Arene Osmium Complexes with Ethacrynic Acid-Modified Ligands: Synthesis, Characterization, and Evaluation of Intracellular Glutathione S-Transferase Inhibition and Antiproliferative Activity

Article abstract of DOI:10.1021/acs.organomet.6b00197

Four arene osmium complexes were prepared containing derivatives of ethacrynic acid, a potent inhibitor of glutathione S-transferases, either by direct coordination or via N- or P-donor ligands. The complexes were characterized by spectroscopic and analytical methods and, for Os(η⁶-p-cymene)(acetylacetonato)(2-(2,3-dichloro-4-(2-ethylenebutanoyl)phenoxy)acetato) and Os(η⁶-p-cymene)Cl₂(2-(2-(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetoxy)ethyl nicotinato), by single-crystal X-ray diffraction. The cytotoxicity of the complexes toward human ovarian cancer cells and nontumorous human embryonic kidney cells was investigated, and two of the complexes, for which ruthenium analogues are known, helped to delineate the influence of the metal ion. Inhibition studies of intracellular glutathione S-transferases (GSTs, detoxification enzymes implicated in drug resistance) indicate that the observed cytotoxicity of the complexes involves GST inhibition, as well as other targets following dissociation of the ethacrynic acid group from the osmium(II) ion.

Full text of DOI:10.1021/acs.organomet.6b00197

Article
pubs.acs.org/Organometallics
Arene Osmium Complexes with Ethacrynic Acid-Modified Ligands:
Synthesis, Characterization, and Evaluation of Intracellular
Glutathione S‑Transferase Inhibition and Antiproliferative Activity
Gabriele Agonigi,† Tina Riedel,‡ M. Pilar Gay,§ Lorenzo Biancalana,† Enrique Onate,§ Paul J. Dyson,‡
̃
Guido Pampaloni,† Emilia Paunescu,‡ Miguel A. Esteruelas,*,§ and Fabio Marchetti*,†
̆
†Dipartimento di Chimica e Chimica Industriale, Universita
̀
di Pisa, Via G. Moruzzi 13, I-56124 Pisa, Italy
ierie Chimiques, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
§Departamento de Química Inorgan
ica, Instituto de Síntesis Química y Catalisis Homogenea (ISQCH), Centro de Innovacion en
‡Institut des Sciences et Ingen
́
́
́
́
́
́
́
Química Avanzada (ORFEO−CINQA), Universidad de Zaragoza-CSIC, 50009 Zaragoza, Spain
S
* Supporting Information
ABSTRACT: Four arene osmium complexes were prepared containing
derivatives of ethacrynic acid, a potent inhibitor of glutathione S-
transferases, either by direct coordination or via N- or P-donor ligands.
The complexes were characterized by spectroscopic and analytical
methods and, for Os(η6-p-cymene)(acetylacetonato)(2-(2,3-dichloro-4-
(2-ethylenebutanoyl)phenoxy)acetato) and Os(η6-p-cymene)Cl2(2-(2-
(2,3-dichloro-4-(2-methylenebutanoyl)phenoxy)acetoxy)ethyl nicotina-
to), by single-crystal X-ray diffraction. The cytotoxicity of the complexes
toward human ovarian cancer cells and nontumorous human embryonic
kidney cells was investigated, and two of the complexes, for which
ruthenium analogues are known, helped to delineate the influence of the
metal ion. Inhibition studies of intracellular glutathione S-transferases
(GSTs, detoxification enzymes implicated in drug resistance) indicate
that the observed cytotoxicity of the complexes involves GST inhibition, as well as other targets following dissociation of the
ethacrynic acid group from the osmium(II) ion.
therapeutic agents, including cisplatin.10 Consequently, in an
INTRODUCTION
■
attempt to overcome drug resistance, ethacrynic acid has been
tethered to the ruthenium(II)-arene framework via two distinct
synthetic approaches, i.e., the modification of the arene ligand11
and the introduction via an appropriate monodentate ligand. In
this respect, ethacrynic acid-functionalized imidazole,12 pyr-
idine, and triphenylphosphine-type13 ligands have been used
(Figure 1). Also ruthenium(III), platinum(II), and platinum-
(IV) complexes with ethacrynic acid-derived appendages have
been prepared and tested as anticancer drugs.14
All the ruthenium and platinum complexes modified with
ethacrynic acid manifest increased ability to inactivate GSTs
and induce apoptosis, even in cisplatin-resistant cell lines.15 To
the best of our knowledge, the inclusion of ethacrynic acid in
osmium complexes has not been reported heretofore. In the
present paper, the synthesis, characterization, and biological
activity of a series of four osmium complexes containing
ethacrynic acid-modified ligands (including two complexes for
which Ru analogues are known) are described.
Arene ruthenium(II) complexes have been intensively inves-
tigated as possible anticancer agents,1 and especially those
complexes containing 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]-
decane (RAPTA complexes) and ethylene-1,2-diamine as
ligands show promising antitumor properties.2 On account of
the proximity of ruthenium and osmium in the same group of
the periodic table, a variety of osmium(II) derivatives have been
considered as a natural extension of this research.3 In particular,
arene osmium complexes usually exhibit slower rates for ligand
exchange than the ruthenium analogues, a desirable property
providing increased drug stability under physiological con-
ditions.4
One of the common approaches aimed at improving the
efficacy of metal drugs consists of incorporating an organic
fragment with a complementary biological function.5 This
strategy has already been applied also to various anticancer
arene ruthenium(II) and osmium(II) complexes, which have
been functionalized with biologically active moieties such as 3-
hydroxyflavones,6 lapachol,7 paullones,8 and lonidamine.9
It has previously been shown that ethacrynic acid, originally
developed as an inhibitor of glutathione S-transferases (GSTs, a
family of cytosolic detoxification enzymes associated with drug
resistance), potentiates the cytotoxic effect of several chemo-
Received: March 8, 2016
© XXXX American Chemical Society
A
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Figure 1. Structures of ethacrynic acid and of arene ruthenium(II) complexes containing ethacrynic acid-modified ligands.
Scheme 1. Synthesis of Osmium(II)−p-Cymene Ethacrynato Complex 1
display a series of absorptions in the range 1759−1518 cm−1,
attributable to the stretching vibrations of different carbonyl
moieties. The NMR spectra of 1−4 (in CD2Cl2) contain single
sets of resonances. The carbonyl carbon atom of the
ethacrynato moiety in 1 exhibits a resonance at 172.5 ppm in
the 13C NMR spectrum, i.e., at comparable frequency to that in
RESULTS AND DISCUSSION
■
The direct reaction of the bis-acetylacetonato complex Os(η6-p-
cymene)(κ(C)-acac)(κ(O,O′)-acac)16 with ethacrynic acid
proceeded at room temperature to give complex 1, which
was isolated as a bright yellow solid in 75% yield (Scheme 1).
