Enantiomerically pure [1,2-diamino-1-(4-fluorophenyl)butane]platinum(II) complexes: Synthesis and antitumor activity against MCF-7 and MDA-MB 231 breast cancer and LnCaP/FGC prostate cancer cell lines

Article abstract of DOI:10.1002/ardp.200400621

Enantiomerically pure 1,2-diamino-1-(4-fluorophenyl)butanes were synthesized by stereoselective procedures. The enantiomeric purity was determined by ¹H NMR spectroscopy after derivatization with (1R)-myrtenal. For the coordination to platinum,

Full text of DOI:10.1002/ardp.200400621

654 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Anja Dullina,
Enantiomerically Pure [1,2-Diamino-1-(4-fluoro-
phenyl)butane]platinum(II) Complexes:
Synthesis and Antitumor Activity against MCF-7
and MDA-MB 231 Breast Cancer and
Francois Dufrasneb,
Michael Gelbckeb,
Ronald Gusta
a Institute of Pharmacy, Free
University of Berlin, Berlin,
Germany
LnCaP/FGC Prostate Cancer Cell Lines
b Laboratoire de Chimie
Pharmaceutique Organique,
Institut de Pharmacie,
Enantiomerically pure 1,2-diamino-1-(4-fluorophenyl)butanes were synthesized
1
by stereoselective procedures. The enantiomeric purity was determined by H
NMR spectroscopy after derivatization with (1R)-myrtenal. For the coordination
to platinum, the diamines were reacted with K2PtI4. Reaction with Ag2SO4
yielded the respective sulfatoplatinum(II) complexes, which were converted into
the dichloroplatinum(II) complexes by treatment with 2 N HCl. The influence of
the configuration and the kind of leaving group on the antitumor activity was
studied on the MCF-7 and MDA-MB 231 breast cancer cell lines, as well as on
the LnCaP/FGC prostate cancer cell line. It was demonstrated that the dichloro-
platinum(II) complexes were more active than the respective diiodoplatinum(II)
derivatives. Conversion into the sulfatoplatinum(II) complexes further enhanced
the antiproliferative effects. The configuration determined the antitumor effects,
dependent on the cell line used: MCF-7: (R,R) > (S,S) > (R,S) > (S,R); MDA-
MB 231: (S,S) > (R,R) > (R,S) = (S,R); LnCaP/FGC: (S,S) > (R,R) > (R,S)
> (S,R).
´
Universite Libre de Bruxelles,
Bruxelles, Belgium
Keywords: Platinum Complexes; MCF-7 and MDA-MB 231 Breast Cancer Cell
Lines; LnCaP/FGC Prostate Cancer Cell Line; Antitumor Activity; Enantiomers
Received: August 12, 2004; Accepted: November 3, 2004 [FP921b]
DOI 10.1002/ardp.200400621
Introduction
efficacy in the treatment of patients with advanced
ovarian [5] and small cell lung cancer [6]. Preliminary
results further indicate that it might be also useful in
the therapy of breast cancer [7].
Since the discovery of the cytotoxicity of platinum
complexes by Rosenberg et al. [1], numerous diamine-
dichloroplatinum(II) complexes have been synthesized
and tested for antitumor activity.
The mode of action includes the building of intrastrand
cross links comparable to other platinum compounds
[8], but DACH-platinum DNA adducts formed by oxali-
platin are generally associated with greater cytoxicity
and inhibition of DNA synthesis.
Cisplatin exhibits high antitumor activity, but its clinical
use is mostly limited by toxic side effects [2]. Ex-
change of both chlorides by cyclobutane-1,1-dicar-
boxylate led to carboplatin, which showed higher water
solubility and reduced toxic side effects; however, my-
elosuppression and the development of resistance are
intolerable disadvantages [3].
The stereochemistry of the 1,2-diaminocyclohexane
moiety significantly influences the toxicity of the plati-
num complexes. Complexes of the two trans isomers
(R,R and S,S) exhibit higher activities than the meso
(R,S and S,R) adducts, and the R,R-complex is more
effective than the S,S one [9Ϫ12].
In order to overcome these drawbacks and to enhance
the tumor selectivity, many carrier ligands were devel-
oped and coordinated to platinum. The most success-
ful ligand, the 1,2-diaminocyclohexane (DACH), made
oxaliplatin to the first platinum-based drug licensed for
the therapy of colorectal cancer [4]. It has also shown
These results encouraged us to investigate the enanti-
oselective antitumor activity of [1,2-diamino-1,2-diaryl-
ethane]dichloroplatinum(II) complexes [13Ϫ15]. On
the human MCF-7 breast cancer cell line, the R,S-con-
figurated complexes were distinctly less active than
their R,R/S,S-diastereomers. Optimization of the anti-
tumor activity due to a separation of the enantiomers
Correspondence: Ronald Gust, Institut für Pharmazie der
FU Berlin, Königin Luise Str. 2 + 4, D-14195 Berlin, Germany.
Phone: +49 30 838-53272, Fax: +49 30 838-53854, e-mail:
rgust@zedat.fu-berlin.de
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Synthesis and Antitumor Activity of Platinum(II) Complexes 655
Scheme 1. Synthesis of 1S,2R-configurated 1,2-diamino-1-(4-fluorophenyl)butanes. Reagents and conditions: a:
BnBr (2 eq.), KI (0.1 eq.), Na2CO3 (2 eq.), THF, reflux; b: DMSO (2 eq.), oxalyl chloride (1.1 eq.), TEA (5 eq.),
CH2Cl2, Ϫ60°C; c: 4F-phenylmagnesium bromide (1.1 eq.), THF, 0°C; d: column chromatography, petroleum
ether(40-60)/acetone (9:1); e: 1. MsCl (1.5 eq.), TEA (3 eq.), Et2O, O°C to room temperature; 2. NaN3 (2 eq.),
H2O; f: column chromatography, petroleum ether(40-60)/acetone (98:2); g: 1. HCOONH4 (4 eq.), Pd on C (10%),
MeOH, reflux ; 2. HCl/Et2O.
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656 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
was less successful. Only small differences between
the enantiomers exist if the aryl rings are unsubsti-
tuted, 3-OH, 4-OH or 4-F substituted.
the antiproliferative effects against human breast
(MCF-7 and MDA-MB 231) and prostate (LnCaP/FGC)
cancer cell lines.
To get more insight into the structural dependence of
enantiomerically pure [1,2-diaminoethane]dichloropla-
tinum(II) complexes, we determined the antiprolifera-
tive effects of [1,2-diamino-1-phenylpropane]dichloro-
platinum(II) (Ph/Me-PtCl2) and [1,2-diamino-1-(4-
fluorophenyl)propane]dichloroplatinum(II) (4F-Ph/Me-
PtCl2) isomers on the MCF-7 cell line [16, 17].
Results
Synthesis
The 1S,2R-configurated 1,2-diamino-1-(4-fluorophe-
nyl)butane (1S,2R)-7 was obtained by an already pub-
lished modified method of O’Brien [18] (Scheme 1).
The cytotoxic potency of Ph/Me-PtCl2 decreased in
the series (R,R) > (S,S) > (S,R) = (R,S), while in the
case of 4F-Ph/Me-PtCl2, the S,S-enantiomer is more
active than its isomers.
