Nitrated indenoisoquinolines as topoisomerase I inhibitors: A systematic study and optimization

Article abstract of DOI:10.1021/jm070361q

The biological activity of indenoisoquinoline topoisomerase I (Top1) inhibitors can be greatly enhanced depending on the choice of substituents on the aromatic rings and lactam side chain. Previously, it was discovered that a 3-nitro group and a 9-methoxy

Full text of DOI:10.1021/jm070361q

J. Med. Chem. 2007, 50, 4419-4430
4419
Nitrated Indenoisoquinolines as Topoisomerase I Inhibitors: A Systematic Study and
Optimization
†
†
†
‡
‡
‡
Andrew Morrell, Michael Placzek, Seth Parmley, Smitha Antony, Thomas S. Dexheimer, Yves Pommier, and
Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and the Purdue Cancer
Center, Purdue UniVersity, West Lafayette, Indiana 47907, and Laboratory of Molecular Pharmacology, Center for Cancer Research, National
Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255
ReceiVed March 27, 2007
The biological activity of indenoisoquinoline topoisomerase I (Top1) inhibitors can be greatly enhanced
depending on the choice of substituents on the aromatic rings and lactam side chain. Previously, it was
discovered that a 3-nitro group and a 9-methoxy group afforded enhanced biological activity. In the present
investigation, indenoisoquinoline analogues were systematically prepared using combinations of nitro groups,
methoxy groups, and hydrogen atoms in an effort to understand the contribution of each group toward
cytotoxicity and Top1 inhibition. Analysis of the biological results suggests that the nitro group is important
for Top1 inhibition and the methoxy group improves cytotoxicity. In addition, previously identified structure-
activity relationships were utilized to select favorable lactam side chain functionalities for incorporation on
the aromatic skeleton of analogues in this study. As a result, this investigation has provided optimal Top1
inhibitors equipotent to camptothecin that demonstrate low nanomolar cytotoxicities toward cancer cells.
Introduction
which results in a hydroxy acid product that has high affinity
12-15
for human serum albumin.
Indenoisoquinoline 1, unlike the
DNA topoisomerases are important for the successful replica-
tion, transcription, and recombination of DNA, as well as
chromatin remodeling.1-5 Thus, the proliferation and survival
of eukaryotic cells are dependent on topoisomerases, which are
therefore potential therapeutic targets for anticancer therapy. The
cellular role of topoisomerase I is to relieve torsional strain in
DNA by creating a single strand nick in the phosphodiester
backbone, a process that renders the enzyme covalently ligated
camptothecins, is hydrolytically stable but alternatively suffers
6
from intrinsically low biological activity. As a result, consider-
able research has been expended to improve the biological
9
,16-26
activity of the indenoisoquinolines.
Novel insights have
been gleaned regarding the contributions of the indenone ring,
isoquinoline ring, and lactam side chain toward biological
20,22,27-30
activity.
Recent efforts have indicated that compounds
3
-5 (Figure 1) represent novel lead compounds with potencies
1
to the DNA until strain is relieved. Thus, a potential therapeutic
comparable to the potencies of camptothecins and with cyto-
toxicities approximately 1000 times more potent than that of
agent could inhibit topoisomerase I (Top1) by two distinct
pathways: (1) “suppressing” its ability to nick the DNA strand
and (2) “poisoning” the enzyme by trapping it as a covalent
complex with DNA.
The identification of indenoisoquinoline 1 as a novel lead
molecule for the inhibition of Top1 resulted from a COMPARE
analysis of its cytotoxicity profile demonstrating a similarity to
3
0
the original lead molecule 1.
In the present study, second-generation analogues were
systematically prepared to examine the contributions of the nitro
group and methoxy group toward Top1 inhibition and cytotox-
icity. Previously observed structure-activity relationships have
indicated that lactam substituents such as amino, morpholino,
dimethylamino, ethanolamino, and imidazolyl afford excellent
biological activity for unsubstituted, dimethoxy-substituted, and
6
that of the known Top1 inhibitor, camptothecin (2). These
molecules intercalate between the DNA base pairs at the site
of single strand cleavage and effectively stabilize a ternary
complex consisting of the drug molecule, DNA, and Top1. This
mechanism of action classifies these inhibitors as Top1 poisons.
17,20,22,24,26,29,31
methylenedioxy-substituted indenoisoquinolines.
Thus, it seemed prudent to select these functionalities for
incorporation in the present study along with analogues pos-
sessing nitrated and methoxy-substituted aromatic rings. Figure
7
,8
Recently published X-ray crystal structures for camptothecins,
indenoisoquinolines,8,9 and an indolocarbazole elegantly dem-
8
2
indicates previously synthesized analogues of interest to the
onstrate this mechanism of action and illustrate the general
features for poisoning Top1 by intercalation at the cleavage
1
7,22,26
present study.
Previously, it has been demonstrated that
1
0
indenoisoquinolines possessing a 3-nitro group display enhanced
biological activity, especially when combined with a 9-methoxy
site.
Clinical validation of Top1 as a drug target has occurred,
with the camptothecin derivatives topotecan and irinotecan
representing the only Top1 inhibitors currently approved by the
U.S. Food and Drug Administration for anticancer therapy.
However, the camptothecins suffer from pharmacokinetic
problems, most notably the hydrolysis of their lactone ring,
2
1,30,32
group.
This enhancement of biological activity has been
hypothesized to result from favorable hydrogen-bonding interac-
tions and electrostatic charge complementarity between Top1,
the indenoisoquinolines, and the DNA base pairs in the cleavage
1
1
2
1,30
complex.
However, a systematic examination of the inde-
pendent effects that these substituents may exert on the
biological activity of the indenoisoquinolines has not been
conducted. As a result, the current study has been designed in
such a way as to improve the current level of understanding
regarding the effects of the nitro and methoxy groups on the
*
To whom correspondence should be addressed. Phone: 765-494-1465.
Fax: 765-494-6790. E-mail: cushman@pharmacy.purdue.edu.
†
‡
Purdue University
National Cancer Institute, NIH.
1
0.1021/jm070361q CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/15/2007

4
420 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Morrell et al.
compound (27, Scheme 2). This procedure was also utilized,
as previously described, for the preparation of compounds
1
7,22,26
6
-13.
With compounds 20, 24, 25, and 6 appropriately function-
alized for analogue synthesis, target compounds were then
prepared according to Scheme 3. The alkyl halide side chain
present in compound 6 was initially converted into the corre-
sponding alkylazide 28 by treatment with sodium azide in
dimethylsulfoxide. Compound 28 was not subjected to reduction
(
analogous to 22) because the corresponding derivative 7 was
2
2
previously prepared by an alternative method. Furthermore,
the corresponding azido- and amino-substituted analogues
derived from 20 and 26 have been previously prepared.2
Compounds 20, 24, and 25 were treated with morpholine in
refluxing 1,4-dioxane to provide target compounds 29-31.
Compounds 32-35 were prepared by treatment of 20, 24, 25,
and 6 with ethanolamine in DMSO and isolated as their
respective hydrochloride salts. Compounds 24 and 25 were
treated with dimethylamine in refluxing 1,4-dioxane to afford
target compounds 36 and 37, which were isolated as their
respective hydrochloride salts. Finally, target compounds 38 and
1,30
Figure 1. Representative topoisomerase I inhibitors.
3
9 were prepared by treatment of 24 and 25 with imidazole in
refluxing 1,4-dioxane.
Biological Results and Discussion
The indenoisoquinolines were examined for antiproliferative
activity against the human cancer cell lines in the National
Cancer Institute screen, in which the activity of each compound
was evaluated with approximately 55 different cancer cell lines
Figure 2. Indenoisoquinolines included in the current study.
3
7,38
of diverse tumor origins.
The GI50 values obtained with
biological activity of the indenoisoquinolines. Furthermore, the
study has been conducted utilizing lactam side chains expected
to confer enhanced biological activity to the analogues in an
effort to provide new lead molecules for anticancer therapy.
selected cell lines, along with the mean graph midpoint (MGM)
values, are summarized 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 GI values below and above the
5
0
-
8
-4
-8
test range (10 -10 M) are taken as the minimum (10 M)
Chemistry
-4
and maximum (10 M) drug concentrations used in the
The syntheses of advanced indenoisoquinoline analogues 20
and 22-25 are described in Scheme 1. Condensation of
homophthalic anhydrides 1433 and 15 with Schiff bases 16
and 17 provided carboxylic acids 18 and 19. The cis stereo-
chemical relationship was established by the observed coupling
screening test. For comparison purposes, the activities of the
previously reported lead compound 1, camptothecin (2), and
34
6,30
compounds 3-5 are also included in the table. The relative
potencies of the compounds in the production of topoisomerase
I mediated DNA cleavage are also listed in the table. These
results were expressed semiquantitatively as follows: 0, no
detectable activity; +, weak activity; ++, similar activity as
compound 1; +++ and ++++, greater activity than compound
1; ++++, similar activity as 1 µM camptothecin.
3
5
constant of ∼6 Hz for the two methine protons. These
carboxylic acids were subjected to oxidative Friedel-Crafts ring
closure with thionyl chloride36 and aluminum chloride
21,30
to
provide indenoisoquinolines 20 and 21. Indenoisoquinoline 3
3
0
was readily available from a previous synthetic endeavor.
Examination of the results in Table 1 indicates that the
substitution pattern at the indenoisoquinoline 3-position, 9-posi-
tion, and the lactam side chain has a pronounced effect on the
biological activities of the molecules. If an “active” compound
is defined as one that displays an MGM value of less than 1
µM or Top1 inhibitory activity of +++ or greater, then the
trends in substitution pattern can be readily evaluated. Four
groups of compounds can be categorized by evaluating the 3-
and 9-positions: group 1, hydrogen-substituted (6-9, 27, 28,
and 35); group 2, 3-position hydrogen and 9-methoxy (21-23,
31, 34, 37, and 39); group 3, 3-nitro and 9-position hydrogen
(10-13, 20, 29, and 32); group 4, 3-nitro and 9-methoxy (3-
5, 24, 30, 33, 36, and 38). These four groups all possess
optimized lactam side chains and differ only in the presence or
absence of a nitro or methoxy substituent at the 3- or 9-positions.
