Synthesis, Biological Activity, and Quantitative Structure-Activity Relationship Study of Azanaphthalimide and Arylnaphthalimide Derivatives

Article abstract of DOI:10.1021/jm0310784

A series of quinoline derivatives as aza analogues of the naphthalene chromophore and a series of "nonfused" tricyclic aromatic systems, in particular 5-arylquinolines and 5- or 6-aryl and heteroaryl naphthalene systems, were synthesized and evaluated for

Full text of DOI:10.1021/jm0310784

2236
J . Med. Chem. 2004, 47, 2236-2242
Syn th esis, Biologica l Activity, a n d Qu a n tita tive Str u ctu r e-Activity
Rela tion sh ip Stu d y of Aza n a p h th a lim id e a n d Ar yln a p h th a lim id e Der iva tives
Miguel F. Bran˜a,*,# Ana Gradillas,*,# Angel Go´mez,# Nuria Acero,§ Francisco Llinares,|
Dolores Mun˜oz-Mingarro,# Cristina Abradelo,‡ Fernanda Rey-Stolle,‡ Mercedes Yuste,‡ J oaqu´ın Campos,†
Miguel AÄ . Gallo,† and Antonio Espinosa†
Departamento de Ciencias Qu´ımicas, Departamento de Ciencias Ambientales y Recursos Naturales,
Departamento de Biologı´a Celular, Bioqu´ımica y Biolog´ıa Molecular, Departamento de Matema´ticas,
Fı´sica Aplicada y Fisicoqu´ımica, Facultad de Ciencias Experimentales y de la Salud,
Universidad San Pablo CEU, Urbanizacio´n Montepr´ıncipe, 28668-Boadilla del Monte, Madrid, Spain, and
Departamento de Qu´ımica Farmace´utica y Orga´nica, Facultad de Farmacia, Universidad de Granada,
C/ Campus de Cartuja s/ n, 18071 Granada, Spain
Received October 24, 2003
A series of quinoline derivatives as aza analogues of the naphthalene chromophore and a series
of “nonfused” tricyclic aromatic systems, in particular 5-arylquinolines and 5- or 6-aryl and
heteroaryl naphthalene systems, were synthesized and evaluated for growth-inhibitory
properties in several human cell lines. The analysis of quantitative structure-antitumor activity
relationships for the growth-inhibitory properties is also reported. Findings suggest that these
compounds may not express their cytotoxicity via interaction on DNA.
In tr od u ction
DNA-intercalating antitumor drugs constitute an
important class of drugs in anticancer therapy.1 Naph-
thalimides are significant examples2 that include com-
pounds in clinical trials such as the mononaphthalimide
amonafide 13 and the bis(naphthalimide) LU 79553
(elinafide) 2.4 Nowadays, it is accepted that the anti-
tumor activity of amonafide is closely related to its
ability to stabilize the DNA-intercalator-topoisomerase
II ternary complex.5 Elinafide is not a poison of topo-
isomerase II (topo II). It is a strong DNA binder, but it
does not stabilize the topo II-DNA complex (or binds
very weakly).6 As part of a program to broaden the scope
of 1 and 2, we have recently reported7,8 the synthesis
and biological evaluation of a new series of mono- and
bis-intercalating agents where heterocyclic systems
have been “fused” to the naphthalimide chromophore.
Previous studies have reported the consequences of
appending “nonfused” aromatic systems. The results
obtained by the Cheng group9 with the “2-phenylnaph-
thalene-type” structural pattern hypothesis and by
Denny and co-workers with tricyclic carboxamides10
without a completely fused chromophore encouraged us
to synthesize the structures proposed herein. In the
present paper we describe the synthesis of new types
of structures related to 1 and 2. The first series has a
quinoline nucleus as aza analogues of the naphthalene
chromophore, 11-17. They were designed to determine
* To whom correspondence should be addressed. For M.F.B.: phone,
34913724771; fax, 34913724009; e-mail mfbrana@ceu.es. For A.G.:
phone, 34913724789; fax, 34913510496; e-mail, gradini@ceu.es.
# Departamento de Ciencias Qu´ımicas, Universidad San Pablo CEU.
§ Departamento Ciencias Ambientales y Recursos Naturales, Uni-
the effect of replacing carbon with nitrogen on antitumor
versidad San Pablo CEU.
activity. On the other hand, several series of “nonfused”
tricyclic aromatic systems, 5- or 6-aryl and hetero-
arylnaphthalene systems 18-22, were also prepared.
The cytotoxic activity and the quantitative structure-
activity relationship (QSAR) analysis for the growth-
| Departamento de Biolog´ıa Celular, Bioqu´ımica y Biolog´ıa Molec-
ular, Universidad San Pablo CEU.
‡ Departamento de Matema´ticas, F´ısica Aplicada y Fisicoqu´ımica,
Universidad San Pablo CEU.
† Departamento de Qu´ımica Farmace´utica y Orga´nica, Universidad
de Granada.
10.1021/jm0310784 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/20/2004

Naphthalimide Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2237
Ta ble 1. Growth-Inhibitory Properties for Azanaphathlimide
and Arylnaphathalimide Derivatives 11-22
meyer synthesis by the reaction of methyl 3-amino-
benzoate with chloral hydrate and hydroxylamine,
yielding the corresponding oxyme, whose cyclization
with a strong acid medium18 produced the desired
product 1. The syntheses of the aryl- or heteroaryl-
substituted naphthalic anhydrides were prepared by a
Stille cross-coupling reaction (Scheme 3) by condensa-
tion of the 3- or 4-bromo-1,8-naphthalic anhydride with
aryl- or heteroaryltributyltin in the presence of tetrakis-
(triphenylphosphine)palladium as a catalyst in dioxane
at refluxing temperature. Details of the synthesis of this
series are reported elsewhere.19 The initial 4-Br product
is commercially available, and the 3-bromo-1,8-naph-
thalic anhydride was prepared by adding bromine to a
solution of 1,8-naphthahlic anhydride in 70% nitric
acid.20 Finally, the monomeric and dimeric compounds
were prepared by reaction between the corresponding
anhydrides and the appropriate commercially available
polyamines. The monomeric compounds contain a
(CH2)2NMe2 side chain, and the corresponding bis
analogues were linked by a (CH2)2NH(CH2)3)NH(CH2)2
chain.
