Photochemical reactivity of RuII(η6-p-cymene) flavonolato compounds

Article abstract of DOI:10.1021/om5006337

Photoactivation is a promising approach for modulating the biological activity of Ru^II compounds. In this work, Ru^II flavonolato compounds, [Ru(η⁶-p-cymene)(L)(3-Hfl)]OTf (8; 3-Hfl = monoanion of 3-hydroxyflavone; L = solvent) and [Ru(η⁶-p-cymene)Cl(3-Hfl-X)] (3a-3c; 3-Hfl-X = p-H, -Cl, or -F on the flavonolato phenyl substituent), have been evaluated for photoinduced reactivity within the flavonolato unit upon irradiation with UV (300 nm) or visible (419 nm) light under aerobic conditions. For each compound, irradiation in CH₃CN was found to result in the loss of the p-cymene ligand and the formation of products resulting from oxidative ring opening of the flavonolato ligand in a dioxygenase-type reaction. This reaction also results in the release of carbon monoxide. The Ru^II products generated in these reactions are [Ru^II(solvent)(carboxylato)]⁺ and [Ru(CO)(solvent)(carboxylato)]⁺ (carboxylato = O-benzoylsalicylato or benzoato) species, as determined by ESI-MS. The amount of free CO generated depends on the wavelength of irradiation, with 300 nm light giving a higher amount of free CO. Evaluation of the photoinduced reactivity of 8 in DMSO/H₂O (10:90) at 300 nm showed similar reactivity to that found in organic solvent, although the reaction occurs more slowly. The products of the photoreactions of 8 and 3a at 419 nm are nontoxic toward human T-lymphocyte (Jurkat) and non-small-cell lung carcinoma (A549) cells. This lack of toxicity versus the starting compounds is likely due to differences in interactions of the [Ru^II(solvent)(carboxylato)]⁺ and [Ru^II(CO)(solvent)(carboxylato)]⁺ species with biomolecules (e.g., serum proteins), thus resulting in reduced bioavailabilty. ¹H NMR studies provide evidence that the photoreaction products coordinate to biologically relevant donors such as histidine and 5′-GMP in d₆-DMSO/D₂O (10:90) and exhibit reactivity with these small molecules that is distinctly different from that exhibited by the starting compounds. Overall, the photoreactivity of 8 and 3a-3c may represent an approach toward altering the biological chemistry of these compounds.

Full text of DOI:10.1021/om5006337

Article
pubs.acs.org/Organometallics
II
6
Photochemical Reactivity of Ru (η ‑p‑cymene) Flavonolato
Compounds
†
‡
‡
§
§
Sushma L. Saraf, Trevor J. Fish, Abby D. Benninghoff, Ashley A. Buelt, Rhett C. Smith,
and Lisa M. Berreau*
,
†
†
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300, United States
‡
Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, Utah 84322-4815, United States
§Department of Chemistry, Clemson University, Clemson, South Carolina 29634-0973, United States
S Supporting Information
*
ABSTRACT: Photoactivation is a promising approach for
II
modulating the biological activity of Ru compounds. In this
II
6
work, Ru flavonolato compounds, [Ru(η -p-cymene)(L)(3-
Hfl)]OTf (8; 3-Hfl = monoanion of 3-hydroxyflavone; L =
6
solvent) and [Ru(η -p-cymene)Cl(3-Hfl-X)] (3a−3c; 3-Hfl-X
p-H, -Cl, or -F on the flavonolato phenyl substituent), have
=
been evaluated for photoinduced reactivity within the
flavonolato unit upon irradiation with UV (300 nm) or visible (419 nm) light under aerobic conditions. For each compound,
irradiation in CH CN was found to result in the loss of the p-cymene ligand and the formation of products resulting from
3
oxidative ring opening of the flavonolato ligand in a dioxygenase-type reaction. This reaction also results in the release of carbon
II
II
+
monoxide. The Ru products generated in these reactions are [Ru (solvent)(carboxylato)] and [Ru(CO)(solvent)-
(
+
carboxylato)] (carboxylato = O-benzoylsalicylato or benzoato) species, as determined by ESI-MS. The amount of free CO
generated depends on the wavelength of irradiation, with 300 nm light giving a higher amount of free CO. Evaluation of the
photoinduced reactivity of 8 in DMSO/H O (10:90) at 300 nm showed similar reactivity to that found in organic solvent,
2
although the reaction occurs more slowly. The products of the photoreactions of 8 and 3a at 419 nm are nontoxic toward human
T-lymphocyte (Jurkat) and non-small-cell lung carcinoma (A549) cells. This lack of toxicity versus the starting compounds is
II
+
II
+
likely due to differences in interactions of the [Ru (solvent)(carboxylato)] and [Ru (CO)(solvent)(carboxylato)] species with
1
biomolecules (e.g., serum proteins), thus resulting in reduced bioavailabilty. H NMR studies provide evidence that the
photoreaction products coordinate to biologically relevant donors such as histidine and 5′-GMP in d -DMSO/D O (10:90) and
6
2
exhibit reactivity with these small molecules that is distinctly different from that exhibited by the starting compounds. Overall, the
photoreactivity of 8 and 3a−3c may represent an approach toward altering the biological chemistry of these compounds.
