Water-Soluble Platinum(II) Complexes Featuring 2-Alkyl-2H-tetrazol-5-ylacetic Acids: Synthesis, Characterization, and Antiproliferative Activity

Article abstract of DOI:10.1002/ejic.201600626

2-R-2H-Tetrazol-5-ylacetic acids (abbreviated as 2-R-taa; R = Me, iPr, tBu) react with K₂[PtCl₄] in 1 m HCl in H₂O at r.t. furnishing trans-platinum(II) complexes trans-[PtCl₂(2-R-taa)₂] (1–3), whereas cis-isomeric species cis-[PtCl₂(2-R-taa)₂] (R = iPr, 4; tBu, 5) are isolated at lower temperature (4–6 °C). In the presence of EtOH in the reaction mixture, esterification of the tetrazol-5-ylacetoxy group of 2-tBu-taa leads to trans-[PtCl₂(ethyl 2-tert-butyl-2H-tetrazol-5-ylacetate)₂] (6). Complexes 1–6 were characterized by elemental analyses (CHN), HRESI⁺-MS,¹H,¹³C{¹H},¹⁹⁵Pt{¹H} NMR and IR spectroscopy, differential scanning calorimetry/thermogravimetry (DSC/TG), and X-ray diffraction (for 1·H₂O, 2, 3·2H₂O, 4, 5·2H₂O, and 6). The generation of the tetrazole-based complexes in solution (1 m DCl in D₂O, 25 °C) was studied by¹H NMR spectroscopy and HPLC-MS. The obtained data indicate the initial formation of anionic [PtCl₃(2-R-taa)]^–complexes that are subsequently converted into disubstituted isomeric platinum(II) species cis- and trans-[PtCl₂(2-R-taa)₂]. By contrast to cis- and trans-[PtCl₂(2-R-taa)₂] that were inactive in two human cancer models in vitro (IC₅₀> 100 μm), complex 6 demonstrated noticeable antiproliferative effects in HT-29 colon and MCF-7 breast carcinoma cell lines with IC₅₀values of 14.2 ± 1.1 and 5.8 ± 1.2 μm, respectively.

Full text of DOI:10.1002/ejic.201600626

DOI: 10.1002/ejic.201600626
Full Paper
Anticancer Complexes
Water-Soluble Platinum(II) Complexes Featuring 2-Alkyl-2H-
tetrazol-5-ylacetic Acids: Synthesis, Characterization, and
Antiproliferative Activity
[a]
[b]
[a]
[c]
Elena A. Popova* Tatiyana V. Serebryanskaya,* Stanislav I. Selivanov, Matti Haukka,
[
d]
[d]
[e]
[a]
Taras L. Panikorovsky, Vladislav V. Gurzhiy, Ingo Ott, Rostislav E. Trifonov, and
Vadim Yu. Kukushkin[
a]
Abstract: 2-R-2H-Tetrazol-5-ylacetic acids (abbreviated as 2-R- tion of the tetrazole-based complexes in solution (1
M
DCl in
1
taa; R = Me, iPr, tBu) react with K [PtCl ] in 1
M
HCl in H O at D O, 25 °C) was studied by H NMR spectroscopy and HPLC-
2
4
2
2
r.t. furnishing trans-platinum(II) complexes trans-[PtCl (2-R-taa) ] MS. The obtained data indicate the initial formation of anionic
2
2
–
(
1–3), whereas cis-isomeric species cis-[PtCl (2-R-taa) ] (R = iPr, [PtCl (2-R-taa)] complexes that are subsequently converted
2 2 3
4
; tBu, 5) are isolated at lower temperature (4–6 °C). In the into disubstituted isomeric platinum(II) species cis- and trans-
presence of EtOH in the reaction mixture, esterification of the [PtCl (2-R-taa) ]. By contrast to cis- and trans-[PtCl (2-R-taa) ]
2
2
2
2
tetrazol-5-ylacetoxy group of 2-tBu-taa leads to trans- that were inactive in two human cancer models in vitro
[
1
PtCl (ethyl 2-tert-butyl-2H-tetrazol-5-ylacetate) ] (6). Complexes (IC50 > 100 μ
M
), complex 6 demonstrated noticeable anti-
2
2
+
–6 were characterized by elemental analyses (CHN), HRESI - proliferative effects in HT-29 colon and MCF-7 breast carcinoma
1
13
1
195
1
MS, H, C{ H}, Pt{ H} NMR and IR spectroscopy, differential cell lines with IC50 values of 14.2 ± 1.1 and 5.8 ± 1.2 μ
scanning calorimetry/thermogravimetry (DSC/TG), and X-ray tively.
M, respec-
diffraction (for 1·H O, 2, 3·2H O, 4, 5·2H O, and 6). The genera-
2
2
2
[
2,3]
Introduction
complicated task.
Apart from the classic case of cis-
[
4a]
[
PtCl (NH ) ],
this concerns platinum–chlorido complexes
bearing bulky amines (such as 1-adamantylamine)
gen-containing heterocycles of low basicity (e.g. pyraz-
2 3 2
Cis–trans isomerism plays a crucial role in the chemistry of
square-planar platinum(II) compounds, strongly affecting their
reactivity and physical properties. Due to a drastic difference
in catalytic activity and therapeutic potential of cis- and trans-
isomeric species, synthesis and characterization of individual
isomers are of great importance whenever investigation of new
compounds is pursued. Although general methods for pre-
paring isomeric platinum(II) compounds are well established, in
some instances, synthesis of the pure cis-isomeric species is a
[2m]
and nitro-
[3a–3e]
[2h,2g]
[2l]
oles,
nitroimidazoles,
acylpyridines, etc.). In some
cases, selectivity can be improved by application of the so-
called Dhara's method.
this method in the case of polydentate nitrogen ligands is not
always feasible due to the formation of silver-containing com-
[4b]
Unfortunately, implementation of
[
1]
[4c–4e]
plexes as by-products.
Being interested in the coordina-
tion chemistry of polynitrogen azoles and their implementation
in metal-based drug design, we have recently studied cis- and
trans-isomeric platinum(II) complexes generated by the reaction
[
[
a] Institute of Chemistry, Saint Petersburg State University,
/9 Universitetskaya nab., Saint Petersburg, 199034, Russia
E-mail: elena.popova@spbu.ru
http://chem.spbu.ru/
b] Research Institute for Physical Chemical Problems of Belarusian State
University,
of K [PtCl ] with N-substituted 5-aminotetrazoles. In this case
2 4
7
we could control the geometry of the derived platinum(II)–
chlorido species by varying the reaction conditions such as, for
[
5]
example, temperature and acidity.
Tetrazoles are polynitrogen five-membered heterocycles that
Leningradskaya 14, 220030, Minsk, Belarus
E-mail: serebryanskaya.t@gmail.com
http://fhp.bsu.by/
can exist in the form of the 1-R-1H- and 2-R-2H-monosubsti-
tuted isomers (A and B in Figure 1, R = H) and also as an NH-
2
unsubstituted form (C). Disubstituted tetrazoles can exist in 1,5-
[
[
[
c] Department of Chemistry, University of Jyväskylä,
Jyväskylä, Finland
d] Institute of Earth Sciences, Saint Petersburg State University,
University Emb. 7/9, 199034, Saint Petersburg, Russia
e] Institute of Medicinal and Pharmaceutical Chemistry,
Technische Universität Braunschweig,
2
and 2,5-forms (A and B, R ≠ H). All these forms are significantly
[
6]
different in their physical, chemical, and biological properties.
