Synthesis, crystal structure and cytotoxicity of new oxaliplatin analogues indicating that improvement of anticancer activity is still possible

Article abstract of DOI:10.1016/j.ejmech.2004.04.003

Oxaliplatin, (trans-R,R-cyclohexane-1,2-diamine)oxalatoplatinum(II), has recently been approved for combination chemotherapy of metastatic colorectal cancer. Oxaliplatin is significantly more active than its trans-S,S isomer and the mixture of both enanti

Full text of DOI:10.1016/j.ejmech.2004.04.003

European Journal of Medicinal Chemistry 39 (2004) 707–714
www.elsevier.com/locate/ejmech
Original article
Synthesis, crystal structure and cytotoxicity of new oxaliplatin analogues
indicating that improvement of anticancer activity is still possible
Markus Galanski *, AfshinYasemi, Susanna Slaby, Michael A. Jakupec, Vladimir B. Arion,
Monika Rausch, Alexey A. Nazarov, Bernhard K. Keppler
Institute of Inorganic Chemistry, University of Vienna, Waehringerstr. 42, A-1090 Vienna, Austria
Received 24 November 2003; received in revised form 26 April 2004; accepted 30 April 2004
Dedicated to Prof. Bernt Krebs on the occasion of his 65th birthday
Abstract
Oxaliplatin, (trans-R,R-cyclohexane-1,2-diamine)oxalatoplatinum(II), has recently been approved for combination chemotherapy of
metastatic colorectal cancer. Oxaliplatin is significantly more active than its trans-S,S isomer and the mixture of both enantiomers. New
oxaliplatin analogues, (SP-4-3)-(4-methyl-trans-cyclohexane-1,2-diamine)oxalatoplatinum(II) and (SP-4-3)-(4-ethyl-trans-cyclohexane-
1,2-diamine)oxalatoplatinum(II), have been synthesized, and their cytotoxicity has been tested in comparison to oxaliplatin, its corresponding
trans-S,S isomer, and the mixture of both enantiomers. In comparison to oxaliplatin, even the trans-R,R/trans-S,S mixture of the 4-methyl and
4-ethyl substituted oxaliplatin analogues have shown an equivalent cytotoxicity in ovarian cancer cells (CH1) and superior antiproliferative
properties in colon cancer cells (SW480) in the case of a predominantly equatorial position of the substituent at position 4 of the
trans-cyclohexane-1,2-diamine ligand, whereas an axial substitution results in decreased cytotoxic potency.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Platinum; Anticancer complexes; Oxaliplatin; Synthesis; Crystal structure; Cytotoxic activity
1. Introduction
Platinum-based coordination complexes had first been
identified as strong inhibitors of bacterial proliferation by
O
H3N
H3N
Cl
Cl
H3N
H3N
O
Barnett Rosenberg in the 1960s [1]. Subsequently, their cyto-
static activity had been examined in tumor-bearing animals
with the encouraging result of a complete inhibition of mu-
rine solid sarcoma-180 by cisplatin [2]. Today, cisplatin,
cis-diamminedichloroplatinum(II), and carboplatin, cis-
diammine(1,1-cyclobutanedicarboxylato)platinum(II), are
routinely used in the clinic as anticancer compounds [3–7].
Apart from the worldwide success of cisplatin and carbopl-
atin,oxaliplatin,(trans-R,R-cyclohexane-1,2-diamine)oxala-
toplatinum(II), a third generation platinum drug, has been
approved for combination chemotherapy of metastatic colo-
rectal cancer in many countries of Asia, Latin America and
Europe (Fig. 1).
Pt
Pt
O
O
a
b
H2
N
O
O
O
Pt
N
O
H2
c
* Corresponding author. Tel.: +43-1-4277-52603;
fax: +43-1-4277-52680.
Fig. 1. Platinum(II) complexes in clinical use, cisplatin (a), carboplatin (b)
and oxaliplatin (c).
E-mail address: markus.galanski@univie.ac.at (M. Galanski).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.04.003

708
M. Galanski et al. / European Journal of Medicinal Chemistry 39 (2004) 707–714
H
H
N2
O
O
N2
O
O
In the United States, oxaliplatin, Eloxatin™ (Sanofi), was
O
O
O
O
approved in 2002, because of its significant benefit in terms
of response rate and time to progression, although a long-
term benefit such as increased survival time has not yet been
demonstrated.1
Pt
Pt
N
N
H2
H2
rac-1(eq/ax)
rac-2(eq/ax)
Oxaliplatin, developed by Kidani et al. [8], first attracted
interest because of its particularly high activity, even in
cisplatin-resistant tumor models. Therefore, it is currently
being explored for its potential as a treatment option after
failure of cisplatin or carboplatin therapy. However, oxali-
platin is primarily used in colorectal cancer in combination
with 5-fluorouracil and leucovorin (5-FU/LV). It shows syn-
ergistic effects with 5-FU even in 5-FU-resistant tumors and
demonstrates excellent safety with a specific and manageable
set of toxicities [9].
The similar or even higher cytotoxicity of oxaliplatin
despite lower levels of initial DNA adduct formation and
similar magnitudes of adduct removal as compared to cis-
platin indicates that the DNA lesions produced by oxaliplatin
are on average more lethal to the cell. This correlates with a
stronger inhibition of DNA chain elongation in the course of
replication [10]. Furthermore, adducts induced by oxaliplatin
are not recognized by the damage recognition complex in-
volved in the mismatch repair system, and cells with
cisplatin-resistance due to enhanced replicative bypass (ac-
companied or not by a loss of the futile and eventually fatal
mismatch repair activity) consistently retain their sensitivity
to oxaliplatin [11].
Fig. 2. Structure of 4-methyl and 4-ethyl substituted (trans-cyclohexane-
1,2-diamine)oxalatoplatinum(II) complexes, rac-1(eq/ax) and rac-
2(eq/ax).
Based on the assumption that the steric demand and/or the
hydrophobicity of the cyclohexane ring are structural re-
quirements for the specific pharmacological properties of
oxaliplatin, we assumed that derivation of the cyclohexane
ring might result in a marked effect on antitumor activity.
Following this concept and in order to explore structure–
activity relationships, we have synthesized new oxaliplatin
analogues, (SP-4-3)-(4-methyl-trans-cyclohexane-1,2-dia-
mine)oxalatoplatinum(II) and (SP-4-3)-(4-ethyl-trans-
cyclohexane-1,2-diamine)oxalatoplatinum(II) (Fig. 2), and
tested their in vitro antitumor activity in comparison to oxali-
platin, its corresponding trans-S,S-isomer, (SP-4-2)-(trans-
S,S-cyclohexane-1,2-diamine)oxalatoplatinum(II), and the
mixture of both enantiomers, (SP-4-2)-(trans-cyclohexane-
1,2-diamine)oxalatoplatinum(II).
2. Results and discussion
Oxaliplatin produces adducts on DNA with a similar ratio
of GG over AG intrastrand cross-links, similar sequence
specificity and similarly low levels of interstrand cross-link
formation compared to cisplatin, but with substantially
slower kinetics. The effects on DNA structure after coordina-
tion are very similar to those of cisplatin with one striking
difference, the presence of the cyclohexane ring. Using mo-
lecular modeling, it has been proposed that in the region of
adduct formation an altered and less polar major groove is
being formed by the methylene units of the cyclohexane-1,2-
diamine ligand [12], which is in accordance with a different
recognition of cisplatin and oxaliplatin DNA adducts by the
mismatch repair system [13]. The X-ray crystal structure of
the cytotoxic (trans-R,R-cyclohexane-1,2-diamine)plati-
num(II) moiety with a DNA dodecanucleotide duplex [14]
was previously reported. Overall geometry and crystal pack-
ing were found to be similar to those of the analogous
cisplatin structure [15], with formation of a 1,2-intrastrand
crosslink, but a novel feature of the oxaliplatin adduct has
been found: a hydrogen bond between the pseudoequatorial
NH of the trans-R,R-cyclohexane-1,2-diamine ligand and
the O6 atom of the 3′-G of the platinated d(GpG) lesion,
demonstrating the importance of chirality which influences
the interaction between oxaliplatin and DNA.
2.1. Syntheses and NMR spectroscopy
The methyl and ethyl substituted trans-cyclohexane-1,2-
diamine ligands have been synthesized via two synthetic
pathways: Method
A
starts from 4-methyl- and
4-ethylcyclohexanone. After bromination, the 2-bromo-4-
alkylcyclohexanones were reacted with hydroxylamine. The
vicinale dioximes were then converted into the correspond-
ing trans-diamine derivatives by reduction with sodium in
ethanol and isolated as the dihydrogensulfates. Method B:
Starting from 4-methyl- or 4-ethyl-1-cyclohexene the corre-
sponding trans-cyclohexane-1,2-diazides were synthesized
by reaction with sodium azide in the presence of
Mn(OAc)3·2H2O. Reduction with hydrogen (3.5 bar, 5%
Pd/CaCO3) resulted in the trans-diamine derivatives, which
were also isolated as the dihydrogensulfates. In both cases,
the trans-cyclohexane-1,2-diamine derivatives are obtained
as (1R,2R/1S,2S, 1:1) mixtures.
Characterization of the ligands was performed by 1H and
13C NMR spectroscopy as well as elemental analysis. Two
sets of resonances were found due to the stereochemistry of
the methyl group in trans-cyclohexane-1,2-diamine dihydro-
gensulfates at position 4 (Fig. 3). Synthesis via Method A
resulted in 4-methyl-cyclohexane-1,2-diamine with the me-
thyl group being mainly in the equatorial position. As a
consequence, the 13C–N resonances C(1) and C(2) (52.4 and
52.5 ppm) were found to be very similar. However, in the
case of an axial methyl group (minor isomer), the chemical
1
In September 2003, the Food and Drug Administration (FDA) has
granted a six-month priority review to eloxatin, for the first line treatment of
metastatic colorectal cancer (MCRC).

M. Galanski et al. / European Journal of Medicinal Chemistry 39 (2004) 707–714
709
4-methyl-trans-chxn
carbon atoms 1, 2 and 4, homonuclear decoupling experi-
ments have been performed. Residual multiplets for protons
H(1) and H(2) with two vicinal coupling constants of 11 Hz
could be found when the equatorial protons H(3) and H(6)
are irradiated. These coupling constants are in accordance
with a coupling of two neighboring protons both being in an
axial position and can only be found in trans-configured
cyclohexane-1,2-diamine derivatives (Karplus curve [16]:
180°, 10–16 Hz, typical values for 3Jax/ax in cyclohexane-1,2-
diamine derivatives: 7–12 Hz). Irradiation in the methyl
resonance H(7) resulted in a broad triplet with two vicinal
coupling constants of about 11 Hz, also proving the axial
position of H(4) being equivalent with an equatorial methyl
substituent at C(4).
axial CH3
4-methyl-trans-chxn
equatorial CH3
4-methyl-cis-chxn
3
2
The 4-methyl and 4-ethyl substituted (SP-4-3)-
dichloro(trans-cyclohexane-1,2-diamine)platinum(II) com-
plexes were synthesized by direct reaction of the diaminedi-
hydrogen sulfates with potassium tetrachloroplatinate(II),
K2PtCl4, in the presence of NaOH and obtained as yellow
powders with yields of 60–95%. The diaminedichloroplati-
num(II) complexes were activated using Ag2SO4 or AgNO3
and were brought to reaction with sodium oxalate, which was
formed in situ by addition of two equivalents of NaOH to
oxalic acid (Scheme 1). The final products precipitated as
white solids (yields: 20–50%) and were characterized by
NMR spectroscopy and elemental analysis.
Dependent on the synthetic route, the title compounds
were obtained as (1R,2R/1S,2S) mixtures rac-1(eq/ax) and
rac-2(eq/ax) with a methyl or ethyl substituent at position
4 being mainly in an equatorial position (ligand synthesis via
Method A) whereas rac-1(eq/ax) and rac-2(eq/ax) were iso-
lated as (1R,2R/1S,2S) mixtures with an excess of an axial
substitution at position 4 (ligand synthesis via Method B).
1
54.0
53.5
53.0
52.5
52.0
51.5
51.0
(ppm)
50.5
50.0
49.5
49.0
48.5
48.0
47.5
Fig. 3. 13C NMR spectra of 4-methyl cis- and trans-cyclohexane-1,2-
diamine (chxn) dihydrogensulfates, region of the 13CHN carbons is shown.