(2R)-2-Aminobutanol (2R)-1 was first N-protected by
two benzyl groups and transformed into the aldehyde
(2R)-3 [19], which was reacted with 4-fluorophe-
nylmagnesiumbromide to give a mixture of the dia-
stereomeric aminoalcohols (1S,2R)-4/(1R,2R)-4 in a
ratio of about 87:13 [20]. After separation by column
chromatography, the alcoholic function of (1S,2R)-4
was activated via mesylate formation. This resulting
Because of these studies, we focussed our attention
on the 4-F derivatives and determined the influence of
the elongation of the C2-alkyl chain (methyl Ǟ ethyl),
as well as the significance of the leaving groups, on
Scheme 2. Synthesis of 1R,2R-configurated 1,2-diamino-1-(4-fluorophenyl)butanes. Reagents and conditions:
a: BOC anhydride (1.1 eq.), TEA (1.1 eq.), CH2Cl2, O°C to room temperature; b: DMSO (2 eq.), oxalyl chloride
(1 eq.), TEA (5 eq.), CH2Cl2, Ϫ60°C; c: benzylhydroxylamine (1 eq.), MgSO4 (1 eq.), CH2Cl2, room temperature;
d: 4F-phenylmagnesium bromide (3 eq.), THF, Ϫ50°C; e: column chromatography, petroleum ether(40-60)/acetone
(95:5); f: HCOONH4 (4 eq.), Pd on C (10%), MeOH; g: 1. HCl/MeOH ; 2. NaOH; 3. D-(Ϫ)-(2S,3S)-tartaric acid.
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Synthesis and Antitumor Activity of Platinum(II) Complexes 657
unstable intermediate could not be isolated because it
Spectroscopic characterization
cyclized readily to afford an aziridinium ion (2R,3R)-5.
The coordination to platinum was confirmed by NMR
Ϫ
Ring opening with N3 yielded mainly the azidoamine
+
spectroscopy. The NH3 resonances of the free 1,2-
(1S,2R)-6 (93%), which was separated, reduced and
deprotected to obtain the diamine (1S,2R)-7 with an
overall yield of 33% [21]. (1R,2S)-7 was available by
the same reaction course starting from (2S)-1.
diaminoethanes were replaced by four resonances
due to a diastereotopically split resulting from the coor-
dination to platinum.
A reciprocal coupling and the coupling to the amine
protons split the methine protons. The signals were
further broadened by platinum satellites (3JCH-Pt). If the
coordination to platinum of the 1,2-diaminoethanes
was performed with K2PtI4 in D2O, an NH/ND-ex-
change took place and the JCH-CH and the JCH-Pt
could be calculated from the spectra. The coupling
constants depended on the dihedral angle and al-
lowed an insight into the conformational behavior of
the platinum complexes [25Ϫ27].
The synthesis of the 1R,2R-configurated 1,2-diamino-
1-(4-fluorophenyl)butane (1R,2R)-13 is presented in
Scheme 2 [22].
(2R)-1 protected with a tert-butoxycarbonyl group
(BOC) [23] was oxydized to the aldehyde (2R)-9 [19],
which reacted with N-benzylhydroxylamine to give the
N-oxide (2R)-10. Addition of 4-fluorophenylmagne-
siumbromide resulted in the diastereoisomeric mixture
of (1R,2R)-11 and (1S,2R)-11 in a ratio of about 90:10.
Separation of (1R,2R)-11, reduction and deprotection
gave (1R,2R)-13 in an overall amount of 10%.
3
3
The five-membered chelate ring was puckered and
could exist in two possible conformations, the δ- and
the λ-form. An exclusive equatorial orientation of the
C1-aryl and the C2-alkyl substituents was realized in
the RR/SS-series, resulting in coupling constants of
Using the (2S)-2-aminobutanol (2S)-1, diamine
(1S,2S)-13 was available.
3
3JCH-CH ഠ 12.0 Hz and JCH-Pt = 0 Hz.
The optical purity of the diamines has been deter-
mined by 1H NMR using (1R)-(Ϫ)-myrtenal as chiral
derivatizing agent, following already published pro-
cedures [24].
On the contrary, the R,S/S,R-configuration enhanced
the dynamic properties. In the spectra of the R,S- and
S,R-configurated complexes, the methine proton at
C2 was split by couplings to the benzylic proton
(3JCH-CH ഠ 4.5 Hz) and to 195Pt (3JCH-Pt = 60 Hz). The
CH-Pt coupling showed a maximum of 80 Hz if the
proton was exclusively equatorially arranged, and a
minimum of 3JCH-Pt = 0 Hz if the CH was axial standing
K2PtI4 was used for the coordination of the enantio-
merically pure 1,2-diaminobutanes 7 and 13 (Scheme
3). The reaction with Ag2SO4 yielded the respective
sulfatoplatinum(II) complexes, which were trans-
formed into the dichloroplatinum(II) complexes by ad-
dition of 2 N HCl (Scheme 3).
3
[25]. The coupling constant of JCH-Pt = 60 Hz indi-
Scheme 3. Synthesis of [1,2-diamino-1-(4-fluorophenyl)butane]platinum(II) complexes. Reagents and conditions:
a: K2PtI4 (1.1 eq.) at 40°C in aqueous solution; b: Ag2SO4 (0.95 eq.) in aqueous solution with protection from
light; c: 2 N HCl.
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658 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Figure 1. Antiproliferative effects of cisplatin on the human MCF-7 and MDA-MB 231 breast cancer cell lines and
on the LnCaP/FGC prostate cancer cell line.
cated an interconversion of the five-membered chelate
ring with a displaced equilibrium in favor of a confor-
mation with an axial-standing aryl ring.
following onset of proliferation indicated the develop-
ment of drug resistance [29].
[1,2 diamino-1-(4-fluorophenyl)butane]platinum(II) com-
plexes showed antiproliferative effects, depending on
the configuration and the kind of leaving group. As
described in Figures 2 and 3, the diiodoplatinum(II)
complexes were less active on the MCF-7 breast
cancer cell line than the sulfatoplatinum(II) and the di-
chloroplatinum(II) derivatives.
The leaving groups influenced the chemical shifts of
the signals. The NH2 as well as the CHmethine signals
of the diiodoplatinum(II) complexes were diamag-
netically shifted compared to their dichloroplatinum(II)
derivatives. This was the consequence of stronger
trans effects of the iodides labilizing the Pt-N bonds.
The spectra of the sulfatoplatinum(II) complexes
showed a strong low-field shift of the same protons,
due to the positive charge on the platinum central
atom. The measurement of the complexes had to be
done in DMSO, since usable spectra were only avail-
able in this solvent [28]. DMSO coordinated to plati-
At the highest concentration, (SS)- and (RR)-4F-Ph/
Et-PtI2 significantly reduced cell growth by 70% and
about 100%, respectively, while (SR)- and (RS)-4F-
Ph/Et-PtI2 were inactive. Exchange of the IϪ leaving
groups increased the antitumor activity in the erythro-
series. In the case of (RS)-4F-Ph/Et-PtCl2, a concen-
tration-dependent antiproliferative effect was observed
(see Figure 3), even comparable to that of cisplatin. It
should be mentioned that the R,S-configurated
complexes were distinctly more active than their S,R-
enantiomers.
num and gave
a well-defined sulfato[sulfinylbis-
methane-S]platinum(II) complex.
Antiproliferative effects
The platinum complexes were tested on the MCF-7
and MDA-MB 231 mammary carcinoma cell lines as
well as the LwCaP/FGC prostate cancer cell line. Cis-
platin was used as reference (Figure 1).
