7-Azaindenoisoquinolines as topoisomerase i inhibitors and potential anticancer agents

Article abstract of DOI:10.1021/jm200719v

A series of 7-azaindenoisoquinoline topoisomerase I (Top1) inhibitors have been prepared to investigate the effect of increased electron affinity of the aromatic system on the ability to stabilize the Top1-DNA cleavage complex. Ab initio calculations suggest that introduction of nitrogen into the aromatic system of the indenoisoquinolines would facilitate charge transfer complex formation with DNA, thus improving the φ-φ stacking interactions. The present study shows that 7-azaindenoisoquinolines demonstrate improved water solubility without any decrease in Top1 inhibitory activity or cytotoxicity. Analysis of the biological results reveals that smaller lactam ring substituents enable intercalation into both free DNA and Top1-DNA cleavage complex, whereas larger substituents only allow binding to the cleavage complex but not free DNA. Free DNA binding suppresses Top1-catalyzed DNA cleavage at high drug concentrations, whereas DNA cleavage and inhibition of religation occurs at low drug concentration.

Full text of DOI:10.1021/jm200719v

ARTICLE
pubs.acs.org/jmc
7-Azaindenoisoquinolines as Topoisomerase I Inhibitors and
Potential Anticancer Agents
Evgeny Kiselev,† Sean DeGuire,† Andrew Morrell,† Keli Agama,‡ Thomas S. Dexheimer,‡ Yves Pommier,‡ and
Mark Cushman*,†
†Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and The Purdue Center for Cancer Research,
Purdue University, West Lafayette, Indiana, 47907, United States
‡Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-4255,
United States
ABSTRACT: A series of 7-azaindenoisoquinoline topoisome-
rase I (Top1) inhibitors have been prepared to investigate the
effect of increased electron affinity of the aromatic system on the
ability to stabilize the Top1ꢀDNA cleavage complex. Ab initio
calculations suggest that introduction of nitrogen into the
aromatic system of the indenoisoquinolines would facilitate
charge transfer complex formation with DNA, thus improving
the πꢀπ stacking interactions. The present study shows that 7-azaindenoisoquinolines demonstrate improved water solubility
without any decrease in Top1 inhibitory activity or cytotoxicity. Analysis of the biological results reveals that smaller lactam ring
substituents enable intercalation into both free DNA and Top1ꢀDNA cleavage complex, whereas larger substituents only allow
binding to the cleavage complex but not free DNA. Free DNA binding suppresses Top1-catalyzed DNA cleavage at high drug
concentrations, whereas DNA cleavage and inhibition of religation occurs at low drug concentration.
’ INTRODUCTION
Human topoisomerase type I (Top1) is a ubiquitous cellular
enzyme. It is a member of the topoisomerase family of enzymes
that solve DNA topological problems associated with super-
coiling.1,2 DNA supercoiling occurs during a number of vital
cellular processes such as replication, transcription, and DNA
repair. Top1 relaxes DNA by producing reversible single-strand
DNA cuts. The generally accepted mechanism of Top1 action
involves formation of a covalent link between the catalytic
tyrosine 723 residue of Top1 and the 30-end of the cut DNA
strand in the Top1ꢀDNA cleavage complex (Top1-DNAcc).
The rotation of the 50-end around the intact strand allows for
relaxation of the supercoils.3 Once the tension caused by super-
coiling has been removed, the backbone of the cut strand is
religated and Top1 released. This mechanism has been substan-
tiated by crystallography of a stable form of the Top1-DNAcc.4
The dependency of living organisms on topoisomerases in
processes like DNA replication during cell division has made
topoisomerases attractive drug targets for anticancer chemother-
apy. The search for Top1 inhibitors was eventually rewarded with
the isolation of camptothecin (1) in 1966 from an extract of the
Chinese tree Camptotheca acuminata (Figure 1).5 It was found
that 1 was capable of inducing DNA cleavage in the presence of
Top1. The ability of 1 to bind to and stabilize the Top1-DNAcc
by forming a drugꢀTop1ꢀDNA ternary complex lies at the
heart of its mechanism of action. Further development of 1 as an
anticancer drug was hindered by its poor water solubility.6
Screening of a number of synthetic analogues of 1 led to the
clinically used Top1 inhibitors, topotecan and irinotecan, which
Figure 1. Representative Top1 inhibitors.
possess basic functionality and thus improved water solubility
and bioavailability. Unfortunately, the derivatives of 1 have
limitations in their clinical use. The lactone form is in equilibrium
with its carboxylate form at physiological pH, which has reduced
bioavailability due to plasma protein binding. Additionally, the
treatment with derivatives of 1 requires long infusion times due
to quick reversibility of the Top1-DNAcc.1,2
Received: June 6, 2011
Published: August 08, 2011
r
2011 American Chemical Society
6106
dx.doi.org/10.1021/jm200719v J. Med. Chem. 2011, 54, 6106–6116
|

Journal of Medicinal Chemistry
ARTICLE
Scheme 1
Replacement of the methyl group on the lactam B-ring of 2
with aminoalkyl chains was found to improve Top1 inhibitory
activity and cytotoxicity of indenoisoquinolines.16ꢀ18 Evaluation
of a number of analogues with diverse substituents on the A- and
D-rings also improved the anticancer properties of the indenoiso-
quinolines.11,19 In particular, the most studied substituents on
the D-ring were the electron-donating alkoxy groups. Only a very
limited number of electron-withdrawing groups on the D-ring have
been evaluated.20 The present report describes a series of indenoi-
soquinolines containing an electron-deficient pyridine D-ring.
The goal of this project was to investigate the abilities of
indenoisoquinolines to form charge-transfer complexes and to
correlate this with their inhibitory potencies against Top1 by
preparing and evaluating a series of 7-azaindenoisoquinoline
derivatives, the 5H-pyrido[30,20:4,5]cyclopenta[1,2-c]isoquinoline-
5-11(6H)-diones (Schemes 1 and 2). One approach to the
modulation of the electron-attracting abilities of the indenoiso-
quinolines was to incorporate additional nitrogen atoms in the
heterocyclic ring system itself. The incorporation of nitrogen into
an aromatic ring system would be expected to increase electron
affinity.21 Thus, indenoisoquinolines containing additional nitro-
gen atoms should have greater capacities for charge-transfer com-
plex formation with neighboring bases in the ternary complex.
Figure 2. Binding mode of 1 within 1ꢀTop1ꢀDNA ternary complex:
hydrogen bonds to Top1 residues (top) and πꢀπ stacking with flanking
DNA base pairs (bottom). The sulfur atom originated from the
phosphorothiolate duplex oligonucleotide substrate used in the crystal-
lography studies.7
Crystallography of the 1ꢀTop1ꢀDNA ternary complex
revealed that 1 is capable of stabilizing the Top1-DNAcc by
binding to it at the site of cleavage.7 The described binding mode
showed an extended region of πꢀπ stacking between the
polycyclic core of 1 and the DNA base pairs, as well as a number
of polar interactions formed between 1 and Top1 residues
(Figure 2). The intercalation of 1 into the Top1-DNAcc
increases the distance between ends of the broken DNA strand
and prevents religation, resulting in prolonged covalent attach-
ment of Top1 to the DNA.
A new class of Top1 inhibitors, the indenoisoquinolines,
emerged with the isolation of 2 (NSC 314622) as a byproduct
of nitidine chloride synthesis (Figure 1).8,9 A COMPARE
analysis revealed similar cytotoxicity profiles between 1 and
2.10 However, observed differences in DNA cleavage site speci-
ficity between camptothecins and indenoisoquinolines suggested
that different genes might be targeted more specifically with
indenoisoquinolines.6 Also and in contrast to 1, the Top1-
DNAcc trapped by 2 was more persistent in both cell- and
enzyme-based assays.6 Moreover, a structural comparison of the
indenoisoquinolines and the camptothecins suggested that in-
denoisoquinolines would likely have enhanced chemical stability
relative to that of the camptothecins. Further structure optimiza-
tion of 2 and biological assessment of synthesized analogues led
to the discovery of a number of potent Top1 inhibitors [e.g., MJ-
III-65 (3a), NSC 724998 (NSC 743400, LMP400, 3b), and NSC
725776 (LMP776, 3c), Figure 1].11,12 The morpholinopropyl
and imidazolylpropyl analogues 3b and 3c13,14 have been pro-
moted to phase I clinical trials at National Cancer Institute.15
’ CHEMISTRY
To date, the most frequently used synthetic approach to
various indenoisoquinolines substituted on the indenone D-ring
has been the condensation of homophthalic anhydrides (e.g., 5ꢀ7,
Scheme 1) with various Schiff bases, followed by cyclization of the
resulting cis-isoquinolonic acids 8a into indenoisoquinolinediones
9a via intramolecular acylation, forming the central C-ring.8,9,16ꢀ18
In the case of 7-azaindenoisoquinolines 9b, a 2-pyridyl Schiff
base would have to be used as a precursor for the D-ring of a new
molecule. Because of extremely low reactivity of pyridines in
reactions of electrophilic aromatic substitution, cyclization of 8b
into 9b would be very difficult (Scheme 1). Therefore, an alter-
native protocol has been followed.22 According to this pathway,
the commercially available 2-cyano-3-picoline 10 was treated
with N-bromosuccinimide (NBS) in refluxing 1,2-dichlor-
oethane in the presence of the radical initiator AIBN. Crude
bromide 11 was subsequently treated with homophthalic anhy-
dride (5), 4,5-dimethoxyhomophthalic anhydride (6),23 or
6107
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116

Journal of Medicinal Chemistry
ARTICLE
Scheme 2a
Scheme 3a
a Reagents and conditions: (a) DIAD, triphenylphosphine, 4-(3-hydro-
xypropyl)morpholine, THF, 23 °C, 3 days (21 62%, 22 68%, 23 30%);
(b) NaH, DMF, ꢀ5 to 23 °C, 3 h, 1,3-dibromopropane, ꢀ5 to 23 °C,
12 h; (c) morpholine, 1,4-dioxane, reflux, 6 h (21 15%, 22 17%, 24
13ꢀ50%, 25 21ꢀ35%).
a Reagents and conditions: (a) NBS, AIBN, 1,2-dichloroethane, reflux,
2 h; (b) 5 (for 12), 6 (for 13) or 7 (for 14), triethylamine, acetonitrile,
reflux 10 h (12 31%, 13 39%, 14 12%); (c) selenium dioxide, 1,4-dioxane,
reflux 16 h (15 89%, 16 26%, 14 59%); (d) DIAD, triphenylphosphine,
3-dimethylamino-1-propanol, THF, 23 °C, 3 days (18 59%, 19 76%, 20
62%).
