8,9-Methylenedioxybenzo[i]phenanthridines: Topoisomerase I-targeting activity and cytotoxicity

Article abstract of DOI:10.1016/S0968-0896(03)00394-8

Substituted benzo[i]phenanthridines that have incorporated within their structure an 8,9-methylenedioxy group can exhibit topoisomerase I-targeting activity. Structure-activity studies were performed to examine the influence of saturation at the 11,12-positions of several substituted 8,9-methylenedioxybenzo[i]phenanthridines. The activities of these dihydro analogues were compared to those of their unsaturated analogues. In addition, the influence of varying substituents at the 2- and 3-positions within the A-ring of these 8,9-methylenedioxybenzo[i]phenanthridines on their relative potency as topoisomerase I-targeting agents and cell proliferation as determined using the MTT assay was investigated. 2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine and its 11,12-dihydro derivative were among the more potent analogues evaluated with regard to topoisomerase I-targeting activity and cytotoxicity.

Full text of DOI:10.1016/S0968-0896(03)00394-8

Bioorganic & Medicinal Chemistry 11 (2003) 3795–3805
8,9-Methylenedioxybenzo[i]phenanthridines:
Topoisomerase I-Targeting Activity and Cytotoxicity
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, Piscataway, NJ 08854-8020, USA
bDepartment of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School,
Piscataway, NJ 08854, USA
cThe Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
Received 31 October 2002; accepted 25 April 2003
Abstract—Substituted benzo[i]phenanthridines that have incorporated within their structure an 8,9-methylenedioxy group can
exhibit topoisomerase I-targeting activity. Structure–activity studies were performed to examine the influence of saturation at the
11,12-positions of several substituted 8,9-methylenedioxybenzo[i]phenanthridines. The activities of these dihydro analogues were
compared to those of their unsaturated analogues. In addition, the influence of varying substituents at the 2- and 3-positions within
the A-ring of these 8,9-methylenedioxybenzo[i]phenanthridines on their relative potency as topoisomerase I-targeting agents and
cell proliferation as determined using the MTT assay was investigated. 2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine
and its 11,12-dihydro derivative were among the more potent analogues evaluated with regard to topoisomerase I-targeting activity
and cytotoxicity.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
derivative that possesses high affinity for human serum
albumin.9À11 The metabolic instability of this lactone
and the observation that both topotecan and irinotecan
are substrates for efflux transporters associated with resis-
tance have prompted further studies on the development
of novel TOP1-targeting agents.8À16
DNA topoisomerases are nuclear enzymes that catalyze
the breaking and rejoining of DNA strands regulating
the topological state of DNA.1À6 Studies suggest that
topoisomerases are also involved in controlling template
supercoiling during RNA transcription.7,8 The anti-
tumor activity of topoisomerase-targeting agents is
associated with their ability to stabilize the enzyme–
DNA cleavable complex. Stabilization of the enzyme–
DNA cleavable complex by such agents effectively
converts the enzyme into a cellular poison.
Benzo[i]phenanthridine derivatives that possess sig-
nificant topoisomerase I (TOP1)-targeting activity have
been identified.17,18 These compounds were developed
on the basis of the structure–activity relationships
observed for analogues of coralyne12,19 and benz[a]acri-
dine.20 These initial studies lead to the discovery of
TOP1-targeting agents, such as MDD-coralyne, 1, and
3,4-methylenedioxy-9,10-dimethoxy-5,6-dihydroacridine,
2 (Fig. 1).
The two camptothecin-based topoisomerase I-targeting
agents in clinical use are topotecan (Hycamtin1) and
irinotecan (CPT-11/Camptosar1). These drugs have
incorporated within their structure the camptothecin-
ring system, which includes the presence of a d-lactone.
Hydrolysis of this lactone moiety results in an inactive
While 2 represents a noncharged analogue that is struc-
turally-related to coralyne and protoberberine alkaloids
and retains significant TOP1-directed activity, its rela-
tive cytotoxic potency against the human lymphoblast
tumor cell line, RPMI8402 was disappointing. Ben-
zo[i]phenanthridine derivatives with a 2,3-methylene-
dioxy substituent within the A-ring and methoxyl
*Corresponding author at: Ernest Mario School of Pharmacy, 160
Frelinghuysen Road, Piscataway, NJ, USA Tel.: +1-732-445-2674;
fax: +1-732-445-6312; e-mail: elavoie@rci.rutgers.edu; internet:
http://www.rci.rutgers.edu/ꢀlayla/faculty/lavoie.htm
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00394-8

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D. Li et al. / Bioorg. Med. Chem. 11 (2003) 3795–3805
These results prompted further study of benzo[i]phe-
nanthridines specifically focusing on those derivatives
that possess a 8,9-methylenedioxy moiety. Earlier stud-
ies with both protoberberine analogues and benz[a]acri-
dines suggest that saturation within their ring systems,
with a consequential loss of planarity, was associated
with increased TOP1-targeting activity and cytotoxicity.
Based upon these observations, several 8,9-methylene-
dioxybenzo[i]phenanthridines with varied substituents in
the A-ring as well as their 11,12-dihydro derivatives were
selected for synthesis and pharmacological evaluation.
The relative importance of each of the methoxy groups
of 4 and their 11,12-dihydroderivatives was specifically
investigated. Phenolic derivatives such as 10e and 10f
have the potential to be modified into prodrugs that
could significantly enhance the water solubility of these
benzo[i]phenanthridine derivatives. Therefore, the
potential of these phenolic derivatives to retain TOP1-
targeting activity was also examined.
Figure 1. Structures of MDD-coralyne: 1, 2,3-methylenedioxy-9,10-
dimethoxy-5,6-dihydrobenz[a]acridine;
dimethoxybenzo[i]phenanthridine; 3, 2,3-dimethoxy-8.9-methylenedi-
oxybenzo[i]phenanthridine; 4, 2,3-dimethoxy-8.9-methylenedioxydi-
benzo[c,h]cinnoline; 5, and the structure and numbering of 8.9-
methylenedioxybenzo[i]phenanthridine derivatives.
2,
2,3-dimethoxy-9,10-
Chemistry
Scheme 1 outlines the general approach that was
employed for the preparation of these 8,9-methylene-
groups at the 8- and 9-positions, such as 3, were identi-
fied as a second generation of noncharged TOP1-tar-
geting agents related to both protoberberine and
benzo[c]phenanthridine alkaloids.12,19,21À25 The initial
structure–activity data associated with the TOP1-tar-
geting activity of benzo[i]phenanthridines suggested that
the presence of 2,3-dimethoxyl substituents within the
A-ring results in a dramatic loss in potency. Based upon
these initial findings, it was surprising that 2,3-dime-
thoxy - 8,9 - methylenedioxybenzo[i]phenanthridine, 4,
exhibited exceptional TOP1-targeting activity and sig-
nificantly greater cytotoxicity than 3. The likely explan-
ation for this apparent contradiction is that 4 adopts a
similar molecular topology to 3, as illustrated in Figure
1. In adopting this orientation, the principle difference
in the molecular arrangement of these two molecules is
in the relative location of the nitrogen heteroatom. In
the case of 4, the nitrogen heteroatom is immediately
adjacent to the ring that contains the methylenedioxy
moiety. This orientation of the ring heteroatom appears
to be linked to increased TOP1-targeted activity and
greater cytotoxicity.
dioxybenzo[i]phenanthridines.
Appropriately
sub-
stituted b-tetralones were converted to their requisite
2-bromo-3,4-dihydronaphthaldehydes, 6a–e, as descri-
bed.27
Compounds 6a–c were prepared from commercially
available 6-methoxy-b-tetralone, 7-methoxy-b-tetralone,
and 6,7-dimethoxy-b-tetralone, respectively. The
benzyloxy b-tetralone intermediates for the preparation
of 6d and 6e were prepared as outlined in Scheme 2.
