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From Sunscreen to Anticancer Agent: Ruthenium(II) Arene Avobenzone Complexes Display Potent Anticancer Activity
Article
pubs.acs.org/Organometallics
From Sunscreen to Anticancer Agent: Ruthenium(II) Arene
Avobenzone Complexes Display Potent Anticancer Activity
Riccardo Pettinari,*,† Fabio Marchetti,‡ Agnese Petrini,† Claudio Pettinari,† Giulio Lupidi,†
Piotr Smoleński,§ Rosario Scopelliti,∥ Tina Riedel,∥ and Paul J. Dyson*,∥
†
School of Pharmacy and ‡School of Science and Technology, University of Camerino, via S. Agostino 1, 62032 Camerino, Macerata,
Italy
§
Faculty of Chemistry, University of Wrocław, ul. F. Joliot-Curie 14, 50-383 Wrocław, Poland
∥
Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
S Supporting Information
*
ABSTRACT: A series of ruthenium(II) arene derivatives (arene = cymene (cym),
hexamethylbenzene (hmb)) containing avobenzone (1-(4-tert-butylphenyl)-3-(4methoxyphenyl)propane-1,3-dione, AVBH) and PTA (1,3,5-triaza-7-phosphaadamantane)
or PTA-Me (N-methyl-1,3,5-triaza-7-phosphaadamantane cation) have been synthesized
and fully characterized. Three types of complexes have been obtained: i.e., neutral
[Ru(arene)(AVB)Cl] (1, arene = cym; 2, arene = hmb), monocationic [Ru(arene)(AVB)(PTA)][SO3CF3] (3, arene = cym; 4, arene = hmb), and dicationic [Ru(arene)(AVB)(PTA-Me)][SO3CF3][BF4] (5, arene = cym; 6, arene = hmb). The solid-state
structures of 1 and 2 were determined by single-crystal X-ray diffraction. The cytotoxicity
of the complexes has been evaluated in vitro against human ovarian carcinoma cells, A2780
and A2780cisR, as well as against nontumorous Human Embryonic Kidney (HEK293)
cells. The ionic complexes with hydrophilic PTA and PTA-Me ligands in 3−6 are
considerably more active than the neutral complexes 1 and 2.
■
INTRODUCTION
In recent years, ruthenium complexes have emerged as
promising antitumor agents with potential uses in platinumresistant tumors and solid metastasis.1−15 The two best known
examples are indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019)10,11,16 and imidazolium trans[tetrachlorido(S-dimethyl sulfoxide)(1H-imidazole)ruthenate(III)] (NAMI-A),17−20 since both have been evaluated in
clinical trials. A prominent example of an organometallic
ruthenium-based compound is [Ru(cymene)Cl 2 (PTA)],
termed RAPTA-C (PTA = 1,3,5-triaza-7-phosphaadamantane),21 which exhibits generally low cytotoxicity in vitro22,23
but relevant antimetastatic24,25 and antiangiogenic26,27 properties in vivo. Moreover, it has recently been shown that RAPTAC reduces the growth of primary tumors in preclinical models
for ovarian and colorectal carcinomas via an antiangiogenic
mechanism.28 In addition to a large number of RAPTA-based
derivatives,29 a wide and diverse range of other ruthenium(II)
arene complexes with N,N-, N,O-, and O,O-chelating ligands
have shown promising therapeutic potential.30−36
A key strategy has been to employ chelating ligands that
exhibit known bioactive properties.37−40 In this respect, here we
investigate the ligating potentials of avobenzone (1-(4-tertbutylphenyl)-3-(4-methoxyphenyl)propane-1,3-dione, AVBH),
a common oil-soluble UVA filter employed extensively in the
formulation of sunscreens and cosmetic products.41 AVBH
absorbs UV rays and converts them into heat that is dispersed
© XXXX American Chemical Society
in the skin. More recently, however, AVBH has been shown to
exhibit promising anticancer properties, especially against a
chemoresistant cancer cell type, where it showed efficacy
similar to that of doxorubicin.42 Doxorubicin is a known DNA
intercalator disrupting topoisomerase II mediated DNA
repair.43 In addition, it generates free radicals and causes
damage to cellular membranes, DNA, and proteins, a
mechanism interesting to explore for the action of AVBH in
cancer cells, as it can induce similar effects. Motivated by the
activity of AVBH against cancer cells, we designed ruthenium(II) AVBH complexes with the focus on enhancing the overall
hydrophilicity to ensure increased cellular uptake of AVBH and
at the same time tuning their activity by introducing PTA or
PTA-Me as a coligand.
■
RESULTS AND DISCUSSION
Complexes 1 and 2 were prepared in high yield from the
reaction of the appropriate dimer [Ru(arene)Cl2]2 (arene =
cymene, hexamethylbenzene) with AVBH and KOH in
methanol (Scheme 1). The complexes are air-stable and are
soluble in alcohols, acetone, acetonitrile, chlorinated solvents,
DMF, and DMSO and sparingly soluble in diethyl ether. The
IR spectra of 1 and 2 show the typical shift of the ν(CO)
vibrations to lower wavenumber upon coordination of the
Received: August 31, 2016
A
DOI: 10.1021/acs.organomet.6b00694
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Scheme 1. Synthesis of 1 and 2
Scheme 3. Synthesis of 5 and 6
AVBH proligand to the ruthenium ion.44 In the far-IR region,
absorptions were observed at 274 and 279 cm−1 which
correspond to ν(Ru−Cl) stretches.45 The electrospray
ionization (ESI) mass spectra of 1 and 2 display peaks
corresponding to the appropriate [Ru(arene)(AVB)]+ ion: i.e.,
generated via dissociation of the chloride ligand. The 1H and
13
C NMR spectra of 1 and 2 recorded in CDCl3 corroborate
the expected structures containing the bidentate AVB ligand.
Conductivity measurements indicate a slight dissociation of the
chloride in CH3CN at room temperature.
The chloride ligand in 1 and 2 is readily replaced by the
amphiphilic, cagelike aminophosphine 1,3,5-triaza-7-phosphaadamantane (PTA), by treatment with AgSO3CF3 in methanol
containing PTA, affording [Ru(cym)(AVB)(PTA)][SO3CF3]
(3) and [Ru(hmb)(AVB)(PTA)][SO3CF3] (4), as depicted in
Scheme 2.
in an acetonitrile solution further confirm the existence of 2:1
electrolyte species.48 The stability of all the complexes in
DMSO was investigated. Solutions of the complexes (c = 20.0
mM) in DMSO were prepared, and their stability was
monitored for 72 h by 1H and 31P NMR spectroscopy.
Complexes 1 and 2 immediately underwent ligand exchange by
ca. 15%, which remained constant during the following 72 h.
