The title compound was isolated by fractional crystallization of the diastereoisomeric mixture of calcium (6R,S)-folinate (Ia-b). This separation has been further improved with the addition of sodium EDTA. The separation of (6R,S) folinic acid has also been described by fractional crystallization of several diamine or polyamine salts of the diastereomeric mixture or by preparative chromatography. Alternatively, the fractional crystallization of 5,10-methenyl-(6R,S)-tetrahydrofolic acid (IIa-b) furnished isomer (III), which was subsequently hydrolyzed to the title compound at a pH of 5.5 to 6.5 in the presence of calcium chloride.

The title compound was isolated by fractional crystallization of the diastereoisomeric mixture of calcium (6R,S)-folinate (Ia-b). This separation has been further improved with the addition of sodium EDTA. The separation of (6R,S) folinic acid has also been described by fractional crystallization of several diamine or polyamine salts of the diastereomeric mixture or by preparative chromatography. Alternatively, the fractional crystallization of 5,10-methenyl-(6R,S)-tetrahydrofolic acid (IIa-b) furnished isomer (III), which was subsequently hydrolyzed to the title compound at a pH of 5.5 to 6.5 in the presence of calcium chloride.

The title compound was isolated by fractional crystallization of the diastereoisomeric mixture of calcium (6R,S)-folinate (Ia-b). This separation has been further improved with the addition of sodium EDTA. The separation of (6R,S) folinic acid has also been described by fractional crystallization of several diamine or polyamine salts of the diastereomeric mixture or by preparative chromatography. Alternatively, the fractional crystallization of 5,10-methenyl-(6R,S)-tetrahydrofolic acid (IIa-b) furnished isomer (III), which was subsequently hydrolyzed to the title compound at a pH of 5.5 to 6.5 in the presence of calcium chloride.

The title compound was isolated by fractional crystallization of the diastereoisomeric mixture of calcium (6R,S)-folinate (Ia-b). This separation has been further improved with the addition of sodium EDTA. The separation of (6R,S) folinic acid has also been described by fractional crystallization of several diamine or polyamine salts of the diastereomeric mixture or by preparative chromatography. Alternatively, the fractional crystallization of 5,10-methenyl-(6R,S)-tetrahydrofolic acid (IIa-b) furnished isomer (III), which was subsequently hydrolyzed to the title compound at a pH of 5.5 to 6.5 in the presence of calcium chloride.

The title compound was isolated by fractional crystallization of the diastereoisomeric mixture of calcium (6R,S)-folinate (Ia-b). This separation has been further improved with the addition of sodium EDTA. The separation of (6R,S) folinic acid has also been described by fractional crystallization of several diamine or polyamine salts of the diastereomeric mixture or by preparative chromatography. Alternatively, the fractional crystallization of 5,10-methenyl-(6R,S)-tetrahydrofolic acid (IIa-b) furnished isomer (III), which was subsequently hydrolyzed to the title compound at a pH of 5.5 to 6.5 in the presence of calcium chloride.

The title compound was isolated by fractional crystallization of the diastereoisomeric mixture of calcium (6R,S)-folinate (Ia-b). This separation has been further improved with the addition of sodium EDTA. The separation of (6R,S) folinic acid has also been described by fractional crystallization of several diamine or polyamine salts of the diastereomeric mixture or by preparative chromatography. Alternatively, the fractional crystallization of 5,10-methenyl-(6R,S)-tetrahydrofolic acid (IIa-b) furnished isomer (III), which was subsequently hydrolyzed to the title compound at a pH of 5.5 to 6.5 in the presence of calcium chloride.

The title compound was isolated by fractional crystallization of the diastereoisomeric mixture of calcium (6R,S)-folinate (Ia-b). This separation has been further improved with the addition of sodium EDTA. The separation of (6R,S) folinic acid has also been described by fractional crystallization of several diamine or polyamine salts of the diastereomeric mixture or by preparative chromatography. Alternatively, the fractional crystallization of 5,10-methenyl-(6R,S)-tetrahydrofolic acid (IIa-b) furnished isomer (III), which was subsequently hydrolyzed to the title compound at a pH of 5.5 to 6.5 in the presence of calcium chloride.

