Antitumor agents. 194. Synthesis and biological evaluations of 4-β- mono-, -di-, and -trisubstituted aniline-4'-O-demethyl-podophyllotoxin and related compounds with improved pharmacological profiles

Article abstract of DOI:10.1021/jm990055f

As a continuation of our structure-activity relationship studies, several new 4-β-substituted 4'-O-demethyl-4-desoxypodophyllotoxins bearing mono-, di-, or trisubstituted anilines have been synthesized and evaluated as inhibitors of DNA topoisomerase II and tumor cell growth in tissue culture. Selected compounds were further evaluated as cytotoxic agents using a clonogenic survival assay. The target compounds include 4'-O-demethyl-4β- [(4''-(benzimidazol-2''-yl)anilino]-4-desoxypodophyllotoxin (21), 4'-O- demethyl-4β-(-)-(4'-camphanamido-anilino)-4-desoxypodophyllotoxin (25), 4- β-disubstituted-anilino-4'-demethyl-4-desoxypodophyllotoxins (18-20, 26), 4- α-disubstituted-anilino-4'-demethyl-4-desoxypodophyllotoxin (27), 4-β- trisubstituted-anilino-4'-demethyl-desoxypodophyllotoxin (22, 23), and 4'-O- demethyl-4β-[4'-(benzimidazol-2''-yl)amino]-4-desoxypodophyllotoxin (24). Among the target series, 19, 21, and 24 displayed significant growth inhibitory action against a panel of tumor cell lines including human epidermoid carcinoma of the nasopharynx (KB) and its etoposide-resistant (KB7B) and vincristine-resistant (vin20c KB) subclones, lung carcinoma (A549), human ileocecal carcinoma (HCT-8), human kidney carcinoma (CAKI-1), breast adenocarcinoma (MCF-7), and human malignant melanoma (SK-MEL-2) cells. Compounds 19, 21, 24, and 25 were 'cleavable-complex'-forming DNA topoisomerase II inhibitors with either improved or similar activity compared with the prototype drug etoposide (VP-16). Compound 21 was the most active analogue, being 10-fold more potent than etoposide in both cell killing and topoisomerase II inhibition in vitro assays. Using mouse models of antitumor activity, 21 was effective against (P388/0) leukemia but not against the growth of a (MCF7) mammary tumor.

Full text of DOI:10.1021/jm990055f

J . Med. Chem. 1999, 42, 2441-2446
2441
An titu m or Agen ts. 194. Syn th esis a n d Biologica l Eva lu a tion s of 4-â-Mon o-, -Di-,
a n d -Tr isu bstitu ted An ilin e-4′-O-d em eth yl-p od op h yllotoxin a n d Rela ted
Com p ou n d s w ith Im p r oved P h a r m a cologica l P r ofiles1
Xiao-Kang Zhu,† J ian Guan,† Yoko Tachibana,† Kenneth F. Bastow,† Sung J in Cho,† Huey-Hwa Cheng,†
Yung-Chi Cheng,‡ Marc Gurwith,§ and Kuo-Hsiung Lee*,†
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599, Department of Pharmacology, Yale University School of Medicine,
New Haven, Connecticut 06510, and Genelabs Technologies, Inc., Redwood City, California 94063
Received February 1, 1999
As a continuation of our structure-activity relationship studies, several new 4-â-substituted
4′-O-demethyl-4-desoxypodophyllotoxins bearing mono-, di-, or trisubstituted anilines have been
synthesized and evaluated as inhibitors of DNA topoisomerase II and tumor cell growth in
tissue culture. Selected compounds were further evaluated as cytotoxic agents using a clonogenic
survival assay. The target compounds include 4′-O-demethyl-4â-[(4′′-(benzimidazol-2′′-yl)-
anilino]-4-desoxypodophyllotoxin (21), 4′-O-demethyl-4â-(-)-(4′′-camphanamido-anilino)-4-des-
oxypodophyllotoxin (25), 4-â-disubstituted-anilino-4′-demethyl-4-desoxypodophyllotoxins (18-
20, 26), 4-R-disubstituted-anilino-4′-demethyl-4-desoxypodophyllotoxin (27), 4-â-trisubstituted-
anilino-4′-demethyl-desoxypodophyllotoxin (22, 23), and 4′-O-demethyl-4â-[4′′-(benzimidazol-
2′′-yl)amino]-4-desoxypodophyllotoxin (24). Among the target series, 19, 21, and 24 displayed
significant growth inhibitory action against a panel of tumor cell lines including human
epidermoid carcinoma of the nasopharynx (KB) and its etoposide-resistant (KB7B) and
vincristine-resistant (vin20c KB) subclones, lung carcinoma (A549), human ileocecal carcinoma
(HCT-8), human kidney carcinoma (CAKI-1), breast adenocarcinoma (MCF-7), and human
malignant melanoma (SK-MEL-2) cells. Compounds 19, 21, 24, and 25 were “cleavable-
complex”-forming DNA topoisomerase II inhibitors with either improved or similar activity
compared with the prototype drug etoposide (VP-16). Compound 21 was the most active
analogue, being 10-fold more potent than etoposide in both cell killing and topoisomerase II
inhibition in vitro assays. Using mouse models of antitumor activity, 21 was effective against
(P388/0) leukemia but not against the growth of a (MCF7) mammary tumor.
