Synthesis of polyamines from ethylenediamine and their platinum(II) complexes, potential antitumor agents

Article abstract of DOI:10.1002/ejic.200501024

This work describes the synthesis and characterization of five new amine ligands and also the preparation and characterization of their respective platinum(II) complexes by reaction with K₂PtCl₄ in water. These ligands were obtained

Full text of DOI:10.1002/ejic.200501024

FULL PAPER
DOI: 10.1002/ejic.200501024
Synthesis of Polyamines from Ethylenediamine and Their Platinum(II
Complexes, Potential Antitumor Agents
)
Mara Rubia Costa Couri,[a] Mauro Vieira de Almeida,[a] Ana Paula Soares Fontes,*[a]
Joana D’Arc Souza Chaves,[a] Eloi Teixeira Cesar,[b] Ricardo José Alves,[c]
Elene Cristina Pereira-Maia,[d] and Arlette Garnier-Suillerot[e]
Keywords: Amines / Platinum / Antitumor agents
This work describes the synthesis and characterization of five
new amine ligands and also the preparation and characteri-
zation of their respective platinum(II) complexes by reaction
with K2PtCl4 in water. These ligands were obtained by treat-
ment of different halides or epoxides with ethylenediamine.
for compounds 12 and 15 were significantly higher than
those found for the reference compounds, cisplatin, carbopla-
tin, or compound 9. This fact suggests that the formation of
adducts between compounds 12 and 15 and the putative
pharmacological target, DNA, is less favored. If these com-
Cytotoxic activity and cellular accumulation of three com- pounds bind more slowly to DNA, interaction with other in-
plexes were investigated in a human small-cell lung carci- tracellular ligands such as sulfur-containing molecules will
noma cell line and its cisplatin resistant subline. The intro-
duction of a spacer (cycle) between the two platinum atoms
leads to a significant decrease in cytotoxic activity. At equi-
toxic doses, the intracellular platinum concentrations found
become relevant and it may be the reason for the elevated
intracellular platinum concentrations.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
pletion of polyamines results in the disruption of a variety
of functions and may, in specific cases, result in cyto-
toxicity.[10] Inhibitors of the polyamine pathway, therefore,
have traditionally been developed as potential antitumor
agents.[11]
Polyamines have multiple cellular functions that can in-
terfere with cell growth and cell death. Therefore, their ana-
logs can play an important role in controlling cancer. Com-
pounds of this class can directly bind to DNA and modu-
late DNA–protein interactions. The production of hydrogen
peroxide from polyamine catabolism might also be respon-
sible for cell death or apoptosis.[12]
In addition to these facts, neutral ligands derived from
amines have been utilized to prepare platinum() complexes
since the discovery of the anticancer activity of cisplatin by
Rosenberg et al.[13] cis-Diaminedichloroplatinum() (cispla-
tin, CDDP)[14] is one of the most widely used and effective
oncological agents against cancers of the testicles, ovaries,
bladder, head, and neck.[14–16] It is also an important ad-
junct for cancers of the lung, cervix, and breast.[16] How-
ever, its clinical usefulness has frequently been limited by
severe side effects,[17–19] such as nephrotoxicity, ototoxicity,
and neurotoxicity, and by the emergence of cancer cells re-
sistant to cisplatin.
Tumor cells having the resistance phenotype exhibit a re-
duced sensitivity to cytotoxic drugs relative to sensitive
cells. The molecular basis for resistance has been the subject
of intensive research. Three main events are frequently ob-
served: (i) decreased accumulation of the drug; the intracel-
Introduction
The polyamines spermidine (1,8-diamino-4-azaoctane)
and spermine (1,12-diamino-4,9-diazadodecane), as well as
the precursor molecule putrescine (1,4-diaminobutane), are
polycation compounds that are found in significant
amounts in nearly every prokaryotic and eukaryotic cell
type.[1]
Many of these bases and their derivatives play key roles
in a number of biological processes and possess a variety of
pharmacological properties.[2] Polyamines may act as carci-
nogenesis promoting factors.[2,3] The connection between
polyamines and cancer growth has been established by bio-
logical and molecular techniques.[4] Polyamines have been
shown to accumulate in cancer tissues, and the concentra-
tion of polyamines and their derivatives increases in cancer
patient fluids.[5–9] These compounds also represent an im-
portant target for chemotherapeutic intervention, since de-
[a] Departamento de Química, Universidade Federal de Juiz de
Fora,
36036-900, Juiz de Fora-MG, Brazil
E-mail: ana.fontes@ufjf.edu.br
[b] Colégio de Aplicação João XXIII,
Juiz de Fora-MG, Brazil
[c] Faculdade de Farmácia, Universidade Federal de Minas Gerais,
Belo Horizonte-MG, Brazil
[d] Departamento de Química, Universidade Federal de Minas
Gerais,
Belo Horizonte-MG, Brazil
[e] Laboratoire de Physicochimie Biomoléculaire et cellulaire (ESA
7033), Université Paris Nord,
Bobigny, France
1868
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2006, 1868–1874

