New Analogues of Amonafide and Elinafide, Containing Aromatic Heterocycles: Synthesis, Antitumor Activity, Molecular Modeling, and DNA Binding Properties

Article abstract of DOI:10.1021/jm0308850

Amonafide- and elinafide-related mono and bisintercalators, modified by the introduction of a π-excedent furan or thiophene ring fused to the naphthalimide moiety, have been synthesized. These compounds have shown an interesting antitumor profile. The best compound, 9, was 2.5-fold more potent than elinafide against human colon carcinoma cells (HT-29). Molecular dynamic simulations and physicochemical experiments have demonstrated that these compounds are capable of forming stable DNA complexes. These results, together with those previously reported by us for imidazo- and pyrazinonaphthalimide analogues, have prompted us to propose that the DNA binding process does not depend on the electronic nature of the fused heterocycle.

Full text of DOI:10.1021/jm0308850

J . Med. Chem. 2004, 47, 1391-1399
1391
New An a logu es of Am on a fid e a n d Elin a fid e, Con ta in in g Ar om a tic Heter ocycles:
Syn th esis, An titu m or Activity, Molecu la r Mod elin g, a n d DNA Bin d in g
P r op er ties
Miguel F. Bran˜a,*,† Mo´nica Cacho,† Mario A. Garc´ıa,† Beatriz de Pascual-Teresa,† Ana Ramos,†
M. Teresa Dom´ınguez,‡ J ose´ M. Pozuelo,‡ Cristina Abradelo,§ Mar´ıa Fernanda Rey-Stolle,§ Mercedes Yuste,§
Mo´nica Ba´n˜ez-Coronel,| and J uan Carlos Lacal|
Departamentos de Ciencias Qu´ımicas, Biolog´ıa Celular, Bioqu´ımica y Biolog´ıa Molecular, Matema´ticas,
Fı´sica Aplicada y Fisicoqu´ımica, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU,
Urbanizacio´n Monteprı´ncipe, 28668-Boadilla del Monte, Madrid, Spain and Instituto de Investigaciones Biome´dicas,
CSIC, Arturo Duperier 4, 28029-Madrid, Spain
Received May 1, 2003
Amonafide- and elinafide-related mono and bisintercalators, modified by the introduction of a
π-excedent furan or thiophene ring fused to the naphthalimide moiety, have been synthesized.
These compounds have shown an interesting antitumor profile. The best compound, 9, was
2.5-fold more potent than elinafide against human colon carcinoma cells (HT-29). Molecular
dynamic simulations and physicochemical experiments have demonstrated that these com-
pounds are capable of forming stable DNA complexes. These results, together with those
previously reported by us for imidazo- and pyrazinonaphthalimide analogues, have prompted
us to propose that the DNA binding process does not depend on the electronic nature of the
fused heterocycle.
In tr od u ction
In our search for analogues of amonafide1 and elin-
afide2 with an improved therapeutic profile, we recently
described the synthesis of new mono- and bisintercala-
tors, where the chromophore consisted of a naphthal-
imide moiety, fused to an imidazole3 or a pyrazine4 ring.
While the introduction of the imidazole did not lead to
a significant improvement in biological activity, bis-
intercalators with activities in the order of 10-8 M were
obtained by introduction of a π-deficient pyrazine ring.
Following this study of the influence that the introduc-
tion of angular fused heterocycles to the naphthalimide
moiety has on the antitumor activity, in the present
article we describe the synthesis, biological activity,
DNA binding properties, and molecular modeling of a
new series of amonafide and elinafide analogues 1-12,
where a π-excedent furan or thiophene ring has been
introduced in the two possible orientations. Patten et
al. synthesized mono- and bisintercalators with a furo-
naphthalimide chromophore, but the nature of the
connecting chains were different, and the compounds
presented lower activity than those reported here.5
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of anhydrides 17-20, from
which the naphthalimides 1-12 were prepared, was
carried out following the method outlined in Scheme 1.
Thus, dimethyl homophthalate was treated with the
corresponding aldehyde in the presence of NaH to yield
* To whom the correspondence should be addressed. Phone
34913724771; fax 34913724009; e-mail mfbrana@ceu.es.
† Departamento de Ciencias Qu´ımicas.
‡ Departamento de Biolog´ıa Celular, Bioqu´ımica y Biolog´ıa Molec-
ular.
§ Departamento de Matema´ticas, F´ısica Aplicada y Fisicoqu´ımica.
| Instituto de Investigaciones Biome´dicas, CSIC.
10.1021/jm0308850 CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/07/2004

1392 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Bran˜a et al.
Ta ble 1. Growth Inhibitory Properties for Mono- and
Sch em e 1a
Bisnaphthalimides 1-12
a
a
IC50
IC50
HT-29 HeLa PC-3
no. HT-29 HeLa PC-3
no.
1
2
3
4
5
6
7
0.55
1.15
2.69
0.75
0.37
NT
0.14 0.164
0.80 3.50
4.30 5.58
0.86 1.19
0.73 > 100 12
NT NT
0.16 0.93
8
9
10
11
0.27
0.0068 0.044
0.71
1.94
0.39
0.21
1.42
0.50
12.58
0.40
2.50
6.38
0.32
2.58
1.04
0.57
2.73
0.07
amonafide 4.67
elinafide 0.017
0.047
a
IC50: concentration of drug (µM) to reduce cell number to 50%
of control cultures.
amonafide. However, dimerization of the chromophore,
using the linker present in elinafide (compound 5), has
not resulted in an increase of activity. However this
result is different from the biological data obtained for
compounds 1 and 5, in a simultaneous work carried out
by Bailly et al.,6 where the dimer proved to be over 100-
times more cytotoxic against CEM leukemia cells than
the monomer, with IC50 values of 0.6 µM and 4.9 nM,
respectively. Similarly, when we carried out dimeriza-
tion of the chromophore bearing the furan ring oriented
toward the inside of the molecule (compound 9), a
remarkable increase in the antitumor potency was
observed compared to the monomeric counterpart (com-
pound 3). Compound 9 presented IC50 ) 6.8 nM against
HT-29, a value that is 2.5-fold lower than the value
found for elinafide under the same experimental condi-
tions.
The effect of the substitution of the oxygen atom
present in the furan moiety by a sulfur atom, has been
analyzed in mononaphthalimides 2 and 4, and bisnaph-
thalimides 7, 8, 11, and 12. These compounds present
also an interesting biological activity, but they show a
different pattern of behavior than their furan counter-
parts. In this case, dimerization of the isomeric chromo-
phore that bears the sulfur atom oriented toward the
outside of the molecule resulted in a compound (7) which
was 25-fold more active against HT-29 (IC50 ) 0.047 µM)
than the corresponding monomer (2). However, the
activity of the monomer (4) and the dimer (11) were
similar for the isomer bearing the sulfur atom oriented
toward the inside of the molecule.
Compounds 6, 8, 10, and 12 with a (CH2)2NCH3(CH2)3-
NCH3(CH2)2 linker were chosen because methylation of
the NH groups in related bis(indeno[1,2-b]-6-carbox-
amides)7 and bis(9-methylphenazine-1-carboxamides)8
led to an increase in potency compared to the unmeth-
ylated counterparts. In our case, methylation has brought
about, in general, a decrease in activity. This effect has
been observed previously in related bisnaphthalimides
such as elinafide.9 Compound 6 could not be evaluated,
as it was not possible to obtain a stable salt, soluble
enough in water to carry out the biological tests.
