Substituted benz[a]acridines and benz[c]acridines as mammalian topoisomerase poisons

Article abstract of DOI:10.1016/S0968-0896(00)00048-1

Coralyne and several other synthetic benzo[a,g]quinolizium derivatives related to protoberberine alkaloids have exhibited activity as topoisomerase poisons. These compounds are characterized by the presence of a positively charged iminium group, which has been postulated to be associated with their pharmacological properties. The objective of the present study was to devise stable noncharged bioisosteres of these compounds. Several similarly substituted benz[a]acridine and benz[c]acridine derivatives were synthesized and their relative activity as topoisomerase poisons was determined. While the benz[c]acridine derivatives evaluated as part of this study were devoid of topoisomerase poisoning activity, several dihydrobenz[a]acridines were able to enhance DNA cleavage in the presence of topo I. In contrast to certain protoberberine derivatives that did exhibit activity as topo II poisons, none of the benz[a]acridines derivatives enhanced DNA cleavage in the presence of topo II. Among the benz[a]acridines studied, 5,6-dihydro-3,4- methylenedioxy-9,10-dimethoxybenz[a]acridine, 13e, was the most potent topo I poison, with comparable potency to coralyne. These data suggest that heterocyclic compounds structurally related to coralyne can exhibit potent topo I posioning activity despite the absence of an iminium cation within their structure. In comparison to coralyne or other protoberberine derivatives, these benz[a]acridine derivatives possess distinctly different physicochemical properties and represent a novel series of topo I poisons. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00048-1

Bioorganic & Medicinal Chemistry 8 (2000) 1171±1182
Substituted Benz[a]acridines and Benz[c]acridines as Mammalian
Topoisomerase Poisons
Darshan Makhey, a Chiang Yu, b Angela Liu, b Leroy F. Liu b
and Edmond J. LaVoie a,*
aDepartment of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, 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
Received 24 August 1999; accepted 2 February 2000
AbstractÐCoralyne and several other synthetic benzo[a,g]quinolizium derivatives related to protoberberine alkaloids have exhibited
activity as topoisomerase poisons. These compounds are characterized by the presence of a positively charged iminium group,
which has been postulated to be associated with their pharmacological properties. The objective of the present study was to devise
stable noncharged bioisosteres of these compounds. Several similarly substituted benz[a]acridine and benz[c]acridine derivatives
were synthesized and their relative activity as topoisomerase poisons was determined. While the benz[c]acridine derivatives
evaluated as part of this study were devoid of topoisomerase poisoning activity, several dihydrobenz[a]acridines were able to
enhance DNA cleavage in the presence of topo I. In contrast to certain protoberberine derivatives that did exhibit activity as topo II
poisons, none of the benz[a]acridines derivatives enhanced DNA cleavage in the presence of topo II. Among the benz[a]acridines
studied, 5,6-dihydro-3,4-methylenedioxy-9,10-dimethoxybenz[a]acridine, 13e, was the most potent topo I poison, with comparable
potency to coralyne. These data suggest that heterocyclic compounds structurally related to coralyne can exhibit potent topo I
posioning activity despite the absence of an iminium cation within their structure. In comparison to coralyne or other proto-
berberine derivatives, these benz[a]acridine derivatives possess distinctly di€erent physicochemical properties and represent a novel
series of topo I poisons. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
indolocarbazoles,14 as well as the fungal metabolites,
bulgarein15 and saintopin,16 have been identi®ed as topo
I poisons.
DNA topoisomerases are nuclear enzymes that catalyze
the breaking and rejoining of DNA strands regulating
the topological state of DNA.1 4 Recent studies suggest
that topoisomerases are also involved in controlling
template supercoiling during RNA transcription.5,6 The
antitumor activity of topoisomerase poisons is asso-
ciated with their ability to stabilize the enzyme±DNA
cleavable complex. This drug-induced stabilization of
the enzyme±DNA cleavable complex e€ectively con-
verts the enzyme into a cellular poison.
The exceptional topoisomerase poisoning observed with
coralyne (1), nitidine (2), MDD-coralyne (3) and related
analogues prompted several studies on those structural
features which are associated with their ability to act
speci®cally as either topo I or II poisons.11,13,17 19
A
common feature associated with all three of these agents
is the presence of a 3-phenylisoquinolinium moiety
within their structure (Chart 1). It has been speculated
in the case of nitidine and related benzo[c]phenan-
thridine alkaloids that the iminium charge is necessary
for biological activity.20 23 Similarly, the charged imi-
nium group is also a common feature of compounds
related to the protoberberine alkaloids, such as cor-
alyne, that exhibit antitumor activity.11,13,17,24,25 It has
been reported, however, that 5,6,7,13a-tetrahydro-
coralyne, in which the iminium group of 5,6-dihydro-
coralyne has been converted to a cyclic tertiary amine,
does retain activity as a topo I poison.18
Camptothecin and its structurally-related analogues are
among the more extensively studied topoisomerase I
(topo I) poisons. Recently, bi- and terbenzimid-
azoles,7 10 certain benzo[c]phenanthridine and proto-
berberine alkaloids and their synthetic analogues,11 13
*Corresponding author. Tel.: +1-732-445-2674; fax: +1-732-445-
6312; e-mail: elavoie@rci.rutgers.edu
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00048-1

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D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
Chart 1. Structures of coralyne, nitidine and MDD-coralyne.
Chart 2. Hydration of coralyne.
The reactivity of the iminium moiety toward nucleo-
philic attack has been proposed as a possible basis for
the antileukemic activity of certain benzo[c]phenan-
thridine alkaloids.26 Janin et al. have successfully
demonstrated, however, that noncharged analogues of
nitidine can be developed that retain their activity as
topoisomerase poisons and possess potent cytotoxic
activity.23
In the present study a series of substituted benz[a]acri-
dines and benz[c]acridines (Chart 3) were synthesized as
bioisosteres of coralyne and structurally-related proto-
berberine alkaloids. To evaluate whether a charged
iminium moiety was linked to topoisomerase poisoning
activity, these benzacridine derivatives, as well as select
N-methyl derivatives, were evaluated as topo I and topo
II poisons. These compounds were also evaluated for
their cytotoxicity against the human lymphoblast cell
line, RPMI-8402, and its camptothecin-resistant variant,
CPT-K5.
Protoberberine alkaloids and related compounds can
exhibit either topo I or topo II poisoning activity. Pre-
vious studies have demonstrated that signi®cant speci®-
city for either of these topoisomerases can be achieved
by subtle alterations in either substituents or substitu-
tion pattern.11,13,17 Despite the observation that several
of these compounds had similar potency to camptothe-
cin as a topo I poison or similar potency to VM-26 as a
topo II poison, they possess only modest cytotoxic
activity.13,17 In general, there has been a poor correla-
tion between cytotoxicity and topoisomerase poisoning
activity among these protoberberine derivatives.
Chemistry
Scheme 1 outlines the synthetic procedures employed
for the preparation of benz[c]acridines structurally rela-
ted to coralyne. Reaction of 2-amino-4,5-dimethoxy-
acetophenone, 4, with 6,7-dimethoxy-1-tetralone, 5,
using similar reaction conditions to those previously
reported31 provided 5,6-dihydro-2,3,9,10-tetramethoxy-
benz[c]acridine, 6, which, when heated as a suspension
in decalin at 190 ꢀC in the presence of Pd/C, could be
converted to 2,3,9,10-tetramethoxybenz[c]acridine, 7.
