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The Pt(IV) prodrug trans, trans, trans -[Pt(pyridine) 2 (N 3 ) 2 (OH) 2 ] ( Pt1 ) and its coumarin derivative trans, trans, trans -[Pt(pyridine) 2 (N 3 ) 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.

Companion diagnostics (CDx) are powerful tests that can provide physicians with crucial biomarker information that can improve treatment outcomes by matching therapies to patients. Here, we report a photoacoustic imaging-based CDx (PACDx) for the selective detection of elevated glutathione (GSH) in a lung cancer model. GSH is abundant in most cells, so we adopted a physical organic chemistry approach to precisely tune the reactivity to distinguish between normal and pathological states. To evaluate the efficacy of PACDx in vivo, we designed a blind study where photoacoustic imaging was used to identify mice bearing lung xenografts. We also employed PACDx in orthotopic lung cancer and liver metastasis models to image GSH. In addition, we designed a matching prodrug, PARx, that uses the same S N Ar chemistry to release a chemotherapeutic with an integrated PA readout. Studies demonstrate that PARx can inhibit tumour growth without off-target toxicity in a lung cancer xenograft model., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Oxoplatin-B, a platinum(IV) complex [Pt(NH 3 ) 2 Cl 2 (L 1 )(OH)] (1) of 4-methylbenzoic acid (HL 1 ) functionalized with 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) was prepared, characterized and its antitumor activity studied. [Pt(NH 3 ) 2 Cl 2 (L 2 )(OH)] (2) of 4-methylbenzoic acid (HL 2 ) was studied as a control. Complex 1 showed an absorption band at 500 nm (ɛ = 4.34 × 10 4  M -1  cm -1 ) and an emission band at 515 nm (λ ex  = 488 nm, Φ F  = 0.64) in 1% dimethyl sulfoxide/Dulbecco's Modified Eagle's Medium (pH = 7.2). Visible light-induced (400-700 nm) generation of singlet oxygen was evidenced from 1,3-diphenylisobenzofuran titration study. Complex 1 showed photo-induced cytotoxicity in visible light (400-700 nm, 10 J cm -2 ) against human breast cancer (MCF-7), cervical cancer (HeLa) and lung cancer (A549) cells (IC 50 : 1.1-3.8 μM) while being less toxic in normal cells. Confocal imaging showed mitochondrial localization with additional evidence from platinum content from isolated mitochondria and 5,5,6,6'-tetrachloro-1,1',3,3' tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) assay. Cellular apoptosis was observed from Annexin-V-FITC (fluorescein isothiocyanate)/propidium iodide assay., (Copyright © 2021. Published by Elsevier Inc.)

Histone deacetylase inhibitors have often been used in combination treatment of various types of cancers due to their non-genotoxic epigenetic potential. Valproic acid (VPA) is a well-known histone deacetylase inhibitor. Conjugate of VPA with a phtoactive platinum diimine complex through an ester bond has been fabricated to potentiate the photocytotoxicity of the photosensitizer. Its capability to generate singlet oxygen, behavior in the presence of esterase, and photocytotoxicity in tumor cells have also been studied. The results revealed that the novel VPA-modified platinum diimine complex could produce singlet oxygen efficiently and release VPA in the presence of porcine liver esterase. The results also suggested that incorporation of VPA moiety into the platinum diimine complex might significantly enhance the cytotoxicity of the complex., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chemotherapy is a powerful tool in the armoury against cancer, but it is fraught with problems due to its global systemic toxicity. Here we report the proof of concept of a chemistry-based strategy, whereby gamma/X-ray irradiation mediates the activation of a cancer prodrug, thereby enabling simultaneous chemo-radiotherapy with radiotherapy locally activating a prodrug. In an initial demonstration, we show the activation of a fluorescent probe using this approach. Expanding on this, we show how sulfonyl azide- and phenyl azide-caged prodrugs of pazopanib and doxorubicin can be liberated using clinically relevant doses of ionizing radiation. This strategy is different to conventional chemo-radiotherapy radiation, where chemo-sensitization of the cancer takes place so that subsequent radiotherapy is more effective. This approach could enable site-directed chemotherapy, rather than systemic chemotherapy, with 'real time' drug decaging at the tumour site. As such, it opens up a new era in targeted and directed chemotherapy., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Hydrogen sulfide (H 2 S) has gained significant attention as a potent bioregulator in the redox metabolome, but it is just one of many reactive sulfur species (RSS). Recently, small molecule persulfides (structure RSSH) have emerged as RSS of particular interest due to their enhanced antioxidant abilities compared to H 2 S and their ability to directly convert protein thiols into protein persulfides, suggesting that persulfides may have distinct physiological functions from H 2 S. However, persulfides exhibit instability and cross-reactivity that hampers the elucidation of their precise biological roles. As such, chemists have designed chemical tools and techniques to facilitate the study of persulfides under various conditions. These molecules and methods include persulfide trapping reagents and sensors, as well as compounds that degrade in response to various triggers to release persulfides, termed persulfide donors. There now exist a variety of persulfide donor classes, some of which possess tissue-targeting capabilities designed to mimic localized endogenous production of RSS. This Review briefly covers the physicochemical properties of persulfides, the endogenous production of small molecule persulfides, and their reactions with protein thiols and other reactive species. These introductory sections are followed by a discussion of chemical tools used in persulfide chemical biology, with critical analysis of recent advancements in the field and commentary on potential directions for future research.

Development of the drug with high therapeutic efficacy and low toxicity is crucial to cancer ablation. In this study, we have demonstrated a red light-responsive prodrug BDP-TK-CPT by connecting the chemotherapeutic agent camptothecin with a boron dipyrromethene (BDP)-based photosensitizer via a reactive oxygen species (ROS)-labile thioketal chain. Since camptothecin is modified by a BDP-based macrocycle at the active site, the formed prodrug displays an extremely low toxicity in dark. However, upon illumination by red light, it can efficiently generate ROS leading to cell death by photodynamic therapy. Meanwhile, the ROS generated can destroy thioketal group to release free camptothecin which further results in local cell death by chemotherapy. The combined antitumor effects of the prodrug have been verified in HepG2, EC109, and HeLa cancer cells and mice bearing H22 tumors. This study may provide an alternative strategy for stimuli-responsive combination treatment of tumors by conjugation of ROS-activatable prodrugs with photosensitizing agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Light is a uniquely powerful tool for controlling molecular events in biology. No other external input (e.g., heat, ultrasound, magnetic field) can be so tightly focused or so highly regulated as a clinical laser. Drug delivery vehicles that can be photonically activated have been developed across many platforms, from the simplest "caging" of therapeutics in a prodrug form, to more complex micelles and circulating liposomes that improve drug uptake and efficacy, to large-scale hydrogel platforms that can be used to protect and deliver macromolecular agents including full-length proteins. In this Review, we discuss recent innovations in photosensitive drug delivery and highlight future opportunities to engineer and exploit such light-responsive technologies in the clinical setting., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Recently, sunscreen-based drug photocages have been introduced to provide UV protection to photoactive drugs, thus increasing their photosafety. Here, combined experimental and theoretical studies performed on a photocage based on the commercial UVA filter avobenzone (AB) and on the photosensitizing non-steroidal anti-inflammatory drug ketoprofen (KP) are presented unveiling the photophysical processes responsible for the light-triggered release. Particular attention is paid to solvent stabilization of the drug and UV filter excited states, respectively, which leads to a switching between the triplet excited state energies of the AB and KP units. Most notably, we show that the stabilization of the AB triplet excited state in ethanol solution is the key requirement for an efficient photouncaging. By contrast, in apolar solvents, in particular hexane, KP has the lowest triplet excited state, hence acting as an energy acceptor quenching the AB triplet manifold, thus inhibiting the desired photoreaction.

