3.2. Preparation of Carboxyethyl β-D-Glycopyranosides by Enzymatic Transglycosylation
Transglycosylation using lactase from
K. lactis as a biocatalyst to synthesis carboxyethyl β-D-glucopyranoside is as follows. To a solution of phenyl β-D-glucopyranoside (0.7 mol; a gift from Prof. Nakajima of Okayama Prefectural University), and hydroxypropionic acid (0.2 mol) in 0.1 M phosphate buffer (pH 7) was added lactase from
K. lactis (200 U) [
11]. The mixture was stirred for 12 h at 35 °C and then was extracted with
n-butanol. The organic layer was concentrated and purified by column chromatography on silica gel to afford carboxyethyl β-D-glucopyranoside (
1a, 0.07 mol). Lactase is also a β-galactosidase. However, lactase from
K. lactis has been reported to be a good biocatalyst for synthesis of β-glucosides [
11]. In this study, it was used for preparation of carboxyethyl β-D-glucopyranoside.
Hydroxypropionic acid (0.2 mol) was galactosylated by β-galactosidase from
A. oryzae as follows. To a solution of phenyl β-D-galactopyranoside (0.7 mol; a gift from Prof. Nakajima of Okayama Prefectural University), and hydroxypropionic acid (0.2 mol) in 0.1 M HEPES-NaOH buffer (pH 6.0) was added β-galactosidase (1000 U) from
A. oryzae. The mixture was stirred for 12 h at 35 °C and then was extracted with
n-butanol. The organic layer was concentrated and purified by column chromatography on silica gel to afford carboxyethyl β-D-galactopyranoside (
1b, 0.04 mol) [
12].
The synthesis of carboxyethyl β-D-xylopyranoside was carried out as follows. To a solution containing hydroxypropionic acid (0.2 mol) and xylobiose (0.5 mol) in 25 mM of HEPES-NaOH buffer (pH 7.5) was added β-xylosidase (200 U) from
Aspergillus sp. After stirring of the reaction mixture for 24 h at rt, the mixture was centrifuged at 3000 × gfor 10 min. The supernatant was subjected on to a Sephadex G-25 column equilibrated with water to remove the enzyme. The fractions containing glycosides were purified by preparative HPLC to give carboxyethyl β-D-xylopyranoside (
1c, 0.05 mol) [
13]. The
1H- and
13C-NMR data of
1a–
1c are as follows.
Carboxyethyl β-D-glucopyranoside (1a): 1H-NMR: δ 3.25–3.85 (10H, m, H-2, H-3, 2', 3', 4', 5', 6'), 4.72 (1H, d, J = 8.0 Hz, H-1'); 13C-NMR: δ 62.2 (C-6'), 68.0 (C-3), 70.4 (C-2), 72.1 (C-4'), 73.5 (C-5'), 74.1 (C-2'), 75.1 (C-3'), 100.5 (C-1') 170.0 (C-1).
Carboxyethyl β-D-galactopyranoside (1b): 1H-NMR: δ 3.20–3.88 (10H, m, H-2, H-3, 2', 3', 4', 5', 6'), 4.85 (1H, d, J = 7.0 Hz, H-1'); 13C-NMR: δ 62.9 (C-6'), 68.0 (C-3), 70.0 (C-2), 70.4 (C-4'), 72.2 (C-2'), 72.5 (C-3'), 76.7 (C-5'), 101.5 (C-1'), 170.2 (C-1).
Carboxyethyl β-D-xylopyranoside (1c): 1H-NMR: δ 3.21–3.88 (9H, m, H-2, H-3, 2', 3', 4', 5'), 4.70 (1H, d, J = 8.0 Hz, H-1'); 13C-NMR: δ 67.2 (C-5'), 68.2 (C-3), 70.4 (C-2), 72.3 (C-4'), 74.1 (C-2'), 75.1 (C-3'), 100.6 (C-1'), 170.4 (C-1).
