Chromophore-modified bis-naphthalimides: Synthesis and antitumor activity of bis-dibenz[de,h]isoquinoline-1,3-diones

Article abstract of DOI:10.1021/jm960295k

The bis-dibenz[de,h]isoquinoline-1,3-diones are a new series of antitumor agents that consist of two chromophores bridged by an alkylamino linker. In the present study we have explored the effect produced by the presence of two dibenz[de,h]isoquinoline-1,3-dione moieties with different polyamine chains on cellular cytotoxicity. Bis-dibenz[de,h]isoquinoline-1,3- diones with the bridge (CH₂)₂-NH-(CH₂)(n)-NH-(CH₂)₂, where n = 2-5, showed optimum cytotoxicity with IC₅₀'s around 10 nM. Compound 16, which has the (CH₂)₂-NH-(CH₂)₃-NH-(CH₂)₂ bridge, altered DNA mobility and topoisomerase I and II activity at approximately 5 μM. When tested in vivo, compound 16 increased the median survival time of mice implanted with M5076 with an optimum %T/C of 154% and produced cures in 50% of mice implanted with Lox melanoma.

Full text of DOI:10.1021/jm960295k

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J . Med. Chem. 1997, 40, 449-454
449
Ch r om op h or e-Mod ified Bis-Na p h th a lim id es: Syn th esis a n d An titu m or Activity
of Bis-Diben z[d e,h ]isoqu in olin e-1,3-d ion es
M. F. Bran˜a,*,† J . M. Castellano,‡ D. Perron,§ C. Maher,§ D. Conlon,§ P. F. Bousquet,§ J . George,§
X.-D. Qian,§ and S. P. Robinson§
Departamento de Quimica Orga´nica y Farmaceu´tica, Universidad San Pablo CEU, Montepr´ıncipe, Ctra. de Boadilla del
Monte, km 5.3, 28668 Madrid, Spain, Laboratorios Knoll S.A., Avenida de Burgos 91, 28050 Madrid, Spain, and
BASF Bioresearch Corp., 100 Research Drive, Worcester, Massachusetts 01605
Received April 19, 1996X
The bis-dibenz[de,h]isoquinoline-1,3-diones are a new series of antitumor agents that consist
of two chromophores bridged by an alkylamino linker. In the present study we have explored
the effect produced by the presence of two dibenz[de,h]isoquinoline-1,3-dione moieties with
different polyamine chains on cellular cytotoxicity. Bis-dibenz[de,h]isoquinoline-1,3-diones with
the bridge (CH2)2-NH-(CH2)n-NH-(CH2)2, where n ) 2-5, showed optimum cytotoxicity with
IC50’s around 10 nM. Compound 16, which has the (CH2)2-NH-(CH2)3-NH-(CH2)2 bridge,
altered DNA mobility and topoisomerase I and II activity at approximately 5 µM. When tested
in vivo, compound 16 increased the median survival time of mice implanted with M5076 with
an optimum %T/C of 154% and produced cures in 50% of mice implanted with Lox melanoma.
DNA intercalators represent one of the most impor-
tant drug classes in cancer therapy. These agents are
characterized by the presence of flat chromophores
bearing π electron-deficient polyconjugated areas bound
to polar groups.1 Their antitumor action results from
DNA distortion and altered nuclear protein interaction
as a consequence of reversible complex formation.2
In our laboratory we have discovered the antitumor
potential of a series of compounds bearing a naphthal-
imide chromophore.3,4 These agents bind to linear DNA,
increase the length of sonicated DNA, and cause un-
winding of closed circular superhelical DNA.5 These
effects are characteristic of intercalating drugs.6 Two
of the most active mononaphthalimides, amonafide (1)
and mitonafide (2) have been selected for phase II
have recently adopted is the formation of a bis-interca-
lator by linking two naphthalimide groups with a
polyamine bridge.16-18 When appropriately optimized
this flexible linker can lead to an improved cellular
cytotoxicity. For example, the bis-naphthalimides have
a higher cellular cytotoxicity than amonafide or mitona-
fide.16 One bis-naphthalimide, LU 79553 (4), has potent
cellular cytotoxicity and excellent in vivo antitumor
activity in several human tumor xenograft models.19
This agent is currently in phase I of clinical develop-
ment.
clinical trials.7,8 Unfortunately, given on some sched-
ules mitonafide had inappropriate central nervous
system (CNS) toxicity and overall has produced limited
clinical activity.9 Amonafide has been examined more
extensively in clinical trials. While some activity has
been identified the effects again have been limited.10-13
Recently approaches have been used to enhance the
potency of the mononaphthalimides. One approach has
been to modify the chromophore to include an an-
thracene moiety rather than the simpler naphthalene.
These 1,2-dihydro-3H-dibenz[de,h]isoquinoline-1,3-di-
ones include a structure unofficially named azonafide
(3), which has shown an increase in cellular cytotoxic
potency over amonafide.14,15 A second approach that we
In the present study we have explored the effect
produced by the presence of two dibenz[de,h]isoquino-
line-1,3-dione moieties with different polyamine chains
(9-24) on cellular cytotoxicity. The influence on activity
of a NO2 group has also been explored.
