Radiation-induced prodrug activation: extending combined modality therapy for some solid tumours.

Combined chemoradiotherapy is the standard of care for locally advanced solid tumours. However, systemic toxicity may limit the delivery of planned chemotherapy. New approaches such as radiation-induced prodrug activation might diminish systemic toxicity, while retaining anticancer benefit. Organic azides have recently been shown to be reduced and activated under hypoxic conditions with clinically relevant doses of radiotherapy, uncaging pazopanib and doxorubicin in preclinical models with similar efficacy as the drug, but lower systemic toxicity. This approach may be relevant to the chemoradiation of glioblastoma and other solid tumours and offers potential for switching on drug delivery from implanted devices. The inclusion of reporters to confirm drug activation, avoidance of off-target effects and synchronisation of irradiation with optimal intratumoral drug concentration will be critical. Further preclinical validation studies of this approach should be encouraged.

Ultrasound-Triggered Delivery of Iproplatin from Microbubble-Conjugated Liposomes.

The PtIV prodrug iproplatin has been actively loaded into liposomes using a calcium acetate gradient, achieving a 3-fold enhancement in drug concentration compared to passive loading strategies. A strain-promoted cycloaddition reaction (azide- dibenzocyclooctyne) was used to attach iproplatin-loaded liposomes L(Pt) to gas-filled microbubbles (M), forming an ultrasound-responsive drug delivery vehicle [M-L(Pt)]. Ultrasound-triggered release of iproplatin from the microbubble-liposome construct was evaluated in cellulo. Breast cancer (MCF-7) cells treated with both free iproplatin and iproplatin-loaded liposome-microbubbles [M-L(Pt)] demonstrated an increase in platinum concentration when exposed to ultrasound. No appreciable platinum uptake was observed in MCF-7 cells following treatment with L(Pt) only or L(Pt)+ultrasound, suggesting that microbubble-mediated ultrasonic release of platinum-based drugs from liposomal carriers enables greater control over drug delivery.

Platinum(IV)-azido monocarboxylato complexes are photocytotoxic under irradiation with visible light.

Complexes trans,trans,trans-[Pt(N3)2(OH)(OCOR)(py)2] where py = pyridine and where OCOR = succinate (1); 4-oxo-4-propoxybutanoate (2) and N-methylisatoate (3) have been synthesized by derivation of trans,trans,trans-[Pt(OH)2(N3)2(py)2] (4) and characterised by NMR and EPR spectroscopy, ESI-MS and X-ray crystallography. Irradiation of 1-3 with green (517 nm) light initiated photoreduction to Pt(ii) and release of the axial ligands at a 3-fold faster rate than for 4. TD-DFT calculations showed dissociative transitions at longer wavelengths for 1 compared to 4. Complexes 1 and 2 showed greater photocytotoxicity than 4 when irradiated with 420 nm light (A2780 cell line IC50 values: 2.7 and 3.7 µM) and complex 2 was particularly active towards the cisplatin-resistant cell line A2780cis (IC50 3.7 µM). Unlike 4, complexes 1-3 were phototoxic under green light irradiation (517 nm), with minimal toxicity in the dark. A pKa(H2O) of 5.13 for the free carboxylate group was determined for 1, corresponding to an overall negative charge during biological experiments, which crucially, did not appear to impede cellular accumulation and photocytotoxicity.

Solvent-Dependent Reactivity and Photochemistry of Dinuclear and Mononuclear Platinum(IV) Azido Triazaolato Complexes.

Reaction between the platinum(IV) azido complex trans,trans,trans-[Pt(py)2(N3)2(OH)2] (1) and 1,4-diphenyl-2-butyne-1,4-dione 2 in MeCN produces the intermediate peroxide-bridged dimeric platinum(IV) azido triazolato species (5), which has been characterised by X-ray crystallography. However, if the reaction between 1 and 2 is conducted in MeOH it results in decomposition. Over time in MeCN, dimer (5) converts into mononuclear complexes trans,trans,trans-[Pt(py)2(N3)(triazole)(OH)2] (3 a/3 b), which are in dynamic exchange. If resuspended in protic solvents (MeOH,H2O), 3 a/3 b undergo a slow (22 d) irreversible rearrangement to a cyclised platinum(IV) species 4 which contains a formally N,O-chelated ligand. Conversion of 3 a/3 b to 4 in d 4-MeOH can be accelerated (384x) by irradiation with visible light, although continued irradiation also produces N3 . and OH. radicals, and the [4-N3]+ species can be readily detected by ESI-MS. Solvent choice significantly effects both the cycloaddition reaction between 1 and 2, and the stability of the resultant complexes.