Complex 3 showed a small amount of ligand exchange (ca.
10%) after 72 h, whereas 5 immediately underwent a ligand
exchange. The ligand exchange process probably involves
breaking a Ru−O bond followed by exchange with a solvent
molecule. Note that the 31 P NMR spectra of 3 and 5 display a
unique resonance for coordinated PTA and remain unchanged
within 96 h. In contrast, the hexamethylbenzene derivatives 4
and 6 were stable during the 72 h period (Figures S1−S6 in the
Supporting Information). The stability of ionic complexes 3−6
was also determined under pseudopharmacological conditions
in NaCl solution (5 mM, corresponding to the low intracellular
NaCl concentration in cells) and in 100 mM NaCl solution
(approximating to the higher chloride levels in blood).
Solutions of the complexes (c = 2.0 mM) in aqueous NaCl
(c = 5 or 100 mM in D2O containing 10% of [D6]DMSO) were
prepared and maintained at 37 °C for 96 h and monitored by
1
H and 31P NMR spectroscopy. None of the complexes were
observed to change under either of the conditions: i.e., the 1H
and 31P NMR spectra of 3−6 remained unchanged after 96 h
(Figures S7−S10 in the Supporting Information). The
molecular structures of 1 and 2 were confirmed by singlecrystal X-ray structure analysis (see the Experimental Section
and Supporting Information for details of the data collection
and structure refinements). The molecular structures of 1 and 2
are shown in Figure 1, and relevant crystallographic parameters
are reported in Table S1 in the Supporting Information.
Both complexes show a typical piano-stool geometry with
central chirality at the metal ion and, despite having in some
cases enantiomerically pure crystals, we made no attempt to
separate and isolate the two enantiomers. From Figure 1 it is
clear that the AVB ligand is O,O′-coordinated to the ruthenium
ion, forming a six-membered metallacycle. The legs of the
aforementioned piano-stool coordination around the metal
center are completed by a Cl ligand and a cymene (Ru−
centroid, 1.653(2) Å) in 1 or a hexamethylbenzene (Ru−
centroid, 1.656(3) Å) in 2. The AVB ligand displays, in both
complexes, a twisted orientation with angles between the
aromatic rings of 24.9° in 1 and 9.8, 24.1, and 15.2° in 2. The
values of the Ru−Oav and Ru−Cl distances are on average
2.067(3) and 2.435(1) Å in 1 and 2.086(5) and 2.428(2) Å in
2, respectively.
Biological Studies. The cytotoxicities of 1−6 were
evaluated in comparison to those of AVBH and cisplatin by
determining the IC50 concentration against human ovarian
Scheme 2. Synthesis of 3 and 4
The substitution of the chloride ligand by PTA and the
formation of the ionic compounds were confirmed by the
disappearance of the ν(M−Cl) band in the IR spectra of 3 and
4. Moreover, a characteristic absorption pattern in the region
1000−1200 cm−1, indicative of a noncoordinated O3SCF3−
anion, is observed.46 The 1H NMR spectra of 3 and 4 in
CD3CN display the expected signals due to the coordinated
arene, AVB, and PTA ligands. The 31P NMR resonances due to
PTA are observed at lower field with respect to those of
uncoordinated PTA, thus confirming coordination to the metal
center.47 The conductance values in acetonitrile confirm the
existence of 1:1 electrolyte species for 3 and 4.48
Similarly, 1 and 2 can be transformed by reaction with the Nmethyl-1,3,5-triaza-7-phosphaadamantane cation (PTA-Me), in
the presence of AgSO3CF3, into the dicationic species
[Ru(cym)(AVB)(PTA-Me)][SO3CF3][BF4] (5) and [Ru(hmb)(AVB)(PTA)][SO3CF3][BF4] (6) (see Scheme 3).
For 5 and 6 the formation of dicationic species, with
characteristic absorption patterns in the region 1000−1200
cm−1 which are indicative of noncoordinated O3SCF3− and
BF4− anions, are observed.46 The conductance values of 5 and 6
B
DOI: 10.1021/acs.organomet.6b00694
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display selectivity in comparison to the noncancerous cell type.
The three types of complexes, neutral [Ru(arene)(AVB)Cl] (1,
arene = cym; 2, arene = hmb), monocationic [Ru(arene)(AVB)(PTA)]+ (3, arene = cym; 4, arene = hmb) and
dicationic [Ru(arene)(AVB)(PTA-Me)]2+ (5, arene = cym;
arene = hmb, 6), show the following trend of activity:
monocationic > dicationic > neutral. Indeed, 4 is almost an
order of magnitude more cytotoxic than cisplatin in the
cisplatin-sensitive A2780 cell line (0.17 ± 0.01 vs 1.5 ± 0.4
μM).
The DNA binding of AVBH and 1−6 was investigated by
UV−visible spectroscopy,49 and the absorption spectra were
recorded in the presence of increasing amounts of calf thymus
DNA (ct-DNA) (Figure S11 in the Supporting Information).
The spectrum of avobenzone possesses a wide band near 370
nm, due to a π−π* transition of the enol form, with a shoulder
near 380 nm and a weak band near 270 nm corresponding
respectively to the n−π* and π−π* transitions of the keto
form.50−52 Complexes 1−6 display intense absorptions due to
the enol form of bonded avobenzone, shifted in the range 330−
350 nm, together with a week band at 280−290 nm and a
shoulder at 380 nm. Moreover, in the case of 1 and 2 a
hypochromism is observed, accompanied by a small red shift of
the band at 330−350 nm, whereas for 3−6 there is negligible
hypochromism without any red shift. These results imply a
relatively strong binding of neutral complexes 1 and 2 via
intercalation, whereas the ionic complexes, 3−6, likely interact
with DNA through an electrostatic interaction.53,54 The binding
constants, Kb, of 1−6 with ct-DNA (Table 2), are on the order
of 104 M−1, with those of 1 and 2 being higher than those of 3−
6. However, such values are smaller than those of classical
intercalators and metallo-intercalators, which are on the order
of 107 M−1.55−57
Competitive binding studies with ethidium bromide (EtBr)
bound DNA were performed to further clarify the interactions
of 1−6 with DNA. The emission spectra of the EB-ct-DNA
system on addition of increasing amounts of 1−6 and AVBH
are shown in Figure S12 in the Supporting Information. The
emission band of the EB-ct-DNA system decreases in all cases,
and the quenching parameters have been analyzed. The
quenching constants Ksv and apparent DNA binding constants
Kapp, reported in Table 3, show that 1 and 2 are able to
compete with EB-ct-DNA more efficiently than 3−6.
