Synthesis, Characterization, Cytotoxicity, and Time-Dependent NMR Spectroscopic Studies of (SP-4-3)-Oxalato[(1R,2R,4R/1S,2S,4S)-(4-trifluoromethyl-cyclohexane-1,2-diamine)]platinum(II)

Article abstract of DOI:10.1002/ejic.201801370

The oxaliplatin derivative (SP-4-3)-oxalato[(1R,2R,4R/1S,2S,4S)-(4-trifluoromethyl-cyclohexane-1,2-diamine)]platinum(II) hosting a trifluoromethyl group at position 4 of the cyclohexane ring is presented. The ligand was synthesized as an 1R,2R,4R/1S,2S,4S racemic mixture starting from 4-trifluoromethyl-cyclohexanol over five steps and coordinated to platinum to yield first the dichlorido and subsequently the oxalato complex. This novel compound and its analogue featuring a (¹³C₂)oxalate ligand were characterized by multinuclear (¹H, ¹³C, ¹⁵N, ¹⁹F, ¹⁹⁵Pt) one- and two-dimensional NMR spectroscopy. Ligand exchange and adduct formation reactions with DMSO, NaCl, CaCl₂, l-methionine, and 5′-GMP were investigated via time-dependent measurements by ¹⁹F and ¹³C NMR spectroscopy. The cytotoxicity of the title complex was investigated in three human cancer cell lines; the IC₅₀ values were found in the low micromolar range, attesting moderate to high antiproliferative activity, depending on the cell line.

Full text of DOI:10.1002/ejic.201801370

DOI: 10.1002/ejic.201801370
Full Paper
Oxaliplatin Analogues
Synthesis, Characterization, Cytotoxicity, and Time-Dependent
NMR Spectroscopic Studies of (SP-4-3)-Oxalato-
[(1R,2R,4R/1S,2S,4S)-(4-trifluoromethyl-cyclohexane-1,2-
diamine)]platinum(II)
Selin Hizal,[a] Michaela Hejl,[a] Christoph Jungmann,[a] Michael A. Jakupec,[a,b]
Markus Galanski,*[a] and Bernhard K. Keppler*[a,b]
Abstract: The oxaliplatin derivative (SP-4-3)-oxalato[(1R,2R,4R/ 15N, 19F, 195Pt) one- and two-dimensional NMR spectroscopy.
1S,2S,4S)-(4-trifluoromethyl-cyclohexane-1,2-diamine)]-
platinum(II) hosting a trifluoromethyl group at position 4 of the
cyclohexane ring is presented. The ligand was synthesized as
an 1R,2R,4R/1S,2S,4S racemic mixture starting from 4-trifluoro-
methyl-cyclohexanol over five steps and coordinated to plati-
num to yield first the dichlorido and subsequently the oxalato
complex. This novel compound and its analogue featuring a
(13C2)oxalate ligand were characterized by multinuclear (1H, 13C,
Ligand exchange and adduct formation reactions with DMSO,
NaCl, CaCl2,
L
-methionine, and 5′-GMP were investigated via
time-dependent measurements by 19F and 13C NMR spectro-
scopy. The cytotoxicity of the title complex was investigated in
three human cancer cell lines; the IC50 values were found in the
low micromolar range, attesting moderate to high antiprolifera-
tive activity, depending on the cell line.
1. Introduction
platin, which can easily be labeled by using 15NH4Cl, the synthe-
sis of the 15N-labeled analogue of trans-1,2-diaminocyclohex-
ane ligand in a diastereomerically or even enantiomerically pure
form is laborious.[4] As with cisplatin, the first step in the activa-
tion of oxaliplatin is believed to be aquation. The bidentate
oxalato leaving group is significantly more inert (with respect
to the release of the complete oxalato ligand) compared to
the chlorido ligands in cisplatin.[5] However, monoaquation of
oxaliplatin to the respective monoaqua complex featuring a
monodentate oxalate ligand is relatively fast, with a half-life of
16 min at 37 °C.[2] Since the reverse reaction of ring closure
back to the bidentate oxalate complex is very fast in water and
NaCl solution, complete loss of the monodentate oxalato ligand
to yield the diaquated species takes place at a very slow rate
(half-life of the formation of the diaqua species: 92 h at 37 °C).[6]
The mono- and dichlorido species of oxaliplatin are formed
at a very slow rate in vitro and this would suggest that the
activation of oxaliplatin in vivo proceeds by other pathways.
On the other hand, it was speculated that the adducts of
oxaliplatin with sulfur-containing molecules such as methionine
may play a role in its activation[7] and mode of action.[1] Conse-
quently, investigation of the time-dependent reactions of oxali-
platin and its derivatives with potential target molecules is still
of great importance in order to understand the mechanisms of
activation and anticancer activity in more detail and to assess
the effectiveness and pharmacological properties of platinum
drugs.
Oxaliplatin is applied, e.g., as first-line treatment in case of
metastatic colorectal cancer in combination with 5-fluorouracil/
leucovorin (5-FU/LV). It is unique in its effectiveness against
colorectal cancer among established platinum drugs owing to
the replacement of the ammine carrier ligands in cisplatin or
carboplatin by (1R,2R)-cyclohexane-1,2-diamine. As a conse-
quence, it possesses distinct pharmacokinetic properties such
as a larger volume of distribution and its faster accumulation.[1]
Moreover, cellular processing and recognition of DNA lesions
as well as side effects are different compared to cisplatin and
carboplatin.[2] Very recently, however, it was shown that oxali-
platin does not kill cells through DNA damage response like
cis- and carboplatin but via induction of ribosome biogenesis
stress.[3]
Despite numerous in vitro studies with cisplatin, e.g. based
on NMR spectroscopy, investigations on the kinetics of the bio-
transformation of oxaliplatin are limited because unlike cis-
[a] Institute of Inorganic Chemistry, University of Vienna,
Waehringer Str. 42, 1090 Vienna, Austria
E-mail: markus.galanski@univie.ac.at
https://anorg-chemie.univie.ac.at/research/bioinorganic-chemistry/
group-markus-galanski/
[b] Research Cluster “Translational Cancer Therapy Research”,
University of Vienna,
Waehringer Str. 42, 1090 Vienna, Austria
E-mail: bernhard.keppler@univie.ac.at
https://anorg-chemie.univie.ac.at/research/bioinorganic-chemistry/
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under https://doi.org/10.1002/ejic.201801370.