Synthesis of Polyamines from Ethylenediamine and Their Platinum() Complexes
FULL PAPER
lular drug concentration falls below the threshold necessary these compounds, one may observe IR spectra absorptions
for cytotoxic activity; (ii) increased levels of sulfur-contain- corresponding to νPt–N and νPt–Cl at 550 and 325 cm–1,
ing molecules, such as glutathione or metallothionein; these respectively, in addition to the absorptions observed for the
1
molecules could play a role in metal detoxification; and ligand. In the H NMR spectra there is a downfield shift
(iii) enhanced repair of DNA damage caused by cisplatin– for the signals corresponding to NH and NH2 relative to
DNA adducts.[20–22]
the free ligands. The 195Pt NMR spectra for the complexes
The need to overcome these problems, as well as to in- show only one signal, and the chemical shift values are ex-
crease the antitumor activity of the platinum complexes, led pected to be close to those of similar compounds in which
to the development of structurally different compounds, the platinum is coordinated to two chlorides and two nitro-
which might act in different ways against the tumor cells.[23] gens.[30]
The family of dinuclear complexes containing two mono-
functional platinum spheres linked by a variable diamine
chain has been extensively studied.[24] The first cytotoxic
dinuclear platinum compounds described in the literature
received special attention because they have shown a degree
of cytotoxicity comparable to that of cisplatin in sensitive
cell lines, but a significantly higher activity in acquired cis-
platin-resistant cell lines.[25,26]
Cell Growth Inhibition
The IC50 values obtained for complexes 9, 12, and 15 in
sensitive and resistant cells, together with the resistant fac-
tor (RF) and the intracellular platinum concentration, Ci,
are shown in Table 1. For the sake of comparison, cisplatin
and carboplatin data are also shown. The GLC4/CDDP
subline is 6.3-fold resistant to cisplatin and 1.4- to carbopla-
tin.
The cytotoxic activity of the compounds decreases in the
order 9 Ͼ 12 Ͼ 15. The sensitivity of the GLC4 cell line to
compound 9 is comparable to that of carboplatin, and the
GLC4/CDDP subline is not cross-resistant to compound 9.
The cytotoxic activity of compounds 12 and 15 is very low
relative to that of the reference compounds. For 12 the RF
obtained is 2.9, and for 15 the RF is 1.2 (Figure 1). In other
words, the GLC4/CDDP cells are 3- and 1.2 times less sen-
sitive than the GLC4 cells to 12 and 15, respectively.
Since some substituted ethylenediamine platinum com-
plexes have shown antitumor activity against a variety of
cell tumors,[27,28] we sought to synthesize platinum()
mono- and dinuclear complexes containing ethylenediamine
derivatives as ligands. The novel compounds that were pre-
pared may show a different reactivity towards DNA with
the possibility of the formation of new adducts; they there-
fore may be active in cisplatin-resistant cell lines. In ad-
dition, compound 12 could possibly intercalate between the
DNA base pairs because of its aromatic ring. Furthermore,
we intended to prepare potentially active antitumor com-
pounds in an attempt to combine the activity of the ligand
to that of the metal.
Platinum Accumulation
Resistance to cisplatin has been intensively studied in
tumor cells repeatedly exposed to the drug in vitro. Among
Results and Discussion
The intermediate 3 was obtained in 92% yield by the the numerous mechanisms of acquired resistance to cispla-
reaction of diol 2 with iodine, imidazole, and triphenylphos- tin that have been reported, decreased accumulation is fre-
phane in toluene at reflux (Scheme 1). The diepoxide 8 (not quently present in resistant cells as a result of reduced drug
isolated) was prepared by treatment of ditosylate 7 with so- uptake. In a previous study, we have determined the uptake
dium methoxide in methanol according to the procedure rate of cisplatin, carboplatin, and aqua-substituted cisplatin
previously described in the literature.[29] The ligand a and in both GLC4 and GLC4/CDDP cell lines.[31] The sensitive
complex 9 were prepared as described in ref.[26] The ligands cell line accumulates approximately twice as much cisplatin
b–g were prepared in satisfactory yields by treating ethyl- as the resistant subline, mainly by an energy-dependent
enediamine with the corresponding intermediate (halide or mechanism. We have postulated that the uptake of cisplatin
epoxide) in ethanol at room temperature for 24 h. In the is composed of passive diffusion of the [Pt(NH3)2Cl2] spe-
preparation of the ligand N,NЈ-bis(2-aminoethyl)-2-xylyl- cies, which is neutral, and of an active uptake of an aqua-
enediamine (e), we have also isolated the ligand N-(2-amino- substituted species, probably the [Pt(NH3)2(H2O)OH]+ or
ethyl)-isoindoline (f). In the IR spectra, absorptions corre- [Pt(NH3)2(H2O)Cl]+ species. For carboplatin, the rate con-
sponding to νN–H at 3300–3100 cm–1 can be observed for stant for the first aquation reaction is 100-fold lower than
1
these ligands. In the H and 13C NMR spectra signals for that for cisplatin. Therefore, the concentration of aquated
the ethylenediamine moiety are observed.
