Photoactivation of the cytotoxic properties of platinum(II) complexes through ligand photoswitching

Article abstract of DOI:10.1021/acs.inorgchem.8b00146

The development of photoactivatable metal complexes with potential anticancer properties is a topical area of current investigation. Photoactivated chemotherapy using coordination compounds is typically based on photochemical processes occurring at the metal center. In the present study, an innovative approach is applied that takes advantage of the remarkable photochemical properties of diarylethenes. Following a proof-of-concept study with two complexes, namely, C1 and C2, a series of additional platinum(II) complexes from dithienylcyclopentene-based ligands was designed and prepared. Like C1 and C2, these new coordination compounds exhibit two thermally stable, interconvertible photoisomers that display distinct properties. The photochemical behavior of ligands L3-L7 has been analyzed by ¹H NMR and UV-vis spectroscopies. Subsequently, the corresponding platinum(II) complexes C3-C7 were synthesized and fully characterized, including by single-crystal X-ray diffraction for some of them. Next, the interaction of each photoisomer (i.e., containing the open or closed ligand) of the metal complexes with DNA was examined thoroughly using various techniques, revealing their distinct DNA-binding modes and affinities, as observed for the earlier compounds C1 and C2. The antiproliferative activity of the two forms of the complexes was then assessed with five cancer cell lines and compared with that of C1 and C2, which supported the use of such diarylethene-based systems for the generation of a new class of potential photochemotherapeutic metallodrugs.

Full text of DOI:10.1021/acs.inorgchem.8b00146

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Photoactivation of the Cytotoxic Properties of Platinum(II)
Complexes through Ligand Photoswitching
Andreu Presa,† Guillem Vazquez,† Leoní A. Barrios,† Olivier Roubeau,‡ Luís Korrodi-Gregorio,§
́
́
Ricardo Perez-Tomas,§ and Patrick Gamez*,†,⊥,∥
́
́
†Department of Inorganic and Organic Chemistry, Inorganic Chemistry Section, University of Barcelona, Martí i Franques
08028 Barcelona, Spain
‡Instituto de Ciencia de Materiales de Aragon
̀
1-11,
́
, CSIC and Universidad de Zaragoza, Plaza San Francisco s/n, 50009 Zaragoza, Spain
§Department of Pathology and Experimental Therapeutics, Faculty of Medicine, University of Barcelona, Campus Bellvitge, Feixa
Llarga s/n, 08907 L’Hospitalet de Llobregat, Spain
∥Catalan Institution for Research and Advanced Studies, Passeig Lluís Companys 23, 08010 Barcelona, Spain
⊥Institute of Nanoscience and Nanotechnology, Martí i Franques
̀
1-11, 08028 Barcelona, Spain
S
* Supporting Information
ABSTRACT: The development of photoactivatable metal complexes with potential anticancer properties is a topical area of
current investigation. Photoactivated chemotherapy using coordination compounds is typically based on photochemical
processes occurring at the metal center. In the present study, an innovative approach is applied that takes advantage of the
remarkable photochemical properties of diarylethenes. Following a proof-of-concept study with two complexes, namely, C1 and
C2, a series of additional platinum(II) complexes from dithienylcyclopentene-based ligands was designed and prepared. Like C1
and C2, these new coordination compounds exhibit two thermally stable, interconvertible photoisomers that display distinct
1
properties. The photochemical behavior of ligands L3−L7 has been analyzed by H NMR and UV−vis spectroscopies.
Subsequently, the corresponding platinum(II) complexes C3−C7 were synthesized and fully characterized, including by single-
crystal X-ray diffraction for some of them. Next, the interaction of each photoisomer (i.e., containing the open or closed ligand)
of the metal complexes with DNA was examined thoroughly using various techniques, revealing their distinct DNA-binding
modes and affinities, as observed for the earlier compounds C1 and C2. The antiproliferative activity of the two forms of the
complexes was then assessed with five cancer cell lines and compared with that of C1 and C2, which supported the use of such
diarylethene-based systems for the generation of a new class of potential photochemotherapeutic metallodrugs.
Photoactivatable prodrugs based on coordination compounds
typically involve activation at the metal center, with concomitant
generation of pharmacologically active species.5 Metal-mediated
PACT can be achieved via distinct mechanisms of action, namely,
photodissociation6 and photosensitization,7 which are often
associated with the redox properties of the metal center,1 and
photothermal reaction.8 A number of research groups worldwide
INTRODUCTION
■
An elegant drug-design approach aimed at enhancing tumor
selectivity while reducing systemic toxicity is the use of light
activation.1 Recent advances in laser and fiber-optic technologies
have driven the development of various medical applications
based on light,2 including the design of metal-containing
prodrugs that can be activated through irradiation.1,3 Photo-
activated chemotherapy (PACT) is a prominent field of
investigation, which provides both temporal and spatial control
over drug activity, and offers tremendous potential for the
treatment of different types of cancer.4
Received: January 20, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.8b00146
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Figure 1. (A) Reversible photocyclization of dithienylcyclopentenes (R = H) and dithienylperfluorocyclopentenes (R = F). (B) Schematic
representation of the platinum(II) complexes C1−C7, obtained with seven different dithienylcyclopentene-based ligands.
are developing elegant systems that are photoactivatable through
the action of light on the metal.9−16
is not.20 Hence, on the basis of these results, a series of new
simple diarylethene-based ligands were designed and prepared,
and the corresponding platinum(II) complexes were synthe-
sized.21 Subsequently, the DNA-interacting properties of their
open and closed forms were examined using various techniques,
and their cytotoxic behavior against various cancer cell lines was
then evaluated and compared with those of C1 and C2.
Promising results were achieved, further indicating that this
innovative mechanism of photochemical control may be applied
to produce a new class of photoactivatable metallodrugs. It can
indeed be stressed that the photoactivation of the cytotoxic
properties of the platinum(II) compounds reported herein is
based on the photomodification of the coordinated ligand and
not on the metal center (as commonly described in the
literature).
The activation of a metallodrug through the photomodifica-
tion of a coordinated ligand has not been investigated
extensively. Different types of organic molecular photoswitches
may be used for this purpose, like azobenzenes, spiropyrans, or
diarylethenes.17 Azobenzenes and spiropyrans are thermally
unstable and gradually revert back to their initial state in the
absence of light.18 In contrast, diarylethenes are more suitable for
such biological application, as they exhibit negligible thermal
relaxation; as a matter of fact, these unique properties have
converted them as one of the most interesting classes of
photoresponsive molecular devices, as reflected by their
numerous applications.19
In the present study, the potential use of diarylethene moieties
to generate potential light-activatable metallodrugs has been
investigated further.20 Indeed, it has recently been shown that the
two thermally stable states of two molecular switches (complexes
C1 and C2 in Figure 1) exhibit distinct biological activities, one
of the two photoisomers being cytotoxic, whereas the other one
EXPERIMENTAL DETAILS
■
Materials and Methods. All reagents and high-performance liquid
chromatography (HPLC)-grade solvents for the synthesis of the ligands
and complexes were purchased from commercial sources and used as
received. Ethidium bromide, sodium cacodylate, tris acetate-ethyl-
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enediaminetetraacetic acid (TAE), and calf thymus DNA (ct-DNA) were
purchased from Sigma-Aldrich. Plasmid pBR322 DNA was purchased
from Roche. All reagents used for the in vitro DNA-interaction studies
were obtained from Sigma-Aldrich and Invitrogen. Anhydrous solvents
were distilled under an inert atmosphere using a PureSolv solvent
purification system from Innovative Technologies.
When required, the reactions were performed under an atmosphere
of dinitrogen using standard procedures. Column chromatography
purifications were performed in air, using ultrapure silica gel (60−200
μm, 60 Å) from Acros Organics, and monitored by analytical thin-layer
chromatography (TLC) using precoated aluminum plates.
Nuclear magnetic resonance (NMR) spectra were recorded at 298 K
on a Varian Mercury 400 MHz spectrometer. Chemical shifts (δ) are
reported in parts per million, and coupling constants (J) are given in
hertz. Proton chemical shifts are referenced to the corresponding
nondeuterated solvent peak (CHCl3: 7.26 ppm; dimethyl sulfoxide
(DMSO): 2.50 ppm); fluorine chemical shifts are referenced to
trifluoroacetic acid (−76.55 ppm). Signal multiplicities are defined as
s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), br
(broad signal), and m (multiplet).
stirred for 2 h, after which pyridine (3.2 mL, 40 mmol) was added
dropwise, producing a brown solution. After 10 min, 1 (7.2 g, 20 mmol)
was added, and the resulting mixture was stirred in the dark overnight.
Next, the solution was cooled in an ice bath and quenched with a 20%
aqueous K2CO3 solution (25 mL), yielding an abundant black
precipitate. After the precipitate was vigorously stirred with diethyl
ether (40 mL), the remaining solid was isolated by filtration over a glass
filter and washed with additional diethyl ether (2 × 25 mL). The
combined organic phase was placed in an ice bath for 30 min and filtered
again to eliminate the resulting undesired white precipitate. The solution
was washed with acidified water (2 × 20 mL), dried over Na2SO4,
filtered, and concentrated under reduced pressure to give an orange oil.
This oil was purified by column chromatography on silica gel with
cyclohexane as the eluent, yielding an oil that slowly solidified to
colorless product 2 (4.75 g, 72%).
1H NMR (400 MHz, CDCl3): δ = 1.88 (s, 6H, Me), 2.02 (m, 2H, J =
7.6 Hz, cyclopentene), 2.71 (t, 4H, J = 7.6 Hz, cyclopentene), 6.57 (s,
2H, thiophene). MS (ESI): m/z = 329 [M]+ (expected: 328.99). Anal.
Calcd for C15H14Cl2S2 (%): C 54.71, H 4.29, S 19.47; found: C 54.89, H
4.37, S 18.78.
Electrospray ionization (ESI) mass spectrometry was performed
using an LC/MSD-TOF spectrometer from Agilent Technologies
equipped with an ESI source, at the Centres Cientifics i Tecnologics de
la Universitat de Barcelona. Samples were eluted with a H2O/CH3CN
1:1 mixture and measured in the positive mode. C, H, N, and S elemental
analyses were performed at the Centres Cientifics i Tecnologics de la
Universitat de Barcelona, using a Thermo EA 1108 CHNS/O analyzer
from Carlo Erba Instruments.
4-Bromo-5-methylthiophene-2-boronic acid (3). n-Butyllithium
(10 mL of a 1.6 M solution in hexane, 16.0 mmol) was added dropwise
to a solution of 3,5-dibromo-2-methylthiophene (2.0 mL, 15.6 mmol) in
anhydrous diethyl ether (25 mL) at −78 °C, under a dinitrogen
atmosphere, resulting in a color change from colorless to bright orange.
The reaction mixture was stirred for 30 min, and then tributyl borate
(8.0 mL, 29.6 mmol) was added. After the resulting solution was stirred
at room temperature overnight, 5% HCl aqueous solution (20 mL) was
added to redissolve the pale yellow precipitate formed. The organic
phase was then separated and extracted with an aqueous NaOH solution
(1 M, 2 × 20 mL); the combined aqueous phase was acidified (HCl,
37%) under vigorous stirring until the apparition of an off-white
precipitate. The product 3 was then filtered, washed with acidified water,
and dried under reduced pressure (3.3 g, 96%).
