Synthesis of N-substituted 5-[2-(N-alkylamino)ethyl]dibenzo[c,h][1,6]naphthyridines as novel topoisomerase I-targeting antitumor agents

Article abstract of DOI:10.1016/j.bmc.2008.09.002

Several N-alkyl and N,N-dialkyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-ones have been identified as topoisomerase I-targeting agents with potent antitumor activity. In the present study, the impact on biological activity of substitution of a trifluoromethyl, cyano, aminocarbonyl, or ethynyl group on a N-methyl substituent of N,N-dimethyl-, N-methyl-N-ethyl-, and N-methyl-N-isopropyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-ones was assessed.

Full text of DOI:10.1016/j.bmc.2008.09.002

Bioorganic & Medicinal Chemistry 16 (2008) 9295–9301
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of N-substituted 5-[2-(N-alkylamino)ethyl]dibenzo[c,h][1,6]-
naphthyridines as novel topoisomerase I-targeting antitumor agents
Wei Feng a, Mavurapu Satyanarayana a, Liang Cheng a, Angela Liu b, Yuan-Chin Tsai b, Leroy F. Liu b,c
,
Edmond J. LaVoie a,c,
*
a Department of Pharmaceutical Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA
b Department of Pharmacology, The University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
c The Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Several N-alkyl and N,N-dialkyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo-
[c,h][1,6]naphthyridin-6-ones have been identified as topoisomerase I-targeting agents with potent anti-
tumor activity. In the present study, the impact on biological activity of substitution of a trifluoromethyl,
cyano, aminocarbonyl, or ethynyl group on a N-methyl substituent of N,N-dimethyl-, N-methyl-N-ethyl-,
and N-methyl-N-isopropyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]-
naphthyridin-6-ones was assessed.
Received 12 April 2008
Revised 26 August 2008
Accepted 2 September 2008
Available online 5 September 2008
Keywords:
Topoisomerase I
Cytotoxicity
Ó 2008 Elsevier Ltd. All rights reserved.
Antitumor
MDA-MB-435
Structure–activity
Non-camptothecin
1. Introduction
N
N
O
O
H3CO
H3CO
Topoisomerases are ubiquitous enzymes that participate in
processes such as DNA replication, repair, transcription, and
recombination as well as chromosome condensation and segrega-
tion.1,2 Topoisomerase I (TOP1) is the target of several antitumor
agents based upon their ability to stabilize the enzyme–DNA
cleavage complex, which results in DNA damage and ultimately
cell death.3–5 Several 5-(2-aminoethyl)dibenzo[c,h][1,6]naphthyri-
din-6-ones have been identified as topoisomerase I-targeting
agents with potent antitumor activity.6,7 One of the more exten-
sively studied of these non-camptothecin TOP1-targeting agents
O
N
CH3
R
1 R = CH3, ARC-111
2 R = CH2CH3
3 R = CH(CH3)2
Figure 1. Structure of ARC-111 and related compounds.
is
5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-meth-
ylenedioxydibenzo[c,h][1,6]-naphthyridin-6-one (ARC-111),
1
(Fig. 1).8,9 Analogs of ARC-111 with various 5-[2-(N,N-dialkylami-
noethyl)] substituents have exhibited potent activity.9 Com-
pounds 2 and 3 (Fig. 1) are among the tertiary alkylamine
analogs that exhibit similar TOP1-targeting activity and cytotoxic
activity in RPMI8402 cells to 1 (IC50 values ranging from 2 to
6 nM).
The effect on biological activity of the addition of a trifluoro-
methyl, cyano, or ethynyl substituent on the N-methyl group of
compounds related to ARC-111 was investigated. These studies
were extended to examining the effect of an aminocarbonyl substi-
tuent on the N-methyl substituent of ARC-111 as well as the
replacement of its 8,9-dimethoxy groups with 8,9-diethoxy sub-
stituents. These data provide further insight into the structure–
activity associated with this family of potent non-camptothecin
TOP1-targeting agents.
* Corresponding author. Tel.: +1 732 445 2674; fax: +1 732 445 6312.
E-mail address: elavoie@rci.rutgers.edu (E.J. LaVoie).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.002

9296
W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 9295–9301
N
N
2. Results
2.1. Chemistry
O
O
H3CO
H3CO
Bromoacetamide, K2CO3, DMF
47%
4
The syntheses of compounds 4–6 were reported previously. The
N-methyl-N-ethyl analog 2 of ARC-111 was prepared by reductive
alkylation of 5. Compounds 4, 5, and 6 were treated with trifluoro-
methanesulfonic acid 2,2,2-trifluoroethyl ester and diisopropyleth-
ylamine (DIEA) to afford 7, 8, and 9, respectively (Scheme 1).
Alkylation of 4, 5, and 6 using bromoacetonitrile provided com-
pounds 10, 11, and 12, respectively (Scheme 1). Conversion of 4, 5,
and 6 to compounds 13, 14, and 15 were carried out in DMF at
80 °C using 3-bromopropyne and anhydrous potassium carbonate
(Scheme 1).
Compound 16 was synthesized from 4 by reaction with bromo-
acetamide and potassium carbonate as outlined in Scheme 2.
The preparation of the diethoxy analog of 1 was performed as
outlined in Scheme 3. Using similar methodology as previously de-
scribed,9 3,4-diethoxybenzoic acid was treated with iodine and sil-
ver trifluoroacetate in CHCl3 to yield 4,5-diethoxy-2-iodobenzoic
acid 17 in 60% yield. Conversion of 17 to its acid chloride 18 was
carried out in anhydrous CH2Cl2 with oxalyl chloride. Without fur-
ther purification 18 was added directly to the solution of appropri-
ate 4-amino-6,7-methylenedioxyquinoline7 and TEA in CH2Cl2.