6
INTRODUCTION
[Ru(η -p-cymene)Cl(3-Hfl-X)] (Figure 1(bottom), 3a−3c)
wherein the 3-Hfl-X ligand is a derivative of 3-hydroxy-
■
Mononuclear ruthenium compounds are being investigated as
alternatives to platinum compounds for anticancer chemo-
1
8−20
flavone.
These compounds were found to exhibit
1
−15
Of particular recent interest are piano-stool RuII
significant in vitro anticancer activity (IC50 values in the low
μM range), with the derivatives containing a p-F or p-Cl
substituent on the flavonolato phenyl substituent exhibiting the
lowest IC values. The anticancer activity of these compounds
is presumed to involve DNA binding, and solution studies of
therapy.
arene compounds such as those shown in Figure 1, with 1 and
being at an advanced stage of preclinical development.
2
6
Compound 1, [Ru(η -p-arene)Cl (PTA)] (PTA = 1,3,5-triaza-
2
50
1
6,17
7
-phosphatricyclo-[3.3.1.1]decane),
is a member of the
ruthenium arene PTA (RAPTA) family. Compounds of this
type strongly interact with proteins in the extracellular matrix
and at the cell surface and exhibit selective antimetastatic
compounds 3a−3c with the DNA model compound 5′-GMP
II
(
guanosine 5′-monophosphate) suggested that the Ru center
can interact with the N7 atom of 5′-GMP while retaining
flavonolato coordination. Notably, these compounds were also
found to inhibit the enzyme topoisomerase IIα in a substituent-
dependent manner that correlates with the antiproliferative
6
activity in vivo. The latter type of compound, [Ru(η -
arene)X(YZ)] (2), contains a YZ chelate ligand (e.g.,
ethylene-1,2-diamine) and a monodentate labile ligand (e.g.,
X = Cl). Aquation of the monodentate ligand enables binding
of the compounds to DNA. The incorporation of biologically
active molecules into structures akin to 1 and 2 is an approach
toward generating compounds capable of targeting cancer cells
in multiple ways. In this regard, Hartinger and co-workers have
recently reported a family of compounds of the general formula
20
activity. This family of compounds thus exhibits a dual
approach toward targeting cancer cells, with the flavonolato
Received: June 14, 2014
©
XXXX American Chemical Society
A
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Photoactivation of ruthenium arene compounds for anti-
cancer activity is an approach of significant current
21−23
interest.
This approach is attractive because it can enable
activation of complexes (e.g., 4, Scheme 1a) that are unreactive
under dark conditions. Most commonly, this approach has been
II
used to enable formation of arene Ru species that can
24−26
II
subsequently coordinate to DNA.
Photoactivatable Ru
compounds that exhibit multiple types of biologically relevant
2
5,27
reactivity have also been reported.
For example, irradiation
II
6
of the dinuclear Ru arene compound [{(η -Indane)RuCl} (μ-
2
2
,3-dpp)](PF ) (5, arene = indane or benzene; Scheme 1b)
6 2
with UVA (365 nm) light results in photoinduced loss of the
arene ligand and the formation of compounds described as
“
reactive ruthenium species”, as well as free arene, which can
27
serve as a fluorescent marker. The “reactive ruthenium
species” show stronger interactions with DNA than 5, with
both mono- and bifunctional adducts with DNA being formed.
Overall, compound 5 thus offers the possibility of photo-
induced cell death via multiple types of interactions with DNA,
along with the opportunity for fluorescence imaging of the
location and efficiency of the photoinduced reaction. Irradiation
II
Figure 1. Mononuclear Ru arene compounds under investigation as
anticancer agents.
portion of the compounds believed to be the key component
for enzyme inhibition.
II
of the Ru arene octreotide conjugate 6 in the presence of five
II
Scheme 1. Photoinduced Reactivity of Ru Arene Species
B
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equivalents of the synthetic oligonucleotide dCATGGCT at
II
4
20 nm results in the formation of two types of Ru -
oligonucleotide adducts (7a and 7b, 7c; Scheme 1c) as
25
determined by mass spectrometry. Compound 6 thus has the
capability of forming multiple types of DNA adducts upon
photoirradiation, which may have important implications for
the biological activity of the compound.
We recently reported that divalent metal compounds
containing a flavonolato ligand undergo photoinduced
dioxygenase-type carbon−carbon bond cleavage within the
flavonolato ligand, resulting in the quantitative release of one
28−30
equivalent of CO when irradiated with UV light (300 nm).
In considering 3a−3c, we hypothesized that these compounds
might be susceptible to photoinduced, dioxygenase-type ring-
opening reactivity involving the flavonolato ligand, a reaction
that would result in the release of CO, a biological signaling
agent. Reactions of this type would also result in the formation
II
of new types of Ru products that could exhibit altered
biological activity. Outlined herein are the results of our initial
investigations of the UV- and visible light-induced photo-
reactions of the Ru flavonolato solvate compound [Ru(η -p-
cymene)(L)(3-Hfl)]OTf (8, L = solvent), as well as those of
Figure 2. Decay of the absorption features of 8 (9.06 × 10− M) upon
5
II
6
irradiation at 300 nm in aerobic CH CN. Spectra were collected at 15
min intervals. The final spectrum was collected after 300 min.
