Synthesis and cytotoxic activity of carboxamide derivatives of benzo[b][1,6]naphthyridines

Article abstract of DOI:10.1021/jm020420u

The reaction of 4-dimethylaminomethylene-6-methyl-4H-pyrano[4,3-b] quinoline-1,3-dione with a range of primary amines gave rise to a series of 2-substituted 6-methyl-1-oxo-1,2-dihydrobenzo-[b] [1,6]naphthyridine-4-carboxylic acids. The derived 4-N-[2-(dim

Full text of DOI:10.1021/jm020420u

J . Med. Chem. 2003, 46, 1049-1054
1049
Syn th esis a n d Cytotoxic Activity of Ca r boxa m id e Der iva tives of
Ben zo[b][1,6]n a p h th yr id in es
Leslie W. Deady,*,† Thomas Rodemann,† Li Zhuang,‡ Bruce C. Baguley,‡ and William A. Denny*,‡
Chemistry Department, La Trobe University, Victoria 3086, Australia, and
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences,
The University of Auckland, Private Bag 92019, Auckland, New Zealand
Received September 25, 2002
The reaction of 4-dimethylaminomethylene-6-methyl-4H-pyrano[4,3-b]quinoline-1,3-dione with
a range of primary amines gave rise to a series of 2-substituted 6-methyl-1-oxo-1,2-dihydrobenzo-
[b][1,6]naphthyridine-4-carboxylic acids. The derived 4-N-[2-(dimethylamino)ethyl]carboxamides
were tested for growth inhibitory properties against murine P388 leukemia, Lewis lung
carcinoma (LLTC), and human J urkat leukemia cell lines. Most compounds were potent
cytotoxins, with some having IC50 values less than 10 nM. Five were tested in vivo against
subcutaneous colon 38 tumors in mice, and a single dose (3.9 mg/kg) proved to be curative for
the 2-methyl and 2-(3,4-dimethoxyphenyl) derivatives in this refractory model.
In tr od u ction
We have now found that 5 reacts readily with a range
of primary amines to form 2-substituted 1-oxo-1,2-
dihydrobenzo[b][1,6]naphthyridine-4-carboxylic acids 6
(Scheme 1). Subsequently, new carboxamides 7 derived
from these were obtained, where the peri arrangement
of amide and central ring nitrogen seen in DACA is
duplicated. Their synthesis, growth inhibitory properties
in a panel of tumor cell lines, and discovery of remark-
ably potent in vivo activity are discussed.
A number of compounds comprising tricyclic chro-
mophores bearing a flexible cationic side chain are
known to show cytotoxic effects. These include benziso-
quinolinediones (e.g., amonafide, 11,2), anthraquinones
(e.g., mitoxantrone, 23), and acridine-4-carboxamides
(e.g., DACA, 34,5). Their utility as anticancer drugs is
considered to be primarily through their ability to
inhibit topoisomerase enzymes. With respect to DACA,
it has been found that the peri arrangement between
the amide function and the central ring nitrogen is
essential for anticancer activity.4
Ch em istr y
N2- and carbon-substituted derivatives of the benzo-
[b][1,6]naphthyridin-2(1H)-one system are known.7-10
Dibenzo[b,h][1,6]naphthyridin-6(5H)-ones have also been
reported,7,11 but the synthetic route in the present work
differs from these previous examples.
Compound 6d was formed previously by refluxing 5b
with phosphoryl chloride for 48 h.6 However, reaction
with methylamine in tetrahydrofuran/dimethylforma-
mide at room temperature brought about the same
conversion in an experimentally preferable procedure
and opened up the possibility of the analogous reaction
with other amines. The related reaction of isocoumarins
with ammonia to give isoquinolones (the Gabriel reac-
tion12) is long known and has been extended in related
systems to reactions with alkylamines13 and ary-
lamines.14 Compounds 5 reacted with a selection of
alkylamines under very mild conditions to give 6a -h
(Scheme 1) in generally good yields, while reaction with
N,N′-bis(2-aminoethyl)-N,N′-dimethylpropane-1,3-di-
amine gave an example of a bis compound 6i.
A slight anomaly occurred when ammonia was used
instead of an alkylamine, since the product that sepa-
rated from the reaction mixture in this case alone
contained some ammonium species; a triplet at 7.1 ppm
(broad or sharp with J ) 51 Hz, depending on solvent
We recently reinvestigated the reaction of homoph-
thalic acid derivatives with Vilsmeier reagent and
extended this to formation of quinoline analogues 5.6
1
and conditions) was evident in the H NMR spectrum,
* To whom correspondence should be addressed. For L.W.D.: phone,
61 3 9479 2561; fax, 61 3 9479 1399; e-mail, L.Deady@latrobe.edu.au.
For W.A.D.: phone, 64 9 3737 599, extension 6144; fax, 64 9 3737 502;
e-mail, b.denny@auckland.ac.nz.
though the NH (δ 12.2) and CO2H (δ 16.0) proton
singlets could also be seen. Liberation of 6c required
treatment of this material with acid (this turned out to
be important to avoid complications in further reac-
† La Trobe University.
‡ The University of Auckland.
10.1021/jm020420u CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/05/2003

1050 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Deady et al.
Sch em e 1a
chloride 8b, and then reaction with an excess of N,N-
dimethylethylenediamine displaced both chlorines to
give compound 9 of Table 1.
Reaction of 5b with the arylamine 3,4-dimethoxya-
niline failed in the absence of triethylamine, and it
seems that moderate base catalysis of this reaction is
required. Alkylamines behave as both nucleophile and
base, but the arylamine required the presence of the
more basic, nonnucleophilic triethylamine. Even then,
reaction did not go to completion probably because of
the insolubility of the initial product 10 (Scheme 3). Hot
pyridine was found to be a suitable solvent, and reflux
of a mixture of 10, more 3,4-dimethoxyaniline, and
triethylamine in this solvent for 8 h gave 6j in 78% yield.
The carboxamides of Table 1 were prepared by reac-
tion of the intermediate acid chlorides (from thionyl
chloride reaction with the appropriate 6 and not iso-
lated) with N,N-dimethylethylenediamine.