Spectroscopic measurements in buffered aqueous media were
performed in cacodylate buffer solution (1 mM sodium cacodylate, 20
mM NaCl, pH = 7.2), which was prepared with ultrapure water and
whose pH was adjusted with HClaq. The UV and visible irradiation of the
samples was performed with a MAX-303 light source from Asahi
Spectra, using appropriate bandpass filters. The UV−vis absorption
spectra were recorded on a Varian Cary 100 Bio spectrophotometer.
Fluorescence and circular dichroism (CD) spectra were collected in 1
cm path length quartz cuvettes, using cacodylate-buffered solutions.
The stock solutions for the DNA-interaction studies were prepared as
follows: the commercial plasmid pBR322 stock solution (250 μg mL−1,
ca. 385 μMbp) was used as received. A 150 μM stock solution of ct-DNA
was prepared by dissolving the highly polymerized sodium salt of the
biomolecule in cacodylate buffer, and the exact concentration was
determined by absorbance at 260 nm (εbp = 13 200 M−1 cm−1). Stock
solutions (5 mM) of the studied complexes were freshly prepared in
DMSO before each experiment and further diluted in DMSO/buffer
mixtures as required. Stock solutions of closed complexes were obtained
by irradiation of the corresponding solutions of the open complexes, at λ
< 365 nm, until reaching the photostationary state (PSS).
Synthesis. 1,5-Bis(5-chloro-2-methyl-3-thienyl)pentane-1,5-
dione (1).22 Aluminum(III) trichloride (29.0 g, 217 mmol) was slowly
added to a mixture of 2-chloro-5-methylthiophene (25.0 g, 189 mmol)
and glutaryl dichloride (12.0 mL, 94 mmol) dissolved in 200 mL of ice-
cold nitromethane. After the addition of AlCl3, the resulting dark red
solution was stirred for 3 h at room temperature, and 150 mL of ice-cold
water was subsequently added in small portions. The reaction mixture
was then placed in an ice bath and vigorously stirred for 1 h, until an
abundant precipitate formed. This precipitate was then poured over a
glass filter, washed with cold n-pentane, and finally dried under reduced
pressure to yield the crude final product 1 as a pale brown solid (27.6 g,
81%).
1H NMR (400 MHz, DMSO-d6): δ = 2.36 (s, 3H, Me), 7.49 (s, 1H,
thiophene), 8.32 (s, 2H, B(OH)2). MS (ESI): m/z = 220.9 [M]+
(expected: 220.94).
4-Bromo-5-methyl-2-(4-pyridyl)thiophene (4). 4-Bromopyridine
hydrochloride (1.3 g, 6.7 mmol) and 3 (1.0 g, 4.5 mmol) were
subsequently added under a nitrogen atmosphere to a solution of
[Pd(PPh3)4] (260 mg, 5% mol) in anhydrous THF (20 mL) and 20%
aqueous K2CO3 (20 mL). The resulting two-phase system was heated to
50 °C, stirred in the dark overnight, and then cooled to room
temperature before dichloromethane (25 mL) and water (15 mL) were
added. The organic layer was separated, washed with brine (25 mL),
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified by column chromatography on silica gel
with dichloromethane as the eluent, yielding the final product 4 as an off-
white solid (0.94 g, 82%).
1H NMR (400 MHz, CDCl3): δ = 2.45 (s, 3H, Me), 7.32 (s, 1H,
thiophene), 7.38 (m, 2H, J1 = 6.0 Hz, J2 = 2.4 Hz, pyridine), 8.59 (m, 2H,
J1 = 6.0 Hz, J2 = 2.4 Hz, pyridine). MS (ESI): m/z = 254.0 [M]+
(expected: 253.96).
4-Bromo-5-methyl-2-(3-quinolyl)thiophene (5). Compound 5 was
prepared following the procedure described for compound 4 but using
3-bromoquinoline (0.9 mL, 6.7 mmol) instead of 4-bromopyridine. The
crude product was purified by column chromatography on silica gel with
dichloromethane as the eluent, to yield the final product as an off-white
solid (0.81 g, 59%).
1H NMR (400 MHz, CDCl3): δ = 2.48 (s, 3H, Me), 7.31 (s, 1H,
thiophene), 7.58 (m, 2H, quinoline), 7.71 (m, 2H, quinoline), 7.84 (d,
2H, J = 8.8 Hz, quinoline), 8.12 (d, 2H, J = 8.8 Hz, quinoline), 8.21 (d,
2H, J = 2.4 Hz, quinoline), 9.11 (d, 2H, J = 2.4 Hz, quinoline). MS (ESI):
m/z = 306.0 [M + H]+ (expected: 305.97).
1H NMR (400 MHz, CDCl3): δ = 2.06 (m, 2H, J = 6.8 Hz, CH2), 2.66
(s, 6H, Me), 2.86 (t, 4H, J = 6.8 Hz, COCH2), 7.18 (s, 2H, thiophene).
Mass spectrometry (MS) (ESI): m/z = 361 [M]+ (expected: 360.98).
Anal. Calcd for C15H14Cl2O2S2 (%): C 49.86, H 3.91, S 17.75; found: C
50.19, H 3.94, S 16.56.
1,2-Bis(5-chloro-2-methyl-3-thienyl)cyclopentene (2). Titanium-
(IV) tetrachloride (4.4 mL, 40 mmol) was carefully added under a
nitrogen atmosphere using a syringe to an ice-cooled suspension of zinc
dust (5.2 g, 80 mmol) in 150 mL of anhydrous tetrahydrofuran (THF).
The resulting gray-blue reaction mixture was slowly heated to 50 °C and
1-(2-Methyl-5-chloro-3-thienyl)-2-(2-methyl-5-phenyl-3-thienyl)-
cyclopentene (6). 2 (1.0 g, 3.0 mmol) was dissolved in anhydrous THF
(10 mL) under a nitrogen atmosphere and treated with n-butyllithium
(2.0 mL of a 1.6 M solution in hexane, 3.2 mmol) at room temperature.
After it was stirred in the dark for ∼45 min, tributyl borate (1.2 mL, 4.5
mmol) was added, and the resulting bright orange solution was then
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stirred in the dark for 1 h; it was subsequently used for the Suzuki cross-
coupling reaction. In the meantime, in a separate flask, [Pd(PPh3)4]
(180 mg, 5% mol) and bromobenzene (0.4 mL, 3.75 mmol) were
dissolved in a solvent mixture containing anhydrous THF (20 mL) and
20% aqueous K2CO3 solution (20 mL), under a dinitrogen atmosphere.
This two-phase system was stirred at 50 °C for 15 min, and the previous
freshly prepared boronic derivative solution was added dropwise with a
syringe. The resulting reaction mixture was stirred in the dark overnight,
after which it was cooled to room temperature; dichloromethane (25
mL) and water (15 mL) were subsequently added. The organic layer was
separated, washed with brine (25 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was purified
by column chromatography on silica gel with a 19:1 mixture of
cyclohexane and ethyl acetate as the eluent, yielding 6 as a slightly
colored oil (0.74 g, 66%).
yield may be because the octafluorocyclopentene is highly volatile (bp =
27 °C).
1H NMR (400 MHz, CDCl3): δ = 2.00 (s, 6H, Me), 7.41 (m, 4H, J1 =
4.4 Hz, J2 = 1.6 Hz, pyridine), 7.47 (s, 2H, thiophene), 8.61 (m, 4H, J1 =
4.4 Hz, J2 = 1.6 Hz, pyridine). 19F NMR (400 MHz, CDCl3): δ =
−131.81 (quint, 2H, J = 5.6 Hz), −110.11 (t, 4H, J = 5.6 Hz). 13C NMR
(100 MHz, CDCl3): δ = 14.9, 111.0 (multiplet), 116.1 (multiplet),
119.7, 124.8, 126.3, 136.3 (multiplet), 139.5, 140.3, 143.8, 150.7. MS
(ESI): m/z = 523.1 [M + H]+ (expected: 523.07). Anal. Calcd for
C25H16F6N2S2 (%): C 57.47, H 3.09, N 5.36, S 12.27; found: C 58.14, H
3.43, N 5.42, S 12.14.
1,2-Bis(2-methyl-5-(3-quinolyl)-3-thienyl)cyclopentane (L3). L3
was prepared following the procedure described for L1 but using 3-
bromoquinoline (1.0 mL, 7.5 mmol) as the bromide derivative in the
Suzuki cross-coupling reaction. The crude product was then purified by
column chromatography on silica gel with a 4:1 mixture of dichloro-
methane and ethyl acetate as the eluent, producing L3 as a bright white
solid (0.87 g, 56%).
1H NMR (400 MHz, CDCl3): δ = 1.88 (s, 3H, Me), 2.00 (s, 3H, Me),
2.05 (m, 2H, 7.6 Hz, cyclopentene), 2.75 (m, 2H, Japp = 7.6 Hz,
cyclopentene), 2.81 (m, 2H, Japp = 7.6 Hz, cyclopentene), 6.62 (s, 1H,
thiophene), 6.99 (s, 1H, thiophene), 7.23 (m, 1H, Japp = 7.2 Hz, phenyl),
7.34 (m, 2H, Japp = 7.2 Hz, phenyl), 7.49 (m, 2H, Japp = 7.2 Hz, phenyl).
MS (ESI): m/z = 371.1 [M]+ (expected: 371.07).
1H NMR (400 MHz, CDCl3): δ = 2.10 (s, 6H, Me), 2.15 (m, 2H, J =
7.6 Hz, cyclopentene), 2.91 (t, 4H, J = 7.6 Hz, cyclopentene), 7.23 (s,
2H, thiophene), 7.53 (m, 2H, J1 = 6.8 Hz, J2 = 1.2 Hz, quinoline), 7.66
(m, 2H, J1 = 6.8 Hz, J2 = 1.2 Hz, quinoline), 7.79 (d, 2H, J = 8.0 Hz,
quinoline), 8.07 (d, 2H, J = 8.0 Hz, quinoline), 8.14 (d, 2H, J = 2.0 Hz,
quinoline), 9.09 (d, 2H, J = 2.0 Hz, quinoline). 13C NMR (100 MHz,
CDCl3): δ = 14.7, 23.2, 38.6, 125.5, 127.3, 127.7, 127.9, 128.1, 129.2,
129.4, 130.6, 135.0, 136.1, 136.4, 137.2, 147.2, 148.4. MS (ESI): m/z =
516.1 [M]+ (expected: 516.17). Anal. Calcd for C33H26N2S2 (%): C
77.01, H 5.09, N 5.44, S 12.46; found: C 76.73, H 5.22, N 5.59, S 12.29.
1,2-Bis(2-methyl-5-(3-quinolyl)-3-thienyl)perfluorocyclopentene
(L4). 5 (1.2 g, 4.0 mmol) was suspended in anhydrous THF (15 mL)
under a nitrogen atmosphere. Next, n-butyllithium (2.6 mL of a 1.6 M
solution in hexane, 4.2 mmol) was added dropwise at −78 °C. After the
resulting pink suspension was stirred for 30 min, octafluorocyclopentene
(1.35 mL of a 20% solution in anhydrous THF, 2.0 mmol) was added,
and the reaction mixture was stirred for 1 h at −78 °C. Afterward, the
cooling bath was removed, and the stirring continued at room
temperature for 3 h, after which dichloromethane (25 mL) and water
(25 mL) were added. The organic layer was separated, washed with
brine, dried over Na2SO4, filtered, and concentrated under reduced
pressure. The crude product was purified by column chromatography on
silica gel using ethyl acetate as the eluent, yielding L4 as a pale blue solid
(0.18 g, 14%).