Intramolecular Heck cyclization of iodobenzamide 20 was per-
formed in refluxing DMF for 2 h to afford 21 in 34% yield, using
Pd(OAc)2, P(o-tolyl)3, and Ag2CO3.
O
O
N
CH3
16
H2N
Scheme 2. Preparation of 16.
are listed in Table 1. The N-(2,2,2-trifluoroethyl) derivatives, 7–9,
did not exhibit appreciable TOP1-targeting activity. The basis for
this loss in activity cannot be explained on the basis of steric fac-
tors in light of the potent TOP1-targeting activity observed for 1–
3.9 In contrast to these data, the cyano derivatives, 10–12, were
highly active TOP1-targeting agents with similar potency to ARC-
111 and camptothecin. The N-methyl-N-propargyl derivative, 13,
and the N-methyl-N-acetamide derivative, 16, also retained potent
TOP1-targeting activity relative to that observed for 1–3. The N-
ethyl-N-propargyl derivative, 14, and the N-isopropyl-N-propargyl
derivative, 15 were much less active as TOP1-targeting agents. The
diethoxy analog of ARC-111, 21, exhibited a dramatic loss in TOP1-
targeting activity.
Those derivatives with potent TOP1-targeting activity also
exhibited the more pronounced cytotoxicity. The cytotoxicity of
compounds 10–12 did not vary dramatically from one another
with IC50 values that ranged from 3 to 7 nM and 2 to 4 nM in
RPMI8402 and P388, respectively. Compound 13 was less cytotoxic
with IC50 values that ranged from 2- to 20-fold higher than 10–12
in these cell lines. Compounds 14 and 15 had similar cytotoxicity
in RPMI8402 to each other, but were 2- to 6-fold less cytotoxic
3. Results and discussion
The relative TOP1-targeting activities of the various N-substi-
tuted 5-[2-(N-alkylamino)ethyl]dibenzo[c,h][1,6]naphthyridines
DIEA, DMF,
F
F
N
O
O
N
N
N
O
O
O
O
F
3-Bromopropyne,
K2CO3, DMF
S
F
O
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
O
O
F
F
N
N
O
O
F
O
H
N
R
N
R
N
R
F
F
4 R= CH3
5 R= CH2CH3
6 R= CH(CH3)2
7 R= CH3
8 R= CH2CH3 31%
9 R= CH(CH3)2 52%
43%
13 R= CH3
65%
14 R= CH2CH3 64%
15 R= CH(CH3)2 58%
Bromoacetonitrile,
K2CO3, DMF
N
O
O
H3CO
H3CO
N
O
N
R
N
10 R= CH3
64%
11 R= CH2CH3 80%
12 R= CH(CH3)2 50%
Scheme 1. Preparation of 7–9, 10–12, and 13–15.

W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 9295–9301
9297
OH
OH
ClOC
(CO)2Cl2
OC2H5
OC2H5
CF3COOAg
OC2H5
OC2H5
OC2H5
OC2H5
O
O
I
I
18
17
CH3
N
N
O
O
H3C
I
HN
C2H5O
C2H5O
TEA, CH2Cl2
CH3
N
CH3
N
O
O
+
18
O
N
19
20
N
N
O
O
Pd(OAc)2, P(o-tolyl)3
Ag2CO3, DMF
C2H5O
C2H5O
O
N
H3C
CH3
21
Scheme 3. Preparation of 21.
shown to be substrates for the MDR1 efflux transporter.13 Topotec-
an, as well as the active metabolite of irinotecan, SN-38, are resis-
tant to tumor cells that express BCRP.15,16 In light of their 7-fold
difference in IC50 values between KB3-1 cells and KBV-1 cells, the
data suggest that 10–12 and 16 are substrates for MDR1. While
not among the more potent cytotoxic agents, 13 does not appear
to be a substrate for either MDR1 or BCRP. It is of interest to note
that, of the compounds evaluated; only 16 appears to be a sub-
strate for BCRP.
Compounds 8 and 10–16 were evaluated for antitumor activity
in vivo in athymic nude mice with MDA-MB-435 human tumor
xenografts. The results of this bioassay are outlined in Table 3
and are illustrated in Figures 2–4.
Figure 2 clearly illustrates that the N-methyl-N-cyanomethyl
analog 10 is less active than the N-methyl-N-propargyl 13 or N-
methyl-N-acetamide 16 at their maximally tolerated doses. The
N-methyl-N-2,2,2-trifluoroethyl analog 8 was also less efficacious
than the N-ethyl-N-cyanomethyl analog 11 or the N-ethyl-N-prop-
argyl derivative 14 as illustrated in Figure 3. While in vitro studies
did indicate that 14 and 15 were less cytotoxic than their structur-
ally related cyano derivatives, 11 and 12, this did result in notable
differences in their antitumor activity in vivo. Both 11 and 14 had
similar antitumor activity, but were less potent and effective than
Table 1
TOP1-targeting activity and cytotoxicity in RPMI8402 and P388 cells and their
camptothecin-resistant variants
Compound
TOP1-mediated
cleavagea
Cytotoxicity IC50 (lM)
RPMI8402
CPT-K5
P388
P388/CPT 45
1
7
8
9
10
11
12
13
14
15
16
21
0.3
0.002
0.25
0.7
0.90
>10
>10
>10
0.98
0.67
1.3
0.001
0.25
0.61
0.23
>10
>10
>10
0.22
0.13
0.06
0.4
2.3
3.7
0.39
0.013
>10
>10
>10
>10
>10
<0.2
<0.2
<0.2
0.1
>10
>10
0.3
1.9
1.1
0.004
0.001
0.002
0.008
0.017
0.065
0.003
0.15
0.003
0.001
0.002
0.02
4.5
>10
>10
3.5
0.14
>10
>10
0.035
0.08
0.009
0.015
0.014
0.045
>10
0.2
1.0
CPT
Topotecan
0.006
0.021
a
Topoisomerase
I cleavage values are reported as REC, relative effective
concentration. These are concentrations relative to topotecan, whose value is
arbitrarily assumed as 1, that are able to produce 10% cleavage of the plasmid DNA
in the presence of human topoisomerase I.
than 13. The aminocarbonyl derivative, 16, exhibited similar cyto-
toxicity to 10–12.
