Targeted delivery of paclitaxel to tumor cells: Synthesis and in vitro evaluation

Article abstract of DOI:10.1021/jm901763f

We previously reported a novel drug delivery system, drug-linker-Phe-Phe- Arg-methylketone (FFR-mk)-factor VIIa (fVIIa). The method utilizes tissue factor (TF), which is aberrantly and abundantly expressed on many cancer cells. The advantage of this delivery system is its ability to furnish a potent anticancer drug specifically to the tumor vasculature and cancer cells. In this paper, we describe the synthesis of paclitaxel (PTX)-Phe-Phe-Arg-chloromethyl ketone (FFR-ck), followed by coupling with fVIIa to form PTX-FFR-mk-fVIIa. FFRck was separately linked to the OH groups at the C2′ or C7 positions of PTX (C2′- or C7-PTX-FFRck), the C2′ analogue exhibiting better activity against human head and neck squamous KB 3-1 cells. The activity order against PTX-sensitive KB 3-1 cells is C2′-PTX-FFRmk-fVIIa > PTX > C2′-PTX-FFRck. The C2′ complex shows an IC₅₀ of 12 nM against the PTX-sensitive cell line and 130 nM against PTX-resistant cells.

Full text of DOI:10.1021/jm901763f

J. Med. Chem. 2010, 53, 3127–3132 3127
DOI: 10.1021/jm901763f
Targeted Delivery of Paclitaxel to Tumor Cells: Synthesis and in Vitro Evaluation
John M. Ndungu,† Yang J. Lu,‡ Shijun Zhu,‡ Chao Yang,† Xu Wang,‡ Georgia Chen,‡ Dong M. Shin,‡ James P. Snyder,†
Mamoru Shoji,*,‡ and Aiming Sun*,†
†Department of Chemistry, 1515 Dickey Drive, Emory University, Atlanta, Georgia 30322, and ‡Winship Cancer Institute, Emory University,
Atlanta, Georgia 30322
Received November 29, 2009
We previously reported a novel drug delivery system, drug-linker-Phe-Phe-Arg-methylketone (FFR-mk)-
factor VIIa (fVIIa). The method utilizes tissue factor (TF), which is aberrantly and abundantly
expressed on many cancer cells. The advantage of this delivery system is its ability to furnish a potent
anticancer drug specifically to the tumor vasculature and cancer cells. In this paper, we describe the
synthesis of paclitaxel (PTX)-Phe-Phe-Arg-chloromethyl ketone (FFR-ck), followed by coupling with
fVIIa to form PTX-FFR-mk-fVIIa. FFRck was separately linked to the OH groups at the C20 or C7
positions of PTX (C20- or C7-PTX-FFRck), the C20 analogue exhibiting better activity against human
head and neck squamous KB 3-1 cells. The activity order against PTX-sensitive KB 3-1 cells is C20-PTX-
FFRmk-fVIIa > PTX > C20-PTX-FFRck. The C20 complex shows an IC50 of 12 nM against the PTX-
sensitive cell line and 130 nM against PTX-resistant cells.
Introduction
a linear polysaccharide whose receptors are overexpressed in
some tumors, has also been used to target paclitaxel to tumor
cells.8 Additionally, Zhao et al. have demonstrated the target-
ing of tumor cells using tripeptide modified liposomes that
home on integrin receptors overexpressed on tumor cells.9
Cancer cells are also known to overexpress GLUT, a family of
membrane proteins, to improve glucose uptake. Chen et al.
