Synthesis and mechanism of action studies of a series of norindenoisoquinoline topoisomerase I poisons reveal an inhibitor with a flipped orientation in the ternary DNA-enzyme-inhibitor complex as determined by X-ray crystallographic analysis

Article abstract of DOI:10.1021/jm050076b

Several norindenoisoquinolines substituted with methoxy or methylenedioxy groups have been prepared and their anticancer properties evaluated in cancer cell cultures and in topoisomerase I inhibition assays. 2,3-Dimethoxy-8,9- methylenedioxy-11H-indeno[1,

Full text of DOI:10.1021/jm050076b

J. Med. Chem. 2005, 48, 4803-4814
4803
Synthesis and Mechanism of Action Studies of a Series of
Norindenoisoquinoline Topoisomerase I Poisons Reveal an Inhibitor with a
Flipped Orientation in the Ternary DNA-Enzyme-Inhibitor Complex As
Determined by X-ray Crystallographic Analysis
Alexandra Ioanoviciu,† Smitha Antony,‡ Yves Pommier,‡ Bart L. Staker,§ Lance Stewart,§ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology and the Purdue Cancer Center, School of Pharmacy and
Pharmaceutical Sciences, Purdue University, West Lafayette, Indiana 47907, Laboratory of Molecular Pharmacology, Center
for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, and
deCODE biostructures, Inc., 7869 Northeast Day Road West, Bainbridge Island, Washington 98110
Received January 27, 2005
Several norindenoisoquinolines substituted with methoxy or methylenedioxy groups have been
prepared and their anticancer properties evaluated in cancer cell cultures and in topoisomerase
I inhibition assays. 2,3-Dimethoxy-8,9-methylenedioxy-11H-indeno[1,2-c]isoquinoline hydro-
chloride (14) is a strong topoisomerase I inhibitor and also displays very high cytotoxicity in
the NCI cancer cell culture screen (mean graph midpoint of 50 nM). The X-ray crystal structure
of norindenoisoquinoline 14 in complex with topoisomerase I and DNA has been solved,
providing insight into the structure-activity relationships within this class of new anticancer
agents. The number and position of the norindenoisoquinoline substituents have a significant
influence on biological activity and demonstrate that substitution on the nitrogen atom is not
an absolute requirement for the antitumor effect of the indenoisoquinolines. Removal of the
11-keto group from the lead compound 1 and replacement of the N-alkyllactam with an
unsubstituted pyridine ring causes the indenoisoquinoline ring system to flip over in the DNA-
enzyme-inhibitor ternary complex. This allows the nitrogen atom to assume the hydrogen
bond acceptor role of the 11-keto group, resulting in hydrogen bonding to Arg364.
Introduction
lactone to a hydroxyacid that binds to plasma pro-
teins.2,3 Moreover, some cancer cells develop resistance
to camptothecin. In this context, there is a need for
chemically stable compounds that act as topoisomerase
I inhibitors and possess cytotoxicity in cancer cell lines.
The prospect of using indenoisoquinolines in cancer
therapy arose as a result of the observation that the
cytotoxicity profile of the indenoisoquinoline 1 (NSC
314622) is similar to the topoisomerase I (top1) inhibitor
camptothecin (2), as indicated by a COMPARE analy-
sis.1 This originally suggested that compound 1 might
be a topoisomerase I inhibitor, and in fact subsequent
studies demonstrated that it inhibits the topoisomerase
I-catalyzed DNA religation reaction in the DNA-
enzyme-1 ternary complex, and it displays moderate
in vitro antiproliferative activity in cancer cells.1 To-
poisomerase I inhibitors such as camptothecin (2) and
the indenoisoquinoline 1 that inhibit the DNA religation
reaction are classified as topoisomerase “poisons”, as
opposed to “suppressors”, which inhibit the enzyme-
catalyzed DNA cleavage reaction. Interest in the po-
tential clinical applications of indenoisoquinolines stems
from the fact that compounds from this class possess
the desired topoisomerase I inhibitory activity and
cancer cell cytotoxicity of camptothecin but potentially
lack some of the disadvantages inherent in campto-
thecin therapy. Although camptothecin is cytotoxic in
a wide range of animal tumors, its duration of action is
limited due to rapid reversibility of the ternary DNA-
enzyme-camptothecin complex and hydrolysis of the
5,11-Diketoindenoisoquinoline analogues of 1 bearing
various substituents on the nitrogen atom have been
previously synthesized and tested for biological activ-
ity.1,4 Also, indenoisoquinolinium salts such as 3 have
been shown to inhibit topoisomerase I and display
cytotoxicity against cancer cells in the in vitro NCI
screen and in the hollow fiber animal model.5,6 A variety
of indenoisoquinolines related to 1 and 3 display dif-
ferent DNA cleavage site specificities relative to camp-
tothecin, raising the prospect that different types of
cancers could be targeted to provide an antitumor
spectrum complementary to the existing therapeutic
agents.7,8
All of the previously studied indenoisoquinolines have
had a substituent on the nitrogen. Herein we evaluate
for the first time the antitumor properties of norinde-
noisoquinolines, which lack a substituent on the nitro-
gen atom. The present study aimed to define the role of
different aromatic ring substitution patterns and to
assess the role of N-6 substitution in the anticancer
properties and topoisomerase I inhibitory activity of
these compounds. Moreover, the indenoisoquinolines in
the present series lack a hydrogen bond-accepting C-11
carbonyl group, which bonds to the Arg364 side chain
of the enzyme.9 Another goal of the present investigation
was to determine whether this group could be deleted
* To whom correspondence should be addressed: Tel: 765-494-1465.
Fax: 765-494-6790. E-mail: cushman@pharmacy.purdue.edu.
† Purdue University.
‡ NIH, Bethesda, MD.
§ deCODE biostructures.
10.1021/jm050076b CCC: $30.25 © 2005 American Chemical Society
Published on Web 06/23/2005

4804 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Ioanoviciu et al.
Scheme 1a
a Reagents: (a) benzene/∆; (b) NaBH4, CH3CH2OH; (c) HCl.
Scheme 2a
in the norindenoisoquinoline series with retention of
activity. To pursue these aims, it was also necessary to
explore the scope and regiochemistry of a modified
Pomeranz-Fritsch strategy for the synthesis of norin-
denoisoquinolines.
Results and Discussion
Chemistry. In the literature there are isolated
reports of norindenoisoquinoline syntheses in low yields.
Compounds 13 and 14 have been prepared by the route
shown in Scheme 1.10,11
The approach investigated here leads to double-
cyclization products resulting from a bimolecular reac-
tion. The first cyclization takes place in an intramolec-
ular fashion and involves the aminoacetal (Scheme 1).
Then a bimolecular reaction with a benzaldehyde de-
rivative occurs followed by a second critical cyclization.
Compounds such as 4, which do not require a second
cyclization, have been prepared successfully using the
Bobbitt conditions.11-13 These arise from the bimolecular
reaction of the aminoacetal-derived intermediates with
benzaldehydes.
a Reagents: (a) benzene/∆; (b) NaBH4, CH3CH2OH; (c) HCl or
TFA.
Scheme 3a
There are numerous reports of intramolecular cy-
clizations of aminoacetals.14 Aminoacetals or acetals
have been widely used to build heterocycles as well as
carbocycles, and there are examples of successful double
intramolecular cyclizations leading to compounds such
as 5 or 6.15,16
The Pomeranz-Fritsch reaction17 and its newer ver-
sions18,19 are generally favored by electron-donating
substituents. The traditional Pomeranz-Fritsch ap-
proach is known to provide low yields of products and
subsequent modifications of the reaction have been
intended to address this difficulty. For example, to
improve the use of aminoacetals in the Pomeranz-
Fritsch-type reactions, a variety of acids have been
employed such as triflic, perchloric,15,16 polyphospho-
ric,20 trifluoroacetic, methanesulfonic, hydrochloric/
acetic, sulfuric,21 and hydrochloric acids.18 The reactions
for simple isoquinolines have been demonstrated to
proceed at lower temperatures as well.15,16 On the other
hand, when aminoacetals were used as starting materi-
a Reagents: (a) CHCl3, MgSO4; (b) NaBH4, CH3CH2OH; (c) HCl.
als for compounds such as the 4, which result from an
intramolecular cyclization followed by a bimolecular
reaction with a benzaldehyde, the bimolecular reactions
were favored by higher temperatures.12
In the present case, harsh conditions were used.
Boiling concentrated hydrochloric acid or trifluoroacetic
acid afforded the oxygenated norindenoisoquinolines,
which were prepared as depicted in Schemes 1-5.
Briefly, the general approach involves the condensation
of aminoacetaldehyde dimethyl acetal with a benzalde-