The ethacrynic acid-functionalized ligands 2-((4-
(diphenylphosphanyl)benzyl)oxy)ethyl-2-(2,3-dichloro-4-(2-
methylenebutanoyl)phenoxy)acetato (LP)13 and 2-(2-(2,3-
dichloro-4-(2-methylenebutanoyl)phenoxy)acetoxy)ethyl-
nicotinato (LN)13 and 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]-
decane (PTA)17 were prepared according to literature
procedures. Then the ligands were allowed to react with the
dimer [OsCl2(η6-p-cymene)]2 to give the corresponding
mononuclear complexes 2 and 3 in 90% and 75% yield,
respectively, and [OsCl2(η6-p-cymene)(PTA)]18 (Scheme 2).
[OsCl2(η6-p-cymene)(PTA)] was further treated with LN, in
the presence of silver tetrafluoroborate, to afford 4 in an overall
yield of 81%.
Complexes 1−4 are air-stable in the solid state and when
dissolved in chlorinated solvents. Complex 4 is a racemate, due
to the presence of a stereogenic osmium center. Complexes 1−
3 in CH2Cl2 exhibit molar conductance values in the range
0.12−0.41 S·cm2·mol−1, while the value found for 4 is 9.32 S·
cm2·mol−1, in agreement with the neutral character of 1−3 and
the ionic character of 4. The IR spectra of 1−4 (solid state)
1
ethacrynic acid itself (172.8 ppm). The H and 13C NMR
spectra of 2 and 3 display resonances due to the LP and LN
ligands close in value to those observed for the same ligands
coordinated in analogous ruthenium complexes.13 As a
consequence of the formal substitution of a chloride with
PTA on going from the neutral complex 3 to the cationic 4, the
resonances of some pyridyl nuclei undergo a significant shift to
1
higher frequency (ΔδC20−H = 0.27 in the H spectra; ΔδC19
=
3.1, ΔδC17 = 1.6, and ΔδC20 = 2.6 in the 13C spectra; see Chart
4 in Experimental Section). The 31P NMR resonance in 2 (at
−12.1 ppm) appears at lower frequency with respect to the free
ligand, LP (−4.9 ppm),13 whereas the 31P NMR resonance due
to the PTA ligand in 4 (−77.4 ppm) resembles the values
previously reported for related arene osmium(II) complexes.19
The expected structures of 1−4 were further corroborated by
mass spectrometry and elemental analysis (see Experimental
Section).
Crystals of 1 and 3 suitable for X-ray diffraction analysis were
obtained from CH2Cl2/pentane and CH2Cl2/hexane mixtures,
respectively. A small amount of hydroquinone was added in
B
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Scheme 2. Synthesis of the Complexes 2−4 Containing Ethacrynic Acid-Modified Ligands
order to facilitate the crystallization of 3.20 The structures of 1
and 3 are shown, respectively, in Figures 2 and 3, while selected
bond distances and angles are given in Tables 1 and 2.
Complexes 1 and 3 adopt the characteristic three-leg piano-
stool geometry, comprising the p-cymene “seat”.3b,7,18,21 The
three “leg” positions are occupied by the bidentate acac ligand
[O(5)−Os−O(6) bite angle 88.14(9)°] and the monodentate
carboxylate [O(1)−Os−O(5) and O(1)−Os−O(6) angles are
80.59(10)° and 77.35(10)°, respectively] in 1 and by LN and
two chlorides in 3 [N(1)−Os−Cl(1), N(1)−Os−Cl(2), and
Cl(1)−Os−Cl(2) angles measure 82.20(10)°, 86.62(10)°, and
85.08(4)°, respectively]. The bonding parameters of the
ethacrynic acid (EA) units are not significantly different from
those found in other reported structures.10a,12,13,22 Compound
1 represents the first example of a crystallographically
characterized osmium p-cymene complex with a monodentate
carboxylate ligand: the Os−O(1) bond length [2.081(3) Å] is
reminiscent of the situation found in other Os(II)-monodentate
carboxylate systems.23 The Os−O(5) and Os−O(6) bond
lengths [2.072(2) and 2.081(2) Å, respectively] in 1 resemble
those previously reported for other Os(II)-acac complex-
es.21b,24
NMR and UV−vis spectroscopy and conductivity measure-
ments were used to assess the stability of the complexes 1−4 in
dmso/water solutions at 37 °C as a function of time. Parallel
NMR and UV−vis analyses were carried out also on 1−4 in 0.1
M sodium chloride dmso/water solutions (this chloride
concentration being similar to that present in blood). The
compounds identified from these studies are depicted in
Scheme 3.
A complicated mixture of compounds form from 1 in dmso/
water, and two sets of resonances have been tentatively
assigned to 1a and 1b; the ethacrynato moiety behaves as a
leaving group, and the NaCl environment accelerates this
dissociation. EA-CO2K was prepared from EA-CO2H/KN-
(SiMe3)2 and characterized in order to identify EA-CO2− in the
studied solutions (see Experimental Section).
Conductivity data suggest that 2−4 undergo rapid solvolysis
in dmso/water, presumably via the release of a chloride ligand.
Figure 2. Molecular structure of 1. Displacement ellipsoids are at the
50% probability level. Hydrogen atoms are omitted for clarity.
C
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Figure 3. Molecular structure of 3. Displacement ellipsoids are at the 50% probability level. Hydrogen atoms are omitted for clarity.
Since both EA-CO2H and EA-(CO)O(CH2)2OH might
be generated from the ligands/complexes, their antiproliferative
activities were also evaluated. The ester EA-(CO)O-
(CH2)2OH shows a 15−20-fold higher cytotoxicity than the
carboxylic acid EA-CO2H, most probably derived from the
better uptake of the former into cells, due to increased
solubility in the cell culture medium.10b Complex 1 has IC50
values similar to those determined for EA-CO2H, whereas 2−4
are significantly more cytotoxic, albeit not too dissimilar from
their corresponding ethacrynic acid-derived ligands LP and LN.
Complex 3 is more cytotoxic than 4, both containing the same
LN ligand, but the latter has a PTA ligand in place of a chloride
ligand. The IC50 values of 2 and 3 are comparable to those
previously described for ruthenium(II)−arene complexes
modified with an ethacrynic acid unit.10a,12,13 Nonetheless, 2
and 3 display a slightly higher selectivity toward cancer cells
with respect to nontumorigenic HEK-293 cells than the
respective Ru analogues (Table 3).13
Intracellular GST Inhibition. Due to the potential of 1−4
to inhibit GSTs inside cells, residual intracellular GST activity
after incubation with the complexes and the ligands at the
respective IC50 concentrations was determined by fluorescence
spectroscopy in A2780 and A2780cisR cells (Figure 4).
Incubation for 18 h led to a modest decrease in GST activity
in both A2780 and A2780cisR cells with all the compounds,
with 1 being the least effective GST inhibitor. This appears to
be in accordance with the low cytotoxicity exhibited by 1, which
is prone to quickly release the ethacrynato anion in aqueous
conditions (see above).
In principle, both the EA-CO2H and EA-(CO)O-
(CH2)2OH fragments might be generated inside the cells
following dissociation of LN and LP from 2−4.15,26 The most
pronounced effects on GST activity were observed for 2 (20%
inhibition) and 4 (30% inhibition) in A2780 cells and to a
lesser extent in A2780cisR cells, which indicates that the GSH/
GST detoxification system in A2780cisR cells is more active,
reflected by 2-fold higher IC50 values, compared to A2780 cells
(Table 3).