In Vivo An tip r olifer a tive Activity. Compound 9
was selected for in vivo antitumor evaluation (due to
its high antiproliferative activity). Tumors were induced
by sc injection of HT-29 cells in the back of inmuno-
supressed mice (Swiss, nu/nu). The compound was
administered ip, dissolved in sterile 0.9% NaCl. Control
mice received an equivalent volume of vehicle alone,
following an identical schedule. Treatment was initiated
when the tumors reached a volume of 0.1 cm3 and tu-
a
Reagents: (a) for 13 2-furanecarbaldehyde, for 14 2-thiophene-
carbaldehyde, in THF, and then NaH (80%); (b) for 15 3-furane-
carbaldehyde, for 16 3-thiophenecarbaldehyde, in THF, and then
NaH (80%); (c) (i) I2/hν, EtOH, (ii) NaOH 1M; (iii) sat. NaHSO3;
(iv) Ac2O, 4.
compounds 13-16. Irradiation of these in the presence
of I2 brought about the oxidative photocyclization of the
stylbene type system. The products formed were not
isolated, but they were directly converted into the
corresponding anhydrides 17-20 by hydrolysis of the
ester group present in the molecule (NaOH, 1 M),
followed by dehydration (Ac2O/∆). Furan-derived anhy-
drides 17 and 19 had been described previously by this
method,5 while thiophene-derived anhydrides 18 and 20
had not been obtained before.
Mononaphthalimides 1-4 were synthesized by treat-
ment of the corresponding anhydride with N1,N1-di-
methyl-1,2-ethanediamine in ethanol at reflux temper-
ature. Bisnaphthalimides 5-7, 9, and 10 were obtained
by a similar procedure, starting from the corresponding
polyamine and anhydride in a 1:2 ratio. In the synthesis
of 8, 11, and 12 an excess of amine was necessary for
the reaction to be completed (TLC). However, the
formation of the corresponding mononaphthalimides
was not observed in the crude reaction. The synthesis
of N,N′-bis(2-aminoethyl)-N,N′-dimethyl-1,3-propanedi-
amine was carried out following the method previously
described by us.3 The rest of amines were commercially
available. Compounds 1 and 5 have been reported
previously.6
In Vitr o An tip r olifer a tive Activity. All analogues
were tested for cytotoxicity against several cell lines.
These include human colon carcinoma (HT-29), human
cervical carcinoma (HeLa), and human prostate carci-
noma (PC-3). All the compounds were tested as their
methanosulfonate or hydrochloride salts.
The results are summarized in Table 1 and compared
with the activity of amonafide and elinafide.
Among all the mononaphthalimides studied, com-
pound 1, bearing a furan ring oriented toward the
outside of the molecule is the most active. Thus, it is
10-fold more active against HT-29, 20-fold against HeLa,
and 40-fold against PC-3 than the leader compound,

New Analogues of Amonafide and Elinafide
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1393
hexamer (Figure 3). Three with the ligand bound
through the major groove and three through the minor
groove. Among these three orientations, two locate the
chromophores in a relative antiparallel orientation,
either with the furan rings stacked between AC or
between TG and the third one with a relative parallel
orientation of the chromophores.
These complexes were refined and initially submitted
to molecular dynamics simulations in a continuum
medium of relative permittivity 4rij for simulating the
solvent environment. Given the instability of the com-
plexes in this environment during the simulation time,
selected complexes were submitted to molecular dynam-
ics simulations with explicit inclusion of the solvent.
There is wide experience in the study of sequence-
dependent structural effects in DNA and ligand-DNA
complexes using this kind of methodology.14-19
The eight model systems initially constructed for
compounds 1 and 3 were submitted to 150 ps MD
simulations in water. After the equilibration period, the
progression of the root-mean-square (rms) deviations of
the coordinates of the solutes with respect to the initial
structures was measured for all complexes and are
shown in Figure 4. All four complexes simulated for
compound 1, were more stable than the corresponding
simulations for compound 3. For both compounds, the
orientation that showed to be more stable along the
simulation time was the one where the side chain
remains in the major groove of the DNA dinucleotide
and the furan ring is stacked between AC, which are
denoted 1b and 3b in Figure 2. This stabilization is
probably due to the fact that these orientations locate
the charged amino group between N7 and O6 of the
guanine base. The distances between N7 and the
nitrogen proton were monitored along the simulation
time, giving average values of 2.41 Å ( 0.46 and 2.34 Å
( 0.30 for 1b and 3b, respectively. The distances
between O6 and the nitrogen proton were also moni-
tored, giving average values of 2.84 Å ( 0.73 and 2.96
Å ( 0.54, for 1b and 3b, respectively. These values,
together with those of the corresponding angles, indicate
the presence of stable hydrogen bonds alternating
between positions N7 and O6 of guanine along the
simulation time. Analysis of the resulting energy-
minimized average structures from the last 50 ps of the
sampling time resulted in a relative stability of complex
1b with respect to 3b. Better stacking interactions
might account for this energy difference. This result is
in agreement with the biological activities reported for
these compounds (Table 1).
F igu r e 1. In vivo antitumoral activity for compound 9 on
human colon cancer xenografts. Tumors were generated by sc
inoculation of HT-29 cells in athymic mice. The compound (5
mg/kg) was administered ip daily for 4 consecutive days,
separated by 10 days without treatment. This schedule was
repeated for up to 60 days. Average tumoral volumes of treated
(n ) 4) and control mice (n ) 4) are shown.
mors were monitored 1-2 times per week. Tumor vol-
ume was calculated using the equation V ) (Dd2)/ 2.
The result obtained for compound 9 is shown in
Figure 1. Average of the tumor volume is represented
for control and treated mice. The therapeutic protocol
consisted of 14-days cycles, administering the compound
daily for 4 consecutive days at a dose of 5 mg/kg, leaving
10 days without treatment.
This schedule caused a reduction of ∼80% of the
tumoral volume at the end of the treatment, showing
an in vivo antitumor effect of the compound tested.
Molecu la r Mod elin g. With the aim of rationalizing
the results obtained for this new series of furonaph-
thalimides, a molecular modeling study was carried out
for compounds 1, 3, 5, and 9. Insight into the mode of
binding of compounds 1, 3, 5, and 9 to DNA was gained
through molecular modeling and molecular mechanics
techniques using the DNA dimer d(TG)2 for the mono-
furonaphthalimides 1 and 3 and the DNA hexamer
d(ATGCAT)2 for the bisfuronaphthalimides 5 and 9. The
sequence was selected according to the experimental
binding preference demonstrated for this type of com-
pounds in a NMR study10 and to modeling experience
with related intercalating agents.3,11,12 A parallel foot-
printing work carried out by Bailly and co-workers6 for
compounds 1 and 5 reveals that this type of compounds
exhibit and enhanced selectivity for GC rich sites if
compared to elinafide.13
Taking into account these results and the computer
time required for this type of simulations, for compounds
5 and 9, only the DNA complexes where these com-
pounds are located in the major groove and in a relative
antiparallel orientation of the chromophores were fur-
ther submitted to molecular dynamics simulations in
water. These complexes (a and b in Figure 3) were
submitted to 150 ps of molecular dynamics, and the rms
deviation of the complexes with respect to the initial
structures were evaluated. These values remained below
2 Å for all complexes. The progression of this parameter
indicates that the complexes do not experience large
conformational changes during the simulation time
(Figure 5). Distances and angles between the protonated
Four different model complexes were constructed for
compounds 1 and 3, by inserting the chromophores into
the d(TG)2 dinucleotide in the four possible orientations
relative to the base pairs (Figure 2). Two through the
major groove: one with the furan ring stacked between
TG and the other with the furan ring stacked between
AC; and the same orientations leaving the side chain
in the minor groove. For dimers 5 and 9, six different
orientations were possible when binding to the DNA

1394 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Bran˜a et al.