1,2-Bis(2-methyl-5-(4-pyridyl)-3-thienyl)cyclopentene (L1). Ligand
L1, whose synthetic pathway was described earlier,20 was prepared as
described below. This procedure was applied for all ligands including a
central cyclopentene ring (ligands L3, L5, L6, and L7).
2 (1.0 g, 3.0 mmol) was dissolved in anhydrous THF (10 mL) under a
nitrogen atmosphere and treated with n-butyllithium (4.0 mL of a 1.6 M
solution in hexane, 6.4 mmol) at room temperature. The reaction
mixture was stirred in the dark for ∼45 min, until a light brown
precipitate appeared, and tributyl borate (2.4 mL, 8.9 mmol) was added
subsequently. The resulting bright orange solution was stirred in the
dark for 1 h and subsequently used for a Suzuki cross-coupling reaction.
Hence, in a separate flask, [Pd(PPh3)4] (360 mg, 5% mol) and 4-
bromopyridine hydrochloride (1.46 g, 7.5 mmol) were dissolved in a
mixture of anhydrous THF (20 mL) and 20% aqueous solution of
K2CO3 (20 mL) under a nitrogen atmosphere. This two-phase system
was stirred at 50 °C for 15 min, and the previous freshly prepared
solution of the boronic derivative of 2 was added dropwise with a
syringe. The resulting reaction mixture was then stirred in the dark
overnight, after which it was cooled to room temperature and treated
with dichloromethane (25 mL) and water (15 mL). The organic layer
was separated, washed with brine (25 mL), dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude product was
purified by column chromatography on silica gel with ethyl acetate
(containing 1% NH3) as the eluent, yielding the final product as a white
solid (0.60 g, 48%).
1H NMR (400 MHz, CDCl3): δ = 2.08 (s, 6H, Me), 7.47 (s, 2H,
thiophene), 7.58 (m, 2H, quinoline), 7.72 (m, 2H, quinoline), 7.84 (d,
2H, J = 8.0 Hz, quinoline), 8.11 (d, 2H, J = 8.0 Hz, quinoline), 8.24 (br,
2H, quinoline), 9.12 (d, 2H, J = 2.0 Hz, quinoline). 19F NMR (400 MHz,
CDCl3): δ = −131.82 (quint, 2H, J = 5.6 Hz), −109.97 (t, 4H, J = 5.6
Hz). 13C NMR (100 MHz, CDCl3): δ = 14.8, 111.1 (multiplet), 116.2
(multiplet), 123.9, 126.3, 126.5, 127.6, 127.9, 128.0, 129.4, 129.9, 131.5,
136.4 (multiplet), 139.0, 142.7, 147.5, 148.1. MS (ESI): m/z = 623.1
[M]+ (expected: 623.1). Anal. Calcd for C33H20F6N2S2 (%): C 63.66, H
3.24, N 4.50, S 10.30; found: C 63.59, H 3.70, N 4.47, S 9.76.
1,2-Bis(2-methyl-5-(5-methyl-3-pyridyl)-3-thienyl)cyclopentene
(L5). L5 was prepared following the procedure described for L1 but
using 3-bromo-5-methylpyridine (0.85 mL, 7.5 mmol) as the bromide
derivative in the Suzuki cross-coupling reaction. The crude product was
then purified by column chromatography on silica gel with a 3:1 mixture
of ethyl acetate and dichloromethane as the eluent to yield the L5 as a
white solid (0.69 g, 52%).
1H NMR (400 MHz, CDCl3): δ = 2.03 (s, 6H, Me), 2.11 (m, 2H, J =
7.6 Hz, cyclopentene), 2.85 (t, 4H, J = 7.6 Hz, cyclopentene), 7.22 (s,
2H, thiophene), 7.35 (m, 4H, Japp = 6.4 Hz, pyridine), 8.53 (m, 4H, Japp
=
6.4 Hz, pyridine). 13C NMR (100 MHz, CDCl3): δ = 14.8, 23.1, 38.6,
119.4, 126.4, 134.9, 136.8, 137.2, 137.4, 141.4, 150.4. MS (ESI): m/z =
415.1 [M + H]+ (expected: 415.13). Anal. Calcd for C25H22N2S2 (%): C
72.43, H 5.35, N 6.76, S 15.47; found: C 72.75, H 5.40, N 6.59, S 15.30.
1,2-Bis(2-methyl-5-(4-pyridyl)-3-thienyl)perfluorocyclopentene
(L2). Ligand L2, whose synthetic pathway was described earlier,20 was
prepared as described below. This procedure was applied for the other
ligand containing a central perfluorocyclopentene ring, namely, L4.
4 (1.0 g, 4.0 mmol) was suspended in anhydrous diethyl ether (15
mL) under a nitrogen atmosphere. Then, n-butyllithium (2.6 mL of a 1.6
M solution in hexane, 4.2 mmol) was added dropwise at −78 °C. After
the resulting pink suspension was stirred for 30 min, octafluorocyclo-
pentene (1.35 mL of a 20% solution in anhydrous Et2O, 2.0 mmol) was
added, and the ensuing reaction mixture was stirred for 1 h at −78 °C.
The cooling bath was subsequently removed, and the stirring continued
at room temperature for 3 h, after which dichloromethane (25 mL) and
water (25 mL) were added. The organic layer was separated, washed
with brine (25 mL), dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was purified by column
chromatography on silica gel using ethyl acetate (containing 1% NH3)
as the eluent, yielding L2 as a pale brown solid (0.27 g, 26%). The low
1H NMR (400 MHz, CDCl3): δ = 2.02 (s, 6H, Me), 2.10 (m, 2H, J =
7.6 Hz, cyclopentene), 2.34 (s, 6H, Me), 2.85 (t, 4H, J = 7.6 Hz,
cyclopentene), 7.05 (s, 2H, thiophene), 7.55 (s, 2H, pyridine), 8.29 (s,
2H, pyridine), 8.56 (s, 2H, pyridine). 13C NMR (100 MHz, CDCl3): δ =
14.6, 18.5, 23.2, 38.6, 125.1, 130.0, 133.0, 133.2, 134.9, 135.7, 136.1,
137.0, 143.9, 148.7. MS (ESI): m/z = 444.1 [M]+ (expected: 444.17).
Anal. Calcd for C27H28N2S2 (%): C 73.26, H 5.92, N 6.33, S 14.49;
found: C 72.31, H 6.17, N 6.33, S 14.09.
1,2-Bis(2-methyl-5-(1-methyl-1H-imidazol-2-yl)-3-thienyl)-
cyclopentane (L6). L6 was prepared following the procedure described
for L1 but using 2-bromo-1-methyl-1H-imidazole (1.0 g, 6.2 mmol) as
D
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the bromide derivative in the Suzuki cross-coupling reaction. The crude
product was then purified by column chromatography on silica gel with
ethyl acetate (containing 1% NH3) as the eluent, to yield L6 as a slightly
pink solid (0.47 g, 37%).
C 28.77, H 2.33, N 2.31, S 10.59; found: C 29.01, H 2.30, N 2.48, S
10.36.
[{trans-PtCl2(DMSO)}2(μ-L3)] (C3). Complex C3 was prepared
following the procedure described for the synthesis of C1 but using
ligand L3 (129 mg, 0.25 mmol) to obtain the corresponding product as a
pale green solid (255 mg, 85%).
1H NMR (400 MHz, CDCl3): δ = 2.08 (m, 2H, J = 7.6 Hz,
cyclopentene), 2.10 (s, 6H, Me), 2.83 (t, 4H, J = 7.6 Hz, cyclopentene),
3.65 (s, 6H, N-Me), 6.84 (d, 2H, J = 1.2 Hz, imidazole), 6.96 (s, 2H,
thiophene), 7.01 (d, 2H, J = 1.2 Hz, imidazole). 13C NMR (100 MHz,
CDCl3): δ = 14.4, 23.2, 34.6, 38.2, 122.4, 126.9, 128.5, 128.9, 135.0,
136.2, 136.2, 142.4. MS (ESI): m/z = 422.1 [M]+ (expected: 422.16).
Anal. Calcd for C23H24N4S2 (%): C 65.68, H 5.75, N 13.32, S 15.25;
found: C 65.38, H 6.07, N 13.21, S 15.43.
1H NMR (400 MHz, CDCl3): δ = 2.09 (s, 6H, Me), 2.17 (m, 2H, J =
7.6 Hz, cyclopentene), 2.93 (t, 4H, J = 7.6 Hz, cyclopentene), 3.47 (s,
12H, DMSO), 7.30 (s, 2H, thiophene), 7.65 (m, 2H, Japp = 7.6 Hz,
quinoline), 7.82−7.88 (m, 4H, quinoline), 8.29 (d, 2H, J = 2.0 Hz,
quinoline), 9.25 (d, 2H, J = 2.0 Hz, quinoline), 9.30 (d, 2H, J = 8.8 Hz,
quinoline). 13C NMR (100 MHz, CDCl3): δ = 14.9, 23.3, 38.7, 43.9,
126.8, 128.3, 128.5, 128.9, 129.2, 129.9, 131.1, 133.7, 134.2, 135.1, 137.6,
137.7, 144.5, 151.5. MS (ESI): m/z = 1225.0 [M + Na]+ (expected:
1224.97). Anal. Calcd for C37H38Cl4N2O2Pt2S4 (%): C 36.94, H 3.18, N
2.33, S 10.66; found: C 36.44, H 3.18, N 2.35, S 10.11.
1-(2-Methyl-5-(4-pyridiyl)-3-thienyl)-2-(2-methyl-5-phenyl-3-
thienyl)-cyclopentene (L7). 6 (0.7 g, 1.9 mmol) was dissolved in
anhydrous THF (10 mL) under a dinitrogen atmosphere and treated
with n-butyllithium (1.3 mL of a 1.6 M solution in hexane, 2.1 mmol) at
room temperature. After it was stirred in the dark for ∼45 min, tributyl
borate (0.8 mL, 3.0 mmol) was added, and the resulting bright red
solution was stirred in the dark for 1 h; it was subsequently used for the
Suzuki cross-coupling reaction. Meanwhile, in a separate flask,
[Pd(PPh3)4] (115 mg, 5% mol) and 4-bromopyridine hydrochloride
(0.47 g, 2.4 mmol) were dissolved in a solvent mixture composed of
anhydrous THF (20 mL) and 20% aqueous K2CO3 solution (20 mL)
under a dinitrogen atmosphere. This two-phase system was stirred at 50
°C for 15 min, and the previously prepared boronic derivative solution
was added dropwise with a syringe. The resulting reaction mixture was
then stirred in the dark overnight, after which it was cooled to room
temperature, and dichloromethane (25 mL) and water (15 mL) were
added. The organic layer was separated, washed with brine (25 mL),
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was purified by column chromatography on silica gel
with ethyl acetate as the eluent, yielding L7 as a brown solid (0.57 g,
72%).
Single crystals of complex C3, suitable for X-ray diffraction studies,
were obtained by slow diffusion of diethyl ether into a concentrated
solution of the complex in dichloromethane.
[{trans-PtCl2(DMSO)}2(μ-L4)] (C4). Complex C4 was prepared
following the procedure described for the synthesis of C1 but using
ligand L4 (156 mg, 0.25 mmol) to obtain the corresponding product as
an off-white solid (260 mg, 79%).
