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[M(arene)(HQ)Cl] complexes (M = Ru II /Os II /Rh III /Ir III ; HQ = 8-hydroxyquinoline) have shown promise as anticancer agents. To assess the effect of conjugating biotin (vitamin B7) to such compounds and improve their tumor-targeting ability through interaction with the sodium-dependent multivitamin transporter (SMVT), the chlorido co-ligand was exchanged with biotinylated 6-aminoindazole. The complexes were characterized by NMR spectroscopy and mass spectrometry, and purity was determined by elemental analysis. The compounds were shown to be stable in aqueous solution but reacted in particular with biologically relevant nitrogen-donor ligands. The biotinylated organometallics were shown to be able to interact with the high-affinity biotin-binding protein streptavidin using molecular modelling. High antiproliferative activity of the biotinylated Rh complex (IC 50 = 1.1-10 μM) and its chlorido precursor (IC 50 = 2.1-7.0 μM) was demonstrated in human HCT116, NCI-H460, COLO 205, SW620, A2780 and A2780cis cancer cells, which feature differing levels of SMVT expression. While there was no clear relationship between the anticancer activity in cells and SMVT expression, the complexes showed similar activity in cisplatin-sensitive and -resistant cells. The most potent was the biotinylated Rh derivative which displayed low toxicity toward zebrafish embryos with >75% survival up to day 4 and after treatment with up to 32 μM complex.

Zwitterionic polymers have been found to be biocompatible alternatives to poly(ethylene glycol) (PEG) for conjugation to proteins. This work reports the site-selective conjugation of poly(caprolactone-carboxybetaine) (pCLZ) to human growth hormone receptor antagonist (GHA) B2036-alkyne and investigation of safety, activity, and pharmacokinetics. Azide-end-functionalized pCLZs were synthesized and conjugated to GHA B2036-alkyne via copper-catalyzed click reaction. The resulting inhibitory bioactivity concentration responses in Ba/F3-GHR cells were compared to those of PEGylated GHA B2036. IgG and IgM antibody production was tested in mice, and no measurable antibody or cytokine production was detected for the pCLZ conjugate. Using 18 F-labeled PET/CT imaging, the pCLZ conjugate showed an increase in circulation time compared to that of GHA B2036. Acute toxicity of the polymer was investigated in vivo and found to be nontoxic. Ex vivo degradation of the polymer on the conjugate was investigated. The results suggest that pCLZ-GHA is a potentially safe alternative to PEG-GHA.

The clinical use of many potent anticancer agents is limited by their non-selective toxicity to healthy tissue. One of these examples is vorinostat (SAHA), a pan histone deacetylase inhibitor, which shows high cytotoxicity with limited discrimination for cancerous over healthy cells. In an attempt to improve tumor selectivity, we exploited the properties of cobalt(III) as a redox-active metal center through stabilization with cyclen and cyclam tetraazamacrocycles, masking the anticancer activity of SAHA and other hydroxamic acid derivatives to allow for the complex to reach the hypoxic microenvironment of the tumor. Biological assays demonstrated the desired low in vitro anticancer activity of the complexes, suggesting effective masking of the activity of SAHA. Once in the tumor, the bioactive moiety may be released through the reduction of the Co III center. Investigations revealed long-term stability of the complexes, with cyclic voltammetry and chemical reduction experiments supporting the design hypothesis of SAHA release through the reduction of the Co III prodrug. The results highlight the potential for further developing this complex class as novel anticancer agents by masking the high cytotoxicity of a given drug, however, the cellular uptake needs to be improved., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Tumour hypoxia promotes poor patient outcomes, with particularly strong evidence for head and neck squamous cell carcinoma (HNSCC). To effectively target hypoxia, therapies require selection biomarkers and preclinical models that can accurately model tumour hypoxia. We established 20 patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of HNSCC that we characterised for their fidelity to represent clinical HNSCC in gene expression, hypoxia status and proliferation and that were evaluated for their sensitivity to hypoxia-activated prodrugs (HAPs). PDX models showed greater fidelity in gene expression to clinical HNSCC than cell lines, as did CDX models relative to their paired cell lines. PDX models were significantly more hypoxic than CDX models, as assessed by hypoxia gene signatures and pimonidazole immunohistochemistry, and showed similar hypoxia gene expression to clinical HNSCC tumours. Hypoxia or proliferation status alone could not determine HAP sensitivity across our 20 HNSCC and two non-HNSCC tumour models by either tumour growth inhibition or killing of hypoxia cells in an ex vivo clonogenic assay. In summary, our tumour models provide clinically relevant HNSCC models that are suitable for evaluating hypoxia-targeting therapies; however, additional biomarkers to hypoxia are required to accurately predict drug sensitivity., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The dominant role of non-homologous end-joining in the repair of radiation-induced double-strand breaks identifies DNA-dependent protein kinase (DNA-PK) as an excellent target for the development of radiosensitizers. We report the discovery of a new class of imidazo[4,5- c ]pyridine-2-one DNA-PK inhibitors. Structure-activity studies culminated in the identification of 78 as a nM DNA-PK inhibitor with excellent selectivity for DNA-PK compared to related phosphoinositide 3-kinase (PI3K) and PI3K-like kinase (PIKK) families and the broader kinome, and displayed DNA-PK-dependent radiosensitization of HAP1 cells. Compound 78 demonstrated robust radiosensitization of a broad range of cancer cells in vitro , displayed high oral bioavailability, and sensitized colorectal carcinoma (HCT116/54C) and head and neck squamous cell carcinoma (UT-SCC-74B) tumor xenografts to radiation. Compound 78 also provided substantial tumor growth inhibition of HCT116/54C tumor xenografts in combination with radiation. Compound 78 represents a new, potent, and selective class of DNA-PK inhibitors with significant potential as radiosensitizers for cancer treatment.

