Ruthenium Complexes are pH-Activated Metallo Prodrugs (pHAMPs

Jun 21, 2017 - Synopsis. New ruthenium complexes use two triggers to target cancer cells. Both light and low pH serve to activate these photolabile ru...
0 downloads 0 Views 5MB Size
Article pubs.acs.org/IC

Ruthenium Complexes are pH-Activated Metallo Prodrugs (pHAMPs) with Light-Triggered Selective Toxicity Toward Cancer Cells Fengrui Qu,† Seungjo Park,‡ Kristina Martinez,§ Jessica L. Gray,† Fathima Shazna Thowfeik,∥ John A. Lundeen,† Ashley E. Kuhn,⊥ David J. Charboneau,†,⊥ Deidra L. Gerlach,† Molly M. Lockart,† James A. Law,† Katherine L. Jernigan,† Nicole Chambers,† Matthias Zeller,# Nicholas A. Piro,g W. Scott Kassel,⊥ Russell H. Schmehl,§ Jared J. Paul,*,⊥ Edward J. Merino,*,∥ Yonghyun Kim,*,‡ and Elizabeth T. Papish*,† †

Department of Chemistry, The University of Alabama, Tuscaloosa, Alabama 35487-0336, United States Department of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, Alabama 35487-0203, United States § Department of Chemistry, Tulane University, New Orleans, Louisiana 70118, United States ∥ Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221, United States ⊥ Department of Chemistry, Villanova University, Villanova, Pennsylvania 19085, United States # Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, United States g Department of Chemistry, Albright College, Reading, Pennsylvania 19612, United States ‡

S Supporting Information *

ABSTRACT: Metallo prodrugs that take advantage of the inherent acidity surrounding cancer cells have yet to be developed. We report a new class of pH-activated metallo prodrugs (pHAMPs) that are activated by light- and pH-triggered ligand dissociation. These ruthenium complexes take advantage of a key characteristic of cancer cells and hypoxic solid tumors (acidity) that can be exploited to lessen the side effects of chemotherapy. Five ruthenium complexes of the type [(N,N)2Ru(PL)]2+ were synthesized, fully characterized, and tested for cytotoxicity in cell culture (1A: N,N = 2,2′-bipyridine (bipy) and PL, the photolabile ligand, = 6,6′-dihydroxybipyridine (6,6′-dhbp); 2A: N,N = 1,10-phenanthroline (phen) and PL = 6,6′-dhbp; 3A: N,N = 2,3dihydro-[1,4]dioxino[2,3-f ][1,10]phenanthroline (dop) and PL = 6,6′-dhbp; 4A: N,N = bipy and PL = 4,4′-dimethyl-6,6′dihydroxybipyridine (dmdhbp); 5A: N,N = 1,10-phenanthroline (phen) and PL = 4,4′-dihydroxybipyridine (4,4′-dhbp). The thermodynamic acidity of these complexes was measured in terms of two pKa values for conversion from the acidic form (XA) to the basic form (XB) by removal of two protons. Single-crystal X-ray diffraction data is discussed for 2A, 2B, 3A, 4B, and 5A. All complexes except 5A showed measurable photodissociation with blue light (λ = 450 nm). For complexes 1A−4A and their deprotonated analogues (1B−4B), the protonated form (at pH 5) consistently gave faster rates of photodissociation and larger quantum yields for the photoproduct, [(N,N)2Ru(H2O)2]2+. This shows that low pH can lead to greater rates of photodissociation. Cytotoxicity studies with 1A−5A showed that complex 3A is the most cytotoxic complex of this series with IC50 values as low as 4 μM (with blue light) versus two breast cancer cell lines. Complex 3A is also selectively cytotoxic, with sevenfold higher toxicity toward cancerous versus normal breast cells. Phototoxicity indices with 3A were as high as 120, which shows that dark toxicity is avoided. The key difference between complex 3A and the other complexes tested appears to be higher uptake of the complex as measured by inductively coupled plasma mass spectrometry, and a more hydrophobic complex as compared to 1A, which may enhance uptake. These complexes demonstrate proof of concept for dual activation by both low pH and blue light, thus establishing that a pHAMP approach can be used for selective targeting of cancer cells.



target all quickly dividing cells, including healthy cells. There is a need for new targeted therapies. Targeted therapies can use the unique redox properties of the cell, the lowered pH of the cells,

INTRODUCTION For certain types of cancer (e.g., testicular cancer), platinumbased drugs are commonly prescribed as one of the most effective treatments. However, the side effects of treatment can include nerve damage, nausea, gastrointestinal distress, hair loss, and other life-threatening side effects. Cisplatin and carboplatin © 2017 American Chemical Society

Received: April 27, 2017 Published: June 21, 2017 7519

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

an IC50 of 20 μM versus a colon carcinoma cell line,7 and it is activated by reduction of RuIII to RuII. Both NKP-1339 and KP1019 (a close analogue) have performed well in clinical trials with minimal side effects.8,9 While these drugs do bind DNA in the cell nuclei, the evidence suggests that DNA binding is not the primary mode of action.9 Rather, NKP-1339 and KP1019 are believed to kill cancer cells by disturbing the cellular redox balance, which can lead to cell cycle arrest and apoptosis.9 NAMI-A also entered clinical trials and was less cytotoxic (IC50 ≈ 400 μM) but had anti-metastasis activity.10 NAMI-A’s activity appears to derive from its ability to scavenge nitric oxide and thereby prevent endothelial cell migration and angiogenesis.11,12 The extensive studies of NAMI-A and NKP-1339 also illustrate that similar complexes can have vastly different modes of action and, furthermore, that in vitro toxicity (in cell culture) often does not predict in vivo toxicity (in animal models) or success in clinical trials.9,13 Most notably, a light-activated ruthenium complex (TLD-1433) is about to enter clinical trials in Canada for bladder cancer, showing the promise of drugs that are visible light-activated via PDT.14,15 TLD-143316,17 and many other PDT agents18−20 act as photosensitizers and generate singlet oxygen to kill cancer cells. Our system is dif ferent, in that it does not require oxygen and can work in hypoxic solid tumors. Our work falls under photoactivated chemotherapy (PACT),21,22 but our system is unique in using both pH and light for activation (Scheme 1). The use of prodrugs that are activated by the low pHe present in cancerous tissue has been rare.4 Tumor-activated prodrugs that use pH for drug activation have been limited to organic drugs and polymers.23−25 Some of the hardest cancers to treat (including “triple negative” breast cancer (TNBC) and cancer stem cells (CSCs)) acidify the extracellular matrix (ECM) to a significant extent26 and should be vulnerable to a strategy that derives its selectivity from inherent pH differences. Cancerous cells typically have a low pHe of 6.2−6.8 (some sources mention pHe values as low as 5),26 whereas normal cells have a higher pHe of 7.2−7.4.27 Cancer cells exhibit fast growth and high aerobic glycolysis that leads to low pHe.27,28 Our hypothesis is that by designing compounds with appropriate features we can ensure both good uptake and light-triggered toxicity (Schemes 1 and 2). Glazer et al. have made great progress in the use of ruthenium complexes that readily photodissociate to bind DNA and cause cytotoxicity.21,29,30 These studies have used steric bulk near the metal center to destabilize the octahedral geometry and facilitate photolabilization of a ligand. The N,N ligands 2,3-dihydro[1,4]dioxino[2,3-f ][1,10]phenanthroline (dop), 2,9-dimethyl1,10-phenanthroline (dmphen), and 2,2′-biquinoline (biq) are known to enhance photodissociation (with blue, red, or near-IR light) by causing strain in octahedral ruthenium complexes.21,29,30 Turro, Dunbar, and co-workers have used cytotoxic ligands (e.g., 5-cyanouracil) on ruthenium for light-triggered release.31−34 Meggers and Gasser have shown that attachment of a drug to a metal center can alter its cytotoxicity and its subcellular localization.19,35−37 McFarland, Sadler, and others have shown that changing the charge of the metal complex (e.g., with cyclometalated C,N bound ligands) can greatly alter the cytotoxicity.6,38−41 We plan to expand upon these studies by investigating new strategies with pH-sensitive hydroxy-substituted ligands, using our synthetic experience to make new ligands and metal complexes.42−46 In a prior publication and patent application we reported a prototype complex, [(bipy)2Ru(6,6′-dhbp)]2+ (1A), where 6,6′-dhbp = 6,6′-dihydroxybipyridine, which was both light- and

and focused light to minimize off target effects. Light is already used to treat certain cancers using the Food and Drug Administration-approved technique of photodynamic therapy (PDT).1 Targeted therapies for cancer often rely on the inherent differences between cancerous and normal tissues.2,3 Each cancer has unique genetic mutations, but certain metabolic characteristics are common to most cancers. These similarities include that most solid tumors are hypoxic and exhibit decreased extracellular pH (pHe) relative to normal tissue.4,5 These metabolic differences are known as the Warburg effect, and they present a unique opportunity in cancer research that has been unexploited thus far in metal-based drugs. Our work aims to target the lowered external pH of cancerous cells through pH-driven dif ferences in photodissociation with visible light. Ruthenium complexes have shown promise for pH and light-activated cytotoxicity (Schemes 1 and 2).2,3 Scheme 1. Conceptual Framework for pH-Activated Metallo Prodrugs

Scheme 2. A Mechanisma for Cancer Selectivity Based upon pH with 1A = [(bipy)2Ru(6,6′-dhbp)]2+

a

A similar mechanism is proposed for derivatives that vary the spectator and photolabile ligands.

Ruthenium anticancer agents have been studied less than platinum agents, but they offer advantages in terms of more sites for binding ligands (six for octahedral Ru(II) or Ru(III) versus four for square planar Pt(II)) and thus more structural diversity. The activity of ruthenium complexes in cells is often governed by their three-dimensional structure, redox potential, and their relative lipophilicity/hydrophilicity, which determines uptake.6 Metal-based prodrugs that are activated in a hypoxic, reducing environment have been developed, and two of these redoxactivated prodrugs have entered clinical trials (Chart 1). For example, NKP-1339, [Na][trans-RuIIICl4(benzimidazole)2] has Chart 1. Ruthenium Complexes That Have Entered Clinical Trials

7520

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

photodissociation rates). The new complexes 2A−4A were fully characterized by spectroscopic and analytical techniques. Synthesis and Characterization of the Ligands and Metal Complexes. The spectator ligands (N,N) were purchased (bipy, phen) or synthesized (dop) using known procedures.49−51 The new photolabile ligand dmdhbp was synthesized by modification of known procedures.52 As shown in Scheme 4, the commercially available 2,6-dichloro-4-methylpyridine

pH-activated by ligand loss and showed modest toxicity (IC50 = 88 μM) toward HeLa (cervical cancer) cells.2,3 In this work, we vary both the spectator and the photolabile ligand to make new ruthenium complexes that are more cytotoxic and show selective toxicity for cancerous versus normal cells. These new complexes also offer improved phototoxicity indices.