Spectrum 1, 4-methyl-trans-chxn·H2SO4 obtained via Method A; spectrum
2, 4-methyl-trans-chxn·H2SO4 obtained via Method B and addition of
4-methyl-cis-chxn·H2SO4; spectrum 3, 4-methyl-cis-chxn·H2SO4.
shifts for C(1) and C(2) (48.7 and 51.2 ppm) show a marked
splitting.
Following Method B, the relation between the equatorial
and axial position of the 4-methyl group is inverted. In order
to undoubtedly exclude the presence of a mixture of the
trans- and cis-cyclohexane-1,2-diamine derivatives, the
analogous 4-methyl-cis-cyclohexane-1,2-diamine dihydro-
gensulfate has also been synthesized starting from 4-methyl-
1,2-cyclohexanedicarboxylic anhydride via hydrolysis and
reaction with sodium azide in the presence of H2SO4. The
resonances of C(1) and C(2) in the pure cis-cyclohexane-1,2-
diamine derivative and in a mixture with the corresponding
trans-isomer from Method B were found to be markedly
different, proving our hypothesis (synthesis of pure trans
isomers via Methods A and B).
2.2. Molecular structure of rac-1(ax)
Crystals suitable for X-ray structure determination were
obtained by slow evaporation of an aqueous solution of
rac-1(eq/ax). The structure of rac-1(ax), a 1:1 mixture of
(SP-4-3)-(4R-methyl-trans-1S,2S-cyclohexane-1,2-diamine)
In order to simplify complex spin systems in proton NMR
spectra and to get further stereochemical information at the
H2
H2
-
N
OSO3
OH2
N
Cl
Cl
+
+ Ag2SO4
- 2 AgCl
Pt
Pt
R
R
N
N
H2
H2
+ (COOH)2 · 2 H2O
+ 2 NaOH
+ 2 AgNO3
- 2 AgCl
R = methyl, ethyl
H2
N
H2
N
O
OH2
OH2
O
O
2+
Pt
-
+ (COOH)2 · 2 H2O
+ 2 NaOH
2 NO3
Pt
R
R
N
N
O
H2
H2
Scheme 1. Synthesis of the 4-methyl and 4-ethyl substituted (trans-cyclohexane-1,2-diamine)oxalatoplatinum(II) complexes.

710
M. Galanski et al. / European Journal of Medicinal Chemistry 39 (2004) 707–714
Table 1
Selected bond lengths (Å) and angles (°) for (SP-4-3)-(4-methyl-trans-
cyclohexane-1,2-diamine)oxalatoplatinum(II), rac-1(ax)
Atom 1–Atom 2
N1–C3
C3–C4
C4–N2
C4–C5
C5–C6
C6–C7
C7–C8
C7–C9
C8–C3
O1–C1
C1–C2
C1–O3
C2–O2
C2–O4
Molecule I
1.498(4)
1.513(4)
1.488(4)
1.515(4)
1.528(5)
1.536(5)
1.545(5)
1.529(5)
1.530(4)
1.296(4)
1.549(4)
1.223(4)
1.305(4)
1.214(4)
Atom 1–Atom 2 Molecule II
N3–C12
C12–C13
C13–N4
C13–C14
C14–C15
C15–C16
C16–C17
C16–C18
C17–C12
O5–C10
C10–C11
C10–O7
C11–O6
C11–O8
1.509(4)
1.517(4)
1.500(4)
1.520(4)
1.531(5)
1.533(5)
1.548(5)
1.555(6)
1.524(4)
1.288(4)
1.548(4)
1.234(4)
1.291(4)
1.226(4)
Atom 1–Atom
2–Atom 3
Molecule I Atom 1–Atom
2–Atom 3
Molecule II
N1–Pt1–N2
O1–Pt1–O2
83.64(10) N3–Pt2–N4
82.16(9) O5–Pt2–O6
83.47(10)
82.27(9)
Fig. 4. Structures of the two independent molecules (SP-4-3)-(4R-methyl-
trans-1S,2S-cyclohexane-1,2-diamine)oxalatoplatinum(II) (top) and (SP-4-
3)-(4S-methyl-trans-1R,2R-cyclohexane-1,2-diamine)oxalatoplatinum(II)
(bottom) in the crystal in rac-1(ax). The displacement ellipsoids are drawn
at 50% probability level.
SW480 (Coloon carcinoma)
120
100
80
oxalatoplatinum(II) and (SP-4-3)-(4S-methyl-trans-1R,2R-
cyclohexane-1,2-diamine)oxalatoplatinum(II), is shown in
Fig. 4.
The platinum(II) atoms in both molecules have a square-
planar coordination geometry. The 4-methyl-trans-
cyclohexane-1,2-diamine ligands act as neutral didentate
ligands and coordinate to PtII through the nitrogen atoms
[Pt1–N1 2.019(3), Pt1–N2 2.016(2), Pt2–N3 2.030(3),
Pt2–N4 2.026(3) Å].
60
40
20
0
0,01
0,1
1
10
100
1000
These bond lengths are not significantly shorter compared
to Pt–N in oxaliplatin, (SP-4-2)-(trans-1R,2R-cyclohexane-
1,2-diamine)oxalatoplatinum(II) [Pt–N1 2.06(2), Pt–N2
2.04(2) Å] [17]. The doubly negatively charged oxalate
ligands are bound to the platinum(II) ions via oxygen atoms
[Pt1–O1 2.027(2), Pt1–O2 2.025(2), Pt2–O5 2.033(2),
Pt2–O6 2.022(2) Å]. These bond lengths are comparable
with those in (SP-4-2)-(trans-1R,2R-cyclohexane-1,2-
diamine)oxalatoplatinum(II). The cyclohexane rings in rac-
1(ax) adopt a chair conformation with the two amino groups
in equatorial positions. The bond distances and angles are
statistically identical between the two molecules within 3r
(Table 1).
concentration [µM]
CH1 (Ovarian carcinoma)
120
100
80
60
40
20
2.3. Cytotoxic activity
0
0,01
0,1
1
10
100
1000
The concentration–effect curves obtained from MTT as-
says of the oxalatoplatinum(II) complexes rac-1(eq/ax) and
rac-2(eq/ax) in comparison with oxaliplatin, its trans-S,S
enantiomer, and the racemic mixture of oxaliplatin and its
trans-S,S enantiomer in colon (SW480) and ovarian (CH1)
cancer cells after exposure for 96 h are shown in Fig. 5.
concentration [µM]
Fig. 5. Concentration–effect curves of the methyl (") and ethyl (●) substi-
tuted oxaliplatin analogues, rac-1(eq/ax) and rac-2(eq/ax), compared with
oxaliplatin (h), its trans-S,S enantiomer (e), and the racemic mixture of its
trans-R,R- and trans-S,S enantiomer (C), in SW480 and CH1 cells after
exposure for 96 h.

