{(1 R,2 R,4 R)-4-Methyl-1,2-cyclohexanediamine}oxalatoplatinum(II): A Novel Enantiomerically Pure Oxaliplatin Derivative Showing Improved Anticancer Activity in Vivo

Article abstract of DOI:10.1021/jm100953c

Novel derivatives of the clinically established anticancer drug oxaliplatin were synthesized. Cytotoxicity of the compounds was studied in six human cancer cell lines by means of the MTT assay. Additionally, most promising complexes were also investigated in cisplatin- and oxaliplatin-resistant human cancer cell models. The therapeutic efficacy in vivo was studied in the murine L1210 leukemia model. Most remarkably, {(1R,2R,4R)-4-methyl-1,2-cyclohexanediamine} oxalatoplatinum(II), comprising an equatorial methyl substituent at position 4 of the cyclohexane ring, was as potent as oxaliplatin in vitro but distinctly more effective in the L1210 model in vivo at the optimal dose. The advantage observed in the in vivo situation was mainly based on a more favorable therapeutic index. The maximum tolerated dose of the novel analogue was higher than that of oxaliplatin and caused a greater increase in life span (>200% versus 152%), with more animals experiencing long-term survival (5/6 versus 2/6). These data support further (pre)clinical development of the methyl-substituted oxaliplatin analogue with improved anticancer activity.

Full text of DOI:10.1021/jm100953c

7356 J. Med. Chem. 2010, 53, 7356–7364
DOI: 10.1021/jm100953c
{(1R,2R,4R)-4-Methyl-1,2-cyclohexanediamine}oxalatoplatinum(II): A Novel Enantiomerically
Pure Oxaliplatin Derivative Showing Improved Anticancer Activity in Vivo
Sergey A. Abramkin,† Ute Jungwirth,‡,^ Seied M. Valiahdi,†,^ Claudia Dworak,† Ladislav Habala,† Kristof Meelich,†
Walter Berger,‡,§ Michael A. Jakupec,† Christian G. Hartinger,† Alexey A. Nazarov,*, Markus Galanski,*,† and
Bernhard K. Keppler*,†,§
†
‡
University of Vienna, Institute of Inorganic Chemistry, Wahringer Strasse 42, A-1090 Vienna, Austria, Department of Medicine I, Institute of
€
Cancer Research, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria, §Research Platform “Translational Cancer Therapy
€
Research” University of Vienna, Wahringer Strasse 42, A-1090 Vienna, Austria, and Institut des Sciences et Ingenierie Chimiques,
ꢀ
Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland. ^ U.J. and S.M.V. contributed equally to this work
ꢀ ꢀ
Received May 20, 2010
Novel derivatives of the clinically established anticancer drug oxaliplatin were synthesized. Cytotoxicity
of the compounds was studied in six human cancer cell lines by means of the MTT assay. Additionally,
most promising complexes were also investigated in cisplatin- and oxaliplatin-resistant human cancer
cell models. The therapeutic efficacy in vivo was studied in the murine L1210 leukemia model. Most
remarkably, {(1R,2R,4R)-4-methyl-1,2-cyclohexanediamine}oxalatoplatinum(II), comprising an equa-
torial methyl substituent at position 4 of the cyclohexane ring, was as potent as oxaliplatin in vitro but
distinctly more effective in the L1210 model in vivo at the optimal dose. The advantage observed in the in
vivo situation was mainly based on a more favorable therapeutic index. The maximum tolerated dose
of the novel analogue was higher than that of oxaliplatin and caused a greater increase in life span
(>200% versus 152%), with more animals experiencing long-term survival (5/6 versus 2/6). These data
support further (pre)clinical development of the methyl-substituted oxaliplatin analogue with improved
anticancer activity.
Introduction
(5-FU/LV); neurotoxicity was found to be dose limiting.6
Clinical trials based on different tumors of the gastrointestinal
tract and breast and lung carcinomas are ongoing. Remark-
ably, oxaliplatin shows a different spectrum of cytotoxicity
compared to cis- and carboplatin as deduced from the 60
cell line panel of the NCI, being in accord with its activity
in the inherently cis- and carboplatin resistant colorectal
carcinoma.7
The cytotoxic properties of cisplatin, cis-diamminedi-
chloridoplatinum(II),1 were found by serendipity in Barnett
Rosenberg’s laboratory in 1965.2,3 Cisplatin was finally ap-
proved by the Food and Drug Administration (FDAa) in
1978. Exchange of the two chloride leaving groups of cisplatin
with the more kinetically inert 1,1-cyclobutanedicarboxylato
ligand resulted in carboplatin, cis-diammine(1,1-cyclobuta-
nedicarboxylato)platinum(II), which received approval in
1989 as second-generation platinum-based agent. In 2004,
the FDA approved the third generation platinum drug,
oxaliplatin (Figure 1), {(1R,2R)-cyclohexanediamine}oxalato-
platinum(II),4,5 for the first line treatment of colorectal carci-
noma in combination with 5-fluorouracil and leucovorin
Compared to cisplatin, the volume of distribution (serves as
a measure, how effectively a drug is cleared from the blood-
stream and going into tissue) of oxaliplatin is considerably
high. Moreover, the terminal elimination phase is longer than
that for cisplatin.8 The mechanism of cellular oxaliplatin
accumulation is still unclear. In contrast to cisplatin, accu-
mulation of oxaliplatin in cancer cells is less dependent on the
copper transporter CTR1.9 Like cisplatin, the ultimate target
of oxaliplatin is the genomic DNA and it forms the same types
of inter- and intrastrand DNA cross-links as cisplatin,10,11
however, the reactivity toward isolated and cellular DNA is
lower.12 Because the cytotoxic potency of oxaliplatin is gen-
erally well comparable or even higher than that of cisplatin in
some cell lines, oxaliplatin-induced DNA adducts have to be
more cytotoxic than those arising from cisplatin.13 Cisplatin-
DNA adducts are effectively recognized by the MMR com-
plex and by HMG1 but not those adducts deriving from
oxaliplatin.14,15 Postreplicative bypass is also different and
more efficient past oxaliplatin-GG adducts.16 On a molecu-
lar level, the adducts of oxaliplatin17 and cisplatin18 with the
same dodecamer duplex were investigated by crystal structure
*To whom correspondence should be addressed. For A.A.N.: phone,
þ41 (0)21 693 98 60; fax, þ41 (0)21 693 98 85; E-mail, alexey.
