Substituted benzo[i]phenanthridines as mammalian topoisomerase-targeting agents

Article abstract of DOI:10.1016/S0968-0896(03)00053-1

Several benzo[c]phenanthridine and protoberberine alkaloids, such as nitidine and berberrubine, are known to induce DNA cleavage in the presence of either topoisomerase I or II. Structure-activity studies performed on various analogues related to benzo[c]phenanthridine and protoberberine alkaloids have provided insights into structural features that influence this topoisomerase-targeting activity. Modifications within the A-ring of benzo[c]phenanthridine and protoberberine alkaloids can significantly alter their ability to enhance the cleavable complex formation that occurs between DNA and topoisomerases. Select benzo[i]phenanthridines were synthesized as potential bioisosteres of nitidine and its analogues. In the present study, 2,3-methylenedioxy-8,9-dimethoxybenzo[i]phenanthridine, 2,3-methylenedioxy-8,9-dimethoxy-5-methylbenzo[i]phenanthridine, 2,3,8,9-tetramethoxybenzo[i]phenanthridine and 5-methyl-2,3,8,9-tetramethoxybenzo[i]phenanthridine were synthesized. These benzo[i]phenanthridine derivatives were evaluated for their ability to enhance cleavable complex formation in the presence of topoisomerases and DNA as well as for their cytotoxicity against the human lymphoblastoma cell line, RPMI8402. 2,3-Methylenedioxy-8,9-dimethoxybenzo[i]phenanthridine (4a) and its 5-methyl derivative (4b) are active as topoisomerase I-targeting agents. In contrast to nitidine, the presence of the 5-methyl substituent in the case of 4b is not associated with enhanced activity. Consistent with previous structure-activity studies on nitidine and protoberberine alkaloids, 2,3,8,9-teramethoxybenzo[i]phenanthridine, 5a, and its 5-methyl derivative, 5b, are inactive as topoisomerase I-targeting agents. These studies were extended to an evaluation of the relative pharmacological activities of 2,8,9-trimethoxybenzo[i]phenanthridine, 3,8,9-trimethoxybenzo[i]phenanthridine, and 2,3-methylenedioxy-8,9-methylenedioxybenzo[i]phenanthridine.

Full text of DOI:10.1016/S0968-0896(03)00053-1

Bioorganic& Medicinal Chemistry 11 (2003) 1809–1820
Substituted Benzo[i]phenanthridines as Mammalian
Topoisomerase-Targeting Agents
Darshan Makhey,a 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, 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 10 July 2002; accepted 3 December 2002
Abstract—Several benzo[c]phenanthridine and protoberberine alkaloids, such as nitidine and berberrubine, are known to induce DNA
cleavage in the presence of either topoisomerase I or II. Structure–activity studies performed on various analogues related to benzo[c]-
phenanthridine and protoberberine alkaloids have provided insights into structural features that influence this topoisomerase-targeting
activity. Modifications within the A-ring of benzo[c]phenanthridine and protoberberine alkaloids can significantly alter their ability to
enhance the cleavable complex formation that occurs between DNA and topoisomerases. Select benzo[i]phenanthridines were synthe-
sized as potential bioisosteres of nitidine and its analogues. In the present study, 2,3-methylenedioxy-8,9-dimethoxybenzo[i]phenan-
thridine, 2,3-methylenedioxy-8,9-dimethoxy-5-methylbenzo[i]phenanthridine, 2,3,8,9-tetramethoxybenzo[i]phenanthridine and
5-methyl-2,3,8,9-tetramethoxybenzo[i]phenanthridine were synthesized. These benzo[i]phenanthridine derivatives were evaluated
for their ability to enhance cleavable complex formation in the presence of topoisomerases and DNA as well as for their
cytotoxicity against the human lymphoblastoma cell line, RPMI8402. 2,3-Methylenedioxy-8,9-dimethoxybenzo[i]phenanthridine
(4a) and its 5-methyl derivative (4b) are active as topoisomerase I-targeting agents. In contrast to nitidine, the presence of the
5-methyl substituent in the case of 4b is not associated with enhanced activity. Consistent with previous structure–activity
studies on nitidine and protoberberine alkaloids, 2,3,8,9-teramethoxybenzo[i]phenanthridine, 5a, and its 5-methyl derivative, 5b,
are inactive as topoisomerase I-targeting agents. These studies were extended to an evaluation of the relative pharmacological
activities of 2,8,9-trimethoxybenzo[i]phenanthridine, 3,8,9-trimethoxybenzo[i]phenanthridine, and 2,3-methylenedioxy-8,9-methy-
lenedioxybenzo[i]phenanthridine.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
Camptothecin and its structurally-related analogues are
among the more extensively studied agents that target
DNA topoisomerases are nuclear enzymes that catalyze
the breaking and rejoining of DNA strands regulating
the topological state of DNA.1À4 Recent studies suggest
that topoisomerases are also involved in controlling
template supercoiling during RNA transcription.5,6 The
antitumor activity of topoisomerase-targeting agents is
associated with their ability to stabilize the enzyme-
DNA cleavable complex. This drug-induced stabili-
zation of the enzyme–DNA cleavable complex effec-
tively converts the enzyme into a cellular poison.
topoisomerase
I
(TOP1). Recently, bi- and
terbenzimidazoles,7À10 certain benzo[c]phenanthridine
and protoberberine alkaloids and their syntheticana-
logues,11À15 indolocarbazoles,16 the fungal metabolites,
bulgarein17 and saintopin,18 as well as derivatives of
indenoisoquinoline,19,20 phenazine21 and benzophena-
zine22 have been identified as TOP1-targeting agents.
Several studies were performed to characterize those
structural features of coralyne (1), nitidine (2), MDD-
coralyne (3) and related analogues associated with their
TOP1-targeting activity.11,13,23À25 As indicated in Figure
1, a common feature associated with all three of these
agents is the presence of a 3-phenylisoquinolinium moi-
*Corresponding author. Tel.: +1-732-445-2674; fax: +1-732-445-
6312; e-mail: elavoie@rci.rutgers.edu
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00053-1

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D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
Previous structure–activity data associated with several
protoberberine alkaloids and related compounds sug-
gest that the replacement of the methylenedioxy moiety
within ring-A with 2,3-dimethoxy substituents results in
a substantial loss of TOP1-targeting activity.11,13,23
Similarly, among various benzo[c]phenanthridines the
presence of 2,3-dimethoxy substituents within ring-A
was also associated with a loss of TOP1-targeting
activity or cytotoxicity.25,29À31 We synthesized similarly
substituted tetramethoxylated benzo[i]phenanthridines,
5a and 5b, to probe the extent to which their structure–
activity paralleled previous results observed with proto-
berberine alkaloids and benzo[c]phenanthridines. These
structure–activity studies were further extended to
include the benzo[i]phenanthridine derivatives, (6–8)
(Fig. 2).
Figure 1. Structures of coralyne, nitidine and MDD-coralyne.
Chemistry
ety within their structure. It has been speculated in the
case of nitidine and related benzo[c]phenanthridine
alkaloids that the iminium charge is necessary for bio-
logical activity.26À29 Similarly, the charged iminium
group is also a common feature of compounds related
to the protoberberine alkaloids, such as coralyne, that
did exhibit modest antitumor activity.11,13,21,30,31 Sev-
eral coralyne analogues with significant TOP1-targeting
activity were developed in our laboratory. These analo-
gues also possessed a charged iminium group and
proved to be difficult to formulate. Their limited solu-
bility and poor bioavailability hindered efforts to assess
their efficacy as antitumor agents in vivo.
The method used for the synthesis of the requisite 2-
bromo-1-naphthaldhydes is outlined in Scheme 1. These
substituted 2-bromo-1-naphthaldehyde and 1-aceto-2-
bromonaphthalene derivatives were employed as inter-
mediates for the preparation of variously substituted
benzo[i]phenanthridines (Schemes 2 and 3).
