Antitumor agents. Part 227: Studies on novel 4′-O-demethyl- epipodophyllotoxins as antitumor agents targeting topoisomerase II

Article abstract of DOI:10.1016/j.bmc.2004.03.067

Eight novel epipodophyllotoxin derivatives (6-13), which were designed to overcome drug resistance and enhance topoisomerase II inhibition, were synthesized and evaluated. Two of these compounds (7 and 8) showed better preclinical activity profiles, inclu

Full text of DOI:10.1016/j.bmc.2004.03.067

Bioorganic & Medicinal Chemistry 12 (2004) 3339–3344
Antitumor agents. Part 227: Studies on novel 40-O-demethyl-
epipodophyllotoxins as antitumor agents targeting
topoisomerase IIq
Zhiyan Xiao,a Kenneth F. Bastow,a John R. Vanceb and Kuo-Hsiung Leea,*
aNatural Products Laboratory, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA
bPlantaceutica Inc., Research Triangle Park, NC 27709-2060, USA
Received 27 August 2003; accepted 23 March 2004
Available online 6 May 2004
Abstract—Eight novel epipodophyllotoxin derivatives (6–13), which were designed to overcome drug resistance and enhance
topoisomerase II inhibition, were synthesized and evaluated. Two of these compounds (7 and 8) showed better preclinical activity
profiles, including cell growth inhibition, cell killing, and in vitro topoisomerase II inhibition, as compared to the prototype
molecule etoposide (1). They also retained the superior drug-resistance profile of GL-331 (4), an epipodophyllotoxin derivative
currently in clinical evaluation.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
R
O
O
O
O
4
Etoposide (1) and teniposide (2) are semisynthetic
derivatives of podophyllotoxin (3), a bioactive compo-
nent of Podophyllum peltatum L (Fig. 1). Although
podophyllotoxin is known as an antimicrotubule agent,
etoposide and teniposide target DNA topoisomerase
II.2;3 Both etoposide and teniposide are clinically useful
against various cancers, including small-cell lung cancer,
testicular carcinoma, lymphoma, and Kaposi’s sar-
coma;4;5 however, their therapeutic uses are often hin-
dered by problems such as acquired drug-resistance. To
obtain better therapeutic agents, extensive synthetic
efforts have been devoted to overcome drug-resistance
and improve topoisomerase II inhibition.
HO
OH
OH
3
O
O
O
A
B
C
D
O
O
O
2
O
O
E
H CO
OCH
3
3
'
4
OR'
H CO
3
OCH
3
OCH
3
'
1. R = CH3; R = H
3
2. R = ; R' = H
S
NO
2
N
N
HN
Molecular area-oriented chemical modification of podo-
phyllotoxin has revealed structural features critical for
the topoisomerase II inhibition: (1) the 4b-configuration
is essential with various substitution accommodated at
C4; (2) the free 40-hydroxy is crucial; (3) the trans-lactone
D ring with 2a, 3b configuration is very important; (4)
the dioxolane A ring is optimal; and (5) the free rotation
CH3
O
O
O
O
O
O
O
O
H CO
3
OCH
H CO
3
OCH
3
3
OH
OH
4
5
q For Part 226, see Ref. 1.
Figure 1. Structures of etoposide (1), teniposide (2), podophyllotoxin
(3), GL-331 (4), and TOP-53 (5).
* Corresponding author. Tel.: +1-919-962-0066; fax: +1-919-966-3893;
e-mail: khlee@unc.edu
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.067

3340
Z. Xiao et al. / Bioorg. Med. Chem. 12 (2004) 3339–3344
of ring E is required.6 Such structure–activity relation-
ships (SAR) unambiguously demonstrate that C4 is the
only molecular area tolerable to significant structural
diversification.
inactive. Compounds 7 and 8 were also examined with a
clonogenic protocol at a concentration of 5 lM (Table
2). Both compounds (especially 7) were much more
cytotoxic than 1 and GL-331 against KB cells. Impres-
sively, a 30 min exposure to compound 7 at 5 lM was
sufficient to kill almost all treated cells. However, the
cell killing effect seemed to be dose-dependent. No sig-
nificant drug effects were observed for either 7 or 8 after
a 30 min treatment at 0.5 lM (data not shown).