In trying to shed more light on the mechanism of action of
oxaliplatin, we present an oxaliplatin derivative hosting a tri-
fluoromethyl group at position 4 of the cyclohexane ring. Time-
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dependent measurements by NMR spectroscopy were per- essary as trans-DACH coordination to platinum results in en-
formed and ligand exchange and adduct formation reactions hanced anticancer activity in comparison with the cis-DACH
with DMSO, NaCl, CaCl2, methionine and 5′-GMP were investi- complex;[8] (2) the synthetic strategy had to include a commer-
gated. Due to the presence of the trifluoromethyl group, time-
dependent measurements should profit from the high sensitiv-
ity of 19F NMR spectroscopy deriving from its high magneto-
gyric ratio and 100 % abundance. Besides, the chemical shift of
the 19F nucleus is highly dependent on the structure of the
molecule. Hence, it was the aim of the present manuscript to
judge if ligand exchange reactions at the platinum center can
easily be followed by 19F NMR spectroscopy, despite the fact
that the CF3 group is rather distant from the reaction center.
cially available source of the trifluoromethyl group; and (3) the
reactions had to be conducted on a low scale owing to the high
costs of fluorinated compounds. For this reason, the platinum
complex was synthesized here as an 1R,2R,4R/1S,2S,4S racemic
mixture. The complex was additionally prepared with 13C2-oxal-
ate in order to get a deeper insight into the ligand exchange
reactions and to judge the suitability of 19F NMR spectroscopy.
The 4-trifluoromethyl–trans-cyclohexane-1,2-diamine ligand
was synthesized starting from 4-trifluoromethyl-cyclohexanol
(1), which was dehydrated to the corresponding cyclohexene
(2) with phosphoric acid (Scheme 1) and converted into the
diol (3) by trans-dihydroxylation with hydrogen peroxide. Upon
formation of the mesylate (4) with mesylchloride, the diazide
(5) was obtained via reaction with sodium azide and reduced
to the amine by Lindlar's catalyst in ethanol, yielding the final
ligand, which was precipitated from solution as the sulfate salt
(6). The ligand was coordinated to platinum while adjusting the
pH to 7 with NaOH in aqueous solution. Subsequent displace-
ment of the chlorido ligands in complex 7 in the presence of
2. Results and Discussion
2.1. Synthesis
Here we present the synthetic strategy that led to a derivative
of oxaliplatin bearing a trifluoromethyl group at position 4 of
the cyclohexane moiety. In addition, the analogous compound
featuring a (13C2)oxalato ligand was also prepared. The syn-
thetic procedure had to satisfy the following three require-
ments: (1) diastereoselectivity, i.e., a synthetic pathway leading, AgNO3 led to the formation of the respective diaqua species
if possible, exclusively to trans-oriented amino groups was nec- in solution. Unlike oxaliplatin, which readily precipitates from
Scheme 1. Reaction sequence and NMR numbering scheme (only one enantiomer is shown). (i) H3PO4; (ii) H2O2/formic acid, 40 °C; (iii) MsCl, py, 0 °C; (iv)
NaN3, DMF, reflux; (v) 1. Lindlar cat. 2. H2SO4; (vi) K2PtCl4, NaOH; (vii) 1. AgNO3 2. IRA402 3. H2C2O4; (viii) Ag213C2O4.
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solution after addition of sodium oxalate, the target complex 2.3. Time-Dependent NMR Spectroscopy Studies
8a possesses a higher water solubility due to the presence of
NMR spectroscopic investigations were undertaken with 8a and
the trifluoromethyl group and the solution needs to be concen-
trated nearly to dryness to obtain the final product. However,
the product thus collected contains nitrate as an impurity. For
this reason, an additional step of ion exchange was conducted.
8b. Featuring the trifluoromethyl group, the oxaliplatin deriva-
tive presented here allows to measure 19F NMR spectroscopy.
1
Besides being very sensitive (relative sensitivity to H of 0.834),
the chemical shift of the 19F nucleus is highly dependent on
The solution containing the diaqua species was treated with a
the structure of the molecule. Thus, ligand exchange reactions
basic preconditioned ion exchange resin, thereby removing
at the platinum center should easily be followed by 19F NMR
nitrate ions and converting the diaqua complex to the respec-
spectroscopy, depending on the position of the 19F substitu-
tive dihydroxido species. Afterwards, the volume of the aque-
ent(s). However, and that was clear from the start of the project,
ous solution was reduced, and oxalic acid was added. Upon
a trifluoromethyl group at position 4 of the cyclohexane ring
evaporation of the solvent, the final product (8a) was obtained
as a mixture of the 1R,2R,4R and 1S,2S,4S enantiomers, in which
the trifluoromethyl substituent is in equatorial position. A very
is rather far away from the reaction center. Consequently, we
synthesized in parallel to complex 8a, the analogue 8b featur-
ing a 13C2-labeled oxalate ligand in order to have an alternative
small quantity (5 %) featuring the axial trifluoromethyl substitu-
for sensitive NMR investigations.
ent was also detected in the 19F NMR spectrum.
