Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2001-05-29
2003-08-26
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S118000, C435S121000, C435S170000, C435S189000
Reexamination Certificate
active
06610518
ABSTRACT:
The following invention relates to a process for oxidising alkyl groups attached directly or via a linker, to a sulfonamide moiety by the use of a cytochrome P450 enzyme.
There are a large number of structurally complex molecules of interest to medicinal chemists, which possess a hydroxylated or carboxylated alkyl group, attached directly or via a linker, to a sulfonamide group. The synthesis of these molecules is complicated by the synthetic steps necessary to hydroxylate or carboxylate such alkyl groups whilst avoiding-undesirable side reactions.
This problem has been solved by the process of the present invention, which teaches the use of a cytochrome P450 enzyme to selectively oxidise an alkyl group attached directly or via a linker to a sulfonamide group. The process which is specific and tolerant of other functional groups in the molecule allows the synthetic chemist to introduce a hydroxy or carboxy group into the synthesis of a molecule at a late stage, in a one step process. This reduces the overall number of steps required to synthesise medicinally important molecules and so improves the efficiency of their preparation.
The term ‘Cytochrome P450’ is used to describe a superfamily of hemoprotein enzymes. They are characterised by the absorption band of their Fe
II
—CO complex form which has an absorption maxima at 447-452 nm, indicative of a thiolate ligated hemoprotein. They are also called heme-thiolate proteins, though this category is much wider than just cytochrome P450. Cytochrome P450 appear to have diversified from a common ancestor and they may be found in almost all forms of living organism, from animals and plants to fungi and bacteria, where they carry out the role of an oxygenase.
Cytochrome P450 catalysed reactions are known to produce a range of metabolites. The reactions may usually be classified as one of three types:
a) Hydroxylation, the insertion of an oxygen atom between the H atom and some other heavier atom such as carbon or nitrogen,
b) Epoxidation, the addition of an oxygen atom to a carbon-carbon double bond,
c) Heteroatom oxidation, the addition of an oxygen atom to the electron pair on a heteroatom.
They are also known to catalyse reduction reactions. Useful reviews of the cytochrome P450 enzyme are provided in Handbook of Drug metabolism, Ed. Thomas Woolf, Publisher Marcel Decker, ISBN 0824702298, March 1999, chap 4, p109 and by T. Omura, Biochemical and Biophysical Research Communications, 1999, 266, 690.
R. A. Johnson et al., Biooganic Chemistry, 1973, 2, 99, discloses the treatment of a number of dialkylbenzenes with the P450 enzyme containing microorganism
Sporotrichum sulfurescens
, to form an alcohol on the alkyl side chain.
The abstract of JP-60258173 discloses the synthesis of an &agr;-(3-t-butyl-5-hydroxy-t-butylbenzylidene)butyrolactone by microbial hydroxylation of the corresponding t-butyl precursor. Suitable microorganisms for the transformation are those from the Mucor and Aspergillus strains.
C. Cerniglia et al., Applied and Environmental Microbiology, 1984, 47, 111, discloses the transformation of 1- and 2-methylnaphthalene by
Cunninghamella elegans
into the corresponding hydroxymethyl derivative. A small amount of further metabolites were also isolated in which the product had been further oxidised to the carboxylic acid or where hydroxylation of the ring system had taken place.
H. L. Holland et al., Tetrahedron Asymm., 1994, 5, 1241, teaches the oxidation of chiral para substituted alkyl benzyl sulfides to the sulfoxide. In cases where the para substituent was i-propyl or t-butyl, hydroxylation at the terminal methyl of the alkyl group was seen, as well as oxidation of the sulfide. In the case of the i-propyl group, the corresponding sulfone was also seen. These transformations were conducted using the microorganism Helminthosporium NRRL-4671.
H. Schwartz et al., Appl. Microbiol. Biotechnol., 1994, 44, 731, investigates the microbial oxidation of ebastine to carebastine. Of 15 microorganisms examined, only the Cunninghamella strains provided the desired biotransformation.
