11H-Isoquino[4,3-c]cinnolin-12-ones: Novel anticancer agents with potent topoisomerase I-targeting activity and cytotoxicity

Article abstract of DOI:10.1016/j.bmc.2003.10.061

Recent studies have identified 2,3-dimethoxy-8,9-methylenedioxy-11-[(2- dimethylamino)ethyl]-11H-isoquino[4,3-c]cinnolin-12-one (1a) as a novel topoisomerase I-targeting agent with potent cytotoxic activity. The effect of varied substituents at the 11-pos

Full text of DOI:10.1016/j.bmc.2003.10.061

Bioorganic & Medicinal Chemistry 12 (2004) 795–806
11H-Isoquino[4,3-c]cinnolin-12-ones:
novel anticancer agents with potent topoisomerase I-targeting
activity and cytotoxicity
Alexander L. Ruchelman,a Sudhir K. Singh,a Abhijit Ray,a Xiaohua Wu,a,b
Jin-Ming Yang,b Nai Zhou,c Angela Liu,c Leroy F. Liub,c and Edmond J. LaVoiea,b,
*
aDepartment of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA
bThe Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
cDepartment of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School,
Piscataway, NJ 08854, USA
Received 21 May 2003; accepted 23 October 2003
Abstract—Recent studies have identified 2,3-dimethoxy-8,9-methylenedioxy-11-[(2-dimethylamino)ethyl]-11H-isoquino[4,3-c]cin-
nolin-12-one (1a) as a novel topoisomerase I-targeting agent with potent cytotoxic activity. The effect of varied substituents at the
11-position of 2,3-dimethoxy-8,9-methylenedioxy-11H-isoquino[4,3-c]cinnolin-12-ones on topoisomerase I-targeting activity and
cytotoxicity was evaluated. Potent TOP1-targeting activity was observed when the 11-position was substituted with either a 2-(N,N-
dimethylamino)ethyl, a 2-(N,N-diethylamino)ethyl, a n-butyl, or a 2-(pyrrolidin-1-yl)ethyl group. The addition of a b-methyl group
to 1a provided an analogue with dramatically reduced TOP1-targeting activity and cytotoxicity. Analogues of 1a wherein the 2-
(N,N-dimethylamino)ethyl group was replaced with a (2-tetrahydrofuranyl)methyl, a 2-(piperidin-1-yl)ethyl, or a 2-(4-methylpi-
perazin-1-yl)ethyl substituent exhibited decreased activity as TOP1-targeting agents. Replacement of the dimethoxy groups of 1a
with hydrogen atoms resulted in an analogue with significantly decreased TOP1-targeting activity and cytotoxicity. Removal of
both the vicinal dimethoxyl groups and the methylenedioxy moiety resulted in a complete loss of TOP1-targeting activity. The
presence of a 9-nitro substituent in place of the 8,9-methylenedioxy group of 1a resulted in a decrease in relative TOP1-targeting
activity and cytotoxicity. Compounds 1a and the 11-n-butyl analogue 1d were evaluated for antitumor activity in the human tumor
xenograft model using athymic nude mice. The non-estrogen responsive breast tumor cell line MDA-MB-435 was used in these
assays. At dose levels that approached its maximum tolerated dose, 1a proved to be effective in inhibiting tumor growth in vivo
when administered orally or by ip injection.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
The drug-induced stabilization of the enzyme–DNA
cleavable complex effectively converts these nuclear
enzymes into cellular poisons.
Topoisomerases are nuclear enzymes that regulate the
topology of DNA and are critical for replication and
transcription. There are two major subtypes, topo-
isomerase I (TOP1) and topoisomerase II (TOP2),
which are distinguished based upon differences in their
initial mechanisms wherein a single or double-stranded
DNA break is implicated.1ꢀ4 Topoisomerase-targeting
agents stabilize the cleavable complex formed between
the enzyme and DNA resulting in the formation of
double-strand breaks. Topoisomerase-targeting agents
are acknowledged as effective in the treatment of cancer.
Camptothecin was the first compound identified as a
TOP1-targeting agent.5 From the extensive studies on
camptothecin and its structurally related analogues, two
clinical agents, topotecan (Hycamtin1) and irinotecan
(CPT-11/Camptosar1), have been developed. As in
camptothecin, these drugs have incorporated within
their structures a g-lactone that is susceptible to hydro-
lysis. Ring-opening of this lactone results in the forma-
tion of an inactive derivative with high affinity for
human serum albumin.6ꢀ8 The instability of this lactone
and the observation that both topotecan and irinotecan
are substrates for efflux transporters associated with
* Corresponding author. Tel.: +1-732-445-2674; fax: +1-732-445-
6312; e-mail: elavoie@rci.rutgers.edu
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.10.061

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A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
resistance have prompted further studies on the
development of novel TOP1-targeting agents.9ꢀ12
8a–h. For those analogues that did not possess a di-
alkylaminoethyl group, relatively poor yields of the
desired amides were obtained. In the case of these vari-
ous 4-aminocinnoline derivatives, competitive acylation
at the N1 position of the cinnoline moiety significantly
lowered the yield. This result is consistent with previous
studies on the reactivity of 4-aminocinnolines,28 and
explains the poor yields obtained in the acylation of 8d
and 8e (19 and 34%, respectively). In the case of 4-[(2-
dialkylaminoethyl)amino]cinnolines, however, acylation
of the 4-amino group occurs exclusively and the yield
obtained for 8a–c ranged from 75 to 87%.
There have been several reports on novel non-camp-
tothecin TOP1-targeting agents. These include
derivatives of bi- and terbenzimidazoles,13,14 benz[a]an-
thracenes,15 benzo[c]phenanthridine and protoberberine
alkaloids,16,17 indolocarbazoles,18 the fungal metabolites
bulgarein19 and saintopin,20 and several indenoisoqui-
nolines,21 benzophenazines22 and benzo[i]phenan-
thridines.23,24 These studies were performed in an effort
to develop TOP1-targeting agents with the potential for
improved efficacy.
These results suggest that the strong tendency for 4-
dialkylaminoethylaminocinnolines to form an intramo-
lecular hydrogen bond through a cyclic five-membered
structure, which has been documented in other stud-
ies,29 promotes the desired acylation of the 4-amino
substituent. The tendency to form such intramolecular
hydrogen bonds would increase the electron density of
the 4-amino group, thereby increasing its nucleophili-
city.
Studies in our laboratory have recently demonstrated that
2,3 - dimethoxy - 8,9 - methylenedioxydibenzo[c,h]cinno-
line,25 5H-dibenzo[c,h][1,6]naphthyridin-6-ones26,27 and
11H-isoquino[4,3-c]cinnolin-12-ones26 can exhibit potent
TOP1-targeting activity and pronounced cytotoxicity
(Chart 1).
In the present study, the synthesis and pharmacological
evaluation of several 11-substituted 2,3-dimethoxy-8,9-
methylenedioxy-11H-isoquino[4,3-c]cinnolin-12-ones and
variously substituted 11-(2-dimethylamino)ethyl-11H-
isoquino[4,3-c]cinnolin-12-ones, as outlined in Fig. 1,
were performed. The results of these studies are
detailed.
Intramolecular cyclization was performed with the o-
iodobenzamides, 8a–h, using the Heck reaction, to pro-
vide the 11-substituted 11H-isoquino[4,3-c]cinnolin-12-
ones, 1a–h. The Heck reaction conditions that were
employed in this study were those that proved optimal
for formation of benzo[c]phenanthridine derivatives.30
The preparation of 11-(2-dimethylaminoethyl)-11H-iso-
quino[4,3-c]cinnolin-12-ones 2–5 was accomplished
using the approach outlined in Scheme 2. 4-Chloro-
cinnoline 9 was prepared as described in the literature
and refluxed with N,N-dimethylethylenediamine to pro-
vide 11. Chlorination of 6-nitro-4-cinnolone with POCl3
(either with or without PCl5) provides poor yields of
4-chloro-6-nitrocinnoline, due to the replacement of the
nitro group by chloride. Under different conditions,
using thionyl chloride and a catalytic amount of PCl5 in
chlorobenzene, 4-chloro-6-nitrocinnoine was prepared
as previously described.31 As 4-chloro-6-nitrocinnoline is
reportedly unstable, it was converted in situ to 4-phen-
oxy-6-nitrocinnoline 10,31 which could be isolated and
served as a useful intermediate for the preparation of 12.