The ligand LN inhibits GST in A2780cisR cells slightly more
than in A2780 cells, which might contribute to the comparable
cytotoxicity in A2780 cells and A2780cisR cells, respectively
(Table 3). The intracellular GST inhibition values of 2−4 do
not correlate directly with the IC50 values (compare Table 3
and Figure 4), thus suggesting that the cytotoxicity of the
Table 1. Selected Bond Distances (Å) and Angles (deg) for 1
Os−C(23)
Os−C(25)
Os−C(28)
Os−O(5)
2.147(3)
2.195(4)
2.150(3)
2.072(2)
2.081(3)
1.224(4)
88.14(9)
77.35(10)
128.1(3)
Os−C(24)
Os−C(27)
Os−C(19)
Os−O(6)
O(1)−C(1)
C(1)−C(2)
O(5)−Os−O(1)
Os−O(1)−C(1)
O(1)−C(1)−C(2)
2.179(4)
2.176(3)
2.188(3)
2.081(2)
1.286(4)
1.521(5)
80.59(10)
126.5(2)
114.0(3)
Os−O(1)
O(2)−C(1)
O(5)−Os−O(6)
O(1)−Os−O(6)
O(1)−C(1)−O(2)
Table 2. Selected Bond Distances (Å) and Angles (deg) for 3
Os−C(22)
Os−C(27)
Os−C(30)
Os−Cl(1)
Os−N(1)
N(1)−Os−Cl(1)
2.193(4)
2.182(5)
2.180(5)
2.4231(11)
2.119(3)
82.20(10)
Os−C(26)
Os−C(28)
Os−C(31)
Os−Cl(2)
N(1)−Os−Cl(2)
Cl(2)−Os−Cl(1)
2.162(4)
2.192(4)
2.149(4)
2.4155(11)
86.62(10)
85.08(4)
According to NMR and UV−vis spectroscopy, this phenom-
enon does not appear to be inhibited in the presence of 0.1 M
NaCl.
It should be noted that the ligands LP and LN in 2 and 3 are
less prone to dissociation than that previously observed for the
analogous ruthenium complexes.13 Indeed, LP in 2 is
substantially inert toward dissociation (5% released after 72 h
in dmso/water and 12% released after 72 h in the chloride-
containing solution). Spectroscopic and conductivity studies
indicate that only minor amounts of 3 and 4 remain unchanged
in the dmso/water and dmso/water/NaCl solutions for 62−72
h and that the LN ligand and chloride ligand compete for the
exchange with the solvent in both cases. Interestingly, the
dissociation of the LN ligand from 3 and to a greater extent
from 4 is partially suppressed in the respective NaCl solutions.
Cytotoxicity Studies. The ability of complexes 1−4 to
inhibit cell growth was evaluated against the cisplatin-sensitive
A2780 and the cisplatin-resistant A2780cisR human ovarian
carcinoma cell lines and nontumoral immortalized human
embryonic kidney HEK-293 cells (Table 3). All the complexes
are considerably more cytotoxic than RAPTA-C on the two
cancer cell lines.
D
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b
Scheme 3. Compounds Detected in dmso/H2O and dmso/H2O/NaCl Solutions of 1−4 Maintained at 37 °C
a
b
1
Not soluble under the experimental conditions. % values are based on H NMR spectroscopy and refer to the identified compounds only.
Table 3. IC50 Values (μM) Determined for RAPTA-C,25 Ethacrynic Acid (EA-CO2H),13 EA-(CO)O(CH2)2OH, Cisplatin,
LP,13 LN,13 and 1−4 on Human Ovarian (A2780 and A2780cisR) Cancer Cells and Human Embryonic Kidney (HEK-293)
a
Cells after Incubation for 72 h
A2780
3
A2780cisR
5
HEK-293
>1000
RAPTA-C
230
40
270
53
EA-CO2H
EA-(CO)O(CH2)2OH
2.0 0.1
3.8 0.7
3.3 0.5
LP
9
13
43
1
2
6
40
11
69
4
1
2
38
7
9
1
4
LN
1
40
2
13 2 (11 2)
20 5 (15 4)
26 8 (26 2)
21 2 (13 4)
13 1 (16 3)
3
8
1 (13 3)
3
4
17
34
25
1
3
25
9
2
2
cisplatin
0.9 0.1
a
Values are given as the mean SD. Values of the corresponding ruthenium analogues of 2 and 3 are given in parentheses.
compounds may only be partially attributed to GST inhibition.
Indeed, these results are consistent with a previous mechanistic
study on a bifunctional arene ruthenium(II)−ethacrynic acid
compound, indicating that the EA unit inhibits GSTs, releasing
the organometallic fragment, which leads to a surge of cell
death.15a
analogues are known, whereas 1 and 4 are unique in that no
ruthenium congeners have been reported. Compounds 2 and 3
are, as expected, more stable than the ruthenium analogues, in
dmso/water and dmso/water/NaCl solutions. To within
experimental error, there is no significant trend indicating
that complexes of one metal (Ru or Os) are substantially more
cytotoxic than the analogues of the other one, as previously
noted.27 The complex with the most stable Os−EA ligand
bond, i.e., 2, in which the ethacrynic acid entity is connected to
the osmium(II) ion via a P-donor ligand, is not the most
cytotoxic compound, indicating that dissociation of the
CONCLUSIONS
■
The first osmium complexes containing ethacrynic acid, a
broad-acting inhibitor of GSTs, have been synthesized and
characterized. For two of the complexes, 2 and 3, ruthenium
E
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by slow diffusion of pentane into a CH2Cl2 solution of 1. Anal. Calcd
for C28H32Cl2O6Os: C, 46.34; H, 4.44. Found: C, 46.34; H, 4.28. ESI-
MS(+): m/z found 749.106 [M + Na]+, calcd for C28H32Cl2NaO6Os+
749.108; the isotopic pattern fits well the calculated one. IR (solid
state) ν: 2966w, 2936w, 2912w, 2876w, 1660m, 1644s, 1587m,
1575m, 1556m, 1518s, 1469m, 1434w, 1383m, 1358vs, 1343w, 1285m,
1270m, 1265s, 1244w, 1219w, 1197w, 1146w, 1120w, 1083w, 1060s,
1031w, 1006m, 962w, 938w, 925w, 891w, 864m, 823w, 803s, 785m,
757w, 729s, 684w, 659w, 633m, 610m cm−1. UV−vis (CH2Cl2): λmax
/
nm (ε/M−1cm−1) = 263 (7.4 × 103), 327 (1.9 × 103). ΛM (CH2Cl2) =
0.12 S·cm2·mol−1. 1H NMR (CD2Cl2): δ = 7.01 (d, 1H, 3JHH = 8.6 Hz,
C11−H); 6.59 (d, 1H, 3JHH = 8.6 Hz, C10−H); 6.06, 5.82 (d, 4H, 3JHH
= 5.6 Hz, arom CHcymene); 5.91, 5.58 (m, 2H, C4−H); 5.04 (s, 1H,
3
CHacac); 4.55 (s, 2H, C12−H); 2.64 (sept, 1H, JHH = 6.9 Hz,
CHMe2); 2.43 (q, 2H, 3JHH = 7.4 Hz, C2−H); 2.17 (s, 3H, MeC6H4);
1.80 (s, 6H, Meacac); 1.24 (d, 6H, 3JHH = 6.9 Hz, CHMe2); 1.12 (t, 3H,
3JHH = 7.4 Hz, C1−H). 13C{1H} NMR (CD2Cl2): δ = 196.3 (C5);
185.8 (COacac); 172.5 (C13); 157.0 (C9); 150.8 (C3); 132.5 (C8);
131.0 (C7); 128.3 (C4); 127.3 (C11); 122.3 (C6); 110.6 (C10); 99.2
(CHacac); 88.6 (CCHMe2); 88.0 (CMecymene); 73.2, 68.5 (arom
CHcymene); 67.7 (C12); 32.0 (CHMe2); 27.0 (Meacac); 24.1 (C2);
22.9 (CHMe2); 18.4 (MeC6H4); 12.8 (C1).