a
IC50
compd
11
12
13
14
15
16
17
18
19
20
21
HT-29
HeLa
3.37
3.09
10.00
>100
>100
6.80
PC-3
11.70
3.44
15.17
NTb
NTb
NTb
NTb
NTb
14.05
5.42
8.70
1.10
2.64
6.42
16.50
6.38
0.32
>100
>100
5.70
0.16
4.81
0.021
1.20
3.60
5.65
4.67
0.017
0.27
2.64
0.23
0.63
1.53
4.17
2.73
0.07
22
amonafide
elinafide
a
IC50: concentration of drug (µM) to reduce the cell number to
b
50% of control cultures. NT: not tested.
Sch em e 1a
Biologica l Activity. In vitro cytotoxic potencies of
the target compounds 11-22 and of the reference drugs
amonafide and elinafide against the human colon adeno-
carcinoma cell line (HT29), human cervical carcinoma
(HeLa), and human prostate carcinoma (PC-3) are
reported in Table 1. The results indicate that all the
compounds possess a good antiproliferative activity in
the micromolar range, with the exception of 14 and 15,
which are the least effective in the series because the
carbonyl form is the tautomeric form preferred by the
quinoline chromophore (see spectral characterizations
in Experimental Section). The dimeric compounds were
generally more potent than the corresponding mono-
meric ones. Compound 19 emerges as the most potent
among the new derivatives with an IC50 value of 0.021
µM against HT-29. This result agrees with the hypoth-
esis that the introduction of “nonfused” tricyclic aro-
matic systems can enhance this activity.9,10 Apart from
19, a noticeable cytotoxic activity is also shown by 17
with an IC50 value of 0.16 µM against HT-29.
a
Reagents: (i) 85% KOH, EtOH, 80 °C, 4-5 days.
inhibitory properties in a panel of tumor cell lines are
included in this study. Two compounds were selected
for the assay of single-cell gel electrophoresis (comet
assay) to further evaluate DNA damage. One of them,
16, was selected because in QSAR studies it was found
to be an outlier. The other was 19 because it emerges
as the most potent among the new derivatives.
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of the compounds listed
in Table 1 required the preparation of the corresponding
anhydrides, which were obtained by the methods out-
lined in Schemes 1-3. The synthesis of some quinoline-
based compounds involved an adaptation of the
Pfitzinger synthesis,11 in which the methyl isatin-4-
carboxylate 1 reacted with a ketone to give the anhy-
drides 2 and 3 (Scheme 1). Many variations of the base
catalysis have been employed12,13 in the Pfitzinger
reaction, and we obtained 2 and 3 with 85% potassium
hydroxide at 80 °C for 4-5 days,14 although the yield
for each was less than 25%. Both compounds showed a
spontaneous loss of water to give the final anhydride
instead of the dicarboxylic compound. A limitation using
this method is the yield obtained in the final step.
The members of the quinoline series were prepared
by the reaction sequence shown in Scheme 2. Methyl
isatin-4-carboxylate 1 reacted with anhydride acetic as
the acylating agent15 to give 4. Then 4 underwent an
intramolecular aldol condensation15 producing the 1,2-
dihydro-2-oxoquinolin-4,5-dicarboxylic acid with spon-
taneous dehydration to the corresponding anhydride 5.
This reacted with POCl3 to give the chloro derivative
6.16 All efforts to dehalogenate 6 by reduction17 proved
to be unsuccessful.
Qu a n tita tive Str u ctu r e-Activity Rela tion sh ip
(QSAR) An a lysis. The data obtained allowed us to
determine quantitative structure-activity relationships.
From the data in Table 2 on HT-29 cells, we have
derived eq 1:
p(IC50 HT-29) ) 9.36 ((0.82) - 2.58 ((0.48) c log P +
0.40 ((0.07) c log2 P (1)
n ) 9, s ) 0.398, r2 ) 0.864, F2,6 ) 19.07,
R < 0.005 (S > 99.5%)
inversion point: c log P0 ) 3.22 (2.97-3.52);
outlier, 16
where n is the number of data points, s is the standard
deviation, r is the correlation coefficient, and the data
within parentheses are for the 95% confidence intervals.
The predictive ability of the models was assessed for
the nine compounds in the training set using the cross-
validation approach and measured in terms of q2 values:
The two above-mentioned syntheses started with 1.
This was prepared using an adaptation of the Sand-
q2 ) (SD - PRESS)/SD
(2)

2238 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Bran˜a et al.
Sch em e 2a
a
Reagents: (i) (CH3CO)2O, reflux; (ii) aqueous NaOH, reflux; (iii) POCl3, dioxane, 80-85 °C.
Sch em e 3a
as the mono- and the bisnaphthalimides, with their
respective bioisosteres and quinoline derivatives, and
(b) the initial negative slope is rarely larger than (1.20,
suggesting something other than a simple hydrophobic
interaction.25
It is of interest to consider the nature of the outlier.
2-Halogenated pyridines are structurally similar to the
very reactive imino halides, which are the nitrogen
analogues of acid chlorides.26 The halogen atom of
2-chloropyridine is very easily displaced by nucleophilic
reagents.26 Quaternization of the quinoline ring brings
about a dramatic increase in the lability of the 4-halo-
quinoline, and it was reported that 4,7-dichloro-1-
methylquinoline methosulfate with piperidine at room
temperature gives 7-chloropiperidinoquinoline.27 It can
be assumed that at physiological pH, the chlorine atom
of 16 could be displaced by nucleophilic centers present
in the bases of DNA, and accordingly, the anticancer
activity of 16 may be the consequence of its alkylating
capacity.28
When we tried to obtain similar QSARs for com-
pounds shown in Table 2 with other cell lines (HeLa
and PC-3), only a moderate statistical fit was achieved
(r2 ) 0.728 and r2 ) 0.562, respectively), compound 16
not being included as we proceeded with eq 1. In the
case of the HeLa cell line, both terms (c log P and
c log2 P) were significant (data not shown), and this
might hint that other variables need to be considered.