2. Chemistry
A series of 11-substituted 2,3-dimethoxy-8,9-methylene-
dioxy-11H-isoquino[4,3-c]cinnolin-12-ones were synthe-
sized as outlined in Scheme 1. Compound 6 was
prepared from 6,7-methylenedioxy-4-cinnolone by
treatment with a mixture of PCl5 and POCl3. The
desired 4-alkylaminocinnolines, 7a–h, were then pre-
pared by reaction of the appropriately substituted
primary alkylamine with 6.
Conversion of 2-iodo-4,5-dimethoxybenzoic acid to its
acid chloride and subsequent treatment with the appro-
priate 4-amino-6,7-methylenedioxycinnoline and TEA
in anhydrous methylene chloride provided the amides
Chart 1. Structure and numbering of 2,3-dimethoxy-8,9-methylenedioxydibenzo[c,h]cinnoline (DBC-74), 5H-dibenzo[c,h][1,6]-naphthyridin-6-ones
and 11H-isoquino[4,3-c]cinnolin-12-ones.

A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
797
Figure 1. The 11-substituted 11H-isoquino[4,3-c]cinnolin-12-ones synthesized and evaluated in this study.
Scheme 1. (i) Reflux or ꢁ135 ꢂC; (ii) (COCl)2, CH2Cl2, then TEA, CH2Cl2; (iii) Pd(OAc)2, P(o-tolyl)3, Ag2CO3, DMF.
Treatment of 7a and 11 with the acid chloride derived
from 2-iodobenzoic acid provided the o-iodobenzamides
13 and 14, respectively. Reaction of 11 and 12 with the
acid chloride formed from 2-iodo-4,5-dimethoxybenzoic
acid yielded the amides, 15 and 16. Using Heck intra-
molecular cyclization conditions, 13–16 were converted
to 2–5.
in the presence of TOP1 and DNA are illustrated in
Figures 2 and 3.
Compounds 1a–h represent several 11-substituted deriva-
tives of 2,3-dimethoxy-8,9-methylenedioxy-11H-iso-
quino[4,3-c]cinnolin-12-one. The presence of either a
2-(N,N-dimethylamino)ethyl, 2-(N,N-diethylamino)ethyl,
a n-butyl, or 2-(pyrrolidin-1-yl)ethyl substituent at the
11-position is associated with potent TOP1-targeting
activity. All four of these analogues, 1a, 1b, 1d, and 1f had
IC50 values <5 nM when evaluated for cytotoxicity in the
human lymphoblast cell line, RPMI8402. Each of these
compounds also exhibited significant cross resistance in
the camptothecin-resistant variant cell line, CPT-K5.
Addition of a b-methyl substituent to the 11-(2-ami-
noethyl) side chain as in 1c adversely affects TOP1-tar-
geting activity and cytotoxicity. Compound 1c is more
than 300-fold less active as a TOP1-targeting agent and
is over two orders of magnitude less cytotoxic than 1a.
3. Pharmacology
3.1. In vitro studies
The 11-substituted 11H-isoquino[4,3-c]cinnolin-12-ones
synthesized in this study were evaluated for their relative
TOP1-targeting activity and for cytotoxicity. These data
are provided in Table 1. Representative gels illustrating
the potential of some of these compounds at various
concentrations to stabilize the cleavable complex formed

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A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
Scheme 2. (i) Reflux or ꢁ135 ꢂC; (ii) (COCl)2, CH2Cl2, then TEA, CH2Cl2; (iii) Pd(OAc)2, P(o-tolyl)3, Ag2CO3, DMF.
The 2-(tetrahydrofuranyl)methyl derivative 1e is less
active as a TOP1-targeting agent than the 2-(pyrrolidin-
1-yl)ethyl analogue 1f and less cytotoxic than 1f to
RPMI8402 and P388 cells. Cross-resistance was
observed with 1e to the camptothecin-resistant variants,
CPT-K5 and P388/CPT45, respectively.32,33
that its cytotoxicity is largely mediated by interaction
with TOP1. Compound 1h is two orders of magnitude
less active as a TOP1-targeting agent than 1f, and is also
significantly less cytotoxic in RPMI8402 or P388 cells
(75–80 nM vs 3.0 nM). In addition to increased steric
bulk at the 11-position, 1h also has an additional tertiary
amine that may be associated with its decreased activity.
Comparison of the relative TOP1-targeting activity of 1f
with either 1g or 1h suggests that additional steric con-
straints, other than substitution at the b-position of the
ethyl side chain, at the 11-position may influence TOP1-
targeting activity. While 1g is 10-fold less potent than 1f
as a TOP1-targeting agent, it exhibits similar cytotoxic
activity. Based upon the cross-resistance observed in
both CPT-K5 and P388/CPT45 cells with 1g, it appears
The effect of removal of the dimethoxyl groups from 1a
was investigated by evaluating the relative activity of 2.
For compound 2, we observed over a thirtyfold decrease
in TOP1-targeting activity and over a hundredfold dif-
ference in cytotoxicity in RPMI8402 relative to 1a.
Replacement of the methylenedioxy moiety of 1a with
hydrogen, as in 4, results in a slight reduction in
Table 1. Relative TOP1-targeting activity and cytotoxicity of 11-substituted 11H-isoquino[4,3-c]cinnolin-12-ones
Compd
TOP1-mediated DNA cleavage
Cytotoxicity IC50 (mM)
RPMI8402
CPT-K5
P388
P388/CPT45
1a
1b
1c
1d
1e
1f
1g
1h
2
3
4
5
0.3
0.3
100
0.5
5.0
0.2
2.0
20
10
0.002
0.004
0.26
0.004
0.01
0.003
0.003
0.08
0.24
3.3
0.03
0.03
0.05
0.005
0.57
0.012
0.22
0.74
0.51
3.6
0.002
0.002
0.11
0.004
0.02
0.003
0.003
0.08
0.09
0.12
0.032
0.033
0.033
0.009
2.3
0.23
0.28
0.56
1.2
10
>10
0.6
1.8
0.26
0.27
1.1
1.1
8.0
2.5
5.0
1.4
0.67
0.11
0.18
0.18
0.55
>10
>10
>100
>10
0.001
>1000
0.8
5.0
DBC-74
CPT
CPT-11
Topotecan
VM-26
8.0
0.5
25
>10
61
>100
>50
1.0
>1000
0.04
0.006
0.28

A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
799
implanted into athymic nude mice. Two separate bioas-
says were performed. Compound 1a, as detailed in the
Experimental, was administered by both gavage (po)
and intraperitoneal injection (ip) in assay at doses that
approached its maximum tolerated dose (MTD). In
assay B, 1a was administered po. As 1d and 2,3-dime-
thoxy-8,9-dimethoxydibenzo[c,h]cinnoline
(DBC-74)
lack sufficient solubility in vehicles suitable for ip
administration, these compounds were given by po
administration as outlined for assay B. The limited
bioavailability of 1d and 2,3-dimethoxy-8,9-methylene-
dioxydibenzo[c,h]cinnoline (DBC-74, Chart 1) pre-
cluded establishing meaningful MTD levels for these
compounds. Table 2 summarizes the results obtained
from these assays. Despite escalation of dose, there was
no effect on tumor growth observed after more than two
weeks of treatment in mice treated with either 1d or
DBC-74. Mice in these experimental groups were ter-
minated on day 16. The total dose reported in Table 2
reflects the total dose administered to each experimental
group for the duration of the study.
Figure 2. Stimulation of enzyme-mediated DNA cleavage by topote-
can (TPT), 1b, 1c, 1g, and 1h 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 mM.
The data from both assay A and assay B suggest that 1a
does exert antitumor activity in vivo. While 1a appears
to be quite potent, its dose-limiting toxic effects appear
to limit its efficacy in these bioassays. Relative to CPT-
11, this analogue as substantially less efficacious when
administered ip. The lack of activity observed with 1d
and DBC-74 are in all likelihood associated with their
poor solubility and limited bioavailability. While both
of these compounds are cytotoxic in vitro, their limited
bioavailability most likely excludes detectable antitumor
activity in vivo. Despite evidence to suggest that 1a
exhibits somewhat greater potency in vitro, as noted in
an earlier communication, it appears to be less effica-
cious in vivo than similarly substituted dibenzo[c,h]-
naphthyridin-6-ones. Differences in the metabolic fate
of members within each of these families of novel
TOP1-targeting agents are likely responsible for this
difference between in vitro and in vivo results.
Figure 3. Stimulation of enzyme-mediated DNA cleavage by topote-
can (TPT), 1a, 2, 3, 4, and 5 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 mM.
