Synthesis and structure-activity relationships of taxuyunnanine C derivatives as multidrug resistance modulator in MDR cancer cells

Article abstract of DOI:10.1016/j.bmcl.2007.04.030

A series of new generation taxoids bearing a bulky group on different positions such as C-2, C-5, C-7, C-9, C-10 or C-14 were obtained by chemical modifications and biotransformation of taxuyunnanine C (1) and its analogs, 4, 5, and 10. Compounds 3, 5, 6, 8, and 9a showed significant activity toward calcein accumulation in MDR 2780AD cells. The most effective compound 9a with a cinnamoyloxy group at C-14 and a hydroxyl group at C-10 was actually efficient for the cellular accumulation of the anticancer agent, vincristine, in MDR 2780AD cells. The enhancing effects of 6 and 9a for taxol, adriamycin, and vincristine were at the same levels as those of verapamil toward MDR 2780AD cells. Thus, compounds 6 and 9a can modulate the multidrug resistance of cancer cells. The cytotoxicity (IC₅₀) of the compounds was examined against human normal cell line, WI-38, and cancer model cell lines, VA-13 and HepG2. Since compounds 6 and 8 had no cytotoxicity, they were expected to be lead compounds of MDR cancer reversal agents. On the contrary, compounds 3, 5, and 9a showed cell growth inhibitory activity toward VA-13 and/or HepG2 as well as accumulation activity of calcein and/or vincristine in MDR 2780AD and they were expected to be lead compounds of new-type anticancer agents.

Full text of DOI:10.1016/j.bmcl.2007.04.030

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3722–3728
Synthesis and structure–activity relationships of taxuyunnanine
C derivatives as multidrug resistance modulator
in MDR cancer cells
Toshiaki Hasegawa,a,c, Jiao Bai, Jungui Dai,b Liming Bai,a Junichi Sakai,b
a,f
*
Shigenori Nishizawa,a Yuhua Bai,a Midori Kikuchi,c Mariko Abe,c Takao Yamori,d
Akihiro Tomida,d Takashi Tsuruo,d Katsutoshi Hirosee and Masayoshi Andob,*
aGraduate School of Science and Technology, Niigata University, Ikarashi, 2-8050, Niigata 950-2181, Japan
bDepartment of Chemistry and Chemical 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 135-8500, Japan
eKNC Laboratories Co. Ltd, 3-2-34, Takatukadai, Nishi-ku, Kobe, Hyogo 651-2271, Japan
fDepartment of Natural Medicine Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Wenhua Road 103, Shenyang 110016, China
Received 28 October 2006; revised 21 March 2007; accepted 10 April 2007
Available online 13 April 2007
Abstract—A series of new generation taxoids bearing a bulky group on different positions such as C-2, C-5, C-7, C-9, C-10 or C-14
were obtained by chemical modifications and biotransformation of taxuyunnanine C (1) and its analogs, 4, 5, and 10. Compounds 3,
5, 6, 8, and 9a showed significant activity toward calcein accumulation in MDR 2780AD cells. The most effective compound 9a with
a cinnamoyloxy group at C-14 and a hydroxyl group at C-10 was actually efficient for the cellular accumulation of the anticancer
agent, vincristine, in MDR 2780AD cells. The enhancing effects of 6 and 9a for taxol, adriamycin, and vincristine were at the same
levels as those of verapamil toward MDR 2780AD cells. Thus, compounds 6 and 9a can modulate the multidrug resistance of cancer
cells. The cytotoxicity (IC50) of the compounds was examined against human normal cell line, WI-38, and cancer model cell lines,
VA-13 and HepG2. Since compounds 6 and 8 had no cytotoxicity, they were expected to be lead compounds of MDR cancer rever-
sal agents. On the contrary, compounds 3, 5, and 9a showed cell growth inhibitory activity toward VA-13 and/or HepG2 as well as
accumulation activity of calcein and/or vincristine in MDR 2780AD and they were expected to be lead compounds of new-type anti-
cancer agents.
Ó 2007 Elsevier Ltd. All rights reserved.
In cancer chemotherapy, occurrence of multidrug resis-
tance (MDR) of cancer cells caused by repeated admin-
istration of anticancer agents is a serious problem. One
of the mechanisms of MDR is overexpression of P-gly-
coprotein (P-gp),1,2 P-gp is originally a transporter for
a wide range of reagents and its physiological role is a
defense mechanism against the toxic materials in cells.