In the presence of the DAPI intercalator specific for the
DNA minor groove (DAPI, 4′,6-diamidino-2-phenylindole58,59), a pronounced decrease in the fluorescence intensity
of the DAPI−ct-DNA complex was observed upon the addition
of increasing concentrations of AVBH or complexes 1−6
(Figure S13 in the Supporting Information). The values of
quenching constants Ksv (Table 2) again indicate that 1 and 2
display affinity higher than that of 3−6 for the minor groove of
DNA.
Since the DNA binding studies do not correlate well with the
observed cytotoxic effects of the compounds, protein binding
studies were performed by tryptophan fluorescence quenching
experiments using bovine serum albumin (BSA) as a model
substrate in phosphate buffer at pH 7.4. Indeed, several recent
studies reported that some cytotoxic Ru(II), Ru(III), Rh(III),
and Ir(III) complexes were found to be able to interact with
some plasma proteins60−65 and with a number of cancer-related
proteins, which may be responsible for the antiangiogenic and
antimetastatic activity of Ru complexes.66,67 The quenching of
the emission intensity of tryptophan residues of BSA at 344 nm
Figure 1. Ortep view of the molecular structures of 1 (top) and 2
(bottom). The probability threshold displayed for the ellipsoids is
50%. The asymmetric unit of 2 contains two independent molecules
(only one is shown, for the sake of clarity). Selected bond distances
(Å) and angles (deg) for 1: Ru1−O1, 2.058(3); Ru1−O2, 2.076(3);
Ru1−Cl1, 2.4348(13); O1−Ru1−O2, 88.13(12); O1−Ru1−Cl1,
84.30(9); O2−Ru1−Cl1, 85.00(9). Selected bond distances (Å) and
angles (deg) for 2: Ru1−O1, 2.088(4); Ru1−O2, 2.083(4); Ru1−Cl1,
2.4267(15); O1−Ru1−O2, 87.16(18); O1−Ru1−Cl1, 84.01(12);
O2−Ru1−Cl1, 86.14(12).
carcinoma A2780 cells and the A2780cisR variant with acquired
resistance to cisplatin, as well as against noncancerous human
embryonic kidney (HEK293) cells. IC50 values for the
compounds determined after 72 h of drug exposure are
presented in Table 1.
In comparison to AVBH alone, which exhibits modest
cytotoxicity but cancer cell selectivity, the mono- and dicationic
complexes display a 10-fold increased potency in vitro against
ovarian carcinoma (A2780 and A2780R) cell lines but do not
Table 1. Cytotoxicities (IC50, μM) of 1−6, AVBH, and
Cisplatin following Incubation for 72 h with Non-Tumorous
Human Embryonic Kidney HEK293 Cells and Human
Ovarian Carcinoma A2780 and A2780R (CisplatinResistant) Cell Lines
IC50 (μM)
compound
A2780
A2780cisR
HEK
1
2
3
4
5
6
AVBH
cisplatin
17.7 ± 2.9
29.2 ± 4.8
0.41 ± 0.10
0.17 ± 0.01
0.70 ± 0.06
2.76 ± 0.24
12.4 ± 1.1
1.4 ± 0.5
46.8 ± 4.1
48.1 ± 2.9
1.25 ± 0.11
1.57 ± 0.05
1.49 ± 0.07
3.74 ± 0.12
15.6 ± 2.4
26.5 ± 2.3
32.5 ± 2.9
27.0 ± 1.5
0.74 ± 0.11
0.21 ± 0.09
1.47 ± 0.08
2.23 ± 0.10
58.8 ± 4.6
11.2 ± 1.8
C
DOI: 10.1021/acs.organomet.6b00694
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Table 2. Binding Constants (Kb), Stern−Volmer Constants (Ksv) and the Apparent Binding Constants (Kapp) for 1−6 and
AVBH with ct-DNA, EB-ct-DNA, and DAPI-ct-DNA
compound
ct-DNA Kb, 104 M−1
EB-ct-DNA Ksv, 104 M−1
EB-ct-DNA Kapp 105 M−1
DAPI-ct-DNA Ksv, 104 M−1
1
2
3
4
5
6
AVBH
13.04 ± 0.41
10.50 ± 0.21
1.52 ± 0.12
1.71 ± 0.04
1.65 ± 0.03
1.43 ± 0.06
4.36 ± 0.12
2.52 ± 0.04
4.36 ± 0.07
1.70 ± 0.08
1.73 ± 0.07
1.66 ± 0.04
1.74 ± 0.09
0.54 ± 0.06
12.6
21.8
8.5
8.65
8.3
8.7
2.7
33.81 ± 0.18
27.05 ± 0.06
22.51 ± 0.07
11.50 ± 0.04
17.68 ± 0.09
22.93 ± 0.11
4.85 ± 0.62
Table 3. Protein Quenching Constants (Ksv), Binding Constants (Kb), and Numbers of Binding Sites (n) for the Interaction of
1−6 and AVB with BSA
compound
Ksv, 105 M−1
Kq, 1013 M−1
Kb, 105 M−1
n
1
2
3
4
5
6
AVBH
2.75 ± 0.09
2.49 ± 0.08
0.89 ± 0.01
0.78 ± 0.03
0.96 ± 0.02
1.18 ± 0.06
7.42 ± 0.37
4.43 ± 0.01
4.01 ± 0.01
1.43 ± 0.02
1.25 ± 0.01
1.54 ± 0.02
1.90 ± 0.01
11.97 ± 0.06
54.95 ± 1.22
38.72 ± 2.35
0.99 ± 0.34
0.11 ± 0.02
0.57 ± 0.12
0.91 ± 0.06
96.38 ± 0.21
1.24 ± 0.02
1.27 ± 0.03
1.10 ± 0.02
0.80 ± 0.02
0.92 ± 0.03
1.06 ± 0.05
1.04 ± 0.09
cytotoxicity of the stable cationic ruthenium compounds over
the neutral complexes.
(excitation wavelength at 285 nm) was monitored after
addition of increasing concentrations of 1−6 or AVBH. The
values of Ksv, Kq, and Kb (Table 3 and Figure S14 in the
Supporting Information) indicate that free AVBH has the
highest affinity for BSA, and 1 and 2 show a slightly higher
affinity in comparison to 3−6.
The values of the bimolecular quenching constant (Kq) fall in
the range (1.25−11.9) × 1013 L mol−1 s−1, higher than the
maximum possible value for dynamic quenching (2.0 × 1010 L
mol−1 s−1),68 suggesting the involvement of a static quenching
mechanism for 1−6. For all complexes, the estimated values of
n (∼1) support the likelihood of a single binding site in BSA for
both AVBH and 1−6. The higher binding affinities observed
for compounds 1 and 2 correlate with the instability of these
two complexes, releasing free AVBH, which can easily be bound
by a cysteine residue of the protein.
