2,3-Dimethoxybenzo[i]phenanthridines: Topoisomerase I-targeting anticancer agents

Article abstract of DOI:10.1016/S0968-0896(02)00530-8

Appropriately substituted benzo[i]phenanthridines structurally related to nitidine, a benzo[c]phenanthridine alkaloid with antitumor activity, are active as topoisomerase I-targeting agents. Studies on benzo[i]phenanthridines have indicated analogues that possess a 2,3-methylenedioxy moiety and at least one and preferably two methoxyl groups at the 8- and 9-positions, such as 8,9-dimethoxy-2,3-methylenedioxybenzo[i]phenanthridine, 2, are active as topoisomerase I-targeting agents. Tetramethoxylated benzo[i]phenanthridines, wherein the 2,3-methylenedioxy moiety is replaced with methoxyl groups at the 2- and 3-position, are inactive as a topoisomerase I-targeting agent. These results initially suggested that the 2,3-methylenedioxy moiety was critical to the retention of potent activity. Further studies revealed that 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine, 7a, is more potent than 2 as a topoisomerase I-targeting agent. The observation that 2,3-dimethoxylated benzo[i]phenanthridines can actually exhibit enhanced activity prompted the present study in which several 8-substituted 2,3-dimethoxybenzo[i]phenanthridines were prepared and their pharmacological activities evaluated. The influence of NH₂, CN, CH₂OH, OBn, OCH₃, OH, and NHCOCH₃substituents at the 8-position on the relative activity of these 2,3-dimethoxybenzo[i]phenanthridines was examined. Relative to these derivatives, 7a was the most potent topoisomerase I-targeting agent, possessing similar cytotoxicity to that of nitidine in the human lymphoblast tumor cell line, RPMI8402.

Full text of DOI:10.1016/S0968-0896(02)00530-8

Bioorganic & Medicinal Chemistry 11 (2003) 521–528
2,3-Dimethoxybenzo[i]phenanthridines: Topoisomerase I-Targeting
Anticancer Agents
Dajie Li,a Baoping Zhao,a Sai-Peng Sim,b Tsai-Kun Li,b Angela Liu,b
Leroy F. Liub,c and Edmond J. LaVoiea,c,*
aDepartment of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, 160Frelinghuysen Road, Piscataway,
NJ 08854-8020, USA
bDepartment of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School,
Piscataway, NJ 08854, USA
cThe Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
Received 28 July 2002; accepted 18 September 2002
Abstract—Appropriately substituted benzo[i]phenanthridines structurally related to nitidine, a benzo[c]phenanthridine alkaloid
with antitumor activity, are active as topoisomerase I-targeting agents. Studies on benzo[i]phenanthridines have indicated analo-
gues that possess a 2,3-methylenedioxy moiety and at least one and preferably two methoxyl groups at the 8- and 9-positions, such
as 8,9-dimethoxy-2,3-methylenedioxybenzo[i]phenanthridine, 2, are active as topoisomerase I-targeting agents. Tetramethoxylated
benzo[i]phenanthridines, wherein the 2,3-methylenedioxy moiety is replaced with methoxyl groups at the 2- and 3-position, are
inactive as a topoisomerase I-targeting agent. These results initially suggested that the 2,3-methylenedioxy moiety was critical to the
retention of potent activity. Further studies revealed that 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine, 7a, is more
potent than 2 as a topoisomerase I-targeting agent. The observation that 2,3-dimethoxylated benzo[i]phenanthridines can actually
exhibit enhanced activity prompted the present study in which several 8-substituted 2,3-dimethoxybenzo[i]phenanthridines were
prepared and their pharmacological activities evaluated. The influence of NH2, CN, CH2OH, OBn, OCH3, OH, and NHCOCH3
substituents at the 8-position on the relative activity of these 2,3-dimethoxybenzo[i]phenanthridines was examined. Relative to these
derivatives, 7a was the most potent topoisomerase I-targeting agent, possessing similar cytotoxicity to that of nitidine in the human
lymphoblast tumor cell line, RPMI8402.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Camptothecin and its structurally-related analogues
that target TOP1 have been extensively studied. Bi-
and terbenzimidazoles,7ꢀ10 certain benzo[c]phenan-
thridine and protoberberine alkaloids and their syn-
thetic analogues,11ꢀ15 indolocarbazoles,16 the fungal
metabolites, bulgarein17 and saintopin,18 and indenoi-
soquinolines,19,20 phenazines21 and benzophenazines22
have been identified as TOP1-targeting agents.
Recently, benz[a]acridine and benzo[i]phenanthridine
derivatives have been identified as TOP1-targeting
agents.23,24
The topological state of DNA is regulated by DNA
topoisomerases that catalyze the breaking and rejoining
of DNA strands.1ꢀ4 These nuclear enzymes are also
involved in controlling template supercoiling during
RNA transcription.5,6 There are two major subtypes,
topoisomerase I (TOP1) and topoisomerase II (TOP2)
based upon differences in their initial mechanisms
wherein a single or double-stranded DNA break is
implicated. The antitumor activity of topoisomerase-
targeting agents is associated with their ability to stabi-
lize the enzyme–DNA cleavable complex. This drug-
induced stabilization of the enzyme–DNA cleavable
complex changes these essential components of the
replication machinery of the cell into a cellular poison.
Initial studies with a select series of benzo[i]phenan-
thridines indicate that 2,3-methylenedioxy-8,9-dimethoxy-
benzo[i]phenanthridine 2 (Fig. 1), was more active than
5,6-dihydro-9,10-dimethoxy-3,4-methylenedioxybenz[a]-
anthracene 1 as a TOP1-targeting agent. While 3 and 4
(Fig. 1) target TOP1, these derivatives are less active
than 2. Compound 6, which possesses both a 2,3- and an
8,9-methylenedioxy group does not exhibit significant
*Corresponding author. Tel. +1-732-445-2674; fax; +1-732-445-
6312; e-mail: elavoie@rci.rutgers.edu; http://www.rci.rutgers.edu/~layla/
Faculty/LaVoie.htm
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00530-8

522
D. Li et al. / Bioorg. Med. Chem. 11 (2003) 521–528
TOP1-targeting activity. The presence of methoxyl
groups at both the 2- and 3-position of benzo[i]phenan-
thridine, such as in the case of 5 is associated with a loss
of topoisomerase-targeting activity. The observation,
therefore, that 7a has greater TOP1-targeting activity
than 2 was initially unexpected and prompted the
synthesis and evaluation of other 2,3-dimethoxy-
benzo[i]phenanthridine derivatives.
