Synthesis and evaluation of [14C]-labelled and fluorescent-tagged paclitaxel derivatives as new biological probes

Article abstract of DOI:10.1016/S0968-0896(98)00158-8

Our present report deals with the preparation of hitherto unreported 7-([carbonyl-¹⁴C]-acetyl)paclitaxel 4 and two new bioactive 7-substituted fluorescent taxoids (FITC 9 and rhodamine 11), as well as evaluation towards their applications as biological probes. The results in this report demonstrate that (a) the new paclitaxel derivatives 4, 9, 11 could be prepared with good yields starting from paclitaxel; (b) the [¹⁴C]acetylation step was found to be better by using [¹⁴C]acetic anhydride rather than [¹⁴C]sodium acetate; (c) the radiochemical purity of 4 was 96% and its specific activity was 48mCi/mmol; (d) the cytotoxicity of 4 was close to that of paclitaxel whereas 9, 11 were far less active than paclitaxel, but these cytotoxic levels were good enough for their biological applications; (e) the drug-quantitation by flow cytometric analysis using 9 and 11 was proved to be equally efficient with respect to the radioactivity-based determination employing 4; (f) the intracellular fluorescence mapping by 9 and 11 was found to be effective and the microtubule network pattern was visible in both the cases; (g) the overall fluorescence imaging efficiency was better with 11 while the intensity of fluorescence was higher with 9; (h) staining of nucleolus was observed in fluorescence studies of both 9 and 11. Based on these results, the newly prepared paclitaxel derivatives can be considered as efficient biological probes and should find further use in relevant applications. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00158-8

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 2193±2204
Synthesis and Evaluation of [14C]-Labelled and Fluorescent-
Tagged Paclitaxel Derivatives as New Biological Probes
Ch. Srinivasa Rao,ab Jao-Jia Chu,a Rai-Shung Liub and Yiu-Kay Lai*a
aDepartment of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China
bDepartment of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China
Received 12 May 1998; accepted 7 August 1998
AbstractÐOur present report deals with the preparation of hitherto unreported 7-([carbonyl-14C]-acetyl)paclitaxel 4 and
two new bioactive 7-substituted ¯uorescent taxoids (FITC 9 and rhodamine 11), as well as evaluation towards their
applications as biological probes. The results in this report demonstrate that (a) the new paclitaxel derivatives 4, 9, 11
could be prepared with good yields starting from paclitaxel; (b) the [14C]acetylation step was found to be better by using
[14C]acetic anhydride rather than [14C]sodium acetate; (c) the radiochemical purity of 4 was 96% and its speci®c activity
was 48 mCi/mmol; (d) the cytotoxicity of 4 was close to that of paclitaxel whereas 9, 11 were far less active than
paclitaxel, but these cytotoxic levels were good enough for their biological applications; (e) the drug-quantitation by ¯ow
cytometric analysis using 9 and 11 was proved to be equally ecient with respect to the radioactivity-based determi-
nation employing 4; (f) the intracellular ¯uorescence mapping by 9 and 11 was found to be e€ective and the micro-
tubule network pattern was visible in both the cases; (g) the overall ¯uorescence imaging eciency was better with 11
while the intensity of ¯uorescence was higher with 9; (h) staining of nucleolus was observed in ¯uorescence studies of
both 9 and 11. Based on these results, the newly prepared paclitaxel derivatives can be considered as ecient biological
probes and should ®nd further use in relevant applications. # 1998 Published by Elsevier Science Ltd. All rights reserved.
Introduction
®laments in 9L rat brain tumor cells.9 These ®ndings
suggest that there may be unexplored cellular targets for
paclitaxel, and many intrinsic factors such as con-
centration, treatment time and intracellular accumula-
tion of drug may contribute to the above mentioned
activities of paclitaxel. In the light of these observations,
we became interested in an investigation of the intracel-
lular kinetics of paclitaxel and the resulting cytoskeletal
alterations by using radio-labelled and ¯uorescent
paclitaxel derivatives. While the [3H]-labelled paclitaxel
derivatives were prepared and used for binding site
localization and drug accumulation studies,10±15 [14C]-
labelled derivatives were thought to be more useful,
particularly in pharmacokinetic studies, because of their
high sensitivity and long half-life of the isotope. The
¯uorescent-conjugated paclitaxel derivatives previously
used were mainly for microscopic studies, there has been
no study to date concerning their utility for drug quan-
titation by ¯ow cytometry. In the context of these
aspects and also to meet our research needs, we felt that
it is worthwhile to introduce new labels on the paclitaxel
structure and evaluate their biological roles.
Paclitaxel (Taxol1), a highly functionalized diterpenoid
plant product, is currently one of the most exciting leads
in cancer chemotherapy.1 Although the microtubule-
targeted mitotic arrest of paclitaxel is well documented,2
there is a lack of clear understanding about a number of
secondary cellular e€ects caused by this drug. For
instance, in macrophages, paclitaxel activates micro-
tubule-associated protein kinase, increases tumor
necrosis factor secretion, and decreases tumor necrosis
factor receptor levels.3±7 Moreover, recent results from
our laboratory indicate that the apoptotic programs in
paclitaxel treated HL-60 human leukemia cells can be
initiated in either the G2/M or S phase through two
di€erent cytotoxic mechanisms,8 and that paclitaxel
induces hyperphosphorylation of vimentin intermediate
Key words: Antitumor compds; chemotherapy; ¯uorescence;
labelling; substituent e€ects.
*Corresponding author. Tel.: 886-3-5731095; Fax: 886-3-
5715934; e-mail: lslyk@life.nthu.edu.tw
0968-0896/98/$ - see front matter # 1998 Published by Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00158-8

2194
Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
However, there are only a few synthetic methods
available for [14C]paclitaxel derivatives: the in vitro
production of minute amounts of [14C]paclitaxel;16,17
preparation of [N30-14C] and [C30-14C]-labelled pacli-
taxel derivatives where the radio-labels were located on
the C-13 side chain of paclitaxel18,19 and a hemisynthesis
of [30-14C]-taxotere.20 It is worth noting that the pre-
paration of these paclitaxel analogues with [14C]-label
on the side chain involved many steps. Moreover, there
has been no report dealing with the synthesis of
[14C]paclitaxel bearing the label on the diterpene ring.
Since it has been shown that paclitaxel and 7-ace-
tylpaclitaxel are similar in their ability to alter cell pro-
liferation and microtubule polymerization,21±23 and also
that 7-epipaclitaxel is as active as paclitaxel,24 we selec-
ted 7-([carbonyl-14C]-acetyl)paclitaxel as our candidate
with a view to examine its capability in the determina-
tion of drug accumulation. Interestingly, this would the
®rst [14C]-paclitaxel derivative bearing the radiolabel on
the diterpene ring.
Figure 1. Structures of paclitaxel (1), [7-14]acetylpaclitaxel (4),
FITC-paclitaxel (9), and rhodamine-paclitaxel (11).
using 4, 9 and 11, as well as the visualization of cytos-
keletal organization in tumor cultured cells with 9 and
11.
