Exploitation of long-lived 3IL excited states for metal-organic photodynamic therapy: verification in a metastatic melanoma model.

Members of a family of Ru(II)-appended pyrenylethynylene dyads were synthesized, characterized according to their photophysical and photobiological properties, and evaluated for their collective potential as photosensitizers for metal-organic photodynamic therapy. The dyads in this series possess lowest-lying (3)IL-based excited states with lifetimes that can be tuned from 22 to 270 µs in fluid solution and from 44 to 3440 µs in glass at 77 K. To our knowledge, these excited-state lifetimes are the longest reported for Ru(II)-based dyads containing only one organic chromophore and lacking terminal diimine groups. These excited states proved to be extremely sensitive to trace amounts of oxygen, owing to their long lifetimes and very low radiative rates. Herein, we demonstrate that (3)IL states of this nature are potent photodynamic agents, exhibiting the largest photocytotoxicity indices reported to date with nanomolar light cytotoxicities at very short drug-to-light intervals. Importantly, these new agents are robust enough to maintain submicromolar PDT in pigmented metastatic melanoma cells, where the presence of melanin in combination with low oxygen tension is known to compromise PDT. This activity underscores the potential of metal-organic PDT as an alternate treatment strategy for challenging environments such as malignant melanoma.

Photophysical properties, DNA photocleavage, and photocytotoxicity of a series of dppn dirhodium(II,II) complexes.

A series of dirhodium(II,II) complexes of the type cis-[Rh(2)(mu-O(2)CCH(3))(2)(dppn)(L)](2+), where dppn = benzo[i]dipyrido[3,2-a:2',3'-h]quinoxaline and L = 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyrido[3,2-f:2'3'-h]quinoxaline (dpq, 3), dipyrido[3,2-a:2',3'-c]phenazine (dppz, 4), and dppn (5), were synthesized and their photophysical properties investigated to probe their potential usefulness as photodynamic therapy agents. The ability of the complexes to bind and photocleave DNA was also probed, along with their toxicity toward human skin cells in the dark and when irradiated with visible light. Nanosecond time-resolved absorption measurements established that the lowest energy excited state in 1-5 is dppn-localized (3)pipi* with lifetimes of 2.4-4.1 micros in DMSO, with spectral features similar to those of the free dppn ligand (tau = 13.0 micros in CHCl(3)). Complexes 1-4 photocleave DNA efficiently via a mechanism that is mostly mediated by reactive oxygen species, including (1)O(2) and O(2)(-). The DNA photocleavage by 5 is significantly lower than those measured for 1-4 in air, and the absence of O(2) in solution or the addition of azide, SOD (superoxide dismutase), or D(2)O does not affect the efficiency of the photocleavage reaction. The toxicity of 1-5 toward Hs-27 human skin fibroblasts is significantly greater upon irradiation with visible light than in the dark. In contrast to the DNA photocleavage results, 5 exhibits the largest increase in toxicity upon irradiation within the series. These results are explained in terms of the known ability of the complexes to transverse cellular membranes, the toxicity of the complexes in the dark, and their photophysical properties.

Photobiological activity of Ru(II) dyads based on (pyren-1-yl)ethynyl derivatives of 1,10-phenanthroline.

Several mononuclear Ru(II) dyads possessing 1,10-phenanthroline-appended pyrenylethynylene ligands were synthesized, characterized, and evaluated for their potential in photobiological applications such as photodynamic therapy (PDT). These complexes interact with DNA via intercalation and photocleave DNA in vitro at submicromolar concentrations when irradiated with visible light (lambda(irr) > or = 400 nm). Such properties are remarkably sensitive to the position of the ethynylpyrenyl substituent on the 1,10-phenanthroline ring, with 3-substitution showing the strongest binding under all conditions and causing the most deleterious DNA damage. Both dyads photocleave DNA under hypoxic conditions, and this photoactivity translates well to cytotoxicity and photocytotoxicity models using human leukemia cells, where the 5- and 3-substituted dyads show photocytotoxicity at 5-10 microM and 10-20 microM, respectively, with minimal, or essentially no, dark toxicity at these concentrations. This lack of dark cytotoxicity at concentrations where significant photoactivity is observed emphasizes that agents with strong intercalating units, previously thought to be too toxic for phototherapeutic applications, should not be excluded from the arsenal of potential photochemotherapeutic agents under investigation.

