Design, synthesis, and antitumor activity of new bis-aminomethylnaphthalenes

Article abstract of DOI:10.1016/j.bmc.2008.07.069

A new series of bis-aminomethylnaphthalenes were synthesized in satisfactory overall yield, through a simple synthetic strategy using reductive amination. The DNA binding properties of these compounds have been examined and compared to those of reference drugs using an UV spectroscopy method. The compounds were evaluated for their in vitro anticancer activity and some of them were studied in vivo. Compound 15 exhibited remarkable antitumor activity and represents a novel template for anticancer chemotherapy and can serve as a new lead compound.

Full text of DOI:10.1016/j.bmc.2008.07.069

Bioorganic & Medicinal Chemistry 16 (2008) 8003–8010
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and antitumor activity of new bis-aminomethylnaphthalenes
Mariela Bollini, Juan José Casal, Ana María Bruno *
Departamento de Química Orgánica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of bis-aminomethylnaphthalenes were synthesized in satisfactory overall yield, through a
simple synthetic strategy using reductive amination. The DNA binding properties of these compounds
have been examined and compared to those of reference drugs using an UV spectroscopy method. The
compounds were evaluated for their in vitro anticancer activity and some of them were studied in vivo.
Compound 15 exhibited remarkable antitumor activity and represents a novel template for anticancer
chemotherapy and can serve as a new lead compound.
Received 16 May 2008
Revised 22 July 2008
Accepted 23 July 2008
Available online 29 July 2008
Keywords:
Bis-aminomethylnaphthalenes
Synthesis
Ó 2008 Elsevier Ltd. All rights reserved.
Antitumor activity
DNA binding
1
. Introduction
The majority of the drugs currently used in the treatment of
cancer reversibly bind to DNA or induce DNA damage, either directly
orviatopoisomeraseinhibition. DNAandassociatedproteinsremain
1,2
valid targets for cancer chemotherapy. Novel DNA intercalating
3
4
5
molecules acridines, indolocarbazoles, naphthalimides, and
6
7
alkylating agents (benzoacronycines, pyrrolobenzodiazepines,
8
distamicin conjugates ) are still being developed. Although it is
well established that DNA binding is not sufficient to confer cyto-
toxic activities, interaction with DNA is often considered as a nec-
essary criterion to maintain a cytotoxic effect, at least for some
series of planar intercalating chromophores such as acridines and
ellipticines.
Symmetrical bis-1-aminomethylnaphthalenes constitute a new
class of molecules (Fig. 1) with cytotoxic activity. Their structure
derives from a long development to determine the minimum ac-
tive molecular structures that result from echinomycine molecule
Figure 1. Symmetrical bis-1-aminomethylnaphthalenes derivatives.
9
,10
simplification.
These compounds show a strong interaction
11,12
2. Results and discussion
with DNA and a potent cytostatic activity.
In order to determine whether the activity was due to a non-
specific siamese structure or it was influenced by the functional
characteristic of the bridge between the two naphthalenes, and
the role of aromatic substituents, both the type and positioning,
a series of compounds was developed.
All the new compounds were evaluated for their ability to bind
DNA and were assayed for antiproliferative properties in vitro and
in vivo by the National Cancer Institute (NCI, USA).
2
.1. Chemistry
The easier procedure to obtain secondary bis-amines is alkyl-
ation of the primary bis-amine with halogenated derivatives. How-
ever, yields are not so good and the reaction requires prolonged
reaction times. Reductive amination is a good method to obtain
these compounds with excellent yields. Several amines condense
with aldehydes to form imine products. Imines are sometimes dif-
ficult to isolate and purify due to their sensitivity to hydrolysis. In
our case, the first step of the synthesis of the compounds was per-
formed by the formation of bis-imines. Derivatives 1–7 were ob-
1
3
*
Corresponding author. Tel.: +54 1149648251.
E-mail address: anabruno@ffyb.uba.ar (A.M. Bruno).
0
968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.069

8
004
M. Bollini et al. / Bioorg. Med. Chem. 16 (2008) 8003–8010
tained starting from 6-methoxy-2-naphthaldehyde or 2-naphthal-
dehyde and the corresponding diamines. All the imines were iso-
lated, crystallized, and characterized by their spectroscopic
properties (IR, NMR) (Scheme 1).
derivatives using thionyl chloride. Finally, alkylation proceeded
under reflux with the corresponding diamine in DMF (Scheme 2).
All the bis-amine compounds were characterized by spectral
data analysis that confirmed the assigned structures.
In the second step, we attempted the synthesis of the secondary
amine by catalytic hydrogenation over Pd/C (10%) in ethanol at a
hydrogen pressure of 0.14 MPa at room temperature, but we recov-
ered the initial imine. However, subsequent reduction of these imi-
2.2. Antineoplastic activity
All the final compounds were evaluated for antiproliferative
properties following the NCI in vitro protocols. They were assayed
in vitro against a panel of approximately 60 human tumor cell
lines, derived from nine cancer types: lung, colon, CNS, melanoma,
ovarian, renal, prostate, breast, and leukemia. Compounds were
tested at five concentrations at 10-fold dilutions starting from
nes with NaBH
4
in methanol14 gave the desired compounds 8–14
in good yields (Scheme 1).
Compounds 15 and 16 were synthesized by alkylation of the
corresponding diamine owing to the impossibility to carry out
reductive amination. As the desired halogenated compounds were
not available, 2-naphthaldehyde and 6-methoxy-2-naphthalde-
hyde were reduced with NaBH in methanol to give the corre-
4
sponding alcohols. After that, we obtained the halogenated
ꢀ4
10 M. A 48 h continuous drug exposure protocol was used and
sulforhodamine B (SRB) protein assay was used to estimate cell
1
5
growth. The antitumoral activity of tested compounds is given
R
R
NaBH4
CH
N
X N CH
CH
CH
2
NH X NHCH
R
2
11 R= OCH
3
R
R
R
X: (CH2)3
N N (CH2)3
4
R= OCH3
H N (CH )
2
N
N
(CH ) NH2
2 3
2 3
5
R= OCH
R= H
3
7
CHO
H
2
N
Z NH
2
N
Z N
CH
Z: (CH ) O(CH ) O(CH )
2 2
2
2
2 2
R
R= H
R= OCH3
NaBH4
H N (CH ) NH2
2
2 n
n= 6, 8, 12
R
R
1
2 R= OCH
3
CH NH Z NHCH2
14 R= H
2
CH N (CH2)n N CH
R
R
1
2
3
6
R= OCH ; n = 6
3
R= OCH ; n = 8
3
R= OCH ; n = 12
3
R= H; n = 6
NaBH
4
R
CH NH (CH ) NHCH
2
2 n
2
R
8
9
R= OCH ; n: 6
3
R= OCH ; n: 8
3
1
1
0 R= OCH ; n: 12
3
3 R= H; n: 6
Scheme 1. Synthesis of compounds 1–14.