Resu lts a n d Discu ssion
The compounds were evaluated for growth inhibitory
properties, measured as IC50 values against murine
P388 leukemia cells, Lewis lung carcinoma cells (LLTC),
and human J urkat leukemia cells (J LC), together with
their amsacrine- and doxorubicin-resistant derivatives
(J LA and J LD, respectively), which were obtained and
cultured as previously described.15,16 The results are
summarized in Table 1, along with comparable data for
some reference compounds.
a
(i) RNH2, DMF, 20 °C; (ii) CH2[CH2NMe(CH2)2NH2]2 (0.5 mol
equiv), DMF, 20 °C; (iii) SOCl2, reflux, then Me2N(CH2)2NH2,
CH2Cl2.
Sch em e 2a
The J LA line is resistant to the DNA intercalator
amsacrine and similar agents because of a reduced level
of topo II enzyme. The J LD line is resistant to doxoru-
bicin, primarily by virtue of altered levels of topo II but
probably also by additional mechanisms. The ratios of
the IC50 values of a drug in the parent line to one of the
sublines (IC50[J LA]/IC50[J LC] and (IC50[J LD]/IC50[J LC])
therefore provide some indication of the mechanism of
cytotoxicity. Classical topo II inhibitors such as amsa-
crine, doxorubicin, and etoposide have large ratios (10-
to 90-fold), whereas topo I inhibitors such as camptoth-
ecin and mixed topo I/II inhibitors such as DACA 3 have
ratios of only about 2-fold. Values of these ratios of less
than about 1.5-2 therefore suggest cytotoxicity by a
non-topo-II-mediated mechanism.
a
(i) POCl3, reflux, aqueous workup; (ii) SOCl2, reflux; (iii) excess
Me2N(CH2)2NH2, CH2Cl2, reflux.
Sch em e 3a
Since the chemistry allowed the introduction of a
range of N2 substituents in the 1-oxo series (Table 1,
structure A), the main interest was in this aspect of
structure-activity relationships and all compounds
were prepared with the same carboxamide function as
DACA. The noteworthy feature is the generally high
level of cytotoxicity compared with that of DACA and 4
(a recently discovered tetracyclic carboxamide with
remarkable in vivo activity in early results;17 see below).
Thus, for a diverse array of substituents from Y ) H
(7c) to Y ) dimethoxyphenyl (7j), potency is high. The
exception is 7h , where branching at the R-carbon is
evidently not tolerated.
a
(i) 4-aminoveratrole, NEt3, DMF, 20 °C; (ii) 4-aminoveratrole,
NEt3, pyridine, reflux 8 h; (iii) SOCl2, reflux, then Me2N(CH2)2NH2,
CH2Cl2.
Two pairs (7b/7d and 7g/7f) allowed the effect of a
6-methyl substituent to be assessed. As expected from
results with DACA analogues where small 5-substitu-
ents gave greater cytotoxicity,18 the methyl group here
too provided enhanced activity.
tions). Further chemistry on 6c was possible (Scheme
2). Reaction with phosphoryl chloride replaced the oxo
function, though complete conversion was not obtained
and prolonged reflux gave complex mixtures. An aque-
ous workup afforded 8a . This was converted to the acid
The bis example 6i was included because there is
much current interest in the anticancer properties of

Derivatives of Naphthyridines
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1051
Ta ble 1. Growth Inhibitory Activity of 1-Oxo-1,2-dihydrobenzo[b][1,6]naphthyridine-4-carboxamides and Reference Compounds
a
IC50 (nM)
LLd
IC50 ratiob
compd
form
Y
Z
P388c
J LC
J LA/J LC
J LD/J LC
e
7b
7c
7d
7e
7f
7g
7h
7j
A
A
A
A
A
A
A
A
Af
B
Me
H
Me
Bu
H
14
11
2.1
14
6.8
54
590
12
22
2.4
15
150
13
71
6-Me
6-Me
6-Me
6-Me
H
6-Me
6-Me
6-Me
10
1.7
15
3.7
50
161
4.4
4.7
26
6.7
51
1.9
5.6
2.3
0.7
0.8
0.8
1.3
0.3
2.6
7.9
3.0
0.9
1.1
1.1
1.5
0.4
Me2N(CH2)2
Me2N(CH2)2
(S)-PhCH(Me)
3,4-(MeO)2C6H3
(CH2)2NMe(CH2)3NMe(CH2)2
8.5
123
1070
9.4
7i
9
1.0
doxorubicin
etoposide
camptothecin
3 (DACA)g
4h
22
180
33
190
101
9.6
160
5.6
580
424
4.4
13
2.0
1.9
2.5
13
90
1.4
2.3
2.5
100
a
IC50: concentration of drug to reduce cell number to 50% of control cultures. b IC50 values for the J urkat lines J LA and J LD, respectively,
relative to J LC. See text. c Murine P388 leukemia. Murine Lewis lung carcinoma. e Human J urkat leukemia. f A bis compound. Data
from ref 18. Data from ref 17.
d
g
h
Ta ble 2. In Vivo Activity of
1-Oxo-1,2-dihydrobenzo[b][1,6]naphthyridine-4-carboxamides
and Reference Compounds against Subcutaneous Colon 38
schedules. Colon 38 is relatively refractory to antime-
Tumors in Mice
the results summarized in Table 2. Noteworthy is the
finding of some curative effects at low, single-dose
tabolites, alkylating agents, and other topoisomerase-
dose,
growth
directed agents; of the established compounds listed in
Table 2, growth delays vary, but none produced cures.