F igu r e 2. Schematic view from the major groove of the four complexes between compounds 1 and 3 and d(TG)2. The black
square in the chromophore represents the orientation of the furan ring, and the sphere represents the protonated dimethylamino
group from the side chain. Left: complexes with the linker in the major groove and the furan ring stacked between TG (a ) or
between AC (b). Right: complexes with the linker in the minor groove and the furan ring stacked between TG (c) or between AC
(d ).
F igu r e 4. Time evolution (ps) of the root-mean-square devia-
tion (Å) between the simulated structures and the correspond-
ing initial structures (all solute non-hydrogen atoms were
included in the comparisons) for complex 1:(TG)2 (top) and
complex 3:(TG)2 (bottom): (a) side chain in the major groove
and the furan ring stacked between TG, (b) side chain in the
major groove and the furan ring stacked between AC, (c) side
chain in the minor groove and the furan ring stacked between
TG, and (d) side chain in the minor groove and the furan ring
stacked between AC.
F igu r e 3. Schematic view of the complexes between com-
pounds 5 and 9 and d(ATGCAT)2. The back square in the
chromophores represent the orientation of the furan rings, and
the spheres account for the protonated amino groups in the
linking chains. Top: complexes with compounds bound through
the major groove. (a ) Relative antiparallel orientation of the
chromophores and the furan rings stacked between AC. (b)
Relative antiparallel orientation of the chromophores and the
furan rings stacked between TG. (c) Relative parallel orienta-
tion of the chromophores. Bottom: complexes in the same
relative orientations and with the ligand bound through the
minor groove of the DNA hexamer (d -f).
differences can be found in the binding affinity of
compounds 5 and 9 to the DNA hexamer through the
major groove. In contrast to what happened with related
imidazobisnaphthalimides,3 the length and nature of the
linking chain seems to be the appropriate for the process
of bisintercalation and the introduction of a furan ring
instead of an imidazole one leads to more stable
complexes, probably due to better stacking interactions.
Although the chromophores are slightly twisted with
respect to the orientation adopted by elinafide in its
complex with the same DNA hexamer, none of the
simulations reported in this work show drastic confor-
mational changes, leading us to propose that the
nitrogens of the linking chains and N7 and O6 of the
guanines were also monitored along the sampling time.
These measurements indicate the presence of either one
or two stable hydrogen bonds for all complexes. Figure
6 shows energy-minimized average structures of the last
50 ps of the molecular dynamics simulation for the four
complexes. Ligand-DNA interaction energies were also
evaluated giving values within the same range for all
four complexes. According to these results, no relevant

New Analogues of Amonafide and Elinafide
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1395
F igu r e 5. Time evolution (ps) of the root-mean-square devia-
tion (Å) between the simulated structures and the correspond-
ing initial structures (all solute non-hydrogen atoms were
included in the comparisons) for complex 5:d(ATGCAT)2 (a)
with the side chain in the major groove and the furan ring
stacked between TG, (b) with the side chain in the major
groove and the furan ring stacked between AC, and complex
9:d(ATGCAT)2 (a) with the side chain in the major groove and
the furan ring stacked between TG, (b) with the side chain in
the major groove and the furan ring stacked between AC.
F igu r e 7. Relative length increase L/L0 of 1 (2) and 5 (b)
DNA complexes as a function of the molar ratio of added
compound to DNA nucleotides (r). The contour lengths in the
presence (L) or absence (Lo) of the compounds were calculated
from viscosity measurements on sonicated calf thymus DNA.
F igu r e 8. Relative length increase L/L0 of 3 (2) and 9 (b)
DNA complexes as a function of the molar ratio of added
compound to DNA nucleotides (r). The contour lengths in the
presence (L) or absence (Lo) of the compounds were calculated
from viscosity measurements on sonicated calf thymus DNA.
F igu r e 6. Side views of the energy minimized average
structures of the last 50 ps of the molecular dynamics
simulations in water for complexes 5a , 5b, 9a , and 9b.
introduction of the furan ring angularly fused to the
naphthalimide moiety, probably leads to better stacking
interactions. This possibility was recently pointed out
by Bailly and co-workers.6 However, the possibility of
the presence of hydrogen bonds between the furan
oxygen and the amino group of the guanine in the minor
groove which was postulated in the same work as the
principal determinant of GC selectivity shown for
compound 5 if compared to elinafide was explored for
all the complexes and discarded in all cases.
With respect to the relative orientation of the furan
ring with respect to the naphthlimide moiety, that
seems to affect the biological activity our simulations
do not allow us, so far, to establish any significant
difference in the binding affinity. Further computer
simulations and analysis of the results will be under-
taken in future works.
Mech a n ism of Action Stu d ies. Compounds 1 and
5 have been extensively studied by Bailly et al.6
Qualitative binding studies by absorption and melting
temperature measurements, as well as quantitative
studies using surface plasmon resonance, have estab-
lished that the dimer binds considerably more tightly
to DNA than the corresponding monomer.6
calators have been studied by viscosimetric titration
with calf thymus DNA. It is known that DNA length
increases when a drug behaves as an intercalator.20
When the relative increase in contour length, L/L0,
versus r is plotted, the slope m of this plot has different
values depending on the functionality of the intercala-
tor. Monofunctional intercalators such as ethidium
bromide, proflavine, and aminoacridines usually have
values of m between 0.8 and 1.5.21 In our case, when
L/L0 is plotted versus r for compounds 1 and 3 (Figures
7 and 8), the least-squares fitting gives a slope of 1.3
for both compounds, indicating that these compounds
provoke DNA helix extension as monofunctional inter-
calators. In the case of compounds 5 and 9, slopes of
2.1 and 3.2 have been obtained, respectively (Figures 7
and 8), showing that 5 and 9 provoke DNA extension
as bifunctional intercalators. For bisintercalators, the
slope is expected to be twice the value observed for
monointercalators, as it has been verified for a variety
of intercalating compounds.22
To further evaluate the mechanism of action, com-
pound 9 was selected to carry out the assay of single-
cell gel electrophoresis (comet assay). The comet assay
detects DNA damage in individual cells embedded in
agarose. It is based on the property of negatively
In this work compounds 1 and 3 as models of
monointercalators and 5 and 9 as models of bisinter-

1396 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Bran˜a et al.
F igu r e 9. Single cell gel electrophoresis assay for a negative control (PBS) (left), for compound 9 (middle) and for a positive
control (doxorubicin) (right).
compound were purged for 1 h with argon and irradiated under
a positive pressure of argon.
charged DNA fragments to migrate when an electric
field is applied to the gel after cell lysis.23 Doxorubicin
was chosen as a positive reference, and PBS (phosphate-
buffered saline, pH ) 7.4) was used as a negative
control. One hour after the treatment, the samples were
observed using a fluorescence microscope, and the DNA
damage of compound 9 was similar to the damage of
the positive reference (Figure 9). This result suggests
that the antitumor activity of this type of mononaph-
thalimides may be related to their ability to induce DNA
damage.