Photodynamic therapy (PDT) has emerged as one of the most up-and-coming non-invasive therapeutic modalities for cancer therapy in rencent years. However, its therapeutic effect was still hampered by the short life span, limited diffusion distance and ineluctable depletion of singlet oxygen ( 1 O 2 ), as well as the hypoxic microenvironment in the tumor tissue. Such problems have limited the application of PDT and appropriate solutions are highly demand. Methods: Herein, a programmatic treatment strategy is proposed for the development of a smart molecular prodrug ( D-bpy ), which comprise a two-photon photosensitizer and a hypoxia-activated chemotherapeutic prodrug. A rhodamine dye was designed to connect them and track the drug release by the fluorescent signal generated through azo bond cleavage. Results: The prodrug ( D-bpy ) can stay on the cell membrane and enrich at the tumor site. Upon light irradiation, the therapeutic effect was enhanced by a stepwise treatment: (i) direct generation of 1 O 2 on the cell membrane induced membrane destruction and promoted the D-bpy uptake; (ii) deep tumor hypoxia caused by two-photon PDT process further triggered the activation of the chemotherapy prodrug. Both in vitro and in vivo experiments, D-bpy have exhabited excellent tumor treatment effect. Conclusion: The innovative programmatic treatment strategy provides new strategy for the design of follow-up anticancer drugs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Two photoactivatable dicarbonyl ruthenium(II) complexes based on an amide-functionalised bipyridine scaffold (4-position) equipped with an alkyne functionality or a green-fluorescent BODIPY (boron-dipyrromethene) dye have been prepared and used to investigate their light-induced decarbonylation. UV/Vis, FTIR and 13 C NMR spectroscopies as well as gas chromatography and multivariate curve resolution alternating least-squares analysis (MCR-ALS) were used to elucidate the mechanism of the decarbonylation process. Release of the first CO molecule occurs very quickly, while release of the second CO molecule proceeds more slowly. In vitro studies using two cell lines A431 (human squamous carcinoma) and HEK293 (human embryonic kidney cells) have been carried out in order to characterise the anti-proliferative and anti-apoptotic activities. The BODIPY-labelled compound allows for monitoring the cellular uptake, showing fast internalisation kinetics and accumulation at the endoplasmic reticulum and mitochondria., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

To enhance the specificity and efficiency of anti-tumor therapies, we have designed a multifunctional nanoparticle platform for photochemotherapy using fluorescence (FL) and photoacoustic (PA) imaging guidance. Nanoparticles (NPs) composed of a eutectic mixture of natural fatty acids that undergo a solid-liquid phase transition at 39 °C were used to encapsulate materials for the rapid and uniform release of the hypoxia-activated prodrug tirapazamine (TPZ) and the photosensitizer IR780, which targets the mitochondria of tumor cells and can be used to induce hypoxic cell death via photodynamic therapy and photothermal therapy. In vitro, the NPs containing TPZ and IR7890 exhibited appreciable cell uptake and triggered drug release when irradiated with a NIR laser. In vivo, photochemotherapy of the NPs achieved the best anti-tumor efficacy under PA and FL imaging guidance and monitoring. By combining IR780 mitochondria-targeting phototherapy with TPZ, we observed improved anti-tumor effectiveness and this has the potential to reduce the side effects of traditional chemotherapy. Herein, we demonstrate a new intracellular photochemotherapy nanosystem that co-encapsulates photosensitizers and hypoxia-activated drugs to enhance the overall anti-tumor effect precisely and efficiently.

Cancer is currently one of the deadliest diseases worldwide. Based on the high incidence of this disease, the side effects associated with current chemotherapies and the appearance of drug resistance, considerable efforts have been directed towards the development of new anticancer drugs with new modes of action. Metal-based compounds are particularly attractive candidates due to their metabolic mechanisms, which differ substantially from those of organic drugs. Of special interest in this context are organometallic ruthenium(II) complexes of the type [Ru(η 6 -arene)(pta)Cl 2 ] (arene: p-cymene, toluene, benzene, etc.; pta: 1,3,5-triaza-7-phosphaadamantane), which are abbreviated to RAPTA. Complementary to chemotherapy, photoactivated chemotherapy is a technique that has received increasing attention towards the development of treatment for numerous kinds of cancer. With this in mind, a photoactive RAPTA-type complex bearing azide ligands has been designed. The diazide complex, [Ru(η 6 -p-cymene)pta-(N 3 ) 2 ], is inert in water, but slowly releases the azide ligand upon exposure to light. Consequently, the in vitro cytotoxicity of the complex in the dark and upon light exposure at λ=450 nm in human cervical carcinoma (HeLa) and noncancerous retinal pigment epithelium (RPE-1) cells was investigated. Although the cytotoxicity of the complex was found to be modest in the dark, an increase in toxicity upon light exposure was observed., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The role of hypoxia in radiation resistance is well established and many approaches to overcome hypoxia in tumours have been explored, with variable success. Two small molecule strategies for targeting hypoxia have dominated preclinical and clinical efforts. One approach has been the use of electron-affinic nitroheterocycles as oxygen-mimetic sensitisers. These agents are best exemplified by the 5-nitroimidazole nimorazole, which has limited use in conjunction with radiotherapy in head and neck squamous cell carcinoma. The second approach seeks to leverage tumour hypoxia as a tumour-specific address for hypoxia-activated prodrugs. These prodrugs are selectively activated by reductases under hypoxia to release cytotoxins, which in some instances may diffuse to kill surrounding oxic tumour tissue. A number of these hypoxia-activated prodrugs have been examined in clinical trial and the merits and shortcomings of recent examples are discussed. There has been an evolution from delivering DNA-interactive cytotoxins to molecularly targeted agents. Efforts to implement these strategies clinically continue today, but success has been elusive. Several issues have been identified that compromised these clinical campaigns. A failure to consider the extravascular transport and the micropharmacokinetic properties of the prodrugs has reduced efficacy. One key element for these 'targeted' approaches is the need to co-develop biomarkers to identify appropriate patients. Hypoxia-activated prodrugs require biomarkers for hypoxia, but also for appropriate activating reductases in tumours, as well as markers of intrinsic sensitivity to the released drug. The field is still evolving and changes in radiation delivery and the impact of immune-oncology will provide fertile ground for future innovation., (Copyright © 2019 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.)