Both 6 and 7 could be converted to their 12-methyl
derivatives by reaction with dimethyl sulfate to form the
quaternary ammonium salts, 8 and 9, respectively.
The presence of the iminium group could have a sub-
stantial in¯uence on cytotoxicity. This charged moiety
could impede cellular uptake and, thereby, limit the
possibility of observing such a correlation with topo-
isomerase poisoning activity. In addition, the iminium
moiety could be linked to cytotoxic e€ects unrelated to
topoisomerase poisoning. It has been suggested that
multiple biochemical e€ects may contribute to the cyto-
toxic activity of coralyne.27,28 In the evaluation of anti-
tumor activity of coralyne and nitidine in laboratory
animals, neither compound has exhibited pronounced
ecacy. It has been speculated that charged compounds
such as coralyne (Chart 2) and nitidine may need to
undergo hydrolysis to permit e€ective transport, with
possible intracellular cyclodehydration to reform the
tetracyclic structure of parent compound.29,30
The preparation of a series of benz[a]acridine analogues
structurally-related to coralyne was accomplished using
the general method outlined in Scheme 2. Knoevenagel
condensation of the appropriate o-nitrobenzaldehyde
with 5,6- or 6,7-disubstituted b-tetralones, provided the
1-(20-nitrobenzylidene)-2-tetralones, 12a±f. Reduction
of 12a±f with zinc in acetic acid gave the desired 5,6-
dihydrobenz[a]acridine derivatives, 13a±f. Heating 13a±d
in decalin at 190 ꢀC in the presence of Pd/C resulted in
This possible hindrance to absorption could explain the
weak antileukemic activity observed in vivo with these
agents. The presence of the iminium moiety within niti-
dine and coralyne could also be linked to nonspeci®c
toxicity in vitro as well as their relatively weak in vivo
antitumor activity.
Chart 3. Structure, numbering, and ring assignments of benz[a]acri-
dine and benz[c]acridine.

D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
1173
Scheme 1.
conversion of these dihydro compounds to their benz-
[a]acridine derivatives, 14a±d. Treatment of either 13a
or 14a with BBr3 in methylene chloride provided the
tetrahydroxy analogues, 13g and 14g, respectively.
Reaction of 13a or 14a with dimethyl sulfate resulted in
the formation of their 7-methyl derivatives, 15a and
16a.
rearrangement by treatment with tri¯uoroacetic acid,
provided 1,2,3,4-tetrahydro-1-methylthio-5,6-methylene-
dioxy-2(1H)-napthalenone, 19.35 Hydrogenolysis of 19
using 10% Pd/C in glacial acetic acid gave 11e.36
Results and Discussion
5,6-Methylenedioxy-2-tetralone (11e) was used as the
requisite intermediate for the preparation of 5,6-dihydro-
9,10-dimethoxy-3,4-methylenedioxybenz[a]acridine, 13e.
This tetralone was prepared in six steps as illustrated in
Scheme 3. 2,3-Methylenedioxybenzaldehyde was con-
densed with malonic acid as previously described32 to
give 2,3-methylenedioxycinnamic acid. 2,3-Methylene-
dioxycinnamic acid was hydrogenated using 10% Pd/C
to give the dihydrocinnamic acid derivative, which was
then transformed into its ethyl ester, 17, as previously
described.33 Ethyl-2,3-methylenedioxydihydrocinnamate
was then converted to its b-ketosulfoxide, 18.34 The b-
ketosulfoxide derivative, 18, when subjected to Pummerer
The relative activity of coralyne (1), nitidine (2), MDD-
coralyne (3), the benz[c]acridine analogues (6±9) and the
benz[a]acridine derivatives (13±16) was determined in a
subcellular assay measuring enzyme±DNA cleavable
complex formation in the presence of topo I and topo
II. In contrast to nitidine and a few select proto-
berberine derivatives,13,19 none of the benzacridines
synthesized as part of this study was active as a topo II
poison.
5,6-Dihydro-2,3,9,10-tetramethoxybenz[c]acridine (6)
and 2,3,9,10-tetramethoxybenz[c]acridine (7), which
have within their A-ring similar methoxyl substitution

1174
D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
Scheme 2.
to coralyne, were devoid of any signi®cant activity as
topo I poisons. The N-methyl quaternary ammonium
derivatives of these benz[c]acridines, 8 and 9, were also
inactive. These data clearly indicated that relocation of
the ring nitrogen of coralyne to its 12 position as in the
case of these benz[c]acridine derivatives results in a loss
of topoisomerase poisoning activity.
tetramethoxybenz[a]acridine (13a) and 2,3,9,10-tetra-
methoxybenz[a]acridine (14a) were synthesized and
evaluated as topo I poisons (Table 1). While 14a was
inactive as a topo I poison, 13a did exhibit modest
activity. These results parallel the structure±activity
relationships observed for 8-desmethyl protoberberine
analogues wherein the 5,6-dihydro derivative of an
active compound exhibited signi®cantly enhanced
potency as topo I poisons. In the case of the benz[a]-
acridines, saturation at the 5,6-postion was a structural
Benz[a]acridines with similar methoxyl substitution to
coralyne within the A-ring such as 5,6-dihydro-2,3,9,10-

D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
1175
Scheme 3.
Table 1. Topo I and topo II mediated DNA cleavage of coralyne
derivatives and related compounds
poisoning activity.13,17 While counterintuitive, studies on
the DNA binding anity demonstrated that for 5,6-
dihydro protoberberine analogues without a 8-methyl
substituents, such as 5,6-dihydro-8-desmethylcoralyne,
there was an increase in relative DNA binding anity
relative to the unsaturated analogue.37 Thus, it is likely
that in the case of 5,6-dihydrobenz[a]acridines that this
increase in DNA binding anity may be essential for
stabilization of the cleaved ternary complex involving
DNA, enzyme, and drug. In recent studies we have also
shown that substituents on the A-ring of 5,6-saturated
protoberberine analogues are engaged in interactions
(e.g., hydrogen bonding) with topo I in the ternary
drug-DNA±enzyme complex.37 It is reasonable to sug-
gest that saturation of the 5,6-bond in benz[a]acridine
may properly position the substituents on the A-ring for
these types of drug-enzyme interactions.
a
Compound
Topoisomerase I
mediated
Cytotoxicity IC50 (mM)
cell lines
DNA cleavageb
RPMI
CPT-K5
1
2
1.0
0.1
4.9
0.4
20
3.9
3
0.1
8.1
7.1
9.0
6.9
1.0
3.0
27
7.1
14.9
6.9
8.6
22.4
14.2
13.6
>24.2
>10
13a
13b
13c
13d
13e
13f
13g
15a
CPT
100
100
Ðc
100
1.0
100
1.0
100
0.1
14.2
20.3
>24.2
0.004
aIC50 has been calculated after 4 days of continuous drug exposure.
bTopo I cleavage values are reported as REC (relative e€ective con-
centration), i.e. concentrations relative to coralyne, whose value is
arbitrarily assumed as 1, that are able to produce the same cleavage on
the plasmid DNA in the presence of human topo I.
cCompound 13c failed to increase cleavage of the plasmid DNA in the
presence of topo I at the concentration range used in this study.