The neutral form of tetrazole species A–C contains three en-
docyclic pyridine-type nitrogen atoms and one pyrrole-type
nitrogen atom. The former N atoms can be bound by various
metal centers, in particular by platinum-group metals, giving
Beethovenstraße 55, 38106, Braunschweig, Germany
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under http://dx.doi.org/10.1002/ejic.201600626.
[
6]
the corresponding complexes.
Eur. J. Inorg. Chem. 0000, 0–0
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Full Paper
acetate was obtained from ethyl cyanoacetic acid, whereupon
it was alkylated with methyl iodide in MeCN; pure ethyl esters
of 1-Me-taa and 2-Me-taa were isolated by flash chromatogra-
phy. Ethyl esters of 2-iPr-taa and 2-tBu-taa were obtained
through selective alkylation of the NH-tetrazole with 2-propanol
and tert-butyl alcohol, correspondingly, in a mixture of glacial
acetic and sulfuric acid. Finally, 2-R-taa (R = Me, iPr, tBu) were
prepared by alkaline hydrolysis of the corresponding esters.
Figure 1. Mono- and disubstituted tetrazole derivatives.
Coordination compounds containing neutral N-substituted
tetrazoles as carrier ligands should be distinguished from metal
tetrazolates featuring the anionic form of the tetrazole cycle
(
deprotonated form of species C). Platinum–tetrazolato com-
plexes are easily accessible by direct deprotonation of NH-
tetrazoles. They can also be generated through [3+2] cycloaddi-
[7]
tion of platinum-coordinated azides to organic nitriles as well
[
8]
as by azidation of platinum-bound nitriles. Recently, a series
of cationic binuclear platinum tetrazolates was shown to be
potent antitumor agents able to overcome cross-resistance to
[
9]
cisplatin.
Scheme 1. Synthesis of 2-alkyl-2H-tetrazol-5-ylacetic acids.
By contrast to the better studied tetrazolate species, only few
platinum(II) complexes with N-substituted tetrazoles have been
[
5,10]
reported to date,
and almost no structural information on
these compounds is available. In view of the recent attention Synthesis and Characterization of Platinum(II) Complexes
to antitumor activity of tetrazole-based cisplatin analogs,[5]
a
It has been shown that configuration of the complexes ob-
tained upon reaction of K [PtCl ] with 2 equiv. of a nitrogen-
containing heterocycle depends on the nature of the ligand
series of cis- and trans-isomeric platinum(II) complexes featuring
N-substituted tetrazoles have been obtained, and a few trans-
2
4
[
5a]
isomeric species have been structurally characterized. Proper
structural characterization of cis-isomeric forms is still lacking
due to their facile cis-to-trans isomerization in solution.
[
2,3,5]
and the reaction conditions.
five-membered heterocycles (such as most of pyridine,
With the majority of six- and
[
2a–2c]
[
2d–2g]
[2h,2i]
[2j]
[2a]
imidazole,
pyrimidine,
pyrazine,
thiazole,
and
The present study is focused on the preparation of cis- and
trans-isomeric platinum(II) complexes with a series of 2-alkyl-
[
2k]
benzimidazole derivatives, etc.) in good agreement with the
higher trans effect of the chlorido ligand, this reaction results
in the formation of cis-isomeric products.
[
14]
2H-tetrazol-5-ylacetic acids (abbreviated as 2-R-taa; R = Me, iPr,
tBu) and comparative structural characterization. Tetrazolyl-
An exception to this general trend is the reaction with pyraz-
ole derivatives. Preparation of pure cis-isomeric complexes cis-
acetic acids may be regarded as amino acid analogs featuring
[
11]
a tetrazole ring in the α position instead of the amino group.
[
PtCl L ] [L = pyrazole, 3,5-dimethylpyrazole, 3,5-diisopropyl-
2 2
Bearing both carboxyl and tetrazolyl fragments, tetrazolylacetic
acids are useful ligands for the molecular design of metal com-
pyrazole, 3,5-pyrazoledicarbonic acid, 1-(2-hydroxyethyl)-3,5-
dimethylpyrazole] was only possible if the reaction was carried
[
12]
plexes.
Particularly, these species can combine increased
[
3a–3e]
out in aqueous HCl solution at pH 1–2.
HCl, the corresponding trans-isomeric products were obtained
for substituted pyrazoles,
In the absence of
lipophilicity with high water solubility, which is extremely im-
portant for the development of new platinum-based antitumor
agents. However, platinum(II) complexes with such ligands are
[
3b–3g]
whereas the reaction with un-
substituted pyrazole led to the analog of Magnus' green salt,
[
12h]
only scarcely known.
[
3a]
that is, [Pt(pyrazole) ][PtCl ].
4
4
The main goals of this study were at least threefold: (i) to
generate cis- and trans-isomeric platinum(II) complexes featur-
ing 2-R-taa and to accomplish their spectroscopic and structural
characterization; (ii) to study ligation of 2-R-taa to platinum(II)
centers in solution in order to reveal the main factors affecting
generation of the cis and trans forms; and (iii) to assess the
antitumor potential of the obtained complexes by studying
their antiproliferative activity in vitro.
Formation of trans-isomeric complexes trans-[PtCl L ] as
2
2
main products of the reaction with K [PtCl ] in aqueous media
2
4
was also reported for several other nitrogen-containing hetero-
[
2h,2g]
cycles such as 2-nitroimidazoles,
3- and 4-acetylpyrid-
[
2l]
[5]
ines, and N-substituted 5-aminotetrazoles (L). In the latter
case, the presence of HCl in the reaction mixture favors forma-
tion of the cis products, presumably due to their decreased
[
5b]
solubility and suppressed isomerization in solution.
In order to study the effect of reaction conditions on the
geometry of the generated complexes, reactions of K [PtCl ]
with the 2-alkyl-2H-tetrazol-5-ylacetic acids were carried out at
different temperatures in the absence and in the presence of
HCl in the reaction mixture, as well as in the presence of EtOH
2
4
Results and Discussion
Synthesis of 2-Alkyl-2H-tetrazol-5-ylacetic Acids
The synthesis of 2-R-taa (Scheme 1) was carried out by using that affected the solubility of both reactants and the formed
[
13]
previously reported methods.
Ethyl 2-(1H-tetrazol-5-yl)- complexes.
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In the absence of HCl, K [PtCl ] reacts with any one of 2-R- formation), and IR spectroscopic analyses, complexes 1 and 3
2
4
taa compounds to give an inseparable mixture of amorphous were isolated as the mono- and dihydrate, respectively, namely
products irrespective of the temperature or the presence of 1·H O and 3·2H O, and the presence of co-crystallized water
2
2
EtOH in the system. On the contrary, in the presence of HCl, molecules was further confirmed by single-crystal X-ray diffrac-
the structure of the products strongly depends on the reaction tion (see later).
conditions (Scheme 2). Particularly in 1
M HCl and at r.t., the
In 1–6, the presence of the tetrazole fragments was
reaction of K [PtCl ] with 2 equiv. of 2-R-taa resulted in the
2
4
confirmed by the characteristic IR bands in the range 900–
generation of the trans species, namely trans-[PtCl (2-R-taa) ]
–1
[15a]
2
2
1600 cm (see Supporting Information).