Scheme 4a
4-nitrohomophthalic anhydride (7)24 to provide 12, 13, or 14,
respectively (Scheme 2). Treatment of 12ꢀ14 with selenium
dioxide in refluxing 1,4-dioxane provided indenoisoquinolines
15ꢀ17.
It was previously shown that addition of the aminoalkyl chain
to the sixth position of the indenoisoquinolines increases Top1
inhibitory activity.11 Treatment of compounds 15, 16, and 17
with 3-dimethylamino-1-propanol, triphenylphosphine, and
DIAD in tetrahydrofuran (Mitsunobu reaction) yielded com-
pounds 18, 19, and 20 (Scheme 2). The overall yields for the
syntheses of 18, 19, and 20 are modest at best (4ꢀ16% from 10)
but are comparable to those that could be achieved for most
indenoisoquinoline systems via the Schiff base/homophthalic
anhydride condensation and FriedelꢀCrafts acylation sequence
(7ꢀ20% from benzaldehydes).11
a Reagents and conditions: (a) (1) NaH, DMF, ꢀ5 to 23 °C, 3 h, 1,3-
dibromopropane, ꢀ5 to 23 °C, 12 h, (2) imidazole, 1,4-dioxane, reflux,
6 h (28 17%, 29 17%); (b) (1) NaH, DMF, ꢀ5 to 23 °C, 3 h, 1,3-
dibromopropane, ꢀ5 to 23 °C, 12 h, (2) 2-ethanolamine, 1,4-dioxane,
reflux, 6 h (30 16%, 31 20%); (c) (1) NaH, DMF, ꢀ5 to 23 °C, 3 h, 1,3-
dibromopropane, ꢀ5 to 23 °C, 12 h, (2) NaN3, DMF, 23 °C, 12 h; (3)
P(OC2H5)3, benzene, reflux, 6 h; (4) HCl, H2O, reflux, 3 h (32 18%).
A number of analogues of 2 with the lactam nitrogen bearing
various aminopropyl substituents were previously prepared and
evaluated as Top1 inhibitors and antiproliferative agents.11,16ꢀ18
Use of such substituents as aminopropyl, ethanolaminopropyl,
morpholinopropyl, and imidazolylpropyl was shown to improve
both cytotoxicity and inhibitory potency of indenoisoquinolines.
The optimization of the aminopropyl group ultimately led to the
identification of 3b and 3c that were advanced to clinical trials.15
It was our intention to explore these aminopropyl substituents in
the context of the 7-azaindenoisoquinoline system. For this
purpose Mitsunobu reaction with 4-(3-hydroxypropyl)morpho-
line was used in order to prepare morpholinopropyl analogues
21ꢀ23 (Scheme 3). Compounds 19 and 20 were also prepared
in an alternative two-step sequence. Treatment of 15 and 16 with
excess of 1,3-dibromopropane in the presence of sodium hydride,
followed by reaction with morpholine, yielded 21 and 22,
respectively. Allyl-substituted indenoisoquinolines 24 and 25
were isolated as minor products along with their morpholino-
propyl analogues 21 and 22. It was noticed that the chromato-
graphic separation of the allyl analogues 24 and 25 was much
easier after the intermediate bromides 26 and 27 were converted
to corresponding morpholine analogues 21 and 22. Therefore,
the conversion of 15 and 16 into their respective aminopropyl
derivatives was carried out without isolation of the intermediate
bromides 26 and 27.
The second approach also provides an easy access to the
aminoalkyl analogues that are difficult to obtain via Mitsunobu
reaction because of the presence of multiple hydroxyl or amino
groups or commercial unavailability of the starting amino alcohols.
6108
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116

Journal of Medicinal Chemistry
ARTICLE
Table 1. Top1 Inhibitory and Antiproliferative Activity of 7-Azaindenoisoquinolines
cytotoxicity GI50, μMc
lung
colon
CNS
melanoma
UACC-62
ovarian
renal
prostate
DU-145
breast
compd
Top1 cleavagea
MGMb
HOP-62
HCT-116
SF-539
OVCAR-3
SN12C
MCF7
1
++++
++
0.04
8.51
1.78
4.50
1.86
15.49
0.30
0.01
0.03
11.48
0.41
1.58
0.44
1.86
0.22
0.01
1.66
1.91
4.07
2.69
5.62
0.29
0.01
0.22
22.39
2.57
3.55
2.29
28.18
0.37
0.02
25.70
0.81
3.16
1.0
0.01
4.79
1.35
1.70
0.93
3.55
0.31
0.013
1.91
0.25
0.44
0.36
2.75
0.052
2
2.82
0.56
18
19
20
21
22
23
28
29
30
31
32
33
34
35
+++
+++
+++(+)
+(+)
++
1.51
2.69
3.392
2.349
35.48
0.30
13.18
3.31
>100
0.095
5.37
0.52
+++
++
7.24
7.24
1.20
5.13
35.48
8.71
3.72
2.88
3.39
++
+
1.29
0.29
2.09
1.86
6.17
9.77
1.41
0.30
3.02
1.74
11.48
5.62
0.30
0.15
0.38
0.58
2.45
6.46
0.87
0.26
2.19
1.86
6.17
3.09
0.12
8.13
0.51
6.61
7.08
1.55
0.34
1.58
1.66
5.89
32.36
0.76
0.23
2.88
0.91
10.96
4.17
0.41
0.32
1.62
1.32
4.47
5.62
0.48
0.074
49
+(+)
+(+)
+++
++(+)
+++
0.55
6.17
0.24
a The relative Top1 inhibitory potencies of the compounds are presented as follows: 0, no detectable activity; +, weak activity; ++, activity similar to that
of 2; +++ and ++++, greater activity than that of 2; ++++, activity similar to that of 1 μM 1. b Mean graph midpoint for growth inhibition of all human
cancer cell lines successfully tested. c The cytotoxicity GI50 values listed are the concentrations corresponding to 50% growth inhibition and are the result
of single determinations.
Examples of such analogues 28ꢀ32 are presented in Scheme 4.
According to this method, reaction of intermediate bromides 26
and 27 with imidazole yielded 28 and 29, respectively. Similarly,
ethanolamine analogues 30 and 31 were isolated after reaction of
26 and 27 with 2-ethanolamine. The reaction of 27 with sodium
azide followed by treatment with triethyl phosphite yielded
amine 32.
’ BIOLOGICAL RESULTS AND DISCUSSION
Figure 3. Representative indenoisoquinolines.
All of the target compounds were tested for induction of DNA
damage in Top1-mediated DNA cleavage assays.25 For this
values of the prepared indenoisoquinolines in selected cell lines
32
purpose, a P 30-end-labeled 117-bp DNA fragment was in-
cubated with human recombinant Top1 and increasing concen-
tration of a tested compound. The DNA fragments were
separated on a denaturing gel. The Top1 inhibitory activity
was assigned based on the visual inspection of the number and
intensities of the DNA cleavage bands and expressed in semi-
quantitative fashion relative to the Top1 inhibitory activities of
compounds 1 and 2: 0, no detectable activity; +, weak activity;
++, activity similar to that of 2; +++, activity greater than that of
2; ++++, equipotent to 1 (Table 1).
are presented in Table 1.
The Top1 inhibitory activities were compared to the pre-
viously prepared indenoisoquinolines 33ꢀ35 (Figure 3).30 The
results clearly show that azaindenoisoquinolines 18ꢀ20 are
equipotent or more potent against Top1 than corresponding
indenoisoquinolines 33ꢀ35 (Table 1). A preservation of the
level of cyctotoxicity in the 7-azaindenoisoquinoline compounds
was also observed relative to the corresponding indenoisoqui-
nolines 33ꢀ35. A slight increase in cytotoxicity was observed in
ethanolaminopropyl 30, 31, and aminopropyl 32 analogues.
Interestingly, the increase in cytotoxicity coincided with the
decrease of Top1 activity and suggested an additional mechanism
of action responsible for the increased cytotoxicity.