species will be 100 times lower. The uptake rate of carbopla-
The platinum() complexes 10–15 were obtained by the tin is similar in both GLC4 and GLC4/CDDP cell lines
reaction of the corresponding ligands with potassium tetra- because the active uptake becomes negligible. Therefore,
chloroplatinate() in water at room temperature for 24 h, carboplatin enters the cells mainly by passive diffusion,
and were isolated by simple filtration (Scheme 2). Even which is in accordance with the low RF observed for car-
though we have used 2 mmol of K2PtCl4 we have observed boplatin (Table 1).
that under these reaction conditions, ligand c was trans-
The cytotoxic activities of compounds 12 and 15 were
formed into the mononuclear platinum() complex 11. For much lower than those exhibited by cisplatin and by com-
Eur. J. Inorg. Chem. 2006, 1868–1874
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
1869

A. P. S. Fontes et al.
FULL PAPER
Scheme 1. Reagents and conditions: (i) NH2CH2CH2NH2/EtOH, reflux; (ii) Ph3P/I2/imidazole, toluene, reflux, 24 h; (iii) MeO–Na+/
MeOH, room temp., 30 min.
pound 9. These results can be explained by two possibilities: platinum concentrations lead to similar intracellular plati-
(i) the uptake of compounds 12 and 15 is very slow, thus a num concentrations.[26]
much higher concentration is needed to force their trans-
Compound 9 exhibits the same behavior, for example, by
port into cells (similar to the case of carboplatin when com- incubating cells with the IC50 dose, the intracellular concen-
pared to cisplatin) or (ii) the interaction of the compounds tration is approximately 10×10–17 mol/cell. The ratio be-
with the pharmacological target, i.e. DNA, is deficient. In tween the IC50 of carboplatin and that of cisplatin is ap-
order to investigate whether the reason for this low activity proximately 25, i.e. more carboplatin is needed to produce
is related to a deficient uptake, we have determined the in- a cytotoxic effect because the former enters cells more
tracellular platinum concentration after 72 h of incubation. slowly. Our results showed that the reason for the low cyto-
Let us first discuss the results obtained with the sensitive toxic activities of 12 and 15 was not an insufficient intracel-
cell line. In a previous study, we found that cytotoxic ac- lular platinum concentration. On the contrary, the intracel-
tivity correlates well with the intracellular drug concentra- lular platinum concentrations found for compounds 12 and
tion for cisplatin and carboplatin, and at IC50, the intracel- 15 were significantly higher than those found for the refer-
lular platinum concentration was about 10×10–17 mol/ ence compounds, cisplatin and carboplatin, and compound
cell.[31] Furthermore, by studying the effect of some dinu- 9. For the sensitive cell line, at IC50 values, the intracellular
clear platinum compounds, we also found that equitoxic platinum concentrations were 14.9- and 9.3-fold higher for
1870
www.eurjic.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2006, 1868–1874

Synthesis of Polyamines from Ethylenediamine and Their Platinum() Complexes
FULL PAPER
Scheme 2. Reagents and conditions: (i) K2PtCl4, H2O, room temp.
compounds 12 and 15, respectively, than that attained with xicity. In a review of structure–activity relationships of
cisplatin. This fact suggests that the formation of adducts some dinuclear platinum compounds, Farrell et al.[33] re-
between compounds 12 and 15 and the putative pharmaco- ported that the cytotoxic activity of a dinuclear compound
logical target DNA is less favored. Knox et al.[32] postulated containing cyclohexane-1,4-diamine as a linker between
that the difference in the kinetics of the interaction with two platinum atoms was very disappointing, probably be-
DNA is responsible for the different sensitivity of the same cause sterically hindered compounds bind more slowly to
cell line to cisplatin and carboplatin. According to these DNA. Assuming that equal binding to DNA would result
authors, 20- to 40-fold larger doses of carboplatin were re- in an equal cytotoxic effect, the fact that the intracellular
quired to produce equal binding to DNA and equal cytoto- concentrations found for compounds 12 and 15 are much
Eur. J. Inorg. Chem. 2006, 1868–1874
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
1871

A. P. S. Fontes et al.
FULL PAPER
Table 1. IC50, RF, and intracellular platinum concentration[a] for
complexes 9, 12, and 15.