Given the lower affinity for DNA and BSA binding but
higher cytotoxic activity of the mono- and dicationic in
comparison to the neutral complexes, the cytotoxic effects of
3−6 are more likely driven by the increased water solubility and
hence higher bioavailability of these compounds.
■
EXPERIMENTAL SECTION
Materials and Methods. The dimers [(arene)RuCl2]2 (arene = pcym and hmb) were purchased from Aldrich, and avobenzone was
purchased from TCI Europe and used as received. [PTA-Me][BF4]
was synthesized as previously described.69 All other materials were
obtained from commercial sources and were used as received. IR
spectra were recorded from 4000 to 30 cm−1 on a PerkinElmer
Frontier FT-IR/FIR spectrometer. 1H and 13C NMR spectra were
recorded on a Varian 400 Mercury Plus instrument operating at room
temperature (400 MHz for 1H, 100 MHz for 13C) relative to TMS. 31P
NMR spectra were recorded on a Varian 400 Mercury Plus instrument
operating at room temperature, 162 MHz relative to 85% H3PO4.
Coupling constants are in Hz. Abbreviations: s, singlet; d, doublet;
sept, septet; m, complex multiplet; vt, virtual triplet; br, broad. Positive
and negative ion electrospray ionization mass spectra (ESI-MS) were
obtained on a Series 1100 MSI detector HP spectrometer using
methanol as the mobile phase. Solutions for analysis (3 mg/mL) were
prepared using reagent-grade methanol. Masses and intensities were
compared to those calculated using an IsoPro Isotopic Abundance
Simulator, version 2.1.28. Melting points are uncorrected and were
recorded on a STMP3 Stuart scientific instrument and on a capillary
apparatus. Samples for microanalysis were dried in vacuo to constant
weight (20 °C, ca. 0.1 Torr) and analyzed on a Fisons Instruments
1108 CHNS-O elemental analyzer. Electrical conductivity measurements (ΛM, reported as S cm2 mol−1) of acetonitrile solutions of the
complexes were recorded using a Eutech Instruments CON2700
apparatus at room temperature. UV−vis spectra of the proligands and
complexes were measured with a Varian Cary1 spectrometer at 20 °C.
The fluorescence of the proligands and complexes was analyzed using
a Hitachi F-4500 spectrofluorimeter at 20 °C.
Synthesis of the Ruthenium Complexes. [(cym)Ru(AVB)Cl] (1).
Avobenzone (285.6 mg, 0.92 mmol) was dissolved in methanol (20
mL), and KOH (52.0 mg, 0,92 mmol) was added. The mixture was
stirred for 1 h at room temperature, and then [(cym)RuCl2]2 (244.9
mg, 0.40 mmol) was added. The mixture was stirred for 24 h at room
temperature, and an orange precipitate formed, which was removed by
filtration and washed with n-hexane (372.6 mg, 0.64 mmol, yield 80%).
The residue was concentrated to ca. 2 mL and stored at 4 °C. Red
crystals formed over several days. Compound 1 is soluble in alcohols,
acetone, acetonitrile, chlorinated solvents, DMF, and DMSO and
■
CONCLUSIONS
We have successfully prepared some novel ruthenium(II) arene
derivatives with avobenzone and ancillary PTA and PTA-Me
ligands conforming to three structural typologies: i.e., neutral
[Ru(arene)(AVB)Cl], monocationic [Ru(arene)(AVB)(PTA)]+, and dicationic [Ru(arene)(AVB)(PTA-Me)]2+. The
ionic complexes 3−6, containing PTA and PTA-Me ligands,
display a potent cytotoxicity in vitro against ovarian carcinoma
(A2780 and A2780R) cell lines, higher than that of the neutral
complexes 1 and 2. This work gives a first insight into
mechanistic studies on DNA and protein binding which show
that attaching AVBH to the ruthenium(II) arene complex
strongly increases the ability to bind DNA but decreases the
affinity for BSA protein binding, two observations that correlate
with the stability of the complexes. Further studies on target
identification are required, however, to explain the high
D
DOI: 10.1021/acs.organomet.6b00694
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slightly soluble in diethyl ether. Mp: 220−221 °C. Anal. Calcd for
C30H35ClO3Ru: C, 62.11; H, 6.08. Found: C, 62.37; H, 6.04. IR
(cm−1): 3066−2838 w ν(C−H), 1605 m, 1588 m, 1532 vs ν(CO;
CC), 1377 s, 1253 s, 1180 s, 1034 s, 836 s, 781 vs, 648 m, 274 s
ν(Ru−Cl). 1H NMR (CDCl3, 293 K): δ, 1.31 (s, 9H, C-(CH3)3 of
AVB), 1.36 (d, 6H, CH3C6H4CH(CH3)2, J = 6.8 Hz), 2.32 (s, 3H,
CH3C6H4CH(CH3)2), 3.01 (sept, 1H, CH3C6H4CH(CH3)2, J = 6.8
Hz), 3.83 (s, 3H, OCH3 of AVB), 5.28 (d, 2H, CH3C6H4CH(CH3)2, J
= 6.0 Hz), 5.56 (m, 2H, CH3C6H4CH(CH3)2), 6.39 (s, 1H, C(7)H of
AVB), 6.87 (d, 2H, C(3,3′)H of AVB, J = 9.2 Hz), 7.38 (d, 2H,
C(11,11′)H of AVB, J = 8.4 Hz), 7.83 (d, 2H, C(10,10′)H of AVB, J =
8.4 Hz), 7.88 (d, 2H, C(4,4′)H of AVB, J = 9.2 Hz). 13C[1H] NMR
(CDCl3, 293 K): δ 18.1 (s, CH3C6H4CH(CH3)2), 22.6 (s,
CH3C6H4CH(CH3)2), 31.1 (s, CH3C6H4CH(CH3)2), 31.4 (s, C(CH3)3 of AVB), 35.1 (s, C-(CH3)3 of AVB), 55.5 (s, OCH3 of AVB),
79.6, 83.3 (s, CH3C6H4CH(CH3)2), 92.7 (s, C(7) of AVB), 97.4, 99.9
(s, CH3C6H4CH(CH3)2), 113.6 (s, C(3,3′) of AVB), 125.2 (s,
C(11,11′) of AVB), 127.3 (s, C(10,10′) of AVB), 129.3 (s, C(4,4′) of
AVB), 131.9 (s, C(5) of AVB), 136.7 (s, C(9) of AVB), 154.4 (s,
C(12) of AVB), 162.2 (s, C(2) of AVB), 180.7 (s, C(8) of AVB), 181.2
(s, C(6) of AVB). ESI-MS (+) CH3OH (m/z [relative intensity, %]):
545 [100] [(cym)Ru(AVB)]+. Λm (CH3CN, 293 K, 10−4 mol/L): 28 S
cm2 mol−1.