nazarov@epfl.ch, For M.G.: phone, þ43-1-4277-52603; fax, þ43-1-
4277-52680; E-mail, markus.galanski@univie.ac.at. For B.K.K.:
phone, þ43-1-4277-52602; fax, þ43-1-4277-52680; E-mail, bernhard.
keppler@univie.ac.at.
a Abbreviations: 5-FU, 5-fluorouracil; Cbz, carbobenzoxy; COSY,
correlated spectroscopy; CTR, copper transporter; DACH, diaminocy-
clohexane; DMF, dimethylformamide; DMSO, dimethyl sulfoxide;
d-OHP, S,S-enantiomer of oxaliplatin; ESI, electrospray ionization;
FDA, Food and Drug Administration; G, guanine; HMG, high-
mobility group protein; ILS, increase in life span; LV, leucovorin;
MEM, minimal essential medium; MMR, mismatch repair; MsCl,
methanesulfonyl chloride; MTD, maximum tolerated dose; MTT,
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NCI,
National Cancer Institute; Py, pyridine.
r
pubs.acs.org/jmc
Published on Web 10/01/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7357
Figure 1. Structures of oxaliplatin and the novel enantiomerically pure oxaliplatin derivatives with either equatorial (3a) or axial (3c) methyl
substituent with NMR numbering scheme.
diffraction, revealing a relatively similar geometry. From that
point of view, it is difficult to understand the differences in
their cytotoxic potential. However, it has to be taken into
account that the situation in solution may vary from that in
the solid state as could be demonstrated recently.19
increased lipophilicity, additionally having an influence on
the structure of adducts formed with the final target DNA.
Consequently, the intention was to improve the biological
properties of oxaliplatin and not to develop completely
different types of complexes (“The most fruitful basis for the
discovery of a new drug is to start with an old drug” Sir James
Black, Nobel Prize in Physiology or Medicine in 1988).25
The oxalato leaving group was left unchanged because it
affects the solubility, the biodistribution, and the systemic
toxicity but of course not the structure of DNA adducts. The
{trans-(1R,2R)-cyclohexanediamine}platinum(II) fragment
binds to DNA in the major groove (Supporting Information,
Figure S1), generating there an unpolar region/steric bulk. From
that point of view, it seems to be reasonable to derivatize the
fragment at position 4 of the cyclohexane ring (preferably in
equatorial position), offering a maximal distance from the
coordination site. Substitution at positions close to the metal
center, or even at the coordinated nitrogen atoms, would
significantly influence the structural changes of DNA adducts
and were therefore not favored.
Synthesis and Characterization. Racemic (1R,2R,4R/
1S,2S,4S)-4-methyl-1,2-cyclohexane-diamine was synthe-
sized as reported recently.26 Shortly, 4-methylcyclohex-1-ene
was converted into the vicinal trans-diol (Scheme 1) by
treatment with hydrogen peroxide in the presence of formic
acid. In a next step, the corresponding mesylate was formed
via reaction with methanesulfonyl chloride/pyridine, which
was then transferred with sodium azide into 4-methyl-
trans-1,2-cyclohexanediazide. The diamine was subsequently
obtained by reduction with hydrogen at Pd on CaCO3
(Lindlar’s catalyst).
Enantiomer separation of racemic (1R,2R,4R/1S,2S,4S)-
4-methyl-1,2-cyclohexanediamine was performed via recrys-
tallization of the respective L- or D-tartrates in an ethanol-
water mixture (Supporting Information, Scheme S1).
Enantiomerical purity was proven by chiral HPLC. Prior
to HPLC analysis, the diamines were derivatized with ben-
zyloxycarbonyl chloride in order to obtain UV-active com-
pounds (6 þ 7, Supporting Information, Scheme S1 and
Figure S2).
Enantiomerically pure (1R,2R,4R)-4-methyl-1,2-cyclo-
hexanediaminium L-tartrate, 1a, was then reacted with
K2PtCl4 in the presence of NaOH, resulting in the yellow
dichloridoplatinum(II) complex 2a. The chlorido ligands
were released with AgNO3 and addition of potassium
oxalate to the formed diaquaplatinum(II) species finally
resulted in the white target complex 3a, {(1R,2R,4R)-4-
methyl-1,2-cyclohexanediamine}oxalatoplatinum(II). The
corresponding 1S,2S,4S-enantiomer 3b was obtained by
following the same procedure and starting from (1S,2S,4S)-
4-methyl-1,2-cyclohexanediaminium D-tartrate 1b.
The cyclohexanediamine carrier ligand in oxaliplatin com-
prises a trans-1R,2R configuration with both amino substi-
tuents in equatorial position; the cyclohexane ring is situated
nearly perfectly within the square planar coordination
plane.20 The same holds true for the trans-1S,2S enantiomer.