In some cases, the intensities of the cleavage bands were
reduced at the highest tested concentration of the drug, 100 μM.
This result can be attributed to the ability of these indenoiso-
quinolines to intercalate into free DNA, preventing DNA cleavage
by Top1. Interestingly, when analogues 19, 22, 29, 31, and 32
were compared side-by-side (Figure 4), it was observed that
ethanolaminopropyl and aminopropyl derivatives 31 and 32
behave similarly to 19. One possible reason for the decrease in
The antiproliferative activity of each compound was deter-
mined in the National Cancer Institute (NCI) screen.26ꢀ29 Cells
of approximately 60 different human cancer cell lines were
incubated for 48 h with five 10-fold dilutions of the tested
compounds starting from 100 μM and then treated with sulfor-
hodamine B dye. The ratios of recorded optical densities relative
to that of the control were plotted as a function of the common
logarithm of the tested compound concentrations. The inter-
polation between the points located above and below the 50%
percentage growth provided respective 50% growth inhibition
(GI50) values. The GI50 and the mean graph midpoint (MGM)
6109
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116

Journal of Medicinal Chemistry
ARTICLE
Figure 4. Lanes 1, 9, 17, 25, 33: DNA alone. Lanes 2, 10, 18, 26, 34: Top1 alone. Lanes 3, 11, 19, 27, 35: Top1 + 1 (1 μM). Lane 4: Top1 + 2 (100 μM).
Lanes 12, 20, 28, 36: Top1 + 3a (1 μM). Lanes 5ꢀ8: Top1 + 19 at 0.1, 1, 10, 100 μM. Lanes 13ꢀ16: Top1 + 22 at 0.1, 1, 10, 100 μM. Lanes 21ꢀ24: Top1
+ 29 at 0.1, 1, 10, 100 μM. Lanes 29ꢀ32: Top1 + 31 at 0.1, 1, 10, 100 μM. Lanes 37ꢀ40: Top1 + 32 at 0.1, 1, 10, 100 μM. Numbers on right and arrows
show the cleavage site positions.
into free DNA. At its highest tested concentration, 100 μM,
compound 19 completely inhibits the Top1-mediated DNA
relaxation, as demonstrated by the fact that there is no more
detectable relaxation by Top1 after addition of the drug. The
results of the experiment with compound 22, on the other hand,
show its inability to unwind/intercalate into DNA in the range of
tested concentrations, 1ꢀ100 μM. These results can presumably
be extrapolated to other aminopropyl derivatives presented in
this work.
This observation might also explain the lack of correlation
Figure 5. DNA unwinding and Top1 inhibition by 19 and 22. Lane 1:
DNA alone. Lanes 2 and 8 were DNA + Top1. Lanes 3ꢀ7: DNA + Top1
+ 19 at 1, 3, 10, 30, 100 μM. Lanes 9ꢀ13: DNA + Top1 + 22 at 1, 3, 10,
30, 100 μM.
between Top1 inhibitory activity and cytotoxicity in the series of
compounds. The ability to intercalate into free DNA can
potentially serve as an off-target effect of the drug, increasing
its cytotoxicity. On the other hand, the Top1 inhibitory activity as
observed in Top1-mediated DNA cleavage assays can decrease as
the ability to intercalate into free DNA increases.
DNA cleavage at high drug concentration is that intercalation of
these indenoisoquinolines into DNA makes it a poorer enzyme
substrate. In contrast, the morpholinopropyl and imidazolylpro-
pyl analogues 22 and 29 express concentration-dependent
increase of intensity of DNA cleavage bands, and therefore,
Top1 inhibitory activity, up to 100 μM.
DNA unwinding assays have been performed in order to test
the DNA intercalation hypothesis for the observed decrease in
DNA cleavage at high drug concentration. Two compounds have
been tested and compared in this assay: dimethylaminopropyl
analogue 19 and morpholinopropyl compound 22 (Figure 5).
DNA unwinding reactions were performed in two steps as
described.31 First, SV40 DNA was reacted with excess of Top1
for 15 min at 37 °C in the absence of drug to fully relax the DNA.
The relaxed DNA was then further incubated for 30 min at 37 °C
in the presence of increasing concentrations of drugs (Figure 5).
Additional incubation of the DNA and Top1 for 30 min at 37 °C
in the absence of drug was performed as a control. The results of
the DNA unwinding assay demonstrated the ability of 19 to
unwind DNA at concentrations above 3 μM in a concentration-
dependent manner, hence showing the ability of 19 to intercalate
Most azaindenoisoquinolines retained the activity of the
“classical” indenoisoquinolines. Introduction of the pyridine
nitrogen (a strong hydrogen bond acceptor) into the core of
the indenoisoquinoline structure should also improve aqueous
solubility. To test this hypothesis, the solubilities of two 7-azain-
denoisoquinolines were assessed and compared to that of closely
related “classical” indenoisoquinolines (Figure 6). Aqueous
solubility was determined by an HPLC method.32 For this test,
the samples of four different indenoisoquinolines (Figure 6)
were shaken with 1 M Tris buffer (pH 7.5) solution. The aliquots
of the supernatants were injected into the HPLC system after
24 h. In order to determine the concentration of the sample, one
point calibration was done against standards with known con-
centrations of the sample compounds. The estimation of the
solubility demonstrated that the introduction of a pyridine ring
into the indenoisoquinoline system led to a significant increase of
the water solubility. Interestingly, the increase in solubility of the
drug did not compromise the Top1 inhibitory activity or
cytotoxicity of the drugs (Figure 6, Table 1). A similar result
was observed when an extra nitrogen was introduced into the
6110
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116

Journal of Medicinal Chemistry
ARTICLE
Figure 8. Derivation of indenoisoquinolineꢀDNA “sandwich” com-
plex models for quantum mechanical calculations.
Figure 6. Solubility of indenoisoquinolines.
Table 2. πꢀπ Staking Interaction Energies of Models
A and B
Eint,vac
(kcal/mol)a
Eint,aq
(kcal/mol)a
Ecorr
(kcal/mol)b
charge transfer
(eꢀ)c
model
A
B
ꢀ6.95
ꢀ7.99
ꢀ15.17
ꢀ15.57
ꢀ26.0284
ꢀ25.7899
0.0115
0.0126
a Eint is derived from MP2/6-31G* single point energy calculations.
b Ecorr = Eint(MP2/6-31G*) ꢀ Eint(HF/6-31G*). c The magnitude of the charge
transfer as estimated by NBO analysis at HF/6-31G** level.
Figure 7. Structure of 14-azacamptothecin.
The CH group in position 7 of the D-ring of the inhibitor was
replaced with nitrogen to produce the 7-azaindenoisoquinoline
model (Figure 8). Before fitting of azaindenoisoquinolines into
their corresponding complexes with DNA base pairs, geometry
optimizations were performed. After the complexes were as-
sembled, MP2/6-31G* single point energy calculations were
performed. The basis set superposition error (BSSE) for each
complex was calculated, specifying two fragments: the first was
two flanking DNA base pairs, and the second was the intercalat-
ing indenoisoquinoline inhibitor. The in vacuo πꢀπ stacking
polycyclic system of 1, resulting in 14-azacamptothecin (36,
Figure 7), which possessed greater water solubility compared to
the parent 1.33,34
Crystallography of the ternary complexes of indenoisoquino-
lines revealed that only one major polar interaction between drug
and the Top1-DNAcc is present, which is a hydrogen bond to the
Arg364.7 It was therefore hypothesized that πꢀπ stacking inter-
actions play a major role in complex stabilization and that these
interactions determine the orientations of the drugs within ternary
complexes. This hypothesis was supported by a series of high level
ab initio calculations on a number of diverse Top1 inhibitors.35ꢀ37
Thus, drug binding orientations and DNA site specificities were
calculated correctly for a number of different ligands.35ꢀ37
To investigate the effect of the introduction of nitrogen into
the aromatic systems of indenoisoquinolines on their abilities to
form strong πꢀπ stacking interactions, a series of hypothetical
indenoisoquinoline structures were modeled and subjected to
MP2 single point energy calculations. In order to derive a model
for such calculations, an inhibitor and adjacent base pairs were
taken from the X-ray structure of 4ꢀTop1ꢀDNA ternary
complex (PDB entry 1SC7).7 The deoxyribose rings of the
DNA bases and the carboxypropyl chain of 4 were replaced with
methyl groups, similar to the previously described model.37 The
geometry optimizations and frequency calculation at the HF/
6-31G** level were performed on the indenoisoquinoline mole-
cule, as well as AꢀT and GꢀC base pairs using the Gaussian 0938
software package. The original complex was then replaced with
the geometry-optimized parts (Figure 8). The MP2 single point
energy calculation with the 6-31G* basis set was done with the
resulting complex.
interaction energy was then calculated as Eint,vac = Ecomplex,vac
ꢀ
Eligand,vac ꢀ Ebp,vac + BSSE, where Ecomplex,vac, Eligand,vac, and Ebp,vac
are the corresponding MP2/6-31G* calculated energies of com-
plex, ligand, and DNA base pairs at their normal distance in the
absence of intercalator (Table 2). Similarly, the energy of the
πꢀπ staking interaction was also calculated in water (Eint,aq
)
using the polarized continuum model. The dispersion and charge
transfer interaction components of the πꢀπ staking have also
been determined. The dispersion (electron correlation) interac-
tion (Ecorr) was defined as the difference of energy calculated by
MP2 and HF methods at the 6-31G* level, Ecorr = Eint(MP2)
ꢀ
Eint(HF) (Table 2). Natural bond orbital (NBO)39 analyses have
been performed at the HF/6-31G** level of theory to determine
the charge transfer component (Table 2). The results of these
calculations showed that the slight decrease in the dispersion
force was outweighed by the increased charge transfer, leading to an
overall greater πꢀπ stacking interaction energy for the 7-azainde-
noiusoquinoline (model B, Figure 8) vs the indenoisoquinoline
(model A, Figure 8) both in vacuo (Eint,vac) and in water (Eint,aq).