In tr od u ction
Etoposide (VP-16, 1) and teniposide (VM-26, 2), two
semisynthetic podophyllotoxin derivatives, are impor-
tant drugs currently used in the treatment of small-
cell lung cancer, testicular carcinoma, lymphoma, and
Kaposi’s sarcoma.2,3 Another podophyllotoxin derivative,
GL-331 (3), discovered and developed in our laboratory,
is currently undergoing phase II clinical trials for
treatment of various cancers.4
In a previous study,5 a comparative molecular field
analysis (CoMFA) and a novel CoMFA/q2-GRS tech-
nique were developed to identify the essential structural
requirements for podophyllotoxin derivatives to form
topoisomerase II-DNA complexes. In the derived model,
steric and electrostatic contour plots were compared
distinct pharmacophoric domains: the DNA intercalat-
with the DNA phosphate backbone environment of the
ing moiety, the minor groove binding site, and a mo-
DNA-4′-O-demethylepipodophyllotoxin analogue com-
lecular region that can accommodate a number of
plex. This comparison revealed that the CoMFA steric
structurally diverse substituents, which might also
and electrostatic fields are compatible with stereochem-
interact with the minor groove (Figure 1). Active
ical properties of the DNA backbone. Examination of
compounds do not have to interact with all three
domains.6 However, based on the stated analysis, the
functional groups have to be sterically and electrostati-
cally compatible with the environment of the DNA
minor groove (i.e., inactive compounds exhibit detri-
mental steric interactions with the DNA backbone, and
a positive charge around the 4-â-substituent is optimal
the contour plots suggested that topoisomerase II
inhibitory activity was related to three structurally
* To whom correspondence should be addressed.
† University of North Carolina.
‡ Yale University.
§ Genelabs Technologies, Inc.
10.1021/jm990055f CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/16/1999

2442 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Zhu et al.
Sch em e 1a
a
Reagents and conditions: (i) PhNO2 (nitrobenzene), DMF, 150
°C, 24 h; (ii) NaBH4, MeOH.
Sch em e 2a
F igu r e 1. Composite pharmacophore model for etoposide-like
analogues expressing topoisomerase II activity (adapted from
ref 6).
to complement the negative rich environment of the
DNA backbone).
As part of a continuing search for potential anticancer
drug candidates, we have synthesized a series of sub-
stituted 4â-anilino-4′-demethyl-4-desoxypodophyllotox-
ins.7 Among them, GL-331, 4, 5, and 6 were potent
topoisomerase II inhibitors with activities comparable
to those of the antitumor drugs VP-16 and VM-26. Most
a
Reagents: (i) pyridine, (CH2Cl)2.
binding agent pibenzimol (Hoechst 33258)8-10 and, thus,
is an ideal target to increase DNA topoisomerase II
activity based on the ComFA analysis.
Ch em istr y
2-(p-Aminophenyl)-benzimidazole (10) was synthe-
sized in two steps: oxidative condensation11 of p-nitro-
benzaldehyde (7) and 1,2-phenylenediamine (8) in DMF
and nitrobenzene to produce intermediate 9, and reduc-
tion of the nitro group of 9 by NaBH4 in MeOH in the
presence of CuCl (Scheme 1). The strong electron-
withdrawing effect of the p-nitro group increases basic-
ity of the imidazole functional group. Thus, the solubility
of 9 decreased dramatically in most commonly used
organic solvents, and therefore, this compound was
quite difficult to handle and separate.
