Structure-based design, synthesis, and biological studies of new anticancer norindenoisoquinoline topoisomerase i inhibitors

Article abstract of DOI:10.1021/jm901649x

On the basis of the superimposition of the crystal structures of norindenoisoquinoline 5 and topotecan (2) bound in the topoisomerase I-DNA covalent complex, as well as molecular docking and quantum chemical calculations, the substituted norindenoisoquino

Full text of DOI:10.1021/jm901649x

J. Med. Chem. 2010, 53, 1979–1989 1979
DOI: 10.1021/jm901649x
Structure-Based Design, Synthesis, and Biological Studies of New Anticancer Norindenoisoquinoline
Topoisomerase I Inhibitors
Yunlong Song,†,‡, Zhiyu Shao,†, Thomas S. Dexheimer,§ Evan S. Scher,§ Yves Pommier,§ and Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and the Purdue University
Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, ‡Department of Medicinal Chemistry, School of Pharmacy,
Second Military Medical University, Shanghai 200433, China, and Laboratory of Molecular Pharmacology, Center for Cancer Research,
§
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. These authors contributed equally to this work.
Received August 1, 2009
On the basis of the superimposition of the crystal structures of norindenoisoquinoline 5 and topotecan
(2) bound in the topoisomerase I-DNA covalent complex, as well as molecular docking and quantum
chemical calculations, the substituted norindenoisoquinoline 14a was designed by transporting the
9-dimethylaminomethyl group of topotecan to the 10-position of the norindenoisoquinoline 5. The
desired compound 14a was synthesized and found to possess topoisomerase I inhibitory activity that
was slightly better than that of the starting compound 5. A focused set of 10-substitued norindenoi-
soquinoline analogues were then synthesized. The imidazole-substituted compound 14c was highly
cytotoxic when evaluated in a series of human leukemia, ovarian, and breast cancer cells.
Introduction
unique pharmacokinetics, toxicity profiles, and resistance
characteristics.1,11,13-17 Several types of non-CPT Top1 inhi-
bitors, including the indolocarbazoles and indenoisoquino-
lines, have been reported.17,18 The discovery of the prototy-
pical indenoisoquinoline NSC314622 (4) as a Top1 inhibitor
originated from a COMPARE analysis.19 Significant progress
has been made on the improvement of the potencies of the
indenoisoquinolines through structure manipulation.20-33
Several indenoisoquinoline compounds such as indimitecan
(NSC 725776) and indotecan (NSC 724998) produce persis-
tent Top1 cleavage complexes and overcome multidrug resis-
tance.34
During the synthetic modification of the indenoisoquino-
lines, a norindenoisoquinoline AI-III-52 (5) was discovered
that demonstrated very high cytotoxicities in the NCI in vitro
anticancer screen (mean graph midpoint of 50 nM) and strong
Top1 inhibitory potency.35 The DNA cleavage patterns of
norindenoisoquinolines are somewhat different from those of
the widely studied indenoisoquinolines. These important
differences indicate that different cancer cell genes could be
targeted moreselectively with onetypeof inhibitor vs another.
Similar to the situation with Top2 or tubulin inhibitors that
share a single target, it can be expected that different Top1
inhibitors will have different spectra of anticancer activity.
Furthermore, norindenoisoquinoline 5 was observed by
X-ray crystallography to adopt a “flipped” orientation
(relative to indenoisoquinolines) in its ternary cleavage com-
plex.35,36 The binding of the norindenoisoquinolines in the
Top1-DNA complex has been determined by high level ab
initio quantum chemical studies to be mainly governed by
π-π stacking forces.37 The data clearly indicate that the
norindenoisoquinolines are novel Top1 inhibitors. However,
their solubility limitations have hindered further preclinical
development. This situation has dictated the challenging
task of designing and synthesizing novel highly potent
norindenoisoquinolines with enhanced water solubility.
DNA topoisomerase I (Top1a) has become an important
target for the design of novel antitumor drugs.1 It is a
ubiquitous and essential nuclear enzyme for DNA replication
and transcription.2 As a class, the Top1 inhibitors in the
camptothecin (CPT, 1; Chart 1) family have historically
suffered from poor water solubility, high toxicity, and meta-
bolic instability.3 Therefore, substantial efforts were made to
improve the water solubilities, toxicity profiles, and pharma-
cokinetics of CPT derivatives. These efforts were eventually
rewarded by the development of the two clinically useful, water-
soluble CPT derivatives topotecan (2) and irinotecan (3).4
However, the clinically useful CPTs are not ideal drug
molecules because they are compromised by the following
inherent limitations: (1) the E-ring of CPT contains an R-
hydroxylactone that opens to a hydroxycarboxylate that
binds tightly to serum albumin;5,6 (2) the drug-target inter-
action is reversible and has to be maintained long enough to
convert Top1 cleavage complexes into DNA damage;7 (3)
several Top1 resistance mutations such as Asn722Ser and
Arg364His occur;8,9 (4) tumor cells that overexpress drug
efflux pumps are resistantto CPTs;10 (5) the side effects caused
by camptothecins limit the doses that can be safely adminis-
tered and, therefore, antitumor efficacy.11-13
To overcome the lactone instability, reversibility of the
drug-target interaction, and drug resistance, extensive efforts
have been made to discover non-CPT Top1 inhibitors that
share similar specificities and potencies with CPT, yet display
*To whom correspondence should be addressed. Phone: 765-494-
1464. Fax: 765-494-6790. E-mail: cushman@purdue.edu.
a Abbreviations: ADME, absorption, distribution, metabolism, and
excretion; AIBN, azobisisobutyronitrile; BSA, bovine serum albumin;
CPT, camptothecin; EDTA, ethylenediaminetetraacetic acid; MGM,
mean graph midpoint; NBS, N-bromosuccinimide; QM, quantum
mechanics; SDS, sodium dodecyl sulfate; TEA, triethylamine; Top1,
topoisomerase I.
r
2010 American Chemical Society
Published on Web 02/15/2010
pubs.acs.org/jmc

1980 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Song et al.
Chart 1. Chemical Structures of Representative Top1 Inhibitors
Figure 1. (A) Overlap of norindenoisoquinoline 5 vs topotecan (2)
based on the superimposition of the target enzyme. (B) Proposed
water-soluble norindenoisoquinoline compound 14a.
exchange of substituents among Top1 inhibitors in order to
maximize substituent effects. In the present study, the two
crystal structures 5-DNA-Top1 (PDB code 1tl8)35 and
topotecan 2-DNA-Top1 (PDB code 1k4t)39 were super-
imposed on the basis of all the heavy atoms of the protein, as
portrayed in Figure 1.
As seen from the overlapped crystal structures, the 9-posi-
tion of topotecan (2) overlaps with the 10-position of the
indenoisoquinoline ring system (see structures 1 and 4 for
atom numbering scheme). Attachment of the 9-dimethyla-
minomethyl substituent of topotecan (2) to the 10-position
of the norindenoisoquinoline 5 results in the potential Top1
inhibitor 14a displayed in Figure 1B, which is expected to
have increased polarity, and could be formulated as a
hydrochloride salt. The design of 14a was also corroborated
by molecular docking studies, in which 14a displayed better
docking scores and more favorable calculated binding
energy than 5 (see Supporting Information). Furthermore,
since substituents on the 10-position of the norindenoisoqui-
nolines and the 7-position of the indenoisoquinolines pro-
ject similarly into the DNA major groove, the 10-dimethyl-
aminomethyl substituent of the compound in Figure 1B
might be advantageously swapped with the types of 7-posi-
tion substituents of the indenoisoquinolines that have been
shown to confer greater Top1 inhibitory activity. This
strategy might result in favorable interactions of the inhi-
bitors with the Top1-DNA covalent complex and thus
enhance antitumor activity.
A further goal of the present study was to investigate the
structure-activity relationships (SAR) of 10-position nitro-
gen-containing side chains and optimize ADME properties.
Starting from 14a, a focused set of 10-substituted norinde-
noisoquinolines were designed. All the resulting structures
were docked into the Top1-DNA covalent complex, and
only those with better docking scores and more favorable
calculated binding energies were kept for further synthesis.
This resulted in norindenoisoquinolines 14b-f.