A variety of ¯uorescent ligands have been attached to
paclitaxel previously, both on the side chain as well as
on the ring skeleton and found to have a variable
degree of success in terms of their cellular imaging e-
ciency.25±32 Among these ¯uorophores, the ¯uor-
escein,27 nitrobenzoyldiazo28 (NBD) and Lissamine
rhodamine B29 dyes have produced good ¯uorescence
images. We became interested in the examination of
¯uorescein isothiocyanate (FITC) because (a) it is a very
commonly used dye in biology; (b) its excitation wave-
length (488 nm) does not interfere with that of cellular
proteins; (c) it is easily accessible and (d) it does not
require either sophisticated microscopic instruments or
special ®lters for ¯uorescence recording. In addition to
FITC, the Lissamine rhodamine B was considered in
order to make comparative study between their ¯uores-
cence abilities. To conjugate these two ¯uorophores on
to the paclitaxel skeleton, esteri®cation of C-7 hydroxyl
position was found to be ideal on the basis of well-
known structure-activity relationship studies.32±34 Based
on the previous ®nding that the C-7 substituted a-amino
acid ester derivatives of paclitaxel are unstable,35±37 6-
aminocaproyl chain was chosen to serve as the linker
between the paclitaxel and the ¯uorescent moiety. We
reasoned that use of this long spacer arm would keep
the bulky ¯uorophore away from the vicinity of the
binding region of the paclitaxel skeleton and facilitate
the drug to exhibit its usual cellular activity. This 6-car-
bon spacer arm was used earlier to conjugate the NBD
dye to docetaxel and provided encouraging results.28
Results and Discussion
Chemistry
The C-20-OH of paclitaxel 1 is normally the most reac-
tive and when it is protected, reaction usually proceeds
selectively at the C-7 hydroxyl group. Thus, it was deci-
ded to replace the C-7 OH group of 2 with ester func-
tions containing either carbon isotope or ¯uorophores
conjugated through an aminocaproyl linker (Fig. 1).
Synthesis of 7-([carbonyl-14C]-acetyl)paclitaxel. Reac-
tion of paclitaxel 1 with triethylsilyl chloride in CH2Cl2
gave 2 in 83% yield (Scheme 1). The acetylation of C-7
OH group was performed by following a similar proce-
dure (Method A),11 wherein the acetic anhydride was
generated in situ from radiolabelled sodium acetate in
the presence of a catalytic amount of acetyl chloride.
Thus, treatment of 20-protected paclitaxel 2 with [14C]
acetic anhydride, obtained as mentioned above, in dry
pyridine and THF produced 3 in 52% yield and the
radiochemical conversion was 38% (Scheme 1). The
®nal step of deprotection at 20-position of 3 was per-
formed easily using a mixture of AcOH-H2O-THF to
obtain the desired 4 in 90% chemical yield. The struc-
ture of 4 was con®rmed by comparing its spectral data
with that of unlabelled 7-acetylpaclitaxel, and its radio-
activity was 14.1 mCi/mmol i.e. 24% of the speci®c
activity of [14C]sodium acetate used. Despite the suc-
cessful preparation of 4, a better method was sought for
the acetylation step as the former procedure requires
elevated temperature (80 ꢀC), and an excess of isotopic
reagent (5 equiv of sodium acetate for 1 equiv of 2).
In an alternate procedure (Method B, Scheme 1),
We herein report the synthesis of 7-([carbonyl-14C]-
acetyl)paclitaxel 4 and two new 7-substituted ¯uorescent
taxoids 9 and 11 (Fig. 1) and their evaluation as biolo-
gical probes to study the intracellular drug quantitation

Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
2195
Scheme 1.
commercially available [14C]acetic anhydride was used
instead of [14C]sodium acetate and it was found that the
reaction could be carried out at room temperature with
only 3 equivalents of isotopic reagent to obtain 3 in
better chemical as well as radiochemical yields (89%
and 45% respectively). Treatment of 3 with a mixture of
AcOH-H2O-THF produced the target compound 4 at
92% chemical yield and its spectral characteristics were
identical to the sample obtained from Method A. The
radiochemical purity of 4 was 96% and its speci®c
activity was 48 mCi/mmol (41% of the isotopic acetic
anhydride). The compound 4 was stored as a methanolic
solution at 20 ꢀC and the product was found to be
stable with very little radioactive decomposition.
room temperature yielded 7-aminocaproylpaclitaxel 7
(91%), wherein both silyl and N-Boc protections were
simultaneously removed (Scheme 2). The intermediate 7
was used to conjugate ¯uorescent labels on its amino
functional group. Thus, the FITC dye 8 was conjugated
to paclitaxel by reacting with 7 in DMF and pyridine at
room temperature over 2 days in dark to a€ord the
desired product 9 at 53% yield (Scheme 3). Similarly,
the sulforhodamine analogue 11 was prepared at 58%
yield from 7 by reacting with sulforhodamine B chloride
10 in 10% aqueous sodium bicarbonate and dioxane
(Scheme 4). The structures of 9 and 11 were con®rmed
by their spectroscopic data and were found to be stable
for at least two weeks when stored at 20 ꢀC.
Synthesis of ¯uorescent-tagged taxoids. The 20-protected
paclitaxel 2 was esteri®ed at position C-7 with 6-(N-ter-
butoxycarbonyl)aminocaproic acid 537 in the presence
of DCC and DMAP to give 6 in 90% yield (Scheme 2).
Treatment of 6 with an excess of 99% formic acid at
Biological evaluation studies
Cytotoxicity assays. In order to evaluate the cytotoxi-
city potentials of the prepared paclitaxel derivatives 4, 7,
9 and 11 relative to paclitaxel, cytotoxic assays were

2196
Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
Scheme 2.
Scheme 3.
performed by testing their ability in the inhibition of the
growth of H460 human non-small cell lung cancer
(NSCLC) cells. We have employed the standard MTT
assay38 and determined the ID50 values (dose that
reduced the ®nal absorbance to 50% of control) from
the survival curves (Fig. 2). The ID50 of 4 was close to
that of paclitaxel thus con®rm that derivatization at the
C-7 position of paclitaxel by acetyl group retains sig-
ni®cant activity. The FITC derivative 9 was found to be
more cytotoxic than 7 and 11, but less than 4. The
cytotoxicity levels of 7 and 11 were almost equal and
apparently the increased hydrophilic character of these
derivatives might reduce their cell-permeability. From
the above cytotoxicity data it was evident that these
derivatives were less active than paclitaxel, but retain
sucient cytotoxicity to be suitable for carrying out
biological applications.
Drug accumulation studies. The drug accumulation stu-
dies were undertaken in order to evaluate the abilities of

Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
2197
Scheme 4.
4 (speci®c activity, 48 mCi/mmol), 9 and 11 in quanti-
tating the intracellular drug accumulation. The assays
were performed by treatment of these derivatives with
the H460 culture cells with variable concentrations or
treatment times.
