Enhanced cytotoxicity of indenyl molybdenum(ii) compounds bearing a thiophene function

Article abstract of DOI:10.1039/c9dt01698h

A series of six indenyl molybdenum compounds bearing a thiophenyl function in the side chain were prepared and characterized by analytical and spectroscopic methods. The structures of [(η⁵-C₉H₆CH₂C₄H₃S)(η³-C₃H₅)Mo(CO)₂] and [(η⁵-C₉H₆CH₂C₄H₃S)Mo(CO)₂(bpy)][BF₄] were determined by single-crystal X-ray diffraction. The compounds bearing N,N-chelating ligands exhibit increased cytotoxic activity against human leukemia cell lines MOLT-4; up to two orders of magnitude lower IC₅₀ values were observed compared to analogues with unsubstituted indenyl, which clearly demonstrates the strong effect of the indenyl ligand modification on the biological activity of the molybdenum(ii) compounds. The highest cytostatic potential was observed for the complex bearing 4,7-diphenyl-1,10-phenanthtoline [(η⁵-C₉H₆CH₂C₄H₃S)Mo(CO)₂(Ph₂phen)][BF₄] with IC₅₀ (MOLT-4) = 0.19 ± 0.02 μM. Detailed regulation of the molecular and cellular mechanism by this derivative was investigated on the lung carcinoma cell line A549 and compared with the lung fibroblast cell line MRC-5. Rather unusual differences in the effects on tumor and non-tumor cell lines provide a unique insight into the cytostatic action of molybdenum(ii) complexes.

Full text of DOI:10.1039/c9dt01698h

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Enhanced cytotoxicity of indenyl molybdenum(II)
compounds bearing a thiophene function†
Cite this: DOI: 10.1039/c9dt01698h
a
b,c
a
d
Ondřej Mrózek,
Lucie Melounková,
Libor Dostál,
Ivana Císařová,
b
c
c,e
f
Aleš Eisner, Radim Havelek,
Eva Peterová, Jan Honzíček * and
a
Jaromír Vinklárek
*
A series of six indenyl molybdenum compounds bearing a thiophenyl function in the side chain were pre-
5
pared and characterized by analytical and spectroscopic methods. The structures of [(η -C
9 6 2 4 3
H CH C H S)
3
5
(
η -C
3
H
5
)Mo(CO)
2
9 6 2 4 3 2 4
] and [(η -C H CH C H S)Mo(CO) (bpy)][BF ] were determined by single-crystal X-ray
diffraction. The compounds bearing N,N-chelating ligands exhibit increased cytotoxic activity against
human leukemia cell lines MOLT-4; up to two orders of magnitude lower IC50 values were observed com-
pared to analogues with unsubstituted indenyl, which clearly demonstrates the strong effect of the indenyl
ligand modification on the biological activity of the molybdenum(II) compounds. The highest cytostatic
5
potential was observed for the complex bearing 4,7-diphenyl-1,10-phenanthtoline [(η -C
9 6 2 4 3
H CH C H S)Mo
Received 23rd April 2019,
Accepted 28th June 2019
(CO) (Ph phen)][BF ] with IC (MOLT-4) = 0.19 ± 0.02 μM. Detailed regulation of the molecular and cellu-
2
2
4
50
lar mechanism by this derivative was investigated on the lung carcinoma cell line A549 and compared with
the lung fibroblast cell line MRC-5. Rather unusual differences in the effects on tumor and non-tumor cell
lines provide a unique insight into the cytostatic action of molybdenum(II) complexes.
DOI: 10.1039/c9dt01698h
rsc.li/dalton
logical applications. For example, tripeptide derivatized Cp
Introduction
complexes of technetium and rhenium serve as an effective
9
The area of biologically active cyclopentadienyl (Cp) com- radiopharmaceutical probe. A promising anticancer activity
1
0
pounds has attracted considerable attention since the anti- was reported for the ferrocene derivative ferrocifen and
1
1
tumor activity of titanocene dichloride (TDC) was discovered monocyclopentadienyl complexes of iridium.
In 2005,
1
in 1979. Despite the promising results in phase I of clinical Romão et al. described the cytotoxic activity for Cp complexes
2
–6
5
2 2 4 2
trials, TDC did not pass through phase II mainly due to its of the type [(η -Cp′)Mo(CO) L ][BF ], where L is a bidentate
7
low efficiency toward metastatic renal cells and breast carci- ligand. Subsequent research implied that intrinsic biological
noma. Subsequent studies have proposed many complexes of activity is rather dependent on the character of the coordinated
8
various transition metals and differently modified Cp for bio- chelating ligand than on substitution in the π-ligand
1
2–15
periphery.
We have recently reported the second gene-
ration of Cp molybdenum complexes with considerably
enhanced cytotoxic activity. The improvement was achieved
a
16
Department of General and Inorganic Chemistry, Faculty of Chemical Technology,
University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic.
E-mail: jaromir.vinklarek@upce.cz
by the functionalization of the π-ligand with the polar
ammonium moiety leading to higher solubility in aqueous
media.
Recently, heterocyclic compounds, e.g. indole or thiophene,
have attracted significant attention due to their pronounced
anti-tumor activities. In the case of the transition metal com-
plexes, the heterocyclic fragment is introduced into the frame-
work of various spectator ligands improving the overall cyto-
static activity considerably. This approach has been used for
the assembly of highly active Cu(II) and Au(I) complexes with
b
Department of Analytical Chemistry, Faculty of Chemical Technology, University of
Pardubice, Studentská 573, 53210 Pardubice, Czech Republic
c
Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové,
Charles University in Prague, Šimkova 870, 500 01 Hradec Králové, Czech Republic
d
Department of Inorganic Chemistry, Faculty of Science, Charles University in
Prague, Hlavova 2030/8, 128 43 Prague 2, Czech Republic
e
2
nd Department of Internal Medicine – Gastroenterology, Charles University,
Faculty of Medicine in Hradec Králové, University Hospital Hradec Králové,
Czech Republic
f
Institute of Chemistry and Technology of Macromolecular Materials, Faculty of
Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice,
Czech Republic
thiophene-derivatized terpyridine and amidophosphine
17,18
ligand, respectively.
The significant anti-cancer activity has
†
Electronic supplementary information (ESI) available: Numbering schemes for
also been described for titanocene dichloride with Cp rings
the ligands. CCDC 1853492 (2) and 1853493 (4). For ESI and crystallographic
1
9
data in CIF or other electronic format see DOI: 10.1039/c9dt01698h
being modified by sulfur-containing functions. Although the
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mechanism regarding how the sulfur atom from the Cp side existence of two conformers arising from a different orien-
3
chain affects cytotoxicity has not been revealed, the substituted tation of the η -bonded allyl ligand (exo, endo). The species
TDC exhibits cytotoxicity toward the pig kidney cell line were assigned based on the strong ring-current effect of the
6
LLC-PK approximately one order of magnitude stronger com- indenyl C -ring shifting particular signals of the allyl ligand
1
9
meso
anti
pared to unsubstituted TDC.
(H
of 2-exo, H
of 2-endo) to higher fields when compared
The aim of this work is to synthesize indenyl molybdenum to the unsubstituted Cp analogue. The exo/endo ratio of 4 : 1
5
N,N
complexes of the type [(η -Ind′)Mo(CO)
2
(
4
L)][BF ], where Ind′ nears the values previously reported for complexes with the
N,N
is indenyl substituted by the thiophene function and
L is unsubstituted indenyl ligand or indenyl modified 1-position
the bidentate N,N-chelating ligand and evaluate the effect of by various functions (e.g. alkyl or amine and methoxy group in
the modification of cytostatic activity. Modification with the the side chain) confirming rather a minor effect of this kind of
1
4,16,22–24
thiophene function seems to be suitable for the suggested modification on the conformational equilibria.
application due to the relatively cheap and straightforward syn-
1
13
1
The H and C{ H} APT NMR spectra were used for the elu-
3
a
7a
thesis, which enables the preparation of the samples on a cidation of indenyl hapticity. Resonances of C and C (for
gram scale.
numbering of the ligands see Scheme S1 in the ESI†) in the
upfield region (∼112 ppm) suggest the expected η -coordi-
5
nation mode. The confirmation of the indenyl hapticity is pro-
2
3
1
vided by the positions of H and H in the H NMR spectrum.
The signal assignment was done using 2D NMR techniques.
Results and discussion
Synthesis of allyl molybdenum precursors
1
13
H– C HMBC revealed the interactions of the methylene
2
Indene, substituted by the thiophene group, C H CH C H S bridge (δ = 4.01 and 4.53 ppm) protons with carbon C (δ =
9
7
2
4
3
1
13
2
2
(
1), was synthesized according to Scheme 1. Firstly, 2-(chloro- 90.7 ppm) and subsequently H– C HSQC shows C –H con-
1
2
methyl)thiophene was prepared by using the protocol nectivity. Thus, the H NMR pattern with H at the higher field
described in the literature using the one-pot reaction of hydro- (δ = 5.18 ppm) than H (δ = 5.31 ppm) well correlates with the
gen chloride, formaldehyde and thiophene. This synthetic literature data for η -indenyl molybdenum compounds. The
approach gives a mixture of mono- and di-substituted thio- infrared spectrum of compound 2 shows two carbonyl stretch-
phenes easily separable by vacuum distillation. Subsequently, ing bands in the range typical of terminal carbonyl ligands
desired indene 1 was prepared by the reaction of 2-(chloro- (Table 1). The wavenumbers are comparable with those
3
2
0
5
25
5
3
methyl)thiophene and sodium indenide. We note that com- reported for the parent indenyl complex [(η -C H )(η -C H )Mo
pound 1 is also accessible by the deconjugation photolysis of (CO)2], suggesting a negligible effect of the indenyl modifi-
9
7
3
5
2
2
(
E)-2-(1-indenylidenemethyl)-thiophene in the presence of cation on the electron density on the molybdenum atom.
