Synthesis and evaluation of indenoisoquinoline topoisomerase I inhibitors substituted with nitrogen heterocycles

Article abstract of DOI:10.1021/jm060564z

In connection with an ongoing investigation of indenoisoquinoline topoisomerase I (Top1) inhibitors as potential therapeutic agents, the pharmacophore possessing di(methoxy) and methylenedioxy substituents was held constant, and new derivatives were synthesized with nitrogen heterocycles appended to the lactam side chain. Compounds were evaluated for Top1 inhibition and for cytotoxicity in the National Cancer Institute's human cancer cell screen. Some of the more potent derivatives were also screened for in vivo activity in a hollow fiber assay. The results of these studies indicate that lactam substituents possessing nitrogen heterocycles can provide highly cytotoxic compounds with potent Topi inhibition. Molecular modeling of these compounds in complex with DNA and Top1 suggests that some of the lactam substituents are capable of interacting with the DNA base pairs above and below the site of intercalation and/or with Topi amino acid residues, resulting in increased biological activity.

Full text of DOI:10.1021/jm060564z

J. Med. Chem. 2006, 49, 6283-6289
6283
Synthesis and Evaluation of Indenoisoquinoline Topoisomerase I Inhibitors Substituted with
Nitrogen Heterocycles
Muthukaman Nagarajan,† Andrew Morrell,† Alexandra Ioanoviciu,† Smitha Antony,‡ Glenda Kohlhagen,‡ Keli Agama,‡
Melinda Hollingshead,§ Yves Pommier,‡ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, and the Purdue Cancer Center, School of Pharmacy and Pharmaceutical
Sciences, Purdue UniVersity, West Lafayette, Indiana 47907, Laboratory of Molecular Pharmacology, Center for Cancer Research, National
Cancer Institute, Bethesda, Maryland 20892-4255, and Biological Testing Branch, DeVelopmental Therapeutics Program, DiVision of Cancer
Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, FairView Center, Suite 205, 1003 West SeVenth Street,
Frederick, Maryland 21701
ReceiVed May 11, 2006
In connection with an ongoing investigation of indenoisoquinoline topoisomerase I (Top1) inhibitors as
potential therapeutic agents, the pharmacophore possessing di(methoxy) and methylenedioxy substituents
was held constant, and new derivatives were synthesized with nitrogen heterocycles appended to the lactam
side chain. Compounds were evaluated for Top1 inhibition and for cytotoxicity in the National Cancer
Institute’s human cancer cell screen. Some of the more potent derivatives were also screened for in vivo
activity in a hollow fiber assay. The results of these studies indicate that lactam substituents possessing
nitrogen heterocycles can provide highly cytotoxic compounds with potent Top1 inhibition. Molecular
modeling of these compounds in complex with DNA and Top1 suggests that some of the lactam substituents
are capable of interacting with the DNA base pairs above and below the site of intercalation and/or with
Top1 amino acid residues, resulting in increased biological activity.
Introduction
In the 1990s, the National Cancer Institute (NCI) developed
a COMPARE algorithm to facilitate the prediction of biological
targets from data generated by the 60-cell human cancer screen.1
Utilizing the COMPARE algorithm, it was predicted that
indenoisoquinoline 1 (NSC 314622, Figure 1) would be a
topoisomerase I (Top1) inhibitor.2 Subsequent in vitro testing
confirmed this prediction, and more potent derivatives (such as
2) have been developed.2-9
Irinotecan (3) and topotecan (4) are the only current Top1
inhibitors approved by the Food and Drug Administration (FDA)
for the treatment of cancer, and they validate Top1 as a
therapeutic target for anticancer drug development. However,
these camptothecin derivatives are not ideal drug molecules.
Camptothecins are compromised by the reversibility of the
Top1-DNA cleavage complex, which necessitates long infusion
times for maximum activity,10 and they are inherently unstable
and suffer from lactone ring opening to form a hydroxy acid
that has a high affinity for human serum albumin.11 As a result
of the pharmacokinetic problems of the camptothecins, there is
Figure 1. Representative indenoisoquinoline and camptothecin Top1
inhibitors.
great interest in the development of noncamptothecin Top1
that different Top1 inhibitors will display different spectra of
anticancer activity as well.13
inhibitors as anticancer agents. Top1 inhibitors such as the
indenoisoquinolines are not limited by the same pharmacokinetic
problems that limit the camptothecins. Some of the more potent
indenoisoquinolines stabilize the Top1-DNA cleavage complex
to a greater extent than camptothecin, they are more stable
chemically, and the in vitro biological activities of certain
derivatives are comparable.1,12 Furthermore, it is known that
clinically useful topoisomerase II (Top2) inhibitors have
preferential activity for different cancers, and it can be expected
The present investigation was undertaken to explore novel
functionalities appended to the lactam side chain of the
indenoisoquinolines, specifically focusing on incorporation of
heterocyclic ring systems. Previous work with an indenoiso-
quinoline system lacking substituents on the aromatic rings
demonstrated that potent biological activity (cytotoxicity and
Top1 inhibition) could be achieved.5 However, the research
demonstrated a small and consistent benefit to having the di-
(methoxy) and methylenedioxy substituents appended to the
isoquinoline and indenone rings, and this has led to their
incorporation in the present investigation.5 Previous work has
also led to the realization that amino, morpholine, and imidazole
motifs appended to the lactam side chain further enhanced
* To whom correspondence should be addressed. Tel: 765-494-1465.
Fax: 765-494-6790. E-mail: cushman@pharmacy.purdue.edu.
† Purdue University.
‡ NIH, Bethesda, MD.
§ Division of Cancer Treatment and Diagnosis, National Institutes of
Health.
10.1021/jm060564z CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/26/2006

6284 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Nagarajan et al.
