Note: Descriptions are shown in the official language in which they were submitted.
CA 02467251 2004-05-14 pCTIEP02112420
WO 031043984
Gabusectin derivatives, processes for preparing them and their use
A large number of antibiotics are used therapeutically for treating infectious
diseases
of bacterial origin. However, the pathogens are becoming increasingly
resistant to the
pharmaceuticals employed; Even what are termed multiresistant organisms, which
have become resistant not only to individual antibiotic groys, such as ~-
lactam
antibiotics, glycopeptides or macrolides, but also carry several resistances
simultaneously, pose a great threat. 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 medicines 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 able to be used as pharmaceuticals.
A relatively large number of antibiotics having a tetramic acid basic
structure have
already been described. Tetramic acid, i.e. 2,4-pyrrolidinedione, is the
parent
compound for a variety of natural products which are formed by some
microorganisms and marine invertebrates.
harzianic acid, an antibiotic which possesses very little activity, was
described in
1994 (R. Sawa et al., J. Antibiotics, 47, 731-732, 1994);
The natural tetramic acid derivatives which were published up until 1994 are
described in a review by B.J. L. Royles CChem. Rev. 95, pages 1981 - 2001,
1995).
Further natural tetramic acids, some of which possess antibacterial
properties, have
been described since 1995:
~ reutericyclin (A. Holtzel et al., Angew. Chem. 112, 2886-2888, 2000), a
compound
which possesses slight antibacterial activity;
~ equisetin and phomasetin (S. S. Singh et al., Tetrahedron Lett. 39, 2243-
2246,
1998) are isomeric inhibitors of HIV-1 integrase;
~ cryptocin (J. Y. Li et al., Org. Lett. 2, 767-770, 2000), which is an
antimycotic
compound;
~ vancoresmycin (N. V. S. Ramakrishna et al., Int. Patent Publication No.
WO 0028064), an antibiotic;
CA 02467251 2004-05-14
2
~ coniosetin (L. Vertesy et al., German patent application No. DE 10060810.8),
a
potent antibiotic composed of a tetramic acid moiety and a naphthyl moiety.
It has been found, surprisingly, that the strain ST 003236 (DSM 14476) is able
to
form the novel antibiotic gabusectin, which is not only very active against
bacteria but
is also well tolerated.
The invention accordingly relates to the compounds which are formed by the
strain
ST 003236 (DSM 14476) and to their physiologically tolerated salts,
stereoisomers,
tautomers, derivatives, in particular ester derivatives, and obvious chemical
equivalents, such as ethers.
The invention relates to compounds of the formula (I)
R3
N
XR
CH3~X4 (I)
R4
HsC CH / R5
where
R, R2 and R3 are, independently of each other:
1. H, or
2. C1-C6-alkyl, C2-Cg-alkenyl or C2-Cg-alkynyl, in which alkyl, alkenyl and
alkynyl
are straight-chain or branched and are optionally substituted, once or twice,
by:
2.1 -OH,
2.2 =O,
2.3 -O-C1-C6-alkyl, in which alkyl is straight-chain or branched,
__ .________~ __
CA 02467251 2004-05-14
3
2.4 -O-C2-C6-alkenyl, in which alkenyl is straight-chain or branched,
2.5 -aryl,
2.6 -NH-C1-C6-alkyl, in which alkyl is straight-chain or branched,
2.7 -NH-C2-C6-alkenyl, in which alkenyl is straight-chain or branched,
2.8 -NH2 or
2.9 halogen,
in which the substituents 2.3 to 2.7 can be further substituted by -CN, -amide
or ~xime functions,
R4 is C1-C6-alkyl or C2-Cg-alkenyl, in which alkyl and alkenyl can be straight-
chain or branched and are optionally substituted once or twice, as described
under 2.1 to 2.9,
R5 is H or methyl,
X, X2, X3, X4 and X5, are, independent of each other O, NH, N-C1-C6-alkyl, N-
C2-C6-alkenyl, N-C2-Cg-alkynyl, N-acyl, N-aryl, N-O-R or S,
or a stereoisomeric form or a tautomeric form of the compound of the formula
(I) or a
mixture of the previously mentioned forms in any ratio, or a physiologically
tolerated
salt of the compound of the formula (I) or of a stereoisomeric form or of a
tautomeric
form of a compound of the formula (1).
