Chemo-enzymatic synthesis of ester-linked taxol-oligosaccharide conjugates as potential prodrugs

Article abstract of DOI:10.1016/j.tetlet.2007.11.156

7-Glycolylpaclitaxel 2″-O-α-glucooligosaccharides, novel taxol (paclitaxel) prodrugs of ester-linked oligosaccharide series compounds, were synthesized by chemo-enzymatic procedures, including enzymatic transglycosylations with α-glucosidase and cyclodext

Full text of DOI:10.1016/j.tetlet.2007.11.156

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 601–604
Chemo-enzymatic synthesis of ester-linked
taxol–oligosaccharide conjugates as potential prodrugs
a,
b
c,
*
*
Kei Shimoda , Hatsuyuki Hamada , Hiroki Hamada
a Department of Pharmacology and Therapeutics, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japan
b National Institute of Fitness and Sports in Kanoya, 1 Shiromizu-cho, Kagoshima 891-2390, Japan
c Department of Life Science, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
Received 26 October 2007; revised 26 November 2007; accepted 27 November 2007
Available online 3 December 2007
Abstract
7-Glycolylpaclitaxel 200-O-a-glucooligosaccharides, novel taxol (paclitaxel) prodrugs of ester-linked oligosaccharide series com-
pounds, were synthesized by chemo-enzymatic procedures, including enzymatic transglycosylations with a-glucosidase and cyclodextrin
glucanotransferase. The water-solubility of 7-glycolylpaclitaxel 200-O-a-glucopentaoside was 2.7 mM, which was 6.8 thousand-fold higher
than that of paclitaxel. C-7 modification of paclitaxel with a longer oligosaccharide chain decreased the in vitro cytotoxicity of paclitaxel
against KF human ovarian cancer cells.
Ó 2007 Elsevier Ltd. All rights reserved.
Taxol (paclitaxel), which is a taxane diterpenoid isolated
from Taxus brevifolia, shows cytotoxic activity against leu-
kemia cells and inhibitory action against a variety of tumors
such as ovarian cancer, and has been recognized as one of the
most effective and widely used anticancer agents.1 It presents
shortcomings such as low solubility in water and toxicity to
normal tissues despite its effective pharmacological activi-
ties. Paclitaxel prodrugs, which incorporate acids or amino
acids, have attracted considerable attention, because ester
and amide linkages improve the water-solubility of paclit-
axel and can be hydrolyzed by hydrolytic enzymes in the liv-
ing body.2 However, acid or amino acid conjugates lack
tumor selectivity. To improve drug selectivity toward tumor
cells, many efforts to chemically synthesize paclitaxel pro-
drugs designed containing a transport system have been
made. An interesting approach for drug delivery is the use
of saccharide based transporters,3,4 and there have been sev-
eral reports on the synthesis of paclitaxel–sugar conjugates.3
In addition, saccharide conjugation drastically enhances the
water-solubility of aglycone molecule.5 In the present Letter,
we describe the synthesis of highly water-soluble ester-linked
oligosaccharide conjugates of paclitaxel, i.e., 7-glycolylpacli-
taxel 200-O-a-glucooligosaccharides [a-maltooligosaccharides;
a-Glc-1?(4-a-Glc-1?)nꢀ14-a-glucosides (n = 1–4)]. We
also show the cytotoxic activity of paclitaxel–sugar conju-
gates toward KF human ovarian cancer cells.
Paclitaxel was used as the starting material for the syn-
thesis of paclitaxel–sugar conjugates 4–8 (Scheme 1).
First, 7-glycolylpaclitaxel 200-O-a-D-glucopyranoside (4)
was synthesized by chemo-enzymatic methods, including
stereoselective a-glucosylation with a-glucosidase. 20-
Hydroxyl group of paclitaxel was initially protected with tri-
ethylsilyl (TES) group to give 20-TES ester of paclitaxel
(Scheme 1). Carboxymethyl a-D-glucopyranoside (1) was
prepared by stereoselective a-transglucosylation to glycolic
acid (0.02 mol) from maltose (0.2 mol) with a-glucosidase
(500 U) (Toyobo Co. Ltd) in 100 mL of DMSO–H2O
(1:4, v/v) at 40 °C for 24 h in 37% yield. Carboxymethyl
a-D-glucopyranoside (1) was benzylated with BnBr/
NaH in DMF at room temperature for 12 h, followed by
stirring with KOH (1.5 equiv) to give carboxymethyl
*
Corresponding authors. Tel.: +81 97 586 5606; fax: +81 97 586 5619
(K.S.); tel.: +81 86 256 9473; fax: +81 86 256 8468 (H.H.).
