Can substitution of chlorides enhance the cytotoxicity of vanadocene dichloride?

Article abstract of DOI:10.1002/ejic.201201505

A series of vanadocene complexes [Cp′₂V(L)][OTf] ₂ (Cp′ = η⁵-C₅H₅, η⁵-C₅H₄Me; L = phen, 5-NO₂-phen, 5-NH₂-phen, 4,7-Ph₂-phen) was prepared and characterized by mass spectrometry and EPR spectroscopy. Structures of two complexes that contain an N,N′-chelating ligand, [(η⁵-C₅H ₄Me)₂V(phen)][OTf]₂·0.5Me₂CO and [(η⁵-C₅H₅)₂V(5-NH ₂-phen)][OTf]₂, and the triflate intermediate [(η⁵-C₅H₅)₂V(OTf)₂] were further confirmed by X-ray diffraction analysis. The cytotoxicity study has shown that complexes that contain phenanthroline ligands have considerably higher in vitro activity toward human leukemia cells MOLT-4 and HL-60 than their parent dichlorides ([Cp′₂VCl₂]). The high activity of these complexes coheres with their higher stability in various aqueous media. This study has further shown that substitution of chloride ligands in the vanadocene dichloride has a considerably higher effect on cytotoxicity than expected on the basis of established mechanistic rules. Copyright

Full text of DOI:10.1002/ejic.201201505

FULL PAPER
DOI:10.1002/ejic.201201505
Can Substitution of Chlorides Enhance the Cytotoxicity of
Vanadocene Dichloride?
[a]
[a]
[b]
ˇ
Jaromír Vinklárek, Hana Hurychová, Jan Honzícek,*
[c]
[a]
[d]
ˇ
ˇ
ˇ
ˇ
ˇ
Lucie Sebestová, Zdenka Padelková, and Martina Rezácová
Keywords: Metallocenes / Vanadium / EPR spectroscopy / Cytotoxicity
A series of vanadocene complexes [CpЈ2V(L)][OTf]2 (CpЈ =
η5-C5H5, η5-C5H4Me; L = phen, 5-NO2-phen, 5-NH2-phen,
4,7-Ph2-phen) was prepared and characterized by mass spec-
trometry and EPR spectroscopy. Structures of two complexes
that contain an N,NЈ-chelating ligand, [(η5-C5H4Me)2V-
(phen)][OTf]2·0.5Me2CO and [(η5-C5H5)2V(5-NH2-phen)]-
[OTf]2, and the triflate intermediate [(η5-C5H5)2V(OTf)2]
were further confirmed by X-ray diffraction analysis. The
cytotoxicity study has shown that complexes that contain
phenanthroline ligands have considerably higher in vitro ac-
tivity toward human leukemia cells MOLT-4 and HL-60 than
their parent dichlorides ([CpЈ2VCl2]). The high activity of
these complexes coheres with their higher stability in various
aqueous media. This study has further shown that substitu-
tion of chloride ligands in the vanadocene dichloride has a
considerably higher effect on cytotoxicity than expected on
the basis of established mechanistic rules.
focused mainly on compounds that have been substituted
on the cyclopentadienyl rings because such an approach can
considerably enhance cytotoxicity, as was already evidenced
in various derivatives.[4] Recently, we have shown that 1 and
its ring-substituted and ansa-bridged derivatives are active
toward leukemia cells MOLT-4.[5] So far, only minor im-
provements have been achieved through the substitution of
the chlorides.[6] It is probably a result of the low hydrolytic
stability of the V–X bonds that results in the formation of
the same “active species” as their chloride parent. Although
the vanadocene framework is stable in various aqueous me-
dia, the hydrolytic chemistry of 1 is relatively complex.[7]
The aqua complex [Cp2V(OH2)2]2+ appears immediately af-
ter dissolution and reacts with carbonates and phosphates
to give chelate complexes [Cp2V(O,O-CO3)][8,9] and
[Cp2V(O,O-PO4H)],[10] respectively. Furthermore, several
structure types were described as the products of the reac-
tion with α-amino acids.[9,11,12] So far, it has been not con-
cluded which of the hydrolytic products is the “active spe-
cies” that reaches the target in the diseased cell.
Our focus on the phenanthroline derivatives follows the
previous study that reports that [Cp2V(phen)][OTf]2 (3) is
stable in phosphate-buffered saline (PBS) and that it shows
high permeability in the liposomal membranes.[13] The un-
usual electrochemical properties of the vanadocene com-
plexes that contain 2,2Ј-bipyridine ligands were described in
detail theoretically[14] and experimentally.[15] High stability,
water solubility, and permeability in membranes, which
were reported for the phenanthroline complexes, could
overcome the problems of the metallocene-based drugs that
are associated with medical applications and with transport
into the diseased cell, thus finally leading to an enhance-
Introduction
Despite our constantly deepening knowledge about pa-
thobiochemical mechanisms of various hematological ma-
lignancies, many leukemias remain incurable and have very
bad survival prognoses. Although targeted therapy, such as
tyrosin kinase inhibitors or specific monoclonal antibodies,
was a great revolution for the therapy of some types of leu-
kemias, others rely fully on classical chemotherapy.[1] For
example, the core of the treatment regimen of acute myeloid
leukemia has remained nearly unchanged for 40 years, and
the prognosis remains poor, mainly in elderly patients.