have synthesized four glycan-based paclitaxel prodrugs to
specifically target GLUT overexpressing cancer cells.10 Inor-
ganic nanoparticles have also been widely used for imaging,
targeting, and drug delivery.11 Gold nanomaterials represent
one approach in which the ability to control the size and shape
of the particles and their surface conjugation with antibodies
allows for both selective imaging and photothermal killing of
cancer cells by using long-wavelength light for tissue
penetration.12-14
Angiogenesis is a crucial process for tumor progression and
metastasis.15 An emerging strategy in cancer treatment is to
target tumor-associated vascular endothelial cells (VECs)
aimed at impeding the growth and survival of solid tumors
by eliminating tumor blood supply. Tissue factor (TF) is
aberrantly overexpressed on tumor VECs and on cancer cells
in many malignant tumors, but not on normal VECs, which
makes it a favorable target for cancer therapy. Several labora-
tories have reported promising results by targeting TF in
tumor cells and VECs and thereby suppressing tumor growth
in animal models.16-18 Tissue factor is a transmembrane
receptor for an endogenous ligand coagulation factor VIIa
(fVIIa). Upon binding to fVIIa, TF forms a high-affinity and
specific complex with fVIIa, and the complex subsequently
initiates the blood clotting sequence.19 Several tripeptide
chloromethyl ketones have been demonstrated to inacti-
vate fVIIa by binding histidine in the serine protease core
and render it a competitive inhibitor of unligated fVIIa.20
Banner and co-workers have reported the crystal structure
of fVIIa-TF. The fVIIa component adopts an extended
Target-specific drug delivery has become an important
strategy to improve the selectivity of cytotoxic drugs against
targeted cells and reduce toxicity against normal cells. The
general method couples a drug carrier and the drug to form a
prodrug, which can be released in the target cells. Then, the
drug is transformed from an inactive to an active form that
performs the cell-killing function. Cancer chemotherapy is
one of the important approaches to treating cancer patients,
although the nonselectivity of many antitumor agents can
cause serious side effects. Paclitaxel (PTX) is a mitotic inhibi-
tor that stabilizes microtubules, thereby interfering with the
normal dissociation of microtubules during cell division.1 The
compound is widely used to treat patients with lung, ovarian,
breast, head, and neckcancer and advanced forms of Kaposi’s
sarcoma.2,3 However, serious drawbacks hamper PTX’sa
clinical usefulness. For instance, paclitaxel lacks selective
cytotoxicity between cancer cells and normal cells, which
frequently leads to serious unwanted side effects.4 The poor
water solubility of paclitaxel is another problem that signifi-
cantly reduces its wider clinical application. Different strate-
gies have been explored to circumvent these side effects. One
of the most attractive tactics is the selective targeting of tumor
cells over normal cells exemplified by employing suitable
monoclonal antibodies.5-7 A number of antigens that are
preferentially expressed on tumor cells have been identified,
and monoclonal antibodies have been developed to specifi-
cally bind to these tumor associated substances. Hyaluronan,
*To whom correspondence should be addressed. Phone: 404-727-6689.
Fax: 404-727-6689. E-mail: asun2@emory.edu (A.S.); mshoji@emory.edu
(M.S.).
a Abbreviations: fVIIa, factor VIIa; TF, tissue factor; FFRck, Phe-
Phe-Arg-chloromethylketone; FFR-mk, Phe-Phe-Arg-methylketone;
PTX, paclitaxel; VECs, vascular endothelial cells; VEGF, vascular
endothelial growth factor; HUVECs, human umbilical vein endothelial
cells; NF-κB, nuclear factor kappa B; NR, Neutral Red.
r
2010 American Chemical Society
Published on Web 03/19/2010
pubs.acs.org/jmc

3128 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Ndungu et al.
Scheme 1
conformation that wraps around TF with the catalytic do-
main of the serine protease distal to the cell membrane.21
Cancer patients are generally in a hypercoagulable state
because cancer cells express TF, which is the cognate receptor
of fVIIa. FFRck binds and inactivates fVIIa to make it a
competitive inhibitor of fVIIa. This is expected to prevent
potential patients from thrombotic complications when
PTX-FFRmk-fVIIa is administered. It requires coupling of
a tripeptide chloromethyl ketone (e.g., FFRck) to the pro-
tein. The active site inactivated fVIIa retains the same bind-
ing affinity as fVIIa to its receptor protein, TF.22 On the
basis of this evidence, we have chosen FFRmk-fVIIa as a
drug carrier with two purposes. First, we intend drugs for
targeted drug delivery to tumor angiogenesis and the tumor
itself because TF is induced to aberrantly express in cancer
VEC by vascular endothelial growth factor (VEGF), and
cancers express TF. Second, this should also prevent or
minimize an increased tendency of blood clotting in cancer
patients.
The use of peptide chloromethyl ketones as the coupling
reagent to generate irreversible inhibitors of serine proteases
was pioneered by Shaw and co-workers.23 Our group has
previously demonstrated the utility of using fVIIa as a selec-
tive anticancer drug carrier. To validate the concept, the
cytotoxic agent EF24 was conjugated to fVIIa through a
tripeptide-chloromethyl ketone linker.24 The experiment suc-
cessfully demonstrated that the conjugate inhibits vascular
endothelial growth factor-induced angiogenesis in rabbit
cornea and Matrigel models in athymic nude mice.25
The potential of this particular delivery method can be max-
imized by inserting highly potent, but highly toxic anticancer
drugs, for instance, paclitaxel, into the tumor cells specifically
to reduce toxic side effects. In this paper, we describe the
synthesis of PTX-FFRck and its conjugation to fVIIa to give
PTX-FFRmk-fVIIa. We expected the latter to exhibit beha-
vior similar to EF24-FFRmk-fVIIa, namely specific targeting
of cancer cells, but with significantly greater potency.