Modi®cation of these benz[a]acridines by replacing
the 9,10-methoxy groups with a 9,10-methylenedioxy
substituent provided consistent results where only the
5,6-dihydro derivative, 13b, was active as a topo I poison.
The fully unsaturated analogue, 14b, was inactive.
Replacement of the methoxy groups at positions 2 and
3 of these 5,6-dihydrobenz[a]acridines with hydrogen
atoms, as in the case of 13c, resulted in a loss of topo I
poisoning activity. The replacement of the methoxy
substituents at the 9,10-positions with hydrogen atoms,
as in the case of 13d, did not diminish the activity of
this dihydrobenz[a]acridine relative to 13a as a topo I
poison.
requirement for topo I poisoning activity within this
series of compounds. None of the fully unsaturated
benz[a]acridines evaluated in this study (14a±d, 14g)
possessed activity as a topo I poison. In the case of
protoberberines derivatives, saturation within the B-
ring was associated with an enhancement of topo I

1176
D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
In certain human tumor cell lines, however, an active
transport mechanism for charged compounds related to
coralyne appears to be operational.38
Several of the benz[a]acridine analogues evaluated in
this study are noncharged compounds for which
enhanced passive di€usion into cells would be expected.
In addition, none of these benz[a]acridine derivatives
are topo II poisons. Nonetheless, the relative cytotoxi-
city of the various benz[a]acridines did not correlate
well with their relative potency as topo I poisons. In
several instances, such as with 13d and 13e, di€erences
in topo I poisoning activity were not re¯ected in their
relative cytotoxic activity. These data suggest that
among benz[a]acridines there may be factors, in addition
to topo I poisoning, that in¯uence cytotoxic activity.
In certain instances, di€erences in physicochemical
properties can explain some of the cytotoxicity data.
The low cytotoxicity observed for both 13g, which is a
highly polar compound, and 16a, which contains the
iminium moiety, could be associated with their
decreased ability to passively di€use into cells.
Figure 1. Stimulation of enzyme-mediated DNA cleavage by coralyne
analogues using human topo I. The top lane is the DNA control
without topo I. The second lane from the top is the control with topo I
alone. The rest of the lanes are with topo I and serially (10-fold each)
diluted coralyne (1), MDD-coralyne (3), and 5,6-dihydro-3,4-methyl-
enedioxy-9,10-dimethoxybenz[a]acridine (13e).
In summary, the primary objective of this study was to
determine whether the presence of an iminium cation
was critical for retaining topoisomerase poisoning
activity. In this investigation, two series of potential
noncharged bioisosteres structurally related to coralyne
and protoberberine alkaloids were investigated. Benz-
[a]acridine and benz[c]acridine derivatives were selected
for initial evaluation in view of their stability and the
absence of any potential for these heterocycles to form
an iminium cation by autooxidation. While benz[c]acri-
dine derivatives appear unsuitable as bioisosteres, sev-
eral dihydrobenz[a]acridines exhibited activity as topo I
poisons. These benz[a]acridine derivatives represent a
new series of topo I poisons. These data support further
research into the development of dihydrobenz[a]acri-
dines and related heterocycles, such as benzo[i]phenan-
thridines, as antitumor agents. Such investigations,
using the structure±activity data derived from coralyne
and protoberberine derivatives could provide a unique
class of topo I poisons that may exhibit a di€erent
toxicity pro®le and an improved potential for ecacy in
vivo.
The structure±activity relationship observed among the
various benz[a]acridines did mirror that observed with
coralyne and various protoberberine derivatives with
regard to their topo I poisoning activity.11,13 Structure±
activity relationships associated with a series of coralyne
derivatives, demonstrated that substitution of the A-
ring with a 3,4-methylenedioxy substituent was asso-
ciated with potent topo I poisoning activity.13 In view
of these data, we prepared 5,6-dihydro-3,4-methylene-
dioxy-9,10-dimethoxybenz[a]acridine, 13e. Consistent
with the structure±activity results previously observed
with coralyne derivatives, 13e was the most potent topo
I poison within this series of benz[a]acridines, exhibiting
comparable activity to coralyne (Fig. 1). Previous studies
with coralyne derivatives also demonstrated that the
presence of 3,4-dimethoxy substituents on the A-ring
diminished activity. Consistent with these results,
3,4,9,10-tetramethoxybenz[a]acridine, 13f, had only 1%
of the relative potency of 13e as a topo I poison. Among
the protoberberine analogues, it had been observed that
demethylation of berberine resulted in an analogue with
enhanced activity as a topo I poison.11 In the case of
13a, conversion of this analogue to its tetrahydroxy
derivative, 13g, did result in an increase in topo I
poisoning activity. Methylation of 13a to form the N-
methyl derivative 15a neither enhanced nor diminished
topo I poisoning activity.
Experimental
Melting points were determined with a Thomas-Hoover
Unimelt capillary melting point apparatus. Column
chromatography refers to ¯ash 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
Perkin±Elmer 1600 Fourier transform spectrophotometer
and are reported in cm 1. Proton (1H NMR) and
carbon (13C NMR) nuclear magnetic resonance were
recorded on a Varian Gemini-200 Fourier Transform
A clear correlation between potency as a topo I poison
and cytotoxicity among coralyne derivatives was not
observed in previous studies. Several factors could be
responsible for this observation. Some coralyne ana-
logues are also topo II poisons or dual topoisomerase
poisons. In addition, coralyne analogues possess an
iminium moiety. Cellular absorption could be viewed as
a limiting factor associated with the cytotoxicity of vari-
ous analogues where only passive di€usion is involved.
1
spectrometer. NMR spectra (200 MHz H and 50 MHz
13C) were recorded in the deuterated solvent indi-
cated with chemical shifts reported in d units down®eld
from tetramethylsilane (TMS). Coupling constants are

D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
1177
reported in hertz (Hz). Mass spectra were obtained from
Washington University Resource for Biomedical and
Bio-organic Mass Spectrometry within the Department
of Chemistry at Washington University, St. Louis, MO.
The purity of all compounds for which HRMS data are
provided was determined by analytical reverse-phase
HPLC. Compounds were analyzed using both of the
following conditions (method A) a Vydac C-18 column
(The Separations Group) using methanol:H2O (80:20)
with a ¯ow rate of 1 mL/min; (method B) a Microsorb
C-8 column (Rainin Instrument Co., Inc.) using metha-
nol:0.1 M potassium phosphate bu€er (pH 7.0) (95:5)
with a ¯ow rate of 1 mL/min. HPLC analyses were
performed with a Hewlett-Packard 1090 liquid chroma-
tograph equipped with a diode array UV detection
monitoring at 254 and 335 nm. The percent purity of
these compounds were calculated from the peak area
assuming that the extinction coecient of the compound
of interest and the impurity are the same. On the basis
of these analyses, all the compounds were found to be
98.0±99% pure in these systems. Combustion analyses
were performed by Atlantic Microlabs, Inc., Norcross,
GA, and were within ‹0.4% of the theoretical value.