E-mail addresses: shimoda@med.oita-u.ac.jp (K. Shimoda), hamada@
das.ous.ac.jp (H. Hamada).
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.11.156

602
K. Shimoda et al. / Tetrahedron Letters 49 (2008) 601–604
O
AcO
OH
Bz
Ph
i
OR1
NH
O
OR1
Paclitaxel
R1O
O
O
O
OR1
OAc
O
OBz
O
OTES
HO
n
1''
O
7
2''
O19
AcO
9
10
11
18
v
OR
6
5
17
16
Bz
Ph
8
RO
OR
OR
12
13
NH
O
15
O
4
3: n=1, R1=Bn, R2=TES
4: n=1, R1,R2=H
ii
O
3
O
20
OAc
2'
vi
HOCH2COOH
2
3'
1'
1
O
O
14
HO
OBz
HO
OR2
1: R=H
vii
iii, iv
5: n=2, R1,R2=H; 6: n=3, R1,R2=H;
7: n=4, R1,R2=H; 8: n=5, R1,R2=H
2: R=Bn
Scheme 1. Reagents and conditions: (i) TESCl, imidazole, DMAP; (ii) a-glucosidase, maltose, DMSO–H2O (1:4, v/v); (iii) BnBr, NaH, DMF; (iv) KOH;
(v) EDCI, DMAP, CH2Cl2; (vi) H2, Pd black, HOAc–H2O (9:1, v/v); (vii) CGTase, starch, Na–Pi buffer (25 mM).
2,3,4,6-tetra-O-benzyl-a-D-glucopyranoside (2). The yield
of 2 was 89%. The coupling of 20-TES ester of paclitaxel with
2 (1.2 equiv) in the presence of EDCI/DMAP in CH2Cl2 at
room temperature for 12 h gave 3 in 95% yield. Deprotec-
tion of both TES and benzyl groups with Pd black in
HOAc–H2O (9:1, v/v) yielded 7-glycolylpaclitaxel 200-O-a-
D-glucopyranoside (4) in 97% yield.
Paclitaxel–sugar conjugates 4–8 were tested for their
water-solubility.7 The water-solubility of 4 was 21 lM,
which was 53-fold higher than that of paclitaxel (Table
1). The water-solubility of paclitaxel–sugar conjugates
increased in the order 4, 5, 6, 7, and 8. The solubility of
8 was about 6.8 thousand-fold higher than that of
paclitaxel.
Next, 7-glycolylpaclitaxel 200-O-a-glucooligosaccharides,
i.e., a-maltooligosaccharides, were synthesized from
7-glycolylpaclitaxel 200-O-a-D-glucopyranoside (4) by enzy-
matic a-(1?4)-glucooligosaccharide formation with cyclo-
dextrin glucanotransferase (CGTase). Glycosylation of 4
(1 mmol) with CGTase (700 U) (Amano Pharmaceutical
Co. Ltd) in the presence of soluble starch (20 g) in
50 mL of 25 mM sodium phosphate buffer (pH 7.0) at
40 °C for 24 h yielded oligosaccharide products, four of
which could be isolated by preparative HPLC [5 (17%
yield), 6 (14%), 7 (11%), and 8 (7%)] (Fig. 1). The structures
of these four new compounds were established by
HRFABMS and NMR analyses. The assignment of each
signal in the NMR spectra of products 5–8 was performed
by H–H COSY, C–H COSY, NOE, HMQC, and HMBC
analyses, and products were identified as 7-glycolylpacli-
taxel 200-O-a-glucobioside (5), 7-glycolylpaclitaxel 200-O-a-
glucotrioside (6), 7-glycolylpaclitaxel 200-O-a-glucotetrao-
side (7), and 7-glycolylpaclitaxel 200-O-a-glucopentaoside
(8).6
The sensitivity of KF (paclitaxel-sensitive) and HAC-2
(paclitaxel-resistant) human ovarian adenocarcinoma cells
to paclitaxel or paclitaxel–sugar conjugates 4–8 was exam-
ined by the MTT assay, and IC50 values of each test com-
pounds are summarized in Table 2.8,9 Two compounds,
7-glycolylpaclitaxel 200-O-a-glucoside (4) and 7-glycolylpac-
litaxel 200-O-a-glucobioside (5), showed strong cytotoxicity
against KF cells. The cytotoxic activity decreased in the
order 4, 5, 6, 7, and 8. On the other hand, no cytotoxic
activity toward HAC-2 cells was found for all derivatives.