TOP1-targeting activity, and a decrease in cytotoxicity
that is greater than an order of magnitude. Replacement
of not only the vicinal methoxyl groups, but also the
methylenedioxy substituent of 1a with hydrogen atoms,
as in 3, results in a loss of TOP1-targeting activity and
significantly decreased cytotoxic activity. Studies in our
laboratory have demonstrated that a nitro substituent at
the 3 position of 5H-8,9-dimethoxy-5-(2-N,N-dimethyl-
aminoethyl)dibenzo[c,h][1,6]-naphthyridin-6-one allows
for retention of significant TOP1-targeting activity and
cytotoxicity (unpublished results). The analogous nitro
analogue of 2,3-dimethoxy-11H-isoquino[4,3-c]cinnolin-
12-one, 5, was prepared in the present study to examine
its activity as a TOP1-targeting agent and its relative
cytotoxic activity. A decrease in TOP1-targeting activity
relative to 1a was observed for 5 similar in magnitude to
that observed for 1e. Compound 5, however, did exhibit
significant cytotoxicity in RMI8402 and P388 cells as
well as significant cross-resistance in their camptothe-
cin-resistant variants.
4. Experimental
Melting points were determined with either a Thomas-
Hoover Unimelt or Meltemp capillary melting point
apparatus. Column chromatography refers to flash
chromatography conducted on SiliTech 32-63 mm, (ICN
Biomedicals, Eschwege, Germany) using the solvent
systems indicated. Proton (1H NMR) and carbon (13C
NMR) nuclear magnetic resonance were recorded on a
Varian Gemini-200 Fourier Transform spectrometer.
Infrared spectral data were obtained using a Thermo-
Nicolet Avatar 360 Fourier transform spectro-
photometer and are reported in cmꢀ1. NMR spectra
1
(200 MHz H and 50 MHz 13C) were recorded in the
deuterated solvent indicated with chemical shifts repor-
ted in d units downfield from tetramethylsilane (TMS).
Coupling constants are reported in hertz (Hz). Mass
spectra were obtained from Washington University
Resource for Biomedical and Bio-organic Mass Spec-
trometry within the Department of Chemistry at
Washington University, St. Louis, MO, USA.
3.2. In vivo assays
Compound 1a and 1d as well as 2,3-dimethoxy-8,9-
methylenedioxydibenzo[c,h]cinnoline (Chart 1, DBC-74)
were selected for evaluation in the human tumor xeno-
graft athymic nude mouse model. Using the human
breast tumor cell line, MDA-MB-435, tumor cells were

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A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
Table 2. Antitumor activity observed in athymic nude mice with the human tumor xenograft MDA-MB-435
Compd
Assaya
# of mice
Route
Average tumor volume (mm3)
Total dose (mg/kg)/mouse
Day 7
Day 16
Day 21
Day 31
1a
1a
Vehicle
CPT-11
A
A
A
A
7
7
7
7
pob
ipb
ipc
159
123
133
99
148
84
152
98
99
65
188
40
62
40
298
14
0.78
0.72
ipd
17.38
1a
1d
DBC-74
Vehicle
CPT-11
B
B
B
B
B
7
7
7
7
7
poe
pof
pof
ipc
139
206
209
199
119
194
345
267
277
65
190
—
—
356
30
76
—
—
472
10
0.29
4.34
4.76
ipd
13.89
a Two separate assays were performed. Both assay A and B used female NCR/NU NU mice.
b Initial dose was 2.0 mg/kg qdꢃ4/week. Administration was adjusted to approximately 3ꢃweek in view of weight loss. Dosing was withheld from
some mice with notable weight loss.
c Vehicle consisted of 0.1% citrate in H2O.
d Administered at a dose of 20 mg/kgꢃ5/week.
e Initial dose was 1.0 mg/kg qdꢃ3/week. Dosing was withheld from some mice with notable weight loss.
f Administered initially at 10 mg/kg qdꢃ3/week, increased to 10 mg/kg qdꢃ7/week. Dose administered was limited by the solubility of compound in
30% Cremophor EL adjusted to pH 3.0.
4.1. General procedure for the preparation of 11-sub-
stituted 11H-isoquino[4,3-c]cinnolin-12-ones via cycli-
zation of o-iodobenzamides
(1c). Prepared from 8c (100 mg, 0.18 mmol); (28%
yield); reaction time 2 h; mp 235–236 ꢂC; IR (KBr) 1659:
1H NMR (CDCl3) d 1.93 (d, 3H, J=8.2), 1.97 (s, 3H),
2.74 (dd, 1H, J=5.8,13.6), 3.27 (dd, 1H, J=7.4,12.8),
4.07 (s, 3H), 4.15 (s, 3H), 4.80 (m, 1H), 6.24 (s,2H), 7.74
(s,1H), 7.81 (s,1H), 8.57 (s,1H); 13C NMR (CDCl3) d
19.4, 45.6, 56.3, 58.6, 63.0, 99.0, 102.6, 104.1, 106.2,
107.9, 114.2, 120.8, 125.6, 128.6, 131.0, 132.5, 132.8,
135.1, 149.2, 150.3, 150.6, 151.3, 154.2, 164.0; HRMS
calcd for C23H24N4O5H 436.1826; found 436.1832.
A mixture of the 4-amino-6,7-methylenedioxycinnoline
o-iodobenzamide derivative (1.0 mmol equiv), Pd(OAc)2
(0.2 mmol equiv), P(o-tolyl)3 (0.4 mmol equiv), and
Ag2CO3 (2.0 mmol equiv) was heated to reflux in DMF
(30 mL per mmol equiv) with stirring. The reaction mix-
ture was allowed to cool to room temperature, diluted
with CHCl3, and filtered through Celite. The sicciate was
extensively washed with 10% CH3OH in CHCl3. The
filtrate was concentrated in vacuo and the residue chro-
matographed on silica gel using chloroform:methanol.
4.1.4. 2,3-Dimethoxy-8,9-methylenedioxy-11-(n-butyl)-
11H-isoquino[4,3-c]cinnolin-12-one (1d). Prepared from
8d (123 mg, 0.2 mmol); (27% yield); reaction time 90
ꢂ
1
min; mp 299 C; IR (KBr) 1654; H NMR (CDCl3) d
1.06 (t, 3H, J=7.4), 1.56 (m, 2H), 2.13 (m, 2H), 4.09 (s,
3H), 4.17 (s, 3H), 4.49 (m, 2H), 6.26 (s, 2H), 7.62 (s,
1H), 7.85 (s, 1H), 7.87 (s, 1H), 8.65 (s, 1H); 13C N MR
(CDCl3) d 13.8, 20.2, 31.1, 48.6, 56.3, 56.6, 98.8,102.7,
104.2, 106.5, 107.9, 119.7, 149.1, 150.1, 150.9, 151.4,
153.6, 154.2, 162.9; HRMS calcd for C22H21N3O5H:
408.1559; found 408.1543.
4.1.1.
2,3-Dimethoxy-8,9-methylenedioxy-11-[(2-dime-
thylamino)ethyl]-11H-isoquino[4,3-c]cinnolin-12-one (1a).
Prepared from 8a (220 mg, 0.40 mmol); (36% yield);
reaction time 25 min; mp 288–289 ꢂC (dec.); IR (CHCl3)
1
1653; H NMR (CDCl3) d 2.42 (s, 6H), 3.04 (t, 2H,
J=7.2), 4.08 (s, 3H), 4.17 (s, 3H), 4.64 (t, 2H, J=7.2),
6.25 (s, 2H), 7.81 (s, 1H), 7.84 (s, 1H), 8.07 (s, 1H), 8.65
(s, 1H); 13C NMR (CDCl3) d 45.9, 47.4, 56.4, 56.7, 57.7,
99.4, 102.8, 104.3, 106.6, 107.9, 113.7, 119.6, 129.1,
131.0, 134.4, 149.4, 150.2, 151.5, 154.4, 163.1; HRMS
calcd for C22H22O5N4H: 423.1668; found: 423.1653.
4.1.5. 2,3-Dimethoxy-8,9-methylenedioxy-11-(2-tetrahy-
drofuranyl)methyl-11H-isoquino[4,3-c]cinnolin-12-one (1e).
Prepared from 8e (140 mg, 0.25 mmol); (22% yield);
reaction time 45 min; mp 300–303 ꢂC (dec.); IR (CHCl3)
1
4.1.2. 2,3-Dimethoxy-8,9-methylenedioxy-11-[(2-diethyl-
amino)ethyl]-11H-isoquino[4,3-c]cinnolin-12-one (1b).