Quantum Chemistry. As described above, a methylene
linker was chosen for 14a and its derivatives. 9-Methyl-
CPT, in which the 9-methyl group occupies space that over-
laps with the 10-methylene group of the norindenoisoquino-
lines in the ternary complex, was reported to demonstrate
better Top1 inhibition than CPT.40 A quantum chemical
investigation was therefore considered to answer the ques-
tion of whether a 10-methyl group would increase the bind-
ing of the norindenoisoquinoline system to DNA as has been
shown with 9-methyl-CPT. High level quantum chemical
MP2 calculations were performed on 10-methyl-norindeno-
isoquinoline 15 using our recently published strategy, and
the results were compared with those of norindenoisoquino-
line 5 from previous calculations.37 The attachment of a
Fortunately, the X-ray crystal structures of ternary com-
plexes containing Top1 and a DNA fragment binding to
several different Top1 inhibitors have been reported. These
include ternary complexes derived from camptothecin,38 topo-
tecan,39 indolocarbazole,38 indenoisoquinoline,38 and norin-
denoisoquinoline35 Top1 inhibitors. This provides a strong
structural basis for the rational design of highly potent
norindenoisoquinolines with enhanced water solubility.
Herein, the structure-based design, chemical synthesis, and
in vitro cytotoxic evaluations of novel potent norindenoiso-
quinolines with enhanced water solubility are reported. The
present study was also conducted to assess the contributions
of different representative nitrogen-containing side chains on
the 10-position of norindenoisoquinoline to their cytotoxic
properties and Top1 inhibitory activities. This has also demon-
strated the power of fragment-based design combined with
computational chemistry to advance the structure-based drug
design process.
Results and Discussion
Molecular Design. The crystal structures of Top1-DNA-
inhibitor complexes allow the superimposition of the inhibi-
tors in the ternary complexes.35,38,39 That, in turn, can suggest
the migration of effective substituents from a region of one
inhibitor to a spatially equivalent location on a different inhi-
bitor. This “mix-and-match” strategy can suggest the rational

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1981
Scheme 2a
Table 1. Interaction and Solvation Energies (kcal/mol) of Norindenoi-
soquinolines Binding to Simplified Cleavage Complex37 Calculated by
the MP2/6-31G*(0.25) Method
compd
parameters
Eint (MP2)a
ΔGsolvation(MP2)b
Eint þ ΔGsolvation
ΔE c
5
15
14ad
-31.56
-40.25
-71.81
0
-32.87
-38.32
-71.19
0.62
-31.77*
-36.53*
-68.3*
3.51*
a Eint = Ecomplex - Eligand - Ebp þ BSSE. b ΔGsolvation(MP2) =
EPCM-MP2 - EMP2.
c ΔE was the relative binding energy difference in
aqueous solution between 15, 14a, and AI-III-52 investigated at the MP2
level, respectively. d The 10-position substituent of 14a forms direct
interactions with neighboring Top1 residues which were not included
in the MP2 calculations because of impractically high computational
cost. Therefore, the values with an asterisk are not meaningful indicators
of binding to the DNA-Top1 covalent complex.
Scheme 1a
a Reagents and conditions: (a) (MeO)2CHCH2NH2, PhH, 23 °C (4 h);
(b) NaBH4, EtOH, reflux (30 min).
methyl group did contribute -1.31 kcal/mol to the calcu-
lated in vacuo binding energy, from -31.56 kcal/mol of
norindenoisoquinoline 5 to -32.87 kcal/mol of 15 (Table 1).
However, the calculated relative binding energy difference in
aqueous solution between 15 (-71.19 kcal/mol) and norinde-
noisoquinoline 5 (-71.81 kcal/mol) of 0.62 kcal/mol suggests
that compound 15 would act as a less potent Top1 inhibitor
compared to the norindenoisoquinoline 5. The QM result
suggests that the same substituent may elicit different calculated
effects on activity if transferred to a new scaffold (in this case a
methyl added to 5 vs CPT) even though it occupies the same
space in the binding site.
a Reagents and conditions: (a) (1) Me2NCH2CH2NMeLi, THF, -78 °C
(1 h), (2) MeI, -78 °C (2 h); (b) MeONH3þCl-, NaOAc, MeOH, 23 °C
(1 h); (c) NBS, AIBN, CCl4, reflux(3h);(d) amine, THF, 23°C;(e) 8, concd
HCl, 100 °C.
Scheme 3a
QM calculations of the binding energy of compound 14a
were also performed. The attachment of a dimethylamino-
methyl substituent to 5 contributes -0.21 kcal/mol to the
calculated DNA interaction energy, which indicates the
attachment of such a bulky group would not interfere with
the binding of the scaffold to DNA. However, it should be
noted that a simplified DNA cleavage complex, which lacks
protein residues, was used in the MP2 calculations because of
the high computational cost.37 Therefore, the calculations
involving 14a cannot be expected to provide an accurate
indicator of binding in the ternary complex because the
bulky dimethylamino substituent makes direct contact with
the neighboring protein residues. On the other hand, the
methyl group of 15 only interacts with the DNA fragment in
the ternary complex.
Chemistry. The syntheses of the desired compounds 14a-f
and 15 are outlined in Schemes 1-3. The general strategy is
based on the classical Pomeranz-Fritsch reaction41 and its
improved versions including the Bobbitt42 and Jackson
modifications.43 The required precursor 8 of the isoquinoline
ring system was prepared as outlined in Scheme 1.35 Con-
densation of veratraldehyde (6) with aminoacetaldehyde
dimethyl acetal resulted in formation of the Schiff base 7,
which was reduced to the amine 8 with sodium borohydride
in ethanol.
a Reagents and conditions: (a) 8, concd HCl, 100 °C (18 h).
As detailed in Scheme 2, regioselective deprotonation of
piperonal (9) with lithium N,N,N0-(trimethyl)ethylenedi-
amine in THF resulted in a phenyllithium intermediate that
was methylated to afford 2-methylpiperonal (10).44 The
aldehyde 10 was converted to its oxime ether 11, which was
subjected to free radical bromination with NBS and AIBN in
refluxing carbon tetrachloride to afford the benzyl bromide
12. Displacement of the bromide with various amines pro-
vided the penultimate intermediates 13a-f. Condensation of
compounds 13a-f with intermediate 8 in concentrated hydro-
chloric acid at 100 °C yielded the final products 14a-f in low
yields (3.1-7.8%). The reaction mixtures were very complex,
and the yields were compromised by formation of uncyclized
4-benzylisoquinolines and by extremely challenging purifica-
tions of the final products. Nevertheless, the compounds were
eventually obtained in pure form in sufficient quantities for
biological testing.

1982 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Song et al.
The crystal structure of the norindenoisoquinoline 14a
(Figure 2) provides a reliable starting conformation for the
quantum chemical calculations and also constitutes an inde-
pendent verification of the structure. The inclusion of one
molecule of chloroform in the crystal structure is consistent
with our long-term experience that solvents are very difficult
to remove from indenoisoquinolines in the solid state.
The 10-methylnorindenoisoquinoline 15 was also pre-
pared in order to evaluate the contributions that the amine
moieties of 14a-f made to their in vitro cytotoxicities and
Top1 inhibitory activities. Condensation of intermediates 8
and 10 under the usual harsh acidic conditions resulted in
formation of the desired product 15 (Scheme 3).
Biological Studies. All of the norindenoisoquinolines
were examined for antiproliferative activity in the National
Cancer Institute’s Developmental Therapeutics Program
(DTP).45,46 Each compound was evaluated against approxi-
mately 55 human cell lines representing leukemia, melanoma,
and cancers of the lung, colon, brain, breast, ovary, prostate, and
kidney. After an initial one-dose assay at a single high dose
(10-5 M), only compounds that satisfied the predetermined
threshold inhibition criteria were selected for further five-
dose assay. The selected compounds were tested at five con-
centrations ranging from 10-8 to 10-4 M. The GI50 values
from each subpanel were obtained with selected cell lines,
and overall antiproliferative effects are quantified as a mean
graph midpoint (MGM, Table 2). The MGM is a measure of
the average GI50 for all of the cell lines tested, in which GI50
values below and above the test range (from 10-8 to 10-4 M)
are taken as the minimum (10-8 M) and maximum (10-4 M)
drug concentrations used in the assay. For comparison, the
activities previously reported for camptothecin (1), indenoi-
soquinoline 4, and norindenoisoquinoline (5) are also listed
in the table.
The introduction of the 10-methyl group into compound
5, resulting in norindenoisoquinoline 15, significantly low-
ered the cytotoxicity about 230-fold according to their
respective MGM values. However, the further introduction
of basic functional groups into compond 15 increased the
cytotoxicity even though it had variable effects on the Top1
inhibitory activity.