Treatment of 6b–d with DDQ in toluene provided 7b–d
in good yield. Stille coupling of 6a–e and 7b–d with tri-
methyl-(2-nitro-4,5-methylenedioxy)stannane,28 8, pro-
vided 9a–e and 11b–d, respectively. Treatment of 9a–e
or 11b–d with zinc dust in acetic acid resulted in reduc-
tion of their nitro substituents and subsequent cycli-
zation to the desired benzo[i]phenanthridines, 10a–e and
12b–d. The benzyloxy derivative 10d proved to be resis-
tant to hydrogenolysis using palladium-on-carbon. It
could be converted to 10f using Raney nickel and
hydrogen at 40–45 psi.
These structure–activity studies were recently extended
to a series of dibenzo[c,h]cinnolines.26 Among the 8,9-
methylenedioxydibenzo[c,h]cinnolines evaluated in this
study, 5 exhibited greater TOP1-targeting activity and
cytotoxicity in both RPMI8402 and U937 cell lines than
4. While 5 exhibits cross-resistance to the camptothecin-
resistant variants, CPT-K5 and U937/CR,26 no cross-
resistance is observed with 4. The ability of benzo[i]-
phenanthridines to overcome camptothecin-resistance
in these cell lines suggests that either they exert their
cytotoxic activity by forming a ternary complex in a
manner that differs from camptothecin or that this ser-
ies of TOP1-targeting agents have, in addition, the
potential to affect cell proliferation by a mechanism that
does not involve TOP1.
The benzyloxy b-tetralone intermediates 21 and 22
required for the preparation of 6d and 6e were prepared
as outlined in Scheme 2. The requisite cinnamic acid
was reduced by catalytic hydrogenation to form com-
pounds 13 and 14. The free phenolic group of 13 and 14
was protected by benzyl bromide in refluxing acetone
with potassium carbonate (K2CO3) as a base. Both the
phenolic group and the carboxylic group were benzyl-
ated under these conditions. The benzyl ester formed in
this step was then subsequently hydrolyzed using KOH
in methanol and water. Acidic work up of the reaction
resulted in the formation of the benzyloxy substituted
phenylpropanoic acid, either 15 or 16 in good yield.
Treatment of 15 and 16 with oxalyl chloride provided

D. Li et al. / Bioorg. Med. Chem. 11 (2003) 3795–3805
3797
the requisite acid chlorides, 17 and 18. Reaction of these
acid chlorides with (trimethylsilyl)diazomethane resul-
ted in the formation of diazoketones 19 and 20.29 Rho-
dium(II) acetate catalyzed cyclization using similar
conditions to those previously reported provided the
benzyloxy substituted 2-tetralones 21 and 22 in 50–55%
yield.30
U937/U937CR,32 P388 and its camptothecin-resistant
variant P388/P388CPT45,33 and KB3-1 and its variant,
KBV-1,34 which overexpresses p-glycoprotein (MDR1).
These data are provided in Table 2.
A representative gel associated with the assays per-
formed to determine TOP1-targeting activity, which
provides an index of the relative potency of individual
compounds to enhance cleavable complex formation
between the enzyme and DNA, is illustrated in Figure 2.
Pharmacology
The relative topoisomerase-targeting activities (both
TOP1 and TOP2) of 11,12-dihydrobenzo[i]phenan-
thridines, 10a–f and the benzo[i]phenanthridines, 4 and
12b–d, are listed in Table 1.
Results and Discussion
The relative TOP1-targeting activity of the various 8,9-
methylenedioxybenzo[i]phenanthridines evaluated in
this study indicates that the presence of methoxyl sub-
stituents at both the 2- and 3-positions is associated
with increased potency relative to those analogues that
possess a single methoxyl group at either of these posi-
tions. In the case of 8,9-methylenedioxybenzo[i]phenan-
thridines, 4 clearly exhibits enhanced TOP1-targeting
Compounds were evaluated for cytotoxicity toward the
human lymphoblastoma tumor cell line, RPMI8402 and
its camptothecin-resistant variant, CPT-K5.31 These
data are provided in Table 1. In addition, these com-
pounds were evaluated for their effects on cell pro-
liferation in U937 and its camptothecin-resistant variant
Scheme 1. Synthetic approach to the preparation of 8,9-methylenedioxydi-benzo[i]phenanthridine derivatives, 10a–10f and 12b–12d: (i) DDQ, tol-
uene; (ii) Pd(PPh3)4, CuBr, THF, reflux; (iii) Zn dust, AcOH, reflux; (iv) Ra–Ni, H2 at 44 psi.

3798
D. Li et al. / Bioorg. Med. Chem. 11 (2003) 3795–3805
activity relative to either 12b or 12c. Among the 11,12-
dihydrobenzo[i]phenanthridines, 10a exhibits sig-
nificantly greater TOP1-targeting activity than either
10b or 10c. There was a slight increase in TOP1-target-
ing activity observed with 10a relative to its unsaturated
derivative, 4. These data parallel the results obtained for
coralyne and its dihydro derivative, MDD-coralyne, in
that the dihydro derivative exhibited enhanced TOP1-
targeting activity and diminished potency as a TOP2-
targeting agent. This trend was not clearly reflected in
the studies performed with the mono-methoxylated
derivatives, 10b, 10c, 12b, and 12c. While 10b was more
active as a TOP1-targeting agent than 12b, it was also
more potent as a TOP2-targeting agent. In the case of
10c, it was actually slightly less active than its unsatu-
rated analogue, 12c, as a TOP1-targeting agent and had
similar TOP2-targeting activity.
The replacement of the 3-methoxy group of either 10a
or 4 with a benzyloxy group, as in the case of 10d or
12d, results in a loss of both TOP1- and TOP2-targeting
activities. While replacement of the 2-methoxyl group of
10a with a hydroxyl group, as in 10e, results in a major
loss of TOP1-targeting activity, substitution of the 3-
methoxyl group of 10a with a hydroxyl group, as in 10f,
did not have a significant impact on TOP1-targeting
activity. Thus, 10f could be used to develop a prodrug
that would allow modification of its physico-chemical
properties.
Table
benzo[i]phenanthridine derivatives
2. Pharmacological
activity
of
8,9-methylenedioxy-
Compd
Cytotoxicity IC50 (mM)a
U937 U937/CR P388 P388/CPT45 KB3-1 KBV-1
1
2
3
4
8
—
2.9
40
—
5.0
0.1
7.5
1.5
0.7
0.45
0.23
0.33
9.0
2.2
0.46
0.26
1.3
1.0
5.5
0.3
0.05
0.16
0.3
7.0
0.18
3.0
0.75
0.23
13
>10
1.0
1.8
b
b
0.1
0.2
5
0.06
0.04
0.2
34
0.09
10
1.0
0.04
8.0
>10
0.12
0.24
0.4
2.2
0.24
0.3
0.36
0.27
2.3
0.25
2.5
10a
10b
10c
10d
10e
10f
12b
12c
12d
0.05 0.19
0.18
0.25
22
—
19.0
0.90
0.52
2.65
0.18
0.63
0.5
>100
0.15
40
0.8
0.16
4.5
—
2.4
0.36
0.06 0.29
60 2.65
0.17 0.1
3.5 0.006
0.65 0.13
0.01 0.006
0.29
0.19
0.06
0.01
0.02
Nitidine 0.8
CPT
VM-26
0.006
0.03
>10
0.001
0.03
2.25
Scheme 2. Preparation of the b-tetralone intermediates used in the
synthesis of 6d and 6e: (i) 10% Pd/C; H2, 40 psi; (ii) BnBr, K2CO3;
(iii) KOH, H2O, CH3OH; (iv) (COCl)2; (v) TMSCHN2; (vi) Rh(OAc)2.
aIC50 has been calculated after 4 days of continuous drug exposure.
bNot determined.
