CA2490570A1 - Polyene carboxylic acid derivatives, method for their production and the use thereof - Google Patents
Polyene carboxylic acid derivatives, method for their production and the use thereof Download PDFInfo
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Abstract
The invention relates to novel compounds, called serpentemycins, of formula (I), in which Y, R, R2, R3, R4, X, X2, X3, n, m and o are defined as per the description, said compounds being formed from the microorganism Actinomycetalessp. DSM 14865 during zymolysis. The invention also relates to chemical derivatives of the serpentemycins, to a method for their production and to the use of said compounds as medicaments, in particular fo r the treatment and prophylaxis of bacterial, infectious diseases.
Description
Description:
Polyene carboxylic acid derivatives, method for their production and the use thereof.
A large number of antibiotics are used therapeutically for treating infectious bacterial diseases. However, the pathogens are becoming increasingly resistant to the pharmaceuticals employed and there is even the threat of a serious risk due to what are termed multiresistant organisms, which have not only become capable of resisting single antibiotic groups, such as ~i-lactam antibiotics, glycopeptides or macrolides, but also carry several resistances at one and the same time. There are even pathogens which have become resistant to all the commercially available antibiotics. Infectious diseases which are caused by these organisms can no longer be treated. There is therefore a great need for novel agents which can be used against resistant organisms. While many thousand antibiotics have been described in the literature, most of them are too toxic to be used as pharmaceuticals.
A relatively large number of polyene antibiotics, most of which belong to the macrocyclic structure type, have already been described. These macrolides act antimycotically by means of interactions with biological membranes and ace therefore toxic to homeotherms. The most important representative of this type is amphotericin B, which is used as a therapeutic agent in humans despite its toxicity.
An example of a nonmacrocyclic polyene antibiotic which has been described (Ritzau et al., Liebigs Ann. Chem. 1993, 433-435) is serpentene, which contains a phenyl ring which is substituted in the 1,2 position by polyene side chains.
In tests directed against Gram-positive and Gram-negative bacteria, serpentene only exhibited a weak antibiotic effect against Bacillus subtilis.
The cell wall of Gram-positive bacteria is composed, inter alia, of murein, which is composed of N-acetyl-D-glucosamine and N-acetylmuramic acid, which are linked to each other like a disaccharide, and contains amino acids and peptides, such as D-glutamine, D- and L-alanine, L-lysine and pentaglycyl units, and which is strongly crosslinked. The biosynthesis of the bacterial cell wall takes place using enzymes which are not found in homeotherm metabolism. These enzymes are therefore suitable sites of attack for developing antibiotics which are well tolerated by homeotherms, and inhibitors of mureinbiosynthesis should not be poisonous to humans. Glycosyltransferase (Transglycosylase, GT) is a key enzyme in peptidoglycanbiosynthesis and consequently of cell wall construction. A
specific inhibitor of this enzyme, i.e. moenomycin (Kurz et al., Eur. J. Biochem. 1998, 252, 500-507), has already been known for a relatively long time. Moenomycin is an antibiotic which exhibits very powerful activity and which is well tolerated;
however, it is not absorbed from the gastrointestinal tract and elimination from the blood following intravenous administration is also disturbed. For these reasons, it has thus far~not been possible to use moenomycin systemically in medicine. Since then, only a very few additional glycosyltransferase inhibitors have been described in the literature; for reasons of pharmacokinetics or tolerance, none of these agents has found its way into therapy. For this reason, novel GT inhibitors are still being sought, as has recently been reported by Goldman & Gange in Current Medicinal Chem.
2000, 7, 801-820. Screening takes place using specific biochemical GT test systems.
These assays have been described repeatedly, for example by Vollmer & Holtje in Antimicrobial Agents and Chemotherapy 2000, 44, 1181-1185.
Darby et al. (J. Org. Chem. 1977, 42, 1960-1967) describe the synthesis of phenyl compounds which are substituted by traps-polyene side chains in the 1,2 position for analyzing the ~ system of macrocyclic annulenes.
It has been found, surprisingly, that the strain Actinomycetales sp. DSM 14865 is able to form novel compounds which are very good inhibitors of glycosyltransferase and very effective antibacterial compounds.
The invention consequently relates to the active compounds which are formed by the strain Actinomycetales sp. DSM 14865, and to their physiologically tolerated salts, esters, ethers and obvious chemical equivalents.
The invention consequently relates to a compound of the formula (I) X~ R~
H H n O
(R)° /
H=H~Y
(I) where Y is a group of the formula (II) O
X282 (II) or of the formula (III) XzR2 ~Ra R4 (III), R is H, C~-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C5-C~4-aryl, halogen, -CN, -OH, -O-C~-C6-alkyl, -O-C2-C6-alkenyl, -O-C5-C~4-aryl, -O-C2-C6-alkynyl, -NH2, -NH-C~-C6-alkyl, -NH-C2-C6-alkenyl, -NH-C2-C6-alkynyl, -NH-C5-C~4-aryl, -N(-C~-alkyl)2, -N(-C2-C6-alkenyl)2, -N(-C2-C6-alkynyl)2, -N(C5-C~4-aryl)2, -NH[-C(=O)-(C~-C6-alkyl)], -NH[-C(=O)-(C5-C~4-aryl)], -NH-O-R~, -SH, -S-C~-C6-alkyl, -S-C2-C6-alkenyl, -S-C~-C6-alkynyl or -O-C5-C~4-aryl, where the abovementioned substituents can be unsubstituted or substituted, once or more than once, by C~-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C5-C~4-aryl, where alkyl, alkenyl, alkynyl and aryl are unsubstituted or substituted, once or twice, by -OH, =O, -O-C~-C6-alkyl, -O-C2-C6-alkenyl, -C~4-aryl, -C5-C~4-aryl, -NH-C~-C6-alkyl, -NH-C2-C6-alkenyl, -NH2, halogen, where alkyl, alkenyl, alkynyl and aryl can be further substituted by a -CN, amide or oxime, R~, R2, R3 and R4 are, independently of each other, H, C~-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C5-C~4-aryl, in which alkyl, alkenyl, alkynyl and aryl are unsubstituted or substituted, once or twice, by -OH, -O-C~-C6-alkyl, -O-C2-C6-alkenyl, -O-C5-C,4-aryl, -C5-C~4-aryl, -NH-C~-C6-alkyl, -NH-C2-C6-alkenyl, -NH2 or halogen, in which alkyl, alkenyl, alkynyl and aryl are unsubstituted or substituted, once or twice, by -OH, =O, -O-C~-C6-alkyl, -O-CZ-C6-alkenyl, -O-C5-C~4-aryl, -C5-C14-aryl, -NH-C~-C6-alkyl, -NH-C2-Cs-alkenyl, -NH2 or halogen, in which alkyl, alkenyl, alkynyl and aryl can be further substituted by a -CN, amide or oxime, X~, X2 and X3 are, independently of each other, -CH2-, -CHR-, -NH-, -N(C~-C6-alkyl}-, -N(C2-C6-alkenyl~, -N(C2-C6-alkynyl~, _N~-C(=O~_(C~_C6_alkyl)J-, -N[-C(=O)-(C5-C~a-aryl)]-, -N(C5-C,a-aryl}-, -N(O-R~, -O- or -S-, n and m are, independently of each other, 2, 3, 4 or 5, and o is 0, 1, 2 or 3, where compounds of the formula (I) are excepted in which o is 0, n is 2, m is 2 or 3, X2 and X3 are O, and R2 and R3 are C2H5, and all double bonds possess the transconfiguration, and/or a stereoisomeric form of the compound of the formula (I) andlor a mixture of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula (I).
C~-C6-Alkyl is a straight-chain or branched alkyl group having from 1 to 6 C
atoms, preferably having from 1 to 4 C atoms, such as methyl, ethyl, i-propyl, tert-butyl and hexyl.
C2-C6-Alkenyl is a straight-chain or branched alkenyl group having from 2 to 6 C
atoms which is unsaturated once, twice or three times, such as allyl, crotyl, propenyl, yenta-1,3-dienyl and pentenyl.
Polyene carboxylic acid derivatives, method for their production and the use thereof.
A large number of antibiotics are used therapeutically for treating infectious bacterial diseases. However, the pathogens are becoming increasingly resistant to the pharmaceuticals employed and there is even the threat of a serious risk due to what are termed multiresistant organisms, which have not only become capable of resisting single antibiotic groups, such as ~i-lactam antibiotics, glycopeptides or macrolides, but also carry several resistances at one and the same time. There are even pathogens which have become resistant to all the commercially available antibiotics. Infectious diseases which are caused by these organisms can no longer be treated. There is therefore a great need for novel agents which can be used against resistant organisms. While many thousand antibiotics have been described in the literature, most of them are too toxic to be used as pharmaceuticals.
A relatively large number of polyene antibiotics, most of which belong to the macrocyclic structure type, have already been described. These macrolides act antimycotically by means of interactions with biological membranes and ace therefore toxic to homeotherms. The most important representative of this type is amphotericin B, which is used as a therapeutic agent in humans despite its toxicity.
An example of a nonmacrocyclic polyene antibiotic which has been described (Ritzau et al., Liebigs Ann. Chem. 1993, 433-435) is serpentene, which contains a phenyl ring which is substituted in the 1,2 position by polyene side chains.
In tests directed against Gram-positive and Gram-negative bacteria, serpentene only exhibited a weak antibiotic effect against Bacillus subtilis.
The cell wall of Gram-positive bacteria is composed, inter alia, of murein, which is composed of N-acetyl-D-glucosamine and N-acetylmuramic acid, which are linked to each other like a disaccharide, and contains amino acids and peptides, such as D-glutamine, D- and L-alanine, L-lysine and pentaglycyl units, and which is strongly crosslinked. The biosynthesis of the bacterial cell wall takes place using enzymes which are not found in homeotherm metabolism. These enzymes are therefore suitable sites of attack for developing antibiotics which are well tolerated by homeotherms, and inhibitors of mureinbiosynthesis should not be poisonous to humans. Glycosyltransferase (Transglycosylase, GT) is a key enzyme in peptidoglycanbiosynthesis and consequently of cell wall construction. A
specific inhibitor of this enzyme, i.e. moenomycin (Kurz et al., Eur. J. Biochem. 1998, 252, 500-507), has already been known for a relatively long time. Moenomycin is an antibiotic which exhibits very powerful activity and which is well tolerated;
however, it is not absorbed from the gastrointestinal tract and elimination from the blood following intravenous administration is also disturbed. For these reasons, it has thus far~not been possible to use moenomycin systemically in medicine. Since then, only a very few additional glycosyltransferase inhibitors have been described in the literature; for reasons of pharmacokinetics or tolerance, none of these agents has found its way into therapy. For this reason, novel GT inhibitors are still being sought, as has recently been reported by Goldman & Gange in Current Medicinal Chem.
2000, 7, 801-820. Screening takes place using specific biochemical GT test systems.
These assays have been described repeatedly, for example by Vollmer & Holtje in Antimicrobial Agents and Chemotherapy 2000, 44, 1181-1185.
Darby et al. (J. Org. Chem. 1977, 42, 1960-1967) describe the synthesis of phenyl compounds which are substituted by traps-polyene side chains in the 1,2 position for analyzing the ~ system of macrocyclic annulenes.
It has been found, surprisingly, that the strain Actinomycetales sp. DSM 14865 is able to form novel compounds which are very good inhibitors of glycosyltransferase and very effective antibacterial compounds.
The invention consequently relates to the active compounds which are formed by the strain Actinomycetales sp. DSM 14865, and to their physiologically tolerated salts, esters, ethers and obvious chemical equivalents.