The title compound was isolated by fractional crystallization of the diastereoisomeric mixture of calcium (6R,S)-folinate (Ia-b). This separation has been further improved with the addition of sodium EDTA. The separation of (6R,S) folinic acid has also been described by fractional crystallization of several diamine or polyamine salts of the diastereomeric mixture or by preparative chromatography. Alternatively, the fractional crystallization of 5,10-methenyl-(6R,S)-tetrahydrofolic acid (IIa-b) furnished isomer (III), which was subsequently hydrolyzed to the title compound at a pH of 5.5 to 6.5 in the presence of calcium chloride.

The stereospecific synthesis for the title chiral compound was developed, based on the enzymatic reduction of dihydrofolate (V). Reduction of folic acid (IV) to dihydrofolate (V) was effected by means of either sodium dithionite or zinc dust and NaOH. Dihydrofolate reductase-catalyzed asymmetric reduction of (V) in the presence of several recycling enzymes for the reducing cofactor NADPH furnished (6S)-tetrahydrofolate (VI), which was subsequently isolated as the 5,10-methenyl derivative (III) upon treatment with formic acid and trifluoroacetic acid. Aqueous hydrolysis of (III) at a pH of 6.5-6.9 led to the title 5-formyl compound. The chiral (6S)-tetrahydrofolate (VI) has also been obtained by fractional crystallization of the sulfate and several sulfonate salts of the diastereomeric mixture of (6R,S)-tetrahydrofolates from a polar medium.

Alternatively, folic acid (IV) was chemically reduced to the (6R,S)-epimeric mixture of tetrahydrofolates (VII) with sodium borohydride in aqueous NaOH. After derivatization of (VII) with (-)-menthyl chloroformate (VIII), the desired (6S) carbamate (IX) was isolated in high diastereoisomeric purity, taking advantage of the different solubility of the diastereoisomers in n-butanol. The menthyloxycarbonyl derivative (IX) was converted to the 5,10-methenyltetrahydrofolate (III) using a mixture of formic acid and acetic acid, saturated with HBr. The cyclic formamidinium salt (III) was then hydrolyzed to the 5-formyl compound, which was finally converted to the corresponding calcium salt. In a closely related method, folic acid (IV) was hydrogenated in the presence of rhodium (I) catalysts supported on an optically active phosphane to provide a diastereomeric mixture of tetrahydrofolate epimers (VII) with moderate diastereomeric excess of the desired (6S) isomer. After derivatization with menthyl chloroformate, the resultant mixture of epimeric mono- and bis-menthyl carbamates was separated using preparative HPLC. The target carbamate (IX) was then converted into the title compound as outlined above.

In an alternative enzymatic synthesis, exposure of the diastereoisomeric (6R,S)-tetrahydrofolates (VIIa-b), with a recombinant enzymatic domain as the source of 10-formyltetrahydrofolate synthetase activity, led to the diastereoselective formylation the (6S) tetrahydrofolate at the 10-N (XXIV), while leaving unchanged the unnatural (6R) isomer (XXIII). The chiral 10-formyl tetrahydrofolate (XXIV) was subsequently isomerized to (6S)-5-formyltetrahydrofolate either under acidic conditions, via formation of the intermediate 5,10-methenyl derivative (III) (22), or in the presence of a source of cyclohydrolase activity.

The cyclic orthoamide (XXVa-b) was prepared from either the diastereomeric mixture of 5,10-methenyl-(6R,S)-tetrahydrofolic acids (IIa-b) or from calcium (6R,S)-folinate (Ia-b) in a formic acid/ammonium formate buffer at a pH of 2.3. After isolation of the pure (6S) diastereoisomer (XXV) by repeated recrystallization from aqueous formic acid, the orthoamide function was hydrolyzed employing a buffered solution of tris(hydroxymethyl)aminomethane at a pH of 7.3 to furnish the title compound.