The 13C-labeled complex 8b was synthesized from the di-
2.3.1. Behavior of 8a in H2O/DMSO
chlorido complex 7 in a one-pot manner. For this purpose, silver
In a first step, the time-dependent fate of complex 8a (2.5 mM)
(13C2)oxalate was prepared from reaction of silver nitrate and
sodium (13C2)oxalate. Afterwards, the dichlorido complex 7 was
treated with silver (13C2)oxalate to yield the labeled complex
8b.
in a 1:1 mixture of H2O/ [D6]DMSO was investigated by 19F NMR
spectroscopy over a period of 70 hours. This reaction, which
was recently investigated by 13C NMR spectroscopy in the case
of oxaliplatin featuring a (13C2)oxalato ligand,[10] served as a first
measure in order to judge the suitability of 19F-NMR investiga-
tions based on a trifluoromethyl group in position 4 of the cy-
clohexane ligand.
2.2. Characterization by NMR Spectroscopy
By integrating the 19F signal of the intact oxalato complex
Platinum(II) complexes 7, 8a and 8b were characterized in de- (Figure 1 and Figure S1, Supporting Information) and the sum
tail by multinuclear (1H, 13C, 15N, 19F, 195Pt) one- and two-di- of signals of the two DMSO species exhibiting a monodentate
mensional NMR spectroscopy. For the dichlorido precursor oxalato ligand, the rate of the reaction was plotted against time.
complex 7 with PtN2Cl2 coordination, the 195Pt signal was de- In ca. 7 hours, 50 % of the title compound was converted into
tected at –652 ppm, whereas the resonance at –351 ppm con-
firms the PtN2O2 coordination of the final oxalato complex.
Both chemical shifts are in accordance with those reported for
the methyl derivative at –657 and –365 ppm.[9] trans-configura-
tion of the amine substituents both in equatorial position is
verified by the axial positions of both H1 and H2. According to
the Karplus relationship, vicinal H-C-C-H coupling constants are
3
large at angles of 180° with JH,H ≈ 11–12 Hz, and small when
the torsion angle is 60° with a coupling of around 5 Hz. H1ax
couples with both H6ax and H2ax with large constants of
around 11 Hz and with H6eq with a small coupling constant of
4 Hz, which proves that H1ax is axially oriented. The same holds
true for H2ax. In the case of proton H3ax, resonating at
1.33 ppm, a pseudo-quartet with a coupling constant of ap-
proximately 12 Hz was found as a result of two neighboring
axial protons (H2ax, and H4ax) in addition to the large geminal
3JH,H coupling to proton H3eq. Consequently, the axial position
of H4ax finally proves that the trifluoromethyl group at C4 is
equatorially oriented (1R,2R,4R or 1S,2S,4S).
Two sets of signals in 1H NMR at 5.20 ppm and 5.87 ppm
(8a) arise from the pseudo-axial and pseudo-equatorial amine
protons, respectively. For the trifluoromethyl group, a doublet
was observed in the 19F NMR spectrum at –72.83 ppm with a
3JH,F coupling constant of 11.9 Hz. In addition, a very small dou-
blet arising from the side product with an axial trifluoromethyl
group at –66.8 ppm (3JH,F ≈ 8 Hz) with an abundance of 5 %
was observed.
Figure 1. Behavior of 8a (2.5 m
M, circles) in H2O/[D6]DMSO (1:1) and in the
presence of NaCl (100 m , 40 equiv.). The squares represent the mono-
M
dentate DMSO-oxalato complex, the triangles represent the chlorido-DMSO
complex. Once the monodentate DMSO-oxalato complex is formed, the for-
mation of the chlorido complex upon addition of NaCl is very fast with a
half-life of about 1.5 h.
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the corresponding DMSO complex (HRMS (ESI+) calcd. for
C11H13D6F3N2O5PtS [M + H]+ 550.11, found 550.1054). In ca. 1.5
days, the conversion was at 90 %.
2.3.2. Behavior of 8a in H2O/DMSO in the Presence of NaCl
Once the DMSO adduct was formed, the monodentate oxalate
ligand was exchanged for chloride after addition of an excess
of NaCl (100 mM, 40 equiv., Figure 1) and the release of the
oxalato ligand was monitored by 19F NMR spectroscopy (Figure
S2, Supporting Information). This reaction is interesting due to
the formation of an analogue of [Pt(DACH)(DMSO)Cl]Cl, a spe-
cies for which antitumor activity has been reported[11] and to
which the potentiating effect of DMSO[12] on oxaliplatin might
be attributed.
Indeed, two additional peaks appeared for the two species
of the chlorido-DMSO complex immediately after adding the
chloride-containing solution and 50 % of the DMSO-oxalato
complex was converted into the chlorido DMSO-complex in
only 1.5 hours (HRMS (ESI+) calcd. for C9H14D6ClF3N2OPtS [M +
H]+ 497.10, found 497.0860). After 10 h, more than 90 % conver-
sion was reached.
Figure 2. Decrease of the integral of 8b in a solution of H2O/[D6]DMSO (1:1)
as measured by 13C NMR spectroscopy (circles) in comparison to the data of
8a (squares) presented in Figure 1 based on 19F NMR spectroscopy.
2.3.3. Behavior of 8b in H2O/DMSO and in the Presence of
NaCl
Although 19F NMR spectroscopy is highly sensitive to structural
variations, the 19F signal of the starting complex 8a at
–71.59 ppm, the resonances of the two species of the DMSO-
oxalato complex at –71.58 and –71.56 ppm as well as the signal
of the chlorido-DMSO species at –71.60 ppm are not well re-
solved. These four 19F resonances are severely overlapping
within a narrow region of only 0.1 ppm and consequently quan-
tification and interpretation of data could be prone to error.
Therefore, the rate of formation of the DMSO-oxalato complex
and subsequent release of oxalate in the presence of chloride
was investigated in addition with 13C2-labeled compound 8b
via 13C NMR spectroscopy.
Figure 3. Plot of the decrease of the monodentate DMSO-oxalato complex of
8b or 8a upon addition of NaCl (100 mM
, 40 equiv.) measured by 13C (circles)
or 19F (squares) NMR spectroscopy.