Scheme 1a
biological activity with respect to compounds lacking these
nitrogenous substituents.5,7,9 To explain this enhancement,
hypotheses such as increased aqueous solubility, DNA targeting,
transporter hijacking, and hydrogen bonding of the side chain
amines with amino acid residues in the ternary cleavage complex
have been proposed and may indeed be involved for some
molecules.5,7
Several recent developments have occurred that allow further
speculation and necessitate a reinvestigation of indenoisoquino-
lines bearing heterocyclic lactam side chains. The crystal
structures of complexes have been reported with small molecule
inhibitors such as topotecan,14 camptothecin,15 an indolocarba-
zole,15 an indenoisoquinoline,15 and a norindenoisoquinoline3,16
intercalated within duplex DNA bound to human Top1. Using
these structures, models of various indenoisoquinolines in
ternary complex with Top1 and cleaved duplex DNA have been
generated and suggest incorporating ammonium cations and
heterocycles on the lactam side chain. If the models are credible,
then appropriate functionalities may be capable of increasing
Top1 inhibition, and ultimately cellular cytotoxicity, by interact-
ing with amino acid residues and/or the DNA base pairs at the
site of intercalation. Thus, the present investigation represents
a new effort toward the development of indenoisoquinoline Top1
inhibitors where the di(methoxy) and methylenedioxy substit-
uents on the aromatic nucleus are combined with previously
utilized and novel heterocyclic lactam side chains in an effort
to improve the anticancer activity of the indenoisoquinolines.
Chemistry
Indenoisoquinolines 6-18 were prepared according to the
methods summarized in Scheme 1. Utilizing compound 517 as
a key advanced intermediate, nucleophilic displacement of the
alkyl bromide functionality with a variety of primary and
secondary amines readily provided the corresponding analogues
possessing heterocyclic lactam substituents. In general, the
conditions utilizing potassium carbonate in 1,4-dioxane to effect
displacement were readily amenable to most of the analogues
synthesized (compounds 7 and 8 being the only exceptions).
However, treatment of the heterocyclic precursors to 7 and 8
with sodium hydride in dimethylformamide readily effected the
desired transformation. Ultimately, compounds 6, 8-10, and
13-16 were treated with a solution of hydrochloric acid in
diethyl ether to provide the corresponding hydrochloride salts.
This was found to both aid purification and provide an improved
solubility profile for the biological assays.
a Reagents and conditions: (a) amine, K2CO3, 1,4-dioxane, 100 °C; (b)
amine, NaH, DMF, 60 °C; (c) 2 M HCl in ether, CHCl3.
similar activity as the parent compound 1; +++ and ++++,
greater activity than the parent compound 1; and ++++, similar
or greater activity than 1 µM camptothecin.
An increase in the biological activity of indenoisoquinolines
resulting from an aminoalkyl chain attached to the lactam
nitrogen has been identified previously.7,17 As the next logical
step, it seemed prudent to investigate the effects of replacing
the amino group with various nitrogen heterocycles (Scheme
1, 6-18) by evaluating the cytotoxicity and Top1 inhibition of
the newly synthesized analogues (Table 1). With multiple
heteroatoms present in these newly synthesized analogues, along
with a flexible three-carbon linker, the development of an
adequate hypothetical structural model that rationalized all of
the biological activity results proved difficult. Depending on
the nature of the heterocyclic side chain, molecular modeling
indicated it could be capable of interacting with the DNA base
pairs and Top1 amino acid residues in several different
orientations within the ternary complex. This variability made
the rationalization of biological activities resulting from minor
changes in the structure of the heterocycle increasingly difficult.
However, the results listed in Table 1 indicate that heterocycles
possessing a heteroatom capable of serving as a hydrogen-bond
acceptor at physiological pH generally provide the best results
regarding Top1 inhibition and cytotoxicity (compounds 6, 8, 9,
11, and 13). Attempts to rationalize this trend using molecular
modeling were generally unsuccessful due to the abundance of
both hydrogen-bond donors and acceptors surrounding the drug
intercalation site and lactam side chain in the ternary complex.
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 in approximately 55 different cancer cell lines
of diverse tumor origins. The GI50 values obtained in 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 molar) are taken as the minimum (10-8 molar) and
maximum (10-4 molar) drug concentrations used in the screen-
ing test. For comparison purposes, the activities of the previously
reported lead compound 1,2 its more potent derivative 2,17 and
previously reported imidazole-appended analogues 19,5 20,9 and
219 are also included in the table. The relative potencies of the
compounds toward the production of topoisomerase I-mediated
DNA cleavage are also listed in the table. The potencies are
expressed semiquantitatively as follows: +, weak activity; ++,

Indenoisoquinoline Topoisomerase I Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6285
Table 1. Cytotoxicities and Topoisomerase I Inhibitory Activities of Indenoisoquinoline Analogues
cytotoxicity (GI50 in µM)a
lung
HOP-62
colon
HCT-116
CNS
SF-539
melanoma
UACC-62
ovarian
OVCAR-3
renal
SN12C
prostate
DU-145
breast
MDA-MB-435
Top 1
compd
MGMb
20.0
0.16 ( 0.02
0.079 ( 0.023
2.16 ( 0.24
3.55
0.112 ( 0.066
0.382 ( 0.119
4.64 ( 1.25
50.1
0.243 ( 0.088
12.0
0.766 ( 0.254
0.715 ( 0.335
7.86 ( 0.27
1.27 ( 0.84
1.86
cleavagec
1
2
1.30
0.06
35.0
0.13
41.0
0.26
4.20
0.25
<0.010
0.269
<0.010
0.014
1.20
73.0
0.31
0.085
56.2
61.7
68.0
0.31
37.0
0.04
96.0
1.21
0.020
7.08
++
+++
++++
++
6
<0.010
0.447
0.079
<0.010
<0.005
1.78
<0.010
0.037
0.398
0.288
<0.010
<0.005
0.040
18.2
<0.005
17.8
0.100
0.145
1.44
0.550
2.34
<0.010
0.316
0.085
<0.010
0.091
0.813
NT
<0.010
0.363
0.085
<0.010
0.015
0.155
>100
<0.005
10.7
0.182
0.081
0.275
0.603
1.41
7
1.99
8
1.91
>100
<0.010
4.57
+++
++++
++
9
<0.010
0.575
1.15
0.041
2.04
10
11
12
13
14
15
16
17
18
19
20
21
0.030
37.2
74.1
67.6
++++
++
26.3
72.4
34.7
>100
0.977
>100
1.74
<0.005
<0.005
5.01
5.75
0.126
11.5
+++
+
18.2
1.48
15.1
15.1
0.427
<0.005
9.77
0.120
0.214
2.34
1.29
0.832
5.01
0.257
0.145
>100
1.48
+
0.457
1.23
2.63
+++
++
15.1
0.525
1.66
< 0.010
0.864
>100
<0.005
2.69
< 0.010
0.19
<0.005
1.41
< 0.010
0.274
0.162
0.79
< 0.010
0.012
1.95
+++
++++
+++
++++
1.66
2.75
< 0.010
0.016
< 0.010
< 0.010
0.017
0.014
2.17
0.059 ( 0.026
0.370 ( 0.28
0.015
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. Compounds with standard deviations listed for the MGM values were tested twice, while those without standard deviations
for the MGM values were tested once. c The compounds were tested at concentrations ranging up to 10 µM. The activity of the compounds to produce
Top1-mediated DNA cleavage was expressed semiquantitatively as follows: +, weak activity; ++, similar activity as the parent compound 1; +++ and
++++, greater activity than the parent compound 1; and ++++, similar activity as 1 µM camptothecin.