The invention consequently relates to a compound of the formula (I) X~ R~
H H n O
(R)° /
H=H~Y
(I) where Y is a group of the formula (II) O
X282 (II) or of the formula (III) XzR2 ~Ra R4 (III), R is H, C~-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C5-C~4-aryl, halogen, -CN, -OH, -O-C~-C6-alkyl, -O-C2-C6-alkenyl, -O-C5-C~4-aryl, -O-C2-C6-alkynyl, -NH2, -NH-C~-C6-alkyl, -NH-C2-C6-alkenyl, -NH-C2-C6-alkynyl, -NH-C5-C~4-aryl, -N(-C~-alkyl)2, -N(-C2-C6-alkenyl)2, -N(-C2-C6-alkynyl)2, -N(C5-C~4-aryl)2, -NH[-C(=O)-(C~-C6-alkyl)], -NH[-C(=O)-(C5-C~4-aryl)], -NH-O-R~, -SH, -S-C~-C6-alkyl, -S-C2-C6-alkenyl, -S-C~-C6-alkynyl or -O-C5-C~4-aryl, where the abovementioned substituents can be unsubstituted or substituted, once or more than once, by C~-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C5-C~4-aryl, where alkyl, alkenyl, alkynyl and aryl are unsubstituted or substituted, once or twice, by -OH, =O, -O-C~-C6-alkyl, -O-C2-C6-alkenyl, -C~4-aryl, -C5-C~4-aryl, -NH-C~-C6-alkyl, -NH-C2-C6-alkenyl, -NH2, halogen, where alkyl, alkenyl, alkynyl and aryl can be further substituted by a -CN, amide or oxime, R~, R2, R3 and R4 are, independently of each other, H, C~-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C5-C~4-aryl, in which alkyl, alkenyl, alkynyl and aryl are unsubstituted or substituted, once or twice, by -OH, -O-C~-C6-alkyl, -O-C2-C6-alkenyl, -O-C5-C,4-aryl, -C5-C~4-aryl, -NH-C~-C6-alkyl, -NH-C2-C6-alkenyl, -NH2 or halogen, in which alkyl, alkenyl, alkynyl and aryl are unsubstituted or substituted, once or twice, by -OH, =O, -O-C~-C6-alkyl, -O-CZ-C6-alkenyl, -O-C5-C~4-aryl, -C5-C14-aryl, -NH-C~-C6-alkyl, -NH-C2-Cs-alkenyl, -NH2 or halogen, in which alkyl, alkenyl, alkynyl and aryl can be further substituted by a -CN, amide or oxime, X~, X2 and X3 are, independently of each other, -CH2-, -CHR-, -NH-, -N(C~-C6-alkyl}-, -N(C2-C6-alkenyl~, -N(C2-C6-alkynyl~, _N~-C(=O~_(C~_C6_alkyl)J-, -N[-C(=O)-(C5-C~a-aryl)]-, -N(C5-C,a-aryl}-, -N(O-R~, -O- or -S-, n and m are, independently of each other, 2, 3, 4 or 5, and o is 0, 1, 2 or 3, where compounds of the formula (I) are excepted in which o is 0, n is 2, m is 2 or 3, X2 and X3 are O, and R2 and R3 are C2H5, and all double bonds possess the transconfiguration, and/or a stereoisomeric form of the compound of the formula (I) andlor a mixture of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula (I).
C~-C6-Alkyl is a straight-chain or branched alkyl group having from 1 to 6 C
atoms, preferably having from 1 to 4 C atoms, such as methyl, ethyl, i-propyl, tert-butyl and hexyl.
C2-C6-Alkenyl is a straight-chain or branched alkenyl group having from 2 to 6 C
atoms which is unsaturated once, twice or three times, such as allyl, crotyl, propenyl, yenta-1,3-dienyl and pentenyl.
5 C2-C6-Alkynyl is a straight-chain or branched alkynyl group having from 2 to atoms which is unsaturated once or twice, such as propinyl, butinyl and pentinyl.
C5-C~4-Aryl is an aryl group having from 5 to 14 C atoms, such as phenyl or 1-naphthyl or 2-naphthyl, which is unsubstituted or substituted by halogen, C~-C6-alkyl, preferably C~-C4-alkyl, for example methyl, hydroxyl, -O-C~-C6-alkyl, preferably-O-C,=C4-alkyl, for example methoxy, or trifluoromethyl.
Aliphatic acyl groups -N[-C(=O)-(C1-C6-alkyl)]- preferably contain a preferably C~-C4-alkyl and are, for example, formyl, acetyl, propionyl, butyryl, hexanoyl, acryloyl, crotonoyl or propioloyl and can be further substituted by halogen, e.g.
chlorine, bromine or fluorine, by NH2, and/or by -NH(C~-C6-alkyl), preferably -NH(C,-C4-alkyl), for example methylamino or ethylamino. Aromatic acyl -N[-C(=O)-(C5-C~a-aryl)]- is, for example, N-benzoyl or N-naphthoyl and can be further substituted by halogen, such as chlorine, bromine or fluorine, by C~-C6-alkyl, preferably C,-C4-alkyl, for example methyl, by hydroxyl, by -NH(C~-C6-alkyl), preferably -NH(C~-C4-alkyl), for example methylamino or ethylamino, or -O-C~-Cs-alkyl, preferably -O-C~-C4-alkyl, for example methoxy.
Halogen is an element in the 7th main group of the periodic system, preferably chlorine, bromine or fluorine.
Unless otherwise indicated, the configuration of the double bonds in the polyene groups of the compounds of the formula (I) can be in the cis or in the traps configuration. The invention relates both to the pure compounds and to stereoisomeric mixtures, such as enantiomeric mixtures and diasteromeric mixtures, in any ratio. Stereoisomeric forms are understood, in particular, as being compounds having different spatial arrangements (configurations) of the atoms or atom groups in a molecule while the atoms are linked in the same way, for example cis-traps isomers at double bonds.
Preferably, at least one polyene group in the compound of the formula (I) contains at least one cis double bond.
R is preferably H.
R~, R2, R3 and R4 are preferably, independently of each other, H or C~-C6-alkyl.
X~, X2 and X3 are preferably, independently of each other, -O-.
m is preferably 3 or 4.
n is preferably 2.
o is preferably 0.
The general definitions of the radicals and the preferred definitions of the radicals are, R, R~, R2, R3 and R4, X~, X2 and X3, m, n and o in the compounds of the formula (I) can, independently of each other, be combined with each other at will.
The invention preferably relates to a compound of the formula (I), where R is H, R~ is H or C~-C6-alkyl, R2 is H or C,-C6-alkyl, R3 is H or C~-C6-alkyl, R4 is C~-C6-alkyl, and X~ and X2 are -O-, and the physiologically tolerated salts thereof.
The invention preferably relates to a compound of the formula (I) which is characterized by a compound of the formula (IV) ORS
O
m H H _~~
OR2 (IV) where m is 3 or 4, and the physiologically tolerated salts thereof.
C5-C~4-Aryl is an aryl group having from 5 to 14 C atoms, such as phenyl or 1-naphthyl or 2-naphthyl, which is unsubstituted or substituted by halogen, C~-C6-alkyl, preferably C~-C4-alkyl, for example methyl, hydroxyl, -O-C~-C6-alkyl, preferably-O-C,=C4-alkyl, for example methoxy, or trifluoromethyl.
Aliphatic acyl groups -N[-C(=O)-(C1-C6-alkyl)]- preferably contain a preferably C~-C4-alkyl and are, for example, formyl, acetyl, propionyl, butyryl, hexanoyl, acryloyl, crotonoyl or propioloyl and can be further substituted by halogen, e.g.
chlorine, bromine or fluorine, by NH2, and/or by -NH(C~-C6-alkyl), preferably -NH(C,-C4-alkyl), for example methylamino or ethylamino. Aromatic acyl -N[-C(=O)-(C5-C~a-aryl)]- is, for example, N-benzoyl or N-naphthoyl and can be further substituted by halogen, such as chlorine, bromine or fluorine, by C~-C6-alkyl, preferably C,-C4-alkyl, for example methyl, by hydroxyl, by -NH(C~-C6-alkyl), preferably -NH(C~-C4-alkyl), for example methylamino or ethylamino, or -O-C~-Cs-alkyl, preferably -O-C~-C4-alkyl, for example methoxy.
Halogen is an element in the 7th main group of the periodic system, preferably chlorine, bromine or fluorine.
Unless otherwise indicated, the configuration of the double bonds in the polyene groups of the compounds of the formula (I) can be in the cis or in the traps configuration. The invention relates both to the pure compounds and to stereoisomeric mixtures, such as enantiomeric mixtures and diasteromeric mixtures, in any ratio. Stereoisomeric forms are understood, in particular, as being compounds having different spatial arrangements (configurations) of the atoms or atom groups in a molecule while the atoms are linked in the same way, for example cis-traps isomers at double bonds.
Preferably, at least one polyene group in the compound of the formula (I) contains at least one cis double bond.
R is preferably H.
R~, R2, R3 and R4 are preferably, independently of each other, H or C~-C6-alkyl.
X~, X2 and X3 are preferably, independently of each other, -O-.
m is preferably 3 or 4.
n is preferably 2.
o is preferably 0.
The general definitions of the radicals and the preferred definitions of the radicals are, R, R~, R2, R3 and R4, X~, X2 and X3, m, n and o in the compounds of the formula (I) can, independently of each other, be combined with each other at will.
The invention preferably relates to a compound of the formula (I), where R is H, R~ is H or C~-C6-alkyl, R2 is H or C,-C6-alkyl, R3 is H or C~-C6-alkyl, R4 is C~-C6-alkyl, and X~ and X2 are -O-, and the physiologically tolerated salts thereof.
The invention preferably relates to a compound of the formula (I) which is characterized by a compound of the formula (IV) ORS
O
m H H _~~
OR2 (IV) where m is 3 or 4, and the physiologically tolerated salts thereof.
The invention particularly preferably relates to a compound of the formula (IV) in which m is 4 and in which the configuration of the,polyenes corresponds to the formula (V):
\ \ \ COORS
/ \
COOR2 (V) Special preference is given to a compound of the formula (V) in which R~ and R2 are H. This compound is termed serpentemycin A (empirical formula: C2oH~804; MW =
322.36) in that which follows.
The invention furthermore particularly preferably relates to a compound of the formula (IV) in which n is 2 and m is 3 and in which the configuration of the polyenes corresponds to the formula (VI):
\ \ \ COORS
/ - \ \ COOR
(VI) Special preference is given to a compound of the formula (VI) in which R~ and R2 are H. This compound is termed serpentemycin B (empirical formula: C~gH~gO4; MW =
296.33) in that which follows:
The invention furthermore particularly preferably relates to a compound of the formula (IV) in which n is 2 and m is 3 and in which the configuration of the polyenes corresponds to the formula (VII):
\ \ \ COORS
/ / / /
(VII) Special preference is given to a compound of the formula (VII) in which R~ and are H. This compound is termed serpentemycin C (empirical formula: CqgH~gO4;
MW
= 296.33) in that which follows.
The invention preferably relates to a compound of the formula (I) which is characterized by a compound of the formula (VIII) ORS
(VIII), and the physiologically tolerated salts thereof.
The invention furthermore particularly preferably relates to a compound of the formula (VIII) in which R2 and R3 are H and in which the configuration of the polyenes corresponds to the formula (IX):
\ \ \ COORS
/ //\ //~ //~
OH (IX) Special preference is given to a compound of the formula (IX) in which R~ is H. This compound is termed serpentemycin D (empirical formula: CZOH2204; MW = 236.40) in that which follows.
The invention furthermore particularly preferably relates to a compound of the formula (VIII) in which R2 and R3 are H and in which the configuration of the polyenes corresponds to the formula (X):
\ \ \ COORS
OH
/ \ \
OH (X) Special preference is given to a compound of the formula (X) in which R~ is H.
This compound is termed serpentemycin E in that which follows.
The invention furthermore relates to a compound having the empirical formula C2QH~gO4 (serpentemycin A), C~gH~gO4 (serpentemycin B and C) or C2oH220a (serpentemycin D) which can be obtained by fermenting Actinomycetales sp. DSM
14865, or one of its variants and/or mutants, in a culture medium until the compounds serpentemycin A, B, C and/or D accrue in the culture solution and by subsequently isolating the compound and, where appropriate, converting it into a pharmacologically tolerated salt.
The invention relates to a compound of the formula (I) which can be obtained by fermenting Actinomycetales sp. DSM 14865 or one of its variants and/or mutants, in a culture medium until one or more of the compounds serpentemycin A, B, C
andlor D accrues in the culture broth, then isolating the compound, where appropriate converting it into a chemical derivative and, where appropriate, converting it into a pharmacologically tolerated salt.
The serpentemycins A, B, C and D differ from substances known from the literature in the structural formulae which are specified. While structurally related polyenecarboxylic acid derivatives are known, they differ from the compounds according to the invention in their chemical structure, in their antimicrobial and/or biochemical activity and in other physical properties.