Figure 4. Residual GST activity in A2780 and A2780cisR cells,
expressed as % activity of control, after incubation at IC50
concentrations for 18 h. The changes in fluorescence over a 20 min
period were used to calculate GST activities.
a
Chart 1. Os(η6-p-cymene)(κ(O,O′)-acac)(κ(O)-O2C-EA), 1
bioactive EA group from the metal ion could be important in
the mechanism of drug action. Indeed, the antiproliferative
activity of 2 may be attributed predominantly to the ethacrynic
acid-derived ligand, as their IC50 values are similar. This could
explain why there is not a direct correlation between
cytotoxicity and inhibition of intracellular GSTs, as the data
suggest that GSTs are relevant targets, but not exclusive targets.
Finally, incorporation of the PTA ligand in 3 to afford 4 does
not improve the cytotoxicity or selectivity of the compounds,
presumably due to the major role of the ethacrynic acid-derived
ligand in the overall biological activity observed.
a
Numbering refers to carbon atoms.
S y n t h e s i s o f O s ( η 6 - p - c y m e n e ) C l 2 ( 2 - ( ( 4 -
(diphenylphosphanyl)benzyl)oxy)ethyl-2-(2,3-dichloro-4-(2-
methylenebutanoyl)phenoxy)acetato), 2. Compound [Os(η6-p-
cymene)Cl2]2 (0.137 g, 0.173 mmol) was added to a solution of LP
(0.265 g, 0.417 mmol) in CH2Cl2 (10 mL). The resulting orange
solution was left stirring for 3 h at room temperature, then the volatile
substances were removed under vacuum to afford an orange, foamy
residue. This solid was dissolved into a small volume of CH2Cl2 and
loaded on top of a silica column (h 4.5 cm, d 2.5 cm). Impurities were
eluted with CH2Cl2; then an orange band was eluted with CH2Cl2/
THF (1:1 v/v). Complex 2 was finally obtained as an orange powder
after solvent removal under vacuum (50 °C). Yield: 0.319 g (90%).
Anal. Calcd for C44H43Cl4O6OsP: C, 51.27; H, 4.20. Found: C, 51.08;
H, 4.33. ESI-MS(+): m/z found 1053.097 [M + Na]+, calcd for
C44H43Cl4NaO6OsP+ 1053.105; the isotopic pattern fits well the
calculated one. IR (solid state) ν: 3058w, 2963w, 2957w, 1759m,
1720s, 1662m, 1584m, 1568w, 1468m, 1435m, 1391w, 1384m, 1339w,
1263s, 1187s, 1111m, 1079vs, 1030m, 1017m, 1000m, 948w, 895w,
858w, 800m, 762m, 749m, 721m, 696vs, 632w, 565w, 541s, 519vs
cm−1. UV−vis (CH2Cl2): λmax/nm (ε/M−1cm−1) = 250 (2.3 × 104),
322 (1.9 × 103). ΛM (CH2Cl2) = 0.25 S·cm2·mol−1. 1H NMR
(CD2Cl2): δ = 7.91−7.73, 7.44 (14H, C18−H + C18′−H + C19−H +
EXPERIMENTAL SECTION
■
OsCl3·3H2O (∼54% purity) was purchased from Heraeus, while the
organic reactants were purchased from Acros Organics and were of the
highest purity available. Solvents were purchased from Scharlau and
then obtained oxygen- and water-free with an MBraun solvent
purification apparatus. The ligands PTA,17 LP,13 and LN13 and the
complexes [OsCl2(η6-p-cymene)]2,28 Os(η6-p-cymene)(κ(C)-acac)(κ-
(O,O′)-acac),16 and OsCl2(η6-p-cymene)(PTA)18 were prepared
according to the respective literature methods. NMR spectra were
recorded on either a Bruker ARX 300 MHz or a Bruker Avance 300
MHz instrument. Chemical shifts (expressed in parts per million) are
referenced to the residual solvent peaks (1H, 13C{1H}) or to external
standard (31P{1H} to 85% H3PO4). Coupling constants are given in
Hz. NMR assignments refer to Charts 1−5 for the numbering of
carbon atoms. Conductivity measurements were carried out using a
Eutech Con 700 instrument (cell constant = 1.0 cm−1).29 UV−vis
spectra were recorded with an Ultraspec 2100 Pro spectrophotometer.
Attenuated total reflection infrared spectra (ATR-IR) of solid samples
were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer.
C, H, and N analyses were carried out with a PerkinElmer 2400
CHNS/O analyzer. High-resolution electrospray mass spectra
(HRMS) were acquired using a MicroTOF-Q hybrid quadrupole
time-of-flight spectrometer (Bruker Daltonics, Bremen, Germany).
Synthesis of Os(η6-p-cymene)(acetylacetonato)(2-(2,3-di-
chloro-4-(2-ethylenebutanoyl)phenoxy)acetato), 1. A solution
of Os(η6-p-cymene)(κ(C)-acac)(κ(O,O′)-acac) (0.097 g, 0.19 mmol)
in CH2Cl2 (5 mL) was treated with EA-CO2H (0.069 g, 0.23 mmol)
and then left stirring at room temperature for 24 h. The solvent was
removed under vacuum to obtain a red, oily residue. Repeated
washings with Et2O (3 × 10 mL) afforded 1 as a bright yellow powder.
Yield: 0.103 g, 75%. Crystals suitable for X-ray analysis were obtained
3
C19′−H + Ph); 7.13 (d, 1H, JHH = 8.5 Hz, C11−H), 6.84 (d, 1H,
3JHH = 8.5 Hz, C10−H); 5.95, 5.58 (m, 2H, C4−H); 5.43, 5.19 (d, 4H,
3JHH = 5.6 Hz, arom CHcymene); 4.80 (s, 2H, C12−H); 4.54 (s, 4H,
C14−H + C15−H); 2.69 (sept, 1H, 3JHH = 6.9 Hz, CHMe2); 2.45 (q,
2H, 3JHH = 7.4 Hz, C2−H); 1.98 (s, 3H, MeC6H4); 1.17 (d, 6H, 3JHH
=
6.8 Hz, CHMe2); 1.14 (t, 3H, JHH = 7.2 Hz, C1−H). 13C{1H} NMR
(CD2Cl2): δ = 196.3 (C5); 168.4 (C13); 166.4 (C16); 156.1 (C9);
151.0 (C3); 140.7, 140.1 (m, C20 + P-C); 135.5−135.3 (m, C19 +
C19′ + arom CHPh); 134.0 (C8); 133.4 (C7); 131.4 (C17); 131.3
(arom CHPh); 129.4 (C4); 129.2, 128.9 (m, C18 + C18′ + arom
CHPh); 127.7 (C11); 123.7 (C6); 111.7 (C10); 103.9 (CCHMe2);
3
F
DOI: 10.1021/acs.organomet.6b00197
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
2
90.0 (arom CMe); 81.4 (d, JPC = 2.8 Hz, arom CHcymene); 81.1 (d,
2JPC = 4.6 Hz, arom CHcymene); 66.9 (C12); 64.1, 63.3 (C14 + C15);
31.0 (CHMe2); 24.2 (C2); 22.8 (CHMe2); 18.4 (MeC6H4); 13.0 (C1).