Unfortunately, with so few points we could not attempt
to use additional parameters.
a
Reagents: (i) aryltrialkyltin, Pd(PPh3)4, dioxane, reflux 48 h.
Ta ble 2. pIC50 Values of Azanaphthalimide and
Arylnaphthalimide Derivatives against the Human Cancer Cell
Lines HT-29, HeLa, and PC-3a
c
pIC50
b
compd
c log P
HT-29
HeLa
PC-3
11
12
13
16d
17
18
19
20
21
22
2.39
3.84
3.61
1.08
1.22
3.34
5.75
2.68
4.42
3.32
4.93
5.46
4.82
5.24
6.80
5.32
7.68
5.92
5.44
5.25
5.47
3.09
5.00
5.17
6.57
5.58
6.64
6.20
5.82
5.38
NT
NT
NT
4.85
5.27
5.06
5.96
5.58
5.19
4.78
a
Compounds 14 and 15 are not included in deriving eq 1.
Predicted by using the PALLAS 2.0 program.21 c pIC50
-log(IC50), bearing in mind that the higher the value of pIC50 the
more potent the compound. Not included in deriving eq 1.
)
b
d
where the PRESS (predictive residual sum of squares)
and SD (standard deviation) values are obtained as
In the present paper, we have a QSAR based on
c log P and c log2 P terms where activity against the HT-
29 cell line first descends to a minimum and then begins
to rise. The outlier 16 could behave as an electrophilic
trap for the bases of DNA instead of an intercalator.
To elucidate whether compound 16 reacts with DNA,
UV-vis spectra and ITC (isothermal titration calorim-
etry) thermograms were carried out. There is no evi-
dence of reaction of compound 16 with DNA or with 2′-
deoxyguanosine because the spectra of the binary
mixtures is the result of the addition of the spectra of
the two pure substances.
Isothermal titration calorimetry is a modern tech-
nique with wide applications to determine the binding
enthalpy and heat capacity change for different com-
pounds as DNA intercalators.29,30 Our results confirm
that there is no reaction between compound 16 and 2′-
deoxyguanosine. The lack of action on DNA does not
justify the above-mentioned hypothesis proposed for 16.
The DNA binding properties of compounds 16 and 19
were also studied by viscosimetric titration with calf
thymus DNA. It is known31,32 that the DNA length
increases when a drug behaves as an intercalator. The
compound could be mono- or bifunctional. One method
to identify the type of intercalator is by viscosimetric
2
PRESS ) (property
- propertypredicted
)
(3)
(4)
∑
observed
2
SD ) (property
- propertymean)
∑
observed
Equation 1 gives a good q2 value of 0.864. q2 will always
be smaller than r2. A model is considered significant21
when q2 > 0.3.
The statistics of QSAR (1) are promising, and only
one compound fitted in so poorly that it had to be
omitted 16. Interestingly, such an equation shows an
inverted parabola; the coefficient with the c log P term
is negative and the squared term is positive. That is,
as lipophilicity increases, activity first decreases to a
minimum and then increases with further increment
in the calculated log P. A constant concern in formulat-
ing a new QSAR is to find as much support as possible.
In fact it is believed that a single QSAR standing alone
cannot be seriously considered until one can find that
it has some generality. Similar inverted parabolas have
been associated with allosteric interactions for enzymes
and receptors23,24 and resistant and sensitive cancer
cells.25 The two following facts are especially note-
worthy: (a) eq 1 includes two structural congeners such

Naphthalimide Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2239
Con clu sion s
The biological results obtained show that antitumor
activity is not limited to those compounds possessing
fused tricyclic or larger chromophores. Apart from
2-phenylnaphthalene, the most interesting chromophore
appears to be 2-chloroquinoline. Both dimeric com-
pounds with these chromophores, 17 and 19, showed
significant growth delays in vitro. The biological activity
of compound 19 was encouraging for the possible further
development of this class of compounds.
Some preliminary results have shown that some
bisnaphthalimides with good inhibitory activity have no
action against topoisomerase II.6 The findings permit
us to suspect an additional action mechanism for these
kinds of compounds. Thus, the preliminary results
obtained by J . C. Lacal and co-workers (personal com-
munication) on the action of elinafide 2 on transcription
factors point in this direction and could open up new
perspectives. It should definitely be noted in future
studies.
F igu r e 1. Relative length increase L/ Lo of 16-DNA (2, solid
lines) and 19-DNA complexes (b, dashed lines) as a function
of the molar ratio of added compound to DNA nucleotides, r.
Exp er im en ta l Section
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
spectrophotometer. 1H and 13C NMR were recorded on a
Bruker 300-AC instrument. Chemical shifts (δ) are expressed
in parts per million relative to internal tetramethylsilane.
Elemental analyses (C, H, N) were performed on a Perkin-
Elmer 2400 CHN apparatus at the Microanalysis Service of
the Complutense University, Madrid. Unless otherwise stated,
all reported values are within (0.4% of the theoretical
compositions. Reactions were monitored by thin-layer chro-
matography (TLC) run on Merck silica gel 60 F-254 plates and
iodine vapor and/or UV light detection. Flash chromatography
was performed using Merck silica gel 60 (size 230-400 mesh
ASTM). Unless stated otherwise, starting materials used were
high-grade commercial products. The tail moment and % DNA
in comet were determined by Vat-Euclid Comet Analysis
Software, St. Louis, MO. 2′-Deoxyguanosine was purchased
from Sigma Aldrich.
