Nitro and amino substitution in the D-ring of 5-(2-dimethylaminoethyl)2,3-methylenedioxy-5H-dibenzo[c,h][1,6] naphthyridin-6-ones: Effect on topoisomerase-I targeting activity and cytotoxicity

Article abstract of DOI:10.1021/jm020498a

5H-8,9 -Dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo [c,h][1,6]naphthyridin-6-one exhibits potent TOP1-targeting activity and pronounced antitumor activity. It was hypothesized that replacement of the two methoxyl groups with a nitro s

Full text of DOI:10.1021/jm020498a

2254
J . Med. Chem. 2003, 46, 2254-2257
Nitr o a n d Am in o Su bstitu tion in th e D-Rin g of 5-(2-Dim eth yla m in oeth yl)-
2,3-m eth ylen ed ioxy-5H-d iben zo[c,h ][1,6]n a p h th yr id in -6-on es: Effect on
Top oisom er a se-I Ta r getin g Activity a n d Cytotoxicity
Sudhir K. Singh,† Alexander L. Ruchelman,† Tsai-Kun Li,‡ Angela Liu,‡ Leroy F. Liu,‡,§ and
Edmond J . LaVoie*,†,§
Department of Pharmaceutical Chemistry, Rutgers, The State University of New J ersey, Piscataway, New J ersey 08854-8020,
Department of Pharmacology, University of Medicine and Dentistry of New J ersey, Robert Wood J ohnson Medical School,
Piscataway, New J ersey 08854, and the Cancer Institute of New J ersey, New Brunswick, New J ersey 08901
Received November 5, 2002
5H-8,9-Dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naph-
thyridin-6-one exhibits potent TOP1-targeting activity and pronounced antitumor activity. It
was hypothesized that replacement of the two methoxyl groups with a nitro substituent would
allow for retention of similar activity. In this study 8-, 9-, and 10-nitro-5H-2,3-methylenedioxy-
5-(2-N,N-dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-one and their amino derivatives
were synthesized and assessed for their relative TOP1-targeting activity and cytotoxicity. In
the case of both the 8- and 9-nitro analogues, their TOP1-targeting activity and cytotoxicity
are greater than that of camptothecin and comparable to that of 5H-8,9-dimethoxy-5-(2-N,N-
dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one.
In tr od u ction
berine alkaloids,15,16 indolocarbazoles,17 the fungal me-
tabolites bulgarein18 and saintopin,19 and several
indenoisoquinolines20 and benzophenazines.21 Studies
in our laboratory have demonstrated that 5H-8,9-
dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methyl-
enedioxydibenzo[c,h][1,6]naphthyridin-6-one, 1, and 11H-
2,3-dim et h oxy-5-(2-N,N-dim et h yla m in oet h yl)-8,9-
methylenedioxy-5,6,11-triazachrysen-12-one, 2, can
exhibit potent TOP1-targeting activity and pronounced
cytotoxicity.22,23 Potent antitumor activity was observed
for 1 administered orally and parenterally to athymic
nude mice bearing the human tumor xenograft, MDA-
MB-435.
Topoisomerases are nuclear enzymes critical to rep-
lication and transcription. There are two major sub-
types, topoisomerase I (TOP1) and topoisomerase II
(TOP2) based upon differences in their initial mecha-
nisms wherein a single- or double-stranded DNA break
is implicated.1-3 Topoisomerase-targeting agents that
stabilize the cleavable complex formed between the
enzyme and DNA have proved to be effective in the
treatment of cancer. Small molecules capable of stabiliz-
ing the enzyme-DNA cleavable complex convert these
nuclear enzymes into cellular poisons.
Camptothecin was the first agent identified as a
TOP1-targeting agent.4 There are two clinical agents,
topotecan (Hycamtin) and irinotecan (CPT-11/Camp-
tosar), that have been developed from the research
performed with camptothecin and its structurally re-
lated analogues. These camptothecin-based drugs have
a γ-lactone incorporated within their structures, which
is susceptible to hydrolysis. Ring-opening of the camp-
tothecin lactone moiety results in the formation of an
inactive derivative that possesses high affinity for
human serum albumin.5-7 The metabolic instability of
this lactone and the observation that both topotecan and
irinotecan are substrates for efflux transporters associ-
ated with resistance have prompted further studies on
the development of novel TOP1-targeting agents.8-11
Structure-activity studies on variously substituted
benzo[i]phenanthridines and dibenzo[c,h]cinnolines have
demonstrated that the presence of a nitrogen heteroa-
tom adjacent to a benzo-ring that possesses a methyl-
enedioxy group is important for maintaining potent
TOP1-targeting activity.24-26 In addition, substitution
of two vicinal methoxyl substituents within the A-ring
of benzo[i]phenanthridine derivatives, specifically at the
2,3-positions, has also proven to be a structural feature
associated with potent TOP1-targeting activity.25 It was
speculated that replacement of the vicinal methoxyl
groups with a nitro substituent could potentially allow
for retention of similar biological activity. The synthetic
methods previously developed in our laboratory for the
preparation of various benzo[i]phenanthridine and diben-
zo[c,h]cinnoline derivatives, however, are incompatible
Several novel non-camptothecin TOP1-targeting agents
have been identified in recent years. These include
derivatives of bi- and terbenzimidazoles,12,13 benz[a]-
anthracenes,14 benzo[c]phenanthridine and protober-
* Correspondence author. Phone 732-445-2674, FAX 732-445-6312.
E-mail elavoie@rci.rutgers.edu. Corresponding address: 160 Freling-
huysen Rd., Piscataway, NJ 08854-8020.
† Rutgers University.
‡ UMDNJ , Robert Wood J ohnson Medical School.
§ Cancer Institute of New J ersey.
10.1021/jm020498a CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/09/2003