The syntheticroute used for the preparation of ocm-
pounds 4a and 5a is outlined in Scheme 2. The 2-bromo-1-
naphthaldehyde intermediates, 13 and 15, were prepared
from appropriately substituted b-tetralones as shown in
Scheme 1. Treatment of 6,7-methylenedioxy-b-tetralone
(9) and 6,7-dimethoxy-b-tetralone (10) with dimethyl-
formamide and phosphorus tribromide gave the respec-
tive 3,4-dihydro-2-bromonaphthaldehyde derivatives in
about 70% yield, which were subsequently oxidized
using DDQ in toluene to the respective bromo-
naphthaldehydes, 13 and 15. Compounds 13 and 15
could be converted into 14 and 16 respectively by treat-
ment with methylmagnesium bromide followed by PCC
oxidation.
Several substituted benz[a]acridine and benz[c]acridines
were synthesized previously in an effort to develop
noncharged analogues of coralyne and those proto-
berberines that are active as topoisomerase-targeting
agents.32 These analogues were prepared in an effort to
develop more suitable topoisomerase-targeting agents
for evaluation in vivo. While none of the benz[c]acri-
dines evaluated in this study was active as either a
TOP1- or TOP2-targeting agent, a benz[a]acridine deri-
vative did exhibit activity as a TOP1-targeting agent.
5,6-Dihydro-9,10-dimethoxy-3,4-methylenedioxybenz[a]-
acridine possesses similar TOP1-targeting activity to
coralyne. Within this series of compounds, a 3-phenyl-
quinoline moiety replaced the 3-phenylisoquinolinium
moiety present in compounds 1–3. These data demon-
strated that benz[a]acridines could serve as noncharged
bioisosteres of protoberberine alkaloids with retention
of TOP1-targeting activity.
Acetals 19 and 20 were obtained in 95% yield by treat-
ment of the respective bromonaphthaldehydes with eth-
ylene glycol in the presence of a catalytic amount of
p-toluenesulfonicacid as outlined in Scheme 2. A Dean–
Stark apparatus was used to remove the water gener-
ated during the acetalization reaction. Lithium-halogen
exchange was performed by treatment of the acetals 19
and 20 with n-butyllithium and then quenched with tri-
methylborate. Acidic work up of the reaction products
resulted in the formation of the boronicacid derivatives,
21 and 22, respectively. Palladium (0) catalyzed cou-
pling of compounds 21 and 22 with the 2-bromo-4,5-
dimethoxynitrobenzene33 resulted in the formation of
the 2-phenylnaphthalene derivatives, 23 and 24, respec-
tively. Reduction of the nitro groups of 23 and 24 with
subsequent intramolecular cyclization resulted in the
formation of the desired benzo[i]phenanthridines, 4a
and 5a, respectively, in 54–60% yield.
In the present study, we investigated whether benzo[i]-
phenanthridines could serve as suitable noncharged
bioisosteres for benzo[c]phenanthridine alkaloids, such
as nitidine. Nitidine is active as a dual toposiomerase-
targeting agent, that is, it is capable of stabilizing the
cleavable complex that forms between DNA and either
TOP1 or TOP2. Benzo[i]phenanthridines 4a and 4b,
which possess a 2,3-methylenedioxy substituent in ring-
A and methoxyl groups at positions 8 and 9 in ring-D,
were viewed as prime candidates for synthesis and
pharmacological evaluation.
The 1-aceto-2-bromonaphthalene derivatives, 14 and
16, were used as intermediates for the preparation of 4b
and 5b, respectively. The use of ortho-nitrophenyl-

D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
1811
Figure 2. Structures of benzo[i]phenanthridine derivatives synthesized and evaluated for pharmacological activity.
Scheme 1. General approach for 2-bromo-1-naphthaldehydes and 2-bromo-1-acetonaphthalene derivatives. (i) DMF, PBr3, CHCl3; (ii) DDQ, tol-
uene; (iii) CH3MgBr, THF; (iv) PCC, CH2Cl2.
Scheme 2. Synthesis of 8,9-dimethoxy-2,3-methylenedioxy benzo[i]phenanthridine, 4a, and 2,3,8,9-tetramethoxybenzo[i]phenanthridine, 5a. (i)
HOCH2CH2OH; TsOH; (ii) n-BuLi; (iii) B(OCH3)3; (iv) HCl; (v) Pd(PPh3)4, CuBr, THF; (vi) Zn, HOAc.

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D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
Scheme 3. Preparation of compounds 4b, 5b, and 6–8. (i) Pd(PPh3)4; CuCN, THF (27 and 28) Pd(PPh3)4; CuBr, THF (29–31); (ii) Zn/HOAc.
stannanes has been reported to be a useful method for
the preparation of benzo[i]phenanthridines.34 Stille
coupling of these ketones with 25, as outlined in Scheme
3, gave the 1-aceto-2-phenylnaphthalenes 27 and 28,
respectively. Reduction and concomitant cyclization of
27 and 28 occurred on treatment with zinc in acetic acid
to provide the desired 5-methyl substituted benzo[i]phe-
nanthridines, 4b and 5b, respectively.
of pyridine was used to form its acid chloride 35.
Treatment of compound 35 with (trimethylsilyl)diazo-
methane, a commercially available safer alternative to
diazomethane,36 resulted in the formation of diazoke-
tone 36 in good yield. Rhodium(II) acetate catalyzed
cyclization led to the formation of 6,7-methlyenedioxy-
2-tetralone 9 in about 90% yield (70% after chromato-
graphy).37 It should be noted that the chromatography
must be rapid since compound 9 readily oxidizes and
substantial loss can occur if this purification step is
prolonged.
The syntheticapproach outlined in Scheme 3 was also
employed for the synthesis of 6–8 based upon the
improved overall yields in formation of 2-(2-nitrophe-
nyl)-1-naphthaldehydes observed using the Stille
couping reaction. Reaction of trimethyl(2-nitro-4,5-
dimethoxyphenyl)stannane, 25, with 17 and 18 provided
the 2-(2-nitrophenyl)-1-naphthaldehydes, 29 and 30.
Trimethyl(2-nitro-4,5-methylenedioxyphenyl)stannane,
26, was coupled with 13 to form the 2-(2-nitrophenyl)-1-
naphthaldehyde, 31. Treatment of 29–31 with zincin
acetic acid provided the desired benzo[i]phenanthridines
(6–8).
The b-tetralone 9 required for the synthesis of 13 was
prepared as illustrated in Scheme 4 using two different
routes. In one approach (Method A), 3,4-methylene-
dioxyphenylpropionic acid was converted to its ethyl
ester, 32. Treatment of 32 with the anion of dimethyl
sulfate resulted in the formation of the b-ketosulfoxide,
33, which cyclized on treatment with trifluoroacetic acid
to the 1-methylthio-b-tetralone, 34.35 Reductive desul-
furization of 34 resulted in the formation of the required
6,7-methylenedioxy-b-tetralone, 9.
An alternative approach (Method B) for the prepara-
tion of 9 involved the formation of the diazoketone, 36.
This method for the preparation of b-tetralones
required fewer steps, and resulted in less difficult reac-
tion work-ups and higher overall yield with better
reproducibility. As shown in Scheme 4, 3,4-methylene-
dioxyphenylpropionicaicd was treated with thionyl
chloride in methylene chloride and a catalytic amount
Scheme 4. Syntheticstrategy employed for the preparation of 6,7-
methylenedioxy-b-tetralone, 9. Method A: (i)C2H5OH, TMSCl; (ii)
Na+CH3SOCH2- ; (iii) CF3COOH; (iv) H2, Pd/C. Method B: (v)
SOCl2, pyridine, CH2Cl2, (vi) TMSCHN2, THF, CH3CN; (vii)
Rh(OAc)2CH2Cl2.

D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
1813
Pharmacology
of both a 2,3- and 8,9-methylenedioxy group on
benzo[i]phenanthridine, as in 8, resulted in a derivative
that had comparatively poor solubility properties and
did not exhibit significant activity as either a TOP1- or
TOP2-targeting agent. Figure 4 illustrates the differ-
ences in the potency between 6 and 7, relative to 8 in the
TOP1-mediated DNA cleavage assay.