Perforin is a pore-forming protein whose normal function enables cytotoxic T and natural killer (NK) cells to kill virus-infected and transformed cells. Conversely, unwanted perforin activity can also result in auto-immune attack, graft rejection and aberrant responses to pathogens. Perforin is critical for the function of the granule exocytosis cell death pathway and is therefore a target for drug development. In this study, by screening a fragment library using NMR and surface plasmon resonance, we identified 4,4-diaminodiphenyl sulfone (dapsone) as a perforin ligand. We also found that dapsone has modest (mM) inhibitory activity of perforin lytic activity in a red blood cell lysis assay in vitro. Sequential modification of this lead fragment, guided by structural knowledge of the ligand binding site and binding pose, and supported by SPR and ligand-detected 19 F NMR, enabled the design of nanomolar inhibitors of the cytolytic activity of intact NK cells against various tumour cell targets. Interestingly, the ligands we developed were largely inert with respect to direct perforin-mediated red blood cell lysis but were very potent in the context of perforin's action on delivering granzymes in the immune synapse, the context in which it functions physiologically. Our work indicates that a fragment-based, structure-guided drug discovery strategy can be used to identify novel ligands that bind perforin. Moreover, these molecules have superior physicochemical properties and solubility compared to previous generations of perforin ligands., 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 © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The cellular accumulation and the underlying mechanisms for the two ruthenium-based anticancer complexes [Ru II (cym)(HQ)Cl] 1 (cym = η 6 -p-cymene, HQ = 8-hydroxyquinoline) and [Ru II (cym)(PCA)Cl]Cl 2 (PCA = N-fluorophenyl-2-pyridinecarbothioamide) were investigated in HCT116 human colorectal carcinoma cells. The results showed that the cellular accumulation of both complexes increased over time and with higher concentrations, and that 2 accumulates in greater quantities in cells than 1. Inhibition studies of selected cellular accumulation mechanisms indicated that both 1 and 2 may be transported into the cells by both passive diffusion and active transporters, similar to cisplatin. Efflux experiments indicated that 1 and 2 are subjected to efflux through a mechanism that does not involve p-glycoprotein, as addition of verapamil did not make any difference. Exploring the influence of the Cu transporter by addition of CuCl 2 resulted in a higher accumulation of 1 and 2 whilst the amount of Pt detected was slightly reduced when cells were treated with cisplatin. Complexes 1 and 2 were further explored in zebrafish where accumulation and distribution were determined with ICP-MS and LA-ICP-MS. The results correlated with the in vitro observations and zebrafish treated with 2 showed higher Ru contents than those treated with 1. The distribution studies suggested that both complexes mainly accumulated in the intestines of the zebrafish., (© 2023. The Author(s), under exclusive licence to Society for Biological Inorganic Chemistry (SBIC).)

Transplantable in vivo CRISPR/Cas9 knockout screens, in which cells are edited in vitro and inoculated into mice to form tumours, allow evaluation of gene function in a cancer model that incorporates the multicellular interactions of the tumour microenvironment. To improve our understanding of the key parameters for success with this method, we investigated the choice of cell line, mouse host, tumour harvesting timepoint and guide RNA (gRNA) library size. We found that high gRNA (80-95%) representation was maintained in a HCT116 subline transduced with the GeCKOv2 whole-genome gRNA library and transplanted into NSG mice when tumours were harvested at early (14 d) but not late time points (38-43 d). The decreased representation in older tumours was accompanied by large increases in variance in gRNA read counts, with notable expansion of a small number of random clones in each sample. The variable clonal dynamics resulted in a high level of 'noise' that limited the detection of gRNA-based selection. Using simulated datasets derived from our experimental data, we show that considerable reductions in count variance would be achieved with smaller library sizes. Based on our findings, we suggest a pathway to rationally design adequately powered in vivo CRISPR screens for successful evaluation of gene function., (© 2023. The Author(s).)

Intracellular accumulation studies are a key step in metallodrug development but often variable results are obtained. Therefore, we aimed here to investigate different protocols for efficient and reproducible lysis of cancer cells in terms of protein content in lysates and in cell uptake studies of the Ru anticancer complex [chlorido(8-oxyquinolinato)(η6-p-cymene)ruthenium(II)] ([Ru(cym)(HQ)Cl]). The physical lysis methods osmosis and sonication were chosen for comparison with chemical lysis with the radioimmunoprecipitation assay (RIPA) buffer. Based on the protein content and the total Ru accumulated in the lysates, the latter determined using inductively coupled plasma-mass spectrometry, RIPA buffer was the most efficient lysis method. Measurements of plastic adsorption blanks revealed that the higher Ru content determined in the RIPA buffer lysis samples may be due a higher amount of Ru extracted from the plastic incubation plates compared with osmosis and sonication. Overall, we found that the choice of lysis method needs to be matched to the information sought and we suggest the least disruptive osmosis method might be the best choice for labile drug-biomolecule adducts. Minimal differences were found for experiments aimed at measuring the overall cell uptake of the Ru complex., (© The Author(s) 2023. Published by Oxford University Press.)

Ruthenium piano-stool complexes have been explored for their anticancer activity and some promising compounds have been reported. Herein, we conjugated a derivative of plecstatin-1 to peptides in order to increase their cancer cell targeting ability. For this purpose, plecstatin-1 was modified at the arene ligand to introduce a functional amine handle ( 3 ), which resulted in a compound that showed similar activity in an in vitro anticancer activity assay. The cell-penetrating peptide TAT 48-60 , tumor-targeting neurotensin 8-13 , and plectin-targeting peptide were functionalized with succinyl or β-Ala-succinyl linkers under standard solid-phase peptide synthesis (SPPS) conditions to spatially separate the peptide backbones from the bioactive metal complexes. These modifications allowed for conjugating precursor 3 to the peptides on resin yielding the desired metal-peptide conjugates (MPCs), as confirmed by high-performance liquid chromatography (HPLC), NMR spectroscopy, and mass spectrometry (MS). The MPCs were studied for their behavior in aqueous solution and under acidic conditions and resembled that of the parent compound plecstatin-1. In in vitro anticancer activity studies in a small panel of cancer cell lines, the TAT-based MPCs showed the highest activity, while the other MPCs were virtually inactive. However, the MPCs were significantly less active than the small molecules plecstatin-1 and 3 , which can be explained by the reduced cell uptake as determined by inductively coupled plasma MS (ICP-MS). Although the MPCs did not display potent anticancer activities, the developed conjugation strategy can be extended toward other metal complexes, which may be able to utilize the targeting properties of peptides.

Decades of published research support a role for growth hormone (GH) in cancer. Accordingly, there is increasing interest in targeting GH in oncology, with GH antagonists exhibiting efficacy in xenograft studies as single agents and in combination with anticancer therapy or radiation. Here we discuss challenges associated with using growth hormone receptor (GHR) antagonists in preclinical models and considerations for translation, such as the identification of predictive biomarkers for selecting patients and for monitoring drug efficacy. Ongoing research will determine whether suppressing GH signalling pharmacologically will also reduce the risk of developing cancer. An increase in GH-targeted drugs in preclinical development will ultimately provide new tools to test anticancer efficacy of blocking the GH signalling pathway.