The variation of the leaving groups in the threo-series
led to complexes that were by far more active than
cisplatin. (RR)-4F-Ph/Et-PtSO4 and (SS)-4F-Ph/
Et-PtSO4 showed cytocidal effects even at the low
concentration of 0.5 µM. Conversion of both com-
plexes into the dichloroplatinum(II) compounds ((RR)-
and (SS)-4F-Ph/Et-PtCl2) decreased the cytotoxicity.
(RR)-4F-Ph/Et-PtCl2 reduced cell growth significantly
stronger than (SS)-4F-Ph/Et-PtCl2 and achieved cyto-
cidal effects at concentrations of 1 and 5 µM.
Cisplatin reduced the growth of the tumor cells, in a
concentration-dependent way. At 5 µM, it suppressed
the proliferation of MDA-MB 231 and MCF-7 cells
completely (T/Ccorr ഠ 0%; see Figure 1). On the
LnCaP/FGC cell line, it was by far less active. At a
concentration of 5 µM, a cell mass reduction of about
75% was achieved after an incubation of 75 h. The
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Synthesis and Antitumor Activity of Platinum(II) Complexes 659
Figure 2. Antiproliferative effects of R,R- and S,S-configurated [1,2-diamino-1-(4-fluorophenyl)butanes]platinu-
m(II) complexes on MCF-7 cells.
[1,2-Diamino-1-(4-fluorophenyl)butane]platinum(II)
complexes also caused antiproliferative effects against
MDA-MB 231 cells. (SR)- and (RS)-4F-Ph/Et-PtCl2 re-
duced the cell growth at the highest concentration
used to about T/Ccorr = 10% (see Figure 4). Their dia-
stereomeres were distinctly more active and reached
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660 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Figure 3. Antiproliferative effects of R,S- and S,R-configurated [1,2-diamino-1-(4-fluorophenyl)butane]platinum(II)
complexes on MCF-7 cells.
cytostatic potency (T/Ccorr ഠ 0%). In this test, (SS)-
4F-Ph/Et-PtCl2 showed slightly better effects than
(RR)-4F-Ph/Et-PtCl2, especially at lower concen-
trations.
On the LnCaP/FGC cell line, (RS)-, (SS)- and (RR)-
4F-Ph/Et-PtCl2 were more active than cisplatin (Fig-
ure 5). (SS)- and (RR)-4F-Ph/Et-PtCl2 demonstrated
cytocidal effects at each concentration used. A some-
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Synthesis and Antitumor Activity of Platinum(II) Complexes 661
Figure 4. Antiproliferative effects of R,S- and S,R-configured [1,2-diamino-1-(4-fluorophenyl)butane]dichloroplati-
num(II) complexes on the MDA-MB 231 cell line.
what higher cytotoxicity of (SS)-4F-Ph/Et-PtCl2 was
deduced from the time activity curves.
It was demonstrated in various pharmacological stud-
ies that the complexes possessed high antiproliferative
effects against MCF-7 and MDA-MB 231 breast can-
cer cells and were also active on the LnCaP/FGC
prostate cancer cell line [30, 31]. The R,R/S,S-con-
figurated compound was by far more active than its
diastereomer. This finding was explained by an ac-
cumulation in the cytoplasm by a stereospecific trans-
port through the cell membrane [32] and different
attachment at the DNA due to the spatial structure of
the diamine ligand.
Discussion
The investigations presented in this paper continue
the structure activity relationship study on [1,2-di-
amino-1-(4-fluorophenyl)ethane]dichloroplatinum(II)
complexes [16, 17] that were synthesized to optimize
the antitumor activity of the parent compounds [(R,S)-
and
(R,R/S,S)-1,2-diamino-1,2-bis(4-fluorophenyl)-
The separation of the enantiomers did not optimize the
antitumor activity [15], although the binding of enanti-
omeric compounds to the double-helical DNA as a chi-
ral structure should lead to diastereomeric adducts.
Furthermore, it has been verified that DNA cross-links
ethane]dichloroplatinum(II) ((RS)-4F-Ph-PtCl2 and
(RR/SS)-4F-Ph-PtCl2) [14].
The 4F-Ph-PtCl2 complexes are cisplatin derivatives
with high selectivity for mammary carcinoma cells [30].
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662 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Figure 5. Antiproliferative effects of [1,2-diamino-1-(4-fluorophenyl)butane]dichloroplatinum(II) complexes on the
LnCaP/FGC cell line.
of platinum complexes with enantiomeric amine li-
gands can not only exhibit different conformational
features but also be differently processed by the cellu-
lar machinery. These results expand the general
knowledge about the influence of the stereochemistry
on the platinum-DNA adduct can influence the cell
response, and contribute to the understanding of the
processes that are crucial for antitumor activity [33].
adducts showed that a substantially greater proportion
of monofunctional adducts remained for the R-enanti-
omer and cisplatin than for the S-enantiomer [34].
4F-Ph-PtCl2 represents a compound with two asym-
metric centres. It is supposed that the identically sub-
stituted benzylic C-atoms might be the reason for the
missing enantioselectivity. Therefore, we tried to op-
timize the antiproliferative activity by exchanging a 4-
fluorophenyl moiety for an alkyl chain. In a previously
published SAR study [16, 17], we demonstrated a
marginal loss of cytotoxicity for the MCF-7 cell line but
an increase of stereoselectivity by use of a C2-methyl
substituent: (SS)-4F-Ph/Me-PtCl2 > (RR)-4F-Ph/Me-
PtCl2 > (SR)-4F-Ph/Me-PtCl2 = (RS)-4F-Ph/Me-PtCl2.
These positive results encouraged us to elongate the
C-alkyl chain. A comparison with the antitumor activity
of [1,2-diamino-1-(4-fluorophenyl)propane]platinum(II)
complexes indicated a distinct increase of activity due
In another study, molecular modeling techniques were
used to investigate the role of steric interactions be-
tween the ligands at the platinum and the DNA in influ-
encing the bifunctional binding of two enantiomers. It
was calculated that the S-configured [3-aminohexa-
hydroazepine]dichloroplatinum(II) should bind more
readily. Indeed, the binding of the S-enantiomer to calf
thymus DNA was slightly greater than the binding of
the R-enantiomer and slightly less than the binding of
cisplatin. Assays of the proportion of monofunctional
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Synthesis and Antitumor Activity of Platinum(II) Complexes 663
to the elongation of the C2-alkyl chain by a methylene
group. However, it is obvious that this modification
also changed the series of activity: (RR)-4F-Ph/Et-
PtCl2 > (SS)-4F-Ph/Et-PtCl2 > (RS)-4F-Ph/Et-PtCl2 >
(SR)-4F-Ph/Et-PtCl2. Interestingly, the cytotoxicity of
(RS)-4F-Ph/Me-PtCl2 and (SR)-4F-Ph/Me-PtCl2 was
identical for the MDA-MB 231 cell line.
Acknowledgments
The technical assistance of S. Bergemann and
I. Schnautz is acknowledged. Thanks are due to the
Deutsche Forschungsgemeinschaft for financial sup-
port (Gu 284/4-1 and FOR 463).