Compounds 4 and 5 were purchased from Aldrich
Chemical Company (Milwaukee, WI). 5,6-Dimethoxy-
2-tetralone, 11f, was synthesized from 1,6-dibromo-2-
hydroxynapthalene.39
5,6-Methylenedioxy-2-tetralone (11e). The thioether
intermediate, 19, (0.67 g, 2.83 mM) was taken up in
10 mL glacial acetic acid in a hydrogenation ¯ask. 0.46 g
of 10% Pd-C was added and this mixture was shaken in
a Parr apparatus at 40 psig of hydrogen for 40 h. The
reaction mixture was ®ltered through a celite bed,
which was washed thrice with 5 mL portions of glacial
acetic acid. The glacial acetic acid was evaporated in
vacuo to give the crude tetralone, 11e. The crude tetra-
lone was then treated with sodium bisul®te to convert it
to the more stable bisul®te adduct. Pure tetralone was
generated as required from its bisul®te adduct by treat-
ment with 10% sodium carbonate solution followed by
extraction with dichloromethane; mp 90±91 ꢀC (lit.36
88±91 ꢀC), IR (Nujol) 1715; H NMR: d 2.50 (2H, t),
1
2.98 (2H, t), 3.50 (2H, s), 5.93 (2H, s), 6.56 (1H, d,
J=6), 6.65 (1H, d, J=6); 13C NMR: d 21.6, 37.9, 45.0,
101.5, 107.4, 118.5, 121.1, 127.8, 144.1, 146.2, 211.2.
Anal. (C11H10O3) C, H.
General procedure for the synthesis of
1-(20-nitrobenzylidene)-2-tetralone derivatives
A glacial acetic acid (10 mL) solution of 2.45 mmol each
of the respective 2-tetralone, 2-nitrobenzaldehyde, and
sodium acetate was re¯uxed for 3±8 h under a nitrogen
atmosphere. The reaction mixture was then allowed to
cool to room temperature. The mixture was carefully
loaded on a silica gel (75 g) column and chromato-
graphed using a 1:1 mixture of ethyl ether and hexanes.
The yellow colored compound generally eluting fourth
from the column was collected to provide the respective
tetralone derivatives in 20±25% yield.
5,6-Dihydro-2,3,9,10-tetramethoxy-7-methylbenz[c]acri-
dine (6). 2-Amino-4,5-dimethoxyacetophenone (1.0 g,
5.1 mmol) was dissolved in 10 mL CH2Cl2 and hydrogen
chloride (1.0 M solution, in anhydrous ether) was added
with vigorous stirring at room temperature. The hydro-
chloride salt of the aminoacetophenone precipitated
out. The solvent was removed in vacuo and the solid
residue obtained dried for 1 h under vacuum. The dry
hydrochloride salt was triturated with 6,7-dimethoxy-1-
tetralone (1.59 g, 7.68 mmol) and the mixture was
transferred into a sealed tube and heated at 140 ꢀC for
1 h. The resulting fused plug was dissolved in boiling
methanol (300 mL). This solution was concentrated to
200 mL and left overnight providing needle-shaped
crystals. These crystals were ®ltered and washed with
three 5 mL portions of acetone, and dried to give golden
yellow needles of the benz[c]acridine hydrochloride
derivative in 99% yield. The hydrochloride salt was
dissolved in 200 mL boiling methanol. After the solution
had cooled to room temperature concentrated NH4OH
was added dropwise until pH 10 was obtained. Light
yellow crystals began to form. The suspension was
then diluted with 200 mL water and extracted thrice
with 100 mL portions of CH2Cl2. The combined
extracts were washed once with 50 mL brine, dried
using anhydrous Na2SO4, ®ltered, and the solvent
removed in vacuo to give the free base; mp 240 ꢀC;
1-(20-Nitro-40,50-dimethoxybenzylidene)-6,7-dimethoxy-2-
tetralone (12a). Prepared from 6,7-dimethoxy-2-tetra-
lone and 6-nitroveratraldehyde; mp 65±66 ꢀC; IR
(Nujol): 2855, 1720, 1540; 1H NMR: d 2.71 (2H, t), 2.98
(2H t), 3.35 (3H, s), 3.66 (3H, s), 3.89 (3H, s), 3.97 (3H,
s), 6.41 (1H, s), 6.62 (1H, s), 6.73 (1H, s), 7.74 (1H, s),
7.91 (1H, s); 13C NMR: d 28.4, 39.7, 56.5, 56.6, 56.8,
56.9, 107.7, 108.6, 111.2, 113.1, 128.1, 129.3, 130.3,
132.2, 133.3, 148.9, 149.0, 150.0, 153.3, 200.7. Anal.
(C21H21NO7) C, H, N.
1-(20-Nitro-40,50-methylenedioxybenzylidene)-6,7-dimeth-
oxy-2-tetralone (12b). Prepared from 6,7-dimethoxy-2-
tetralone and 6-nitropiperonal; mp 76±77 ꢀC; IR
(Nujol): 2875, 1723, 1553; 1H NMR: d 2.68 (2H, t),
3.00 (2H, t), 3.41 (3H, s), 3.90 (3H, s), 6.08 (2H, s),
6.37 (1H, s), 6.51 (1H, s), 6.73 (1H, s), 7.68 (1H, s),
7.83 (1H, s); 13C NMR: d 28.0, 38.0, 56.1, 56.4, 103.7,
105.9, 110.1, 111.3, 112.6, 124.3, 130.8, 131.1, 132.2,
133.8, 147.6, 148.4, 149.7, 152.5. Anal. (C20H17NO7) C,
H, N.
1
IR (Nujol): 2922, 1620; H NMR: d 2.52 (3H, s), 2.86
(2H s), 3.02 (2H, t), 3.90 (3H, s), 3.98 (3H, s), 4.03
(3H, s), 4.05 (3H, s), 6.70 (1H, s), 7.08 (1H, s), 7.43
(1H, s), 8.04 (1H, s); 13C NMR: d 16.5, 25.4, 28.0, 56.6,
56.7, 57.8, 58.3, 102.3, 102.7, 110.9, 111.8, 119.5, 123.2,
127.7, 134.7, 135.6, 146.3, 148.5, 149.7, 151.3, 153.5,
155.2; HRMS calcd for C22H23NO4: 365.1630; found:
365.1628.
1-(20-Nitrobenzylidene)-6,7-dimethoxy-2-tetralone (12c).
Prepared from 6,7-dimethoxy-2-tetralone and 2-nitro-
ꢀ
1
benzaldehyde; mp 63±64 C; IR (Nujol): 1712, 1540; H
NMR: d 2.69 (2H, t), 3.01 (2H, t), 3.25 (3H, s), 3.88
(3H, s), 6.26 (1H, s), 6.72 (1H, s), 7.25±7.26 (1H, m),
7.44±7.50 (2H, m), 7.87 (1H, s), 8.12±8.17 (1H, m); 13C
NMR: d 28.0, 37.9, 55.7, 56.3, 108.6, 111.3, 112.5, 124.2,

1178
D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
124.9, 125.4, 129.3, 130.1, 131.8, 133.5, 133.9, 134.7,
147.5, 149.6, 200.7. Anal. (C19H17NO5) C, H, N.
5,6-Dihydro-2,3,-dimethoxy-9,10-methylenedioxybenz-
[a]acridine (13b). Prepared from 1-(20-nitro-40,50-meth-
ylenedioxybenzylidene)-6,7-dimethoxy-2-tetralone; mp
1-(20-Nitro-40,50-dimethoxybenzylidene)-2-tetralone (12d).