Table 1. Pharmacological activity of 8,9-methylenedioxybenzo[i]phenanthridine derivatives
Compd
Topo I-mediated
DNA cleavageb
Topo II-mediated
DNA cleavagec
Cytotoxicity IC50 (mM)a cell lines
RPMI8402
CPT-K5
1
2
3
4
5
10a
10b
10c
10d
10e
10f
12b
400
50
100
10
8
8
10
>1000
10
>1000
500
>1000
500
500
>1000
500
>1000
>1000
500
>1000
1
>1000
>1000
1
5.9
6.8
4.5
0.4
0.07
0.4
0.6
27
22
11
0.4
5.5
0.2
0.8
200
200
>1000
500
10
>1000
100
>1000
10
33
70
0.16
7.6
1.4
0.2
18
0.3
0.4
0.012
0.005
0.2
0.14
25
1.1
0.2
12c
12d
>100d
0.3
4.0
Nitidine
Topotecan
Camptothecin
VM-26
1.0
0.5
>1000
>10
>60
0.3
aIC50 has been calculated after 4 days of continuous drug exposure.
bTopoisomerase I cleavage values are reported as REC, relative effective concentration, that is concentrations relative to topotecan, whose value is
arbitrarily assumed as 1, that are able to produce the same cleavage on the plasmid DNA in the presence of human topoisomerase I.
cTopoisomerase II cleavage values are reported as REC, relative effective concentration, that is concentrations relative to VM-26, whose value is
arbitrarily assumed as 1, that are able to produce the same cleavage on the plasmid DNA in the presence of human topoisomerase II.
dIC50 values were substantially greater than the highest doses assayed.

D. Li et al. / Bioorg. Med. Chem. 11 (2003) 3795–3805
3799
proliferation as determined using the MTT assay. These
data as outlined in Table 2 indicate that mechanisms
other than TOP1-targeting activity are associated with
the effects that several benzo[i]phenanthridines have on
cell proliferation (some of these derivatives have modest
TOP2-targeting activity). Three of the benzo[i]phenan-
thridines evaluated in this study, 10d, 12b, and 12d, did
not exhibit significant TOP1- or TOP2-targeting activ-
ity. Nonetheless, these derivatives had significant cyto-
toxicity (IC50 values ranging from 40 nmol to 300 nM).
These data again suggest that mechanisms other than
stabilization of TOP1-cleavable complex formation do
contribute to the cytotoxicity observed with substituted
benzo[i]phenanthridines.
Several cytotoxic agents are resistant to cells that over-
express the efflux transporter p-glycoprotein or MDR1.
In cells that overexpress p-glycoprotein, much higher
concentrations of the drug are required to achieve leth-
ality if it is a substrate for this efflux transported. The
absence of a significant difference between KB3-1 cells
and its variant, KBV-1, which is known to overexpress
p-glycoprotein, indicates that these benzo[i]phenan-
thridines are not substrates for this efflux transporter
and are capable of overcoming this form of multidrug
resistance.
Figure 2. Stimulation of enzyme-mediated DNA cleavage by camp-
tothecin (CPT), 4, 10a, 10c, and 10b using human TOP1. The first lane
is DNA control without enzyme. The second lane is the control with
enzyme alone. The rest of the lanes contain human TOP1 and serially
(10-fold each) diluted compounds from 0.01 to 1.0 mM for CPT and
1.0 to 100 for compounds 4, 10a, 10b and 10c.
The influence of structural modification on the cyto-
toxicity of these 8,9-methylenedioxybenzo[i]phenan-
thridines as determined using the MTT assay was also
assessed. Previous studies in our laboratory have noted
that cytotoxic activity and TOP1-targeting activity do
not correlate well unless the compound is exclusively a
TOP1-targeting agent (i.e., does not exhibit significant
TOP2-targeting activity), its potency as a TOP1-target-
ing agent is at least 10% of that of camptothecin, and
there are no significant differences in their potential for
cellular absorption. The three more potent TOP1-tar-
geting benzo[i]phenanthridines were 4, 10a and 10f. The
IC50 values obtained for 4 and 10a ranged from 40 to
500 nM and for 10f from 360 to 1400 nM. As previously
observed for 4, there was no significant cross-resistance
(ꢁ10-fold difference) observed between either
RPMI8402 and its camptothecin-resistant variant CPT-
K5 or the leukemia cell line, U937 and its camptothecin-
resistant variant U937/CR with any of the benzo[i]phe-
nanthridines evaluated. Both of these camptothecin-
resistant cell lines have a functional, but mutant form of
TOP1 that forms a cleavable complex with DNA that is
not stabilized by the presence of camptothecin.31,32
Therefore, the potential role of TOP1-targeting being
associated with the effects observed with these benzo[i]-
phenanthridines in these camptothecin-resistant cell
lines cannot be ruled out. These data are, however, in
marked contrast to the results obtained with similarly
substituted benzo[c,h]cinnolines, which are 5-aza ana-
logues of benzo[i]phenanthridine. In this series of
TOP1-targeting agents, there is clear evidence of cross-
resistance observed in both CPT-K5 cells and in U937/
CR cells.26 In light of these differences benzo[i]phenan-
thridines were also evaluated for their effect on P388
murine leukemia cells and its camptothecin-resistant
variant, P388/CPT45. As the P388/CPT45 cell line does
not functionally express topoisomerase-I, these cells
provide a good indication as to whether or not TOP1-
targeting activity is likely to play a dominant role on cell
Conclusions
Benzo[i]phenanthridines represent a series of novel
topoisomerase I-targeting agents. Unlike the camp-
tothecin-based TOP1-targeting agents available in the
clinic, these agents do not have an unstable lactone
moiety incorporated within their structures. Improved
potency has been observed for certain 11,12-dihy-
drobenzo[i]phenanthridines as TOP1-targeting agents.
Benzo[i]phenanthridines with
a 8,9-methylenedioxy
moiety in the D-ring together with a 2-methoxyl group
and either a hydroxyl or a second methoxyl group at the
3-position of the A-ring are among the more potent
analogues. The data also suggest that mechanisms other
than TOP-1 targeting contribute significantly to the
effects observed by several benzo[i]phenanthridines on
cell proliferation as obtained using the MTT assay. In
contrast to results obtained with 2,3-dimethoxy-8,9-
methylenedioxydibenzo[c,h]cinnoline, 5, several ben-
zo[i]phenanthridines did not exhibit significant differ-
ences with respect between their effect on parent and
variants cell lines, which have either mutant topoisome-
rase I or do not express topoisomerase I. Studies are in
progress to develop derivatives with increased potency as
TOP1-targeting agents that specifically exert their cyto-
toxic effect through stabilization of the TOP1 cleavable
complex.
Experimental
General
Melting points were determined with a Thomas-Hoover
Unimelt capillary melting point apparatus. Column

3800
D. Li et al. / Bioorg. Med. Chem. 11 (2003) 3795–3805
1
chromatography refers to flash chromatography con-
ducted on SiliTech 32–63 mm, (ICN Biomedicals,
Eschwegge, Germany) using the solvent systems indi-
cated. Infrared spectral data (IR) were obtained on a
Perkin-Elmer 1600 Fourier transform spectrophotometer
and are reported in cmÀ1. Proton (1H NMR) and carbon
(13C NMR) nuclear magnetic resonance were recorded on
a Varian Gemini-200 Fourier Transform spectrometer.