1H NMR (400 MHz, CDCl3): δ = 2.12 (s, 6H, Me), 3.52 (s, 12H,
DMSO), 7.51 (s, 2H, thiophene), 7.71 (m, 2H, Japp = 7.6 Hz, quinoline),
7.89−7.93 (m, 4H, quinoline), 8.39 (d, 2H, J = 2.0 Hz, quinoline), 9.27
(d, 2H, J = 2.0 Hz, quinoline), 9.37 (d, 2H, J = 9.2 Hz, quinoline). 19F
NMR (400 MHz, CDCl3): δ = −131.84 (quint, 2H, J = 5.6 Hz),
−109.87 (t, 4H, J = 5.6 Hz). 13C NMR (100 MHz, CDCl3): δ = 15.1,
44.0, 125.3, 126.5, 127.9, 128.4, 128.7, 129.3, 129.7, 131.8, 135.1, 136.3,
144.2, 145.0, 151.3. MS (ESI): m/z = 1332.9 [M + Na]+ (expected:
1332.92). Anal. Calcd for C37H32Cl4F6N2O2Pt2S4 (%): C 33.90, H 2.46,
N 2.14, S 9.78; found: C 33.86, H 2.47, N 2.33, S 9.64.
Single crystals of complex C4, suitable for X-ray diffraction studies,
were obtained by slow diffusion of diethyl ether into a concentrated
solution of the complex in dichloromethane.
[{trans-PtCl2(DMSO)}2(μ-L5)] (C5). Complex C5 was prepared
following the procedure described for the synthesis of C1 but using
ligand L5 (111 mg, 0.25 mmol) to obtain the corresponding product as
an off-white solid (235 mg, 83%).
1H NMR (400 MHz, CDCl3): δ = 1.99 (s, 3H, Me), 2.04 (s, 3H, Me),
2.05−2.14 (m, 2H, Japp = 7.6 Hz, cyclopentene), 2.85 (t, 4H, J = 7.4 Hz,
cyclopentene), 7.02 (s, 1H, thiophene), 7.20−7.25 (m, 2H, thiophene +
phenyl), 7.31−7.36 (m, 4H, phenyl + pyridine), 7.48−7.51 (m, 2H, Japp
= 8.4 Hz, phenyl), 8.52 (dd, 2H, J = 1.5 Hz, J = 4.7 Hz, pyridine). 13
C
NMR (100 MHz, CDCl3): δ = 14.6, 14.9, 23.2, 38.6, 38.6, 119.5, 124.0,
125.5, 126.9, 127.2, 129.0, 134.1, 134.5, 134.6, 135.7, 136.3, 136.6, 137.6,
137.9, 140.1, 142.0, 149.9. MS (ESI): m/z = 413.1 [M]+ (expected:
413.13).
1H NMR (400 MHz, CDCl3): δ = 2.02 (s, 6H, Me), 2.11 (m, 2H, J =
7.6 Hz, cyclopentene), 2.38 (s, 6H, Me), 2.84 (t, 4H, J = 7.6 Hz,
cyclopentene), 3.46 (s, 12H, DMSO), 7.10 (s, 2H, thiophene), 7.66 (s,
2H, pyridine), 8.35 (s, 2H, pyridine), 8.66 (d, 2H, J = 1.6 Hz, pyridine).
13C NMR (100 MHz, CDCl3): δ = 13.6, 17.5, 22.0, 37.4, 43.2, 125.5,
131.3, 132.2, 133.9, 134.8, 135.1, 136.2, 136.5, 144.7, 148.4. MS (ESI):
m/z = 1154.0 [M + Na]+ (expected: 1153.97). Anal. Calcd for
C31H38Cl4N2O2Pt2S4 (%): C 32.93, H 3.39, N 2.48, S 11.34; found: C
33.03, H 3.64, N 2.62, S 11.12.
[{trans-PtCl2(DMSO)}2(μ-L1)] (C1). cis-[PtCl2(DMSO)2]23 (211 mg,
0.5 mmol) was suspended in methanol (40 mL), and the system was
refluxed until the complex was completely dissolved. After the resulting
yellow solution was hot filtered to remove any Pt(0) impurities, ligand
L1 (104 mg, 0.25 mmol) was immediately added, and the mixture was
stirred in the dark at room temperature overnight, yielding C1 as a pale
green solid, which was filtered, washed with methanol, and dried under
reduced pressure (260 mg, 94%).
[{trans-PtCl2(DMSO)}2(μ-L6)] (C6). Complex C6 was prepared
following the procedure described for the synthesis of C1 but using
ligand L6 (105 mg, 0.25 mmol) to obtain the corresponding product as
an off-white solid (205 mg, 74%).
1H NMR (400 MHz, CDCl3): δ = 2.03 (s, 6H, Me), 2.13 (m, 2H, J =
7.6 Hz, cyclopentene), 2.85 (t, 4H, J = 7.6 Hz, cyclopentene), 3.47 (s,
12H, DMSO), 7.28 (s, 2H, thiophene), 7.39 (m, 4H, Japp = 6.4 Hz,
pyridine), 8.59 (m, 4H, Japp = 6.4 Hz, pyridine). 13C NMR (100 MHz,
CDCl3): δ = 15.0, 23.0, 38.6, 44.3, 120.5, 128.9, 134.5, 135.1, 137.9,
140.8, 144.5, 151.7. MS (ESI): m/z = 1124.9 [M + Na]+ (expected:
1124.94). Anal. Calcd for C29H34Cl4N2O2Pt2S4 (%): C 31.58, H 3.11, N
2.54, S 11.63; found: C 31.11, H 3.09, N 2.52, S 11.53.
[{trans-PtCl2(DMSO)}2(μ-L2)] (C2). Complex C2 was prepared
following the procedure described for the synthesis of C1 but using
ligand L2 (136 mg, 0.25 mmol) to obtain the corresponding product as
an off-white solid (295 mg, 95%).
1H NMR (400 MHz, CDCl3): δ = 2.12 (m, 2H, cyclopentene), 2.23
(s, 6H, Me), 2.89 (t, 4H, cyclopentene), 3.33 (s, 12H, DMSO), 3.68 (s,
6H, N-Me), 6.95 (d, 2H, J = 1.2 Hz, imidazole), 7.15 (d, 2H, J = 1.2 Hz,
imidazole), 7.25 (s, 2H, thiophene). 13C NMR (100 MHz, CDCl3): δ =
14.9, 23.2, 36.0, 38.4, 44.1, 123.1, 123.3, 128.4, 132.5, 134.9, 135.9,
139.8, 142.3. MS (ESI): m/z = 1108 [M]+ (expected: 1107.97). Anal.
Calcd for C27H36Cl4N4O2Pt2S4 (%): C 29.25, H 3.27, N 5.05, S 11.57;
found: C 29.21, H 3.37, N 5.04, S 11.69.
trans-[PtCl2(DMSO)(L7)] (C7). Complex C7 was prepared following
the procedure described for the synthesis of C1 but using equimolar
amounts of cis-[PtCl2(DMSO)2] (105 mg, 0.25 mmol) and ligand L7
(103 mg, 0.25 mmol) to obtain the corresponding mononuclear product
as a dark green solid (80 mg, 42%).
1H NMR (400 MHz, CDCl3): δ = 2.03 (s, 6H, Me), 3.48 (s, 12H,
DMSO), 7.50 (m, 4H, J1 = 5.6 Hz, J2 = 1.6 Hz, pyridine), 7.55 (s, 2H,
thiophene), 8.71 (m, 4H, J1 = 5.6 Hz, J2 = 1.6 Hz, pyridine). 19F NMR
(400 MHz, CDCl3): δ = −131.78 (quint, 2H, J = 5.6 Hz), −110.10 (t,
4H, J = 5.6 Hz). 13C NMR (100 MHz, CDCl3): δ = 15.1, 44.4, 121.0,
126.8, 127.3, 137.0, 143.4, 146.5, 152.3. MS (ESI): m/z = 1231.9 [M +
Na]+ (expected: 1231.88). Anal. Calcd for C29H28Cl4F6N2O2Pt2S4 (%):
1H NMR (400 MHz, CDCl3): δ = 1.97 (s, 3H, Me), 2.07 (s, 3H, Me),
2.11 (m, 2H, 7.6 Hz, cyclopentene), 2.84 (m, 4H, cyclopentene), 3.47 (s,
6H, DMSO), 6.99 (s, 1H, thiophene), 7.24 (m, 1H, J1 = 7.6 Hz, J2 = 1.2
Hz, phenyl), 7.31 (s, 1H, thiophene), 7.34 (m, 2H, Japp = 7.6 Hz,
E
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phenyl), 7.39 (m, 2H, J1 = 5.6 Hz, J2 = 1.6 Hz, pyridine), 7.48 (m, 2H, Japp
adenocarcinoma (SW620), and ovarian adenocarcinoma (SKOV3)
cell lines used in this study were purchased from the American Type
Culture Collection (ATCC). All cell lines were tested and authenticated
by ATCC using short tandem repeat analysis and were cultured (passage
number 10−25) following ATCC recommended media. A549, A375,
SW620, and SKOV3 cells were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) medium supplemented with 10% (v/v) fetal bovine
serum (FBS), 100 unit/mL penicillin, 100 μg/mL streptomycin, and 2
mM L-glutamine. MCF7 cells were cultured in DMEM-F12 (Ham)
media (1:1) supplemented with 5% horse serum (v/v), 100 μM sodium
pyruvate, 10 μg/mL insulin, 100 unit/mL penicillin, 100 μg/mL
streptomycin, and 2 mM L-glutamine. All cell lines were grown at 37 °C
under a 5% CO2 atmosphere. The cell lines were routinely tested using a
specific standard PCR to control mycoplasma contamination.
Cell Viability Assays. Cell proliferation was evaluated by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay.
Cells were plated in 96-well sterile plates at a density of 1 × 105 cells per
mL (100 μL) and allowed to grow for 24 h. After attachment to the
surface, the cells were then incubated with various concentrations of the
studied complexes (10 and 50 μM for single-point experiments and
within the range of 0.5−5 μM for dose−response curves), freshly
dissolved in DMSO and diluted in the corresponding culture medium
(DMSO final concentration = 1%), for 48 h at 37 °C. Control cells were
cultured in the corresponding culture medium plus the carrier (DMSO
final concentration = 1%). Following the treatment, 10 μM MTT was
added to each well for an additional 4 h. Afterward, the medium was
aspirated, the purple formazan precipitate was dissolved in 100 μL of
DMSO, and the absorbance at 570 nm was measured in a multiwell plate
reader Multiskan FC (Thermo Scientific). The cell viability was
expressed as percentage values with respect to control cells, and the data
= 7.6 Hz, phenyl), 8.56 (m, 2H, J1 = 5.6 Hz, J2 = 1.6 Hz, pyridine). 13
C
NMR (100 MHz, CDCl3): δ = 14.5, 15.1, 23.1, 38.4, 38.6, 44.3, 120.5,
123.8, 125.4, 127.3, 129.0, 129.3, 133.5, 134.0, 134.3, 134.6, 136.3, 136.5,
138.4, 140.3, 141.0, 144.8, 151.7. MS (ESI): m/z = 780.0 [M + Na]+
(expected: 780.03). Anal. Calcd for C28H29Cl2NOPtS3 (%): C 44.38, H
3.86, N 1.85, S 12.69; found: C 43.28, H 3.94, N 2.04, S 12.46.