Prepared from 8b (578 mg, 1.0 mmol); (18% yield);
1653; H NMR (CDCl3) d 1.79 (m, 1H), 2.00 (m, 2H),
2.25 (m, 1H), 3.87 (m, 2H), 4.09 (s, 3H), 4.18 (s, 3H),
4.65 (m, 3H), 6.25 (s, 2H), 7.80 (s, 1H), 7.84 (s, 1H), 8.32
(s, 1H), 8.63 (s, 1H); 13C NMR (CDCl3) d 25.7, 30.8,
53.0, 56.4, 56.7, 68.4, 77.8, 100.0, 102.7, 104.3, 106.3,
108.0, 114.1, 119.7, 129.1, 131.4, 134.5, 149.5, 150.2,
150.8, 151.4, 154.4, 163.7; HRMS calcd for
C23H21O6N3: 435.1430; found: 435.1427.
reaction time 25 min; mp 245–247 ꢂC (dec.); IR (CHCl3)
1
1652; H NMR (CDCl3) d 1.08 (t, 6H, J=7.0), 2.67 (q,
4H, J=7.0), 3.14 (t, 2H, J=7.1), 4.08 (s, 3H), 4.17 (s,
3H), 4.64 (t, 2H, J=7.1), 6.25 (s, 2H), 7.80 (s, 1H), 7.84
(s, 1H), 8.18 (s, 1H), 8.63 (s, 1H); 13C NMR (CDCl3) d
11.8, 47.7, 48.0, 51.5, 56.4, 56.6, 99.7, 102.7, 104.3,
106.4, 108.0, 113.7, 119.7, 129.1, 131.1, 134.4, 149.4,
150.3, 151.2, 151.5, 154.4, 163.2; HRMS calcd for
C24H26O5N4H: 451.1952; found: 451.1960.
4.1.6. 2,3-Dimethoxy-8,9-methylenedioxy-11-[2-(pyrroli-
din-1-yl)ethyl]-11H-isoquino[4,3-c]cinnolin-12-one (1f).
Prepared from 8f (150 mg, 0.2 mmol); (24% yield);
reaction time 30 min; mp 229 ꢂC; IR (KBr) 1644; H
1
4.1.3. 2,3-Dimethoxy-8,9-methylenedioxy-11-[(2-dimethy-
lamino)-1-methylethyl]-11H-isoquino[4,3-c]cinnolin-12-one
NMR (CDCl3) d 1.83 (m, 4H), 2.71 (m, 4H), 3.23 (t, 2H,
J=7.0), 4.06 (s, 3H), 4.61 (s, 3H), 4.63 (t, 2H, J=7.0),

A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
801
6.23 (s, 2H), 7.74 (s, 1H), 7.80 (s, 1H); 13C N MR
(CDCl3) d 23.7, 54.0, 54.2, 56.3, 56.6, 99.4, 102.7, 104.2,
106.3, 107.7, 113.5, 119.4, 129.0, 134.1, 140.2, 150.2,
151.4, 154.3, 154.3, 163.0; HRMS calcd for
C24H24N4O5H: 449.1825; found: 449.1822.
108.0, 116.1, 119.8, 123.6, 129.2, 129.6, 130.4, 130.5,
131.4, 134.2, 150.0, 151.7, 154.6, 162.8; HRMS calcd for
C21H22N4O3H: 379.1771; found; 379.1772.
4.1.12.
11-[(2-Dimethylamino)ethyl]-2,3-dimethoxy-9-
nitro-11H-isoquino[4,3-c]cinnolin-12-one (5). Prepared
from 16 (220 mg, 0.4 mmol); (25% yield); reaction time
4.1.7. 2,3-Dimethoxy-8,9-methylenedioxy-11-[2-(piperi-
din-1-yl)ethyl]-11H-isoquino[4,3-c]cinnolin-12-one (1g).
Prepared from 8g (295 mg, 0.5 mmol); (32.4% yield);
reaction time 30 min; mp 294–295 ꢂC; IR (KBr) 1662;
1H NMR (CDCl3) d 1.59 (s, 6H), 2.51 (s, 4H), 3.02 (t,
2H, J=6.6), 4.08 (s, 3H), 4.17 (s, 3H), 4.64 (t, 2H,
J=6.6), 6.26 (s, 2H), 7.81 (s,1H), 7.85 (s, 1H), 8.36 (s,
1H), 8.65 (s, 1H); 13C NMR (CDCl3) d 24.3, 26.0, 47.5,
55.0, 56.3, 56.6, 57.4, 99.9, 102.7, 104.2, 106.3, 107.9,
113.7, 119.6, 129.0, 131.1, 134.3, 149.3, 150.2, 151.1,
151.4, 154.3, 163.1; HRMS calcd for C25H26N4O5H:
463.1981; found: 463.1986.
2 h; mp 262–264 ꢂC (dec.); H NMR (CDCl3) d 2.49 (s,
1
6H), 3.20 (t, 2H, J=7.0), 4.11 (s, 3H), 4.20 (s, 3H), 4.70
(t, 2H, J=7.0), 7.89 (s, 1H), 8.59 (dd, 1H, J=9.2,
J=1.8), 8.69 (s, 1H), 8.78 (d, 1H, J=9.0), 9.91 (d, 1H,
J=1.8); 13C NMR (CDCl3) d 46.1, 48.2, 56.5, 56.8, 57.3,
104.5, 108.1, 115.0, 120.0, 122.0, 123.1, 128.5, 131.5,
133.2, 135.4, 147.7, 149.9, 152.4, 154.9, 162.4; IR
(CHCl3) 1347, 1533, 1663; HRMS calcd for
C21H22N5O5H: 424.1621; found: 424.1616.
4.1.13. 4-Chloro-6,7-methylenedioxycinnoline (6). 6,7-
Methylenedioxy-4-cinnolone (1.0 g, 5.3 mmol) was
added in small portions to a stirred mixture of phos-
phorus pentachloride (1.4 g, 6.7 mmol) and phosporus
oxychloride (4 mL, 6.6 mmol) at room temperature. The
reaction flask was heated to 80 ꢂC for 4 h, then cooled to
room temperature and poured onto 50 g of crushed ice.
Following neutralization of the solution with solid
sodium acetate, the precipitate was removed by fil-
tration and recrystallized from ethanol to give 800 mg
of the chlorocinnoline as an off-white solid, in 73%
4.1.8. 2,3-Dimethoxy-8,9-methylenedioxy-11-[2-(4-me-
thylpiperazin-1-yl)ethyl]-11H-isoquino[4,3-c]cinnolin-12-
one (1h). Prepared from 8h (726 mg, 1.2 mmol); (23%
ꢂ
1
yield); reaction time 30 min; mp 292–295 C (dec.); H
NMR (CDCl3) d 2.29 (s, 3H), 2.40 (m, 4H), 2.61 (m,
4H), 3.04 (t, 2H, J=6.6), 4.08 (s, 3H), 4.17 (s, 3H), 4.64
(t, 2H, J=6.6), 6.25 (s, 2H), 7.78 (s, 1H), 7.82 (s, 1H),
8.16 (s, 1H), 8.61 (s, 1H); 13C NMR (CDCl3) d 46.1,
47.2, 53.5, 55.1, 56.4, 56.5, 56.7, 99.6, 102.7, 104.3,
106.4, 108.0, 113.7, 119.6, 129.0, 131.1, 134.4, 149.3,
150.2, 151.2, 151.5, 154.4, 163.2; IR (CHCl3) 1653;
HRMS calcd for C25H27N5O5H: 478.2090; found:
478.2088.
ꢂ
1
yield; mp 203.5–204.5 C; H NMR (CDCl3) d 6.25 (s,
2H), 7.39 (s, 1H), 7.73 (s, 1H), 9.14 (s, 1H); 13C N MR
(CDCl3) d 97.8, 102.9, 105.1, 124.2, 133.4, 144.0, 150.0,
152.3, 152.7; HRMS calcd for C9H5O2N2Cl: 208.0040;
found; 208.0042.