In the case of the cis-1R,2S counterpart, one amino substi-
tuent is in an equatorial and the other in an axial position,
leading to a nearly perpendicular arrangement of the cyclo-
hexane ring with respect to the coordination plane.21 The
antiproliferative properties of all three (cyclohexanediamine)-
oxalatoplatinum(II) complexes have been investigated with
the following structure-activity relationship (DACH =
diaminocyclohexane): trans-1R,2R-DACH enantiomer >
trans-1R,2R/1S,2S-DACH racemate > trans-1S,2S-DACH
enantiomer>cis-1R,2S-DACH diastereomer.22,23 The differ-
ence between trans- and cis-configured complexes is easily
explainable by the shape of the cis analogue (perpendicular
arrangement of the cyclohexane ring). Indeed, due to steric
hindrance, adduct formation with DNA was slow and also the
conversion of the monoadduct to the bisadduct.24 However,
in the case of both trans-isomers (same shape), the DNA
seems to act as chiral discriminator as could be shown by
Lippard and colleagues.17 Formation of a hydrogen bond
between the pseudoequatorial NH hydrogen of the (1R,2R)-
cyclohexanediamineplatinum(II) fragment and O6 of a neighbor-
ing guanine was found, which is not possible in the case of
trans-1S,2S-configuration.
Finally, what we really do know about the mode of action
ofoxaliplatin andwhatmay serveasbasis forthe development
of novel oxaliplatin derivatives with improved cytotoxic and
anticancer properties are: (i) distribution in the body and
cellular accumulation of cisplatin and oxaliplatin are different
and (ii) recognition and processing of DNA adducts are not
the same. These features are, at least in part, the result of the
trans-(1R,2R)-cyclohexanediamine carrier ligand, providing
lipophilicity along with steric bulk to oxaliplatin. Conse-
quently, we have focused on further increasing the lipophili-
city/steric bulk of the cyclohexanediamine moiety. Here, we
report for the first time on the synthesis, characterization, in
vitro cytotoxicity, and in vivo anticancer activity of a novel
enantiomerically pure oxaliplatin derivative, {(1R,2R,4R)-4-
methyl-1,2-cyclohexanediamine}oxalatoplatinum(II), ex-
hibiting an equatorial methyl group at position 4 of the
cyclohexane ring. We also demonstrate that bigger substitu-
ents (ethyl or t-butyl) do not lead to an improvement of the
cytotoxic and anticancer properties.
Oxalatoplatinum(II) complexes 3a and 3b were fully char-
acterized by elemental analysis and multinuclear (1H, 13C,
and 195Pt) one- and two-dimensional NMR spectroscopy.
The coordination sphere around the platinum atom can best
Results and Discussion
The aim of the present work was to synthesize close
analogues of oxaliplatin, which are equipped with an

7358 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Abramkin et al.
be judged by measuring a 195Pt NMR spectrum. The reso-
nance at -365 ppm for 3a (Figure 2) is indicative for a
PtN2O2 coordination and is in accordance with the chemical
shift found for oxaliplatin at -361 ppm.27 The correspond-
ing 195Pt signal for the dichlorido precursor complex 2a with
PtN2Cl2 coordination was detected significantly upfield
at -644 ppm.
In 3a, all substituents (two amino and one methyl group)
of the cyclohexane ring are in an equatorial position; conse-
quently, protons at carbon atoms 1, 2, and 4 are axially
oriented, which can be proven by the Karplus relationship
for NMR spin-spin coupling constants (vicinal H-C-
C-H coupling constants are large at angles of 180ꢀ with
3JH,H ≈ 11-12 Hz, and small when the torsion angle is 60ꢀ
with a coupling of around 5 Hz).
Scheme 1. Synthesis of {(1R,2R,4R)-4-Methyl-1,2-cyclohex-
anediamine}oxalatoplatinum(II), 3aa
Indeed, large couplings of the axial proton H-1ax at 2.31
ppm with the neighboring protons H-2ax and H-6ax with
3JH,H = 11.5 Hz, and a small 3JH,H coupling to H-6eq of 4 Hz
were detected (Figure 2), proving the equatorial position of
the amino substituent. The same holds true for proton H-2ax,
demonstrating the trans-configuration at C1-C2. In the case
of proton H-3ax, resonating at 0.89 ppm, a quartet with a
coupling constant of 12 Hz was found as a result of two
neighboring axial protons (H-2ax, and H-4ax) and addition-
ally the large geminal 3JH,H coupling to proton H-3eq. Axial
position of H-4ax finally proves that the methyl group at C-4 is
equatorially oriented. Signal assignment of all remaining pro-
tons is based on two-dimensional H,1H- and H,13C corre-
lated NMR spectra. Of note is the diastereotopic splitting of
the methylene protons at C-3, C-4, and C-6, giving rise to six
single shift correlation signals in the 1H,13C NMR spectrum.
Synthesis and characterization of racemic 4-ethyl and 4-t-
butyl-1,2-cyclohexanediamine}oxalatoplatinum(II) com-
plexes 4 and 5 (all substituents at the cyclohexane ring
equatorial position, Scheme 1) were described recently.28
With the intention of further optimizing structure-activity
relationships, the platinum complex 3c, {(1R,2R,4S)-4-methyl-
1,2-cyclohexanediamine}oxalatoplatinum(II), exhibiting
1
1
a (i) H2O2/HCOOH, (ii) MsCl/Py, NaN3, (iii) Pd/CaCO3, H2 (3 bar),
(iv) L-tartaric acid, (v) K2PtCl4/NaOH (0.25 M), and (vi) AgNO3,
potassium oxalate. Structures of 4-ethyl- and 4-t-butyl oxaliplatin
derivatives 4 and 5, synthesized as enantiomeric mixtures (all substitu-
ents at the cyclohexane ring in equatorial positions).