Resu lts a n d Discu ssion
Ch em istr y. The preparation of bis-dibenz[de,h]iso-
quinoline-1,3-diones 9-23 was carried out by reaction
of the corresponding polyamine with 2 equiv of the
necessary anthracene-1,9-dicarboxylic acid anhydride,
using toluene as solvent, as is shown in Scheme 1. In
Table 1 are shown all the bis-dibenz[de,h]isoquinoline-
1,3-diones synthesized and their physical properties.
The anthracene-1,9-dicarboxylic acid anhydride was
obtained following essentially the procedure already
described.20 Nitration of the anthracene-1,9-dicarboxy-
† Universidad San Pablo CEU.
‡ Laboratorios Knoll S.A.
§ BASF Bioresearch Corp.
X Abstract published in Advance ACS Abstracts, J anuary 15, 1997.
S0022-2623(96)00295-6 CCC: $14.00 © 1997 American Chemical Society

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450 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Ta ble 1. Bis-Dibenz[de,h]isoquinoline-1,3-diones 9-24
Bran˜a et al.
compd
X
R
yield (%) mp (°C) recrystn solvent
formula
C37H27N3O4
C37H27N3O4
C38H29N3O4
C39H32N3O4
anal.
IC50 (µM)
9
H
H
H
H
H
H
H
H
H
H
H
H
H
(CH2)2-NH-(CH2)2
(CH2)2-NH-(CH2)3
(CH2)3-NH-(CH2)3
(CH2)3-NCH3-(CH2)3
53
78
83
35
41
66
78
50
40
25
61
41
46
53
46
271
260
184
194
244
203
255
191
183
122
180
140
183
340
340
DMF-H2O
DMF
toluene
toluene
DMF
DMF-H2O
toluene
toluene
toluene
toluene
toluene
toluene
toluene
toluene
toluene
C, H, N
C, H, N
C, H, N
C, H, N
0.030
2.000
0.020
0.019
0.070
0.009
0.075
0.004
0.041
0.009
0.095
0.090
0.200
0.095
0.035
10
11
12
13
14
15
16
17
18
19
20
21
22
23
(CH2)4-NH-(CH2)3
C39H32N3O4‚0.5H2O C, H, N
(CH2)2-NH-(CH2)2-NH-(CH2)2
(CH2)2-NCH3-(CH2)2-NCH3-(CH2)2
(CH2)2-NH-(CH2)3-NH-(CH2)2
(CH2)2-NH-(CH2)4-NH-(CH2)2
(CH2)2-NH-(CH2)5-NH-(CH2)2
(CH2)3-NH-(CH2)2-NH-(CH2)3
(CH2)3-NH-(CH2)3-NH-(CH2)3
(CH2)3-NH-(CH2)4-NH-(CH2)3
C38H30N4O4
C40H34N4O4
C39H32N4O4
C40H34N4O4
C42H38N4O4
C40H34N4O4
C41H36N4O4
C42H38N4O4
C38H28N6O8
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
8-NO2 (CH2)2-NH-(CH2)2-NH-(CH2)2
8-NO2 (CH2)2-NH-(CH2)3-NH-(CH2)2
C39H30N6O8‚0.5H2O C, H, N
24
H
60
222
DMF-H2O
C40H32N4O4 C, H, N
0.002
amonafide
3.000
0.600
0.140
0.014
mitonafide
azonafide
LU 79553
Sch em e 1
Sch em e 3a
a
Reagents: (i) ClCH2CN, K2CO3, CH3CN; (ii) H4LiAl, diethyl
ether.
Sch em e 4
Sch em e 2a
a
Reagents: (i) N-Boc-glycine, CDI, CH2Cl2; (ii) HCl gas, MeOH;
(iii) BH3-THF, THF.
of carbonyldiimidazole, and the resulting diamide was
hydrolyzed and reduced with BH3-THF complex.
The synthesis of N,N′-bis(2-aminoethyl)-N,N′-dim-
ethyl-1,2-ethylenediamine (8), used in the preparation
of 15, was performed following an analogous procedure
to that described by Alcock.22 N,N′-Dimethylethylene-
diamine was reacted with chloroacetonitrile, and the
dinitrile 8a formed was reduced with lithium aluminum
hydride (Scheme 3).
The cyclization of 16 was accomplished by reaction
of the NH groups with formaldehyde23 yielding the
acidic unstable compound 24 (Scheme 4).
Biology. The compounds were initially evaluated for
in vitro cytotoxicity against the HT-29 human colon
carcinoma cell line. We have previously used this cell
lic acid anhydride with 1 equiv of nitric acid in sulfuric
acid yielded a mixture of 5- and 8-mononitro deriva-
tives15 (Scheme 1). In our hands, the more soluble
5-nitroanhydride was separated from the mixture by
treatment with boiling toluene. This allowed removal
of the 8-nitro isomer as an insoluble precipitate.