Results and Discussion
The synthesis of C20-PTX-FFRck is based on the observa-
tion that paclitaxel can be esterified at C20 without protection
of other functional groups. The preparation began with the
global deprotection of tripeptide 120 to furnish the tripeptide
chloromethylketone 2. The C20 succinic acid derivative of
PTX,26 namely compound 3, was treated with EEDQ27 and
then coupled with 2 to afford the desired C20-PTX-FFRck, 4,
as a TFA salt. (Scheme 1)
To explore the possible biological differences between C20-
FFRck and C7-FFRck, tripeptide chloromethyl ketone
(FFRck) was also coupled with the C7-position hydroxyl
group of PTX as illustrated in Scheme 2. The C20-hydroxyl
group of PTX was selectively protected with TBSCl in the
presence of imidazole to obtain 6,28 which was combined with
succinic anhydride to give coupling precursor 7. The conjuga-
tion of acid 7 to the TFA salt of FFR-ck 2 yielded 8.
Deprotection of the TBS group under acidic conditions
afforded the TFA salt of C7-PTX-tripeptide chloromethyl
ketone 9.
Conjugation of PTX-FFRck to fVIIa. The procedure is
essentially the same as that described for conjugating
EF24.21 Briefly, C20-PTX-FFRck in 100% DMSO is added
dropwise to factor VIIa solution in a molar ratio of 3:1 at
room temperature for 1-2 h and then gently stirred at 4 °C
overnight. The unconjugated excess C20-PTX-FFRck is
removed by dialysis in 10 mM Tris-HCl, pH 7.5, with several

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3129
Scheme 2a
a Reagents and conditions: (a) TBSCl, imidazole, 92%; (b) succinic anhydride, Et3N, 86%; (c) EEDQ, 2, Et3N, 46%; (d) HCl, MeOH, 69%.
changes of the buffer. The resulting C20-PTX-FFRmk-fVIIa
is a competitive inhibitor of fVIIa because PTX-FFRck
binds the core of serine protease of fVIIa but retains the
same binding activity as fVIIa to TF.22 The fVIIa activity of
the PTX-FFRmk-fVIIa is less than 4-5% of fVIIa as deter-
mined according to the manufacturer’s instruction using an
ST art 4 device to measure factor VIIa coagulation activity
(Diagnostica Stargo, Asnieres-sur-Seine, France). The result
indicates that approximately 95% of fVIIa is bound by C20-
PTX-FFRck to make C20-PTX-FFRmk-fVIIa.
Neutral Red Dye Cell Viability Assays for Testing Drug
Activity. The activities of C20- and C7-PTX-FFRck at
various concentrations were assessed against KB 3-1 head
and neck cancer cells and compared with PTX. Both C20 and
C7 analogues decrease the viability of cancer cells in a dose-
dependent manner, although the former is more active than
Figure 1. Cell viabilities for C20-PTX-FFRck and C7-PTX-FFRck.
Varying concentrations of C20-PTX-FFRck, C7-PTX-FFRck, and
PTX were incubated with human head and neck cancer cells (KB 3-1)
in triplicate for 72 h. Cell viability was determined using the Neutral
Red dye assay.
the latter. The cytotoxicities of both PTX-analogues were
less than that of free paclitaxel. The IC50 of C20-PTX-FFRck
is approximately 12 nM, whereas that of C7-analogue is
130 nM. Thus, the cytotoxic activity of the C20 compound is
approximately 10-fold superior to that of the C7 variation.
This undoubtedly arises because the C20 ester bond is more
susceptible to hydrolysis and release of PTX by comparison
with the C7 ester (Figure 1).29 Therefore, C20-PTX-FFRck
(4) was chosen for conjugation with fVIIa to form C20-PTX-
FFRmk-fVIIa (abbreviated as PTX-FFRmk-fVIIa, herein).
Activity of PTX-FFRmk-fVIIa in Vitro against PTX-
Sensitive and PTX-Resistant Cell Lines. PTX-sensitive (KB
3-1 and TU212) and PTX-resistant (KB-V1) cells were
treated with varying concentrations of PTX, C20-PTX-
FFRck, and C20-PTX-FFRmk-fVIIa. The rank order of
activities against PTX-sensitive KB 3-1 and TU212 cells is
C20-PTX-FFRmk-fVIIa > PTX > C20-PTX-FFRck. The
apparent IC50 values of C20-PTX-FFRmk-fVIIa, PTX, and
C20-PTX-FFRck against KB 3-1 cell lines are 0.6, 1.2, and
12 nM, respectively (Supporting Information Figure 1SA
and 1SB). PTX, PTX-FFRmk-fVIIa, and PTX-FFRck all
show a weaker effect on the PTX-resistant cell line KB-V1 by
comparison with the PTX-sensitive cell lines. (Supporting
Information Figure 1SC).
Activity of PTX, PTX-FFRck, and PTX-FFRmk-fVIIa on
Human Umbilical Vein Endothelial Cells (HUVECs). HU-
VECs were cultured in the presence or absence of TPA
(phorbol ester 100 nM) overnight in order to induce TF
because normal HUVECs do not express this protein. HU-
VECs were incubated with varying concentrations of PTX-
FFRmk-fVIIa, PTX, PTX-C20-FFRck, or PTX-C7-FFRck
for 72 h, and then cell viability was determined by Neutral
Red dye assays in triplicate. TPA-treated HUVECs express
TF, PTX-FFRmk-fVIIa binds this protein, the TF-fVIIa

3130 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Ndungu et al.