Experimental
It should be noted that the antitumor activity depended
on the kind of cells used in the in vitro assays. The
4F-Ph/Et-PtCl2 complexes showed the highest activity
against LnCaP/FGC prostate cancer cells, superior to
cisplatin. The hormone-dependent MCF-7 cells were
more sensitive to 4F-Ph/Et-PtCl2 complexes than the
hormone-independent MDA-MB 231 cells. With the
exception of (SR)-4F-Ph-PtCl2 on the MCF-7 cell line
and both (SR)-4F-Ph-PtCl2 and (RS)-4F-Ph-PtCl2 on
the MDA-MB 231 cell line, the complexes were more
active than cisplatin.
1H and 13C NMR spectra were taken at 293 K on a Bruker
Avance 300 MHz spectrometer with TMS as internal stand-
ard. IR analyses were performed with a Shimadzu IR-470
spectrophotometer. Melting points (uncorrected) were meas-
ured with a Mettler FP1 apparatus. All CC purifications were
done using silica gel Kieselgel® 100 (Merck). TLC was per-
formed on Kieselgel® 60 F254 plates (Merck). Mass spectra
were recorded on a Thermo-Fisons VG Auto Spec (70 eV).
Specific rotations were measured with a Perkin-Elmer 141
polarimeter. All reagents were obtained from Aldrich, except
benzylhydroxylamine which was synthesized following pub-
lished procedures [40, 41]. Experimental procedures were
performed with either (2R)- or (2S)-2-aminobutane-1-ol ((2R)-
1 or (2S)-1) as starting material. The synthesis was described
on the example of (2R)-1.
The antitumor activity was determined, besides the
asymmetric C-atoms, by the kind of leaving group. It
is expected that after the transport into the cytoplasm,
the platinum complexes hydrolize into the aqua- and
the diaquaplatinum(II) complexes, which then bind to
nucleobases [35].
Synthesis
(2R)-N,N-dibenzyl-2-aminobutan-1-ol ((2R)-2)
To a solution of (2R)-2-aminobutan-1-ol ((2R)-1) (10 g,
0.112 mol) in THF (200 mL), Na2CO3 (23.7 g, 0.224 mol), KI
(1.9 g, 11.2 mmol) and benzyl bromide (38.3 g, 0.224 mol)
were added. The suspension was stirred and refluxed for
10 h. After cooling and filtration, the solvent was evaporated
under reduced pressure. The residue was dissolved in
CH2Cl2 (200 mL). This solution was washed with NaOH 10%
(wt/vol) (100 mL), brine (50 mL), then dried over MgSO4, fil-
tered and evaporated under reduced pressure. Yellow oil:
30.2 g (yield: quantitative).
In previous studies [28, 36Ϫ38] on the leaving group
derivatives of [1,2-diamino-1,2-bis(4-fluorophenyl)-
ethane]platinum(II), we demonstrated a correlation be-
tween the strength of the Pt-X bond (X = leaving
group) and the cytotoxic properties. The same trend
can be estimated from the time activity curves de-
picted in Figures 2 and 3. (SS)- and (RR)-4F/Et-PtI2
were marginally active. The exchange of I- by Cl- en-
hanced the influence on tumor cell growth. Iodide is a
leaving group that is strongly bound to platinum.
1H NMR (CDCl3) (base): δ = 0.88 (t, 3H, J = 7.5 Hz, CH3),
1.21 and 1.76 (m and m, 1H and 1H, CH3CH2), 2.69 (m, 1H,
CHN), 3.14 (bs, 1H, OH), 3.38 (superimposed) and 3.50 (dd
and dd, 1H and 1H, J = 5.0 and 10.6 Hz, CH2OH), 3.40 and
3.79 (d and d, 2H and 2H, J = 13.2 Hz, ArCH2), 7.18Ϫ7.32
(10H, Ar). 13C NMR (CDCl3) (base): δ = 12.3 (CH3CH2), 18.5
(CH3CH2), 53.8 (ArCH2), 61.1 (CH2OH), 61.2 (CHN), 127.7
(C4Ј), 129.5 (C2ЈC6Ј), 129.6 (C3ЈC5Ј), 140 (C1Ј). IR (film)
The hydrolysis as prerequisite for DNA binding is re-
tarded. In contrast, the Pt-Cl bond is hydrolyzed in the
cytoplasm in sufficient amounts to achieve high cyto-
toxicity. Interestingly, the aquasulfatoplatinum(II) com-
plexes which are rapidly transformed into the diaqua-
platinum(II) form [39] were more active than the di-
chloroplatinum(II) complexes. This correlates very well
with the findings obtained with the parent compound
4F-Ph-PtSO4. (RR/SS)-4F-Ph-PtSO4 was enriched in
the MCF-7 cells, although it represented a cationic
Pt(H2O)2 form under the conditions used (Cl-free me-
dium) [32]. It was proposed that the platinum complex
was accumulated by an active transport. This might
also be possible for [1,2-diamino-1-(4-fluorophenyl)-
butane]platinum(II) complexes, which will be evalu-
ated in a further study.
(base): 3410 (ν O-H), 2840, 1445, 1044, 744, 692 cmϪ1
.
(2R)-N,N-dibenzyl-2-aminobutanal ((2R)-3)
In a three-necked flask maintained under N2, oxalyl chloride
(11 g, 87 mmol) was dissolved in dry CH2Cl2 (100 mL) and
cooled to Ϫ60°C. At this temperature, a solution of anhy-
drous DMSO (12.3 g, 157 mmol) in dry CH2Cl2 (25 mL) was
added. After 2 min of stirring, a solution of (2R)-N,N-dibenzyl-
2-aminobutan-1-ol ((2R)-2) (21.2 g, 79 mmol) in dry CH2Cl2
(50 mL) was added dropwise during 5 min. This mixture was
stirred for 15 min, and TEA (40 g, 395 mmol) was added. The
suspension formed was stirred at about Ϫ30°C for 5 min and
then warmed to room temperature. Water (200 mL) was ad-
ded, and the mixture was decanted. The organic layer was
washed with brine (50 mL), dried over MgSO4, filtered and
evaporated under reduced pressure. The crude product (dark
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

664 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
yellow oil: 19.4 g) (yield: 92% estimated by 1H NMR) was not
purified before analysis and directly used for the next step.
(1S,2R)-1,2-diamino-1-(4-fluorophenyl)butane ((1S,2R)-7)
To a solution of (1S,2R)-N,N-dibenzyl-2-azido-1-(4-fluorophen-
yl)butane ((1S,2R)-6) (1.4 g, 3.6 mmol) in MeOH (75 mL)
was added palladium on charcoal 10% (0.3 g). This suspen-
sion was stirred and refluxed for 30 min. Afterwards am-
monium formate (0.45 g, 7.2 mmol) was added, and the mix-
ture was stirred for an additional 10 h. An equal amount of
ammonium formate was then added, and the reaction was
carried on for 10 h. The reaction medium was filtered on ce-
lite, and the solvent evaporated under reduced pressure. The
oily residue was dissolved in CH2Cl2 (100 mL). This solution
was washed with a solution of sodium carbonate 20% (wt/
vol) (50 mL) and brine (50 mL). It was further dried over
MgSO4, filtered and evaporated under reduced pressure to
give 0.51 g of yellow oil. Upon addition of HCl in diethyl ether,
a yellowish solid precipitated. It was filtered and recrystallized
from iPrOH/MeOH/Et2O (5:5:20). White solid, 0.66 g (di-
hydrochloride); mp: 290.1°C with some decomposition.
(Yield: 72%). ee (%) for the two enantiomers: 98.