M. Bollini et al. / Bioorg. Med. Chem. 16 (2008) 8003–8010
8005
CHO
CH
OH
2
CH Cl
2
NaBH4
Cl SO
2
R
CH OH
R
R
3
R= OCH3
R= H
15a R= OCH3
16a R= H
15b R= OCH
3
16b R= H
HN
NH
R
R
H C
2
N
N
CH2
1
1
5 R= OCH3
6 R= H
Scheme 2. Synthesis of compounds 15–16.
by three parameters for each cell line: log GI50 value (GI50 = molar
concentration of the compound that inhibits 50% net cell growth),
log TGI value (TGI = molar concentration of the compound leading
to the total inhibition), and log LC50 value (LC50 = molar concentra-
tion of the compound leading to 50% net cell death). Furthermore, a
mean graph-midpoint (MG-MID) is calculated for each response
parameter, which indicates the average sensitivity of all tested cell
lines to each tested compound. The results collected in Table 1
Table 1
Average values (MG-MID) for in vitro antitumor activity on 60 human cell lines
Linker
Linker
R
R
R
R
Series I
Series II
Compound (NSC code)
R
Linker
MG-MIDa
log TGIc
Panel sensitivity
log GI50b
log LC50d
Series I
8
9
1
(676426)
(676427)
0 (676428)
O–CH
O–CH
O–CH
3
3
3
–NH(CH
–NH(CH
–NH(CH
2
)
2
)
2
)
6
NH–
NH–
12NH–
ꢀ6.22
ꢀ5.94
ꢀ4.81
ꢀ5.76
ꢀ5.62
ꢀ4.53
ꢀ5.39
ꢀ5.20
ꢀ4.26
Leukemia, colon, breast
Leukemia, colon, CNS, prostate
Leukemia, colon, CNS
8
1
1 (676429)
O–CH
3
HN(CH2)3
N
N
(CH ) NH
ꢀ5.96
ꢀ5.61
ꢀ5.25
Leukemia, colon
2 3
1
1
2 (695801)f
4 (695800)g
O–CH
H
3
NH(CH
NH(CH
2
)
)
2
O(CH
O(CH
2
)
)
2
O(CH
2
)
)
2
NH
NH
ꢀ6.14
ꢀ5.77
ꢀ5.72
ꢀ5.43
ꢀ5.28
ꢀ5.00
Leukemia, colon, melanoma, breast, CNS
Leukemia, colon
2
2
2
2
O(CH
2
2
1
5 (683792)
6 (696885)
O–CH
3
N
N
N
ꢀ5.53
ꢀ5.54
ꢀ5.01
ꢀ5.08
ꢀ4.39
ꢀ4.60
Leukemia, melanoma, colon, breast
Melanoma, colon, leukemia
1
H
N
Series II11,12
629732
629733
629734
H
H
H
–NH(CH
–NH(CH
–NH(CH
2
)
)
)
6
NH–
NH–
12NH–
ꢀ5.27
ꢀ5.62
ꢀ5.66
ꢀ4.91
ꢀ5.28
ꢀ5.36
ꢀ4.53
ꢀ4.94
ꢀ5.07
Leukemia, colon, melanoma
Leukemia, colon, melanoma
Leukemia, colon, melanoma
2
2
8
6
29735
H
H
HN(CH2)3
N
N
(CH ) NH
ꢀ5.18
ꢀ4.71
ꢀ4.36
Leukemia, melanoma, colon
2 3
6
29736
N
N
ꢀ5.71
ꢀ7.32
ꢀ6.64
ꢀ5.37
ꢀ6.16
ꢀ5.47
ꢀ5.02
ꢀ5.14
ꢀ4.68
Leukemia, melanoma, colon, breast, lung
Leukemia, lung, CNS, renal
Leukemia, CNS, renal, lung
e
Mitox
m-AMSA
a
MG-MID mean graph-midpoint: arithmetical mean value for all tested cancer cell lines.
b
c
d
e
f
The response parameter: log GI50 is interpolated value representing the molar concentration at which percentage growth is +50.
The response parameter: log TGI is interpolated value representing the molar concentration at which percentage growth is 0.
The response parameter: log LC50 is interpolated value representing the molar concentration at which percentage growth is ꢀ50.
Mitox, mitoxantrone.
As hydrochloride.
As hydrochloride.
g

8
006
M. Bollini et al. / Bioorg. Med. Chem. 16 (2008) 8003–8010
refer the mean graph-midpoint (MG-MID) values at each of princi-
pal response parameters: GI50, TGI, and LC50
Table 3
The in vivo activity (hollow fiber assay) for compounds 8, 12, 15, 16
.
Compound (NSC code)
(676426)
IP scorea
SC scoreb
Total score
Cell kill
Tested compounds demonstrated a relatively broad spectrum of
tumor cell growth inhibition. Compounds displayed selectivity on
panel of leukemia, melanoma, colon, breast, and prostate cancers
tumor cell lines. In series I, an increase in the number of methylene
units between nitrogen atoms resulted in a significant drop in anti-
cancer potency. In series II, antineoplastic activity did not decrease
with the enlargement of the linker chain.
Introduction of a methoxy group at 6-position of naphthalene
chromophores in compound 14 (NSC 695800) results in a signifi-
cant increase of antineoplastic activity.
8
4
6
8
0
4
6
6
4
8
12
14
4
N
N
Y
c
12 (695801)
15 (683792)
16 (696885)
N
% T/C, reduction percentage of the viable cell mass in treated mice relative to a
control group.
a
IP score, % T/C for each of two compounds doses for intraperitoneal samples.
SC score, % T/C for each of two compounds doses for subcutaneous samples.
As hydrochloride.
b
c
Even though the compound 15 (NCS 696885, series I) has a GI50
lower than the compound NCS 629736 (series II), this last one
shows an LC50 of the same order as GI50. Furthermore, compound
belonging to the standard agents database,18 the compound of
interest may have the same mechanism of action as those agents
with known mechanism. A correlation coefficient of 0.55–0.6 is
considered the lowest correlation that suggests a relationship with
1
5 is of special interest because there is a substantial difference be-
tween the cytostatic and the cytotoxic activities.