The role of hypoxic tumour cells in resistance to radiotherapy, and in suppression of immune response, continues to endorse tumour hypoxia as a bona fide, yet largely untapped, drug target. Radiotherapy innovations such as stereotactic body radiotherapy herald new opportunities for classical oxygen-mimetic radiosensitisers. Only nimorazole is used clinically as a radiosensitiser, and there is a dearth of new radiosensitisers in development. In this report, we augment previous work to present new nitroimidazole alkylsulfonamides and we document their cytotoxicity and ability to radiosensitise anoxic tumour cells in vitro. We compare radiosensitisation with etanidazole and earlier nitroimidazole sulfonamide analogues and we identify 2-nitroimidazole and 5-nitroimidazole analogues with marked tumour radiosensitisation in ex vivo assays of surviving clonogens and with in vivo tumour growth inhibition.

Half-sandwich M II (cym)Cl (cym = η 6 -p-cymene; M = Ru, Os) complexes of pyridinecarbothioamide (PCA) ligands have demonstrated potential as orally active anticancer agents. In order to investigate the impact of the substitution of the labile chlorido ligand with phosphorous donor ligands on the antiproliferative properties, the triphenylphosphine (PPh 3 ) and 1,3,5-triaza-7-phophaadamantane (pta) analogues were prepared and characterized by spectroscopic techniques and the molecular structures of several complexes were determined by X-diffraction analysis. Interestingly, the molecular structures contained the PCA ligand deprotonated, presumably driven by the reduction in overall charge of the complex. Density Functional Theory (DFT) calculations suggested minor energy differences between the protonated and deprotonated forms. The aqueous stability and the reactivity with the amino acids l-histidine and l-cysteine were investigated by 1 H NMR spectroscopy of representative examples. The most potent anticancer agents featured Ru or Os centers and a PPh 3 ligand and showed IC 50 values in the submicromolar range against four cancer cell lines. This suggests that the antiproliferative activity was mainly dependent on the lipophilic properties of the phosphine ligand with PPh 3 having a significantly higher clog P value than pta., 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 © 2023 Elsevier Inc. All rights reserved.)

Pt(terpyridine) complexes are well-known DNA intercalators. The introduction of an NHC co-ligand rendered such a complex highly antiproliferative in cancer cells compared to its chlorido derivative. Despite the high potency, zebrafish embryos tolerated the compound well, especially compared to cisplatin. DNA interaction studies support a mode of action related to intercalation.

A new [PdPtL 4 ] 4+ heterobimetallic cage containing hydrazone linkages has been synthesised using the sub-component self-assembly approach. 1 H and DOSY nuclear magnetic resonance (NMR) spectroscopy and electrospray ionisation mass spectrometry (ESIMS) data were consistent with the formation of the [PdPtL 4 ] 4+ architecture. The cage was stimulus-responsive and could be partially disassembled and reassembled by the addition of dimethylaminopyridine (DMAP) and p -tolenesulfonic acid (TsOH), respectively. Additionally, the stability of the hydrazone cage against hydrolysis in the presence of water and nucleophilic decomposition in the presence of guest molecules was compared to a previously synthesised imine-containing [PdPtL 4 ] 4+ cage. It was established that the hydrazone linkage was more resistant to hydrolysis. Furthermore, the host-guest (HG) chemistry with a series of drug and drug-like molecules was examined. The hydrazone cage was shown to interact with cisplatin while the smaller imine cage was shown to interact with 5-fluorouracil and oxaliplatin in CD 3 CN. No HG interactions were observed in the more polar d 6 -DMSO. In vitro antiproliferative activity studies demonstrated both cages were active against the cancer cell lines tested and displayed half-maximal inhibitory (IC 50 ) values in the range of 25-35 μM. Most [PdPtL 4 ] 4+ -drug mixtures tested had higher IC 50 values than the hosts. However, the [PdPtL 4 ] 4+ cages, and [PdPtL 4 ] 4+ :drug mixtures were less cytotoxic than the well established anticancer drugs cisplatin, oxaliplatin and 5-fluorouracil.

The substitution of phenyl rings in established drugs with ferrocenyl moieties has been reported to yield compounds with improved biological activity and alternative modes of action, often involving the formation of reactive oxygen species (ROS). Translating this concept to N-heterocyclic carbene (NHC) complexes, we report here organometallics with a piano-stool structure that feature di- or tridentate ligand systems. The ligands impacted the cytotoxic activity of the NHC complexes, but the coordination modes seemed to have a limited influence, which may be related to the propensity of forming the same species in solution. In general, the stability of the complexes in an aqueous environment and their reactivity to selected biomolecules were largely dominated by the nature of the metal center. While the complexes promoted the formation of ROS, the levels did not correlate with their cytotoxic activity. However, the introduction of ferrocenyl moieties had a significant impact on the antiproliferative potency of the complexes and, in particular, some of the ferrocenyl-functionalized compounds yielded IC 50 values in the low μM range.

While delivery of chemotherapeutics to cancer cells by nanomedicines can improve therapeutic outcomes, many fail due to the low drug loading (DL), poor cellular uptake and endosomal entrapment. This study investigated the potential to overcome these limitations using pH-sensitive liposomes (PSL) empowered by the use of calcium acetate. An acidic dinitrobenzamide mustard prodrug SN25860 was used as a model drug, with non pH-sensitive liposomes (NPSL) as a reference. Calcium acetate as a remote loading agent allowed to engineer PSL- and NPSL-SN25860 with DL of > 31.1% (w/w). The IC 50 of PSL-SN25860 was 21- and 141-fold lower than NPSL and free drug, respectively. At 48 h following injection of PSL-SN25860, NPSL-SN25860 and the free drug, drug concentrations in EMT6-nfsB murine breast tumors were 56.3 µg/g, 6.76 µg/g and undetectable (< 0.015 µg/g), respectively (n = 3). Meanwhile, the ex vivo tumor clonogenic assay showed 9.1%, 19.4% and 42.7% cell survival in the respective tumors. Live-cell imaging and co-localization analysis suggested endosomal escape was accomplished by destabilization of PSL followed by release of Ca 2+ in endosomes allowing induction of a proton sponge effect. Subsequent endosomal rupture was observed approximately 30 min following endocytosis of PSL containing Ca 2+ . Additionally, calcium in liposomes promoted internalization of both PSL and NPSL. Taken together, this study demonstrated multifaceted functions of calcium acetate in promoting drug loading into liposomes, cellular uptake, and endosomal escape of PSL for efficient cytoplasmic drug delivery. The results shed light on designing nano-platforms for cytoplasmic delivery of various therapeutics., (© 2022. The Author(s).)

