5-(2-Aminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: Variation of N-alkyl substituents modulates sensitivity to efflux transporters associated with multidrug resistance

Article abstract of DOI:10.1021/jm049447z

5H-8,9-Dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c, h]-[1,6]naphthyridin-6-one (ARC-111) has potent TOP1-targeting activity and pronounced antitumor activity. Several analogues of ARC-111 were synthesized with NH₂, N-alkyl, N,N-dialkyl, pyrrolidinyl, piperidinyl, and piperazinyl substituents at the 2-position of the 5-ethyl group. The relative TOP1-targeting activity and cytotoxicity of these structural analogues were assessed in RPMI8402 and P388 tumor cells and their camptothecin-resistant variants CPT-K5 and P388/CPT45, respectively. Potent TOP1-targeting activity was retained within a series of mono N-alkyl analogues that included NHCH ₂CH₃, NHCH(CH₃)₂, and NHC(CH ₃)₃. TOP1-targeting activity was diminished by the presence of a N-benzyl moiety. In a comparison of a series of N-alkyl-N-isopropyl analogues, activity decreased in the order CH₃ > CH₂CH₃ > CH-(CH₃)₂. Cytotoxicity in RPMI8402 and P388 did correlate with TOP1-targeting activity. Cytotoxic activity was also determined in KB3-1 cells and its variants KB/V-1 and KBH5.0. As KB/V-1 cells overexpress MDR1 and KBH5.0 cells overexpress BCRP, decreased cytotoxicity in these cell lines relative to the parent cell line is indicative of compounds that are substrates for these efflux transporters. In view of their diminished cytotoxicity in KB/V-1 cells, it appears that the likely demethylated metabolites of ARC-111, i.e., where NH₂ or NHCH₃ replaces the N(CH₃)₂ at the 2-position of the 5-ethyl substituent, are substrates for MDR1. In contrast, no significant difference in cytotoxicity among these three cell lines was observed with other N-alkyl analogues, including NHC₂H₅, NHCH(CH ₃)₂, NHC(CH₃)₃, N(CH ₃)₂, N(CH₂CH₃)₂, NCH ₃(CH-(CH₃)₂), and either the pyrrolidinyl or the piperidinyl analogues. The 2-(piperazinyl) analogues were associated with diminished cytotoxicity in KB/V-1 cells, suggesting that the second basic amino substituent is associated with their recognition as substrates by MDR1. Comparative studies on the antitumor activity of ARC-111 and its N-demethylated derivatives (the NHCH₃ and NH₂ analogues) against SJ-BT45 medulloblastoma xenografts in seid mice revealed that the secondary amine metabolite is at least as active as ARC-111 in vivo, although the primary amine derivative was significantly less potent.

Full text of DOI:10.1021/jm049447z

792
J. Med. Chem. 2005, 48, 792-804
5-(2-Aminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones: Variation of N-Alkyl
Substituents Modulates Sensitivity to Efflux Transporters Associated with
Multidrug Resistance
Alexander L. Ruchelman,† Peter J. Houghton, Nai Zhou,‡ Angela Liu,‡ Leroy F. Liu,‡,§ and
Edmond J. LaVoie*,†,§
Department of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road,
Piscataway, New Jersey 08854-8020, Department Molecular Pharmacology, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105-2794, Department of Pharmacology, University of Medicine and Dentistry of New Jersey,
Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, and The Cancer Institute of New Jersey,
New Brunswick, New Jersey 08901
Received July 13, 2004
5H-8,9-Dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h]-
[1,6]naphthyridin-6-one (ARC-111) has potent TOP1-targeting activity and pronounced
antitumor activity. Several analogues of ARC-111 were synthesized with NH2, N-alkyl, N,N-
dialkyl, pyrrolidinyl, piperidinyl, and piperazinyl substituents at the 2-position of the 5-ethyl
group. The relative TOP1-targeting activity and cytotoxicity of these structural analogues were
assessed in RPMI8402 and P388 tumor cells and their camptothecin-resistant variants CPT-
K5 and P388/CPT45, respectively. Potent TOP1-targeting activity was retained within a series
of mono N-alkyl analogues that included NHCH2CH3, NHCH(CH3)2, and NHC(CH3)3. TOP1-
targeting activity was diminished by the presence of a N-benzyl moiety. In a comparison of a
series of N-alkyl-N-isopropyl analogues, activity decreased in the order CH3 > CH2CH3 > CH-
(CH3)2. Cytotoxicity in RPMI8402 and P388 did correlate with TOP1-targeting activity. Cytotoxic
activity was also determined in KB3-1 cells and its variants KB/V-1 and KBH5.0. As KB/V-1
cells overexpress MDR1 and KBH5.0 cells overexpress BCRP, decreased cytotoxicity in these
cell lines relative to the parent cell line is indicative of compounds that are substrates for
these efflux transporters. In view of their diminished cytotoxicity in KB/V-1 cells, it appears
that the likely demethylated metabolites of ARC-111, i.e., where NH2 or NHCH3 replaces the
N(CH3)2 at the 2-position of the 5-ethyl substituent, are substrates for MDR1. In contrast, no
significant difference in cytotoxicity among these three cell lines was observed with other N-alkyl
analogues, including NHC2H5, NHCH(CH3)2, NHC(CH3)3, N(CH3)2, N(CH2CH3)2, NCH3(CH-
(CH3)2), and either the pyrrolidinyl or the piperidinyl analogues. The 2-(piperazinyl) analogues
were associated with diminished cytotoxicity in KB/V-1 cells, suggesting that the second basic
amino substituent is associated with their recognition as substrates by MDR1. Comparative
studies on the antitumor activity of ARC-111 and its N-demethylated derivatives (the NHCH3
and NH2 analogues) against SJ-BT45 medulloblastoma xenografts in scid mice revealed that
the secondary amine metabolite is at least as active as ARC-111 in vivo, although the primary
amine derivative was significantly less potent.
Introduction
poisons. Camptothecin (CPT) was the first molecule
identified as a TOP1-targeting agent.5 It has recently
been shown that formation of the TOP1-DNA cleavable
complex in the presence of camptothecin (CPT) induces
downregulation of TOP1 via an ubiquitin/26S proteo-
somal pathway.6,7 However, this CPT-induced down-
regulation of TOP1 is impaired to a variable degree in
many tumor cell lines. Therefore, it is believed that
detoxification by the ubiquitin/26S proteosomal pathway
might be responsible for the tumor selectivity as well
as resistance observed with CPT in some tumor cells.
Topotecan (Hycamtin) and irinotecan (CPT-11/Camp-
tosar) are two clinical anticancer agents developed from
the research performed with camptothecin and its
structurally related analogues. These camptothecin-
based drugs have incorporated within their structures
a δ-lactone, which is susceptible to hydrolysis. Hydroly-
sis of this lactone results in the formation of an inactive
derivative that possesses high affinity for human serum
Topoisomerases are enzymes that regulate the topol-
ogy of DNA and are critical to replication and transcrip-
tion. Two major subtypes, topoisomerase I (TOP1) and
topoisomerase II (TOP2), are distinguished based upon
differences in their primary sequence and initial mech-
anisms, wherein either a single- or double-stranded
DNA break is implicated.1-4 Topoisomerase-targeting
agents that stabilize the cleavable complex formed
between the enzyme and DNA have proven to be
effective in the treatment of cancer. Molecules capable
of stabilizing the topoisomerase-DNA cleavable com-
plex in effect convert these enzymes into cellular
* Correspondence author. Phone: 732-445-2674. Fax: 732-445-6312.
E-mail: elavoie@rci.rutgers.edu.
† Rutgers University.
St. Jude Children’s Research Hospital.
‡ UMDNJ, Robert Wood Johnson Medical School.
§ The Cancer Institute of New Jersey.
10.1021/jm049447z CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/15/2005

N-Alkyl Substituent Modulation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 793
albumin.8-10 Susceptibility to transporter-mediated cel-
lular efflux limits intracellular accumulation of several
camptothecin analogues. Overexpression of MDR1 (P-
glycoprotein, P-gp) confers resistance to camptothecins,
although to a lesser degree than that observed for TOP2-
targeting drugs such as doxorubicin or etoposide.11,12
The camptothecins that are in clinical use are good
substrates for the efflux transporter, breast cancer
resistance protein (BCRP),13,14 which may represent a
more clinically relevant source of resistance.15 Although
topotecan and irinotecan have good utility as a result
of their extraordinary potency, their metabolic instabil-
ity coupled with their susceptibility to efflux transport
have prompted further studies on the development of
novel TOP1-targeting agents. A chemotherapeutic agent
structurally unrelated to CPT could in theory avoid the
pharmacological drawbacks outlined above and might
also realize a different pattern of biodistribution. Such
a drug could be clinically useful in the treatment of
tumor types not affected by camptothecins or could be
used in combination with a CPT derivative.
Several novel non-camptothecin TOP1-targeting agents
have been identified. These include derivatives of bi- and
terbenzimidazoles,16-18 benz[a]anthracenes,19 benzo[c]-
phenanthridine and protoberberine alkaloids,20,21 in-
dolocarbazoles,22 the fungal metabolites bulgarein23 and
saintopin,24 and several indenoisoquinolines25-27 and
benzophenazines.28 Previous studies in our laboratory
identified 2,3-dimethoxy-8,9-methylenedioxybenzo[i]-
phenanthridine, 1,29 and 2,3-dimethoxy-8,9-methylene-
dioxydibenzo[c,h]cinnoline, 2,30 as lead compounds with
good TOP1-targeting activities (Figure 1). Further work
led to the development of solubilized derivatives such
as 8,9-dimethoxy-2,3-methylenedioxy-5-[2-(N,N-dime-
thylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-
one, 3, and 2,3-dimethoxy-8,9-methylenedioxy-11-[2-
(N,N-dimethylamino)ethyl]-11H-isoquino[4,3-c]-cinnolin-
12-one, 4.31 Studies of structure-activity relationships
(SAR) among these families of compounds elucidated
those structural features that were associated with
potent TOP1-targeting activity. Enhanced TOP1-target-
ing activity and cytotoxicity are associated with (1) the
presence of methoxy substituents at both the 2- and
3-position of the A-ring (benzo[i]phenanthridine num-
bering), (2) a 8,9-methylenedioxy moiety within the
D-ring, and (3) heteroatom substitution adjacent to the
benzo ring that incorporates the methylenedioxy sub-
stituent. For families of compounds represented by 3
and 4, the alkyl linker between the dialkylamino moiety
and the fused-ring polycyclic nucleus should be two
carbons in length for optimal activity. The dialkylamino
group can be a cyclic amine such as pyrrolidine, but
increasing the size beyond piperidine results in ana-
logues with decreased activity.
Figure 1. Structure and numbering of 2,3-dimethoxy-8,9-
methylenedioxybenzo[i]phenanthridine, 1, 2,3-dimethoxy-2,3-
methylenedioxydibenzo[c,h]cinnoline, 2, 5H-8,9-dimethoxy-5-
(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydiben-
zo[c,h][1,6]naphthyridin-6-one, 3, and 11H-2,3-dimethoxy-11-
(2-N,N-dimethylaminoethyl)-8,9-methylenedioxy-5,6,11-
isoquino[4,3-c]cinnolin-12-one, 4.
min postdosing in vivo. The M1 metabolite resulting
from N-dealklyation retains very potent activity in cell-
based assays. However, this minor structural modifica-
tion renders the M1 metabolite sensitive to BCRP-
mediated cellular clearance. Although unlikely to trigger
clinical resistance to 3, the ability of the M1 metabolite
to be a substrate for BCRP-mediated transport could
potentially have a detrimental impact on its activity.
The focus of the present study is to develop analogues
of 3 in which the dialkylamino moiety has been modified
to reduce the rate of N-dealkylation or to provide
N-dealkylation metabolites that are not substrates for
efflux transporters.
Compounds synthesized to investigate the “structure-
sensitivity relationships” relevant to the BCRP trans-
porter are listed in Figure 2. The variable within this
series is the identity of the N-alkyl, N,N-dialkylamino,
or cyclic amino group. Compounds 5e-g contain identi-
cal N,N-dialkyl substituents (benzyl, ethyl, and isopro-
pyl groups). Several unsymmetrical dialkyl substitu-
tients were also evaluated including 5a-d and 7a-b.
Benzylamines 5a-e and 6e provide a means for assess-
ing the effect of benzyl substitution on cytotoxicity,
TOP1-targeting activity, and sensitivity to MDR1 and
BCRP. These benzylamines also served as useful pre-
cursors to the respective secondary amines 6a-e and
the primary amine 6f. The secondary amines 6a-e as
Compound 3 is not a substrate for either MDR1 or
BCRP (see below for further discussion). When assayed
in NCR-nude-nude mice against the breast tumor xe-
nograft MDA-MB-435, 3 was found to have in vivo
efficacy comparable to that of CPT-11.32 Results from
an unpublished study revealed the secondary amine (M1
metabolite) resulting from N-dealkylation is a major
metabolite in Sprague-Dawley rats. In total, 46.5% of
3 is converted to the M1 metabolite, which reaches a
maximum plasma concentration (8.54 ng/mL) at 240