All the amines were commercially available, except
N,N′-bis(2-aminoethyl)-1,4-butanediamine (6) and N,N′-
bis(2-aminoethyl)-1,5-pentanediamine (7) used for the
synthesis of compounds 17 and 18. These compounds
were initially prepared by Israel et al. from the corre-
sponding diamine and aziridine.21 To avoid the use of
aziridine, we have now modified this synthetic proce-
dure (Scheme 2). The central diamine segments were
acylated at each end with Boc-glycine in the presence

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Chromophore-Modified Bis-Naphthalimides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 451
Ta ble 2. Selected IR and 1H-NMR Data of Compounds 9-24
IR (KBr,
compd
ν
max, cm-1
)
1H-NMR δ (ppm)
9
1670, 1640,
(CF3CO2D) 4.07 (br s, 4H), 4.81 (br s, 4H), 7.28 (t, 2H, J ) 8 Hz), 7.55 (t, 2H, J ) 8 Hz), 7.76 (t, 2H, J ) 8 Hz),
8.00 (d, 2H, J ) 8 Hz), 8.40 (d, 2H, J ) 8 Hz), 8.63-8.68 (m, 4H), 8.96 (d, 2H, J ) 8 Hz)
(CF3CO2D) 2.60 (m, 2H), 3.55 (m, 2H), 3.95 (m, 2H), 4.52 (m, 2H), 4.91 (m, 2H), 7.32-7.41 (m, 1H),
7.46-7.54 (m, 1H), 7.60 (t, 1H, J ) 8 Hz), 7.68-7.75 (m, 1H), 7.81-7.92 (m, 2H), 8.00 (d, 1H, J ) 8 Hz),
8.17 (d, 1H, J ) 8 Hz), 8.39 (d, 2H, J ) 8 Hz), 8.53 (d, 1H, J ) 8 Hz), 8.75 (s, 1H), 8.84-8.88 (m, 1H),
8.90 (s, 1H), 9.25 (d, 1H, J ) 8 Hz), 9.58 (d, 1H, J ) 8 Hz)
1560, 1430
1680, 1640,
1560, 1430
10
11
12
13
1680, 1640,
1560, 1430
1690, 1640,
1560, 1430
1680, 1630,
1550, 1430
(CF3CO2D) 2.57 (t, 4H, J ) 7 Hz), 3.52 (t, 4H, J ) 2 Hz), 4.62 (t, 4H, J ) 6 Hz), 7.53 (t, 2H, J ) 7 Hz),
7.70-7.85 (m, 4H), 8.00 (d, 2H, J ) 8 Hz), 8.46 (d, 2H, J ) 8 Hz), 8.80 (m, 4H), 9.54 (d, 2H, J ) 8 Hz)
(CF3CO2D) 2.59 (m, 4H), 3.29 (s, 3H), 3.55 (m, 2H), 3.71 (m, 2H), 4.54 (t, 4H, J ) 7 Hz), 7.44 (t, 2H, J ) 8 Hz),
7.65-7.83 (m, 4H), 7.94 (d, 2H, J ) 8 Hz), 8.44 (d, 2H, J ) 8 Hz), 8.76 (m, 4H), 9.47 (d, 2H, J ) 8 Hz)
(CF3CO2D) 2.13 (br s, 4H), 2.53 (br s, 2H), 3.51 (m, 4H), 4.46 (m, 2H), 4.50 (m, 2H), 7.12 (t, 1H, J ) 8 Hz),
7.43-7.77 (m, 6H), 7.93 (d, 1H, J ) 8 Hz), 8.24 (d, 2H, J ) 8 Hz), 8.39 (m, 2H), 8.61 (d, 1H, J ) 7 Hz),
8.70 (m, 2H), 9.26 (d, 1H, J ) 9 Hz), 9.45 (d, 1H, J ) 9 Hz)
14
15
16
1685, 1650,
1580, 1470
1680, 1640,
1550, 1420
1680, 1645,
1560, 1430
(CDCl3) 2.99 (s, 4H), 3.11 (t, 4H, J ) 6 Hz), 4.33 (t, 4H, J ) 6 Hz), 7.47 (m, 4H), 7.65 (m, 2H), 7.82 (d, 2H,
J ) 8 Hz), 8.07 (d, 2H, J ) 8 Hz), 8.45 (s, 2H), 8.49 (dd, 2H, J ) 8, 1 Hz), 9.70 (d, 2H, J ) 8 Hz)
(CF3CO2D) 3.64 (s, 6H), 3.95-4.36 (m, 6H), 4.81 (m, 6H), 7.14 (t, 2H, J ) 7.9 Hz), 7.47-7.71 (m, 6H),
8.30 (d, 2H, J ) 8 Hz), 8.47 (s, 2H), 8.51 (d, 2H, J ) 7 Hz), 9.03 (d, 2H, J ) 9 Hz)