Norindenoisoquinoline Topoisomerase I Poisons
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4805
Scheme 4a
Scheme 6
a Reagents: (a) CHCl3, MgSO4; (b) NaBH4, CH3CH2OH; (c) HCl.
Scheme 5a
quinoline synthesis.17 The method provides a relatively
direct means of building the norindenoisoquinoline
system when electron-donating groups such as a meth-
ylenedioxy bridge or methoxy substituents are present
on both aromatic rings. It is also complementary to other
methods of indenoisoquinoline synthesis.4,22
a Reagents: (a) benzene/∆; (b) NaBH4, CH3CH2OH; (c) HCl.
hyde derivative to afford the corresponding imine.
Reduction of the Schiff base was found to proceed best
using sodium borohydride in ethanol at reflux. The
secondary amine obtained in this step was reacted with
a substituted benzaldehyde in a strongly acidic environ-
ment, either concentrated hydrochloric acid or trifluo-
roacetic acid at 100 °C, conditions that led to the
formation of tars. The target compounds were then
isolated as hygroscopic hydrochloride salts. In all cases,
only one of the theoretically possible regioisomers was
isolated, and the structures of the products were readily
apparent from the appearance of the aromatic protons
Reaction Mechanism. The proposed mechanism for
the key reaction is depicted in Scheme 6. The first
cyclization begins with the elimination of methanol from
the protonated intermediate 16, followed by the attack
of the aromatic ring on the resulting cation 37. This first
step is the same as in the Pomeranz-Fritsch isoquino-
line synthesis as well as in its improved versions such
as the Bobbitt13 or Jackson modifications.19 In the
traditional Pomeranz-Fritsch synthesis the presence
of the protonated imine group results in partial deac-
tivation of the phenyl ring,17 an effect that was overcome
in the later versions such as in the Bobbitt reaction by
using the corresponding amine, which is more stable to
hydrolysis. The formation of the first ring is favored by
electron-donating substituents para or ortho to the
position of ring closure.23
As already mentioned, methoxy groups are strongly
activating in this process. As reported previously,13,24-27
we find that both methylenedioxy or methoxy groups
cause ring closure to occur preferentially in the para
position as opposed to ortho. In all cases, during the first
cyclization, when two products are possible, only the
para cyclized isomer was isolated. Thus compounds 13,
14, 18, 19, and 20 formed.
1
as singlets in the H NMR spectra.
Compounds 13 and 14 have been synthesized previ-
ously using the route outlined in Scheme 1.10 However,
their antitumor effects had not been investigated before.
The remaining norindenoisoquinolines are novel com-
pounds. The present investigation demonstrates that
this synthetic approach is versatile and can be extended
to obtain a wide variety of analogues possessing oxygen-
ated substituents. This route provides rapid access to
the synthetic targets and requires the use of starting
materials that are either commercially available or can
be obtained quantitatively from commercially available
substances. The overall yields for the synthesis of
norindenoisoquinolines are determined by the last step,
which is a modification of the Pomeranz-Fritsch iso-

4806 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Ioanoviciu et al.
Table 1. Cytotoxicities and Topoisomerase I Inhibitory Activities of Indenoisoquinoline Analogues
cytotoxicity (GI50 in µM)a
lung
HOP-62
colon
HCT-116
CNS
SF-539
melanoma
UACC-62
ovarian
OVCAR-3
renal
SN12C
prostate
DU-145
breast
MDA-MB-435
Top 1
compd
MGMb
cleavagec
1
1.30
0.01
0.0721
NTd
35
41
4.2
73
68
37
96
20.0
0.0405
0.0505
44.7
33.9
0.272
20.9
19.1
2.01
24
18.6
24.5
13.2
26.3
26.3
++
++++
+++
++
+++
++
0
2
0.03
>100
>50
11.1
0.283
18.8
31.8
0.0694
33.5
17.8
0.249
8.81
22.1
27.8
0.01
<0.01
>50
23.4
0.598
NTd
14.0
0.869
NTd
14.0
NTd
NTd
NTd
NTd
0.01
<0.01
>50
0.22
NTd
>50
61.2
0.0232
15.5
15.9
0.403
19.1
15.4
26.9
0.529
NTd
21.5
0.02
>100
>50
0.01
0.0281
>50
25.3
NTd
23.2
21.7
>100
26.6
28.1
48.4
NTd
26.9
29.6
0.04
<0.01
>50
14
13
18
19
20
23
24
25
29
30
34
35
36
NTd
>100
0.190
19.3
7.42
0.439
17.4
18.0
>100
3.84
22.4
21.1
>100
0.0414
13.4
14.4
0.602
17.6
14.8
>100
NTd
NTd
63.4
<0.01
13.2
16.2
0.178
20.8
23.4
>100
NTd
>100
23.0
0.227
21.7
34.8
0.234
35.4
21.4
<0.01
16.0
NTd
+
++
0
0
++
++
++
++
28.5
20.3
a The cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition. b Mean graph midpoint for growth inhibition
of all human cancer cell lines successfully tested. c The compounds were tested at concentrations ranging up to 10 µM. The activity of the
compounds to produce top 1-mediated DNA cleavage was expressed semiquantitatively as follows: 0: no activity; +: weak activity; ++:
similar activity as the parent compound 2; +++ and ++++: greater activity than the parent compound 2; ++++: similar activity as 1
µM camptothecin. d NT ) not tested.
The rearomatization of the phenyl ring then leads to
intermediate 39. Although intermediate 39 is shown as
a 4-methoxy-1,2,3,4-tetrahydroisoquinoline, it is likely
that the corresponding 4-hydroxy tetrahydroisoquino-
line is present as well. Such hydroxyl derivatives are
unstable products that have been isolated before when
the reaction was run at low (room) temperature.28 Loss
of methanol from intermediate 39 results in the forma-
tion of the 1,2-dihydroisoquinoline 40, an unstable
compound that is considered to be an intermediate in
the modified Pomeranz-Fritsch reactions.12,13,19 The
first cyclization does not depend on the presence of the
nitrogen atom in the 2 position. Precedents exist when
the nitrogen atom was either acylated,21 tosylated (the
Jackson modification),19 or altogether absent from the
molecule,16 and yet the first step proceeded successfully
for phenyl rings activated by electron-donating substit-
uents.
Scheme 7
It is in the next step leading to intermediate 41 that
the presence of the nitrogen is essential. The lone
electron pair on the nitrogen atom facilitates the nu-
cleophilic attack on the protonated benzaldehyde de-
rivative. The protonation of the substituted benzalde-
hyde also further deactivates the aromatic ring and
prevents its attack by electrophiles, thus minimizing
competing reactions at this stage.
Subsequent attack of the iminium ion 41 by the
second aromatic ring leads to the formation of the
second ring, as previously hypothesized.29 Due to their
electrophilic character, iminium ions similar to 41 were
considered to be intermediates in other reactions.30 It
can be inferred that activation of the second aromatic
ring from the substituted benzaldehyde is important,
and the present results demonstrate that methoxy and
methylenedioxy groups have a para-directing effect.
Again, whenever there is a choice between ortho or para
ring closure, the second cyclization affords para-cyclized
products, namely 13, 14, 18, 19, 23, 24, 22, 29, 30, 34,
and 35. The rearomatization of 42 to 43 is then followed
by dehydration and dehydrogenation to afford the final
norindenoisoquinoline.
44 and the second cyclization could occur then as the
benzylic hydroxyl is lost in the acidic medium10 to form
intermediate 45, which can be rearomatized to 46 and
dehydrogenated to the norindenoisoquinoline 19.10
Biological Results. All norindenoisoquinolines were
tested at the National Cancer Institute for their ability
to inhibit the proliferation of approximately 55 human
cancer cell lines. The GI50 values for several selected
cell lines and the mean graph midpoints (MGM) are
presented in Table 1. The MGM is based on a calculation
of the average GI50 for all of the cell lines tested
(approximately 55) in which GI50 values below and
above the test range (10-8 to 10-4 molar) are taken as
the minimum (10-8 molar) and maximum (10-4 molar)
drug concentrations used in the screening test. There-
fore, the MGM value represents an overall assessment
of toxicity of the compound across numerous cell lines.
The results of topoisomerase I DNA cleavage experi-
ments are expressed semiquantitatively and provide a
means of comparison with the biological activity of
A secondary mechanism that may also be involved is
shown in Scheme 7. Intermediate 41 may isomerize to