C1-C6-alkyl is a straight-chain or branched alkyl having from 1 to 6 C atoms,
preferably having from 1 to 4 C atoms, e.g. methyl, ethyl, i-propyl, tert-
butyl and
hexyl.
C2-C6-alkenyl is a straight-chain or branched alkenyl which has from 2 to 6 C
atoms,
and which is unsaturated once, twice or three times, e.g. allyl, crotyl, 1-
propenyl,
yenta-1,3-dienyl and pentenyl.
_ __. ~~ _. .~._ . _ _... _. _ . __ . _ ~_. _____ ____..__ ~_.___
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4
C2-C6-alkynyl is a straight-chain or branched alkynyl which has from 2 to 6 C
atoms,
and which is saturated once or twice, e.g. propynyl, butynyl and pentynyl.
Aryl is phenyl, benzyl or 1- or 2-naphthyl, which can also be additionally
substituted,
for example by halogen, such as chlorine, bromine, fluorine, by alkyl having 1-
4 C
atoms, preferably methyl-, by hydroxyl, by alkoxy having 1-4 C atoms, in
particular
methoxyl, or by trifluoromethyl.
Acyl can be aliphatic or aromatic acyl radicals. Aliphatic acyl has 1-7,
preferably 1-4,
C atoms, such as formyl, acetyl, propionyl, butyryl, hexanoyl, acryloyl,
crotonoyl, or
propioloyl, which can be still further substituted, for example by halogen,
such as
chlorine, bromine or fluorine, by amino, or by alkylamino having 1-4 C atoms,
preferably methyl or ethylamino groups. Aromatic acyl can, for example, be
benzoyl
or naphthoyl which can also be additionally substituted, for example by
halogen, such
as chlorine, bromine or fluorine, by alkyl having 1-4 C atoms, preferably
methyl, by
hydroxyl, by amino groups, such as ethylamino, or by alkoxy groups having 1-7,
preferably 1-4, C atoms, in particular methoxy.
The invention preferably relates to a compound of the formula (I), where
R is 1.0 H, or
2.0 C1-Cg-alkyl, C2-C6-alkenyl or C2-Cg-alkynyl, in which alkyl, alkenyl and
alkynyl are straight-chain or branched and are optionally substituted once or
twice by:
2.1-OH,
2.2=O,
2.3-O-C1-Cg-alkyl, in which alkyl is straight-chain or branched,
2.4-O-C2-C6-alkenyl, in which alkenyl is straight-chain or branched,
2.5-aryl,
2.6-NH-C1-C6-alkyl, in which alkyl is straight-chain or branched,
2.7-NH-C2-C6-alkenyl, in which alkenyl is straight-chain or branched,
2.8-NH2 or
2.9 halogen,
' CA 02467251 2004-05-14
in which the substituents 2.3 to 2.7 can be additionally substituted by
-CN, -amide or-oxime functions,
R2 is H,
R3 is CH3,
5 R4 is-CH=CH-CH3,
R5 is CH3, and ~ ' '
X, X2, X3, Xq. and X5 are O.
Particularly preferably, the invention relates to a compound of the formula
(I), where
R is H,
R2 is H or CH3,
R3 is CH3,
R4 is -CH=CH-CH3,
R5 is CH3, and
X, X2, X3, X4 and X5 are O.
Tautomeric forms of the compound (I) are, for example, a compound of the
formula (II)
R3
I
R2X3 N Xs
_. 1 XR
CH3 ~X4 (II)
~ Ra
HsC CH / R5
where the radicals R, R2, R3, R4, R5, X, X2, X3, X4 and X5 are defined as
above,
where tautomeric forms of the compounds of the formula (I) result, for
example, from
the hydrogen-bonded tetramic acid structural moiety,
CA 02467251 2004-05-14
6
Rs Rs
H~O N ,O N
' I ~ H
O~ O
X4 X4
and are converted into each other in solution in dependence on parameters such
as
pH and solvent polarity.
Unless otherwise indicated, chiral centers in the compounds of the formulae
(I) and
(II) can be present in the R configuration or in the S configuration. The
invention
relates both to the optically pure compounds and to stereoisomeric mixtures,
such as
enantiomeric mixtures and diasteromeric mixtures, in any ratio.
