Organometallic Ru(II) Photosensitizers Derived from "-Expansive Cyclometalating Ligands: Surprising Theranostic PDT Effects

Article abstract of DOI:10.1021/acs.inorgchem.5b01838

The purpose of the present study was to investigate the influence of "-expansive cyclometalating ligands on the photophysical and photobiological properties of organometallic Ru(II) compounds. Four compounds with increasing " conjugation on the cyclometalating ligand were prepared, and their structures were confirmed by HPLC, 1D and 2D ¹H NMR, and mass spectrometry. The properties of these compounds differed substantially from their Ru(II) polypyridyl counterparts. Namely, they were characterized by red-shifted absorption, very weak to no room temperature phosphorescence, extremely short phosphorescence state lifetimes (<10 ns), low singlet oxygen quantum yields (0.5-8%), and efficient ligand-centered fluorescence. Three of the metal complexes were very cytotoxic to cancer cells in the dark (EC₅₀ values = 1-2 μM), in agreement with what has traditionally been observed for Ru(II) compounds derived from small C^N ligands. Surprisingly, the complex derived from the most "-expansive cyclometalating ligand exhibited no cytotoxicity in the dark (EC₅₀ > 300 μM) but was phototoxic to cells in the nanomolar regime. Exceptionally large phototherapeutic margins, exceeding 3 orders of magnitude in some cases, were accompanied by bright ligand-centered intracellular fluorescence in cancer cells. Thus, Ru(II) organometallic systems derived from "-expansive cyclometalating ligands, such 4,9,16-triazadibenzo[a,c]napthacene (pbpn), represent the first class of potent light-responsive Ru(II) cyclometalating agents with theranostic potential.

Full text of DOI:10.1021/acs.inorgchem.5b01838

Article
pubs.acs.org/IC
Organometallic Ru(II) Photosensitizers Derived from π‑Expansive
Cyclometalating Ligands: Surprising Theranostic PDT Effects
Tariq Sainuddin, Julia McCain, Mitch Pinto, Huimin Yin, Jordan Gibson, Marc Hetu,
and Sherri A. McFarland*
Department of Chemistry, Acadia University, Wolfville, Nova Scotia B4P 2R6, Canada
S
* Supporting Information
ABSTRACT: The purpose of the present study was to investigate the influence of π-expansive cyclometalating ligands on the
photophysical and photobiological properties of organometallic Ru(II) compounds. Four compounds with increasing π
conjugation on the cyclometalating ligand were prepared, and their structures were confirmed by HPLC, 1D and 2D 1H NMR,
and mass spectrometry. The properties of these compounds differed substantially from their Ru(II) polypyridyl counterparts.
Namely, they were characterized by red-shifted absorption, very weak to no room temperature phosphorescence, extremely short
phosphorescence state lifetimes (<10 ns), low singlet oxygen quantum yields (0.5−8%), and efficient ligand-centered
fluorescence. Three of the metal complexes were very cytotoxic to cancer cells in the dark (EC50 values = 1−2 μM), in agreement
with what has traditionally been observed for Ru(II) compounds derived from small C^N ligands. Surprisingly, the complex
derived from the most π-expansive cyclometalating ligand exhibited no cytotoxicity in the dark (EC50 > 300 μM) but was
phototoxic to cells in the nanomolar regime. Exceptionally large phototherapeutic margins, exceeding 3 orders of magnitude in
some cases, were accompanied by bright ligand-centered intracellular fluorescence in cancer cells. Thus, Ru(II) organometallic
systems derived from π-expansive cyclometalating ligands, such 4,9,16-triazadibenzo[a,c]napthacene (pbpn), represent the first
class of potent light-responsive Ru(II) cyclometalating agents with theranostic potential.
part, from the very short excited state lifetimes that characterize
the cyclometalated systems,33 which are generally up to 2
orders of magnitude shorter than the hallmark 1 μs lifetime of
the prototype [Ru(bpy)3]2+ (bpy = 2,2′-bipyridine) complex.10
The strong σ-donating capacities of cyclometalating ligands,
such as phpy−, raise the energy of the metal-based dπ HOMO
orbitals to a greater extent than the diimine-based LUMOs,
leading to unusually low-energy triplet metal-to-ligand charge
transfer (MLCT) excited states that are governed by the energy
gap law. The ensuing poor photoluminescence and attenuated
metal-based oxidation potentials have limited the utility of
cyclometalated Ru(II) complexes as luminescent sensors and as
sensitizers in other photonic applications. Similarly, Turro and
co-workers noted that cyclometalated complexes do not appear
to be good candidates for the design of new PCT agents that
operate via photoinduced ligand substitution,34 presumably due
to the increased energy required to reach dissociative triplet
metal-centered (MC) states that would otherwise undergo
ligand substitution to form covalent DNA adducts. Never-
theless, their characteristic robustness and red-shifted absorp-
tion relative to the analogous polypyridyl systems has prompted
1. INTRODUCTION
Ru(II) polypyridyl complexes have been widely studied for
more than 30 years due to their attractive photophysical and
electrochemical properties. Their utility has been explored
across a variety of technological areas: dye-sensitized solar
cells,1 solar fuels photochemistry,2 light-emitting electro-
chemical cells,3 photoluminescence sensors,4 biophotonics,5
photochromics,6 water-oxidation catalysts,7 water-reduction
catalysts,8 low-power photon upconversion,9 and in more
fundamental studies of photoinduced electron and energy
transfer.10 More recently, Ru(II) polypyridyl complexes have
been investigated as light-responsive agents in a range of
photobiological applications−notably as mediators of photo-
dynamic therapy (PDT) or photoactivated cancer therapy
(PACT),11−23 and as covalent modifiers of DNA in photo-
chemotherapy (PCT).24−30 A 2015 review by Knoll and Turro
highlights some of the most recent and important contributions
related to phototoxic Ru(II) polypyridyl complexes.28
By comparison, the related class of cyclometalated Ru(II)
complexes has received very little focus over the same time
period. Fewer than 20 [Ru(LL)2(C^N)]+ complexes appear in
the Cambridge Structural Database as of 2015, and these are
based on C^N = phpy− (deprotonated 2-phenylpyridine) or
derivatives of phpy−.31,32 This lack of attention may stem, in
Received: August 17, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
continued effort toward adapting these structures for use as
panchromatic light-harvesting sensitizers for a variety of light-
based applications.35−37
Chart 2. Ru(II) Polypyridyl Complexes Used as Reference
Compounds in this Study
An additional challenge to the development of coordinatively
saturated tris-bidentate cyclometalated Ru(II) complexes as
light-responsive prodrugs, lies in their inherent cytotoxicity,
which is often significantly greater than that of the well-known
anticancer drug cisplatin.36−38 High dark cytotoxicities
compounded by modest light cytotoxicities have produced
suboptimal phototherapeutic margins of less than 10-fold for
this class of organometallic Ru(II) systems to date.36 Similar to
the parent [Ru(bpy)2(phpy−)]+, all of these compounds appear
to possess lowest-energy 3MLCT states with pure Ru →
diimine character and derive from a similar scaffold: a
cyclometalating phpy− or benzo[h]quinoline (bhq−) ancillary
ligand combined with diimine coligands, such as bpy, 2,2′-
biquinoline (biq), [1,10]phenanthroline[5,6]dione, or benzo-
[i]dipyrido[3,2-a:2′,3′-c]phenazine (dppn).
To the best of our knowledge, cyclometalation with π-
expansive ligands that could serve as the site of excited-state
charge localization (or reduction) has not been explored. We
hypothesized that such systems might combine the best
attributes of cyclometalated Ru(II) complexes (thermal stability
with broad, low-energy absorption into the PDT window) and
polypyridyl Ru(II) metal−organic dyads (potent PDT effects
with low dark cytotoxicities) and be particularly well-suited for
use as PDT agents. Herein, we report that certain π-expansive
cyclometalating ligands do, in fact, turn cytotoxic organo-
metallic Ru(II) complexes into attractive photosensitizers for
PDT (or PACT) (compounds 1−4, Chart 1) and highlight that
the number of fused rings is critical.
2. EXPERIMENTAL PROCEDURES
2.1. Materials. 2,2′-Bipyridine (bpy), 2,3-diaminonaphthalene,
benzo[h]quinoline (bhq), and RuCl3·xH2O were purchased from
Sigma-Aldrich and used without further purification. [Ru(bpy)2Cl2]·
2H2O was prepared by an established procedure.39 Characterized fetal
bovine serum (FBS) (VWR), RPMI 1640 (Corning Cellgro), and
Eagle’s Minimum Essential Medium (EMEM) (Corning Cellgro) were
purchased from VWR. Human promyelocytic leukemia cells (HL-60)
and human malignant melanoma cells (SK-MEL-28) were procured
from the American Type Culture Collection. Prior to use, FBS was
divided into 40 mL aliquots that were heat inactivated (30 min, 55 °C)
and subsequently stored at −20 °C. Water for biological experiments
was deionized to a resistivity of 18 MΩ cm using a Barnstead filtration
system.
Chart 1. Cyclometalated Ru(II) Complexes Investigated in
1
this Study and the Labeling Used for H NMR Assignments
2.2. Instrumentation. Microwave reactions were performed in a
CEM Discover microwave reactor. NMR spectra were collected using
Bruker AVANCE 500 (Dalhousie University Nuclear Magnetic
Resonance Research Resource) or 300 (Acadia Centre for Micro-
structural Analysis) MHz spectrometers, and ESI mass spectra were
obtained using a Bruker microTOF focus mass spectrometer
(Dalhousie University Mass Spectrometry Laboratory). HPLC
analyses were carried out on an Agilent/Hewlett-Packard 1100 series
instrument (ChemStation Rev. A. 10.02 software) using a Hypersil
GOLD C18 reversed-phase column with an A-B gradient (98% →
40% A; A = 0.1% formic acid in H2O, B = 0.1% formic acid in
MeOH). Reported retention times are correct to within 0.1 min.
2.3. Synthesis. The preparation and characterization of compound
1 has been previously reported but using a different synthetic
method.33 Reference polypyridyl Ru(II) compounds 5−8 were
synthesized according to modified literature protocols reported
previously15,40−43 using microwave irradiation at 180 °C for 10 min
1
and characterized by TLC, H NMR, and mass spectrometry. The
metal complexes for this study were isolated and purified as PF6− salts
and subsequently subjected to anion metathesis on Amberlite IRA-410
with MeOH to yield the more water-soluble Cl− salts for biological
1
experiments. H NMR and electrospray ionization ESI (+ve) mass
−
spectra were collected on PF6 salts in MeCN-d and MeCN,
respectively.
B
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1
Benzo[h]quinoline (bhq). H NMR (500 MHz, chloroform-d): δ
9.33 (dd, J = 8.3, 1.3 Hz, 1H, x), 9.02 (dd, J = 4.4, 1.7 Hz, 1H, a), 8.18
(dd, J = 8.0, 1.9 Hz, 1H, c), 7.91 (d, J = 7.8 Hz, 1H, g), 7.82 (d, J = 8.8
Hz, 1H, f), 7.76 (ddd, J = 8.3, 6.9, 1.5 Hz, 1H, d), 7.72 (dd, J = 7.7, 1.4
Hz, 1H, h), 7.70−7.66 (m, 1H, e), 7.53 (dd, J = 8.0, 4.2 Hz, 1H, b).