CPT-K5 cell line, a variant of RPMI8402 cells, and P388/CPT45 a
variant of P388 have been used to investigate the role of TOP1 in
the mechanism of cytotoxicity of suspect TOP1-targeting
agents.10,11 Resistance to CPT-K5 cells is associated with its mutant
form of the enzyme TOP1 as well as its ability to expresses the ef-
flux transporter BCRP.12 Resistance to P388/CPT45, which has min-
imal expression of TOP1, is consistent with TOP1-targeting as the
primary mechanism associated with cytotoxic activity. With the
notable exception of 21, these results suggest that TOP1-targeting
is associated with cytotoxicity, even in the case of those derivatives
with comparatively weak TOP1-targeting activity.
Data are provided in Table 2 on the relative cytotoxicity in the
parent cell line, KB3-1, KBV-1, a variant that overexpresses the ef-
flux transporter MDR1 (ABCB1/P-glycoprotein/PGY1/CD243), and
KBH5.0, a variant that overexpresses BCRP (ABCG2/MXR/ABCP1).
Differences in relative cytotoxicity between these variant cell lines
and the parent cell line, KB3-1, may be indicative of a compound
that is a substrate for an efflux transporter.13,14 Topotecan has been
Table 2
Cytotoxicity of N-substituted 5-[2-(N-alkylamino)ethyl]dibenzo[c,h][1,6]naphthyri-
dines in KB3-1 cells and its variant cell lines, KBV-1 and KBH5.0
Compound
Cytotoxicity IC50
KBV-1
(lM)
KB3-1
KBH5.0
1
7
8
9
10
11
12
13
14
15
16
21
0.005
0.41
0.65
1.0
0.005
0.004
0.006
0.02
0.07
0.10
0.01
0.07
0.04
0.005
1.0
2.9
0.006
0.63
0.75
1.1
0.027
0.007
0.009
0.034
0.11
0.07
0.32
0.1
4.4
0.043
0.034
0.043
0.07
0.1
0.43
0.38
0.19
0.44
Topotecan
0.44

9298
W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 9295–9301
Table 3
Tumor Volume
Antitumox activity in athymic nude mice with the human tumor xenograft MDA-MB-
435
1000
900
800
700
600
500
400
300
200
100
0
12
Compound Route
Average tumor volume (mm3)
Total dose
(mg/kg)/
mouse
Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 49
15
8
10
11
12
13
14
15
16
I.P.a 127 176
268
370
178
213
218
170
165
230
332
145
310
392
202
220
284
204
170
319
408
158
435
528
293
250
317
331
234
344
573
175
543
669
361
286
358
349
310
434
747
189
718
919
435
314
433
488
357
527
966
242
111
111
117
168
223
117
117
301
Vehicle
ARC-111
I.P.a 149 222
I.P.a 100 156
I.P.b 142 175
I.P.c 144 167
I.P.a 115 106
I.P.a
99 112
I.P.d 147 178
I.P.e 126 181
Vehicle
1
I.P.f
120 123
37.5
7
14
21
28
35
42
49
a
Initial dose was 2.0 mg/kg qd ꢁ 5/week for
2 weeks and was gradually
Day
increased to 6.0 mg/kg ꢁ 5/week.
b
Initial dose was 5.0 mg/kg qd ꢁ 5/week and was increased to 6.0 mg/kg qd ꢁ 5/
week for 2 weeks, then adjusted to 6.0 mg/kg qd ꢁ 3/week for one week. This dose
was then again modified to the initial dose of 5.0 mg/kg qd ꢁ 5/week.
Figure 4. Antitumor activity of compounds 12 and 15, vehicle, and ARC-111.
c
however, appear from these in vivo studies to be less potent and
less efficacious than ARC-111 (Table 3).
Initial dose was 5.0 mg/kg qd ꢁ 5/week for 1 week and was gradually increased
to 7.0 mg/kg ꢁ 5/week.
d
Initial dose was 5.0 mg/kg qd ꢁ 5/week for one and a half weeks and was
These comparative studies did not result in the identification of
an analog of ARC-111 with comparable in vivo potency and effi-
cacy. The presence of electron-withdrawing substituents on the
N-methyl substituents of these various analogs of compounds 1–
3 did negatively affect the relative ease with which they could be
formulated for injection. The decreased basicity of these deriva-
tives lessened the solubility of their citrate salts and may have also
impacted their absorption and distribution. Further studies are in
process to assess the influence of other more polar substituents
on the amino group of the 5-(2-aminoethyl) substituent of ARC-
111 and related compounds.
increased to 6.0 mg/kg qd ꢁ 5/week for two and a half weeks. Administration was
increased to 14.0 mg/kg qd ꢁ 5/week for one and a half weeks then adjusted back to
10.0 mg/kg qd ꢁ 5/week for 1 week and finally adjusted to 12.0 mg/kg qd ꢁ 5/week.
e
Vehicle consisted of 0.1% citrate in H2O and was administered qd ꢁ 5/week.
f
Initial dose was 1.5 mg/kg, which was administered qd ꢁ 3/5 days.
