Investigation of the lactam side chain length necessary for optimal indenoisoquinoline topoisomerase I inhibition and cytotoxicity in human cancer cell cultures

Article abstract of DOI:10.1021/jm0613119

Indenoisoquinolines with lactam substituents such as ethylamino, propylamino, and butylamino have previously demonstrated potent biological activity, but an optimal length has never been established. In the present study, a series of simplified indenoisoquinoline analogues possessing a linker spacing of 0-12 carbon atoms between the lactam nitrogen and the terminal amino group have been prepared, determining that 2-4-atom lengths are optimal for topoisomerase I inhibition and cytotoxicity. Using these lengths, analogues were prepared with the amino group and portions of the linker replaced by a pyridine ring. A three-carbon spacer within the pyridine series still demonstrated potent topoisomerase I inhibition.

Full text of DOI:10.1021/jm0613119

2040
J. Med. Chem. 2007, 50, 2040-2048
Investigation of the Lactam Side Chain Length Necessary for Optimal Indenoisoquinoline
Topoisomerase I Inhibition and Cytotoxicity in Human Cancer Cell Cultures
Andrew Morrell,† Michael S. Placzek,† Jamin D. Steffen,† Smitha Antony,‡ Keli Agama,‡ 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, Bethesda, Maryland 20892-4255
ReceiVed NoVember 11, 2006
Indenoisoquinolines with lactam substituents such as ethylamino, propylamino, and butylamino have
previously demonstrated potent biological activity, but an optimal length has never been established. In the
present study, a series of simplified indenoisoquinoline analogues possessing a linker spacing of 0-12
carbon atoms between the lactam nitrogen and the terminal amino group have been prepared, determining
that 2-4-atom lengths are optimal for topoisomerase I inhibition and cytotoxicity. Using these lengths,
analogues were prepared with the amino group and portions of the linker replaced by a pyridine ring. A
three-carbon spacer within the pyridine series still demonstrated potent topoisomerase I inhibition.
Introduction
Topoisomerases are nature’s solution to the topological
problems associated with manipulating double-stranded, helical
DNA during essential cellular processes.1-4 The main clas-
sification of topoisomerases is dependent upon the enzyme’s
ability to create a single-stranded (type I) or double-stranded
(type II) nick in DNA to alleviate torsional strain.1-4 Interest
Figure 1. Representative topoisomerase I inhibitors.
in topoisomerase I (top1) as a potential therapeutic target
originated from the characterization of camptothecin (1; Figure
1) as a selective mammalian topoisomerase I inhibitor.5-7
Validation of drug development for this target occurred with
the approval of two camptothecin derivatives, topotecan and
irinotecan, for the treatment of various forms of cancer.7,8
However, these drugs are less than optimal, each possessing a
hydrolytically unstable lactone ring that when hydrolyzed affords
a product with high affinity for serum albumin.9-12 Therefore,
novel, hydrolytically stable top1 inhibitors would represent an
improvement in top1-targeted anticancer chemotherapy.8
The top1 inhibitory activity of indenoisoquinoline 2 (Figure
1) was determined after a COMPARE analysis of its cytotoxicity
profile in the National Cancer Institute’s (NCI’s) 60-cell screen
indicated a high degree of correlation with camptothecin.13
Subsequent experimentation confirmed the activity of 2 as a
top1 inhibitor with modest potency. The mechanism of action
of the indenoisoquinolines involves the stabilization of the
ternary cleavage complex by intercalation between the DNA
base pairs upon single-strand cleavage by top1 and formation
of a network of hydrogen bonds with top1, thus preventing
religation of the broken phosphodiester backbone, a mechanism
identical to that of the camptothecins. Recent crystallographic
analyses elegantly demonstrate the inhibition of top1 by these
compounds and illustrate the general features of interfacial top1
inhibition by intercalation and specific contacts with top1 amino
acid residues.14-17 In addition, ab initio calculations have
underscored the primary importance of π-stacking interactions
in the stabilization of the ternary complex.18
Previously, it was demonstrated that ethylamino-, propyl-
amino-, and butylamino-substituted lactam side chains conferred
potent biological activity for unsubstituted indenoisoquinolines.19
Indeed, the influence of these three substituents on biological
activity has made them a standard functionality for incorporation
into variously substituted indenoisoquinoline analogues. Fur-
thermore, the 2-4-carbon spacing has been, and is currently,
utilized for the development of highly potent indenoisoquino-
lines with various functional groups at the end of this linker.19-30
However, the determination of optimal spacing between the
terminal amino substituent and the lactam nitrogen has not yet
been confirmed experimentally, and its establishment could lead
to the improvement of indenoisoquinoline top1 inhibitors.
Therefore, a complete series of simplified indenoisoquinoline
homologues possessing a linker spacing of 0-12 carbon atoms
between the lactam nitrogen and the terminal amino group have
been prepared and evaluated for both cytotoxicity and top1
inhibition in an effort to determine the optimal chain length for
biological activity.
Chemistry
Preparation of analogues for this study began with the
monoprotection of diaminoalkanes 3-11 with Boc2O according
to standard methods (Scheme 1). With compounds 12-20
suitably protected, analogues 25-36 were prepared by the
treatment of 12-24 with benz[d]indeno[1,2-b]pyran-5,11-dione
(21)29 (Scheme 2). Through experimentation, it was determined
that the monoprotection of diaminoalkanes with lengths of less
than seven carbons was not entirely necessary. Employment of
excess diaminoalkanes provided a suitable method to prevent
large quantities of dimerized product from forming, and the
diaminoalkanes were sufficiently water soluble to allow their
* 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.
10.1021/jm0613119 CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/03/2007

Optimal Indenoisoquinoline Top1 Inhibition
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2041
Scheme 1. Preparation of Mono-Boc-Protected Diaminoalkanesa
reported compounds 37-3919 are included in the table. The
relative potencies of the compounds in the production of top1-
mediated DNA cleavage are listed in the table and summarized
as follows: +, weak activity; ++, activity similar to that of
the parent compound 2; +++ and ++++, activity greater than
that of the parent compound 2; ++++, activity similar to that
of 1 µM 1.