Of the compounds of the formulae (I) and (II) according to the invention,
preference
is given to those compounds in which the configuration corresponds to the
substituted hydrogenated naphthyl backbone of the formula (III):
H ~,,. CH3 R
4
H C~~~~ _ /
3 CH3 R5
The invention furthermore relates to a compound of the formula (IV),
CH3
HO ~ OH
CH30 (IV)
CHs
H3C' v CH ~CH3 .
3
CA 02467251 2004-05-14
7
to a compound of the formula (V),
CH3
O N O
HO ~ ~OH
H = CH3 O (V)
CH3
H3C,,, CH / CH3
3
to a compound of the formula (VI),
CH3
N.
HO ~ O-CH3
CH3 \ CH (VI)
/'w s
H3C' ~ ICH ~CH3
3
to a compound of the formula (VII),
CH3
O N O
HO ~ O-CH3
O
H ' CH3 \ CH (VII)
3
H3C'~ CH ~ CH3
3
CA 02467251 2004-05-14
or to a stereoisomeric form or a tautomeric form of a compound of the formula
(IV),
(V), (VI) or (VII) or to a mixture of the respective previously mentioned
forms in any
ratio, or to a physiologically tolerated salt of a compound of the formula
(IV), (V), (VI)
or (VII) or of a stereoisomeric form or of a tautomeric form of a compound of
the
formula (IV), (V), (VI) or (VII).
The inventive compounds differs from substances which are known from the
literature, for example in their polarity, their chemical structure or their
antimicrobial
activity or other physical properties. In particular, as compared with the
compounds in
the prior art, the compounds according to the invention contain an additional
methyl
group in the naphthyl moiety.
The invention furthermore relates to obvious chemical equivalents of the
compounds
of the formulae (I) to (VII).
Obvious chemical equivalents of the compounds according to the invention are
compounds which possess the same activity as the compounds according to the
invention and exhibit a trivial chemical difference or which are converted,
under mild
conditions, into the compounds according to the invention. Said equivalents
include,
for example, esters, azomethines (Schiff~s bases), ketals, oximes,
hydrogenation
products, reduction products, complexes or addition compounds of or with the
compounds according to the invention.
For example, an activated acid, for example acid chlorides or other acid
derivatives,
can be reacted with the hydroxyl group of the compound of the formula (I), or
of one
or more double bonds and/or carbonyl groups of the compound of the formula (I)
can
be reduced with a reducing agent, with double bonds being reduced, for
example,
using H2/Pd and carbonyl groups being reduced, for example, using NaBH4. The
abovementioned methods for derivatizing are described in text books such as
Jerry
March, Advanced Organic Chemistry, John Wiley & Sons, 4th Edition, 1992. In
order
to carry out reactions selectively, it can 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
subsequently
purified.
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9
The invention furthermore relates to gabusectin, a compound which has the
empirical
formula C25H35N04, as demonstrated by ESI and FAB mass spectroscopy, and
which is characterized by the 1 H NMR and 13C NMR data given in table 2, or to
a
stereoisomeric form or a tautomeric form of the compound gabusectin, or to a
mixture of the respective previously mentioned forms in any ratio, or to a
physiologically tolerated salt of the compound gabusectin or of a
stereoisomeric form
or of a tautomeric form of the compound gabusectin.
The invention furthermore relates to gabusectin methyl ester, a compound of
the
empirical formula C27H3gN05, demonstrated by ESI and FAB mass spectroscopy,
and characterized by the 1 H NMR and 13C NMR data given in table 3, or to a
stereoisomeric form or a tautomeric form of the compound gabusectin methyl
ester,
or to a mixture of the respective previously mentioned forms in any ratio, or
to a
physiologically tolerated salt of the compound gabusectin methyl ester or of a
stereoisomeric form or a tautomeric form of the compound gabusectin methyl
ester.
The invention furthermore relates to a compound of the formula (I) which can
be
obtained by fermenting ST 003236 (DSM 14476), or a variant and/or mutants of
ST 003236 (DSM 14476), in a culture medium until the compound of the formula
(I)
accumulates in the culture broth, then isolating the compound of the formula
(I) and,
where appropriate, converting it into a pharmacologically tolerated salt.
The invention also relates to a compound of the empirical formula C26H37N05
(Gabusectin) which can be obtained by fermenting ST 003236 (DSM 14476), or a
variant and/or mutant of ST 003236 (DSM 14476) in a culture medium until the
compound gabusectin accumulates in the culture broth, subsequently isolating
the
compound Gabusectin and, where appropriate, converting it into a
pharmacologically
tolerated salt.