The invention furthermore relates to a process for preparing the compound of the formula (I), which comprises 1. culturing the microorganism Actinomycetales sp. DSM 14865, or one of its variants andlor mutants, in an aqueous nutrient medium until one or more of the compounds serpentemycin A, B, C andlor D accrues in the culture broth, 2. isolating and purifying serpentemycin A, B, C or D, 5 3. where appropriate, using a suitable reagent to convert serpentemycin A, B, C or D into a compound of the formula {l), 4. and, where appropriate, converting the compound of the formula (I) into a pharmacologically tolerated salt.
10 Examples of suitable reagents are alkylating agents, which can be used to convert carboxyl groups of the serpentemycins A, B, C and D into the corresponding ester.
Examples of alkylating agents are methyl iodide, diethyl sulfate, diazomethane and other alkyl derivatives as has been described, for example, by Jerry March im Buch Advanced Organic Chemistry, John Wiley & Sons, 4t" Edition, 1992. Double bonds can, for example, be isomerized photochemically or in the added presence of a free radical former. In order to carry out reactions selectively, it may be advantageous to introduce suitable protecting groups, in a manner known per se, prior to the reaction.
The protecting groups are eliminated after the reaction and the reaction product is then purified.
In addition to this, the invention relates to obvious chemical equivalents of the compounds of the formula (I). Obvious chemical equivalents are compounds which exhibit a slight chemical difference, that is have the same activity or are converted into the compounds according to the invention under mild conditions. Said equivalents include, for example, esters, amides, hydrazides, anhydrides, hydrogenation products, reduction products, complexes or adducts of or with the compounds according to the invention.
Methods known to the skilled person can be used to convert a compound of the formula (I) into the corresponding pharmacologically tolerated salts.
Pharmacologically tolerated salts of the compounds according to the invention are understood as meaning both inorganic and organic salts, as are described in Remingtons Pharmaceutical Sciences (17th Edition, page 1418 [1985]).
Particularly suitable salts are alkali metal, ammonium or alkaline earth metal salts, salts with physiologically tolerated amines and salts with inorganic or organic acids, such as HCI, HBr, H2S04, malefic acid and fumaric acid.
The strain Actinomycetales sp. DSM 14865 forms the compounds Serpentemycins A, B, C and D on nutrient solutions which contain glucose, starch, yeast extract or glycerol.
In addition to this, Actinomycetales sp. DSM 14865 forms numerous byproducts possessing slightly varying polyene chains.
An isolate of Actinomycetales sp. was deposited in the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM) [German collection of microorganisms and cell cultures], Mascheroder Weg 1 B, 38124 Brunswick, Germany, on 18.03.2002 under the number DSM 14865, in accordance with the rules of the Budapest treaty.
On oat-flake medium, Actinomycetales sp. DSM 14865 possesses a brown-beige substrate mycelium and a sparse aerial mycelium. In culture, it does not form any of the fruiting bodies which are characteristic of the Actinomycetes.
Said process comprises culturing Actinomycetales sp. DSM 14865, its mutants and/or variants, under aerobic conditions in culture media which contain a carbon and nitrogen source, inorganic salts and, where appropriate, trace elements.
Instead of the strain Actinomycetales sp. DSM 14865, it is also possible to use its mutants and variants provided they synthesize the compounds according to the invention.
A mutant is a microorganism in which one or more genes in the genome have been modified, with the gene or genes which is/are responsible for the ability of the organism to produce the inventive compound being retained such that it/they is/are functional and heritable.
Such mutants can be produced, in a manner known per se, by physical means, for example irradiation, such as using ultraviolet rays or X-rays, or by using chemical mutagens, such as ethyl methanesulfonate (EMS); 2-hydroxy-4-methoxybenzophenone (MOB) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or as described by Brock et al. in "Biology of Microorganisms", Prentice Hall, pages 238-247 (1984).
A variant is a phenotype of the microorganism. Microorganisms have the ability to adapt to their environment and therefore exhibit highly developed physiological flexibility. All the cells of the microorganism are involved in the phenotypic adaptation, with the nature of the change not being conditioned genetically and being reversible under altered conditions (H. Stolp, Microbial ecology: organismus, habitats, activities. Cambridge University Press, Cambridge, GB, page 180, 1988).
The screening for mutants and variants which produce the antibiotic according to the invention can be effected by determining the biological activity of the active compound which has accrued in the culture broth, for example by determining its antibacterial effect, or else by detecting compounds in the fermentation broth which are known to possess antibacterial activity, for example using HPLC or t_C-MS
methods.
Suitable and preferred carbon sources for the fermentation are assimilable carbohydrates and sugar alcohols, such as glucose, lactose, sucrose or D-mannitol, and also carbohydrate-containing natural products, such as malt extract or yeast extract. Suitable nitrogen sources are: amino acids, peptides and proteins and their breakdown products, such as casein, peptones or tryptones, and also meat extracts, yeast extracts, ground seeds, for example corn, wheat, beans, soybean or the cotton plant, distillation residues from alcohol production, meat meals or yeast extracts, and also ammonium salts and nitrates, in particular peptides which have been obtained synthetically or biosynthetically, as well. Examples of inorganic salts and trace elements which the nutrient solution can contain are chlorides, carbonates, sulfates or phosphates of the alkali metals or alkaline earth metals, iron, zinc, cobalt and manganese.
The formation of the compounds according to the invention proceeds particularly well, for example, in a nutrient solution which contains about 0.05 to 5%, preferably from 0.5 to 2%, of starch, from 0.05 to 3%, preferably from 0.1 to 1 %, of yeast extract, from 0.05 to 5%, preferably from 0.1 to 2%, of glucose, from 0.2 to 5%, preferably from 0.5% to 2%, of glycerol, from 0.05 to 3%, preferably from 0.1 to 1 %, of Consteep, from 0.05 to 3%, preferably from 0.1 to 1 %. of peptone, from 0.05 to 3%, preferably from 0.05 to 0.5%, of NaCI and from 0.05 to 3%, preferably from 0.1 to 1 %, of CaC03. The percentage values are in each case based on the weight of the entire nutrient solution.
In the nutrient solution, Actinomycetales sp. DSM 14865 forms a mixture of the serpentemycins A, B, C and D and byproducts. The quantitative proportion of one or more of the compounds according to the invention can vary in dependence on the composition of the nutrient solution. In addition, the synthesis of individual polyenecarboxylic acids can be controlled by the composition of the medium such that the microorganism does not produce an antibiotic at all or produces it in a quantity which is below the detection limit.
The microorganism is cultured aerobically, that is, for example, submerged while being shaken or stirred in shaking flasks or fermenters, or on solid medium, where appropriate while passing in air or oxygen. The culture can be carried out in a temperature range of from about 15 to 35°C, preferably at from about 25 to 32°C, particularly at from 27 to 30°C. The pH range should be between 4 and 10, preferably between 6.5 and 7.5. The microorganism is generally cultured under these conditions over a period of from 48 to 720 hours, preferably from 72 to hours. The culture is advantageously carried out in several steps; i.e. one or more preliminary cultures are initially prepared in a liquid nutrient medium, with these preliminary cultures then being inoculated, for example in a volume ratio from 1:10 to1:100, into the actual production medium, i.e. the main culture. The preliminary culture is obtained, for example, by inoculating the mycelium into a nutrient solution and allowing it to grow for from about 20 to 120 hours, preferably from 48 to hours. The mycelium can be obtained, for example, by allowing the strain to grow for from about 1 to 40 days, preferably from 14 to 21 days, on a solid or liquid culture medium, for example yeast-malt-glucose agar, oat flake agar or starch agar.
The course of the fermentation, and the formation of the antibiotics according to the invention, can be monitored using methods which are known to the skilled person, for example by means of testing the biological activity in bioassays or by means of chromatographic methods such as thin layer chromatography (TLC) or high performance liquid chromatography (HPLC).
Actinomycetales sp. DSM 14865 is able to form the compound according to the invention by means of submerged fermentation. The compounds according to the invention can be present both in the mycelium and in the culture filtrate; the major quantity is usually present in the culture filtrate. It is therefore expedient to separate off 'the fermentation solution by filtration or centrifugation. The filtrate is extracted using an adsorption resin as a solid phase. The mycelium, and also the surface culture, is expediently extracted with a suitable solvent, for example methanol or 2-propanol.
While the extraction can be carried out in a broad pH range, it is expedient to carry it out in a neutral or weakly acidic medium, preferably between pH 3 and pH 7.
The extracts can, for example, be concentrated in vacuo and dried.
One method of isolating the antibiotic according to the invention is solution partition, carried out in a manner known per se.
Another purification method is that of chromatography on adsorption resins, such as Diaion~ HP-20 (Mitsubishi Casei Corp., Tokyo, Japan), Amberlite~ XAD 7 (Rohm and Haas, USA), Amberchrom~ CG (Toso Haas, Philadelphia, USA) or the like. In addition to this, a large number of reverse phase supports, e.g. RP$ and RP~8, as have become well-known, for example, within the context of high pressure liquid chromatography (HPLC), are also suitable.
Another option for purifying the antibiotic according to the invention consists in using what are termed normal phase chromatography supports, such as silica gel or AI203, or others, in a manner known per se.
An alternative isolation method is that of using molecular sieves such as Fractogel~
TSK HW-40 (Merck KGaA, Darmstadt, Germany), Sephadex~ G-25 (Amersham Biosciences, Uppsala, Sweden), and others, in a manner known per se. In addition to this, it is also possible to obtain the novel polyenecarboxylic acids by crystallizing 5 them from enriched material. Organic solvents and their mixtures, either water-free or with water having been added, or various salts, are, for example, suitable for this purpose. An additional method for isolating and purifying the antibiotics according to the invention is that of using anion exchangers, preferably in a pH range from 4 to 10. The use of buffer solutions to which quantities of organic solvents have been 10 added is particularly suitable for this purpose.
It has been found, surprisingly, that the compounds according to the invention are powerful inhibitors of glycosyltransferase. Selective and powerful inhibitors of glycosyltransferase, such as the antibiotics of the moenomycin group, always exhibit 15 antibacterial effects, with a low inhibitory concentration (ICSO) being associated with powerful bacteriostatic activity. The compounds according to the invention possess bacteriostatic activity because they inhibit the growth and replication of the bacteria;
they are therefore suitable for treating diseases which are caused by bacterial infections. Table 1 summarizes, by way of example, the inhibitory concentrations (ICSO) of some compounds according to the invention. A special test system was used for finding glycosyltransferase inhibitors which are specific and very effective.
This system makes use of a glycosyltransferase/3H-moenomycin complex, which is brought into contact with substances which are to be tested. The determination of the GT-inhibitory effect is based on the displacement of radioactively labeled moenomycin A from the glycosyltransferase (PBP 1 b)/3H-moenomycin complex, with it being possible to separate off the released 3H-moenomycin, as a soluble compound, from the fixed complex and to measure the remaining radioactivity using a Geiger counter.
Table 1: The inhibitory constants (ICSO values) of the antibiotics according to the invention in the glycosyltransferase assay.
serpentemycin A: 0.2,uM
serpentemycin B: 0.1 ,uM
serpentemycin C: 21 ,uM
serpentemycin D: 19 NM
The inhibitory constant of serpentene (Ritzau et al., Liebigs Ann. Chem. 1993, 435) was determined for comparison: the ICSO value was 18 NM.
The present invention consequently also relates to the use of a compound of the formula (I) as a pharmaceutical, preferably for the treatment and/or prophylaxis of bacterial infections.
The invention furthermore also relates to the use of a compound of the formula (I) for prdducing a pharmaceutical, preferably for the treatment and/or prophylaxis of bacterial infections.
In addition, the present invention relates to a pharmaceutical having a content of one or more of the compounds according to the invention.
Said pharmaceutical is produced by mixing at least one compound of the formula (I) with one or more physiologically suitable auxiliary substances and bringing this mixture into a suitable form for administration.
In general, the pharmaceuticals according to the invention are administered orally, locally or parenterally; however, it is also possible in principle to use them rectally.
Examples of suitable solid or liquid galenic preparation forms are granules, powders, tablets, sugar-coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, aerosols, drops or injectable solutions in ampoule form, and also preparations giving a protracted release of active compound, in the production of which physiologically suitable adjuvants such as disintegrants, binding agents, coating agents, swelling agents, glidants, lubricants, flavorings, sweeteners and solubilizers are customarily used. Examples of frequently employed auxiliary substances which may be mentioned are magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal or vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols.