An intriguing point to be considered is that a substan-
tially higher intracellular platinum concentration is required
to achieve a comparable cytotoxic effect in resistant cells by
compounds 12 and 15 than in sensitive cells. Increased
levels of sulfur-containing molecules have been found in
various resistant cell lines. Hospers et al.[34] found that the
GLC4/CDDP cell line possesses a significantly higher
amount of sulfur-containing molecules than the GLC4 cell
line, especially glutathione. Increased levels of sulfhydryl
compounds are pointed as a possible mechanism for resis-
tance. These thiols could bind to platinum and prevent it
from attaining its target, DNA. If resistant cells possess
higher levels of sulfur-containing compounds, one could ex-
pect to find a higher platinum concentration within resist-
ant cells, because these compounds could bind to platinum
in a detoxification mechanism. This is the case for com-
pounds 12 and 15. A possible explanation is that the inter-
actions of these compounds with DNA are very slow, thus
interactions of other intracellular ligands such as glutathi-
one become determinant. They are able to accumulate in
cells but are not very effective in binding to DNA and in-
hibiting cell growth.
Com-
Cell Line
IC50 [µ₎
RF Ci×1016 [mol/cell]
pound
DDP[b]
GLC4
0.40 0.05
2.5 0.2
0.98 0.09
1.07 0.11
CDDP[b] GLC4/
CDDP
6.3
1.4
0.9
CBDCA[b] GLC4
CBDCA[b] GLC4/
CDDP
9
9
9.9 1.1
13.5 1.3
0.79 0.08
1.02 0.11
GLC4
GLC4/
CDDP
GLC4
GLC4/
CDDP
GLC4
GLC4/
CDDP
9.9 0.9
8.9 0.9
0.96 0.10
1.05 0.10
12
12
28.10 2.80
81.0 8.1
14.92 1.50
2.88 65.90 6.70
15
15
84.0 8.3
102.5 10.3 1.22 24.10 2.50
9.28 0.90
[a] IC50 is the complex concentration required to inhibit 50% of
cell growth. [b] ref.[31] RF value was calculated as resistant cell IC50/
sensitive cell IC50. The values represent the mean SD of triplicate
determinations;·Ci is the mol number of platinum per cell at IC50
doses after 3 d.
In the cases of cisplatin, carboplatin, and compound 9
we have observed that the equal intracellular platinum
amounts are responsible for equal cytotoxicity. Thus, the
larger doses required in the case of resistant cells are neces-
sary to force the uptake, and, once inside cells, equal plati-
num amounts can produce equal binding to DNA and
equal cytotoxic effects. Sadowitz et al.[35] have shown that at
low concentrations of cisplatin, endogenous thiols intercept
cellular platinum, but this mechanism is not relevant at
CDDP concentrations within the therapeutic range.
Conclusion
This work describes the synthesis and characterization
of different amine-ligand derivatives of 1,2-ethylenediamine,
and also the preparation and characterization of their re-
spective platinum() complexes.
Figure 1. Growth inhibition of GLC4 and GLC4/CDDP cells as a
function of the intracellular platinum concentration after 3 d of
incubation with equitoxic doses of complexes 12 and 15. The values
are the mean values of two independent experiments. The platinum
Concerning the dinuclear platinum complexes, the intro-
content was assessed by flameless atomic absorption spectroscopy. duction of a spacer (cycle) between the two platinum atoms
leads to a significant decrease in cytotoxic activity. This
finding is very important in guiding further design of dinu-
clear platinum compounds aimed at treating cancer.
higher than those of the reference compounds indicates that
12 and 15 bind to DNA more slowly or with a lower affin-
ity, probably because of steric hindrance.
If these compounds bind more slowly to DNA, interac-
tion with other intracellular ligands such as sulfur-contain-
Experimental Section
ing molecules will become relevant and this may be the
reason for the elevated intracellular platinum concentra-
tions.
Reagents: All chemicals were of reagent grade and were used with-
out further purification.
Starting Materials
Considering 9 and 12, these dinuclear platinum com-
pounds are expected to form interstrand cross-links with
DNA. The structural difference between them lies in the
diamine linker. One can speculate that the insertion of a
spacer (cycle) between the two platinum atoms engenders a
steric hindrance to form DNA adducts. It is also possible
that 12 only forms monoadducts with DNA.
