Bioreduction activated prodrugs of camptothecin: Molecular design, synthesis, activation mechanism and hypoxia selective cytotoxicity

Article abstract of DOI:10.1039/b502813b

Several water-soluble derivatives (CPT3, CPT3a-d) of camptothecin (CPT) were synthesized, among which CPT3 bearing an N,N′-dimethyl-1- aminoethylcarbamate side-chain was further conjugated with reductively eliminating structural units of indolequinone, 4-nitrobenzyl alcohol and 4-nitrofuryl alcohol to produce novel prodrugs of camptothecin (CPT4-6). All CPT derivatives were of lower cytotoxicity than their parent compound of CPT. In contrast, CPT4 and CPT6 showed higher hypoxia selectivity of cytotoxicity towards tumor cells than CPT. A mechanism by which a representative prodrug CPT4 is activated in the presence of DT-diaphorase to release CPT was also discussed. The bioreduction activated CPT prodrugs including CPT4 and CPT6 are identified to be promising for application to the hypoxia targeting tumor chemotherapy. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b502813b

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A R T I C L E
Bioreduction activated prodrugs of camptothecin: molecular design,
synthesis, activation mechanism and hypoxia selective cytotoxicity†
Zhouen Zhang, Kazuhito Tanabe, Hiroshi Hatta and Sei-ichi Nishimoto*
Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto
University, Katsura Campus, Nishikyo-ku, Kyoto, 615-8510, Japan.
E-mail: nishimot@scl.kyoto-u.ac.jp
Received 24th February 2005, Accepted 4th April 2005
First published as an Advance Article on the web 14th April 2005
Several water-soluble derivatives (CPT3, CPT3a–d) of camptothecin (CPT) were synthesized, among which CPT3
ꢀ
bearing an N,N -dimethyl-1-aminoethylcarbamate side-chain was further conjugated with reductively eliminating
structural units of indolequinone, 4-nitrobenzyl alcohol and 4-nitrofuryl alcohol to produce novel prodrugs of
camptothecin (CPT4–6). All CPT derivatives were of lower cytotoxicity than their parent compound of CPT. In
contrast, CPT4 and CPT6 showed higher hypoxia selectivity of cytotoxicity towards tumor cells than CPT. A
mechanism by which a representative prodrug CPT4 is activated in the presence of DT-diaphorase to release CPT
was also discussed. The bioreduction activated CPT prodrugs including CPT4 and CPT6 are identified to be
promising for application to the hypoxia targeting tumor chemotherapy.
Introduction
chain was conjugated with three types of well documented
15
structural units with bioreduction reactivity (indolequinone,
20(S)-Camptothecin (CPT) is a potent inhibitor of DNA
16
17
4
-nitrobenzyl and 4-nitrofuryl motifs ) to obtain the cor-
topoisomerase I (topo I) first isolated by Wall and coworkers
responding prodrugs CPT4–6, as shown in Fig. 1. We also
evaluated in vitro cytotoxicity of the CPT derivatives towards
tumor cells under both aerobic and hypoxic conditions, and
bioreductive activation of a representative prodrug CPT4 by
1,2
from Camptotheca acuminate. CPT has been identified to
stabilize covalent binding of topo I to DNA, thereby resulting
in irreversible and lethal strand breaks of DNA during its
2
replication. The clinical application of CPT to cancer treatment
was however suspended because of its unfavorable properties
3
such as non-specific toxicity and negligible water solubility.
Another key problem is the structural instability of the 20-
hydroxy lactone ring moiety, which is easily hydrolyzed into an
inactive E-ring-opened carboxylate form under physiological
4
conditions (Scheme 1). To improve these drawbacks, a great
deal of effort has been made to develop various modifications
5
,6
7,8
such as CPT analogues, CPT prodrugs, and combination
9,10
with drug delivery systems, some of which have recently been
subjected to clinical trials.