Paclitaxel–sugar conjugates 4–8 were individually incu-
bated with KF cells.10 Compounds 4 and 5 were converted
to a small amount of paclitaxel in 3% and 1%, respectively,
whereas trace or no paclitaxel was obtained in the cases of
6–8. These findings indicated that all derivatives were cyto-
toxic themselves, and that C-7 modification of paclitaxel
with a longer oligosaccharide chain decreased its cyotoxi-
city. The oligosaccharide based transporters have been
reported to serve to target drug to a specific organ in the
living body, liver,4 in which oligosaccharide moiety could
be cleaved by hydrolytic enzymes. Thus, paclitaxel deriva-
tives having a longer oligosaccharide, which show low
cytotoxicity, would be potential antitumor prodrugs.2d
The ester-linked paclitaxel–sugar conjugates, novel taxol
prodrugs of oligosaccharide-linked series compounds with a
Table 1
Water-solubility of paclitaxel and paclitaxel–sugar conjugates 4–8
Compound
Water-solubilitya (lM)
Fold
Paclitaxel
0.4
1
4
5
6
7
8
a
21
53
3.0 ꢁ 102
7.6 ꢁ 102
1.6 ꢁ 103
2.7 ꢁ 103
7.5 ꢁ 102
1.9 ꢁ 103
4.1 ꢁ 103
6.8 ꢁ 103
Fig. 1. HPLC analysis of 7-glycolylpaclitaxel 200-O-a-glucooligosaccha-
rides 5–8 produced by CGTase-catalyzed glycosylation of 4.
Water-solubility was measured at 25 °C.

K. Shimoda et al. / Tetrahedron Letters 49 (2008) 601–604
603
Table 2
(3H, s, H-18), 2.00 (1H, dd, J = 15.6, 9.2 Hz, H-14a), 2.15 (3H, s, CH3
in 10Ac), 2.23 (1H, dd, J = 15.6, 9.2 Hz, H-14b), 2.37 (3H, s, CH3 in
4Ac), 2.58 (1H, m, H-6a), 3.29–3.78 (8H, m, H-200, 2a, 3a, 4a, 5a, 6a),
3.90 (1H, d, J = 7.2 Hz, H-3), 4.18 (3H, m, H-7, 20), 4.75 (1H, d,
J = 5.2 Hz, H-20), 4.95 (1H, d, J = 3.2 Hz, H-1a), 5.01 (1H, d,
J = 9.2 Hz, H-5), 5.63 (2H, m, H-2, 30), 6.15 (1H, t, J = 9.2 Hz, H-13),
6.21 (1H, s, H-10), 7.28 (1H, t, J = 7.6 Hz, p-H in Ph), 7.39–7.58 (9H,
m, m-H in NBz, p-H in NBz, m-H in OBz, o-H in Ph, m-H in Ph), 7.65
(1H, t, J = 7.6 Hz, p-H in OBz), 7.85 (2H, d, J = 8.0 Hz, o-H in NBz),
8.10 (2H, d, J = 8.0 Hz, o-H in OBz); 13C NMR (100 MHz, CD3OD,
d in ppm): d 11.3 (C-19), 14.7 (C-18), 20.6 (CH3 in 10Ac), 22.1 (C-16),
23.1 (CH3 in 4Ac), 26.7 (C-17), 34.1 (C-6), 36.3 (C-14), 44.5 (C-3,
C-15), 57.2 (C-30), 57.7 (C-8), 62.4 (C-6a), 65.9 (C-200), 71.4 (C-7,