RESULTS Research Design. Our prototype molecule, [(bipy)2Ru(6,6′-dhbp)]2+ (1A), is an active prodrug in vitro due to photodissociation in blue light to give a complex that binds to biological targets (Scheme 2). The photolabile ligand 6,6′-dhbp and closely related ligands (e.g., 4,4′-dimethyl-6,6′-dihydroxybipyridine (dmdhbp) in Scheme 3) were specifically designed for this

Scheme 4. Synthesis of 4,4′-Dimethyl-6,6′dihydroxybipyridine

Scheme 3. New Complexes Designed as pHAMPs

was treated with a large excess of sodium metal in dry methanol under air-free conditions. The resulting 2-chloro-6-methoxy-4methylpyridine was used without purification in the coupling reaction with itself in the presence of Zn, Bu4NBr, and NiCl2(PPh3)2 as the catalyst in dry dimethylformamide (DMF) to afford the 6,6′-dimethoxy-4,4′-dimethyl-2,2′-bipyridine. A deprotection procedure involving treatment with HBr/ AcOH yielded the final product, dmdhbp. The synthesis of 1A has been reported previously.2 Using a similar procedure, RuCl3·(H2O)x was treated with cycloocta-1,5diene (cod) to afford the intermediate (η4-cod)RuCl2. This intermediate was then treated with 2 equiv of the spectator ligand (N,N) in 1,2-dichlorobenzene to yield cis-(N,N)2RuCl2. After purification, treatment of cis-(N,N)2RuCl2 with the photolabile ligand (PL = 6,6′-dhbp or dmdhbp) in aqueous and/or alcoholic solution led to complexes 1A−4A as the chloride salt, [(N,N)2Ru(PL)]Cl2 (Scheme 5). The final products were

project for three reasons. (1) The ligand provides steric bulk near the metal center, which distorts the metal geometry and facilitates light-triggered ligand loss due to weakened M−N bonds. Upon photoexcitation, an electron will be promoted into a metal−ligand antibonding molecular orbital that disrupts the metal−ligand interaction and causes ligand dissociation. Lighttriggered excitation leads to occupation of this metal-centered excited state that weakens metal−ligand bonding (Figure 7). (2) The ligand is pH-sensitive and will have a different protonation state in cancerous versus normal tissue. These protonation states will differ with respect to how readily they undergo [light-triggered] ligand loss. (3) The ligand is new in transitionmetal chemistry, with the Papish group reporting some of the first metal complexes of this ligand in 2011.44,46−48 Thus, its biochemistry and potential uses were unknown when we began this project. We varied the spectator bidentate (N,N) ligand from bipy (1A) to phenanthroline (phen in 2A) to 2,3-dihydro-[1,4]dioxino[2,3-f ][1,10]phenanthroline (dop in 3A; Scheme 3). The dop ligand is described in the literature as enhancing photodissociation by creating a twist via the nonplanar six-membered ring containing sp3 atoms (O−CH2−CH2−O).21 Thus, we synthesized a series of new ruthenium complexes (2A−4A) that are both pH and light activated (Scheme 3). These new complexes are only photoactive under conditions in which the dhbp ligand is protonated. Throughout this paper, XA indicates the acidic form of the complex (e.g., 2A = [(phen)2Ru(6,6′-dhbp)]2+, isolated as the chloride salt), and XB is the deprotonated form (e.g., 2B). In this work, we varied the photolabile ligand to look at the impact of methyl electron donor groups in dmdhbp. Complex 4A was initially designed for its higher pKa value (to give a larger proportion of 4A versus 4B in solution and thereby better

Scheme 5. Synthesis of the Metal Complexes

recrystallized by slow diffusion of diethyl ether in a solution of dry ethanol to afford the pure product. In a similar fashion, [(phen)2Ru(4,4′-dhbp)]Cl2 (5A) was synthesized to test the impact of moving the position of the hydroxyl groups (4,4′-dhbp = 4,4′-dihydroxybipyridine). 7521

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

Figure 1. ORTEP diagrams (with ellipsoids at 50% probability) of the cation of 2A (left) and the neutral species 2B (right) with hydrogen atoms (except OH) and counteranions (for 2A) omitted for clarity. Selected bond lengths (Å) and angles (deg): 2A: Ru1−N1, 2.0855(17); Ru1−N2, 2.0872(17); Ru1−N3, 2.0752(17); Ru1−N4, 2.0627(17); Ru1−N5, 2.0549(17); Ru1−N6, 2.0520(17); 2B: Ru1−N1, 2.0940(14); Ru1−N2, 2.0516(15); Ru1−N3, 2.0613(15). A full list of structural parameters is included in the Supporting Information.

Table 1. Selected Bond Lengths and Angles of the Complexes 1A, 2A, 3A, 1B, 2B, and 4B Ru−N1 Ru−N2 Ru−N3 Ru−N4 Ru−N5 Ru−N6 avg Ru−N avg_Ru−N (PL) avg Ru−N (spectator) N−Ru−N1 N−Ru−N2 N−Ru−N3 avg trans angle L1 benda (PL) avg |L1 bend| L2 benda (spectator) L3 benda (spectator) avg |L2 or L3 bend|

1A

1B

2A

2B

3A

4B

5A

2.091(2) 2.094(2) 2.046(2) 2.043(2) 2.066(2) 2.053(2) 2.066(1) 2.093(3) 2.052(1) 169.4(1) 173.9(1) 175.7(1) 173.0(1) 10.2(3) −21.6(3) 15.9(4) 2.6(3) 2.4(3) 6.4(3) −12.4(3) 6.0(2)

2.097(2) 2.102(2) 2.044(1) 2.057(2) 2.051(1) 2.047(2) 2.066(1) 2.100(2) 2.050(1) 174.7(1) 173.4(1) 172.5(1) 173.5(1) −16.6(2) −3.1(2) 9.9(3) −3.1(2) 2.8(2) −1.2(2) −1.4(2) 2.1(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) −4.5(2) −6.0(2) 5.0(2) 4.9(1)

2.094(1) 2.094(1) 2.052(2) 2.052(2) 2.061(2) 2.061(2) 2.069(1) 2.094(2) 2.056(1) 172.5(1) 171.9(1) 171.9(1) 172.1(1) −8.1b −8.1b 8.1b 0.7b −4.3b 0.7b −4.3b 2.5b

2.096(2) 2.096(2) 2.048(2) 2.048(2) 2.065(2) 2.066(2) 2.070(1) 2.096(2) 2.057(1) 171.0(1) 173.6(1) 173.6(1) 172.7(1) −7.4b −7.4b 7.4b 4.1b −5.5b 4.1b −5.5b 4.8b

2.083(1) 2.091(1) 2.055(1) 2.048(1) 2.047(1) 2.040(1) 2.061(1) 2.087(2) 2.048(1) 175.9(1) 170.6(1) 176.1(1) 174.2(1) −12.9(2) 16.6(2) 14.8(3) 8.1(2) −0.3(2) 2.3(2) 1.8(2) 3.1(1)

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) 4.5(3) −5.6(3) −5.2(3) 5.1(3) 5.1(2)

a

Bends are defined as the torsion angles of Ru−N−C-C′ as well as Ru−N′−C′−C. bThe estimated standard deviations (esds) for these are estimated at zero by Mercury software, because C and C′ (or N and N′) are symmetry-related. In practice, the true esd should be 0.1 or 0.2.

This suggests that the 6,6′-dhbp is more weakly bound to the metal relative to the phen ligand. Significantly, the 6,6′-dhbp ligand is distorted away from a planar geometry, as illustrated by viewing the photolabile ligand side on in Figure 2 (red wireframe = 2A) and Figure 5. For example, the oxygen atoms of the dhbp ligand are 0.723 and 0.733 Å out of the plane defined by Ru1, N1, and N2. This “bowing” type of distortion suggests that the 6,6′-dhbp ligand introduces strain that may help promote photodissociation. Similar bowing of the photolabile ligand was seen for 1A (Figure 5).2 When 2A was deprotonated with Bu4NOH in ethanol solution, a color change from bright red to black was observed. Crystals suitable for X-ray diffraction were grown by slow diffusion of diethyl ether into an ethanol solution of the complex. The crystal structure confirmed that the acidic hydrogens were removed to yield the neutral complex 2B, as shown in Scheme 3 and Figure 1.

Complexes 6−8, [(N,N)2Ru(H2O)2]SO4, where (N,N) = bipy (6), phen (7), dop (8), represent the metal-containing products of photodissociation with 1A−4A. We independently synthesized complex 6 to test its cytotoxicity and to confirm that it is the product of photodissociation with 1A and 4A. Complex 6 was synthesized by treating cis-(bipy)2RuCl2 with 1 equiv of Ag2SO4 in water, followed by filtration to remove precipitated AgCl. These complexes (1−6) were spectroscopically and analytically characterized by 1H NMR, 13C NMR, IR, UV−vis spectroscopy, and MS and/or elemental analysis. X-ray Crystallography. Quality crystals of 2A for singlecrystal X-ray diffraction were obtained by slow diffusion of diethyl ether into an ethanol solution of 2A. The structure (Figure 1 and Table 1) shows a distorted octahedral geometry, with the dhbp ligand showing longer Ru−N bond distances (by 0.025(2) Å on average) than those for the phen ligand. 7522

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

Similarly, crystals of 3A were grown by diffusion of diethyl ether into an ethanol solution and these were used for X-ray diffraction. Complex 3A features a distorted octahedral geometry, and again, Ru−N distances were significantly longer (by 0.03− 0.05 Å) involving the dhbp ligand versus the dop ligand. Here, the average Ru−N(dhbp) distance (2.096(2) Å) is 0.040(2) Å longer than the average bond distance for Ru−N(dop) (2.057(1) Å; Table 1). As a comparison, the bond distances differ by 0.025 Å (2.086(2)Å − 2.061(1) Å) for 2A. The other important feature of the structure of 3A is that the dop ligands are not planar, as expected. The O−CH2−CH2−O containing ring imparts a slight twist in the ligand (torsion angle, defined by O−CH2−CH2−O chain = 61.3(4)°), as shown for other Ru complexes of the dop ligand.21 The complex 3A also shows a twist away from planarity in the photolabile 6,6′-dhbp ligand (Figure 5). Unfortunately, we were unable to obtain X-ray quality crystals for 3B, the deprotonated analogue. X-ray quality crystals of compound 4A were not obtained despite several attempts. However, when the ethanol solution of 4A was treated with excess base (Bu4NOH), the bright red color turned to black. Single crystals were grown by slow diffusion of diethyl ether into the solution. A crystal structure of 4B is shown in Figure 3. The central ruthenium ion features a mildly distorted octahedral geometry, as seen with the other three ruthenium compounds discussed above. The dmdhbp ring is mildly bowed/ puckered, similar to the structure in 2A, but with less strain. For example, the oxygen atoms are 0.551 and 0.662 Å off the plane defined by N5−Ru1−N6. As a comparison, in 2A the oxygen atoms are 0.723 and 0.733 Å off the plane defined by Ru1, N1, and N2. It is interesting to note that the dmdhbp ligand is bowed in 4B in a similar way as for the photolabile ligand in 2A, as opposed to the twisting of the photolabile ligand in 2B (Figure 5). The dmdhbp ligand also shows a substantial downward tilt of the hydroxyl groups below the plane of the Ru−N bonds (Figure 5). In contrast to the other crystal structures above, the structure of 5A (Figure 4) shows that the plane defined by Ru, N1, and N2 also contains most of the atoms in the 4,4′-dhbp ligand, including the hydrogens at the 6 and 6′ positions. Clearly, upon moving the hydroxy groups away from the metal center and to the 4,4′-positions, there is now no distortion at the metal center (Figure 5) or in the bond lengths and angles involving the 4,4′-dhbp ligand (Ru−N distances are similar between the phen

Figure 2. Crystal structures of 2B (ellipsoids) and 2A (overlaid in red wireframe) to show the changes upon protonation.