Recently, two C4 variable 1-analogs, GL-331 (4)7 and
TOP-53 (5),8 have been developed. GL-331 (4), a 4b-
arylamino derivative, was more active than 1 both in
vitro and in vivo,9 and retained cytotoxicity against 1-
resistant cells.10 It is currently under phase II clinical
evaluation against several forms of cancer, especially 1-
resistant malignancies.11 TOP-53 (5), a 4b-alkylated 1-
analog, was a more potent topoisomerase II inhibitor
than 1. It exhibited high activity to nonsmall cell lung
cancer in both tumor cells and animal tumor models,12
and showed nearly wild-type potency against a mutant
yeast type II enzyme highly resistant to 1.13 Compound
5 is currently in phase I clinical trials.6 Both GL-331 and
TOP-53 showed topoisomerase II inhibitory activity,
antitumor spectra, and drug-resistance profiles signifi-
cantly different from those of 1, which suggested an
important role of various C4 substitution to the activity
profiles of 1-related analogs and the feasibility of opti-
mizing this compound class through rational C4 modi-
fication.
Because topoisomerase II is the pharmacological target
of clinical relevance, mechanistic approaches targeting
the enzyme should be more appropriate for the evalua-
tion of these analogs. Both in vitro topoisomerase II
inhibition and cellular protein-linked DNA breakage
(PLDB) induction assays were applied to further assess
compounds 6–13. When all compounds were tested at
the 50 lg/mL concentration, compounds 7, 10, and 11
showed comparable in vitro inhibition of topoisomerase
II catalytic activity to 1 and GL-331 (Table 1). The
minimum concentrations for 7 and 8 to induce detect-
able linear DNA were determined as 5 lM, the same
concentration at which 7 and 8 showed significant cell
killing activity (Table 2), while those for 1 and GL-331
were 20 and 5 lM, respectively. In the cellular protein–
DNA complex formation assay, only compounds 7, 8,
and 10 induced marked levels of cellular protein-linked
DNA breaks at the tested condition, although none of
them were superior to etoposide or GL-331 (Table 1).
This postulation coincides with the composite pharma-
cophore model proposed by MacDonald et al.14 and the
comparative molecular field analysis (CoMFA) models
generated by us.15;16 Both models indicated that the C4
molecular area could accommodate considerable struc-
tural diversity. The CoMFA model further demon-
strated that bulky substituents at C4 might be favorable
for topoisomerase II inhibition. Accordingly, we de-
signed several novel 40-O-demethyl-epipodophyllotoxin
derivatives (6–13, Scheme 1) bearing bulky tails at the
C4 side chain with the aim to overcome drug resistance
and enhance topoisomerase II inhibition simulta-
neously. Because it was previously observed that the
linking groups to the C4 atom might be important for
the activity of this compound class,17 different types of
C4 linkages, including p-carbonyl anilino, p-amino ani-
lino, amino, and carbonyl, were explored. For most
compounds, protected a-amino acids were introduced to
modulate the water-solubility of this compound class
and potentially address another problem associated with
1 and 2, poor water-solubility.
The most promising compound, 7, was further examined
in parallel with GL-331 for its ability to induce double-
strand (ds) DNA breaks and reduce KB cell colonies
after short exposure (Fig. 2). Although the induction of
ds DNA breaks did not correlate with cytotoxicity for
either GL-331 or 7 at a concentration of 5 lM, com-
pound 7 was superior to GL-331 in both assays.
3. Discussion and conclusion
The results in Table 1 revealed preliminary structure–
activity relationships (SAR) for the tumor cell growth
inhibitory activity of these novel epipodophyllotoxins.