The benzo[i]phenanthridine derivatives (4–8) synthe-
sized in this study were evaluated for their potential to
stabilize the cleavable complex that forms in the pre-
sence of either TOP1 or TOP2 and DNA. The results of
these assays are summarized in Table 1. The data clearly
indicate that appropriately substituted benzo[i]phenan-
thridines can stabilize the cleavable complex formed in
the presence of DNA and both TOP1 and TOP2. The
more active analogues were those that possessed a
methylenedioxy substituent at the 2,3-position. Among
the benzo[i]phenanthridine evaluated in this study, 4a
and 4b were the more potent TOP1-targeting deriva-
tives. Both 4a and 4b had similar TOP1-targeting activ-
ity, exhibiting approximately 10% of the potency of
nitidine. There was a notable difference between 4a and
4b with regard to their TOP2-targeting activity. While
4a possesses approximately 10% of the potency of niti-
dine in stabilizing cleavable complex formed in the pre-
sence of DNA and TOP2, 4b was approximately two
orders of magnitude less potent than nitidine. As
observed for coralyne derivatives and for various sub-
stituted benz[a]acridines, the presence of methoxyl sub-
stituents at both the 2- and 3-positions of these
benzo[i]phenanthridines (which are equivalent to the 3
and 4-positions of coralyne and protoberberine alka-
loids) was associated with a loss of topoisomerase-tar-
geting activity. Figure 3 illustrates the differences in the
potency of 4a and 4b, relative to 5a and 5b in the TOP1-
mediated DNA cleavage assay.
The relative cytotoxic activities of these compounds,
determined in the human lymphoblast cell line,
RPMI8402, and its camptothecin-resistant variant, CPT-
K5, are provided in Table 1. The resistance of CPT-K5
cells to camptothecin is related to a mutant form of
TOP1 where aspartate-533 is replaced by glycine.
Figure 3. Stimulation of enzyme-mediated DNA cleavage by 4a, 4b,
5a, 5b 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 DNA, 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 4a, 4b, 5a, and 5b. A triangle on top
of the lanes is used to indicate the direction of increasing concen-
tration of the compound. The narrow end of the triangle indicates the
lowest concentration of the compound.
The effect of a single methoxyl group within the A-ring
of these benzo[i]phenanthridines on activity was also
evaluated. In contrast to 5a and 5b, the trimethoxylated
derivatives, 6 and 7, did exhibit modest TOP1- and
TOP2-targeting activity. While 6 and 7 had similar
activities, they were substantially less potent than 4a or
4b as TOP1- and TOP2-targeting agents. The presence
Table 1. Pharmacological activities of benzo[i]phenanthridine derivatives
TOP1-mediated DNA cleavageb
TOP2-mediated DNA cleavagec
Cytotoxicity IC50 (mM)a cell lines
Compd
RPMI8402
CPT-K5
1
2
10
1
(—)d
1
3.9
0.4
17
4.0
3
4a
4b
5a
5b
6
7
40
10
10
(—)d
(—)d
80
100
(—)d,e
0.1
(—)d
10
10
5.9
4.5
7.6
14
22
5.0
27
10.8
9.7
16
11
7.5
100
(—)d
(—)d
500
500
(—)d,e
(—)d
1
5.0
8.5
8
>40e
0.005
0.22
>40e
>40
0.28
CPT
VM-26
aIC50 has been calculated after 4 days of continuous drug exposure.
bTopoisomerase I cleavage values are reported as REC, relative effective concentration, i.e., concentrations relative to nitidine, whose value is
arbitrarily assumed as 1, that are able to produce approximately 10% cleavage on the plasmid DNA in the presence of human topoisomerase I.
cTopoisomerase II cleavage values are reported as REC, relative effective concentration, i.e., concentrations relative to VM-26 (teniposide), whose
value is arbitrarily assumed as 1, that are able to produce approximately 10% cleavage on the plasmid DNA in the presence of human topoisome-
rase II.
dCompound with a REC value indicating a potency of less than 1% of that of nitidine (2) as a TOP1-targeting agent and less than 0.1% of nitidine
as a TOP2-targeting agent were designated as not having significant activity, (—).
eThe extremely poor solubility of this derivative could have limited accurate assessment of its pharmacological activity.

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D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
In general, there is a poor correlation between cyto-
toxicity and topoisomerase-targeting activity among
these protoberberine derivatives. Several factors could
be responsible for this observation. Some coralyne ana-
logues are dual topoisomerase poisons while others are
not. In addition, several coralyne analogues possess the
iminium moiety. Differences in cellular absorption,
therefore, could be a limiting factor associated with the
cytotoxicity of various analogues.38
The reactivity of the charged iminium moiety toward
nucleophilic attack has been proposed as a possible
basis for the antileukemic activity of certain benzo[c]-
phenanthridine alkaloids.26À28,39 It has been successfully
demonstrated, however, that stable noncharged analo-
gues of nitidine can be developed that retain activity as
topoisomerase poisons and possess potent cytotoxic
activity.19,29 Benz[a]acridines structurally-related to
protoberberine alkaloids, with the distinct difference of
not possessing the cationic iminium group, have been
developed that possess topoisomerase I poisoning
activity comparable to coralyne.32 These results promp-
ted our investigation into whether benzo[i]phenan-
thridines could be developed to serve as noncharged
analogues of benzo[c]phenanthridine alkaloids, such as
nitidine, that retain the ability to act as potent dual
topoisomerase poisons.
Figure 4. Stimulation of enzyme-mediated DNA cleavage by 6, 7, 8
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, DNA, and serially
(10-fold each) diluted compound from 0.01 to 1.0 mM for CPT and 1.0
to 100 mM for compounds 6, 7, and 8. A triangle on top of the lanes is
used to indicate the direction of increasing concentration of the com-
pound. The narrow end of the triangle indicates the lowest concen-
tration of the compound.
Camptothecin is not able to stabilize cleavable complex
formation between this mutant enzyme and DNA. The
least cytotoxic derivatives in RPMI8402 were those that
were devoid of any significant topoisomerase-targeting
activity. Thus, 5a, 5b, and 8 had IC50 values ꢀ14 mM.
The differences in topoisomerase-targeting activity
among the other benzo[i]phenanthridines evaluated,
were not reflected in their relative cytotoxicity. As these
analogues are able to target both TOP1 and TOP2,
major differences in cytotoxicity were not observed for
4a, 4b, 6, and 7 between RPMI8402, and its camp-
tothecin-resistant variant, CPT-K5. The more potent
topoisomerase-targeting agents such as nitidine, camp-
tothecin, and VM-26 did exhibit more pronounced
cytotoxicity in RPMI8402. In the case of camptothecin
was there was a 10,000-fold decrease in its relative
cytotoxic potency observed with CPT-K5 relative to
RPMI8402 cells. As would be expected in the case of
nitidine, the difference in its relative cytotoxicity
between these two cell lines is comparatively minor as it
can also exert a cytotoxic effect by targeting either
TOP1 or TOP2. In the case of VM-26, which acts
exclusively as a TOP2-targeting agent, no difference in
its relative cytotoxicity was observed between these two
cell lines.
Structure–activity data on coralyne derivatives, proto-
berberine alkaloids, benzo[c]phenanthridines and var-
iously substituted benz[a]acridines provided direction in
the selection of compounds for evaluation. The 3,4-
methylenedioxy substituent on the A-ring of benz[a]-
acridines and protoberberine alkaloids analogues, such
as MDD-coralyne, is similarly positioned in regard to
molecular topology to a 2,3-methylenedioxy substituent
on the A-ring of benzo[i]phenanthridines and nitidine.
In the case of nitidine and coralyne, the presence of
methoxyl groups at the 8,9- and 10,11-positions,
respectively, was associated with enhanced TOP1-tar-
geting activity. Based on our studies and previous
structure–activity data on benzo[c]phenanthridines, it
was anticipated that benzo[i]phenanthridines with simi-
lar substitution and molecular topology would be active
as TOP1-targeting agents.