Cell-permeable lanthanide-platinum(IV) anti-cancer prodrugs.

Platinum compounds are a vital part of our anti-cancer arsenal, and determining the location and speciation of platinum compounds is crucial. We have synthesised a lanthanide complex bearing a salicylic group (Ln = Gd, Eu) which demonstrates excellent cellular accumulation and minimal cytotoxicity. Derivatisation enabled access to bimetallic lanthanide-platinum(ii) and lanthanide-platinum(iv) complexes. Luminescence from the europium-platinum(iv) system was quenched, and reduction to platinum(ii) with ascorbic acid resulted in a "switch-on" luminescence enhancement. We used diffusion-based 1H NMR spectroscopic methods to quantify cellular accumulation. The gadolinium-platinum(ii) and gadolinium-platinum(iv) complexes demonstrated appreciable cytotoxicity. A longer delay following incubation before cytotoxicity was observed for the gadolinium-platinum(iv) compared to the gadolinium-platinum(ii) complex. Functionalisation with octanoate ligands resulted in enhanced cellular accumulation and an even greater latency in cytotoxicity.

Oxaliplatin and [Pt(R,R-DACH)(panobinostat-2H)] show nanomolar cytotoxicity towards diffuse intrinsic pontine glioma (DIPG).

We report the synthesis of two novel platinum(ii) complexes which incorporate histone deacetylase (HDAC) inhibitors: [PtII(R,R-DACH)(Sub-H)] (1), [PtII(R,R-DACH)(panobinostat-2H)] (2), where SubH = suberoyl-bis-hydroxamic acid; DACH = (1R,2R)-(-)-1,2-diaminocyclohexane and panobinostat = (E)-N-hydroxy-3-[4-[[2-(2-methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]prop-2-enamide. Complexes 1 and 2 were characterised by 1H, 13C, 195Pt NMR spectroscopy and ESI-MS. Whilst oxaliplatin demonstrated considerable cytotoxicity in two patient-derived low-passage paediatric glioma DIPG cell lines (IC50 values of 0.333 µM in SU-DIPG-IV, and 0.135 µM in SU-DIPG-XXI), complex 2 showed even greater cytotoxicities, with IC50 values of 0.021 µM (SU-DIPG-IV), 0.067 µM (BIOMEDE 194) and 0.009 µM (SU-DIPG-XXI). Complex 2 also demonstrated superior aqueous solubility in comparison to panobinostat. Complex 2 released free intact panobinostat under HPLC conditions, as determined by ESI-MS. Incubation of solutions of oxaliplatin (H2O) and panobinostat (DMF) resulted in instantaneous reactivity and precipitation of a panobinostat derivative which was not a platinum complex; the same reactivity was not observed between carboplatin and panobinostat.

Enhancing 31P NMR relaxation rates with a kinetically inert gadolinium complex.

The kinetically stable heptadentate gadolinium complex Gd.pDO3A (1.Gd) demonstrates significant 31P nuclear magnetic resonance (NMR) relaxation enhancement of biologically relevant phosphate species; adenosine triphosphate (ATP), phosphocreatine (PCr) and inorganic phosphate. Gd.pDO3A (1.Gd) binds these species in fast exchange, enabling the relaxation of the bulk phosphate species in solution. This gives rise to 31P relaxation enhancements up to 250-fold higher than those observed for 31P relaxation enhancements with the commercial MRI contrast agent Gd.DOTA (DOTAREM), 2. Gd.pDO3A-like complexes may have potential applications as 31P magnetic resonance contrast agents, since shortening the T1 relaxation time of phosphate species would reduce the time needed to acquire 31P-MR spectra.

