Chemo-Enzymatic Synthesis of Ester-Linked Docetaxel-Monosaccharide Conjugates as Water-Soluble Prodrugs

Three new docetaxel prodrugs, i.e., 7-propionyldocetaxel 3''-O-β-D-glycopyranosides, which contain ester-linked monosaccharides, were synthesized by a chemo-enzymatic procedure involving enzymatic transglycosylations with lactase, β-galactosidase, or β-xylosidase. The water-solubility of 7-propionyldocetaxel 3''-O-β-D-glucopyranoside was 52-fold higher than that of docetaxel. 7-Propionyldocetaxel 3''-O-β-D-glucopyranoside and 7-propionyldocetaxel 3''-O-β-D-xylopyranoside were effectively hydrolyzed by the relevant enzyme(s) of human cancer cells to release docetaxel, whereas 7-propionyldocetaxel 3''-O-β-D-galactopyranoside was relatively resistant under similar conditions. 7-Propionyldocetaxel 3''-O-β-D-glucopyranoside and 7-propionyldocetaxel 3''-O-β-D-xylopyranoside showed in vitro cytotoxic activity against human cancer cells, whereas 7-propionyldocetaxel 3''-O-β-D-galactopyranoside exerted low cytotoxicity.


Introduction
Docetaxel is a taxane diterpenoid, which shows cytotoxic activity against leukemia cells and inhibitory action against a variety of tumors [1]. It has been recognized as one of the most effective OPEN ACCESS and widely used drugs for the treatment of ovarian, breast, and lung cancers. Despite its effective pharmacological activities, docetaxel has shortcomings such as low solubility in water and toxicity to normal tissues. Its prodrugs, which incorporate acids or amino acids, have attracted much attention, because ester and amide linkages improve the water-solubility of docetaxel and can be hydrolyzed to release docetaxel by hydrolytic enzymes in the living body [2][3][4][5]. However, acid or amino acid conjugates lack tumor selectivity. A number of docetaxel prodrugs have been designed and chemically prepared in order to improve drug selectivity toward tumor cells. One of the most important approaches for drug delivery is the use of saccharide based transporters [6][7][8]. Notably, saccharide conjugation drastically enhances the water-solubility of aglycones [9]. We report here the synthesis of highly water-soluble new ester-linked monosaccharide conjugates of docetaxel, i.e., 7-propionyldocetaxel 3''-O-β-D-glycosides, and their cytotoxic activity toward human cancer cells.
Product 4 was assigned a Mr of 1064.2820 [M+Na] + in the HRFABMS spectrum, which suggested a molecular formula of C 52 H 67 NO 21 . In the 13 C-NMR spectrum of 4, the chemical shifts of the sugar carbon signals indicated that the sugar component in 4 was β-D-glucopyranose. Correlations were observed in the HMBC spectrum of 4 between the proton signal at δ 4.70 (H-1a) and the carbon signal at δ 68.0 (C-3'') and between the proton signal at δ 4.15 (H-7) and the carbon signal at δ 170.8 (C-1''). These results confirmed that the β-D-glucopyranosyl residue was attached to the hydroxyl group at C-3'' and that propionyl group was linked at C-7 of docetaxel. HMBC spectrum showed correlations between the proton signal at δ 1.75 (H-19) and the carbon signal at δ 57.7 (C-8) and between the proton signal at δ 1.08 (H-16) (δ 1.15 (H-17)) and the carbon signal at δ 44.5 (C-15). The proton signals and carbon resonances at docetaxel moiety of 4 were in good agreement with those of 7-glycolylpaclitaxel 2''-O-β-D-glucopyranoside [9], except for the t-butyloxyl group. Paclitaxel is a structural analog of docetaxel, the t-butyloxyl group of which is replaced by a benzoyl group. The NMR data of the t-butyloxyl moiety of 4 were identified by comparison with those of docetaxel [10]. Thus, compound 4 was identified as 7-propionyldocetaxel 3''-O-β-D-glucopyranoside. The HRFABMS spectrum of product 5 showed a peak at m/z 1064.2815 [M+Na] + suggesting a molecular formula of C 52 H 67 NO 21 . The 13 C-NMR data of the sugar moiety of 5 agreed with those of β-D-galactopyranose. In the HMBC spectrum of 5, correlations were observed between the proton signal at δ 4.87 (H-1a) and the carbon signal at δ 68.0 (C-3'') and between the proton signal at δ 4.15 (H-7) and the carbon signal at δ 170.8 (C-1''). These results confirmed that the β-D-galactopyranosyl residue was attached to the hydroxyl group at C-3'' and that propionyl group was linked at C-7 of docetaxel. Thus, compound 5 was identified as 7-propionyldocetaxel 3''-O-β-D-galactopyranoside.
Product 6 showed a pseudo molecular ion peak at m/z 1034.2770 [M+Na] + (HRFABMS) consistent with a molecular formula of C 51 H 65 NO 20 . The sugar component in 6 was shown to be β-D-xylopyranose based on the chemical shifts of the sugar carbon signals. HMBC correlations between the proton signal at δ 4.70 (H-1a) and the carbon signal at δ 68.2 (C-3'') and between the proton signal at δ 4.15 (H-7) and the carbon signal at δ 170.8 (C-1'') established that the β-D-xylopyranosyl residue was attached to the hydroxyl group at C-3'' and that propionyl group was linked at C-7 of docetaxel. Thus, compound 6 was identified as 7-propionyldocetaxel 3''-O-β-D-xylopyranoside.

