Characterization and initial demonstration of in vivo efficacy of a novel heat-activated metalloenediyne anti-cancer agent.

BACKGROUND: Enediynes are anti-cancer agents that are highly cytotoxic due to their propensity for low thermal activation of radical generation. The diradical intermediate produced from Bergman cyclization of the enediyne moiety may induce DNA damage and cell lethality. The cytotoxicity of enediynes and difficulties in controlling their thermal cyclization has limited their clinical use. We recently showed that enediyne toxicity at 37 °C can be mitigated by metallation, but cytotoxic effects of 'metalloenediynes' on cultured tumor cells are potentiated by hyperthermia. Reduction of cytotoxicity at normothermia suggests metalloenediynes will have a large therapeutic margin, with cell death occurring primarily in the heated tumor. Based on our previous in vitro findings, FeSO4-PyED, an Fe co-factor complex of (Z)-N,N'-bis[1-pyridin-2-yl-meth-(E)-ylidene]oct-4-ene-2,6-diyne-1,8-diamine, was prioritized for further in vitro and in vivo testing in normal human melanocytes and melanoma cells. METHODS: Clonogenic survival, apopotosis and DNA binding assays were used to determine mechanisms of enhancement of FeSO4-PyED cytotoxicity by hyperthermia. A murine human melanoma xenograft model was used to assess in vivo efficacy of FeSO4-PyED at 37 or 42.5 °C. RESULTS: FeSO4-PyED is a DNA-binding compound. Enhancement of FeSO4-PyED cytotoxicity by hyperthermia in melanoma cells was due to Bergman cyclization, diradical formation, and increased apoptosis. Thermal enhancement, however, was not observed in melanocytes. FeSO4-PyED inhibited tumor growth when melanomas were heated during drug treatment, without inducing normal tissue damage. CONCLUSION: By leveraging the unique thermal activation properties of metalloenediynes, we propose that localized moderate hyperthermia can be used to confine the cytotoxicity of these compounds to tumors, while sparing normal tissue.

The Outliers: Metal-Mediated Radical Reagents for Biological Substrate Degradation.

The predictable and controllable interaction of small organic or peptidic molecules with biological substrates is the primary reason most pharmaceuticals are narrowly decorated carbon frameworks. The inhibition or activation binding models are measurable and without side reactions that can cause pathological angst. Yet many diseases, especially those involving rapid proliferation of cells (i.e., cancer) or aggregation of peptides (e.g., heart disease, Alzheimer's disease) have not yet been cured by inhibition therapeutics. Additionally, interventional medicine is often required to alleviate such maladies by physical removal first, followed by molecular-level therapy as a second stage. Thus, there appears to be a niche for more aggressive therapeutics that may employ harsher chemical processes to realize clinical efficacy, albeit without causing catastrophic side effects. Molecules that may be considered for this challenge are not typically biomimetic, nor do they fit the traditional pharmaceutical paradigm. They may have unusual modes of action or undesired reactivity that can be lethal if not controlled. These are the outliers; potential pharmacophores that biology does not know how to manage or adapt to. This is why they may be an intriguing class of agents that needs continuous development. In this Account, we connect the under-developed enediyne family of compounds and our metalloenediyne derivatives to existing radical-based therapeutics such as bleomycin and doxorubicin to illustrate that controlled diradical reactivity, although an outlier mechanism, has a place in the therapeutic portfolio. This is self-evident in that of the 11 natural product enediynes known, 2 have clinical impact, a strong ratio. We expand on the chemical diversity of potential enediyne constructs and focus on the accessible trigger mechanisms to activate diradical formation as a method to control toxicity. Moreover, we further illustrate how electromagnetic fields can be employed to activate both molecular and larger nanomaterial constructs that carry highly concentrated payloads of reactive reagent. Finally, we describe how controlled diradical reactivity can reach beyond traditional therapeutic targets such as DNA, to peptide aggregates found in blood clots, neural fibrils, and membrane scaffolds. It is our belief that cleverly constructed frameworks with well-designed and controlled activation/reaction schemes can lead to novel therapeutics that can challenge evolving viral and bacterial invaders. From this evangelical perspective, our hope is that the conceptual framework, if not the specific designs in this Account, stimulate the readership to develop out-of-the-box therapeutic designs that may combat resistant disease targets.

Metal-mediated diradical tuning for DNA replication arrest via template strand scission.

