Synthesis and Characterization of Ru(II) Complexes with π-Expansive Imidazophen Ligands for the Photokilling of Human Melanoma Cells

Article abstract of DOI:10.1111/php.13177

Ru(II) complexes were synthesized with π-expanding (phenyl, fluorenyl, phenanthrenyl, naphthalen-1-yl, naphthalene-2-yl, anthryl and pyrenyl groups) attached at a 1H-imidazo[4,5-f][1,10]phenanthroline ligand and 4,4′-dimethyl-2,2′-bipyridine (4,4′-dmb) coligands. These Ru(II) complexes were characterized by 1D and 2D NMR, and mass spectroscopy, and studied for visible light and dark toxicity to human malignant melanoma SK-MEL-28 cells. In the SK-MEL-28 cells, the Ru(II) complexes are highly phototoxic (EC₅₀?=?0.2–0.5?μm) and have low dark toxicity (EC₅₀?=?58–230?μm). The highest phototherapeutic index (PI) of the series was found with the Ru(II) complex bearing the 2-(pyren-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline ligand. This high PI is in part attributed to the π-rich character added by the pyrenyl group, and a possible low-lying and longer-lived ³IL state due to equilibration with the ³MLCT state. While this pyrenyl Ru(II) complex possessed a relatively high quantum yield for singlet oxygen formation (Φ_??=?0.84), contributions from type-I processes (oxygen radicals and radical ions) are competitive with the type-II (¹O₂) process based on effects of added sodium azide and solvent deuteration.

Full text of DOI:10.1111/php.13177

DR. ALEXANDER GREER (Orcid ID : 0000-0003-4444-9099)
DR. S. MCFARLAND (Orcid ID : 0000-0002-8028-5055)
Article type
: Special Issue Research Article
Synthesis and Characterization of Ru(II) Complexes with π-Expansive
Imidazophen Ligands for the Photokilling of Human Melanoma Cells†
Goutam Ghosh1,3, Huimin Yin1, Susan M. A. Monro,1 Tariq Sainuddin1, Lloyd Lapoot3,4,
Alexander Greer3,4*, and Sherri A. McFarland1,2,*
1Department of Chemistry, Acadia University, Wolfville, Nova Scotia B4P 2R6, Canada
2Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro,
Greensboro, North Carolina 27402, United States
3Department of Chemistry, Brooklyn College of the City University of New York, Brooklyn, New
York 11210, United States
4Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, 365 Fifth
Avenue, New York, New York 10016, United States
*Corresponding authors’ e-mails: samcfarl@uncg.edu (Sherri A. McFarland);
agreer@brooklyn.cuny.edu (Alexander Greer)
†This article is part of a Special Issue dedicated to Dr. Jarod Finlay.
This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: 10.1111/PHP.13177
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ABSTRACT
Ru(II) complexes were synthesized with π-expanding (phenyl, fluorenyl, phenanthrenyl,
naphthalen-1-yl, naphthalene-2-yl, anthryl, and pyrenyl groups) attached at a 1H-imidazo[4,5-
f][1,10]phenanthroline ligand and 4,4′-dimethyl-2,2′-bipyridine (4,4′-dmb) co-ligands. These Ru(II)
complexes were characterized by 1D and 2D NMR, and mass spectroscopy, and studied for visible-
light and dark toxicity to human malignant melanoma SK-MEL-28 cells. In the SK-MEL-28 cells, the
Ru(II) complexes are highly phototoxic (EC50 = 0.2-0.5 µM), and have low dark toxicity (EC50 = 58-
230 µM). The highest phototherapeutic index (PI) of the series was found with the Ru(II) complex
bearing the 2-(pyren-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline ligand. This high PI is in part
attributed to the π-rich character added by the pyrenyl group, and a possible low-lying and longer-
3
3
lived IL state due to equilibration with the MLCT state. While this pyrenyl Ru(II) complex
possessed a relatively high quantum yield for singlet oxygen formation (∆ = 0.84), contributions
from type-I processes (oxygen radicals and radical ions) are competitive with the type-II (1O2) process
based on effects of added sodium azide and solvent deuteration.
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INTRODUCTION
A number of contributions have been made to understanding metal-organic dyad sensitizers in
photodynamic therapy (PDT) and synergy in cancer cell killing (1-4), where ligand substitution on the
metal serves to increase light absorption and triplet lifetime (5). In systems based on Ru(II) as well as
other metals (6,7), excited states with intraligand charge transfer (IL) character have been shown to
increase the triplet lifetime (5). Whether these photosensitizers involve predominantly a type II
1
reaction producing O2 as the key cytotoxic agent, or whether there is competition with the type I
reaction (oxygen radicals or radical ions) (8,9) has not been fully examined. It is plausible that both
pathways contribute to the observed photocytotoxic effects due to the various types of excited states
that are accessible in metal complexes (Figure 1).