4.1.9. 8,9-Methylenedioxy-11-[2-(N,N-dimethylamino)-
ethyl]-11H-isoquino[4,3-c]cinnolin-12-one (2). Prepared
from 13 (343 mg, 0.7 mmol); (22% yield); reaction time
4.1.14. 6,7-Methylenedioxy-4-cinnolone. A mixture of 60-
amino-30,40-methylenedioxyacetophenone (2.40 g, 13.4
mmol) in concentrated hydrochloric acid (92 mL) and
water (13 mL) was cooled to ꢀ5 ꢂC and a diazotized by
the dropwise addition of a solution of sodium nitrite
(0.925 g, 13.4 mmol) in water (4 mL). After stirring for
an additional 1 h at ꢀ5 ꢂC the mixture was transferred
to a bath preheated at 75 ꢂC and left to stir at this tem-
perature overnight. The reaction mixture was cooled to
5 ꢂC to induce crystallization. This material was filtered
and then added to 10% aqueous NaOH (100 mL),
which was again filtered and dried under vacuum to
yield 2.37 g of the cinnoline as a colorless solid, in 93%
25 min; mp 229–230 ꢂC; IR (CHCl3) 1663; H N MR
1
(DMSO-d6) d 2.26 (s, 6H), 2.92 (t, 2H, J=6.6), 4.58 (t,
2H, J=6.6), 6.38 (s, 2H), 7.79 (m, 2H), 7.99 (s, 1H),
8.00 (m, 1H), 8.34 (d, 1H, J=8.4), 9.04 (d, 1H, J=8.0);
13C NMR (DMSO-d6) d 45.1, 47.8, 56.9, 99.9, 104.1,
105.9, 113.9, 123.7, 125.8, 127.1, 128.4, 130.7, 134.1,
134.6, 139.6, 150.1, 151.5, 151.9, 163.5; HRMS calcd for
C20H18O3N4H: 363.1457; found: 363.1453.
4.1.10. 11-[(2-Dimethylamino)ethyl]-11H-isoquino[4,3-c]-
cinnolin-12-one (3). Prepared from 14 (140 mg, 0.4
mmol); (15% yield); reaction time 30 min; mp 179 ꢂC;
yield; mp 318–320 ꢂC; H NMR (DMSO-d6) d 6.21 (s,
1
1
IR (KBr) 1664; H NMR (CDCl3) d 2.41 (s, 6H), 3.06
2H), 6.97 (s, 1H), 7.30 (s, 1H), 7.63 (s, 1H); 13C N MR
(DMSO-d6) d 94.9, 100.3, 103.3, 120.1, 139.7, 139.9,
147.4, 153.5, 169.4; HRMS calcd for C9H6O3N2:
190.0378; found: 190.0372.
(t, 2H, J=7.4), 4.76 (t, 2H, J=6.8), 7.72–8.00 (m, 4H),
8.50 (d, 1H, J=8.0), 8.65–8.70 (m, 2H), 9.30 (d, 1H,
J=7.8); 13C NMR (CDCl3) d 45.9, 47.4, 57.3, 123.6,
124.0, 125.5, 127.9, 130.0, 130.5, 131.3, 133.8, 163.4;
HRMS calcd for C19H18N4OH: 319.1551; found
319.1560.
4.2. General procedure for the formation of 4-amino-6,7-
methylenedioxycinnolines (7a–h)
4.1.11. 11-[(2-Dimethylamino)ethyl]-2,3-dimethoxy-11H-
isoquino[4,3-c]cinnolin-12-one (4). Prepared from 15
(150 mg, 0.3 mmol); (40% yield); reaction time 25 min;
The appropriate primary amine (2 mL/mmol of 6 unless
noted otherwise) is added with stirring to intermediate
6. Copper powder was added in certain reactions as
indicated. The reaction was then allowed to stir at
100 ꢂC, unless otherwise noted, for several hours. The
reaction mixture was then concentrated in vacuo.
The residue was partitioned between CHCl3 and 10%
mp 218–219 ꢂC; IR (CHCl3) 1655; H NMR (CDCl3) d
1
2.42 (s, 6H), 3.06 (t, 2H, J=7.5), 4.09 (s, 3H), 4.19 (s,
3H), 4.76 (t, 2H, J=7.5), 7.85 (m, 3H), 8.67 (m, 3H);
13C NMR (CDCl3) d 45.9, 47.3, 56.4, 56.7, 57.4, 104.5,

802
A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
NaOH. The aqueous layer was repeatedly extracted
with CHCl3. All of the CHCl3 solutions (initial partition
and extracts) were combined and dried (MgSO4).
(m, 4H), 2.63 (m, 4H), 2.90 (t, 2H, J=6.0), 3.42 (t, 2H,
J=6.0), 5.63 (s, 1H), 6.14 (s, 2H), 7.04 (s, 1H), 7.57 (s,
1H), 8.53 (s, 1H); 13C NMR (DMSO-d6) d 23.9, 42.0, 54.5,
54.7, 97.0, 102.9, 104.4, 112.7, 126.8, 140.8, 149.3, 151.0;
HRMS calcd for C15H18N4O2: 293.1590; found 293.1579.
4.2.1. N0-(6,7-Methylenedioxycinnolin-4-yl)-N,N-dime-
thylethane-1,2-diamine (7a). Prepared from 6 (350 mg,
1.7 mmol); (74% yield); reaction time 3 h; mp 228–
4.2.7. 1-[2-[N-(6,7-Methylenedioxycinnolin-4-yl)]amino]-
ethylpiperidine (7g). This was prepared from 6 (1.04 g,
5.0 mmol); (37% yield); reaction time 2 h; mp 238–
230 ꢂC; H NMR (CDCl3) d 2.33 (s, 6H), 2.70 (t, 2H,
1
J=5.9), 3.38 (m, 2H), 6.15 (s, 2H), 7.03 (s, 1H), 7.56 (s,
1H), 8.53 (s, 1H); 13C NMR (CDCl3) d 39.5, 45.1, 57.0,
94.7, 102.1, 105.3, 112.7, 128.8, 139.8, 147.8, 149.5,
150.7; HRMS calcd for C13H16O2N4: 260.1273; found:
260.1267.
ꢂ
1
239 C; H NMR (CD3OD) d 1.56 (d, 2H, J=5.2), 1.70
(d, 2H, J=4.6), 2.87 (t, 2H, J=7.0), 3.65 (t, 2H,
J=6.6), 6.20 (s, 2H), 7.32 (s, 1H), 7.43 (s, 1H), 8.46 (s,
1H); 13C NMR (CD3OD) d 23.1, 24.7, 38.5, 53.6, 56.1,
94.7, 101.7, 102.1, 112.4, 126.6, 141.1, 146.7, 149.4,
151.2 (CDCl3); HRMS calcd for C16H20N4O2H:
300.1586; found 300.1586.
4.2.2. N0-(6,7-Methylenedioxycinnolin-4-yl)-N,N-diethyl-
ethane-1,2-diamine (7b). Prepared from 6 (1.0 g, 4.8
mmol); (70% yield); reaction time 3 h; mp 230–232 ꢂC;
1H NMR (CDCl3) d 1.10 (t, 6H, J=7.2), 2.63 (q, 4H,
J=7.2), 2.84 (t, 2H, J=5.7), 3.35 (q, 2H, J=5.7), 5.78
(br, 1H), 6.15 (s, 2H), 6.96 (s, 1H), 7.57 (s, 1H), 8.52 (s,
1H); 13C NMR (CDCl3) d 12.2, 39.5, 46.6, 50.8, 94.4,
102.0, 105.4, 112.8, 129.0, 139.8, 147.8, 149.5, 150.7;
HRMS calcd for C15H20O2N4: 288.1586; found:
288.1575.
4.2.8. 1-[2-[N-(6,7-Methylenedioxycinnolin-4-yl)]amino]-
ethyl-4-methylpiperazine (7h). This was prepared from 6
(1.0 g, 4.8 mmol); (52% yield); reaction time 2 h at
120 ꢂC; mp 227–229 ꢂC; H NMR (CDCl3) d 2.30 (s,
1
3H), 2.48 (m, 4H), 2.54 (m, 4H), 2.76 (t, 2H, J=7.2),
3.37 (m, 2H), 5.73 (br, 1H), 6.11 (s, 2H), 6.99 (s, 1H),
7.51 (s, 1H), 8.47 (s, 1H); 13C NMR (CDCl3) d 38.8,
46.1, 52.7, 55.3, 55.6, 94.5, 102.1, 105.2, 112.7, 128.7,
139.4, 147.8, 149.5, 150.7; HRMS calcd for
C16H21N5O2: 315.1695; found: 315.1699.
4.2.3. N2-(6,7-Methylenedioxycinnolin-4-yl)-N1,N1-dime-
thylpropane-1,2-diamine (7c). Prepared from 6 (0.52 g,
2.5 mmol); (42% yield), reaction time 4 h, mp 196–
ꢂ
1
197 C; H NMR (CD3OD) d 1.31 (d, 3H, J=6.6), 2.33
(s, 6H), 2.45 (dd, 1H, J=5.4, 12.8), 2.74 (dd, 1H,
J=8.2, 12.6), 4.12 (dd, 1H, J=5.8, 13.8), 6.19 (s, 2H),
7.32 (s, 1H), 7.56 (s, 1H), 8.51 (s, 1H); 13C N MR
(CD3OD) d 17.1, 44.0, 45.3, 63.5, 95.1, 101.6, 102.0,
112.6, 126.7, 140.8, 149.3, 151.2; HRMS calcd for
C14H18O2N4: 274.1430; found: 274.1429.