M. Galanski et al. / European Journal of Medicinal Chemistry 39 (2004) 707–714
711
Compared to the enantiomerically pure oxaliplatin, the
derivatives rac-1(eq/ax) and rac-2(eq/ax), prepared as trans-
R,R/trans-S,S mixtures with a substituent at C(4) being
mainly in an equatorial position, show slightly higher cyto-
toxic potency in SW480 cells and equivalent potency in CH1
cells. This is remarkable, and it should be pointed out that a
direct comparison with the racemic mixture of oxaliplatin
reveals that the new derivatives display significantly superior
activity in both cell lines. Since trans-R,R-cyclohexane-1,2-
diamine-containing platinum complexes are usually more
active than their trans-R,R/trans-S,S isomeric mixtures
[18,19], it is expected that the antitumor activity should be
even more markedly improved by use of the pure trans-R,R
isomers.
ents at C(4) predominantly being in an axial position, indi-
cating a marked dependency of cytotoxicity on the stereo-
chemistry at position 4 of the cyclohexane-1,2-diamine
ligand.
At present, we focus on the synthesis of (i) trans-
cyclohexane-1,2-diamine derivatives bearing exclusively
equatorial substituents at position 4 and (ii) on the synthesis
of the trans-R,R isomers. A marked and positive effect on
antitumor activity is expected. Moreover, since oxaliplatin
and other cyclohexane-1,2-diamine-containing platinum
complexes exhibit a lack of cross-resistance in certain
cisplatin-resistant tumors, the new derivatives might not only
be more cytotoxic, but also may be endowed with an im-
proved capacity of overcoming this resistance.
The cytotoxic properties of the analogous oxalatoplatinu-
m(II) complexes rac-1(eq/ax) and rac-2(eq/ax), synthesized
as trans-R,R/trans-S,S mixtures with predominantly axial
substitution at position 4, have also been investigated in
colon (SW480) and ovarian (CH1) cancer cells. Interestingly,
the activity was markedly different in comparison to rac-
1(eq/ax) and rac-2(eq/ax) and was comparable to the trans-
R,R/trans-S,S-isomeric mixture of oxaliplatin in the case of
complex rac-1(eq/ax) and comparable to the cytotoxicity of
the trans-S,S derivative of oxaliplatin in case of compound
rac-2(eq/ax) (data not shown).
The cyclohexane ring is an essential structural require-
ment for the high cytotoxicity of oxaliplatin and probably
also for its different pharmacodynamic properties in com-
parison to the diammine complexes cisplatin and carboplatin.
It seems reasonable to assume that the steric demand of the
cyclohexane ring is either directly or indirectly responsible
for the stronger replication-inhibiting properties of oxalipl-
atin. The assumption, that increasing this steric demand is a
promising strategy to improve activity, is supported by our
findings with derivatives carrying small substituents on posi-
tion 4 at the cyclohexane-1,2-diamine ligand. However, it
could also be demonstrated, that this assumption can only be
confirmed by the use of oxaliplatin-derivatives with equato-
rial substitution at position 4, whereas axial substitution at
this position decreases the cytotoxic properties.
4. Experimental
4.1. Syntheses
The new compounds reported in this study are illustrated
in Fig. 2. Potassium tetrachloroplatinate was obtained from
Degussa (Germany).All other chemicals obtained from com-
mercial suppliers were used as received and were of analyti-
cal grade. Water was used doubly distilled. The synthetic
procedures were carried out in a light protected environment
when platinum complexes were involved. The methyl substi-
tuted cis- [20,21] and the methyl and ethyl substituted trans-
cyclohexane-1,2-diamine ligands (Method A [22,23] and B
[24,25]), the corresponding (SP-4-3)-dichloro(trans-cyclo-
hexane-1,2-diamine)platinum(II) complexes, oxaliplatin,
(SP-4-2)-(trans-S,S-cyclohexane-1,2-diamine)oxalatoplati-
num(II) and (SP-4-2)-(trans-cyclohexane-1,2-diamine)
oxalatoplatinum(II) [26] have been synthesized according to
standard literature methods. Analyses indicated by the sym-
bols of the elements or functions were within 0.4%.
4.1.1. Synthesis of trans-cyclohexane-1,2-diamine
dihydrogensulfates
In preliminary in vivo experiments in mice bearing L1210
leukemia, even rac-1(eq/ax) and rac-2(eq/ax) were found to
be more tolerable and displayed a higher anticancer activity
in comparison to oxaliplatin at analogous dosage.
4.1.1.1. Method A.
4.1.1.1.1. 4-Methyl-trans-cyclohexane-1,2-diamine dihy-
1
drogensulfate. H NMR in D2O: d = 0.85 [d, 3H, H(7),
3JH,H = 6.5 Hz], 0.99 [m, 1H, H(5)], 1.15 [m, 1H, H(3)],
1.42–1.55 [m, 2H, H(4), H(6)], 1.66–1.77 [m, 1H, H(5)],
1.99–2.10 [m, 2H, H(6), H(3)], 3.23–3.39 [m, 2H, H(1),
H(2)]; minor isomer: d = 0.90 [d, 3H, H(7′), 3JH,H = 7.0 Hz].
13C NMR in D2O: d = 20.7 [C(7)], 29.5 [C(6)], 30.2 [C(4)],
31.5 [C(5)], 37.6 [C(3)], 52.4 [C(1) or C(2)], 52.5 [C(1) or
C(2)]; minor isomer: d = 17.9 [C(7′)], 24.0 [C(6′)], 25.8
[C(4′)], 27.8 [C(5′)], 33.8 [C(3′)], 48.7 [C(1′) or C(2′)], 51.2
[C(1′) or C(2′)]. Anal. C7H16N2·H2SO4 (C, H, N).