Tumor Volume
1000
10
900
800
700
600
500
400
300
200
100
0
13
16
4. Experimental
Vehicle
ARC-111
Melting points were determined with either a Thomas-Hoover
Unimelt or Meltemp capillary melting point apparatus. Column
chromatography refers to flash chromatography conducted on Sil-
iTech 32–63 lm, (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-
2000 Fourier Transform spectrometer. Infrared spectral data were
obtained using a Thermo-Nicolet Avatar 360 Fourier transform
7
14
21
28
Day
35
42
49
spectrophotometer and are reported in cmꢀ1
. NMR spectra
Figure 2. Antitumor activity of compounds 10, 13, 16, vehicle, and ARC-111.
(200 MHz 1H and 50 MHz 13C) were recorded in the deuterated sol-
vent indicated with chemical shifts reported in d units downfield
from tetramethylsilane (TMS). Coupling constants are reported in
hertz (Hz). Mass spectra were obtained from Washington Univer-
sity Resource for Biomedical and Bio-organic Mass Spectrometry
within the Department of Chemistry at Washington University,
St. Louis, MO, USA. All starting materials and reagents were pur-
chased from Aldrich. Solvents were purchased from Fisher Scien-
tific, and were A.C.S. grade or HPLC grade. Methylene chloride
was freshly distilled from calcium hydride. All other solvents were
used as provided without further purification. Compounds 5–7
were prepared as previously described.9 Methods for the prepara-
tion of intermediate 19 have also been previously reported.7 Tri-
fluoromethanesulfonic acid 2,2,2-trifluoroethyl ester was
purchased from TCI America (Portland, OR).
Tumor Volume
1000
8
900
800
700
600
500
400
300
200
100
0
11
14
Vehicle
ARC-111
7
14
21
28
Day
35
42
49
4.1. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-ethyl-N-methyl
aminoethyl]dibenzo[c,h][1,6]naphthyridin-6-one (2)
Figure 3. Antitumor activity of compounds 8, 11, 14, vehicle, and ARC-111.
A mixture of 5 (150 mg, 0.37 mmol), acetaldehyde (0.2 mL,
3.38 mmol), and sodium cyanoborohydride (78 mg, 1.1 mmol) in
ethanol (15 mL) was heated to 75 °C in a sealed tube for 6 h. The
ARC-111 in this bioassay. The N-isopropyl-N-cyanomethyl and the
N-isopropyl-N-propargyl derivatives, 12 and 15, respectively, also
had similar antitumor activity (Fig. 4). Both of these derivatives,

W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 9295–9301
9299
mixture was filtered and the filtrate was evaporated. The residue
was partitioned between chloroform (50 mL) and 10% NaOH
(30 mL), and the aqueous phase was extracted with chloroform
(2ꢁ 50 mL). The combined organic phases were evaporated and
chromatographed through a short column of silica eluting with
97:3 chloroform/methanol, to provide 89 mg (55% yield); mp
251–253 °C; IR (neat) 1650; 1H NMR (CDCl3): d 1.01 (t, J = 7.4,
3H), 2.27 (s, 3H), 2.46 (q, J = 7.4, 2H), 3.01 (t, J = 7, 2H), 4.04 (s,
3H), 4.10 (s, 3H), 4.60 (t, J = 7, 2H), 6.15 (s, 2H); 7.44 (s, 1H), 7.65
(s, 1H), 7.87 (s, 1H), 7.92 (s, 1H), 9.34 (s, 1H), 13C NMR
(CDCl3 + CD3OD) d 11.1, 40.9, 47.8, 50.8, 53.9, 55.1, 55.3, 100.0,
101.0, 101.3, 105.2, 107.6, 110.0, 113.7, 118.1, 126.5, 139.9,
142.1, 145.6, 146.8, 149.1, 149.3, 153.3, 163.2; HRMS (M++H) Calcd
for C24H25N3O5H: 436.1872; found: 436.1874.
101.3, 106.2, 107.7, 110.8, 113.8, 118.3, 124.6 (q, J = 279.6),
126.8, 139.9, 142.5, 146.2, 146.8, 149.0, 149.3, 153.2, 163.3; HRMS
m/z Calcd for C26H26F3N3O5H: 518.1903; found: 518.1903.
4.3. General procedure for the preparation of 2,3-
methylenedioxy-8,9-dimethoxy-5-[N-alkyl-N-cyanomethyl
aminoethyl]dibenzo[c,h][1,6]naphthyridin-6-ones (10–12)
To
a solution of 2,3-methylenedioxy-8,9-dimethoxy-5-[2-
alkylaminoethyl]dibenzo[c,h][1,6]naph-thyridin-6-one (1.0 mmol
equiv) in DMF (8 mL per mmol equiv) was added potassium car-
bonate (20.0 mmol equiv) and bromoacetonitrile (3.0 mmol equiv)
at room temperature. The resulting reaction mixture was heated at
75 °C with stirring. The reaction mixture was allowed to cool to
room temperature, and then diluted with CHCl3. The CHCl3 solu-
tion was washed with water, and then brine, dried over Na2SO4, fil-
tered, and concentrated under reduced pressure. The residue was
purified by flash chromatography using dichloromethane:
methanol.