2
1
protic acid. Nevertheless, the method reported here includes
The molecular structure of 2 was determined by single-
high yields and cheap starting material as the main advantages. crystal X-ray diffraction analysis (see Fig. 1). The selected bond
Lithiation of 1 with nBuLi in THF gives substituted indenyl lengths and angles are summarized in the caption of Fig. 1.
lithium 1-Li, which readily reacts with the molybdenum The X-ray data confirm the absence of the interaction between
3
3
5
complex [(η -C H )Mo(CO) (NCMe) Cl] to afford the mixed thiophene and molybdenum. Thus, the η -allyl, η -indenyl and
3
5
2
5
2
3
allyl-indenyl compound [(η -C
9
H
6
CH
2
C
4
H
3
S)(η -C
3
H
5
)Mo(CO)
2
]
two carbonyls adopt a pseudo-tetrahedral environment around
(
2) in a moderate yield (63%) (Scheme 2). Complex 2 is a yellow the molybdenum atom. Hapticity of the indenyl in the crystal
crystalline powder well soluble in hot hexane, aromatic hydro- structure was quantified using the structural parameter Δ(M–C)
carbons and polar organic solvents. The solution structure of 2 and envelope fold angle Ω (for definition see the footnotes of
1
13
1
was evaluated by NMR spectroscopy. H and C{ H} NMR Table 2). Low values of Δ(M–C) [0.109(17) Å] and Ω [4.4(2)°]
5
spectra of 2 revealed two series of sharp signals reflecting the prove the η -coordination mode with a small contribution of
3
+2
5
η
that is typical of η -indenyl molybdenum complexes
1
4,16,24
shown in Table 2.
a
Table 1 IR and MS data of selected molybdenum compounds
Scheme 1 Synthesis of substituted indene 1.
a
ν (CO)
s
ν (CO)
ν
a
(BF)
m/z
2
3
4
5
6
7
1938
1971
1962
1963
1962
1966
1963
1970
1860
1889
1886
1883
1884
1892
1887
1880
—
—
1054
1055
1056
1052
1062
—
406
521
545
697
560
—
5
3
22
[(η -C
(η -C
9
H
H
7
)(η -C
7
3
H
5
)Mo(CO)
2
]
5
33
[
9
)Mo(CO)
2
(NCMe)
2
][BF
4
]
—
—
5
3
a
Wavenumbers are given in cm−1
.
Scheme 2 Synthesis of [(η -C
9
H
6
CH
2
C
4
H
3
S)(η -C
3
H
5
)Mo(CO)
2
] (2).
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Scheme 3 Synthesis of complexes 3–7.
1
The H NMR spectrum of the final product 3, measured in
CD Cl , revealed resonances of two coordinated acetonitrile
2
2
ligands, which appear at a considerably lower-field (2.47 and
2
7
2.41 ppm) than those of free acetonitrile (1.97 ppm).
Chemical shifts of the indenyl protons and quaternary carbons
5
confirm its η -coordination mode analogously as in the case of
1
3
2
. Two C resonances at 248 and 247 ppm evidence the pres-
ence of two carbonyl ligands in the coordination sphere of 3.
2
8–32
As usual for molybdenum carbonyl complexes,
the pro-
posed solution structure of 3 satisfies the 18-electron rule.
Thus, the S → Mo interaction was excluded despite the accessi-
ble lone pairs located on the sulfur atom. As recently demon-
2
4
strated on pyridyl-substituted Cp complexes, such an intra-
molecular interaction should kinetically stabilize η -indenyl,
3
5
3
9 6 2 4 3 3 5 2
Fig. 1 ORTEP view of [(η -C H CH C H S)(η -C H )Mo(CO) ] (2). Only
one position of the disordered thiophene function and allyl is shown for which is formed via the well-known acetonitrile-induced hap-
2
5
clarity. Thermal ellipsoids are drawn at the 30% probability level.
Selected bond length (Å) and angles (°): Mo1–Cg(Cp) = 2.0303(8), Mo1–
Cg(allyl) = 2.0441(15), Mo1–C15 = 1.9541(17), Mo1–C16 = 1.9436(17),
C15–Mo1–C16 = 80.8(7), Cg(Cp)–Mo–Cg(allyl) = 128.2(5).
totropic rearrangement.
3
The behavior of 3 was further investigated in MeCN-d in
order to evaluate the ability of the complex to undergo the hap-
totropic rearrangement. In the H NMR spectrum, sharp
1
signals were observed only for the protons of the thiophene
fragment and methylene bridge whereas the remaining
protons appear as broad unresolved resonances suggesting
that the thiophene function is not involved in the indenyl
Table 2 Structural parameters Δ(M–C) and Ω of indenyl molybdenum
complexes
a
b
Δ(M–C)
Ω
rearrangement. The character of the spectrum implies the
3
dynamic haptotropic shift
3
⇆
[(η -C H CH C H S)Mo
9
6
2
4
3
2
4
0.109(17)
0.136(3)
0.115(3)
0.128(2)
4.4(2)
5.0(4) (CO)
2 3 4
(NCMe) ][BF ] in acetonitrile. The IR spectrum of 3
3
5
32
[
(η -C
(η -C
3
H
H
5
)(η -Ind)Mo(CO)
2
]
3.3(3)
5.4(3)
shows two carbonyl stretching bands shifted significantly to
higher energies compared to the vibration modes of 2. Such
5
14
[
9
7
)Mo(CO) (bpy)][BF
2
4
]
a
Δ(M–C) is defined as the difference between the averages of the Mo– an observation, together with the typical broad B–F vibration
C4 and Mo–C5 distances and that of the Mo–C1, Mo–C2, and M–C3
−
−1
band of BF
4
at 1054 cm , confirms the successful protona-
3
4 b
distances.
Ω is the fold angle between planes defined by C1, C2,
34
tion and exchange of negatively charged allyl and the appear-
ance of the cationic complex. Moreover, the wavenumbers of
carbonyl vibration bands are practically identical with those
and C3 and that defined by C1, C3, C4, and C5.
5
33
described for the parent [(η -C H )Mo(CO) (NCMe) ][BF ],
9
6
2
2
4
Synthesis and characterization of the acetonitrile complex
providing further relevant evidence that the thiophene func-
tion is not involved in the donor–acceptor interaction with the
central metal. Complex 3 was further characterized by mass
spectroscopy (MS). The base peak (m/z = 406) was assigned to
The allyl ligand of 2 is readily cleaved by fluoroboric acid in
the presence of acetonitrile to afford the cationic complex [(η -
5
C
9
H
6
2 4 3 2 2 4
CH C H S)Mo(CO) (NCMe) ][BF ] (3) in excellent yield
+
species with one acetonitrile cleaved [M–MeCN] . The
(
91%) (Scheme 3). Although we expected that protonation of
3
2
spectrum also gives two peaks corresponding to the loss of
η -allyl should produce the η -propene intermediate similarly
as described for the parent complex [(η -C
(
+
+
5
3
carbonyl: 419 [M–CO] , 378 [M–CO–MeCN] .
5
H
5
)(η -C
CO)2], the suggested intermediate was not isolated even at
30 °C. The attempt to follow the reaction in situ in CD Cl by
3 5
H )Mo
2
6
Synthesis of complexes with N,N-chelating ligands
−
2
2
1
H NMR led only to free propene, which appears as the only As expected, the acetonitrile ligands of 3 are readily exchanged
traceable decomposition product.
by various proligands, including 1,10-phenanthroline (phen),
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4
2
,7-diphenyl-1,10-phenanthroline (Ph phen), 1,10-phenanthro-
line-5-amine (NH phen) or 2,2′-bipyridine (bpy), to afford com-
2
5
plexes of type [(η -C
bpy (4), L = phen (5), L
Scheme 3). Considering the cationic character of 4–7 and
9
H
6
CH
2
C
4
H
3
S)Mo(CO)
2
L
2
][BF
4
], where L
2
=
2
2
= Ph
2
phen (6) and L
2
= NH
2
phen (7)
(
stronger Mo–N bonds, the MS technique was used to prove the
coordination of the chelating ligands. The base peaks in the
spectra revealed the formation of appropriate species
(
for details see Table 1) and a progression in the isotope
pattern by m/z = 1 proving the monoanionic character of 4–7.
1
13
1
The H and C{ H} APT NMR spectra of 4–7 confirm both
5
the η -coordination of the indenyl and successful acetonitrile
exchange by N,N-chelating ligands. In the case of 7, the NMR
spectra contain two series of sharp signals in a ratio of 1 : 1.
The spectra reflected the existence of two enantiomers in a
solution formed due to the coordinated unsymmetrical
NH phen ligand. IR spectra of 4–7 showed two carbonyl
2
stretching bands slightly shifted to lower energies compared to
those of 3. The shift can be rationalized through the somewhat
stronger σ-donating ability of the bidentate ligands compared
to that of acetonitrile, further confirming the presence of the
N,N-chelating ligand in the coordination sphere of 4–7.
The solid-state structure of compound 4 was determined by
single-crystal X-ray diffraction analysis (Fig. 2). Selected bond
distances and bond angles are listed in the caption of Fig. 2.
The coordination sphere around the central molybdenum
adopts a distorted square-pyramidal molecular geometry with
indenyl in the apical position. The basal plane contains two
cis-arranged carbonyls and nitrogen donors of 2,2′-bipyridine.
The bidentate ligand is located below the annulated benzene
ring of the indenyl representing the most common confor-
5
+
Fig. 2 ORTEP view of [(η -C
9
H
6
CH
2
C
4
H
3
S)Mo(CO)
2
(bpy)] present in
crystal structure of 4. Thermal ellipsoids are drawn at the 20% probability
level. Selected bond length (Å) and angles (°): Mo1–Cg(Cp) = 1.9965(17),
Mo1–C16 = 1.970(3), Mo1–C15 = 1.962(3), Mo1–N1 = 2.190(3), Mo1–N2 =
2
.183(3), C15–Mo1–C16 = 73.3(1), N1–Mo1–N2 = 72.8(1).
5
mation of the indenyl complexes of the type [(η -Ind′)Mo
+
14,16,30,35
(CO)
2
L
2
]
reported for various chelates so far.