Prepared from 2-tetralone and 2-nitro-4,5-dimethoxy-
218±219 ꢀC; IR (Nujol): 2780, 1630; H NMR: d 2.97
1
(2H, t), 3.17 (2H, t), 3.93 (3H, s), 4.00 (3H, s), 6.07 (2H,
s), 6.79 (1H, s), 7.07 (1H, s), 7.29 (1H, s), 7.32 (1H, s),
8.07 (1H, s); 13C NMR: d 29.0, 33.1, 56.5, 56.7, 102.1,
103.3, 105.7, 107.6, 111.8, 125.2, 125.9, 127.0, 128.0,
130.6, 145.0, 147.9, 148.8, 149.6, 150.8, 156.9; HRMS
calcd for C20H17NO4: 335.1158; found: 335.1162.
ꢀ
1
benzaldehyde; mp 58±60 C; IR (Nujol): 1724, 1540; H
NMR: d 2.71 (2H, t), 3.08 (2H, t), 3.56 (3H, s), 4.02
(3H, s), 6.51 (1H, s), 6.92±6.96 (2H, m), 7.17±7.24 (2H,
m), 7.73 (1H, s), 8.00 (1H, s); 13C NMR: d 28.3, 27.8,
56.7, 56.9, 108.3, 112.5, 126.7, 127.6, 128.5, 128.8, 129.9,
132.3, 133.4, 134.5, 138.8, 141.6, 149.4, 153.5. Anal.
(C19H17NO5) C, H, N.
5,6-Dihydro-2,3-dimethoxybenz[a]acridine (13c). Pre-
pared from 1-(20-nitrobenzylidene)-6,7-dimethoxy-2-tet-
1-(20 -Nitro-40,50 -dimethoxybenzylidene)-5,6-methylene-
dioxy-2-tetralone (12e). Prepared from 5,6-methylen-
ralone; mp 55±56 ꢀC; IR (Nujol): 2815, 1615; H NMR:
1
d 2.96 (2H, t), 3.23 (2H, t), 3.90 (3H, s), 3.98 (3H, s),
6.75 (1H, s), 7.30 (1H, s), 7.44 (1H, t), 7.60 (1H, t), 7.80
(1H, d, J=10.2), 8.0 (1H, d, J=10.2), 8.19 (1H, s); 13C
NMR: d 27.3, 28.9, 56.5, 56.6, 106.2, 110.7, 125.2, 125.5,
127.6, 127.8, 131.2, 132.4, 133.7, 133.9, 134.2, 136.3,
151.3, 153.6, 157.8; HRMS calcd for C19H17NO2:
291.1259; found: 291.1246.
dioxy-2-tetralone
and
2-nitro-4,5-dimethoxybenz-
aldehyde; mp 66±68 ꢀC; IR (Nujol): 1710, 1553; 1H
NMR: d 2.98 (2H, t), 3.19 (2H, t), 4.02 (3H, s), 4.03
(3H, s), 6.03 (2H, s), 6.82 (1H, d, J=8.1), 7.07 (1H, s),
7.29 (1H, d, J=8.1), 7.49 (1H, s), 8.20 (1H, s); 13C
NMR: d 21.8, 30.8, 56.5, 56.7, 101.8, 105.7, 107.6,
117.9, 119.1, 123.9, 125.9, 126.9, 128.7, 143.4, 145.4,
147.7, 150.1, 152.9, 156.1, 176.4. Anal. (C20H17NO7)
C, H, N.
5,6-Dihydro-9,10-dimethoxybenz[a]acridine (13d). Pre-
pared from 1-(20-nitro-40,50-dimethoxybenzylidene)-2-
tetralone; mp 95±96 ꢀC; IR (Nujol): 1633, 1516; 1H
NMR: d 3.06 (2H, t), 3.24 (2H, t), 4.02 (3H, s), 4.03
(3H, s), 7.10 (1H, s), 7.28±7.37 (3H, m), 7.50 (1H, s),
7.82 (1H, d, J=7.0), 8.29 (1H, s); 13C NMR: d 29.2,
32.3, 56.5, 56.7, 105.9, 107.4, 124.0, 124.3, 127.3, 127.7,
128.5, 128.9, 129.1, 133.8, 137.6, 144.2, 150.5, 153.1;
HRMS calcd for C19H17NO2: 291.1259; found:
291.1250.
1-(20Nitro-40,50-dimethoxybenzylidene)-5,6-dimethoxy-2-
tetralone (12f). Prepared from 5,6-dimethoxy-2-tetra-
lone and 2-nitro-4,5-dimethoxybenzaldehyde; mp 70±
ꢀ
1
72 C; IR (Nujol): 1715, 1550; H NMR: d 2.59 (2H, t),
3.06 (2H, t), 3.78 (3H, s), 3.86 (3H, s), 3.88 (3H, s), 3.92
(3H, s), 6.47 (1H, s), 6.77 (1H, d, J=8.2), 7.41 (1H, d,
J=8.2), 7.67 (1H, s); 13C NMR: d 19.7, 32.0, 56.3, 56.7,
56.8, 56.85, 108.1, 121.9, 125.6, 126.1, 128.0, 129.2,
132.1, 132.9, 133.7, 140.3, 141.4, 149.3, 152.9, 153.5,
200.9. Anal. (C21H21NO7) C, H, N.
5,6-Dihydro-9,10-dimethoxy-3,4-methylenedioxybenz[a]-
acridine (13e). Prepared from 1-(20-nitro-40,50-dimethoxy-
benzylidine)-5,6-methylenedioxy-2-tetralone; mp 220±
222 ꢀC; IR (Nujol): 1715, 1532; 1H NMR: d 3.02 (2H, t),
3.19 (2H, t), 4.02 (3H, s), 4.03 (3H, s), 6.03 (2H, s), 6.83
(1H, d, J=8.1), 7.07 (1H, s), 7.34±7.38 (2H, m), 8.19
(1H, s); 13C NMR: d 30.2, 32.3, 56.5, 56.6, 101.8, 105.8,
107.5, 107.8, 117.9, 119.2, 123.8, 126.9, 128.3, 128.7,
143.2, 145.4, 147.6, 149.9, 152.7, 156.3; Anal (C20H17
NO4) C, H, N.
General procedure for the synthesis of
5,6-dihydrobenz[a]acridine derivatives
The respective 1-(20-nitrobenzylidene)-2-tetralone deri-
vative (0.3 mmol) was dissolved in 10 mL glacial acetic
acid and re¯uxed with zinc dust (1.64 mmol) under a
nitrogen atmosphere for 1±4 h. The reaction mixture
was allowed to cool to room temperature and then the
entire mixture was loaded carefully on silica gel (100 g)
column and chromatographed ®rst with 500 mL of ethyl
ether to remove acetic acid followed by elution with a
1:1 mixture of hexanes and ethyl acetate. The polarity of
the mobile phase was reduced, if necessary, by mixing
suitable proportions of hexanes. The relevant fractions
were pooled and concentrated in vacuo to yield 83±95%
of corresponding 5,6-dihydrobenz[a]acridines.
5,6-Dihydro-3,4,9,10-tetramethoxybenz[a]acridine (13f).