C-13), 72.1 (C-4a), 73.6 (C-5a), 74.1 (C-2a), 74.8 (C-20), 75.1 (C-3a),
75.7 (C-2), 76.6 (C-10), 77.2 (C-20), 78.8 (C-1), 81.8 (C-4), 85.0 (C-5),
100.6 (C-1a), 128.3 (o-C in NBz, o-C in Ph), 128.9 (p-C in NBz), 129.5
(m-C in OBz, m-C in Ph), 131.1 (m-C in NBz, q-C in OBz), 132.7 (o-C
in OBz, p-C in Ph), 134.1 (C-11), 134.5 (q-C in Ph), 135.4 (p-C in
OBz), 139.8 (q-C in NBz), 142.1 (C-12), 167.4 (C@O in OBz), 170.1
(C@O in NBz), 170.9 (C-100), 171.3 (C@O in 4Ac), 171.9 (C@O in
10Ac), 174.3 (C-10), 203.2 (C-9). Product 5: HRFABMS: m/z
1258.3451 [M+Na]+; 1H NMR (CD3OD): d 1.10 (3H, s, H-16), 1.16
(3H, s, H-17), 1.78 (3H, s, H-19), 1.81 (1H, m, H-6b), 1.87 (3H, s,
H-18), 2.01 (1H, dd, J = 15.4, 9.2 Hz, H-14a), 2.15 (3H, s, CH3 in
10Ac), 2.23 (1H, dd, J = 15.4, 9.2 Hz, H-14b), 2.37 (3H, s, CH3 in
4Ac), 2.58 (1H, m, H-6a), 3.30–3.79 (14H, m, H-200, 2a, 2b, 3a, 3b, 4a,
4b, 5a, 5b, 6a, 6b), 3.90 (1H, d, J = 7.2 Hz, H-3), 4.19 (3H, m, H-7,
20), 4.75 (1H, d, J = 5.2 Hz, H-20), 4.97 (1H, d, J = 3.2 Hz, H-1a),
5.01 (1H, d, J = 9.0 Hz, H-5), 5.22 (1H, d, J = 3.6 Hz, H-1b), 5.62
(2H, m, H-2, 30), 6.15 (1H, t, J = 9.0 Hz, H-13), 6.21 (1H, s, H-10),
7.28 (1H, t, J = 7.2 Hz, p-H in Ph), 7.38–7.59 (9H, m, m-H in NBz,
p-H in NBz, m-H in OBz, o-H in Ph, m-H in Ph), 7.65 (1H, t,
J = 7.6 Hz, p-H in OBz), 7.85 (2H, d, J = 7.6 Hz, o-H in NBz), 8.10
(2H, d, J = 7.6 Hz, o-H in OBz); 13C NMR (CD3OD): d 11.3 (C-19),
14.7 (C-18), 20.6 (CH3 in 10Ac), 22.2 (C-16), 23.1 (CH3 in 4Ac), 26.7
(C-17), 34.2 (C-6), 36.3 (C-14), 44.5 (C-3, C-15), 57.2 (C-30), 57.7
(C-8), 62.4 (C-6b), 62.5 (C-6a), 65.9 (C-200), 71.4 (C-7, C-13), 72.0
(C-4b), 73.3 (C-5a), 73.9 (C-2a), 74.8 (C-20), 75.1 (C-2b, C-3a), 75.3
(C-3b, C-5b), 75.7 (C-2), 76.6 (C-10), 77.2 (C-20), 78.8 (C-1), 81.0
(C-4a), 81.8 (C-4), 85.0 (C-5), 100.5 (C-1a), 100.7 (C-1b), 128.3 (o-C in
NBz, o-C in Ph), 128.9 (p-C in NBz), 129.6 (m-C in OBz, m-C in Ph),
131.2 (m-C in NBz, q-C in OBz), 132.7 (o-C in OBz, p-C in Ph), 134.1
(C-11), 134.5 (q-C in Ph), 135.4 (p-C in OBz), 139.9 (q-C in NBz),
142.2 (C-12), 167.5 (C@O in OBz), 170.0 (C@O in NBz), 170.9 (C-100),
171.3 (C@O in 4Ac), 172.0 (C@O in 10Ac), 174.3 (C-10), 203.2 (C-9).