In H2O/[D6]DMSO (1:1), the half-life of the conversion of 8b
2.3.4. Time-Dependent Exchange of the Complete Oxalato
Ligand of 8b
(2.5 m
M) to the monodentate DMSO complex is ca. 6 hours, as
determined by integrating the 13C peak of the chelating
(13C2)oxalato ligand and the two doublets for the two none- In an aqueous solution of NaCl, oxaliplatin readily reacts with
quivalent carbon atoms of the monodentately coordinated ox- chloride via ring-opening to a species featuring a monoden-
alate (Figure 2, Figure S3, Supporting Information). This half-life tately coordinated oxalato ligand forming [Pt(dach)oxCl]–,
is in approximate conformance to the one determined for 8a which is efficiently converted back to oxaliplatin.[13] Conse-
via 19F NMR spectroscopy (Figure 2, 7 hours).
quently, the complexes are kinetically inert with respect to for-
mation of the dichlorido species and release of the complete
oxalate ligand is very slow in aqueous solution, with a half-life
of ca. 90 h.[4] With complex 8b in hand, it was now possible to
investigate the complete exchange of a chelating (13C2)oxalato
ligand by NMR spectroscopy with unlabeled oxalate added to
the solution.
Release of the monodentately coordinated (13C2)oxalato li-
gand upon addition of NaCl (100 mM, 40 equiv.) was followed
in comparison to complex 8a by integrating the singlet of free
oxalate as well as the decrease of the sum of integrals of the
two doublets arising from monodentately coordinated oxalate
by 13C NMR spectroscopy (Figure 3).
In ca. 1 h, half of the coordinated oxalate was released to
The rate of complete exchange of the (13C2)oxalato ligand
form the chlorido-DMSO complex which is in good accordance was investigated by adding unlabeled oxalic acid (8 m
M, 2
with the rate determined by 19F NMR spectroscopy with com- equiv.) to a solution of 8b (4 m
M
) in D2O. As expected, due to
pound 8a (1.5 h, compare overlay of data derived from 13C or the fast reverse reaction of ring closure, the complete oxalato
19F NMR spectroscopy in Figure 3). Thus, it can be stated that, ligand is released only at a very slow rate, with an apparent
despite the overlap of 19F NMR resonances, 19F NMR spectro- half-life of around 10 days (250 hours) (Figure 4). At that point, it
scopy can be used as powerful tool in such type of investiga- should be kept in mind that the attack of released (13C2)oxalate
tions.
proceeds in parallel.
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Figure 6. Plot of the rate of formation of 5′-GMP adduct (squares) of 8a
(circles).
Figure 4. Plot of the decrease of 8b (circles) and increase of free (13C2)oxalate
(squares).
2.3.5. Reaction of 8a with Chloride Ions
2.3.7. Reaction of 8a with -Methionine
L
In that line, we also investigated the reaction of 8a (2.5 m
M) in
According to the HSAB (Hard and Soft Acids and Bases) princi-
ple, platinum(II) is a soft metal ion and reacts preferentially with
soft bases. There is sufficient evidence that sulfur-containing
D2O in the presence of chloride (100 m , 40 equiv.) and moni-
M
tored the formation of the dichlorido species by 19F NMR spec-
troscopy. The reaction proceeded with a half-life of ≈ 75 h,
where the peak area of 8a was decreased by half (Figure 5
and Figure S4, Supporting Information). The total integral of the
chlorido species increased within the first 16 h with decreasing
8a, after which it decreased as a consequence of precipitation
of the dichlorido complex. Chlorido-oxalato complex: HRMS
(ESI–) calcd. for C9H14ClF3N2O4Pt [M – H]– 501.02, found
501.0160. Dichlorido complex: HRMS (ESI+) calcd. for
C7H13Cl2F3N2NaPt [M + Na]+ 469.99, found 470.8801.
compounds like the α-amino acid L-methionine react with plati-
num drugs[15] and may play a role in the activation, detoxifica-
tion and side-effects of oxaliplatin. Jerremalm et al. showed that
oxaliplatin reacts about twice as a fast as cisplatin with
methionine,[1] the reason being that the strain of the five-mem-
bered ring is relieved by the nucleophilic attack of sulfur and
the formation of the monoadduct.[16] Hence, we investigated
the reaction of 8a with 2.5 equiv. of L-Met in D2O (Figure 7)
and observed an apparent half-life of 40 min by integrating the
decreasing peak area of 8a in 19F NMR spectra. Expectedly, the
reaction with methionine proceeds much faster than with
5′-GMP. Further, this half-life is in accordance with the half-life
of adduct formation of oxaliplatin with L-methionine, which was
found to be smaller than 2 h.[4] In addition to the 19F signal for
8a, several overlapping peaks were observed, which belong to
the species of the ring-opened methionine adduct [Pt(CF3-
DACH)(oxalate-O)(
L-Met-S)] and to the respective chelate
[Pt(CF3-DACH)(
L-Met-S,N)], representing the final product of the
reaction.[4,17]
Figure 5. Plot of 8a (circles) and the total integral of chlorido species (squares)
vs. time. As 8a is degraded, the integral of the chlorido species increases at
first, but then decreases as the dichlorido complex precipitates from solution.
2.3.6. Reaction of 8a with 5′-GMP
In a next step, we investigated the interaction of the nucleotide
5′-GMP with 8a as a typical model reaction used in literature to
judge the reactivity towards DNA. Integration of the peak areas
in the 19F NMR spectra revealed a half-life of 41 h for 8a (Fig-
ure 6) to form the final product [Pt(CF3-DACH)(5′-GMP-N7)2]2+
.
This is in proximity with the half-life of 65 h reported for the
incubation of oxaliplatin with 5′-GMP[14] and of 58 h for the
labeled derivative of oxaliplatin.[4]
Figure 7. Decrease of the peak area of 8a upon reaction with
L-methionine.