Coordination of 2-
(
[
R = Me, 1; iPr, 2; tBu, 3; Scheme 2). The cis congeners cis-
PtCl (2-R-taa) ] (R = iPr, 4; tBu, 5) were obtained from the reac-
R-taa resulted in the respective changes of the tetrazole cycle
and the vibration bands of the substituents. In particular, in
good agreement with the electron-withdrawing effect of coor-
dination, the endocyclic ν(C=N) band of 2-R-taa shifted to
2
2
tion of 2-iPr-taa and 2-tBu-taa, correspondingly, by using
K [PtCl ] in 1 HCl at 4–6 °C, upon slow evaporation of the
M
2
4
solvent (Scheme 2). In the case of 2-Me-taa, even at lower tem-
perature only the trans product was isolated.
–1
–1
higher frequency by 5–10 cm (from 1516–1517 cm in the
–1
spectrum of free 2-R-taa to 1521–1526 cm in the spectra of
1
–5). Besides, a slight decrease in frequency of the ν(C=O) band
–
1
was observed upon the coordination (by 3–12 cm , corre-
spondingly). The difference between the position of the bands
in the spectra of trans and cis isomers was only marginal,
–1
namely 2–4 cm . In the far-IR region, cis isomers displayed two
bands associated with ν(Pt–Cl) stretching vibrations (at 341 and
52 cm– for 4 and at 330 and 345 cm for 5), whereas the
1
–1
3
spectra of trans-isomeric complexes, as expected, exhibited
–1
only one ν(Pt–Cl) band in the range 340–355 cm . This is in
agreement with the theoretically expected number of ν(Pt–Cl)
bands for the idealized C2v (cis geometry) and D (trans geome-
2h
[
15b]
try) point symmetry groups.
The coordination led to substantial changes of the chemical
shifts in the H and C{ H} NMR spectra. Generally, H and
1
13
1
1
1
3
1
C{ H} NMR signals in the spectra of the complexes were no-
ticeably deshielded with respect to those in the spectra of un-
complexed 2-R-taa, the effect being more pronounced for the
1
spectra of the trans isomers. In the H NMR spectra, the most
significant changes were observed for the signals of the CH2
groups: the coordination resulted in large downfield shifts, by
up to 0.59 ppm (for trans complexes 1–3) and 0.54 ppm (for cis
isomers 4 and 5). The shifts of the signals of the alkyl substitu-
ents were smaller. Consequently, the peaks of the CH groups
3
in the spectra of trans-isomeric complexes 1–3 were shifted
downfield by 0.07 ppm as compared with those of metal-free
Scheme 2. Synthesis of platinum(II) complexes featuring 2-alkyl-2H-tetrazol-
2
-R-taa. On the contrary, formation of cis products 4 and 5 was
5
-ylacetic acids 1–6.
accompanied by a slight upfield shift of the same resonances
1
3
1
by 0.02 ppm. The most significant changes in the C{ H} NMR
spectra upon formation of the complexes were observed for
the signals of the endocyclic C(5) atoms that were shifted
downfield from approximately 161.3 to 162.5 ppm. The
In the presence of HCl, the rate of the reaction drastically
decreased (see below). EtOH was added to the reaction mixture
to increase the solubility of the tetrazoles and to enhance the
complexation, but no acceleration of the reaction was ob-
1
95
1
Pt{ H} NMR spectra of the obtained platinum(II) complexes
display signals at –2175 to –2165 ppm for trans-[PtCl (2-R-taa) ]
served. Instead, trans-[PtCl (ethyl 2-tert-butyl-tetrazol-5-ylacet-
2
2
2
and at about –2145 ppm for cis complexes 4 and 5.
ate) ] (6) was formed along with 3 due to esterification of the
2
tetrazol-5-ylacetoxy group in the acidic medium (Scheme 2). It
The thermal stability of the complexes was studied by the
is worth mentioning that esterification of 2-tBu-taa did not pro- DSC/TG method (Figures S14–S17 in the Supporting Informa-
ceed under the indicated conditions (1 HCl in EtOH, r.t., tion). Both cis and trans isomers are thermally stable at least up
weeks) in the absence of K [PtCl ]. Under the same conditions, to 150 °C. In the case of 4 (Figure S15), decomposition was
M
2
2
4
esterification of 2-Me-taa and 2-iPr-taa was also not observed, preceded by endothermic transformation assigned to melting
and only trans-isomeric complexes 1 and 2 were isolated in followed by the exothermic process of cis-to-trans isomerization
trace amounts.
(formation of the trans isomer was confirmed by NMR spectro-
According to elemental analyses, differential scanning calor- scopy, Figure S18). These transformations were not observed
imetry/thermogravimetry studies (DSC/TG, see Supporting In- for tert-butyl-substituted derivative 5.
Eur. J. Inorg. Chem. 0000, 0–0
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Full Paper
Crystal Structure Analysis
ters. Tetrazolylacetic acids contain several potential coordina-
tion sites such as the endocyclic N atoms of the tetrazolyl moi-
ety and the O atoms of the carboxyl group. In this regard, tet-
razolylacetic acids are potentially multifunctional ligands and
Molecular and crystal structures of 1–6 were elucidated by X-
ray diffraction. The crystals of 1·H O and 3·2H O belong to the
triclinic system, and crystals of 2, 4, 5·2H O, and 6 are mono-
2
2
2
[11]
can form a variety of coordination patterns. However, our X-
clinic. The unit cell parameters and the refinement parameters
are given in Table S1. Table 1 lists selected bond lengths and
bond angles for the studied species. The platinum coordination
environment and molecular structures of 1–6 showing the la-
beling of the atoms are given in Figure 2.
Among platinum(II) complexes featuring neutral N-substi-
tuted tetrazoles, complexes of cis geometry are significantly less
studied than their trans congeners. For example, only cis-
ray diffraction data demonstrate that in 1–6 the N(4) atoms of
the tetrazolyl moieties are exclusively involved in coordination
with the platinum(II) centers.
In all cases, the tetrazole cycles are planar within a range of
0
.004(1) Å. The endocyclic N–N bonds are marginally different
in length, the shortest bond between the N(2) and N(3) atoms
being 1.304(5)–1.317(4) Å in length except 3. The lengths of the
adjacent N(1)–N(2) [1.316(12)–1.342(17) Å] and N(3)–N(4)
[1.314(16)–1.330(4) Å] bonds are similar. The observed distribu-
tion of bond lengths demonstrates a moderate degree of elec-
tron-density delocalization in the tetrazole rings.
[
PtCl (5-amino-2-methyltetrazole) ] has been structurally char-
2
2
[
16]
acterized to date with use of powder X-ray diffraction. There-
fore, compounds 4 and 5·2H O are the first cis-isomeric com-
plexes of this type whose structures have been elucidated by
single-crystal X-ray diffraction.