NMR spectra (200MHz 1H and 50MHz 13C) were
recorded in the deuterated chloroform, unless otherwise
indicated, with chemical shifts reported in d units down-
field from tetramethylsilane (TMS). Coupling constants
are reported in hertz (Hz). Mass spectra were obtained
from Washington University Resource for Biomedical
and Bio-organic Mass Spectrometry within the Depart-
ment of Chemistry at Washington University, St. Louis,
MO, USA. Tetrahydrofuran was freshly distilled from
sodium and benzophenone prior to use. Tetra-
kis(triphenylphosphine)palladium(0) was purchased as
bright yellow powder or crystal from Aldrich Chemical
Company (Milwaukee, WI, USA) or Acros Organics
(Fisher Scientific, Pittsburgh, PA, USA). It is very sensi-
tive to air-oxidation and the resulting Pd(II) with brown
color loses catalytic activity. 6-Methoxy-, 7-methoxy-, and
6,7-dimethoxy-b-tetralone were obtained from Aldrich
Chemical Company (Milwaukee, WI, USA). Compounds
4 and 8 were prepared as previously described.24
in 73% yield (1.11 g); mp 69–70 ꢂC; H NMR d 2.83
(2H, t, J=7.3), 3.02 (2H, t, J=7.3), 3.81 (3H, s), 6.79
(1H, dd, J=8.3, 2.7), 7.06 (1H, d, J=8.3), 7.64 (1H, d,
J=2.7), 10.32 (1H, s); 13C NMR d 28.0, 38.4, 55.5,
111.5, 114.1, 126.7, 128.2, 130.8, 132.6, 146.2, 158.4,
192.8; HRMS calcd for C12H11BrO2: 265.9942; found:
265.9946.
7-Benzyloxy-2-bromo-3,4-dihydro-6-methoxy-1-naphthal-
dehyde (6d). Prepared from 6-methoxy-7-benzyloxy-2-
tetralone, 21 (950 mg, 3.4 mmol) in 20% yield; (254 mg);
mp 88–89 ꢂC; 1H NMR d 2.84 (2H, t, J=7.6), 2.98 (2H,
t, J=7.6), 3.90 (3H, s), 5.18 (2H, s), 6.69 (1H, s), 7.34–
7.53 (5H, m), 7.80 (1H, s), 10.29 (1H, s); 13C NMR d
28.5, 38.1, 56.1, 71.2, 111.3, 112.2, 122.6, 127.8, 128.0,
128.2, 128.7, 132.2, 137.2, 143.7, 146.4, 149.4, 193.1;
HRMS calcd for C19H17BrO3: 372.0361; found:
372.0380.
2-Bromo-3,4-dihydro-6-hydroxy-7-methoxy-1-naphthal-
dehyde (6e). Prepared from 6-methoxy-7-benzyloxy-2-
tetralone 22 (950 mg, 3.4 mmol in 50% yield (481 mg);
mp >250 C H NMR d 2.82 (2H, t, J=7.2), 3.00 (2H,
t, J=7.2), 3.93 (3H, s), 5.71 (1H, s), 6.74 (1H, s), 7.72
(1H, s), 10.33 (1H, s); 13C NMR d 28.2, 38.1, 56.3,
109.1, 113.8, 128.5, 130.5, 132.3, 143.9, 144.9, 145.5,
193.3; HRMS calcd for C12H11BrO3: 281.9892; found:
281.9887.
ꢂ
1
General procedure for the preparation of 2-bromo-3,4-di-
hydro-1-naphthaldehydes (6a–e)
General procedure for the synthesis of 2-bromo-1-naph-
thaldehydes (7b, 7c, 7d)
2-Bromo-3,4-dihydro-6,7-dimethoxy-1-naphthaldehyde (6a).
Dimethylformamide (1.6 mL, 21.4 mmol) was added
dropwise to a solution of phosphorus tribromide (1.6
mL, 16.9 mmol) in dry chloroform (20 mL) at 0 ꢂC . The
mixture was stirred at 0 ꢂC for 1 h to give a pale yellow
suspension. A solution of 6,7-dimethoxy-2-tetralone
(1.0 g, 4.85 mmol) in dry chloroform (20 mL) was added
to the yellow suspension and the mixture was heated at
reflux for 1 h. The reaction mixture was cooled to 0 ꢂC
and made basic with saturated aqueous NaHCO3 solu-
tion. The resulting mixture was extracted with di-
chloromethane, dried (anhyd Na2SO4), and evaporated
in vacuo. The residue was chromatographed over 100 g
of silica gel using a 1:3 mixture of ethyl acetate/hexanes
to give 72 (1.15 g) as yellow crystalline solid in 80% yield;
mp 82–83 ꢂC; 1H NMR d 2.83 (2H, t, J=7.6), 3.01 (2H, t,
J=7.6), 3.88 (3H, s), 6.66 (1H, s), 7.71 (1H, s), 10.31 (1H,
s); 13C NMR d 28.9, 38.5, 56.4, 56.5, 110.0, 111.1, 123.1,
128.0, 132.7, 144.2, 147.7, 149.1, 193.6; HRMS calcd for
C13H13BrO3: 296.0048; found: 296.0042.
The appropriate 2-bromo-3,4-dihydro-1-naphthalde-
hyde (1.0 mmol) and DDQ (272 mg, 1.2 mmol) was
refluxed in toluene (40 mL) for 15 h. After cooling to
room temperature, the mixture was filtered through a
Celite bed and the filtrate was evaporated to dryness.
The residue obtained was chromatographed on 75 g
silica gel using a 1:5 mixture of ethyl acetate/hexanes.
2-Bromo-6-methoxy-1-naphthaldehyde (7b). Prepared
from 6b (370 mg, 1.39 mmol) in 80% yield (294 mg); mp
105–107 ꢂC; IR (KBr) 1672; H NMR d 3.92 (3H, s),
1
7.09 (1H, d, J=2.6), 7.29 (1H, dd, J=9.4, 2.6), 7.61
(1H, d, J=8.8), 7.73 (1H, d, J=8.8), 8.99 (1H, d,
J=9.4), 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 calcd for C12H9BrO2: 263.9786; found:
263.9782.
2-Bromo-7-methoxy-1-naphthaldehyde (7c). Prepared
from (6c) (850 mg, 3.18 mmol) in 85% yield as yellow
solid (714 mg); mp 132–134 ꢂC; IR(KBr) 1670; 1H
NMR d 3.97 (3H, s), 7.21 (1H, dd, J=8.9, 2.6), 7.54
(1H, d, J=8.7), 7.71 (1H, d, J=8.9), 7.77 (1H, d,
J=8.7), 8.69 (1H, d, J=2.6), 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 calcd for C12H9BrO2:
263.9786; found: 263.9783.
2-Bromo-3,4-dihydro-6-methoxy-1-naphthaldehyde (6b).
Prepared from 6-methoxy-2-tetralone (950 mg, 5.4
1
mmol) in 50% yield (718 mg); H NMR d 2.88 (2H, t,
J=6.5), 3.01 (2H, t, J=6.5), 3.81 (3H, s), 6.70 (1H, d,
J=2.7), 6.78 (1H, dd, J=8.5, 2.7), 7.95 (1H, d, J=8.5),
10.30 (1H, s); 13C NMR d 29.2, 37.8, 55.3, 111.3, 113.8,
123.0, 127.3, 132.5, 136.8, 142.5, 159.5, 193.0; HRMS
calcd for C12H11BrO2: 265.9942; found: 265.9924.
7-Benzyloxy-2-bromo-6-methoxy-1-naphthaldehyde (7d).
Prepared from 6d (190 mg, 0.51 mmol) in 84% yield as
light yellow powder (160 mg); mp 147–148 ꢂC; 1H NMR
2-Bromo-3,4-dihydro-7-methoxy-1-naphthaldehyde (6c).