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2.3.8. Reaction of 8a with CaCl2
Table 1. Cytotoxicity of the investigated platinum(II) complex 8a in compari-
son with oxaliplatin, its 1S,2S-enantiomer, and KP1537, (SP-4-3)-[(1R,2R,4R)-(4-
methyl-cyclohexane-1,2-diamine)]oxalatoplatinum(II), an oxaliplatin analogue
featuring an equatorial methyl group at position 4 of the cyclohexane ligand
in human cancer cell lines, tested with the MTT assay (exposure time: 96 h).
Further, in order to compare the rate of formation of chlorido
species in the presence of CaCl2 with that of NaCl, 8a (2.5 m
M)
was treated with CaCl2 (50 m , 20 equiv.) in D2O (Figure 8). In
M
addition to the dichlorido complex, calcium oxalate was
formed, shifting the reaction equilibrium to the side of the
products, thus favoring the formation of chlorido species. As a
result, the decay of 8a due to formation of chlorido species is
much faster in CaCl2 solution (≈ 10 h) compared to a solution
of NaCl (≈ 75 h) which might be the explanation for activation
of oxaliplatin in vivo.
IC50 [μM]
CH1 (PA-1)
Compound
SW480
A549
8a
0.64 0.13
0.18 0.01
1.03 0.04
0.28 0.09
5.5 0.8
0.29 0.05
1.3 0.3
20
2
Oxaliplatin[20]
0.98 0.21
–
–
1S,2S-enantiomer of oxaliplatin[9]
KP1537[9]
0.67 0.31
Figure 9. Concentration–effect curves based on the MTT assay (exposure
time: 96 h) of compound 8a in three human cancer cell lines.
Figure 8. Degradation of 8a upon reaction with CaCl2 is much faster with a
half-life of ca. 10 h compared to a solution of NaCl (ca. 75 h).
Accordingly, substitution at position 4 of the cyclohexane
ring with a trifluoromethyl group seems to have a negative im-
pact with respect to cytotoxic properties of the investigated
complexes.
2.4. Cytotoxicity
Although the antiproliferative potential was not in focus of the
present study, the cytotoxic activity of the trifluoromethyl deriv-
ative 8a was compared with that of oxaliplatin, the 1S,2S-
enantiomer of oxaliplatin and KP1537, (SP-4-3)-[(1R,2R,4R)-(4-
methyl-cyclohexane-1,2-diamine)]oxalatoplatinum(II), featuring
3. Conclusion
an equatorial methyl group at position 4 of the cyclohexane A novel oxaliplatin derivative (SP-4-3)-[(1R,2R,4R/1S,2S,4S)-(4-tri-
ligand. Consequently, compound 8a was tested for its capacity fluoromethyl-cyclohexane-1,2-diamine)]oxalatoplatinum(II)
of inhibiting proliferation of the human cancer cell lines A549 hosting a trifluoromethyl group at position 4 of the cyclohex-
(non-small cell lung cancer), CH1(PA-1) (ovarian teratocarci- ane ring was synthesized and characterized. Investigation of its
noma) and SW480 (colon carcinoma) by the MTT assay (Table 1, cytotoxicity in three human cancer cell lines revealed moderate
Figure 9). As in the case of related compounds, CH1(PA-1) is to high antiproliferative activity, depending on the cell line. The
the most sensitive, the P-glycoprotein-expressing SW480 cells unique potential of the novel compound, and this was the origi-
intermediate and the highly multidrug-resistant A549 cells the nal reason for its synthesis, is the presence of the CF3 group,
least sensitive of these cell lines. Compared to oxaliplatin, com- which is favorable for very sensitive 19F NMR investigations. A
pound 8a is hence 3.6–20 times less potent in these cell lines, further advantage is the lack of fluorine species in biological
with the smallest difference in the most sensitive CH1(PA-1) samples so that there is no background, which could interfere
cells. In part, the higher IC50 values are a consequence of testing with the measurements. Although the CF3 group is rather dis-
an enantiomeric mixture (1R,2R,4R/1S,2S,4S) instead of the pure tant from the reaction center, it could be used to follow time-
(1R,2R,4R)-enantiomer of 8a, which can be inferred from the dependent reactions under various conditions, proving the suit-
comparison of cytotoxicity data of oxaliplatin and its (1S,2S)- ability of the title complex for such type of investigations as
enantiomer (Table 1), with the differences being due to the compared to oxaliplatin (see Scheme 2 for an overview of the
chiral differentiation of DNA adducts and DNA binding pro- performed investigations).
teins.[18]
In addition, we have gained first evidence how oxaliplatin
In comparison, the IC50 values of KP1537, the methyl deriva- could be activated in vivo as the speed of the reaction of the
tive of oxaliplatin and close analogue of 8a, were found in the title complex with CaCl2 was significantly different in compari-
range of oxaliplatin; KP1537 was shown to be highly active in son to NaCl suggesting an important role of calcium ions in the
vivo.[19]
rapid biotransformation of oxaliplatin.
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Scheme 2. Overview and half-lives of time-dependent reactions of title complexes 8a and 8b studied by 19F and 13C NMR spectroscopy (only one enantiomer
of the diamine ligand is shown, only one possible isomeric product is shown, * = 13C labeled variant).
trospray ionization quadrupole–quadrupole time-of-flight) spec-
trometer in the positive mode with ACN/MeOH 1 % H2O as the
solvent (ACN = acetonitrile). Elemental analyses were performed at
4. Experimental Section
Materials and Methods: All chemicals were obtained from com-
mercial suppliers and used as received. 4-(Trifluoromethyl)cyclo-
the microanalytical laboratory of the faculty of chemistry of the
hexanol (> 98 %) was purchased from TCI, mesyl chloride (99 %)
University of Vienna with a Eurovector EA3000 elemental analyzer.