The imidazole-substituted norindenoisoquinoline com-
pound 14c is the most cytotoxic compound in this series,
with an MGM of 0.52 μM. Notably, the hydrophobic
prototype 5 is inactive in colon HCT-116 cancer cell and
renal SN12C cancer cell cultures, whereas compound 14c is
potent against these two tumor cell types, with GI50 values of
0.16 and 0.60 μM, respectively. All of the 10-aminomethylene
norindenoisoquinolines except 14f demonstrated good acti-
vity against these two tumor cell lines. The 10-aminomethyl-
ene norindenoisoquinolines have different anticancer
spectra compared to their hydrophobic counterpart 5. As
norindenoisoquinoline 14c was the most cytotoxic in the
series, yet demonstrates moderate Top1 inhibition, addi-
tional cellular targets mediating the cytotoxic effects possibly
exist.35
Norindenoisoquinoline 14c was highly cytotoxic against a
selected series of human cancer cells, especially against
leukemia, breast, and ovarian cancer cells (Table 3). The
GI50 values of 14c against leukemia SR, CCRF-CEM,
RPMI-8226, and MOLT-4 are 12, 37, 41, and 60 nM, res-
pectively. It also displayed strong cytotoxicity against the
human breast cancer cell MCF-7 with a GI50 of 90 nM. This
compound demonstrated a GI50 of 121 nM against human
ovarian cancer cell IGROVI. Clearly, the norindenoiso-
quinoline 14c represents an interesting chemical entity that
merits further preclinical investigation.
Norindenoisoquinolines 5, 14a-f, and 15 were examined
for induction of Top1-mediated DNA cleavage using a 117-
bp DNA oligonucleotide encompassing the previously iden-
tified Top1 cleavage sites in the 161-bp fragment from
pBluescript SK(-) phagemid DNA.13 The resulting gel
electrophoresis DNA cleavage patterns are displayed in
Figure 3, along with those resulting from CPT and the
indenoisoquinoline MJ-III-65.43 As often found in the case
of indenoisoquinoline series of Top1 inhibitors, a close
Figure 2. Crystal structure of norindenoisoquinoline 14a (the neigh-
boring solvent molecule is chloroform).
Table 2. Cytotoxicities and Topoisomerase I Inhibitory Activities of Norindenoisoquinolines
cytotoxicity (GI50 in μM)a
lung colon
HOP-62 HCT-116
CNS SF-
539
melanoma
UACC-62
ovarian
OVCAR-3
renal
SN12C
prostate
breast
Top1
cleavagec
compd
DU-145 MDA-MB-435 MGMb
CPT
4
0.01
1.30
0.072
6.7
0.03
35
0.01
41
0.01
4.2
0.22
73
0.02
68
0.01
37
0.04
96
0.0405
20
þþþþ
þþ
5
>100
1.5
<0.01
14
1.9
<0.01
12
6.7
NTd
1.4
>100
13
4.4
0.028
1.6
<0.01
16
2.5
NTd
0.050
4.4
2.3
þþ(þ)
þþþ
þþþ
þ(þ)
þþ
14a
14b
14c
14de
14e
14f
15
1.2
0.35
0.64
0.16
0.73
0.21
0.73
0.26
0.28
0.21
0.60
0.52
2.1
1.5
7.3
1.4
1.9
1.8
11
100
1.7
7.4
5.8
2.3
>100
>100
1.9
3.4
NTd
NTd
>100
2.4
6.3
þ
0.73
0.70
þ(þ)
þþ
0.89
4.6
>100
11.8
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 Top1-mediated DNA cleavage was expressed semiquantitatively as follows: 0, no activity; þ, weak activity; þþ, moderate activity; þþþ, good
activity; þþþþ, similar activity as 1 μM CPT. d NT=not tested. e Not selected for further testing; refer to text for details.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1983
Table 3. Cytotoxicities of 14c against a Series of Selected Human Cancer Cells (GI50 in μM)
colon cancer
renal cancer
CNS cancer
non-small-
cell
leukemia lung cancer melanoma cancer
ovarian prostate
cancer
breast
cancer
SR
0.012
NCI-H460
0.16
0.11
UACC-62 IGROVI DU-145
MCF7
0.09
HCC-2998 COLO 205 CAKI-1 ACHN U251 SF-295
1st run
2nd run
mean
0.21
0.18
0.20
0.033
0.21
0.12
0.26
0.41
0.33
0.11
2.1
0.76
0.28
0.51
0.17
0.40
0.29
0.27
0.30
0.28
0.15 0.16
0.17 0.13
0.16 0.15
0.089
0.090
0.012
0.13
1.1
Figure 3. Top1-mediated DNA cleavage induced by norindenoisoquinolines 14a-f and 15: (lane 1) DNA alone; (lane 2) Top1 alone; (lane 3)
camptothecin (1), 1 μM; (lane 4) MJ-III-65, 1 μM; (lanes 5-36) (for 5, 15, 14a-f) Top1 þ indicated compound at 0.1, 1, 10, and 100 μM,
respectively. The numbers on the right and arrows indicate cleavage site positions. A 117-bp DNA substrate was used in this assay, but the
cleavage sites are numbered to be consistent with the 161-bp DNA substrate traditionally used in this assay in order to facilitate comparison.
correlation between Top1 inhibition and antiproliferative
activity was not observed.20-33 Compound 14a displayed
slightly better Top1 inhibition than the starting lead com-
pound, norindenoisoquinoline 5. However, the cytotoxic
activity of 14a was not as good as expected, which might
due to many factors besides the interaction with target enzyme,
such as cellular permeability, distribution, and metabolism.
Compound 15 has a þþ Top1 inhibition, whereas 5 has a
þþ(þ) Top1 inhibition. This is consistent with the QM
prediction that 15 would be a less potent Top1 inhibitor
than 5.
The norindenoisoquinolines produced different DNA
cleavage patterns. For example, the 106 (C-T) cleavage
observed with norindenoisoquinolines 5 and 15 was absent
in 14d and 14e. Also, some of the cleavage sites induced with
the norindenoisoquinolines more closely resemble the inde-
noisoquinolines than the camptothecins. For example, the 44
(C-G) cleavage site was seen with the indenoisoquinoline
MJ-III-65 and the norindenoisoquinolines 5, 14a-e, and 15
but not with CPT, whereas the 37 (T-G) site is observed with
CPT but not with the indenoisoquinolines and norindeno-
isoquinolines.
but distinguishing differences also exist. For example, the
62 (C-A) site is observed with MJ-III-65 while not with the
norindenoisoquinolines. The 119 (T-G) site is found in the
norindenoisoquinolines 14a,b but not with MJ-III-65. In-
troduction of the 10-position nitrogen-containing side chains
seems to result in preferential binding to the 44 (C-G) site in
comparison with the hydrophobic norindenoisoquinolines 5
and 15. The 97 (T-A) site observed in 5 and 15 is hardly
detected in the norindenoisoquinolines 14a-e. These obser-
vations are important because they indicate that there could
be selectivity in cancer gene targeting with the norindenoi-
soquinolines vs indenoisoquinolines and CPT. It can there-
fore be expected that different Top1 inhibitors may selecti-
vely target different tumor types.
Molecular Modeling. The pKa values of the amines 14a-f
were calculated to determine the dominant species at physio-
logical pH 7.4 using the online program ADME Boxes
(http://www.pharma-algorithms.com/webboxes/) from Pharma
Algorithms, which has been described as “the most predic-
tive, high throughput pKa predictor” based on a recent com-
parison of current in silico pKa prediction tools by Balogh
et al.47 The method employs the pKa prediction tool of
Pharma Algorithms used in the present study. The algorithm
utilizes ∼18000 experimental pKa data points, a database of
The DNA cleavage patterns induced by the norindeno-
isoquinolines share some similarity with indenoisoquinolines,

1984 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Song et al.
Figure 4. Binding model of 14b binding to Top1-DNA cleavage complex.
∼4600 ionization centers, ∼500 interaction constants, and
four interaction calculation methods to produce microcon-
stants. In addition, the log P and log D values were also
calculated with the Pharma Algorithms online service, and
the data are included in the Supporting Information. From
the data, it is clear that by the introduction of 10-position
nitrogen-containing side chain substitutions, the log D va-
lues were decreased significantly. Obviously, the aqueous
solubilities of 14a-f can be further enhanced through salt
formation, and in fact clear aqueous solutions of hydro-
chloride salts were observed during workup of the reaction
mixtures that produced these products.