Table 2 shows the antitumor activity of compound 15 in most
sensitive human cell lines.
19
another compound. Using GI50 values of compound 15 (NSC
6
83792) as seed, COMPARE analysis showed that compounds in
Because of this promising in vitro activity, compounds 8 (NSC
Table 4 had a Pearson’s correlation coefficient (PCC) <0.6. The
weakly correlated compounds, shows in Table 4, are cytotoxic
through diverse mechanisms of action, including DNA alkylation
6
76426), 12 (NCS 695801), 15 (NSC 683792), and 16 (NCS
6
96885) were selected at NCI Biological Testing Branch for preli-
16
minary hollow fiber in vivo testing (see Section 3). Among the
twelve cell lines forming the standard panel routinely used in such
assays, compound 15 showed IP+SC score of 14, an SC score of 6,
but demonstrated a net cell kill. Table 3 shows the results of the
four compounds tested.
(
CCNU), induction of apoptosis (anguidine), and alteration of cell
cycle progression involving the MAPK pathway (tetrandine). All
in all the COMPARE analysis for the representative compound 15
against the standard agent database showed poor or no correlation
indicating that mechanism of action for the novel bis-aminometh-
ylnaphthalenes may differ from that of the standard antitumor
drugs. Therefore, antitumoral activity of the novel compound
may be caused by a new and unknown mechanism.
1
7
A COMPARE analysis was performed with the more active
compound 15 to investigate whether it resembles anticancer drugs
of the NCI standard agent database and to probably predict its
mechanism of action. The COMPARE algorithm was developed to
determine the degree of similarity of mean graph fingerprints ob-
tained from the in vitro anticancer screen with patterns of activity
of standard agents. The hypothesis is that if the data pattern of a
compound correlates well with the data pattern of compounds
2
.3. DNA binding properties
As the bis-aminomethylnaphthalenes synthesized previously11
displayed close affinity to calf thymus DNA, we expected the syn-
thesized compounds to conserve this property.
All the final products were tested for their ability to bind DNA.
The binding capacity of these compounds was evaluated by mea-
suring the hypochromic and bathochromic effects of their absor-
bance in the UV spectra. The typical experiment was enhanced
by means of a slow rotation of DNA/drug mixture stirring, in a
5:1 ratio for 24 h. The procedure was validated by repeating assays
with well-known intercalating agents (m-AMSA and mitoxan-
trone) and a compound which binds closely in the minor groove
Table 2
Antitumor activity of compound 15 in most sensitive human tumor cell lines
2
0
Panel/cell line
log10 GI50
Leukemia
HL-60(tb)
K-562
MOLT-4
RPMI-8226
SR
ꢀ5.58
ꢀ6.30
ꢀ5.66
ꢀ5.76
ꢀ5.91
(
bis-benzimide, Hoechst No. 33258). The degree of interaction
was expressed by the ratio between the final absorbance area after
Colon cancer
COLO 205
HCT-116
HT29
KM12
SW-620
ꢀ5.79
ꢀ5.46
ꢀ5.47
ꢀ5.49
ꢀ5.50
Table 4
Compare correlation coefficients (PCC) using GI50 values of compound 15 (NSC
683792) as seed
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
ꢀ5.55
ꢀ5.64
ꢀ5.61
ꢀ5.19
ꢀ5.58
ꢀ5.83
ꢀ5.69
ꢀ5.72
Rank
NSC
PCC
Num common cell lines
Compound
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
68075
192965
77037
322921
45441
118994
180973
83265
303861
141537
409962
133100
332598
95441
0.600
0.595
0.488
0.485
0.481
0.465
0.464
0.460
0.459
0.458
0.412
0.408
0.408
0.405
0.404
50
60
60
59
49
60
60
47
60
60
60
60
59
60
60
Thalicarpine
Spirogermanium
D-Tetrandrine
Hoechst dye
CCNU
IdA
Tamoxifen
Tritylthioalanine
L-Cysteine
Anguidine
Carmustine
Rifamycin
Rhizoxin
Semustine
Pancratistatin
Breast cancer
MCF7
MCF7/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
MDA-N
T-47D
ꢀ5.28
ꢀ4.88
ꢀ5.48
ꢀ4.97
ꢀ5.72
ꢀ5.77
ꢀ5.75
349156

M. Bollini et al. / Bioorg. Med. Chem. 16 (2008) 8003–8010
8007
2
4 h (a24) and that of the compound at the same concentration (a
0
),
When plotting absorbance against the different ratios of drug–
DNA mixture at 1 and 24 h the observed process is in one step
which indicates a single mode of interaction of compound 15 with
centered at maximal absorbance. Values of 1 indicate a total lack of
affinity and value 0 shows that the whole compound was bound to
DNA. The coefficient a24/a
2
1
0
values obtained are summarized in Ta-
DNA (Fig. 3).
ble 5.
A medium correlation between degree of affinity and antineo-
plastic activity was observed (R = 0.62), so a different mechanism
2
Under such experimental conditions, all these compounds
exhibited a bathochromic and hypochromic effects. The DNA bind-
ing assay showed that compounds tested are excellent DNA ligands
with higher affinity than m-AMSA and bis-benzimide, except com-
pounds 10 and 16.
of action is suggested, like an inhibition of specific enzymes such as
2
2
DNA-topoisomerases.
2.4. Cell cycle effects
A large number of relatively small organic molecules are known
to bind to DNA by various mechanisms such as intercalation and
ionic attraction. It is important to characterize such phenomena
because the mechanism of action of many carcinogenic, mutagenic,
and antitumor substances presumably relates to binding with
DNA. Additionally, as the interaction is best described as a dynamic
equilibrium, we performed a kinetic study of compound 15 using
different drug/DNA ratios (1:2; 1:2.5; 1:3; 1:3.5; 1:4 and 1:5). This
assay showed that the rate of the process was proportional to rel-
ative concentration of drug/DNA ratio. Moreover, Figure 2 shows
that in drug/DNA (1:2) ratio, the rate of occupying the sites of un-
ion to DNA is faster than in the others ratios.
In the search of a possible mechanism of action responsible for
compound 15 antitumor activity we have investigated its effect on
the cell cycle by fluorescence activated cell sorting (FACS). TSU-Pr1
cell line derived from prostate carcinoma was used in this assay.
