Design, synthesis and structure-activity relationships of novel taxane-based multidrug resistance reversal agents

Article abstract of DOI:10.1021/jm049483y

A series of novel taxane-based multidrug resistance (MDR) reversal agents (TRAs) has been designed and synthesized. Structure - activity relationship (SAR) study clearly indicates that modification of the C-7 position with hydrophobic arenecarbonylcinnamoyl groups brings about high potency against drug efflux mediated by P-glycoprotein (P-gp). Six TRAs exhibit ability to modulate a wide range of ATP-binding cassette (ABC) transporters, such as P-gp, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), which may serve as novel broad-spectrum modulators of ABC transporters.

Full text of DOI:10.1021/jm049483y

2218
J. Med. Chem. 2005, 48, 2218-2228
Design, Synthesis and Structure-Activity Relationships of Novel Taxane-Based
Multidrug Resistance Reversal Agents
Iwao Ojima,*,† Christopher P. Borella,† Xinyuan Wu,† Pierre-Yves Bounaud,† Cecilia Fumero Oderda,†
Matthew Sturm,† Michael L. Miller,† Subrata Chakravarty,† Jin Chen,† Qing Huang,† Paula Pera,‡
Tracy A. Brooks,‡ Maria R. Baer,‡ and Ralph J. Bernacki‡
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, and
Department of Experimental Therapeutics, Grace Cancer Center, Roswell Park Cancer Institute, Elm and Carlton Streets,
Buffalo, New York 14263
Received June 29, 2004
A series of novel taxane-based multidrug resistance (MDR) reversal agents (TRAs) has been
designed and synthesized. Structure-activity relationship (SAR) study clearly indicates that
modification of the C-7 position with hydrophobic arenecarbonylcinnamoyl groups brings about
high potency against drug efflux mediated by P-glycoprotein (P-gp). Six TRAs exhibit ability
to modulate a wide range of ATP-binding cassette (ABC) transporters, such as P-gp, multidrug
resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), which
may serve as novel broad-spectrum modulators of ABC transporters.
Introduction
FK506).14-18 However, undesirable side effects limited
their use in clinical trials. Thus, potent noncytotoxic
reversal agents with minimum undesirable side effects
are in demand.
Clinical resistance to drugs is a major obstacle in
cancer chemotherapy today. Although there are many
antineoplastic drugs in use, only a few are effective in
the treatment of each specific tumor type because of
intrinsic or acquired drug resistance. A broad-spectrum
resistance to structurally and mechanistically diverse
antitumor agents is known as multidrug resistance
(MDR).1 Classical MDR results from the overexpression
of ATP-binding cassette (ABC) transporters.2 The two
best-known and extensively studied ABC transporters
are P-glycoprotein (P-gp) and multidrug resistance-
associated protein 1 (MRP1).3 A large number of struc-
turally unrelated compounds are known to be P-gp
substrates, only having in common high hydrophobicity,
an amphiphilic nature, and a net positive charge.4
MRP1 also effluxes a broad range of substrates, either
by a glutathione cotransport mechanism or after their
conjugation to glutathione.5,6 P-gp and MRP1 are typi-
cally coexpressed with other ABC transporters such as
breast cancer resistance protein (BCRP).7-11
As soon as P-gp and sister proteins were recognized
to be responsible for MDR, blocking the efflux of drugs
by inhibiting the functions of these transporters became
a realistic way to overcome MDR.12 The use of noncy-
totoxic chemosensitizers (MDR-reversal agents, MDR
modulators) which can block the anticancer drug bind-
ing sites, thus preventing their exclusion from the cells,
has received considerable attention to date. Tsuruo and
co-workers13 were the first to demonstrate the ability
of the calcium channel blocker, verapamil, to reverse
MDR. Since then, many other reversal agents were
brought to light, such as calmodulin antagonists (tri-
fluoperazine), antiarrhythmics (amiodarone), antihy-
pertensive agents (reserpine), antipsychotics (phenothi-
azines), and immunosuppressants (cyclosporine A,
Kobayashi et al.19-22 reported that natural taxanes
isolated from the Japanese yew tree, Taxus cuspidata,
could increase the cellular accumulation of vincristine
in MDR tumor cells as potent as verapamil. This
discovery inspired us to investigate the taxane structure
for the design of new MDR reversal agents. Our
research is based on the derivatization of 10-deacetyl-
baccatin III (DAB) (Figure 1) and 14â-hydroxybaccatin
III (14-OH-DAB). Previous structure-activity relation-
ship (SAR) studies of structurally different classes of
MDR reversal agents pointed out the importance of a
hydrophobic, conjugated, planar ring.23,24 Accordingly,
benzophenone, naphthalene-containing carboxylic acids
and other hydrophobic groups were chosen to modify
the hydroxyl groups of DAB. The SAR study of the
taxane-based MDR reversal agents (TRAs) focused on
two areas: (1) exploring the structural and flexibility
requirements of a key hydrophobic pendant group; (2)
the position of the hydrophobic group.