2-[(1-Meth oxycar bon yl)-2-(3-th ien yl)vin yl]ben zoic Acid
(16). To a solution of dimethyl homophthalate24 (1.0 g, 4.80
mmol) in dry THF (9 mL) at 0 °C under argon was added
3-thiophenecarbaldehyde (0.54 g, 4.80 mL). Then, sodium
hydride (0.16 g, 5.00 mmol of a 80% oil dispersion) was added
in portions. When the addition was complete, the reaction was
allowed to warm to room temperature and was stirred for 24
h. The reaction crude was diluted with diethyl ether (14 mL)
and filtered. The residue was dissolved in water (16 mL) and
the pH adjusted to 5 with 1 N aqueous oxalic acid. The
precipitate formed was collected by filtration to give 16 (1.29
g, 93%) as a white solid, mp 148-150 °C (AcOEt/hexane). IR
Con clu sion s
1
(KBr): 3100, 1720, 1675 cm-1. H NMR (CDCl3): δ 8.21 (dd,
In conclusion, the introduction of a π-excedent furan
or thiophene ring fused to the naphthalimide moiety has
led to chromophore modified naphthalimides suitable
for dimerization. Recently we reported the poor anti-
tumor activities obtained in the dimerization of struc-
turally related imidazonaphthalimides. In that case,
molecular modeling techniques put forward the low
stability of the corresponding DNA-drug complexes.
The present molecular dynamics simulations show that
the synthesized bisfuronaphthalimides are capable of
forming stable DNA complexes, which is in accordance
with their improved in vitro activities, further supported
by in vivo experiments. These results, together with the
encouraging activities found for the related electron poor
pyrazinonaphthalimides, have prompted us to propose
that the electronic nature of the heterocycle fused to the
naphthalimide moiety does not affect the binding affin-
ity. Most probably, the van der Waals contribution to
the staking interactions might be governing the DNA
binding process for this type of compounds. At this point,
further experimental and theoretical work becomes
necessary in order to support this statement.
1H, J ) 7.7 and 1.1 Hz, ArH), 7.81 (s, 1H, CHdC), 7.54 (m,
2H, ArH), 7.25 (d, 1H, J ) 7.7 Hz, ArH), 7.02 (m, 2H, ArH),
6.34 (dd, 1H, J ) 4.9 and 1.1 Hz, ArH), 3.70 (s, 3H, CH3). 13C
NMR (CDCl3): δ 171.4, 168.0, 138.6, 136.5, 133.4, 132.2, 131.7,
131.6, 130.9, 129.2, 129.0, 128.3, 127.9, 125.5, 52.2. Anal.
(C15H12O4S) C, H, S.
Na p h th o[1,2-b]th iop h en e-5,6-d ica r boxylic An h yd r id e
(20). A mixture of 16 (300 mg, 1.05 mmol) and iodine (275
mg, 1.10 mmol) in absolute EtOH (400 mL) was photolyzed
for 8 h. Then, a solution of aqueous NaOH (2.2 mL, 1 M) was
added, followed by saturated NaHSO3 (1.1 mL). The solvent
was removed under reduced pressure and the residue sus-
pended in water (3.8 mL). After standing overnight, the
precipitate was collected by filtration and washed with water.
The solid was suspended in acetic anhydride (2.0 mL) and
heated on a steam bath for 15 min. Then, the solution was
allowed to stand at room-temperature overnight. The product
was isolated by filtration and washed with AcOEt to give 20
(146 mg, 55%) as a yellow solid, mp >300 °C (CHCl3). IR
(KBr): 1770, 1730 cm-1. 1H NMR (DMSO-d6/CF3CO2D): δ 9.63
(s, 1H, ArH), 9.33 (d, 1H, J ) 8.5 Hz, ArH), 9.11 (d, 1H, J )
7.3 Hz, ArH), 8.73 (d, 1H, J ) 5.0 Hz, thiopheneH), 8.57 (m,
1H, ArH), 8.51 (d, 1H, J ) 5.0 Hz, thiopheneH). 13C NMR
(CDCl3): δ 161.0, 160.8, 152.1, 138.0, 131.8, 130.7, 130.5, 129.3,
128.1, 127.9, 127.8, 126.2, 118.7, 107.6. Anal. (C14H6O3S) C,
H, S.
Exp er im en ta l Section
Na p h th o[2,1-b]th iop h en e-5,6-d ica r boxylic An h yd r id e
(18). The procedure described above was used for the synthesis
of 18. From 1425 (300 mg, 1.05 mmol) and iodine (275 mg, 1.10
mmol) in absolute EtOH (400 mL), 18 (30 mg, 11%) was
obtained as a yellow solid, mp >300 °C (CHCl3). IR (KBr):
Gen er a l Met h od s. Melting points (uncorrected) were
determined on a Stuart Scientific SMP3 apparatus. Infrared
(IR) spectra were recorded with a Perkin-Elmer 1330 infrared
1
spectrophotometer. H and 13C NMR: δ values were recorded
1
1770, 1730 cm-1. H NMR (CDCl3): δ 9.11 (s, 1H, ArH), 8.76
on a Bruker 300-AC instrument. Chemical shifts (δ) are ex-
pressed in parts per million relative to internal tetramethyl-
silane; coupling constants (J ) are in hertz. Mass spectra were
run on a HP 5989A spectrometer. Elemental analyses (C, H,
N) were performed on a Perkin-Elmer 2400 CHN apparatus
at the Microanalyses Service of the University Complutense
of Madrid; unless otherwise stated all reported values are
within (0.4% of the theoretical compositions. Thin-layer
chromatography (TLC) was run on Merck silica gel 60 F-254
plates. Unless stated otherwise, starting materials used were
high-grade commercial products. The photolyses were carried
out in a quartz immersion well apparatus with a Pyrex filter
and a 400-W medium-pressure Hg arc lamp. Solutions of the
(d, 1H, J ) 8.2 Hz, ArH), 8.66 (d, 1H, J ) 7.2 Hz, ArH), 8.15
(d, 1H, J ) 5.5 Hz, thiopheneH), 8.08 (d, 1H, J ) 5.5 Hz,
thiopheneH), 7.93 (m, 1H, ArH). 13C NMR (CDCl3): δ 161.0,
160.9, 151.0, 144.7, 134.1, 131.6, 131.0, 130.9, 129.6, 129.0,
127.7, 122.3, 121.7, 112.6. Anal. (C14H6O3S) C, H, S.
Gen er a l P r oced u r e for th e P r ep a r a tion of Mon on a p h -
th a lim id es 1-4 a n d Th eir Sa lts. A suspension of the
adequate anhydride (1 equiv) in toluene was treated with
N1,N1-dimethyl-1,2-ethanediamine (1 equiv) in absolute EtOH.
The mixture was heated at reflux temperature until the
reaction was completed (TLC). The precipitated solid was
filtered and recrystallized from the appropriate solvent to

New Analogues of Amonafide and Elinafide
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1397
provide the mononaphthalimide as a free base. This compound
was converted into the corresponding hydrochloride (1 and 3)
or methanosulfonate (2 and 4). For the synthesis of hydro-
chlorides, a suspension of the free base in absolute EtOH was
saturated with HCl (g) for 2 h, and the solid formed was
filtered. For the synthesis of methanesulfonates, the free base
was suspended in absolute EtOH and methanesulfonic acid
(2.2 equiv) was added. The monoimide salt was isolated by
filtration and washed with diethyl ether.
corresponding polyamine (1 equiv) in ethanol. The mixture was
heated at reflux temperature until the reaction was completed
(TLC). The precipitated solid was filtered and recrystallized
from the appropriate solvent to provide the bisnaphthalimide
as a free base. This compound was suspended in absolute
EtOH and methanesulfonic acid (2.5 equiv) was added. The
bisimide salt was isolated by filtration and washed with diethyl
ether.