Prepared from 7-methoxy-2-tetralone (1.0 g, 5.7 mmol)

D. Li et al. / Bioorg. Med. Chem. 11 (2003) 3795–3805
3801
d 4.02 (3H, s), 5.36 (2H, s), 7.12 (1H, s), 7.34–7.42 (4H,
m), 7.57 (1H, d, J=8.6), 7.60 (1H, d, J=5.2), 7.73 (1H,
d, J=8.6), 8.86 (1H, s), 10.75 (1H, s); 13C NMR d 55.9,
70.7, 105.4, 106.8, 126.1, 127.9, 128.0, 128.2, 128.7,
129.1, 129.4, 129.6, 134.0, 136.2, 150.3, 151.7, 195.5;
HRMS calcd for C19H15BrO3: 370.0205; found:
370.0187.
m), 3.93 (3H, s), 5.20 (2H, s), 6.21 (2H, s), 6.70 (1H, s),
6.77 (1H, s), 7.31–7.43 (5H, m), 7.76 (1H, s), 7.98 (1H,
s), 9.68 (1H, s); 13C NMR d 27.8, 33.1, 56.1, 71.3, 103.7,
106.2, 110.2, 111.4, 113.1, 122.5, 127.8, 128.2, 128.6,
129.4, 131.6, 132.1, 137.4, 148.5, 149.4, 152.3, 157.3,
157.4, 190.8; HRMS calcd for C26H21NO7: 459.1318;
found: 459.1304.
3,4-Dihydro-6-hydroxy-7-methoxy-2-(4,5-methylenedioxy-
2-nitrophenyl)-1-naphthaldehyde (9e). Prepared from 6e
(50 mg, 0.18 mmol) in 38% yield as orange solid (25
General procedure for the synthesis of 9a–9e and 11b–
11d
mg); mp >250 ꢂC H NMR d 2.60–2.88 (4H, m), 3.95
1
Tetrakis(triphenylphosphine)palladium (0) (60 mg, 0.05
mmol) and cuprous bromide (10 mg, 0.07 mmol) was
added to a solution of 8 (400 mg, 1.2 mmol) and the
(3H, s), 5.73 (1H, s), 6.22 (2H, s), 6.72 (1H, s), 6.80 (1H,
s), 7.77 (1H, s), 7.90 (1H, s), 9.70 (1H, s); 13C NMR d
27.5, 33.0, 56.3, 103.6, 106.2, 110.2, 113.8, 129.7, 131.8,
132.1, 138.1, 144.9, 145.6, 148.4, 148.6, 152.3, 157.5,
190.9; HRMS calcd for C19H15NO7: 369.0849; found:
369.0856.
appropriate
2-bromo-3,4-dihydronaphthalene
(1.0
mmol) in THF (25 mL) at room temperature. The mix-
ture was refluxed for 48 h. After cooling, THF was
evaporated and ethyl acetate (30 mL) was added to the
residue. The solution was washed with water (20 mL).
The organic layer was separated and passed through a
Celite bed to remove suspended particles. The organic
layer was then washed with brine, dried (anhyd
Na2SO4), and evaporated in vacuo. The residue was
chromatographed using 75 g of silica gel and a 1:4 mix-
ture of ethyl acetate/hexanes.
2-(4,5-Methylenedioxy-2-nitrophenyl)-6-methoxy-1-naph-
thaldehyde (11b). Prepared from 8 (350 mg, 1.1 mmol)
and 7b (260 mg, 1.0 mmol) as yellow solid in 84% yield;
ꢂ
1
mp 153–154 C H NMR d 3.98 (3H, s), 6.23 (2H, s),
6.78 (1H, s), 7.22 (1H, s), 7.25 (1H, d, J=8.8), 7.38 (1H,
d, J=9.6), 7.70 (1H, s), 7.97 (1H, d, J=9.6), 9.15 (1H,
d, J=8.8), 10.28 (1H, s); 13C NMR d 55.5, 103.5, 105.6,
106.6, 111.9, 121.9, 126.1, 127.3, 128.3, 131.1, 133.2,
135.2, 142.0, 142.7, 148.2, 151.4, 158.4, 192.9; HRMS
calcd for C19H13NO6: 351.0743; found: 351.0753.
3,4-Dihydro-2-(3,4-methylenedioxy-6-nitrophenyl)-6,7-di-
methoxy-1-naphthaldehyde (9a). Prepared from 6a in
90% yield as a bright yellow solid (234 mg); mp 188–
189 ꢂC H NMR d 2.69 (2H, t, J=5.9), 2.82 (2H, t,
1
J=5.9), 3.92 (3H, s), 3.93 (3H, s), 6.20 (2H, s), 6.71 (1H,
s), 6.74 (1H, s), 7.76 (1H, s), 7.90 (1H, s), 9.70 (1H, s);
13C NMR d 28.2, 33.5, 56.4, 56.5, 104.0, 106.6, 110.6,
111.0, 111.2, 122.9, 129.2, 132.1, 132.5, 141.8, 147.6,
148.9, 149.1, 152.7, 157.9, 191.2; HRMS calcd for
C20H17NO7: 383.1005; found: 383.1014.
2-(4,5-Methylenedioxy-2-nitrophenyl)-7-methoxy-1-naph-
thaldehyde (11c). Prepared from 8 (350 mg, 1.1 mmol)
and 7c (300 mg, 1.1 mmol) as light yellow solid in 40%
yield; mp 142–143 ꢂC 1H NMR d 4.02 (3H, s), 6.23 (2H,
s), 6.78 (1H, s), 7.14 (1H, d, J=8.0), 7.28 (1H, dd,
J=9.2, 2.6), 7.70 (1H, s), 7.82 (1H, d, J=9.2), 8.00 (1H,
d, J=8.0), 8.81 (1H, d, J=2.6), 10.26 (1H, s); 13C NMR
d 55.6, 103.5, 104.2, 105.6, 111.7, 120.1, 124.3, 126.8,
129.1, 130.0, 131.2, 132.5, 134.4, 142.5, 145.9, 148.2,
151.4, 161.2, 193.0; HRMS calcd for C19H13NO6:
351.0743; found: 351.0752.
3,4-Dihydro-2-(4,5-methylenedioxy-2-nitrophenyl)-6-
methoxy-1-naphthaldehyde (9b). Prepared from 6b (267
mg, 1.0 mmol) in 84% yield as golden solid (297 mg);
mp 156–157 ꢂC; 1H NMR d 2.62–3.25 (4H, m), 3.86
(3H, s), 6.21 (2H, s), 6.72 (1H, s), 6.79 (1H, s), 6.82 (1H,
dd, J=8.6, 3.0), 7.77 (1H, s), 8.14 (1H, d, J=8.6), 9.72
(1H, s); 13C NMR d 28.5, 32.8, 55.4, 103.7, 106.2, 110.2,
111.3, 113.7, 122.8, 128.2, 131.9, 132.2, 138.0, 148.4,
152.3, 156.7, 159.5, 190.7; HRMS calcd for C19H15NO6:
353.0899; found: 353.0916.
7-Benzyloxy-6-methoxy-2-(4,5-methylenedioxy-2-nitro-
phenyl)-1-naphthaldehyde (11d). Prepared from 8 (126
mg, 0.38 mmol) and 7d (130 mg, 0.35 mmol) as yellow-
ꢂ
1
green solid in 90% yield; mp 192–193 C H NMR d
4.05 (3H, s), 5.37 (2H, s), 6.22 (2H, s), 6.78 (1H, s), 7.13
(1H, d, J=8.0), 7.20 (1H, s), 7.30–7.63 (5H, m), 7.68
(1H, s), 7.91 (1H, d, J=8.0), 8.94 (1H, s), 10.20 (1H, s);
13C NMR d 56.0, 70.8, 103.5, 105.6, 106.3, 106.9, 112.0,
124.9, 126.6, 126.9, 128.0, 128.2, 128.7, 130.0, 131.2,
132.9, 136.4, 142.7, 143.3, 148.1, 150.4, 151.3, 151.8,
193.2; HRMS (FAB) calcd for C26H19NO7+ Li:
464.1322; found: 464.1315.