Cytotoxicity of Docetaxel Prodrugs
The sensitivity of KB and MCF-7 human cancer cells to doceaxel or docetaxel-sugar conjugates 4-6 was examined by the MTT assay, and IC 50 values of each test compounds are summarized in Table 2. All three docetaxel derivatives showed cytotoxicity against KB and MCF-7 human cancer cells. It has been reported that esterification with hyaluronic acid at C-7 of paclitaxel, a taxane anticancer drug, slightly reduced the cytotoxicity of paclitaxel [6,7]. Galactosyl conjugation relatively decreased cytotoxicity of docetaxel and IC 50 value of 5 for KB cells was 41 µg/mL. The results of these experiments show that docetaxel-sugar conjugates with a monosaccharide at C-7 position would be useful water-soluble docetaxel derivatives.

Release of Docetaxel from Docetaxel Prodrugs by Hydrolysis with KB Cells
Docetaxel-sugar conjugates 4-6 were individually incubated with KB human cancer cells. Compounds 4 and 6 were converted to docetaxel in 88 and 76%, respectively. On the other hand, a relatively low hydrolysis activity of 45% was found in the case of 5. These findings suggest that 7-propionyldocetaxel 3''-O-β-D-glucopyranoside and 7-propionyldocetaxel 3''-O-β-D-xylopyranoside were effectively hydrolyzed by KB cells to release docetaxel and that 7-propionyldocetaxel 3''-O-β-Dgalactopyranoside was relatively resistant against the hydrolysis by KB human cancer cells.

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 × g for 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 1 H-and 13 C-NMR data of 1a-1c are as follows.

Water-Solubility of 7-Propionyldocetaxel 3''-O-β-D-Glycosides
Water-solubility of 7-propionyldocetaxel 3''-O-β-D-glycosides was examined as follows: each compound was stirred in water for 24 h at 25 °C. The mixture was centrifuged at 100,000 g for 30 min at 25 °C. The concentration of test compounds was estimated on the basis of their peak areas using calibration curves prepared by HPLC analyses of authentic samples.

Cytotoxicity Assay In Vitro
The sensitivity of KB and MCF-7 cells to docetaxel or 4-6 was determined according to the previously reported method [10]. Cells were diluted with culture medium to the seeding density (10 5 cells/mL), suspended in 96-well tissue culture plates (100 µL/well), preincubated at 37 °C for 4 h, and then treated for 24 h with docetaxel or 4-6 at various concentrations to obtain a dose-response curve for each compound. After incubation, 20 µL MTT solution (2.5 mg/mL) was added to each well and the plates were further incubated for 4 h. Absorbance at 570 nm was measured with a microplate reader model 450 (BIO-RAD). Dose-response curves were plotted on a semi-log scale as percentage of the cell numbers in control cultures not exposed to test compounds.

Hydrolysis of 7-Propionyldocetaxel 3''-O-β-D-Glycosides by KB Cells
To a 5-mL vial containing RPMI 1640 medium (1 mL, Nissui Pharmaceutical Co. Ltd.) and KB cells (20 mg) was added 5 µmol of each compound. The mixture was incubated at 37 °C for 24 h. The cells and medium were separated by centrifugation at 10,000 g for 5 min. The cells were extracted with MeOH. MeOH extract was concentrated, and the residue was partitioned between H 2 O and CH 2 Cl 2 . The medium was extracted with CH 2 Cl 2 . The CH 2 Cl 2 fractions were combined, concentrated, and analyzed by HPLC. The yield of docetaxel was calculated on the basis of the peak area from HPLC using a calibration curve provided by HPLC analyses of authentic docetaxel.

Conclusions
Three new docetaxel prodrugs were successfully synthesized by the chemo-enzymatic procedures. The glycosyl derivatives (carboxyethyl) of glucose, galactose, xylose are prepared through enzymatic glycosylation. The intermediates are useful for the preparation of docetaxel-monosaccharide conjugates. These docetaxel prodrugs were effectively hydrolyzed by the relevan enzymes of human cancer cells releasing docetaxel. The present chemo-enzymatic synthesis is useful for the practical preparation of docetaxel prodrugs. Studies on the antitumor activity in vivo of the prodrugs prepared are now in progress.