From the data, the two most potent compounds 14a and
14b against Top1 have tertiary amines with pKa values of
about 8.6. This pKa value means the nitrogen atom attached
to 10-benzyl group of norindenoisoquinoline is 95% proto-
nated at physiological pH. The dominant species of 14d and
14f are monoprotonated on their distal nitrogens. For
compounds containing two nitrogen atoms such as pipera-
zine, one nitrogen atom is protonated first, making the other
nitrogen less basic. For example, the pKa values of piperazine
are 9.81 and 5.59, respectively.45 As for 14c and 14e, the
dominant species are unprotonated forms (88% and 95%,
respectively) at physiological pH.
To further explore the influence of protonation states of
14a and 14b on Top1 inhibition, the protonated form and
also the free bases of these two compounds were docked
into Top1-DNA covalent complex (PDB code 1tl8). Clearly,
the protonated species is calculated to form stronger
interactions with the Top1-DNA covalent complex com-
pared to the free bases. For example, in Figure 4, the
“additional” hydrogen atom attached to the pyrrolidine
nitrogen atom forms a direct hydrogen bond with Glu356
of Top1. Moreover, the isoquinoline nitrogen atom at the
6-position of the norindenoisoquinoline forms a charac-
teristic hydrogen bond with Arg364 and one oxygen atom
of the methylenedioxy group neighboring the 10-position
of norindenoisoquinoline forms a hydrogen bond with
Lys425.
The most potent compound 14c exists predominantly in
its neutral form (88%) at physiological pH based on
calculated pKa values. From molecular docking studies,
the neutral form afforded a less favorable binding energy
(-71.7130 kcal/mol) than all the other nitrogen-contain-
ing side chain substituted norindenoisoquinolines (Sup-
porting Information). This is consistent with the Top1
inhibition data that 14c is a poor Top1 inhibitor. On the
other hand, compound 14c exhibited high cytotoxicity,
indicating that additional molecular targets likely exist for
this compound.
Conclusions
10-Substituted norindenoisoquinolines with improved aque-
ous solubilities were designed and synthesized. The design
was based on superimposition of two crystal structures of
norindenoisoquinoline 5 and topotecan (2) binding to the
Top1-DNA covalent complex, molecular docking, and
quantum mechanics studies. The 10-dimethylaminomethyl
norindenoisoquinoline 14a was designed by moving the
9-dimethylaminomethyl group of topotecan to the 10-position
of the norindenoisoquinoline, resulting in slightly better Top1
inhibition relative to the parent compound 5. Molecular
docking studies subsequently led to the design of a focused
set of 10-substituted norindenoisoquinolines 14b-e. Among
them, the imidazole-substituted compound 14c demonstrated
nanomolar cytotoxicity against a series of human leukemia,
ovarian, and breast cancer cells and is worthy of further
preclinical investigation. The new norindenoisoquinolines
14a-e and 15 act as Top1 poisons, stabilizing the Top1-
DNA cleavage complex by inhibiting the religation reaction.
The pattern of DNA cleavages observed with the norindenoi-
soquinolines was different from the indenoisoquinolines and
camptothecins, suggesting one possible explanation for the
unique cytotoxicity profiles observed in each case (e.g., com-
pare cytotoxicity data for camptothecin, indenoisoquinoline
4, and the norindenoisoquinoline 5 in Table 2). The mean
graph midpoint (MGM) values of the norindenoisoquinolines
14a-c, 14e, 14f, and 15 are obviously higher than those of
camptothecin (4) and the previously reported norindenoiso-
quinoline 5. However, the new norindenoisoquinolines are
significantly more cytotoxic than 5 in selected cell lines (e.g.,
14a-c, 14e, 14f, and 15 vs colon HCT-116 and 14a-c, 14e vs
renal SN12C cells, Table 2). With regard to the general topic
of overall relative cytotoxicities (MGM values), it is worth
noting that the Top1 inhibitor indotecan, which has out-
performed many of the more cytotoxic indenoisoquinolines
in animal models and is presently undergoing clinical trials in
cancer patients at the National Cancer Institute, has a rela-
tively unimpressive MGM of 4.64 μM.17,31 This seems to
indicate the possibility that, in some cases, MGM values are
being overemphasized when decisions are made in the anti-
cancer drug development process.
The novelty and significance of this work stem from testing
a hypothetical design strategybased on exchange of optimized
fragments that occupy the same space in the ternary drug-
DNA-enzyme complexes.
Experimental Section
Melting points were determined in capillary tubes and are
uncorrected. Infrared spectra were obtained using KBr pellets

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1985
and were recorded using a Perkin-Elmer 1600 series FTIR. Except
where noted, H NMR spectra were obtained using CDCl3 as
and AIBN (0.4078 g, 2.49 mmol) in carbon tetrachloride (100 mL)
was stirred at reflux under argon for 3 h. The reaction mixture
was cooled to room temperature, filtered, and evaporated to
give the crude product 12. Purification by preparative flash
chromatography (SiO2, EtOAc-hexane 15:1) and then recrys-
tallization from hexane gave the final pure product (3.27 g, 58%)
as a white solid: mp 122-123 °C. IR (KBr) 2902, 1480, 1455,
1
solvent and the solvent peak as internal standard. 1H NMR
spectra were determined at 300 MHz on a Bruker ARX-300
spectrometer. Electrospray mass spectra were obtained using a
FinniganMATT LCQ (Thermoquest Corp., San Jose, CA).
Microanalyses were performed at the Purdue University Micro-
analysis Laboratory. Mostchemicalsand solvents were analytical
grade and used without further purification. Commercial rea-
gents were purchased from Aldrich Chemical Co. Purities of
g95% were established by elemental analysis except for com-
pound 14f, which was estimated to be g95% pure by HPLC.
(3,4-Dimethoxybenzylidene)(2,2-dimethoxyethyl)amine (7).
3,4-Dimethoxybenzaldehyde (12.20 g, 73.42 mmol) was dis-
solved in benzene (200 mL), and aminoacetaldehyde dimethyl
acetal (12.0 mL, 11.7 g, 111 mmol) was added. The mixture was
stirred at reflux for 4 h using a Dean-Stark trap. The reaction
mixture was then concentrated and dissolved in CHCl3 (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
(18.41 g, 99.1%) as a light-yellow solid: mp 50-52 °C. 1H NMR
(300 MHz, CDCl3) δ 8.18 (s, 1 H), 7.42 (s, 1 H), 7.15 (d, J =
8.4 Hz, 1 H), 6.86 (d, J=8.4 Hz, 1 H), 4.65 (t, J=5.1 Hz, 1 H),
3.92 (s, 3 H), 3.90 (s, 3 H), 3.74 (d, J=5.1 Hz, 2 H), 3.40 (s, 6 H).
(3,4-Dimethoxybenzyl)(2,2-dimethoxyethyl)amine (8).The imine
7 (12.01 g, 47.47 mmol) was dissolved in ethanol (200 mL), and
NaBH4 (3.61 g, 95.4 mmol) was added over 0.5 h while the reaction
mixture was stirred at reflux. The reaction mixture was diluted with
water (200 mL). The amine was extracted into chloroform
(250 mL) and the extract washed with water (2 ꢀ 150 mL) and
brine (150 mL), dried (Na2SO4), and concentrated to provide a
1
1265, 1048, 927 cm-1; H NMR (300 MHz, CDCl3) δ 8.19 (s,
1 H), 7.09 (d, J=8.1 Hz, 1 H), 6.75 (d, J=8.1 Hz, 1 H), 6.05 (s,
2 H), 4.73 (s, 2 H), 3.97 (s, 3 H); low resolution ESIMS m/z
(rel intensity) 272 (MHþ, 37), 192 [(M - Br)þ, 100]. Anal.
(C10H10BrNO3) C, H, N.
(E)-4-[(Dimethylamino)methyl]benzo[d][1,3]dioxole-5-carbal-
dehyde O-Methyloxime (13a). A solution of the oxime 12 (0.40 g,
1.47 mmol) in THF (10 mL) was added by syringe during 15 min
to dimethylamine (2 M in THF, 3 mL, 6 mmol) at 0 °C. The
reaction mixture was stirred at room temperature for 2 h. The
organic solvent was removed by evaporation under reduced
pressure. A 5% NaHCO3 aqueous solution (20 mL) was added
to the residue, which was further extracted with EtOAc (40 mLꢀ
2). The organic layer was washed with brine (5 mL) and dried
(Na2SO4). Filtration and evaporation under reduced pressure
gave 13a as a yellow oil (0.3122 g, 90%). IR (KBr) 2939, 1474,
1455, 1253, 1053 cm-1; 1H NMR δ (300 MHz, CDCl3) 8.39 (s,
1 H), 7.33(d, J=8.1 Hz, 1 H), 6.73(d, J=8.1 Hz, 1 H), 5.96 (s,
2 H), 3.93 (s, 3 H), 3.47 (s, 2 H), 2.22 (s, 6 H); low resolution
ESIMS m/z (rel intensity) 237 (MHþ, 100), 192 [(M-NHMe2)þ,
55]. Anal. (C12H16N2O3) C, H, N.