Fluorescence studies. To establish the ¯uorescent map-
ping eciency of 9 and 11, drug-treatment protocols
were performed using H460 NSCLC culture cells. Dif-
ferent doses and treatment periods were followed in the
reported protocols to get the ideal results. While one
group used 10 mM during 6 h exposure, another group
required 20 mM for 2 h to draw better results.29 Both of
these reported scales were not suitable for our experi-
ments. As the tumor cultures under study were not the
same, these di€erences could be regarded as the proto-
col variations. The cells were treated with 9 or 11 and
then ¯uorescence was observed after ®xing the inter-
phasic cells in ethanol. It was observed that the con-
centration and the treatment time have profound e€ects
on the ¯uorescence pattern of cellular organization as
well as the illumination levels. Also, 9 and 11 required
di€erent doses and treatment periods in order to obtain
optimal results. The FITC-paclitaxel showed good
¯uorescence pattern when the tumor cells were incu-
bated with 0.1 mM of 9 for 2 h at 4 ꢀC, whereas a much
higher dose (10 mM) of 11 was needed for obtaining
good images with rhodamine-paclitaxel. This di€erence
in the corresponding doses of 9 and 11 may be due to
variations in their cytotoxicity and drug uptake level.
Under these optimal conditions, the cells displayed
¯uorescent microtubule networks (Fig. 5) and both 9
and 11 have exhibited the microtubule organizing cen-
ters (MTOCs). However, the microtubule ®lamentous
The quantitation was conducted by using the radio-
active counting for 4 or ¯ow cytometric analysis for 9
and 11. In the time-dependent experiments, the values of
drug amounts retained by the H460 tumor cells were
plotted against the time-scale and the drug saturation
points were then determined (Figs 3A and 4A). The
accumulation of 4 was increased steadily during a 1 h
period and the saturation point was reached after 6 h
of treatment (Fig. 3A). Similarly, the saturation level
for 9 and 11 was attained after 6 h (Fig. 4A). In the
case of the dose-dependent experiments, 4 needed
0.1 mM for its saturation level (Fig. 3B) while saturation
concentration was 0.1 and 0.01 mM for 9 and 11
respectively (Fig. 4B). The ®nite variations in the con-
centrations required for saturation points of 9 and 11
may be either due to the di€erences in their sensitivities
or because of their variable drug-uptake levels. How-
ever, the results clearly demonstrate that the ¯uores-
cence-based quantitation is almost similar when
compared to radioactive determination and could be
useful as a complementary tool for the drug quantita-
tion studies.

2198
Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
Figure 2. Inhibition of the growth of H-460 cells by 4, 7, 9 and
11 relative to paclitaxel: Cells were grown in culture in the
presence of various concentrations of paclitaxel derivatives for
4 days and an MTT assay was performed. One hundred percent
represents the number of cells present in the control culture.
structures appeared to be slightly di€erent from the
corresponding immuno¯uorescence stain.
It is interesting to note that two aspects of our results
are similar to that of Lissamine rhodamine taxoid as
used by Nicolaou's group.29 The ®rst and most striking
similarity is regarding the staining of nucleoli by 9 and
11 and the second indication is related to the presence of
few di€used background signals. This identical mode of
¯uorescence pattern could be expected for our rhoda-
mine paclitaxel 11 as it is the same ¯uorophore as that
used by the above group except that it is linked to the
paclitaxel skeleton by a much longer carbon chain in
our studies. Surprisingly, the FITC-labelled paclitaxel 9
also exhibited similar phenomena and thus supports the
view that there may be undetected targets for paclitaxel
other than tubulin. However, a di€erent reason was put
forward by Guenard et al. for the staining of nuclei
when they used the NBD-labelled docetaxel, and they
attributed the ®ndings thereof due to cytological tech-
niques.28 While these e€ects were observed only under
cell ®xation procedures with NBD-docetaxel, the repor-
ted results with rhodamine-conjugated paclitaxel
demonstrated under both living and ®xation protocols.
Interestingly, the ¯uorescein-labelled paclitaxel used by
another group did not display the ¯uorescence of the
nucleolus.27 Further investigations are necessary to
clarify these discrepancies and to provide a better
understanding about these observations. The ®ndings
from previous reports and our present study reveal that
Figure 3. Drug accumulation using radioactive paclitaxel 4. (A)
Time-dependent accumulation: Treatment of H-460 cells with
1 mM of 4 for di€erent time periods. The cells were collected at
the indicated time periods and the radioactivity was determined
using b-scintillation counting. (B) Dose-dependent accumula-
tion: Treatment of H-460 cells for 3 h with di€erent indicated
doses of 4.
the staining patterns of ¯uorescent taxoids may not
necessarily depend on the structure of the ¯uorophore.
This is clearly evident from the observations that (a) the
structure of FITC closely resembles the ¯uorescein dye
but the performance of FITC-taxoid 9 was similar to the
rhodamine analogue 11 (b) while the change in the
spacer arm linking the ¯uorophore to the paclitaxel
provided di€erent results when ¯uorescein dye was
employed,27,29 not much e€ect was observed in the case
of the rhodamine ¯uorophore.
Our results from 9 and 11 indicate that the ¯uorescence
pattern appeared to be similar in both cases but 11

Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
2199
Figure 5. Fluorescence microscopy image of permealized and
®xed H-460 cells at interphase. (A) FITC-paclitaxel 9 staining;
(B) rhodamine paclitaxel 11 staining; m, microtubule organiz-
ing center (MTOC); n, nucleolus.
Figure 4. Drug accumulation using ¯uorescent taxoids 9 and
11. (A) Time-dependent accumulation: Treatment of H-460
cells with 1 mM of 9 and 11 for indicated time periods. The cells
were collected at these intervals and ¯ow cytometry analysis
was performed for quantitation. (B) Dose-dependent accumu-
lation: Treatment of H-460 cells for 3 h with indicated doses of
9 and 11.
actions of paclitaxel have been observed recently.
Understanding of these new actions of paclitaxel is very
important for its better practical therapy and therefore,
warrants further research in this direction. The intracel-
lular localization studies are thus essential to elucidate
the molecular mechanism(s) involved in these processes.
The bio-tracer investigations using radioactive and
¯uorescent-labelled paclitaxel derivatives attain sig-
ni®cance in this respect as valuable tools to determine
the cellular events. In pursuit of this goal, our present
e€orts are directed to seek for novel potential labelling
groups to conjugate to paclitaxel. The synthetic results
show that the desired paclitaxel derivatives 4, 9 and 11
could be prepared in good yields starting from pacli-
o€ers a better overall ¯uorescence image for the micro-
tubule network than 9. However, the ¯uorescence level
of 9 was found to be much higher than that of 11. Both
9 and 11 have delivered good ¯uorescence abilities and
thus suitable for further sub-cellular localization studies.