The crystal structures for 2A and 2B are overlaid in Figure 2 to illustrate the differences in geometry that occur with ligand deprotonation. Similar to its acidic congener, the bond distances of Ru−N in the deprotonated dhbp ligand are slightly longer (by 0.038(2) Å on average, see Table 1) than for the phen ligand, indicating weaker bonds, and deprotonated dhbp is predicted to be the more labile ligand. Interestingly, the deprotonated dhbp, [O2-bipy]2−, also features distortion away from a planar ligand structure, but it is less severe than with the neutral 6,6′-dhbp ligand in 2A (Figure 5). For example, the two oxygen atoms of the dhbp ligands are 0.219 Å out of the plane defined by the N1−Ru1−N2, and these two oxygen atoms are on the opposite sides of this plane. This resulted in the ring twisting around the C2−C2′ bond in the dhbp ligand (torsion angle 10.7(2)− 10.9(3)°), as compared to the ring “bowing” in the case of 2A. The lesser distortion as well as the mild twisting (vs severe bowing/puckering) suggest that 2A and 2B may have significant differences in their tendencies to photodissociate (vide infra). Strain that distorts the geometry of the ligand and the octahedral geometry at the metal center is known to lower the energy of the triplet metal-centered (3MC) excited state, from which photodissociation typically occurs.32,53 Thereby, strain can increase the quantum yields for photodissociation.

Figure 3. ORTEP diagrams (with ellipsoids at 50% probability) of the cations of 3A (left) and 4B (right) with hydrogen atoms (except OH) and counteranions (for 3A) omitted for clarity. Selected bond lengths (Å) and angles (deg): 3A: Ru1−N1, 2.0962(17); Ru1−N2, 2.0477(16); Ru1−N3, 2.0655(16); 4B: Ru1−N1, 2.0548(13); Ru1−N2, 2.0483(13); Ru1−N3, 2.0403(13); Ru1−N4, 2.0473(13); Ru1−N5, 2.0906(13); Ru1−N6, 2.0832(13). A full list of structural parameters is included in the Supporting Information. 7523

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

Figure 4. ORTEP diagram (with ellipsoids at 50% probability) of the dication of 5A with hydrogen atoms (except OH) and counteranions omitted for clarity. Selected bond lengths (Å) and angles (deg): Ru1−N1, 2.062(2); Ru1−N2, 2.058(2); Ru1−N3, 2.058(3); Ru1−N4, 2.070(2); Ru1−N5, 2.061(2); Ru1−N6, 2.050(3). A full list of structural parameters is included in the Supporting Information.

ligands and 4,4′-dhbp in Table 1, in contrast to significant differences in Ru−N for complexes 1A, 1B, 2A, 2B, 3A, and 4B). Not surprisingly, complex 5A does not undergo photodissociation with blue light. Figure 5 shows that complex 5A shows the least distortion in the dhbp-derived ligand of all the complexes we studied. Thermodynamic Acidity. The pKa values for removing two protons from 1A−4A are shown in Table 2, in which the acidity data for 1A were previously reported.2 Complexes 2A−5A were dissolved in aqueous solution, and these were treated with 4 equiv of HCl(aq) to ensure that the complexes were fully protonated. This solution was then titrated with NaOH to determine the equivalence point. These experiments were done in low lighting conditions to avoid photodissociation (with 2A−4A). Although these complexes are all diprotic acids, after neutralizing the excess HCl only one equivalence point was observed (with 2 equiv of base) showing that an averaged (avg) pKa value is observed. Thus, pKa1 and pKa2 are close enough that we could not observe individual protonation events using the titration method. Similar results have been seen with other dhbp metal complexes.47,48 However, spectrophotometric investigation of the pH-dependent absorbance allowed determination of pKa1 values for several complexes. We examined the spectra of each complex from pH 4 to pH 8 to determine a pH range in which the isosbestic points are clearly defined. For example, for complex 2A, a clean isosbestic point is observed at 351 nm (Figure 6) when the spectra from pH 4 to pH 5.66 are plotted. By plotting the absorbance values at 359 nm as a function of pH, we were able to estimate pKa1 for 2A at 5.2(1) from two experiments. Similarly, we estimated pKa1 for complexes 3A and 4A in the same manner (Table 2). While clean isosbestic points were not observed in the UV−vis spectra above pH 6 due to peaks shifting with pH, we were able to estimate pKa2 from pKa avg and pKa1 (pKa2 = 2pKa avg − pKa1). See the Supporting Information for further experimental details. Thus, the average pKa value is known more precisely than the values of pKa1 and pKa2. Focusing therefore on pKa avg, the trend is that the pKa avg values increase in this order: 3A < (2A = 5A) < 1A < 4A. This trend is expected because electron-withdrawing groups (of phen and dop) tend to lower the pKa values.

Figure 5. A comparison of the photolabile ligand geometry in complexes 1A, 2A, 3A, 1B, 2B, and 4B.

Not surprisingly, 2A and 5A have very similar pKa values as the hydroxy groups are simply moved to a different position on the rings but are still in conjugation with the N donor atoms. Complex 4A has the highest pKa values of the series (due to the electron-donating methyl groups) and is expected to have the highest concentration of protonated species at pH 7.2−7.4 in cell culture. Quantum Yields. The first step in photodissociation involves exciting an electron into an metal-to-ligand charge transfer (MLCT) excited state that is most likely localized on the photolabile 6,6′-dhbp ligand (Figure 7). (While the spectator 7524

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

Table 2. A Comparison of Thermodynamic Acidity, Quantum Yields for Photodissociation upon Irradiation at 450 nm, and log(Do/w) Valuesa for Compounds 1A−5A compound 1Ac 2A 3A 4A 5A

structure 2+

[(bipy)2Ru(6,6′-dhbp)] [(phen)2Ru(6,6′-dhbp)]2+ [(dop)2Ru(6,6′-dhbp)]2+ [(bipy)2Ru(dmdhbp)]2+ [(phen)2Ru(4,4′-dhbp)]2+

pKa1

pKa2b

pKa avg

ϕ at pH = 5.0 (mostly XA)

ϕ at pH = 7.5 (mostly XB)

log(Do/w) at pH = 7.4

5.26 5.2(2) 5.0(2) 5.6(2)

7.27 6.8(2) 6.8(2) 7.2(2)

6.3 6.0(1) 5.9(1) 6.4(1) 6.01(8)

0.0058(5) 0.0020(2) 0.001(1) 0.0048(3)

0.0012(1) 0.000036(1) 0.00022(3) 0.00076(5)

0.76(3) 1.5(1) 1.2(1) 1.4(2)

a Estimated standard deviations are in parentheses. bThe value of pKa2 was computed mathematically from pKa avg and pKa1. cThe pKa data for complex 1A have been previously reported.

for reacting with DNA or other biomolecules.2 These results give us qualitative measures of how pH influences photodissociation; below we present quantum yields for a more quantitative approach. We measured the quantum yields for the photodissociation reaction starting from complexes 1A−4A (Table 2). The quantum yield is defined as moles of photoproduct ((N,N)2Ru(OH2)2)2+ (6, 7, or 8) per mole of photons. These experiments were done at pH = 5.0, because this pH is below pKa1 in most cases, and therefore the complexes should be mostly in the acidic form (XA). Similarly, quantum yields were also measured at pH = 7.5 to represent the complexes in mostly the basic form (XB). Here the trends are more important than the absolute numbers. The quantum yield measurements are complicated by the fact that the excited states can potentially undergo deprotonation/protonation events. Our data support that XA is more photolabile than XB, with the ratio of the quantum yields (ϕpH5/ϕpH7.5) ranging from 4.5 (for 3A) to 4.8 (for 1A) to 6.3 (for 4A) to 56 (for 2A; Table 2). The greater quantum yields for the acidic forms are in line with the crystal structures of complexes 1A, 2A, and 3A, which show significant distortion away from an ideal octahedral geometry at the metal center and also distortions in the 6,6′-dhbp ligands. In viewing the crystal structures of 2A and 2B (Figure 1), it is clear that simply removing a proton changes the distortion at the metal center and thereby the efficiency of photodissociation, as evident from ϕpH5/ϕpH7.5. Complex 4A was initially designed for its relatively high pKa value (to give a larger proportion of 4A vs 4B in solution and thereby better photodissociation rates). However, 4A has not led to an improvement in quantum yields. The quantum yield for 4 at pH 7.5 is lower than that of 1 (mostly 4B and 1B at pH 7.5), and at pH 5.0 the quantum yields for 1 and 4 are similar (mostly 4A and 1A at pH 5.0). The low quantum yield for 4B may be due to the electron-donating methyl groups perturbing the relative energies of the excited-state molecular orbitals (Figure 7, computational studies are ongoing). Cytotoxicity toward Cancerous and Normal Cells. Cells were treated with varied concentrations of 1A−5A in both the dark and with irradiation by 450 nm blue light. The resulting IC50 values and phototoxicity indices (the ratio of dark IC50 to light IC50) are shown in Table 2. These complexes are isolated and administered in the acidic form (XA), but given that the pH external is typically 7−7.5 in cell culture, these complexes would exist as the neutral species (XB) in all but the most acidic organelles. Treatment with 3A gives the best results with IC50 values of ∼4 μM in two breast cancer cell lines with light treatment and phototoxicity indices up to 120 (red box, Table 2). Importantly, complex 3A also displays selective toxicity toward cancer cells (sevenfold more active toward breast cancer cells vs normal cells). The toxicity of complex 3A is more moderate toward MOLM-13 cells, but again some selectivity is seen for cancerous versus normal umbilical cord blood (UCB) cells.

Figure 6. pH dependent UV−vis spectra of 2A as used to determine pKa1 from the change in absorbance at λ = 359 nm.