3
3
a−3c. The photochemical studies were performed in CH CN
3
for all of the compounds, with additional studies of 8 being
performed in DMSO/H O (10:90). The combined results of
2
analysis of the products in CH CN indicated the formation of
3
II
2
detailed product identification, CO quantification, and isotope
three new carboxylato-bound Ru species: [Ru(CH CN) (η -
3
4
II
+
1
+
labeling studies demonstrate that the Ru flavonolato
depside)] (9), [Ru(CH CN) (CO)(η -depside)] (10), and
Ru(CH CN) (η -benzoato)] (11) (Scheme 2; Figures S3
3
4
2
+
compounds undergo photoinduced dioxygenase-type ring
[
3 4
opening of the flavonol with CO release, as well as
and S4; depside = O-benzoylsalicylato). The benzoato product
likely results from hydrolysis of the depside ligand due to the
presence of water during the ESI-MS experiment. Analysis of
the headspace gas revealed the release of 0.7 equivalent of CO.
These combined results indicate a photochemical process
wherein displacement of the arene and oxidative cleavage
involving the flavonolato ligand both occur, with a portion of
II
photoinduced arene release. The Ru products generated
were found to be nontoxic toward human T-lymphocyte
(
Jurkat) and non-small-cell lung carcinoma (A549) cells and to
exhibit notably different solution chemistry with 5′-GMP and
histidine.
II
RESULTS
the CO generated being trapped by the Ru center.
■
Compound Preparation and Characterization. The
Irradiation of 8 in CH CN at 419 nm in the presence of O
3
2
II
6
Ru triflate compound [Ru(η -p-cymene)(3-Hfl)]OTf (8) was
prepared via treatment of 3a with AgOTf and was characterized
also results in the decay of the absorption features of the
compound and results in a similar mixture of products (Scheme
3). The growth of a Ru(CO) moiety is clearly observed by IR
1
13
by H and C NMR, IR, UV−vis, mass spectrometry (Figure
S1), and elemental analysis. Compounds 3a−3c were prepared
using a synthetic procedure that is slightly modified from that
previously reported, but results in similar isolated yields of
−1
during the reaction (νCO = 2010 cm ; Figure 3). Mass spectral
analysis of the final product mixture indicates the presence of
2
+
[Ru(CH
species, [Ru(CH CN) (CO)(η -depside)] (10) and [Ru-
3 4
CN) (η -depside)] (9), along with two Ru(CO)
3
4
2
0
1
+
∼
60%.
2
+
Free 3-hydroxyflavone (3-HflH) dissolved in methanol
(CH CN) (CO)(η -depside)] (12) (Figure S5). Analysis of
3 3
exhibits absorption features at 302 and 350 nm, respectively.
The lower energy feature (band I) has been assigned to a π−π*
HOMO to LUMO transition, while band II corresponds to a
the headspace gas from the reaction revealed 0.4 equivalent of
free CO, which is less than that generated at 300 nm. Use of
18
O gas in the reaction produced isotope clusters consistent
2
31
HOMO−1 to LUMO transition. The absorption spectrum of
with the quantitative incorporation of two labeled oxygen
atoms into 9, 10, and 12 (Figure S6). This labeling study
provides evidence for a dioxygenase-type pathway for the
photoinduced CO-release reaction involving the flavonolato
8
in CH CN is shown in Figure S2a. As is characteristic for
3
2
8−30
metal flavonolato compounds,
the two absorption features
of the coordinated flavonolato ligand are red-shifted relative to
the free flavonol. The absorption spectra of 3a−3c are similar
ligand.
II
(Figure S2a). Excitation into the lowest energy band of each
Irradiation of a CH CN solution of the Ru flavonolato
3
compound produces an emission feature with a Stokes shift of
chloride compound 3a at 419 nm in aerobic CH CN produced
3
∼
74−87 nm (Figure S2b). The florescence quantum yield and
a similar final product mixture to that obtained for the triflate
compound 8, with compounds 9, 10, and 11 being identified by
mass spectrometry (Figure S7), along with 0.4 equivalent of
free CO being detected by GC analysis. The quantum yield for
this reaction is Φ = 0.001(1). The solid-state IR spectrum of
the final product mixture contains a single Ru(CO) vibration at
lifetime associated with the excited state for 3a are Φ = 0.01(1)
and 1.1(3) ns, respectively.
Photochemistry in CH CN. Irradiation of 8 in aerobic
3
CH CN with 300 nm light results in a decrease in the intensity
3
of the absorption features associated with the flavonolato ligand
1
−1
(
Figure 2). H NMR analysis of the final product mixture
2009 cm (Figure S8), consistent with the presence of the
indicated release of the p-cymene ligand. ESI mass spectral
Ru(CO) species 10 and 12. Over the course of the irradiation
C
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Scheme 2. Photoinduced Reactivity of 8 upon Irradiation at 300 nm in Aerobic CH CN
3
Scheme 3. Photoinduced Reactivity of 8 upon Irradiation at 419 nm in Aerobic CH CN
3
Figure 4. Metal carbonyl region of the reaction mixture generated after
irradiation of 3a for 13 h at 419 nm in aerobic CH CN. The energies
3
II
of the νCO vibrations are consistent with the presence of Ru carbonyl
chloride species. The spectrum was obtained as a KBr pellet.