Perforin is a key effector of lymphocyte-mediated cell death pathways and contributes to transplant rejection of immunologically mismatched grafts. We have developed a novel series of benzenesulfonamide (BZS) inhibitors of perforin that can mitigate graft rejection during allogeneic bone marrow/stem cell transplantation. Eight such perforin inhibitors were tested for their murine pharmacokinetics, plasma protein binding, and their ability to block perforin-mediated lysis in vitro and to block the rejection of major histocompatibility complex (MHC)-mismatched mouse bone marrow cells. All compounds showed >99% binding to plasma proteins and demonstrated perforin inhibitory activity in vitro and in vivo . A lead compound, compound 1 , that showed significant increases in allogeneic bone marrow preservation was evaluated for its plasma pharmacokinetics and in vivo efficacy at multiple dosing regimens to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship. The strongest PK/PD correlation was observed between perforin inhibition in vivo and time that total plasma concentrations remained above 900 μM, which correlates to unbound concentrations similar to 3× the unbound in vitro IC 90 of compound 1 . This PK/PD relationship will inform future dosing strategies of BZS perforin inhibitors to maintain concentrations above 3× the unbound IC 90 for as long as possible to maximize efficacy and enhance progression toward clinical evaluation., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

The number of donor atoms available on peptides that can competitively coordinate to metal centers renders the site-selective generation of advanced metal-peptide conjugates in high purity a challenging venture. Herein, we present a transmetalation-based synthetic approach on solid support in which an imidazolium pro-ligand can be used to selectively anchor a range of transition metal half-sandwich complexes onto peptides in the presence of multiple coordinative motifs. Amenable to solid support, a range of N-terminus and/or lysine conjugated metal-peptide conjugates were obtained in high purity after cleavage from the resin. The metalated peptides were evaluated for their anticancer properties against human cancer cell lines. While no cytotoxic activity was observed, this platform has the potential to i) provide a pathway to site-selective peptide labelling, ii) be explored as a biorthogonal handle and/or iii) generate a new strategy for ligand design in transition metal catalysts., (© 2021 Wiley-VCH GmbH.)

Redox-active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore leads to low ROS generation. In this work, we demonstrate that an exchange of the α-amino acid Gly with the β-amino acid β-Ala at position 2 (Gly2→β-Ala2) of the ATCUN motif reinstates ROS production ( • OH and H 2 O 2 ). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these β-Ala2-containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for β-Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where β-Ala2 peptide complexes had lower IC 50 values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP-MS measurements., (© 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Multimetallic complexes have been shown in several examples to possess greater anticancer activity than their monometallic counterparts. The increased activity has been attributed to altered modes of action. We herein report the synthesis of a series of heterodimetallic compounds based on a ditopic ligand featuring 2-pyridylimine chelating motifs and organometallic half-sandwich moieties. The complexes were characterized by a combination of 1 H NMR spectroscopy, electrospray ionization mass spectrometry, elemental analysis and single crystal X-ray diffraction. Investigations into the stability of representative complexes in DMSO- d 6 and 10% DMSO- d 6 /D 2 O revealed the occurrence of solvent-chlorido ligand exchange. Proliferation assays in four human cancer cell lines showed that the Os-Rh complex possessed minimal activity, while all other complexes were inactive., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Green, Steel, Riisom, Hanif, Söhnel, Jamieson, Wright, Crowley and Hartinger.)

We report investigations on the anticancer activity of organometallic [M II/III (η 6 -p-cymene/η 5 -pentamethylcyclopentadienyl)] (M=Ru, Os, Rh, and Ir) complexes of N-heterocyclic carbenes (NHCs) substituted with a triazolyl moiety. Depending on the precursors, the NHC ligands displayed either mono- or bidentate coordination via the NHC carbon atom or as N,C-donors. The metal complexes were investigated for their stability in aqueous solution, with the interpretation supported by density functional theory calculations, and reactivity to biomolecules. In vitro cytotoxicity studies suggested that the nature of both the metal center and the lipophilicity of the ligand determine the biological properties of this class of compounds. The Ir III complex 5 d bearing a benzimidazole-derived ligand was the most cytotoxic with an IC 50 value of 10 μM against NCI-H460 non-small cell lung carcinoma cells. Cell uptake and distribution studies using X-ray fluorescence microscopy revealed localization of 5 d in the cytoplasm of cancer cells., (© 2021 Wiley-VCH GmbH.)

N-Heterocyclic carbene (NHC) ligands are widely investigated in medicinal inorganic chemistry. Here, we report the preparation and characterization of a series of half-sandwich [M(L)(NHC)Cl 2 ] (M = Ru, Os, Rh, Ir; L = cym/Cp*) complexes with a N-flanking anthracenyl moiety attached to imidazole- and benzimidazole-derived NHC ligands. The anticancer activity of the complexes was investigated in cell culture studies where, in comparison to a Rh derivative with an all-carbon-donor-atom-based ligand ( 5a ), they were found to be cytotoxic with IC 50 values in the low micromolar range. The Ru derivative 1a was chosen as a representative for stability studies as well as for biomolecule interaction experiments. It underwent partial chlorido/aqua ligand exchange in DMSO- d 6 /D 2 O to rapidly form an equilibrium in aqueous media. The reactions of 1a with biomolecules proceeded quickly and resulted in the formation of adducts with amino acids, DNA, and protein. Hen egg white lysozyme crystals were soaked with 1a , and the crystallographic analysis revealed an interaction with an l-aspartic acid residue (Asp119), resulting in the cleavage of the p -cymene ligand but the retention of the NHC moiety. Cell morphology studies for the Rh analog 3a suggested that the cytotoxicity is exerted via mechanisms different from that of cisplatin.