3,4-Dihydro-2-(4,5-methylenedioxy-2-nitrophenyl)-7-
methoxy-1-naphthaldehyde (9c). Prepared from 6c (270
mg,ꢂ1.0 mmol) in 45% yield as yellow solid; mp 133–
134 C; 1H NMR d 2.62–3.22 (4H, m), 3.86 (3H, s), 6.22
(2H, s), 6.71 (1H, s), 6.84 (1H, dd, J=8.2, 2.6), 7.14
(1H, d, J=8.2), 7.78 (1H, s), 7.84 (1H, d, J=2.6), 9.74
(1H, s); 13C NMR d 27.3, 33.4, 55.5, 103.7, 106.2, 109.9,
112.3, 114.2, 127.9, 128.2, 130.7, 131.9, 132.1, 141.3,
148.5, 152.4, 158.3, 159.8, 190.5; HRMS calcd for
C19H15NO6: 353.0899; found: 353.0893.
General procedure for the preparation of 11,12-dihydro-
2,3-dimethoxy-8,9methylenedioxybenzo[i]phenanthridines
(10a, 10b, 10c, 10d) and 2,3-dimethoxy-8,9methylene-
dioxybenzo[i]phenanthridines (12b, 12c, 12d)
7-Benzyloxy-3,4-Dihydro-6-methoxy-2-(4,5-methylene-
dioxy-2-nitrophenyl)-1-naphthaldehyde (9d). Prepared
from 6d (100 mg, 0.27 mmol) in 73% yield as yellow
solid (90 mg); mp 151–152 ꢂC 1H NMR d 2.65–3.30 (4H,
The appropriate 3,4-dihydro-2-(6-nitrophenyl)-1-naph-
thaldehyde (0.26 mmol) was dissolved in glacial acetic
acid (15 mL) and heated under reflux with zinc dust

3802
D. Li et al. / Bioorg. Med. Chem. 11 (2003) 3795–3805
70% yield; mp >250 ꢂC H NMR (DMSO-d6) d 2.80
1
(180 mg, 2.8 mmol) for 2.5 h. Acetic acid was evapor-
ated in vacuo and the residue was dissolved in chloro-
form. The solution was filtered through a Celite bed and
the filtrate washed successively with saturated sodium
bicarbonate solution and brine. The organic layer was
dried (anhyd Na2SO4) and evaporated in vacuo. The
residue was chromatographed using 50 g of silica gel
and a 1:1 mixture of ethyl acetate/hexanes.
(2H, t, J=7.0), 3.20 (2H, t, J=7.0), 3.89 (3H, s), 6.28
(1H, s), 6.78 (1H, s), 7.43 (1H, s), 7.56 (1H, s), 7.70 (1H,
s), 9.24 (1H, s); 13C NMR (DMSO-d6) d 24.2, 26.9, 56.7,
100.3, 103.0, 104.0, 109.0, 109.2, 115.9, 122.6, 124.0,
126.6, 129.9, 144.5, 145.0, 145.4, 147.7, 149.3, 151.0;
HRMS calcd for C19H15NO4: 321.1001; found:
321.0992.
11,12-Dihydro-2,3-dimethoxy-8,9-methylenedioxybenzo[i]-
phenanthridine (10a). Prepared from 9a in 60% yield as
2-Methoxy - 8,9 - methylenedioxybenzo[i]phenanthridine
(12b). Prepared from 11b in 50% yield as pale yellow
pale yellow solid (52 mg); mp 210–211 ꢂC H NMR d
solid (29 mg); mp 232–233 ꢂC; H NMR (DMSO-d6) d
1
1
2.91 (2H, t, J=7.8), 3.13 (2H, t, J=7.8), 3.94 (3H, s),
3.99 (3H, s), 6.10 (2H, s), 6.81 (1H, s), 7.30 (1H, s), 7.37
(2H, s), 9.03 (1H, s); 13C NMR 24.4, 28.0, 56.5, 56.7,
99.5, 102.2, 106.9, 107.5, 111.9, 123.8, 125.3, 126.1,
129.6, 140.1, 144.5, 145.8, 148.8, 149.4, 150.3; HRMS
calcd for C20H17NO4: 335.1158; found: 335.1145.
3.95 (3H, s), 6.27 (2H, s), 7.42 (1H, dd, J=9.2, 2.6), 7.54
(1H, s), 7.60 (1H, d, J=2.6), 8.20 (1H, d, J=9.2), 8.31
(1H, s), 8.68 (1H, d, J=9.2), 9.00 (1H, d, J=9.2), 10.08
(1H, s); 13C NMR (DMSO-d6) d 55.9, 100.4, 102.6, 107.0,
109.1, 119.5, 120.8, 121.4, 121.7, 124.4, 124.7, 130.4,
131.6, 133.6, 142.7, 145.0, 146.1, 148.8, 149.7; HRMS
calcd for C19H13NO3: 303.0895; found: 303.0899.
11,12-Dihydro-2-methoxy-8,9-methylenedioxybenzo[i]-
phenanthridine (10b). Prepared from 9b (100 mg, 0.28
mmol) in 35% yield as pale yellow solid (30 mg); mp
187–188 ꢂC 1H NMR d 2.95 (2H, t, J=6.6), 3.13 (2H, t,
J=6.6), 3.87 (3H, s), 6.10 (2H, s), 6.84 (1H, d, J=2.6),
6.85 (1H, s), 6.90 (1H, dd, J=8.4, 2.6), 7.37 (1H, s), 7.79
(1H, d, J=8.4), 9.06 (1H, s); 13C NMR d 23.8, 28.4,
55.5, 99.2, 101.8, 106.5, 112.6, 113.9, 123.4, 124.8, 125.3,
125.7, 138.1, 139.5, 144.3, 145.3, 148.3, 149.8, 159.5;
HRMS (FAB) calcd for C19H15NO3+H: 306.1130;
found: 306.1129.
3-Methoxy - 8,9 - methylenedioxybenzo[i]phenanthridine
(12c). Prepared from 11c (50 mg, 0.14 mmol) in 56%
ꢂ
1
yield as pale yellow solid (24 mg); mp 239–240 C H
NMR (DMSO-d6) d 4.05 (3H, s), 6.28 (2H, s), 7.36 (1H,
dd, J=8.8, 1.8), 7.56 (1H, s), 8.05 (1H, d, J=8.8), 8.19
(1H, d, J=9.2), 8.32 (1H, s), 8.46 (1H, d, J=1.8), 8.54
(1H, d, J=9.2), 10.18 (1H, s); 13C NMR (DMSO-d6) d
56.3, 100.7, 102.7, 103.6, 107.0, 118.4, 118.5, 120.6,
126.8, 130.9, 131.4, 131.7, 131.8, 132.4, 143.3, 146.7,
148.8, 150.0, 159.8; HRMS calcd for C19H13NO3:
303.0895; found: 303.0892.
11,12-Dihydro-3-methoxy-8,9-methylenedioxybenzo[i]-
phenanthridine (10c). Prepared from 9c (70 mg, 0.20
mmol) in 50% yield as pale yellow solid (30 mg); mp
190–191 ꢂC 1H NMR d 2.94 (2H, t, J=7.8), 3.17 (2H, t,
J=7.8), 3.91 (3H, s), 6.14 (2H, s), 6.86 (1H, dd, J=8.4,
2.6), 7.24 (1H, d, J=8.4), 7.36 (1H, s), 7.42 (1H, s), 7.44
(1H, s), 9.11 (1H, s); 13C NMR d 24.2, 27.1, 55.6, 99.3,
101.9, 106.5, 109.6, 113.0, 125.7, 128.6, 129.0, 133.5,
141.2, 144.6, 145.9, 148.4, 150.3, 151.5, 159.1; HRMS
(FAB) calcd for C19H15NO3+H: 306.1130; found:
306.1143.