F igu r e 2. Tail moment for compounds 16 and 19 was 0, and
the % DNA in comet was 0% (7% and 17%, respectively, for
doxorubicin) in HT-29 cells.
Syn th etic Ch em istr y. Meth yl 4-Isa tin ca r boxyla te (1).
A solution of 13.6 g (0.09 mol) of methyl 3-aminobenzoate, 16.1
g (0.09 mol) of chloral hydrate, 234.4 g of sodium sulfate, and
19.8 g (0.29 mol) of hydroxylamine hydrochloride in 145 mL
of water and 8 mL of concentrated HCl was refluxed for 2 h
and then kept at room temperature overnight. The cream-
colored isonitroso intermediate was filtered off, washed with
water, and dried (16.0 g, 80%). This compound, 16.0 g (0.07
mol), was added in portions, while stirring over 30 min, to
concentrated H2SO4 (70 mL) maintained at 70-75 °C. The
mixture was then heated at 80 °C until the reaction was
complete (TLC, CHCl3/EtOH, 8:2) and poured onto ice. The
aqueous phase was extracted with ethyl acetate. The organic
layer was dried over MgSO4, filtered, and evaporated to
dryness to yield the isatin derivative, typically as an orange
solid, which was purified by gradient flash column chroma-
tography on silica gel, eluting with CHCl3/EtOH, 8:2, to yield
the title compound (5 g, 34%), mp 215-217 °C. IR (KBr): 3250,
titrations. With these measurements the relative in-
crease in contour length, L/Lo, against r is plotted. The
slope of this plot has different values depending on the
functionality of the intercalator.31,33 In Figure 1 L/Lo is
plotted against r for 16 and 19. Least-squares fitting
gives a slope of 0.49 and 0.40, respectively. These values
reveal that there is no evidence of intercalative binding
in the viscosimetric titration assay.
Nevertheless, to further evaluate the mechanism of
action, compounds 16 and 19 were selected for single-
cell gel electrophoresis assay (comet assay). It is based
on the property of negatively charged DNA fragments
migrating when an electrical field is applied to the gel
after cell lysis.34 Doxorubicin was chosen as a positive
reference, and PBS (phosphate-buffered saline, pH 7.4)
was used as a negative control. One hour after treat-
ment, the samples were observed using fluorescence.
The comet assay detected no DNA damage in the cells
treated with these compounds, similar to that observed
in the negative control. Parent DNA damage was
observed with the positive reference (Figure 2). Thus,
we suggest these compounds may not express their
cytotoxicity via DNA degradation.
1
1765, 1740, 1720, 1200 cm-1. H NMR (DMSO-d6): δ 3.92 (s,
3H, OCH3), 7.07 (d, 1H, ArH), 7.22 (d, 1H, ArH), 7.66 (t, 1H,
ArH), 11.20 (s, 1H, NH). 13C NMR (DMSO-d6): δ 52.60, 115.42,
123.21, 129.60, 131.61, 138.05, 151.52, 158.56, 166.75, 181.93.
Anal. (C10H7NO4) C, H, N.
P r ep a r a tion of 2-Ar yl-4,5-qu in olin ed ica r boxylic An -
h yd r id es 2 a n d 3. Gen er a l P r oced u r e for P fit zin ger
Rea ction . A mixture of methyl 4-isatincarboxylate (2.5 mmol),
ethanol (5 mL), and 85% potassium hydroxide (7.3 mmol) was
stirred at room temperature for 30 min. The appropriate
ketone (1.7 mmol), dissolved in EtOH (1 mL), was added, and
the reaction was stirred at room temperature for 1 h and at

2240 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Bran˜a et al.
80 °C for 72 h. Evaporation of the solvent produced a residue
that was dissolved in H2O (2 mL), and the solution was washed
twice with Et2O (5 mL). The ice-cold aqueous phase was
acidified to pH 1 with 37% HCl, and the precipitate was
collected by suction filtration, washed with H2O, and dried.
Typically, yields of this reaction were very poor. The following
were prepared in this manner.
8.31 (d, 1H, ArH), 8.34 (d, 1H, ArH) 8.48 (s, 1H, ArH), 8.62 (t,
1H, ArH), 8.88 (s, 1H, ArH). 13C NMR (CDCl3): δ 118.66,
119.24, 127.01, 127.39, 127.41, 127.97, 129.27, 132.41, 132.99,
133.35, 135.27, 135.37, 138.31, 140.71, 160.38. Anal. (C18H10O3)
C, H, N.
3-(2′-F u r yl)n a p h th a lic An h yd r id e (9). This was prepared
from 3-bromonaphthalic anhydride 7b.20 The residue was
purified by gradient flash column chromatography on silica
gel, eluting with (CH2Cl2/hexane, 7:3) to yield the correspond-
ing anhydride (80%), mp 188-190 °C. IR (KBr): 1780, 1740
cm-1. 1H NMR (CDCl3): δ 6.60 (t, 1H, ArH), 6.97 (d, 1H, ArH),
7.61 (s, 1H, ArH), 7.81 (t, 1H, ArH) 8.31 (d, 1H, ArH), 8.54 (d,
1H, ArH), 8.57 (d, 1H, ArH), 8.87 (s, 1H, ArH). Anal. (C16H8O4)
C, H, N.
4-P h en yln a p h th a lic An h yd r id e (10). This was prepared
from 4-bromonaphthalic anhydride 7b. The residue was puri-
fied by gradient flash column chromatography on silica gel,
eluting with (CH2Cl2/hexane, 8:2) to yield the corresponding
anhydride (89%), mp >190-200 °C. IR (KBr): 1770, 1749
cm-1. 1H NMR (CDCl3): δ 7.52 (m, 5H, ArH), 7.74 (d, 1H, ArH),
7.79 (t, 1H, ArH), 8.39 (d, 1H, ArH), 8.65 (d, 1H, ArH), 8.68
(d, 1H, ArH). 13C NMR (CDCl3): δ 117.67, 118.95, 127,32,
128.32, 128.84, 128.93, 129.72, 130.30, 133.05, 133.40, 134.20,
148.55, 160.40. Anal. (C18H10O3) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of Mon om er ic
Com p ou n d s. A mixture of the corresponding anhydride (2.0
mmol) and N,N-dimethylethylenediamine (2.0 mmol) in 10 mL
of DMF (for quinoline derivatives) or CHCl3 (for naphthalene
derivatives) was heated to reflux for 24 h. The solvent in the
reaction mixture was evaporated.