From the biological data presented in Table 1, several general
features are evident from the investigation of optimal lactam
chain length. First, carbamate-protected indenoisoquinolines
such as compounds 31-36 demonstrated poor cytotoxicity and
top1 inhibition relative to those of 1 and poor top1 inhibition
relative to that of the parent indenoisoquinoline 2. This
structure-activity relationship has been previously identified
with carbamate-protected bisindenoisoquinolines33 and is be-
lieved to arise from inefficient targeting of DNA (relative to
that of the deprotected, positively charged derivatives), detri-
mental steric interactions in the ternary complex, and general
hydrophobicity of the molecules relative to that of the depro-
tected derivatives.
a Reagents and conditions: (a) Boc2O (1 equiv), CHCl3, room temper-
ature.
removal during aqueous workup. Hence, compounds 25, 29,
and 30 were prepared without utilization of mono-Boc-protected
diaminoalkanes. Beyond a six-carbon length, however, aqueous
solubility of the unprotected diaminoalkanes was negligible, and
purification of 31-36 was greatly facilitated by the use of a
tert-butyl carbamate to protect the terminal amine on the lactam
side chain. Deprotection of carbamates 26-28 and 31-36
readily occurred upon exposure to hydrochloric acid, providing
the remainder of the analogues (37-47) for this study.
Upon evaluation of the analogues prepared in Scheme 2, the
optimal chain length for lactam substituents was determined and
utilized in the preparation of pyridine-substituted indenoiso-
quinolines 48-51 (Scheme 3). Unlike analogues 37-47,
compounds 48-51 should not be protonated at physiological
pH, and their biological activities, when compared to those of
analogues 37-39, should be different due to the different roles
in hydrogen bonding (donor versus acceptor) of the analogue
side chains. Treatment of 2129 with the appropriate aminoalkyl-
pyridine derivatives readily provided compounds 48-51, with
the isolation of 49 and 50 greatly facilitated by the formation
of their corresponding hydrochloride salts.
In general, potent biological activity was observed for the
deprotected, (alkylamino)indenoisoquinolines 37-47 relative to
their carbamate-protected precursors (Table 1). From the data
reported in Table 1, it is clear that biological activity is greatly
improved, for both cytotoxicity and top1 inhibition, with the
utilization of shorter lactam side chains. Indeed, maximum
biological activity was observed for the previously reported19
compounds 37-39, with 2-4-carbon chain lengths. Thus, the
historical use of two- and three-carbon lactam substituent chain
lengths has been validated and experimentally confirmed to be
optimal for developing potent indenoisoquinoline top1 inhibitors.
Interestingly, there are two anomalies in the generalization that
“a shorter chain length is better” for biological activity. First,
compound 25, with a hydrazine-derived lactam side chain was
poorly cytotoxic and an inactive top1 inhibitor (MGM ) 39.8
µM; top1 inhibition 0). This result is believed to be due to
inefficient targeting of DNA (since the side chain of 25 should
not have a fixed positive charge at physiological pH). Further-
more, hypothetical binding models of 25 in a ternary complex
with DNA and top1 did not show any stabilizing hydrogen-
bonding interaction between the amino group and either the
DNA base pairs at the site of intercalation or top1. This finding
was in stark contrast to that of the terminal amines of compounds
37-39, which displayed stabilizing hydrogen-bonding interac-
tions in hypothetical models.19 The second anomaly worth
mentioning is the discrepancy between the cytotoxicity of 40
and its top1 inhibition (MGM ) 0.471 µM; top1 inhibition 0).
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
of diverse tumor origins.31,32 The GI50 values obtained with
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 GI50 values below and above the
test range (10-8 to 10-4 M) are taken as the minimum (10-8
M) and maximum (10-4 M) drug concentrations used in the
screening test. For comparison purposes, the activities of
camptothecin (1), parent indenoisoquinoline 2,13 and previously
Scheme 2. Preparation of Indenoisoquinoline Analoguesa
a Reagents and conditions: (a) CHCl3, reflux; (b) 3 M HCl in MeOH, room temperature.

2042 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Morrell et al.
Table 1. Cytotoxicities and Topoisomerase I Inhibitory Activities of Indenoisoquinoline Analogues
cytotoxicity (GI50, µM)a
lung
HOP-62
colon
HCT-116
CNS
SF-268
melanoma
UACC-62
ovarian
OVCAR -3
renal
SN12C
prostate
DU-145
breast
MDA-MB-435
top1
compd
MGMb
cleavagec
1
2
0.010
1.30
24.5
NT
0.030
35.0
>50.1
17.4
25.7
NT
60.3
35.5
>100
0.270
0.180
0.10
0.200
0.912
1.26
1.66
6.46
2.75
1.91
NT
0.010
NT
0.010
4.20
>50.1
20.0
15.5
17.4
43.6
24.0
27.5
0.920
0.26
0.05
1.35
1.62
2.00
3.80
9.33
13.8
1.70
14.8
10.7
8.71
27.5
89.1
2.6
0.220
73.0
>50.1
33.9
0.020
68.0
>50.1
74.1
>50.1
77.6
>100
>100
>100
0.490
0.160
0.04
0.010
37.0
>50.1
31.6
>50.1
>100
97.7
0.040
96.0
>50.1
81.3
>50.1
>100
>100
>100
>100
0.920
0.78
0.84
1.35
2.04
2.24
5.62
9.55
11.7
11.0
19.5
3.09
42.7
5.13
4.57
2.7
0.0405 ( 0.0187
++++
20.0
++
0
25
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
28.2
20.9
8.71
17.8
42.7
29.5
20.4
0.210
0.25
0.10
0.871
1.23
1.78
2.82
9.77
1.82
2.04
18.2
12.3
6.76
17.4
27.5
39.8
30.9
19.9
50.1
52.5
42.6 ( 5.35
51.3
0.530 ( 0.320
0.32 ( 0.23
0.16 ( 0.01
0.471 ( 0.054
1.32
1.66 ( 0.155
3.71
8.13
5.50
5.13
18.2
15.1
11.5
53.7
44.7
2.4
3.0
0
0
10.7
24.5
28.8
20.9
32.4
0.620
0.200
0.08
0.288
1.29
1.20
2.14
6.76
4.79
2.19
17.0
11.2
7.59
21.4
28.8
2.8
28.8
>100
70.8
>100
0.710
1.38
0.52
0.708
2.00
1.70
3.47
9.55
10.7
2.09
17.8
13.8
13.8
>100
>100
1.7
0
91.2
91.2
0.760
0.22
0.01
0.347
0.603
0.832
3.39
6.03
3.47
4.57
19.1
13.5
13.8
74.1
81.3
1.2
4.6
+++
+++
+++
0
++
0
+
0
0
+
0.398
1.32
1.66
3.47
8.51
3.31
2.00
13.5
28.2
0/+
0
+++
0
NT
NT
NT
NT
0.9
1.3
23
43
13.8
77.6
>100
0
2.5
1.7
20
33
++++
++
++++
++
2.9
1.9
37
44
8.1
20
0.88
3.4
38
68
52
>100
41
59
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 100 µM. The activity of the compounds to produce
top1-mediated DNA cleavage was expressed semiquantitatively as follows: +, weak activity; ++, activity similar to that of the parent compound 2; +++
and ++++, activity greater than that of the parent compound 2; ++++, activity similar to that of 1 µM 1.