MDR reversal activity of these taxanes was evaluated
by testing the cytotoxicity of paclitaxel (Taxol) coad-
ministered with a TRA against drug-resistant human
breast cancer cell line MCF7-R or MDA-435/LCC6-
MDR. In our previous study,25-28 the taxanes with
modifications at the C-7 position of DAB proved to be
very active with >95% reversal activity in most cases.
The best activity reached 99.8% recovery of the original
antitumor activity of paclitaxel at 1 µM level of a TRA.25
The majority of the previous studies in our laborato-
ries as well as others have focused on specific MDR
inhibitors, only targeting P-gp, despite the fact that
additional ABC transport proteins contribute to clinical
MDR. It has been shown that P-gp and BCRP are
significantly coexpressed, and P-gp expressed with
MRP-1 greatly increases the efflux of chemotherapeutic
* To whom correspondence should be addressed. Phone (631) 632-
7890; Fax (631) 632-7942; E-mail iojima@notes.cc.sunysb.edu.
† State University of New York at Stony Brook.
‡ Roswell Park Cancer Institute.
10.1021/jm049483y CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/25/2005

Taxane-Based Multidrug Resistance Reversal Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2219
Scheme 1. Preparation of Modified Baccatin Cores31 a
Figure 1. Structure of DAB and paclitaxel.
Figure 2. Design of TRAs.
agents.7-11 Accordingly, the efficacy of broad spectrum
modulation of these transporters by TRAs are worthy
to explore.
A subset of the TRAs was selected for further testing
to check their ability to block the efflux of mitoxantrone
mediated by MRP-1, P-gp and BCRP. Then, six TRAs
were found to be able to modulate all three efflux
pumps, which would serve as broad-spectrum modula-
tors of ABC transporters.
a Reagents and conditions: (a) Ac2O, CeCl3, THF, rt; (b) TESCl,
Imidazole, DMF, 25 °C; (c) Ac2O, DMAP, CH2Cl2, 25 °C; (d) HF-
pyridine, pyridine/CH3CN, 0 °C to 25 °C; (e) TESCl, pyridine/DMF,
25 °C; (f) Cl3COCOOCCl3, pyridine/CH2Cl2, 0 °C; (g) TPAP, NMO,
CH2Cl2, 25 °C.
We describe here a full account of our work on the
design, synthesis and SAR of TRAs.
TES-protection of the C-7 hydroxyl group of DAB,
oxidation of the C-13 hydroxyl group, and acetylation
of the C-10 hydroxyl group, followed by deprotection.
TRA 1-1a to TRA 1-6a were obtained through acyla-
tions at the C-7 position of I-1 with two coupling
methods. One was the preparation of the acyl chloride
of the corresponding acid and subsequent coupling with
the 7-OH of a baccatin in the presence of DMAP. The
other was a direct coupling of the acid with the 7-OH of
a baccatin using EDC and DMAP. Acylations of the C-7
hydroxyl group of I-2 and I-3 were carried out with
appropriate carboxylic acids in the presence of EDC and
DMAP to afford the corresponding TRA 2-1a to TRA
2-14a, TRA 2-1b to TRA 2-5b, TRA 3-1a to TRA 3-13a,
TRA 3-3b, TRA 3-4b, TRA 3-11b, TRA 4-1a, TRA 4-2a,
TRA 4-7a to TRA 4-9a, TRA 4-2b to TRA 4-6b, TRA
4-10b, TRA 9-1a, and TRA 9-2a (“a” and “b” means the
corresponding TRA is derived from DAB and 14-OH-
DAB respectively). TRA 3-14a and I-5, bearing a phenol
moiety in the side chain, were obtained by treating the
silyl-protected intermediates with HF/pyridine. Acyla-
tion of the phenolic hydroxyl groups of TRA 3-14a and
I-5 with different acids led to TRA 4-11a to TRA 4-15a,
and TRA 4-11b to TRA 4-14b. (Scheme 2).
Results and Discussion
Rational Design. Our design is based on the deriva-
tization of DAB or 14-OH-DAB, which provides the
crucial component of paclitaxel and taxoids, but is
noncytotoxic by itself. These naturally occurring com-
plex diterpenes possess several hydroxyl groups that can
easily be modified with hydrophobic side chains by
esterification (Figure 2). Benzophenone, naphthalene-
containing carboxylic acids, and other hydrophobic
groups were selected to modify C-2, C-7, C-10, and/or
C-13 positions of either DAB or 14-OH-DAB. The
structural requirements for the hydrophobic modifiers
and the positional preference have been investigated in
our SAR study.