The enhancing effect of a 3-nitro group and a 9-methoxy group
on biological activity has previously been identified, and an
electrostatic charge complementarity hypothesis has been offered
It was quickly recognized that the choice of halide leaving
group on the lactam side chain was critical for successful SN2
displacement with organic amines such as dimethylamine,
ethanolamine, morpholine, and imidazole. Azide, however, was
a suitably active nucleophile for displacement of the alkylchlo-
ride present in 21 to provide analogue 22, which was subse-
quently reduced to provide compound 23 upon isolation as the
hydrochloride salt. Unsuccessful efforts to displace the alkyl
chloride motifs present in compounds 3 and 21 using organic
amines as nucleophiles prompted their conversion to the
corresponding alkyl iodides 24 and 25, thereby providing
advanced intermediates amenable to the preparation of the
desired analogues. Subsequent experiments determined that
lactam-substituted alkyl bromides, such as 20, were suitably
functionalized for nucleophilic displacement to provide the
desired analogues.
Benz[d]indeno[1,2-b]pyran-5,11-dione 2626 was treated with
the corresponding primary amine to provide the desired target
3
0
to help rationalize the effects. However, a detailed analysis

Indenoisoquinolines as Topoisomerase I Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4421
Scheme 1a
a
Reagents and conditions: (a) CHCl3, room temp; (b) (i) SOCl2, benzene, reflux; (ii) AlCl3, nitrobenzene, 100 °C; (c) NaN3, DMSO, 100 °C; (d) (i)
P(OEt)3, benzene, reflux; (ii) 3 M HCl in MeOH, reflux; (e) NaI, acetone, reflux.
Scheme 2
in the NCI 55 cell screen are not always strongly correlated
within the indenoisoquinoline class of Top1 inhibitors and
several of these molecules may be interacting with other cellular
targets leading to the observed cytotoxicity.
Previously derived structure-activity relationships predicted
that the group 3 analogues, with their 3-nitro substituent, would
greatly outperform the group 2 analogues in the biological
2
1,30
assays.
than predicted. In group 3, four compounds (11, 13, 29, and
2) displayed submicromolar MGM values. This was the same
Interestingly, the magnitude of this trend was less
of the extent to which each substituent modulates the biological
activity of the indenoisoquinolines has not been performed.
Thus, examination of these groups in the context of this study
should provide insight into the importance of each substituent
regarding cytotoxicity and Top1 inhibition.
3
number of compounds as that observed for group 2, thereby
indicating that the presence of the 3-nitro group did not improve
the relative cytotoxic nature of the analogues to a greater extent
than the 9-methoxy group. Five compounds (11, 12, 13, 29,
and 32) displayed Top1 inhibition of +++ or greater. This was
a slight improvement over the groups 1 and 2 analogues and
suggests a role for the 3-nitro group in improving Top1
inhibition. Furthermore, group 3 demonstrated an improvement
for both cytotoxicity and Top1 inhibition relative to group 1.
Thus, it appears that the 3-postion nitro group and the 9-methoxy
group are both independently capable of affording potent
biological activity. If this speculation is correct, then preparing
analogues with the 3- and 9-position both substituted with a
nitro and methoxy group, respectively, should provide more
potent analogues than in situations in which only one of the
Investigation of the results for group 1 (3- and 9-positions
substituted with hydrogen) reveals that only two compounds
(7 and 35) satisfy the criterion for active compounds as defined
by having an MGM value of less than 1 µM. Furthermore, four
compounds (7, 8, 9, and 35) possessed Top1 inhibitory activities
of +++ or greater. However, one can readily conclude from
Table 1 that substituting the 3- or 9-position with hydrogen
atoms is less optimal (relative to the other compounds in Table
1) for biological activity. It is noted that the compounds designed
and evaluated in group 1 were not intended to be the most active
compounds because this distinction was intended for group 4
on the basis of previously gleaned structure-activity relation-
3
0
2
1,30
two substituents is present.
ships.
However, the biological results for the compounds
Analysis of the analogues in group 4 appears to confirm this
hypothesis. In group 4 all eight analogues (3, 4, 5, 24, 30, 33,
in group 1 do serve to provide a benchmark for evaluating the
efficacy of the nitro and methoxy substituents at the 3- and
3
6, and 38) possessed submicromolar MGM values. Addition-
9-positions in the other groups.
ally, all of the compounds in group 4 displayed potent Top1
inhibition greater than or equal to +++. Furthermore, com-
pounds 5, 30, 33, 36, and 38 were all 2-3 times more cytotoxic
than camptothecin (2). Except for compound 4, all of the group
4 analogues were at least equipotent to camptothecin in their
abilities to inhibit the enzyme. A comparison of the analogues
in group 4 with those in group 1 provides an even more striking
example of the substituents’ ability to modulate biological
activity. The possession of both the nitro and methoxy substit-
uents provided 4 times as many “active” analogues based on
their MGM values. Additionally, those molecules were 20-
1000 times more cytotoxic. Thus, when it comes to the
In contrast to group 1, the analogues classified according to
group 2 (3-position hydrogen atom and 9-methoxy group)
displayed improved cytotoxicity profiles. Four compounds (23,
4, 37, and 39) possessed submicromolar MGM values (a 2-fold
improvement versus group 1) and four compounds (21, 22, 31,
and 39) demonstrated potent Top1 inhibition (+++). Therefore,
it can be concluded that the 9-methoxy substituent contributes
greatly to the improvement in cellular cytotoxicity relative to
the hydrogen-substituted analogues in group 1. Interestingly,
only analogue 39, with its imidazolyl-substituted lactam side
chain, fits the criterion established for an “active” compound
in both assays. This implies that Top1 inhibition and cytotoxicity
3

4
422 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Morrell et al.
Scheme 3a
compound 7 as the benchmark (since it possesses hydrogen
atoms substituted at the 3- and 9- positions), one can clearly
see that substituting a nitro group at the 3-position (analogue
1
1) resulted in a 2-fold increase in cytotoxicity with a
concomitant 20% increase in Top1 inhibition. Substituting a
methoxy group at the 9-position (analogue 23) resulted in a
7-fold increase in cytotoxicity but at the cost of Top1 inhibition.
Aromatic rings with both substituents (analogue 5) resulted in
a 12-fold increase in cytotoxicity and a 60% increase in Top1
inhibition. Therefore, it is possible to make the generalization
that the 3-nitro group tends to improve Top1 inhibition to a
greater extent than cytotoxicity and the 9-methoxy group
improves cytotoxicity more than Top1 inhibition.
Compounds 8 (MGM, 1.86 µM; Top1, +++), 12 (MGM,
.77 µM; Top1, +++), 36 (MGM, 0.020 µM; Top1, ++++),
9
and 37 (MGM, 0.300 µM; Top1, 0) all possess dimethylami-
nopropyl-substituted lactam nitrogens and displayed biological
activities similar to the previous group. Surprisingly, the trend
observed for the previous group with aminopropyl-substituted
lactam nitrogens was not exactly similar to the current group,
especially given the similarity of the lactam side chains. In the
current group, substituting the 3-position with a nitro group was
not as beneficial to the cytotoxicity (analogue 12) because it
was 5 times less active than the benchmark compound 8.
However, it did retain Top1 inhibition. Substituting the 9-posi-
tion with a methoxy group (analogue 37) resulted in a compound
that was 6 times more cytotoxic but provided a poor Top1
inhibitor. This was similar to the previously identified trend for
the 9-methoxy group. Accordingly, when the 3- and 9-positions
were substituted with the nitro and methoxy groups, respectively,
analogue 36 demonstrated a 93-fold improvement in cytotoxicity
and provided a potent Top1 inhibitor.
From continuation of the analysis, compounds 27 (MGM,
1
5.1 µM; Top1, ++), 29 (MGM, 0.632 µM; Top1, +++), 30
(MGM, 0.014 µM; Top1, ++++), and 31 (MGM, 2.70 µM;
Top1, +++) all possess morpholine-substituted lactam nitro-
gens. By return to a previously identified trend, substituting a
nitro group at the 3-position (analogue 29) resulted in a 24-
fold increase in cytotoxicity relative to the benchmark compound
27. Interestingly, the 9-methoxy-substituted analogue 31 did not
display the typical large increase in cytotoxicity as previously
observed, although a small increase in activity did occur and
good Top1 inhibition was maintained. Exactly as before,
substituting both the 3- and 9-positions resulted in an exceed-
ingly potent compound 30 that was over 1000 times more
cytotoxic than 27 and was a potent Top1 inhibitor.
a
Reagents and conditions: (a) NaN3, DMSO, 100 °C; (b) morpholine,
K2CO3, 1,4-dioxane, reflux; (c) 3 M HCl in MeOH; (d) ethanolamine,
DMSO, room temp; (e) dimethylamine, K2CO3, 1,4-dioxane, reflux; (f)
imidazole, K2CO3, 1,4-dioxane, reflux.
biological activity of these indenoisoquinoline analogues, a
definite synergistic benefit results from possessing both a 3-nitro
group and a 9-methoxy group.
The results in Table 1 also provide for an alternative method
of analysis by evaluating analogues with the same lactam side
chain but different 3- and 9-position substituents. From this
method, it follows that there are five groups represented in Table
The DNA cleavage patterns produced by camptothecin (2,
lane 3), the indenoisoquinoline NSC 314622 (lane 4), and
compounds 27 and 29-31 are displayed in Figure 3. The
following points are apparent from inspection of the gels: (1)
The potencies of the indenoisoquinolines as Top1 inhibitors are
reflected in the intensities of the DNA cleavage bands. The
bands produced by compounds 27 (Top1, ++), 29 (Top1,
+++), and 31 (Top1, +++) are slightly weaker in comparison
with 30 (Top1, ++++) and camptothecin (Top1, ++++). (2)
Top1 inhibitors can be classified as Top1 suppressors, which
inhibit DNA cleavage, and as Top1 poisons, which inhibit the
religation reaction after DNA cleavage. Many of the Top1-
mediated DNA cleavages are trapped at lower compound
concentrations and suppressed at higher concentrations, and
therefore, the indenoisoquinolines act as Top1 poisons at lower
concentrations and as Top1 suppressors at higher concentrations.