Figure 3. Metal carbonyl region of the IR spectra (KBr) of aliquots
collected over 6 h of the photoirradiation of 8 at 419 nm in aerobic
CH CN. The signal is associated with the formation of Ru(CO)
species.
10, 12, and one additional significant m/z envelope at 576.0835
(13, Figure S9). The new species is best fit as an acetonitrile
cluster ion of the Ru(CO) species 10 (+41 Da), suggesting that
a significant amount of Ru(CO) compounds is present at this
point in the reaction.
The reaction mixtures generated upon irradiation of 3b and
3c were also evaluated after 12 h. Similar to the reactivity found
for 3a, corresponding aryl-substituted analogues of 9, 10, 12,
and 13 were detected by ESI-MS (Figures S10 and S11) and IR
(Figures S12 and S13). The amount of free CO generated in
each photoreaction is also commensurate with the reactivity
3
the chloride ligand also dissociates from the RuII center.
Investigation of the reaction mixture after 12 h of irradiation
revealed two terminal Ru(CO) vibrations at 1982 and 1927
−1
cm (Figure 4). The lower energy of these terminal νCO
vibrations relative to that of 10/12 suggests the presence of
32,33
chloride-bound Ru(CO) species.
ESI mass spectral analysis
of this intermediate reaction mixture showed molecular ions
−
consistent with ionization via loss of Cl and the presence of 9,
D
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Scheme 4. Photoinduced Reactivity of 3a−3c upon Irradiation at 419 nm in Aerobic CH CN
3
a
Scheme 5. Photoinduced Reactivity of 8 upon Irradiation at 300 nm for 45 h in DMSO/H O (10:90) in the Presence of O
2
2
a
II
ESI-MS analysis was performed in CH CN to identify the Ru products.
3
II
observed for 3a. The overall reactivity of 3a−3c is outlined in
Scheme 4.
reactivity of the Ru acetonitrile compound 8 under two sets of
conditions in DMSO/H O (10:90). First, irradiation of 8
dissolved in aerobic DMSO/H O (10:90) at 300 nm for 45 h
2
Photochemistry in Aqueous Solution. The RuII
2
compounds 3a−3c are typically dissolved in DMSO/H O
produced a reaction mixture that is primarily starting material
(8), along with solvent-ligated Ru depside (14), benzoato (15
and 16), and salicylato (17) species (Scheme 5; Figure S14) as
2
II
(
10:90) for anticancer studies, conditions under which aquation
II
of the Ru−Cl bond occurs to give a Ru aqua species. Of
relevance to such species, we have examined the photoinduced
determined by ESI-MS in CH CN. One additional isotope
3
E
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1
−3
Figure 5. H NMR spectra of 8 (4.3−4.7 × 10 M) in d -DMSO/D O (10:90) under the following conditions: (a) immediately following solution
6
2
preparation, with signals assigned; after (b) 1 day and (c) 10 days of exposure of the NMR tube to sunlight in the presence of O ; and (d) after 72 h
2
of irradiation (419 nm) of an independently prepared sample. The singlet at ∼5.51 ppm is residual CH Cl in the sample of 8. The asterisks indicate
2
2
6
+
signals for [Ru (η -p-cymene) (μ-OH) ] .
2
2
3
Figure 6. Plots of human leukemia (Jurkat) and non-small-cell lung carcinoma (A549) cell viability versus compound concentration and
determination of IC50 values (μM) for 8 and its photoinduced reaction products (generated in CH CN in the absence of cells). IC values were
3
50
calculated using a four-parameter nonlinear regression.
+
envelope in the ESI mass spectrum has been assigned as the
OH) )] , a known hydrolytic decomposition product of
3
III
III
1
19
Ru salicylato derivative [Ru (DMSO) (CH CN) (η -salicy-
molecules of this type. After 10 days, these doublets are no
2
3
2
+
lato) ] (18). Analysis of the headspace gas of the reaction
longer present, and a precipitate was observed in the NMR
tube. Changes in several signals associated with the p-cymene
and flavonolato resonances are also apparent that suggest
photoinduced reactivity. Specifically, the spectral changes
present are very similar to those observed for irradiation of a
2
indicated the formation of 0.2 equivalent of free CO. No
Ru(CO) species were detected by IR, which is consistent with
photoirradiation at 300 nm promoting the release of a metal-
34
bound carbonyl ligand. Overall, these results indicate that
while photoinduced flavonolato oxidation chemistry is possible
solution of 8 at 419 nm for 72 h in the presence of O (Figure
2
for 8 in DMSO/H O (10:90), the reaction is significantly
5d) in d -DMSO/D O (10:90). Thus, compounds such as 8
2
6
2
slower and the depside product undergoes an enhanced
amount of hydrolysis.
can exhibit both hydrolytic as well as photoinduced
decomposition in DMSO/H O (10:90).