HPLC retention time: 31.598 min, 33.842 min. TLC of commercial
bhq showed 4 spots (1% MeOH:CH2Cl2).
f), 9.08 (dd, J = 4.6, 1.8 Hz, 1H, c), 8.96 (s, 1H, g), 8.93 (s, 1H, l),
8.25−8.15 (m, 2H, h, k), 7.94−7.83 (m, 2H, d, e), 7.71 (dd, J = 8.1,
4.6 Hz, 1H, b), 7.66−7.58 (m, 2H, i, j). HPLC retention time: 34.02
min.
[Ru(bpy)2(bhq)]PF6 (1). [Ru(bpy)2(bhq)]PF6 was synthesized using
an adapted literature procedure.47 Ru(bpy)2Cl2·2H2O (100 mg, 0.19
mmol) and bhq (37 mg, 0.21 mmol) were added to a microwave vessel
containing triethylamine (1 mL) and ethylene glycol (3 mL). The
mixture was subjected to microwave irradiation at 120 °C for 40 min.
TLC indicated that not enough Ru(bpy)2Cl2·2H2O had been added to
consume the bhq ligand; thus, additional Ru(bpy)2Cl2·2H2O (20 mg,
0.04 mmol) was added to the mixture, and it was microwaved for an
additional 20 min at 120 °C. The purple mixture was pipetted into
approximately 30 mL of stirring saturated KPF6 solution. The resulting
precipitate was vacuum filtered with a fine glass-sintered frit and
purified by silica gel column chromatography with 5% H2O and 0.5%
Benzo[h]quinoline-5,6-dione. Benzo[h]quinoline-5,6-dione was
synthesized following a literature method.44 Benzo[h]quinoline (3.6
g, 20 mmol) and iodopentoxide (8.2 g, 25 mmol) were added to a 250
mL round-bottom flask with glacial acetic acid (50 mL). The orange
mixture was heated at reflux (118 °C) for 3 h, resulting in a dark
purple solution. The reaction was determined to have gone to
1
completion by H NMR spectroscopy. The product was precipitated
by the addition of deionized water (∼75 mL) and left to stand
overnight at room temperature. The precipitate was isolated by
vacuum filtration using a medium glass-sintered frit and subsequently
dissolved in chloroform (300 mL) to give a dark red solution that was
washed with 100 mL of sat. NaHCO3 and 100 mL of sat. Na2S2O3.
The organic layer was dried with anhydrous NaSO4, and the solvent
was removed by rotary evaporation to give a dark brown solid (4.1 g,
97%). Rf = 0.22 (10% EtOAc in hexanes). 1H NMR (300 MHz,
chloroform-d): δ 8.92 (dd, J = 4.7, 1.9 Hz, 1H, a), 8.73 (ddd, J = 7.9,
1.3, 0.6 Hz, 1H, x), 8.43 (dd, J = 7.9, 1.9 Hz, 1H, c), 8.23 (ddd, J = 7.8,
1.4, 0.6 Hz, 1H, f), 7.83 (ddd, J = 7.9, 7.4, 1.4 Hz, 1H, d), 7.61 (td, J =
7.6, 1.2 Hz, 1H, e), 7.45 (dd, J = 7.9, 4.7 Hz, 1H, b). HPLC retention
time: 24.30 min.
4,9,12-Triazadibenzo[a,c]naphthalene (pbpq). Pbpq was synthe-
sized using a procedure adapted from the literature.45 Benzo[h]-
quinoline-5,6-dione (209 mg, 1.0 mmol) was added to a 100 mL
round-bottom flask with methanol (30 mL). Ethylene diamine (67 mg,
1.1 mmol) was added dropwise to the round-bottom flask, resulting in
an orange solution that was heated at reflux (65 °C) for 4.5 h. The
reaction was then cooled to room temperature, concentrated to 3 mL
by rotary evaporation, and left to stand at 4 °C overnight. The
resulting precipitate was removed by vacuum filtration, and a yellow
powder was isolated from the filtrate by rotary evaporation. The
product was purified by silica gel chromatography with 10% EtOAc in
hexanes to yield a yellow powder (69 mg, 30%). Rf = 0.68 (30%
EtOAc in hexanes). 1H NMR (500 MHz, chloroform-d): δ 9.57 (d, J =
6.0 Hz, 1H, a), 9.44 (d, J = 5.4 Hz, 1H, x), 9.24 (d, J = 6.9 Hz, 1H, f),
9.17 (d, J = 4.5 Hz, 1H, c), 9.00 (d, J = 2.0 Hz, 1H, g), 8.95 (d, J = 2.1
Hz, 1H, h), 7.97−7.87 (m, 2H, d, e), 7.80−7.74 (m, 1H, b). HPLC
retention time: 22.99, 28.26, 32.18 min.
−
−
sat. KNO3 in MeCN to yield a mixture of PF6 and NO3 salts. The
resulting solid was dissolved in 5−10 mL of water, and 1−2 mL of sat.
−
KPF6 was added to precipitate the desired PF6 salt. Subsequent
extraction of the aqueous solution with dichloromethane and
−
concentration under reduced pressure gave the pure PF6 salt as a
purple solid (40 mg, 26%). Rf = 0.73 (10% H2O + 2.5% sat. KNO3 in
1
MeCN). H NMR (500 MHz, MeCN-d3): δ 8.48 (dd, J = 8.26, 1.18
Hz, 1H, 3A), 8.39−8.32 (m, 2H, 3B, 3C), 8.29 (d, J = 8.1 Hz, 1H, 3D),
8.20 (dd, J = 8.0, 1.4 Hz, 1H, c), 8.04−7.96 (m, 2H, 4A, 6A), 7.91−
7.77 (m, 5H, 4B, 6B, 6C, a, g), 7.76−7.67 (m, 3H, 4C, 4D, h), 7.63 (d,
J = 5.8 Hz, 1H, 6D), 7.52−7.45 (m, 1H, 5A), 7.42 (d, J = 7.8 Hz, 1H,
f), 7.33−7.19 (m, 3H, 5B, b, e), 7.04−7.08 (m, 2H, 5C, 5D), 6.69 (dd,
J = 7.0, 1.0 Hz, 1H, d). MS (ESI+) m/z 592.2 [M − PF6]+. HRMS ESI
+ m/z for C33H24N5Ru: calcd 592.1070, found 592.1060. HPLC
retention time: 26.05 min.
[Ru(bpy)2(pbpq)]PF6 (2). [Ru(bpy)2(pbpq)]PF6 was synthesized
using an adapted literature procedure.47 Ru(bpy)2Cl2·2H2O (135 mg,
0.26 mmol) and pbpq (46 mg, 0.20 mmol) were added to a microwave
vessel containing triethylamine (1 mL) and ethylene glycol (3 mL).
The mixture was subjected to microwave irradiation at 120 °C for 1 h
and then pipetted into approximately 30 mL of stirring saturated KPF6
solution. The resulting precipitate was vacuum filtered with a fine
glass-sintered frit and purified by silica gel column chromatography
with 10% MeOH in DCM. TLC indicated impurities in the purified
product (62 mg), so the column was repeated with 1% MeOH in
DCM to give a single spot by TLC. Purple solid (40 mg, 25%). Rf =
1
0.69 (10% H2O + 2.5% sat. KNO3 in MeCN). H NMR (500 MHz,
MeCN-d3): δ 9.21 (dd, J = 8.1, 1.4 Hz, 1H, c), 9.00 (d, J = 2.1 Hz, 1H,
g), 8.94 (d, J = 2.1 Hz, 1H, h), 8.50 (d, J = 8.2 Hz, 1H, 3A), 8.47 (dd, J
= 8.0, 1.0 Hz, 1H, f), 8.41−8.36 (m, 2H, 3B, 3C), 8.32 (dd, J = 9.0, 1.3
Hz, 1H, 3D), 8.04 (td, J = 7.9, 1.6 Hz, 1H, 4A), 8.02−7.94 (m, 3H, 6A,
6C, a), 7.91−7.84 (m, 2H, 4B, 6B), 7.79−7.72 (m, 3H, 4C, 4D, 6D),
7.48 (ddd, J = 7.5, 5.4, 1.2 Hz, 1H, 5A), 7.43 (dd, J = 8.1, 5.3 Hz, 1H,
b), 7.36 (t, J = 7.5 Hz, 1H, e), 7.29 (ddd, J = 7.3, 5.7, 1.4 Hz, 1H, 5B),
7.09−7.02 (m, 2H, 5C, 5D), 6.87 (dd, J = 7.1, 1.0 Hz, 1H, d). 13C
NMR (75 MHz, CD3CN): δ 187.38, 155.62, 154.76, 154.59, 153.96,
153.60, 152.29, 151.11, 148.54, 147.27, 147.09, 146.17, 141.63, 140.35,
139.99, 138.91, 136.81, 133.31, 132.70, 131.88, 130.87, 130.61, 128.22,
128.01, 125.52, 123.76, 123.09, 122.93, 122.73, 122.15, 120.16, 120.01,
119.74, 119.09, 112.75. MS (ESI+) m/z: 644.2 [M − PF6]+. HRMS
ESI+ m/z for C35H24N7Ru: calcd 644.1131, found 644.1159. HPLC
retention time: 26.46 min.
4,9,14-Triazadibenzo[a,c]anthracene (pbpz). Benzo[h]quinoline-
5,6-dione (174 mg, 0.83 mmol) and o-phenylenediamine (101 mg,
0.93 mmol) were combined in 30 mL of ethanol. The orange mixture
was heated at reflux (78 °C) for 4 h, resulting in a dark red solution.
The mixture was cooled to room temperature, and the resulting
precipitate was vacuum filtered with a medium glass-sintered frit and
washed with 50 mL of cold deionized water, 50 mL of cold ethanol,
and 100 mL of cold diethyl ether to give a pale yellow powder (114
1
mg, 49%). Rf = 0.63 (30% EtOAc in hexanes). H NMR (500 MHz,
chloroform-d): δ 9.62 (dd, J = 8.1, 1.8 Hz, 1H, a), 9.39 (dd, J = 7.8, 1.7
Hz, 1H, x), 9.26 (dd, J = 7.7, 1.6 Hz, 1H, f), 9.09 (dd, J = 4.4, 1.8 Hz,
1H, c), 8.40−8.31 (m, 2H, h, i), 7.93−7.85 (m, 4H, d, e, i, j), 7.70 (dd,
J = 8.0, 4.5 Hz, 1H, b). HPLC retention time: 26.70 min.