Natural lipid nanocarriers, exosomes, carry cell-signaling materials such as DNA and RNA for intercellular communications. Exosomes derived from cancer cells contribute to the progression and metastasis of cancer cells by transferring oncogenic signaling molecules to neighboring and remote premetastatic sites. Therefore, applying the unique properties of exosomes for cancer therapy has been expected in science, medicine, and drug discovery fields. Herein, we report that an exosome-targeting prodrug system, designated MARCKS-ED-photodoxaz, could spatiotemporally control the activation of an exquisitely cytotoxic agent, doxazolidine (doxaz), with UV light. The MARCKS-ED peptide enters a cell by forming a complex with the exosomes in situ at its plasma membrane and in the media. MARCKS-ED-photodoxaz releases doxaz under near-UV irradiation to inhibit cell growth with low nanomolar IC 50 values. The MARCKS-ED-photodoxaz system targeting exosomes and utilizing photochemistry will potentially provide a new approach for the treatment of cancer, especially for highly progressive and invasive metastatic cancers.

An adamantane-containing zwitterionic copolymer poly(2-(methacryloyloxy)ethyl phosphorylcholine)- co-poly(2-(methacryloyloxy)ethyl adamantane-1-carboxylate) (poly(MPC- co-MAda)) was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. The hydrophobic photosensitizer chlorin e6 (Ce6) was conjugated to β-cyclodextrin (β-CD) by glutathione (GSH)-sensitive disulfide bonds. The Ce6 conjugated supramolecular prodrug nanocarriers were fabricated due to the host-guest interaction between adamantane and β-CD, which was confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The Ce6 conjugated prodrug nanocarriers showed reduction-responsive release of Ce6, which could result in the activation of Ce6. The generation of cytotoxic reactive oxygen species (ROS) was significantly enhanced due to the activation of Ce6. In additiona, the Ce6 conjugated prodrug nanocarriers could effectively inhibit the proliferation of cancer cells upon light irradiation.

A series of dinuclear octahedral Pt IV complexes trans, trans, trans-[{Pt(N 3 ) 2 (py) 2 (OH)(OC(O)CH 2 CH 2 C(O)NH)} 2 R] containing pyridine (py) and bridging dicarboxylate [R = -CH 2 CH 2 - (1), trans-1,2-C 6 H 10 - (2), p-C 6 H 4 - (3), -CH 2 CH 2 CH 2 CH 2 - (4)] ligands have been synthesized and characterized, including the X-ray crystal structures of complexes 1·2MeOH and 4, the first photoactivatable dinuclear Pt IV 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 (N 3 → Pt) band at ca. 300 nm. The dinuclear complexes are photocytotoxic to human cancer cells (465 nm, 4.8 mW/cm 2 , 1 h), including A2780 human ovarian and esophageal OE19 cells with IC 50 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 IC 50 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 Pt IV 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.

The delivery of controlled amounts of carbon monoxide (CO) to biological targets is of significant current interest. Very few CO-releasing compounds are currently known that can be rigorously controlled in terms of the location and amount of CO released. To address this deficiency, we report herein a new metal-free, visible-light-induced CO-releasing molecule (photoCORM) and its prodrug oxidized form, which offer new approaches to controlled, localized CO delivery. The new photoCORM, based on a 3-hydroxybenzo[ g]quinolone framework, releases 1 equiv of CO upon visible-light illumination under a variety of biologically relevant conditions. This nontoxic compound can be tracked prior to CO release using fluorescence microscopy and produces a nontoxic byproduct following CO release. An oxidized prodrug form of the photoCORM is reduced by cellular thiols, providing an approach toward activation in the reducing environment of cancer cells. Strong noncovalent affinity of the nonmetal photoCORM to albumin enables use of an albumin:photoCORM complex for targeted CO delivery to cancer cells. This approach produced cytotoxicity IC 50 values among the lowest reported to date for CO delivery to cancer cells by a photoCORM. This albumin:photoCORM complex is also the first CO delivery system to produce significant anti-inflammatory effects when introduced at nanomolar photoCORM concentration.

CHS-828 (N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N″-4-pyridyl guanidine) is an anticancer agent with low bioavailability and high systemic toxicity. Here we present an approach to improve the therapeutic profile of the drug using photolabile ruthenium complexes to generate light-activated prodrugs of CHS-828. Both prodrug complexes are stable in the dark but release CHS-828 when irradiated with visible light. The complexes are water-soluble and accumulate in tumour cells in very high concentrations, predominantly in the mitochondria. Both prodrug complexes are significantly less cyototoxic than free CHS-828 in the dark but their toxicity increases up to 10-fold in combination with visible light. The cellular responses to light treatment are consistent with release of the cytotoxic CHS-828 ligand., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A unique, dual-function, photoactivatable anticancer prodrug, RuEuL, has been tailored that features a ruthenium(II) complex linked to a cyclen-europium chelate via a π-conjugated bridge. Under irradiation at 488 nm, the dark-inactive prodrug undergoes photodissociation, releasing the DNA-damaging ruthenium species. Under evaluation-window irradiation (λ irr = one-photon 350 nm or two-photon 700 nm), the drug delivery process can be quantitatively monitored in real-time because of the long-lived red europium emission. Linear relationships between released drug concentration and ESI-MS or luminescence responses are established. Finally, the efficiency of the new prodrug is demonstrated both in vitro RuEuL anticancer prodrug over some existing ones and open the way for decisive improvements in multipurpose prodrugs.

A FRET donor-acceptor xanthene-coumarin conjugate has been designed for redox-regulated synergic treatment of photodynamic therapy and chemotherapy with real-time monitoring. The "locked" FRET pair was selectively "unlocked" by biological reducing thiols via rupture of a sacrificial disulfide linker. A distinct change in fluorescence color and selective cancer cell toxicity were observed in vitro.