We recently reported that 4 at 65 mg/kg in a single
repeat-dose schedule was therefore strikingly effective
in this regard,17 but compounds in the present set are
even more potent. Thus, the 2-methyl compound 7d
produced 100% cures at a single dose of 3.9 mg/kg. The
2-butyl analogue 7e also produced cures but was slightly
less potent, while the 2-H compound 7c was much less
active. Compound 7f, bearing the extra cationic side
chain, was also less effective. The wide tolerance of the
nature of the 2-substituent noted in the in vitro testing
was retained in the colon 38 results; the aryl-substituted
7j also produced cures at a low dosage, though the
indication is that this compound is more toxic than is
7d . Compound 9, with the cationic side chain in place
of the 1-oxo group of compounds 7, produced only a
small growth delay at the highest tolerated dose.
drug
mg kg-1 day-1 schedulea delay days curesb
7c
7d
7d
7d
7d
7e
7e
7f
7j
30c
8.9c
5.9
3.9
sd
sd
sd
sd
sd
sd
sd
sd
14
>20
>20
>20
10
>20
18
14
0/5
4/4
10/10
4/4
0/5
5/5
0/5
0/5
4/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
5/5
2.6
13.3c
5.9
5.9
3.9c
2.6
sd
sd
sd
>20
16
7j
9
8.9c
2.6
6
8
0
2
2
13
1.5
7
doxorubicin
daunorubicin
amsacrine
mitoxantrone
DACA
q4d × 3
q4d × 3
q4d × 3
q4d × 3
q7d × 2
q4d × 3
q4d × 3
q2d × 2
3.9
13.3
3.9
200
etoposide
irinotecan
4d
45
65
65
>20
a
q2d × 2 ) every 2 days × 2; q4d × 3 ) every 4 days × 3; q7d
b
× 2 ) every 7 days × 2; sd ) single dose. Number of mice with
Con clu sion s
no measurable tumor 20 days after commencement of treatment
divided by total number of mice. c Highest dose that did not
The 4-(2-(dimethylamino)ethylcarboxamide deriva-
tives of 1-oxo-1,2-dihydrobenzo[b][1,6]naphthyridines
are an interesting new class of antitumor agents, with
some showing curative in vivo activity in the sc 38 model
in a single dosing protocol. The range of 2-substituents
that produce significant activity is apparently wide.
Further investigation of the scope of this variable and
the mechanism of action of these compounds is in
progress.
d
produce evidence of toxicity. Data from ref 17.
compounds comprising two DNA-intercalating chro-
mophores joined by a flexible cationic or dicationic linker
chain.19
Compound 9, the lone example of structure B, is a
direct 2-aza analogue of an acridine derivative reported
recently to have high in vitro cytotoxicity against colon
adenocarcinoma and ovarian carcinoma cell lines.20 The
P388 result indicates that 9 too is a potent cytotoxic
agent.
Exp er im en ta l Section
Melting points are uncorrected. NMR spectra were recorded
on a Bruker AM-300 spectrometer operating at 300.13 MHz
(1H) and 75.47 MHz (13C) and on a Bruker DRX-400 spectrom-
Five examples were then evaluated in vivo against
subcutaneously implanted colon 38 tumors in mice with

1052 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
Deady et al.
eter operating at 400.13 MHz (1H) and 100.62 MHz (13C).
Chemical shifts are reported as δ values (ppm) relative to Me4-
Si. Various standard techniques were used to identify proton-
bound carbons in 13C NMR spectra. Full assignments of the
spectra for 6e were obtained from further COSY, HMQC, and
HMBC experiments using appropriate pulse programs from
the Bruker library. Electrospray mass spectra (ESMS) were
recorded on a VG Bio-Q triple quadrupole mass spectrometer
using methanol or acetonitrile with formic acid (1%) as mobile
phase. EI and LSI (3-nitrobenzyl alcohol as liquid matrix)
mode high-resolution mass spectra were obtained by Dr. N.
Davies, University of Tasmania, Australia. Microanalyses,
indicated by symbols of the elements, were confined to the final
products, compounds of Table 1, along with one example of a
precursor acid (6d ) and were within (0.4% of the theoretical
values. They were carried out at the Campbell Microanalytical
Laboratory, University of Otago, New Zealand.
2-[(2-D i m e t h y la m i n o )e t h y l]-6-m e t h y l-1-o x o -1,2-
dih ydr oben zo[b][1,6]n aph th yr idin e-4-car boxylic Acid (6f).
This was prepared from 5b and N,N-dimethylethylenediamine,
as for 6d , and obtained as a yellow solid (78%), mp 254-255
1
°C. H NMR (DMSO-d6): δ 2.21 (s, 6 H, N(CH3)2), 2.60 (t, J )
6.0 Hz, 2 H), 2.73 (s, 3 H, CH3), 4.23 (t, J ) 6.0 Hz, 2 H), 7.65
(t, J ) 7.6 Hz, 1 H, H-8), 7.93 (d, J ) 6.8 Hz, 1 H), 8.21 (d, J
) 8.3 Hz, 1 H), 8.74 (s, 1 H, H-3), 9.48 (s, 1 H, H-10).
2-[(2-Dim eth yla m in o)eth yl]-1-oxo-1,2-d ih yd r oben zo[b]-
[1,6]n a p h th yr id in e-4-ca r boxylic Acid (6g). This was pre-
pared from reaction of N,N-dimethylethylenediamine with 5a ,
as for 6d , and obtained as a yellow solid (52%), mp 232-233
1
°C. H NMR (DMSO-d6): δ 2.20 (s, 6 H, N(CH3)2), 2.58 (t, J )
5.9 Hz, 2 H), 4.22 (t, J ) 6.0 Hz, 2 H), 7.77 (t, J ) 7.4 Hz, 1
H), 8.06 (t, J ) 7.6 Hz, 1 H), 8.23 (d, J ) 8.6 Hz, 1 H), 8.39 (d,
J ) 8.3 Hz, 1 H), 8.74 (s, 1 H, H-3), 9.54 (s, 1 H, H-10).
(S)-6-Meth yl-1-oxo-2-(1-ph en yleth yl)-1,2-dih ydr oben zo-
[b][1,6]n a p h th yr id in e-4-ca r boxylic Acid (6h ). This was
prepared from 5b and (S)-(-)-R-methylbenzylamine, as for 6d ,
except that the product remained in solution. After 16 h at 20
°C, water was added and the resulting precipitate was collected
by filtration and washed with water to give the product as a
yellow solid (79%), mp 234-235 °C. 1H NMR (DMSO-d6): δ
1.84 (d, J ) 7.1 Hz, 3 H, CH-CH3), 2.73 (s, 3 H, C6-CH3),
6.29 (q, J ) 7.1 Hz, 1 H, N-CH), 7.3-7.5 (m, 5 H), 7.66 (t, J
) 7.7 Hz, 1 H, H-8), 7.94 (d, J ) 6.8 Hz, 1 H), 8.23 (d, J ) 8.2
Hz, 1 H), 8.43 (s, 1 H), 9.53 (s, 1 H, H-10), 16.00 (br s, 1 H,
COOH).