Two new di(2,2'-bipyridine) ligands, 2,6-bis([2,2'-bipyridin]-5-ylethynyl)pyridine ( L1 ) and bis(4-([2,2'-bipyridin]-5-ylethynyl)phenyl)methane ( L2 ) were synthesized and used to generate two metallosupramolecular [Fe 2 ( L ) 3 ](BF 4 ) 4 cylinders. The ligands and cylinders were characterized using elemental analysis, electrospray ionization mass spectrometry, UV-vis, 1 H-, 13 C and DOSY nuclear magnetic resonance (NMR) spectroscopies. The molecular structures of the [Fe 2 ( L ) 3 ](BF 4 ) 4 cylinders were confirmed using X-ray crystallography. Both the [Fe 2 ( L1 ) 3 ](BF 4 ) 4 and [Fe 2 ( L2 ) 3 ](BF 4 ) 4 complexes crystallized as racemic ( rac ) mixtures of the ΔΔ (P) and ΛΛ (M) helicates. However, 1 H NMR spectra showed that in solution the larger [Fe 2 ( L2 ) 3 ](BF 4 ) 4 was a mixture of the rac -ΔΔ/ΛΛ and meso -ΔΛ isomers. The host-guest chemistry of the helicates, which both feature a central cavity, was examined with several small drug molecules. However, none of the potential guests were found to bind within the helicates. In vitro cytotoxicity assays demonstrated that both helicates were active against four cancer cell lines. The smaller [Fe 2 ( L1 ) 3 ](BF 4 ) 4 system displayed low μM activity against the HCT116 (IC 50 = 7.1 ± 0.5 μM) and NCI-H460 (IC 50 = 4.9 ± 0.4 μM) cancer cells. While the antiproliferative effects against all the cell lines examined were less than the well-known anticancer drug cisplatin, their modes of action would be expected to be very different., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lisboa, Riisom, Vasdev, Jamieson, Wright, Hartinger and Crowley.)

The development of bifunctional platinum complexes with the ability to interact with DNA via different binding modes is of interest in anticancer metallodrug research. Therefore, we report platinum(II) terpyridine complexes to target DNA by coordination and/or through a tethered alkylating moiety. The platinum complexes were evaluated for their in vitro antiproliferative properties against the human cancer cell lines HCT116 (colorectal), SW480 (colon), NCI-H460 (non-small cell lung), and SiHa (cervix) and generally exhibited potent antiproliferative activity although lower than their respective terpyridine ligands. 1 H NMR spectroscopy and/or ESI-MS studies on the aqueous stability and reactivity with various small biomolecules, acting as protein and DNA model compounds, were used to establish potential modes of action for these complexes. These investigations indicated rapid binding of complex PtL3 to the biomolecules through coordination to the Pt center, while PtL4 in addition alkylated 9-ethylguanine. PtL3 was investigated for its reactivity to the model protein hen egg white lysozyme (HEWL) by protein crystallography which allowed identification of the N δ1 atom of His15 as the binding site.

Growth hormone (GH) has been implicated in cancer progression andis a potential target for anticancer therapy. Currently, pegvisomant is the only GH receptor (GHR) antagonist approved for clinical use. Pegvisomant is a mutated GH molecule (B2036) which is PEGylated on amine groups to extend serum half-life. However, PEGylation significantly reduces the bioactivity of the antagonist in mice. To improve bioactivity, we generated a series of B2036 conjugates with the site-specific attachment of 20, 30, or 40 kDa methoxyPEG maleimide (mPEG maleimide) by introduction of a cysteine residue at amino acid 144 (S144C). Recombinant B2036-S144C was expressed in Escherichia coli , purified, and then PEGylated using cysteine-specific conjugation chemistry. To avoid issues with dimerization due to the introduced cysteine, B2036-S144C was PEGylated while immobilized on an Ni-nitrilotriacetic (Ni-NTA) acid column, which effectively reduced disulfide-mediated dimer formation and allowed efficient conjugation to mPEG maleimide. Following PEGylation, the IC 50 values for the 20, 30, and 40 kDa mPEG maleimide B2036-S144C conjugates were 66.2 ± 3.8, 106.1 ± 7.1, and 127.4 ± 3.6 nM, respectively. The circulating half-life of the 40 kDa mPEG conjugate was 58.3 h in mice. Subcutaneous administration of the 40 kDa mPEG conjugate (10 mg/kg/day) reduced serum insulin-like growth factor I (IGF-I) concentrations by 50.6%. This in vivo reduction in serum IGF-I was at a considerably lower dose compared to the higher doses required to observe comparable activity in studies with pegvisomant. In conclusion, we have generated a novel PEGylated GHR antagonist by the solid-phase site-specific attachment of mPEG maleimide at an introduced cysteine residue, which effectively reduces serum IGF-I in vivo .

Ru II (cym)Cl (cym = η 6 - p -cymene) complexes of pyridinecarbothioamides have shown potential for development as orally active anticancer metallodrugs, underlined by their high selectivity towards plectin as the molecular target. In order to investigate the impact of the metal center on the anticancer activity and their physicochemical properties, the Os(cym), Rh- and Ir(Cp*) (Cp* = pentamethylcyclopentadienyl) analogues of the most promising and orally active compound plecstatin 2 were prepared and characterized by spectroscopic techniques and X-ray diffraction analysis. Dissolution in aqueous medium results in quick ligand exchange reactions; however, over time no further changes in the 1 H NMR spectra were observed. The Rh- and Ir(Cp*) complexes were investigated for their reactions with amino acids, and while they reacted with Cys, no reaction with His was observed. Studies on the in vitro anticancer activity identified the Ru derivatives as the most potent, independent of their halido leaving group, while the Rh derivative was more active than the Ir analogue. This demonstrates that the metal center has a significant impact on the anticancer activity of the compound class.

Hydroxypyr(id)ones are a pharmaceutically important class of compounds that have shown potential in diverse areas of drug discovery. We investigated the 3-hydroxy-4-pyridones 1a - 1c and 3-hydroxy-4-thiopyridones 1d - 1f as well as their Ru(η 6 - p -cymene)Cl complexes 2a - 2f , and report here the molecular structures of 1b and 1d as determined by X-ray diffraction analysis. Detailed cell biological investigations revealed potent cytotoxic activity, in particular of the 3-hydroxy-4-thiopyridones 1d - 1f , while the Ru complexes of both compound types were less potent, despite still showing antiproliferative activity in the low μM range. The compounds did not modulate the cell cycle distribution of cancer cells but were cytostatic in A549 and cytotoxic in NCI-H522 non-small lung cancer cells, among other effects on cancer cells.

Patient survival from head and neck squamous cell carcinoma (HNSCC), the seventh most common cause of cancer, has not markedly improved in recent years despite the approval of targeted therapies and immunotherapy agents. Precision medicine approaches that seek to individualise therapy through the use of predictive biomarkers and stratification strategies offer opportunities to improve therapeutic success in HNSCC. To enable precision medicine of HNSCC, an understanding of the microenvironment that influences tumour growth and response to therapy is required alongside research tools that recapitulate the features of human tumours. In this review, we highlight the importance of the tumour microenvironment in HNSCC, with a focus on tumour hypoxia, and discuss the fidelity of patient-derived xenograft and organoids for modelling human HNSCC and response to therapy. We describe the benefits of patient-derived models over alternative preclinical models and their limitations in clinical relevance and how these impact their utility in precision medicine in HNSCC for the discovery of new therapeutic agents, as well as predictive biomarkers to identify patients' most likely to respond to therapy.