31P NMR (CD2Cl2): δ = −12.1.
with AgBF4 (0.048 g, 0.25 mmol). The mixture was stirred for 72 h at
room temperature under protection from light. The final mixture was
filtered through Celite; then the volatiles substances were removed
under vacuum. The residue was precipitated with Et2O (30 mL) and
then washed with hexane (20 mL). Complex 4 was obtained as a pale
yellow solid. Yield: 0.156
g (81%). Anal. Calcd for
a
Chart 2. Os(η6-p-cymene)Cl2(LP), 2
C37H45BCl3F4N4O6OsP: C, 42.08; H, 4.29; N, 5.30. Found: C,
42.70; H, 4.36; N, 5.47. ESI-MS(+): m/z found 969.177 [M]+, calcd
for C37H45Cl3N4O6OsP+ 969.175; the isotopic pattern fits well the
calculated one. IR (solid state) ν: 2967w, 2936w, 2880w, 1757m-sh,
1733s, 1662m, 1585m, 1469m, 1444w-m, 1385w-m, 1288s, 1242m,
1197s, 1138m, 1117s-sh, 1054vs-br, 973s, 947s, 896m, 803m, 766w-m,
744s, 695w-m, 606w, 577s cm−1. UV−vis (CH2Cl2): λmax/nm (ε/
M−1cm−1) = 258 (1.4 × 104), 317 (5.2 × 103). ΛM (CH2Cl2) = 9.32 S·
1
cm2·mol−1. H NMR (CD2Cl2): δ = 9.55 (s, 1H, C18−H); 9.09 (d,
3
3
1H, JHH = 5.5 Hz, C19−H); 8.43 (d, 1H, JHH = 8.0 Hz, C21−H);
7.74 (m, 1H, C20−H); 7.15 (d, 1H, 3JHH = 8.6 Hz, C11−H); 6.89 (d,
3
3
a
1H, JHH = 8.6 Hz, C10−H); 6.03, 5.79 (d, 4H, JHH = 5.7 Hz, arom
Numbering refers to carbon atoms.
2
CHcymene); 5.96, 5.58 (m, 2H, C4−H); 4.88 (d, 2H, JHH = 1.5 Hz
C12−H); 4.71−4.58 (m, 6H, NCH2N); 4.62 (s, 4H, C14 + C15);
Synthesis of Os(η6-p-cymene)Cl2(2-(2-(2,3-dichloro-4-(2-
methylenebutanoyl)phenoxy)acetoxy)ethylnicotinato), 3. This
compound was prepared by the same procedure described for the
synthesis of 2, from [OsCl2(η6-p-cymene)]2 (0.100 g, 0.126 mmol)
and LN (0.139 g, 0.307 mmol). Yellow solid, yield: 0.153 g (75%).
Crystals of 3·CH2Cl2·1/2[1,4-C6H4(OH)2] suitable for X-ray analysis
were obtained by slow diffusion of hexane into a CH2Cl2 solution of 3,
with a few grains of hydroquinone added. Anal. Calcd for
C31H33Cl4NO6Os: C, 43.93; H, 3.92; N, 1.65. Found: C, 44.25; H,
4.10; N, 1.62. ESI-MS(+): m/z found 812.096 [M − Cl]+, calcd for
C31H33Cl3NO6Os+ 812.099; the isotopic pattern fits well the calculated
one. IR (solid state) ν: 2964w, 2940w, 2893w, 1751m, 1728s, 1662m,
1584m, 1467w, 1432w, 1383w, 1339w, 1286vs, 1194vs, 1137s, 1116s,
1079vs, 1062s, 1025m, 1000m, 941w, 891w, 876w, 820w, 803m, 767w,
745s, 702w, 689m, 637w cm−1. UV−vis (CH2Cl2): λmax/nm (ε/
M−1cm−1) = 259 (1.0 × 104), 282 (6.8 × 103), 322 (4.3 × 103). ΛM
2
3
4.19 (q, 6H, JHP = 15.4 Hz, NCH2P); 2.54 (sept, 1H, JHH = 6.6 Hz,
CHMe2); 2.44 (q, 2H, 3JHH = 7.3 Hz, C2−H); 2.05 (s, 3H, MeC6H4);
1.26 (d, 6H, 3JHH = 6.8 Hz, CHMe2); 1.13 (t, 3H, 3JHH = 7.4 Hz, C1−
H). 13C{1H} NMR (CD2Cl2): δ = 196.1 (C5); 168.4 (C13); 163.2
(C16); 161.2 (C19); 156.7 (C18); 155.9 (C9); 150.8 (C3); 140.8
(C21); 134.6 (C8); 131.8 (C7); 129.2 (C4); 129.0 (C17); 127.6
(C11); 127.6 (C20); 123.3 (C6); 111.7 (C10); 104.3 (CCHMe2);
95.0 (arom CMe); 75.9, 73.4 (arom CHcymene); 72.9 (NCH2N); 66.8
(C12); 64.5, 63.5 (C14, C15); 49.5 (d, 1JCP = 21.1 Hz, NCH2P); 31.6
(CHMe2); 24.0 (C2); 23.0, 22.4 (CHMe2); 18.4 (MeC6H4); 12.8
(C1). 31P NMR (CD2Cl2): δ = −77.4.
a
Chart 4. [(η6-p-Cymene)OsCl(LN)(PTA)][BF4], 4
(CH2Cl2) = 0.41 S·cm2·mol−1. H NMR (CD2Cl2): δ = 9.51 (s, 1H,
1
C18−H); 9.13 (m, 1H, C19−H); 8.29 (m, 1H, C21−H); 7.47 (m, 1H,
3
3
C20−H); 7.18 (d, 1H, JHH = 8.6 Hz, C11−H); 6.95 (d, 1H, JHH
=
=
8.6 Hz, C10−H); 5.99, 5.61 (m, 2H, C4−H); 5.86, 5.64 (d, 4H, 3JHH
5.7 Hz, arom CHcymene); 4.94 (s, 2H, C12−H); 4.62 (s, 4H, C14 +
C15); 2.80 (sept, 1H, 3JHH = 6.9 Hz, CHMe2); 2.47 (q, 2H, 3JHH = 7.4
3
Hz, C2−H); 2.03 (s, 3H, MeC6H4); 1.31 (d, 6H, JHH = 6.9 Hz,
CHMe2); 1.16 (t, 3H, JHH = 7.4 Hz, C1−H). 13C{1H} NMR
3
(CD2Cl2): δ = 196.0 (C5); 168.3 (C13); 163.7 (C16); 158.1 (C19);
155.9 (C18); 155.8 (C9); 150.7 (C3); 139.1 (C21); 134.3 (C8); 131.6
(C7); 129.1 (C4); 127.6 (C11); 127.4 (C17); 125.0 (C20); 123.3
(C6); 111.5 (C10); 94.1 (CCHMe2); 89.4 (arom CMe); 75.9, 73.4
(arom CHcymene); 66.6 (C12); 63.9, 63.3 (C14, C15); 31.5 (CHMe2);
23.9 (C2); 22.8 (CHMe2); 18.4 (MeC6H4); 12.8 (C1).
a
Numbering refers to carbon atoms.