Figure 7. Typical Jablonski diagram for photodissociation reaction involving octahedral Ru(II) with imine ligands.

ligands also show MLCT bands, these excitations do not lead to photodissociation due to stronger M−N bonds.) The 1MLCT state is then typically converted (with near unity efficiency) to a 3 MLCT state via intersystem crossing, and then conversion to the 3MC (metal centered) state can lead to photodissociation products (Figure 7). During much of this process, back reaction to the ground-state starting material competes with product formation.22 Our past work has shown that 1A has pKa values of 5.26 and 7.27, which indicate that the complex is predominantly (64%) in the form of 1A at pH 5 and predominantly (63%) in the form of 1B at pH 7.5.2 At pH 7.5, UV−visible spectroscopy studies after irradiating the sample with blue light (photoexcitation at 450 nm) show that ligand loss does not occur to a significant extent. The resonance structures in Scheme 2 offer an explanation in terms of a dianionic ligand binding strongly to the ruthenium(II) center.2 Thus, at normal physiological pH values the ruthenium center does not have free sites for DNA binding. The data also suggest that the mono-deprotonated form, [(bipy)2Ru(6,6′-O,OH-bipy)]+, which is the minor component (37%) at pH 7.5, is not photolabile.2 In contrast, at pH 5, upon irradiation with blue light complete 6,6′-dhbp ligand loss occurs over 1 h. This indicates that, in an acidic environment, exposure to light would cause complex (1A) to form 6, which has free sites 7525

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

ruthenium complexes are localized in any particular organelles. Furthermore, the uptake of 3A (dosed at the concentration that gave 50% viability, incubated, and the cells were washed with media before being lysed) was 7.2 and 3.9 μg/L with MDA-MB231 and MCF10A cell lines, respectively, as measured by inductively coupled plasma mass spectrometry (ICP-MS). In a separate experiment, cells were dosed with 10 μM (chosen to be well below the dark IC50 values) of 1A−4A, and Ru uptake was measured in the dark (Table 3, in green). Uptake is best for 3A (Figure 9 and Table 3), but even in this case it is at most 0.32% in MCF10A (normal cells) and ∼0.02% in MDA-MB-231 and MCF7. Clearly, improving uptake is an appropriate goal for future studies.59

Complex 4A appears to have low toxicity in most cases and is not light-activated to any appreciable extent (PI values ∼1 for most cell lines). In conclusion, it appears that the order of increasing cytotoxicity, phototoxicity, and cancer selectivity is 4A ≤ 1A < 2A ≪ 3A. The reasons for this trend appear to be a combination of drug uptake and light activation. Complex 5A has only been tested in one cell line, but like 4A it also shows poor cytotoxicity and is not light-activated to any significant extent. This underscores the need for both distortion at the metal center and efficient photodissociation. Some pyridone derivatives are drugs,54−56,75 but 6,6′-dhbp is nontoxic as the free ligand (IC50 > 100 μM) versus the breast cell lines above. We cannot exclude the possibility that 6,6′-dhbp has better uptake when bound to Ru versus as the free ligand. We synthesized one product of photodissociation, [(bipy)2Ru(OH2)2]SO4 (6), and this complex was nontoxic (>100 μM), but cellular uptake has not been measured yet. Ru complexes with labile sites typically have poor uptake and may react with molecules outside the cell.6,57,75 Octanol/Water Partition Coefficients. The hydrophilic versus lipophilic (hydrophobic) nature of the complexes gives an estimate as to how readily these complexes will penetrate the cell membrane by passive diffusion. Ideally the complexes should be more lipophilic while still maintaining sufficient water solubility for drug delivery (log(Do/w) should ideally be 4−6).58 We measured the octanol−water partition coefficient of complexes 1A−4A at pH 7.4, and we report log(Do/w) in Table 2. At pH = 7.4, the major species in solution should be the basic form XB, which is a neutral species, but there can still be a significant fraction of the singly deprotonated monocation, and therefore total ruthenium complex concentration was measured. Lipophilicity increases in the order of 1 < 3 < 2, as expected because of increased hydrophobic rings on the spectator ligands. The methyl groups of the dmdhbp ligand serve to increase the lipophilicity of 4 (cf. 1). Here, the species 1 (or 2−4) refers to all protonation states that result when 1A is dissolved at pH 7.4. Cellular Uptake. We measured cellular uptake of 3A and other drugs both qualitatively and quantitatively. Most ruthenium complexes are phosphorescent and can be visualized by microscopy. Figure 8 shows that 3A is in the cytoplasm of the cells. Further studies would be needed to determine if these



DISCUSSION Initially, we anticipated that pKa would be a key factor in determining which complex would be most effective at killing cancer cells, and we expected that a higher pKa value would be beneficial in giving a greater proportion of the protonated form XA, which is more photolabile. Thereby, we expected that complex 4A with higher pKa values (pKa avg = 6.4) would be the most cytotoxic compound, but actually complex 4A is among the least cytotoxic complexes we studied (cytotoxicity increases as 4A ≲ 1A < 2A ≪ 3A). Putting this factor in perspective, the pKa values range from 5.9 to 6.4 for pKa avg and from 5.0 to 5.6 for pKa1, and thus all of these values are pretty similar. In cell culture at pH 7.0−7.5, these complexes will exist mostly as the monodeprotonated and doubly deprotonated (XB) species. Relatively little of these complexes will be in the fully protonated and more photolabile form XA. Building up the protonated species in a cellular environment will require localization of the ruthenium prodrug in an acidic organelle. Complex 2 shows the largest change in quantum yields between their acidic and basic forms (ϕpH5/ϕpH7.5 = 56 for predominantly 2A vs 2B). The crystal structure for 2A shows significant bowing of the photolabile ligand, whereas 2B shows twisting but no bowing. For the complexes for which we crystallized both the acidic and basic form, it is clear that the acidic form (1A and 2A) always involves a greater twist (as illustrated by the torsion angle, L1 bend, Table 1) than the basic form (1B and 2B). The quantum yields for the acidic forms, XA, were greatest for 1 and 4 at pH 5 (predominantly 1A or 4A in solution). At pH 7.5, complex 1 is most photolabile. This may suggest that greater light-triggered activity is expected for complexes 1 and 4; however, all of the quantum yields are low (ϕ = 1 × 10−3 or less),53 and the quantum yields are all relatively similar (ranging from 5.8 × 10−3 for 1 to 1 × 10−3 for 3 at pH 5). Thus, the uptake factor appears to be more important than either the quantum yields or the pKa values. Complex 3 leads to the highest ruthenium concentration in cells and is most cytotoxic and shows the best phototoxicity indices. Only if these complexes enter the cells does the photodissociation lead to drug activation and binding to biological targets. The products of photodissociation (e.g., 6) appear to have low cytotoxicity, perhaps due to worse drug uptake in this form. Thus, it appears that complex 3 is most cytotoxic due favorable uptake in cancer cells and sufficient quantum yields. Selective toxicity toward cancer cells is seen, but this effect cannot be explained by uptake results, because uptake of 3 is actually greater in normal cells versus cancer cells. It appears that the cancer cells are more vulnerable to ruthenium treatment, perhaps due to complex 3 localizing in an acidic environment within the cancer cells (and thereby forming 3A rather than 3B). Further studies will

Figure 8. MDA-MB-231 cells treated with 3A at 3.65 μM and stained with DAPI. (blue) The nucleus. (red) The Ru complex in the cytoplasm. (top) 10×. (bottom) 40×. 7526

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

Table 3. Cell Viability Data for Treatment with 1A−5A in the Dark and upon Irradiation for One Hour with Blue Light (450 nm)a vs MDA-MB-231 (breast CSC) com-pound

IC50 dark

1A 2A 3A 4A 5A

1010 280 190 >1000

IC50 light

PI

290 83 3.7 1140

3.5 3.3 52 ∼1

vs MCF7 (breast cancer)

% uptake

IC50 dark

0.0016 0.0071 0.022 0.0048

>500 490 490 >500 >300

IC50 light

>500 ∼1 180 2.8 4.1 120 >100 ∼5 194 1.5 vs UCB (normal)

vs MOLM-13 (leukemia)

a

com-pound

IC50 dark

IC50 light

1A 2A 3A 4A

>100 60 53 250

>100 27 29 18

PI ∼1 2.2 1.8 14

PI

vs MCF10A (normal) % uptake

IC50 dark

IC50 light

PI

% uptake

0.0021 0.14 0.025 0.0033

>500 110 58 >800

210 13 29 >460

>2.4 9 2 ∼1.7

0.0086 0.022 0.32 0.011

vs HeLa (cervical cancer)

IC50 dark

IC50 light

PI

IC50 dark

IC50 light

PI

114 120 110 >100

122 132 115 >100

0.93 0.91 0.96 ∼1

148 1440 730 >100

202 383 120 >100

0.73 3.8 6.0 ∼1

All IC50 values are in μM. Phototoxicity index (PI) is defined as IC50 dark/IC50 light. CSC: cancer stem cells, UCB: umbilical cord blood. The 4,4′-dhbp was synthesized according to a previously published procedure.63 Dry solvents were obtained by passing through a column of activated alumina using a Glass Contour Solvent Purification System built by Pure Process Technology, LLC. General Specifications: Instruments and Analysis (Complexes 1A−4A and 6−8, Papish Group). 1H, 13C, and 19F NMR spectra were acquired at room temperature on a Bruker AV360 360 MHz or AV500 500 MHz spectrometer, as designated. Chemical shifts are reported in parts per million and referenced to residual solvent resonance peaks. Abbreviations for the multiplicity of NMR signals are s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad). UV−visible spectra were recorded on a PerkinElmer Lambda 35 UV−visible spectrometer. Mass spectrometric data were collected on a Waters AutoSpec-Ultima NT spectrometer with electron ionization method. Electrospray ionization mass spectrometry (ESI-MS) was provided by the University of Alabama Mass Spectrometry Resource. Elemental analyses were performed by Nu-Mega Resonance Laboratories, Inc., San Diego, CA. Quantum yields were measured at Tulane University; see the Supporting Information for further details. General Specifications: Instruments and Analysis (Complex 5A, Paul Group). UV−visible absorption spectra were collected on a Scinco S-3100 diode-array spectrophotometer at a resolution of 1 nm. IR spectra were collected on a PerkinElmer Spectrum One FTIR with ATR accessory. Cyclic voltammetry measurements were performed on a Bioanalytical Systems CW-50 potentiostat. For studies performed in 0.1 M tetrabutylammonium hexafluorophosphate (TBAPF6) electrolyte in acetonitrile, a standard three-electrode setup with platinum wire auxiliary electrode, glass carbon working electrode, and Ag/Ag+ reference electrode filled with 0.1 M TBAPF6 was used with a scan rate of 200 mV/s. The acetonitrile was dried over molecular sieves and filtered prior to use to remove any particulates. Ferrocene was used as an internal standard with E1/2 = +0.40 V versus saturated calomel electrode (SCE).64 Thermodynamic Acidity Measurements. The pH measurements were performed using a VWR SympHony pH meter, utilizing a threepoint calibration at pH = 4, 7, and 10. Aqueous solutions were prepared using a Millipore DirectQ UV water purification system. Buffer solutions were made according to previous published methods.65 (cod)RuCl2. A patent procedure66 was followed. A 250 mL ovendried Schlenk flask equipped with a stir bar was evacuated and refilled with nitrogen three times. RuCl3(H2O)x (2.074 g, 10.0 mmol) was measured into the flask. Absolute EtOH (50 mL) was injected to the flask. 1,5-Cyclooctadiene (3.70 mL, 30.0 mmol, 3.0 equiv) was injected. A condenser was attached while flushing with N2. The final dark brown/ black solution was heated to reflux in an oil bath at 85 °C under N2 for 20 h. After it cooled, the product was collected by suction filtration. The brown powder was washed with a small amount of EtOH, then 50 mL of diethyl ether, then air-dried. Yield: 2.223 g, 7.935 mmol, 79.4%. (phen)2RuCl2. A procedure67 reported for the synthesis of cis-[Ru(bpy)2Cl2] was adapted. A 100 mL oven-dried Schlenk flask equipped with a stir bar was evacuated and refilled with nitrogen three

Figure 9. Ru uptake as measured by ICP-MS for cells treated with 1A−4A (10 μM) in the dark. Blue = MDA-MB-231. Red = MCF7, Green = MCF10A.

be needed to clarify this matter and the pathway by which 3A causes cell death.