Where appropriate, the dosage units for oral administration can be microencapsulated in order to delay release or extend it over a relatively long period of time, for example by means of coating or embedding the active compound, in particle form, in suitable polymers, waxes or the like.
From 0.1 to 1000, preferably from 0.2 to 100, mg/kg of bodyweight is/are administered as an expedient dose. These amounts are expediently administered in dosage units which at least contain the effective daily quantity of the compounds according to the invention, e.g. 30 - 3000, preferably 50 - 1000, mg.
The following examples are intended to clarify the invention without in any way limiting its scope.
Example 1 Preparing a glycerol culture of Actinomycetales sp. DSM 14865 30 ml of nutrient solution (malt extract 1.0%, yeast extract 0.4%, glucose, 0.4%, pH
7.0 in water) were inoculated, in a sterile 100 ml Erlenmeyer flask, with the strain Actinomycetales sp, DSM 14865 and incubated for 7 days at 28°C and 180 rpm on a rotating shaker. In each case 1.5 ml of this culture were then diluted with 2.5 ml of 80% glycerol and stored at -135°C.
Example 2 Preparing a preliminary culture of Acfinomycetales sp, DSM 14865 in an Erlenmeyer flask In each case 100 ml of nutrient solution (glucose 1.5%, soybean meal, 1.5%, Cornsteep, 0.5%, CaC03, 0.2% and NaCI, 0.5%, pH 7.0) were inoculated, in a sterile 300 ml Erlenmeyer flask, with the strain Actinomycefales sp, DSM 14865 and incubated for 4 days, at 28°C and 180 rpm, on a rotating shaker. This preliminary culture was then used for inoculating the main cultures.
Example 3 Kinetics of the formation of the aromatic polyenecarboxylic acids in the shaken cultures of Actinomycetales sp, DSM 14865.
Cultures of Actinomycetales sp, DSM 14865 were started, as described in Example 2, in 20 Erlenmeyer flasks and the cultures were incubated on a shaker for a period of up to 2 weeks. Shaking flasks were removed after 48, 72, 96, 102, 120, 240 and 312 hours. After the mycelium has been separated off, and after the solid-phase extraction of the aromatic polyenecarboxylic acids from the culture filtrate with MCI-Gel~ (Mitsubishi Chemical Industries) and elution with methanol, the extracts were analyzed by HPLC as described in Example 8. The changes in the HPLC surface area units which occurred in dependence on the incubation time are listed in the following table. The surface area units are proportional to the concentrations of the products.
Table 2: Change in the concentrations of the products occurring over the time during which Actinomycetales sp, DSM 14865 is incubated in shaken cultures. The product concentrations are proportional to the given HPLC surface area units.
Incubation timeSerpentemycins Serpentene A B C D
48h 174 132 151 54 574 72h 313 284 372 94 1948 96h 727 443 891 242 2530 102h 698 424 814 358 1878 120h 816 470 1098 430 1212 240h 917 1216 1786 1437 47 312h .882 1411 1877 734 35 Whereas serpentene has already reached its maximum formation after 96 hours, the highest concentrations of serpentemycins A and D are only to be observed after days. The products serpentemycin B and C are even present at their highest concentrations after 13 days.
Example 4 Fermenting Actinomycetales sp. DSM 14865 in order to produce serpentemycin A.
In order to produce the antibiotics according to the invention, the strain Actinomycetales sp. DSM 14865 was fermented in 30 L fermenters. The nutrient solution employed was the following: 10 g of soluble starch/L; 10 g of glucoselL; 10 g of glycerol/L; 2.5 g of Cornsteep, Iiquid/L; 5 g of peptonelL; 2 g of yeast extract/L; 1 g of NaCI/L; 3 g of CaC03/L in water; pH prior to sterilization, 7.2. Good growth and rapid productivity were observed in this nutrient medium. The fermentation was carried out under the following conditions:
Sterilization : in-situ, 20 min at T=121 °C
Inoculum: 6%
Stirring speed: 180 rpm T: 28°C
Air: 0.45 m3/h p02 : not regulated, pH, during fermentation: about 6.2; not regulated.
Under these conditions, good growth was seen within the first 70h; at this time, the C
source in the medium had been consumed and growth was stagnating. After 80 h, it was possible to detect the first formation of the product of the compounds according to the invention. The fermenters were harvested after 98 h.
Example 5: Isolating serpentemycin A from the Actinomycetales sp. DSM 14865 culture solution.
After the fermentation of Actinomycetales sp. DSM 14865 had come to an end, 27.5 liters of culture broth from the fermenter, obtained as described in Example 4, were filtered in the added presence of approx. 2% of filtration accelerator (Celite~) and the cell mass (1890 g) was extracted with 6 liters of methanol. The active compound-containing, methanolic solution was freed from the mycelium by filtration and concentrated in vacuo. The concentrate was combined with 25 L of the culture filtrate, after which the pH was adjusted to 7 and the whole was loaded onto a previously prepared 3 titer ~MCI GEL, CHP20P column. The column was eluted with a gradient of 50 mM NH4 acetate buffer in water after 2-propanol. The column flow-through (7 liters per hour) was collected in fractions (in each case 2 liters) and the antibiotic-containing fractions 4 to 11 and 16 to 19 were in each case combined and concentrated in vacuo. The fractions were analyzed using HPLC. Fractions 4 to contain the more polar serpentemycins while fractions 16 to 19 contain the serpentenes. The serpentene-containing fractions were purified twice 5 chromatographically on LiChrospherfl 100 RP-18e, 250-25, HPLC columns firstly using 50 mM NH4 acetate buffer/acetonitrile mixtures and then using 0.05%
trifluoroacetic acid/acetonitrile gradients. Freeze-drying the fractions containing pure antibiotic resulted in 19 mg of pure serpentene.
After they had been concentrated in vacuo, the more polar compounds in fractions 4 10 to 11 were purified once again by column chromatography on 160 mL MCI GEL~
CHP20P (column dimensions: 26 mm x 300 mm) using a gradient method. The gradient ranged from 10% to 60% acetonitrile in 50 mM NH4 acetate buffer over hours. The column flow-through was 25 mL per minute while the fraction size was 50 mL. The compounds serpentemycin B and D were located in fractions 15 to 35 15 while fractions 36 to 39 contained serpentemycin C and fractions 40 to 43 contained serpentemycin A. The latter were freed from organic solvent in vacuo, after which 0.01 % ascorbic acid was added and purification by isocratic chromatography was carried out on a LiChrospherfl 100 RP-18e, 250-25, HPLC column using 45%
acetonitrile in 0.05% trifluoroacetic acid. The fractions which contained pure 20 serpentemycin A (fractions 31 to 35) were freeze-dried in the dark and the serpentemycin A was stored air-tight in an argon atmospheric (37 mg). ESI-mass spectrometry: 321.4 (M - H), ESI'' mass spectrometry: 305.3 (MH - H20).
UV maxima: 315 and 355 nm. The NMR data are listed in Table 3, with the C
atoms being numbered as follows:
\ \ \ COORS
/ \
COOR2 (V) Special preference is given to a compound of the formula (V) in which R~ and R2 are H. This compound is termed serpentemycin A (empirical formula: C2oH~804; MW =
322.36) in that which follows.
The invention furthermore particularly preferably relates to a compound of the formula (IV) in which n is 2 and m is 3 and in which the configuration of the polyenes corresponds to the formula (VI):
\ \ \ COORS
/ - \ \ COOR
(VI) Special preference is given to a compound of the formula (VI) in which R~ and R2 are H. This compound is termed serpentemycin B (empirical formula: C~gH~gO4; MW =
296.33) in that which follows:
The invention furthermore particularly preferably relates to a compound of the formula (IV) in which n is 2 and m is 3 and in which the configuration of the polyenes corresponds to the formula (VII):
\ \ \ COORS
/ / / /
(VII) Special preference is given to a compound of the formula (VII) in which R~ and are H. This compound is termed serpentemycin C (empirical formula: CqgH~gO4;
MW
= 296.33) in that which follows.
The invention preferably relates to a compound of the formula (I) which is characterized by a compound of the formula (VIII) ORS
(VIII), and the physiologically tolerated salts thereof.
The invention furthermore particularly preferably relates to a compound of the formula (VIII) in which R2 and R3 are H and in which the configuration of the polyenes corresponds to the formula (IX):
\ \ \ COORS
/ //\ //~ //~
OH (IX) Special preference is given to a compound of the formula (IX) in which R~ is H. This compound is termed serpentemycin D (empirical formula: CZOH2204; MW = 236.40) in that which follows.
The invention furthermore particularly preferably relates to a compound of the formula (VIII) in which R2 and R3 are H and in which the configuration of the polyenes corresponds to the formula (X):
\ \ \ COORS
OH
/ \ \
OH (X) Special preference is given to a compound of the formula (X) in which R~ is H.
This compound is termed serpentemycin E in that which follows.
The invention furthermore relates to a compound having the empirical formula C2QH~gO4 (serpentemycin A), C~gH~gO4 (serpentemycin B and C) or C2oH220a (serpentemycin D) which can be obtained by fermenting Actinomycetales sp. DSM
14865, or one of its variants and/or mutants, in a culture medium until the compounds serpentemycin A, B, C and/or D accrue in the culture solution and by subsequently isolating the compound and, where appropriate, converting it into a pharmacologically tolerated salt.
The invention relates to a compound of the formula (I) which can be obtained by fermenting Actinomycetales sp. DSM 14865 or one of its variants and/or mutants, in a culture medium until one or more of the compounds serpentemycin A, B, C
andlor D accrues in the culture broth, then isolating the compound, where appropriate converting it into a chemical derivative and, where appropriate, converting it into a pharmacologically tolerated salt.
The serpentemycins A, B, C and D differ from substances known from the literature in the structural formulae which are specified. While structurally related polyenecarboxylic acid derivatives are known, they differ from the compounds according to the invention in their chemical structure, in their antimicrobial and/or biochemical activity and in other physical properties.
The invention furthermore relates to a process for preparing the compound of the formula (I), which comprises 1. culturing the microorganism Actinomycetales sp. DSM 14865, or one of its variants andlor mutants, in an aqueous nutrient medium until one or more of the compounds serpentemycin A, B, C andlor D accrues in the culture broth, 2. isolating and purifying serpentemycin A, B, C or D, 5 3. where appropriate, using a suitable reagent to convert serpentemycin A, B, C or D into a compound of the formula {l), 4. and, where appropriate, converting the compound of the formula (I) into a pharmacologically tolerated salt.
10 Examples of suitable reagents are alkylating agents, which can be used to convert carboxyl groups of the serpentemycins A, B, C and D into the corresponding ester.
Examples of alkylating agents are methyl iodide, diethyl sulfate, diazomethane and other alkyl derivatives as has been described, for example, by Jerry March im Buch Advanced Organic Chemistry, John Wiley & Sons, 4t" Edition, 1992. Double bonds can, for example, be isomerized photochemically or in the added presence of a free radical former. In order to carry out reactions selectively, it may be advantageous to introduce suitable protecting groups, in a manner known per se, prior to the reaction.
The protecting groups are eliminated after the reaction and the reaction product is then purified.
In addition to this, the invention relates to obvious chemical equivalents of the compounds of the formula (I). Obvious chemical equivalents are compounds which exhibit a slight chemical difference, that is have the same activity or are converted into the compounds according to the invention under mild conditions. Said equivalents include, for example, esters, amides, hydrazides, anhydrides, hydrogenation products, reduction products, complexes or adducts of or with the compounds according to the invention.
Methods known to the skilled person can be used to convert a compound of the formula (I) into the corresponding pharmacologically tolerated salts.
Pharmacologically tolerated salts of the compounds according to the invention are understood as meaning both inorganic and organic salts, as are described in Remingtons Pharmaceutical Sciences (17th Edition, page 1418 [1985]).
Particularly suitable salts are alkali metal, ammonium or alkaline earth metal salts, salts with physiologically tolerated amines and salts with inorganic or organic acids, such as HCI, HBr, H2S04, malefic acid and fumaric acid.
The strain Actinomycetales sp. DSM 14865 forms the compounds Serpentemycins A, B, C and D on nutrient solutions which contain glucose, starch, yeast extract or glycerol.