[(hmb)Ru(AVB)Cl] (2). Avobenzone (249.9 mg, 0.81 mmol) was
dissolved in methanol (30 mL), and KOH (45.2 mg, 0.81 mmol)) was
added. The mixture was stirred for 1 h at room temperature, and then
[(hmb)RuCl2]2 (234.0 mg, 0.35 mmol) was added. The resulting
mixture was stirred for 24 h at room temperature. The solvent was
removed under reduced pressure, dichloromethane (10 mL) was
added, and the mixture was filtered to remove the KCl precipitate. The
solution was concentrated to 2 mL, and then addition of an excess of
n-hexane resulted in precipitation of an orange powder (315.0 mg,
0.52 mmol, yield 74%). Compound 2 is soluble in alcohols, acetone,
acetonitrile, chlorinated solvents, DMF, and DMSO and slightly
soluble in diethyl ether. Mp: 261−263 °C. Anal. Calcd for
C32H39ClO3Ru: C, 63.20; H, 6.46. Found: C, 63.02; H, 6.41. IR
(cm−1): 3066−2838 w ν(C−H), 1604 m, 1589 m, 1532 vs, 1494 vs
ν(CO; CC), 1422 s, 1349 s, 1255 s, 1180 s, 1035 s, 844 s, 787 vs,
625 m, 549 m, 516 m, 279 s ν(Ru−Cl). 1H NMR (CDCl3, 293 K): δ,
1.33 (s, 9H, C-(CH3)3 of AVB), 2.15 (s, 18H, CH3(hmb)), 3.85 (s,
3H, OCH3 of AVB), 6.40 (s, 1H, C(7)H of AVB), 6.88 (d, 2H,
C(3,3′)H of AVB, J = 8.8 Hz), 7.39 (d, 2H, C(11,11′)H of AVB, J =
8.4 Hz), 7.90 (d, 2H, C(10,10′)H of AVB, J = 8.4 Hz), 7.95 (d, 2H,
C(4,4′)H of AVB, J = 8.8 Hz). 13C[1H] NMR (CDCl3, 293 K): δ 15.4
(s, CH3(hmb)), 31.4 (s, C-(CH3)3 of AVB), 35.0 (s, C-(CH3)3 of
AVB), 55.5 (s, OCH3 of AVB), 90.5 (s, C6(hmb)), 92.1 (s, C(7) of
AVB), 113.5 (s, C(3,3′) of AVB), 125.1 (s, C(11,11′) of AVB), 127.1
(s, C(10,10′) of AVB), 129.1 (s, C(4,4′) of AVB), 132.3 (s, C(5) of
AVB), 137.0 (s, C(9) of AVB), 154.1 (C(12) of AVB), 161.9 (C(2) of
AVB), 179.9 (C(8) of AVB), 180.4 (s, C(6) of AVB). ESI-MS (+)
CH3OH (m/z [relative intensity, %]): 573 [100] [(hmb)Ru(AVB)]+.
Λm (CH3CN, 293 K, 10−4 mol/L): 22 S cm2 mol−1.
[(cym)Ru(AVB)(PTA)][CF3SO3] (3). Compound 1 (101.5 mg, 0.175
mmol) was dissolved in methanol (20 mL), and AgCF3SO3 (45.0 mg,
0.175 mmol) was added. The mixture was stirred for 1 h at room
temperature and filtered to remove AgCl. PTA (PTA = 1,3,5-triaza-7phosphaadamantane; 27.5 mg, 0.175 mmol) was then added to the
filtrate, and the resulting mixture was stirred for 24 h at room
temperature. Then the solution was dried by rotary evaporation and
dichloromethane (2 mL) and an excess of n-hexane were added. The
mixture was left at 4 °C until a yellow precipitate formed. The powder
was recovered by filtration and air-dried. Compound 3 (110.2 mg, 0.09
mmol, yield 74%), is soluble in alcohols, acetone, acetonitrile,
chlorinated solvents, DMF, and DMSO and slightly soluble in diethyl
ether and water. Mp: 150−153 °C. Anal. Calcd for
C37H47F3N3O6PRuS: C, 52.23; H, 5.57; N, 4.94; S, 3.77. Found: C,
52.20; H, 5.62; N, 4.87; S, 3.73. IR (cm−1): 3070−2962 m ν(C−H),
1603 m, 1584 m, 1524 vs ν(CO; CC), 1373 s, 1253 vs, 1152 s,
1029 vs ν(SO3CF3), 971 s, 946 vs, 786 s, 741 m, 637 s. 1H NMR
(CD3CN, 293 K): δ, 1.31 (d, 6H, CH3C6H4CH(CH3)2, J = 6.8 Hz),
1.37 (s, 9H, C(CH3)3 of AVB), 2.15 (s, 3H, CH3C6H4CH(CH3)2),
2.71 (sept, 1H, CH3C6H4CH(CH3)2, J = 6.8 Hz), 3.89 (s, 3H, OCH3
of AVB), 4.13 (s, 6H, NCH2P, PTA), 4.44 (s, 6H, NCH2N, PTA),
5.86 (d, 2H, CH3C6H4CH(CH3)2, J = 6.0 Hz), 5.91 (m, 2H,
CH3C6H4CH(CH3)2), 6.82 (s, 1H, C(7)H of AVB), 7.04 (d, 2H,
C(3,3′)H of AVB, J = 9.2 Hz), 7.56 (d, 2H, C(11,11′)H of AVB, J =
8.4 Hz), 7.93 (d, 2H, C(10,10′)H of AVB, J = 8.4 Hz), 7.99 (d, 2H,
C(4,4′)H of AVB, J = 9.2 Hz). 13C[1H] NMR (CD3CN, 293 K): δ
17.4 (s, CH3C6H4CH(CH3)2), 22.4 (s, CH3C6H4CH(CH3)2), 31.3 (s,
CH3C6H4CH(CH3)2), 31.4 (s, C-(CH3)3 of AVB), 35.7 (s, C-(CH3)3
of AVB), 52.2 (d, PCH2N, PTA, JCP = 13.7 Hz), 56.4 (s, OCH3 of
AVB), 73.1 (d, NCH2N, PTA, JCP = 7.7 Hz), 89.3 and 90.6 (s,
CH3C6H4CH(CH3)2), 95.4 (s, C(7) of AVB), 98.4 and 105.3 (s,
CH3C6H4CH(CH3)2), 114.9 (s, C(3,3′) of AVB), 126.7 (s, C(11,11′)
of AVB), 128.2 (s, C(10,10′) of AVB), 130.5 (s, C(4,4′) of AVB),
130.8 (s, C(5) of AVB), 135.9 (s, C(9) of AVB), 156.6 (s, C(12) of
AVB), 164.0 (s, C(2) of AVB), 183.2 (s, C(8) of AVB), 183.3 (s, C(8)
of AVB). 31P[1H] NMR (CD3CN, 293 K): δ − 28.4 (s, PTA). ESI-MS
(+) CH3OH (m/z [relative intensity, %]): 702 [100] [(cym)Ru(AVB)(PTA)]+, 545 [5] [(cym)Ru(AVB)]+. Λm (CH3CN, 293 K,
10−4 mol/L): 101 S cm2 mol−1.
