Targeting the delivery of glycan-based paclitaxel prodrugs to cancer cells via glucose transporters

Article abstract of DOI:10.1021/jm8006257

This report describes the synthesis of four novel paclitaxel based prodrugs with glycan conjugation (1-4). Glycans were conjugated using an ester or ether bond as the linker between 2′-paclitaxel and the 2′-glucose or glucuronic acid moiety. These prodrug

Full text of DOI:10.1021/jm8006257

7428
J. Med. Chem. 2008, 51, 7428–7441
Targeting the Delivery of Glycan-Based Paclitaxel Prodrugs to Cancer Cells via Glucose
Transporters
Yih-Shyan Lin,†,‡ Rudeewan Tungpradit,†,§ Supachok Sinchaikul,† Feng-Ming An,† Der-Zen Liu,| Suree Phutrakul,§ and
Shui-Tein Chen*,†,‡
Institute of Biological Chemistry and Genomics Research Center, Academia Sinica, Taipei, 11529, Taiwan, Institute of Biochemical Sciences,
National Taiwan UniVersity, Taipei, 10617, Taiwan, Department of Chemistry, Faculty of Science, Chiang Mai UniVersity,
Chiang Mai, 50200, Thailand, and Graduate Institute of Biomedical Materials, Taipei Medical UniVersity, Taipei, 110, Taiwan
ReceiVed May 26, 2008
This report describes the synthesis of four novel paclitaxel based prodrugs with glycan conjugation (1-4).
Glycans were conjugated using an ester or ether bond as the linker between 2′-paclitaxel and the 2′-glucose
or glucuronic acid moiety. These prodrugs showed good water solubility and selective cytotoxicity against
cancer cell lines, but showed reduced toxicity toward normal cell lines and cancer cell lines with low
expression levels of GLUTs. The ester conjugated prodrug 1 showed the most cytotoxicity among the prodrugs
examined and could be transported into cells via GLUTs. Fluorescent and confocal microscopy demonstrated
that targeted cells exhibited morphological changes in tubulin and chromosomal alterations that were similar
to those observed with paclitaxel treatment. Therefore, these glycan-based prodrugs may be good drug
candidates for cancer therapy, and the glycan conjugation approach is an alternative method to enhance the
targeted delivery of other drugs to cancer cells that overexpress GLUTs.
Introduction
coding for an enzyme is delivered to cancer cells specifically,
and the expressed enzyme can convert a nontoxic prodrug into
a cytotoxic species that can kill the cell. Likewise, prodrug
monotherapy (PMT) is based on elevated tumor enzymes. For
example, ꢀ-glucuronidase plays a role in the degradation of
glycosaminoglycans that contain glucuronic acid, and it can
activate low-toxic prodrugs to become highly cytotoxic agents
specifically at the tumor site.5-8 These enzymes are overex-
pressed in many malignancies, yet present at relatively low
concentrations in normal tissues, thus, curbing activation of the
prodrug in nontargeted tissues. Indeed, this enzymatic pathway
has been a promising candidate in PMT.4,9-11
Glucose uptake into tissues or cells is mainly performed by
GLUTs, a family of membrane proteins. Cancer cells generally
express higher levels of GLUTs than normal cells.12-16 This is
because the efficiency of ATP production in normal cells does
not satisfy the high energy requirements of cancer cells, so
GLUTs are overexpressed in cancer cells to improve glucose
uptake. This differential rate of glucose uptake has been used
in positron emission tomography (PET), whereby 2-[18F]-2-
deoxy-D-glucose (FDG) (a glucose analogue) accumulates in
cancer cells and indicates the tumor position.17,18 The ratio of
GLUT expression in malignant verses normal cells is about
100-300, which makes the noise peaks and background signal
of the tumor analysis very reliable. FDG uptake and GLUTs
expression is well-correlated in multiple malignant cells, such
as colon, breast, and lung carcinomas.
Paclitaxel, a diterpenoid taxane derivative, is one of most
potent anticancer agents used in clinical practice today.19,20 It
possesses a unique mechanism of action by binding tubulin and
stabilizing microtubule formation, which ultimately disrupts
mitosis and causes cell death.21-23 However, some drawbacks
of paclitaxel hamper its clinical usefulness. For example,
paclitaxel lacks selective cytotoxicity toward cancer cells. Thus,
both normal and cancerous cells may be affected, and this may
lead to unwanted side effects.24 The poor water solubility of
paclitaxel is another problem that seriously reduces its wider
clinical application. Although many attempts have been made
Recently, target-specific drug delivery has become a promis-
ing strategy to improve the selectivity of cytotoxic drugs toward
targeted cells. In this system, a carrier for a specific cellular
target is conjugated with the drug to form a prodrug which is
less toxic than that of the parent drug. The carrier has two roles.
One is to reduce the toxicity of the drug to normal cells, and
the other is to enhance the specificity of the drug for candidate
cells. After the prodrug gets to the target cell, the drug is released
from its inactive form (or less toxic form) to an active form
that performs its physiological functions. This concept has been
successfully applied to the development of many chemotherapy
agents. One such example is antibody directed enzyme prodrug
therapy (ADEPTa), in which an antibody bound enzyme is
targeted to tumor cells. For cancer therapy, this allows the
nontoxic prodrug to be selectively activated at the tumor site,
and the site-selective prodrug activation results in reduced side
effects in remote tissues.1-3 Another approach is gene-directed
enzyme prodrug therapy (GDEPT), which is a gene-based, two-
step treatment for cancer.4 In this case, an exogenous gene
* To whom correspondence should be addressed. Dr. Shui-Tein Chen,
Institute of Biological Chemistry, Academia Sinica, 128 Academia Rd.,
Sec. II, Nankang, Taipei, 11529, Taiwan. Tel: +886-2-27886230. Fax:
+886-2-27883473. E-mail: bcchen@gate.sinica.edu.tw.
†
Institute of Biological Chemistry and Genomics Research Center,
Academia Sinica.
‡
National Taiwan University.
Chiang Mai University.
Taipei Medical University.
§
|
a Abbreviations: ADEPT, antibody directed enzyme prodrug therapy;
DCC, dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine; DMF,
dimethyl formamide; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; FBS,
fetal bovine serum; FCS, fetal calf serum; GDEPT, gene-directed enzyme
prodrug therapy; FDG, 2-[18F]-2-deoxy-D-glucose; GLUTs, glucose trans-
porters; MeOH, methanol; MEME, minimum essential medium eagle; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NaH, sodium
hydride; PBS, phosphate buffer saline; PDC, pyridium dichromate; PET,
positron emission tomography; PMT, prodrug monotherapy; Pyr, pyridine;
RP-HPLC, reverse phase-high performance liquid chromatography; TBAF,
tetrabutylammonium fluoride; TBAI, tetrabutylammonium iodide; TESCl,
triethylchlorosilane; THF, tetrahydrofuran
10.1021/jm8006257 CCC: $40.75
2008 American Chemical Society
Published on Web 11/16/2008

DeliVery of Glycan-Based Paclitaxel Prodrugs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7429
droxy group, saponification of the 2-hydroxy, and esterification
at the 6-carboxylic acid of 12 gave the glucuronic acid-derivative
13 with a yield of 23% for the three steps. 13 was converted to
14 by treatment with succinic anhydride in pyridine with a yield
of 96%. 14 was coupled with paclitaxel in DCC/DMAP/THF
(75% yield), which was followed by hydrogenolysis of the
protecting groups to give 3, with a yield of 95%. For synthesis
of 4, 7 was treated with AlCl3 and NEt3BF3 in toluene to give
16 (62% yield). Saponification of 16 in NaOH/MeOH followed
by Jones oxidation gave 17 (60% yields in both steps). 17 was
coupled with paclitaxel by DCC/DMAP in THF to give 18 with
a yield of 73%. Hydrogenolyis of 18 generated 4 with a yield
of 90%. The prodrugs 1-4 were purified by RP-HPLC and the
molecular weight was determined by mass spectrometry (m/z)
(Figures S1-S4, Supporting Information). The structures of
Figure 1. Structures of glucose-based (1 and 2) and glucuronic acid-
based (3 and 4) paclitaxel prodrugs.
to improve the solubility of paclitaxel, such as using detergents
or Cremophor EL, these have caused hypersensitivity or related
side effects in patients.25
1
prodrugs were confirmed by H NMR and 13C NMR.
Physical tests found that glucose and glucuronic acid con-
jugation significantly increased the solubility of paclitaxel in
aqueous solution. The solubility of prodrugs 1, 2, 3, and 4 in
PBS buffer were 53, 80, 104, and 137 µg/mL, respectively, while
the solubility of paclitaxel is less than 0.4 µg/mL. Thus, the
solubility of glycan-based prodrugs increased between 130- and
340-fold compared with paclitaxel. The fluorescently labeled
paclitaxel derivative was prepared by first protecting the
2-hydroxy group of paclitaxel with TES (84% yield). This was
followed by the conjugation of an m-aminobenzoic acid to the
7′-position of 19 to give 20 with a yield of 46%. TBAF/THF
released the TES protecting group from 20 to generate 21 with
a quantitative yield. 23 was obtained by coupling 21 and 5 with
DCC/DMAP (85% yield), followed by hydrogenolysis to give
23 (97% yield) (Scheme 5).
In this study, we developed two glucose and two glucuronic
acid-derived paclitaxel prodrugs: 1, 2, 3, and 4 (for structures,
see Figure 1). These prodrugs were designed to take advantage
of the increased GLUT expression and ꢀ-glucuronidase activity
of cancer cells. We expected that these prodrugs would be
similar to FDG, which is specifically delivered into cells via
GLUT uptake. We used seven cancer cell lines (NCI-H838,
Hep-3B, A498, MES-SA, HCT-116, NPC-TW01, and MKN-
45) and two normal cell lines (HUV-E-C and CHO-K1) to
determine the toxicity and specificity of these prodrugs com-
pared to paclitaxel. To examine the targeted delivery of the
prodrugs, their internalization into cells was analyzed by
observing the morphological changes of tubulin and the
chromosomal alterations in cancer cells before and after the drug
treatment using fluorescent and confocal microscopy.
Biological Assay Results
Chemistry
Cytotoxicity of Prodrugs against Cancer and Normal
Cell Lines. The prodrugs were designed to improve their
selective targeting to cancer cells which overexpressed GLUTs,
but not to normal cells, which express fewer GLUTs. We chose
two normal cells (HUV-EC-C and CHO-K1) and seven cancer
cell lines (NCI-H838, Hep-3B, A498, MES-SA, HCT-116,
NPC-TW01, and MKN-45) to test the toxicity of prodrugs and
compare the results to paclitaxel (Table 1). From the Human
Protein Atlas (HPA) database, the GLUTs were highly expressed
on NCI-H838, MES-SA, HCT-116, and NPC-TW01 cancer cell
lines, but not on Hep-3B, A498, or MKN-45, nor on the normal
cells, HUV-EC-C and CHO-K1. In addition, the gene expression
levels of GLUT1 and GLUT4 were determined in all cell lines
by RT-PCR and the results showed that the expression level of
GLUT1, the major GLUT in cancer cells, was higher than its
expression level in normal cells (Table 1 and Figure S5,
Supporting Information). However, it is also dependent upon
the selective cytotoxicity of prodrug against each type of cancer
cells.
Among these cell lines, the cytotoxicity of the prodrugs was
less than that of paclitaxel. The IC50 (concentration for half
inhibition of cell growth) of the prodrugs was modulated by
the amount of GLUTs expressed on both the cancer and normal
cells. As shown in Table 1, the growth of NCI-H838, MES-
SA, HCT-116, and NPC-TW01 cells was greatly inhibited.
However, normal cells and the Hep-3B, A498, and MKN-45
cells were unaffected. The nine cell lines also have different
cytotoxic responses to paclitaxel, as shown by their IC50.
Interestingly, none of the prodrugs (except prodrug 4) had any
effect on the HUV-EC-C and CHO-K1 cells, whereas paclitaxel
inhibited their proliferation. The selectivity index (SI) was
Two glycans, glucose and glucuronic acid, were conjugated
at the 2′-position of paclitaxel through an ester or ether linker
at the C2′-hydroxyl group of the glycans. The following four
glycan-based paclitaxel prodrugs were obtained: 1, 2, 3, and 4
(Figure 1). In vivo, the covalent bonds of the glycans can be
cleaved by intracellular or extracellular enzymes and subse-
quently expose the carboxylic or hydroxyl group at the terminal
position of the spacer. The lone-pair electrons in these functional
groups will then react with the carboxylic group at the other
side of linker and release paclitaxel.
Procedures for the syntheses of prodrugs 1-4 are shown in
Schemes 1-4, respectively. A protected glucose, C14, was used
as the starting material. For synthesis of 1, the 2′-OH position
of C-14 was treated with succinic anhydride to give 5 with a
yield of 71%. 5 was directly conjugated with paclitaxel using
DCC/DMAP in pyridine to give 6 with a yield of 71%. In the
synthesis of 2, C-14 was coupled with a benzoic acid 4-bromo-
butyl ester. This reaction was catalyzed by NaH/TBAI in DMF
to form the ether, 7, with a yield of 72%. The free hydroxyl
group of 8 was obtained by treatment of 7 with 4 N NaOH/
MeOH at room temperature for 1.5 h to give 8 in quantitative
yield. 8 was oxidized to the carboxylic acid 9 by PDC in DMF
with a 50% yield, and 9 was then conjugated with paclitaxel
by DCC/DMAP coupling to give 10 with an 80% yield.
Hydrogenolysis of 6 and 10 using Pd/C in MeOH generated 1
and 2 with yields of 93% and 95%, respectively. For synthesis
of 3, the 2-hydroxy group of C-14 was treated with benzoic
chloride in pyridine to yield 11 (100% yield). This was followed
by the selective release of the 6-hydroxy of 11 catalyzed by
aluminum chloride (75% yield). Jones oxidation of the 6-hy-