Prepared from 1-(20-nitro-40,50-dimethoxybenzylidine)-
5,6-dimethoxy-2-tetralone; mp 195±196 ꢀC; IR (Nujol):
1
1730, 1515; H NMR: d 2.82 (2H, t), 3.20 (2H, t), 3.86
(3H, s), 3.91 (3H, s), 4.03 (3H, s), 4.04 (3H, s), 6.93 (1H,
d, J=8.6), 7.10 (1H, s), 7.52 (1H, s), 7.56 (1H, d,
J=8.6), 8.27 (1H, s); 13C NMR: d 21.8, 31.6, 56.4, 56.6,
56.8, 61.1, 105.8, 106.4, 106.5, 107.6, 111.4, 111.9, 120.4,
124.1, 127.1, 129.3, 131.6, 150.3, 150.4, 153.4, 156.3.
Anal. (C21H21NO4) C, H, N.
5,6-Dihydro-2,3,9,10-tetramethoxybenz[a]acridine (13a).
Prepared from 1-(20-nitro-40,50-dimethoxybenzylidene)-
6,7-dimethoxy-2-tetralone; mp 182±183 ꢀC; IR (Nujol):
General procedure for the synthesis of benz[a]acridines
and benz[c]acridines from their 5,6-dihydro derivatives
1
3210, 1615; H NMR: d 2.99 (2H, t), 3.23 (2H, t), 3.94
(3H, s), 4.00 (3H, s), 4.02 (3H, s), 4.04 (3H, s), 6.79 (1H,
s), 7.10 (1H, s), 7.31 (1H, s), 7.54 (1H, s), 8.17 (1H, s);
13C NMR: d 28.8, 32.3, 56.5, 56.7, 105.7, 107.2, 107.5,
111.8, 123.9, 125.8, 127.1, 128.0, 130.5, 143.1, 148.8,
149.6, 152.8, 156.7, 176.7; HRMS calcd for C21H21NO4:
351.1471; found: 351.1475.
The respective 5,6-dihydrobenz[a]acridine or 5,6-di-
hydrobenz[c]acridine derivatives (0.22 mmol) were
re¯uxed in 15 mL decalin with 76 mg of 10% palladium
on carbon under nitrogen atmosphere for 2±9 h. The
reaction mixture was then quickly suction ®ltered while

D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
1179
hot through a celite bed using a sintered glass funnel.
The ®lter bed was washed thoroughly thrice using
20 mL portions of boiling chloroform followed by
two 20mL portions of boiling ethyl acetate. The com-
bined ®ltrate was then concentrated in vacuo and dried
under vacuum to give the respective benz[a]acridine
derivatives.
chilled to 50 ꢀC using a cooling bath of isopropanol
and dry ice. 1.95 mmols of boron tribromide (1.0 M)
solution in CH2Cl2 was added under a nitrogen atmo-
sphere. The reaction mixture was stirred at 50 ꢀC for
1 h and then slowly allowed to come to room tempera-
ture over a period of 4 h. The reaction mixture was then
cooled to 10 ꢀC and was quenched by addition of 5 mL
saturated ammonium chloride solution. The resulting
solution was evaporated to dryness and the residue
obtained was extracted thrice with 20 mL portions of
boiling acetone. The resulting yellow suspensions were
®ltered each time. The undissolved precipitate was dis-
solved in 5 mL boiling methanol and set aside overnight.
Needle shaped crystals of the respective tetrahydroxy-
benz[a]acridines were formed in 95% yield.
2,3,9,10-Tetramethoxy-7-methylbenz[c]acridine (7). Pre-
pared from 4; mp >250 ꢀC; IR (Nujol): 3520, 1633,
1610; 1H NMR: d 2.87 (3H, s), 4.03 (3H, s), 4.04 (3H, s),
4.13 (3H, s), 4.23 (3H, s), 7.17 (1H, s), 7.20 (1H, s), 7.52
(1H, d, J=9.2), 7.59 (1H, s), 7.80 (1H, d, J=9.2), 8.87
(1H, s); 13C NMR: d 14.4, 56.4, 56.7, 101.7, 106.0,
108.2, 108.3, 120.8, 122.1, 122.4, 125.5, 125.8, 126.9,
128.6, 135.7, 139.0, 145.2, 149.8, 150.9, 153.3; HRMS
calcd for C22H21NO4: 363.1470; found: 363.1472.
5,6-Dihydro-2,3,9,10-tetrahydroxybenz[a]acridine (13g).
Prepared from 13a; mp >220 ꢀC; IR (Nujol): 3361,
1
2,3,9,10-Tetramethoxybenz[a]acridine (14a). Prepared
from 13a; mp 247±249 ꢀC; IR (Nujol): 2810, 1630; H
3164, 2719, 1620; H NMR (CD3OD): d 2.97 (2H, t),
1
3.35 (2H, t), 6.77 (1H, s), 7.45 (3H, m), 8.83 (1H, s); 13C
NMR (CD3OD): d 27.4, 29.2, 102.9, 110.9, 112.5, 116.6,
122.9, 126.5, 128.5, 129.4, 134.8, 135.6, 146.6, 148.6, 151.1,
152.9, 156.0; HRMS calcd for C17H13NO4: 295.0845;
found: 295.0842.
NMR: d 3.99 (3H, s), 4.01 (3H, s), 4.03 (3H, s), 4.08
(3H, s), 7.08 (1H, s), 7.12 (1H, s), 7.41 (1H, s), 7.74 (1H,
d, J=9.3), 7.81 (2H, m), 8.82 (1H, s); 13C NMR: d 56.3,
56.4, 56.5, 56.6, 103.9, 104.8, 106.8, 109.2, 122.8, 123.0,
124.5, 126.2, 126.4, 127.7, 130.7, 145.7, 147.2, 149.8,
149.9, 150.3, 153.9; HRMS calcd for C21H19NO4:
349.1314; found: 349.1314.
2,3,9,10-Tetrahydroxybenz[a]acridine (14g). Prepared
from 14a; mp >270 ꢀC; IR (KBr): 3361, 3164, 1620,
1
1516; H NMR (CD3OD): d 7.26 (1H, s), 7.37 (1H, s),
2,3-Dimethoxy-9,10-methylenedioxybenz[a]acridine (14b).
Prepared from 13b; mp 245±246 ꢀC; IR (Nujol): 2790,
1630; 1H NMR: d 4.06 (3H, s), 4.16 (3H, s), 6.15 (2H, s),
7.25 (2H, s), 7.48 (1H, s), 7.84 (1H, d, J=8.2), 7.99 (1H,
s), 8.99 (1H, s); 13C NMR: d 56.5, 56.6, 102.3, 102.4,
104.1, 104.9, 109.4, 112.8, 122.9, 124.4, 126.5, 126.7,
128.5, 130.9, 135.7, 147.0, 147.4, 148.4, 150.1, 152.0;
HRMS calcd for C20H15NO4 calcd: 333.1002; found:
333.1004.
7.56 (1H, s), 7.61 (1H, d, J=9.2), 8.05 (2H, m), 9.61
(1H, s); 13C NMR (CD3OD): d 101.1, 108.7, 110.3,
114.3, 114.7, 122.9, 124.4, 124.8, 126.7, 137.6, 137.8,
138.3, 139.0, 149.4, 150.7, 150.8, 159.3; HRMS calcd for
C17H11NO4: 293.0688; found: 293.0685.