[(hmb)Ru(AVB)(PTA)][CF3SO3] (4). Compound 4 was prepared
following a procedure similar to that reported for 3 by using precursor
2 (84.0 mg, 0.095 mmol, yield 74%). Compound 4 is soluble in
alcohols, acetone, acetonitrile, chlorinated solvents, DMF, and DMSO
and slightly soluble in diethyl ether and water. Mp: 200−202 °C. Anal.
Calcd for C39H51F3N3O6PRuS: C, 53.29; H, 5.85; N, 4.78; S, 3.65.
Found: C, 53.18; H, 5.83; N, 4.70; S, 3.57. IR (cm−1): 2959 br ν(C−
H), 1603 m, 1583 m, 1523 vs ν(CO; CC), 1258 vs, 1175 m, 1030
vs ν(SO3CF3), 972 s, 947 vs, 787 s, 637 s, 573 m. 1H NMR (CDCl3,
293 K): δ, 1.37 (s, 9H, C-(CH3)3 of AVB), 2.16 (s, 18H, CH3(hmb)),
3.90 (s, 3H, OCH3 of AVB), 4.11 (s, 6H, NCH2P, PTA), 4.50 (s, 6H,
NCH2N, PTA), 6.71 (s, 1H, C(7)H of AVB), 7.00 (d, 2H, C(3,3′)H
of AVB, J = 8.8 Hz), 7.49 (d, 2H, C(11,11′)H of AVB, J = 8.4 Hz),
7.86 (d, 2H, C(10,10′)H of AVB, J = 8.4 Hz), 7.93 (d, 2H, C(4,4′)H
of AVB, J = 8.8 Hz). 13C[1H] NMR (CDCl3, 293 K): δ 16.1 (s,
CH3(hmb)), 31.4 (s, C-(CH3)3 of AVB), 35.3 (s, C-(CH3)3 of AVB),
49.7 (d, PCH2N, PTA, JCP = 12.2 Hz), 55.8 (OCH3 of AVB), 73.0 (d,
NCH2N, PTA, JCP = 6.8 Hz),), 93.7 (s, C6(hmb)), 98.5, (s, C(7) of
AVB), 114.5 (s, C(3,3′) of AVB), 126.1 (s, C(11,11′) of AVB), 126.8
(s, C(10,10′) of AVB), 129.1 (s, C(4,4′) of AVB), 130.4 (s, C(5) of
AVB), 135.3 (s, C(9) of AVB), 156.1 (s, C(12) of AVB), 163.4 (s,
C(2) of AVB), 181.8 (s, C(6,8) of AVB). 31P[1H] NMR (CHCl3, 293
K): δ − 34.0 (s, PTA). ESI-MS (+) CH3OH (m/z [relative intensity,
%]): 730 [100] [(hmb)Ru(AVB)(PTA)]+, 573 [5] [(hmb)Ru(AVB)]+. Λm (CH3CN, 293 K, 10−4 mol/L): 113 S cm2 mol−1.
[(cym)Ru(AVB)(PTA-Me)][BF4][CF3SO3] (5). Compound 5 was
prepared following a procedure similar to that reported for 3 by
using [PTA-Me][BF4] (1-methyl-1-azonia-3,5-diaza-7phosphatricyclo[3.3.1.1]decane tetrafluoroborate) (67.8 mg, 0.07
mmol, yield 70%). Compound 5 is soluble in alcohols, acetone,
acetonitrile, chlorinated solvents, DMF, and DMSO and slightly
soluble in water. Mp: 159−161 °C. Anal. Calcd for
C38H50BF7N3O6PRuS: C, 47.91; H, 5.29; N, 4.41; S, 3.37. Found:
C, 47.76; H, 5.33; N, 4.35; S, 3.31. IR (cm−1): 3611−2966 m ν(C−
H), 1604 m, 1584 m, 1524 s ν(CO; CC), 1360 m, 1251 s, 1175 s,
1029 vs ν(SO3CF3), 791 m, 750 s, 638 vs, 573 s, 550 m, 518 s. 1H
NMR (CD3CN, 293 K): δ, 1.32 (d, 6H, CH3C6H4CH(CH3)2, J = 6.8
Hz), 1.37 (s, 9H, C(CH3)3 of AVB), 2.17 (s, 3H, CH3C6H4CH(CH3)2), 2.71 (s, 3H, N+CH3), 2.75 (sept, 1H, CH3C6H4CH(CH3)2, J
= 6.8 Hz), 4.04 and 4.17 (J(HAHB) = 15.0 Hz, 2J(HA-P) = 15 Hz,
2
J(HB-P) = 5 Hz, 4H, PCHAHBN, PTA-Me), 3.91 (s, 3H, OCH3 of
AVB), 4.38 (s, 2H, PCH2N+, PTA-Me), 4.36 and 4.52 (J(HAHB) = 15
Hz, 2H, NCHAHBN, PTA-Me), 4.81 and 4.97 (J(HAHB) = 13 Hz, 4H,
NCHAHBN+, PTA-Me), 6.02 (m, 2H, CH3C6H4CH(CH3)2), 6.06 (m,
2H, CH3C6H4CH(CH3)2), 6.87 (s, 1H, C(7)H of AVB), 7.05 (d, 2H,
C(3,3′)H of AVB, J = 8.8 Hz), 7.58 (d, 2H, C(11,11′)H of AVB, J =
8.4 Hz), 7.97 (d, 2H, C(10,10′)H of AVB, J = 8.4 Hz), 8.03 (d, 2H,
C(4,4′)H of AVB, J = 8.8 Hz). 13C[1H] NMR (CDCl3, 293 K): δ 18.7
(s, CH3C6H4CH(CH3)2), 22.5 (s, CH3C6H4CH(CH3)2), 30.9 (s,
E
DOI: 10.1021/acs.organomet.6b00694
Organometallics XXXX, XXX, XXX−XXX
Article
Organometallics
CH3C6H4CH(CH3)2), 31.3 (s, C-(CH3)3 of AVB), 35.4 (s, C-(CH3)3
of AVB), 47.0 (dd, vt, 1JCP = 15.7 Hz, PCH2N, PTA-Me), 49.2 (s,
N+CH3, PTA-Me), 55.9 (OCH3 of AVB), 58.7 (s, PCH2N+, PTA-Me),
70.2 (s, NCH2N, PTA-Me) 80.8 (s, NCH2N+, PTA-Me), 88.3, 89.0,
90.3, 90.7 (s, CH3C6H4CH(CH3)2), 94.4 (s, C(7) of AVB), 99.9 and
107.1 (s, CH3C6H4CH(CH3)2), 114.6 (s, C(3,3′) of AVB), 126.3 (s,
C(11,11′) of AVB), 127.2 (s, C(10,10′) of AVB), 129.1 (s, C(5) of
AVB), 129.6 (s, C(4,4′) of AVB), 134.1 (s, C(9) of AVB), 156.7 (s,
C(12) of AVB), 163.6 (s, C(2) of AVB), 182.7 (s, C(6,8) of AVB).