Conclusion
taxel. The 7-([carbonyl-14C]-acetyl)paclitaxel
4 was
Although the microtubule-targeted activity of paclitaxel
has been extensively studied, a number of non-mitotic
obtained in very good chemical yield as well as radio-
chemical activity when the acetylation was carried out

2200
Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
using [14C]acetic anhydride in place of [14C]sodium
acetate. The speci®c activity of 4 (obtained from
Method B) was determined to be 48 mCi/mmol and it
was found to be sucient enough for drug accumulation
studies. This is the ®rst report for the preparation of
[14C]paclitaxel having its radiolabel on the taxane ring
skeleton. The ¯uorescent paclitaxel derivatives 9 and 11
were prepared from a common intermediate 7 and they
were chemically stable when stored at 20 ꢀC. The
cytotoxicity levels for these derivatives were lower than
that of paclitaxel, but sucient for their use as biologi-
cal probes. We have demonstrated for the ®rst time that
the ¯uorescent taxoids could be used for drug quantita-
tion. The drug accumulation experiments reveal that a
close homology exists between the results of radioactive
and ¯ow cytometric analyses and prove that these
probes could be of more utility for further related stu-
dies. The ¯uorescence microscopy results from 9 and 11
were successful in displaying the microtubule network,
and a noteworthy feature is the staining of nucleolus.
The results also indicate that the ¯uorescence-mapping
pattern of labelled taxoids is not completely identical to
the corresponding anti-tubulin antibody stain and thus
helpful in determining unexplored targets of paclitaxel.
the speci®c activity was determined with an IN/US b-
RAM System (2 mL aliquots in 1 mL of Aquasol II, New
England Nuclear). NMR spectra were recorded on
Bruker AMX-300 and AMX-400 instruments and cali-
brated using TMS as internal standard. Multiplicities
were recorded by the following abbreviations: s, singlet;
d, doublet; dd, double doublet; t, triplet; q, quartet, m,
multiplet; br, broad; band, overlapping signals. Chemical
shifts were expressed in parts per million (ppm). Cou-
pling constants (J) were given in Hertz (Hz).
20-Triethylsilylpaclitaxel 2. To a well-stirred solution of
paclitaxel 1 (527 mg, 0.62 mmol) and imidazole (460 mg,
6.75 mmol) in CH2Cl2 (12 mL) at room temperature was
added Et3SiCl (0.15 mL, 0.99 mmol) slowly under a
nitrogen atmosphere. After stirring the reaction mixture
for 30 min, the reaction was quenched with water
(15 mL), and the organic layer was separated, dried and
concentrated under reduced pressure. The crude pro-
duct was then passed through a short silica gel column
(hexane/EtOAc, 2:3; Rf=0.58) to give 20-triethylsilylox-
ypaclitaxel 2 (496 mg, 83%) as a colorless powder: 1H
NMR (400 MHz, CDCl3)
d
0.37±0.49 (m, 6H,
SiCH2CH3), 0.80 (t, J=7.9, 9H, SiCH2CH3), 1.12 (s,
3H, H16), 1.23 (s, 3H, H17), 1.67 (s, 3H, H18), 1.71±1.74
(m, 1H, H6b), 1.92 (s, 3H, H19), 2.11±2.19 (m, 2H, H14),
2.23 (s, 3H, C10-CH3CO), 2.32±2.55 (m, H6a), 2.54 (s,
3H, C4-CH3CO), 3.80 (d, J=7.0, 1H, H3), 4.21 (d,
J=8.5, 1H, H20), 4.30 (d, J=8.5, 1H, H20), 4.41 (dd,
Experimental
0
J=10.6, 6.6, 1H, H7), 4.67 (d, J=1.9, 1H, H2 ), 4.96 (d,
0
J=8.0, 1H, H5), 5.67±5.70 (m, 2H, H3 and H2), 6.25 (t,
Chemical materials and methods. All reactions were car-
ried out in a nitrogen atmosphere under anhydrous
conditions, unless otherwise mentioned. Commercially
available reagents were used without further puri®ca-
tion. [1-14C]Acetic acid, sodium salt and [1-14C]acetic
anhydride were purchased from Amersham Interna-
tional plc (England). The [1-14C]acetic acid, sodium salt
was supplied as an aqueous solution (speci®c activity,
59.0 mCi/mmol; radioactive concentration, 200 mCi/ml;
radiochemical purity, 98.6%) and was stored at 2 ꢀC. The
[1-14C]acetic anhydride was supplied in borosilicate
glass break-seal ampules sealed under vacuum [speci®c
activity, 118 mCi/mmol; radiochemical purity, 98.6%]
and stored at 20 ꢀC. Fluorescein isothiocyanate (FITC),
isomer 1 (5 isomer) and sulforhodamine B were pur-
chased from Aldrich Chemical Company Inc (USA) and
they were stored under prescribed conditions. All the
prepared paclitaxel derivatives were stored at 20 ꢀC.
Solvents were dried and puri®ed before use. Thin-layer
chromatography was carried out using aluminum-
backed silica gel plates (Merck-60F254). Developed TLC
plates were viewed under UV detector (Spectroline;
model, ENF 240). Radiochemical purity was done by
analytical thin-layer chromatography and the radio-
active TLC plates were scanned on a Berthold Model
LB 511. Radioactive measurements were performed using
a PACKARD 1600 TR liquid scintillation analyzer and
J=8.8, 1H, H13), 6.28 (s, 1H, H10), 7.09 (d, J=8.6, 1H,
NH), 7.31±7.58 (m, 11H, ArH), 7.64 (d, J=8.0, 2H,
ArH), 8.13 (d, J=8.0, 2H, ArH); 13C NMR (100 MHz,
CDCl3) d 4.4, 6.5, 14.8, 20.8, 22.3, 23.0, 26.8, 35.6, 35.9,
43.3, 45.6, 55.7, 58.6, 71.5, 72.1, 74.9, 75.2, 75.6, 76.5,
79.2, 81.2, 84.5, 126.5, 127.0, 128.0, 128.6, 128.7, 128.8,
129.3, 130.2, 131.7, 133.0, 133.6, 134.2, 138.5, 142.5,
166.9, 167.0, 170.1, 171.2, 171.5, 203.7. Anal. calcd for
C53H65NSiO14: C, 65.75; H, 6.77; N, 1.44. Found: C,
65.59; H, 6.61; N, 1.24.
20-Triethylsilyl-7-([carbonyl-14C]-acetyl)paclitaxel 3.
(Method A): The commercial [14C]-sodium acetate [sup-
plied as an aqueous solution; 101 mmol; speci®c activity,
59 mCi/mmol] was introduced into a two-neck round-
bottom ¯ask and water was removed under vacuum. It
was heated at 190 ꢀC for 1 h under reduced pressure
(0.01 mm/Hg) and then the heating was stopped and the
material was allowed to reach room temperature. Then,
THF (1 mL) and acetyl chloride (8 mL) were added and
the mixture was stirred overnight at room temperature.
To the milky suspension that resulted, 2 (19 mg,
20 mmol) dissolved in dry pyridine (0.2 mL) was added
and the mixture was heated at 80 ꢀC for 20 h. The
solvents were removed by ¯ushing with a stream of

Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
2201
nitrogen and the crude material was treated with water
(3Â2 mL) and extracted with CH2Cl2 (3Â5 mL). The
combined organic extract was concentrated under
vacuum and the preparative TLC on silica gel (EtOAc/
hexanes, 1:2; Rf=0.65) a€orded the product 3 as a col-
orless solid (11 mg, 52%). (Method B): To a solution of
2 (31 mg, 30 mmol) in dry pyridine (0.5 mL) at room
temperature under nitrogen atmosphere were added
DMAP (0.1 mg, catalytic) and [14C]acetic anhydride
(90 mmol; speci®c activity, 118 mCi/mmol). The reaction
mixture was stirred at room temperature for 20 h and
evaporated to dryness. The residue was treated with
water (3Â5 mL) and extracted with CH2Cl2 (3Â8 mL).
