Chemo-enzymatic synthesis of ester-linked docetaxel-monosaccharide conjugates as water-soluble prodrugs

Article abstract of DOI:10.3390/molecules16086769

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, b-galactosidase, or β-xy

Full text of DOI:10.3390/molecules16086769

Molecules 2011, 16, 6769-6777; doi:10.3390/molecules16086769
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Chemo-Enzymatic Synthesis of Ester-Linked
Docetaxel-Monosaccharide Conjugates as
Water-Soluble Prodrugs
Kei Shimoda * and Naoji Kubota
Department of Chemistry, Faculty of Medicine, Oita University, 1-1 Hasama-machi,
Oita 879-5593, Japan
* Author to whom correspondence should be addressed; E-Mail: shimoda@med.oita-u.ac.jp;
Tel.: +81-97-586-5606; Fax: +81-97-586-5619.
Received: 24 June 2011; in revised form: 28 July 2011 / Accepted: 3 August 2011 /
Published: 9 August 2011
Abstract: 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-
52-fold higher than that of docetaxel. 7-Propionyldocetaxel 3''-O-β-
7-propionyldocetaxel 3''-O-β- -xylopyranoside were effectively hydrolyzed by the relevant
enzyme(s) of human cancer cells to release docetaxel, whereas 7-propionyldocetaxel
3''-O-β- -galactopyranoside was relatively resistant under similar conditions.
7-Propionyldocetaxel 3''-O-β- -glucopyranoside and 7-propionyldocetaxel 3''-O-β-
xylopyranoside showed in vitro cytotoxic activity against human cancer cells, whereas
7-propionyldocetaxel 3''-O-β- -galactopyranoside exerted low cytotoxicity.
D
-glucopyranoside was
D
-glucopyranoside and
D
D
D
D-
D
Keywords: docetaxel; chemo-enzymatic synthesis; glycoside; ester-linker; prodrug;
cytotoxicity
1. 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

Molecules 2011, 16
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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 [2-5]. 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 [6-8]. 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.
2. Results and Discussion
2.1. Chemo-Enzymatic Synthesis of Docetaxel Prodrugs
7-Propionyldocetaxel 3''-O-β-D-glucopyranoside was synthesized by chemo-enzymatic methods,
including stereoselective β-glucosylation with lactase from K. lactis (Figure 1). Glucosylation of
hydroxypropionic acid was catalyzed by lactase from K. lactis and the yield of carboxyethyl
β-
D
D
-glucopyranoside (1a) was 35%. Carboxyethyl β-
D-galactopyranoside (1b) and carboxyethyl
β-
-xylopyranoside (1c) were prepared in 22 and 27% yields using β-galactosidase from A. oryzae and
β-xylosidase from Aspergillus sp. as biocatalysts, respectively. Carboxyethyl β-glucopyranoside (1a)
was benzylated with BnBr/NaH in DMF at room temperature for 12 h, followed by stirring with
aqueous KOH (1.5 equiv.) to give carboxyethyl 2,3,4,6-tetra-O-benzyl-β-D-glucopyranoside (2a) in
89% yield after silica gel column chromatography. The C2' hydroxyl group of docetaxel was
selectively protected as a triethylsilyl ether using triethylsilyl chloride (TESCl) and imidazole in DMF
affording the 2'-TES ester of docetaxel in 85% yield. The coupling of 2'-TES ester of docetaxel with 2a
(1.2 equiv.) in the presence of EDCI/DMAP in CH2Cl2 at room temperature for 12 h gave 3a in 92%
yield. Deprotection of both TES and benzyl groups with Pd black in HOAc-H2O (9:1, v/v) gave
7-propionyldocetaxel 3''-O-β-
galactopyranoside (5) and 7-propionyldocetaxel 3''-O-β-
same method employing carboxyethyl 2,3,4,6-tetra-O-benzyl-β-
2,3,4,6-tetra-O-benzyl-β- -xylopyranoside in coupling reactions.
D
-glucopyranoside (4) in 95% yield. 7-Propionyldocetaxel 3''-O-β-
-xylopyranoside (6), were synthesized by the
-galactopyranoside and carboxyethyl
D-
D
D
D
Product 4 was assigned a Mr of 1064.2820 [M+Na]+ in the HRFABMS spectrum, which suggested
13
a molecular formula of C52H67NO21. In the 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