4
p-Camphanoylaminoaniline (13) was synthesized by
monoacylation of 1,4-diaminoaniline with camphanic
chloride (Scheme 2). Although diacylation does lower the
yield, this procedure is simple, easy to process, and
needs no protective groups.
The intermediate 15 was synthesized by reacting 4′-
demethyl-epipodophyllotoxin (14) and HBr.12 As shown
in Scheme 3, the preparation of 18-24, 26, and 27 was
achieved by treatment of 15 with appropriately substi-
tuted arylamines. Although this reaction occurred via
a SN1 mechanism,13 the bulky C-1R pendent aromatic
ring probably directed the stereoselective substitution,
resulting in preferential formation of C-4â-oriented
products, accompanied by trace amounts of the C-4R-
oriented isomers (i.e., 27).
of the analogues studied to date bear a monosubstituted
aniline component at the 4â-position of podophyllotoxin.
In this paper, we report the synthesis and biological
evaluation of analogues with di- and trisubstituted
anilines at the 4â-position as well as an aniline bearing
a benzimidazole group. Benzimidazole is a structural
component of the cytotoxic and DNA minor groove

Substituted Aniline-4′-O-demethyl-podophyllotoxins
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2443
Sch em e 3
antitumor epipodophyllotoxin analogue, may warrant
further preclinical evaluation in other species.
Exp er im en ta l Section
All melting points were taken on Fisher-J ohns and Mel-
Temp II melting point apparatus and are uncorrected. IR
spectra were recorded on a Perkin-Elmer 1320 spectropho-
tometer. 1H NMR spectra were obtained using Bruker AC-
300 and WM 250 NMR spectrometers with TMS as the
internal standard. All chemical shifts are reported in ppm.
FAB MS and HR-FAB MS spectral analyses were determined
on a J EOL HX-110 instrument. Analytical thin-layer chroma-
tography (TLC) was carried out on Merck precoated aluminum
silica gel sheets (Kieselgel 60 F-254). Optical rotations were
measured with a J ASCO DIP-1000 polarimeter. All new target
compounds were characterized by 1H and IR spectral analyses
and MS analyses.
2-(p-Nitr op h en yl)-ben zim id a zole (9). A mixture of 2.42
g (16 mmol) of 4-nitrobenzaldehyde, 1.78 g (16 mmol) of 1,2-
diaminobenzene, 2.5 mL of nitrobenzene, and 20 mL of DMF
was stirred at 150 °C under N2 for 24 h. After the mixture
cooled to room temperature, the crude product was purified
by column chromatography to give 2.72 g (58.8%), mp 318-
321 °C (DMF).
2-(p-Am in op h en yl)-ben zim id a zole (10). To a mixture of
1.82 g (7.6 mmol) of 9, 151 mg (1.5 mmol) of CuCl, and 30 mL
of MeOH was added 575 mg (15.2 mmol) of NaBH4 in portions
at room temperature. After the reaction was completed as
checked by TLC, the reaction mixture was filtered and
evaporated to dryness. The residue was purified via chroma-
totron to give product (727 mg) in a 45.8% yield. The structure
was verified by spectroscopic and analytical data.
Compounds 18-27 were prepared in an analogous manner.
NMR data were similar, and complete data are given only for
18, 21, and 25.
Biologica l Resu lts a n d Discu ssion
As illustrated in Table 1, compared to etoposide and
GL-331, most 4â-substituted-anilino-podophyllotoxin
compounds bearing di- and trisubstituted anilines (18-
20, 22, 23, 26, 27) showed significantly reduced or
comparable activity in inhibiting human DNA topo-
isomerase II and causing cellular protein-DNA break-
age. The most active compounds, 21 and 25, were almost
10-fold more potent than etoposide in inhibiting human
DNA topoisomerase II and induced similar (25) or more
(21) cellular protein-DNA strand breaks than etoposide
under similar testing conditions. Removing the phenyl
ring linking the amino aryl benzimidazole group of 21
afforded a less active compound (24). Generally, inhibi-
tion of tumor cell growth correlated with activity against
topoisomerase II, with 21 showing the highest activity
in the series in both assay types (Table 2). Compound
21 showed promise for further development since growth
inhibitory activity varied no more than 7-fold against a
panel of human tumor cell lines and drug-resistant KB
subclones (Table 2). Furthermore, 21 was 5-fold more
cytotoxic than GL-331 in a clonogenic cell survival assay
(Table 1). On the basis of the in vitro testing results,
21 was compared to etoposide against implanted tumor
cell growth in mouse models: P388/00, an etoposide-
sensitive tumor, and MCF-7 mammary tumor, an eto-
poside-resistant tumor (Tables 3 and 4, respectively).