C20-PTX-FFRmk-fVIIa, is approximately 30-fold lower
than that of the C20-PTX-FFRck against the TF-expressing
cells. The results are consistent with cell surface paclitaxel-
conjugate fVIIa binding to TF to deliver a complex
subsequently endocytosed by the target. Once inside the
cells, it is hypothesized that the PTX-FFRmk-fVIIa complex
is enzymatically cleaved to free paclitaxel, which is locally
available for cytotoxic action by ablating the dynamic
properties of microtubules during cell division. The strategy
of using the tripeptide (Phe-Phe-Arg)-ck as a linker and
fVIIa as a vehicle for target delivery of highly potent and
toxic drugs (e.g., paclitaxel) specifically to cancer cells,
further demonstrates the utility of the previously described
drug-targeting concept. This delivery mechanism opens a
useful approach to reducing the side effects of chemotherapy
treatment. Further research in our laboratory is directed
toward more efficient methodologies to deliver paclitaxel
and other highly toxic anticancer drugs to critical sites of
action.
Conclusion
Conjugation of FFRck separately with the C20-position
and C7-hydroxyl groups of paclitaxel leads to the successful
preparation of C20-PTX-FFRck and C7-PTX-FFRck, re-
spectively, in fairly good yields by means of a short synthetic
procedure. The C20 derivative exhibits considerably better
activity against KB 3-1 head and neck cancer cells than the C7
analogue. Thus, C20-PTX-FFRck was coupled with fVIIa
using known methods. Cell viability assays for paclitaxel, C20-
PTX-FFRck, and C20-PTX-FFRmk-fVIIa employing pacli-
taxel-sensitive and -resistant cancer cells demonstrate two
important features: (1) PTX and C20-PTX-FFRmk-fVIIa
are nearly equally efficient for cell killing of paclitaxel sensitive
cancercells invitro, whileC20-PTX-FFRck is significantly less
active. Intuitively, it might be imagined that the active trans-
port of C20-PTX-FFRmk-fVIIa in complex with TF should
be faster than the corresponding passive diffusion of free
paclitaxel across the cell membrane and, thereby, occasion
higher potency for the protein conjugate. However, the
apparent IC50 values estimating a diminutive 2-fold advan-
tage for the complex suggests that the two transport mechan-
isms may be competitive. The important difference, of course,
is that while hydrophobic PTX can diffuse across almost all
membranes with equal ease, the complex will be actively
transported only across cell membranes rich in overexpressed
TF. The latter is aberrantly expressed in the most malignant
cancers because nuclear factor kappa B (NF-κB) is constitu-
tively activated, while normal cells do not express TF. We
previously demonstrated a proof of principle that VEGF
attracts VECs and induces TF on VECs. An anticancer
reagent, namely EF24-FFRmk-fVIIa, binds to TF expressed
on VECs and tumors in vivo, endocytoses, releases the drug,
and inhibits TF-expressing VECs and cancer cells in vivo.25 In
the present work, we applied this concept by treatment of
cancer cells (KB 3-1 head and neck cancer cells), HUVECs
and TPAþ HUVECs with PTX, PTX-FFRck and PTX-
FFRmk-fVIIa, respectively. (Figures 1 and 2) These experi-
ments further support our hypothesis on the targeted drug
delivery to TF-expressing VECs and tumor cells. The minimal
effect observed in vitro will be maximized in vivo under
conditions of rapid blood flow. In this way, the specific cell-
surface targeting character of C20-PTX-FFRmk-fVIIa offers
promise for both therapeutic efficacy and reduction of side
Figure 2. (A) Cell viabilities for PTX, C20-PTX-FFRck, and PTX-
FFRmk-fVIIa on HUVECs. (B) Cell viabilities for PTX, C20-PTX-
FFRck, C7-PTX-FFRck, and PTX-FFRmk-fVIIa on TPA-treated
HUVECs.
complex is internalized (endocytosed), and the correspond-
ing HUVECs were killed. By contrast, normal HUVECs
do not express TF preventing PTX-FFRmk-fVIIa from
binding and subsequent cell kill. PTX alone does not require
ligand-receptor-mediated endocytosis to enter cells. Thus,
PTX kills both normal HUVECs and TPA-treated HUVECs
because it diffuses into the cells and suppresses microtubule
dynamics. Moderate cell killing capability was detected for
C20-PTX-FFRck on TPA treated HUVEC cells, which is
consistent with our hypothesis that C20 ester bond is more
susceptible to hydrolysis and release of free PTX by compar-
ison with the C7 ester (Figure 2).
