ࡱ> BDA9 R 4bjbj2Nl<<<<J&p |<  h# % % % % % % $ (bI < I L <<Z L L L H<<# L # L 0L |  <<  ] `  $p 0 0  L <<<<Experimental. General remarks and materials 1H NMR and 13C NMR spectra were recorded on a Bruker AM 300 (300 MHz) NMR spectrometer using tetramethylsilane as the internal standard. IR spectra were obtained with a Bruker Equinox 55 FT-IR spectrometer. High-resolution mass spectra were recorded on a JEOL JMX-HX110/110A by the Korea Basic Science Center, Taejon Korea. Flash chromatography was performed on silica-gel 60 (230-400 mash). Perkin-Elmer Lambda 15 UV-Vis spectrometer was used for kinetic study. 2-methoxymethoxy-3-phenylpropanic acid (1a) n-Butyllitium (3.95 mL, 6 mmol, 1.6 M in hexane) was added to a solution of phenyllactic acid (0.5 g, 3 mmol) in THF at 0 oC and stirred under nitrogen atmosphere for 5 min. Chloromethyl methyl ether (266 mg, 3.3 mmol) was added to the reaction mixture, then stirred for 8 h at room temperature. The reaction mixture was acidified to pH 3 with 10% citric acid, then extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO4 and evaporated. The residue was purified on column chromatography (silica gel, chloroform:methanol, 10:1) to afford an oily product. (0.21 g, 35%); IR (KBr) 2750 ~ 3600, 1718 cm-1; 1H NMR 300 MHz (CDCl3) d 3.00 (q, 1H), 3.09 (s, 3H), 3.19 (q, 1H), 4.39 (q, 1H), 4.49 4.67 (q, 2H) 7.21 7.34 (m, 5H); 13C NMR 300 MHz (CDCl3) d 39.3, 56.3, 76.6, 96.7, 127.3, 128.8, 129.9 137.1, 177.0. The product was converted into a dicyclohexylamine salt, mp 109-110 oC. Anal. Calcd for C23H37NO4: C, 70.55; H, 9.52; N, 3.58. Found: C, 70.59; H, 9.82; N, 3.83. 2-benzyloxymethoxyphenylpropanic acid (1b) n-Butyllitium (3.95 mL, 6 mmol, 1.6 M in hexane) was added to a solution of phenyllactic acid (0.5 g, 3 mmol) in THF at 0 oC and stirred under nitrogen atmosphere for 5 min. Benzyl chloromethyl ether (516 mg, 3.3 mmol) was added to the reaction mixture, then stirred for 8 h at room temperature. The reaction mixture was acidified to pH 3 with 10% citric acid, then extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous MgSO4 and evaporated. The residue was purified on column chromatography (silica gel, chloroform:methanol, 10:1) to afford an oily product. (0.28 g, 32%); IR (KBr) 2750 ~ 3600, 1733 cm-1; 1H NMR 300 MHz (CDCl3) d 3.01 (q, 1H), 3.19 (s, 3H), 4.16 4.32 (q, 1H), 4.64 4.67 (q, 2H), 7.12-7.33 (m, 10H); 13C NMR 300 MHz (CDCl3) d 39.3, 70.2, 76.4, 94.4, 127.3, 128.3, 128.8, 128.9, 130.0, 137.3, 137.6, 177.4; HRMS EI m/z 286.1206 (M+; Calcd. 286.1205). The product was converted into a dicyclohexylamine salt, mp 134-135 oC. Anal. Calcd for C29H41NO4: C, 74.48; H, 8.84; N, 3.00. Found: C, 74.71; H, 8.99; N, 3.03. (S)-2-benzyloxymethoxyphenylpropanic acid ((S)-1b) was prepared from L-phenyllactic acid by as described above.; IR (KBr) 2750 ~ 3600, 1733 cm-1; 1H NMR 300 MHz (CDCl3) d 3.01 (q, 1H), 3.19 (s, 3H), 4.16 4.32 (q, 1H), 4.64 4.67 (q, 2H), 7.12 7.33 (m, 10H); 13C NMR 300 MHz (CDCl3) d 39.3, 70.2, 76.4, 94.4, 127.3, 128.2, 128.3, 128.8, 128.9, 130.0, 137.3, 137.6, 177.4; [a]D + 64.8( (c 1, CH2Cl2). The product was converted into a dicyclohexylamine salt, mp 147-148.5 oC. Anal. Calcd for C29H41NO4: C, 74.48; H, 8.84; N, 3.00. Found: C, 74.75; H, 9.14; N, 3.02. (R)-2-Benzyl-2-benzyloxymethoxyacetic acid ((R)-1b) was prepared from D-phenyllactic acid by as described above.; IR (KBr) 2750 ~ 3600, 1733 cm-1; 1H NMR 300 MHz (CDCl3) d 3.01 (q, 1H), 3.19 (s, 3H), 4.16 4.32 (q, 1H), 4.64 4.67 (q, 2H), 7.12 7.33 (m, 10H); 13C NMR 300 MHz (CDCl3) d 39.3, 70.2, 76.4, 94.4, 127.3, 128.2, 128.3, 128.8, 128.9, 130.0, 137.3, 137.6, 177.4; [a]D - 62.1( (c 1, CH2Cl2). The product was converted into a dicyclohexylamine salt, mp 147-149 oC. Anal. Calcd for C29H41NO4: C, 74.48; H, 8.84; N, 3.00. Found: C, 74.32; H, 9.08; N, 2.99. 