4,9,16-Triazadibenzo[a,c]napthacene (pbpn). Pbpn was synthe-
sized according to a procedure adapted from a literature preparation of
3-pyrid-2′-yl-4,9,16-triazadibenzo[a,c]naphthacene (pyHdbn).46
Benzo[h]quinoline-5,6-dione (209 mg, 1.00 mmol) and 2,3-
diaminonaphthalene (177 mg, 1.12 mmol) were added to a 100 mL
round-bottom flask with ethanol (30 mL). The orange mixture was
heated at reflux (78 °C) for 4.5 h, resulting in a dark red mixture. The
mixture was cooled to room temperature, and the resulting precipitate
was vacuum filtered with a fine glass-sintered frit and washed with 50
mL of cold deionized water, 50 mL of cold ethanol, and 100 mL of
cold diethyl ether to give a brown powder (221 mg, 67%). Rf = 0.36
(10% EtOAc in hexanes). 1H NMR (300 MHz, chloroform-d): δ 9.64
(d, J = 7.9 Hz, 1H, a), 9.43−9.36 (m, 1H, x), 9.24 (d, J = 7.4 Hz, 1H,
[Ru(bpy)2(pbpz)]PF6 (3). [Ru(bpy)2(pbpz)]PF6 was synthesized
using an adapted literature procedure.47 Ru(bpy)2Cl2·2H2O (135 mg,
0.26 mmol) and pbpz (56 mg, 0.20 mmol) were added to a microwave
vessel containing a mixture of triethylamine (1 mL) and ethylene
glycol (3 mL). The purple solution was subjected to microwave
irradiation at 120 °C for 1 h and then pipetted into 30 mL of a stirring
saturated KPF6 solution. The resulting precipitate was vacuum filtered
with a fine glass-sintered frit and purified by silica gel column
chromatography (5% MeOH:DCM). Purple solid (41 mg, 25%). Rf =
1
0.70 (10% H2O + 2.5% sat. KNO3 in MeCN). H NMR (500 MHz,
MeCN-d3): δ 9.33 (dd, J = 8.0, 1.5 Hz, 1H, c), 8.61 (dd, J = 7.8, 1.1
Hz, 1H, f), 8.51 (d, J = 8.2, 1.1 Hz, 1H, 3A), 8.44−8.37 (m, 2H, 3B,
C
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
3C), 8.37−8.31 (m, 3H, 3D, g, j), 8.07−8.02 (m, 2H, 4A, 6C), 8.01−
7.93 (m, 4H, 6A, a, h, i), 7.91−7.85 (m, 2H, 4B, 6B), 7.82 (d, J = 5.7,
1H, 6D), 7.78−7.75 (m, 2H, 4C, 4D), 7.48 (ddd, J = 7.7, 5.4, 1.2 Hz,
1H, 5A), 7.44 (dd, J = 8.0, 5.4 Hz, 1H, b), 7.36 (t, J = 7.5 Hz, 1H, e),
7.29 (ddd, J = 7.4, 5.6, 1.4 Hz, 1H, 5B), 7.12−7.07 (m, 2H, 5C, 5D),
6.88 (dd, J = 7.2, 1.1 Hz, 1H, d). 13C NMR (75 MHz, CD3CN): δ
187.90, 157.25, 154.61, 154.15, 153.96, 153.63, 152.45, 152.29, 152.18,
151.18, 148.95, 147.35, 147.08, 146.17, 140.77, 139.27, 138.43, 138.21,
133.66, 133.33, 131.93, 130.91, 130.67, 128.60, 128.29, 127.40, 126.90,
126.04, 126.00, 125.66, 123.78, 123.11, 122.98, 122.77, 120.19, 120.02,
119.77, 119.31, 113.66. MS (ESI+) m/z 694.3 [M − PF6]+. HRMS
(ESI+) m/z for C39H26N7Ru: calcd 694.1288, found 694.1274. HPLC
retention time: 29.20 min.
fluorometer with an R928 stroboscopic detector with excitation from a
GL-3300 nitrogen/GL-301 dye laser (2−3 nm fwhm). Excited-state
lifetimes were extracted from the observed data using PTI Felix32
fitting software. Emission and excitation spectra were corrected for the
wavelength dependence of lamp output and detector response.
2.5. Cellular Assays. 2.5.1. Metal Compound Solutions. Stock
solutions of the chloride salts of the Ru(II) complexes were prepared
at 5 mM in 10% DMSO in water and kept at −20 °C prior to use.
Working dilutions were made by diluting the aqueous stock with pH
7.4 Dulbecco’s phosphate buffered saline (DPBS). DPBS is a balanced
salt solution of 1.47 mM potassium phosphate monobasic, 8.10 mM
sodium phosphate dibasic, 2.68 mM potassium chloride, and 0.137 M
sodium chloride (no Ca2+ or Mg2+). DMSO in the assay wells was
under 0.1% at the highest complex concentration.
2.5.2. Cell Culture. HL-60. HL-60 human promyelocytic leukemia
cells (ATCC CCL-240) were cultured at 37 °C under 5% CO2 in
RPMI 1640 (Mediatech Media MT-10-040-CV) supplemented with
20% FBS (PAA Laboratories, A15-701) and were passaged 3−4 times
per week according to standard aseptic procedures. Cultures were
started at 200,000 cells mL−1 in 25 cm2 tissue culture flasks and were
subcultured when growth reached 800,000 cells mL−1 to avoid
senescence associated with prolonged high cell density. Complete
growth medium was prepared in 200 mL portions as needed by
combining RPMI 1640 (160 mL) and FBS (40 mL, prealiquoted and
heat inactivated) in a 250 mL Millipore vacuum stericup (0.22 μm)
and filtering.
SK-MEL-28. Adherent SK-MEL-28 malignant melanoma cells
(ATCC HTB-72) were cultured in Eagle’s Minimum Essential
Medium (EMEM, Mediatech Media MT-10-009-CV) supplemented
with 10% FBS, were incubated at 37 °C under 5% CO2, and were
passaged 2−3 times per week according to standard aseptic
procedures. SK-MEL-28 cells were started at 200,000 cells mL−1 in
75 cm2 tissue culture flasks and were subcultured when growth
reached 550,000 cells mL−1 by removing old culture medium and
rinsing the cell layer once with Dulbecco’s phosphate buffered saline
(DPBS 1×, Mediatech, 21-031-CV), followed by dissociation of the
cell monolayer with trypsin-EDTA solution (0.25% w/v Trypsin/0.53
mM EDTA, ATCC 30-2101). Complete growth medium was added to
the cell suspension to allow appropriate aliquots of cells to be
transferred to new cell vessels. Complete growth medium was
prepared in 150 mL portions as needed by combining EMEM (135
mL) and FBS (15 mL, prealiquoted and heat inactivated) in a 250 mL
Millipore vacuum stericup (0.22 μm) and filtering.
2.5.3. Cytotoxicity and Photocytotoxicity. Cell viability experi-
ments were performed in triplicate in 96-well ultra-low attachment flat
bottom microtiter plates (Corning Costar, Acton, MA), where outer
wells along the periphery contained 200 μL of DPBS (2.68 mM
potassium chloride, 1.47 mM potassium phosphate monobasic, 0.137
M sodium chloride, and 8.10 mM sodium phosphate dibasic) to
minimize evaporation from sample wells. Cells growing in log phase
(HL-60 cells: ∼800,000 cells mL−1. SK-MEL-28 cells: ∼550,000 cells
mL−1) with at least 93% viability were transferred in 50 μL aliquots to
inner wells containing warm culture medium (25 μL) and placed in a
37 °C, 5% CO2 water-jacketed incubator (Thermo Electron Corp.,
FormaSeries II, Model 3110, HEPA Class 100) for 3 h to equilibrate
(and allow for efficient cell attachment in the case of SK-MEL-28
adherent cells). Metals compounds were serially diluted with DPBS
and prewarmed at 37 °C before 25 μL aliquots of the appropriate
dilutions were added to cells. PS-treated microplates were incubated at
37 °C under 5% CO2 for 16 h drug-to-light intervals. Control
microplates not receiving a light treatment were kept in the dark in an
incubator, and light-treated microplates were irradiated under one of
the following conditions: visible light (400−700 nm, 34.2 mW cm−2)
using a 190 W BenQ MS 510 overhead projector or red light (625 nm,
29.1 mW cm−2) from an LED array (PhotoDynamic Inc., Halifax, NS).
Irradiation times using these two light sources were approximately 49
and 57 min, respectively, to yield total light doses of 100 J cm−2. Both
untreated and light-treated microplates were incubated for another 48
h before 10 μL aliquots of prewarmed Alamar Blue reagent (Life
Technologies DAL 1025) were added to all sample wells and
[Ru(bpy)2(pbpn)]PF6 (4). [Ru(bpy)2(pbpn)]PF6 was synthesized
using an adapted literature procedure.47 Ru(bpy)2Cl2·2H2O (150 mg,
0.29 mmol) and pbpn (68 mg, 0.21 mmol) were added to a microwave
vessel containing a mixture of triethylamine (1 mL) and ethylene
glycol (3 mL). The solution was subjected to microwave irradiation at
120 °C for 1 h and then pipetted into 30 mL of a stirring saturated
KPF6 solution. The resulting precipitate was collected under vacuum
on a fine glass-sintered frit and purified by silica gel column
chromatography with 5% MeOH in DCM. Purple solid (42 mg,
1
23%). Rf = 0.76 (10% H2O + 2.5% sat. KNO3 in MeCN). H NMR
(500 MHz, MeCN-d3): δ 9.30 (dd, J = 8.0, 1.4 Hz, 1H, c), 8.94 (d, J =
5.5 Hz, 2H, g, l), 8.60 (dd, J = 7.8, 1.1 Hz, 1H, f), 8.51 (d, J = 8.3, 1H,
3A), 8.42−8.37 (m, 2H, 3B, 3C), 8.34 (d, J = 8.2 Hz, 1H, 3D), 8.30−
8.25 (m, 2H, h, k), 8.13−8.07 (m, 1H, 6C), 8.05 (td, J = 7.9, 1.6 Hz,
1H, 4A), 8.00−7.94 (m, 2H, 6A, a), 7.92−7.85 (m, 3H, 4B, 6B, 6D),
7.81−7.76 (m, 2H, 4C, 4D), 7.70−7.63 (m, 2H, i, j), 7.48 (ddd, J =
7.7, 5.3, 1.2 Hz, 1H, 5A), 7.40 (dd, J = 8.0, 5.4 Hz, 1H, b), 7.33 (t, J =
7.5 Hz, 1H, e), 7.30 (ddd, J = 7.4, 5.7, 1.4 Hz, 1H, 5B), 7.15−7.10 (m,
2H, 5C, 5D), 6.88 (dd, J = 7.2, 1.1 Hz, 1H, d). 13C NMR (75 MHz,
MeCN-d3): δ 188.19, 157.95, 154.73, 154.62, 153.96, 153.63, 152.28,
151.27, 149.13, 147.40, 147.07, 146.15, 141.66, 139.50, 139.40, 135.86,
135.62, 135.01, 134.20, 133.34, 131.97, 131.20, 130.92, 130.81, 130.69,
128.67, 128.38, 125.71, 125.57, 125.11, 124.38, 124.14, 124.00, 123.78,
123.77, 123.57, 123.11, 123.03, 122.81, 120.22, 120.03, 119.78, 119.39.
MS (ESI+) m/z 744.4 [M − PF6]+. HRMS ESI+ m/z for
C43H28N7Ru: calcd 744.1444, found 744.1450. HPLC retention
time: 30.92 min.