Scheme 3. Pyridine-Substituted Indenoisoquinolinesa
7 and 8), the predominant top1 cleavage site (marker 44) is
trapped at lower compound concentrations and suppressed at
higher concentrations, and therefore, indenoisoquinoline 39 acts
as a top1 poison at lower concentrations and top1 suppressor
at higher concentrations. The suppression could result from
binding of the drug to the DNA, rendering it a poorer enzyme
substrate at high drug concentration, or from a direct effect on
the enzyme to suppress its ability to cleave DNA. (3) There are
differences in the cleavage pattern of camptothecin vs the
indenoisoquinolines. For example, the cleavage at base pair 44
seen with the indenoisoquinolines is absent with camptothecin.
This difference may indicate that the indenoisoquinolines might
display antitumor spectra different from those of camptothecin
or its clinically useful derivatives irinotecan and topotecan.
a Reagents and conditions: (a) aminoalkylpyridine, Et3N, CHCl3, reflux;
(b) (i) aminoalkylpyridine, Et3N, CHCl3; (ii) 3 M HCl in MeOH.
This result is not entirely without precedence, and it is obvious
that the cytotoxicity of this molecule is not related to its ability
to inhibit top1, but must be dependent upon another, unknown
biological target.19,33
Although the knowledge that a shorter alkyl side chain is
important for furthering drug development efforts for inde-
noisoquinolines, the cause of this result is equally important.
Molecular modeling of compounds 37-47 in a ternary complex
with DNA and top1 (PDB code 1SC7)15 indicated that the
majority of these molecules should exert similar top1 inhibitory
activities, since the side chains project out into the major groove
and can be readily accommodated sterically.15 However, the
results from Table 1 show that, as the length of the lactam side
chain increases, there is a general decrease in top1 inhibition
after a four-carbon atom length. Rationalization of the biological
data was therefore initially sought without the use of molecular
models of the ternary complexes. Upon further inspection of
Table 1 regarding only the compounds assumed to be protonated
at physiological pH (37-47), it was realized that, as the lactam
chain length increased, there was an increase in the MGM values
of the compounds (as well as the previously observed decrease
in top1 inhibition). Since these compounds differed only by the
The DNA cleavage patterns produced by 1 (lane 3 of each
gel), the indenoisoquinoline NSC 314622, and compounds 39,
41, 43, and 45 are displayed in Figure 2. 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
compound 39 (top1 inhibition +++) are slightly weaker in
comparison with those produced by camptothecin, but stronger
in intensity than the bands produced by 41 (top1 inhibition ++),
43 (top1 inhibition +), and 45 (top1 inhibition 0). Thus, a
comparison of the gels in Figure 2 indicates that as the lactam
side chains of the indenoisoquinolines increase, a corresponding
decrease in the ability to inhibit top1 is observed. (2) Top1
inhibitors can be classified as top1 suppressors, which inhibit
DNA cleavage, and top1 poisons, which inhibit the religation
reaction after DNA cleavage. As compund 39 illustrates (lanes

Optimal Indenoisoquinoline Top1 Inhibition
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2043
Figure 2. Key: lane 1, DNA alone; lane 2, top1 alone; lane 3, + CPT (1 µM); first panel, lane 4, top1 + NSC 314622 (100 µM); lanes 5-8 (for
compound 39) and lanes 4-7 (for compounds 41, 43, and 45), top1 + indicated compound at 0.1, 1, 10, and 100 µM, respectively. Numbers on
the left and arrows indicate cleavage site positions.
Figure 3. C log P vs log(MGM).
incremental increase of methylene units, the effect could
possibly be hydrophobic in nature. Indeed, calculation of the C
log P values of compounds 37-47 (performed in ChemDraw
Ultra, version 9.0.1) and plotting against log(MGM) resulted
in a linear dependence with an r2 value of 0.90, indicating a
high degree of correlation (Figure 3). Thus, for this series of
compounds general hydrophobicity, as measured by C log P
values, appears to be predictive of cytotoxicity in the NCI’s
60-cell screen. Inspection of the ternary complex of topotecan
bound to DNA and top1 (PDB code 1K4T)14 reveals the
presence of a hydrated binding pocket and appears to provide
an essential clue supporting both the data in Table 1 and the
relationship between C log P and cytotoxicity (Figure 3). A
hypothetical model was subsequently developed utilizing the
topotecan crystal structure and the experimentally determined
structural overlay between topotecan and the indenoisoquinolines
(Figure 4).14,15,17 Overlaying the hypothetical models for
compounds 37, 39, 41, 43, 45, and 47 (colored by atom type)
in a ternary complex with DNA (red) and top1 (blue) allows
the visualization of the extensive hydrophilic binding pocket
(water molecules are indicated in green) for the indenoisoquino-
line side chain. Increasing the number of methylene units beyond
four in the lactam side chain of the indenoisoquinolines results
in an increasingly hydrophobic linker with increasingly negative
interactions with the hydrated binding pocket. In agreement with
this hypothesis, polyamine-substituted indenoisoquinolines 52-
55 retain significant top1 inhibitory activity in the ++ to
++++ range.24
Having experimentally confirmed that the optimal lactam
chain length for cytotoxicity and top1 inhibition for the
indenoisoquinolines was 2-4 carbon atoms between the lactam
nitrogen and the terminal amino group, this relationship was
further scrutinized by preparing analogues 48-51 that utilize a
pyridine ring in place of a portion of the alkyl linker and the
terminal amino group. However, the newly designed analogues
still maintained a 2-4-carbon atom linker between the lactam
nitrogen and the nitrogen atom in the pyridine ring. Although
all of the analogues were less cytotoxic than their corresponding
alkyl chain analogues, compound 49 displayed potent top1
inhibition equal in magnitude to that of compounds 37-39. With
the discrepancy between cytotoxicity and top1 inhibition, it
seems reasonable to infer that protonation of the terminal amino
group of unsubstituted indenoisoquinolines is important for
cellular cytotoxicity (perhaps by improved DNA targeting) but
is not necessarily a requirement for potent top1 inhibition.
Indeed, this result has been observed in another indenoiso-
quinoline study involving heterocycles appended to the lactam
side chain, and the present study of pyridine heterocycles

2044 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Morrell et al.