Chemical Synthesis. Baccatin III (I-1), 13-Ac-bac-
catin III (I-2), 13-Ac-1,14-CO-baccatin III (I-3), and 13-
oxo baccatin III (I-4) were prepared using previously
established procedures (Scheme 1).29 Baccatin III (I-1)
was synthesized through acetylation of the C-10 hy-
droxyl group of DAB.30,31 13-Ac-baccatin III (I-2) was
prepared by the selective TES-protection of the C-7
hydroxyl group of DAB and acetylation of the C-10 and
C-13 hydroxyl groups, followed by deprotection of the
C-7 silyl ether.30 13-Ac-1,14-CO-baccatin III (I-3) was
prepared from 14-OH-DAB through the selective TES-
protection of the C-7 hydroxyl group, carbonate forma-
tion between the C-1 and C-14 hydroxyl groups, acety-
lation of the C-10 and C-13 hydroxyl groups, and
subsequent deprotection of the C-7 silyl ether. 13-Oxo-
baccatin III (I-4)32 was obtained through the selective
Syntheses of C-10 modified TRAs are shown in
Scheme 3. Preparation of TRA 5-1a started from 7-TES-
DAB I-6,33 and the C-10 modification was carried out
with the use of N-(4-benzoylcinnamoyloxy)succinimide.
Subsequent removal of the TES group with HF/pyridine
afforded TRA 5-1a. For the syntheses of other C-10
modified TRAs, 7,13-diacetyl-10-DAB (I-7) was used.

2220 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Scheme 2. Synthesis of 7-Modified TRAsa
Ojima et al.
a Reagents and conditions: (a) RCO2H, EDC, DMAP, CH2Cl2, 25 °C; (b) RCOCl, Et3N, DMAP, CH2Cl2, 25 °C; (c) HF-pyridine, pyridine/
CH3CN, 0 °C to 25 °C.
Scheme 3. Synthesis of 10-Modified TRAsa
a Reagents and conditions: (a) TESCl, pyridine, room temperature; (b) N-hydroxysuccinimide ester of RCO2H, LiHMDS, THF, -40 °C;
(c) HF-pyridine, pyridine/CH3CN, 0 °C to room temperature; (d) CF3C(OTMS)NTMS, THF, 0 °C; (e) Ac2O, DMAP, CH2Cl2, 25 °C; (f)
0.1N HCl, EtOH, 25 °C; (g) RCO2H, EDC, DMAP, CH2Cl2, 25 °C.
Syntheses started from the selective protection of DAB
at C-10, followed by acetylation at C-7 and C-13.
Deprotection of C-10 and coupling with the correspond-
ing acid afforded the desired TRA 5-2a to TRA 5-6a
(Scheme 3).
C-7,C-10 dimodified TRAs were also prepared using
previously established procedures.33,34 Starting from
7-TES-DAB I-6, the C-10 position was modified by
reacting with the corresponding succinimide esters.
Removal of the 7-TES group with HF/pyridine, followed

Taxane-Based Multidrug Resistance Reversal Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2221
Scheme 4. Synthesis of 7,10-Modified and 7,10,13-Modified TRAsa
a Reagents and conditions: (a) N-hydroxysuccinimide ester of RCOOH, LiHMDS, THF, -40 °C; (b) HF-pyridine, pyridine/CH3CN, 0
°C to room temperature; (c) RCOOH, DCC, DMAP, CH2Cl2, room temperature; (d) RCOCl, LiHMDS, THF, -40 °C; (e) Ac2O, DMAP,
CH2Cl2, 25 °C; (f) p-benzoylcinnamic acid, EDC, DMAP, CH2Cl2, 25 °C.
Scheme 5. Synthesis of 13-Modified TRAsa
Scheme 6. Synthesis of 2-Modified TRAsa
a Reagents and conditions: (a) RCOOH, DCC, DMAP, CH2Cl2,
room temperature; (b) Zn, AcOH/MeOH, 70 °C.
by coupling with the corresponding acids, provided TRA
6-1a to TRA 6-3a. TRA 7-7a was synthesized in the
same manner with the third modifier at the C-13
position. TRAs 6-4a to TRA 6-9a were prepared in
parallel using a slightly different method. Selective
protection of the C-7 hydroxyl group was followed by
acylation of the C-10 hydroxyl group with the corre-
sponding acid chloride. Acetylation of the C-13 alcohol
and deprotection of the silyl ether gave alcohols I-8 to
I-13. Coupling of the resulting alcohols with p-benzoyl-
cinnamic acid in the presence of EDC and DMAP
yielded C-10 modified TRA 6-4a to TRA 6-9a (Scheme
4).