In a further method for the separation of the diastereomeric mixture, (6R,S)-folinic acid (XXVIa-b) was converted to the alpha 2,6-dichlorobenzyl ester upon treatment with 2,6-dichlorobenzyl bromide (XXVII) and Na2CO3. The resultant diastereomeric esters were easily separated by column chromatography to provide the (6S)-isomer (XXVIII), which was finally converted to the title compound by saponification of the benzyl ester group.

The stereospecific synthesis for the title chiral compound was developed, based on the enzymatic reduction of dihydrofolate (V). Reduction of folic acid (IV) to dihydrofolate (V) was effected by means of either sodium dithionite or zinc dust and NaOH. Dihydrofolate reductase-catalyzed asymmetric reduction of (V) in the presence of several recycling enzymes for the reducing cofactor NADPH furnished (6S)-tetrahydrofolate (VI), which was subsequently isolated as the 5,10-methenyl derivative (III) upon treatment with formic acid and trifluoroacetic acid. Aqueous hydrolysis of (III) at a pH of 6.5-6.9 led to the title 5-formyl compound. The chiral (6S)-tetrahydrofolate (VI) has also been obtained by fractional crystallization of the sulfate and several sulfonate salts of the diastereomeric mixture of (6R,S)-tetrahydrofolates from a polar medium.

The stereospecific synthesis for the title chiral compound was developed, based on the enzymatic reduction of dihydrofolate (V). Reduction of folic acid (IV) to dihydrofolate (V) was effected by means of either sodium dithionite or zinc dust and NaOH. Dihydrofolate reductase-catalyzed asymmetric reduction of (V) in the presence of several recycling enzymes for the reducing cofactor NADPH furnished (6S)-tetrahydrofolate (VI), which was subsequently isolated as the 5,10-methenyl derivative (III) upon treatment with formic acid and trifluoroacetic acid. Aqueous hydrolysis of (III) at a pH of 6.5-6.9 led to the title 5-formyl compound. The chiral (6S)-tetrahydrofolate (VI) has also been obtained by fractional crystallization of the sulfate and several sulfonate salts of the diastereomeric mixture of (6R,S)-tetrahydrofolates from a polar medium.

The stereospecific synthesis for the title chiral compound was developed, based on the enzymatic reduction of dihydrofolate (V). Reduction of folic acid (IV) to dihydrofolate (V) was effected by means of either sodium dithionite or zinc dust and NaOH. Dihydrofolate reductase-catalyzed asymmetric reduction of (V) in the presence of several recycling enzymes for the reducing cofactor NADPH furnished (6S)-tetrahydrofolate (VI), which was subsequently isolated as the 5,10-methenyl derivative (III) upon treatment with formic acid and trifluoroacetic acid. Aqueous hydrolysis of (III) at a pH of 6.5-6.9 led to the title 5-formyl compound. The chiral (6S)-tetrahydrofolate (VI) has also been obtained by fractional crystallization of the sulfate and several sulfonate salts of the diastereomeric mixture of (6R,S)-tetrahydrofolates from a polar medium.

The stereospecific synthesis for the title chiral compound was developed, based on the enzymatic reduction of dihydrofolate (V). Reduction of folic acid (IV) to dihydrofolate (V) was effected by means of either sodium dithionite or zinc dust and NaOH. Dihydrofolate reductase-catalyzed asymmetric reduction of (V) in the presence of several recycling enzymes for the reducing cofactor NADPH furnished (6S)-tetrahydrofolate (VI), which was subsequently isolated as the 5,10-methenyl derivative (III) upon treatment with formic acid and trifluoroacetic acid. Aqueous hydrolysis of (III) at a pH of 6.5-6.9 led to the title 5-formyl compound. The chiral (6S)-tetrahydrofolate (VI) has also been obtained by fractional crystallization of the sulfate and several sulfonate salts of the diastereomeric mixture of (6R,S)-tetrahydrofolates from a polar medium.

