Design, Synthesis, and Biological Evaluation of Indenoisoquinoline Topoisomerase I Inhibitors Featuring Polyamine Side Chains on the Lactam Nitrogen

Article abstract of DOI:10.1021/jm030313f

The indenoisoquinolines are a class of noncamptothecin topoisomerase I inhibitors that display significant cytotoxicity in human cancer cell cultures. They offer a number of potential advantages over the camptothecins, including greater chemical stability, formation of more persistent cleavage complexes, and induction of a unique pattern of DNA cleavage sites. Molecular modeling has suggested that substituents on the indenoisoquinoline lactam nitrogen would protrude out of the DNA duplex in the ternary cleavage complex through the major groove. This indicates that relatively large substituents in that location would be tolerated without compromising biological activity. As a strategy for increasing the potencies and potential therapeutic usefulness of the indenoisoquinolines, a series of compounds was synthesized containing polyamine side chains on the lactam nitrogen. The rationale for the synthesis of these compounds was that the positively charged ammonium cations would increase DNA affinity through electrostatic binding to the negatively charged DNA backbone, and the polyamines might also facilitate cellular uptake by utilization of polyamine transporters. The key step in the synthesis involved the condensation of Schiff bases, containing protected amine side chains, with substituted homophthalic anhydrides, to afford cis-3-aryl-4-carboxy-1-isoquinolones. These isoquinolones were then converted to indenoisoquinolines with thionyl chloride. Although monoamines were much more potent than the lead compound, no significant increase in potency was observed through incorporation of additional amino groups in the side chain. However, one of the monoamine analogues, which features a bis(2-hydroxyethyl)amino group in the side chain, proved to be one of the most cytotoxic indenoisoquinoline synthesized to date, with a GI50 mean-graph midpoint (MGM) of 0.07 μM in the NIH human cancer cell culture screen, and topoisomerase I inhibitory activity comparable to that of camptothecin.

Full text of DOI:10.1021/jm030313f

5712
J . Med. Chem. 2003, 46, 5712-5724
Design , Syn th esis, a n d Biologica l Eva lu a tion of In d en oisoqu in olin e
Top oisom er a se I In h ibitor s F ea tu r in g P olya m in e Sid e Ch a in s on th e La cta m
Nitr ogen
Muthukaman Nagarajan,† Xiangshu Xiao,† Smitha Antony,‡ Glenda Kohlhagen,‡ Yves Pommier,‡ and
Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences,
Purdue University, West Lafayette, Indiana 47907, and Laboratory of Molecular Pharmacology,
Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892-4255
Received J une 26, 2003
The indenoisoquinolines are a class of noncamptothecin topoisomerase I inhibitors that display
significant cytotoxicity in human cancer cell cultures. They offer a number of potential
advantages over the camptothecins, including greater chemical stability, formation of more
persistent cleavage complexes, and induction of a unique pattern of DNA cleavage sites.
Molecular modeling has suggested that substituents on the indenoisoquinoline lactam nitrogen
would protrude out of the DNA duplex in the ternary cleavage complex through the major
groove. This indicates that relatively large substituents in that location would be tolerated
without compromising biological activity. As a strategy for increasing the potencies and potential
therapeutic usefulness of the indenoisoquinolines, a series of compounds was synthesized
containing polyamine side chains on the lactam nitrogen. The rationale for the synthesis of
these compounds was that the positively charged ammonium cations would increase DNA
affinity through electrostatic binding to the negatively charged DNA backbone, and the
polyamines might also facilitate cellular uptake by utilization of polyamine transporters. The
key step in the synthesis involved the condensation of Schiff bases, containing protected amine
side chains, with substituted homophthalic anhydrides, to afford cis-3-aryl-4-carboxy-1-
isoquinolones. These isoquinolones were then converted to indenoisoquinolines with thionyl
chloride. Although monoamines were much more potent than the lead compound, no significant
increase in potency was observed through incorporation of additional amino groups in the side
chain. However, one of the monoamine analogues, which features a bis(2-hydroxyethyl)amino
group in the side chain, proved to be one of the most cytotoxic indenoisoquinoline synthesized
to date, with a GI50 mean-graph midpoint (MGM) of 0.07 µM in the NIH human cancer cell
culture screen, and topoisomerase I inhibitory activity comparable to that of camptothecin.
The discovery of the topoisomerase I inhibitory activ-
ity of the indenoisoquinoline 1 (NSC 314622) has
naturally generated interest in its potential clinical
utility as an anticancer agent.1 Comparison of the
properties of 1 with those of the camptothecins, which
are the only topoisomerase I inhibitors in clinical use,
has revealed a number of differences that might be
advantageous for the indenoisoquinolines. First, in
contrast to the camptothecins, which are inactivated by
lactone hydrolysis at physiological pH,2 the indenoiso-
quinolines are chemically stable. Second, the enzyme-
DNA cleavage complexes stabilized by indenoisoquin-
oline 1 are more persistent than those induced by
camptothecin.1 Long camptothecin infusion times are
necessary to compensate for the reversibility of the
camptothecin cleavage complexes during chemotherapy
to achieve maximal activity.3 Third, the indenoisoquino-
lines induce a unique pattern of DNA cleavage sites
relative to the camptothecins, indicating that they may
target the human genome differently and therefore
potentially exhibit a different spectrum of anticancer
activity from the camptothecins.1,4,5 However, the po-
tential clinical utility of the lead compound 1 itself is
limited by its moderate activity, both as a cytotoxic
agent in cancer cell cultures and as a topoisomerase I
poison.
In a search for more potent and useful indenoiso-
quinolines based on the lead structure 1, a series of
analogues were synthesized in which the N-methyl
group was replaced by various substituents.4-6 One of
the most cytotoxic topoisomerase I inhibitors proved to
be compound 2, having a 3-aminopropyl substituent on
the indenoisoquinoline nitrogen. Compound 2 was more
cytotoxic than 1 in human cancer cell cultures by a
factor of 125, and it was also more potent as a topo-
* To whom correspondence should be addressed: Tel: 765-494-1465.
Fax: 765-494-6790. E-mail: cushman@pharmacy.purdue.edu.
† Purdue University.
‡ National Cancer Institute.
10.1021/jm030313f CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/21/2003

Indenoisoquinoline Topoisomerase I Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5713
isomerase I inhibitor.5 The reasons for the enhanced
potency of the 3-aminopropyl analogue 2 remain to be
established. One possibility is that the positively charged
ammonium cation could possibly increase the affinity
of 2 for DNA by an electrostatic attraction to the
negatively charged phosphodiester linkages of the DNA.
This would be similar to the situation established for
the polyamines and polyamine derivatives related to
spermine and spermidine.7-10 Another possible reason
is that the 3-aminopropyl analogue 2 could be actively
transported into cells similarly to the polyamines.9-14
However, in general, the affinities of polyamines for the
“polyamine transporter” are in the order tetraamines
> triamines > diamines > monoamines, so monoamines
such as the 3-aminopropyl analogue 2 are the least
likely to utilize the polyamine transport system ef-
ficiently.11,15,16 Since both DNA affinity and transport
of polyamines are facilitated by an increase in the
number of basic nitrogens in the polyamine structure,
the cytotoxicity and topoisomerase I inhibitory activity
of the 3-aminopropyl analogue 2 might be enhanced
further by the incorporation of additional basic nitro-
gens in the side chain.7,11,17 In addition, since polyamines
have a high affinity for DNA but are “motionally free”
so that they can move rapidly along the DNA backbone,
the attachment of a polyamine to an intercalator could
allow the conjugate to slide along the DNA until it finds
an ideal location for formation of the cleavage complex.18
Accordingly, the goal of the present study was to
synthesize indenoisoquinolines with polyamine side
chains on the indenoisoquinoline nitrogen and to test
them for topoisomerase I inhibitory activity as well as
for cytotoxicity in human cancer cell cultures.
Sch em e 1
Sch em e 2
Ch em istr y
sequence of reactions, it was necessary to employ
tertiary amines or protected amines 4 to avoid simple
aminolysis of the anhydride at the expense of conden-
sation with the Schiff base moiety. The FMOC protect-
ing group was chosen since it was stable to the hydro-
chloric acid generated during the treatment of the
isoquinolones with thionyl chloride to produce indeno-
isoquinoline 6. The FMOC-protected amine necessary
to prepare 4c was obtained from 9, which in turn was
made from the BOC-protected amine 8 (Scheme 2).23,24
It was anticipated that side chains containing two,
three, or four basic nitrogens could be attached to the
isoquinolone nitrogen by reaction of appropriately pro-
tected polyamines with the N-(3-bromopropyl) interme-
diate 105 (Scheme 3). This strategy proved to be
successful in the synthesis of the BOC-protected inter-
mediates 11a and 11b (Scheme 3), as well as 18a -d
(Scheme 5) and 22 (Scheme 7). The BOC protecting
groups were then removed under acidic conditions to
afford the hydrochloride salts 12a and 12b (Scheme 3),
19a -d (Scheme 5), and 23 (Scheme 7). In the case of
the bis(hydrochloride) salt 12b, the diamine 13 was
obtained after neutralization with sodium hydroxide
(Scheme 3).
Scheme 1 outlines our general strategy for the syn-
thesis of indenoisoquinolines. A key reaction in this
sequence is the condensation of Schiff bases with
homophthalic anhydrides to afford substituted isoqui-
nolones.19 Thus, the reaction of 4,5-dimethoxyhomo-
phthalic anhydride (3)20 with Schiff bases 4a -c in
chloroform at room temperature afforded the expected
racemic mixtures of the isoquinolones 5a -c in moderate
yields. The 5-6 Hz coupling constant observed for the
two methine protons in the 1H NMR spectra of the
products 5a -c is consistent with the assigned cis
stereochemistry.21 In contrast, these two protons usually
1
appear as broad singlets in the H NMR spectra of the
trans diastereomers in closely related systems.21 The
(methylenedioxy)phenyl ring and the carboxyl group are
both pseudoaxial in the trans diastereomers, and one
pseudoaxial proton and one pseudoequatorial proton
(most likely at C-3) are present in the cis diastereomers.
Treatment of the cis diastereomers 5a -c with thionyl
chloride afforded the corresponding indeno[1,2-c]isoqui-
nolones 6a -c.22 The BOC-protected compound 6d was
prepared by removal of the FMOC group in a 1:1
mixture of chloroform and piperidine to provide the
corresponding primary amine, which was converted in
situ to the BOC-protected intermediate 6d . To increase
aqueous solubility, the formate salt 7b was made by
treatment of 6b with formic acid, while the hydrochlo-
ride salt 7e was obtained by subjection of the BOC-
protected compound 6d to hydrochloric acid. In this
The BOC-protected polyamines 17a -d necessary for
the synthesis of the triamine salts 19a -d were synthe-
sized as outlined in Scheme 4. The two BOC-protected
amino alcohols 14a and 14b were converted to their
mesylates, reacted with ethanolamine or propanola-
mine, and the resulting secondary amines were then

5714 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Nagarajan et al.
Sch em e 3a
Sch em e 5a
a
a
Reagents: (a) K2CO3, 1,4-dioxane, 100 °C, 4 h; (b) 2 M HCl in
Reagents: (a) K2CO3, 1,4-dioxane, 100 °C, 4 h; (b) 2 M HCl in
ether, RT, 8 h; (c) 2 M NaOH, CHCl3, RT.
ether, RT, 8 h.
Sch em e 4a
Sch em e 6a
a
Reagents: (a) Acrylonitrile, CH3OH, RT, 16 h; (b) BOC2O,
Et3N, CHCl3, 0 °C-RT, 12 h; (c) H2, Raney Ni, EtOH-2 M NaOH,
RT, 16 h.
The BOC-protected amine 21 necessary for the prepa-
ration of the tetraamine salt 23 was obtained by
addition of the primary amino group of 17d to acrylo-
nitrile, followed by BOC protection of the resulting
secondary amine (Scheme 6). Catalytic hydrogenation
of the nitrile 20 using Raney nickel then afforded the
primary amine 21. Alternative approaches to the syn-
thesis of BOC-protected polyamines that are struc-
turally related to 17a -d and 21 have also been
reported.25-27
Finally, an assortment of additional analogues 24-
26 were obtained by displacement of bromide from 10
using various amines. Two of the amines were isolated
as their hydrochloride salts 24 and 26, while amine 25
was obtained as the free base. In the case of the reaction
with aminopyrazine, the trifluoroacetate 28 was ob-
tained in low yield instead of the expected trifluoro-
acetate salt 27. Evidently, the aniline-type nitrogen of
aminopyrazine is not nucleophilic enough to displace the
a
Reagents: (a) CH3SO2Cl, Et3N, CHCl3, 0 °C, 4 h; (b) ethanol-
amine or propanolamine (neat), 80 °C, 8 h; (c) BOC2O, Et3N,
CHCl3, 0 °C-RT, 8 h; (d) DEAD, PPh3, phthalimide, dry THF, 0
°C-RT, 5 h; (e) NH2NH2‚nH2O, CH3OH, reflux, 12 h.
BOC-protected to afford intermediates 15a -d . Mitsu-
nobu reaction of the primary alcohols 15a -d with
phthalimide afforded intermediates 16a -d , which were
then converted to the primary amines 17a -d by hy-
drazinolysis.