Cancer-specific anticancer drugs are still an elusive goal. Using light as the temporal control to generate cytotoxic species from photo-activated prodrug in the presence or absence of molecular oxygen has shown potential application targeted chemotherapy as in photodynamic therapy (PDT). In the present work we explored the chemistry of several photo-active (μ-oxo)diiron(III) complexes of the following formulation [{Fe(μ-O) (L-his)(B)} 2 ](ClO 4 ) 2 (1a-1c), [Fe 2 (μ-O)(H 2 O) 2 B 4 ](ClO 4 ) 4 (2b, 2c) and [Fe 2 (μ-O)(μ-O 2 CMe)B 4 ](ClO 4 ) 3 (3b, 3c), L-his = l-histidine, B is 2,2'-bipyridine, 1,10-phenanthroline (phen) and dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) complexes for tumor-specific anticancer activity. Facile redox chemistry and photochemical aspects of the complexes prompted us to investigate the cytotoxic as well as the photo-activated cytotoxic properties of the complexes to the cancer cells. In the present investigation we explored the cancer-specific condition of excess concentration of H 2 O 2 for our approach to targeted chemotherapy. Cytotoxic effect of the complexes to the cancer cells was found to be significantly higher than in normal cells indicating tumor-specific anticancer activity of the complexes. Cytotoxic effect was even more pronounced when the cancer cells treated with the complexes were exposed to the visible light (400-700 nm). There was >12 fold increase in cytotoxicity of the photoactivated complexes in cancer cells (MCF-7) in comparison to the normal cells (MCF-10a). We have defined a factor viz. cancer cell specificity factor (f) describing the targeted photochemotherapeutic effect of the complexes at their specific concentration. The factor (f) > 1 indicated the cancer cell specificity of the complexes, while f > 2.5 for the complexes under the visible light exposure suggested photodynamic effect. DCFDA assay indicated the presence of excess of ROS in the treated HeLa cells. ROS concentration was found to increase even more on visible light exposure. Increased ROS in the cancer cells disturb the cellular redox mechanism inducing oxidative stress to lethality. Decarboxylation of photo-activated diiron(III) complexes generate OH radical responsible for cell death. Overall, the high efficacy and selectivity of the (μ-oxo)diiron(III) complexes potentially make them suitable for in vivo applications and extensive testing toward transfer into the clinical arena., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Metal ions are essential for a wide range of physiological processes, but they can also be toxic if not appropriately regulated by a complex network of metal trafficking proteins. Intervention in cellular metal distribution with small-molecule or peptide chelating agents has promising therapeutic potential to harness metals to fight disease. Molecular outcomes associated with forming metal-chelate interactions in situ include altering the concentration and subcellular metal distribution, inhibiting metalloenzymes, enhancing the reactivity of a metal species to elicit a favorable biological response, or passivating the reactivity of a metal species to prevent deleterious reactivity. The systemic administration of metal chelating agents, however, raises safety concerns due to the potential risks of indiscriminate extraction of metals from critical metalloproteins and inhibition of metalloenzymes. One can estimate that chelators capable of complexing metal ions with dissociation constants in the submicromolar range are thermodynamically capable of extracting metal ions from some metalloproteins and disrupting regular function. Such dissociation constants are easily attainable for multidentate chelators interacting with first-row d-block metal cations in relevant +1, + 2, and +3 oxidation states. To overcome this challenge of indiscriminate metal chelation, we have pursued a prodrug strategy for chelating agents in which the resulting "prochelator" has negligible metal binding affinity until a specific stimulus generates a favorable metal binding site. The prochelator strategy enables conditional metal chelation to occur preferentially in locations affected by disease- or therapy-associated stimuli, thereby minimizing off-target metal chelation. Our design of responsive prochelators encompasses three general approaches of activation: the "removal" approach operates by eliminating a masking group that blocks a potential metal chelation site to reveal the complete binding site under the desired conditions; the molecular "switch" approach involves a reversible conformational change between inactive and active forms of a chelator with differential metal binding affinity under specific conditions; and the "addition" approach adds a new ligand donor arm to the prochelator to constitute a complete metal chelation site. Adopting these approaches, we have created four categories of triggerable prochelators that respond to (1) reactive oxygen species, (2) light, (3) specific enzymes, and (4) biological regulatory events. This Account highlights progress from our group on building prochelators that showcase these four categories of responsive metal chelating agents for manipulating cellular metals. The creation and chemical understanding of such stimulus-responsive prochelators enables exciting applications for understanding the cell biology of metals and for developing therapies based on metal-dependent processes in a variety of diseases.

In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG) to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV) irradiation of the prodrugs original inhibitory activity was completely restored and even distinctly reinforced, as was the case for the prodrug 4. The presented results are a further evidence for caging technique being an interesting approach in the protein kinase field. It could enable spatial and temporal control for the inhibition of VEGFR-2. The described photoactivatable prodrugs might be highly useful as biological probes for studying the VEGFR-2 signal transduction.

[Pt(cur)(NH3)2](NO3) (1), a curcumin-bound cis-diammineplatinum(II) complex, nicknamed Platicur, as a novel photoactivated chemotherapeutic agent releases photoactive curcumin and an active platinum(II) species upon irradiation with visible light. The hydrolytic instability of free curcumin reduces upon binding to platinum(II). Interactions of 1 with 5'-GMP and ct-DNA indicated formation of platinum-bound DNA adducts upon exposure to visible light (λ=400-700 nm). It showed apoptotic photocytotoxicity in cancer cells (IC50 ≈ 15 μM), thus forming (⋅)OH, while remaining passive in the darkness (IC50 >200 μM). A comet assay and platinum estimation suggest Pt-DNA crosslink formation. The fluorescence microscopic images showed cytosolic localization of curcumin, thus implying possibility of dual action as a chemo- and phototherapeutic agent., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new anticancer agent based on the conjugation of a photoactivatable Pt(IV) pro-drug to a cyclic RGD-containing peptide is described. Upon visible light irradiation, phototoxicity was induced preferentially in SK-MEL-28 melanoma cancer cells overexpressing αVβ3 integrin compared to control DU-145 human prostate carcinoma cells.