The 4b-anilino moiety at the C4 position might be
important for inhibition against tumor cell growth (GL-
331 and 7–9 vs 10–13). Extending the 4b-anilino ring
with bulky groups containing another aryl ring might
retain the superior activity, particularly the drug-resis-
tance profile, of the 4b-arylamino analogs (GL-331 vs 7–
9). However, the spacer (e.g., distance and linkage)
between the aryl rings might be critical (6 vs 7–9). De-
protection to free the amine residue markedly reduced
activity (10 vs 11). The structural requirement for
topoisomerase II inhibition was less obvious according
to the available data. Although these SAR postulates
are rudimentary, the good activity profiles of com-
pounds 7 and 8 support the molecular design of
extending the 4b-anilino moiety with bulky tails.
2. Results
Compounds 6–13 were first evaluated for their inhibi-
tory activity against multiple tumor cell lines (Table 1).
Compounds 7, 8, and 9 showed cell growth inhibitory
activity (expressed as ED50) superior or comparable to
that of 1 and GL-331 against the tested cell lines.
Notably, they retained inhibitory activity against the
KB/7d 1-resistant tumor cell line, and thus shared the
superior drug-resistance profile of GL-331.
In the cytotoxicity assay (Table 1, data given as LD50),
compound 7 was more potent than GL-331 against
MCF-7, KB, and KB-7d cells, but compound 9 was

Z. Xiao et al. / Bioorg. Med. Chem. 12 (2004) 3339–3344
3341
Scheme 1. Preparation of compounds 6–13.
Consistent with the previous observations,18 in vitro
topoisomerase II inhibition correlated reasonably well
with the ability to induce cellular protein-linked DNA
breaks (PLDB). (The only exception, compound 11,
failed to induce significant amount of PLDB and also
showed no marked inhibition of cell growth. Because it
was much more polar than the other analogs, poor
cellular uptake might be responsible for the lack of
activity.) The in vitro topoisomerase II inhibitory
activity of these compounds did not correlate with their
inhibitory activity against cell growth. In spite of their
weak inhibition against cell growth, compounds 10 and

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Z. Xiao et al. / Bioorg. Med. Chem. 12 (2004) 3339–3344
Table 1. Biological evaluation of compounds 6–13
Com-
pound
In vitro
topo II
% PLDB
forma-
ED50 (lM)c
LD50 (lM)c
A549d
MCF-7
KB
KB-7d
A549
MCF-7
KB
KB-7d
inhibitiona tionb
1
P
100
244
ND
76
5.2
0.7
>20
12.0
ND
4.5
0.3
0.3
17.2
1.9
>50
6.6
21
5.3
49.0
9.9
>50
<50
ND
<20
ND
>50
ND
ND
ND
ND
4
P
6
ND
P
ND
2.4
>25
0.7
ND
4.5
ND
12.5
ND
NA
ND
ND
ND
ND
ND
<3.1
ND
45
ND
1.7
7
8
WP
WP
P
58
16
ND
0.8
ND
1.9
0.7
3.5
ND
>10
ND
ND
ND
ND
9
0.49
3.9
0.41
11.9
30.2
16.2
5.6
10
11
12
13
94
8
15.9
54.3
>10
NA
17.4
41.7
ND
ND
ND
ND
ND
ND
P
18.8
11.0
10.4
ND
NA
ND
4
ND ¼ not determined; NA ¼ not active at 10 lM under test condition.
a All compounds were tested at 50 lg/mL. Compounds that resulted in a level of linear DNA comparable to 1 and 4 were classified as poisons (P), and
those compounds that resulted in a level of linear DNA inferior to 1 and 4 were classified as weak poisons (WP).
b % PLDB formation, percentage of cellular protein-linked DNA breaks formed relative to 1. All compounds were tested at 10 lg/mL.
c ED50 is the concentration that inhibits 50% cell replication after 3 days of continuous treatment; LD50 is the concentration that kills 50% of the cells
after 30 min of treatment.
d Cell lines: A549 (lung adenocarcinoma); MCF-7 (breast adenocarcinoma); KB (nasopharyngeal carcinoma), and KB-7d (1-resistant KB sub-clone).