Low
5
values of structural parameters Δ(M–C) and Ω confirm the η
coordination mode of the indenyl, as summarized in Table 2.
Cytotoxicity of chelate complexes
In the first step, the cytotoxic effects of all synthesized molyb-
denum complexes 3–7 were evaluated against human
T-lymphocytic leukemia cells MOLT-4 in the exponential
growth phase at 24 h interval of treatment. Our working group
uses the suspension cell line MOLT-4 as a standard cancer line
1
4–16,36
for the initial cytotoxicity assessment of complexes.
The
cytotoxicity of compounds in terms of 50% maximal inhibitory
concentration (IC50) values was determined using the standard
WST-1 cell proliferation and viability assays. This assay is
based on the reduction of the tetrazolium salt 2-(4-iodo-
Fig. 3 Cytotoxicity effect of complexes 3–7 determined in the MOLT-4
phenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium cells after 24 h of treatment using WST-1 assay. The results shown are
sodium salt (WST-1) to a colored substance by mitochondrial mean values ± SD of three independent replications.
dehydrogenases of viable cells, and thus reflects combined
drug treatment-induced changes in proliferation, growth and
viability. The dose-dependent curves illustrating the effect of than the parent acetonitrile complex 3. By considering the
the increasing complex concentrations on the viability of the structure and cytotoxicity of the newly prepared complexes 4–7
MOLT-4 cells relative to untreated control cells are presented versus parent compound 3 is evident that the activity is mark-
in Fig. 3 together with the obtained IC50 values. Our results edly improved through the acetonitrile ligand exchange by the
show that all complexes 4–7 show considerably higher activity 2,2′-bypyridine or 1,10-phenanthroline framework. However, it
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is crucial to point out that complexes bearing the thiophene
The non-cancer cells MRC-5 were the least effective. It was
function show a remarkable improvement in the cytotoxic found that the cytotoxicity was dependent on the dose and
activity toward the MOLT-4 cells compared to the analogous incubation time, but the values of GP were higher than other
compounds with the unsubstituted indenyl ligand. Previously tested cell lines. The lung adenocarcinoma cell line A549 and
5
5
reported complexes [(η -Ind)Mo(CO) (phen)][BF ] and [(η -Ind) non-cancer foetal lung cells MRC-5 in 2 and 5 µM concen-
2
4
Mo(CO)
72 ± 11 μM, respectively. The highest overall cytotoxicity is 24 h for the study of the mechanism of effect.
exhibited by compound 6 bearing Ph phen with the IC value Cytotoxicity of complex 6 was also measured throughout
2 4
(bpy)][BF ] show IC50 values of 16.9 ± 0.7 μM and trations were chosen for comparison with a treatment time of
1
4
1
2
50
of 0.19 ± 0.02 μM. This complex has about two orders of mag- the concentration range on the chosen cell lines A549 and
nitude higher cytotoxic activity than that reported previously MRC-5. It was determined that the IC50 value is 1.0 ± 0.2 µM
1
5
for cisplatin (15.8 ± 1.9 μM). Due to the notable cytotoxic pro- for the A549 cells and 3.7 ± 0.4 µM for the MRC-5 cells after
perties of 6 among other complexes, it was used as a hit com- 24 h incubation (Fig. 4). These results indicate that complex 6
pound in the following experiments, in which we aimed to is more cytotoxic to the cancer cells A549 than to the non-
clarify possible cellular and molecular mechanisms.
cancer cell line MRC-5.
In the next phase, we evaluated the overall cytotoxic activity
In the next experiments (Fig. 5), it was determined that
and potency of complex 6 against cancer cells and compared it complex 6 causes considerable inhibition of A549 cell prolifer-
with non-cancer cells. Cytotoxic activity of 6 at selected concen- ation to 65.9% at 2 µM concentration and to 16.9% at 5 µM
trations (0.1, 2 and 5 µM) were determined at 24 and 48 h after concentration with the significantly decreased amount of
treatment against several human-derived cancer cells (MOLT-4, viable A549 cells and to 68.7% at 5 µM concentration after
A2780, A549, HT-29 and A2780cis) by WST-1 assay. MRC-5 cells 24 h of exposure. The different mechanism of the effect of
were chosen as non-cancer cells in parallel experiments. The compound 6 on the cell proliferation and viability occurred
results are shown in Table 3. The suspension leukemic cells after treatment of MRC-5 cells. Inconsistent with the previous
MOLT-4 was the most sensitive cell line to complex 6 in the results, the MRC-5 cells were more sensitive to the tested
experiments using 24 h treatment.
The obtained mean growth percent (GP) values for the
MOLT-4 cells were 44.1% (24 h) for the group treated with a
concentration of 0.1 µM. The cytotoxic effect against the
MOLT-4 cells increased in a dose-dependent manner for
complex 6 (2 and 5 µM) but the cytotoxicity was independent
of the incubation time. The A2780 cells showed GP values of
7
0.9% (24 h) and 16.9% (48 h) after treatment with 0.1 µM
complex 6. The GP values of the cancer cells A549 and HT-29
were 77.2% and 103.4%, respectively, after 24 h and 41.6%
and 63.0%, respectively, after 48 h treatment with 0.1 µM 6.
The cytotoxic effect against the suspension cell line MOLT-4
was dependent on the dose and incubation time. At the end of
the 48 h incubation period, it was observed that 2 and 5 µM
concentrations show very low GP values. The A549 cells
showed GP values of only 2.6% and 0.1% after treatment with
2
and 5 µM 6, respectively. Complex 6 was effective on the
A2780cis cell after treatment with 2 and 5 µM and the effect determined using WST-1 assay after 24 h of treatment. The results are
was higher after 48 h of treatment.
the mean values ± SD of three independent replications.
Fig. 4 The cytotoxic effect of complex 6 on the A549 and MRC-5
Table 3 The value of growth percent of the cells MOLT-4 (I), A2780 (II), A549 (III), HT-29 (IV), A2780cis (V) and MRC-5 (VI) after treatment with
a
complex 6 during incubation time 24 and 48 h
2
0
4 h
48 h
.1 µM
2 µM
5 µM
0.1 µM
2 µM
5 µM
I
II
III
IV
V
44.1 ± 9.7
70.9 ± 5.9
77.2 ± 6.2
103.4 ± 4.5
108.0 ± 54
109.1 ± 2.5
1.4 ± 0.1
20.3 ± 3.2
35.2 ± 70.1
66.4 ± 3.8
56.4 ± 2.8
68.4 ± 4.2
0.8 ± 0.8
7.9 ± 1.3
17.9 ± 2.0
41.3 ± 2.6
27.3 ± 1.1
39.3 ± 2.1
48.6 ± 1.3
16.9 ± 0.1
41.6 ± 0.8
63.0 ± 1.3
118.0 ± 9.2
79.9 ± 7.3
0.2 ± 0.1
1.0 ± 1.0
2.6 ± 1
19.0 ± 5.8
4.1 ± 0.5
46.1 ± 7.8
0.6 ± 0.2
1.2 ± 0.9
0.1 ± 0.2
5.6 ± 0.4
1.7 ± 0.3
5.9 ± 4.2
VI
a
Determined using WST-1 as means ± SD from three independent experiments.
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most studied transcriptional activator of the cell proliferation
3
7
and apoptosis controlling gene. Its upregulation was deter-
mined with the quantitative polymerase chain reaction (qPCR)
and western blotting analysis and the obtained data showed
that the amount of the p53 is maintained at a constant level
for the A549 cells (Fig. 6B). Similarly, the measurement of the
level of the p53 negative regulator, an E3 ubiquitin ligase the
mouse double minute 2 (MDM2), proved that the expression of
the p53 is not affected. The p53 contains a binding side for
the MDM2 E3 ubiquitin ligase, which is responsible for the
3
8
negative regulation of the p53. However, ubiquitination and
degradation activity controlled through the antagonizing
protein MDM2 is not affected for the tested concentrations of
complex 6. Regulation of the MDM2 protein is not affected sig-
nificantly by the expression of the MDM2 protein either as
shown by the results of the western blotting analysis (Fig. 6C)
and qPCR. It is well known that the regulation of the p53
activity is driven by posttranslational modifications. However,
the increased expression of the p53 protein stabilized by the
posttranslational modification affected by phosphorylation on
the serine 392 was not detected at 2 and 5 µM concentrations
of complex 6.
In contrast to the A549 cell exposure, the activation of the
programmed cell death in the MRC-5 cells appears after 24 h
treatment with 5 µM complex 6, as shown in Fig. 7A. The apop-
totic caspase cascade is induced via the intrinsic pathway by
activation of the caspases 9. Subsequent activation of the
downstream caspase 3/7 is likely associated with cellular
events, ultimately leading to a reduction in the viability of the
Fig. 5 The amount of A549 and MRC-5 cells after treatment with 6 for
24 h. The results are shown as mean ± SD of three independent experi-
ments. *Significantly different to control (P < 0.05).
complex showing a dose-dependent decrease of the prolifer-
ation and amount of the viable cells more obviously than
those observed for the A549 cells. The viability of the MRC-5
cells was inhibited to 79.7% and 8.2% by the treatment with 2
and 5 µM, respectively. The proliferation of the MRC-5 cells
reached only 58.6% and 5.7% relative to the untreated control
at the same determined concentrations.
The following experiments were performed in order to
obtain a preliminary insight into the mechanism of the cyto-
toxic effect of complex 6. A different effect on the viability of
the A549 and MRC-5 cells was explored by the evaluation of
the apoptotic process in both cell types using caspase activity.
As shown in the Fig. 6A, the apoptotic death is not activated in
A549 cells. The tumor suppressor protein p53 (p53) is the
Fig. 6 Effect of 6 on the programmed cell death of A549 cells after
Fig. 7 Effect of 6 on the programmed cell death of MRC-5 cells after
24 h of incubation. (A) Activity of caspase 3/7, caspase 8 and caspase 9.
The results are shown as mean ± SD of three independent experiments.