Cells were treated with 10 lM of compound 15 and after 48 h fixed
and labeled with propidium iodide. The different phases of cell cy-
cle were analyzed by cytometry. TSU-Pr1 cells treated with com-
pound 15 did not show changes of the cell cycle profiles as
shown by the histograms depicted in Figure 4. Flow cytometry of
propidium labeled cells indicates that treatment with compound
15 does not provoke a significative induction of cell apoptosis.
In conclusion, we have synthesized a series of bis-aminometh-
ylnaphthalenes. The new compounds were evaluated for their abil-
ity to bind DNA and were assayed for antiproliferative properties
both in vitro and in vivo. Compound 15 exhibited remarkable anti-
tumor activity, being an appropriate ligand (in structure and func-
tion) for interaction with DNA, and represents a novel template for
Table 5
DNA affinity assay
a
Compound (NSC code)
a24/a
0
8
9
1
1
1
1
1
1
1
(676426)
(676427)
0.51
0.34
0.77
0.52
0.47
0.53
0.47
0.51
0.61
0.54
0.00
0.57
0 (676428)
1 (676429)
2 (695801)
anticancer chemotherapy and could serve as
compound.
a new lead
b
c
3 (—)
d
4 (695800)
5 (683792)
6 (696885)
m-AMSA (249992)
Mitoxantrone (301739)
Bis-benzimide (322921)
a
0
a24, final absorbance area (DNA-compound) after 24 h; a , initial absorbance
area (compound) at the same concentration.
b
As hydrochloride.
Not studied by NCI.
As hydrochloride.
c
d
Figure 3. Absorbance (kmax = 232 nm) versus different ratios of drug–DNA at 1 and
4 h.
2
Figure 4. DNA fragmentation analysis for determination of apoptosis by FACS. TSU-
Pr1 cells were treated with compound 15 and DNA content in the cells was
determined after 48 h. Drug treated cells did not show any change in the histogram
indicating that compound 15 is not able to induce apoptosis.
Figure 2. Absorbance (kmax = 232 nm) versus times of drug–DNA (–r– 1:2;
:2.5; N 1:3; 1:3.5; 1:4; -.-r-.- 1:5).
1

8
008
M. Bollini et al. / Bioorg. Med. Chem. 16 (2008) 8003–8010
3
3
. Experimental
The mixture was stirred at reflux temperature for 3 h. 3/4 parts
of solvent were evaporated under reduced pressure; the white
crude product was filtered off and recrystallized from EtOH. Yield:
.1. Chemistry
5
8.9%, mp: 121–123 °C.
IR (KBr): 2900, 2850, 1595, 1100, 850, 780 cm 1. 1H NMR
(CDCl ) d: 8.30 (s, 2H, CH@N), 7.61–7.65 (m, 6H, b-naphthyl-H),
ꢀ
Melting points were determined in a capillary with an Electro-
thermal 9100 SERIES-Digital apparatus and are uncorrected. IR
spectra were recorded with a FT Perkin-Elmer Spectrum One from
KBr discs. UV spectra were measured with a Jasco V-570 UV/vis/
3
7.30 (dd, J = 8.0, 1.2 Hz, 2H, b-naphthyl-H), 7.08 (d, J = 6.9 Hz, 2H,
b-naphthyl-H), 7.06 (d, J = 7.0 Hz, 2H, b-naphthyl-H), 3.82 (s, 6H,
1
NIR spectrophotometer. H NMR (200 MHz) spectra were obtained
O–CH
HC@N–CH
3
), 3.60 (t, J = 4.5 Hz, 8H, O–CH –), 2.82 (t, J = 5.3 Hz, 4H,
32 2 4
–). Anal. Calcd for C30H N O : C, 74.18; H, 6.23; N,
2
with a Bruker spectrometer at room temperature with tetrameth-
ylsilane as internal standard. Chemical shifts (d) are reported in
ppm and coupling constants (J) in Hz. Elemental analysis was car-
ried out in our laboratories with a Coleman Analyzer.
2
5.97. Found: C, 73.98; H, 6.40; N, 5.7.
0
3.1.1.6. N,N -Bis-naphthalen-2-ylmethylene-hexane-1,6-diamine
(6). To a solution of 2-naphthaldehyde (4.6 g, 29.7 mmol) in 20 mL
3
.1.1. General procedure for the synthesis of compounds 1–4
Toa solutionof 6-methoxy-2-naphthaldehyde(4.6 g, 24.7 mmol)
of dry EtOH, a solution of 1,6-hexanediamine (1.72 g, 14.6 mmol)
in 10 mL of dry EtOH was added dropwise. The reaction mixture
was heated at reflux for 15 min. The formed precipitate was fil-
tered off and recrystallized from EtOH. Yield: 95%, mp: 137–
in 20 mL of dry EtOH, the appropriate diamine (12.6 mmol) in 10 mL
ofdry EtOHwas added dropwise. Thereaction mixturewas heated at
reflux for 30 min; the crude products were filtered off and were crys-
tallized from an appropriate solvent.
139 °C.
IR (KBr): 2900, 1595, 850, 780 cm 1. 1H NMR (DMSO-d
ꢀ
) d: 8.06
6
(
s, 2H, CH@N), 7.82–7.90 (m, 8H, b-naphthyl-H), 7.50–7.68 (m, 6H,
b-naphthyl-H), 3.00 (t, J = 6.1 Hz, 4H, HC@N–CH –), 0.90–1.44
m, 8H, –CH ). Anal. Calcd for C28 : C, 85.67; H, 7.19; N,
.14. Found: C, 85.40; H, 7.40; N, 6.90.
0
3
.1.1.1.
N,N -Bis((6-methoxynaphthalen-2-yl)methylene)hex-
2
ane-1,6-diamine (1). Yield: 96%, white solid, mp: 178–180 °C (eth-
(
7
2
28 2
H N
ꢀ
1 1
anol). IR (KBr): 2950, 2890, 1590, 1100, 850, 780 cm
CDCl ): d 8.20 (s, 2H, CH@N), 7.66–7.73 (m, 6H, b-naphthyl-H),
.31 (dd, J = 8.3, 1.7 Hz, 2H, b-naphthyl-H), 7.06 (d, J = 6.7 Hz, 2H,
b-naphthyl-H), 7.04 (d, J = 6.7 Hz, 2H, b-naphthyl-H), 3.57 (s, 6H,
O–CH ), 2.56 (t, J = 6.9 Hz, 4H, HC@N–CH –), 1.20–1.60 (m, 8H,
–CH ). Anal. Calcd for C30 : C, 79.61; H, 7.13; N, 6.19.