Figure 4. Overlay of the hypothetical models derived from PDB code 1K4T for compounds 37, 39, 41, 43, 45, and 47 (colored by atom type) in
a ternary complex with DNA (red) and top1 (blue). Water molecules are indicated in green, and the hydrogen bond between the analogues and
Arg364 is shown in yellow. The figure is programmed for wall-eyed (relaxed) viewing.
reaffirms previously derived structure-activity relationships.34
However, the absence of protonation of the pyridine nitrogen
at physiological pH should not be construed as the only reason
for the discrepancies in biological activity between compounds
37-39 and 48-51. Furthermore, the pyridine-substituted ana-
logues do not provide strong support of the currently established
optimal length of the linker chain. This could be a result of
several factors including, but not limited to, reduced flexibility
of the side chain, increased steric bulk, disrupted hydrogen-
bonding networks with the aqueous environment, and the
aforementioned protonation state of the pyridine nitrogen at
physiological pH and DNA targeting.
excretion within the cancer cell. Hence, discrepancies will
ultimately arise between enzyme-based assays and the NCI 60-
cell-line screen for cytotoxicity.
In conclusion, a series of indenoisoquinolines have been
prepared to determine the optimal carbon chain length between
the lactam nitrogen and the terminal amine of the side chain.
The results of this study confirmed that the optimal carbon linker
for biological activity (both top1 inhibition and cytotoxicity) is
2-4 atoms, thereby validating the use of this length in past
and future efforts to develop indenoisoquinolines as top1
inhibitors. Interestingly, the study indicated a strong correlation
between C log P values and MGM values for indenoisoquino-
lines with protonated terminal amines on the lactam side chain.
Furthermore, the substitution of a pyridine ring in indenoiso-
quinoline 49 in place of a portion of the linker and the terminal
amino group resulted in a discrepancy between top1 inhibition
and cytotoxicity, indicating that a protonated terminal amino
group on the indenoisoquinolines is more important for main-
taining cytotoxicity than top1 inhibition.
Compounds 37-39 are more cytotoxic than compounds 52-
55 and 48-51. Thus, incorporating polar motifs (such as
polyamino groups) on the lactam side chain of the indenoiso-
Experimental Section
General Procedures. Melting points were determined using
capillary tubes with a Mel-Temp apparatus and are uncorrected.
Infrared spectra were obtained using CHCl3 as the solvent unless
otherwise specified. The proton nuclear magnetic resonance (1H
NMR) spectra were recorded using an ARX300 300 MHz Bruker
NMR spectrometer. 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 the compounds were visualized
with short-wavelength UV light. Silica gel flash chromatography
was performed using 230-400 mesh silica gel. Compounds 12-
14, 26-28, and 37-39 were prepared as previously described.19
General Procedure for the Preparation of Mono-Boc-
Protected Diamines 12-20. Boc2O (0.500 g, 2.291 mmol) was
dissolved in CHCl3 (10 mL), and the solution was added dropwise
to a solution of diamine (11.45 mmol) in CHCl3 (50 mL). The
reaction mixture was allowed to stir at room temperature for 24 h,
concentrated, and purified by flash column chromatography (SiO2),
eluting with a solution of 1% Et3N/10% MeOH in CHCl3, to provide
the mono-Boc-protected diamine.
quinolines does not necessarily translate into increased cyto-
toxicity but does improve top1 inhibition. Additionally, structural
changes to the lactam side chain that reduce its flexibility, such
as the present case for the pyridine-containing analogues, result
in discrepancies between enzyme inhibition and cytotoxicity.
The presently observed relationship between C log P and
cytotoxicity indicates that drug design and molecular modeling
of the indenoisoquinolines should consider the environment of
the lactam side chain in the ternary complex. Multiple variables
in addition to lipophilicity are expected to be involved in cellular
uptake, distribution, and ultimately cytotoxicity of the inde-
noisoquinolines (as well as other DNA-targeting drug mol-
ecules). However, lipophilicity has previously been demonstrated
to affect the nonspecific binding of anthracycline antitumor
drugs to serum albumin, and it seems reasonable to assume that
nonspecific binding (or unintended localization) could occur
intracellularly with the indenoisoquinolines and result in
discrepant biological activities.35,36 Differences in structure,
such as the replacement of the indenoisoquinoline side chain
with a pyridine ring or polyamino groups, are also expected to
result in different rates of uptake, distribution, metabolism, and
Mono-Boc-1,7-diaminoheptane (15).37 The general procedure
provided the desired compound as a colorless semisolid (0.473 g,
90%): 1H NMR (CDCl3) δ 4.52 (br s, 1 H), 3.12 (q, J ) 6.2 Hz,
2 H), 2.70 (t, J ) 6.8 Hz, 2 H), 1.43-1.23 (m, 19 H).
Mono-Boc-1,8-diaminooctane (16).38 The general procedure
provided the desired compound as a colorless semisolid (0.492 g,

Optimal Indenoisoquinoline Top1 Inhibition
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2045
88%): 1H NMR (CDCl3) δ 4.51 (br s, 1 H), 3.12 (q, J ) 6.5 Hz,
2 H), 2.69 (t, J ) 6.8 Hz, 2 H), 1.43-1.23 (m, 21 H).
1H NMR (DMSO-d6) δ 8.58 (d, J ) 7.9 Hz, 1 H), 8.23 (dd, J )
8.1 and 0.7 Hz, 1 H), 7.84 (dt, J ) 7.2 and 1.4 Hz, 1 H), 7.71 (d,
J ) 7.5 Hz, 1 H), 7.63-7.47 (m, 4 H), 6.76 (m, 1 H), 4.50 (t, J )
7.4 Hz, 2 H), 2.90 (q, J ) 6.5 Hz, 2 H), 1.77 (m, 2 H), 1.46-1.23
(m, 23 H); ESIMS m/z (rel intens) 525 (MNa+, 100). Anal.
(C31H38N2O4) C, H, N.
6-(11′-tert-Boc-aminoundecyl)-5,6-dihydro-5,11-dioxo-11H-in-
deno[1,2-c]isoquinoline (35). The general procedure provided the
desired compound as a yellow-orange solid (0.445 g, 86%): mp
111-114 °C; IR (KBr) 3364, 2918, 2850, 1678, 1660, 1534, 1505,
758 cm-1; 1H NMR (DMSO-d6) δ 8.58 (d, J ) 8.1 Hz, 1 H), 8.23
(d, J ) 7.4 Hz, 1 H), 7.84-7.79 (m, 1 H), 7.71 (d, J ) 7.5 Hz, 1
H), 7.62-7.50 (m, 4 H), 6.74 (m, 1 H), 4.50 (t, J ) 7.4 Hz, 2 H),
2.90 (q, J ) 6.5 Hz, 2 H), 1.78 (m, 2 H), 1.46-1.22 (m, 25 H);
ESIMS m/z (rel intens) 539 (MNa+, 100). Anal. (C32H40N2O4) C,
H, N.