This working hypothesis was validated by the observa-
tion that compounds 4a and 4b, are able to stabilize
the cleavable complex formed between topoisomerase
and DNA. As was observed for nitidine, 4a and 4b
stabilized cleavable complex formation with both
TOP1 and TOP2. Previous studies have demonstrated
that replacement of the methylenedioxy moiety in
similarly substituted protoberberine or benzo[c]phe-
nanthridine alkaloids with two methoxyl substituents
results in a loss of topoisomerase-targeting activity.
The structure–activity relationships observed with these
benzo[i]phenanthridine derivatives also appears to par-
allel this phenomenon. Thus, as would be anticipated,
evaluation of the 2,3,8,9-tetramethoxy analogues 5a
and 5b demonstrated that neither of these compounds
is active in targeting either TOP1 or TOP2. In contrast
to the structure–activity relationships observed with
Discussion
Protoberberine alkaloids and related compounds can
exhibit significant activity as either TOP1- or TOP2-
targeting agents. Previous studies have demonstrated
that significant specificity for either of these topoisome-
rases can be achieved by subtle alterations in either
substituents or substitution pattern.11,13,23 Despite the
observation that several of these compounds can exhibit
similar potency to camptothecin as TOP1-targeting
agents, they possess only modest cytotoxic activity.13,23

D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
1815
nitidine analogues, the presence of a 5-methyl sub-
stituent in the case of these benzo[i]phenanthridine
derivatives did not significantly enhance topoiso-
merase-targeting activity.
on a Varian Gemini-200 Fourier Transform spectro-
1
meter. NMR spectra (200 MHz H and 50 MHz 13C)
were recorded in the CDCl3 with chemical shifts repor-
ted in d units downfield from tetramethylsilane (TMS).
Coupling constants are reported in hertz (Hz). Mass
spectra were obtained from Washington University
Resource for Biomedical and Bio-organic Mass Spec-
trometry within the Department of Chemistry at
Washington University, St. Louis, MO. Combustion
analyses were performed by Atlantic Microlabs, Inc.,
Norcross, GA, and were within Æ0.4% of the theore-
tical value. Compounds 10–12 were purchased from
Aldrich Chemical Co. Compounds 25 and 26 were pre-
pared as previously described.34
It has been suggested that either active transport or
hydrolysis of the iminium group of nitidine and cor-
alyne derivatives may be required for cellular absorp-
tion.38,40 In addition, it has been speculated that the
cationic charge associated with nitidine and related
benzo[c]phenanthridine alkaloids is linked to the occur-
rence of several adverse effects.40,41 It has also been
speculated that the reactivity of the iminium charge on
the benzo[c]phenantridine ring toward nucleophilic
attack may provide an explanation for the weak anti-
tumor activity observed in vivo for various nitidine
analogues.42 As compounds 4a and 4b are noncharged,
it was hoped that their decreased intrinsic activity as
topoisomerase-targeting agents relative to nitidine
would be off-set by increased cellular absorption and
cytotoxicity. This, however, was not observed in the
present studies performed using RPMI8402. Com-
pounds 4a and 4b had only one-tenth the relative cyto-
toxicity of nitidine in this assay. While there were
significant differences between the intrinsic topoisome-
rase-targeting activities of 6 and 7 as compared to 4a,
these derivatives had similar cytotoxicity. These data
suggest that in the absence of potent intrinsic topoi-
somerase-targeting activity other mechanisms may
begin to substantially contribute to the observed cyto-
toxicity. Studies in our laboratory suggest an improved
correlation between cytotoxic activity and intrinsic
TOP1-targeting activity is generally realized with com-
pounds that (1) possess at least 10% of the intrinsic
potency of camptothecin as a TOP1-targeting agent and
(2) are selective as TOP1-targeting agents.
General procedure for the synthesis of benzo[i]phenan-
thridine derivatives (4a, 4b, 5a, 5b, 6, 7, 8). The respec-
tive 2-(ortho-nitrophenyl)-1-naphthaldehyde or 2-(ortho-
nitrophenyl)-1-acetonaphthalene (100 mg) was dissolved
in glacial acetic acid (8 mL) and heated to reflux with
zinc dust (200 mg) for 4 h. Acetic acid was evaporated in
vacuo and the resulting residue extracted with chloro-
form. The chloroform solution was filtered through a
Celite bed. The filtrate was washed successively with
saturated sodium bicarbonate solution and brine and
evaporated to dryness. The residue obtained was chro-
matographed on 75 g of silica using a 1:1 mixture of
hexanes:ethyl acetate as eluent.
2,3-Methylenedioxy-8,9-dimethoxybenzo[i]phenanthridine
(4a). Prepared from compound 23 in 60% yield; mp
>250 ꢁC; IR (Nujol): 1680; UV (CHCl3): 285, 360,
1
380 nm (log e=3.01, 2.21, 2.47); H NMR: d 4.10 (3H,
s), 4.14 (3H, s), 6.15 (2H, s), 7.28 (1H, s), 7.68 (1H, s),
7.83 (1H, s), 7.97 (1H, d, J=9.2), 8.16 (1H, s), 8.28 (1H,
d, J=9.2), 9.85 (1H, s); 13C NMR: d 56.7, 100.3, 102.1,
102.2, 106.1, 109.2, 118.4, 127.5, 128.9, 131.2, 131.7,
140.8, 145.6, 145.7, 148.4, 149.9, 150.4, 151.7, 176.1;
HRMS m/z calcd for C20H15NO4: 333.1001; found:
333.0999.
Coralyne and nitidine have not proved to be highly
effective as antitumor agents in vivo. It is likely that
these charged compounds are subject to several dynam-
ics within mammalian systems that limit their absorp-
tion and bioavailability. Noncharged compounds, such
as substituted benzo[i]phenanthridines, may represent
an alternative class of TOP1-targeting agents that is free
of some of the limitations associated with these posi-
tively charged alkaloids. Studies are in progress to
develop benzo[i]phenanthridines with enhanced intrinsic
TOP1-targeting activity and cytotoxicity.
8,9-Dimethoxy-5-methyl-2,3-methylenedioxybenzo[i]phe-
nanthridine (4b). Prepared from compound 27 in 63%
yield; mp >250 ꢁC; IR (Nujol): 1685; UV (CHCl3): 280,
1
365, 385 nm (log e=2.79, 1.90, 2.03); H NMR: d 3.36
(3H, s), 4.08 (3H, s), 4.13 (3H, s), 6.15 (2H, s), 7.31 (1H,
s), 7.51 (1H, s), 7.80 (1H, s), 7.93 (1H, d, J=9.1 Hz),
8.29–8.35 (2H, m); 13C NMR: d 31.7, 56.6, 102.1, 105.8,
106.3, 108.8, 118.5, 119.1, 122.9, 127.9, 130.2, 131.3,
132.7, 137.1, 140.3, 147.3, 148.8, 149.7, 151.7, 155.2;
HRMS m/z calcd for C21H17NO4: 347.1158; found:
347.1156.
Experimental
Chemistry
2,3,8,9-Tetramethoxybenzo[i]phenanthridine (5a). Pre-
pared from compound 24 in 54% yield; mp 267–268 ꢁC;
IR (Nujol): 1621, 1513, 1282; H NMR: d 4.04 (3H, s),
4.06 (3H, s), 4.10 (3H, s), 4.13 (3H, s), 7.22 (1H, s), 7.55
(1H, s), 7.74 (1H, s), 7.89 (1H, d, J=8.8 Hz), 8.05 (1H,
s), 8.18 (1H, d, J=8.8 Hz), 9.82 (1H, s); 13C NMR: d
56.4, 56.6, 102.0, 102.3, 108.5, 109.9, 118.3, 118.4, 120.7,
120.8, 125.7, 127.5, 130.7, 141.5, 145.9, 150.0, 150.1,
150.9, 151.4; HRMS m/z calcd for C21H19NO4:
349.1314; found: 349.1321.
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,
Eschwege, Ger.) using the solvent systems indicated.
Infrared spectral data (IR) were obtained on a Perkin-
Elmer 1600 Fourier transform spectrophotometer and
are reported in cmÀ1. Proton (1H NMR) and carbon
(13C NMR) nuclear magnetic resonance were recorded
1

1816
D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
5-Methyl-2,3,8,9-tetramethoxybenzo[i]phenanthridine (5b).