Therefore, constant research continues on novel cytostatics
that would provide fewer undesirable side effects while
maintaining potent antitumor activity.[2]
Vanadocene dichloride (1, [Cp2VCl2]; Cp = η5-C5H5) is
known as a potent antitumor agent with low general toxic-
ity.[3] In the last few years, drug design in this area has been
[a] Department of General and Inorganic Chemistry, Faculty of
Chemical Technology, University of Pardubice,
Studentská 573, 53210 Pardubice, Czech Republic
[b] Institute of Chemistry and Technology of Macromolecular
Materials, Faculty of Chemical Technology, University of
Pardubice,
Studentská 573, 53210 Pardubice, Czech Republic
Fax: +420-46-603-7068
E-mail: jan.honzicek@upce.cz
Homepage: http://www.upce.cz/en/fcht/uchtml.html
[c] Department of Biological and Biochemical Sciences, Faculty of
Chemical Technology, University of Pardubice,
Studentská 573, 53210 Pardubice, Czech Republic
[d] Department of Medical Biochemistry, Faculty of Medicine in
Hradec Králové, Charles University in Prague,
ˇ
Simkova 870, 50001 Hradec Králové, Czech Republic
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201201505.
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ment of their activity. This study reports the synthesis, char- 69.5ϫ10–4 cm–1, giso = 1.991), after about one hour, proved
acterization, and a detailed stability study of vanadocene– that the first reaction step is complete (compare spectra A
phenanthroline complexes. Their cytotoxicity was evaluated and B in Figure 1). The reaction mixture was filtered from
in vitro on human leukemia cells MOLT-4 and HL-60.
the precipitated silver chloride. The addition of 1,10-phen-
anthroline to the filtrate led to the appearance of the signal
of 3 (|Aiso| = 62.8ϫ10–4 cm–1, giso = 1.986) and slow forma-
tion of a brown precipitate. The reaction was complete after
18 h when the signal of triflate complex had disappeared
(see spectrum C in Figure 1). The precipitate of 3 was iso-
lated and characterized. The obtained analytical and spec-
troscopic data are in line with those published elsewhere.[13]
A similar reaction process was observed for substituted
vanadocene dichloride 1a and for substituted phenan-
throline ligands (5-NO2-phen and 5-NH2-phen). In the case
of reactions with 4,7-Ph2-phen only the products do not
precipitate from solution because compounds 6 and 6a are
much more soluble in THF than the other derivatives.
The triflate intermediates 2 and 2a were isolated from
the reaction mixture and characterized by elemental analy-
sis. We have checked that the species were not changed dur-
ing isolation. The samples dissolved in acetone display the
EPR spectra with the same |Aiso| and giso parameters as the
species in the reaction mixture. The |Aiso| values of com-
pounds 2 and 2a are in range of the vanadocene complexes
that contain two monodentate O-donor ligands (73.5–
74.2ϫ10–4 cm–1).[16] It suggests that both triflate ligands
are coordinated through an oxygen atom rather than by
forming some type of chelate or ionic structure. The X-ray
analysis of compound 2 proves that this structure remains
in the solid state (see Figure 2).
Results and Discussion
Preparation and Characterization
Phenanthroline complexes [Cp2V(L)][OTf]2 (L = 5-NO2-
phen (4), 5-NH2-phen (5), 4,7-Ph2-phen (6)) and [(η5-
C5H4Me)2V(L)][OTf]2 (L = phen (3a), 5-NO2-phen (4a), 5-
NH2-phen (5a), 4,7-Ph2-phen (6a)) were prepared in a two-
step procedure according to the method developed for
[Cp2V(phen)][OTf]2 (3)[13] (see Scheme 1). The reaction of
dichloride complexes 1 and [(η5-C5H4Me)2VCl2] (1a) with
silver triflate gives reactive intermediates [Cp2V(OTf)2] (2)
and [(η5-C5H4Me)2V(OTf)2] (2a), respectively. The weakly
bonded ligands are exchanged in the second step with the
appropriate phenanthroline to give cationic vanadocene
complexes 3–6 and 3a–6a.
The reaction process was followed by EPR spectroscopy.
The addition of silver triflate to the solution of vanadocene
dichloride 1 in THF was accompanied with the precipi-
tation of silver chloride and the appearance of a new signal
in the EPR spectrum (|Aiso| = 73.7ϫ10–4 cm–1, giso = 1.980)
that was assigned to triflate complex 2. The disappearance
of the signal of the starting compound (1: |Aiso| =
Figure 2. ORTEP drawing of the molecule [Cp2V(OTf)2] present in
the crystal structure of 2 (ellipsoids: 30% probability). Numbering
of all non-hydrogen atoms is shown.
Figure 1. EPR spectra of the solutions of (A) 1, (B) 2, and (C) 3
in THF. The spectra were measured at ν = 9.45 GHz.
Scheme 1. Preparation of phenanthroline complexes 3–6 and 3a–6a.
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Phenanthroline complexes 3–6 and 3a–6a were charac-
terized by positive-ion ESI mass spectrometry. These com-
pounds give peaks that were assigned to the parent dicat-
ionic species [M]2+. The EPR spectra of the phenanthroline
complexes were measured in acetone and methanol. The
obtained |Aiso| and giso values are summarized in Table 1.
Although these solvents might coordinate, the negligible
differences in the |Aiso| and giso values show that they are
inert for this type of species. The complexes of 5-nitro-1,10-
phenanthroline (4 and 4a) and 4,7-diphenyl-1,10-phenan-
throline (6 and 6a) display a slightly lower |Aiso| parameter
than complexes of 1,10-phenanthroline (3 and 3a) and 1,10-
phenanthroline-5-amine (5 and 5a). The effect of the elec-
tron-withdrawing substituents in the phenanthroline frame-
work lowers the spin density on the metal. The substitution
of the cyclopentadienyl ring with a methyl group has a neg-
ligible effect on these parameters because the unpaired elec-
tron occupies the orbital that is anti bonding to the V–N
bonds.[17,18] Structures of the complexes [(η5-C5H4Me)2V-
(phen)][OTf]2·0.5Me2CO (3a·0.5Me2CO) and [Cp2V(5-
NH2-phen)][OTf]2 (5) were determined by X-ray diffraction
analysis.