CONCLUSIONS These experiments (with IC50 as low as 4 μM with 450 nm light) show the potential of our prodrugs, which are already comparable in cytotoxicity to cisplatin (IC50 < 2 μM)60,61 and NKP-1339 (IC50 = 20 μM),7 which have entered clinical trials (p 1).8,9,62 We have demonstrated that at lower pH values (pH 5) these complexes exist in their acidic form (XA) and that the quantum yields for photodissociation are higher in this form. The change in quantum yields upon lowering the pH from 7.5 to 5 is greatest for complexes 2 (56-fold), and changes in geometric distortion are evident in the crystal structures of 2A and 2B. This illustrates that pH can activate metallo prodrugs toward photodissociation. Significantly, drug uptake is also enhanced by the use of ligands with an extended ring structure for greater hydrophobicity. These complexes are selectively cytotoxic toward cancer cells (as compared with normal cells), and they show efficient light activation with phototoxicity indices as high as 120. These complexes are activated by two triggers (light and pH) to minimize off target effects, thus showing the promise of a pHAMP approach. Most metal-based drugs are prodrugs that are activated by light or the reducing environment in a cancer cell, but here we have used pH to activate these prodrugs, which represents a new approach in metal-based chemotherapy.



EXPERIMENTAL SECTION

General Specifications: Materials. All experiments were performed under a nitrogen atmosphere using glovebox or standard Schlenk techniques if not indicated otherwise. All commercially available reagents were purchased from Sigma-Aldrich, Acros, Strem, or Pressure Chemical and were used as received without further purification. 7527

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry times. (cod)RuCl2 (0.50 g, 1.785 mmol) and 1,10-phenanthroline (0.643 g, 3.57 mmol, 2.0 equiv) were measured into the flask, followed by 25 mL of anhydrous o-dichlorobenzene. The final dark brown suspension was heated to reflux in a sand bath at 180 °C under N2 for 3 h. After it cooled, the product was collected by suction filtration. The dark purple/black powder was washed with Et2O, then air-dried. Yield: 1.011 g, 1.778 mmol, 99.6%. To remove potential [(phen)3Ru]Cl2 the crude product was stirred in 200 mL of deionized (DI) water (200 mL/g of crude product) for a few hours. The product was then collected and washed with ether and dried. 1H NMR (360 MHz, DMSO) δ 10.28 (dd, J = 5.2, 1.4 Hz, 2H), 8.71 (m, 2H), 8.29 (d, J = 8.9 Hz, 2H), 8.23 (m, 4H), 8.14 (d, J = 8.9 Hz, 2H), 7.75 (m, 2H), 7.33 (dd, J = 8.1, 5.4 Hz, 2H). [(phen)2Ru(6,6′-dhbp)]Cl2 (2A). A procedure2 reported for the synthesis of [(bipy)2Ru(6,6′dhbp)](PF6)2 was adapted. The whole process was protected from light, as the final product is sensitive to light. A 100 mL oven-dried Schlenk flask equipped with a stir bar was charged with DI water (30 mL) and was purged with nitrogen for 20 min. (phen)2RuCl2 (0.200 g, 0.375 mmol) and 6,6′-dihydroxy-2,2′bipyridine (0.776 g, 0.412 mmol, 1.1 equiv) were measured into the flask. The final dark brown suspension was heated to reflux in an oil bath at 110 °C under N2 for 22 h. After it cooled, the solution was filtered to remove any excess dhbp ligand. To the filtrate, a few drops of concentrated HCl were added. Water was removed by rotary evaporation. The product was collected by suction filtration with the help of diethyl ether. The red powder was washed with Et2O, then air-dried. Yield: 0.220 g, 0.3053 mmol, 81.4%. 1H NMR (360 MHz, dimethyl sulfoxide (DMSO)) δ 11.88 (s, 2H), 8.82 (dd, J = 8.4, 1.3 Hz, 2H), 8.54 (dd, J = 8.3, 1.3 Hz, 2H), 8.44 (dd, J = 5.3, 1.2 Hz, 2H), 8.34 (d, J = 8.9 Hz, 2H), 8.27 (d, J = 8.9 Hz, 2H), 8.19 (dd, J = 7.9, 1.1 Hz, 2H), 8.00 (dd, J = 8.3, 5.2 Hz, 2H), 7.86 (t, J = 8.0 Hz, 2H), 7.70 (dd, J = 5.4, 1.2 Hz, 2H), 7.49 (dd, J = 8.2, 5.3 Hz, 2H), 6.77 (d, J = 8.2 Hz, 2H). 13C NMR (126 MHz, DMSO) δ 168.64, 156.13, 152.80, 152.06, 148.25, 147.90, 139.90, 136.07, 135.10, 129.94, 129.64, 127.73, 127.34, 125.97, 125.03, 115.34, 111.70. Elem. Anal.: Found: C, 55.04; N, 10.64; and H, 4.28%. Calc. for C36H32Cl2N6O4Ru·EtOH·H2O: C, 55.11; N, 10.71; and H, 4.11%. MALDI-MS (m/z): 650.2, Calcd for [(phen)2Ru(dhbp)]+, [C34H24N6O2Ru]+: 650.1. pKa1: 5.2; pKa2: 6.8; average: 6.0. (dop)2RuCl2. A procedure 67 reported for the synthesis of cis-[Ru(bpy)2Cl2] was adapted. A 100 mL oven-dried Schlenk flask equipped with a stir bar was evacuated and refilled with nitrogen three times. CODRuCl2 (0.081 g, 0.2896 mmol) and dop (0.138 g, 0.579 mmol, 2.0 equiv) were measured into the flask, followed by 15 mL of anhydrous o-dichlorobenzene. The final dark brown suspension was heated to reflux in a sand bath at 180 °C under N2 for 3 h. After it cooled, the product was collected by suction filtration. The black powder was washed with Et2O and then air-dried. Yield: 0.170 g, 0.262 mmol, 90.5%. To remove potential [(dop)3Ru]Cl2: the crude product was stirred in 10 mL of DI water (200 mL/g of crude product) for a few hours. Then the product was collected, washed with ether, and air-dried. [(dop)2Ru(6,6′-dhbp)]Cl2 (3A). A procedure2 reported for the synthesis of [(bipy)2Ru(6,6′dhbp)](PF6)2 was adapted. The whole process was protected from light, as the final product is sensitive to light. A 100 mL oven-dried Schlenk flask equipped with a stir bar was charged with 5 mL of DI water with 5 mL of EtOH and was purged with nitrogen for 20 min. (dop)2RuCl2 (0.089 g, 0.137 mmol) and 6,6′-dihydroxy-2,2′bipyridine (0.028 g, 0.151 mmol, 1.1 equiv) were measured into the flask. The final dark brown suspension was heated to reflux in an oil bath at 110 °C under N2 for 30 h. After it cooled, the solution was filtered to remove any excess dhbp ligand. To the filtrate, a few drops of concentrated HCl were added. Water was removed by rotary evaporation. The product was collected by suction filtration with the help of diethyl ether. The brown-red powder was washed with Et2O and then air-dried. Yield: 0.075 g, 0.0896 mmol, 65.4%. 1H NMR (500 MHz, DMSO) δ 11.89 (s, 2H), 8.67 (dd, J = 8.5, 1.2 Hz, 2H), 8.42 (dd, J = 8.3, 1.2 Hz, 2H), 8.30 (d, J = 5.3, 2H), 8.18 (d, J = 7.8 Hz, 2H), 7.93 (dd, J = 8.4, 5.2 Hz, 2H), 7.87 (t, J = 8.0 Hz, 2H), 7.60 (dd, J = 5.3, 1.2 Hz, 2H), 7.46 (dd, J = 8.4, 5.3 Hz, 2H), 6.81 (d, J = 8.3 Hz, 2H), 4.66 (d, J = 5.0 Hz, 8H). 13 C NMR (126 MHz, DMSO) δ 168.53, 156.05, 150.84, 150.31, 143.86, 143.57, 140.01, 134.05, 133.73, 128.74, 127.76, 125.67, 124.81, 123.98, 123.56, 115.51, 111.71, 64.96, 64.91. Elem. Anal.: Found: C, 53.65; N,