The suppression could result from binding of the drug to the
DNA, rendering it a poorer enzyme substrate at high drug
1
that differ by their respective lactam side chains (each of which
has been proven to improve the biological activity of the
indenoisoquinolines)1
group. Compounds 5 (MGM, 0.027 µM; Top1, ++++), 7
MGM, 0.320 µM; Top1, +++), 11 (MGM, 0.143 µM; Top1,
+++), and 23 (MGM, 0.048 µM; Top1, +) all possess
7,20,22,24,26,29,31
and four members in each
(
+
propylamino-substituted lactam nitrogens but have different
aromatic ring substituents. Thus, evaluating the differences in
the biological activities between these molecules should allow
conclusions to be drawn regarding specific contributions to the
biological activity by the 3- and 9-position substituents. Using

Indenoisoquinolines as Topoisomerase I Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4423
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
Top1
cleavage
b
c
compd
MGM (µM)
1
2
3
4
5
6
7
8
9
1.3
0.01
0.295
<0.001
<0.010
4.59
0.20
1.74
2.69
35
0.03
0.794
<0.001
<0.010
4.47
0.18
0.58
1.41
8.13
41
0.01
0.027
<0.001
<0.010
4.61
0.25
1.86
2.34
79.4
4.2
0.01
<0.010
73
68
0.02
<0.010
37
0.01
0.036
<0.001
96
20.0
++
0.22
3.39
0.155
2.82
23.5
1.38
1.7
0.04
3.24
0.251
3.31
21.5
0.78
2.82
2.75
>100
0.832
>100
2.17
>100
0.0405 ( 0.0187
0.178 ( 0.012
0.198 ( 0.18
0.027 ( 0.008
13.3 ( 4.80
0.32 ( 0.23
1.86
1.86
13.5
0.143 ( 0.097
9.77
++++
++++
+++
++++
+
<0.010
10.1
0.26
0.51
0.79
44.7
0.372
7.08
0.012
>100
<0.010
7
13
0.16
0.91
1.66
72.4
0.102
4.17
0.015
>100
0.22
1.32
1.41
>100
0.148
5.62
0.017
>100
+++
+++
++++
++
++++
+++
++++
0
+++
+++
+
++++
++
0
+++
++++
+++
++++
++++
0
++++
++++
0
1.66
10
11
12
13
20
21
22
23
24
27
28
29
30
31
32
33
34
35
36
37
38
39
>100
0.275
5.62
0.085
6.46
0.274
3.47
0.302
0.112
25.7
0.864
>100
0.19
0.016
>100
0.370 ( 0.28
40.0
33.9
<0.010
26.9
<0.010
0.046
44.7
75.9
52.5
0.028
3.02
16.6
7.94
>100
<0.010
0.309
21.4
61.7
64.6
0.059
1.48
>100
7.76
1.23
<0.010
4.68
38.9
0.038
0.058
3.47
0.014
0.034
7.24
29.5
0.085
0.048 ( 0.024
0.328 ( 0.046
15.1
0.148
5.37
19.5
3.72
7.59
0.021
<0.010
1.41
0.031
<0.010
0.026
0.195
<0.010
0.078
<0.010
0.056
4.90
25.1
12.3
0.038
<0.010
1.26
0.095
0.380
<0.010
1.58
0.200
<0.010
0.550
0.178
<0.010
1.00
0.309
<0.010
4.07
0.632 ( 0.029
0.014 ( 0.001
2.70 ( 0.125
0.296 ( 0.067
0.016
0.124 ( 0.014
0.339
0.020 ( 0.001
0.300 ( 0.072
0.019 ( 0.004
0.416 ( 0.134
<0.010
2.69
1.35
<0.010
0.417
0.550
0.028
0.427
0.020
1.66
1.95
2.29
0.027
>100
<0.010
0.112
0.550
<0.010
0.240
<0.010
0.178
0.229
0.012
0.158
0.269
<0.010
0.245
<0.010
0.676
>100
<0.010
0.055
0.174
<0.010
0.257
<0.010
0.204
1.07
<0.010
0.389
0.490
<0.010
0.617
<0.010
0.646
0.044
<0.010
0.102
<0.010
0.110
<0.010
0.071
++++
+++
a
The cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition. b Mean graph midpoint for growth inhibition of all human
c
cancer cell lines successfully tested. 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: +, weak activity; ++ and +++, modest activity; ++++, similar activity as 1 µM
camptothecin.
indenoisoquinolines. For example, the cleavages at base pairs
4
4 and 126 seen with the indenoisoquinolines are absent with
camptothecin. Alternatively, the cleavage at base pair 97 is much
more intense for camptothecin than for any of the indenoiso-
quinoline analogues. These differences may indicate that the
indenoisoquinolines might display antitumor spectra different
from camptothecin or its clinically useful derivatives irinotecan
and topotecan.
For compounds 32 (MGM, 0.296 µM; Top1, ++++), 33
(
0
MGM, 0.016 µM; Top1, ++++), 34 (MGM, 0.124 µM; Top1,
), and 35 (MGM, 0.339 µM; Top1, ++++), all possessing
an ethanolamino-substituted lactam nitrogen, the trends in
biological activity were less pronounced than the previous
groups because of the increased potency of the benchmark
compound 35. Substituting the 3-position with a nitro group
(analogue 32) had very little effect on the cytotoxicity, whereas
substituting the 9-position with a methoxy group resulted in
the typical increase in cytotoxic activity (although diminished
relative to the previously examined groups). Yet again, substi-
tuting both the 3- and 9-position with the nitro and methoxy
groups yielded an exceedingly potent compound 33 that is 21-
fold more cytotoxic than the benchmark compound 35. This
compound is even more interesting because of the ethanolamine-
containing side chain, which is envisioned to be a viable handle
for formulating the indenoisoquinoline via pegylation to improve
its solubility profile for advanced biological testing.
Figure 3. Lane 1: DNA alone. Lane 2: Top1 alone. Lane 3: +CPT
1 µM). Lane 4: Top1 + NSC 314622 (100 µM). Lanes 5-20 (for
compounds 27, 29, 30, and 31): Top1 + indicated compound at 0.1,
(
1
, 10, and 100 µM, respectively. Number on right and arrows indicate
cleavage site positions.
concentration, or from a direct effect on the enzyme to suppress
its ability to cleave DNA. Interestingly, compound 27 (the least
potent analogue of the series) was the only analogue presented
in Figure 3 that did not demonstrate this effect. (3) There are
differences in the cleavage pattern of camptothecin vs the
The final group of compounds to examine are those that
possess imidazolyl-substituted lactam nitrogens. Analogues 9
(MGM, 1.86 µM; Top1, ++++), 13 (MGM, 0.370 µM; Top1,
++++), 38 (MGM, 0.019 µM; Top1, ++++), and 39 (MGM,

4
424 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Morrell et al.
Figure 4. Hypothetical model of compound 5 (red) in ternary complex with DNA and Top1. Top1 amino acid residues not involved in bonding
interactions have been removed to improve clarity. The stereoview is programmed for wall-eyed (relaxed) viewing.
Figure 5. Hypothetical model of compound 23 (red) in ternary complex with DNA and Top1. Top1 amino acid residues not involved in bonding
interactions have been removed to improve clarity. The stereoview is programmed for wall-eyed (relaxed) viewing.
0
.416 µM; Top1, +++) were in general very potent compounds,
the effect results solely from the presence or absence of the
nitro group. Last, a hydrogen bond is proposed to exist between
the 11-position carbonyl oxygen of 5 and the guanidine protons
of Arg364 (heavy atom distance of 2.65 Å). Interestingly,
compound 23 displays a similar hydrogen-bonding interaction
with Arg364, but the interaction is envisioned to be weaker
(relative to 5) because of the increased heavy atom distance
(3.88 Å). This increase, along with the absence of a hydrogen-
bonding interaction with Asn722, allows compound 23 to orient
in such a way as to allow the 9-methoxy group to achieve a
nonplanar orientation extending into the nonscissile DNA strand.
Prior observations with camptothecins and indenoisoquinolines
have confirmed the detrimental effects on Top1 inhibition for
analogues possessing nonplanar substituents protruding into the
performing well in both the cytotoxicity and Top1 inhibition
assays. This tendency has been previously observed and was a
major determinant for the inclusion of the imidazole group in
2
0,26,29
the present study.
Unlike the previously analyzed group
of compounds (those possessing an ethanolamino-substituted
lactam nitrogen), introduction of a nitro group at the 3-position
had an improving effect on cytotoxicity, providing a compound
that was 5 times more cytotoxic than the benchmark compound
9. However, the Top1 inhibition remained unchanged (yet equal
in magnitude to camptothecin). In the present case, introducing
a methoxy group at the 9-position (analogue 39) resulted in a
less pronounced effect on cytotoxicity, providing a compound
only 4-fold more active than the benchmark analogue. Interest-
ingly, Top1 inhibition remained potent for 39 (Top1, +++),
which is in contrast to the other analogues with a similar
aromatic ring substitution investigated in this study. Thus, the
result highlights the ability of the imidazole group on the lactam
side chain to confer potent Top1 inhibition in the absence of
aromatic ring substituents that tend to promote such activity.
Last, substituting both the 3- and 9-positions with nitro and
methoxy groups, respectively, afforded an exceedingly potent
compound 38 similar to those in the previously observed groups.
In an attempt to further understand the contribution toward
Top1 inhibition by the 3-nitro group, hypothetical binding
models were developed using compounds 5 and 23 and a
12,30,39
nonscissile area of the cleavage complex.
Thus, the present
observations are significant because they rationalize the data
in Table 1 where Top1 inhibition is typically decreased for
analogues possessing a 3-position hydrogen atom and a 9-meth-
oxy group. Alternatively, the observations help to explain why
Top1 inhibition is significantly enhanced when a nitro group
replaces the 3-position hydrogen atom. From examination of
the data for compound 7 in Table 1, it appears to fit these
hypotheses because its cytotoxicity was less potent than 5 and
23 (predicted because it lacks the 9-methoxy group) and it was
a less potent topoisomerase I inhibitor than 5 (predicted because
7 lacks the 3-nitro group) but a more potent inhibitor than 23
(a less predictable result but still in line with the observation
that the 9-methoxy group is not the sole determining factor for
improving, or limiting, enzyme inhibition).
8
previously reported crystal structure of an indenoisoquinoline
in ternary complex with DNA and Top1 (Figures 4 and 5).
Compound 5 is capable of making three hydrogen-bonding
contacts to enhance the stabilization of the ternary complex,
illustrated as yellow lines in Figure 4. One oxygen atom of the
nitro group is shown to participate in hydrogen bonding with
the amide protons of Asn722 (heavy atom distance of 2.37 Å),
thereby providing one potential explanation for the increased
Top1 inhibition of indenoisoquinoline 5 relative to compound
Previous molecular modeling studies have indicated that
π-stacking plays a definitive role in the binding of indenoiso-
2
7
quinolines in the ternary complex. Utilizing charge comple-
mentarity to gain insight into the π-stacking interactions, a
hypothetical binding model has been developed that emphasizes
another role for the 3-nitro group (Figures 6 and 7). The models
illustrated in Figures 6 and 7 were constructed from the energy-
minimized complexes of 5 and 23 in ternary complex with DNA
and Top1. Replacement of the aminopropyl lactam side chains
present in compounds 5 and 23 with a methyl group was
performed in order to improve the overall contrast between areas
of net negative and positive charge in the model. This
simplification seemed justified because the present emphasis is
2
3. The amino group on the lactam side chain of 5 is suitably
positioned in the hypothetical model for hydrogen bonding with
an oxygen atom of thymine (heavy atom distance of 2.58 Å).
However, compound 23 is capable of interacting in a similar
manner (heavy atom distance 2.60 Å, Figure 5). This implies
that the lactam side chain interactions are not responsible for
the difference in biological activities between 5 and 23 and that

Indenoisoquinolines as Topoisomerase I Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4425
Figure 6. Electrostatic potential surfaces for truncated analogue 5 (solid surface) intercalated between DNA base pairs (line surface). The left
model is a view of the -1 base pair, and the right model is a view of the +1 base pair. Red indicates regions of negative charge, and blue indicates
regions of positive charge. All generated surfaces were globally scaled to a range of +25 to -25 kcal/mol and used MMFF94 charges.
Figure 7. Electrostatic potential surfaces for truncated analogue 23 (solid surface) intercalated between DNA base pairs (line surface). The left
model is a view of the -1 base pair, and the right model is a view of the +1 base pair. Red indicates regions of negative charge, and blue indicates
regions of positive charge. All generated surfaces were globally scaled to a range of +25 to -25 kcal/mol and used MMFF94 charges.
on the aromatic ring substituents and not the lactam side chains,
which would be expected to have similar interactions for both
compounds in the ternary complex. Last, the deoxyribose sugars
of the DNA base pairs at the site of intercalation were replaced
with methyl groups, a modification previously demonstrated to
still allow accurate calculations predicting the binding of an
indenoisoquinoline.27 Electrostatic potential surfaces [globally
scaled to a range of -25 kcal/mol (red) to +25 kcal/mol (blue)]
were then generated in Sybyl 6.9 using MMFF94 charges for
the DNA base pairs (illustrated as lines) and 5 and 23 (both
shown as opaque). Each figure depicts orientations that allow
viewing of charge complementarity between the indenoiso-
quinoline and the -1 base pair (left) and +1 base pair (right).
base pairs). This deficiency results in good electrostatic comple-
mentarity of 5 with the DNA base pairs and contributes to the
stabilization of the complex from π-stacking interactions. This
rationale helps to explain the substantial loss in Top1 inhibition
reported in Table 1 from the replacement of the 3-nitro group
with a hydrogen atom (compound 23, Figure 7). Examination
of Figure 7 indicates that there is a substantial decrease in
beneficial electrostatic complementarity between the isoquino-
line ring (left edge of both orientations) and the DNA base pairs
in the cleavage complex. Thus, electrostatic charge comple-
mentarity, in conjunction with hydrogen-bonding to Asn722,
appears to rationalize the enhanced biological activity of Top1
inhibitors that possess a nitro substituent at the 3-position.
In Figure 6, the nitro group illustrates two beneficial effects
toward Top1 inhibition. First, the electron-rich oxygen atoms
of the nitro groups present in 5 complements the electron-
deficient outer edges of the scissile strand DNA base pair (left
edge of both orientations). Second, the electron-withdrawing
nature of the nitro group results in a relatively electron-deficient
indenoisoquinoline aromatic ring system (compared to the DNA
In conclusion, the present study provides a detailed analysis
of the effects of the 3-nitro group and the 9-methoxy group.
Trends observed from the biological activities reported in Table
1 indicate that the 9-methoxy group typically improves the
cytotoxicity of the indenoisoquinoline analogues at the expense
of Top1 inhibition. Alternatively, the 3-nitro group was found
to improve Top1 inhibition to a greater extent than cytotoxicity

4
426 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Morrell et al.
1573, 1514, 1479, 1258, and 1173 cm- ; H NMR (CD
1
1
(
although many compounds exhibited significant increases in
both assays). Furthermore, a comparison of analogues 5 and
3, utilizing hypothetical models, indicates the importance of
3
OD) δ
8.10 (dd, J ) 7.6 and 1.4 Hz, 1 H), 7.63 (d, J ) 7.6 Hz, 1 H), 7.55
(
2
6
dt, J ) 7.4 and 1.5 Hz, 1 H), 7.50-7.43 (m, 1 H), 6.97-6.95 (m,
2
H), 6.75-6.71 (m, 2 H), 5.13 (d, J ) 6.3 Hz, 1 H), 4.76 (d, J )
.2 Hz, 1 H), 3.98-3.92 (m, 1 H), 3.70 (s, 3 H), 3.61-3.57 (m, 2
the 3-nitro group in both hydrogen-bonding interactions with
Top1 and π-stacking interactions with the DNA base pairs in
the cleavage complex. Thus, by systematic examination of the
nitro and methoxy groups and by observation of differences in
the hypothetical models of the selected compounds, the en-
hanced biological activity previously observed for nitrated
indenoisoquinolines has been rationalized.21,26,28,30 The resulting
models will be useful for the future design of effective
topoisomerase I inhibitors. Additionally, the use of optimal
lactam side chains that tend to confer enhanced biological
activity to the indenoisoquinolines has succeeded in providing
new lead compounds with exceptionally potent biological
activity.
H), 3.22-3.17 (m, 1 H), 2.13-2.01 (m, 2 H); ESIMS m/z (rel
+
intensity) 374/376 (MH , 100/33). Anal. (C20
-(3-Bromopropyl)-5,6-dihydro-5,11-dioxo-3-nitro-11H-indeno-
1,2-c]isoquinoline (20). Thionyl chloride (5 mL) was added to a
4
H20ClNO ) C, H, N.
6
[
solution of cis-4-carboxy-3,4-dihydro-N-(3-bromopropyl)-3-phenyl-
7-nitro-1(2H)isoquinolone (18) (1.000 g, 2.308 mmol) in benzene
(50 mL). The reaction mixture was heated at reflux for 30 min,
allowed to cool to room temperature, and concentrated. The residue
was diluted with nitrobenzene (30 mL) and chilled in an ice bath,
and aluminum chloride (0.616 g, 4.616 mmol) was added. The
reaction mixture was removed from the bath and heated at 100 °C
for 1 h. Ice-water (100 mL) was added, and the solution was
extracted with CHCl
3
(3 × 100 mL). The combined organic layer
was washed with saturated NaHCO
3
(3 × 50 mL) and saturated
Experimental Section
NaCl (50 mL) and dried over sodium sulfate. The solution was
concentrated, hexanes (900 mL) were added, and liquid was
decanted. The obtained solid was washed with hexanes (100 mL),
and the liquid was again decanted. The crude solid was purified
General. Melting points were determined using capillary tubes
with a Mel-Temp apparatus and are uncorrected. Infrared spectra
were obtained using CHCl as the solvent unless otherwise
3
1
specified. The proton nuclear magnetic resonance ( H NMR) spectra
were recorded using an ARX300 300 MHz Bruker NMR spec-
trometer. IR spectra were recorded using a Perkin-Elmer 1600 series
FTIR spectrometer. Combustion microanalyses were performed at
the Purdue University Microanalysis Laboratory, and the reported
values were within 0.4% of the calculated values. Analytical thin-
layer chromatography was carried out on Baker-flex silica gel IB2-F
plates, and compounds were visualized with short-wavelength UV
light. Silica gel flash chromatography was performed using 230-
by flash column chromatography (SiO ), eluting with chloroform,
to provide an orange solid (0.432 g, 45%): mp 258-260 °C (dec).
IR (film) 1672, 1612, 1560, 1503, 1428, and 1337 cm ; H NMR
2
-
1 1
(CDCl ) δ 9.20 (d, J ) 2.4 Hz, 1 H), 8.89 (d, J ) 8.9 Hz, 1 H),
3
8.52 (dd, J ) 9.0 and 2.4 Hz, 1 H), 7.92-7.90 (m, 1 H), 7.75-
7.72 (m, 1 H), 7.57-7.52 (m, 2 H), 4.76-4.70 (m, 2 H), 3.70 (t,
J ) 6.2 Hz, 2 H), 2.54-2.48 (m, 2 H); CIMS m/z (rel intensity)
+
413/415 (MH , 100/82). Anal. (C H BrN O ) C, H, N.
19
13
2
4
6-(3-Chloropropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-
indeno[1,2-c]isoquinoline (21). Thionyl chloride (2 mL) was added
to a solution of cis-4-carboxy-N-(3-chloropropyl)-3,4-dihydro-3-
(4-methoxyphenyl)-1(2H)isoquinolone (19) (0.510 g, 1.364 mmol)
in benzene (40 mL). The reaction mixture was heated at reflux for
30 min, allowed to cool to room temperature, and concentrated.
The residue was diluted with nitrobenzene (20 mL) and chilled in
an ice bath, and aluminum chloride (0.364 g, 2.728 mmol) was
added. The reaction mixture was removed from the bath and heated
at 100 °C for 1.5 h. Ice-water (100 mL) was added, and the solution
4
00 mesh silica gel.
Benzylidene-(3-bromo-1-propylamine) (17). The hydrobromide
salt of 3-bromopropylamine (5.364 g, 24.50 mmol) was treated with
triethylamine (4 mL) in CHCl (100 mL), and the reaction mixture
was allowed to stir at room temperature for 5 min. Benzaldehyde
2.000 g, 18.85 mmol) and magnesium sulfate (6.000 g) were added,
3
(
and the reaction mixture was allowed to stir at room temperature
for 16 h. The reaction mixture was filtered, and the filter pad was
washed with CHCl
3 × 50 mL) and saturated NaCl (50 mL), dried over sodium sulfate,
and concentrated to provide a yellow oil (4.262 g, 100%). IR (film)
3
(50 mL). The filtrate was washed with water
(
was extracted with CHCl (3 × 50 mL). The combined organic
3
layer was washed with saturated NaHCO3 (3 × 50 mL) and
-
1 1
1
7
2
645, 754, and 693 cm ; H NMR (CDCl
3
) δ 8.34 (s, 1 H), 7.76-
saturated NaCl (50 mL) and dried over sodium sulfate. The solution
was concentrated, hexanes (250 mL) were added, and liquid was
decanted. The obtained solid was washed with hexanes (100 mL),
and the liquid was again decanted. The solid was purified by flash
.71 (m, 2 H), 7.44-7.40 (m, 3 H), 3.78 (dt, J ) 6.3 and 1.3 Hz,
H), 3.52 (t, J ) 6.5 Hz, 2 H), 2.31 (pent, J ) 6.4 Hz, 2 H);
+
ESIMS m/z (rel intensity) 226/228 (MH , 100/91). Anal. (C10
H
12
-
BrN) C, H, N.
column chromatography (SiO ), eluting with chloroform, to provide
2
cis-4-Carboxy-3,4-dihydro-N-(3-bromopropyl)-3-phenyl-7-ni-
tro-1(2H)-isoquinolone (18). 4-Nitrohomophthalic anhydride (14)
a purple-red solid (0.082 g, 17%) that was precipitated from EtOAc/
hexanes: mp 195-198 °C. IR (KBr) 1662, 1611, 1505, 1481, 1432,
-
1 1
(3.664 g, 17.69 mmol) was added to a chloroform (125 mL) solution
3
and 1299 cm ; H NMR (CDCl ) δ 8.67 (d, J ) 8.1 Hz, 1 H),
of benzylidene-(3-bromo-1-propylamine) (17) (4.000 g, 17.69
mmol), and the reaction mixture was allowed to stir at room
temperature for 1.25 h. The precipitate was filtered, washed with
chloroform (150 mL), and dried to provide a yellow solid (6.278
g, 82%): mp 158-160 °C. IR (KBr) 3435, 3061, 1743, 1638, 1520,
8.31 (dd, J ) 8.2 and 0.7 Hz, 1 H), 7.73-7.71 (m, 1 H), 7.66 (d,
J ) 8.4 Hz, 1 H), 7.45-7.40 (m, 1 H), 7.22 (d, J ) 2.6 Hz, 1 H),
6.86 (dd, J ) 8.4 and 2.6 Hz, 1 H), 4.67-4.62 (m, 2 H), 3.89 (s,
3 H), 3.83-3.79 (m, 2 H), 2.43-2.36 (m, 2 H); CIMS m/z (rel
+
intensity) 354/356 (MH , 100/30). Anal. (C20
3
H16ClNO ) C, H, N.
349, and 1191 cm- ; H NMR (CD
1
1
OD) δ 8.90 (d, J ) 2.5 Hz,
1
1
7
3
6-(3-Azidopropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-in-
deno[1,2-c]isoquinoline (22). Thionyl chloride (2 mL) was added
to a solution of cis-4-carboxy-N-(3-chloropropyl)-3,4-dihydro-3-
(4-methoxyphenyl)-1(2H)isoquinolone (21) (0.500 g, 1.337 mmol)
in benzene (40 mL). The reaction mixture was heated at reflux for
30 min, allowed to cool to room temperature, and concentrated.
The residue was diluted with CH Cl (30 mL) and chilled in an ice
H), 8.38 (dd, J ) 8.7 and 2.6 Hz, 1 H), 7.98-7.95 (m, 1 H),
.25-7.19 (m, 3 H), 7.07-7.03 (m, 2 H), 5.32 (d, J ) 6.2 Hz, 1
H), 4.96 (d, J ) 6.2 Hz, 1 H), 3.99-3.96 (m, 1 H), 3.52-3.46 (m,
2
m/z (rel intensity) 431/433 [(M - H ) , 12/9]. Anal. (C19
BrN ‚1.0H O) C, H, N.
H), 3.31-3.22 (m, 1 H), 2.26-2.12 (m, 2 H); negative ion ESIMS
+
-
17
H -
2
O
5
2
2
2
cis-4-Carboxy-N-(3-chloropropyl)-3,4-dihydro-3-(4-methox-
yphenyl)-1(2H)-isoquinolone (19). Homophthalic anhydride (15)
bath, and aluminum chloride (0.357 g, 2.675 mmol) was added.
The reaction mixture was removed from the bath and heated at
reflux for 1.5 h. Ice-water (100 mL) was added, and the solution
(
3.065 g, 18.90 mmol) was added to a chloroform (125 mL) solution
34
of 4-methoxybenzylidene-(3-chloro-1-propylamine) (16) (4.000
g, 18.90 mmol), and the reaction mixture was allowed to stir at
room temperature for 3 h. The precipitate was filtered, washed with
chloroform (100 mL), and dried to provide an off-white solid (4.723
g, 67%): mp 180-181 °C. IR (KBr) 3437, 2957, 1740, 1622, 1598,
was extracted with CHCl
3
(3 × 50 mL). The combined organic
layer was washed with saturated NaHCO
3
(3 × 50 mL) saturated
NaCl (50 mL) and dried over sodium sulfate. The solution was
concentrated, and sodium azide (0.087 g, 1.337 mmol) was added.
The reaction mixture was diluted with DMSO (50 mL) and heated

Indenoisoquinolines as Topoisomerase I Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4427
at 100 °C for 2 h. The reaction mixture was diluted with CHCl
3
MeOH (10 mL) was added dropwise. The reaction mixture was
allowed to stir at room temperature for 24 h, and the precipitate
was filtered to provide an orange solid (0.338 g, 98%): mp 310-
315 °C. IR (film) 3434, 2929, 2559, 2462, 1695, 1663, 1609, 1549,
(
175 mL), washed with water (4 × 30 mL) and saturated NaCl (30
mL), and dried over sodium sulfate. The solution was concentrated
to provide a crude solid that was purified by precipitation from
EtOAc/hexanes to afford a red solid (0.340 g, 71%): mp 172-
-
1 1
6
1504, 758 cm ; H NMR (DMSO-d ) δ 10.80 (s, 1 H), 8.60 (d, J
1
73 °C. IR (KBr) 2099, 1661, 1611, 1505, 1481, 1432, and 1300
) 8.0 Hz, 1 H), 8.24 (d, J ) 7.0 Hz, 1 H), 7.87-7.79 (m, 2 H),
7.60-7.49 (m, 4 H), 4.58-4.54 (m, 2 H), 3.96 (d, J ) 11.0 Hz, 2
H), 3.77 (t, J ) 11.9 Hz, 2 H), 3.36-3.30 (m, 2 H), 3.11-3.06
-
1 1
cm ; H NMR (CDCl
7
7
3
) δ 8.68 (d, J ) 8.1 Hz, 1 H), 8.32 (d, J )
.3 Hz, 1 H), 7.74-7.68 (m, 1 H), 7.61 (d, J ) 8.4 Hz, 1 H),
.46-7.40 (m, 1 H), 7.24 (d, J ) 2.6 Hz, 1 H), 6.89 (dd, J ) 8.4
+
(m, 2 H), 2.32-2.28 (m, 2 H); ESIMS m/z (rel intensity) 375 (MH ,
and 2.6 Hz, 1 H), 4.61 (t, J ) 7.5 Hz, 2 H), 3.90 (s, 3 H), 3.66 (t,
100). Anal. (C23
2 3 2
H23ClN O ‚1.0H O) C, H, N.
J ) 6.1 Hz, 2 H), 2.19-2.10 (m, 2 H); EIMS m/z (rel intensity)
6-(3-Azidopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (28). A solution of 6-(3-bromopropyl)-5,6-dihydro-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline (6) (0.256 g, 0.695
mmol) and sodium azide (0.090 g, 1.390 mmol) in DMSO (20 mL)
was allowed to stir at room temperature for 20 h. The reaction
+
3
60 (M , 4). Anal. (C20
H
16
N
4
3
O ) C, H, N.
6
-(3-Aminopropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-in-
deno[1,2-c]isoquinoline Hydrochloride (23). Triethyl phosphite
0.12 mL) was added to a solution of 6-(3-azidopropyl)-5,6-dihydro-
-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (22) (0.100
(
9
3
mixture was diluted with CHCl (100 mL), washed with water (3
g, 0.277 mmol) in benzene (50 mL), and the reaction mixture was
heated at reflux for 16 h. The reaction mixture was allowed to cool
to room temperature, 3 M HCl in methanol (10 mL) was added,
and the reaction mixture was heated at reflux for 3 h. The reaction
mixture was allowed to cool to room temperature, and the obtained
precipitate was filtered to provide an orange solid (0.085 g, 83%):
mp 272-275 °C (dec). IR (KBr) 3430, 1705, 1639, 1611, 1480,
× 50 mL) and saturated NaCl (50 mL), and dried over sodium
sulfate. The solution was concentrated and the crude solid was
purified by flash column chromatography (SiO
2
), eluting with
chloroform, to provide an orange solid (0.199 g, 87%): mp 144-
-
1
145 °C. IR (film) 2099, 1698, 1662, 1610, 1503, and 1318 cm
;
1
H NMR (DMSO-d ) δ 8.58 (d, J ) 8.1 Hz, 1 H), 8.23 (d, J ) 8.2
6
Hz, 1 H), 7.84-7.80 (m, 2 H), 7.61-7.50 (m, 4 H), 4.58 (t, J )
-
1 1
1
435, 1305, and 1229 cm ; H NMR (DMSO-d
6
) δ 8.54 (d, J )
7.6 Hz, 2 H), 3.63 (t, J ) 6.5 Hz, 2 H), 2.07-2.02 (m, 2 H); CIMS
+
8
.1 Hz, 1 H), 8.20 (d, J ) 8.1 Hz, 1 H), 7.88 (bs, 2 H), 7.83 (dt,
m/z (rel intensity) 331 (MH , 100). Anal. (C19
H
14
N O
4 2
) C, H, N.