Coupling of the appropriate pendant groups with 7,
10-diTroc-DAB afforded 13 modified TRA 7-2a, TRA
7-4a, and TRA 7-6a (Scheme 5). Further deprotection
of the Troc groups was carried out using zinc in AcOH/
MeOH at 70 °C to produce TRA 7-1a, TRA 7-3a, and
TRA 7-5a.
a Reagents and conditions: (a) TESCl, imidazole, DMF, room
temperature; (b) Red-Al, THF, 0 °C; (c) RCOOH, DCC, DMAP,
CH2Cl2, room temperature; (d) HF-pyridine, pyridine/CH3CN, 0
°C to room temperature; (e) Ac2O, DMAP, CH2Cl2, room temper-
ature.
Hydrogenation of the C-2 benzoate of 13-Ac-baccatin
III (I-2) over Pt/C catalyst followed by acylation of the
C-7 alcohol with p-benzoylcinnamic acid afforded C-2,C-7
modified TRA 8-4a (Scheme 8).
Acylation of the C-7 hydroxyl of 13-oxo baccatin III
(I-4) with p-benzoylcinnamic acid in the presence of
EDC and DMAP gave 7-modified-13-keto analogue, TRA
8-5a (Scheme 9).
Evaluation of Biological Activities. MDR-Rever-
sal Activity in Combination with Paclitaxel. For
the assessment of MDR-reversal activity of these TRAs,
the cytotoxicity of paclitaxel in combination with a TRA
is evaluated against the resistant human breast tumor
cell lines MCF7-R and/or MDA-435/LCC6-MDR. We
have found that all TRAs, except for the ones bearing a
side chain at the C-13 position, possess very weak
cytotoxicities (i.e., IC50 > 10 µM) or are noncytotoxic up
to the solubility limit (ca. 30 µM).
MDR-reversal activity of the six TRAs, TRA 1-1a to
TRA 1-6a, bearing different side chains at C-7 and a
free hydroxyl group at C-13, is listed in Table 1. Four
of them, TRA 1-1a to TRA 1-4a, show high level of
activity in the range of 96-99.8% at 1-3 µM concentra-
tion. These results clearly indicate that the modification
To prepare C-2 modified analogues, the hydroxyl
groups at the C-7, C-10, and C-13 positions were
suitably protected with TES groups (Scheme 6). Deben-
zoylation at C-2 using Red-Al proceeded smoothly to
afford 2-debenzoyl-triTES-DAB (I-14)35 which was then
coupled with acids in the presence of DCC and DMAP.
Finally, the TES groups were replaced by acetyl groups
in two steps to give TRA 8-1a and TRA 8-2a.
Acylation of the C-2 hydroxyl group with m-anisic acid
followed by deprotection and selective protection of the
resulting C-7 alcohol yielded C-2 modified baccatin I-15.
Acetylation of the C-10 and C-13 positions, removal of
the silyl group, and acylation of the resulting C-7 alcohol
with p-benzoylcinnamic acid in the presence of EDC and
DMAP yielded the C-2,C-7 modified TRA 8-3a (Scheme
7).

2222 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Scheme 7. Synthesis of 2,7-Modified TRA 8-3aa
Ojima et al.
Table 1. MDR-reversal Activity of TRAs Bearing a
Hydrophobic Moiety at C-7 and a Free Hydroxyl Group at the
C-13
a Reagents and conditions: (a) m-anisic acid, DIC, DMAP,
CH2Cl2, 37 °C; (b) HF-pyridine, pyridine/CH3CN, 0 °C to 25 °C;
(c) TESCl, imidazole, DMF, 25 °C; (d) Ac2O, DMAP, CH2Cl2, 25
°C; (e) HF-pyridine, pyridine/CH3CN, 0 °C to 25 °C; (f) p-
benzoylcinnamic acid, EDC, DMAP, CH2Cl2, 25 °C.
Scheme 8. Synthesis of 2,7-Modified TRA 8-4aa
a % Reduction ) [1 - {IC50(0.1 µM reversal agent + paclitaxel)/
IC50(paclitaxel)}] × 100. For example, for TRA 1-1a, in MCF7-R
cell line, IC50(3 µM reversal agent + paclitaxel) ) 1.6 nM,
IC50(paclitaxel) ) 860 nM, % reduction ) [1-(1.6/860)] × 100 )
99.8%. b See ref 36.