Figure 3. ORTEP drawing of the dicationic complex [(η5-C5H4-
Me)2V(phen)]2+ present in the crystal structure of 3a·0.5Me2CO
(ellipsoids: 30% probability). Numbering of all non-hydrogen
atoms is shown.
Table 1. Isotropic g factors and isotropic hyperfine coupling con-
stants [10–4 cm–1] of the vanadocene complexes.
giso
|Aiso
|
giso
|Aiso|
1
2
3
4
5
6
1.991
1.980
1.986
1.986
1.986
1.987
69.5
73.7
62.8
61.9
62.5
62.1
1a
2a
3a
4a
5a
6a
1.990
1.981
1.986
1.986
1.986
1.987
69.5
73.5
62.9
62.1
62.7
62.0
Figure 4. ORTEP drawing of the dicationic complex [Cp2V(5-NH2-
phen)]2+ present in the crystal structure of 5 (ellipsoids: 30% prob-
ability). Numbering of all non-hydrogen atoms is shown.
X-ray Structures of Compounds 2, 3a·0.5Me2CO and 5
Molecular structures of compounds 2, 3a·0.5Me2CO,
and 5 are shown in Figures 2, 3, and 4. Important structural
parameters are listed in Table 2. These complexes have a
typical bent metallocene structure in which two η5-bonded
cyclopentadienyl rings and two other donor atoms occupy
the pseudotetrahedral coordination sites around the vanadi-
um(IV) center. The Cg–V distances [1.949(2)–1.963(2) Å]
and Cg–V–Cg angles [132.63(9)–134.38(7)°] lay in the range
common for the other known vanadocene(IV) complexes
(Cg–V: 1.95–1.97 Å; Cg–V–Cg: 131–135°).[19]
Table 2. Structural parameters (bond lengths in Å, angles in °) of
vanadocene compounds describing the coordination around the
vanadium.
2[a]
3[b]
3a[c]
5[d]
V–Cg1
V–Cg2
V–X1
V–X2
Cg1–V–Cg2 132.63(9)
X1–V–X2 82.88(12)
1.954(2)
1.963(2)
2.044(3)
2.057(3)
1.96
1.96
2.134(2)
2.139(2)
133.63
76.66(7)
1.965(2)
1.957(2)
2.136(4)
2.131(3)
134.38(7) 133.40(10)
76.66(12) 76.46(13)
1.954(2)
1.949(2)
2.152(4)
2.125(3)
Compound 2 has two OTf ligands bonded through oxy-
gen atoms to vanadium. The V–O bonds [2.044(3),
2.057(3) Å] are longer than in the vanadocene complexes of
carboxylic acids {[CpЈ2V(OOCR)2]: V–O 2.014(2)–
2.042(3) Å}.[12,16] The O–V–O angle was found to be
[a] X1 = O1, X2 = O2, Cg1 = C1–C5, Cg2 = C6–C10. [b] X = N,
data reported in the literature.[13] [c] Compound 3a·0.5Me2CO: X1
= N1, X2 = N2, Cg1 = C1–C5, Cg2 = C7–C11. [d] X1 = N1, X2
= N2; Cg1 = C1–C5, Cg2 = C6–C10.
smaller [82.88(12)°] than the O–Ti–O angle in the titano- angles [76.46(13)–76.66(12)°] were found to be close to the
cene counterpart [Cp2Ti(OTf)2] [90.74(8)°].[20] The M–O–S values reported for unsubstituted analogue 3[13] and the
angles in these compounds are very similar {2: V–O–S 2,2Ј-bipyridine complexes [Cp2V(bpy)][BPh4] (bpy = 2,2Ј-
146.7(2), 151.3(2)°; [Cp2Ti(OTf)2]: 145.7(1), 152.3(1)°}.
bipyridyl)[15] and [Cp2V(bpy)][OTf]2.[13] The small effect of
Complexes 3a·0.5Me2CO and 5 have the chelating phen- substitution and the oxidation state of vanadium on these
anthroline ligand bonded to the vanadocene moiety. The structural parameters is in line with rigid chelate structure
V–N bond lengths [2.125(3)–2.152(4) Å] and N–V–N bond in these complexes.
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Cytotoxicity Studies
Table 3. Cytotoxicity of the vanadocene complexes and cisplatin
toward MOLT-4 and HL-60 cells expressed as the IC50 values [μm].
The cytotoxic effect of phenanthroline complexes 3–6
and 3a–6a was evaluated on human T-lymphocytic leuke-
mia cells MOLT-4 and human promyelocytic leukemia cells
HL-60 in exponential grow phase, 24 h after the incubation
with the cytostatic drugs.
The IC50 values were obtained from the standard WST-
1 viability assays. The assay is based on reduction of the
tetrazolium salt WST-1 to a colored substance by mito-
chondrial dehydrogenases of viable cells, and thus reflects
combined changes in proliferation and viability. The ob-
MOTL-4
HL-60
MOTL-4 HL-60
1
3
4
5
6
70Ϯ7[a]
7.8Ϯ1.4
15.2Ϯ1.8
3.1Ϯ0.4
5.6Ϯ0.5
60.5Ϯ3.3
9.6Ϯ1.3
26.2Ϯ1.6
2.8Ϯ0.4
7.1Ϯ0.7
11.3Ϯ2.5
1a
3a
4a
5a
6a
96Ϯ12[a] 88.6Ϯ5.2
5.0Ϯ0.7 7.6Ϯ0.5
17.4Ϯ 3.1 28.2Ϯ3.3
15.5Ϯ1.0 20.3Ϯ3.0
6.0Ϯ0.7 16.8Ϯ4.1
cisplatin 15.8Ϯ1.9
[a] Data reported in literature.[5]
tained IC50 values are listed in Table 3. The effect of the amino-substituted phenanthroline ligand, has an IC50 value
drug concentration on the viability of the leukemic cells rel- that is about two times lower for MOLT-4 cells and about
ative to untreated control cells is given in Figures 5 and 6.