In addition to this, Actinomycetales sp. DSM 14865 forms numerous byproducts possessing slightly varying polyene chains.
An isolate of Actinomycetales sp. was deposited in the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM) [German collection of microorganisms and cell cultures], Mascheroder Weg 1 B, 38124 Brunswick, Germany, on 18.03.2002 under the number DSM 14865, in accordance with the rules of the Budapest treaty.
On oat-flake medium, Actinomycetales sp. DSM 14865 possesses a brown-beige substrate mycelium and a sparse aerial mycelium. In culture, it does not form any of the fruiting bodies which are characteristic of the Actinomycetes.
Said process comprises culturing Actinomycetales sp. DSM 14865, its mutants and/or variants, under aerobic conditions in culture media which contain a carbon and nitrogen source, inorganic salts and, where appropriate, trace elements.
Instead of the strain Actinomycetales sp. DSM 14865, it is also possible to use its mutants and variants provided they synthesize the compounds according to the invention.
A mutant is a microorganism in which one or more genes in the genome have been modified, with the gene or genes which is/are responsible for the ability of the organism to produce the inventive compound being retained such that it/they is/are functional and heritable.
Such mutants can be produced, in a manner known per se, by physical means, for example irradiation, such as using ultraviolet rays or X-rays, or by using chemical mutagens, such as ethyl methanesulfonate (EMS); 2-hydroxy-4-methoxybenzophenone (MOB) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or as described by Brock et al. in "Biology of Microorganisms", Prentice Hall, pages 238-247 (1984).
A variant is a phenotype of the microorganism. Microorganisms have the ability to adapt to their environment and therefore exhibit highly developed physiological flexibility. All the cells of the microorganism are involved in the phenotypic adaptation, with the nature of the change not being conditioned genetically and being reversible under altered conditions (H. Stolp, Microbial ecology: organismus, habitats, activities. Cambridge University Press, Cambridge, GB, page 180, 1988).
The screening for mutants and variants which produce the antibiotic according to the invention can be effected by determining the biological activity of the active compound which has accrued in the culture broth, for example by determining its antibacterial effect, or else by detecting compounds in the fermentation broth which are known to possess antibacterial activity, for example using HPLC or t_C-MS
methods.
Suitable and preferred carbon sources for the fermentation are assimilable carbohydrates and sugar alcohols, such as glucose, lactose, sucrose or D-mannitol, and also carbohydrate-containing natural products, such as malt extract or yeast extract. Suitable nitrogen sources are: amino acids, peptides and proteins and their breakdown products, such as casein, peptones or tryptones, and also meat extracts, yeast extracts, ground seeds, for example corn, wheat, beans, soybean or the cotton plant, distillation residues from alcohol production, meat meals or yeast extracts, and also ammonium salts and nitrates, in particular peptides which have been obtained synthetically or biosynthetically, as well. Examples of inorganic salts and trace elements which the nutrient solution can contain are chlorides, carbonates, sulfates or phosphates of the alkali metals or alkaline earth metals, iron, zinc, cobalt and manganese.
The formation of the compounds according to the invention proceeds particularly well, for example, in a nutrient solution which contains about 0.05 to 5%, preferably from 0.5 to 2%, of starch, from 0.05 to 3%, preferably from 0.1 to 1 %, of yeast extract, from 0.05 to 5%, preferably from 0.1 to 2%, of glucose, from 0.2 to 5%, preferably from 0.5% to 2%, of glycerol, from 0.05 to 3%, preferably from 0.1 to 1 %, of Consteep, from 0.05 to 3%, preferably from 0.1 to 1 %. of peptone, from 0.05 to 3%, preferably from 0.05 to 0.5%, of NaCI and from 0.05 to 3%, preferably from 0.1 to 1 %, of CaC03. The percentage values are in each case based on the weight of the entire nutrient solution.
In the nutrient solution, Actinomycetales sp. DSM 14865 forms a mixture of the serpentemycins A, B, C and D and byproducts. The quantitative proportion of one or more of the compounds according to the invention can vary in dependence on the composition of the nutrient solution. In addition, the synthesis of individual polyenecarboxylic acids can be controlled by the composition of the medium such that the microorganism does not produce an antibiotic at all or produces it in a quantity which is below the detection limit.
The microorganism is cultured aerobically, that is, for example, submerged while being shaken or stirred in shaking flasks or fermenters, or on solid medium, where appropriate while passing in air or oxygen. The culture can be carried out in a temperature range of from about 15 to 35°C, preferably at from about 25 to 32°C, particularly at from 27 to 30°C. The pH range should be between 4 and 10, preferably between 6.5 and 7.5. The microorganism is generally cultured under these conditions over a period of from 48 to 720 hours, preferably from 72 to hours. The culture is advantageously carried out in several steps; i.e. one or more preliminary cultures are initially prepared in a liquid nutrient medium, with these preliminary cultures then being inoculated, for example in a volume ratio from 1:10 to1:100, into the actual production medium, i.e. the main culture. The preliminary culture is obtained, for example, by inoculating the mycelium into a nutrient solution and allowing it to grow for from about 20 to 120 hours, preferably from 48 to hours. The mycelium can be obtained, for example, by allowing the strain to grow for from about 1 to 40 days, preferably from 14 to 21 days, on a solid or liquid culture medium, for example yeast-malt-glucose agar, oat flake agar or starch agar.
The course of the fermentation, and the formation of the antibiotics according to the invention, can be monitored using methods which are known to the skilled person, for example by means of testing the biological activity in bioassays or by means of chromatographic methods such as thin layer chromatography (TLC) or high performance liquid chromatography (HPLC).
Actinomycetales sp. DSM 14865 is able to form the compound according to the invention by means of submerged fermentation. The compounds according to the invention can be present both in the mycelium and in the culture filtrate; the major quantity is usually present in the culture filtrate. It is therefore expedient to separate off 'the fermentation solution by filtration or centrifugation. The filtrate is extracted using an adsorption resin as a solid phase. The mycelium, and also the surface culture, is expediently extracted with a suitable solvent, for example methanol or 2-propanol.
While the extraction can be carried out in a broad pH range, it is expedient to carry it out in a neutral or weakly acidic medium, preferably between pH 3 and pH 7.
The extracts can, for example, be concentrated in vacuo and dried.
One method of isolating the antibiotic according to the invention is solution partition, carried out in a manner known per se.
Another purification method is that of chromatography on adsorption resins, such as Diaion~ HP-20 (Mitsubishi Casei Corp., Tokyo, Japan), Amberlite~ XAD 7 (Rohm and Haas, USA), Amberchrom~ CG (Toso Haas, Philadelphia, USA) or the like. In addition to this, a large number of reverse phase supports, e.g. RP$ and RP~8, as have become well-known, for example, within the context of high pressure liquid chromatography (HPLC), are also suitable.
Another option for purifying the antibiotic according to the invention consists in using what are termed normal phase chromatography supports, such as silica gel or AI203, or others, in a manner known per se.
An alternative isolation method is that of using molecular sieves such as Fractogel~
TSK HW-40 (Merck KGaA, Darmstadt, Germany), Sephadex~ G-25 (Amersham Biosciences, Uppsala, Sweden), and others, in a manner known per se. In addition to this, it is also possible to obtain the novel polyenecarboxylic acids by crystallizing 5 them from enriched material. Organic solvents and their mixtures, either water-free or with water having been added, or various salts, are, for example, suitable for this purpose. An additional method for isolating and purifying the antibiotics according to the invention is that of using anion exchangers, preferably in a pH range from 4 to 10. The use of buffer solutions to which quantities of organic solvents have been 10 added is particularly suitable for this purpose.
It has been found, surprisingly, that the compounds according to the invention are powerful inhibitors of glycosyltransferase. Selective and powerful inhibitors of glycosyltransferase, such as the antibiotics of the moenomycin group, always exhibit 15 antibacterial effects, with a low inhibitory concentration (ICSO) being associated with powerful bacteriostatic activity. The compounds according to the invention possess bacteriostatic activity because they inhibit the growth and replication of the bacteria;
they are therefore suitable for treating diseases which are caused by bacterial infections. Table 1 summarizes, by way of example, the inhibitory concentrations (ICSO) of some compounds according to the invention. A special test system was used for finding glycosyltransferase inhibitors which are specific and very effective.
This system makes use of a glycosyltransferase/3H-moenomycin complex, which is brought into contact with substances which are to be tested. The determination of the GT-inhibitory effect is based on the displacement of radioactively labeled moenomycin A from the glycosyltransferase (PBP 1 b)/3H-moenomycin complex, with it being possible to separate off the released 3H-moenomycin, as a soluble compound, from the fixed complex and to measure the remaining radioactivity using a Geiger counter.
Table 1: The inhibitory constants (ICSO values) of the antibiotics according to the invention in the glycosyltransferase assay.
serpentemycin A: 0.2,uM
serpentemycin B: 0.1 ,uM
serpentemycin C: 21 ,uM
serpentemycin D: 19 NM
The inhibitory constant of serpentene (Ritzau et al., Liebigs Ann. Chem. 1993, 435) was determined for comparison: the ICSO value was 18 NM.
The present invention consequently also relates to the use of a compound of the formula (I) as a pharmaceutical, preferably for the treatment and/or prophylaxis of bacterial infections.
The invention furthermore also relates to the use of a compound of the formula (I) for prdducing a pharmaceutical, preferably for the treatment and/or prophylaxis of bacterial infections.
In addition, the present invention relates to a pharmaceutical having a content of one or more of the compounds according to the invention.
Said pharmaceutical is produced by mixing at least one compound of the formula (I) with one or more physiologically suitable auxiliary substances and bringing this mixture into a suitable form for administration.
In general, the pharmaceuticals according to the invention are administered orally, locally or parenterally; however, it is also possible in principle to use them rectally.
Examples of suitable solid or liquid galenic preparation forms are granules, powders, tablets, sugar-coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, aerosols, drops or injectable solutions in ampoule form, and also preparations giving a protracted release of active compound, in the production of which physiologically suitable adjuvants such as disintegrants, binding agents, coating agents, swelling agents, glidants, lubricants, flavorings, sweeteners and solubilizers are customarily used. Examples of frequently employed auxiliary substances which may be mentioned are magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal or vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols.
Where appropriate, the dosage units for oral administration can be microencapsulated in order to delay release or extend it over a relatively long period of time, for example by means of coating or embedding the active compound, in particle form, in suitable polymers, waxes or the like.
From 0.1 to 1000, preferably from 0.2 to 100, mg/kg of bodyweight is/are administered as an expedient dose. These amounts are expediently administered in dosage units which at least contain the effective daily quantity of the compounds according to the invention, e.g. 30 - 3000, preferably 50 - 1000, mg.
The following examples are intended to clarify the invention without in any way limiting its scope.
Example 1 Preparing a glycerol culture of Actinomycetales sp. DSM 14865 30 ml of nutrient solution (malt extract 1.0%, yeast extract 0.4%, glucose, 0.4%, pH
7.0 in water) were inoculated, in a sterile 100 ml Erlenmeyer flask, with the strain Actinomycetales sp, DSM 14865 and incubated for 7 days at 28°C and 180 rpm on a rotating shaker. In each case 1.5 ml of this culture were then diluted with 2.5 ml of 80% glycerol and stored at -135°C.
Example 2 Preparing a preliminary culture of Acfinomycetales sp, DSM 14865 in an Erlenmeyer flask In each case 100 ml of nutrient solution (glucose 1.5%, soybean meal, 1.5%, Cornsteep, 0.5%, CaC03, 0.2% and NaCI, 0.5%, pH 7.0) were inoculated, in a sterile 300 ml Erlenmeyer flask, with the strain Actinomycefales sp, DSM 14865 and incubated for 4 days, at 28°C and 180 rpm, on a rotating shaker. This preliminary culture was then used for inoculating the main cultures.
Example 3 Kinetics of the formation of the aromatic polyenecarboxylic acids in the shaken cultures of Actinomycetales sp, DSM 14865.
Cultures of Actinomycetales sp, DSM 14865 were started, as described in Example 2, in 20 Erlenmeyer flasks and the cultures were incubated on a shaker for a period of up to 2 weeks. Shaking flasks were removed after 48, 72, 96, 102, 120, 240 and 312 hours. After the mycelium has been separated off, and after the solid-phase extraction of the aromatic polyenecarboxylic acids from the culture filtrate with MCI-Gel~ (Mitsubishi Chemical Industries) and elution with methanol, the extracts were analyzed by HPLC as described in Example 8. The changes in the HPLC surface area units which occurred in dependence on the incubation time are listed in the following table. The surface area units are proportional to the concentrations of the products.