3. Conclusions
Even the trans-R,R/trans-S,S mixtures of the methyl and
ethyl substituted oxaliplatin analogues rac-1(eq/ax) and rac-
2(eq/ax) with substituents at C(4) being mainly in the equa-
torial position have demonstrated a superior in vitro antitu-
mor activity in colon cancer cells (SW480) and equivalent
potency in CH1 cells in comparison to oxaliplatin. On the
other hand, the cytotoxic properties are decreased in the case
of complexes rac-1(eq/ax) and rac-2(eq/ax) with substitu-
4.1.1.1.2. 4-Ethyl-trans-cyclohexane-1,2-diamine dihy-
1
drogensulfate. H NMR in D2O: d = 0.77 [t, 3H, H(8),
3JH,H = 7.3 Hz], 0.96 [m, 1H, H(5)], 1.05–1.20 [m, 1H, H(3)],
1.11–1.28 [m, 2H, H(7)], 1.21–1.38 [m, 1H, H(4)], 1.38–

712
M. Galanski et al. / European Journal of Medicinal Chemistry 39 (2004) 707–714
1.56 [m, 1H, H(6)], 1.72–1.84 [m, 1H, H(5)], 2.04–2.13 [m,
2H, H(6), H(3)], 3.23–3.43 [m, 2H, H(1), H(2)]; minor iso-
mer: d = 0.79 [t, 3H, H(8′), 3JH,H = 6.8 Hz]. 13C NMR in D2O:
d = 10.8 [C(8)], 28.3 [C(7)], 29.1 [C(5)], 29.5 [C(6)], 35.6
[C(3)], 36.8 [C(4)], 52.5 [C(1) or (2)], 52.7 [C(1) or C(2)];
minor isomer: d = 11.4 [C(8′)], 24.2 [C(7′)], 24.8 [C(5′)],
25.6 [C(6′)], 31.8 [C(3′)], 32.8 [C(4′)], 48.8 [C(1′) or C(2′)],
51.3 [C(1′) or C(2′)]. Anal. C8H20N2·H2SO4 (C, H, N).
4.1.3.2. (SP-4-3)-Dichloro(4-ethyl-trans-cyclohexane-1,2-
diamine)platinum(II). Ligand from Method A was used;
yield 95%. Anal. C8H18Cl2N2Pt (C, H, N).
4.1.3.3. (SP-4-3)-Dichloro(4-methyl-trans-cyclohexane-1,2-
diamine)platinum(II). Ligand from Method B was used;
yield 60%. Anal. C7H16Cl2N2Pt (C, H, N).
4.1.3.4. (SP-4-3)-Dichloro(4-ethyl-trans-cyclohexane-1,2-
diamine)platinum(II). Ligand from Method B was used;
yield 66%. Anal. C8H18Cl2N2Pt (C, H, N).
4.1.1.2. Method B.
4.1.1.2.1. 4-Methyl-trans-cyclohexane-1,2-diamine dihy-
1
drogensulfate. H NMR in D2O: d = 0.88 [d, 3H, H(7),
3JH,H = 7.0 Hz], 1.32–1.44 [m, 1H, H(5)], 1.56 [m, 1H, H(5)],
1.62–1.82 [m, 3H, H(3), H(6)], 1.82–1.98 [m, 2H, H(6),
H(4)], 3.37 [m, 1H, H(1)], 3.54 [m, 1H, H(2)]; minor isomer:
4.1.4. Synthesis of oxalatoplatinum complexes
4.1.4.1. (SP-4-3)-(4-Methyl-trans-cyclohexane-1,2-diami-
ne)oxalatoplatinum(II), rac-1(eq/ax). (SP-4-3)-Dichloro(4-
methyl-trans-cyclohexane-1,2-diamine)platinum(II) (803.6 mg,
2.039 mmol, from 4.1.3.1.) was suspended in 30 ml of water and
Ag2SO4 (643.3 mg, 2.00 mmol) was added in one portion. The
mixture was stirred for a period of 2 days at room temperature.
Silver chloride precipitated, was filtered off, and the remaining
solution was evaporated to dryness. Oxalic acid dihydrate
(193 mg, 1.53 mmol), 10 ml of water and NaOH (3 ml 0.5 M,
1.5 mmol) were added to aqua(4-methyl-trans-cyclohexane-1,2-
diamine)sulfatoplatinum(II) (669 mg, 1.53 mmol) and stirred
over night. A white precipitate formed, which was filtered off
and dried under reduced pressure over P2O5 to obtain 310 mg
of [Pt(4-methyl-trans-chxn)(ox)] as a white solid; yield
50%.1H NMR in H2O/D2O (9/1): d = 0.75–0.87 [m, 1H,
H(5)], 0.83 [d, 3H, H(7), 3JH,H = 6.6 Hz], 0.88–0.99 [m, 1H,
H(3)], 1.19–1.40 [m, 2H, H(4), H(6)], 1.41–1.51 [m, 1H,
H(5)], 1.86–1.97 [m, 2H, H(6), H(3)], 2.18–2.41 [m, 2H,
H(1), H(2)], 5.03 NH2, 5.73 NH2. 13C NMR in H2O/D2O
(9/1): d = 20.4 [C(7)], 31.0 [C(6)], 31.3 [C(4)], 32.6 [C(5)],
39.9 [C(3)], 62.5–62.7 [2C, C(1), C(2)], 168.7 [2C, C=O];
minor isomer: d = 17.1 [C(7′)], 26.9 [C(6′)], 27.4 [C(4′)],
29.7 [C(5′)], 39.8 [C(3′)]. Anal. C9H16N2O4Pt (C, H, N).
d = 0.86 [d, 3H, H(7′), JH,H = 6.7 Hz]. 13C NMR in D2O:
3
d = 18.0 [C(7)], 24.0 [C(6)], 25.8 [C(4)], 27.8 [C(5)], 33.8
[C(3)], 48.7 [C(1) or C(2)], 51.2 [C(1) or C(2)]; minor iso-
mer: d = 20.7 [C(7′)], 29.5 [C(6′)], 30.2 [C(4′)], 31.5 [C(5′)],
37.7 [C(3′)], 52.4 [C(1′) or C(2′)], 52.5 [C(1′) or C(2′)].Anal.
C7H16N2·H2SO4 (C, H, N).