Ispinesib is a potent inhibitor of kinesin spindle protein (KSP), which has been identified as a promising target for antimitotic anticancer drugs. Herein, we report the synthesis of half-sandwich complexes of Ru, Os, Rh, and Ir bearing the ispinesib-derived N , N -bidentate ligands ( R )- and ( S )-2-(1-amino-2-methylpropyl)-3-benzyl-7-chloroquinazolin-4(3H)-one and studies on their chemical and biological properties. Using the enantiomerically pure ( R )- and ( S )-forms of the ligand, depending on the organometallic moiety, either the S M , R or R M , S diastereomers, respectively, were observed in the molecular structures of the Ru- and Os(cym) (cym = η 6 - p -cymene) compounds, whereas the R M , R or S M , S diastereomers were found for the Rh- and Ir(Cp*) (Cp* = η 5 -pentamethylcyclopentadienyl) derivatives. However, density functional theory (DFT) calculations suggest that the energy difference between the diastereomers is very small, and therefore a mixture of both will be present in solution. The organometallics exhibited varying antiproliferative activity in a series of human cancer cell lines, with the complexes featuring the ( R )-enantiomer of the ligand being more potent than the ( S )-configured counterparts. Notably, the Rh and Ir complexes demonstrated high KSP inhibitory activity, even at 1 nM concentration, which was independent of the chirality of the ligand, whereas the Ru and especially the Os derivatives were much less active.

Regulation of human growth hormone (GH) signaling has important applications in the remediation of several diseases including acromegaly and cancer. Growth hormone receptor (GHR) antagonists currently provide the most effective means for suppression of GH signaling. However, these small 22 kDa recombinantly engineered GH analogues exhibit short plasma circulation times. To improve clinical viability, between four and six molecules of 5 kDa poly(ethylene glycol) (PEG) are nonspecifically conjugated to the nine amines of the GHR antagonist designated as B2036 in the FDA-approved therapeutic pegvisomant. PEGylation increases the molecular weight of B2036 and considerably extends its circulation time, but also dramatically reduces its bioactivity, contributing to high dosing requirements and increased cost. As an alternative to nonspecific PEGylation, we report the use of genetic code expansion technology to site-specifically incorporate the unnatural amino acid propargyl tyrosine (pglY) into B2036 with the goal of producing site-specific protein-polymer conjugates. Substitution of tyrosine 35 with pglY yielded a B2036 variant containing an alkyne functional group without compromising bioactivity, as verified by a cellular assay. Subsequent conjugation of 5, 10, and 20 kDa azide-containing PEGs via the copper-catalyzed click reaction yielded high purity, site-specific conjugates with >89% conjugation efficiencies. Site-specific attachment of PEG to B2036 is associated with substantially improved in vitro bioactivity values compared to pegvisomant, with an inverse relationship between polymer size and activity observed. Notably, the B2036-20 kDa PEG conjugate has a molecular weight comparable to pegvisomant, while exhibiting a 12.5 fold improvement in half-maximal inhibitory concentration in GHR-expressing Ba/F3 cells (103.3 nM vs 1289 nM). We expect that this straightforward route to achieve site-specific GHR antagonists will be useful for GH signal regulation.

The combination of more than one bioactive moiety in a multitargeted anticancer agent may result in synergistic activity of its components. Using this concept, bioorganometallic compounds were designed to feature a metal center, a 2-pyridinecarbothioamide (PCA), and a hydroxamic acid, which is found in the anticancer drug vorinostat (SAHA). The organometallics showed inhibitory activity in the nanomolar range against histone deacetylases (HDACs) as the key target for SAHA. In particular, the Rh complex was a potent inhibitor of HDAC6 over HDAC1 and HDAC8. Whereas this complex was highly cytotoxic in human cancer cells, it showed low toxicity in hemolysis studies and zebrafish, demonstrating the role of the metal center. For this complex a slightly reduced expression of vascular endothelial growth factor receptor 2 (VEGFR2) was established, which was upregulated by SAHA. This finding indicates that the new organometallics display different modes of action than their bioactive components., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Thiones have been investigated as ligands in metal complexes with catalytic and biological activity. We report the synthesis, characterization, and biological evaluation of a series of M II/III complexes of the general formulae [M II (cym)(L)Cl]X (cym = η 6 - p -cymene) or [M III (Cp*)(L)Cl]X (Cp* = η 5 -pentamethylcyclopentadienyl), where X = Cl - or PF 6 - , and L represents heterocyclic derivatives of thiourea. The thiones feature a benzyl-triazolyl pendant and they act as bidentate ligands via N , S -coordination to the metal centers. Several derivatives have been investigated by single-crystal X-ray diffraction analysis. NMR investigations showed a counterion-dependent shift of several protons due to the interaction with the counterions. These NMR investigations were complemented with X-ray diffraction analysis data and the effects of different counterions on the secondary coordination sphere were also investigated by DFT calculations. In biological studies, the Ir benzimidazole derivative was found to accumulate in the cytoplasm and it was the most cytotoxic derivative investigated.

Perforin is a key effector protein in the vertebrate immune system and is secreted by cytotoxic T lymphocytes and natural killer cells to help eliminate virus-infected and transformed target cells. The ability to modulate perforin activity in vivo could be extremely useful, especially in the context of bone marrow stem cell transplantation where early rejection of immunologically mismatched grafts is driven by the recipient's natural killer cells, which overwhelmingly use perforin to kill their targets. Bone marrow stem cell transplantation is a potentially curative treatment for both malignant and nonmalignant disorders, but when the body recognizes the graft as foreign, it is rejected by this process, often with fatal consequences. Here we report optimization of a previously identified series of benzenesulfonamide-based perforin inhibitors for their physicochemical and pharmacokinetic properties, resulting in the identification of 16 , the first reported small molecule able to prevent rejection of transplanted bone marrow stem cells in vivo by blocking perforin function.