Figure 2. 1H and 195Pt (small insert) NMR spectrum of {(1R,2R,4R)-4-methyl-1,2-cyclohexanediamine}oxalatoplatinum(II) 3a with NMR
numbering scheme and signal assignment. The oxalatoplatinum(II) fragment has been omitted for clarity.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7359
Table 1. Cytotoxicity of Oxaliplatin Derivatives Compared to Oxaliplatin in Six Human Cancer Cell Lines
IC50 (μM)a
compd
CH1
HeLa
HCT-15
HCT-116
SW480
U-2 OS
oxaliplatin
d-OHPb
0.33 ( 0.09
1.0 ( 0.04
0.28 ( 0.09
0.69 ( 0.10
0.10 ( 0.01
0.38 ( 0.08
4.1 ( 0.8
0.35 ( 0.07
1.1 ( 0.4
1.5 ( 0.6
4.4 ( 1.6
1.6 ( 0.1
4.6 ( 0.7
0.33 ( 0.07
3.4 ( 1.0
39 ( 3
0.38 ( 0.03
1.7 ( 0.1
0.81 ( 0.31
2.9 ( 0.7
0.32 ( 0.05
1.6 ( 0.1
31 ( 2
0.30 ( 0.08
1.3 ( 0.3
0.67 ( 0.31
2.3 ( 1.2
0.33 ( 0.14
0.92 ( 0.30
24 ( 3
0.72 ( 0.24
2.5 ( 0.7
0.71 ( 0.41
1.8 ( 0.6
0.52 ( 0.03
4.0 ( 0.3
24 ( 5
3a
3b
3c
4
0.21 ( 0.06
0.88 ( 0.38
0.24 ( 0.02
0.66 ( 0.13
11 ( 2
5
a 50% inhibitory concentrations in the MTT assay (96 h exposure). Values are means ( standard deviations obtained from at least two (mostly three)
independent experiments. b d-OHP is the (1S,2S)-enantiomer of oxaliplatin.
Figure 3. Concentration-effect curves of ring-substituted oxaliplatin derivatives in comparison to oxaliplatin and its S,S analogue (d-OHP)
in CH1, HeLa, U-2 OS, HCT-15, HCT-116, and SW480 cells, obtained by the MTT assay (96 h exposure).
an axial methyl substituent at position 4 of the cyclohexane
ring, was synthesized (Supporting Information, Scheme S2).
Starting from enantiomerically pure (S)-citronellal, (S)-4-
methylcyclohexene was prepared, which was converted to
the corresponding diazide exhibiting trans-configuration at
C1/C2. After reduction, (1R,2R,4S)-4-methyl-1,2-cyclohex-
anediaminium L-tartrate, 1c, was obtained (diastereomeric
purity was proven by chiral HPLC in the form of the Cbz
derivative, Supporting Information, Figure S3). In analogy
to 3a and 3b, the oxalato complex 3c was synthesized and
characterized. Besides a distinctly different 195Pt chemical
shift (3c, -381 ppm; 3a, -365 ppm), the axial position of the
methyl group could be judged by comparison of 1H
(Supporting Information, Figure S4) and 13C NMR spectra
of 3a and 3c, respectively. As expected, the chemical shift
difference of protons H-1ax and H-2ax (Δδ = 0.27 ppm) in
3c is significantly bigger compared to 3a (Δδ = 0.08 ppm).
The same holds true for chemical shift differences of carbon
atoms C1 and C2 in 3c (Δδ = 4.9 ppm) and 3a (Δδ = 0.2
ppm). However, most indicative are the differences (shapes
and chemical shifts, compare Figures 2 and Supporting
Information, Figure S4) in the proton NMR spectra for 1H
resonances at C3, C4 and C5.
Cytotoxicity in Cancer Cell Lines and Structure-Activity
Relationships. Cytotoxicity of oxaliplatin and the here pre-
sented derivatives with equatorial alkyl substituents in posi-
tion 4 of the cyclohexane ring was compared by means of a
colorimetric microculture assay (MTT assay) in six human
cancer cell lines representing four tumor entities: ovarian
carcinoma (CH1), cervical carcinoma (HeLa), osteosarcoma
(U-2 OS), and colon carcinoma (HCT-15, HCT-116,
SW480), yielding IC50 values mostly in the low micromolar
or even submicromolar range (Table 1, Figure 3).
Oxaliplatin and the 4-methyl derivative 3a show quite
similar cytotoxic potencies in all six cell lines. Notably, 3a
is at least as active as oxaliplatin in four of them, except for
the two colon cancer cell lines HCT-116 and SW480. Gen-
erally, the (1R,2R,4R)-enantiomer 3a is by factors of 2-4 more
cytotoxic than the (1S,2S,4S)-enantiomer 3b, indicating that
the well-known structure-activity relationship observed

7360 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Abramkin et al.
Table 2. Cytotoxicity of Oxaliplatin Derivatives Compared to Oxaliplatin in Cisplatin- And Oxaliplatin-Resistant Human Cancer Cell Models
IC50 (μM)a
compd
GLC4
GLC4/CDDP -fold resistance
A2780
A2780/cis -fold resistance HCT116 HCT116 oxR -fold resistance
cisplatin
1.7 ( 0.5
18.0
10.6
2.0
2.6
2.0
1.8 ( 0.2 >10
>5
1.8
4.7 ( 0.1
0.6 ( 0.1
1.5 ( 0.1
0.5 ( 0.1
7.3 ( 0.1
22.0 ( 0.1
11.7 ( 0.1
9.3 ( 0.1
1.6
36.7
7.8
oxaliplatin 0.6 ( 0.2
3a
3c
1.2 ( 0.3
2.1 ( 0.1
0.6 ( 0.1
0.4 ( 0.2
0.5 ( 0.1
0.2 ( 0.1
0.7 ( 0.4
1.6 ( 0.8
0.5 ( 0.3
0.8 ( 0.1
0.3 ( 0.2
3.2
2.5
18.6
a 50% inhibitory concentrations in the MTT assay (72 h exposure). Values are means ( standard deviations obtained from at least two (mostly three)
independent experiments.
with oxaliplatin and its (1S,2S)-enantiomer (d-OHP) also
applies to the alkyl-substituted derivatives. The other 4-methyl
derivative 3c (methyl substituent in axial position) is even
more cytotoxic than oxaliplatin in the majority of cell lines
and thus the most potent of the studied compounds, whereas
the racemic 4-ethyl derivative 4 yields IC50 values that are
mostly intermediate between those of 3a and 3b (with the
exception of U-2 OS cells).