In conclusion, a series of novel azaindenoisoquinolines have
been prepared. Two alternative derivatization pathways have
6111
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116

Journal of Medicinal Chemistry
ARTICLE
been explored, and the established protocols were then used to
synthesize diverse products. All synthesized compounds were
evaluated in Top1-mediated DNA cleavage reactions in order to
establish the extent of their abilities to stabilize Top1-DNAcc and
hence their Top1 inhibitory activities. The NCI screening for
antiproliferative activity demonstrated a slight but consistent
improvement of the cytotoxic properties of the azaindenoiso-
quinoline series over identically substituted, previously described
indenoisoquinolines. The role of the aminopropyl chain was also
investigated and demonstrated to play a crucial role in biological
activity, consistent with prior results for indenoisoquinolines.
The results of the DNA unwinding assay presented in this work
unambiguously show the effect this group has on the ability of the
drug to intercalate into both free DNA and Top1-DNAcc.
Smaller amines (e.g., 17) allow intercalation into DNA in the
absence of Top1, while larger ones (e.g., 20) do not. This
explains why indenoisoquinolines with smaller amines suppress
DNA cleavage by Top1 at high drug concentrations.
[3-dimethylamino-1-propanol (0.17ꢀ0.21 mL, 1.46ꢀ1.82 mmol,
3 equiv) or 3-morpholinopropan-1-ol (145ꢀ435 mg, 1.0ꢀ3.0 mmol,
3 equiv)] was added, followed by DIAD (202ꢀ606 mg, 1.0ꢀ3.0 mmol,
3 equiv). The solution was stirred at room temperature for 64 h. As the
reaction reached completion, all of the solid material dissolved. The
reaction mixture was concentrated to dryness. The solid was purified by
flash column chromatography (SiO2), eluting with 1% MeOH in
CHCl3, to provide a dark-orange solid. The solid was further purified
by treating it with 3 M HCl in methanol (15 mL) for 2 h at room
temperature with stirring. The solution was concentrated to dryness.
The orange solid was diluted in ethyl ether (50 mL). The hydrochloride salt
of the product precipitated and was collected using vacuum filtration,
washing with ethyl ether (3 ꢁ 15 mL), to provide an orange solid.
General Procedure for the Preparation of 7-Azaindenoi-
soquinolines 21ꢀ25 and 29ꢀ31. Sodium hydride (95%, 50 mg,
2 mmol, 2 equiv) was added to a mixture of the appropriate 7-aza-5,6-
dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (15, 243 mg, 1 mmol,
1 equiv, or 16, 308 mg, 1 mmol, 1 equiv) and DMF (6 mL) at ꢀ5 °C, and
the resulting mixture was allowed to warm to room temperature over 3 h.
The resulting clear dark red solution was cooled to ꢀ5 °C, and 1,3-
dibromopropane (788 mg, 4 mmol, 4 equiv) was added. The solution
was stirred at room temperature for 12 h. The reaction mixture was
quenched with water (50 mL). The products were extracted with
chloroform (3 ꢁ 10 mL). The combined extracts were washed with
water (3 ꢁ 10 mL), brine (10 mL), dried with sodium sulfate, and
concentrated under reduced pressure. The residue was subjected to flash
column chromatography (silica gel, CHCl3). The appropriate amine
(140.3ꢀ200 mg, 2.3 mmol, 2.3 equiv) was added to a solution of crude
intermediate in 1,4-dioxane (10 mL). The resulting mixture was heated
to reflux for 6 h. The solvent was evaporated under reduced pressure,
and the residue was redissolved in chloroform (30 mL). The chloroform
solution was washed with water (3 ꢁ 5 mL), brine (5 mL), dried with
sodium sulfate, and evaporated to dryness. The solid residue was
subjected to flash column chromatography (silica gel), eluting with
chloroform, to get 24 and 25, and then with 5ꢀ15% methanol in
chloroform to obtain 21ꢀ23 and 29ꢀ31.
’ EXPERIMENTAL SECTION
General. Melting points were determined using capillary tubes with
a Mel-Temp apparatus and are uncorrected. The nuclear magnetic
resonance spectra (1H and 13C NMR) were recorded using ARX300 300
MHz and DRX500 500 MHz Bruker NMR spectrometers. IR spectra
were recorded using a Perkin-Elmer 1600 series FTIR spectrometer.
Purity of all tested compounds was g95%, as established by combustion
analysis. Combustion microanalyses were performed at the Purdue
University Microanalysis Laboratory or Galbraith Laboratories Inc.,
and the reported values were within 0.4% of the calculated values.
HPLC analyses were performed on a Waters 1525 binary HPLC pump/
Waters 2487 dual λ absorbance detector system. 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 230ꢀ400 mesh silica gel.
General Procedure for the Preparation of 7-Aza-5,6-dihy-
dro-5-oxo-11H-indeno[1,2-c]isoquinolines 12ꢀ14. 3-Methyl-
picolonitrile (10, 3.0ꢀ4.0 g, 25.4ꢀ33.9 mmol, 1 equiv), NBS (6.78ꢀ
9.04 g, 38.1ꢀ50.8 mmol, 1.5 equiv), and AIBN (0.42ꢀ0.56 g, 2.5ꢀ3.4
mmol, 0.1 equiv) were diluted with 1,2-dichloroethane (80ꢀ100 mL),
and the reaction mixture was heated at reflux for 2 h. The reaction
mixture was concentrated to half its original volume, filtered, and the
filtrate was concentrated to dryness to provide crude 11. Compound 11
was diluted with acetonitrile (100ꢀ125 mL). The appropriate homo-
phthalic anhydride (5, 6, or 7, 6.8ꢀ12.4 g, 41.9ꢀ55.9 mmol, 1.65 equiv)
was added, followed by triethylamine (18ꢀ24 mL, 127.0ꢀ169.5 mmol,
5 equiv), and the solution was heated at reflux for 10 h. The solution was
allowed to cool to room temperature and the precipitate was filtered and
washed with hot acetonitrile (2 ꢁ 35 mL) to provide the desired
compound.
7-Aza-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinoline
(12). The general procedure provided the desired compound as a gray
solid (2.49 g, 31%): mp 267ꢀ269 °C. IR (KBr) 3098, 1665, 1573, 1478,
767, and 756 cmꢀ1; 1H NMR (DMSO-d6) δ 12.18 (s, 1 H), 8.56 (dd, J =
4.9 and 1.3 Hz, 1 H), 8.30 (d, J = 8.0 Hz, 1 H), 8.02 (dd, J = 7.5 and
1.1 Hz, 1 H), 7.82ꢀ7.80 (m, 2 H), 7.57ꢀ7.51 (m, 1 H), 7.36 (dd, J =
7.5 and 5.0 Hz, 1 H), 3.94 (s, 2 H); ESIMS m/z (rel intensity) 235
(MH+, 100). Anal. Calcd for C15H10N2O: C, 76.91; H, 4.30; N, 11.96.
Found: C, 76.58; H, 4.16; N, 11.80.
7-Aza-5,6-dihydro-2,3-dimethoxy-5-oxo-11H-indeno[1,2-
c]isoquinoline (13). The general procedure provided the desired
compound as a purple solid (3.87 g, 39%): mp 284ꢀ286 °C. IR (KBr)
3399, 3276, 1635, 1604, 1479, 1215, and 803 cmꢀ1; 1H NMR (DMSO-
d6) δ 11.99 (s, 1 H), 8.52 (dd, J = 4.9 and 1.1 Hz, 1 H), 7.95 (d, J = 6.9
Hz, 1 H), 7.67 (s, 1 H), 7.32 (dd, J = 7.5 and J = 4.9, 1 H), 7.24 (s, 1 H),
3.97 (s, 3 H), 3.91 (s, 2 H), 3.89 (s, 3 H); ESIMS m/z (rel intensity) 295
(MH+, 100).
General Procedure for the Preparation of 7-Aza-5,6-dihy-
dro-5,11-dioxo-11H-indeno[1,2-c]isoquinolines 15ꢀ17. The
appropriate 7-aza-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinoline
(12ꢀ14, 0.50ꢀ1.0 g, 1.8ꢀ4.27 mmol, 1 equiv) and SeO2 (0.4ꢀ
0.95 g, 3.6ꢀ8.54 mmol, 2 equiv) were diluted with 1,4-dioxane
(125ꢀ250 mL) and heated at reflux for 16 h. The reaction mixture
was filtered, the filtrate was concentrated, and the precipitate was washed
with MeOH (2 ꢁ 50 mL) to provide the desired product.