. H NMR
(
7
3
0
3
.1.1.7. 2,2 -(Ethane-1,2-diylbis(oxy))bis(N-((naphthalen-2-yl)-
methylene)ethanamine) (7). A solution of 2-[2-(2-aminoeth-
oxy)-ethoxy]-ethylamine (0.7 mL, 4.7 mmol) in 10 mL of dry EtOH
was added dropwise to a solution of 2-naphthaldehyde (1.5 g,
3
2
(
)
2 4
32 2 2
H N O
Found: C, 79.95; H, 7.01; N, 6.20.
8
.1 mmol) in dry EtOH (20 mL). The mixture was stirred at reflux
temperature for 3 h. 3/4 of solvent volume was evaporated under re-
duced pressure; the product was filtered, washed with EtOH. Yield:
0
3
1
.1.1.2. N,N -Bis((6-methoxynaphthalen-2-yl)methylene)octane-
,8-diamine (2). Yield: 97%, white solid, mp: 132–138 °C (ethanol).
6
0%, mp: 108–110 °C.
ꢀ
1 1
IR (KBr): 2950, 2840, 1595, 1300, 850, 780 cmꢀ1. 1H NMR (DMSO-
IR (KBr): 2900, 2890, 1595, 1100, 850, 780 cm . H NMR (CDCl
3
): d
.20 (s, 2H, CH@N), 7.62–7.80 (m, 6H, b-naphthyl-H), 7.51 (dd, J = 8.1,
.4 Hz, 2H, b-naphthyl-H), 7.10 (d, J = 6.6 Hz, 2H, b-naphthyl-H), 7.00
), 2.64 (t, J = 7.0 Hz,
). Anal. Calcd for
: C, 79.96; H, 7.55; N, 5.83. Found: C, 79.82; H, 7.81; N, 5.63.
8
1
(
d
(
8
6
)d: 8.00(s, 2H, CH@N), 7.70–7.90(m, 8H, b-naphthyl-H), 7.50–7.60
m, 6 H, b-naphthyl-H), 3.20–3.24 (m, 4H, O–CH –CH –O), 3.00 (m,
: C, 79.22; H,
2
2
d, J = 7.0 Hz, 2H, b-naphthyl-H), 3.90 (s, 6H, O–CH
3
H, HC@N–CH
2 2 28 2 2
–CH –O–). Anal. Calcd for C28H N O
4
2 2 6
H, HC@N–CH –), 1.20–1.49 (m, 12H, (–CH )
6
.65; N, 6.6. Found: C, 79.12; H, 6.81; N, 6.44.
32 36 2 2
C H N O
3
.1.2. General procedure for the synthesis of compound 8–12
NaBH (1.4 g, 39.6 mmol) was added in small portions over sev-
eral minutes to a suspension of corresponding compounds 1–7
3.15 mmol) in 10 mL of dry methanol. The mixture was heated
at reflux for 3 h and then was allowed to come to room tempera-
ture and stirred overnight. The products were filtered, washed with
0
3
.1.1.3. N,N -Bis((6-methoxynaphthalen-2-yl)methylene)dode-
4
cane-1,12-diamine (3). Yield: 76%, white solid, mp: 139–141 °C
ꢀ1
1
(
ethanol). IR (KBr): 2950, 2850, 1595, 1100, 850, 780 cm . H
(
3
NMR (CDCl ): d 8.00 (s, 2H, CH@N), 7.60–7.79 (m, 6H, b-naphthyl-
H), 7.46 (dd, J = 8.3, 1.2 Hz, 2H, b-naphthyl-H), 7.10 (d, J = 6.6 Hz,
H, b-naphthyl-H), 6.90 (d, J = 6.9 Hz, 2H, b-naphthyl-H), 3.95
s, 6H, O–CH ), 2.62 (t, J = 6.9 Hz, 4H, HC@N–CH –), 1.20–1.52
m, 20H, (–CH 10). Anal. Calcd for C36 : C, 80.56; H, 8.26;
2
2
H O, and were crystallized from an appropriate solvent.
(
(
3
2
2
)
H
44
N
2
O
2
3
1
.1.2.1. N,N -Bis-(6-methoxy-naphthalen-2-ylmethyl)-hexane-
,6-diamine (8). Compound 8 was recrystallized from cyclohexane/
0
N, 5.22. Found: C, 80.43; H, 8.05; N, 5.38.
benzene (1:1) (1.03 g, 79% yield), mp: 117–119 °C.
0
IR (KBr): 3330, 2900, 2850, 1100, 850, 780 cmꢀ1. 1H NMR
3
2
.1.1.4. 3,3 -(Piperazine-1,4-diyl)bis(N-((6-methoxynaphthalen-
-yl)methylene)propan-1-amine) (4). Yield: 86%, white solid,
(
CDCl
Hz, 2H, b-naphthyl-H), 7.04 (d, J = 6.9 Hz, 2H, b-naphthyl-H), 7.00
d, J = 7.0 Hz, 2H, b-naphthyl-H), 3.83 (s, 6H, O–CH ), 3.80 (s, 4H,
naphthyl-CH –N), 2.52 (t, J = 6.8 Hz, 4H, N–CH –), 1.25–1.47 (m,
and NH). Anal. Calcd for C30 : C, 78.91; H,
3
) d: 7.30–7.60 (m, 6H, b-naphthyl-H), 7.10 (dd, J = 8.0, 1.6
mp: 165–167 °C (ethanol). IR (KBr): 2900, 2850, 1590, 1100, 850,
ꢀ
1 1
7
3
80 cm . H NMR (CDCl ) d: 8.10 (s, 2H, CH@N), 7.61–7.65 (m, 6H,
(
3
b-naphthyl-H), 7.32 (dd, J = 8.3, 1.7 Hz, 2H, b-naphthyl-H), 7.08 (d,
J = 6.9 Hz, 2H, b-naphthyl-H), 7.06 (d, J = 7.0 Hz, 2H, b-naphthyl-H),
.85 (s, 6H, O–CH
m, 12H, piperazine-H and –CH
CH ). Anal. Calcd for C34 : C, 76.09; H, 7.51; N, 10.44. Found:
2
2
1
7
0H, –(CH
2
)
4
36 2 2
H N O
3
(
–
3
), 2.62 (t, J = 6.8 Hz, 4H, HC@N–CH
2
–), 2.27–2.38
.95; N, 6.13. Found: C, 78.73; H, 8.10; N, 6.28.