in the minor groove with Arg364 and on the scissile side of the
DNA with Asn722, have been previously reported for inde-
noisoquinoline analogues.3,15,16 However, the model indicates
a potential hydrogen bond between the 3-position nitrogen of
the lactam heterocycle in compound 6 and Lys436. This
hydrogen bond provides a rationale for the low Top1 inhibition
of 7, which does not have an appropriately placed heteroatom
Figure 2. Previously synthesized heterocyclic indenoisoquinoline
derivatives.
in the heterocycle for hydrogen bonding. The model also
explains the Top1 activity of 8, which has the 4-position nitrogen
of the 1,2,4-tetrazole motif oriented in a similar position to
accept a hydrogen bond from Lys436. The slight decrease in
the Top1 inhibitory activity of 8 (Top1 +++) compared to 6
(Top1 ++++) could be due to a decrease in the basicity of
the 4-position nitrogen of the 1,2,4-triazole motif and a
subsequent decrease in participation as a hydrogen-bond ac-
ceptor with Lys436. An additional 1,3-nitrogen-substituted
analogue 9 (MGM 0.112 µM, Top1 ++++) with unshared
electron density between the 1,3-related nitrogen atoms (exo-
cyclic and endocyclic) also provided a potent analogue. These
results led to the realization that a 1,3 relationship between
hydrogen-bond donors/acceptors appears to be optimal for
biological activity in five-membered heterocyclic ring systems.
Six-membered heterocyclic rings were also investigated
(Table 1, compounds 10-15, 17, and 18), with potent biological
activity still observed for some of the compounds. For these
ring systems, however, a 1,4-relationship between heteroatoms
capable of donating and accepting hydrogen bonds was observed
to be necessary for optimal biological activity. Compound 10
(MGM 0.382 µM, Top1 ++), with a pyrazine ring serving as
its appended heterocycle, showed potent cytotoxicity, but poor
Top1 inhibition. However, replacing the 4-position nitrogen
atom in the heterocycle with an oxygen atom (thereby providing
morpholine derivative 11) reversed the biological activity profile
to provide a potent Top1 inhibitor (++++), but a poorly
cytotoxic molecule (MGM 4.64 µM). Incorporation of a sulfur
atom, which forms weaker hydrogen bonds than oxygen or
nitrogen, in place of the aforementioned heteroatoms in the
4-position, provided compound 12. This compound was a poor
Top1 inhibitor (++) and a weakly cytotoxic molecule (MGM
50.1 µM). Thus, compound 12 emphasizes that a 4-position
heteroatom capable of strong hydrogen bonding is important
for biological activity, but there is an imperfect correlation
With regard to compounds 6-9, each of which possess a
five-membered heterocycle appended to the lactam side chain,
the biological results suggest that the types of ring systems
involved still confer significant activity. Compound 6, with its
imidazole motif, was the most potent compound synthesized in
the present investigation with an MGM of 0.079 µM and Top1
inhibition of ++++, making it comparable to 1 µM camp-
tothecin in the assay. This outcome could be expected with the
activities of compounds 19, 20, and 21 serving as a precedent
(Table 1). These previously reported compounds are potent Top1
inhibitors that display unusually potent cytotoxicity, with the
exception of unsubstituted compound 19.5,9 As mentioned above,
there is a small, yet consistent benefit to possessing dimethoxy-
and methylenedioxy-substituted isoquinoline and indenone
rings.5 Gratifyingly, incorporating this substitution pattern in
the present study produced an analogue 6 that retained the potent
Top1 inhibition seen with compound 19, but improved the
cytotoxicity, making it similar to compounds 20 and 21 (Figure
2).
Altering the 1,3 nitrogen relationship of imidazole in 6 to
the 1,2 relationship present in compound 7 resulted in a
significant loss of biological activity. Inclusion of an additional
nitrogen heteroatom (as for 8) resulted in a decrease in
cytotoxicity, but still maintained significant Top1 inhibition. A
hypothetical model of 6 in ternary complex highlights the
importance of the 1,3 nitrogen atom relationship regarding Top1
inhibition that is illustrated by compounds 6, 7, and 8 (Figure
3). The hypothetical model illustrated in Figure 3 was developed
utilizing the previously reported crystal structure of an inde-
noisoquinoline in complex with DNA and Top1.3,15,16 It
postulates that compound 6 has the potential to make three
hydrogen-bonding contacts with Top1. The first two contacts,

6286 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Nagarajan et al.
Figure 3. Stereoview of compound 6 (red) in ternary complex with DNA and Top1. The lactam side chain is pointing into the major groove, and
potential hydrogen bonds are indicated in yellow. The diagram is programmed for wall-eyed (relaxed) viewing.
between cytotoxicity and Top1 inhibition in this series of
compounds. Compounds 13 and 14 further demonstrate the
importance of the 4-position and further support a specific
hydrogen-bonding role for atoms in the 4-position. Compound
13 (MGM 0.243 µM, Top1 +++), with an exocyclic alcohol,
demonstrated potent cytotoxicity and good Top1 inhibition.