3.3. Synthesis of 7-Propionyldocetaxel 3''-O-β-D-Glycosides
A typical procedure is described for the synthesis of 7-propionyldocetaxel 3''-O-β-D-glucopyranoside to exemplify the synthesis of the 7-propionyldocetaxel 3''-O-β-D-glycosides. To a solution of BnBr/NaH (0.15 mol) in DMF (5 mL) was added carboxyethyl β-D-glucopyranoside (1a, 0.03 mol). The mixture was stirred at rt for 12 h, followed by stirring with aqueous KOH (1.5 equiv.). The reaction mixture was quenched with saturated aqueous NaHCO3 and extracted with ethyl acetate (20 mL × 3). The ethyl acetate layer was concentrated in vacuo and purified by silica gel column chromatography (hexane/ethyl acetate = 2/1) to give carboxyethyl 2,3,4,6-tetra-O-benzyl-β-D-glucopyranoside (2a, 0.027 mol). To a solution of docetaxel (0.03 mol) and imidazole (0.12 mmol) in dry DMF (4 mL) was added chlorotriethylsilane (0.1 mol) dropwise at rt. The reaction mixture was stirred at rt for 2 h and diluted with ethyl acetate. The mixture was washed with water and brine, dried over MgSO4, and concentrated in vacuo. Column chromatography of the residue on silica gel (hexane/ethyl acetate = 2/1 to 1/1) gave the 2'-TES ester of docetaxel (0.025 mol). To a mixture of the 2'-TES ester of docetaxel (0.015 mol) in the presence of EDCI/DMAP (0.022 mol) in CH2Cl2 (10 mL) was added 2a (1.2 equiv.). The mixture was stirred at rt for 12 h. The reaction mixture was extracted with ethyl acetate. The organic layer was concentrated in vacuo and purified by column chromatography on silica gel (hexane/ethyl acetate = 2/1) to give 3a (0.014 mol). To a solution of Pd black (0.001 mol) in HOAc-H2O (9:1, v/v) was added 3a (0.01 mol). The suspension was stirred at room temperature for 24 h. Extraction of the reaction mixture with n-butanol followed by column chromatography on silica gel (chloroform/methanol = 4/1) yielded 7-propionyldocetaxel 3''-O-β-D-glucopyranoside (4, 0.01 mol). HRFABMS: calcd for C52H67NO21Na [M+Na]+m/z 1064.2812, found 1064.2820; 1H-NMR: δ 1.08 (3H, s, H-16), 1.15 (3H, s, H-17), 1.35 (9H, s, CH3 in t-Bu), 1.75 (3H, s, H-19), 1.81 (1H, m, H-6β), 1.87 (3H, s, H-18), 2.02 (1H, dd, J = 15.6, 9.0 Hz, H-14a), 2.28 (1H, dd, J = 15.6, 9.0 Hz, H-14b), 2.37 (3H, s, CH3 in 4Ac), 2.58 (1H, m, H-6α), 3.20–3.88 (10H, m, H-2'', H-3'', 2a, 3a, 4a, 5a, 6a), 3.90 (1H, d, J = 7.2 Hz, H-3), 4.15 (1H, m, H-7), 4.18 (2H, m, H-20), 4.70 (1H, d, J = 8.0 Hz, H-1a), 4.79 (1H, d, J = 5.1 Hz, H-2'), 5.01 (1H, d, J = 9.0 Hz, H-5), 5.62 (2H, m, H-2, 3'), 6.15 (1H, t, J = 9.0 Hz, H-13), 6.21 (1H, s, H-10), 7.27 (1H, t, J = 7.6 Hz, p-H in Ph), 7.29–7.68 (6H, m, m-H in OBz, o-H in Ph,m-H in Ph), 7.65 (1H, t, J = 7.6 Hz, p-H in OBz), 8.10 (2H, d, J = 8.0 Hz, o-H in OBz); 13C-NMR: δ 10.9 (3CH3 in t-Bu), 11.3 (C-19), 14.5 (C-18), 22.1 (C-16), 22.8 (CH3 in 4Ac), 26.7 (C-17), 34.1 (C-6), 36.0 (C-14), 44.5 (C-3, C-15), 57.0 (C-3'), 57.7 (C-8), 62.4 (C-6a), 68.0 (C-3''), 70.1 (C-2''), 71.5 (C-7, C-13), 72.1 (C-4a), 73.6 (C-5a), 74.1 (C-2a), 74.8 (C-2'), 75.1 (C-3a), 75.5 (C-2), 76.6 (C in t-Bu, C-10), 77.0 (C-20), 78.8 (C-1), 81.5 (C-4), 85.1 (C-5), 100.6 (C-1a), 128.3 (o-C in Ph), 129.7 (m-C in OBz, m-C in Ph), 131.0 (q-C in OBz), 132.7 (o-C in OBz, p-C in Ph), 134.1 (C-11), 134.5 (q-C in Ph), 135.2 (p-C in OBz), 142.1 (C-12), 167.4 (C=O in OBz), 170.1 (t-BuOC=O), 170.8 (C-1''), 171.2 (C=O in 4Ac), 174.3 (C-1'), 203.2 (C-9).
The other two new docetaxel prodrugs, i.e., 7-propionyldocetaxel 3''-O-β-D-galactopyranoside (5) and 7-propionyldocetaxel 3''-O-β-D-xylopyranoside (6), were synthesized in the same manner using the corresponding carboxyethyl β-D-glycosides. The characterization data are shown below.