2
For all trans complexes, the N–Pt–N and Cl–Pt–Cl angles are
All studied compounds are molecular complexes exhibiting 180°, except for 6 [177.9(2)° and 175.58(5)°, respectively]. In the
the typical square-planar environment of the platinum(II) cen- cis complexes, all angles around the platinum(II) centers are
Table 1. Selected bond lengths [Å] and bond angles [°] for trans-[PtCl
2
(2-Me-taa)
2
]·H
2
O (1·H
2
O), trans-[PtCl
2
(2-iPr-taa)
2
] (2), trans-[PtCl
] (6).
2 2 2
(2-tBu-taa) ]·2H O
(
3·2H
2
O), cis-[PtCl
2
(2-iPr-taa)
2
] (4), cis-[PtCl
2
(2-tBu-taa)
2
]·2H
2
O (5·2H
2
O), and trans-[PtCl
2
(ethyl 2-tert-butyl-tetrazol-5-ylacetate)
2
1·H
2
O
2
3·2H
2
O
4
5·2H
2
O
6
Pt1–N4
Pt1–Cl1
C1–C2
C2–C3
C4–C5
C3–O2
N1–C1
N1–N2
N2–C4
N2–N3
N3–N4
N4–C1
O1–C3
C4–C6
C4–C7
O2–C8
2.008(3)
2.2979(10)
1.490(5)
1.519(5)
–
1.188(5)
1.333(5)
1.324(5)
1.462(5)
1.311(5)
1.330(4)
1.349(5)
1.320(5)
–
2.0040(15)
2.2975(5)
1.493(3)
1.516(3)
1.520(3)
1.198(2)
1.328(2)
1.332(2)
1.486(2)
1.305(2)
1.329(2)
1.351(2)
1.334(2)
1.519(3)
–
2.001(10)
2.300(3)
1.49(2)
1.50(2)
1.55(2)
2.010(8)
2.282(2)
1.49(2)
1.50(2)
1.55(2)
1.256(12)
1.306(16)
1.316(12)
1.468(16)
1.305(12)
1.322(11)
1.336(19)
1.281(11)
1.511(5)
–
2.016(3)
2.2889(9)
1.487(5)
1.511(5)
1.513(5)
1.211(4)
1.328(4)
1.330(4)
1.508(4)
1.317(4)
1.325(4)
1.354(4)
1.310(4)
1.527(6)
1.528(5)
–
1.994(4)
2.2915(13)
1.481(6)
1.506(7)
1.512(7)
1.388(15)
1.319(6)
1.335(5)
1.505(5)
1.304(5)
1.324(5)
1.353(6)
1.193(6)
1.509(7)
1.504(7)
1.567(15)
1.48(3)
1.20(2)
1.290(19)
1.342(17)
1.474(17)
1.319(15)
1.314(16)
1.349(17)
1.300(19)
1.300(19)
1.51(2)
–
–
–
–
–
–
–
–
–
C8–C9
–
N1–C1–C2
N1–C1–N4
N4–C1–C2
N1–N2–C4
N3–N2–C4
N3–N2–N1
N4–N3–N2
C1–C2–C3
O1–C3–C2
O2–C3–O1
C1–N4–Pt1
N3–N4– C1
N3–N4–Pt1
C6–C4–C5
C7–C4–C5
C7–C4–C6
N2–C4–C5
N2–C4–C6
N4–Pt–Cl1
N2–C4–C7
C1–N1–N2
N2–C4–C7
Cl1′–Pt–Cl1
122.9(4)
110.9(4)
126.3(4)
123.1(3)
121.5(4)
115.3(3)
104.4(3)
113.8(3)
110.5(3)
125.3(4)
133.2(3)
107.6(3)
119.2(3)
–
123.74(16)
110.48(16)
125.78(17)
123.24(15)
121.75(16)
115.00(15)
104.45(15)
110.64(15)
111.29(16)
124.29(19)
131.58(13)
107.90(15)
120.52(12)
113.62(18)
–
125.5(13)
110.5(13)
124.0(12)
124.1(11)
123.2(11)
112.6(11)
104.7(10)
111.1(12)
113.7(13)
123.5(14)
131.7(10)
108.3(10)
120.0(8)
110.4(11)
112.1(13)
110.1(13)
108.1(12)
107.2(11)
90.1(3)
122.4(15)
111.0(12)
126.6(12)
115.4(9)
128.9(10)
115.5(8)
103.3(8)
115.3(11)
114.4(9)
124.1(11)
130.9(8)
108.1(9)
120.9(7)
119.9(14)
–
124.4(3)
109.9(3)
125.7(3)
123.5(3)
121.8(3)
114.4(3)
104.4(3)
113.8(3)
111.8(3)
125.9(4)
129.7(2)
108.4(3)
121.9(2)
111.7(3)
111.7(4)
111.4(3)
108.0(3)
106.4(3)
89.80(8)
107.2(3)
102.9(3)
107.2(3)
91.12(3)
127.3(4)
109.9(4)
122.7(4)
123.1(4)
122.6(4)
114.2(4)
104.6(4)
111.3(4)
124.5(5)
131.6(8)
129.4(3)
108.2(4)
122.2(3)
110.0(4)
111.6(5)
112.3(5)
107.0(4)
108.3(4)
90.82(11)
107.4(4)
103.0(4)
107.4(4)
175.58(5)
–
–
–
–
–
–
108.79(16)
108.87(16)
91.54(5)
–
102.17(15)
–
114.5(13)
107.6(11)
88.5(3)
–
102.0(11)
–
89.11(9)
–
101.9(3)
–
108.8(11)
103.9(11)
–
180.00(7)
180.0
180.0
90.45(9)
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and C–H···Cl H bonds were found in the crystal structure of 6
(Figure S6).
1
Combined H NMR and HPLC-MS Study of Complexation in
Solution
Although we isolated pure complexes 1–6, their yields were
moderate (Scheme 2). Moreover, slight alteration of the reaction
conditions led to dramatic changes in the structures of the iso-
lated products. In order to investigate how the structure of the
products depends on the reaction conditions and to detect the
minor products and intermediates, we studied the complexa-
tion by NMR spectroscopy accompanied by HPLC-MS detection
of the main products.
We monitored the reaction of K [PtCl ] with any one of 2-R-
2
4
taa (molar ratio 1:2) in 1
M DCl in D O at 25 °C by recording the
2
1
H NMR spectra of the reaction mixtures until equilibrium was
reached. Owing to the proton–deuterium exchange of the CH2
protons, monitoring on the basis of the signals of the CH2
groups was not possible. Therefore, the reaction was monitored
in the area of the CH protons of methyl, isopropyl, or tert-butyl
3
substituents (Figures 3, S10, and S12). The assignment of the
1
signals appearing in the H NMR spectra during the reaction
Figure 2. Molecular structures of 1–6 with the key atoms labeled.
was performed by addition of the respective complexes to the
1
mixture. Thus, for example, a fragment of the H NMR spectrum
of the reaction mixture of 3 with K [PtCl ] at different reaction
times is given in Figure 3. The signal at δ = 1.73 ppm is assigned
to the CH3 protons of the free 2-tBu-taa, the one at δ =
2
4
close to 90° [for 4 and 5·2H O, the N–Pt–N angles equal 91.8(4)
and 91.39(11)°, whereas the Cl–Pt–Cl angles are 90.45(9) and
2
91.12(5)°, correspondingly]. For all studied species, the Pt–Cl
1
.70 ppm belongs to 5 (cis isomer), and that at δ = 1.79 ppm
[
2.282(2)–2.300(3) Å] and Pt–N [1.994(4)–2.016(3) Å] bond
II
II
[17]
is assigned to 3 (trans isomer) (Figure 3).
lengths are characteristic of Pt –Cl and Pt –N bonds. A slight
difference in the Pt–Cl bond lengths was observed for cis and
trans isomers. As can be inferred from the inspection of Table 1,
for cis isomers 4 and 5·2H O the Pt–Cl bonds are somewhat
2
shorter in comparison with those of their trans congeners 2 and
3
·2H O. This difference can be explained in terms of the
2
ground-state trans influence, assuming that the trans-labilizing
action of the chlorido ligands is higher than that of the tetraz-
ole ligands. The observed difference in the Pt–Cl bond lengths
between the cis and trans isomers (ca. 0.01 Å) is close to the
[
2c,2l,2n]
values reported earlier for pyridine derivatives.