Thus, by comparing its activity with 10 and 11, it can be inferred
that the 4-position heteroatom needs to be capable of serving
as a hydrogen-bond donor to attain potent cytotoxicity (the ether
motif present in 11 can only serve as a hydrogen-bond acceptor).
However, Top1 inhibition was enhanced by having a 4-position
heteroatom capable of serving as a hydrogen-bond acceptor (10
is doubly protonated at physiological pH and can only serve as
a hydrogen-bond donor). The exocyclic alcohol in 13 can act
as both a hydrogen-bond acceptor and a hydrogen-bond donor,
and compound 13 performed well in both assays, while
compound 12 was inactive in both assays. Extending the length
of the lactam side chain had no significant effect on the role of
the 4-position of the heterocycle. Compound 18 (MGM 1.27
µM, Top1 +++), with its pendant morpholine ring, would be
expected to act better as a Top1 inhibitor than as a cytotoxic
agent, according to the rationale mentioned above, and it indeed
supported the general trend.
Alkylation of the 4-position of the heterocycle was found to
be detrimental to biological activity, with compounds 14 and
17 being relatively inactive in the assays when compared to
several of the other molecules. The cytotoxicities of these
molecules were rather interesting because they would have been
expected to be more active in light of the 4-position being a
hydrogen-bond donor at physiological pH.
The relationship between the 4-position of the heterocycle
and its effect on biological activity was further investigated with
analogues 15 and 16. Expanding the size of the heterocycle by
a single carbon atom to afford compound 16 (MGM 0.715 µM,
Top1 +++) provided a moderately active Top1 inhibitor and
cytotoxic molecule. Comparing the biological activities of
analogues 16 and 10, whose lactam side chains are a homopy-
razine ring and a pyrazine ring, respectively, a 2-fold loss in
cytotoxicity was observed for the ring-expanded analogue.
However, the loss in cytotoxicity was accompanied by a slight
increase in Top1 inhibition. Compound 15 (MGM 0.766 µM,
Top1 +) was a moderately cytotoxic analogue and a poor Top1
inhibitor. According to the structure-activity relationships for
the studied analogues, this result was consistent because the
exocyclic primary amine present in 15 would be expected to
be protonated at physiological pH and, thereby, serve only as a
hydrogen-bond donor.
results being compared to 1 µM camptothecin. Differences in
cleavage sites and band intensities between camptothecin and
the indenoisoquinolines in Figure 4 are noteworthy. For
example, the camptothecin band at site 37 was not prominent
in the indenoisoquinoline assays (the exception being 8).
Conversely, the cleavage site at 44 was intense for most of the
indenoisoquinolines. As a result of different cleavage site
specificities, it may be possible to target different cancer cell
genes selectively using a camptothecin or an indenoisoquinoline.
Figure 4 also indicates that several cleavage sites are conserved
between camptothecin and the indenoisoquinolines. Cleavage
sites 70, 92, 97, and 119 were prominent for camptothecin and
most of the indenoisoquinolines, especially compounds 11, 19,
and 21. It is also worth highlighting the fact that several cleavage
bands were more prominent at intermediate drug concentrations
as opposed to high drug concentrations, especially for com-
pounds 8 and 9. This could be due to inhibition of Top1 cleavage
activity at higher drug concentrations due to a direct effect on
the enzyme, or alternatively, to DNA intercalation, which could
result in DNA unwinding to render it a poorer substrate for
Top1.
Several of the active analogues (10, 11, 13, 16, and 17) were
evaluated as anticancer agents in an in vivo animal model in
which polyvinylidene fluoride hollow fibers containing various
cancer cell cultures were implanted intraperitoneally (IP) and
subcutaneously (SC) into mice, and the compounds were
administered by the IP route. The effects of the compounds on
the reduction of viable cancer cell mass compared to those of
controls were determined. Each compound was tested in the
hollow fiber assay against a panel of twelve human tumor cell
lines as described previously.18 The compounds were solubilized
in 10% DMSO in saline/Tween-80R and administered IP once
daily for a total of four doses at each of two dose levels. The
two doses were selected based on single dose toxicity studies
for each derivative. A score of 2 was assigned each time the
compound produced a 50% or greater reduction in viable cell
mass compared to vehicle-treated controls. The score for each
compound was summed for the intraperitoneal fibers and the
subcutaneous fibers to provide the total score for each derivative
(Table 2). For comparative purposes, the performances of
compounds 1 and 2 are included in Table 2. Three of the five
newly tested analogues (indenoisoquinolines 2, 10, and 16) were
more potent than lead compound 1. All of the newly tested
compounds were more active at the IP implant site than at the
SC implant site. This could imply problems with absorption,
distribution, or metabolism of these compounds. None of the
newly synthesized compounds were more potent than 2, but
compound 6 is currently undergoing hollow fiber testing and
xenograft studies at the National Cancer Institute.
All of the newly synthesized compounds were examined for
induction of DNA cleavage in the 3′-end-labeled PvuII/HindIII
fragment of pBluescript SK(-) phagemid DNA in the presence
of Top1.2 The resulting cleavage patterns of some of the more
potent indenoisoquinolines are displayed in Figure 4, with the
In conclusion, several potent indenoisoquinolines have been
synthesized with lactam side chains containing nitrogen het-
erocycles. Several structural motifs have been found to increase

Indenoisoquinoline Topoisomerase I Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6287
Figure 4. Comparison of the Top1-mediated DNA cleavages at different drug concentrations. The DNA used corresponds to the 3′-end-labeled
PvuII/HindIII fragment of pBluescript SK (-) phagemid DNA. Top1 was present in all reaction mixtures except Lane 1. Lane 1, DNA; Lane 2,
DNA + Top1; Lane 3, 1 µM camptothecin; Lanes 4-7, inhibitor concentrations of 0.1, 1.0, 10, and 100 µM, respectively. The figure is comprised
of five gels that are placed side by side to facilitate comparison.