N,N′-Bis(2-a m in oet h yl)-N,N′-dim et h ylpr opa n e-1,3-di-
amine was available,19 and the other amines were commercial
samples.
4-Dim eth yla m in om eth ylen e-6-m eth yl-4H-p yr a n o[4,3-
b]qu in olin e-1,3-d ion e (5b). Phosphoryl chloride (13 mL) was
added, with stirring at 0 °C, to dimethylformamide (40 mL).
After a further 20 min, a solution of ethyl (3-carboxy-8-
methylquinolin-2-yl)acetate6 (10.0 g, 0.037 mol) in dimethyl-
formamide (10 mL) was added in a single portion and the
mixture was stirred at room temperature for a further 1.5 h.
The precipitate was collected by filtration and washed with
cold acetone to give the product as an orange solid (10.0 g,
97%), mp >295 °C (decomposed, formed needles above 280 °C).
1H NMR (DMSO-d6): δ 2.69 (s, 3 H, CH3), 3.30 (s, 3 H, NCH3),
3.59 (s, 3 H, NCH3), 7.36 (t, J ) 7.6 Hz, 1 H, H-3), 7.68 (d, J
) 7.0 Hz, 1 H), 7.89 (d, J ) 8.1 Hz, 1 H), 8.81 (s, 1 H), 8.93 (s,
1 H).
2 -( 3 ,4 -D i m e t h o x y p h e n y l ) -6 -m e t h y l -1 -o x o -1 ,2 -
dih ydr oben zo[b][1,6]n aph th yr idin e-4-car boxylic Acid (6j).
A mixture of 4-aminoveratrole (0.12 g, 0.78 mmol), 5b (0.20
g, 0.7 mmol), dimethylformamide (5 mL), and triethylamine
(1 mL) was stirred at room temperature for 16 h. Solid was
present at all times, and it was collected by filtration and
washed with a little cold dichloromethane to give the inter-
mediate 4-[(3,4-dimethoxyphenyl)aminomethylene]-6-methyl-
4H-pyrano[4,3-b]quinoline-1,3-dione (10) as a yellow solid (0.25
4-Dim eth ylam in om eth ylen e-4H-pyr an o[4,3-b]qu in olin e-
1,3-d ion e (5a ). 5a was similarly prepared from acetanilide,
1
as an orange solid (87%), mp >300 °C. H NMR (DMSO-d6):
1
δ 3.33 (s, 3 H, NCH3), 3.64 (s, 3 H, NCH3), 7.55 (t, J ) 7.4 Hz,
1 H), 7.91 (t, J ) 7.3 Hz, 1 H), 8.08 (d, J ) 7.9 Hz, 1 H), 8.15
(d, J ) 7.8 Hz, 1 H), 8.94 (s, 1 H), 9.13 (s, 1 H).
g, 90%), mp 280-281 °C. H NMR (DMSO-d6): δ 2.81 (s, 3 H,
CH3), 3.79 (s, 3 H, OCH3), 3.87 (s, 3 H, OCH3), 7.06 (s, 2 H),
7.21 (s, 1 H), 7.52 (t, J ) 7.5 Hz, 1 H, H-8), 7.83 (d, J ) 7.0
Hz, 1 H), 8.02 (d, J ) 7.9 Hz, 1 H), 8.81 (s, 1 H, J ) 13.2 Hz,
1 H, CHN), 9.09 (s, 1 H, H-10), 13.83 (d, J ) 13.2 Hz, 1 H,
NH).
2 ,6 -D i m e t h y l -1 -o x o -1 ,2 -d i h y d r o b e n z o [ b ] [ 1 ,6 ] -
n a p h th yr id in e-4-ca r boxylic Acid (6d ). A solution of 2 M
methylamine in tetrahydrofuran (7.0 mL, 14 mmol) was added
to a suspension of 5b (0.8 g, 2.8 mmol) in dimethylformamide
(20 mL), and the whole was stirred for 16 h at room temper-
ature. The reactant dissolved and was soon replaced by a new
solid. This was collected by filtration and washed with a little
cold acetone to give the product as a bright-yellow solid (0.60
A mixture of 10 (0.23 g), 4-aminoveratrole (0.46 g), pyridine
(15 mL), and triethylamine (1 mL) was heated under reflux
for 8 h (dissolution occurred during the heating). The reaction
mixture was allowed to stand at -10 °C overnight, and the
resulting precipitate was collected by filtration and washed
with a little cold acetone to give the product as a yellow solid
(0.18 g, 78%), mp 297-298 °C (after forming needles above
1
g, 79%), mp >300 °C (formed cubic crystals above 290 °C). H
NMR (DMSO-d6): δ 2.75 (s, 3 H, CH3), 3.67 (s, 3 H, NCH3),
7.67 (t, J ) 7.7 Hz, 1 H, H-8), 7.95 (d, J ) 6.6 Hz, 1 H), 8.25
(d, J ) 8.1 Hz, 1 H), 8.83 (s, 1 H), 9.52 (s, 1 H), 16.03 (s, 1 H,
COOH). Anal. (C15H12N2O3‚0.2H2O) C, H, N.