2-P h en yl-4,5-qu in olin ed ica r boxylic An h yd r id e (2). The
precipitate formed was filtered and dried to give 2 as a light-
cream solid (22%), mp 198-200 °C. IR (KBr): 1780, 1750 cm-1
.
1H NMR (DMSO-d6): δ 7.59 (m, 3H, ArH), 7.71 (t, 1H, ArH),
7.86 (t, 1H, ArH), 8.19 (d, 1H, ArH), 8.29 (d, 1H, ArH), 8.46
(s, 1H, ArH), 8.65 (d, 1H, ArH). 13C NMR (DMSO-d6): δ 128.74,
130.01, 136.93, 138.65, 138.79, 138.89, 139.87, 140.70, 142.60,
147.03, 150.14, 158.08, 165.77, 177.50, 178.43. Anal. (C17H9NO3)
C, H, N.
2-(2′-Na p h th yl)-4,5-qu in olin ed ica r boxylic An h yd r id e
(3). The precipitate formed was filtered and dried to give 3 as
a light-cream solid (15%), mp 218-220 °C. IR (KBr): 1760,
1730 cm-1. 1H NMR (DMSO-d6): δ 7.60 (m, 4H, ArH), 7.79 (t,
1H, ArH), 7.99 (m, 3H, ArH), 8.10 (d, 1H, ArH), 8.32 (d, 1H,
ArH), 8.35 (s, 1H, ArH). 13C NMR (DMSO-d6): δ 118.51,
122.93, 123.82, 124.50, 125.91, 125.94, 126.05, 126.20, 128.01,
128.09, 128.51, 130.52, 132.11, 132.96, 134.13, 137.10, 149.95,
154.63, 166.00, 177.03, 179.02. Anal. (C21H11NO3) C, H, N.
2-Oxo-1,2-d ih yd r o-4,5-q u in olin ed ica r b oxylic An h y-
d r id e (5). N-Acetylisatin derivative 4 was prepared by re-
fluxing 1.5 g of 1 (7.3 mmol) in 3.5 mL of anhydride acetic for
2 h (TLC, ethyl acetate/hexane, 11:1). The solvent was removed
to dryness to obtain a crude that was purified by gradient flash
column chromatography on silica gel, eluting with ethyl
acetate/hexane, 11:1, 1 g (55%), mp 121-123 °C. IR (KBr):
N,N-Dim et h yl-N′-2-{5-p h en yl-1,3,-d ioxo-1,3-d ih yd r o-
p yr id o[3,4,5-d e]qu in olin -2-yl}eth a n a m in e (11). This was
prepared from 2. The residue was purified by gradient flash
column chromatography on silica gel, eluting with (CHCl3/
EtOH, 10:1) to yield the corresponding monointercalator as a
1
1750, 1720, 1705, 1670 cm-1. H NMR (CDCl3): δ 2.75 (s, 3H,
OCH3), 3.99 (s, 3H, OCH3), 7.63 (d, 1H, ArH), 7.76 (t, 1H, ArH),
8.62 (d, 1H, ArH). 13C NMR (CDCl3): δ 24.28, 53.16, 116.55,
120.73, 126.61, 130.68, 138.04, 148.92, 156.66, 165.24, 168.61,
177.41. Anal. (C12H9NO5) C, H, N. Compound 4, 1 g (4 mmol),
was added, with stirring, to NaOH solution (8 mmol) at 90 °C
for 1 h. The pH was taken to 2 with concentrated HCl to give
a white solid, which was collected by suction filtration to yield
5 (0.82 g, 93%), mp >250 °C; IR (KBr): 3450, 1760, 1700, 1650
1
free base (34%), mp >250 °C. IR (KBr): 1720, 1680 cm-1. H
NMR (CD3OD): δ 2.36 (s, 6H, CH3), 2.71 (t, 2H, CH2), 4.28 (t,
2H, CH2), 7.54 (m, 5H, ArH), 7.97 (m, 1H, ArH), 8.18 (m, 1H,
ArH), 8.41 (m, 1H, ArH), 8.62 (m, 1H, ArH). 13C NMR
(CD3OD): δ 41.10, 43.92, 57.30, 117.71, 125.20, 127.11, 127.14,
127.19, 129.00, 131.33, 131.72, 134.51, 138.45, 138.80, 139.70,
145.37, 159.79, 175.91, 171.01. Anal. (C21H19N3O2) C, H, N.
N,N-Dim eth yl-N′-2-{5-(2′-n a p h th yl)-1,3,-d ioxo-1,3-d ih y-
d r op yr id o[3,4,5-d e]qu in olin -2-yl}eth a n a m in e (13). This
was prepared from 3 in 24% yield, mp 246-248 °C. IR (KBr):
1719, 1675 cm-1. 1H NMR (DMSO-d6): δ 2.28 (s, 6H, CH3), 2.59
(t, 2H, CH2), 4.23 (t, 2H, CH2), 7.44 (m, 2H, ArH), 7.82 (m,
5H, ArH), 8.24 (d, 1H, ArH), 8.37 (t, 1H, ArH), 8.49 (s, 1H,
ArH), 8.71 (s, 1H, ArH). Anal. (C25H21N3O2) C, H, N.
1
cm-1. H NMR (DMSO-d6): δ 6.77 (s, 1H, ArH), 7.54 (m, 3H,
ArH), 12.18 (s, 1H, NH). 13C NMR (DMSO-d6): δ 113.30,
118.97, 123.39, 123.70, 130.09, 131.91, 140.01, 140.28, 160.65,
167.36, 168.70. Anal. (C11H5NO4) C, H, N.