0.25 µM doxorubicin is used as the positive control (Ctrl+). *Significantly
different to control (Ctrl−) (P < 0.05). (B) Analysis of the expression levels
of proteins using the western blotting analysis. As a positive control, the
MRC-5 cells treated with 0.25 µM doxorubicin were used. To confirm
equal protein loading, the membranes were reincubated with β-actin.
Representative results of one of three independent experiments are
shown. (C) Analysis of cell gene using qPCR. Representative results of
one of three independent experiments are shown.
2
4 h of incubation. (A) Activity of caspase 3/7, caspase 8 and caspase 9.
The results shown are mean ± SD of three independent experiments.
.25 µM doxorubicin is used as positive control (Ctrl+). *Significantly
0
different to control (Ctrl−) (P < 0.05). (B) Analysis of the expression levels
of proteins using western blotting analysis. As a positive control, the
A549 cells treated with 0.25 µM doxorubicin were used. To confirm
equal protein loading, the membranes were reincubated with β-actin.
Representative results of one of three independent experiments are
shown. (C) Analysis of cell gene using qPCR. Representative results of
one of three independent experiments are shown.
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MRC-5 cells. To explore this, we determined the amount of the
gene for MDM2 following complex 6 treatment and found sig-
nificantly up-regulated expression at 2 µM concentration and
down-regulated expression at 5 µM concentration (Fig. 7B).
This outcome corresponds well with the change in the level of
the protein MDM2 determined by western blotting analysis
(Fig. 7C). The down-regulation of the MDM2 allows the stabi-
lization of the p53 protein via posttranslational modifications,
transfer of the p53 into the nucleus and the increase of its
3
9,40
transcriptional function.
Since the treatment with complex 6 significantly decreased
the amount of the A549 and MRC-5 cells, we became curious
whether this treatment also influences cell cycle progression.
The effect of complex 6 on cell cycle progression was evaluated
by propidium iodide staining and flow cytometry analysis. As
shown in Fig. 8A, exposure of the A549 cells to 2 µM 6 caused a
decrease in the S phase cells to 16.5% against the non-treated
control cells (23.6%) and arrested cell cycle in the G2 phase
(21.2%) compared to the non-treated cells (13.2%). For the dose
effect, the treatment with a higher dose was determined.
Complex 6 at 5 µM concentration does not have any impact on
all G1, S, and G2 phase populations compared to the negative
Fig. 9 Analysis of gene expression (A) using qPCR and protein levels
using western blotting analysis (B) in A549 cells after treatment with 6
for 24 h. As a positive control, the A549 cells treated with 0.25 µM doxo-
control. Only the G1 phase decreased from 63.1% to 55.9%. rubicin were used. To confirm equal protein loading, the membranes
were reincubated with β-actin. Representative results of one of the three
independent experiments are shown.
These results strongly suggest that the effect of 6 on cell-cycle
distribution changes in A549 cells is not dependent on the
applied concentration. The cell cycle distribution of the MRC-5
cells did not change after the application of complex 6 (Fig. 8B).
From the aforementioned results, it is obvious that the (ser) 345 leads to the activation of the Chk1. The amount of
main mechanism of complex 6 is the inhibition of the prolifer- the Chk1_ser345 also increases with complex 6 in a dose-
ation of A549 cells. Blocking of the cell proliferation is a very dependent fashion. The growth of the checkpoint activity is
complicated process induced and controlled by multiple mole- associated with an increase in the p21 transcription, of which
cular changes. The gene for cyclin B and the cyclin-dependent the activity increases with the concentration of complex 6 as
kinase inhibitor-interacting protein 1 (p21) was analyzed with proved by western blotting analysis and confirmed by qPCR
the qPCR (Fig. 9A) and the alterations in the protein levels analysis. Western blotting analysis also proves that the amount
were established by western blotting analysis (Fig. 9B). of the protein proliferating cell nuclear antigen (PCNA)
Western blotting analysis shows dose-dependent up-regulation decreases with increasing concentration (from 2 to 5 µM) of
check point kinases 1 (Chk1). Phosphorylation on the serine complex 6. Interaction of the Chk1 with the p21 prevents the
PCNA from forming the active complex with the DNA polymer-
ase. This leads to the inhibition of the DNA synthesis and pro-
vokes the blocking of the cell cycle at the G1/S checkpoint
4
1
alternatively in later phases of the cycle. Simultaneously, the
expression of gene CCNB1 for cyclin B is inhibited in a dose-
dependent manner by complex 6. The combination of impact
of these proteins leads to the anti-proliferative effect of
complex 6 as was proved by the previous analysis of the viabi-
lity and the proliferation.
It was proved that the MRC-5 cells have a different effect
mechanism compared to the A549 cells. The qPCR analysis of
the gene for the cyclin B1 and p21 was also employed in the
MRC-5 cells (Fig. 10A). The results at the protein level were
confirmed by western blotting analysis (Fig. 10B). The analysis
of the p21 protein showed the same result as the gene and the
MDM2 protein estimation. Since a lower concentration of
complex 6 (2 µM) causes an increase in the expression of the
p21, the use of the 5 µM concentration leads to a decrease in
the amount of gene for the p21. Similar to the A549 cells, the
Fig. 8 Cell cycle analysis of 6 in A549 (A) and MRC-5 (B) cells after 24 h
incubation period. Results are shown as mean of three independent
experiments. 0.25 µM doxorubicin is used as a positive control (Ctrl+).
*
Significantly different to control (Ctrl−) (P < 0.05).
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complex on the tumor A549 in the lung carcinoma cell line.
Moreover, a considerable advantage is the observation of
different effect on the non-tumor MRC-5 lung fibroblast cell
line. The unusual properties of complex 6 are very promising
for future investigation that could reveal further selective anti-
tumor effects.
Experimental
Methods and materials
All experiments were performed under argon and dried using
4
2
standard methods. Starting materials were available com-
mercially or prepared according to the literature procedures:
3
22
[
(η -C
3 5 2 2
H )Mo(CO) (NCMe) Cl] (10).
Measurements
Infrared spectra were recorded in the 4000–400 cm− region
1
with a Nicolet iS50 FTIR spectrometer using a diamond smart
Fig. 10 Analysis of gene expression (A) using qPCR and protein levels
using western blotting analysis (B) in MRC-5 cells after treatment with 6
for 24 h. As a positive control, the MRC-5 cells treated with 0.25 µM
doxorubicin were used. To confirm equal protein loading, the mem-
1
13
1
orbit ATR. H and C{ H} NMR spectra were recorded at 300 K
on Bruker 400 Avance and Bruker 500 Avance spectrometers.
1
13
The H and C resonances were assigned using 2D techniques
1
1
1
13
1
13
branes were reincubated with β-actin. Representative results of one of including H– H COSY, H– C HSQC or H– C HMBC.
the three independent experiments are shown.
Chemical shifts are given in ppm relative to the external stan-
dard (TMS). Deuterated solvents were purchased from Acros
Organics and purified by standard methods. Mass spec-
4
2
expression of the gene for the synthesis of the cyclin B1 is trometry was performed with a quadruple mass spectrometer
inhibited. Simultaneously, it was shown that the amount of (LCMS 2010, Shimadzu, Japan). The sample was injected into
the Chk1 in the MRC-5 cells is maintained at a constant level the mass spectrometer with an infusion mode at a constant
at 2 and 5 µM concentrations but the increasing concentration flow rate of 10 μl min− . Electrospray ionization-mass spec-
causes an increase in the expression of the phosphorylated trometry (ESI-MS) was used for the identification of the ana-
1
form of the Chk1 on the ser345.
lyzed samples.
Synthesis of C H CH C H S (1)
9
7
2
4
3
1
50 mL of concentrated HCl was pre-cooled to 0 °C in an
Conclusions
Erlenmeyer flask and 50 mL of thiophene was added. The HCl
A series of indenyl molybdenum(II) compounds bearing a thio- gas was bubbled through the mixture for 5 minutes. After that,
phene function 2–7 were described and characterized using 50 mL of 38% formaldehyde was added and the solution was
multinuclear NMR and IR spectroscopy, mass spectrometry, bubbled with gaseous HCl for further 20 minutes. Then, the
and elemental analysis and in the case of 2 and 4 by X-ray mixture was diluted with 200 mL of distilled water and
diffraction. Despite the molybdenum in the oxidation state II extracted three times with 50 mL of ether. Combined organic
can be considered as a soft Lewis acid and analogously sulfur phases were dried over anhydrous MgSO and the solvents
4
represent soft Lewis base, no compound with expected S → Mo were vacuum evaporated. Finally, the crude product was
intramolecular interaction was obtained. The acetonitrile vacuum distilled (65 °C, 22 mmHg) to afford 2-(chloromethyl)
complex 3 thus acts similarly to the compound with an unsub- thiophene (20.5 g, 24.8%). Calcd for C14
stituted indenyl ligand and exhibits a rapid dynamic haptotro- S, 15.10. Found: C, 79.43; H, 5.81; S, 15.22. H NMR (CDCl
H12S: C, 79.20; H, 5.70;
1
3
;
3
1
1
pic rearrangement in acetonitrile solution. The ligand 400 MHz; δ): 4.84 (s, 2H, CH ), 6.98 (dd, J( H, H) = 5.3 Hz,
2
3
4
3
1
1
4
3
1
1
exchange reaction of 3 using various N,N-chelating ligands
gives compounds 4–7, which represents potential anticancer
drugs with considerably increased cytotoxic activity compared
J( H, H) = 3.5 Hz, H , C
4
H
H
3
3
S), 7.11 (dd, J( H, H) = 3.5 Hz,
S), 7.33 (dd, J( H, H) = 5.3 Hz,
J( H, H) = 1.2 Hz, H , C H S). The solution of 5.9 g of inde-
1
1
3
3
1
1
J( H, H) = 1.2 Hz, H , C
4
1
1
5
4
3
to the complex with the unsubstituted indenyl ligand. The nide sodium (45 mmol) in 15 mL THF pre-cooled to −70 °C
highest cytotoxicity was observed for compound 6 bearing was added dropwise into the solution of 2-(chloromethyl)thio-
Ph phen. This complex has about two orders of magnitude phene in 10 mL of THF also cooled to −70 °C. Then, the
2
higher cytotoxic activity toward MOLT-4 than cisplatin.
mixture was slowly allowed to warm to room temperature and
Our results further provide a unique insight into the stirred overnight. After that, the solution was poured into ice-
regulation of mechanism of effect of the molybdenum cooled distilled water and extracted with ether (3 × 50 mL).