three times lower for HL-60 relative to unsubstituted ana-
All phenanthroline complexes under study (3–6 and 3a– logue 3. Substitution of the cyclopentadienyl ring with a
6a) showed considerably higher activity than the parent di- methyl group has a rather negative effect. Compounds 1a,
chloride complexes (1 and 1a). High cytotoxicity was ob- 4a, 5a, and 6a have activity that is similar to or lower than
served against both leukemic cell lines. It was further shown their unsubstituted analogues 1, 4, 5, and 6, respectively.
that the activity of the vanadocene complexes is markedly Only the methyl-substituted derivative 3a has higher cytoto-
improved through the substitution of the phenanthroline xicity than its unsubstituted counterpart 3; but even here
framework. For example, complex 5, which contained an the improvement nears the accuracy of the assay.
Figure 5. Cytotoxicity curves from WST-1 assays showing the effect of compounds 3–6 and 3a–6a on the viability of MOLT-4 cells. The
representation of viable cells was determined by cell proliferation reagent WST-1 24 h after the application of the compounds. To calculate
cell viability, the value of the signal from the treated culture well was expressed as a part of that of the control well with untreated cells.
Figure 6. Cytotoxicity curves from WST-1 assays showing the effect of compounds 3–6 and 3a–6a on the viability of HL-60 cells. The
representation of viable cells was determined by cell proliferation reagent WST-1 24 h after the application of the compounds. To calculate
cell viability, the value of the signal from the treated culture well was expressed as a part of that of the control well with untreated cells.
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The highest cytotoxic effect was detected with the com- dia used in the preclinical in vitro testing. These compounds
plex of 1,10-phenanthroline-5-amine (5). The IC50 values should be able to enter into the organism without hydrolyz-
against both MOLT-4 [(3.1Ϯ0.4) μm] and HL-60 ing the phenanthroline or cyclopentadienyl ligand. The in-
[(2.8Ϯ0.4) μm] are considerably lower than those observed ertness toward ionic components of the human plasma at
for cisplatin (Table 3). The large improvement is observed physiological pH contrasts with dichloride complexes 1 and
mainly for the HL-60 cells because cisplatin exhibits a very 1a. This might be the main reason for higher activity of
slow decrease in the cytotoxicity curve (see Figure S1 in the phenanthroline complexes presented here.
Supporting Information).
Conclusion
Stability of the Phenanthroline Complexes
Vanadocene complexes that contain phenanthroline li-
The behavior of phenanthroline complexes 3–5 and 3a– gands are promising cytotoxic agents, mainly owing to their
5a was monitored in water, physiological saline, PBS, high cytotoxicity, high water solubility, and stability in the
Iscove’s Modified Dulbecco’s Medium (IMDM), and application media. Their cytotoxic properties are easily
Krebs–Ringer solution for seven days. Complexes 6 and 6a modified through substitution of the phenanthroline frame-
were rejected from this part of the study owing to their very work. Substitution of the cyclopentadienyl ring described
low solubility in water. Complexes 3–5 and 3a–5a were fully here has only a minor effect on the cytotoxicity. Neverthe-
dissolved without addition of organic solvents. The dissol- less, it is possible that the attachment of substituents with
ution was checked by eye and by solution EPR spec- functional groups can bring additional advantages, such as
troscopy.
was previously successfully done with various metallocene
The EPR spectra of compounds 3–5 and 3a–5a, obtained compounds.[21]
immediately after dissolution in these media, show the same
The high activity of the phenanthroline compounds
|Aiso| and giso values as the solutions in the inert solvents proves that stabilization of the vanadocene complexes
such as acetone. It proves that the dicationic species stay through the substitution of chlorides can lead to consider-
unchanged because all expected hydrolysis products display able improvement of their cytotoxic properties. This finding
very different values of these parameters.[8–10] The spectra contrasts with established mechanistic rules, which suggests
in water, physiological saline, and PBS stay unchanged for that the modification of the putative active Cp2M fragment
seven days. Hence, the signal does not lower the intensity, is the only way to develop new, highly active metallocene
and the appearance of another species was also not ob- anticancer drugs.
served. Slightly lower stability of phenanthroline complexes
3–5 and 3a–5a was observed in IMDM and Krebs–Ringer
Experimental Section
solution. These species were found to be stable in these me-
dia for six days. After seven days, a trace of carbonate com-
plex [Cp2V(O,O-CO3)] and [(η5-C5H4Me)2V(O,O-CO3)]
were detected for compounds 3–5 and 3a–5a, respectively
(for example, see Figure 7).
Methods and Materials: All operations were performed under nitro-
gen using conventional Schlenk-line techniques. The solvents were
purified and deoxygenated by standard methods.[22] Complexes
1,[23] 1a,[17] and 3[13] were prepared according literature procedures.
1,10-Phenanthroline, 5-nitro-1,10-phenanthroline, 1,10-phenan-
throline-5-amine, 4,7-diphenyl-1,10-phenanthroline, and Iscove’s
Modified Dulbecco’s Medium were obtained from Sigma–Aldrich
and used without further purification. Physiological saline
(0.154 mm NaCl), PBS (137 mm NaCl, 2.7 mm KCl, 10 mm
Na2HPO4, 2.0 mm KH2PO4, pH = 7.4), and Krebs–Ringer solution
(115 mm NaCl, 5.9 mm KCl, 1.2 mm MgCl2, 1.2 mm NaH2PO4,
1.2 mm Na2SO4, 2.5 mm CaCl2, 25 mm NaHCO3, 10 mm glucose,
pH = 7.4) were prepared from analytical-grade chemicals and de-
ionized redistilled water.