2
In a second experiment, an NMR tube solution of 8 dissolved
in O -purged d -DMSO/D O (10:90) was exposed to direct
sunlight and evaluated by H NMR after 1 and 10 days. As
shown in Figure 5, after 1 day, a new set of doublets appears at
Biological Evaluation. A colorimetric MTT assay was
employed to determine the effect of 8 and 3a, and their
photoinduced reaction product mixtures, on the growth of two
human cancer cell lines. After 96 h, compounds 8 and 3a
inhibited proliferation of both A549 and Jurkat cells in a
concentration-dependent manner. Results of this cell viability
2
6
2
1
5
.20 and 5.45 ppm. These signals are consistent with the
6
presence of the hydrolysis product [Ru (η -p-cymene) (μ-
2
2
F
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Figure 7. Plots of human leukemia (Jurkat) and non-small-cell lung carcinoma (A549) cell viability versus compound concentration and
determination of IC50 values (μM) for 3a and its photoinduced reaction products (generated in CH CN in the absence of cells). IC values were
3
50
calculated using a four-parameter nonlinear regression.
1
Figure 8. H NMR spectra of the reaction of 8 with histidine in d -DMSO/D O (10:90) at ambient temperature. (a) Analytically pure 8;
6
2
assignments for the aromatic resonances are as given in Figure 5; aliphatic proton assignments are given; (b) 8 + 1 equiv His; (c) 8 + 5 equiv + 24 h
at 37 °C. Signals associated with D O and d -DMSO are marked with an s.
2
6
assay showed that virtually no viable A549 cells were present
after 96 h (Figures 6 and 7), with IC50 values for the
compounds determined as 33.6 ± 1.16 μM (8) and 47.0 ± 1.26
μM (3a), respectively. These values are similar to that
previously reported by Hartinger et al. for 3a (20 ± 2 μM)
for the A549 cell line. The viability of Jurkat cells treated with 8
and 3a was similar, with IC values of 27.2 ± 1.23 μM (8) and
because it serves as a model for the aquated form of 3a.
II
Histidine was chosen as a model for interactions of the Ru
II
photoproducts with proteins, as mononuclear Ru compounds
have been shown to bind to proteins via interactions with
3
5,36
histidine residues.
releasing molecule CORM-3 ( fac-Ru(CO) Cl(κ -
For example, the carbon monoxide
2
3
35
H NCH CO )) binds to hen egg white lysozyme (HEWL)
protein via formation of a Ru −His15 interaction to give a fac-
Ru(H O) (CO) (His) motif.
2 3 2
5
0
2
2
2
II
30.4 ± 1.2 μM (3a), respectively. Notably, cytotoxic effects
36
were not observed for the photoinduced reaction products of 8
and 3a (generated in CH CN in the absence of cells) at any of
Treatment of a d -DMSO/D O (10:90) solution of 8 with
3
6 2
the compound concentrations examined (Figures 6 and 7).
These combined results suggest that the changes produced in
the primary coordination sphere of the Ru center upon
one equivalent of histidine results in the formation of a
precipitate, along with spectral changes for the remaining
soluble species suggesting the formation of a mixture of Ru
II
II
photoirradiation strongly influence the reactivity of the metal
compounds having histidine, p-cymene, and flavonolato ligation
(Figure 8b). The pH of this mixture is approximately 5.6.
Increasing the amount of histidine relative to 8 (∼5:1) and
heating of the sample for 24 h at 37 °C (Figure 8c) results in
the complete loss of the resonances associated with the
flavonolato ligand, suggesting that it has been displaced from
9
compounds with biological molecules.
1
H NMR Studies of 8 and Photoinduced Reaction
Products with Histidine and 5′-GMP. On the basis of the
results of the biological studies, we comparatively examined the
histidine and 5′-GMP binding properties of 8 and its
photoproducts generated at 419 nm. Compound 8 was chosen
II
the Ru center and precipitated out of solution. Loss of the
G
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1
Figure 9. H NMR spectra of the reaction of the photogenerated mixture of 9, 10, and 12 (generated in CH CN as shown in Scheme 3) with
3
II
histidine in d -DMSO/D O (10:90) at ambient temperature. (a) Ru product mixture; (b) + 0.6 equiv His; (c) + 3 equiv His and 24 h at 37 °C; (d)
6
2
+
8 equiv His and 24 h at 37 °C. Signals associated with D O and d -DMSO are marked with an s.
2 6
flavonolato ligand is consistent with the previously reported
histidine H2 resonance at 7.84 ppm (Figure 9c; free His H2
7.76 ppm). Increasing the amount of histidine present to an 8:1
ratio results in the His H2 resonance shifting slightly upfield.
Analysis of the O-benzoylsalicylato aromatic proton region of
this 8:1 sample (Figure S16e) indicates differences versus the 9,
10, 12 compound mixture in d -DMSO/D O (10:90), again
6
+
reactivity of [(Ru(η -p-cymene)(3-Hfl)(H O)] , which was
2
reported to undergo flavonolato displacement in the presence
1
8
of histidine. The spectral features of the species remaining in
solution following treatment of 8 with five equivalents of His
II
are consistent with the presence of p-cymene-ligated Ru
6
2
species that coordinate a single histidine ligand. Specifically,
shifted histidine H2 and H5 resonances at 8.58 and 7.03 ppm,
respectively, integrate to one proton each relative to the sum of
the p-cymene resonances, indicating compounds having a
coordinated p-cymene:histidine ratio of 1:1.
suggesting interaction with the heterocycle. Overall, our
assessment is that the mixture of Ru species undergoes ligand
exchange with solvent and that the species present do interact
with histidine, although a definitive number of histidine ligands
could not be determined.