A series of M(PyED)·X (X = 2Cl-, SO42-) pyridine-metalloenediyne complexes [M = Cu(II), Fe(II), or Zn(II)] and their independently synthesized, cyclized analogs have been prepared to investigate their potential as radical-generating DNA-damaging agents. All complexes possess a 1:1 metal-to-ligand stoichiometry as determined by electronic absorption spectroscopy and X-ray diffraction. Solution structural analysis reveals a pπ Cl [Formula: see text] Cu(II) LMCT (22,026 cm-1) for Cu(PyED)·2Cl, indicating three nitrogens and a chloride in the psuedo-equatorial plane with the remaining pyridine nitrogen and solvent in axial positions. EPR spectra of the Cu(II) complexes exhibit an axially elongated octahedron. This spectroscopic evidence, together with density functional theory computed geometries, suggest six-coordinate structures for Cu(II) and Fe(II) complexes and a five-coordinate environment for Zn(II) analogs. Bergman cyclization via thermal activation of these constructs yields benzannulated product indicative of diradical generation in all complexes within 3 h at 37 °C. A significant metal dependence on the rate of the reaction is observed [Cu(II) > Fe(II) > Zn(II)], which is mirrored in in vitro DNA-damaging outcomes. Whereas in situ chelation of PyED leads to considerable degradation in the presence of all metals within 1 h under hyperthermia conditions, Cu(II) activation produces >50% compromised DNA within 5 min. Additionally, Cu(II) chelated PyED outcompetes DNA polymerase I to successfully inhibit template strand extension. Exposure of HeLa cells to Cu(PyBD)·SO4 (IC50 = 10 µM) results in a G2/M arrest compared with untreated samples, indicating significant DNA damage. These results demonstrate metal-controlled radical generation for degradation of biopolymers under physiologically relevant temperatures on short timescales.

Anion Control of Lanthanoenediyne Cyclization.

A suite of lanthanoenediyne complexes of the form Ln(macrocycle)X3 (Ln = La3+, Ce3+, Eu3+, Gd3+, Tb3+, Lu3+; X = NO3-, Cl-, OTf-) was prepared by utilizing an enediyne-containing [2 + 2] hexaaza-macrocycle (2). The solid-state Bergman cyclization temperatures, measured via DSC, decrease with the denticity of X (bidentate NO3-, T = 267-292 °C; monodentate Cl-, T = 238-262 °C; noncoordinating OTf-, T = 170-183 °C). 13C NMR characterization shows that the chemical shifts of the acetylenic carbon atoms also rely on the anion identity. The alkyne carbon closest to the metal binding site, CA, exhibits a Δδ > 3 ppm downfield shift, while the more distal alkyne carbon, CB, displays a concomitant Δδ ≤ 2.5 ppm upfield shift, reflecting a depolarization of the alkyne on metal inclusion. For all metals studied, the degree of perturbation follows the trend 2 < NO3- < Cl- < OTf-. This belies a greater degree of electronic rearrangement in the coordinated macrocycle as the denticity of X and its accompanying shielding of the metal's Lewis acidity decrease. Computationally modeled structures of LnX3 show a systematic increase in the lanthanide-2 coordination number (CNLa-mc = 2 (NO3-), 4 (Cl-), 5 (H2O, model for OTf-)) and a decrease in the mean Ln-N bond length (La-Naverage = 2.91 Å (NO3-), 2.78 Å (Cl-), 2.68 Å (H2O)), further suggesting that a decrease in the anion coordination number correlates with an increase in the metal-macrocycle interaction. Taken together, these data illustrate a Bergman cyclization landscape that is influenced by the bonding of metal to an enediyne ligand but whose reaction barrier is ultimately dominated by the coordinating ability of the accompanying anion.

The role of ligand covalency in the selective activation of metalloenediynes for Bergman cyclization.

One of the key concerns with the development of radical-generating reactive therapeutics is the ability to control the activation event within a biological environment. To that end, a series of quinoline-metal-loenediynes of the form M(QuiED)·2Cl (M = Cu(II), Fe(II), Mg(II), or Zn(II)) and their independently synthesized cyclized analogs have been prepared in an effort to elucidate Bergman cyclization (BC) reactivity differences in solution. HRMS(ESI) establishes a solution stoichiometry of 1:1 metal to ligand with coordination of one chloride counter ion to the metal center. EPR spectroscopy of Cu(QuiED)·2Cl and Cu (QuiBD)·2Cl denotes an axially-elongated tetragonal octahedron (g║ > g⊥ > 2.0023) with a dx2-y2 ground state, while the electronic absorption spectrum reveals a pπ Cl→Cu(II) LMCT feature at 19,000 cm -1, indicating a solution structure with three nitrogens and a chloride in the equatorial plane with the remaining quinoline nitrogen and solvent in the axial positions. Investigations into the BC activity reveal formation of the cyclized product from the Cu(II) and Fe(II) complexes after 12 h at 45 °C in solution, while no product is observed for the Mg(II) or Zn(II) complexes under identical conditions. The basis of this reactivity difference has been found to be a steric effect leading to metal-ligand bond elongation and thus, a retardation of solution reactivity. These results demonstrate how careful consideration of ligand and complex structure may allow for a degree of control and selective activation of these reactive agents.