[Figure 1 here]
Depending on the natue of the metal complex, some of these excited state configurations
include: metal-to-ligand charge transfer (MLCT), metal centered (MC), metal-to-metal charge
transfer (MMCT), in the case of multimetallic complexes, IL, intraligand charge transfer (ILCT), and
ligand-to-ligand charge transfer (LLCT). We previously found that the ligand centered (3IL) state (10)
in the π-expansive organic unit extended the triplet lifetime 10-fold due to an equilibrium with
3MLCT state (11). When the energy of the 3IL state is sufficiently low compared to 3MLCT state, its
interaction with the 3MLCT state becomes almost negligible, thereby forming a pure 3IL state with an
even longer lifetime. For example, a triplet lifetime of >100 µs was found by Kozlov et al. when
pyrenylethynylene-conjugated bipyridine was incorporated in a Ru complex (12). In a similar vein,
Ru(II) complexes that incorporated pyrenylethynylene conjugated to phenanthroline into the dyad
were reported to extend the lifetime of the triplet state to 240 µs, which may be the longest lifetime
reported to date for Ru complexes (13). Previous reports on Ru(II) dyads derived from 2,2′-bipyridine
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(bpy) and 4,4′-dimethyl-2,2′-bipyridine (4,4′-dmb) showed high phototoxicity with low dark toxicity
(14,15), making these metal-organic dyads attractive photosensitizers for PDT.
In the present study, we explore complexes of the type [Ru(4,4′-dmb)2(ip-R)]2+, where
imidazo[4,5-f][1,10]phenanthroline (ip) is fused to an aromatic R group. We hypothesized that
3
increasing the π-conjugation in the R group will systematically lower the energy of the IL state
3
relative to the MLCT state and thus improve phototoxic effects toward human melanoma SK-MEL-
28 cells, particularly when the 3IL energy drops below that of the 3MLCT. The R groups investigated
were: 2-phenyl (1), 2-(9H-fluoren-2-yl) (2), 2-(phenanthren-9-yl) (3), 2-(naphthalen-1-yl) (4), 2-
(naphthalene-2-yl) (5), 2-(anthracene-10-yl) (6), and 2-(pyren-1-yl) (7) along with ruthenium 4,4-
dimethyl-2,2-bipyridine (4,4′-dmb) co-ligands (Figure 2). We determined whether complexes 1-7: (i)
could be synthesized in high yield, (ii) would lead to high phototherapeutic indicies (PI) in malignant
melanoma SK-MEL-28 cells, (iii) would have high singlet oxygen quantum yields (∆), and (iv)
would show evidence in both type I and type II sensitized oxidation mechanisms. Our results have
identified 7, with pyrenyl as the R group, as a potentially useful photosensitizer against melanoma
cells.
[Figure 2 here]
MATERIALS AND METHODS
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Materials. 1,10-Phenanthroline-5,6-dione, NH4OAC, glacial acetic acid, benzaldehyde, 9H-
fluorene-2-carbaldehyde, phenanthrene-9-carbaldehyde, 1-napthaldehyde, 2-napthaldehyde, 9-
anthraldehyde, pyrene-1-carbaldehyde, 9H-fluorene-2-carbaldehyde, phenanthrene-9-carbaldehyde,
4,4̕-dimethyl-2,2̕-bipyridine (4,4̕-dmb), potassium nitrate (KNO3), acetonitrile (MeCN), potassium
hexafluoro phosphate (KPF6), dichloromethane (DCM), methanol (MeOH), and deuterated solvents
(DMSO-d6 and CD3CN) were purchased from Sigma-Aldrich and used without further purification.
Fetal bovine serum (FBS), RPMI 1640 (Corning Cellgro), and Eagle’s Minimum Essential Medium
(EMEM) (Corning Cellgro) were purchased from VWR. Human malignant melanoma cells (SK-
MEL-28) were obtained from the American Type Culture Collection (ATCC). Prior to use, FBS was
divided into 40 mL aliquots, heat inactivated (30 min, 55 °C), and then stored at −20 °C. Deionized
water of 18 MΩ cm resistivity from a Barnstead filtration system was used for the biological
experiments.
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 MHz spectrometers (Acadia Centre for Microstructural
Analysis), and ESI mass spectra were obtained using a Bruker microTOF focus mass spectrometer
(Dalhousie University Mass Spectrometry Laboratory). HPLC analyses were performed with an
Agilent 1100 series instrument (ChemStation Rev. A 10.02 software) using a Hypersil GOLD C18
reverse phase column with an A-B gradient (98% → 5% A; A = 0.1% formic acid in H2O, B = 0.1%
formic acid in MeOH).
Synthesis. The ligands 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (bip), 2-(naphthalen-
1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline
f][1,10]phenanthroline (2-nip), 2-(9H-fluoren-2-yl)-1H-imidazo [4,5-f][1,10] phenanthroline (fip), 2-
(pyren-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (ippy), and 2-(anthracene-10-yl)-1H-
imidazo[4,5-f][1,10]phenanthroline (aip) were synthesized according to literature procedures (31-36).
(1-nip),
2-(naphthalen-2-yl)-1H-imidazo[4,5-
Compounds [Ru(4,4′-dmb)2Cl2]∙2H2O (37), Ru(4,4-dmb)2(bip)](PF6)2
1
(38), Ru(4,4-
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dmb)2(ippy)](PF6)2 7 (22), and 9,10-anthracene dipropionate dianion 8 (28,29) were synthesized as
reported in the literature. For our in vitro study, Ru(II) complexes 1-7 were converted to their
corresponding Cl– salts.
Synthesis of 2-(phenanthren-9-yl)-1H-imidazo [4,5-f][1,10] phenanthroline (pcip). Yield 247
mg (78%): purity >80%. A mixture of phen-dione (166.6 mg, 0.8 mmol), phenanthrene-9-
carbaldehyde (165 mg, 0.8 mmol), NH4OAC (1.23 g, 16 mmol), and glacial acetic acid (4 mL) was
subjected to microwave irradiation for 15 min at 180 °C. The resulting solution was diluted with
water in a beaker, and ammonium solution was added dropwise until the pH of the solution reached 8.