Alternatively, folic acid (IV) was chemically reduced to the (6R,S)-epimeric mixture of tetrahydrofolates (VII) with sodium borohydride in aqueous NaOH. After derivatization of (VII) with (-)-menthyl chloroformate (VIII), the desired (6S) carbamate (IX) was isolated in high diastereoisomeric purity, taking advantage of the different solubility of the diastereoisomers in n-butanol. The menthyloxycarbonyl derivative (IX) was converted to the 5,10-methenyltetrahydrofolate (III) using a mixture of formic acid and acetic acid, saturated with HBr. The cyclic formamidinium salt (III) was then hydrolyzed to the 5-formyl compound, which was finally converted to the corresponding calcium salt. In a closely related method, folic acid (IV) was hydrogenated in the presence of rhodium (I) catalysts supported on an optically active phosphane to provide a diastereomeric mixture of tetrahydrofolate epimers (VII) with moderate diastereomeric excess of the desired (6S) isomer. After derivatization with menthyl chloroformate, the resultant mixture of epimeric mono- and bis-menthyl carbamates was separated using preparative HPLC. The target carbamate (IX) was then converted into the title compound as outlined above.

Alternatively, folic acid (IV) was chemically reduced to the (6R,S)-epimeric mixture of tetrahydrofolates (VII) with sodium borohydride in aqueous NaOH. After derivatization of (VII) with (-)-menthyl chloroformate (VIII), the desired (6S) carbamate (IX) was isolated in high diastereoisomeric purity, taking advantage of the different solubility of the diastereoisomers in n-butanol. The menthyloxycarbonyl derivative (IX) was converted to the 5,10-methenyltetrahydrofolate (III) using a mixture of formic acid and acetic acid, saturated with HBr. The cyclic formamidinium salt (III) was then hydrolyzed to the 5-formyl compound, which was finally converted to the corresponding calcium salt. In a closely related method, folic acid (IV) was hydrogenated in the presence of rhodium (I) catalysts supported on an optically active phosphane to provide a diastereomeric mixture of tetrahydrofolate epimers (VII) with moderate diastereomeric excess of the desired (6S) isomer. After derivatization with menthyl chloroformate, the resultant mixture of epimeric mono- and bis-menthyl carbamates was separated using preparative HPLC. The target carbamate (IX) was then converted into the title compound as outlined above.

Alternatively, folic acid (IV) was chemically reduced to the (6R,S)-epimeric mixture of tetrahydrofolates (VII) with sodium borohydride in aqueous NaOH. After derivatization of (VII) with (-)-menthyl chloroformate (VIII), the desired (6S) carbamate (IX) was isolated in high diastereoisomeric purity, taking advantage of the different solubility of the diastereoisomers in n-butanol. The menthyloxycarbonyl derivative (IX) was converted to the 5,10-methenyltetrahydrofolate (III) using a mixture of formic acid and acetic acid, saturated with HBr. The cyclic formamidinium salt (III) was then hydrolyzed to the 5-formyl compound, which was finally converted to the corresponding calcium salt. In a closely related method, folic acid (IV) was hydrogenated in the presence of rhodium (I) catalysts supported on an optically active phosphane to provide a diastereomeric mixture of tetrahydrofolate epimers (VII) with moderate diastereomeric excess of the desired (6S) isomer. After derivatization with menthyl chloroformate, the resultant mixture of epimeric mono- and bis-menthyl carbamates was separated using preparative HPLC. The target carbamate (IX) was then converted into the title compound as outlined above.