Mono-Boc-1,9-diaminononane (17).39 The general procedure
provided the desired compound as a colorless semisolid (0.125 g,
21%): 1H NMR (CDCl3) δ 4.50 (br s, 1 H), 3.12 (q, J ) 6.5 Hz,
2 H), 2.70 (t, J ) 6.8 Hz, 2 H), 1.44-1.22 (m, 23 H).
Mono-Boc-1,10-diaminodecane (18).39 The general procedure
provided the desired compound as a colorless semisolid (0.192 g,
31%): 1H NMR (CDCl3) δ 4.50 (br s, 1 H), 3.13 (q, J ) 6.3 Hz,
2 H), 2.71 (t, J ) 6.9 Hz, 2 H), 1.44-1.18 (m, 27 H).
Mono-Boc-1,11-diaminoundecane (19). The general procedure
provided the desired compound as a colorless solid (0.555 g,
85%): mp 30-34 °C; IR (film) 3370, 2919, 2851, 1687, 1522 cm-1
;
1H NMR (CDCl3) δ 4.49 (br s, 1 H), 3.11 (q, J ) 6.5 Hz, 2 H),
2.71 (t, J ) 6.8 Hz, 2 H), 1.44-1.27 (m, 29 H); ESIMS m/z (rel
intens) 287 (MH+, 100). Anal. (C16H34N2O2) C, H, N.
Mono-Boc-1,12-diaminododecane (20).39,40 The general pro-
cedure provided the desired compound as a colorless semisolid
(0.191 g, 28%): 1H NMR (CDCl3) δ 4.48 (br s, 1 H), 3.11 (q, J )
6.2 Hz, 2 H), 2.76 (t, J ) 6.9 Hz, 2 H), 1.44-1.26 (m, 31 H).
6-Amino-5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquino-
line (25). Benz[d]indeno[1,2-b]pyran-5,11-dione (21)29 (0.150 g,
0.604 mmol) was treated with hydrazine (22) (0.255 g, 7.964 mmol)
in CHCl3 (50 mL), and the reaction mixture was heated at reflux
for 16 h. The reaction mixture was allowed to cool to room
temperature, diluted with CHCl3 (150 mL), and washed with
saturated NaHCO3 (2 × 50 mL). The solution was dried over
sodium sulfate and concentrated to provide a red-orange solid (0.120
g, 76%): mp 272-274 °C; IR (film) 3448, 3305, 1686, 1663, 1610,
1507, 1312, 762 cm-1; 1H NMR (CDCl3) δ 8.54 (d, J ) 7.8 Hz, 1
H), 8.51 (d, J ) 7.2 Hz, 1 H), 8.24 (d, J ) 7.4 Hz, 1 H), 7.85 (m,
1 H), 7.60-7.45 (m, 4 H), 6.19 (s, 2 H); EIMS m/z (rel intens)
262 (M+, 100). Anal. (C16H10N2O2‚0.25H2O) C, H, N.
General Procedure for the Preparation of Mono-Boc-
Protected Indenoisoquinolines 31-36. Mono-Boc-protected di-
amine (2.054 mmol) was added to a solution of 2129 (0.255 g, 1.027
mmol) in CHCl3 (100 mL). The reaction mixture was heated at
reflux for 24 h, concentrated, and purified by flash column
chromatography (SiO2), eluting with CHCl3, to provide the mono-
Boc-protected indenoisoquinoline.
6-(12′-tert-Boc-aminododecyl)-5,6-dihydro-5,11-dioxo-11H-in-
deno[1,2-c]isoquinoline (36). The general procedure provided the
desired compound as a yellow-orange solid (0.177 g, 66%): mp
129-134 °C; IR (film) 3369, 2926, 1698, 1666, 1504, 1172 cm-1
;
1H NMR (DMSO-d6) δ 8.58 (d, J ) 8.0 Hz, 1 H), 8.22 (d, J ) 7.4
Hz, 1 H), 7.84-7.78 (m, 1 H), 7.71 (d, J ) 7.5 Hz, 1 H), 7.62-
7.50 (m, 4 H), 6.74 (m, 1 H), 4.50 (t, J ) 7.3 Hz, 2 H), 2.90 (q, J
) 6.6 Hz, 2 H), 1.77 (m, 2 H), 1.46-1.22 (m, 27 H); ESIMS m/z
(rel intens) 553 (MNa+, 100). Anal. (C33H42N2O4) C, H, N.
6-(5-Aminopentyl)-5,6-dihydro-5,11-diketo-11H-indeno[1,2-
c]isoquinoline Hydrochloride (40). Lactone 2129 (0.100 g, 0.403
mmol) was treated with diamine 23 (0.206 g, 2.014 mmol) in CHCl3
(40 mL), and the reaction mixture was heated at reflux for 16 h.
The reaction mixture was allowed to cool to room temperature and
washed with water (3 × 15 mL). The solution was dried over
sodium sulfate, filtered, and treated with 2 M HCl in Et2O (5 mL).
After 30 min, the reaction mixture was filtered, and the filter pad
was washed with CHCl3 (50 mL) and hexanes (50 mL) to provide
an orange solid (0.122 g, 82%): mp 265-268 °C; IR (film) 3432,
3077, 2856, 1707, 1635, 1611, 1549, 1504 cm-1; 1H NMR (CDCl3)
δ 8.59 (d, J ) 8.1 Hz, 1 H), 8.23 (d, J ) 8.1 Hz, 1 H), 7.85-7.80
(m, 3 H), 7.74 (d, J ) 7.4 Hz, 1 H), 7.63-7.51 (m, 4 H), 4.52 (t,
J ) 7.3 Hz, 2 H), 2.81 (m, 2 H), 1.83 (m, 2 H), 1.65-1.52 (m, 4
H); ESIMS m/z (rel intens) 333 (MH+, 100). Anal. (C21H21ClN2O2‚
0.75H2O) C, H, N.
6-(7′-tert-Boc-aminoheptyl)-5,6-dihydro-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (31). The general procedure provided the
desired compound as a yellow-orange solid (0.451 g, 95%): mp
6-(6-Aminohexyl)-5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]-
isoquinoline Hydrochloride (41). Lactone 2129 (0.100 g, 0.403
mmol) was treated with diamine 24 (0.234 g, 2.014 mmol) in CHCl3
(40 mL), and the reaction mixture was heated at reflux for 16 h.
The reaction mixture was allowed to cool to room temperature and
washed with water (3 × 25 mL). The solution was dried over
sodium sulfate, filtered, and treated with 2 M HCl in Et2O (5 mL).