Table 2: Change in the concentrations of the products occurring over the time during which Actinomycetales sp, DSM 14865 is incubated in shaken cultures. The product concentrations are proportional to the given HPLC surface area units.
Incubation timeSerpentemycins Serpentene A B C D
48h 174 132 151 54 574 72h 313 284 372 94 1948 96h 727 443 891 242 2530 102h 698 424 814 358 1878 120h 816 470 1098 430 1212 240h 917 1216 1786 1437 47 312h .882 1411 1877 734 35 Whereas serpentene has already reached its maximum formation after 96 hours, the highest concentrations of serpentemycins A and D are only to be observed after days. The products serpentemycin B and C are even present at their highest concentrations after 13 days.
Example 4 Fermenting Actinomycetales sp. DSM 14865 in order to produce serpentemycin A.
In order to produce the antibiotics according to the invention, the strain Actinomycetales sp. DSM 14865 was fermented in 30 L fermenters. The nutrient solution employed was the following: 10 g of soluble starch/L; 10 g of glucoselL; 10 g of glycerol/L; 2.5 g of Cornsteep, Iiquid/L; 5 g of peptonelL; 2 g of yeast extract/L; 1 g of NaCI/L; 3 g of CaC03/L in water; pH prior to sterilization, 7.2. Good growth and rapid productivity were observed in this nutrient medium. The fermentation was carried out under the following conditions:
Sterilization : in-situ, 20 min at T=121 °C
Inoculum: 6%
Stirring speed: 180 rpm T: 28°C
Air: 0.45 m3/h p02 : not regulated, pH, during fermentation: about 6.2; not regulated.
Under these conditions, good growth was seen within the first 70h; at this time, the C
source in the medium had been consumed and growth was stagnating. After 80 h, it was possible to detect the first formation of the product of the compounds according to the invention. The fermenters were harvested after 98 h.
Example 5: Isolating serpentemycin A from the Actinomycetales sp. DSM 14865 culture solution.
After the fermentation of Actinomycetales sp. DSM 14865 had come to an end, 27.5 liters of culture broth from the fermenter, obtained as described in Example 4, were filtered in the added presence of approx. 2% of filtration accelerator (Celite~) and the cell mass (1890 g) was extracted with 6 liters of methanol. The active compound-containing, methanolic solution was freed from the mycelium by filtration and concentrated in vacuo. The concentrate was combined with 25 L of the culture filtrate, after which the pH was adjusted to 7 and the whole was loaded onto a previously prepared 3 titer ~MCI GEL, CHP20P column. The column was eluted with a gradient of 50 mM NH4 acetate buffer in water after 2-propanol. The column flow-through (7 liters per hour) was collected in fractions (in each case 2 liters) and the antibiotic-containing fractions 4 to 11 and 16 to 19 were in each case combined and concentrated in vacuo. The fractions were analyzed using HPLC. Fractions 4 to contain the more polar serpentemycins while fractions 16 to 19 contain the serpentenes. The serpentene-containing fractions were purified twice 5 chromatographically on LiChrospherfl 100 RP-18e, 250-25, HPLC columns firstly using 50 mM NH4 acetate buffer/acetonitrile mixtures and then using 0.05%
trifluoroacetic acid/acetonitrile gradients. Freeze-drying the fractions containing pure antibiotic resulted in 19 mg of pure serpentene.
After they had been concentrated in vacuo, the more polar compounds in fractions 4 10 to 11 were purified once again by column chromatography on 160 mL MCI GEL~
CHP20P (column dimensions: 26 mm x 300 mm) using a gradient method. The gradient ranged from 10% to 60% acetonitrile in 50 mM NH4 acetate buffer over hours. The column flow-through was 25 mL per minute while the fraction size was 50 mL. The compounds serpentemycin B and D were located in fractions 15 to 35 15 while fractions 36 to 39 contained serpentemycin C and fractions 40 to 43 contained serpentemycin A. The latter were freed from organic solvent in vacuo, after which 0.01 % ascorbic acid was added and purification by isocratic chromatography was carried out on a LiChrospherfl 100 RP-18e, 250-25, HPLC column using 45%
acetonitrile in 0.05% trifluoroacetic acid. The fractions which contained pure 20 serpentemycin A (fractions 31 to 35) were freeze-dried in the dark and the serpentemycin A was stored air-tight in an argon atmospheric (37 mg). ESI-mass spectrometry: 321.4 (M - H), ESI'' mass spectrometry: 305.3 (MH - H20).
UV maxima: 315 and 355 nm. The NMR data are listed in Table 3, with the C
atoms being numbered as follows:
8 \6 \ \ COOH
9 ~ / \ - /
COOH
COOH
Table 3: NMR-chemical shifts of serpentemycin A in DMSO at 300° K.
H C
1 167.43 2 6.02 122.54 3 7.35 144.35 4 7.04 128.09 7.19 137.16 6 134.33 7 7.73 125.93 8 7.35 127.90 9 7.35 128.57 7.27 130.22 11 135.78 12 6.81 130.33 13 6.63 131.24 14 6.59 132.61 7.15 130.33 16 6.41 136.57 17 6.21 127.45 18 7.70 138.40 19 5.95 123.07 167.48 Serpentemycin A:
Appearance: lemon-yellow substance which is soluble in medium-polar and polar organic solvents and not particularly soluble in water. Stable in neutral and mildly 5 acidic media but unstable in strongly acidic and strongly alkaline solution.
Serpentemycin A is sensitive to light and air.
Empirical formula: C2pH~gO4 Molecular weight: 322.36 10 Example 6: Isolating and describing serpentemycin B.
The serpentemycin B-containing fractions 36 to 39 from the 160 mL MCI GEL~
CHP20P column, obtained as described in Example 4, were concentrated in vacuo and the aqueous solution, which still contained a little acetonitrile, was loaded onto a LiChrospher~ 100 RP-18e, 250-25, HPLC column. The column was eluted isocratically using 42% acetonitrile in 0.05% trifluoroacetic acid (pH 2.4).
The column flow through was 39 mL/minute; fractions were removed every 19.5 mL and analyzed by HPLC. Fractions 22 to 24 contained the antibiotic serpentemycin B;
they were rechromatographed on the same column with the acetonitrile concentration in the eluent being reduced to 40%. Fractions 38 to 42 contained pure serpentemycin B; they were freeze-dried and yielded 10.2 mg of the antibiotic. ESI- mass spectrometry: 295.0985 (M - H), ESI+ mass spectrometry: 297.1164 (M + H), corresponding to the empirical formula C~gH~gO4. UV maxima: 306 and 332 nm in acetonitrile/0.1 % phosphoric acid in water. The NMR data are listed in Table 4; the numbering of the atoms is analogous to the serpentemycin A numbering.
Table 4: NMR-chemical shifts of serpentemycin B in DMSO at 300° K.
H C
1 167.42 2 6.02 122.59 3 7.35 144.32 4 7.05 128.23 5 7.19 137.09 6 134.40 7 7.74 126.00 8 7.36 128.10 9 7.36 128.61 10 7.28 130.31 11 135.40 12 6.90 131.86 13 6.53 130.54 14 6.81 135.71 15 6.65 132.89 16 7.19 143.79 17 5.93 122.69 18 167.33 Serpentemycin B:
Appearance: pale yellow substance which is soluble in medium-polar and polar organic solvents and not particularly soluble in water. Stable in neutral and mildly acidic media but unstable in strongly acidic and strongly alkaline solution.
Serpentemycin B is sensitive to light and air.
Empirical fiormula: C~gH~gO4 Molecular weight: 296.33 Example 7: Isolating and describing serpentemycin C.
450 mg of ascorbic acid were added to 45 liters of culture filtrate, obtained as described in Example 3, and the whole was loaded, at pH 4.5, onto a 3.5 liter MCi GEL~ CHP20P column (15 cm x 20 cm). The column was eluted with a gradient of 0.1 % NH4 acetate buffer, pH 4.5, after 2-propanol. The flow through was 15 liters/hour. The propanol-containing efflux was collected in fractions (in each case 7 liters); fraction 6 contained the polar aromatic polyenedicarboxylic acids.
They were concentrated in vacuo; after which 50 mg of ascorbic acid were added and the pH of the aqueous solution was adjusted to 3; the solution was then loaded onto a 490 mL
MCI GEL~ CHP20P column (5 cm x 30 cm). The desorption was carried out using a gradient of 10% acetonitrile in 0.5% acetic acid after 100% acetonitrile; at a flow rate of 25 mL per minute, the fraction time was 10 minutes (in each case 250 mL).
The polar aromatic polyenedicarboxyiic acids were located in fractions 12 to 19;
they were pooled, concentrated in vacuo and further purified by means of gel permeation chromatography. The support employed was 3.9 L of Fractogel~ TSK HW-40 (column dimensions: 10 x 50 cm), while the mobile phase employed was a mixture consisting of 60% acetonitrile, 20% methanol and 20% 25 mM NH4 acetate buffer, pH 7. At a flow through of 4.5 mL/minute, fractions were collected every half hour (in each case 135 mL). Fractions 37 to 40, which were examined by HPLC, contained the compound serpentemycin C. The final purification took place on LiChrospher~
100 RP-18e, 250-25 using the mobile phase 50 mM NH4 acetate buffer, pH
7/acetonitrile. The fractions containing pure serpentemycin C were pooled and desalted through LiChrospher~ RP-18e, 250-10, using waterlacetonitrile. Freeze-drying yielded 15 mg of serpentemycin C ammonium salt. ESI~ mass spectrometry:
295.1 (M - H), ESI+ mass spectrometry: 279.2 (MH - H20), corresponding to the empirical formula C~gH1604~ UV maxima: 212, 308 and 356 nm in acetonitrile/0.1 phosphoric acid in water (1:1 ). The NMR data are listed in Table 5; the numbering of the atoms is analogous to the serpentemycin A numbering.
Table 5: NMR-chemical shifts of serpentemycin C in DMSO at 300° K.
C
' 1 167.94 2 6.03 124.53 3 7.37 143.06 4 6.98 129.04 5 7.45 135.64 6 134.29 7 7.65 126.29 8 7.29 128.25 9 7.32 128.70 10 7.66 125.99 11 135.01 12 7.26 132.17 13 6.97 130.76 14 6.87 139.75 15 6.59 131.34 16 7.18 142.39 17 5.95 124.21 18 167.33 Serpentemycin C:
Appearance: pale yellow substance which is soluble in medium-polar and polar organic solvents and not particularly soluble in water. Stable in neutral and mildly acidic medium but unstable in strongly acidic and strongly alkaline solution.
Serpentemycin C is sensitive to light and air.
Empirical formula: C~$H~gO4 Molecular weight: 296.33 5 Example 8: Isolating and describing serpentemycin D.
The serpentemycin D-containing fractions 36 to 39 from the 160 mL MCI GEL~
CHP20P column, obtained as described in Example 4, were concentrated in vacuo and the aqueous solution, which still contained a little acetonitrile, was loaded onto a 10 LiChrospher~ 100 RP-18e, 250-25, HPLC column. The column was eluted isocratically using 42% acetonitrile in 0.05% trifluoroacetic acid (pH 2.4).
The column flow through was 39 mL/minute; fractions of in each case 19.5 mL were taken and analyzed by HPLC, as described in Example 9. Fractions 51 to 53 contained the antibiotic serpentemycin D; they were rechromatographed on the same column, as 15 described, except that the concentration of acetonitrile in the eluent was reduced to 40%. Fractions 13 to 15 contained pure serpentemycin D; they were freeze-dried and yielded 3 mg of the antibiotic. ESI- mass spectrometry: 325.5 (M - H), ESI+
mass spectrometry: 349.2 (M + Na), corresponding to the empirical formula C2oH2204. UV maxima: 299 and 338 nm in acetonitrile/0.1 % phosphoric acid in 20 water. The NMR data are listed in Table 6; the atoms were numbered in analogy with the compound serpentemycin A.
Table 6: NMR-chemical shifts of serpentemycin D in DMSO at 300° K.
