Photosubstitution and photoreduction of a diazido platinum(IV) anticancer complex.

The hyphenation of HPLC with its high separation ability and ICP-MS with its excellent sensitivity, allows the analysis of Pt drugs in biological samples at the low nanomolar concentration levels. On the other hand, LC-MS provides molecular structural confirmation for each species. Using a combination of these methods, we have investigated the speciation of the photoactive anticancer complex diazido Pt(IV) complex trans, trans, trans-[Pt(N3)2(OH)2(py)2] (FM-190) in aqueous solution and biofluids at single-digit nanomolar concentrations before and after irradiation. FM-190 displays high stability in human blood plasma in the dark at 37 °C. Interestingly, the polyhydroxido species [{PtIV(py)2(OH)4} + Na]+ and [{PtIV(py)2(N3)(OH)3} + Na]+ resulting from the replacement of azido ligands, as determined by LC-MS, were the major products after photoirradiation of FM-190 with blue light (463 nm). This finding suggests that such photosubstituted Pt(IV) tri- and tetra-hydroxido species could play important roles in the biological activity of this anticancer complex. Density Functional Theory (DFT) and Time-Dependent DFT (TDDFT) calculations show that these Pt(IV) species arising from FM-190 in aqueous media can be formed directly from a singlet excited state. The results highlight how speciation analysis (metallomics) can shed light on photoactivation pathways for FM-190 and formation of potential excited-state pharmacophores. The ability to detect and identify photoproducts at physiologically-relevant concentrations in cells and tissues will be important for preclinical development studies of this class of photoactivatable platinum drugs.

A Photodynamic and Photochemotherapeutic Platinum-Iridium Charge-Transfer Conjugate for Anticancer Therapy.

The novel hetero-dinuclear complex trans,trans,trans-[PtIV(py)2(N3)2(OH)(µ-OOCCH2CH2CONHCH2-bpyMe)IrIII(ppy)2]Cl (Pt-Ir), exhibits charge transfer between the acceptor photochemotherapeutic Pt(IV) (Pt-OH) and donor photodynamic Ir(III) (Ir-NH2) fragments. It is stable in the dark, but undergoes photodecomposition more rapidly than the Pt(IV) parent complex (Pt-OH) to generate Pt(II) species, an azidyl radical and 1O2. The Ir(III)* excited state, formed after irradiation, can oxidise NADH to NAD⋅ radicals and NAD+. Pt-Ir is highly photocytotoxic towards cancer cells with a high photocytotoxicity index upon irradiation with blue light (465 nm, 4.8 mW/cm2), even with short light-exposure times (10-60 min). In contrast, the mononuclear Pt-OH and Ir-NH2 subunits and their simple mixture are much less potent. Cellular Pt accumulation was higher for Pt-Ir compared to Pt-OH. Irradiation of Pt-Ir in cancer cells damages nuclei and releases chromosomes. Synchrotron-XRF revealed ca. 4× higher levels of intracellular platinum compared to iridium in Pt-Ir treated cells under dark conditions. Luminescent Pt-Ir distributes over the whole cell and generates ROS and 1O2 within 1 h of irradiation. Iridium localises strongly in small compartments, suggestive of complex cleavage and excretion via recycling vesicles (e.g. lysosomes). The combination of PDT and PACT motifs in one molecule, provides Pt-Ir with a novel strategy for multimodal phototherapy.

Tuning the photoactivated anticancer activity of Pt(iv) compounds via distant ferrocene conjugation.