Norindenoisoquinoline Topoisomerase I Poisons
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4807
Figure 1. Comparison of the topoisomerase I-mediated DNA
cleavage products of pBluescript SK(-) phagemid DNA at
various drug concentrations as indicated. The assays were
carried out at room temperature for 30 min and stopped by
adding 0.5% SDS. The resulting DNA fragments were sepa-
rated using 16% denaturing polyacrylamide gel electrophore-
sis. Topoisomerase I was present in all reaction mixtures
except for the one corresponding to the first lane.
Figure 2. Top: Electron density (|Fo| - |Fc| at 3.1 Å,
contoured at 2σ) showing the initial unbiased experimental
density used for the placement of compound 14 into the
topo70-DNA covalent complex crystal structure. Bottom:
Diagram of the topo70-DNA complex bound with indenoiso-
quinoline 14. The topoisomerase I inhibitor 14 is displayed as
a space-filling model (CPK). The N-terminal fragment is
colored in cyan, the core subdomain I yellow, subdomain II
blue, subdomain III red, the linker is orange and the C-
terminus is magenta. The oligonucleotide is represented as
capped sticks and is colored according to atom type.
camptothecin (2) (++++) and with the parent com-
pound 1 (++).
The cytotoxicities of most of the compounds were in
the micromolar range. Compound 14 was quite active
with an average MGM of 50 nM. The norindenoiso-
quinoline 14 is also a good topoisomerase I inhibitor
(+++). Interestingly, compound 18, in which the loca-
tions of the di(methoxy) and methylenedioxy substitu-
ents are exchanged relative to 14, inhibits topoisomer-
ase I to the same extent as norindenoisoquinoline 14
(Figure 1). However, the cytotoxicity of 18 (33.9 µM) is
much lower than that of 14. This suggests either that
compound 18 does not reach its biological target as
efficiently as 14 in intact cells, or that compound 14 may
have an alternate cellular target. Differences in me-
tabolism could also possibly explain the observed re-
sults.
Replacement of the methylenedioxy substituent in
positions 8 and 9 of 14 with two methoxy groups
drastically reduced biological activity. Compound 13 has
a low cytotoxicity (44.7 µM) and it inhibits topoisomer-
ase I moderately (++).
Compound 19 displays submicromolar cytotoxicity,
yet it appears to be only a moderately active topoisom-
erase I poison. Most of the remaining norindenoiso-
quinolines had moderate cytotoxicities ranging from
13.2 µM to 26.3 µM, with the exception of compound
24, which had an MGM in the low micromolar range
(2.0 µM).
All of the compounds were examined for induction of
DNA cleavage in the 3′-end-labeled PvuII/HindIII frag-
ment of pBluescript SK(-) phagemid DNA in the
presence of top1.1 The cleavage patterns for the norin-
denoisoquinolines 13, 14, and 18 are displayed in Figure
1, along with those of the lead indenoisoquinoline 1
(NSC 314622), indenoisoquinoline 494,8 (MJ-III-65) with
enhanced potency relative to 1, and camptothecin (2,
CPT). Some, but not all, of the DNA cleavage sites
observed with the norindenoisoquinolines were different
from each other, and there were also some differences
relative to the indenoisoquinolines and camptothecin.
For example, the camptothecin band at site 37 was not
seen with the indenoisoquinolines or with the norinde-
noisoquinolines in Figure 1, and the band at site 44 was
observed with the indenoisoquinolines and norinde-

4808 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Ioanoviciu et al.
Figure 3. Crystal structure of norindenoisoquinoline 14 in complex with topoisomerase I and DNA.
noisoquinolines but not with camptothecin. Some dif-
ferences are also apparent in comparison of the norin-
denoisoquinolines vs the indenoisoquinolines. These
differences are important because they indicate that
different cancer cell genes could be targeted more
selectively with one type of inhibitor vs another. Also,
similar to the situation with other anticancer drugs that
share a single target (e.g. top2 and tubulin inhibitors),
it can be expected that different top1 inhibitors will have
different spectra of antitumor activity.31 Similar conclu-
sions have been reached in prior top1-DNA cleavage
studies involving other indenoisoquinolines.4-6,19,32
Crystal Structure and Molecular Modeling. Norin-
denoisoquinoline 14, as the biological assays indicate,
is an efficient topoisomerase I poison. Its mechanism
of action, involving inhibition of the DNA religation
reaction in the ternary complex, is similar to that
proposed for topotecan.33 The crystal structure of the
ternary cleavage complex involving 14 was determined
and the resulting electron density omit map for the drug
molecule 14 is shown in Figure 2 (top). The asymmetry
of the electron density makes a clear argument for the
correct placement of the indenoisoquinoline 14 in the
ternary complex (Figure 2, bottom). As shown in Figure
3, the structure reveals that the norindenoisoquinoline
inserts between the DNA base pairs in the ternary
cleavage complex at the cleavage site. Stacking interac-
tions as well as two direct hydrogen bonds to enzyme
residues Arg364 and Asn722 then stabilize the ternary
complex. These interactions may help to explain why
norindenoisoquinoline 14 is a stronger topoisomerase I
poison than 47, which uses the oxygen atom from the
Figure 4. Overlay of the norindenoisoquinoline 14 (white
carbon atoms) and the 5,11-diketoindenoisoquinoline 47 (green
carbon atoms) in their ternary cleavage complexes as deter-
mined by X-ray crystallography.
The crystal structure of the camptothecin (2) ternary
complex shows that Arg364 participates in hydrogen
bonding to the N1 of camptothecin.9 Camptothecin also
forms two hydrogen bonds to topoisomerase I while
intercalating between the DNA bases pairs.9 The cyto-
toxicities of camptothecin and norindenoisoquinoline 14
are in the same range (40.5 nM vs 50 nM), although
the topoisomerase I DNA cleavage assays suggest that
norindenoisoquinoline 14 is a weaker topoisomerase I
inhibitor than camptothecin.
As compared to topotecan (48), norindenoisoquinoline
14 has a larger number of direct contacts with the
topoisomerase I residues. Topotecan has only one direct
hydrogen bond to the enzyme, between the C20 hydroxyl
and the carboxylate oxygen of Asp533.9 Residue Asp533
is in fact located in the immediate vicinity of Arg364,
to which it hydrogen bonds. Therefore, both norinde-
noisoquinoline 14 and topotecan (48) have a point of
attachment to the enzyme in the minor groove region
of the DNA. The hydrolyzed, hydroxyacid form of
topotecan also participates in water-mediated hydrogen
bonding with residues Asn722 and Tyr723.
C11 carbonyl to hydrogen bond to residue Arg364, but
lacks a second hydrogen bond to the protein.9 The
overlay of these two ligands in their respective ternary
complex crystal structures (Figure 4) indicates that in
fact the heterocyclic nitrogen atom of the norindenoiso-
quinoline 14 is placed in the same position as the C-11
oxygen atom of the ketone carbonyl group of 47, so that
14 is flipped over relative to 47 in the cleavage complex.
This positioning implies that the nitrogen atom of 14 is
not protonated and is free to use its electon pair to
stabilize the interaction with topoisomerase I. At neu-
tral pH, norindenoisoquinoline 14 exists as the free
base.
In the case of norindenoisoquinoline 14, there is a
direct interaction between one methoxy oxygen atom
and the side chain amide nitrogen of Asn722, which is

Norindenoisoquinoline Topoisomerase I Poisons
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4809
Figure 5. Overlay of Asn722 from the topotecan ternary complex (Asn722 carbon atoms magenta) with the norindenoisoquinoline
14 cleavage complex (Asn722 colored by atom type). The figure documents the movement of Asn722 toward 14 in its cleavage
complex relative to the position of Asn722 in the topotecan complex. The indenoisoquinoline 14 is displayed in green. The figure
is programmed for wall-eyed (relaxed) viewing.
placed in the region between the two methoxy substit-
uents. The methyl groups are pointed away from
Asn722, and this key feature allows the hydrogen bond
to form. Figure 5 displays the region around the
norindenoisoquinoline 14 in its crystal structure, and
Asn722 from the topotecan complex structure is over-
layed and shown in magenta. In the case of norindeno-
isoquinoline 14, Asn722 moves toward the ligand and
attains the correct conformation to participate in hy-
drogen bonding. It is also interesting that the carbon
atoms of the two methoxy substituents on the norinde-
noisoquinoline lie in the same plane as the heterocycle.
This is not in fact the lowest energy conformation for
the isolated ligand. Most likely the methoxys become
coplanar with the aromatic rings of the norindenoiso-
quinoline as a result of steric interactions with the DNA
base pairs above and below. Computer modeling in
Sybyl of the other norindenoisoquinolines has indicated
that when the ligand is prepositioned in the binding site
according to the information provided by the crystal
structure, during energy minimization there is a ten-
dency to bring methoxy substituents in the plane of the
norindenoisoquinoline heterocycle whenever possible.
The crystal structure of norindenoisoquinoline 14
suggests that stacking interactions are crucial for ef-
ficient bonding to the covalent DNA topoisomerase I
cleavage complex. The biological assays reveal that the
larger substituents around the indenoisoquinoline
nucleus are not well tolerated and result in decreased
biological activity. The methoxy groups in the structures
of most norindenoisoquinoline have the potential to act
as hydrogen bond acceptors. A larger number of bulky
substituents, however, especially the presence of three
adjacent methoxy groups, is difficult to accommodate
in the cleavage complex. As the van der Waals surface
of the energy minimized inactive compound 25 in Figure
6 indicates, repulsive interactions among the three
substituents on the same phenyl ring result in some
being placed above the plane of the heterocycle while
some are located below. The steric clashes among the
adjacent methoxy groups confer increased bulk to the
molecule, which in turn is expected to disrupt stacking
with the DNA base pairs in the cleavage complex and
is likely the cause of its poor activity.
Figure 6. Top: norindenoisoquinoline 25 is displayed accord-
ing to atom types in a capped sticks model. Bottom: the
translucent van der Waals surface of norindenoisoquinoline
25 is displayed in green.
the phosphodiester backbone. Comparison of the crystal
structures of ternary complexes derived from a variety
of topoisomerase I inhibitors has indicated a very snug
fit on the side of the binding pocket next to the
nonscissile DNA strand.9 This fact is consistent with the
inactivity of the more bulky trimethoxylated compounds
20 and 25 as topoisomerase I inhibitors.
Last, a methoxy substituent in position 7 has a
shielding effect on the nitrogen atom and interferes with
the hydrogen bond formation between the isoquinoline
heterocyclic nitrogen and Arg364. The methoxy sub-
stituent could be considered as a hydrogen bond accep-
tor, yet it is itself prevented from forming a hydrogen
bond to Arg364 due to repulsive van der Waals interac-
tions, which are calculated to orient it with the methyl
protruding toward the arginine ꢀ-nitrogen. The resulting
effect of the six methoxy substituents in the case of
compound 25 is that during energy minimization the
ligand is moved away from Arg364 by about 1 Å.
Interference of the 7-methoxy group with hydrogen
bonding to Arg364 may also contribute to the inactivity
of 20 as a topoisomerase I inhibitor.
Steric bulk in the ligand is especially poorly tolerated
in the vicinity of the nonscissile phosphodiester bonds.
Replacement of the methylenedioxy bridge in 14 with
two methoxy groups in compound 13 leads to diminished
biological activity, most likely due to unfavorable van
der Waals contacts between the additional methoxy and
Norindenoisoquinoline 19 constitutes a particular
case, since it is the least voluminous of all of the
compounds reported here. Although it is quite cytotoxic,
it is a moderately potent inhibitor of topoisomerase I.
Figure 7 depicts the electrostatic potential surfaces
of indenoisoquinolines 1, 3 and 14 calculated using

4810 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Ioanoviciu et al.
the 11-ketone oxygen. These features allow norinde-
noisoquinolines to interact differently with topoisom-
erase I than 5,11-diketoindenoisoquinolines such as 1
and 47. For this reason, the structure-activity relation-
ships derived for this series are not expected to correlate
exactly with the other subclasses of indenoisoquinolines.
Conclusion
The modified Pomeranz-Fritsch strategy is generally
applicable to the synthesis of norindenoisoquinolines
and affords the desired products in yields ranging from
5% to 52%. The methoxy or methylenedioxy groups
display a para-directing effect in both cyclizations
involved in the formation of the indenoisoquinoline
system. Only para-substituted products were isolated
whenever both ortho and para positions to the activating
substituent were free. This method allows the rapid
assembly of norindenoisoquinolines that would be dif-
ficult to synthesize otherwise.
Structural requirements for antitumor properties in
the norindenoisoquinoline series are very strict. Sub-
stitution with two methoxy groups in positions 2 and 3
and a methylenedioxy bridge in positions 8 and 9 confers
the highest anticancer activity. Compound 14 is in fact
more cytotoxic than the alkyl-substituted indenoiso-
quinolium salts5,6 and it also compares favorably with
many compounds in the 5,11-diketoindenoisoquinoline
series.1,4,5 From this evidence, it can be concluded that
a substituent on the nitrogen atom is not essential for
biological activity. However, the presence of such a
substituent atom is likely to alter the binding mode of
the molecule to its cellular targets. It is also apparent
that the 11-keto group is not essential for maximum
activity, since the nitrogen atom in the norindenoiso-
quinolines can play the role of the 11-keto oxygen atom
of the 5,11-diketo-N-alkylindenoisoquinolines when bound
in the ternary cleavage complex. This fact is demon-
strated in the “flipped over” orientations of the two
systems in the cleavage complex as shown in Figure 4.
The inactivities of the trimethoxylated indenoisoquino-
lines 20 and 25 as topoisomerase I inhibitors can be
explained as the result of (1) steric interference with
the requirement for planarity in the intercalation site;
(2) lack of sufficient space next to the nonscissile DNA
strand to accommodate increased steric bulk, and (3)
interference of the C-7 methoxy group with hydrogen
bonding between the 11-ketone and the side chain of
Arg364.
Figure 7. Electrostatic potential surfaces for 14 (top), 1
(middle), and 3 (lower) calculated using MMFF94 charges.
MMFF94 charges. Red contours correspond to regions
that are relatively electron-rich, whereas blue areas
indicate relatively electron-poor regions within each
molecule. There are obvious differences in the electron
distributions of these three compounds in the regions
containing the nitrogen atom and the C-11 carbon atom.
As compared to the indenoisoquinolinium salts, the
nitrogen atom in the norindenoisoquinolines is not
expected to be charged at physiological pH. A neutral
norindenoisoquinoline uses the nonprotonated nitrogen
as a hydrogen bond acceptor from Arg364. The electron
density on the nitrogen atom in the case of norinde-
noisoquinolines is higher than in the 5,11-diketoinde-
noisoquinolines, like 1, where the nitrogen is part of a
lactam moiety. Also the nitrogen atom, being unsubsti-
tuted in norindenoisoquinolines, is not sterically hin-
dered from participating in bonding interactions with
biological macromolecules, resulting in the “flipped”
orientation of 14 in the cleavage complex relative to 47
(Figure 4).9 It is clear from Figure 7 (top) that the region
of high electron density in 14 corresponds to the
nitrogen, while in the lead compound 1 (middle) it is
Experimental Section
Melting points were determined in capillary tubes and are
uncorrected. Except where noted, 1H NMR spectra were
obtained using CDCl3 as solvent and the solvent peak as
1
internal standard. H NMR spectra were determined at 300
MHz. Electrospray mass spectra were obtained using a Finni-
ganMATT LCQ (Thermoquest Corp., San Jose, CA) instrument
at the Purdue Campus-Wide Mass Spectrometry Center.
Microanalyses were performed at the Purdue University
Microanalysis Laboratory. Analytical thin-layer chromatog-
raphy was carried out on Baker-Flex silica gel IB2-F flexible
sheets. Compounds were visualized with short wavelength UV
light.
(3,4-Dimethoxybenzylidene)-(2,2-dimethoxyethyl)-
amine (9). 3,4-Dimethoxybenzaldehyde (7, 12.17 g, 0.07323
mol) was dissolved in benzene, (200 mL) and aminoacetalde-
hydedimethylacetal (8, 12.0 mL, 11.7 g, 0.111 mol) was added.