We describe here the synthesis of both C20- and C7-PTX-
substituted PTX-FFRcks using succinic acid as a linker
between paclitaxel and FFRck. Both paclitaxel and the
conjugates exhibit cytotoxic action against the KB 3-1 head
and neck cancer cell line. Certain substitutions of the
C20-hydroxyl functionality of the prodrug are known to
cause reduction in cyctotoxic activity.30-32 The C20-conju-
gate is ca. 10-fold more active than the corresponding C7-
conjugate, which is due to the C20 ester bond being more
susceptible to hydrolysis and release of PTX by comparison
with the C7 ester. Paclitaxel itself is slightly more potent than
both conjugates against the same cell line. Although esterify-
ing the C20-hydroxyl group of paclitaxel with succinic acid
appeared to slightly reduce cancer cell toxicity relative to free
paclitaxel, the linker preserves the activity of paclitaxel and
was therefore chosen to implement the coupling chemistry
with fVIIa. Subsequently, the paclitaxel sensitive KB 3-1 and
TU212 cell lines and the paclitaxel resistant KB-V1 SCC cell
line were treated with PTX, C20-PTX-FFRck and C20-PTX-
FFRmk-fVIIa. The apparent IC50 of the fVIIa-conjugate,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3131
effects in the living organism. (2) Paclitaxel and the conjugates
(C20-PTX-FFRck and PTX-FFRmk-fVIIa) are all much less
effective against the paclitaxel resistant KB-V1 cell line by
comparison with treatment of paclitaxel sensitive cell lines.
of Dr. Michael M. Gottesman, the NCI.34 D-MEM/F12 (a 1:1
Dulbecco’s modified Eagle’s medium: Ham’s F12) medium,
D-MEM/high glucose medicum, and penicillin G/streptomycin
and L-glutamine were purchased from Mediatech (Herndon,
VA), Hyclone (Logan, UT), and GIBCO-BRL/Invitrogen
(Rockville, MD), respectively. Fetal bovine serum (FBS) was
purchased from Atlanta Biologicals (Atlanta, GA).
Experimental Section
KB 3-1 cells were maintained in D-MEM/F12 and KB-V1
cells were maintained in D-MEM/F12 containing vinblastine
(1 μg/mL). MDA-MB-231 cells were maintained in D-MEM/
high glucose. All media contained 10% FBS, penicillin (100
units/mL), streptomycin (100 μg/mL), and 2 mM L-glutamine.
Cells were incubated at 37 °C in 5% CO2, 95% air in a
humidified atmosphere.
General. All amino acids were obtained from Chem Impex
International, while all solvents and other reagents were pur-
chased from Aldrich Chemical Co., Milwaukee, WI, and used as
received. Thin layer chromatography (TLC) was performed on
precoated, glass-backed plates (silica gel 60 F254; 0.25 mm
thickness) from EM Science, and the plates were visualized by
UV lamp. Column chromatography was performed with silica
gel (230-400 mesh ASTM) using the “flash” method. Elemental
analyses were carried out by Atlantic Microlab Inc. Norcross,
GA. All reactions were performed under anhydrous nitrogen
atmosphere in oven-dried glassware.
Neutral Red (NR) Dye Cell Viability Assay for Testing Effi-
cacy of Drugs. The efficacies of C20-PTX-FFRck, C7-PTX-
FFRck, PTX-FFRmk-fVIIa, and paclitaxel were determined
with the cell viability assay in triplicate employing NR dye
uptake by surviving cells as previously described.33 The NR
dye is taken up only by viable cells. Briefly, cells were plated into
a 96-well plate at 20000 cells/200 μL/well, incubated, and
allowed to adhere for 24 h. Then, drugs were added to appro-
priate wells in various concentrations in triplicate. Subse-
quently, cells were incubated for 72 h. At the termination of
culture, medium was removed and 200 μL of fresh, warm
medium containing 50 μg of NR/mL was added to each well
in a 96-well plate. Cells were incubated at 37 °C for 30 min,
followed by two washes with 200 μL of PBS. The NR taken up
by cells was dissolved by adding 200 μL of 0.5 N HCl containing
35% ethanol. Then, the amount of the dye in each well was read
at 570 nm by a Universal Microplate Reader (EL800, Bio-Tek,
Winooski, VT). Results from triplicate samples were recorded as
optical density units (OD570) and averaged after subtraction of
the blank.
Synthesis of phe-phe-arg trifluoroacetic acid salt (2). Boc-phe-
phe-arg (Mtr) chloromethyl ketone, 120 (390.0 mg, 0.480 mmol),
was dissolved in TFA (5.0 ML). Thioanisole (200 μL) and water
(200 μL) were added and the mixture stirred at RT for 36 h. TFA
was removed under vacuum and the crude product washed
with ether and then ethyl acetate. The resultant solid was washed
with dichloromethane to give 2 (150.0 mg, 43% yield) as a white
solid. MS calcd for C25H33ClN6O3, 500.2303; found, 501.2370
[M þ H]þ.