The molecular packing in the crystals of 1–6 is realized
through the system of intermolecular hydrogen bonds or weak
hydrogen bonding (see Tables S2–S6 and Figures S1–S6 in the
[
18]
Supporting Information).
In the crystal structure of 1·H O,
2
the H-bond system includes four O–H···O, one O–H···N, and six
C–H···Cl bonds, Figure S1 indicating the strongest of the O–
H···O bonds. In the structures of 1·H O, 3·2H O, and 5·2H O,
1
Figure 3. Fragment of the H NMR spectrum of the reaction mixture of 2-tBu-
taa (L) with K
2
2
2
2 4 2
[PtCl ] in 1 M DCl in D O at 25 °C: (a) 160 (red line), (b) 5520
a number of intra- and intermolecular H-bonds involving co-
crystallized water molecules give rise to infinite 1D chains (in
(
green line), and (c) 10240 min (blue line) after the reaction started.
3
·2H O and 5·2H O, Figures S3 and S5) or 2D layers (in 1·H O,
Using this approach, we were unable to assign the resonance
2
2
2
Figure S1). In 2, complex molecules are linked into a supramo- at δ = 1.75 ppm, and therefore, we carried out an additional
lecular 3D structure by a system of rather strong O–H···Cl bonds HPLC-MS study of the reaction mixture. According to the ob-
and weak C–H···Cl bonds (Figure S2). In the crystal structure of tained data (Figures S7 and S8), a compound with an m/z value
–
7
, the carboxy groups of both tetrazole-containing ligands form of 484.9580 was found in the reaction mixture in the ESI ioniza-
–
the intermolecular O1–H1···O1′ and O2′–H2′···O2 H-bonds with tion mode, and it was identified as [PtCl (2-tBu-taa)] (7). Fur-
3
the adjacent complex, leading to the formation of infinite thermore, peaks with m/z values of 183.0853 and 633.0736
chains running along the b axis (Figure S4). Only weak C–H···O were assigned to 2-tBu-taa and to disubstituted species
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[
PtCl (2-tBu-taa) ], respectively. Based on these results, the sig- 6 °C – when the substitution is even more selective – in the
2
2
nal at δ = 1.75 ppm was assigned to the CH group of anionic isolation of pure cis isomers 4 and 5.
3
complex 7.
The results of our qualitative kinetic studies also explain the
As follows from Figures 3, S10, and S12, generation of the low yields of the final disubstituted platinum complexes 1–5.
complexes can be monitored by a decrease of the integral in- Due to the low basicity of the starting tetrazoles, their conver-
tensity of the CH -proton signals of the metal-free 2-R-taa as sion never exceeded 70 %. It is worth mentioning that the ratio
3
well as by appearance and growth of the proton signals of the of cis to trans isomer in the reaction mixture also correlated
complexes. As integral intensities of the signals were propor- with the basicity of the starting tetrazoles, decreasing in the
tional to the concentrations of the corresponding compounds order 2-tBu-taa > 2-iPr-taa > 2-Me-taa (Figures 4, S11, and S13).
in the reaction mixture, we plotted the accumulation and con- Notably, for 2-Me-taa, generation of both isomers proceeds at
sumption kinetic curves (Figures 4, S11, and S13).
comparable rates even in 1 M DCl (Figure S13), which explains
the fact that we were never able to isolate the respective cis-
isomeric complex from the reaction mixture (see synthetic part).
Antiproliferative Activity
Tetrazolylacetic acids are good candidates for medicinal chemis-
[
11,19]
try studies.
Tetrazolyl fragments are included in drugs with
a wide range of therapeutic action such as antibacterial, anti-
viral, and cytotoxic agents as well as antihistamines, agents act-
ing on the central nervous and the cardiovascular systems, and
[
11]
so on. In some cases, the use of metal complexes of biologi-
cally active compounds was shown to increase the initial activ-
[
19]
ity. The antiproliferative activity of tetrazolylacetic acid com-
plexes of platinum has never been studied.
3
Figure 4. Normalized integral intensities of the CH signals as a function of
the reaction time (L = 2-tBu-taa).
To assess the therapeutic potential of the obtained water-
soluble complexes, their antiproliferative activity was evaluated
Monosubstituted complex 7 appeared in the reaction mix- in vitro against three human cancer cell lines (HT-29 colon carci-
ture 2 hours after mixing the reactants (at concentrations noma, MCF-7 and MDA-MB-231 breast carcinomas). We found
–
1
–1
0
.04 mol L of 2-tBu-taa and 0.02 mol L of K [PtCl ]) (Fig- that both uncomplexed 2-R-taa and their platinum complexes,
2 4
ure 4). Accumulation of disubstituted complexes 3 and 5 regardless of their geometry and the nature of the alkyl substit-
started after 21 hours; the cis complex accumulated faster than uent R, were inactive with IC50 values exceeding 100 μ
. How-
M
the trans isomer. After 164 hours, the conversion of 2-tBu-taa ever, esterification of the tetrazol-5-ylacetoxy group leads to a
reached 60 %. Then the reaction proceeded very slowly and drastic increase in antiproliferative activity observed for 6
practically stopped when 30 % of the tetrazole was still uncom- (Table S11). This fact is consistent with the data previously ob-
plexed. The reactions between K [PtCl ] and any one of 2-R-taa tained for platinum(II) complexes bearing pyridinecarboxylic
2
4
[
20a]
(
R = Me and iPr) in 1
M
DCl in D O at 25 °C proceeded in a acids, which did not show cytotoxicity.
We assume that, in
2
similar way. In the beginning, monosubstituted species [PtCl - the case of complex 6, the uncharged ester group increases the
3
–
(
2-R-taa)] were formed, whereupon they were transformed into lipophilicity, and it may facilitate transport across the cellular
disubstituted cis- and trans-[PtCl (2-R-taa) ] (Figures S10–S13).
membrane. Having trans geometry, 6 demonstrated noticeable
showed that the geometry of the antiproliferative effect in two human cancer cell lines with IC50
generated platinum complexes can be affected by the presence values (14.2 ± 1.1 μ in HT-29 cells and 5.8 ± 1.2 μ in MCF-7
of HCl in the reaction mixture, we carried out the reaction of cell line) comparable to those of cisplatin (4.1 0.3 and
2
2
[
3,5]
Since previous studies
M
M
[
20b]
K [PtCl ] with 2-tBu-taa in D O. In this case, the reaction pro- 1.6 ± 0.5 μ
M
in HT-29 and MCF-7 cells,
ceeded much faster, and mono- (7) and disubstituted com- tetrazole-containing analog cis-[PtCl
plexes (3, 5) appeared in the reaction mixture almost instantly ole) ] (11.1 ± 2.0 μ in HT-29 cells). The latter complex was in-
after mixing the reactants (Figure S9). Notably, in the absence cluded into the test since it had already demonstrated high
respectively) and its
2
4
2
(5-amino-1-phenyltetraz-
2
M
2
[
5b]
of DCl, trans complex 3 accumulated faster than its cis isomer levels of cytotoxic activity.