6
,11
In this work, an attempt was made to develop a new class of
water-soluble bioreduction activated prodrugs of CPT that are
subject to metabolism by intracellular reductases and thereby
release a potent topo I inhibitor of CPT selectively under
hypoxic conditions. The strategy employed herein is closely
12
related to tumor hypoxia targeting therapy, antibody-directed
13
enzyme prodrug therapy (ADEPT) and gene-directed enzyme
14
prodrug therapy (GDEPT). Firstly, we synthesized a series
of water-soluble CPT derivatives (CPT3, CPT3a–d) to explore
appropriate linker structures for constructing efficient biore-
duction activated CPT prodrugs. Based on this exploration,
ꢀ
CPT3 bearing an N,N -dimethyl-1-aminoethylcarbamate side-
†
Electronic supplementary information (ESI) available: Experimen-
tal procedures for syntheses of CPT derivatives, CPT1–3, CPT3a–
ꢀ
ꢀ
d, CPT3a and CPT3b . See http://www.rsc.org/suppdata/ob/b5/
b502813b/
Fig. 1 Structures of bioreduction activated CPT prodrugs CPT4–6.
Scheme 1 Hydrolytic ring-opening reaction of CPT under physiological conditions.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 0 5 – 1 9 1 0
1 9 0 5

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ꢀ
ꢀ
Fig. 2 Structures of water-soluble CPT derivatives CPT3 and CPT3a–d, and the representative carboxylate forms CPT3a ,3b .
DT-diaphorase (DTD). These in vitro assays demonstrated that
the bioreduction activated prodrugs such as CPT4 and CPT6
have higher hypoxia selectivity of cytotoxicity towards tumor
cells than CPT.
was readily converted into the corresponding E-ring opened
carboxylate form CPT3a at the lactone ring moiety in tris buffer,
ꢀ
accompanying the release of free CPT. CPT3b was similarly less
stable in tris buffer (t1/2 = 10.2 h) and was partly converted into
ꢀ
its carboxylate form CPT3b , along with the release of free CPT.
In contrast, CPT3 was relatively more stable (t1/2 = 57.2 h),
undergoing slow and spontaneous cyclization to release CPT
with the highest selectivity, as shown in Scheme 3. The other
derivatives CPT3c (t1/2 = 67.3 h), CPT3d (t1/2 = 36.5 h) and
CPT4 (t1/2 = 67.3 h) were also slowly hydrolyzed by a mechanism
different from cyclization to release CPT in tris buffer, in which
several unknown products were simultaneously produced.
Results and discussion
7,8a
Synthesis of CPT prodrugs
For screening of water-soluble linker structures appropriate
for construction of bioreduction activated CPT prodrugs,
several CPT derivatives listed in Fig. 2 were synthesized.
According to the previous reports, modification of the 20-
hydroxy group in the lactone ring moiety has the merit of
enhancing the stability of CPT. In this light, reaction of CPT
at the 20-hydroxy group with 4-nitrophenyl choroformate was
18
Bioreductive activation of CPT4 by DT-diaphorase
We further characterized a mechanism by which a represen-
tative CPT prodrug CPT4 is bioreductively activated by DT-
diaphorase (DTD) in tris buffer. It is well known that DTD
can two-electron reduce various quinoid compounds, includ-
ing indolequinones, which have been applied to bioactivated
performed to obtain 4-nitrophenyl camptothecin carbonate
7
(
CPT1), which was readily converted to water-soluble CPT
derivatives, CPT3 and CPT3a–c, upon treatment by mono-Boc-
ꢀ
N,N -dimethylethylenediamine, mono-Boc-ethylenediamine, N-
Boc-1,3-diaminopropane, and N-a-Boc-L-lysine, respectively,
followed by deprotection: a representative synthesis route of
CPT3 is outlined in Scheme 2. Similarly, treatment of CPT1
by 1-amino-1-deoxy-b-D-galactose gave CPT3d. For reference,
E-ring opened analogues of CPT3a,b (CPT3a ,b ) were also
derived from reactions of CPT1 with ethylenediamine and 1,3-
diaminopropane, respectively.