SN30000 is a benzotriazine di-N-oxide which is selectively toxic to radio-resistant hypoxic cells in tumours. Given the complex photochemistry of some aromatic N-oxides, we evaluated the potential for photodegradation of SN30000 solutions. Initial studies demonstrated significant oxygen-insensitive degradation under normal laboratory lighting conditions. The kinetics of photodegradation showed marked concentration dependence of the form predicted by Beer's law, with a quantum yield of 0.016. The photoproducts could be rationalised as arising from an oxaziridine intermediate. The major stable product (cmpd 6; yield ∼50% of SN30000 loss under either UV or visible light) was characterised as resulting from intra-molecular oxygen transfer to the morpholine side chain of SN30000. This mechanism is consistent with lack of formation of the corresponding morpholine N-oxide from an analogue (SN29751) in which the proposed six-membered-ring transition state cannot form. Cmpd 6 was less cytotoxic than SN30000 to human tumour cells in culture, under either hypoxic or aerobic conditions, and was not toxic when administered intra-peritoneally to NIH-III nude mice at a dose (750 μmol/kg) above the maximal tolerated dose of SN30000 itself. In conclusion, SN30000 solutions are significantly photosensitive at low concentration, requiring protection from light, but the major photoproduct is less toxic than the parent., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Two molecules of an antitumor agent, 5-fluorodeoxyuridine (5-FdUrd), were connected by a 2-oxoalkyl linker (Oxo-linker) at the N(3) position to obtain radiation-activated prodrugs, FdUrd(2) A and FdUrd(2) B. The prodrugs in this study released 5-FdUrd via one-electron reduction initiated by hypoxic X-irradiation. The release of 5-FdUrd from FdUrd(2) A and FdUrd(2) B proceeded more efficiently than that of previous prodrug, Oxo-FdUrd, which possessed one molecule of 5-FdUrd. FdUrd(2) A exhibited increased cytotoxicity against A549 cells when the FdUrd(2) A solution had been irradiated with a large dose of X-rays before administration to the cells. However, we observed no effect on cytotoxicity when the cells were X-irradiated under hypoxic conditions in the presence of FdUrd(2) A because the amount of 5-FdUrd released in the cells seemed to be too low to induce cytotoxic activity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We prepared a 5-fluorodeoxyuridine (5-FdUrd) derivative possessing azide methyl group (N(3)-FdUrd) as a novel radiation-activated prodrug. The parent antitumor agent, 5-FdUrd, was released efficiently from N(3)-FdUrd by hypoxic X-irradiation. On the other hand, the activation of N(3)-FdUrd was suppressed upon X-irradiation under aerobic conditions. A biological assay using A549 cells revealed that the cytotoxicity of N(3)-FdUrd was significantly enhanced by hypoxic X-irradiation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: Doxorubicin (DOX) is a very effective anticancer agent. However, in its pure form, its application is limited by significant cardiotoxic side effects. The purpose of this study was to develop a controllably activatable chemotherapy prodrug of DOX created by blocking its free amine group with a biotinylated photocleavable blocking group (PCB)., Methods: An n-hydroxy succunamide protecting group on the PCB allowed selective binding at the DOX active amine group. The PCB included an ortho-nitrophenyl group for photo cleavability and a water-soluble glycol spacer arm ending in a biotin group for enhanced membrane interaction., Results: This novel DOX-PCB prodrug had a 200-fold decrease in cytotoxicity compared to free DOX and could release active DOX upon exposure to UV light at 350 nm. Unlike DOX, DOX-PCB stayed in the cell cytoplasm, did not enter the nucleus, and did not stain the exposed DNA during mitosis. Human liver microsome incubation with DOX-PCB indicated stability against liver metabolic breakdown., Conclusions: The development of the DOX-PCB prodrug demonstrates the possibility of using light as a method of prodrug activation in deep internal tissues without relying on inherent physical or biochemical differences between the tumor and healthy tissue for use as the trigger.

A series of metal complexes were prepared as potential prodrugs of the extremely toxic DNA minor groove alkylator 1-(chloromethyl)-5-hydroxy-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (seco-6-azaCBI-TMI) and close analogues. The pyrrolo[3,2-f]quinoline cytotoxins were prepared from 2-methoxy-4-nitroaniline in a nine-step synthesis involving a Skraup construction of a quinoline intermediate, its appropriate functionalization, and a final radical cyclization. The metal complexes were prepared from these and the labile metal complex synthons [Co(cyclen)(OTf)(2)](+), [Cr(acac)(2)(H(2)O)(2)](+), and [Co(2)(Me(2)dtc)(5)](+). The cobalt complexes were considerably more stable than the free effectors and showed significant attenuation of the cytotoxicity of the latter, with IC(50) ratios (complex/effector) of 50- to 150-fold, and substantial hypoxic cell selectivity, with IC(50) ratios (oxic/hypoxic cells) of 20- to 40-fold. The cobalt complexes were also efficiently activated by ionizing radiation, with G values for loss of the compound close to the theoretical value for one-electron reduction of 0.68 micromol/J. This work extends earlier observations that cobalt cyclen complexes are suitable for both the bioreductive and radiolytic release of potent pyrrolo[3,2-f]quinoline effectors.

An anti-tumour agent of cytarabine (ara-C) was conjugated with a 2-oxopropyl group at the N(4) position to obtain a radiation-activated prodrug (oxo-ara-C) that targeted hypoxic tumour tissues with selective cytotoxicity. The parent anti-tumour agent, ara-C, was confirmed to be released from oxo-ara-Cvia one-electron reduction upon hypoxic X-ray treatment. The prodrug oxo-ara-C had dramatically reduced cytotoxicity against human lung adenocarcinoma A549 cells relative to ara-C because of the effect of 2-oxopropyl substituent. In contrast, X-ray treatment of hypoxic A549 cells containing oxo-ara-C enhanced the cytotoxic effect, indicating that toxic ara-C was preferentially released in hypoxic cells via radiolytic one-electron reduction by hydrated electrons (e(aq)(-)).

5-Fluorouracil (5-FU) is used widely as an anticancer drug to treat solid cancers, such as colon, breast, rectal, and pancreatic cancers, although its clinical application is limited because 5-FU has gastrointestinal and hematological toxicity. Many groups are searching for prodrugs with functions that are tumor selective in their delivery and can be activated to improve the clinical utility of 5-FU as an important cancer chemotherapeutic agent. UV and ionizing radiation can cause chemical reactions in a localized area of the body, and these have been applied in the development of site-specific drug activation and sensitization. In this review, we describe recent progress in the development of novel 5-FU prodrugs that are activated site specifically by UV light and ionizing radiation in the tumor microenvironment. We also discuss the chemical mechanisms underlying this activation.

A novel coumarin-based highly water-soluble photocleavable protective group was designed and synthesized, and then this photosensitive protecting group was used to design paclitaxel prodrugs. These novel paclitaxel conjugates demonstrated excellent water solubility, over 100mgmL(-1). Thus, the use of a detergent in the formulation can be omitted completely, even at high doses. Phototaxel 11 released the parent drug, paclitaxel, quickly and efficiently by minimal tissue-damaging 365nm UV light irradiation at low power, while laser activation at 355nm led to extensive decomposition of the prodrug. The carbamate-type prodrug, phototaxel 11, was stable in the dark prior to activation, whereas carbonate-type phototaxel 9 demonstrated poor stability under aqueous conditions. For such prodrugs, tumor-tissue targeting after administration could be achieved by selective light delivery, similar to that used in photodynamic therapy. In addition, newly designed coumarin derivative 8 can be applied in organic chemistry as a photosensitive protective group and for the design of caged compounds.