Table 2. Clonogenic assay of compounds 1, 4, 7, and 8
inhibition of tubulin polymerization) and differences in
cellular uptake and chromosomal DNA breakage pat-
terns could contribute to the lack of correlation between
these parameters.
Compounda
Colony number
% Inhibitionb
Control
110, 98
85, 93
64, 48
0, 1
––
1
4
7
8
0c
46%
99.5%
82%
In summary, based on previous structure–activity rela-
tionship and molecular modeling studies, eight novel C4
modified epipodophyllotoxin derivatives were designed
and synthesized to overcome drug resistance and
enhance topoisomerase II inhibition simultaneously.
Two compounds (7 and 8) showed superior preclinical
activity profiles to the etoposide (1) prototype. They
were potent inhibitors of tumor cell growth and potent
cytotoxic agents (Tables 1 and 2). These activities are
likely to be mediated by DNA topoisomerase II
according to the plasmid cleavage and PLDB induction
assays (Table 1 and Fig. 2). Compounds 7 and 8 also
exhibited a good drug-resistance profile similar to that
of GL-331. These results agree with the hypothesis that
C4 derivation is an effective approach to optimize the
activity profiles of this compound class. The preparation
and biological testing of additional 40-O-demethyl-
epipodophyllotoxin derivatives with bulky substitutions
at C4 position are underway and will be reported upon
completion of the studies.
17, 21
a All compounds were tested at 5 lM.
b 250 cells were plated and the plating efficiency of cells was 41%. The
inhibition percentage was calculated by comparing the number of
colonies surviving drug treatment with that in the control.
c Not markedly different from control.
100
90
80
70
60
50
40
30
20
10
0
300000
200000
100000
GL331
GL331
0
7
0
10
20
30
7
min
Figure 2. ds DNA break induction and KB cytotoxicity of GL-331 (4)
and 7. Both compounds were tested 5 lM. Double-stranded DNA
breaks: KB cells were mock treated (medium change only) or treated
with GL-331 (4) or 7. The increases in intensities of drug-induced
double-stranded DNA breaks were quantified by measuring the fluo-
rescence intensity of the corresponding bands as compared to that in
the control. Inhibition of clonogenic potential: the data were based on
the colony-forming ability of cells after short treatment (30 min) with
the tested compounds. The inhibition percentage was calculated by
comparing the number of colonies surviving drug treatment with that
in the control.
4. Experimental
4.1. Chemistry
Compounds 6–13 were prepared from podophyllotoxin
(3, Scheme 1) according to previously published meth-
ods. Briefly, 40-demethyl-epipodophyllotoxin (DMEP,
14) was synthesized from 3 stereoselectively through
11 were characterized as topoisomerase II poisons in
vitro. In contrast, the potent cell growth inhibitors,
compounds 8 and 9, were found to be only weak enzyme
poisons. Other possible mechanisms of action (e.g.,
successive
40-demethylation,
4-iodination
with
methanesulfonic acid/sodium iodide, and nucleophilic
substitution with water.19 This important intermediate
was subjected to nucleophilic displacement by 4-amino

Z. Xiao et al. / Bioorg. Med. Chem. 12 (2004) 3339–3344
3343
1
benzoic acid, p-nitroaniline, sodium azide, and tri-
methylsilyl cyanide to provide intermediates 15, 16, 17,
and 18, respectively. Compound 15 was condensed with
the appropriate amines in the presence of N,N0-di-
cyclohexylcarbodiimide (DCC) and 4-(dimethyl-
amino)pyridine (DMAP) to provide compounds 6–8.