1
285 °C). H NMR (DMSO-d6): δ 2.73 (s, 3 H, CH3), 3.78 (s, 3
H, OCH3), 3.84 (s, 3 H, OCH3), 7.12 (s, 2 H), 7.23 (s, 1 H), 7.67
(t, J ) 7.4 Hz, 1 H, H-8), 7.95 (d, J ) 6.7 Hz, 1 H), 8.23 (d, J
) 8.2 Hz, 1 H), 8.45 (s, 1 H), 9.50 (s, 1 H, H-10), 15.98 (br s,
1 H, COOH).
2-Meth yl-1-oxo-1,2-dih ydr oben zo[b][1,6]n aph th yr idin e-
4-ca r boxylic Acid (6b). This was prepared from 5a and
methylamine, as for 6d , and obtained as a yellow solid (69%),
mp >305 °C (formed needles above 230 °C). 1H NMR (DMSO-
d6): δ 3.61 (s, 3 H, N-CH3), 7.71 (t, J ) 7.7 Hz, 1 H), 8.00 (t,
J ) 8.2 Hz, 1 H), 8.16 (d, J ) 8.6 Hz, 1 H), 8.33 (d, J ) 8.1 Hz,
1 H), 8.66 (s, 1 H), 9.46 (s, 1 H), 16.00 (s, 1 H, COOH).
2,2′-[1,3-P r opan ediylbis[(m eth ylim in o)-2,1-eth an ediyl]]-
bis[6-methyl-1-oxo-1,2-dihydrobenzo[b][1,6]naphthyridine-4-
ca r boxylic Acid ] (6i). N,N′-Bis(2-aminoethyl)-N,N′-dimeth-
ylpropane-1,3-diamine (0.10 g, 0.54 mmol) was added to a
suspension of 5b (0.30 g, 1.06 mmol) in dimethylformamide
(7.5 mL) and triethylamine (0.4 mL), and the resulting solution
was stirred for 16 h at room temperature. The precipitate,
which formed during the reaction, was collected by filtration
and was stirred in ethyl acetate (10 mL) for 5 min. The solid
was collected by filtration to give the product as a yellow solid
2-B u t y l-6-m e t h y l-1-o x o -1,2-d ih y d r o b e n zo [b ][1,6]-
n a p h th yr id in e-4-ca r boxylic Acid (6e). This was prepared
from 5b and butylamine, as for 6d , and obtained as a bright-
1
yellow solid (82%), mp 259 °C. H NMR (CDCl3): δ 0.98 (t, J
) 7.2 Hz, 3 H, δ-CH3), 1.42 (sextet, J ) 7.6 Hz, 2 H, γ-CH2),
1.81 (quintet, J ) 7.7 Hz, 2 H, â-CH2), 2.83 (s, 3 H, C6-CH3),
4.10 (t, J ) 7.4 Hz, 2 H, N-CH2), 7.57 (t, J ) 7.7 Hz, 1 H,
H-8), 7.81 (d, J ) 7.0 Hz, 1 H, H-7), 7.93 (d, J ) 8.3 Hz, 1 H,
H-9), 8.59 (s, 1 H, H-3), 9.35 (s, 1 H, H-10), 16.09 (br s, 1 H,
COOH). 13C NMR (CDCl3): δ 13.6 (δ-CH3), 18.2 (C6-CH3), 19.9
(γ-CH2), 31.3 (â-CH2), 49.8 (N-CH2), 105.2 (C-4), 119.1 (C-
10a), 126.4 (C-9a), 127.4 (C-8), 127.6 (C-9), 134.2 (C-7), 134.9
(C-6), 141.3 (C-10), 145.2 (C-3), 146.7 (C-5a), 148.7 (C-4a),
161.8 (C-1), 166.3 (COOH).
1
(0.15 g, 43%), mp 224-228 °C. H NMR (DMSO-d6): δ 1.45-
1.49 (m, 2 H), 2.13 (s, 6 H, 2 × NCH3, 2.28-2.34 (m, 4 H),
2.48-2.55 (m, 10 H), 4.01-4.05 (m, 4 H), 7.36 (t, J ) 7.4 Hz,
2 H), 7.62 (d, J ) 6.5 Hz, 2 H), 7.93 (d, J ) 7.7 Hz, 2 H), 8.49
(s, 2 H), 9.16 (s, 2 H).
6-Meth yl-1-oxo-1,2-dih ydr oben zo[b][1,6]n aph th yr idin e-
4-ca r boxylic Acid (6c). A mixture of 5b (1.0 g) in dimethyl-
formamide (25 mL) was stirred under a stream of ammonia
for 4 h at room temperature. The precipitate, which formed

Derivatives of Naphthyridines
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6 1053
after dissolution of the starting material, was collected by
filtration and washed thoroughly with acetone to give a yellow
solid (0.70 g).
(7e). 7e was prepared from acid 6e and was obtained as a
yellow solid (89%), mp 127-128 °C [light petroleum (bp 90-
1
120 °C)]. H NMR (CDCl3): δ 0.95 (t, J ) 7.3 Hz, 3 H, CH2-
CH3), 1.41 (sextet, J ) 7.3 Hz, 2 H, CH2-CH3), 1.80 (quintet,
J ) 7.4 Hz, 2 H, N2-CH2CH2), 2.29 (s, 6 H, N(CH3)2), 2.62 (t,
J ) 6.5 Hz, 2 H, CH2-N(CH3)2), 2.85 (s, 3 H, C6-CH3), 3.72
(q, J ) 6.3 Hz, 2 H, CONH-CH2), 4.08 (t, J ) 7.4 Hz, 2 H,
N2-CH2), 7.47 (t, J ) 7.9 Hz, 1 H, H-8), 7.71 (d, J ) 6.8 Hz,
1 H), 7.85 (d, J ) 8.2 Hz, 1 H), 8.59 (s, 1 H, H-3), 9.27 (s, 1 H,
H-10), 10.97 (br s, 1 H, CONH). 13C NMR (CDCl3): δ 13.6
(CH2-CH3), 18.5 (C6-CH3), 19.8 (CH2), 31.3 (CH2), 37.6 (CH2),
45.4 (N(CH3)2), 49.4 (CH2), 58.8 (CH2), 109.5 (C), 119.5 (C),
125.9 (C), 126.5 (CH), 127.3 (CH), 132.8 (CH), 135.9 (C), 140.0
(CH), 143.0 (CH), 148.2 (C), 148.8 (C), 162.4 (C), 164.6 (C).
ESMS m/z: 381 (M + 1, 100%), 191 [(M + 2)/2, 20%]. Anal.
(C22H28N4O2) C, H, N.
This ammonium-containing species (see text) (0.15 g) in 5%
hydrochloric acid (20 mL) was heated under reflux until a clear
solution was obtained (10 min). After being cooled, the solution
was adjusted to pH 4 with 10% sodium hydroxide. The
precipitate was collected by filtration to give the acid as a
yellow solid (0.13 g), mp >300 °C (after forming needles above
1
255 °C). H NMR (DMSO-d6): δ 2.67 (s, 3 H, CH3), 7.58 (br s,
1 H, H-8), 7.89 (d, J ) 4.9 Hz, 1 H), 8.14 (d, J ) 7.0 Hz, 1 H),
8.31 (s, 1 H, H-3), 9.35 (s, 1 H, H-10), 12.19 (br s, 1 H, NH),
15.90 (br s, 1 H, COOH).