Using a photosensitization-singlet oxygenation-dioxetane cleavage strategy, a photodynamic prodrug system has been developed, whereby drugs bearing carbonyl groups can first be attached to a photosensitizer to give a photosensitizer-drug complex and then released from the complex upon visible light irradiation. Visible light, which has good penetration through tissue, generates singlet oxygen via the photosensitizer, which then releases the prodrug when and where required. With this system, drug mimics and methyl esters of NSAIDs have been successfully incorporated with photosensitizers related to verteporfin and then released by visible light illumination in high to quantitative yields within minutes.

Purpose: The purpose of this study is to demonstrate the rational design and behaviour of the first dual-mode optical and chemical prodrug, exemplified by an acetyl salicylic acid-based system., Methods: A cyclic 1,4-benzodioxinone prodrug was synthesised by reaction of 3,5-dimethoxybenzoin and acetyl salicoyl chloride with pyridine. After purification by column chromatography and recrystallization, characterization was achieved using infrared and NMR spectroscopies, mass spectrometry, elemental analysis and single crystal X-ray diffraction. Light-triggered drug liberation was characterised via UV-visible spectroscopy following low-power 365 nm irradiation for controlled times. Chemical drug liberation was characterised via UV-visible spectroscopy in pH 5.5 solution., Results: The synthetic method yielded pure prodrug, with full supporting characterisation. Light-triggered drug liberation proceeded at a rate of 8.30x10(-2) s-1, while chemical, hydrolytic liberation proceeded independently at 1.89x10(-3) s-1. The photochemical and hydrolytic reactions were both quantitative., Conclusions: This study demonstrates the first rational dual-mode optical and chemical prodrug, using acetyl salicylic acid as a model, acting as a paradigm for future dual-mode systems. Photochemical drug liberation proceeds 44 times faster than chemical liberation, suggesting potential use in drug-eluting medical devices where an additional burst of drug is required at the onset of infection.

The cytotoxicity and photocytotoxicity of trans-[RuCl2(DMSO)4] and cis-[RuCl2(DMSO)4] complexes was tested in two melanoma cell lines, human (SK-MEL 188) and mouse (S91). The trans isomer was found to be more effective for cell growth inhibition than its cis analogue both in the presence and in the absence of illumination. However, the antiproliferative activity of both isomers was significantly enhanced after irradiation with UVA light in comparison with their activity observed in the dark. The influence of light on the reaction of both ruthenium(II) isomers with the single-stranded hexanucleotide d(T2GGT2), chosen as a model system for DNA, was also studied using chromatography and mass spectrometry techniques. The photochemical reaction of the ruthenium(II) complexes with the oligonucleotide d(T2GGT2) resulted in the formation of Ru(G-N7)2 adducts, which was not observed in the same time scale in thermal reactions. The initial short irradiation of the inert cis isomer was found to facilitate the covalent adduct formation with d(T2GGT2) in the secondary thermal reactions and with a rate comparable to that found for the trans isomer, which is ca. 5-10 times more reactive in the dark.

A new spectrofluorimetric method for the enalapril maleate monitoring was studied. Enalapril maleate was found to be highly photolabile. This drug was evaluated according to photodegradation assay at pH 2.5 and 6. Enalapril maleate was exposed to UVA-UVB radiations. Under these specific conditions was found as degradation product, the diketopiperazine. The modification of the fluorescent properties of enalapril maleate in solution after exposure UV-radiation and the degradation mechanisms were studied. The photodegradation was followed by the developed spectrofluorimetric assay.

We have developed two photolabile insulin prodrugs, insulin-2P and insulin-3P. These prodrugs were synthesized by protecting GlyA1 (N(alphaA1)), and one or both of the PheB1 (N(alphaB1)) and LysB29 (N(epsilonB29)) amino groups in insulin using 5'-(alpha-methyl-nitro-piperonyl)oxy-carbonyl as the protecting group. These insulin prodrugs were efficiently activated by exposure to longwave UV light to produce insulin quantitatively. Using 2-deoxyglucose uptake assays, both di- and tri-protected compounds were less active than native insulin in the protected state, and showed comparable activity to native insulin upon photoactivation.

Tumour hypoxia is a negative factor in cancer radiotherapy. In order to overcome the problem, various pharmacotherapies have been investigated as an adjunct to radiotherapy. The use of hypoxic cell sensitisers is a classical strategy, and many new compounds have been developed and investigated. Development of more efficient compounds than those currently available seems difficult and clinical studies to prove the efficacy of the existing compounds are encouraged, especially in combination with radiosurgery, intraoperative radiotherapy, and interstitial irradiation, in which a single high dose of radiation is used. Following the advent of hypoxic cell sensitisers, hypoxic cytotoxins have become available. Among them, tirapazamine has already gained success when combined with cisplatin in non-small cell lung cancer. The beneficial effect of tirapazamine when combined with radiation needs to be determined. As a third-generation compound in this field, antitumour prodrugs that are activated by irradiation under hypoxic conditions via one-electron reduction have been proposed. Prodrugs of 5-fluorouracil and 5-fluoro-2'-deoxyuridine have shown in vivo as well as in vitro activity. Although clinical evaluation of the compounds is not warranted due to a relatively low in vivo effect, this strategy appears promising if the prodrug design can be applied to more potent agents that shall be developed in future.

A potential approach for activating prodrugs in hypoxic regions of tumors is to use ionizing radiation, rather than bioreductive enzymes, to effect reduction. This study investigates radiolytic release of 8-hydroxyquinoline (8-HQ), as a model for hydroxyaza-chloromethylbenzindoline DNA minor groove alkylators, from Co(III) complexes under hypoxia. 8-HQ release, measured by HPLC, showed higher efficiency (one-electron stoichiometry) when the auxiliary ligand was a tetraazamacrocycle [e.g. 1,4,7,10-tetraazacyclododecane (cyclen)] rather than a triazamacrocycle [1,4,7-triazacyclononane (TACN)]. These complexes differ from the bioreductive cobalt complex SN 24771 in that their reduction provides stable cobalt-containing products rather than free (aquated) Co(2+). Radiolytic release of 8-HQ from Co(cyclen)(8-HQ) and Co(TACN)(CN)(8-HQ) was also demonstrated in deoxygenated human plasma, selectively in the absence of oxygen, again with higher efficiency for the cyclen system. The cobalt complexes were >1000-fold less potent than free 8-HQ as inhibitors of cell proliferation and were metabolically stable in aerobic and hypoxic cell cultures. Investigation of cell uptake of total cobalt, by inductively coupled plasma mass spectrometry, showed that these complexes enter cells but do not accumulate to the high concentrations seen with SN 24771. The results demonstrate the feasibility of masking the cytotoxicity of hydroxyquinoline-based cytotoxins as Co(III) complexes and demonstrate the utility of cyclen-based auxiliary ligands for optimizing radiolytic activation of these novel prodrugs under hypoxia.