Live cell cytotoxicity studies: documentation of the interactions of antitumor active dirhodium compounds with nuclear DNA.

The promising antitumor activity of dirhodium complexes has been known for over 30 years. There remains, however, a general lack of understanding of their activity in cellulo. In this study, we report the DNA interactions and activity in living cells of six monosubstituted dirhodium(II,II) complexes of general formula [Rh(2)(mu-O(2)CCH(3))(2)(eta(1)-O(2)CCH(3))(L)(CH(3)OH)](+), where L = bpy (2,2'-bipyridine) (1), phen (1,10-phenanthroline) (2), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline) (3), dppz (dipyrido[3,2-a:2',3'-c]phenazine) (4), dppn (benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) (5), and dap (4,7-dihydrodibenzo[de,gh][1,10]phenanthroline) (6). DNA interactions were investigated by UV/visible spectroscopy, relative viscosity measurements, and electrophoretic mobility shift assay. These measurements indicate that compound 5 exhibits the strongest interaction with DNA. Compound 5 also causes the most damage to DNA after cellular internalization, as evaluated by the alkaline comet assay. Compound 5, however, is not the most effective at inhibiting cell viability of the human cancer cells HeLa and COLO-316. The greater hydrophobicity of 5 as compared to that of 4, which is the most effective compound in the series, hinders its ability to reach its cellular target(s). Data from modulation studies of glutathione using N-acetylcysteine and L-buthionine-sulfoximine indicate that changes in glutathione levels do not affect the activity of these particular dirhodium complexes. These results suggest that glutathione is not the only agent involved in the deactivation of these dirhodium complexes.

Ultrafast ligand exchange: detection of a pentacoordinate Ru(II) intermediate and product formation.

The ultrafast kinetics of ligand exchange of cis-[Ru(bpy)(2)(CH(3)CN)(2)](2+) were measured in H(2)O and CH(3)CN. The formation of the (3)MLCT excited-state and a five-coordinate intermediate are observed in both solvents within 2 ps after excitation (310 nm, fwhm approximately 300 fs). The (3)MLCT excited-state undergoes vibrational cooling (5-6 ps), then decays to regenerate the ground-state with a lifetime of approximately 50 ps. In CH(3)CN, ligand recombination takes place in 28 ps, while the formation of cis-[Ru(bpy)(2)(CH(3)CN)(H(2)O)](2+) in H(2)O takes place with tau = 77 ps.

Design, synthesis, characterization, and biological evaluation of triazine dendrimers bearing paclitaxel using ester and ester/disulfide linkages.

The design, synthesis, characterization, and preliminary biological assessment of three dendrimers are reported. All three dendrimers, 1-3, present twelve paclitaxel groups linked by acylation of the 2'-hydroxyl group. The linker for dendrimers 2 and 3 also includes a disulfide. Installation of the paclitaxel group relies on reacting twelve primary amines of a second generation triazine dendrimer, a scaffold available on kilogram scale, with a dichlorotriazine bearing the drug. This dichlorotriazine is available in four steps by (i) reacting paclitaxel with glutaric anhydride, (ii) activating with N-hydroxysuccinimide (NHS), (iii) treating the resulting ester with either 1,3-diaminopropane (for 1) or cystamine (for 2 and 3), and (iv), finally, reacting with cyanuric chloride. After reaction with the dendrimer, the resulting monochlorotriazine groups are reacted with 4-aminomethylpiperidine (AMP) and then a poly(ethylene glycol) (PEG) group of molecular weight 2 kDa. Two different PEG-NHS esters are employed that differ in lability. For 1 and 2, the PEG incorporates an ester-linked succinic acid group. For 3, the PEG incorporates an ether-linked acetic acid group. Both mass spectrometry and 1H NMR spectroscopy prove valuable for determining the final ratios of dendrimer:paclitaxel:AMP:PEG. These values are typically 1:12:12:9. Cytotoxicity of these constructs using an MTT-based assay and PC-3 cells reveals IC(50) values in the low nanomolar range with dithiothreitol and glutathione enhancing the toxicity of the disulfide-containing constructs 2 and 3. Preliminary toxicology assessment of 1 suggests that it is well tolerated in vivo with preferential renal clearance. The elimination half-lives of all of the dendrimers appear shorter than predicted from the previous results. Tumor localization is observed for all the three dendrimers.