The invention additionally relates to a process for preparing a compound of
the
formula (I), which comprises culturing the microorganism ST 003236 {DSM
14476),
or a variant and/or mutant of ST 003236 (DSM 14476), in an aqueous nutrient
medium, isolating and purifying a compound of the formula (I) and, where
CA 02467251 2004-05-14
appropriate, converting it into an obvious chemical equivalent or a
pharmacologically
tolerated salt.
The invention furthermore relates to a process for preparing a compound of the
5 formula (1), which comprises esterifying gabusectin of the formula (IV) with
a C1-Cg-
alkyl-, C2-C5-alkenyl- or C2-Cg-alkynyl-alcohol derivative, or ~r.~ith a C1-Cb-
alkyl-,
C2-Cg-alkenyl- or C2-Cg-alkynyl-alkylating agent, to give a compound of the
formula
(I), in which alkyl, alkenyl and alkynyl are straight-chain or branched and
can
optionally be substituted, once or twice, by the radicals 2.1 to 2.9 in
accordance with
10 formula (I) in claim 1, in which the substituents 2.3 to 2.7 can be further
substituted
by -CN, -amide or -oxime functions, and R2 is H, R3 is CH3, R4 is -CH=CH-CH3,
R5 is CH3, and X, X2, X3, X4 and X5 are O, preferably using a C1-Cg-alkyl-
alkylating agent, particularly preferably using a C1-alkylating agent.
C1-C6-Alkyl-, C2-Cg-alkenyl- or C2-Cg-alkynyl-alcohol derivatives are straight-
chain
or branched and optionally substituted once or twice by the radicals 2.1 to
2.9, see
above, in which the substituents 2.3 to 2.7 can be further substituted by -CN,
-amide
or -oxime functions, for example methanol, ethanol, n-propanol, isopropanol,
n-butanol, sec-butanol, tert-butanol and n-hexanol, 2-buten-1-of (crotyl
alcohol),
1-propen-3-of (allyl alcohol), 1,3-pentadien-5-ol, 1,4-pentadien-3-of and 2-
penten-1-
ol, 1-penten-4-of (allylmethylcarbinol), 1-penten-3-of (ethylvinylcarbinol), 2-
propyn-1-
of (propargyl alcohol), 1-butyn-3-ol, 2-butyn-1-ol, 3-butyn-1-ol, 1-pentyn-3-
ol,
2-pentyn-1-ol, 3-pentyn-1-of and 4-pentyin-1-ol, preferably methanol.
C1-C6-Alkyl-, C2-C6-alkenyl- or C2-C6-alkynyl-alkylating agents are straight-
chain or
branched and optionally substituted once or twice by the radicals 2.1 to 2.9,
see
above, for example diazomethane derivatives as C1-alkylating agents, for
example
trimethylsilyldiazomethane.
Methods for esterifying are described, for example, in Jerry March, Advanced
Organic Chemistry, John Wiley & Sons, 4th Edition, 1992.
CA 02467251 2004-05-14
11
The strain ST 003236 has been deposited in the Deutsche Sammlung von
Microorganismen and Zellkulturen [German collection of microorganisms and cell
cultures] GmbH (DSM), Mascheroder Weg 1 B, 38124 Braunschweig, Germany, in
accordance with the rules of the Budapest Treaty, under the following number
DSM 14476.
Said process comprises culturing ST 003236 (DSM 14476), its mutants or
variants,
under aerobic conditions in a culture media containing one or more carbon and
nitrogen sources, inorganic salts and, where appropriate, trace elements.
The course of the fermentation, and the formation of the antibiotics according
to the
invention, can be monitored using methods known to a skilled person, for
example by
testing the biological activity in bioassays or by means of chromatographic
methods
such as thin layer chromatography (TLC) or high performance liquid
chromatography
(HPLC).
A mutant is a microorganism in which one or more genes in the genome haslhave
been modified, with the gene or genes which islare responsible for the ability
of the
organism to produce the compound according to the invention remaining
functional
and inheritable.
Such mutants can be generated, in a manner known per se, by physical means,
for
example irradiation, such as with ultraviolet rays or X-rays, or using
chemical
mutagens, such as ethyl methanesulfonate (EMS); 2-hydroxy-4-methoxy-benzo-
phenone (MOB) or N-methyl-N'-vitro-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 demonstrate pronounced physiological
flexibility. In phenotypic adaptation, all the cells in the microorganism are
involved,
with the nature of the change not being genetically conditioned and being
reversible
under altered circumstances (H. Stolp, Microbial ecology: organism, habitats,
activities. Cambridge University Press, Cambridge, GB, page 180, 1988).