The combined organic extract was concentrated under
reduced pressure and the crude material was puri®ed by
preparative TLC on silica gel (EtOAc/hexanes, 1:2;
Rf=0.65) to give 3 (27 mg, 89%). The TLC Rf of 3 from
both the methods was found to be identical and the
chromatographic behavior was similar to the authentic
unlabelled sample. The radiochemical purity of 3 was
determined to be 98% and the NMR spectra were found
to be identical by comparing with an authentic unla-
6H, H18 and H19), 1.77±1.89 (m, 1H, H6b), 2.06 (s, 3H,
C7-CH3CO), 2.14 (s, 3H, C10-CH3CO), 2.29±2.38 (m,
2H, H14), 2.40 (s, 3H, C4-CH3CO), 2.42±2.58 (m, 1H,
H6a), 3.92 (d, J=7.0, 1H, H3), 4.18 (d, J=8.0, 1H, H20),
0
4.34 (d, J=8.0, 1H, H20), 4.79 (d, J=2.4, 1H, H2 ), 4.94
(d, J=7.0, 1H, H5), 5.55 (dd, J=10.0, 4.5, 1H, H7), 5.68
0
(d, J=7.0, 1H, H3 ), 5.80 (d, J=7.0, 1H, H2), 6.18 (t,
J=8.5, 1H, H13), 6.26 (s, 1H, H10), 7.16 (d, J=8.0, 1H,
NH), 7.28±7.68 (m, 11H, ArH), 7.74 (d, J=8.0, 2H,
ArH), 8.10 (d, J=7.3, 2H, ArH); 13C NMR (100 MHz,
CDCl3) d 10.8, 14.6, 20.7, 20.8, 21.0, 22.5, 26.6, 33.4,
35.6, 43.2, 47.0, 54.9, 56.1, 71.4, 72.0, 73.2, 73.3, 74.3,
75.5, 76.4, 76.5, 76.6, 78.4, 81.1, 84.0, 84.1, 127.0, 128.2,
128.7, 128.9, 129.1, 130.1, 131.9, 133.0, 133.7, 134.0,
138.0, 140.3, 150.4, 166.9, 168.9, 170.3, 170.4, 172.4,
201.8.
6-N-ter-Butoxycarbonylaminocaproic acid 5. Prepared
from 6-aminocaproic acid and di-ter-butyl dicarbonate
in MeOH in the presence of NEt3 using reported proce-
dure.37 1H NMR (300 MHz, CDCl3) d 1.31±1.46 (m,
13H, ter-butyl-CH3 and CH2), 1.55±1.64 (m, 2H, CH2),
2.29 (br t, 2H, CH2), 3.01±3.07 (m, 2H, NCH2), 4.63 (br
s, 1H, NH).
1
belled sample. H NMR (400 MHz, CDCl3) d 0.37±0.48
(m, 6H, 6H, SiCH2CH3), 0.80 (t, J=8.0, 9H,
SiCH2CH3), 1.14 (s,3H, H16), 1.19 (s,3H, H17), 1.74 (s,
3H, H18), 1.77±1.83 (m, 1H, H6b), 1.95 (s, 3H, H19), 2.01
(s, 3H, C7-CH3CO), 2.12±2.19 (m, 1H, H14), 2.24 (s, 3H,
C10-CH3CO), 2.38±2.48 (m, 1H, H14), 2.52 (s, 3H, C4-
CH3CO), 2.53±2.64 (m, 1H, H6a), 3.90 (d, J=7.0, 1H,
H3), 4.18 (d, J=8.5, 1H, H20), 4.32 (d, J=8.5, 1H, H20),
20-Triethylsilyl-7-(tert-butoxycarbonylaminocaproyl)pacl-
itaxel 6. To a solution of 2 (48 mg, 0.05 mmol, 1 equiv)
and DMAP (6 mg, 0.05 mmol, 1 equiv) in CH2Cl2
(5 mL) at room temperature under nitrogen was
sequentially treated with
5
(52 mg, 0.225 mmol,
0
4.68 (d, J=1.6, 1H, H2 ), 4.94 (d, J=8.0, 1H, H5), 5.60
0
(dd, J=10.6, 6.6, 1H, H7), 5.67 (d, J=6.0, 2H, H3 and
4.5 equiv) and DCC (198 mg, 0.225 mmol, 4.5 equiv).
The mixture was stirred at room temperature for 20 h
and then diluted with CH2Cl2 (10 mL) and ®ltered to
remove the urea. The ®ltrate was concentrated under
reduced pressure and the resulting residue was passed
through a silica gel column (EtOAc/hexanes, 1:1) to
a€ord 6 as a colorless solid (53 mg, 90%). 1H NMR
H2), 6.20 (t, J=8.5, 1H, H13), 6.26 (s, 1H, H10), 7.10 (d,
J=8.0, 1H, NH), 7.30±7.61 (m, 11H, ArH), 8.11 (d,
J=8.0, 2H, ArH); 13C NMR (100 MHz, CDCl3) d 4.3,
6.4, 10.8, 14.5, 20.7, 21.0, 22.9, 26.4, 33.3, 35.6, 41.2,
43.4, 46.9, 55.7, 56.0, 71.3, 74.5, 74.7, 75.3, 76.3, 76.5,
76.7, 76.8, 77.6, 81.0, 84.0, 126.5, 127.0, 128.1, 128.8,
129.1, 130.2, 131.7, 132.7, 133.7, 140.9, 166.9, 167.1,
168.9, 169.8, 170.4, 171.8, 202.6.