4.3. General procedure for the formation of 2-Iodo-4,5-
dimethoxybenzamides (8a–h)
A 2.0 M solution of oxalyl chloride in CH2Cl2 (1.3
equiv) was added to
a solution of 2-iodo-4,5-
dimethoxybenzoic acid (1.0 equiv) in anhydrous CH2Cl2
(60 mL per 10 mmol benzoic acid) and the solution
stirred at reflux for 3 h. The mixture was allowed to cool
and was then concentrated to dryness in vacuo. To the
residue was added a solution of the appropriate
4-amino-6,7-methylenedioxycinnoline (1.0 equiv), tri-
ethylamine (2 equiv) in CH2Cl2 (60 mL per 4 mmol
aminocinnoline). The reaction mixture was then stirred
at reflux under N2. The reaction mixture was cooled and
washed with satd NaHCO3. The reaction mixture was
concentrated in vacuo. The product was isolated using
column chromatography for those analogues not pos-
sessing a tertiary amine. In the case of those analogues
containing a tertiary amine, the residue was extracted
with 3% HCl. The aqueous layer was neutralized with
20% NaOH and extracted with CHCl3, dried (MgSO4)
and evaporated.
4.2.4. N-(6,7-Methylenedioxycinnolin-4-yl)-n-butylamine
(7d). Prepared from 6 (1.0 g, 4.8 mmol), n-butylamine
(25 mL) and copper powder (250 mg); (32.5% yield);
reaction time 18 h at 80 ꢂC, mp 247–248 ꢂC; H N MR
1
(CDCl3) d 1.02 (t, 3H, J=7.4), 1.50 (m, 2H), 1.73 (m,
2H), 3.40 (m, 2H), 4.59 (s, 1H), 6.14 (s, 2H), 6.96 (s,
1H), 7.57 (s, 1H), 8.59 (s, 1H); 13C NMR (CDCl3) d
13.8, 20.2, 29.7, 31.3, 42.8, 94.2, 102.1, 105.3, 112.7,
126.7, 140.6, 149.6, 150.7; HRMS calcd for
C13H15N3O2H: 246.1243; found 246.1237.
4.2.5. 2-[[N-(6,7-Methylenedioxycinnolin-4-yl)]amino]me-
thyl]tetrahydrofuran (7e). This was prepared from 6
(500 mg, 2.4 mmol); (78% yield); reaction time 2 h; mp
196–198 ꢂC; H NMR (CDCl3) d 1.74 (m, 1H), 2.11 (m,
1
3H), 3.30 (m, 1H), 3.58 (m, 1H), 3.92 (m, 2H), 4.29 (m,
1H), 5.22 (br, 1H), 6.12 (s, 2H), 6.98 (s, 1H), 7.52 (s,
1H), 8.54 (s, 1H); 13C NMR (CDCl3) d 25.9, 29.2, 46.9,
68.4, 76.9, 94.4, 102.2, 105.2, 112.8, 128.7, 139.8, 147.9,
149.6, 150.8; HRMS calcd for C14H15O3N3: 273.1130;
found: 273.1130.
4.3.1. N-(6,7-Methylenedioxycinnnolin-4-yl)-N-[(2-N,N-
dimethylamino)ethyl] - 2 - iodo - 4,5 - dimethoxybenzamide
(8a). This was prepared from 7a (1.0 g, 3.84 mmol);
1
(75% yield); reaction time 4 h; IR (CHCl3) 1655; H
NMR (CDCl3) d 2.27 (s, 6H), 2.53 (m, 2H), 3.43 (s, 3H),
3.75 (s, 3H), 3.97 (m, 1H), 4.44 (m, 1H), 6.24 (s, 1H),
6.25 (s, 1H), 6.43 (s, 1H), 7.02 (s, 1H), 7.43 (s, 1H), 7.68
(s, 1H), 9.18 (s, 1H); 13C NMR (CDCl3) d 45.5, 47.1,
55.7, 56.1, 56.7, 82.8, 96.7, 102.9, 105.4, 110.6, 121.9,
123.2, 133.1, 136.0, 144.8, 148.2, 149.9, 150.9, 151.7,
152.4, 169.8; HRMS calcd for C22H23O5N4IH:
551.0791; found: 551.0795.
4.2.6. 1-[2-[N-(6,7-Methylenedioxycinnolin-4-yl)]amino]-
ethylpyrrolidine (7f). This was prepared from 6 (750
mg, 3.5 mmol), 1-(2-aminoethyl)pyrrolidine (3 mL) and
copper powder (300 mg); (75% yield); reaction time 18
h at 90 ꢂC; mp 215 ꢂC (dec); H NMR (CDCl3) d 1.85
1

A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
803
4.3.2. N-(6,7-Methylenedioxycinnolin-4-yl)-N-[2-(N,N-
diethylamino)ethyl]-2-iodo-4,5-dimethoxybenzamide (8b).
This was prepared from 7b (640 mg, 2.2 mmol); (87%
4.3.7. N-(6,7-Methylenedioxycinnolin-4-yl)-N-[2-(piperi-
din-1-yl)ethyl]-2-iodo-4,5-dimethoxybenzamide (8g). This
was prepared from 7g (500 mg, 1.66 mmol); (85%
yield); reaction time 16 h; mp 93–94 ꢂC; IR (KBr) 1655;
1H NMR (CDCl3) d 1.43 (m, 6H), 2.35 (m, 4H), 2.50–
2.71 (m, 2H), 3.43 (s, 3H), 3.73 (s, 3H), 3.78–3.93 (m,
1H), 4.32–4.42 (m, 1H), 6.22 (d, 2H, J=1.6), 6.42 (s,
1H), 7.02 (s, 1H), 7.47 (s, 1H), 7.66 (s, 1H), 9.19 (s, 1H);
13C NMR (CDCl3) d 24.3, 25.9, 46.0, 46.4, 54.5, 55.6,
56.0, 56.4, 82.9, 97.0, 102.8, 105.3, 110.8, 122.0, 113.7,
123.2, 133.1, 136.3, 145.0, 148.2, 149.9, 150.8, 151.6,
152.1, 169.8; HRMS calcd for C23H25IN4O5H:
591.1105; found 591.1108.
1
yield); reaction time 16 h; IR (CHCl3) 1656; H N MR
(CDCl3) d 0.92 (t, 6H, J=7.0), 2.50 (q, 4H, J=7.0), 2.80
(t, 2H, J=6.8), 3.39 (s, 3H), 3.71 (s, 3H), 3.94 (m, 1H),
4.41 (m, 1H), 6.21 (d, 2H, J=1.4), 6.39 (s, 1H), 7.01 (s,
1H), 7.39 (s, 1H), 7.64 (s, 1H), 9.11 (s, 1H); 13C N MR
(CDCl3) d 11.6, 46.9, 47.8, 51.1, 55.7, 56.1, 82.9, 96.9,
102.9, 105.5, 110.9, 122.1, 122.9, 133.0, 136.5, 144.9,
148.3, 150.1, 150.9, 151.7, 152.3, 169.8; HRMS calcd for
C24H27O5N4IH: 579.1105; found: 579.1105.
4.3.3. N-(6,7-Methylenedioxycinnolin-4-yl)-N-[2-(N,N-di-
methylamino)-1-methylethyl)-2-iodo-4,5-dimethoxybenza-
mide (8c). This was prepared from 7c (240 mg, 0.87
mmol); (83% yield); reaction time 16 h; mp 110–111 ꢂC;
1H NMR (CDCl3) was a mixture of atropisomer iso-
mers: #1 d 1.03–1.36 (m, 3H), 2.21–2.37 (m, 6H), 2.74–
3.07 (m, 1H), 3.43–3.65 (m, 6H), 3.84–3.91 (m, 1H), 5.15
(m, 1H), 6.18 (s, 2H), 6.59 (s, 1H), 6.91 (s, 1H), 7.56 (s,
1H), 8.04 (s, 1H), 9.34 (s, 1H); isomer #2 d 1.03–1.36 (m,
3H), 2.31-2.37 (m, 6H), 2.74–3.07 (m, 1H), 3.43–3.65
(m, 6H), 3.84–3.91 (m,1H), 5.15 (m, 1H), 6.18 (s, 2H),
6.59 (s, 1H), 6.91 (s, 1H), 7.56 (s, 1H), 8.04 (s, 1H), 9.34
(s, 1H); HRMS calcd for C23H25O5N4IH: 565.0870;
found: 565.0926.