Results
Chemistry
The synthetic approach employed for the preparation of
7a and the 8-substituted 2,3-dimethoxybenzo[i]phenan-
thridines 7b–7h is outlined in Scheme 1. Treatment of
6,7-dimethoxy-b-tetralone under bromo-Vilsmeier con-
ditions provided 8 in 80% yield.25 Treatment of 8 with
Figure 1. Benz[a]acridine and benzo[i]phenanthridine derivatives evaluated as TOP1-targeting agents.
Scheme 1. (i) DMF, PBr3,CHCl3, reflux; (ii) DDQ, toluene, reflux; (iii) (CH3)3SnSn(CH3)3, Pd(PPh3)4, THF, reflux; (iv) Zn, AcOH, reflux; (v)
Ac2O, pyridine; (vi) H2, 45 psi, Ra–Ni, MeOH; EtOAc/THF.

D. Li et al. / Bioorg. Med. Chem. 11 (2003) 521–528
523
DDQ in toluene provided 3-bromo-6,7-dimethoxy-1-
naphthldehyde 9 in 95% yield. Trimethyl(ortho-nitro-
phenyl)stannanes have proved useful intermediates for
the preparation of phenanthridines and benzo[i]phe-
nanthridines by reaction with the appropriate benzalde-
hyde or naphthaldehyde.26 Stille coupling of 9 with
various trimethyl(othro-nitrophenyl)stannanes 10a–f gave
the 11a–f in yields ranging from 70 to 95%. Treatment
of 11a–f with zinc dust in acetic acid provided the ben-
zo[i]phenanthridines 7a–f. Hydrogenolysis of 7e using
H2 and Raney–Ni gave 7g in good yield. Reaction of 7b
with acetic anhydride in pyridine provided the acetamide
derivative 7h.
Pharmacology
The relative TOP1-targeting activities of the various 2,3-
dimethoxybenzo[i]phenanthridines 7a–h are provided in
Table 1. Of the benzo[i]phenanthridines evaluated in
this study, 7a was the most potent TOP1-targeting
agent. This result was unanticipated in view of previous
studies with benzo[i]phenanthridine derivatives, such as
5, wherein the presence of methoxyl groups at the 2- and
3-positions was associated with a loss of topoisomerase-
targeting activity. Of the 8-substituted 2,3-dimethoxy-
benzo[i]phenanthridines evaluated, only 7c, 7f and 7g
were active in stabilizing the cleavable complex formed
between TOP1 and DNA. Steric factors associated with
substituents at the 8-position appear to influence TOP1-
targeting activity as the 8-hydroxy derivative 7g was
more active than the methoxy derivative 7f which in
turn was more active than the benzyloxy derivative 7e.
A representative gel map of the TOP1-mediated DNA
cleavage assay observed with compounds 4, 7a, 7d and
7g is illustrated in Figure 2. Each of the compounds was
also evaluated for TOP2-targeting activity. Nitidine and
VM-26, which have comparable potency as TOP2-tar-
geting agents, were used as positive controls in these
assays. Compound 2 did exhibit activity in stabilizing
the cleaved complex formed between TOP2 and DNA.
Figure 2. Stimulation of enzyme-mediated DNA cleavage by 4, 7a, 7d,
7g and camptothecin (CPT) using human TOP1. The first lane is DNA
control without enzyme. The second lane is the control with enzyme
alone. The rest of the lanes contain human TOP1 and serially (10-fold
each) diluted compound from 0.01 to 1.0 mM for CPT and 1.0 to
100 mM for compounds 4, 7a, 7d and 7g.
It had approximately 10% of the potency of nitidine as
a TOP2-targeting agent (data not shown). None of the
other benzo[i]phenanthridine derivatives was active as
TOP2-targeting agents.
The cytotoxic activities of 7a–h are provided in Table 1.
Compound 7a had similar cytotoxicity to nitidine in the
human lymphoblastoma cell line RPMI8402. CPT-K5 is
the camptothecin-resistant variant of RPMI8402. The
basis for this resistance is a mutant but functional
TOP1, which does not form a stabilized cleaved com-
plex in the presence of camptothecin.27 While nitidine
exhibited marginal cross-resistance, 7a did not exhibit
cross-resistance in CPT-K5.
Table 1. TOP1-targeting activity and cytotoxicity of 1, 2, and the 2,3-
dimethoxybenzo[i]phenanthridines 7a–h
Discussion
Compound
TOP1-mediated
DNA cleavagea
Cytotoxicity IC50 (m M)b
cell lines
Previous studies indicate that the presence of methoxyl
substituents at the 2- and 3-positions of benzo[i]phe-
nanthridines was associated with a loss of TOP1-target-
ing activity. The fact that 7a is not only active as a
TOP1-targeting agent, but also an order of magnitude
more potent than 2 was, therefore, unexpected. This
result prompted the study of a series of 2,3-dimethoxy-
8-substituted benzo[i]phenanthridines. Comparative
studies on the relative potency of the 2,3-dimethoxy-
benzo[i]phenanthridines synthesized and evaluated as
TOP1-targeting agents showed the relative potencies to
be 7a > >7g >7f > >7c > >7b,7d,7e,7h.
RPMI8402
CPT-K5
1
2
7a
7b
7c
7d
7e
7f
7g
7h
Nitidine
CPT
VM-26
5
10
1
(–)
80
(–)
(–)
10
2
(–)
1
0.1
(–)
6.8
4.5
0.4
2.3
8.0
13
8.5
3.9
1.6
4.0
0.4
0.005
0.2
22
11
0.4
5.6
>40
40
23
25
14
30
4.0
>40
0.3
Consistent with its greater potency as a TOP1-targeting
agent, 7a was also an order of magnitude more cyto-
toxic than 2. Compound 7a had an IC50 of 400 nM in
the human lymphoblastoma cell line RPMI8402, while
compound 2 had an IC50 of 4.5 mM. In addition to 7a,
only 7f and 7g had greater or comparable TOP1-targeting
aIC50 was calculated after 4 days of continuous drug exposure.
bTOP1 cleavage values are reported as REC, relative effective concen-
tration, that is, concentrations relative to camptothecin (CPT), whose
value is arbitrarily assumed as 1, that are able to produce the same
cleavage on the plasmid DNA in the presence of human TOP1.