Indenoisoquinoline Topoisomerase I Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5715
Sch em e 7a
Sch em e 8a
a
Reagents: (a) K2CO3, 1,4-dioxane, 100 °C, 4 h; (b) 2 M HCl in
ether, RT, 8 h.
bromide from 10 even after prolonged heating at 100
°C in 1,4-dioxane, and the trifluoroacetate 28 subse-
quently results from the reaction of 10 with the trifluo-
roacetic acid that was intended to form the salt 27.
Biologica l Resu lts a n d Discu ssion . The indenoiso-
quinolines were examined for antiproliferative activity
against the human cancer cell lines in the National
Cancer Institute screen, in which the activity of each
compound was evaluated with approximately 55 differ-
ent cancer cell lines of diverse tumor origins. The GI50
values obtained with selected cell lines, along with the
mean graph midpoint (MGM) values, are summarized
in Table 1. The MGM is based on a calculation of the
average GI50 for all of the cell lines tested (approxi-
mately 55) in which GI50 values below and above the
test range (10-8 to 10-4 M) are taken as the minimum
(10-8 M) and maximum (10-4 M) drug concentrations
used in the screening test. Therefore, the MGM value
represents an overall assessment of toxicity of the
compound across numerous cell lines. For comparison
purposes, the activities of the previously reported lead
compound 11 and its more potent N-3′-aminoalkyl
derivative 25 are also included in the table. The relative
potencies of the compounds in the production of topo-
isomerase I-mediated DNA cleavage are also listed in
the table.
a
Reagents: (a) (i) K2CO3, 1,4-dioxane, 100 °C, 4 h; (ii) 2 M HCl
in ether, RT, 8 h; (b) K2CO3, 1,4-dioxane, 100 °C, 4 h; (c) (i) K2CO3,
1,4-dioxane, 100 °C, 4 h; (ii) CF3COOH (neat), RT, 30 min.
ase I inhibitors, although 19b and 19d did not display
increased potency, and the tetraamine 23 was inactive.
However, in contrast to prior literature reports correlat-
ing the cytotoxicities of polyamine derivatives to the
number of basic nitrogens in the polyamine chain,11,12,17
the incorporation of additional amino groups in the
polyamine side chain beyond the one amino group in 2
did not result in increased cytotoxicity in the present
series of compounds. It therefore seems likely that these
polyamines are not actively transported by the polyamine
uptake system. Although the present series of polyamine
derivatives is not based on the exact structures of
spermine and spermidine, the selectivity of the polyamine
transport systems is not strictly limited to the naturally
occurring polyamines, as a wide variety of synthetic
polyamines are also transported.12 This would argue
that the failure to observe increased cytotoxicity with
an increase in the number of basic nitrogens in the side
chain in the present series of compounds should not be
strictly attributed to the use of synthetic polyamines
instead of spermine or spermidine. On the other hand,
During the design of the present series of indenoiso-
quinolines, the ring system and its appendages were
held constant except for the substituent on the lactam
nitrogen. This was done to focus on the biological effects
of side chain variation through the incorporation of
amino groups.
It is apparent from the MGM values listed in Table 1
that most of the indenoisoquinolines containing amine
side chains were more cytotoxic than the lead compound
1. They were also generally more potent as topoisomer-
1
at least in the series of N -anthracenylmethyl-poly-
amine conjugates studied by Phanstiel et al., the
architecture of the polyamine vector presented to the

5716 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Nagarajan et al.
Ta ble 1. Cytotoxicities and Topoisomerase I Inhibitory Activities of Indenoisoquinoline Analogues
cytotoxicity (GI50 in µM)a
lung
colon
melanoma
UACC-62
ovarian
renal
prostate
breast
topoisomerase I
cleavagec
compd HOP-62 HCT-116 CNSSF-539
OVCAR-3 SN12C DU-145 MDA-MB-435 MGMb
1
2
6a
7b
7e
12a
13
19a
19b
19c
19d
23
24
25
26
28
29
1.3
35
0.13
0.12
0.66
0.068
0.56
1.3
0.9
1.3
23
43
1.5
0.62
0.18
41
0.26
0.19
0.90
1.3
0.43
1.2
0.55
8.6
4.2
0.25
0.54
1.2
0.14
0.65
3.4
2.6
1.9
37
44
73 68 37 96 20
0.31 0.31 0.04 1.21 0.16
1.4
10
++
0.06
0.018
1.1
0.58
0.38
1.6
+++
++++
+++++
++++
+++
+++
++++
++
0.9
4.4
0.14
1.9
0.65
6.1
0.35
3.1
0.86
0.70
3.1
0.36
0.37
2.3
0.40
0.72
3.1
1.04
1.6
2.5
0.34
0.88
3.2
2.8
2.9
1.7
2.5
1.2
2.7
2.4
8.1
20
0.88
1.6
1.7
4.6
3.4
3.0
38
>100
20
33
1.6
52
>100
1.4
38
68
1.4
41
59
2.0
++++
++
0
+++
++++
++++
++++
++++
3.0
0.14
0.45
<0.01
<0.01
<0.01
1.7
0.36
6.7
<0.01
<0.01
<0.01
0.28
2.86
<0.01
<0.01
<0.01
15.2
6.9
0.18
2.51
0.54
7.2
10.8
1.6
2.1
<0.01
0.018
0.03
0.13
0.07
0.22
<0.01
<0.01
0.02
<0.01
0.08
0.03
0.04
0.07
0.15
0.04
0.66
<0.01
a
The cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition. b Mean graph midpoint for growth inhibition
of all human cancer cell lines successfully tested. c The compounds were tested at concentrations ranging up to 10 µM. The activity of the
compounds to produce topoisomerase I-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; ++++: similar activity as 1
µM camptothecin.
polyamine transporter was crucial for uptake.28,29 There-
fore, the optimal use of the polyamine transporter is not
open to an indiscriminate array of structural motifs, and
this may explain the results observed with the present
series of polyamine conjugates.
and then falling off at higher drug concentration. This
bell-shaped dose-response pattern was observed pre-
viously with the amines 2 and 30.5 Compound 25
was an exception, with the intensities of the cleavage
bands reaching a maximum at a drug concentration of
10 µM. This biphasic pattern of response was not
observed with the indenoisoqinolines 28 and 1, which
do not contain amino groups. These results indicate that
the amines suppress topoisomerase I-mediated DNA
cleavage at high drug concentration, which is similar
to the situation observed with DNA unwinding or inter-
calating inhibitors.30-32 Prior results with the amino
alcohol 30 demonstrated a low affinity DNA intercala-
tion that could be responsible for suppression of DNA
cleavage at higher drug concentration.33 Alternatively,
higher concentrations of the amines may suppress
topoisomerase I-mediated DNA cleavage through a
direct effect on the enzyme resulting in a conformational
change, as has been proposed with saintopin E.31
As mentioned above, the tetraamine 23 was cytotoxic
even though it was inactive as a topoisomerase I
inhibitor. Obviously, the cytotoxicity of this compound
has nothing to do with poisoning of topoisomerase I.
This raises the question of whether additional targets
could be involved in the cytotoxicities of the other
compounds as well. However, the previously published
COMPARE analysis of the cytotoxicity profile of the lead
compound 1 provides convincing evidence that inhibition
of topoisomerase I is likely to be responsible for the
cytotoxicity of this compound and many of the other
indenoisoquinolines as well.1
Within the series of triamines 19, the two compounds
having central ethylenediamine linkages [19a (MGM 2.4
µM) and 19b (MGM 3.0 µM), m ) 1] were significantly
more cytotoxic than the two corresponding propylene-
diamines 19c (MGM 41 µM) and 19d (MGM 59 µM)
(m ) 2). Also, the two compounds having terminal
ethylenediamine moieties (n ) 1, 19a and 19c) were
more potent topoisomerase I inhibitors than the two
having terminal propylenediamine moieties (n ) 2, 19b
and 19d ).
In general, the intensities of the DNA cleavage
bands produced by the amines in this study varied as a
function of drug concentration, reaching a maximum
intensity at 1 µM indenoisoquinoline concentration,

Indenoisoquinoline Topoisomerase I Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5717
force field and MMFF94 charges. During energy mini-
mization, the structures of the protein, nucleic acid, and
the surrounding water molecules were frozen while the
inhibitor was allowed to move. According to this model,
the C-11 ketone is hydrogen bonded directly to the
Arg364 side chain. In addition, the lactam oxygen and
the amino group of the side chain are hydrogen bonded
to a network of water molecules that hydrogen bond
with the protein and the nucleic acid residues. The
stabilization of the side chain through hydrogen bonding
would provide an explanation for the more potent
topoisomerase I inhibitory activity of 2 and related
amines and amino alcohols relative to the lead com-
pound 1. The model indicates that it is possible for the
indenoisoquinoline ring system to intercalate into the
DNA structure of the cleavage complex, with the ring
system occupying the space that would normally be
filled with a base pair. We assume that, like the
camptothecins, the indenoisoquinolines effectively re-
place a base pair in the duplex at the cleavage site in
the ternary cleavage complex, thereby sterically hinder-
ing the religation reaction.34
F igu r e 1. Hypothetical model of the orientation of indenoiso-
quinoline 2 relative to DNA in the ternary complex containing
topoisomerase I, DNA, and the inhibitor 2.
Dimethylation of the terminal amino group of 2
(MGM 0.16 µM) did not have a large effect on cytotox-
icity (see 6a , MGM 0.35 µM). Shortening the side chain
by one methylene unit decreased cytotoxicity (7b, MGM
3.1 µM), but increased topoisomerase I inhibitory activ-
ity. Overall, these results, in conjunction with those
obtained with 19a -d and 23, reveal that there is not
an exact correlation of topoisomerase I inhibition po-
tency and cytotoxicity in the present series of polyamine
derivatives. In this regard, it is interesting to note that
in a related series of tetraamine-(DNA-intercalator)
conjugates, the less potent topoisomerase II inhibitors
proved to be more cytotoxic to L1210 cells than the more
potent topoisomerase II inhibitors.13,14 In this previously
reported case, as well as in the present case with the
tetraamine 23, differences in cellular uptake and the
possibility of additional targets may play a role. Prior
investigations with tetraamine-(DNA-intercalator) con-
jugates have indicated that strong binding of tetra-
amines in the cell membrane may disrupt its structure
and function.29
With respect to the molecular modeling, it is worth
noting that the indenoisoquinoline 32 (MGM 0.34 µM),
which has a C-11 aminoalkenyl substituent on the
opposite side of the molecule, also has enhanced activity
relative to the lead compound 1 (MGM 20 µM).2 We
assume that in the case of 32, the aminoalkyl group
projects outward from the minor groove. Taken together,
these results indicate that the C-11 region and the
lactam region of the indenoisoquinolines will be fruitful
locations for the placement of large substituents, since
they will not clash with the surrounding DNA structure
in the ternary complex.
In addition to being an unexpected product formed
during the attempted synthesis of the aminopyrazine
derivative 27, the trifluoroacetate 28 displayed unex-
pectedly potent cytotoxicity and topoisomerase I inhibi-
tory activity. Its cytotoxicity MGM was 0.15 µM, re-
flecting a potency 133 times that of the lead compound
1. The reason for the high potency of 28 remains to be
established, but it is obvious that the trifluoroacetate
is a good leaving group that provides a potential for
alkylation of nucleophiles in DNA and the enzyme in
the ternary complex. In this regard, it is worth noting
that the cytotoxic potency of 28 is 91 times greater than
the previously reported 3-bromopropyl compound 10
(MGM 13.7 µM), both of which bear a good leaving
group.5 The gain in both cytotoxicity and topoisomerase
I inhibitory activity of compound 28 in comparison to
10 will undoubtedly stimulate additional work on
structurally related topoisomerase I inhibitors.
Compound 26, having two 2-hydroxyethyl substitu-
ents on the side chain nitrogen atom, is the most
cytotoxic compound in the present series (MGM 0.07
µM). Its potency approaches that of camptothecin (MGM
0.04), and it is also as potent as camptothecin as a
topoisomerase I poison. The previously reported com-
pound 30, having a single 2-hydroxyethyl substituent,
was also very cytotoxic (MGM 0.11 µM).5
Assuming the lactam ring of the 3-aminopropyl
analogue 2 is oriented in the ternary cleavage complex
in the same way that it is in the published crystal
structure of the camptothecin derivative topotecan (31),
the aminopropyl side chain of 2 points out from the
major groove of DNA (Figure 1).2,34 This would also
suggest that the polyamine side chains of the indenoiso-
quinolines in the present series are also directed out of
the duplex through the major groove, which would
explain the bulk tolerance for the biological effect of
replacing the N-methyl group in the lead compound 1
with a variety of relatively large polyamine chains. A
hypothetical model (Figure 2) was constructed by over-
lapping the structure of the 3-aminopropyl analogue 2
with the structure of topotecan (31) in the published
crystal structure of the ternary complex.34 During this
operation, the inhibitor was oriented relative to DNA
according to the schematic representation of the struc-
ture displayed in Figure 1. The structure of topotecan
(31) was then removed and the energy of the new
complex was minimized with Sybyl, using the MMFF94s
In summary, a new series of indenoisoquinolines has
been synthesized with aminoalkyl and polyamine side
chains on the lactam nitrogen. These compounds are
more potent as topoisomerase I poisons and as cytotoxic
agents in human cancer cell cultures than the lead
compound 1. The structure-activity relationships and
the molecular models displayed in Figures 1 and 2
provide a possible explanation for the enhanced activity
of the amines, and they also provide a framework for
the design of future indenoisoquinoline topoisomerase
I inhibitors.

