Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
1995-04-18
2002-12-31
Raymond, Richard L. (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Heterocyclic carbon compounds containing a hetero ring...
C544S258000
Reexamination Certificate
active
06500829
ABSTRACT:
The present invention relates to the preparation of substantially pure diastereoisomers of derivatives of tetrahydrofolate and the use of such diastereoisomers.
Methotrexate (N-(4-((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-L-glutamic acid) is an inhibitor of the enzyme dihydrofolate reductase (DHFR) which prevents the conversion of deoxyuridylate into thymidylate. It thus prevents the biosynthesis of DNA and is commonly used in cancer chemotherapy. However, in common with most anticancer agents, it is toxic to normal cells as well as cancerous cells and hence a “rescue agent” is often administered some 12 to 24 hours after treatment with a high dose of methotrexate. Leucovorin (5-formyltetrahydrofolic acid) is the commonly used rescue agent for methotrexate.
Leucovorin has two chiral centres and the product commercially available (Wellcovorin (RTM) of the Wellcome Foundation Ltd) is composed of equal amounts of the compounds of formulae (Ia) and Ib) (in the form of their calcium salts) which compounds have the (R) and (S) stereochemistry respectively at C-6.
It has been reported (Montgomery et al, Cancer Treatment Reports, 1981, 65, 1117-1119) that only the (6S) diastereoisomer (Ib), which is the natural diastereoisomer, is effective in restoring one-carbon metabolism and thus of use in methotrexate rescue.
Indeed there are reports (Leary et al, Biochem, Biophys. Res. Commun., 1973, 56, 484) that thymidylate synthase from
L. casei
is inhibited by the non-natural dia stereoisomer of 5,10-methylene tetrahydrofolate and that 5,10-methylene tetrahydrofolate dehydrogenase from
E. coli
is also inhibited by the same diastereoisomer (Scott and Donaldson, Biochem. Biophys. Res. Commun., 1964, 14, 523). Moreover, the non-natural diastereoisomer of 10-formyltetrahydrofolate is a potent competitive inhibitor of glycinamide ribonucleotide (GAR) formyl-transferase from chicken liver (Smith, Benkovic and Benkovic, Biochemistry, 1981, 20, 4034).
These results imply inhibition of both pyrimidine and purine biosynthesis, and thus of DNA biosynthesis also, by the non-natural diastereoisomers of one-carbon derivatives of tetrahydrofolate. If this inhibition is also present in mammalian systems there is a potential clinical requirement for the natural (6S) diastereoisomer of leucovorin.
Separation of the two diastereoisomers of leucovorin has been carried out by fractional crystallisation (Cosulich et al, J Amer.Chem.Soc, 1952, 74,4215) and by chromatography (Feeney et al., Biochemistry, 1981, 20, 1837) and the (6S) diastereoisomer has been obtained by the enzyme catalysed reduction of dihydrofolate followed-by formylation (Rees, Valente, Suckling and Wood, Tetrahedron, 1986, 42, 117) but the yields are low in the latter and the isomers difficult to obtain in reasonable purity in the former. It is an object of the present invention to avoid or minimize one or more of the above disadvantages.
It has now been found that the introduction of a chiral auxiliary group into tetrahydrofolate or a tetrahydrofolate derivative close to the epimeric centre at C-6 enables ready separation of the new pair of diastereoisomers and the conversion of the separated diastereoisomers into pure (Ia) and (Ib) in good yield.
REFERENCES:
patent: 2688018 (1954-08-01), Cosulich
patent: 4959472 (1990-09-01), Wood et al.
patent: 5010194 (1991-04-01), Mueller
patent: 5134235 (1992-07-01), Mueller et al.
patent: 5347005 (1994-09-01), Mueller et al.
Temple et al. Canc. Treat. Reports vol. 65 pp. 1117-9 (1981) Merck Index 10thEdition, 1983 entry 4111.*
Rees et al. J. Chem Soc. Chem. Comm., 1987, p 470-2.*
Fontelilla-Camps et al. J. Am. Chem. Soc. vol. 101, 614-5 (1979).*
Rees et al., “Asymmetric Reduction of Dihydrofolate Using Dihydrofolate Reductase and Chiral Boron-Containing Compounds”, Tetrahedron, vol. 42, No. 1, pp. 117-136 (1986).
Couslich et al. Jour. Am. Chem. Soc vol. 74 p 4215-6 (1952.*
Rees et al., “Asymmetric Reduction of Dihydrofolate Using Dihydrofolate Reductase and Chiral Boron-Containing Compounds”, Tetrahedron, vol. 42, No. 1, pp. 117-118 (1986).
Blakley et al., “Folates and Pterins”, Wiley Interscience, (New York, 1984) vol. 1, p. 99.
Rees Lilias G.
Suckling Colin James
Wood Hamish Christopher Swan
Alston & Bird LLP
Raymond Richard L.
University of Strathclyde
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