[Study on synthesis and purification of calcium 5-formyltetrahydrofolate].

OBJECTIVE: To establish a new approach to synthesis and purification of calcium 5-formytetrahydrofolate. METHODS: Target compound was synthesized by the use of folic acid as starting material via reduction, formylation, hydrolysis and salt formation RESULTS: The structure of calcium 5-formytetrahydrofolate was confirmed by UV, 1H-NMR and elemental analysis and the overall yield was 54%-59%, 14%-17% higher than the reported yield. CONCLUSION: A convenient synthetic route was developed and it would be suitable for industrial production.

Syntheses of labeled vitamers of folic acid to be used as internal standards in stable isotope dilution assays.

[2H4]Folic acid was synthesized by deuterating p-aminobenzoic acid, which was then coupled to glutamic acid and 6-formylpterin. Using [2H4]folic acid as starting component enabled the preparation of labeled vitamers tetrahydrofolate, 5-formyltetrahydrofolate, 5-methyltetrahydrofolate, and 10-formylfolate which were characterized by electrospray mass spectrometry and collision-induced dissociation. The mass spectrometric studies confirmed that the compounds could be used as internal standards in stable isotope dilution assays.

Desarrollo tecnológico y estudio de estabilidad de folinato cálcico 10 mg inyectable liofilizado / Technological development and stability study of 10mg injectable freeze-dried calcium folinate

El folinato cálcico es un antídoto indicado para prevenir la toxicidad grave debido a sobredosis de metotrexato, o la terapia con dosis elevadas de este para tratar las reacciones severas a dosis bajas o moderadas de metotrexato, y como parte de los programas quimioterapéuticos en el tratamiento de varias formas de cáncer, así como en el tratamiento de anemias megaloblásticas. Se empleó la liofilización para la obtención de un producto con la calidad farmacéutica requerida según las especificaciones de la USP 23; además, se desarrolló una técnica analítica para el estudio de la estabilidad del liofilizado con la utilización de la cromatografía líquida de alta resolución, mediante la cual se cuantificó el contenido del principio activo. Se propuso como fecha de vencimiento un período de 3 años a temperatura ambiente(AU)

Biological properties of fluoroglutamate-containing analogs of folates and methotrexate with altered capacities to form poly (gamma-glutamate) metabolites.

Fluoroglutamate-containing analogs of folates and methotrexate (MTX) with altered capacities for poly (gamma-glutamate) metabolism were synthesized to probe the biological roles of polyglutamates. Compared to folic acid, DL-e,t-gamma-fluorofolic acid, a compound that is a poor substrate for polyglutamylation, was approximately 25-fold less potent in promoting growth of folate-depleted H35 rat hepatoma cells. DL-beta,beta-Difluorofolic acid, a compound that forms diglutamates more readily than does folic acid, was at least equivalent to folic acid in potency. Leucovorin (LV), a reduced folate, was 30-fold more potent than folic acid in promoting growth, whereas the analogous form of DL-e,t-gamma-fluorofolate, DL-e,t-gamma-fluoroleucovorin (DL-e,t-gamma-FLV) was only 4-fold more potent than folic acid. Both LV and DL-e,t-gamma-FLV protected or "rescued" cells from the growth inhibitory effects of MTX; however a 37- to 46-fold higher concentration of the fluoro analog was required. Folic acid, DL-e,t-gamma-fluorofolic acid, LV, and DL-e,t-gamma-FLV each potentiated the growth inhibitory effect of 5-fluoro-2'-deoxyuridine on CCRF-CEM human leukemia cells; higher concentrations of fluorinated analogs again were required. Stereochemically pure L-t-gamma-fluoromethotrexate (L-t-gamma-FMTX), a poor substrate for polyglutamylation, was evaluated as a cell growth inhibitor. In continuous exposure, L-t-gamma-FMTX), was 7-fold less potent than MTX as an inhibitor of CCRF-CEM growth. Results with these fluorinated folate and MTX analogs offer insight into the importance of polyglutamate metabolism to these biological and pharmacological effects.

Chemo-enzymatic synthesis of optically pure l-leucovorin, an augmentor of 5-fluorouracil cytotoxicity against cancer.

Optically pure l-leucovorin was synthesized on a large scale by the combination of chemical and enzymatic processes. After reduction of folate with zinc, dihydrofolate was reduced asymmetrically to (6)-tetra-hydrofolate by use of dihydrofolate reductase from E. coli C600/pTP600, with simultaneous NADPH cofactor recycling using glucose dehydrogenase from Gluconobacter scleroideus KY3613. Calcium l-leucovorin.4H2O (113 g) was obtained from (6S)-tetrahydrofolate via 5,10-methyenyltetrahydrofolate by formylation, reflux, addition of calcium ions and floricil column chromatography, with an overall yield of 50% based on folate. The l-leucovorin showed optical purity of 99.9% de as (6S)-form.

Preparation of (6R)-tetrahydrofolic acid and (6R)-5-formyltetrahydrofolic acid of high stereochemical purity.

Commercially available 5-formyltetrahydrofolate (5-CHO-H4PteGlu) is chemically prepared in a reaction that introduces an asymmetric center at the 6 carbon, and hence is the mixture of diastereomers differing in chirality about this position. (6R)-5-CHO-H4PteGlu, the diastereomer that is not normally found in vivo, was prepared from folic acid. Folic acid was chemically reduced and (6R)-tetrahydrofolate (H4PteGlu) was obtained from the resultant (6R,S)-H4PteGlu by enzymatic consumption of the natural diastereomer of (6R,S)-5,10-CH2-H4PteGlu (reversibly formed from (6R,S)-H4PteGlu in the presence of formaldehyde) with Lactobacillus casei thymidylate synthase. The 5 position of purified (6R)-H4PteGlu was directly formylated in a carbodiimide-catalyzed reaction. The level of contamination of these preparations with the corresponding 6S diastereomers was estimated using the binding of fluorodeoxyuridylate to thymidylate synthase promoted by folate cofactor (for H4PteGlu) and by the growth of folate requiring bacteria (for 5-CHO-H4PteGlu). Purified preparations of (6R)-H4PteGlu promoted the binding of fluorodeoxyuridylate to L. casei thymidylate synthase (in the presence of formaldehyde) only at concentrations greater than 1000-fold higher than equiactive levels of (6S)-H4PteGlu. Likewise, the (6R)-5-CHO-H4PteGlu made by this method was 600 times less active as a growth factor for Pediococcus cerevisiae than was authentic (6S)-5-CHO-H4PteGlu. Hence, the minimum stereochemical purity of these preparations was 99.9% for (6R)-H4PteGlu and 99.8% for (6R)-5-CHO-H4PteGlu.