9.06; and H, 4.40%. Calc. for C38H28Cl2N6O6Ru·EtOH·H2O: C, 53.34; N, 9.33; and H, 4.03%. ESI-MS (m/z): 383.1, Calcd for [(dop)2Ru(dhbp)]2+: 383.1. pKa1: 4.9; pKa2: 6.9; average: 5.9. 2-Chloro-6-methoxy-4-methylpyridine. A patent procedure68 was followed with adaptation. A 250 mL oven-dried Schlenk flask equipped with a stir bar was evacuated and refilled with nitrogen three times. Anhydrous MeOH (150 mL) was injected to the flask. Na (0.92 g, 40 mmol, 4.0 equiv) was added portionwise while flushing with N2. To the resultant pale white emulsion the 2,6-dichloro-4-methyl-pyridine (1.62 g, 10 mmol) was added. The final clear faint yellow solution was heated to reflux in an oil bath at 80 °C under N2 for 5 d. After it cooled, solvent was removed by rotary evaporation. The residue was diluted in dichloromethane and washed with water. The organic layer was isolated, washed with brine, and dried over MgSO4. Solvent was removed to give the final product as a pale, yellow oil. Yield: 1.427 g, 9.05 mmol, 90.5%. 1 H NMR (360 MHz, CDCl3) δ 6.74 (s, 1H), 6.46 (s, 1H), 3.91 (s, 3H), 2.28 (s, 3H). 6,6′-Dimethoxy-4,4′-dimethyl-2,2′-bipyridine. A patent procedure52 was followed with adaptation. A 250 mL oven-dried Schlenk flask equipped with a stir bar was evacuated and refilled with nitrogen three times. Dry DMF (150 mL) was injected to the flask. 2-Chloro-6methoxy-4-methylpyridine (2.0 g, 12.69 mmol), Zn (0.83 g, 12.69 mmol, 1 equiv), NiCl2(PPh3)2 (2.49 g, 3.807 mmol, 0.3 equiv), and n Bu4NBr (4.091 g, 12.69 mmol, 1 equiv) were added while flushing with N2. The final black solution was heated to reflux in an oil bath at 55 °C under N2 for 5 d. After it cooled, the solvent was removed by rotary evaporation. The residue was diluted in dichloromethane and washed with water. The organic layer was isolated, washed with brine, and dried over MgSO4. The solvent was removed to give the final product as a light yellow residue. Yield: 0.276 g, 1.13 mmol, 17.8%. 1 H NMR (500 MHz, CDCl3) δ 7.83 (dd, J = 1.3, 0.7 Hz, 1H), 6.56 (dq, J = 1.7, 0.8 Hz, 1H), 4.03 (s, 3H), 2.38 (t, J = 0.7 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 164.06 (s), 153.40 (s), 150.67 (s), 115.53 (s), 110.93 (s), 53.44 (s), 21.44 (s). 4,4′-Dimethyl-[2,2′-bipyridine]-6,6′-diol (dmdhbp). A literature procedure69 reported for the synthesis of analogous 6,6′dihydroxyl-bipyridine was followed. To a 25 mL round-bottom flask equipped with a stir bar 6,6′-dimethoxy-4,4′-dimethyl-bipyridine (0.244 g, 1.0 mmol) was added, followed by 5 mL of HBr/AcOH (33 wt %). The final suspension was heated to reflux in a sand bath for 3 d. After it cooled, the white solid was collected by filtration, which was washed with small amounts of water and acetone. Then the solid was dispersed in 10 mL of boiling water and brought to neutral pH using a KOH solution. The resultant solid was collected by filtration and dried. Yield: 0.1646 g, 0.761 mmol, 76.1%. 1H NMR (360 MHz, DMSO) δ 10.86 (s, 2H), 7.10 (s, 2H), 6.37 (s, 2H), 2.24 (s, 6H). [(bpy)2Ru(dmdhbp)]Cl2 (4A). A procedure2 reported for the synthesis of [(bipy)2Ru(6,6′dhbp)](PF6)2 was adapted. The whole process was protected from light, as the final product was sensitive to light. A 100 mL oven-dried Schlenk flask equipped with a stir bar was charged with 10 mL of DI water with 10 mL of EtOH and was degassed for 20 min. (bpy)2RuCl2 (0.1275 g, 0.261 mmol) and 4,4′-dimethyl-6,6′dihydroxy-2,2′-bipyridine (0.062 g, 0.2867 mmol, 1.1 equiv) 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 it cooled, the solution was filtered to remove any excess dmdhbp ligand. To the filtrate, a few drops of concentrated aqueous HCl were added. Water was removed by rotary evaporation. The product was collected by suction filtration with the help of diethyl ether. The brown-red powder was washed with Et2O and then air-dried. The crude product was purified by ether diffusion into an ethanol solution to crystallize the product. Yield: 0.1497 g, 0.2137 mmol, 65.4%. 1H NMR (500 MHz, DMSO) δ 11.76 (s, 2H), 8.77 (d, J = 8.2 Hz, 2H), 8.65 (d, J = 8.1 Hz, 2H), 8.13 (t, J = 7.9, 2H), 8.04 (s, 2H), 7.96 (m, 4H), 7.58 (t, J = 6.3 Hz, 2H), 7.53 (d, J = 5.4 Hz, 2H), 7.31 (t, J = 6.7 Hz, 2H), 6.56 (s, 2H), 2.36 (s, 6H). 13C NMR (126 MHz, DMSO) δ 167.91 (s), 157.83 (s), 157.05 (s), 155.14 (s), 151.48 (s), 151.34 (s), 150.89 (s), 136.88 (s), 136.13 (s), 127.16 (s), 126.18 (s), 123.58 (s), 122.98 (s), 116.93 (s), 111.67 (s), 20.52 (s). ESI-MS (m/z): 315.1, Calcd for [(bpy) 2 Ru(dmdhbp)] 2 + , [C32H28N6O2Ru]2+: 315.1. Elem. Anal.: Found: C, 48.81; N, 10.67; 7528

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

these regions were unstable, so the data were treated using a solvent mask as implemented in OLEX2.72−74 The procedure found two voids of 600 Å3, each containing the equivalent of 150 e−, for approximately four molecules of methanol per asymmetric unit. More details regarding disorder and refinement details for all structures are given in the Supporting Information. Cell Culture. MDA-MB-231, MCF7, and MCF10A cell lines were purchased from American Type Culture Collection (ATCC). MDAMB-231 and MCF7 of less than 10 passages were grown using DMEM (Gibco, 21063−029) supplemented with 10% FBS (Gibco, 26140079). MCF10A was cultured with MEBM (Lonza) supplemented with MEGM kit according to manufacturer’s protocol (Lonza). Human umbilical cord blood (UCB) and MOLM-13 cells were cultured in IMDM supplemented with 20% FBS. UCB required SCF, IL-3, IL-6, Flt-3L, and TPO growth factors at 10 ng mL−1. IC50 Measurement. MDA-MB-231, MCF7, and MCF10A were seeded at a density of 5000 cells per well in 100 μL of media in 96-well plates and incubated for 24 h to adhere to plate. Compounds were dissolved in DMSO and diluted in media to avoid cytotoxic effect from DMSO on cells. Final concentration of DMSO was set to less than 1% (v/v). Cells were treated with 100 μL of serially diluted compounds and incubated for 48 h in the dark. After 48 h, cells were washed with Hank’s Balanced Salt Solution (HBSS; Gibco) and irradiated for an hour with blue light (Philips, goLITE BLU) in the dark. Cells were then provided with 100 μL of fresh media per well and incubated overnight. Cytotoxic effects of the compounds were measured using a Cell Counting Kit-8 according to the manufacturer’s protocol (Enzo Life Sciences). The IC50 of each cell line was determined using a Minitab 17. Immunocytochemistry. After irradiation with blue light as described above, cells were fixed by 4% paraformaldehyde (PFA) and permeabilized by Triton X-100 (Fisher Scientific). Cells were stained with DAPI (1:5,000; Invitrogen). Ruthenium compounds were detected by mCherry channel. ICP-MS. Cells were treated with 10 μM of compound concentration to analyze the uptake rate of each cell line for each compound. After irradiation, the HBSSs were aspirated and washed with PBS. Nitric acid (70%) was added to lysate the cells. The cell lysates were diluted with 5% nitric acid in distilled water. The concentration of ruthenium was analyzed using ICP-MS (PerkinElmer).

and H, 4.86%. Calc. for C32H28Cl2N6O2Ru.5H2O: C, 48.61; N, 10.63; and H, 4.84%. pKa1: 5.6; pKa2: 7.2; average: 6.4. [Ru(phen)2(4,4′-dhbp)][PF6]2 (5A). A round-bottom flask containing 30 mL of a 1:1 ethanol and water solution was degassed for 30 min. A 0.5532 g (0.001 001 mol) sample of Ru(phen)2(Cl)2 and a 0.2251 g (0.001 196 mol) sample of 4,4′-dhbp were added to the reaction flask and heated at 80 °C under argon for 5 h. After the reaction was completed, the solution was removed from heat and allowed to cool to room temperature, then filtered to remove insoluble impurities. The filtered solution was added to 150 mL of H2O. A few drops of concentrated HCl was added to ensure protonation of the complex, and an excess of NH4PF6 in 20 mL of water was added to precipitate the orange complex. The complex was collected on a fritted flask and washed with water, followed by ether. Crystals of [Ru(phen)2(4,4′dhbp)][PF6]2 for X-ray diffraction were grown by the slow diffusion of ether into a methanol solution. Yield: 0.8431 g, 88%. 1H NMR (300 MHz, CD3CN): δ 8.6 (d, 2H), 8.5 (d, 2H), 8.3 (d, 2H), 8.2 (m, 4H), 7.8 (m, 6H), 7.5 (dd, 2H), 7.2 (d, 2H), 6.7 (d, 2H). FT-IR (ATR, cm−1): 3634 (w), 3087 (w), 2161 (w), 1975 (w), 1619 (m), 1577 (m), 1490 (m), 1449 (m), 1427 (m), 1412 (m), 1311 (m), 1204 (m), 1146 (m), 1095 (m), 1057 (m), 1026 (m), 972 (m), 823 (s), 719 (s), 555 (s). Anal. Calcd for RuC34N6O2H24P2F12·1H2O: C, 42.65%; N, 8.78%; H, 2.74%. Found: C, 42.73%; N, 8.74%; H, 2.83%. Once the characterization of [Ru(phen)2(4,4′-dhbp)][PF6]2 was complete, a salt metathesis was performed to isolate the chloride salt, [Ru(phen)2(4,4′dhbp)]Cl2, for cell studies. [(bpy)2Ru(H2O)2]SO4 (6). Ru(bpy)2Cl2 (132.1 mg, 0.273 mmol) was added to a flask and transferred to the glovebox, where Ag2SO4 (85 mg, 0.274 mmol) was added. Milli-Q water (6 mL) was added, and the solution was stirred at room temperature for 18 h. The solution was filtered and washed with DI H2O (3 × 10 mL). The filtrate was collected and dried under vacuum to yield a maroon solid (136.0 mg, 91% yield). 1 H NMR 360 MHz (D2O) δ 9.33 (d, 2H), 8.54 (d, 2H), 8.33 (d, 2H), 8.20 (t, 2H), 7.85 (t, 2H), 7.72 (m, 4H), 7.06 (t, 2H). MALDI-TOF MS (m/z): 547.0, Calcd for [(bpy)2Ru(H2O)2SO4 + H+]+ 547.0, C20H21N4O6SRu. Elem. Anal.: Found: C, 41.26; N, 9.68; and H, 3.97%. Calc. for C20H20N4O6SRu·2H2O: C, 41.30; N, 9.63; and H, 4.16%. Single-Crystal X-ray Diffraction Structure Determinations of 2A, 2B, 3A, 4B, 5A. Crystals of appropriate dimension were mounted on a Mitgen 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 = 0.710 73 Å) operated at 2000 W power (50 kV, 40 mA). The X-ray intensities were measured at low temperature (100(2) K or 173(2) K). The collected frames were integrated with the Saint70 software using a narrow-frame algorithm. Data were corrected for absorption effects using the multiscan method in SADABS.71 The space groups were assigned using XPREP of the Bruker ShelXTL72 package, solved with ShelXT72 and refined with ShelXL72 and the graphical interface ShelXle73 (for 2A, 2B, 3A, 4B) or OLEX2 (5A).73,74 All nonhydrogen atoms were refined anisotropically. H atoms attached to carbon were positioned geometrically and constrained to ride on their parent atoms. The structure of 2A was found to have two occupationally disordered ethanol molecules, with two-component disorder at each site. The structure of 2B was also found to have two occupationally disordered ethanol molecules, and both of them are located on symmetry elements. The structure of 3A was found to contain several regions of residual density. Part of them was modeled as one occupationally disordered ethanol molecule. Attempts to model the remaining residual density were not successful, so the residual density was corrected for using a reverse Fourier transform approach (the SQUEEZE procedure) using the PLATON program. The solvent-accessible volume was found to be 1193 Å3. The electrons found in the solvent-accessible void is 294 e−, which corresponds to approximately three ethanol molecules. The structure of 4B was found to contain two occupationally disordered ethanol molecules, with one of the molecules being threefold disordered. For structure 5A, large regions of residual density were again associated with disordered solvent molecules. Attempted refinements of



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01065. 1 H, 13C NMR, IR, UV−vis spectra; methods and details of thermodynamic acidity (pKa) measurements; quantum yield measurement details; methodology for partition coefficient determination; structural parameters for X-ray crystallography (PDF) Accession Codes

CCDC 1545429−1545433 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (J.J.P.) *E-mail: [email protected]. (E.J.M.) *E-mail: [email protected]. (Y.K.) *E-mail: [email protected]. (E.T.P.) ORCID