(CDCl3) 1.84 (q, 2H, J ) 6 Hz), 2.74 (br s, 2H, interchange with D2O), 2.89 (t, 4H, J ) 6 Hz), 3.02 (t, 4H,
J ) 6 Hz), 4.31 (t, 4H, J ) 6 Hz) 7.54 (m, 4H), 7.73 (m, 2H), 7.89 (d, 2H, J ) 8 Hz), 8.11 (d, 2H,
J ) 8 Hz), 8.51 (s, 2H), 8.57 (d, 2H, J ) 8 Hz), 9.78 (d, 2H, J ) 8 Hz)
17
18
1680, 1640,
1560, 1430
1680, 1650,
1560, 1440
(CDCl3) 1.58 (br s, 4H), 2.76 (m, 4H), 3.06 (t, 4H, J ) 6 Hz), 4.38 (t, 4H, J ) 6 Hz), 7.49-7.69 (m, 4H),
7.73-7.78 (m, 2H), 7.95 (d, 2H, J ) 8 Hz), 8.19 (d, 2H, J ) 8 Hz), 8.62 (m, 4H), 9.83 (d, 2H, J ) 8 Hz)
(CDCl3) 1.50 (m, 8H), 2.68 (t, 4H, J ) 7 Hz), 3.02 (t, 4H, J ) 7 Hz), 4.39 (t, 4H, J ) 7 Hz), 7.53-7.85 (m, 6H),
8.06 (d, 2H, J ) 8 Hz), 8.30 (dd, 2H, J ) 8, 1.6 Hz), 8.71 (dd, 2H, J ) 7.2, 1.2 Hz), 8.75 (s, 2H),
9.96 (d, 2H, J ) 8 Hz)
19
20
21
22
23
1680, 1640,
1560, 1430
1680, 1640,
1560, 1430
1680, 1640,
1560, 1430
1690, 1650,
1520, 1340
1690, 1650,
1540, 1350
(CDCl3) 1.63 (br s, 2H), 2.04 (m, 4H), 2.78 (m, 8H), 4.32 (t, 4H, J ) 7 Hz), 7.53-7.82 (m, 6H), 8.03 (d, 2H,
J ) 8 Hz), 8.27 (dd, 2H, J ) 8, 1 Hz), 8.69-8.72 (m, 4H), 9.96 (d, 2H, J ) 8 Hz)
(CDCl3) 1.76 (m, 4H), 2.06 (m, 4H), 2.77 (m, 8H), 4.33 (t, 4H, J ) 7 Hz), 4.39 (t, 4H, J ) 7 Hz), 7.46-7.76
(m, 6H), 7.95 (d, 2H, J ) 8 Hz), 8.22 (dd, 2H, J ) 8, 1.6 Hz), 8.62-8.66 (m, 4H), 9.88 (dd, 2H, J ) 8, 1 Hz)
(CDCl3) 1.67 (br s, 4H), 2.06 (t, 4H, J ) 6 Hz), 2.79 (m, 10H), 4.27 (t, 4H, J ) 7 Hz), 7.56 (m, 4H), 7.74 (m, 2H),
7.92 (d, 2H, J ) 8 Hz), 8.15 (d, 2H, J ) 8 Hz), 8.55 (s, 2H), 8.59 (dd, 2H, J ) 8, 1 Hz), 9.82 (d, 2H, J ) 8 Hz)
(CF3CO2D) 3.93 (s, 8H), 4.82 (s, 4H), 7.82-7.91 (m, 4H), 8.35 (d, 2H, J ) 7.4 Hz), 8.52 (d, 2H, J ) 8 Hz),
8.83 (d, 2H, J ) 7 Hz), 9.59 (s, 2H), 10.08 (d, 2H, J ) 9.2 Hz)
(CDCl3) 1.75 (c, 2H), 2.80 (t, 4H, J ) 6.6 Hz), 3.01 (t, 4H, J ) 6.4 Hz), 4.33 (t, 4H, J ) 6.6 Hz), 7.76-7.85
(m, 4H), 8.25 (dd, 2H, J ) 7.2, 1 Hz), 8.35 (d, 2H, J ) 7.6 Hz), 8.76 (dd, 2H, J ) 7, 1.2 Hz), 9.41 (s, 2H),
10.35 (d, 2H, J ) 7 Hz)
24
1670, 1640,
1550, 1420
(CDCl3) 1.89 (t, 2H, J ) 6 Hz), 2.81 (t, 8H, J ) 8 Hz), 3.40 (s, 2H), 4.32 (t, 4H, J ) 6 Hz), 7.17-7.37 (m, 6H),
7.53 (d, 2H, J ) 7.6 Hz), 7.73 (dd, 2H, J ) 8.4, 1.2 Hz), 7.87 (dd, 2H, J ) 7, 1 Hz), 8.08 (s, 2H),
9.28 (d, 2H, J ) 9 Hz)
line to guide bis-naphthalimide development.16-18 IC50
values were compared with those of the monomeric
compounds amonafide, mitonafide, and azonafide as
well as the bis-naphthalimide LU 79553 (see Table 1).
The bis-dibenz[de,h]isoquinoline-1,3-diones 9-24 had,
in general, improved cytotoxic activity over the parental
monomers. In optimized compounds this represented
up to a 70-fold increase in activity over azonafide.