31 1
P[ H] NMR (CDCl3, 293 K): δ, −13.2. ESI-MS (+) CH3OH (m/z
[relative intensity, %]): 545 [100] [(cym)Ru(AVB)]+, 172 [45] [PTAMe]+. Λm (CH3CN, 293 K, 10−4 mol/L): 262 S cm2 mol−1.
[(hmb)Ru(AVB)(PTA-Me)][BF4][CF3SO3] (6). Compound 6 was
prepared following a procedure similar to that reported for 4 by
using [PTA-Me][BF4] (61.2 mg, 0.06 mmol, yield 61%). Compound 6
is soluble in alcohols, acetone, acetonitrile, chlorinated solvents, DMF,
and DMSO and slightly soluble in water. Mp: 208−210 °C. Anal.
Calcd for C40H54BF7N3O6PRuS: C, 48.99; H, 5.55; N, 4.28; S, 3.27.
Found: C, 49.10; H, 5.65; N, 4.36; S, 3.33. IR (cm−1): 3610−2962 w
ν(C−H), 1603 m, 1583 m, 1523 s ν(CO; CC), 1356 m, 1252 vs,
1175 s, 1023 vs ν(SO3CF3), 814 m, 790 m, 750 m, 638 vs, 572 s, 551
m, 518 s. 1H NMR (CD3CN, 293 K): δ, 1.37 (s, 9H, C-(CH3)3 of
AVB), 2.18 (s, 18H, CH3, hmb), 2.64 (s, 3H, N+CH3), 3.92 (s, 3H,
OCH3 of AVB), 3.92 and 4.22 (J(HAHB) = 15.0 Hz, 2J(HA-P) = 5 Hz,
2
J(HB-P) = 0, 4H, PCHAHBN, PTA-Me), 4.04 (s, 2H, PCH2N+, PTAMe), 4.36 and 4.49 (J(HAHB) = 12 Hz, 2H, NCHAHBN, PTA-Me),
4.75 and 4.82 (J(HAHB) = 12 Hz, 4H, NCHAHBN+, PTA-Me), 6.90 (s,
1H, C(7)H of AVB), 7.07 (d, 2H, C(3,3′)H of AVB, J = 8.8 Hz), 7.59
(d, 2H, C(11,11′)H of AVB, J = 8.4 Hz), 8.04 (d, 2H, C(10,10′)H of
AVB, J = 8.8 Hz), 8.10 (d, 2H, C(4,4′)H of AVB, J = 8.8 Hz). 13C[1H]
NMR (CDCl3, 293 K): δ 15.9 (CH3(hmb)), 31.3 (C(CH3)3 of AVB),
35.3 (C(CH3)3 of AVB), 45.6 (d, 1JCP = 25.4 Hz, PCH2N, PTA-Me),
49.2 (s, N+CH3, PTA-Me), 54.6, (d, 1JCP = 19.3 Hz, PCH2N+, PTAMe), 55.8 (s, OCH3 of AVB), 69.8 (s, NCH2N, PTA-Me), 80.8 (s,
NCH2N+, PTA-Me), 93.5 (C6(hmb)), 99.6 (s, C(7) of AVB), 114.6 (s,
C(3,3′) of AVB), 126.2 (s, C(11,11′) of AVB), 127.0 (s, C(10,10′) of
AVB), 129.4 (s, C(5) of AVB), 129.9 (s, C(4,4′) of AVB), 134.8 (s,
C(9) of AVB), 156.5 (s, C(12) of AVB), 163.6 (s, C(2) of AVB), 181.9
(s, C(6,8) of AVB).31P[1H] NMR (CDCl3, 293 K): δ, −18.6. ESI-MS
(+) CH3OH (m/z [relative intensity, %]): 573 [55] [(cym)Ru(AVB)]+, 172 [100] [PTA-Me]+. Λm (CH3CN, 293 K, 10−4 mol/L):
192 S cm2 mol−1.
X-ray Crystallography. Diffraction data were recorded at low
temperature (100(2) K) using Mo Kα radiation on a Bruker APEX II
CCD diffractometer equipped with a κ geometry goniometer. The data
sets were reduced by EvalCCD70 and then corrected for absorption.71
The solutions and refinements were performed by SHELX.72 The
crystal structures were refined using full-matrix least squares based on
F2 with all non-hydrogen atoms anisotropically defined. Hydrogen
atoms were placed in calculated positions by means of the “riding”
model. In the crystal structure of 2, one of the two independent
molecules displayed, during the last stages of refinement, disorder
problems (tert-butyl and −OMe substituents may occupy the same
position). In order to handle these problems correctly and to retain a
reasonable geometry and acceptable ADPs, a split model, in
combination with some soft restraints (SIMU and SADI cards), was
employed. A very small twinning (TWIN 100, 0−10, 00−1)
component was found for 2, and its refined BASF parameter was
0.0189(6).
Cell Culture and Inhibition of Cell Growth. The human A2780
and A2780cisR ovarian carcinoma and HEK293 (human embryonic
kidney) cells were obtained from the European Collection of Cell
Cultures (Salisbury, U.K.). A2780 and A2780cisR cells were grown
routinely in RPMI-1640 medium, while HEK293 cells were grown in
DMEM medium, with 10% fetal bovine serum (FBS) and 1%
antibiotics at 37 °C and 5% CO2. Cytotoxicity was determined using
the MTT assay (MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium·bromide). Cells were seeded in 96-well plates as
monolayers with 100 μL of cell suspension (approximately 10000
cells) per well and preincubated for 24 h in medium supplemented
with 10% FBS. Compounds were prepared as DMSO solutions and
then dissolved in the culture medium and serially diluted to the
appropriate concentration, to give a final DMSO concentration of
0.5%. A 100 μL portion of the drug solution was added to each well,
and the plates were incubated for another 72 h. Subsequently, MTT (5
mg/mL solution) was added to the cells and the plates were incubated
for a further 2 h. The culture medium was aspirated, and the purple
formazan crystals formed by the mitochondrial dehydrogenase activity
of vital cells were dissolved in DMSO. The optical density, directly
proportional to the number of surviving cells, was quantified at 590 nm
using a multiwell plate reader, and the fraction of surviving cells was
calculated from the absorbance of untreated control cells. Evaluation is
based on means from at least two independent experiments, each
comprising quadruplicates per concentration level.