J ) 6.9 and 1.3 Hz, 1 H), 7.52 (t, J ) 8.0 Hz, 1 H), 7.15 (d, J )
.6 Hz, 1 H), 7.02 (dd, J ) 8.4 and 2.6 Hz, 1 H), 4.56 (t, J ) 6.8
5,6-Dihydro-6-(3-morpholinyl-1-propyl)-3-nitro-5,11-dioxo-
11H-indeno[1,2-c]isoquinoline Hydrochloride (29). 6-(3-Bro-
mopropyl)-5,6-dihydro-5,11-dioxo-3-nitro-11H-indeno[1,2-c]iso-
quinoline (20) (0.200 g, 0.484 mmol) and K CO (0.201 g, 1.452
2
Hz, 2 H), 3.89 (s, 3 H), 2.99-2.94 (m, 2 H), 2.13-2.08 (m, 2 H);
+
2 3
ESIMS m/z (rel intensity) 335 (MH , 100). Anal. (C20H19ClN O )
2
3
C, H, N.
mmol) were diluted with 1,4-dioxane (50 mL). Morpholine (0.21
mL, 2.42 mmol) was added, and the reaction mixture was heated
at reflux for 16 h. The reaction mixture was concentrated, diluted
5
,6-Dihydro-6-(3-iodopropyl)-9-methoxy-3-nitro-5,11-dioxo-
1H-indeno[1,2-c]isoquinoline (24). Sodium iodide (1.300 g, 8.673
mmol) and 6-(3-chloropropyl)-5,6-dihydro-9-methoxy-3-nitro-5,-
1-dioxo-11H-indeno[1,2-c]isoquinoline (3) (0.200 g, 0.502 mmol)
1
with CHCl
saturated NaCl (50 mL). The organic layer was dried over sodium
sulfate and concentrated. The obtained solid was dissolved in CHCl
3
(150 mL), and washed with water (3 × 50 mL) and
1
were diluted with acetone (125 mL), and the mixture was heated
at reflux for 48 h. The reaction mixture was concentrated, diluted
3
(50 mL), 3 M HCl in MeOH was added, and the reaction mixture
was allowed to stir at room temperature for 1 h. The precipitate
with CHCl
and purified by flash column chromatography (SiO
chloroform, to provide a red solid (0.090 g, 37%): mp 243-246
C. IR (film) 1678, 1609, 1554, 1479, 1432, 1336, and 1232 cm-1
3
(300 mL), and filtered. The filtrate was concentrated
2
), eluting with
3
was filtered and washed with CHCl and hexanes to provide an
orange solid (0.152 g, 69%): mp 280-285 °C (dec). IR (KBr) 3428,
-1
1
°
;
2346, 1697, 1679, 1616, 1560, 1506, 1431, and 1339 cm ; H
NMR (DMSO-d ) δ 8.85 (d, J ) 2.4 Hz, 1 H), 8.70 (d, J ) 9.0
1
H NMR (DMSO-d
Hz, 1 H), 8.51 (dd, J ) 9.0 and 2.5 Hz, 1 H), 7.83 (d, J ) 8.5 Hz,
H), 7.14 (d, J ) 2.6 Hz, 1 H), 7.07 (dd, J ) 8.5 and 2.6 Hz, 1
H), 4.52 (t, J ) 7.6 Hz, 2 H), 3.90 (s, 3 H), 3.50 (t, J ) 7.1 Hz, 2
6
) δ 8.81 (d, J ) 2.2 Hz, 1 H), 8.60 (d, J ) 8.9
6
Hz, 1 H), 8.58 (dd, J ) 9.0 and 2.5 Hz, 1 H), 7.91 (d, J ) 7.5 Hz,
1 H), 7.68-7.56 (m, 3 H), 4.60 (t, J ) 6.8 Hz, 2 H), 3.96 (d, J )
12.0 Hz, 2 H), 3.80 (t, J ) 12.0 Hz, 2 H), 3.41-3.34 (m, 4 H),
1
+
H), 2.35-2.30 (m, 2 H); ESIMS m/z (rel intensity) 363 (MH
HI, 100). Anal. (C20 ) C, H, N.
,6-Dihydro-6-(3-iodopropyl)-9-methoxy-5,11-dioxo-11H-in-
-
3.08-3.04 (m, 2 H), 2.32-2.30 (m, 2 H); ESIMS m/z (rel intensity)
+
H15IN O
2 5
420 (MH , 100). Anal. (C23
H
22ClN
3
O
5
2
‚0.75H O) C, H, N.
5
5,6-Dihydro-9-methoxy-6-(3-morpholinyl-1-propyl)-3-nitro-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (30).
5,6-Dihydro-6-(3-iodopropyl)-9-methoxy-3-nitro-5,11-dioxo-11H-
deno[1,2-c]isoquinoline (25). Sodium iodide (2.338 g, 15.60 mmol)
and 6-(3-chloropropyl)-5,6-dihydro-9-methoxy-5,11-dioxo-11H-in-
deno[1,2-c]isoquinoline (21) (0.311 g, 0.879 mmol) were diluted
with acetone (500 mL), and the mixture was heated at reflux for
2 3
indeno[1,2-c]isoquinoline (24) (0.200 g, 0.408 mmol) and K CO
(0.169 g, 1.224 mmol) were diluted with 1,4-dioxane (50 mL).
Morpholine (0.18 mL, 2.04 mmol) was added, and the reaction
mixture was heated at reflux for 16 h. The reaction mixture was
4
8 h. The reaction mixture was concentrated, diluted with CHCl
300 mL), and filtered. The filtrate was concentrated and purified
by flash column chromatography (SiO ), eluting with chloroform,
to provide a red solid (0.240 g, 61%): mp 206-209 °C. IR (KBr)
3
(
2
3
concentrated, diluted with CHCl (200 mL), and washed with water
(4 × 50 mL) and saturated NaCl (50 mL). The organic layer was
651, 1610, 1478, 1433, 1300, and 1228 cm-1; H NMR (CDCl
1
)
dried over sodium sulfate and concentrated. The obtained solid was
1
3
δ 8.67 (d, J ) 7.8 Hz, 1 H), 8.31 (dd, J ) 8.2 and 0.7 Hz, 1 H),
3
dissolved in CHCl (100 mL), 3 M HCl in MeOH (10 mL) was
added, and the reaction mixture was allowed to stir at room
temperature for 3 h. The precipitate was filtered and washed with
7
7
.73 (dt, J ) 7.2 and 1.4 Hz, 1 H), 7.64 (d, J ) 8.4 Hz, 1 H),
.45-7.43 (m, 1 H), 7.22 (d, J ) 2.6 Hz, 1 H), 6.87 (dd, J ) 8.4
and 2.6 Hz, 1 H), 4.59-4.54 (m, 2 H), 3.90 (s, 3 H), 3.39 (t, J )
CHCl
3
and hexanes to provide a red solid (0.159 g, 80%): mp
6
.7 Hz, 2 H), 2.45-2.40 (m, 2 H); ESIMS m/z (rel intensity) 446
213-215 °C (dec). IR (KBr) 3422, 1682, 1611, 1554, 1504, 1480,
+
-1
1
(MH , 45). Anal. (C20
H
16INO
3
) C, H, N.
6
1435, and 1335 cm ; H NMR (DMSO-d ) δ 10.52 (bs, 1 H),
5
,6-Dihydro-6-(3-morpholinyl-1-propyl)-5,11-dioxo-11H-in-
8.85 (d, J ) 2.4 Hz, 1 H), 8.66 (d, J ) 9.0 Hz, 1 H), 8.56 (dd, J
) 9.0 and 2.5 Hz, 1 H), 7.82 (d, J ) 8.6 Hz, 1 H), 7.20 (d, J ) 2.5
Hz, 1 H), 7.08 (dd, J ) 8.5 and 2.6 Hz, 1 H), 4.56-4.54 (m, 2 H),
3.97-3.91 (m, 5 H), 3.75 (t, J ) 11.6 Hz, 2 H), 3.40-3.26 (m, 4
deno[1,2-c]isoquinoline Hydrochloride (27). 4-(3-Aminopropyl)-
morpholine (0.35 mL, 2.417 mmol) was added to a solution of
benz[d]indeno[1,2-b]pyran-5,11-dione (26) (0.200 g, 0.806 mmol)
in CHCl
room temperature for 48 h. The reaction mixture was diluted with
CHCl (100 mL), washed with H
O (3 × 25 mL) and saturated
aqueous NaCl (25 mL), dried over Na SO , and concentrated. The
obtained residue was diluted with CHCl (50 mL), and 3M HCl in
(50 mL). The reaction mixture was allowed to stir at
H), 3.08-3.06 (m, 2 H), 2.29-2.26 (m, 2 H); ESIMS m/z (rel
3
+
intensity) 450 (MH , 100). Anal. (C24
N.