3-Benzyloxy-2-methoxy-8,9-methylenedioxybenzo[i]phe-
nanthridine (12d). Prepared from 11d (100 mg, 0.22
mmol) as white solid in 50% yield (45 mg); H NMR d
1
4.10 (3H, s), 5.47 (2H, s), 6.18 (2H, s), 7.34 (1H, s),
7.38–7.48 (3H, m), 7.57 (1H, s), 7.58–7.62 (2H, m), 7.91
(1H, s), 7.99 (1H, d, J=9.2), 8.19 (1H, s), 8.25 (1H, d,
J=9.2), 9.76 (1H, s); 13C NMR d 56.1, 71.3, 99.4, 101.9,
104.7, 107.2, 108.4, 118.3, 120.8, 124.8, 125.0, 127.4,
128.3, 129.0, 130.5, 130.8, 131.1, 136.7, 142.3, 145.5,
148.4, 149.4, 149.6, 150.2; HRMS calcd for C26H19NO4:
409.1314; found: 409.1300.
3-Benzyloxy-11,12-dihydro-2-methoxy-8,9-methylene-
dioxybenzo[i]phenanthridine (10d). Prepared from 9d (80
mg, 0.17 mmol in 20% yield as pale yellow solid (15
mg); 1H NMR d 2.90 (2H, t, J=8.0), 3.12 (2H, t,
J=8.0), 3.96 (3H, s), 5.27 (2H, s), 6.10 (2H, s), 6.84 (1H,
s), 7.35–7.56 (8H, m), 8.86 (1H, s); 13C NMR d 24.0,
27.7, 56.2, 71.7, 99.1, 101.8, 106.5, 110.4, 111.9, 123.4,
124.8, 125.7, 127.5, 128.1, 128.8, 129.8, 137.3, 139.7,
144.1, 145.4, 147.4, 148.4, 149.7, 149.9; HRMS calcd for
C26H21NO4: 411.1471; found: 411.1471.
11,12-Dihydro-3-hydroxy-2-methoxy-8,9-methylenedi-
oxybenzo[i]phenanthridine (10f). To a solution of 10d
(70 mg, 0.17 mmol) in a mixture of methanol (20 mL)
and ethyl acetate (15 mL) was added Raney–Nickel (70
mg) and the resulted solution was shaken in a Parr1
apparatus at 44 psi of hydrogen for 18 h. A magnetic
rod was used to get rid of most of the nickel. Tri-
fluoroacetic acid (5 mL) was added dropwise to the
reaction mixture until the suspension turned to clear
yellow solution. The solution was filtered through a
Celite bed and the filtrate was evaporated in vacuo to
give 10f (54 mg) as yellow solid in quantitative yield; mp
11,12-Dihydro-2-hydroxy-3-methoxy-8,9-methylenedioxy-
benzo[i]phenanthridine (10e). Prepared from 9e (15 mg,
0.04 mmol) was dissolved in glacial acetic acid (10 mL)
and heated under reflux with zinc dust (25 mg, 0.38
mmol) for 2 h. Trifluoroacetic acid (5 mL) was added
until the suspension turned clear. The solution was fil-
tered through a Celite bed and the filtrate was evapor-
ated in vacuo. The residue was recrystallized using
ethanol/hexanes to give 10e (9 mg) as yellow solid in
>250 ꢂC; H NMR (CD3OD) d 2.99 (2H, t, J=7.0),
1
3.40 (2H, t, J=7.0), 3.93 (3H, s), 6.34 (2H, s), 6.96 (1H,
s), 7.36 (1H, s), 7.41 (1H, s), 7.77 (1H, s), 9.07 (1H, s);
13C NMR (DMSO-d6) d 24.2, 26.9, 56.7, 100.3, 103.0,
104.0, 109.0, 116.0, 122.6, 124.0, 126.6, 129.9, 147.6,
147.8, 149.2, 149.5, 151.0; HRMS calcd for C19H15NO4:
321.1001; found: 321.0987.

D. Li et al. / Bioorg. Med. Chem. 11 (2003) 3795–3805
3803
3-(4-Hydroxy-3-methoxyphenyl)propionic acid (13). To a
solution of trans-4-hydroxy-3-methoxy cinnamic acid
(5.0 g, 25.7 mmol) in ethyl acetate (20 mL) was carefully
added palladium on carbon (10%, 500 mg). Methanol
(20 mL) was then added. The reaction mixture was
shaken in a Parr1 apparatus for 5 h at 40 psi of hydro-
gen. The solution was passed through a Celite bed and
the catalyst was washed carefully with methanol. Con-
centration in vacuo gave pure 13 (5.0 g) as white solid in
After cooling to room temperature, the reaction mixture
was washed with chloroform (30 mLÂ3) and con-
centrated hydrochloric acid (HCl) was added dropwise
until the pH of the solution was 2.0. The mixture was
extracted with chloroform (40 mLÂ3) and the organic
layer was washed with water (20 mLÂ2) and brine (30
mL), dried (anhyd Na2SO4), and evaporated in vacuo.
The residue was triturated with hexanes several times
and 16 (1.02 g) was obtained in 70% yield as a beige
solid; mp 115–116 C; H NMR d 2.62 (2H, t, J=7.0),
2.88 (2H, t, J=7.0), 3.88 (3H, s), 5.15 (2H, s), 6.76–6.87
(3H, m), 7.31–7.49 (5H, m); 13C NMR d 30.2, 35.8, 56.2,
71.2, 112.1, 114.7, 120.9, 127.5, 127.9, 128.6, 132.8,
137.3, 148.2, 148.4, 178.8; HRMS calcd for C17H18O4:
286.1205; found: 286.1194.
ꢂ
1
1
quantitative yield; H NMR (CD3OD) d 2.56 (2H, t,
J=7.3), 2.83 (2H, t, J=7.3), 3.83 (3H, s), 6.62–6.81
(3H, m); 13C NMR (CD3OD) d 32.0, 37.5, 56.6, 113.3,
116.4, 121.9, 134.0, 146.1, 149.2, 177.3.
3-(3-Hydroxy-4-methoxyphenyl)propionic acid (14). To a
solution of 3-hydroxy-4-methoxy cinnamic acid (1.0 g,
5.1 mmol) in ethyl acetate (20 mL) was carefully added
palladium on carbon (10%, 500 mg). Methanol (30 mL)
was then added. The reaction mixture was shaken in a
Parr1 apparatus for 5 h at 42 psi of hydrogen. The
solution was passed through a Celite bed and the cata-
lyst was washed carefully with methanol. Concentration
in vacuo gave pure 14 (1.0 g) as white solid in quantita-
7-Benzyloxy-6-methoxy-2-tetralone (21). To a refluxing
solution of rhodium (II) acetate dimer (6 mg) in di-
chloromethane (15 mL) was added dropwise a solution
of diazoketone (19) (400 mg, 1.3 mmol) in dichloro-
methane (10 mL). The reaction mixture was refluxed for
1 h and was then concentrated in vacuo. The residue
obtained was chromatographed over 75 g of silica gel
and a 1:3 mixture of ethyl acetate/hexanes to give 21
tive yield; mp 143–144 ꢂC; H NMR (CD3OD) d 2.55
1
1
(2H, t, J=7.4), 2.83 (2H, t, J=7.4), 3.82 (3H, s), 6.64-
6.71 (2H, m), 6.83 (1H, d, J=8.0); 13C NMR (CD3OD)
d 30.1, 35.7, 55.1, 111.6, 115.1, 119.1, 133.8, 146.2,
175.7; HRMS calcd for C10H12O4: 196.0736; found:
196.0734.
(183 mg) in 50% yield; H NMR d 2.56 (2H, t, J=7.0),
3.01 (2H, t, J=7.0), 3.47 (2H, s), 3.91 (3H, s), 5.14 (2H,
s), 6.67 (1H, s), 6.79 (1H, s), 7.35–7.44 (5H, m); 13C
NMR d 28.3, 38.6, 44.3, 56.3, 71.4, 111.8, 114.3, 125.2,
127.4, 128.0, 128.7, 129.3, 137.2, 147.2, 148.6, 210.9.