After 30 min, the reaction mixture was filtered, and the filter pad
was washed with CHCl3 (50 mL) and hexanes (50 mL) to provide
an orange solid (0.125 g, 81%): mp 195 °C dec; IR (film) 3435,
1
112-116 °C; IR (film) 3369, 1697, 1664, 1503, 1172 cm-1; H
NMR (DMSO-d6) δ 8.58 (d, J ) 8.1 Hz, 1 H), 8.23 (d, J ) 8.2
Hz, 1 H), 7.84-7.79 (m, 1 H), 7.71 (d, J ) 7.7 Hz, 1 H), 7.63-
7.50 (m, 4 H), 6.76 (m, 1 H), 4.50 (t, J ) 7.4 Hz, 2 H), 2.90 (q, J
) 6.2 Hz, 2 H), 1.77 (m, 2 H), 1.46-1.28 (m, 17 H); ESIMS m/z
(rel intens) 483 (MNa+, 100). Anal. (C28H32N2O4) C, H, N.
6-(8′-tert-Boc-aminooctyl)-5,6-dihydro-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (32). The general procedure provided the
desired compound as a yellow-orange solid (0.466 g, 97%): mp
140-143 °C; IR (film) 3368, 2929, 1698, 1665, 1504, and 1172
1
1660, 1630, 1610, 1504 cm-1; H NMR (CDCl3) δ 8.59 (d, J )
7.8 Hz, 1 H), 8.23 (d, J ) 8.1 Hz, 1 H), 7.85-7.71 (m, 4 H),
7.61-7.51 (m, 4 H), 4.52 (t, J ) 7.3 Hz, 2 H), 2.78 (m, 2 H), 1.79
(m, 2 H), 1.59-1.39 (m, 6 H); ESIMS m/z (rel intens) 347 (MH+,
100). Anal. (C22H23ClN2O2‚0.5H2O) C, H, N.
1
cm-1; H NMR (DMSO-d6) δ 8.58 (d, J ) 7.9 Hz, 1 H), 8.23 (d,
J ) 8.1 Hz, 1 H), 7.84-7.78 (m, 1 H), 7.71 (d, J ) 7.5 Hz, 1 H),
7.63-7.47 (m, 4 H), 6.75 (m, 1 H), 4.50 (t, J ) 7.4 Hz, 2 H), 2.91
(q, J ) 6.6 Hz, 2 H), 1.78 (m, 2 H), 1.46-1.26 (m, 19 H); ESIMS
m/z (rel intens) 497 (MNa+, 100). Anal. (C29H34N2O4) C, H, N.
6-(9′-tert-Boc-aminononyl)-5,6-dihydro-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (33). The general procedure provided the
desired compound as an orange solid (0.145 g, 77%): mp
General Procedure for the Preparation of Indenoisoquinoline
Hydrochloride Salts 42-47. HCl (3 M) in MeOH (10 mL) was
slowly added to a solution of mono-Boc-protected indenoisoquino-
line (0.100 g, 0.188-0.217 mmol) in CHCl3 (50 mL) at room
temperature. After 2 h, the reaction mixture was concentrated, and
the residue was triturated with Et2O. Filtration of the obtained solid
provided the indenoisoquinoline as a hydrochloride salt.
91-95 °C; IR (film) 3371, 2928, 1698, 1666, 1504, 1172 cm-1
;
1H NMR (DMSO-d6) δ 8.58 (d, J ) 8.0 Hz, 1 H), 8.23 (d, J ) 7.4
Hz, 1 H), 7.84-7.79 (m, 1 H), 7.71 (d, J ) 7.4 Hz, 1 H), 7.63-
7.50 (m, 4 H), 6.74 (m, 1 H), 4.50 (t, J ) 7.3 Hz, 2 H), 2.91 (q, J
) 6.6 Hz, 2 H), 1.78 (m, 2 H), 1.47-1.24 (m, 21 H); ESIMS m/z
(rel intens) 511 (MNa+, 100). Anal. (C30H36N2O4) C, H, N.
6-(10′-tert-Boc-aminodecyl)-5,6-dihydro-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (34). The general procedure provided the
desired compound as a yellow-orange solid (0.220 g, 78%): mp
6-(7-Aminoheptyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline Hydrochloride (42). The general procedure provided
the desired compound as a yellow-orange solid (0.085 g, 99%):
mp 228-231 °C; IR (KBr) 3436, 2931, 1702, 1650, 1611, 1549,
1
1504, 759 cm-1; H NMR (DMSO-d6) δ 8.60 (d, J ) 8.1 Hz, 1
H), 8.24 (d, J ) 8.2 Hz, 1 H), 7.85-7.80 (m, 1 H), 7.73 (d, J )
7.4 Hz, 1 H), 7.63-7.49 (m, 6 H), 4.53 (t, J ) 7.0 Hz, 2 H), 2.78
135-137 °C; IR (film) 3368, 2927, 1698, 1666, 1504, 1172 cm-1
;

2046 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Morrell et al.
(t, J ) 7.1 Hz, 2 H), 1.80 (m, 2 H), 1.55-1.35 (m, 8 H); ESIMS
m/z (rel intens) 361 (MH+, 100). Anal. (C23H25ClN2O2‚0.5H2O)
C, H, N.
residue was diluted with CHCl3 (40 mL), 3 M HCl in MeOH (10
mL) was added, and the reaction mixture was allowed to stir at
room temperature for 2 h. The reaction mixture was concentrated,
and the residue was washed with CHCl3 to provide a pink solid
(0.146 g, 97%): mp 274 °C dec; IR (KBr) 2343, 2106, 1695, 1655,
6-(8-Aminooctyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]i-
soquinoline Hydrochloride (43). The general procedure provided
the desired compound as an orange solid (0.083 g, 95%): mp 182-
1
1610, 1551, 1501, 754 cm-1; H NMR (DMSO-d6) δ 8.98 (s, 1
1
185 °C; IR (KBr) 3436, 2930, 1661, 1505, 761 cm-1; H NMR
H), 8.78 (d, J ) 5.2 Hz, 1 H), 8.64 (d, J ) 8.1 Hz, 1 H), 8.36 (d,
J ) 9.1 Hz, 1 H), 8.23 (d, J ) 7.5 Hz, 1 H), 7.90 (m, 2 H), 7.59-
7.39 (m, 5 H), 5.89 (s, 2 H); ESIMS m/z (rel intens) 339 (MH+,
100). Anal. (C22H15ClN2O2) C, H, N.
(DMSO-d6) δ 8.60 (d, J ) 8.5 Hz, 1 H), 8.24 (d, J ) 7.0 Hz, 1 H),
7.86-7.81 (m, 1 H), 7.73 (d, J ) 7.6 Hz, 1 H), 7.63-7.52 (m, 6
H), 4.52 (t, J ) 7.9 Hz, 2 H), 2.78 (t, J ) 7.3 Hz, 2 H), 1.79 (m,
2 H), 1.50 (m, 4 H), 1.31 (m, 6 H); ESIMS m/z (rel intens) 375
(MH+, 100). Anal. (C24H27ClN2O2‚0.75H2O) C, H, N.