Compound 21, at highest tolerated dose, caused sub-
stantially less tumor reduction than etoposide in (P388/
00) leukemia growth at treatment end (Table 3). The
discrepancy between in vitro and in vivo results is not
yet explained, but may be related to metabolic factors.
Compound 21 gave no significant antitumor effect
(results not shown). Thus, 21, a novel and potent
Syn th esis of 4â-P olysu bstitu ted -a n ilin o-4′-d em eth yl-
d esoxyp od op h yllotoxin s (18-27). A solution containing 4′-
O-demethyl-4â-bromo-4-desoxypodophyllotoxin (15) (695 g, 1.5
mmol), anhydrous barium carbonate (590 g, 3.0 mmol), and
the appropriate arylamine (1.65 mmol) in 15 mL of dry
dichloroethane under nitrogen was stirred overnight at room
temperature. The reaction mixture was filtered, diluted with
EtOAc, washed with water, dried over anhydrous magnesium
sulfate, and purified via silica gel column chromatography
using CH2Cl2-acetone-EtOAc (100:5:5) as an eluent. NMR
data for 18-27 were similar, and complete data are given only
for 18, 21, and 25.
4′-O-Dem eth yl-4â-(2′′-n itr o-4′′-m eth oxya n ilin o)-4-d es-
oxyp od op h yllotoxin (18): yield 85.5%; crystals from acetone;
mp 233-235 °C; [R]25D -87.2 (c ) 0.11, CHCl3); IR (KBr) 3350
(OH), 2900 (aliphatic C-H), 1758 (lactone), 1600, 1510, 1480
1
(aromatic CdC) cm-1; MS m/e 550 [M]+; H NMR (DMSO-d6,
D2O exchange) δ 8.32 (b, 1 H, exchangeable, 4′-OH), 7.89 (d, J
) 6.0 Hz, exchangeable, 4-NH), 7.56 (d, J ) 2.91 Hz, 1 H, 3′′-
H), 7.30 (dd, J ) 2.91, 9.31 Hz, 1 H, 5′′-H), 7.24 (d, J ) 9.31
Hz, 1 H, 6′′-H), 6.95 (s, 1 H, 5-H), 6.59 (s, 1 H, 8-H), 6.25 (s, 2
H, 2′,6′-H), 6.02 (d, 2 H, 12-H), 5.23 (dd, J ) 4.7, 12.1, 1 H,
4-H), 4.56 (d, J ) 5.06 Hz, 1 H, 1-H), 4.38 (t, J ) 7.6 Hz, 1 H,
11-H), 3.79 (s, 3 H, 4′′-OCH3), 3.65 (s, 6 H, 3′,5′-OCH3), 3.61
(t, J ) 7.6 Hz, 1 H, 11-H), 3.28 (m, 1 H, 2-H), 3.07 (m, 1 H,
3-H); Anal. (C28H26N2O10‚11/2H2O) C, H, N.
4′-O-Dem eth yl-4â-(2′′-m eth oxy-4′′-n itr oa n ilin o)-4-d es-
oxyp od op h yllotoxin (19): yield 61.8%; crystals from MeOH;
mp 199-200.5 °C; [R]25D -117 (c ) 0.07, CHCl3); IR (KBr) 3390
(OH), 2900 (aliphatic C-H), 1760 (lactone), 1590, 1500, 1475
(aromatic CdC) cm-1; MS m/e 551 [M + 1]+; Anal. (C28H26N2O10)
C, H, N.
4′-O-Dem eth yl-4â-(2′′-m eth oxy-5′′-n itr oa n ilin o)-4-d es-
oxyp od op h yllotoxin (20): yield 77.1%; crystals from MeOH;
mp 223-224 °C; [R]25 -97 (c ) 0.225, CHCl3); IR (KBr) 3390
D
(OH), 2900 (aliphatic C-H), 1770 (lactone), 1610, 1580, 1475
(aromatic CdC) cm-1; MS m/e 550 [M]+; Anal. (C28H26N2O10
1/2H2O) C, H, N.