P-gp utilizes energy induced by hydrolysis of ATP for
their transport from inside to the outside of cells.
Many taxane derivatives with MDR reversal activity
have been reported.3–11 Effect of the substituents of
baccatin III on the MDR reversal activity was widely
investigated. However, since baccatin III has no func-
tional group at C-14, only a few reports appeared about
the effect of the substituent at C-14 on the MDR reversal
activity. Ojima et al. reported structure–activity rela-
tionships of 14b-hydroxybaccatin III derivatives with
1,14-carbonate moiety. In this case, hydrophobic groups
at C-7 are effective in MDR reversal activity.12
Taxuyunnanine C (1) and its 14-acyloxy analogs (2–5)
are the major metabolites from callus cultures of Taxus
species in high yields. We reported their activity toward
the accumulation of vincristine in MDR 2780AD cells
Keywords: Taxoid; Taxuyunnanine C; 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 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.030

T. Hasegawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3722–3728
3723
Figure 1. Acyloxy groups r–y in the structures of taxuyunnanine C
derivatives.
and deduced that a hydrophobic less-hindered alkyl side
chain of the C-14 acyloxy group of these compounds
played some role to increase the activity.13
Scheme 1. Reagents and conditions: (i) 1, 1 M K2CO3 (5 equiv), 1:3
THF/MeOH, 45 °C, 7 h (23.9% for 6, 4.3% for 7, 0.5% for 8, 20.7% for
9); (ii) 6, CH3(CH2)2COCl (13 equiv), DMAP, dry Py, 80 °C, 12.5 h
(87.6% for 6a); (iii) 6, CH3(CH2)3COCl (13 equiv), DMAP, dry Py,
80 °C, 13.5 h (88.0% for 6b); (iv) 6, PhCOCl (10 equiv), DMAP, dry
Py, 80 °C, 15 h (92.7% for 6c); (v) 6, PhCH@CHCOCl (8 equiv),
DMAP, dry Py, 85 °C, 16 h (92.8% for 6d); (vi) 7, PhCH@CHCOCl (8
equiv), DMAP, dry Py, 85 °C, 13 h (91.5% for 7a); (vii) 8,
PhCH@CHCOCl (20 equiv), DMAP, dry Py, 85 °C, 19h (80% for
8a); (viii) 9, PhCH@CHCOCl (2.2 equiv), DMAP, dry Py, 85–90 °C,
23 h (8.4% for 9a, 29.1% for 9b, 5.9% for 9c).
As a part of our study of structure–activity relationships
(SAR) of taxoids as MDR reversal agent, we further
investigated effect of the substitution of the functional
groups at C-14 position on MDR reversal activity. For
this purpose, we synthesized a series of new 14-oxygen-
ated taxoids derived from taxuyunnanine C (1) and its
analogs which are produced by callus cultures of Taxus
cuspidata.13–15 (Fig. 1).
We obtained 14-deacetyltaxuyunnanine C (6), 10-deac-
etyltaxuyunnanine C (7), 5-deacetyltaxuyunnanine C
(8), and 10,14-dideacetyltaxuyunnanine C (9) by hydro-
lysis of 1. 14-Butanoyloxy- (6a), 14-pentanoyloxy- (6b),
14-benzoyloxy- (6c), and 14-cinnamoyloxy- (6d) deriva-
tives of 6 were obtained by the acylation of 6 by the
corresponding acylchloride. Cinnamoylation of 10-deac-
etyltaxuyunnanine C (7) and 5-deacetyltaxuyunnanine
C (8) gave 7a and 8a bearing a cinnamoyloxy group
at C-10 or C-5, respectively. Cinnamoylation of
10,14-dideacetyltaxuyunnanine C (9) gave 10- and
14-monocinnamoyloxy analogs, 9b and 9a, and 10,14-
dicinnamoyloxy analog 9c, respectively (Scheme 1). 14-
Scheme 2.