X-ray Structure Determination. Data for the open and closed
forms of complex C3 and the open form of complex C4 were collected
on a Bruker PHOTON100 CMOS diffractometer at beamline 11.3.1 of
the Advanced Light Source (synchrotron radiation, λ = 0.7749 Å),
respectively, on a dark purple/orange (depending on crystal
orientation) plate at 200 K and a colorless plate at 100 K. Data
reduction was done with SAINT.24 The structures were solved by
intrinsic phasing using SHELXT25 and refined by full-matrix least-
squares on F2 with SHELXL-2014.26 Absorption corrections for the
structure of C4 were done with SADABS.24 The crystal of C3 was found
to contain both the open and closed forms. It is unclear if this is a
consequence of cocrystallization or solid-state transformation upon
exposure to light. The structure was refined as a 2-component twin using
cells and twin law determined with CELL_NOW.27 Absorption
corrections were done with TWINABS.27 The heavy disorder of the
central part of the ligand associated with the presence of both
photoisomers in the same crystal, initially defined from electron-density
peaks, was refined with both 1,2- and 1,3- distances and displacement
parameters restraints. Crystallographic and refinement parameters are
summarized in Tables S1 and S3, for complexes C3 and C4, respectively.
Additional crystallographic information is available in the Supporting
Information.
Agarose Gel Electrophoresis. Cacodylate-buffered solutions (20
μL) containing 15 μM (in base pair) pBR322 plasmid DNA, 0.5−2.0
equiv of the complexes (7.5−30 μM), and 5% DMSO were incubated in
the dark for 24 h at 37 °C. Analogous sample solutions of free plasmid
DNA and DNA bound to cisplatin (0.5 equiv, without DMSO) were
also prepared and used as controls. After the incubation, all samples were
treated with 4 μL of a xylene cyanol 1X aqueous solution (containing
30% glycerol) and subsequently electrophoretized in agarose gel (1% in
TAE buffer) for 1 h at 6.25 V cm−1, using a Bio-Rad horizontal tank
connected to a Consort EV231 variable potential power supply. Next,
the gel was treated with SYBR Safe DNA gel stain and subsequently
photographed with a Bio-Rad Gel Doc EZ imager.
Ethidium Bromide Displacement Assays. Samples containing 15
μM (in base pair) ct-DNA and 75 μM ethidium bromide in cacodylate
buffer were incubated for 1 h at 37 °C, after which they were treated with
increasing amounts of the complex stock solutions (see Materials and
Methods). The ratio of DNA/ethidium bromide (EB) of 1:5 (15 μMbp
DNA, 75 μM EB) was determined experimentally, by fluorescence
spectroscopy. It corresponds to the saturation of the emission signal
(i.e., when a plateau is reached), which indicates that EB occupies all
possible intercalation sites. The resulting samples, containing 1−25 μM
of the studied complexes and up to 5% DMSO in a final volume of 3 mL,
were then incubated in the dark for 24 h at 37 °C. Following the
incubation, the fluorescence emission spectra of all samples were
recorded at room temperature in the range of 530−800 nm, using a
Horiba Jovin Yvon SPEX Fluorolog 3−22 spectrofluorometer and
applying an excitation wavelength of λexc = 514 nm. Cacodylate-buffered
solutions of ct-DNA/EB (i.e., without complex) were used as control
references.
Circular Dichroism Spectroscopy. Samples containing 50 μM ct-
DNA (in base pair), 5−25 μM of the studied complexes, and up to 5%
DMSO in a final volume of 3 mL of cacodylate-buffered solution were
incubated for 24 h at 37 °C. Following the incubation, the circular
dichroism spectra of all samples were recorded at room temperature
using a Jasco J-815 CD spectropolarimeter. A wavelength range of 235−
315 nm was used to minimize DMSO interference, using a bandwidth of
2 nm, a data pitch of 0.5 nm, scanning speed of 50 nm min−1, response
time of 1 s, and 4 accumulations. Cacodylate-buffered solutions of ct-
DNA were used as control references.
are shown as the mean value
standard deviation (SD) of three
independent experiments. Dose−response curves and the correspond-
ing IC50 values were obtained by means of nonlinear regression (curve
fit), calculated with the GraphPad Prism 5.0 software. For comparison
purposes, the cytotoxic effect of cisplatin was also evaluated under
identical experimental conditions.
RESULTS AND DISCUSSION
■
Preparation of the Ligands and Their Respective
Platinum(II) Complexes. The diarylethene-based ligands
were prepared using common synthetic procedures (Figure
S1).20,22,28 Ligands L1, L3, and L5−L7 were obtained in
moderate-to-good yields, ranging from 37% to 72%, by Suzuki
cross-coupling reaction between the in situ-generated bis-
boronic ester derivative of 1,2-bis(5-chloro-2-methyl-3-thienyl)-
cyclopentane and the corresponding bromo(hetero)arene (see
Experimental Section).29 Ligands L2 and L4, containing a
perfluorinated cyclopentene ring, were synthesized in low yields
(26% and 14%, respectively), by reaction of octafluorocyclo-
pentene with the corresponding bromo heteroarylthiophene
(see Experimental Details).
The photoswitching abilities of the ligands can be assessed by
UV−vis and 1H NMR spectroscopies. For example, the colorless,
open form of L1 exhibits intense absorption bands in the UV
region of the spectrum (Figures S2). The absorption observed at
λ = 322 nm corresponds to the highest occupied molecular
orbital (HOMO) → lowest unoccupied molecular orbital
(LUMO) transition, which, according to the Woodward−
Hoffmann rules, is required for the photocyclization process to
occur.30 UV irradiation of open L1 gives rise to the development
of an intense new absorption band in the visible region, that is, at
λ = 544 nm (Figures S2), as reflected by the deep purple color of
the solution. It can be pointed out here that a photostationary
state (PSS) is reached, which corresponds to a final equilibrium
situation of the photocyclization reaction; although complete
photoconversion is rarely achieved, the PSS is assumed to
Cell Lines and Culture. Human lung adenocarcinoma (A549),
melanoma (A375), breast adenocarcinoma (MCF7), colorectal
F
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Figure 4. Plots of I0/I vs [complex] for the titration of ct-DNA/EB
samples ([DNA]bp = 15 μM, [EB] = 75 μM) treated with the open (red)
and closed (blue) forms of C1 at λexc = 514 nm and λem = 610 nm.
Experimental data points obtained in triplicate and linear fitting of the
data.
Table 1. Calculated Stern−Volmer Quenching Constants for
the Titration of ct-DNA/EB ([DNA]bp = 15 μM, [EB] = 75
μM) with the Different Complexes (open and closed forms),
at λexc = 514 nm and λem = 610 nm
complex KSV (1 × 103 M−1), open form KSV (1 × 103 M−1), closed form
C1
C2
C3
C4
C5
C6
C7
6.6 0.8
4.8 0.9
13.7 0.7
18.4 0.7
9.9 0.4
17.1 0.9
1.4 0.3
42.5 0.6
26.8 0.9
58.0 3.0
45.0 3.0
37.4 0.8
Figure 2. Agarose gel electrophoresis images for pBR322 DNA
incubated for 24 h at 37 °C with increasing quantities of the open and
closed forms of complexes C1−C7 (solely the open form in the case of
photoinactive C6). Lane 1: native plasmid DNA; lane 2: DNA + 0.5
equiv of cisplatin; lanes 3−6: DNA + 0.5 to 2.0 equiv of the open
complex; lanes 7−10: DNA + 0.5 to 2.0 equiv of the closed complex.
a
21.7 0.6
a
This complex does not undergo photocyclization.
cannot undergo photocyclization; this distinctive behavior was
investigated theoretically and was ascribed to the presence of a
non-photoreactive HOMO−2 → LUMO transition with a
higher computed oscillator strength value than that of the
HOMO → LUMO one, therefore impeding the photocyclization
process.32 The photochemical transformation of all ligands,
except L6, can also be followed by 1H NMR. For instance, NMR
spectra of L2 after different photoirradiation times are illustrated
in Figure S3. These spectra are representative of the other
compounds inspected in the present study (excluding L6, which
does not cyclize). Upon UV exposure, a gradual disappearance of
the original NMR signals (open form of L2) is observed, which is
accompanied by the concomitant, progressive apparition of a
new set of peaks ascribed to closed L2 (see blue asterisks in
Figure S3). The upfield shift of the proton at the 4-position of the
thiophene, from 7.47 to 6.85 ppm, corroborates the occurrence
of the expected electrocyclic reaction, generating a new
intramolecular bond at the proximal 2-position. For irradiation
times longer than 5 min, no further variation of the peaks was
noticed, therefore indicating that the PPS was reached. At this
equilibrium situation, a photoconversion ratio of 0.86 could be
determined from the relative intensity of the two sets of signals
(i.e., from the open and closed forms of L2). Comparable
features are observed for ligands L1, L3−L5, and L7.
Figure 3. Fluorescent emission spectra of ct-DNA/EB samples
([DNA]bp = 15 μM, [EB] = 75 μM) treated with increasing quantities
(1−25 μM) of open C1 (top) and closed C1 (bottom), registered after
incubation for 24 h at 37 °C.
After full characterization, platination of the ligands was
performed, generating complexes C1−C7 with good yields, from
42% to 95% (Figure 1). Hence, reaction of 2 equiv of cis-
dichlorobis(dimethyl sulfoxide)platinum(II) (cis-
[PtCl2(DMSO)2]) with 1 equiv of L1−L6 produced the
corresponding complexes C1−C6, while the reaction between
1 equiv of the platinum(II) salt and 1 equiv of L7 yielded C7.
represent the closed form of the molecule.31 Similar features
were observed for all other ligands, except for ligand L6, which
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Figure 5. Single-point cell viability assays for the open and closed forms of C2, at constant complex concentrations (i.e., 10 and 50 μM) with incubation
time of 48 h. The results are given in % of cell viability, using five different cancer cell lines, namely, A549, A375, MCF7, SW620, and SKOV3. The data
shown are means SD of three independent experiments.
Figure 6. Single-point cell viability assays for photoinactive C6, at constant complex concentrations (i.e., 10 and 50 μM) with an incubation time of 48 h.
The results are given in % of cell viability, using five different cancer cell lines, namely, A549, A375 (melanoma), MCF7, SW620, and SKOV3. The data
shown are means SD of three independent experiments.
a
state structure of C3 contains both the open and closed forms of
the metal complex, in a 31.6/68.4 (Figure S4) ratio, which is not
commonly observed with such photoswitching molecules.
It can also be stressed that all complexes contain a DMSO,
which is trans to the N-coordinated ligand (strong trans effect of
S-coordinated DMSO33,34); in principle, this solvent molecule
may be used to further functionalize the platinum compounds,35
hence increasing the versatility of the system (since numerous
monodentate ligands may be used to replace this DMSO).
Moreover, one may expect that the high chloride concentration
in blood (95−105 mM) would convert the compounds into
more hydrophilic “[PtLCl3]−” species (for instance, a complex of
the type [PtCl2(DMSO)L], where L is phosphane ligand, was
reported that could convert into [PtCl3L]− in the presence of
NBu4Cl36), which could be hydrolyzed inside cells (with
subsequent DNA binding).