1
(
5). After 2 hours, the released precipitate prevented further H
High antiproliferative activity of trans-platinum complexes
NMR spectroscopic measurements.
with some heterocyclic ligands has been known since the early
Thus, our observations confirmed that the selectivity of sub- 1990s,[2a,21] and molecular bases of their antitumor action are
[
21e–21h]
stitution is strongly affected by the presence of HCl in the reac- still intensively studied.
For some N-containing hetero-
tion mixture. Apparently, in the presence of HCl, rates of all cycles,[ trans-isomeric complexes appeared to be more cyto-
the reactions associated with substitution of chloride ions with toxic than the corresponding cis isomers; however, our previous
tetrazole ligands decrease, leading to the higher yield of cis studies demonstrated that, in the case of 5-amino-N-R-tetraz-
isomers as kinetically controlled products. Although we were oles, trans-isomeric Pt complexes were at least tenfold less ac-
21]
[
5b]
unable to isolate cis-isomeric species at r.t., we succeeded at 4– tive than their cis congeners.
In the present study, trans-iso-
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–
1
1
13
1
195
1
meric complex 6 turned out to be almost as potent as the 5- at 4000–400 cm . H, C{ H}, and Pt{ H} NMR spectra were re-
corded with Bruker 300 MHz DPX and Bruker 400 MHz Avance spec-
trometers. H and C{ H} chemical shifts were determined with re-
sidual signals of the deuterated solvents (CDCl , D O, CD OD, and
DCl) at 298 K and were referenced to that of SiMe (δ = 0 ppm).
Pt{ H} chemical shifts were determined indirectly and referenced
to that of Na [PtCl ] (δ = 0 ppm) as an external standard. Analytical
aminotetrazole-based species featuring cis geometry. Thus, one
1
13
1
can conclude that the antiproliferative activity of tetrazole-
based platinum complexes is strongly affected by the nature of
the substituent in the 5-position of the tetrazole ring, and by
changing it one can increase the antitumor potency of trans-
isomeric species.
3
2
3
4
195
1
2
6
HPLC measurements were performed with an Agilent 1200 Zorbax-
SBD-C18 column (2.1 × 150 mm, 3.5 μm) by using gradients of sol-
vents, 10–90 % solvent A (MeCN) and solvent B (H O).
2
Conclusions
Synthetic Work
In summary, we have synthesized and characterized water-solu-
ble cis- and trans-platinum(II) complexes featuring 2-alkyl-2H-
tetrazol-5-ylacetic acids. Both geometric isomers were obtained
trans-[PtCl (2-Me-taa) ]·H O (1·H O): K [PtCl ] (42 mg, 0.1 mmol)
2
2
2
2
2
4
and 2-Me-taa (28 mg, 0.2 mmol) were dissolved in 1
M HCl (2 mL)
and stirred at r.t. for 4 h. The formed yellow solution was kept at
r.t. for 2 weeks, and two portions of the yellow crystalline product
by a reaction of K [PtCl ] with 2-R-taa in 1
M HCl. At r.t., only
2
4
trans isomers were isolated, whereas the cis-isomeric products were separated. The yellow precipitate was filtered off and washed
+
were synthesized by lowering the temperature of the reaction with water (10 mL). Yield: 24 mg (44 %). HRESI -MS: m/z = 587.9663
mixture to 4–6 °C. However, solution 1H NMR spectroscopic [M + K] ; 571.9931 [M + Na] . H NMR (300 MHz, 1
+
+ 1
M
DCl in D
O):
2
195
1
δ = 4.16 (s, 4 H, CH ), 4.44 (s, 6 H, CH ) ppm. Pt{ H} NMR (86 MHz,
CD OD): δ = –2174.7 ppm. Crystals of 1·H O suitable for X-ray dif-
fraction were obtained by slow evaporation of the reaction mixture
at r.t. for 5–10 d.
studies showed that upon reaction of K [PtCl ] with 2-R-tetra-
zol-5-ylacetic acids at r.t. generation of the cis-platinum com-
2
3
2
4
3
2
plexes occurs along with their trans isomers. This reaction pro-
–
ceeds sequentially through formation of the [PtCl (2-R-taa)]
3
species.
trans-[PtCl
taa (34 mg, 0.2 mmol) were dissolved in 1
2
(2-iPr-taa)
2
] (2): K
2
[PtCl
4
] (42 mg, 0.1 mmol) and 2-iPr-
HCl (2 mL) and stirred
M
Rather unexpectedly, in the presence of EtOH trans-
at r.t. for 4 h. The formed yellow solution was kept at r.t. for 2 weeks,
and two portions of the yellow crystalline product were separated
during that period. The yellow precipitate was collected by filtration
and washed with water (10 mL). Yield: 34 mg (56 %).
C H Cl N O Pt (606.34): calcd. C 23.77, H 3.32, N 18.48; found C
[
PtCl (ethyl 2-tert-butyl-tetrazol-5-ylacetate) ] (6) was isolated
2
2
instead of trans-[PtCl (2-tBu-taa) ]. The reason for this is esterifi-
2
2
cation of the 2-tert-butyl-2H-tetrazol-5-ylacetoxy ligand in the
acidic medium. The latter complex, despite its trans geometry,
exhibits marked antiproliferative activity in HT-29 colon and
MCF-7 breast carcinoma cell lines.
12
20
2 8 4
+
+ 1
23.27, H 2.89, N 18.60. HRESI -MS, m/z: 629.0530 [M + Na] . H NMR
(400 MHz, 1 DCl in D O): δ = 1.69 (d, J = 6.7 Hz, 6 H, CH from
M
2
H,H
3
1
Previous studies indicated that the antiproliferative effect of iPr), 4.57 (s, 2 H, CH ) ppm. H NMR (400 MHz, CD OD): δ = 5.23
2
3
tetrazole-based platinum complexes depends on their geome- (sept, JH,H = 6.7 Hz, 1 H, CH from iPr), 4.53 (s, 2 H, CH
), 1.70 (d, JH,H
=
2
13
1
6
.7 Hz, 6 H, CH from iPr) ppm. C{ H} NMR (101 MHz, CD OD): δ =
try (cis complexes are supposed to be more cytotoxic than trans
3
3
[
5b]
22.0 (CH from iPr), 32.8 (CH ), 61.0 (CH from iPr), 162.5 (CN ), 170.2
3
2
4
isomers) and strongly correlates with their lipophilicity.
The
195
1
(
COOH) ppm.
Pt{ H} NMR (86 MHz, CD OD): δ = –2169.8 ppm.