Utilizing redox-mediated Bergman cyclization toward the development of dual-action metalloenediyne therapeutics.

Reaction of 2 equiv of 1,2-bis((diphenylphosphino)ethynyl)benzene (dppeb, 1) with Pt(cod)Cl2 followed by treatment with N2H4 yields the reduced Pt(0) metalloenediyne, Pt(dppeb)2, 2. This complex is stable to both air oxidation and metal-mediated Bergman cyclization under ambient conditions due to the nearly idealized tetrahedral geometry. Reaction of 2 with 1 equiv of I2 in the presence of excess 1,4-cyclohexadiene (1,4-CHD) radical trap rapidly and near-quantitatively generates the cis-Bergman-cyclized, diiodo product 3 ((31)P: δ = 41 ppm, J(Pt-P) = 3346 Hz) with concomitant loss of 1 equiv of uncyclized phosphine chelate ((31)P: δ = -33 ppm). In contrast, addition of 2 equiv of I2 in the absence of additional radical trap instantaneously forms a metastable Pt(dppeb)2(2+) intermediate species, 4, that is characterized by δ = 51 ppm in the (31)P NMR (J(Pt-P) = 3171 Hz) and ν(C≡C) = 2169 cm(-1) in the Raman profile, indicating that it is an uncyclized, bis-ligated complex. Over 24 h, 4 undergoes ligand exchange to form a neutral, square planar complex that spontaneously Bergman cyclizes at ambient temperature to give the crystalline product Pt(dppnap-I2)I2 (dppnap-I2 = (1,4-diiodonaphthalene-2,3-diyl)bis(diphenylphosphine)), 5, in 52% isolated yield. Computational analysis of the oxidation reaction proposes two plausible flattened tetrahedral structures for intermediate 4: one where the phosphine core has migrated to a trans-spanning chelate geometry, and a second, higher energy structure (3.3 kcal/mol) with two cis-chelating phosphine ligands (41° dihedral angle) via a restricted alkyne-terminal starting point. While the energies are disparate, the common theme in both structures is the elongated Pt-P bond lengths (>2.4 Å), indicating that nucleophilic ligand substitution by I(-) is on the reaction trajectory to the cyclized product 5. The efficiency of the redox-mediated Bergman cyclization reaction of this stable Pt(0) metalloenediyne prodrug and resulting cisplatin-like byproduct represents an intriguing new strategy for potential dual-threat metalloenediyne therapeutics.

Gating the mechanistic pathway to the elusive 4-membered ring azeteoporphyrin.

Photolysis of metalated (Cu and Ni) and free base 2-diazo-3-oxochlorins within a frozen matrix (λ = 457.9 nm, toluene, 80 K) generates a single photointermediate with a hypsochromically shifted electronic absorption spectrum relative to the starting diazochlorins. The appearance of ketene (~2131 cm(-1)) and azete (~1670 cm(-1)) vibrations in infrared absorption and Raman spectra, respectively, identifies this intermediate as resulting from the Wolff rearrangement of the diazochlorins upon N(2) loss. Computational modeling of the vibrational spectra and TDDFT simulation of the electronic transitions of potential photointermediates corroborate this assignment. Isolation and analysis of photoproducts of these diazochlorins formed within n-butanol-doped frozen toluene matrices indicate near exclusive formation of azeteoporphyrins. In sharp contrast, room temperature laser photolysis of these materials yields a mixture of photoproducts deriving from the presence of both carbene and ketene intermediates. Computational modeling of the intramolecular reactivity of the proposed sp(2) carbene intermediate shows exclusive bond insertion to the adjacent phenyl group, and no evidence of Wolff rearrangement. Computational reaction profile analyses reveal that the barrierless Wolff rearrangement proceeds via an out-of-plane carbene electronic configuration that is generated directly during the loss of N(2). The formation of out-of-plane carbene, resulting in the exclusive formation of the observed ketene photointermediate at low temperatures, is consistent with orbital symmetry considerations and by the geometric constraints imposed by the frozen matrix. Combined, this leads to a model showing that azeteoporphyrin formation via the Wolff rearrangement is dependent upon the structural disposition of the adjacent framework, and the specific reaction intermediate formed is very sensitive to this feature.