Redox-regulated Inhibition of T7 RNA polymerase via establishment of disulfide linkages by substituted Dppz dirhodium(II,II) complexes.

The series of dirhodium(II) complexes cis-[Rh(2)(O(2)CCH(3))(2)(R(1)R(2)dppz)(2)](2+) 1-6 (R(1) = R(2) = H, MeO, Me, Cl, NO(2) for 1-4, 6, respectively, and R(1)= H, R(2) = CN for 5), coordinated to R(1)R(2)dppz ligands with electron-donating or -withdrawing substituents at positions 7,8 of dppz (dppz = dipyrido[3,2-a:2',3'-c]phenazine), were synthesized and their effect on the transcription process in vitro was monitored. Complexes 1-6 are easily reduced, readily oxidize cysteine, and engage in redox-based reactions with T7-RNA Polymerase (T7-RNAP), which contains accessible thiol groups. Transcription is inhibited in vitro by 1-6 via formation of intra- and inter-T7-RNAP disulfide bonds that affect the enzyme critical sulfhydryl cysteine groups. The progressively increasing electron-withdrawing character of the dppz substituents (MeO < Me < H < Cl < CN < NO(2)) gives rise to the order 2 < 3 < 1 < 4 < 5 < 6 for the measured IC(50) values of 1-6. The ease of reduction for 1-6 is consistent with the energies of the dppz-centered lowest unoccupied molecular orbitals (LUMOs), which decrease with the electron-withdrawing character of the dppz substituents. The ligand-centered reductions for 1-6 are supported by electron paramagnetic resonance (EPR) studies which support the conclusion that reduction of 1-6 leads to the formation of dppz centered radicals [Rh(2)(O(2)CCH(3))(2)(R(1)R(2)dppz)(2)](*+) with isotropic g values approximately 2.003 which are essentially identical to the reported value for the free radical dppz anions. The EPR results are corroborated by density functional theory (DFT) calculations, which indicate that the complexes contain dppz-based LUMOs primarily phenazine (phz) in character; the unpaired electron is completely delocalized in the phenazine orbitals in 4-6. The low IC(50) values for 1-6 lend further support to the fact that they exhibit redox-based activity with the enzyme and lead to the conclusion that the complexes constitute a sensitive redox-regulated series of T7-RNAP inhibitors with the potential to control or inhibit other important biochemical processes.

Synthesis and Characterization of Anionic Triazine Dendrimers with a Labile Disulfide Core.

Anionic dendrimers based on melamine with disulfide bonds at the core were prepared to investigate the solubility of these architectures, the ability of these molecules to solubilize pyrene as a model drug, and the ability of these architectures to undergo thiol-disulfide exchange. The ability to solubilize pyrene is directly correlated with molecular weight of the dendrimer-aggregation of dendrons does not occur. Thiol-disulfide exchange occurs rapidly using dithiothreitol as the reductant to yield dendrimers with thiol cores that can undergo oxidation in air to yield the original dendrimer.

Intercalation is not required for DNA light-switch behavior.