5718 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Nagarajan et al.
F igu r e 2. Model of the binding of the indenoisoquinoline 2 in the ternary complex consisting of DNA, topoisomerase I, and the
inhibitor. The diagram is programmed for wall-eyed viewing.
of the imine 4b (2.0 g, 9.1 mmol) and the reaction mixture
was stirred at room temperature for 16 h. The reaction mixture
was concentrated to give an oily residue. The residue was
dissolved in a minimum amount of methanol (20 mL) and ether
(70 mL) was added slowly with stirring to obtain a white
precipitate. The resultant solid was filtered off and washed
with CH3OH-ether (2:1, 30 mL) to afford pure indenoisoqui-
nolone 5b (2.8 g, 70%) as a pale white solid: mp 188-190 °C.
1H NMR (DMSO-d6) δ 7.49 (s, 1 H), 7.23 (s, 1 H), 6.76 (d, J )
8.0 Hz, 1 H), 6.58 (d, J ) 7.8 Hz, 1 H), 6.50 (s, 1 H), 5.93 (s, 2
H), 5.10 (d, J ) 5.7 Hz, 1 H), 4.47 (d, J ) 5.3 Hz, 1 H), 3.94-
3.88 (m, 2 H), 3.81 (s, 3 H), 3.74 (s, 3 H), 2.91-2.88 (m, 1 H),
2.49 (m, 1 H, merged with DMSO-d6 peak), 2.22 (s, 6 H);
ESIMS m/z (relative intensity) 444 (23), 443 (MH+, 100), 398
(38). Anal. (C23H26N2O7‚0.6H2O) C, H, N.
Exp er im en ta l Section
Melting points were determined in capillary tubes and are
uncorrected. Infrared spectra were obtained using CHCl3 as
the solvent unless otherwise specified. Except where noted,
1H NMR spectra were obtained using CDCl3 as solvent and
1
TMS as internal standard. H NMR spectra were determined
at 300 MHz. 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 with
short wavelength UV light. Silica gel flash chromatography
was performed using 230-400 mesh silica gel.
cis-2-[3-(N,N-Dim eth yla m in o)-1-p r op yl]-4-ca r boxy-3,4-
d ih yd r o-6,7-d im et h oxy-3-(3,4-m et h ylen ed ioxyp h en yl)-
1(2H)-isoqu in olon e (5a ). Piperonal (4.00 g, 26.6 mmol) was
added to a stirred solution of commercially available 3-(di-
methylamino)propylamine (3.2 g, 32.0 mmol) and anhydrous
magnesium sulfate (10.0 g) in chloroform (100 mL) and the
mixture was stirred at room temperature for 24 h. The reaction
mixture was filtered through a Celite pad and the residue was
washed with chloroform (40 mL). The combined organic layer
was washed with water (100 mL), brine, and dried (Na2SO4).
The organic solution was concentrated on a rotary evaporator
and the resulting Schiff base was used in the next reaction
without purification. 4,5-Dimethoxyhomophthalic anhydride
3 (4.10 g, 18.4 mmol) was added to a chloroform (100 mL)
solution of the crude imine 4a (4.30 g, 18.4 mmol) and the
reaction mixture was stirred at room temperature for 16 h.
The reaction mixture was concentrated to give an oily residue.
The residue was dissolved in a minimum amount of methanol
(20 mL) and ether (70 mL) was added slowly with stirring to
obtain a white precipitate. The resultant solid was filtered off
and washed with CH3OH-ether (2:1, 30 mL) to yield pure
indenoisoquinolone 5a (4.8 g, 57%) as a pale white solid: mp
230-232 °C. 1H NMR (CDCl3) δ 7.52 (s, 1 H), 6.64 (s, 1 H),
6.63 (bs, 2 H), 6.57 (s, 1 H), 5.86 (s, 1 H), 5.84 (s, 1 H), 5.28 (s,
1 H), 4.30 (t, J ) 9.5 Hz, 1 H), 3.82 (bs, 7 H), 3.61 (s, 1 H),
3.15-3.13 (m, 1 H), 3.04-2.96 (m, 1 H), 2.63 (s, 6 H), 2.11 (bs,
1 H), 1.89 (bs, 1 H); ESIMS m/z (relative intensity) 458 (24),
457 (MH+, 100), 413 (7), 412 (28); HRESIMS calcd for
cis-2-(2-F MOCa m in oeth yl)-4-ca r boxy-3,4-d ih yd r o-6,7-
d im eth oxy-3-(3,4-m eth ylen ed ioxyp h en yl)-1(2H)-isoqu i-
n olon e (5c). Piperonal (1.3 g, 8.5 mmol) was added to a stirred
solution of FMOC-protected aminoethylamine hydrochloride
923,24 (3.0 g, 9.4 mmol), Et3N (2.00 mL, 14.2 mmol) and
anhydrous magnesium sulfate (5.0 g) in chloroform (100 mL)
and the mixture was stirred further at room temperature for
24 h. The reaction mixture was filtered through a Celite pad
and the residue was washed with chloroform (40 mL). The
combined organic layer was washed with water (100 mL),
brine, and dried (Na2SO4). The organic solution was concen-
trated on a rotary evaporator and used in the next reaction
without purification. 4,5-Dimethoxyhomophthalic anhydride
3 (1.6 g, 7.2 mmol) was added slowly in portions to a chloroform
(100 mL) solution of the imine 4c (3.0 g, 7.2 mmol) at 0 °C
and the reaction mixture was further stirred at room temper-
ature for 12 h. The precipitated solid was filtered off through
a sintered glass funnel, washed with chloroform (50 mL), and
dried to give pure indenoisoquinolone 5c (2.6 g, 48%) as a white
1
solid: mp 210-212 °C. H NMR (DMSO-d6) δ 7.86 (d, J ) 7.2
Hz, 2 H), 7.65 (bs, 2 H), 7.50 (s, 1 H), 7.39 (bs, 2 H), 7.30 (t, J
) 7.0 Hz, 2 H), 7.12 (s, 1 H), 6.76 (d, J ) 7.8 Hz, 1 H), 6.54 (d,
J ) 7.6 Hz, 1 H), 6.45 (s, 1 H), 5.94 (s, 2 H), 5.04 (d, J ) 5.7
Hz, 1 H), 4.69 (d, J ) 5.2 Hz, 1 H), 4.25 (bs, 2 H), 4.17 (d, J )
6.0 Hz, 2 H), 3.99 (t, J ) 6.0 Hz, 1 H), 3.78 (s, 3 H), 3.74 (s, 3
H), 3.24 (bs, 2 H); ESIMS m/z 637 (MH+, 24), 434 (11), 399
(12), 398 (45), 369 (22), 368 (100), 355 (10), 354 (42). Anal.
(C36H32N2O9‚1.5H2O) C, H, N.
C
24H28N2O7: 457.1975. Found: 457.1979.
cis-2-[2-(N,N-Dim et h yla m in o)-1-et h yl]-4-ca r b oxy-3,4-
d ih yd r o-6,7-d im et h oxy-3-(3,4-m et h ylen ed ioxyp h en yl)-
1(2H)-isoqu in olon e (5b). Piperonal (1.3 g, 8.5 mmol) was
added to a stirred solution of commercially available 2-(dim-
ethylamino)ethylamine (3.0 g, 9.4 mmol) and anhydrous
magnesium sulfate (5.0 g) in chloroform (100 mL) and the
mixture was stirred at room temperature for 24 h. The reaction
mixture was filtered through a Celite pad and the residue was
washed with chloroform (40 mL). The combined organic layer
was washed with water (100 mL), brine, and dried (Na2SO4).
The organic solution was concentrated on a rotary evaporator
and the resulting Schiff base was used in the next reaction
without purification. 4,5-Dimethoxyhomophthalic anhydride
3 (2.0 g, 9.0 mmol) was added to a chloroform (100 mL) solution
6-[3-(N,N-Dim et h yla m in o)-1-p r op yl]-5,6-d ih yd r o-2,3-
d im et h oxy-8,9-m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o-
[1,2-c]isoqu in olin e (6a ). Thionyl chloride (100 mL) was
added to the indenoisoquinolone 5a (3.2 g, 7.02 mmol), and
the reaction mixture was stirred at room temperature for 6 h.
Benzene (50 mL) was added to the red solution and the
mixture was concentrated under reduced pressure. Et3N (20
mL) was added to a mixture of the resultant purple solid in
chloroform (50 mL) and the mixture was stirred at room
temperature for 2 h. The mixture was concentrated on a rotary
evaporator and methanol (50 mL) was added to the resultant
solid, which was filtered off through a sintered glass funnel.