2
-piperazine) 1.60–1.72 (m, 4H,
2
H
40
N
4
O
2
3
.1.2.2. N,N -Bis((6-methoxynaphthalen-2-yl)methyl)octane-1,8-
0
C, 75.88; H, 7.69; N, 10.58.
diamine (9). Compound9 wascrystallizedfrommethanol/ciclohex-
ane (1:1) (1.25 g, 82% yield), mp: 93–94 °C.
0
IR (KBr): 3330, 2900, 2850, 1100, 850, 780 cmꢀ1. 1H NMR
3
.1.1.5. 2,2 -(Ethane-1,2-diylbis(oxy))bis(N-((6-methoxynaphth-
alen-2-yl)methylene)ethanamine) (5). A solution of 2-[2-(2-
aminoethoxy)-ethoxy]-ethylamine (0.7 mL, 4.7 mmol) in 10 mL of
dry EtOH was added dropwise to a solution of 6-methoxy-2-naph-
thaldehyde (1.5 g, 8.1 mmol) in dry EtOH (20 mL).
(
CDCl
Hz, 2H, b-naphthyl-H), 7.00 (d, J = 7.0 Hz, 2H, b-naphthyl-H), 6.90
d, J = 7.0 Hz, 2H, b-naphthyl-H), 3.98 (s, 6H, O–CH ), 3.85 (s, 4H,
naphthyl-CH –N), 2.68 (t, J = 6.8 Hz, 4H, N–CH –), 1.25–1.47 (m,
3
) d: 7.30–7.60 (m, 6H, b-naphthyl-H), 7.18 (dd, J = 8.1, 1.6
(
3
2
2

M. Bollini et al. / Bioorg. Med. Chem. 16 (2008) 8003–8010
8009
1
4H, –(CH
2
)
6
and NH). Anal. Calcd for C32
H
40
N
2
O
2
: C, 79.30; H,
2 2
CH Cl was added gradually at 0 °C. Afterwards, the mixture was
8
.32; N, 5.78. Found: C, 79.12; H, 8.49; N, 5.89.
heated at reflux for 1 h and the solvent was evaporated to dryness
under reduced pressure. The residue was washed with cyclohexane
and dried to give 15b, mp: 57–59 °C (mp lit.: 58–59 °C). and 16b,
mp: 48–49 °C (mp lit.: 48–49 °C).
A solution of 4,4 -bipiperidine (1.3 g, 7.7 mmol) in 5 mL of dry
EtOH was added dropwise to a solution of the select compound
0
3
1
.1.2.3. N,N -Bis((6-methoxynaphthalen-2-yl)methyl)dodecane-
,12-diamine (10). Compound 10 was recrystallized from etha-
0
nol (1.4 g, 82% yield), mp: 110–115 °C.
IR (KBr): 3330, 2950, 2890, 1100, 850, 780 cm 1. 1H NMR
CDCl ) d: 7.30–7.60 (m, 6H, b-naphthyl-H), 7.15 (dd, J = 8.1 Hz,
J = 1.6 Hz, 2H, b-naphthyl-H), 7.10 (d, J = 6.9 Hz, 2H, b-naphthyl-
H), 7.00 (d, J = 7.0 Hz, 2H, b-naphthyl-H), 3.95 (s, 6H, O–CH ),
–),
ꢀ
(
3
2 3
15b or 16b (15 mmol) in 20 mL of dry EtOH and K CO (0.7 g,
7.7 mmol). The mixture was heated at reflux for 5 h. The white pre-
cipitate formed was filtered to afford the crude product. Purifica-
tion was effected as noted.
3
3
1
.90 (s, 4H, naphthyl-CH
.25–1.47 (m, 22H, –(CH
2
–N), 2.65 (t, J = 6.9 Hz, 4H, N–CH
48 2 2
10 and NH). Anal. Calcd for C36H N O :
2
2
)
C, 79.96; H, 8.95; N, 5.18. Found: C, 79.78; H, 9.05; N, 5.0.
0
0
3
.1.3.1. 1,1 -Bis-(6-methoxy-naphthalen-2-ylmethyl)-[4,4 ]-bipi-
peridine (15). Compound 15 was recrystallized from acetone
1.05 g, 66%), mp: 212–213 °C.
IR (KBr): 3300, 2950, 2850, 1100, 850, 780 cm . H NMR (CDCl
0
3
2
.1.2.4. 3,3 -(Piperazine-1,4-diyl)bis(N-((6-methoxynaphthalen-
-yl)methyl)propan-1-amine) (11). Compound 11 was recrys-
(
ꢀ1 1
3
)
tallized from ethanol (1.02 g, 60% yield), mp: 75–80 °C.
d: 7.61–7.65 (m, 6H, b-naphthyl-H), 7.38 (dd, J = 8.0, 1.6 Hz, 2H, b-
naphthyl-H), 7.08 (d, J = 6.9 Hz, 2H, b-naphthyl-H), 7.06
d, J = 7.0 Hz, 2H, b-naphthyl-H), 3.85 (s, 6H, O–CH
ꢀ1 1
3
IR (KBr): 3330, 2950, 2800, 1100, 850, 780 cm . H NMR (CDCl )
d: 7.60–7.66 (m, 6H, b-naphthyl-H), 7.31 (dd, J = 8.4, 1.7 Hz, 2H, b-
naphthyl-H), 7.19 (d, J = 6.9 Hz, 2H, b-naphthyl-H), 7.09 (d,
(
3
), 3.80 (s, 4H,
H
CH ), 2.27–2.38 (m, 8H, piperazine-H) 1.65 (d, 2H,
2
N
N
),
J = 6.9 Hz, 2H, b-naphthyl-H), 3.98 (s, 6H, O–CH
thyl-CH –N), 2.62 (m, 16H, N–CH – and CH -piperazine), 1.90 (s, 2H,
NH), 1.60–1.72 (m, 4H, –CH ). Anal. Calcd for C34 : C, 75.52;
3
), 3.83 (s, 4H, naph-
H
2
2
2
1
8
.10–1.27 (m, 8H, piperazine-H). Anal Calcd for C34
0.28; H, 7.28; N, 5.51. Found: C, 80.07; H, 7.44; N, 5.79.