4.1.1.2.2. 4-Ethyl-trans-cyclohexane-1,2-diamine dihy-
1
drogensulfate. H NMR in D2O: d = 0.80 [t, 3H, H(8),
3JH,H = 7.5 Hz], 1.20–1.38 [m, 2H, H(7)], 1.44–1.77 [m, 5H,
H(5), H(6), H(3)], 1.80–1.96 [m, 2H, H(6), H(4)], 3.41 [m,
1H, H(1) or H(2)], 3.53 [m, 1H, H(1) or H(2)]; minor isomer:
d = 0.78 [t, 3H, H(8′), JH,H = 7.5 Hz]. 13C NMR in D2O:
3
d = 11.3 [C(8)], 24.2 [C(7)], 24.9 [C(5)], 25.6 [C(6)], 31.8
[C(3)], 32.8 [C(4)], 48.8 [C(1) or C(2)], 51.3 [C(1) or C(2)];
minor isomer: d = 10.8 [C(8′)], 28.3 [C(7′)], 29.1 [C(5′)],
29.5 [C(6′)], 35.6 [C(3′)], 36.8 [C(4′)], 52.5 [C(1′) or (2′)],
52.7 [C(1′) or C(2′)]. Anal. C8H20N2·H2SO4 (C, H, N).
4.1.2. 4-Methyl-cis-cyclohexane-1,2-diamine dihydrogen-
sulfate
1H NMR in D2O: d = 0.85 [d, 3H, CH3, 3JH,H = 6.5 Hz],
0.90 [d, 3H, CH3,3JH,H= 6.0 Hz], 0.97–1.18 [m, 2H, CH2],
1.28–1.52 [m, 2H, CH2], 1.52–2.08 [m, 10H, CH2 and CH],
3.47–3.64 [m, 2H, CHN], 3.75–3.91 [m, 2H, CHN].
13C NMR in D2O: d = 20.6 [CH3], 21.0 [CH3], 24.0 [CH2],
24.6 [CHCH3], 26.1 [CH2], 27.9 [CH2], 30.8 [CHCH3], 31.4
[CH2], 31.9 [CH2], 35.8 [CH2], 48.8 [CHN], 49.5 [CHN],
50.9 [CHN], 51.0 [CHN]. Anal. C7H16N2·H2SO4 (C, H, N).
4.1.4.2. (SP-4-3)-(4-Ethyl-trans-cyclohexane-1,2-diamine)
oxalatoplatinum(II), rac-2(eq/ax). Starting with 805.2 mg
(1.972 mmol, from 4.1.3.2.) of (SP-4-3)-dichloro(4-ethyl-
trans-cyclohexane-1,2-diamine)platinum(II), 170 mg of
[Pt(4-ethyl-trans-chxn)(ox)] were obtained as a white solid;
yield 20%. 1H NMR in H2O/D2O (9/1): d = 0.69 – 0.85 [m,
1H, H(5)], 0.75 [t, 3H, H(8), 3JH,H = 7.1 Hz], 0.86–0.99 [m,
1H, H(3)], 1.07–1.31 [m, 4H, H(4), H(6), H(7)], 1.49–1.60
[m, 1H, H(5)], 1.89–2.03 [m, 2H, H(6), H(3)], 2.21–2.41 [m,
2H, H(1), H(2)], 5.75 NH2, second NH2 group under the
water signal. 13C NMR in H2O/D2O (9/1): d = 11.1 [C(8)],
27.9 [C(7)], 30.1 [C(5)], 30.8 [C(6)], 37.5 [C(3)], 37.9
[C(4)], 62.5 [2C, C(1), C(2)], C=O could not be detected.
Anal. C10H18N2O4Pt (C, H, N).
4.1.3. Synthesis of dichloroplatinum complexes
4.1.3.1. (SP-4-3)-Dichloro(4-methyl-trans-cyclohexane-1,2-
diamine)platinum(II). To a solution of K2PtCl4 (3.944 g,
9.5 mmol) in 50 ml of water, 4-methyl-trans-cyclohexane-
1,2-diamine dihydrogensulfate (2.15 g, 9.50 mmol, ligand
from Method A) was added. The pH was adjusted to 7 with
0.5 M NaOH and was kept constant during the reaction at this
value using 0.1 M NaOH. A yellow precipitate formed which
was filtered off and dried under reduced pressure over P2O5
to obtain 3.0 g of [Pt(4-methyl-trans-chxn)Cl2]; yield 80%.
Anal. C7H16Cl2N2Pt (C, H, N).
4.1.4.3. (SP-4-3)-(4-Methyl-trans-cyclohexane-1,2-diamine)
oxalatoplatinum(II), rac-1(eq/ax). (SP-4-3)-Dichloro(4-me-
thyl-trans-cyclohexane-1,2-diamine)platinum(II) (1.81 g,

M. Galanski et al. / European Journal of Medicinal Chemistry 39 (2004) 707–714
713
Table 2
4.60 mmol, from 4.1.3.3.) was suspended in 70 ml of water
and AgNO3 (1.48 g, 8.74 mmol) was added in one portion.
The mixture was stirred for 24 h at room temperature. Silver
chloride precipitated, was filtered off, and the remaining
solution was reduced by evaporation to 30 ml. Oxalic acid
(550 mg, 4.37 mmol) dissolved in 8.7 ml of 1 N NaOH
(8.7 mmol) was added to the diaqua(4-methyl-trans-
cyclohexane-1,2-diamine)platinum(II) complex and stirred
over night. A white precipitate formed which was filtered off
and dried under reduced pressure over P2O5 to obtain 770 mg
of [Pt(4-methyl-trans-chxn)(ox)] as a white solid; yield 43%.
Crystal data and details of data collection for (SP-4-3)-(4-methyl-trans-
cyclohexane-1,2-diamine)oxalatoplatinum(II) dihydrate, rac-1(ax) (synthe-
sis via Method B) a
Chemical formula
M (g mol–1
C9H16PtN2O4·2H2O
447.36
)
Temperature (K)
Crystal size
Crystal color, habit
Crystal system
Space group
a (Å)
120
0.29 × 0.23 × 0.20
White, block
Monoclinic
P21/c
15.809 (3)
12.566 (3)
14.863 (3)
102.57 (3)
2689.6 (9)
8
3
1H NMR in D2O: d = 0.83 [d, 3H, H(7), JH,H = 6.6 Hz],
b (Å)
c (Å)
1.22–1.54 [m, 4H, H(5), H(3), H(6)], 1.72–1.89 [m, 3H,
H(6), H(3), H(4)], 2.23 [m, 1H, H(1) or H(2)], 2.49 [m, 1H,
H(1) or H(2)]. 13C NMR in D2O: d = 17.0 [C(7)], 26.8 [C(6)],
27.4 [C(4)], 29.7 [C(5)], 37.6 [C(3)], 58.2 [C(1) or C(2)],
63.1 [C(1) or C(2)], 168.7 [2C, C=O]; minor isomer: d = 20.4
[C(7′)], 31.0 [C(6′)], 31.2 [C(4′)], 32.5 [C(5′)], 39.1 [C(3′)],
62.3 [C(1′) or C(2′) 62.5 [C(1′) or C(2′)].Anal. C9H16N2O4Pt
(C, H, N).