3 6 2
H24ClN O ‚1.0H O) C, H,
3
2
2
4
5,6-Dihydro-9-methoxy-6-(3-morpholinyl-1-propyl)-5,11-di-
oxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (31). 5,6-Di-
3

4
428 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Morrell et al.
hydro-6-(3-iodopropyl)-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (25) (0.200 g, 0.449 mmol) and K CO (0.620 g, 4.492
sodium sulfate. The solution was concentrated and diluted with
CHCl (100 mL). Then 3 M HCl in MeOH (10 mL) was added,
3
2
3
mmol) were diluted with 1,4-dioxane (50 mL). Morpholine (0.39
mL, 4.492 mmol) was added, and the reaction mixture was heated
at reflux for 2 h. The reaction mixture was concentrated, diluted
and the reaction mixture was allowed to stir at room temperature
for 3 h. The reaction mixture was concentrated and the solid residue
was washed with CHCl
(0.086 g, 92%): mp 275 °C. IR (KBr) 3426, 1704, 1637, 1610,
3
and filtered to provide an orange solid
with CHCl
saturated NaCl (50 mL). The organic layer was dried over sodium
sulfate and concentrated. The obtained solid was dissolved in CHCl
100 mL), 3 M HCl in MeOH (10 mL) was added, and the reaction
mixture was allowed to stir at room temperature for 3 h. The
obtained precipitate was filtered and washed with CHCl and
3
(150 mL), and washed with water (4 × 50 mL) and
-
1 1
6
1477, 1437, 1308, and 1232 cm ; H NMR (DMSO-d ) δ 8.60
3
(bs, 2 H), 8.57 (d, J ) 8.3 Hz, 1 H), 8.21 (d, J ) 8.1 Hz, 1 H),
7.84-7.79 (m, 1 H), 7.73 (d, J ) 8.4 Hz, 1 H), 7.53-7.48 (m, 1
H), 7.17 (d, J ) 2.6 Hz, 1 H), 7.03 (dd, J ) 8.4 and 2.5 Hz, 1 H),
5.26 (t, J ) 4.8 Hz, 1 H), 4.56 (t, J ) 6.4 Hz, 2 H), 3.89 (s, 3 H),
3.64-3.60 (m, 2 H), 3.12-3.10 (m, 2 H), 3.01-2.98 (m, 2 H),
(
3
hexanes to provide a red solid (0.149 g, 75%): mp 265 °C (dec).
IR (KBr) 3428, 1659, 1506, 1479, 1458, 1430, 1301, 1227, and
+
2.22-2.19 (m, 2 H); ESIMS m/z (rel intensity) 379 (MH , 100).
-
1 1
1
7
(
126 cm ; H NMR (DMSO-d
6
) δ 10.65 (bs, 1 H), 8.54 (d, J )
Anal. (C22
2 4 2
H23ClN O ‚1.25H O) C, H, N.
.8 Hz, 1 H), 8.20 (d, J ) 8.1 Hz, 1 H), 7.83-7.78 (m, 1 H), 7.71
5,6-Dihydro-6-[3-(2-hydroxyethyl)amino-1-propyl]-5,11-dioxo-
11H-indeno[1,2-c]isoquinoline (35). Ethanolamine (0.333 g, 5.445
mmol) was added to a solution of 6-(3-bromopropyl)-5,6-dihydro-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline (6) (0.150 g, 0.363
mmol) in DMSO (100 mL), and the reaction mixture was allowed
to stir at room temperature for 35 h. The reaction mixture was
d, J ) 8.4 Hz, 1 H), 7.52-7.47 (m, 1 H), 7.15 (d, J ) 2.6 Hz, 1
H), 7.01 (dd, J ) 8.4 and 2.6 Hz, 1 H), 4.53-4.51 (m, 1 H), 3.95-
3
3
(
.89 (m, 5 H), 3.76-3.68 (m, 2 H), 3.42-3.35 (m, 4 H), 3.09-
.06 (m, 2 H), 2.28-2.25 (m, 2 H); ESIMS m/z (rel intensity) 405
+
MH , 100). Anal. (C24
H25ClN
2
4 2
O ‚0.75H O) C, H, N.
5
,6-Dihydro-6-[3-(2-hydroxyethyl)amino-1-propyl]-3-nitro-5,-
diluted with CHCl
and saturated NaCl (100 mL), and dried over sodium sulfate. The
solution was concentrated and diluted with CHCl (100 mL). Then
3
(200 mL), washed with water (4 × 100 mL)
11-dioxo-11H-indeno[1,2-c]isoquinoline (32). Ethanolamine (0.222
g, 3.630 mmol) was added to a solution of 6-(3-bromopropyl)-5,6-
dihydro-5,11-dioxo-3-nitro-11H-indeno[1,2-c]isoquinoline (20) (0.100
g, 0.242 mmol) in DMSO (100 mL), and the reaction mixture was
allowed to stir at room temperature for 16 h. The reaction mixture
3
3 M HCl in MeOH (10 mL) was added, and the reaction mixture
was allowed to stir at room temperature for 3 h. The reaction
mixture was concentrated, and the solid residue was washed with
was diluted with CHCl
mL) and saturated NaCl (100 mL), and dried over sodium sulfate.
The solution was concentrated and diluted with CHCl (100 mL).
3
(200 mL), washed with water (4 × 100
CHCl
3
and filtered to provide an orange solid (0.140 g, 99%): mp
280-283 °C (dec). IR (KBr) 3439, 2944, 2798, 1712, 1638, 1610,
1550, and 1503 cm ; H NMR (DMSO-d ) δ 8.61 (d, J ) 8.0 Hz,
6
-
1 1
3
Then 3 M HCl in MeOH (10 mL) was added, and the reaction
mixture was allowed to stir at room temperature for 3 h. The
reaction mixture was concentrated and the solid residue was washed
1 H), 8.25 (d, J ) 8.1 Hz, 1 H), 7.88-7.81 (m, 2 H), 7.63-7.53
(m, 4 H), 5.22-5.19 (m, 1 H), 4.60 (t, J ) 6.7 Hz, 2 H), 3.64-
3.61 (m, 2 H), 3.12-3.09 (m, 2 H), 3.00-2.98 (m, 2 H), 2.22-
+
with CHCl
3
and filtered to provide an orange solid (0.088 g,
2.18 (m, 2 H); ESIMS m/z (rel intensity) 349 (MH , 100). Anal.
8
1
2
5%): mp 265 °C (dec). IR (KBr) 3414, 1698, 1677, 1615, 1559,
(C21
H
21ClN
2
O
3
2
‚0.25H O) C, H, N.
-
1 1
504, 1428, and 1339 cm ; H NMR (DMSO-d
.4 Hz, 1 H), 8.77 (d, J ) 8.9 Hz, 1 H), 8.63 (bs, 1 H), 8.60 (dd,
6
) δ 8.92 (d, J )
5,6-Dihydro-9-methoxy-6-(3-dimethylamino-1-propyl)-3-nitro-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (36).
5,6-Dihydro-6-(3-iodopropyl)-9-methoxy-3-nitro-5,11-dioxo-11H-
indeno[1,2-c]isoquinoline (24) (0.121 g, 0.246 mmol) and K CO
(0.155 g, 1.12 mmol) were diluted with 1,4-dioxane (50 mL).
Dimethylamine (2 M solution in THF) (1.12 mL, 2.24 mmol) was
added, and the reaction mixture was heated at reflux. After 2 h,
dimethylamine (2 M solution in THF) (1.12 mL, 2.24 mmol) was
added and the reaction mixture was heated at reflux for 16 h. The
J ) 9.0 and 2.5 Hz, 1 H), 7.94 (d, J ) 7.0 Hz, 1 H), 7.71-7.62
(
m, 3 H), 5.27 (t, J ) 4.8 Hz, 1 H), 4.63 (t, J ) 7.2 Hz, 2 H), 3.65
2
3
(q, J ) 5.2 Hz, 2 H), 3.15 (bs, 2 H), 3.00 (bs, 2 H), 2.26-2.22 (m,
+
2
H); ESIMS m/z (rel intensity) 394 (MH , 100). Anal. (C21
H
20
-
ClN
,6-Dihydro-6-[3-(2-hydroxyethyl)amino-1-propyl]-9-methoxy-
-nitro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (33). Ethano-
lamine (0.187 g, 3.060 mmol) was added to a solution of
,6-dihydro-6-(3-iodopropyl)-9-methoxy-3-nitro-5,11-dioxo-11H-
indeno[1,2-c]isoquinoline (24) (0.100 g, 0.204 mmol) in DMSO
100 mL), and the reaction mixture was allowed to stir at room
temperature for 16 h. The reaction mixture was diluted with CHCl
3
5 2
O ‚0.75H O) C, H, N.
5
3
3
reaction mixture was concentrated, diluted with CHCl (200 mL),
5
and washed with water (4 × 50 mL) and saturated NaCl (50 mL).
The organic layer was dried over sodium sulfate and concentrated.
The crude product was purified by flash column chromatography
(SiO
CHCl
(
3
2
), eluting with a gradient of CHCl
3
/1% Et
3
N to 6% MeOH/
(50
(
(
250 mL), washed with water (3 × 100 mL) and saturated NaCl
50 mL), and dried over sodium sulfate. The solution was
(100 mL). Then 3 M HCl in
3
/1% Et N. The obtained solid was dissolved in CHCl
3
3
mL), 3 M HCl in MeOH (10 mL) was added, and the reaction
mixture was allowed to stir at room temperature for 3 h. The
concentrated and diluted with CHCl
3
MeOH (10 mL) was added, and the reaction mixture was allowed
to stir at room temperature for 2 h. The reaction mixture was
precipitate was filtered and washed with CHCl
3
and hexanes to
provide an orange solid (0.072 g, 65%): mp 280-285 °C (dec).
IR (KBr) 1681, 1609, 1554, 1478, and 1333 cm-
1
; H NMR
1
concentrated and the solid residue was washed with CHCl
3
and
filtered to provide a red solid (0.086 g, 91%): mp 255-258 °C
(DMSO-d ) δ 9.99 (bs, 1 H), 8.85 (d, J ) 2.4 Hz, 1 H), 8.67 (d, J
6
(
dec). IR (KBr) 3347, 2941, 2786, 1679, 1611, 1556, 1504, 1480,
) 9.0 Hz, 1 H), 8.57 (dd, J ) 8.9 and 2.4 Hz, 1 H), 7.82 (d, J )
8.6 Hz, 1 H), 7.21 (d, J ) 2.6 Hz, 1 H), 7.08 (dd, J ) 8.4 and 2.6
Hz, 1 H), 4.57 (t, J ) 6.6 Hz, 2 H), 3.92 (s, 3 H), 3.28-3.16 (m,
-
1 1
1
2
433, 1335, and 1229 cm ; H NMR (DMSO-d
6
) δ 8.81 (d, J )
.4 Hz, 1 H), 8.76 (bs, 1 H), 8.61 (d, J ) 9.0 Hz, 1 H), 8.52 (dd,
J ) 9.0 and 2.4 Hz, 1 H), 7.80 (d, J ) 8.5 Hz, 1 H), 7.14 (d, J )
2 H), 2.77 (s, 3 H), 2.75 (s, 3 H), 2.27-2.22 (m, 2 H); ESIMS m/z
+
2
4
2
4
.5 Hz, 1 H), 7.06 (dd, J ) 8.4 and 2.5 Hz, 1 H), 5.26 (bs, 1 H),
(rel intensity) 408 (MH , 100). Anal. (C22
H, N.