3
drastic increase in activity of 6 in comparison with its congeners
bearing 2-alkyl-2H-tetrazol-5-ylacetic acids might also be
caused by their difference in lipophilicity. However, to verify this
hypothesis and to reveal the role that the nature of the 5-R-
substituent plays in the antiproliferative activity of tetrazole-
based platinum complexes, additional experimental data are re-
quired. To achieve this goal, further studies focused on the syn-
thesis of cis- and trans-isomeric platinum complexes bearing N-
R-tetrazol-5-ylacetic acid esters and evaluation of their antipro-
liferative activity are on the way in our group.
–
1
far-IR: ν˜ = 347 (ν, Pt–Cl) cm . Crystals of 2 suitable for X-ray diffrac-
tion were obtained by slow evaporation of the reaction mixture at
r.t. for 5–10 d.
trans-[PtCl (2-tBu-taa) ]·2H O (3·2H O): K [PtCl ] (104 mg,
2
2
2
2
2
4
0.25 mmol) and 2-tBu-taa (92 mg, 0.50 mmol) were dissolved in 1 M
HCl (5–7 mL) and stirred at r.t. for 24 h. The formed yellow solution
was kept at r.t. for 2 weeks, and three portions of the yellow crystal-
line product were separated during that period. Yield: 86 mg (54 %).
C H Cl N O Pt (670.42): calcd. C 25.08, H 4.21, N 16.71; found C
1
4
28
2 8 6
+
+ 1
2
5.06, H 4.47, N 16.73. HRESI -MS, m/z: 782.2230 [M – Cl + L] . H
NMR (400 MHz; 1 DCl in D O): δ = 4.57 (s, 2 H, CH ), 1.79 [s, 9 H,
M
2
2
1
C(CH ) ] ppm. H NMR (400 MHz, CD OD): δ = 4.54 (s, 2 H, CH ),
3
3
3
2
13
1
Experimental Section
1.81 (s, 9 H, tBu) ppm. C{ H} NMR (101 MHz, CD OD): δ = 29.1
3
(
CH3 from tBu), 32.8 (CH ), 69.0 (C from tBu), 162.3 (CN ), 170.4
2 4
Materials and Instruments: K [PtCl ] and all solvents were pur-
2
4
195
1
(COOH) ppm.
Pt{ H} NMR (86 MHz, CD OD): δ = –2165.3 ppm.
3
chased from commercial sources and used without further purifica-
tion. 2-Methyl-, 2-isopropyl-, and 2-tert-butylacetic acids were syn-
thesized as described elsewhere.[ Elemental analyses (CHN) were
performed with a EuroVector EuroEA3000 CHN analyzer. Mass spec-
tra were recorded with Bruker MicroTOF and Shimadzu MALDI-TOF
–
1
far-IR: ν˜ = 343 (ν, Pt–Cl) cm . Crystals of 3·2H O suitable for X-ray
2
diffraction were obtained by slow evaporation of the reaction mix-
ture at r.t. for 5–10 d.
13]
cis-[PtCl (2-iPr-taa) ] (4): K [PtCl ] (208 mg, 0.5 mmol) and 2-iPr-
2
2
2
4
AXIMA resonance spectrometers as well as a Bruker Daltonik GmbH taa (170 mg, 1 mmol) were dissolved in 1
M HCl (7 mL) and kept at
“
MaXis” LC-MS spectrometer equipped with a diode array detector
4–6 °C for 3 weeks. A single portion of the yellow crystalline product
was separated. Yield: 30 mg (9.3 %). C H Cl N O Pt (606.34): calcd.
and a HRMS ESI Q-TOF detector. DSC/TG measurements were per-
formed with a Netzsch STA 449 F1 Jupiter instrument under argon
flow (100 mL min ) at a heating rate of 10 °C min . The IR spectra
of powder samples were recorded with an IRAffinity-1 spectrometer
12
20
2 8 4
+
C 23.77, H 3.32, N 18.48; found C 23.47, H 2.94, N 18.19. HRESI -MS,
–1
–1
+
+ 1
m/z: 628.0528 [M + Na] , 740.1805 [M – Cl + L] . H NMR (400 MHz,
DCl in D O): δ = 1.61 (d, J = 6.7 Hz, 6 H, CH from iPr), 4.54
1
M
2
H,H
3
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1
(
6
s, 2 H, CH ) ppm. H NMR (400 MHz, CD OD): δ = 5.12 (sept, J =
difference Fourier maps and included in the refinement without any
restrictions. Other hydrogen atoms were positioned geometrically
and constrained to ride on their parent atoms, with C–H = 0.98–
1.00 Å, O–H = 0.84 Å, and Uiso = 1.2–1.5 Ueq (parent atom). High
2
3
H,H
.7 Hz, 1 H, CH from iPr), 4.49 (s, 2 H, CH ), 1.62 (d, J = 6.7 Hz, 6
2 H,H
H, CH from iPr) ppm. C{ H} NMR (101 MHz, CD OD): δ = 21.9 (CH
1
3
1
3
3
3
from iPr), 32.5 (CH ), 60.95 (CH from iPr), 162.6 (CN ), 170.3 (COOH)
2
4
ppm. 1 Pt{ H} NMR (86 MHz, CD OD): δ = –2145.6 ppm. far-IR: ν˜ = values of the residual density as well as the refinement parameters
95
1
3
–
1
3
41, 352 (ν, Pt–Cl) cm . Crystals of 4 suitable for X-ray diffraction
and rather low bonds precision in the structural model of 3·2H O
2
were obtained directly from the reaction mixture.
were due to the small size and low quality of the crystals.
CCDC 1401577 (for 1·H O), 1401576 (for 2), 1040854 (for 3·2H O),
cis-[PtCl (2-tBu-taa) ] (5): K [PtCl ] (42 mg, 0.1 mmol) and 2-tBu-
2
2
2
2
2
4
1
400470 (for 4), 1401575 (for 5·2H O), and 1040853 (for 6) contain
taa (37 mg, 0.20 mmol) were dissolved in 1
M
HCl (4 mL) and kept
2
the supplementary crystallographic data for this paper. These data
can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre.
1H NMR Spectroscopic Study of the Complexation in Solution
without stirring at 6–8 °C for 6 weeks. A single portion of the yellow
crystalline product was separated. Yield: 21 mg (33 %).
C H Cl N O Pt (634.39): calcd. C 26.51, H 3.81, N 17.66; found C
1
4
24
2 8 4
+
+
1
2
6.26, H 3.90, N 17.68. HRESI -MS, m/z: 672.0567 [M + K] . H NMR
(
400 MHz, 1 DCl in D O): δ = 4.53 (s, 2 H, CH ), 1.70 (s, 9 H, tBu)
M
2
2
2
-Me-taa and 2-iPr-taa: A solution of 2-Me-taa (0.14 mmol) or 2-
iPr-taa in 1 DCl in D O (1 mL) was added to a solution of K [PtCl ]
0.07 mmol) in 1
mixed.
2-tBu-taa: A solution of 2-tBu-taa (0.04 mmol) in of 1
1 mL) was added to a solution of K [PtCl ] (0.02 mmol) in 1 M DCl
1
ppm. H NMR (400 MHz, CD OD): δ = 4.48 (s, 2 H, CH ), 1.72 (s, 9
3
2
M
H, tBu) ppm. 1 C{ H} NMR (101 MHz, CD OD): δ = 29.1 (CH from
3
1
2
2
4
3
3
(
M DCl in D O (0.5 mL), and the solutions were
2
tBu), 32.6 (CH ), 68.9 (C from tBu), 162.5 (CN ), 170.4 (COOH) ppm.