General procedure for N-methylation of benz[a]acridines
and benz[c]acridines
Dimethyl sulfate (4 mL) was added to 0.27 mmol of the
respective benz[a]acridine or benz[c]acridine and the
mixture heated under a nitrogen atmosphere in an oil
bath at 150 ꢀC for between 20 min and 5 h. Anhydrous
ethyl ether (10 mL) was added to the reaction mixture
with vigorous stirring after it had cooled to room tem-
perature. The precipitated quaternary salt was suction
®ltered and washed thrice with 10 mL portions of
anhydrous ethyl ether and dried. The quaternary salts
were crystallized from boiling methanol in 90% yield.
2,3-Dimethoxybenz[a]acridine (14c). Prepared from 13c;
mp 190±192 ꢀC; IR (Nujol): 2881, 1632; H NMR: d
1
4.02 (3H, s), 4.13 (3H, s), 7.18 (1H, s), 7.51±7.59 (1H,
m), 7.72±7.81 (1H, m), 7.83 (1H, s), 7.89 (1H, s), 7.93±
8.22 (2H, m), 8.23 (1H, d, J=8.5), 9.13 (1H, s); 13C
NMR: d 56.5, 56.6, 104.4, 109.5, 124.2, 124.5, 126.3,
126.4, 126.8, 126.9, 128.5, 129.4, 130.0, 130.1, 130.2,
132.3, 148.1, 149.2, 150.1, 150.2; HRMS calcd for
C19H17NO2: 289.1104; found: 289.1099.
9,10-Dimethoxybenz[a]acridine (14d). Prepared from
13d; mp 181±182 ꢀC; IR (Nujol): 2883, 1621; H NMR:
5,6-Dihydro-7,12-dimethyl-2,3,9,10-tetramethoxybenz[c]-
acridinium methosulfate (8). Prepared from 6; mp
>250 ꢀC; IR (Nujol): 3510, 1645, 1613; 1H NMR
(CD3OD): d 2.90 (3H, s), 2.96 (2H, t), 3.08 (2H, t), 3.93
(3H, s), 4.00 (3H, s), 4.10 (3H, s), 4.18 (3H, s), 4.62 (3H,
s), 7.17 (1H, s), 7.42 (1H, s), 7.61 (1H, s), 7.63 (1H, s);
13C NMR (CD3OD): d 16.6, 27.2, 29.0, 46.2, 57.0, 57.2,
57.3, 57.4, 100.7, 105.5, 112.5, 115.2, 121.2, 125.1, 133.4,
138.5, 139.7, 149.7, 150.5, 152.7, 152.9, 154.8, 157.4;
HRMS calcd for C23H26NO4+: 380.1858; found:
380.1856.
1
d 4.09 (6H, s), 7.26 (1H, s), 7.54 (1H, s), 7.60±7.71 (2H,
m), 7.89±7.96 (3H, m), 8.69 (1H, d, J=8.1), 9.23 (1H, s);
13C NMR: d 56.6, 56.7, 105.1, 107.1, 122.9, 123.3, 123.4,
125.5, 127.6, 128.5, 128.7, 129.3, 130.4, 131.6, 135.7,
146.4, 148.0, 150.6, 154.3; HRMS calcd for C19H17NO2:
289.1100; found: 289.1104.
General procedure for the synthesis of
5,6-dihydro-2,3,9,10-tetrahydroxybenz[a]acridine (13g)
and 2,3,9,10-tetrahydroxybenz[a]acridine (14g)
7,12-Dimethyl-2,3,9,10-tetramethoxy-benz[c]acridinium
methosulfate (9). Prepared from 7; mp >240 ꢀC; IR
The respective benz[a]acridine derivatives (0.195 mmol)
were dissolved in 2 mL CH2Cl2 and the solution was
1
(Nujol): 3495, 1640, 1615; H NMR (DMSO-d6): d 2.53

1180
D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
(3H, s), 3.39 (3H, s), 3.97 (3H, s), 4.03 (3H, s), 4.16 (3H,
s), 4.17 (3H, s), 7.69 (1H, s), 7.75 (1H, s), 7.91 (1H, s),
7.99 (1H, d, J=9.5), 8.24 (1H, d, J=9.5); 13C (DMSO-
d6) NMR: d 15.5, 55.4, 56.4, 56.5, 56.6, 56.7, 102.1,
106.3, 110.3, 111.2, 125.8, 123.2, 122.5, 128.8, 129.0,
130.9, 133.3, 139.0, 141.1, 147.6, 152.1, 153.8, 155.3;
HRMS calcd for C23H24NO4+: 378.1698; found:
378.1695.
white crystalline solid; mp 79±80 ꢀC (lit.36 79±80 ꢀC); 1H
NMR: d 2.69 (2H, t), 2.91 (2H, t,), 5.94 (2H, s), 6.68±
6.79 (3H, m), 11.43 (1H, s); 13C NMR: d 25.2, 34.2,
101.2, 107.5, 122.0, 122.1, 122.8, 146.0, 147.7, 179.9.
Trimethylsilyl chloride 2.5 mL (18.2 mM) was added to
a solution of 1.5 g (7.73 mM) of 2,3-(methylenedioxy)-
dihydrocinnamic acid in 70 mL dry ethanol 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 hexanes to give a quantitative yield of the ester
5,6-Dihydro-2,3,9,10-tetramethoxy-7-methylbenz[a]acri-
dinium methosulfate (15a). Prepared from 13a; mp
>250 ꢀC; IR (Nujol): 3490, 1620; H NMR (DMSO-
1
d6): d 3.06 (2H, t), 3.37 (5H, t), 3.85 (3H, s), 3.93 (3H, s),
4.02 (3H, s), 4.05 (3H, s), 7.04 (1H, s), 7.45 (1H, s), 7.62
(1H, s), 7.63 (1H, s), 9.29 (1H, s); 13C NMR (DMSO-
d6): d 25.9, 27.8, 53.1, 56.0, 56.1, 56.7, 56.8, 99.6, 106.6,
106.7, 107.9, 112.2, 112.3, 122.0, 124.4, 127.3, 129.1,
148.8, 150.3, 151.1, 153.0, 155.3; HRMS calcd for
C22H24NO4+: 366.1706; found: 366.1706.
1
(17) as a colorless liquid; H NMR: d 1.23 (3H, t), 2.63
(2H, t), 2.91 (2H, t), 4.14 (2H, q, J1=7.1, J2=14.3), 5.92
(2H, s), 6.64±6.79 (3H, m); 13C NMR: d 14.7, 25.5, 34.4,
60.9, 101.1, 107.3, 122.0, 122.4, 122.8, 145.9, 147.6,
173.2.