Norindenoisoquinoline Topoisomerase I Poisons
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4811
The mixture was stirred at reflux for 4 h using a Dean-Stark
trap. The reaction mixture was then concentrated and dis-
solved in chloroform (250 mL), the solution was washed with
water (4 × 150 mL) and brine (150 mL), dried (Na2SO4), and
concentrated, and the last traces of solvent were removed
under vacuum to provide the imine as a light yellow solid
8,9-Dimethoxy-2,3-methylenedioxy-11H-indeno[1,2-c]-
isoquinoline Hydrochloride (18). Compound 18 was ob-
tained from the amine 16 (1.79 g, 7.49 mmol) and veratral-
dehyde (11, 3.04 g, 0.018 mol) following the same method as
for 13. The yellow precipitate that formed (0.7649 g, 29%) was
collected by filtration. An analytical sample was prepared by
recrystallizing 0.3655 g from methanol (50 mL) to form a
hygroscopic solid: mp 257-259 °C. 1H NMR (300 MHz, TFA)
δ 8.88 (s, 1 H), 7.69 (s, 1 H), 7.39 (s, 1 H), 7.33 (s, 2 H), 6.21
(s, 2 H), 4.18 (s, 2 H), 4.02 (s, 3 H), 4.01 (s, 3 H); low resolution
ESIMS m/z (rel intensity) 322 (MH+, 100); high-resolution
ESIMS m/z (rel intensity) 322.1084 (MH+, 100) (calculated
mass 322.1079). Anal. (C19H16NO4Cl‚0.7H2O) C, H, N.
1
(18.55 g, 100%): mp 51-52 °C. H NMR (300 MHz, CDCl3) δ
8.09 (s, 1 H), 7.33 (d, J ) 1.8 Hz, 1 H), 7.07 (dd, J1 ) 1.8 Hz,
J2 ) 8.3 Hz, 1 H), 6.77 (d, J ) 8.1, 1 H), 4.57 (t, J ) 5.1 Hz, 1
H), 3.83 (s, 3 H), 3.80 (s, 3 H), 3.65 (dd, J1 ) 5.1 Hz, J2) 1.2
Hz, 2 H), 3.32 (s, 6 H); low resolution ESIMS m/z (rel intensity)
254 (MH+, 66), 222 (100). Anal. (C13H19NO4‚0.1CHCl3) C, H,
N.
(3,4-Dimethoxybenzyl)-(2,2-dimethoxyethyl)amine (10).
The imine 9 (13.90 g, 0.05488 mol) was dissolved in ethanol
(50 mL), and NaBH4 (4.20 g, 0.111 mol) was added over 1 h
while the reaction mixture was stirred at reflux. The reaction
mixture was diluted with water (250 mL). The amine was
extracted into chloroform (300 mL) and the extract washed
with water (2 × 200 mL) and brine (200 mL), dried (Na2SO4),
and concentrated to provide a clear colorless oil (12.88 g,
2,3-Methylenedioxy-7,8-methylenedioxy-11H-indeno-
[1,2-c]isoquinoline Hydrochloride (19). The amine 16
(6.22, 0.0224 mol) and piperonal (12, 10.10 g, 0.06728 mol)
were reacted as described previously for compound 13, but
trifluoroacetic acid was used in place of concentrated hydro-
chloric acid. The pure base (1.501 g, 22%) was obtained. An
analytical sample (0.75 g) was purified further by dissolving
the sample in trifluoroacetic acid (50 mL) and adding hydro-
chloric acid (2 M in diethyl ether, 30 mL). The hydrochloride
salt was then washed with ether (200 mL) and recrystallized
from methanol (75 mL) to obtain a green-yellow hygroscopic
1
91.7%): bp 148 °C (0.35 mmHg). H NMR (300 MHz, CDCl3)
δ 6.81 (s, 1 H), 6.78 (d, J ) 8.1 Hz, 1 H), 6.74 (d, J ) 8.1, 1 H),
4.41 (t, J ) 5.4 Hz, 1 H), 3.81 (s, 3 H), 3.79 (s, 3 H), 3.67 (s, 2
H), 3.29 (s, 6 H), 2.67 (d, J ) 5.4 Hz, 2 H); low resolution
ESIMS m/z (rel intensity) 256 (MH+, 34), 151 (100). Anal.
(C13H21NO4) C, H, N.
1
solid: mp 237-239 °C. H NMR (300 MHz, TFA) δ 8.74 (s, 1
H), 7.51 (s, 1 H), 7.35 (s, 1 H), 7.31 (s, 1 H), 7.16 (s, 1 H), 6.20
(s, 2 H), 6.05 (s, 2 H), 4.12 (s, 3 H); low resolution ESIMS m/z
(rel intensity) 306 (MH+, 100). Anal. (C18H12NO4Cl‚0.85H2O)
C, H, N.
2,3,8,9-Tetramethoxy-11H-indeno[1,2-c]isoquinoline Hy-
drochloride (13). The amine 10 (2.93 g, 0.0115 mol) and
veratraldehyde (4.11 g, 0.0247 mol) were mixed with concen-
trated HCl and stirred at 100 °C for 3 h. The reaction mixture
was then cooled and washed with ether (50 mL × 3). Then it
was brought to a basic pH with NH4OH. The mixture was
extracted with chloroform (4 × 50 mL), and the solution was
washed with water (100 mL), dried (Na2SO4), and concen-
trated. The residue was dissolved in chloroform (200 mL). The
mixture was filtered, and HCl (2 M in ether, 40 mL) was added
to the filtrate. The precipitate that formed was recrystallyzed
from methanol (300 mL) to provide a bright yellow hygroscopic
solid (0.5938 g, 14%): mp 254-255 °C. 1H NMR (300 MHz,
TFA) δ 9.09 (s, 1 H), 7.77 (s, 1 H), 7.62 (s, 1 H), 7.47 (s, 2 H),
7.40 (s, 1 H), 4.30 (s, 2 H), 4.25 (s, 3 H), 4.18 (s, 3 H), 4.09 (s,
3 H), 4.08 (s, 3 H); low resolution ESIMS m/z (rel intensity)
338 (100, MH+). Anal. (C20H20NO4Cl‚1.85H2O) C, H, N.
2,3-Dimethoxy-8,9-methylenedioxy-11H-indeno[1,2-c]-
isoquinoline Hydrochloride (14). The amine 10 (3.26 g,
0.0128 mol) was reacted with piperonal (12, 4.0 g, 0.0267 mol)
following a procedure similar to the one described above for
compound 13. Compound 14 was obtained as a yellow hygro-
scopic precipitate (2.37 g, 52%): mp 238-240 °C. 1H NMR (300
MHz, D2O) δ 8.30 (s, 1 H), 6.91 (s, 1 H), 6.69 (s, 1 H), 6.44 (s,
1 H), 6.39 (s, 1 H), 5.94 (s, 2 H), 3.78 (s, 3 H), 3.77 (s, 3 H),
3.00 (s, 2 H); low resolution ESIMS m/z (rel intensity) 322
(MH+, 100). Anal. (C19H16NO4Cl‚0.85H2O) C, H, N.
(3,4-Methylenedioxybenzylidene)-(2,2-dimethoxyeth-
yl)amine (15). Piperonal (12, 15.19 g, 0.1001 mol) was reacted
in benzene (200 mL) with aminoacetaldehydedimethylacetal
(8, 15.0 mL, 14.6 g, 0.139 mol) according to the procedure
described for compound 9. The imine was isolated as a clear
colorless oil (23.71 g, 100%): bp 147 °C (0.4 mmHg). 1H NMR
(300 MHz, CDCl3) δ 8.13 (s, 1 H), 7.33 (s, 1 H), 7.07 (d, J )
8.10 Hz, 1 H), 6.78 (d, J ) 8.10 Hz, 1 H), 5.96 (s, 2 H), 4.62 (t,
J ) 5.4 Hz, 1 H), 3.70 (d, J ) 4.2 Hz, 2 H), 3.37 (s, 6 H); low
resolution ESIMS m/z (rel intensity) 238 (MH+, 100), 206 (98).
Anal. (C12H15NO4) C, H, N.
(3,4-Methylenedioxybenzyl)-(2,2-dimethoxyethyl)-
amine (16). The imine 15 (15.97 g, 0.06731 mol) was dissolved
in ethanol (150 mL) and reduced with NaBH4 (4.12 g, 0.109
mol) as described previously for the amine 10. The amine was
obtained as a clear colorless oil (15.82 g, 99%): bp 142-143
°C (0.75 mmHg). 1H NMR (300 MHz, CDCl3) δ 6.79-6.69 (m,
3 H), 5.86 (s, 2 H), 4.41 (t, J ) 5.4 Hz, 1 H), 3.64 (s, 2 H), 3.30
(s, 6 H), 2.65 (d, J ) 5.4 Hz, 2 H); low resolution ESIMS m/z
(rel intensity) 240 (MH+, 100). Anal. (C12H17NO4) C, H, N.
7,8,9-Trimethoxy-2,3-methylenedioxy-11H-indeno[1,2-
c]isoquinoline Hydrochloride (20). The amine 16 (2.28 g,
9.52 mmol) and 3,4,5-trimethoxybenzaldehyde (17, 3.77 g, 19.2
mmol) were reacted in trifluoracetic acid (25 mL) with stirring
at 100 °C for 3 h. The reaction mixture was cooled and
hydrochloric acid (2 M in ether, 10 mL) and ether (200 mL)
were added. The precipitate was isolated and dissolved in
methanol-water-acetone 1:1:1 (250 mL) and NH4OH was used
to bring the mixture to a basic pH. The mixture was extracted
with chloroform (350 mL). The organic layer was washed with
brine (200 mL), dried (Na2SO4), and concentrated. The residue
was dissolved in chloroform (60 mL) and hydrochloric acid (2
M in dry ether, 10 mL) was added. The precipitate was
collected and washed with hot methanol (10 mL) and ether
20 (mL) to provide a yellow hygroscopic solid (1.65 g, 45%):
1
mp 257-259 °C. H NMR (300 MHz, CD3OD) δ 9.04 (s, 1 H),
7.63 (s, 1 H), 7.51 (s, 1 H), 7.18 (s, 1 H), 6.32 (s, 2 H), 4.28 (3
H), 4.25 (2 H), 3.98 (3 H), 3.90 (s, 3 H); low resolution ESIMS
m/z (rel intensity) 352 (MH+, 100). Anal. (C20H18NO5Cl‚1.9H2O)
C, N, H.
(3,4,5-Trimethoxybenzylidene)-(2,2-dimethoxyethyl)-
amine (21). Aminoacetaldehydedimethylacetal (8, 20.0 mL,
19.5 g, 0.185 mol) was dissolved in chloroform (80 mL), and
MgSO4 (15 g) was added. Then 3,4,5-trimethoxybenzaldehyde
(17, 10.03 g, 0.05112 mol) was also added, and the mixture
was stirred at room temperature for 24 h. The reaction mixture
was diluted with chloroform (70 mL), washed with water (4 ×
150 mL) and brine (150 mL), dried (Na2SO4), and concentrated
to provide the pure imine as a light yellow oil (14.47 g, 100%):
1
bp > 180 °C (0.4 mmHg). H NMR (300 MHz, CDCl3) δ 8.10
(s, 1 H), 6.91 (s, 2 H), 4.58 (t, J ) 5.4 Hz, 1 H), 3.81 (s, 6 H),
3.79 (s, 3 H), 3.69 (d, J1 ) 5.4 Hz, J2 ) 1.2 Hz, 2 H), 3.33 (s,
6 H); low resolution ESIMS m/z (rel intensity) 284 (MH+, 100).
Anal. (C14H21NO5‚0.35 CHCl3)
(3,4,5-Trimethoxybenzyl)-(2,2-dimethoxyethyl)-
amine (22). The imine 21 (11.59 g, 0.04090 mol) was reduced
in ethanol (150 mL) with NaBH4 (2.50 g, 0.0661 mol) following
the previous procedure (compound 10). The amine was isolated
as a clear light yellow oil (11.66 g, 100%): bp 175-176 °C (1.4
1
mmHg). H NMR (300 MHz, CDCl3) δ 6.47 (s, 2 H), 4.39 (t,
J ) 5.4 Hz, 1 H), 3.76 (s, 6 H), 3.73 (s, 3 H), 3.65 (s, 2 H), 3.28
(s, 6 H), 2.65 (d, J ) 5.4 Hz, 2 H); low resolution ESIMS m/z
(rel intensity) 286 (MH+, 79), 181 (100). Anal. Calcd for C14H23-
NO5: C, 58.93; H, 8.12; N, 4.91. Found: C, 58.66; H, 8.17; N,
4.63.