2-Ch lor o-4,5-qu in olin ed ica r boxylic An h yd r id e (6). A
mixture of 0.1 g (0.5 mmol) of 5 and 0.06 mL (0.064 mmol) of
POCl3 in 1,4-dioxane (3 mL) was maintained at 80-85 °C while
being stirred for about 15 min until most of the solid had
dissolved and was then warmed for an additional 15 min until
dissolution was complete. The unreacted POCl3 was then
removed under reduced pressure. Ice was added to the residue,
and the precipitate that separated was filtered and washed
with water to give 6 as a yellow solid (0.047 g, 40%), mp >250
N,N-Dim et h yl-N′-{2-[1,3,5-t r ioxo-1,3,5,6-t et r a h yd r o-
p yr id o[3,4,5-d e]qu in olin -2-yl}eth a n a m in e (14). This was
prepared from 5. The residue was purified by gradient flash
column chromatography on silica gel, eluting with (CHCl3/
EtOH, 10:1) to yield the corresponding monointercalator as a
free base (63%), mp 210-218 °C. IR (KBr): 3410, 1670, 1650,
1
°C. IR (KBr): 1782, 1760 cm-1. H NMR (DMSO-d6): δ 8.18
1620 cm-1 1H NMR (D2O): δ 2.52 (s, 6H, CH3), 2.98 (t, 2H,
.
(t, 1H, ArH), 8.37 (s, 1H, ArH), 8.51 (d, 1H, ArH), 8.57 (d, 1H,
ArH). 13C NMR (DMSO-d6): δ 115.50, 119.02, 122.11, 123.90,
131.11, 131.92, 133.01, 141.02, 149.40, 167.40, 169.70. Anal.
(C11H4ClNO3) C, H, Cl, N.
CH2), 3.97 (t, 2H, CH2), 6.01 (s, 1H, ArH), 7.10 (d, 1H, ArH),
7.29 (t, 1H, ArH), 7.53 (d, 1H, ArH). Anal. (C15H15N3O3) C, H,
N.
P r ep a r a tion of 3- or 4-Ar yl a n d Heter oa r yl-1,8-Na p h -
th a lic An h yd r id es (8-10). Gen er a l P r oced u r e for Stille
Cou p lin g Rea ction . A mixture of the bromo-1,8-naphthalic
anhydride derivative (12.6 mmol), aryl or heteroaryltributiltin
derivative (15.3 mmol), and tetrakis(triphenylphosphine)-
palladium(0) (0.41 mmol) in 50 mL of dioxane was heated to
reflux for 48 h. After the mixture was cooled to room temper-
ature, the solvent was evaporated to dryness. The following
anhydrides were prepared in a similar manner.
N,N-Dim et h yl-N′-2-{5-ch lor o-1,3,-d ioxo-1,3-d ih yd r o-
p yr id o[3,4,5-d e]qu in olin -2-yl}eth a n a m in e (16). This was
prepared from 6. The residue was purified by gradient flash
column chromatography on silica gel, eluting with (CHCl3/
EtOH, 10:1) to yield the corresponding monointercalator as a
free base (60%), mp 238-240 °C. IR (KBr): 1720, 1675 cm-1
.
1H NMR (DMSO-d6): δ 2.93 (s, 6H, CH3), 3.48 (t, 2H, CH2),
4.42 (t, 2H, CH2), 8.18 (t, 1H, ArH), 8.30 (s, 1H, ArH), 8.45 (d,
1H, ArH), 8.58 (d, 1H, ArH). Anal. (C15H14ClN3O2) C, H, Cl,
N.
3-P h en yln a p h th a lic An h yd r id e (8). This was prepared
from 3-bromonaphthalic anhydride 7a .20 The residue was
purified by gradient flash column chromatography on silica
gel, eluting with (CH2Cl2/hexane, 8:2) to yield the correspond-
N,N-Dim et h yl-N-{2-[1,3-d ioxo-5-p h en yl-2,3-d ih yd r o-
1H-ben zo[d e]isoqu in ol-5-yl)eth yl}eth a n a m in e (18). This
was prepared from 8. The residue was purified by gradient
flash column chromatography on silica gel, eluting with
(CHCl3/EtOH/NH3, 10:1:0.1%) to yield the corresponding
ing anhydride (84%), mp >250 °C. IR (KBr): 1775 1740 cm-1
.
1H NMR (CDCl3): δ 7.56 (m, 2H, ArH), 7.80 (m, 3H, ArH),

Naphthalimide Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9 2241
monointercalator as a free base (60%), mp 150-154 °C. IR
(KBr): 1700, 1655 cm-1. 1H NMR (CDCl3): δ 2.36 (s, 6H, CH3),
2.67 (t, 2H, CH2), 4.34 (t, 2H, CH2), 7.48 (m, 2H, ArH), 7.52
(m, 4H, ArH), 8.18 (d, 1H, ArH), 8.31 (s, 1H, ArH), 8.52 (d,
1H, ArH), 8.80 (s, 1H, ArH). 13C NMR (CDCl3): δ 38.11,
45.70, 56.92, 122.41, 122.95, 127.10, 127.27, 128.26, 129.01,
130.55, 131.03, 131.94, 133.90, 138.99, 139.81, 167.40. Anal.
(C22H20N2O2) C, H, N.
(t, 4H, CH2), 3.23 (t, 4H, CH2), 4.30 (t, 4H, CH2), 6.56 (s, 2H,
ArH), 6.86 (s, 2H, ArH), 7.26 (m, 2H, ArH), 7.57 (dd, 2H, ArH),
7.62 (d, 2H, ArH), 8.11 (d, 2H, ArH), 8.35 (d,2 H, ArH),
8.55 (s, 2H, ArH). 13C NMR (CDCl3): δ 28.76, 37.55, 46.81,
47.86, 77.01, 106.70, 11.32, 121.01, 121.37, 125.71, 126.43,
128.40, 129.71, 130.63, 133.79, 142.39, 150.87, 163.33. Anal.