Deacetoxytaxuyunnanine
C
(6f) was obtained by
For the preparation of 7- or 9-oxygenated derivatives of
1, 4, and 5, we used a biotransformation. Thus, 14-acyl-
oxytaxoids, 11–13, bearing a hydroxyl group at the C-7
or C-9 position were obtained by treatment of 1, 4, and
5 with Absidia coerulea IFO 4011. Compounds 11–13
Chugaev reaction of 6 (Scheme 2). 2-Benzoyloxy analog,
10a, was prepared from 2-deacetyltaxuyunnanine C
(10), which was isolated from the callus culture of
T. cuspidata (Scheme 3) as a minor product.16

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T. Hasegawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3722–3728
C-14 or C-5 of 1 with a more polar substituent such as a
hydroxyl group is desirable to increase the activity to-
ward calcein accumulation in MDR 2780AD cells. On
the other hand, the substitution of the acetoxyl group
at C-2, C-10 or one hydrogen at C-9 of 1 or at C-7 of
4 and 5 with a more polar substituent such as a hydroxyl
group is an undesirable change to increase the activity.
Scheme 3.
Then, the activities of 1, its seven kinds of C-14 acyloxy
analogs (2–5 and 6a–d), 14-deacetyltaxuyunnanine
C (6), and 14-deacetoxytaxuyunnanine C (6f) were
compared. The activity order of the compounds
was 5 > 6 = 3 > 6c P 1 = 6d P 2 = 4 P 6a P 6b ꢀ 6f.
These results suggested that the compounds with hydro-
philic substituents at C-14 such as 5 and 6, and the com-
pounds with bulky acyloxy substituents at C-14 such as
3 and 6c are desirable to increase the activity. On the
contrary, the compounds possessing acyloxy groups
with less-hindered hydrophobic propyl and butyl side
chains such as 6a and 6b showed lower activity than
the parental compound 1. Compound 6f, in which the
acetoxy group at C-14 of 1 was displaced by hydrogen,
lost the activity completely.
were further converted to the corresponding cinnamoyl
esters, 11a, 12a, and 13a (Scheme 4).
The effects of 14-oxygenated taxoids mentioned above
on the cellular accumulation of calcein in MDR human
ovarian cancer 2780AD cells were examined (Table 1).
The calcein derived from calcein AM by endogenous
esterase was used as an easily operated functional fluo-
rescent probe for this drug efflux protein. Taxuyunna-
nine C (1), its monodeacetyl analogs, 6–8 and 10, and
its 9-hydroxylated analog (11) showed weak to signifi-
cant activity toward calcein accumulation in MDR
2780AD cells. In them, compounds 6 and 8, in which
the acetoxyl groups at C-14 or C-5 of 1 were displaced
by a hydroxyl group, respectively, showed stronger
activity than that of 1. On the other hand, compounds
7, 10, and 11, in which the acetoxy groups at C-10 or
C-2, or one hydrogen at C-9, of 1 were displaced by a
hydroxyl group, respectively, showed weaker activity
than 1. Taxoid 6f in which the acetoxy group at C-14
of 1 was displaced by hydrogen, showed no activity.
Compounds 12 and 13 with a hydroxyl group at the
C-7 position showed lower activity than those of their
parental compounds, 4 and 5. These results of bioassay
suggested that the substitution of the acetoxyl group at
We examined whether functional group at C-14 influ-
enced the effect of structural change at the different po-
sition of taxane skeleton on the calcein accumulating
activity. When the functional group at C-14 is acetoxyl
group such as 7 and 7a, the activity of 7 with hydroxyl
group at C-10 was less than that of 7a with cinnamoyl-
oxy group at C-10. On the other hand, when functional
group at C-14 is cinnamoyl group such as 9a and 9c, the
activity of 9a with hydroxyl group at C-10 was higher
than that of 9c with cinnamoyloxy group at C-10. When
the functional groups at C-14 are 2-methylbutanoyloxy
group such as 12 and 12a, and 2-methyl-3-hydroxybuta-
noyloxy group such as 13 and 13a, the activity of 12 and
13 with hydroxyl group at C-7 and 12a with cinnamoyl-
oxy group at C-7 was at same level. On the other hand,
the activity of 13a with cinnamoyloxy group at C-7 was
higher than that of 13 with hydroxyl group at C-7. Thus,
the influences of the functional groups at C-14 on the
calcein accumulating activity seemed to be complicated.