Alternatively, folic acid (IV) was chemically reduced to the (6R,S)-epimeric mixture of tetrahydrofolates (VII) with sodium borohydride in aqueous NaOH. After derivatization of (VII) with (-)-menthyl chloroformate (VIII), the desired (6S) carbamate (IX) was isolated in high diastereoisomeric purity, taking advantage of the different solubility of the diastereoisomers in n-butanol. The menthyloxycarbonyl derivative (IX) was converted to the 5,10-methenyltetrahydrofolate (III) using a mixture of formic acid and acetic acid, saturated with HBr. The cyclic formamidinium salt (III) was then hydrolyzed to the 5-formyl compound, which was finally converted to the corresponding calcium salt. In a closely related method, folic acid (IV) was hydrogenated in the presence of rhodium (I) catalysts supported on an optically active phosphane to provide a diastereomeric mixture of tetrahydrofolate epimers (VII) with moderate diastereomeric excess of the desired (6S) isomer. After derivatization with menthyl chloroformate, the resultant mixture of epimeric mono- and bis-menthyl carbamates was separated using preparative HPLC. The target carbamate (IX) was then converted into the title compound as outlined above.

Alternatively, folic acid (IV) was chemically reduced to the (6R,S)-epimeric mixture of tetrahydrofolates (VII) with sodium borohydride in aqueous NaOH. After derivatization of (VII) with (-)-menthyl chloroformate (VIII), the desired (6S) carbamate (IX) was isolated in high diastereoisomeric purity, taking advantage of the different solubility of the diastereoisomers in n-butanol. The menthyloxycarbonyl derivative (IX) was converted to the 5,10-methenyltetrahydrofolate (III) using a mixture of formic acid and acetic acid, saturated with HBr. The cyclic formamidinium salt (III) was then hydrolyzed to the 5-formyl compound, which was finally converted to the corresponding calcium salt. In a closely related method, folic acid (IV) was hydrogenated in the presence of rhodium (I) catalysts supported on an optically active phosphane to provide a diastereomeric mixture of tetrahydrofolate epimers (VII) with moderate diastereomeric excess of the desired (6S) isomer. After derivatization with menthyl chloroformate, the resultant mixture of epimeric mono- and bis-menthyl carbamates was separated using preparative HPLC. The target carbamate (IX) was then converted into the title compound as outlined above.

In a different synthetic strategy, L-serine methyl ester (X) was protected as the N-benzyl derivative (XI) by reductive alkylation with benzaldehyde and NaBH4. Ammonolysis of ester (XI) then produced N-benzyl serinamide (XII). Subsequent reduction of the amide function of (XII) employing borane-dimethyl sulfide complex gave diamine (XIII). This was condensed with the chloropyrimidine (XIV) to afford adduct (XV). After protection of the exocyclic amine with di-tert-butyl dicarbonate, alcohol (XVI) oxidation with Dess-Martin periodinane reagent yielded aldehyde (XVII). Reductive coupling of (XVII) with p-aminobenzoyl glutamic acid (XVIII), followed by acidic Boc group deprotection, led to the triaminopropane derivative (XIX). Catalytic hydrogenation of the nitro group of (XIX) and N-debenzylation over Pd/C yielded the pteridine ring precursor (XX).

Oxidation of the triamino pyrimidinone system (XX) by means of iodine produced the intermediate ortho-iminoquinone (XXI), which underwent hydrolysis to dione (XXII). Subsequent cyclization of (XXII) under controlled conditions led to the chiral tetrahydrofolic acid (VI). Finally, regioselective N5-formylation of (VI) was accomplished by treatment with formic acid and carbonyl diimidazole.

In an alternative enzymatic synthesis, exposure of the diastereoisomeric (6R,S)-tetrahydrofolates (VIIa-b), with a recombinant enzymatic domain as the source of 10-formyltetrahydrofolate synthetase activity, led to the diastereoselective formylation the (6S) tetrahydrofolate at the 10-N (XXIV), while leaving unchanged the unnatural (6R) isomer (XXIII). The chiral 10-formyl tetrahydrofolate (XXIV) was subsequently isomerized to (6S)-5-formyltetrahydrofolate either under acidic conditions, via formation of the intermediate 5,10-methenyl derivative (III) (22), or in the presence of a source of cyclohydrolase activity.