2.4. Spectroscopy. Photophysical characterization was carried out
on dilute solutions (5 μM) of the PF6− salts of the metal complexes in
spectroscopic-grade MeCN unless otherwise noted. Molar extinction
coefficients at wavelength maxima were determined from the slopes ε
of linear fits of absorption versus concentration plots (A = εbc). Five
concentrations (20 μM and four serial dilutions of 25%) were used
with points measured in duplicate. Quantum yields for emission (Φem)
and singlet oxygen (ΦΔ) were measured relative to [Ru(bpy)3](PF6)2
according to eq 1, where I, A, and η are integrated emission intensity,
absorbance at the excitation wavelength, and refractive index of the
solvent, respectively. Reference values used for [Ru(bpy)3](PF6)2 as
the standard are as follows: Φem = 0.012 at 298 K in aerated MeCN,48
Φem = 0.062 at 298 K in deaerated MeCN, Φem = 0.38 at 77 K in
frozen 4:1 v/v EtOH:MeOH,10 and ΦΔ = 0.56 in aerated MeCN.49
2
⎛
⎝
⎞
⎠
⎛
⎜
⎝
⎞
⎟
⎠
As
⎛
⎜
⎞
⎟
I
A
η
⎜
⎜
⎟
⎟
Φ
= Φ
em
s
η2
⎝
⎠
I
s
(1)
s
Where relevant, oxygen was removed from room temperature samples
in long-neck cuvettes (Luzchem SC-10L) by purging with argon at a
pressure of 50
10 mmHg for 30 min. Samples for 77 K
measurements were prepared in 4:1 EtOH:MeOH in a 5 mm i.d.
NMR tube that was placed in a quartz-tipped cold finger Dewar
(fabricated by Wilmad Labglass) filled with liquid nitrogen. Absorption
spectra were recorded with a Jasco V-530 spectrophotometer. Steady-
state luminescence spectra were measured on a PTI Quantamaster
equipped with a K170B PMT for measuring ultraviolet to visible
emission and a Hamamatsu R5509-42 near-IR PMT for measuring
near-infrared (near-IR) emission (<1400 nm). Phosphorescence
lifetimes (<1 μs) were measured on a PTI LaserStrobe spectro-
D
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
subsequently incubated for another 15−16 h. Cell viability was
determined on the basis of the ability of the Alamar Blue redox
indicator to be metabolically converted to a fluorescent dye by only
live cells. Fluorescence was quantified with a Cytofluor 4000
fluorescence microplate reader with the excitation filter set at 530
all the treatments were 200 μm. The images were collected and
analyzed using the Zeiss LSM Image Browser Version 4.2.0.121
software (Carl Zeiss Inc.).
Intracellular production of reactive oxygen species (ROS) was
detected in HL-60 cells and SK-MEL-28 cells using the ROS
fluorescent dye dihydroethidium (DHE) (VWR, 38483-26-0)
according to the manufacturer’s protocol. Cells loaded with the
metal compound were then washed with DPBS and incubated with 3
μM DHE at 37 °C for 15 min before receiving a dark or light (visible
or red, 50 J cm−2) treatment. Visible and red light treatments were
delivered from the sources described in section 2.5.3. Production of
ROS was imaged using a Carl Zeiss LSM 510 laser scanning confocal
microscope with a 40× oil objective lens. Excitation was delivered at
458/488 nm from an argon−krypton laser, and signals were acquired
through a 510 nm long-pass filter and a 475−525 nm band-pass filter.
The pinhole diameter for all of the treatments was 200 μm. The
images were collected and analyzed using Zeiss LSM Image Browser
Version 4.2.0.121 software (Carl Zeiss Inc.).
25 nm and emission filter set at 620
40 nm. EC50 values for
cytotoxicity (dark) and photocytotoxicity (light) were calculated from
sigmoidal fits of the dose−response curves using Graph Pad Prism 6.0
according to eq 2, where yi and yf are the initial and final fluorescence
signal intensities. For cells growing in log phase and of the same
passage number, EC50 values are generally reproducible to within
25% in the submicromolar regime, 10% below 10 μM, and 5%
above 10 μM. Phototherapeutic indices (PIs), a measure of the
therapeutic window, were calculated from the ratio of dark to light
EC50 values obtained from the dose−response curves.
y − y
i
f
y = y +
i
1 + 10(log EC
50−x)×(Hillslope)
(2)
2.6. DNA Photocleavage Assays. DNA photocleavage experi-
ments were performed according to a general plasmid DNA gel
mobility shift assay16,50,51 with 30 μL total sample volumes in 0.5 mL
microfuge tubes. Transformed pUC19 plasmid (3 μL, >95% form I)
was added to 15 μL of 5 mM Tris-HCl buffer supplemented with 50
mM NaCl (pH 7.5). Serial dilutions of the Ru(II) compounds were
prepared in ddH2O and added in 7.5 μL aliquots to the appropriate
tubes to yield final Ru(II) concentrations ranging from 1 to 100 μM.
Then, ddH2O (4.5 μL) was added to bring the final assay volumes to
30 μL. Control samples with no metal complex received 12 μL of
water. Sample tubes were kept at 37 °C in the dark or irradiated. Light
treatments employed visible light (14 J cm−2) delivered from a
Luzchem LZC-4X photoreactor over the course of 30 min. After
treatment, all samples (dark and light) were quenched by the addition
of 6 μL of gel loading buffer (0.025% bromophenol blue, 40%
glycerol). Samples (11.8 μL) were loaded onto 1% agarose gels cast
with 1× TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.2) containing
ethidium bromide (0.75 μg mL−1) and electrophoresed for 30 min at
80 V cm−1 in 1× TAE. The bands were visualized using the Gel Doc-It
Imaging system (UVP) with Vision Works software and further
processed with the GNU Image Maniupulation Program (GIMP).
2.7. Confocal Microscopy. Sterile glass-bottom Petri dishes
(MatTek) were coated with 200 μL poly-L-lysine (Ted Pella) in a
laminar flow hood under standard aseptic conditions. After a 1 h
incubation period at 37 °C, 5% CO2 in a water-jacketed incubator
(Thermo Electron Corp., Forma Series II, Model 3110, HEPA class
100), the dishes were washed three times with sterile Dulbecco’s
phosphate buffered saline (DPBS 1×, Mediatech, 21-031-CV)
containing 2.68 mM potassium chloride, 1.47 mM potassium
phosphate monobasic, 0.137 M sodium chloride, and 8.10 mM
sodium phosphate dibasic, pH 7.4, and were left to dry uncovered at
room temperature for approximately 15 min. HL-60 human
promyelocytic leukemia cells (ATCC CCL-240) or SK-MEL-28
malignant melanoma cells (ATCC HTB-72) were then transferred
in aliquots of 500 μL (approximately 100,000 cells) to the poly-L-
lysine-coated glass bottom Petri dishes and were allowed to adhere for
15 min or 2 h, respectively, in a 37 °C, 5% CO2 water-jacketed
incubator. Metal compound (500 μL of a 10 μM solution in sterile
PBS prewarmed to 37 °C) was added to sample dishes (destined to
receive either a dark or light treatment), which were returned to the
incubator for 30 min prior to further treatment; control dishes that did
not contain the metal compound were also prepared. Light-treated
samples were irradiated with visible light for 24 min from a 190 W
BenQ MS 510 overhead projector (400−700 nm, power density =
34.2 mW cm−2, total light dose ≈ 50 J cm−2) or with red light for 28
min from an LED array (625 nm, power density = 29.1 mW cm−2,
total light dose ≈ 50 J cm−2). Dark samples were covered with foil and
placed in a drawer for the same amount of time. Cells were then
imaged at 15 min post-treatment using a Carl Zeiss LSM 510 laser
scanning confocal microscope with a 40x oil objective lens. Excitation
was delivered at 458/488 nm from an argon−krypton laser, and signals
were acquired through a 475 nm long-pass filter. Pinhole diameters for
3. RESULTS AND DISCUSSION
3.1. Synthesis and Characterization. Following the
seminal discovery that electron-withdrawing groups added to
cyclometalating ligands of the type phpy− could position the
RuIII/II couple at the same energy as that of the champion N3
photosensitizer,35 a growing number of functionalized cyclo-
metalated ligands are now being explored for dye-sensitized
solar cells.1,52 Functionalization of phpy− has generally involved
only mono- or disubstitution with electron-withdrawing atoms,
groups, or rings rather than fused π-systems. Even bhq− as an
alternate cyclometalating ligand remains relatively underutilized
owing to the ease with which phpy− can be substituted and
subsequently coordinated to Ru(II). Thus, incorporation of
extended π-systems directly into cyclometalating bidentate
frameworks has not been explored.
Therefore, we chose to investigate the novel cyclometalating
analogue of the benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine
(dppn) ligand, pbpn, out of our interest in π-expansive ligands
that contribute low-lying triplet intraligand (IL) excited states
that are highly photosensitizing.15 To probe the effects of π-
conjugation on the photophysics and photobiological activities
of the cyclometalated systems (and to compare with their
Ru(II) polypyridyl counterparts), we also prepared pbpz and
pbpq. These three new ligands were synthesized from
benzo[h]quinoline-5,6-dione via a condensation reaction with
the corresponding diamines in alcoholic solvent at reflux. Yields
ranged from 30% for pbpq to 67% for pbpn and increased with
the number of fused rings. The starting benzo[h]quinoline-5,6-
dione was obtained in almost quantitative yield by oxidation of
commercial bhq with iodopentoxide in glacial acetic acid.44,53
1
1H NMR and two-dimensional H−1H COSY NMR spectros-
copy were used to characterize these new ligands as well as bhq
and benzo[h]quinoline-5,6-dione. The chemical shifts for bhq
were reported previously with hydrogens f-h assigned
incorrectly using 1D NMR.54 With 2D techniques, we have
corrected these assignments (Figure S1).
A recent method for generating a variety of cycloruthenated
compounds involves the preparation of a Ru π-arene, dimeric
[π-C6H6RuCl2]2 in the solid state,55 from 1,3-cyclohexadiene
and RuCl3·xH2O, and its subsequent reaction with the
cyclometalating ligand in MeCN to produce the corresponding
[Ru(CH3CN)4(C^N)]PF6 complex.34,56 This air-sensitive
intermediate,57 which is then reacted with the desired
coligands, normally requires purification and is only stable
under ambient conditions for a few hours. Older methods that
E
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1
Figure 1. H NMR spectra collected for compounds 1 (a), 2 (b), 3 (c), and 4 (d) in d3-MeCN.
employ the more stable cis-Ru(bpy)2Cl2 require long reaction
times at reflux and the use of silver(I) salts to abstract chloride
ions.58 We sought an alternate method for preparing bis-
heteroleptic Ru(II) C^N systems that could be carried out in
air without Ag(I) ions using microwave irradiation to ensure
short reaction times while avoiding oxygen-sensitive inter-
mediates. Use of 1:3 NEt3:ethylene glycol47 for reacting cis-
Ru(LL)2Cl2 (LL = bpy or some other diimine ligand) with a
wide range of cyclometalating ligands led to identifiable product
by TLC in 1 h or less at 120 °C with unoptimized yields of
approximately 25%. These yields are as good or better than
those previously reported with more cumbersome steps.
Compounds 1−4 were synthesized using this methodology,
distortions bring ring F and its hydrogens closer to the metal
center while pushing rings A, B, and E and their respective
hydrogens farther from Ru(II) (Chart 1). The impact was
greatest for hydrogen d next to the Ru−C bond, which was the
most shielded proton in all of the complexes investigated, and
for hydrogen c in 2−4, which was the most deshielded proton.