‚

2444 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Ta ble 1. Cytotoxic Activities and Topoisomerase II Inhibition
Zhu et al.
cytotoxicitya
inhibition of
topo IIb IC100
cellular protein-DNAc
complex formn (%)
compd
R
LD50
18
>20
>100
>100
>100
15 ( 6
19
20
>20
>20
83 ( 25
12
21
0.2
10
128 ( 12
22
23
NDd
ND
>100
>100
4.4 ( 0.5
3.5 ( 0.7
24
25
ND
ND
100
10
58 ( 15
88 ( 22
26
27
ND
ND
>100
>100
20 ( 1
8 ( 0.5
VP-16
20
1
100
10
100
GL-331
122 ( 7
a
Cytotoxicity was determined by a clonogenic “cell survival” assay under conditions described in the Experimental Section. The LD50
b
is the drug concentration (µM) that reduced plating efficiency by 50% relative to control. Inhibition of human DNA topoisomerase II
was determined by the P4 DNA unknotting assay described in the Experimental Section. IC100 is the drug concentration (µM) that completely
inhibited enzyme activity relative to the DNA control reaction without enzyme. c Cellular protein-DNA complex formation was determined
by the SDS/potassium precipitation method (Experimental Section). VP-16 at 50 µm induced DNA complexes 50-56-fold over control
d
levels. Percent values are levels induced by drug treatment (50 µm) relative to the VP-16 control set arbitrarily at 100%. Not determined.
4′-O-Dem et h yl-4â-[4′′-(b en zim id a zol-2′′-yl)a n ilin o]-4-
d esoxyp od op h yllotoxin (21): yield 2.1%; crystals from
4′-O-Dem eth yl-4â-(2′′,5′′-d im eth oxy-4′′-n itr oa n ilin o)-4-
d esoxyp od op h yllotoxin (22): yield 45.8%; crystals from
EtOAc; mp 82-84 °C; [R]25 -75 (c ) 0.02, CHCl3); IR (KBr)
acetone; mp 174-177 °C; [R]25.4 -123.3 (c ) 0.15, CHCl3); IR
D
D
3360 (OH), 2905 (aliphatic C-H), 1760 (lactone), 1600, 1490,
1470 (aromatic CdC) cm-1; FAB MS m/e 592 [M + 1]+; 1H
NMR (CD3OD) δ 7.90 (d, J ) 8.7 Hz, 2H, 3′′,5′′-H), 7.53 (m,
2H, 3′′,6′′-H), 7.19 (m, 2 H, 4′′,5′′-H), 6.83 (d, J ) 8.7 Hz, 2 H,
2′′,6′′-H), 6.79 (s, 1 H, 5-H), 6.51 (s, 1 H, 8-H), 6.38 (s, 2 H,
2′,6′-H), 5.93 (s, 2 H, 12-H), 4.97 (d, J ) 3.8 Hz, 1 H, 4-H),
4.59 (d, J ) 5.1 Hz, 1 H, 1-H), 4.45 (t, J ) 7.7 Hz, 1 H, 11-H),
3.91 (t, J ) 7.7 Hz, 1 H, 11-H), 3.74 (s, 6 H, 3′,5′-OCH3), 3.12
(m, 2 H, 2,3-H); HR-FAB MS (M + H; obtained in glycerol
matrix) calcd for C34H30N3O7 592.2084, found 592.2073.
(KBr) 3350 (OH), 2900 (aliphatic C-H), 1750 (lactone), 1600,
1575, 1510, 1475 (aromatic CdC) cm-1; MS m/e 580 [M]+; Anal.
(C29H28N2O11) C, H, N.
4′-O-Dem eth yl-4â-(2′′-m eth yl-4′′-n itr o-5′′-m eth oxya n ili-
n o)-4-d esoxyp od op h yllot o-xin (23): yield 79.7%; crystals
from CH2Cl2; mp 199-201 °C; [R]26D -136.7 (c ) 0.06, CHCl3);
IR (KBr) 3400 (OH), 2900 (aliphatic C-H), 1770 (lactone),
1610, 1570, 1520 (aromatic CdC) cm-1; MS m/e 565 [M + 1]+;
Anal. (C29H28N2O10) C, H, N.