Evaluation of the polyamine bridge indicated that
compounds with the bridge Z-NH-Y, where Z and Y
were equal alkyl chains (9 and 11), were more potent
cytotoxic agents than those where Z and Y were differ-
ent alkyl chains (10 and 13). Furthermore, methylation
of the basic nitrogen in compound 11 was tolerated and
resulted in no loss of cytotoxic potency (compound 12).
Compounds with two nitrogen atoms in the bridge had
better activity if the alkyl chain between the aminic-
imidic nitrogens was ethylene (14 and 16-18) rather
than propylene (19-21). The distance between the
basic nitrogens, however, did not appear to influence
cytotoxicity from two to five methylenes. N,N′-Dim-
ethylation of the potent cytotoxic agent 14, to give
compound 15, resulted in approximately a 10-fold loss
of cytotoxic potency. Similarly the 8-nitro substitution
of 14 and 16 gave compounds 22 and 23, respectively,
10-fold less active. In order to confer a certain rigidity
to the bridge with respect to 16, compound 24 was
synthesized. These compounds had similar cytotoxic
activity.
F igu r e 1. Effect of compound 16 on the mobility of supercoiled
DNA. Incubation of compound 16 with pBR322-supercoiled
DNA reduced its mobility on a 1% agarose gel to that of relaxed
DNA.
Compound 16 was selected for additional testing.
This compound altered DNA mobility on agarose gel at
5 µM (see Figure 1) and inhibited topoisomerase II at 5
µM (see Figure 2) and topoisomerase I at 5 µM (see
Figure 3). In mice implanted ip with M5076 murine
sarcoma, compound 16 produced a moderate increase
in life span (see Table 3). In contrast, in a separate
study the bis-naphthalimide LU 79553 (4) was inactive
in this tumor model. Compound 16 had good activity
against sc implanted Lox melanoma (see Figure 4) with
some tumor cures.

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452 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Bran˜a et al.
F igu r e 4. Effect of compound 16 on Lox melanoma growth
in athymic mice. Following sc implantation of tumor, mice
were treated as control (9) or iv with compound 16 at 2 mg/
kg (O) or 1 mg/kg (4) on days 3-7 or at 5 mg/kg (b) or 2.5
mg/kg (0) on days 3, 7, and 11. The highest dose with each
treatment schedule produced 3/6 animals that were tumor free
on day 60 and considered cured.
F igu r e 2. Inhibition of topoisomerase II decatenation of
kDNA by compound 16. kDNA was decatenated by incubating
with topoisomerase II and ATP. This effect was inhibited in a
concentration-related manner by compound 16.
nonsubstituted bis-naphthalimides with a (CH2)2-NH-
Z-NH-(CH2)2 bridge need Z to be an alkyl chain of at
least three methylene groups for optimum cellular
cytotoxicity.18 In the present series of bis-dibenz[de,h]-
isoquinoline-1,3-diones, optimum activity was achieved
even if Z was as short as ethylene. This is comparable
to our earlier results with 3-nitro-substituted bis-
naphthalimides.18 We previously speculated that the
3-nitro may influence the orientation of the chro-
mophore during DNA intercalation compared to the
nonsubstituted form. This may also be true of the 1,2-
benzo substitution in the dibenz[de,h]isoquinoline-1,3-
diones. Compound 16 was demonstrated to bind DNA
and alter its gel mobility and to inhibit topoisomerase
activity. These effects are comparable to our previous
findings for LU 79553.19 This compound has the same
polyamine bridge as 16 and similar in vitro activity. The
in vivo activity of LU 79553 against a number of human
tumor xenografts has been impressive and prompted
clinical development. Compound 16 had antitumor
activity against Lox melanoma comparable to effects we
have reported for LU 79553 (Figure 4). In the M5076
murine tumor model, however, compound 16 produced
a moderate increase in life span whereas LU 79553 was
inactive. These studies suggest compound 16 and LU
79553 may have some differences in their antitumor
profile.
F igu r e 3. Inhibition of the relaxation activity of topoi-
somerase I by compound 16. Supercoiled DNA was relaxed by
incubating with topoisomerase I. This effect was inhibited in
a concentration-related manner by compound 16.
Ta ble 3. Effect of Compound 16 and LU 79553 on the Life
Span of C57B1/6 Mice Implanted ip with M5076
agent
dosea (mg/kg)
MSTb (days)
%T/C
control
LU 79553
23.5
21
24
21.5
21.5
22
31.2
34
100
89
102
91
7.5
5.0
3.3
2.2
91
control
16
100
143
155
143
1.8
1.2
0.8
Exp er im en ta l Section
31.5
Melting points were determined using a Bu¨chi 535 and an
Electrothermal capillary melting point apparatus and are
uncorrected. 1H-NMR spectra at 200 MHz were recorded on
a Bruker AC-200 spectrometer, with SiMe4 as internal stan-
dard. IR spectra were determined with a Perkin Elmer 1310
spectrophotometer. Analyses indicated by the symbols of the
elements or functions were within (0.4% of theoretical values.
N,N′-Bis[[(ter t-b u t oxyca r b on yl)a m in o]a cet yl]-1,4-d i-
a m in obu ta n e (6a ). A solution of N-(tert-butoxycarbonyl)-
glycine (10 g, 57 mmol) in 100 mL of dichloromethane was
treated at 0 °C with 1,1′-carbonyldiimidazole (9.3 g, 57 mmol).