(E)-4-(Pyrrolidin-1-ylmethyl)benzo[d][1,3]dioxole-5-carbalde-
hyde O-Methyloxime (13b). A solution of the oxime 12 (0.95 g,
3.49 mmol) in THF (10 mL) was added by syringe to a solution
of pyrrolidine (0.72 mL, 0.62 g, 8.7 mmol) in THF (10 mL) at
0 °C. The reaction mixture was stirred at room temperature for
2 h. The organic solvent was removed by evaporation under
reduced pressure. An aqueous solution of 5% NaHCO3 (20 mL)
was added to the residue, and the mixture was extracted with
EtOAc (40 mL ꢀ 2). The organic layer was washed with brine
(20 mL) and dried (Na2SO4). Filtration and evaporation under
reduced pressure gave 13b as a yellow oil (0.801 g, 88%). IR
(KBr) 2960, 1473, 1455, 1253, 1051 cm-1; 1H NMR (300 MHz,
CDCl3) δ 8.48 (s, 1 H), 7.35 (d, J = 8.1 Hz, 1 H), 6.74 (d, J =
8.1 Hz, 1 H), 5.97 (s, 2 H), 3.95 (s, 3 H), 3.70 (s, 2 H), 2.54 (broad
s, 4 H), 1.72 (broad s, 4 H); ESIMS m/z (rel intensity) 263 (MHþ,
100). Anal. (C14H18N2O3) C, H, N.
1
clear colorless oil (10.65 g, 88%). H NMR (300 MHz, CDCl3)
δ 6.78-6.84 (m, 3 H), 4.44 (t, J=5.4 Hz, 1 H), 3.88 (s, 3 H), 3.86 (s,
3 H), 3.70 (s, 2 H), 3.33 (s, 6 H), 2.69 (d, J=5.4 Hz, 2 H).
2-Methylpiperonal (10). n-BuLi (2.5 M in THF, 26.4 mL,
66 mmol) was added dropwise to a solution of N,N,N0-(tri-
methyl)ethylenediamine (8.6 mL, 6.79 g, 66.4 mmol) in THF
(120 mL) at -78 °C (acetone-dry ice bath). After 15 min,
piperonal (9.0 g, 60 mmol) was added, the mixture was stirred
for 15 min, and n-BuLi (2.5 M in THF, 72 mL, 180 mmol) was
added via syringe. After the reaction mixture was stirred for
1 h at -78 °C, CH3I (22.4 mL, 50.92 g, 360 mmol) was added at
-78 °C. The mixture was stirred for 2 h at room temperature,
poured into cold stirred 10% hydrochloric acid (prepared with
100 mL of concentrated hydrochloric acid and 360 mL of water),
extracted with CHCl3 (400 mLꢀ3), washed with brine (400 mL),
dried (Na2SO4), filtered, and concentrated to give the crude
product. Purification by preparative flash chromatography
(SiO2, EtOAc-hexane 10:1) gave the white crystalline product
(E)-4-[(1H-Imidazol-1-yl)methyl]benzo[d][1,3]dioxole-5-carbalde-
hyde O-Methyloxime (13c). A THF solution (3 mL) of the oxime
12 (0.40 g, 1.47 mmol) was added by syringe during 15 min to a
solution of imidazole (0.40 g, 5.88 mmol) in THF (10 mL) at
0 °C. The reaction mixture was stirred at room temperature for
2 h. The organic solvent was removed by evaporation under
reduced pressure. An aqueous 5% NaHCO3 solution (10 mL)
was added to the residue, and the mixture was extracted with
EtOAc (40 mL ꢀ 2). The organic layer was washed with brine
(20 mL) and dried (Na2SO4). Filtration and evaporation under
reduced pressure gave 13c as a yellow solid (0.32 g, 84%): mp
1
(6.71 g, 68.1%): mp 70-71 °C. H NMR (300 MHz, CDCl3)
δ 9.96 (s, 1 H), 7.34 (d, J=8.1 Hz, 1 H), 6.78 (d, J=8.1 Hz, 1 H),
6.04 (s, 2 H), 2.50 (s, 3 H).
(E)-2-Methylpiperonal O-Methyloxime (11). 2-Methylpiper-
onal (10, 6.00 g, 36.5 mmol), O-methylhydroxyamine hydro-
chloride (6.11 g, 73.2 mmol), and sodium acetate hydrate (19.92 g,
146.4 mmol) were dissolved in methanol (200 mL), and the
mixture was stirred at room temperature for 1 h. The reaction
mixture was concentrated to dryness, ethyl acetate (200 mL) was
added to the residue, and then 5% NaHCO3 was added to
neutralize acid. The solution was washed with brine (100 mL),
dried (Na2SO4), filtered, and evaporated under vacuum to
afford 11 (5.92 g, 84%) as a solid: mp 34 °C. IR (KBr) 2894,
1642, 1598, 1458, 1343, 1270, 1055 cm-1; 1H NMR (300 MHz,
CDCl3) δ 8.18 (s, 1 H), 7.17 (d, J = 8.1 Hz, 1 H), 6.66 (d, J =
8.1 Hz, 1 H), 5.96 (s, 2 H), 3.93 (s, 3 H), 2.28 (s, 3 H); low
resolution ESIMS m/z (rel intensity) 194 (MHþ, 100). Anal.
(C10H11NO3) C, H, N.
102 °C. IR (KBr) 2964, 1462, 1271, 1051 cm-1 1H NMR
;
(300 MHz, CDCl3) δ 8.06 (s, 1 H), 7.63 (s, 1 H), 7.03 (brs,
1 H), 6.97 (brs, 1 H), 6.90 (d, J=8.1 Hz, 1 H), 6.78 (d, J=8.1 Hz,
1 H), 6.04 (s, 2 H), 5.43 (s, 2 H), 3.96 (s, 3 H); ESIMS m/z (rel
intensity) 260 (MHþ, 100). Anal. (C13H13N3O3) C, H, N.
4-[(4-Methylpiperazin-1-yl)methyl]benzo[d][1,3]dioxole-5-car-
baldehyde O-Methyloxime (13d). A solution of the oxime 12
(360.0 mg, 1.324 mmol) in THF (10 mL) was added by syringe
during 30 min to a solution of 4-methylpiperazine (400.0 mg,
4.00 mmol) in THF (10 mL) at 0 °C. The reaction mixture was
then stirred at room temperature for 4 h. The organic solvent
was removed by evaporation under reduced pressure. An aqu-
eous 5% NaHCO3 solution (20 mL) was added to the residue,
and the mixture was extracted with EtOAc (40 mLꢀ2). The orga-
nic layer was washed with brine (20 mL) and dried (Na2SO4).
(E)-2-Bromomethylpiperonal O-Methyloxime (12). A solution
of the oxime 11 (4.00 g, 20.7 mmol), NBS (4.43 g, 2.49 mmol),

1986 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Song et al.
Filtration and evaporation under reduced pressure gave 13d as a
yellow solid (303.1 mg, 78%): mp 100-102 °C. IR 2937, 1473,
1455, 1295, 1050 cm-1; 1H NMR (300 MHz, CDCl3) δ 8.47 (s,
1 H), 7.39 (d, J=8.2 Hz, 1 H), 6.74 (d, J=8.2 Hz, 1 H), 5.97 (s,
2 H), 3.96 (s, 3 H), 3.96 (s, 2 H), 3.53 (s, 3 H), 2.46 (brs, 8 H), 2.26
(s, 3 H); ESIMS m/z (rel intensity) 292 (MHþ, 100), 192 (10).