7-Aza-5,6-dihydro-3-nitro-5-oxo-11H-indeno[1,2-c]isoquino-
line (14). The general procedure provided the desired compound as a green
solid (878 mg, 12%): mp 323 °C. IR (KBr) 3096, 1691, 1607, 1508, 1330,
1
and 834 cmꢀ1; H NMR (DMSO-d6, 500 MHz) δ 12.74 (s, 1 H), 8.95
(d, J = 2.3 Hz, 1 H), 8.60 (d, J = 4.9 Hz, 1 H), 8.52 (d, J = 2.3 Hz, 1 H), 8.51
(d, J = 2.6 Hz, 1 H), 8.06 (d, J = 8.0 Hz, 1 H), 8.00 (d, J = 8.0 Hz, 1 H), 7.41
(dd, J = 7.6 and 4.9 Hz, 1 H), 3.97 (s, 3 H), 3.91 (s, 2 H), 3.89 (s, 3 H);
negative ion ESIMS m/z (rel intensity) 278 [(M ꢀ H+)ꢀ, 100].
General Procedure for the Preparation of 7-Azaindenoi-
soquinolines 18ꢀ23. The appropriate 7-aza-5,6-dihydro-5,11-di-
oxo-11H-indeno[1,2-c]isoquinoline (15ꢀ17, 94ꢀ248 mg, 0.31ꢀ1.0
mmol, 1 equiv) and triphenylphosphine (262ꢀ786 mg, 1.0ꢀ3.0 mmol,
3 equiv) were diluted in THF (16ꢀ50 mL). The appropriate alcohol
7-Aza-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline
(15). The general procedure provided the desired compound as a red-
orange solid (0.94 g, 89%): mp 337 °C (dec). IR (KBr) 2979, 1678,
6112
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116

Journal of Medicinal Chemistry
ARTICLE
1575, 1085, and 770 cmꢀ1; 1H NMR (DMSO-d6) δ 13.12 (s, 1 H), 8.61
(dd, J = 6.2 and 1.4 Hz, 1 H), 8.44 (d, J = 7.9 Hz, 1 H), 8.23 (d, J = 7.3 Hz,
1 H), 7.89ꢀ7.81 (m, 2 H), 7.58ꢀ7.53 (m, 1 H), 7.46 (dd, J = 7.3 and 5.2
Hz, 1 H); negative ion ESIMS m/z (rel intensity) 247 [(M ꢀ H+)ꢀ,
100].
7-Aza-5,6-dihydro-6-(3-morpholinopropyl)-2,3-dimethoxy-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (22).
The general procedure provided the desired compound as a red solid (109
mg, 68%): mp 322ꢀ324 °C. IR (KBr) 1735, 1704, 1655, 1642, 1607, 1552,
1513 cmꢀ1; 1H NMR (CDCl3) δ 8.37 (dd, J = 5.4 and 1.5 Hz, 1 H), 7.86
(s, 1 H), 7.61 (dd, J = 7.2 and 1.5 Hz, 1 H), 7.54 (s, 1 H), 7.11 (dd, J = 7.2
and 5.1 Hz, 1 H), 4.91 (m, 2 H), 3.97 (s, 3 H), 3.91 (s, 3 H), 3.43 (m, 4 H),
2.44 (m, 2 H), 2.34 (m, 4 H), 1.95 (m, 2 H); positive ion ESIMS m/z (rel
intensity) 436 [(MH+), 100]. Anal. Calcd for C24H25N3O5 HCl 2.5H2O:
7-Aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (16). The general procedure provided the
desired compound as a dark red solid (272 mg, 26%): mp 358ꢀ
1
359 °C. IR (KBr) 3129, 2988, 1676, 1504, 1277, and 777 cmꢀ1; H
3
3
C, 55.76; H, 6.04; N, 8.13. Found: C, 55.83; H, 5.87; N, 8.33.
NMR (DMSO-d6) δ 12.96 (s, 1 H), 8.56 (dd, J = 6.2 and 1.4 Hz, 1 H),
7.86ꢀ7.81 (m, 2 H), 7.59 (s, 1 H), 7.40 (t, J = 6.8 Hz, 1 H); negative ion
ESIMS m/z (rel intensity) 307 [(M ꢀ H+)ꢀ, 100].
7-Aza-5,6-dihydro-6-(3-morpholinopropyl)-3-nitro-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (23).
The general procedure provided the desired compound as a light-red
solid (69 mg, 30%): mp 266ꢀ228 °C. IR (KBr) 1705, 1679, 1614, 1598,
1556, 1502 cmꢀ1; 1H NMR (DMSO-d6) δ 10.28 (s, 1 H), 8.96 (d, J =
2.4 Hz, 1 H), 8.78ꢀ8.75 (m, 2 H), 8.67 (dd, J = 9.0 and 2.4 Hz, 1 H), 8.08
(d, J = 7.2 Hz, 1 H), 7.60 (dd, J = 7.2 and 5.1 Hz, 1 H), 4.98 (m, 2 H),
3.98 (m, 2 H), 3.40 (m, 2 H), 3.09 (m, 4 H), 2.29 (m, 2 H). Anal. Calcd
7-Aza-5,6-dihydro-3-nitro-5,11-dioxo-11H-indeno[1,2-
c]isoquinoline (17). The general procedure provided the desired
compound as a red-orange solid (310 mg, 59%): mp 351ꢀ353 °C. IR
1
(KBr) 3187, 1692, 1567, 1335, and 785 cmꢀ1; H NMR (DMSO-d6)
δ 13.75 (s, 1 H), 8.86 (s, 1 H), 8.69 (dd, J = 5.2 and 1.3 Hz, 1 H), 8.55
(s, 2 H), 7.97 (dd, J = 7.4 and 1.3 Hz, 1 H), 7.53 (dd, J = 7.4 and 5.2 Hz
1 H); negative ion ESIMS m/z (rel intensity) 292 [(M ꢀ H+)ꢀ, 100].
7-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-5,11-dioxo-
11H-indeno[1,2-c]isoquinoline Hydrochloride (18). The gen-
eral procedure provided the desired compound as an orange solid
(160 mg, 59%): mp 268ꢀ270 °C. IR (KBr) 3446, 1703, 1670, 1505,
and 770 cmꢀ1; 1H NMR (DMSO-d6) δ 10.05 (s, 1 H), 8.66 (dd, J = 1.5
and 5.2 Hz, 1 H), 8.56 (d, J = 7.8 Hz, 1 H), 8.27 (d, J = 7.5 Hz, 1 H), 7.95
(dd, J = 1.5 Hz and 7.4, Hz 1 H), 7.90ꢀ7.84 (m, 1 H), 7.63ꢀ7.58
(m, 1 H), 7.50 (dd, J = 5.2 and 7.3 Hz, 1 H), 4.92 (t, J = 6.8 Hz, 2 H), 3.23
(p, J = 5.3 and 10.1 Hz, 2 H), 2.75 (s, 3 H), 2.74 (s, 3 H), 2.22ꢀ2.17 (m,
2 H); positive ion ESIMS m/z (rel intensity) 333 [(MH+), 100]. Anal.
Calcd for C20H19N3O2 HCl 1.5H2O: C, 60.53; H, 5.84; N, 10.59.
for C22H20N4O5 0.8H2O: C, 60.77; H, 5.01; N, 12.88. Found: C, 60.92;
3
H, 4.71; N, 12.56.
7-Aza-5,6-dihydro-6-allyl-5,11-dioxo-11H-indeno[1,2-c]iso-
quinoline (24). The general procedure provided the desired com-
pound as a red solid (37ꢀ150 mg, 13ꢀ52%): mp 222ꢀ224 °C. IR
(KBr) 1700, 1666, 1644, 1611, 1568, 1548, 1501 cmꢀ1 1H NMR
;
(CDCl3) δ 8.66 (d, J = 7.8 Hz, 1 H), 8.52 (dd, J = 5.1 and 1.5 Hz, 1 H),
8.38 (d, J = 7.5 Hz, 1 H), 7.81ꢀ7.73 (m, 2 H), 7.52 (t, J = 8.1 Hz, 1 H),
7.24 (dd, J = 7.5 and 5.4 Hz, 1 H), 6.11 (m, 1 H), 5.67 (d, J = 5.7 Hz, 1
H), 5.32ꢀ5.19 (m, 2 H). Anal. Calcd for C18H12N2O2 0.2H2O: C,
3
74.06; H, 4.28; N, 9.60. Found: C, 73.97; H, 4.14; N, 9.53.
7-Aza-5,6-dihydro-6-allyl-2,3-dimethoxy-5,11-dioxo-11H-
indeno[1,2-c]isoquinoline (25). The general procedure provided
the desired compound as a dark-red solid (73ꢀ122 mg, 21ꢀ35%): mp
266ꢀ268 °C. IR (KBr) 1701, 1659, 1608, 1570, 1552, 1515 cmꢀ1; 1H
NMR (CDCl3) δ 8.47 (dd, J = 5.4, 1.5 Hz, 1 H), 8.03 (s, 1 H), 7.74ꢀ7.71
(m, 2 H), 7.18 (dd, J = 7.5, 5.4 Hz, 1 H), 6.01 (m, 1 H), 5.62 (d, J =
5.7 Hz, 2 H), 5.3ꢀ5.17 (m, 2 H), 4.06 (s, 3 H), 3.99 (s, 3 H). Anal. Calcd
3
3
Found: C, 60.36; H, 5.62; N, 10.45.