1H NMR (CDCl3) (base): δ = 0.95 (t, 3H, J = 7.4 Hz, CH3),
1.67 (m, 2H, CH3CH2), 3.06 (t, 1H, J = 6.8 Hz, NCHC=O),
3.69 and 3.78 (d and d, 2H and 2H, J = 13.7 Hz, ArCH2),
7.18Ϫ7.38 (10H, Ar), 9.70 (s, 1H, CH=O). 13C NMR (CDCl3)
(base): δ = 12.3 (CH3), 18.0 (CH3CH2), 55.4 (ArCH2), 69.0
(CHN), 127.8 (C4Ј), 128.9 (C2ЈC6Ј), 129.4 (C3ЈC5Ј), 139.8
(C1Ј), 204.2 (C=O). IR (film) (base): 2795, 2690, 1712 (C=O),
1444, 1361, 1135, 1066, 730, 692 cmϪ1
.
(1S,2R)-N,N-dibenzyl-2-amino-1-(4-fluorophenyl)butan-1-ol
((1S,2R)-4)
To a solution of (2R)-N,N-dibenzyl-2-aminobutanal ((2R)-3)
(10 g, 37.4 mmol) cooled to 0°C, an 1 M solution of 4-fluoro-
phenylmagnesium bromide in THF (41 mL, 41 mmol) was
added dropwise. The solution was stirred for 2 h at 0°C and
poured into saturated NH4Cl solution (100 mL). The mixture
was extracted twice with diethyl ether (100 mL). The organic
layers were collected, washed with brine (50 mL), dried over
MgSO4, filtered and evaporated under reduced pressure (vis-
cous yellow oil, 13.52 g). The diastereoisomeric mixture
(87:13) was separated by column chromatography
(petroleum ether40-60/acetone 95 : 5). Light yellow viscous oil,
9.6 g ((1S,2R)-4, yield: 81%).
1H NMR ([D6]-DMSO) (hydrochloride): δ = 1.01 (t, 3H, J = 7.3
Hz, CH3), 1.60 and 1.73 (m and m, 1H and 1H, CH3-CH2),
3.66 (m, 1H, CH2CHN), 4.73 (d, 1H, J = 4.5 Hz, ArCH), 7.35
(m, 2H, ArH3ЈH5Ј), 7.74 (m, 2H, ArH2ЈH6Ј), 8.65 and 9.14
(bs and bs, 3H and 3H, NH3+). 13C NMR ([D6]-DMSO) (hydro-
chloride): δ = 9.4 (CH3), 21.7 (CH2), 53.8 (CH2CHN), 54.4
(ArCH), 115.5 (d, JC-F
= 22 Hz, ArC3ЈC5Ј), 129 (d,
1H NMR (CDCl3) (base): δ = 0.99 (t, 3H, J = 7.4 Hz, CH3),
1.20 and 1.42 (m and m, 1H and 1H, CH3CH2), 2.70 (dt, 1H,
CHN), 3.57 and 3.71 (d and d, 2H and 2H, J = 13.8 Hz,
ArCH2), 4.94 (d, 1H, J = 3.9 Hz, ArCH), 6.92 (m, 2H, F-
ArH3ЈH5Ј), 7.12 (m, 2H, F-ArH2ЈH6Ј), 7.17Ϫ7.31 (10H, Ar).
IR (film) (base): 3220 (ν O-H), 2915, 1455, 1219 (ν C-F), 842
(ν ArF), 740, 695 cmϪ1. α2D0: (base): (1R,2S)-4: α2D0 = +36.3
(c = 0.6, MeOH), (1S,2R)-4: α2D0 = Ϫ37 (c = 1, MeOH).
JC-F = 3Hz, ArC1Ј), 130.3 (d, JC-F = 9 Hz, ArC2’C6’), 162.2
(d, JC-F = 245 Hz, ArC4Ј). IR (KBr, 1%) (hydrochloride): 2905
(ν N-H), 2720 (ν C-H), 1603, 1230 (ν C-F), 1075, 852 (ν Ar-
F) cmϪ1. α2D0 (hydrochloride): (1R,2S)-7: α2D0 = +24.4 (c = 0.8,
MeOH), (1S,2R)-7: α2D0 = Ϫ24.2 (c = 2, MeOH). MS (base):
m/z (%) = 183 (41) [M+], 166 (30), 124 (21), 97 (6), 58 (100).
(2R)-N-(tert-butoxycarbonyl)-2-aminobutan-1-ol ((2R)-8)
(1S,2R)-N,N-dibenzyl-2-amino-1-azido-1-(4-fluorophenyl)-
butane ((1S,2R)-6)
A solution of (2R)-2-aminobutan-1-ol ((2R)-1) (5 g, 56.1
mmol) and TEA (6.24 g, 62 mmol) in CH2Cl2 (50 mL) was
cooled to 0°C. A solution of di-tert-butyl dicarbonate (13.5 g,
62 mmol) in CH2Cl2 (10 mL) was added dropwise. The reac-
tion was carried on for 12 h at room temperature. The sol-
vents and reagents were evaporated under reduced pressure
(60°C/66 Pa). Yellow oil: 10.6 g (yield: quantitative).
(1S,2R)-N,N-dibenzyl-2-amino-1-(4-fluorophenyl)butan-1-ol
((1S,2R)-4) (9.6 g, 26.4 mmol) and TEA (8.02 g, 79.2 mmol)
were dissolved in diethyl ether (200 mL). This solution was
cooled in an ice bath. Methanesulfonyl chloride (4.5 g, 39.6
mmol) was added dropwise. The mixture was stirred for 10 h
at room temperature. Water (100 mL) and sodium azide (3.4
g, 52.8 mmol) were added. After 12 h, the organic layer was
taken up and washed with brine (50 mL). After drying over
MgSO4 and filtration, the solvent was evaporated in vacuo.
The mixture of regioisomers ((1S,2R)-6 and (1R,2S)-6)
(93:7) was separated by column chromatography (petroleum
ether40-60/acetone, 98:2). White solid, 6.25 g; mp: 84°C.
(yield: 61%).
1H NMR (CDCl3): δ = 0.96 (t, 3H, J = 7.4 Hz, CH3CH2), 1.45
(s, 9H, C(CH3)3), 1.54 (m, 2H, CH3CH2), 3.56 (m, 2H super-
imposed, CHN and CH2OH), 3.66 (m, 1H, CH2OH), 4.67 (bs,
1H, O=CNH). 13C NMR (CDCl3): δ = 11.2 (CH3CH2), 25.2
(CH3CH2), 29.0 (C(CH3)3), 55.0 (CHN), 66.2 (COH), 80.2
(C(CH3)3), 157.3 (C=O).