II
II
To study the coordination chemistry of His with the Ru
compounds in the photochemical product mixture, 8 was
The nucleotide binding properties of 8 and its photoinduced
reaction products were also investigated using the DNA model
compound 5′-GMP. Kurzenwernhart et al. have previously
reported that treatment of 8 with 5′-GMP results in an upfield
shift of the H8 signal of 5′-GMP from 8.1 to 7.6 ppm, which
irradiated in CH CN to produce a mixture of 9, 10, and 12 as
3
outlined in Scheme 3. After removal of the solvent under
1
reduced pressure, H NMR analysis of the residue dissolved in
d -DMSO/D O (10:90) at ambient temperature provided
was taken as evidence for 5′-GMP coordination. They
6
2
6
evidence for exchange of the coordinated CH CN ligands in
additionally noted that the hydrolysis product [Ru (η -p-
3
2
+
19
9, 10, and 12. Specifically, signals associated with coordinated
cymene) (μ-OH) ] was formed. We found that treatment
2 3
CH CN molecules (2.30−2.70 ppm; Figure S15a−c) decrease
of 8 with one equivalent of 5′-GMP in d -DMSO/D O (10:90)
3
6
2
in intensity over time, with an increase in the integrated
results in the formation of a precipitate, portions of which are
6
+
intensity of the signal for free CH CN. Additionally, subtle
likely [Ru (η -p-cymene) (μ-OH) ] and free flavonol. For the
3
2 2 3
changes are observed for the O-benzoylsalicylato protons
remaining soluble species, there are shifts in the resonances
(
7.20−8.10 ppm; Figure S16), suggesting changes within the
associated with the flavonolato and p-cymene ligands (Figure
S17b), which suggest changes within the Ru coordination
II
II
Ru coordination sphere. Addition of histidine to this
photoproduct mixture at ambient temperature and within 30
min of generation of the d -DMSO/D O (10:90) solution gives
distinctly different results (Figure 9) from those found for 8. In
the presence 0.6 equiv of histidine, a slightly downfield-shifted
histidine H2 resonance appears (Figure 9b), suggestive of the
sphere. However, our data are inconclusive with regard to
identifying an H8 signal for coordinated 5′-GMP, as over-
lapping flavonolato resonances are present, making a definitive
assignment of the signal impossible, even in the presence of
excess 5′-GMP (Figure S17c).
6
2
II
interaction of the Ru center with the heterocycle. Increasing
The results of performing a similar 5′-GMP experiment with
II
38
the histidine:Ru ratio to ∼3:1 and heating of the sample at 37
the photoproduct mixture of 9, 10, and 12 are shown in
°C for 24 h yielded a spectrum with a slightly downfield-shifted
Figure S18. Some of the observed spectral changes may be due
H
dx.doi.org/10.1021/om5006337 | Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
to solvent exchange in d -DMSO/D O (10:90) as described
above. No significant shifts are initially evident in the 5′-GMP
signals versus those of the free molecule. However, after 24 h at
EXPERIMENTAL SECTION
6
2
■
Instrumentation. 1H NMR data were measured at ambient
temperature using a 300 or 400 MHz NMR spectrometer. Chemical
3
7 °C, a small downfield-shifted signal, integrating to ∼0.3
shifts are referenced to the residual signal of the solvent (CD HCN:
H: 1.94 (quintet) ppm). J values are given in Hz. IR spectra were
2
1
proton relative to the O-benzoylsalicylato proton region, is
present at 8.8 ppm, suggesting the formation of a Ru -5′-GMP
species for a portion of the sample.
II
collected at ambient temperature as KBr pellets. Absorption spectral
data were collected at ambient temperature using a diode array
spectrophotometer. Fluorescence emission spectra were collected in
the range of 250−800 nm. The excitation and emission slit widths
were set at 2 mm for all experiments. The fluorescence quantum yield
Overall, the 1H NMR studies demonstrate that the
photoproduct mixture of 9, 10, and 12 exhibits distinctly
different solution chemistry and interactions with heterocyclic
donors than the starting compound 8. Combined with the
results of the MTT assays, it is clear that photoinduced
reactivity of the Ru flavonolato species could have a profound
influence on the biological properties of these compounds.
28
and lifetime for 3a were determined as previously described. CO was
detected and quantified using gas chromatography. Photochemical
reactions were performed using a Rayonet photoreactor equipped with
RPR-3000A lamps or RPR-4190A lamps. Reaction quantum yields
were determined by ferrioxalate actinometry using an integrative
II
4
9,50
analysis method.
The output measured during the quantum yield
1
7
16
determination for 3a was 1.046 37 × 10 and 4.001 94 × 10
DISCUSSION
■
photons/s for 300 and 419 nm lamps, respectively.