The DNA light-switch complex [Ru(bpy)2(tpphz)]2+ (1, bpy = 2,2'-bipyridine, tpphz = tetrapyrido[3,2-a:2',3'-c:3'',2''-h:2''',3'''-j]phenazine) is luminescent when bound to DNA and in organic solvents and weakly emissive in water. To date, light-switch behavior by transition metal complexes has generally been regarded as confirmation of DNA intercalation. In contrast, the present work demonstrates that the nonintercalating bimetallic complex [(bpy)2Ru(tpphz)Ru(bpy)2]4+ (2) behaves as a DNA light-switch. Weak emission from the 3MLCT excited state of 2 is observed in water with lambda(em) = 623 nm (phi(em) = 1.4 x 10(-4)), and a red shift (lambda(em) = 702 nm) and 40-fold increase in intensity are observed upon addition of 100 microM calf thymus DNA (ct-DNA). Addition of increasing concentrations of 2 to 1 mM herring sperm DNA does not result in an increase in the viscosity of the solution, indicating that the complex is not an intercalator. Additionally, experiments were conducted to ensure that the emission enhancement did not arise from threading intercalation of the complex. The in situ generation of 2 intercalated between the base pairs of ct-DNA in a threading fashion, however, exhibits emission maximum at 685 nm, which is blue-shifted from that of surface-bound 2. DFT calculations show low-lying orbitals in 2 that are expected to exhibit nonemissive character when contributing to the MLCT state, in accord with the lower emission intensity observed for 2 relative to that for 1. To our knowledge, the present work is the first example of a nonintercalating light-switch metal complex, thus showing that light-switch behavior cannot be used exclusively as confirmation of intercalation.

Conversion of a porous material based on a Mn(II)-TCNQF(4) honeycomb net to a molecular magnet upon desolvation.

Removal of methanol molecules from the interstices of a metal-organic framework based on a 2-D hexagonal Mn(II)-TCNQF(4) net results in stronger magnetic interactions and leads to a glassy magnetically ordered state; the magnetic behavior can be reversibly cycled upon solvation-desolvation of the material.

Dirhodium(II,II) complexes: molecular characteristics that affect in vitro activity.

In the series Rh2(O2CR)4 (R=CH3, 1; R=CF3, 2), [Rh2(O2CR)2(phen)2]2+ (R=CH3, 3; R=CF3, 4), and [Rh2(O2CR)2(dppz)2]2+ (R=CH3, 5; R=CF3, 6), 2, 4, and 6 are twice as cytotoxic as 1, 3, and 5, respectively. The substitution reactions of 2 with 9-ethylguanine at various temperatures take place at faster rates than those of 1, and the activation energy Ea(1)=69+/-4 kJ/mol is twice Ea(2)=35+/-2 kJ/mol. The higher cytotoxicities of [Rh2(micro-O2CCH3)2(eta1-O2CCH3)L(MeOH)]+ (L=dppz, 7; L=dppn, 8) relative to [Rh2(micro-O2CCH3)2(bpy)L]2+ (L=dppz, 10; L=dppn, 11) are attributed to the labile equatorial groups in 7 and 8 not present in 10 and 11. The toxicities of complexes 1-8 are not related to their charge or the ease by which they transverse the cellular membrane but to the lability of the ligands on the dirhodium core.

Mimicking PAMAM Dendrimers with Ampholytic, Hybrid Triazine Dendrimers: A Comparison of Dispersity and Stability.