7-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-2,3-di-
methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Hydro-
chloride (19). The general procedure provided the desired compound
as a light-orange solid (158 mg, 76%): mp 278ꢀ281 °C (dec). IR (KBr)
1
3407, 2456, 1702, 1654, 1476, and 779 cmꢀ1; H NMR (DMSO-d6)
for C20H16N2O4 0.2H2O: C, 68.25; H, 4.70; N, 7.96. Found: C, 61.21;
δ 9.99 (s, 1 H), 8.59 (dd, J = 1.2 and 5.2 Hz, 1 H), 7.88 (s, 1 H), 7.85
(d, J = 7.3 Hz, 1 H), 7.54 (s, 1 H), 7.43 (dd, J = 5.3 and 7.3 Hz, 1 H), 4.86
(t, J = 6.4 Hz, 1 H), 3.93 (s, 3 H), 3.88 (s, 3 H), 3.21ꢀ3.16 (m, 3 H), 2.76
(s, 3 H), 2.74 (s, 3 H), 2.19ꢀ2.15 (m, 2 H); positive ion ESIMS m/z (rel
intensity) 394 [(MH+), 100]. Anal. Calcd for C22H23N3O4 2HCl 0.5
3
H, 4.63; N, 7.90.
7-Aza-5,6-dihydro-6-(3-(1H-imidazol-1-yl)propyl)-5,11-dioxo-
11H-indeno[1,2-c]isoquinoline (28). The general procedure pro-
vided the desired compound as an orange solid (59 mg, 17%): mp
200ꢀ202 °C. IR (KBr) 1698, 1664, 1610, 1570, 1549, 1506 cmꢀ1; 1H
NMR (CDCl3) δ 8.64 (d, J = 8.1 Hz, 1 H), 8.43 (dd, J = 5.1, 1.5 Hz, 1 H),
8.37 (d, J = 7.2 Hz, 1 H), 7.80ꢀ7.72 (m, 2 H), 7.58 (s, 1 H), 7.53 (m,
2 H), 7.23 (dd, J = 7.5, 5.4 Hz, 1 H), 7.09 (s, 1 H), 7.01 (s, 1 H), 5.00
(t, J = 7.5 Hz, 2 H), 4.17 (t, J = 6.9 Hz, 2 H), 2.36 (quint, J = 7.5 Hz, 2 H);
positive ESIMS m/z (rel intensity) 357 (MH+, 100). Anal. Calcd for
3
3
H2O: C, 55.47; H, 5.45; N, 10.59. Found: C, 55.59; H, 5.51; N, 8.84.
7-Aza-5,6-dihydro-6-(3-dimethylaminopropyl)-3-nitro-
5,11-dioxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (20).
The general procedure provided the desired compound as a bright
orange solid (130 mg, 62%): mp 280ꢀ282 °C (dec). IR (KBr) 3393,
1705, 1677, 1501, 1338, and 791 cmꢀ1; 1H NMR (DMSO-d6) δ 9.88
(s, 1 H), 8.94 (d, J = 2.3 Hz, 1 H), 8.74 (dd, J = 3.9 and 4.4 Hz, 2 H), 8.67
(dd, J = 2.5 and 8.9 Hz, 1 H), 8.08 (dd, J = 1.7 and 6.6 Hz, 1 H), 7.60 (dd,
J = 5.2 and 7.4 Hz, 1 H), 4.97 (t, J = 6.6, 2 H), 3.26ꢀ3.21 (m, 2 H), 2.77
(s, 3 H), 2.75 (s, 3 H), 2.25 (m, 2 H); positive ion ESIMS m/z (rel
intensity) 379 (MH+, 100). Anal. Calcd for C20H18N4O4 HCl 1.5
C21H16N4O2 0.6H2O: C, 68.69; H, 4.72; N, 15.26. Found: C, 68.98; H,
3
4.62; N, 14.82.
7-Aza-5,6-dihydro-6-(3-(1H-imidazol-1-yl)propyl)-2,3-di-
methoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (29).
The general procedure provided the desired compound as a purple solid
(69 mg, 17%): mp 238ꢀ240 °C. IR (KBr) 1699, 1649, 1609, 1570, 1552,
1516 cmꢀ1;1H NMR(CDCl3) δ8.38 (dd, J= 5.1, 1.5 Hz, 1 H), 8.0 (s, 1 H),
7.72 (dd, J=7.5,1.8Hz,1H),7.67(s,1H),7.56(s,1H),7.19(dd,J= 7.5, 5.4
Hz, 1 H), 7.07 (s, 1 H), 6.99 (s, 1 H), 4.96 (t, J = 7.5 Hz, 2 H), 4.15 (t, J = 7.2
Hz, 2 H), 4.04 (s, 3 H), 3.99 (s, 3 H), 2.33 (quint, J = 7.2 Hz, 2 H); positive
ESIMS m/z (rel intensity) 349 (100), 417 (MH+, 61). Anal. Calcd for
3
3
H2O: C, 54.36; H, 5.02; N, 12.68. Found: C, 54.13; H, 4.69; N, 12.52.
7-Aza-5,6-dihydro-6-(3-morpholinopropyl)-5,11-dioxo-
11H-indeno[1,2-c]isoquinoline Hydrochloride (21). The gen-
eral procedure provided the desired compound as an orange solid
(255 mg, 62%): mp 316ꢀ318 °C. IR (KBr) 1704, 1669, 1612, 1570,
1550, 1506, 779 cmꢀ1 1H NMR (DMSO-d6) δ 10.40 (s, 1 H),
;
C23H20N4O4 0.5H2O: C, 64.93; H, 4.98; N, 13.17. Found: C, 65.13; H, 4.88;
N, 12.70.
7-Aza-5,6-dihydro-6-(3-(2-hydroxyethylamino)propyl)-5,
11-dioxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (30).
The general procedure provided the desired compound. The product
was redissolved in chloroform (5 mL), and HCl solution in methanol
8.61 (d, J = 4.5 Hz, 1 H), 8.54 (d, J = 7.8 Hz, 1 H), 7.92 (dd, J = 7.2, 1.5
Hz, 1 H), 7.86 (t, J = 7.2 Hz, 1 H), 7.56 (t, J = 7.2 Hz, 1 H), 7.45 (dd,
J = 7.5 and 6.6 Hz, 1 H), 4.91 (m, 2 H), 3.90 (m, 2 H), 3.69 (m, 2 H),
3.37 (m, 2 H), 3.05 (m, 4 H), 2.24 (m, 2 H). Anal. Calcd for
C22H21N3O3 HCl 1.5H2O: C, 60.20; H, 5.74; N, 9.57. Found: C,
3
3
3
60.37; H, 5.83; N, 9.62.
6113
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116

Journal of Medicinal Chemistry
ARTICLE
(3 M, 1 mL) was added. The resulting solution was evaporated to
dryness to obtain an orange solid (58 mg, 16%): mp 258ꢀ262 °C (dec).
IR (KBr) 1711, 1665, 1611, 1569, 1549, 1503 cmꢀ1; 1H NMR (CDCl3)
δ 8.73 (s, 2 H), 8.64 (dd, J = 5.1, 1.5 Hz, 1 H), 8.51 (d, J = 7.8 Hz, 1 H),
8.24 (d, J = 8.1 Hz, 1 H), 7.91 (dd, J = 7.5, 1.5 Hz, 1 H), 7.86 (t, J =
7.5 Hz, 1 H), 7.59 (t, J = 8.1 Hz, 1 H), 7.46 (dd, J = 7.2, 5.1 Hz, 1 H), 4.89
(t, J = 6.6 Hz, 2 H), 3.63 (t, J = 5.1 Hz, 2 H), 3.05ꢀ2.98 (m, 4 H), 2.19
(m, 2 H); positive ESIMS m/z (rel intensity) 350 (MH+ 100), 289 (74).
Anal. Calcd for C20H19N3O3 HCl H2O: C, 59.22; H, 5.52; N, 10.36.
(quint, J = 7.0 Hz, 2 H); positive ESIMS m/z (rel intensity) 349 (100),
366 (MH+, 72). Anal. Calcd for C20H19N3O4 HCl 0.6H2O: C, 58.22;
3
3
H, 4.96; N, 9.95. Found: C, 58.21; H, 5.18; N, 10.18.
Topoisomerase I Mediated DNA Cleavage Reactions. Hu-
man recombinant Top1 was purified from baculovirus as previously
described.40 DNA cleavage reactions were prepared as previously
reported with the exception of the DNA substrate.25 Briefly, a 117-bp
DNA oligonucleotide (Integrated DNA Technologies) encompassing
the previously identified Top1 cleavage sites in the 161-bp fragment
from pBluescript SK(ꢀ) phagemid DNA was employed. This 117-bp
oligonucleotide contains a single 50-cytosine overhang, which was 30-
end-labeled by fill-in reaction with [R-32P]dGTP in React 2 buffer
(50 mM Tris-HCl, pH 8.0, 100 mM MgCl2, 50 mM NaCl) with 0.5 unit
of DNA polymerase I (Klenow fragment, New England BioLabs).