Infrared spectra were recorded in the 4000–400 cm–1 region with a
Nicolet Magna 550 FTIR spectrometer using Nujol mull between
KBr windows. The EPR spectra were recorded with Miniscope MS
300 spectrometers at the X band at ambient temperature. Mass
spectrometry was performed with a quadruple mass spectrometer
(LCMS 2010, Shimadzu, Japan). The sample was injected into the
mass spectrometer with infusion mode at a constant flow rate of
10 μLmin–1, and electrospray ionization-mass spectrometry (ESI-
MS) was used for the identification of analyzed samples.
Figure 7. EPR spectra of (A) 3 in IMDM obtained 7 d after dissol-
ution; (B) aqueous solution of [Cp2V(O,O-CO3)] prepared accord-
ing to the literature.[8] The spectra were measured at ν = 9.45 GHz.
[Cp2V(OTf)2] (2): Complex 1 (0.253 g, 1.00 mmol) was dissolved in
THF (20 mL), treated with AgOTf (0.515 g, 2.00 mmol), and
stirred for 90 min in the dark. The reaction mixture was filtered
The behavior observed here in water, therapeutic, and
physiological solutions proves that phenanthroline com-
plexes 3–5 and 3a–5a stay unchanged in the application me- through Celite to remove the fine precipitate of silver chloride. The
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clear green solution was evaporated under vacuum and recrys-
tallized from a mixture of acetone/diethyl ether to give a grass-
green powder. Yield: 144 mg (30%, 0.30 mmol). C12H10F6O6S2V
(Mr = 479.27): C 30.07, H 2.10; found C 30.01; H 2.14. EPR (ace-
tone): |Aiso| = 79.7 G; giso = 1.980. Single crystals suitable for X-
ray diffraction analysis were prepared by careful overlayering of the
solution of compound 2 in acetone with diethyl ether.
[(η5-C5H4Me)2V(OTf)2] (2a): The reaction was carried out as de-
scribed for 2 but with 1a (0.253 g, 0.90 mmol) and AgOTf (0.464 g,
1.80 mmol). Yield: 162 mg (35%, 0.31 mmol). C14H14F6O6S2V (Mr
= 507.32): calcd. C 33.15, H 2.78; found C 33.32, H 2.89. EPR
(acetone): |Aiso| = 79.5 G; giso = 1.981.
suitable for X-ray diffraction analysis were prepared by careful
overlayering of the solution of compound 5 in acetone with diethyl
ether.
[(η5-C5H4Me)2V(5-NH2-phen)][OTf]2 (5a): The reaction was car-
ried out as described for 3a but with 5-NH2-phen (0.264 g,
1.35 mmol). Yield: 216 mg (34%, 0.31 mmol). C26H23F6N3O6S2V
(Mr = 702.54): calcd. C 44.45, H 3.30, N 5.98; found C 44.52, H
3.34, N 6.01. Positive-ion MS (acetone): m/z = 202 [M]2+. EPR
(acetone): |Aiso| = 67.6 G, giso = 1.986; EPR (water): |Aiso| = 67.6 G,
giso = 1.986; EPR (physiological saline): |Aiso| = 67.6 G, giso = 1.986;
EPR (IMDM): Aiso = 67.7 G, giso = 1.986; EPR (Krebs–Ringer
solution): |Aiso| = 67.5 G; giso = 1.986; EPR (PBS): |Aiso| = 67.6 G;
giso = 1.986.
[(η5-C5H4Me)2V(phen)][OTf]2 (3a): Complex 1a (0.253 g,
0.90 mmol) was dissolved in THF (20 mL), treated with AgOTf
(0.464 g, 1.80 mmol), and stirred for 90 min in the dark. The reac-
tion mixture was filtered through Celite to remove the fine precipi-
tate of silver chloride. The clear green solution was treated with
1,10-phenanthroline (0.244 g, 1.35 mmol), which caused an imme-
diate color change from green to brown. The reaction mixture was
stirred for 18 h. The solid product was separated by decantation.
It was washed with THF and diethyl ether and dried under vacuum
[Cp2V(4,7-Ph2-phen)][OTf]2 (6): The reaction was carried out as de-
scribed for 3a but with 1 (0.253 g, 1.00 mmol), AgOTf (0.515 g,
2.00 mmol), and 4,7-Ph2-phen (0.499 g, 1.50 mmol). The product
did not precipitate from THF solution. The solvent was vacuum-
evaporated, then the crude product was washed with diethyl ether
and dried under vacuum to give a green-brown powder. Yield:
269 mg (33%, 0.33 mmol). C36H26F6N2O6S2V (Mr = 811.66):
calcd. C 53.27, H 3.23, N 3.45; found C 53.35, H 3.09, N 3.61.
to give
a |
brown powder. Yield: 247 mg (40%, 0.36 mmol). Positive-ion MS (acetone): m/z = 256.5 [M]2+. EPR (acetone) |Aiso
C26H24VS2N2F6O6 (Mr = 689.54): calcd. C 45.29, H 3.51, N 4.06;
found C 45.42, H 3.47, N 4.10. Positive-ion MS (acetone): m/z =
194.5 [M]2+. EPR (acetone): |Aiso| = 67.8 G, giso = 1.986; EPR
(water): |Aiso| = 67.8 G, giso = 1.985; EPR (physiological saline):
= 66.9 G, giso = 1.987.