4.2. General procedure for the preparation of 2,3-methyl-
enedioxy-8,9-dimethoxy-5-[N-alkyl-N-(2,2,2-trifluoro
ethyl)aminoethyl]dibenzo[c,h][1,6]naphthyridin-6-ones (7–9)
To
a mixture of 2,3-methylenedioxy-8,9-dimethoxy-5-[2-
alkylaminoethyl]dibenzo[c,h]-[1,6]naphthyridin-6-one (1.0 mmol
equiv) in DMF (8 mL per mmol equiv) was added DIEA (20.0 mmol
4.3.1. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-cyanomethyl-
N-methylaminoethyl]dibenzo[c,h][1,6]naphthyridin-6-one (10)
Prepared from 4 (50 mg, 0.12 mmol); (64% yield); reaction time
35 min; mp 235–236 °C; IR (neat): 2239, 1639; 1H NMR (CDCl3): d
2.32 (s, 3H), 3.07 (t, 2H, J = 6.6 Hz), 3.43 (s, 2H), 4.08 (s, 3H), 4.14 (s,
3H), 4.70 (t, 2H, J = 6.6 Hz), 6.20 (s, 2H), 7.50 (s, 1H), 7.66 (s, 1H),
7.69 (s, 1H), 7.89 (s, 1H), 9.39 (s, 1H); 13C NMR (CDCl3 + CD3OD):
d 35.9, 40.8, 44.7, 53.0, 55.4, 99.4, 101.1, 105.3, 107.7, 111.1,
113.7, 116.1, 118.1, 126.3, 140.0, 142.2, 145.5, 147.0, 149.2,
149.4, 153.2, 163.5; HRMS m/z Calcd for C24H22N4O5H 447.1668;
found: 447.1660.
equiv)
and
2,2,2-trifluoroethyl
trifluoromethanesulfonate
(5.0 mmol equiv) at room temperature. The resulting reaction mix-
ture was heated to 80 °C with stirring. The reaction mixture was al-
lowed to cool to room temperature, and then diluted with CHCl3.
The CHCl3 solution was washed with water, then brine, dried over
Na2SO4, and concentrated under reduced pressure. The residue was
chromatographed on silica gel using dichloromethane:methanol.
4.2.1. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-methyl-N-
(2,2,2-trifluoroethyl)aminoethyl]dibenzo[c,h][1,6]
naphthyridin-6-one (7)
4.3.2. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-cyanomethyl-
N-ethylaminoethyl]dibenzo[c,h][1,6]naphthyridin-6-one (11)
Prepared from 5 (100 mg, 0.24 mmol); (80% yield); reaction
time 40 min; mp 226–227 °C; IR (neat): 2285, 1634; 1H NMR
(CDCl3): d 0.83 (t, 3H, J = 7.2), 2.49 (q, 2H, J = 7.2), 3.05 (t, 2H,
J = 6.6), 3.42 (s, 2H), 4.08 (s, 3H), 4.14 (s, 3H), 4.72 (t, 2H, J = 6.6),
6.20 (s, 2H), 7.51 (s, 1H), 7.67 (s, 1H), 7.68 (s, 1H), 7.89 (s, 1H),
9.38 (s, 1H); 13C NMR (CDCl3 + CD3OD): d 11.4, 40.8, 47.2, 47.3,
50.9, 55.2, 55.4, 99.4, 100.0, 101.6, 104.9, 107.6, 111.2, 113.7,
114.0, 118.0, 126.2, 140.1, 141.7, 144.8, 147.0, 149.4, 153.3,
163.3; HRMS m/z Calcd for C25H24N4O5H 461.1825; found:
461.1826.
Prepared from 4 (50 mg, 0.12 mmol); (43% yield); reaction time
4 h; mp 237–239 °C; IR (neat): 1647; 1H NMR (CDCl3) d 2.43 (s,
3H), 3.04 (q, 2H, J = 9.4), 3.22 (t, 2H, J = 6.8), 4.04 (s, 3H), 4.11 (s,
3H), 4.62 (t, 2H, J = 6.8), 6.16 (s, 2H), 7.44 (s, 1H), 7.64 (s, 1H),
7.70 (s, 1H), 7.84 (s, 1H), 9.33 (s, 1H); 13C NMR (CDCl3) d 42.2,
47.9, 55.2, 55.4, 55.4, 57.3 (q, J = 30.8), 99.8, 101.0, 101.3, 106.2,
107.7, 110.9, 113.8, 118.3, 124.6 (q, J = 279.6), 126.7, 140.0,
142.5, 146.2, 146.8, 149.0, 149.3, 153.2, 163.3; HRMS m/z Calcd
for C25H27N3O5H: 490.1590; found: 490.1567.
4.2.2. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-ethyl-N-(2,2,2-
trifluoroethyl)aminoethyl]dibenzo[c,h][1,6]naphthyridin-6-one
(8)
4.3.3. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-cyanomethyl-
N-isopropyl-aminoethyl]dibenzo[c,h][1,6]naphthyridin-6-one
(12)
Prepared from 5 (100 mg, 0.20 mmol); (31% yield); reaction
time 7 h; mp 246–248 °C; IR (neat): 1644; 1H NMR (CDCl3) d
0.98 (t, 3H, J = 7.0), 2.72 (q, 2H, J = 7.0), 3.11 (q, 2H, J = 9.6), 3.26
(t, 2H, J = 7.4), 4.05 (s, 3H), 4.12 (s, 3H), 4.60 (t, 2H, J = 7.4), 6.17
(s, 2H), 7.44 (s, 1H), 7.64 (s, 1H), 7.70 (s, 1H), 7.85 (s, 1H), 9.34
(s, 1H); 13C NMR (CDCl3) d 11.1, 48.1, 48.2, 52.1, 53.8 (q, J = 30.4),
55.4, 55.4, 99.8, 100.9, 101.4, 106.2, 107.7, 110.8, 113.7, 118.3,
124.7 (q, J = 279.4), 126.7, 139.8, 142.5, 146.3, 146.8, 149.0,
149.3, 153.2, 163.3; HRMS m/z Calcd for C25H24F3N3O5H:
504.1746; found: 504.1748.