N,N′-Bis[2-(4,6-d ioxo-5,6-d ih yd r o-4H-ben z[d e]fu r o[3,2-
g]isoq u in olin -5-yl)et h yl]-N,N′-d im et h yl-1,3-p r op a n ed i-
a m in e (6). To a suspension of compound 175 (168 mg, 0.70
mmol) in DMF (3 mL), was added dropwise N,N′-bis(2-
aminoethyl)-N,N′-dimethyl-1,3-propanediamine (60 mg, 0.32
mmol) in DMF (2 mL). The mixture was heated at 80 °C for 7
h and then was allowed to stand at room-temperature over-
night. The precipitated solid was isolated by filtration to give
6 (92 mg, 46%) as a yellow solid, mp 167-170 °C (DMF). IR
(KBr): 1695, 1655 cm-1. 1H NMR (DMSO-d6/CF3CO2D): δ 9.68
(br s, 2H, 2NH+), 8.83 (d, 2H, J ) 7.9 Hz, ArH), 8.72 (s, 2H,
ArH), 8.52 (m, 4H, furanH, ArH), 7.96 (dd, 2H, J ) 7.9 and
7.3 Hz, ArH), 7.87 (m, 2H, furanH), 4.46 (br s, 4H, 2CH2N),
3.44 (br s, 4H, 2CH2N), 3.28 (br s, 4H, 2CH2N), 3.00 (s, 6H,
2CH3), 2.13 (br s, 2H, CH2). 13C NMR (DMSO-d6/CF3CO2D):
δ 164.3, 164.2, 151.6, 150.7, 130.8, 129.4, 127.5, 125.9, 125.0,
122.8, 118.8, 117.1, 116.9, 107.2, 53.0, 52.2, 52.1, 35.1, 30.8.
MS (ESI): m/z 629 [M + H]+. Anal. (C37H32N4O6) C, H, N.
N,N′-Bis[2-(4,6-d ioxo-5,6-d ih yd r o-4H -b en z[d e]t h ien o-
[3,2-g]isoqu in olin -5-yl)eth yl]-1,3-pr opan ediam in e (7). Fol-
lowing the general procedure, from 18 (100 mg, 0.39 mmol) in
toluene (2.5 mL) and N,N′-bis(2-aminoethyl)-1,3-propane-
diamine (32 mg, 0.20 mmol) in absolute EtOH (1 mL) was
obtained 7 (83 mg, 67%) as a yellow solid, mp 204 °C (dec)
(absolute EtOH/toluene). IR (KBr): 3400, 3090, 1690, 1650
cm-1. 1H NMR (DMSO-d6/CF3CO2D): δ 9.15 (s, 2H, ArH), 9.02
(d, 2H, J ) 7.9 Hz, ArH), 8.90 (br s, 4H, 2NH2+), 8.51 (d, 2H,
J ) 7.9 Hz, ArH), 8.45 (d, 2H, J ) 4.9 Hz, thiopheneH), 8.40
(d, 2H, J ) 4.9 Hz, thiopheneH), 7.97 (t, 2H, J ) 7.9 Hz, ArH),
4.39 (br s, 4H, 2CH2N), 3.34 (br s, 4H, 2CH2N), 3.06 (br s, 4H,
2CH2N), 1.93 (br s, 2H, CH2). The free base was converted into
the corresponding dimethanesulfonate trihydrate (96%), mp
188 °C (dec). IR (KBr): 3400, 2800, 1690, 1650 cm-1. 1H NMR
(DMSO-d6): δ 9.18 (s, 2H, ArH), 9.05 (d, 2H, J ) 7.9 Hz, ArH),
8.64 (br s, 4H, 2NH2+), 8.53 (d, 2H, J ) 7.9 Hz, ArH), 8.49 (d,
2H, J ) 4.9 Hz, thiopheneH), 8.41 (d, 2H, J ) 4.9 Hz,
thiopheneH), 7.99 (t, 2H, J ) 7.9 Hz, ArH), 4.40 (br s, 4H,
2CH2N), 3.34 (br s, 4H, 2CH2N), 3.07 (br s, 4H, 2CH2N), 2.31
(s, 6H, 2CH3), 1.94 (br s, 2H, CH2). 13C NMR (DMSO-d6): δ
164.3, 164.1, 140.1, 137.2, 134.8, 130.7, 130.5, 129.2, 127.5,
127.1, 125.4, 123.2, 122.6, 118.4, 45.4, 44.3, 39.7, 36.6, 22.1.
Anal. (C35H28N4O4S2‚2CH3SO3H‚3H2O) C, H, N, S.
5-[2-(Dim et h yla m in o)et h yl]b en z[d e]t h ien o[3,2-g]iso-
qu in olin e-4,6(5H)-d ion e (2). From 18 (100 mg, 0.39 mmol)
in toluene (2.5 mL) and N1,N1-dimethyl-1,2-ethanediamine (34
mg, 0.39 mmol) in absolute EtOH (1 mL) yielded 2 (103 mg,
81%) as a yellow solid, mp 181-182 °C (absolute EtOH/
1
toluene). IR (KBr): 1690, 1660 cm-1. H NMR (DMSO-d6): δ
9.24 (s, 1H, ArH), 9.10 (d, 1H, J ) 8.5 Hz, ArH), 8.60 (d, 1H,
J ) 6.7 Hz, ArH), 8.55 (d, 1H, J ) 5.5 Hz, thiopheneH), 8.48
(d, 1H, J ) 5.5 Hz, thiopheneH), 8.06 (m, 1H, ArH), 4.28 (t,
2H, J ) 7.3 Hz, CH2), 2.62 (t, 2H, J ) 7.3 Hz, CH2), 2.31 (s,
6H, 2CH3). The free base was converted into the corresponding
methanesulfonate trihydrate (70%), mp 197-199 °C. IR
(KBr): 2650, 1690, 1650 cm-1 1H NMR (DMSO-d6): δ 9.23
.
(s, 1H, ArH), 9.09 (d, 1H, J ) 7.9 Hz, ArH), 8.59 (d, 1H, J )
7.3 Hz, ArH), 8.54 (d, 1H, J ) 4.9 Hz, thiopheneH), 8.47 (d,
1H, J ) 4.9 Hz, thiopheneH), 8.05 (dd, 1H, J ) 7.9 and 7.3
Hz, ArH), 4.49 (br s, 2H, CH2), 3.55 (br s, 2H, CH2), 3.00 (s,
6H, 2CH3N), 2.36 (s, 3H, CH3SO3-). 13C NMR (DMSO-d6): δ
164.3, 164.1, 140.1, 137.2, 134.8, 130.7, 129.2, 127.5, 127.3,
127.1, 125.4, 123.2, 122.6, 118.3, 55.0, 42.9, 39.7, 35.3. Anal.
(C18H16N2O2S‚CH3SO3H‚3H2O) C, H, N, S.
5-[2-(Dim eth yla m in o)eth yl]ben z[d e]fu r o[2,3-g]isoqu in -
olin e-4,6(5H)-d ion e (3). From 195 (200 mg, 0.84 mmol) in
toluene (5 mL) and N1,N1-dimethyl-1,2-ethanediamine (74 mg,
0.84 mmol) in absolute EtOH (2 mL) yielded 3 (160 mg, 62%)
as a yellow solid, mp 178-180 °C (absolute EtOH/toluene).