4-(Morpholinomethyl)benzo[d][1,3]dioxole-5-carbaldehyde O-
Methyloxime (13e). A solution of the oxime 12 (400.0 mg, 1.47
mmol) in THF (10 mL) was added by syringe during 30 min to a
solution of morpholine (510.0 mg, 5.86 mmol) in THF (10 mL)
at 0 °C. The reaction mixture was stirred at room temperature
for 2 h. The organic solvent was removed by evaporation under
reduced pressure. An aqueous 5% NaHCO3 solution (20 mL)
was added to the residue, and the mixture was extracted with
EtOAc (40 mL ꢀ 2). The organic layer was washed with brine
(20 mL) and dried (Na2SO4). Filtration and evaporation under
reduced pressure gave 13e as a yellow oil (369.0 mg, 90.2%). IR
2896, 1473, 1455, 1254, 1050 cm-1; 1H NMR (300 MHz, CDCl3)
δ 8.46 (s, 1 H), 7.38 (d, J=8.4 Hz, 1 H), 6.74 (d, J=8.4 Hz, 1 H),
5.98 (s, 2 H), 3.95 (s, 3 H), 3.67 (t, J=4.6 Hz, 4 H), 3.53 (s, 2 H),
2.44 (t, J=4.6 Hz, 4 H); ESIMS m/z (rel intensity) 279 (MHþ,
100), 192 (30).
4-(((2-(Dimethylamino)ethyl)(methyl)amino)methyl)benzo[d]-
[1,3]dioxole-5-carbaldehyde O-Methyloxime (13f). A solution of
the oxime 12 (908.1 mg, 3.34 mmol) in THF (10 mL) was added
by syringe during 15 min to N,N,N0-trimethylethylenediamine
(1021.2 mg, 9.994 mmol) in THF (5 mL) at 0 °C. The reaction
mixture was stirred at room temperature for 4 h. The organic
solvent was removed by evaporation under reduced pressure.
An aqueous 5% NaHCO3 solution (10 mL) was added to the
residue, which was further extracted with EtOAc (20 mL ꢀ 2).
The organic layer was washed with brine (10 mL) and dried
(Na2SO4). Filtration and evaporation under reduced pressure
gave 13f as a yellow oil (485.2 mg, 49.4%). IR 2939, 1473, 1455,
1254, 1051 cm-1; 1H NMR (300 MHz, CDCl3) δ 8.51 (s, 1 H),
7.37 (d, J=8.1 Hz, 1 H), 6.73 (d, J=8.1 Hz, 1 H), 5.97 (s, 2 H),
3.94 (s, 3 H), 3.59 (s, 3 H), 2.52 (t, J=6.5 Hz, 2 H), 2.43 (t, J=
6.5 Hz, 2 H), 2.21 (s, 9 H); ESIMS m/z (rel intensity) 294 (MHþ,
100), 249 [(M -Me2NH)þ, 80].
2,3-Dimethoxy-8,9-methylenedioxy-10-(dimethylamino)methyl-
11H-indeno[1,2-c]isoquinoline (14a). The amine 8 (0.30 g, 1.18
mmol) and oxime ether 13a (0.126 g, 0.534 mmol) were mixed
with concentrated hydrochloric acid (7 mL), and the mixture
was stirred at 100 °C for 18 h. The reaction mixture was cooled
and washed with ether (3ꢀ20 mL). It was then brought to basic
pH with NH4OH. The mixture was extracted with chloroform
(40 mL ꢀ 3), and the solution was washed with brine (50 mL),
dried (Na2SO4), and concentrated. The residue was diluted in
chloroform (40 mL) and filtered. Hydrochloric acid (2 M HCl in
ether, 5 mL) was added to the filtrate. The precipitate that
formed was recrystallized from methanol to provide a yellow
solid. The solid was dissolved in water, basified with NH4OH,
and extracted with chloroform. The organic layer was concen-
trated to dryness. The residue was purified by preparative flash
chromatography (SiO2, methanol-chloroform-TEA=3:97:2)
to afford the final product 14a as a light-yellow solid (0.0162 g,
7.8%): mp 205-207 °C. IR (KBr) 2940, 1449, 1408, 1326, 1246,
1159, 1061 cm-1; 1H NMR (300 MHz, CDCl3) δ 8.98 (s, 1 H),
7.62 (s, 1 H), 7.23 (s, 1 H), 7.16 (s, 1 H), 6.11 (s, 2 H), 4.21 (s, 2 H),
4.10 (s, 3 H), 4.05 (s, 3 H), 2.77 (s, 6 H); low resolution ESIMS
m/z (rel intensity) 379 (MHþ, 100), 334 (90). Anal. (C22H22-
dried (Na2SO4), and concentrated. The residue was dissolved in
chloroform (40 mL) and filtered. Hydrochloric acid (2 M HCl in
ether, 5 mL) was added to the filtrate. The precipitate that
formed was recrystallized from methanol to provide a yellow
solid. The solid was dissolved in water, basified with NH4OH,
and extracted with chloroform. The organic layer was concen-
trated to dryness. The residue was purified by preparative flash
chromatography (SiO2, methanol-chloroform-TEA=3:97:2)
to yield the final pure product 14b as a light-yellow solid (84.9
mg, 6.1%): mp 210 °C (dec). IR (KBr) 2958, 1449, 1406, 1326,
1
1246 cm-1; H NMR (300 MHz, CDCl3) δ 8.94 (s, 1 H), 7.44
(s, 1 H), 7.16 (s, 1 H), 7.01 (s, 1 H), 6.04 (s, 2 H), 4.07 (s, 3 H), 4.02
(s, 3 H), 3.85 (s, 2 H), 3.79 (s, 2 H), 2.67 (broad s, 4 H), 1.82
(broad s, 4 H); low resolution ESIMS m/z (rel intensity) 405
(MHþ, 15), 334 (100). Anal. (C24H24N2O4 1.75H2O) C, H, N.
3
2,3-Dimethoxy-8,9-methylenedioxy-10-(1H-imidazol-yl)methyl-
11H-indeno[1,2-c]isoquinoline (14c). The amine 8 (0.905 g, 3.55
mmol) and oxime ether 13c (0.41 g, 1.6 mmol) were mixed with
concentrated hydrochloric acid (8 mL), and the mixture was
stirred at 100 °C for 18 h. The reaction mixture was then cooled
and washed with ether (3ꢀ20 mL). It was then brought to basic
pH with NH4OH. The mixture was extracted with chloroform
(40 mL ꢀ 3), and the solution was washed with brine (50 mL),
dried (Na2SO4), and concentrated. The residue was diluted in
chloroform (40 mL) and filtered. Hydrochloric acid (2 M HCl in
ether, 5 mL) was added to the filtrate. The precipitate that
formed was recrystallized from methanol to provide a yellow
solid. The solid was dissolved in water, basified with NH4OH,
and extracted with chloroform. The organic layer was concen-
trated to dryness. The residue was purified by preparative flash
chromatography (SiO2, methanol-chloroform-TEA =10:90:2)
to afford the final pure product 14c as a light-brown solid
(0.0419 g, 6.6%): mp 250 °C (dec). IR (KBr) 2961, 1496, 1413,
1207, 1020 cm-1; 1H NMR (300 MHz, DMSO) δ 9.01 (s, 1 H),
7.84 (s, 1 H), 7.54 (s, 1 H), 7.39 (s, 1 H), 7.31 (s, 1 H), 7.24 (s, 1 H),
6.90 (s, 1 H), 6.15 (s, 2 H), 5.33 (s, 2 H), 4.08 (s, 2 H), 3.99 (s, 3 H),
3.92 (s, 3 H); low resolution ESIMS m/z (rel intensity) 402
(MHþ, 100), 334 (15). Anal. (C23H19N3O4 1.5H2O) C, H, N.
3
2,3-Dimethoxy-8,9-methylenedioxy-10-(4-methylpiperazin-1-yl)-
methyl-11H-indeno[1, 2-c]isoquinoline (14d). The amine 8 (560.3
mg, 2.19 mmol) and oxime ether 13d (320.2 mg, 1.10 mmol) were
mixed together with concentrated hydrochloric acid (10 mL),
and the mixture was stirred at 100 °C for 3 h. The reaction
mixture was then cooled and washed with ether (3ꢀ 20 mL). It
was then brought to basic pH with 28% aqueous NH4OH. The
mixture was extracted with CHCl3 (3ꢀ40 mL), and the organic
layer was washed with brine (50 mL), dried with anhydrous
Na2SO4, and concentrated. The residue was diluted in CHCl3
(40 mL) and filtered. Hydrochloric acid (2 M HCl in ether,
10 mL) was added to the filtrate. A dark-yellow precipitate was
obtained and recrystallized from methanol to provide a bright-
yellow solid. NH4OH (2 mL) was added to the yellow solid, and
the mixture was concentrated to dryness. The residue was puri-
fied by preparative flash chromatography (SiO2, methanol-
chloroform-TEA=10:90:2) to yield the final pure product 14d
as a yellow solid (16.7 mg, 3.5%): mp 296 °C (dec). IR 3400,
1
1489, 1455, 1245, 1159 cm-1; H NMR (300 MHz, CDCl3) δ
8.98 (s, 1 H), 7.47 (s, 1 H), 7.24 (s, 1 H), 7.08 (s,1 H), 6.04 (s, 2 H),
4.11 (s, 3 H), 4.04 (s, 3 H), 3.91 (s, 2 H), 3.68 (s, 2 H), 2.65 (brs,
4 H), 2.56 (brs, 4 H), 2.34 (s, 3 H); ESIMS m/z (rel intensity) 434
(MHþ, 100), 334 (25). Anal. (C25H27N3O4 3.5H2O) C, H, N.