(2R)-N-(tert-butoxycarbonyl)-2-aminobutanal ((2R)-9)
1H NMR (CDCl3) (base): δ = 0.97 (t, 3H, J = 7.3 Hz, CH3),
1.46 and 1.81 (m and m, 1H and 1H, CH3CH2), 2.73 (dt, 1H,
CHNBn2), 3.60 and 3.92 (d and d, 2H and 2H, J = 13.8 Hz,
CH2Ar), 4.77 (d, 1H, J = 5.5 Hz, ArCH), 6.94Ϫ7.05 (m, 2H,
F-ArH3ЈH5Ј), 7.18Ϫ7.37 (m, 12H, Ar + 4F-Ar). 13C NMR
(CDCl3) (base): δ = 13.2 (CH3), 19.6 (CH3CH2), 54.8 (ArCH2),
64.2 (CH2CHN), 67.1 (ArCH), 116.0 (d, JC-F = 21 Hz, F-
ArC3ЈC5Ј), 127.7 (ArC4Ј), 128.9 (ArC2ЈC6Ј), 129.5
(ArC3ЈC5Ј), 129.6 (d, JC-F = 8 Hz, F-ArC2ЈC6Ј), 139.8 (F-
ArC1Ј), 140.2 (ArC1Ј), 162.8 (d, JC-F = 246 Hz, F-ArC4Ј). IR
(film) (base): 2900, 2075 (ν N3), 1591, 1496, 1444, 1220 (C-
Oxalyl chloride (13.4 g, 106 mmol) in dry CH2Cl2 (200 mL)
was placed into a three-necked flask under N2 and cooled
to Ϫ60°C. This temperature was maintained during all the
reaction. Anhydrous DMSO (15 g, 193 mmol) in dry CH2Cl2
(50 mL) was added dropwise. After 2 min of stirring, a
solution of (2R)-N-(tert-butoxycarbonyl)-2-aminobutan-1-ol
((2R)-8) (18.23 g, 96 mmol) in dry CH2Cl2 (50 mL) was added
dropwise during 5 min. After 5 min of stirring, TEA (48.8 g,
483 mmol) was added. The suspension was stirred at Ϫ30°C
for 5 min, then allowed to warm to room temperature. Water
(200 mL) was added. After decantation, the organic layer was
washed with brine (50 mL), dried over MgSO4, filtered and
evaporated under reduced pressure. The yellowish oil
F), 823 (ν ArF), 737, 694 cmϪ1. α2D0 (base); (1R,2S)-6: α2D0
=
+17.4 (c = 0.2, MeOH), (1S,2R)-6: α2D0 = Ϫ17.9 (c = 0.4,
MeOH).
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Synthesis and Antitumor Activity of Platinum(II) Complexes 665
(1R,2R)-N2-(tert-butoxycarbonyl)-1,2-diamino-1-(4-fluoro-
1
(16.91 g, yield: 90% estimated by H NMR) was not purified
before analysis and directly used for the next step.
phenyl)butane ((1R,2R)-12)
(1R,2R)-N1-benzyl-N2-(tert-butoxycarbonyl)-2-amino-1-(4-
fluorophenyl)-1-hydroxyaminobutane (1R,2R)-11 (8.26 g,
21.3 mmol) was dissolved in MeOH (250 mL), and palladium
on charcoal 10% (0.8 g) was added. The suspension was
refluxed for 30 min. Ammonium formate (2.68 g, 42.5 mmol)
was added. The suspension was refluxed for 12 h. An equal
amount of ammonium formate was then added, and the reac-
tion was carried on for 12 h. The cooled reaction mixture was
filtered on celite, and the solvent evaporated under reduced
pressure. The residue was extracted with CH2Cl2 (100 mL).
This solution was washed with brine (50 mL), dried over
MgSO4, filtered and evaporated under reduced pressure. The
oily residue (6 g) was directly used for the next step.
1H NMR (CDCl3): δ = 0.97 (t, 3H, J = 7.5 Hz, CH3CH2), 1.46
(s, 9H, C(CH3)3), 1.67 and 1.95 (m and m, 1H and 1H,
CH3CH2), 4.20 (m, 1H, CHN), 5.12 (bs, 1H, O=CNH), 9.58
(s, 1H, O=CH). IR (film): 3320 (ν N-H), 2940 (ν C-H), 2705 (ν
C-H), 1680 large (ν C=O aldehyde and carbamate), 1502,
1159 cmϪ1
.
(2R)-[N-(tert-butoxycarbonyl)-2-aminobutylidene]benzyl-
amine N-oxide ((2R)-10)
To a solution of (2R)-N-(tert-butoxycarbonyl)-2-aminobutanal
((2R)-9) (5.92 g, 31.6 mmol) and benzylhydroxylamine
(3.9 g, 31.7 mmol) in dry CH2Cl2 (150 mL) MgSO4 was added
(3.8 g, 31.7 mmol). The suspension was stirred for 15 h at
room temperature. After filtration, the solvent was evaporated
under reduced pressure, and the obtained yellow solid was
purified by column chromatography (diethyl ether) to afford
6.14 g of yellow solid; mp: 129.2°C (yield: 66%).
(1R,2R)-1,2-diamino-1-(4-fluorophenyl)butane ((1R,2R)-13)
(1R,2R)-12 was dissolved in a solution of HCl in anhydrous
MeOH (10%, wt/vol) (50 mL). This solution was stirred at
room temperature for 8 h. Then, the solvent was evaporated
under reduced pressure. The yellow solid obtained was
treated with water (50 mL), NaOH 40% (wt/vol) (10 mL) and
extracted two times with CH2Cl2 (100 mL). The organic solu-
tion was washed with brine (50 mL), dried over MgSO4, fil-
tered and evaporated under reduced pressure. The liquid ob-
tained was dissolved in MeOH (20 mL) and a solution of D-
(Ϫ)-(2S,3S)-tartaric acid (1.54 g, 10.3 mmol) in MeOH (20
mL) was added. This mixture was heated to reflux for 10 min
and then let to cool to room temperature. The white crystals
(1.41 g) were collected by filtration, washed two times with
MeOH and dried at 60°C under 13.3 kPa. White crystals
(yield: 20%); mp: 281.7°C with some decomposition. A small
fraction of these salts were transformed into base and hydro-
chloride for analysis. ee (%) for the two enantiomers: 98.
1H NMR (CDCl3): δ =0.91 (t, 3H, J = 7.4 Hz, CH3CH2), 1.41
(s, 9H, C(CH3)3), 1.78 (m, 2H, CH3CH2), 4.32 (m, 1H,
CHNBOC), 4.87 (s, 2H, ArCH2), 5.89 (bs, 1H, O=CNH), 6.82
(bs, 1H, N=CH), 7.39Ϫ7.42 (5H, Ar). 13C NMR (CDCl3): δ =
11.1 (CH3CH2), 24.6 (CH3CH2), 30.0 (C(CH3)3), 50.6
(CH2CHN), 70.4 (ArCH2), 80.2 (C(CH3)3), 129.7 (ArC2ЈC6Ј),
129.9 (ArC3ЈC4ЈC5Ј), 133.3 (ArC1Ј), 139.0 (C=N), 156.1
(C=O). IR (KBr, 1%): 3323 (ν N-H), 2922 (ν C-H), 1677
(ν C=O, C=N), 1518, 1250, 1163, 700 cmϪ1
.