7430 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lin et al.
Scheme 1. Synthesis of Glycan-Based Paclitaxel Prodrug 1 Using an Ester Bond as the Linker between Paclitaxel and Glucosea
a Reagents and conditions: (i) DMAP, Pyr, rt, 12 h, 71%; (ii) Paclitaxel, DCC, DMAP, Pyr, rt, 20 h, 71%; (iii) H2, Pd/C, MeOH, rt, 12 h, 93%.
Scheme 2. Synthesis of Glycan-Based Paclitaxel Prodrug 2 Using an Ether Bond as the Linker between Paclitaxel and Glucosea
a Reagents and conditions: (i) NaH, TBAI, DMF, rt, 36 h, 72%; (ii) NaOH, MeOH, rt, 1.5 h, 100%; (iii) PDC, DMF, rt, 18 h, 50%; (iv) Paclitaxel, DCC,
DMAP, THF, rt, 20 h, 80%; (v) H2, Pd/C, MeOH, rt, 12 h, 95%.
determined by the proportion of the IC50 of cancer cells relative
to that of normal cells. The SI values of prodrugs 1, 2, 3, and
4 against the NPC-TW01 cell line and two normal cell lines
were relatively low (HUV-EC-C cells, <0.25 × 103, <0.78 ×
103, <0.67 × 103, and 1.52 × 103, respectively; CHO-K1 cells,
<0.25 × 103, <0.78 × 103, <0.67 × 103, and 2.79 × 103,
respectively) as compared to that of paclitaxel (HUV-EC-C cells,
1.16 × 103; CHO-K1 cells, 0.38 × 103).
The low toxicity of prodrugs 1-3 in normal cells indicated
that the conjugations (glucose-ester or ether linkage and
glucuronic acid-ester linkage) could reduce the toxicity of
paclitaxel and demonstrated the safety of these prodrugs in
normal human cells. Among these prodrugs, prodrug 1, which
used a glucose-ester linkage, had the highest cytotoxicity specific
to cancer cells and may represent a potent candidate prodrug
for further investigation.
Internalization of a Fluorescently Labeled Prodrug into
Cancer Cells. To investigate the delivery of prodrugs into
cancer cells, compound 23 was conjugated with m-aminoben-
zoic acid (a chromophore group) at the 7′-position on
paclitaxel (Scheme 5). This conjugation at the 7′-position of
paclitaxel, instead of glucose, was to avoid any physiological
changes in the uptake via GLUTs. In addition, this type of
modification reportedly does not affect the biological activity
of paclitaxel. After the treatment of NPC-TW01 cells with
the fluorescently labeled prodrug 1 for 5 h, prodrug 1 could
be detected in the cytoplasmic region of NPC-TW01 cells
by fluorescence microscopy (Figure 2). According to the
colocalization of prodrug 1 and tubulin in NPC-TW01 cells
under fluorescence microscopy (Figure 3), this suggested that
cancer cells can uptake this glucose-based prodrug into cells.
In addition, the rate of internalization of prodrug 1 was very
fast (within 5 h), while there were no fluorescent signal in

DeliVery of Glycan-Based Paclitaxel Prodrugs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7431
Scheme 3. Synthesis of Glycan-Based Paclitaxel Prodrug 3 Using an Ester Bond as the Linker between Paclitaxel and Glucuronic
Acida
a Reagents and conditions: (i) BzCl, Pyr, rt, 12 h, 100%; (ii) AlCl3, NEt3BF3, toluene, 0 °C, 20 min, 75%; (iii) Jones reagent, rt, 18 h; (iv) NaOH, MeOH,
rt, 4 h; (v) BnOH, DCC, DMAP, THF, rt, 18 h, 3 steps, 23%; (vi) succinic anhydride, Pyr, rt, 12 h, 96%; (vii) paclitaxel, DCC, DMAP, THF, rt, 18 h, 75%;
(viii) H2, Pd/C, MeOH, rt, 12 h, 95%.
Scheme 4. Synthesis of Glycan-Based Paclitaxel Prodrug 4 Using an Ether Bond as the Linker between Paclitaxel and Glucuronic
Acida
a Reagents and conditions: (i) AlCl3, NEt3BF3, toluene, 0 °C, 20 min, 62%; (ii) NaOH, MeOH, rt, 2 h; (iii) Jones reagent, rt, 18 h, 2 steps 60%; (iv)
paclitaxel, DCC, DMAP, THF, rt, 20 h, 73%; (v) H2, Pd/C, MeOH, rt, 18 h, 90%.
cells either without prodrug treatment or with treatment of
100 µg/mL paclitaxel.
determined by RP-HPLC and the results showed that phloretin
can block the prodrug delivery into NPC-TW01 cells (Figures
S7 and S8, Supporting Information). In addition, the internaliza-
tion of fluorescently labeled prodrug 1 was detected under
fluorescence microscopy. The fluorescence microscopy analysis
revealed that inhibitor-treated NPC-TW01 cells could only take
up a tiny amount of the glucose-conjugated prodrug 1, possibly
due to passive diffusion (Figure 4). These results suggest that
the delivery of the glucose-based paclitaxel prodrug is glucose
To study the modified prodrug’s delivery into NPC-TW01
cells via GLUTs, we treated NPC-TW01 cells with a glucose
transporter inhibitor, phloretin.36-38 This inhibitor blocks the
transporter and is not affect to the cell viability of NPC-TW01
cells (Figure S6, Supporting Information). We were able to
observe its influence on the prodrug uptake. The prodrug uptake
in NPC-TW01 cells with and without phloretin treatment was

7432 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Scheme 5. Synthesis of Fluorescently Labeled Prodrug 1a
Lin et al.
a Reagents and conditions: (i) TESCl, Pyr, rt, 3 h, 84%; (ii) DCC, DMAP, THF, rt, 18 h, 46%; (iii) TBAF, THF, rt, 2 h, 100%; (iv) 5, DCC, DMAP, THF,
rt, 18 h, 85%; (v) H2, Pd/C, MeOH, rt, 12 h, 97%.
Table 1. Cytotoxic Activities of Paclitaxel and Four Glycan-Based Paclitaxel Prodrugs against Seven Cancer Cell Lines and Two Normal Cell Linesa
GLUT protein expressionc
IC50 (µM)e
cancer
tissues
normal
tissues
GLUT
cells
GLUTsb
gene expressiond Paclitaxel
1
2
3
4
1. NCI-H838 lung adenocarcinoma
GLUT-126
GLUT-4
7/12
-
2/12
-
4/12
-
10/12
-
weak
-
weak
-
weak
-
moderate
-
0.843
0.572
0.764
0.772
0.973
0.189
0.902
0.332
0.961
0.159
0.867
0.026
0.082
0.374
0.267
0.181
2. Hep-3B hepatocellular carcinoma GLUT-127
GLUT-4
3. A498 renal carcinoma
7.26
100
>100 >100 >100
GLUT-128
GLUT-4
8.81
>100 >100 >100 >100
4. MES-SA uterine carcinoma
5. HCT-116 colon carcinoma
GLUT-129
GLUT-4
0.008
0.061
0.006
0.082
0.100
0.025
0.174
0.480
0.078
0.095
0.452
0.067
0.201
0.469
0.067
GLUT-130
GLUT-4
7/12
-
NS
strong
-
-
6. NPC-TW01 nasopharyngeal
carcinoma
GLUT-131
GLUT-4
-
-
0.115
0.910
0.036
0.252
7. MKN-45 gastric carcinoma
GLUT-132
GLUT-432
GLUT-133
1/12
NS
NS
moderate
NS
89.06
5.14
>100 >100 >100 >100
>100 >100 >100 44
8. HUV-EC-C human umbilical
cord endothelial cell
line
NS
GLUT-4
-
6/12
-
0.000
0.398
9. CHO-K1 Chinese hamster
ovary cell line
GLUT-134
negative
15.99
>100 >100 >100 24
GLUT-334
GLUT-435
3/12
NS
negative
NS
-
0.354
a Also Indicated is the Relative Expression Levels of GLUT Proteins among the Tissues and GLUT Genes among the Tested Cell Lines. Symbol: NS,
non-observed; -, no expression. b GLUTs, according to references of GLUTs in each cell line in the first column. c The GLUT protein expression levels in
human cancer and normal tissues were searched on the Human Protein Atlas (HPA) database (http://www.proteinatlas.org/), which detected using
immunohistochemistry. The GLUT protein expression showed the number of positive staining per 12 tissues in cancer and the evaluated value in normal
tissues, respectively. d GLUT gene expression level was determined in 9 cell lines by RT-PCR and also shows in Figure S5, Supporting Information. e The
value of IC50 (50% inhibition concentration) represents the means of at least three independent experiments with SD less than 10%.
transporter-dependent. Thus, a paclitaxel-derived prodrug con-
jugated with glucose as a carrier can be selectively delivered to
cancer cells which overexpress GLUTs. This approach can
minimize the side effects of prodrugs on normal tissues.
Confocal Microscopy Analysis of NPC-TW01 Cells
Treated with Prodrugs. Paclitaxel is an anticancer drug that
inhibits mitosis. This drug stabilizes microtubule polymers and
leads to G2/M phase cell cycle arrest and subsequent cell death.
We designed the carbohydrate modifications to block the active
site of paclitaxel (2′-OH) in our prodrugs, leading them to
temporarily lose their microtubule-stabilizing ability. We as-
sumed that paclitaxel was released after carbohydrate cleavage
and linker autocatalysis inside the cells. To prove that these
prodrugs caused cell death by the same biological mechanism
as paclitaxel, we investigated the morphological changes in
tubulin in NPC-TW01 cells that were treated with paclitaxel or
prodrugs (1 µM). These changes were visualized by indirect
immunofluorescent staining using a ꢀ-tubulin antibody (Figure
5a-f). After 24 h treatment, many microtubules were aggregated
around the nuclei of these NPC-TW01 cells. We also observed
the relative cellular location of prodrug 1 and tubulin by treating
cells with fluorescently labeled prodrug 1 and immunostaining