Figure 4A). Although in equal doses the efficacy of the
4-methyl derivative 3a is not higher than that of oxaliplatin,
the lower toxicity of this compound enables the application
of a higher dose, resulting in a clearly superior therapeutic
effect with as much as five long-term survivors and an ILS of
more than 200% (Figure 4B). Surprisingly, the analogous
complex 3c (with the 4-methyl substituent in axial instead of
equatorial position) causes an ILS of only 122%. Thus, this
compound is much less active than 3a and even somewhat
less active than oxaliplatin despite a cytotoxic potency super-
ior to all other compounds studied in vitro. The 4-ethyl
derivative 4 is also slightly less effective than oxaliplatin and
causes an ILS of 129%, with one long-term surviving animal
at the optimal dose. However, this compound is a racemic
mixture, and improved therapeutic efficacy might still be
expected from the pure (1R,2R,4R)-enantiomer. The lower
efficacy of (1S,2S)-enantiomers is confirmed by data obtained
with d-OHP and 3b, which are no more than half as effective
as oxaliplatin and 3a, respectively. Finally, further increasing
the size of the substituent to t-butyl (compound 5) results in a
tremendous loss of activity in applicable doses, which par-
allels the structure-activity relationships observed in vitro.
A synopsis of in vitro and in vivo data reveals that small
alkyl substituents (methyl, perhaps also ethyl) on the cyclo-
hexane ring result in an optimum with respect to the ther-
apeutic index mainly due to a higher tolerability than
unsubstituted oxaliplatin and a higher activity than deriva-
tives with larger substituents. However, the geometric orien-
tation (axial vs equatorial) of the substituent obviously
plays a decisive role, having opposite effects on cytotoxicity
in vitro and efficacy in vivo. Apart from that, structure-
activity relationships in vitro and in vivo nicely parallel each
other within the 4-substituted oxaliplatin derivatives, but
this aberrant example demonstrates that caution should be
exercised when selecting candidate compounds for drug
development on the basis of in vitro data. The L1210
leukemia model employed may seem remote from the actual
clinical indication of oxaliplatin, i.e. colorectal cancer, but it
should be called in mind that it was one of the few tumor
models in which the promising activity of oxaliplatin was
originally recognized and therefore proved particularly valu-
able in this context.30,31 Nevertheless, further evaluation of
the compounds will require additional models, in particular,
solid tumors.
In contrast, the IC50 values of the racemic 4-t-butyl
derivative 5 are on average an order of magnitude higher
than those of 4. These data support a previous study, which
showed that large alkyl substituents will result in a decrease
of cytotoxicity.29 Generally, the cytotoxicity profiles of these
compounds resemble each other, with CH1 and HeLa being
the most sensitive cell lines and HCT-15 being the least
sensitive cell line. Therefore, fundamental differences in the
underlying molecular mechanisms of action are unlikely.
The cytotoxic properties of compounds 3a and 3c were
also analyzed in cisplatin- and oxaliplatin-resistant cell
models derived from small cell lung (GLC4), ovarian cancer
(A2780), and colon cancer (HCT-116) (Table 2). Both highly
cisplatin-resistant cell models (GLC4/CDDP and A2780/cis)
were (comparable to oxaliplatin) significantly but very mod-
erately (up to 3.2-fold) cross-resistant to the novel deriva-
tives. The resistance of oxaliplatin-insensitive HCT-116 oxR
against the new derivatives was, as expected, more distinct
(up to 18.6-fold) as compared to that of cisplatin. Never-
theless, it was significantly weaker for both tested derivatives
(3a and 3c) as compared to oxaliplatin (36.7-fold).
In Vivo Anticancer Activity. Anticancer activity in vivo was
investigated in the murine L1210 leukemia model. Com-
pounds were administered intraperitoneally on three con-
secutive days. A transient decrease of body weight to not less
than 85% was considered tolerable, and for each compound
the tolerable dose resulting in the highest increase in life span
(ILS) was considered optimal. Except for 5, which did not
allow dose escalation beyond 12 mg/kg/day because of much
lower solubility, the maximum tolerated dose (MTD) was
reached for all compounds. The optimal dose in terms of
therapeutic efficacy is mostly identical with the MTD, except
for d-OHP, the (S,S)-analogue of oxaliplatin (Table 3).
Remarkably, a mere 50% dose increase over the MTD
(6 mg/kg/day) turned oxaliplatin in a 100% lethal com-
pound, indicating a steep lethality curve, whereas in the case
of the new derivatives, a 50% increase over the respective
MTD resulted only in 0-2 toxic deaths (in groups of six
animals each). With the exception of 3b, all compounds were
tolerated in at least 50% higher doses (9 mg/kg/day) than
oxaliplatin.
As the cytotoxicity profiles of the studied compounds
argue against fundamental differences in the mode of action,
the reasons for the differences in therapeutic index and the
favorable cross-resistance patterns may be found in the
combination of characteristics such as lipophilicity and
bulkiness and their subtle consequences for drug transport,
biotransformation, and interactions with DNA as well as
DNA repair systems.