Prepared from compound 28 in 60% yield; mp 255–
dropwise to a solution of rhodium(II) acetate in di-
chloromethane (15 mL). The resulting mixture was
refluxed under nitrogen for 1 h. The reaction mixture
was evaporated in vacuo to give 9 as deep yellow crys-
tals in 90% yield, which wasꢁtypically used without fur-
ther purification, mp 91–92 C (lit43 mp 88–91 ꢁC); IR
ꢁ
1
257 C; IR (Nujol): 1620, 1513, 1209; H NMR: d 3.42
(3H, s), 4.08 (6H, s), 4.13 (6H, s), 7.33 (1H, s), 7.52 (1H,
s), 7.82 (1H, s), 7.98 (1H, d, J=8.8 Hz), 8.32 (1H, s),
8.35 (1H, d, J=8.8 Hz); 13C NMR: d 31.6, 56.4, 56.5,
56.6, 102.1, 108.7, 108.8, 108.9, 118.6, 119.0, 122.2,
126.5, 129.0, 130.9, 132.6, 140.4, 149.1, 149.6, 149.7,
151.7, 154.9; HRMS m/z calcd for C22H21NO4:
363.1470; found: 363.1471.
1
(Nujol): 1727; H NMR: d 2.52 (2H, t, J=6.9 Hz), 2.96
(2H, t, J=6.9 Hz), 3.48 (2H, s), 5.93 (2H, s), 6.60 (1H,
s), 6.71 (1H, s); 13C NMR: d 28.7, 38.7, 45.4, 101.4,
108.7, 118.9, 121.7, 126.6, 130.3, 147.0, 210.9; anal.
calcd for C11H10O3: C, 69.46, H, 5.30; found: C, 69.40,
H, 5.29.
2,8,9-Trimethoxybenzo[i]phenanthridine (6). Prepared
from compound 29 in 50% yield; mp 207–209 ꢁC; IR
1
(KBr): 1620, 1487, 1215; H NMR: d 3.99 (3H, s), 4.10
General procedure for the synthesis of 2-bromo-1-naph-
thaldehydes (13, 15, 17, 18). Dimethylformamide (3.0 g,
41 mmol) was added dropwise to solution of phospho-
rus tribromide (3.3 mL, 35 mmol) in dry chloroform
(50 mL) at 0 ꢁC. The mixture was stirred at 0 ꢁC for 1 h
to give a pale yellow suspension. A solution of the
appropriately substituted b-tetralone (2.0 g, 9.7 mmol)
in chloroform 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 saturated aqueous
NaHCO3 solution was added dropwise until no effer-
vescence was obtained. The resulting mixture was
extracted with dichloromethane, dried (Na2SO4) and
evaporated to provide the respective 2-bromo-3,4-dihy-
dro-1-naphthaldehyde derivatives as yellow solids. Each
of the 2-bromo-3,4-dihydro-1-naphthaldehyde deriva-
tives were chromatographed on silica gel using a 3:1
mixture of hexanes:ethyl acetate as the eluent to give the
respective 2-bromo-3,4-dihydro-1-naphthaldehydes as
(3H, s), 4.14 (3H, s), 7.33 (1H, d, J=2.6 Hz), 7.40 (1H,
dd, J=9.2, 2.6 Hz), 7.62 (1H, s), 7.84 (1H, s), 8.02 (1H, d,
J=9.0 Hz), 8.41 (1H, d, J=9.0Hz), 8.77 (1H, d,
J=9.2Hz), 9.99 (1H, s); 13C NMR: d 56.0, 56.5, 56.6,
101.9, 108.8, 110.0, 119.5, 120.8, 121.7, 124.0, 125.1, 130.4,
131.3, 133.6, 141.8, 146.0, 151.5, 158.8; HRMS m/z calcd
for C20H17NO3+H: 320.1287; found: 320.1288.
3,8,9-Trimethoxybenzo[i]phenanthridine (7). Prepared
from compound 30 in 52% yield; mp 224 ꢁC; IR (KBr):
1
1615, 1502, 1220; H NMR: d 4.07 (3H, s), 4.10 (3H, s),
4.14 (3H, s), 7.30 (1H, dd, J=8.8, 2.4 Hz), 7.63 (1H, s),
7.86 (1H, s), 7.90 (1H, d, J=8.8 Hz), 8.03 (1H, d,
J=8.8 Hz), 8.19 (1H, d, J=2.4 Hz), 8.27 (1H, d,
J=8.8 Hz), 9.97 (1H, s); 13C NMR: d 56.0, 56.6, 102.2,
103.0, 109.9, 117.6, 117.9, 120.0, 121.5, 127.0, 130.8,
131.6, 132.3, 142.3, 146.1, 150.4, 152.1, 159.9; HRMS m/z
calcd for C20H17NO3+H: 320.1287; found: 320.1293.
1
determined by H NMR. Without further characteriza-
2,3 - Methylenedioxy - 8,9 - methylenedioxybenzo[i]phen-
anthridine (8). Prepared from compound 31 in 40%
yield; mp >250 ꢁC; IR (KBr) 1474, 1386, 1279, 1194; 1H
NMR: d 6.17 (2H, s), 6.18 (2H, s), 7.31 (1H, s), 7.58
(1H, s), 7.92 (1H, s), 7.98 (1H, d, J=9.2 Hz), 8.20 (1H,
s), 8.27 (1H, d, J=9.2 Hz), 9.88 (1H, s); 13C NMR: d
99.8, 100.4, 102.1, 102.3, 106.2, 107.7, 118.8, 123.9,
127.0, 127.9, 130.9, 131.3, 146.2, 148.3, 149.7, 151.3,
152.0, 152.9, 154.4; HRMS m/z calcd for C19H11NO4:
317.0688: found: 317.0686.
tion, these 2-bromo-3,4-dihydro-1-naphthaldehydes
(2.4 g, 8.1 mmol) were mixed with DDQ (2.2 g, 97 mmol)
in toluene (50 mL) and refluxed for 15 h. After cooling
to room temperature, the mixture was subjected to fil-
tration through a Celite bed and the filtrate was evapo-
rated to dryness. The residue obtained was
chromatographed on 75 g silica gel using a 3:1 mixture
of hexanes–ethyl acetate as eluent to give the respective
2-bromo-1-naphthaldehydes in good yield.
2-Bromo-6,7-methylenedioxy-1-naphthaldehyde
(13).
6,7-Methylenedioxy-2-tetralone (9): Method A. A solu-
tion of 34 (669 mg, 2.83 mmol) in glacial acetic acid
(10 mL) was placed in a hydrogenation flask. To this
mixture 460 mg of 10% Pd-C was added and the result-
ing mixture was shaken in a Parr apparatus at 40 psig of
hydrogen for 40 h. The reaction mixture was filtered
through a Celite bed, which was washed thrice with
5 mL portions of glacial acetic acid. The glacial acetic
acid was rotaevaporated to give the crude tetralone, 9.
The crude tetralone was then stirred vigorously with
sodium bisulfite (2.0 g in 7.0 mL H2O and 4.0 mL EtOH)
to convert it to the more stable bisulfite adduct. Pure
tetralone was generated as required from its bisulfite
adduct by treatment with 10% sodium carbonate solu-
tion followed by extraction with dichloromethane; mp
91–92 ꢁC (lit43=88–91 ꢁC).
Prepared from 9 in 96% yield; mp 165–166 ꢁC; IR
1
(Nujol): 1680; H NMR: d 6.09 (2H, s), 7.05 (1H, s),
7.51 (1H, d, J=8.7 Hz), 7.63 (1H, d, J=8.7 Hz), 8.60
(1H, s), 10.68 (1H, s); 13C NMR: d 102.1, 102.3, 104.6,
127.4, 129.5, 129.6, 130.1, 131.1, 134.6, 148.6, 151.7,
195.5; anal. calcd for C12H7O3Br: C, 51.60, H, 2.51;
found: C, 51.98, H, 2.48.