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5
5 4 2 4 3 2 2 4
The combined organic phases were dried over anhydrous Synthesis of [(η -C H CH C H S)Mo(NCMe) (CO) ][BF ] (3)
MgSO and the solvents were vacuum evaporated. The crude
4
A flame-dried Schlenk tube was charged with 808 mg of 2
2 mmol) and the complex was dissolved in a mixture of 10 mL
CH Cl and 209 μL (4 mmol) MeCN. The solution was cooled
product was vacuum distilled (105 °C, 10 Pa) to afford yellow
(
1
3
oily compound 1 (7.5 g, 35 mmol, 79%). H NMR (CDCl ;
2
2
3
1
1
3
4
00 MHz; δ): 3.48 (d, J( H, H) = 2.0 Hz, 2H, H , C H ), 4.22 (s,
9
7
4 2
to 0 °C and 274 μL of HBF ·Et O (2 mmol) was added dropwise
3
1
1
2
2
H, CH
2
), 6.40 (t, J( H, H) = 2.0 Hz, 1H, H , C
9
H
7
), 7.00 (dd,
within 30 minutes. After that, the mixture was allowed to warm
to room temperature and stirred overnight. Then, the solvents
were vacuum evaporated and oily residue was washed three
3
1
1
4
1
1
3
J( H, H) = 3.5 Hz, J( H, H) = 1.2 Hz, H , C
4
H
3
S), 7.04 (dd,
3
3
3
1
1
3
1
1
4
J( H, H) = 5.3 Hz, J( H, H) = 3.5 Hz, H , C H S), 7.25 (dd,
4
3
1
1
3
1
1
5
J( H, H) = 5.3 Hz, J( H, H) = 1.2 Hz, H , C
4
H
3
S), 7.32 (t,
times with 7 mL of Et
2
O. Finally, the crude product was recrys-
1
1
5/6
3
1
1
J( H, H) = 7.3 Hz, 1H, H , C
H, H , C H ), 7.45 (d, J( H, H) = 7.3 Hz, 1H, H , C H ),
9 7 9 7
.58 (d, J( H, H) = 7.3 Hz, 1H, H , C H ).
9 7
9
H
7
), 7.38 (t, J( H, H) = 7.3 Hz,
tallized from the CH Cl /Et O mixture to afford an orange
2
2
2
5
/6
3
1
1
4/7
1
7
powder of
3 (968 mg, 1.81 mmol, 91%). Calcd for
3
1
1
4/7
C H N O SMoBF : C, 44.94; H, 3.21; N, 5.24; S, 5.99. Found:
2
0
17
2
2
4
C, 44.58; H, 3.31; N, 5.42; S 5.73. Positive-ion MS (MeCN): m/z
+
+
3
5
(%) = 406 (100) [M–MeCN] , 419 [M–CO] , 378 [M–CO–MeCN].
Synthesis of [(η -C H )(η -C H CH C H S)Mo(CO) ] (2)
3
5
9
6
2
4
3
2
1
H NMR (CD CN; 500 MHz; δ ppm): 2.41 (s, 3H, CH CN), 2.47
3
3
Solution of C
9
H
7
CH
2
C
4
H
3
S (1; 1.06 g, 5 mmol) in 20 ml of
2
1
1
(
s, 3H, CH CN), 4.01 (d, J( H, H) = 16.2 Hz, 1H, CH ), 4.53 (d,
3 2
THF was cooled to −70 °C and 3.1 mL of nBuLi was added
dropwise (1.6 mol l
mixture was slowly allowed to warm to room temperature
within two hours. After that the mixture was added dropwise
via a cannula into a solution of [(η -C
1.55 g, 5 mmol) in 10 mL THF, pre-cooled to −70 °C. The reac-
tion mixture was stirred at room temperature overnight and
then vacuum evaporated to dryness. The solid residue was 13
extracted with hexane (3 × 20 mL). The vacuum evaporation
gives a yellow viscous liquid. The product was purified by
recrystallization from the hexane–ether mixture at −80 °C and
then vacuum dried. Yield: 1.36 g (3.35 mmol, 67%) to afford
yellow powder of 2. Calcd for C H O SMo: C, 56.16; H, 3.97;
2
1
1
3
1
1
2
J( H, H) = 16.2 Hz, 1H, CH ), 5.14 (d, J( H, H) = 2.7 Hz, 1H,
−1
solution in hexane, 5 mmol). The
2
3
1
1
3
H , C H ), 5.99 (d, J( H, H) = 2.7 Hz, 1H, H , C H ), 6.92 (dd,
9
1
6
9 6
3
3
1
3
1
1
J( H, H) = 5.1 Hz, J( H, H) = 3.5 Hz, 1H, H , C H S), 6.94
4
3
3
1
1
4
1
1
4
(
4 3
dd, J( H, H) = 3.5 Hz, J( H, H) = 1.2 Hz, 1H, H , C H S),
3
3
H
5
)Mo(CO)
2
(NCMe)
2
Cl]
3
1
1
4
1
1
2
7
7
H
.18 (t, J( H, H) = 5.1 Hz, J( H, H) = 1.2 Hz, 1H, H , C H S),
4
3
5/6
(
3
1
1
5/6
.54 (t, J( H, H) = 8.3 Hz, 1H, H , C H ), 7.60 (m, 2H, H
H ), 7.69 (d, J( H, H) = 8.3 Hz, 1H, H , C H ).
9 6 9 6
C NMR (C D ; 125.77 MHz; δ ppm): 4.8 (1C, CH CN), 4.9 (1C,
,
9
6
4/7
3
1
1
4/7
, C
6
6
3
3
2
CH CN), 28.4(1C, CH ), 76.3 (1C, C , C H ), 90.7 (1C, C ,
3
2
9
6
1
3a/7a
C
C
9
H
6
), 98,7 (1C, C , C
9
H
6
), 119.1 (1C, C
, C H ), 125.0 (1C, C , C H S), 125.3 (1C, C , C H ),
26.3 (1C, C , C H S), 127.51 (1C, C , C H ), 127.57 (1C, C ,
4 3 9 6
4 3 9 6 9 6
H S), 131.3 (1C, C , C H ), 131.6 (1C, C , C H ), 139.3
1C, CH CN), 139.9 (1C, CH CN), 142.6 (1C, C , C H S), 247.5
3 3 4 3
1C, CO), 248.7 (1C, CO). IR(ATR; cm ): 1971 vs. [ν (CO)], 1889
9 6
, C H ), 119.8 (1C,
3a/7a
4
4/7
9
6
4
3
9
6
3
2
5/6
1
C
(
(
5/6
4/7
1
9
16 2
1
S, 7.87. Found: C, 56.41; H, 4.01; S 7.81. H NMR [C
00 MHz; δ ppm; 4 : 1 mixture of 2-exo (a) and 2-endo (b)]:
6
D
6
;
1
5
−
−1
a
3
1
1
anti
0.91 and −0.80 [2 × (d, J( H, H) = 11.0 Hz, 1H of b, H
,
vs. [ν
s
(CO)], 1054 vs., br. [ν(BF)].