4.3.8. N- (6,7-Methylenedioxycinnolin-4- yl)- N -[2 -(4 -
methyl-1-piperazinyl)ethyl]-2-iodo-4,5-dimethoxybenza-
mide (8h). This was prepared from 7h (700 mg, 2.2
1
mmol) (82% yield); H NMR (CDCl3) d 2.20 (s, 3H),
2.32 (m, 4H), 2.42 (m, 4H), 3.37 (s, 3H), 3.68 (s, 3H),
3.86 (m, 3H), 4.34 (m, 1H), 6.18 (s, 1H), 6.19 (s, 1H),
6.37 (s, 1H), 6.97 (s, 1H), 7.42 (s, 1H), 7.60 (s, 1H), 9.13
(s, 1H); 13C NMR (CDCl3) d 45.9, 46.1, 53.0, 55.1, 55.6,
55.7, 56.1, 82.9, 97.0, 102.9, 105.3, 110.7, 121.9, 123.2,
132.9, 136.1, 145.0, 148.2, 149.9, 150.9, 151.7, 152.3,
169.8; IR (CHCl3) 1654; HRMS calcd for
C25H28IN5O5H: 606.1213; found 606.1213.
4.3.9. 4-Chlorocinnoline (9). A mixture of 4-hydroxy-
cinnoline (2.0 g, 13.7 mmol), phosphorus oxychloride
(1.94 mL, 20.5 mmol), and pyridine (0.33 mL, 4.1
mmol) in chlorobenzene (50 mL) was refluxed for 1 h.
Then the mixture was cooled and the solvent evaporated
under vacuum, water was added, and the mixture was
neutralized with solid sodium bicarbonate and extracted
with chloroform (3ꢃ100 mL), washed with water
(3ꢃ100 mL), dried (MgSO4) and evaporated under
vacuum, giving 1.84 g, in 82% yield; mp 76–77 ꢂC (lit.28
4.3.4. N-(6,7-Methylenedioxycinnolin-4-yl)-N-(n-butyl)-
2-iodo-4,5-dimethoxybenzamide (8d). This was prepared
from 7d (350 mg, 1.4 mmol); (19% yield) reaction time
18 h; mp 133–134 ꢂC; IR (KBr) 1654; 1H NMR (CDCl3)
d 0.87 (t, 3H, J=7.2), 1.20–1.90 (m, 4H), 3.33 (s, 3H),
3.68 (s, 3H), 3.90 (m, 1H), 4.35 (m, 1H), 6.19 (d, 2H,
J=3.2), 6.34 (s, 1H), 6.98 (s, 1H), 7.25 (s, 1H), 7.62 (s,
1H), 9.01 (s, 1H); 13C NMR (CDCl3) d 13.7, 20.1, 30.0,
49.4, 55.7, 56.1, 82.9, 96.5, 102.9, 105.6, 110.5, 121.9,
133.1, 148.3, 150.0, 151.8, 152.5, 169.7; HRMS calcd for
C22H22IN3O5Li 542.0764; found 542.0757.
mp 78 ꢂC); H NMR (CDCl3) d 7.91 (m, 2H), 8.20 (dd,
1
1H, J=6.2, J=2.1), 8.57 (dd, 1H, J=6.6, J=1.8), 9.34
(s, 1H); 13C NMR (CDCl3) d 123.0, 124.9, 130.2, 131.6,
132.3, 133.8, 134.9, 144.4.
4.3.5. N-(6,7-Methylenedioxycinnolin-4-yl)-N-[2-(tetrahy-
drofuranyl)methyl]-2-iodo-4,5-dimethoxybenzamide (8e).
This was prepared from 7e (400 mg, 1.5 mmol); (34%
yield); reaction time 16 h; IR (CHCl3) 1654; 1H N MR, a
mixture of atropisomers, (CDCl3) isomer #1: d 1.94 (m,
4H), 3.70 (m, 4H), 3.73 (s, 3H), 3.94 (s, 3H), 4.34 (m,
1H) 6.23 (s, 2H), 7.00 (s, 1H), 7.40 (s, 1H), 7.70 (s, 1H),
9.31 (s, 1H); isomer #2 d 1.94 (m, 4H), 3.70 (m, 4H),
3.73 (s, 3H), 3.94 (s, 3H), 4.34 (m, 1H) 6.46 (s, 2H), 7.36
(s, H), 7.49 (s, 1H), 7.65 (s, 1H), 9.17 (s, 1H); HRMS
calcd for C23H22O6N3IH: 564.0632; found: 564.0650.
4.3.10. 4-[2-(Dimethylamino)ethylamino]cinnoline (11).
Intermediate 9 (1.0 g, 6.1 mmol) was stirred in neat
refluxing N,N-dimethylethylenediamine (6.25 g, 70.9
mmol) for 3 h, then the mixture was cooled and the
solvent evaporated under reduced pressure. The crude
material was partitioned between water (100 mL) and
chloroform (100 mL), and the aqueous phase was
washed with chloroform (2ꢃ100 mL). The combined
organic phases were washed with brine (75 mL), dried
(MgSO4) and evaporated under vacuum, giving 890 mg,
1
4.3.6. N-(6,7-Methylenedioxycinnolin-4-yl)-N-[(2-pyrroli-
din-1-yl)ethyl]-2-iodo-4,5-dimethoxybenzamide (8f). This
was prepared from 7f (400 mg, 0.4 mmol); (42% yield);
reaction time 4 h; IR (KBr) 1655; 1H NMR (CDCl3) d 1.60
(m, 4H), 2.40 (m, 4H), 2.67 (m, 2H), 3.28 (s, 3H), 3.60 (s,
3H), 4.32 (m, 1H), 6.11 (d, 2H, J=2.2), 6.32 (s, 1H), 6.91
(s, 1H), 7.37 (s, 1H), 7.50 (s 1H), 9.04 (s, 1H); 13C N MR
(CDCl3) d 23.6, 29.7, 47.6, 52.9, 53.9, 55.7, 56.0, 56.4, 82.8,
96.7, 102.9, 105.4, 110.6, 121.9, 123.1, 132.8, 135.9, 144.7,
148.2, 149.9, 150.9, 151.7, 152.4, 169.9; HRMS calcd for
C24H25IN4O5HLi 584.1108; found 584.1115.
in 68% yield; mp 146–148 ꢂC; H NMR (CDCl3) d 2.31
(s, 6H), 2.69 (t, 2H, J=5.9), 3.88 (m, 2H), 6.08 (br, 1H),
7.57 (ddd, 1H, J=8.3, J=7.4, J=0.8), 7.72 (ddd, 1H,
J=8.4, J=7.4, J=0.6), 7.82 (dd, 1H, J=8.3, J=0.6),
8.30 (dd, J=8.4, J=0.8), 8.63 (s, 1H); 13C N MR
(CDCl3) 39.6, 45.1, 57.0, 115.8, 119.2, 128.2, 128.7,
129.5, 129.9, 139.9, 148.7; HRMS calcd for C12H16N4:
216.1375; found 216.1376.
4.3.11. N0 -(6-Nitrocinnolin-4-yl)-N,N-dimethylethane-
1,2-diamine (12). A mixture of 10 (2.0 g, 7.5 mmol) and

804
A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
N,N-dimethylethylenediamine (1.33 g, 15.2 mmol) in
DMF (20 mL) was heated to 90 ꢂC with stirring for 1 h.