Unincorporated [32P]dGTP was removed using mini Quick Spin
DNA columns (Roche, Indianapolis, IN), and the eluate containing
the 30-end-labeled DNA substrate was collected. Approximately 2 nM
radiolabeled DNA substrate was incubated with recombinant Top1 in
20 μL of reaction buffer [10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM
MgCl2, 0.1 mM EDTA, and 15 μg/mL BSA] at 25 °C for 20 min in the
presence of various concentrations of compounds. The reactions were
terminated by adding SDS (0.5% final concentration) followed by the
addition of two volumes of loading dye (80% formamide, 10 mM sodium
hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1%
bromphenol blue). Aliquots of each reaction mixture were subjected
to 20% denaturing PAGE. Gels were dried and visualized by using a
phosphoimager and ImageQuant software (Molecular Dynamics). For
simplicity, cleavage sites were numbered as previously described in the
161-bp fragment.40
DNA Unwinding Reactions. These assays were performed in two
steps as described.31 First, SV40 DNA was reacted with excess Top1 for
15 min at 37 °C in the absence of drug to fully relax the DNA. Second,
the relaxed DNA was further incubated for 30 min at 37 °C in the
presence of increasing (1, 3, 10, 30, 100 μM) concentrations of drugs.
Determination of Drug Water Solubility. Water solubility for
18, 19, 33, or 34 was determined by HPLC according to a previously
published protocol.32 Solid samples of 18, 19, 33, or 34 (4ꢀ10 mg) were
weighed and added to 1 M Tris buffer solution (250 μL, pH 7.5). The
suspensions were shaken for 24 h at 25 °C and then centrifuged, and
the supernatants were filtered. Aliquots (10 μL) of the supernatants
were injected into the HPLC system equipped with a C18 reverse-phase
column (5 μm, 100 Å, 15 cm ꢁ 4.6 cm, ES Industries, West Berlin, NJ),
eluting with methanol (1% trifluoroacetic acid)/water [90:10 (v:v)].
One point calibration was done by injecting 10 μL aliquots of the
corresponding buffer solutions of 18, 19, 33, or 34 with known
concentrations.
3
3
Found: C, 58.98; H, 5.02; N, 10.10.
7-Aza-5,6-dihydro-6-(3-(2-hydroxyethylamino)propyl)-2,
3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline
Hydrochloride (31). The general procedure provided the desired
compound. The product was redissolved in chloroform (5 mL), and
HCl solution in methanol (3 M, 1 mL) was added. The resulting
solution was evaporated to dryness to obtain a purple solid (83 mg,
20%): mp 280ꢀ282 °C (dec). IR (KBr) 1703, 1639, 1609, 1594, 1570,
1553, 1516 cmꢀ1; 1H NMR (DMSO-d6) δ 8.72 (s, 2 H) 8.57 (d, J =
5.1 Hz, 1 H), 7.83 (s, 1 H), 7.81 (d, J = 7.5 Hz, 1 H), 7.49 (s, 1 H), 7.56
(s, 1 H), 7.39 (dd, J = 7.2, 5.4 Hz, 1 H), 5.25 (s, 1 H), 4.82 (t, J = 6.3 Hz,
2 H),, 3.90 (s, 3 H), 3.86 (s, 3 H), 3.63 (s, 2 H), 2.98 (s, 4 H), 2.16 (m,
2 H); positive ESIMS m/z (rel intensity) 410 (MH+, 100). Anal. Calcd
for C22H23N3O5 HCl H2O: C, 56.96; H, 5.65; N, 9.06. Found: C,
3
3
56.65; H, 5.24; N, 9.08.
7-Aza-5,6-dihydro-6-(3-aminopropyl)-2,3-dimethoxy-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline Hydrochloride (32).
Sodium hydride (95%, 50 mg, 2 mmol) was added to a mixture of 7-
aza-5,6-dihydro-2,3-dimethoxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline
(16, 308 mg, 1 mmol) and DMF (6 mL) at ꢀ5 °C, and the resulting
mixture was allowed to warm to room temperature over 3 h. The
resulting clear dark red solution was cooled to ꢀ5 °C, and 1,3-
dibromopropane (788 mg, 4 mmol) was added. The solution was stirred
at room temperature for 12 h. The reaction mixture was quenched with
water (50 mL). The products were extracted with chloroform (3 ꢁ
10 mL). The combined extracts were washed with water (3 ꢁ 10 mL),
brine (10 mL), dried with sodium sulfate, and concentrated under
reduced pressure. The residue was subjected to flash column chroma-
tography (silica gel, CHCl3) to provide a mixture of 25 and 27 that was
further used without additional purification or separation. Sodium azide
(650 mg, 10 mmol) was added to a solution of the mixture of 25 and 27
in DMF (5 mL). The resulting mixture was stirred at room temperature
for 12 h. Water (50 mL) was added to the mixture, and the products were
extracted with chloroform (3 ꢁ 10 mL). The chloroform solution was
washed with water (3 ꢁ 10 mL), brine (10 mL), dried with sodium
sulfate, and evaporated to dryness. The residue was redissolved in
benzene (10 mL), and triethyl phosphite (332 mg, 2 mmol) was added.
The resulting solution was heated to reflux for 6 h. Diluted hydrochloric
acid (1 mL) was added to the solution, and the mixture was heated for
3 h and cooled down to room temperature. Potassium hydroxide
solution (2%) was added to the mixture to pH 10ꢀ12. The organic
layer was separated, and the aqueous solution was extracted with ethyl
acetate (3 ꢁ 5 mL). The combined organic layers were washed with
water (3 ꢁ 10 mL), concentrated potassium carbonate solution (10 mL),
dried with sodium sulfate, and evaporated to dryness. The residue was
subjected to flash column chromatography (silica gel), eluting with
chloroform, to get unreacted 25, and then with 15% methanol in
chloroform to obtain a solid poduct. The product was redissolved in
chloroform (5 mL), and HCl solution in methanol (1 mL) was added.
The resulting solution was evaporated to dryness to obtain 32 as a purple
solid (69 mg, 18%): mp 298ꢀ300 °C (dec). IR (KBr) 1703, 1658, 1617,
1570, 1555, 1516 cmꢀ1; 1H NMR (DMSO-d6) δ 8.60 (dd, J = 5.4, 1.5
Hz, 1 H), 7.95 (s, 1 H), 7.87 (dd, J = 7.5, 1.5 Hz, 1 H), 7.75 (s, 1 H), 7.59
(s, 1 H), 7.41 (dd, J = 7.5, 5.4 Hz, 1 H), 4.89 (t, J = 6.3 Hz, 2 H),
3.94 (s, 3 H), 3.89 (s, 3 H), 2.88 (t, J = 7.2 Hz, 2 H), 2.42 (s, 3 H), 2.08
Quantum Mechanics Calculations. All calculations were per-
formed in Gaussian 09. The structures of model indenoisoquinoline and
the AꢀT and GꢀC base pairs from models A and B were optimized at
the HF/6-31G** level of theory. The single point energy calculation was
performed at the MP2/6-31G* and HF/6-31G* levels of theory. The
NBO analysis was performed at HF/6-31G** level of theory.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 765-494-1465. Fax: 765-494-6790. E-mail: cushman@
purdue.edu.
’ ACKNOWLEDGMENT
This work was made possible by the National Institutes of
Health (NIH) through support of this work with Research Grant
UO1 CA89566, as well as through support by the Intramural
6114
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116

Journal of Medicinal Chemistry
ARTICLE
Program of the National Cancer Institute, Center for Cancer
Research.
Length Necessary for Optimal Indenoisoquinoline Topoisomerase I
Inhibition and Cytotoxicity in Human Cancer Cell Cultures. J. Med.
Chem. 2007, 50, 2040–2048.
(17) Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.; Kohlhagen,
G.; Agama, K.; Hollingshead, M.; Pommier, Y.; Cushman, M.
Synthesis and Evaluation of Indenoisoquinoline Topoisomerase I Inhibi-
tors Substituted with Nitrogen Heterocycles. J. Med. Chem. 2006, 49,
6283–6289.
(18) Nagarajan, M.; Xiao, X. S.; 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, 5712–5724.
(19) Morrell, A.; Placzek, M.; Parmley, S.; Antony, S.; Dexheimer,
T. S.; Pommier, Y.; Cushman, M. Nitrated Indenoisoquinolines as
Topoisomerase I Inhibitors: A Systematic Study and Optimization.
J. Med. Chem. 2007, 50, 4419–4430.
(20) Morrell, A.; Placzek, M.; Parmley, S.; Grella, B.; Antony, S.;
Pommier, Y.; Cushman, M. Optimization of the Indenone Ring of
Indenoisoquinoline Topoisomerase I Inhibitors. J. Med. Chem. 2007,
50, 4388–4404.
(21) Dillow, G. W.; Kebarle, P. Electron-Affinities of Aza-Substituted
Polycyclic Aromatic-Hydrocarbons. Can. J. Chem. 1989, 67, 1628–1631.
(22) Jagtap, P. G.; Baloglu, E.; Southan, G.; Williams, W.; Roy, A.;
Nivorozhkin, A.; Landrau, N.; Desisto, K.; Salzman, A. L.; Szabo, C.
Facile and Convenient Syntheses of 6,11-Dihydro-5H-indeno[1,2-c]-
isoquinolin-5-ones and 6,11-Dihydro-5H-indolo[3,2-c]isoquinolin-5-
one. Org. Lett. 2005, 7, 1753–1756.