Fengrui Qu: 0000-0002-9975-2573 7529

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

(13) Antonarakis, E. S.; Emadi, A. Ruthenium-based chemotherapeutics: are they ready for prime time? Cancer Chemother. Pharmacol. 2010, 66, 1−9. (14) http://theralase.com/pressrelease/health-canada-approvesclinical-trial-application-anti-cancer-drug/ (accessed 2/16/17). (15) Fong, J.; Kasimova, K.; Arenas, Y.; Kaspler, P.; Lazic, S.; Mandel, A.; Lilge, L. A novel class of ruthenium-based photosensitizers effectively kills in vitro cancer cells and in vivo tumors. Photochem. Photobiol. Sci. 2015, 14, 2014−23. (16) Stephenson, M.; Reichardt, C.; Pinto, M.; Wächtler, M.; Sainuddin, T.; Shi, G.; Yin, H.; Monro, S.; Sampson, E.; Dietzek, B.; McFarland, S. A. Ru(II) Dyads Derived from 2-(1-Pyrenyl)-1 Himidazo[4,5- f][1,10]phenanthroline: Versatile Photosensitizers for Photodynamic Applications. J. Phys. Chem. A 2014, 118, 10507−10521. (17) 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. Ru(II) dyads derived from αoligothiophenes: A new class of potent and versatile photosensitizers for PDT. Coord. Chem. Rev. 2015, 282−283, 127−138. (18) Naik, A.; Rubbiani, R.; Gasser, G.; Spingler, B. Visible-LightInduced Annihilation of Tumor Cells with Platinum-Porphyrin Conjugates. Angew. Chem., Int. Ed. 2014, 53, 6938−6941. (19) Mari, C.; Huang, H.; Rubbiani, R.; Schulze, M.; Würthner, F.; Chao, H.; Gasser, G. Evaluation of Perylene Bisimide-Based Ru IIand Ir IIIComplexes as Photosensitizers for Photodynamic Therapy. Eur. J. Inorg. Chem. 2017, 2017, 1−9. (20) Pierroz, V.; Rubbiani, R.; Gentili, C.; Patra, M.; Mari, C.; Gasser, G.; Ferrari, S. Dual mode of cell death upon the photo-irradiation of a RuII polypyridyl complex in interphase or mitosis. Chemical Science 2016, 7, 6115−6124. (21) Hidayatullah, A. N.; Wachter, E.; Heidary, D. K.; Parkin, S.; Glazer, E. C. Photoactive Ru(II) Complexes With Dioxinophenanthroline Ligands Are Potent Cytotoxic Agents. Inorg. Chem. 2014, 53, 10030−10032. (22) White, J. K.; Schmehl, R. H.; Turro, C. An overview of photosubstitution reactions of Ru(II) imine complexes and their application in photobiology and photodynamic therapy. Inorg. Chim. Acta 2017, 454, 7−20. (23) Chari, R. Targeted delivery of chemotherapeutics: tumoractivated prodrug therapy. Adv. Drug Delivery Rev. 1998, 31, 89−104. (24) Zhou, L.; Cheng, R.; Tao, H.; Ma, S.; Guo, W.; Meng, F.; Liu, H.; Liu, Z.; Zhong, Z. Endosomal pH-Activatable Poly(ethylene oxide)graft-Doxorubicin Prodrugs: Synthesis, Drug Release, and Biodistribution in Tumor-Bearing Mice. Biomacromolecules 2011, 12, 1460−1467. (25) Denny, W. A. Tumor-activated ProdrugsA New Approach to Cancer Therapy. Cancer Invest. 2004, 22, 604−619. (26) Montcourrier, P.; Silver, I.; Farnoud, R.; Bird, I.; Rochefort, H. Breast cancer cells have a high capacity to acidify extracellular milieu by a dual mechanism. Clin. Exp. Metastasis 1997, 15, 382−392. (27) Cardone, R. A.; Casavola, V.; Reshkin, S. J. The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat. Rev. Cancer 2005, 5, 786−795. (28) Gatenby, R. A.; Gillies, R. J. Why do cancers have high aerobic glycolysis? Nat. Rev. Cancer 2004, 4, 891−899. (29) Howerton, B. S.; Heidary, D. K.; Glazer, E. C. Strained Ruthenium Complexes Are Potent Light-Activated Anticancer Agents. J. Am. Chem. Soc. 2012, 134, 8324−8327. (30) Wachter, E.; Heidary, D. K.; Howerton, B. S.; Parkin, S.; Glazer, E. C. Light-activated ruthenium complexes photobind DNA and are cytotoxic in the photodynamic therapy window. Chem. Commun. 2012, 48, 9649. (31) Garner, R. N.; Gallucci, J. C.; Dunbar, K. R.; Turro, C. [Ru(bpy) 2(5-cyanouracil) 2] 2+as a Potential Light-Activated Dual-Action Therapeutic Agent. Inorg. Chem. 2011, 50, 9213−9215. (32) Arora, K.; White, J. K.; Sharma, R.; Mazumder, S.; Martin, P. D.; Schlegel, H. B.; Turro, C.; Kodanko, J. J. Effects of Methyl Substitution in Ruthenium Tris(2-pyridylmethyl)amine Photocaging Groups for Nitriles. Inorg. Chem. 2016, 55, 6968−6979.

Nicholas A. Piro: 0000-0003-4219-0909 W. Scott Kassel: 0000-0002-6764-9045 Jared J. Paul: 0000-0003-0641-1671 Elizabeth T. Papish: 0000-0002-7937-8019 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank NSF CAREER for past support (Grant Nos. CHE0846383 and CHE-1360802 to E.T.P. and her group), the Undergraduate Creativity and Research Academy at Univ. of Alabama (UA), the Research Grants Committee at UA to E.T.P. and Y.K., and NSF EPSCoR Track 2 Grant to E.T.P. and R.H.S. for support (PI N. Hammer, Grant No. OIA-1539035), the UA (E.T.P. and Y.K.), and Villanova Univ. (J.J.P.) for generous financial support. E.J.M. and his group are supported by an NIH grant (R21A185370) and U.S. Army Medical Research (CA150055) grants. We also thank Q. Liang (UA) for MS analysis. Finally, we thank the members of the Papish group for assistance and suggestions, including S. E. Brown, K. Hughes, and S. Reed for preliminary experiments on this project.



REFERENCES

(1) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3, 380−387. (2) Hufziger, K. T.; Thowfeik, F. S.; Charboneau, D. J.; Nieto, I.; Dougherty, W. G.; Kassel, W. S.; Dudley, T. J.; Merino, E. J.; Papish, E. T.; Paul, J. J. Ruthenium dihydroxybipyridine complexes are tumor activated prodrugs due to low pH and blue light induced ligand release. J. Inorg. Biochem. 2014, 130, 103−111. (3) Papish, E. T.; Paul, J. J.; Merino, E. J. Ruthenium Complexes as Tumor Activated Metallo-Prodrugs. 2014, Patent Application filed with US Patent Office. (4) Denny, W. A. Prodrug strategies in cancer therapy. Eur. J. Med. Chem. 2001, 36, 577−595. (5) Stubbs, M.; McSheehy, P. M.; Griffiths, J. R.; Bashford, C. L. Causes and consequences of tumour acidity and implications for treatment. Mol. Med. Today 2000, 6, 15−19. (6) Gaiddon, C.; Pfeffer, M. The Fate of Cycloruthenated Compounds: From C-H Activation to Innovative Anticancer Therapy. Eur. J. Inorg. Chem. 2017, 2017, 1−17. (7) Flocke, L. S.; Trondl, R.; Jakupec, M. A.; Keppler, B. K. Molecular mode of action of NKP-1339 − a clinically investigated ruthenium-based drug − involves ER- and ROS-related effects in colon carcinoma cell lines. Invest. New Drugs 2016, 34, 261−268. (8) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.; Zorbas, H.; Keppler, B. K. From bench to bedside − preclinical and early clinical development of the anticancer agent indazolium trans[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A). J. Inorg. Biochem. 2006, 100, 891−904. (9) Trondl, R.; Heffeter, P.; Kowol, C. R.; Jakupec, M. A.; Berger, W.; Keppler, B. K. NKP-1339, the first ruthenium-based anticancer drug on the edge to clinical application. Chemical Science 2014, 5, 2925−2932. (10) Bergamo, A.; Sava, G. Ruthenium anticancer compounds: myths and realities of the emerging metal-based drugs. Dalton Trans. 2011, 40, 7817. (11) Morbidelli, L.; Donnini, S.; Filippi, S.; Messori, L.; Piccioli, F.; Orioli, P.; Sava, G.; Ziche, M. Antiangiogenic properties of selected ruthenium(III) complexes that are nitric oxide scavengers. Br. J. Cancer 2003, 88, 1484−1491. (12) Bencini, A.; Failli, P.; Valtancoli, B.; Bani, D. Low molecular weight compounds with transition metals as free radical scavengers and novel therapeutic agents. Cardiovasc. Hematol. Agents Med. Chem. 2010, 8, 128−146. 7530