Then the mixture was cooled and the solvent was
removed under vacuum. The residue was partitioned
between 10% NaOH (150 mL) and CHCl3 (100 mL),
and the aqueous phase was extracted with CHCl3
(4ꢃ100 mL). The combined organic extracts were
washed with 10% NaOH (2ꢃ150 mL), dried (MgSO4),
and evaporated under vacuum, yielding 1.4 g, in 72%
yield; mp 200–202 C; IR (CHCl3) 1345, 1518, 3349; H
NMR (CDCl3) d 2.36 (s, 3H), 2.77 (t, 2H, J=5.9), 3.54
(m, 2H), 6.87 (br, 1H), 8.40 (m, 2H), 8.82 (s, 1H), 8.95
(d, 1H, J=1.8); 13C NMR (CDCl3) d 40.0, 45.2, 56.7,
114.5, 118.2, 123.5, 130.4, 131.4, 141.5, 145.7, 149.3;
HRMS calcd for C12H15N5O2: 261.1226; found:
261.1233.
anhydrous methylene chloride (40 mL), and the stirred
mixture was refluxed for 4 h. The mixture was then
concentrated to dryness under reduced pressure. The
acid chloride was redissolved in 40 mL of anhydrous
methylene chloride, and this solution was added to a
solution of 11 (870 mg, 4.05 mmol) and triethylamine
(4.0 g, 40.0 mmol) in methylene chloride (30 mL), and
the resulting mixture was stirred at reflux overnight. The
reaction mix was cooled and washed with saturated
sodium bicarbonate (3ꢃ75 mL), and brine (75 mL),
dried (MgSO4) and evaporated, and the crude product
mixture was chromatographed in 98:2 chloroform–
methanol, to provide 165 mg of the desired amide, in
8% yield; IR (CHCl3) 1658; 1H NMR (CDCl3) d 2.21 (s,
6H), 2.63 (m, 2H), 3.19 (s, 3H), 3.70 (s, 3H), 3.94 (m,
1H), 4.59 (m, 1H), 6.28 (s, 1H), 7.01 (s, 1H), 7.88 (m,
2H), 8.20 (dd, 1H, J=6.6, J=3.4), 8.54 (dd, 1H, J=6.6,
J=3.0), 9.35 (s, 1H); 13C NMR (CDCl3) d 45.5, 47.3,
55.4, 56.1, 56.8, 82.9, 110.4, 121.9, 122.0, 123.8, 130.7,
131.0, 132.0, 133.0, 136.6, 145.2, 148.2, 149.9, 151.7,
169.9; HRMS calcd for C21H23IN4O3H: 507.0894;
found 507.0880.
ꢂ
1
4.3.12. N-(6,7-Methylenedioxycinnolin-4-yl)-N-[(2-N,N-
dimethylamino)ethyl]-2-iodobenzamide (13). Oxalyl chlo-
ride (1.05 g, 8.1 mmol) was added to a solution of
o-iodobenzoic acid (0.57 g, 2.3 mmol) in methylene
chloride (20 mL) under nitrogen, and the stirred mixture
was refluxed for 4 h. Then the mixture was concentrated
to dryness under vacuum. The acid chloride was redis-
solved in methylene chloride (20 mL) and added to a
solution of 7a and triethylamine (2.0 g, 20.0 mmol) in
methylene chloride (20 mL). The reaction was stirred at
reflux under nitrogen for 2 h. The mixture was then
cooled and washed with a saturated solution of sodium
bicarbonate (3ꢃ75 mL), and extracted into dilute HCl
(3ꢃ75 mL), and the combined aqueous extracts were
washed with chloroform (75 mL) and basified using
30% NaOH, then extracted with chloroform (3ꢃ75
mL). The combined organic extracts were washed with
brine (100 mL), dried (anhydrous MgSO4), and con-
centrated in vacuo, to give 652 mg as a gum, in 70%
4.3.15. N-(6-Nitrocinnolin-4-yl)-N-[2-(N,N-dimethylami-
no)ethyl]-2-iodo-4,5-dimethoxybenzamide (16). Oxalyl
chloride (3.0 g, 23.2 mmol) was added to a mixture of
2-iodo-4,5-dimethoxybenzoic acid (1.5 g, 4.9 mmol) in
methylene chloride (40 mL), and the stirred mixture
was heated to reflux under nitrogen for 4 h. The mix-
ture was concentrated under reduced pressure and the
acid chloride was redissolved in methylene chloride (40
mL) and added to a mixture of 12 (1.0 g, 4.0 mmol) and
TEA (2.5 g, 25.0 mmol) in methylene chloride (40 mL).
The resulting mixture was heated to reflux overnight
and then cooled and washed with saturated sodium
bicarbonate (3ꢃ100 mL) and brine (150 mL), dried
(MgSO4) and evaporated, and the residue was chroma-
tographed in 99:1 CHCl3–MeOH to provide 590 mg as
a glue, in 29% yield; IR (CHCl3) 1654; 1H N MR
(CDCl3) d 2.11 (s, 6H), 2.58 (m, 2H), 3.40 (s, 3H), 3.64
(s, 3H), 3.85 (m, 1H), 4.16 (m, 1H), 6.51 (s, 1H), 6.85 (s,
1H), 8.50 (m, 1H), 8.60 (m, 1H), 9.28 (s, 1H), 9.61 (m,
1H); 13C NMR (CDCl3) d 45.2, 48.1, 52.3, 56.2, 57.0,
82.5, 111.3, 121.0, 121.7, 123.8, 124.3, 131.0, 132.7,
138.2, 145.8, 148.8, 149.0, 150.2, 151.4, 169.6; HRMS
calcd for C21H23IN5O5H: 552.0744; found: 552.0743.
1
yield; IR (CHCl3) 1658; H NMR (CDCl3) d 2.20 (s,
6H), 2.58 (m, 2H), 3.59 (m, 1H), 4.53 (m, 1H), 6.21 (s,
1H), 6.23 (s, 1H), 6.81 (m, 3H), 7.36 (s, 1H), 7.61 (s,
1H), 7.68 (m, 1H), 9.11 (s, 1H); 13C NMR (CDCl3) d
45.4, 46.8, 56.7, 93.7, 96.7, 102.9, 105.4, 123.1, 126.8,
127.4, 128.4, 135.7, 139.9, 140.9, 144.9, 150.1, 151.9,
152.6, 169.9; HRMS calcd for C20H19O3N4IH:
491.0581; found 491.0592.
4.3.13. N-(Cinnolin-4-yl)-N-[2-(N,N-dimethylamino)-
ethyl]-2-iodobenzamide (14). This was prepared from 11
(740 mg, 3.4 mmol); (33% yield); reaction time 18 h at
50 ꢂC from the acid chloride prepared using 20 mmol of
oxalyl chloride and 4.0 mmol of 2-iodobenzoic acid; IR
4.4. Topoisomerase-mediated DNAcleavage assays
Human topoisomerase I was expressed in Escherichia
coli and isolated as a recombinant fusion protein using a
T7 expression system as described previously.14 Plasmid
YepG was also purified by the alkali lysis method fol-
lowed by phenol deproteination and CsCl/ethidium iso-
pycnic centrifugation method as described.34 The 30 end-
labeling of the plasmid was accomplished by digestion
with a restriction enzyme followed by end-filling with
Klenow polymerase as previously described.35 The clea-
vage assays were performed as previously reported.36,37
The drug and the DNA in presence of topoisomerase I
was incubated for 30 min at room temperature. The
reactions were terminated by the addition of 5 mL of 5%
SDS and 1 mg/mL protein kinase K with an additional
1
(CHCl3) 1660; H NMR (CDCl3) d 2.11 (s, 6H), 2.43–
2.53 (m, 1H), 2.56–2.66 (m, 1H), 3.52–3.68 (m, 1H),
4.51–4.64 (m, 1H) 6.69–6.76 (m, 3H), 7.30–7.57 (m, 2H),
7.75–7.82 (m, 3H), 8.09–8.14 (m, 1H), 8.39–8.44 (m,
1H), 9.29 (s, 1H); 13C NMR (CDCl3) d 45.4, 47.1, 56.7,
93.8, 122.1, 123.4, 126.9, 127.3, 128.4, 130.4, 130.6,
131.2, 136.2, 139.7, 140.8, 145.1, 151.6, 169.7; HRMS
calcd for C19H19IN4OH: 447.0682; found 447.0687.
4.3.14. N-(Cinnolin-4-yl)-N-[2-(N,N-dimethylamino)e-
thyl]-2-iodo-4,5-dimethoxybenzamide (15). Oxalyl chlo-
ride (2.1 g, 16.2 mmol) was added to a solution of
3,4-dimethoxy-6-iodobenzoic acid (1.5 g, 4.87 mmol) in

A. L. Ruchelman et al. / Bioorg. Med. Chem. 12 (2004) 795–806
805
1 h of incubation at 37 ꢂC. Samples were then alkali
denatured by the addition of NaOH, EDTA, sucrose,
and bromophenol blue to final concentrations of 75
mM, 2.5%, and 0.05 mg/mL, respectively, prior to
loading onto a neutral agarose gel. After development
of the gels, typically 24-h exposure was used to obtain
autoradiograms outlining the extent of DNA fragmen-
tation. Topoisomerase I-mediated DNA cleavage values
are reported as Relative Effective Concentration (REC),
that is concentrations relative to topotecan, 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.
weighed individually on a daily basis. Tumor volume
was determined for each individual mouse every other
day.
Acknowledgements
This study was supported by AVAX Technologies, Inc.
(E.J.L.) and Grant CA39662 (L.F.L.) and Grant
CA077433 (L.F.L.) from the National Cancer Institute.
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