After the mixture was stirred in an ice bath for 1.5 h, 1,4-
diaminobutane (2.5 g, 28 mmol) in 50 mL of dichloromethane
was added and stirred for an additional 1 h at 0 °C. After
keeping at room temperature overnight, the solution was
washed with a saturated solution of Na2CO3 (2 × 100 mL)
followed by water (2 × 50 mL). This was dried on anhydrous
magnesium sulfate and filtered, the solvent was evaporated
and the residue was crystallized from ethyl acetate to give the
title compound 6a (5.5 g, 74%) as a white solid: mp 145 °C;
a
b
Administered ip on days 1, 5, and 9. Median survival time.
Con clu sion s
The cytotoxic potency of azonafide on the HT-29 cell
line in the present study was similar to that previously
reported by Sami.14 Our findings also supported the
previous suggestion that azonafide was more potent
than amonafide and indicated a increased potency over
mitonafide. Appropriate polyamine linkers bridging the
imidic nitrogens of two dibenz[de,h]isoquinoline-1,3-
dione chromophores, however, were capable of increas-
ing the cytotoxic potency of azonafide further. This
result is comparable to our previous findings that the
presence of two chromophores of naphthalimide can
result in agents with cellular cytotoxic activity greater
than that of amonafide and mitonafide.16 Interestingly

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Chromophore-Modified Bis-Naphthalimides
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 453
1
IR (KBR) 3380, 3300, 1690, 1650, 1530, 1170 cm-1; H-NMR
(DMSO-d6) δ 1.36 (s, 22H, 6CH3, 2CH2), 3.03 (m, 4H, 2CH2),
3.46 (d, 4H, J ) 6.2 Hz, 2CH2), 6.89 (t, 2H, J ) 6 Hz, 2NH),
7.73 (t, 2H, J ) 5.3 Hz, 2NH).
g, 78 mmol) in tetrahydrofuran (100 mL). The reaction
mixture was refluxed for 3 h and then cooled. Water was
carefully added to decompose the excess hydride followed by
100 mL of diethyl ether. The organic layer was separated and
concentrated; then the title compound 8 (2.5 g, 79%) was
isolated as a colorless oil by Kugelrohr distillation: bp 115
°C/0.3 mmHg (lit.22 bp 94-96 °C/0.7 mmHg); IR (neat) 3330,
N,N′-Bis(a m in oa cet yl)-1,4-d ia m in ob u t a n e Dih yd r o-
ch lor id e (6b). A solution of 6a (7 g, 17 mmol) in 200 mL of
dry ethanol, at 0 °C, was saturated with hydrochloric acid
(gas). After 1 h at 0 °C, the mixture was stirred at room
temperature overnight. The solvent was evaporated, and the
residue was washed with diethyl ether and dried, to give the
title compound 6b (4.7 g, 98%) as a white solid: mp 240 °C;
IR (KBR) 3500-2500, 1650, 1600, 1460, 1120, 1100 cm-1; 1H-
NMR (D2O) δ 1.33 (m, 4H, 2CH2), 3.04 (m, 4H, 2CH2), 3.58 (s,
4H, 2CH2).
N,N′-Bis(2-a m in oet h yl)-1,4-d ia m in ob u t a n e Tet r a h y-
d r och lor id e (6c). 6b (4.5 g, 16 mmol) in 150 mL of anhy-
drous tetrahydrofuran was added to 350 mL of 1 M borane-
THF complex solution. This mixture was refluxed for 24 h
and cooled to room temperature, and then 200 mL of methanol
was added slowly and refluxed for an additional 15 h. After
filtration, the solvents were evaporated and the residue
dissolved in 50 mL of methanol. After the mixture cooled in
an ice bath, 4 mL of concentrated hydrochloric acid was added.
This mixture was treated with diethyl ether, and the solid
formed was filtered to give the title compound 6c (3.0 g, 57%)
as a hygroscopic solid.
2940, 2760, 1600, 1450, 1030 cm-1 1H-NMR (CDCl3) δ 1.46
;
(br s, 4H, 2NH2), 2.19 (s, 6H, 2CH3), 2.37 (t, 4H, J ) 6 Hz,
2CH2), 2.43 (s, 4H, 2CH2), 2.71 (t, 4H, J ) 6 Hz, 2CH2).
Gen er a l P r oced u r e for th e P r ep a r a tion of Bis(1,2-
d ih yd r o-3H-d iben z[d e,h ]isoqu in olin e-1,3-d ion es) 9-23.
To a mixture of the anthracene-1,9-dicarboxylic acid anhydride
(6 mmol) in 40 mL of toluene was added the corresponding
polyamine (3 mmol) dissolved in 10 mL of toluene. The
suspension was heated under reflux for 4 h and then cooled
to room temperature. The solid was filtered, washed with
toluene, dried, and crystallized from the appropriate solvent
to yield a yellow solid. Crystallization solvents were toluene
dimethylformamide and mixtures of dimethylformamide and
water. See Tables 1 and 2.