Conformational and electronic consequences in crafting extended, π-conjugated, light-harvesting macrocycles.

The synthesis of a new series of free-base, Ni(II) and Zn(II) 2,3,12,13-tetra(ethynyl)-5,10,15,20-tetraphenyl porphyrins is described. Upon heating, two of the four ethynyl moieties undergo Bergman cyclization to afford the monocyclized 2,3-diethynyl-5,20-diphenylpiceno[10,11,12,13,14,15-jklmn]porphyrin in 30 %, 10 %, and trace yields, respectively. The structures of all products were investigated by using quantum chemical calculations and the free-base analogue was isolated and crystallized; all compounds show significant deviation from the idealized planar structure. No fully-cyclized bispiceno[20,1,2,3,4,5,10,11,12,13,14,15-fghij]porphyrin was isolated from the reaction mixture. To understand why only two of the four enthynyl groups undergo Bergman cyclization, the reaction coordinates were examined by using DFT at the PWPW91/cc-pVTZ(-f) level coupled to a continuum solvation model. The barrier to cyclization of the second pair of ethynyl groups was found to be 5.5 kcal mol(-1) higher than the first, suggesting a negative cooperative effect and significantly slower rate for the second cyclization. Cyclization reactions for model porphyrin-enediynes with ethene- and H-functionality substitutions at the meso-phenyl rings were also examined, and found to have a similar barrier to diradical formation for the second cyclization event as for the first in these highly planar molecules. By enforcing an artificial 30° cant in two of the pyrrole rings of the porphyrin, the second barrier was increased by 2 kcal mol(-1) in the ethene model system; this suggests that the disruption of the π conjugation of the extended porphyrin structure is the cause of the increased barrier to the second cyclization event.

Potentiation of metalloenediyne cytotoxicity by hyperthermia.

PURPOSE: Enediynes are potent inducers of DNA damage, but their clinical usefulness has been limited. Here we report the thermal enhancement of cytotoxicity of two novel metalloenediyne compounds at concentrations that are either not or minimally cytotoxic at 37°C, and present evidence regarding possible mechanisms for enhanced cytotoxicity. MATERIALS AND METHODS: HeLa cells were exposed to (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine (PyED) (which becomes metallated in culture medium) or ((Z)-N,N'-bis[quinolin-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine)zinc(II) chloride (QuinED · ZnCl(2)) at 37°C or 42.5°C for 1 h, and clonogenic survival was compared after treatment at each temperature. Analyses of cell cycle progression and mode of death were performed after treatments. RESULTS: Treatment with PyED or QuinED · ZnCl(2) resulted in a significant decrease in cell survival when cells were treated with drug at 42.5°C compared to 37°C. Enhanced cytotoxicity was attributed to increased apoptosis. However, perturbation of the cell cycle may also play a role. Cells which were only heated or exposed to PyED at 37°C experienced significant G(2)/M blocks that were eliminated when PyED and heat were administered simultaneously, suggesting that combined treatments override cell cycle arrests normally observed with each agent individually. Conversely, cells heated during treatment with QuinED · ZnCl(2) displayed an increased G(2)/M arrest compared to treatment at 37°C. CONCLUSIONS: With improvements in site-specific heat delivery to tumours, systemic administration of non-toxic metalloenediynes coupled with localised hyperthermia may improve selective enediyne activation/targeting. Therefore PyED and QuinED · ZnCl(2), which show significantly enhanced cytotoxicity at elevated temperatures, may represent viable candidates for thermochemotherapy.

Modulating the light switch by (3)MLCT-(3)ππ* state interconversion.