Metal complexes provide a versatile platform to develop novel anticancer pharmacophores, and they form stable compounds with N -heterocyclic carbene (NHC) ligands, some of which have been shown to inhibit the cancer-related selenoenzyme thioredoxin reductase (TrxR). To expand a library of isostructural NHC complexes, we report here the preparation of Rh III - and Ir III (Cp*)(NHC)Cl 2 (Cp* = η 5 -pentamethylcyclopentadienyl) compounds and comparison of their properties to the Ru II - and Os II (cym) analogues (cym = η 6 - p -cymene). Like the Ru II - and Os II (cym) complexes, the Rh III - and Ir III (Cp*) derivatives exhibit cytotoxic activity with half maximal inhibitory concentration (IC 50 ) values in the low micromolar range against a set of four human cancer cell lines. In studies on the uptake and localization of the compounds in cancer cells by X-ray fluorescence microscopy, the Ru and Os derivatives were shown to accumulate in the cytoplasmic region of treated cells. In an attempt to tie the localization of the compounds to the inhibition of the tentative target TrxR, it was surprisingly found that only the Rh complexes showed significant inhibitory activity at IC 50 values of ∼1 μM, independent of the substituents on the NHC ligand. This indicates that, although TrxR may be a potential target for anticancer metal complexes, it is unlikely the main target or the sole target for the Ru, Os, and Ir compounds described here, and other targets should be considered. In contrast, Rh(Cp*)(NHC)Cl 2 complexes may be a scaffold for the development of TrxR inhibitors.

The HER2-targeted antibody-drug conjugate trastuzumab emtansine (T-DM1) is approved for the treatment of metastatic, HER2-positive breast cancer after prior trastuzumab and taxane therapy, and has also demonstrated efficacy in the adjuvant setting in incomplete responders to neoadjuvant therapy. Despite its objective activity, intrinsic and acquired resistance to T-DM1 remains a major clinical challenge. T-DM1 mediates its activity in a number of ways, encompassing HER2 signalling blockade, Fc-mediated immune response and payload-mediated microtubule poisoning. Resistance mechanisms relating to each of these features have been demonstrated, and we outline the findings of these studies in this review. In our overview of the substantial literature on T-DM1 activity and resistance, we conclude that the T-DM1 resistance mechanisms most strongly supported by the experimental data relate to dysfunctional intracellular metabolism of the construct and subversion of DM1-mediated cell killing. Loss of dependence on signalling initiated by HER2-HER2 homodimers is not substantiated as a resistance mechanism by clinical or experimental studies, and the impact of EGFR expression and tumour immunological status requires further investigation. These findings are instructive with respect to strategies that might overcome T-DM1 resistance, including the use of second-generation anti-HER2 antibody-drug conjugates that deploy alternative linker-payload chemistries.

Introduction: Medicinal mushrooms have been used for the treatment of diseases and general promotion of health for many centuries. Recent pharmacological research into medicinal mushrooms has identified various therapeutic properties, with applications in modern medicine. Aim: To evaluate the anti-cancer activities of Fomitopsis pinicola ( F. pinicola ) alcoholic extract in an in vivo setting. Methods: The anti-tumour effect of the F. pinicola extract was tested in a xenograft immune-compromised Rag-1 mouse model. This was followed by RT-PCR and metabolomics analyses. Results: There were no observable differences in tumor growth between treated and non-treated groups. The bioactive components were not detected in the mouse plasma or the tumor site. Conclusions: The extract was poorly absorbed; this is likely due to the timing of treatment, dosage levels and modifications made to the extract where the alcohol-based solvent was replaced with water. This, in combination with fractionation studies which identified most anti-cancer compounds to be hydrophobic, largely explained the lack of anti-cancer activities in vivo.

Background and Objectives: Excessive adipose tissue macrophage accumulation in obesity has been implicated in mediating inflammatory responses that impair glucose homeostasis and promote insulin resistance. Colony-stimulating factor 1 (CSF1) controls macrophage differentiation, and here we sought to determine the effect of a CSF1 receptor inhibitor, PLX3397, on adipose tissue macrophage levels and understand the impact on glucose homeostasis in mice., Methods: A Ten-week-old mice were fed a chow or high-fat diet for 10 weeks and then treated with PLX3397 via oral gavage (50 mg/kg) every second day for 3 weeks, with subsequent monitoring of glucose tolerance, insulin sensitivity and assessment of adipose tissue immune cells., Results: PLX3397 treatment substantially reduced macrophage numbers in adipose tissue of both chow and high-fat diet fed mice without affecting total myeloid cell levels. Despite this, PLX3397 did not greatly alter glucose homeostasis, did not affect high-fat diet-induced increases in visceral fat cytokine expression (Il-6 and Tnfa) and had limited effect on the phosphorylation of the stress kinases JNK and ERK and macrophage polarization., Conclusions: Our results indicate that macrophage infiltration of adipose tissue induced by a high-fat diet may not be the trigger for impairments in whole body glucose homeostasis, and that anti-CSF1 therapies are not likely to be useful as treatments for insulin resistance.

Redox-modulating anticancer drugs allow the exploitation of altered redox biology observed in many cancer cells. We discovered dinuclear RhIII(Cp*) and IrIII(Cp*) complexes that have in vitro anticancer activity superior to cisplatin and the investigational drug IT-139, while being less toxic in haemolysis and in vivo zebrafish models. The mode of action appears to be related to DNA damage and ROS-mediated stress pathways.