J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2255
Sch em e 1a
a
(a) (COCl)2, CH2Cl2, 11, and then TEA, CH2Cl2; (b) Pd(OAc)2, P(o-tolyl)3, Ag2CO3, DMF; (c) RaNi, H2NNH2‚H2O.
with the formation of such nitro analogues.24-26 The
synthetic approach employed herein for the preparation
of 5-substituted 5H-8,9-dimethoxy-2,3-methylenedioxy-
dibenzo[c,h][1,6]naphthyridin-6-ones permits the intro-
duction of a nitro moiety at or adjacent to the sites
occupied by the methoxyl substituents. The present
study was undertaken to determine if one or more such
nitro derivatives could retain similar TOP1-targeting
activity as 1. In this study we synthesized 8-, 9-, and
10-nitro-5H-2,3-methylenedioxy-5-(2-N,N-dimethylami-
noethyl)dibenzo[c,h][1,6]naphthyridin-6-one as well as
their amino derivatives to assess their relative TOP1-
targeting activity and cytotoxicity.
benzamide, 15, which under Heck cyclization conditions
only formed the desired methylenedioxydibenzo[c,h][1,6]-
naphthyridin-6-one, 16a . This byproduct is most likely
formed by intramolecular nucleophilic substitution by
the tertiary alkylamine on the electrophilic p-nitroaryl
bromide, which is sterically unhindered in the case of
12d , followed by elimination of methyl bromide. In the
case of both 6a and 16a , treatment with Ra-Ni and
hydrazine provides the corresponding amino analogues
6b and 16b.
Biologica l Resu lts a n d Discu ssion
Results from the evaluation of the relative TOP1-
targeting activity and cytotoxicity of 3 and its nitro
derivatives, 4a -6a , and amino derivatives, 4b-6b, are
provided in Table 1. In comparing their intrinsic TOP1-
targeting activity relative to the 8,9-dimethoxy analogue
1, it is evident that both 5a and 6a are more potent.
Both 5a and 6a are more potent than camptothecin as
TOP1-targeting agents. In contrast, 3 is at least 1 order
of magnitude less active than 1 as a TOP1-targeting
agent and the 10-nitro derivative 4a does not exhibit
any significant TOP1-targeting activity. The side-
product 13, formed in the synthesis of 6a , does not
possess significant TOP1-targeting activity.
Compounds 5a and 6a have comparable cytotoxicity
to that of 1 toward the human lymphoblast tumor cell
line, RPMI8402. Both 5a and 6a possess greater cyto-
toxicity than either camptothecin or topotecan in this
cell line. They also exhibit similar cross-resistance to
the camptothecin-resistant variant of RPMI8402, CPT-
K5. Consistent with its lower potency as a TOP1-
targeting agent, 3 is significantly less cytotoxic toward
RPMI8402 than 1. Compound 4a is more than 3 orders
of magnitude less cytotoxic toward RPMI8402 than
either 1, 5a , or 6a , consistent with its lack of significant
TOP1-targeting activity. The extent of DNA fragmenta-
Ch em istr y
The synthetic methods for the preparation of 5H-2,3-
methylenedioxy-5-(2-N,N-dimethylaminoethyl)dibenzo-
[c,h][1,6]naphthyridin-6-one, 3, and its 8-, 9-, and 10-
nitro derivatives, 4a , 5a , and 6a are outlined in Schemes
1 and 2. The o-bromobenzoic acids 7-10 were available
commercially or prepared as reported in the literature.27
Reaction of 4-chloroquinoline with N,N-dimethylethyl-
enediamine in the presence of phenol provided the
4-alkylaminoquinoline, 11. As outlined in Schemes 1
and 2, the o-bromobenzoic acids, 7-9, were converted
to their acid chlorides and reacted with 11 to form the
requisite o-iodobenzamide intermediates, 12a -d . Treat-
ment of 12a -c under Heck cyclization conditions28
resulted in the formation of 3, 4a , or 5a as the major
product in yields ranging from 34 to 38%. Reduction of
4a and 5a with Ra-Ni in the presence of hydrazine
provided the amino derivatives, 4b and 5b.
Reaction of 12d under identical cyclization conditions,
as shown in Scheme 2, resulted in the formation of 6a
together with 13 as a major side product. The involve-
ment of the tertiary amine in the formation of this side-
product was demonstrated using the N-butyl-o-bromo-