40 2 2
H N O
: C,
2
44 4 2
H N O
H, 8.20; N, 10.36. Found: C, 72.30; H, 8.4; N, 10.46.
0
0
3
(
.1.3.2. 1,1 -Bis-(naphthalen-2-ylmethyl)-[4,4 ]-bipiperidine
0
3
.1.2.5. 2,2 -(Ethane-1,2-diylbis(oxy))bis(N-((6-methoxynaphth-
16). Compound 16 was recrystallized from acetone (1.05 g, 75%),
alen-2-yl)methyl)ethanamine) (12). Compound 12 was crystal-
lized from acetone, mp: 200–202 °C (d). 12 was obtained as
hydrochloride using ethanol/HCl to give 1.42 g, 86% yield, mp 222–
23 °C. IR (KBr) (as free base): 3320, 2950, 2820, 1260, 850,
3
80 cm . H NMR (CDCl ) (as free base) d: 7.80–7.84(m, 6H, b-naph-
thyl-H), 7.40 (dd, J = 7.9, 1.5 Hz, 2H, b-naphthyl-H), 7.31 (d, J = 6.9 Hz,
H, b-naphthyl-H), 7.20 (d, J = 6.9 Hz, 2H, b-naphthyl-H), 3.98 (s, 6H,
O–CH ),3.90(s,4H, naphthyl-CH –N),3.1–3.15(m,8H, CH –O), 2.89–
.00 (m, 4H, N–CH –), 1.90 (s, 2H, NH). Anal. Calcd for C30 : C,
mp: 139–140 °C.
IR (KBr): 3300, 2950, 850, 780 cmꢀ1. 1H NMR (CDCl
3
) d: 7.61–7.65
(
m, 6H, b-naphthyl-H), 7.38–7.49 (m, 6H, b-naphthyl-H), 3.85 (s, 6H,
2
7
O–CH
3
2
), 3.80 (s, 4H, CH ), 1.96–2.27 (m, 8H, piperazine-H), 1.65 (d,
ꢀ1 1
H
2
H,
), 1.14–1.27 (m, 8H, piperazine-H). Anal. Calcd for
H
2
C
32
36 2
H N : C, 85.71; H, 8.04; N, 6.25. Found: C, 85.68; H, 8.07; N, 6.24.
3
2
2
3
7
2
36 2 4
H N O
3
.2. DNA affinity assay
3.74; H, 7.43; N, 5.73. Found: C, 73.54; H, 7.60; N, 5.86.
0
DNA solution: Calf thymus DNA (12.5 mg) was slowly magnet-
3
.1.2.6. N,N -Bis-((naphthalen-2-yl)methyl)hexane-1,6-diamine
ically stirred in 10 mM Tris–HCl buffer, pH 7.4 (5 mL), for 24 h at
4 °C. 0.6 mL was taken from this solution and diluted to 25 mL with
the same buffer.
(
13). Compound13 waswashedwithH
IR (KBr): 3330, 2900, 850, 780 cm
2
O(1.04 g, 84%), mp:74–76 °C.
ꢀ1
1
6
. H NMR (DMSO-d ) d:
7
.73–7.82 (m, 8H, b-naphthyl-H), 7.41–7.48 (m, 6H, b-naphthyl-
–N), 2.64 (t, J = 6.8 Hz, 4H, N–CH –),
and NH). Anal. Calcd for C28 : C,
The test compound solution was prepared at a 10 4 M concen-
ꢀ
H), 3.93 (s, 4H, naphthyl-CH
1
8
2
2
tration using a minimal volume of ethanol and then diluted by
.31–1.40 (m, 10H, (CH
2
)
4
32 2
H N
ꢀ5
adding water to a concentration of 2 ꢁ 10 M. Three milliliters
sample of this solution was mixed with 3 mL of the DNA solution.
The mixture was slowly rotated for 24 h and, then, its UV spectra
were recorded at 20 °C using a 1 cm cell.
4.80; H, 8.13; N, 7.06. Found: C, 84.65; H, 8.36; N, 7.25.
0
3
.1.2.7. 2,2 -(Ethane-1,2-diylbis(oxy))bis(N-(naphthalen-2-ylmethyl)-
ethanamine) (14). Compound 14 was recrystallized from ethanol/
HCl to give compound 14 as hydrochloride (1.28 g, 88%), mp: 232–
2
34 °C.
IR (KBr) (as free base): 3300, 2900, 2830, 1100, 850, 780 cmꢀ1.
) (as free base) d: 7.73–7.79 (m, 8H, b-naph-
3.3. Antineoplastic activity
1
H NMR (DMSO-d
6
3.3.1. Primary disease-oriented in vitro antitumor screen
The evaluation of antiproliferative activity was performed at the
National Cancer Institute (NCI, USA), following the well-known in
vitro disease-oriented primary antitumor screening against a panel
about 60 different cell lines derives from nine clinically isolated
thyl-H), 7.38–7.46 (m, 6H, b-naphthyl-H), 3.93 (s, 4H, naphthyl-
CH –N), 3.59–3.64 (m, 8H, CH –O), 2.80–2.85 (m, 4H, N–CH –),
.90 (s, 2H, NH). Anal. Calcd for C28 : C, 78.47; H,7.53; N,
.54. Found: C, 78.33; H, 7.22; N, 6.69.
2
2
2
1
6
32 2 4
H N O
1
5
human tumors. The tested compounds were assayed in range
ꢀ4
ꢀ8
3
1
.1.3. General procedure for the synthesis of compounds 15 and
6
To a solution of the appropriate aldehyde (10 mmol) in 30 mL of
of doses from 10 –10 . Results are reported as GI50 (concentra-
tion that produces a 50% growth inhibition on each cell line), TGI
(concentration that produces a total growth inhibition on each cell
line), and LC50 (concentration that produces cytotoxic effect in 50%
on each cell line) parameters in Table 1.
dry EtOH was added in a small portion 0.2 g NaBH
0
4
over 30 min at
°C. Afterwards the mixture was stirred at 50 °C for 3 h. Then, the
mixture was filtered, the alcohol solution was evaporated, and the
residue was washed with EtOH to yield the compound 15a, mp:
3.3.2. Preliminary in vivo antitumor test (hollow fiber assay)
Twelve cell lines derived from human tumors are cultivated in
polyvinylidene fluoride (PVDP) hollow fibers and a sample of each
cell line is implanted into each of two physiologic compartments
1
21–122 °C, and compound 16a, mp: 79–80 °C (mp lit.: 79–81 °C).