A yellow precipitate was formed, which was filtered through a fine glass-sintered frit. Single spot in
1
TLC indicated pure product was formed. H NMR (300 MHz, DMSO-d6) δ 9.08 (d, J = 5.0 Hz, 2H;
a,d), 8.99 (m, 4H; c,f,o,l), 8.48 (s, 1H; g), 8.19 (d, J = 7.7 Hz, 1H; k), 7.92–7.73 (m, 6H; b,e,n,m,i,j).
Synthesis of [Ru(4,4-dmb)2(fip)](PF6)2 2. Yield 55 mg (48%): purity 98%. In a microwave
vessel, 63.4 mg (0.11 mmol) of [Ru(4,4′-dmb)2Cl2]∙2H2O was mixed with 38.4 mg (0.10 mmol) of
FIP. Ethylene glycol (3.0 mL) was added to the vessel. The mixture was microwaved for 15 min at
180 °C. The resulting dark red color solution was directly loaded on to a silica column and eluted
using 100% MeCN followed by 5% H2O in MeCN to remove the excess starting material and other
impurities. Finally, Ru(II) complex 2 was eluted as an orange band with 5–10% H2O and 0.5–2.5%
–
KNO3 in MeCN to give an orange solid as a mixture of Cl– and NO3 salts. This mixture was
–
converted to the PF6 salt by adding 5–10 mL of water and 2–4 mL of saturated KPF6. Evaporation of
–
DCM under reduced pressure gave the pure PF6 salt as an orange solid: Rf = 0.51 (10% H2O + 2.5%
KNO3 in MeCN). HPLC retention time: 25.6 min. 1H NMR (300 MHz, acetonitrile-d3) δ 9.00 (s, 2H;
f, c), 8.52 (s, 1H; g), 8.40 (s, 2H; 3’), 8.36 (s, 2H; 3), 8.32 (s, 1H, n), 8.09 (d, J = 7.9 Hz, 1H; m), 8.04
(d, J = 5.2 Hz, 2H; a,d), 7.97 (d, J = 6.7 Hz, 1H; l), 7.79 (dd, J = 8.3, 5.2 Hz, 2H; b,e), 7.67 (m, 3H;
6’, i), 7.51 – 7.38 (m, 4H; 6, k, j), 7.30 (d, J = 5.8 Hz, 2H; 5’), 7.06 (d, J = 7.1 Hz, 2H; 5), 4.13 (s, 2H;
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h), 2.59 (s, 6H, 4’-Me), 2.48 (s, 6H, 4-Me). MS (ESI+) m/z: 999.13 [M-PF6]+, 853.47 [M-PF6-H]+,
427.40 [M2PF6]2+. HRMS (ESI+) m/z for C50H40N8Ru: calcd 427.1209, found 427.1204.
Synthesis of [Ru(4,4-dmb)2(pcip)](PF6)2 3. Yield 38 mg (33%): purity 98%. In a microwave
vessel, 63.4 mg (0.11 mmol) of [Ru (4,4′-dmb)2Cl2]∙2H2O was mixed with 39.6 mg (0.10 mmol) of
PCIP. Ethylene glycol (3.0 mL) was added to the vessel. The mixture was microwaved at 180 °C for
15 min. The resulting dark red color solution was directly loaded on to a silica column and eluted
using 100% MeCN followed by 5% H2O in MeCN to remove the excess starting material and other
impurities. Finally, Ru(II) complex 3 was eluted as an orange band with 5–10% H2O and 0.5–2.5%
–
KNO3 in MeCN to give an orange solid as a mixture of Cl– and NO3 salts. This mixture was
–
converted to the PF6 salt by adding 5–10 mL of water and 2–4 mL of saturated KPF6. Evaporation of
–
DCM under reduced pressure gave the pure PF6 salt as an orange solid: Rf = 0.64 (10% H2O + 2.5%
KNO3 in MeCN). HPLC retention time: 24.9 min. 1H NMR (500 MHz, acetonitrile-d3) δ 9.29 (d, J =
8.2 Hz, 1H; o), 9.10 (d, J = 8.2 Hz, 2H; f, c), 8.93 (d, J = 8.2 Hz, 1H; l), 8.86 (d, J = 8.4 Hz, 1H; k),
8.55 (s, 1H; g), 8.43 (s, 2H; 3’), 8.38 (s, 2H; 3), 8.15 (d, J = 7.8 Hz, 1H; h), 8.07 (dd, J = 5.2, 1.3 Hz,
2H; a, d), 7.87–7.75 (m, 6H; b, e, n, m, i, j), 7.71 (d, J = 5.7 Hz, 2H; 6’), 7.46 (d, J = 5.9 Hz, 2H; 6),
7.33 (dd, J = 6.4, 1.5 Hz, 2H; 5’), 7.10 (dd, J = 5.8, 1.5 Hz, 2H; 5), 2.61 (s, 6H; 4’-Me), 2.51 (s, 6H;
4-Me). MS (ESI+) m/z: 1011.00 [M-PF6]+, 865.33 [MPF6H]+, 433.33 [M2PF6]2+. HRMS (ESI+)
m/z for C51H40N8Ru: calcd 433.1209, found 433.1209.