Exploiting azide-alkyne click chemistry in the synthesis, tracking and targeting of platinum anticancer complexes.

Click chemistry is fundamentally important to medicinal chemistry and chemical biology. It represents a powerful and versatile tool, which can be exploited to develop novel Pt-based anticancer drugs and to better understand the biological effects of Pt-based anticancer drugs at a cellular level. Innovative azide-alkyne cycloaddition-based approaches are being used to functionalise Pt-based complexes with biomolecules to enhance tumour targeting. Valuable information in relation to the mechanisms of action and resistance of Pt-based drugs is also being revealed through click-based detection, isolation and tracking of Pt drug surrogates in biological and cellular environments. Although less well-explored, inorganic Pt-click reactions enable synthesis of novel (potentially multimetallic) Pt complexes and provide plausible routes to introduce functional groups and monitoring Pt-azido drug localisation.

Platinum(iv) dihydroxido diazido N-(heterocyclic)imine complexes are potently photocytotoxic when irradiated with visible light.

A series of trans-di-(N-heterocyclic)imine dihydroxido diazido PtIV complexes of the form trans,trans,trans-[Pt(N3)2(OH)2(L1)(L2)] where L = pyridine, 2-picoline, 3-picoline, 4-picoline, thiazole and 1-methylimidazole have been synthesised and characterised, and their photochemical and photobiological activity evaluated. Notably, complexes 19 (L1 = py, L2 = 3-pic) and 26 (L1 = L2 = 4-pic) were potently phototoxic following irradiation with visible light (420 nm), with IC50 values of 4.0 µM and 2.1 µM respectively (A2780 cancer cell line), demonstrating greater potency than the previously reported complex 1 (L1 = L2 = py; 6.7 µM); whilst also being minimally toxic in the absence of irradiation. Complexes with mixed N-(heterocyclic)imine ligands 19 and 20 (L1 = py, L2 = 4-pic) were particularly photocytotoxic towards cisplatin resistant (A2780cis) cell lines. Complex 18 (L1 = py, L2 = 2-pic) was comparatively less photocytotoxic (IC50 value 14.5 µM) than the other complexes, despite demonstrating the greatest absorbance at the irradiation wavelength and the fastest half-life for loss of the N3 → Pt LMCT transition upon irradiation (λ irr = 463 nm) in aqueous solution. Complex 29 (X1 = X2 = thiazole) although potently phototoxic (2.4 µM), was also toxic towards cells in the absence of irradiation.

A visible-light photoactivatable di-nuclear PtIV triazolato azido complex.

A novel PtIV triazolato azido complex [3]-[N1,N3] has been synthesised via a strain-promoted double-click reaction (SPDC) between a PtIV azido complex (1) and the Sondheimer diyne (2). Photoactivation of [3]-[N1,N3] with visible light (452 nm) in the presence of 5'-guanosine monophosphate (5'-GMP) produced both PtIV and PtII 5'-GMP species; EPR spectroscopy confirmed the production of both azidyl and hydroxyl radicals. Spin-trapping of photogenerated radicals - particularly hydroxyl radicals - was significantly reduced in the presence of 5'-GMP.

A novel Pt(iv) mono azido mono triazolato complex evolves azidyl radicals following irradiation with visible light.

The platinum(iv) azido complex trans,trans,trans-[PtIV(N3)2(OH)2(py)2] (1) undergoes cycloaddition with 1,4-diphenyl-2-butyne-1,4-dione (2) under mild, catalyst-free conditions, affording a number of mono and bis click products. The major mono click product (3) exists in MeCN as an equilibrium mixture between two species; 3a and 3b rapidly interconvert through nucleophilic attack of the axial Pt-OH group at the adjacent Ph-CO group. The kinetic and thermodynamic parameters for this interconversion have been measured by selective saturation-transfer NMR spectroscopic experiments and are consistent with cyclisation at the Pt centre. Complex 3b was also characterised by X-ray crystallography. Visible light irradiation (440-480 nm) of 3 in d3-MeCN produces azidyl radicals (N3˙), as demonstrated by EPR spin-trapping with DMPO; no generation of hydroxyl radicals was observed. 1H-195Pt HMBC NMR confirmed that the photoproducts were PtIV rather than PtII species, and HPLC was consistent with these being [3-N3]+ species; no facile photoejection of the triazolato ligand was observed, consistent with MS/MS fragmentation of 3. When 3 was irradiated in the presence of 5'-GMP, no 5'-GMP photoproducts were observed, suggesting that complex 3 is likely to exhibit significantly simplified biological activity (release of azidyl radicals but not DNA binding) compared with complex 1.