524
D. Li et al. / Bioorg. Med. Chem. 11 (2003) 521–528
Figure 3. Comparison of the two-dimensional structures of 2 and 7a after alignment of similar functionality and molecular topology.
activity to 2. While 7g had 5-fold greater intrinsic
potency as a TOP1-targeting agent than 7f, it had only
slightly above a 2-fold greater cytotoxic activity in the
human lymphoblastoma cell line RPMI402, with an
IC50 of 1.6 mM. The IC50 of 7f in RPMI8402 cells was
3.9 mM.
Perkin-Elmer 1600 Fourier transform spectrophoto-
meter and are reported in cmꢀ1. Proton (1H NMR,
200 MHz) and carbon (13C NMR, 50 MHz) nuclear
magnetic resonance were recorded on a Varian Gemini-
200 Fourier Transform spectrometer on compounds
dissolved in deuterated chloroform, unless otherwise
indicated, with chemical shifts reported in d units
downfield from tetramethylsilane (TMS). Coupling
constants are reported in hertz (Hz). Mass spectra were
obtained by the Washington University Resource for
Biomedical and Bio-organic Mass Spectrometry within
the Department of Chemistry at Washington Uni-
versity, St. Louis, MO, USA. Tetrahydrofuran was
freshly distilled from sodium and benzophenone prior
to use. Tetrakis(triphenylphosphine)palladium(0) was
purchased as bright yellow powder or crystal from
Aldrich Chemical Company (Milwaukee, WI, USA) or
Acros Organics (Fisher Scientific, Pittsburgh, PA,
USA). Compound 10a was synthesized as previously
described.26
Structure–activity data had initially suggested that 2
was among the more potent benzo[i]phenanthridine
derivatives as TOP1-targeting agents. In addition,
replacement of the methylenedioxy moiety of 2 with two
methoxy groups results in a complete loss of activity.
These data raised the speculation that 2,3-dimethoxy-
lated benzo[i]phenanthridines would not possess potent
TOP1-targeting activity. The exceptional TOP1-target-
ing activity and cytotoxicity of 7a proved this is not the
case. The data in this study do suggest that the methyl-
enedioxy moiety in the case of both 2 and 7a is a sig-
nificant structural component for maintaining potent
TOP1-targeting activity. In light of these results, the
optimal molecular topology for effective TOP1-target-
ing activity among these benzo[i]phenanthridines may
consist of a methylendioxy moiety at one end of the
molecule with dimethoxy groups on the benzo-ring dis-
tal to this substituent. Arranging both 2 and 7a such
that methylenedioxy moieties are on the same side for
each molecule (which requires rotating 7a clockwise by
180ꢁ as illustrated in Fig. 3), so that each adopts a
similar two-dimensional molecular topology. In com-
paring these two molecules, as illustrated in Figure 3,
the only obvious difference between 2 and 7a is the
location of the nitrogen heteroatom. In the case of 7a, it
appears that having the nitrogen heteroatom adjacent to
the benzo-ring containing the methylenedioxy sub-
stituent results in an enhancement of the TOP1-target-
ing activity of these benzo[i]phenanthridines.
General procedure for the preparation of benzo[i]phenan-
thridines (7a–f)
The appropriate 2-(o-nitrophenyl)-6,7-dimethoxy-1-
naphthaldehyde (100 mg, 0.26 mmol) was dissolved in
glacial acetic acid (15 mL) and heated to reflux with zinc
dust (160 mg, 2.5 mmol) for 2-3.5 h. Acetic acid was
evaporated in vacuo and the residue was dissolved in
chloroform. The solution was filtered through a Celite
545 bed and the filtrate washed successively with satu-
rated aqueous sodium bicarbonate and brine. The
organic layer was dried (anhyd. Na2SO4) and evapo-
rated in vacuo. The residue was chromatographed using
50 g of silica gel and 1:1 ethyl acetate/hexanes to give as
follows.
It is our intention to use 7a as a molecular template for
further studies to gain additional insight into those
structural features that are associated with enhancement
of the TOP1-targeting activity of substituted benzo[i]-
phenanthridines and related compounds.
2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine
(7a). This was prepared from 11a (100 mg, 0.26 mmol)
and isolated as a beige solid after chromatography using
1:1 ethyl acetate/hexanes in 80% yield (40 mg), mp 171–
173 ꢁC; IR (KBr) 2226, 1610, 1461, 1277; H NMR d
1
4.06 (3H, s), 4.15 (3H, s), 6.15 (2H, s), 7.27 (1H, s), 7.54
(1H, s), 7.85 (1H, s), 7.94 (1H, d, J=6.7) 8.09 (1H,s),
8.15 (1H, d, J=8.7), 9.88 (1H, s); 13C NMR d 56.4,
56.6, 99.7, 102.3, 102.4, 107.7, 108.5, 118.5, 121.0, 121.2,
125.6, 127.7, 130.8, 131.2, 142.8, 145.9, 148.8, 149.7,
150.1, 151.0; HR-MS calcd for C20H15NO4+H:
334.1079; found: 334.1079.
Experimental
General
Melting points were determined with a Thomas-Hoover
Unimelt capillary melting point apparatus. Column
chromatography refers to flash chromatography con-
ducted on SiliTech 32–63 mm, (ICN Biomedicals,
Eschwegge, Germany) using the solvent systems indi-
cated. Infrared spectral data (IR) were obtained on a
8-Amino-2,3-dimethoxybenzo[i]phenanthridine (7b). This
was prepared from 11b (100 mg, 0.26 mmol) and iso-
lated as an orange–yellow solid after chromatography
using a 1:8 mixture of methanol/ethyl acetate in 50%

D. Li et al. / Bioorg. Med. Chem. 11 (2003) 521–528
525
yield (40 mg); mp 224–225 ꢁC; 1H NMR (CD3OD) d
3.95 (3H, s), 4.04 (3H, s), 7.15 (1H, dd, J=8.8, 2.3), 7.23
(1H, d, J=2.3), 7.26 (1H, s), 7.88 (1H, d, J=9.0), 7.98
(1H, s), 8.20 (1H, d, J=9.0), 8.32 (1H, d, J=8.8),
9.68 (1H, s); 13C NMR (CD3OD) d 59.3, 59.5, 105.8,
112.4, 112.9, 120.9, 121.4, 122.6, 123.2, 127.7, 129.7,
131.2, 135.7, 136.2, 149.5, 151.3, 153.5, 153.7, 155.1;
HR-MS calcd for C19H16N2O2: 304.1212; found:
304.1206.