Indenoisoquinoline Topoisomerase I Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5719
The solid was further washed with CH3OH-CHCl3 (9:1, 30
mL) to obtain pure indenoisoquinoline 6a (2.0 g, 65%) as a
purple solid: mp 235-240 °C. 1H NMR (CDCl3) δ 8.00 (s, 1
H), 7.62 (s, 1 H), 7.47 (s, 1 H), 7.03 (s, 1 H), 6.06 (s, 2 H), 4.47
(t, J ) 8.2 Hz, 2 H), 4.03 (s, 3 H), 3.96 (s, 3 H), 2.48 (t, J ) 6.2
Hz, 2 H), 2.29 (s, 6 H), 2.02-1.92 (m, 2 H); ESIMS m/z (relative
intensity) 437 (MH+, 100), 392 (5). Anal. (C24H24N2O6‚0‚3H2O)
C, H, N.
30 min. The reaction mixture was then concentrated on a
rotary evaporator and the resultant solid was mixed with
chloroform (30 mL) and filtered off through a sintered glass
funnel to obtain pure compound 7b (270 mg, 82%) as a pale
purple solid: mp 296-298 °C. 1H NMR (DMSO-d6) δ 8.32 (bs,
1 H, COOH), 6.82 (bs, 1 H), 6.58 (bs, 2 H), 6.26 (bs, 1 H), 5.95
(bs, 2 H), 4.31 (bs, 2 H), 3.61 (s, 3 H), 3.53 (s, 3 H), 3.37 (bs, 2
H), 2.98 (bs, 6 H); ESIMS m/z (relative intensity) 424 (22), 423
(MH+-HCOOH, 100), 379 (15), 378 (70). Anal. (C24H24N2O6‚
HCOOH‚0.6H2O) C, H, N.
6-[2-(N ,N -Dim e t h yla m in o)-1-e t h yl]-5,6-d ih yd r o-2,3-
d im et h oxy-8,9-m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o-
[1,2-c]isoqu in olin e (6b). Thionyl chloride (80 mL) was added
to the indenoisoquinolone 5b (2.5 g, 5.7 mmol) and the mixture
was stirred at room temperature for 6 h. Benzene (50 mL) was
added to the red solution and the mixture was concentrated
under reduced pressure. Et3N (20 mL) was added to a mixture
of the resultant purple solid in chloroform (50 mL) and the
mixture was stirred at room temperature for 2 h. The mixture
was concentrated on a rotary evaporator and methanol (50 mL)
was added to the resultant solid, which was filtered off through
a sintered glass funnel. The solid was further washed with
CH3OH-CHCl3 (9:1, 30 mL) to obtain pure compound 6b (1.53
6-(2-Am in oeth yl)-5,6-d ih yd r o-2,3-d im eth oxy-8,9-m eth -
ylen ed ioxy-5,11-d ioxo-11H-in d en o[1,2-c]isoqu in olin e Hy-
d r och lor id e (7e). BOC-protected indenoisoquinoline 6d (0.5
g, 1.01 mmol) was dissolved in chloroform (50 mL) and an
anhydrous solution of HCl in ether (2 M, 10.1 mL, 20.2 mmol)
was added at 0 °C. The reaction mixture was then stirred at
room temperature for 6 h. The precipitated product was
filtered off and washed with chloroform (50 mL) and dried over
P2O5 for 24 h to afford pure indenoisoquinoline hydrochloride
1
7e (0.33 g, 76%) as a purple solid: mp 294-296 °C. H NMR
(DMSO-d6) δ 8.16 (bs, 2 H, -NH2), 7.79 (s, 1 H), 7.47 (s, 1 H),
7.42 (s, 1 H), 7.02 (s, 1 H), 6.19 (s, 2 H), 4.60 (bs, 2 H), 3.89 (s,
3 H), 3.84 (s, 3 H), 3.19 (bs, 2 H); ESIMS m/z (relative
intensity) 397 (7), 396 (24), 395 (MH+, 100), 394 (7), 380 (7),
379 (20), 378 (79), 355 (9), 277 (14), 255 (11), 215 (24). Anal.
(C21H19N2O6Cl‚0.4H2O) C, H, N.
1
g, 64%) as a purple solid: mp 300-302 °C. H NMR (CDCl3)
δ 8.00 (bs, 1 H), 7.63 (bs, 1 H), 7.24 (bs, 1 H), 7.06 (bs, 1 H),
6.07 (bs, 2 H), 4.54 (bs, 2 H), 4.03 (s, 3 H), 3.96 (s, 3 H), 2.72
(bs, 2 H), 2.39 (bs, 6 H); ESIMS m/z (relative intensity) 424
(23), 423 (MH+, 100), 378 (32), 306 (39), 118 (12); HRESIMS
calcd for C23H22N2O6: 423.1556. Found: 423.1558.
Gen er a l P r oced u r e for th e Syn th esis of Am in o In d e-
n oisoqu in olin es 11a , 11b, 18a -18d , a n d 22. A mixture of
indenoisoquinoline bromide 10 (0.3 g, 0.63 mmol), BOC-
protected amines (1.89 mmol), and anhydrous K2CO3 (0.44 g,
3.2 mmol) in anhydrous 1,4-dioxane (25 mL) was heated to
100 °C and kept at that temperature for 4 h. The reaction
mixture was cooled and concentrated on a rotary evaporator.
The reaction mixture was diluted with chloroform (100 mL)
and washed with a 1% HCl solution (50 mL), water, and brine
and dried (Na2SO4). The reaction mixture was concentrated,
loaded on a silica gel column, and eluted with a 0-1% gradient
of methanol in chloroform to provide indenoisoquinolines 11a ,
11b, 18a -18d , and 22 in 25-68% yields.
6-[2-(N-F MOCa m in oeth yl)-5,6-d ih yd r o-2,3-d im eth oxy-
8,9-m eth ylen ed ioxy-5,11-dioxo-11H-in d en o[1,2-c]isoqu in -
olin e (6c). Thionyl chloride (50 mL) was added to the
indenoisoquinolone 5c (2.0 g, 3.15 mmol) and the mixture was
stirred at room temperature for 6 h. Benzene (50 mL) was
added to the red solution and the reaction mixture was
concentrated under reduced pressure. The resultant solid was
directly loaded on a silica gel column and eluted with a 0-1%
gradient of methanol in CHCl3 to give FMOC-protected ami-
noindenoisoquinoline 6c (1.3 g, 67%) as a purple solid: mp
1
242-244 °C (dec). H NMR (CDCl3) δ 8.00 (s, 1 H), 7.74 (d, J
) 7.6 Hz, 2 H), 7.59 (s, 1 H), 7.54 (d, J ) 3.3 Hz, 2 H), 7.51 (s,
1 H), 7.37 (t, J ) 7.4 Hz, 2 H), 7.25 (m, 2 H), 6.99 (s, 1 H), 5.91
(s, 2 H), 5.45 (bs, 1 H), 4.55 (t, J ) 6.4 Hz, 2 H), 4.40 (d, J )
7.2 Hz, 2 H), 4.17 (t, J ) 7.2 Hz, 1 H), 4.02 (s, 3 H), 3.95 (s, 3
H), 3.68 (m, 2 H); ESIMS m/z (relative intensity) 617 (MH+,
3), 611 (3), 444 (24), 443 (MH+ - FMOC, 100), 426 (78), 395
(26), 378 (21); HRESIMS calcd for C36H28N2O8: 617.1924.
Found: 617.1921. Anal. (C36H28N2O8‚1.7H2O) C, H, N.
6-[3-(2-ter t-BOCa m in oeth yl)a m in o-1-p r op yl]-5,6-d ih y-
d r o-2,3-d im et h oxy-8,9-m et h ylen ed ioxy-5,11-d ioxo-11H -
in d en o[1,2-c]isoqu in olin e (11a ). The indenoisoquinoline
11a (87 mg, 25%) was isolated as a purple solid: mp 199-201
°C. IR (thin film) 3450 (br), 2970, 2965, 1712, 1699, 1693, 1650,
1
1481, 1280 cm-1; H NMR (CDCl3) δ 7.96 (s, 1 H), 7.60 (s, 1
H), 7.40 (s, 1 H), 7.00 (s, 1 H), 6.06 (s, 2 H), 5.06 (bs, 1 H),
4.49 (t, J ) 7.3 Hz, 2 H), 4.02 (s, 3 H), 3.96 (s, 3 H), 3.25 (q, J
) 5.6 Hz, 2 H), 2.77 (q, J ) 6.3 Hz, 4 H), 2.01 (m, 2 H), 1.42
(s, 9 H); ESIMS m/z (relative intensity) 554 (5), 553 (28), 552
(MH+, 100), 496 (12), 453 (14), 452 (MH+ - BOC, 57), 392 (21).
Anal. (C29Η33Ν3Ã8‚1.2H2O) C, H, N.
6-[2-(N -t er t -B O C a m in o )-1-e t h y l]-5,6-d ih y d r o -2,3-
d im et h oxy-8,9-m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o-
[1,2-c]isoqu in olin e (6d ). Compound 6c (1.5 g, 2.4 mmol) was
dissolved in chloroform-piperidine (1:1, 100 mL) and the
reaction mixture was stirred at room temperature for 24 h.
The reaction mixture was then concentrated on a rotary
evaporator to dryness to give purple solid. BOC2O (0.76 g, 3.5
mmol) in chloroform (20 mL) was added dropwise to a stirred
solution of crude product (0.91 g, 2.3 mmol) and Et3N (0.65
mL, 4.6 mmol) in chloroform (100 mL) at 0 °C and the reaction
mixture was further stirred at room temperature for 8 h. The
reaction mixture was diluted with water (100 mL), washed
with 1% HCl (100 mL), brine and dried (Na2SO4). The reaction
mixture was concentrated under reduced pressure and purified
by silica gel column chromatography, eluting with a 0-1%
gradient of methanol in chloroform to afford compound 6d
(0.84 g, 70%) as a purple solid: mp 252-254 °C. 1H NMR
(CDCl3) δ 7.92 (s, 1 H), 7.56 (s, 1 H), 7.50 (s, 1 H), 7.00 (s, 1
H), 6.06 (s, 2 H), 5.25 (bs, 1 H, -NH), 4.52 (t, J ) 6.7 Hz, 2
H), 4. 01 (s, 3 H), 3.96 (s, 3 H), 3.61 (q, J ) 6.4 Hz, 2 H), 1.41
(s, 9 H); ESIMS m/z (relative intensity) 517 (MNa+, 20), 495
(MH+, 9), 455 (10), 395 (MH+ - BOC, 31), 379 (21), 378 (100),
352 (18). Anal. (C26H26N2O8‚0.3H2O) C, H, N.
6-[3-(3-ter t-BOCa m in op r op yl)a m in o-1-p r op yl]-5,6-d i-
h ydr o-2,3-dim eth oxy-8,9-m eth ylen edioxy-5,11-dioxo-11H-
in d en o[1,2-c]isoqu in olin e (11b). The indenoisoquinoline
11b (108 mg, 30%) was isolated as a purple solid: mp 197-
1
199 °C. H NMR (CDCl3) δ 8.02 (s, 1 H), 7.63 (s, 1 H), 7.40 (s,
1 H), 7.06 (s, 1 H), 6.07 (s, 2 H), 4.98 (bs, 1 H), 4.52 (t, J ) 7.8
Hz, 2 H), 4.03 (s, 3 H), 3.97 (s, 3 H), 3.22 (m, 2 H), 2.78 (t, J
) 6.3 Hz, 2 H), 2.69 (t, J ) 6.7 Hz, 2 H), 2.00 (m, 2 H), 1.70
(m, 2 H), 1.40 (s, 9 H); ESIMS m/z (relative intensity) 568 (6),
567 (29), 566 (MH+, 100), 510 (8), 467 (10), 466 (MH+ - BOC,
39). Anal. (C30Η35Ν3Ã8‚0.5H2O) C, H, N.
6-{3-[2-(2-ter t-BOCa m in o)-eth yl-(ter t-BOCa m in o)-eth -
yla m in o]-1-p r op yl}-5,6-d ih yd r o-2,3-d im eth oxy-8,9-m eth -
ylen edioxy-5,11-dioxo-11H-in den o[1,2-c]isoqu in olin e (18a).
The indenoisoquinoline 18a (760 mg, 52%) was isolated as a
purple solid: mp 123-125 °C. 1H NMR (CDCl3) δ 8.07 (s, 1
H), 7.61 (s, 1 H), 7.32 (bs, 1 H), 7.04 (s, 1 H), 6.07 (s, 2 H),
5.49 (bs, 1 H), 5.31 (bs, 1 H), 4.50 (t, J ) 7.4 Hz, 2 H), 4.02 (s,
3 H), 3.96 (s, 3 H), 3.34-3.29 (bs, 6 H), 2.80 (t, J ) 5.3 Hz, 4
H), 2.01 (m, 2 H), 1.44 (s, 9 H), 1.38 (s, 9 H); ESIMS m/z
(relative intensity) 696 (34), 695 (MH+, 100), 639 (24), 595
6-[2-(N ,N -Dim e t h yla m in o)-1-e t h yl]-5,6-d ih yd r o-2,3-
d im et h oxy-8,9-m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o-
[1,2-c]isoqu in olin e Hyd r ofor m a te (7b). The indenoisoquin-
oline 6b (0.3 g, 0.7 mmol) was dissolved in 98% formic acid
(10 mL) and the solution was stirred at room temperature for
(MH+ - BOC), 495 (MH+ - 2 × BOC). Anal. (C36H46N4O10
‚
0.2H2O) C, H, N.