Hydrogen d, adjacent to the chelating carbon of the C^N
ligand and shielded due to the shortened Ru−C bond distance
of ring F, was diagnostic for all of the complexes, giving rise to a
doublet at substantially lower frequencies (6.7−6.9 ppm) than
the other aromatic proton signals (Figure 1). The other
diagnostic peak for complexes 2−4 was produced by proton c
and occurred as a doublet at substantially higher frequencies
(9.15−9.35 ppm) than the rest of the aromatic signals owing to
the lengthening of the Ru−N bond of ring E combined with its
proximity to the nitrogen of the electron-deficient pyrazine
ring. When pyrazine was absent, as for compound 1, the signal
for proton c occurred upfield at 8.2 ppm, and no signals were
shifted higher than 8.5 ppm. This proximity effect was further
supported by the appearance of deshielded signals correspond-
ing to protons g and h near 9.0 ppm for 2, and g and l near 8.9
ppm for 4. Interestingly, the signals for protons g and j of 3,
which correspond to protons g and l of 4, occurred at much
lower frequencies (8.3−8.4 ppm), underscoring the influence of
increased π-conjugation and the necessity for detailed 2D NMR
analysis for each new complex. All other protons of the C^N
ligands were assigned based on their coupling profiles in the
1H−1H COSY spectra obtained for the complexes and their
corresponding ligands and by comparing these profiles between
different ligands and complexes within the series (Figures S1−
7, S9, and S11).
1
and their structures were confirmed by 1D H NMR and 2D
1H−1H COSY NMR as well as HPLC and mass spectrometry.
1
Two-dimensional H−1H COSY NMR has previously been
used to assign all 20+ protons of certain cyclometalated
[Ru(bpy)2(phpy−)]+ derivatives.31,59,60 The low microsymme-
try of the overall pseudo octahedral complex imparted by the
asymmetric cyclometalating ligand translates to nonequivalence
of all 16 bpy protons alongside those of the C^N ligand.
Therefore, a complex such as 4 would be expected to yield 28
unique signals (26 for 3, 24 for 2, and 24 for 1). To our
knowledge, the proton spectrum of previously reported 1 has
not been assigned.61 A close analysis of the similarities and
1
differences in the 1D and 2D H NMR spectra obtained for
compounds of the present series scrutinized alongside the
published [Ru(bpy)2(phpy−)]+ assignments afforded the
opportunity to decipher otherwise rather complex spectra
characterized by overlapping multiplets.
X-ray crystallographic evidence31 of shortened Ru−C bond
distances that lead to elongated Ru−N bonds trans and cis
relative to the Ru−C coordination axis, with the trans effect
being greater, provided the basis for our interpretation and
assignments (Figures S6−7, S9, and S11). Qualitatively, these
The resonant frequencies for all 16 nonequivalent bpy
protons of 1−4 (Chart 1) were assigned based on established
trends for cis-[Ru(bpy)2LL]2+ complexes (whereby, in general,
H3 > H4 > H6 > H5),41,59 the bond distances reported by
F
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Sasaki et al.60 for cyclometalated phpy− complexes, H−1H
correlations, and coupling constants. The chemical shifts of the
bpy proton signals increased by ring in the order A > B > C >
D. The ring protons of A and B were the most deshielded of
the bpy protons due to the longer Ru−N bond distance
reported for ring A (and to a lesser extent ring B), whereas the
protons of ring D were shielded the most due to their close
proximity to C^N ring E. Within any one bpy ring, the chemical
shifts followed H3 > H4 > H6 > H5 with a few exceptions: 1,
ring C, 6 > 4; 2, ring C, 6 > 4; 3, rings C and D, 6 > 4; and 4,
rings C and D, 6 > 4.
1
3.2. Photophysical Properties. 3.2.1. Absorption. Elec-
tronic absorption spectra for complexes 1−4 were collected in
MeCN at room temperature (Figure 2). The spectroscopic
Figure 3. Ground-state absorption spectrum of 4 in MeCN compared
to that of its free cyclometalating ligand in CHCl3 at room
temperature.
spectrum of 4 with only slight differences in the peak maxima of
the fingerprint vibronic progression.
3.2.2. Emission. Static and dynamic photoluminescence
measurements were made for compounds 1−4 at room
temperature (argon-saturated MeCN) and at 77 K (4:1
EtOH:MeOH glass). Compounds 1−3 produced very weak
broad, structureless phosphorescence in solution at room
temperature (Φem ∼10−4) centered near 800 nm (Table 1), and
4 displayed no discernible 3MLCT emission in this region. The
excitation spectra for MLCT emission from 1−3 superimposed
the corresponding absorption profiles in the MLCT regions.
Quantum efficiencies for MLCT emission are listed in Table 1,
but their yields were too small to make reliable quantitative
comparisons. As expected, emission lifetimes measured near
800 nm at 298 K for 1−3 were extremely short (5−20 ns)
compared to related Ru(II) polypyridyl complexes (∼1 μs)10
and agreed with the reported lifetime for 1 (20 ns). For
Figure 2. Ground-state absorption spectra of 1−4 in MeCN at room
temperature.
signature of 1 has been reported,33 with the sharp bands in the
250−300 nm region assigned to ππ* transitions involving the
bpy and bhq− ligands, and the two sets of low-energy bands
between 320 and 700 nm assigned to MLCT transitions. The
previous report designated the lower energy MLCT band
(450−800 nm) as possessing Ru2+ → bpy parentage and the
higher energy band (345−450 nm) of Ru2+ → C^N parentage.
Numerous spectroscopic and electrochemical techniques
employed in that study confirmed that the lowest energy
charge-transfer excited state for 1 retains pure Ru2+ → bpy
character and that the C^N unit serves as an ancillary ligand.
CT transitions were characterized by maximum extinction
coefficients near 10,000 M−1 cm−1, but we observed slightly
smaller ε values (7,400−9,400 M−1 cm−1) for 1.
3
example, in deoxygenated MeCN, the MLCT lifetime of the
prototype [Ru(bpy)3]2+ is as long as 1 μs in fluid solution at
room temperature,10 whereas that of the analogous [Ru-
(bpy)2phpy−]+ complex is almost 2 orders of magnitude
shorter.33 Radiative and nonradiative decay rate constants were
not calculated for the present series owing to the error
associated with Φem values <0.005.
Relative to room temperature emission, the phosphorescence
from 1−3 at low temperature (77 K in 4:1 EtOH:MeOH glass)
shifted to higher energies, increased in intensity, and displayed
3
Fusing a pyrazine ring to the cyclometalating bhq− to form
the more π-expansive pbpq ligand (complex 2) gave rise to a
qualitatively similar absorption profile with the Ru2+ → bpy
transitions occurring at slightly shorter wavelengths and ε being
marginally larger throughout the spectrum. However, the
introduction of additional fused benzene rings beyond pyrazine
produced substantial changes in the absorption spectra, as
exemplified for compounds 3 and 4. Extinction coefficients for
3 and 4 were larger at all wavelengths, and notably, new bands
characteristic MLCT vibronic structure (Figure 4). Quantum
yields and lifetimes increased up to 127- and 89-fold,
respectively, at low temperature but were still substantially
reduced relative to their Ru(II) diimine counterparts according
to the energy gap law. The vibronic intervals were 1140−1220
cm−1, which is diagnostic of diimine involvement in the
emissive excited state.62 Together, these factors support the
notion that the lowest energy MLCT states are of Ru2+ → bpy
origin. Further, the thermally induced Stokes shifts (ΔEs) for
complexes 1−3 ranged from 1230 cm−1 for 1 to 1435 cm−1 for
2, which is consistent with ΔEs for [Ru(bpy)3]2+ under
identical conditions (1127 cm−1). This relatively large value for
ΔEs is in agreement with what is expected from polar excited
states of 3MLCT character.63 The onset of the structured
emission from 2 and 3 was blue-shifted by approximately 248
cm−1 relative to 1, suggesting that the pyrazine ring may
facilitate delocalization of the HOMO orbital onto the
cyclometalating ligand, reducing structural distortion between
the ground and emissive excited state.35
formed between 300 and 450 nm that overlapped the Ru2+
→
C^N MLCT transitions. These new features were attributed to
longer wavelength ππ* transitions (with possible contribution
from nπ* transitions) centered on the more π-expansive
cyclometalating ligands. A comparison shown for compound 4
and its corresponding C^N ligand (pbpn) supports this
interpretation (Figure 3). Although the absorption spectra for
the complex and its C^N ligand were collected in different
solvents (owing to solubility differences), the characteristic
pattern produced by pbpn was clearly observable in the
G
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Table 1. Photophysical Properties of 1−4
Φem ( ×
ab
ab
,
ab
,
,
λem [nm]
10−4
298 K
)
τ [ns]
ac
,
compound
λabs [nm] (log ϵ)
298 K
77 K
730
77 K
298 K
77 K
ΦΔ (×10−2
)
1
2
3
4
542 (3.90), 486 (3.87), 382 (3.97), 332 (3.89), 294 (4.60)
532 (3.95), 480 (3.96), 376 (4.00), 294 (4.70), 250 (4.67)
528 (4.01), 482 (4.00), 382 (4.32), 366 (4.33), 294 (4.76)
532 (4.06), 476 (4.12), 414 (4.42), 392 (4.35), 294 (5.02)
802
798
800
2.3
5.8
1.1
91
100
140
110
20
20
388
498
588
6.8
7.6
716
718
6.6
1.3
718, 800
527, 404
0.56
a
b
Measured with excitation at the longest-wavelength absorption maximum corresponding to the Ru2+ → bpy transition. Measurements at 298 K
c
were performed on argon-purged samples in MeCN; 77 K measurements were performed in air-saturated EtOH:MeOH (4:1) glasses. Air-saturated
MeCN at 298 K assuming 21% O2.
Figure 4. Normalized low-temperature emission spectra of 1−4 in 4:1
EtOH:MeOH glass at 77 K with λex = 542 (1), 532 (2), 528 (3), and
532 (4) nm.
Figure 5. Normalized room-temperature emission spectra of 4 and its
free cyclometalating ligand pbpn in MeCN and CHCl3, respectively.
(λem max = 548 nm); complexation to Ru(II) broadened this
emission slightly with a concomitant bathochromic shift of 14
nm. Free and complexed LC lifetimes were too short to
quantify reliably in our experiments (<10 ns), which also
supports the assignment as a singlet emission. Similar LC
emission occurred at 375 (λex max = 290 nm) and 523 nm
(λex max = 294 nm) for compounds 2 and 3, respectively. Green
fluorescence from π-expansive cyclometalated complexes such
as 4 could be exploited for diagnostic purposes in photo-
biological applications.