'H C
1 167.54 2 6.02 122.38 3 7.45 144.49 4 6.99 128.31 5 7.49 136.82 6 133.56 7 7.64 126.22 8 7.25 127.52 9 7.31 128.84 10 7.62 125.68 11 135.74 12 7.07 128.14 13 6.89 131.72 14 6.48 132.12 15 6.45 134.02 16 6.30 129.68 17 5.90 136.71 18 3.82 75.31 18-OH 4.81 (broad) 19 3.48 69.77 19-OH 4.48 (broad) 20 1.03 19.10 Serpentemycin D:
Appearance: pale yellow substance which is soluble in medium-polar and polar organic solvents and not particularly soluble in water. Stable in neutral and mildly acidic media but unstable in strongly acidic and strongly alkaline solution.
Serpentemycin D is sensitive to light and air.
Empirical formula: C2oH22Oa Molecular weight: 326.40 Example 9: High pressure liquid chromatography (HPLC) of the serpentemycins.
The HPLC was carried out under the following conditions:
Column: Superspher 100 RP-18e~, 250-4, with precolumn, Mobile phase: 50% acetonitrile in 0.1 % phosphoric acid, Flow rate: 1 mL per minute, Column temperature: 40°C, Detection by means of UV absorption at 210 nm.
The following retention times were observed:
Serpentemycin A 10.1 minutes, Serpentemycin B 5.6 minutes, Serpentemycin C 7.3 minutes, Serpentemycin D 5.2 minutes.
Example 10: Determining the inhibition of glycosyltransferase by serpentemycins.
The assay was carried out as described by Vollmer & Holtje (Antimicrobial Agents and Chemotherapy 2000, 44, 1181-1185), except that purified penicillin-binding protein 1 b (PBP 1 b; 5 pM), carrying 3H-labeled moenomycin at the glycosyltransferase site, was used in the present example instead of [3H]benzylpenicillin. 3H-Moenomycin is obtained from moenomycin A (Kurt et al., Eur. J. Biochem., 1998, 252, 500-507) by hydrogenating it with 3H2:
6 mg of moenomycin, dissolved in 300 NL methanol, are added to a 1 cm3 flask, after which 2 mg of palladium-charcoal (Degussa Type E10N/D) are added. The flask is then gassed, while excluding air, with 1 cm3 3H2 and the reaction solution is left to hydrogenate for 15 minutes. After the reaction has come to an end, the catalyst is filtered off from the reaction mixture, which is diluted to 100 mL with ethanol. This solution can be used directly for preparing the glycosyltransferase/3H-moenomycin complex. Total radioactivity of the reaction product: 6.56 GBq (177mCi), specific activity: 1.9 TBq/mmol. The radioactive complex is attached to SPA PVT Copper His-Tag beads. The radioactive moenomycin which is displaced by the inhibitors is measured.
SPA PVT Copper His-Tag beads: Amersham RPN 0095;
PBS: GIBCO BRL 14200-067;
BSA: Calbiochem 12657;
NOG: SIGMA O-8001 (n-octyl f3-D-glucopyranoside);
Tween 20: Acros 23336-067;
Microtiter plates: Greiner Labortechnik;
The determination was carried out in microtiter plates. 10 NL of the test solution were added to the microtiter plates, followed by 10,uL of 3H-moenomycin (25 nM, 3.1 kBq/well) and 40,uL of SPA beads loaded with PBP 1 b (100 Ng of beads, 45 nM
enzyme). The plates were sealed and left to stand at room temperature for 8 hours.
After that, the beads were separated off from the test solution by centrifuging for 3 minutes at 1300 rpm and the distribution of the radioactivity was measured in a WALLAC MicroBeta~ 1450 Counter.
The inhibitory values were calculated in accordance with the formula:
(1 - (cPm sample-CPm low ctr~~( CPm high ctr - CPm low ctr)] X 100 (%~.
H C
1 167.43 2 6.02 122.54 3 7.35 144.35 4 7.04 128.09 7.19 137.16 6 134.33 7 7.73 125.93 8 7.35 127.90 9 7.35 128.57 7.27 130.22 11 135.78 12 6.81 130.33 13 6.63 131.24 14 6.59 132.61 7.15 130.33 16 6.41 136.57 17 6.21 127.45 18 7.70 138.40 19 5.95 123.07 167.48 Serpentemycin A:
Appearance: lemon-yellow substance which is soluble in medium-polar and polar organic solvents and not particularly soluble in water. Stable in neutral and mildly 5 acidic media but unstable in strongly acidic and strongly alkaline solution.
Serpentemycin A is sensitive to light and air.
Empirical formula: C2pH~gO4 Molecular weight: 322.36 10 Example 6: Isolating and describing serpentemycin B.
The serpentemycin B-containing fractions 36 to 39 from the 160 mL MCI GEL~
CHP20P column, obtained as described in Example 4, were concentrated in vacuo and the aqueous solution, which still contained a little acetonitrile, was loaded onto a LiChrospher~ 100 RP-18e, 250-25, HPLC column. The column was eluted isocratically using 42% acetonitrile in 0.05% trifluoroacetic acid (pH 2.4).
The column flow through was 39 mL/minute; fractions were removed every 19.5 mL and analyzed by HPLC. Fractions 22 to 24 contained the antibiotic serpentemycin B;
they were rechromatographed on the same column with the acetonitrile concentration in the eluent being reduced to 40%. Fractions 38 to 42 contained pure serpentemycin B; they were freeze-dried and yielded 10.2 mg of the antibiotic. ESI- mass spectrometry: 295.0985 (M - H), ESI+ mass spectrometry: 297.1164 (M + H), corresponding to the empirical formula C~gH~gO4. UV maxima: 306 and 332 nm in acetonitrile/0.1 % phosphoric acid in water. The NMR data are listed in Table 4; the numbering of the atoms is analogous to the serpentemycin A numbering.
Table 4: NMR-chemical shifts of serpentemycin B in DMSO at 300° K.
H C
1 167.42 2 6.02 122.59 3 7.35 144.32 4 7.05 128.23 5 7.19 137.09 6 134.40 7 7.74 126.00 8 7.36 128.10 9 7.36 128.61 10 7.28 130.31 11 135.40 12 6.90 131.86 13 6.53 130.54 14 6.81 135.71 15 6.65 132.89 16 7.19 143.79 17 5.93 122.69 18 167.33 Serpentemycin B:
Appearance: pale yellow substance which is soluble in medium-polar and polar organic solvents and not particularly soluble in water. Stable in neutral and mildly acidic media but unstable in strongly acidic and strongly alkaline solution.
Serpentemycin B is sensitive to light and air.
Empirical fiormula: C~gH~gO4 Molecular weight: 296.33 Example 7: Isolating and describing serpentemycin C.
450 mg of ascorbic acid were added to 45 liters of culture filtrate, obtained as described in Example 3, and the whole was loaded, at pH 4.5, onto a 3.5 liter MCi GEL~ CHP20P column (15 cm x 20 cm). The column was eluted with a gradient of 0.1 % NH4 acetate buffer, pH 4.5, after 2-propanol. The flow through was 15 liters/hour. The propanol-containing efflux was collected in fractions (in each case 7 liters); fraction 6 contained the polar aromatic polyenedicarboxylic acids.
They were concentrated in vacuo; after which 50 mg of ascorbic acid were added and the pH of the aqueous solution was adjusted to 3; the solution was then loaded onto a 490 mL
MCI GEL~ CHP20P column (5 cm x 30 cm). The desorption was carried out using a gradient of 10% acetonitrile in 0.5% acetic acid after 100% acetonitrile; at a flow rate of 25 mL per minute, the fraction time was 10 minutes (in each case 250 mL).
The polar aromatic polyenedicarboxyiic acids were located in fractions 12 to 19;
they were pooled, concentrated in vacuo and further purified by means of gel permeation chromatography. The support employed was 3.9 L of Fractogel~ TSK HW-40 (column dimensions: 10 x 50 cm), while the mobile phase employed was a mixture consisting of 60% acetonitrile, 20% methanol and 20% 25 mM NH4 acetate buffer, pH 7. At a flow through of 4.5 mL/minute, fractions were collected every half hour (in each case 135 mL). Fractions 37 to 40, which were examined by HPLC, contained the compound serpentemycin C. The final purification took place on LiChrospher~
100 RP-18e, 250-25 using the mobile phase 50 mM NH4 acetate buffer, pH
7/acetonitrile. The fractions containing pure serpentemycin C were pooled and desalted through LiChrospher~ RP-18e, 250-10, using waterlacetonitrile. Freeze-drying yielded 15 mg of serpentemycin C ammonium salt. ESI~ mass spectrometry:
295.1 (M - H), ESI+ mass spectrometry: 279.2 (MH - H20), corresponding to the empirical formula C~gH1604~ UV maxima: 212, 308 and 356 nm in acetonitrile/0.1 phosphoric acid in water (1:1 ). The NMR data are listed in Table 5; the numbering of the atoms is analogous to the serpentemycin A numbering.
Table 5: NMR-chemical shifts of serpentemycin C in DMSO at 300° K.
C
' 1 167.94 2 6.03 124.53 3 7.37 143.06 4 6.98 129.04 5 7.45 135.64 6 134.29 7 7.65 126.29 8 7.29 128.25 9 7.32 128.70 10 7.66 125.99 11 135.01 12 7.26 132.17 13 6.97 130.76 14 6.87 139.75 15 6.59 131.34 16 7.18 142.39 17 5.95 124.21 18 167.33 Serpentemycin C:
Appearance: pale yellow substance which is soluble in medium-polar and polar organic solvents and not particularly soluble in water. Stable in neutral and mildly acidic medium but unstable in strongly acidic and strongly alkaline solution.
Serpentemycin C is sensitive to light and air.
Empirical formula: C~$H~gO4 Molecular weight: 296.33 5 Example 8: Isolating and describing serpentemycin D.
The serpentemycin D-containing fractions 36 to 39 from the 160 mL MCI GEL~
CHP20P column, obtained as described in Example 4, were concentrated in vacuo and the aqueous solution, which still contained a little acetonitrile, was loaded onto a 10 LiChrospher~ 100 RP-18e, 250-25, HPLC column. The column was eluted isocratically using 42% acetonitrile in 0.05% trifluoroacetic acid (pH 2.4).
The column flow through was 39 mL/minute; fractions of in each case 19.5 mL were taken and analyzed by HPLC, as described in Example 9. Fractions 51 to 53 contained the antibiotic serpentemycin D; they were rechromatographed on the same column, as 15 described, except that the concentration of acetonitrile in the eluent was reduced to 40%. Fractions 13 to 15 contained pure serpentemycin D; they were freeze-dried and yielded 3 mg of the antibiotic. ESI- mass spectrometry: 325.5 (M - H), ESI+
mass spectrometry: 349.2 (M + Na), corresponding to the empirical formula C2oH2204. UV maxima: 299 and 338 nm in acetonitrile/0.1 % phosphoric acid in 20 water. The NMR data are listed in Table 6; the atoms were numbered in analogy with the compound serpentemycin A.
Table 6: NMR-chemical shifts of serpentemycin D in DMSO at 300° K.
'H C
1 167.54 2 6.02 122.38 3 7.45 144.49 4 6.99 128.31 5 7.49 136.82 6 133.56 7 7.64 126.22 8 7.25 127.52 9 7.31 128.84 10 7.62 125.68 11 135.74 12 7.07 128.14 13 6.89 131.72 14 6.48 132.12 15 6.45 134.02 16 6.30 129.68 17 5.90 136.71 18 3.82 75.31 18-OH 4.81 (broad) 19 3.48 69.77 19-OH 4.48 (broad) 20 1.03 19.10 Serpentemycin D:
Appearance: pale yellow substance which is soluble in medium-polar and polar organic solvents and not particularly soluble in water. Stable in neutral and mildly acidic media but unstable in strongly acidic and strongly alkaline solution.
Serpentemycin D is sensitive to light and air.
Empirical formula: C2oH22Oa Molecular weight: 326.40 Example 9: High pressure liquid chromatography (HPLC) of the serpentemycins.
The HPLC was carried out under the following conditions:
Column: Superspher 100 RP-18e~, 250-4, with precolumn, Mobile phase: 50% acetonitrile in 0.1 % phosphoric acid, Flow rate: 1 mL per minute, Column temperature: 40°C, Detection by means of UV absorption at 210 nm.