CA 02467251 2004-05-14
12
Screening for mutants and variants which produce the antibiotic according to
the
invention can be carried out by determining the biological activity of the
active
compound which has accumulated in the culture broth, for example by
determining its
antibacterial effect, or by detecting compounds, which are known to be
antibacterially
active, in the fermentation broth using HPLC or LC-MS methods, for example.
The compound gabusectin is found both in the mycelium and in the culture
filtrate. It
is therefore expedient to separate the fermentation solution into the culture
filtrate
and the mycelium by means of filtration and to dry these fractions separately.
The
dried culture filtrate and the dried mycelium are expediently extracted
separately with
an organic solvent, for example methanol or 2-propanol.
While the extraction can be carried out over a wide pH range, it is expedient
to carry
it out in a neutral or weakly acidic medium, preferably between pH 3 and pH 7.
The
extract can, for example, be concentrated and dried in vacuo.
One method of isolating the antibiotic according to the invention proceeds in
accordance with the polarity separation principle, in a manner known per se.
Another method of purification is chromatography on adsorption resins, for
example
on Diaion~ HP-20 (Mitsubishi Casei Corp., Tokyo), on Amberlite~ XAD 7 (Rohm
and
Haas, USA), on Amberchrom~ CG, (Toso Haas, Philadelphia, USA) or on similar
materials. A large number of reverse-phase supports, such as RPg and RPIg, as
have become well known, for example, within the context of high pressure
liquid
chromatography (HPLC), are also suitable.
Another possibility for purifying the compound according to the invention is
that of
using what are termed normal-phase chromatography supports, such as silica gel
or
AI203, or other supports, in a manner known per se.
An alternative isolation method is that of using molecular sieves, such as
Fractogel~
TSK HW-40 (Merck, Germany) and others, in a manner known per se. In addition
to
this, it is also possible to isolate the gabusectin by crystallization from
enriched
material. Organic solvents and their mixtures, either anhydrous or containing
added
CA 02467251 2004-05-14
13
water, 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 of from 4 to 10, and cation exchangers,
preferably in a pH range of from 2 to 5. The use of buffer solutions to which
quantities
of organic solvents have been added is particularly suitable for this purpose.
Gabusectin, the said chemical derivatives thereof, and the obvious chemical
equivalents thereof, can be converted into the corresponding pharmacologically
tolerated salts using methods known to a skilled person.
Pharmacologically tolerated salts of the compounds according to the invention
are
understood as being both inorganic and organic salts, as are described in
Remington's Pharmaceutical Sciences (17th edition, page 1418 [1985]). Suitable
salts are, in particular, alkali metal salts, ammonium salts, 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.
It has been found, surprisingly, that the compounds of the formula (I)
according to the
invention exhibit antibacterial effects and are therefore suitable for the
treatment of
diseases which are caused by bacterial infection. Table 1 summarizes the
minimum
inhibitory concentrations (MICs) of gabusectin, by way of example.
Table 1: In-vitro antibacterial activity of the compound gabusectin in a
serial
dilution test.
Bacterium (strain) MIC values (,ug/ml)
S.aureus (SG511 ) 5
S.aureus (Exp54146) 20
S.pyogenes {A561 ) 20
E.faecium (M78L) 40
Gabusectin is well-tolerated at and above its effective concentration.
The present invention therefore also relates to the use of one or more of the
compounds of the formula (I) to (VII) according to the invention as
pharmaceuticals,
and the use of one or more of the compounds of the formula (I) to (VII)
according to
__.~._ _.___.__ ~._____ _._. _ __ . ...__._
CA 02467251 2004-05-14
14
the invention for producing pharmaceuticals, in particular for the treatment
and/or
prophylaxis of bacterial infections.
The present invention furthermore relates to a pharmaceutical which has a
content of
one or more compounds according to the invention.
Said pharmaceutical comprising a compound of the formula (I) is produced using
one
or more physiological auxiliary substances and brought into a suitable
administration
form.