[(η5-C5H4Me)2V(4,7-Ph2-phen)][OTf]2 (6a): The reaction was car-
ried out as described for 6 but with 4,7-Ph2-phen (0.450 g,
1.35 mmol). Yield: 245 mg (32%, 0.29 mmol). C38H30F6N2O6S2V
(Mr = 839.72): calcd. C 54.35, H 3.60, N 3.34; found C 54.53, H
3.55, N 3.22. Positive-ion MS (acetone): m/z = 270.5 [M]2+. EPR
(acetone): |Aiso| = 66.8 G; giso = 1.987.
|Aiso| = 67.8 G, giso = 1.985; EPR (IMDM): |Aiso| = 67.9 G, giso
=
1.985; EPR (Krebs–Ringer solution): |Aiso| = 67.9 G; giso = 1.986;
EPR (PBS): |Aiso| = 67.9 G; giso = 1.986. Single crystals of
3a·0.5Me2CO suitable for X-ray diffraction analysis were prepared
by careful overlayering of the solution of compound 3a in acetone
with diethyl ether.
Stability Studies: The appropriate compound (50 μmol) was dis-
solved in the medium (e.g., water, physiological saline, PBS,
IMDM, or Krebs–Ringer solution; 5 mL) under an air atmosphere.
The composition and concentration of the vanadium(IV) species
were monitored by EPR spectroscopy (Miniscope MS 300) imme-
diately after dissolution, and then after each 12 h for 7 d. Samples
were measured in 50 μL capillaries (fixed adapter) at room tem-
perature. The concentration was calculated by the integration
method.
[Cp2V(5-NO2-phen)][OTf]2 (4): The reaction was carried out as de-
scribed for 3a but with 1 (0.253 g, 1.00 mmol), AgOTf (0.515 g,
2.00 mmol), and 5-NO2-phen (0.338 g, 1.5 mmol). Yield: 225 mg
(32%; 0.32 mmol). C24H17F6N3O8S2V (Mr = 704.47): calcd. C
40.92, H 2.43, N 5.96; found C 41.04, H 2.39, N 5.92. Positive-ion
MS (acetone): m/z = 203 [M]2+. EPR (acetone): |Aiso| = 66.8 G, giso
= 1.986; EPR (water): |Aiso| = 67.0 G, giso = 1.985; EPR (physiolog-
ical saline): |Aiso| = 67.0 G, giso = 1.985; EPR (IMDM): |Aiso| =
66.9 G, giso = 1.986; EPR (Krebs–Ringer solution): |Aiso| = 67.0 G;
giso = 1.985; EPR(PBS): |Aiso| = 67.0 G; giso = 1.985.
[(η5-C5H4Me)2V(5-NO2-phen)][OTf]2 (4a): The reaction was car-
ried out as described for 3a but with 5-NO2-phen (0.305 g,
1.35 mmol). Yield: 228 mg (35%, 0.31 mmol). C26H21F6N3O8S2V
(Mr = 732.52): calcd. C 42.63, H 2.89, N 5.74; found C 42.76, H
3.02, N 5.91. Positive-ion MS (acetone): m/z = 217 [M]2+. EPR
(acetone): |Aiso| = 66.9 G; giso = 1.986; EPR (water): |Aiso| = 67.0 G,
giso = 1.985; EPR (physiological saline): |Aiso| = 67.0 G, giso = 1.985;
EPR (IMDM): |Aiso| = 67.0 G, giso = 1.986; EPR (Krebs–Ringer
solution): |Aiso| = 67.0 G; giso = 1.986; EPR (PBS): |Aiso| = 66.9 G;
giso = 1.985.
Cytotoxicity Studies: The studies were performed on the human T-
lymphocytic leukemia cells MOLT-4 obtained from the American
Type Culture Collection (USA) and human promyelocytic leuke-
mia cells HL-60 obtained from the European Collection of Cell
Cultures (Porton Down, Salisbury, Great Britain). The cells were
cultured in IMDM supplemented with a 20% fetal calf serum and
0.05% l-glutamine (Sigma–Aldrich) in a humidified incubator at
37 °C and a controlled 5% CO2 atmosphere. Cell lines in the maxi-
mal range of up to 20 passages were used for this study. The cytoto-
xicity of compounds 1, 3–6, 1a, 3a–6a, and cisplatin were evaluated
by the WST-1 cell viability test (Roche) according to the manufac-
turer’s instructions. The assay was based on the reduction of WST-
1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benz-
ene disulfonate} by viable cells. The reaction produced a colored
soluble formazan salt.[24] The absorbance at 440 nm was measured
[Cp2V(5-NH2-phen)][OTf]2 (5): The reaction was carried out as de-
scribed for 3a but with 1 (0.253 g, 1.00 mmol), AgOTf (0.515 g, with a multiplate reader (Tecan Infinite 200). Compounds 1, 3–5,
2.00 mmol), and 5-NH2-phen (0.293 g, 1.5 mmol). Yield: 202 mg
(30%; 0.30 mmol). C24H19F6N3O6S2V (Mr = 674.49): calcd. C
42.74, H 2.84, N 6.23; found C 42.80, H 2.77, N 6.18. Positive-ion
MS (acetone): m/z = 188 [M]2+. EPR (acetone): |Aiso| = 67.4 G, giso
= 1.986; EPR (water): |Aiso| = 67.4 G, giso = 1.986; EPR (physiolog-
1a, 3a–5a, and cisplatin were dissolved in cultivation medium to
the desired concentrations. Compounds 6 and 6a were dissolved in
DMSO and diluted by cultivation medium to the desired concen-
trations. The cells were seeded in a 96-well plate, incubated in 1–
1000 μm solutions of the evaluated compound for 24 h, then
ical saline): |Aiso| = 67.4 G, giso = 1.986; EPR (IMDM): |Aiso| = washed in pure media and incubated for 180 min in WST-1 solu-
67.5 G, giso = 1.986; EPR (Krebs–Ringer solution): |Aiso| = 67.4 G;
giso = 1.986; EPR (PBS): |Aiso| = 67.4 G; giso = 1.986. Single crystals
tion. The same cells incubated in the cultivation media only were
used as the control. Absorbance data were normalized to 100%
Eur. J. Inorg. Chem. 2013, 2665–2672
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FULL PAPER
cell viability for untreated cells. Half-inhibiting concentration
(IC50), which is defined as the concentration of the drug-reducing
cell viability by 50%, and standard deviations were calculated from
three independent measurements. They were obtained from the
dose–response sigmoid using Origin Pro 8.0 (Microcal Software
Inc., Northampton, MA, USA).