Synthesis of EA-CO2K. KN(SiMe3)2 (0.36 mL, 0.33 mmol) was
added to a solution of EA-CO2H (0.100 g, 0.330 mmol) in Et2O (10
mL), under a nitrogen atmosphere. The mixture was left stirring for an
additional 24 h. The resulting colorless solid was separated, washed
with pentane (20 mL), and dried under vacuum. Yield: 0.075 g, 67%.
Anal. Calcd for C13H11Cl2KO4: C, 45.76; H, 3.25; Cl, 20.78. Found: C,
45.71; H, 3.18; Cl, 20.82. IR (solid state): ν = 2965w, 2938w, 2922w,
2878w, 2550w, 1657m (CO), 1598vs (COO), 1586vs, 1554m,
1471m-s, 1417s, 1385m, 1339m, 1267m-s, 1202m, 1120w, 1054vs,
1002m, 941m, 908w, 824w, 799s, 760m, 737m, 724m, 698w, 680w,
Synthesis of Os(η6-p-cymene)Cl(2-(2-(2,3-dichloro-4-(2-
methylenebutanoyl)phenoxy)acetoxy)ethylnicotinato)(1,3,5-
triaza-7-phosphaadamantane)tetrafluoroborato, 4. A stirred
solution of OsCl2(η6-p-cymene)(PTA) (0.100 g, 0.181 mmol) in
CH2Cl2 (6 mL) was treated with LN (0.098 g, 0.216 mmol) and then
a
Chart 3. (η6-p-Cymene)OsCl2(LN), 3
627m, 590w, 570w, 531w cm−1. 1H NMR (D2O): δ = 7.26 (d, 3JHH
=
3
8.80 Hz, 1H, C10−H); 6.92 (d, JHH = 8.80 Hz, 1H, C11−H); 6.08,
5.69 (s, 2 H, C4−H); 4.58 (s, 2 H, C12−H); 2.36 (m, 2 H, C2−H);
1.06 (m, 3 H, C1−H). 13C NMR (D2O): δ = 199.2 (C5); 174.9
(C13); 156.1 (C9); 150.1 (C6); 131.8 (C7), 131.6 (C8); 130.3 (C3);
128.0 (C11); 122.0 (C4); 111.1 (C10); 67.8 (C12); 23.2 (C2); 12.0
(C1).
Stability Studies. General Procedure. Complexes 1−4 were
dissolved in d6-dmso/D2O mixture (2.0 mL) ([Os] = 2.1 × 10−2 mol
L−1). An aliquot of the resulting solution (0.40 mL) was transferred
into a NMR tube, maintained at 37 °C, and analyzed by NMR
spectroscopy as a function of time. The remaining solution was diluted
up to 4.2 mL with the appropriate dmso/water mixture (final [Os] =
a
Numbering refers to carbon atoms.
G
DOI: 10.1021/acs.organomet.6b00197
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
Compound 2. ΛM (dmso/water, 9:1) = 8.9 S·cm2·mol−1 (t = 5 min
to 72 h). UV−vis (dmso/water, 9:1), λmax/nm (ε/M−1·cm−1) = 322
(2.8 × 103), 423 (4.3 × 102) (t = 5 min to 72 h). UV−vis (dmso/
water, 9:1, + NaCl 0.1 M) = 320 (2.8 × 103), 423 (4.0 × 102) (t = 5
a
Chart 5. EA-CO2K
1
min); 320 (2.4 × 103), 423 (4.0 × 102) (t = 72 h). H NMR (d6-
dmso/D2O, 9:1, or d6-dmso/D2O, 9:1, + NaCl 0.1 M): δ = 7.85 (m,
2H), 7.79 (m, 2H), 7.64 (m, 4H), 7.43 (m, 6H), 7.18, 7.11 (d, 2H),
6.00 (s, 1H), 5.50, 5.37 (d, 4H), 5.47 (s, 1H), 5.01 (s, 2H), 4.45 (s,
4H), 2.41 (m, 1H), 2.31 (q, 2H), 1.83 (s, 3H), 1.02 (t, 3H), 0.98 (d,
6H) (t = 5 min to 72 h). 13C{1H} NMR (d6-dmso/D2O, 9:1, or d6-
dmso/D2O, 9:1, + NaCl 0.1 M): δ = 195.7, 168.4, 165.7, 155.5, 149.7,
134.8−134.5, 133.3, 133.0, 132.9, 131.1, 130.1, 128.6−128.4, 127.8,
112.1, 88.9, 81.3, 80.3, 66.0, 63.4, 63.2, 30.1, 23.3, 22.1, 17.5, 12.7 (t =
5 min to 72 h). 31P NMR (d6-dmso/D2O, 9:1): δ = −12.8 (t = 5 min
to 22 h); δ = −12.8 (major, 2a), 26.6 (ca. 6%, LPO) (t = 51−72 h).
31P NMR (d6-dmso/D2O, 9:1, + NaCl 0.1 M): δ = −12.8 (major, 2a),
26.6 (ca. 9%, LPO) (t = 5 min to 23 h); −12.8 (major, 2a), −6.3
(LP, ca. 7%), 26.6 (ca. 5%, LPO) (t = 72 h).
a
Numbering refers to carbon atoms.
7.8 × 10−3 mol L−1), maintained at 37 °C, and analyzed by
conductivity measurements and UV−vis spectroscopy as a function of
time (t). Parallel analyses were carried out on dmso/water mixtures
prepared by the same procedure described above and with the addition
of NaCl (ca. 0.1 M). CH3OD (2.5 × 10−2 mol L−1) was used as a
Compound 3. ΛM (dmso/water, 9:1) = 2.2 S·cm2·mol−1 (t = 5
min); 3.2 S·cm2·mol−1 (t = 50 min); 3.4 S·cm2·mol−1 (t = 6 h); 3.3 S·
cm2·mol−1 (t = 24 h); 3.9 S·cm2·mol−1 (t = 114 h). UV−vis (dmso/
water, 9:1), λmax/nm (ε/M−1·cm−1) = 322 (2.2 × 103), 418 (3.6 × 102)