In view of the stability and hydrolysis reactivity of the above
water-soluble CPT derivatives (see below), CPT3 was identified
as a favorable lead compound that was readily conjugated with
well-known bioreductive motifs of indolequinone, 4-nitrobenzyl
alcohol and 4-nitrofuryl alcohol in their p-nitrophenyl carbonate
forms to obtain the corresponding prodrugs CPT4–6 (see also
Scheme 2 for the synthesis of CPT4).
12a,19
chemotherapy.
In the presence of DTD and 2-nicotinamide
adenine dinucleotide disodium salt (NADH), CPT4 was quickly
reduced to eliminate the conjugated indolequinone moiety and
produce CPT3 in tris buffer, as shown in Fig. 3a (see also
Scheme 3). As characterized in a separate experiment, the
resulting CPT3 effectively underwent intramolecular cyclization
to release CPT. In a control solution without DTD, CPT4 was
quite slowly hydrolyzed to release CPT3 followed by formation
of CPT in considerably low yield, as shown in Fig. 3b.
ꢀ
ꢀ
Cytotoxicity of CPT and its derivatives
In order to evaluate the anticancer ability, the cytotoxicity (IC50
0% inhibitory concentration) of CPT derivatives towards three
kinds of tumor cell lines of HT-1080, EMT6/KU and HT-29
;
5
Stability and hydrolysis reactivity of CPT derivatives
20
were determined by an MTT method. The IC50 value of CPT5
was failed to be measured because of its very weak solubility.
As summarized in Table 1, the bioreduction activated prodrugs
CPT4 and CPT6 showed at least one order of magnitude (about
10∼27 times) lower cytotoxicity than the parent anticancer agent
CPT, while the water-soluble derivatives CPT3 and CPT3a–
d were of slightly reduced cytotoxicity: exceptionally CPT3c,d
All the modified CPT derivatives except CPT5 showed higher
water-solubility than CPT. These CPT derivatives underwent
hydrolysis in tris buffer (pH 7.4) at 37 C, following the pseudo-
first-order kinetics with their characteristic reactivity. CPT3a
was the most unstable with a half life time (t1/2) of 3.6 hours. The
results of analytical HPLC and FAB-MS indicated that CPT3a
◦
1
9 0 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 0 5 – 1 9 1 0

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Scheme 2 Synthesis of bioreduction activated prodrug CPT4.
Scheme 3 Mechanism of CPT4 activation by a reductase of DTD.
Fig. 3 (a) Time course profiles of CPT4 (ꢀ), CPT3 (᭺) and CPT (ꢁ) in
the hydrolysis of 0.1 mM CPT4 in 0.1 M tris buffer (pH 7.4) containing
showed one order of magnitude lower cytotoxicity towards
EMT6/KU cells.
Table 1 also compares the IC50 values ofCPT, CPT4 and
CPT6 towards HT-29 cells without and with 90 min hypoxia
◦
−
1
DTD (2.5 units mL ) and NADH (1.25 mM) at 37 C; (b) time course
profiles of CPT4 (ꢀ), CPT3 (᭺) and CPT (ꢁ) in the hydrolysis of 0.1 mM
CPT4 in 0.1 M tris buffer (pH 7.4) containing NADH (1.25 mM) but
◦
not DTD at 37 C.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 0 5 – 1 9 1 0
1 9 0 7

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Table 1 Cytotoxicity of CPT and its derivatives towards various tumor cells
IC50/lM, mean ± SD, n = 5
Compound
HT-1080
EMT6/KU
HT-29
HT-29 with hypoxia treatment for 90 min
CPT
0.07 ± 0.01
0.15 ± 0.02
0.10 ± 0.02
0.13 ± 0.02
0.14 ± 0.02
0.09 ± 0.01
0.10 ± 0.01
0.10 ± 0.02
0.20 ± 0.02
0.30 ± 0.02
0.70 ± 0.10
2.5 ± 0.2
0.06 ± 0.01
a
CPT3
CPT3a
CPT3b
CPT3c
CPT3d
CPT4
CPT6
–
a
a
–
–
–
a
a
a
–
–
a
0.30 ± 0.01
0.50 ± 0.05
0.75 ± 0.05
1.0 ± 0.1
1.5 ± 0.1
1.9 ± 0.1
2.1 ± 0.2
1.4 ± 0.1
–
a
–
1.40 ± 0.02
2.1 ± 0.2
0.50 ± 0.01
a
Not determined.