3
1
1
3
1
1
3 5
C H )], 0.00 (tt, J( H, H) = 11.0 Hz, J( H, H) = 7.4 Hz, 1H of
meso
3
1
1
a, H
3 5
, C H ), 0.90 and −0.97 [2 × (d, J( H, H) = 11.0 Hz, 1H
5
anti
3
1
1
syn
Synthesis of [(η -C
5
H
4
CH
2
C
4
H
3
S)Mo(CO)
2
(bpy)][BF
4
] (4)
Cl
), 4.06 (m, 2H of added dropwise via a cannula into a solution of bpy
of a, H , C H )], 2.13 (d, J( H, H) = 7.4 Hz, 2H of a, H
,
3
5
3
1
1
3
1
1
C
3
H
5
), −3.17 (tt, J( H, H) = 11.0 Hz, J( H, H) = 6.4 Hz, 1H The solution of 3 (266 mg, 0.5 mmol) in 5 mL of CH
2
2
was
meso
syn
of a, H
, C
3
H
5
), 3.35 (m, 2H of b, H , C
3
H
5
2
1
1
a, CH ), 4.14 (ABq, J( H, H) = 16.2 Hz, Δδ = 0.12 ppm, 2H of (78.1 mmol, 0.5 mg) in 3 mL of CH Cl . The mixture was
2
2
2
2
2
b, CH
2
), 5.18 (m, 1H of a, H , C
.31 (m, 1H of a, H , C
5
H
4
, 1H of b, H , C
, 1H of b, H , C
5
H
4
), stirred at room temperature overnight. After that, the solvents
), 6.38–6.74 (m, were vacuum evaporated and the red solid residue was washed
H of a, C H CH C H S; 7H of b, C H CH C H S). C NMR three times with 7 mL of Et O. Finally, the crude product was
3
3
5
7
5
H
4
5
H
4
1
3
9
6
2
4
3
9
6
2
4
3
2
(
C
6
D
6
; 125.77 MHz; δ ppm; 2-exo (a) and 2-endo (b)): 28.9 (1C recrystallized from the CH
2
Cl
(263 mg, 0.43 mmol, 87%). Calcd for:
9.6 (1C of a, C , C H ), 55.2 (1C of b, C , C H ), 55.5 (1C C H N O SMoBF C, 51.31; H, 3.15; N, 4.61; S, 5.26. Found:
2 2
/Et O mixture to afford a red
1
/3
2 2 3 5
of a, CH ), 29.5 (1C of b, CH ), 48.7 (1C of a, C , C H ), powder of 4
4
of b, C , C
C
1
/3
1/3
3
5
3
5
26 19
2
2
4
1
/3
3
3
3 5 9 6
H ), 75.9 (1C of b, C , C H ), 78.5 (1C of a, C , C, 51.17; H, 3.54; N, 4.42; S 5.18. Positive-ion MS (MeCN): m/z
2
2
+ 1
9
H
6
), 84.6 (1C of a, C , C H ), 90.0 (1C of b, C , C H ), (%) = 521 (100) [M] . H NMR (CD CN; 500 MHz; δ ppm): 4.17
3 5 9 6 3
2
2
3
1
1
3
1
1
9
0.07 (1C of b, C , C H ), 90.08 (1C of a, C , C H ), 98.2 (1C of (d, J( H, H) = 15.7 Hz, 1H, CH ), 4.77 (d, J( H, H) = 15.7 Hz,
3
5
9
6
2
3
3
3a/7a
3
1
1
2
b, C , C
C
C H ), 112.2 (1C of b, C
1
1
9
H
6
), 100.8 (1C of a, C , C
), 111.7 (1C of a, C
9
H
6
), 111.5 (1C of a, C
), 112.0 (1C of b, C
,
,
2 9 6
1H, CH ), 5.64 (d, J( H, H) = 2.8 Hz, 1H, H , C H ), 6.48 (d,
3
a/7a
3a/7a
3
1
1
3
5,6
9
H
6
, C
9
H
6
9 6 9 6
J( H, H) = 2.8 Hz, 1H, H , C H ), 6.71 (m, 2H, H , C H ),
3
a/7a
3
1
1
4
1
1
4
, C H ), 122.3, 123.7, 124.2, 6.85 (dd, J( H, H) = 7.4 Hz, J( H, H) = 1.3 Hz 1H, H , C H S),
9 6 4 3
9
6
3
1
1
3
3
1
1
4 3
24.3, 124.4, 124.6, 124.68, 124.73, 124.8, 125.3, 125.4, 6.91 (d, J( H, H) = 3.8 Hz, 1H, H , C H S), 6.98 (d, J( H, H) =
25.8, 127.3, 128.7 (7C of a, C
2
3
1
1
4/7
9
H
6
CH
2
C
4
H
3
S; 7C of b, 3.8 Hz, 1H, H , C
4
H
3
S), 7.13 (d, J( H, H) = 7.9 Hz, 1H, H
,
1
1
3
1
1
4/7
C H CH C H S), 144.2 (1C of a, C , C H S), 144.5 (1C of b, C , C H ), 7.21 (d, J( H, H) = 7.9 Hz, 1H, H , C H ), 7.59 (t,
C
CO), 241.5 (1C of b, CO). IR(ATR; cm ): 1938 vs. [ν
1
9
6
2
4
3
4
3
9
6
9 6
3
3
1
1
3/8
1
1
4
H
3
S), 237.9 (1C of a, CO), 238.5 (1C of a, CO), 240,8 (1C of b,
8 2
J( H, H) = 5.7 Hz, 1H, H , C10H N ), 7.63 (t, J( H, H) =
−1
3/8
3
1
1
a 8 2
(CO)], 5.7 Hz, 1H, H , C10H N ), 8.10, (t, J( H, H) = 8.1 Hz, 2H,
4
,7
3
1
1
5,6
860 vs. [ν (CO)].
H , C H N ), 8.33 (d, J( H, H) = 8.1 Hz, 2H, H , C H N ),
10 8 2 10 8 2
s
This journal is © The Royal Society of Chemistry 2019
Dalton Trans.

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Paper
.21 (d, J( H, H) = 5.7 Hz, 1H, H , C10
Dalton Transactions
3
1
1
2/9
13
9
H
8
N
2
), 9.37 (d, (3 × 7 mL) and recrystallized from a CH
2
Cl
2
/Et
2
O mixture to
3
1
1
2/9
1
J( H, H) = 5.7 Hz, 1H, H , C H N ). C { H} NMR (CD CN; afford a red powder of 6 (301 mg, 0.38 mmol, 77%). Calcd
1
0
8
2
3
3
1
25.77 MHz; δ ppm): 27.9 (1C, CH
2
), 79.6 (1C, C , C
9
H
6
27 2 2 4
), 93.3 forC40H N O SMoBF : C, 61.22; H, 3.47; N, 3.57; S, 4.08.
, C H ), Found: C, 61.31; H, 3.58; N, 3.73; S 4.29. Positive-ion MS
9 6
2
1
3a/7a
(1C, C , C
9
H
6
), 99.9 (1C, C , C
9
H
6
), 118.9 (1C, C
3
a/7a
2
4/7
+ 1
1
19.1 (1C, C
, C H ), 124.8 (1C, C , C H S), 125.6 (1C, C
,
(MeCN): m/z (%) = 697 (100) [M] . H NMR (CD CN; 500 MHz;
3
9
4
6
/7
4
3
5/5′
2
1
1
C
9
H
6
), 126.1 (1C, C , C
9
H
6
), 126.6 (1C, C , C10
H
8
N
2
), 126.7 δ ppm): 4.28 (d, J( H, H) = 15.8 Hz, 1H, CH
2
), 4.88 (d,
5
/5′
3/3′
3/3′
2
1
1
3
1
1
(1C, C , C10
H
8
N
2
), 127.1 (1C, C , C10
H
8
N
2
), 127.2 (1C, C
,
J( H, H) = 15.8 Hz, 1H, CH ), 5.75 (d, J( H, H) = 2.8 Hz, 1H,
2
3
5/6
2
3
1
1
5/6
C H N ), 128.1 (1C, C , C H S), 130.4 (1C, C , C H ), 130.6 H , C H ), 6.38 (t, J( H, H) = 8.4 Hz, 1H, H , C H ), 6.45 (t,
1
0
8
2
/6
4
3
9
6
9
1
6
9 6
1
5
4
4/4′
3
1
5/6
3
1
(1C, C , C
9
H
6
), 144.1 (1C, C , C
4
H
3
S), 144.2 (1C, C
,
9 6
J( H, H) = 8.4 Hz, 1H, H , C H ), 6.62 (d, J( H, H) = 2.8 Hz,
4
/4′
4
3
3
1
1
4/7
C
10
H
8
N
2
), 144.5 (1C, C , C10H N ), 147.1 (1C, C , C H S), 1H, H , C H ), 6.82 (d, J( H, H) = 8.4 Hz, 1H, H , C H ), 6.91
8 2 4 3 9 6 9 6
2
/2′
2/2′
3
1
1
3
1
1
3
1
53.4 (1C, C , C H N ), 153.6 (1C, C , C H N ), 156.6 (1C, (dd, J( H, H) = 5.3 Hz, J( H, H) = 3.6 Hz, 1H, H , C H S),
1
0
8
2
10
8
2
4
3
6
/6′
6/6′
3
1
1
4
1
1
2
C
, C10
H
8
N
2
), 157.5 (1C, C , C10
H
8
N
2
), 250.7 (1C, CO), 7.00 (dd, J( H, H) = 3.6 Hz, J( H, H) = 1.0 Hz, 1H H , C
4
1
H
3
1
S),
−
1
3
1
1
4/7
3
2
53.3 (1C, CO). IR(ATR; cm ): 1962 vs. [ν
a
(CO)], 1886 vs. 7.09 (d, J( H, H) = 8.4 Hz, 1H, H , C
9
H
6
), 7.21 (dd, J( H, H)
4
1
1
4
[νs(CO)], 1055 vs., br. [ν(BF)].
= 5.3 Hz, J( H, H) = 1.0 Hz, 1H, H , C H S), 7.36 [m, 10 H,
4
3
3
1
1
3/8
(
(
C
C
6
6
H
H
5
)
2
C
12
H
H
6
6
N
N
2
2
], 7.91 [d, J( H, H)
=
5.6 Hz,1H,
5.6 Hz,1H,
H
H
,
,
5
Synthesis of [(η -C H CH C H S)Mo(CO) (phen)][BF ] (5)
3
1
1
3/8
5
4
2
4
3
2
4
5
)
2
C
12
)], 7.93 [d, J( H, H)
=
5
,6
The reaction was performed in the same way as that described (C H ) C H N )], 8.00 [s, 2H, H , (C H ) C H N )], 9.63 [d,
6
5 2 12
6
2
6
5 2 12
6
2
3
1
1
2/9
for 4 but with 3 (266 mg, 0.5 mmol) and phen (90.1 mg,
.5 mmol). The crude product was washed with diethyl ether
J( H, H)
J( H, H) = 5.6 Hz,1H, H , (C
=
5.6 Hz,1H,
H
,
(C
6
H
5
)
2
C
12
H
6
N
2
)], 9.74 [d,
3
1
1
2/9
13
1
0
6
H
5
)
2
C
12
H
6
N
2
)]. C { H} NMR
3
(3 × 7 mL) and recrystallized from a CH Cl /Et O mixture to (CD CN; 125.77 MHz; δ ppm): 28.0 (1C, CH ), 79.6 (1C, C ,
2 2 2 3 2
2
1
3a/
afford a red powder of 5 (233 mg, 0.37 mmol, 73%). Calcd
forC28 SMoBF : C, 53.16; H, 3.03; N, 4.43; S, 5.06.