The pharmaceuticals according to the invention can be used enterally (orally),
parenterally (intramuscularly or intravenously), rectally or locally
(topically). They can
be administered in the form of solutions, powders (tablets and capsules,
including
microcapsules), ointments (creams or gels), or suppositories. Suitable
auxiliary
substances for such formulations are the pharmaceutically customary liquid or
solid
fillers and extenders, solvents, emulsifiers, glidants, taste corrigents, dyes
and/or
buffering substances. 0.1 - 1 000, preferably 0.2 - 100, mg/kg of body weight
is/are
administered as an expedient dose. The doses are expediently administered in
dosage units which contain at least the effective daily quantity of the
compounds
according to the invention, for example 30 - 3 000, preferably 50 - 1 000, mg.
The following examples are intended to be used for clarifying the invention
without in
any way restricting its scope.
Example 1 Preparing a glycerol culture of ST 003236 (DSM 14476).
ml of nutrient solution (malt extract, 2.0%, yeast extract, 0.2%, glucose,
1.0%,
(NH4)2HP04, 0.05%, pH 6.0) were inoculated with the strain ST 003236 (DSM
14476)
in a sterile 100 ml Erlenmeyer flask and incubated for 6 days, at 25°C
and 140 rpm,
30 on a rotating shaker. 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 ST 003236 (DSM 14476) in an
Erlenmeyer flask.
CA 02467251 2004-05-14
100 ml of nutrient solution (malt extract, 2.0%, yeast extract, 0.2%, glucose,
1.0%,
(NH4)2HP04, 0.05%, pH 6.0) were inoculated with an ampoule of the strain
ST 003236 (DSM 14476) in a sterile 300 ml Erlenmeyer flask and incubated for
5 6 days at 25°C and 140 rpm. 2 ml of this preliminary culture were
subsequently
inoculated for preparing the main cultures.
Example 3 Preparing a liquid main culture of ST 003236 (DSM 14476).
10 A sterile 300 ml Erlenmeyer flask containing 100 ml of the following
nutrient solution:
potato dextrose, 2.4%, yeast extract, 0.2%, was inoculated with a culture
grown on a
sloping tube (same nutrient solution but containing 2% agar) or with 2 ml of a
preliminary culture (see example 2) and incubated, at 140 rpm and 25°C,
on a
shaker. The maximum production of one or more compounds of the formula (I)
15 according to the invention was reached after approx. 144 hours. A 96 hour-
old
submerged culture from the same nutrient solution (inoculation quantity,
approx.
10%) was adequate for inoculating fermenters of from 10 to 200 I in volume.
The
conditions for these fermenters were:
Temperature: 25°C
Stirrer speed: 200 rpm
Aeration: 15 I. Min-.
It was possible to suppress foam formation by repeatedly adding ethanolic
polyol
solution. The production maximum was achieved after approx. 96 to 144 hours.
Example 4: Isolating the compound gabusectin.
3 I of culture solution, obtained as described in example 3, were filtered and
the
culture filtrate and the mycelium were freeze-dried separately. The dried
culture
filtrate was extracted with 3 liters of methanol. The clear liquid phase was
concentrated down to 200 ml in vacuo and filtered. This methanol solution was
mixed
with water in a ratio of 9:1 in an HPLC high pressure gradient unit and loaded
onto a
300 ml-capacity column filled with the adsorption resin MCI Gel~ CHP20P
(Mitsubishi Casei Corp., Tokyo). Column dimensions: width x height: 5 cm x 15
cm.
The column was eluted with a solvent gradient of water to 100% methanol and
the
CA 02467251 2004-05-14
16
outflow from the column (50 ml/minute) was collected in fractions of in each
case
25 ml in volume. The gabusectin-containing fractions 65 to 75, which were
checked
by HPLC analyses, were collected and concentrated in vacuo and freeze-dried
(0.23 g).
Example 5: Purifying gabusectin by high pressure liq«id chromatography (NPLC).
Column: Purospher O STAR RP-18 a 3,urn, 30-2,
(Merck, Germany)
Mobile phase buffer A: 5% acetonitrile + 0.1 % ammonium acetate,
Mobile phase buffer B: 95% acetonitrile + 0.1 % ammonium acetate,
Gradient: 15 min
Flow rate: 0.25 ml per minute
Detection by UV absorption at 210 nm.
Gabusectin was found to have a retention time of 6.5 min.
Example 6: Final purification of gabusectin.