(t = 5 min to 72 h). UV−vis (dmso/water, 7:3, + NaCl 0.1 M) = 322
(1.6 × 103), 418 (2.4 × 102) (t = 5 min); 322 (1.8 × 103), 418 (2.4 ×
1
reference for the H NMR spectra. For NMR assignments refer to
Scheme 3. The NMR spectra of LP, LN, EA-CO2H, EA-C(
O)O(CH2)2OH, PTA, and acacH were recorded in d6-dmso/D2O
(9:1 v/v) and used for comparison for NMR assignments. FeCp2 was
selected as an organometallic standard for conductivity data (ΛM
=
0.69 S·cm2·mol−1 in 9:1 dmso/H2O at 37 °C).
1
102) (t = 72 h). H NMR (d6-dmso/D2O, 9:1): δ = 9.30 (s, 3 + 3a),
Compound 1. ΛM (dmso/water, 9:1) = 3.4 S·cm2·mol−1 (t = 5
min); 5.3 S·cm2·mol−1 (t = 50 min); 4.6 S·cm2·mol−1 (t = 6 h); 5.3 S·
cm2·mol−1 (t = 24 h); 3.8 S·cm2·mol−1 (t = 114 h). UV−vis (dmso/
water, 9:1), λmax/nm (ε/M−1cm−1) = 327 (2.6 × 103) (t = 2−114 h).
UV−vis (dmso/water, 9:1, + NaCl 0.1 M) = 330 (2.7 × 103) (t = 2 h);
9.04 (s, LN), 8.99 (t, 3a), 8.98 (d, 3), 8.78 (d, LN), 8.47 (d, 3a), 8.30
(d, 3), 8.24 (d, LN), 7.77 (m, 3a), 7.59 (dd, 3), 7.54 (dd, LN), 7.23 (d,
3 + 3a), 7.16 (d, LN), 7.14 (m, 3a), 7.10 (d, LN), 6.38 (d, 3a), 6.25
(d, 3a), 6.17 (d, 3a), 6.08 (d, 3a), 6.04 (d, 3b), 6.03 (s, LN), 5.94 (d,
3b), 5.92 (d, 3), 5.74 (d, 3), 5.52 (s, 3a), 5.49 (s, LN), 5.04 (s, LN),
4.50 (s, LN), 2.70 (m, 3b), 2.33 (q, LN), 2.14 (s, 3a), 2.11 (s, 3b),
1.89 (s, 3), 1.17 (d, 3b), 1.04 (t, LN). Ratio 3:3a:3b:LN = 13:0:87:88
(t = 5 min), 10:7:79:82 (t = 1 h), 10:5:83:87 (t = 3 h), 11:6:82:77 (t =
330 (2.1 × 103) (t = 72 h). H NMR (d6-dmso/D2O, 9:1): δ = 7.23
1
−
−
(d, EACO2 ), 7.16 (d, 1), 6.91 (d, EACO2 ), 6.67 (d, 1), 6.24 (s, 1a),
6.13 (s, 1a), 6.03 (s, 1 + EACO2 ), 5.86 (s, 1a), 5.55 (s, EACO2 ),
5.54 (s, 1), 4.97 (s, 1), 4.52 (s, 1), 4.47 (s, EACO2 ), 2.33 (q, 1 +
−
−
−
1
−
48 h), 11:6:83:81 (t = 114 h). H NMR (d6-dmso/D2O, 7:3, + NaCl
EACO2 ), 1.95 (s, 1), 1.69 (s, 1 + 1a), 1.20 (d, 1), 1.14 (d, 1a), 1.04
−
−
(t, 1 + EACO2 ). Ratio 1:EACO2 :1a = 1:1:1 (t = 1−72 h). 1H NMR
0.1 M): 3, 3a, 3b, and LN, ratio 25:10:70:60 (t = 5 min to 72 h).
13C{1H} NMR (d6-dmso/D2O, 9:1): δ = 195.7 (LN), 168.4 (LN),
165.1 (LN), 155.5 (LN), 154.1 (LN), 150.4 (LN), 149.7 (LN), 137.5
(LN), 133.0 (LN), 130.1 (LN), 127.8 (LN), 125.8 (LN), 124.4 (LN),
−
(d6-dmso/D2O, 9:1, + NaCl 0.1 M): 1, EACO2 , and 1b [δ = 6.00 (d),
5.78 (d), 5.31 (s), 2.08 (s), 1.80 (s)], ratio 2:8:7 (t = 5 min), 0:5:3 (t =
2−72 h).
Table 4. Crystal Data and Measurement Details for 1 and 3·CH2Cl2·1/2[1,4-C6H4(OH)2]
1
3·CH2Cl2·1/2[1,4-C6H4(OH)2]
formula
C28H32Cl2O6Os
725.64
C35H38Cl6NO7Os
987.56
fw
T, K
100(2)
100(2)
λ, Å
0.71073
0.71073
cryst syst
orthorhombic
Pbca
monoclinic
P21/n
space group
a, Å
19.6723(19)
11.5289(11)
24.095(2)
17.0370(17)
13.3798(13)
17.2905(17)
103.3800(10)
3834.4(7)
4
b, Å
c, Å
β, °
cell volume, Å3
5464.8(9)
8
Z
Dc, g·cm−3
μ, mm−1
1.764
1.711
4.903
3.791
F(000)
2864
1956
Crystal size, mm
θ limits, deg
reflns collected
independent reflns
data/restraints/params
goodness of fit on F2
R1 (I > 2σ(I))
wR2 (all data)
largest diff peak and hole, e·Å−3
0.10 × 0.09 × 0.06
1.7−29.6
57 473
0.19 × 0.10 × 0.06
1.9−28.6
45 640
7332
9208
7332/0/340
1.011
9208/4/457
1.001
0.0299
0.0389
0.0621
0.0821
1.501, −1.111
1.146, −1.313
H
DOI: 10.1021/acs.organomet.6b00197
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
121.5 (LN), 112.0 (LN), 98.4 (3b), 93.6 (3b), 78.5 (3b), 78.4 (3b),
65.9 (LN), 63.5 (LN), 63.1 (LN), 30.3 (3b), 23.3 (LN), ): 22.1 (3b),
18.1 (3b), 12.7 (LN) (t = 72 h).
(Molecular Devices), and the fraction of surviving cells was calculated
from the absorbance of untreated control cells. Evaluation is based on
means from three independent experiments, each comprising four
testings per concentration level.