Substituted Aniline-4′-O-demethyl-podophyllotoxins
Ta ble 2. Tumor Cell Growth Inhibition
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2445
a
cell line/ED50
HCT-8
compd
KB
KB-7d
KB-vin20c
A-549
1.70
0.63
2.25
CAKI-1
MCF-7
2.50
5.00
2.40
0.85
ND
ND
ND
SK-MEL-2
18
19
20
21
22
23
24
25
26
27
VP-16
GL331
0.85
0.32
1.90
0.74
4.00
1.80
0.45
37.00
ND
0.90
5.60
17.50
7.60
23.80d
ND
0.40
6.70
1.30
1.40
20.00
ND
2.00
>10(46)c
4.80
0.63(25-30)b
1.10
3.00
4.80
2.40
2.5(34-39)b
0.35
0.20
0.23
0.63
0.45
18.50
44.50
2.00
21.50
>50(38)c
>50(0)c
3.30
45.00
>50(46)c
>50(7)c
>25(38)c
ND
>50(18)c
3.50
>50(26)c
17.50
1.00
0.80
1.90
2.20
20.00
26
>1.25
10.00
>5.00
10.00
10.00
0.200d
0.49e
>40(47)c
>40(42)c
30.60d
ND
>40(46)c
>5.00(20-29)b
1.95
>5.00(33-42)b
>2.50(36-42)b
>5(34)c
28.50
ND
ND
ND
16.5
>5.00(27-30)b
>5(47)c
8.50
>10.00(20-21)b
2.20
ND
1.50
a
Cytotoxicity as ED50 for each cell line is the concentration of compound that causes a 50% reduction in adsorbance at 562 nm relative
to the untreated cell using the SRB assay (Experimental Section). b Survival curves plateaued at percentage values indicated in parentheses
(up to 10 mM). c When the inhibition was below 50% at the highest test concentration, the growth inhibition value observed is indicated
d
in the parentheses. See ref 14. e See ref 15.
Ta ble 3. Antitumor Activity of Compound 21 against (P388/0)
Leukemia in Mice
4′-O-Dem eth yl-4â-(3′′,5′′-d in itr o-a n ilin o)-4-d esoxyp od o-
p h yllotoxin (26): yield 67%; crystals from EtOAc; mp 206-
208 °C; [R]24 -96.9 (c ) 0.23, CHCl3); IR (KBr) 3350 (OH),
D
therapeutic response
2950 (aliphatic C-H), 1750 (lactone), 1600, 1500, 1475 (aro-
matic CdC) cm-1; Anal. (C27H23N3O11‚13/4MeOH) C, H, N.
4′-O-Dem eth yl-4â-(3′′,5′′-d in itr o-a n ilin o)-4-d esoxyp od o-
p h yllotoxin (27): yield 3%; crystals from EtOAc; mp 192-
net log
change
dosage
median day
of death
in tumor
agent
(mg/kg/dose)a
%ILS burdenb
194 °C; [R]24.4 -34.0 (c ) 0.62, CHCl3); IR (KBr) 3350 (OH),
D
2910 (aliphatic C-H), 1760 (lactone), 1600, 1530, 1500 (aro-
matic CdC) cm-1; Anal. (C27H23N3O11‚3/4EtOAc) C, H, N.
Biologica l Assa ys. The cell growth inhibition assay using
sulforhodamine B (SRB) was carried out according to proce-
dures described in Rubinstein et al.16 Drug stock solutions were
prepared in DMSO and the final solvent concentration was
e2% DMSO (v/v), a concentration without effect on cell
replication. The human tumor cell line panel consisted of
epiderimoid carcinoma of the nasopharynx (KB), two subclones
resistant to etoposide (KB7B) and vincristine (KB vin20c), lung
carcinoma (A549), ileocecal carcinoma (HCT-8), renal cancer
(CAKI-1), breast cancer (CF-7), and melanoma (SKMEL-2).
Cells were cultured at 37 °C in RPMI-1640 with 100 mm/mL
kanamycin and 10% (v/v) fetal bovine serum in a humidified
atmosphere containing 5% CO2. Initial seeding densities varied
among the cell lines to ensure a final absorbance reading in
control (untreated) cultures in the range 1-2.5 A562 units. Drug
exposure was for 3 days, and the ED50 value, the drug
concentration that reduced the absorbance by 50%, was
interpolated from dose-response data. Each test was per-
formed in triplicate, and absorbance readings varied no more
than 5%. ED50 values determined in independent tests varied
no more than 30%.
control (saline)
etoposide
10
26
23
19
17
12
40.0
27.0
10.0
5.0
+160
+130
+90
-5.5
-3.4
-0.7
+0.7
+1.9
21
+70
2.5
+20
a
b
Treatment: IV, Q4DX3. Measured at end of treatment, e.g.,
a -6 log change means that there was 99.9999% reduction and
+3 log change means there was a 1000-fold increase in tumor
burden.