2,3-Dimethoxy-8-hydroxybenzo[i]phenanthridine
(7g).
Compound 7e (15 mg, 0.038 mmol) was dissolved
40 mL of 3:3:2 methanol/ethyl acetate/THF. Raney–Ni
(40 mg) was added to this solution. The mixture was
shaken in a Parr1 apparatus for 40 h at 45 psi of
hydrogen. The reaction mixture was then concentrated
in vacuo and the residue was chromatographed using
50 g of silica gel and a 1:10 methanol/ethyl acetate as a
light-yellow powder in 86% yield (10 mg); mp 238–
ꢁ
1
240 C H NMR (CD3OD) d 4.03 (3H, s), 4.12 (3H, s),
7.30 (1H, dd, J=9.0, 2.4), 7.44 (1H, s), 7.47 (1H, d,
J=2.4), 8.09 (1H, d, J=9.0), 8.22 (1H, s), 8.43 (1H, d,
J=9.0), 8.58 (1H, d, J=9.0), 9.95 (1H, s); HR-MS calcd
for C19H15NO3: 305.1052; found: 305.1060.
8-Cyano-2,3-dimethoxybenzo[i]phenanthridine (7c). This
was prepared from 11c (80 mg, 0.22 mmol) and isolated
as a light yellow solid as after chromatography using 2:1
mixture of ethyl acetate/hexanes in 40% yield (28 mg);
mp >250 ꢁC; IR (KBr) 2226, 1610, 1516; 1H NMR
(DMSO-d6) d 4.00 (3H, s), 4.12 (3H, s), 7.67 (1H, s),
8.06 (1H, d, J=8.0), 8.31 (1H, d, J=8.9), 8.51 (1H, s),
8.67 (1H, s), 8.73 (1H, d, J=8.0), 9.01 (1H, d, J=8.9),
10.46 (1H, s); 13C NMR (DMSO-d6) d 56.0, 56.4, 103.4,
108.6, 110.9, 118.5, 119.0, 122.3, 124.9, 125.3, 127.5,
128.6, 128.9, 129.4, 132.2, 134.9, 143.5, 150.6, 151.1,
156.2; HR-MS calcd for C20H14N2O2: 314.1055; found:
314.1067.
8-Acetamino-2,3-dimethoxybenzo[i]phenanthridine (7h).
A solution of 7b (15 mg, 0.049 mmol) in pyridine
(6 mL) was treated dropwise with acetic anhydride
(1.5 mL) at 0 ꢁC. The mixture was gradually warmed up
to room temperature and stirred for 2 h. The solvent
was evaporated and the residue was taken up in metha-
nol (15 mL). Evaporation of the solvent afford 7h
(15 mg) as a light yellow solid in mp >250 ꢁC 90%
1
yield. H NMR (CD3OD) d 2.24 (3H, s), 4.03 (3H, s),
2,3 - Dimethoxy - 8 - hydroxymethylbenzo[i]phenanthridine
(7d). This was prepared from 11d (80 mg, 0.22 mmol)
and isolated as a yellow solid after chromatography
using ethyl acetate in 50% yield (35 mg); mp >250 ꢁC
IR (KBr) 3429, 2366, 1732; 1H NMR (DMSO-d6) d 3.98
(3H, s), 4.11 (3H, s), 7.64 (1H, s), 7.73 (1H, d, J=8.4),
8.16 (1H, s), 8.24 (1H, d, J=8.8), 8.49 (1H, s), 8.66 (1H,
d, J=9.0), 8.83 (1H, d, J=8.8), 10.36 (1H, s); 13C NMR
(DMSO-d6) d 55.9, 56.3, 65.4, 103.1, 108.6, 118.3, 121.2,
123.7, 123.8, 125.0, 127.0, 128.0, 128.9, 130.1, 131.7,
136.8, 144.4, 149.4, 150.1; HR-MS calcd for
C20H17NO3: 319.1208; found: 319.1196.
4.13 (3H, s), 7.47 (1H, s), 7.86 (1H, dd, J=8.8, 1.9), 8.12
(1H, d, J=9.2), 8.24 (1H, s), 8.47 (1H, d, J=8.8), 8.48
(1H, d, J=1.9), 8.63 (1H, d, J=9.2), 10.00 (1H, s); 13C
NMR (DMSO-d6) d 24.5, 55.8, 56.3, 103.0, 108.7, 117.4,
118.1, 119.9, 120.1, 120.4, 123.8, 125.1, 127.5, 130.3,
131.4, 139.6, 145.3, 149.4, 149.8, 150.9, 169.0; HR-MS
calcd for C21H18N2O3: 346.1317; found: 346.1319.
2-Bromo-3,4-dihydro-6,7-dimethoxy-1-naphthaldehyde (8).
Dimethylformamide (1.6 mL, 21.4 mmol) was added
dropwise to a solution of phosphorus tribromide
(1.6 mL, 16.9 mmol) in dry chloroform (20 mL) at 0 ꢁC.
The mixture was stirred at 0 ꢁC for 1 h to give a pale
yellow suspension. A solution of 6,7-dimethoxy-2-tetra-
lone (1.0 g, 4.85 mmol) in dry chloroform (20 mL) was
added to the yellow suspension and the mixture was
heated at reflux for 1 h. The reaction mixture was cooled
to 0 ꢁC and made basic with saturated aqueous
NaHCO3. The resulting mixture was extracted with di-
chloromethane, dried (anhyd Na2SO4), and evaporated
in vacuo. The residue was chromatographed over 100 g
of silica gel using 1:3 ethyl acetate/hexanes to give 8
(1.15 g) as a yellow crystalline solid in 80% yield; mp
8-Benzoxyl-2,3-dimethoxybenzo[i]phenanthridine (7e). This
was prepared from 11e (100 mg, 0.23 mmol) and isolated
as a white solid after chromatography using a 2:1 mix-
ture of eꢁthyl acetate/hexanes in 56% yield (51 mg); mp
1
227–228 C H NMR d 4.08 (3H, s), 4.18 (3H, s), 5.29
(2H, s), 7.33 (1H, s), 7.36–7.48 (5H, m), 7.55 (1H, dd,
J=7.9, 1.7), 7.73 (1H, d, J=1.7), 8.04 (1H, d, J=8.9),
8.17 (1H, s), 8.41 (1H, d, J=8.8), 8.54 (1H, d, J=8.9),
10.04 (1H, s); 13C NMR d 56.5, 56.6, 70.8, 102.3, 108.6,
110.9, 118.4, 119.5, 119.6, 120.7, 124.2, 125.8, 127.7,
128.2, 128.6, 129.2, 131.4, 131.7, 137.1, 146.8, 148.6,
150.1, 151.2, 159.5; HR-MS calcd for C26H21NO3:
395.1521; found: 395.1522.