While the removal of the ethylene linker (TRA 2-2a and
TRA 2-2b) was well tolerated, shortening the linker by
one carbon (TRA 2-3a and TRA 2-3b), or increasing the
chain length (TRA 2-4a, TRA 2-5a, TRA 2-4b, and TRA
2-5b), resulted in severely muted reversal activity. Para
substituents on the benzophenone moiety were gener-
ally well tolerated (TRA 2-6a, TRA 2-10a, TRA 2-11a,
TRA 2-13a, and TRA 2-14a), resulting in retained or
slightly increased activities. Formation of a C-C bond
between the two ortho positions of the benzophenone
(TRA 2-9a) was well tolerated, and the insertion of a
carbonyl linker and reduction of the double bond (TRA
2-8a) resulted in loss of activity. Replacement of the
benzophenone carbonyl with an alkene resulted in
muted activity (TRA 2-12a). A cyclic carbonate func-
tionality at the C-1 and C-14 positions of the baccatin
does not seem to change the activity [compare (TRA 2-1b
to TRA 2-5b) with (TRA 2-1a to TRA 2-5a)].
MDR-reversal activity of TRAs bearing a variety of
hydrophobic side chains at the C-7 position is sum-
marized in Table 3. TRA 3-11a bearing a naphthalene
moiety at the C-7 position exhibited very high activity
against MCF7-R cell line, while 14-OH-DAB analogue
(TRA 3-11b) gave fairly good activity against MDA-435/
LCC6-MDR cell line. The reduction of the ketone moiety
to a racemic alcohol and saturation of the double bond
(TRA 3-1a) resulted in about a 50% loss in activity.
Further reduction of the alcohol to an alkane (TRA 3-2a)
drastically reduced activity. Replacing the benzophe-
none moiety with a diphenyl ether (TRA 3-3a and TRA
3-3b) retained activity, while replacement with a thio-
ether (TRA 3-4a and TRA 3-4b) reduced activity.
Replacement of the benzophenone with an indole ring
(TRA 3-8a) or a naphthyl ether (TRA 3-12a) was well
tolerated, but replacement with a C-1 naphthyl ether
(TRA 3-13a) resulted in muted activity.
a Reagents and conditions: (a) Pt/C, H2, EtOAc, 25 °C; (b)
p-benzoylcinnamic acid, EDC, DMAP, CH2Cl2, 25 °C.
Scheme 9. Synthesis of 13-Oxo-TRA 8-5aa
a Reagents and conditions: (a) p-benzoylcinnamic acid, EDC,
DMAP, CH2Cl2, 25 °C.
of C-7 position is suitable for strong MDR reversal
activity. Benzophenone and naphthalene side chains are
appropriate pendant groups, and the former appears to
be slightly better than the latter. Shortening the side
chain by two carbons does not have influence on the
reversal activity (TRA 1-4a compared with TRA 1-3a).
However, increasing the hydrophilicity of the TRA by
introducing a glycine linker (TRA 1-5a, 42%), or reduc-
ing the aromatic groups to saturated cycloalkyl groups
(TRA 1-6a, 81%), results in dramatic or considerable
reduction of activity. This implies the need of a conju-
gated planar ring structure in the pharmacophore of
TRAs.
Further evaluation of the TRAs containing a ben-
zophenone moiety in the C-7 side chain is summarized
in Table 2. TRA 2-1a gave 99.7% reversal activity,
similar to TRA 1-1a. This means that the C-13 acety-
lation does not affect MDR-reversal activity. The length
of the linker plays an important role in reversal activity.

Taxane-Based Multidrug Resistance Reversal Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2223
Table 2. MDR-Reversal Activity of TRAs Bearing a
Benzophenone Moiety with a Variable Chain Length at the C-7
Position
Table 3. MDR-Reversal Activity of TRAs Bearing Various
Hydrophobic Pendant Groups at the C-7 Position
a % Reduction ) [1 - {IC50(0.1 µM reversal agent + paclitaxel)/
IC50(paclitaxel)}] × 100. c Taxanes derived from 14-OH DAB. d See
ref 36.
a % Reduction ) [1 - {IC50(0.1 µM reversal agent + paclitaxel)/
IC50(paclitaxel)}] × 100. c Taxanes derived from 14-OH DAB. d See
ref 36.
ments for good activity. Introduction of a heteroaromatic
group (TRA 4-5b, TRA 4-6b, and TRA 4-10b) gave
significantly reduced activities, in all cases as expected.
This can be abscribed to a reduction of hydrophobic
nature of the group. Introduction of a hydroxyl group
to the benzoyloxy moiety (TRA 4-15a) led to a significant
loss in activity, so as that of a methoxy substituent (TRA
4-12a to TRA 4-14a, TRA 4-12b to TRA 4-14b).