5720 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Nagarajan et al.
6-{3-[2-(3-ter t-BOCam in o)-pr opyl-(ter t-BOCam in o)-eth -
yla m in o]-1-p r op yl}-5,6-d ih yd r o-2,3-d im eth oxy-8,9-m eth -
ylen edioxy-5,11-dioxo-11H-in den o[1,2-c]isoqu in olin e (18b).
The indenoisoquinoline 18b (510 mg, 68%) was isolated as a
purple solid: mp 155-158 °C. 1H NMR (CDCl3) δ 8.01 (s, 1
H), 7.62 (s, 1 H), 738 (s, 1 H), 7.05 (s, 1 H), 6.07 (s, 2 H), 4.50
(t, J ) 6.8 Hz, 2 H), 4.03 (s, 3 H), 3.97 (s, 3 H), 3.29 (bs, 4 H),
3.10 (apparent t, J ) 6.4 Hz, 2 H), 2.80-2.77 (m, 4 H), 2.01-
1.98 (m, 2 H), 1.66 (m, 2 H), 1.44 (s, 9 H), 1.41 (s, 9 H); ESIMS
m/z (relative intensity) 709 (MH+, 100), 652 (10), 610 (6), 609
(MH+ - BOC, 18), 509 (MH+ - 2 × BOC, 10). Anal.
(C37Η48Ν4Ã10‚0.3H2O) C, H, N.
[1,2-c]isoqu in olin e Hyd r och lor id e (12b). The indenoiso-
quinoline 11b was converted to the hydrochloride 12b (251
mg, 75%), which was obtained as a purple solid: mp 273-276
1
°C (dec). H NMR (DMSO-d6) δ 9.15 (bs, 1 H), 8.79 (bs, 2 H),
7.68 (s, 1 H), 7.36 (s, 1 H), 7.29 (s, 1 H), 6.96 (s, 1 H), 6.18 (s,
2 H), 4.42 (bs, 2 H), 3.85 (s, 3 H), 3.82 (s, 3 H), 2.98 (bs, 4 H),
1
2.85 (bs, 2 H), 2.12 (bs, 2 H), 1.69 (bs, 2 H); H NMR (D2O) δ
6.63 (bs, 1 H), 6.47 (bs, 1 H), 6.36 (bs, 1 H), 6.08 (bs, 1 H),
5.89 (bs, 2 H), 3.94 (bs, 2 H), 3.42 (s, 3 H), 3.29 (s, 3 H), 3.04-
2.95 (bs, 6 H), 1.98 (bs, 2 H), 1.74 (bs, 2 H); ESIMS m/z (relative
intensity) 467 (18), 466 (MH+ - 2 × HCl, 72), 393 (25), 392
(100). Anal. (C24H25N3O6‚2.2H2O) C, H, N.
6-{3-[3-(2-t er t -BOCa m in o)-e t h yl-(t er t -BOCa m in o)-
p r op yla m in o]-1-p r op yl}-5,6-d ih yd r o-2,3-d im et h oxy-8,9-
m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o[1,2-c]isoq u in o-
lin e (18c). The indenoisoquinoline 18c (750 mg, 50%) was
isolated as a purple solid: mp 115-118 °C. 1H NMR (CDCl3) δ
8.02 (s, 1 H), 7.63 (s, 1 H), 7.44 (s, 1 H), 7.05 (s, 1 H), 6.07 (s,
2 H), 5.04 (bs, 1 H), 4.85 (bs, 1 H), 4.51 (t, J ) 7.4 Hz, 2 H),
4.03 (s, 3 H), 3.97 (s, 3 H), 3.27 (bs, 6 H), 2.78 (t, J ) 6.1 Hz,
2 H), 2.62 (t, J ) 6.8 Hz, 2 H), 1.99 (m, 2 H), 1.75 (m, 2 H),
1.44 (s, 9 H), 1.40 (s, 9 H); ESIMS m/z (relative intensity) 710
(35), 709 (MH+, 100), 653 (6), 609 (MH+ - BOC, 16), 594 (8),
509 (MH+ - 2 × BOC, 67), 494 (6). Anal. (C37H48N4O10) C, H, N.
6-{3-[2-(2-Am in oet h yla m in o)-et h yla m in o]-1-p r op yl}-
5,6-dih ydr o-2,3-dim eth oxy-8,9-m eth ylen edioxy-5,11-dioxo-
11H-in d en o[1,2-c]isoqu in olin e Hyd r och lor id e (19a ). The
indenoisoquinoline 18a was converted to the hydrochloride 19a
(305 mg, 70%), which was obtained as a dark purple solid: mp
1
257-260 °C (dec). H NMR (D2O) δ 6.66 (bs, 1 H), 6.52 (bs, 2
H), 6.18 (bs, 1 H), 5.90 (bs, 2 H), 3.97 (bs, 2 H), 3.52 (s, 3 H),
3.49 (s, 3 H), 3.45-3.37 (m, 6 H), 3.32-3.27 (m, 2 H), 3.19 (bs,
2 H), 2.06 (bs, 2 H); ESIMS m/z (relative intensity) 496 (27),
495 (MH+ - 3 × HCl, 100), 478 (7), 435 (19), 409 (6), 393 (15),
392 (68). Anal. (C26H33N4O6Cl3‚2.7H2O) C, H, N.
6-{3-[2-(3-(Am in opr opyl)am in o)-eth ylam in o]-1-pr opyl}-
5,6-dih ydr o-2,3-dim eth oxy-8,9-m eth ylen edioxy-5,11-dioxo-
11H-in d en o[1,2-c]isoqu in olin e Hyd r och lor id e (19b). The
indenoisoquinoline 18b was converted to the hydrochloride 19b
(340 mg, 83%), which was obtained as a purple solid: mp 270-
6-{3-[3-(3-t er t -BOCa m in o)-p r op yl-(t er t -BOCa m in o)-
p r op yla m in o]-1-p r op yl}-5,6-d ih yd r o-2,3-d im et h oxy-8,9-
m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o[1,2-c]isoq u in o-
lin e (18d ). The indenoisoquinoline 18d (360 mg, 47%) was
isolated as a purple solid: mp 96-99 °C. 1H NMR (CDCl3) δ
8.01 (s, 1 H), 7.61 (s, 1 H), 7.31 (s, 1 H), 7.04 (s, 1 H), 6.06 (s,
2 H), 5.25 (bs, 1 H), 4.51 (t, J ) 7.4 Hz, 2 H), 4.02 (s, 3 H),
3.96 (s, 3 H), 3.24 (bs, 4 H), 3.08 (apparent t, J ) 5.7 Hz, 2 H),
2.79 (t, J ) 5.9 Hz, 2 H), 2.64 (t, J ) 6.1 Hz, 2 H), 2.04 (bs, 2
H), 1.76 (bs, 2 H), 1.65 (bs, 2 H), 1.43 (s, 9 H), 1.41 (s, 9 H);
ESIMS m/z (relative intensity) 724 (35), 723 (MH+, 100), 624
(14), 623 (MH+ - BOC, 40), 524 (17), 523 (MH+ - 2× BOC,
67), 332 (10). Anal. (C38H50N4O10‚0.2H2O) C, H, N.
1
273 °C (dec). H NMR (DMSO-d6) δ 9.52 (bs, 1 H), 9.39 (bs, 1
H), 8.02 (bs, 2 H), 7.86 (s, 1 H), 7.49 (s, 1 H), 7.40 (s, 1 H),
7.09 (s, 1 H), 6.20 (s, 2 H), 4.50 (bs, 2 H), 3.90 (s, 3 H), 3.86 (s,
3 H), 3.15 (bs, 4 H), 3.11 (bs, 2 H), 3.03 (bs, 2 H), 2.90 (bs, 2
H), 2.16 (bs, 2 H), 1.96 (bs, 2 H); ESIMS m/z (relative intensity)
510 (28), 509 (MH+ - 3 × HCl, 100), 435 (6), 392 (14). Anal.
Calcd (C27H35Cl3N4O6‚1.8H2O) C, H, N.
6-{3-[3-(2-Am in oeth yla m in o)-p r op yla m in o]-1-p r op yl}-
5,6-dih ydr o-2,3-dim eth oxy-8,9-m eth ylen edioxy-5,11-dioxo-
11H-in d en o[1,2-c]isoqu in olin e Hyd r och lor id e (19c). The
indenoisoquinoline 18c was converted to the hydrochloride 19c
(270 mg, 65%), which was obtained as a dark purple solid: mp
6-{3-[3-3-(3-ter t-BOCa m in o)-p r op yl-(ter t-BOCa m in o)-
p r op yl-(ter t-BOCa m in o)-p r op yla m in o]-1-p r op yl}-5,6-d i-
h ydr o-2,3-dim eth oxy-8,9-m eth ylen edioxy-5,11-dioxo-11H-
in d en o[1,2-c]isoqu in olin e (22). The indenoisoquinoline 22
(467 mg, 50%) was isolated as a dark purple solid: mp 102-
1
263-265 °C (dec). H NMR (D2O) δ 6.59 (bs, 1 H), 6.48 (bs, 1
H), 6.40 (bs, 1 H), 6.13 (bs, 1 H), 5.89 (bs, 2 H), 3.90 (bs, 2 H),
3.48 (s, 3 H), 3.44 (s, 3 H), 3.36-3.24 (m, 4 H), 3.16-3.07 (m,
6 H), 2.13-1.97 (m, 4 H); ESIMS m/z (relative intensity) 510
(21), 509 (MH+ - 3 × HCl, 88), 495 (28), 494 (100), 393 (11),
392 (48). Anal. (C27H35Cl3N4O6‚1.3H2O) C, H, N.
1
104 °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 (bs, 2 H), 4.03 (s, 3 H),
3.96 (s, 3 H), 3.22 (bs, 4 H), 3.13 (bs, 6 H), 2.84 (bs, 2 H), 2.68
(bs, 2 H), 1.73 (bs, 4 H), 1.63 (bs, 4 H), 1.43 (s, 18 H), 1.41 (s,
9 H); ESIMS m/z (relative intensity) 881 (45), 880 (MH+, 100),
803 (40), 780 (MH+ - BOC, 20), 747 (14), 691 (10), 680 (MH+
6-{3-[3-(3-(Am in op r op yl)a m in o)-p r op yla m in o]-1-p r o-
p yl}-5,6-d ih yd r o-2,3-d im eth oxy-8,9-m eth ylen ed ioxy-5,11-
dioxo-11H-in den o[1,2-c]isoqu in olin e Hydr och lor ide (19d).
The indenoisoquinoline 18d was converted to the hydrochloride
19d (205 mg, 66%), which was obtained as a dark purple
- 2 × BOC, 18), 580 (MH+ - 3 × BOC, 9). Anal. (C46H65N5O12
‚
0.8H2O) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Am in o In d e-
n oisoqu in olin e Hyd r och lor id es 12a , 12b, 19a -19d , a n d
23. Indenoisoquinolines 11a , 11b, 18a -18d , and 22 (1.0 mmol)
were dissolved separately in chloroform (50 mL) and an
anhydrous solution of HCl in ether (2 M, 30.0 mmol) was added
at 0 °C. The reaction mixture was then stirred at room
temperature for 8 h. The precipitated product was filtered off
and washed with chloroform (50 mL), methanol (20 mL), and
dried over P2O5 for 24 h to afford pure amino indenoisoquino-
line hydrochlorides 12a , 12b, 19a -19d , and 23 in 51-83%
yield as dark purple solids.
6-[3-(2-Am in oet h yla m in o)-1-p r op yl]-5,6-d ih yd r o-2,3-
d im et h oxy-8,9-m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o-
[1,2-c]isoqu in olin e Hyd r och lor id e (12a ). The indenoiso-
quinoline 11a was converted to the hydrochloride 12a (304 mg,
80%), which was obtained as a purple solid: mp 280-283 °C
(dec). 1H NMR (DMSO-d6) δ 9.29 (bs, 1 H), 8.26 (bs, 2 H), 7.86
(s, 1 H), 7.48 (s, 1 H), 7.40 (s, 1 H), 7.09 (s, 1 H), 6.20 (s, 2 H),
4.50 (bs, 2 H), 3.89 (s, 3 H), 3.85 (s, 3 H), 3.14 (bs, 6 H), 2.16
(bs, 2 H); ESIMS m/z (relative intensity) 453 (24), 452 (MH+
- 2 × HCl, 95), 393 (22), 392 (100). Anal. (C24H27Cl2N3O6‚
0.5H2O) C, H, N.
1
solid: mp 288-291 °C (dec). H NMR (D2O) δ 6.57 (bs, 1 H),
6.46 (bs, 1 H), 6.38 (bs, 1 H), 6.11 (bs, 1 H), 5.88 (bs, 2 H),
3.89 (bs, 2 H), 3.47 (s, 3 H), 3.43 (s, 3 H), 3.11-3.04 (m, 8 H),
2.98 (t, J ) 7.8 Hz, 2 H), 2.11-1.93 (m, 6 H); ESIMS m/z
(relative intensity) 525 (22), 524 (63), 523 (MH+- 3 × HCl, 100),
392 (37). Anal. (C28H37Cl3N4O6‚1.2H2O) C, H, N.
6-{3-[3-3-(3-(Am in op r op yl)a m in o)-(p r op yla m in o)-
p r op yla m in o]-1-p r op yl}-5,6-d ih yd r o-2,3-d im et h oxy-8,9-
m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o[1,2-c]isoq u in o-
lin e Hyd r och lor id e (23). The indenoisoquinoline 22 was
converted to the hydrochloride 23 (232 mg, 51%), which was
obtained as a dark brown solid: mp 280-282 °C. 1H NMR
(D2O) δ 6.79 (bs, 1 H), 6.63 (bs, 2 H), 6.29 (bs, 1 H), 5.96 (bs,
2 H), 4.06 (bs, 2 H), 3.61 (s, 3 H), 3.58 (s, 3 H), 3.10-2.99 (bs,
14 H), 2.06 (bs, 8 H); ESIMS m/z (relative intensity) 581 (32),
580 (MH+, 100), 523 (9), 466 (7), 392 (24), 290 (12). Anal.
(C27H35Cl3N4O6‚1.8H2O) C, H, N.
6-[3-(3-Am in op r op yl)a m in o-1-p r op yl]-5,6-d ih yd r o-2,3-
d im et h oxy-8,9-m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o-
[1,2-c]isoqu in olin e (13). A 2 M NaOH solution was added
to a stirred solution of compound 12b (160 mg, 0.30 mmol) in
a 1:1 mixture of chloroform-water (40 mL) at 0 °C, until a
basic pH of 7-8 obtained. The organic portion was then
6-[3-(3-(Am in op r op yl)a m in o)-1-p r op yl]-5,6-d ih yd r o-
2,3-dim eth oxy-8,9-m eth ylen edioxy-5,11-dioxo-11H-in den o-