Two strategies are applied to mimic the ampholytic nature of the surfaces of half-generation PAMAM dendrimers and yet retain the very narrow dispersity inherent of triazine dendrimers. Both strategies start with a monodisperse, single-chemical entity, generation two triazine dendrimer presenting twelve surface amines that is available at the kilogram scale. The first method relies on reaction with methyl bromoacetate. Complete conversion of the surface primary amines to tertiary amines occurs to provide 24 surface esters. Extended reaction times lead to quarternization of the amines while other unidentified species are also present. The resulting polyester can be quantitatively hydrolyzed using 4M aqueous HCl to yield a dendrimer with 12 tertiary amines and 24 carboxylic acids about a hydrophobic triazine core. The second method utilizes Michael additions of methyl acrylate to yield 24 surface esters. This reaction proceeds more rapidly and more cleanly than the former strategy. Hydrolysis of this material proceeds quantitatively using 4M aqueous HCl to yield desired dendrimer. In both cases, MALDI-TOF mass spectrometry provides compelling evidence of reaction progress. Electrophoretic analysis confirms the ampholytic nature of these materials with the former targets having a pI value in the 1.8 < pI < 3.4 range, and the latter having a pI value in the 4.7 < pI < 5.9. These ranges bookend the pH range within which PAMAM dendrimers become zwitterionic, 3.4 < pI < 4.7. The strategy of using monodisperse amine-terminated dendrimer constructs as core offers significant advantage over PAMAM homopolymers including dispersity, ease of characterization and batch-to-batch reproducibility. These triazine dendrimers could ultimately be adopted into materials with applications wherein the demands of purity have hitherto remained unsatisfied.

Electrophoretic Behavior of Anionic Triazine and PAMAM Dendrimers: Methods for Improving Resolution and Assessing Purity Using Capillary Electrophoresis.

The synthesis and characterization of second- and third-generation triazine dendrimers bearing carboxylic acid groups on the periphery are reported. These materials were synthesized by exhaustive succinylation of amine-terminated dendrimers. (1)H and (13)C NMR spectra are consistent with the desired products, but these techniques are limited by degeneracy in signals. MALDI-TOF mass spectrometry confirms the presence of the desired material. These materials display pH-dependent solubility in water. Capillary electrophoresis proves to be valuable in multiple elements of this work, and general protocols emerge that appear to be useful for the characterization of lower-generation anionic dendrimers. Specifically, capillary electrophoresis provides a convenient method for monitoring the removal of excess succinic anhydride/succinic acid and offers additional clues to the chemical nature of the impurities in these samples. Optimization of the background electrolyte and instrumental parameters allows for the assessment of the purity of these triazine targets as well as comparison with two sets of commercially available anionic poly(amidoamine) (PAMAM) dendrimers. Corroborative information from the different orthogonal analytical techniques employed supports the hypothesis that triazine dendrimers exist as very narrowly disperse mixtures of macromolecules approaching, in some cases, single chemical entities.

Anticancer activity of heteroleptic diimine complexes of dirhodium: a study of intercalating properties, hydrophobicity and in cellulo activity.

The series of complexes cis-[Rh(2)(mu-O(2)CCH(3))(2)(dppn)(L)](2+), where dppn = benzo[i]dipyrido[3,2-a:2',3'-c] phenazine, and L = bpy (2,2'-bipyridine) (1), phen (1,10-phenanthroline) (2), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline) (3), dppz (dipyrido[3,2-a:2',3'-c]phenazine) (4), and dppn (5) were synthesized and their effect on the human cancer cells HeLa and COLO-316 was monitored. Complexes 1 and 2 interact with DNA through intercalation, whereas compounds 3-5 bind only electrostatically. It was found that the dirhodium complex 4 is the most effective compound at inhibiting cell viability of the human cancer cells HeLa and COLO-316. A general conclusion is that the hydrophobicity of the compounds correlates with their in cellulo activity in both cell lines. The ability of the compounds to reach nuclear DNA and form adducts was explored using the comet assay. The results indicate that compounds 1-5 either do not form adducts with DNA that are detrimental to the cell or that they are successfully repaired by the cellular machinery. The results of an annexin V assay indicate that compounds 1-4 trigger apoptosis, whereas compound 5 clearly does not. These findings are significant because they support the contention that dirhodium complexes can be tuned to direct their effect to cellular targets other than nuclear DNA.

Kilogram-scale synthesis of a second-generation dendrimer based on 1,3,5-triazine using green and industrially compatible methods with a single chromatographic step.