Photoactive prodrugs offer potential for spatially-selective antitumour activity with minimal effects on normal tissues. Excited-state chemistry can induce novel effects on biochemical pathways and combat resistance to conventional drugs. Photoactive metal complexes in particular, have a rich and relatively unexplored photochemistry, especially an ability to undergo facile intersystem crossing and populate triplet states. We have conjugated the photoactive octahedral Pt(iv) complex trans, trans, trans-[Pt(N3)2(OH)2(py)2] to ferrocene to introduce novel features into a candidate photochemotherapeutic drug. The X-ray crystal structure of the conjugate Pt-Fe confirmed the axial coordination of a ferrocene carboxylate, with Pt(iv) and Fe(ii) 6.07 Å apart. The conjugation of ferrocene red-shifted the absorption spectrum and ferrocene behaves as a light antenna allowing charge transfer from iron to platinum, promoting the photoactivation of Pt-Fe with light of longer wavelength. Cancer cellular accumulation is enhanced, and generation of reactive species is catalysed after photoirradiation, introducing ferroptosis as a contribution towards the cell-death mechanism. TDDFT calculations were performed to shed light on the behaviour of Pt-Fe when it is irradiated. Intersystem spin-crossing allows the formation of triplet states centred on both metal atoms. The dissociative nature of triplet states confirms that they can be involved in ligand detachment due to irradiation. The Pt(ii) photoproducts mainly retain the trans-{Pt(py)2}2+fragment. Visible light irradiation gives rise to micromolar activity for Pt-Fe towards ovarian, lung, prostate and bladder cancer cells under both normoxia and hypoxia, and some photoproducts appear to retain Pt(iv)-Fe(ii) conjugation.

Reactions of cisplatin and oxaliplatin with penicillin G: implications for drug inactivation and biological activity.

Determination of the toxicity of compounds toward cancer cells is a frequent procedure in drug discovery. For metal complexes, which are often reactive prodrugs, care has to be taken to consider reactions with components of the cell culture medium that might change the speciation of the metal complex before it is taken up by the cells. Here, we consider possible reactions between the clinical platinum drugs cisplatin and oxaliplatin with penicillin G, an antibiotic added routinely to cell culture media to prevent bacterial contamination. Platinum has a high affinity for ligands with sulfur donors. Penicillin G is an unstable thioether that degrades in a range of pathways. Nuclear magnetic resonance (NMR) and UV-Vis absorption spectroscopic studies show that reactions with cisplatin can occur within minutes to hours at 310 K, but more slowly with oxaliplatin. The identities of the Pt- adducts were investigated by mass spectrometry. The marked effect on cytotoxicity of co-incubation of cisplatin with penicillin G was demonstrated for the HeLa human cervical cancer cell line. These studies highlight the possibility that reactions with penicillin G might influence the cytotoxic activity of metal complexes determined in culture media.

Single-Cell Chemistry of Photoactivatable Platinum Anticancer Complexes.

The Pt(IV) prodrug trans, trans, trans-[Pt(pyridine)2(N3)2(OH)2] (Pt1) and its coumarin derivative trans, trans, trans-[Pt(pyridine)2(N3)2(OH)(coumarin-3-carboxylate)] (Pt2) are promising agents for photoactivated chemotherapy. These complexes are inert in the dark but release Pt(II) species and radicals upon visible light irradiation, resulting in photocytotoxicity toward cancer cells. Here, we have used synchrotron techniques to investigate the in-cell behavior of these prodrugs and visualize, for the first time, changes in cellular morphology and Pt localization upon treatment with and without light irradiation. We show that photoactivation of Pt2 induces remarkable cellular damage with extreme alterations to multiple cellular components, including formation of vacuoles, while also significantly increasing the cellular accumulation of Pt species compared to dark conditions. X-ray absorption near-edge structure (XANES) measurements in cells treated with Pt2 indicate only partial reduction of the prodrug upon irradiation, highlighting that phototoxicity in cancer cells may involve not only Pt(II) photoproducts but also photoexcited Pt(IV) species.

DNA-Intercalative Platinum Anticancer Complexes Photoactivated by Visible Light.

Photoactivatable agents offer the prospect of highly selective cancer therapy with low side effects and novel mechanisms of action that can combat current drug resistance. 1,8-Naphthalimides with their extended π system can behave as light-harvesting groups, fluorescent probes and DNA intercalators. We conjugated N-(carboxymethyl)-1,8-naphthalimide (gly-R-Nap) with an R substituent on the naphthyl group to photoactive diazido PtIV complexes to form t,t,t-[Pt(py)2 (N3 )2 (OH)(gly-R-Nap)], R=H (1), 3-NO2 (2) or 4-NMe2 (3). They show enhanced photo-oxidation, cellular accumulation and promising photo-cytotoxicity in human A2780 ovarian, A549 lung and PC3 prostate cancer cells with visible light activation, and low dark cytotoxicity. Complexes 1 and 2 exhibit pre-intercalation into DNA, resulting in enhanced photo-induced DNA crosslinking. Complex 3 has a red-shifted absorption band at 450 nm, allowing photoactivation and photo-cytotoxicity with green light.