IR (KBr): 1690, 1650 cm-1 1H NMR (DMSO-d6): δ 8.84 (s,
.
1H, ArH), 8.69 (d, 1H, J ) 8.6 Hz, ArH), 8.51 (d, 1H, J ) 7.3
Hz, ArH), 8.39 (d, 1H, J ) 2.4 Hz, furanH), 7.98 (m, 1H, ArH),
7.39 (d, 1H, J ) 2.4 Hz, furanH), 4.18 (t, 2H, J ) 6.7 Hz, CH2),
2.51 (m, 2H, CH2), 2.21 (s, 6H, 2CH3). The free base (140 mg,
0.46 mmol) was converted into the corresponding hydrochloride
2.5hydrate (116 mg, 74%), mp >300 °C. IR (KBr): 3400, 2810,
2760, 1690, 1655 cm-1. 1H NMR (DMSO-d6): δ 9.73 (br s, 1H,
NH+), 8.90 (s, 1H, ArH), 8.76 (d, 1H, J ) 8.5 Hz, ArH), 8.56
(d, 1H, J ) 7.3 Hz, ArH), 8.42 (d, 1H, J ) 2.5 Hz, furanH),
8.02 (m, 1H, ArH), 7.43 (d, 1H, J ) 2.5 Hz, furanH), 4.43 (dd,
2H, J ) 6.1 and 5.5 Hz, CH2), 3.47 (br s, 2H, CH2), 2.90 (s,
6H, 2CH3). 13C NMR (D2O): δ 165.4, 165.0, 153.4, 148.0, 130.6,
127.9, 127.7, 124.8, 124.7, 120.5, 118.2, 115.7, 109.1, 55.8, 44.3,
36.1. MS (ESI): m/z 309 [M + H]+. Anal. (C18H16N2O3‚HCl‚
2.5H2O) C, H, N.
N,N′-Bis[2-(4,6-d ioxo-5,6-d ih yd r o-4H -b en z[d e]t h ien o-
[3,2-g]isoqu in olin -5-yl)eth yl]-N,N′-d im eth yl-1,3-p r op a n e-
d ia m in e (8). To a hot suspension of compound 18 (67 mg, 0.26
mmol) in DMF (2.5 mL) was added dropwise N,N′-bis(2-
aminoethyl)-N,N′-dimethyl-1,3-propanediamine (25 mg, 0.13
mmol) in DMF (1 mL). After the mixture was refluxed for 2 h,
an excess of N,N′-bis(2-aminoethyl)-N,N′-dimethyl-1,3-pro-
panediamine (12 mg, 0.06 mmol) was added. The reaction
mixture was refluxed for an additional 3 h, and then the
solution was allowed to stand at room-temperature overnight.
The precipitate was isolated by filtration to give 8 (49 mg, 57%)
as a yellow solid, mp 172-174 °C (DMF). IR (KBr): 1700, 1660
cm-1. 1H NMR (DMSO-d6/CF3CO2D): δ 9.18 (s, 2H, ArH), 9.02
(d, 2H, J ) 7.9 Hz, ArH), 8.56 (d, 2H, J ) 7.3 Hz, ArH), 8.46
(d, 2H, J ) 5.5 Hz, thiopheneH), 8.40 (d, 2H, J ) 5.5 Hz,
thiopheneH), 7.98 (dd, 2H, J ) 7.9 and 7.3 Hz, ArH), 4.54 (br
s, 4H, 2CH2N), 3.62 (br s, 4H, 2CH2N), 3.51 (br s, 2H, CH2N),
3.34 (br s, 2H, CH2N), 3.05 (s, 6H, 2CH3), 2.18 (br s, 2H, CH2).
The free base was converted into the corresponding dimethane-
sulfonate dihydrate (63%), mp 154 °C (dec). IR (KBr): 2620,
1690, 1650 cm-1. 1H NMR (DMSO-d6): δ 9.47 (br s, 2H, 2NH+),
9.15 (s, 2H, ArH), 9.00 (d, 2H, J ) 7.9 Hz, ArH), 8.51 (d, 2H,
J ) 7.3 Hz, ArH), 8.44 (d, 2H, J ) 5.5 Hz, thiopheneH), 8.39
(d, 2H, J ) 5.5 Hz, thiopheneH), 7.95 (m, 2H, ArH), 4.47 (br
5-[2-(Dim et h yla m in o)et h yl]b en z[d e]t h ien o[2,3-g]iso-
qu in olin e-4,6(5H)-d ion e (4). From 20 (200 mg, 0.79 mmol)
in toluene (5 mL) and N1,N1-dimethyl-1,2-ethanediamine (70
mg, 0.79 mmol) in absolute EtOH (2 mL) yielded 4 (204 mg,
80%) as a yellow solid, mp 179-181 °C (toluene). IR (KBr):
1695, 1655 cm-1. 1H NMR (DMSO-d6/CF3CO2D): δ 8.92 (s, 1H,
ArH), 8.60 (d, 1H, J ) 7.9 Hz, ArH), 8.49 (d, 1H, J ) 7.3 Hz,
ArH), 8.09 (d, 1H, J ) 5.5 Hz, thiopheneH), 7.94 (m, 1H, ArH),
7.89 (d, 1H, J ) 5.5 Hz, thiopheneH), 4.44 (t, 2H, J ) 5.5 Hz,
CH2), 3.52 (t, 2H, J ) 5.5 Hz, CH2), 2.96 (s, 6H, 2CH3). The
free base was converted into the corresponding methane-
sulfonate dihydrate (82%), mp 217-220 °C. IR (KBr): 2620,
2450, 1700, 1650 cm-1. 1H NMR (DMSO-d6): δ 9.24 (br s, 1H,
NH+), 9.00 (s, 1H, ArH), 8.70 (d, 1H, J ) 7.9 Hz, ArH), 8.55
(d, 1H, J ) 6.7 Hz, ArH), 8.18 (d, 1H, J ) 4.3 Hz, thiopheneH),
8.00 (m, 2H, thiopheneH, ArH), 4.48 (br s, 2H, CH2), 3.50 (br
s, 2H, CH2), 3.00 (s, 6H, 2NCH3), 2.35 (s, 3H, CH3SO3-). 13C
NMR (DMSO-d6): δ 164.0, 163.9, 142.2, 137.4, 129.6, 129.3,
129.0, 127.9, 127.7, 126.6, 124.6, 122.9, 119.3, 54.9, 42.8, 39.6,
35.3. Anal. (C18H16N2O2S‚CH3SO3H‚2H2O) C, H, N, S.
Gen er a l P r oced u r e for th e P r ep a r a tion of Bisn a p h -
th a lim id es 5-12 a n d Th eir Sa lts. A suspension of the
adequate anhydride (2 equiv) in toluene was treated with the

1398 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Bran˜a et al.
s, 4H, 2CH2N), 3.45 (br s, 4H, 2CH2N), 3.28 (br s, 4H, 2CH2N),
2.99 (s, 6H, 2CH3N), 2.29 (s, 6H, 2CH3SO3-), 2.14 (br s, 2H,
CH2). 13C NMR (DMSO-d6): δ 164.2, 164.0, 140.1, 137.1, 134.8,
130.7, 129.2, 127.4, 127.3, 127.1, 125.3, 123.1, 122.4, 118.2,
56.0, 52.8, 52.1, 40.1, 39.7, 34.8. Anal. (C37H32N4O4S2‚
2CH3SO3H‚2H2O) C, H, N, S.
a m in e (12). To a suspension of compound 20 (200 mg, 0.79
mmol) in toluene (5 mL) was added dropwise N,N′-bis(2-
aminoethyl)-N,N′-dimethyl-1,3-propanediamine (74 mg, 0.39
mmol) in absolute EtOH (2 mL). After the mixture was
refluxed for 4 h, an excess of N,N′-bis(2-aminoethyl)-N,N′-
dimethyl-1,3-propanediamine (39 mg, 0.20 mmol) in DMF (4
mL) was added. The reaction mixture was refluxed for 3 days
and then was allowed to stand at room-temperature overnight.