Intermediates 16 and 17 underwent Pd–C catalyzed
hydrogenation to afford 19 and 20, respectively, which
were subsequently condensed with the corresponding
acids to give compounds 9 and 10. Deprotection of
compound 10 through catalytic hydrogenation provided
compound 11. Intermediate 18 was easily hydrolyzed to
the carboxylic acid 21 in the presence of acetic acid and
hydrochloric acid.20 Condensation of 21 with the
appropriate amines provided compounds 12 and 13,
respectively. The structures of compounds 6–13 were
confirmed by spectroscopic and analytical data.
MS m=e: 787 [M]þ; H NMR (acetone) d 9.01 (s, 1H,
–OH), 7.40 (d, J ¼ 8:7 Hz, 2H, 300,500-H), 7.32 (m, 5H,
20000-60000-H), 7.12 (d, J ¼ 8:4 Hz, 2H, 20,60-H), 6.82 (s, 1H,
5-H), 6.75 (d, J ¼ 8:4 Hz, 2H, 30,50-H), 6.69 (d,
J ¼ 8:7 Hz, 2H, 200,600-H), 6.52 (s, 1H, 8-H), 6.39 (s, 2H,
20,60-H), 5.96 (s, 2H, –OCH2O–), 5.03 (d, J ¼ 6:9 Hz,
2H, –OCH2–Ph), 4.88 (m, 1H, 1-H), 4.55 (d, J ¼ 4:5 Hz,
1H, 4-H), 4.41 (m, 1H, –CO–CH–N–), 4.39 (t,
J ¼ 8:1 Hz, 1H, 11-H), 3.91 (t, J ¼ 8:1 Hz, 1H, 11-H),
3.70 (s, 6H, 30,50-OCH3), 3.31–3.10 (m, 4H, 2, 3-H,
–CO–CH–CH2–), 2.92 (m, 1H, 3-H).
25
D
Compound 10: yield 80%; mp 155–157 °C; ½aꢀ )24.0 (c
0.05, acetone); IR (film) 1745 (lactone), 1735 (amides)
1475, 1450, 1420 (aromatic C@C) cmꢁ1; MS m=e: 697
[M+1]þ; 1H NMR (DMSO) d 9.15 (s, 1H, –OH), 8.37 (d,
J ¼ 8:1 Hz, 1H, –NH), 8.23 (s, 1H, –OH), 7.46 (d,
J ¼ 7:5 Hz, 1H, –NH), 7.30 (m, 5H, 20-60-H), 7.05 (d,
J ¼ 8:4 Hz, 2H, 200,600-H), 6.65 (d, J ¼ 8:4 Hz, 2H, 30,500-
H), 6.57 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6.24 (s, 2H, 20,60-
H), 5.99 (d, J ¼ 15:3 Hz, 2H, –OCH2O–), 5.13 (m, 1H,
1-H), 4.96 (d, J ¼ 5:1 Hz, 2H, –OCH2–Ph), 4.50 (d,
J ¼ 5:1 Hz, 1H, 4-H), 4.19 (t, J ¼ 8:1 Hz, 1H, 11-H),
4.11 (m, 1H, –CO–CH–N–), 3.85 (t, J ¼ 8:1 Hz, 1H, 11-
H), 3.63 (s, 6H, 30,50-OCH3), 3.32 (m, 1H, 2-H), 3.05 (d,
J ¼ 5:4 Hz, 2H, –CO–CH–CH2–), 2.82 (m, 1H, 3-H).