Product 6: HRFABMS: m/z 1420.3877 [M+Na]+; 1H NMR
(CD3OD): d 1.09 (3H, s, H-16), 1.16 (3H, s, H-17), 1.78 (3H, s, H-
19), 1.81 (1H, m, H-6b), 1.87 (3H, s, H-18), 2.00 (1H, dd, J = 15.6,
9.2 Hz, H-14a), 2.15 (3H, s, CH3 in 10Ac), 2.24 (1H, dd, J = 15.6,
9.2 Hz, H-14b), 2.37 (3H, s, CH3 in 4Ac), 2.58 (1H, m, H-6a), 3.29–
3.80 (20H, m, H-200, 2a, 2b, 2c, 3a, 3b, 3c, 4a, 4b, 4c, 5a, 5b, 5c, 6a, 6b,
6c), 3.90 (1H, d, J = 7.0 Hz, H-3), 4.17 (3H, m, H-7, 20), 4.75 (1H, d,
J = 5.4 Hz, H-20), 4.98 (1H, d, J = 3.2 Hz, H-1a), 5.00 (1H, d,
J = 9.2 Hz, H-5), 5.19 (1H, d, J = 3.6 Hz, H-1b), 5.23 (1H, d,
J = 3.6 Hz, H-1c), 5.63 (2H, m, H-2, 30), 6.15 (1H, t, J = 9.2 Hz, H-
13), 6.21 (1H, s, H-10), 7.28 (1H, t, J = 7.3 Hz, p-H in Ph), 7.40–7.59
(9H, m, m-H in NBz, p-H in NBz, m-H in OBz, o-H in Ph, m-H in
Ph), 7.66 (1H, t, J = 7.6 Hz, p-H in OBz), 7.85 (2H, d, J = 8.0 Hz, o-H
in NBz), 8.10 (2H, d, J = 7.6 Hz, o-H in OBz); 13C NMR (CD3OD): d
11.3 (C-19), 14.7 (C-18), 20.7 (CH3 in 10Ac), 22.1 (C-16), 23.1 (CH3 in
4Ac), 26.7 (C-17), 34.1 (C-6), 36.3 (C-14), 44.6 (C-3, C-15), 57.1
(C-30), 57.7 (C-8), 62.3 (C-6b, C-6c), 62.5 (C-6a), 66.0 (C-200), 71.4
(C-7, C-13), 71.9 (C-4c), 73.5 (C-5a), 74.0 (C-2a), 74.4 (C-5b), 74.7
(C-2b), 74.8 (C-20), 75.1 (C-2c, C-3a), 75.3 (C-3b, C-3c, C-5c), 75.7
(C-2), 76.6 (C-10), 77.2 (C-20), 78.8 (C-1), 81.1 (C-4a, C-4b), 81.8
(C-4), 85.0 (C-5), 100.5 (C-1a), 100.6 (C-1b, C-1c), 128.3 (o-C in NBz,
o-C in Ph), 129.0 (p-C in NBz), 129.5 (m-C in OBz, m-C in Ph), 131.2
(m-C in NBz, q-C in OBz), 132.7 (o-C in OBz, p-C in Ph), 134.1
IC50 values of paclitaxel and paclitaxel–sugar conjugates 4–8a
Cell line Paclitaxel (nM) 4 (nM) 5 (nM) 6 (nM) 7 (nM) 8 (nM)
KF
131
170
855
1320
1906
2152
HAC-2 >3000
>3000 >3000 >3000 >3000 >3000
a
IC50 values in KF or HAC-2 ovarian cancer cells.
high water-solubility, were synthesized by chemo-enzymatic
procedures, including enzymatic glycosylations with a-glu-
cosidase and CGTase. Recently, Mizukami and co-workers
reported that the water-solubility of curcumin was drasti-
cally enhanced by a glycosyl conjugation.5 The water-solu-
bility of curcumin digentiobioside was 20 million-fold
higher than that of curcumin. In this study, glycosyl conju-
gation effectively enhanced the water-solubility of paclitaxel,
for example, the water-solubility of 7-glycolylpaclitaxel 200-
O-a-glucopentaoside, which has five glucose residues, was
6.8 thousand-fold higher than that of paclitaxel. We should
emphasize that paclitaxel–sugar conjugates with a shorter
saccharide at C-7 position would be useful water-soluble
antitumor agents, that is, 7-glycolylpaclitaxel 200-O-a-gluco-
side and 7-glycolylpaclitaxel 200-O-a-glucobioside showed
strong cytotoxicity against KF human ovarian cancer cells.