82–83 ꢁC H NMR d 2.83 (2H, t, J=7.6), 3.01 (2H, t,
1
J=7.6), 3.88 (3H, s), 6.66 (1H, s), 7.71 (1H, s), 10.31
(1H, s); 13C NMR d 28.9, 38.5, 56.4, 56.5, 110.0, 111.1,
123.1, 128.0, 132.7, 144.2, 147.7, 149.1, 193.6; HR-MS
calcd for C13H13BrO3: 296.0048; found: 296.0042.
2,3,8-Trimethoxybenzo[i]phenanthridine (7f). This was
prepared from 11f (70 mg, 0.19 mmol) as a light yellow
solid after chromatography using ethyl acetate as elut-
2-Bromo-6,7-dimethoxy-1-naphthaldehyde (9). 2-Bromo-
3,4-dihydro-6,7-dimethoxy-1-naphthaldehyde (8) (960 mg,
3.23 mmol) and DDQ (880 mg, 3.88 mmol) was refluxed
in toluene (45 mL) for 12 h. After cooling to room tem-
perature, the mixture was filtered through a Celite 545
bed and the filtrate was evaporated to dryness. The
residue obtained was chromatographed on 75 g of silica
gel using 1:3 ethyl acetate/hexanes to give 9 (902 mg) as
a light yellow solid in 95% yield; mp 141–142 ꢁC IR
ing solvent in 70% yield (42 mg); mp 200–201 ꢁC; H
1
NMR d 4.02 (3H, s), 4.08 (3H, s), 4.18 (3H, s), 7.32 (1H,
s), 7.35 (1H, dd, J=9.2, 2.7), 7.64 (1H, d, J=2.7), 8.03
(1H, d, J=8.8), 8.16 (1H, s), 8.40 (1H, d, J=8.8), 8.52
(1H, d, J=9.2), 10.04 (1H, s); 13C NMR d 56.1, 56.5,
56.6, 102.3, 108.6, 109.7, 118.4, 119.2, 119.3, 120.7,
124.2, 125.8, 127.7, 131.4, 131.7, 146.9, 148.6, 150.1,
151.1, 160.4; HR-MS calcd for C20H17NO3: 319.1208;
found: 319.1204.
1
(KBr) 1671; H NMR d 4.01 (3H, s), 4.06 (3H, s), 7.09

526
D. Li et al. / Bioorg. Med. Chem. 11 (2003) 521–528
(1H, s), 7.56 (1H, d, J=8.6), 7.72 (1H, d, J=8.6), 8.74
(1H, s), 10.76 (1H, s); 13C NMR d 56.3, 56.6, 104.2,
107.0, 126.5, 128.9, 129.5, 129.8, 129.9, 134.4, 150.4,
153.1, 196.0; HR-MS calcd for C13H11BrO3: 293.9892;
found: 293.9896.
J=2.6); 13C NMR d ꢀ7.1, 56.7, 107.7, 117.3, 133.9,
146.8, 149.6, 154.1; HR-MS calcd for C10H15NO3Sn–
CH3: 301.9839; found: 301.9832.
4-Benzyloxy-1-bromo-2-nitrobenzene.
A
solution of
4-bromo-3-nitrophenol (1.0 g, 4.6 mmol) and benzyl
bromide (0.82 mL, 7.0 mmol) in 40 mL of 2:1 acetoni-
trile/acetone was treated with potassium carbonate
(970 mg, 7.0 mmol). The resulted mixture was heated
under reflux for 17 h. The reaction mixture was filtered
and the filtrate was evaporated in vacuo. The residue
was dissolved in ethyl acetate and the solution was
washed successively with H2O (30 mL), 1 M HCl (30 mL
ꢂ 3) and brine. The organic layer was dried (anhyd
Na2SO4) and evaporated in vacuo. The residue was
chromatographed using 75 g of silica gel and 1:5 ethyl
acetate/hexanes to give 4-benzyloxy-1-bromo-2-nitro-
benzene (1.34 g)ꢁ as a yellow crystalline solid in 95%
Trimethyl(3,4-methylenedioxy-6-nitro)stannane (10a). This
was prepared from 1-bromo-3,4-methylenedioxy-6-nitro-
benzene28 as previously detailed.26
General procedure for the preparation of trimethyl-
nitroarylstannanes (10b–10e)
Trimethyl(2,4-dinitrophenyl)stannane (10b). A mixture
of hexamethylditin (1.0 g, 3.1 mmol), 1-iodo-2,4-dini-
trobenzene (588 mg, 2.0 mmol) and Pd(PPh3)4 (95 mg) in
anhydrous THF (25 mL) was heated to reflux for 10 h.
After cooling to room temperature, THF was evapo-
rated and methylene chloride (30 mL) was added to the
residue. Aqueous potassium fluoride (7.0 M, 2 mL) was
added dropwise to this mixture with vigorous stirring.
The mixture was passed through a Celite 545 bed and
the filtrate was washed with brine. The methylene chlo-
ride layer was dried (anhyd. Na2SO4), filtered and eva-
porated in vacuo. The residue was chromatographed
over 100 g of silica gel using 1:6 ethyl acetate/hexanes to
give 10b (350 mg) in 53% yield; 1H NMR d 0.43 (9H, s),
7.95 (1H, d, J=8.0), 8.44 (1H, dd, J=8.0, 2.0), 9.11
(1H, d, J=2.0); 13C NMR d ꢀ6.9, 119.2, 127.2, 139.0,
149.4, 150.0, 154.1; HR-MS calcd for C9H12N2O4Sn–
CH3: 316.9584; found: 316.9591.