794 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Ruchelman et al.
Figure 2. Structures of 5-(2-aminoethyl)dibenzo[c,h][1,6]naphthyridin-6-one and variously N-alkyl substituted analogues
synthesized and evaluated for TOP1-targeting activity and cytotoxicity.
Scheme 1a
a Conditions: (a) 9a-b ClCH2CN, TEA, PhCH3; 9c-e, 9j, 9k ClCH2CN or BrCH2CN, NaI, K2CO3, CH3CN; (b) LiAlH4, THF.
well as the primary amine 6f were synthesized to
explore the sensitivity of steric bulk on efflux transport.
In addition, the effect of replacement of the 2-N,N-
dialkylamino substituent with pyrrolidine as well as
piperidine and piperazine derivatives (8a-f) was also
evaluated.
of phenol40 gave the 4-aminoquinolines 14a-14k. These
intermediates, upon reaction with 2-iodo-4,5-dimethoxy-
benzoyl chloride, yielded o-iodobenzamides 15a-k. Pal-
ladium-assisted biaryl coupling41-43 then provided the
dibenzo[c,h][1,6]naphthyridin-6-ones (5a-g, 8a-d, and
8f). Secondary amines 6a-6e and 8e were obtained by
debenzylation of the corresponding tertiary benzyl-
amines (Scheme 3). Deprotection proved refractory to
most reaction conditions tried, including catalytic hy-
drogenolysis over 10% Pd-C or Raney nickel using 70
psi of H2 gas at ambient temperature or with heating
as well as transfer hydrogenolysis using cyclohexadiene/
10% Pd-C, cyclohexene/20% Pd(OH) (Pearlman’s cata-
lyst), and ammonium formate/10% Pd-C. Benzyl groups
were removed in near-quantitative yield using pal-
ladium black in acetic acid with formic acid as the
hydrogen source.44 In the hydrogenation of the diben-
zylamino compound, allowing the reaction to proceed
for 90 min gave the primary amine 6f (85%). Alterna-
tively, quenching the reaction after 15 min gave pre-
dominantly the monobenzyl analogue 6e (54%). N-Me-
thyl- and N-ethyl-N-isopropyl-5-(2-aminoethyl)dibenzo-
[c,h][1,6]naphthyridin-6-ones, 7a and 7b, were obtained
by reductive alkylation of the secondary isopropylamine
6c with paraformaldehyde and acetaldehyde, respec-
tively (Scheme 4).
Chemistry
The synthetic methodology reported previously for the
synthesis of 3 was applicable to the new compounds
described herein. Dialkylaminoethylamines 11a-e, 1-(2-
aminoethyl)-4-methylipiperidine, 11j, and the 1-(2-amino-
ethyl)-4-benzylpiperazine, 11k, were not commercially
available and were synthesized as described previ-
ously,33-38 (Scheme 1). Alkylation of dialkylamines or
a nitrogen heterocycle 9a-e, 9j, and 9k with chloroac-
etonitrile or bromoacetonitrile provided intermediates
10a-e, 10j, and 10k, which were reduced to the
corresponding dialkylaminoethylamines. N,N-Diethyl-
ethylenediamine, 11f, N,N-diisopropylethylenediamine,
11g, 1-(2-aminoethyl)pyrrolidine, 11h, and 1-(2-amino-
ethyl)piperidine, 11i were available commercially.
The targeted 5-[2-(N,N-dialkylamino)ethyl]-5H-dibenzo-
[c,h][1,6]naphthyridin-6-ones were synthesized as shown
in Scheme 2. An improved synthesis of 4-hydroxy-6,7-
methylenedioxyquinoline, 12, recently developed in our
laboratory, involves treating the o-aminoacetophenone
with sodium hydride in ethyl formate. This process is
considerably simpler to execute than the previously
reported literature procedure39 and the yield is improved
(89% versus 41% over four steps starting from 3,4-
methylenedioxyaniline). Chlorination with phosphoryl
chloride provided 4-chloroquinoline, 13, which when
heated with ethylenediamines 11a-k in the presence
Results and Discussion
The relative TOP1-targeting activities of several 5-[(2-
monoalkylamino)ethyl]-dibenzo[c,h][1,6]naphthyridin-
6-ones 6a-e and 5-(2-aminoethyl)dibenzo[c,h][1,6]naph-
thyridin-6-one, 6f, are listed in Table 1. The pharma-
cological activities of ARC-111 3 and its N,N-diethyl
analogue 5f are also provided in Table 1. The DNA

N-Alkyl Substituent Modulation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 795
Scheme 2a
a Conditions: (a) NaH, ethyl formate; (b) POCl3, reflux; (c) refluxing phenol, then cool to 135 °C and add 11a-l; (d) 4,5-dimethoxy-2-
iodobenzoyl chloride, TEA, CH2Cl2; (e) Pd(OAc)2, P(o-tol)3, Ag2CO3, DMF.
Scheme 3a
a Conditions: (a) AcOH, formic acid, Pd black.
Scheme 4a
fragmentation pattern observed for those analogues that
possessed significant TOP1-targeting activity was simi-
lar to that observed with topotecan.31,32 The unsubsti-
tuted 2-aminoethyl analogue 6f exhibited the most
potent TOP1-targeting activity. With the exception of
the 2-(N-benzylamino)ethyl analogue 6e, the other
2-(monoalkylamino)ethyl analogues, 6a-d, had compa-
rable TOP1-targeting activity to 3 and 5f. The relative
CH3CHO for 7b.
TOP1-targeting activity of a series of N-isopropyl-N-
a Conditions: (a) NaCNBH3, EtOH, with (CH2O)n for 7a or
alkyl derivatives, 7a, 7b, and 5g in Table 2, revealed
that activity decreases with the increase in size of the
second alkyl group, i.e., CH3 > CH2CH3 > CH(CH3)2.
In a series of N-benzyl-N-alkyl derivatives, 5a-e, the

796 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Ruchelman et al.
Table 1. Topoisomerase I-Targeting Activity and Cytotoxicity of 5-[2-(Amino)ethyl)]-, 5-[2-(N-Alkylamino)ethyl)]-,
5-[2-(N,N-Dimethylamino)ethyl)]-, and 5-[2-(N,N-Diethylamino)ethyl)dibenzo[c,h][1,6]naphthyridin-6-ones
cytotoxicity IC50 (µM)b
TOP1-mediated
cleavagea
KB3-1
P388/CPT45 parent
KBV-1 KBH5.0
compd
R1
R2
RPMI8402 CPT-K5
P388
mdr-1
bcrp
6f
H
H
H
H
H
H
H
0.02
0.1
0.1
0.3
0.1
0.5
0.3
1.4
1.0
0.004
0.0003
0.002
0.003
0.002
0.016
0.002
0.006
0.021
0.25
0.25
0.17
0.18
0.22
0.28
0.900
0.85
> 10
0.003
0.0003
0.0007
0.001
0.002
0.016
0.001
0.004
0.045
0.027
0.07
0.03
0.03
0.037
0.18
0.23
0.15
0.005
0.0004
0.001
0.005
0.003
0.006
0.005
0.004
0.040
0.015
0.022
0.016
0.005
0.016
0.035
0.005
0.015
0.440
0.012
0.0005
0.005
0.006
0.006
0.010
0.006
0.005
0.440
6a
CH3
6b
CH2CH3
CH(CH3)2
C(CH3)3
CH2C6H5
CH3
6c
6d
6e
3
CH3
5f
CH2CH3
CH2CH3
topotecan
> 10
a Topoisomerase I cleavage values are reported as REC, relative effective concentrations, i.e., concentrations relative to topotecan,
whose value is arbitrarily assumed as 1, that are able to produce the same cleavage on the plasmid DNA in the presence of human
topoisomerase I. b See Experimental Section for details.
Table 2. Topoisomerase I-Targeting Activity and Cytotoxicity of 5-[2-(N-Alkyl-N-benzylamino)ethyl)]dibenzo[c,h][1,6]naphthyridin-
6-ones
cytotoxicity IC50 (µM)b
TOP1-mediated
cleavagea
KB3-1
parent
KBV-1
mdr-1
KBH5.0
bcrp
compd
R1
R2
CH3
CH2CH3
CH(CH3)2
CH3
CH2CH3
CH(CH3)2
C(CH3)3
CH2C6H5
RPMI 8402
CPT-K5
P388
P388/CPT45
7a
7b
5g
5a
5b
5c
5d
5e
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
0.03
0.17
4.7
15.8
12.3
7.4
0.008
0.005
0.008
0.053
0.077
0.30
0.63
0.31
0.60
2.0
7.5
5.9
0.006
0.004
0.006
0.038
0.05
0.071
0.12
0.20
0.45
0.36
0.65
1.85
0.50
0.003
0.003
0.005
0.05
0.005
0.021
0.04
0.30
0.40
4.0
0.004
0.008
0.04
0.15
0.33
0.5
0.18
0.23
0.80
>1000
15
0.85
0.38
5.75
1.95
0.20
0.19
2.5
0.36
0.5
0.30
0.25
0.30
a Topoisomerase I cleavage values are reported as REC, relative effective concentrations, i.e., concentrations relative to topotecan,
whose value is arbitrarily assumed as 1, that are able to produce the same cleavage on the plasmid DNA in the presence of human
topoisomerase I. b See Experimental Section for details.
presence of a second alkyl group significantly decreased
activity relative to 6e, with the N-benzyl-N-tert-butyl
analogue, 5d, not exhibiting any significant TOP1-
targeting activity. Table 3 summarizes the results
observed for several cyclic amines at the 2-position of
the 5-ethyl substituent within a series of dibenzo[c,h]-
[1,6]naphthyridin-6-ones. In the case of either a piperi-
dine or a piperazine moiety at this position, the presence
of a 4-methyl group on these heterocycles resulted in
decreased cytotoxic activity, i.e., 8c and 8e relative to
8b and 8f, respectively. In the case of 8e and 8f, there
was also a precipitous drop in TOP1-targeting activity.
This observation is consistent with the difference in
activity observed between 8d and 8e and suggests that
an unfavorable steric interaction may, in general, be
associated with substitution at the 4-position of the
2-piperazine moiety.
respectively. In the case of CPT-K5, resistance to
camptothecin is associated with a mutant form of
topoisomerase I.45 The camptothecin resistance of P388/
CPT45 is associated with the decreased expression of
TOP1 in this variant cell line.46 The cytotoxicity data
summarized in Table 1 reflects a good correlation
between relative TOP1-targeting activity and cytotox-
icity. The most cytotoxic analogue in this table is 6a and
the least is 6e. As would be anticipated for compounds
that primarily exert their cytotoxicity by stabilizing
TOP1-DNA cleavable complexes, all of these analogues
were less active in the camptothecin-resistant variants
CPT-K5 or P388/CPT45 as compared to their parent cell
lines.
The cytotoxicity data for KB3-1 cells and for the
variants KB/V-1 and KBH5.0 are also listed in Tables
1-3. KB/V-1 cells overexpress the efflux transporter
47
Tables 1-3 also list the relative cytotoxicity in
RPMI8402 and P388 cell lines as well as their camp-
tothecin-resistant variants CPT-K5 and P388/CPT45,
MDR1,
and KBH5.0 cells overexpress the efflux
transporter BCRP.48 The decreased cytotoxicity against
KB/V-1 and KBH5.0 cells observed with CPT-11 and

N-Alkyl Substituent Modulation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 797
Table 3. Topoisomerase I-Targeting Activity and Cytotoxicity of 5-[2-Pyrrolidin-1-yl]ethyl]-, 5-[2-Piperidin-1-yl]-, and
5-[2-Piperazin-1-yl]ethyl]dibenzo[c,h][1,6]naphthyridin-6-ones
a Topoisomerase I cleavage values are reported as REC, relative effective concentrations, i.e., concentrations relative to topotecan,
whose value is arbitrarily assumed as 1, that are able to produce the same cleavage on the plasmid DNA in the presence of human
topoisomerase I. b See Experimental Section for details.
topotecan relative to KB3-1 cells is indicative of drugs
that are substrates for both MDR1 and BCRP efflux
transporters. Among the dibenzo[c,h][1,6]naphthyridin-
6-ones in Table 1, the absence of any significant differ-
ence in the cytotoxicity observed in KB3-1, KBV-1, and
KBH5.0 cells for 3, 5f, and 6c-f indicates that these
analogues are not substrates for either MDR1 or BCRP
efflux transporters. In the case of 6a and 6b, there were
significant differences (>10-fold) in their relative cyto-
toxicity between KB3-1 and KBV-1 cell lines, indicating
that these specific analogues are substrates for the
efflux transporter, MDR1. The absence of a significant
difference in the cytotoxicity of 6a and 6b in KBV-1 and
KBH5.0 cells, however, indicates that these analogues
are not substrates for BCRP.
The only compounds in Table 2 that were cytotoxic
below 10 nM in RPMI8402 and P388 cells were 7a, 7b,
and 5g. This is consistent with their greater TOP1-
targeting activity relative to 5a-e. As indicated by the
data in Table 2, as relative TOP1-targeting activity
decreases, the differences in cytotoxicity in camptoth-
ecin-sensitive and camptothecin-resistant cell lines are
also reduced. For 5d and 5e, it is likely that mechanisms
other than targeting TOP1 contribute to their cytotox-
icity. In the case of 5g, the data do suggest that as steric
bulk or lipophilicity are increased these compounds may
tend toward becoming substrates for MDR1 and BCRP
efflux transporters. Among the cyclic amines listed in
Table 3, it is again evident that cytotoxicity of individual
analogues correlates with its relative potency as a
TOP1-targeting drug. Although both 8e and 8f are not
potent TOP1-targeting agents, it is evident that the
presence of a second basic nitrogen atom within these
heterocycles is associated with their recognition as
substrates by the MDR1 efflux transporter based upon
their decreased cytotoxicity in KBV-1 relative to KB3-1
cells.
It is anticipated that both 6a, a known major me-
tabolite of ARC-111, and 6f contribute to the antitumor
activity observed with ARC-111. Although 6a is a
substrate for MDR1, 6f is not. To assess the relative
potency of these N-demthylated analogues of ARC-111,
the antitumor activity of these compounds was evalu-
ated against SJ-BT45 medulloblastoma xenografts in
scid mice. The results of this study are illustrated in
Figure 3.
Both 3 and 6a had significant antitumor activity
when administered at a dose of 9 mg per week. It is
evident that the primary amine 6f was significantly less
potent in vivo than either ARC-111 or its monodem-
ethylated metabolite, 6a, at this dose. This is likely
associated with an increased metabolic instability as-
sociated with 6f. These data suggest that 6a could
contribute to a greater extent to the overall antitumor
activity observed with ARC-111, especially in tumors
that do not overexpress MDR1.
In light of the data summarized in Tables 1 and 2,
analogues such as 5f, 7a, and 7b may not readily
undergo N-dealkylation to form active metabolites that
would be substrates for MDR1. To the degree that
N-dealkylation may occur in the case of 7a and 7b, one
would most likely observe the N-isopropyl analogue 6c,
which is not a substrate for MDR1, as a primary
metabolite. Such analogues may represent a potential
therapeutic advantage relative to ARC-111 in those
instances where the ability of one of its major metabo-
lites to serve as a substrate for the MDR1 efflux
transporter has an adverse impact on overall antitumor
activity.
Experimental Section
Melting points were determined with a Meltemp capillary
melting point apparatus. Column chromatography refers to
flash chromatography conducted on SiliTech 32-63 µm (ICN
Biomedicals, Eschwege, Germany) using the solvent systems