INDIANA: An in-cell diffusion method to characterize the size, abundance and permeability of cells.

NMR and MRI diffusion experiments contain information describing the shape, size, abundance, and membrane permeability of cells although extracting this information can be challenging. Here we present the INDIANA (IN-cell DIffusion ANAlysis) method to simultaneously and non-invasively measure cell abundance, effective radius, permeability and intrinsic relaxation rates and diffusion coefficients within the inter- and intra-cellular populations. The method couples an experimental dataset comprising stimulated-echo diffusion measurements, varying both the gradient strength and the diffusion delay, together with software to fit a model based on the Kärger equations to robustly extract the relevant parameters. A detailed error analysis is presented by comparing the results from fitting simulated data from Monte Carlo simulations, establishing its effectiveness. We note that for parameters typical of mammalian cells the approach is particularly effective, and the shape of the underlying cells does not unduly affect the results. Finally, we demonstrate the performance of the experiment on systems of suspended yeast and mammalian cells. The extracted parameters describing cell abundance, size, permeability and relaxation are independently validated.

Platinum(iv) azido complexes undergo copper-free click reactions with alkynes.

We report our investigations into the first examples of copper-free 1,3-dipolar cycloaddition (click) reactions of electrophiles with a PtIV azido complex. The Pt-IV azido complex trans, trans, trans-[PtIV(py)2(N3)2(OH)2] (1) was reactive towards dimethyl acetylenedicarboxylate (DMAD) (2), diethyl acetylenedicarboxylate DEACD (3), N-[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane (BCN) (11) and dibenzocyclooctyne-amine (DBCO) (12) resulting in formation of the corresponding mono (a) and bis-substituted (b) complexes. Complexes of 2 undergo further reactions between the Pt centre and the carbonyl group to form 2a' and 2b'. This is not seen for the products of the corresponding PtII azido complex trans-[Pt(py)2(N3)2] with acetylene 2. Novel complexes 2a', 2b', 11a and 11b have been characterised by multinuclear NMR, IR and UV-vis spectroscopy and ESI-MS. These reactions represent new synthetic routes to novel Pt(iv) complexes.

De novo generation of singlet oxygen and ammine ligands by photoactivation of a platinum anticancer complex.

Worth the excitement: Highly reactive oxygen and nitrogen species are generated by photoactivation of the anticancer platinum(IV) complex trans,trans,trans-[Pt(N3 )2 (OH)2 (MA)(Py)] (MA=methylamine, Py=pyridine). Singlet oxygen is formed from the hydroxido ligands and not from dissolved oxygen, and ammine ligands are products from the conversion of azido ligands to nitrenes. Both processes can induce oxidation of guanine.

Diazido mixed-amine platinum(IV) anticancer complexes activatable by visible-light form novel DNA adducts.