(1R,2R)-N1-benzyl-N2-(tert-butoxycarbonyl)-2-amino-1-(4-
fluorophenyl)-1-hydroxyaminobutane ((1R,2R)-11)
1H NMR ([D6]-DMSO) (hydrochloride): δ = 1.01 (t, 3H, J = 5.5
Hz, CH3), 1.62 and 1.73 (m and m, 1H and 1H, CH2), 3.67
(m, 1H, CH2CHN), 4.76 (d, 1H, J = 4.1 Hz, ArCH), 7.34 (m,
2H, ArH3’H5’), 7.76 (m, 2H, ArH2H6’), 8.59 and 9.32 (bs and
bs, 3H and 3H, NH3+). 13C NMR ([D6]-DMSO) (hydrochlo-
ride): δ = 9.4 (CH3), 21.7 (CH2), 53.8 (CH2CHN), 54.4 (ArCH),
115.5 (d, JC-F = 22 Hz, ArC3ЈC5Ј), 129.0 (d, JC-F = 3 Hz,
A solution of (2R)-[N-(tert-butoxycarbonyl)-2-aminobutylid-
ene]benzylamine N-oxide ((2R)-10) (6.14 g, 21 mmol) in an-
hydrous THF (100 mL) was cooled to Ϫ40°C. A 2 M solution
of 4-fluorophenylmagnesium bromide in diethyl ether (31.5
mL, 63 mmol) was added dropwise at Ϫ40°C. The reaction
medium was stirred at this temperature for 4 h. Then, it was
let to warm to room temperature and poured into a saturated
solution of NH4Cl (200 mL). After three extractions with di-
ethyl ether (100 mL), the organic layer was washed with brine
(50 mL), dried over MgSO4, filtered and evaporated under
reduced pressure. The brown solid obtained (8.49 g: mixture
of diastereoisomers (1R,2R)-11 and (1S,2R)-11: 90:10) from
which (1R,2R)-11 was separated by column chromatography
(petroleum ether(40-60)/acetone, 95:5). Yellow solid, 4.5 g; mp:
127.5°C (yield: 68%).
ArC1Ј), 130.3 (d, JC-F = 9 Hz, ArC2ЈC6Ј), 162.2 ( d, JC-F
=
246 Hz, ArC4Ј). IR (KBr, 1%) (hydrochloride): 2905 (ν N-H),
2720 (ν C-H), 1603, 1230 (ν C-F), 1075, 852 (ν Ar-F) cmϪ1
.
α2D0 (hydrochloride), (1R,2R)-13: α2D0 = +7.5 (c = 0.4, MeOH),
(1S,2S)-13: α2D0 = Ϫ7.7 (c = 0.2, MeOH). MS (base): m/z
(%) = 183 (41) [M+], 166 (30), 124 (21), 97 (6), 58 (100).
[1,2-Diamino-1-(4-fluorphenyl)butane]diiodoplatinum(II)
1H NMR (CDCl3): δ = 0.86 (t, 3H, J = 7.6 Hz, CH3CH2), 1.35
(m, 2H, CH3CH2), 1.53 (s, 9H, C(CH3)3), 3.24 (d, 1H, J = 10.2
Hz, ArCH), 3.55 and 3.60 (d and d, 1H and 1H, J = 13.9 Hz,
ArCH2), 4.06 (m, 1H, CH2CHN), 4.47 (d, 1H, J = 9.7, O=
CNH), 6.82 (bs, 1H, NOH), 7.06 (m, 2H, F-ArH3ЈH5Ј),
7.20Ϫ7.35 (7H, Ar). 13C NMR (CDCl3): δ = 11.0 (CH3CH2),
25.4 (CH3CH2), 29.2 (C(CH3)3), 54.3 (CH2CHN), 61.0
(ArCH2), 74.2 (ArCH), 80.9 (C(CH3)3), 115.7 (d, JC-F = 21 Hz,
F-ArC3ЈC5Ј), 127.5 (ArC4Ј), 128.8 (ArC2ЈC6Ј), 129.1
(ArC3ЈC5Ј), 131.0 (d, J = 3Hz, F-ArC1Ј), 132.2 (ArC1Ј), 132.3
To an aqueous solution (5 mL) of the diamine (0.25 mmol),
K2PtI4 (0.25 mmol) dissolved in 5 mL water was added after
the pH was adjusted to 6.5Ϫ7.5 with 0.1 N NaOH. The reac-
tion mixture was stirred in the dark for 24 h. Subsequently, it
was acidified with 0.1 N HCl, and the yellow precipitate was
sucked off and dried over P2O5 in vacuo.
(RR)-4F-Ph/Et-PtI2/(SS)-4F-Ph/Et-PtI2
1H NMR ([D7]-DMF): δ = 0.85 (t, 3H, CH3), 1.30Ϫ1.51 (m,
2H, CH2), 3.15 (1H, CH-alkyl), 3.80 (m, 1H, Ar-H), 5.05 (br,
1H, NH), 5.38 (br, 1H, NH), 5.72 (br, 1H, NH), 6.00 (br, 1H,
NH), 7.21Ϫ7.32 (m, 2H, H3’H5’), 7.67Ϫ7.79 (m, 2H, H2’H6’).
Brownish powder. (RR)-4F-Ph/Et-PtI2; yield: 29%. (SS)-4F-
Ph/Et-PtI2; yield: 29%. C10H15N2FPtI2 (633.0): calc: C 18.95,
H 2.37, N 4.43; found: C 18.47, H 2.19, N 4.16 ((RR)-4F-Ph/
(d, JC-F = 8 Hz, F-ArC2ЈC6Ј), 159.1 (C=O), 163.2 (d, JC-F
246 Hz, F-ArC4Ј). IR (KBr, 1%): 3350 (ν N-H O-H), 2960 (ν
C-H), 1677 (ν C=O), 1538, 1219 (ν C-F), 1155, 728 cmϪ1
2D0; (1R,2R)-11: α2D0 = +2.3 (c = 0.9, MeOH), (1S,2S)-11:
α2D0 = Ϫ2.1 (c = 0.5, MeOH).
=
.
α
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

666 Gust et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 654−667
Et-PtI2); found: C 18.55, H 2.36, N 4.16 ((SS)-4F-Ph/Et-PtI2).
found: C 26.76, H 3.29, N 6.43 ((RS)-4F-Ph/Et-PtCl2); found:
C 26.74, H 3.37, N 6.11 ((SR)-4F-Ph/Et-PtCl2).
(RS)-4F-Ph/Et-PtI2/(SR)-4F-Ph/Et-PtI2
1H NMR ([D7]-DMF): δ = 0.95 (t, 3H, CH3), 1.52 (m, 2H, CH2),
3.19 (1H, CH-alkyl), 4.13 (m, 1H, Ar-H), 4.80 (br, 1H, NH),
5.50 (br, 2H, NH), 6.00 (br, 1H, NH), 7.20Ϫ7.29 (m, 2H,
H3ЈH5Ј), 8.12Ϫ8.24 (m, 2H, H2’H6’). Brownish powder. (RS)-
4F-Ph/Et-PtI2; yield: 29%. (SR)-4F-Ph/Et-PtI2; yield: 30%.
C10H15N2FPtI2 (633.0): calc: C 18.95, H 2.37, N 4.43; found:
C 18.55, H 2.35, N 4.20 ((RS)-4F-Ph/Et-PtI2); found: C 18.57,
H 2.21, N 4.78 ((SR)-4F-Ph/Et-PtI2).
Biological methods
Cell culture
The human MCF-7 and MDA-MB 231 breast cancer cell lines
as well as the LwCaP/FGC cell line were obtained from the
American Type Culture Collection (ATCC, USA). Cell line
banking and quality control were performed according to the
seed stock concept reviewed by Hay [42]. The MCF-7 cells
were maintained in L-glutamine containing Eagle’s MEM
(Sigma, Germany) supplemented with NaHCO3 (2.2 g/L), so-
dium pyruvate (110 mg/L), gentamycin (50 mg/L) and 10%
fetal calf serum (FCS; Gibco, Germany), using 75-cm2 culture
flasks in a humidified atmosphere (95% air/5% CO2) at 37°C.