(C39H32N4O6) C, H, N.
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 incubated at
37 °C in a 5% CO2 atmosphere.
N,N-Dim eth yl-N-{2-[1,3-d ioxo-5-(2-fu r yl)-2,3-d ih yd r o-
1H-ben zo[d e]isoqu in ol-5-yl)eth yl}eth a n a m in e (20). This
was prepared from 9. The residue was purified by gradient
flash column chromatography on silica gel, eluting with
(CHCl3/EtOH, 8:2) to yield the corresponding anhydride (87%),
mp 130-132 °C. IR (KBr): 1700, 1655 cm-1 1H NMR
.
(CDCl3): δ 2.51 (s, 6H, CH3), 2.90 (t, 2H, CH2), 4.34 (t, 2H,
CH2), 6.56 (s, 1H, ArH), 7.26 (m, 4H, ArH), 7.70 (t, 1H, ArH),
8.12 (d, 1H, ArH). 13C NMR (CDCl3): δ 38.12, 45.71, 56.91,
107.41, 112.18, 122.48, 122.92, 126.96, 127.05, 129.40, 131.86,
133.85, 143.24, 152.07, 163.97. Anal. (C20H18N2O3) C, H, N.
N,N-Dim et h yl-N-{2-[1,3-d ioxo-6-p h en yl-2,3-d ih yd r o-
1H-ben zo[d e]isoqu in ol-5-yl)eth yl}eth a n a m in e (22). This
was prepared from 10. The residue was purified by gradient
flash column chromatography on silica gel, eluting with
(CHCl3/EtOH, 10:1) to yield the corresponding anhydride
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 × 104 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.
1
(90%), mp 188-190 °C. IR (KBr): 1710, 1655 cm-1. H NMR
After a 72 h exposure period, cells were washed twice with
PBS (phosphate-buffered saline, pH 7.4), and then 50 µL/well
of MTT reagent (1 mg/mL in PBS; Sigma) and 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 micro-
plate reader (Opsys MR). All experiments were performed at
least three times, and the average of the percentage absorb-
ance was plotted against concentration. The concentration of
drug required to inhibit 50% of cell growth (IC50) was then
calculated for each compound.
(CDCl3): δ 2.36 (s, 6H, CH3), 2.67 (t, 2H, CH2), 4.32 (t, 2H,
CH2), 7.48 (m, 2H, ArH), 7.52 (m, 4H, ArH), 8.18 (d, 1H, ArH),
8.31 (s, 1H, ArH), 8.52 (d, 1H, ArH), 8.80 (s, 1H, ArH). 13C
NMR (CDCl3): δ 38.12, 45.72, 56.91, 121.65, 122.77, 126.76,
127.78, 128.61, 129.82, 130.81, 131.16, 132.62, 138.74, 146.86,
164.10. Anal. (C22H20N2O2) C, H, N.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Dim er ic
Com p ou n d s. A mixture of the corresponding anhydride (2.0
mmol) and N,N-bis(2-aminoethyl)-1,3-propanediamine (1.0
mmol) in 10 mL of DMF (for quinoline derivatives) or CHCl3
(for naphthalene derivatives) was heated to reflux for 24 h.
The solvent in the reaction mixture was evaporated. The
remaining residue was flash-chromatographed to provide the
bis intercalator as a free base.
Alk a lin e Sin gle-Cell Gel Electr op h or esis Assa y. The
alkaline single-cell gel electrophoresis assay (comet assay)
detects DNA damage in individual cells embedded in agarose.
The test was performed on HT-29 cells following the method
described by Moinet-Hedin et al.34 After 1 h of treatment with
the drug, cells were centrifuged and resuspended in low-
melting-point (LMP) agarose at 37 °C. Doxorubicin was chosen
as a positive reference, and PBS was used as a negative
control. 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.
Isoth er m a l Titr a tion Ca lor im etr y. Experiments were
carried out at 37 °C using a Microcal VP-ITC microcalorimeter
(Microcal Inc., Northampton, MA). The Origin software (Micro-
cal) was used for data acquisition and analysis. 2′-Deoxy-
guanosine, 0.057 mM, in Tris buffer (50 mM, pH 6.9) was
titrated using a 2.16 mM compound 16 solution in the same
buffer (30 injections of 3 µL each), using a 250 µL syringe
rotating at 450 rpm. The injection time was 6 s, and the delay
between injections was 4 min. The peaks produced during
titration were converted to heat output per injection by
integration and correction for the cell volume and sample
concentration.
N,N′-Bis{2-[5-p h en yl-1,3-d ioxo-1,3-d ih yd r o-2H-p yr id o-
[3,4,5-d e]qu in olin -2-yl]eth yl}p r op ylen ed ia m in e (12). This
was prepared from 2 in 32% yield, mp 240-244 °C. IR (KBr):
1710, 1675 cm-1 1H NMR (DMSO-d6): δ 1.67 (q, 2H, CH2),
.
2.74 (t, 4H, CH2), 2.92 (t, 4H, CH2), 4.18 (t, 4H, CH2), 7.42 (m,
10H, ArH), 7.82 (t, 2H, ArH), 8.12 (d, 2H, ArH), 8.37 (dd, 2H,
ArH), 8.60 (s, 2H, ArH). Anal. (C41H34N6O4) C, H, N.
N,N′-Bis{2-[1,3,5-tr ioxo-1,3,5,6-tetr a h yd r op yr id o[3,4,5-
d e]qu in olin -2-yl]eth yl}p r op ylen ed ia m in e (15). This was
prepared from 5 in 35% yield, mp 242-244 °C. IR (KBr): 3410,
1710, 1680, 1650 cm-1. 1H NMR (DMSO-d6 + TFA): δ 2.07 (q,
2H, CH2), 3.05 (t, 4H, CH2), 3.10 (dd, 4H, CH2), 3.21 (t, 4H,
CH2), 6.95 (s, 2H, ArH), 7.60 (m, 6H, ArH). Anal. (C29H26N6O6)
C, H, N.