treatment. A trend is obvious that the bioreduction activated
prodrugs CPT4 and CPT6 caused higher hypoxia selectivity
of cytotoxicity (the enhancement ratio of hypoxia selective
cytotoxicity at 90 min were 1.7 and 4.1, respectively) than their
parent CPT. The higher cytotoxicity of CPT under hypoxia is
attributed to environment acidification induced by tumor cell
oxygen, the latter two-electron reduction pathway is independent
of oxygen effect.
Conclusion
A series of water-soluble CPT derivatives CPT3 and CPT3a–
d were synthesized. CPT3 was a useful lead compound that
could be readily conjugated with three kinds of typical biore-
ductive motifs to produce the bioreduction activated prodrugs
CPT4–6, respectively. All the modified CPT derivatives, except
CPT5, had greater water-solubility than their parent CPT, thus
undergoing hydrolysis to release free CPT in tris buffer at
pH 7.4 according to the pseudo-first-order kinetics with their
characteristic reactivity. A mechanism by which a representative
CPT prodrug, CPT4 is bioreductively activated by DTD in tris
buffer was characterized and discussed. As determined by in vitro
studies, the bioreduction activated prodrugs CPT4 and CPT6
had at least one magnitude of lower cytotoxicity (measured by
IC50) than CPT, showing hypoxia selective cytotoxicity. CPT4
and CPT6 are identified as promising bioreduction activated
anticancer prodrugs that may be applicable to the hypoxia
targeting tumor chemotherapy and the gene-directed enzyme
prodrug therapy (GDEPT).
21
glycolysis, in accord with the recent reports that cytotoxicity
22
of CPT and its analogues is enhanced at acidic pH. The HT-29
cells employed for the assay are among various tumor cell lines
23
over-expressing bioreductive enzymes of DTD and NADPH
24
cytochrome P450 reductase. It is predictable that the apparent
hypoxia selective cytotoxicity of CPT4 and CPT6 will be more
enhanced, as the concentration of released CPT increases upon
prolonged hypoxic incubation.
These behaviors demonstrate that bioreductive motifs in-
volved in the CPT4 and CPT6 are responsible for the hypoxia
selective cytotoxicity. Evidently, nitroaromatics as in CPT6
are more effective motifs than indolequinones as in CPT4
for enhancing hypoxia selective cytotoxicity. This is in accord
with previously reported bioreductive activation mechanisms
for prodrug systems with nitroaromatics and quinines includ-
ing indolequinone. In the nitroaromatics prodrug system (see
12a,16,17
Scheme 4),
the first reduction step from nitro to nitroso
anion may be inhibited by back-oxidation in the presence of
oxygen. On the other hand, the quinone (Q) prodrug system
Experimental
(
(
Scheme 4) is activated through either a pathway of semiquinone
SQ) intermediate (one-electron reduction process such as biore-
Materials
duction by NADPH cytochrome P450 reductase) or a direct
20(S)-Camptothecin (CPT) was obtained from Tokyo Ka-
sei (Tokyo, Japan). 4-Dimethylaminopyridine (DMAP), ace-
tonitrile, trifluoroacetic acid (TFA), 2-propanol, triethylamine
(TEA), b-nicotinamide adenine dinucleotide disodium salt
pathway to hydroquinone (HQ) (two-electron reduction process
12a,15,19
such as bioreduction by DTD) (Scheme 4).
Whereas the
former one-electron reduction pathway may be inhibited by
(
NADH), culture medium PRMI-1640, and Eagle’s minimal
essential medium (MEM) were purchased from Nacalai Tesque
Kyoto, Japan). Indolequinone was a gift from Mr Arim-
(
ichi Okazaki. N-a-Boc-L-lysine was purchased from Watanabe
Chemical Ind. (Hiroshima, Japan). Fetal bovine serum (FBS)
was obtained from Thermo Trace (Melbourne, Australia).