To the appropriate compound 15a or 16a (4.5 mmol) in 25 mL
2 2
of CH Cl , a solution of thionyl chloride (6.8 mmol) in 5 mL of

8
010
M. Bollini et al. / Bioorg. Med. Chem. 16 (2008) 8003–8010
(
intraperitoneal-IP and subcutaneous-SC) in athymic mice.16 Each
References and notes
test mouse receives a total of 6 fibers (3 intraperitoneally and 3
subcutaneously) representing 3 distinct cancer cell lines. Three
mice are treated with potential antitumor compounds at each of
two test doses by intraperitoneal route using a QDx4 treatment
schedule. Vehicle controls consist of 6 mice receiving the diluent
only. The fiber cultures are collected on the day following the last
day of treatment. To assess anticancer effects, viable cell mass is
determined for each of the cell lines using a formazan dye (MTT)
conversion assay. The data are reported as %T/C for each of the 2
compounds doses against each of the cell lines with separate val-
ues calculated for the intraperitoneal and subcutaneous samples.
To simplify evaluation, a points system has been adopted which re-
sults in a 50% or greater reduction in viable cell mass. Compounds
with a combined IP+SC score P20, a SC score P8 or a net cell kill of
one or more cell lines are referred for further xenograft testing.
1
2
3
.
.
.
Thurston, D. E. Br. J. Cancer 1999, 80, 65.
Hurley, L. H. Nat. Rev. Cancer 2002, 2, 188.
Charmantray, F.; Demeunynck, M.; Carrez, D.; Croisy, A.; Lansiaux, A.; Bailly, C.;
Colson, P. J. Med. Chem. 2003, 46, 967.
Pindur, U.; Kim, Y.-S.; Mehrabani, F. Curr. Med. Chem. 1999, 6, 29.
Braña, M. F.; Cacho, M.; Gradillas, A.; de Pascual-Teresa, B.; Ramos, A. Curr.
Pharm. Des. 2001, 7, 1745.
4
5
.
.
6. Doan Thi Mai, H.; Gaslonde, T.; Michel, S.; Tillequin, F.; Koch, M.; Bongui, J.-B.;
Elomri, A.; Seguin, E.; Pfeiffer, B.; Renard, P.; David-Cordonnier, M.-H. ; Tardy,
C.; Laine, W.; Bailly, C.; Kraus-Berthier, L.; Léonce, S.; Hickman, J.-A.; Pierré, A. J.
Med. Chem. 2003, 46, 3072.
7
.
Gregson, S. J.; Howard, P. W.; Hartley, J. A.; Brooks, N. A.; Adams, L. J.; Jenkins, T.
C.; Kelland, L. R.; Thurston, D. E. J. Med. Chem. 2001, 44, 737.
Cozzi, P. Farmaco 2001, 56, 57.
8
9
.
.
Waring, M. J.; Wakelin, L. P. G. Nature 1974, 252, 653.
10. Atwell, G. J.; Baguley, B. C.; Wilmanska, D.; Denny, W. A. J. Med. Chem. 1984, 29,
69.
1
1
1. Bruno, A. M.; Gaeta, J.; Gaozza, C. H. An. Quim. 1992, 88, 267.
2. Bruno, A. M.; Asís, S. E.; Lo Balbo, A.; Molina, D.; Conti, G. M.; Gaozza, C. H. Boll.
Chim. Farma. 1996, 135, 374.
3
.4. Cell cycle analysis
13. Sprung, M. M. Chem. Rev. 1940, 26, 297.
1
1
1
4. Horii, Z.; Sakai, T.; Inoi, T. J. Pharm. Soc. Jpn. 1955, 75, 1161.
5. Boyd, M. R. Princ. Pract. Oncol. 1989, 3, 1.
6. Hollingshead, M. G.; Alley, M. C.; Camalier, R. F.; Abbott, B. J.; Mayo, J. G.;
Malspeis, L.; Grever, M. R. Life Sci. 1995, 57, 131.
5
For flow cytometry of DNA content, 5 ꢁ 10 TSU-Pr1 cells in
exponential growth were treated at 10 M of compound 15 for
8 h. After the incubation period, cells were centrifuged, fixed in
l
1
7. (a) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero, D. A.;
Rubinstein, L.; Plowman, J.; Boyd, M. R. J. Natl. Cancer Inst. 1989, 81, 1088; (b)
http://dtp.nci.nih.gov/docs/compare/compare.html.
4
ice-cold ethanol (70%), treated with lysis buffer containing RNase,
and finally stained with propidium iodide. Samples were analyzed
18. Data concerning the NCI screening are accessible from the NCI via the Internet
from the following address: http://dtp.nci.nih.gov/webdata.html.
on a Becton Coulter Epics XL-MCL flow cytometer. For cell cycle
analysis, DNA histograms were analyzed using MultiCycleÒ for
19.
Weinstein, J. N.; Myers, T. G.; O’ Connor, P. M.; Friend, S. H.; Fornace, A. J.; Kohn,
K. W.; Fojo, T.; Bates, S. E.; Rubinstein, L. V.; Anderson, N. L.; Buolamwini, J. K.;
van Osdol, W. W.; Monks, A.; Scudiero, D. A.; Sausville, E. A.; Zaharevitz, D. W.;
Bunow, B.; Viswanadhan, V. N.; Johnson, G. S.; Wittes, R. E.; Paull, K. D. Science
Windows (Phoenix Flow Systems, San Diego, CA).
1
997, 275, 343.
Acknowledgment
2
0. (a) Asís, S. E.; Bruno, A. M.; Martínez, A. R.; Sevilla, M. V.; Gaozza, C. H.;
Romano, A. M.; Coussio, J. D.; Ciccia, G. Farmaco 1999, 54, 517; (b) Seshadri, R.;
Israel, M.; Pegg, W. J. Med. Chem. 1983, 26, 11.
1. Mc Pherson, D. D.; Pezzuto, J. M. J. Chromatogr. 1983, 281, 348.
2. Romano, A.; Mongelli, E.; Bruno, A. M.; Gaozza, C.; Coussio, J.; Ciccia, G.
Pharmazie 2000, 55, 612.
We thank the Development Therapeutics Program of the Na-
tional Cancer Institute, Bethesda, MD, for performing biological as-
says and SECyT-UBA for financial support.
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