The following retention times were observed:
Serpentemycin A 10.1 minutes, Serpentemycin B 5.6 minutes, Serpentemycin C 7.3 minutes, Serpentemycin D 5.2 minutes.
Example 10: Determining the inhibition of glycosyltransferase by serpentemycins.
The assay was carried out as described by Vollmer & Holtje (Antimicrobial Agents and Chemotherapy 2000, 44, 1181-1185), except that purified penicillin-binding protein 1 b (PBP 1 b; 5 pM), carrying 3H-labeled moenomycin at the glycosyltransferase site, was used in the present example instead of [3H]benzylpenicillin. 3H-Moenomycin is obtained from moenomycin A (Kurt et al., Eur. J. Biochem., 1998, 252, 500-507) by hydrogenating it with 3H2:
6 mg of moenomycin, dissolved in 300 NL methanol, are added to a 1 cm3 flask, after which 2 mg of palladium-charcoal (Degussa Type E10N/D) are added. The flask is then gassed, while excluding air, with 1 cm3 3H2 and the reaction solution is left to hydrogenate for 15 minutes. After the reaction has come to an end, the catalyst is filtered off from the reaction mixture, which is diluted to 100 mL with ethanol. This solution can be used directly for preparing the glycosyltransferase/3H-moenomycin complex. Total radioactivity of the reaction product: 6.56 GBq (177mCi), specific activity: 1.9 TBq/mmol. The radioactive complex is attached to SPA PVT Copper His-Tag beads. The radioactive moenomycin which is displaced by the inhibitors is measured.
SPA PVT Copper His-Tag beads: Amersham RPN 0095;
PBS: GIBCO BRL 14200-067;
BSA: Calbiochem 12657;
NOG: SIGMA O-8001 (n-octyl f3-D-glucopyranoside);
Tween 20: Acros 23336-067;
Microtiter plates: Greiner Labortechnik;
The determination was carried out in microtiter plates. 10 NL of the test solution were added to the microtiter plates, followed by 10,uL of 3H-moenomycin (25 nM, 3.1 kBq/well) and 40,uL of SPA beads loaded with PBP 1 b (100 Ng of beads, 45 nM
enzyme). The plates were sealed and left to stand at room temperature for 8 hours.
After that, the beads were separated off from the test solution by centrifuging for 3 minutes at 1300 rpm and the distribution of the radioactivity was measured in a WALLAC MicroBeta~ 1450 Counter.
The inhibitory values were calculated in accordance with the formula:
(1 - (cPm sample-CPm low ctr~~( CPm high ctr - CPm low ctr)] X 100 (%~.
Claims (24)
1. A compound of the formula (I) where Y is a group of the formula (II) or of the formula (III) R is H, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C5-C14-aryl, halogen, -CN, -OH, -O-C1-C6-alkyl, -O-C2-C6-alkenyl, -O-C5-C14-aryl, -O-C2-C6-alkynyl, -NH2, -NH-C1-C6-alkyl, -NH-C2-C6-alkenyl, -NH-C2-C6-alkynyl, -NH-C5-C14-aryl, -N(-C1-alkyl)2, -N(-C2-C6-alkenyl)2, -N(-C2-C6-alkynyl)2, -N(C5-C14-aryl)2, -NH[-C(=O)-(C1-C6-alkyl)], -NH[-C(=O)-(C5-C14-aryl)], -NH-O-R1, -SH, -S-C1-C6-alkyl, -S-C2-C6-alkenyl, -S-C1-C6-alkynyl or -O-C5-C14-aryl, where the abovementioned substituents can be unsubstituted or substituted, once or more than once, by C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C5-C14-aryl, where alkyl, alkenyl, alkynyl and aryl are unsubstituted or substituted, once or twice, by -OH, =O, -O-C1-C6-alkyl, -O-C2-C6-alkenyl, -C14-aryl, -C5-C14-aryl, -NH-C1-C6-alkyl, -NH-C2-C6-alkenyl, -NH2, halogen, where alkyl, alkenyl, alkynyl and aryl can be further substituted by a -CN, amide or oxime, R1, R2, R3 and R4 are, independently of each other, H, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl or C5-C14-aryl, in which alkyl, alkenyl, alkynyl and aryl are unsubstituted or substituted, once or twice, by -OH, -O-C2-C6-alkyl, -O-C2-C6-alkenyl, -O-C5-C14-aryl, -C5-C14-aryl, -NH-C1-C6-alkyl, -NH-C2-C6-alkenyl, -NH2 or halogen, in which alkyl, alkenyl, alkynyl and aryl are unsubstituted or substituted, once or twice, by -OH, =O, -O-C1-C6-alkyl, -O-C2-C6-alkenyl, -O-C5-C14-aryl, -C5-C14-aryl, -NH-C1-C6-alkyl, -NH-C2-C6-alkenyl, -NH2 or halogen, in which alkyl, alkenyl, alkynyl and aryl can be further substituted by a -CN, amide or oxime, X1, X2 and X3 are, independently of each other, -CH2-, -CHR-, -NH-, -N(C1-C6-alkyl, -N(C2-C6-alkenyl)-, -N(C2-C6-alkynyl-, -N[-C(=O)-(C1-C6-alkyl)]-, -N[-C(=O)-(C5-C14-aryl)]-, -N(C5-C14-aryl)-, -N(O-R)-, -O- or -S-, n and m are, independently of each other, 2, 3, 4 or 5, and o is 0, 1, 2 or 3, where compounds of the formula (I) are excepted in which o is 0, n is 2, m is 2 or 3, X2 and X3 are O, and R2 and R3 are C2H5, and all double bonds possess the transconfiguration, and/or a stereoisomeric form of the compound of the formula (I) and/or a mixture of these forms in any ratio, and/or a physiologically tolerated salt of the compound of the formula (I).
2. A compound of the formula (I) as claimed in claim 1, wherein at least one polyene group contains at least one cis double bond.
3. A compound of the formula (I) as claimed in claim 1 or 2, wherein R is H, R1 is H or C1-C6-alkyl, R2 is H or C1-C6-alkyl, R3 is H or C1-C6-alkyl, R4 is C1-C6-alkyl, and X1 and X2 are -O-, and the physiologically tolerated salts thereof.
4. A compound of the formula (I) as claimed in one or more of claims 1 to 3, which is a compound of the formula (IV) where m is 3 or 4, and the physiologically tolerated salts thereof.
5. A compound of the formula (I) as claimed in one or more of claims 1 to 4, which is a compound of the formula (V)
6. A compound of the formula (V) as claimed in claim 5, where R1 and R2 are H.
7. A compound of the formula (I) as claimed in one or more of claims 1 to 4, which is a compound of the formula (VI)
8. A compound of the formula (VI) as claimed in claim 7, where R1 and R2 are H.
9. A compound of the formula (I) as claimed in one or more of claims 1 to 4, which is a compound of the formula (VII)
10. A compound of the formula (VII) as claimed in claim 9, where R1 and R2 are H.
11. A compound of the formula (I) as claimed in one of claims 1 to 3, which is a compound of the formula (VIII)
12. A compound of the formula (VIII) as claimed in claim 11, which is a compound of the formula (IX)
13. A compound of the formula (IX) as claimed in claim 12, where R1 is H.
14. A compound of the formula (VIII) as claimed in claim 11, which is a compound of the formula (X)
15. A compound of the formula (X) as claimed in claim 14, where R1 is H.
16. A process for preparing a compound of the formula (I) as claimed in claim 1, which comprises 1. culturing the microorganism Actinomycetaies sp. DSM 14865, or one of its variants and/or mutants, in an aqueous nutrient medium until one or more of the compounds serpentemycin A, B, C and/or D accrues in the culture broth, 2. isolating and purifying serpentemycin A, B, C or D, 3. where appropriate, using a suitable reagent to convert serpentemycin A, B, C or D into a compound of the formula (I), 4. and, where appropriate, converting the compound of the formula (I) into a pharmacologically tolerated salt.
17. The process as claimed in claim 16, wherein the suitable reagent is an alkylating agent.
18. A process for preparing a compound as claimed in one or more of claims 12 to 15, which comprises fermenting the microorganism Actinomycetales sp. DSM
14865, or one of its variants and/or mutants, in a culture medium which contains a carbon and nitrogen source and also the customary inorganic salts and trace elements, isolating serpentemycins A, B, C and/or D and, where appropriate, converting serpentemycins A, B, C and/or D into a pharmacologically tolerated salt.
14865, or one of its variants and/or mutants, in a culture medium which contains a carbon and nitrogen source and also the customary inorganic salts and trace elements, isolating serpentemycins A, B, C and/or D and, where appropriate, converting serpentemycins A, B, C and/or D into a pharmacologically tolerated salt.
19. A process as claimed in one or more of claims 16 to 18, wherein the fermentation is carried out under aerobic conditions at a temperature of between 20 and 35°C and at a pH between 4 and 10.
20. The use of a compound as claimed in one or more of claims 1 to 15 for producing a pharmaceutical.
21. The use of a compound as claimed in claim 20 for producing a pharmaceutical for the treatment and/or prophylaxis of infectious bacterial diseases.
22. A pharmaceutical having a content of at least one compound as claimed in one or more of claims 1 to 15 and one or more physiologically suitable auxiliary substances.
23. A process for producing a pharmaceutical as claimed in claim 22, which comprises bringing at least one compound as claimed in one or more of claims 1 to 15, together with one or more physiologically suitable auxiliary substances, into a suitable form for administration.
24. The microorganism Actinomycetales sp., DSM 14865.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10229713.4 | 2002-07-02 | ||
| DE10229713A DE10229713A1 (en) | 2002-07-02 | 2002-07-02 | Polyenecarboxylic acid derivatives, process for their preparation and their use |
| PCT/EP2003/006407 WO2004005236A1 (en) | 2002-07-02 | 2003-06-18 | Polyene carboxylic acid derivatives, method for their production and the use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2490570A1 true CA2490570A1 (en) | 2004-01-15 |
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ID=29723635
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002490570A Abandoned CA2490570A1 (en) | 2002-07-02 | 2003-06-18 | Polyene carboxylic acid derivatives, method for their production and the use thereof |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP1519909B1 (en) |
| JP (1) | JP4500163B2 (en) |
| AT (1) | ATE490228T1 (en) |
| AU (1) | AU2003281344A1 (en) |
| BR (1) | BR0312337A (en) |
| CA (1) | CA2490570A1 (en) |
| DE (2) | DE10229713A1 (en) |
| MX (1) | MXPA04012309A (en) |
| WO (1) | WO2004005236A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080222120A1 (en) * | 2007-03-08 | 2008-09-11 | Nikolaos Georgis | System and method for video recommendation based on video frame features |
-
2002
- 2002-07-02 DE DE10229713A patent/DE10229713A1/en not_active Withdrawn
-
2003
- 2003-06-18 AT AT03740270T patent/ATE490228T1/en active
- 2003-06-18 CA CA002490570A patent/CA2490570A1/en not_active Abandoned
- 2003-06-18 WO PCT/EP2003/006407 patent/WO2004005236A1/en active Application Filing
- 2003-06-18 AU AU2003281344A patent/AU2003281344A1/en not_active Abandoned
- 2003-06-18 EP EP03740270A patent/EP1519909B1/en not_active Expired - Lifetime
- 2003-06-18 MX MXPA04012309A patent/MXPA04012309A/en active IP Right Grant
- 2003-06-18 DE DE50313293T patent/DE50313293D1/en not_active Expired - Lifetime
- 2003-06-18 BR BR0312337-5A patent/BR0312337A/en not_active IP Right Cessation
- 2003-06-18 JP JP2004518540A patent/JP4500163B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| WO2004005236A1 (en) | 2004-01-15 |
| ATE490228T1 (en) | 2010-12-15 |
| JP4500163B2 (en) | 2010-07-14 |
| JP2006502983A (en) | 2006-01-26 |
| AU2003281344A1 (en) | 2004-01-23 |
| DE50313293D1 (en) | 2011-01-13 |
| BR0312337A (en) | 2005-04-12 |
| EP1519909A1 (en) | 2005-04-06 |
| EP1519909B1 (en) | 2010-12-01 |
| DE10229713A1 (en) | 2004-01-15 |
| MXPA04012309A (en) | 2005-02-25 |
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| FZDE | Discontinued |