Indenoisoquinoline Topoisomerase I Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5721
separated from the aqueous portion and the organic layer was
concentrated, loaded on a silica gel column, eluted with a
5-10% gradient of methanol in chloroform, along with 1 mL
of aqueous NH4OH to provide pure diamino indenoisoquinoline
13 (105 mg, 76%) as a purple solid: mp 204-206 °C. 1H NMR
(CDCl3) δ 7.99 (s, 1 H), 7.61 (s, 1 H), 7.37 (s, 1 H), 7.03 (s, 1
H), 6.06 (s, 2 H), 4.99 (bs, 1 H), 4.51 (t, J ) 7.1 Hz, 2 H), 4.02
(s, 3 H), 3.97 (s, 3 H), 3.23 (apparent t, J ) 5.8 Hz, 2 H), 2.78
(t, J ) 5.9 Hz, 2 H), 2.70 (t, J ) 6.4 Hz, 2 H), 2.01 (m, 2 H),
1.70 (m, 2 H); ESIMS m/z (relative intensity) 467 (27), 466
(MH+, 100), 393 (17), 392 (74). Anal. (C25H27N3O6‚0.6H2O) C,
H, N.
phthalimide (5.4 g, 36.0 mmol) in dry THF (100 mL). The
reaction mixture was slowly brought to room temperature and
stirred at that temperature for 5 h. THF was evaporated under
vacuum and the residue was directly loaded on a silica gel
column and purified by flash chromatography (230-400 mesh,
8:2 benzene-EtOAc as an eluant) to afford compounds 16a -
16d in 69-85% yield.
(2-ter t-BOCam in o-eth yl)-[2-(1,3-dioxo-1,3-dih ydr o-isoin -
d ol-2-yl)-eth yl]-ca r ba m ic Acid ter t-Bu tyl Ester (16a ). The
protected triamine 16a (11.76 g, 82%) was isolated as a white
solid: mp 41-43 °C. IR (neat) 3375 (br), 2977, 2935, 1773,
1714, 1515, 1250, 1156 cm-1 1H NMR (CDCl3) δ 7.81 (m, 2
;
H), 7.69 (m, 2 H), 5.13 (bs, 1 H), 3.81 (bs, 2 H), 3.48 (bs, 2 H),
3.30 (m, 4 H), 1.40 (s, 9 H), 1.20 (s, 9 H); ESIMS m/z 457 (24),
456 (MNa+, 100), 356 (MNa+ - BOC, 26), 400 (17). Anal.
(C22Η31Ν3Ã6) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
15a -15d . The BOC-protected amino alcohols 14a and 14b35
(1.0 mmol) and Et3N (1.5 mmol) were separately dissolved in
CHCl3 (100 mL) and cooled to 0 °C. Methanesulfonyl chloride
(1.1 mmol) was added dropwise to this ice-cold solution and
the reaction mixture was further stirred at that temperature
for 4 h. The excess methanesulfonyl chloride was quenched
with 1 N NaOH solution (5 mL). The organic portion was then
washed with water (2 × 200 mL), brine, dried over Na2SO4,
concentrated and used in the next step without purification.
The above crude product (1.0 mmol) and ethanolamine (10.0
mmol) and/or propanolamine (10.0 mmol) were heated at 80
°C for 8 h. The reaction mixture was cooled to room temper-
ature, diluted with water (50 mL), and extracted with CH2Cl2
(3 × 100 mL). The organic portion was washed with brine,
dried over Na2SO4, concentrated, and used in the next reaction
without purification. BOC2O (1.1 mmol) was added to the
above crude product (1.0 mmol) and Et3N (2.0 mmol) in CHCl3
(100 mL) and the reaction mixture was stirred further at 23
°C for 8 h. The reaction mixture was washed with water, brine,
dried over Na2SO4, and concentrated. The product was further
purified by silica gel column and eluted with a 1-3% gradient
of methanol in chloroform to afford compounds 15a -15d as
colorless, viscous oils in 70-75% overall yield in three steps.
(3-ter t-BOCa m in o-p r op yl)-[3-(1,3-d ioxo-1,3-d ih yd r o-
isoin d ol-2-yl)-p r op yl]-ca r b a m ic Acid ter t-Bu t yl E st er
(16b). The protected triamine 16b (12.02 g, 85%) was isolated
as a semisolid: IR (neat) 3376 (br), 2977, 2936, 1800, 2770,
1714, 1505, 1393, 1250 cm-1 1H NMR (CDCl3) δ 7.82 (m, 2
;
H), 7.68 (m, 2 H), 5.26 (bs, 1 H), 3.81 (bs, 2 H), 3.44 (m, 2 H),
3.26 (m, 2 H), 3.10 (bs, 2 H), 1.63 (m, 2 H), 1.40 (s, 9 H), 1.25
(s, 9 H); ESIMS m/z 471 (23), 470 (MNa+, 100), 370 (MNa+
BOC, 5). Anal. (C23Η33Ν3Ã6‚0.4H2O) C, H, N.
-
(2-ter t-BOCam in o-eth yl)-[3-(1,3-dioxo-1,3-dih ydr o-isoin -
d ol-2-yl)-p r op yl]-ca r b a m ic Acid ter t-Bu t yl E st er (16c).
The protected triamine 16c (12.6 g, 69%) was isolated as a
viscous oil: IR (neat) 3358 (br), 2976, 2933, 2874, 1692 (br),
1
1688, 1524, 1420, 1366, 1170, 1059 cm-1; H NMR (CDCl3) δ
7.83-7.77 (m, 2 H), 7.71-7.66 (m, 2 H), 5.01 (bs, 1 H), 3.65 (t,
J ) 7.2 Hz, 2 H), 3.30-3.28 (m, 4 H), 3.23 (t, J ) 4.7 Hz, 2 H),
1.93-1.78 (m, 2 H), 1.39 (s, 9 H), 1.36 (s, 9 H); 13C NMR
(CDCl3) δ 168.3, 156.2, 133.9, 131.9, 123.2, 80.1, 79.2, 50.3,
44.2, 35.6, 28.2; ESIMS m/z (relative intensity) 471 (22), 470
(MNa+, 100), 414 (11), 370 (12), 292 (13); HRESIMS calcd for
(2-ter t-BOCa m in o-et h yl)-(2-h yd r oxy-et h yl)-ca r b a m ic
Acid ter t-Bu tyl Ester (15a ).36 The amino alcohol 15a (16.0
g, 73%, three steps) was isolated as a viscous, colorless oil:
IR (neat) 3356 (br), 2976, 2932, 1670, 1249 and 1146 cm-1; 1H
NMR (CDCl3) δ 3.72 (bs, 2 H), 3.34 (bs, 6 H), 1.46 (s, 9 H),
1.42 (s, 9 H); ESIMS m/z (relative intensity) 328 (12), 327
(MNa+, 100), 279 (17), 271 (35), 227 (42).
C
23H33N3O6: 448.2448. Found: 448.2442.
(3-ter t-BOCa m in op r op yl)-[3-(1,3-d ioxo-1,3-d ih yd r o-
isoin d ol-2-yl)-p r op yl]-ca r ba m ic Acid ter t-Bu tyl Ester
(16d ). The protected triamine 16d (11.2 g, 77%) was isolated
as a semisolid: IR (neat) 3364, 2976, 2933, 1770, 1714, 1694,
1504, 1249, 1172 cm-1 1H NMR (CDCl3) δ 7.82-7.73 (m, 2
;
H), 7.72-7.68 (m, 2 H), 5.24 (bs, 1 H), 3.66 (t, J ) 7.1 Hz, 2
H), 3.24 (m, 4 H), 3.06 (m, 2 H), 1.88 (m, 2 H), 1.60 (m, 2 H),
1.40 (s, 18 H); ESIMS m/z 485 (23), 484 (MNa+, 100), 428 (11),
384 (MNa+ - BOC, 17). Anal. (C24Η35Ν3Ã6) C, H, N.
(3-ter t-BOCa m in o-p r op yl)-(2-h yd r oxy-eth yl)-ca r ba m ic
Acid ter t-Bu tyl Ester (15b). The amino alcohol 15b (20.5 g,
75%, three steps) was isolated as a viscous, colorless oil: IR
1
(neat) 3358 (br), 2975, 2931, 1681, and 1169 cm-1; H NMR
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
17a -17d . Hydrazine hydrate (40.0 mmol) was added sepa-
rately to a solution of compounds 16a -16d (20.0 mmol) in
methanol (100 mL) at room temperature and the mixture was
stirred further under reflux for 12 h, after which the TLC
showed the complete disappearance of the starting phthalim-
ide derivative. The reaction mixture was cooled and the
byproduct phthalhydrazide separated out as a white solid. The
reaction mixture was then filtered off through a sintered glass
funnel and the solid washed with cold methanol and the
filtrate was concentrated. The reaction product was purified
by silica gel column chromatography, eluting with a 2-10%
gradient of methanol in chloroform, to provide pure BOC-
protected triamines 17a -17d in 65-72% yield.
(2-Am in o-eth yl)-(2-ter t-BOCam in o-eth yl)-car bam ic Acid
ter t-Bu tyl Ester (17a ).37 The BOC-protected triamine 17a
(3.52 g, 72%) was isolated as a white solid: mp 74-76 °C. IR
(neat) 3363 (br), 2976, 2932, 1693 (br), 1517, 1479, 1414, 1366,
1250, 1165, 1067 cm-1; 1H NMR (CDCl3) δ 5.27 (bs, 1 H), 3.29-
3.24 (m, 8 H), 2.81 (bs, 2 H), 1.43 (s, 9 H), 1.40 (s, 9 H); 13C
NMR (CDCl3) δ 155.9, 79.6, 78.7, 50.7, 47.5, 40.6, 39.3, 28.2;
ESIMS m/z (relative intensity) 326 (MNa+, 37), 305 (12), 304
(MH+, 100), 247 (27), 204 (MH+ - BOC, 26), 104 (MH+ - 2 ×
BOC, 10). Anal. (C14H29N3O4‚0.22H2O) C, H, N.
(CDCl3) δ 3.73 (bs, 2 H), 3.33 (bs, 4 H), 3.10 (bs, 2 H), 1.63
(quin, J ) 6.6 Hz, 2 H), 1.44 (s, 9 H), 1.40 (s, 9 H); ESIMS m/z
342 (16), 341 (MNa+ - 100), 285 (10), 266 (10), 241 (10). Anal.
(C15Η30Ν2Ã5) C, H, N.
(2-ter t-BOCa m in o-eth yl)-(3-h yd r oxy-p r op yl)-ca r ba m ic
Acid ter t-Bu tyl Ester (15c). The amino alcohol 15c (19.4 g,
70%, three steps) was isolated as a viscous oil: IR (neat) 3363
(br), 2976, 2933, 1695 (br), 1523, 1480, 1366, 1250, 1170, 1061
1
cm-1; H NMR (CDCl3) δ 3.52 (bs, 2 H), 3.30 (bs, 2 H), 3.23
(bs, 4 H), 1.64 (bs, 2 H), 1.44 (s, 9 H), 1.40 (s, 9 H); 13C NMR
(CDCl3) δ 156.3, 155.8, 80.1, 78.9, 58.2, 46.4, 43.2, 39.1, 30.4,
28.1; ESIMS m/z (relative intensity) 320 (13), 319 (MH+, 100),
219 (MH+ - BOC, 48), 119 (MH+ - 2 × BOC, 3); HRESIMS
calcd for C15H30N2O5: 319.2233. Found: 319.2234.
(3-ter t-BOCam in o-pr opyl)-(3-h ydr oxy-pr opyl)-car bam ic
Acid ter t-Bu tyl Ester (15d ). The amino alcohol 15d (10.43
g, 73%, three steps) was isolated as a viscous oil: IR (neat)
1
3361 (br), 2976, 2933, 1685, 1681, 1251, 1165 cm-1; H NMR
(CDCl3) δ 4.75 (bs, 1 H), 3.50 (bs, 2 H), 3.31-3.16 (m, 4 H),
3.06 (apparent t, J ) 5.8 Hz, 2 H), 1.64 (m, 4 H), 1.42 (s, 9 H),
1.39 (s, 9 H); ESIMS m/z 356 (18), 355 (MNa+, 100), 303 (10),
289 (8), 281 (6), 255 (MNa+ - BOC, 11). Anal. (C16Η32Ν2Ã5)
C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
16a -16d . Diethyl azodicarboxylate (5.7 g, 36.0 mmol) was
added separately, over 10 min, to an ice-cold mixture of
compounds 15a -15d (33.0 mmol), PPh3 (9.6 g, 36.0 mmol) and
3-[(2-Am in o-et h yl)-ter t-BOCa m in o]-p r op yl-ca r b a m ic
Acid ter t-Bu tyl Ester (17b). The BOC-protected triamine
17b (6.9 g, 65%) was isolated as a viscous yellow oil: IR (neat)
3360 (br), 2976, 2932, 2871, 1693 (br), 1516, 1480, 1417, 1366,