Synthesis of [Ru(4,4-dmb)2(1-nip)](PF6)2 4. Yield 44 mg (40%): purity 96%. In a microwave
vessel, 63.4 mg (0.11 mmol) of [Ru(4,4′-dmb)2Cl2]∙2H2O was mixed with 34.63 mg (0.10 mmol) of
1-NIP. Ethylene glycol (3 mL) was added to the vessel. The mixture was microwaved for 15 min at
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180 °C. The resulting dark red color solution was directly loaded on to a silica column and eluted
using 100% MeCN followed by 5% H2O in MeCN to remove the excess starting material and other
impurities. Finally, Ru(II) complex 4 was eluted as an orange band with 5–10% H2O and 0.5–2.5%
–
KNO3 in MeCN to give an orange solid as a mixture of Cl– and NO3 salts. This mixture was
–
converted to the PF6 salt by adding 5–10 mL of water and 2–4 mL of saturated KPF6. Evaporation of
–
DCM under reduced pressure gave the the pure PF6 salt as an orange solid: Rf = 0.53 (10% H2O +
2.5% KNO3 in MeCN). HPLC retention time: 23.7 min.1H NMR (500 MHz, acetonitrile-d3) δ 9.18 (d,
J = 8.6 Hz, 1H; m), 9.05 (s, 2H; c, f), 8.42 (s, 2H, 3’), 8.38 (s, 2H; 3), 8.18 (t, J = 10 Hz, 2H; g, i),
8.12–8.07 (m, 3H; k, a, d), 7.82 (dd, J = 8.3, 5.3 Hz, 2H; b, e), 7.77 (t, J = 10 Hz, 1H; h), 7.75–7.68
(m, 4H; 6’, l, j), 7.44 (d, J = 5.8 Hz, 2H; 6), 7.33 (dd, J = 6.4, 1.5 Hz, 2H; 5’), 7.08 (dd, J = 5.8, 1.4
Hz, 2H; 5), 2.61 (s, 6H; 4’-Me), 2.51 (s, 6H; 4-Me). MS (ESI+) m/z: 961.2 [M-PF6]+, 815.2 [M–PF6–
H]+, 408.1 [M–2PF6]2+. HRMS (ESI+) m/z for C47H38N8Ru: calcd 408.1131, found 408.1126.
Synthesis of [Ru(4,4-dmb)2(2-nip)](PF6)2 5. Yield 50 mg (45%): purity 99%. In a microwave
vessel, 63.4 mg (0.11 mmol) of [Ru(4,4′-dmb)2Cl2]∙2H2O was mixed with 34.63 mg (0.10 mmol) of
2-NIP. Ethylene glycol (3 mL) was added to the vessel. The mixture was placed in a microwave for
15 min at 180 °C. The resulting dark red color solution was directly loaded on to a silica column and
eluted using 100% MeCN followed by 5% H2O in MeCN to remove the excess starting material and
other impurities. Finally, Ru(II) complex 6 was eluted as an orange band with 5–10% H2O and 0.5–
–
2.5% KNO3 in MeCN to give an orange solid as a mixture of Cl– and NO3 salts. This mixture was
–
converted to the PF6 salt by adding 5–10 mL of water and 2–4 mL of saturated KPF6. Evaporation of
–
DCM under reduced pressure gave the pure PF6 salt as an orange solid: Rf = 0.58 (10% H2O + 2.5%
KNO3 in MeCN). HPLC retention time: 24.4 min. 1H NMR (500 MHz, acetonitrile-d3) δ 9.05 (d, J =
8.2 Hz, 2H; c, f), 8.85 (d, J = 1.6 Hz, 1H; m), 8.45 (dd, J = 8.6, 1.8 Hz, 1H; g), 8.42 (s, 2H; 3’), 8.38
(s, 2H; 3), 8.17 (d, J = 8.7 Hz, 1H; h), 8.15–8.12 (m, 1H; l), 8.06 (dd, J = 5.2, 1.2 Hz, 2H; a, d), 8.05–
8.03 (m, 1H; i), 7.82 (dd, J = 8.2, 5.3 Hz, 2H; b, e), 7.70–7.67 (m, 4H; 6’, j, k), 7.43 (d, J = 5.8 Hz,
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2H; 6), 7.32 (dd, J = 6.1, 1.1 Hz, 2H; 5’), 7.07 (dd, J = 5.8, 1.0 Hz, 2H; 5), 2.61 (s, 6H; 4’-Me), 2.50
(s, 6H; 4-Me). MS (ESI+) m/z: 961.2 [MPF6]+, 815.3 [MPF6H]+, 408.1 [M2PF6]2+. HRMS
(ESI+) m/z for C47H38N8Ru: calcd 408.1131, found 408.1126.
Synthesis of [Ru(4,4-dmb)2(aip)](PF6)2 6. Yield 64 mg (55%): purity 90%. In a microwave
vessel, 63.4 mg (0.11 mmol) of [Ru(4,4′-dmb)2Cl2]∙2H2O was mixed with 39.61 mg (0.10 mmol) of
AIP. Ethylene glycol (3 mL) was added to the vessel. The mixture was microwaved for 15 min at 180
°C. The resulting dark red color solution was directly loaded on to a silica column and eluted using
100% MeCN followed by 5% H2O in MeCN to remove the excess starting material and other
impurities. Finally, the desired Ru(II) complex was eluted as an orange band with 5–10% H2O and
–
0.5–2.5% KNO3 in MeCN to give an orange solid as a mixture of Cl– and NO3 salts. This mixture
–
was converted to the PF6 salt by adding 5–10 mL of water and 2–4 mL of saturated KPF6.
–
Evaporation of DCM under reduced pressure gave the pure PF6 salt as an orange solid: Rf = 0.48
(10% H2O + 2.5% KNO3 in MeCN). HPLC retention time: 24.0 min. 1H NMR (500 MHz,
acetonitrile-d3) δ 8.88 (s, 1H; k), 8.44 (s, 2H; 3’), 8.40 (s, 2H; 3), 8.37 (d, J = 1.6 Hz, 1H; f), 8.32 (d, J
= 1.6 Hz, 1H; c), 8.26 (d, J = 8.5 Hz, 2H; a, d), 8.11 (dd, J = 5.3, 1.3 Hz, 2H; o, l), 7.90 (t, J = 8.6 Hz,
1H; m), 7.85 (dd, J = 8.8, 1.1 Hz, 2H; g, j), 7.81 (t, J = 6.8 Hz, 1H; n), 7.73 (d, J = 5.7 Hz, 2H; 6’),
7.63 (m, 2H; b, e), 7.56 (m, 2H; h, i), 7.49 (d, J = 4.7 Hz, 2H; 6), 7.34 (dd, J = 5.7, 1.0 Hz, 2H; 5’),
7.14 (dd, J = 6.1, 1.2 Hz, 2H; 5), 2.62 (s, 6H; 4’-Me), 2.54 (s, 6H; 4-Me). MS (ESI+) m/z: 1011.2
[MPF6]+, 433.1 [M2PF6]2+. HRMS (ESI+) m/z for C51H40N8Ru: calcd 433.1209, found 433.1204.
Cell assays for dark and phototoxicity. Solutions of the Ru(II) complexes were prepared as
follows: 5 mM stock solutions of the Ru(II) chloride complexes were prepared in 10% DMSO-H2O
and stored at 20 °C prior to use. Dilutions of the stock solutions were carried out with Dulbeco’s
phosphate buffered saline (DPBS) (pH = 7.4). The SK-MEL-28 cells were cultured according to the
procedure described in the literature (16). Cell viability experiments were conducted in triplicate
using a 96-well plate, which had 200 µL of DPBS buffer added. Aliquotes (50 µL) of cells in the log
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phase (SK-MEL-28 cells: ∼550,000 cells mL–1) were transferred to the inner well containing 25 µL
of warm culture medium. The plate was placed in a 5% CO2 water-jacketed incubator at 37 °C for 3 h
to allow the cells to equilibrate. The Ru(II) complexes were diluted with DPBS several times and
prewarmed at 37 °C before adding 25 µL of aliquots of appropriate concentration to the cells. Ru(II)
complexes 1-7 treated microplates were incubated under 5% CO2 at 37 °C for 16 h drug-to-light
intervals. Control cells that are not treated with light were kept in the dark. Cells in the microplate,
those receiving light treatment, were treated with visible light (400−700 nm, 34.2 mW cm–2) using a
190 W BenQ MS 510 overhead projector for 49 min to apply the total light doses of 100 J cm–2.
Control cells and light treated cell in the microplate both were kept inside the incubator for another 48
h. Then, 10 µL aliquots of Alamar blue was added to each well plate and incubated for another 15–16
h. Viability of the cells was quantified based on the fluorescence of Alamar blue dye that emits from
live cells, whereas it does not fluoresce in dead cells. Fluorescence was quantified using a Cytofluor
4000 fluorescence microplate reader. Excitation filter and emission filter of the microplate reader
were set in 530 ± 25 nm and 620 ± 40 nm, respectively. EC50 values of the dark toxicity and
photocytotoxicity were derived from eq 1 from the dose response curve of Graph Pad Prism 6.0. The
yi and yf represent the initial and final fluorescence signal intensities, respectively. EC50 values are
reproducible within the range of ±25% in the submicromolar, ±10% below 10 μM, and ±5% above 10
μM region. PIs were calculated from the ratio of the dark EC50-to-light EC50 values.
1
Singlet oxygen quantum yield measurements. The quantum yields for O2 production (ΦΔ)
were measured relative to [Ru(bpy)3](PF6)2 according to eq 2 (see footnote “a” in Table 2), where I,
A, and η are the integrated emission intensity, the absorbance at the excitation wavelength, and the
refractive index of the solvent, respectively, in which the subscript “s” denotes the standard. The
reference value for ΦΔ used for [Ru(bpy)3](PF6)2 was 0.56 in aerated MeCN (39).
RESULTS AND DISCUSSION
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Synthesis and characterization
A two-step synthesis was carried out to reach Ru(II) complexes 1-7 (Figure 3). Step 1: 1,10-
phenanthroline-5,6-dione reacted with NH4OAc, in glacial acetic acid, and an appropriately
substituted aldehyde under microwave irradiation at 180 °C for 15 min to obtain ip–R1-7 in 50-80%
yields. Step 2: [Ru(4,4-dmb)2Cl2]∙2H2O reacted with ip–R1-7 under microwave irradiation in ethylene
glycol at 180 °C for 15 min to obtain a crude chloride salt of Ru(II) complexes 1-7. These chloride
salts were purified by column chromatography using acetonitrile and water with KNO3, which led to
–
their isolation as mixtures of Cl– and NO3 salts. The mixtures were then converted to the
–
–
corresponding PF6 salts by introducing a saturated KPF6 solution. The Ru(II) complexes 1-7 as PF6
salts were formed in 23-55% yields as red or orange solids.
[Figure 3 here]
Proton NMR assignments for the phenanthroline unit of ip have been reported earlier (16).
However, 2-(phenanthren-9-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (pcip) was not previously
characterized. Here, we assign the pcip protons a and d as the most downfield protons because these
are connected to a carbon which is directly attached to nitrogen (Figure 4 and Figure S1). The close
proximity to imidazolium nitrogen brings f and c signals downfield compared to the b and e signals.
The singlet for g appeared at 8.48 ppm, and a doublet at 8.19 was tentatively assigned for k. Protons b
and e appeared as a multiplet within 7.92–7.73 ppm. The other protons o, l, n, m, i, and j appeared as a
multiplet.
[Figure 4 here]
The characterization of Ru(II) complex 1 and 7 has been previously reported (16). Therefore,
the characterization of Ru(II) complexes 2-6 are reported here. Based on HPLC data, the purity of 2
was 98%, of 3 was 98%, of 4 was 96%, of 5 was 99%, and of 6 was 90%. The LCMS data indicated
that 2 contained a fluorenyl group [(ESI+) m/z for C50H40N8Ru: calcd 427.1209, found 427.1204], 3
contained a phenanthrenyl group [(ESI+) m/z for C51H40N8Ru: calcd 433.1209, found 433.1209], 4
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contained a naphthyl group [(ESI+) m/z for C47H38N8Ru: calcd 408.1131, found 408.1126], 5
contained a naphthyl group [(ESI+) m/z for C47H38N8Ru: calcd 408.1131, found 408.1126], and 6
contained an anthryl group [(ESI+) m/z for C51H40N8Ru: calcd 433.1209, found 433.1204].
For Ru(II) complexes 1-7, protons c and f in phenanthroline moiety were assigned as the most
downfield protons at 9.13–8.89 ppm, because they are far from Ru electron cloud and closer to
imidazolium nitrogens (Figures S2-S7). However, the signals for c and f were slightly upfield shifted
for 6 and appeared within 8.37–8.32 ppm. The protons b and e in phenanthroline moiety of complexes
1-7 that appeared within 7.93–7.63 ppm were assigned based on their correlation with c and f. For all
complexes, a and d appeared as a doublet within 8.26–7.97 ppm. Downfield chemical shifts were
observed for the aromatic protons attached to the 4,4-dmb unit in the Ru(II) complexes in the
following order: 3,3 > 6,6 > 5,5 as reported previously in the literature (17). For 2, methylene
proton h appeared as a singlet at 4.14–4.13 ppm. Other aromatic protons on the fluorinone moiety of
these complexes appeared in the range 8.55–7.38 ppm. Doublet for m attached to napthalene
chromophore in 5 appeared at 8.85 ppm, although this proton does not show an interaction with other
protons in 1H-1H COSY, this doublet could be the result of long-range four bond coupling. A singlet
for g in 3 appeared at 8.55 ppm, and indeed all other aromatic protons of the phenanthrene unit
in 3 appeared within 8.29–7.75 ppm. Protons o, m, and k were assigned as the most downfield protons
and appeared as doublet, doublet, and singlet at 9.29 (for 3), 9.18 (for 4), and 8.88 ppm (for 6),
respectively. Protons k does not show correlation with another proton in 6; whereas, o and m showed
correlation with n and l, respectively.
Photokilling of melanoma SK-MEL-28 cells
Table 1 shows the phototoxic effects of Ru(II) complexes 1-7 in human malignant melanoma
SK-MEL-28 cells. The cells were incubated with 1 nM to 300 µM complex 1-7 prior to irradiation
with visible light (400-700 nm) in a 16 h drug-to-light interval. The phototoxicity results were
obtained with a fluence of 100 J cm-2, in which the highest concentration tested was 300 µM. Dark
toxicities of Ru(II) complexes 1-7 were fairly low (ranging from 59–230 µM). We remind the reader
that compounds with EC50 dark toxicities of >300 µM are generally considered to be nontoxic (4). In
our case, the phototoxicities of Ru(II) complexes 1-7 were found to be in the tenths of micromolar
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range (0.24–0.46 µM). Ru(II) complex 7 had a particularly low dark toxicity and a fairly good
phototoxicity resulting in the highest PI (of 657) in the series. The PI obtained for Ru(II) complex 6 is
409, which had an anthracene substituent. Subtle structural differences between naphth-1-yl and
naphth-2-yl led to quite large differences in phototoxicities in the SK-MEL-28 cells, where the PI of 4
was about double that of 5. The PI value for Ru(II) complex 1 was 558, which was second highest in
the series. The relationship between the size of the substituent and phototoxicity of Ru(II) complexes
1-7 was only marginally correlated. For example, the pyrenyl-substituted Ru(II) complex 7 had a PI
of only ~100 greater than the phenyl-substituted Ru(II) complex 1. Another example is the slightly
improved dark toxicity of 7 compared to 4, where the phototoxicity is slightly better for 4 than 7.
We believe there is value in comparing the new Ru(II) complexes 1-7 to several Ru(II)
complexes reported in the literature (19-21). Compounds 8-11 are Ru-based dyads containing π-
expansive ligands combined with relatively small and substitutionally inert coligands bpy or 4,4′-dmb
(Figure 5). As seen in Table 1, changing either the coligand and/or the π-expansive functional ligand
can have a marked effect on the dark and phototoxicities as well as the PIs, but not necessarily in a
systematic way. Ru(II) complex 7 ranks among the least dark toxic in this comparison (surpassed only
by 8) and displays similar phototoxic effects as 10 and 11 (surpassed only by 9). While its PI is not as
large as that of 9, the PI measured for 7 compares favorably with 10, being among the two largest in
the comparison. Ru(II) complex 7 does have a clear advantage over its counterpart with a similar PI in
that 7 is almost four times less dark toxic, which is of consideration for a phototherapeutic agent.
[Table 1 here]
[Figure 5 here]
Quantum yield for singlet oxygen formation (∆)
Next, the singlet oxygen quantum yields for Ru(II) complexes 1-7 were measured. Table 2
shows that the ∆ values ranged from a high of 0.87 for 6 to a low of 0.58 for 1, which compare
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favorably to literature values for 8 (0.17) (19,20), 9 (0.79) (19,20), 10 (0.78) (20), and 11 (0.74) (21).
For the new Ru(II) complexes, interestingly, we found that the quantum yields increase in the order 1
≈ 2 ≈ 3 < 4 ≈ 5 < 6 ≈ 7 and very roughly correlate with the decrease of the triplet energies (ET) of the
π-group (R1-R7) alone. The triplet energies of the π-groups (R1-R7) are in the range of 3.66–1.82 eV.
These π-groups (R1-R7) decrease in the order benzene > fluorene > phenanthrene ≈ naphthalene >
3
pyrene > anthracene. For comparison, to the π-groups (R1-R7), the energy of the MLCT transition
state for [Ru(bpy)3]2+ complex is reported to be 3.29 eV (18). While the high ∆’s for Ru(II)
complexes 1-7 and low ET’s for the π-groups (R1-R7) point to a type-II process, contributions from
type-I processes may also exist, as was investgated next with Ru(II) complex 7.
[Table 2 here]
Mechanistic considerations
We next carried out experiments with Ru(II) complex 7 to assess whether its mechanism is
dominated by type I (oxygen radicals or radical ions) or type II (1O2) processes. Data were collected
1
with 9,10-anthracene dipropionate dianion 12 as a trapping agent for O2 by a [4 + 2] cycloaddition
reaction, in which the product of the trapping reaction is 9,10-endoperoxide 13 (Figure 6) (28).
Anthracene 12 has been used to verify the presence of 1O2 in aqueous solution and is a good trapping
agent based on a comparison to trans-2-methyl-2-pentenoate anion that undergoes an ‘ene’ reaction
and is considered to be a fingerprint for 1O2 (28,29). Here, anthracene 12 was used as it requires UV-
vis instead of NMR and also a much lower concentration than the trans-2-methyl-2-pentenoate anion.
Our studies were carried out in H2O or D2O, and in the presence or absence of sodium azide (Table
3). We find that the efficiency of 1O2 trapping by 12 increased by changing the solvent from H2O and
in D2O. However, the increase in yield of endoperoxide 13 was 2-fold in D2O compared to H2O.
1
1
Because the lifetime of O2 (∆) is 30-fold higher in D2O vs H2O (30), a greater difference in O2
trapping in D2O vs H2O would have been expected if the Ru(II) complex 7 was mainly serving as a
type-II sensitizer. We also found only a modest decrease in the 1O2 trapping by 12 when sodium azide
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1
(a known O2 quencher) was added to the reaction. Based on these results, we propose that Ru(II)
complex 7 contains contributions from both type-I and type II processes.
What are the mechanistic implications of these results? Our finding for Ru(II) complex 7 is
similar to an earlier study that showed Ru(II) complexes with α-oligothiophene groups contained
contributions from both type I and type II processes (14). While type-I and type II processes are
oxygen dependent, contributions from Ru(II) complexes may also include oxygen-independent
reactions. However in the current study, we did not explore the photokilling of the SK-MEL-28 cells
under oxygen-free conditions to assess its potential contribution.
[Table 3 here]
[Figure 6 here]
CONCLUSION
Herein, we report on Ru(II) dyads tethered to R groups that differ in π-conjugation in an
attempt to increase the PI in a rational manner. Specifically, we synthesized Ru(II) complexes 1-7 by
covalent attachment of phenyl, fluorenyl, phenanthrenyl, naphthalen-1-yl, naphthalene-2-yl, anthryl,
and pyrenyl groups to ip, which led to stable Ru(II) complexes 1-7. These complexes were
1
synthesized in modest yields (33-78%). As expected, the higher O2 quantum yields roughly
correlated with lower triplet-state energies of the R groups. We found Ru(II) complex 7 to be the most
promising dyad of the series, with a relatively high value for ∆ (0.84) and a large PI toward SK-
MEL-28 cells, which included contributions from both type I and type II processes based on the
effects of added sodium azide and solvent deuteration.
SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the
end of the article:
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1
Figure S1: H NMR spectrum of 2-(phenanthren-9-yl)-1H-imidazo [4,5-f][1,10] phenanthroline
(pcip).
Figure S2: 1H NMR spectrum (upper) and 1H-1H COSY spectrum (lower) of 1.
Figure S3: 1H NMR spectrum of 2.
Figure S4: 1H NMR spectrum (upper) and 1H-1H COSY spectrum (lower) of 3.
Figure S5: 1H NMR spectrum (upper) and 1H-1H COSY spectrum (lower) of 4.
Figure S6: 1H NMR spectrum (upper) and 1H-1H COSY spectrum (lower) of 5.
Figure S7: 1H NMR spectrum (upper) and 1H-1H COSY spectrum (lower) of 6.