Table 2. Hollow Fiber Activities of Indenoisoquinoline Analogues
with short wavelength UV light. Silica gel flash chromatography
was performed using 230-400 mesh silica gel.
compd
IP scorea
SC scorea
total score
cell killb
General Procedure for the Synthesis of Indenoisoquinolines
6 and 9-18. A mixture of indenoisoquinoline 517 (0.500 g, 1.06
mmol), amine (2.11 mmol), and anhydrous K2CO3 (0.584 g, 4.23
mmol) in anhydrous 1,4-dioxane (30 mL) was heated at 100 °C
for 4 h. The reaction mixture was cooled and then concentrated.
The residue was diluted with water (50 mL), extracted with CHCl3
(2 × 50 mL), washed with 1% aq HCl (50 mL), water (50 mL),
and satd NaCl (50 mL), and dried over Na2SO4. The crude product
was purified by flash column chromatography (SiO2), eluting with
a 0-5% gradient of methanol in chloroform, to provide the pure
indenoisoquinoline.
1
2
10
11
13
16
17
2
16
16
4
6
4
2
2
0
2
0
8
20
18
6
Y
N
N
N
N
N
N
6
6
12
8
14
8
a The IP and SC scores listed are the sums of all the IP and SC scores
for each compound. b A net cell kill at one or more implant sites is indicated
with a Y.
the potency of Top1 inhibition with imidazole and morpholine
substituents the most active in this aspect of biological evalu-
ation. Furthermore, a hypothetical model has been constructed
that rationalizes the Top1 inhibitory activity of the imidazole,
pyrazole, and 1,2,4-tetrazole substituted analogues. It was
observed that heteroatom placement within the lactam side chain
has a significant impact on both Top1 inhibition and cytotox-
icity, with 3- and 4-position heteroatoms providing optimal
activity for 5- and six-membered heterocyclic ring systems,
respectively. Furthermore, the ability to participate as a hydrogen-
bonding donor or acceptor at the 4-position was found to
differentiate molecules that were either potent Top1 inhibitors
or highly cytotoxic agents.
3-(Imidazolyl-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-meth-
ylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Hydro-
chloride (6). The desired analogue was obtained as a dark purple
1
solid (245 mg, 63%): mp 316-318 °C. H NMR (CDCl3) δ 8.01
(s, 1 H), 7.63 (s, 1 H), 7.60 (s, 1 H), 7.14 (s, 1 H), 7.04 (s, 2 H),
6.40 (s, 1 H), 6.07 (s, 2 H), 4.45 (t, J ) 5.8 Hz, 2 H), 4.20 (t, J )
6.6 Hz, 2 H), 4.03 (s, 3 H), 3.98 (s, 3 H), 2.33 (t, J ) 6.9 Hz, 2 H);
ESI MS m/z (rel intensity) 460 (MH+, 100). Anal. (C25H21N3O6‚
0.2 H2O) C, H, N. The hydrochloride salt was formed by dissolving
the product in chloroform (50 mL), and an anhydrous solution of
2 M HCl in diethyl ether (15 mL, 30.0 mmol) was added at 0 °C.
The reaction mixture was stirred at room temperature for 6 h, and
the precipitated product was filtered and washed with chloroform
(50 mL) and methanol (20 mL), and dried over P2O5 for 24 h to
afford the product as a dark purple solid (170 mg, 79%): mp 270-
Experimental Section
1
272 °C. H NMR (DMSO-d6-CD3OD, 2:1) δ 9.07 (s, 1 H), 7.78
(s, 2 H), 7.60 (s, 1 H), 7.42 (s, 1 H), 7.14 (s, 1 H), 6.96 (s, 1 H),
6.13 (s, 2 H), 4.41 (t, J ) 6.6 Hz, 2 H), 4.36 (t, J ) 7.3 Hz, 2 H),
3.86 (s, 3 H), 3.82 (s, 3 H), 2.35 (t, J ) 6.1 Hz, 2 H); ESI MS m/z
(rel intensity) 494 (MH+, 100). Anal. (C25H22N3O6Cl) C, H, N.
Melting points were determined in capillary tubes and are
uncorrected. Infrared spectra were obtained using CHCl3 as the
solvent (unless otherwise specified) and were recorded using a
1
Perkin-Elmer 1600 series FTIR. Except where noted, H NMR
spectra were obtained using CDCl3 as solvent and the residual
solvent peak as an internal standard. 1H NMR spectra were recorded
on an ARX300 300 MHz Bruker NMR spectrometer. Electrospray
mass spectra were determined using a FinniganMATT LCQ
(Thermoquest Corp., San Jose, CA) instrument at the Purdue
Campus-Wide Mass Spectrometry Center. Microanalyses were
performed at the Purdue University Microanalysis Laboratory.
Analytical thin-layer chromatography was carried out on Analtech
silica gel GF 1000-micron glass plates. Compounds were visualized
6-[3-Pyrazolyl-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-me-
thylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (7). In-
denoisoquinoline 517 (0.2113 g, 0.448 mmol) was added to sodium
hydride (86.8 mg of a 60% suspension in mineral oil, 2.17 mmol)
and pyrazole (0.1749 g, 2.57 mmol) in DMF (50 mL), and the
reaction mixture was heated at 60 °C for 4 h. The reaction mixture
was diluted with water (200 mL) and extracted with chloroform
(200 mL). The organic layer was washed with water (7 × 200 mL)
and concentrated. Benzene was added (2 × 30 mL), and the mixture

6288 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Nagarajan et al.
was concentrated again. The residue was dissolved in chloroform
(4 mL), and diethyl ether (50 mL) was added. The precipitate was
washed with diethyl ether (100 mL), and a dark red solid (118.5
mg, 57.6%) was obtained: mp 262-264 °C (dec). IR (film) 3462,
3104, 2918, 1693, 1640, 1557, 1495, 1488, 1430, 1394, 1308, 1284,
2.09 (br s, 2 H); ESI MS m/z (rel intensity) 495 (MH+, 100). Anal.
(C26H26N2O6S‚0‚3 H2O) C, H, N.
