ are both OH, and R is stearoyl or linoleoyl. The antibiotic is brought into contact with the crude enzyme solution. The manufacturing method according to scope item 9. 12 In the formula, R ¹ , R ² , R ³ and R ⁴ are H
10. The production method according to claim 9, wherein the antibiotic in which R is stearoyl or linoleoyl is brought into contact with the crude enzyme solution. 13 In the formula, R ¹ and R ⁴ are OH,
10. The production method according to claim 9, characterized in that an antibiotic in which R ² is H, R ³ is CH ₃ O, and R is stearoyl or linoleoyl is brought into contact with the crude enzyme solution. 14 In the formula, R ¹ and R ⁴ are OH,
R ² is H, R ³ is C ₂ -C ₆ alkoxy,
10. The production method according to claim 9, wherein the antibiotic in which R is stearoyl or linoleoyl is brought into contact with the crude enzyme solution. 15 In the formula, R ¹ , R ³ and R ⁴ are OH, R ² is [Formula], and R is myristoyl. The antibiotic is brought into contact with the crude enzyme solution. The manufacturing method according to item 9. 16. The production method according to any one of claims 9 to 15, wherein the crude enzyme solution is a culture medium for the microorganism according to claim 9. 17 Claims 9 to 16 further including the step of isolating the cyclic peptide or a salt thereof
The manufacturing method described in any of paragraphs. 18 Claims 9 to 17 further including the step of purifying the cyclic peptide or a salt thereof
The manufacturing method described in any of paragraphs.