798 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Ruchelman et al.
Figure 3. Comparison of the antitumor activity against SJ-BT45 medulloblastoma xenografts in scid mice. (A) Vehicle-treated
control (10 mice). (B) Compound 3, 3 mg/(kg/dose) (10 mice). (C) Compound 3, 2 mg/(kg/dose) (5 mice). (D) Compound 6f, 3 mg/
(kg/dose) (10 mice). (E) Compound 6a, 3 mg/(kg/dose) (5 mice). (F) Compound 6a, 2 mg/(kg/dose) (5 mice). Each compound was
administered by intravenous injection 3 times per week (Monday, Wednesday, Friday) for 2 consecutive weeks. The cycle of therapy
was repeated at 21 days for a total of two cycles. Each curve shows the growth of an individual tumor in a female scid mouse.
indicated. Proton (1H NMR) and carbon (13C NMR) nuclear
magnetic resonance were recorded on a Varian Gemini-200
2H), 7.20 (m, 5H), 7.45 (s, 1H), 7.67 (s, 1H), 7.86 (s, 1H), 7.89
(s, 1H), 9.36 (s, 1H).13C NMR (CDCl3) δ: 42.4, 49.2, 56.0, 56.3,
56.4, 63.1, 101.4, 101.9, 102.2, 107.1, 108.8, 111.7, 114.9, 119.3,
127.1, 127.7, 128.2, 129.0, 138.8, 141.0, 143.5, 147.3, 147.7,
149.9, 150.2, 154.1, 164.1. HRMS Calcd for C29H27O5N3H:
498.2029. Found: 498.2005. Anal. (C29H27O5N3) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-benzyl-N-
ethylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-
one (5b) was prepared from 15b (639 mg, 1.0 mmol), reaction
time 30 min. Chromatographic purification using chloroform
gave 204 mg (40%) as a white solid: mp 237.5-238.5 °C. IR
(CHCl3): 1648. 1H NMR (CDCl3) δ: 0.92 (t, 3H, J ) 7.1), 2.46
(q, 2H, J ) 7.1), 3.09 (t, 2H, J ) 6.4), 3.57 (s, 2H), 4.07 (s,
3H), 4.15 (s, 3H), 4.67 (t, 2H, J ) 6.4), 6.21 (s, 2H), 7.17 (m,
5H), 7.47 (s, 1H), 7.69 (s, 1H), 7.88 (s, 1H), 7.90 (s, 1H), 9.38
(s, 1H). 13C NMR (CDCl3) δ: 11.6, 47.9, 49.4, 52.3, 56.3, 56.4,
58.9, 101.4, 101.9, 102.2, 107.0, 108.9, 111.8, 115.0, 119.4,
126.9, 127.7, 128.1, 128.8, 139.3, 141.1, 143.5, 147.2, 147.7,
149.9, 150.2, 154.1, 164.1. HRMS Calcd for C30H29N3O5H:
512.2185. Found: 512.2195. Anal. (C30H29N3O5‚0.5H2O) C, H,
N.
1
Fourier transform spectrometer. NMR spectra (200 MHz H
and 50 MHz 13C) were recorded in the deuterated solvent
indicated with chemical shifts reported in δ units downfield
from tetramethylsilane (TMS). Coupling constants are re-
ported in hertz (Hz). Mass spectra were obtained from Wash-
ington University Resource for Biomedical and Bioorganic
Mass Spectrometry within the Department of Chemistry at
Washington University, St. Louis, MO. Elemental analyses
were obtained from Atlantic Microlabs, Norcross, GA, and were
within 0.4% of theory. All starting materials and reagents were
purchased from Aldrich. Solvents were purchased from Fisher
Scientific and were ACS or HPLC grade. Methylene chloride
was freshly distilled from calcium hydride. All other solvents
were used as provided without further purification. The
preparation of compounds 3, 8a, and 8f have been reported.31,32
Compounds 11a,33 11b,34 11c,35 11d,36 11e,34 11j,37 and 11k38
were synthesized as previously described. N,N-Diethylethyl-
enediamine, 11f, N,N-diisopropylethylenediamine, 11g, 1-(2-
aminoethyl)pyrrolidine, 11h, and 1-(2-aminoethyl)piperidine,
11i, were commercially available from Aldrich.
General Procedure for the Preparation of 5-[2-(N,N-
Dialkylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-
6-ones 5a-5g and the Piperaidinyl and Piperazinyl
Derivatives 8b-e. 8,9-Dimethoxy-2,3-methylenedioxy-5-
[2-(N-benzyl-N-methylamino)ethyl]-5H-dibenzo[c,h]-
[1,6]naphthyridin-6-one (5a). A mixture of 15a (720 mg, 1.2
mmol), Pd(OAc)2 (54 mg, 0.24 mmol), P(o-tolyl)3 (147 mg, 0.48
mmol), and Ag2CO3 (660 mg, 2.4 mmol) in dimethylformamide
(DMF) (36 mL) was heated to reflux with stirring for 30 min.
The reaction mixture was cooled, diluted with chloroform, and
filtered through Celite, and the solvent was removed under
vacuum. The crude residue was chromatographed in 99:1
chloroform/methanol to provide 210 mg (35%) of the cyclized
product as a white solid: mp 224-226 °C. IR (CHCl3): 1648.
1H NMR (CDCl3) δ: 2.19 (s, 3H), 3.06 (t, 2H, J ) 6.4), 3.51 (s,
2H), 4.06 (s, 3H), 4.14 (s, 3H), 4.67 (t, 2H, J ) 6.9), 6.21 (s,
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-benzyl-N-
isopropylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-
6-one (5c) was prepared from 15c (653 mg, 1.0 mmol), reaction
time 30 min. Chromatographic purification eluting with
chloroform gave 225 mg (43%) as a white solid: mp 218.5-
1
220.0 °C. IR (CHCl3): 1647. H NMR (CDCl3) δ: 0.91 (d, 6H,
J ) 6.7), 2.80 (m, 1H), 3.08 (t, 2H, J ) 6.5), 3.58 (s, 2H), 4.06
(s, 3H), 4.14 (s, 3H), 4.56 (t, 2H, J ) 6.5), 6.19 (s, 2H), 7.12
(m, 5H), 7.45 (s, 1H), 7.67 (s, 1H), 7.81 (s, 1H), 7.86 (s, 1H),
9.36 (s, 1H). 13C NMR (CDCl3) δ: 17.7, 48.5, 50.1, 50.6, 55.0,
56.3, 56.4, 101.4, 102.0, 102.1, 107.0, 108.9, 111.8, 114.9, 119.5,
126.6, 127.7, 128.1, 128.4, 140.4, 141.1, 143.4, 147.3, 147.5,
149.8, 150.2, 154.1, 164.0. HRMS Calcd for C31H31N3O5Li:
532.2424. Found: 532.2402. Anal. (C31H31N3O5‚0.5H2O) C, H,
N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-benzyl-N-
tert-butylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-