CCDC-904714 (for 2), -904715 (for 3a·0.5Me2CO), and -904716
(for 5) contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
Supporting Information (see footnote on the first page of this arti-
cle): Cytotoxicity curves of the WST-1 assays on MOLT-4 for cis-
platin and on HL-60 for 1, 1a, and cisplatin.
Crystallography: The X-ray data for crystals of compounds 2,
3a·0.5Me2CO, and 5 were obtained at 150 K with an Oxford Cryos-
tream low-temperature device on a Nonius Kappa CCD dif-
fractometer with Mo-Kα radiation (λ = 0.71073 Å), a graphite mon-
ochromator, and the φ and χ scan mode. Data reductions were per-
formed with DENZO-SMN.[25] Structures were solved by direct
methods (SIR92)[26] and refined by full-matrix least-squares on the
basis of F2 (SHELXL97).[27] Crystallographic data are summarized
in Table 4. Hydrogen atoms were mostly localized on a difference
Fourier map. However, to ensure uniform treatment of the crystal,
all hydrogen were recalculated into idealized positions (riding
model) and assigned temperature factors Hiso(H) = 1.2 Ueq (pivot
atom) or of 1.5 Ueq for the methyl moiety with C–H = 0.96, 0.97,
and 0.93 Å for methyl, methylene, and hydrogen atoms in the Cp
ring, respectively.
Acknowledgments
This study was supported by the Charles University in Prague (pro-
gram PRVOUK P37/01).
[1] D. Hoelzer, Hematology Am. Soc. Hematol. Educ. Program
2011, 243–249.
[2] G. J. Roboz, Hematology Am. Soc. Hematol. Educ. Program
2011, 43–50.
[3] a) P. Köpf-Maier, H. Köpf, Z. Naturforsch. B 1979, 34, 805–
807; b) V. J. Moebus, R. Stein, D. G. Kieback, I. B. Runne-
baum, G. Sass, R. Kreienberg, Anticancer Res. 1997, 17, 815–
821; c) J. Aubrecht, R. K. Narla, P. Ghosh, J. Stanek, F. M.
Uckun, Toxicol. Appl. Pharmacol. 1999, 154, 228–235; d) J. H.
Toney, L. N. Rao, M. S. Murthy, T. J. Marks, Breast Cancer
Res. Treat. 1985, 6, 185; e) S. Gómez-Ruiz, D. Maksimovic-
Ivanic, S. Mijatovic, G. N. Kaluderovic, Bioinorg. Chem. and
Appl. 2012, DOI: 10.1155/2012/140284.
Table 4. Crystallographic data of the compounds 1, 3a·0.5Me2CO,
and 5.
2
3a·0.5Me2CO
5
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α [°]
β [°]
γ [°]
Z
monoclinic
P21/c
8.2350(3)
12.5260(7)
17.4559(9)
90
115.327(5)
90
4
triclinic
P1
monoclinic
P21/c
16.9301(12)
9.7960(8)
16.0840(11)
90
107.336(6)
90
4
¯
[4] a) B. Gleeson, J. Claffey, M. Hogan, H. Müller-Bunz, D. Wal-
lis, M. Tacke, J. Organomet. Chem. 2009, 694, 1369–1374; b)
B. Gleeson, J. Claffey, A. Deally, M. Hogan, L. M. M. Méndez,
H. Müller-Bunz, S. Patil, D. Wallis, M. Tacke, Eur. J. Inorg.
Chem. 2009, 2804–2810; c) B. Gleeson, A. Deally, H. Müller-
Bunz, S. Patil, M. Tacke, Aust. J. Chem. 2010, 63, 1514–1520.
[5] J. Honzícˇek, J. Vinklárek, I. Klepalová, Z. Padeˇlková, I. Císa-
8.43701(5)
8.6150(7)
22.5601(16)
80.896(7)
79.383(5)
73.252(6)
2
ˇ
ˇ
rˇová, P. Siman, M. Rezácˇová, Inorg. Chim. Acta 2011, 373, 1–
μ [mm–1]
Dcalcd. [gcm–3]
Crystal color
Crystal shape
Size [mm]
θ range [°]
h range
0.962
1.956
dark green
plate
0.546
1.615
brown
rod
0.646
1.759
red
block
7.
[6] I. Fichtner, J. Claffey, A. Deally, B. Gleeson, M. Hogan, M. R.
Markelova, H. Müller-Bunz, H. Weber, M. Tacke, J. Or-
ganomet. Chem. 2010, 695, 1175–1181.
[7] a) J. H. Toney, T. J. Marks, J. Am. Chem. Soc. 1985, 107, 947–
953; b) J. H. Toney, C. P. Brock, T. J. Marks, J. Am. Chem. Soc.
1986, 108, 7263–7274.
[8] J. Vinklárek, J. Honzícˇek, J. Holubová, Magn. Reson. Chem.
2004, 42, 870–874.