Compound 3: Imidazole (2.82 g, 41.6 mmol), triphenylphosphane
(10.92 g, 41.6 mmol), and iodine (10.6 g, 41.6 mmol) were added
to a solution of trans-1,4-bis(hydroxymethyl)cyclohexane (2) (2.0 g,
14 mmol) in toluene (50 mL). The reaction mixture was stirred at
60 °C in an oil bath for 2 h. The solution was cooled, treated with
an aqueous solution of sodium bisulfite and extracted with toluene
(3×50 mL). The organic phases were combined and concentrated.
1872
www.eurjic.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2006, 1868–1874

Synthesis of Polyamines from Ethylenediamine and Their Platinum() Complexes
FULL PAPER
The crude product was purified on silica gel using n-hexane as the
eluent to give 3 in 92% yield (4.65 g, 12.77 mmol).
dark at room temperature, the solid that formed was filtered off,
washed with water, and dried. In the case of complexes 14 and 15
only 1 mmol of K2PtCl4 was used. Yields: (10) 0.47 g (62%), (11)
0.25 g (50%), (12) 0.69 g (92%), (13) 0.32 g (42%), (14) 0.17 g
(40%), (15) 0.364 g (75%).
Compound 8:[29] Methanolic sodium methoxide (25 mmol of so-
dium in 10 mL of methanol) was slowly added to a solution of
ditosylate 7 (5 g, 10.35 mmol) in methanol (15 mL). The reaction
was stirred for 30 min at room temperature, concentrated under
reduced pressure and added to chloroform (10 mL). The resulting
precipitate was collected and washed, and the filtrate was concen-
trated under reduced pressure to give 8 in quantitative yield.
(10): IR (KBr): νmax = 3270, 2910, 2846, 1628, 1313, 1045, 827, 549,
˜
326 cm–1. 195Pt NMR (86 MHz, [D6]DMSO) (DMSO = dimethyl
sulfoxide): δ = –2380 ppm. C12H28N4Pt2Cl4 (760.18): calcd. C
18.95, H 3.68, N 7.37; found C 19.15, H 3.86, N 7.35.
Synthesis of Ligands
(11): IR (KBr): ν
= 3207, 3114, 2923, 2847, 1639, 1541, 1462,
˜
max
1306, 1188, 1060, 1014, 861, 759, 569, 317 cm–1. 1H NMR
(400 MHz, [D6]DMSO): δ = 2.83, 3.59 (m, 4 H, CH2NH,
CH2NH2), 4.25 (m, 4 H, CH2Ph), 5.08, 6.29 (s, 3 H, NH, NH2),
7.95 (m, 4 H, Ar) ppm. 13C NMR (100 MHz, [D6]DMSO): δ =
54.3–45.6 (CH2NH, CH2NH2), 61.1 (CH2Ph); 128.0–130.4
(Ar) ppm. 195Pt NMR (86 MHz, [D6]DMSO): δ = –2018 ppm.
C12H20N4PtCl2 (486.21): calcd. C 28.69, H 3.98, N 11.15; found C
29.03, H 3.86, N 11.45.
Ligand a: This ligand was prepared by following the procedure de-
scribed in ref.[26]
General procedure for the preparation of ligands b–g is shown in
Scheme 1.
The corresponding halide or epoxide (10 mmol) was slowly added
to a solution of ethylenediamine (6.7 mL, 100 mmol) in ethanol
(20 mL) over 5 h. The reaction mixture was stirred for 12–24 h un-
der reflux, evaporated under reduced pressure, and the residue puri-
fied on silica gel using dichloromethane/methanol as the eluent.
Yields: (b) 1.37 g (60%), (c) 1.18 g (50%), (d) 1.56 g (70%), (e)
1.20 g (54%), (f) 0.18 g (11%), and (g) 1.54 g (70%)
(12): IR (KBr): ν
= 3130, 3056, 2984, 2952, 2883, 1643, 1451,
˜
max
1287, 1189, 1064, 1018, 954, 853, 759, 576, 501, 324 cm–1. 1H NMR
(400 MHz, [D6]DMSO): δ = 2.53 (m, 8 H, CH2NH, CH2NH2),
3.98 (s, 4 H, CH2Ph), 5.30, 6.25 (s, 4 H, NH2), 6.38 (s, 2 H, NH),
7.56 (m, 4 H, Ph) ppm. 13C NMR (100 MHz, [D6]DMSO): δ =
43.8, 44.7 (CH2NH2), 45.6, 46.9 (CH2NH), 54.3, 54.9 (CH2Ph),
130.4–136.7 (Ar) ppm. C12H22N4Pt2Cl4 (754,14): calcd. C 19.10, H
2.92, N 7.43; found C 19.45, H 2.86, N 7.75.
