Structure-activity relationships of some taxoids as multidrug resistance modulator

Article abstract of DOI:10.1016/j.bmcl.2006.09.068

1,7-Deoxy-4-deacetylbaccatin III (12) and its five analogues 6-9, 13, and their oxetane ring opened derivatives 14, 16, and 17, which were synthesized from taxinine, showed significant activity as MDR reversal agent by the assay of the calcein accumulatio

Full text of DOI:10.1016/j.bmcl.2006.09.068

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1122–1126
Structure–activity relationships of some taxoids as
multidrug resistance modulator
a
a
a
a
Toshiaki Hasegawa,a,c, Jao Bai, Shujun Zhang, Jinlan Wang, Junichi Matsubara,
Junichi Kawakami,b Akihiro Tomida,d Takashi Tsuruo,d Katsutoshi Hirose,e
Junichi Sakai,b Midori Kikuchi,c Mariko Abec and Masayoshi Andob,*
*
aGraduate School of Science and Technology, Niigata University, Ikarashi, 2-8050, Niigata 950-2181, Japan
bDepartment of Chemistry and Chemical Engineering, Faculty of Engineering, Niigata University, Ikarashi, 2-8050,
Niigata 950-2181, Japan
cMitsubishi Gas Chemical Company Inc., Niigata Research Laboratory, 182, Shinwari, Tayuhama, Niigata 950-3112, Japan
dCancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan
eKobe Natural Products and Chemicals Co., Ltd., 527 Nisikawanobe, Ichikawa-Cho, Kanzaki-Gun, Hyogo 679-2315, Japan
Received 1 June 2006; revised 21 September 2006; accepted 23 September 2006
Available online 18 January 2007
Abstract—1,7-Deoxy-4-deacetylbaccatin III (12) and its five analogues 6–9, 13, and their oxetane ring opened derivatives 14, 16, and
17, which were synthesized from taxinine, showed significant activity as MDR reversal agent by the assay of the calcein accumula-
tion toward MDR human ovarian cancer 2780AD cells. The most effective compound 12 in this assay is actually efficient for the
recovery of cytotoxic activity of paclitaxel (taxolÒ), adriamycin (ADM), and vincristine (VCR) toward MDR 2780AD cells at
the same level toward parental 2780 cells. This activity of 12 is very interesting because baccatin III (4) has no such MDR reversal
activity but has cytotoxic activity. The essential functional groups inducing such a difference in biological activity between 4 and 12
are 4a-acetoxyl for 4 and 4a-hydroxyl for 12. In seven compounds possessing MDR reversal activity, compound 12 is the most desir-
able compound for anti-MDR cancer reversal agent, because it has the highest accumulation ability of anticancer agent in MDR
cancer cells and weak cytotoxic activity. Compounds 8 and 13 showed significant cytotoxic activity toward HepG2 and VA-13,
respectively, as well as MDR reversal activity. They are expected to become lead compounds for new types of anticancer agent
or anti-MDR cancer agent.
Ó 2006 Elsevier Ltd. All rights reserved.
In cancer chemotherapy, occurrence of multidrug resis-
tance (MDR) in cancer cells caused by repeated admin-
istration of anticancer agents is a serious problem. One
mechanism of MDR is overexpression of the P-glyco-
protein (P-gp), which is the efflux pump of anticancer
agent.1,2 P-gp is a transporter for a wide range of re-
agents utilizing energy by hydrolysis of ATP. P-gp is ex-
pressed on the small intestine, capillary of brain, kidney,
and liver. Its physiological role is considered as a defense
mechanism against the toxic materials in the cell. When
P-gp is expressed on the cell membrane of a cancer cell,
it transports various kinds of anticancer agents from in-
side of the cell to the outside. Many taxane derivatives,
which showed MDR reversal activity, have been report-
ed.3–11 We have previously reported the isolation of
MDR reversal agents from Taxus caspidata,12 their pro-
duction by callus culture,13 and the synthesis of taxinine
NN-1, the most efficient MDR reversal agents in natural
taxane.12a,14 (Fig. 1).
Structure–activity relationship (SAR) studies of the
functional groups of taxanes revealed that substitutions
of the OH group at the C-7 position to hydrophobic side
groups are effective for MDR reversal activity.15
Many taxane derivatives for SAR studies were synthe-
sized from 10-deacetyl-baccatin III or 14b-hydroxybacc-
atin III with an acetoxy group at the C-4 position.7,8,10,11
It has been reported that deacetylation at the C-4 posi-
tion results in the loss of cytotoxicity in pacritaxel.16
Keywords: Taxoid; Taxinine; 1,7-Deoxy-4-deacetylbaccatin III; MDR;
Cytotoxicity.
*
Corresponding authors. Tel.: +81 25 259 8226; fax: +81 25 259 8218
(T.H.), Tel./fax: +81 25 262 7326 (M.A.); e-mail addresses: toshiaki-
hasegawa@mgc.co.jp; mando@eng.niigata-u.ac.jp
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.068

T. Hasegawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1122–1126
1123
MDR human ovarian cancer 2780AD cells were exam-
ined (Table 1).
Baccatin III (4) and 10-deacetyl baccatin III (3) showed
no activity toward calcein accumulation in MDR
2780AD cells. Among the compounds possessing an
oxetane ring, compounds 6–8 with a benzoyloxy group
at C-2 and an acetoxy group at C-9 and/or C-10 showed
significant effect on the calcein accumulation and the
strength of their activities is almost the same. However
the corresponding compound 5 with hydroxyl groups
at both C-9 and C-10 showed weaker activity than those
of 6–8. These results suggested that compound 5 pos-
sessing hydroxyl groups at both C-9 and C-10 positions
decreased the activity of the cellular accumulation of
calcein in MDR 2780AD cells.
Compound 9 with a 13-carbonyl, 5b,20-epoxy ring (4,5-
oxetane ring), and three acetoxyl groups at C-2, C-9,
and C-10 showed significant activity toward calcein
accumulation in MDR 2780AD cells. On the other
hand, the corresponding 13a- and 13b-hydroxyl deriva-
tives, 10 and 11, the reduction products of the carbonyl
group at C-13 of 9, lost the activity.
Figure 1.
From the viewpoint of MDR reversal agent, it is desir-
able that the compound has no cytotoxicity and has high
MDR reversal activity at the time when it is used with
an anticancer reagent toward MDR cancer cells.
The structure change of 6 to 13 is the hydroxyl group at
C-9 of 6 to a carbonyl group of 13. Since the activity of 6
and 13 is almost the same, the effect of carbonyl and
hydroxyl groups at C-9 showed the same efficiency.
Compound 12 with structure change of the 13-carbonyl
group of 13 to a 13a-hydroxyl group showed further
stronger activity. These results showed that the 13a-hy-
droxyl group is more efficient than the 13-carbonyl
group in this case. The structure–activity relationship
of compounds 9–13 probably indicated that the exis-
We synthesized some taxane derivatives with a 5b,20-ep-
oxy ring (4,5-oxetane ring) and with hydroxyl group at
the C-4 position from taxinine (1) by the method shown
in Scheme 1. The effects of 15 kinds of taxane deriva-
tives, 3–17, on the cellular accumulation of calcein in
Scheme 1.