4812 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
Ioanoviciu et al.
1
2,3,4,7,8-Pentamethoxy-11H-indeno[1,2-c]isoquino-
line Hydrochloride (23). The amine 22 (3.56 g, 0.0125 mol)
and veratraldehyde (11, 4.00 g, 0.0241 mol) were reacted in
concentrated hydrochloric acid (25 mL) as described previously
for compound 13. The product was isolated as a yellow
hygroscopic solid (0.75 g, 15%): mp 218-220 °C (dec). 1H NMR
(300 MHz, CD3OD) δ 9.24 (s, 1 H), 7.63 (s, 1 H), 7.57 (s, 1 H),
7.34 (s, 1 H), 4.36 (s, 2 H), 4.16 (s, 3 H), 4.12 (s, 3 H), 4.08 (s,
3 H), 3.95 (s, 3 H), 3.94 (s, 3 H); low resolution ESIMS m/z
(rel intensity) 368 (MH+, 100). Anal. (C21H22NO5Cl‚1.6H2O) C,
H, N.
2,3,4-Trimethoxy-7,8-methylenedioxy-11H-indeno[1,2-
c]isoquinoline Hydrochloride (24). The amine 22 (2.59 g,
9.08 mmol) and piperonal (12, 3.50 g, 0.023 mol) were reacted
in concentrated hydrochloric acid as described previously for
compound 13. The product was isolated as a yellow solid (0.62
g, 18%). Reprecipitation of the sample (68.7 mg) from methanol
(50 mL) afforded analytically pure material as a hygroscopic
solid: mp 230-232 °C (dec). 1H NMR (300 MHz, CD3OD) δ
9.29 (s, 1 H), 7.62 (s, 1 H), 7.53 (s, 1 H), 7.26 (s, 1 H), 6.11 (s,
2 H), 4.41 (s, 2 H), 4.16 (s, 3 H), 4.11 (s, 3 H), 4.08 (s, 3 H); low
resolution ESIMS m/z (rel intensity) 352 (MH+, 100). Anal.
(C20H18NO5Cl‚0.45H2O) C, H, N.
2,3,4,7,8,9-Hexamethoxy-11H-indeno[1,2-c]isoquino-
line Hydrochloride (25). The amine 22 (2.62 g, 9.19 mmol)
and 3,4,5-trimethoxybenzaldehyde (17, 3.70 g, 18.9 mmol) were
reacted in concentrated hydrochloric acid as described previ-
ously for compound 13. The product was isolated as a yellow
hygroscopic solid (1.612 g, 40%): mp 197-199 °C (dec). 1H
NMR (300 MHz, CD3OD) δ 9.20 (s, 1 H), 7.69 (s, 1 H), 7.22 (s,
1 H), 4.51 (s, 2 H), 4.29 (s, 3 H), 4.17 (s, 3 H), 4.12 (s, 3 H),
4.08 (s, 3 H), 3.99 (s, 3 H), 3.90 (s, 3 H); low resolution ESIMS
m/z (rel intensity) 398 (MH+, 100). Anal. (C22H24NO6Cl‚
1.95H2O) C, H, N.
scopic solid (1.6081 g, 52%): mp 245-247 °C (dec). H NMR
(300 MHz, CD3OD) δ 9.50 (s, 1 H), 7.56 (s, 1 H), 7.48 (d, J )
8.7 Hz, 1 H), 7.28 (s, 1 H), 7.17 (d, J ) 8.7 Hz, 1 H), 6.13 (s, 2
H), 4.47 (s, 2 H), 4.10 (s, 6 H); low resolution ESIMS m/z (rel
intensity) 322 (MH+, 100). Anal. (C19H16NO4Cl‚0.5H2O) C, H,
N.
(2,3,4-Trimethoxybenzylidene)-(2,2-dimethoxyethyl)-
amine (32). 2,3,4-Trimethoxybenzaldehyde (31, 11.48 g, 0.05851
mol) was dissolved in benzene (200 mL) and aminoacetalde-
hydedimethylacetal (8, 10.0 mL, 9.75 g, 0.0927 mol) was added.
The same procedure was applied as for 9. The product was
isolated as a clear light yellow oil (16.58 g, 100%): bp 153-
155 °C (0.45 mmHg). 1H NMR (300 MHz CDCl3) 8.49 (s, 1 H),
7.64 (d, J ) 8.7 Hz, 1 H), 6.65 (d, J ) 8.7 Hz, 1 H), 4.61 (t,
J ) 5.4 Hz, 1 H), 3.88 (s, 3 H), 3.84 (s, 3 H), 3.82 (s, 3 H), 3.71
(d, J ) 5.4 Hz, 2 H), 3.36 (s, 6 H); low resolution ESIMS m/z
(rel intensity) 284 (MH+, 100), 252 (45). Anal. (C14H21NO5)
(2,3,4-Trimethoxybenzyl)-(2,2-dimethoxyethyl)am-
ine (33). The imine 32 (14.28 g, 0.05040 mol) was dissolved
in ethanol (100 mL) and reacted with NaBH4 (3.00 g, 0.0793
mol) as described previously for compound 10. A colorless oil
1
(14.38 g, 100%) was obtained. H NMR (300 MHz, CDCl3) δ
6.89 (d, J ) 8.4 Hz, 1 H), 6.57 (d, J ) 8.4 Hz, 1 H), 4.45 (t, J
) 5.4 Hz, 1 H), 3.87 (s, 3 H), 3.82 (s, 3 H), 3.80 (s, 2 H), 3.70
(s, 3 H), 3.31 (s, 6 H), 2.68 (d, J ) 5.4 Hz, 2 H); low resolution
ESIMS m/z (rel intensity) 286 (MH+, 100), 181 (69). Anal.
(C14H23NO5) C, H, N
1,2,3,7,8-Pentamethoxy-11H-indeno[1,2-c]isoquino-
line Hydrochloride (34). The amine 33 (2.67, 9.36 mmol)
and 3,4-dimethoxybenzaldehyde (11, 3.23 g, 0.195 mol) were
reacted in concentrated hydrochloric acid as described previ-
ously for compound 13. The product was isolated as a yellow
1
hygroscopic solid (135.5 mg, 5%): mp 201-203 °C. H NMR
(300 MHz, CD3OD) δ 9.26 (s, 1 H), 7.67 (s, 1 H), 7.39 (s, 1 H),
7.27 (s, 1 H), 4.26 (s, 3 H), 4.22 (s, 2 H), 4.17 (s, 3 H), 3.98 (s,
3 H), 3.97 (s, 3 H), 3.96 (s, 3 H); low resolution ESIMS m/z
(rel intensity) 368 (MH+, 100). Anal. (C21H22NO5Cl‚1.8H2O) C,
H, N.
(2,5-Dimethoxybenzylidene)-(2,2-dimethoxyethyl)-
amine (27). Aminoacetaldehydedimethylacetal (8, 20.0 mL,