Prepared from 6 (100 mg, 0.23 mmol); (49% yield); reaction
time 90 min; mp 219–220 °C; IR (neat): 2287, 1644; 1H NMR
(CDCl3): d 0.90 (d, 6H, J = 6.6), 2.78 (m, 1H), 3.12 (t, 2H, J = 6.6),
3.41 (s, 2H), 4.07 (s, 3H), 4.14 (s, 3H), 4.68 (t, 2H, J = 6.6), 6.19 (s,
2H), 7.48 (s, 1H), 7.66 (s, 2H), 7.88 (s, 1H), 9.36 (s, 1H); 13C NMR
(CDCl3): d 18.3, 37.7, 47.3, 47.6, 52.4, 55.4, 99.4, 100.9, 101.4,
105.7, 107.9, 111.1, 113.7, 116.1, 118.3, 126.3, 140.0, 142.0,
145.5, 146.9, 149.2, 149.4, 153.2, 163.3; HRMS m/z Calcd for
C26H26N4O5H 475.1981; found: 475.1982.
4.2.3. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-isopropyl-N-
(2,2,2-trifluoroethyl)aminoethyl]dibenzo[c,h][1,6]
4.4. General procedure for the preparation of 2,3-
naphthyridin-6-one (9)
methylenedioxy-8,9-dimethoxy-5-[N-alkyl-N-(prop-2-
ynyl)aminoethyl]dibenzo[c,h][1,6]naphthyridin-6-ones (13–15)
Prepared from 6 (50 mg, 0.12 mmol); (52% yield); reaction time
7 h; mp 258–261 °C; IR (neat): 1650; 1H NMR (CDCl3) d 1.01 (d, 6H,
J = 6.6), 3.0 (m, 1H), 3.07 (q, 2H, J = 9.6), 3.24 (t, 2H, J = 7.6), 4.05 (s,
3H), 4.12 (s, 3H), 4.58 (t, 2H, J = 7.6), 6.17 (s, 2H), 7.46 (s, 1H), 7.67
(s, 1H), 7.73 (s, 1H), 7.87 (s, 1H), 9.37 (s, 1H); 13C NMR (CDCl3) d
17.4, 48.9, 49.2, 51.3 (q, J = 31.9), 51.8, 55.4, 55.4, 99.8, 100.9,
To
a mixture of 2,3-methylenedioxy-8,9-dimethoxy-5-[2-
alkylaminoethyl]dibenzo[c,h][1,6]naph-thyridin-6-one (1.0 mmol
equiv) in DMF (8 mL per mmol equiv) was added potassium car-
bonate (20.0 mmol equiv) and 3-bromopropyne (3.0 mmol equiv)

9300
W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 9295–9301
at room temperature. The resulting reaction mixture was heated
up to 80 °C with stirring. The reaction mixture was allowed to cool
to room temperature, and then diluted with CHCl3. The CHCl3 solu-
tion was washed with water, then brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel using dichloromethane:methanol.
added I2 (2.29 g, 9.0 mmol) portionwise at 0 °C. The resulting reac-
tion mixture was warmed up to room temperature with stirring
overnight. The reaction mixture was quenched with 10 mL water.
The organic layer was washed with satd Na2S2O3 solution (2ꢁ
10 mL), brine (1ꢁ 10 mL), dried over Na2SO4, and concentrated un-
der reduced pressure. The residue was chromatographed on silica
gel using ethyl acetate/hexanes (1:1), yielding 1.4 g in 50% yield
as a light brown solid; mp 195–197 °C; IR (neat): 1694; 1H NMR
(CDCl3) d 1.49 (m, 6H), 4.14 (m, 4H), 7.44 (s, 1H), 7.63 (s, 1H), 9.2
(s, 1H); 13C NMR (CDCl3) d 13.7, 13.7, 63.8, 64.0, 84.7, 115.4,
124.4, 124.4, 147.2, 151.6, 170.5; HRMS m/z Calcd for C11H13IO4H:
334.9775; found: 334.9784.
4.4.1. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-methyl-N-
(prop-2-ynyl)aminoethyl]dibenzo[c,h][1,6]naphthyridin-6-one
(13)
Prepared from 4 (100 mg, 0.25 mmol); (65% yield); reaction
time 30 min; mp 239–241 °C; IR (neat): 1648; 1H NMR (CDCl3) d
2.22 (t, 1H, J = 2.2), 2.34 (s, 1H), 3.09 (t, 2H, J = 6.6), 3.30 (d, 2H,
J = 2.2), 4.04 (s, 3H), 4.11 (s, 3H), 4.61 (t, 2H, J = 6.6), 6.16 (s, 2H),
7.42 (s, 1H), 7.65 (s, 1H), 7.77 (s, 1H), 7.85 (s, 1H), 9.31 (s, 1H);
13C NMR (CDCl3) d 42.0, 46.4, 48.9, 54.0, 56.3, 56.3, 73.4, 78.6,
101.0, 102.0, 102.2, 107.1, 108.8, 111.7, 114.8, 119.3, 127.6,
140.9, 143.4, 147.2, 147.7, 149.9, 150.2, 154.1, 164.1; HRMS m/z
Calcd for C25H23N3O5H: 446.1716; found: 446.1716.