Synthesis of 20-succinyl PTX-phe-phe-arg chloromethyl ketone,
C20-PTX-FFRck, (4). To a solution of C20-succinyl paclitaxel,
3 (34.0 mg, 0.0357 mmol), in CH2Cl2 (500 μL) was added EEDQ
(11.0 mg, 0.0445 mmol) and Et3N (6.0 μL, 0.043 mmol) and
the mixture stirred for 30 min. To the solution was added
Et3N (18.0 μL, 0.043 mmol), FFRck (30.0 mg, 0.0412 mmol),
and DMF (200 μL). The mixture was stirred for 3 h and the
solvent removed in vacuo. The crude product was dried under
vacuum to remove DMF and then purified by column chloro-
matography (5-8% MeOH/ CH2Cl2) to give 4 (35 mg, 64%
yield) as a white solid. Anal. Calcd for C78H86ClF3N7O20: C%,
61.07; H%, 5.65; N%, 6.39. Found: C%, 60.69; H%, 5.61; N%,
6.20. MS calcd for C76H87ClN7O19, 1435.5667; found, 1436.5756
[M þ H]þ.
Acknowledgment. This work was financially supported by
the National Institutes of Health (NIH) grants R21 CA82995-
01A1 and U.S. Department of Defense, the Division of U.S.
Army DAMD17-00-1-0241 (to M. Shoji), and P50 CA128613
“Developmental Research Program pilot project” (to D. M.
Shin and M. Shoji).
Synthesis of C20-TBS-C7-succinyl-paclitaxel-phe-phe-arg chloro-
methyl ketone (8). To a solution of C20-TBS-C7-succinyl pacli-
taxel 7 (94 mg) in 2.5 mL of CH2Cl2 was added EEDQ (48 mg).
After stirring at room temperature for 30 min, Et3N (40 μL)
was added. The reaction mixture was transferred to a suspen-
sion of FFRck (94 mg) in 0.4 mL of DMF and allowed to stir
overnight. The reaction mixture was concentrated under va-
cuum and the residue purified by column chromatography
(3-8% MeOH/CH2Cl2) to give C20-TBS-7-succinyl FFRck
paclitaxel 8 (84 mg, 56%) as a white solid. The product was
used in the next step without further characterization.
Supporting Information Available: Activities of PTX, C20-
PTX-FFRck, and C20-PTX-FFRmk-fVIIa against PTX-sensi-
tive (human head and neck cancer cells KB 3-1 and TU212)
and PTX-resistant (KB-V1) cells) and 1H NMR of com-
pounds 4 (C20-PTX-FFRck) and 9 (C7-PTX-FFRck). This
material is available free of charge via the Internet at http://
pubs.acs.org.
Synthesis of 7-succinyl PTX-phe-phe-arg chloromethyl ketone
C7-PTX-FFRck (9). C20-TBS-7-succinyl-paclitaxel-FFRck, 8
(84 mg) was treated with 2 mL of 5% HCl in MeOH. The
reaction was monitored by LC-MS. After completion of reac-
tion, the mixture was diluted with CH2Cl2 and quenched
with NaHCO3 (sat. aq.). The aqueous layer was separated and
extracted with CH2Cl2 and the combined organic phase washed
with brine, dried over sodium sulfate, and concentrated in
vacuo. The residue was purified by column chromatography
(8% MeOH/DCM) to give C7-PTX-FFRck, 9 (61 mg, 78%) as
a white solid. Anal. Calcd for C78H86ClF3N7O20: C%, 61.07;
H%, 5.65; N%, 6.39. Found: C%, 59.71; H%, 5.93; N%,
6.41. MS calcd for C76H87ClN7O19, 1435.5667; found, 1436.5746
[M þ H]þ.
References
(1) (a) Kumar, N. Taxol-Induced Polymerization of Purified Tubulin.
J. Biol. Chem. 1981, 256, 10435–10441. (b) Horwitz, S. B. Mec-
hanism of action of taxol. Trends Pharmacol. Sci. 1992, 13, 134–
136.
(2) Rooseboom, M.; Commandeur, J. N.; Vermeulen, N. P. Enzyme-
catalyzed activation of anticancer prodrugs. Pharmacol. Rev. 2004,
56, 53–102.
(3) Abraham, S.; Guo, F.; Li, L. S.; Rader, C.; Liu, C.; Barbas, C. F.;
Lerner, R. A.; Sinha, S. C. Synthesis of the next-generation
therapeutic antibodies that combine cell targeting and antibody-
catalyzed prodrug activation. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 5584–5589.
(4) Michaud, L. B.; Valero, V.; Hortobagyi, G. Risks and Benefits
of Taxanes in Breast and Ovarian Cancer. Drug Safety 2000, 23,
401–428.