Platinum diam(m)ine complexes, such as cisplatin, are successful anticancer drugs, but suffer from problems of resistance and side-effects. Photoactivatable Pt(IV) prodrugs offer the potential of targeted drug release and new mechanisms of action. We report the synthesis, X-ray crystallographic and spectroscopic properties of photoactivatable diazido complexes trans,trans,trans-[Pt(N3)2(OH)2(MA)(Py)] (1; MA=methylamine, Py=pyridine) and trans,trans,trans-[Pt(N3)2(OH)2(MA)(Tz)] (2; Tz=thiazole), and interpret their photophysical properties by TD-DFT modelling. The orientation of the azido groups is highly dependent on H bonding and crystal packing, as shown by polymorphs 1p and 1q. Complexes 1 and 2 are stable in the dark towards hydrolysis and glutathione reduction, but undergo rapid photoreduction with UVA or blue light with minimal amine photodissociation. They are over an order of magnitude more potent towards HaCaT keratinocytes, A2780 ovarian, and OE19 oesophageal carcinoma cells than cisplatin and show particular potency towards cisplatin-resistant human ovarian cancer cells (A2780cis). Analysis of binding to calf-thymus (CT), plasmids, oligonucleotide DNA and individual nucleotides reveals that photoactivated 1 and 2 form both mono- and bifunctional DNA lesions, with preference for G and C, similar to transplatin, but with significantly larger unwinding angles and a higher percentage of interstrand cross-links, with evidence for DNA strand cross-linking further supported by a comet assay. DNA lesions of 1 and 2 on a 50 bp duplex were not recognised by HMGB1 protein, in contrast to cisplatin-type lesions. The photo-induced platination reactions of DNA by 1 and 2 show similarities with the products of the dark reactions of the Pt(II) compounds trans-[PtCl2(MA)(Py)] (5) and trans-[PtCl2(MA)(Tz)] (6). Following photoactivation, complex 2 reacted most rapidly with CT DNA, followed by 1, whereas the dark reactions of 5 and 6 with DNA were comparatively slow. Complexes 1 and 2 can therefore give rapid potent photocytotoxicity and novel DNA lesions in cancer cells, with no activity in the absence of irradiation.

Photo-isomerisation of alkenyl complexes of platinum(II): structural, spectroscopic, kinetic and computational investigations.

In this work UVA and blue light have been used to study photo-isomerisation about the C=C double bond in complexes of the type [PtCl(-CH=CHAr)(tmeda)] [Ar = C6H5, (E)-2a; 4-CH3O-C6H4, (E)-2b; 3-NO2-C6H4, (E)-2c; and 3-CH3O-C6H4, (E)-2d]. The progress of the reaction has been monitored by NMR spectroscopy following irradiation of the NMR sample. The NMR data have been complemented with X-ray diffractometric analysis of compounds (E)-2a-c and (Z)-2a. The kinetic data clearly indicate that a monomolecular mechanism is operating with the energy of the irradiating light influencing the rate of isomerisation but not the equilibrium composition, which is only slightly in favour of the Z isomer. DFT and TD-DFT theoretical investigations have been carried out to elucidate the nature of the main electronic transitions in the UV-Vis region and the mechanism of the photo-isomerisation reaction appears to proceed through a C=C bond twist process similar to that involved in purely organic molecules such as stilbene. In the Z isomer, one ortho proton of the phenyl group can come close to platinum (Pt···H(ortho) distance of 2.632 Å in (Z)-2a). In the case of 2c, the difference in chemical shift between the two ortho protons varies from 3.30 ppm in the Z isomer, where interaction with Pt is possible, to 0.60 ppm in the E isomer, where such interaction cannot take place. The analysis of the DFT orbitals indicates that the most shifted H(ortho) is that with a greater positive charge, pointing to an H-bond type of interaction.

Two-photon-activated ligand exchange in platinum(II) complexes.

Two photons are better than one: a square-planar Pt(II) complex with derivatized pyridine ligands was synthesized, which undergoes two-photon-induced ligand substitution with 600-740 nm light. Linear and quadratic density functional response theory allowed identification of the electronic transitions involved.

Tryptophan switch for a photoactivated platinum anticancer complex.

The octahedral Pt(IV) complex trans,trans,trans-[Pt(N(3))(2)(OH)(2)(py)(2)] (1) is potently cytotoxic to cancer cells when irradiated with visible (blue) light. We show that the acute photocytotoxicity can be switched off by low doses (500 µM) of the amino acid l-tryptophan. EPR and NMR spectroscopic experiments using spin traps show that l-Trp quenches the formation of azidyl radicals, probably by acting as an electron donor. l-Trp is well-known as a mediator of electron transfer between distant electron acceptor/donor centers in proteins, and such properties may make the free amino acid clinically useful for controlling the activity of photochemotherapeutic azido Pt(IV) drugs. Since previous work has demonstrated the ability of photoactivated 1 to platinate DNA, this suggests that the high potency of such photoactive platinum complexes is related to their dual attack on cancer cells by radicals and Pt(II) photoproducts.