25
D
Compound 6: yield 45%; mp 152–154 °C (dec); ½aꢀ
)36.0 (c 0.05, acetone); IR (film) 1727 (lactone and
amide) 1475, 1455, 1273 (aromatic C@C) cmꢁ1; MS m=e:
709 [M)1]þ; H NMR (CDCl3) d 7.52 (d, J ¼ 8:7 Hz,
1
2H, 200,600-H), 7.43 (d, J ¼ 8:7 Hz, 2H, 20,60-H), 7.41 (m,
2H, 40000,70000-H), 6.72 (s, 1H, 5-H), 6.63 (d, J ¼ 8:7 Hz,
2H, 30,50-H), 6.62 (m, 50000,60000-H), 6.54 (s, 1H, 8-H), 6.53
(d, J ¼ 8:7 Hz, 2H, 300,500-H), 6.33 (s, 2H, 20,60-H), 5.90
(dd, 2H, –OCH2O–), 4.74 (m, 1H, 4-H), 4.60 (d,
J ¼ 4:8 Hz, 1H, 1-H), 4.37 (t, J ¼ 7:8 Hz, 1H, 11-H),
3.78 (m, 1H, 11-H), 3.80 (s, 6H, 30,50-OCH3), 3.50 (m,
1H, 2-H), 3.06 (m, 1H, 3-H).
25
Compound 11: yield 30%; mp 172–175 °C; ½aꢀ )66.0 (c
D
0.05, acetone); IR (film) 1770 (lactone), 1701 (amide),
1465, 1458, 1365 (aromatic C@C), 1122 (phenol) cmꢁ1
;
MS m=e: 563[M+1]þ; 1H NMR (CD3OD) d 7.04 (d,
J ¼ 8:4 Hz, 2H, 200,600-H), 6.68 (d, J ¼ 8:4 Hz, 2H, 300,500-
H), 6.32 (s, 1H, 5-H), 6.20 (s, 2H, 20,60-H), 6.19 (s, 1H, 8-
H), 5.93 (dd, J ¼ 2:4, 0.9 Hz, 2H, –OCH2O–), 4.98 (d,
J ¼ 2:1 Hz, 1H, 1-H), 4.42 (d, J ¼ 5:1 Hz, 1H, 4-H),
4.23 (t, J ¼ 8:1 Hz, 1H, 11-H), 3.96 (t, J ¼ 8:1 Hz, 1H,
11-H), 3.70 (s, 1H, –CO–CH–N–), 3.60 (s, 6H, 30,50-
OCH3), 3.18 (m, 1H, 2-H), 2.88 (m, 1H, 3-H).
25
D
Compound 7: yield 31%; mp 203 °C (dec); ½aꢀ )183.3 (c
0.03, acetone); IR (film) 1737 (lactone) 1727 (amide and
ester) 1462, 1445, 1427 (aromatic C@C) 1217 (phe-
nol) cmꢁ1; MS m=e: 696 [M]þ; 1H NMR (acetone) d 8.38
(s, 1H, –OH), 7.71 (d, J ¼ 8:7 Hz, 2H, 300,500-H), 7.51 (d,
J ¼ 8:1 Hz, 1H, –NH), 7.21 (s, 1H, –OH), 7.12 (d,
J ¼ 8:7 Hz, 2H, 20,60-H), 6.82 (s, 1H, 5-H), 6.75 (d,
J ¼ 8:7 Hz, 2H, 30,50-H), 6.74 (d, J ¼ 8:7 Hz, 2H, 200,600-
H), 6.52 (s, 1H, 8-H), 6.40 (s, 2H, 20,60-H), 5.96 (dd, 2H,
–OCH2O–), 5.03 (m, 1H, 1-H), 4.80 (m, 1H, –CO–CH–
N–), 4.55 (d, J ¼ 4:5 Hz, 1H, 4-H), 4.39 (t, J ¼ 8:1 Hz,
1H, 11-H), 3.85 (t, J ¼ 8:1 Hz, 1H, 11-H), 3.70 (s, 6H,
30,50-OCH3), 3.25–3.02 (m, 4H, 2, 3-H, –CO–CH–CH2–).