5722 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Nagarajan et al.
1250, 1167, 1068 cm-1; 1H NMR (CDCl3) δ 5.26 (bs, 1 H), 4.80
(bs, 2 H), 3.27 (bs, 2 H), 3.20 (bs, 2 H), 3.09 (apparent t, J )
6.0 Hz, 2 H), 2.81 (t, J ) 6.2 Hz, 2 H), 1.66 (m, 2 H), 1.45 (s,
9 H), 1.42 (s, 9 H); 13C NMR (CDCl3) δ 155.8, 79.4, 78.6, 49.7,
44.3, 40.4, 37.2, 28.1; ESIMS m/z (relative intensity) 318 (MH+,
100), 302 (21), 258 (17), 218 (MH+ - BOC, 31), 118 (MH+ - 2
× BOC, 15). Anal. (C15H31N3O4‚0.1H2O) C, H, N.
K2CO3 (0.44 g, 3.2 mmol) in anhydrous DMF (30 mL) was
heated to 100 °C and kept at that temperature for 4 h. The
hot reaction mixture was filtered, diluted with ethanol (5 mL),
and cooled in an ice bath. The precipitated product was filtered
off through a sintered glass funnel, washed with ethanol (15
mL), and dried. The product was then crystallized in ethyl
acetate-chloroform (3:1) to give a purple solid, which was
further treated with 2 M HCl in ether (6.3 mL, 12.7 mmol) in
chloroform at room temperature for 8 h to provide indenoiso-
quinoline aminol hydrochloride 24 (140 mg, 43%) as a dark
purple solid: mp 274-276 °C. 1H NMR (DMSO-d6) δ 8.50 (bs,
1 H), 7.80 (s, 1 H), 7.44 (s, 1 H), 7.36 (s, 1 H), 7.05 (s, 1 H),
6.20 (s, 2 H), 5.59 (bs, 1 H), 4.46 (bs, 2 H), 3. 88 (s, 3 H), 3.84
(s, 3 H), 3.41 (bs, 2 H), 3.05 (bs, 2 H), 2.16 (bs, 2 H), 1.20 (s, 6
H); ESIMS m/z (relative intensity) 482 (27), 481 (MH+ - HCl,
100), 478 (9), 393 (11), 392 (47); HRESIMS calcd for (MH+)
(3-Am in o-p r op yl)-(2-ter t-BOCa m in o-et h yl)-ca r b a m ic
Acid ter t-Bu tyl Ester (17c).38 The BOC-protected triamine
17c (7.3 g, 68%) was isolated as a viscous yellow oil: IR (neat)
3361 (br), 2976, 2933, 2871, 1694 (br), 1519, 1417, 1366, 1251,
1168, 1068 cm-1; 1H NMR (CDCl3) δ 5.13-4.97 (bs, 1 H), 3.18
(bs, 6 H), 2.61 (bs, 2 H), 2.01 (bs, 2 H, NH2), 1.62-1.53 (m,
2 H), 1.37 (s, 9 H), 1.30 (s, 9 H); 13C NMR (CDCl3) δ 155.9,
79.8, 79.1, 46.2, 44.4, 39.3, 32.2, 28.3; ESIMS m/z (relative
intensity) 319 (16), 318 (MH+, 100), 218 (MH+ - BOC).
HRESIMS Calcd for C15H31N3O4: 318.2393. Found: 318.2393.
Anal. (C15H31N3O4‚0.6CH2Cl2) C, H, N.
C
26H29N2O7Cl: 481.1975; Found: 481.1978. Anal. (C26H29
-
ClN2O7‚1.3CHCl3) C, H, N.
6-[(3-Dim eth oxyeth yl)a m in o-1-p r op yl]-5,6-d ih yd r o-2,3-
d im et h oxy-8,9-m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o-
[1,2-c]isoqu in olin e (25). A mixture of indenoisoquinoline
bromide 10 (0.4 g, 0.85 mmol), aminoacetaldehyde dimethyl
acetal (0.2 g, 1.86 mmol) and anhydrous K2CO3 (0.47 g, 3.40
mmol) in anhydrous 1,4-dioxane (25 mL) was heated to 100
°C and kept at that temperature for 4 h. The reaction mixture
was cooled and then concentrated. The reaction mixture was
diluted with water (50 mL) and extracted with CHCl3 (2 × 50
mL), and washed with brine and dried (Na2SO4). The reaction
mixture was concentrated and loaded on the silica gel column
(50 g), eluting with a 1-2% gradient of methanol in chloroform,
to provide pure indenoisoquinoline 25 (0.23 g, 55%) as a purple
(3-Am in o-p r op yl)-(3-ter t-BOCa m in o-p r op yl)-ca r ba m ic
Acid ter t-Bu tyl Ester (17d ).39,40 The BOC-protected triamine
17d (7.3 g, 68%) was isolated as a viscous yellow oil: IR (neat)
3360 (br), 2976, 2933, 2870, 1694, 1683, 1519, 1480, 1419,
1
1366, 1275, 1251, 1170, 1083 cm-1; H NMR (CDCl3) δ 5.25
(bs, 1 H), 4.71 (bs, 2 H), 3.22 (bs, 4 H), 3.07 (apparent t, J )
6.0 Hz, 2 H), 2.67 (t, J ) 5.8 Hz, 2 H), 1.63 (m, 4 H), 1.43 (s,
9 H), 1.41 (s, 9 H); 13C NMR (CDCl3) δ 155.9, 79.5, 78.5, 43.5,
39.2, 37.3, 32.3, 28.3; ESIMS m/z (relative intensity) 333 (16),
332 (MH+, 100), 276 (16), 232 (MH+ - BOC, 62), 132 (MH+
2 × BOC, 27). Anal. (C16H33N3O4) C, H, N.
-
(3-ter t-BOCam in o-pr opyl)-{3-[ter t-BOC-(2-cyan o-eth yl)-
am in o]-pr opyl}-car bam ic Acid ter t-Bu tyl Ester (20). Acry-
lonitrile (1.5 mL, 23.0 mmol) was added to a solution of BOC-
protected amine 17d (5.8 g, 18.0 mmol) in methanol (30 mL)
at 0 °C and the reaction mixture was stirred for 16 h, protected
from light. The solvent was evaporated in vacuo, and the
residue was used in the next reaction without purification.
BOC2O (3.3 g, 15.0 mmol) was added slowly to the above crude
product (4.9 g, 13.0 mmol) and Et3N (3.5 mL, 25.0 mmol) in
CHCl3 (40 mL) at 0 °C and the reaction mixture was stirred
further at RT for 12 h. The reaction mixture was washed with
water, brine, dried over Na2SO4, and concentrated. The reac-
tion mixture was purified by silica gel column using CHCl3-
CH3OH (95:5) as an eluant to afford compound 20 (4.1 g, 67%)
as a viscous oil: IR (neat) 3364 (br), 3004, 2976, 2933, 2249,
1694 (br), 1513, 1479, 1417, 1303, 1250 cm-1; 1H NMR (CDCl3)
δ 3.44 (t, J ) 6.6 Hz, 2 H), 3.27-3.22 (m, 4 H), 3.16-3.07 (m,
4 H), 2.60 (bs, 2 H), 1.79-1.70 (m, 2 H), 1.65-1.58 (m, 2 H),
1.44 (s, 18 H), 1.41 (s, 9 H); ESIMS m/z (relative intensity)
508 (25), 507 (MNa+, 100), 485 (MH+, 23), 385 (MH+ - BOC,
7), 329 (5), 285 (MH+ - 2 × BOC, 3), 185 (MH+ - 3 × BOC,
3). Anal. (C24H44N4O6) C, H, N.
1
solid: mp 206-208 °C. H NMR (CDCl3) δ 7.99 (s, 1 H), 7.61
(s, 1 H), 7.33 (s, 1 H), 7.03 (s, 1 H), 6.06 (s, 2 H), 4.57 (t, J )
5.4 Hz, 1 H), 4.51 (t, J ) 6.9 Hz, 2 H), 4.02 (s, 3 H), 3.96 (s, 3
H), 3.39 (s, 6 H), 2.84 (t, J ) 6.1 Hz, 2 H), 2.79 (d, J ) 5.6 Hz,
2 H), 2.07 (m, 2 H); 13C NMR (CDCl3) δ 189.9, 162.7, 154.9,
153.3, 151.5, 149.1, 148.7, 132.5, 130.4, 128.3, 116.8, 107.9,
105.3, 103.2, 102.9, 102.6, 56.3, 56.0, 54.2, 51.0, 46.5, 42.5;
ESIMS m/z 498 (26), 497 (MH+, 100), 465 (26). Anal.
(C25H26N2O8) C, H, N.
6-[3-(Bis-h yd r oxyet h yla m in o)-1-p r op yl]-5,6-d ih yd r o-
2,3-dim eth oxy-8,9-m eth ylen edioxy-5,11-dioxo-11H-in den o-
[1,2-c]isoqu in olin e Hyd r och lor id e (26). A mixture of in-
denoisoquinoline bromide 10 (0.3 g, 0.63 mmol), bis-(2-tert-
butyldimethylsilyloxyethyl)amine43 (0.63 g, 1.9 mmol) and
anhydrous K2CO3 (0.44 g, 3.2 mmol) in anhydrous 1,4-dioxane
(25 mL) was heated to 100 °C and kept at that temperature
for 4 h. The reaction mixture was then cooled and concen-
trated. The reaction mixture was diluted with chloroform (50
mL), washed with water, brine and dried (Na2SO4). The
product was purified by silica gel column and eluted with a
1-5% gradient of methanol in chloroform to give silyl protected
indenoisoquinoline aminol (0.24 g, 0.33 mmol, 52%), which was
further treated with 2 M HCl in ether (7.0 mL, 13.2 mmol) in
chloroform (30 mL) at room temperature for 8 h to afford pale
purple solid. The resultant solid was filtered through a sintered
glass funnel, and washed with chloroform (30 mL) to provide
indenoisoquinoline aminol 26 (105 mg, 61%) as a pale purple
[3-({3-[(3-Am in op r op yl)-ter t-BOCa m in o]-p r op yl}-ter t-
BOCa m in o)-p r op yl]-ca r b a m ic Acid ter t-Bu t yl E st er
(21).41,42 Raney nickel slurry in water (pore size ∼50 µ, 80-
100 m2/g) (∼2.0 g) and 2 M NaOH solution (30 mL) were added
to a solution of compound 20 (3.2 g, 6.6 mmol) in ethanol (70
mL) at room temperature. The reaction mixture was stirred
at room temperature for 16 h under atmospheric hydrogen
pressure (∼20 psi). The reaction mixture was carefully filtered
through a Celite pad and the solvent was evaporated. The
product was passed through a short pad of silica gel column
using CHCl3-CH3OH-NH4OH (6:3:1) as an eluant to give
amine 21 (1.7 g, 53%) as a viscous oil: IR (neat) 3361 (br),
1
solid: mp 270-272 °C (dec). H NMR (DMSO-d6) δ 7.72 (s, 1
H), 7.37 (s, 1 H), 7.27 (s, 1 H), 6.97 (s, 1 H), 6.17 (s, 2 H), 5.27
(bs, 2 H, -OH groups), 4.39 (bs, 2 H), 3.86 (s, 3 H), 3.82 (s, 3
H), 3.73 (bs, 4 H), 3.24 (bs, 6 H), 2.19 (bs, 2 H); ESIMS m/z
498 (27), 497 (MH+ - HCl, 100), 392 (20), 335 (6), 334 (25).
Anal. (C26H29N2O8Cl‚0.6CHCl3) C, H, N.
1
2975, 2932, 1686 (br), 1478, 1419, 1365, 1249, 1163 cm-1; H
NMR (CDCl3) δ 3.22 (bs, 4 H), 3.11 (bs, 6 H), 2.70 (bs, 2 H),
2.30 (bs, 2 H), 1.74-1.63 (m, 6 H), 1.42 (bs, 18 H), 1.40 (s, 9
H); ESIMS m/z (relative intensity) 490 (25), 489 (MH+, 100),
389 (MH+ - BOC, 19), 289 (MH+ - 2 × BOC, 18), 189 (MH+
- 3 × BOC, 8). Anal. (C24H48N4O6) C, H, N.
6-[(3-Tr iflu or oa ce t yloxy)-1-p r op yl]-5,6-d ih yd r o-2,3-
d im et h oxy-8,9-m et h ylen ed ioxy-5,11-d ioxo-11H -in d en o-
[1,2-c]isoqu in olin e (28). A mixture of indenoisoquinoline
bromide 10 (0.3 g, 0.63 mmol), aminopyrazine (0.12 g, 1.3
mmol), and anhydrous K2CO3 (0.35 g, 2.5 mmol) in anhydrous
1,4-dioxane (25 mL) was heated to 100 °C and kept at that
temperature for 4 h. The reaction mixture was cooled and
concentrated on a rotary evaporator. The reaction mixture was
diluted with chloroform (100 mL) and washed with 1% HCl
solution (50 mL), water, and brine and dried (Na2SO4). The
6-[3-(1,1′-Dim et h yl-2-h yd r oxyet h yl)a m in o-1-p r op yl]-
5,6-dih ydr o-2,3-dim eth oxy-8,9-m eth ylen edioxy-5,11-dioxo-
11H-in den o[1,2-c]isoqu in olin e Hydr och lor ide (24). A mix-
ture of indenoisoquinoline bromide 10 (0.3 g, 0.63 mmol),
2-amino-2-methyl-1-propanol (0.17 g, 1.9 mmol) and anhydrous