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T. Hasegawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1122–1126
Table 1. Effects of compounds on the accumulation of calcein in 2780AD cells, and cytotoxicities of compounds against WI-38, VA-13, and HepG2
cells
Calcein accumulationa,b (% control)
Cytotoxicity IC50 (lg/ml)
c
Compound
0.25lg/ml
2.5lg/ml
25lg/ml
WI-38
VA-13
HepG2
Verapamil
99
—
103
—
140
—
—
0.034
—
—
0.0043
—
—
2
3
6.90
—
95
93
90
84
93
81
4
16.1
>100
42.3
0.67
0.82
77.0
8.39
76.2
54.2
7.38
57.4
85.3
>100
68.1
0.84
>100
>100
67.0
51.9
>100
81.5
>100
77.0
7.93
59.6
9.73
8.47
82.3
9.91
67.7
77.7
43.2
7.32
>100
>100
>100
69.9
30.1
33.4
>100
>100
2.14
5
105
99
110
102
118
119
120
95
106
140
134
135
123
95
6
7
115
97
8
9
115
94
10
11
12
13
14
15
16
17
82
96
95
96
129
120
114
94
163
137
112
88
101
88
79
88
97
116
126
145
88
a The amount of calcein accumulated in multidrug-resistant human ovarian cancer 2780AD cells was determined compared with the control in the
presence of 0.25, 2.5, and 25 lg/ml of test compounds and verapamil (positive control).
b The values are the relative amount of calcein accumulated in the cell compared with the control experiment. The values represent the mean of
triplicate determination.
c IC50 represents the mean of duplicate determination.
tence of one carbonyl group at C-13 or C-9 is desirable
for the expression of the activity.
In 15 taxane derivatives tested in this research, 10 com-
pounds showed moderate to strong activity on calcein
accumulation in MDR 2780AD cells. These results
seemed to indicate that the taxane skeleton has special
meaning toward the MDR reversal activity although
the strength of activity depends on the combination of
the functional groups on the taxane skeleton.
Compound 12 showed the strongest activity among the
compounds tested but 4 had no activity for calcein accu-
mulation in MDR 2780AD cells. The structure changes
from 4 to 12 occur at the C-1, C-4, and C-7 positions.
Two hydroxyl groups at C-1 and C-7 of 4 were lost in
12 and the acetoxyl group at C-4 of 4 was displaced
by a hydroxyl group in 12. These changes of functional
groups of 4 to 12 enhanced remarkably the accumula-
tion of calcein in MDR 2780AD cells. Compound 14
We tested the effect of the compound 12 on the cytotox-
icity of taxol, adriamycin (ADM), and vincristine (VCR)
toward MDR 2780AD cells and its parental cells
(A2780) comparing with verapamil, which is a well-
known MDR reversal agent. The IC50 values of taxol
for A2780 and MDR 2780AD cells were 0.7 and
535 nM, respectively (Table 2). When compound 12
was added at a final concentration of 0.2, 2.0, and
10 lM, the IC50 values of taxol for MDR 2780AD cells
were shifted to 375, 10, and 1 nM, respectively. The
enhancing effect of 12 for taxol was comparable to that
of verapamil. Compound 12 is also effective for ADM
and VCR. The IC50 values of ADM for A2780 and
MDR 2780AD cells were 13 and 909 nM, respectively.
The IC50 values for MDR 2780AD cells were shifted
to 556, 107, and 17 nM in the presence of 12 at a final
concentration of 0.2, 2.0, and 10 lM, respectively. The
IC50 values of VCR for A2780 and 2780AD cells were
0.9 and 895 nM, respectively. The IC50 values for
MDR 2780AD cells were shifted to 336, 30, and 7 nM
in the presence of 12 at a final concentration of 0.2,
2.0, and 10 lM, respectively. The enhancing effects of
12 for taxol, ADM, and VCR were the same levels as
that of verapamil toward MDR 2780AD cells. On the
other hand, compound 12 showed no enhancing effect
for taxol, ADM, and VCR toward the parental 2780
cells. Thus, compound 12 can modulate the multidrug
resistance of cancer cells as well as verapamil in vitro.
possessing 5a-,20-dihydroxyl groups instead of
a
5b,20-epoxy ring (4,5-oxetane ring) in 13 showed weaker
activity than that of 13. Analogously, compound 15 pos-
sessing 5a-,20-dihydroxyl groups instead of 5b,20-epoxy
ring (4,5-oxetane ring) in 9 showed no activity of accu-
mulation of calcein in 2780 AD cells, although 9 had sig-
nificant activity. These results indicated that the 5b,20-
epoxy ring (4,5-oxetane ring) is an important functional
group and 5a-,20-dihydroxyl group has no effect for the
expression of activity of the taxane derivatives above
mentioned. On the contrary, compound 16 possessing
5a-hydroxy-20-acetoxy groups instead of the 5a,20-
dihydroxyl groups of 15 showed significant activity.
Compound 17, 2-cinnamoyloxy derivative of 16, showed
more efficient activity than that of 16. This enhancement
of the activity was induced by the change of the func-
tional group at C-2 from hydroxyl group to a cinna-
moyloxyl group.
The above-mentioned compounds, 5–17, are all oxygen-
ated at C-2, C-4, C-5, C-9, C-10, C-13, and C-20 posi-
tions and synthesized for this research from taxinine,
which is the major component of the Japanese yew tree,
T. caspidata.

T. Hasegawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1122–1126
1125
Table 2. Effect of compound 12 on the cytotoxicity of anticancer
agents toward A2780 and MDR 2780AD cellsa
Compound 13 with a carbonyl group at C-13 showed
both MDR reversing and cytotoxic activities in vitro.
On the other hand, the corresponding 13a-hydroxy
derivative 12 showed efficient MDR reversing activity
but its cytotoxic activity decreased drastically. Introduc-
tion of an isoserine moiety to the 13a-hydroxyl group of
taxane derivatives increases cytotoxic activity remark-
ably as shown in the change from baccatin III (4) to tax-
ol (2) in Table 1. Compound 12 is also expected to be a
lead compounds of anti-MDR cancer agents or antican-
cer agents after introduction of the isoserine moiety on
the13a-hydroxyl group of 12.
Cell lines
MDR modulator
(lM)
IC50 (nM) of anticancer
agents
Taxol
ADM
VCR
2780
No modulator
Verapamil
0.7
0.7
0.8
0.9
0.7
0.9
0.8
13
11
9
0.9
1
0.2
2
10
0.8
1.1
1
14
8
12
0.2
2
10
10
7
0.8
0.8
2780AD
No modulator
Verapamil
535
197
11
909
615
109
59
895
563
29
7
0.2
Acknowledgment
2
10
0.9
375
10
12
0.2
556
107
17
336
30
7
We thank Ms. Sachiko Shimizu for her technical assis-
tance in biological evaluation.
2
10
1
The values represent the mean of triplicate determination.
a Enhancing effects of verapamil and compound 12 on the cytotoxicity
of Taxol, adriamycin (ADM), and vincristin (VCR) toward A2780
cells and MDR A2780 (2780AD ) cells were determined in the
presence of 0.2, 2.0, and 10 lM of each compound.
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Cell growth inhibitory activity (IC50) of compounds 4–
17 to three different cell lines was examined (Table 1).
The three cell lines employed in this experiment are hu-
man lung fibroblast cells (WI-38), malignant lung tumor
cells (VA-13) induced from WI-38, and human liver can-
cer Hepatoma G2 cells (HepG2). Compound 8 showed
the smallest IC50 values toward VA-13 and HepG2 in
compounds 5–8. The results suggested that displacement
of the acetoxyl group at C-9 and/or C-10 of 8 with a
hydroxyl group induced the decrease of activity in 5,
6, and 7. Cytotoxic activity of 9 against WI-38, VA-
13, and HepG2 decreased remarkably on displacement
of the 2a-benzoyloyl group of 8 with an acetoxyl group.
Compound 13 showed moderate and weak activities to
VA-13 and HepG2, respectively. Compound 14 with
5a-,20-dihydroxyl groups instead of 5a,20-oxetane ring
in 13 showed weaker activity to VA-13 than that of
13. Compounds 15 and 16 with a 5a-,20-dihydroxyl
and 5a-hydroxy-20-acetoxy groups, respectively,
showed significant activity toward VA-13. On the con-
trary, compound 17, 2-cinnamoyloxy derivative of 16,
showed significant activity not to VA-13 but HepG2.
These results suggested that some modifications of the
functional groups on the taxane skeleton induced a
new cytotoxicity to a different cell line.
Compounds 6, 9, and 12 showed MDR reversal activities
but have no cytotoxicity. These compounds are expected
to be lead compounds of MDR cancer reversal agents.
On the other hand, cytotoxic activity of compounds 8
and17 against HepG2 is the same level or more than that
of taxol (2) or baccatin III (4). Since compounds 8 and 17
also showed significant MDR reversal activity, they are
expected as lead compounds of new type anticancer
agents. Compounds 13, 15, and 16 showed significant
cytotoxic activity toward VA-13. Among them, 13 and
16 showed significant MDR reversal activity and are
expected to be a new type of anticancer agents.

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