ß (°)
V (Å3)
Z
Dc (g cm–3
)
2.210
µ (cm–1
F (0 0 0)
)
104.55
1712
h range for data collection (°)
2.15–30.50
–22/22
h range
k range
–17/17
l range
–21/21
4.1.4.4. (SP-4-3)-(4-Ethyl-trans-cyclohexane-1,2-diamine)
oxalatoplatinum(II), rac-2(eq/ax). Starting with 1.48 g
(3.61 mmol, from 4.1.3.4.) of (SP-4-3)-dichloro(4-ethyl-
trans-cyclohexane-1,2-diamine)platinum(II), 650 mg of
[Pt(4-ethyl-trans-chxn)(ox)] were obtained as a white solid;
yield 45%. 1H NMR in D2O: d = 0.94 [t, 3H, H(8),
3JH,H = 7.1 Hz], 1.17–2.11 [m, 9H, H(3), H(4), H(5), H(6),
H(7)], 2.20–2.52 [m, 2H, H(1), H(2)], 5.31 NH2, 6.02 NH2.
13C NMR in D2O: d = 11.6 [C(8)], 23.8 [(C7)], 30.7 [C(5)],
31.1 [C(6)], 33.3 [(C4)], 35.6 [C(3)], 58.6 [C(1) or C(2)],
63.4 [C(1) or C(2)], 168.7 [2C, C=O]; minor isomer: d = 10.9
[C(8′)], 27.2 [(C7′)], 27.9 [C(5′)], 28.4 [(C6′)], 37.9 [(C3′)],
38.4 [(C4′)], 62.8 [C(1′) or C(2′)], 63.1 [C(1′) or C(2′)].Anal.
C10H18N2O4Pt (C, H, N).
Total no. measured reflections
16 385
No. unique measured reflections
8195
No. reflections used in refinement
8195
No. parameters
Rint
360
0.024
R1 (obs.)
wR2 (all data)
S
0.0219
0.0537
1.208
Largest diff. peak and hole (eÅ–3
)
1.560 and –1.579
a Refinement by full-matrix least-squares F2 for all reflections,
͑
͒
R = R||F | – |F ||/Rw|F |, wR = R F2–F2 2⁄RwF4o 1⁄2, w = 1⁄ r2 F2
+
͒
o
͓
͑
͒
͔
͓
͑
͑
1
o
c
o
2
o
c
o
0.0173P + 5.64P, with P = F2 + 2F2c ⁄3 . Goodness of fit, S = R
2
͑
͑
͒
͓
͒
͓
͕
͖
o
F2–F2 ⁄ n–p
.
2
1⁄2
͒
c
͔
͑
͒
o
structure refinement details are given in Table 2. The struc-
ture was solved by direct methods and refined by full-matrix
least-squares techniques. Non-hydrogen atoms were refined
with anisotropic displacement parameters. All hydrogen at-
oms (except those of four water molecules per asymmetric
unit, which were located on difference Fourier maps) were
placed at calculated positions and refined as riding atoms in
the subsequent least squares model refinement. Their isotro-
pic thermal parameters were estimated to be 1.2 times the
value of the equivalent isotropic thermal parameter of the
atom to which hydrogen was bound. Computer programs:
data reduction, DATAP [27]; structure solution, SHELXS-97
[28]; refinement SHELXL-97 [29], molecular diagrams,
ORTEP [30], computer: PENTIUM II; scattering factors
[31].
4.2. Physical measurements
1H, 13C{1H},1H,1H-COSY, and 13C,1H,-COSY spectra
were recorded in H2O/D2O (9:1) or D2O at 298 K (2D in a
gradient enhanced mode) using a Bruker Avance DPX
400 instrument (UltraShield™ Magnet) and standard pulse
programs at 400.13 (1H) and 100.62 MHz (13C). Chemical
shifts were measured relative to the solvent peak or to exter-
nal NH4Cl. Elemental analyses were performed by the mi-
croanalytical laboratory at the University of Vienna.
4.3. Structure determination
X-ray diffraction measurements were performed on a
Nonius Kappa CCD diffractometer with Mo Ka (k = 0.71073
Å) radiation. The single crystal was positioned at 30 mm
from the detector. The 341 frames were measured, each for
65 s over a 2° x-scan. The data were processed using the
Denzo-SMN software, no empirical absorption correction
was applied. Crystal data, data collection parameters, and
4.4. Cell lines and culture conditions
SW480 cells (adenocarcinoma of the colon) were ob-
tained from the American Type Culture Collection (ATCC)
and kindly provided by Brigitte Marian (Institute of Cancer
Research, University of Vienna, Austria). The CH1 cell line

714
M. Galanski et al. / European Journal of Medicinal Chemistry 39 (2004) 707–714
has been established from an ascites sample of a patient with
a papillary cystadenocarcinoma of the ovary and was kindly
provided by Lloyd R. Kelland (CRC Centre for Cancer
Therapeutics, Institute of Cancer Research, Sutton, UK).
Cells were grown as adherent monolayer cultures in Minimal
Essential Medium (MEM) containing 10% heat-inactivated
fetal bovine serum, 1 mM sodium pyruvate, 2 mM
L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin
(all purchased from Gibco). Cultures were maintained at
37 °C in a humidified atmosphere containing 5% CO2.
Compagnie Translational Cancer Research GmbH is grate-
fully acknowledged. We are thankful to Prof. G. Giester for
the collection of X-ray data.
References
[1] B. Rosenberg, L. Van Camp, T. Krigas, Nature 205 (1965) 698–699.
[2] B. Rosenberg, L. Van Camp, J.E. Trosko, V.H. Mansour, Nature 222
(1969) 385–386.