N-Alkyl Substituent Modulation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 799
6-one (5d) was prepared from 15d (500 mg, 0.75 mmol),
reaction time 30 min. Chromatographic purification eluting
with chloroform gave 223 mg (55%) as a white solid: mp 212-
(t, 2H, J ) 6.6), 6.19 (s, 2H), 7.45 (s, 1H), 7.67 (s, 1H), 7.89 (s,
1H), 8.00 (s, 1H), 9.36 (s, 1H). 13C NMR (CDCl3) δ: 22.0, 30.6,
34.2, 49.0, 54.3, 56.3, 56.4, 57.0, 101.5, 101.9, 102.2, 107.0,
108.8, 111.8, 114.9, 119.4, 127.7, 141.1, 146.4, 147.2, 147.7,
149.9, 150.2, 154.1, 164.3. IR (CHCl3): 1648. HRMS Calcd for
C27H29N3O5H: 476.2185. Found: 476.217. Anal. (C27H32O5N3‚
0.5H2O) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(4-benzylpip-
erazinyl)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-
one (8d) was prepared from 15k (1.02 g, 1.5 mmol), reaction
time 30 min. Chromatographic purification eluting with 99:1
chloroform/methanol gave 365 mg (44%) of a colorless crystal-
line solid: mp 230.5-231 °C. 1H NMR (CDCl3) δ: 2.34 (t, 4H,
J ) 4.4), 2.49 (t, 4H, J ) 4.4,), 2.96 (t, 2H, J ) 6.6), 3.46 (s,
2H), 4.07 (s, 3H), 4.14 (s, 3H), 4.65 (t, 2H, J ) 6.6), 6.17 (s,
2H), 7.30 (m, 5H), 7.45 (s, 1H), 7.67 (s, 1H), 7.88 (s, 1H), 7.93
(s, 1H), 9.35 (s, 1H). 13C NMR (CDCl3) δ: 48.8, 53.1, 53.4, 56.3,
56.4, 56.6, 63.1, 101.3, 101.9, 102.2, 107.2, 108.9, 111.9, 115.0,
119.5, 127.1, 127.6, 128.3, 129.2, 138.2, 141.1, 143.4, 147.2,
147.7, 149.9, 150.3, 154.1, 164.3. IR (CHCl3): 1649. HRMS
Calcd for C32H32N4O5H: 553.2451. Found: 553.2443. Anal.
(C26H32O5N4) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(piperazinyl)-
ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-one (8e). For-
mic acid (1.2 mL) and then palladium black (25 mg) were
added to a solution of 8d (50 mg, 0.074 mmol) in acetic acid
(30 mL). The mixture was stirred at room temperature for 10
h, then filtered, and evaporated under vacuum. The residue
was partitioned between CHCl3 (50 mL) and 10% NaOH (50
mL), and the aqueous phase was extracted with additional
CHCl3 (2 × 25 mL). The combined organic phases were washed
with water (2 × 75 mL) and brine (75 mL), dried (MgSO4),
and evaporated under vacuum, yielding 27 mg (79%) of a white
solid: mp 229-230 °C. 1H NMR (CDCl3) δ: 1.75 (1H, br), 2.40
(t, J ) 4.6, 4H), 2.73 (t, J ) 4.6, 4H), 2.93 (t, J ) 6.4, 2H),
4.07 (s, 3H), 4.14 (s, 3H), 4.67 (t, J ) 6.4, 2H), 6.19 (s, 2H),
7.46 (s, 1H), 7.68 (s, 1H), 7.89 (s, 1H), 7.99 (s, 1H), 9.37 (s,
1H). 13C NMR (CDCl3) δ: 46.1, 48.7, 54.7, 56.3, 56.4, 57.2,
101.3, 101.9, 102.2, 107.0, 108.8, 111.9, 115.0, 119.4, 127.6,
141.2, 143.4, 147.2, 147.7, 149.9, 150.2, 154.1, 164.4. IR
(CHCl3): 1648. HRMS Calcd for C25H25N4O5H: 463.1981.
Found: 463.1962. Anal. (C25H26O5N4‚0.5CHCl3) C, H, N.
General Procedure for the Preparation of 5-[2-(N-
Alkylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-
ones 6a-f. 8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-
methylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-
6-one (6a). Compound 5a (100 mg, 0.02 mmol) was dissolved
in acetic acid (60 mL). Formic acid (2.4 mL) was added, and
then palladium black (50 mg) was added. The mixture was
stirred at room temperature for 90 min, and was then filtered
through Celite, and the filtrate was evaporated under vacuum.
The residue was partitioned between CHCl3 (75 mL) and 10%
NaOH (75 mL), and the aqueous phase was extracted with
additional CHCl3 (2 × 30 mL). The combined organic phases
were evaporated under vacuum, and the residue was chro-
matographed through a short column of silica eluting with 97:3
chloroform/methanol, yielding 78 mg of a light yellow solid,
in 95% yield: mp 254-257 °C (dec). IR (CHCl3): 3330, 1648.
1H NMR (CDCl3) δ: 2.50 (s, 3H), 3.30 (t, 2H, J ) 6.6), 4.07 (s,
3H), 4.14 (s, 3H), 4.61 (t, 2H, J ) 6.6), 6.18 (s, 2H), 7.47 (s,
1H), 7.70 (s, 2H), 7.89 (s, 1H), 9.38 (s, 1H). 13C NMR (CDCl3)
δ: 36.5, 50.6, 51.2, 56.3, 56.4, 100.8, 102.0, 102.2, 107.2, 108.7,
111.7, 114.8, 119.3, 127.7, 140.9, 143.5, 147.3, 147.6, 150.0,
150.3, 154.2, 164.3. HRMS Calcd for C22H21N3O5H: 408.1559.
Found: 408.1550. Anal. (C22H21O5N3‚0.5H2O) C, H, N.
1
213 °C. IR (CHCl3): 1648. H NMR (CDCl3) δ: 1.21 (s, 9H),
3.20 (t, 2H, J ) 7.2), 3.78 (s, 2H), 4.06 (s, 3H), 4.12 (s, 3H),
4.25 (t, 2H, J ) 6.5), 6.22 (s, 2H), 7.13 (m, 5H), 7.44 (s, 1H),
7.65 (s, 1H), 7.81 (s, 1H), 7.86 (s, 1H), 9.32 (s, 1H). 13C NMR
(CDCl3) δ: 27.2, 50.0, 51.9, 55.1, 55.7, 56.3, 56.4, 101.6, 102.0,
102.1, 106.9, 108.9, 111.6, 114.8, 119.5, 126.2, 127.6, 127.8,
127.9, 140.7, 142.5, 143.2, 147.2, 147.4, 149.9, 150.2, 154.1,
163.9. HRMS Calcd for C32H33N3O5H: 540.2498. Found:
540.2492. Anal. (C32H33N3O5) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N,N-dibenzyl-
amino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-one (5e)
was prepared from 15e (701 mg, 1.0 mmol), reaction time 30
min. Chromatographic purification eluting with chloroform
gave 361 mg (63%) as a white solid: mp 213-215 °C. IR
(CHCl3): 1647. 1H NMR (CDCl3) δ: 3.06 (t, 2H, J ) 5.9), 3.49
(s, 4H), 4.05 (s, 3H), 4.14 (s, 3H), 4.68 (t, 2H, J ) 5.9), 6.23 (s,
2H), 7.15 (m, 10H), 7.46 (s, 1H), 7.66 (s, 1H), 7.72 (s, 1H), 7.79
(s, 1H), 9.35 (s, 1H). 13C NMR (CDCl3) δ: 49.0, 53.1, 56.3, 56.4,
59.2, 101.3, 102.0, 102.3, 106.6, 109.0, 111.8, 114.8, 119.4,
127.0, 127.5, 128.2, 128.9, 138.7, 141.2, 142.9, 146.6, 147.7,
150.0, 150.4, 154.2, 163.8. HRMS Calcd for C35H31N3O5H:
574.2342. Found: 574.2314. Anal. (C35H31N3O5‚0.5H2O) C, H,
N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N,N-diethyl-
amino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-one (5f)
was prepared from 15f (577 mg, 1.0 mmol), reaction time 30
min. Chromatographic purification eluting with 99:1 chloroform/
methanol gave 250 mg (56%) as a white solid: mp 221-223
°C (dec). IR (CHCl3): 1648. 1H NMR (CDCl3) δ: 0.95 (t, 6H, J
) 7.0), 2.80 (q, 4H, J ) 7.0), 3.04 (t, 2H, J ) 6.7), 4.06 (s, 3H),
4.13 (s, 3H), 4.63 (t, 2H, J ) 6.7), 6.17 (s, 2H), 7.46 (s, 1H),
7.68 (s, 1H), 7.90 (s, 1H), 7.96 (s, 1H), 9.37 (s, 1H). 13C NMR
(CDCl3) δ: 12.0, 47.6, 49.6, 51.7, 56.3, 101.4, 102.0, 102.2,
107.0, 108.9, 111.8, 115.0, 119.5, 127.7, 141.1, 143.5, 147.3,
147.7, 149.9, 150.3, 154.2, 164.2. HRMS Calcd for C25H27-
O5N3H: 450.2029. Found: 450.2032. Anal. (C25H27O5N3) C, H,
N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N,N-diisopro-
pylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-
one (5g) was prepared from 15g (605 mg, 1.0 mmol), reaction
time 35 min. Chromatographic purification eluting with 98:2
chloroform/methanol gave 240 mg (50%) as a light beige
solid: mp 257-260 °C (dec). IR (CHCl3): 1650. 1H NMR
(CDCl3) δ: 0.88 (d, 12H, J ) 6.2), 2.94 (m, 4H), 4.07 (s, 3H),
4.14 (s, 3H), 4.61 (t, 2H, J ) 6.7), 6.18 (s, 2H), 7.46 (s, 1H),
7.68 (s, 1H), 7.91 (s, 1H), 7.96 (s, 1H), 9.37 (s, 1H). 13C NMR
(CDCl3) δ: 20.7, 43.8, 48.4, 51.0, 56.3, 56.4, 101.6, 101.9, 102.2,
107.0, 108.9, 111.9, 114.9, 119.6, 127.7, 141.2, 143.4, 147.2,
147.5, 149.9, 150.2, 154.1, 164.2. HRMS Calcd for C27H31O5N3-
Li: 484.2424. Found: 484.2438. Anal. (C27H31O5N3) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(piperidinyl)-
ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-one (8b) was
prepared from 15i (294.5 mg, 0.5 mmol), reaction time 25 min.
Chromatograpic purification with 99:1 chloroform/methanol
1
gave 82 mg (36%) of a light beige solid: mp 269-270 °C. H
NMR (CDCl3) δ: 1.41 (t, 6H, J ) 7.4), 2.38 (t, 4H, J ) 5.4),
2.92 (t, 2H, J ) 6.6), 4.06 (s, 3H), 4.13 (s, 3H), 4.65 (t, 2H, J
) 6.6), 6.18 (s,2H), 7.46 (s,1H), 7.68 (s,1H), 7.89 (s,1H), 8.04
(s,1H), 9.36 (s,1H). 13C NMR (CDCl3) δ: 24.3, 26.0, 49.0, 54.8,
56.3, 57.4, 101.5, 101.9, 102.1, 106.9, 108.9, 111.8, 115.0, 119.5,
127.6, 141.2, 143.4, 147.2, 147.6, 149.8, 150.2, 154.1, 164.3.
HRMS Calcd for C26H28N3O5H: 462.2029. Found: 462.2029.
Anal. (C26H27O5N3‚0.5H2O) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-ethylamino)-
ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-one (6b) was
prepared from 5b (20 mg, 0.04 mmol), reaction time 90 min.
Short column chromatographic purification of the crude prod-
uct using 97:3 chloroform/methanol gave 14 mg (85%) as a
white solid: mp 245-247 °C (dec). IR (CHCl3): 3308, 1647.
1H NMR (CDCl3) δ: 1.14 (t, 3H, J ) 7.0), 2.06 (br, 1H), 2.75
(q, 2H, J ) 7.1), 3.35 (t, 2H, J ) 6.4), 4.07 (s, 3H), 4.14 (s,
3H), 4.59 (t, 2H, J ) 6.4), 6.18 (s, 2H), 7.46 (s, 1H), 7.68 (s,
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(4-methylpip-
eridin-1-yl)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-
one (8c) was prepared from 15j (603 mg, 1.0 mmol), reaction
time 45 min. Chromatograpic purification with 99:1 chloroform/
methanol gave 166 mg (34%) of a colorless crystalline solid:
mp 247.0-248.5 °C. 1H NMR (CDCl3) δ: 0.89 (d, 3H, J ) 5.8),
1.12 (m, 3H), 1.55 (d, 2H, J ) 11.0), 2.08 (m, 2H), 2.82 (d, 2H,
J ) 11.0), 2.96 (t, 2H, J ) 6.6), 4.06 (s, 3H), 4.13 (s, 3H), 4.66

800 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Ruchelman et al.
1H), 7.70 (s, 1H), 7.89 (s, 1H), 9.37 (s, 1H). 13C NMR (CDCl3)
δ: 15.3, 44.0, 48.8, 50.8, 56.3, 56.4, 100.9, 102.0, 102.2, 107.2,
108.8, 111.7, 114.8, 119.3, 127.7, 140.9, 143.5, 147.4, 147.7,
149.9, 150.4, 154.3, 164.3. HRMS Calcd for C23H23N3O5H:
422.1716. Found: 422.1705. Anal. (C23H23N3O5) C, H, N.
6.6), 2.27 (s, 3H), 2.80 (m, 1H), 3.06 (t, 2H, J ) 6.9), 4.06 (s,
3H), 4.13 (s, 3H), 4.60 (t, 2H, J ) 6.9), 6.18 (s, 2H), 7.47 (s,
1H), 7.69 (s, 1H), 7.91 (s, 1H), 7.99 (s, 1H), 9.38 (s, 1H). 13C
NMR (CDCl3) δ: 18.0, 37.5, 49.9, 51.7, 54.6, 56.3, 56.4, 101.5,
102.0, 102.2, 107.0, 108.9, 111.7, 115.0, 119.5, 127.7, 141.2,
143.5, 147.3, 147.7, 149.9, 150.3, 154.2, 164.2. HRMS Calcd
for C25H27O5N3Li: 456.2111. Found: 456.2106. Anal. (C25H27-
O5N3‚0.5H2O) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-isopropy-
lamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-one (6c)
was prepared from 5c (30 mg, 0.06 mmol), reaction time 90
min. Short column chromatographic purification of the crude
product using 97:3 chloroform/methanol gave 23 mg (94%) as
a white solid: mp 275-278 °C (dec). IR (CHCl3): 3306, 1647.
1H NMR (CDCl3) δ: 1.16 (d, 6H, J ) 6.2), 2.96 (m, 1H), 3.40
(t, 2H, J ) 6.4), 4.07 (s, 3H), 4.14 (s, 3H), 4.58 (t, 2H, J ) 6.4),
4.79 (br, 1H), 6.18 (s, 2H), 7.47 (s, 1H), 7.66 (s, 1H), 7.69 (s,
1H), 7.89 (s, 1H), 9.39 (s, 1H). 13C NMR (CDCl3) δ: 22.6, 46.3,
48.8, 50.9, 56.3, 56.4, 100.9, 102.1, 102.2, 107.1, 108.9, 111.7,
114.8, 119.2, 127.7, 141.0, 143.4, 147.3, 147.8, 150.0, 150.4,
154.4, 164.4. HRMS Calcd for C24H25N3O5H: 436.1872.
Found: 436.1855. Anal. (C24H25N3O5) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-ethyl-N-iso-
propylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-
6-one (7b). A mixture of 6c (44 mg, 0.1 mmol), acetaldehyde
(44 mg, 1.0 mmol), and sodium cyanoborohydride (21 mg, 0.3
mmol) in ethanol (15 mL) was heated to 75 °C in a sealed tube
for 6 h. The mixture was filtered, and the filtrate was
evaporated. The residue was partitioned between chloroform
(30 mL) and 10% NaOH (30 mL), and the aqueous phase was
extracted with chloroform (2 × 30 mL). The combined organic
phases were evaporated and chromatographed through a short
column of silica eluting with in 97:3 chloroform/methanol to
provide 33 mg (76%) as a white solid: mp 223-225 °C. IR
(CHCl3): 1647. 1H NMR (CDCl3) δ: 0.91 (d, 6H, J ) 6.2), 1.20
(m, 3H), 2.46 (m, 2H), 2.84 (m, 1H), 3.01 (t, 2H, J ) 6.6), 4.07
(s, 3H), 4.14 (s, 3H), 4.63 (t, 2H, J ) 6.6), 6.18 (s, 2H), 7.47 (s,
1H), 7.69 (s, 1H), 7.91 (s, 1H), 7.99 (s, 1H), 9.38 (s, 1H). 13C
NMR (CDCl3) δ: 13.9, 18.1, 44.2, 48.0, 48.2, 50.4, 56.3, 56.4,
101.5, 102.0, 102.2, 107.0, 109.0, 111.9, 115.0, 119.6, 127.7,
141.2, 143.5, 147.3, 147.7, 149.9, 150.3, 154.2, 164.3. HRMS
Calcd for C26H29O5N3Li: 470.2267. Found: 470.2277. Anal.
(C26H29O5N3‚0.5H2O) C, H, N.
6,7-Methylenedioxy-4-quinolone (12). To a mixture of
6′amino-3′4′-methylenedioxyacetophenone (16.0 g, 89.4 mmol)
in ethyl formate (300 mL) in a 1 L flask equipped with a
condenser was added sodium hydride (14.3 g, 0.358 mol). After
a short induction period, the mixture began to react vigorously
and soon began refluxing. The mixture was stirred for an
additional 30 min and was then cooled to 0 °C. The reaction
was quenched by the addition of water (3 mL) and then acetic
acid (5 mL). The solid material that had precipitated during
the reaction was filtered and washed with water, ethanol, ethyl
acetate, and ethyl ether, consecutively. Drying under high
vacuum provided 15.0 g of an off-white solid in 89% yield: mp
285-289 °C (lit.36 mp 276 °C). 1H NMR (DMSO-d6) δ: 5.95 (d,
1H, J ) 7.3), 6.13 (s, 2H), 6.97 (s, 1H), 7.38 (s, 1H), 7.77 (d,
1H, J ) 7.3). 13C NMR (DMSO-d6) δ: 97.5, 102.1, 102.6, 108.7,
119.4, 122.0, 130.8, 138.7, 145.8, 151.7.
4-Chloro-6,7-methylenedioxyquinoline (13). Compound
12 (5.0 g, 26.5 mmol) was boiled in POCl3 (75 mL) for 2 h and
then cooled. Excess phospohoryl chloride was removed under
reduced pressure, and ice-water (100 mL) was added to
hydrolyze any residual phosphorus oxychloride. The mixture
was basified (pH 9) with ammonium hydroxide, and the solid
precipitate was filtered. This material was dissolved in chlo-
roform (250 mL), washed with water (3 × 250 mL), dried
(MgSO4), and evaporated to provide 4.98 g of a white solid in
91% yield: mp 127.5-128 °C (lit.36 mp 129 °C). 1H NMR
(CDCl3) δ: 6.15 (s, 2H), 7.35 (d, 1H, J ) 4.7), 7.39 (s, 1H),
7.49 (s, 1H), 8.56 (d, 1H, J ) 4.7). 13C NMR (CDCl3) δ: 99.8,
102.2, 106.1, 119.9, 123.7, 129.8, 141.2, 147.7, 149.1, 151.4.
General Procedure for the Preparation of 4-[[2-(Di-
alkylamino)ethyl]amino]-6,7-methylenedioxyquino-
lines 14a-k. 4-[[2-(N-Benzyl-N-methylamino)ethyl]amino]-
6,7-methylenedioxyquinoline (14a). Compound 13 (1.0 g,
4.83 mmol) was stirred in refluxing phenol for 2.5 h. The bath
temperature was lowered to 128° C, and 11a (1.7 g, 9.66 mmol)
was added. The mixture was stirred at this temperature for
20h, then phenol was removed by Kugelrohr distillation, and
the resulting crude residue was partitioned between chloro-
form (150 mL) and 10% NaOH (100 mL). The organic phase
was washed with water (3 × 100 mL) and was then extracted
with dilute aqueous HCl (3 × 100 mL). The combined aqueous
layers were washed with chloroform (2 × 100 mL), basified
(30% NaOH), and back-extracted into chloroform (3 × 100 mL).
The combined organic layers were dried (MgSO4) and evapo-
rated, and the residue was triturated with ethyl ether and
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-tert-buty-
lamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-one
(6d) was prepared from 5d (30 mg, 0.06 mmol), reaction time
90 min. Short column chromatographic purification of the
crude product using 97:3 chloroform/methanol gave 22 mg
(88%) as a white solid: mp 258-260 °C. IR (CHCl3): 3298,
1
1650. H NMR (CDCl3) δ: 1.19 (s, 9H), 3.36 (t, 2H, J ) 6.7),
4.07 (s, 3H), 4.14 (s, 3H), 4.54 (t, 2H, J ) 6.7), 6.18 (s, 2H),
7.47 (s, 1H), 7.67 (s, 1H), 7.77 (s, 1H), 7.90 (s, 1H), 9.40 (s,
1H). 13C NMR (CDCl3) δ: 28.9, 42.0, 51.0, 51.5, 56.3, 56.4,
101.2, 102.0, 102.2, 107.1, 108.9, 111.6, 114.8, 119.2, 127.8,
141.0, 143.5, 147.4, 147.8, 149.9, 150.4, 154.3, 164.3. HRMS
Calcd for C25H27N3O5H: 450.2029. Found: 450.2027. Anal.
(C25H27N3O5‚0.5H2O) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-benzylami-
no)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-6-one (6e)
was prepared from 5e (100 mg, 0.17 mmol), reaction time 15
min. Chromatographic purification of the crude product eluting
with 97:3 chloroform/methanol gave 46 mg (54%) as a pale
yellow solid: mp 244-247 °C. IR (CHCl3): 3307, 1648. 1H
NMR (CDCl3) δ: 3.33 (t, 2H, J ) 6.5), 3.84 (s, 2H), 4.07 (s,
3H), 4.14 (s, 3H), 4.64 (t, 2H, J ) 6.5), 6.20 (s, 2H), 7.28 (s,
5H), 7.47 (s, 1H), 7.69 (s, 1H), 7.76 (s, 1H), 7.88 (s, 1H), 9.38
(s, 1H). 13C NMR (CDCl3) δ: 48.4, 50.7, 53.9, 56.3, 56.4, 101.1,
102.0, 102.2, 107.2, 108.9, 111.8, 114.9, 119.3, 127.1, 127.7,
128.2, 128.4, 140.0, 141.0, 143.5, 147.3, 147.7, 149.9, 150.3,
154.3, 164.3. HRMS Calcd for C28H25N3O5H: 484.1872.
Found: 484.1854. Anal. (C28H25N3O5) C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-(2-aminoethyl)-
5H-dibenzo[c,h][1,6]naphthyridin-6-one (6f) was prepared
from 5e (200 mg, 0.35 mmol), reaction time 90 min. Short
column chromatographic purification of the crude product
using 96:4 chloroform/methanol gave 110 mg (80%) as a white
solid: mp 298-299 °C (dec). IR (CHCl3): 3391, 1649. 1H NMR
(CDCl3) δ: 1.48 (br, 2H), 3.38 (t, 2H, J ) 6.6), 4.07 (s, 3H),
4.14 (s, 3H), 4.58 (t, 2H, J ) 6.6), 6.18 (s, 2H), 7.47 (s, 1H),
7.65 (s, 1H), 7.69 (s, 1H), 7.90 (s, 1H), 9.38 (s, 1H). 13C NMR
(DMSO-d6) δ: 40.7, 52.4, 56.4, 57.1, 101.7, 103.1, 104.2, 107.0,
109.0, 112.1, 114.9, 119.2, 128.0, 140.9, 145.0, 147.5, 147.8,
150.3, 150.6, 154.8, 163.8. HRMS Calcd for C21H19-
N3O5H: 394.1403. Found: 394.1402; Anal. (C21H19N3O5‚H2O)
C, H, N.
8,9-Dimethoxy-2,3-methylenedioxy-5-[2-(N-isopropyl-
N-methylamino)ethyl]-5H-dibenzo[c,h][1,6]naphthyridin-
6-one (7a). A mixture of 6c (44 mg, 0.1 mmol), paraformal-
dehyde (29 mg), and sodium cyanoborohydride (21 mg, 0.3
mmol) in ethanol (35 mL) was heated to reflux under nitrogen
for 20 h. The mixture was filtered, and the filtrate was
evaporated. The residue was partitioned between chloroform
(30 mL) and 10% NaOH (30 mL), and the aqueous phase was
extracted with chloroform (2 × 30 mL). The combined organic
phases were evaporated and chromatographed through a short
column of silica eluting with in 97:3 chloroform/methanol to
provide 41 mg (91%) as a cream colored solid: mp 238-240
1
°C. IR (CHCl3): 1648. H NMR (CDCl3) δ: 0.98 (d, 6H, J )