9 6 9 6 9 6
C H ), 93.1 (1C, C , C H ), 100.2 (1C, C , C H ), 119.0 (1C, C
7
a
3a/7a
4/7
H
19
N
2
O
2
4
, C
9
H
6
), 119.2 (1C, C
9 6 9 6
, C H ), 124.7 (1C, C , C H ), 125.7
4
5/6
Found: C, 53.32; H, 3.21; N, 4.54; S 5.13. Positive-ion MS (1C, C , C H S), 126.1 [1C, C , (C H ) C H N )], 126.3 [1C,
4
3
6
5 2 12
3/8
6
2
+
1
5/6
(
MeCN): m/z (%) = 545 (100) [M] . H NMR (CD
3
CN; 500 MHz;
C
, (C
6
H
5
)
2
C
12
H
6
N
2
)], 126.40 [1C, C , (C
6
2
H
5
)
2
C
12
H
6
N
2
)],
2
1
1
3/8
δ ppm): 4.25 (d, J( H, H) = 15.7 Hz, 1H, CH ), 4.86 (d, 126.45 [1C, C , (C
J( H, H) = 15.7 Hz, 1H, CH ), 5.70 (d, J( H, H) = 2.7 Hz, 1H, (1C, C , C H ), 128.1 (1C, C , C H S), 130.1–130.3 [5C,
2
6
H
5
)
2
C
12
H
6
N
2
)], 126.6 (1C, C , C
4
H
3
S), 127.0
2
1
1
3
1
1
4/7
3
2
9
6
4
3
2
3
1
1
5
5/6
4a,6a
H , C
9
H
6
), 6.26 (t, J( H, H) = 7.8 Hz, 1H, H , C
9
1
H
6
), 6.34 (t, (C
6
H
H
5
)
)
2
C
12
H
H
6
N
N
2
); 1C,
C
,
C
9
H
6
], 130.7 [2C,
C
,
,
3
1
1
6
3
1
5/6
J( H, H) = 7.8 Hz, 1H, H , C
9
1
H
6
), 6.57 (d, J( H, H) = 2.7 Hz, (C
6
5
2
C
12
6
2
)], 130.8–131.1 [5C, (C
6
H
5
)
2
C
12
H
6
N
2
); 1C, C
3
3
1
4
q
1
1
(
6
7
1
H, H , C H ), 6.73 (d, J( H, H) = 7.8 Hz, 1H, H , C H ), 6.91 C H ], 136.3 [2C, C , (C H ) C H N )], 144.3 (1C, C , C H S),
9 6 9 6 9 6 6 6 2 4 3
5 2 12
3
3
1
1
3
4/7
4/7
dd, J(1H,1H) = 5.3 Hz, J( H, H) = 3.6 Hz, 1H, H , C
4
H
3
S), 145.5 [1C,
C
,
(C
)], 152.0 [2C, C
.8 Hz, 1H, H , C H ), 7.21 (dd, J( H, H) = 5.3 Hz, J( H, H) = [1C, C , (C H ) C H N )], 157.4 [1C, C , (C H ) C H N )],
6
H
5
)
2
C
12
H
6
N
2
)], 145.8 [1C,
C
,
3
1
1
2
3
1
1
10a,1a
.99 (d, J( H, H) = 3.6 Hz, 1H, H , C
4
H
3
S), 7.03 (d, J( H, H) = (C
6
H
5
)
2
C
12
H
6
N
2
, (C
6
H
5
)
2
C
12
H
6
N
2
)], 156.1
7
3
1
1
3
1
1
2/9
2/9
9
6
6
5 2 12
6
2
6
5 2 12
6 2
4
3
1
1
3
1
1
−1
.0 Hz, 1H, H , C
5.3 Hz, 2H, H , C12
4
3
H
3
S), 7.95 (dd, J( H, H) = 8.2 Hz, J( H, H) 251.0 (1C, CO), 253.5 (1C, CO).IR(ATR; cm ): 1962 vs. [ν
), 7.99 (dd, J( H, H) = 8.2 Hz, 1884 vs. [ν
J( H, H) = 5.3 Hz, 2H, H , C H N ), 8.07 (s, 2H, H
), 8.67 (dd, J( H, H) = 8.2 Hz, J( H, H) = 0.9 Hz, 2H,
8 2
, C12H N ), 9.60 (dd, J( H, H) = 5.4 Hz, J( H, H) = 0.9 Hz, The reaction was performed in the same way as that described
H, H C H N ), 9.71 (dd, J( H, H) = 5.4 Hz, J( H, H) = 0.9 for 4 but with 3 (266 mg, 0.5 mmol) and NH phen (97.6 mg,
a
(CO)],
/8
3
1
1
=
H
8
N
2
3/8
s
(CO)], 1052 vs., br. [ν(BF)].
3
1
1
5/6
,
1
2 8 2
5
3
1
1
4
1
1
Synthesis of [(η -C H CH C H S)Mo(CO) (NH phen)][BF ] (7)
5
4
2
4
3
2
2
4
C
H
1
12
,7
H
8
N
2
4
3
1
1
4
1
1
2
/9
3
1
1
4
1
1
1
2
8
2
2
2
/9
13
1
Hz 1H, H , C12
δ ppm): 28.0 (1C, CH
9
C
C
H
8
N
2
). C { H} NMR (CD
3
CN; 125.77 MHz; 0.5 mmol). The crude product was washed with diethyl ether
3
2
2
), 79.3 (1C, C , C
9
H
6
), 92.9 (1C, C , C
9
H
6
), (3 × 7 mL) and recrystallized from a MeCN/Et
2
O mixture to
afford a red powder of 7 (206 mg, 0.32 mmol, 64%). Calcd
S), 125.9 (1C, forC28 SMoBF : C, 52.11; H, 3.12; N, 6.51; S, 4.46.
), 126.6 (1C, C , Found: C, 52.23; H, 3.34; N, 6.70; S 4.29. Positive-ion MS
1
3a/7a
3a/7a
9.9 (1C, C , C H ), 118.9 (1C, C
, C H ), 119.1 (1C, C
,
9
6
9
6
4
7
9
3
H
, C12
6
), 124.8 (1C, C , C
9
H
6
), 125.7 (1C, C , C
4
H
3
H
20
N
3
O
2
4
/8
3/8
2
H
8
N
2
), 126.2 (1C, C , C12
H
8
N
2
4
3
+ 1
C H S), 126.9 (1C, C , C H ), 128.1 (1C, C , C H S), 128.4 (2C, (MeCN): m/z (%) = 560 (100) [M] . H NMR (CD CN; 400 MHz;
4
5
3
9
6
4
3
3
,6
5/6
5/6
2
1
1
C
1
(
C
C
8 2 9 6 9 6
, C12H N ), 129.9 (1C, C , C H ), 130.1 (1C, C , C H ), 1 : 1 mixture of conformers a and b; δ ppm): 4.18 (d, J( H, H)
4
a,6a
4,7
2
1
1
32.9 (2C, C
8 2 8 2 2
, C12H N ), 139.8 (2C, C , C12H N ), 144.3 = 6.5 Hz, 1H of a/b, CH ), 4.22 (d, J( H, H) = 6.5 Hz, 1H of a/
1
10a/11a
2
1
1
1C, C , C H S), 144.7 (1C, C
, C H N ), 145.0 (1C, b, 1H, CH ), 4.79 (d, J( H, H) = 9.5 Hz, 1H of a/b, 1H, CH ),
4
3
12
8
2
2
1
2
1
0a/C11a
2/9
2/9
2
1
, C12
H
8
N
2
), 156.9 (1C, C , C12
H
8
N
2
), 157.7 (1C, C
,
4.83 (d, J( H, H) = 9.5 Hz, 1H of a/b,1H, CH
2
), 5.47 (s-br, 2H
2
/9
3
1
1
12
H
8
N
2
), 156.9 (1C, C , C12
H
8
N
2
), 250.9 (1C, CO), 253.5 (1C, of a, 2H of b, NH , C12H N ), 5.65 (d, J( H, H) = 2.8 Hz, 1H of
2 9 3
−
1
2
5
CO). IR(ATR; cm ): 1963 vs. [ν (CO)], 1883 vs. [ν (CO)], 1056 a, 1H of b, H , C H ), 6.30 (m, 1H of a, 1H of b H , C H ), 6.38
a
s
9
6
9 6
6
3
vs., br. [ν(BF)].
9 6
(m, 1H of a, 1H of b H , C H ) 6.51 (m, 1H of a, 1H of b, H ,
3
1
1
4/7
C
9
H
6
), 6.69 (d, J( H, H) = 8.3 Hz, 1H of a/b, H , C
9
H
7
), 6.72
5
Synthesis of [(η -C
5
H
4
CH
2
C
4
H
3
S)Mo(CO)
2
(Ph
2
phen)][BF
4
] (6)
3
1
1
4
(
d, J( H, H) = 8.3 Hz, 1H of a, 1H of b, H , C H ), 6.89 (t,
9
7
3
1
1
3
The reaction was performed in the same way as that described
for 4 but with 3 (266 mg, 0.5 mmol) and Ph phen (166.2 mg, a/b, H , C12
.5 mmol). The crude product was washed with diethyl ether
J( H, H) = 5.0 Hz, 1H of a, 1H of b, H , C
4
H
3
S), 6.93 (s, 1H of
), 6.94 (s, 1H of a/b, H , C12 ), 6.97 (d,
J( H, H) = 5.0 Hz, 1H of a, 1H of b, H , C H S), 7.01 (d,
5
5
2
H
9
N
3
9 3
H N
3
1
1
2
0
4
3
Dalton Trans.