DeliVery of Glycan-Based Paclitaxel Prodrugs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7433
Figure 2. Internalization of prodrug 1 into NPC-TW01 cells under fluorescence microscopy. Fluorescence microscopy images represent the NPC-
TW01 cells in the absence of any drugs (a, d, and g), paclitaxel (b, e, and h), and fluorescently labeled prodrug 1 (c, f, and i). Panels a-c are
observed under white light, panels d-f are observed under fluorescence, and panels g-i are the merged images. After cell treatment with fluorescently
labeled prodrug 1 for 5 h, the fluorescent drug was observed in the plasma regions of cells under fluorescent microscopy.
them for tubulin. The results showed that prodrug 1 is
colocalized with tubulin. Thus, we demonstrated that the prodrug
was internalized into cells and exhibited the same mechanism
of cell death as paclitaxel. However, after prodrug treatment
for 24 h, there were still small microtubule bundles in the
cytoplasm. Compared with paclitaxel alone, it is possible that
the prodrugs may need more time to release paclitaxel and
inhibit microtubule disassembly. Thus, the degree of microtubule
aggregation induced by prodrugs may be weaker than that
induced by paclitaxel. In addition, the chromosomal condensa-
tion, which is a special characteristic of apoptotic cells, was
dispersed around the nuclei of viable NPC-TW01 cells. How-
ever, the chromosomes of viable cells treated with paclitaxel
or prodrugs were condensed into fragments (Figure 5g-l). These
results indicate that these prodrugs can inhibit microtubule
disassembly and induce cell death through apoptosis.
linkages were the following: (i) the conjugated glucose at 2′-
position of paclitaxel facilitated the delivery of paclitaxel to
specific cancer cells via GLUTs; (ii) the glucuronic acid
analogue can be digested by ꢀ-glucuronidase and then release
the paclitaxel into target cells; (iii) the glucose and glucuronic
acid improve paclitaxel’s solubility; and (iv) the ester and ether
linkages are easily hydrolyzed and readily release paclitaxel into
cells. Therefore, we investigated the physiological changes of
targeted cancer cells treated with these novel glycan-based
paclitaxel prodrugs, which can be transported into cancer cells
quickly through GLUTs and release paclitaxel into specific target
cells.
According to previous studies, the C2′-hydroxyl group is
critical to the biological activity of paclitaxel, and modifications
at this position reduce the cytotoxicity of the compound
dramatically.11,40,41 We examined the cytotoxic effects of
modifying the 2′-position of paclitaxel using succinic acid as a
linker between paclitaxel and glucose. The results showed a
concentration-dependent inhibition of MCF-7 cancer cell growth
with the IC50 of 25 ng/mL (Figure 6). Although modifying the
2′-hydroxyl group of paclitaxel with succinic acid appeared to
reduce the toxicity of paclitaxel to cancer cells, this linker
preserved the activity of paclitaxel and could be used for further
conjugation with 2′-glucopyranose. Thus, we chose to modify
this position in the prodrugs. This position was blocked by
conjugation with a four-carbon spacer between paclitaxel and
the target glycan. Prodrugs 1 and 2 were glucose-conjugated
paclitaxel derivatives that were apparently internalized into
cancer cells through GLUTs on cell membrane surfaces. In
contrast, 3 and 4 are glucuronic acid-conjugated paclitaxels, in
which the glucose analogue apparently allows the prodrug to
diffuse into cells. If this is the case, the cytotoxicity values of
prodrugs 1 and 2 should be better than those of prodrugs 3 and
4. However, the cytotoxicity values of prodrugs 3 and 4 were
higher than that of prodrug 2. These results suggest that the
Discussion
Specific characteristics of cancer cells, such as high GLUT
expression levels, fast glucose uptake, high energy consumption,
and intracellular digestion,8,39 can be exploited to develop novel
prodrugs that overcome certain limitations of anticancer agents,
like low solubility and low selectivity. In our previous report,
prodrug 1 was synthesized using a glucose-ester conjugate,
and its cytotoxicity against various cell lines was examined.6
However, the mechanism of drug targeting was unknown, and
it was not clear if the bulky structure of paclitaxel could pass
through the GLUT. To develop a practical synthetic method
for glycan-based paclitaxel prodrugs, the synthesis of prodrug
1 was slightly modified as shown in Scheme 1. The total yield
was increased to 45%, compared with the previously reported
total yield of 25%. In addition, the current study avoided the
low yield (27%) synthetic step in coupling 2′-succinyl paclitaxel
with 3-O-benzyl-4,6-O-benzylidene methyl-D-glucopyranoside
(Scheme 2). The reasons for using the different glycans and

7434 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lin et al.
1. After 4, 8, and 24 h, the quantification of prodrug 1 in the
medium, serum free medium and cell cultured medium was
analyzed by RP-HPLC. The RP-HPLC analysis revealed that
the prodrug 1 was not cleaved by any extracellular enzymes,
for example, ꢀ-glucuronidase from FBS, outside the cells
(Figures S9 and S10, Supporting Information). The prodrug
uptake and the release paclitaxel were detected at 8 h and
significantly increased at 24 h. We suggest that the prodrug may
be cleaved by the intracellular enzymes, in which the ꢀ-glucu-
ronidase may be secreted from other organelles inside the cells.
Furthermore, the drug localization in cancer cells was
examined with fluorescent microscopy. The fluorescently labeled
prodrug 1 was synthesized by conjugation of a chromophore,
m-aminobenzoic group, at the 7′-position of paclitaxel. Here,
the C7′-hydroxyl group was not involved in paclitaxel’s
modification, but it is a good future candidate for fluorescent
labeling. After treatment for 5 h, light blue fluorescence appeared
in the cytoplasm of NPC-TW01 cells. This indicated that
prodrug 1 was rapidly transported into cancer cells. In addition,
the tubulin distribution and chromosome morphology were
examined by indirect immunofluorescent staining with confocal
microscopy. The tubulin in untreated cells appeared as dispersed
bundled structures. After treatment with paclitaxel and the
prodrugs, the tubulin structure in the NPC-TW01 cells was
aggregated around the nuclei. These results indicate that the
free paclitaxel can be released from the prodrugs and affect
targeted cells. However, the extent of tubulin aggregation during
prodrug treatment was not as strong as that seen with paclitaxel
treatment. This difference in tubulin aggregation may reflect
the relative release efficiency of paclitaxel. Moreover, changes
in chromosome condensation were observed in cells treated with
prodrugs. These results indicate that the prodrugs may lead to
cell cycle arrest or apoptosis of cancer cells. In future studies,
we plan to study the precise mechanisms of drug delivery, the
pharmacokinetics of different prodrugs, and their therapeutic
applications.
Conclusions
In this study, four glycan-based paclitaxel prodrugs were
synthesized using either ester or ether bonds as linkers between
2′-paclitaxel and glucose or glucuronic acid. These prodrugs
are not only more water soluble than paclitaxel, they also have
enhanced delivery to specifically targeted cells. In addition,
prodrug 1 showed the most cytotoxicity against cancer cells
and could induce tubulin aggregation and chromosomal con-
densation in NPC-TW01 cells, which could lead to cell cycle
arrest or apoptosis of cancer cells. This study proved that the
expression of GLUTs not only contributes to glucose uptake,
but also allows for the uptake of glucose derivatives with bulky
parent structures such as paclitaxel. However, it is also depended
on the selective cytotoxicity of prodrug against each type of
cancer cells. Therefore, these prodrugs may potentially be used
for further therapeutic applications, and the design of prodrugs
using a glucose conjugate with an ester linkage may be used as
a technique for the targeted delivery of other drugs.
Figure 3. Colocalization of prodrug 1 and tubulin in NPC-TW01 cells
under fluorescence microcopy. Fluorescence microscopy images rep-
resent the relative position of fluorescence-labeled prodrug 1 and
tubluin. The distributions of tubulin and fluorescence-labeled prodrug
1 are represented in panels a and b, respectively. Panel c is the merged
images. These images show the colocalization of prodrug 1 and tubulin.
glucuronic acid conjugate, which has a similar structure to
glucose, may be transported via GLUTs.
In addition, another difference between prodrugs 1 and 3, as
compared to prodrugs 2 and 4, was the type of linkage with
glucose or glucuronic acid. The glycan linkage of prodrugs 1
and 3 was an ester bond, while the glycan linkage of prodrugs
2 and 4 was an ether bond. The ether bond in prodrugs 2 and
4 was designed to imitate the glycosidic bond and enhance the
rates of enzyme-cleavage. We hypothesized that the order of
cytotoxicity among the prodrugs should be 2 > 1 > 4 > 3, but
the experimental results showed the order of cytoxicity was 1
> 2 > 3 > 4. The labile property of the ester bond may be the
reason for the greater cytotoxicity of prodrug 1.
Experimental Section
Materials. Paclitaxel was purchased from Apin Chemical Ltd.
(Oxford, U.K.). C14 was obtained from CNH Technologies, Inc.
(Woburn, MA). Succinic anhydride, palladium on 10% activated
carbon, 1,4-butandiol, benzoyl chloride, pyridinum dichromate
(PDC), chromium(III) oxide, aluminum chloride, boran-trimethy-
lamine, chlorotriethylsolane, tetrabutyl-ammonium iodide, and
sodium hydroxide were purchased from Acros Organics (Spring-
field, NJ). High Pure RNA Isolation kit was purchased from Roche
To investigate the possibility of prodrug cleavage outside
cells, the NPC-TW01 cells were cultured in the medium with
and without FBS (serum free medium) and treated with prodrug

DeliVery of Glycan-Based Paclitaxel Prodrugs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7435
Figure 4. Inhibition of phloretin on internalization of prodrug 1 into NPC-TW01 cells under fluorescence microscopy. Fluorescence microscopy
images represent the NPC-TW01 cells treated with fluorescence-labeled prodrug 1 in the absence (a, c, and e) or presence (b, d, and f) of phloretin.
Panels a and b are observed under white light, panels c and d are observed under fluorescence, and panels e and f are the merged images.
Figure 5. Morphological observations of NPC-TW01 cells treated with paclitaxel and glycan-based paclitaxel prodrugs under confocal microscopy.
Confocal microscopy images represent the tubulin distribution (a-f) and the chromosome morphology (g-l) of NPC-TW01 cell. These images
were taken in the absence of both drugs (a and g), and in the presence of either 1 µM paclitaxel (b and h), prodrug 1 (c and i), prodrug 2 (d and
j), prodrug 3 (e and k), or prodrug 4 (f and l). Nuclei were stained with Hoechst 33258 dye. The chromosomal aggregation in cells treated with
paclitaxel and prodrugs was detected under confocal microscopy.
(Mannheim, Germany). Sodium hydride was obtained from Alfa
Aesar (Ward Hill, MA). Dicyclohexylcarbodiimide (DCC) was
purchased from Fluka Chemie AG (Buchs, Switzerland). 4-Dim-
ethylaminopyridine (DMAP) was obtained from Aldrich Chemical
Co. (Milwaukee, WI). 3-Aminobenzoic acid was purchased from
Merck (Darmstadt, Germany). NCI-H838 (lung), MCF-7 (breast),
Hep-3B (liver), A-498 (kidney), HCT-116 (colon), MES-SA
(uterus), NPC-TW01 (nasopharynx), and MKN-45 (stomach) cancer
cell lines were obtained from American Type Culture Collection
(ATCC; Rockville, MD). Phloretin (3-[4-hydroxyphenyl-]-1-[2,4,6-
trihydroxyphenyl]-1-propanone), RPMI-1640, Minimum Essential
Medium Eagle (MEME), McCoy’s 5A, Dulbecco’s Modified
Eagle’s Medium (DMEM), Ham’s F12K medium, and antibiotics
were purchased from Sigma (St. Louis, MO). Fetal bovine serum
(FBS), glutamine and ProLong Gold antifade reagent were pur-
chased from Invitrogen (Carlsbad, CA).
Synthesis of Prodrugs 1-4. Prodrugs 1, 2, 3, and 4 were
synthesized by following the procedures described in Schemes 1,
2, 3, and 4, respectively.