The enriched antibiotic gabusectin (0.23 g), obtained as described in example
4, was
fractionated on a LUNA~ 10 Nm C 18(2) HPLC column (Phenomenex, USA) (width x
height = 2.1 cm x 25 cm) by the gradient method using from 5% to 95%
acetonitrile in
0.05% trifluoroacetic acid. Flow rate: 25 ml/min. Fraction size: 25 ml.
Fraction 48,
which was examined by analytical HPLC (see example 5) was freeze-dried. It
yielded
50 mg of gabusectin at 98% purity.
Example 7: Characterizing gabusectin.
The physicochemical and spectroscopic properties of the antibiotic according
to the
invention can be summarized as follows:
Appearance:
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Colorless to pale yellow substance which is soluble in medium-polar and polar
organic solvents but not particularly soluble in water. Stable in neutral and
mildly
acidic medium but unstable in strongly acidic and strongly alkali solution.
Empirical formula: C26H37N05
Molecular weight: 443.59
1H NMR and 13C NMR: see table 2
UV maxima: 236 nm and 294 nm
Determining the molar peak:
The mass of 443 is assigned to the sought-after molecule on the basis of the
following findings: ESI+ spectrum and FAB+ spectra exhibit peaks at 444 amu
(M+H)+. ESI spectrum exhibits a peak at 442 amu (M-H) . High resolution of the
quasi molecule ion: FAB+ 444.27424 (M+H)+. 443.59 was calculated for the
empirical formula C26H37NO5.
Table 2: 1 H- and 13C-chemical shifts of gabusectin in CDC13 at 275K.
CH3
O N 17 2o p
16 18 ~22
19
HO 14 15 21 OH
H =: CH3 O
9 . ~ CH3
8 10 1 2 113
12
5 4
HsC ,,,.~ s CH / 3 CH3
3
Position 13C g (ppm)1 H 8 (ppm)HMBC correlations (13C-1 H)
1 49.01 H3-Me(w), H1-Me, H2(w), H10,
14-OH
1-Me 20.77 1.24 s -
2 45.69 3.36 H3-Me, H9',H1-Me, H10, H4, H12(s),
H11(w)
3 132.86 H11(w), H2, H3-Me(s), H6'(w)
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Position 13C g (ppm)1 H g (ppm)HMBC correlations (13C-1 H)
3-Me 23.43 1.69 s H4
4 130.04 5.06 H2, H10, H3-Me(s), H6', H5-Me(s)
37.61 H6', H7-Me(w), H5-Me(s), H10,
H4(s)
5-Me 31.91 1 0.70 H3-Me, H6', H10(w)
6 51:62 1.36, 0.92H3-Me(w), H9', H7-Me, H5-Me,
H4(w)
7 29.46 1.26 H6(w), H6', H9(w), H7-Me(s),
H5-Me(w)
7-Me 22.41 0.81 d -
8 34.97 1.65, 0.87H9(w), H9'(w), H6(w), H6', H7-Me(s)
9 25.52 1.84, 1.29H 10
42.17 2.66 dd H9', H1-Me, H5-Me, H2(w), H4
11 132.25 5.31 H12, H2(w), H13(s)
12 128.95 5.44 H 11, H2, H 13(s)
13 17.90 1.69 H12, H11
14 203.37 14-OH, H2, H10, H1-Me
14-OH 17.73 -
98.81 14-OH
16 177.05 14-OH, H18(s), H17-Me(s)
17-Me 27.20 3.02 s
18 64.27 3.76 dd H17-Me, H20, H20'
19 190.36 H 18, H20(w), H20'
23.27 2.32, 2.08H21, H 18
21 27.44 2.30, 2.30H20, H20', H18
22 178.18 H20, H21
22-COON 7.1 br -
Example 8: Inhibitory effect of gabusectin in the agar diffusion test.
Agar plates containing 2 ml of Staphylococcus aureus inoculum in 200 ml of
agar
5 solution were prepared. gabusectin was applied, in a 10 mM solution, to 6 mm-
diameter paper disks, which were then laid on the agar plate. The inoculated
Staphylococcus plates were incubated at 37°C for 16 hours. Inhibition
halos having
the following diameters (mm) were then observed:
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Quantity Inhibition halo size~(mm)
,uL 8
NL 14
40 ~L 17
Example 9: Methylation, and subsequent purification of the gabusectin methyl
ester.