Materials. All reagents and solvents were obtained from
commercial sources and were used as received unless otherwise
noted. Anaerobic procedures were performed in a Vacuum
Photoactivation is an approach of current interest for
controlling the timing and location of the release of biologically
active species from RuII compounds.21−23 For Ru arene
II
Atmospheres glovebox in an atmosphere of dry N . Solvents for
2
derivatives, photoactivation has previously been used to break
metal−ligand bonds and open coordination sites for DNA
binding, with irradiation in some cases also resulting in loss of
glovebox use were dried using standard methods and distilled under
5
1
N . The flavone derivatives 3-hydroxy-2-(4-fluorophenyl)-4H-chro-
2
men-f(1H)-one and 3-hydroxy-2-(4-chlorophenyl)-4H-chromen-
II
20
the arene. We have demonstrated herein that the Ru arene
4(1H)-one were prepared according to literature procedures.
General Procedure for the Preparation of 3a−3c. In a N -
flavonolato compounds will undergo dioxygenase-type oxida-
tive cleavage of carbon−carbon bonds within the coordinated
flavonolato ligand, resulting in CO release, as well as
photoinduced arene release, when irradiated with UV or visible
light. While examples of photoinduced loss of arene ligands in
Ru compounds to give Ru (solvent) species have been
previously reported,
previously been shown to exhibit photoinduced reactivity.
2
filled glovebox, lithium bis(trimethylsilyl)amide (1 mmol) was
dissolved in ether (5 mL) and added to solid flavone derivative (1
mmol). The reaction mixture was allowed to stir for 3 h at ambient
temperature. The solvent was then removed under reduced pressure,
leaving the corresponding lithium flavonolato salt. This salt was
dissolved in acetonitrile (5 mL), the solution was added to solid
II
II
n
39−45
II
6
Ru flavonolato compounds have not
[Ru(η -p-cymene)Cl ] (0.5 mmol), and the resulting mixture was
2
2
4
6,47
stirred overnight. Removal of the solvent under vacuum yielded a red
solid. This material was dissolved in CH Cl (5 mL), and the solution
The flavonolato oxidative ring-opening reactivity of 3a occurs
with a relatively low reaction quantum yield (Φ = 0.001(1)
2
2
was filtered through a Celite/glass wool plug. The solvent was then
removed from the filtrate under vacuum. The remaining residue was
then washed with acetonitrile and dried under vacuum. The final
product was obtained by recrystallization from methanol. Compounds
(
419 nm)). This means that 3a−3c can be handled in air under
normal lighting with limited degradation. That being said, the
observed photoinduced reactivity offers the possibility of
photogenerating new species with significantly different
coordination chemistry properties.
3
a−3c were judged to be analytically pure based on elemental analysis
1
and the congruence of H NMR features and melting points with
previously reported values.
1
9
3
a: Yield: 71%. Mp: 230 °C (dec). UV−vis λ (CH CN)/nm (ε/
The mechanism by which the observed photoinduced
oxidative ring-opening reactivity within the flavonolato ligand
occurs for compounds 8 and 3a−3c is currently unknown. This
pathway could involve the flavonol acting as a photosensitizer
for the generation of singlet O , which is subsequently trapped,
or could involve a singlet to triplet conversion followed by
reaction with O . The feasibility of this reactivity within a
biological environment is yet to be investigated. However, the
results presented herein suggest that if photoinduced reactivity
does occur, the Ru center could serve as a trap for a portion of
the CO that is liberated, especially when visible light is
employed.
max
3
M− cm ): 324 (13 800), 472 (9700). H NMR (CD CN, 300
1
−1
1
3
MHz): δ 8.63 (d, J = 7.2 Hz, 2H), 8.11 (d, J = 8.3 Hz, 1H), 7.76−7.66
3
(
4
m, 2H), 7.59−7.52 (m, 2H), 7.47−7.40 (m, 2H), 5.68 (dd, J(H,H) =
3
3
3
.8 Hz, J(H,H) = 4.8 Hz, 2H), 5.39 (dd, J(H,H) = 4.8 Hz, J(H,H) =
2
4.8 Hz, 2H), 2.93 (sept, 1H), 2.32 (s, 3H), 1.39 (m, 6H). FTIR (KBr,
−
1
cm ): 1612 (ν
). ESI-MS m/z (relative intensity): 473.0704
CO
6
+
[(Ru(η -p-cymene)(3-Hfl)] , calcd 473.0685. Anal. Calcd for
2
C H O ClRu: C, 59.11; H, 4.56. Found: C, 58.72; H, 4.54.
25 23
3
3
b: Yield: 69%. Mp: 214−216 °C (dec). UV−vis λ (CH CN)/
max
3
−1 −1 1
II
nm (ε/M cm ): 331 (15200), 478 (11 200). H NMR (CD CN,
3
3
2
00 MHz): 8.63 (m, 2H), 8.10 (d, J = 8.3 Hz, 1H), 7.76−7.66 (m,
3
H), 7.46−7.40 (m, 1H), 7.32−7.24 (m, 2H), 5.68 (dd, J(H,H) = 5.1
3
3
3
Hz, J(H,H) = 5.1 Hz, 2H), 5.40 (dd, J(H,H) = 5.1 Hz, J(H,H) = 5.1
The photoproducts generated upon irradiation of 8 and 3a−
Hz, 2H), 2.93 (sept, 1H), 2.31 (s, 3H), 1.38 (m, 6H). FTIR (KBr,
II
II
−1
3
c are Ru carboxylato and Ru carboxylato carbonyl species
cm ): 1528 (ν
). Anal. Calcd for C H O Cl Ru: C, 55.36; H,
CO
25 22 3 2
containing multiple solvent-occupied coordination positions.