from Fluka, sodium azide (99 %) from Fischer, Lindlar's catalyst (5 %
Pd) from Fluka, silver nitrate (99.85 %) from Acros, oxalic acid (99 %)
4-(Trifluoromethyl)-1-cyclohexene (2):[21] 4-(Trifluoromethyl)cyc-
from Fluka, sodium (13C2)oxalate (99 % 13C) was purchased from
Sigma–Aldrich, and K2[PtCl4] was purchased from Johnson Matthey
(Switzerland). Water was purified by reversed osmosis and double
distillation before use. Reactions involving platinum compounds
were performed under light protection and with glass-coated mag-
netic stirring bars. NMR spectroscopy measurements were per-
formed with a Bruker AVANCE NEO 500 MHz or a Bruker AVANCE III
HD 700 MHz spectrometer at 500.32 (1H), 125.81 (13C), 50.70 (15N),
470.56 or 659.03 (19F), and 107.55 MHz (195Pt) at 25 °C. The solvent
resonances were used as internal references for 1H {[D6]-
dimethylsulfoxide ([D6]DMSO), δ = 2.51 ppm; [D7]dimethylforma-
mide ([D7]DMF), δ = 2.93 ppm (lowfield methyl signal); D2O, δ =
4.70 ppm; [D4]methanol, δ = 3.33 ppm} and 13C {[D6]DMSO,
40.0 ppm; [D7]DMF, 34.6 (lowfield methyl signal); [D4]methanol,
47.6 ppm}. The 195Pt and 15N NMR resonances were referenced rela-
tive to external K2[PtCl4] or NH4Cl, respectively. 19F chemical shifts
are given relative to CCl3F. High-resolution electrospray ionization
mass spectra were measured with a Bruker maXis ESI-QqTOF (elec-
lohexanol (5 g, 29.73 mmol) was mixed with 3 mL of phosphoric
acid and heated to 160 °C using a short path distillation kit. The
product was distilled into collecting flasks. The combined phases
were dried with MgSO4. Yield: 3.737 g (84 %), colorless liquid.
4-(Trifluoromethyl)-trans-1,2-cyclohexanediol (3): To a mixture
of 13 mL of formic acid (98 %) and 5 mL of hydrogen peroxide
(30 %) was added 4-(trifluoromethyl)-1-cyclohexene (3.737 g,
24.89 mmol) dropwise at 40 °C. The mixture was stirred at 40 °C for
1 h, then at room temperature for 3 h. The solvents were distilled
off under reduced pressure and NaOH (8
M, 10 mL) was added
dropwise at 0 °C. The reaction mixture was stirred overnight at r.t.
and the crude product was extracted with ethyl acetate (3x10 mL).
The combined organic phases were washed with brine, dried with
MgSO4 and the solvent was evaporated in vacuo. Yield: 3.68 g
(80 %), white solid.
4-(Trifluoromethyl)-trans-1,2-cyclohexanediol-O,O′-dimeth-
anesulfonate (4): To a solution of 4-(trifluoromethyl)-trans-1,2-cy-
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clohexanediol (3.68 g, 19.98 mmol) in pyridine was added mesyl
1.227 mmol, 1 equiv.) was suspended in 15 mL of water, and AgNO3
chloride (4.5 mL, 58.10 mmol) dropwise at 0 °C. The reaction mix- (396.10 mg, 2.33 mmol, 1.9 equiv.) was added. The mixture was
ture was stirred for 2 h before 10 mL of water were added cau-
tiously. The product was extracted with CH2Cl2 (3x10 mL) and the
combined organic phases were washed with water twice, dried with
MgSO4 and the solvent was distilled off in vacuo. Excess pyridine
was removed by azeotropic distillation with toluene. Yield: 4.57 g
(67 %), white solid. 1H NMR (CDCl3): δ = 1.70–1.82 (m, 1H, H5), 1.84–
1.92 (m, 1H, H3), 1.99–2.10 (m, 2H, H5/H6), 2.10–2.24 (m, 2H, H3/
H6), 2.43–2.56 (m, 1H, H4), 3.13 (s, 6H, 2CH3), 4.87–4.92 (m, 1H, H1),
4.97–5.03 (m, 1H, H2) ppm.
stirred for 24 h and shaken from time to time in order to guarantee
wetting with the solvent. Silver chloride was filtered off using a MN-
GF-3 filter paper in a filter crucible. The complex thus activated
was treated with an ion exchange resin, before forming the oxalate
complex [thereby removing nitrate ions from solution and generat-
ing the dihydroxidoplatinum(II) species]. For this purpose, the
IRA402 resin in the chloride form was preconditioned by stirring
with 2
M NaOH (200 mL) for 30 min. Then, it was washed with water
until achieving neutral pH, followed by washing two more times.
Then, the filtrate from the first step of the reaction containing the
activated complex was treated with this resin by shaking at room
temperature for 24 h. Next, the solution was decanted off and the
resin was washed with water three times. The combined aqueous
solutions were filtered through a G4 sintered glass crucible and the
volume was reduced to ca. 100 mL. After adding oxalic acid
(99.42 mg, 1.10 mmol, 0.9 equiv.), the mixture was stirred for 24 h.
Then the volume was further reduced to ca. 50 mL, whereupon the
product began to precipitate, which was collected by filtration. The
volume of the filtrate was further reduced, and the precipitate col-
lected. Both fractions were dried in vacuo. Yield: 230 mg (45 %),
white solid. 1H NMR (D2O): δ = 1.17–1.27 (m, 1H, H5ax), 1.27–1.38
(m, 2H, H6ax, H3ax), 1.70–1.78 (m, 1H, H5eq), 1.99–2.07 (m, 1H,
H6eq), 2.11–2.25 (m, 2H, H3eq, H4ax), 2.29–2.47 (m, 2H, H1, H2),
5.02–5.37 (m, 2H, C1NH, C2NH), 5.74–6.00 (m, 2H, C1NH, C2NH)
4-(Trifluoromethyl)-trans-1,2-cyclohexanediazide (5): 4-(Tri-
fluoromethyl)-trans-1,2-cyclohexanediol-O,O′-di(methanesulfonate)
(4.57 g, 13.42 mmol, 1 equiv.) and sodium azide (3.60 g, 55.24 mmol,
4.1 equiv.) were dissolved in DMF (dry) in a 250 mL round-bottom
flask flushed with argon and stirred under reflux at 130 °C for 2
days. Thereafter, the mixture was cooled down to room tempera-
ture and diluted with 70 mL of water. The product was extracted
with hexane (4x25 mL), the combined organic extracts were washed
with water and dried with MgSO4. The solvent was removed under
reduced pressure. Yield: 1.55 g (49 %), reddish brown liquid. The
product was directly used without further characterization.