6-[3-(3-Hydroxypiperidinyl)-1-propyl]-5,6-dihydro-2,3-
dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]i-
soquinoline Hydrochloride (13). The product (220 mg, 0.45 mmol,
70%) was treated with 2 M HCl in diethyl ether (4.0 mL, 6.69
mmol) in chloroform at room temperature to afford the desired
1
1251, 1205, 868, 785, 769 cm-1; H NMR (DMSO-d6) δ 7.97 (s,
1 H), 7.65 (d, J ) 1.5 Hz, 1 H), 7.61 (s, 1 H), 6.57 (s, 1 H), 6.97
(s, 1 H), 6.68 (s, 1 H), 6.33 (s, 1 H), 6.05 (s, 2 H), 4.40 (m, 4 H),
4.01 (s, 3 H), 3.96 (s, 3 H), 2.45 (m, 2 H); ESI MS m/z (rel intensity)
460 (MH+, 100). Anal. (C25H21N3O6‚0.75 H2O) C, H, N.
1
analogue as a purple solid (210 mg, 89%): mp 288-290 °C. H
NMR (D2O) δ 6.54 (br s, 1 H), 6.41 (s, 1 H), 6.29 (br s, 1 H), 6.06
(s, 1 H), 5.88 (s, 2 H), 3.82 (br s, 2 H), 3.45 (s, 3 H), 3.37 (br s,
7 H), 3.15 (br s, 3 H), 1.99 (br s, 4 H), 1.68 (br s, 2 H); ESI MS
m/z (rel intensity) 493 (MH+, 100). Anal. (C27H29ClN2O7‚1.4 H2O)
C, H, N.
6-{3-[2-(1,2,4)]-Triazolyl-1-propyl}-5,6-dihydro-2,3-dimethoxy-
8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Hy-
drochloride (8). Indenoisoquinoline 517 (0.2538 g, 0.538 mmol)
was added to sodium hydride (124.8 mg of a 60% suspension in
mineral oil, 3.12 mmol) and 1,2,4-triazole (0.2673 g, 0.566 mol)
in DMF (50 mL), and the reaction mixture was heated at 60 °C for
3 h. The reaction mixture was diluted with water (200 mL), and
the precipitate was separated by filtration and washed with water
(50 mL). The precipitate was partially dissolved in methanol-
chloroform 1:1 (200 mL). Diethyl ether (100 mL) was added, and
the precipitate was separated by filtration and washed with
additional diethyl ether (100 mL) to provide the product as the free
base. The residue was dissolved in trifluoroacetic acid (2 mL), and
hydrochloric acid (4 mL of a 2 M solution in diethyl ether) was
added, followed by more diethyl ether (30 mL). The product was
collected as a dark red solid (159.5 mg, 57%): mp > 240 °C. IR
(KBr) 3429, 1694, 1647, 1553, 1500, 1487, 1431, 1394, 1311, 1254,
1207, 1032, 928, 873, 800, 786, 722, 617 cm-1; 1H NMR (DMSO-
d6) δ 8.56 (s, 1 H), 7.99 (s, 1 H), 7.90 (s, 1 H), 7.52 (s, 1 H), 7.15
(s, 1 H), 7.10 (s, 1 H), 6.19 (s, 2 H), 4.44-4.38 (m, 4 H), 3.90 (s,
3 H), 3.86 (s, 3 H), 2.25 (m, 2 H); ESI MS m/z (rel intensity) 461
(MH+, 53), 392 (MH+ - C2N3H3, 100). High-resolution ESI MS
m/z (rel intensity) 461.1464 (100, MH+; calculated mass 461.1461).
6-(3-Thiazolylamino-1-propyl)-5,6-dihydro-2,3-dimethoxy-
8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Di-
hydrochloride (9). The product (213 mg, 41%) was dissolved in
chloroform (50 mL) and treated with an anhydrous solution of 2
M HCl in diethyl ether (15 mL, 30.0 mmol) at 0 °C. The reaction
mixture was stirred at room temperature for 6 h, and the precipitated
product was filtered, washed with chloroform (50 mL) and methanol
(10 mL), and dried over P2O5 to provide the desired analogue as a
pale purple solid (140 mg, 61%): mp 298-300 °C (dec). 1H NMR
(DMSO-d6) δ 7.82 (s, 1 H), 7.44 (s, 1 H), 7.38 (s, 1 H), 7.04 (s, 1
H), 6.18 (s, 2 H), 4.42 (br s, 2 H), 4.07 (br s, 2 H), 3.88 (s, 3 H),
3.83 (s, 3 H), 3.76 (br s, 4 H), 2.07 (br s, 2 H); ESI MS m/z (rel
intensity) 494 (MH+, 100). Anal. (C25H25N3O6SCl2‚0.6 CHCl3) C,
H, N.
3-[(1-Methylpiperazinyl)-1-propyl]-5,6-dihydro-2,3-dimethoxy-
8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (14).
The desired analogue was isolated as a dark purple solid (160 mg,
1
51%): mp 254-256 °C. H NMR (CDCl3) δ 7.99 (s, 1 H), 7.60
(s, 1 H), 7.30 (s, 1 H), 7.03 (s, 1 H), 6.08 (s, 2 H), 4.47 (t, J ) 6.0
Hz, 2 H), 4.02 (s, 3 H), 3.96 (s, 3 H), 2.55 (br s, 10 H), 2.30 (s, 3
H), 1.99 (br s, 2 H); ESI MS m/z (rel intensity) 492 (MH+, 100).
Anal. (C27H29N3O6‚0.5 CHCl3) C, H, N.
6-[3-(4-Aminopiperidinyl)-1-propyl]-5,6-dihydro-2,3-dimethoxy-
8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Di-
hydrochloride (15). The product (205 mg, 66%) was dissolved in
chloroform (30 mL) and treated with 2 M HCl in diethyl ether
(5.2 mL, 10.40 mmol) at room temperature for 8 h. The precipitate
was filtered and washed with chloroform (30 mL) to provide the
desired analogue as a dark purple solid (165 mg, 85%): mp 262-
1
264 °C (dec). H NMR (D2O) δ 6.62 (s, 1 H), 6.50 (s, 1 H), 6.44
(s, 1 H), 6.17 (s, 1 H), 5.92 (s, 2 H), 3.92 (br s, 2 H), 3.64 (br s,
2 H), 3.50 (s, 4 H), 3.45 (s, 3 H), 3.23 (br s, 2 H), 3.08 (br s, 2 H),
2.25 (m, 2 H), 2.06 (br s, 2 H), 1.90 (m, 2 H); ESI MS m/z (rel
intensity) 492 (MH+, 70).