Molecules 2011, 16
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signals and carbon resonances at docetaxel moiety of 4 were in good agreement with those of
7-glycolylpaclitaxel 2''-O-β- -glucopyranoside [9], except for the t-butyloxyl group. Paclitaxel is a
D
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.
Figure 1. Synthesis of the docetaxel prodrug 7-propionyldocetaxel 3''-O-β-
D
-gluco-
pyranoside (4).
O
O
HO
HO
OH
OH
t-BuO
t-BuO
i
NH
O
NH
O
O
O
O
O
OR1
Ph
Ph
OAc
OAc
O
OBz
OBz
OR
R1O
OR1
O
OTES
OH
HO
HO
O
OR1
1''
O
O19
HO
10
11
2
Docetaxel
9
7
18
v
6
5
17
t-BuO
O
8
16
12
13
NH
O
15
RO
4
OR
O
ii
O
3
O
2'
HO(CH2)2COOH
Hydroxypropionic acid
HO
2
OAc20
3'
1'
1
O
OR
Ph
2
14
HO
OBz
OR2
3a: R1=Bn, R2=TES
4: R1,R2=H
1a: R=H
vi
iii, iv
2a: R=Bn
Reagents and conditions: (i) TESCl, imidazole, DMAP; (ii) lactase from K. lactis; (iii) BnBr, NaH,
DMF; (iv) KOH; (v) EDCI, DMAP, CH2Cl2; (vi) H2, Pd black, HOAc-H2O (9:1, v/v).
The HRFABMS spectrum of product 5 showed a peak at m/z 1064.2815 [M+Na]+ suggesting a
13
molecular formula of C52H67NO21. The C-NMR data of the sugar moiety of 5 agreed with those of
β-
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- -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- -galactopyranoside.
Product 6 showed a pseudo molecular ion peak at m/z 1034.2770 [M+Na]+ (HRFABMS) consistent
with a molecular formula of C51H65NO20. The sugar component in 6 was shown to be β- -xylopyranose
D-galactopyranose. In the HMBC spectrum of 5, correlations were observed between the proton
D
D
D
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.
2.2. Water-Solubility of Docetaxel Prodrugs
The 7-propionyldocetaxel 3''-O-β-
their water-solubility. The water-solubility of compound 4 was 27 µM, which was 52-fold higher than
that of docetaxel (Table 1). The water-solubility of 7-propionyldocetaxel 3''-O-β- -glycosides
D-glycoside docetaxel-sugar conjugates 4–6 were next tested for
D
increased in the order 6 > 5 > 4. Among the three glycosylation derivatives the glucosyl conjugation
most effectively enhanced the water-solubility of docetaxel.

Molecules 2011, 16
Table 1. Water-solubility of 7-propionyldocetaxel 3''-O-β-
6772
D
-glycosides 4–6.
Compound
Docetaxel
Water-solubility (µM) a
Fold
0.5
1
4
5
6
27.0
52
49
39
25.5
20.2
a Water-solubility was measured at 25 °C.
2.3. 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 IC50 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 IC50 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.
Table 2. IC50 values of docetaxel and docetaxel prodrugs 4–6.
IC50 (µg/mL)
Compound
KB cells
12
MCF-7 cells
Docetaxel
4
5
29
15
5
6
41
25
28
12
2.4. 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-D-
galactopyranoside was relatively resistant against the hydrolysis by KB human cancer cells.
3. Experimental
3.1. General
Lactase from Kluyveromyces lactis and β-galactosidase from Aspergillus oryzae were gifts from
Okayama University of Science. The NMR spectra were recorded in MeOH-d4 using a Varian XL-400
13
spectrometer (1H at 400 MHz, C at 100 MHz, respectively). The chemical shifts were expressed in
δ (ppm) referring to tetramethylsilane. The HRFABMS spectra were measured using a JEOL MStation

Molecules 2011, 16
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JMS-700 spectrometer. HPLC was carried out on a Crestpak C18S column (150 × 30 mm) [solvent:
MeOH:H2O (2:3, v/v); detection: UV (228 nm); flow rate: 1.0 mL/min].
3.2. Preparation of Carboxyethyl β-D-Glycopyranosides by Enzymatic Transglycosylation
Transglycosylation using lactase from K. lactis as a biocatalyst to synthesis carboxyethyl
-glucopyranoside is as follows. To a solution of phenyl β- -glucopyranoside (0.7 mol; a gift from
β-D
D
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- -galactopyranoside (0.7 mol; a gift from Prof. Nakajima of Okayama
D
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-
0.05 mol) [13]. The 1H- and 13C-NMR data of 1a–1c are as follows.
D-xylopyranoside (1c,
1
-glucopyranoside (1a): H-NMR: δ 3.25–3.85 (10H, m, H-2, H-3, 2', 3', 4', 5', 6'),
Carboxyethyl
β
-D
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).
1
-galactopyranoside (1b): H-NMR: δ 3.20–3.88 (10H, m, H-2, H-3, 2', 3', 4', 5', 6'),
Carboxyethyl
β
-D
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).
1
-xylopyranoside (1c): H-NMR: δ 3.21–3.88 (9H, m, H-2, H-3, 2', 3', 4', 5'), 4.70
Carboxyethyl
β
-D
(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

Molecules 2011, 16
6774
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,
13
o-H in OBz); C-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-Propionyldocetaxel 3''-O-β-D-galactopyranoside (5): HRFABMS: calcd for C52H67NO21Na
1
[M+Na]+ m/z 1064.2812, found 1064.2815; H-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,

Molecules 2011, 16
6775
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-Propionyldocetaxel 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, CH3
in 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,
13
d, J = 8.0 Hz, o-H in OBz); C-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).
3.4. Water-Solubility of 7-Propionyldocetaxel 3''-O-
β
-
D
-Glycosides
-glycosides was examined as follows: each
Water-solubility of 7-propionyldocetaxel 3''-O-β-
D
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.
3.5. 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
(105 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

Molecules 2011, 16
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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.
3.6. 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 H2O and CH2Cl2.
The medium was extracted with CH2Cl2. The CH2Cl2 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.
4. 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.
Conflict of Interest
The authors declare no conflict of interest.
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1
Sample Availability: Samples of the compounds described in the present paper as well as the H- and
13C-NMR data are available from the authors.
© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).