Organometallic compounds based on bioactive ligand systems have shown promising antiproliferative properties. The use of 8-hydroxyquinoline and its derivatives as bioactive ligands resulted in organometallic complexes with potent anticancer activity, but they lack aqueous solubility for further development. We report here the preparation of a series of M II/III (cym/Cp*)Cl complexes (η 6 -p-cymene (cym): M = Ru, Os; η 5 -pentamethylcyclopentadienyl (Cp*): M = Rh, Ir) with hydroxyquinoline-derived co-ligands and in a subsequent step the substitution of the chlorido ligands for amphiphilic 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane (PTA). Solubility studies indicated that the introduced PTA ligand significantly improved the aqueous solubility of all complexes. The complexes were shown to be stable in aqueous and DMSO solution over a period of at least 3 d. As would be expected for such modification of complexes, the higher solubility resulted in significantly decreased cytotoxicity in cancer cells. The antiproliferative activity was still more pronounced than that of RAPTA-C [Ru(cym)(PTA)Cl] which, however, has been demonstrated to have antimetastatic and antiangiogenic properties in vivo., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tumour hypoxia is a marker of poor prognosis and failure of chemoradiotherapy in head and neck squamous cell carcinoma (HNSCC), providing a strategy for therapeutic intervention in this setting. To evaluate the utility of the hypoxia-activated prodrug evofosfamide (TH-302) in HNSCC, we established ten early passage patient-derived xenograft (PDX) models of HNSCC that were characterised by their histopathology, hypoxia status, gene expression, and sensitivity to evofosfamide. All PDX models closely resembled the histology of the patient tumours they were derived from. Pimonidazole-positive tumour hypoxic fractions ranged from 1.7-7.9% in line with reported HNSCC clinical values, while mRNA expression of the Toustrup hypoxia gene signature showed close correlations between PDX and matched patient tumours, together suggesting the PDX models may accurately model clinical tumour hypoxia. Evofosfamide as a single agent (50 mg/kg IP, qd × 5 for three weeks) demonstrated antitumour efficacy that was variable across the PDX models, ranging from complete regressions in one p16-positive PDX model to lack of significant activity in the three most resistant models. Despite all PDX models showing evidence of tumour hypoxia, and hypoxia being essential for activation of evofosfamide, the antitumour activity of evofosfamide only weakly correlated with tumour hypoxia status determined by pimonidazole immunohistochemistry. Other candidate evofosfamide sensitivity genes- MKI67 , POR , and SLFN11 -did not strongly influence evofosfamide sensitivity in univariate analyses, although a weak significant relationship with MKI67 was observed, while SLFN11 expression was lost in PDX tumours. Overall, these data confirm that evofosfamide has antitumour activity in clinically-relevant PDX tumour models of HNSCC and support further clinical evaluation of this drug in HNSCC patients. Further research is required to identify those factors that, alongside hypoxia, can influence sensitivity to evofosfamide and could act as predictive biomarkers to support its use in precision medicine therapy of HNSCC.

Replacing one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 with a variety of sulfonamide-linked solubilizing substituents produced a new class of active and potent PI3Kα inhibitors, with several derivatives demonstrating high PI3Kα enzyme potency and good cellular potency in two human derived cell lines. The overall results suggest a preference for linear and somewhat flexible solubilizing functions. From this series, compound 16, also known as SN32976, was selected for advanced preclinical evaluation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Benzoylthiourea derivatives feature several donor atoms capable of coordinating to metal centers. We report here a series of Ru(η 6 -p-cymene) complexes employing benzoylthiourea derivatives as ligands. Such ligands often coordinate to metal centers through their S and O donor atoms. We isolated complexes where the ligands were mono- or bidentately coordinated to Ru involving the S donor atom and surprisingly in bidentate coordination mode a deprotonated thiourea nitrogen resulting in a 4-membered ring structure around the metal center. DFT calculations were used to explain the differences in coordination behavior. These were complemented by stability studies and biological investigations of the compounds as anticancer agents. Several of the synthesized derivatives exhibited significant cell growth inhibitory activity, with the complexes featuring bidentate ligands being more potent than their monodentate counterparts. This can be explained by the higher stability of the former under the conditions employed in cell culture assays., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tumour hypoxia plays an important role in tumour progression and resistance to therapy. Under hypoxia unfolded proteins accumulate in the endoplasmic reticulum (ER) and this stress is relieved through the protein kinase R-like ER kinase (PERK) signalling arm of the unfolded protein response (UPR). Targeting the UPR through PERK kinase inhibitors provides tumour growth inhibition, but also elicits on-mechanism normal tissue toxicity. Hypoxia presents a target for tumour-selective drug delivery using hypoxia-activated prodrugs. We designed and prepared hypoxia-activated prodrugs of modified PERK inhibitors using a 2-nitroimidazole bioreductive trigger. The new inhibitors retained PERK kinase inhibitory activity and the corresponding prodrugs were strongly deactivated. The prodrugs were able to undergo fragmentation following radiolytic reduction, or bioreduction in HCT116 cells, to release their effectors, albeit inefficiently. We examined the effects of the prodrugs on PERK signalling in hypoxic HCT116 cells. This study has identified a 2-substituted nitroimidazole carbamate prodrug with potential to deliver PERK inhibitors in a hypoxia-selective manner., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The promise of the metal(arene) structure as an anticancer pharmacophore has prompted intensive exploration of this chemical space. While N-heterocyclic carbene (NHC) ligands are widely used in catalysis, they have only recently been considered in metal complexes for medicinal applications. Surprisingly, a comparatively small number of studies have been reported in which the NHC ligand was coordinated to the Ru II (arene) pharmacophore and even less with an Os II (arene) pharmacophore. Here, we present a systematic study in which we compared symmetrically substituted methyl and benzyl derivatives with the nonsymmetric methyl/benzyl analogues. Through variation of the metal center and the halido ligands, an in-depth study was conducted on ligand exchange properties of these complexes and their biomolecule binding, noting in particular the stability of the M-C NHC bond. In addition, we demonstrated the ability of the complexes to inhibit the selenoenzyme thioredoxin reductase (TrxR), suggested as an important target for anticancer metal-NHC complexes, and their cytotoxicity in human tumor cells. It was found that the most potent TrxR inhibitor diiodido(1,3-dibenzylbenzimidazol-2-ylidene)(η 6 -p-cymene)ruthenium(II) 1b I was also the most cytotoxic compound of the series, with the antiproliferative effects in general in the low to middle micromolar range. However, since there was no clear correlation between TrxR inhibition and antiproliferative potency across the compounds, TrxR inhibition is unlikely to be the main mode of action for the compound type and other target interactions must be considered in future.

Ru II (η 6 -arene) compounds carrying bioactive flavonol ligands have shown promising anticancer activity against tumor cells via a multitargeting mode of action, i.e., through interaction with DNA and inhibition of topoisomerase IIα. By introducing a novel arene ligand based on the amino acid l-phenylalanine (Phe), we aimed to alter the pharmacological properties of the complexes. We report here a series of novel Ru II (η 6 -arene)Cl complexes with different substituents on the phenyl ring of the flavonol which should maintain the multitargeting capability of the parent η 6 - p-cymene (cym) complexes. Studies with selected examples revealed stability in aqueous solution after quickly forming aqua complexes but rapid decomposition in pure DMSO. The reactions with protein and DNA models proceeded quickly and resulted in cleavage of the flavonol or adduct formation, respectively. The compounds were found to be cytotoxic with significant antiproliferative activity in cancer cells with IC 50 values in the low μM range, while not following the same trends as observed for the cym analogues. Notably, the cellular accumulation of the new derivatives was significantly higher than for their respective cym complexes, and they induced DNA damage in a manner similar to that of cisplatin but to a lesser extent.