All compounds tested prolonged the survival of leukemic
mice with high statistical significance (p < 0.001) but to
different extents. The optimal therapeutic effect of the parent
compound oxaliplatin caused an increase in life-span (ILS)
by 152% and long-term survival of two animals (Table 3,
Comparing both methyl derivatives 3a (equatorial methyl
group) and 3c (axial methyl group), it is remarkable that 3c is

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7361
Table 3. Tolerability and Efficacy of Intraperitoneal Treatment with Oxaliplatin Derivatives Compared to Oxaliplatin in L1210 Leukemia-Bearing
Mice (Therapeutic Effects at the Respective Optimum Dose Are Marked in Bold Type)
compd
dose (mg/kg/day)
min body weight (%)
toxic deaths
ILS (%)a
long-term survivorsb
oxaliplatin
9
6/6
0/6
0/6
0/6
0/6
2/6
1/6
1/6
6 (MTD)c
93.2
91.3
94.1
152
89
4.5
3
94
d-OHP
12
9 (MTD)c
1/6
0/6
0/6
0/6
0/6
0/6
1/6
0/6
85.9
93.1
93.6
63
73
26
6
4.5
3a
12
9 (MTD)c
2/6
0/6
0/6
0/6
0/6
4/6
5/6
0/6
1/6
0/6
88.6
97.5
94.6
97.3
> 200
119
84
6
4.5
3
90
3b
3c
4
9
1/6
0/6
0/6
0/6
0/6
1/6
2/6
1/6
6 (MTD)c
92.0
93.4
97.9
103
68
4.5
3
68
12
9 (MTD)c
2/6
0/6
0/6
0/6
1/6
2/6
1/6
2/6
86.4
87.3
87.9
122
78
6
4.5
111
12
9 (MTD)c
83.2
89.9
93.6
98.4
0/6
0/6
0/6
0/6
144
129
89
2/6
1/6
0/6
0/6
6
3
61
5
12
6
100.0
100.0
98.5
0/6
0/6
0/6
33
25
17
0/6
0/6
0/6
3
a Increase in life span compared to untreated controls, based on median survival; all values indicate life prolongation with high statistical significance
(p < 0.001). b Animals with ILS > 200% without any signs of leukemia. c Maximum tolerated dose.
equipped with a higher cytotoxicity in vitro, but in parallel
showing a lower anticancer activity in vivo. Differences
between 3a and 3c in the cellular accumulation and DNA
adduct formation are expected to be very similar in vitro and
in vivo. Nevertheless, one might assume the existence of
interacting factors like altered transport by drug uptake and/
or efflux pumps which might impact on the pharmacological
characteristics of these stereoisomeric complexes in vivo but
not in vitro. The identification of these interacting factors is a
matter of ongoing investigations.
primarily focus on colorectal cancer, where oxaliplatin is used
as therapeutic standard, but also on other tumors recently
suggested to be sensitive against this third-generation plati-
num compound like certain leukemias32 or esophago-gastric
cancer.33
Experimental Section
If necessary, the reactions were carried out in dry solvents and
under argon atmosphere. Potassium tetrachloridoplatinate(II) was
obtained from Johnson Matthey (Switzerland). All other chemicals
obtained from commercial suppliers were used as received and
were of analytical grade. Water was prior to use doubly distilled.
The synthetic procedures were carried out in light protected
environment when platinum complexes were involved. 1H, 13C,
15N, 195Pt, and two-dimensional COSY NMR spectra were re-
corded with a Bruker Avance DPX 400 instrument (Ultrashield
Magnet) at 400.13 MHz (1H) and 100.63 MHz (13C) or with a
Bruker Avance III 500 MHz NMR spectrometer at 500.32 (1H),
125.81 (13C), 107.55 (195Pt), and 50.70 MHz (15N) in DMF-d7,
D2O, or CDCl3 at 298 K. 15N chemical shifts were referenced
relative to external NH4Cl, whereas 195Pt chemical shifts were
referenced relative to external K2[PtCl4]. HPLC analysis was
carried out using a Dionex Summit system. The sample concentra-
tion was 1.0 mg/mL, with an injection volume of 10 μL introduced
onto a Chiralcel OD column (250 mm ꢀ 4.6 mm) protected by a
guard column (50 mm ꢀ 4.6 mm) at 5 ꢀC. The flow rate of the
mobile phase (hexane:i-propyl alcohol 9:1) was 1 mL/min. Melting
Conclusions
In this study, we successfully developed an oxaliplatin
derivative with enhanced antitumor activity compared to
the parental drug by introducing a small alkyl substituent
on the cyclohexane ring in equatorial position. Using the
L1210 murine leukemia model, the highest anticancer activity
was proven for {(1R,2R,4R)-4-methyl-1,2-cyclohexanedi-
amine}oxalatoplatinum(II), resulting in both increased life
span and enhanced long-term survival. Although in vitro this
compound is not consistently more cytotoxic than oxaliplatin,
the better tolerability enables application of higher doses,
resulting in enhanced efficacy in vivo. Considering that
adverse effects are a central problem of clinical oxaliplatin
application, the evaluation of the new derivative in
(pre)clinical studies is highly desirable. These studies should
€
points were determined with a Buchi B-540 apparatus and are

7362 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Abramkin et al.
3JH,H = 4.0 Hz, 1H, H-2ax). 13C NMR in D2O: δ = 20.0 (C-7),
30.6 (C-6), 30.9 (C-4), 32.2 (C-5), 39.5 (C-3), 62.0 (C-2), 62.2 (C-1),
168.3 (COO). 195Pt NMR in D2O: δ = -365.