Agarose Gel Electrophoresis. After full characterization of
complexes C1−C7, the interaction of their open and closed
photoisomers with pBR322 DNA was investigated. The
corresponding gel images are depicted in Figure 2. It can be
mentioned here that one may expect that complexes C1−C7 will
exhibit a mechanism of action related to that of cisplatin. Hence,
hydrolysis of the chlorides of C1−C7 should lead to the
formation of aquated species,37,38 which will bind to DNA by
substitution of the water molecules for purine bases.39 Because of
the trans disposition of the chlorides, it is expected that each
platinum ion will mostly produce 3-intrastand cross-links and
Table 2. Half-Maximal Inhibitory Concentrations (IC50, μM)
of Cisplatin and Complexes C2 and C6 for the A375
(Melanoma) and SW620 (Colorectal Adenocarcinoma)
Human Cell Lines, after Incubation for 48 h
complex
A375 cells
SW620 cells
cisplatin
open C2
closed C2
6.6 0.6
>75
13.5 1.9
>75
3.1 0.2
3.4 0.6
2.4 0.2
1.8 0.3
b
C6
a
The results are expressed as mean values
SD out of three
independent experiments. The dose-response curves are shown in
Figure S8. This complex does not undergo photocyclization.
b
Single crystals, suitable for X-ray diffraction studies, could be
obtained for C2, C3, C4, and C6. The crystal structures of C220
and C632 were described earlier. The X-ray structures of C3 and
C4 are reported herein. Crystallographic and refinement
parameters are summarized in Tables S1 and S3 for C3 and
C4, respectively, and selected coordination bond lengths and
angles for C3 are listed in Table S2, whereas those for C4 are
shown in Table S4. The molecular structures of C3 and C4
(Figures S4 and S5) are related to those of C2 and C6. In all
cases, the platinum atom is in a square-planar environment
defined by two trans chlorido ligands, an S-coordinated DMSO
solvent molecule, and a nitrogen atom from the heteroaromatic
moiety of the ligand. It can be pointed out here that the solid-
H
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Figure 7. CD spectra of ct-DNA samples ([DNA]bp = 50 μM) treated with increasing concentrations (5−25 μM) of open complexes C1−C7, registered
after incubation for 24 h at 37 °C.
monofunctional adducts, which will undergo conversion to
interstrand cross-links;40 the diplatinum complexes should
therefore show strong DNA interactions. It can be pointed out
that polynuclear platinum complexes (PPCs) have shown
remarkable biological activities.41 Furthermore, one may
anticipate that the photoinduced closing/opening of the ligand
will alter the DNA-binding properties of the corresponding
complex isomers. Indeed, the electronic changes taking place
upon ring closure will affect the hydrolysis of the chlorido ligands
(and therefore their DNA-binding aptitude), and the change of
flexibility between the closed and open species will modify their
DNA binding; for instance, interstrand binding may be less
favorable for the closed, more rigid complexes.
As a matter of fact, for complexes C1−C5, a clearly distinct
DNA-interacting behavior of the two isomeric forms is observed.
At low concentration, open C1 appears to produce a decrease of
the electrophoretic mobility of supercoiled DNA (lane 3), which
may be explained by the unwinding of the double helix. However,
this effect becomes less pronounced with the increase of the
concentration of open C1 (lanes 4 to 6). In contrast to lane 3,
DNA form I is detected in lanes 4−6, and the diminution of its
mobility with the increase of [open C1] resembles that of
supercoiled DNA treated with cisplatin (lane 2).42,43 This
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concentration-dependent interaction of open C1 with DNA
suggests that it exhibits different binding modes, most likely
arising from cooperative effects between the platinum centers
and the organic ligand. It seems that with only 0.5 equiv of open
C1, noncovalent interactions play a role (lane 3), whereas for
[open C1] ≥ 1 equiv, typical platination effects are taking place
(lanes 4 to 6).
are comparable to those observed with cisplatin, therefore
suggesting metal binding to DNA.
Closed C5 exhibits the same behavior as its open form (see
lanes 3−6 and lanes 7−10 in Figure 2). Thus, closed C5 does not
seem to show the intercalative properties observed with the
closed isomers of the previous complexes (see above). This
feature may arise from (i) steric hindrance caused by the methyl
substituents of the pyridine ring of ligand L5 and/or (ii) the
relative disposition of the metal centers (meta position instead of
para position for the previous complexes). This drastic alteration
of the DNA-interacting properties of closed C5 (compared, for
instance, to those of closed C1 and C2), triggered by a subtle
modification of the metal-coordinating unit, again indicates that
the binding activity of the two photoisomers proceeds via two
different mechanisms.
As reported earlier, complex C6 does not display photo-
switching properties.32 Consequently, only the DNA interaction
of open C6 could be investigated. Hence open C6 behaves as
open C1; namely, 0.5 equiv of the complex leads to the formation
of DNA form II (lane 3). Higher [open C6] result in effects
comparable with those of the interaction of cisplatin with DNA,
that is, metal binding to the biomolecule. Actually, this covalent
binding of open C6 appears to be particularly strong, since a
vanishing of the bands is observed with the increase of its
concentration (lanes 5−6), which is indicative of the
precipitation of the resulting complex−DNA adducts.
The mononuclear complex C7 has completely distinct
behavior. No photoisomeric differentiation is observed; in both
cases, a comparable retardation of the DNA mobility takes place
(lanes 3−6 and lanes 7−10). Surprisingly, closed C7 does not
exhibit better DNA-intercalating properties than open C7, even
though one may have expected that substitution of one of the
metal-binding units by a phenyl ring (ligand L1 to ligand L7)
would have somewhat favored the insertion of closed C7
between DNA base pairs. Consequently, it can be inferred that
the strong DNA-unwinding ability displayed by most closed
complexes of the present study is due to not only their insertion
between DNA base pairs but also to the subsequent binding of
the two platinum atoms.
Fluorescent Intercalator Displacement Assays. Com-
petitive binding studies were performed subsequently, using EB
bound to ct-DNA and the open and closed forms of the
complexes, as fluorescence quenchers (through the release of
EB). Fluorescence spectra were therefore recorded at constant
concentrations of ct-DNA and EB, respectively, 15 and 75 μM),
by adding increasing amounts of both photoisomers of the
platinum(II) complexes, specifically, in the concentration range
of 1−25 μM. Representative fluorescence spectra for open and
closed C1 are shown in Figure 3. In all cases, a decrease of the
fluorescent emission of the DNA/EB adduct45 is observed, which
is indicative of the displacement of the dye resulting from the
interaction of the complex with the double helix.
On the contrary, closed C1 behaves as a strong DNA
intercalator (lanes 7 to 10), probably as the result of its higher
planarity and electronic conjugation. It is well-known that DNA
intercalation induces a stronger local unwinding of the double
helix than covalent binding; this may result in the relaxation of
negatively supercoiled DNA sequences until reaching an open
circular conformation, whereas further addition of the
intercalating agent will lead to positive supercoiling.44 Such
phenomenon is clearly occurring with closed C1 (lanes 7 to 10).
At lane 7, an initial decrease of the mobility is observed, as the
result of a partial twist reduction of the biomolecule. When the
amount of closed C1 is increased, this effect is enhanced, so that
all initial DNA form I is relaxed and comigrates with DNA form II
(lane 8). Upon further increase of [closed C1], the DNA
mobility is improved (lanes 9 and 10), which is likely due to a
subsequent induction of positive supercoiling to the previously
relaxed structure (lane 8). Finally, note that a vanishing of the
band intensities takes place when the amount of closed C1 is
increased (lanes 8 to 10). This feature points to the precipitation
of highly platinated DNA species.
Replacement of the cyplopentene ring by a perfluorocyclo-
pentene one (from C1 to C2) clearly modifies the DNA-
interacting properties of the resulting complex. Like open C1,
open C2 also induces the unwinding of DNA, the total
conversion to form II now being observed for the highest
complex concentration (lane 6). This feature may be explained
by single-strand cleavage of the plasmid upon platinum binding,
producing the complete relaxation of supercoiled DNA frag-
ments through free rotation around the nicked strand.
However, closed C2 exhibits intercalating properties as closed
C1, but higher concentrations are required to give the same
effect. Indeed, 1.0 equiv of closed C1 is sufficient to provoke
complete relaxation of supercoiled circular DNA (lane 8),
whereas 2.0 equiv are required with closed C2 (lane 10). This
weaker intercalating behavior exhibited by closed C2 may be due
to repulsive interactions between the fluorine atoms of ligand L2
and the phosphate backbone.
For complexes C3 and C4, minor differences were observed
between their open and closed forms (Figure 2). In all cases, the
treatment of plasmid DNA with increasing quantities of the two
photoisomers of each compound gives rise to a noticeable
decrease of the electrophoretic mobility of supercoiled DNA; the
open and closed forms of C3 are even capable of inducing the
comigration of supercoiled and open circular DNA (lanes 4−6
and lanes 8−10). The analogous activities of both forms of the
complexes may be attributed to the larger, planar quinoline
moieties of ligands L3 and L4, compared to the pyridine rings in
L1 and L2, which apparently confers a comparable intercalating
behavior to the photoisomers. It can also be noticed that C4 is
less active than C3, confirming the negative influence of the
fluorinated backbone of ligand L4 on the DNA interaction.
Open C5 follows a dose-dependent pattern that is like that of
open C1 (lanes 3−6). 0.5 equiv of open C5 produces the
complete unwinding of supercoiled circular DNA, generating its
form II (lane 3). Higher amounts of open C5 induce effects that
It can be noticed that the quenching effect is clearly more
pronounced for closed C1 than for open C1 (Figure 3), hence
suggesting that the closed photoisomer has a higher capacity to
expel DNA-bound EB, thus confirming its better intercalating
properties (see Gel Electrophoresis results).46
To properly compare the DNA affinity of all studied species,
the fluorescence emission data obtained were used to determine
the corresponding Stern−Volmer quenching constants (KSV) for
intermolecular deactivations, by applying the Stern−Volmer eq
1:47
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IO
I
fading observed by gel electrophoresis for this complex (Figure
2).
= 1 + KSV[Q]
(1)
Cell Viability Assays. The potential application of such
metalloswitches as a novel type of photochemotherapeutic
agents was examined through the evaluation of the effect of the
open and closed isomers of C1−C7 (only the open form in the
case of C6) on cancer cell proliferation and survivability. Five
cancer cell lines were selected for these studies, namely, lung
adenocarcinoma (A549), melanoma (A375), breast carcinoma
(MCF7), colorectal adenocarcinoma (SW620), and ovarian
adenocarcinoma (SKOV3).
In this expression, I0 is the initial fluorescence intensity of the
DNA/EB system, I represents the fluorescence emission
intensity after the addition of a quencher, and Q is the quenching
molecule, which is the metal complex in the present study. A plot
of I0/I versus [complex] at the maximum emission wavelength
(i.e., λmax = 610 nm) gives a straight line, whose slope is equal to
KSV. Applying this procedure, Stern−Volmer constants of KSV
=
6.6 0.8 × 103 M−1 and KSV = 42.5 0.6 × 103 M−1 were
determined for open and closed C1, respectively (Figure 4).
Similar trends were observed for the open and closed
photoisomers of all compounds investigated, whose respective
Stern−Volmer constants are listed in Table 1.
In a first instance, single-point screening assays at constant
complex concentrations, that is, 10 and 50 μM, were performed
to obtain a general comparative estimation of the cytotoxic
behavior of both photoisomers of each complex. The results
achieved after an incubation time of 48 h are depicted in Figure 5
for complex C2, Figure 6 for C6, and Figure S6 for C1, C3−C5,
and C7. Surprisingly (considering the DNA-binding studies),
most of the screened platinum(II) complexes exhibit very poor
cytotoxic properties; moreover, there is no discrimination
between the activity of their open and closed isomers. Hence,
treatment of the cells with open/closed C1, C3, C4, and C5 gives
rise to above 75% survival of the cell populations (Figure S6).