Purpose: We previously developed a novel antitumor prodrug that has a 2-oxopropyl substituent at the N(1) position of 5-fluorouracil (5-FU) and releases 5-FU via one-electron reduction on hypoxic irradiation. Although the compound was effective in vivo, its activity against murine tumors was not high enough to warrant clinical studies. Therefore, we developed a similar family of radiation-activated prodrugs of 5-fluoro-2'-deoxyuridine (FdUrd), which is generally more potent than 5-FU, and investigated their radiation chemical reactivity and in vitro and in vivo effects., Methods and Materials: Compounds bearing various 2-oxoalkyl substituents at the N(3) position of FdUrd were synthesized and investigated. After aerobic or hypoxic irradiation to the prodrugs dissolved in water or culture medium, release of FdUrd was measured using high-performance liquid chromatography. To investigate in vitro cytotoxicity, SCCVII and EMT6 cells in culture were irradiated in the presence of the prodrug under aerobic or hypoxic conditions, and then kept with the compound for 24 h. Cell survival was then measured using a colony assay. To investigate in vivo effects, the drug was injected intraperitoneally at a dose of 100 or 300 mg/kg into Balb/c mice bearing EMT6 tumors 30 min before irradiation. The tumor growth delay-time was then assessed., Results: In vitro, the prodrugs released FdUrd at G-values (molar numbers of molecules produced by 1 J of radiation energy) of 1.6-2.0 x 10(-7) mol/J after hypoxic irradiation. The G-values for FdUrd release with hypoxic irradiation were about 100-fold greater than those with aerobic irradiation. Among the prodrugs tested, OFU106 bearing a 2-oxocyclopentyl substituent released the highest amount of FdUrd in the culture medium, and it was subjected to further in vitro and in vivo assays. Although OFU106 administered alone showed no cytotoxicity up to a concentration of 0.2 mM, it produced an enhanced cytotoxic effect when administered before hypoxic irradiation and kept with the cells for 24 h. The enhancement ratios calculated at the surviving fraction of 1% were 1.35-1.4 at 0.04 mM and 1.45-1.5 at 0.2 mM. In vivo, however, administration of OFU106 (100 or 300 mg/kg) before 20 Gy of irradiation did not produce marked growth delays compared with 20 Gy of radiation alone., Conclusion: On hypoxic irradiation in vitro, the prodrugs of FdUrd were activated as efficiently as were the prodrugs of 5-FU, but marked in vivo effects could not be detected. This strategy of prodrug design should be used in further development of radiation-activated prodrugs of more potent anticancer agents.

Previously a series of 2- and 3-substituted indolequinone phosphoramidate prodrugs was synthesized, and the compounds were shown to be nanomolar inhibitors of cell proliferation. The activation of these compounds following both one- and two-electron reduction has been investigated. (31)P NMR experiments demonstrated that both series of compounds undergo rapid activation following two-electron reduction. Additionally, the 3-series of compounds undergo rapid activation following one-electron reduction, while activation of the 2-series of compounds via this mechanism is very slow. The activation of these prodrugs by direct displacement using sulfur nucleophiles such as glutathione has been examined. Activation via this route is rapid for the 3-regioisomers, but is considerably slower for the 2-substituted analogues under similar conditions. Together these findings suggest that drug delivery via two-electron reduction from the 2-position is the more selective prodrug strategy.

The purpose of this study was to clarify whether 5-fluoro-2'-deoxyuridine (FdUrd) is superior to 5-fluorouracil (5-FU) as an effector in the radiation-activated prodrugs which we have been developing. The in vitro cytotoxicity of 5-FU and FdUrd was compared in two murine tumor and four human pancreatic cancer cell lines using a colony assay and in vivo efficacy was compared with SCCVII tumor using a growth delay time assay. FdUrd was slightly more hydrophilic than 5-FU. In vitro, FdUrd was more efficient than 5-FU in two lines, whereas 5-FU was more efficient in two lines and the two drugs were almost equal in efficacy in the remaining two. The concentration to reduce tumor cell survival to 50% after 24-h drug exposure was 5-32 microM for both 5-FU and FdUrd in murine lines, while it was 30-210 microM in human pancreatic cancer cell lines. The difference in relative efficacy of the two drugs among these cell lines could not be attributed to the rate of intracellular uptake of the compounds. FdUrd was less toxic than 5-FU in C3H/He mice, and FdUrd was less efficient than 5-FU in SCCVII tumors in vivo. These results suggest that FdUrd is not necessarily more potent than 5-FU, and development of the FdUrd prodrugs may not necessarily turn out to be fruitful.

L-Leucyl-L-leucine methyl esters (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. Glucose-carrying 2-nitrobenzyl (2-NB) and 2-nitrophenethyl (2-NPE) caged LeuLeuOMe, 1a and b, were synthesized and their photochemical and immunological properties were studied. Caged glycine methyl esters (GlyOMe), 2a,b, were also prepared to examine the cytotoxic activity of the photolytic byproducts from 1a,b. All the caged compounds were soluble in PBS containing 1% DMSO more than 400 microM, and efficiently released the substrates upon irradiation at 350 nm. While both 1a and 1b were not toxic to HL60 cells, 1b released LeuLeuOMe more quickly and induced apoptosis of HL60 cells far more efficiently than 1a. Although GlyOMe was not cytotoxic, 2a and b were slightly toxic before and after irradiation almost to the similar extent. Therefore, the photolytic products from the caging groups appear to be not toxic to the cells, and the apoptosis inducing activity of 1a and b may be for the most part due to LeuLeuOMe.