25
Compound 12: yield 35%; mp 131–133 °C; ½aꢀ )100.0
D
(c 0.2, acetone); IR (film) 1740, 1735 (lactone and amide)
1455, 1365 (aromatic C@C) 1217 (phenol) cmꢁ1; MS
m=e: 574 [M)1]þ; H NMR (CDCl3) d 7.37 (m, 5H, 200-
1
600 H), 7.08 (s, 1H, 5-H), 6.39 (s, 1H, 8-H), 6.07 (s, 2H,
20,60-H), 5.91 (d, J ¼ 1:8 Hz, 2H, –OCH2O–), 5.17 (d,
J ¼ 2:7 Hz, 1H, 1-H), 4.11 (m, 5H, 4, 11-H, –NH–CH2–),
3.72 (s, 6H, 30,50-OCH3), 3.13 (m, 1H, 3-H), 2.80 (m, 1H,
2-H).
25
D
Compound 8: yield 91%; mp 177–179 °C (dec); ½aꢀ
)48.0 (c 0.05, acetone); IR (film) 1736 (lactone) 1727
(amide and ester) 1478, 1461, 1433 (aromatic C@C)
1217 (phenol) cmꢁ1; MS m=e: 720 [M+1]þ; 1H NMR
(acetone, D2O exchange) d 7.67 (d, J ¼ 8:7 Hz, 2H, 300,
500-H), 7.58 (d, J ¼ 8:1 Hz, 1H, 10-H), 7.35 (d,
J ¼ 8:1 Hz, 1H, 20-H), 7.22 (s, 1H, –CO–NH–), 7.02 (m,
4H, 40-70-H), 6.77 (s, 1H, 5-H), 6.73 (d, J ¼ 8:7 Hz, 2H,
200,600-H), 6.49 (s, 1H, 8-H), 6.35 (s, 2H, 20,60-H), 5.94 (d,
J ¼ 2:1 Hz, 2H, –OCH2O–), 5.00 (m, 1H, 1-H), 4.90 (m,
1H, –CO–CH–N–), 4.54 (d, J ¼ 4:8 Hz, 1H, 4-H), 4.37
(t, J ¼ 7:8 Hz, 1H, 11-H), 3.78 (t, J ¼ 7:8 Hz, 1H, 11-H),
3.71 (s, 6H, 30,50-OCH3), 3.64 (s, 3H, COOCH3), 3.43–
3.16 (m, 4H, 2, 3-H, –NH–CH–CH2–).
Compound 13: yield 41%; MS m=e: 627 [M)1]þ; 1H
NMR (CDCl3) d 7.46 (d, J ¼ 7:5 Hz, 1H, 10-H), 7.27 (d,
J ¼ 7:8 Hz, 1H, 20-H), 7.18 (s, 1H, 5-H), 7.07 (m, 5H, 8-
H, 40-70-H), 6.88 (s, 2H, 20, 60-H), 5.99 (d, J ¼ 9:9 Hz,
2H, –OCH2O–), 4.88 (m, 2H, 1-H, –NH–CH–CO–),
4.12–3.70 (m, 3H, 4, 11-H), 3.61 (s, 6H, 30,50-OCH3),
3.60 (s, 3H, COOCH3), 3.30–3.17 (m, 4H, 2,3-H, –NH–
CH–CH2–).
4.2. Biology
25
Compound 9: yield 48%; mp 192–195 °C; ½aꢀ )76.0 (c
D
0.05, acetone); IR (film) 1738 (lactone, with shoulder)
Cell growth inhibition assay. Cell growth inhibition was
assayed using the sulforhodamine B (SRB) protocol
1475, 1450, 1420 (aromatic C@C) 1216 (phenol) cmꢁ1
;

3344
Z. Xiao et al. / Bioorg. Med. Chem. 12 (2004) 3339–3344
developed by Rubinstein et al.21 Drug exposure was for
3 days, and the ED50 value was interpolated from dose–
response data.
References and notes
1. Xia, Y.; Yang, Z. Y.; Xia, P.; Bastow, K. F.; Nakanishi, Y.;
Nampoothiri, P.; Hackl, T.; Hamel, E.; Brossi, A.; Kuo, S.