2-Bromo-6,7-dimethoxy-1-naphthaldehyde (15). Prepared
from 10 in 95% yield; mp 141–142 ꢁC; H NMR: d 4.00
1
(3H, s), 4.05 (3H, s), 7.07 (1H, s), 7.54 (1H, d, J=8.5 Hz),
7.79 (1H, d, J=8.5 Hz), 8.71 (1H, s), 10.74 (1H, s); 13C
NMR: d 56.3, 56.6, 104.2, 107.0, 126.5, 128.8, 129.4,
129.8, 129.9, 134.3, 150.4, 153.1, 195.9; HRMS m/z calcd
for C13H11O3Br: 293.9891; found: 293.9896.
2-Bromo-6-methoxy-1-naphthaldehyde (17). Prepared
from compound 11 in 50% yield; mp 105–107 ꢁC; IR
(KBr): 1672; 1H NMR: d 3.92 (3H, s), 7.09 (1H, d,
6,7-Methylenedioxy-2-tetralone (9): Method B. A di-
chloromethane solution of compound 36 was added

D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
1817
J=2.6 Hz), 7.29 (1H, dd, J=9.4, 2.6 Hz), 7.61 (1H, d,
J=8.8 Hz), 7.73 (1H, d, J=8.8 Hz), 8.99 (1H, d,
J=9.4 Hz), 10.70 (1H, s); 13C NMR: d 56.8, 106.9,
122.3, 126.8, 127.5, 128.2, 128.7, 131.6, 134.6, 135.1,
158.7, 195.4; HRMS m/z calcd for C12H9BrO2:
263.9786; found: 263.9782.
General procedure for the synthesis of 2-bromonaphthal-
dehyde-1-acetals (19, 20). The respective 2-bromo-1-
naphthaldehyde derivatives (1.95 mmol), ethylene glycol
(0.7 mL) and p-toluenesulfonicaicd (10 mg) were dis-
solved in dry toluene (30 mL). This reaction mixture
refluxed under nitrogen in a flask fitted with a Dean-
Stark apparatus to remove the water formed during
acetal formation. At the end of the reaction (15 h), the
solvent from the cooled reaction mixture was evapo-
rated in vacuo and the residue obtained was dissolved in
ethyl acetate (50 mL). The ethyl acetate solution was
washed with a saturated solution of sodium bicarbo-
nate, dried and the solvent was removed to give the
crude acetals. Chromatography on silica gel using a 3:17
mixture of ethyl acetate–hexanes afforded the pure
acetals as clear viscous liquids in good yield.
2-Bromo-7-methoxy-1-naphthaldehyde (18). Prepared
from compound 12 in 62% yield; mp 132–134 ꢁC; IR
1
(KBr): 1670; H NMR: d 3.97 (3H, s), 7.21 (1H, dd,
J=8.9, 2.6 Hz), 7.54 (1H, d, J=8.7 Hz), 7.71 (1H, d,
J=8.9 Hz), 7.77 (1H, d, J=8.7 Hz), 8.69 (1H, d,
J=2.6 Hz), 10.77 (1H, s); 13C NMR: d 56.0, 103.6,
120.5, 126.8, 128.9, 129.1, 130.3, 132.8, 134.2, 135.8,
161.6, 195.7; HRMS m/z calcd for C12H9BrO2:
263.9786; found: 263.9783.
General procedure for the synthesis of 1-aceto-2-bromo-
naphthalene derivatives (14, 16). A 1.4 M solution of
2-Bromo-6,7-methylenedioxynaphthaldehyde-1-ethylace-
tal (19). Prepared from 13 in 95% yield; IR (Nujol):
1665, 1617; H NMR: d 4.11–4.18 (2H, m), 4.36–4.43
1
methylmagnesium
bromide
in
tetrahydrofuran
(16.8 mL, 16.8 mmol) was added to a solution of the
respective naphthaldehydes (13 and 15; 715 mg;
2.56 mmol) in dry tetrahydrofuran (20 mL) at 0 ꢁC
under nitrogen. The reaction mixture was stirred for 1 h
at 0 ꢁC. The reaction was quenched by dropwise addi-
tion of water (100 mL) followed by rapid addition of
0.1 N hydrochloric acid (50 mL). The reaction mixture
was extracted with ethyl acetate, dried and the solvent
was evaporated in vacuo to give an oily residue. The oil
was triturated with chloroform to give the respective
alcohol derivatives as white needle shaped crystalline
solids in 95% and 97% yield respectively. These benzyl
alcohols were not characterized, but were treated as
formed with pyridinium chlorochromate (857 mg,
3.98 mmol) suspended in dry dichloromethane (10 mL).
The respective alcohols (2.4 mmol) were added to this
suspension and the resulting mixture was stirred under
nitrogen at room temperature for 6 h. Ether (100 mL)
was added to the reaction mixture and the suspension
obtained was filtered through a Celite bed. The filtrate
was evaporated to dryness and the residue obtained was
chromatographed on 75 g silica gel using a 1:9 mixture
of ethyl acetate–hexanes to give the respective 1-aceto-2-
bromonaphthalene derivatives (14 and 16) as fluffy
bright white needles in 85% and 90% yield, respectively.
(2H, m), 6.03 (2H, s), 6.56 (1H, s), 7.05 (1H, s), 7.41–
7.48 (2H, m), 7.73 (1H, s); 13C NMR: d 65.6, 101.8,
102.6, 104.8, 106.3, 123.0, 128.0, 129.3, 130.4, 131.1,
131.5, 147.9, 148.6; anal. calcd for C14H11O4Br: C,
52.10, H, 3.41; found: C, 52.52, H, 3.48.
2- Bromo - 6,7 - dimethoxynaphthaldehyde - 1 - ethylacetal
(20). Prepared from 15 in 95% yield; IR (Nujol): 1660,
1
1635; H NMR: d 4.02 (3H, s), 4.06 (3H, s), 4.13–4.19
(2H, m), 4.38–4.48 (2H, m), 7.09 (1H, s), 7.54–7.68 (2H,
m), 7.72 (1H, s), 7.78 (1H, s); 13C NMR: d 56.2, 56.6,
65.7, 102.8, 104.6, 106.5, 122.9, 128.4, 129.1, 130.5,
131.2, 131.3, 148.0, 148.5; anal. calcd for C15H15O4Br:
C, 53.11, H, 4.48; found: C, 52.93, H, 4.51.
Preparation of 2-(2-nitro-3,4-dimethoxyphenyl)-6,7-me-
thylenedioxy-1-naphthaldehyde, 23, and 2-(2-nitro-3,4-di-
methoxyphenyl)-6,7-dimethoxy-1-naphthaldehyde, (24).
Acetals (19 and 20, 1.4 mmol) were each dissolved in
anhydrous tetrahydrofuran (10 mL). This solution was
stirred under nitrogen at À78 ꢁC. A hexanes solution of
n-butyllithium (1.2 mL, 2.8 mmol) was added slowly and
the reaction mixture was stirred at À78 ꢁC for 30 min. A
pale yellowish brown solution was obtained. To this
yellow reaction mixture trimethylborate (0.5 mL,
4.2 mmol) was added and the resulting mixture was
stirred at À78 ꢁC for 1 h prior to allowing it to come to
room temperature. A 5% solution of hydrochloric acid
(20 mL) was added to the reaction mixture and stirred
for 30 min at room temperature. The tetrahydrofuran
was evaporated in vacuo and the water layer was
extracted with dichloromethane. The combined organic
layer was washed once with brine, dried and evaporated
to give the respective crude boronic acid derivatives (21
and 22). In a 3 neck flask, 2-bromo-4,5-dimethoxy-
nitrobenzene (876 mg, 3.34 mmol) was taken with
430 mg of tetrakis(triphenylphosphine)palladium (0) in
dimethoxyethane (20 mL) and the resulting solution was
stirred at room temperature under nitrogen for 30 min.
The solution changed color from brown to yellow. A
solution of the respective crude boronates (1.1 mmol) in
dimethoxyethane (5 mL) was added to the reaction
mixture followed by the addition of 2 M sodium
1-Aceto-2-bromo-6,7-methylenedioxynaphthalene
(14).