7436 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lin et al.
or C12), 129.10 (C or CH, aromatic or C11 or C12), 129.02 (C or
CH, aromatic or C11 or C12), 128.69 (C or CH, aromatic or C11
or C12), 128.48 (C or CH, aromatic or C11 or C12), 128.26 (C or
CH, aromatic or C11 or C12), 127.65 (C or CH, aromatic or C11
or C12), 127.21 (C or CH, aromatic or C11 or C12), 126.53 (C or
CH, aromatic or C11 or C12), 126.00 (C or CH, aromatic or C11
or C12), 101.36 (CH, C7′′), 97.54 (CH, C1′′), 84.44 (CH, C5), 82.09
(CH, C4′′), 81.05 (C, C4), 79.12 (C, C1), 75.96 (CH, C3′′), 75.58
(CH, C10), 75.09 (CH, C2), 74.73 (CH2, -CH2Ph), 74.25 (CH, C2′),
73.14 (CH, C2′′), 72.11 (CH, C7), 71.86 (CH, C13), 68.90 (CH2,
C6′′), 62.25 (CH, C5′′), 58.51 (C, C8), 55.20 (CH3, -OCH3), 52.71
(CH, C3′), 45.53 (CH, C3), 43.17 (C, C15), 35.53 (CH2, C6 or
C14), 28.99 (CH2, Cb or Cc), 28.88 (CH2, Cb or Cc), 26.79 (CH3,
C16), 22.66 (CH3, 4-OCOCH3), 22.13 (CH3, C17), 20.81 (CH3, 10-
OCOCH3), 14.76 (CH3, C18), 9.58 (CH3, C19); m/z 1308.5008 (M
+ H)+, 1330.4829 (M + Na)+.
Figure 6. Cell cytotoxicity of paclitaxel and 2′-succinyl paclitaxel
against MCF-7 cells at 72 h. The concentrations of paclitaxel and 2′-
succinyl paclitaxel used were 1.57, 3.125, 6.25, 12.5, 25, and 50 ng/
mL. The cell proliferation was examined by the MTT assay.
Prodrug 1 (1). The mixture of 6 (46 mg, 0.035 mmol) and Pd/C
(25 mg) in MeOH (4 mL) was stirred at room temperature in
hydrogen for 12 h. After filtering by celite and silica gel, the residue
was concentrated and recrystallized to give a white solid of prodrug
Succinic Acid Mono-(8-benzyloxy-6-methoxy-2-phenyl-hexahy-
dro-pyrano[3,2-d][1,3]dioxin-7-yl) Ester (5). The succinic acid
mono-(8-benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-
d][1,3]dioxin-7-yl) ester (5) was synthesized as described below.
Briefly, the mixture of succinic anhydride (1.345 g, 13.45 mmol),
DMAP (33 mg, 0.27 mmol) and C14 (1.0 g, 2.69 mmol) in Pyr
(15 mL) was stirred at room temperature for 12 h. After quenching
with brine (15 mL), the mixture was extracted with EtOAc (15
mL × 3), dried over MgSO4, and concentrated. The residue was
purified by silica gel chromatography (CH2Cl2/MeOH ) 20/1) and
dried under vacuum to produce a white solid 5 (898 mg, 71%). 1H
NMR (400 MHz, CDCl3) δ 7.25-7.50 (m, 10 H), 5.59 (s, 1 H,
H7), 4.86-4.93 (m, 3 H, H1, H2, and sOCH2Ph), 4.70 (d, 1 H, J
) 11.8 Hz, -OCH2Ph), 4.30 (dd, 1 H, J ) 9.9, 4.4 Hz, H6),
4.01-4.07 (m, 1 H, H3), 3.85 (ddd, 1 H, J ) 9.9, 4.4 Hz, H5),
3.78 (t, 1 H, J ) 9.9 Hz, H6), 3.71 (t, 1 H. J ) 9.5 Hz, H4), 3.38
(s, 3 H, -OCH3), 2.57-2.70 (m, 4 H, Hb and Hc); 13C NMR (100
Hz, CDCl3) δ 176.71 (C, Ca or Cd), 171.60 (C, C1′ or C4′), 137.25
and 138.44 (C, aromatic), 126.02-129.5 (CH, aromatic), 101.36
(CH, C7), 97.67 (CH, C1), 82.03 (CH, C4), 76.09 (CH, C3), 74.78
(CH2, -OCH2Ph), 73.26 (CH, C2), 68.92 (CH2, C6), 62.27 (CH,
C5), 55.30 (CH3, OCH3), 28.76 (CH2, Cb or Cc), 28.62 (CH2, Cb
or Cc).
1
1 (37 mg, 93.1%). H NMR (400 MHz, CDCl3) δ 7.29-8.13 (m,
15H, aromatic), 7.13 (d, 1H, J ) 9.0 Hz, NH), 6.30 (s, 1H, H10),
6.17 (t, 1H, J ) 8.7 Hz, H13), 5.92 (dd, 1H, J ) 9.0, 4.2 Hz, H3′),
5.66 (d, 1H, J ) 7.0 Hz, H2), 5.48 (d, 1H, J ) 4.2 Hz, H2′), 4.96
(m, 1H, H5), 4.82 (d, 1H, J ) 3.6 Hz, H1′′), 4.67 (dd, 1H, J ) 9.7,
3.6 Hz, H2′′), 4.41 (m, 1H, H7), 4.30 (d, 1H, J ) 8.5 Hz, H20),
4.13 (d, 1H, J ) 8.5 Hz, H20), 3.92 (ddd, 1H, J ) 9.7, 9.7, 4.4 Hz,
H3′′), 3.80-3.85 (m, 2H, H6′′), 3.77 (d, 1H, J ) 7.0 Hz, H3),
3.52-3.63 (m, 2H, H4′′ and H5′′), 3.35 (s, 3H, -OCH3), 3.24 (d,
1H, J ) 4.4 Hz, 3′′-OH), 3.18 (d, 1H, J ) 3.0 Hz, 4′′-OH),
2.64-2.76 (m, 5H, Hb, Hc, and 7-OH), 2.50-2.57 (m, 1H, H6),
2.41 (s, 3H, 4-OAc), 2.24-2.34 (m, 2H, H14 and 6′′-OH), 2.22 (s,
3H, 10-OAc), 2.04-2.10 (m, 1H, H14), 1.84-1.90 (m, 5H, H6,
H10, and H18), 1.67 (s, 3H, H19), 1.21 (s, 3H, H15 or H16), 1.13
(s, 3H, H15 or H16); 13C NMR (100 MHz, CDCl3) δ 203.69 (C,
C9), 171.84 (C, Ca), 171.68 (C, Cd), 171.25 (C, 4 or 10-OCOCH3),
170.00 (C, 4 or 10-OCOCH3), 168.11 (C, C1′), 167.27 (C,
3′-NHCOC6H5), 166.97 (C, 2-OCOC6H5), 142.45 (C, C12), 136.74
(C, aromatic or C11), 133.68 (CH, aromatic or C11), 132.86 (C,
aromatic or C11), 132.01 (CH, aromatic or C11), 130.19 (C,
aromatic or C11), 129.10 (CH, aromatic or C11), 128.68
(CH, aromatic or C11), 127.21 (CH, aromatic or C11), 126.73 (CH,
aromatic or C11), 97.00 (CH, C1′′), 84.38 (CH, C5), 81.12 (C,
C4), 79.06 (C, C1), 75.57 (CH, C10), 75.02 (CH, C2), 74.52 (CH,
C2′), 73.70 (CH, C2′′), 71.96 (CH, C7 and C3′′), 71.63 (CH, C13),
70.91 (CH, C4′′ or C5′′), 70.72 (CH, C4′′ or C5′′), 62.08 (CH2,
C6′′), 58.41 (C, C8), 55.15 (CH3, -OCH3), 52.97 (CH, C3′), 45.71
(CH, C3), 43.13 (C, C15), 35.60 (CH, C6 or C14), 35.39 (CH, C6
or C14), 29.08 (CH2, Cb and Cc), 26.73 (CH3, C17), 22.62 (CH3,
4-OCOCH3), 22.01 (CH3, C16), 20.82 (CH3, 10-OCOCH3), 14.79
(CH2 or CH3, C18), 14.08 (CH2 or CH3, C6 or C18), 9.61 (CH3,
C19); m/z 1130.4227 (M+H)+, 1152.4047 (M+Na)+.
C15 (6). C15 (6) was synthesized as described below. Briefly,
the mixture of paclitaxel (50 mg, 0.06 mmol), DCC (36 mg, 0.18
mmol), and DMAP (3 mg, 0.02 mmol) in THF (2 mL) was added
into a solution of 5 (55 mg, 0.12 mmol) in THF (2 mL). After
stirring at room temperature for 20 h, and subsequent concentration,
the residue was purified by silica gel chromatography (EtOAc/Hex
) 4/1) and dried under vacuum to produce a white solid 6 (66.8
1
mg, 87.3%). H NMR (400 MHz, CDCl3) δ 7.20-8.16 (m, 25H,
aromatic), 7.01 (d, 1H, J ) 9.1 Hz, NH), 6.22-6.28 (m, 2H, H10
and H13), 5.98 (dd, 1H, J ) 9.1, 3 Hz, H3′), 5.69 (d, 1H, J ) 7.0
Hz, H2), 5.58 (s, 1H, H7′′), 5.49 (d, 1H, J ) 3.0 Hz, H2′), 4.97
(m, 1H, H5), 4.80-4.87 (m, 3H, H1′′ and H2′′ and sOCH2Ph),
4.65 (d, 1H, J ) 11.9 Hz, -OCH2Ph), 4.44 (dd, 1H, J ) 7.3, 6.6
Hz, 7H), 4.27-4.33 (m, 2H, H20 and H6′′), 4.20 (d, 1H, J ) 8.4
Hz, H20), 3.99 (t, 1H, J ) 9.2 Hz, H3′′), 3.75-3.86 (m, 2H, H3
and H5′′ and H6′′), 3.64 (t, 1H, J ) 9.2 Hz, H4′′), 3.34 (s, 3H,
-OCH3), 2.52-2.78 (m, 5H, Hb and Hc and H6′), 2.44 (s, 3H,
4-OAc), 2.34-2.41 (m, 1H, H14′), 2.23 (s, 3H, 10-OAc), 2.14-2.20
(m, 1H, H14), 1.92 (s, 3H, H18), 1.85-1.90 (m, 1H, H6), 1.68 (s,
3H, H19), 1.14 (s, 3H, H15 or H16), 0.98 (s, 3H, H15 or H16);
13C NMR (100 MHz, CDCl3) δ 203.80 (C, C9), 171.43 (C,
-COOH), 171.23 (C, -COOH), 171.02 (C, -COOH), 169.79 (C,
-COOH), 167.88 (C, -COOH), 167.15 (C, -COOH), 167.03 (C,
-COOH), 142.72 (C or CH, aromatic or C11 or C12), 138.41 (C or
CH, aromatic or C11 or C12), 137.19 (C or CH, aromatic or C11
or C12), 136.93 (C or CH, aromatic or C11 or C12), 133.66 (C or
CH, aromatic or C11 or C12), 133.57 (C or CH, aromatic or C11
or C12), 132.79 (C or CH, aromatic or C11 or C12), 131.99 (C or
CH, aromatic or C11 or C12), 130.23 (C or CH, aromatic or C11
Benzoic Acid 4-(8-Benzyloxy-6-methoxy-2-phenyl-hexahydro-
pyrano[3,2-d][1,3] dioxin-7-yloxy)-butyl Ester (7). The benzoic
acid 4-(8-benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-
d][1,3]dioxin-7-yloxy)-butyl ester (7) was synthesized as described
below. Briefly, the mixture of C14 (1000 mg, 2.69 mmol), benzoic
acid 4-bromo-butyl ester (1380 mg, 5.37 mmol), sodium hydride
(300 mg, 8.0 mmol), and TBAI in DMF (15 mL) was stirred at
room temperature for 36 h. After quenching with water (10 mL),
the mixture was extracted with EtOAc, dried over MgSO4 and
concentrated. The residue was purified by silica gel chromatography
(EtOAc/Hex ) 1/2) and dried under vacuum to produce a white
solid, 9 (1080 mg, 73.3%). 1H NMR (400 MHz, CDCl3) δ
7.25-8.07 (m, 15H, aromatic), 5.59 (s, 1H, H7), 4.81-4.93 (m,
3H, H1 and sOCH2Ph), 4.30-4.36 (m, 3H, H6 and Hd), 4.00 (t,
1H, J ) 9.3 Hz, H3), 3.86 (ddd, 1H, J ) 9.3, 9.3, 4.6 Hz, H5),
3.74-3.79 (m, 3H, H6 and H1a), 3.60-3.65 (m, 3H, H4 and Hd),
3.50 (dd, 1H, J ) 9.3, 3.6 Hz, H2), 3.46 (s, 3H, -OCH3), 1.80-1.88
(m, 4H, Hb and Hc); 13C NMR (100 Hz, CDCl3) δ 166.6 (C,
-OCOPh), 138.73 (C, aromatic), 137.42 (C, aromatic), 132.90 (CH,