Thiazolyl blue tetrazolium bromide (MTT), 1-amino-1-deoxy-
b-D-galactose, 4-nitrophenyl choroformate and E. coli DT-
diaphorase (DTD) were obtained from Sigma (Steinheim,
Germany). Dimethyl sulfoxide (DMSO), 4-nitrofuryl alcohol,
4
-nitrobenzyl alcohol and other chemicals were purchased from
Wako (Osaka, Japan). The cell line of HT-1080 human fibrosar-
coma was supplied by the Cell Resource Center for Biomedical
Research, Tohoku University (Sendai, Japan). The cell line of
EMT6/KU murine mammary carcinoma was subcultured in
Kyoto University. The cell line of HT-29 human colorectal
adenocarcinoma was obtained from the American Type Culture
Collection (www.attc.org).
General methods
Thin-layer chromatography (TLC) was performed on a silica
gel coated glass sheet (Silica Gel 60 F254 TLC plates, Merck &
Co., Germany). Preparative layer chromatography (PLC) was
Scheme 4 Bioreductive activation mechanisms of (a) the nitroaro-
matics prodrug system and (b) the quinone prodrug system releasing
anticancer agent.
1
9 0 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 0 5 – 1 9 1 0

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◦
performed on a Silica Gel 60 F254 PLC plate (Merck & Co.
Germany). A Wakogel C-300 silica gel (45–75 lm, Wako Pure
Chemical Industries Ltd.) was used for column chromatography.
0.6 mmol) were dissolved in dichloromethane (3 mL) at 0 C. The
resulting solution was stirred for 2 hours at room temperature
under nitrogen to produce p-nitrophenyl nitrofuryl carbonate
(NF1). CPT2 (50 mg, 0.089 mmol) was treated by 1 mL of
trifluoroacetic acid for 15 min at room temperature. After
removal of trifluoroacetic acid the crude product CPT3 was
added to the solution containing NF1, followed by the addition
of 0.2 mL triethylamine. The reaction mixture was kept under
stirring for 20 min and concentrated in vacuo. The residue
was purified by PLC (ethyl acetate) to give camptothecin–
nitrofuryl conjugate CPT6 (45 mg, 80%) as a yellow powder.
CN)/nm 247 (e/dm mol cm 19 700), 310 (15 500),
360 (12 500), 425 (6800). d (300 MHz, CDCl
) 0.93 (t, J =
5.4 Hz, 3 H), 2.02–2.24 (m, 2H), 2.75–3.50 (m, 10 H), 5.05–5.64
(m, 6 H), 6.58 (m, 1 H), 7.15–8.34 (m, 7 H). d (75 MHz, CDCl
7.7, 31.9, 34.9, 35.1, 46.6, 46.8, 50.0, 66.8, 67.0, 96.8, 112.1,
1
Proton nuclear magnetic resonance ( H NMR) spectra and
1
3
carbon nuclear magnetic resonance ( C NMR) spectra were
recorded on a JEOL JNM-EX270J (270 MHz) spectrometer and
a JNM-AL300 (300 MHz) spectrometer, respectively. Fast atom
bombardment mass spectrometry (FAB-MS) was performed on
a JEOL JMS-SX102A mass spectrometer, using a glycerol or
3
-nitrobenzyl alcohol (NBA) matrix. High performance liquid
chromatography (HPLC) was performed on a Hitachi D-7000
ꢀ
R
3
−1
−1
HPLC system equipped with an Interstil ODS-3 column
4.6φ × 250 mm, GL Science Inc., Japan). The samples were
eluted with an aqueous CH CN (30%) solution containing 0.1 M
kmax (CH
3
(
H
3
3
−
1
TEEA buffer (pH 6.4) at a flow rate of 0.6 mL min and detected
by UV absorbance at wavelength of 360 nm.