The MDA-MB 231 cells (McCoy’s 5A medium supplemented
with NaHCO3 (2.2 g/L), sodium pyruvate (110 mg/L), genta-
mycin (50 mg/L), 5% FCS) and the LwCaP/FGC cells (L-glu-
tamine-containing RPMI 1640 supplemented with NaHCO3
(2.0 g/L), gentamycin (50 mg/L), 7.5% FCS) were maintained
under the same conditions. The cell lines were weekly pas-
saged after treatment with 0.05% trypsin/0.02% ethylene-
diaminetetraacetic acid (EDTA; Boehringer, Germany). Myco-
plasma contamination was regularly monitored, and only
mycoplasma-free cultures were used.
Aqua[1,2-diamino-1-(4-fluorphenyl)butane]sulfatoplatinum(II)
The corresponding sulfatoplatinum(II) complexes were ob-
tained by addition of Ag2SO4 (0.195 mmol) to the aqueous
suspensions (30 mL) of the diiodoplatinum(II) complexes (0.2
mmol). After stirring for 3 days, the precipitated AgI was sepa-
rated by filtration and the resulting clear solution was lyophil-
ized.
(RR)-4F-Ph/Et-PtSO4/(SS)-4F-Ph/Et-PtSO4
1H NMR ([D6]-DMSO): δ = 0.68 (t, 3H, CH3), 1.20Ϫ1.35 (m,
2H, CH2), 3.20 (1H, CH-alkyl), 3.77 (m, 1H, Ar-H), 5.82 (br,
1H, NH), 6.17 (br, 1H, NH), 6.30 (br, 1H, NH), 6.42 (br, 1H,
NH), 7.21Ϫ7.30 (m, 2H, H3ЈH5Ј), 7.45Ϫ7.58 (m, 2H,
H2ЈH6Ј). Colorless powder. (RR)-4F-Ph/Et-PtSO4; yield:
20%. (SS)-4F-Ph/Et-PtSO4; yield: 21%. C10H15N2FPtSO5 ϫ1/2
H2O (500.3): calc: C 23.98, H 3.20, N 5.60, found: C 24.22,
H 3.40, N 5.40 ((RR)-4F-Ph/Et-PtSO4); found: C 24.06, H
3.50, N 5.64 ((SS)-4F-Ph/Et-PtSO4).
In vitro chemosensitivity assays
The in vitro testing of the platinum complexes for antitumor
activity was carried out on exponentially dividing human can-
cer cells, according to a previously published microtiter assay
[29, 43]. Briefly, in 96-well microtiter plates, 100 µL of a cell
suspension at 7700 cells/mL culture medium (MCF-7) or
3200 cells/mL (MDA-MB 231) or 2700 cells/mL (LwCaP/FGC)
was plated into each well and incubated at 37°C for 3 days
(MCF-7, MDA-MB 231) or 5 days (LwCaP/FGC) in a humidi-
fied atmosphere (5% CO2). By addition of an adequate vol-
ume of a stock solution of the respective compound (solvent:
DMF) to the medium, the aimed test concentration was ob-
tained. For each test concentration and for the control, which
contained the corresponding amount of DMF, 16 wells were
used. After the proper incubation time, the medium was re-
moved, the cells were fixed with a glutardialdehyde solution
and stored under phosphate buffered saline (PBS) at 4°C.
Cell biomass was determined by a crystal violet staining tech-
nique, as described earlier [29, 43]. The efficiency of the com-
plexes is expressed as corrected% T/Ccorr values according
to the following equations:
(RS)-4F-Ph/Et-PtSO4/(SR)-4F-Ph/Et-PtSO4
1H NMR ([D6]-DMSO): δ = 0.85 (t, 3H, CH3), 1.23 (m, 2H,
CH2), 3.16 (1H, CH-alkyl), 4.20 (m, 1H, Ar-H), 6.05 (br, 1H,
NH), 6.26 (br, 1H, NH), 6.52 (br, 2H, NH), 7.21Ϫ7.32 (m, 2H,
H3’H5’), 7.51Ϫ7.60 (m, 2H, H2’H6’). Colorless powder. (RS)-
4F-Ph/Et-PtSO4; yield: 29%. (SR)-4F-Ph/Et-PtSO4; yield:
25%. C10H15N2FPtSO5 (491.3): calc: C 24.42, H 3.05, N
5.69; found: C 23.71, H 3.75, N 5.53 ((RS)-4F-Ph/Et-PtSO4
ϫ 1 H2O); found: C 24.28, H 3.24, N 5.56 ((SR)-4F-Ph/Et-
1
PtSO4 ϫ /2 H2O).
[1,2-Diamino-1-(4-fluorphenyl)butane]dichloroplatinum(II)
The respective sulfatoplatinum(II) complex (0.2 mmol) was
converted into the dichloroplatinum(II) complex by treatment
with 2 N HCl in aqueous solution. The yellow precipitate was
collected and dried over P2O5 in vacuo.
Cytostatic effect: T/Ccorr [%] = [(TϪCo)/(CϪCo)] ϫ 100
where T (test) and C (control) were the optical densities at
590 nm of the crystal violet extract of the cells in the wells
(i.e. the chromatin-bound crystal violet extracted with ethanol
70%), and Co was the density of the cell extract immediately
before treatment.
(RR)-4F-Ph/Et-PtCl2/(SS)-4F-Ph/Et-PtCl2
1H NMR ([D7]-DMF): δ = 0.85 (t, 3H, CH3), 1.30 (m, 2H, CH2),
3.30 (1H, CH-alkyl), 4.15 (m, 1H, Ar-H), 5.21 (br, 1H, NH),
5.50 (br, 1H, NH), 5.89 (br, 1H, NH), 6.08 (br, 1H, NH),
7.21Ϫ7.30 (m, 2H, H3ЈH5Ј), 7.70Ϫ7.80 (m, 2H, H2ЈH6Ј). Yel-
low powder. (RR)-4F-Ph/Et-PtCl2; yield: 38%. (SS)-4F-Ph/
Et-PtCl2; yield: 31%. C10H15FCl2N2Pt (450.1): calc: C 26.66,
H 3.33, N 6.22; found: C 26.64, H 3.36, N 6.42 ((RR)-4F-
Ph/Et-PtCl2); found: C 26.69, H 3.32, N 6.33 ((SS)-4F-Ph/
Et-PtCl2).
Cytocidal effect: τ [%] = [(TϪCo)/Co] ϫ 100.
A microplate reader at 590 nm (Flashscan AnalytikJena AG)
was used for the automatic estimation of the optical density
of the crystal violet extract in the wells.
(RS)-4F-Ph/Et-PtCl2/(SR)-4F-Ph/Et-PtCl2
1H NMR ([D7]-DMF): δ = 0.95 (t, 3H, CH3), 1.48 (m, 2H, CH2),
3.50 (1H, CH-alkyl), 4.20 (m, 1H, Ar-H), 4.89 (br, 1H, NH),
5.72 (br, 2H, NH), 6.15 (br, 1H, NH), 7.21Ϫ7.33 (m, 2H,
H3ЈH5Ј), 8.20Ϫ8.32 (m, 2H, H2’H6’). Yellow powder. (RS)-
4F-Ph/Et-PtCl2; yield: 36%. (SR)-4F-Ph/Et-PtCl2; yield:
38%. C10H15FCl2N2Pt (450.1): calc: C 26.66, H 3.33, N 6.22;
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