(SP-4-3)-[(1S,2S,4S)-4-Methyl-1,2-cyclohexanediamine- K2N,
N0][ethanedioato(2-)-K2O1,O2]platinum(II) 3b. Following the
same procedure as described for 3a, complex 3b was obtained
from (SP-4-3)-dichlorido[(1S,2S,4S)-4-methyl-1,2-cyclohexane-
diamine-κ2N,N0]platinum(II) 2b (200 mg, 0.5 mmol). Yield: 0.82
mg(38%), mp275-280 ꢀC(decomp). Elemental analysis, Found:
C, 26.07; H, 3.66; N, 6.83. Calcd for C9H16N2O4Pt: C, 26.28; H,
3.92; N, 6.81. MS (ESIþ) m/z 434 [M þ Na]þ. 1H NMR in D2O:
δ = 0.85 (dq, 3JH,H=2JH,H = 13 Hz, 3JH,H = 3.5Hz, 1H, H-5ax),
0.86 (d, 3JH,H = 6.5 Hz, 3H, H-7), 0.97 (q, 3JH,H = 2JH,H=12 Hz,
1H, H-3ax), 1.28 (dddd, JH,H = 12.5 Hz, JH,H = 12.5 Hz, JH,H =
12.0Hz, JH,H =3.5 Hz, 1H, H-6ax), 1.36 (m,1H, H-4ax), 1.50(m,
1H, H-5eq), 1.88-2.00 (m þ m, 2H, H-3eq, H-6eq), 2.30 (dt,
3JH,H = 11.5 Hz, 3JH,H = 4.0 Hz, 1H, H-1ax), 2.38 (dt, 3JH,H
=
11.5 Hz, 3JH,H = 4.0 Hz, 1H, H-2ax). 13C NMR in D2O: δ =20.0
(C-7), 30.6 (C-6), 30.9 (C-4), 32.2 (C-5), 39.5 (C-3), 62.0 (C-2),
62.2 (C-1), 168.3 (COO). 195Pt NMR in D2O: δ=-365.
(SP-4-3)-[(1R,2R,4S)-4-Methyl-1,2-cyclohexanediamine- K2N,N0]-
[ethanedioato(2-)-K2O1,O2]platinum(II) 3c. (SP-4-3)-{Dichlorido-
[(1R,2R,4S)-4-methyl-1,2-cyclohexanediamine-κ2N,N0]platinum(II)}
(1.767 g, 4.48 mmol) was suspended in 80 mL of water and a solution
of AgNO3 (1.462 g; 8.60 mmol) in 10 mL of water was added. The
suspension was stirred for 24 h at room temperature. The AgCl
formed was filtered off with a G4 glass sinter filter with an additional
MN GF3 filter. The clear, slightly yellowish solution was treated with
conditioned basic ion-exchange resin IRA402 for 24 h at room
temperature (shaking). [Conditioning of IRA402: 150 g of IRA402
chloride form was stirred with 2 M NaOH (400 mL) for 30 min. Then
the resin was washed with deionized water until achieving neutral pH
followed by washing with distilled water twice.] The ion-exchange
resin was separated from the solution by decantation and washed
with water three times. The combined aqueous solutions were filtered
through a G4 glass sinter filter, and oxalic acid dihydrate (0.565 g,
4.48 mmol) was added. The mixture was put into a round flask, and
the volume was reduced to ca. 80 mL within 3 h, whereupon the
liquid became cloudy. The latter was kept at room temperature for
12 h and was further reduced to ca. 40 mL. A white solid formed
which was finally filtered with a G4 glass sinter filter after 1 h, washed
with water twice, and dried in vacuum over P4O10. Yield: 0.534 g
(29%). Elemental analysis, Found: C, 26.07; H, 3.88; N, 6.66. Calcd
for C9H16N2O4Pt: C, 26.28; H, 3.92; N, 6.81. 1H NMR in D2O: δ =
0.84 (d, 3JH,H = 7.5 Hz, 3H, H-7), 1.22-1.55 (m þ m þ m þ m, 4H,
H-5eq, H-5ax, H-3ax, H-6ax) 1.71-1.90 (m þ m þ m, 3H, H-3eq,
Figure 4. Kaplan-Meier plots showing the survival (days after
tumor implantation) of L1210 leukemia-bearing mice treated in-
traperitoneally with different doses of oxaliplatin or 3a in compar-
ison to untreated controls, in groups of six animals each. Note that
mortality in animals treated with 9 mg/kg/day oxaliplatin is drug-
related in all cases (A), whereas the same dose of 3a results in long-
term survival of five animals (B).
uncorrected. Electrospray ionization mass spectra were recorded
on a Bruker esquire3000 in positive ion mode. Elemental analyses of
prepared compounds were carried out on a Perkin-Elmer 2400
CHN elemental analyzer or a Carlo Erba microanalyzer at the
Microanalytical Laboratory of the University of Vienna and are
within (0.4% of the calculated values, confirming their g95%
purity. Silica gel (Polygram SIL G/UV254) was used for thin layer
chromatography.
3
3
H-6eq, H-4eq), 2.24 (dt, JH,H = 12.0 Hz, JH,H = 4.5 Hz, 1H,
3
3
(SP-4-3)-[(1R,2R,4R)-4-Methyl-1,2-cyclohexanediamine- K2N,
N0][ethanedioato(2-)-K2O1,O2]platinum(II) 3a. AgNO3 (167 mg,
0.98 mmol) was added to a suspension of (SP-4-3)-dichlorido-
[(1R,2R,4R)-4-methyl-1,2-cyclohexanediamine-κ2N,N0]platinum-
(II) 2a (200 mg, 0.5 mmol) in water (3 mL). The reaction mixture
was stirred for 24 h at room temperature, the precipitated silver
chloride was filtered off, and an in situ prepared solution of
potassium oxalate from oxalic acid dihydrate (61 mg, 0.48 mmol)
and KOH(0.96 mL, 0.5 M, 0.48 mmol) inwater (2 mL) was added
tothe diaqua complex. The reaction mixture was stirred for 24 h at
room temperature, the solvent was removed in vacuum, and the
pure white complex was obtained after recrystallization in water
(1 mL). The product was filtered off and dried under reduced
pressure over P4O10. Yield: 0.82 mg (39%), mp 282-290 ꢀC
(decomp). Elemental analysis, Found: C, 26.05; H, 3.65; N,
6.55. Calcd for C9H16N2O4Pt: C, 26.28; H, 3.92; N, 6.81. MS
H-1ax), 2.51 (dt, JH,H =12.0 Hz, JH,H = 4.0 Hz, 1H, H-2ax).