A computational approach to tuning the photochemistry of platinum(IV) anticancer agents.

Diazido Pt(IV) complexes are inert stable prodrugs that can be photoactivated to produce Pt(II) species with promising anticancer activity. Our studies of the photochemistry of Pt(IV) complexes, [Pt(X)(2) (Y)(2) (Z)(2) ](0/-1) (X=N-ligands (NH(3) , pyridine, etc.)/S(CH(3) )(2) /H(-) , Y=(pseudo)halogen (N(3) (-) , I(-) ), Z=OR(-) , R=H, Ac) by time-dependent density functional theory (TDDFT) show close agreement with spectroscopic data. Broad exploration of cis/trans geometries, trans influences, the nature of the OR(-) and (pseudo)halogen ligands, electron-withdrawing/donating/delocalising substituents on the N-ligands, and intramolecular H bonds shows that: 1) the design of platinum(IV) complexes with intense bands shifted towards longer wavelengths (from 289 to ∼330 nm) can be achieved by introducing intramolecular H bonds involving the OH ligands and 2-hydroxyquinoline or by iodido ligands; 2) mesomeric electron-withdrawing substituents on pyridine result in low-energy absorption with significant intensity in the visible region; and 3) the distinct makeup of the molecular orbitals involved in the electronic transitions for cis/trans-{Pt(N(3) )(2) } isomers results in different photoproducts. In general, the comparison of the optimised geometries shows that Pt(IV) complexes with longer PtL bonds are more likely to undergo photoreduction with longer-wavelength light. The novel complex trans,trans,trans-[Pt(N(3) )(2) (OH)(2) (NH(3) )(4-nitropyridine)] with predicted absorption in the visible region has been synthesised. The experimental UV/Vis spectrum in aqueous solution correlates well with the intense band in the computed spectrum, whereas the overlay in the low-energy region can be improved by a solvent model. This combined computational and experimental study shows that TDDFT can be used to tune the coordination environment for optimising photoactive Pt(IV) compounds as anticancer agents.

Trans,trans,trans-[PtIV(N3)2(OH)2(py)(NH3)]: a light-activated antitumor platinum complex that kills human cancer cells by an apoptosis-independent mechanism.

Photoactivatable Pt(IV) diazido complexes have unusual photobiologic properties. We show here that trans,trans,trans-[Pt(IV)(N(3))(2)(OH)(2)(py)(NH(3))] complex 3 is a potent photoactivated cytotoxin toward human cancer cells in culture, with an average IC(50) value in 13 cell lines of 55 ± 28 µmol/L after 30 minutes (0.12 mW/cm(2)) photoactivation with UVA, although visible light was also effective. Photoactivated complex 3 was noncross-resistant to cisplatin in 3 of 4 resistant cell lines. Cell swelling but very little blebbing was seen for HL60 cells treated with irradiated complex 3. Unlike cisplatin and etoposide, both of which cause apoptosis in HL60 cells, no apoptosis was observed for UVA-activated complex 3 by the Annexin V/propidium iodide flow cytotometry assay. Changes in the levels of the autophagic proteins LC3B-II and p62 in HL60 cells treated with UVA-activated complex 3 indicate autophagy is active during cell death. In a clonogenic assay with the SISO human cervix cancer cell line, 3 inhibited colony formation when activated by UVA irradiation. Antitumor activity of complex 3 in mice bearing xenografted OE19 esophageal carcinoma tumors was photoaugmented by visible light. Insights into the novel reaction pathways of complex 3 have been obtained from (14)N{(1)H} nuclear magnetic resonance studies, which show that photoactivation pathways can involve release of free azide in buffered solution. Density functional theory (DFT) and time-dependent DFT calculations revealed the dissociative character of singlet and triplet excited states of complex 3, which gives rise to reactive, possibly cytotoxic azidyl radicals.