4-(Trifluoromethyl)-trans-1,2-cyclohexanediaminium sulfate (6):
4-(Trifluoromethyl)-trans-1,2-cyclohexanediazide (1.55 g,
6.62 mmol) was dissolved in ethanol and transferred to a dry auto-
clave tube equipped with a magnetic stirring bar. The solution was
flushed with argon for 5 min. via a needle. Then, Lindlar's catalyst
(190 mg) was added. The tube was placed into the autoclave, which
was then filled with hydrogen gas at 10 bar. The mixture was stirred
overnight at room temperature, after which the catalyst was filtered
off and the solvent removed under reduced pressure. The product
was precipitated by addition of half-concentrated H2SO4 and the
suspension was filtered. The product was washed with a small
amount of ethanol and dried in vacuo. Yield: 1.071 g (58 %), off-
3
ppm. 13C{1H} NMR (D2O): δ = 22.8 (q, JF,C = 2 Hz, C5), 28.7 (C6),
3
2
29.7 (q, JF,C = 2 Hz, C3), 39.7 (q, JF,C = 26 Hz, C4), 60.6 (C2), 61.3
(C1), 126.6 (q, JF,C = 280 Hz, CF3), 168.3 (2 C=O) ppm. 15N NMR
1
(D2O): δ = –36.0 (C1N), –35.1 (C2N) ppm. 19F NMR (D2O): δ = –72.8
3
3
(d, JH,F = 8 Hz), –66.8 (d, JH,F = 12 Hz, signal of around 5 % of
the isomer featuring an axial trifluoromethyl substituent) ppm. 195Pt
NMR (D2O): δ = –351 ppm. HRMS: (pos) m/z 488.04 [M + Na+]+,
953.08 [2M + Na+]+; (neg) 463.85 [M – H+]–, 928.89 [2M – H+]–.
C9H13F3N2O4Pt; calcd. C 23.23, H 2.82, N 6.02; found C 23.26, H 2.80,
N 5.75.
1
white solid. H NMR (D2O): δ = 1.40–1.52 (m, 1H, H5), 1.55–1.68 (m,
2H, H3/H6), 1.99–2.09 (m, 1H, H5), 2.21–2.29 (m, 1H, H3), 2.29–2.39
(m, 1H, H6), 2.40–2.53 (m, 1H, H4), 3.36–3.46 (m, 1H, H1), 3.46–3.56
(m, 1H, H2).
Silver (13C2)oxalate: Sodium (13C2)oxalate (133.0 mg, 0.97 mmol, 1
equiv.) and silver nitrate (331.5 mg, 1.95 mmol, 2 equiv.) were dis-
solved in 3 mL of H2O and stirred at r.t. for 15 min. The precipitated
product was collected by filtration and dried in vacuo. Yield:
208.5 mg (70 %), white solid.
(SP-4-3)-Dichlorido[(1R,2R,4R/1S,2S,4S)-(4-trifluoromethyl-cy-
clohexane-1,2-diamine)] platinum(II) (7): 4-(Trifluoromethyl)-
trans-1,2-cyclohexanediaminium sulfate (1070.0 mg, 3.82 mmol)
and potassium tetrachloroplatinate (1584.76 mg, 3.82 mmol) were
(SP-4-3)-(13C2)Oxalato[(1R,2R,4R/1S,2S,4S)-(4-trifluoromethyl-cy-
clohexane-1,2-diamine)]platinum(II) (8b): Complex 7 (320.0 mg,
0.71 mmol, 1 equiv.) and silver (13C2)oxalate (196.0 mg, 0.64 mmol,
0.9 equiv.) were suspended in 5 mL of H2O and the mixture was
stirred overnight. AgCl was filtered off using a MN-GF-3 filter paper
in a filter crucible. The filtrate was concentrated, cooled and the
precipitating product was filtered off. 1H NMR ([D6]DMSO): δ = 1.11–
1.39 (m, 3H, H3, H5, H6), 1.67–1.75 (m, 1H, H5), 1.85–1.94 (m, 1H,
H6), 1.94–2.02 (m, 1H, H3), 2.07–2.18 (m, 1H, CHNH2), 2.18–2.30 (m,
1H, CHNH2), 2.37–2.47 (m, 1H, H4), 5.40–5.62 (m, 2H, NHeq), 6.11–
6.30 (m, 2H, NHax) ppm. 13C{1H} NMR (D2O): δ = 22.9 (q, 3JF,C = 3 Hz,
suspended in 30 mL of water. A solution of 0.5
M NaOH was slowly
added until a pH of 7 was reached and the product began to form
as a yellow precipitate. The pH was adjusted at regular intervals
using 0.1
M NaOH solution until after a few hours it did not change
significantly. Finally, the product was filtered off and dried in vacuo.
Yield: 1.133 g (66 %), yellow solid. 1H NMR ([D7]DMF): δ = 1.25–1.40
(m, 1H, H5ax), 1.61–1.75 (m, 2H, H3ax, H6ax), 1.80–1.89 (m, 1H,
H5eq), 2.14–2.24 (m, 1H, H6eq), 2.27–2.35 (m, 1H, H3eq), 2.40–2.54
(m, 1H, H4ax), 2.74–2.84 (m, 1H, H1), 2.90–3.00 (m, 1H, H2), 4.96–
5.50 (m, 2H, C1NH, C2NH), 5.50–5.97 (m, 2H, C1NH, C2NH) ppm.