P r ep a r a tion of N,N′-Bis[2-(1,2-d ih yd r o-1,3-d ioxo-3H-
d ib e n z[d e,h ]isoq u in olin -2-yl)e t h yl]p e r h yd r op yr im i-
d in e (24). To a suspension of 16 (0.5 g, 0.4 mmol) in ethanol
(75 mL) was added formaldehyde (0.5 mL, 35% in water) in
ethanol (25 mL). The mixture was heated at reflux temper-
ature for 4 h. After cooling the precipitated solid was filtered,
washed with water, and crystallized from DMF-H2O to give
the title compound 24 (0.320 g, 60%) as a yellow solid: mp
222 °C.
DNA Mobility Assa y. The interaction of the bis-dibenz-
[de,h]isoquinoline-1,3-diones with DNA was assessed on the
basis of altered agarose gel mobility of supercoiled DNA
pBR322. Supercoiled pBR322 (680 ng) was mixed with drug
and the 20 µL final volume incubated at 37 °C for 30 min.
Following the addition of 4 µL of loading buffer (5% sarkosyl,
0.0025% bromophenol blue, 25% glycerol), the sample was
loaded onto a 1% agarose gel. The gel was run at 6 V/cm for
2.5 h in TAE buffer (40 mM Tris, 20 mM sodium acetate, 1
mM EDTA-Na2, pH 8.5) and then stained with ethidium
bromide and photographed under UV illumination using
Polaroid instant film, type 55.
N,N′-Bis(2-a m in oeth yl)-1,4-d ia m in obu ta n e (6). 6c (3g,
10 mmol) was added to a freshly prepared solution of sodium
ethoxide in ethanol [sodium (0.92 g, 40 mmol) in 75 mL of
ethanol]. After the mixture stirred overnight at room tem-
perature, the sodium chloride was removed by filtration and
the solvent evaporated. The title compound 6 (0.7 g, 43%) was
isolated by Kugelrohr distillation as a colorless oil: bp 160
°C/0.2 mmHg; IR (neat) 3250, 2920, 2800, 1590, 1470, 1130
cm-1
;
1H-NMR (CDCl3) δ 1.50 (m, 10H, 2CH2, 2NH2, 2NH),
2.70 (m, 12H, 6CH2).
N,N′-Bis[[(ter t-b u t oxyca r b on yl)a m in o]a cet yl]-1,5-d i-
a m in op en ta n e (7a ). The procedure described for 6a was
followed, using 1,5-diaminopentane (2.8 g, 27 mmol). The title
compound 7a (8.4 g, 73% crude yield) was obtained as a
colorless oil: IR (neat) 3390, 3320, 1680, 1640, 1530, 1160
cm-1 1H-NMR (CDCl3) δ 1.33 (m, 2H, CH2), 1.44 (s, 18H,
;
6CH3), 1.50 (m, 4H, 2CH2), 3.22 (m, 4H, 2CH2), 3.80 (d, 4H, J
) 6 Hz, 2CH2), 5.64 (m, 2H, 2NH), 6.70 (m, 2H, 2NH).
N,N′-Bis(a m in oa cetyl)-1,5-d ia m in op en ta n e Dih yd r o-
ch lor id e (7b). The procedure described above for 6b was
followed, using 7a (8.4 g, 20 mmol). The title compound 7b
(5.4 g, 93%) was obtained as a white solid: mp 215 °C; IR
Top oisom er a se II Deca ten a tion Assa y. Inhibition of the
decatenation activity of topoisomerase II was measured by the
method of Sahai and Kaplan.24 Briefly, 450 ng of C. fasciculata
kDNA was incubated with 6 units of purified human topoi-
somerase II in 20 µL of reaction buffer (0.05 M Tris HCl, pH
8.0, 0.12 M KCl, 0.01 M MgCl2, 0.5 mM ATP, 0.5 mM DTT)
for 30 min at 37 °C in the presence or absence of drug.
Following the addition of 4 µL of stop buffer (5% sarkosyl,
0.0025% bromophenol blue, 25% glycerol), proteinase K was
added to a final concentration of 50 µg/mL and the mixture
incubated at 37 °C for 1 h. This reaction mixture was then
loaded on an 1% agarose gel containing 0.5 µg/mL ethidium
bromide and run for 3.5 h at 5 V/cm in TAE buffer. After
destaining, the gel was photographed under UV illumination.
Top oisom er a se I Rela xa tion Assa y. Inhibition of DNA
relaxation by topoisomerase I was measured by the method
outlined by Chen.25 Calf thymus topoisomerase I (4 units) was
mixed with supercoiled DNA pBR322 (680 ng) in the presence
or absence of drug and the 20 mL final volume incubated at
37 °C for 30 min. Following the addition of 128 µL of stop
solution (containing 0.3% SDS, 75% EtOH, 10 mM MgCl2, 30
mM sodium acetate, pH 5.2, to remove intercalated drug), the
mixture was microfuged for 10 min and the pellet washed twice
with 200 µL of 70% EtOH. The pellet was air-dried, resolu-
bilized in 20 µL of TE buffer plus 4 µL of gel loading buffer,
and run on a 1% agarose gel. Gels were stained with ethidium
bromide and photographed under UV illumination.