19.5 g, 0.185 mol) was dissolved in chloroform (100 mL) and
MgSO4 (10 g) was added. Then 2,5-dimethoxybenzaldehyde
(26, 7.00 g, 0.0421 mol) was also added, and the mixture was
stirred at room temperature for 24 h. The reaction mixture
was diluted with chloroform (100 mL), washed with water (2
× 200 mL) and brine (200 mL), dried (Na2SO4), and concen-
trated to provide the pure imine as a light yellow oil (10.64 g,
1,2,3-Trimethoxy-7,8-methylenedioxy-11H-indeno[1,2-
c]isoquinoline Hydrochloride (35). The amine 33 (3.03,
10.7 mmol) and piperonal (12, 3.96 g, 0.0264 mol) were were
reacted in concentrated hydrochloric acid as described previ-
ously for compound 13. The product was isolated as an yellow
1
1
100%): bp 151 °C (0.4 mmHg). H NMR (300 MHz CDCl3) δ
hygroscopic solid (0.353 g, 8.5%): mp 206-207 °C. H NMR
8.66 (s, 1 H), 7.47 (d, J ) 3.0 Hz, 1 H), 6.91 (dd, J1 ) 9.0 Hz,
J2 ) 3.0 Hz, 1 H), 6.81 (d, J ) 9.0 Hz, 1 H), 4.65 (t, J ) 5.1
Hz, 1 H), 3.78 (s, 3 H), 3.77 (s, 3 H), 3.76 (dd, J1 ) 5.4 Hz,
J2 ) 1.2 Hz, 2 H), 3.38 (s, 6 H); low resolution ESIMS m/z (rel
intensity) 254 (MH+, 100). Anal. (C13H19NO4) C, H, N.
(2,5-Dimethoxybenzyl)-(2,2-dimethoxyethyl)amine (28).
The imine 27 (10.64 g, 0.04201 mol) was dissolved in ethanol
(100 mL) and reduced with NaBH4 (2.60 g, 0.0687 mol) as
described previously for compound 10. The product was
isolated as a clear colorless oil (10.71 g, 100%): bp 155 °C (1.3
mmHg). 1H NMR (300 MHz CDCl3) δ 6.79-6.67 (m, 3 H), 4.43
(t, J ) 5.7 Hz, 1 H), 3.72 (s, 3 H), 3.71 (s, 3 H), 3.68 (d, J ) 2.7
Hz, 2 H), 3.28 (s, 6 H), 2.7 (d, J ) 5.7 Hz, 2 H); low resolution
ESIMS m/z (rel intensity) 256 (MH+, 100). Anal. (C13H21NO4)
C, H, N.
1,4,7,8-Tetramethoxy-11H-indeno[1,2-c]isoquinoline Hy-
drochloride (29). The amine 28 (2.82 g, 11.1 mmol) and
veratraldehyde (11, 4.00 g, 0.030 mol) were reacted in con-
centrated hydrochloric acid as described previously for com-
pound 13. The product was isolated as a yellow-orange
hygroscopic solid (1.01 g, 24%): mp 242-244 °C. 1H NMR (300
MHz, TFA) δ 9.52 (s, 1 H), 7.75 (s, 1 H), 7.43 (d, J ) 7.8 Hz,
1 H), 7.38 (s, 1 H), 7.08 (d, J ) 8.7 Hz, 1 H), 4.56 (s, 2 H), 4.10
(s, 3 H), 4.09 (s, 3 H), 4.04 (s, 6 H). Anal. (C20H20NO4Cl‚1.7H2O)
C, H, N.
1,4-Dimethoxy-7,8-methylenedioxy-11H-indeno[1,2-c]-
isoquinoline Hydrochloride (30). The amine 28 (2.22 g,
8.69 mmol) and piperonal (12, 3.96 g, 0.0264 mol) were reacted
in concentrated hydrochloric acid as described previously for
compound 13. The product was isolated as an orange hygro-
(300 MHz, CD3OD) δ 9.26 (s, 1 H), 7.52 (s, 1 H), 7.27 (s, 2 H),
6.11 (s, 2 H), 4.25 (s, 3 H), 4.21 (s, 2 H), 4.16 (s, 3 H), 3.97 (s,
3 H); low resolution ESIMS m/z (rel intensity) 352 (MH+, 100).
Anal. (C20H18NO5Cl‚0.15H2O) C, H, N.
1,2,3,7,8,9-Hexamethoxy-11H-indeno[1,2-c]isoquino-
line Hydrochloride (36). The amine 33 (3.23, 11.4 mmol)
and 3,4,5-trimethoxybenzaldehyde (17, 4.04 g, 0.0206 mol)
were reacted in concentrated hydrochloric acid as described
previously for compound 13. The product was isolated as a
hygroscopic yellow solid (0.5203 g, 10.5%): mp 196-198 °C.
1H NMR (300 MHz, CD3OD) δ 9.12 (s, 1 H), 7.27 (s, 1 H), 7.20
(s, 1 H), 4.30-4.26 (8 H), 4.18 (s, 2 H), 3.99 (s, 3 H), 3.98 (s, 3
H), 3.90 (s, 3 H); low resolution ESIMS m/z (rel intensity) 398
(MH+, 100). Anal. (C22H24NO6Cl‚1.4H2O) C, H, N.
Topoisomerase I-Mediated DNA Cleavage Reactions
Using 3′-End-Labeled 161 BP Plasmid DNA. The assays
were conducted as previously described.5 Briefly, the 161 bp
fragment obtained after the cleavage of pBluescript SK(-)
phagemid DNA (Stratagene, La Jolla. CA) with restriction
endonuclease Pvu II and Hind III (New England Biolabs,
Beverly, MA) was endlabeled at the Hind III site by fill-in
reaction with [R-32P]-dCTP and dATP, dGTP and dTTP. The
labeled DNA fragment was incubated with top1 for 30 min at
30 °C in the presence of the synthetic compound to be tested.
After precipitation with ethanol, the samples in loading buffer
were separated on a denaturing gel (16% polyacrylamide, 7
M urea) at 51 °C. The gel was visualized by using Phospho-
imager.
DNA Cleavage Semiquantitative Analysis. One of the
most abundant cleavage products, specifically the sequence