DeliVery of Glycan-Based Paclitaxel Prodrugs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7437
aromatic), 130.38 (CH, aromatic), 129.57 (CH, aromatic), 128.93
(CH, aromatic), 128.36 (CH, aromatic), 128.29 (CH, aromatic),
128.24 (CH, aromatic), 127.91 (CH, aromatic), 127.58 (CH,
aromatic), 126.06 (CH, aromatic), 101.27 (CH, C7), 98.87 (CH,
C1), 82.14 (CH, C4), 80.50 (CH,C2), 78.39 (CH, C3), 75.28 (CH2,
-OCH2Ph), 71.39 (CH2, C6), 69.10 (CH2, Cd), 64.70 (CH, C5),
62.37 (CH2, Ca), 55.33 (CH3, -OCH3), 26.70 (CH2, Cb or Cc), 25.47
(CH2, Cb or Cc); m/z 549.2483 (M + H)+, 571.2302 (M + Na)+.
Prodrug 2 (2). The mixture of 10 (55 mg, 0.044 mmol) and
Pd/C (28 mg) in MeOH (4 mL) was stirred at room temperature in
hydrogen for 12 h. After filtering by celite and silica gel, the residue
was concentrated and recrystallized to give a white solid of prodrug
3,4-Bis-benzyloxy-5-hydroxy-6-methoxy-tetrahydro-pyran-2-
carboxylic Acid Benzyl Ester (13). A solution of 12 (500 mg,
1.05 mmol) in acetone (5 mL) was added with Jones reagent (0.43
M CrO3 in H2SO4, 7.3 mL) on ice. The mixture was stirred at room
temperature for 18 h, quenched with water (10 mL), extracted with
EtOAc (100 mL × 2), and dried over MgSO4. The crude product
was added to 1% NaOH in MeOH and stirred at room temperature
for 4 h. The pH of the mixture was adjusted to 2-4 by addition of
0.1 M H2SO4. The mixture was extracted with CHCl3 (100 mL ×
2), concentrated, and dried under vacuum. The mixture of the crude
residue, DCC (336 mg, 1.625 mmol), and DMAP (catalytic amount)
in DMF (4 mL) was added to benzyl alcohol (170 µL, 1.625 mmol)
and stirred at room temperature for 18 h. After purification by silica
gel chromatography (EtOAc/Hex ) 1/2), the residue was dried
1
2 (47 mg, 95%). H NMR (400 MHz, CDCl3) δ 7.34-8.15 (m,
15H, aromatic), 7.23 (d, 1H, J ) 9.2 Hz, NH), 6.31 (s, 1H, H10),
6.24 (t, 1H, J ) 8.5 Hz, H13), 5.99 (dd, 1H, J ) 9.2, 3.5 Hz, H3′),
5.69 (d, 1H, J ) 7.1 Hz, H2), 5.51 (d, 1H, J ) 3.5 Hz, H2′),
4.96-4.99 (m, 1H, H5), 4.79 (d, 1H, J ) 3.4 Hz, H1′′), 4.44 (dd,
1H, J ) 10.6, 6.6 Hz, H7), 4.32 (d, 1H, J ) 8.6 Hz, -CH2Ph), 4.20
(d, 1H, J ) 8.6 Hz, -CH2Ph), 3.75-3.82 (m, 4H, H3, H3′′, and
H6′′), 3.67-3.72 (m, 1H, Ha), 3.54-3.59 (m, 1H, H5′′), 3.37-3.49
(m, 5H, H4′′, Ha, and sOCH3), 3.20 (dd, 1H, J ) 9.6, 3.4 Hz,
H2′′), 2.66-2.74 (m, 1H, Hc), 2.52-2.60 (m, 1H, H6), 2.47 (s,
3H, 4-OCOCH3), 2.33-2.44 (m, 2H, H14 and Hc), 2.23 (s, 3H,
10-OCOCH3), 2.14-2.20 (m, 1H, H14), 1.95 (s, 3H, H18),
1.86-1.92 (m, 3H, H6 and Hb), 1.68 (s, 3H, H19), 1.24 (s, 3H,
H17), 1.14 (s, 3H, H16); 13C NMR (100 Hz, CDCl3) δ 203.75 (C,
C9), 172.76 (C, -COOR), 171.26 (C, -COOR), 169.94 (C, -COOR),
168.46 (C, -COOR), 167.34 (C, -COOR), 166.99 (C, -COOR),
142.54 (C or CH, aromatic or C11 or C12), 136.88 (C or CH,
aromatic or C11 or C12), 133.69 (C or CH, aromatic or C11 or
C12), 132.85 (C or CH, aromatic or C11 or C12), 132.04 (C or
CH, aromatic or C11 or C12), 130.21 (C or CH, aromatic or C11
or C12), 129.18 (C or CH, aromatic or C11 or C12), 129.07 (C or
CH, aromatic or C11 or C12), 128.69 (C or CH, aromatic or C11
or C12), 128.48 (C or CH, aromatic or C11 or C12), 127.23 (C or
CH, aromatic or C11 or C12), 126.63 (C or CH, aromatic or C11
or C12), 97.35 (CH, C1′′), 84.42 (CH, C5), 81.07 (C, C4), 80.04
(CH, C2′′), 79.07 (C, C1), 76.40 (CH2, C20), 75.59 (CH, C10),
75.03 (CH, C2), 74.15 (CH, C2′), 73.00 (CH or CH2, C3′′ or C6′′),
71.99 (CH, C7 or C13), 71.92 (CH, C7 or C13), 70.75 (CH, C4′′
or C5′′), 70.62 (CH, C4′′ or C5′′), 68.63 (CH2, Ca), 62.20 (CH or
CH2, C3′′ or C6′′), 58.41 (C, C8), 55.16 (CH3, -OCH3), 52.76 (CH,
C3′), 45.67 (CH, C3), 43.16 (C, C15), 35.54 (CH, C6 or C14),
35.60 (CH, C6 or C14), 29.69 (CH2, Cc), 26.78 (CH3, C17), 24.86
(CH2, Cb), 22.68 (CH3, 4-OCOCH3), 22.07 (CH3, C16), 20.86 (CH3,
10-OCOCH3), 14.87 (CH3, C18), 9.64 (CH3, C19); m/z 1138.4254
(M+Na)+.
Benzoic Acid 4,5-Bis-benzyloxy-6-hydroxymethyl-2-methoxy-
tetrahydro-pyran-3-yl Ester (12). The mixture of 11 (700 mg,
1.45 mmol) and boran-trimethylamine (635 mg, 8.7 mmol) in
toluene (5 mL) was added with aluminum chloride (1.14 g, 8.7
mmol) to ether (5 mL) on ice, and was then stirred on ice for 20
min. After quenching with water, the mixture was extracted with
EtOAc, dried over MgSO4, and concentrated. The residue was
purified by silica gel chromatography (CH2Cl2/MeOH ) 40/1) and
dried under vacuum to produce a white solid (517 mg, 74.5%). 1H
NMR (400 MHz, CDCl3) 7.18-8.08 (m, 15H, aromatic), 5.08 (dd,
1H, J ) 9.7, 3.7 Hz, H2), 5.03 (d, 1H, J ) 3.7 Hz, H1), 4.90 (d,
1H, J ) 11.0 Hz, -CH2Ph), 4.83 (s, 2H, -CH2Ph), 4.69 (d, 1H, J )
11.0 Hz, -CH2Ph), 4.22 (t, 1H, J ) 9.7 Hz, H3), 3.68-3.87 (m,
4H, H4, H5, and H6), 3.37 (s, 3H, -OCH3); 13C NMR (100 Hz,
CDCl3) δ 165.98 (C, -COOPh), 138.16 (C, aromatic), 138.03 (C,
aromatic), 133.05 (CH, aromatic), 129.84 (CH, aromatic), 129.69
(CH, aromatic), 128.48 (CH, aromatic), 128.45 (CH, aromatic),
128.31 (CH, aromatic), 128.02 (CH, aromatic), 127.86 (CH,
aromatic), 127.64 (CH, aromatic), 97.26 (CH, C1), 80.01 (CH, C3),
77.65 (CH, C4 or c5), 75.54 (CH2, -CH2Ph), 75.13 (CH2, -CH2Ph),
74.20 (CH, C2), 70.87 (CH, C4 or C5), 61.79 (CH2, C6), 55.22
(CH3, -OCH3); m/z 479.2064 (M + H)+, 501.1884 (M + Na)+.
1
under vacuum to produce a white solid (154 mg, 23%). H NMR
(400 MHz, CDCl3) δ7.14-7.33 (m, 15H, aromatic), 5.14-5.22 (m,
2H, -CH2Ph), 4.79-4.85 (m, 3H, H1 and -CH2Ph), 4.72 (d, 1H, J
) 10.8 Hz, -CH2Ph), 4.48 (d, 1H, J ) 10.8 Hz, -CH2Ph), 4.28-4.30
(m, 1H, H3), 3.75-3.80 (m, 3H, H2, H4, and H5), 3.47 (s, 3H,
-OCH3), 2.27 (d, 1H, J ) 7.6 Hz, -OH); 13C NMR (100 Hz, CDCl3)
δ 169.39 (C, COOBn), 138.28 (C, aromatic), 137.74 (C, aromatic),
135.05 (C, aromatic), 128.58 (CH, aromatic), 126.46 (CH, aro-
matic), 128.33 (CH, aromatic), 127.89 (CH, aromatic), 127.77 (CH,
aromatic), 99.63 (CH, C1), 81.88 (CH, C2 or C4 or C5), 79.04
(CH, C2 or C4 or C5), 75.25 (CH2, -CH2Ph), 74.74 (CH2, -CH2Ph),
72.17 (CH, C2 or C4 or C5), 70.87 (CH, C3), 67.33 (CH2, -CH2Ph),
55.80 (CH3, -OCH3); m/z 479.2064 (M + H)+, 501.1884 (M +
Na)+.
Prodrug 3 (3). The mixture of 15 (33 mg, 0.023 mmol) and
Pd/C (15 mg) in MeOH (2 mL) was stirred at room temperature in
hydrogen for 12 h. After filtering by celite and silica gel, the residue
was concentrated and recrystallized to give the white solid of
prodrug 3 (25 mg, 95%). 1H NMR (400 MHz, CDCl3) δ7.13-8.14
(m, 15H, aromatic), 7.08 (d, 1H, J ) 9.2 Hz, NH), 6.29 (s, 1H,
H10), 6.20 (t, 1H, J ) 8.3 Hz, H13), 5.95 (dd, 1H, J ) 9.2, 3.9
Hz, H3′), 5.67 (d, 1H, J ) 6.7 Hz, H2), 5.49 (d, 1H, J ) 3.9 Hz,
H2′), 4.95-4.97 (m, 1H, H5), 4.91 (d, 1H, J ) 3.6 Hz, H1′′), 4.71
(dd, 1H, J ) 9.8, 3.6 Hz, H2′′), 4.42 (dd, 1H, J ) 11.2, 6.9 Hz,
H7), 4.30 (d, 1H, J ) 8.4 Hz, H20), 4.19 (d, 1H, J ) 8.4 Hz,
H20), 4.12 (d, 1H, J ) 9.8 Hz, H5′′), 3.96 (t, 1H, J ) 9.8 Hz,
H3′′), 3.79 (d, 1H, J ) 6.7 Hz, H3), 3.70 (t, 1H, J ) 9.8 Hz, H4′′),
3.40 (s, 3H, -OCH3), 2.67-2.79 (m, 4H, Hb and Hc), 2.51-2.57
(m, 1H, H6), 2.42 (s, 3H, 4-OCOCH3), 2.26-2.34 (m, 1H, H14),
2.23 (s, 3H, 10-OCOCH3), 2.04-2.14 (m, 1H, H14), 1.90 (s, 3H,
H18), 1.85-1.89 (m, 1H, H6), 1.68 (s, 3H, H19), 1.24 (s, 3H, H16),
1.13 (s, 3H, H17); 13C NMR (100 Hz, CDCl3) δ 203.73 (C, C9),
171.60 (C, -COOR, 171.33 (C, -COOR), 171.22 (C, -COOR),
170.47 (C, -COOR), 169.95 (C, -COOR), 168.14 (C, -COOR),
167.99 (C, -COOR), 167.30 (C, -COOR), 166.98 (C, -COOR),
136.78 (C or CH, aromatic or C11 or C12), 133.61 (C or CH,
aromatic or C11 or C12), 132.86 (C or CH, aromatic or C11 or
C12), 132.01 (C or CH, aromatic or C11 or C12), 130.20 (C or
CH, aromatic or C11 or C12), 129.10 (C or CH, aromatic or C11
or C12), 128.69 (C or CH, aromatic or C11 or C12), 127.21 (C or
CH, aromatic or C11 or C12), 126.78 (C or CH, aromatic or C11
or C12), 126.67 (C or CH, aromatic or C11 or C12), 97.31 (CH,
C1′′), 84.41 (CH, C5), 81.11 (C, C4), 79.09 (C, C1), 75.59 (CH,
C10), 75.07 (CH, C2), 74.45 (CH, C2′), 72.82 (CH, C2′′), 72.02
(CH, C7 or/and C13 or/and C4′′), 71.95 (CH, C7 or/ and C13 or/
and C4′′), 70.74 (CH, C3′′), 69.73 (CH, C5′′), 58.47 (C, C8), 55.78
(CH3, -OCH3), 52.82 (CH, C3′), 45.80 (CH,C3), 43.15 (C, C15),
35.54 (CH, C6 and C14), 29.07 (CH2, Cb and Cc), 26.78 (CH3,
C16), 22.63 (CH3, 4-OCOCH3), 22.06 (CH3, C17), 20.81 (CH3,
10-OCOCH3), 14.76 (CH3, C18), 9.60 (CH3, C19); m/z 1166.3840
(M + Na)+.
Benzoic Acid 4-(4,5-Bis-benzyloxy-6-hydroxymethyl-2-meth-
oxy-tetrahydro-pyran-3-yloxy)-butyl Ester (16). The mixture of
7 (240 mg, 0.449 mmol) and boran-trimethylamine (196.5 mg, 2.694
mmol) in toluene (2 mL) was added to aluminum chloride (353.8
mg, 2.694 mmol) in ether (2 mL) on ice and stirred on ice for 20
min. After quenching with water, the mixture was extracted with