2-phenethoxymethoxyphenylpropanic acid (1c) n-Butyllitium (3.95 mL, 6 mmol, 1.6 M in hexane) was added to a solution of phenyllactic acid (0.5 g, 3 mmol) in THF at 0 oC and stirred under nitrogen atmosphere for 5 min. Chloromethyl phenethyl ether (563 mg, 3.3 mmol) was added to the reacting mixture, then stirred for 8 h at room temperature. The reaction mixture was acidified to pH 3 with 10% citric acid, then extracted with ethyl acetate. The combined extract was washed with brine, dried over anhydrous MgSO4 and evaporated. The residue was purified on column chromatography (silica gel, chloroform:methanol, 10:1) to afford an oily product (0.31 g, 34%); IR (KBr) 2750 ~ 3750, 1723 cm-1; 1H NMR 300 MHz (CDCl3) d 2.69 (t, 2H), 2.99 (q, 1H), 3.15 (q, 1H), 3.40 (m, 1H), 3.57 (m, 1H), 4.40 (q, 1H), 4.57 4.73 (q, 2H), 7.11 7.33 (m, 10H); 13C NMR 300MHz (CDCl3) d 36.3, 39.3, 69.5, 76.4, 127.3, 128.7, 128.8, 129.3, 129.9, 137.2, 138.9, 177.4. The product was converted into a dicyclohexylamine salt, mp 139-140 oC. Anal. Calcd for C30H43NO4: C, 74.81; H, 9.00; N, 2.91. Found: C, 74.97; H, 8.81; N, 2.85. 2-Benzyl-4-(2-phenylethoxy)butanoic acid t-butyl ester (4) A mixture of 2-phenylethanol (5.16 g, 42.3 mmol), benzene (15 mL), 50% aqueous NaOH (10 mL) and Bu4NHSO4 (2.86 g, 8.45 mmol) was stirred vigorously at room temperature. To the mixture was added a solution of 2-benzyl-4-iodobutanoic acid t-butyl ester (3) (3 g, 8.45 mmol) in benzene (5 mL) and the reaction mixture was stirred for 24 h. Water (50 mL) and hexane (100 mL) were then added, and the mixture was stirred for 10 min. The organic layer was washed with 5% citric acid (2 ( 15 mL) and water (20 mL), dried over anhydrous MgSO4, and evaporated. The residue was purified on column chromatography (silica gel, hexane:ethyl acetate, 25:1) to afford an oily product (0.84 g, 28%); IR (KBr) 1725 cm-1; 1H NMR 300 MHz (CDCl3) d 1.32 (s, 10H) 1.72 1.76 (m, 1H), 1.87 1.92 (m, 1H), 2.71 2.77 (m, 2H), 2.83 2.89 (m, 3H), 3.40 3.50 (m, 2H), 3.57 3.62 (t, 2H) 7.15 7.31 (m, 10H); 13C NMR 300 MHz (CDCl3) d 28.3, 32.5, 36.6, 39.1, 45.5, 69.0, 72.3, 77.0, 77.4, 77.8, 80.6, 126.6, 128.6, 128.7, 129.3, 129.5, 139.2, 139.7, 175.0; HRMS EI m/z 354.2212 (M+; Calcd. 354.2195). 2-Benzyl-4-(2-phenylethoxy)butanoic acid (2) 2-benzyl-4-(2-phenylethoxy)butanoic acid t-butyl ester (4) (0.5 g, 1.41 mmol) was dissolved in TFA (2 mL). The solution was stirred for 30 min and was diluted with ethyl acetate (50 mL), and washed with water (5 mL) and brine. The organic layer was dried over anhydrous MgSO4 and evaporated. The residue was purified on column chromatography (silica gel, hexane:ethyl acetate, 8:1) to afford the product as an oil (0.39 g, 93%); IR (KBr) 3028, 1783, 1705 cm-1; 1H NMR 300 MHz (CDCl3) d 1.76 1.85 (m, 1H), 1.90 2.02 (m, 1H), 2.74 2.89 (m, 3H), 2.98 3.06 (m, 1H), 3.48 3.52 (t, 2H), 3.58 3.64 (m, 2H) 7.16 7.30 (m, 10H); 13C NMR 300 MHz (CDCl3) d 31.7, 35.5, 36.6, 44.7, 68.9, 72.3, 126.3, 126.6, 126.9, 128.8, 129.3, 129.4, 139.2, 139.3, 181.6; HRHS EI m/z 298.1568 (M+; Calcd. 298.1569). Kinetic Studies CPA (Allan form, twice crystallized, from bovine pancreas, aqueous suspension.) was purchased from Sigma Chemical Co. All stock solutions were prepared using doubly distilled and deionized water, and filtered before use. The DMSO solution (90 mL, 40 mM) of inhibitor was added to a solution (90 mL, 3.80 mM) of CPA in pH 7.5, 0.5 M NaCl, 0.05 M Tris buffer. Aliquots (20 mL) of the inactivation mixture were taken at specified time intervals and diluted with the assay mixture (980 mL) containing ClCPL (200 mM). The rate of hydrolysis of ClCPL was monitored at 320 nm. The velocity of CPA-catalyzed substrate hydrolysis in the presence of inhibitor (1a, (S)-1b, (R)-1b, rac-1b, 1c) was obtained from the linear portion of absorbance change with respect to reaction time, and kobs values were calculated from the plot of ln (v/vo) vs incubation time as exemplified in Figure 1. Ki values were determined according to the method of Dixon as exemplified in Figure 2. 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