H
22ClN
3
O
5
2
‚1.0H O) C,
.55 (t, J ) 6.4 Hz, 2 H), 3.90 (s, 3 H), 3.65 (bs, 2 H), 3.15 (bs,
H), 2.99 (bs, 2 H), 2.23-2.21 (m, 2 H); ESIMS m/z (rel intensity)
5,6-Dihydro-9-methoxy-6-(3-dimethylamino-1-propyl)-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (37). 5,6-
Dihydro-6-(3-iodopropyl)-9-methoxy-5,11-dioxo-11H-indeno[1,2-
+
24 (MH , 100). Anal. (C22
H
22ClN
3
O
6
2
‚1.0H O) C, H, N.
5,6-Dihydro-6-[3-(2-hydroxyethyl)amino-1-propyl]-9-methoxy-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline (34). Ethanolamine (0.137
2 3
c]isoquinoline (25) (0.200 g, 0.449 mmol) and K CO (0.620 g,
g, 2.250 mmol) was added to a solution of 5,6-dihydro-6-(3-
iodopropyl)-9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquino-
line (25) (0.100 g, 0.225 mmol) in DMSO (50 mL), and the reaction
mixture was allowed to stir at room temperature for 16 h. The
4.492 mmol) were diluted with 1,4-dioxane (50 mL). Dimethy-
lamine (2 M solution in THF) (2.25 mL, 4.492 mmol) was added,
and the reaction mixture was heated at reflux for 4 h. The reaction
3
mixture was concentrated, diluted with CHCl (200 mL), and
reaction mixture was diluted with CHCl
water (3 × 50 mL) and saturated NaCl (50 mL), and dried over
3
(250 mL), washed with
washed with water (4 × 50 mL) and saturated NaCl (50 mL). The
organic layer was dried over sodium sulfate and concentrated. The

Indenoisoquinolines as Topoisomerase I Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4429
obtained solid was dissolved in CHCl
10 mL) was added, and the reaction mixture was allowed to stir
at room temperature for 2 h. The precipitate was filtered and washed
with CHCl to provide a purple solid (0.155 g, 90%): mp 275 °C
dec). IR (KBr) 3427, 1670, 1611, 1504, 1481, 1456, 1294, and
3
(50 mL), 3 M HCl in MeOH
mixed with 30 µL of loading buffer (80% formamide, 10 mM
sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and
0.1% bromophenol blue, pH 8.0). Aliquots were separated in
denaturing gels (16% polyacrylamine, 7 M urea). Gels were dried
and visualized by using a Phosphoimager and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA). The results of the assay
were expressed semiquantitatively as follows: 0, no detectable
activity; +, weak activity; ++, similar activity as compound 1;
+++ and ++++, greater activity than compound 1; ++++,
similar activity as 1 µM camptothecin.
(
3
(
-
1 1
1
7
7
2
2
2
6
243 cm ; H NMR (DMSO-d ) δ 10.35 (bs, 1 H), 8.53 (d, J )
.8 Hz, 1 H), 8.20 (d, J ) 8.0 Hz, 1 H), 7.83 (t, J ) 7.3 Hz, 1 H),
.72 (d, J ) 8.4 Hz, 1 H), 7.51 (t, J ) 7.4 Hz, 1 H), 7.14 (d, J )
.4 Hz, 1 H), 7.01 (dd, J ) 8.2 and 2.4 Hz, 1 H), 4.53-4.50 (m,
H), 3.89 (s, 3 H), 3.36-3.27 (m, 2 H), 2.75-2.74 (m, 6 H), 2.22-
+
.18 (m, 2 H); ESIMS m/z (rel intensity) 363 (MH , 100). Anal.
Molecular Modeling. The structure of the ternary complex,
containing topoisomerase I, DNA, and an indenoisoquinoline, was
(
C
22
H
23ClN
2
O
3
‚1.75H
,6-Dihydro-6-(3-imidazolyl-1-propyl)-9-methoxy-3-nitro-5,-
1-dioxo-11H-indeno[1,2-c]isoquinoline (38). 5,6-Dihydro-6-(3-
2
O) C, H, N.
8
5
downloaded from the Protein Data Bank (PDB code 1SC7). Several
1
of the atoms were then fixed according to the Sybyl atom types.
Hydrogens were added and minimized using the MMFF94s force
field and MMFF94 charges. Modeled analogues were constructed
in Sybyl 7.1, energy-minimized with the MMFF94s force field and
MMFF94 charges, and overlapped with the crystal structure ligand
in the ternary complex, and the crystal structure ligand was then
deleted. The new complex was subsequently subjected to energy
minimization using MMFF94s force field with MMFF94 charges.
During the energy minimization, the structure of the indenoiso-
quinoline was allowed to move while the structures of the protein
and nucleic acids were frozen. The energy minimization was
iodopropyl)-9-methoxy-3-nitro-5,11-dioxo-11H-indeno[1,2-c]iso-
quinoline (24) (0.135 g, 0.275 mmol) and K CO (0.190 g, 1.377
2
3
mmol) were diluted with 1,4-dioxane (50 mL). Imidazole (0.094
g, 1.377 mmol) was added, and the reaction mixture was heated at
reflux for 16 h. The reaction mixture was concentrated, diluted with
CHCl
3
(200 mL), and washed with water (3 × 50 mL) and saturated
NaCl (50 mL). The organic layer was dried over sodium sulfate
and concentrated. The crude product was purified by flash column
chromatography (SiO
to 4% MeOH/CHCl /1% Et
1%): mp 215 °C (dec). IR (KBr) 1678, 1610, 1554, 1504, 1478,
2
), eluting with a gradient of CHCl
3 3
/1% Et N
3
3
N, to provide a red solid (0.061 g,
-
1
-1
5
1
8
performed using the Powell method with a 0.05 kcal mol
Å
-
1 1
432, and 1335 cm ; H NMR (DMSO-d
6
) δ 8.85 (s, 1 H), 8.62-
energy gradient convergence criterion and a distance-dependent
dielectric function. Molecular surfaces were created using the
MOLCAD module implemented in Sybyl 6.9 with MMFF94
charges.
.61 (m, 1 H), 8.54 (d, J ) 9.2 Hz, 1 H), 7.74 (s, 1 H), 7.29 (s, 1
H), 7.25-7.23 (m, 1 H), 7.18 (s, 1 H), 6.97 (s, 1 H), 6.94 (d, J )
7
.2 Hz, 1 H), 4.46-4.44 (m, 2 H), 4.25 (t, J ) 6.8 Hz, 2 H), 3.90
+
(s, 3 H), 2.25-2.22 (m, 2 H); ESIMS m/z (rel intensity) 431 (MH ,
Acknowledgment. This work was made possible by the
National Institutes of Health (NIH) through support of this work
with Research Grant UO1 CA89566 and Training Grant ST32
CA09634-12 and by an ACS Medicinal Chemistry Predoctoral
Fellowship sponsored by Pfizer Global Research and Develop-
ment (A.M.). The in vitro testing was conducted through the
Developmental Therapeutics Program, DCTD, NCI, under
Contract NO1-CO-56000. This research was supported in part
by the Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research. This research was
conducted in a facility constructed with support from Research
Facilities Improvement Program Grant C06-14499 from the
National Center for Research Resources of the National
Institutes of Health.
1
00). Anal. (C23
H
18
N
4
O
5
2
‚1.25H O) C, H, N.
5
,6-Dihydro-6-(3-imidazolyl-1-propyl)-9-methoxy-5,11-dioxo-
11H-indeno[1,2-c]isoquinoline (39). 5,6-Dihydro-6-(3-iodopropyl)-
9-methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (25) (0.200
2 3
g, 0.449 mmol) and K CO (0.620 g, 4.492 mmol) were diluted
with 1,4-dioxane (50 mL). Imidazole (0.306 g, 4.492 mmol) was
added, and the reaction mixture was heated at reflux for 4 h. The
3
reaction mixture was concentrated, diluted with CHCl (150 mL),
and washed with water (3 × 50 mL) and saturated NaCl (50 mL).
The organic layer was dried over sodium sulfate and concentrated.
The crude product was purified by flash column chromatography
(
SiO
CHCl
50 °C. IR (KBr) 1650, 1610, 1505, 1479, 1459, 1431, 1299, 1226,
2
), eluting with a gradient of CHCl
3 3
/1% Et N to 6% MeOH/
3
/1% Et N, to provide a red solid (0.112 g, 65%): mp 145-
3
1
-
1 1
and 788 cm ; H NMR (DMSO-d
.29 (d, J ) 8.2 Hz, 1 H), 7.72-7.66 (m, 2 H), 7.44 (t, J ) 7.1
Hz, 1 H), 7.19 (s, 1 H), 7.15 (d, J ) 2.5 Hz, 1 H), 7.07 (s, 1 H),
.66 (dd, J ) 8.4 and 2.5 Hz, 1 H), 6.59 (d, J ) 8.3 Hz, 1 H), 4.50
t, J ) 7.3 Hz, 2 H), 4.25 (t, J ) 6.4 Hz, 2 H), 3.85 (s, 3 H),
6
) δ 8.63 (d, J ) 8.1 Hz, 1 H),
8
Supporting Information Available: Elemental analysis results
for compounds 17-23 and 25-39. This material is available free
of charge via the Internet at http://pubs.acs.org.
6
(
2
+
.39-2.32 (m, 2 H); ESIMS m/z (rel intensity) 386 (MH , 63).
References
Anal. (C23 ‚0.5H O) C, H, N.
H N
19 3
O
3
2
(
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JM070361Q