7438 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lin et al.
EtOAc, dried over MgSO4, and concentrated. The residue was
purified by silica gel chromatography (EtOAc/Hex ) 1/1) and dried
under vacuum to produce a white solid (153.1 mg, 62%). 1H NMR
(400 MHz, CDCl3) δ 7.25-8.04 (m, 15H, aromatic), 4.81-4.96
(m, 4H, H1 and -CH2Ph), 4.65 (d, 1H, J ) 11.1 Hz, -CH2Ph), 4.32
(t, 2H, J ) 6.0 Hz, Hd), 3.95 (t, 1H, J ) 9.2 Hz, H3), 3.63-3.82
(m, 5H, H5, H6, and Ha), 3.54 (t, 1H, J ) 9.2 Hz, H4), 3.40-3.43
(m, 4H, H2 and sOCH3), 1.79-1.85 (m, 4H, Hb and Hc); 13C
NMR (100 Hz, CDCl3) δ 166.58 (C, -COPh), 138.75 (C, aromatic),
138.15 (C, aromatic), 132.86 (CH, aromatic), 130.36 (CH, aro-
matic), 129.54 (CH, aromatic), 128.53 (CH, aromatic), 128.46 (CH,
aromatic), 128.36 (CH, aromatic), 128.34 (CH, aromatic), 128.03
(CH, aromatic), 127.84 (CH, aromatic), 127.76 (CH, aromatic),
127.57 (CH, aromatic), 126.94 (CH, aromatic), 97.85 (CH, C1),
81.79 (CH, C3), 81.06 (CH, C2), 75.64 (CH2, -CH2Ph), 75.02 (CH2,
-CH2Ph), 70.83 (C5 or C6), 70.75 (C5 or C6), 64.63 (CH2, Cd),
61.89 (CH2, Ca), 55.15 (CH3, -OCH3), 26.72 (CH2, Cb or Cc), 25.47
(CH2, Cb or Cc); m/z 551.2639 (M + H)+, 573.2459 (M + Na)+.
or C12), 98.06 (CH, C1′′), 84.42 (CH,C5), 81.13 (C, C4), 79.80
(CH, C2′′), 79.13 (C,C1), 75.56 (CH,C10), 75.07 (CH, C2), 74.09
(CH, C2′), 72.75 (CH, C3′′ and C4′′), 72.07 (CH, C7 and C13),
71.99 (CH, C7 and C13), 68.73 (CH2, Ca), 68.13 (CH, C5′′), 58.41
(C, C8), 55,91 (CH3, -OCH3), 52.77 (CH, C3′), 45.69 (CH, C3),
43.17 (C, C15), 35.62 (CH, C6 and C14), 29.84 (CH2, Cc), 26.84
(CH3, C16), 24.75 (CH2, Cb), 22.64 (CH3, 4-OCOCH3), 22.05 (CH3,
C17), 20.84 (CH3, 10-OCOCH3), 14.89 (CH3, C18), 9.64 (CH3,
C19).
Synthesis of Fluorescence-Labeled Prodrug. The synthesis of
fluorescence-labeled prodrug, C16-F, was performed in five steps
(Scheme 5).
Paclitaxel-2′TES (19). The mixture of paclitaxel (100 mg, 0.117
mmol) and chlorotriethylsilane (19.4 mg, 0.1287 mmol) in pyridine
was stirred at room temperature for 3 h. After quenching by the
addition of water, the mixture was extracted with EtOAc (15 mL
× 3), dried over MgSO4, and concentrated. The residue was purified
by silica gel chromatography (EtOAc/Hex ) 3/2) and dried under
1
vacuum to produce a white solid (190 mg, 84%). H NMR (400
3,4-Bis-benzyloxy-5-(3-carboxy-propoxy)-6-methoxy-tetrahy-
dro-pyran-2-carboxylic Acid (17). The mixture of 16 (150 mg,
0.28 mmol) and NaOH (17 mg, 0.42 mmol) in MeOH (2 mL) was
stirred at room temperature for 2 h. After evaporation of MeOH
and subsequent addition of water, the residue was extracted with
ether, dried over MgSO4, and concentrated. The crude residue was
dissolved in acetone (3 mL), added CrO3 (0.34 M, 4 mL) in H2SO4
on ice, and stirred at room temperature for 18 h. After extraction
with CHCl3, the organic layer was collected and extracted with
NaOH solution (pH 14), followed by HCl, and CHCl3. The organic
layer was dried over MgSO4 and concentrated as a white solid (80
mg, 60%). 1H NMR (400 MHz, CDCl3) δ 7.24-8.12 (m, 10H,
aromatic), 4.89-4.91 (m, 2H, H1 and -CH2Ph), 4.80-4.83 (m, 2H,
-CH2Ph), 4.66 (d, 1H, J ) 10.8 Hz, -CH2Ph), 4.24 (d, 1H, J ) 9.6
Hz, H5), 3.96 (t, 1H, J ) 9.6 Hz, H3), 3.75 (t, 1H, J ) 9.6 Hz,
H4), 3.70 (t, 2H, J ) 6.4, Ha), 3.46-3.50 (m, 3H, H2 and sOCH3),
2.46-2.50 (m, 2H, Hc), 1.95 (quintet, 2H, J ) 6.4, Hb); 13C NMR
(100 Hz, CDCl3) δ 178.33 (C, -COOH), 173.18 (C, -COOH),
138.31 (C, aromatic), 137.43 (C, aromatic), 130.18 (CH, aromatic),
128.41 (CH, aromatic), 128.12 (CH, aromatic), 127.94 (CH,
aromatic), 127.81 (CH, aromatic), 127.74 (CH, aromatic), 98.23
(CH, C1), 81.18 (CH, C3), 80.24 (CH, C2), 79.06 (CH, C4), 75.80
(CH2, -CH2Ph), 75.25 (CH2, -CH2Ph), 70.16 (CH2, Ca), 69.54 (CH1,
C5), 55.76 (CH3, -OCH3), 30.37 (CH2, Cc), 25.03 (CH2, Cb); m/z
475.1963 (M + H)+, 497.1782 (M + Na)+.
Prodrug 4 (4). The mixture of 18 (15 mg, 0.0115 mmol) and
Pd/C (15 mg) in MeOH (2 mL) was stirred at room temperature in
hydrogen for 18 h. After filtering by celite and silica gel, the residue
was concentrated and recrystallized to give the white solid of
prodrug 4 (12 mg, 90%). 1H NMR (400 MHz, CDCl3) δ 7.26-8.14
(m, 15H, aromatic), 7.10 (d, 1H, J ) 9.2 Hz, NH), 6.33 (s, 1H,
H10), 6.22 (t, 1H, J ) 8.9 Hz, H13), 5.97 (dd, 1H, J ) 9.2, 2.8
Hz, H3′), 5.69 (d, 1H, J ) 7.0 Hz, H2), 5.54 (d, 1H, J ) 3.8 Hz,
H2′), 4.96-4.98 (m, 1H, H5), 4.83 (d, 1H, J ) 3.6 Hz, H1′′),
4.41-4.43 (m, 2H, H7 and H5′′), 4.31 (d, 1H, J ) 8.4 Hz, H20),
4.20 (d, 1H, J ) 8.4 Hz, H20), 3.75-3.82 (m, 3H, H3, H3′′, and
H4′′), 3.66-3.71 (m, 1H, Ha), 3.38-3.44 (m, 4H, Ha and sOCH3),
3.31 (dd, 1H, J ) 8.8, 3.6 Hz), 2.67-2.70 (m, 1H, Hc), 2.54-2.60
(m, 1H, H6), 2.45 (s, 3H, 4-OCOCH3), 2.32-2.45 (m, 2H, H14
and Hc), 2.25 (s, 3H, 10-OCOCH3), 2.13-2.17 (m, 1H, H14), 1.93
(s, 3H, H18), 1.90-1.93 (m, 3H, H6 and Hb), 1.69 (s, 3H, H19),
1.24 (s, 3H, H16), 1.14 (s, 3H, H17); 13C NMR (100 Hz, CDCl3)
δ 203.70 (C, C9), 176.76 (C, -COOR), 172.61 (C, -COOR), 171.20
(C, -COOR), 169.88 (C, -COOR), 168.51 (C, -COOR), 167.17 (C,
-COOR), 167.01 (C, -COOR), 152.71 (C or CH, aromatic or C11
or C12), 142.37 (C or CH, aromatic or C11 or C12), 136.86 (C or
CH, aromatic or C11 or C12), 133.65 (C or CH, aromatic or C11
or C12), 133.01 (C or CH, aromatic or C11 or C12), 132.00 (C or
CH, aromatic or C11 or C12), 130.20 (C or CH, aromatic or C11
or C12), 129.22 (C or CH, aromatic or C11 or C12), 129.08 (C or
CH, aromatic or C11 or C12), 128.70 (C or CH, aromatic or C11
or C12), 128.51 (C or CH, aromatic or C11 or C12), 127.20 (C or
CH, aromatic or C11 or C12), 126.60 (C or CH, aromatic or C11
MHz, CDCl3) δ 7.26-8.15 (m, 15H, aromatic, 7.12 (d, 1H, J )
8.9 Hz, NH), 6.25-6.30 (m, 2H, H10 and H13), 5.69-5.73 (m,
2H, H2 and H3′), 4.98 (dd, 1H, J ) 9.6, 2.0 Hz, H5), 4.70 (d, 1H,
J ) 3.4 Hz, H2′), 4.43 (m, 1H, H7), 4.33 (d, 1H, J ) 8.4 Hz,
H20), 4.23 (d, 1H, J ) 8.4 Hz, H20), 3.83 (d, 1H, J ) 7.1 Hz,
H3), 2.53-2.58 (m, 4H, H6 and 4-OCOCH3.), 2.38-2.44 (m, 1H,
H14), 2.23 (s, 3H, 10-OCOCH3), 2.14-2.20 (m, 1H, H14),
1.86-1.93 (m, 4H, H6 and H18), 1.69 (s, 3H, H19), 1.25 (s, 3H,
H17), 1.14 (s, 3H, H16), 0.82 (t, 9H, J ) 7.9 Hz, SiCH2CH3),
0.41-0.53 (m, 6H, SiCH2CH3); 13C NMR (100 Hz, CDCl3) δ
203.74 (C, C9), 171.53 (C, RCOOR′), 171.27 (C, RCOOR′), 170.06
(C, RCOOR′), 166.99 (C, RCOOR′), 142.49 (C, C11 or C13 or
aromatic), 138.39 (C, C11 or C13 or aromatic), 134.07 (C, C11 or
C13 or aromatic), 133.65 (CH, aromatic), 132.94 (C, C11 or C13
or aromatic), 131.76 (CH, aromatic), 130.21 (CH, aromatic), 129.17
(C, C11 or C13 or aromatic), 128.69 (CH, aromatic), 128.00 (C,
C11 or C13 or aromatic), 127.02 (CH, aromatic), 126.45 (CH,
aromatic), 84.44 (CH,C5), 81.16 (C, C4), 79.13 (C, C1), 75.55 (CH,
C10), 75.10 (CH, C2), 74.87 (CH, C2′), 72.11 (CH,C7), 71.45 (CH,
C13), 58.53 (C, C8), 55.69 (CH, C3′), 45.50 (CH, C3), 43.24 (C,
C15), 35.82 (CH, C14), 35.55 (CH, C6), 26.76 (CH3, C17), 22.97
(CH3, 4-OCOCH3), 22.26 (CH3, C16), 20.81 (CH3, 10-OCOCH3),
14.82 (CH3, C18), 9.61 (CH3, C19), 6.49 (SiCH2CH3), 4.37
(SiCH2CH3); m/z 968.4247 (M + H)+.
Paclitaxel-2′TES-7F (20). The mixture of 19 (60 mg, 0.062
mmol), m-NHCbz benzoic acid (74 mg, 0.27 mmol), DCC (55 mg,
0.267 mmol), and a catalytic amount of DMAP in THF (2 mL)
was stirred at room temperature for 18 h. After concentration, the
residue was purified by silica gel chromatography (EtOAc/Hex )
1/2) and dried under vacuum to produce a white solid (35 mg, 46%).
1H NMR (400 MHz, CDCl3) δ 7.31-8.16 (m, 23H, aromatic), 7.13
(d, 1H, J ) 8.9 Hz, NH), 6.82 (s, 1H), 6.40 (s, 1H, H10), 6.25 (t,
1H, J ) 9.0 Hz, H13), 5.71-5.79 (m, 3H, H2, H7, and H3′), 5.22
(s, 2H, -CH2Ph), 5.02 (m, 1H, H5), 4.72 (d, 1H, J ) 1.9 Hz, H2′),
4.38 (d, 1H, J ) 8.4 Hz, H20), 4.16 (d, 1H, J ) 8.4 Hz, H20),
4.05 (d, 1H, J ) 7.0 Hz, H3), 2.73-2.79 (m, 1H, H6), 2.56 (s, 3H,
4-OCOCH3), 2.43-2.49 (m, 1H, H14), 2.17-2.23 (m, 1H, H14),
2.01 (s, 3H, H18), 1.97 (s, 3H, 10-OCOCH3), 1.92 (s, 3H, H19),
1.21 (s, 3H, H17), 1.18 (s, 3H, H16), 0.83 (t, 9H, J ) 3.4 Hz,
SiCH2CH3), 0.44-0.54 (m, 6H, SiCH2CH3); 13C NMR (100 Hz,
CDCl3) δ 202.54 (C, C9), 171.59 (C, RCOOR′), 169.85 (C,
RCOOR′), 168.41 (C, RCOOR′), 166.99 (C, RCOOR′), 164.81 (C,
RCOOR′), 141.06 (CH, aromatic), 138.43 (CH, aromatic), 137.77
(CH, aromatic), 135.96 (CH, aromatic), 134.14 (CH, aromatic),
133.73 (CH, aromatic), 132.93 (CH, aromatic), 131.74 (CH,
aromatic), 130.21 (CH, aromatic), 129.10 (CH, aromatic), 128.79
(CH, aromatic), 128.69 (CH, aromatic), 128.62 (CH, aromatic),
128.31 (CH, aromatic), 127.97 (CH, aromatic), 127.03 (CH,
aromatic), 126.46 (CH, aromatic), 124.88 (CH, aromatic), 123.13
(CH, aromatic), 83.99 (CH, C5), 80.95 (C,C4), 78.75 (C, C1), 74.77
(CH, C10 and C2′), 74.64 (CH, C2 or C7 or C3′), 72.23 (CH, C2
or C7 or C3′), 71.33 (CH, C13), 67.12 (CH2, -CH2Ph), 56.25 (C,