A kilogram scale, divergent and iterative synthesis of a second generation, triazine dendrimer with 12 protected amines on the periphery using common laboratory equipment is reported. The route benefits from common reaction conditions, inexpensive reagents, and aqueous solvents. From the monomers, the desired product dendrimer--the last uncommitted intermediate that leads to a range of committed, generation three targets--can be obtained in 70% overall yield. Of critical importance in the execution of this divergent synthesis is the differential reactivity of chlorine atoms of trichlorotriazine. The stepwise, nucleophilic aromatic substitution of these atoms with amine nucleophiles is both the basis for the dendrimer growth as well as incorporation of solubilizing piperidine groups. Intermediates are obtained and purified through precipitation and/or extraction protocols with the exception of the final product. Isolation of the target dendrimer requires a single silica gel plug filtration. The purity of this material is assessed at >93%, a level consistent with and/or exceeding other commercially available targets.

Influence of anions on the dimensionality of extended networks based on Cu I cations and 1,4,5,8,9,12-hexaazatriphenylene (HAT) ligands.

Reactions of Cu I salts with 1,4,5,8,9,12-hexaazatriphenylene (HAT) afford three types of cationic coordination polymers depending on the anion present in the reaction solution. In the crystal structure of {[Cu(HAT)][BF4]x1/3(C6H6)}infinity, (1), Cu ions and HAT molecules form extended layers that are best described as strongly distorted honeycomb nets. The space between the layers is occupied by [BF4]- anions and solvent molecules. {[Cu(HAT)][PF6]}infinity, (2), crystallizes as a chiral (10,3)-a net with [PF6]- anions residing in the cavities of the three-dimensional metal-organic framework. The crystal structure of {[Cu4(HAT)3][SbF6]4x3C6H6}infinity, (3), is based on unique extended [Cu4(HAT)3]infinity "nanotubules" filled with solvent molecules and [SbF6]- anions.

Anion-pi interactions as controlling elements in self-assembly reactions of Ag(I) complexes with pi-acidic aromatic rings.

Reactions of 3,6-bis(2'-pyridyl)-1,2,4,5-tetrazine (bptz) and 3,6-bis(2'-pyridyl)-1,2-pyridazine (bppn) with the AgX salts (X = [PF6]-, [AsF6]-, [SbF6]-, and [BF4]-) afford complexes of different structural motifs depending on the pi-acidity of the ligand central ring and the outer-sphere anion. The bptz reactions lead to the polymeric [[Ag(bptz)][PF6]]infinity (1) and the dinuclear compounds [Ag2(bptz)2(CH3CN)2][PF6]2 (2) and [Ag2(bptz)2(CH3CN)2][AsF6]2 (3), as well as the propeller-type species [Ag2(bptz)3][AsF6]2 (4) and [Ag2(bptz)3][SbF6]2 (5a and 5b). Reactions of bppn with AgX produce the grid-type structures [Ag4(bppn)4][X]4 (6-9), regardless of the anion present. In 6-9, pi-pi stacking interactions are maximized, whereas multiple and shorter (therefore stronger) anion-pi interactions between the anions and the tetrazine rings are established in 1-5b. These differences reflect the more electron-rich character of the bppn pyridazine ring as compared to the bptz tetrazine ring. The evidence gleaned from the solid-state structures was corroborated by density functional theory calculations. In the electrostatic potential maps of the free ligands, a higher positive charge is present in the bptz as compared to the bppn central ring. Furthermore, the electrostatic potential maps of 3, 4, and 5b indicate an electron density transfer from the anions to the pi-acidic rings. Conversely, upon addition of the [AsF6]- ions to the cation of 7, there is negligible change in the electron density of the central pyridazine ring, which supports the presence of weaker anion-pi interactions in the bppn as compared to the bptz complexes. From the systems studied herein, it is concluded that anion-pi interactions play an important role in the outcome of self-assembly reactions.