N-Alkyl Substituent Modulation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 801
filtered to provide 1.05 g as a white solid in 65% yield: mp
162-165° C. 1H NMR (CDCl3) δ: 2.24 (s, 3H), 2.59 (t, 2H, J )
7.2), 3.16 (m, 2H), 3.55 (s, 2H), 5.75 (d, 1H, J ) 5.6), 5.98 (s,
2H), 6.80 (s, 1H), 7.29 (m, 5H), 7.58 (s, 1H), 7.72 (d, 1H, J )
5.6). 13C NMR (CDCl3) δ: 40.0, 42.1, 54.6, 62.4, 96.2, 98.8,
101.72, 106.4, 114.43, 127.55, 128.6, 129.1, 138.83, 146.2,
146.8, 148.8, 149.6, 150.1. HRMS Calcd for C20H21O2N3:
335.1634. Found: 335.1633.
12 h, to provide 600 mg of a light brown solid in 40% yield:
mp 203-204 °C. 1H NMR (CDCl3) δ: 1.25-1.50 (m, 2H), 1.59-
1.64 (m, 4H), 2.42 (t, 4H, J ) 5.2), 2.67 (t, 2H, J ) 5.4), 3.23
(m, 2H), 5.7 (br, 1H), 6.0 (s, 2H), 6.32 (d, 1H, J ) 5.2), 7.06
(s,1H), 7.29 (s,1H), 8.37 (d, 1H, J ) 5.6). 13C NMR (CDCl3) δ:
24.4, 26.2, 30.9, 39.3, 54.1, 56.4, 96.1, 98.9, 101.5, 106.6, 114.4,
146.5, 146.7, 149.1, 149.5, 149.9. HRMS Calcd forC17H21N3O2:
299.1634. Found: 299.1629.
4-[[2-(N-Benzyl-N-ethylamino)ethyl]amino]-6,7-meth-
ylenedioxyquinoline (14b) was prepared from 13 (1.0 g, 4.83
mmol) and 11b (1.78 g, 10.0 mmol), reaction time 20 h, to
provide 1.0 g of a light beige solid in 60% yield: mp 143-144
°C. 1H NMR (CDCl3) δ: 1.16 (t, 3H, J ) 7.1), 2.66 (q, 2H, J )
7.1), 2.85 (t, 2H, J ) 5.7), 3.22 (m, 2H), 3.65 (s, 2H), 5.42 (br,
1H), 6.12 (s, 2H), 6.28 (d, 1H, J ) 5.1), 6.96 (s, 1H), 7.34 (m,
6H), 8.37 (d, 1H, J ) 5.1). 13C NMR (CDCl3) δ: 12.1, 40.0, 47.4,
51.0, 58.0, 96.1, 98.9, 101.6, 106.7, 114.4, 127.4, 128.7, 129.1,
139.5, 146.5, 146.7, 149.2, 149.5, 149.9. HRMS Calcd for
C21H23N3O2: 349.1790. Found: 349.1779.
4-[[2-(N-Benzyl-N-isopropylamino)ethyl]amino]-6,7-
methylenedioxyquinoline (14c) was prepared from 13 (1.0
g, 4.83 mmol) and 11c (1.90 g, 10.0 mmol), reaction time 20 h,
to provide 1.19 g of a light beige solid in 68% yield: mp 155-
156 °C. 1H NMR (CDCl3) δ: 1.14 (d, 6H, J ) 6.6), 2.86 (t, 2H,
J ) 5.7), 3.11 (m, 3H), 3.60 (s, 2H), 5.36 (br, 1H), 6.10 (s, 2H),
6.23 (d, 1H, J ) 5.2), 6.90 (s, 1H), 7.32 (m, 6H), 8.35 (d, 1H, J
) 5.2). 13C NMR (CDCl3) δ: 18.1, 40.2, 47.4, 49.4, 53.6, 96.2,
98.9, 101.6, 106.7, 114.5, 127.3, 128.7, 128.8, 140.5, 146.5,
146.7, 149.1, 149.5, 149.9. HRMS Calcd for C22H25N3O2:
363.1947. Found: 363.1934.
4-[[2-(4-Methylpiperidin-1-yl)ethyl]amino]-6,7-methyl-
enedioxyquinoline (14j) was prepared from 13 (1.0 g, 4.8
mmol) and 11j (1.56 g, 10.0 mmol), reaction time 20 h, to
provide 1.29 g of an off-white solid in 85% yield: mp 193.5-
194 °C. 1H NMR (CDCl3) δ: 0.97 (d, J ) 6.2, 3H), 1.29 (m,
3H), 1.67 (d, J ) 11.4, 2H), 2.05 (m, 2H), 2.73 (t, J ) 5.9, 2H),
2.90 (d, J ) 11.4, 2H), 3.28 (t, J ) 5.9, 2H), 6.09 (s, 2H), 6.34
(d, J ) 5.2, 1H), 7.05 (s, 1H), 7.30 (s, 1H), 8.40 (d, J ) 5.2,
1H). HRMS Calcd for C18H23N3O2: 313.1790. Found: 313.1780.
4-[[2-(4-Benzylpiperazin-1-yl)ethyl]amino]-6,7-methyl-
enedioxyquinoline (14k) was prepared from 13 (1.2 g, 5.8
mmol) and 11k (10 g, 106 mmol), reaction time 2.5 h, to provide
1.65 g of a light brown solid in 73% yield: mp 244-247 °C. 1H
NMR (CDCl3) δ: 2.56 (s, 8H), 2.77 (t, J ) 5.9, 2H), 3.30 (m,
2H), 3.56 (s, 2H), 5.61 (br, 1H), 6.10 (s, 2H), 6.35 (d, J ) 5.2,
1H), 7.06 (s, 1H), 7.32 (m, 6H), 8.40 (m, J ) 5.2, 1H). 13C NMR
(CDCl3) δ: 39.2, 52.8, 53.4, 55.8, 63.1, 96.1, 99.0, 101.6, 106.7,
114.4, 127.2, 128.3, 129.2, 138.3, 146.5, 146.8, 149.1, 149.5,
150.0. HRMS Calcd for C23H26N4O2H: 391.2134. Found:
391.2135.
General Procedure for the Preparation of N-(6,7-
Methylenedioxyquinolin-4-yl)-N-[2-(N,N-dialkylamino)-
ethyl]-2-iodo-4,5-dimethoxybenzamides 15a-l. N-(6,7-
Methylenedioxyquinolin-4-yl)-N-[2-(N-benzyl-N-meth-
ylamino)ethyl]-2-iodo-4,5-dimethoxybenzamide (15a).
Oxalyl chloride (1.37 g, 10.8 mmol) was added to a mixture of
2-iodo-4,5-dimethoxybenzoic acid (1.5 g, 3.25 mmol) in meth-
ylene chloride (40 mL), and the mixture was heated to reflux
under nitrogen with stirring for 4 h. The mixture was
concentrated to dryness under vacuum. The acid chloride was
redissolved in 40 mL of methylene chloride, and a solution of
14a (906 mg, 2.7 mmol) added, then triethylamine (2.0 mL,
27 mmol) was added. The mixture was heated to reflux with
stirring for 16 h and was then cooled to room temperature.
The mixture was washed with saturated NaHCO3 (3 × 100
mL) and extracted into dilute aqueous HCl (3 × 100 mL). The
combined aqueous phases were washed with chloroform (2 ×
100 mL), basified (30% NaOH), and extracted with chloroform
(3 × 100 mL). The combined organic layers were washed with
brine (100 mL), dried (MgSO4), and evaporated, yielding 1.44
g in 85% yield as a light brown sticky glue. IR (CHCl3): 1652.
1H NMR (CDCl3) δ: 2.20 (s, 3H), 2.77 (m, 2H), 3.31 (s, 3H),
3.46 (m, 1H), 3.61 (s, 2H), 4.60 (m, 1H), 6.13 (s, 2H), 6.39 (s,
1H), 7.02 (s, 1H), 7.32 (m, 8H), 8.50 (d, 1H, J ) 4.8). 13C NMR
(CDCl3) δ: 42.3, 44.3, 46.8, 55.5, 56.0, 62.5, 82.8, 98.4, 102.2,
106.7, 110.4, 120.3, 121.6, 122.8, 127.1, 128.2, 129.1, 133.7,
138.7, 145.9, 148.0, 148.3, 148.4, 149.0, 149.6, 151.0, 169.9.
HRMS Calcd for C29H28IN3O5H: 626.1152. Found: 626.1117.
4-[[2-(N-Benzyl-N-tert-butylamino)ethyl]amino]-6,7-
methylenedioxyquinoline (14d) was prepared from 13 (1.0
g, 4.83 mmol) and 11d (2.06 g, 10.0 mmol), reaction time 20
h, to provide 1.24 g of a light beige solid in 69% yield: mp
1
112-115 °C. H NMR (CDCl3) δ: 1.24 (s, 9H), 2.97 (m, 4H),
3.75 (s, 2H), 5.08 (br, 1H), 6.01 (d, 1H, J ) 5.4), 6.10 (s, 2H),
6.82 (s, 1H), 7.24 (m, 6H), 8.26 (d, J ) 5.4, 1H). 13C NMR
(CDCl3) δ: 27.6, 42.8, 49.5, 55.5, 55.7, 96.1, 98.9, 101.5, 106.7,
114.5, 126.9, 128.0, 128.5, 142.3, 146.5, 146.6, 149.0, 149.4,
149.8. HRMS Calcd for C23H27N3O2: 377.2103. Found: 377.2090.
4-[[2-(N,N-Dibenzylamino)ethyl]amino]-6,7-methylene-
dioxyquinoline (14e) was prepared from 13 (1.0 g, 4.83
mmol) and 11e (2.40 g, 10.0 mmol), reaction time 20 h, to
provide 1.48 g of a light beige solid in 75% yield: mp 132-
133 °C. 1H NMR (CDCl3) δ: 2.87 (t, 2H, J ) 5.5), 3.23 (m,
2H), 5.19 (br, 1H), 6.14 (s, 2H), 6.23 (d, 1H, J ) 5.4), 6.92 (s,
1H), 7.33 (m, 11H), 8.34 (d, 1H, J ) 5.4). 13C NMR (CDCl3) δ:
40.2, 51.4, 58.7, 96.2, 98.8, 101.6, 106.6, 114.3, 127.6, 128.7,
129.2, 139.1, 146.4, 146.8, 149.0, 149.4, 150.0. HRMS Calcd
for C26H25N3O2: 411.1947. Found: 411.1953.
4-[[2-(N,N-Diethylamino)ethyl]amino]-6,7-methylene-
dioxyquinoline (14f) was prepared from 13 (1.0 g, 4.83
mmol) and N,N-diethylethylenediamine (1.16 g, 10.0 mmol),
reaction time 20 h, to provide 793 mg of an off-white solid in
8% yield: mp 201-202 °C. 1H NMR (CDCl3) δ: 1.09 (t, 6H, J
) 7.2), 2.61 (q, 4H, J ) 7.2), 2.82 (t, 2H, J ) 5.8), 3.26 (m,
2H), 5.71 (br, 1H), 6.08 (s, 2H), 6.35 (d, 1H, J ) 5.2), 7.03 (s,
1H), 7.31 (s, 1H), 8.40 (d, 1H, J ) 5.2). 13C NMR (CDCl3) δ:
12.2, 40.1, 46.7, 51.0, 96.1, 99.0, 101.5, 106.7, 114.5, 146.5,
146.7, 149.1, 149.6, 149.9. HRMS Calcd for C16H21N3O2:
287.1634. Found: 287.1631.
4-[[2-(N,N-Diisopropylamino)ethyl]amino]-6,7-methyl-
enedioxyquinoline (14 g) was prepared from 13 (1.0 g, 4.83
mmol) and N,N-diisopropylethylenediamine (1.44 g, 10.0
mmol), reaction time 20 h, to provide 943 mg of an off-white
solid in 62% yield: mp 195-196 °C. 1H NMR (CDCl3) δ: 1.11
(d, 6H, J ) 6.6), 2.90 (t, 2H, J ) 5.8), 3.13 (m, 4H), 5.77 (br,
1H), 6.08 (s, 2H), 6.35 (d, 1H, J ) 5.2), 7.00 (s, 1H), 7.31 (s,
1H), 8.40 (d, J ) 5.2, 1H). 13C NMR (CDCl3) δ: 21.0, 40.4, 42.3,
47.4, 95.9, 99.1, 101.5, 106.8, 114.6, 146.6, 146.7, 149.2, 149.6,
149.9. HRMS Calcd for C18H25N3O2: 315.1947. Found: 315.1944.
4-[[2-(Piperidin-1-yl)ethyl]amino]-6,7-methylenedioxy-
quinoline (14i) was prepared from 13 (1.0 gm, 5.0 mmol) and
2-aminoethylpiperidine, 11i (0.9 gm, 7.0 mmol), reaction time
N-(6,7-Methylenedioxyquinolin-4-yl)-N-[2-(N-benzyl-N-
ethylamino)ethyl]-2-iodo-4,5-dimethoxybenzamide (15b)
was prepared from 2-iodo-4,5-dimethoxybenzoic acid (0.9 g,
2.60 mmol) and 14b (0.75 g, 2.14 mmol), reaction time 16 h,
to provide 916 mg of a light brown sticky glue in 89% yield.
1
IR (CHCl3): 1649. H NMR (CDCl3) δ: 1.04 (t, 3H, J ) 7.0),
2.57 (q, 2H, J ) 7.0), 2.87 (m, 2H), 3.30 (s, 3H), 3.47 (m, 1H),
3.59 (s, 2H), 3.73 (s, 3H), 4.55 (m, 1H), 6.14 (s, 2H), 6.36 (s,
1H), 7.02 (s, 1H), 7.14 (d, 1H, J ) 4.8), 7.17 (m, 5H), 7.32 (s,
1H), 7.36 (s, 1H), 8.47 (d, 1H, J ) 4.8). 13C NMR (CDCl3) δ:
11.9, 47.2, 47.9, 51.2, 55.5, 56.1, 58.3, 82.8, 98.3, 102.2, 106.8,
110.4, 120.2, 121.6, 122.6, 126.9, 128.1, 129.0, 133.6, 139.2,
146.1, 148.0, 148.3, 148.4, 149.0, 149.6, 151.0, 169.9. HRMS
Calcd for C30H30IN3O5H: 640.1308. Found: 640.1248.
N-(6,7-Methylenedioxyquinolin-4-yl)-N-[2-(N-benzyl-N-
isopropylamino)ethyl]-2-iodo-4,5-dimethoxybenzam-
ide (15c) was prepared from 2-iodo-4,5-dimethoxybenzoic acid
(1.1 g, 3.30 mmol) and 14c (1.0 g, 2.75 mmol), reaction time