Although not emissive at room temperature, compound 4
displayed structured phosphorescence at 77 K with an onset
similar to 2 and 3 at 718 nm (Figure 4). The largest peak in the
vibronic progression of 4, however, was the sharp transition
centered at 800 nm (overlapping what looks to be the expected
v
0−1 band), making the 77 K spectrum for 4 quite distinct from
the rest of the series. Nevertheless, its low-temperature Φem of
0.011% and lifetime of 527 ns were similar to other members of
the series with Ru2+ → bpy emissive character. ΔEs could not
be determined for complex 4 because it produced no
measurable 3MLCT emission at room temperature. It is
tempting to speculate that the low-energy MLCT phosphor-
escence from 4 at 77 K may have contributions from both Ru2+
→ bpy and Ru2+ → C^N states. In fact, the lifetime measured at
800 nm was 100 ns shorter than that measured at 718 nm, and
the two emission wavelengths gave rise to excitation spectra
with some differences as well. If this is the case, then π-
expansion on the cyclometalating ligand should have the ability
to significantly impact the ensuing photophysical dynamics
given the mixed orbital parentage of the excited state(s) that
presumably dominates the trajectory. The observation that
similar scaffolds in tridentate cyclometalated Ru(II) systems
can lengthen excited state lifetimes by more than 5 orders of
magnitude relative to [Ru(tpy)2]2+ (τ = 120 ps) attests to the
profound influence that π-expansive ligands can have on
photophysical dynamics.46
3.2.3. Singlet Oxygen Sensitization. Quantum yields for
singlet oxygen production (ΦΔ) by 1−4 were measured in air-
1
saturated MeCN solution and calculated from sensitized O2
emission (centered at 1268 nm) relative to [Ru(bpy)3]2+ as the
standard, which has been reported as 56% in aerated MeCN.49
The cyclometalated compounds were poor 1O2 generators with
ΦΔ values generally less than 8% (Table 1). Compounds 1 and
2 gave the largest yields in the range of 7−8%, 3 was near 1%,
and 4 was less than 1%. These values did not change
appreciably when measured in water. By comparison, the
related Ru(II) polypyridyl complexes, such as 5, 6 and 8,
15,64,65
1
typically yield O2 with 80−90% quantum efficiencies
with [Ru(bpy)2dppz]2+ (7) known to be lower at 15−20%.64 It
is not surprising that the cyclometalated Ru(II) compounds
yield less singlet oxygen given that their excited state decay is
heavily influenced by the energy gap law. Presumably efficient
3MLCT−1MLCT nonradiative coupling afforded by low-energy
3MLCT states competes effectively with other modes of excited
Interestingly, the π-extended cylcometalated complexes
exhibited ligand-centered (LC) fluorescence at room temper-
ature; this phenomenon was not observed for 1. Although 528
1
state deactivation, namely, O2 sensitization and phosphor-
escence.
3
nm excitation of 4 produced no MLCT emission at room
3.3. Photobiological Activity. 3.3.1. Cytotoxicity and
Photocytotoxicity. Previous investigations involving cyclo-
metalated Ru(II) complexes overwhelmingly indicate that in
vitro cytotoxicity for this class is extremely high, categorizing
these compounds more accurately as potential chemo-
temperature, singlet emission from an LC state was readily
apparent at 562 nm (Figure 5) and was greatest with λex = 312
nm. The assignment was made based on the similarity of the
LC emission from 4 to the free pbpn ligand measured in CHCl3
H
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Table 2. In Vitro Cytotoxicity and Photocytotoxicity Data for Cyclometalated Ru(II) Complexes 1−4 and Reference Ru(II)
Polypyridyl Complexes 5−8 Obtained in Two Cell Lines
EC50 (μM)
SK-MEL-28
vis PDT
HL60
vis PDT
a
b
a
b
compd
dark
PI
dark
PI
1
2
3
4
5
6
7
8
1.94 0.04
1.16 0.01
1.92 0.02
>300
0.258 0.009
0.142 0.002
0.208 0.003
0.206 0.003
8.86 0.12
7.5
8.3
9.1
1.29 0.01
3.06 0.10
1.14 0.01
>300
0.151 0.001
0.167 0.003
0.211 0.004
0.741 0.016
19.52 1.32
253 11
8.6
18
5.4
>1,400
>34
>410
>15
>1.2
>1.8
>930
>300
>300
>300
237
172
7
5
>1.3
>300
>300
>1.7
>300
166
4
265
5
0.182 0.005
1,500
282 19
0.303 0.020
a
b
Vis PDT: 16 h drug-to-light interval followed by 100 J cm−1 visible-light irradiation. PI = phototherapeutic index (ratio of dark EC50 to light EC50).
therapeutics rather than photodynamic or photoactivatable
agents. For example, [Ru(bpy)(dppn)(phpy−)]+ gave an EC50
value (concentration of compound required to kill 50% of a cell
population) of 7 μM in HeLa cells in the dark,36 and the very
closely related compounds of the type [Ru(bpy)(phpy−)(LL)]+
(where LL = dpq or dppn) (dpq = dipyrido-3,2-d:2′,3′-
f]quinoxaline) were even more potent than cisplatin against all
cancer cell lines screened (<10 μM).38 For comparison, Ru(II)
polypyridyl compounds of the type [Ru(bpy)2LL]2+ (where LL
= phen (5), dpq (6), dppz (7), or dppn (8)) (dppz =
dipyrido[3,2-a:2′,3′-c]phenazine) were essentially nontoxic in
the dark (EC50 > 100 μM) under comparable conditions in the
two cells lines used in the present study (Table 2).
As might be expected, compounds 1−3, which are the
cyclometalated counterparts to 5−7, were highly toxic to cancer
cells in the dark. Their dark EC50 values were 1−2 μM in SK-
MEL-28 cells and 1−3 μM in HL-60 cells (Table 2), making
1−3 conventional anticancer agents. Notably, compound 4 was
nontoxic to cells (dark EC50 > 300 μM in both cells lines
investigated) without light activation. In fact, 4 was less
cytotoxic compared to its Ru(II) diimine counterpart 8 in both
cell lines, which defies the conventional notion that the tris
bidentate cyclometalated Ru(II) analogues are inherently more
toxic. It is interesting to note the striking difference in dark
cytotoxicity for compounds 3 and 4 (Figure 6), which differ by
only one fused benzene ring. Compound 3 was >150 times
more cytotoxic than 4 in SK-MEL-28 cells and almost 270
times more toxic in HL-60 cells. Therefore, π-expansion on at
least one coordinating ligand of cyclometalating Ru(II) systems
appears to be a requirement for suppressing dark cytotoxicity,
but more importantly, the π-expansion must be on the C^N
ligand in this limited series. The related [Ru(bpy)(phpy−)-
(dppn)]+ complex,36 with the π-extended dppn and cyclo-
metalating phpy−, is over 40× more toxic than compound 4. At
present, it is not clear why [Ru(bpy)(phpy−)(dppn)]+ is
substantially more cytotoxic. Differences in cellular uptake,
localization, relocalization, efflux, and/or metabolism must play
a role.
Photocytotoxicities of the cyclometalated Ru(II) compounds
were submicromolar with a 100 J·cm−2 visible light treatment.
Light potencies varied from 142 nM (compound 2 in SK-MEL-
28 cells) to 741 nM (compound 4 in HL-60 cells). Compounds
2 and 4 were slightly more phototoxic toward SK-MEL-28 cells,
whereas 1 and 3 showed a slight phototoxic preference for HL-
60 cells. Generally, the cytotoxicities of 1−3 were amplified less
than 10-fold in the presence of a light trigger (compound 2 in
HL-60 cells was an exception at almost 20-fold). These
phototherapeutic indices (PIs), ratios of dark EC50 values to
light EC50 values, were marginal compared to the light
enhancements that were achieved for the more π-expansive 4.
Cyclometalated 4 was almost as potent toward SK-MEL-28
cells when compared to its diimine counterpart 8 (200 vs 180
nM) and was slightly less phototoxic toward HL-60 cells (740
vs 300 nM). Thus, π-expansion on the cyclometalating ligand
can achieve similar light potencies as the π-expansive
polypyridyl cogeners that utilize highly photosensitizing, low-
lying 3IL states (<2.1 eV). Given that the photocytotoxicities of
Ru(II) polypyridyl complexes 5−7 (3IL energy > 2.1 eV) are
significantly attenuated relative to 8 (and assuming that the 3IL
energies of 5−7 are also >2.1 eV),64 one can infer that the
apparent photocytotoxicity observed for 1−3 is derived mainly
3
from inherent dark cytotoxicity rather than contributing IL
excited-state reactivity. Compounds 1−3 yielded low PIs in
both cell lines, underscoring that the contribution of light-
activation to photocytotoxicity is minimal for the less π-
expansive systems. Compound 4, on the other hand, produced
some of the largest PIs reported to date: >1,400 in SK-MEL-28
cells and >410 in HL-60 cells.11,25 It is clear that compound 4 is
a highly effective light-responsive biological agent, and low-
3
lying IL states combined with a low dark cytotoxicity may be
key. As reported previously for 8 (and attributed to the
presence of low-energy IL states), activation with mono-
chromatic red light (625 nm) also produced photocytotoxicity
Figure 6. In vitro dose−response curves for compounds 3 (right
panel) and 4 (left panel) in SK-MEL-28 (a) and HL-60 cells (b) with
(red) and without (black) visible-light activation.
15
3
I
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
but with attenuated PIs relative to activation with broad-band
visible light of the same dose.
3.3.2. Mechanistic Studies. DNA Interactions. An estab-
lished cell-free DNA photocleavage assay16,50,51 was used to
examine whether cyclometalated Ru(II) complexes could affect
the structural integrity of DNA. Figure 7 outlines the dose−
produce 100% form II DNA. Compounds 1 and 2 produced
some DNA aggregation at the highest concentrations tested,
but overall, the changes to plasmid DNA induced by 1 and 2
were less substantial than those observed for the more π-
expansive compounds 3 and 4.
When probed over the same concentration range as
compounds 1 and 2, metal complexes 3 and 4 acted as DNA
condensation agents (Figure 7, lane 5). Notably, the DNA
bands disappeared with higher concentrations of 3 or 4, which
suggests that ethidium cannot intercalate aggregated DNA
effectively. In general, nonfluorescing DNA can be attributed to
one or more of the following: (i) the DNA helix is significantly
distorted (or made inaccessible) by metal-complex binding,
preventing ethidium from binding, (ii) ethidium has been
displaced from its binding sites by the metal complex, or (iii)
the metal complex quenches ethidium fluorescence.
Experiments performed with metal complex concentrations
from 1 to 50 μM (r = 0.035−1.75) showed traces of single-
strand breaks (form II DNA) in parallel with mostly DNA
aggregation/precipitation (Figures S13 and S14). For 3, three
forms of DNA were present when pUC19 plasmid was dosed
with 18−27 μM metal complex: undamaged form I, nicked
form II, and aggregates (form IV). At 30 μM MC, only form IV
was observed on the gel, and higher concentrations of metal
complex resulted in no fluorescence from ethidium bromide.
The formation of DNA aggregates was confirmed by phase-
contrast light microscopy (Figure S15), and precipitation was
also visible by eye. Thus, we infer that DNA aggregation makes
the DNA binding sites for ethidium inaccessible, resulting in no
fluorescent bands on the gel. Compound 4 facilitated DNA
aggregation/precipitation (Figure S15) at r values as low as
0.45, producing almost exclusively form IV DNA at metal
complex concentrations greater than 20 μM (r = 0.70) with
bands becoming invisible above 30 μM (r = 1.05). The
disappearance of fluorescent bands with increasing concen-
trations of 3 or 4 alongside DNA aggregation/precipitation also
occurred in the absence of a light trigger as demonstrated by
lane 11 (Figure 7). It is possible that π-expansive cyclo-
metalated complexes with fused systems of phenazine and
longer interact with DNA through a templating effect that
drives DNA aggregation followed by precipitation. It is known
that chemical agents can cause DNA condensation by
modifying electrostatic interactions between DNA segments,
by modifying DNA-solvent interactions, by causing volume
reductions of the helix, by causing local bending or distortion of
the helical structure, or by combinations of these effects.66 In
the present series, the ability of the metal compound to
facilitate DNA aggregation directly correlates with DNA
intercalating power (where pbpn is predicted to be the best
intercalating ligand because it has the largest π surface area),
which in turn reflects the relative abilities of the compounds to
act as DNA unwinders. Any further speculation on a definitive
mechanism would require a detailed analysis of the DNA-metal
complex structures, particularly with regard to whether they are
of finite size and orderly morphology (true DNA condensation)
or bulk aggregates of random size and shape.