1
yield; mp 46–47 C; H NMR d 5.11 (2H, s), 7.05 (1H,
dd, J=9.0, 3.0), 7.36–7.45 (5H, m), 7.47 (1H, d, J=3.0),
7.60 (1H, d, J=9.0); 13C NMR d 71.4, 105.4, 112.4,
121.1, 128.1, 128.9, 129.1, 129.3, 129.5, 135.8, 136.0,
138.2, 158.7; HR-MS calcd for C13H10BrNO3: 306.9844;
found: 306.9846.
4-Bromo-3-nitrophenol. A solution of 4-bromo-3-nitro-
anisole (2.32 g, 10 mmol) in dichloromethane (25 mL)
was added dropwise to a solution of boron tribromide
(BBr3, 1.0 M in dichloromethane, 25 mL) at ꢀ78 ꢁC.
The solution was gradually brought to room tempera-
ture and stirred for 30 h. H2O (30 mL) was added drop-
wise and resulting precipate was taken up in ethyl
acetate (40 mL). The aqueous layer was extracted twice
with ethyl acetate (25 mL ꢂ 2). The combined organic
layer was washed successively with saturated aqueous
sodium bicarbonate and brine. The organic layer was
dried (anhyd Na2SO4) and evaporated in vacuo. The
residue was chromatographed using 75 g of silica gel and
a 1:3 mixture of ethyl acetate/hexanes to give 4-bromo-
3-nitrophenol (1.52 g) as a yellow solid in 70% yield; mp
Trimethyl(4-cyano-2-nitrophenyl)stannane (10c). This
was prepared from 4-chloro-3-nitrobenzonitrile (460 mg,
2.5 mmol) and hexamethylditin (1.0 g, 3.1 mmol) as a
1
yellow solid in 40% yield; H NMR d 0.40 (9H, s), 7.87
(2H, s), 8.57 (1H, s); 13C NMR d ꢀ6.9, 114.2, 117.5, 127.5,
135.9, 138.9, 147.9; HR-MS calcd for C10H12N2O2Sn–
CH3: 296.9686; found: 296.9693.
1
Trimethyl(4-formyl-2-nitrophenyl)stannane (10d). This
was prepared from 4-chloro-3-nitrobenzaldehyde (490 mg,
2.5 mmol) and hexamethylditin (1.0 g, 3.1 mmol) as a
144–145 ꢁC; H NMR d 6.95 (1H, dd, J=8.8, 2.9), 7.36
(1H, d, J=2.9), 7.58 (1H, d, J=8.8); 13C NMR d 105.2,
113.4, 116.4, 121.4, 136.3, 155.9; HR-MS calcd for
C6H4BrNO3: 216.9375; found: 216.9380.
light yellow solid in 70% yield; mp 97–98 ꢁC; H NMR
1
d 0.30 (9H, s), 7.90 (1H, d, J=7.4), 8.09 (1H, d, J=7.4),
8.76 (1H, s), 10.11 (1H, s); 13C NMR d ꢀ7.0, 125.2,
133.1, 138.1, 138.8, 149.1, 154.2, 190.7; HR-MS calcd
for C10H13NO3Sn–CH3: 299.9683; found: 299.9684.
General procedure for the synthesis of 11a–11f
2-(2,4-Dinitrophenyl)-6,7-dimethoxy-1-naphthaldehyde
(11b). Pd(Ph3P)4 (60 mg, 0.05 mmol) and cuprous bro-
mide (10 mg, 0.07 mmol) were added to a solution of
10b (345 mg, 1.0 mmol) and 9 (186 mg, 0.63 mmol) in
THF (20 mL) at room temperature. The mixture was
heated to reflux under N2 for 15 h. After cooling, THF
was evaporated and ethyl acetate (30 mL) was added to
the residue. The solution was washed with water
(20 mL). The organic layer was separated and passed
through a Celite 545 bed to remove suspended particles.
The organic layer was washed with brine, dried (anhyd
Na2SO4) and evaporated in vacuo. The residue was
chromatographed using 75 g of silica gel and 1:3 ethyl
acetate/hexanes to give 11b (229 mg) in 95% yield; mp
Trimethyl(4-benzyloxy-2-nitrophenyl)stannane (10e). This
was prepared from 4-benzyloxy-1-bromo-2-nitrobenzene
(462 mg, 1.5 mmol) and hexamethylditin (1.0 g, 3.1 mmol)
1
as a light yellow oil in 70% yield; H NMR d 0.32 (9H,
s), 5.16 (2H, s), 7.27 (1H, dd, J=8.1, 2.5), 7.36–7.65
(5H, m), 7.58 (1H, d, J=8.1), 7.95 (1H, d, J=2.5); 13C
NMR d ꢀ7.3, 70.9, 110.4, 121.8, 128.0, 128.8, 129.2,
130.7, 136.4, 138.2, 154.6, 160.3.
Trimethyl(4-methoxy-2-nitrophenyl)stannane (10f). This
was prepared from 4-bromo-3-nitroanisole (1.6 g,
6.9 mmol) in 55% yield; mp 93–95 ꢁC; H NMR d 0.32
1
ꢁ
(9H, s, [119Sn-H, d, 2JSn-H=54] ), 3.89 (3H, s), 7.21 (1H,
dd, J=8.0, 2.6), 7.57 (1H, d, J=8.0), 7.86 (1H, d,
185–186 C; H NMR d 4.06 (3H, s), 4.10 (3H, s), 7.12
(1H, d, J=8.3), 7.24 (1H, d, J=9.5), 7.66 (1H, d,
1

D. Li et al. / Bioorg. Med. Chem. 11 (2003) 521–528
527
J=8.3), 7.98 (1H, d, J=8.3), 8.53 (1H, dd, J=8.3, 2.2),
8.63 (1H, s), 8.97 (1H, d, J=2.2), 10.30 (1H, s); 13C
NMR d 56.4, 56.7, 104.3, 107.1, 120.5, 124.5, 126.9,
128.0, 131.0, 133.4, 134.8, 139.3, 142.2, 148.0, 149.3,
151.0, 153.4, 191.9; HR-MS calcd for C19H14N2O7:
382.0801; found: 382.0787.
J=8.3), 8.85 (1H, s), 10.17 (1H, s); 13C NMR d 56.3,
56.4, 56.6, 105.2, 107.1, 109.8, 119.4, 125.9, 126.8, 127.5,
130.3, 133.2, 134.7, 143.2, 149.8, 150.4, 153.1, 160.4,
194.0; HR-MS calcd for C20H17NO6: 367.1056; found:
367.1050.