C
3
)
1
1
1
6
12.2, 112.8, 115.6, 126.1, 128.2, 128.3, 129.2, 130.8, 131.4,
40.9, 145.9, 147.1, 148.7, 152.2, 153.3, 153.6, 155.6, 157.5,
Camptothecin–indolequinone conjugate CPT4
62.7, 168.0. FAB-MS (positive mode, NBA as matrix): m/z
Indolequinone (58 mg, 0.25 mmol), 4-nitrophenyl choroformate
+
32.1991 [MH ], C31
H
30
5
N O10 requires 632.1993.
(
50 mg, 0.25 mmol) and triethylamine (0.04 mL, 0.5 mmol)
◦
were dissolved in dichloromethane (5 mL) at 0 C. The resulting
solution was stirred for 2 hours at room temperature under
nitrogen to produce p-nitrophenyl indolequinone carbonate
Stability and hydrolyses of CPT derivatives
Solutions of CPT derivatives CPT3 and CPT3a–d (0.1 lmol) in
0.1 M tris buffer containing 5% acetonitrile (1 mL, pH 7.4)
were incubated at 37 C. Aliquots (10 lL) were sampled at
various time intervals, and the concentrations of CPT derivatives
and hydrolysates during hydrolysis were quantified by analytical
HPLC, using the respective calibration curves prepared by
reference to the authentic samples. Since the hydrolytic decom-
positions of CPT derivatives were well represented in terms of
the first-order kinetics, the hydrolysis rate constants (k) were
evaluated from eqn. (1):
(
IQ1). CPT2 (50 mg, 0.089 mmol) was treated by 1 mL of
◦
trifluoroacetic acid for 15 min at room temperature. After
removal of trifluoroacetic acid the residual crude product CPT3
was added to the solution containing IQ1, followed by the
addition of 0.5 mL triethylamine. The reaction mixture was
kept under stirring for another 15 min, and concentrated in
vacuo. The residue was purified by PLC (ethyl acetate) to give
camptothecin–indolequinone conjugate CPT4 (60 mg, 93%) as
3
−1
−1
a red powder. kmax (CH
88 (20 500), 360 (17 300), 440 (2300). d
m, 3 H), 2.06–2.24 (m, 5 H), 2.71–3.79 (3 m, 16 H), 5.08–5.60 (m,
3
CN)/nm 253 (e/dm mol cm 27 300),
2
H
(300 Hz, CDCl ) 0.92
3
C
t
= Ct = 0exp (−kt)
(1)
(
7
8
7
1
H), 7.16 (m, 2 H), 7.56–8.29 (m, 4 H). d
.4, 29.6, 31.9, 32.3, 35.4, 36.0, 45.6, 47.4, 47.7, 49.9, 57.5, 66.9,
5.9, 96.3, 106.6, 116.4, 127.9, 128.1, 128.2, 128.5, 129.5, 130.5,
C
(75 MHz, CDCl
3
) 7.7,
Accordingly, the half-life periods (t1/2) of CPT derivatives were
then evaluated as the measures of their stability from eqn. (2):
t
1/2 = ln 2/k
(2)
31.1, 138.0, 145.9, 148.7, 152.5, 157.4, 159.6, 168.1, 178.8. FAB-
+
HRMS (positive mode, NBA as matrix): m/z 724.2631 [MH ],
C
38
H
38
N O10 requires 724.2619.
5
Bioreductive activation of CPT4 by DT-diaphorase
A solution of CPT4 (0.1 mM), NADH (1.25 mM), and DT-
Camptothecin–nitrobenzyl conjugate CPT5
-Nitrobenzyl alcohol (50 mg, 0.3 mmol), 4-nitrophenyl cho-
−
1
diaphorase (2.5 units mL ) in 0.1 M tris buffer containing
◦
4
5% acetonitrile (1 mL, pH 7.4) was incubated at 37 C under
roformate (60 mg, 0.3 mmol) and triethylamine (0.05 mL,
aerobic conditions. Aliquots were sampled for analytical HPLC
at appropriate time intervals. A control solution of CPT4
(0.1 mM) and NADH (1.25 mM) in 0.1 M tris buffer was
similarly incubated and analyzed.