As Table 5 shows, modification of the C-10 hydroxyl
group with a benzophenone side chain (TRA 5-1a)
results in a very good reversal activity (95%). However,
in all other instances, the attachment of a hydrophobic
side chain containing diphenyl ether, diphenyl thio-
ether, benzamide, benzoate to the C-10 position (TRA
5-2a to TRA 5-6a) resulted in significant loss of activity.
As Table 4 illustrates, replacement of the benzoyl
group with a benzamide (TRA 4-1a) is well tolerated,
and the corresponding ester isosteres (TRA 4-11a, TRA
4-11b) also retain high levels of potency. Reversing the
ester orientation, however, resulted in decreased activity
for the TRA derived from DAB (TRA 4-2a), while the
corresponding TRA based on 14-OH DAB (TRA 4-2b)
retained activity. Introduction of para-substituted ben-
zoyloxy substituents (both electron donating and with-
drawing) (TRA 4-3b, TRA 4-4b, TRA 4-14a, TRA 4-14b,
and TRA 4-15a) resulted in drastically reduced activi-
ties, indicating that there are definite steric require-

2224 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Ojima et al.
Table 4. MDR-Reversal Activitya of TRAs Bearing Aromatic or
Heteroaromatic Benzoate or Benzamide Moiety at the C-7
Position
Table 5. MDR-Reversal Activity of TRAs Bearing a
Hydrophobic Moiety at the C-10 Position
a % Reduction ) [1 - {IC50(0.1 µM reversal agent + paclitaxel)/
IC50(paclitaxel)}] × 100.
Table 6. MDR-reversal Activity of TRAs Bearing Various
Pendant Groups at the C-7 and C-10 Positions
a MDA-435/LCC6-MDR cell line was used. b % Reduction )
[1 - {IC50(0.1 µM reversal agent + paclitaxel)/IC50(paclitaxel)}]
× 100. c Taxanes derived from 14-OH DAB. d See ref 36.
As Table 6 indicates, the effects of introducing two
hydrophobic side chains at the C-7 and C-10 positions
on the reversal activity are more complicated. An
activity ranging from 0% (TRA 6-2a, Table 6) to 99.7%
(TRA 6-1a) is observed depending on the substitution
pattern. TRA 6-1a with modification at both C-7 and
C-10 positions with benzophenone moiety proved to be
very active (99.7%). Replacement of the C-10 benzophe-
none moiety with either a methyl formate (TRA 6-8a)
or a propanoyl group (TRA 6-9a) was well tolerated,
resulting in only minimal loss of activity. However,
replacement with larger aromatic substituents (TRA
6-4a to TRA 6-7a), or replacement of the C-7 benzophe-
none side chain with a naphthalene moiety (TRA 6-2a
and TRA 6-3a), resulted in significant loss of activity.
Thus, hydrophobicity is not the only requirement for an
efficient MDR reversal activity. These results strongly
suggest that there is a specific binding site for TRAs on
P-gp that has rather strict steric/shape requirements.
a % Reduction ) [1 - {IC50(0.1 µM reversal agent + paclitaxel)/
IC50(paclitaxel)}] × 100.
Table 7 shows the activity of TRAs bearing a hydro-
phobic moiety at the C-13 position. In general, C13-
modified TRAs showed little activity except for TRA 7-2a
and TRA 7-3a, which exhibited relatively strong rever-
sal activity (80% and 88%, respectively). Additionally,
C7,C10,C13-trisubstituted TRA 7-7a (Figure 3) showed
no activity.
Modification of the C-2 position with a benzophenone
or a naphthalene side chain also resulted in very little

Taxane-Based Multidrug Resistance Reversal Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2225
Table 7. MDR-reversal Activitya of TRAs Bearing a
Hydrophobic Moiety at the C-13 Position
Figure 4. Structure of TRA 8-5a.
is not appropriate for achieving good MDR reversal
activity.
We should add that besides paclitaxel, the sensitiza-
tion of MCF7-R cells by the TRAs was also observed
with another commonly used anthracycline-class anti-
cancer agent, doxorubicin. TRA 1-2a exhibited 92%
MDR reversal activity (at 1 µM concentration) when
coadministered with doxorubicin.25
As mentioned above, we hypothesized that TRAs
would block the efflux pump of P-gp and other ABC
transporters, thereby restoring the drug sensitivity in
the MDR-expressing cancer cells by allowing cytotoxic
drugs to accumulate in the cancer cells. To verify this
hypothetical mechanism, we investigated the effects of
TRA 1-2a on the intracellular accumulation of paclitaxel
in the drug-resistant cancer cell MCF7-R. As a control
experiment, we also looked at the effects of TRA 1-2a
on the concentration of paclitaxel in the parent drug
sensitive cancer cells MCF7. As Figure 5 shows, TRA
1-2a does not have any appreciable effects on the
accumulation of paclitaxel in the drug sensitive cells.