6-(3-Homopiperazinyl-1-propyl)-5,6-dihydro-2,3-dimethoxy-
8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Di-
hydrochloride (16). The obtained product (390 mg, 0.66 mmol,
69%) was dissolved in chloroform and treated with 2 M HCl in
diethyl ether (10.0 mL, 19.8 mmol) to afford the desired analogue
as a purple solid (305 mg, 82%): mp 264-266 °C (dec). 1H NMR
(D2O) δ 6.71 (br s, 1 H), 6.56 (br s, 2 H), 6.21 (br s, 1 H), 5.92 (s,
2 H), 3.98 (br s, 2 H), 3.63-3.57 (br s, 6 H), 3.55 (s, 3 H), 3.50 (s,
3 H), 3.36-3.25 (br s, 4 H), 2.19 (br s, 2 H), 2.09 (br s, 2 H); ESI
MS m/z (rel intensity) 492 (MH+, 100). Anal. (C27H31Cl2N3O6‚0.7
H2O) C, H, N.
3-[(1-Hydroxyethyl-piperazine)-1-propyl]-5,6-dihydro-2,3-
dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]i-
soquinoline (17). The desired analogue was isolated as a dark
1
6-(3-Piperazinyl-1-propyl)-5,6-dihydro-2,3-dimethoxy-8,9-me-
thylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline Dihydro-
chloride (10). The product (350 mg, 72%) was dissolved in
chloroform and treated with 2 M HCl in diethyl ether (9.0 mL,
18.2 mmol) at room temperature to afford the desired analogue as
brown solid (258 mg, 47%): mp 262-264 °C. H NMR (CDCl3)
δ 8.00 (s, 1 H), 7.60 (s, 1 H), 7.32 (s, 1 H), 7.04 (s, 1 H), 6.06 (s,
2 H), 4.52 (br s, 2 H), 4.03 (s, 3 H), 3.96 (s, 3 H), 3.22 (br s, 4 H),
3.13 (br s, 6 H), 2.84 (br s, 2 H), 2.68 (br s, 2 H), 1.73 (br s, 4 H),
1.63 (br s, 4 H), 1.43 (s, 18 H), 1.41 (s, 9 H); ESI MS m/z (rel
intensity) 522 (MH+, 100). Anal. (C28H31N3O7‚0.8 H2O) C, H, N.
6-[(3-Morpholylethylamino)-1-propyl]-5,6-dihydro-2,3-
dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]i-
soquinoline (18). The desired analogue was isolated as a pale purple
1
a pale purple solid (280 mg, 84%): mp 276-278 °C (dec). H
NMR (D2O) δ 6.63 (br s, 1 H), 6.53 (s, 1 H), 6.47 (br s, 1 H), 6.18
(s, 1 H), 5.91 (s, 2 H), 3.90 (br s, 2 H), 3.51 (s, 3 H), 3.46 (br s,
11 H), 3.20 (br s, 2 H), 2.02 (br s, 2 H); ESI MS m/z (rel intensity)
478 (MH+, 100). Anal. (C26H29Cl2N3O6‚2.3 H2O) C, H, N.
3-[(Morpholinyl)-1-propyl]-5,6-dihydro-2,3-dimethoxy-8,9-
methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (11).
The product was isolated as a dark purple solid (0.220 g, 72%):
mp 290-292 °C. 1H NMR (CDCl3) δ 7.98 (s, 1 H), 7.59 (s, 1 H),
7.36 (s, 1 H), 7.02 (s, 1 H), 6.07 (s, 2 H), 4.48 (t, J ) 7.39 Hz, 2
H), 4.02 (s, 3 H), 3.95 (s, 3 H), 3.76 (br s, 4 H), 2.54 (br s, 6 H),
2.01 (br s, 2 H); ESI MS m/z (rel intensity) 479 (MH+, 100). Anal.
(C26H26N2O7‚0.2 H2O) C, H, N.
1
solid (245 mg, 59%): mp 215-217 °C. H NMR (CDCl3) δ 8.01
(s, 1 H), 7.62 (s, 1 H), 7.42 (s, 1 H), 7.05 (s, 1 H), 6.06 (s, 2 H),
4.52 (br s, 2 H), 4.03 (s, 3 H), 3.97 (s, 3 H), 3.70 (br s, 4 H), 2.81
(br s, 2 H), 2.73 (br s, 2 H), 2.53 (br s, 2 H), 2.46 (br s, 4 H), 2.02
(br s, 2 H); ESI MS m/z (rel intensity) 522 (MH+, 100). Anal.
(C28H31N3O7‚1.0 H2O) C, H, N.
Top1-Mediated DNA Cleavage Reactions. Human recombinant
Top1 was purified from Baculovirus, as described previously.19 The
161 bp fragment from pBluescript SK(-) phagemid DNA (Strat-
agene, La Jolla, CA) was cleaved with the restriction endonuclease
Pvu II and Hind III (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 1X TBE
buffer. The 161 bp fragment was eluted from the gel slice using
the QIAEX II kit (QIAGEN Inc., Valencia, CA). Approximately
3-[(Thiomorpholinyl)-1-propyl]-5,6-dihydro-2,3-dimethoxy-
8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (12).
The product was isolated as a dark purple solid (275 mg, 53%):
mp 306-308 °C. 1H NMR (CDCl3) δ 7.80 (s, 1 H), 7.60 (s, 1 H),
7.33 (s, 1 H), 7.04 (s, 1 H), 6.08 (s, 2 H), 4.48 (t, J ) 6.4 Hz, 2 H),
4.02 (s, 3 H), 3.96 (s, 3 H), 2.84-2.78 (br s, 8 H), 2.67 (br s, 2 H),

Indenoisoquinoline Topoisomerase I Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6289
(5) Nagarajan, M.; Morrell, A.; Fort, B. C.; Meckley, M. R.; Antony,
S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Synthesis and
Anticancer Activity of Simplified Indenoisoquinoline Topoisomerase
I Inhibitors Lacking Substituents on the Aromatic Rings. J. Med.