3-(4-Benzyloxy-3-methoxyphenyl)propionic acid (15). A
solution of 13 (1.0 g, 5.1 mmol), benzyl bromide (3.0
mL, 25.2 mmol), and potassium carbonate (2.0g, 14.5
mmol) in acetone (15 mL) and acetonitrile (15 mL) was
refluxed for 15 h. The reaction mixture was con-
centrated in vacuo. To the residue was added methanol
(3 mL), water (30 mL) and potassium hydroxide (4.0 g,
71 mmol). The mixture was heated at 70 ꢂC for 10 h.
After cooling to room temperature, the reaction mixture
was washed with chloroform (30 mLÂ3) and con-
centrated hydrochloric acid (HCl) was added dropwise
until the pH of the solution was 2.0. The mixture was
extracted with chloroform (40 mLÂ3) and the organic
layer was washed with water (20 mLÂ2) and brine (30
mL), dried (anhyd Na2SO4), and evaporated in vacuo.
The residue was triturated with hexanes several times
and 15 (1.26 g) was obtained in 86% yield as a beige
3-(4-Benzyloxy-3-methoxyphenyl)-1-diazo-butan-2-one
(19). To a solution of trimethylsilyldiazomethane (2.0 M
in hexanes, 1.5 mL), in THF and acetonitrile (1:1, 10 mL)
was added dropwise a solution of 17 (400 mg, 1.3 mmol)
in THF and acetonitrile (1:1, 10 mL) at 0 ꢂC. The reac-
tion mixture was stirred for 4 h and evaporated in vacuo.
The crude diazoketone was used for the next step without
further purification and identification.
3-(4-Benzyloxy-3-methoxyphenyl)propionic acid chloride
(17). A solution of 15 (500 mg, 1.75 mmol) in dichloro-
methane (30 mL) was treated with oxalyl chloride (2.5
mL of 2.0 M solution in dichloromethane, 5.0 mmol)
and the resulted mixture was stirred vigorously at room
temperature for 20 h. The solution turned to dark yel-
low-green. The reaction mixture was then concentrated
in vacuo and the crude acid chloride was used for the
solid; mp 94–95 ꢂC H NMR (CD3OD) d 2.57 (2H, t,
next step without further purification; H NMR d 2.97
(2H, t, J=7.9), 3.20 (2H, t, J=7.9), 3.91 (3H, s), 5.15
(2H, s), 6.65–6.86 (5H, m), 7.35–7.49 (3H, m).
1
1
J=7.0), 2.86 (2H, t, J=7.0), 3.84 (3H, s), 5.06 (2H, s),
6.72 (1H, dd, J=8.1, 1.8), 6.87 (1H, s), 6.89 (1H, d,
J=8.1), 7.29–7.46 (5H, m); 13C NMR (CD3OD) d 31.9,
37.2, 56.8, 72.8, 114.2, 116.4, 121.8, 129.0, 129.1, 129.7,
136.2, 139.2, 148.2, 151.5, 177.1; HRMS calcd for
C17H18O4: 286.1205; found: 286.1218.
6-Benzyloxy-7-methoxy-2-tetralone (22). To a refluxing
solution of rhodium (II) acetate dimer (5 mg) in di-
chloromethane (15 mL) was added dropwise to a solu-
tion of diazoketone (20) (400 mg, 1.3 mmol) in
dichloromethane (10 mL). The reaction mixture was
refluxed for 1 h and was then concentrated in vacuo.
The residue obtained was chromatographed over 75 g of
silica gel and a 1:3 mixture of ethyl acetate/hexanes to
3-(3-Benzyloxy-4-methoxyphenyl)propionic acid (16). A
solution of 14 (1.0 g, 5.1 mmol), benzyl bromide (3.0
mL, 25.2 mmol), and potassium carbonate (2.0 g, 14.5
mmol) in acetone (15 mL) and acetonitrile (15 mL) was
refluxed for 25 h. The reaction mixture was con-
centrated in vacuo. To the residue was added methanol
(3 mL), water (30 mL) and potassium hydroxide (4.0 g,
71 mmol). The mixture was heated at 70 ꢂC for 10 h.
1
give 21 (202 mg) in 55% yield; H NMR d 2.54 (2H, t,
J=7.0), 2.96 (2H, t, J=7.0), 3.53 (2H, s), 3.89 (3H, s),
5.17 (2H, s), 6.67 (1H, s), 6.79 (1H, s), 7.36–7.51 (5H,
m); 13C NMR d 28.1, 38.7, 44.4, 56.3, 71.4, 112.1, 114.1,

3804
D. Li et al. / Bioorg. Med. Chem. 11 (2003) 3795–3805
125.9, 127.4, 128.0, 128.6, 128.7, 137.2, 147.0, 148.8,
210.9; HRMS calcd for C18H18O3: 282.1256; found:
282.1263.
cells/well, in 200 mL of growth medium. Cells were
grown in suspension at 37 ꢂC in 5% CO2 and main-
tained by regular passage in RPMI medium supple-
mented with 10% heat-inactivated fetal bovine serum,
l-glutamine (2 mM), penicillin (100 U/mL), and strep-
tomycin (0.1 mg/mL). For determination of IC50, cells
were exposed continuously for 3–4 days to varying
concentrations of drug, and MTT assays were per-
formed at the end of the fourth day. Each assay was
performed with a control that did not contain any drug.
All assays were performed at least twice in six replicate
wells.
3-(3-Benzyloxy-4-methoxyphenyl)-1-diazo-butan-2-one
(20). To
a solution of trimethylsilyldiazomethane
(2.0 M in hexanes, 1.5 mL), in THF and acetonitrile
(1:1, 10 mL) was added dropwise a solution of 18 (400
mg, 1.3 mmol) in THF and acetonitrile (1:1, 10 mL) at
0 ꢂC. The reaction mixture was stirred for 4 h and
evaporated in vacuo. The crude diazoketone was used
for the next step without further purification and
characterization.
3-(4-Benzyloxy-3-methoxyphenyl)propionic acid chloride
(18). A solution of 16 (400 mg, 1.40 mmol) in dichloro-
methane (30 mL) was treated with oxalyl chloride (1.4
mL of 2.0 M solution in dichloromethane, 2.8 mmol)
and the resulting mixture was stirred vigorously at room
temperature for 20 h. The reaction mixture was then
concentrated in vacuo and the crude acid chloride was
used for the next step without further purification; H
NMR d 2.92 (2H, t, J=7.0), 3.14 (2H, t, J=7.0), 3.89 (3H,
s), 5.16 (2H, s), 6.74–6.88 (3H, m), 7.32-7.49 (5H, m).
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). We thank Mr. Nai Zhou for
performing the nucleosomal DNA fragmentation assays
as an indicator of apoptosis. 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.
1
Topoisomerase-mediated DNA cleavage assays
Human topoisomerase I was expressed in Escherichia
coli and isolated as a recombinant fusion protein using a
T7 expression system as described previously.35 DNA
topoisomerase II was purified as reported previously.36
Plasmid YepG was also purified by the alkali lysis
method followed by phenol deproteination and CsCl/
ethidium isopycnic centrifugation method as descri-
bed.37 The end-labeling of the plasmid was accom-
plished by digestion with a restriction enzyme followed
by end-filling with Klenow polymerase as previously
described.38 The cleavage assays were performed as
previously reported.39 The drug and the DNA in pre-
sence of topoisomerase I was incubated for 30 min at
37 ꢂC. After development of the gels, typically 24-h
exposure was used to obtain autoradiograms outlining
the extent of DNA fragmentation. Topoisomerase I-
mediated DNA cleavage values are reported as REC,
Relative Effective Concentration, that is, concentrations
relative to topotecan for TOP1 and VM-26 for TOP2,
whose value is arbitrarily assumed as 1.0, that are able
to produce the same cleavage on the plasmid DNA in
the presence of either TOP1 or TOP2. Relative potency
was based upon the amount of drug needed to induce
approximately 10% DNA fragmentation.
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