(300 MHz, CDCl3±CD3OD)
d 0.33±0.46 (m, 6H,
SiCH2CH3), 0.82 (t, J=7.5, 9H, SiCH2CH3), 1.06±1.28
(m, 4H, CH2), 1.14 (s, 3H, H16), 1.21 (s, 3H, H17), 1.29±
1.34 (m, 9H, ter-butyl CH3), 1.57±1.62 (m, 2H, CH2),
1.64±1.68 (m, 1H, H6b), 1.76 (s, 3H, H19), 1.95±1.99 (m,
2H, CH2), 2.02 (s, 3H, H18), 2.11 (s, 3H, C10-CH3CO),
2.12±2.23 (m, 2H, H14), 2.28±2.39 (m, 1H, H6a), 2.47 (s,
3H, C4-CH3CO), 3.02±3.08 (m, 2H, NCH2), 3.92 (d,
J=6.0, 1H, H3), 4.14 (d, J=8.0, 1H, H20), 4.30 (d,
7-([Carbonyl-14C]-acetyl)paclitaxel 4. To 20-Triethylsilyl-
7-([carbonyl-14C]-acetyl)paclitaxel
3 [obtained from
method A or method B; 10 mg, 9.9 mmol), was added to
a mixture of acetic acid (0.6 mL), water (0.3 mL), and
THF (0.1 mL) and the contents were stirred at room
temperature for 24 h (monitored by TLC). The reaction
solution was then treated with aqueous NaHCO3 (3 mL)
and extracted with EtOAc (3Â4 mL). The combined
organic extract was dried (Na2SO4), and the crude
compound was puri®ed by preparative TLC (EtOAc/
hexanes, 1:2; Rf=0.44) to a€ord 7-([carbonyl-14C]-acet-
yl)paclitaxel 4 as a white powder (7.0 mg, 90%). The
TLC Rf and NMR spectra of 4 were consistent with that
of unlabelled 7-acetylpaclitaxel. 1H NMR (400 MHz,
CDCl3) d 1.12 (s, 3H, H16), 1.16 (s, 3H, H17), 1.78 (s,
0
J=8.0, 1H, H20), 4.59±4.68 (m, 1H, H2 ), 4.93 (d,
J=8,0, 1H, H5), 5.54 (dd, J=6.5, 1.2, 1H, H7), 5.68 (d,
0
J=7.0, 1H, H2), 5.91 (m, 1H, H3 ), 6.21 (t, J=6.0, 1H,
H13), 6.26 (s, 1H, H10), 7.16 (d, J=8.0, 1H, NH), 7.29±
7.58 (m, 11H, ArH), 7.72 (d, J=8.0, 2H, ArH), 8.10 (d,
J=8.0, 2H, ArH); 13C NMR (100 MHz, CDCl3±
CD3OD) d 4.4, 6.6, 10.9, 14.2, 20.4, 20.8, 22.0, 24.2,
26.5, 26.8, 28.4, 30.0, 30.2, 33.5, 33.9, 35.8, 43.1, 43.2,
43.9, 46.7, 54.7, 56.1, 56.6, 71.8, 72.0, 73.5, 74.4, 75.2,
76.4, 76.6, 78.6, 80.2, 81.1, 83.8, 84.2, 126.9, 127.4,

2202
Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
128.8, 129.3, 130.2, 131.5, 133.0, 133.5, 134.0, 140.2,
150.6, 167.2, 168.5, 170.2, 172.2, 202.3. Anal. calcd for
C64H84N2SiO17: C, 65.06; H, 7.16; N, 2.37. Found: C,
65.18; H, 7.01; N, 2.45.
J=9.8, 3.2, 1H, H7), 5.58 (d, J=7.6, 1H, H2), 5.66 (m,
0
1H, H3 ), 6.16 (t, J=8.6, 1H, H13), 6.23 (s, 1H, H10),
6.44 (d, J=8.0, 2H, ArH), 6.52±6.71 (m, 4H, ArH and
NH), 7.15 (d, J=8.0, 1H, NH), 7.23±7.38 (m, 14H, ArH
and NH), 7.64 (d, J=8.0, 2H, ArH), 7.91±8.03 (m, 2H,
ArH), 8.18 (d, J=8.0, 2H, ArH); 13C NMR (100 MHz,
CD3OD) d 11.0, 14.3, 20.5, 20.7, 22.0, 24.0, 26.2, 30.2,
33.0, 33.2, 33.6, 35.4, 43.1, 43.2, 43.5, 47.1, 50.5, 50.8,
54.9, 56.0, 56.6, 71.9, 72.2, 73.3, 74.6, 75.6, 76.3, 78.8,
81.0, 83.5, 84.4, 101.8, 127.1, 127.3, 128.3, 128.6, 128.7,
129.2, 130.8, 131.8, 133.1, 133.5, 134.0, 138.2, 140.2,
142.0, 167.0, 168.5, 170.1, 172.8, 191.9, 202.2. Anal.
calcd for C74H73N3SO20: C, 65.52; H, 5.42; N, 3.09.
Found: C, 65.39; H, 5.69; N, 2.83.
7-(Aminocaproyl)paclitaxel 7. A mixture of 6 (50 mg,
0.04 mmol) and 99% formic acid (2 mL) was stirred at
room temperature under nitrogen for 36 h. The excess of
the reagent was removed under vacuum and 10% aqu-
eous NaHCO3 (2Â10 mL) was added to the residue.
This was then extracted with EtOAc (2Â15 dried
(Na2SO4) and concentrated. The preparative TLC of the
crude material on silanized silica gel (EtOAc/petroleum
ether, 2:1; Rf=0.39) a€orded 7 as a colorless solid
(35 mg, 91%). 1H NMR (CDCl3±CD3OD) d 1.13 (s, 3H,
H16), 1.22 (s, 3H, H17), 1.19±1.29 (m, 4H, CH2), 1.52±
1.56 (m, 2H, CH2), 1.58±1.61 (m, 1H, H6b), 1.70 (s, 3H,
H19), 1.73 (s, 3H, H18), 1.74±1.87 (m, 2H, CH2), 2.16 (s,
3H, C10-CH3CO), 2.19±2.31 (m, 2H, H14), 2.39 (s, 3H,
C4-CH3CO), 2.36±2.51 (m, 1H, H6a), 3.05±3.14 (m, 2H,
NCH2), 3.90 (d, J=7.0, 1H, H3), 4.16 (d, J=8.0, 1H,
Lissamine rhodamine B conjugated paclitaxel 11. To a
solution of 7 (16 mg, 0.016 mmol, 1 equiv) in dioxane
and saturated aqueous NaHCO3 (1:1, 2 mL) at room
temperature under inert atmosphere was added Lissa-
mine rhodamine
B sulfonyl chloride 10 (12 mg,
0.020 mmol, 1.25 equiv) and the reaction mixture was
stirred for 36 h. This was then diluted with aqueous
NaCl solution (5 mL) and extracted with EtOAc
(10Â10 mL). The combined organic extract was con-
centrated under reduced pressure and the residue was
puri®ed by TLC on silica gel (1% AcOH and 15%
MeOH in CH2Cl2). The major band (Rf=0.6) was
scrapped, eluted with EtOAc, and concentrated to give
0
H16), 4.25 (d, J=8.0, 1H, H16), 4.78±4.81 (m, 1H, H2 ),
4.95 (d, J=8.0, 1H, H5), 5.58 (dd, J=9.0, 1.5, 1H, H7),
0
5.64 (d, J=7.0, 1H, H2), 5.84 (dd, J=7.5, 1.0, 1H, H3 ),
6.20 (t, J=7.0, 1H, H13), 6.25 (s, 1H, H10), 7.29±7.58
(m, 12H, ArH and NH), 7.62 (d, J=8.0, 2H, ArH), 8.09
(d, J=8.0, 1H, ArH); 13C NMR (100 MHz, CDCl3±
CD3OD) d 11.0, 14.4, 20.6, 20.8, 22.2, 24.1, 26.4, 30.1,
33.3, 33.5, 33.8, 35.6, 43.1, 43.2, 43.8, 46.9, 54.9, 56.2,
56.6, 71.6, 72.1, 73.3, 74.5, 75.4, 76.6, 76.8, 78.5, 81.0,
83.9, 84.2, 127.0, 127.3, 128.6, 128.8, 129.1, 130.1, 131.7,
133.1, 133.5, 134.2, 138.2, 140.4, 158.0, 167.0, 168.8,
170.5, 172.6, 202.0. Anal. calcd for C53H60N2O15: C,
65.96; H, 6.26; N, 2.90. Found: C, 65.77; H, 6.44; N,
2.71.