In sharp contrast to this, the addition of TRA 1-2a (1
µM) dramatically increased the accumulation of pacli-
taxel in the drug resistant cells although only a very
low level of paclitaxel is detected without TRA 1-2a.
These results strongly support that TRAs block the P-gp
efflux, allowing the anticancer drug to accumulate in
the drug-resistant cancer cells and exert its chemo-
therapeutic effect.
a MCF7-R cell line was used. b % Reduction ) [1 - {IC50(0.1
µM reversal agent + paclitaxel)/IC50(paclitaxel)}] × 100.
Figure 3. Structure of TRA 7-7a.
Table 8. MDR-reversal Activitya of TRAs Bearing Hydrophobic
Moieties at the C-2 or C-7 and C-2 Positions
Broad Spectrum Modulation of ABC Transport
Proteins by TRAs and Mitoxantrone.36,37 Since the
majority of previous studies on MDR-reversal agents
have only focused on P-gp modulators, clinical trials of
the known MDR-reversal agents have been disappoint-
ing to date.38-42 The true measure of an effective MDR-
reversal agent is its ability to modulate drug efflux
mediated by a broad spectrum of ABC transporters.
Recently, Okumura et al.43 found that a newly synthe-
sized taxoid, 5-O-benzoylated taxinine K (BTK), has the
ability to reverse P-gp- and MRP-mediated multidrug
resistance. Our group also examined the broad-spectrum
modulatory activity of TRAs synthesized in our labora-
tories. Among the large number of TRAs synthesized,
the best 20 compounds in terms of their MDR reversal
activity in combination with paclitaxel against P-gp-
overexpressing MDA435/LCC6-MDR cell lines were
chosen for further testing to check their ability to block
the efflux of mitoxantrone mediated by MRP-1, P-gp,
and BCRP.36 Cell lines used for this study were drug-
resistant human myelogenous HL60-ADR (overexpress-
es MRP-1), drug-resistant human myeloma line 8226-
Dox6 (overexpresses P-gp), and 8226-MR20 (over-
expresses BCRP). The bioassay results showed that four
TRAs were able to modulate all three efflux pumps (TRA
2-10a, TRA 2-11a, TRA 4-1b, TRA 2-2a). Based on these
results, two more new TRAs, TRA 9-1a and TRA 9-2a,
a MDA-435/LCC6-MDR cell line was used. b % Reduction ) [1
- {IC50 (0.1 µM reversal agent + paclitaxel)/IC50(paclitaxel)}] ×
100. c See ref 36.
activity (TRA 8-1a and TRA 8-2a) as shown in Table 8.
When the C-7 hydroxyl group was substituted by an
arenecarbonylcinnamoyl group, replacement of the C-2
benzoyl group with a m-methoxybenzoyl group (TRA
8-3a) resulted in very good activity (Table 8). Replace-
ment with a cyclohexanoyl group (TRA 8-4a) instead of
a benzoyl group was tolerated, causing only a minor loss
of activity. Oxidation of the C-13 position to a ketone
(TRA 8-5a, Figure 4) resulted in a significant loss in
activity (37% reduction at 0.1 µM against MDA-435/
LCC6-MDR cell line). These results indicate that a C-2
modification with a bulky group or a C-13 modification

2226 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Ojima et al.
Figure 5. Modulation of tritiated paclitaxel accumulation by MDR reversal TRA 1-2a in MCF7 cells, resistant (a) and sensitive
(b). Square, 10 nM of paclitaxel; triangle, 10 nM of paclitaxel and 1 µM of TRA 1-2a.
Figure 7. Effect of TRAs on drug efflux in resistant cell
lines.36 D-values represent the separation of the distribution
histograms for uptake in RPMI 1640 + 3 µM mitoxantrone
and uptake in RPMI 1640 + 3 µM mitoxantrone + 10 µM TRA
in Kolmogorov-Smirnov (KS) statistics. Screening of TRAs for
modulation of mitoxantrone efflux mediated by Pgp (8226-
Dox6), MRP-1 (HL60-ADR) and BCRP (8226-MR20) is dem-
onstrated. D-value g 0.20 indicates modulatory activity. The
graph shows the mean values from triplicate experiments, with
standard errors.
leading broad-spectrum modulator with the highest
reversal activity in all three cell lines. TRA 9-2a is the
Figure 6. TRAs that modulate all three efflux pumps.
next active compound, which is less active than TRA
2-10a against HL60-ADR cell line, overexpressing MRP-
1, but comparable against the P-gp- and BCRP-overex-
pressing cell lines.
were also synthesized as analogues of TRA 2-10a and
TRA 2-11a, which were the best two TRAs so far, and
evaluated for their broad-spectrum modulatory activi-
ties. The structures of these six TRAs, TRA 2-10a, TRA
9-1a, TRA 2-11a, TRA 9-2a, TRA 4-1b and TRA 2-2a,
are shown in Figure 6, and their broad-spectrum
modulatory activities are illustrated in Figure 7.