Norindenoisoquinoline Topoisomerase I Poisons
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15 4813
Table 2. Refinement Statisticsb
previously labeled 44,8 was chosen for semiquantitation using
ImageQuant TL v2003.3. The rubberband baseline correction
was applied with band detection sensitivity set at 90. In the
case of each norindenoisoquinoline the absolute density value
for the band corresponding to the above product was compared
to the value for the standard substance, NSC 314622. The ratio
of the norindenoisoquinoline band density to the NSC 314622
band was multiplied by 100 to obtain percentages. Assign-
ments were performed as follows: 0-25% 0; 25-75% +; 75-
175% ++; 175-325% +++; camptothecin ++++.
Molecular Modeling of the Crystal Structure. The
diagram of the crystal structure (Figure 3) was prepared
starting from the pdb file and the Sybyl 6.9 software package
from Tripos. Missing bonds were added in the phosphodiester
backbone, in T10 as well as the bond to the sulfur atom of the
DNA oligonucleotide. The atom types of the DNA strands were
corrected according to Sybyl types. The atom types for the end
groups were also corrected according to Sybyl types and
assigned as charged residues. The software correctly assigned
atom types in the protein structure except for the amino
terminus. No missing bonds were identified in the enzyme.
Crystallization and Structure Determination of the
Ternary Complex. A 70 kDa construct of human topoisom-
erase I (topo70), residues Lys175 to Phe765, was expressed
and purified from baculovirus-insect cells (SF9) as described
previously.34 The covalent complex of topo70-DNA was pre-
pared using a 5′ bridging phosphorothiolate duplex oligonu-
cleotide previously described.35 The oligonucleotide sequence
of the cleavable strand of the duplex oligomer was 5′-AAAAA-
GACTTsCGAAAAATTTTT-3′ where ‘s’ represents the 5′ bridg-
ing phosphorothiolate of the cleaved strand. Ternary complex
crystals were grown by sitting drop vapor diffusion by prepar-
ing drops containing 2.0 µL of precipitant, 1.5 µL of 50 µM
duplex DNA, 0.5 µL of compound, and 1.5 µL of of 4.2 mg/mL
protein. Precipitant was 10-12% PEG 8000, 100mM MES pH
6.4, 200 mM lithium sulfate. Compound 14 was dissolved in
water and diluted with preciptant to final concentrations of
1, 2, 5, and 10 mM for crystallization trials. Crystals were
cryoprotected for data collection by passing them through
precipitant plus 30% v/v PEG 400. Data were collected at 100
K at beamline 5.0.3, Advanced Light Source, Lawrence Berkely
National Laboratory. Crystals were isomorphous with previous
topo70-DNA complex crystal structures33 and the protein
model was positioned by rigid body refinement. Refinements
were conducted using CNX36 and iterative model adjustments
with XtalView.37 DNA’s were placed into the |Fo|-|Fc| electron
density and refined. Compound 14 model was then placed into
the |Fo|-|Fc| electron density using XtalView and refined. Data
and refinement statistics are presented in Table 2. The highest
resolution of the X-ray data obtained was 3.1 Å and did not
allow the placement of water molecules.
inhibitor bound
PDBID
AI-III-52
1TL8
resolution (Å)
no. reflections
50-3.1 (3.21-3.10)
16738 (1581)
10.0 (52.2)
97.8 (93.1)
13.7 (2.3)
P21
a
Rsym
completeness
I/σI
space group
a (Å)
56.949
b (Å)
114.140
73.499
c (Å)
â
94.18
reflections used in RFREE
no. of protein atoms
no. of DNA atoms
no. of inhibitor atoms
no. of solvent atoms
Rfactor
5%, 808
4703
892
24
0
22.9 (33.2)
30.5 (39.5)
Rfree
rms deviations from ideal stereochemistry
bond lengths (Å)
bond angles (deg)
impropers (deg)
dihedrals (deg)
mean Bfactor, all atoms (Å3)
0.017
1.9
3.94
23.8
60.4
a Rsym )Σ |Ii - Im|/Σ Im where Ii is the intensity of the measured
reflection and Im is the mean intensity of all symmetry related
reflections. b Numbers in parentheses represent final shell of data.
were also detected between the sugar-phosphate backbone
of the nonscissile DNA strand and the methoxy group in
position 8. Stacking interactions appeared to be decreased as
in the resulting complex there was little contact between the
norindenoisoquinoline 25 and the neighboring pyrimidine
bases. These are regions where in the crystal structure there
is good overlap and favorable van der Waals interaction due
to dispersion forces. During energy minimization the ligand
moved about 1 Å away from Arg364 and there is no hydrogen
bonding interaction between the norindenoisoquinoline 25 and
Arg364.
The second minimization was carried out in the same way
except an aggregate was used that included both the protein
backbone atoms and all the DNA substructures. In the
resulting complex, since the DNA bases had been frozen, the
neighboring bases could not move away to avoid steric clashes
and additional unfavorable contacts were detected between the
norindenoisoquinoline 25 (the methoxy substituents in posi-
tions 2 and 7) and the neighboring thymine (T10), adenine
(ADE113), and guanine (GUA112).
Acknowledgment. This work was made possible by
the National Institutes of Health (NIH) through support
of this work with Research Grant UO1 CA89566. The
in vitro and in vivo testing was conducted through the
Developmental Therapeutics Program, DCTD, NCI
under Contract NO1-CO-56000. The Advanced Light
Source is supported by the Director, Office of Science,
Office of Basic Energy Sciences, Materials Sciences
Division, of the U.S. Department of Energy under
Contract No. DE-AC03-76SF00098 at Lawrence Berke-
ley National Laboratory. We are grateful to Prof. V. Jo
Davisson for allowing the use of the densitometer and
ImageQuant TL v2003.3 software. This research was
conducted in a facility constructed with support from
Research Facilities Improvement Program Grant Num-
ber C06-14499 from the National Center for Research
Resources of the National Institutes of Health.
Computer Modeling. Norindenoisoquinoline 25 was mini-
mized prior to its inclusion in the ternary complex using the
MMFF94s force field, MMFF94 charges, and the conjugate
gradient method, with no initial optimization, to a gradient
of less than 0.05 kcal/mol. Then norindenoisoquinoline 25 was
placed in the ternary complex of the crystal structure in the
same orientation as norindenoisoquinoline 14, and the ligand
14 was deleted. The resulting complex was minimized in two
different manners. First it was minimized as a subset using
the Powell method, MMFF94s force field and MMFF94 charges
to a gradient of 0.5 kcal/mol or less. The protein backbone
atoms were included into an aggregate and their positions
remained fixed. The atoms not included in the aggregate in a
sphere of 6 Å around the ligand were allowed to move during
energy minimization; atoms included in a shell stretching from
6 to 12 Å were also allowed to move yet to a lesser extent
(radius of hot region 6 Å, radius of interesting region 12 Å).
The result indicated that the adjacent DNA base, thiocyti-
dine, was flipped in part to avoid steric interference with the
methoxy substituents. Steric conflicts still appeared to persist
between Arg364 and the methoxy group in position 7 and
between the ligand methoxy in position 3 and the deoxyribose
(C1′, C5′) of thiocytidine 11 (TCP 11). Unfavorable contacts
Supporting Information Available: Elemental analysis
data. This material is available free of charge via the Internet
at http://pubs.acs.org.

4814 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 15
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JM050076B