The spectroscopic, electronic, and DNA-binding characteristics of two novel ruthenium complexes based on the dialkynyl ligands 2,3-bis(phenylethynyl)-1,4,8,9-tetraaza-triphenylene (bptt, 1) and 2,3-bis(4-tert-butyl-phenylethynyl)-1,4,8,9-tetraaza-triphenylene (tbptt, 2) have been investigated. Electronic structure calculations of bptt reveal that the frontier molecular orbitals are localized on the pyrazine-dialkynyl portion of the free ligand, a property that is reflected in a red shift of the lowest energy electronic transition (1: λ(max) = 393 nm) upon substitution at the terminal phenyl groups (2: λ(max) = 398 nm). Upon coordination to ruthenium, the low-energy ligand-centered transitions of 1 and 2 are retained, and metal-to-ligand charge transfer transitions (MLCT) centered at λ(max) = 450 nm are observed for [Ru(phen)(2)bptt](2+)(3) and [Ru(phen)(2)tbptt](2+)(4). The photophysical characteristics of 3 and 4 in ethanol closely parallel those observed for [Ru(bpy)(3)](2+) and [Ru(phen)(3)](2+), indicating that the MLCT excited state is primarily localized within the [Ru(phen)(3)](2+) manifold of 3 and 4, and is only sparingly affected by the extended conjugation of the bptt framework. In an aqueous environment, 3 and 4 possess notably small luminescence quantum yields (3: ϕ(H(2)O) = 0.005, 4: ϕ(H(2)O) = 0.011) and biexponential decay kinetics (3: τ(1) = 40 ns, τ(2) = 230 ns; 4: τ(1) ∼ 26 ns, τ(2) = 150 ns). Addition of CT-DNA to an aqueous solution of 3 causes a significant increase in the luminescence quantum yield (ϕ(DNA) = 0.045), while the quantum yield of 4 is relatively unaffected (ϕ(DNA) = 0.013). The differential behavior demonstrates that tert-butyl substitution on the terminal phenyl groups inhibits the ability of 4 to intercalate with DNA. Such changes in intrinsic luminescence demonstrate that 3 binds to DNA via intercalation (K(b) = 3.3 × 10(4) M(-1)). The origin of this light switch behavior involves two competing (3)MLCT states similar to that of the extensively studied light switch molecule [Ru(phen)(2)dppz](2+). The solvent- and temperature-dependence of the luminescence of 3 reveal that the extended ligand aromaticity lowers the energy of the (3)ππ* excited state into competition with the emitting (3)MLCT state. Interconversion between these two states plays a significant role in the observed photophysics and is responsible for the dual emission in aqueous environments.

Au-Cu@PANI Alloy Core Shells for Aerobic Fibrin Degradation under Visible Light Exposure.

Fibrin plays a critical role in wound healing and hemostasis, yet it is also the main case of cardiovascular diseases and thrombosis. Here, we show the unique design of Au-Cu@PANI alloy core-shell rods for fibrin clot degradation. Microscopic (transmission electron microscopy (TEM), scanning transmission electron microscopy-energy-dispersive X-ray (STEM-EDX)) and structural characterizations (powder X-ray diffraction (PXRD), X-ray photoelectron spectroscopy (XPS)) of the Au-Cu@PANI hybrid material reveal the formation of Au-Cu heterogeneous alloy core rods (aspect ratio = 3.7) with thin Cu2O and PANI shells that create a positive surface charge (ζ-potential = +22 mV). This architecture is supported by the survey XPS spectrum showing the presence of Cu 2p, N 1s, and C 1s features with binding energies of 934.8, 399.7, and 284.8 eV, respectively. Upon photolysis (λ ≥ 495 or 590 nm), these hybrid composite nanorods provide sufficient excited-state redox potential to generate reactive oxygen species (ROS) for degradation of model fibrin clots within 5-7 h. Detailed scanning electron microscopy (SEM) analysis of the fibrin network shows significant morphology modification including formation of large voids and strand termini, indicating degradation of fibrin protofibril by Au-Cu@PANI. The dye 1,3-diphenylisobenzofuran (DPBF) used to detect the presence of 1O2 shows a 27% bleaching of the absorption at λ = 418 nm within 75 min of irradiation of an aqueous Au-Cu@PANI solution in air. Moreover, electron paramagnetic resonance (EPR) spin-trapping experiments reveal a hyperfine-coupled triplet signature at room temperature with intensities 1:1:1: and g-value = 2.0057, characteristic of the reaction between the spin probe 4-Oxo-TEMP and 1O2 during irradiation. Controlled 1O2 scavenging experiments by NaN3 show 82% reduction in the spin-trapped EPR signal area. Both DPBF bleaching and EPR spin trapping indicate that in situ generated 1O2 is responsible for fibrin strand scission. This unique nanomaterial function via use of ubiquitous oxygen as a reagent could open creative avenues for future in vivo biomedical applications to treat fibrin clot diseases.