N,N′-Bis{2-[5-ch lor o-1,3-d ioxo-1,3-d ih yd r o-2H-p yr id o-
[3,4,5-d e]qu in olin -2-yl]eth yl}p r op ylen ed ia m in e (17). This
was prepared from 6 in 28% yield, mp 222-225 °C. IR (KBr):
1720, 1670 cm-1 1H NMR (DMSO-d6): δ 2.25 (q, 2H, CH2),
.
3.07 (t, 4H, CH2), 3.33 (t, 4H, CH2), 4.38 (t, 4H, CH2), 8.20 (m,
2H, ArH), 8.42 (s, 2H, ArH), 8.60 (m, 4H, ArH). Anal. (C29H24
Cl2N4O4) C, H, Cl, N.
-
N,N′-Bis{2-[5-p h en yl-1,3-d ioxo-2,3-d ih yd r o-1H-ben zo-
[d e]isoqu in ol-5-yl]eth yl}-1,3-p r op a n od ia m in e (19). This
was prepared from 8 in 62% yield, mp 218-220 °C. IR (KBr):
1
1720, 1670 cm-1. H NMR (CDCl3): δ 1.77 (q, 2H, CH2), 2.84
(t, 4H, CH2), 3.01 (t, 4H, CH2), 4.27 (t, 4H, CH2), 7.47 (m, 4H,
ArH), 7.64 (s, 6H, ArH), 8.06 (m, 3H, ArH), 8.09 (d, 2H, ArH),
8.21 (dd, 3H, ArH), 8.44 (d, 2H, ArH). 13C NMR (CDCl3): δ
28.54, 39.32, 47.49, 48.07, 77.21, 122.41, 122.95, 126.72,
127.11, 128.23, 129.01, 130.79, 131.05, 131.90, 133.79, 138.81,
139.67, 164.17. Anal. (C43H36N4O4) C, H, N.
N,N′-Bis{2-[5-(2-fu r yl)-1,3-d ioxo-2,3-d ih yd r o-1H-ben zo-
[d e]isoqu in ol-5-yl]eth yl}-1,3-p r op a n od ia m in e (21). This
was prepared from 9 in 62% yield, mp 222-225 °C. IR (KBr):
UV-Visible Sp ectr a . For spectrometric determinations we
have used a spectrophotometer Perkin-Elmer Lambda 14 with
a digital temperature controller PTP-6. The spectra of com-
1
1700, 1655 cm-1. H NMR (CDCl3): δ 2.13 (q, 2H, CH2), 3.13

2242 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 9
Bran˜a et al.
(11) J oule, J . A.; Mills, K.; Smith, G. F. Heterocyclic Chemistry, 3rd
ed. Chapman & Hall: London, 1995; pp 135-136.
pound 16, 2′-deoxyguanosine, sonicated DNA, 16-sonicated
DNA, and 16-2′-deoxyguanosine have been registered at 37
°C.
DNA. Calf thymus DNA was purchased from Sigma Chemi-
cal Co. as the highly polymerized sodium salt. For viscosimetric
experiments the DNA has been sonicated to fragments of
approximately 4.5 × 105 Da determined as described by Eigner
and Doty.35
The sonicated DNA sample displayed an A260/A280 ratio of
1.92. This spectral result is consistent with published values.36
Viscosim etr ic Titr a tion s. The viscosimetric measure-
ments were performed in an Ubbelohde microviscometer at
25 ( 0.05 °C. Solutions of sonicated DNA and the selected
compound have been prepared in Tris buffer (50 mM, pH 6.9).
These solutions have a different molar ratio, r, of added
compound to DNA nucleotides. Flow times were measured
with a Schott-Gera¨te Viscosystem AVS 350 to an accuracy of
0.01%. Time readings were recorded in triplicate to 0.01 s.
(12) Waldmann, H. Isatincarboxilic Acid. J . Prakt. Chem. 1937, 147,
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(13) Raveglia, F. L.; Giardina, M. A. G.; Grugni, M.; Rigoli, R.; Farina,
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(14) Giardina, M. A. G.; Sarau, M. H.; Farina, C.; Medhurst, D. A.;
Grugni, M.; Raveglia, L. F.; Schmidt, D. B.; Rigolio, R.; Lutt-
mann, M.; Vecchietti, V.; Hay, D. W. P. Discovery of a Novel
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E. F.; Seymour, D. 2-Hydroxycinchoninic Acid. Org. Synth. 1955,
456-458 (collective volume III).
(16) Kaslow, C. E.; Lauer, W. M. 2-Chlorolepidine. Org. Synth. 1955,
194-195 (collective volume III).
(17) Neumann, F. W.; Sommer, N. B.; Kaslow, C. E.; Shriner, R. L.
Lepidine. Org. Synth. 1955, 519-522 (collective volume III).
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(collective volume I).
(19) Sun, J .-H.; Seitz, P. S. 3-Aromatic and 3-Heteroaromatic Sub-
stituted Bisnaphthalimides. Patent WO9625400, 1997.
(20) Amegadzie, A. K.; Carey, M. E.; Domagala, J . M.; Micetich, R.
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(21) PALLAS FRAME MODULE, a prediction tool of physicochemical
parameters, is supplied by CompuDrug Chemistry Ltd., P.O. Box
23196, Rochester, NY 14692.
Ack n ow led gm en t. We are grateful to the Direccio´n
General de Estudios Superiores (Grant No. PB98-0055),
CYTED, and the Universidad San Pablo CEU (Grant
No. 4/01 U.S.P.) for financial support and to Brian Crilly
for his assistance in the preparation of this manuscript.
(22) Wermuth, C. G. The Practice of Medicinal Chemistry; Academic
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