The precipitate was isolated by filtration to give 12 (71 mg,
28%) as a yellow solid, mp 164-168 °C (DMF). IR (KBr): 1700,
1660 cm-1.1H NMR (DMSO-d6/CF3CO2D): δ 8.95 (s, 2H, ArH),
8.63 (d, 2H, J ) 7.9 Hz, ArH), 8.53 (d, 2H, J ) 6.7 Hz, ArH),
8.09 (d, 2H, J ) 5.0 Hz, thiopheneH), 7.93 (m, 2H, ArH), 7.87
(d, 2H, J ) 5.0 Hz, thiopheneH), 4.53 (br s, 4H, 2CH2N), 3.60
(br s, 4H, 2CH2N), 3.48 (br s, 2H, CH2N), 3.34 (br s, 2H, CH2N),
3.05 (s, 6H, 2CH3), 2.19 (br s, 2H, CH2). The free base was
converted into the corresponding dimethanesulfonate tetra-
hydrate (61%), mp 163-168 °C (absolute EtOH/toluene). IR
N,N′-Bis[2-(4,6-d ioxo-5,6-d ih yd r o-4H-ben z[d e]fu r o[2,3-
g]isoqu in olin -5-yl)eth yl]-1,3-p r op a n ed ia m in e (9). Follow-
ing the general procedure, from 195 (150 mg, 0.63 mmol) in
toluene (7 mL) and N,N′-bis(2-aminoethyl)-1,3-propanediamine
(51 mg, 0.32 mmol) in absolute EtOH (2 mL) was obtained 9
(138 mg, 72%) as a yellow solid, mp 126-129 °C (absolute
EtOH/toluene). IR (KBr): 3480, 1690, 1645 cm-1 1H NMR
.
(CF3CO2D): δ 9.00 (s, 2H, ArH), 8.90 (d, 2H, J ) 7.4 Hz, ArH),
8.71 (d, 2H, J ) 6.0 Hz, ArH), 8.01 (m, 4H, furanH, ArH), 7.19
(d, 2H, J ) 2.0 Hz, furanH), 4.84 (m, 4H, 2CH2N), 3.88 (m,
4H, 2CH2N), 3.59 (m, 4H, 2CH2N), 2.56 (m, 2H, CH2). The free
base was converted into the corresponding dimethanesulfonate
trihydrate (56%), mp 217-220 °C. IR (KBr): 3400, 2750, 1690,
1645 cm-1. 1H NMR (D2O): δ 7.41 (s, 2H, ArH), 7.31 (d, 2H, J
) 7.3 Hz, ArH), 7.25 (m, 4H, furanH, ArH), 6.84 (dd, 2H, J )
8.3 and 7.3 Hz, ArH), 6.37 (m, 2H, furanH), 3.80 (m, 4H,
2CH2N), 3.05 (m, 8H, 4CH2N), 2.53 (s, 6H, 2CH3), 1.97 (m,
2H, CH2). 13C NMR (D2O): δ 165.2, 164.8, 152.9, 147.8, 130.1,
127.6, 127.3, 127.1, 124.3, 124.0, 119.9, 117.5, 115.1, 108.7,
45.8, 44.7, 39.4, 37.0, 21.6. Anal. (C35H28N4O6‚2CH3SO3H‚
3H2O) C, H, N, S.
(KBr): 2400, 1700, 1650 cm-1 1H NMR (DMSO-d6): δ 9.59
.
(br s, 2H, 2NH+), 9.03 (br s, 2H, ArH), 8.73 (br s, 2H, ArH),
8.59 (br s, 2H, ArH), 8.18 (br s, 2H, ArH), 8.01 (br s, 2H, ArH),
7.96 (br s, 2H, ArH), 4.55 (br s, 4H, 2CH2N), 3.56 (br s, 4H,
2CH2N), 3.37 (br s, 4H, 2CH2N), 3.08 (s, 6H, 2CH3N), 2.37 (s,
6H, 2CH3SO3-), 2.21 (br s, 2H, CH2). 13C NMR (DMSO-d6): δ
164.1, 163.9, 142.3, 137.4, 129.8, 129.4, 129.1, 128.0, 127.8,
126.7, 126.6, 124.7, 122.9, 119.3, 52.7, 52.1, 52.0, 40.1, 39.6,
34.8. Anal. (C37H32N4O4S2‚2CH3SO3H‚4H2O) C, H, N, S.
In Vitr o Cytotoxicity Assa ys. The cell lines used were
human colon carcinoma (HT-29) (ATCC, HTB 38), human
cervical carcinoma (HeLa) (ATCC, CCL 2), and human pros-
tate carcinoma (PC-3) (ECACC, 90112714). For each experi-
ment, cultures were seeded from frozen stocks. Each cell line
was maintained in its appropriate medium and was incubated
at 37 °C in a 5% CO2 atmosphere.
All cell lines were in the logarithmic phase of growth when
the assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) was carried out. Cells were harvested
and seeded into 96-well tissue culture plates at a density of
2.5 × 103 cells/well in 150 µL aliquots of medium. The
concentrations tested were serial dilutions of a stock solution
(1 × 10-5 M in DMSO) with phosphate-buffered saline (PBS)
and were added 24 h later. The assay was ended after 72 h of
drug exposure and PBS was used as a negative control and
doxorubicin as a positive control.
After a 72 h exposure period, cells were washed twice with
PBS, and then 50 µL/well of MTT reagent (1 mg/mL in PBS;
Sigma) together with 150 µL/well of prewarmed medium were
added. The plates were returned to the incubator for 4 h.
Subsequently, DMSO was added as solvent. Absorbance was
determined at 570 nm with a Microplate reader (Opsys MR).
All experiments were performed at least three times, and
the average of the percentage absorbance was plotted against
concentration. Then, the concentration of drug required to
inhibit 50% of cell growth (IC50) was calculated for each
compound.
In Vivo An titu m or a l Eva lu a tion . Human tumor xeno-
grafts were established by s.c. injection of HT-29 cell line in
athymic nu/ nu nude mice. Mice were kept under standard
laboratory conditions according to the guidelines of the Span-
ish Government. Cells were trypsinized and resuspended in
DMEM just before inoculation (106 cells/0.1 mL). When tumors
reached a volume of ∼0.1 cm3, mice were randomized to a
control and treated group (four mice each). The compound was
administered ip, dissolved in sterile 0.9% NaCl. Control mice
received an equivalent volume of vehicle alone, following an
identical schedule. Tumor size was measured 1-2 times per
week, and the volumes were calculated using the equation V
) (Dd2)/ 2, where V (nm3) is tumoral volume, D is longest
diameter in mm, and d is shortest diameter in mm.