Ta ble 4. Antitumor Activity of Compound 21 against
Subcutaneous Mammary Tumor MCF-7
days to
dosage
nonspecific tumor-free tumor
(mg/kg/dose) deaths/total survival doubling
agent
control (saline)
etoposide
1/15
0/8
0/8
0/8
0/8
1/8
17.1
>24.1
18.5
40.0
27.0
10.0
5.0
2/8
0/8
8/8
2/8
8/8
21
21.5
18.7
2.5
Cytotoxicity Assa y. LD50 concentrations were determined
by treating cells for 3 h with selected compounds at various
concentrations and then replating cells to measure colony
formation and plating efficiency, the percentage of total cells
forming colonies. Compounds were evaluated in three inde-
pendent experiments. The plating efficiencies of untreated cells
varied between experiments with values ranging from 14 to
31% for KB cells. The concentration of compound that reduced
plating efficiency by 50% relative to control, the LD50, was
determined graphically as stated in the preceding section. LD50
values determined in independent tests varied by no more than
15%.
En zym e Assa y. Inhibition of topoisomerase II activity was
examined using the P4 DNA unknotting assay.17 Reactions
contained 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM
MgCl2, nuclease-free bovine serum albumin (30 mg/mL), 0.5
mM DTT, 0.5 mM EDTA, 1 mM ATP, P4 DNA substrate (10
mg/mL), 0.25 U enzyme (human p170 isoform; TopoGen,
Columbus, OH), and various amounts of test compounds. The
dimethyl sulfoxide carrier did not interfere with catalytic
activity at the highest concentration used. After 1 h at 37 °C,
reactions were terminated by the addition of SDS-sucrose stop
solution and analyzed after horizontal agarose (0.7%, v/v) gel
4′-O-Dem eth yl-4â-[4′′-ben zim id a zol-2′′-yl)a m in o]-4-d es-
oxyp od op h yllotoxin (24): yield 46.7%; crystals from EtOAc;
mp 273-276 °C; [R]25 -75 (c ) 0.02, CHCl3); IR (KBr) 3340
D
(OH), 2910 (aliphatic C-H), 1770, 1700 (lactone), 1620, 1595,
1575, 1500, 1475 (aromatic CdC) cm-1; MS m/e 516 [M + 1]+;
Anal. (C28H25N3O7‚1/2EtOAc) C, H, N.
4′-O-Dem et h yl-4â-(4′′-ca m p h a n a m id o-a n ilin o)-4-d es-
oxyp od op h yllotoxin (25): yield 69.6%; crystals from MeOH;
mp 210-212 °C; [R]24.7 -134.0 (c ) 0.19, CHCl3); IR (KBr)
D
3350 (OH), 2950 (aliphatic C-H), 1760, 1650 (lactone), 1610,
1500, 1475 (aromatic CdC) cm-1 1H NMR (CDCl3, D2O
;
exchange) δ 7.43 (d, J ) 8.7 Hz, 2 H, 3′′,5′′-H), 6.75 (s, 1 H,
5-H), 6.54 (d, J ) 8.7 Hz, 2H, 2′′,6′′-H), 6.53 (s, 1 H, 8-H), 6.33
(s, 2 H, 2′,6′-H), 5.97 (d, J ) 5.3 Hz, 2 H, 12-H), 5.45 (b, 1 H,
exchangeable, 4-NH), 4.66 (d, J ) 4.9 Hz, 1 H, 4-H), 4.60 (d,
J ) 4.8 Hz, 1 H, 1-H), 4.38 (t, J ) 8.0 Hz, 1 H, 11-H), 3.98 (t,
J ) 8.0 Hz, 1 H, 11-H), 3.80 (s, 6 H, 3′,5′-OCH3), 3.14 (d,d,
J ) 4.7, 14 Hz, 1 H, 2-H), 3.0 (m, 1 H, 3-H), 2.6, 2.05, 1.75
(each m, 4 H in total, camphanoyl-CH2), 1.18 (s, 3 H, cam-
phanoyl-CH3), 1.16 (s, 3 H, camphanoyl-CH3), 1.0 (s, 3 H,
camphanoyl-CH3); Anal. (C27H23N3O11) C, H, N‚1/2H2O.

2446 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 13
Zhu et al.
Cancer; Potmesil, M., Kohn, K. W., Eds; Oxford University
Press: New York, 1991; pp 119-214.
electrophoresis by staining with ethidium bromide and pho-
tography (Polaroid Type 667). Inhibition was quantified by eye,
by comparing catalytic activity in test reactions to the DNA
substrate and enzyme control reaction.