Development of Magnesium Oxide-Silver Hybrid Nanocatalysts for Synergistic Carbon Dioxide Activation to Afford Esters and Heterocycles at Ambient Pressure.

Multi-metallic hybrid nancatalysts consisting of a porous metal oxide host and metal satellite guests serve as a scaffold for multi-step transformations of divergent and energy-challenging substrates. Here we have developed a 3D porous MgO framework (Lewis basic host) with Ag0 nanoparticles (noble metal guest) for ambient pressure activation and insertion of CO2 into unsaturated alkyne substrates. The hybrid MgO@Ag-x (x = 2, 5, 7, 8 at % Ag) catalysts are synthesized by impregnating Ag+ ions in porous MgO cubes followed by reduction using NaBH4. Morphological (SEM, TEM, EDX mapping) and structural (PXRD, XPS) characterization reveal that the micron-sized hybrid cubes derive from self-assembly of ~100 nm (edge length) MgO cubes decorated with ~ 5 to 25 nm Ag0 NPs. Detailed XPS analysis illustrates Ag0 is present in two forms, <10 nm NPs and ~25 nm aggregates. The MgO@Ag-7 catalyst is effective for inserting CO2 into aryl alkynes followed by SN2 coupling with allylic chlorides to afford a wide range of ester and lactone heterocycles in excellent yields (61-93%) and with low E-factor (2.8). The proposed mechanism suggests a CO2 capture and substrate assembly role for 3D porous MgO while Ag0 performs the key activation of alkyne and CO2 insertion steps. The catalyst is recyclable (5x) with no significant loss of product yield. Overall, these results demonstrate viable approaches to hybrid catalyst development for challenging conversions such as CO2 utilization in a green and sustainable manner.

Enhancement of Cytotoxicity of Enediyne Compounds by Hyperthermia: Effects of Various Metal Complexes on Tumor Cells.

Enediyne natural products are a class of compounds that were recognized for their potential as chemotherapeutic agents many years ago, but found to be highly cytotoxic due to their propensity for low thermal activation. Bergman cyclization of the enediyne moiety produces a diradical intermediate, and may subsequently induce DNA damage and account for the extreme cytotoxicity. While difficulties in controlling the thermal cyclization reaction have limited the clinical use of cyclic enediynes, we have previously shown that enediyne activity, and thus toxicity at physiological temperatures can be modulated by metallation of acyclic enediynes. Furthermore, the cytotoxicity of "metalloenediynes" can be potentiated by hyperthermia. In this study, we characterized a suite of novel metallated enediyne motifs that usually induced little or no cytotoxicity when two different human cancer cell lines were treated with the compounds at 37°C, but showed a significant enhancement of cytotoxicity after cells were exposed to moderate hyperthermia during drug treatment. Cultured U-1 melanoma or MDA-231 breast cancer cells were treated with various concentrations of Cu, Fe and Zn complexes of the enediyne (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine (PyED) and clonogenic survival was assessed to determine the effects of the drugs at 37°C and 42.5°C. Toxicity at 37°C varied for each compound, but hyperthermia potentiated the cytotoxicity of each compound in both cell lines. Cytotoxicity was concentration-, time- and temperature-dependent. Heating cells during drug treatment resulted in enhanced apoptosis, but the role of cell cycle perturbation in the response of the cells to the drugs was less clear. Lastly, we showed that hyperthermia enhanced the number of DNA double-strand breaks (DSBs) induced by the compounds, and inhibited their repair after drug treatment. Thus, thermal enhancement of cytotoxicity may be due, at least in part, to the propensity of the enediyne moiety to induce DSBs, and/or a reduction in DSB repair efficiency. We propose that "tuning" of metalloenediyne toxicity through better-controlled reactivity could have potential clinical utility, since we envision that such compounds could be administered systemically as relatively non-toxic agents, but cytotoxicity could be enhanced in, and confined to a tumor volume when subjected to localized heating.

Density functional analysis of ancillary ligand electronic contributions to metal-mediated enediyne cyclization.