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View Article Online
Dalton Transactions
Paper
3
3
1
1
7
J( H, H) = 8.3 Hz, 1H of a, 1H of b, H , C
9
H
6
), 7.19 (d, Screening of cytotoxicity of the test complexes and cytotoxicity
1
1
4
J( H, H) = 5.0 Hz, 1H of a, 1H of b, H , C H S), 7.69 (m, 1H of assay over the broad concentration range
4
3
3
/8
3/8
2/9
2/9
a, 1H of b, H , C12
C H N ), 8.21 (d, J( H, H) = 8.0 Hz, 1H of a, 1H of b, H
12 9 3
C H N ), 8.64 (d, J( H, H) = 8.4 Hz, 1H of a, 1H of b, H
C
H
9
N
3
), 7.91 (m, 1H of a, 1H of b, H
,
,
,
Cytotoxicity was quantified using the ability of mitochondria
dehydrogenase to reduce of WST-1 to a water-soluble forma-
zan. WST-1 Cell Proliferation Reagent was obtained from
Roche Applied Science, Switzerland. The MOLT-4, A549 and
3
1
1
3
1
1
1
2
9
3
3
1
1
12
H
9
N
3
), 9.18, 9.29, 9.54, 9.64 (d, J( H, H) = 5.3 Hz, 2H of a,
4
,7
13
1
2
H of b, H , C12
H
9
N
3
). C { H} NMR (CD
3
CN; 125.77 MHz; δ
3
MRC-5 cells were cultivated at a concentration 25 × 10 cells per
3
ppm): 26.9 (1C of a, 1C of b, CH ), 78.1 (1C of a/b, C , C H ),
9
C
2
9
6
1
well of culture medium. The A549 and MRC-5 cells were culti-
vated at a concentration of 700 and 500 cells per well of culture
medium overnight, respectively. The MOLT-4 cells were treated in
a concentration range of 0.1, 0.4, 0.7, 1.5, 2.5, 4, 9, 15, 27, 50 µM
in a total volume of 200 µL, for test complexes 3–7 and they were
used in the concentration range of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5,
2
1.8 (1C of a, 1C of b, C , C
H
9
H
6
), 98.7 (1C of a, 1C of b, C ,
6
6
9
6
), 102.4 (1C of a/b, C , C12
H
9
N
3
), 102.5 (1C of a/b, C ,
, C H ), 118.1 (1C of a,
9 6
3
a/7a
C H N ), 117.9 (1C of a, 1C of b, C
1
2
9
3
3
a/7a
3/8
1
1
1
C
C of b, C
, C
9
H
6
), 123.4 (1C of a, 1C of b, C , C12
H
9
N
3
),
),
7
5
9 6 9
23.5 (1C of a, 1C of b, C , C H ), 123.6 (1C of a/b, C , C H
23.7 (1C of a/b, C , C H ), 124.6 (1C of a, 1C of b, C
6
5
3/8
,
9
6
1, 2, 5, 10 µM in a total volume 200 µL for complex 6 on the A549
4
12
H
9
N
3
2
), 124.8 (1C of a, 1C of b, C , C
4
H
3
S), 125.5 (1C of a, 1C
), 125.9 (1C of a/b,
C , C H ), 127.0 (1C of a, 1C of b, C , C H S), 128.8 (1C of a,
and MRC-5 cells. The well-plate was incubated for 24 h. WST-1
assay was carried out after the incubation. 50 µL WST-1 solution
was pipetted into each well and the plate was incubated for 3 h
and then absorbance at 440 nm was measured using a multiple
reader Tecan Infinite M200 (Tecan Group, Switzerland).
Cytotoxicity was calculated as the IC50 value using the statistic
software Origin Pro (version 9, OriginLab Corporation, USA).
Three independent experiments were performed; the results are
expressed as mean ± standard deviation (SD).
4
4 3 9 6
of b, C , C H S), 125.8 (1C of a/b, C , C H
4
3
9
6
4
3
5
/6
5/6
1
C of b, C , C
9 6 9 6
H ), 129.0 (1C of a, 1C of b, C , C H ), 133.7
2
/9
2/9
(1C of a, 1C of b, C , C12
H
9
N
3
), 135.2 (1C of a, 1C of b, C
,
5
5
C H N ), 138.3 (1C of a/b, C , C H N ), 138.6 (1C of a/b, C ,
C
C
a, 1C of b, C
C
C
1
2
9
3
12
9 3
1
12
H
9
N
3
), 143.3 (1C of a, 1C of b, C , C
4
H
3
S), 143.4 (1C of a/b,
, C12H N ), 144.3 (1C of
, C H N ), 144.6 (1C of a, 1C of b, C
4
a/6a
4a/6a
, C12
H
9
N
3
), 143.5 (1C of a/b, C
9 3
1
0a/11a
10a/11a
,
1
2
9 3
4
/7
12
H
/7
9
N
3
), 151.2 (1C of a/b, C , C12
H
9
N
3
), 152.1 (1C of a/b,
4
4/7
9 3 9 3
, C12H N ), 155.6 (1C of a/b, C , C12H N ), 156.5 (1C of
Screening of cytotoxicity on the cell lines
4
/7
a/b, C , C H N ), 250.3, 250.5, 252.8, 253.0 (2C of a, 2C of b,
1
2
9 3
CO). IR(ATR; cm− ): 1966 vs. [ν
1
Selected human tumor cell lines (MOLT-4 – leukemic lympho-
blastoid, HT-29 – colon adenocarcinoma, A549 – lung adeno-
carcinoma, A2780 – ovarian adenocarcinoma, A2780cis – cis-
platin-resistant ovarian adenocarcinoma) and human non-
cancer cell line MRC-5 (human foetal lung) were purchased
a
s
(CO)], 1892 vs. [ν (CO)], 1062
vs., br. [ν(BF)].
Crystallography
The X-ray data for the single-crystals of compounds 2 and 4 from either ATCC, USA, or Sigma Aldrich, USA, and cultured
were collected on Bruker D8 VENTURE Kappa Duo according to the provider’s culture method guidelines. The
a
PHOTON100 equipped with a IμS micro-focus sealed tube cells were cultivated with a final concentration 0.1, 2 and 5 µM
MoKα (λ = 0.77015 Å) at a temperature of 150(2) K. The struc- of complex 6 after 24 h and 48 h, in a previously established
4
3
tures were solved by direct methods (SHELXS 2014/7) and optimal density in a 96-well plate. After the incubation time,
2
44
refined by full matrix least squares based on F (SHELXL97).
WST-1 proliferation assay (Roche Applied Science, Switzerland)
The position of atoms in the disordered parts in both struc- was performed according to the manufacturer’s instructions
tures was found on the difference Fourier map; however the and the absorbance was measured using Tecan Infinite M200
atoms could be refined only with the restraints on their pos- (Tecan Group, Switzerland). The value of cytotoxicity was
itional and displacement parameters. The allyl ligand of 2 is expressed as the percentage of proliferation/viability of control,
disordered. The hydrogen with a high occupancy could be dis- non-treated cells (100%), expressed as GP ± SD of the absor-
cerned on the difference Fourier map, but the hydrogen with bance measured from three different experiments.
2
2% occupancy has to be placed into the calculated position,
which is in the plane of the allyl, thus not reflecting the Determination of viability and proliferation
coordination to Mo. CCDC 1853493 (for 2) and 1853492 (for 4)
contain the supplementary crystallographic data for this
paper.
4
5
1
5 × 10 A549 cells and 3 × 10 MRC-5 cells in 5 µL of cultiva-
tion medium were treated with 0.1, 2 and 5 µM complex 6.
Non-treated A549 and MRC-5 cells were used as controls. The
cells were incubated for 24 h. The amount of viable and death
cells were counted after trypsinization of the cells followed by
coloring of the cells with Trypan blue. The results from the
three independent experiments are expressed as mean ± SD.
Preparing of complexes for biological tests
The culture media and dimethyl sulfoxide (DMSO; Sigma-
Aldrich, USA) were used to prepare the solution of the test
complexes. Each test complex was dissolved just before the
cells were affected. The maximum used concentration of
Determination of caspases activity
DMSO was 0.5% w/v because this concentration did not have The activity of caspases 3/7, 8 and 9 was measured for detec-
any significant negative effect on cells.
tion apoptosis using the commercial kit Caspase-Glo Assays
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Dalton Trans.

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Paper
Dalton Transactions
6
(
Promega, USA). 2 × 10 A549 and MRC-5 cells in 5 mL were The chemiluminescent signal was detected using the BM
4
incubated with complex 6 for 24 h. Then 1 × 10 cells in 10 µL Chemiluminescence Western Blotting Substrate (POD) from
cultivation medium were pipetted into a 96 well-plate with Sigma-Aldrich, USA, using the PXi imaging system (Syngene,
4
0 µL medium and then 50 µL reagent was added. The lumi- Cambridge, UK). β-Actin was detected in each membrane to
nescent signal was measured using a multiple reader Tecan ensure equal protein loading.
Infinite M200 (Tecan Group, Switzerland) after 30 min at room
Statistical analysis
temperature. Doxorubicin in 0.25 µM concentration was used
as a positive control. The results from three independent The measured results of WST-1 were processed as mean ± SD
experiments are expressed as mean ± SD.
of three independent experiments in the Microsoft Office Excel
003 (Microsoft Inc., Redmond, USA). Using software Origin
Pro (version 8, Microcal Software, Inc. Northampton, MA,
2
Analysis of the cell cycle
The principle of cell cycle analysis using a flow cytometer is USA), in the block “Analysis” by statistical method “ANOVA”
based on the stereochemical bond of propidium iodide to the graph of the normalized cell viability of cells depending on
5
cell’s DNA. 6 × 10 A549 and MRC-5 cells in 5 mL medium the concentration of the test complexes was constructed.
were treated with 2 and 5 µM 6 and incubated for 24 h. The
The other experiments were evaluated as mean ± SD of
cells were washed with cold PBS and fixed with 70% ethanol. three independent experiments in the Microsoft Office Excel
Detection of low molecular-weight fragments of DNA was done 2003 (Microsoft Inc., Redmond, USA) again. The statistical
after incubation for 5 min at room temperature in a buffer analysis of significant values between the groups was evaluated
2 4
(192 mL 0.2 M Na HPO + 8 mL of 0.1 M citric acid, pH 7.8) using Student’s t-test and P values ≤0.05 were considered to be
and then labelled with propidium iodide in Vindelov’s solu- significant.
tion for 1 h at 37 °C. The analysis was determined using the
flow cytometer CyAn ADP (Beckman Coulter, USA). Data were
analyzed using Multicycle AV software (Phoenix Flow systems, Conflicts of interest
USA). All reagents were obtained from Sigma-Aldrich, USA.
Three independent experiments were performed; the results
are expressed as mean ± SD.
The authors declare no conflicts of interest.
RNA isolation and quantitative polymerase chain reaction
Acknowledgements
(qPCR)
This work was supported by the Ministry of Education of the
Czech Republic (UPA SG380006 and Progres/UK Q40/01).
The cells were scraped in PBS and dissolved in RTL lysis
buffer. Total cellular RNA was extracted using an RNeasy Mini
kit according to manufacturer’s instructions (Qiagen, Hilde,
Germany). RNA was reverse transcribed using a cDNA Reverse
Transcription kit and quantified with TaqMan Gene
Notes and references
Expression
Hs00234753_m1,
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Hs00233365_m1,
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Dalton Trans.