Unexpected photoactivation pathways in a folate-receptor-targeted trans-diazido Pt(IV) anticancer pro-drug.

A conjugate between a photoactive trans-diazido Pt(iv) pro-drug, trans,trans,trans-[Pt(N3)2(OH)2(py)2], and folic acid has been synthesized and fully characterized by high resolution ESI-MS, NMR and UV-vis spectroscopy. Photoactivation of the Pt-folate conjugate with visible light confirmed the generation of cytotoxic Pt(ii) species capable of binding to guanine nucleobases. Importantly, photoreduction of the Pt(iv) complex triggered the photodecomposition of the folate vector into a p-aminobenzoate-containing fragment and several pterin derivatives, including 6-formylpterin. Besides exhibiting high dark stability in physiological-like conditions, the Pt-folate conjugate was ca. 2× more photocytotoxic towards MCF-7 breast cancer cell line than its parent Pt(iv) complex with a high photoselectivity index (PI > 6.9). The higher photocytotoxicity of the conjugate may be a consequence of its higher cellular accumulation and of the generation of a set of different cytotoxic species, including Pt(ii) photoproducts and several pterin derivatives, which are known to generate ROS.

How promising is phototherapy for cancer?

Oncological phototherapy, including current photodynamic therapy (PDT), developmental photoactivated chemotherapy (PACT) and photothermal therapy (PTT), shows promising photo-efficacy for superficial and internal tumours. The dual application of light and photochemotherapeutic agents allows accurate cancer targeting, low invasiveness and novel mechanisms of action. Current advances in new light sources and photoactive agents are encouraging for future development.

Biotinylated photoactive Pt(iv) anticancer complexes.

Novel biotinylated diazido-Pt(iv) complexes exhibit high visible light photocytotoxicity while being stable in the dark. Photocytotoxicity and cellular accumulation of all-trans-[Pt(py)2(N3)2(biotin)(OH)] (2a) were enhanced significantly when bound to avidin; irradiation induced dramatic cellular morphological changes in human ovarian cancer cells treated with 2a.

Ligand-Controlled Reactivity and Cytotoxicity of Cyclometalated Rhodium(III) Complexes.

We report the synthesis, characterisation and cytotoxicity of six cyclometalated rhodium(III) complexes [CpXRh(C^N)Z]0/+, in which CpX = Cp*, Cpph, or Cpbiph, C^N = benzo[h]quinoline, and Z = chloride or pyridine. Three x-ray crystal structures showing the expected "piano-stool" configurations have been determined. The chlorido complexes hydrolysed faster in aqueous solution, also reacted preferentially with 9-ethyl guanine or glutathione compared to their pyridine analogues. The 1-biphenyl-2,3,4,5,-tetramethylcyclopentadienyl complex [CpbiphRh(benzo[h]quinoline)Cl] (3a) was the most efficient catalyst in coenzyme reduced nicotinamide adenine dinucleotide (NADH) oxidation to NAD+ and induced an elevated level of reactive oxygen species (ROS) in A549 human lung cancer cells. The pyridine complex [CpbiphRh(benzo[h]quinoline)py]+ (3b) was the most potent against A549 lung and A2780 ovarian cancer cell lines, being 5-fold more active than cisplatin towards A549 cells, and acted as a ROS scavenger. This work highlights a ligand-controlled strategy to modulate the reactivity and cytotoxicity of cyclometalated rhodium anticancer complexes.

Metallodrugs are unique: opportunities and challenges of discovery and development.