1
11 as a purple layer (13 mg, 58%). H NMR (400 MHz,
CD3OD) d 1.05 (s, 3H, H16), 1.07 (s, 3H, H17), 1.08±1.31
(m, 12H, NCH2CH3), 1.31±1.48 (m, 2H, CH2), 1.53±
1.69 (m, 4H, CH2), 1.81 (s, 3H, H19), 1.82±1.84 (m 1H,
H6b), 1.86 (s, 3H, H18), 2.12 (s, 3H, C10-CH3CO), 2.14±
2.31 (m, 2H, CH2), 2.33±2.42 (m, 2H, H14), 2.44 (s, 3H,
C4±CH3CO), 2.49±2.65 (m, 1H, H6a), 3.31±3.39 (m, 2H,
NCH2), 3.81 (t, 8H, J=6, NCH2), 3.94 (d, J=7.5, 1H,
0
H3), 4.19 (d, J=7.0, 2H, H20), 4.79 (d, J=3.0, 1H, H2 ),
0
4.91 (d, J=8.0, 1H, H5), 5.51±5.73 (m, 3H, H2, H3 , and
FITC-conjugated paclitaxel 9. 7-Aminocaproylpaclitaxel
7 (25 mg, 0.025 mmol, 1 eq) was dissolved in a mixture
of dry pyridine:DMF (1:2, 2 mL). To this solution was
added FITC (15 mg, 0.037 mmol, 1.5 equiv) and the
contents were stirred at room temperature under nitro-
gen and in darkness for 2 days. The solution was eva-
porated to dryness under vacuum at 20 ꢀC and the
residual material was washed successively with acetone
(3Â5 mL) and diethylether (3Â5 mL). The crude mixture
was puri®ed by preparative TLC on silica gel (EtOAc/
CH2Cl2, 1:5; Rf=0.45) to give the product 8 as a bright
yellow solid (18 mg, 53%). 1H NMR (400 MHz,
CD3OD) d 1.02 (s, 3H, H16), 1.05 (s, 3H, H17), 1.27±1.34
(m, 2H, CH2), 1.49±1.54 (m, 4H, CH2), 1.68 (s, 3H,
H19), 1.69±1.71 (m, 1H, H6b), 1.78 (s, 3H, H18), 2.03 (s,
3H, C10-CH3CO), 2.01±2.18 (m, 2H, CH2), 2.24±2.39
(m, 2H, H14), 2.26 (s, 3H, C4-CH3CO), 2.41±2.59 (m,
1H, H6a), 3.22±3.28 (m, 2H, NCH2), 3.91 (d, J=6.0, 1H,
H3), 4.16 (d, J=7.9, 1H, H20), 4.28 (d, J=7.9, 1H, H20),
H7), 6.22 (t, J=9.5, 1H, H13), 6.28 (s, 1H, H10), 7.01±
7.04 (m, 2H, ArH), 7.15 (d, J=7.6, 1H, NH), 7.21±7.38
(m, 5H, ArH and NH), 7.40±7.68 (m, 12H, ArH), 7.69±
7.81 (m, 1H, ArH), 8.06 (d, J=8.0, 1H, ArH), 8.19 (brt,
1H, ArH), 8.68 (brs, 1H, ArH); 13C NMR (100 MHz,
CD3OD) d 11.7, 13.1, 14.7, 20.6, 20.9, 22.3, 23.5, 24.0,
24.4, 25.0, 26.2, 27.1, 30.7, 31.0, 33.1, 34.5, 34.8, 35.6,
36.8, 41.1, 43.0, 43.2, 43.5, 47.0, 50.6, 50.8, 55.0, 56.4,
56.9, 58.1, 72.3, 75.1, 76.0, 76.9, 77.6, 79.1, 82.2, 83.5,
85.4, 97.1, 115.2, 115.3, 115.9, 116.1, 127.3, 128.6, 129.3,
129.9, 130.5, 131.4, 131.8, 133.1, 134.1, 134.2, 134.7,
134.9, 140.2, 142.0, 146.8, 156.9, 157.0, 159.5, 159.9,
167.5, 170.6, 170.9, 171.0, 172.9, 174.7, 176.8, 202.5.
Anal. calcd for C80H91N4S2O21: C, 63.68; H, 6.07; N,
3.71. Found: C, 63.92; H, 6.22; N, 3.44.
Cell culture and ¯uorescent staining. The human non-
small cell lung cancer cells H460 were obtained from
0
4.74 (m, 1H, H2 ), 4.87 (d, J=8.0, 1H, H5), 5.45 (dd,

Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
2203
American Type Culture Collection (ATCC; MD). These
References
cells were grown in RPMI-1640 supplemented with 10%
FBS and subcultured 2±3 times/week. The cells were
seeded on a cover glass in a density of 1Â105 ml and
allowed to grow overnight for attachment. The culture
medium was removed and cells on the cover glass were
washed three times with PBS. Then, the cells were ®xed
with ethanol at 20 ꢀC for 30 min. After ®xation, the
ethanol was removed and cells were washed three times
with PBS. Now 0.25 mM of 9 or 11 was added to the
sample and stained for 1 h at 4 ꢀC in darkness. The
coverslips were washed extensively with PBS and then
mounted in an anti-bleaching mountant (5% propy-
gallate dissolved in 15% PBS/85% glycerol). The sam-
ples were examined under a Zeiss Axiophot microscope
equipped with epi¯uorescence optics and all images
were recorded under a Plan-neo¯ar 100Â (N.A. 1.3)
objective.
1. Georg, G. I.; Chen, T. T.; Ojima, I.; Vyas, D. M.; Eds.;
Taxane Anticancer Agents: Basic Science and Current Status;
ACS Symposium Series 583; Washington, DC American Che-
mical Society, 1995; 1±343.
2. Horwitz, S. B. Trends Pharmacol. Sci. 1992, 13, 134±136.
3. Ding, A. H.; Porteu, F.; Sanchez, E.; Nathan, C. F. Science
1990, 248, 370±372.
4. Bogdan, C.; Ding, A. H. J. Leuk. Biol. 1992, 52, 119.
5. Manthey, C. L.; Brandes, M. E.; Perera, P.-Y.; Vogel, S. N.
J. Immunol. 1992, 149, 2459±2465.
6. Manthey, C. L.; Perera, P.-Y.; Salkowski, C. A.; Vogel,
S. N. J. Immunol. 1994, 152, 825±831.
7. Henricson, B. E.; Carboni, J. M.; Burkhardt, A. L.; Vogel,
S. N. Mol. Med. 1995, 1, 428±435.
8. Lieu, C.-H.; Chang, Y.-N.; Lai, Y.-K. Biochem. Pharmacol.
1997, 53, 1583±1596.