DNA Interaction Studies. Stock solutions of calf thymus DNA
(ct-DNA) (Sigma-Aldrich) were prepared by dissolving the DNA
powder overnight in 20 mM phosphate buffer pH 7.4 containing 5
mM NaCl and incubated with stirring at 4 °C for 12 h. The UV
absorbance at 260 and 280 nm of the DNA solution gave an A260/
A280 solution ratio of ct-DNA of ca. 1.9, indicating that the DNA was
sufficiently free from protein.68 The concentration of ct-DNA solution
was determined by UV absorbance at 260 nm. The molar absorption
coefficient, ε260, was taken as 6600 M−1 cm−1.73 All AVB complexes
and ligands were dissolved in DMSO. Electronic absorption titrations
of different complexes with DNA were recorded at 25 °C by addition
of ct-DNA in small aliquots (from 0 to 63 μM) to buffer solution (50
mM phosphate buffer pH 7.4) containing the complexes 1−6 or
AVBH.. Absorption spectra were recorded from 200 to 600 nm, and
changes on the intensity of the bands were monitored. The intrinsic
binding constant Kb for the interaction of 1−6 or AVBH with ct-DNA
was determined from a plot of [DNA]/(εa − εf) versus [DNA], by
using absorption spectral titration data and eq 1:
[DNA]/|εa − εf | = [DNA]/|εb − εf | + 1/Kb|εb − εf |
(1)
where [DNA] is the concentration of DNA, the apparent absorption
coefficients εa, εf, and εb correspond to Aobsd/[complex], the extinction
coefficient for the free metal complex, and the extinction coefficient for
the metal complex in the fully bound form, respectively.73 The Kb
value is given by the ratio of the slope to the intercept.
Major Groove Displacement Assay. Competitive binding
experiments were performed with the ethidium bromide (EB) and
ct-DNA concentrations maintained at 5 and 55.7 μM, respectively,
while the concentrations of different complexes added to the buffer
solution were increased. Fluorescence quenching spectra were
recorded using a Hitachi 4500 spectrofluorimeter with an excitation
wavelength of 490 nm and emission spectrum of 500−700 nm. The
fluorescence spectra were recorded, and the fluorescence values of
decrease in emission spectra were corrected according to the
relationship given by eq 2:
Fc = Fm × e(A1 + A2)/2
(2)
where Fc and Fm are the corrected and measured fluorescences,
respectively. A1 and A2 are the absorbances of the tested compounds
at the excitation and emission wavelengths. For fluorescence
quenching experiments, the Stern−Volmer equation was used (eq 3):
F0/Fc = 1 + kqτ0[C] = 1 + K sv[C]
(3)
where F0 and Fc represent the fluorescence intensities in the absence
and in the presence of the metal complex, respecitvely, [C] is the
concentration of the metal complex, and Ksv is the Stern−Volmer
constant that can be obtained from fluorescence data, plotted as F0/Fc
vs the metal complex concentration [C].74 All experiments involving
ct-DNA were performed in buffer solution (50 mM phosphate buffer
pH 7.4) at room temperature. The apparent binding constant Kapp of
AVBH and 1−6 was calculated from eq 4:
KEB[EB] = K app[C]
F
(4)
DOI: 10.1021/acs.organomet.6b00694
Organometallics XXXX, XXX, XXX−XXX
Organometallics
■
where KEB and [EB] are the binding constant and concentration of EB,
respectively, and [C] is the concentration of the metal complex at 50%
reduction of fluorescence.75
Minor Groove Displacement Assay. Samples were prepared in
triplicate for each concentration, and at least seven different
concentrations were used. In a typical experiment, the changes in
the emission spectra of the DAPI complex with the DNA in 50 mM
phosphate buffer pH 7.4 were monitored upon addition of increasing
concentrations of AVB and complexes at room temperature.
Fluorescence quenching data were evaluated after excitation of the
DAPI-ct-DNA complex at 338 nm and recording of the spectra from
400 to 600 nm. Values of Ksv constants were evaluated using eq 3, and
the quenching fluorescence values obtained were corrected according
to the relationship given by eq 2.
BSA Binding Studies. The protein-binding studies were
performed by tryptophan fluorescence quenching experiments using
bovine serum albumin (BSA) prepared in 50 mM potassium
phosphate buffer pH 7.4. Fluorescence measurements were recorded
on a Hitachi 4500 spectrofluorometer with the concentration of BSA
kept constant (15 × 10−6 M) while increasing complex concentrations
were added to the protein solution at room temperature. Protein
fluorescence intensities were recorded after each successive addition of
complex solution and equilibration (ca. 5 min). Fluorescence spectra
were recorded from 300 to 450 nm at an excitation wavelength of 285
nm. The values of Stern−Volmer constants of the different metal
complexes to HSA were evaluated following eq 3 ,and fluorescence
values were corrected by eq 2. Ksv, the Stern−Volmer quenching
constant, was determined by linear regression of a plot of F0/F vs [C];
Kq is the bimolecular quenching rate constant, and τ0 is the average
fluorescence lifetime of the fluorophore in the absence of drug, having
a value of 6.2 ns for the biopolymer.76 Considering the existence of
similar and independent binding sites in the BSA for the static
quenching interaction, the binding constant (Kb) and the number of
the binding sites (n) for the different complexes on BSA can be
determined according to eq 5:77
log[(F0 − F )/F ] = log Kb + n log[Q]
(5)
ASSOCIATED CONTENT
* Supporting Information
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.organomet.6b00694.
NMR, absorption, emission, and fluorescence spectra and
crystal data (PDF)
Crystallographic data (CIF)
Crystallographic data (CIF)
■
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versus log[C].
■
Article
AUTHOR INFORMATION
Corresponding Authors
*R.P.: e-mail, riccardo.pettinari@unicam.it; tel. +39
0737402338.
*P.D.: e-mail, paul.dyson@epfl.ch.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We thank the Swiss National Science Foundation, EPFL, the
University of Camerino, and the NCN program (Grant No.
2012/07/B/ST/00885) of Poland for financial support.
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