◦
0
.6 mmol) were mixed in dichloromethane (5 mL) at 0 C. The
resulting solution was stirred for 2 hours at room temperature
under nitrogen to produce p-nitrophenyl nitrobenzyl carbonate
(
NB1). CPT2 (40 mg, 0.071 mmol) was treated by 1 mL of
trifluoroacetic acid for 15 min at room temperature. After
removal of trifluoroacetic acid the crude product CPT3 was
added to the solution containing NB1, followed by the addition
of 0.2 mL triethylamine. The reaction mixture was kept under
stirring for 20 min and concentrated in vacuo. The residue
was purified by PLC (ethyl acetate) to give camptothecin–
nitrobenzyl conjugate CPT5 (40 mg, 87%) as a yellow powder.
In vitro cytotoxicity assays
HT-1080 and EMT6/KU cells were cultured in MEM con-
taining 1% non-essential amino acids and 10% FBS, while
HT-29 cells were cultured in RPMI-1640 medium containing
1
0% FBS. All the cells were grown in an atmosphere of 5%
◦
CO
2
and 90% relative humidity at 37 C. Inhibition of the
3
−1
−1
cellular growth was evaluated using a method of MTT assay
k
3
2
7
4
1
1
max (CH
3
CN)/nm 217 (e/dm mol cm 31 800), 250 (25 100),
62 (14 500). d (270 MHz, CDCl
) 0.98 (t, J = 7.3 Hz, 3 H),
.04–2.25 (m, 2 H), 2.80–3.51 (m, 10 H), 5.17–5.70 (m, 6 H),
.21–8.37 (m, 10 H). d (68 MHz, CDCl ) 7.8, 32.0, 35.0, 46.5,
20
described by Mosmann. Suspensions of 5000 cells (50 lL)
were seeded in each well of a 96-well cell culture microplate and
treated by either 50 lL fresh drug–medium solution at various
H
3
C
3
−
10
−3
−1
concentrations from 10 to 10 mol L or 50 lL control
6.9, 49.9, 65.7, 67.2, 68.4, 96.2, 120.1, 123.5, 123.8, 125.1,
27.7, 127.9, 128.1, 128.2, 128.4, 129.3, 130.6, 131.1, 144.0,
45.8, 147.1, 148.7, 150.7, 152.5, 154.6, 157.2, 167.9. FAB-
medium solution. These plates were incubated for 72 hours
◦
at 37 C in an atmosphere of humidified 5% CO
2
, and then
−
1
+
10 lL of 5 mg mL MTT in PBS was added to each well.
HRMS (positive mode, NBA as matrix): m/z 642.2201 [MH ],
◦
After MTT cleavage for 4 hours at 37 C, 100 lL of 0.04 N
C
33
H
32
N
5
9
O requires 642.2200.
HCl in 2-propanol was added to each well to dissolve the dark
blue crystals. The absorbance at a test wavelength of 570 nm
was measured on a Model-550 Microplate Reader (Bio-Rad
Laboratories, Hercules, California). The concentration of drug
Camptothecin–nitrofuryl conjugate CPT6
-Nitrofuryl alcohol (50 mg, 0.34 mmol), 4-nitrophenyl cho-
roformate (60 mg, 0.3 mmol) and triethylamine (0.05 mL,
4
to reduce the cell survival to half of the control value (IC50
)
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 1 9 0 5 – 1 9 1 0
1 9 0 9

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was calculated from a regression analysis of cell survival vs
drug concentration. Hypoxia selective cytotoxicity of CPT4 and
CPT6 towards HT-29 cells were determined by a similar MTT
assay, except that the microplates containing HT-29 cells were
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Acknowledgements
The authors thank Mr Arimichi Okazaki for his kindly supply-
ing the sample of indolequinone. The authors also would like
to acknowledge Ms Hiromi Ushitora for her kind help with MS
measurements.
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