These compounds are similar to Ru -(DMSO) and Ru -
4.09. Found: C, 54.59; H, 4.16.
II
II
3c: Yield: 60%. Mp: 234−235 °C (dec). UV−vis λ (CH CN)/
max
3
n
−1
−1
1
nm (ε/M cm ): 310 (13 880), 458 (9800). H NMR (CD CN, 300
3
(
DMSO) (O,O-chelate) species that have generally shown no
n
48
MHz): 8.69 (m, 2H), 8.10 (d, J = 8.3 Hz, 1H), 7.76−7.66 (m, 2H),
in vitro cytotoxicity. The lack of toxicity for the photo-
products generated from 8 and 3a−3c suggests that photo-
inactivation is possible in this family of compounds, although
the in vivo reactivity of the compounds may differ significantly
from the in vitro results.
3
7
.46−7.40 (m, 1H), 7.32−7.24 (m, 2H), 5.68 (dd, J(H,H) = 5.1 Hz,
3
3
3
J(H,H) = 5.1 Hz, 2H), 5.39 (dd, J(H,H) = 5.1 Hz, J(H,H) = 5.1 Hz,
−1
2
H), 2.93 (sept, 1H), 2.31 (s, 3H), 1.38 (m, 6H). FTIR (KBr, cm ):
1539 (ν
Found: C, 56.81; H, 4.30.
). Anal. Calcd for C H O ClFRu: C, 57.09; H, 4.22.
CO
25 22 3
I
dx.doi.org/10.1021/om5006337 | Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
II
6
Preparation [Ru (η -p-cymene)(3-Hfl)]OTf (8). In an N -filled
three times, and each experimental treatment was performed in
triplicate.
2
−2
glovebox, AgOTf (25 mg, 9.9 × 10 mmol) was dissolved in
−2
acetonitrile (5 mL) and added to solid 3a (50 mg, 9.9 × 10 mmol).
The reaction mixture was allowed to stir for 3 h at ambient
temperature. The solvent was then removed under vacuum, and the
remaining residue was dissolved in dichloromethane. Filtration of the
solution through a Celite/glass wool plug was followed by removal of
the solvent from the filtrate under vacuum. The product was
recrystallized via hexanes diffusion into acetonitrile, which resulted
in the deposition of a red-orange solid (53 mg, 86%). Mp: 68−70 °C.
ASSOCIATED CONTENT
■
*
S
Supporting Information
1
UV−vis, fluoresence, IR, ESI-MS, and H NMR data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
−1
−1
UV−vis λ (CH CN)/nm (ε/M cm ): 310 (13 988), 458 (11
21). FTIR (KBr, cm ): 1540 (ν
MHz): 8.66 (d, J = 8.6 Hz, 2H), 8.18 (d, J = 8.6 Hz, 1H), 7.84−7.77
m, 2H), 7.62−7.49 (m, 4H), 5.90 (d, J = 6.2 Hz, 2H), 5.61 (d, J = 6.2
Hz, 2H), 2.94 (sept, 1H), 2.35 (s, 3H), 1.39 (d, J = 7.2, 6H). ESI-MS
m/z (relative intensity): 473.0704 [(Ru(η -p-cymene)(3-Hfl)] , calcd
73.0685. Anal. Calcd for C H F O RuS: C, 50.23; H, 3.73. Found:
max
3
−1
1
AUTHOR INFORMATION
2
). H NMR (CD CN, 300
■
CO
3
Corresponding Author
E-mail: lisa.berreau@usu.edu.
(
*
6
+
Notes
4
The authors declare no competing financial interest.
26
23
3
6
C, 49.61; H, 3.94.
Fluorescence Quantum Yield and Lifetime Determinations.
The fluorescence quantum yield (Φ) for 3a was determined using an
acetonitrile solution and was calculated relative to quinine bisulfate in
ACKNOWLEDGMENTS
■
We thank the United States National Science Foundation
CHE-1301092 to L.M.B.; CHE-0847132 to R.C.S.) and the
Utah Agricultural Experiment Station (Project UTA00172 to
A.D.B.).
29
(
0
.1 M H SO (aq) (Φ = 0.546). The quantum yield was corrected for
2
4
the refractive index of the solvent. Lifetime data was collected using an
Easylife II lifetime fluorimeter from Photon Technology International.
Photoirradiation of Ruthenium Compounds. Solutions of
6
6
[
Ru(η -p-cymene)(3-Hfl)]OTf (8) and [Ru(η -p-cymene)Cl(3-Hfl-
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dx.doi.org/10.1021/om5006337 | Organometallics XXXX, XXX, XXX−XXX