Cellu la r P r otein -DNA Com p lex F or m a tion . Stimula-
tion of intracellular protein-associated DNA breaks was
measured using a standard assay method.18 Briefly, KB cells
were labeled overnight with tritiated thymidine (0.5 mCi/mL,
60 Ci/mmol), chased for 2 h, and then treated in triplicate with
test compounds at 15 mm. After 1 h, samples were processed,
and protein-DNA complexes were measured as potassium-
SDS precipitable radioactivity.
Activity in Im p la n ted Tu m or Bea r in g Mice. Com-
pounds were tested for antitumor activity in mouse models
by Southern Research Institute (Birmingham, AL) under a
contracted agreement. Briefly, female CD 2F1 or athymic nude
mice were implanted (ip) with P388/0 leukemia or (sc) with
MCF-7 human mammary tumor cell, respectively. Implanted
mice were treated (iv) with either saline (control), etoposide
(in saline), or compound 21 in 10% DMSO + 35% PEG 300 +
5% Tween 80 + 50% D5W. Injection volume was 0.1 cm3/10 g
body weight, and treatment schedule was Q4D × 3.
(7) Zheng, J .; Wang, H.-K.; Bastow, K. F.; Zhu, X.-K.; Cho, S. J .;
Cheng, Y.-C.; Lee, K. H. Antitumor Agents. 177. Design,
Syntheses, and Biological Evaluation of Novel Etoposide Ana-
logues Bearing Pyrrolecarboxamidino Group as DNA Topo-
isomerase II Inhibitors. Bioorg. Med. Chem. Lett. 1997, 7, 607-
612.
(8) Pjura, P. E.; Grzeskowiak, K.; Dicherson, R. E. Binding of
Hoechst 33258 to the minor groove of B-DNA. J . Mol. Biol. 1987,
197, 257-271.
(9) Teng, M.; Usman, N.; Frederick, C. A.; Wang, A. H.-J . The
molecular structure of the complex of Hoechst 33258 and the
DNA dodecamer d(CGCGAATTCGCG). Nucleic Acids Res. 1988,
16, 2671-2690.
(10) Carrondo, M.; Coll, M.; Ayami, J .; Wang, A. H.-J .; van der Marel,
G. A.; van Boom, J . H.; Rich, A. Binding of a Hoechst dye to
d(CGCGATATCGCG) and its influence on the conformation of
the DNA fragment. Biochemistry 1989, 28, 7849-7859.
(11) Su, Q.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J . Sythesis
and Evaluation of Terbenzimidazoles as Topoisomerase I Inhibi-
tors. J . Med. Chem. 1995, 38, 3638-3644.
(12) Kuhn, M.; Keller-J uslen, C.; von Warburg A. Partial synthese
von 4′-Demethylepipodophyllotoxin. Helv. Chim. Acta. 1969, 52,
944-947.
(13) Lee, K. H.; Beers, S. A.; Mori, M.; Wang, Z.-Q.; Kuo, Y.-H.; Li,
L.; Liu, S.-Y.; Chang, J .-Y.; Han, F.-S.; Cheng, Y.-C. Antitumor
Agents. 111. New 4-Hydroxylated Anilino Derivatives of 4′-
Demethylepipodophyllotoxin as Potent Inhibitors of Human
DNA Topoisomerase II. J . Med. Chem. 1990, 33, 1364-1368.
(14) Ferguson, P. J .; Fisher, M. H.; Stephenson, J .; Li, D.-H.; Zhou,
B.-S.; Cheng, Y. C. Combined modalities of Resistance in
Etoposide-resistant Human KB Cell Lines. Cancer Res. 1988,
48, 5956-5964.
(15) Wang, Z. Q.; Hu, H.; Chen, H. X.; Cheng, Y. C.; Lee, K. H.
Antitumor Agents. 124. New 4â-Substituted Aniline Derivatives
of 6,7-O,O,-Demethylene-4′-O-Demethylpodophyllotoxin and Re-
lated Compounds as potent Inhibitors of Human DNA Topo-
isomerase II. J . Med. Chem. 1992, 35, 871-877.
(16) Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.;
Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, M. R. Comparison
of In Vitro Anticancer-Drug-Screening Data Generated With a
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Ack n ow led gm en t . This investigation was sup-
ported in part by grant CA 17625 from the National
Cancer Institute awarded to K.H.L.
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