13C{1H} NMR ([D7]DMF): δ = 23.5 (C5), 29.2 (C6), 30.3 (C3), 40.1 (q,
3
2
C5), 28.8 (C6), 29.8 (C3, JF,C = 2 Hz), 39.8 (q, JF,C = 27 Hz, C4), 60.7
(C2), 61.4 (C1), 126.6 (q, 1JF,C = 278 Hz, CF3), 168.3 (2 C=O) ppm. 15
NMR ([D6]DMSO): δ = –33.4, –33.1 ppm. 19F NMR ([D6]DMSO): δ =
1
2JF,C = 27 Hz, C4), 61.8 (C2), 62.7 (C1), 127.5 (q, JF,C = 279 Hz, CF3)
N
ppm. 15N NMR ([D7]DMF): δ = –21.9 (C1N), –21.4 (C2N) ppm. 19F
3
3
3
3
–71.6 (d, JH,F = 8 Hz), –65.6 (d, JH,F = 12 Hz, signal of around 5 %
of the isomer featuring an axial trifluoromethyl substituent) ppm.
195Pt NMR ([D6]DMSO): δ = –353 ppm. Yield: 79.9 mg (17 %). HRMS:
(pos) m/z 490.04 [M + Na+]+, 957.09 [2M + Na+]+; (neg) 466.05 [M
– H+]–, 933.10 [2M – H+]–.
NMR ([D7]DMF): δ = –74.0 (d, JH,F = 8 Hz), –68.0 (d, JH,F = 12 Hz,
signal of around 5 % of the isomer featuring an axial trifluoromethyl
substituent) ppm. 195Pt NMR ([D7]DMF): δ = –652 ppm. HRMS (ESI+):
m/z 470.97 [M + Na+]+; (neg) 446.80 [M – H+]–. C7H13Cl2F3N2Pt:
calcd. C 18.76, H 2.92, N 6.25; found C 19.12, H 2.86, N 6.18.
(SP-4-3)-Oxalato[(1R,2R,4R/1S,2S,4S)-(4-trifluoromethyl-cyclo-
hexane-1,2-diamine)]platinum(II) (8a): Complex 7 (550 mg,
Time-Dependent NMR Spectroscopy Measurements: Time-de-
pendent measurements by NMR spectroscopy were conducted with
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complex 8a (2.5 m
[D6]DMSO (1:1) or in D2O; with CaCl2 (50 m
M) with NaCl (100 mM, 40 equiv.) in H2O/
Keywords: Platinum · Antitumor agents · Cytotoxicity ·
Synthesis design
M, 20 equiv.); 5′-GMP
(12.5 m
M
, 5 equiv.) and
L-methionine (7.5 m
M, 3 equiv.) in D2O and
integration of 19F signals. Time-dependent investigations with the
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Formation of the unlabeled oxalate complex (8a) starting from the
13C-labeled analogue (8b, 4 m ) in D2O was followed by 13C NMR
spectroscopy upon addition of unlabeled oxalic acid (8 m
equiv.).
M, 40 equiv.) in H2O/[D6]DMSO (1:1).
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M, 3
Cytotoxicity Tests in Human Cancer Cell Lines
CH1(PA-1) (provided by L. R. Kelland, CRC Centre for Cancer Thera-
peutics, Institute of Cancer Research, Sutton, UK; confirmed by STR
profiling as PA-1 ovarian teratocarcinoma cells at Multiplexion, Hei-
delberg, Germany), as well as SW480 colon carcinoma and A549
non-small cell lung cancer cells (both provided by B. Marian, Insti-
tute of Cancer Research, Department of Medicine I, Medical Univer-
sity of Vienna, Austria) were grown as monolayer cultures in 75 cm2
culture flasks (Starlab) in complete medium, i.e., minimal essential
medium (MEM) supplemented with 1 m
M sodium pyruvate, 4 mM
L-glutamine, 1 % (v/v) nonessential amino acids from 100-fold stock
solution (all purchased from Sigma-Aldrich) and 10 % heat-inacti-
vated fetal bovine serum (BioWest). Cultures were maintained at
37 °C in a humidified atmosphere containing 5 % CO2 in air.
Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay in at least three inde-
pendent experiments as follows: Cells were harvested from culture
flasks by using trypsin-EDTA (Sigma-Aldrich), and the following cell
numbers were seeded into 96-well microculture plates (Starlab):
1 × 103 [CH1(PA-1)], 2 × 103 (SW480), 3 × 103 (A549) cells per well
(each in 100 μL). Plates were incubated for 24 h, then the test com-
pound was dissolved and serially diluted in complete MEM and
each dilution added to plates in triplicate (100 μL per well). After
exposure for 96 h, the medium was replaced with 100 μL of drug-
free RPMI 1640 medium and 20 μL of MTT solution (5 mg/mL) in
phosphate-buffered saline. After incubation for 4 h, these mixtures
were removed, and the formazan products were dissolved in 150 μL
of DMSO per well. Optical densities at 550 nm (and at 690 nm as a
reference) were measured with a microplate reader (Biotek ELx808),
and IC50 values were calculated from concentration–effect curves
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Received: November 8, 2018
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Oxaliplatin Analogues
A racemic mixture of an analogue of
the clinically established anticancer
drug oxaliplatin featuring a CF3 group
at position 4 of the cyclohexane ring
was synthesized and fully character-
ized. Its binding to DMSO, chloride,
S. Hizal, M. Hejl, C. Jungmann,
M. A. Jakupec, M. Galanski,*
B. K. Keppler* .................................... 1–10
Synthesis, Characterization, Cyto-
toxicity, and Time-Dependent NMR
Spectroscopic Studies of (SP-4-3)-
Oxalato[(1R,2R,4R/1S,2S,4S)-(4-tri-
fluoromethyl-cyclohexane-1,2-di-
amine)]platinum(II)
5′-GMP, and -methionine was investi-
L
gated by 19F and 13C NMR spectro-
scopy.
DOI: 10.1002/ejic.201801370
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