Also paclitaxel derivatives with a longer oligosaccharide
chain at C-7 position would act as potential prodrugs with
a low cytotoxicity. The present chemo-enzymatic method
is very useful for the practical preparation of ester-linked
drug-oligosaccharide conjugates as highly water-soluble
and inactive derivatives. Further studies on the thera-
peutic value of paclitaxel–sugar conjugates and on their
hydrolysis process in the living body are currently in
progress.
References and notes
1. Nicolaou, K. C.; Dai, W.-M.; Guy, R. K. Angew. Chem., Int. Ed.
Engl. 1994, 33, 15.
2. (a) Greenwald, R. B.; Gilbert, C. W.; Pendri, A.; Conover, C. D.; Xia,
J.; Martinez, A. J. Med. Chem. 1996, 39, 424; (b) Hayashi, Y.;
Skwarczynski, M.; Hamada, Y.; Sohma, Y.; Kimura, T.; Kiso, Y. J.
Med. Chem. 2003, 46, 3782; (c) Bradley, M. O.; Webb, N. L.;
Anthony, F. H.; Devanesan, P.; Witman, P. A.; Hemamalini, S.;
Chander, M. C.; Baker, S. D.; He, L. F.; Horwitz, S. B.; Swindell, C.
S. Clin. Cancer Res. 2001, 7, 3229; (d) Battaglia, A.; Guerrini, A.;
Baldelli, E.; Fontana, G.; Varchi, G.; Samori, C.; Bombardelli, E.
Tetrahedron Lett. 2006, 47, 2667.
3. (a) Khmelnitsky, Y. L.; Budde, C.; Arnold, J. M.; Usyatinsky, A.;
Clark, D. S.; Dordick, J. S. J. Am. Chem. Soc. 1997, 119, 11554; (b)
Luo, Y.; Prestwich, G. D. Bioconjugate Chem. 1999, 10, 755; (c) Luo,
Y.; Ziebell, M. R.; Prestwich, G. D. Biomacromolecules 2000, 1, 208;
(d) Skwarczynski, M.; Hayashi, Y.; Kiso, Y. J. Med. Chem. 2006, 49,
7253.
4. (a) Gonsho, A.; Irie, K.; Susaki, H.; Iwasawa, H.; Okuno, S.;
Sugawara, T. Biol. Pharm. Bull. 1994, 17, 275; (b) Hashida, M.;
Hirabayashi, H.; Nishikawa, M.; Takakura, Y. J. Control. Release
1997, 46, 129.
5. Kaminaga, Y.; Nagatsu, A.; Akiyama, T.; Sugimoto, N.; Yamazaki,
T.; Maitani, T.; Mizukami, H. FEBS Lett. 2003, 555, 311.
6. Spectral data for 4–8; product 4: HRFABMS: m/z 1096.3050
[M+Na]+; 1H NMR (400 MHz, CD3OD, d in ppm): d 1.09 (3H, s,
H-16), 1.15 (3H, s, H-17), 1.78 (3H, s, H-19), 1.81 (1H, m, H-6b), 1.87

604
K. Shimoda et al. / Tetrahedron Letters 49 (2008) 601–604
(C-11), 134.5 (q-C in Ph), 135.4 (p-C in OBz), 139.8 (q-C in NBz),
(C-16), 23.3 (CH3 in 4Ac), 26.7 (C-17), 34.1 (C-6), 36.3 (C-14), 44.5
(C-3, C-15), 57.3 (C-30), 57.7 (C-8), 62.4 (C-6b, C-6c, C-6d, C-6e), 62.5
(C-6a), 65.8 (C-200), 71.4 (C-7, C-13), 72.0 (C-4e), 73.5 (C-5a), 73.9
(C-2a), 74.4, 74.5 (C-2b, C-2c, C-2d, C-5b, C-5c, C-5d), 74.8 (C-20),
75.1, 75.2, 75.3 (C-2e, C-3a, C-3b, C-3c, C-3d, C-3e, C-5e), 75.7 (C-2),
76.6 (C-10), 77.2 (C-20), 78.5 (C-1), 81.0, 81.1 (C-4a, C-4b, C-4c,
C-4d), 81.8 (C-4), 85.0 (C-5), 100.5 (C-1a), 100.6, 100.7 (C-1b, C-1c,
C-1d, C-1e), 128.3 (o-C in NBz, o-C in Ph), 129.1 (p-C in NBz), 129.5
(m-C in OBz, m-C in Ph), 131.0 (m-C in NBz, q-C in OBz), 132.7 (o-C
in OBz, p-C in Ph), 134.0 (C-11), 134.5 (q-C in Ph), 135.4 (p-C in
OBz), 139.9 (q-C in NBz), 142.2 (C-12), 167.5 (C@O in OBz), 170.3
(C@O in NBz), 170.9 (C-100), 171.3 (C@O in 4Ac), 172.0 (C@O in
10Ac), 174.3 (C-10), 203.2 (C-9).