DeliVery of Glycan-Based Paclitaxel Prodrugs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7439
C8), 55.72 (CH, C2 or C7 or C3′), 46.63 (CH, C3), 43.32 (C, C15),
35.70 (CH, C14), 33.33 (CH, C6), 26.34 (CH3, C17), 22.93 (CH3,
4-OCOCH3), 21.48 (CH3, C16), 20.42 (CH3, 10-OCOCH3), 14.67
(CH3, C18), 11.10 (CH3, C19), 6.48 (CH3, SiCH2CH3), 4.37 (CH2,
SiCH2CH3); m/z 1221.4986 (M + H)+, 1243.4805 (M + Na)+.
Paclitaxel-2′OH-7F (21). The mixture of 20 (30 mg, 0.025
mmol) and Bu4NF (30 µL of 1 M solution in THF, 0.03 mmol) in
THF (1 mL) was stirred at room temperature for 2 h. After
concentration, the residue was dissolved in EtOAc, extracted with
NaCl, dried over MgSO4, and concentrated. The residue was
purified by silica gel chromatography (EtOAc/Hex ) 3/2) and dried
(C, C1), 76.34 (CH2, C20), 74.78 (CH, C10), 74.62 (CH, C2), 74.39
(CH, C2′), 73.79 (CH, C2′′), 72.22 (CH, C7), 71.88 (CH, C13),
71.67 (CH, C3′′), 71.19 (CH, C4′′), 70.84 (CH, C5′′), 62.29 (CH,
C6′′), 56.28 (C, C8), 55.18 (CH3, -OCH3), 53.14 (CH, C3′), 46.73
(CH, C3), 43.19 (C, C15), 35.31 (CH2, C14), 33.23 (CH2, C6),
29.16 (CH2, Cb or Cc), 29.07 (CH2, Cb or Cc), 26.38 (CH3, C17),
22.59 (CH3, 4-OCOCH3), 21.26 (CH3, C16), 20.45 (CH3, 10-
OCOCH3), 14.53 (CH3, C18), 11.03 (CH3, C19).
C16-F (23). The mixture of 22 (46 mg, 0.029 mmol) and Pd/C
(25 mg) in MeOH (4 mL) was stirred at room temperature in
hydrogen for 12 h. The solution was filtered by celite and silica
gel. The residue was concentrated and recrystallized to give a white
solid of fluorescence-labeled prodrug 23 (35 mg, 96.6%). 1H NMR
(400 MHz, CDCl3) δ 7.24-8.14 (20H, m, aromatic), 7.09 (1H, d,
J ) 9.0 Hz, NH), 6.43 (1H, s, H10), 6.16 (1H, t, J ) 8.2 Hz, H13),
5.93 (1H, dd, J ) 9.0, 4.1 Hz, H3′), 5.73 (1H, d, J ) 6.9 Hz, H2),
5.68 (1H, dd, J ) 10.4, 7.2 Hz, H7), 5.54 (1H, d, J ) 4.1 Hz,
H2′), 5.00 (1H, d, J ) 8.3 Hz, H5), 4.82 (1H, d, J ) 3.6 Hz, H1′′),
4.70 (1H, dd, J ) 9.7 Hz, H2′′), 4.34 (1H, d, J ) 8.4 Hz, H20),
4.22 (1H, d, J ) 8.4 Hz, H20), 4.00 (1H, d, J ) 6.9 Hz, H3), 3.94
(1H, t, J ) 9.7 Hz, H3′′), 3.79-3.83 (2H, m, H6′′), 3.61v3.65 (1H,
m, H5′′), 3.52 (1H, t, J ) 9.7 Hz, H4′′), 3.37 (3H, s, -OCH3),
2.66-2.86 (5H, m, H6 and succinic), 2.43 (3H, s, 4-OCOCH3),
2.28-2.34 (1H, m, H14), 2.08-2.17 (1H, m, H14), 2.01 (3H, s,
H18), 1.99 (3H, s, 10-OCOCH3), 1.88-1.91 (4H, m, H6 and H19),
1.18 (6H, s, H16 and H17); 13C NMR (100 Hz, CDCl3) δ 202.36
(C, C9), 171.80 (C, RCOOR′), 171.64 (C, RCOOR′), 169.83 (C,
RCOOR′), 168.38 (C, RCOOR′), 168.11 (C, RCOOR′), 167.20 (C,
RCOOR′), 166.98 (C, RCOOR′), 166.22 (C, RCOOR′), 150.26 (C),
141.15 (C or CH, aromatic or C11 or C12), 136.81 (C or CH,
aromatic or C11 or C12), 133.76 (C or CH, aromatic or C11 or
C12), 132.88 (C or CH, aromatic or C11 or C12), 132.00 (C or
CH, aromatic or C11 or C12), 130.56 (C or CH, aromatic or C11
or C12), 130.18 (C or CH, aromatic or C11 or C12), 129.09 (C or
CH, aromatic or C11 or C12), 128.71 (C or CH, aromatic or C11
or C12), 128.58 (C or CH, aromatic or C11 or C12), 127.18 (C or
CH, aromatic or C11 or C12), 126.76 (C or CH, aromatic or C11
or C12), 117.95 (C or CH, aromatic or C11 or C12), 116.87 (C or
CH, aromatic or C11 or C12), 113.81 (C or CH, aromatic or C11
or C12), 96.99 (CH,C1′′), 83.96 (CH, C5), 80.95 (C, C4), 78.76
(C, C1), 76.34 (CH2, C20), 74.78 (CH, C10), 74.62 (CH, C2), 74.39
(CH, C2′), 73.79 (CH, C2′′), 72.22 (CH, C7), 71.88 (CH, C13),
71.67 (CH, C3′′), 71.19 (CH, C4′′), 70.84 (CH, C5′′), 62.29 (CH,
C6′′), 56.28 (C, C8), 55.18 (CH3, -OCH3), 53.14 (CH, C3′), 46.73
(CH, C3), 43.19 (C, C15), 35.31 (CH2, C14), 33.23 (CH2, C6),
29.16 (CH2, Cb or Cc), 29.07 (CH2, Cb or Cc), 26.38 (CH3, C17),
22.59 (CH3, 4-OCOCH3), 21.26 (CH3, C16), 20.45 (CH3, 10-
OCOCH3), 14.53 (CH3, C18), 11.03 (CH3, C19).
Biology. Cell Cultures. NCI-H838 nonsmall lung cancer cells
(lung adenocarcinoma) were maintained in DMEM medium with
10% FBS, 2 mM glutamine, and antibiotics (100 units/mL penicillin
and 100 µg/mL streptomycin). Hep-3B hepatoma cells were
maintained in MEME containing 10% FBS. A498 renal cancer cells
were maintained in 90% MEME medium supplemented with 1 mM
sodium pyruvate, 10% FCS, and antibiotics. MES-SA uterine cancer
cells were maintained in McCoy’s 5A medium supplemented with
10% FCS, 25 mM HEPES, 2 mM L-glutamine, and antibiotics.
HCT-116 colon cancer cells were maintained in McCoy’s 5A
medium supplemented with 10% heat-inactivated FBS. NPC-TW01
nasopharynx cancer cells were maintained in DMEM medium
supplemented with 10% FBS and antibiotics. MKN-45 gastric
cancer cells were maintained in RPMI-1640 medium supplemented
with 10% FBS and antibiotics. HUV-EC-C human umbilical vein
endothelial cells were maintained in Ham’s F12K medium supple-
mented with 50 µg/mL endothelial growth factors, 12% FBS, and
antibiotics. CHO-K1 Chinese hamster ovary cells were maintained
in Ham’s F12K medium with 10% FBS and antibiotics. All cell
lines were cultured in an incubator with humidified 5% CO2/95%
air at 37 °C.
1
under vacuum to produce a white solid (27 mg, 100%). H NMR
(400 MHz, CDCl3 δ7.33-8.14 (m, 23H, aromatic), 7.10 (d, 1H, J
) 8.9 Hz, NH), 6.36 (s, 1H, H10), 6.19 (t, 1H, J ) 8.8 Hz, H13),
5.82 (dd, 1H, J ) 8.9, 2.4 Hz, H3′), 5.69-5.74 (m, 2H, H2 and
H7), 5.21 (s, 2H, -CH2Ph), 4.98 (d, 1H, J ) 8.6 Hz, H5), 4.83 (t,
1H, J ) 2.4 Hz, H2′), 4.35 (d, 1H, J ) 8.4 Hz, H20), 4.24 (d, 1H,
J ) 8.4 Hz, H20), 4.01 (d, 1H, J ) 6.9 Hz, H3), 3.71 (br, 1H,
OH), 2.72-2.80 (m, 1H, H6), 2.40 (s, 3H, 4-OCOCH3), 2.35-2.37
(m, 1H, H14), 1.97 (s, 3H, 10-OCOCH3), 1.95 (s, 3H, H16),
1.87-1.90 (m, 4H, H14 and H19), 1.20 (s, 3H, H17), 1.19 (s, 3H,
H16); 13C NMR (100 Hz, CDCl3) δ 202.42 (C, C9), 172.52 (C,
RCOOR′), 170.35 (C, RCOOR′), 168.41 (C, RCOOR′), 167.02 (C,
RCOOR′), 166.91 (C, RCOOR′), 164.82 (C, RCOOR′), 153.24 (C,
aromatic), 140.46 (C, aromatic), 138.02 (C, aromatic), 137.81
(C, aromatic), 135.97 (C, aromatic), 133.77 (C, aromatic), 133.68
(C, aromatic), 133.22 (C, aromatic), 131.94 (C, aromatic), 130.82
(C, aromatic), 130.18 (C, aromatic), 129.09 (C, aromatic),
128.99 (C, aromatic), 128.71 (C, aromatic), 128.62 (C, aromatic),
128.30 (C, aromatic), 127.04 (C, aromatic), 124.83 (C, aromatic),
123.12 (C, aromatic), 119.74 (C, aromatic), 83.90 (CH, C5), 81.03
(C, C4), 78.57 (C, C1), 74.86 (CH, C10), 74.43 (CH, C2), 73.17
(CH, C2′), 72.26 (CH, C7 or C13), 72.28 (CH, C7 or C13), 67.11
(CH2, -CH2Ph), 56.37 (C, C8), 54.90 (CH, C3′), 46.78 (CH, C3),
43.22 (C, C15), 35.65 (CH, C14), 33.38 (CH, C6), 26.49 (CH3,
C17), 22.54 (CH3, 4-OCOCH3), 20.94 (CH3, C16), 20.45 (CH3,
10-OCOCH3), 14.09 (CH3, C18), 11.03 (CH3, C19); m/z 1107.4121
(M + H)+, 1129.3941 (M + Na)+.
Fluorescence-Labeled Prodrug 1 (22). The mixture of 21 (30
mg, 0.027 mmol), 5 (17 mg, 0.035 mmol), DCC (9 mg, 0.041
mmol), and a catalytic amount of DMAP in THF (1 mL) was stirred
at room temperature for 18 h. After concentration, the residue was
purified by silica gel chromatography (EtOAc/Hex ) 1/1) and dried
1
under vacuum to produce a white solid (36 mg, 85%). H NMR
(400 MHz, CDCl3) δ 7.24-8.14 (20H, m, aromatic), 7.09 (1H, d,
J ) 9.0 Hz, NH), 6.43 (1H, s, H10), 6.16 (1H, t, J ) 8.2 Hz, H13),
5.93 (1H, dd, J ) 9.0, 4.1 Hz, H3′), 5.73 (1H, d, J ) 6.9 Hz, H2),
5.68 (1H, dd, J ) 10.4, 7.2 Hz, H7), 5.54 (1H, d, J ) 4.1 Hz,
H2′), 5.00 (1H, d, J ) 8.3 Hz, H5), 4.82 (1H, d, J ) 3.6 Hz, H1′′),
4.70 (1H, dd, J ) 9.7 Hz, H2′′), 4.34 (1H, d, J ) 8.4 Hz, H20),
4.22 (1H, d, J ) 8.4 Hz, H20), 4.00 (1H, d, J ) 6.9 Hz, H3), 3.94
(1H, t, J ) 9.7 Hz, H3′′), 3.79-3.83 (2H, m, H6′′), 3.61-3.65
(1H, m, H5′′), 3.52 (1H, t, J ) 9.7 Hz, H4′′), 3.37 (3H, s, -OCH3),
2.66-2.86 (5H, m, H6 and succinic), 2.43 (3H, s, 4-OCOCH3),
2.28-2.34 (1H, m, H14), 2.08-2.17 (1H, m, H14), 2.01 (3H, s,
H18), 1.99 (3H, s, 10-OCOCH3), 1.88-1.91 (4H, m, H6 and H19),
1.18 (6H, s, H16 and H17); 13C NMR (100 Hz, CDCl3) δ 202.36
(C, C9), 171.80 (C, RCOOR′), 171.64 (C, RCOOR′), 169.83 (C,
RCOOR′), 168.38 (C, RCOOR′), 168.11 (C, RCOOR′), 167.20 (C,
RCOOR′), 166.98 (C, RCOOR′), 166.22 (C, RCOOR′), 150.26 (C),
141.15 (C or CH, aromatic or C11 or C12), 136.81 (C or CH,
aromatic or C11 or C12), 133.76 (C or CH, aromatic or C11 or
C12), 132.88 (C or CH, aromatic or C11 or C12), 132.00 (C or
CH, aromatic or C11 or C12), 130.56 (C or CH, aromatic or C11
or C12), 130.18 (C or CH, aromatic or C11 or C12), 129.09 (C or
CH, aromatic or C11 or C12), 128.71 (C or CH, aromatic or C11
or C12), 128.58 (C or CH, aromatic or C11 or C12), 127.18 (C or
CH, aromatic or C11 or C12), 126.76 (C or CH, aromatic or C11
or C12), 117.95 (C or CH, aromatic or C11 or C12), 116.87 (C or
CH, aromatic or C11 or C12), 113.81 (C or CH, aromatic or C11
or C12), 96.99 (CH,C1′′), 83.96 (CH, C5), 80.95 (C, C4), 78.76
Cell Proliferation Assay. Cell proliferation assays were con-
ducted to measure and compare the cytotoxicity and selectivity of