C.; Lee, K.H. Bioorg. Med. Chem. Lett., 2003, 13, 2891.
2. Osheroff, N.; Zechiedrich, E. L.; Gale, K. C. Bio. Essays
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Clonogenic assay. The detailed protocol for clonogenic
assay has been described previously.22 Briefly, 250
freshly trypsinized KB cells were exposed to drug at
various concentrations for 30 min, and then cells were
immediately plated and left undisturbed for 12 days.
Colonies were fixed with formalin, stained with toluidine
blue, and then scored. The plating efficiency and per-
centage of total cells forming colonies were calculated.
LD50 concentrations were determined by interpolating
from graphed data.
8. Terada, T.; Fujimoto, K.; Nomura, M.; Yamashita, J.;
Wierzba, K.; Yamazaki, R.; Shibata, J.; Sugimoto, Y.;
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In vitro DNA topoisomerase II assay. The P4 DNA
unknotting assay was carried out according to the pro-
cedure described previously.18 Assays were performed
with drug concentrations of 50 lg/mL.
9. Lee, K. H. Med. Res. Rev. 1999, 19, 569.
K+-SDS precipitation assay for cellular protein–DNA
complexes. Stimulation of intracellular protein-associ-
ated DNA breaks was measured using a standard assay
method,23 and the procedure was described in our pre-
vious publication.18 All compounds were tested in
duplicate at 10 lg/mL.
10. Chang, J. Y.; Han, F. S.; Liu, S. Y.; Wang, Z. Q.; Lee,
K. H.; Cheng, Y. C. Cancer Res. 1991, 51, 1755.
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Wierzba, K.; Kobunai, T.; Terada, T.; Ohhara, T.;
Tsuruo, T.; Yamada, Y. Cancer Res. 1996, 56, 2809.
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Yamada, Y.; Osheroff, N. Biochemistry 2001, 40, 712.
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C.; Ross, W. E. DNA Topoisomerase in Cancer; Oxford
University: New York, 1991; p 119.
Gel lysis for detection of cellular DNA breaks. A pub-
lished gel-based semi-quantitative assay was used.22
Briefly, KB cells were treated with drugs for various
times and were then harvested, cast in agarose plugs,
and de-proteinized. Gel plugs were subjected to aga-
rose–gel electrophoresis and levels of ethidium-stained
broken DNA fragments were determined using a
STORM phosphor-Images (Molecular Dynamics, Sun-
nyvale, Calif.), and the supplied Image Plant software.
The relative amounts of double-stranded DNA were
compared by measuring the fluorescence intensity of the
corresponding ‘ds DNA fragment’ bands on the gel.
15. Cho, S. J.; Tropsha, A.; Suffness, M.; Cheng, Y. C.; Lee,
K. H. J. Med. Chem. 1996, 39, 1383.
16. Xiao, Z.; Xiao, Y. D.; Feng, J.; Golbraikh, A.; Tropsha,
A.; Lee, K. H. J. Med. Chem. 2002, 45, 2294.
17. Zhou, X. M.; Wang, Z. Q.; Chang, J. Y.; Chen, H. X.;
Cheng, Y. C.; Lee, K. H. J. Med. Chem. 1991, 34, 3346.
18. Lee, K. H.; Imakura, Y.; Haruna, M.; Beers, S. A.;
Thurston, L. S.; Dai, H. J.; Chen, C. H. J. Nat. Prod.
1989, 52, 606.
19. Kamal, A.; Laxman, N.; Ramesh, G. Bioorg. Med. Chem.
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R. M.; Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks,
M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
22. Krishnan, P.; Bastow, K. F. Cancer Chemother. Pharma-
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Acknowledgement
This investigation was supported by a NIH grant CA
17625 awarded to K. H. Lee.
23. Rowe, T. D.; Chen, G. L.; Hsiang, Y. H.; Liu, L. F.
Cancer Res. 1986, 46, 2021.