2,3-(Methylenedioxy)phenethyl methylsul®nylmethyl
ketone (18). The anion of dimethyl sulfoxide was pre-
pared by adding 6.0 mL dry dimethyl sulfoxide to 0.6 g
sodium hydride 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 trans-
ferred to a water bath maintained at 5±10 ꢀC. 1.0 g
(4.54 mM) of 17 was separately dissolved in 6.0 mL dry
dimethyl sulfoxide and this solution was added drop-
wise, over a period of 15 min, to the dimethyl sulfoxide
anion generated previously. The reaction mixture was
then slowly allowed to come to room temperature and
stirred for 2 h. The reaction mixture was poured into
100 mL cold water and acidi®ed to pH 3±4 using 1.2 N
hydrochloric acid and extracted ®ve times with 40 mL
portions of chloroform. The combined organic layer
was washed twice with 100 mL portions of distilled
water, dried over anhydrous sodium sulfate, ®ltered and
evaporated in vacuo to give quantitative yield of 17 as a
low melting bu€ colored solid. Compound 17 was found
to be unstable to column chromatography using silica
7-Methyl-2,3,9,10-tetramethoxybenz[a]acridinium metho-
sulfate (16a). Prepared from 14a; mp >250 ꢀC; IR
(Nujol): 2820, 1620; H NMR (DMSO-d6): d 3.39 (3H,
s), 3.96 (3H, s), 3.97 (3H, s), 4.03 (3H, s), 4.07 (3H, s),
7.59 (1H, s), 7.67 (1H, s), 7.85 (1H, d, J=9.2), 8.25 (1H,
s), 8.31 (1H, d, J=9.2), 8.75 (1H, s), 10.18 (1H, s); 13C
NMR (DMSO-d6): d 54.4, 56.8, 56.9, 57.5, 57.9, 105.7,
106.7, 107.9, 110.4, 123.2, 125.3, 125.4, 127.8, 127.9,
128.3, 131.4, 146.8, 149.3, 152.0, 158.5, 159.9; HRMS
calcd for C22H22NO4+: 364.1549; found: 364.1542.
1
Ethyl-2,3-(methylenedioxy)dihydrocinnamate (17). This
intermediate was prepared in three steps (Scheme 3)
from 2,3-(methylenedioxy)benzaldehyde. Malonic 6.93 g
(67 mM) acid was placed in a 125 mL 3-neck ¯ask ®tted
with a re¯ux condenser, into which a pyridine solution
of 5.0 g (67 mM) of 2,3-(methylenedioxy)benzaldehyde
was added. The reaction mixture was heated at 80±85 ꢀC
for 30 min and then the temperature was raised to 110±
115 ꢀC when it began to re¯ux. The reaction mixture
was re¯uxed for 3 h. After cooling to room temperature
the reaction mixture was poured into 150 mL cold water
and slowly acidi®ed by dropwise addition of con-
centrated hydrochloric acid to pH 1. The crystals
obtained were collected by ®ltration under vacuum and
washed thrice with 10 mL cold water and air-dried. The
crystals were recrystallized from methanol and decolor-
ized using charcoal to give a quantitative yield of the
white crystalline cinnamic acid; mp 194±195 ꢀC (lit.36
194±195 ꢀC); 1H NMR: d 6.08 (2H, s), 6.66 (1H, d,
J=16.1), 6.84±6.97 (3H, m), 7.72 (1H, d, J=16.1); 13C
NMR: d 102.0, 110.7, 117.5, 120.2, 122.3, 123.2, 141.9,
147.3, 148.5, 171.9.
1
gel, however, H NMR of the crude compound indi-
cated that it could be used directly in the next step
without further puri®cation; mp 62±63 ꢀC; H NMR: d
1
2.60 (3H, s), 2.79±3.01 (4H, m), 3.53±3.82 (2H, q,
J1=13.8, J2=34.3), 5.89 (2H, s), 6.59±6.86 (3H, m); 13
C
NMR: d 23.8, 39.4, 45.1, 64.4, 101.1, 107.4, 121.9, 122.0,
123.0, 145.8, 147.6, 201.9.
1,2,3,4-Tetrahydro-1-methylthio-5,6-methylenedioxy-
2(1H)-napthalenone (19). Tri¯uoroacetic acid (0.3 mL,
3.7 mM) was dissolved in 25 mL benzene and added to
the reaction ¯ask containing 0.47 g (1.85 mM) of 18.
The reaction mixture was then heated to re¯ux 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 then dried over
anhydrous sodium sulfate, ®ltered 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 19 in 60% yield; mp
2,3-(Methylenedioxy)cinnamic acid 6.0 g (26 mM) was
placed in a hydrogenation ¯ask and dissolved in 80 mL
absolute ethanol. 0.3 g of 10% Pd-C was added and this
mixture was shaken in a Parr apparatus at 60 psig of
hydrogen for 5 h. The reaction mixture was ®ltered
through a celite bed to remove the catalyst and the
clear ®ltrate was evaporated in vacuo to give a quanti-
tative yield of the dihydrocinnamic acid. An analytical
sample was crystallized from benzene to provide a
ꢀ
1
56 C; H NMR: d 2.08 (3H, s), 2.78±3.18 (4H, m), 4.02
(1H, s), 5.89 (1H, s), 6.63 (1H, d, J=8), 6.72 (1H, d,

D. Makhey et al. / Bioorg. Med. Chem. 8 (2000) 1171±1182
1181
J=8); 13C NMR: d 16.5, 21.3, 34.6, 54.3, 101.7, 107.8,
118.8, 122.9, 127.9, 145.2, 147.3, 203.2.
3. Chen, A. Y.; Liu, L. F. Annu. Rev. Pharmacol. Toxicol.
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Topoisomerase mediated DNA cleavage assays
5. Liu, L. F.; Wang, J. C. Proc. Natl. Acad. Sci. USA 1987, 84,
7024.
Human topoisomerase was isolated as a recombinant
fusion protein using a T7 expression system (unpub-
lished results). DNA topoisomerase I was isolated as a
recombinant fusion protein using a T7 expression sys-
tem (unpublished results). DNA topoisomerase II was
puri®ed from calf thymus gland as reported pre-
viously.40 Plasmid YEpG was also puri®ed by the alkali
lysis method followed by phenol deproteination and
CsCl/ethidium isopycnic centrifugation as described.41
The end-labeling of the plasmid was accomplished by
digestion with a restriction enzyme followed by end-®lling
with Klenow polymerase as previously described.42 The
cleavage assays were performed as previously reported.8
Assessment of relative potency was performed by visual
comparison of the extent of cleavage of DNA in the
presence of enzyme and varying concentrations of drug.
Topo I cleavage values are reported as REC (relative
e€ective concentrations), i.e., concentrations relative
to coralyne, whose value is arbitrarily assumed as 1.
Potencies were determined at doses that were required
to cause conversion of approximately 10% of the DNA
substrate into the cleavage products.
6. Tsao, Y.-P.; Wu, H.-Y.; Liu, L. F. Cell 1989, 56, 111.
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Cytotoxicity assay
The cytotoxicity was determined using the as MTT-
microtiter plate tetrazolinium cytotoxicity assay
(MTA).43 45 The human lymphoblast RPMI 8402 and
its camptothecin-resistant variant cell line, CPT-K5
were provided by Dr. Toshiwo Andoh (Aichi Cancer
Center Research Insitute, Nagoya, Japan).46 The cyto-
toxicity 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 med-
ium supplemented with 10% heat inactivated fetal
bovine serum, l-glutamine (2 mM), penicillin (100 U/mL),
and streptomycin (0.1 mg/mL). For determination of
IC50, cells were exposed continuously with varying con-
centrations of drug concentrations and MTT assays
were performed at the end of the fourth day.
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Mass spectrometry was provided by the Washington
University Mass Spectrometry Resource, an NIH
Research Resource (Grant No. P41RR0954). This study
was supported by Grant CA 39662 from the National
Cancer Institute (L.F.L.) and a fellowship grant from
the Johnson & Johnson Discovery Research Fund
(E.J.L.).
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a