3
N2O4 1.25H2O) C, H, N.
2,3-Dimethoxy-8,9-methylenedioxy-10-(4-morpholino)methyl-
11H-indeno[1,2-c]isoquinoline (14e). The amine 8 (660.0 mg, 2.59
mmol) and oxime 13e (320.0 mg, 1.15 mmol) were mixed
together with concentrated hydrochloric acid (10 mL) and stir-
red at 100 °C for 3 h. The reaction mixture was cooled and
washed with ether (3ꢀ20 mL). It was brought to basic pH with
28% aqueous NH4OH. The mixture was extracted with CHCl3
(3ꢀ40 mL), and the organic layer was washed with brine (50 mL),
dried with anhydrous Na2SO4, and concentrated. The residue
3
2,3-Dimethoxy-8,9-methylenedioxy-10-(pyrrolidin-1-yl)methyl-
11H-indeno[1,2-c]isoquinoline (14b). The amine 8 (1.90 g, 7.45
mmol) and oxime ether 13b (0.90 g, 3.44 mmol) were mixed with
concentrated hydrochloric acid (15 mL), and the mixture was
stirred at 100 °C for 18 h. The reaction mixture was then cooled
and washed with ether (3ꢀ20 mL). It was then brought to basic
pH with NH4OH. The mixture was extracted with chloroform
(40 mL ꢀ 3), and the solution was washed with brine (50 mL),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 1987
was diluted in CHCl3 (40 mL) and filtered. Hydrochloric acid
(2 M HCl in ether, 10 mL) was added to the filtrate. A dark-
yellow precipitate was obtained and then recrystallized from
methanol to provide a bright-yellow solid. NH4OH (2 mL) was
added to the yellow solid, and the mixture was extracted with
CHCl3 (3ꢀ20 mL) and concentrated to dryness. The residue was
purified by preparative flash chromatography (SiO2, metha-
nol-chloroform-TEA = 5:95:2) to afford the final pure pro-
duct as a golden crystalline solid 14e (32.2 mg, 6.7%): mp 269 °C
DNA was employed. This 117-bp oligonucleotide contains a
single 50-cytosine overhang, which was 30-end-labeled by fill-in
reaction with [R-32P]dGTP in React 2 buffer (50 mM Tris-HCl,
pH 8.0, 100 mM MgCl2, 50 mM NaCl) with 0.5 units of DNA
polymerase I (Klenow fragment, New England BioLabs). Un-
incorporated [32P]dGTP was removed using mini Quick Spin
DNA columns (Roche, Indianapolis, IN), and the eluate con-
taining the 30-end-labeled DNA substrate was collected. App-
roximately 2 nM of radiolabeled DNA substrate was incu-
bated with recombinant Top1 in 20 μL of reaction buffer
[10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2,
0.1 mM EDTA, and 15 μg/mL BSA] at 25 °C for 20 min in
the presence of various concentrations of compounds. The
reactions were terminated by adding SDS (0.5% final con-
centration) followed by the addition of two volumes of loading
dye (80% formamide, 10 mM sodium hydroxide, 1 mM sodium
EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue).
Aliquots of each reaction mixture were subjected to 20%
denaturing PAGE. Gels were dried and visualized by using a
phosphoimager and ImageQuant software (Molecular Dynamics).
For simplicity, cleavage sites were numbered as previously des-
cribed in the 161-bp fragment.47
1
(dec). IR 3400, 1489, 1455, 1249, 1115 cm-1; H NMR (300
MHz, CDCl3) δ 8.99 (s, 1 H), 7.49 (s, 1 H), 7.11 (s, 1 H), 6.04 (s,
2 H), 4.12 (s, 3 H), 4.05 (s, 3 H), 3.97 (s, 2 H), 3.72 (t, J=4.5 Hz,
4 H), 3.68 (s, 2 H), 2.56 (t, J = 4.5 Hz, 4 H); ESIMS m/z
(rel intensity) 421 (MHþ, 100), 334 (32). Anal. (C24H24N2O5
0.4H2O) C, H, N.
3
2,3-Dimethoxy-8,9-methylenedioxy-10-(N,N,N0-trimethylethyl-
enediamino)methyl-11H-indeno[1,2-c]isoquinoline (14f). The
amine 8 (1020 mg, 4.00 mmol) and oxime ether 13f (458.1 mg,
1.56 mmol) were mixed together with concentrated hydrochloric
acid (10 mL), and the mixture was stirred at 100 °C for 18 h. The
reaction mixture was cooled and washed with ether (3ꢀ20 mL).
It was then brought to basic pH with NaOH (10% aqueous) at
0 °C (water-ice bath). The mixture was extracted with CHCl3
(3 ꢀ 40 mL), and the organic layer was washed with brine
(50 mL), dried with anhydrous Na2SO4, and concentrated.
The residue was diluted in CHCl3 (40 mL) and filtered. Hydro-
chloric acid (2 M HCl in ether, 10 mL) was added to the filtrate.
The dark-yellow precipitate was recrystallized from methanol to
provide a bright-yellow solid. NH4OH (2 mL) was added to the
yellow solid, and the mixture was concentrated to dryness. The
residue was purified by preparative flash chromatography
(SiO2, methanol-chloroform-TEA = 10:90:2) to provide the
final pure product as a yellow solid 14f (21.1 mg, 3.1%): mp 192 °C.
Molecular Docking. Norindenoisoquinoline 14b was built on
the basis of the crystal structure of 14a in the SYBYL 8.0.3
software from Tripos.48 The protonated form of 14b was further
built by modifying the atom type of the pyrrolidine nitrogen
atom from N3 to N4 and then adding hydrogen atoms. Both
molecules were minimized using the MMFF94s force field,
MMFF94 charges, and the conjugate gradient method, with
simplex initial optimization, to a gradient of less than 0.001 kcal/
mol. The conformations were docked to Top1-DNA complex
(PDB code 1tl8) by CCDC’s molecular docking software
GOLD49 3.0.1 using default settings. The binding region was
IR 3400, 2944, 1488, 1247, 1060 cm-1 1H NMR (300 MHz,
;
˚
defined as all the atoms that are 12 A around the centroid of
norindenoisoquinoline 5 in the crystal structure. Early termina-
CDCl3) δ 8.95 (s, 1 H), 7.45 (s, 1 H), 7.20 (s, 1 H), 7.04 (s, 1 H),
6.03 (s, 2 H), 4.09 (s, 3 H), 3.91 (s, 3 H), 3.84 (s, 2 H), 3.62 (s, 2 H),
2.58 (t, J=6.3 Hz, 2 H), 2.50 (t, J=6.3 Hz, 2 H), 2.29 (s, 3 H), 2.22
(s, 6 H); ESIMS m/z (rel intensity) 436 (MHþ, 85), 391(MHþ -
Me2NH, 22), 334 (MHþ-CH3NHCH2CH2N(CH3)2, 100); HRE-
SIMS calculated 435.2236, found 436.2240 (MHþ); purity 98.6%
(HPLC).