Metals play vital roles in nutrients and medicines and provide chemical functionalities that are not accessible to purely organic compounds. At least 10 metals are essential for human life and about 46 other non-essential metals (including radionuclides) are also used in drug therapies and diagnostic agents. These include platinum drugs (in 50% of cancer chemotherapies), lithium (bipolar disorders), silver (antimicrobials), and bismuth (broad-spectrum antibiotics). While the quest for novel and better drugs is now as urgent as ever, drug discovery and development pipelines established for organic drugs and based on target identification and high-throughput screening of compound libraries are less effective when applied to metallodrugs. Metallodrugs are often prodrugs which undergo activation by ligand substitution or redox reactions, and are multi-targeting, all of which need to be considered when establishing structure-activity relationships. We focus on early-stage in vitro drug discovery, highlighting the challenges of evaluating anticancer, antimicrobial and antiviral metallo-pharmacophores in cultured cells, and identifying their targets. We highlight advances in the application of metal-specific techniques that can assist the preclinical development, including synchrotron X-ray spectro(micro)scopy, luminescence, and mass spectrometry-based methods, combined with proteomic and genomic (metallomic) approaches. A deeper understanding of the behavior of metals and metallodrugs in biological systems is not only key to the design of novel agents with unique mechanisms of action, but also to new understanding of clinically-established drugs.

Axial functionalisation of photoactive diazido platinum(iv) anticancer complexes.

Mono-axial functionalised octahedral diazido Pt(iv) complexes trans, trans, trans-[Pt(py)2(N3)2(OR1)(OR2)] (OR1 = OH and OR2 = anticancer agent coumarin-3 carboxylate (cou, 2a), pyruvate dehydrogenase kinase (PDK) inhibitors 4-phenylbutyrate (PhB, 2b) or dichloroacetate (DCA, 2c)), and their di-axial functionalised analogues with OR1 = DCA and OR2 = cou (3a), PhB (3b), or DCA (3c) have been synthesised and characterised, including the X-ray crystal structures of complexes 2a, 3a, 3b and 3c. These complexes exhibit dark stability and have the potential to generate cytotoxic Pt(ii) species and free radicals selectively in cancer cells when irradiated. Mono-functionalised complexes 2a-2c showed higher aqueous solubility and more negative reduction potentials. Mono- and di-functionalised complexes displayed higher photocytotoxicity with blue light (1 h, 465 nm, 4.8 mW cm-2) than the parent dihydroxido complex 1 (OR1 = OR2 = OH) in A2780 human ovarian (IC50 0.9-2.9 µM for 2a-2c; 0.11-0.39 µM for 3a-3c) and A549 human lung cancer cells (5.4-7.8 µM for 2a-2c; 1.2-2.6 µM for 3a-3c) with satisfactory dark stability. Notably, no apparent dark cytotoxicity was observed in healthy lung MRC-5 fibroblasts for all complexes (IC50 > 20 µM). Significantly higher platinum cellular accumulation and photo-generated ROS levels were observed for the di-functionalised complexes compared with their mono-functionalised analogues when cancer cells were treated under the same concentrations.

Photoactive platinum(iv) complex conjugated to a cancer-cell-targeting cyclic peptide.

A conjugate of cancer-cell targeting cyclic disulphide nona-peptide c(CRWYDENAC) consisting of nine l-amino acids with the photoactive succinate platinum(iv) complex trans,trans-[Pt(N3)2(py)2(OH)(succinate)] (Pt-cP) has been synthesised and characterised. The conjugate was stable in dark, but released succinate-peptide and Pt(ii) species upon irradiation with visible light, and formed photoproducts with guanine. Conjugate Pt-cP exhibited higher photocytotoxicity than parent complex trans,trans,trans-[Pt(N3)2(OH)2(py)2] (FM-190) towards cancer cells, including ovarian A2780, lung A549 and prostate PC3 human cancer cells upon irradiation with blue light (465 nm, 17.28 J cm-2) with IC50 values of 2.8-22.4 µM and the highest potency for A549 cells. Even though the dark cellular accumulation of Pt-cP in A2780 cells was lower than that of parent FM-190, Pt from Pt-cP accumulated in cancer cells upon irradiation to a level >3× higher than that from FM-190. In addition, the cellular accumulation of Pt from Pt-cP was enhanced ca. 47× after irradiation.