Density functional theory (DFT) has been used to study electronic perturbations induced by ancillary halogen ligation within metalloenediyne constructs, and the subsequent affect upon thermal Bergman cyclization temperatures. To isolate electronic from geometric components of Bergman cyclization thermodynamics, model diamine- and diphosphine-enediynes (L = 1,6-diamino- or 1,6-diphosphino-cis-1,5-hexadiyne-3-ene) of Mn(II), Cu(II), Zn(II), and Pd(II) with ancillary chloride ligands have been examined computationally and compared to more complex ethylenediamine-based metalloenediyne frameworks of the form MLX(2) (X = Cl, Br, I; L = 1,4-dibenzyl-1,4-diaza-cyclododec-8-ene-6,10-diyne) with distorted square-planar (Cu(II)), T(d) (Zn(II)), and D(4h) (Pd(II)) geometries. In the latter systems, the ethylenediamine linkage restricts the conformation of the enediyne backbone, causing the alkyne termini separation to be nearly independent of metal geometry (3.75-3.82 A). Within the Zn(II) family, steric effects are shown to induce conformational changes on the cyclization potential energy surface (PES) prior to the Bergman transition state, introducing distinct electron-electron repulsive interactions. Multiple metal and ligand conformations are also observed on the Cu(II) metalloenediyne cyclization PES. In contrast, square-planar Pd(II) compounds exhibit overlap between the out-of-plane halogen lone pairs and metal d orbitals, as well as the enediyne pi system, reminiscent of an organometallic "push-pull" reaction mechanism. These systems have significantly higher predicted activation barriers toward cycloaromatization due to enhanced electron repulsion.

Structure modulated electronic contributions to metalloenediyne reactivity: synthesis and thermal Bergman cyclization of MLX2 compounds.

The synthesis of novel metalloendiyne complexes MLRX(2) (where L = 1,4-dibenzyl/diethyl-1,4-diaza-cyclododec-8-ene-6,10-diyne, X = halogen) are reported with their X-ray crystal structures and thermal Bergman cyclization temperatures. Two distinct types of constructs are obtained; the Zn(II) compounds are tetrahedral, while the Cu(II) and the Pd(II) compounds are all distorted- or square-planar. Each possesses structurally similar enediyne conformations and critical distances (3.75-3.88 A). The tetragonal Cu(II) species all exhibit Bergman cyclization temperatures between 140 and 150 degrees C in the solid state, while the square-planar Pd(II) analogues possess similar critical distances but cyclize at significantly higher temperatures (205-220 degrees C). In contrast, the Zn(II) derivatives show a marked halogen dependence, with X = Cl having the highest Bergman cyclization temperature, which is comparable to the Pd(II) square-planar set, while the ZnLX(2) compound with X = I shows the lowest Bergman cyclization temperature (144 degrees C), similar to the Cu(II) derivatives. Moreover, for the planar constructs, the R group has little influence on the cyclization temperatures; however, for the tetrahedral ZnLX(2) compounds, the steric influence of the R group plays a more significant role in the cyclization reaction coordinate by influencing the stability of the precyclized intermediate. This complex set of results is best interpreted by a combination of steric contributions and electronic interactions between the halogen through space (in the case of Zn(II)) and through bonds (in the case of Pd(II)) and the pi orbitals of the endiyne fragment. In contrast, for Cu(II) systems, the distorted square-planar geometry permits neither direct through space nor symmetry-allowed through bond communication between the orbital partners, and thus little variation in Bergman cyclization reactivity is observed.

Expansion by contraction: diversifying the photochemical reactivity scope of diazo-oxochlorins toward development of in situ alkylating agents.

Irradiation of 2-diazo-3-oxochlorins (200 W, lambda > or = 345 nm, 10 degrees C) in the presence of nucleophilic and biomimetic substrates 1-butanol, tosylhydrazine, or tetrahydrofurfuryl alcohol generates Wolff-rearranged, pyrrole ring-contracted azeteoporphyrinoids in 11-34% yield, with the corresponding hydroxyporphyrins in up to 55% yield. For metalated diazo-oxochlorins, these products compete with intramolecular exocyclic ring formation by meso-phenyl ring addition, which occurs in up to 76% yield in the absence of substrate. The dependence of product distribution on substrate is established by photolysis in neat dichlorometane. Under these conditions, formation of the Wolff-rearranged product is inhibited and the phenyl addition product dominates (76%) due to the absence of a good nucleophile. A conceptually analogous dependence is also observed for the free-base derivative, with the exocyclic ring-containing dimerization product isolated in 42% yield. The third reaction pathway, formation of the hydroxyporphyrin, is enhanced by the presence of non-nucleophilic, oxidizable substrates such as 1,4-cyclohexadiene (M = Cu; 55%); however, in the presence of the bulky and oxidatively more stable tert-butyl alcohol, intramolecular exocyclic ring-quenching is observed in 51% yield with no detection of the hydroxyporphyrin. X-ray structure characterization of the azeteoporphyrinoids reveals a planar macrocycle, illustrating the strong influence of periphery contraction. Specifically, the copper-containing azeteoporphyrinoids show remarkably short Cu-N(azete) distances of 1.88-1.90 A. All porphyrinoid photoproducts possess intense absorption bands throughout the visible spectral region, indicating that ring-contracted substrate adducts, as well as phenyl ring addition products, maintain porphyrinoid aromaticity. Overall, the ability of these chromophores to photochemically react under substrate control may make unimolecular porphyrinoid photoreagents such as these useful for applications in photobiology or O2-independent photodynamic therapy.