7-Propionyl docetaxel 3''-O-β-D-galactopyranoside (5): HRFABMS: calcd for C52H67NO21Na [M+Na]+m/z 1064.2812, found 1064.2815; 1H-NMR: δ 1.08 (3H, s, H-16), 1.15 (3H, s, H-17), 1.35 (9H, s, CH3 in t-Bu), 1.75 (3H, s, H-19), 1.80 (1H, m, H-6β), 1.87 (3H, s, H-18), 2.02 (1H, dd, J = 15.6, 9.2 Hz, H-14a), 2.28 (1H, dd, J = 15.6, 9.2 Hz, H-14b), 2.38 (3H, s, CH3 in 4Ac), 2.58 (1H, m, H-6α), 3.19-3.89 (10H, m, H-2'', H-3'', 2a, 3a, 4a, 5a, 6a), 3.90 (1H, d, J = 7.2 Hz, H-3), 4.15 (1H, m, H-7), 4.18 (2H, m, H-20), 4.77 (1H, d, J = 5.1 Hz, H-2'), 4.87 (1H, d, J = 7.0 Hz, H-1a), 5.01 (1H, d, J = 9.2 Hz, H-5), 5.60 (2H, m, H-2, 3'), 6.15 (1H, t, J = 9.2 Hz, H-13), 6.21 (1H, s, H-10), 7.27 (1H, t, J = 7.6 Hz, p-H in Ph), 7.30–7.68 (6H, m, m-H in OBz, o-H in Ph,m-H in Ph), 7.65 (1H, t, J = 7.6 Hz, p-H in OBz), 8.10 (2H, d, J = 8.0 Hz, o-H in OBz); 13C-NMR: δ 10.9 (3CH3 in t-Bu), 11.3 (C-19), 14.5 (C-18), 22.0 (C-16), 22.8 (CH3 in 4Ac), 26.7 (C-17), 34.1 (C-6), 36.0 (C-14), 44.5 (C-3, C-15), 57.0 (C-3'), 57.7 (C-8), 62.8 (C-6a), 68.0 (C-3''), 70.1 (C-2''), 70.4 (C-4a), 71.5 (C-7, C-13), 72.0 (C-2a), 72.5 (C-3a), 74.8 (C-2'), 75.5 (C-2), 76.4 (C-5a), 76.7 (C in t-Bu, C-10), 77.0 (C-20), 78.8 (C-1), 81.5 (C-4), 85.1 (C-5), 101.5 (C-1a), 128.3 (o-C in Ph), 129.7 (m-C in OBz, m-C in Ph), 131.0 (q-C in OBz), 132.8 (o-C in OBz, p-C in Ph), 134.1 (C-11), 134.5 (q-C in Ph), 135.1 (p-C in OBz), 142.1 (C-12), 167.4 (C=O in OBz), 170.1 (t-BuOC=O), 170.8 (C-1''), 171.2 (C=O in 4Ac), 174.3 (C-1'), 203.2 (C-9).
7-Propionyl docetaxel 3''-O-β-D-xylopyranoside (6): HRFABMS: calcd for C51H65NO20Na [M+Na]+m/z 1034.2780, found 1034.2770; 1H-NMR: δ 1.08 (3H, s, H-16), 1.15 (3H, s, H-17), 1.35 (9H, s, CH3in t-Bu), 1.75 (3H, s, H-19), 1.80 (1H, m, H-6β), 1.87 (3H, s, H-18), 2.01 (1H, dd, J = 15.6, 9.0 Hz, H-14a), 2.28 (1H, dd, J = 15.6, 9.0 Hz, H-14b), 2.37 (3H, s, CH3 in 4Ac), 2.58 (1H, m, H-6α), 3.21–3.89 (9H, m, H-2'', H-3'', 2a, 3a, 4a, 5a), 3.90 (1H, d, J = 7.2 Hz, H-3), 4.15 (1H, m, H-7), 4.18 (2H, m, H-20), 4.70 (1H, d, J = 8.0 Hz, H-1a), 4.79 (1H, d, J = 5.1 Hz, H-2'), 5.00 (1H, d, J = 9.0 Hz, H-5), 5.62 (2H, m, H-2, 3'), 6.15 (1H, t, J = 9.0 Hz, H-13), 6.20 (1H, s, H-10), 7.27 (1H, t, J = 7.6 Hz, p-H in Ph), 7.29–7.68 (6H, m, m-H in OBz, o-H in Ph,m-H in Ph), 7.65 (1H, t, J = 7.6 Hz, p-H in OBz), 8.10 (2H, d, J = 8.0 Hz, o-H in OBz); 13C-NMR: δ 10.9 (3CH3 in t-Bu), 11.3 (C-19), 14.5 (C-18), 22.2 (C-16), 22.8 (CH3 in 4Ac), 26.7 (C-17), 34.1 (C-6), 36.0 (C-14), 44.5 (C-3, C-15), 57.0 (C-3'), 57.7 (C-8), 67.0 (C-5a), 68.2 (C-3''), 70.1 (C-2''), 71.5 (C-7, C-13), 72.3 (C-4a), 74.1 (C-2a), 74.8 (C-2'), 75.1 (C-3a), 75.5 (C-2), 76.6 (C in t-Bu, C-10), 77.0 (C-20), 78.8 (C-1), 81.5 (C-4), 85.1 (C-5), 100.6 (C-1a), 128.3 (o-C in Ph), 129.7 (m-C in OBz, m-C in Ph), 130.9 (q-C in OBz), 132.7 (o-C in OBz, p-C in Ph), 134.1 (C-11), 134.5 (q-C in Ph), 135.2 (p-C in OBz), 142.1 (C-12), 167.4 (C=O in OBz), 170.1 (t-BuOC=O), 170.8 (C-1''), 171.2 (C=O in 4Ac), 174.3 (C-1'), 203.2 (C-9).