802 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Ruchelman et al.
16 h, to provide 1.34 g of a light brown sticky glue in 75%
yield. IR (CHCl3): 1650. 1H NMR (CDCl3) δ: 1.05 (m, 6H),
2.76 (m, 1H), 3.00 (m, 2H), 3.27 (s, 3H), 3.55 (s, 2H), 3.71 (s,
3H), 3.89 (m, 1H), 4.40 (m, 1H), 6.13 (s, 2H), 6.31 (s, 1H), 7.07
(s, 1H), 7.05 (d, 1H, J ) 4.8), 7.09 (m, 5H), 7.20 (s, 1H), 7.35
(s, 1H), 8.45, (d, 1H, J ) 4.8). 13C NMR (CDCl3) δ: 18.4, 48.0,
48.9, 51.5, 55.1, 55.5, 56.0, 82.8, 98.3, 102.2, 106.7, 110.6, 120.0,
121.8, 122.4, 126.6, 128.0, 128.6, 133.7, 140.6, 146.2, 148.1,
148.2, 148.3, 148.9, 149.7, 150.9, 169.8. HRMS Calcd for
C31H32IN3O5Li: 660.1547. Found: 660.1531.
N-(6,7-Methylenedioxyquinolin-4-yl)-N-[2-(N-benzyl-N-
tert-butylamino)ethyl]-2-iodo-4,5-dimethoxybenza-
mide (15d) was prepared from 2-iodo-4,5-dimethoxybenzoic
acid (1.5 g, 3.25 mmol) and 14d (1.02 g, 2.7 mmol), reaction
time 16 h, to provide 1.48 g of a white solid in 82% yield. IR
(CHCl3): 1649. 1H NMR (CDCl3) δ: 1.19 (s, 9H), 2.58 (m, 1H),
2.83 (m, 1H), 3.23 (s, 3H), 3.41 (m, 2H), 3.69 (s, 3H), 3.90 (m,
2H), 4.13 (m, 1H), 6.12 (s, 2H), 6.24 (s, 1H), 6.84 (m, 5H), 7.00
(m, 3H), 7.33 (s, 1H), 8.42, (d, 1H, J ) 4.6). 13C NMR (CDCl3)
δ: 27.5, 47.1, 50.5, 54.8, 55.1, 55.5, 56.0, 82.8, 98.1, 102.1,
106.6, 110.4, 119.6, 121.7, 122.3, 126.2, 127.6, 127.7, 128.5,
133.7, 141.5, 145.9, 148.1, 148.2, 148.8, 149.7, 150.9, 169.8.
HRMS Calcd for C32H34IN3O5Li: 674.1703. Found: 674.1706.
N-(6,7-Methylenedioxyquinolin-4-yl)-N-[2-(N,N-diben-
zylamino)ethyl]-2-iodo-4,5-dimethoxybenzamide (15e).
This reaction was considerably more sluggish than the other
acylations described in this section, and the procedure was
modified accordingly. The acid chloride prepared from 2-iodo-
4,5-dimethoxybenzoic acid (1.5 g, 3.25 mmol) was added to 14e
(1.11 g, 2.7 mmol). After 16 h at reflux, an equivalent amount
of acid chloride was added, and after an additional 16 h
refluxing another equivalent of acid chloride was added. The
workup was performed as described above, except that it was
followed by purification by column chromatography eluting
with chloroform, to provide 1.70 g of a fluffy white solid in
90% yield. IR (CHCl3): 1650. 1H NMR (CDCl3) δ: 2.79 (m, 1H),
2.94 (m, 1H), 3.30 (s, 3H), 3.64 (s, 4H), 3.72 (s, 3H), 3.96 (m,
1H), 4.67 (m, 1H), 6.15 (s, 2H), 6.34 (s, 1H), 6.90 (d, 1H, J )
4.8), 7.18 (m, 13H), 8.39, (d, 1H, J ) 4.8). 13C NMR (CDCl3) δ:
46.8, 51.6, 55.6, 56.1, 58.7, 82.8, 98.2, 102.2, 106.8, 110.6, 120.1,
121.7, 122.5, 127.2, 128.3, 128.5, 129.2, 133.6, 138.8, 145.8,
148.1, 148.3, 149.0, 149.8, 151.0, 169.9. HRMS Calcd for
C35H32I N3O5H: 702.1465. Found: 702.1465.
N-(6,7-Methylenedioxyquinolin-4-yl)-N-[2-(N,N-dieth-
ylamino)ethyl]-2-iodo-4,5-dimethoxybenzamide (15f) was
prepared from 2-iodo-4,5-dimethoxybenzoic acid (820 mg, 2.6
mmol) and 14f (640 mg, 2.2 mmol), reaction time 16 h, to
provide 1.10 g of a light brown sticky glue in 86% yield. IR
(CHCl3): 1651. 1H NMR (CDCl3) δ: 0.96 (t, 6H, J ) 7.2), 2.54
(q, 4H, J ) 7.2), 2.82 (m, 2H), 3.29 (s, 3H), 3.71 (s, 3H), 3.92
(m, 1H), 4.46 (m, 1H), 6.12 (s, 2H), 6.37 (s, 1H), 7.00 (s, 1H),
7.27 (d, 1H, J ) 4.8), 7.33 (s, 1H), 7.39 (s, 1H), 8.52 (d, 1H, J
) 4.8). 13C NMR (CDCl3) δ: 11.8, 47.1, 47.5, 50.7, 55.5, 56.1,
82.7, 98.5, 102.2, 106.7, 110.6, 120.1, 121.8, 122.7, 133.7, 146.3,
148.1, 148.3, 148.5, 149.0, 149.7, 151.0, 170.0. HRMS Calcd
for C25H28O5N3IH: 578.1152. Found: 578.1153.
1.42-1.52 (m, 6H), 2.42 (m, 4H), 2.65 (t, 2H, J ) 6.8), 3.33 (s,
3H), 3.73 (s, 3H), 3.94 (m, 1H), 4.42-4.52 (m, 1H), 6.15 (s, 2H),
6.41 (s, 1H), 7.02 (s, 1H), 7.35 (s, 1H), 7.38 (s, 1H), 7.45 (s,
1H), 8.54 (d, 1H, J ) 4.8). 13C NMR (CDCl3) δ: 24.3, 25.9, 46.2,
54.6, 55.5, 56.0, 56.1, 56.4, 98.6, 102.2, 106.6, 110.4, 120.2,
121.5, 122.9, 133.7, 146.1, 148.0, 148.2, 148.3, 148.9, 149.5,
150.9, 169.9. HRMS Calcd for C26H29IN3O5H: 590.1152.
Found: 590.1157.
N-(6,7-Methylenedioxyquinolin-4-yl)-N-[2-(4-methylpi-
peridin-1-yl)ethyl]-2-iodo-4,5-dimethoxybenzamide (15j)
was prepared from 2-iodo-4,5-dimethoxybenzoic acid (1.4 g,
4.54 mmol) and 14j (1.0 g, 3.19 mmol) reaction time 16 h, to
provide 1.40 g of a white solid in 73% yield as a sticky glue.
1
IR (CHCl3): 1648. H NMR (CDCl3) δ: 0.86 (d, J ) 6.2, 3H),
1.17 (m, 3H), 1.54 (d, J ) 12.8, 2H), 1.96 (m, 2H), 2.61 (m,
2H), 2.81 (d, J ) 12.8, 2H), 3.30 (s, 3H), 3.69 (s, 3H), 3.92 (m,
1H), 4.42 (m, 1H), 6.10 (s, 1H), 6.37 (s, 1H), 6.98 (s, 1H), 7.27
(m, 2H), 7.43 (s, 1H), 8.52 (d, J ) 4.8, 1H). 13C NMR (CDCl3)
δ: 22.0, 30.8, 34.5, 46.5, 53.7, 54.5, 55.5, 56.0, 82.7, 98.7, 102.2,
106.6, 110.4, 120.2, 121.5, 122.9, 133.8, 146.1, 148.0, 148.2,
148.3, 148.9, 149.5, 150.9, 169.9. HRMS Calcd for C27H30-
IN3O5H: 604.1308. Found: 604.1288.
N-(6,7-Methylenedioxyquinolin-4-yl)-N-[2-(4-benzylpip-
erazinyl)ethyl]-2-iodo-4,5-dimethoxybenzamide (15k) was
prepared from 2-iodo-4,5-dimethoxybenzoic acid (1.4 g, 4.61
mmol) and 14k (1.5 g, 3.84 mmol), reaction time 16h, to
provide 1.32 g in 51% yield as a light brown sticky glue. IR
(CHCl3): 1650. 1H NMR (CDCl3) δ: 2.46 (s, 8H), 2.62 (m, 2H),
3.31 (s, 3H), 3.50 (s, 2H), 3.70 (s, 3H) 3.87 (m, 1H), 4.43 (m,
1H), 6.11 (s, 1H), 6.12 (s, 1H), 6.39 (s, 1H), 7.00 (s, 1H), 7.25
(m, 5H), 7.33 (d, J ) 4.7, 1H), 7.46 (s, 1H), 8.53 (d, J ) 4.7,
1H). 13C NMR (CDCl3) δ: 45.9, 53.0, 53.2, 55.5, 56.0, 56.1, 63.1,
82.8, 98.7, 102.2, 106.6, 110.6, 120.3, 121.7, 123.0, 127.1, 128.3,
129.3, 133.8, 138.0, 146.0, 148.0, 148.2, 148.3, 148.9, 149.6,
151.0, 169.9. HRMS Calcd for C32H33IN4O5H: 681.1574.
Found: 681.1590.
Topoisomerase-Mediated DNA Cleavage Assays. Hu-
man topoisomerase I was expressed in E. coli and isolated as
a recombinant fusion protein using a T7 expression system
as described previously.49 Plasmid YepG was purified by the
alkali lysis method followed by phenol deproteination and
CsCl/ethidium bromide isopycnic centrifugation method as
described.50 The end labeling of the plasmid was accomplished
by digestion with a restriction enzyme followed by end filling
with Klenow polymerase as previously described.51 The cleav-
age assays were performed as previously reported.49,52 The
drug and the DNA in the presence of topoisomerase I were
incubated for 30 min at 37 °C. After development of the gels,
typically 24 h exposure was used to obtain autoradiograms
outlining the extent of DNA fragmentation. Topoisomerase
I-mediated DNA cleavage values are reported as REC, relative
effective concentration, i.e., concentrations relative to topote-
can, whose value is arbitrarily assumed as 1.0, that are able
to produce the same cleavage on the plasmid DNA in the
presence of human topoisomerase I.
Cytotoxicity Assays. The cytotoxicity was determined
using the MTT-microtiter plate tetrazolinium cytotoxicity
assay (MTA).53-55 The human lymphoblast RPMI 8402 and its
camptothecin-resistant variant cell line, CPT-K5, were pro-
vided by Dr. Toshiwo Andoh (Aichi Cancer Center Research
Institute, Nagoya, Japan).56 The P388 mouse leukemia cell line
and its CPT-resistant TOP1-deficient variant P388/CPT45
were obtained from Michael R. Mattern and Randal K.
Johnson (GlaxoSmithKline, PA).57 The U937 cell line and its
CPT-resistant variant U937/CR were obtained from Dr. Eric
H. Rubin (The Cancer Institute of New Jersey, NJ).58 The
KB3-1 cell line and its multidrug-resistant variant KBV-1 were
obtained from K. V. Chin (The Cancer Institute of New Jersey,
NJ).59 The KBH5.0 cell line was derived from KB3-1 by
stepwise selection against Hoechst 33342.48 The CEM cell line
and its VP-16-resistant variants CEM/V-1 and CEM/V-5 were
obtained from William Beck (University of Illinois, Chicago).60
The cytotoxicity assay was performed using 96-well microtiter
plates. Cells were grown in suspension at 37 °C in 5% CO2
N-(6,7-Methylenedioxyquinolin-4-yl)-N-[2-(N,N-diiso-
propylamino)ethyl]-2-iodo-4,5-dimethoxybenzamide (15g)
was prepared from 2-iodo-4,5-dimethoxybenzoic acid (820 mg,
2.6 mmol) and 14g (700 mg, 2.2 mmol), reaction time 16 h, to
1
provide 1.15 g of a light brown sticky glue in 86% yield. H
NMR (CDCl3) δ: 0.95 (m, 12H), 2.70 (m, 1H), 2.97 (m, 2H),
3.27 (s, 3H), 3.71 (s, 3H), 3.90 (m, 2H), 4.37 (m, 1H), 6.12 (s,
2H), 6.35 (s, 1H), 7.00 (s, 1H), 7.23 (d, 1H, J ) 4.8), 7.33 (s,
2H), 8.52 (d, 1H, J ) 4.8). 13C NMR (CDCl3) δ: 20.8, 42.9, 49.4,
51.3, 55.5, 56.1, 82.8, 98.3, 102.3, 106.7, 110.4, 119.7, 121.7,
122.5, 133.7, 146.5, 148.1, 148.3, 148.5, 149.0, 149.7, 151.0,
169.9. HRMS Calcd for C27H32O5N3ILi: 612.1547. Found:
612.1573.
N-(6,7-Methylenedioxyquinolin-4-yl)-N-[2-(piperidin-
1-yl)ethyl]-2-iodo-4,5-dimethoxybenzamide (15i) was pre-
pared from 2-iodo-4,5-dimethoxybenzoic acid and 14i (450 mg,
1.5 mmol), reaction time 16 h, to provide 350 mg of a light
1
brown solid in 40% yield: mp 91-92 °C. H NMR (CDCl3) δ:

N-Alkyl Substituent Modulation
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 803
(14) Yang, C. H.; Schneider, E.; Kuo, M. L.; Volk, E. L.; Roccchi; E.;
Chen, Y. C.; BCRP/MXR/ABCP expression in topotecan-resistant
human breast cancer cells, Biochem. Pharmacol. 2000, 60, 831-
837.
(15) Scehllens, J. H. M., Melirpaard, M., Scheper, R. J., Scheffer, G.
L., Jonker, J. W., Smit, J. W., Beijnen, J. H., and Schinkel, A.
H. Transport of topoisomerase I inhibitors by the breast cancer
resistance protein. Ann. N. Y. Acad. Sci. 2000, 922, 188-194.
(16) Kim, J. S.; Gatto B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J.
Terbenzimidazoles: Influence of 2′′-, 4-, and 5-substituents on
cytotoxicity and relative potency as topoisomerase I poisons. J.
Med. Chem. 1997, 40, 2818-2824.
(17) Ranagarajan, M.; Kim, J. S.; Sim, S.-P.; Liu, A.; Liu, L. F.;
LaVoie, E. J. Topoisomerase I inhibition and cytotoxicity of
5-bromo and 5-phenylterbenzimidazoles. Bioorg. Med. Chem.
2000, 11, 2591-2600.
(18) Jin, S.; Kim, J. S.; Sim, J. S.; Sim, S.-P.; Liu, A.; Pilch, D. S.;
Liu, L. F.; LaVoie, E. J. Heterocyclic bibenzimidazole derivatives
as topoisomerase I inhibitors. Bioorg. Med. Chem. Lett. 2000,
10, 719-723.
(19) Makhey, D.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J. Substituted
benz[a]acridines and benz[c]acridines as mammalian topo-
isomerase poisons. Bioorg. Med. Chem. 2000, 8, 1171-1182.
(20) Janin, Y. L.; Croisy, A.; Rious, J.-L.; Bisagni, E. Synthesis and
evaluation of new 6-amino-substituted benzo[c]phenanthridine
derivatives. J. Med. Chem. 1993, 36, 3686-3692.
(21) Makhey, D.; Gatto, B.; Yu, C.; Liu, A.; Liu, L. F.; LaVoie, E. J.
Protoberberine alkaloids and related compounds as dual inhibi-
tors of mammalian topoisomerase I and II. Med. Chem. Res.
1995, 5, 1-12.
(22) Yamashita, Y.; Fujii, N.; Murakaya C.; Ashizawa, T.; Okabe, M.;
Nakano, H. Induction of mammalian DNA topoisomerase I
mediated DNA cleavage by antitumor indolocarbazole deriva-
tives. Biochemistry 1992, 31, 12069-12075.
(23) Fujii, N.; Yamashita, Y.; Saitoh; Y.; Nakano H. Induction of
mammalian DNA topoisomerase I-mediated DNA cleavage and
DNA winding by bulgarein. J. Biol. Chem. 1993, 268, 13160-
13165.
and maintained by regular passage in RPMI medium supple-
mented with 10% heat-inactivated fetal bovine serum, L-
glutamine (2 mM), penicillin (100 U/mL), and streptomycin
(0.1 mg/mL). For determination of IC50 values, cells were
exposed continuously for 4 days to varying concentrations of
drug, and MTT assays were performed at the end of the fourth
day. Each assay was performed with a control that did not
contain any drug. All assays were performed at least twice in
six replicate wells.
Antitumor Activity Against Scid Mice Carrying Hu-
man Tumor Xenografts. CB17/Icr female scid mice were
subcutaneously implanted with a single tumor fragment.
Tumor-bearing mice were randomized into groups of 5 or 10
animals prior to therapy. All mice were maintained under
barrier conditions. Mice bearing subcutaneous xenografts were
treated with D5W when tumors were approximately 0.2-1 cm
in diameter. The procedures have been reported previously.61
Briefly, two perpendicular diameters were determined at 7-day
intervals using digital Vernier calipers interfaced with a
Macintosh computer. Tumor volumes were calculated assum-
ing tumors to be spherical using the formula [(π/6)d3], where d
is the mean diameter, and mice were followed for up to 12
weeks after starting treatment. Compounds 3, 6a, and 6f were
administered 3 times per week for 2 consecutive weeks as their
monocitrate salts dissolved in 5% dextrose solution U. S. P.
Cycles of treatment were repeated every 21 days for a total of
two cycles of treatment.
Acknowledgment. This study was supported by
Grants CA098127 (E.J.L.), CA39662 (L.F.L.), and
CA077433 (L.F.L.) from the National Cancer Institute.
Supporting Information Available: Elemental analysis
data on new compounds evaluated for biological activity. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(24) Yamashita, Y.; Kawada, S.; Fujii, N.; Nakano, H. Induction of
mammalian DNA topoisomerase I and II mediated DNA cleav-
age by saintopin, a new antitumor agent from fungus. Biochem-
istry 1991, 30, 5838-5845.
References
(25) Jayaraman, M.; Fox, B. M.; Hollingshead, M.; Kohlhagen, G.;
Pommier, Y.; Cushman, M. Synthesis of new dihydroindeno[1,2-
c]indeno[1, 2-c]isoquinoline and indenoisoquinolinium chloride
topoisomerase I inhibitors having high in vivo anticancer activity
in the hollow fiber animal model. J. Med. Chem. 2002, 45, 242-
249.
(26) Cushman, M.; Jayaraman, M.; Vroman, J. A.; Fukunaga, A. K.;
Foc, B. M.; Kohlhagen, G.; Strumberg, D.; Pommier, Y. Synthesis
of new indeno[1,2-c]isoquinolines; cytotoxic noncamptothecin
topoisomerase I inhibitors. J. Med. Chem. 2000, 43, 3688-3698.
(27) Ruchelman, A. L.; Singh, S. K.; Liu, A.; Zhou, N.; Liu, L. F.;
LaVoie E. J. Cytotoxicity of 5H-Dibenzo[c,h][1,6]naphthyridin-
6-ones and 6H-indeno[1,2-c]isoquinolin-5, 11-diones in tumor
cells sensitive and resistant to camptothecin analogues. Lett.
Drug Des. Disc. 2004, 1, 198-202.
(28) Vicker, N.; Burgess, L.; Chuckowree, I. S.; Dodd, R.; Folkes, A.
J.; Hardick, D.; Hancox, T. C.; Miller, W.; Milton, J.; Sohal, S.;
Wang, S.; Wren, S. P.; Charlton, P. A.; Dangerfield, W.; Liddle,
C.; Mistry, P.; Stewart, A. J.; Denny, W. A. Novel angular
benzophenazines: dual topoisomerase I and topoisomerase II
inhibitors as potential anticancer agents. J. Med. Chem. 2002,
45, 721-739.
(29) Li, D.; Zhao, B.; Sim, S.-P.; Li, T.-K.; Liu, A.; Liu, L. F.; LaVoie,
E. J. 8,9-Methylenedioxybenzo[i]phenanthridines: Topoisomerase
I-targeting activity and cytotoxicity. Bioorg. Med. Chem. 2003,
11, 3795-3805.
(30) Yu, Y.; Singh, S. K.; Liu, A.; Li, T.-K.; Liu, L. F.; LaVoie, E. J.
Substituted Dibenzo[c,h]cinnolines: Topoisomerase I-targeting
anticancer agents. Bioorg. Med. Chem. 2003, 11, 1475-1491.
(31) Ruchelman, A. L.; Singh, S. K.; Wu, X.; Ray, A.; Yang, J.-M.; Li,
T.-K.; Liu, A.; Liu, L. F.; LaVoie, E. J. Diaza- and triazachry-
senes: Potent topoisomerase-targeting agents with exceptional
antitumor activity against the human tumor xenograft, MDA-
MB-435. Bioorg. Med. Chem. Lett. 2002, 12, 3333-3336.
(32) Ruchelman, A. L.; Singh, S. K.; Ray, A.; Wu, X.; Yang, J.-M.; Li,
T.-K.; Liu, A.; Liu L. F.; LaVoie, E. J. 5H-Dibenzo[c,h]1,6-
naphthyridin-6-ones: Novel topoisomerase I-targeting antican-
cer agents with potent cytotoxic activity. Bioorg. Med. Chem.
2003, 11, 2061-2073.
(1) Li, T.-K.; Liu, L. F. Tumor cell death induced by topoisomerase-
targeting drugs. Annu. Rev. Pharmacol. Toxicol. 2002, 41, 53-
77.
(2) Wang, J. C. DNA topoisomerases. Annu. Rev. Biochem. 1985,
54, 665-697.
(3) Liu, L. F. DNA topoisomerase poisons as antitumor drugs. Annu.
Rev. Biochem. 1989, 58, 351-375.
(4) Chen, A. Y.; Liu, L. F. DNA topoisomerases: essential enzymes
and lethal targets. Annu. Rev. Pharmacol. Toxicol. 1994, 34,
191-218.
(5) Hsiang, Y. H.; Lihou, M.; Liu, L. Arrest or replication forks by
drug-stabilized topoisomerase I-DNA cleavable complexes as a
mechanism of cell killing by camptothecin. Cancer Res. 1989,
49, 5077-5082.
(6) Desai, S. D.; Li, T.-K.; Rodriguez-Bauman, A., Rubin, E. H., Liu,
L. F. Ubiquitin/26S proteosome-mediated degradation of topo-
isomerase I as a resistance mechanism to camptothecin in tumor
cells. Cancer Res. 2001, 61, 5926-5932.
(7) Liu, L. F., Desai, S. D., Li, T.-K., Mao, Y., Sun, M., Sim, S.-P.
Mechanism of Action of Camptothecins. Ann. N. Y. Acad. Sci.
2000, 922, 1-8.
(8) Wall, J. G.; Burris, H. A.; Von-Hoff, D. D.; Rodriquez, G.;
Kneuper-Hall R.; Shaffer, D.; O’Rourke, T.; Brown, T.; Weiss,
G.; Clark, G. A phase I clinical and pharmacokinetic study of
the topoisomerase I inhibitor topotecan (SK&F 104864). Anti-
cancer Drugs 1992, 3, 337-345.
(9) Burke, T. G.; Mi, Z. Ethyl substitution at the 7 position extends
the half-life of 10-hydroxycamptothecin in the presence of human
serum albumin. J. Med. Chem. 1993, 36, 2580-2582.
(10) Burke, T. G.; Mi, Z. Preferential binding of the carboxylate form
of camptothecin by human serum albumin. Anal. Biochem. 1993,
212, 285-287.
(11) Chen, A. Y.; Yu, C., Potmesil, M.; Wall, M. E.; Wani, M. C.; Liu,
L. F. Camptothecin overcomes MDR1-mediated resistance in
human KB carcinoma cells. Cancer Res. 1991, 51, 6039-6044.
(12) Chu, X., Kato, Y., Sugiyama, Y. Multiplicity of biliary excretion
mechanisms for irinotecan, CPT-11, and its metabolites in rats.
Cancer Res. 1997 57, 1934-1938.
(13) Kawabata, S.; Oka, M.; Shiozawa, K.; Tsukamoto, K.; Nakatomi,
K.; Soda, H.; Fududa; M.; Ikegami, Y.; Sugahara, K.; Yamada,
Y.; Kamihira, S.; Doyle, L. A.; Ross, D. D. Kohno, S. Breast
cancer resistance protein directly confers SN-38 resistance of
lung cancer cells. Biochem. Biophys. Res. Commun. 2001, 280,
1216-1223.
(33) Contreras, J.-M.; Rival, Y. M.; Chayer, S.; Bourguignon, J.-J.;
Wermuth. C. G.; Aminopyridazines as acetylcholinesterase
inhibitors. J. Med. Chem. 1999, 42, 730-741.