Systemic cytotoxic (anti-proliferative) anticancer drugs rely primarily for their therapeutic effect on cytokinetic differences between cancer and normal cells. One approach aimed at improving the selectivity of tumour cell killing by such compounds is the use of less toxic prodrug forms that can be selectively activated in tumour tissue (tumour-activated prodrugs; TAP). There are several mechanisms potentially exploitable for selective activation. Some utilise unique aspects of tumour physiology such as selective enzyme expression, hypoxia, and low extracellular pH. Others are based on tumour-specific delivery techniques, including activation of prodrugs by exogenous enzymes delivered to tumour cells via monoclonal antibodies (ADEPT), or generated in tumour cells from DNA constructs containing the corresponding gene (GDEPT). Because only a small proportion of the tumour cells may be competent to activate the prodrug, whichever activating mechanism is used, TAP need to be capable of killing activation-incompetent cells as well via a "bystander effect", in order to fully exploit these "activator" cells. A wide variety of chemistries have been explored for the selective activation of TAP. These include reduction of quinones, N-oxides, nitroaromatics and metal complexes by endogenous enzymes or radiation, amide cleavage by endogenous peptidases, and metabolism by a variety of exogenous enzymes, including phosphatases, kinases, amidases and glycosidases.

A series of 5-fluoro-1-(2'-oxocycloalkyl)uracils (3-11) that are potentially novel radiation-activated prodrugs for the radiotherapy of hypoxic tumor cells have been synthesized to evaluate a relationship between the molecular structure and the reactivity of one-electron reductive release of antitumor 5-fluorouracil (1) in anoxic aqueous solution. All the compounds 3-11 bearing the 2'-oxo group were one-electron reduced by hydrated electrons (eaq-) and thereby underwent C(1')-N(1) bond dissociation to release 5-fluorouracil 1 in 47-96% yields upon radiolysis of anoxic aqueous solution, while control compounds (12, 13) without the 2'-oxo substituent had no reactivity toward such a reductive C(1')-N(1) bond dissociation. The decomposition of 2-oxo compounds in the radiolytic one-electron reduction was more enhanced, as the one-electron reduction potential measured by cyclic voltammetry in N,N-dimethylformamide became more positive. The efficiency of 5-fluorouracil release was strongly dependent on the structural flexibility of 2-oxo compounds. X-ray crystallographic studies of representative compounds revealed that the C(1')-N(1) bond possesses normal geometry and bond length in the ground state. MO calculations by the AM1 method demonstrated that the LUMO is primarily localized at the pi* orbital of C(5)-C(6) double bond of the 5-fluorouracil moiety, and that the LUMO + 1 is delocalized between the pi* orbital of 2'-oxo substituent and the sigma* orbital of adjacent C(1')-N(1) bond. The one-electron reductive release of 5-fluorouracil 1 in anoxic aqueous solution was presumed to occur from the LUMO + 1 of radical anion intermediates possessing a partial mixing of the antibonding C(2')=O pi* and C(1')-N(1) sigma* MO's, that may be facilitated by a dynamic conformational change to achieve higher degree of (pi* + sigma*) MO mixing.

A chiral HPLC method to quantify in vivo enantiomeric inversion of prodrug CI-1010 (IR) or its drug IIR (PD 146923), a radiosensitizer, upon X-irradiation of dosed rats was developed. These polar enantiomers were separated only by using normal-phase chiral HPLC. A Chiralpak AS column provided the best separation. Isolation of analytes from plasma employed solid-phase extraction (SPE), and required conditions that were compatible with normal-phase HPLC. Options for SPE were restricted by the chemically reactive nature of both prodrug and drug, which produced analyte losses as high as 100%. Acceptable recoveries using SPE required evaluation of conditions for analyte chemical stability. The validated method gave a lower-limit of quantitation (LLOQ) of 200 ng/ml for each enantiomer extracted from 0.15 ml of plasma. The LLOQ of the inverted enantiomer could be detected in the presence of 10,000 ng/ml of the dosed enantiomer. Precision (RSD) ranged from 14.2 to 4.4%, and from 24.2 to 5.1% for IIS and IIR, respectively. Accuracy (RE) was +/- 13.1 and +/- 13.2%, respectively. Recoveries ranged from 44.3 to 71.4%, and from 40.7 to 67.9%, for IIS and IIR, respectively.

Bioreductive drugs are designed to be activated by enzymatic reduction in hypoxic regions of tumours, but activation of these drugs is not always fully suppressed by oxygen in normal tissues. A further limitation is that bioreductive drug activation depends on suitable reductases being expressed in the hypoxic zone. This essay proposes an alternative approach in which prodrugs are reduced, and thereby activated, in hypoxic regions by ionizing radiation rather than by enzymes. This strategy is theoretically attractive, but design requirements for such radiation-activated cytotoxins are challenging. In particular, the reducing capacity of radiation at clinically relevant doses is small, which necessitates the development of prodrugs capable of releasing very potent cytotoxins efficiently in hypoxic tissue. It is shown that nitroarylmethyl quaternary (NMQ) salts possess many of the features required of a radiation-activated prodrug. In some heterocyclic NMQ compounds the cytotoxicity of the latent cytotoxic amine effector is suppressed by > 100-fold in the prodrug form, and the effector is released rapidly by fragmentation following reduction by a single electron. Appreciable cytotoxic activation of NMQ prodrugs can be achieved by irradiation at clinically relevant doses in anoxic plasma. Some of the further drug design challenges required to develop a clinical agent based on this approach are outlined.

Nitroarylmethyl quaternary ammonium nitrogen mustards are bioreductive drugs designed to release mechlorethamine, when reduced metabolically, via fragmentation of the initial nitro radical anion to a benzylic-type radical. This proposed mechanism (termed Type I) is analogous to the well-known reductive fragmentation of 2-nitrobenzyl halides. The lead nitroarylmethyl quaternary mustard SN 25246 (NSC 656581), which contains a 2-nitrobenzyl electron acceptor, was shown previously to release mechlorethamine in hypoxic cell cultures and to be a highly selective hypoxic cytotoxin. In the present work the mechanism of reductive release of mechlorethamine from nitroarylmethyl quaternary prodrugs was investigated by steady-state radiolysis with product analysis by high-performance liquid chromatography. SN 25246 releases mechlorethamine in high yield upon reduction, but several reducing equivalents are required (Type II mechanisms). Investigation of other nitroarylmethyl units identified two heterocyclic analogues, the 1-methyl-4-nitro-5-imidazolyl derivative SN 25341 and the 1-methyl-5-nitro-2-pyrrolyl derivative SN 26581, which have a reduction stoichiometry of about one reducing equivalent and which release mechlorethamine efficiently. The other products from reduction of SN 25341 are also consistent with Type I fragmentation, via intramolecular electron transfer, to give the 1-methyl-4-nitroimidazole-5-CH2. radical. The sensitivity of the 4-nitroimidazole and 5-nitropyrrole nitroarylmethyl quaternary mustards to Type I reductive fragmentation suggests that these electron acceptor units may be well suited to development of prodrugs which release tertiary amine effectors after metabolic or radiolytic reduction in hypoxic regions of tumors.