’ ABBREVIATIONS USED
AIBN, azobisisobutyronitrile; DIAD, diisopropyl azodicarboxy-
late; DMF, dimethylformamide; DMSO-d6, dimethyl-d6 sulfox-
ide; NBS, N-bromosuccinimide; THF, tetrahydrofuran; Top1,
topoisomerase type I; Top1-DNAcc, topoisomerase type IꢀDNA
cleavage complex
’ REFERENCES
(1) Pommier, Y. Topoisomerase I Inhibitors: Camptothecins and
Beyond. Nat. Rev. Cancer 2006, 6, 789–802.
(2) Pommier, Y.; Pourquier, P.; Fan, Y.; Strumberg, D. Mechanism
of Action of Eukaryotic DNA Topoisomerase I and Drugs Targeted to
the Enzyme. Biochim. Biophys. Acta, Gene Struct. Expression 1998,
1400, 83–106.
(3) Koster, D. A.; Croquette, V.; Dekker, C.; Shuman, S.; Dekker,
N. H. Friction and Torque Govern the Relaxation of DNA Supercoils by
Eukaryotic Topoisomerase Ib. Nature 2005, 434, 671–674.
(4) Redinbo, M. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.; Hol,
W. G. J. Crystal Structures of Human Topoisomerase I in Covalent and
Noncovalent Complexes with DNA. Science 1998, 279, 1504–1513.
(5) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; Mcphail,
A. T.; Sim, G. A. Plant Antitumor Agents. I. Isolation and Structure of
Camptothecin a Novel Alkaloidal Leukemia and Tumor Inhibitor from
Camptotheca Acuminata. J. Am. Chem. Soc. 1966, 88, 3888–3890.
(6) Kohn, K. W.; Pommier, Y. Molecular and Biological Determi-
nants of the Cytotoxic Actions of Camptothecins: Perspective for the
Development of New Topoisomerase I Inhibitors of Article. Ann. N.Y.
Acad. Sci. 2000, 922, 11–26.
(23) Potts, K. T.; Robinson, R. Synthetical Experiments Related to
the Indole Alkaloids. J. Chem. Soc. 1955, 2675–2686.
(24) Whitmore, W. F.; Cooney, R. C. The Preparation of Homo-
phthalyl Cyclic Hydrazide and 4-Aminohomophthalyl Cyclic Hydrazide.
J. Am. Chem. Soc. 1944, 66, 1237–1240.
(25) Dexheimer, T. S.; Pommier, Y. DNA Cleavage Assay for the
Identification of Topoisomerase I Inhibitors. Nat. Protoc. 2008, 3, 1736–1750.
(26) Boyd, M. R.; Pauli, K. D. Some Practical Considerations and
Applications of the National-Cancer-Institute in-Vitro Anticancer Drug
Discovery Screen. Drug Dev. Res. 1995, 34, 91–109.
(7) Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.;
Zembower, D.; Stewart, L.; Burgin, A. B. Structures of Three Classes
of Anticancer Agents Bound to the Human Topoisomerase IꢀDNA
Covalent Complex. J. Med. Chem. 2005, 48, 2336–2345.
(27) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; Mcmahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New
Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. J. Natl.
Cancer Inst. 1990, 82, 1107–1112.
(28) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New
Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. J. Natl.
Cancer Inst. 1990, 82, 1107–1112.
(29) Boyd, M. R.; Paull, K. D. Some Practical Considerations and
Applications of the National Cancer Institute in Vitro Anticancer Drug
Discovery Screen. Drug Dev. Res. 1995, 34, 91–109.
(30) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman,
M. Synthesis of Benz[d]indeno[1,2-b]pyran-5,11-diones: Versatile In-
termediates for the Design and Synthesis of Topoisomerase I Inhibitors.
Bioorg. Med. Chem. Lett. 2006, 16, 1846–1849.
(31) Pommier, Y.; Covey, J. M.; Kerrigan, D.; Markovits, J.; Pham, R.
DNA Unwinding and Inhibition of Mouse Leukemia-L1210 DNA Topoi-
somerase-I by Intercalators. Nucleic Acids Res. 1987, 15, 6713–6731.
(32) Vogel, G. H. Determination of Solubility by Hyphenated
HPLC Methods. In Drug Discovery and Evaluation: Safety and Pharma-
cokinetiks Assay; Springer: New York, 2006; pp 400ꢀ402.
(33) Cheng, K. J.; Rahier, N. J.; Eisenhauer, B. M.; Gao, R.; Thomas,
S. J.; Hecht, S. M. 14-Azacamptothecin: A Potent Water-Soluble
Topoisomerase I Poison. J. Am. Chem. Soc. 2005, 127, 838–839.
(34) Rahier, N. J.; Cheng, K. J.; Gao, R.; Eisenhauer, B. M.; Hecht,
S. M. Synthesis of 14-Azacamptothecin, a Water-Soluble Topoisomerase
I Poison. Org. Lett. 2005, 7, 835–837.
(8) Cushman, M.; Cheng, L. Stereoselective Oxidation by Thionyl
Chloride Leading to Indeno[1,2-c]isoquinoline System. J. Org. Chem.
1978, 43, 3781–3783.
(9) Cushman, M.; Cheng, L. Total Synthesis of Nitidine Chloride.
J. Org. Chem. 1978, 43, 286–288.
(10) Kohlhagen, G.; Paull, K. D.; Cushman, M.; Nagafuji, P.;
Pommier, Y. Protein-Linked DNA Strand Breaks Induced by NSC
314622, a Novel Noncamptothecin Topoisomerase I Poison. Mol.
Pharmacol. 1998, 54, 50–58.
(11) Cushman, M.; Jayaraman, M.; Vroman, J. A.; Fukunaga, A. K.;
Fox, B. M.; Kohlhagen, G.; Strumberg, D.; Pommier, Y. Synthesis of
New Indeno[1,2-c]isoquinolines: Cytotoxic Non-Camptothecin Topoi-
somerase I Inhibitors. J. Med. Chem. 2000, 43, 3688–3698.
(12) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman,
M. A Systematic Study of Nitrated Indenoisoquinolines Reveals a Potent
Topoisomerase I Inhibitor. J. Med. Chem. 2006, 49, 7740–7753.
(13) Antony, S.; Agama, K. K.; Miao, Z. H.; Takagi, K.; Wright,
M. H.; Robles, A. I.; Varticovski, L.; Nagarajan, M.; Morrell, A.;
Cushman, M.; Pommier, Y. Novel Indenoisoquinolines NSC 725776
and NSC 724998 Produce Persistent TopoIsomerase I Cleavage
Complexes and Overcome Multidrug Resistance. Cancer Res. 2007,
67, 10397–10405.
(14) Pommier, Y.; Cushman, M. The Indenoisoquinoline Non-
camptothecin Topoisomerase I Inhibitors: Update and Perspectives.
Mol. Cancer Ther. 2009, 8, 1008–1014.
(15) Clinical Study: 10-C-0056, a Phase I Study of Indenoisoquino-
lines LMP400 and LMP776 in Adults with Relapsed Solid Tumors and
Lymphomas. http://clinicalstudies.info.nih.gov/cgi/detail.cgi?A_2010-
C-0056.html (accessed July 2011).
(35) Song, Y. L.; Cushman, M. The Binding Orientation of a
Norindenoisoquinoline in the Topoisomerase IꢀDNA Cleavage Com-
plex Is Primarily Governed by PiꢀPi Stacking Interactions. J. Phys.
Chem. B 2008, 112, 9484–9489.
(16) Morrell, A.; Placzek, M. S.; Steffen, J. D.; Antony, S.; Agama, K.;
Pommier, Y.; Cushman, M. Investigation of the Lactam Side Chain
6115
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116

Journal of Medicinal Chemistry
ARTICLE
(36) Xiao, X. S.; Antony, S.; Pommier, Y.; Cushman, M. On the
Binding of Indeno[1,2-c]isoquinolines in the DNAꢀTopoisomerase I
Cleavage Complex. J. Med. Chem. 2005, 48, 3231–3238.
(37) Xiao, X. S.; Cushman, M. An Ab Initio Quantum Mechanics
Calculation That Correlates with Ligand Orientation and DNA Cleavage
Site Selectivity in CamptothecinꢀDNAꢀTopoisomerase I Ternary
Cleavage Complexes. J. Am. Chem. Soc. 2005, 127, 9960–9961.
(38) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.;
Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.;
Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.;
Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.;
Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.;
Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich,
S.; Daniels, A. D.; Farkas; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox,
D. J. Gaussian 09, revision A.02; Gaussian, Inc.: Wallingford, CT, 2009.
(39) Glendening, E. D.; Reed, A. E.; Carpenter, J. E.; Weinhold, F.
NBO, version 3.1.
(40) Pourquier, P.; Ueng, L. M.; Fertala, J.; Wang, D.; Park, H. J.;
Essigmann, J. M.; Bjornsti, M. A.; Pommier, Y. Induction of Reversible
Complexes between Eukaryotic DNA Topoisomerase I and DNA-
Containing Oxidative Base Damages—7,8-Dihydro-8-oxoguanine and
5-Hydroxycytosine. J. Biol. Chem. 1999, 274, 8516–8523.
6116
dx.doi.org/10.1021/jm200719v |J. Med. Chem. 2011, 54, 6106–6116