6-(9-Aminononyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline Hydrochloride (44). The general procedure provided
the desired compound as an orange solid (0.082 g, 94%): mp 204-
207 °C; IR (KBr) 3435, 2927, 1702, 1662, 1610, 1549, 1504, 1427,
759 cm-1; 1H NMR (DMSO-d6) δ 8.60 (d, J ) 8.3 Hz, 1 H), 8.24
(d, J ) 9.3 Hz, 1 H), 7.83-7.81 (m, 1 H), 7.73 (d, J ) 7.8 Hz, 1
H), 7.63-7.51 (m, 6 H), 4.52 (t, J ) 8.3 Hz, 2 H), 2.78 (t, J ) 7.3
Hz, 2 H), 1.79 (m, 2 H), 1.51 (m, 4 H), 1.28 (m, 8 H); ESIMS m/z
(rel intens) 389 (MH+, 100). Anal. (C25H29ClN2O2‚0.75H2O) C,
H, N.
5,6-Dihydro-5,11-dioxo-6-(2-pyridylethyl)-11H-indeno[1,2-c]-
isoquinoline Hydrochloride (50). 2-(2-Aminoethyl)pyridine (0.098
g, 0.806 mmol) was added to a solution of 2129 (0.100 g, 0.403
mmol) in CHCl3 (50 mL), and the reaction mixture was heated at
reflux for 16 h. The reaction mixture was allowed to cool to room
temperature, washed with H2O (3 × 25 mL) and saturated NaCl
(25 mL), dried over sodium sulfate, and concentrated. The residue
was diluted with CHCl3 (40 mL), 3 M HCl in MeOH (10 mL) was
added, and the reaction mixture was allowed to stir at room
temperature for 2 h. The reaction mixture was concentrated, and
the residue was washed with CHCl3 to provide a yellow solid (0.146
g, 93%): mp 240 °C dec; IR (KBr) 2307, 1698, 1659, 1610, 1548,
1
1504, 1429, 760 cm-1; H NMR (DMSO-d6) δ 8.78 (d, J ) 5.5
6-(10-Aminodecyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline Hydrochloride (45). The general procedure provided
the desired compound as an orange solid (0.087 g, 91%): mp 189-
192 °C; IR (KBr) 3443, 2925, 2851, 1705, 1646, 1611, 1550, 1504,
Hz, 1 H), 8.57 (d, J ) 8.0 Hz, 1 H), 8.33 (m, 1 H), 8.02 (d, J )
8.0 Hz, 1 H), 7.97 (d, J ) 8.0 Hz, 1 H), 7.90 (d, J ) 8.0 Hz, 1 H),
7.80 (m, 2 H), 7.61-7.44 (m, 4 H), 4.92 (t, J ) 6.5 Hz, 2 H), 3.59
(t, J ) 6.3 Hz, 2 H); ESIMS m/z (rel intens) 353 (MH+, 100).
Anal. (C23H17ClN2O2) C, H, N.
1
1467, 759 cm-1; H NMR (DMSO-d6) δ 8.60 (d, J ) 7.9 Hz, 1
H), 8.23 (d, J ) 7.5 Hz, 1 H), 7.83 (m, 1 H), 7.73 (d, J ) 8.0 Hz,
1 H), 7.63-7.51 (m, 6 H), 4.52 (t, J ) 7.4 Hz, 2 H), 2.76 (m, 2
H), 1.79 (m, 2 H), 1.49 (m, 4 H), 1.27 (m, 10 H); ESIMS m/z (rel
intens) 403 (MH+, 100). Anal. (C26H31ClN2O2‚0.5H2O) C, H, N.
6-(11-Aminoundecyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-
c]isoquinoline Hydrochloride (46). The general procedure pro-
vided the desired compound as an orange solid (0.085 g, 88%):
mp 125-129 °C; IR (KBr) 3436, 2922, 2851, 1662, 1610, 1549,
5,6-Dihydro-5,11-dioxo-6-(3-pyridylethyl)-11H-indeno[1,2-c]-
isoquinoline (51). 3-(2-Aminoethyl)pyridine (0.172 g, 0.604 mmol)
was added to a solution of 2129 (0.100 g, 0.403 mmol) in CHCl3
(50 mL). Triethylamine (0.224 mL, 1.612 mmol) was added, and
the reaction mixture was heated at reflux for 16 h. The reaction
mixture was allowed to cool to room temperature, washed with
H2O (3 × 25 mL) and saturated NaCl (25 mL), dried over sodium
sulfate, and concentrated. The obtained precipitate was washed with
EtOAc and hexanes and dried to provide an orange solid (0.140 g,
99%): mp 220 °C dec; IR (KBr) 1691, 1660, 1609, 1549, 1504,
1
1504, 1426, 758 cm-1; H NMR (DMSO-d6) δ 8.60 (d, J ) 8.0
Hz, 1 H), 8.23 (d, J ) 7.5 Hz, 1 H), 7.85 (m, 1 H), 7.72 (d, J )
7.5 Hz, 1 H), 7.63-7.51 (m, 6 H), 4.51 (t, J ) 8.0 Hz, 2 H), 2.77
(t, J ) 7.6 Hz, 2 H), 1.78 (m, 2 H), 1.48 (m, 4 H), 1.25 (m, 12 H);
ESIMS m/z (rel intens) 417 (MH+, 100). Anal. (C26H31ClN2O2‚
H2O) C, H, N.
1
1424, 765 cm-1; H NMR (DMSO-d6) δ 8.90 (s, 1 H), 8.73 (d, J
) 5.6 Hz, 1 H), 8.59 (d, J ) 8.1 Hz, 1 H), 8.44 (d, J ) 7.9 Hz, 1
H), 8.10 (d, J ) 8.1 Hz, 1 H), 7.88 (m, 3 H), 7.61-7.48 (m, 4 H),
4.87 (t, J ) 6.7 Hz, 2 H), 3.37 (t, J ) 6.3 Hz, 2 H); ESIMS m/z
(rel intens) 353 (MH+, 100). Anal. (C23H16N2O2‚0.55H2O) C, H,
N.
6-(12-Aminododecyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-
c]isoquinoline Hydrochloride (47). The general procedure pro-
vided the desired compound as a yellow solid (0.087 g, 91%): mp
175-178 °C; IR (KBr) 3435, 2927, 2850, 1704, 1644, 1506, 1466,
762 cm-1; 1H NMR (DMSO-d6) δ 8.59 (d, J ) 7.8 Hz, 1 H), 8.23
(d, J ) 7.5 Hz, 1 H), 7.83 (m, 1 H), 7.72 (d, J ) 7.8 Hz, 1 H),
7.63-7.51 (m, 6 H), 4.51 (t, J ) 7.5 Hz, 2 H), 2.76 (m, 2 H), 1.78
(m, 2 H), 1.51 (m, 4 H), 1.24 (m, 14 H); ESIMS m/z (rel intens)
431 (MH+, 100). Anal. (C28H35ClN2O2‚1.25H2O) C, H, N.