Characterization of Thermally Activated Metalloenediyne Cytotoxicity in Human Melanoma Cells.

Enediynes are a highly cytotoxic class of compounds. However, metallation of these compounds may modulate their activation, and thus their cytotoxicity. We previously demonstrated that cytotoxicity of two different metalloenediynes, including (Z)-N,N'-bis[1-pyridyl-2-yl-meth-(E)-ylidene]octa-4-ene-2,6-diyne-1,8-diamine] (PyED), is potentiated when the compounds are administered to HeLa cells during hyperthermia treatment at concentrations that are minimally or not cytotoxic at 37°C. In this study, we further characterized the concentration, time and temperature dependence of cytotoxicity of PyED on human U-1 melanoma cells. We also investigated the potential mechanisms by which PyED cytotoxicity is enhanced during hyperthermia treatment. Cell killing with PyED was dependent on concentration, temperature during treatment and time of exposure. Potentiation of cytotoxicity was observed when cells were treated with PyED at temperatures ≥39.5°C, and enhancement of cell killing increased with temperature and with increasing time at a given temperature. All cells treated with PyED were shown to have DNA damage, but substantially more damage was observed in cells treated with PyED during heating. DNA repair was also inhibited in cells treated with the drug during hyperthermia. Thus, potentiation of PyED cytotoxicity by hyperthermia may be due to enhancement of drug-induced DNA lesions, and/or the inhibition of repair of sublethal DNA damage. While the selective thermal activation of PyED supports the potential clinical utility of metalloenediynes as cancer thermochemotherapeutic agents, therapeutic gain could be optimized by identifying compounds that produce minimal toxicity at 37°C but which become activated and show enhancement of cytotoxicity within a tumor subjected to localized hyperthermic or thermal ablative treatment, or which might act as bifunctional agents. We thus also describe the development and initial characterization of a novel cofactor complex of PyED, platinated PyED (Pt-PyED). Pt-PyED binds to DNA-like cisplatin, and much like PyED, cytotoxicity is greatly enhanced after treatment with the drug at elevated temperatures. However, in contrast to PyED, Pt-PyED is only minimally cytotoxic at 37°C, at concentrations at which cytotoxicity is enhanced by hyperthermia. Further development of cisplatin-based enediynes may result in compounds which, when activated, will possess multiple DNA binding modalities similar to cisplatin, but produce less side effects in tissues at normothermic temperatures.

Metal complex-DNA interactions: from transcription inhibition to photoactivated cleavage.

Metal ions and complexes, because of their cationic character, three-dimensional structural profiles, and propensity for performing hydrolysis, redox, or photoreactions, have a natural aptitude for interacting with DNA. Indeed, the need for cellular regulation of DNA led to the evolution of metallonucleases to catalyze and repair DNA strand breaks. Moreover, inorganic constructs such as cisplatin and bimetallic rhodium acetate exert antitumor activity by inner-sphere coordination to DNA. Because binding and cleavage of DNA is at the heart of cellular transcription and translation, it is an obvious target for therapeutic intervention and the development of diagnostic structural probes. To this end, new metal complexes have been designed that utilize or create open coordination positions for DNA binding and hydrolysis, generate reactive oxygen-containing species or other radicals for DNA oxidation, or perform direct redox reactions with DNA. The recent emerging themes are the development of bifunctional architectures containing multiple metal-binding or reactive sites, specialized ligand implementation, or incorporation of site-specific targeting substructures. This review describes their employment in novel reaction strategies that do not require bimolecular cofactors and as site-specific probes or cleavage agents.

Elucidation of the extraordinary 4-membered pyrrole ring-contracted azeteoporphyrinoid as an intermediate in chlorin oxidation.

Reaction of 2,3-dioxochlorins with benzeneselenic anhydride (BSA) results in the formation of unusual ring-contracted azetine derivatives that further react with BSA to afford porpholactones.