N-[(2-Dim eth yla m in o)eth yl]-1-[2-((d im eth yla m in o)et-
h yl)a m in o]-6-m et h ylb en zo[b][1,6]n a p h t h yr id in e-4-ca r -
boxa m id e (9). Acid 6c (0.5 g) was heated under reflux in
phosphoryl chloride (50 mL) for 8 h. The excess of phosphoryl
chloride was removed at reduced pressure and ice/water (30
mL) was carefully added to the residue. The precipitate was
collected by filtration, washed with water, and dried. The solid
was heated under reflux in thionyl chloride (20 mL) for 1 h.
The thionyl chloride was removed at reduced pressure, and a
solution of N,N-dimethylethylenediamine (1 mL) in dichlo-
romethane (20 mL) was added to the residue. The resulting
solution was heated under reflux for 16 h and then cooled and
washed with 10% sodium carbonate and twice with water. The
solvent was removed, and preparative TLC of the residue
(alumina; chloroform/methanol, 20:1) gave the crude product,
Rf ) 0.6. This was recrystallized from toluene to give the
product as a yellow solid (0.27 g, 35%), mp 219-220 °C. 1H
NMR (CDCl3): δ 2.33 (s, 6 H, N(CH3)2), 2.36 (s, 6 H, N(CH3)2),
2.64 (t, J ) 4.8 Hz, 2 H), 2.70 (t, J ) 5.9 Hz, 2 H), 2.86 (s, 3 H,
CH3), 3.71-3.78 (m, 4 H), 6.89 (br s, 1 H, NH), 7.45 (dd, J )
7.9, 6.9 Hz, 1 H, H-8), 7.70 (d, J ) 7.0 Hz, 1 H), 7.86 (d, J )
8.3 Hz, 1 H, H-9), 8.75 (s, 1 H), 9.26 (s, 1 H, H-10), 11.02 (br
s, 1 H, CONH). ESMS m/z: 198 [(M + 2)/2, 100%], 395 (M + 1,
25%). Anal. (C22H30N6O) C, H, N.
P r ep a r a tion of N-[2-(Dim eth yla m in o)eth yl]-2,6-d im -
et h yl-1-oxo-1,2-d ih yd r ob en zo[b][1,6]-n a p h t h yr id in e-4-
ca r boxa m id e (7d ). An Exa m p le of th e Gen er a l Meth od
for Am id e F or m a tion . Acid 6d (0.40 g) was heated under
reflux in thionyl chloride (20 mL) for 30 min. The excess of
thionyl chloride was removed at reduced pressure, and a
solution of N,N-dimethylethylenediamine (0.5 mL) in dichlo-
romethane (20 mL) was added to the residue. The resulting
solution was stirred at room temperature for 16 h, and the
solution was washed with 10% sodium carbonate and water
(× 2). The solvent was removed at reduced pressure to give
the product as a yellow solid (0.48 g, 95%), mp 167-170 °C
(acetonitrile). 1H NMR (CDCl3): δ 2.28 (s, 6 H, N(CH3)2), 2.61
(t, J ) 6.3 Hz, 2 H), 2.82 (s, 3 H, CH3), 3.67-3.73 (m, 5 H),
7.46 (t, J ) 7.6 Hz, 1 H, H-8), 7.70 (d, J ) 6.9 Hz, 1 H), 7.82
(d, J ) 8.2 Hz, 1 H), 8.56 (s, 1 H, H-3), 9.21 (s, 1 H, H-10),
10.91 (br s, 1 H, CONH). 13C NMR (CDCl3): δ 18.5 (C6-CH3),
37.2 (N-CH3), 37.6 (CH2), 45.4 (N(CH3)2), 58.8 (CH2), 109.5
(C), 119.3 (C), 125.9 (C), 126.6 (CH), 127.3 (CH), 132.8 (CH),
135.9 (C), 139.8 (CH), 143.6 (CH), 148.2 (C), 148.8 (C), 162.8
(C), 164.5 (C). ESMS m/z: 339 (M + 1). Anal. (C19H22N4O2) C,
H, N.
N-[2-(Dim et h yla m in o)et h yl]-2-[2-(d im et h yla m in o)et -
h y l ] -6 -m e t h y l -1 -o x o -1 , 2 -d i h y d r o b e n z o [ b ] [ 1 , 6 ] -
n a p h th yr id in e-4-ca r boxa m id e (7f). 7f was prepared from
acid 6f and was obtained as a yellow solid (84%), mp 141-
1
143 °C [light petroleum (bp 90-120 °C)]. H NMR (CDCl3): δ
2.30 (s, 6 H, N(CH3)2), 2.38 (s, 6 H, N(CH3)2), 2.67-2.76 (m, 4
H), 2.86 (s, 3 H, C6-CH3), 3.78 (q, J ) 6.3 Hz, 2 H, CH2), 4.18
(t, J ) 6.6 Hz, 2 H, CH2), 7.49 (t, J ) 7.4 Hz, 1 H, H-8), 7.73
(d, J ) 6.8 Hz, 1 H), 7.87 (d, J ) 8.2 Hz, 1 H), 8.61 (s, 1 H,
H-3), 9.29 (s, 1 H, H-10), 11.07 (br s, 1 H, CONH). 13C NMR
(CDCl3): δ 18.6 (C6-CH3), 37.1 (CH2), 45.0 (N(CH3)2), 45.6
(N(CH3)2), 47.2 (CH2), 57.7 (CH2), 58.5 (CH2), 109.3 (C), 119.5
(C), 126.0 (C), 126.6 (CH), 127.4 (CH), 133.0 (CH), 135.9 (C),
140.1 (CH), 143.4 (CH), 148.3 (C), 148.8 (C), 162.5 (C), 165.0
(C). ESMS m/z: 198.5 [(M + 2)/2, 100%], 396 (M + 1, 60%).
Anal. (C22H29N5O2‚0.25H2O) C, H, N.