Mononuclear C7 shows slightly better results, especially for the
SKOV3 cell line, for which the closed form is more active than
the open one (Figure S6). At [C7] = 10 μM, closed C7 can
induce 50% growth inhibition, whereas cell viability of ∼95% is
observed with the open form. Much more favorable results were
achieved with compound C2. Indeed, whereas the open form of
this complex is not active in all tested cell lines, its closed
photoisomer presents a remarkably high cytotoxic activity against
most of them (Figure 5). For instance, incubation of the cells
with 50 μM C2 led to survival levels below 35% in all cases,
except for the ovarian SKOV3 line. Even more interesting is the
fact that comparable cancer-cell death ratios were also obtained
with the same cell lines using a lower amount of the complex,
namely, with a concentration of 10 μM (Figure 5).
The cell-viability results achieved with photochemically inert
C6 were also noteworthy. In accordance with the DNA-
interaction studies (see above), open C6 is highly cytotoxic, as
reflected by the cell-death ratios of over 85% observed in all cell
lines with a complex concentration of 50 μM (Figure 6). Related
high antiproliferative levels were even achieved using a complex
concentration of 10 μM, thus highlighting the different mode of
action of this imidazole-based compound compared with the
open forms of the pyridine- and -quinoline-containing ones
(which are not cytotoxic).
Subsequently, half-maximal inhibitory concentrations (IC50)
were determined for the two most interesting platinum(II)
compounds, namely, C2 and C6 (Figure S7), using the two cell
lines A375 and SW620, carefully selected from the single-point
results (see Figures 5 and 6) and specific characteristics. The
melanoma A375 cells constitute the most suitable target for
PACT drug candidates such as those herein reported, owing to
the inherent accessibility to the treatment area.49 The choice of
the colorectal cancer line SW620 arises from its bad prognosis
and current shortfall of efficient therapies.50 The IC50 for
cisplatin with these two cell lines was also determined, for
comparison purposes.
In all cases, a significantly higher KSV value was obtained for the
closed isomer, hence corroborating their higher intercalative
behavior. Thus, one may expect that the closed forms of the
platinum(II) complexes will induce higher growth inhibitory
effects than their open counterparts. It can also be noticed that
lower quenching constants were systematically achieved for the
fluorinated closed-ring species, compared with their correspond-
ing nonhalogenated analogues (see C1 and C2, and C3 and C4
in Table 1). These features agree with the data achieved by gel
electrophoresis (see above) and confirm the negative effect of the
fluorinated cyclopentane ring on the intercalation of the
corresponding coordination compounds. These differences
may result from a slight destabilization of the DNA/complex
adducts through electrostatic repulsion between the fluorine
atoms and the negatively charged phosphate backbone of the
double helix.48
A noticeably reduced dye-displacement ability is exhibited by
the mononuclear complex C7 (in both its forms; see Table 1).
For instance, closed C7 shows a KSV value that is twice lower than
that of closed C1. These results are in line with the earlier data
obtained with plasmid DNA and confirm that the binding of
dinuclear species like C1 to neighboring nucleobases (after the
insertion of the compound between base pairs) is crucial
regarding the stabilization of the resulting DNA/complex
adducts.
This new set of data again shows that the closed form of the
diplatinum complexes are inducing stronger alterations of the
double-helix conformation than the open one, apparently
through a combination of intercalative and covalent interactions.
It may therefore be expected that such closed species can be more
efficient in inhibiting gene expression and DNA-replication
processes, which may be reflected by a higher antiproliferative
efficiency.
The results achieved with photoinert C6 requires some
attention. As indicated by its KSV value (Table 1), this imidazolic
species exhibits a far superior degree of interaction with DNA
than open C1 and C2 (Table 1). Its dye-displacement activity is
comparable to or even greater than that of quinoline-containing
complexes C3 and C4. Contrary to these two compounds, C6
does not have potentially intercalating structural motifs; hence,
C6 can expel ethidium bromide through a different mode of
action. Most likely, the binding of C6 strongly alters the
conformation of the double helix. Local modifications of the
secondary or tertiary structure of DNA, like those produced by
1,3-interstrand cross-links, may indeed force the dye to leave the
nearby interbase positions as the result of the destabilization of
the π-stacking interactions. This phenomenon may also explain
the apparent higher degree of platination and consequent band
The IC50 data listed in Table 2 corroborate the single-point cell
viability assays (see above) and the cytotoxic potential of the
selected compounds, which are more efficient than cisplatin for
the two cell lines. The IC50 values for closed C2 and open C6 are
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in the low micromolar range, the compounds being up to 8 times
more active than cisplatin (Table 2).
metal complexes prepared were investigated, which showed that
the ligand L6 and the corresponding complex C6 were
photoinactive.32
Circular Dichroism Measurements. The distinct results
achieved with open C6, compared with those of all other open
complexes, were analyzed further by circular dichroism (CD)
spectroscopy. Thus, increasing quantities of the open form of
C1−C7 were added to solutions of ct-DNA, and the
corresponding CD spectra were recorded in the near-UV region,
specifically, between 235 and 315 nm, after incubation for 24 h at
37 °C (see Experimental Section for details). The spectra for C6
and open C1−C5 and C7 are shown in Figure 7.
Direct comparison of the CD spectra of C1 and C6 interacting
with B-DNA reveals noticeable differences. The addition of
increasing amounts of C1 to ct-DNA results in a minor
bathochromic shift of both the characteristic positive and
negative bands, and a progressive slight increase of ellipticity of
the positive signal, up to saturation. Such CD features are
indicative of the monofunctional covalent binding of platinum-
(II) drugs to DNA, and therefore agree with the previously
proposed metal-mediated activity of the open photoisomers.51
Furthermore, these spectroscopic data corroborate the minor
impact of the complex binding on the helical conformation nor
on the base-pair stacking of the biomolecule. Apart from C6,
similar spectra were obtained for all other open complexes (see
Figure 7); the spectral changes induced upon treatment of ct-
DNA with the highest concentration of each complex are listed in
Table S5.
Open C6 induces a drastically distinct alteration of the
conformation of the double helix (Figure 7 and Table S5). The
progressive binding of this imidazolic species leads to a severe
decrease of the whole CD signal of B-DNA, particularly for the
negative band. These spectral changes suggest that C6 has a
distinct effect on the secondary structure of the biomolecule,
most likely generating local disruptions of the helical turn, and
destabilization of the alignment of adjacent base pairs. This effect
is consistent with the efficient expulsion of ethidium bromide
noted by fluorescence assays (see above).
Since C6 is the sole metal complex of the series exhibiting this
behavior, it may be attributed to the methylimidazole units;
however, the exact mechanism through which C6 induces such a
distinct structural alteration of B-DNA remains to be elucidated.
Probably, the specific electronic structure of ligand H632 has a
direct effect on the ligand-exchange kinetics and/or the binding
affinity of the platinum(II) centers, which may thus become
more prone to mediate DNA cross-links. These different DNA-
interacting properties may explain the higher cytotoxicity of C6,
compared with that of the other open complexes (Table 2 and
Figures 5, 6, and S7).
DNA-interaction studies performed with all complexes
revealed that the activity of these photoisomerisable agents can
be efficiently modified through their light-mediated conversion.
Clearly distinct DNA-interacting properties for the open and
closed forms of the coordination compounds were observed. For
instance, different metal covalent binding and ligand intercalative
association were noticed for the two forms. These remarkable
results suggest that the activity of each photoisomer may be
tailored through appropriate structural modifications, thus
opening design possibilities for the future development of
more effective DTC-based metallodrugs (DTC stands for
dithienylcyclopentene).
The antiproliferative activity of the open and closed forms of
the complexes were examined, which showed that most of the
compounds surprisingly were not cytotoxic; actually, the distinct
DNA-interacting ability of each isomer did not translate into
interesting results with cells. Remarkable data were though
obtained with complex C2, whose closed form is highly cytotoxic
against melanoma and colorectal cancer cells, whereas its open
form is nontoxic. These drastically distinct behaviors of open/
closed C2 further strengthen the innovative approach proposed
in a previous proof of concept study,20 consisting in using DTC
photoswitches to develop novel PACT agents.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b00146.
The schematic representations of the structures of L1−L7;
1
UV−vis spectra of open/closed C1; H NMR spectra of
L2 upon UV irradiation; crystal data and structure
refinement for C3 and C4; representations of the crystal
structures of C3 and C4; selected bond lengths and angles
for C3 and C4; CD data of the open complexes; CD
spectra of the closed complexes; cell viability assays and
dose-response curves (PDF)
Accession Codes
CCDC 1565000−1565001 contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2
1EZ, UK; fax: +44 1223 336033.
Finally, the CD spectra for the closed form of complexes C1−
C5 and C7 were recorded (Figure S6); the spectroscopic data
obtained are comparable to those of the corresponding open
complexes (see Figures 7 and S6), indicating that the closed
compounds are also not capable of significantly altering the
secondary structure of DNA.
AUTHOR INFORMATION
Corresponding Author
*E-mail: patrick.gamez@qi.ub.es.
ORCID
Olivier Roubeau: 0000-0003-2095-5843
Patrick Gamez: 0000-0003-2602-9525
Notes
■
CONCLUSIONS
■
Following earlier promising results,20 a series of photoswitchable
platinum(II) complexes based on photoresponsive dithienylcy-
clopentene moieties were prepared and fully characterized. In
particular, the connectivity of the metal ions was clearly
established by single-crystal X-ray diffraction, which revealed a
trans disposition of the chlorido ligands and the S-coordination
of DMSO. The photochemical properties of the ligands and
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial support from the Spanish Ministerio de Economia y
Competitividad/FEDER (Project Nos. CTQ2015-70371-
REDT, CTQ2014-55293-P, and CTQ2017-88446-R) is ac-
■
́
L
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(18) Irie, M. Photochromism of Diarylethene Molecules and Crystals.
Proc. Jpn. Acad., Ser. B 2010, 86, 472−483.
́
knowledged. P.G. thanks the Institucio Catalana de Recerca i
Estudis Avanca
a grant from the Spanish government and the EU (FIS PI13/
00089), a grant from La Marato de TV3 Foundation
̧ ts (ICREA). This work was partially supported by
(19) Feringa, B. L.; Browne, W. R. Molecular Switches, 2nd ed.; Wiley-
VCH, 2011.
́
(20) Presa, A.; Brissos, R. F.; Caballero, A. B.; Borilovic, I.; Korrodi-
Gregorio, L.; Perez-Tomas, R.; Roubeau, O.; Gamez, P. Photoswitching
the Cytotoxic Properties of Platinum(II) Compounds. Angew. Chem.,
Int. Ed. 2015, 54, 4561−4565.
(21) Presa, A. Diarylethene-Based Pt(II) Molecular Switches: A Novel
Approach to Photoactivated Chemotherapy; University of Barcelona,
Barcelona, Spain, 2016.
(20132730). Also, an individual contract to L.K.G. supported
by a program from the Barcelona Univ. in collaboration with
́
Obra Social de la Fundacio Bancaria “La Caixa” is acknowledged.
This research used resources of the Advanced Light Source,
which is a Department of Energy Office of Science User Facility
under Contract No. DE-AC02-05CH11231.
(22) Lucas, L. N.; van Esch, J.; Kellogg, R. M.; Feringa, B. L. A New
Class of Photochromic 1,2-Diarylethenes; Synthesis and Switching
Properties of Bis(3-thienyl)cyclopentenes. Chem. Commun. 1998,
2313−2314.
(23) Price, J. H.; Schramm, R. F.; Wayland, B. B.; Williamson, A. N.
Palladium(II) and Platinum(II) Alkyl Sulfoxide Complexes - Examples
of Sulfur-Bonded, Mixed Sulfur-Bonded and Oxygen-Bonded, and
Totally Oxygen-Bonded Complexes. Inorg. Chem. 1972, 11, 1280−
1284.