WO-A-99/47693 discloses a method for making fexofenadine from terfenadine by a biotransformation performed using a microorganism culture of the genus Streptomyces at a pH ranging between 5 and 8.
The oxidation by a cytochrome P450 enzyme of alkyl groups attached directly, or via a linker, to a sulfonamide moiety, is new. Sulfonamides are known to exhibit antimicrobial activity. Accordingly, molecules containing a sulfonamide group have not been viewed as suitable candidates for use in a biotransformation process as it was thought they would kill any microorganism used. Surprisingly, it has now been found that molecules containing a sulfonamide group may be used in certain oxidative biotransformations.
According to a first aspect of the present invention there is provided a process for synthesising the compounds of formula (I)
R—NHSO
2
—X—Y—Z (I)
wherein
R is an organic radical;
X is selected from
a) a 5- or 6-membered monocyclic aromatic ring optionally containing one or two heteroatoms, each independently selected from O, N and S;
b) a C
1
-C
6
alkylene group, straight chain or branched chain; and
c) a direct link;
Y is —C(CH
3
)
2
— or —CH(CH
3
)—; and
Z is —CH
2
OH or —COOH;
which comprises oxidising a compound of formula (II)
R—NHSO
2
—X—Y—CH
3
(II)
wherein
R, X and Y are as defined above, with a cytochrome P450 enzyme.
The process described may be used to oxidise a suitable alkyl group to the corresponding hydroxy or carboxy derivative. These oxidations proceed from the alkyl to the hydroxy and in the latter case, onwards from the hydroxy to the corresponding carboxylic acid. It will be appreciated that by careful manipulation of the reaction conditions, one may selectively isolate a product at the desired oxidation level and maximise the yield of the desired product. The oxidation may be stopped at the hydroxy derivative or allowed to continue onwards to the carboxylic acid. To produce the corresponding carboxylic acid no modification to the process is required other than that the process be given sufficient time to further oxidise the substrate. It should be noted that the nature of the R substituent may influence the speed of the reaction and hence the yields of the desired products. Whilst all P450 enzymes are considered suitable for conducting the disclosed invention, certain microorganisms will be particularly suitable for specific levels of oxidation.
This transformation is effected by a cytochrome P450 enzyme. Particularly favoured are microorganisms which contain the P450 enzyme. Preferred microorganisms are unicellular bacteria, exemplified by species such as
Escherichia coli
, filamentous bacteria exemplified by such strains as Actinomyces and Streptomyces and filamentous fungi. Suitable microorganisms are specifically noted in this text by their name and the American Type Culture Collection (ATCC) number assigned to them when deposited with a recognised International Depository Authority under the terms of the Budapest Treaty. Three new microorganisms were identified as being effective in this transformation and deposits were made with the American Type Culture Collection in Manassas, USA, under the terms of the Budapest Treaty. All three are gram positive filamentous bacteria, belonging to the Actinomycetales: Streptomyces species PTA-1685,
Streptomyces cyaneus
PTA-1686 and
Streptomyces lydicus
PTA-1687 and were cultivated on quarter strength ATCC172 agar slope.
The new microorganisms assigned Accession Numbers PTA-1685, PTA-1686 and PTA-1687 were all deposited with the ATCC at 10801 University Boulevard, Manassas, Va., 20110-2209, USA, on Apr. 11, 2000.
In a preferred embodiment X is selected from:
a) a 5- or 6-membered monocyclic aromatic ring optionally containing one or two heteroatoms, each independently selected from O, N and S; and
b) a C
1
-C
6
alkylene group, straight chain or branched chain;
When X is as defined in (a) above, X may suitably be phenylene or a 5 or 6 membered aromatic hete
Ginsburg Paul H.
Kohn & Associates PLLC
Lilling Herbert J.
Munchhof Martha G.
Pfizer Inc.
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