5,6-Dihydro-5,11-dioxo-6-(2-pyridylmethyl)-11H-indeno[1,2-
c]isoquinoline (48). 2-(Aminomethyl)pyridine (0.054 g, 0.504
mmol) was added to a solution of 2129 (0.100 g, 0.403 mmol) in
CHCl3 (50 mL), and the reaction mixture was heated at reflux for
16 h. The reaction mixture was allowed to cool to room temperature,
washed with H2O (3 × 25 mL) and saturated NaCl (25 mL), dried
over sodium sulfate, and concentrated. The residue was washed
with EtOAc and hexanes, and dried to provide a yellow solid (0.110
g, 81%): mp 240-242 °C; IR (KBr) 1698, 1655, 1618, 1501, 1427,
755 cm-1; 1H NMR (DMSO-d6) δ 8.62 (d, J ) 8.0 Hz, 1 H), 8.56
(d, J ) 4.9 Hz, 1 H), 8.22 (d, J ) 8.1 Hz, 1 H), 7.97 (dt, J ) 7.8
and 1.7 Hz, 1 H), 7.89 (m, 1 H), 7.61-7.37 (m, 7 H), 5.91 (s, 2
H); ESIMS m/z (rel intens) 339 (MH+, 100). Anal. (C22H14N2O2)
C, H, N.
Top1-Mediated DNA Cleavage Reactions. Human recombinant
Top1 was purified from baculovirus as described previously.41 The
161 bp fragment from pBluescript SK(-) phagemid DNA (Strat-
agene, La Jolla, CA) was cleaved with the restriction endonucleases
PVuII and HindIII (New England Biolabs, Beverly, MA) in supplied
NE buffer 2 (50 µL reactions) for 1 h at 37 °C and separated by
electrophoresis in a 1% agarose gel made in 1× TBE buffer. The
161 bp fragment was eluted from the gel slice using the QIAEX II
kit (QIAGEN Inc., Valencia, CA). Approximately 200 ng of the
fragment was 3′-end-labeled at the HindIII site by fill-in reaction
with [R-32P]dGTP and 0.5 mM dATP, dCTP, and dTTP in React
2 buffer (50 mM Tris-HCl, pH 8.0, 100 mM MgCl2, 50 mM NaCl)
with 0.5 unit of DNA polymerase I (Klenow fragment). Unincor-
porated [32P]dGTP was removed using miniature Quick Spin DNA
columns (Roche, Indianapolis, IN), and the eluate containing the
3′-end-labeled 161 bp fragment was collected. Aliquots (ap-
proximately 50000 dpm/reaction) were incubated with top1 at 22 °C
for 30 min in the presence of the tested drug. Reactions were
terminated by adding SDS (0.5% final concentration). The samples
(10 µL) were 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% polyacrylamide, 7 M urea). The
gels were dried and visualized by using a phosphoimager and
ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Molecular Modeling. The structure of the ternary complex,
containing topoisomerase I, DNA, and topotecan, was downloaded
5,6-Dihydro-5,11-dioxo-6-(3-pyridylmethyl)-11H-indeno[1,2-
c]isoquinoline Hydrochloride (49). 3-(Aminomethyl)pyridine
(0.054 g, 0.504 mmol) was added to a solution of 2129 (0.100 g,
0.403 mmol) in CHCl3 (50 mL), and the reaction mixture was
heated at reflux for 16 h. The reaction mixture was allowed to cool
to room temperature, washed with H2O (3 × 25 mL) and saturated
NaCl (25 mL), dried over sodium sulfate, and concentrated. The

Optimal Indenoisoquinoline Top1 Inhibition
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2047
from the Protein Data Bank (PDB code 1K4T).14 One molecule of
PEG and the topotecan carboxylate form were deleted. Several of
the atoms were then fixed according to Sybyl atom types.
Hydrogens were added and minimized using the MMFF94s force
field and MMFF94 charges. The structure of indenoisoquinolines
37-47, constructed in Sybyl and energy minimized with the
MMFF94s force field and MMFF94 charges, was overlapped with
the structure of topotecan according to the proposed structural
similarity16,17 in the ternary complex, and the structure of topotecan
was then deleted. The new whole complex was subsequently
subjected to energy minimization using the MMFF94s force field
with MMFF94 charges. During energy minimization, the structure
of the indenoisoquinoline was allowed to move while the structures
of the protein, nucleic acid, and water molecules were frozen. The
energy minimization was performed using the Powell method with
a 0.05 kcal/(mol‚Å) energy gradient convergence criterion and a
distance-dependent dielectric constant.
a Camptothecin Analog. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
15387-15392.
(15) Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.; Zembower,
D. L. S.; Burgin, A. B. Structures of Three Classes of Anticancer
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Fiber Animal Model. J. Med. Chem. 2002, 45, 242-249.
(23) 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.
(24) Nagarajan, M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.;
Cushman, M. Design, Synthesis, and Biological Evaluation of
Indenoisoquinoline Topoisomerase I Inhibitors Featuring Polyamine
Side Chains on the Lactam Nitrogen. J. Med. Chem. 2003, 46, 5712-
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(25) Antony, S.; Jayaraman, M.; Laco, G.; Kohlhagen, G.; Kohn, K. W.;
Cushman, M.; Pommier, Y. Differential Induction of Topoisomerase
I-DNA Cleavage Complexes by the Indenoisoquinoline MJ-III-65
(NSC 706744) and Camptothecin: Base Sequence Analysis and
Activity against Camptothecin-Resistant Topoisomerase I. Cancer
Res. 2003, 63, 7428-7435.
(26) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M.
Synthesis of Nitrated Indenoisoquinolines as Topoisomerase I Inhibi-
tors. Bioorg. Med. Chem. Lett. 2004, 14, 3659-3663.
(27) Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M.
Design, Synthesis, and Biological Evaluation of Cytotoxic 11-
Aminoalkenylindenoisoquinoline and 11-Diaminoalkenylindenoiso-
quinoline Topoisomerase I Inhibitors. Bioorg. Med. Chem. 2004, 12,
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Acknowledgment. This work was supported by Research
Grant UO1 CA89566, Training Grant ST32 CA09634-12, the
Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research, and the American
Chemical Society Division of Medicinal Chemistry and Pfizer
Global Research and Development (predoctoral support of
A.M.). This research was conducted in a facility constructed
with support from Research Facilities Improvement Program
Grant C06-14400 from the National Institutes of Health.
Supporting Information Available: Elemental analyses for
compounds 19, 25, 31-36, and 40-51. This material is available
free of charge via the Internet at http://pubs.acs.org.
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