N-[2-(Dim et h yla m in o)et h yl]-2-[2-(d im et h yla m in o)et -
h yl]-1-oxo-1,2-d ih yd r oben zo[b][1,6]n a p h th yr id in e-4-ca r -
boxa m id e (7g). 7g was prepared from acid 6g and was
obtained as a yellow solid (80%), mp 143-144 °C (toluene).
1H NMR (CDCl3): δ 2.29 (s, 6 H, N(CH3)2), 2.42 (s, 6 H,
N(CH3)2), 2.65-2.71 (m, 4 H), 3.70 (q, J ) 6.0 Hz, 2 H, CH2),
4.17 (t, J ) 6.5 Hz, 2 H, CH2), 7.60 (dd, J ) 8.4 Hz, 1 H, 7.4
Hz, 1 H), 7.88 (t, J ) 7.3 Hz, 1 H), 8.02 (d, J ) 8.2 Hz, 1 H),
8.12 (d, J ) 8.6), 8.60 (s, 1 H, H-3), 9.32 (s, 1 H, H-10), 11.32
(br s, 1 H, CONH). ESMS m/z: 382 (M + 1, 100%), 191.5 [(M
+ 2)/2, 90%]. Anal. (C21H27N5O2) C, H, N.
(S)-N-[2-(Dim et h yla m in o)et h yl]-6-m et h yl-1-oxo-2-(1-
p h en yleth yl)-1,2-d ih yd r oben zo[b][1,6]n a p h th yr id in e-4-
ca r boxa m id e (7h ). 7h was prepared from acid 6h and was
obtained as a yellow semisolid (86%). 1H NMR (CDCl3): δ 1.05
(d, J ) 7.2 Hz, 3 H, CH-CH3), 2.27 (s, 6 H, N(CH3)2), 2.59 (t,
J ) 6.5 Hz, 2 H, CH2-N(CH3)2), 2.85 (s, 3 H, C6-CH3), 3.67
(q, J ) 6.7 Hz, 2 H, CONH-CH2), 6.45 (q, J ) 7.1 Hz, 1 H,
N2-CH), 7.27-7.41 (m, 5 H), 7.45 (dd, J ) 8.1, 7.3 Hz, 1 H,
H-8), 7.69 (d, J ) 7.0 Hz, 1 H), 7.84 (d, J ) 8.2 Hz, 1 H), 8.62
(s, 1 H, H-3), 9.30 (s, 1 H, H-10), 10.96 (br s, 1 H, CONH). 13C
NMR (CDCl3): δ 18.5 (CH3), 19.1 (CH3), 37.4 (CH2), 45.4
(N(CH3)2), 53.4 (CH), 58.7 (CH2), 110.0 (C), 119.4 (C), 126.0
(C), 126.5 (CH), 127.2 (CH), 127.3 (CH), 128.2 (CH), 128.9
(CH), 132.8 (CH), 135.9 (C), 139.56 (CH), 139.61 (CH), 140.3
(CH), 148.2 (C), 148.4 (C), 162.4 (C), 164.6 (C). HRMS (LSI)
calcd for C26H29N4O2: ((M + H)+) 429.2292. Found: 429.2298.
The following amides were made in a similar manner.
N-[2-(Dim eth yla m in o)eth yl]-2-(3,4-d im eth oxyp h en yl)-
6-m eth yl-1-oxo-1,2-d ih yd r oben zo[b][1,6]n a p h th yr id in e-
4-ca r boxa m id e (7j). 7j was prepared from acid 6j and was
obtained as a yellow solid (92%), mp 199-201 °C (toluene).
1H NMR (CDCl3): δ 2.35 (s, 6 H, N(CH3)2), 2.70 (t, J ) 6.3 Hz,
2 H, CH2-N(CH3)2), 2.89 (s, 3 H, C6-CH3), 3.77 (q, J ) 6.4
Hz, 2 H, CONH-CH2), 3.90 (s, 3 H, OCH3), 3.94 (s, 3 H, OCH3),
6.96-6.99 (m, 3 H), 7.52 (t, J ) 7.5 Hz, 1 H, H-8), 7.76 (d, J )
7.0 Hz, 1 H), 7.89 (d, J ) 8.1 Hz, 1 H), 8.71 (s, 1 H, H-3), 9.34
(s, 1 H, H-10), 11.07 (br s, 1 H, CONH). 13C NMR (CDCl3): δ
18.6 (C6-CH3), 37.3 (CH2), 45.2 (N(CH3)2), 56.1 (2 × OCH3),
58.5 (CH2), 109.7 (C), 110.6 (CH), 111.2 (CH), 118.9 (CH), 119.8
(C), 126.1 (C), 126.9 (CH), 127.4 (CH), 133.0 (C), 133.2 (CH),
136.0 (C), 140.5 (CH), 143.8 (CH), 148.4 (C), 148.6 (C), 149.3
(C), 149.4 (C), 162.5 (C), 164.7 (C). ESMS m/z: 461 (M + 1,
N -[2-(D i m e t h y la m i n o )e t h y l]-2-m e t h y l-1-o x o -1,2-
d ih yd r oben zo[b][1,6]n a p h th yr id in e-4-ca r boxa m id e (7b).
7b was prepared from acid 6b and was obtained as yellow
plates (67%), mp 192-194 °C (acetonitrile). 1H NMR (CDCl3):
δ 2.40 (s, 6 H, N(CH3)2), 2.63 (t, J ) 6.1 Hz, 2 H), 3.6-3.7 (m,
5 H), 7.60 (t, J ) 7.1 Hz, 1 H), 7.87 (t, J ) 7.5 Hz, 1 H), 8.01
(d, J ) 8.2 Hz, 1 H), 8.10 (d, J ) 8.5 Hz, 1 H), 8.58 (s, 1 H),
9.31 (s, 1 H, H-10), 11.30 (br s, 1 H, CONH). 13C NMR
(CDCl3): δ 36.8 (N-CH3), 37.0 (CH2), 44.9 (N(CH3)2), 57.7
(CH2), 109.0 (C), 119.1 (C), 125.5 (C), 126.4 (CH), 128.0 (CH),
128.8 (CH), 132.5 (CH), 139.1 (CH), 143.2 (CH), 148.5 (C),
149.3 (C), 162.4 (C), 164.0 (C). Anal. (C18H20N4O2) C, H, N.