Since taxinine derivatives, taxinine NN-1 and taxinine
NN-11 bearing a cinnamoyloxy group, exhibited strong
MDR reversal activity, we expected the special effect of
an aromatic acyloxy group such as a cinnamoyloxy
group or a benzoyloxy group on taxuyunnanine C deriv-
atives in calcein accumulation in MDR 2780AD cells.
We examined the activity of ten kinds of cinnamoyloxy
analogs of 1. The order of activity was
9a > 13a > 7a = 6d = 10a = 11a P8a > 9b > 12a ꢀ 9c.
In them, compound 9a with a cinnamoyloxy group at
C-14 and a hydroxyl group at C-10 showed the strongest
activity. On the contrary, 9b, regioisomer of 9a, with a
cinnamoyloxy group at C-10 and a hydroxyl group at
C-14 showed very weak activity. Compound 13a with
a cinnamoyloxy group at C-7 and a polar acyloxy
group, 2-methyl-3-hydroxybutanoyloxy group at C-14
showed significant activity. Interestingly, 12a with a cin-
namoyloxy group at C-7 and less polar 2-methylbuta-
noyloxy group at C-14 lost activity. Other analogs of
Scheme 4. Reagents and conditions: (i) 11, PhCH@CHCOCl (10
equiv), DMAP, dry Py, 85–90 °C, 9 h (84.6% for 11a); (ii) 12,
PhCH@CHCOCl (10 equiv), DMAP, dry Py, 85–90 °C, 10 h (88.8%
for 12a); (iii) 13, PhCH@CHCOCl (6 equiv), DMAP, dry Py, 85–
90 °C, 5.5 h (13.9% for 13a).

T. Hasegawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3722–3728
3725
Table 1. Effects of compounds on the accumulation of calcein in multidrug resistant cells 2780AD and cell growth inhibitory activities of compounds
against WI-38, VA-13, and HepG2 cells
Compound
Calcein accumulation (% of control)a,b
Cell growth inhibitory activity (IC50 lM)c
WI-38 VA-13 HepG2
0.005
0.38
53.1
0.25 lg/ml
2.5 lg/ml
25 lg/ml
Paclitaxel
Adriamycin
0.04
0.66
8.1
1.2
1
2
130
104
133
100
81
141
98
127
119
148
119
168
148
114
111
133
125
91
130
18.5
86.4
28.0
41.7
18.5
>193
>188
>183
93.7
90.9
61.1
3
139
86
4
5
94
86
79.4
133
73.4
9.29
6
104
97
80.0
17.2
56.7
216
6a
6b
6c
6d
6f
98
88
18.5
16.6
94
90
>183
>176
154
107
126
105
98
>176
>169
12.2
12.6
>169
144
>169
10.3
123
89
8.93
7
103
92
111
126
141
121
190
113
80
105
>169
149
108
106
183
7a
8
123
118
95
118
102
107
109
83
105
125
8a
9a
9b
9c
10
10a
11
11a
12
12a
13
13a
99.0
9.95
15.7
15.3
>147
122
51.9
132
113
80
13.5
71.7
37.4
11.5
>147
>147
99
98
99
96
111
125
110
125
110
102
107
130
130.4
125
129
104
175
102
102
94
101
103
98
>192
>154
128
>154
151
81.6
79.1
75.2
96
87
98
>144
140
62.4
>144
>173
>144
153
104
102
97
49.9
80.4
a The amount of calcein accumulated in multidrug-resistant human ovarian cancer 2780AD cells was determined with the control in the presence of
0.25, 2.5 and 25 lg/ml of test compounds.
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.
1, 6d, 7a, 8a, and 11a which possess one cinnamoyloxy
group at C-14, C-10, C-5, and C-9, and compound 10a
with one benzoyloxy group at C-2 showed almost the
same activity as that of 1. Compound 9c with two cinna-
moyloxy groups at C-10 and C-14 showed no activity. In
conclusion, compound 9a with a cinnamoyloxy group at
C-14 and a hydroxyl group at C-10 has the strongest
activity in 26 test samples of taxuyunnanine C analogs
toward calcein accumulation in MDR 2780AD cells.
lar accumulation of vincristine in MDR 2780AD cells
among the taxoids subjected to the test. Compound
9b, regioisomer of 9a, with a cinnamoyloxy group at
C-10 and a hydroxyl group at C-14 showed weaker
activity than 9a. Taxoid 9c bearing cinnamoyloxy
groups at both C-10 and C-14 positions showed very
weak activity. The activity of taxoid 7a with a cinna-
moyloxy group at the C-10 position was higher than
that of taxioid 7 with a hydroxyl group at the corre-
sponding positions. The results were similar to the calce-
in assay though there were some little differences.