142.1 (C-12), 167.5 (C@O in OBz), 170.1 (C@O in NBz), 171.0 (C-100),
171.3 (C@O in 4Ac), 172.1(C@O in 10Ac), 174.3 (C-10), 203.2 (C-9).
Product 7: HRFABMS: m/z 1582.4395 [M+Na]+; 1H NMR
(CD3OD): d 1.10 (3H, s, H-16), 1.16 (3H, s, H-17), 1.78 (3H, s,
H-19), 1.80 (1H, m, H-6b), 1.87 (3H, s, H-18), 2.00 (1H, dd, J = 15.6,
9.0 Hz, H-14a), 2.15 (3H, s, CH3 in 10Ac), 2.23 (1H, dd, J = 15.6,
9.0 Hz, H-14b), 2.37 (3H, s, CH3 in 4Ac), 2.59 (1H, m, H-6a), 3.25–
3.80 (26H, m, H-200, 2a, 2b, 2c, 2d, 3a, 3b, 3c, 3d, 4a, 4b, 4c, 4d, 5a, 5b,
5c, 5d, 6a, 6b, 6c, 6d), 3.90 (1H, d, J = 7.6 Hz, H-3), 4.18 (3H, m, H-7,
20), 4.75 (1H, d, J = 5.2 Hz, H-20), 4.99 (1H, d, J = 3.2 Hz, H-1a),
5.02 (1H, d, J = 9.2 Hz, H-5), 5.20 (2H, d, J = 3.6 Hz, H-1b, 1c), 5.25
(1H, d, J = 4.0 Hz, H-1d), 5.63 (2H, m, H-2, 30), 6.15 (1H, t,
J = 9.2 Hz, H-13), 6.21 (1H, s, H-10), 7.28 (1H, t, J = 7.6 Hz, p-H in
Ph), 7.39–7.58 (9H, m, m-H in NBz, p-H in NBz, m-H in OBz, o-H in
Ph, m-H in Ph), 7.66 (1H, t, J = 7.6 Hz, p-H in OBz), 7.85 (2H, d,
J = 7.6 Hz, o-H in NBz), 8.11 (2H, d, J = 7.2 Hz, o-H in OBz); 13C
NMR (CD3OD): d 11.3 (C-19), 14.7 (C-18), 20.7 (CH3 in 10Ac), 22.1
(C-16), 23.1 (CH3 in 4Ac), 26.7 (C-17), 34.1 (C-6), 36.3 (C-14), 44.5
(C-3, C-15), 57.3 (C-30), 57.7 (C-8), 62.4 (C-6b, C-6c, C-6d), 62.5
(C-6a), 66.0 (C-200), 71.5 (C-7, C-13), 71.9 (C-4d), 73.6 (C-5a), 74.1
(C-2a), 74.4, 74.5 (C-2b, C-2c, C-5b, C-5c), 74.8 (C-20), 75.1, 75.2, 75.3
(C-2d, C-3a, C-3b, C-3c, C-3d, C-5d), 75.9 (C-2), 76.6 (C-10), 77.2
(C-20), 78.9 (C-1), 81.0, 81.1 (C-4a, C-4b, C-4c), 81.8 (C-4), 85.2
(C-5), 100.5 (C-1a), 100.6, 100.7 (C-1b, C-1c, C-1d), 128.5 (o-C in
NBz, o-C in Ph), 128.9 (p-C in NBz), 129.5 (m-C in OBz, m-C in Ph),
131.2 (m-C in NBz, q-C in OBz), 132.7 (o-C in OBz, p-C in Ph), 134.1
(C-11), 134.6 (q-C in Ph), 135.4 (p-C in OBz), 140.0 (q-C in NBz),
142.2 (C-12), 167.4 (C@O in OBz), 170.2 (C@O in NBz), 170.9 (C-100),
171.3 (C@O in 4Ac), 172.1 (C@O in 10Ac), 174.3 (C-10), 203.2 (C-9).