˚
tion was allowed if the top three solutions were within 1.5 A rms
deviation. To rank docking results, the GoldScore fitness scores
were used. The top three energy-ranked structures for each
ligand were saved for graphic analysis. The resulting complexes
were further minimized using MMFF94s force field, MMFF94
charges as above. The binding energies were calculated by the
following equation:
2,3-Dimethoxy-8,9-methylenedioxy-10-methyl-11H-indeno[1,2-c]-
isoquinoline (15). The amine 8 (0.640 g, 2.51 mmol) and 2-methyl-
piperonal (10, 0.450 g, 2.74 mmol) were mixed with concentrated
hydrochloric acid (8 mL), and the mixture was stirred at 100 °C for
18 h. The reaction mixture was then cooled and washed with ether
(3ꢀ 20 mL). It was then brought to basic pH with NH4OH. The
mixture was extracted with chloroform (40 mL ꢀ 3), and the
solution was washed with brine (50 mL), dried (Na2SO4), and
concentrated. The residue was dissolved in chloroform (40 mL)
and filtered. Hydrochloric acid (2 M HCl in ether, 8.7 mL) was
added to the filtrate. The precipitate that formed was recrystallized
from methanol to provide a yellow solid. The solid was dissolved in
water, basified with NH4OH, and extracted with chloroform. The
organic layer was concentrated to dryness. The residue was
purified by preparative flash chromatography (SiO2, aceto-
ne-chloroform-TEA=20:80:2) to afford the final pure product
as a light-yellow solid (0.025 g, 3.0%): mp 272-274 °C (dec). IR
(KBr) 2902, 1487, 1240, 1075 cm-1; 1H NMR (300 MHz, CDCl3)
δ 8.96 (s, 1 H), 7.41 (s, 1 H), 7.22 (s, 1 H), 7.21 (s, 1 H), 6.02 (s, 2 H),
4.07 (s, 3 H), 4.02 (s, 3 H), 3.78 (s, 2 H), 2.33 (s, 3 H); low resolution
Ebinding ¼ Ecomplex - Eprotein - Eligand
Quantum Chemistry Calculation. The DNA-binding energies
of 14a and 15 to the simplified DNA cleavage site of Top1, and
the solvation energies, were calculated using the methods and
strategies described in detail in our earlier publication.37 First,
the molecules were built starting from the crystal structure of
14a in SYBYL and geometry optimizations and frequency
calculations were carried out for each compound at the HF/
6-31G** level. The energy-minimized structures displayed no
imaginary frequencies and were therefore utilized to replace the
original ligand in the simplified DNA model. The model was
then subjected to single-point energy calculations using the MP2
method at the modified 6-31G*(0.25) level using the quantum
chemical program package Gaussian 03.50 The effect of solva-
tion was investigated using the polarizable continuum model
(PCM) at the MP2/6-31G*(0.25) level with the default radii
scheme.
The intermolecular interaction energy was calculated using
the supermolecular approach. The energy of interaction be-
tween the ligand and the neighboring DNA base pairs is defined
as the difference between the energy of the complex Ecomplex and
the energies of the monomers Eligand and Ebp. The basis set
superposition error (BSSE) was also corrected using the Boys
and Bernardi counterpoise method because of the use of an
incomplete basis set in practical applications of the supermole-
cular approach. Therefore, the interaction energies of the two
ESIMS m/z (rel intensity) 336 (MHþ, 100). Anal. (C20H17NO4
3
0.5H2O) C, H, N.
Topoisomerase I Mediated DNA Cleavage Reactions. Human
recombinant Top1 was purified from baculovirus as previously
described.46 DNA cleavage reactions were prepared as pre-
viously reported with the exception of the DNA substrate.13
Briefly, a 117-bp DNA oligonucleotide (Integrated DNA Tech-
nologies) encompassing the previously identified Top1 cleavage
sites in the 161-bp fragment from pBluescript SK(-) phagemid

1988 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Song et al.
compounds 14a and 15 were calculated at the MP2/6-31G*(0.25)
level through the following equation:
of Edotecarin, a Novel Topoisomerase I Inhibitor, Administered
Once Every 3 Weeks in Patients with Solid Tumors. Cancer Che-
mother. Pharmacol. 2006, 58, 173–182.
Eint ¼ Ecomplex - Eligand - Ebp þ BSSE
(19) Kohlhagen, G.; Paull, K.; Cushman, M.; Nagafuji, P.; Pommier, Y.
Protein-Linked DNA Strand Breaks Induced by NSC 314622, a
Novel Noncamptothecin Topoisomerase I Poison. Mol. Pharma-
col. 1998, 54, 50–58.
(20) Cushman, M.; Jayaraman, M.; Vroman, J. A.; Fukunaga, A. K.;
Fox, B. M.; Kohlhagen, G.; Strumberg, D.; Pommier, Y. Synthesis
of New Indeno[1,2-c]isoquinolines: Cytotoxic Non-Camptothecin
Topoisomerase I Inhibitors. J. Med. Chem. 2000, 43, 3688–3698.
(21) Fox, B. M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.;
Staker, B. L.; Stewart, L.; Cushman, M. Design, Synthesis, and
Biological Evaluation of Cytotoxic 11-Alkenylindenoisoquinoline
Topoisomerase I Inhibitors and Indenoisoquinoline-Camptothecin
Hybrids. J. Med. Chem. 2003, 46, 3275–3282.
Acknowledgment. This work was made possible by the
National Institutes of Health (NIH) through support of this
work with Research Grant UO1 CA89566. Some of this re-
search was conducted in a facility constructed with the finan-
cial support of a Research Facilities Improvement Program
Grant C06-14499 from the National Institutes of Health. We
thank the Rosen Center for Advanced Computing (RCAC),
Purdue University, for providing computing facilities.
(22) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman,
M. Synthesis of Nitrated Indenoisoquinolines as Topoisomerase I
Inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 3659–3663.
(23) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman,
M. A Systematic Study of Nitrated Indenoisoquinolines Reveals a
Potent Topoisomerase I Inhibitor. J. Med. Chem. 2006, 49, 7740–
7753.
(24) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman,
M. Synthesis of Benz[d]indeno[1,2-b]pyran-5,11-diones: Versatile
Intermediates for the Design and Synthesis of Topoisomerase I
Inhibitors. Bioorg. Med. Chem. Lett. 2006, 16, 1846–1849.
(25) Morrell, A.; Jayaraman, M.; Nagarajan, M.; Fox, B. M.; Meckley,
M. R.; Ioanoviciu, A.; Pommier, Y.; Antony, S.; Hollineshead, M.;
Cushman, M. Evaluation of Indenoisoquinoline Topoisomerase I
Inhibitors Using a Hollow Fiber Assay. Bioorg. Med. Chem. Lett.
2006, 16, 4395–4399.
(26) Morrell, A.; Placzek, M.; Parmley, S.; Antony, S.; Dexheimer,
T. S.; Pommier, Y.; Cushman, M. Nitrated Indenoisoquinolines as
Topoisomerase I Inhibitors: A Systematic Study and Optimization.
J. Med. Chem. 2007, 50, 4419–4430.
(27) Morrell, A.; Placzek, M.; Parmley, S.; Grella, B.; Antony, S.;
Pommier, Y.; Cushman, M. Optimization of the Indenone Ring
of Indenoisoquinoline Topoisomerase I Inhibitors. J. Med. Chem.
2007, 50, 4388–4404.
(28) Morrell, A.; Placzek, M. S.; Steffen, J. D.; Antony, S.; Agama, K.;
Pommier, Y.; Cushman, M. Investigation of the Lactam Side
Chain Length Necessary for Optimal Indenoisoquinoline Topoi-
somerase I Inhibition and Cytotoxicity in Human Cancer Cell
Cultures. J. Med. Chem. 2007, 50, 2040–2048.
(29) Nagarajan, M.; Morrell, A.; Antony, S.; Kohlhagen, G.; Agama,
K.; Pommier, Y.; Ragazzon, P. A.; Garbett, N. C.; Chaires, J. B.;
Hollingshead, M.; Cushman, M. Synthesis and Biological Evalua-
tion of Bisindenoisoquinolines as Topoisomerase I Inhibitors.
J. Med. Chem. 2006, 49, 5129–5140.
(30) Nagarajan, M.; Morrell, A.; Fort, B. C.; Meckley, M. R.; Antony,
S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Synthesis and
Anticancer Activity of Simplified Indenoisoquinoline Topoisome-
rase I Inhibitors Lacking Substituents on the Aromatic Rings.
J. Med. Chem. 2004, 47, 5651–5661.
(31) Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.; Kohlhagen,
G.; Agama, K.; Hollingshead, M.; Pommier, Y.; Cushman, M.
Synthesis and Evaluation of Indenoisoquinoline Topoisomerase I
Inhibitors Substituted with Nitrogen Heterocycles. J. Med. Chem.
2006, 49, 6283–6289.
Supporting Information Available: Elemental analysis results
for compounds 11, 12, 13a-c, 14a-e, and 15; HPLC trace for
compound 14f; docking scores; binding energies; and calculated
physical chemical properties including log P, log D, pKa. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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