(b): IR (KBr): νmax = 2925, 2853, 1634, 1401, 1350, 1037, 772 cm–1.
˜
1H NMR (200 MHz, CD3OD): δ = 3.25 (m, 8 H, CH2N), 1.38 (m,
6 H, CH, CH2 cyclohexyl) ppm. 13C NMR (50 MHz, CD3OD): δ
= 54.0 (CH2NHCH2CH2NH2), 44.7 (NHCH2), 36.5 (CH2NH2),
35.4, 28.6 (cyclohexyl) ppm.
(13): IR (KBr): ν
= 3259, 3192, 3109, 2954, 2885, 2858, 1586,
˜
max
1441, 1191, 1164, 1064, 762, 531, 314 cm–1. 1H NMR (400 MHz,
[D6]DMSO): δ = 3.40 (m, 4 H, CH2NH2); 3.80 (m, 4 H, CH2NH);
4.30 (s, 4 H, CH2Ph); 5.52 (s, 4 H, NH2); 5.80, 6.30 (s, 2 H, NH);
7.50 (m, 4 H, Ar) ppm.
(c): IR (KBr): ν
= 2933, 1646, 1543, 1506, 1313, 1160, 1108,
˜
max
1019, 859, 757 cm–1. 1H NMR (200 MHz, CD3OD): δ = 7.79 (d, 2
H, H-2, H-6, J = 8.0 Hz), 7.55 (d, 2 H, H-3, H-5, J = 8.0 Hz) ppm.
13C NMR (50 MHz, CD3OD): δ = 170.6 (C=O), 134.6, 134.0,
130.1, 128.1 (Ph), 51.0 (CH2Ph), 44.0, 39.3 (CH2NH), 37.4, 35.7
(CH2NH2) ppm.
(14): IR (KBr): ν
= 3200, 3116, 2949, 1622, 1313, 1158, 1049,
˜
max
978, 926, 894, 823, 749, 609, 409 cm–1. H NMR (400 MHz, [D6]-
DMSO): δ = 2.51, 2.84 (2m, 4 H, CH2NH2); 4.25, 4.45 (2d, 2 H,
CH2N); 4.25, 4.45, 4.87, 4.97 (4d, 4 H, CH2Ph), 5.52 (s, 1 H, NH2),
6.31 (s, 1 H, NH2), 7.28 (m, 4 H, Ph) ppm. 13C NMR (100 MHz,
1
(d): IR (KBr): ν
= 2948, 2821, 1594, 1384, 1352, 1247, 1055,
˜
max
774, 669 cm–1. 1H NMR (200 MHz, CD3OD): δ = 7.32 (s, 4 H,
CH), 3.74 (s, 4 H, CH), 3.74 (s, 4 H, CH2Ph), 2.71 (t, 8 H, NHCH2,
CH2NH2) ppm. 13C NMR (50 MHz, CD3OD): δ = 139.7, 129.6
(Ph), 54.1 (CH2Ph), 51.8 (NHCH2), 41.7 (CH2NH2) ppm.
[D6]DMSO):
δ = 44.7 (CH2NH2), 46.2 (NCH2), 64.4, 65.4
(CH2Ph), 122.9–136.7 (Ar). C10H14N2PtCl2 (428.13): calcd. C
28.04, H 3.27, N 6.54; found C 28.40, H 2.96, N 6.75.
(e): IR (KBr): ν
= 2950, 2820, 1592, 1386, 1352, 1248, 1055,
˜
max
772, 669 cm–1. 1H NMR (200 MHz, CD3OD): δ = 7.44 (m, 4 H,
Ph), 4.17 (s, 4 H, CH2Ph), 3.11 (m, 8 H, NHCH2, CH2NH2) ppm.
13C NMR (50 MHz, CD3OD): δ = 136.6, 132.9, 130.4 (Ph), 52.2
(CH2Ph), 46.6 (CH2NH), 39.5 (CH2NH2) ppm.
(15): IR (KBr): ν
= 3415, 3269, 3197, 2958, 2928, 1140, 1034,
˜
max
1018, 716, 549, 326 cm–1. C9H20N2O4PtCl2 (486.17): calcd. C 22.20,
H 4.12, N 5.76; found C 22.15, H 3.86, N 5.75.
Cell Lines and Cultures
(f): IR (KBr): ν
= 2948, 1594, 1383, 1352, 1058, 775, 667 cm–1.
˜
max
The GLC4 cell line was derived from pleural effusion of a patient
with small-cell lung carcinoma in the laboratory of Prof. E. G. E.
de Vries (Department of Internal Medicine, University Hospital,
Groningen, The Netherlands). The GLC4/CDDP was obtained in
the same laboratory by continuous exposure to CDDP. No amplifi-
cation of the MDR gene or expression of P-glycoprotein was found
in the GLC4/CDDP subline.