The effects of compounds 6 and 9a on the cytotoxicity of
taxol, adriamycin (ADM), and vincristine (VCR) to-
ward MDR 2780AD cells and its parental cells
(A2780) were tested comparing with verapamil, which
is a well-known MDR reversal agent (Table 3).
The IC50 values of taxol for A2780 and MDR 2780AD
cells were 2.8 and 183 nM, respectively. When com-
pound 6 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 21, 26, and 5.3 nM, respec-
tively. At this time, cytotoxicity of compound 6 against
2780AD cells was equal to that of verapamil, showing
more than 70% of cell viability at the concentration of
Then, we tested the effects of several taxoids, 6, 7, 10, 11,
6d, 7a, 9a–c, on the accumulation of a widely used anti-
cancer agent, vincristine (VCR), in MDR 2780AD cells
comparing with the effects of verapamil as a positive
control (Table 2). Compound 9a possessing a hydroxyl
group at the C-10 position and a cinnamoyloxy group
at the C-14 position showed the highest activity of cellu-

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T. Hasegawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3722–3728
Table 2. Effects of compounds on the accumulation of vincristine (VCR) in multidrug-resistant cells 2780ADa
Concd (lg/mL)
Averageb (dpm/well)
% of controlc
Activitiesd
Verapamile (%)
6
0.1
1
360
517
853
336
494
985
324
420
805
404
641
1385
425
959
1855
402
669
1749
320
325
429
306
436
786
303
379
592
102
147
242
95
94
79
+
+
10
0.1
1
53
87
6d
7
140
280
92
+
+
75
62
10
0.1
1
84
64
119
229
132
209
451
121
272
527
114
190
497
91
+
10
0.1
1
+
51
7a
9a
9b
9c
+
130
118
81
+
10
0.1
1
++
+
111
146
116
105
102
110
83
+
10
0.1
1
+++
+
+
++
10
0.1
1
92
49
27
10
0.1
1
122
101
144
259
86
+
10
89
84
+
10
0.1
1
+
—
74
79
11
108
168
58
37
10
+
aThe amount of VCR accumulated in MDR2780AD cells was determined with the control in the presence of 0.1, 1, and 10 lg/ml of taxoids.
b The values represents triplicated determinations.
c The values are the relative amount of VCR accumulated in the cell compared with the control experiment.
d The indices are expressed on a scale of seven by the range of the relative amount of VCR accumulation as compared with the control experi-
ment(%): +++, 501–1000%; ++, 301–500%; +, 111–300%; , 91–100%; —, <90%.
e The values are expressed as the relative amount of vincristine(VCR) accumulation in the cell as compared with that of verapamil.
Table 3. Effect of the compounds on the cytotoxicity of anticancer agents toward A2780 and MDR 2780AD cells
Cell lines
MDR modulator (lM)
Viability of the cell (%)a
IC50 (nM) of anticancer agentsb
Taxol
ADM
VCR
A2780
No modulator
Verapamil
0
100
93
82
53
97
95
20
89
74
27
2.8
2.7
2.9
3.5
2.6
2.3
6.6
2.6
2.3
6.6
5.7
5.8
6.0
7.7
7.0
6.0
7.8
7.0
6.0
7.8
3.1
3.4
2.0
0.5
2.6
1.6
4.8
2.6
1.6
4.8
0.2
2
10
6
0.2
2
10
9a
0.2
2
10
2780AD
No modulator
Verapamil
0
100
84
183
36
5.5
4.9
21
26
5.3
22
4.2
3.9
298
269
201
164
215
256
181
284
138
73
311
0.2
2
41
25
90
71
10
3.9
6
0.2
2
101
96
55
26
11
51
39
23
10
77
9a
0.2
2
105
86
10
38
a Cyhotoxicity of the compounds was evaluated in the absence of the anticancer agents.
b Enhancing effects of the compounds 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 mM of each compound. The values represent means of triplicate determination.

T. Hasegawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3722–3728
3727
10 lM, while compound 6 showed significant cytotoxic-
ity against A2780 cells showing 20% of cell viability at
the same concentration. From these results, compound
6 enhanced the sensitivity to taxol in MDR 2780AD
cells. The IC50 of taxol for 2780AD cells in the presence
of 10 lM of compound 6 was equal to that for parental
A2780 cells (Table 4).
for MDR 2780AD cells were shifted to 284, 138, and
73 nM in the presence of 9a at a final concentration of
0.2, 2.0, and 10 lM, respectively. The IC50 values of
VCR for A2780 and 2780AD cells were 3.1 nM and
311 nM, respectively. The IC50 values for MDR
2780AD cells were shifted to 51, 39, and 23 nM in the
presence of 9a at a final concentration of 0.2, 2.0, and
10 lM, respectively. Thus, compound 9a can modulate
the multidrug resistance of cancer cells as well as verap-
amil in vitro.
Compound 6 is also effective for ADM and VCR. The
IC50 values of ADM for A2780 and MDR 2780AD cells
were 5.7 nM and 298 nM, respectively. The IC50 values
for MDR 2780AD cells were shifted to 215, 256, and
181 nM in the presence of 6 at a final concentration of
0.2, 2.0, and 10 lM, respectively. The IC50 values of
VCR for A2780 and 2780AD cells were 3.1 nM and
311 nM, respectively. The IC50 values for MDR
2780AD cells were shifted to 55, 26, and 11 nM in the
presence of 6 at a final concentration of 0.2, 2.0, and
10 lM, respectively. The enhancing effects of 6 for taxol,
ADM, and VCR were at the same levels as that of
verapamil toward MDR 2780AD cells. Thus, compound
6 can modulate the multidrug resistance of cancer cells
as well as verapamil in vitro.
Cell growth inhibitory activity (IC50) of taxoids to three
different cell lines was examined (Table 1). The three cell
lines employed in this experiment are human lung fibro-
blast cells (WI-38), malignant lung tumor cells (VA-13)
induced from WI-38, and human liver cancer, Hepa-
toma G2 (HepG2) cells.
Taxuyunnanine C (1) and its analogs 6–8, 10, 11 showed
weak activity in many cases. Only taxuyunnanine C (1)
and its C-10 deacetyl taxuyunnanine C (7) showed sig-
nificant activity against HepG2 and VA-13, respectively.
On the contrary, 14-deacetoxytaxuyunnanine C (6f)
showed significant activities to all three cell lines. This
result contrasted with the result of calcein assay and sug-
gested that the modification of the C-14 position of the
taxane skeleton significantly affected the biological
activities.
Cytotoxicity of compound 9a against 2780AD cells in
the absence of anticancer agents was more significant
than that of compound 6, showing 38% of cell viability
at the concentration of 10 lM, while the viability of the
cell was 27% in the case of parental A2780 cells at the
same concentration of compound 9a. IC50 of com-
pound9a for MDR 2780AD and parental A2780 was
calculated as 8.0 and 6.1 lM, respectively. Thus, com-
pound 9a showed MDR reversal activity against
2780AD cells.
The cytotoxic activity on HepG2 of taxuyunnanine C
analogs, 2–4, 6a, and 6b, bearing different kinds of acyl-
oxy groups at C-14 increased according to the increase
of hydrophobicity of their acyloxy group at C-14. In
them, 6a also showed significant cytotoxic activity on
VA-13 in addition to the activity on HepG2. However,
these compounds showed very weak or no cytotoxic
activity against normal cell line, WI-38. This is a desir-
able result in the screening of anticancer agents.
When compound 9a 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 22, 4.2, and 3.9 nM, respec-
tively. These results showed that compound 9a enhanced
the sensitivity to taxol in MDR 2780AD cells.
The cytotoxicity of 7a was examined, in the results of
in vitro primary screening of 7a based on the 39 human
cancer cell lines17,18 shown in Table 4. Although the
effective concentration of 7a is rather high, differential
growth inhibition is recognized. Since the result of
COMPARE of 7a is marginal (0.5 < r < 0.75), it possi-
bly belongs to a unique mechanistic class and is a new
member of anticancer agents. On the other hand, cell
growth inhibition effect (MG-MID of GI50 = À4.41)
and differential growth inhibition of compound 7a are
weak. The result suggests that compound 7a is not effec-
tive as an anticancer agent.