[9] J. Vinklárek, H. Palácˇková, J. Honzícˇek, J. Holubová, M. Hol-
cˇapek, I. Císarˇová, Inorg. Chem. 2006, 45, 2156–2162.
[10] J. Vinklárek, J. Honzícˇek, J. Holubová, Inorg. Chim. Acta 2004,
357, 3765–3769.
[11] a) J. Vinklárek, H. Palácˇková, J. Honzícˇek, Collect. Czech.
Chem. Commun. 2004, 69, 811–821; b) H. Palácˇková, J.
Vinklárek, J. Holubová, I. Císarˇova, M. Erben, J. Organomet.
Chem. 2007, 692, 3758–3764; c) J. Honzícˇek, H. Palácˇková, J.
Vinklárek, I. Císarˇová, J. Organomet. Chem. 2006, 691, 202–
207.
0.29; 0.22; 0.07 0.76; 0.38; 0.19 0.35; 0.22; 0.10
2.08–27.49
–10/10
–15/16
–21/22
16609
3707
0.0514
2842
244
2.48–27.50
–10/10
–11/11
–29/29
28140
6914
0.0779
5708
388
2.43–27.40
–21/20
–10/12
–19/20
15321
5683
0.0650
3788
379
k range
l range
Reflns. measured
Unique reflections
[a]
Rint
Obsd. reflections
Parameters
S[b] (all data)
R[c], wR[c]
1.074
1.101
1.148
0.0494, 0.1272 0.0554, 0.1333 0.0654, 0.1441
0.591/–0.484 0.788/–0.598
Δρ max./min. [eÅ–3] 1.138/–0.608
[a] Rint = Σ|F2o – Fo2(mean)|/ΣF2o. [b] S = {Σ[w(F2o – Fc2)2]/(Ndiff.
–
Nparams.)}1/2 for all data. [c] R(F) = Σ||Fo| – |Fc||/Σ|Fo| for observed
data, wR(F2) = {Σ[w(F2o – Fc2)2]/[Σw(F2o)2]}1/2 for all data.
[12] J. Vinklárek, T. Deˇdourková, J. Honzícˇek, A. Ru˚zicˇka, J. Inorg.
ˇ
Biochem. 2010, 104, 936–943.
[13] P. Ghosh, A. T. Kotchevar, D. D. DuMez, S. Ghosh, J. Peiter-
son, F. M. Uckun, Inorg. Chem. 1999, 38, 3730–3737.
[14] C. C. Scarborough, K. Wieghardt, Inorg. Chem. 2011, 50,
9773–9793.
[15] M. Jordan, W. Saak, D. Haase, R. Beckhaus, Eur. J. Inorg.
Chem. 2007, 5168–5172.
There is disordered solvent (acetone) in the structure of
3a·0.5Me2CO. Attempts were made to model this disorder or split
it into two positions, but were unsuccessful. Platon/Squezee[28] was
used to correct the data for the presence of disordered solvent. A
potential solvent volume of 240 Å3 was found. Forty-four electrons
per unit cell worth of scattering were located in the void. The calcu-
lated stoichiometry of solvent was calculated to be one molecule
of acetone per unit cell, which results in 32 electrons per unit cell.
[16] a) J. Honzícˇek, P. Nachtigall, I. Císarˇova, J. Vinklárek, J. Or-
ganomet. Chem. 2004, 689, 1180–1187; b) J. Vinklárek, J.
Honzícˇek, I. Císarˇová, M. Pavlisˇta, J. Holubová, Cent. Eur. J.
Chem. 2005, 3, 157–168.
[17] J. Petersen, F. Dahl, J. Am. Chem. Soc. 1975, 97, 6422–6433.
Eur. J. Inorg. Chem. 2013, 2665–2672
2671
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.eurjic.org
FULL PAPER
[18] J. Honzícˇek, J. Vinklárek, P. Nachtigall, Chem. Phys. 2004, 305,
291–298.
[19] Cambridge Structural Database: F. H. Allen, Acta Crystallogr.,
Sect. B 2002, 58, 380.
[20] G. B. Deacon, P. C. Junk, T. J. Ness, R. Schwering, G. Meyer,
J. Chem. Crystallogr. 2006, 36, 167–174.
[23]
[24]
[25]
G. Wilkinson, J. M. Birmingham, J. Am. Chem. Soc. 1954, 76,
4281–4284.
M. Ishiyama, M. Shiaga, K. Sasamoto, M. Mizoguchi, P. He,
Chem. Pharm. Bull. 1993, 41, 1118–1122.
Z. Otwinowski, W. Minor, Methods Enzymol. 1997, 276, 307–
326.
[21] a) O. R. Allen, A. L. Gott, J. A. Hartley, J. M. Hartley, R. J.
Knox, P. C. McGowan, Dalton Trans. 2007, 5082–5090; b) K.
Strohfeldt, M. Tacke, Chem. Soc. Rev. 2008, 37, 1174–1187; c)
J. R. Boyles, M. C. Baird, B. G. Campling, N. Jain, J. Inorg.
Biochem. 2001, 84, 159–162; d) J. Honzícˇek, J. Vinklárek, Z.
[26] A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi, J.
Appl. Crystallogr. 1993, 26, 343–350.
[27] G. M. Sheldrick, SHELXL97, University of Göttingen, Ger-
many, 1997.
[28] A. L. Spek, Acta Crystallogr., Sect. A 1990, 46, C34.
Received: December 12, 2012
ˇ
ˇ
Padeˇlková, L. Sebestová, K. Foltánová, M. Rezácˇová, J. Or-
ganomet. Chem. 2012, 716, 258–268.
Published Online: March 25, 2013
[22] W. L. F. Armarego, D. D. Perrin, in: Purification of Laboratory
Chemicals, Butterworth-Heinemann, Oxford, UK, 1996.
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