1H NMR (200 MHz, CD3OD): δ = 7.21 (m, 4 H, Ph), 4.04 (s, 4 H,
CH2Ph), 3.09 (m, 4 H, NHCH2, CH2NH2) ppm. 13C NMR
(50 MHz, CD3OD): δ = 140.4, 130.0, 124.6 (Ph), 59.9 (CH2Ph),
53.5 (CH2NH), 39.1 (CH2NH2) ppm.
(g): IR (KBr): ν
= 3403, 2936, 2862, 1648, 1573, 1460, 1343,
˜
max
1301, 1151, 1052, 958 cm–1. 1H NMR (200 MHz, CD3OD): δ =
3.92 (m, 1 H, H-3), 3.70 (t, 1 H, H-4), 3.13 (t, 1 H, H-5), 2.64 (m,
4 H, CH2NH, CH2NH2), 1.80 (m, 4 H, H-2, H-2Ј, H-6, H-6Ј) ppm.
13C NMR (50 MHz, CD3OD): δ = 76.7, 71.1, 68.5, 65.6 (C-1, C-3,
C-4, C-7), 63.8, 59.8, 44.0 (C-5, CH2N), 38.7, 36.7 (C-2, C-6) ppm.
The cell lines were cultured in an RPMI 1640 (Sigma Chemical
Co.) medium supplemented with 10% fetal calf serum (Biomedia
Co.) at 37 °C in a humidified 5% CO2 atmosphere. Cultures grow
exponentially from 105 cells/mL to about 106 cells/mL in 3 d. Cell
viability was checked by Trypan blue exclusion. The cell number
was determined by Coulter counter analysis. For the cytotoxicity
assessment, 1×105 cells/mL were cultured for 72 h in the absence
and the presence of various concentrations of each platinum-based
compound. The sensitivity to the drug was evaluated by the drug
concentration that inhibits cell growth by 50%, IC50. A resistance
Synthesis of Complexes 9–15
Complex 9: Prepared following the procedure described in ref.[26]
Complexes 10–15 (Scheme 2): The appropriate ligand (1 mmol), dis-
solved in water (5 mL), was slowly added to a solution of K2PtCl4
(0.830 g, 2 mmol) in water (10 mL). After stirring for 24 h in the
Eur. J. Inorg. Chem. 2006, 1868–1874
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
1873

A. P. S. Fontes et al.
FULL PAPER
[12] T. Thomas, T. J. Thomas, Cell. Mol. Life Sci. 2001, 58, 244–
258.
[13] B. Rosenberg, L. Van Camp, J. E. Trosco, V. H. Mansour, Na-
ture 1969, 222, 385–386.
[14] Bristol-Myers Squibb & Johnson Matthey, Drugs of the Future
1990, 15, 739.
[15] W. B. Pratt, R. W. Ruddon, W. D. Ensminger, J. Maybaum, in:
The Anticancer Drugs, Oxford University Press, New York, 2nd
Ed., 1994, 133–139.
factor (RF) was obtained by dividing the IC50 of resistant cells by
the IC50 of sensitive cells. The RF to cisplatin is 6.3.
Stock solutions of compounds 9, 12, and 15 were prepared in a
mixture of water/DMSO (90:10). In the cytotoxic assays, the final
concentration of DMSO was below 1.5%.
Cellular Accumulation
[16] T. W. Hambley, Coord. Chem. Rev. 1997, 166, 181–223.
[17] A. G. Sykes, Platinum Met. Rev. 1988, 32, 170–178.
[18] A. Pasini, F. Zunino, Angew. Chem. Int. Ed. Engl. 1987, 26,
615–624.
Cells were incubated with different complex concentrations for 3 d.
After incubation, an aliquot was removed, washed twice with ice-
cold PBS (phosphate-buffered saline), and the pellet mineralized in
65% HNO3. The platinum concentration was determined by
atomic absorption spectroscopy with a Varian model Zeeman 220
spectrophotometer equipped with a graphite tube atomizer and an
autosampler. The heating program consisted of sequential drying
(85 °C for 5 s, 95 °C for 40 s, and 95 °C for 20 s), an ashing stage
[19] N. Farrell, Y. Qu, M. P. Hacker, J. Med. Chem. 1990, 33, 2179–
2184.
[20] F. M. Muggia, G. Los, Stem Cells 1993, 11, 182–193.
[21] Z. H. Siddik, Oncogene 2003, 22, 7265–7279.
[22] R. P. Wernyj, P. J. Morin, Drug Resist Updat. 2004, 7, 227–232.