Prepared from 13 in 90% yield; mp 138 ꢁC; IR (Nujol):
1695; 1H NMR: d 2.67 (3H, s), 6.06 (2H, s), 6.89 (1H, s),
7.09 (1H, s), 7.41 (1H, d, J=8.8 Hz), 7.56 (1H, d,
J=8.8 Hz); 13C NMR: d 32.4, 101.0, 102.1, 104.9, 113.6,
127.6, 128.4, 129.6, 130.1, 139.8, 148.6, 149.7, 205.2;
HRMS m/z calcd for C13H9O3Br: 291.9735; found:
291.9730.
1-Aceto-2-bromo-6,7-dimethoxynaphthalene (16). Pre-
pared from 15 in 85% yield; mp 102–103 ꢁC; IR (Nujol):
1693; 1H NMR: d 2.70 (3H, s), 3.95 (3H, s), 3.99 (3H, s),
6.84 (1H, s), 7.10 (1H, s), 7.43 (1H, d, J=8.4 Hz), 7.56
(1H, d, J=8.4 Hz); 13C NMR: d 32.4, 56.4, 103.1, 107.2,
113.3, 126.3, 128.2, 128.7, 129.1, 139.0, 150.4, 151.4,
205.5; HRMS m/z calcd for C14H13O3Br: 308.0048;
found: 308.0059.

1818
D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
carbonate (5 mL). The reaction mixture was refluxed for
18 h. The reaction mixture was allowed to come to room
temperature and the reaction mixture was filtered
through a Celite bed. The filtrate was evaporated to
dryness and the residue obtained was column chroma-
tographed on 75 g silica gel using 1:9 mixture of ethyl
acetate–hexanes to give the respective 2-phenyl-1-naph-
thaldehyde derivatives, 23 and 24 in 25 and 22% overall
yield respectively, based on the acetals.
1-Aceto-2-(3,4-dimethoxy-6-nitrophenyl)-6,7-dimethoxy-
naphthalene (28). Prepared from 16 in 30% yield; mp
ꢁ
1
185–187 C; IR (Nujol): 1689, 1503, 1260; H NMR: d
2.25 (3H, s), 3.90 (3H, s), 3.98 (3H, s), 4.02 (3H, s), 4.03
(3H, s), 6.76 (1H, s), 7.08 (1H, d, J=8.4 Hz), 7.12 (1H,
s), 7.18 (1H, s), 7.70 (1H, s), 7.71 (1H, d, J=8.4 Hz); 13C
NMR: d 32.7, 56.4, 56.9, 57.0, 57.1, 103.8, 107.2, 108.5,
115.7, 124.9, 125.1, 128.2, 129.5, 130.1, 131.6, 136.9,
141.1, 149.0, 150.5, 151.2, 152.8, 208.0; HRMS m/z
calcd for C21H19NO7-C2H3O: 368.1134; found:
368.1135.
2-(3,4-Dimethoxy-6-nitrophenyl)-6,7-methylenedioxy-1-
naphthaldehyde (23). Prepared from 19 in 25% yield;
ꢁ
1
mp 225 C; IR (Nujol): 1670, 1515, 1309; H NMR: d
3.92 (3H, s), 4.04 (3H, s), 6.13 (2H, s), 6.74 (1H, s), 7.13
(1H, d, J=8.3 Hz), 7.18 (1H, s), 7.77 (1H, s), 7.87 (1H,
d, J=8.3 Hz), 8.76 (1H, s), 10.14 (1H, s); 13C NMR: d
57.0, 57.1, 102.1, 103.1, 104.7, 108.2, 114.9, 125.7, 127.9,
128.8, 129.5, 131.5, 133.6, 141.2, 144.1, 148.6, 149.2,
151.6, 152.9, 193.6.
2-(2-Nitro-3,4-dimethoxyphenyl)-6-methoxy-1-naphthal-
dehyde (29). Prepared from compound 17 in 70% yield;
mp 158–160 ꢁC; IR (KBr): 1677, 1518, 1328; H NMR:
1
d 3.92 (3H, s), 3.97 (3H, s), 4.05 (3H, s), 6.74 (1H, s),
7.20 (1H, d, J=2.7 Hz), 7.25 (1H, d, J=8.4 Hz), 7.35
(1H, dd, J=9.2, 2.7 Hz), 7.77 (1H, s), 7.94 (1H, d,
J=8.4 Hz), 9.15 (1H, d, J=9.2 Hz), 10.20 (1H, s); 13C
NMR: d 55.8, 57.0, 57.1, 107.0, 108.3, 115.0, 122.3,
126.4, 127.7, 127.9, 128.7, 129.4, 133.5, 135.5, 141.2,
143.0, 149.2, 153.0, 158.7, 193.6; HRMS m/z calcd for
C20H17NO6+H: 368.1134; found: 368.1129.
2- (3,4 - Dimethoxy - 6 - nitrophenyl) - 6,7 - dimethoxy - 1 -
naphthaldehyde (24). Prepared from 20 in 22% yield;
mp 228–230 ꢁC; IR (Nujol): 1676, 1513, 1259. 1H NMR:
d 3.93 (3H, s), 4.05 (3H, s), 4.09 (3H, s), 6.76 (1H, s),
7.16 (1H, d, J=8.1 Hz), 7.19, (1H, s). 7.78 (1H, s), 7.92
(1H, d, J=8.1 Hz), 8.66 (1H, s), 10.17 (1H, s); 13C
NMR: d 56.3, 56.6, 57.0, 57.1, 105.2, 107.1, 108.2, 115.0,
125.5, 127.0, 127.4, 129.6, 130.3, 133.2, 141.3, 144.3,
149.2, 150.4, 152.9, 153.1, 193.9; HRMS m/z calcd for
C21H19NO7-CHO: 368.1134; found: 368.1130.
2-(2-Nitro-3,4-dimethoxyphenyl)-7-methoxy-1-naphthal-
dehyde (30). Prepared from compound 18 in 70% yield;
mp 203–205 ꢁC; IR (KBr): 1677, 1513, 1325; H NMR:
1
d 3.91 (3H, s), 4.00 (3H, s), 4.04 (3H, s), 6.75 (1H, s),
7.15 (1H, d, J=8.2 Hz), 7.26 (1H, dd, J=8.9, 2.6 Hz),
7.79 (1H, s), 7.81 (1H, d, J=8.9 Hz), 8.00 (1H, d,
J=8.2 Hz), 8.81 (1H, d, J=2.6 Hz), 10.20 (1H, s); 13C
NMR: d 55.5, 56.5, 56.6, 104.0, 107.7, 114.2, 119.9,
124.3, 126.6, 128.9, 129.1, 129.8, 132.3, 134.2, 140.5,
146.3, 148.7, 152.4, 161.0, 193.2; HRMS m/z calcd for
C20H17NO6+H: 368.1134; found: 368.1127.
General procedure for the synthesis of 1-aceto-2-(3,4-di-
methoxy-6-nitrophenyl)naphthalene derivatives (27,28),
2-(2-nitro-3,4-dimethoxyphenyl)-1-naphthaldehydes (29–
30), and 2-(nitro-3,4-methylenedioxyphenyl)-6,7-methyle-
nedioxy-1-naphthaldehyde (31). A mixture containing
either 25 or 26 (0.98 mmol) and the respective 1-aceto-2-
bromonaphthalene derivatives (14 or 16, 0.89 mmol),
(Ph3P)4Pd (106 mg) together with copper cyanide
(17 mg) or in the case of the 2-bromonaphthaldehdes
(13, 17 or 18, 0.89 mmol) with cuprous bromide (10 mg)
were refluxed in toluene (20 mL) under nitrogen for 20–
24 h. After cooling to room temperature, ethyl acetate
(20 mL) was added to the reaction mixture and the
organiclayer was poured into a separating funnel. The
organiclayer was washed with distilled water (20 mL).
The two phases were allowed to separate as much as
possible and the aqueous layer was discarded. The
remaining emulsion was passed through a Celite bed.
The organiclayer was separated from the filtrate and
evaporated to dryness. The residue obtained was chro-
matographed on 75 g silica gel using a 3:2 mixture of
hexanes:ethyl acetate.