5 20 mg of the antibiotic gabusectin (0.045 mmol), obtained as described in
example 6,
were dissolved in 5 ml of MeOH, after which trimethylsilyldiazomethane was
added in
a 6-fold molar excess. The reaction mixture was left to stand at room
temperature for
60 min and then concentrated to dryness. The resulting mixture was
fractionated
chromatographically on a LUNA~ 5 Nm C 18(2) HPLC column (Phenomenex, USA)
10 (width x height = 1 cm x 25 cm) by the gradient method using from 5% to 95%
acetonitrile in 0.05% trifluoroacetic acid. Flow rate: 6.5 ml/min. Fraction
size: 6.5 ml.
Fraction 61, which was examined by analytical HPLC (see example 5), was freeze-
dried. It yielded 7.4 mg of gabusectin methyl ester at 97% purity.
15 Example 10: Characterizing gabusectin methyl ester.
The physicochemical and spectroscopic properties of the antibiotic according
to the
invention can be summarized as follows:
20 Appearance:
Colorless to pale-yellow substance which is soluble in medium-polar and polar
organic solvents but not particularly soluble in water. Stable in neutral and
mildly
acidic medium but unstable in strongly acidic and strongly alkaline solution.
Empirical formula: C27H3gN05
Molecular weight: 457.62
~H NMR and'3C NMR: see table 3
UV maxima: 236 nm and 294 nm
~_ _ _~_~ . _ ~. __~._ _ _ . _ _ _ _ ____ . _.__.__. __ _. ~__ __. _.
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Determination of the molar peak:
The mass of 457.6 is assigned to the sought-after molecule on the basis of the
following findings: ESI+ spectrum and FAB+ spectra exhibit peaks at 457 amu
(M+H)+. ESI- spectrum exhibits a peak at 458.5 amu (M-H)- .
5
Table 3: 1 H and 13C chemical shifts of gabusectin methyl ester in CDCI3 at
275K
CH3
O N1~ ~ O
16 16 ~22
1g
HO 14~ 15 21 ~-CHg
H =; CH30
- - \ CH3
8 10 1 2 113
12
H C ~~~' 6 =5 ~ 3 CH
3 CH3 3
Position 13C b (ppm)1 H 8 (ppm)HMBC correlations (13C- 1 H)
1 48.89 H3-Me(w), H1-Me, H10(w)
1-Me 20.83 1.24 -
2 45.70 3.36 H3-Me, H1-Me, H9', H10(w), H12,
H4 -
3 132.86 H3-Me
3-Me 23.44 1.68 H4
4 130.08 5.05 H3-Me, H6', H5-Me, H10(w)
5 37.63 H6', H5-Me, H10(w), H4(w)
5-Me 31.91 0.70 H10(w), H6', H3-Me(w)
6 51.65 1-35, 0.92H9', H7-Me, H5-Me, H4(w)
7 29.48 1.25 H6', H7-Me
7-Me 22.42 0.80 -
8 35.00 1.64, 0.88H6', H7-Me(s)
9 25.55 1.83, 1.30H 10
10 42.17 2.67 H1-Me, H5-Me, H9', H4(w)
11 132.35 5.30 H 12, H 13
12 128.87 5.43 H 11, H 13
_.......T.__ .__._. ____.__.....,__._.
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Position 13C g (ppm)1 H 8 (ppm)HMBC correlations (13C- 1 H)
13 17.90 1.69 H 12, H 11
14 202.86 H1-Me
14-OH 17.75 -
-
15 98.91 -
16 177.06 H 17-Me
17-Me 27.13 3.02 -
18 64.49 3.71 H20, H20', H21, H17-Me
19 190.33 H18, H20'
20 23.52 2.29, 2.11H21
21 27.42 2.23, 2.23-
22 173.05 22-OMe, H20, H20', H21
22-OMe 51.93 3.66 -
Example 11: Inhibitory effect of the gabusectin methyl ester in the agar
diffusion test.
Rgar plates containing 2 ml of Staphylococcus aureus inoculum in 200 ml of
agar
solution were prepared. gabusectin methyl ester is applied, in a 10 mM
solution, to
6 mm-diameter paper disks, which are then laid on the agar plate. The
inoculated
Staphylococcus plates were incubated at 37°C for 16 hours. Inhibition
halos having
the following diameters (mm) were then observed.
Quantity Inhibition halo size
(mm)
NL 0
NL 7
40 NL 8
._~. _ ~_~ . ~ __ _ __ _.