13C NMR in D2O: δ = 16.7 (C-7), 26.4 (C-6), 27.0 (C-4), 29.3 (C-5),
37.2 (C-3), 57.8 (C-2), 62.7 (C-1), 168.3 (COO). 195Pt NMR in D2O:
δ=-381.
Cell Lines and Culture Conditions. CH1 (ovarian carcinoma,
human) cells were kindly provided by Lloyd R. Kelland (CRC
Centre for Cancer Therapeutics, Institute of Cancer Research,
Sutton, UK). HeLa (cervical carcinoma, human) and U-2 OS
(osteosarcoma, human) cells were donated by Thomas Czerny
(Institute of Genetics, University of Veterinary Medicine Vienna,
Austria). SW480, HCT116, and HCT15 (all colon carcinoma,
human) cells were kindly provided by Brigitte Marian (Institute of
Cancer Research, Medical University of Vienna, Austria). In addi-
tion, cell models comprising a parental drug-sensitive and respective
cisplatin- or oxaliplatin-resistant subline were used. GLC4 small cell
lung cancer cells together with cisplatin-resistant GLC4/CDDP
cells were kindly supplied by Elisabeth de Vries, University of
Groningen. The ovarian carcinoma cell line A2780 together with
the cisplatin-resistant subline A2780/cis were obtained from Sigma.
The oxaliplatin-resistant subline HCT-116 oxR was established by
stepwise drug selection of HCT-116 colon cancer cells.
1
(ESIþ) m/z 434 [M þ Na]þ. H NMR in D2O: δ = 0.85 (dq,
2
3
3JH,H = JH,H = 13 Hz, JH,H = 3.5 Hz, 1H, H-5ax), 0.86 (d,
3JH,H = 6.5 Hz, 3H, H-7), 0.98 (q, 3JH,H=2JH,H = 12 Hz, 1H,
H-3ax), 1.29 (dddd, JH,H = 12.5 Hz, JH,H = 12.5 Hz, JH,H = 12.0
Hz, JH,H = 3.5 Hz, 1H, H-6ax), 1.37 (m, 1H, H-4ax), 1.51 (m, 1H,
H-5eq), 1.88-2.00 (m þ m, 2H, H-3eq, H-6eq), 2.31 (dt, 3JH,H
=
11.5 Hz, 3JH,H = 4.0 Hz, 1H, H-1ax), 2.39 (dt, 3JH,H = 11.5 Hz,
GLC4 and A2780 cell lines and derivatives were grown in
RPMI1680 medium with 10% fetal bovine serum. All other cells

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7363
were cultured in complete culture medium, i.e., Minimal Essen-
tial Medium (MEM) supplemented with 10% heat-inactivated
fetal bovine serum, 1 mM sodium pyruvate, 4 mM L-glutamine,
and 1% nonessential amino acids (100ꢀ) (all purchased from
Sigma-Aldrich). Cultures were maintained at 37 ꢀC in a humid-
ified atmosphere containing 5% CO2 and 95% air.
of {(1R,2R,4S)-4-methyl-1,2-cyclohexanediamine}oxalato-
platinum(II), figure with the confirmation of diastereomeric
purity of (1R,2R,4S)-4-methyl-1,2-cyclohexanediamine after
transformation into the CBz-derivative, figure with 1H- and
195Pt NMR spectra of {(1R,2R,4S)-4-methyl-1,2-cyclohexanedi-
amine}oxalatoplatinum(II) 3c, synthesis and characterization
of (S)-4-methylcyclohexene, 4-methyl-1,2-cyclohexanediaminium
tartrates 1a-1c, (SP-4-3)-dichlorido[4-methyl-1,2-cyclohexanedia-
mine-κ2N,N0]platinum(II) complexes 2a-2c, and N,N0-bis-
(benzyloxycarbonyl)-4-methyl-1,2-cyclohexanedicarbamates 6
and 7. This material is available free of charge via the Internet
at http://pubs.acs.org.
Cytotoxicity Tests in Cancer Cell Lines. Antiproliferative
activity was determined by a colorimetric microculture assay
(MTT assay, MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphen-
yl-2H-tetrazolium bromide). For this purpose, cells were har-
vested from culture flasks by trypsinization and seeded in
100-μL aliquots into 96-well microculture plates (Iwaki/Asahi
Technoglass) in the following densities: 1.5 ꢀ 103 (CH1, HeLa),
2.0 ꢀ 103 (GLC4 cell model), 2.5 ꢀ 103 (SW480, HCT-116 cell
model, HCT-15), and 3.2 ꢀ 103 (U2OS, A2780 cell model) viable
cells/well. Cells were allowed to settle and resume adherent
growth in drug-free complete culture medium for 24 h, followed
by the addition of dilutions of the test compounds in 100 μL/
well complete culture medium and incubation for 96 h. In
experiments involving drug-resistance models, the incubation
time was 72 h. At the end of exposure, medium was replaced by
100 μL/well RPMI 1640 medium (supplemented with 10%
heat-inactivated fetal bovine serum and 4 mM L-glutamine) plus
20 μL/well MTT solution in phosphate-buffered saline (5 mg/
mL). After incubation for 4 h, medium was removed and the
formazan product formed by viable cells was dissolved in
DMSO (150 μL/well). Optical densities at 550 nm were mea-
sured with a microplate reader (Tecan Spectra Classic). The
quantity of viable cells was expressed in terms of T/C values by
comparison to untreated controls, and 50% inhibitory concen-
trations (IC50) were calculated from concentration-effect
curves by interpolation. Evaluation is based on means from
at least two (mostly three) independent experiments, each
comprising six replicates per concentration level.
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