Figure 7. DNA photocleavage of pUC19 DNA (28.6 μM bases) dosed
with metal complex (MC) 1 (a), 2 (b), 3 (c), or 4 (d) and visible light
(14 J cm−2). Gel mobility shift assays employed 1% agarose gels (0.75
μg mL−1 ethidium bromide) electrophoresed in 1× TAE at 8 V cm−1
for 30 min. Lane 1, DNA only (−hν); lane 2, DNA only (+hν); lane 3,
5 μM MC (+hν); lane 4, 20 μM MC (+hν); lane 5, 40 μM MC (+hν);
lane 6, 60 μM MC (+hν); lane 7, 80 μM MC (+hν); lane 8, 100 μM
MC (+hν); lane 9, 200 μM MC (+hν); lane 10, 300 μM MC (+hν);
lane 11, 300 μM MC (−hν).
response profiles of 1−4 for damaging pUC19 plasmid DNA
when activated with visible light. Lanes 1 and 2 are controls and
demarcate mostly undamaged, supercoiled plasmid (form I).
Lane 11 is also a control lane and represents the effect of the
highest concentration of metal complex tested but without a
light trigger. Two features were readily apparent. First, the
cyclometalated compounds interfered with ethidium bromide
staining in the order 4 > 3 > 1 ≈ 2, and for 3 and 4, this
interference occurred at high concentration even without a light
treatment. Second, the Ru(II) cyclometalated compounds
caused DNA aggregation/precipitation at the loading well
(with virtually no migration) in the same order. These factors
precluded an assessment of the DNA photocleaving capacities
of 3 and 4 but did point toward very strong DNA interactions
that significantly altered the topological form of DNA.
Compounds 1 and 2 induced single-strand breaks in DNA
when activated with visible light, which resulted in the slower
migrating, nicked circular DNA (form II). Compound 2 was
the more potent DNA-photodamaging agent but neither was
considered particularly potent given that concentrations as high
as 300 μM (drug-to-nucleotide ratio (r) = 10.5) did not
We infer that the DNA aggregation/precipitation discerned
for 4 by gel electrophoretic analysis does not account for the
stark difference in dark and light cytotoxicity (PIs > 1,400 for 4
in some cases) measured for this π-expansive cyclometalated
complex. Likewise, the ability to cause DNA aggregation/
precipitation in the absence of light activation is not directly
linked to dark cytotoxicity because the gel electrophoretic
J
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
patterns for 3 and 4 are strikingly similar, yet 3 is cytotoxic in
the dark (EC50 = 1−2 μM) and 4 is not (EC50 > 300 μM).
Others have demonstrated that increased hydrophobicity
(positive log Po/w values) leads to increased cellular uptake,
which in turn enhances cytotoxicity.38 Following this logic, 4
should be the most lipophilic and therefore the most cytotoxic
in the present series. However, the increased lipophilicity of the
pbpn cyclometalating ligand could also facilitate the formation
of extracellular π-stacked metal-complex assemblies with
reduced cell penetration and dark cytotoxicity. The light trigger
likely makes the cell membrane more permeable (known as
photoactivated uptake67,68) while simultaneously disrupting π-
stacking interactions (through the formation of more highly
polarized excited states). Factors other than cellular uptake
(e.g., efflux, localization, redistribution, and metabolism) may
also contribute to the dark cytotoxicity profiles for the Ru(II)
cyclometalated complexes, and we are currently investigating a
larger library of compounds to better understand this
phenomenon.
obtained when HL-60 cells were treated with 4 under various
conditions (SK-MEL-28 results were similar). No fluorescent
product was detected from cells that were exposed to 4 in the
dark. Strikingly, ethidium emission was evident from all cells
that were dosed with 4 followed by a visible (Figure 8e) or red
(Figure 8f) light treatment. The intensity of red emission
detected from visible and red light-treated cells correlated
directly with the relative magnitudes of phototoxicity elicited by
these two different light conditions (red light gave less
•−
potency). Consequently, O2 may be a mediator of photo-
cytotoxicity in addition to other mechanistic pathways that are
influenced by cell uptake, efflux, localization, and relocalization.
Nevertheless, these results indicate that π-expansive cyclo-
metalating compounds such as 4 may act as powerful
photoreductants in cells.
3.3.3. Diagnostic Imaging. To probe whether 1LC emission
could serve as a diagnostic tool, compound 4 was incubated
with SK-MEL-28 or HL-60 cells for 30 min prior to a dark or
light treatment. Representative data taken 15 min post-
treatment is shown for melanoma cells in Figure 9, where
ROS Detection. Plasmid DNA (in particular, changes to its
gel mobility) serves as a convenient probe for detecting
whether compounds can photodamage biological macro-
molecules in general, often via the production of ROS and
other reactive intermediates that convert form I plasmid DNA
to forms II or III. However, strong interactions between the
more π-expansive cyclometalated complexes and DNA (vide
supra) prevented a detailed analysis of any cleavage products.
Therefore, an in vitro fluorescence-based assay for ROS
detection was employed to scrutinize further the photactivity
of 4.
1
Given that O2 production by the cyclometalated complexes
was quite low (ΦΔ = 0.56% for 4), other reactive intermediates
are likely responsible for the photocytotoxicity of this
nondissociative cyclometalated system. In fact, the increased
electron density on the metal in phpy− complexes has been
implicated in the cathodic shift of the RuIII/RuII potential38 and
could lead to direct photoreduction of biological macro-
molecules as well as dioxygen. To test the latter, we used the
dihydroethidium (DHE) fluorescence assay for superoxide
(O2•−).69
Figure 9. Comparison of the green fluorescence emitted from SK-
MEL-28 cells dosed with 4 in the dark (a) or with a visible (b) or red
(c) light treatment (50 J cm−2). The scale bar corresponds to 10 μm.
Briefly, cells were dosed with 4 (10 μM), washed, and then
incubated with DHE (3 μM) for 15 min. The production of
•−
O2 was inferred from the oxidation of the nonfluorescent
DHE to ethidium, which emits red fluorescence (λmax = 610
nm) with blue-green excitation. Figure 8 shows the results
green emission is clearly visible from cells treated with the π-
expansive cyclometalated compound under all three conditions
(left panel, fluorescence). No red phosphorescence could be
discerned, which is in agreement with the lack of red emission
in steady-state luminescence measurements at room temper-
ature.
Differential interference contrast (DIC) microscopy (center
panel) was used to highlight cellular shape and morphology to
ascertain the subcellular distribution of the photosensitizer
before and after a light treatment. With no light treatment,
compound 4 appeared to localize in the nucleus of SK-MEL-28
cells (Figure 9a). With observation at sublethal time points
after a visible or red light dose (Figure 9b, c), 4 relocalized to
the cytoplasm concomitant with the gross changes in cellular
morphology that accompany cell death pathways. These
changes can be seen best by comparing the overlays of
fluorescence and DIC images; fluorescence indicates where the
compound is located within the DIC-imaged cell. The green
signal was brightest in cell populations of the lowest viability
(i.e., in those that received the most potent light treatment).
Figure 8. DIC (a−c) and fluorescence images (d−f) of HL-60 cells
treated with 4 in the dark (left panel) or with a visible (middle panel)
or red (right panel) light treatment (50 J cm−2). Fluorescence
emission was collected through a 510 nm long-pass filter. The scale bar
corresponds to 10 μm.
K
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
The polypyridyl cousin of 4, 8, did not produce any
ACKNOWLEDGMENTS
■
detectable green or red luminescence in cells, underscoring the
We acknowledge financial support from the Natural Sciences
and Engineering Council of Canada, the Canadian Foundation
for Innovation, the Nova Scotia Research and Innovation Trust,
and Acadia University.
1
importance of cyclometalation in providing an accessible LC
state for cellular imaging. Despite the fact that luminescence
directly competes with photosensitization (and any other light-
mediated cytotoxicity pathways) for excited-state deactivation,
this radiative channel observed for 4 (and not 8) did not
compromise the photocytotoxicity for 4 in comparison to 8.
Both gave submicromolar light EC50 values in both cell lines
investigated. Therefore, one advantage afforded by the
cyclometalating scaffold is the ability to turn potent photo-
sensitizers into diagnostic agents.
REFERENCES
■
(1) Bomben, P.; Robson, K.; Koivisto, B.; Berlinguette, C. Coord.
Chem. Rev. 2012, 256, 1438−1450.
(2) Alstrum-Acevedo, J. H.; Brennaman, M. K.; Meyer, T. J. Inorg.
Chem. 2005, 44, 6802−6827.
(3) Wu, A.; Yoo, D.; Lee, J.-K.; Rubner, M. F. J. Am. Chem. Soc. 1999,
121, 4883−4891.
4. SUMMARY AND CONCLUSIONS
(4) McFarland, S. A.; Magde, D.; Finney, N. S. Inorg. Chem. 2005, 44,
4066−4076.
Four organometallic Ru(II) compounds that systematically
increase π-conjugation on the cyclometalating ligand were
prepared and fully characterized. To our knowledge, this is the
first investigation of tris-bidentate systems where π-expansion
was probed on the cyclometalating framework rather than the
diimine ligands. In agreement with previous reports of Ru(II)
organometallics derived from non-π-expansive cyclometalating
ligands, 1−3 did not act as potent light-responsive agents.
Rather, they were extremely cytotoxic toward cancer cells in the
dark, and light activation did not appreciably amplify this
toxicity. Compound 4, on the other hand, was completely
nontoxic to cells in the dark, but was extremely phototoxic to
cancer cells when activated with a moderate light treatment.
This photoactivity was as potent as that previously reported for
the related π-expansive Ru(II) polypyridyl system.
Despite the fact that the energy gap law might be invoked to
explain red-shifted absorption, a lack of room-temperature
phosphorescence, and low singlet oxygen quantum yields from
cyclometalated Ru(II) complexes in this study, it does not
compromise the photodynamic activity of 4 (which is
comparable to 8). In addition to excellent photocytotoxicity,
compound 4 displayed intense green intracellular fluorescence
that was not produced by the analogous Ru(II) polypyridyl
complex 8. This orthogonal functionality as a diagnostic tool
makes cyclometalated 4 an attractive starting point for the
investigation of whether triplet intraligand (3IL) excited states
play a role in these cyclometalated systems that act as
theranostic agents.
(5) Lakowicz, J. R.; Malak, H.; Gryczynski, I.; Castellano, F. N.;
Meyer, G. J. Biospectroscopy 1995, 1, 163−168.
(6) McClure, B. A.; Rack, J. J. Angew. Chem., Int. Ed. 2009, 48, 8556−
8558.
(7) Karkas, M. D.; Verho, O.; Johnston, E. V.; Åkermark, B. Chem.
̈
̈
Rev. 2014, 114, 11863−12001.
(8) White, T. A.; Whitaker, B. N.; Brewer, K. J. J. Am. Chem. Soc.
2011, 133, 15332−15334.
(9) Islangulov, R. R.; Kozlov, D. V.; Castellano, F. N. Chem. Commun.
2005, 3776−3778.
(10) Juris, A.; Balzani, V.; Barigelletti, F.; Campagna, S.; Belser, P.;
von Zelewsky, A. Coord. Chem. Rev. 1988, 84, 85−277.