Cell Culture. Human breast cancer cells MDA-MB-231
were obtained from American Type Cell Collection (ATCC:
Rockville, MD). KB 3-1 (paclitaxel-sensitive, parental line of
KB V1) and KB V1 (paclitaxel resistant) cells were the kind gifts
(5) Ojima, I.; Geng, X.; Wu, X.; Qu, C.; Borella, C. P.; Xie, H.;
Wilhelm, S. D.; Leece, B. A.; Bartle, L. M.; Goldmacher, V. S.;
Chari, R. V. Tumor-Specific Novel Taxoid-Monoclonal Anti-
body Conjugates. J. Med. Chem. 2002, 45, 5620–5623.

3132 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Ndungu et al.
(6) Guillemard, V.; Saragovi, H. U. Taxane-antibody conjugates
afford potent cytotoxicity, enhanced solubility, and tumor target
selectivity. Cancer Res. 2001, 61, 694–699.
(7) Jaime, J.; Page, M. Paclitaxel immunoconjugate for the specific
treatment of ovarian cancer in vitro. Anticancer Res. 2001, 21,
1119–1128.
(8) Banzato, A.; Bobisse, S.; Rondina, M.; Renier, D.; Bettella, F.;
Esposito, G.; Quintieri, L.; Melendez-A, L.; Mazzi, U.; Zanovello,
P.; Rosato, A. A. Paclitaxel-Hyaluronan Bioconjugate Targeting
Ovarian Cancer Affords a Potent In vivo Therapeutic Activity.
Clin. Cancer Res. 2008, 14, 3598–3606.
(9) Zhao, H.; Wang, J.-C.; Sun, Q.-S.; Luo, C.-L.; Zhang, Q. RGD-
based strategies for improving antitumor activity of paclitaxel-
loaded liposomes in nude mice xenografted with human ovarian
cancer. J. Drug Targeting 2009, 17, 10–18.
(10) Lin, Y.; Tungpradit, R.; Sinchaikul, S.; An, F.; Liu, D.; Phutrakul,
S.; Chen, S. Targeting the Delivery of Glycan-Based Paclitaxel
Prodrugs to Cancer Cells via Glucose Transporters. J. Med. Chem.
2008, 51, 7428–7441.
(11) Liong, M.; Lu, J.; Kovochich, M.; Xia, T.; Ruehm, S. G.; Nel,
A. E.; Tamanoi, F.; Zink, J. I. Multifunctional Inorganic Nano-
particles for Imaging, Targeting, and Drug Delivery. ACS Nano
2008, 2, 889–896.
(12) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. Cancer Cell
Imaging and Photothermal Therapy in the Near-Infrared Region
by Using Gold Nanorods. J. Am. Chem. Soc. 2006, 128, 2115–2120.
(13) Chen, J.; Wang, D.; Xi, J.; Au, L.; Siekkinen, A.; Warsen, A.; Li, Z.;
Zhang, H.; Xia, Y.; Li, X. Immuno Gold Nanocages with Tailored
Optical Properties for Targeted Photothermal Destruction of
Cancer Cells. Nano Lett. 2007, 7, 1318–1322.
(14) Gobin, A. M.; Lee, M.; Halas, N.; James, W. D.; Drezek, R. A.;
West, J. L. Near-Infrared Resonant Nanoshells for Combined
Optical Imaging and Photothermal Cancer Therapy. Nano Lett.
2007, 7, 1929–1934.
(22) Sorensen, B. B.; Persson, E.; Freskgard, P.-O.; Kjalke, M.; Ezban,
M.; Williams, T.; Rao, L.V. M. Incorporation of an active site
inhibitor in factor VIIa alters the affinity for tissue factor. J. Biol.
Chem. 1997, 272, 11863–11868.
(23) (a) Schoellmann, G.; Shaw, E. Direct evidence for the presence of
histidine in the active center of chymotrypsin. Biochemistry 1963, 2,
252–255. (b) Kettner, C.; Shaw, E. Inactivation of trypsin-like enzymes
with peptides of arginine chloromethyl ketone. Methods Enzymol.
1981, 80, 826–842.
(24) Sun, A.; Shoji, M.; Lu, Y. J.; Liotta, D. C.; Snyder, J. P. Synthesis
of EF24-tripeptide chloromethyl ketone: a novel curcumin-related
anticancer drug delivery system. J. Med. Chem. 2006, 49, 3153–
3158.
(25) Shoji, M.; Sun, A.; Kisiel, W.; Lu, Y.; Shim, H.; Mccarey, B. E.;
Nichols, C.; Parker, E.; Pohl, J.; Mosley, C. A.; Alizadeh, A. R.;
Liotta, D. C.; Snyder, J. P. Targeting tissue factor-expressing
tumor angiogenesis and tumors with EF24 conjugated to factor
VIIa. J. Drug Targeting 2008, 16, 185–197.
(26) Deutsch, H. M.; Glinski, J. A.; Hernandez, M.; Haugwitqt, R. D.;
Narayanan, V. L.; Suffness, M.; Zalkow., L. H. Synthesis of
Congeners and Prodrugs; Water-Soluble Prodrugs of Taxol with
Potent Antitumor Activity. J. Med. Chem. 1989, 32, 788–792.