(KBR) 3500-2500, 1650, 1560, 1490, 1280 cm-1 1H-NMR
;
(D2O) δ 1.14 (m, 2H, CH2), 1.31 (m, 4H, 2CH2), 3.01 (t, 4H, J
) 7 Hz, 2CH2), 4.59 (s, 4H, 2CH2).
N,N′-Bis(2-a m in oeth yl)-1,5-d ia m in op en ta n e Tetr a h y-
d r och lor id e (7c). The procedure described above for 6c was
followed, using 7b (5.3 g, 18 mmol). The title compound 7c
(3.0 g, 50%) was obtained as a white solid: mp 242 °C
(ethanol-diethyl ether).
N,N′-Bis(2-a m in oeth yl)-1,5-d ia m in op en ta n e (7). The
procedure described above for 6 was followed, using 7c (2.0 g,
5.9 mmol). The title compound 7 (0.26 g, 23%) was obtained
as a colorless oil: bp 185 °C/ 0.1 mmHg; IR (neat) 3300, 2900,
1590, 1450, 1120 cm-1; 1H-NMR (CDCl3) δ 1.50 (m, 12H, 3CH2,
2NH2, 2NH), 2.10 (m, 12H, 6CH2).
N,N′-Bis(cya n om et h yl)-N,N′-d im et h yl-1,2-d ia m in oet -
h a n e (8a ). Chloroacetonitrile (20 g, 255 mmol) in 250 mL of
acetonitrile was added to N,N′-dimethyl-1,2-diaminoethane (11
g, 125 mmol) and anhydrous potassium carbonate (35 g, 255
mmol) in acetonitrile (250 mL). The reaction mixture was
stirred at room temperature overnight. The solid was removed
by filtration and the solvent evaporated. The residue was
crystallized from methanol, to give the title compound 8a (12
g, 57%) as a white solid: mp 77-78 °C (lit.22 mp 79-80 °C); IR
(KBR) 2840, 2780, 2210, 1450, 1030 cm-1; 1H-NMR (CDCl3) δ
2.40 (s, 6H, 2CH3), 2.62 (s, 4H, 2CH2), 3.62 (s, 4H, 2CH2).
N,N′-Bis(2-a m in oet h yl)-N,N′-d im et h yl-1,2-d ia m in oet -
h a n e (8). 8a (3 g, 18 mmol) in tetrahydrofuran (50 mL) was
added slowly to a suspension of lithium aluminum hydride (3
Cytotoxicity Assa y. The cytotoxicity of the bis-dibenz-
[de,h]isoquinoline-1,3-diones was measured using a standard
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. Human colon carcinoma cell line HT-29 was
obtained from American Type Culture Collection and cultured
in the recommended media. Exponentially growing cells were
plated at 3000/well into 96-well plates in 150 µL of complete

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454 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Bran˜a et al.
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DMEM media containing 10% FBS. Cells were allowed to
attach for 24 h before the addition of a serial (1:4) dilution of
drug in 50 µL of fresh media. After 72 h of incubation at 37
°C, 5% CO2, MTT (50 µL, 3 mg/mL in PBS) was added to each
well and the plates were incubated for 4 h. Formazan crystals
formed by MTT metabolism were solubilized by the addition
of 50 µL of 25% SDS (pH 2) to each well and incubating
overnight. The cellular metabolism of MTT was then quanti-
fied by reading the absorbance of the solubilized product at
550 nm with
a 96-well plate reader. IC50 values were
calculated as the concentration of drug needed to inhibit cell
growth to 50% of controls.
In Vivo An titu m or Eva lu a tion . M5076 murine sarcoma
was kindly provided by Don Dykes (Southern Research
Institute, Birmingham, AL). Cells (106) were implanted ip in
C57Bl/6 mice. Antitumor activity was determined on the basis
of median survival time (MST) of treated (T) mice compared
to controls (C) expressed as a percentage (%T/C). An agent
was considered active if it produced a %T/C of >125%. Lox
melanoma was obtained from the tumor repository of the
National Cancer Institute (Bethesda, MD) and grown in female
athymic Ncr-nude mice (Taconic Farms, Germantown, NY).
Tumor was implanted sc and passaged as tumor fragments
(50 mg) dissected from the non-necrotic portion of a donor
tumor. Tumor size was determined by caliper measurement
and weight estimated according to a published method.26
For cytotoxicity testing the free bases of compounds were
dissolved in DMSO and then diluted into media to give a final
DMSO concentration that did not interfere with the assay
(<1%). Compound 16 was converted into the dimethane-
sulfonate salt and solubilized in buffer for DNA and topoi-
somerase assays and water for in vivo testing. LU 79553 was
used as the dimethanesulfonate salt and solubilized in water
for in vivo testing.
Ack n ow led gm en t. This work was supported in part
by Plan de Fomento de la Investigacio´n de la Industria
Farmace´utica of Ministry of Industry of Spain.
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