Topoisomerase mediated DNA cleavage assays
2-(3,4-Methylenedioxy-6-nitrophenyl)-6,7-dimethoxy-1-
naphthaldehyde (11a). This was prepared from 10a
(122 mg, 0.37 mmol) and 9 (100 mg, 0.34 mmol in 83%
Human topoisomerase I was isolated as a recombinant
fusion protein using a T7 expression system.29 DNA
topoisomerase II was isolated and purified as previously
described.30 Plasmid YEpG was also purified by the
alkali lysis method followed by phenol deproteination
and CsCl/ethidium isopycnic centrifugation as descri-
bed.31 The 30 end-labeling of the plasmid was accom-
plished by digestion with a restriction enzyme followed
by end-filling with Klenow polymerase as previously
described.32 The cleavage assays were performed as
previously reported.33,34 The reactions were terminated
by the addition of 5 mL of 5% SDS and 1 mg/mL pro-
tein kinase K with an additional 1 h of incubation at
37 ꢁC. Samples were then alkali denatured by the addition
of NaOH, EDTA, sucrose, and bromophenol blue to
final concentrations of 75 mM, 2.5%, and 0.05 mg/mL,
respectively, prior to loading onto a neutral agarose gel.
yield). Mp 223–225 ꢁC; IR (KBr) 1677, 1502, 1256; H
1
NMR d 4.04 (3H, s), 4.09 (3H, s), 6.21 (2H, s), 6.78 (1H,
s), 7.13 (1H, d), J=8.4), 7.18 (1H, s), 7.66 (1H, s), 7.91
(1H, d, J=8.4) 8.84 (1H, s), 10.22 (1H, s); 13C NMR d
56.3, 56.6, 103.9, 105.2, 105.9, 107.1, 112.3, 125.4, 127.0,
127.5, 130.4, 131.6, 133.3, 143.8, 148.5, 150.1, 151.7,
153.1, 193.6; HR-MS calcd for C20H15NO7+ H:
382.0927; found: 382.0927.
2-(4-Cyano-2-nitrophenyl)-6,7-dimethoxy-1-naphthalde-
hyde (11c). This was prepared from 10c (150 mg,
0.51 mmol) and 9 (147 mg, 0.50 mmol) in 95% yield; mp
ꢁ
1
186–187 C; H NMR (DMSO-d6) d 3.99 (6H, s), 7.27
(1H, d, J=8.3), 7.56 (1H, s), 7.78 (1H, d, J=8.1), 8.14
(1H, d, J=8.3), 8.27 (1H, d, J=8.1), 8.54 (1H, s), 8.78
(1H, s), 10.31 (1H, s); 13C NMR (DMSO-d6) d 55.8,
55.9, 103.5, 107.6, 112.5, 117.2, 124.9, 126.2, 127.0,
128.6, 130.2, 133.1, 134.1, 136.5, 139.0, 139.6, 149.0,
150.2, 152.5, 193.1; HR-MS calcd for C20H14N2O5:
362.0903; found: 362.0918.
Cytotoxicity assay
Cytotoxicity was determined using the MTT-microtiter
plate tetrazolinium assay (MTA).35ꢀ37 The human lym-
phoblast RPMI 8402 and its camptothecin-resistant
variant cell line CPT-K5 were provided by Dr. Toshiwo
Andoh (Aichi Cancer Center Research Institute,
Nagoya, Japan).27 The cytotoxicity assay was per-
formed using 96-well microtiter plates. Cells were grown
in suspension at 37 ꢁC in 5% CO2 and maintained by
regular passage in RPMI medium supplemented with
10% heat-inactivated fetal bovine serum, l-glutamine
(2 mM), penicillin (100 U/mL), and streptomycin
(0.1 mg/mL). Each well was plated with either 2000
RPMI8402 cells or 4000 CPT-K5 cells. For determina-
tion of IC50, cells were exposed continuously with vary-
ing concentrations of drug and MTT assays were
performed at the end of the fourth day.
2-(4-Formyl-2-nitrophenyl)-6,7-dimethoxy-1-naphthalde-
hyde (11d). This was prepared from 10d (157 mg,
0.50 mmol) and 9 (147 mg, 0.50 mmol) as a bright yellow
solid in 80% yield; mp 175–176 ꢁC; H NMR (DMSO-
1
d6) d 3.88 (6H, s), 7.29 (1H, d, J=8.2), 7.56 (1H, s), 7.79
(1H, d, J=7.9), 8.15 (1H, d, J=8.2), 8.28 (1H, d,
J=7.9), 8.56 (1H, s), 8.69 (1H, s), 10.22 (1H, s), 10.30
(1H, s); 13C NMR (DMSO-d6) d 55.9, 103.6, 107.6,
124.9, 125.4, 126.2, 126.9, 130.1, 132.9, 133.1, 134.1, 136.8,
139.9, 140.1, 149.2, 150.1, 152.5, 191.8, 193.0; HR-MS
calcd for C20H15NO6: 365.0899; found: 365.0909.
2-(4-Benzyloxy-2-nitrophenyl)-6,7-dimethoxy-1-naphth-
aldehyde (11e). This was prepared from 10e (320 mg,
0.82 mmol) and 9 (204 mg, 0.69 mmol) as a light yellow
solid in 92% yield; mp 143–144 ꢁC; H NMR d 4.05
Acknowledgements
1
(3H, s), 4.09 (3H, s), 5.21 (2H, s), 7.14 (1H, d, J=8.3),
7.18 (1H, s), 7.29 (1H, d, J=2.3), 7.30 (1H, s), 7.42–7.49
(5H, m), 7.72 (1H, d, J=2.3), 7.90 (1H, d, J=8.3), 8.86
(1H, s), 10.18 (1H, s); 13C NMR d 56.3, 56.6, 71.4,
105.2, 107.1, 110.9, 114.1, 120.0, 123.9, 125.8, 126.4,
127.1, 127.4, 128.1, 129.1, 129.4, 130.3, 133.2, 134.7,
135.9, 150.3, 150.9, 168.8, 194.0; HR-MS calcd for
C26H21NO6: 443.1369; found: 443.1370.
We are grateful to Washington University Resource for
Biomedical and Bio-Organic Mass Spectrometry for
providing mass spectral data and for the partial support
of this facility by the National Institutes of Health
(Grant No. P41RR0954). This study was supported by
AVAX Technologies, Inc, (E.J.L.) and Grant CA39662
(L.F.L.) and Grant CA077433 (L.F.L.) from the
National Cancer Institute.