Indenoisoquinoline Topoisomerase I Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26 5723
(2) Fox, B. M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.;
Staker, B. L.; Stewart, L.; Cushman, M. Design, Synthesis, and
Biological Evaluation of Cytotoxic 11-Alkenylindenoisoquinoline
Topoisomerase I Inhibitors and Indenoisoquinoline-Campto-
thecin Hybrids. J . Med. Chem. 2003, submitted.
(3) Pommier, Y. Eukaryotic DNA Topoisomerase I: Genome Gate
Keeper and Its Intruders, Camptothecins. Semin. Oncol. 1996,
23, 1-10.
(4) Strumberg, D.; Pommier, Y.; Paull, K.; J ayaraman, M.; Nagafuji,
P.; Cushman, M. Synthesis of Cytotoxic Indenoisoquinoline
Topoisomerase I Poisons. J . Med. Chem. 1999, 42, 446-457.
(5) Cushman, M.; J ayaraman, M.; Vroman, J . A.; Fukunaga, A. K.;
Fox, B. M.; Kohlhagen, G.; Strumberg, D.; Pommier, Y. Synthesis
of New Indeno[1,2-c]isoquinolines: Cytotoxic Non-Camptothecin
Topoisomerase I Inhibitors. J . Med. Chem. 2000, 43, 3688-3698.
(6) J ayaraman, M.; Fox, B. M.; Hollingshead, M.; Kohlhagen, G.;
Pommier, Y.; Cushman, M. Synthesis of New Dihydroindeno-
[1,2-c]isoquinoline and Indenoisoquinolinium Chloride Topo-
isomerase I Inhibitors Having High in Vivo Anticancer Activity
in the Hollow Fiber Animal Model. J . Med. Chem. 2002, 45,
242-249.
(7) Edwards, M. L.; Snyder, R. D.; Stemerick, D. M. Synthesis and
DNA-Binding Properties of Polyamine Analogues. J . Med. Chem.
1991, 34, 2414-2420.
(8) Cohen, G. M.; Cullis, P. M.; Hartley, J . A.; Mather, A.; Symons,
M. C. R.; Wheelhouse, R. T. Targeting of Cytotoxic Agents by
Polyamines: Synthesis of a Chlorambucil-Spermidine Conjugate.
J . Chem. Soc., Chem. Commun. 1992, 298-300.
(9) Cullis, P. M.; Green, R. E.; Merson-Davies, L.; Travis, N. Probing
the Mechanism of Transport and Compartmentalization of
Polyamines in Mammalian Cells. Chem. Biol. 1999, 6, 717-729.
(10) Delcros, J .-G.; Tomasi, S.; Carrington, S.; Martin, B.; Renault,
J . Effect of Spermine Conjugation on the Cytotoxicity and
Cellular Transport of Acridine. J . Med. Chem. 2002, 45, 5098-
5111.
(11) Bergeron, R. J .; McManis, J . S.; Weimar, W. R.; Schreier, K. M.;
Gao, F.; Wu, Q.; Ortiz-Ocasio, J .; Luchetta, G. R.; Porter, C.;
Vinson, J . R. T. The Role of Charge in Polyamine Analogue
Recognition. J . Med. Chem. 1995, 38, 2278-2285.
reaction mixture was concentrated, loaded on the silica gel
column, and eluted with a 2-5% gradient of methanol in
chloroform to provide indenoisoquinoline derivative, which was
further treated with neat CF3COOH (10 mL) at room temper-
ature for 30 min to provide indenoisoquinoline trifluoroacetate
ester 28 (46 mg, 15%) as a pale purple solid: mp 280-282 °C.
1H NMR (CDCl3) δ 8.00 (s, 1 H), 7.61 (s, 1 H), 7.06 (s, 1 H),
7.02 (s, 1 H), 6.08 (s, 2 H), 4.57 (t, J ) 6.2 Hz, 4 H), 4.03 (s, 3
H), 3.97 (s, 3 H), 2.35 (m, 2 H); ESIMS m/z (relative intensity)
507 (26), 506 (MH+, 100), 505 (6), 481 (14), 465 (6), 433 (32),
410 (7), 393 (6), 392 (1). Anal. (C24H18F3NO8) C, H, N.
Top oisom er a se I-Med ia ted DNA Clea va ge Rea ction s.
Human recombinant topoisomerase I was purified from Bacu-
lovirus as described previously.44 The 161 bp fragment from
pBluescript SK(-) phagemid DNA (Stratagene, La J olla, 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 1× TBE buffer.
The 161-bp fragment was eluted from the gel slice using the
QIAEX II kit (QIAGEN Inc., Valencia, CA). Approximately
200 ng of the fragment was 3′-end labeled at the 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 topoisomerase I at 22 °C
for 30 min in the presence of the tested drug. Reactions were
terminated by adding SDS (0.5% final concentration).1 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% polyacryl-
amide, 7 M urea). Gels were dried and visualized by using a
Phosphoimager and ImageQuant software (Molecular Dynam-
ics, Sunnyvale, CA).
Molecu la r Mod elin g. The structure of the ternary com-
plex, containing topoisomerase I, DNA, and topotecan, was
downloaded from the Protein Data Bank (PDB code 1K4T).34
One molecule of PEG and the topotecan carboxylate form were
deleted. All of the atoms were then fixed according to Sybyl
atom types. Hydrogens were added and minimized using the
MMFF94s force field and MMFF94 charges. The structure of
the indenoisoquinoline 2, constructed in Sybyl and energy
minimized with the Tripos force field and Gasteiger-Hu¨ckel
charges, was overlapped with the structure of topotecan
according to the proposed structural similarity2 in the ternary
complex, and the structure of topotecan was then deleted. The
new whole complex was subsequently subjected to energy
minimization using the MMFF94s force field with MMFF94
charges. During energy minimization, the structure of the
indenoisoquinoline was allowed to move, while the structures
of the protein, nucleic acid, and water molecules were frozen.
The energy minimization was performed using the Powell
method with a 0.05 kcal/mol Å energy gradient convergence
criterion and a distance-dependent dielectric constant.
(12) Seiler, N.; Delcros, J . G.; Moulinoux, J . P. Polyamine Transport
in Mammalian Cells. An Update. Int. J . Biochem. Cell. Biol.
1996, 28, 843-861.
(13) Phanstiel, O., IV; Price, H. L.; Wang, L.; J uusola, J .; Kline, M.;
Shah, S. M. The Effect of Polyamine Homologation on the
Transport and Cytotoxicity Properties of Polyamine-(DNA-
Intercalator) Conjugates. J . Org. Chem. 2000, 65, 5590-5599.
(14) Wang, L.; Price, H. L.; J uusola, J .; Kline, M.; Phanstiel, O., IV.
Influence of Polyamine Architecture on the Transport and
Topoisomerase II Inhibitory Properties of Polyamine DNA-
Intercalator Conjugates. J . Med. Chem. 2001, 44, 3682-3691.
(15) Siddiqui, A. Q.; Merson-Davies, L.; Cullis, P. M. The Synthesis
of Novel Polyamine-Nitroimidazole Conjugates to Probe the
Structural Specificities of the Polyamine Untake System in A549
Lung Carcinoma Cells. J . Chem. Soc., Perkin Trans. 1 1999,
3243-3252.
(16) Porter, C. W.; Miller, J .; Bergeron, R. J . Aliphatic Chain Length
Specificity of the Polyamine Transport System in Acites L1210
Leukemia Cells. Cancer Res. 1984, 44, 126-128.
(17) Bergeron, R. J .; Feng, Y.; Weimar, W. R.; McManis, J . S.;
Dimova, H.; Porter, C.; Raisler, B.; Phanstiel, O. A Comparison
of Structure-Activity Relationships between Spermidine and
Spermine Analogue Antineoplastics. J . Med. Chem. 1997, 40,
1475-1494.
(18) Cullis, P. M.; Symons, C. R.; Cohen, G. M.; Wardman, P. A
General Method for Efficient Drug Delivery to DNA. Med. Sci.
Res. 1990, 18, 87-88.
(19) Cushman, M.; Gentry, J .; Dekow, F. W. Condensation of Imines
with Homophthalic Anhydrides. A Convergent Synthesis of cis-
and trans-13-Methyltetrahydroprotoberberines. J . Org. Chem.
1977, 42, 1111-1116.
(20) Rastogi, S. N.; Bindra, J . S.; Anand, N. R-Methyldopa in a Rigid
Framework: 3-Amino-3-methyl-6,7-dihydroxy-3,4-dihydroxycou-
marins and 2-Amino-6,7-dihydroxytetralin-2-carboxylic Acids.
Indian J . Chem. 1971, 9, 1175-1182.
Ack n ow led gm en t. This work was made possible by
the National Institutes of Health (NIH) through support
of this work with Research Grant UO1 CA89566 and
Training Grant ST32CA09634. The in vitro and in vivo
testing was conducted through the Developmental
Therapeutics Program, DCTD, NCI under Contract
NO1-CO-56000.
(21) Cushman, M.; Cheng, L. Total Synthesis of Nitidine Chloride.
J . Org. Chem. 1978, 43, 286-288.
(22) Cushman, M.; Cheng, L. Stereoselective Oxidation by Thionyl
Chloride Leading to the Indeno[1,2-c]isoquinoline System. J .
Org. Chem. 1978, 43, 3781-3783.
(23) Boeijin, A.; Ameijde, J . V.; Liskamp, R. M. Solid-Phase Synthesis
of Oligourea Peptidomimetics Employing the Fmoc Protection
Strategy. J . Org. Chem. 2001, 66, 8454-8462.
(24) Adamcyzk, M.; Grote, J . A. A Practical Method for the Synthesis
of N-Fluorenylmethoxycarbonyl Diamines. Org. Prep. Proc. Int.
1995, 27, 239-242.
(25) Blagbrough, I. S.; Geall, A. J . Practical Synthesis of Unsym-
metrical Polyamine Amides. Tetrahedron Lett. 1998, 39, 439-
442.
Refer en ces
(1) 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. Phar-
macol. 1998, 54, 50-58.

5724 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 26
Nagarajan et al.
(26) Geall, A. J .; Blagbrough, I. S. Homologation of Polyamines in
the Synthesis of Lipo-Spermine Conjugates and Related Lipo-
plexes. Tetrahedron Lett. 1998, 39, 443-446.
(36) Schutte, M. T.; Schumacher, P.; Unger, C.; Mulhaupt, R.; Kratz,
F. Synthesis of Two Maleimide Derivatives of Cis-Configurated
Platinum(II) Complexes for the Preparation of Chemoimmuno-
conjugates. Inorg. Chem. 1998, 267, 133-136.
(37) Verheijen, J . C.; Deiman, B. A. L. M.; Yeheskiely, E.; van der
Marcel, G. A.; Van Boom, J . H. Efficient Hydrolysis of RNA by
a PNA-Diethylenetriamine Adduct. Angew. Chem., Int. Ed. 2000,
39, 369-372.
(38) Lochner, M.; Geneste, H.; Hesse, M. General Access to Polyamines
Containing Ethane-1,2-diamine Units. Synthesis of Unnatural
Homologs and Isomeric N1,4-Di(4-coumaroyl)spermines. Helv.
Chim. Acta 1998, 81, 2270-2281.
(39) Garrett, S. W.; Davies, O. R.; Milroy, D. A.; Wood, P. J .; Pouton,
C. W.; Threadgill, M. D. Synthesis and Characterisation of
Polyamine-Poly(ethylene glycol) Constructs for DNA Binding
and Gene Delivery. Bioorg. Med. Chem. 2000, 8, 1779-1797.
(40) Nakamura, Y. Actinocynyl Bis(Hemin): DNA-intercalating Cleav-
age Agent. Heterocycles 1988, 27, 1873-1879.
(27) Kuksa, V.; Buchan, R.; Lin, P. K. T. Synthesis of Polyamines,
Their Derivatives, Analogues and Conjugates. Synthesis 2000,
1189-1207.
(28) Wang, C.; Delcros, J .-G.; Biggerstaff, J .; Phanstiel, O., IV.
Synthesis and Biological Evaluation of N 1-(Anthracen-9-yl-
methyl)triamines as Molecular Recognition Elements for the
Polyamine Transporter. J . Med. Chem. 2003, 46, 2663-2671.
(29) Wang, C.; Delcros, J . G.; Biggerstaff, J .; Phanstiel, O., IV.
Molecular Requirements for Targeting the Polyamine Transport
System. Synthesis and Biological Evaluation of Polyamine-
Anthracene Conjugates. J . Med. Chem. 2003, 46, 2672-2682.
(30) Chen, A. Y.; Yu, C.; Gatto, B.; Liu, L. F. DNA Minor Groove-
binding Ligands: A Different Class of Mammalian DNA Topo-
isomerase I Inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1993, 90,
8131-8135.
(31) Fujii, N.; Yamashita, Y.; Mizukami, T.; Nakano, H. Correlation
Between the Formation of Cleavable Complex with Topo-
isomerase I and Growth Inhibitory Activity for Saintopin-Type
Antibiotics. Mol. Pharmacol. 1997, 51, 269-276.
(32) Fujii, N.; Yamashita, Y.; Saitoh, Y.; Nakana, H. Induction of
Mammalian DNA Topoisomerase I-Mediated DNA Cleavage and
DNA Winding by Bulgarein. J . Biol. Chem. 1993, 268, 13160-
13165.
(41) Geall, A. J .; Al-Hadithi, D.; Blagbrough, I. S. Efficient Calf
Thymus DNA Condensation upon Binding with Novel Bile Acid
Polyamine Amides. Bioconj. Chem. 2002, 13, 481-490.
(42) Geall, A. J .; Taylor, R. J .; Earll, M. E.; Eaton, M. A. W.;
Balgbrough, I. S. Synthesis of Cholesteryl Polyamine Carbam-
ates: pKa Studies and Condensation of Calf Thymus DNA.
Bioconj. Chem. 2000, 11, 314-326.
(43) Grote, G.; Marcel, H. B.; Steemers, F. J .; Verboom, W.; Rein-
houdt, D. N. Synthesis of Neutral, Water-Soluble Calix[4]arenes.
Synthesis 1997, 6, 643-648.
(33) Kim, D.-K.; Kim, Y.-W.; Gam, J .; Kim, G.; Lim, J .; Lee, N.;
Kim, H.-T.; Kim, K. H. Synthesis and Anti-HIV-1 Activity of a
Series of 1-(Alkoxymethyl)-5-alkyl-6-(arylselenenyl)uracils and
-2-thiouracils). J . Heterocyclic Chem. 1996, 33, 1275-1283.
(34) Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin
J r., A. B.; Stewart, L. The Mechanism of Topoisomerase I
Poisoning by a Camptothecin Analogue. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 15387-15392.
(44) 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.
(35) Mattingly, P. G. Monoprotected Diamines. NR-(tert-Butoxycar-
bonyl)-R,ω-Alkanediamine Hydrochlorides from Amino Alcohols.
Synthesis 1990, 4, 366-368.
J M030313F