Product 8: HRFABMS: m/z 1744.4733 [M+Na]+; 1H NMR
(CD3OD): d 1.10 (3H, s, H-16), 1.16 (3H, s, H-17), 1.78 (3H, s,
H-19), 1.82 (1H, m, H-6b), 1.87 (3H, s, H-18), 2.01 (1H, dd, J = 15.6,
9.2 Hz, H-14a), 2.15 (3H, s, CH3 in 10Ac), 2.23 (1H, dd, J = 15.6,
9.2 Hz, H-14b), 2.37 (3H, s, CH3 in 4Ac), 2.59 (1H, m, H-6a), 3.26–
3.83 (32H, m, H-200, 2a, 2b, 2c, 2d, 2e, 3a, 3b, 3c, 3d, 3e, 4a, 4b, 4c, 4d,
4e, 5a, 5b, 5c, 5d, 5e, 6a, 6b, 6c, 6d, 6e), 3.90 (1H, d, J = 7.2 Hz, H-3),
4.19 (3H, m, H-7, 20), 4.75 (1H, d, J = 5.2 Hz, H-20), 4.99 (1H, d,
J = 3.2 Hz, H-1a), 5.01 (1H, d, J = 9.6 Hz, H-5), 5.20 (3H, m, H-1b,
1c, 1d), 5.25 (1H, d,J = 4.0 Hz, H-1e), 5.63 (2H, m, H-2, 30), 6.15 (1H,
t, J = 9.0 Hz, H-13), 6.21 (1H, s, H-10), 7.28 (1H, t, J = 7.6 Hz, p-H
in Ph), 7.37–7.58 (9H, m, m-H in NBz, p-H in NBz, m-H in OBz, o-H
in Ph, m-H in Ph), 7.65 (1H, t, J = 7.6 Hz, p-H in OBz), 7.85 (2H, d,
J = 8.2 Hz, o-H in NBz), 8.11 (2H, d, J = 8.0 Hz, o-H in OBz); 13C
NMR (CD3OD): d 11.3 (C-19), 14.7 (C-18), 20.6 (CH3 in 10Ac), 22.1
7. Each test compound was stirred in water for 24 h at 25 °C. The
mixture was centrifuged at 12,000g for 30 min at 25 °C. Concentra-
tion of each compound was estimated on the basis of the peak area
from HPLC using calibration curves prepared by HPLC analyses of
respective samples on a Crestpak C18S column (4.6 ꢁ 150 mm,
JASCO) [solvent: MeOH–H2O (2:3, v/v); detection: UV (228 nm);
flow rate: 1.0 mL/min].
8. The sensitivity of KF and HAC-2 cells to paclitaxel or 4–8 was
determined according to the previously reported method.9 Cells were
diluted with culture medium to the seeding density (105 cells/mL),
suspended in 96-well tissue culture plates (100 lL/well), preincubated
at 37 °C for 4 h, and then treated for 24 h with paclitaxel or 4–8 at
various concentrations to obtain a dose–response curve for each
compound. After incubation, 20 lL 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.
9. Mosmann, T. J. Immunol. Meth. 1983, 65, 55.
10. To a 5-mL vial containing 1 mL of RPMI 1640 medium (Nissui
Pharmaceutical Co. Ltd) and 20 mg of KF cells was added 5 lmol of
each compound. The mixture was incubated at 37 °C for 24 h. The
cells and medium were separated by centrifugation at 10,000g 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 the product, paclitaxel, was calculated on the basis of the peak
area from HPLC using a calibration curve provided by HPLC
analyses of authentic paclitaxel. Yields of paclitaxel from compounds
4–8 were 3%, 1%, trace, 0%, and 0%, respectively.