(11) Lincoln, R.; Kohler, L.; Monro, S.; Yin, H.; Stephenson, M.;
Zong, R.; Chouai, A.; Dorsey, C.; Hennigar, R.; Thummel, R. P.;
McFarland, S. A. J. Am. Chem. Soc. 2013, 135, 17161−17175.
(12) Arenas, Y.; Monro, S.; Shi, G.; Mandel, A.; McFarland, S.; Lilge,
L. Photodiagn. Photodyn. Ther. 2013, 10, 615−625.
(13) Shi, G.; Monro, S.; Hennigar, R.; Colpitts, J.; Fong, J.; Kasimova,
K.; Yin, H.; DeCoste, R.; Spencer, C.; Chamberlain, L.; Mandel, A.;
Lilge, L.; McFarland, S. A. Coord. Chem. Rev. 2015, 282−283, 127−
138.
(14) Stephenson, M.; Reichardt, C.; Pinto, M.; Wachtler, M.;
̈
Sainuddin, T.; Shi, G.; Yin, H.; Monro, S.; Sampson, E.; Dietzek, B.;
McFarland, S. A. J. Phys. Chem. A 2014, 118, 10507−10521.
(15) Yin, H.; Stephenson, M.; Gibson, J.; Sampson, E.; Shi, G.;
Sainuddin, T.; Monro, S.; McFarland, S. A. Inorg. Chem. 2014, 53,
4548−4559.
(16) Monro, S.; Scott, J.; Chouai, A.; Lincoln, R.; Zong, R.;
Thummel, R. P.; McFarland, S. A. Inorg. Chem. 2010, 49, 2889−2900.
(17) Holder, A. A.; Zigler, D. F.; Tarrago-Trani, M. T.; Storrie, B.;
Brewer, K. J. Inorg. Chem. 2007, 46, 4760−4762.
(18) Holder, A. A.; Swavey, S.; Brewer, K. J. Inorg. Chem. 2004, 43,
303−308.
(19) Swavey, S.; Brewer, K. J. Inorg. Chem. 2002, 41, 6196−6198.
(20) Higgins, S. L. H.; Brewer, K. J. Angew. Chem., Int. Ed. 2012, 51,
11420−11422.
(21) Higgins, S. L. H.; Tucker, A. J.; Winkel, B. S. J.; Brewer, K. J.
Chem. Commun. 2012, 48, 67−69.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.5b01838.
1D 1H and 2D 1H−1H COSY NMR spectra for
cyclometalating ligands and their corresponding Ru(II)
complexes, 13C NMR spectra for the metal complexes,
narrow-range gel electrophoretic data, and microscopy
images of DNA aggregates (PDF)
(22) Mari, C.; Gasser, G. Chimia 2015, 69, 176−181.
(23) Mari, C.; Pierroz, V.; Ferrari, S.; Gasser, G. Chem. Sci. 2015, 6,
2660−2686.
(24) Glazer, E. C. Isr. J. Chem. 2013, 53, 391−400.
(25) Howerton, B. S.; Heidary, D. K.; Glazer, E. C. J. Am. Chem. Soc.
2012, 134, 8324−8327.
(26) Wachter, E.; Howerton, B. S.; Hall, E. C.; Parkin, S.; Glazer, E.
C. Chem. Commun. 2014, 50, 311−313.
AUTHOR INFORMATION
■
(27) Wachter, E.; Heidary, D. K.; Howerton, B. S.; Parkin, S.; Glazer,
E. C. Chem. Commun. 2012, 48, 9649−9651.
Corresponding Author
(28) Knoll, J. D.; Turro, C. Coord. Chem. Rev. 2015, 282−283, 110−
126.
*E-mail: sherri.mcfarland@acadiau.ca.
Notes
(29) Sgambellone, M. A.; David, A.; Garner, R. N.; Dunbar, K. R.;
The authors declare no competing financial interest.
Turro, C. J. Am. Chem. Soc. 2013, 135, 11274−11282.
L
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(30) Sears, R. B.; Joyce, L. E.; Ojaimi, M.; Gallucci, J. C.; Thummel,
R. P.; Turro, C. J. Inorg. Biochem. 2013, 121, 77−87.
(31) Ertl, C. D.; Ris, D. P.; Meier, S. C.; Constable, E. C.; Housecroft,
C. E.; Neuburger, M.; Zampese, J. A. Dalton Trans.. 2015, 44, 1557−
1570.
(62) Hissler, M.; Connick, W. B.; Geiger, D. K.; McGarrah, J. E.;
Lipa, D.; Lachicotte, R. J.; Eisenberg, R. Inorg. Chem. 2000, 39, 447−
457.
(63) Goze, C.; Kozlov, D. V.; Tyson, D. S.; Ziessel, R.; Castellano, F.
N. New J. Chem. 2003, 27, 1679−1683.
(64) Sun, Y.; Joyce, L. E.; Dickson, N. M.; Turro, C. Chem. Commun.
2010, 46, 2426−2428.
(32) Allen, F. H. Acta Crystallogr., Sect. B: Struct. Sci. 2002, 58, 380−
388.
(65) Foxon, S. P.; Alamiry, M. A. H.; Walker, M. G.; Meijer, A. J. H.
M.; Sazanovich, I. V.; Weinstein, J. A.; Thomas, J. A. J. Phys. Chem. A
2009, 113, 12754−12762.
(33) Muro-Small, M. L.; Yarnell, J. E.; McCusker, C. E.; Castellano,
F. N. Eur. J. Inorg. Chem. 2012, 2012, 4004−4011.
(34) Albani, B. A.; Pena, B.; Dunbar, K. R.; Turro, C. Photochem.
̃
(66) Bloomfield, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334−341.
Photobiol. Sci. 2014, 13, 272−280.
(67) Svensson, F. R.; Abrahamsson, M.; Stromberg, N.; Ewing, A. G.;
̈
(35) Bessho, T.; Yoneda, E.; Yum, J.-H.; Guglielmi, M.; Tavernelli, I.;
Lincoln, P. J. Phys. Chem. Lett. 2011, 2, 397−401.
(68) Svensson, F. R.; Matson, M.; Li, M.; Lincoln, P. Biophys. Chem.
2010, 149, 102−106.
Imai, H.; Rothlisberger, U.; Nazeeruddin, M. K.; Gratzel, M. J. Am.
̈
Chem. Soc. 2009, 131, 5930−5934.
(36) Pena, B.; David, A.; Pavani, C.; Baptista, M. S.; Pellois, J.-P.;
Turro, C.; Dunbar, K. R. Organometallics 2014, 33, 1100−1103.
(37) Palmer, A. M.; Pena, B.; Sears, R. B.; Chen, O.; El Ojaimi, M.;
Thummel, R. P.; Dunbar, K. R.; Turro, C. Philos. Trans. R. Soc., A
2013, 371, 20120135.
̃
(69) Gomes, A.; Fernandes, E.; Lima, J. L. F. C. J. Biochem. Biophys.
Methods 2005, 65, 45−80.
̃
(38) Huang, H.; Zhang, P.; Chen, H.; Ji, L.; Chao, H. Chem. - Eur. J.
2015, 21, 715−725.
(39) Sullivan, B. P.; Salmon, D. J.; Meyer, T. J. Inorg. Chem. 1978, 17,
3334−3341.
(40) Sun, Y.; Joyce, L. E.; Dickson, N. M.; Turro, C. Chem. Commun.
2010, 46, 6759−6761.
(41) Schatzschneider, U.; Niesel, J.; Ott, I.; Gust, R.; Alborzinia, H.;
Wolfl, S. ChemMedChem 2008, 3, 1104−1109.
̈
(42) Friedman, A. E.; Chambron, J. C.; Sauvage, J. P.; Turro, N. J.;
Barton, J. K. J. Am. Chem. Soc. 1990, 112, 4960−4962.
(43) Zhou, Q.-X.; Lei, W.-H.; Chen, J.-R.; Li, C.; Hou, Y.-J.; Wang,
X.-S.; Zhang, B.-W. Chem. - Eur. J. 2010, 16, 3157−3165.
(44) Kosuge, T.; Senoo, A.; Ohrui, H.; Muratsubaki, M. Azafluorene
Derivative and Organic Light-Emitting Device Using the Derivative.
US Patent 8,129,899 B1, March 6, 2012.
(45) Delgadillo, A.; Romo, P.; Leiva, A. M.; Loeb, B. Helv. Chim. Acta
2003, 86, 2110−2120.
(46) Sun, Y.; El Ojaimi, M.; Hammitt, R.; Thummel, R. P.; Turro, C.
J. Phys. Chem. B 2010, 114, 14664−14670.
(47) Koizumi, T.; Tomon, T.; Tanaka, K. J. Organomet. Chem. 2005,
690, 1258−1264.
(48) Foxon, S. P.; Metcalfe, C.; Adams, H.; Webb, M.; Thomas, J. A.
Inorg. Chem. 2007, 46, 409−416.
(49) DeRosa, M. C.; Crutchley, R. J. Coord. Chem. Rev. 2002, 233/
234, 351−371.
(50) Croke, D. T.; Perrouault, L.; Sari, M. A.; Battioni, J.-P.; Mansuy,
D.; Helene, C.; Le Doan, J. J. Photochem. Photobiol., B 1993, 18, 41−
50.
(51) Praseuth, D.; Gaudemer, A.; Verlhac, J.-B.; Kraljic, I.; Sissoeff, I.;
̈
Guille,
́
E. Photochem. Photobiol. 1986, 44, 717−724.
(52) Robson, K. C. D.; Koivisto, B. D.; Yella, A.; Sporinova, B.;
Nazeeruddin, M. K.; Baumgartner, T.; Gratzel, M.; Berlinguette, C. P.
̈
Inorg. Chem. 2011, 50, 5494−5508.
(53) Braven, J.; Hanson, R. W.; Smith, N. G. J. Heterocycl. Chem.
1995, 32, 1051−1055.
(54) Saeki, K.; Tomomitsu, M.; Kawazoe, Y.; Momota, K.; Kimoto,
H. Chem. Pharm. Bull. 1996, 44, 2254−2258.
(55) Zelonka, R. A.; Baird, M. C. Can. J. Chem. 1972, 50, 3063−3072.
(56) Bomben, P. G.; Gordon, T. J.; Schott, E.; Berlinguette, C. P.
Angew. Chem., Int. Ed. 2011, 50, 10682−10685.
(57) Fernandez, S.; Pfeffer, M.; Ritleng, V.; Sirlin, C. Organometallics
1999, 18, 2390−2394.
(58) Constable, E. C.; Holmes, J. M. J. Organomet. Chem. 1986, 301,
203−208.
(59) Reveco, P.; Medley, J. H.; Garber, A. R.; Bhacca, N. S.; Selbin, J.
Inorg. Chem. 1985, 24, 1096−1099.
(60) Sasaki, I.; Vendier, L.; Sournia-Saquet, A.; Lacroix, P. G. Eur. J.
Inorg. Chem. 2006, 2006, 3294−3302.
(61) Bomben, P. G.; Robson, K. C. D.; Sedach, P. A.; Berlinguette, C.
P. Inorg. Chem. 2009, 48, 9631−9643.
M
DOI: 10.1021/acs.inorgchem.5b01838
Inorg. Chem. XXXX, XXX, XXX−XXX