(24) SADABS and SAINT; Bruker AXS Inc.: Madison, WI, 2015.
(25) Sheldrick, G. M. SHELXT - Integrated space-group and crystal-
structure determination. Acta Crystallogr., Sect. A: Found. Adv. 2015, 71,
3−8.
(26) Sheldrick, G. M. Crystal Structure Refinement with SHELXL.
Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71, 3−8.
(27) Sheldrick, G. M. Cell_Now and Twinabs; Bruker AXS Inc.:
Madison, WI, 2008.
(28) Hanazawa, M.; Sumiya, R.; Horikawa, Y.; Irie, M. Thermally
Irreversible Photochromic Systems - Reversible Photocyclization of 1,2-
Bis(2-methylbenzo[b]thiophen-3-yl)perfluorocycloalkene Derivatives.
J. Chem. Soc., Chem. Commun. 1992, 206−207.
(29) de Jong, J. J. D.; Lucas, L. N.; Hania, R.; Pugzlys, A.; Kellogg, R.
M.; Feringa, B. L.; Duppen, K.; van Esch, J. H. Photochromic Properties
of Perhydro- and Perfluorodithienylcyclopentene Molecular Switches.
Eur. J. Org. Chem. 2003, 2003, 1887−1893.
(30) Woodward, R. B.; Hoffmann, R. Stereochemistry of Electrocyclic
Reactions. J. Am. Chem. Soc. 1965, 87, 395−397.
(31) Feringa, B. L. Molecular Switches; Wiley-VCH Verlag GmbH:
Weinheim, Germany, 2001.
(32) Presa, A.; Barrios, L.; Cirera, J.; Korrodi-Gregorio, L.; Perez-
Tomas, R.; Teat, S. J.; Gamez, P. Non-Switching 1,2-Dithienylethene-
based Diplatinum(II) Complex Showing High Cytotoxicity. Inorg.
Chem. 2016, 55, 5356−5364.
(33) Elding, L. I.; Groning, O. Kinetics and Mechanism for Ligand
Substitution-Reactions of Square-Planar (Dimethyl Sulfoxide)-
Platinum(II) Complexes - Stability and Reactivity Correlations. Inorg.
Chem. 1978, 17, 1872−1880.
(34) Kukushkin, Y. N. Contribution of Investigations of Dimethylsulf-
oxide Complexes in Coordination Chemistry Theory. Koord. Khim.
1997, 23, 163−174.
(35) Zheng, F.; Hutton, A. T.; van Sittert, C.; Moss, J. R.; Mapolie, S. F.
Synthesis, Structural Characterization and Cis-trans Isomerization of
Novel (Salicylaldiminato)platinum(II) Complexes. Dalton Trans. 2013,
42, 11163−11179.
(36) Snelders, D. J. M.; Siegler, M. A.; von Chrzanowski, L. S.; Spek, A.
L.; van Koten, G.; Gebbink, R. Coulombic Inter-Ligand Repulsion
Effects on the Pt(II) Coordination Chemistry of Oligocationic,
Ammonium-functionalized Triarylphosphines. Dalton Trans. 2011, 40,
2588−2600.
REFERENCES
■
(1) Farrer, N. J.; Salassa, L.; Sadler, P. J. Photoactivated Chemotherapy
(PACT): The Potential of Excited-state d-Block Metals in Medicine.
Dalton Trans. 2009, 10690−10701.
(2) Phillips, D. Light Relief: Photochemistry and Medicine. Photochem.
Photobiol. Sci. 2010, 9, 1589−1596.
(3) Farrer, N. J.; Sadler, P. J. Photochemotherapy: Targeted Activation
of Metal Anticancer Complexes. Aust. J. Chem. 2008, 61, 669−674.
(4) Benov, L. Photodynamic Therapy: Current Status and Future
Directions. Med. Princ. Pract. 2015, 24, 14−28.
(5) Crespy, D.; Landfester, K.; Schubert, U. S.; Schiller, A. Potential
Photoactivated Metallopharmaceuticals: From Active Molecules to
Supported Drugs. Chem. Commun. 2010, 46, 6651−6662.
(6) Smith, N. A.; Sadler, P. J. Photoactivatable Metal Complexes: From
Theory to Applications in Biotechnology and Medicine. Philos. Trans. R.
Soc., A 2013, 371, 13.
(7) Doherty, R. E.; Sazanovich, I. V.; McKenzie, L. K.; Stasheuski, A. S.;
Coyle, R.; Baggaley, E.; Bottomley, S.; Weinstein, J. A.; Bryant, H. E.
Photodynamic Killing of Cancer Cells by a Platinum(II) Complex with
Cyclometallating Ligand. Sci. Rep. 2016, 6.10.1038/srep22668
(8) Abadeer, N. S.; Murphy, C. J. Recent Progress in Cancer Thermal
Therapy Using Gold Nanoparticles. J. Phys. Chem. C 2016, 120, 4691−
4716.
(9) Sun, Y. J.; Joyce, L. E.; Dickson, N. M.; Turro, C. DNA
Photocleavage by An Osmium(II) Complex in the PDT Window. Chem.
Commun. 2010, 46, 6759−6761.
(10) Mari, C.; Pierroz, V.; Ferrari, S.; Gasser, G. Combination of Ru(II)
Complexes and Light: New Frontiers in Cancer Therapy. Chem. Sci.
2015, 6, 2660−2686.
(11) van Rixel, V. H. S.; Siewert, B.; Hopkins, S. L.; Askes, S. H. C.;
Busemann, A.; Siegler, M. A.; Bonnet, S. Green Light-induced Apoptosis
in Cancer Cells by a Tetrapyridyl Ruthenium Prodrug Offering Two
Trans Coordination Sites. Chem. Sci. 2016, 7, 4922−4929.
(12) Respondek, T.; Garner, R. N.; Herroon, M. K.; Podgorski, I.;
Turro, C.; Kodanko, J. J. Light Activation of a Cysteine Protease
Inhibitor: Caging of a Peptidomimetic Nitrile with Ru-II(bpy)(2). J. Am.
Chem. Soc. 2011, 133, 17164−17167.
(13) Albani, B. A.; Pena, B.; Leed, N. A.; de Paula, N.; Pavani, C.;
Baptista, M. S.; Dunbar, K. R.; Turro, C. Marked Improvement in
Photoinduced Cell Death by a New Tris-heteroleptic Complex with
Dual Action: Singlet Oxygen Sensitization and Ligand Dissociation. J.
Am. Chem. Soc. 2014, 136, 17095−17101.
(14) Brabec, V.; Pracharova, J.; Stepankova, J.; Sadler, P. J.;
Kasparkova, J. Photo-Induced DNA Cleavage and Cytotoxicity of a
Ruthenium(II) Arene Anticancer Complex. J. Inorg. Biochem. 2016, 160,
149−155.
(15) Loganathan, D.; Morrison, H. Effect of Ring Methylation on the
Photophysical, Photochemical and Photobiological Properties of Cis-
dichlorobis(1,10-phenanthroline)rhodium(III)chloride. Photochem.
Photobiol. 2006, 82, 237−247.
(37) Anderson, C. M.; Taylor, I. R.; Tibbetts, M. F.; Philpott, J.; Hu, Y.
F.; Tanski, J. M. Hetero-multinuclear Ruthenium(III)/Platinum(II)
Complexes That Potentially Exhibit Both Antimetastatic and Antineo-
plastic Properties. Inorg. Chem. 2012, 51, 12917−12924.
(38) Molas Saborit, J.; Caubet, A.; Brissos, R. F.; Korrodi-Gregorio, L.;
Perez-Tomas, R.; Martinez, M.; Gamez, P. pH-Driven Preparation of
Two Related Platinum(II) Complexes Exhibiting Distinct Cytotoxic
Properties. Dalton Trans. 2017, 46, 11214−11222.
(16) Cuello-Garibo, J.-A.; James, C. C.; Siegler, M. A.; Bonnet, S.
Ruthenium-Based PACT Compounds Based on an N,S Non-Toxic
Ligand: A Delicate Balance Between Photoactivation and Thermal
Stability. Chem. Sq. 2017, 1, 2.
(17) Brieke, C.; Rohrbach, F.; Gottschalk, A.; Mayer, G.; Heckel, A.
Light-Controlled Tools. Angew. Chem., Int. Ed. 2012, 51, 8446−8476.
M
DOI: 10.1021/acs.inorgchem.8b00146
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(39) Baik, M. H.; Friesner, R. A.; Lippard, S. J. Theoretical Study of
Cisplatin Binding to Purine Bases: Why Does Cisplatin Prefer Guanine
Over Adenine? J. Am. Chem. Soc. 2003, 125, 14082−14092.
(40) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The Next
Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle
Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016, 116, 3436−3486.
(41) Farrell, N. P. Multi-Platinum Anti-Cancer Agents. Substitution-
inert Compounds for Tumor Selectivity and New Targets. Chem. Soc.
Rev. 2015, 44, 8773−8785.
(42) Cohen, G. L.; Bauer, W. R.; Barton, J. K.; Lippard, S. J. Binding of
C i s - d i c h l o r o d i a m m i n e p l a t i n u m ( I I ) a n d T r a n s -
dichlorodiammineplatinum(II) to DNA - Evidence for Unwinding
and Shortening of the Double Helix. Science 1979, 203, 1014−1016.
(43) Sherman, S. E.; Lippard, S. J. Structural Aspects of Platinum
Anticancer Drug-Interactions With DNA. Chem. Rev. 1987, 87, 1153−
1181.
(44) Bates, A. D.; Maxwell, A. DNA Topology; Oxford University Press:
Oxford, U.K., 2005.
(45) Meyeralmes, F. J.; Porschke, D. Mechanism of Intercalation into
the DNA Double Helix by Ethidium. Biochemistry 1993, 32, 4246−4253.
(46) Nejat Dehkordi, M.; Lincoln, P. Comprehensive Study on the
Binding of Iron Schiff Base Complex with DNA and Determining the
Binding Mode. J. Fluoresc. 2013, 23, 813−821.
(47) Permyakov, E. A. Luminescent Spectroscopy of Proteins; CRC Press:
Boca Raton, FL, 1993.
(48) Martin-Pintado, N.; Yahyaee-Anzahaee, M.; Deleavey, G. F.;
Portella, G.; Orozco, M.; Damha, M. J.; Gonzalez, C. Dramatic Effect of
Furanose C2 ′ Substitution on Structure and Stability: Directing the
Folding of the Human Telomeric Quadruplex with a Single Fluorine
Atom. J. Am. Chem. Soc. 2013, 135, 5344−5347.
(49) Horbert, R.; Pinchuk, B.; Davies, P.; Alessi, D.; Peifer, C.
Photoactivatable Prodrugs of Antimelanoma Agent Vemurafenib. ACS
Chem. Biol. 2015, 10, 2099−2107.
(50) Aklilu, M.; Eng, C. The Current Landscape of Locally Advanced
Rectal Cancer. Nat. Rev. Clin. Oncol. 2011, 8, 649−659.
(51) Macquet, J. P.; Butour, J. L. Circular-Dichroism Study of DNA
Platinum Complexes - Differentiation Between Monofunctional, Cis-
bidentate and Trans-Bidentate Platinum Fixation on a Series of DNAs.
Eur. J. Biochem. 1978, 83, 375−387.
N
DOI: 10.1021/acs.inorgchem.8b00146
Inorg. Chem. XXXX, XXX, XXX−XXX