804 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Ruchelman et al.
(34) Iwanami, S.; Takashima, M.; Hirata, Y.; Hasegawa, O.; Usuda,
S. Synthesis and neuroleptic activity of benzamides. cis-N-(1-
benzyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4-(methyl-
amino)benzamide and related compounds. J. Med. Chem. 1981,
24, 1224-1230.
(35) Meyer, R. F.; Stratton, C. D.; Hastings, S. G.; Corey, R. M.
â-Adrenergic blocking agents. Nitrogen heteroaryl-substituted
2-propanolamines and ethanolamines. J. Med. Chem. 1973, 16,
1113-16.
(36) Leclerc, E.; Vrancken, E.; Mangeney, P. Diastereoselective
aldolization of R-aminonitriles. diastereoselective synthesis of
â-amino alcohols and â,γ-diamino alcohols. J. Org. Chem. 2002,
67, 8928-8937.
(37) Pasini, C., Coda, S.; Colo, V.; Ferrini, R.; Glaesser, A. H.
Pharmacological effects of some guanidine and guanethidine
derivatives. Farmaco Ed. Sci. 1965, 20, 673-685.
(38) Saxena, M.; Agarwal, A.; Shiv, K.; Patnaik, G. K.; Saxena, A.
K. Synthesis, biological evaluation, and quantitative structure-
activity relationship analysis of [â-(aroylamino)ethyl]piperazines
and -piperidines and [2-[(aryamino)carbonyl]ethyl]piperazines,
-piperidines, -pyrazinopyridoindoles, and -pyrazinoisoquinolines.
A new class of potent H1 antagonists. J. Med. Chem. 1990, 33,
2970-2976.
(48) Li, T.-K.; Houghton, P. J.; Desai, S. D.; Daroui, P.; Liu, A. A.;
Hars, E. S.; Ruchelman, A. L.; LaVoie, E. J.; Liu, L. F.
Characterization of ARC-111 as a novel topoisomerase I-target-
ing anticancer agent, Cancer Res. 2003, 63, 8400-8407.
(49) Gatto, B.; Sanders, M. M.; Yu C.; Wu, H.-Y.; Makhey, D.; LaVoie,
E. J.; Liu, L. F. Identification of topoisomerase I as the cytotoxic
target for the protoberberine alkaloid coralyne. Cancer Res. 1996,
56, 2795-2800.
(50) Maniatis, T.; Fritsch, E. F.; Sambrook, J. Molecular Cloning, a
Laboratory Manual; Cold Spring Harbor Laboratory: Cold
Spring Harbor, NY 1982; pp 149-185.
(51) Tewey, K. M.; Rowe, T. C.; Yang, L.; Hallogan, B. C.; Liu, L. F.
Adriamycin-induced DNA damage mediated by mammalian
DNA topoisomerase II. Science 1984, 226, 466-468.
(52) Wang, H.; Mao, Y.; Chen, A.; Zhou, N.; LaVoie, E. J.; Liu, L. F.
Stimulation of topoisomerase II-mediated DNA damage via a
mechanism involving protein thiolation. Biochemistry 2001, 40,
3316-3323.
(53) Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J.
Immunol. Methods 1983, 65, 55-63.
(54) Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.;
Mitchell, J. B. Evaluation of a tetrazolium-based semiautomated
colorimetric assay: assessment of chemosensitivity testing.
Cancer Res. 1987, 47, 936-942.
(55) Denizot, F.; Lang, R. J. Rapid colorimetric assay for cellular
growth and survival. Modifications to the tetrazolium dye
procedure giving improved sensitivity and reliability. J. Immu-
nol. Methods 1986, 89, 271-277.
(56) Andoh, T.; Ishii, K.; Suzuki, Y.; Ikegami, Y.; Kusunoki, Y.;
Takemoto, Y.; Okada, K. Characterization of a mammalian
mutant with a camptothecin-resistant DNA topoisomerase I.
Proc. Natl. Acad. Sci., U.S.A. 1987, 84, 5565-5569.
(57) Woessner, R. D.; Eng, W. K.; Hofmann, G. A.; Rieman, D. J.;
McCabe, F. L.; Hertzberg, R. P.; Mattern, M. R.; Tan, K. B.;
Johnson, R. K. Camptothecin hyper-resistant P388 cells: drug-
dependent reduction in topoisomerase I content. Oncol. Res.
1992, 4, 481-488.
(58) Rubin, E.; Pantazis, P.; Bharti, A.; Toppmeyer, D.; Giovanella,
B.; Kufe, D. Identification of a mutant human topoisomerase I
with intact catalytic activity and resistance to 9-nitro-camptoth-
ecin. J. Biol. Chem. 1994, 269, 2433-2439.
(39) Gall-Istok, K.; Sterk, L.; Deak, G. Notes on the Synthesis of
4-Amino-6,7-di-sec-butoxyquinoline, -6,7-Methylenedioxyquino-
line and its N-alkylaminoacetyl derivatives. Acta Chim. Hung.
1983, 112, 241-247.
(40) Wright, G. C.; Watson, E. J.; Ebetino, F. F.; Lougheed, G.;
Stevenson, B. F.; Winterstein, A. Synthesis and hypotensive
properties of new 4-aminoquinolines. J. Med. Chem. 1971, 14,
1060-1066.
(41) Grigg, R.; Sridharan, V.; Stevenson, P.; Sukirthalingham, S.;
Worakun, T. The synthesis of fused ring nitrogen heterocycles
via regiospecific intramolecular Heck reactions. Tetrahedron
1990, 46, 4003-4018.
(42) Harayama, T.; Akiyama, H.; Kawano, K.; Abe, H.; Takeuchi, Y.
Total synthesis of benzo[c]phenanthridine alkaloids, cheleryth-
rine and 12-methoxydihydrochelerythrine, by
a palladium-
assisted internal biaryl coupling reaction. Synthesis 2001, 444-
450.
(43) Harayama, T.; Sato, T.; Nakano, Y.; Abe, H.; Takeuchi, Y.
Concise synthesis of fagaridine and decarine, phenolic benzo[c]-
phenanthridine alkaloids, using the palladium-assisted biaryl
coupling reaction. Heterocycles 2003, 59, 293-301.
(59) Gervasoni, J. E., Jr.; Fields, S. Z.; Krishna, S.; Baker, M. A.;
Rosado, M.; Thuraisamy, K.; Hindenburg, A. A., and Taub, R.
N. Subcellular distribution of daunorubicin in P-glycoprotein-
positive and -negative drug-resistant cell lines using laser-
assisted confocal microscopy. Cancer Res. 1991, 51, 4955-4963.
(60) Fernandes, D. J.; Danks, M. K.; Beck, W. T. Decreased nuclear
matrix DNA topoisomerase II in human leukemia cells resistant
to VM-26 and m-AMSA. Biochemistry 1990, 29, 4235-4241.
(61) Houghton, P. J.; Cheshire, P. J.; Hallman, J. D.; Lutz, L.;
Friedman, H. S.; Danks, M. K.; Houghton, J. A. Efficacy of
topoisomerase I inhibitors, topotecan and irinotecan, adminis-
tered at low dose levels in protracted schedules to mice bearing
xenografts of human tumors. Cancer Chem.Pharm. 1995, 36,
393-403.
(44) Jacobi, P. A.; Martinelli, M. J.; Polanc, S. Total synthesis of (()-
saxitoxin. J. Am. Chem. Soc. 1984, 106, 5594-5598.
(45) Tamura, H.; Kohchi, C.; Yamada, R., Ikeda, T.; Koiwai, O.;
Patterson, E.; Keene, J. D.; Okada, K.; Kjeldsen, E.; Nishikawa,
K.; Andoh, T. Molecular cloning of a cDNA of a camptothecin-
resistant human DNA topoisomerase I and identification of
mutation sites, Nucleic Acids Res. 1990, 19, 69-75.
(46) Woessner, R. D.; Eng, W. K.; Hofmann, G. A.; Rieman, D. J.;
McCabe, F. L.; Hertzberg, R. P.; Mattern, M. R.; Tan, K. B.,
Johnson, R. K. Camptothecin hyper-resistant P388 cells: drug-
dependent reduction in topoisomerase I content. Oncol. Res.
1992, 4, 481-488.
(47) Shen, D. W.; Fojo, A.; Chin, J. E.; Roninson, I. B.; Richert, N.;
Pastan, I.; Gottesman, M. M. Human multidrug-resistant cell
lines: increased mdr1 expression can precede gene amplification,
Science 1986, 232, 643-645.
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