The ability of these TRAs to increase the uptake of
mitoxantrone was examined. At 10 µM, all six TRAs
exerted significant effects on increasing the amount of
mitoxantrone taken up into the cancer cells overex-
pressing either P-gp or BCRP. However, MRP-1 was
found to be rather difficult to modulate efficiently
(Figure 7).36
The ability of TRAs to enhance mitoxantrone cyto-
toxicity was also studied. Cells were treated either with
mitoxantrone alone or in combination with each of the
four TRAs, TRA 2-10a, TRA 2-11a, TRA 4-1b, and TRA
2-2a, at different doses (0.1 µM, 1 µM, 10 µM). Figure 8
shows the effects of TRAs on mitoxantrone cytotoxicity
for 96 h continuous exposure to 8226-Dox6 (A), HL60-
ADR (B), and 8226-MR20 (C) cell lines.
All four TRAs effectively enhanced cytotoxicity of
mitoxantrone in the P-gp-overexpressing and BCRP-
overexpressing cell lines. However, the effect on the
MRP-1-overexpressing cell line was more limited. Nev-
ertheless, a significant improvement in the IC50 was still
observed with TRA 2-10a and TRA 2-11a at the con-
Among the six TRAs that possess broad-spectrum
reversal activity, demonstrating modulation of P-gp,
MRP-1, and BCRP, TRA 2-10a has been selected as the

Taxane-Based Multidrug Resistance Reversal Agents
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 2227
Figure 8. Effect of TRAs on mitoxantrone cytotoxicity 96 h continuous exposure to cell lines A: 8226-Dox6 (overexpresses P-gp);
B: HL60-ADR (overexpresses MRP 1); C: 8226-MR20 (overexpresses BCRP).
centration of 10 µM. The best result for the reduction
of the IC50 of mitoxantrone against HL60-ADR (MRP-
1+) cell line was observed with TRA 2-10a at 10 µM
concentration (lowering the IC50 by 40% to 73 ( 9 nM).
Comparison of activity at 1 µM concentration also shows
that TRA 2-10a is the most effective agent against 8226-
Dox6 (P-gp+) cell line, lowering the IC50 by 87% to 0.27
( 0.04 nM, and 8226-MR20 (BCRP+) cell line, lowering
the IC50 by 96% to 5.02 ( 0.5 nM. TRA 2-11a is also
very effective against these three cell lines.
fectively modulate mitoxantrone efflux from drug-
resistant cancer cell lines overexpressing P-gp, MDR-
1, and BCRP. Among these, TRA 2-10a exhibited the
best activity as broad-spectrum modulator and has been
identified as the lead drug candidate for further phar-
macological evaluations directed toward clinical applica-
tions.
Acknowledgment. This research was supported by
grants from the National Institutes of Health (GM417980
and CA103314 to I.O. and CA13038 to R. J.B.). Gener-
ous support from Indena, SpA (to I.O.) is also gratefully
acknowledged.
Conclusion
Libraries of novel taxane-based MDR reversal agents
(TRAs) were designed, synthesized, and evaluated for
their modulating ability against P-glycoprotein overex-
pressed in the drug-resistant cancer cell lines, MCF7-R
and MDA-435/LCC6-MDR. These TRAs exhibited MDR-
reversal activities up to 99.8% when coadministered
with paclitaxel, acting as efficient sensitizing agents.
These TRAs are noncytotoxic and can potentially revi-
talize known and well-studied anticancer agents toward
the treatment of resistant tumors.
The SAR studies of TRAs indicate that the C-7
position of the baccatin skeleton is the most effective
position for introducing hydrophobic pendant group as
compared to the C-10, C-13, and C-2 positions. Among
the hydrophobic pendant groups designed and exam-
ined, the one consisting of two aromatic rings, spaced
by a 1- or 2-atom linker and bearing a carbonyl or ether
group, was identified as the highly effective unit struc-
ture. For example, an arenecarbonylcinnamoyl group is
found to be the best pendant group so far to date. While
the P-gp transporter is known for its ability to nonse-
lectively eliminate hydrophobic contaminants from cells,
our SAR study suggests the presence of a discrete
recognition site of TRAs in the P-gp system and a strict
steric requirement for the TRAs to be effective.
Supporting Information Available: Synthetic proce-
dures and characterization data for all TRAs and intermedi-
ates; procedures for biological evaluations. This material is
available free of charge via the Internet at http://pubs.acs.org.
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