N-[2-(Dim et h yla m in o)et h yl]-2-b u t yl-6-m et h yl-1-oxo-
1,2-d ih yd r ob en zo[b][1,6]n a p h t h yr id in e-4-ca r b oxa m id e

1054 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 6
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100%), 231 [(M + 2)/2, 25%). Anal. (C26H28N4O4‚0.5H2O) C, H.
N requires 11.9; found 11.3.
2,2′-[1,3-P r opan ediylbis[(m eth ylim in o)-2,1-eth an ediyl]]-
bis[N-(2-(dimethylamino)ethyl)-6-methyl-1-oxo-1,2-dihydrobenzo
[b][1,6]n a p h th yr id in e-4-ca r boxa m id e] (7i). 7i was pre-
pared from acid 6i and was obtained as a yellow solid (75%),
mp 97-102 °C [light petroleum (bp 90-120 °C)]. 1H NMR
(CDCl3): δ 1.55-1.63 (m, 2 H), 2.23 (s, 6 H), 2.42-2.84 [m, 30
H, including 2.50 (s, 12 H), 2.72 (s, 6 H)], 3.78 (q, J ) 6.2 Hz,
4 H, CH2), 4.08 (t, J ) 5.9 Hz, 4 H, CH2), 7.31 (t, J ) 7.8 Hz,
2 H), 7.53 (d, J ) 6.8 Hz, 2 H), 7.71 (d, J ) 8.2 Hz, 2 H), 8.51
(s, 2 H), 9.11 (s, 2 H), 10.96 (br s, 2 H, 2 × CONH). 13C NMR
(CDCl3): δ 18.5 (CH3), 24.8 (CH2), 36.9 (CH2), 42.1 (NCH3),
44.9 (N(CH3)2), 46.8 (CH2), 54.9 (CH2), 56.5 (CH2), 58.2 (CH2),
108.6 (C), 119.2 (C), 125.6 (C), 126.4 (CH), 127.1 (CH), 132.6
(CH), 135.6 (C), 139.7 (CH), 143.8 (CH), 147.8 (C), 148.4 (C),
162.1 (C), 164.8 (C). ESMS m/z: 402.3 [(M + 2)/2, 100%], 803.5
(M + 1, 50%). Anal. (C45H58N10O4‚1.5H2O) C, H. N requires
16.9; found 16.3.
N-[(2-Dim et h yla m in o)et h yl]-6-m et h yl-1-oxo-1,2-d ih y-
d r oben zo[b][1,6]-n a p h th yr id in e-4-ca r boxa m id e P er ch lo-
r a te Sa lt (7c). 7c was prepared from the ammonium salt of
6c and was obtained in crude form as a yellow solid (69%).
The perchlorate salt was prepared in ethanol, recrystallized
from “moist” methanol, and obtained as a brown solid, mp
218-219 °C (explosive decomposition above 250 °C). 1H NMR
(MeOD-d4): δ 2.91 (s, 9 H, C6-CH3 + N(CH3)2), 3.19 (t, J )
6.2 Hz, 2 H, CH2), 3.45 (t, J ) 6.2 Hz, 2 H), 6.96-7.00 (m, 2
H), 7.21 (br s, 1 H), 7.37 (br s, 1 H), 7.67 (br s, 1 H). ESMS
m/z: 325 (M + 1, 100%), 163 [(M + 2)/2, 12%]. Anal.
(C18H20N4O2‚HClO4‚2H2O) C, H, N.
In Vitr o Cytotoxicity Assa ys. Murine P388 leukemia
cells, Lewis lung carcinoma cells (LL), and human J urkat
leukemia cells (J LC), together with their amsacrine and
doxorubicin-resistant derivatives (J LA and J LD, respectively),
were obtained and cultured as described.21 Growth inhibition
assays were performed by culturing cells at 4.5 × 103 (P388),
103 (LL), and 3.75 × 103 (J urkat lines) per well in microculture
plates (150 mL per well) for 3 (P388) or 4 days in the presence
of drug. Cell growth was determined by [3H]TdR uptake
(P388)22 or the sulforhodamine assay.23 Independent assays
were performed in duplicate.
In Vivo Tu m or Assa ys. Colon 38 tumors were grown
subcutaneously from 1 mm3 fragments implanted in one flank
of BDF1 mice (anesthetized with pentobarbitone, 90 mg/kg).
When tumors reached a diameter of approximately 4-6 mm
(7-8 days), mice were divided into control and drug treatment
groups (five mice per group) with similar average tumor
volumes in each group. Drugs were administered as solutions
of the hydrochloride salts in distilled water (perchlorate salt
for 7c) and were injected intraperitoneally in a volume of 0.01
mL/g of body weight, using either single-dose or intermittent
(q4d × 3 or qd2 × 2) schedules. The mice were monitored
closely, and tumor diameters were measured with calipers
three times a week. Tumor volumes were calculated as 0.52a2b,
where a and b are the minor and major tumor axes and data
are plotted on
a semilogarithmic graph as mean tumor
volumes ((SEM) versus time after treatment. The growth
delay was calculated as the time taken for tumors to reach a
mean volume 4-fold higher than their pretreatment volume.
(21) Finlay, G. J .; Riou, J .-F.; Baguley, B. C. From amsacrine to
DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide): se-
lectivity for topoisomerases I and II among acridine derivatives.
Eur. J . Cancer 1996, 32A, 708-714.
(22) Marshall, E. S.; Finlay, G. J .; Matthews, J . H. L.; Shaw, J . H.
F.; Nixon, J .; Baguley, B. C. Microculture-based chemosensitivity
testing: a feasibility study comparing freshly explanted mela-
noma cells with human melanoma cell lines. J . Natl. Cancer Inst.
1992, 84, 340-345.
Ack n ow led gm en t. The work was supported by an
Australian Research Council Small Grant and by the
Auckland Division of the Cancer Society of New Zealand.
T.R. is grateful for an Australian Postgraduate Award.
We thank Ms. Debbie Greenhalgh and Dr. Graeme
Finlay for carrying out the in vitro assays.
(23) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J . T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New colorimetric cytotoxicity assay for anticancer-drug screen-
ing. J . Natl. Cancer Inst. 1990, 82, 1107-1112.
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