7440 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Lin et al.
these prodrugs and their parent drug paclitaxel. Cells (2 × 104 cells/
well) were seeded onto a 96-well plate before 12 h of drug
treatment. Stock drug solutions were serially diluted and admin-
istrated to cells at final concentrations of 0.5 nM to 10 µM. After
72 h, the medium was removed and replaced with fresh media.
Ten microliters of MTT (5 mg/mL) solution was added to each
well. After 4 h incubation at 37 °C, the MTT solution was aspirated
and 100 µL of DMSO was added to dissolve the dye precipitated
in cells. The absorbance of each sample was recorded at a
wavelength of 570 nm on a DYNEX MRX II. The percentage of
viable cells at different concentrations of prodrugs was plotted
relative to control values, and the IC50 values were computed. The
IC50 value was defined as the concentration required to inhibit cell
growth by 50% relative to untreated cancer growth. This value was
determined after 72-h treatment.
RNA Extraction and RT-PCR of GLUTs. Total RNA from 9
cell lines was isolated by High Pure RNA Isolation Kit, according
to the manufacturer’s instruction. The sequence primers used were
(1)GLUT1,forward5′-CGGGCCAAGAGTGTGCTAAA-3′(846-865),
reverse 5′-TGACGATACCGGAGCCAATG-3′ (1109-1128), 283
bp PCR product; (2) GLUT4, forward 5′-CCCCCTCAGCAGC-
GAGTGA-3′ (188-206), reverse 5′-GCACCGCCAGGACAT-
TGTTG-3′ (487-506), 319 bp PCR product; (3) ꢀ-actin, forward
5′-GTGGGGCGCCCCAGGCACCA-3′ (176-196), reverse 5′-
TCCTTAATGTCACGCACGATTTC-3′ (692-714), 539 bp PCR
product. PCR was carried out with a Px2 Thermal Cycler. The PCR
condition was to heat at 94 °C for 1 min, followed by denaturation
at 94 °C for 1 min, annealing at 60 °C for 1 min, extension at 72
°C for 1 min for 35 cycles, and finally incubation at 72 °C for 10
min. The PCR products were separated on 1.5% agarose gel and
visualized with ethidium bromide.
Analysis of prodrug 1 or paclitaxel in medium was performed using
a RP-HPLC column (4.6 × 150 mm, Supelco Ascentis C18 column,
5 µm particle size). The mobile phase was prepared as follows:
mobile phase A, 95% distilled water and 5% ACN; mobile phase
B, 100% ACN, then filtered through a 0.22-µm Millipore filter and
degassed before use. The sample was eluted with an isocratic elution
of 60% A and 40% B. The HPLC condition was used for 20 min
running time at a flow rate of 1 mL/min. The eluent was monitored
at 220 nm. For the quantification of prodrug uptake in NPC-TW01
cells in the presence and absence of 100 µM phloretin, the cells
were pretreated with 100 µM phloretin for 1 h and followed with
100 µM prodrug 1. After 24 h, the prodrug 1 was extracted and
analyzed on RP-HPLC as described above. The standard mixture
of prodrug 1 and paclitaxel with different equal concentrations at
1.25, 2.5, 5 and 10 µM were used to calculate the prodrug 1 or
paclitaxel content in cell cultured medium or serum-free medium.
Acknowledgment. We gratefully thank Dr. C. C. Hung for
his helpful instruction with the confocal microscopy and Dr.
S. C. Jao for his helpful instruction with the nuclear magnetic
resonance (NMR) spectroscopy. This work is supported by the
Graduate School, Chiang Mai University, Chiang Mai, Thailand.
Supporting Information Available: Analysis of prodrugs 1-
4 by RP-HPLC and MS; RT-PCR result of GLUT expression level
in 9 cell lines; cytotoxic activity of prodrug 1 with/without phloretin
against NPC-TW01 cells. This material is available free of charge
via the Internet at http://pubs.acs.org.
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