Compound 9a is also effective for ADM and VCR. The
IC50 values of ADM for A2780 and MDR 2780AD cells
were 5.7 nM and 298 nM, respectively. The IC50 values
Table 4. Summary of evaluation of compound 7a based on the 39
human cancer cell lines
GI50
TGI
LC50
Parameters of effective concentrations
MG-MID
Rank
À4.41
À4.04
À4.00
ra
Molecular
targets/drug type
Compounds 6, 6c, and 8 showed the activity toward cal-
cein accumulation in MDR 2780AD cells 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 6f and 8a
against HepG2 is at the same level of paclitaxel. Since
compound 8a showed moderate activity toward calcein
accumulation in MDR 2780AD cells, it is expected as
lead compound of new-type anticancer agents. Com-
pounds 5, 6a, 7, and 9b showed significant cytotoxic
activity toward VA-13.
Compound
Results of the COMPARE analysis
1
2
3
ICRF-154
ICRF-193
TNP-470
0.581
0.580
0.552
Topoisomerase
Topoisomerase
The mean graph of compound 7a was compared with those of 200
standard compounds using the COMPARE analysis. Drugs were
ordered according to the correlation coefficient. Drugs with correlation
coefficients higher than 0.5 (P < 0.001) were included.
a Pearson’s correlation coefficient.

3728
T. Hasegawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3722–3728
5. Kobayashi, J.; Shigemori, H.; Hosoyama, H.; Chen, Z.;
Akiyama, S.; Naito, M.; Tsuruo, T. Jpn. J. Cancer Res.
2000, 91, 638.
6. Kobayashi, J.; Hosoyama, H.; Shigemori, H.; Koiso, Y.;
Iwasaki, S. Experientia 1995, 51, 592.
7. Ojima, I.; Borella, C. P.; Wu, X.; Bounaud, P. Y.; Oderda,
C. F.; Sturm, M.; Miller, M. L.; Chakravarty, S.; Chen, J.;
Huang, Q.; Pera, P.; Brooks, T. A.; Baer, M. R.; Bernacki,
R. J. J. Med. Chem. 2005, 48, 2218.
8. Brooks, T. A.; Kennedy, D. R.; Gruol, D. J.; Ojima, I.;
Baer, M. R.; Bernacki, R. J. Anticancer Res. 2004, 24,
409.
Compound 5 showed significant cytotoxic activity to-
ward VA-13 and HepG2 along with the strong activity
toward calcein accumulation in MDR 2780AD cells.
Compound 3 showed significant cytotoxic activity to-
ward HepG2 along with the strong activity toward cal-
cein accumulation in MDR 2780AD cells. Compound
9a showed significant cytotoxic activity toward WI-13
and HepG2 along with the strongest activity in tested
samples toward calcein accumulation in MDR
2780AD cells. They are expected to be lead compounds
of anti-MDR cancer agents or anticancer agents.
9. Minderman, H.; Brooks, T. A.; O’Loughlin, K. L.; Ojima,
I.; Bernacki, R. J.; Baer, M. R. Cancer Chemother.
Pharmacol. 2004, 53, 363.
10. Brooks, T. A.; Minderman, H.; O’Loughlin, K. L.; Pera,
P.; Ojima, I.; Baer, M. R.; Bernacki, R. J. Mol. Cancer
Ther. 2003, 2, 1195.
Acknowledgments
The biological activity of compounds was examined by
the Screening Committee of New Anticancer Agents
supported by a Grant-in-Aid for Scientific Research
on Priority Area ‘Cancer’ from The Ministry of Educa-
tion, Science, Sports, and Culture, Japan. We thank our
colleagues in Niigata Research Laboratory: Dr. Sinyo
Gayama and Ryuichiro. Harada for their considerable
cooperation and Ms. Sachiko Shimizu for her technical
assistance in biological evaluation.
11. Geney, R.; Ungureanu, M.; Li, D.; Ojima, I. Clin. Chem.
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12. Ojima, I.; Bounaud, P. Y.; Takeuchi, C.; Pera, P.;
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13. Bai, J.; Kitabatake, M.; Toyoizumi, K.; Fu, L.; Zhang, S.;
Dai, J.; Sakai, J.; Hirose, K.; Yamori, T.; Tomida, A.;
Tsuruo, T.; Ando, M. J. Nat. Prod. 2004, 67, 58.
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