2
4
1
95
1
Pt{ H} NMR (86 MHz, CD OD): δ = –2144.9 ppm. far-IR: ν˜ = 330,
3
–1
M DCl in D O
2
3
45 (ν, Pt–Cl) cm . Single crystals of 5·2H O suitable for X-ray dif-
2
(
fraction were obtained directly from the reaction mixture.
2
4
in D O (0.5 mL), and the solutions were mixed.
2
trans-[PtCl (ethyl 2-tBu-2H-tetrazol-5-ylacetate) ] (6): A solution
2
2
The time of mixing was taken as the starting point of the reaction,
and the solution was transferred to a 5 mm NMR tube. The tube
of 2-tBu-taa (92 mg, 0.50 mmol) in of EtOH (5 mL) was added to a
solution of K [PtCl ] (104 mg, 0.25 mmol) in of 1 HCl (5 mL), and
M
2
4
1
was kept at 25 °C, and H NMR spectra were recorded at fixed time
the reaction mixture was stirred for 24 h at r.t. The formed yellow
solution was kept at r.t. for 2 weeks, and three portions of the
yellow crystalline product were separated during that period. Yield:
intervals with a Bruker 300 MHz DPX spectrometer. The peak areas
were measured by cutting and weighing. The spectra were col-
lected until the equilibrium state was reached and no further
changes in the spectra were detected.
1
1
–
4
10 mg (65 %). C H Cl N O Pt (690.50): calcd. C 31.31, H 4.67, N
18 32 2 8 4
+
6.23; found C 31.13, H 4.71, N 16.41. HRESI -MS, m/z: 866.3158 [M
+
1
Cl + L] . H NMR (400 MHz, CDCl ): δ = 4.54 (s, 2 H, CH CO Et), Cell Culture and Cytotoxicity Assay in MCF-7, MDA-MB-231, and
3
2
2
.27 (q, 2 H, CH CH ), 1.78 (s, 9 H, tBu), 1.30 (t, 3 H, CH CH ) ppm.
HT-29 Cells: The antiproliferative effects in MCF-7, MDA-MB-231,
C{ H} NMR (101 MHz, CDCl ): δ = 166.8 (C5), 160.7 (CO Et), 67.7 and HT-29 cells after 72 h (HT-29) or 96 h (MCF-7, MDA-MB-231)
2
3
2
3
1
3
1
3
2
(
C from tBu), 62.1 (CH CO Et), 32.5 (CH from Et), 29.2 (CH3 from exposure to the platinum complexes were evaluated with use of
2 2 2
1
95
1
tBu), 14.2 (CH from Et) ppm.
Pt{ H} NMR (86 MHz, CDCl ): δ =
the crystal violet assay. In short, HT-29 human colon carcinoma cells,
MCF-7 and MDA-MB-231 breast cancer cells were maintained in cell
culture medium (minimum essential eagle medium supplemented
3
3
–2168.9 ppm. Crystals of 6 suitable for X-ray diffraction were ob-
tained by slow evaporation of the reaction mixture at r.t. for 5–10 d.
–
1
with NaHCO (2.2 g), sodium pyruvate (110 mg L ) and gentamicin
3
X-ray Crystal Structure Determinations
–1
sulfate (50 mg L ), adjusted to pH 7.4, containing 10 % (v/v) fetal
The crystals of 1·H O, 2, and 5·2H O were immersed in cryo-oil,
calf serum at 37 °C/5 % CO and passaged once a week according
2
2
2
mounted in a MiTeGen loop, and measured at a temperature of
to standard procedures. For the experiments, the compounds were
freshly prepared as stock solutions in DMF and diluted with the cell
culture medium to the final assay concentrations (0.1 % v/v DMF).
The assay procedure is described in more detail in our recent pa-
1
20–170 K with Bruker Kappa Apex II diffractometer (Mo-Kα radia-
tion). The crystals of 3·2H O, 4, and 6 were fixed on a micro mount
2
and placed into an Agilent Technologies Excalibur Eos (for 6) or
[
23]
Agilent Technologies Supernova Atlas (for 3·2H O and 4) diffrac-
pers.
The IC50 value is described as the concentration reducing
2
tometer; the diffraction was measured at 100 K by using monochro-
the proliferation of untreated control cells by 50 %.
[
22a]
mated Mo-K or Cu-K radiation, respectively. The CrysAlisPro
Denzo-Scalepack
or
α
α
[22b]
program packages were used for cell refine-
Acknowledgments
program or by di- The authors acknowledge the Russian Foundation for Basic Re-
ments and data reductions. The structures were solved by the
[22c]
charge flipping method with the SUPERFLIP
[22d]
rect methods with the SHELXS-2014
program. An analytical search and Belarusian Republican Foundation for Fundamental
CrysAlisPro[ ) or numerical absorption correction (SADABS[22e]
was applied to the data. Structural refinement was carried out by
22a]
(
)
Research for financial support of a joint project (grants 16-53-
0047 Bel_a and X16P-036). Physicochemical measurements
0
using SHELXL-2014[
22d]
with the Olex2
[22f,22g]
and SHELXL
[22h]
graphi-
were carried out at the X-ray Diffraction Centre, the Centre for
Magnetic Resonance, the Center for Thermal Analysis and Calor-
imetry, and the Centre for Chemical Analysis and Materials (all
belong to Saint Petersburg State University).
cal user interfaces. In the structure of 5·2H O, the CH groups in
2
3
one of the tBu substituents were disordered over two sites with an
occupancy ratio of 0.65/0.35. The C–C bonds in this disordered
group were constrained to be similar. Also, the anisotropic displace-
ment parameters were set to be similar.
Keywords: Platinum · N ligands · Tetrazoles · Biological
activity · Antiproliferation
The carbon-bound H atoms were placed in the calculated positions
and included in the refinement in the “rider” model approximation
with Uiso(H) set to 1.5 U (C), C–H 0.96 Å for the CH groups and
eq
3
Uiso(H) set to 1.2 U (C), C–H 0.97 Å for the CH groups. Hydrogen
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2
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2
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Received: May 26, 2016
Published Online: ■
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Anticancer Complexes
E. A. Popova,* T. V. Serebryanskaya,*
S. I. Selivanov, M. Haukka,
T. L. Panikorovsky, V. V. Gurzhiy,
I. Ott, R. E. Trifonov,
V. Y. Kukushkin ................................. 1–10
Water-Soluble Platinum(II) Com-
plexes
Featuring
2-Alkyl-2H-
tetrazol-5-ylacetic Acids: Synthesis,
Characterization, and Antiprolifera-
tive Activity
1
Water-soluble platinum(II) complexes DCl in D O, 25 °C) was studied by H
2
featuring 2-alkyl-2H-tetrazol-5-ylacetic NMR spectroscopy and HPLC-MS. Anti-
acids have been synthesized and char- proliferative activity of the new tetraz-
acterized. The generation of the tetraz- ole-containing platinum(II) complexes
ole-based complexes in solution (1
M
is discussed.
DOI: 10.1002/ejic.201600626
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim