Sterically demanding methoxy and methyl groups in ruthenium complexes lead to enhanced quantum yields for blue light triggered photodissociation

Article abstract of DOI:10.1039/C8DT03295E

Ruthenium complexes containing a sterically congested metal center can serve as light activated prodrugs through photo-activated chemotherapy (PACT). In this work, we modified PACT agents containing 6,6′-dihydroxybipyridine (6,6′-dhbp) (Papish et al., Inorg. Chem., 2017, 56, 7519) by replacing it with a sterically bulky isoelectronic ligand, 6,6′-dimethoxybipyridine (6,6′-dmbp). The resulting complexes, [(phen)₂Ru(6,6′-dmbp)]Cl₂ (2^OMe, phen = 1,10-phenanthroline) and [(dop)₂Ru(6,6′-dmbp)]Cl₂ (3^OMe, dop = 2,3-dihydro-[1,4]dioxino[2,3-f][1,10]phenanthroline), have been fully characterized and display enhanced quantum yields for blue light triggered photodissociation of 0.024(6) and 0.0030(2), respectively. We have also synthesized 4^OH = [(dmphen)₂Ru(4,4′-dhbp)]Cl₂ wherein dmphen = 2,9-dimethyl-1,10-phenanthroline and 4,4′-dhbp = 4,4′-dihydroxybipyridine. These ligands enhance steric bulk near the metal center and move the hydroxy groups further from the metal center, respectively. Complex 4^OH displays a relatively low quantum yield of 0.0014(2). All of the new complexes (2^OMe, 3^OMe, 4^OH) were tested in breast cancer cells (MDA-MB-231) and were non-toxic (IC₅₀ > 100 μM). This has been interpreted in terms of unfavorable log(D_o/w) values and furthermore photodissociation alone is insufficient for cytotoxicity. We also report the crystal structures of 4^OH and 2^OMe, the thermodynamic acidity of complex 4^OH, and the redox potentials for all new complexes.

Full text of DOI:10.1039/C8DT03295E

Showcasing research from a collaboration between the
laboratories of Professors Papish and Kim (The University of
Alabama, USA); Schmehl (Tulane University, USA); and
Paul (Villanova University, USA).
As featured in:
Volume 47 Number 44 28 November 2018 Pages 15619–15996
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An international journal of inorganic chemistry
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Sterically demanding methoxy and methyl groups in ruthenium
complexes lead to enhanced quantum yields for blue light
triggered photodissociation
Bulky groups on ruthenium complexes lead to faster
photodissociation but they do not increase toxicity towards
breast cancer cells. Rather, toxicity is related to the lipophilicity
of neutral ruthenium complexes bearing O- groups and an
alternative mode of action.
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PAPER
Sterically demanding methoxy and methyl groups
in ruthenium complexes lead to enhanced
quantum yields for blue light triggered
photodissociation†
Cite this: Dalton Trans., 2018, 47,
15685
Fengrui Qu, a Kristina Martinez, b Ashley M. Arcidiacono,c Seungjo Park,d
Matthias Zeller,e Russell H. Schmehl,b Jared J. Paul, *c Yonghyun Kim *d and
a
Elizabeth T. Papish
*
Ruthenium complexes containing a sterically congested metal center can serve as light activated pro-
drugs through photo-activated chemotherapy (PACT). In this work, we modified PACT agents containing
6,6’-dihydroxybipyridine (6,6’-dhbp) (Papish et al., Inorg. Chem., 2017, 56, 7519) by replacing it with a
sterically bulky isoelectronic ligand, 6,6’-dimethoxybipyridine (6,6’-dmbp). The resulting complexes,
[(phen)2Ru(6,6’-dmbp)]Cl2 (2OMe, phen = 1,10-phenanthroline) and [(dop)2Ru(6,6’-dmbp)]Cl2 (3OMe
,
dop = 2,3-dihydro-[1,4]dioxino[2,3-f][1,10]phenanthroline), have been fully characterized and display
enhanced quantum yields for blue light triggered photodissociation of 0.024(6) and 0.0030(2), respect-
ively. We have also synthesized 4OH = [(dmphen)2Ru(4,4’-dhbp)]Cl2 wherein dmphen = 2,9-dimethyl-
1,10-phenanthroline and 4,4’-dhbp = 4,4’-dihydroxybipyridine. These ligands enhance steric bulk near
the metal center and move the hydroxy groups further from the metal center, respectively. Complex 4OH
displays a relatively low quantum yield of 0.0014(2). All of the new complexes (2OMe, 3OMe, 4OH) were
tested in breast cancer cells (MDA-MB-231) and were non-toxic (IC50 > 100 µM). This has been inter-
preted in terms of unfavorable log(Do/w) values and furthermore photodissociation alone is insufficient for
cytotoxicity. We also report the crystal structures of 4OH and 2OMe, the thermodynamic acidity of complex
4OH, and the redox potentials for all new complexes.
Received 11th August 2018,
Accepted 29th September 2018
DOI: 10.1039/c8dt03295e
rsc.li/dalton
et al. have made great progress in the use of Ru complexes that
readily photodissociate a ligand to bind DNA and cause
Introduction
Ruthenium complexes with sterically bulky ligands are fre- cytotoxicity.2–4 These studies have used steric bulk near the
quently used to trigger photodissociation for anticancer appli- metal center to destabilize the octahedral geometry and facili-
cations. Such ligand loss via photodissociation can lead to tate photolabilization of a ligand.3,4 The ligands dop, dmphen,
prodrug activation which can be used for photo-activated and 2,2′-biquinoline are known to enhance photodissociation
chemotherapy (PACT). (Abbreviations are listed here.1) Glazer (with blue, red, or near-IR light) by causing strain in octa-
hedral ruthenium complexes. Turro, Dunbar, and co-workers
have used cytotoxic ligands (e.g. 5-cyanouracil with a ligand
aDepartment of Chemistry & Biochemistry, The University of Alabama, Tuscaloosa,
AL 35487-0336, USA. E-mail: etpapish@ua.edu
exchange quantum yield of 0.16) on Ru for light triggered
release.5–8 McFarland, Sadler, and others have shown that
changing the charge of the metal complex (e.g. with cyclo-
metallated C,N bound ligands) can greatly alter the
cytotoxicity.9–13 Collectively, these studies show that photodis-
sociation to generate toxic byproducts (free metal, free ligand
or both)14,15 plays a key role in the cytotoxicity of ruthenium
prodrugs. However, another important factor is the uptake of
these complexes as governed by charge and lipophilicity.16–18
Our initial effort in this area was the design of [(N,N)2Ru
(6,6′-dhbp)]Cl2 complexes for PACT vs. cancerous cells wherein
6,6′-dhbp is a pH sensitive photolabile ligand (all ligands and
bDepartment of Chemistry, Tulane University, New Orleans, Louisiana 70118, USA
cDepartment of Chemistry, Villanova University, Villanova, PA 19085, USA.
E-mail: jared.paul@villanova.edu
dDepartment of Chemical and Biological Engineering, The University of Alabama,
Tuscaloosa, AL 35487-0203, USA. E-mail: ykim@eng.ua.edu
eDepartment of Chemistry, Purdue University, West Lafayette, IN 47907, USA
†Electronic supplementary information (ESI) available: 1H, 13C NMR, IR, UV-vis
spectra; methods and details of thermodynamic acidity (pKa) measurements;
quantum yield measurement details; methodology for partition coefficient deter-
mination; structural parameters for X-ray crystallography. CCDC 1861548 and
1861549. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c8dt03295e
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metal complexes used in this work are shown in were at pH 5.0 (at this pH the complexes have OH groups and
Scheme 1).19–21 Upon irradiation with blue light (λ = 450 nm), a +2 charge) (Table 1 and Scheme 1). Admittedly, this pH value
[(N,N)2Ru(6,6′-dhbp)]Cl2 forms the aqua complex, [(N,N)2Ru is below that found in the extracellular matrix in hypoxic
(OH2)2]2+, and 6,6′-dhbp. The spectator ligand, (N,N), was tumors (pH 6.0–6.8),22 and while this pH value is not biologi-
varied to include bipy (1OH), phen (2OH), and dop (3OH
(Scheme 1). While complex 3OH was the most cytotoxic (IC50
)
=
cally relevant it illustrates how the compound behaves as the
more photolabile diprotic acid. These quantum yields are pH
∼4 μM vs. breast cancer cell lines) upon activation with blue sensitive. For the most cytotoxic complexes 2OH and 3OH, the
light, this complex had a low quantum yield for photodissocia- two pKa values (reported in our prior work19) can be used to
tion.19 In fact, all three complexes (1OH–3OH) have low calculate the fraction which is fully protonated and more
quantum yields on the order of 1 × 10−3 when the complexes photolabile at pH 7.5. The Henderson–Hasselbach equation
predicts that only 0.08% of 2OH and 0.05% of 3OH bears OH
groups (with a 2+ charge) at pH 7.5; the major species is
neutral with O− groups (2O− and 3O−, Scheme 1). Thus, under
physiological conditions, the quantum yields are 1–2 orders of
magnitude lower at pH 7.5 (vs. pH 5.0) (Table 1). Therefore, we
postulated that low quantum yields were limiting cytotoxicity
and we aimed to synthesize complexes with increased
quantum yields for photodissociation. Replacing OH groups
with OMe groups affords 100% of the complex in a photolabile
form at physiological pH. In this work we describe the
synthesis and properties of [(phen)2Ru(6,6′-dmbp)]Cl2 (2OMe
and [(dop)2Ru(6,6′-dmbp)]Cl2 (3OMe) (Scheme 1).
)
Furthermore, we have also synthesized 4OH = [(dmphen)2Ru
(4,4′-dhbp)]Cl2 to provide steric bulk from the dmphen ligand
with four methyl groups near the metal center (Scheme 1). Six
bulky groups cannot be accommodated near the metal center
as such a complex (with 6,6′-dhbp) would be unstable; thus
using the dmphen ligand necessitated moving the hydroxyl
groups away from the metal center with 4,4′-dhbp. This
complex 4OH was designed to determine if such derivatives
would have similar quantum yields or cytotoxicity to analogous
complexes like 2OH. All of the new complexes (2OMe, 3OMe, 4OH
)
were tested in breast cancer cells (MDA-MB-231) and were non-
toxic (IC50 > 100 µM) despite greatly improved quantum yields.
This information has been interpreted in terms of unfavorable
log(Do/w) values reported herein that may lead to poor cellular
uptake and furthermore photodissociation alone is insufficient
for toxicity. Herein, octanol/water partition coefficients (log
(Do/w)) are used to measure the hydrophilic (negative log(Do/w))
vs. lipophilic (positive log(Do/w)) character of our complexes
and to estimate cellular uptake by passive diffusion. We also
Scheme 1 Protic ruthenium anticancer compounds and their novel
methylated (2OMe, 3OMe) and 4,4’-substituted (4OH) analogs. Complexes
5 and 6 were reported previously and are used for structural compari-
son. The ligands used in this study are also illustrated.
Table 1 Comparison of acidity, quantum yields for photodissociation upon irradiation at 450 nm, and log(Do/w) values, and E1/2 values for selected
compounds discussed herein
φPD at pH 5.0
φPD at pH 7.5
log(Do/w)
at pH 7.4
Compound
Structure
pKa avg
(mostly XOH if protic ligand)
(mostly XO− if protic ligand)
E1/2 (V)
1OH a
2OH a
3OH a
2OMe
3OMe
4OH
[(bipy)2Ru(6,6′-dhbp)]2+
[(phen)2Ru(6,6′-dhbp)]2+
[(dop)2Ru(6,6′-dhbp)]2+
[(phen)2Ru(6,6′-dmbp)]Cl2
[(dop)2Ru(6,6′-dmbp)]Cl2
[(dmphen)2Ru(4,4′-dhbp)]Cl2
6.3
0.0058(5)
0.0020(2)
0.001(1)
0.024(6)
0.0030(2)
0.0014(2)
0.0012(1)
0.000036(1)
0.00022(3)
—
—
—
1.4(1)a
1.6(1)a
1.8(1)a
−1.3(2)
−1.1(1)
0.58(2)
1.12b
c
6.0(1)
5.9(1)
N/A
N/A
6.08(2)
—
—
c
0.99d
0.98d
0.66d
a All measurements on 1OH, 2OH, and 3OH are from our prior work.19,20 An improved procedure for measuring log(Do/w) led to small changes in
the observed values relative to our published prior work.19 b Versus SCE in acetonitrile.20 c Redox potentials for 2OH and 3OH were not measured.19
d Versus Ag/AgCl in aqueous solution at pH = 7.0 at a scan rate of 200 mV s−1
.
15686 | Dalton Trans., 2018, 47, 15685–15693
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Paper
report the structures of 4OH and 2OMe as determined by Single
Crystal X-Ray Diffraction and we report the thermodynamic
acidity of complex 4OH
.
Results and discussion
Synthesis and characterization of the metal complexes
The new metal complexes (2OMe, 3OMe, 4OH) were synthesized
by modifying known procedures (Scheme 2).19,20 The N,N co-
ligands (in blue, Scheme 2) were purchased (phen, dmphen)
or synthesized (dop) using literature protocols.3 These co-
ligands are readily added to the 1,5-cyclooctadiene complex of
ruthenium ((COD)RuCl2). The cis product, [(N,N)2RuCl2], was
then treated with the oxygenated bipy ligand (6,6′-dmbp or
4,4′-dhbp, in red, Scheme 2). The final products were recrystal-
lized by slow diffusion of diethyl ether into a solution of
corresponding complexes in dry ethanol (for 4OH) or aceto-
Fig. 1 The ORTEP diagrams (with ellipsoids at 50% probability) of the
cations of 4OH (left) and 2OMe (right). Hydrogen atoms (except that on
oxygen) and chloride counter anions were omitted for clarity. Complex
4OH contains no solvent molecules, but complex 2OMe contains one
water molecule per each Ru molecule, which is omitted for clarity. A full
list of structural parameters is included in the ESI.†
nitrile (for 2OMe and 3OMe) to afford the pure product. For 4OH
,
the complex was isolated as hexafluorophosphate salt, and
then was converted to chloride salt by treating with excess of
Et4NCl in acetone. All three products were fully characterized
1
by H- and 13C-NMR, ESI-MS, IR, UV-Vis (see Fig. S7–9 in the
ESI†), and elemental analysis. Single crystal X-ray diffraction
structures were obtained for compounds 4OH and 2OMe
.
X-Ray crystallography
Crystals suitable for single crystal XRD analysis of 2OMe were
obtained by slow diffusion of diethyl ether into a MeCN solu-
tion of 2OMe (Fig. 1–3 and Table 2). The structure features a
distorted octahedral geometry around the central metal,
similar to its previously reported hydroxyl analogue 2OH
,
[(phen)2Ru(6,6′-dhbp)]Cl2 (Fig. 2 and 3).19 The substitution of
6,6′-OH (2OH) with 6,6′-OMe on 2OMe did not disturb the geo-
metric configuration around the central metal significantly.
For example, the average Ru–N(6,6′-dmbp) distance in com-
pound 2OMe (2.095(1)) is only slightly longer than the corres-
ponding distances in the hydroxy analogue 2OH (Ru–N =
2.086(2)), suggesting a slight steric bulk increase for methoxy
groups. All other bond distances, angles and torsion angles
are similar between 2OH and 2OMe
.
Crystals suitable for single crystal XRD analysis of 4OH were
obtained by slow diffusion of diethyl ether into an ethanol
solution of 4OH (Fig. 1–3 and Table 2). Compared to 2OH, the
Fig. 2 A comparison of crystal structures of the complexes 2OMe, 2OH
,
4OH, 5 and 6. Structure 6 was reproduced from literature.24 Note:
Thermal ellipsoid data are not available for 6.
compound 4OH, [(dmphen)2Ru(4,4′-dhbp)]Cl2, shows some
significant changes. Effectively, steric distortions are present
in 6,6′-dhbp in 2OH, but these distortions are moved to the
Scheme 2 Synthesis of new photolabile ruthenium complexes.
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due to the OH groups being in a less crowded position in 4,4′-
dhbp. This can be clearly seen in the side-by-side comparison
presented in Fig. 2. The pyridinol rings are coplanar in 4OH
and 4,4′-dhbp lines up with the equatorial Ru–N plane, while
the 6,6′-dhbp in 2OH is tilted below the equatorial plane.
Conversely, the torsion angle on the spectator ligand (17.8(2)°)
in 4OH is now larger than those in 2OH (4.9(1)°), due to the
steric hindrance from the methyl groups of dmphen. Thus,
4OH is significantly more distorted from an octahedral
geometry vs. 2OH
.
We can also compare 4OH to 5, [(phen)2Ru(4,4′-dhbp)]Cl2,
which was reported by us previously and only differs in
removal of the methyl groups from the spectator ligands.
Complex 4OH is much more distorted than 5 as shown in the
torsion angles of the spectator ligands (Fig. 2). This can also
be seen in the trans angles (Table 2), which vary more for 4OH
as is evident from Fig. 3. The highly-distorted structure of 4OH
favors photodissociation (vide infra), which is in direct contrast
to 5 (non-dissociative upon irradiation).
A crystal structure similar to 4OH has been reported before
(see Fig. 2, compound 6), namely, [(AFO)Ru(dmphen)2](ClO4)2,
where AFO = 4,5-diazafluore-9-one.24 Selected bond distances
and angles of interest are summarized in Table 2 and pre-
sented in Fig. 3 for comparison. The average Ru–N(dmphen)
bond distance in 4OH is essentially the same as the corres-
ponding Ru–N(dmphen) bond distance in 6, which are
2.101(1) and 2.097(3), respectively. As a contrast, the average
Ru–N(dhbp) bond distance in 4OH is significantly shorter than
the corresponding Ru–N(AFO) bond distance in 6, which are
2.092(3) and 2.143(5), respectively. The longer Ru–N(AFO)
bond distances are probably due to electronic factors unique
to the diazafluorenone ligand.
Thermodynamic acidity
Complex 4OH has two acidic protons on the hydroxyl groups,
so it can equilibrate between the protonated (dicationic) and
deprotonated (neutral) forms depending on the solution pH.
Therefore, we were interested in measuring the pKa values for
4OH by potentiometric titration. Briefly, a sample of 4OH was
dissolved in deionized water to prepare the analyte solution,
which was then pre-treated with four equivalents of HCl(aq) to
Fig. 3 A comparison of crystal structures of complexes showing only
the coordination spheres, with bond distances listed in Å. Structure 6
was reproduced from literature.24 Note: Thermal ellipsoid data are not
available for 6.
dmphen ligands in 4OH. The average Ru–N (dmphen) bond ensure that the analyte is fully protonated. The solution was
distances = 2.101(1) Å are longer in 4OH due to the methyl then titrated with standardized base (aq. NaOH solution).
groups’ steric bulk, cf. the Ru–N (phen) = 2.061(1) Å in 2OH. In Despite the fact that the compound is diprotic, only one equiv-
addition, the average Ru–N (4,4′-dhbp) bond distances = alence point was observed and hence the average of two pKa
2.092(3) in 4OH
, which are slightly longer than Ru–N values was determined to be 6.08(2). This result is common in
(6,6′-dhbp) = 2.086(2) in 2OH. Complex 4OH does have OH⋯Cl− the literature for closely spaced pKa values, as similar results
hydrogen bonds, but these do not alter the bond lengths have been reported by us and others.25–27 This value is similar
appreciably (e.g. the C–O bond distances are ∼1.34 Å in 4OH
which are similar to analogous C–O distances in 2OMe, 2OH
,
,
to literature analogs including pKa(avg) = 6.0(1) for 2OH and
6.01(8) for 5.19 Thus, moving the hydroxy groups to a different
and other pyridinol derived Ru complexes23). In 4OH, the steric position (6,6′ vs. 4,4′) or adding methyl groups to the phen
bulk is less for the oxygenated bipy derivative, but the crowd- ligand does not appreciably change the pKa values.
ing from the methyl groups of the dmphen ligand serves to
Cyclic voltammetry
lengthen Ru–N (of 4,4′-dhbp) distances (cf. 5 = [(phen)2Ru(4,4′-
dhbp)]2+ in Table 2). The torsion angle (5.6(4)°, Table 2) within The electrochemical properties of the Ru polypyridyl com-
the 4,4′-dhbp ring is much smaller than that for 2OH (16.6(3)), plexes were investigated by cyclic voltammetry. The measure-
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Table 2 Selected Bond Lengths and Angles of the Complexes 2OMe and 4OH, as compared to their structural analogues 2OH, 5, and 6
Paper
2OMe
2OH a
4OH
5 b
6 c
Ru−N1
2.087(1)
2.103(1)
2.060(1)
2.054(1)
2.064(1)
2.074(1)
2.074(1)
2.095(1)
2.063(1)
174.86(5)
171.15(4)
175.42(5)
173.81(4)
16.4(1)
−14.5(1)
15.5(1)
−4.5(2)
−0.5(2)
−2.9(1)
−2.3(1)
2.6(1)
2.086(2)
2.087(2)
2.075(2)
2.063(2)
2.055(2)
2.052(2)
2.070(1)
2.086(2)
2.061(1)
172.0(1)
172.8(1)
177.1(1)
174.0(1)
16.8(2)
−16.4(2)
16.6(3)
4.1(2)
2.089(2)
2.094(2)
2.095(2)
2.105(2)
2.113(2)
2.089(2)
2.098(1)
2.092(3)
2.101(1)
170.71(8)
171.73(8)
176.10(8)
172.85(7)
−7.4(3)
2.062(2)
2.058(2)
2.058(2)
2.070(2)
2.061(2)
2.050(2)
2.060(1)
2.060(3)
2.060(1)
173.1(1)
173.4(1)
175.9(1)
174.1(1)
−2.9(3)
−5.2(3)
4.1(4)
2.148(4)
2.138(3)
2.109(5)
2.080(3)
2.102(4)
2.098(5)
2.113(2)
2.143(5)
2.097(3)
171.0(2)
173.9(2)
176.9(2)
173.9(2)
−5.0(6)
3.0(6)
4.0(8)
−13.5(6)
11.0(6)
15.9(6)
−20.1(5)
15.1(4)
Ru−N2
Ru−N3
Ru−N4
Ru−N5
Ru−N6
Avg Ru−N
Avg Ru−N (oxygenated N,N)
Avg Ru−N (“spectator” N,N)
N−Ru−N1
N−Ru−N2
N−Ru−N3
Avg trans angle
L1 torsiond (oxygenated N,N)
−3.8(3)
Avg |L1 torsion|
5.6(4)
−21.5(3)
15.0(3)
−20.6(3)
13.9(3)
17.8(2)
L2 torsiond (“spectator” N,N)
4.5(3)
−4.5(2)
−6.0(2)
5.0(2)
−5.6(3)
−5.2(3)
5.1(3)
L3 torsiond (“spectator” N,N)
Avg |L2 or L3 torsiond|
4.9(1)
5.1(2)
a Data for 2OH = [(phen)2Ru(6,6′-dhbp)]Cl2 has been previously reported.19 b Data for 5 = [(phen)2Ru(4,4′-dhbp)]Cl2 has been previously reported.19
c Data for 6 = [Ru(dmphen)2(AFO)]2+ was reproduced from the literature.24 d These torsion angles are for Ru–N–C–C′ and Ru–N′–C′–C and they
involve the C and C′ atoms which connect the pyridine derived rings.
ments were performed in aqueous solutions with a Britton– mine quantum yields as shown in Table 1 (see the ESI† for
Robinson buffer system as electrolyte. All complexes studied further details). To facilitate comparison, the quantum yields
exhibited reversible waves assigned to RuII/III couple (see the of the parent analogues 2OH and 3OH are also shown in
ESI† for CV spectra and further details). The E1/2 values vs. Ag/ Table 1. All compounds were studied under acidic conditions
AgCl of all three new complexes are shown in Table 1. The (pH 5.0–5.1) to ensure that the protic complexes are present as
metal based redox potentials are a great probe for the electron the more photolabile dicationic species bearing OH groups.
density on the central metal. Compared to 2OMe and 3OMe (E1/2 This work shows that the quantum yields increase in the
= 0.99 and 0.98 V, respectively), the significantly lower E1/2 order: 3OH < 4OH < 2OH < 3OMe ≪ 2OMe. Comparing these
(RuII/III) for 4OH (0.66 V) reflects the higher electron density on values, the quantum yields for 3OH, 4OH, and 2OH are all
Ru due to the electron-donating OH groups which are predo- similar and quite low (∼10−3). Most surprising in this series is
minantly deprotonated under the experimental CV conditions that 2OH and 4OH have similar quantum yields, despite the
(pH = 7.0, which was chosen to model physiological con- crystal structure of 4OH showing more distortion in the specta-
ditions). Similarly, the metal complexes [(bipy)2Ru(6,6′- tor ligands. Comparing 3OH to 3OMe, we observe a three-fold
dhbp)]2+ (1OH) and [(bipy)2Ru(4,4′-dhbp)]2+ show RuII/III redox increase in φPD by using the more-bulky methoxy group.
potentials of 1.12 V and 1.16 V vs. SCE respectively, in aceto- Furthermore, electronics may play a role in that 3OH is
nitrile.20 Deprotonation is expected to shift these potentials expected to have some (49.6%) mono-deprotonated species
lower, and for [(bipy)2Ru(6,6′-(O)2-bipy] (1O− in Scheme 1) an present in solution at pH 5.0, which is expected to be less
irreversible wave at 0.45
observed.20 Despite differences in solvent, the general trend is highest for complex 2OMe at 0.024(6). Thus, a twelve-fold
suggests that the differences in redox potentials between 2OMe increase in quantum yield occurs in moving from 2OH to 2OMe
V
vs. SCE in acetonitrile was photolabile with a monoanionic ligand.19 The quantum yield
.
and 3OMe vs. 4OH are largely due to ligand deprotonation In our prior work, we showed a 56-fold increase in the
which is only possible for 4OH
.
quantum yield (Table 1) by going from predominantly 2O− to
OH by lowering the pH from 7.5 to 5.0. Thus, the change from
2OH to 2OMe can be attributed to both sterics and to eliminat-
2
Photodissociation
All three new ruthenium complexes undergo blue light (λ = ing the protic ligand, which at pH 5.0 for 2OH leads to 38.5%
450 nm) induced ligand dissociation in aqueous solution. The of the mono-deprotonated species based on the pKa values.19
photodissociation rate is known to be directly related to the In summary, it appears from comparing 2OMe and 4OH that
steric bulk close to the metal center, as increasing steric strain steric bulk on the 6,6′-dmbp ligand (with two methoxy groups
is known to lower the energy of the 3MC state, thus making near the metal) is a more effective strategy for favoring photo-
the pathway to photodissociation products more accessible.28 dissociation vs. the use of dmphen (with four methyl groups
The process was followed by UV-vis spectroscopy using actino- near the metal). We speculate that this may be due to the
metry to measure photons absorbed which allowed us to deter- presence of steric bulk in a ligand that already has weaker
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Ru–N bonds due to the electronic effects of the methoxy similar to 2OMe and 3OMe, can be deprotonated at pH 7.4 to
group. It is known that the addition of OH or OMe groups give more lipophilic neutral species. This deprotonation
ortho to nitrogen results in a less basic bipyridine ligand appears to increase log(Do/w) by over two orders of magnitude.
which implies a weaker Ru–N bond strengths.25,29,30 The ease We postulate that the low toxicity observed for these new com-
of ligand distortion is evident in terms of the tilting of the 6,6′- pounds is both due to poor uptake and low toxicity for the pro-
dmbp ligand below the equatorial plane in 2OMe (Fig. 2).
ducts of photodissociation (see further discussion below).
Cytotoxicity towards breast cancer cells
Using the same protocol as reported previously,19 breast
cancer cells were incubated with the ruthenium complexes for
one hour and then they were exposed to blue light or kept in
the dark as a comparison. The resulting IC50 values are shown
Conclusions
By installing methoxy groups at the ortho to nitrogen posi-
tions, we have made ruthenium complexes that photodissoci-
ate more readily than any of the compounds reported in our
prior work. In one exemplary example, by adjusting the func-
tional groups from 2O− to 2OH to 2OMe, we are able to increase
the quantum yields by 56-fold (with pH changes19) and then
another 12-fold (with synthesis), for a combined quantum
yield enhancement of 667-fold (to φPD = 0.024(6) for 2OMe). It
appears that the quantum yield enhancement is due to both
steric and electronic factors: namely that it is important to
have the sterically bulky ligand be a weak sigma donor with
inherently weak Ru–N bond strengths. Illustrating this trend,
4OH (with the 4,4′-dhbp ligand) displayed a relatively low
quantum yield despite having four methyl groups near the
metal center from the two dmphen ligands. Despite the rela-
tively large quantum yield for 2OMe, it was non-toxic. This can
be (partially) attributed to an unfavorable log(Do/w) value
which would lead to poor drug uptake by passive diffusion.
Furthermore, note that 2OMe photodissociation leads to the
in Table 3. All of the new compounds reported (2OMe, 3OMe
,
4
OH) are essentially non-toxic vs. MDA-MB-231 breast cancer
cells. This stands in marked contrast to complex 2OH which
had some light triggered toxicity toward certain cell lines and
3
OH which displayed good cytotoxicity (IC50 light = 3.7 μM) and
photo-toxicity indices against several cell lines.19 In our prior
work, cellular uptake played a key role in determining toxicity,
and thus we suspected that poor cellular uptake may be
responsible for the low toxicity of 2OMe, 3OMe, and 4OH
.
Octanol/water partition coefficients
Octanol/water partition coefficients (log(Do/w) provide a con-
venient measure by which to estimate cellular uptake by
passive diffusion. Lipophilic complexes display positive log
(Do/w) values that are ideally between 2–6 for good cellular
uptake with sufficient water solubility for drug adminis-
tration.16,31,32 Conversely, without appreciable cellular uptake
it is unlikely that a drug will generate appreciable cytotoxicity,
unless the cellular membrane is compromised upon light
irradiation. We measured the log(Do/w) values of these com-
pounds at pH 7.4 to simulate physiological conditions, as
shown in Table 1. Complexes 2OMe and 3OMe are dicationic at
all pH values due to the aprotic ligands. Thus, these complexes
are quite hydrophilic which leads to negative log(Do/w) values.
In contrast, 4OH is majority deprotonated at pH 7.4 which
leads to a positive (but low) log(Do/w) value of 0.58(2).
same photoproduct as 2OH
,
namely [(phen)2Ru(OH2)2]2+.
Similarly, 3OMe photodissociation leads to [(dop)2Ru(OH2)2]2+
(same photoproduct as for 3OH). Therefore, this work also
leads us to question whether, in our prior work with 2OH and
3OH
, these photoproducts were the true toxic species.
Alternative hypotheses can include that the photo-released free
ligand (e.g. 6,6′-dhbp)14 was in fact toxic (we note that 6,6′-
dhbp was non-toxic when administered directly to cells,19 but
we cannot rule out that ruthenium acts as a drug delivery
vehicle) or that we were generating singlet oxygen13 as a toxic
species in our past work.19 We note that 3OH had a very low
quantum yield (2.2 × 10−4) for photodissociation at physiologi-
cal pH (Table 1). More effective photo dissociating ruthenium
complexes will typically have quantum yields of 10−1 to
Nonetheless, all three compounds appear to have log(Do/w
)
values that are too low (cf. 2OH and 3OH in Table 1) to lead to
appreciable cytotoxicity. Again, 2OH and 3OH, while structurally
10−2 6,33
We will clarify this matter with a separate publication
.
addressing the cause of toxicity in 3OH. We summarize our
Table 3 Breast cancer (MDA-MB-231) cell cytotoxicity data when
treated with ruthenium complexes at pH 7.4 in the dark or upon studies of 2OMe, 3OMe, and 4OH by stating that log(Do/w) values
irradiation for one hour with blue light (λ = 450 nm). All IC50 values are
in µM
are not ideal for uptake by passive diffusion and, despite excel-
lent quantum yields, the species generated by photodissocia-
tion are not sufficiently toxic to trigger cell death from outside
Compound
IC50 dark
IC50 light
the cell membrane. Importantly, we have elucidated a key
factor in controlling quantum yields by installing sterically
bulky groups on weak donor ligands. Furthermore, these
changes help to separate the contributions from sterics (2OMe
vs. 2OH) vs. electronics (2OH vs. 2O−) in determining the
quantum yields and this study suggests that electronics play a
dominant role in this case.
2OMe
3OMe
4OH
>100
>100
>100
280
>100
>100
>100
83
2OH a
3OH a
190
3.7
a The IC50 values for both 2OH and 3OH have been reported
previously.19
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Paper
(0.0454 g, 0.2066 mmol, 1.1 eq.) were measured into the flask.
The final dark brown suspension was heated to reflux in an oil
bath at 110 °C under N2 for 24 h. After cooling down, the solu-
Experimental section
General specifications: materials
All syntheses were carried out under a nitrogen atmosphere tion was filtered to remove any excess 6,6′-dmbp ligand.
using glovebox or standard Schlenk techniques if not other- Solvent was removed by rotary evaporation. The product was
wise indicated. All commercially available reagents were pur- collected by vacuum filtration with a diethyl ether wash. Yield:
chased from Sigma-Aldrich, Acros, Strem or Pressure Chemical 0.0784 g, 0.1047 mmol, 56%. The crude product was purified
and were used as received without further purification. (COD) by recrystallization from acetonitrile solution by slow diffusion
RuCl2, (phen)2RuCl2, (dop)2RuCl2, 6,6′-dmbp and 4,4′-dhbp of ether. 1H NMR (500 MHz, CD3CN) δ 8.70 (dd, J = 8.3, 1.3 Hz,
were synthesized according to previously published pro- 2H), 8.44 (dd, J = 8.2, 1.3 Hz, 2H), 8.38 (dd, J = 5.3, 1.3 Hz, 2H),
cedures.19,26,30 Dry solvents were obtained by passing through 8.27 (m, 4H), 8.19 (d, J = 8.9 Hz, 2H), 8.03 (dd, J = 8.5, 7.8 Hz,
a column of activated alumina using a Glass Contour Solvent 2H), 7.84 (dd, J = 8.3, 5.2 Hz, 2H), 7.64 (dd, J = 5.3, 1.3 Hz, 2H),
Purification System built by Pure Process Technology, LLC. pH 7.40 (dd, J = 8.2, 5.3 Hz, 2H), 6.82 (dd, J = 8.5, 1.3 Hz, 2H), 2.86
measurements were carried out using a VWR SympHony pH (s, 6H). 13C NMR (126 MHz, CD3CN) δ 170.48 (s), 158.00 (s),
meter, utilizing a three-point calibration at pH = 4, 7, and 10. 154.61 (s), 153.18 (s), 149.83 (s), 149.35 (s), 142.04 (s), 137.38
Britton–Robinson buffer solutions were made from a stock (s), 136.49 (s), 131.75 (s), 131.28 (s), 128.87 (s), 128.62 (s),
solution of 0.04 M acetic acid, 0.04 M boric acid, and 0.04 M 126.79 (s), 125.88 (s), 118.55 (s), 56.99 (s). Elem. anal.: found:
phosphoric acid with the addition of 0.2 M sodium hydroxide C, 53.16; N, 10.40; and H, 4.44%. Calc. for [(phen)2Ru(6,6′-
to achieve an approximate pH of 7 for the solution after the dmbp)]Cl2·4H2O, C36H36Cl2N6O6Ru: C, 52.69; N, 10.24; and H,
addition of the metal complex.
4.42%. ESI-MS (m/z): 339.1, calcd for [(phen)2Ru(6,6′-dmbp) ]2+,
[C36H28N6O2Ru]2+: 339.1.
General specifications: instruments and analysis
1H and 13C spectra were acquired at room temperature on a
[(dop)2Ru(6,6′-dmbp)]Cl2 (3OMe
)
Bruker AV360 360 MHz or AV500 500 MHz spectrometer, as A similar procedure reported for the synthesis of [(phen)2Ru
designated. Chemical shifts are reported in ppm and refer- (6,6′-dhbp)Cl2] was used.19 The whole process was protected
enced to residual solvent resonance peaks. Abbreviations for from light as the final product is sensitive to light. An oven
the multiplicity of NMR signals are s (singlet), d (doublet), dried Schlenk flask equipped with a stir bar charged with 1 : 1
t (triplet), q (quartet), m (multiplet), br (broad). UV-visible volume ratio of DI water and ethanol (20 mL overall) and was
spectra were recorded on a PerkinElmer Lambda 35 UV-visible purged with nitrogen for 20 minutes. (dop)2RuCl2 (0.0455 g,
spectrometer. Mass spectrometric data were collected on a 0.0694 mmol) and 6,6′-dimethoxy-2,2′-bipyridine (0.0178 g,
Waters AutoSpec-Ultima NT spectrometer with electron ioniza- 0.0763 mmol, 1.1 eq.) were measured into the flask. The final
tion method. ESI mass-spectrometry was provided by the dark brown suspension was heated to reflux in an oil bath at
University of Alabama Mass Spectrometry Resource. Elemental 110 °C under N2 for 24 h. After cooling down, the solution was
analyses were performed by Atlantic Microlab, Inc., Norcross, filtered to remove any excess 6,6′-dmbp ligand. Solvent was
GA. Cyclic voltammetry measurements were performed on a removed by rotary evaporation. The product was collected by
Bioanalytical Systems CW-50 potentiostat. Typical concen- vacuum filtration with a diethyl ether wash. The crude product
trations for metal complexes ranged from 0.64 to 0.84 mM. was purified by recrystallization from acetonitrile solution by
Studies were carried out in aqueous Britton–Robinson buffer slow diffusion of ether. Yield: 0.0490 g, 0.0567 mmol, 81.6%.
solutions as the supporting electrolyte. The pH of each solu- 1H NMR (500 MHz, CD3CN) δ 8.63 (dd, J = 8.4, 1.3 Hz, 2H),
tion was checked after the dissolution of metal complex for 8.40 (dd, J = 8.4, 1.3 Hz, 2H), 8.22 (m, 4H), 8.03 (t, J = 8.3, 2H),
study. A three-electrode setup with an Ag/AgCl reference elec- 7.75 (dd, J = 8.4, 5.2 Hz, 2H), 7.50 (dd, J = 5.3, 1.3 Hz, 2H), 7.35
trode in 3 M aqueous sodium chloride (electrode model (dd, J = 8.3, 5.3 Hz, 1H), 6.83 (d, 2H), 4.62 (m, 4H), 2.91 (s,
MF-2052 from Bioanalytical Systems), platinum wire auxiliary 6H). 13C NMR (126 MHz, CD3CN) δ 170.41 (s), 157.93 (s),
electrode, and glassy carbon working electrode was used. In all 152.44 (s), 151.05 (s), 145.39 (s), 144.92 (s), 141.98 (s), 135.60
studies, the solutions were degassed for approximately (s), 135.41 (s), 130.05 (s), 129.18 (s), 126.28 (s), 125.95 (s),
30 minutes with argon prior to data collection and the glassy 125.43 (s), 125.41 (s), 109.33 (s), 66.20 (s), 57.04 (s). Elem.
carbon electrode was polished before each scan.
anal.: found: C, 50.75; N, 8.98; and H, 4.37%. Calc. for
[(dop)2Ru(6,6′-dmbp)]Cl2·5H2O, C40H42Cl2N6O11Ru: C, 50.32;
N, 8.80; and H, 4.43%. ESI-MS (m/z): 829.1, calcd for [(dop)2Ru
[(phen)2Ru(6,6′-dmbp)]Cl2 (2OMe
)
A similar procedure reported for the synthesis of [(phen)2Ru (6,6′-dmbp) Cl]+, [C40H32ClN6O6Ru]+: 829.1; 779.2, calcd for
(6,6′-dhbp)Cl2] was used.19 The whole process was protected [(dop)2Ru(6,6′-dmbp) − CH3]+, [C39H29N6O6Ru]+: 779.1; 397.2,
from light as the final product is sensitive to light. An oven calcd for [(dop)2Ru(6,6′-dmbp)]2+, [C40H32N6O6Ru]2+: 397.1.
dried Schlenk flask equipped with a stir bar was charged with
[(dmphen)2Ru(4,4′-dhbp)]Cl2 (4OH
)
1 : 1 volume ratio of DI water and ethanol (40 mL overall) and
was purged with nitrogen for 20 minutes. (phen)2RuCl2 The compound [(dmphen)2Ru(4,4′-dhbp)](PF6)2 was made by
(0.1010 g, 0.1878 mmol) and 6,6′-dimethoxy-2,2′-bipyridine following a similar procedure as reported previously.20 40 mL
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of ethylene glycol was degassed with argon for 30 min in a
The structure of 4OH was found to contain several regions
round-bottom flask. To the flask, 4,4′-dhbp (0.1762 g, of residual density. Attempts to model the remaining residual
0.936 mmol) and previously synthesized Ru(dmphen)2Cl2 density as ethanol molecules were not successful, so the
(0.4076 g, 0.693 mmol) were added. The solution was heated residual density was “SQUEEZED” (i.e., applied a “solvent
to 190 °C under argon for 4 h. The solution turned a deep red mask”) out using the PLATON program.38 The solvent accessi-
as the reaction proceeded. The solution was removed from ble volume was found to be 520 Å3. The electrons found in
heat and cooled to room temperature, followed by filtration solvent accessible void is 135e−, which corresponds to approxi-
through a Büchner funnel to remove insoluble impurities. mately 5 ethanol molecules per asymmetric unit. Hydroxyl H
Five-fold excess (0.8128 g) of ammonium hexafluorophosphate atom positions were restrained based on hydrogen bonding
was dissolved in 20 mL of water and added to the filtrate to considerations (DFIX and DANG).
−
precipitate the PF6 salt. The solution was filtered and left to
sit on vacuum overnight. The next day the remaining powder
Cell culture
was rinsed with ether. [Ru(dmphen)2(4,4′-dhbp)][PF6]2·4H2O
MDA-MB-231 cell line was purchased from American Type
(0.6206 g, 0.581 mmol) was collected (83.8% yield). It was then
−
Culture Collection (ATCC). MDA-MB-231 cells of less than 10
passages were grown using DMEM (Gibco, 21063-029) sup-
plemented with 10% FBS (Gibco, 26140079).
converted to the chloride salt in the following way: the PF6
salt (210 mg) was measured into a 100 mL flask with 50 mL of
acetone. The solution was stirred in dark for 20 minutes. The
solution was filtered to remove the undissolved powder. To the
clear red filtrate, saturated Et4NCl in acetone was added, until Determination of the IC50 values for 2OMe, 3OMe, and 4OH
no further precipitate was formed. The red precipitate was col-
MDA-MB-231 (ATCC, HTB-26) cells were grown with DMEM
lected by filtration, washed with acetone, ether, and then dried
(Gibco, 21063-029) supplemented with 10% fetal bovine serum
under vacuum. Crystals suitable for X-ray analysis were grown
(Gibco, 26140079) at 37 °C with 5% CO2. IC50 values were
by slow diffusion of ether into the ethanol solution of the com-
determined as reported previously.19 Briefly, MDA-MB-231
cells were seeded in 96-well plates with 5000 cells per well and
incubated for 24 h. The ruthenium compounds were dissolved
in dimethyl sulfoxide (DMSO) and serially diluted in media.
Final DMSO concentration was lower than 0.1% to prevent any
cytotoxicity from DMSO. After 24 h of incubation, the diluted
compounds were added to each well and incubated for 48 h.
Cells were washed with phosphate buffer saline (PBS) twice
pound. 1H NMR (500 MHz, DMSO) δ 12.03 (br, 2H), 8.79 (d, J =
8.3 Hz, 2H), 8.52 (d, J = 8.3 Hz, 2H), 8.34 (d, J = 8.7 Hz, 2H),
8.23 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 8.3 Hz, 2H), 7.68 (s, 2H),
7.61 (d, J = 8.3 Hz, 2H), 6.51 (m, 4H), 1.99 (s, 6H), 1.83 (s, 6H).
13C NMR (126 MHz, DMSO) δ 167.60 (s), 165.90 (s), 165.84 (s),
157.85 (s), 151.54 (s), 149.00 (s), 148.11 (s), 137.38 (s), 136.42
(s), 129.28 (s), 127.32 (s), 126.97 (s), 126.90 (s), 126.56 (s),
114.63 (s), 111.34 (s), 25.74 (s), 24.32 (s). Elem. anal.: found: C,
and fed with fresh media. The plate was irradiated by blue
54.75; N, 9.72; and H, 4.84%. Calc. for [(dmphen)2Ru(4,4′-
light (Phillips, goLITE BLU) for an hour and incubated over-
night. Cell Counting Kit-8 (Enzo Life Sciences) was used to
determine cell viability. IC50 values were determined using
nonlinear regression in Minitab 17.
dhbp)]Cl2·EtOH·3H2O, C44H44Cl2N6O6Ru: C, 54.79; N, 9.59;
and H, 5.06%. ESI-MS (m/z): 353.2, calcd for [(dmphen)2Ru
(4,4′-dhbp) ]2+, [C38H32N6O2Ru]2+: 353.1.
Single crystal X-ray diffraction (SC-XRD) structure
determinations
Conflicts of interest
Crystals of appropriate dimension were mounted on a Mitgen
There are no conflicts to declare.
cryoloop or glass filament in
a
random orientation.
Preliminary examination and data collection were performed
on a Bruker ApexII CCD-based X-ray diffractometer equipped
with an Oxford N-Helix Cryosystem low temperature device
and a fine focus Mo-target X-ray tube (λ = 0.71073 Å) operated
Acknowledgements
at 1500 W power (50 kV, 30 mA). The X-ray intensities were We thank the Undergraduate Creativity and Research Academy
measured at low temperature (223(2) K). The collected frames (UCRA) at UA, the Research Grants Committee (RGC) at UA to
were integrated with the Saint34 software using a narrow-frame ETP and YK, and NSF EPSCoR Track 2 Grant to ETP and RHS
algorithm. Data were corrected for absorption effects using the for support (PI: N. Hammer, Grant OIA-1539035), the
multi-scan method in SADABS.35 The space groups University of Alabama (ETP and YK), and Villanova University
were assigned using XPREP of the Bruker ShelXTL package,36 (JJP) for generous financial support. We also thank Qiaoli
solved with ShelXT36 and refined with ShelXL36 and the Liang (the University of Alabama) for MS analysis. Finally, we
graphical interface ShelXle.37 All non-hydrogen atoms were thank M. K. Thompson and S. A. McFarland for helpful discus-
refined anisotropically. H atoms attached to carbon were sion and the members of the Papish group for assistance and
positioned geometrically and constrained to ride on their suggestions. We thank Jessica L. Gray for assistance with
parent atoms.
measuring log(Do/w) values.
15692 | Dalton Trans., 2018, 47, 15685–15693
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Notes and references
1 Abbreviations: PACT
= photo-activated chemotherapy,
6,6′-dhbp = 6,6′-dihydroxybipyridine, 6,6′-dmbp = 6,6′-
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Dalton Trans., 2018, 47, 15685–15693 | 15693