Chem. 2004, 47, 5651-5661.
(6) 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,
5147-5160.
(7) 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-
5724.
(8) Fox, B. M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.;
Staker, B.; 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.
200 ng of the fragment was 3′-end labeled at the Hind III site by
fill-in reaction with [alpha-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 units of DNA polymerase I (Klenow
fragment). Unincorporated 32P-dGTP was removed using mini
Quick Spin DNA columns (Roche, Indianapolis, IN), and the eluate
containing the 3′-end-labeled 161 bp fragment was collected.
Aliquots (approximately 50 000 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 concentra-
tion). 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).
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 an indenoisoquinoline, was
utilized as follows.15 All of the atoms were fixed according to Sybyl
atom types, and hydrogens were added and minimized using the
MMFF94s force field and MMFF94 charges. The structure of the
indenoisoquinoline 6, constructed in Sybyl and energy minimized
with the Tripos force field and MMFF94 charges, was overlapped
with the structure of the crystallized indenoisoquinoline according
to structural similarities of the aromatic rings in the ternary complex,
and the structure of the crystallized indenoisoquinoline 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 inde-
noisoquinoline was allowed to move while the structures of the
protein and nucleic acids 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.
(9) 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.
(10) Pommier, Y. Eukaryotic DNA Topoisomerase I: Genome Gate
Keeper and Its Intruders, Camptothecins. Semin. Oncol. 1996, 23,
1-10.
(11) (a) Jaxel, C.; Kohn, K. W.; Wani, M. C.; Pommier, Y. Structure-
Reactivity Study of the Actions of Camptothecin Derivatives on
Mammalian Topoisomerase I: Evidence for a Specific Receptor Site
and a Relation to Antitumor Activity. Cancer. Res. 1989, 49, 1465-
1469. (b) Minami, H.; Beijnen, J. H.; Verweij, J.; Ratain, M. J.
Limited Sampling Model for the Area under the Concentration Time
Curve of Total Topotecan. Clin. Cancer Res. 1996, 2, 43-46. (c)
Danks, M. K.; Pawlik, C. A.; Whipple, D. O.; Wolverton, J. S.
Intermittant Exposure of Medulloblastoma Cells to Topotecan
Produces Growth Inhibition equivalent to Continuous Exposure. Curr.
Top. Med. Chem. 1997, 3, 1731-1738. (d) Haas, N. B.; LaCreta, F.
P.; Walczak, J.; Hudes, G. R.; Brennan, J. M.; Ozols, R. F.; O’Dwyer,
P. J. PhaseI/Pharmaco-kinetic Study of Topotecan by 24-Hour
Continuous Infusion Weekly. Cancer Res. 1994, 54, 1220-1226.
(12) Antony, S.; Kohlhagen, G.; Agama, K.; Jayaraman, M.; Cao, S.;
Durrani, F.; Rustum, Y.; Cushman, M.; Pommier, Y. Cellular
Topoisomerase I Inhibition and Antiproliferative Activity by MJ-
III-65 (NSC 706744), an Indenoisoquinoline Topoisomerase I Poison.
Mol. Pharmacol. 2005, 67, 523-530.
Acknowledgment. This work was made possible by the
National Institutes of Health (NIH) through support of this work
with Research Grant UO1 CA89566, Training Grant ST32
CA09634-12, and an American Chemical Society Division of
Medicinal Chemistry Predoctoral Fellowship (A.M.). The in
vitro and in vivo testing was conducted through the Develop-
mental Therapeutics Program, DCTD, NCI under Contract NO1-
CO-56000.
(13) Meng, L. H.; Liao, Z. Y.; Pommier, Y. Non-Camptothecin DNA
Topoisomerase I Inhibitors in Cancer Therapy. Curr. Top. Med.
Chem. 2003, 3, 305-320.
(14) Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin, A.
B., Jr.; Stewart, L. The Mechanism of Topoisomerase I Poisoning
by a Camptothecin Analogue. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 15387-15392.
Supporting Information Available: Elemental analyses for
compounds 6, 7, 9-14, and 16-18. This material is available free
of charge via the Internet at http://pubs.acs.org.
(15) 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 I-DNA
Covalent Complex. J. Med. Chem. 2005, 48, 2336-2345.
(16) Marchand, C.; Antony, S.; Kohn, K. W.; Cushman, M.; Ioanoviciu,
A.; Staker, B. L.; Burgin, A. B.; Stewart, L.; Pommier, Y. A Novel
Norindenoisoquinoline Structure Reveals a Common Interfacial
Inhibitor Paradigm for Ternary Trapping of the Topoisomerase
I-DNA Covalent Complex. Mol. Cancer Ther. 2006, 5, 287-295.
(17) 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.
(18) Hollingshead, M.; Plowman, J.; Alley, M.; Mayo, J.; Sausville, E.
The Hollow Fiber Assay. Contrib. Oncol. 1999, 54, 109-120.
(19) Pourquier, P.; Ueng, L.-M.; Fertala, J.; Wang, D.; Park, H.-J.;
Essigman, J.; 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.
References
(1) Paull, K. D.; Hamel, E.; Malspeis, L. Prediction of Biochemical
Mechanism of Action from the In Vitro Antitumor Screen of the
National Cancer Institute. Cancer Chemotherapeutic Agents; Ameri-
can Chemical Society: Washington, DC, 1995; pp 9-45.
(2) Kohlhagen, G.; Paull, K.; Cushman, M.; Nagafuji, P.; Pommier, Y.
Protein-Linked DNA Strand Breaks Induced by NSC 314622, a Novel
Noncamptothecin Topoisomerase I Poison. Mol. Pharmacol. 1998,
54 50-58.
(3) Ioanoviciu, A.; Antony, S.; Pommier, Y.; Staker, B. L.; Stewart, L.;
Cushman, M. Synthesis and Mechanism of Action Studies of a Series
of Norindenoisoquinoline Topoisomerase I Poisons Reveal an Inhibi-
tor with a Flipped Orientation in the Ternary DNA-Enzyme-Inhibitor
Complex As Determined by X-ray Crystallographic Analysis. J. Med.
Chem. 2005, 48, 4803-4814.
(4) 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.
JM060564Z