2.3- Difluoro-6-nitrophenol

The second-resolution approach relied on enzymatic resolution of acetate esters 62 (Scheme 4.7) (Hayakawa et ah, 1991). The sequence opened with the alkylation of 2,3-difluoro-6-nitrophenol (59) with l-acetoxychloro-2-propane (60) to deliver ether 61. Reduction of the nitro group of 61 gave an intermediate anihne that cyclized to give racemic benzoxazine 62 in 62% yield. A variety of lipases were then examined for the resolution. The best results arose from use of LPL Amano 3, derived from P. aeruginosa, which gave a ratio of 73 23 in favor of the desired (—)-enantiomer. Benzoylation of the enantiomerically-enriched mixture followed by chromatography of the aryl amides delivered enantiomerically pure 63. 

Difluoro-6-nitrophenol Nickel Raney Thionyl chloride Acetic anhydride N-Methylpiperazine... 

Acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[l,4]benzoxazine (m.p. 73-74°C) was synthesized by hydrogenation of a compound prepared from 2,3-difluoro-6-nitrophenol, l-acetoxy-3-chloro-2-propane and potassium iodide. The hydrogenation was carried out on Raney nickel. The resulting compound was dissolved in THF, and 3,5-dinitrobenzoyl chloride and pyridine were added thereto, followed by heating at 60°C for 3 hours. The mixture was concentrated, and the concentrate was dissolved in ethyl acetate, washed successively with diluted hydrochloric acid, an aqueous solution of sodium bicarbonate and water, dried over anhydrous sodium sulfate and concentrated. Addition of n-hexane to the concentrate caused precipitation of yellow crystals of a racemate. The yield of 3,5-dinitrobenzoyl derivative of the ()-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[l,4]benzoxazine 3.93 g. 

Applied are fluorogenic compounds such as 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) or compounds changing the absorption spectra after dephosphorylation, such phospho-tyrosine, paro-nitrophenol phosphate (pNPP) or 3-0-methylfluorescein phosphate (OMFP) [22, 26] (Figure 3). In a phosphatase inhibition assay, the phosphatase is first incubated with the inhibitor and then the phosphatase activity assay is carried out. Reduced activity due to inhibition is measured by one of the above-mentioned assay types, which are chosen depending on the phosphatase. A parameter often obtained in inhibition assays is the inhibitor concentration 50 (ICjq) (inhibition constant), which is the concentration where 50% of the enzyme is inhibited. 

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