[3] B. Rosenberg, Interdiscip. Sci. Rev 3 (1978) 134–147.
[4] B. Lippert (Ed.), Cisplatin: Chemistry and Biochemistry of a Leading
Anticancer Drug, Verlag Helvetica Chimica Acta, Zürich
(Switzerland) and WILEY-VCH, Weinheim (Germany), 1999.
[5] P.J. O’Dwyer, J.P. Stevenson, S.W. Johnson, Drugs 59 (Suppl. 4)
(2000) 19–27.
4.5. Cytotocicity tests in cancer cell lines
Cytotoxicity was determined by means of a colorimetric
microculture assay (MTT assay, MTT = 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). SW480
and CH1 cells were harvested from culture flasks by
trypsinization and seeded into 96-well microculture plates.
Cell densities of 1.5 × 104 cells/ml (3 × 103 cells/well) and
1.25 × 104 cells/ml (2.5 × 103 cells/well) were chosen for
SW480 and CH1 cells, respectively, in order to ensure expo-
nential growth throughout drug exposure. After a 24 h pre-
incubation, cells were exposed to solutions of the test com-
pounds in complete culture medium for 96 h. At the end of
exposure, drug solutions were replaced by mixtures of com-
plete culture medium and aqueous MTT solution. After incu-
bation for 4 h the medium/MTT mixtures were removed and
the formazan crystals formed by the mitochondrial dehydro-
genase activity of living cells were dissolved in DMSO.
Optical densities at 550 nm were measured with a microplate
reader (Tecan Spectra Classic), and the quantity of living
cells was expressed in terms of T/C values by comparison to
untreated control microcultures. Evaluation is based on
means from at least three independent experiments, each
comprising eight microcultures per concentration level.
[6] P.J. O’Dwyer, J.P. Stevenson, S.W. Johnson, in: B. Lippert (Ed.),
Cisplatin: Chemistry and Biochemistry of a LeadingAnticancer Drug,
Verlag Helvetica Chimica Acta, Zürich (Switzerland) and WILEY-
VCH, Weinheim (Germany), 1999, pp. 31–69.
[7] M.A. Jakupec, M. Galanski, B.K. Keppler, Rev. Physiol. Biochem.
Pharmacol. 146 (2003) 1–53.
[8] Y. Kidani, M. Noji, T. Tashiro, Gann 71 (1980) 637–643.
[9] E. Cvitkovic, M. Bekradda, Semin. Oncol. 26 (1999) 647–662.
[10] J.M. Woynarowski, S. Faivre, M.C.S. Herzig, B. Arnett, W.G. Chap-
man, A.V. Trevino, E. Raymond, S.G. Chaney, A. Vaisman, M. Varch-
enko, P.E. Juniewicz, Mol. Pharmacol. 58 (2000) 920–927.
[11] S.G. Chaney, A. Vaisman, in: L.R. Kelland, N.P. Farrell (Eds.),
Platinum-Based Drugs in Cancer Therapy, Humana Press, Totowa,
NJ, 2000, pp. 129–148.
[12] E.D. Scheeff, J.M. Briggs, S.B. Howell, Mol. Pharmacol. 56 (1999)
633–643.
[13] S.G. Chaney, A. Vaisman, J. Inorg. Biochem. 77 (1999) 71–81.
[14] B. Spingler, D.A. Whittington, S.J. Lippard, Inorg. Chem. 40 (2001)
5596–5602.
[15] P.M. Takahara, A.C. Rosenzweig, C.A. Frederick, S.J. Lippard,
Nature 377 (1999) 649–652.
[16] H. Friebolin, Basic One- and Two-Dimensional NMR Spectroscopy,
VCH, New York, 1993.
[17] M.A. Bruck, R. Bau, M. Noji, K. Inagaki,Y. Kidani, Inorg. Chim.Acta
92 (1984) 279–284.
[18] L. Pendyala, Y. Kidani, R. Perez, J. Wilkes, R.J. Bernacki,
P.J. Creaven, Cancer Lett. 97 (1995) 177–184.
[19] Z.H. Siddik, S. Al-Baker, G. Thai, A.R. Khokhar, J. Cancer Res. Clin.
Oncol. 120 (1994) 409–414.
[20] N.W. Werner, J. Casanova, Org. Synth. Coll. V (1973) 273.
[21] R. Saito, Y. Kidani, Bull. Chem. Soc. Jpn. 59 (1986) 1689–1694.
[22] R. Belcher, W. Hoyle, T.S. West, J. Chem. Soc. (1958) 2743–2745.
[23] R. Belcher, W. Hoyle, T.S. West, J. Chem. Soc. (1961) 667–670.
[24] B.B. Snyder, H. Lin, Synthetic Commun. 28 (1998) 1913–1922.
[25] E.J. Corey, K.C. Nicolaou, R.D. Balanson, Y. Machida, Synthesis 9
(1975) 590–591.
[26] Y. Kidani, K. Inagaki, M. Iigo, A. Hoshi, K. Kuretani, J. Med. Chem.
21 (1978) 1315–1318.
[27] P. Coppens, L. Leiserowitz, P. Rabinovich, Acta Crystallogr. 18
(1965) 1035–1038.
5. Supplementary material
Crystallographic data for the structural analysis have been
deposited with the Cambridge Crystallographic Data Centre,
CCDC No. 223975. Copies may be obtained free of charge
from the Director Cambridge Crystallographic Data Centre,
12, Union Road, Cambridge CB2 1EZ, UK (fax (interna-
tional): +44-1223-336-033; e-mail: deposit@ccdc.cam.
ac.uk or www.ccdc.cam.ac.uk/conts/retrieving.html).
[28] G.M. Sheldrick, SHELXS-97, Program for Crystal Structure Solu-
tion, University Göttingen, Germany, 1997.
[29] G.M. Sheldrick, SHELXL-97, Program for Crystal Structure Refine-
ment, University Göttingen, Germany, 1997.
[30] C.K. Johnson, Rep. ORNL-5138, OAK Ridge National Laboratory,
OAK, Ridge, TN, 1976.
[31] International Tables for X-ray Crystallography, C, Kluwer Academic
Press, Dordrecht, The Netherlands, 1992 Tables 4.2.6.8 and 6.1.1.4.
Acknowledgements
The support of the FWF (Fonds zur Foerderung der wis-
senschaftlichen Forschung), COST and Faustus Forschungs