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry

Hydroxy Groups During Basic Electrocatalysis. Inorg. Chem. 2014, 53, 12689−12698. (49) Keegstra, E. M. D.; Zwikker, J. W.; Roest, M. R.; Jenneskens, L. W. A highly selective synthesis of monodisperse oligo(ethylene glycols). J. Org. Chem. 1992, 57, 6678−6680. (50) Paw, W.; Eisenberg, R. Synthesis, Characterization, and Spectroscopy of Dipyridocatecholate Complexes of Platinum. Inorg. Chem. 1997, 36, 2287−2293. (51) Rice, C. R.; Guerrero, A.; Bell, Z. R.; Paul, R. L.; Motson, G. R.; Jeffery, J. C.; Ward, M. D. New hybrid ditopic ligands containing fused phenanthroline and crown ether units. New J. Chem. 2001, 25, 185−187. (52) Dubreuil, D. M.; Pipelier, M. G.; Pradere, J. P.; Bakkali, H.; Lepape, P.; Delaunay, T.; Tabatchnik, A. Pyridazine and pyrrole compounds, processes for obtaining them and uses. WO2008012440A2, 2008. (53) Ross, H. B.; Boldaji, M.; Rillema, D. P.; Blanton, C. B.; White, R. P. Photosubstitution in Tris Chelate Complexes of Ruthenium(II) Containing the Ligands 2,2′Bipyrazine, 2,2′-Bipyrimidine, 2,2′-Bipyridine, and 4,4′-Dimethyl-2,2′-bipyridine: Energy Gap Control. Inorg. Chem. 1989, 28, 1013−1021. (54) Groutas, W. C.; Stanga, M. A.; Brubaker, M. J.; Huang, T. L.; Moi, M. K.; Carroll, R. T. Substituted 2-Pyrones, 2-Pyridones, and Other Congeners of Elasnin as Potential Agents for the Treatment of Chronic Obstructive Lung Diseases? J. Med. Chem. 1985, 28, 1106−1109. (55) Jacinto Demuner, A.; Moreira Valente, V. M.; Almeida Barbosa, L. C.; Rathi, A.; Donohoe, T. J.; Thompson, A. L. Synthesis and Phytotoxic Activity of New Pyridones Derived from 4-Hydroxy-6-Methylpyridin2(1H)-one. Molecules 2009, 14, 4973−4986. (56) Dutta, U.; Deb, A.; Lupton, D. W.; Maiti, D. The regioselective iodination of quinolines, quinolones, pyridones, pyridines and uracil. Chem. Commun. 2015, 51, 17744−17747. (57) Wachter, E.; Zamora, A.; Heidary, D. K.; Ruiz, J. x.; Glazer, E. C. Geometry matters: inverse cytotoxic relationship for cis/trans-Ru(ii) polypyridyl complexes from cis/trans-[PtCl2(NH3)2]. Chem. Commun. 2016, 52, 1−4. (58) Tardito, S.; Bassanetti, I.; Bignardi, C.; Elviri, L.; Tegoni, M.; Mucchino, C.; Bussolati, O.; Franchi-Gazzola, R.; Marchiò, L. Copper Binding Agents Acting as Copper Ionophores Lead to Caspase Inhibition and Paraptotic Cell Death in Human Cancer Cells. J. Am. Chem. Soc. 2011, 133, 6235−6242. (59) Kalaivani, P.; Prabhakaran, R.; Poornima, P.; Dallemer, F.; Vijayalakshmi, K.; Padma, V. V.; Natarajan, K. Versatile Coordination Behavior of Salicylaldehydethiosemicarbazone in Ruthenium(II) Carbonyl Complexes: Synthesis, Spectral, X-ray, Electrochemistry, DNA Binding, Cytotoxicity, and Cellular Uptake Studies. Organometallics 2012, 31, 8323−8332. (60) Graf, N.; Mokhtari, T. E.; Papayannopoulos, I. A.; Lippard, S. J. Platinum(IV)-chlorotoxin (CTX) conjugates for targeting cancer cells. J. Inorg. Biochem. 2012, 110, 58−63. (61) Lovejoy, K. S.; Lippard, S. J. Non-traditional platinum compounds for improved accumulation, oral bioavailability, and tumor targeting. Dalton Trans. 2009, 10651. (62) Trondl, R.; Heffeter, P.; Jakupec, M. A.; Berger, W.; Keppler, B. K. NKP-1339, a first-in-class anticancer drug showing mild side effects and activity in patients suffering from advanced refractory cancer. BMC Pharmacol. Toxicol. 2012, 13, A82. (63) Hong, Y.-R.; Gorman, C. B. Synthetic Approaches to an Isostructural Series of Redox-Active, Metal Tris(bipyridine) Core Dendrimers. J. Org. Chem. 2003, 68, 9019−9025. (64) Connelly, N. G.; Geiger, W. E. Chemical Redox Agents for Organometallic Chemistry. Chem. Rev. 1996, 96, 877−910. (65) Shugar, G. J.; Bauman, R. S.; Shugar, R.; Bauman, L. Chemical Technicians’ Ready Reference Handbook, 2nd ed.; McGraw-Hill Book Company: New York, NY, 1981. (66) Saitou, R.; Chung, K.-G.; Nishimura, H.; Sakai, T.; Komiya, S.; Noda, N.; Nishiguchi, M. Method for producing ruthenium compound. US20120101290A1, 2012. (67) Norris, M. R.; Concepcion, J. J.; Glasson, C. R. K.; Fang, Z.; Lapides, A. M.; Ashford, D. L.; Templeton, J. L.; Meyer, T. J. Synthesis of

(33) Li, A.; White, J. K.; Arora, K.; Herroon, M. K.; Martin, P. D.; Schlegel, H. B.; Podgorski, I.; Turro, C.; Kodanko, J. J. Selective Release of Aromatic Heterocycles from Ruthenium Tris(2-pyridylmethyl)amine with Visible Light. Inorg. Chem. 2016, 55, 10−12. (34) Loftus, L. M.; White, J. K.; Albani, B. A.; Kohler, L.; Kodanko, J. J.; Thummel, R. P.; Dunbar, K. R.; Turro, C. New Ru IIComplex for Dual Activity: Photoinduced Ligand Release and 1O 2Production. Chem. Eur. J. 2016, 22, 3704−3708. (35) Debreczeni, J. É.; Bullock, A. N.; Atilla, G. E.; Williams, D. S.; Bregman, H.; Knapp, S.; Meggers, E. Ruthenium Half-Sandwich Complexes Bound to Protein Kinase Pim-1. Angew. Chem., Int. Ed. 2006, 45, 1580−1585. (36) Kastl, A.; Wilbuer, A.; Merkel, A. L.; Feng, L.; Di Fazio, P.; Ocker, M.; Meggers, E. Dual anticancer activity in a single compound: visiblelight-induced apoptosis by an antiangiogenic iridium complex. Chem. Commun. 2012, 48, 1863−1865. (37) Streib, M.; Kräling, K.; Richter, K.; Xie, X.; Steuber, H.; Meggers, E. An Organometallic Inhibitor for the Human Repair Enzyme 7,8Dihydro-8-oxoguanosine Triphosphatase. Angew. Chem., Int. Ed. 2014, 53, 305−309. (38) Liu, Z.; Sadler, P. J. Organoiridium Complexes: Anticancer Agents and Catalysts. Acc. Chem. Res. 2014, 47, 1174−1185. (39) Zeng, L.; Chen, Y.; Huang, H.; Wang, J.; Zhao, D.; Ji, L.; Chao, H. Cyclometalated Ruthenium(II) Anthraquinone Complexes Exhibit Strong Anticancer Activity in Hypoxic Tumor Cells. Chem. - Eur. J. 2015, 21, 15308−15319. (40) Rico Bautista, H.; Saavedra Díaz, R. O.; Shen, L. Q.; Orvain, C.; Gaiddon, C.; Le Lagadec, R.; Ryabov, A. D. Impact of cyclometalated ruthenium(II) complexes on lactate dehydrogenase activity and cytotoxicity in gastric and colon cancer cells. J. Inorg. Biochem. 2016, 163, 28−38. (41) Sainuddin, T.; McCain, J.; Pinto, M.; Yin, H.; Gibson, J.; Hetu, M.; McFarland, S. A. Organometallic Ru(II) Photosensitizers Derived from π-Expansive Cyclometalating Ligands: Surprising Theranostic PDT Effects. Inorg. Chem. 2016, 55, 83−95. (42) Papish, E. T.; Taylor, M. T.; Jernigan, F. E., III; Rodig, M. J.; Shawhan, R. R.; Yap, G. P. A.; Jove, F. A. Synthesis of Zinc, Copper, Nickel, Cobalt, and Iron Complexes Using Tris(pyrazolyl)methane Sulfonate Ligands: A Structural Model for N,N,O Binding in Metalloenzymes. Inorg. Chem. 2006, 45, 2242−2250. (43) Jernigan, F. E., III; Sieracki, N. A.; Taylor, M. T.; Jenkins, A. S.; Engel, S. E.; Rowe, B. W.; Jove, F. A.; Yap, G. P. A.; Papish, E. T.; Ferrence, G. M. Sterically Bulky Tris(triazolyl)borate Ligands as WaterSoluble Analogues of Tris(pyrazolyl)borate. Inorg. Chem. 2007, 46, 360−362. (44) Nieto, I.; Livings, M. S.; Sacci, J. B.; Reuther, L. E.; Zeller, M.; Papish, E. T. Transfer Hydrogenation in Water via a Ruthenium Catalyst with OH Groups near the Metal Center on a bipy Scaffold. Organometallics 2011, 30, 6339−6342. (45) Dixon, N. A.; McQuarters, A. B.; Kraus, J. S.; Soffer, J. B.; Lehnert, N.; Schweitzer-Stenner, R.; Papish, E. T. Dramatic tuning of ligand donor properties in (Ttz)CuCO through remote binding of H+ (Ttz = hydrotris(triazolyl)borate). Chem. Commun. 2013, 49, 5571−5573. (46) Marelius, D. C.; Bhagan, S.; Charboneau, D. J.; Schroeder, K. M.; Kamdar, J. M.; McGettigan, A. R.; Freeman, B. J.; Moore, C. E.; Rheingold, A. L.; Cooksy, A. L.; Smith, D. K.; Paul, J. J.; Papish, E. T.; Grotjahn, D. B. How Do Proximal Hydroxy or Methoxy Groups on the Bidentate Ligand Affect [(2,2′;6′,2″-Terpyridine)Ru(N,N)X] WaterOxidation Catalysts? Synthesis, Characterization, and Reactivity at Acidic and Near-Neutral pH. Eur. J. Inorg. Chem. 2014, 2014, 676−689. (47) DePasquale, J.; Nieto, I.; Reuther, L. E.; Herbst-Gervasoni, C. J.; Paul, J. J.; Mochalin, V.; Zeller, M.; Thomas, C. M.; Addison, A. W.; Papish, E. T. Iridium Dihydroxybipyridine Complexes Show That Ligand Deprotonation Dramatically Speeds Rates of Catalytic Water Oxidation. Inorg. Chem. 2013, 52, 9175−9183. (48) Gerlach, D. L.; Bhagan, S.; Cruce, A. A.; Burks, D. B.; Nieto, I.; Truong, H. T.; Kelley, S. P.; Herbst-Gervasoni, C. J.; Jernigan, K. L.; Bowman, M. K.; Pan, S.; Zeller, M.; Papish, E. T. Studies of the Pathways Open to Copper Water Oxidation Catalysts Containing Proximal 7531

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532

Article

Inorganic Chemistry Phosphonic Acid Derivatized Bipyridine Ligands and Their Ruthenium Complexes. Inorg. Chem. 2013, 52, 12492−12501. (68) Payne, L. J.; Downham, R.; Sibley, G. E. M.; Edwards, P.; Davies, G. M. 2-Oxo-2-(2-phenyl-5,6,7,8-tetrahydroindolizin-3-yl)acetamide derivatives as antifungal agents and their preparation. WO2008145963A1, 2008. (69) Zhang, T.; Wang, C.; Liu, S.; Wang, J.-L.; Lin, W. A Biomimetic Copper Water Oxidation Catalyst with Low Overpotential. J. Am. Chem. Soc. 2014, 136, 273−281. (70) Apex2, v2008.2−4 (2008) or Apex2, v2009.7−0 (2009) Bruker Advanced X-ray Solutions; Bruker AXS Inc.: Madison, WI, 2009. (71) SADABS: An Empirical Absorption Correction Program, v. 2008/1; Bruker AXS: Madison, WI, 2009. (72) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, A64, 112−122. (73) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 2009, 42, 339−341. (74) Sheldrick, G. M. Crystal structure refinement with SHELXL. Acta Crystallogr., Sect. C: Struct. Chem. 2015, A71, 3−8. (75) Cuello-Garibo, J.-A.; Meijer, M. S.; Bonnet, S. To cage or to be caged? The cytotoxic species in ruthenium-based photoactivated chemotherapy is not always the metal. Chem. Commun. 2017, DOI: 10.1039/c7cc03469e.

7532

DOI: 10.1021/acs.inorgchem.7b01065 Inorg. Chem. 2017, 56, 7519−7532