2-(4-Methoxy-2-nitrophenyl)-6,7-dimethoxy-1-naphth-
aldehyde (11f). This was prepared from 10f (348 mg,
1.1 mmol) and 9 (295 mg, 1.0 mmol) as a orange solid in
70% yield; mp 200–202 ꢁC; IR (KBr) 1672, 1523, 1256;
1H NMR d 3.95 (3H, s), 4.04 (3H, s), 4.09 (3H, s), 7.14
(1H, d, J=8.3), 7.17 (1H, s), 7.21 (1H, dd, J=8.6, 2.6),
7.31 (1H, d, J=8.6), 7.62 (1H, d, J=2.6), 7.89 (1H, d,
References and Notes
1. Wang, J. C. Annu. Rev. Biochem. 1985, 54, 665.
2. Liu, L. Annu. Rev. Biochem. 1989, 58, 351.
3. Chen, A. Y.; Liu, L. F. Annu. Rev. Pharmacol. Toxicol.
1994, 34, 191.

528
D. Li et al. / Bioorg. Med. Chem. 11 (2003) 521–528
4. Li, T.-K.; Liu, L. F. Annu. Rev. Pharmacol. Toxicol. 2001,
41, 53.
5. Liu, L. F.; Wang, J. C. Proc. Natl. Acad. Sci. U.S.A. 1987,
84, 7024.
6. Tsao, Y.-P.; Wu, H-Y.; Liu, L. F. Cell 1989, 56, 111.
7. Sun, Q.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J.
Bioorg. Med. Chem. Lett. 1994, 4, 2871.
8. Chen, A. Y.; Yu, C.; Bodley, A. L.; Peng, L. F.; Liu, L. F.
Cancer Res. 1993, 53, 1332.
9. Sun, Q.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J.
J. Med. Chem. 1995, 38, 3638.
10. Kim, J. S.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie,
E . J.J. Med. Chem. 1996, 39, 992.
21. Spicer, J. A.; Gamage, S. A.; Rewcastle, G. W.; Funlay,
G. J.; Bridewell, D. J.; Baguley, B. C.; Denny, W. A. J. Med.
Chem. 2000, 43, 1350.
22. Vicker, N.; Burgess, L.; Chuckowree, I. S.; Dodd, R.;
Folkes, A. J.; Hardick, D.; Hancox, T. C.; Miller, W.; Milton,
J.; Sohal, S.; Wang, S.; Wren, S. P.; Charlton, P. A.; Danger-
field, W.; Liddle, C.; Mistry, P.; Stewart, A. J.; Denny, W. A.
J. Med. Chem. 2002, 45, 721.
23. Makhey, D.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J.
Bioorg. Med. Chem. 2000, 8, 1171.
24. Li, D.; Makhey, D. B.; Zhao, B.; Sim, S-P.; Liu, A.; Li,
L. F.; LaVoie, E. J. Proc. Amer. Assoc. Cancer Res. 2001, 42,
720.
11. Makhey, D.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.;
LaVoie, E. J. Med. Chem. Res. 1995, 5, 1.
25. Gilchrist, T. L.; Summersell, R. J. J. Chem. Soc., Perkin
Trans. 1988, 2595.
12. Stermitz, F. R.; Gillespie, J. P.; Amoros, L. G.; Romero,
R.; Stermitz, T. A.; Larson, K. A.; Earl, S.; Ogg, J. E. J. Med.
Chem. 1975, 18, 708.
13. Makhey, D.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.;
LaVoie, E. J. Bioorg. Med. Chem. 1996, 4, 781.
14. Cushman, M.; Mohan, P. J. Med. Chem. 1985, 28, 544.
15. Cushman, M.; Mohan, P.; Smith, E. C. J. Med. Chem.
1984, 27, 1031.
16. Yamashita, Y.; Fujii, N.; Murakaya, C.; Ashizawa, T.;
Okabe, M.; Nakano, H. Biochemistry 1992, 31, 12069.
17. Fujii, N.; Yamashita, Y.; Saitoh, Y.; Nakano, H. J. Biol.
Chem. 1993, 268, 13160.
26. Li, D.; Zhao, B.; LaVoie, E. J. J. Org. Chem. 2000, 65, 2802.
27. Andoh, T.; Okada, K. Adv. in Pharmacol. 1994, 29B, 93.
28. Dallacker, F.; Wagner, A. Z. Naturforsch. 1984, 39b, 936.
29. Gatto, B.; Sanders, M. M.; Yu, C.; Wu, H-Y.; Makhey,
D.; LaVoie, E. J.; Liu, L. F. Cancer Res. 1996, 56, 2795.
30. Wasserman, R. A.; Austin, C. A.; Fisher, L. M.; Wang,
J. C. Cancer Res. 1993, 53, 3591.
31. Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular
Cloning, a Laboratory Manual; Cold Spring Harbor Labora-
tory: Cold Spring Harbor, NY, 1982; p. 149.
32. Liu, L. F.; Rowe, T. C.; Yang, L.; Tewey, K. M.; Chen,
G. L. J. Biol. Chem. 1983, 258, 15365.
18. Yamashita, Y.; Kawada, S-Z.; Fujii, N.; Nakano, H. Bio-
chemistry 1991, 30, 5838.
33. Chen, A. Y.; Yu, C.; Bodley, A. L.; Peng, L. F.; Liu, L. F.
Cancer Res. 1993, 53, 1332.
19. Cushman, M.; Jayaraman; Vroman, J. A.; Fukunaga,
A. K.; Fox, B.; Kolhagen, G.; Strumberg, D.; Pommier, Y. J.
Med. Chem. 2000, 43, 3688.
20. Jayaraman, M.; Fox, B. M.; Hollingshead, M.; Kohlha-
gen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2000, 45,
242.
34. Li, T.-K.; Chen, A. C.; Yu, C.; Mao, T.; Wang, H.; Liu,
L. F. Genes Dev.elop. 1999, 13, 1553.
35. Mosmann, T. J. J. Immunol. Meth. 1983, 65, 55.
36. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna,
J. D.; Mitchell, J. B. Cancer Res. 1987, 47, 936.
37. Denizot, F.; Lang, R. J. Immunol. Meth. 1986, 89, 271.