(500 °C for 5.0 s followed by 15 s at 1100 °C), and atomizing at [23] N. Farrell, S. G. de Almeida, K. A. Skov, J. Am. Chem. Soc.
1988, 110, 5018–5019.
2500 °C for 5 s.
[24] N. Farrell, T. G. Appleton, Y. Qu, J. D. Roberts, A. P. S. Fontes,
K. A. Skov, P. Wu, Y. Zou, Biochemistry 1995, 34, 15480–
15486, and references therein.
[25] J. D. Roberts, B. V. Houten, Y. Qu, N. Farrell, Nucleic Acids
Res. 1989, 17, 9719–9733.
[26] E. T. Cesar, M. V. de Almeida, A. P. S. Fontes, E. C. P. Maia,
A. Garnier-Suillerot, M. R. C. Couri, E. C. A. Felício, J. Inorg.
Biochem. 2003, 95, 297–305.
Acknowledgments
We acknowledge CNPq, CAPES, and FAPEMIG for the financial
support and fellowships.
[27] E. Wong, G. M. Giandomenico, Chem. Rev. 1999, 99, 2451–
2466.
[28] K. P. Beaumont, C. A. Mcauliffe, M. J. Cleare, Chem. Biol. In-
teract. 1976, 14, 179–193.
[29] D. Mercier, J. Leboul, J. Cleophax, S. D. Gero, Carbohydr. Res.
1971, 20, 299–304.
[30] M. V. de Almeida, E. T. Cesar, E. C. A. Felício, A. P. S. Fontes,
J. Braz. Chem. Soc. 2000, 11, 154–158.
[1] A. E. Pegg, Cancer Res. 1988, 48, 759–774.
[2] M. L. Edwards, N. J. Prakash, D. M. Stemerick, S. P. Sunkara,
A. J. Bitonti, G. F. Davis, J. A. Dumont, P. Bey, J. Med. Chem.
1990, 33, 1369–1375.
[3] M. L. Edwards, D. M. Stemerick, R. D. Snyder, J. Med. Chem.
1991, 34, 2414–2420.
[4] P. S. Sunkara, S. B. Baylin, G. D. Luk in Inhibitors of polyamine
biosynthesis: cellular and in vivo effects on tumor proliferation. [31] E. Pereira-Maia, A. Garnier-Suillerot, J. Biol. Inorg. Chem.
Inhibition of Polyamine Metabolism: Biological Significance and
Basis for New Therapies (Eds.: P. P. MacCann, A. E. Pegg, A.
Sjoerdsma), Academic Press, New York, 1987, 121.
[5] D. H. Russel, Nature 1971, 233, 144–145.
[6] B. Cipolla, J. P. Moulinoux, V. Quemener, R. Havouis, L. A.
Martin, F. Guille, B. Lobel, J. Urol. 1990, 144, 1164–1166.
[7] T. Matsumoto, O. Suzuki, in The Physiology of Polyamines
(Eds.: U. Bacharach, Y. M. Heimer), CRC Press, Boca Raton,
1989, vol. 2, 219–234.
2003, 8, 626–634.
[32] R. J. Knox, F. Friedlos, D. A. Lydall, J. J. Roberts, Cancer Res.
1986, 46, 1972–1979.
[33] N. Farrell, Y. Qu, U. Bierbach, M. Valsecchi, E. Menta, in:
Cisplatin: Chemistry and Biochemistry of a Leading Anticancer
Drug (Ed.: B. Lippert), VHCA and Wiley-VCH, Zurich, 1999,
479–496.
[34] G. A. P. Hospers, N. H. Mulder, B. de Jong, L. de Ley, D. R. A.
Uges, A. M. J. Fichtinger-Schepman, R. J. Scheper, E. G. E.
de Vries, Cancer Res. 1988, 48, 6803–6807.
[35] P. D. Sadowitz, B. A. Hubbard, J. C. Dabrowiak, J. Goodis-
man, K. A. Tacka, M. K. Aktas, M. J. Cunningham, R. L. Du-
bowy, A. Souid, Drug Metabolism and Disposition 2002, 30,
183–190.
[8] U. Bacharach, in: The Physiology of Polyamines (Eds.: U. Bach-
arach, Y. M. Heimer), CRC Press, Boca Raton, 1989, vol. 2,
235–250.
[9] C. W. Porter, J. R. Sufrin, Anticancer Res. 1986, 6, 525–542.
[10] I. D. Algranati, S. H. Goldemberg, Contrib. Gynecol. Obstet.
TIBS 1977, 2, 272–274.
Received: November 16, 2005
Published Online: March 16, 2006
[11] R. A. Casero, A. E. Pegg, FASEB J. 1993, 7, 653–661.
1874
www.eurjic.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2006, 1868–1874