Photoactivatable platinum anticancer complex can generate tryptophan radicals.

l-Tryptophan (Trp), melatonin (MLT) and the Trp-peptide pentagastrin quenched the formation of azidyl radicals generated on irradiation of the anticancer complex trans,trans,trans-[Pt(pyridine)2(N3)2(OH)2] with visible light, giving rise to C3-centred indole radicals which were characterized for Trp and MLT using an EPR spin-trap; indole, together with azidyl and hydroxyl radicals, have potential roles in a multitargeting mechanism of action against resistant cancers.

Photoactivatable Cell-Selective Dinuclear trans-Diazidoplatinum(IV) Anticancer Prodrugs.

A series of dinuclear octahedral PtIV complexes trans, trans, trans-[{Pt(N3)2(py)2(OH)(OC(O)CH2CH2C(O)NH)}2R] containing pyridine (py) and bridging dicarboxylate [R = -CH2CH2- (1), trans-1,2-C6H10- (2), p-C6H4- (3), -CH2CH2CH2CH2- (4)] ligands have been synthesized and characterized, including the X-ray crystal structures of complexes 1·2MeOH and 4, the first photoactivatable dinuclear PtIV complexes with azido ligands. The complexes are highly stable in the dark, but upon photoactivation with blue light (420 nm), they release the bridging ligand and mononuclear photoproducts. Upon irradiation with blue light (465 nm), they generate azidyl and hydroxyl radicals, detected using a 5,5-dimethyl-1-pyrroline N-oxide electron paramagnetic resonance spin trap, accompanied by the disappearance of the ligand-to-metal charge-transfer (N3 → Pt) band at ca. 300 nm. The dinuclear complexes are photocytotoxic to human cancer cells (465 nm, 4.8 mW/cm2, 1 h), including A2780 human ovarian and esophageal OE19 cells with IC50 values of 8.8-78.3 µM, whereas cisplatin is inactive under these conditions. Complexes 1, 3, and 4 are notably more photoactive toward cisplatin-resistant ovarian A2780cis compared to A2780 cells. Remarkably, all of the complexes were relatively nontoxic toward normal cells (MRC5 lung fibroblasts), with IC50 values >100 µM, even after irradiation. The introduction of an aromatic bridging ligand (3) significantly enhanced cellular uptake. The populations in the stages of the cell cycle remained unchanged upon treatment with complexes in the dark, while the population of the G2/M phase increased upon irradiation, suggesting that DNA is a target for these photoactivated dinuclear PtIV complexes. Liquid chromatography-mass spectrometry data show that the photodecomposition pathway of the dinuclear complexes results in the release of two molecules of mononuclear platinum(II) species. As a consequence, DNA binding of the dinuclear complexes after photoactivation in cell-free media is, in several respects, qualitatively similar to that of the photoactivated mononuclear complex FM-190. After photoactivation, they were 2-fold more effective in quenching the fluorescence of EtBr bound to DNA, forming DNA interstrand cross-links and unwinding DNA compared to the photoactivated FM-190.

Supramolecular Photoactivatable Anticancer Hydrogels.

A photoactivatable dopamine-conjugated platinum(IV) anticancer complex (Pt-DA) has been incorporated into G-quadruplex G4K+ borate hydrogels by using borate ester linkages (Pt-G4K+B hydrogel). These were characterized by 11B NMR, attenuated total reflection Fourier transform infrared spectroscopy, circular dichroism, scanning electron microscopy and transmission electron microscopy. Microscopy investigations revealed the transformation of an extended fiber assembly into discrete flakes after incorporation of Pt-DA. Pt-DA showed photocytotoxicity against cisplatin-resistant A2780Cis human ovarian cancer cells (IC50 74 µM, blue light) with a photocytotoxic index <2, whereas Pt-G4K+B hydrogels exhibited more potent photocytotoxicity (IC50 3 µM, blue light) with a photocytotoxic index >5. Most notably, Pt-DA and Pt-G4K+B hydrogels show selective phototoxicity for cancer cells versus normal fibroblast cells (MRC5).