Figure S8: In vitro PDT dose−response curves for Ru(II) complexes 1-7 in SK-MEL-28 cells.
ACKNOWLEDGEMENTS
Research reported in this publication was supported by the National Cancer Institute of the National
Institutes of Health under Award Number R01CA222227 (to SAM). The content is solely the
responsibility of the authors and does not necessarily represent the official views of the National
Institutes of Health. SAM also acknowledges financial support from the University of North Carolina
at Greensboro, the Natural Sciences and Engineering Council of Canada, the Canadian Institutes of
Health Research, the Canadian Foundation for Innovation, the Nova Scotia Research and Innovation
Trust, and Acadia University. GG, LL, and AG acknowledge support from the National Science
Foundation (CHE-1856765). We thank Oliver Turque for help with the table of contents graphic.
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FIGURE CAPTIONS
Figure 1. Type I and type II photosensitized oxidations. The type I reaction forms oxygen radicals
and radical ions, and the type II reaction mainly forms singlet oxygen by energy transfer from an
excited photosensitizer to triplet oxygen. Both reactions lead to oxidized and/or oxygenated products.
Oxygenated products can include hydroperoxides and endoperoxides.
Figure 2. Structures of Ru(II) complexes 1–7 bearing functional ligands (ip-R).
Figure 3. Two-step synthesis of Ru(II) complexes 1-7.
Figure 4. Proton NMR assignments of 2-(phenanthren-9-yl)-1H-imidazo[4,5-f][1,10]phenanthroline
(pcip).
Figure 5. Structures of Ru(II) complexes 8–11.
Figure 6. Reactive oxygen intermediates are generated by Ru(II) complex 7 upon illumination with
1
white light in D2O. The O2 produced is detected by its reaction with 9,10-anthracene dipropionate
dianion 12 to reach endoperoxide 13.
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TABLE CAPTIONS
Table 1. SK-MEL-28 cell phototoxicities of the new Ru(II) complexes 1-7 and Ru(II) complexes 8-
11 previously reported in the literature.
Table 2. Singlet oxygen quantum yields (∆) for Ru(II) complexes 1-7, and literature triplet energies
(ET) of the π-group (R1-R7) alone.
Table 3. H2O/D2O solvent and sodium azide quenching photochemical effects with Ru(II) complex 7
on the trapping of 1O2 by antracene 12.
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Table 1. SK-MEL-28 cell photocytotoxicities of the new Ru(II) complexes 1-7 and Ru(II)
complexes 8-11 previously reported in the literature.
Ru(II)
complexes
1
PI b
dark EC50 (M) a
light EC50 (M) a
558
179
232
505
277
409
657
>20
2071
650
400
143  10
82  1
0.26  0.01
0.46  0.02
0.25  0.01
0.26  0.04
0.35  0.02
0.24  0.01
0.35  0.02
14.9 ± 0.2
2
3
59  4
4
133  8
96  8
5
6
98  21
230  11
>300
7
8 c,d
9 c,d
10 d
11e
140 ± 10
58.5 ± 0.5
49.9 ± 0.3
0.07 ± 0.01
0.35 ± 0.01
0.125 ± 0.003
a Experiments were conducted in triplicate. Dark EC50 and light EC50 values were derived from
eq 1 using a dose-response curve of Graph Pad Prism 6.0, which are shown in the Supporting
Information.
푦푖 − 푦푓
푦 = 푦푖 +
(1)
(
)
logEC −푥 ×(Hillslope)
50
1 + 10
b
Phototherapeutic indicies (PIs) were calculated as the ratio of the dark EC50 and light EC50
values.
c Reference 19.
d Reference 20.
e Reference 21.
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Table 2. Singlet oxygen quantum yields (∆) for Ru(II) complexes 1-7, and literature triplet
energies (ET) of the π-group (R1-R7) alone.
Ru(II)
π-group (R1-R7)
ET (eV) b
a
∆
complex
alone
1
2
3
4
5
6
7
0.58
0.59
0.60
0.69
0.67
0.87
0.84
benzene
fluorene
3.66
2.92
2.69
2.64
2.64
1.82
2.08
phenanthrene
naphthalene
naphthalene
anthracene
pyrene
a
Singlet oxygen quantum yields (∆) for 1-7 were measured in aerated acetonitrile using eq 2
relative to [Ru(bpy)3](PF6)2 (∆ = 0.57 in aerated acetonitrile) (22,23) and contains 5% error.
See Materials and Methods section for more information on the ∆ measurement.
=
b ET values of benzene (24), fluorene, phenanthrene, napthalene (25), pyrene (26), and anthracene
(27) were obtained from the literature.
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Table 3. H2O/D2O solvent and sodium azide quenching photochemical effects with Ru(II)
complex 7 on the trapping of 1O2 by antracene 8.
solvent
yield 9
(µM) a
yield 9 (µM) b
ratio
9D2O/9H2O
2.0
ratio
9D2O/9D2O+NaN3 9H2O/9H2O+NaN3
1.5 0.80
ratio
(solvent+NaN3)
19.8±1.0
16.7±2.6
D2O
H2O
28.1±0.9
13.9±3.7
a Irradiation (15 min) of a biphasic system containing 82 µM of 8 in D2O or H2O, and 63.7 µM
of Ru(II) complex 7 in CDCl3.
b
Endoperoxide 9 yield in presence of 1 mM sodium azide (NaN3) added as a singlet oxygen
quencher.
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