N,N′-Bis[2-(4,6-d ioxo-5,6-d ih yd r o-4H-ben z[d e]fu r o[2,3-
g]isoq u in olin -5-yl)et h yl]-N,N′-d im et h yl-1,3-p r op a n ed i-
a m in e (10). Following the general procedure, from 195 (190
mg, 0.80 mmol) in toluene (5 mL) and N,N′-bis(2-aminoethyl)-
N,N′-dimethyl-1,3-propanediamine (75 mg, 0.40 mmol) in
absolute EtOH (2 mL) was obtained 10 (68 mg, 27%) as an
unstable brown solid. The free base was converted into the
corresponding dimethanesulfonate dihydrate (75%), mp 255-
257 °C. IR (KBr): 2650, 1695, 1650 cm-1.1H NMR (DMSO-
d6): δ 9.45 (br s, 2H, 2NH+), 8.83 (s, 2H, ArH), 8.68 (d, 2H, J
) 7.9 Hz, ArH), 8.51 (d, 2H, J ) 7.3 Hz, ArH), 8.38 (m, 2H,
furanH), 7.97 (m, 2H, ArH), 7.36 (m, 2H, furanH), 3.43 (m,
4H, 2CH2N), 3.42 (m, 4H, 2CH2N), 3.40 (br s, 4H, 2CH2N),
2.95 (s, 6H, 2CH3N), 2.28 (s, 6H, 2CH3SO3-), 2.09 (m, 2H, CH2).
13C NMR (DMSO-d6): δ 164.0, 163.7, 152.6, 147.8, 129.4, 127.7,
126.9, 126.3, 125.5, 124.1, 122.5, 118.5, 117.8, 108.8, 72.3, 60.2,
52.8, 52.4, 39.6, 35.1. MS (ESI): m/z 629 [M + H]+. Anal.
(C37H32N4O6‚2CH3SO3H‚2H2O) C, H, N, S.
N,N′-Bis[2-(4,6-d ioxo-5,6-d ih yd r o-4H -b en z[d e]t h ien o-
[2,3-g]isoqu in olin -5-yl)eth yl]-1,3-p r op a n ed ia m in e (11).
To a suspension of compound 20 (200 mg, 0.79 mmol) in
toluene (5 mL) was added dropwise N,N′-bis(2-aminoethyl)-
1,3-propanediamine (63 mg, 0.39 mmol) in absolute EtOH (2
mL). After the mixture was refluxed for 4 h, an excess of N,N′-
bis(2-aminoethyl)-1,3-propanediamine (10 mg, 0.06 mmol) was
added. The reaction mixture was refluxed for 2 additional
hours, and then the precipitate was isolated by filtration to
give 11 (207 mg, 84%) as a yellow solid, mp 168-170 °C
(absolute EtOH/toluene). IR (KBr): 3325, 1690, 1650 cm-1. 1H
NMR (DMSO-d6/ CF3CO2D): δ 8.94 (s, 2H, ArH), 8.84 (br s,
4H, 2NH2+), 8.64 (d, 2H, J ) 7.9 Hz, ArH), 8.49 (d, 2H, J )
7.3 Hz, ArH), 8.11 (d, 2H, J ) 4.9 Hz, thiopheneH), 7.93 (m,
4H, thiopheneH, ArH), 4.39 (br s, 4H, 2CH2N), 3.35 (br s, 4H,
2CH2N), 3.07 (br s, 4H, 2CH2N), 1.93 (br s, 2H, CH2). The free
base was converted into the corresponding dimethanesulfonate
monohydrate (92%), mp 212 °C (dec). IR (KBr): 3400, 2800,
1700, 1650 cm-1 1H NMR (DMSO-d6): δ 8.93 (s, 2H, ArH),
.
8.64 (d, 6H, J ) 7.9 Hz, ArH, 2NH2+), 8.48 (d, 2H, J ) 7.3 Hz,
ArH), 8.11 (d, 2H, J ) 4.9 Hz, thiopheneH), 7.94 (dd, 2H, J )
7.9 and 7.3 Hz, ArH), 7.91 (d, 2H, J ) 4.9 Hz, thiopheneH),
4.38 (br s, 4H, 2CH2N), 3.38 (br s, 4H, 2CH2N), 3.07 (br s, 4H,
2CH2N), 2.27 (s, 6H, 2CH3), 1.94 (br s, 2H, CH2). 13C NMR
(DMSO-d6): δ 165.2, 165.1, 143.3, 138.0, 130.5, 130.4, 129.8,
128.7, 128.6, 128.5, 127.3, 125.1, 123.1, 119.5, 46.5, 44.9, 40.1,
37.4, 22.8. Anal. (C35H28N4O4S2‚2CH3SO3H‚H2O) C, H, N, S.
DNA Bin d in g Exp er im en ts. DNA. Calf thymus DNA was
purchased from Sigma Chemical Co. as the highly polymerized
sodium salt. For viscometric experiments, the DNA was
sonicated to fragments of approximately 4.5 × 105 D deter-
mined as described by Eigner and Doty.26 The sonicated DNA
N,N′-Bis[2-(4,6-d ioxo-5,6-d ih yd r o-4H -b en z[d e]t h ien o-
[2,3-g]isoqu in olin -5-yl)eth yl]-N,N′-dim eth yl-1,3-pr opan edi-

New Analogues of Amonafide and Elinafide
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1399
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sample displayed an A260/A280 ratio of 1.92. This spectral data
is consistent with published values.27
Viscom etr ic Titr a tion s. The viscometric measurements
were performed in an Ubbelohde microviscometer at 25 ( 0.05
°C. Solutions of sonicated DNA and the selected compound
were prepared in Tris buffer (50 mM, pH ) 6.9). These
solutions had different molar ratio, r, of added compound to
DNA nucleotides. Flow rates were measured with a Schott-
Gera¨te Viscosystem AVS 350 to an accuracy of 0.01%. Time
readings were recorded in triplicate to 0.01s.
Alk a lin e Sin gle Cell Gel Electr op h or esis Assa y. The
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described by Moinet-Hedin et al.23 After 1 h of treatment with
the drug, cells were centrifuged and resuspended in low-
melting-point (LMP) agarose at 37 °C. The cell suspension was
put on a slide precoated with normal agarose, and a glass cover
slip was added. After solidification at 0 °C, the glass cover slip
was gently removed, and a third layer of 0.5% of LMP agarose
in PBS was added and run for solidification.
The slides were put in a lysis solution (2.5 M NaCl, 0.1 M
EDTA, 10 mM Tris, pH 10, with freshly added 1% Triton X-100
and 10% DMSO) for 1 h and were rinsed in the electrophoresis
buffer (0.3 M NaOH, 1 mM EDTA, pH 13) for 40 min.
Electrophoresis (300 mA, 0.7 V/cm) was then performed for
24 min in fresh buffer. The slides were washed twice in
neutralization buffer (0.4 M Tris, pH 7.5) and stained with
ethidium bromide (20 µg/mL). They were observed using a
fluorescence microscope (Nikon) with an excitation filter of
515-560 nm and a barrier filter of 580 nm.
Ack n ow led gm en t. We thank Federico Gago for
helpful comments and discussion. This work was sup-
ported by the Direccio´n General de Estudios Superiores
(Grant No. PB98-0055), CYTED and Universidad San
Pablo-CEU (Grant No 5-99/01). M.C. thanks Fundacio´n
Ramo´n Areces for finantial support and M. A. G.
acknowledges Fundacio´n Universitaria San Pablo CEU
for a predoctoral fellowship. We thank the CIEMAT
(Spain) for computer time and facilities.
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