4.7. N-([1,3]Dioxolo[4,5-g]quinolin-8-yl)-N-(2-
(dimethylamino)ethyl)-4,5-diethoxy-2-iodobenzamide (20)
Oxalyl chloride (1.3 mL, 11.3 mmol) was added to a mixture of
2-iodo-4,5-diethoxybenzoic acid (1.95 g, 5.7 mmol) in methylene
chloride (40 mL), and the mixture was heated to reflux under nitro-
gen with stirring for 4 h. The mixture was concentrated to dryness
under vacuum to provide crude 18. The acid chloride was used
without purification and redissolved in 40 mL of methylene chlo-
ride, and a solution of 197 (1.24 g, 4.78 mmol) added, then triethyl-
amine (2.0 mL, 27 mmol) was added. The mixture was heated to
reflux with stirring for 16 h and was then cooled to room temper-
ature. The mixture was washed with saturated NaHCO3 (3ꢁ
100 mL) and extracted into dilute aqueous HCl (3ꢁ 100 mL). The
combined aqueous phases were washed with chloroform (2ꢁ
100 mL), basified (30% NaOH), and extracted with chloroform
(3ꢁ 100 mL). The combined organic layers were washed with brine
(100 mL), dried (Mg2SO4), and evaporated, yielding 2.6 g in 94%
yield as a light brown sticky glue; IR (neat): 1654. 1H NMR (CDCl3)
d 1.00 (t, 3H, J = 7.0), 1.28 (t, 3H, J = 7.0), 2.19 (s, 6H), 2.55 (m, 2H),
3.25 (m, 1H), 3.52 (q, 2H, J = 7.0), 3.85 (q, 2H, J = 7.0), 4.44 (m, 1H),
6.09 (m, 2H), 6.31 (s, 1H), 6.97 (s, 1H), 7.25 (d, 1H, J = 4.6), 7.29 (s,
1H), 7.35 (s, 1H), 7.25 (d, 1H, J = 4.6); 13C NMR (CDCl3) d 14.3, 14.6,
45.6, 46.9, 56.6, 64.3, 64.6, 82.8, 98.5, 102.2, 106.7, 112.3, 120.2,
123.0, 123.2, 133.6, 146.0, 147.5, 148.3, 148.4, 149.0, 149.6,
151.0, 170.0; HRMS m/z Calcd for C25H28IO5N3H: 578.1146; 2029;
found: 578.1149.
4.4.2. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-ethyl-N-(prop-
2-ynyl)aminoethyl]dibenzo[c,h][1,6]naphthyridin-6-one (14)
Prepared from 5 (100 mg, 0.20 mmol); (64% yield); reaction
time 60 min; mp 227–229 °C; IR (neat): 1648; 1H NMR (CDCl3) d
0.95 (t, 3H, J = 7.4), 2.18 (t, 1H, J = 2.2), 2.56 (q, 2H, J = 7.4), 3.12
(t, 2H, J = 7.0), 3.33 (d, 2H, J = 2.2), 4.04 (s, 3H), 4.10 (s, 3H), 4.56
(t, 2H, J = 7.0), 6.15 (s, 2H), 7.41 (s, 1H), 7.59 (s, 1H), 7.78 (s, 1H),
7.84 (s, 1H), 9.28 (s, 1H); 13C NMR (CDCl3) d 12.7, 42.1, 47.9,
49.1, 51.6, 56.3, 56.3, 73.0, 78.7, 101.1, 101.9, 102.2, 107.0, 108.8,
111.6, 114.7, 119.3, 127.4, 140.8, 143.3, 147.1, 147.6, 149.8,
150.2, 154.0, 164.0; HRMS m/z Calcd for C26H25N3O5H: 460.1872;
found: 460.1890.
4.4.3. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-isopropyl-N-
(prop-2-ynyl)aminoethyl]dibenzo-[c,h][1,6]naphthyridin-6-one
(15)
Prepared from 6 (100 mg, 0.23 mmol); (58% yield); reaction
time 60 min; mp 217–219 °C; IR (neat): 1649; 1H NMR (CDCl3) d
0.98 (t, 6H, J = 6.6), 2.13 (t, 1H, J = 1.8), 2.93 (m, 1H,), 3.16 (t, 2H,
J = 7.0), 3.33 (d, 2H, J = 1.8), 4.04 (s, 3H), 4.11 (s, 3H), 4.58 (t, 2H,
J = 7.0), 6.15 (s, 2H), 7.41 (s, 1H), 7.60 (s, 1H), 7.80 (s, 1H), 7.85
(s, 1H), 9.29 (s, 1H); 13C NMR (CDCl3) d 19.5, 39.6, 48.1, 49.7,
52.4, 56.3, 56.3, 72.4, 80.8, 101.2, 101.9, 102.2, 107.0, 108.8,
111.7, 114.8, 119.4, 127.6, 141.0, 143.4, 147.1, 147.6, 149.8,
150.2, 154.1, 164.1; HRMS m/z Calcd for C27H28N3O5H: 474.2029;
found: 474.2031.
4.8. 2,3-Methylenedioxy-8,9-diethoxy-5-(2-N,N-
dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-one (21)
A
mixture of 19 (1.5 g, 2.6 mmol), Pd(OAc)2 (117 mg,
0.52 mmol), P(o-tolyl)3 (390 mg, 1.3 mmol), and Ag2CO3 (1.43 g,
5.2 mmol) in dimethylformamide (DMF) (35 mL) was heated to re-
flux with stirring for 30 min. The reaction mixture was cooled, di-
luted with chloroform, and filtered through Celite, and the solvent
was removed under vacuum. The crude residue was chromato-
graphed in 99:1 chloroform/methanol to provide 500 mg (34%) of
the cyclized product as a white solid; mp 229–232 °C; IR (neat):
1649; 1H NMR (CDCl3) d 1.58 (m, 6H), 2.38 (s, 6H), 2.99 (t, 2H,
J = 6.6), 4.30 (m, 4H), 4.58 (t, 2H, J = 6.6), 6.16 (s, 2H), 7.43 (s,
1H), 7.63 (s, 1H), 7.85 (s, 1H), 9.31 (s, 1H); 13C NMR (CDCl3) d
14.7, 14.7, 45.8, 49.1, 57.9, 64.7, 64.9, 101.2, 102.2, 103.2, 107.0,
109.8, 111.7, 114.8, 119.1, 127.3, 140.8, 143.5, 147.2, 147.6,
149.8, 149.9, 153.8, 164.1; HRMS m/z Calcd for C29H27O5N3H:
450.2029; found: 450.2030.