9. Chu, J.-J.; Chen, K.-D.; Lin, Y.-L.; Fei, C.-Y.; Chiang,
A.-S.; Chiang, C.-D.; Lai, Y.-K. J. Cell. Biochem. 1998, 68,
472±483.
Drug accumulation assays. H460 cells were grown to 70±
80% con¯uence and seeded in 35 mm dishes (Falcon,
CA). The cell monolayers were washed with PBS and
then incubated with serum free RPMI-1640 medium
and 4 (speci®c activity, 48 mCi/mmol) at 37 ꢀC. At the
end of the treatment, the cell monolayers were washed
three times with an ice-cold PBS and the cells were lysed
with 20 mM Tris-HCl, pH 7.7, containing 0.2% SDS.
Radioactivity in the cell lysates was determined by b-
scintillation counting.
10. Parness, J. J.; Horwitz, S. B. J. Cell Biol. 1981, 91, 479±
487.
11. Chenu, J.; Takoudju, M.; Wright, M.; Senilh, V.;
Guenard, D. J. Labelled Compd. Radiopharm. 1987, 24, 1245±
1255.
12. Rao, S.; Horwitz, S. B.; Ringel, F. J. Natl. Cancer Inst.
1992, 84, 785±788.
13. Taylor, G. F.; Thornton, S. S.; Tallent, C. R.; Kepler, J. A.
J. Labelled Compd. Radiopharm. 1993, 33, 501±515.
14. Rimoldi, J. M.; Kingston, D. G.; Chaudhary, A. G.;
Samaranayake, G.; Grover, S.; Hamel, E. J. Nat. Prod. 1993,
56, 1313.
Flow cytometric analysis of ¯uorescent paclitaxel accu-
mulation. H460 cells were seeded on a 35 mm dish at
5Â105 ml and incubated overnight to attach. The old
medium was removed and replaced by fresh medium.
The ¯uorescent-paclitaxel (9 or 11) was added to the
culture and incubated at 37 ꢀC. At the end of treatment,
the cells were washed three times with PBS and followed
by trypsinization. The cell suspension samples were kept
on ice until they were analyzed. The ¯uorescence inten-
sity of each sample was quantitated by ¯ow cytometry
(FACStar plus, Becton Dickinson, San Jose, CA). The
wavelength for excitation was set at 488 nm. The optical
®lter for emission signal of FITC and rhodamine is
519 nm and 576 nm, respectively. Cell populations of
interest were gated according to the forward/side scatter
dot plot. The mean channel number of ¯uorescence
intensities of 5,000 cells in each sample tube was deter-
mined.
15. Rao, S.; Orr, G. A.; Ghaudary, A. G.; Kingston, D. G. I.;
Horwitz, S. B. J. Biol. Chem. 1995, 270, 20235±20238.
16. Zamir, L. O.; Nedea, M. E.; Garneau, F. X. Tet. Lett.
1992, 33, 5235±5236.
17. Strobel, G. A.; Stierle, A.; van Kuijk, F. J. G. M. Plant
Science 1992, 84, 65±74.
18. Walker, D. G.; Swigor, J. E.; Kant, J.; Schroeder, D. R. J.
Labelled. Compd. Radiopharm. 1994, 34, 973±980.
19. Walker, D. G.; Standridge, R. T.; Swigor, J. E. J. Labelled
Compds. Radiopharm. 1995, 36, 479±492.
20. de Suzzoni, S.; Boullais, C.; Vandais, A.; Noel, J. P. J.
Labelled Compds. Radiopharm. 1995, 36, 739±743.
21. Parness, J.; Kingston, D. G. I.; Powell, R. G.; Harrack-
singh, C.; Horwitz, S. B. Biochem. Biophys. Res. Comm. 1982,
105, 1082±1089.
22. Mellado, W.; Magri, N. F.; Kingston, D. G. I.; Garcia-
Arenas, R.; Orr, G. A.; Horwitz, S. B. Biochem. Biophys. Res.
Comm. 1984, 124, 329±336.
Acknowledgements
23. Takoudju, M.; Wright, M.; Chenu, J.; Gueritte-Voegelein,
F.; Guenard, D. FEBS Lett. 1988, 234, 177±180.
This work was supported in parts by research grants
from the ROC National Science Council (NSC87-2311-
B007-028 and NSC87-2311-B007-001-B12) to Y.-K.L.
The authors wish to thank Drs. C. Yu and J. Hammond
for their valuable comments on this manuscript.
24. Ringel, I.; Horwitz, S. B. J. Pharmacol. Exp. Ther. 1987,
242, 692±698.
25. Kingston, D. G. I. Pharmacol. Ther. 1991, 52, 1±34.

2204
Ch. S. Rao et al./Bioorg. Med. Chem. 6 (1998) 2193±2204
26. Sengupta, S.; Boge, T. C.; George, G. I.; Himes, R. H.
Biochemistry 1995, 34, 11889±11894.
Le Go€, M.-T.; Maangatal, L.; P. J. Med. Chem. 1991, 34,
992±998.
27. Souto, A. A.; Acuna, A. U.; Andreu, J. M.; Barasoain, I.;
Abal, M.; Amat-Guerri, F. Angew. Chem. Int. Ed. Engl. 1995,
34, 2710±2712.
33. Grover, S.; Rimoldi, J. M.; Molinero, A. A.; Chaudhary,
A. G.; Kingston, D. G. I.; Hamel, E. Biochemistry 1995, 34,
3927±3934.
28. Dubois, J.; Le Go€, M-T.; Gueritte-Voegelein, F.; Gue-
nard, D.; Tollon, Y.; Wright, M. Bioorg. Med. Chem. 1995, 3,
1357±1368.
34. Deutsch, J. A.; Glinski, J. A.; Hernandez, M.; Haugwitz,
R. D.; Narayana, V. L.; Suness, M.; Zalkow, L. H. J. Med.
Chem. 1989, 32, 788±792.
29. Guy, R. K.; Scott, Z. A.; Sloboda, R. D.; Nicolaou, K. C.
Chem. Biol. 1996, 3, 1021±1031.
35. Mathew, A. E.; Mejillano, M. R.; Nath, J. P.; Himes, R.
H.; Stella, V. J. J. Med. Chem. 1992, 35, 145±151.
30. Han, Y.; Chaudhary, A. G.; Chordia, M. D.; Sackett, D.
L.; Perez-Ramirez, B. Kingston, D. G. I.; Bane, S. Biochemistry
1996, 35, 14173±14183.
36. Nicolaou, K. C.; Dai, W. M.; Guy, R. K. Angew. Chem.
Int. Ed. Engl. 1994, 106, 15±44.
37. Ponnusamy, E.; Fotadar, U.; Spisni, A.; Fiat, D. Synthesis
1986, 48±49.
31. Sengupta, S.; Boge, T. C.; Liu, Y.; Hepperle, M, Georg, G.
I.; Himes, R. H. Biochemistry 1997, 36, 5179±5184.
32. Potier Gueritte-Voegelein, F.; Guenard, D.; Lavelle, F.;
38. Mosmann, T. J. Immunol. Methods 1983, 65, 55±63.