2-(2- Nitro-3,4-methylenedioxyphenyl)-6,7-methylene-
dioxy-1-naphthaldehyde (31). Prepared from compound
ꢁ
1
13 in 36% yield; mp 192–193 C; H NMR: d 6.12 (2H,
s), 6.20 (2H, s), 6.76 (1H, s), 7.11 (1H, d, J=8.4 Hz),
7.17 (1H, s), 7.66 (1H, s), 7.85 (1H, d, J=8.4 Hz), 8.73
(1H, s), 10.19 (1H, s); 13C NMR: d 102.2, 103.1, 103.9,
104.7, 106.0, 112.3, 125.5, 127.9, 128.9, 131.5, 131.6,
133.7, 143.0, 143.6, 148.5, 148.6, 151.6, 151.7, 193.3.
Ethyl 3,4-(methylenedioxy)dihydrocinnamate (32). Tri-
methylsilyl chloride (2.5 mL, 18.2 mmol) was added to a
solution of 3,4-(methylenedioxy)dihydrocinnamic acid
(1.5 g, 7.7 mmol) in dry ethanol (70 mL) and this mixture
was stirred at room temperature under nitrogen for 12 h.
The excess ethanol was evaporated in vacuo and the
residue obtained was chromatographed on 100 g silica gel
using 1:9 mixture respectively of ethyl acetate and hex-
anes to give a quantitative yield of the ester 32 as a col-
orless liquid; 1H NMR: d 1.21 (3H, t), 2.60 (2H, t), 2.87
(2H, t), 4.08 (2H, q, J=14.1, 7.3 Hz), 5.90 (2H, s), 6.56–
6.69 (3H, m); 13C NMR: d 14.5, 25.1, 34.2, 60.7, 100.8,
106.9, 121.8, 122.2, 122.5, 145.3, 147.1, 173.2; HRMS
m/z calcd for C12H14O4: 222.0892; found: 222.0895.
1-Aceto-2-(3,4-dimethoxy-6-nitrophenyl)-6,7-methylene-
dioxynaphthalene (27). Prepared from 14 in 20% yield;
mp 190–192 ꢁC; IR (Nujol): 1680, 1523, 1300; 1H NMR:
d 2.23 (3H, s), 3.89 (3H, s), 4.01 (3H, s), 6.07 (2H, s),
6.75 (1H, s), 7.06 (1H, d, J=8.4 Hz), 7.10 (1H, s), 7.16
(1H, s), 7.66 (2H, m); 13C NMR: d 32.6, 56.9, 57.1,
101.7, 102.6, 105.0, 108.4, 115.6, 125.1, 126.0, 128.7,
129.5, 130.8, 131.5, 133.8, 144.5, 149.6, 150.5, 150.7,
152.8, 207.6.
3,4-(Methylenedioxy)phenethylmethylsulfinylmethyl ke-
tone (33). The anion of dimethyl sulfoxide was pre-
pared by adding dry dimethyl sulfoxide (6.0 mL) to

D. Makhey et al. / Bioorg. Med. Chem. 11 (2003) 1809–1820
1819
sodium hydride (600 mg) and heating the mixture at 70–
75 ꢁC for 45 min under nitrogen. This reaction mixture
was allowed to cool to room temperature and then
transferred to a water bath maintained at 5–10 ꢁC. A
solution of 32 (1.0 g, 4.54 mmol) in dry dimethyl sulf-
oxide (6.0 mL) was added dropwise, over a period of
15 min, to the dimethyl sulfoxide anion generated pre-
viously. The reaction mixture was slowly allowed to
come to room temperature and stirred for 2 h. The
reaction mixture was poured into cold water (100 mL)
and acidified to pH 3–4 using 1.2 N hydrochloric acid
and extracted five times with 40 mL portions of chloro-
form. The combined organic layer was washed twice
with 100 mL portions of distilled water, dried over
anhydrous sodium sulfate, filtered and evaporated in
vacuo to give quantitative yield of 33 as a low melting
buff colored solid. Compound 33 was found to be
unstable to column chromatography using silica gel,
THF–acetonitrile (1:1) was added dropwise at 0 ꢁC to a
solution of TMSCHN2 (3 mL, 6 mmol, 2M in hexane) in
5 mL THF–acetonitrile (1:1). The resulting mixture was
stirred at 0 ꢁC for 4 h. After the reaction was completed,
the reaction mixture was evaporated in vacuo to give 36
as a red viscous oil in 95% yield which was used without
further purification.
Topoisomerase mediated DNA cleavage assays
Human topoisomerase I was isolated as a recombinant
fusion protein using a T7 expression system.23 DNA
topoisomerase II was purified from calf thymus gland as
reported previously.44 Plasmid YEpG was also purified
by the alkali lysis method followed by phenol
deproteination and CsCl/ethidium isopycnic centrifuga-
tion as described.45 The end-labeling of the plasmid was
accomplished by digestion with a restriction enzyme
followed by end-filling with Klenow polymerase as pre-
viously described.46 The cleavage assays were performed
as previously reported.47
1
however, H NMR of the crude compound indicated
that it could be used for the synthesis of 34 without
further purification; mp 60–62 ꢁC; 1H NMR: d 2.55 (3H,
s), 2.75–2.82 (4H, m), 3.56–3.76 (2H, q, J1=13.8,
J2=34.3 Hz), 5.82 (2H, s), 6.53–6.65 (3H, m); 13C
NMR: d 29.3, 41.4, 47.4, 64.4, 101.3, 108.7, 109.3, 121.6,
134.4, 146.4, 148.1, 202.1.
Cytotoxicity assays. The cytotoxicity was determined
using the MTT-microtiter plate tetrazolinium cytotoxi-
city assay (MTA).48À50 The human lymphoblast
RPMI8402 and its camptothecin-resistant variant cell
line, CPT-K5 were provided by Dr. Toshiwo Andoh
(Aichi Cancer Center Research Institute, Nagoya,
Japan).51 The cytotoxicity assay was performed 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- inacti-
vated fetal bovine serum, l-glutamine (2 mM), penicillin
(100 U/mL), and streptomycin (0.1 mg/mL). For deter-
mination of IC50, cells were exposed continuously with
varying concentrations of drug and MTT assays were
performed at the end of the fourth day.
1,2,3,4 - Tetrahydro - 1 - methylthio - 6,7 - methylenedioxy -
2(1H)-napthalenone (34). Trifluoroacetic acid (0.3 mL,
3.7 mmol) was dissolved in benzene (25 mL) and added
to the reaction flask containing 34 (470 mg, 1.85 mmol).
The reaction mixture was heated to reflux for 1.5 h. On
cooling to room temperature the reaction mixture was
transferred to a separating funnel and washed twice
using 10 mL portions of a saturated solution of sodium
bicarbonate. The benzene layer was dried over anhy-
drous sodium sulfate, filtered and evaporated in vacuo
to give a red syrup which was chromatographed over
100 g of silica gel using 1:9 mixture respectively of ethyl
acetate and hexanes to give 34 in 60% yield; mp 52 ꢁC;
1H NMR: d 2.05 (3H, s), 2.70–3.10 (4H, m), 4.02 (1H,
s), 5.85 (1H, s), 6.65 (1H, d, J=8.1 Hz), 6.72 (1H, d,
J=8.1 Hz); 13C NMR: d 16.3, 21.1, 34.2, 54.1, 101.6,
107.9, 118.5, 123.0, 127.6, 145.1, 147.3, 203.5; HRMS
m/z calcd for C12H12O3S: 236.0507; found: 236.0505.
Acknowledgements
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.
3-(3,4-Methylenedioxyphenyl)propionyl chloride (35). 3-
(3,4-Methylenedioxyphenyl)propionicidac (600 mg,
3.0 mmol) was suspended in methylene chloride (10 mL)
and a catalytic amount of pyridine (1 drop) was added.
Thionyl chloride (0.27 mL, 3.6 mmol) was then added
dropwise and the reaction mixture was stirred vigor-
ously under nitrogen at room temperature for 20 h.
After the reaction was completed (monitored by TLC),
the reaction mixture was evaporated in vacuo to give a
quantitative yield of 35 as a yellow oil which was used
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