4.5. 2,3-Methylenedioxy-8,9-dimethoxy-5-[N-(2-amino-2-
oxoethyl)-N-methylaminoethyl]dibenzo[c,h][1,6]naphthyridin-
6-ones (16)
Prepared from 4 (150 mg, 0.37 mmol) and bromoacetamide
(56 mg, 0.41 mmol); reaction time 40 min; (47% yield); mp 228–
229 °C; IR (neat): 3423, 1671, 1647; 1H NMR (CDCl3 + CD3OD): d
2.17 (s, 3H), 2.93 (s, 2H), 3.00 (t, 2H, J = 6.6 Hz), 4.00 (s, 3H), 4.08
(s, 3H), 4.14 (s, 3H), 4.63 (t, 2H, J = 6.6 Hz), 6.14 (s, 2H), 7.39 (s,
1H), 7.63 (s, 1H), 7.65 (s, 1H), 7.80 (s, 1H), 9.30 (s, 1H); 13C NMR
(CDCl3 + CD3OD): d 41.9, 47.2, 47.6, 55.2, 60.2, 99.4, 101.1, 101.5,
105.2, 107.6, 111.5, 113.7, 116.0, 118.0, 126.3, 141.0, 142.0,
144.8, 147.0, 149.5, 153.4, 163.3, 171.4; HRMS m/z Calcd for
C24H24N4O6H 465.1774; found: 465.1791.
4.9. Topoisomerase-mediated DNA cleavage assays
4.6. 4,5-Diethoxy-2-iodobenzoic acid (17)
Human topoisomerase I was expressed in Escherichia coli and
isolated as a recombinant fusion protein using a T7 expression sys-
tem as described previously.17 Plasmid YepG was also purified by
the alkali lysis method followed by phenol deproteination and
To a suspension of 3,4-diethoxybenzoic acid (1.8 g, 8.6 mmol)
and silver trifluoroacetate (2.0 g, 9.02 mmol) in 15 mL CHCl3 was

W. Feng et al. / Bioorg. Med. Chem. 16 (2008) 9295–9301
9301
CsCl/ethidium isopycnic centrifugation method as described.18 The
30 endlabeling of the plasmid was accomplished by digestion with
a restriction enzyme followed by end filling with Klenow polymer-
ase as previously described.19 The cleavage assays were performed
as previously reported.20,21 The drug and the DNA in presence of
topoisomerase I was incubated for 30 min at room temperature.
were inoculated on the right flank with 1.5 ꢁ 106 MDA-MB-435 tu-
mor cells in 0.1 mL of RPMI 1640 Media by sc injection (25 gauge
needle ꢁ 5/800). The MDA-MB-435 cells were grown in 75 cm2
flasks using RPMI 1640 Media and 10% fetal bovine serum. Tumors
were of sufficient size at 19–20 days after inoculation. Tumor-
bearing mice were evenly matched in each experimental group
based on tumor volume. Tumor volume was calculated by measur-
ing the tumor with a microcaliper. The length (l) is the maximum
two dimensional distance of the tumor and the width (w) is the
maximum distance perpendicular to this length measured in
mm. Tumor volume was calculated using the formula (l * w2)/2.
Every mouse in this study was weighed individually on a daily ba-
sis. Dose adjustments for each experimental group, as indicated in
Table 3, were made throughout the study based upon the effect or
lack of an effect of treatment on average body weights. Tumor vol-
ume was determined for each individual mouse every other day.
The reactions were terminated by the addition of 5 lL of 5% SDS
and 1 mg/mL protein kinase K with an additional 1 h of incubation
at 37 °C. Samples were then alkali denatured by the addition of
NaOH, EDTA, sucrose, and bromophenol blue to final concentra-
tions of 75 mM, 2.5%, and 0.05 mg/mL, respectively, prior to load-
ing onto a neutral agarose gel. After development of the gels,
typically 24-h exposure was used to obtain autoradiograms outlin-
ing the extent of DNA fragmentation. Topoisomerase I-mediated
DNA cleavage values are reported as relative effective concentra-
tion (REC), that is, concentrations relative to camptothecin, whose
value is arbitrarily assumed as 0.2, that are able to produce the
same 10% cleavage on the plasmid DNA in the presence of human
topoisomerase I.
Acknowledgments
This study was supported by National Cancer Institute Grants
CA098127 (E.J.L.) and CA39662 (L.F.L.) and Genzyme Corporation.
4.10. Cytotoxicity assays
The cytotoxicity was determined using the MTT-microtiter
plate tetrazolinium cytotoxicity assay (MTA). The human lympho-
blast RPMI 8402 and its camptothecin-resistant variant cell line,
CPT-K5 was provided by Dr. Toshiwo Andoh (Aichi Cancer Center
Research Institute, Nagoya, Japan).10 The P388 mouse leukemia cell
line and its CPT-resistant TOP1-deficient variant P388/CPT4511
were obtained from Michael R. Mattern and Randal K. Johnson
(GlaxoSmithKline, King of Prussia, PA). The KB3-1 cell line and its
multidrug-resistant variant KBV-113 were obtained from K.V. Chin
(The Cancer Institute of New Jersey, New Brunswick, NJ). The
KBH5.0 cell line as noted previously8 was derived from KB3-1 by
stepwise selection against Hoechst 33342. The cytotoxicity assay
was performed using 96-well microtiter plates. Cells were grown
in suspension at 37 °C in 5% CO2 and maintained by regular passage
in RPMI medium supplemented with 10% heat inactivated fetal bo-
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