2256 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Singh et al.
Sch em e 2a
(a) (COCl)2, CH2Cl2, 11, and then TEA, CH2Cl2; (b) Pd(OAc)2, P(o-tolyl)3, Ag2CO3, DMF; (c) RaNi, H2NNH2‚H2O.
Ta ble 1. TOP1-Targeting Activity and Cytotoxicity
cytotoxicity (IC50; µM)b
TOP1-mediated
compound
DNA cleavage (REC)a RPMI8402
CPT-K5
1
2
3
4a
4b
5a
5b
6a
6b
0.5
0.3
6
0.002
0.001
0.06
4.8
0.34
0.002
0.025
0.003
0.08
3.0
0.24
1.25
0.005
0.57
0.012
0.22
0.9
0.6
0.95
> 10
3.2
1000
15
0.1
2.8
1.0
0.5
0.2
8.0
0.33
0.38
> 10
> 10
> 10
61
>100
> 50
0.28
13
16a
16b
Camptothecin
CPT-11
Topotecan
VM-26
1000
10
500
0.5
25
1
>1000
F igu r e 1. Stimulation of enzyme-mediated DNA cleavage by
topotecan (TPT), 6a , 5a , 4a using human TOP1. The first lane
is DNA control without enzyme. The second lane is the control
with enzyme alone. The rest of the lanes contain human TOP1
and serially (10-fold each) diluted compound from 0.001 to 1.0
µM.
a
TOP1 cleavage values are reported as REC, relative effective
concentration, i.e., concentrations relative to topotecan, whose
value is arbitrarily assumed as 1, that produce the same degree
of cleavage of the plasmid DNA in the presence of human TOP1.
b
IC50 was calculated after 4 days of continuous drug exposure and
are the average of two or more replicate assays.
in TOP1-targeting activity and cytotoxicity relative to
its nitro analogue, 4a . The steric interactions between
the nitro group that resides within the bay-region
(position 10-10a-10b-11) and the hydrogen atom
attached at C11 may force the nitro group out of the
plane of the benzo-ring. This alteration in topology may
not be favored for retention of TOP1-targeting activity.
Reduction of the nitro group reduces the steric inter-
action that can occur within this bay-region and could
be responsible for the enhancement observed for 4b
tion that occurs in the presence of TOP1 and various
concentrations of 4a , 5a , and 6a can be seen in Figure
1.
The TOP1-targeting activities of the amino analogues
5b and 6b are 5- and 40-fold less, respectively, than
their nitro derivatives 5a and 6a . This difference is
reflected in their decreased cytotoxic activity toward
RPMI8402 relative to their nitro analogues. In the case
of 4b, however, there was a significant enhancement

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These data indicate that a nitro substituent at either
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Ack n ow led gm en t. We thank Ms. Rebekah Burr for
performing the HPLC analyses. This study was sup-
ported by AVAX Technologies, Inc, (E.J .L.) and Grant
CA39662 (L.F.L.) and Grant CA077433 (L.F.L.) from the
National Cancer Institute.
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
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experimental details of in vitro assays. This material is
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