(27) Zakharian, T. Y.; Seryshev, A.; Balaji Sitharaman, B.; Gilbert,
B. E. A Fullerene-Paclitaxel Chemotherapeutic: Synthesis, Char-
acterization, and Study of Biological Activity in Tissue Culture. J.
Am. Chem. Soc. 2005, 127, 12508–12509.
(28) Turunen, B. J.; Ge, H.; Oyetunji, J.; Desino, K. E.; Vasandani, V.;
€
Guthe, S.; Himes, R. H.; Audus, K. L.; Seelig, A.; Georg, G. I.
Paclitaxel succinate analogs: anionic and amide introduction as a
strategy to impart blood-brain barrier permeability. Bio. Med.
Chem. Lett. 2008, 18, 5971–5974.
(29) Liu, C.; Strobl, J. S.; Bane, S.; Schilling, J. K.; McCracken, M.;
Chatterjee, S. K.; Rahim-Bata, R.; Kingston, D. G. I. Design,
Synthesis, and Bioactivities of Steroid-Linked Taxol Analogues as
Potential Targeted Drugs for Prostate and Breast Cancer. J. Nat.
Prod. 2004, 67, 152–159.
(15) Folkman, J. Angiogenesis: an organizing principle for drug dis-
covery. Nat. Rev. Drug Discovery 2007, 6, 273–286.
(16) Zhang, Y.; Deng, Y.; Wendt, T.; Liliensiek, B.; Bierhaus, A.;
Greten, J.; He, W.; Chen, B.; Hach-Wunderle, V.; Waldherr, R.;
Ziegler, R.; Mannel, D.; Stern, D. M.; Nawroth, P. P. Intravenous
somatic gene transfer with antisense tissue factor restores blood
flow by reducing tumor necrosis factor expression and fibrin
deposition in mouse meth-A sarcoma. J. Clin. Invest. 1996, 97,
2213–2224.
(17) Huang, X.; Molema, G.; King, S.; Watkins, L.; Edington, T. S.;
Thorpe, P. E. Tumor infarction in mice by antibody-directed
targeting of tissue factor to tumor vasculature. Science 1997, 275,
547–550.
(18) Hu, Z.; Sun, Y.; Garen, A. Targeting tumor vasculature endothe-
lial cells and tumor cells for immunotherapy of human melanoma
in a mouse xenograft model. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
8161–8166.
(19) Nemerson, Y. Tissue factor and hemostasis. Blood 1988, 71, 1–8.
(20) Williams, E. B.; Krishnaswamy, S.; Mann, K. G. Zymogen/enzyme
discrimination using peptide chloromethyl ketones. J. Biol. Chem.
1989, 264, 7536–7545.
(30) Mellado, W.; Magri, N. F.; Kingston, D. G. I.; Garcia-Arenas, R.;
Orr, G. A.; Horwitz, S. B. Preparation and biological activity of
taxol acetates. Biochem. Biophys. Res. Commun. 1984, 124, 329–
336.
(31) Magri, N. F.; Kingston, D. G. I. Modified taxols, 4. Synthesis and
biological activity of taxols modified in the side chain. J. Nat. Prod.
1988, 51, 298–306.
(32) Kant, J.; Huang, S.; Wong, H.; Fairchild, C.; Vyas, D.; Farina, V.
Studies toward structure-activity relationships of Taxol. Synthesis
and cytotoxicity of Taxol. Analogues with C-20 modified phenyli-
soserine side chains. Bioorg. Med. Chem. Lett. 1993, 3, 2471–2474.
(33) (a) Zhang, S. Z.; Lipsky, M. M.; Trump, B. F.; Hsu, I. C. Neutral
Red (NR) Assay for Cell Viability and Xenobiotic-Induced Cyto-
toxicity in Primary Cultures of Human and Rat Hepatocytes. Cell
Biol. Toxicol. 1990, 6, 219–234. (b) Ciapetti, G.; Granchi, D.; Verri, E.;
Savarino, L.; Cavedagna, D.; Pizzoferrato, A. Application of a combi-
nation of neutral red and amido black staining for rapid, reliable
cytotoxicity testing of biomaterials. Biomaterials 1996, 17, 1259–
1261.
(21) Banner, D. W.; D’Arcy, A.; Chene, C.; Winkler, F. K.; Guha, A.;
Konigsberg, W. H.; Nemerson, Y.; Kirchhofer, D. The crystal
structure of the complex of blood coagulation factor VIIa with
soluble tissue factor. Nature 1996, 380, 41–46.
(34) Akiyama, S.; Fojo, A.; Hanover, J. A.; Pastan , I.; Gottesman,
M. M. Isolation and genetic characterization of human KB cell
lines resistant to multiple drugs . Som. Cell. Mol. Genet. 1985, 11
(2), 117–126.