Compound 4. ΛM (dmso/water, 9:1) = 67 S·cm2·mol−1 (t = 5
min); 60 S·cm2·mol−1 (t = 2 h); 65 S·cm2·mol−1 (t = 24 h); 71 S·cm2·
mol−1 (t = 62 h). UV−vis (dmso/water, 9:1), λmax/nm (ε/M−1·cm−1)
= 317 (8.2 × 103) (t = 5 min); 317 (4.1 × 103) (t = 62 h). UV−vis
(dmso/water, 1:1, + NaCl 0.1 M) = 317 (8.3 × 103) (t = 5 min); 317
(6.9 × 103) (t = 62 h). 1H NMR (d6-dmso/D2O, 9:1): δ = 9.31 (s, 4a),
9.04 (m, LN), 8.92 (d, 4a), 8.75 (m, LN), 8.41 (d, 4a), 8.24 (m, LN),
7.62 (m, 4a), 7.54 (m, LN), 7.24 (d, 4a), 7.16 (d, LN), 7.11 (d, 4a),
7.09 (d, LN), 6.57 (d, 4b), 6.39 (d, 4b), 6.27 (d, 4b), 6.15 (m, 4a),
6.07 (d, 4b), 6.06 (m, 4a), 6.03 (s, LN), 5.96 (d, 4a), 5.51 (s, 4a), 5.48
(s, LN), 5.02 (s, 4a), 4.50 (m-br, 4a + LN), 4.26 (q, 4b), 4.06 (q, 4a),
2.43 (m, 4a), 2.33 (q, 4a), 2.25 (s, 4b), 1.93 (s, 4a), 1.15 (d, 4a), 1.04
(t, 4a). Ratio 4a:4b:LN = 100:0:0 (t = 5 min), 45:50:55 (t = 24 h),
GST Activity Assay. A2780 and A2780cisR cells were plated in six-
well plates (2 × 106 cells/well) and incubated for 24 h in a CO2
incubator at 37 °C. The cells were then exposed to compounds LN,
LP, EA-(CO)O(CH2)2OH, and 1−4 for 18 h, at final
concentrations according to the IC50 concentration for each respective
cell line. The cells were then washed twice with PBS, harvested, lysed
by repetitive freeze−thaw cycles, and centrifuged at 10000g for 15 min
at 4 °C. The supernatants were used for the analysis of GST activity
according to a fluorometric GST detection kit (Abnova). The changes
in fluorescent intensities at Ex/Em 380/460 nm were recorded in a
kinetic mode, every 5 min over 60 min, on a microplate reader
(Molecular Devices). Two independent experiments were performed
in which the GST activities were measured in duplicate. The GST
activity expressed as U/min/mL per mg protein was converted to %
activity of control (nontreated cells). The GST activity of the test
samples was calculated by applying the equation ΔRFU = RFU2 −
RFU1 to the GST standard curve to get B [mU] during the reaction
time (ΔT = T2 − T1). The sample GST activity is calculated by the
following formula: sample GST activity = B/(ΔT × V) × dilution
factor [mU/min/mL], where B is sample GST activity from the GST
standard curve [mU], ΔT is the reaction time (min), and V is the
sample volume added into the reaction well [mL].
1
23:60:63 (t = 46 h), 17:65:67 (t = 62 h). H NMR (d6-dmso/D2O,
1:1, + NaCl 0.1 M): 4a, 4b, and 4c [δ = 8.92 (m), 8.64 (d), 8.21 (d),
7.49 (m), 7.01 (d), 6.92 (d), 6.97, 6.02, 5.98, 5.90, 5.41 (s), 4.91 (s),
2.01 (s)]. Ratio 100:0:0 (t = 5 min), 100:31:11 (t = 24 h), 100:70:20
(t = 46 h), 100:100:20 (t = 62 h). LN is not soluble in these
conditions. 13C{1H} NMR (d6-dmso/D2O, 9:1): δ = LN and 4a [δ =
86.6, 83.8, 71.8, 51.3, 30.8, 22.2, 22.0, 18.6] (t = 62 h). 31P NMR (d6-
dmso/D2O, 9:1): δ = −76.5 (4a), −74.8 (4b). Ratio 100:0 (t = 2 h),
42:58 (t = 24 h), 23:76 (t = 46 h), 17:82 (t = 62 h). 31P NMR (d6-
dmso/D2O, 1:1, + NaCl 0.1 M): 4a, 4b, and 4c [δ = −72.0]. Ratio
100:0:0 (t = 2 h), 64:35:0 (t = 24 h), 46:46:7 (t = 46 h), 35:47:12 (t =
62 h).
Protein Determination. The protein concentration was deter-
mined by a Bradford assay (Bio-Rad) using BSA as a standard.
X-ray Crystallography. Crystal data and collection details for 1
and 3·CH2Cl2·1/2[1,4-C6H4(OH)2] are reported in Table 4. X-ray
data were collected on Bruker Smart APEX CCD (3) and Smart Apex
CCD DUO (1) diffractometers equipped with a normal focus, 2.4 kW
sealed tube source (molybdenum radiation, λ = 0.710 73 Å), operating
at 50 kV and 30 (1) or 40 (3) mA. Data were collected over the
complete sphere by a combination of four sets. Each frame exposure
time was 30 s (1) or 10 s (3), covering 0.3° in ω. Data were corrected
for absorption by using a multiscan method applied with the Sadabs
program.30 The structures were solved by the Patterson method or by
direct methods. Refinement, by full-matrix least-squares on F2 with
SHELXL97,31 was similar for the two complexes, including isotropic
and subsequently anisotropic displacement parameters. The hydrogen
atoms were observed or calculated and refined freely or refined using a
restricted riding model. In the case of 3, a half-molecule of
hydroquinone (added to facilitate the crystallization, vide infra) and
one molecule of dichloromethane were observed in the asymmetric
unit. All the highest electronic residuals were observed in close
proximity to the Os centers and do not hold any chemical significance.
Cell Culture. Human A2780 and A2780cisR ovarian carcinoma
cells were obtained from the European Centre of Cell Cultures
(ECACC, UK). Nontumorigenic HEK-293 cells were obtained from
ATCC (Sigma, Switzerland). A2780 and A2780cisR were routinely
grown in RPMI 1640 GlutaMAX (Lifetechnologies, Switzerland),
while HEK-293 cells were grown in DMEM medium, both containing
10% heat-inactivated fetal bovine serum (FBS, Pan Biotech, Germany)
and 1% antibiotics (penicillin/streptomycin), at 37 °C and CO2 (5%).
Determination of Antiproliferative Activity. Cytotoxicity was
determined using the MTT assay (MTT = 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). Cells were seeded
in 96-well plates as monolayers with 100 μL of cell solution per well
and preincubated for 24 h in the cell culture medium. Compounds
were prepared as dmso solutions that were rapidly dissolved in the
culture medium and serially diluted to the appropriate concentration
to give a final dmso concentration of 0.5%. A 100 μL amount of the
drug solution was added to each well, and the plates were incubated
for another 72 h. Subsequently, MTT (5 mg/mL solution) was added
to the cells, and the plates were incubated for a further 4 h. The culture
medium was aspirated, and the purple formazan crystals formed by the
mitochondrial dehydrogenase activity of vital cells were dissolved in
dmso. The optical density, directly proportional to the number of
surviving cells, was quantified at 590 nm using a multiwell plate reader
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.organo-
met.6b00197. CCDC reference numbers 1421240 (1) and
1421241 (3) contain the supplementary crystallographic data
for the X-ray studies reported in this paper. These data can be
obtained free of charge at www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:
(internat.) + 44-1223/336-033; e-mail: deposit@ccdc.cam.ac.
uk).
Crystallographic data for 1 (CIF)
Crystallographic data for 3 (CIF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: maester@unizar.es.
*E-mail: fabio.marchetti1974@unipi.it.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the Swiss National Science Foundation, MINECO of
Spain (Projects CTQ2014-52799-P and CTQ2014-51912-
■
́
́
REDC), the Diputacion General de Aragon (E-35), the
European Social Fund (FSE and FEDER), and the University
of Pisa (PRA 2015) for financial support. M.P.G. (FPI grant)
thanks MINECO for funding.
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