No

Yes / We report the encapsulation of highly hydrophobic 16-electron organometallic ruthenium and osmium carborane complexes [Ru/Os(p-cymene)(1,2-dicarba-closo-dodecarborane-1,2-dithiolate)] (1 and 2) in Pluronic® triblock copolymer P123 core–shell micelles. The spherical nanoparticles RuMs and OsMs, dispersed in water, were characterized by dynamic light scattering (DLS), cryogenic transmission electron microscopy (cryo-TEM), and synchrotron small-angle X-ray scattering (SAXS; diameter ca. 15 and 19 nm, respectively). Complexes 1 and 2 were highly active towards A2780 human ovarian cancer cells (IC50 0.17 and 2.50 μM, respectively) and the encapsulated complexes, as RuMs and OsMs nanoparticles, were less potent (IC50 6.69 μM and 117.5 μM, respectively), but more selective towards cancer cells compared to normal cells. / We thank the Leverhulme Trust (Early Career Fellowship no. ECF-2013-414 to NPEB), the University of Warwick (Grant no. RDF 2013-14 to NPEB), the Swiss National Science Foundation (Grant no. PA00P2_145308 to NPEB and PBNEP2_142949 to APB), the ERC (Grant no. 247450 to PJS), EPSRC (EP/G004897/ 1 to APB, and EP/F034210/1 to PJS), Institute of Advanced Study (IAS) – University of Warwick (Fellowship to JJSB), and Science City (AWM/ERDF) for support. We thank the Wellcome Trust (055663/Z/98/Z) for funding to the Electron Microscopy Facility, School of Life Sciences, University of Warwick.

Yes / The archetypal prodrug of the imidazotetrazine class is the anticancer agent temozolomide (TMZ). The prodrug activation kinetics of TMZ show an unusual pH dependence: it is stable in acid and rapidly hydrolyses in alkali (Denny, B.J., et al. Biochemistry 1994, 33, 9045–9051). The incipient drug MTIC has the opposite properties—relatively stable in alkali but unstable in acid. In this study, the mechanism of prodrug activation was probed in greater detail to determine whether the reactions are specific or general acid or base catalysed. Three prodrugs and drugs were investigated, TMZ, MTIC and the novel dimeric imidazotetrazine EA27. Hydrolysis in a range of citrate-phosphate buffers (pH 8.0, 7.4, 4.0) was measured by UV spectrophotometry. Reaction of TMZ and MTIC obeyed single-phase, pseudo-first order kinetics (Figure 1). EA27 was more complex, showing biphasic but approximately pseudo-first order kinetics, Figure. General acid or base catalysis indicates that protonation or deprotonation is the rate-limiting step (rls). In biological milieu, the nature and concentration of other acidic or basic solutes may affect the prodrug activation reaction. In contrast, specific acid or base catalysis indicates that protonation or deprotonation occurs before the rls, so catalysis depends only on the local concentration of hydroxide or hydronium ion (i.e., pH) so the reaction kinetics are not expected to change appreciably in a biological medium.

Yes / To develop potential next-generation metal anticancer agents, we designed and synthesised five Cu(II) 2-pyridine-thiosemicarbazone complexes by modifying the hydrogen atom at the N-4 position of ligands, and then investigated their structure-activity relationships and anticancer mechanisms. Modification of the N-4 position with different groups caused significant differences in cellular uptake and produced superior antitumor activity. Cu complexes arrested the cell cycle at S phase, leading to down-regulation of levels of cyclin and cyclin-dependent kinases and up-regulation of expression of cyclin-dependent kinase inhibitors. Cu complexes exerted chemotherapeutic effects via activating p53 and inducing production of reactive oxygen species to regulate expression of the B-cell lymphoma-2 family of proteins, causing a change in the mitochondrial membrane potential and release of cytochrome c to form a dimer with apoptosis protease activating factor-1, resulting in activation of caspase-9/3 to induce apoptosis. In addition, Cu complexes inhibited telomerase by down-regulating the c-myc regulator gene and expression of the human telomerase reverse transcriptase. / Natural ScienceFoundation of China (31460232, 21431001, 21561017, 21462004),the Natural Science Foundation of Guangxi (2017GXNSFEA198002,AD17129007), IRT_16R15, Guangxi“Bagui”scholar program to HBSun, and High-Level Innovation Team and Distinguished Scholarprogram of Guangxi universities to F Yang.

No / Herein we report a library of new ruthenium(II) complexes which incorporate a range of functionalised β -ketoiminate ligands. The complexes undergo an unusual reduction from Ru(III) to Ru(II), and consequently incorporate carbonyl ligands from the 2-ethoxyethanol solvent, forming ruthenium dicarbonyl complexes. In order to address the potential applications of these complexes, we have screened the library against a range of tumour cell lines, however, all compounds exhibit low cellular activity and this is tentatively assigned to the decomposition of the compounds in aqueous media. Studies to establish the antifungal and antibacterial potential of these complexes was addressed and show increased growth inhibitions for C. neoformans and S. aureus species. / The authors would like to thank the universities of Leeds, Huddersfield and Bradford for internal financial support.

Dans ce travail de thèse, plusieurs groupes de nouveaux complexes d'or(I) à base de carbènes N-hétérocyclique (NHC)contenant des bras amino-aliphatiques et aromatiques avec un potentiel intéressant dans des applications biomédicales ont été synthétisés et entièrement caractérisés. En outre, une série de complexes d'iridium(III) contenant des ligands NHC avec des activités anticancéreuses prononcées pour une application en thérapie photodynamique, a étépréparée et entièrement caractérisée. Le premier groupe représente une famille de complexes cationiques or(I) bis(NHC) contenant des bras latéraux amino-aliphatiques. Ces complexes ont étésynthétisés et étudiés pour leurs activités antiprolifératives vis-à-vis de quatre lignées cellulaires cancéreuses humaines et de la lignée cellulaire non cancéreuse MDCK. Dans cette série, la lipophilie est directement liée àl'activitécytotoxique contre les cellules cancéreuses. La deuxième famille de composés concerne les complexes cationiques or(I) bis(NHC) contenant des bras latéraux amino-aromatiques. La cytotoxicitéin vitro de ces complexes et de leurs proligands sur les lignées cellulaires représentatives du cancer de la prostate PC-3 et de la vessie T24 a étéévaluée. Tous ces complexes présentent des valeurs de Log P (lipophilie) supérieures àcelles de la première série de complexes en accord avec leur cytotoxicité plus élevée, mais une lipophilie trop élevée peut également conduire àune sélectivitéplus faible. Afin de développer un candidat-médicament avec une activitéet une sélectivité optimisées, nous avons conçu et synthétisé la troisième famille de complexes cationiques or(I) bis(NHC). Les valeurs de log P de cette série se situent entre la première série et la deuxième série. Ces complexes moins lipophiles sont moins cytotoxiques envers les lignées cellulaires saines (NIH3T3) et montre des activités anticancéreuses un peu plus faibles sur les cellules PC-3 que la deuxième série, avec néanmoins des valeurs de GI50 dans la gamme du nanomolaire. Les études mécanistiques sur deux complexes d'or(I) ont été réalisées. Les mesures d'absorption cellulaire ont montré une accumulation cellulaire rapide et une bonne biodisponibilitédes complexes, en accord avec l'activitéantiproliférative de ces deux complexes. De plus, les deux complexes inhibent la thiorédoxine reductase (TrxR), une cible commune pour les complexes d'or(I). La mort cellulaire induite par ces deux complexes est dépendante des espèces réactives de l'oxygène. En plus des activités anticancéreuses, nous avons également testédes complexes d'or(I) mono-NHC pour une autre application biomédicale, la leishmaniose, maladie parsitaire. Ils ont été testés in vitro sur les formes promastigotes et amastigotes axéniques de L. infantum. De plus, leur cytotoxicitéa étéévaluée sur les macrophages murins J774A.1 afin de déterminer leur sélectivitéd'action. Un autre sujet de cette thèse concerne les complexes d'iridium(III)-NHC. Trois familles de complexes ont étépréparées et caractérisées. La cytotoxicitéin vitro de tous les complexes sur les cellules cancéreuses de la prostate PC-3 et de la vessie T24, et les cellules non cancéreuses NIH3T3 a étéévaluée. De plus, tous les complexes sont des agents théranostiques, et les expériences de microscopie confocale d'un complexe ont montréqu'il pouvait être rapidement et efficacement absorbédans les cellules PC-3 et se localiser spécifiquement dans les mitochondries. De manière intéressante, ces complexes peuvent agir comme des photosensibilisateurs efficaces. La cytotoxicitéde ces complexes a étéaugmentée substantiellement après une irradiation lumineuse de 365 nm, ce qui suggère le potentiel élevéde ces agents anticancéreux théranostiques ciblant les mitochondries pour la thérapie photodynamique. / In this work of thesis, several groups of novel NHC-based gold(I) complexes containing aliphatic and aromatic amino-side arms with interesting potential in biomedical applications have been synthesized and fully characterized. Also, a series of iridium(III) complexes containing NHC ligands with pronounced PDT anticancer activities has been prepared and fully characterized. The first group represents a family of cationic bis(NHC)-gold(I) complexes containing aliphatic amino-side arms. These complexes have been synthesized and investigated for their antiproliferative activities towards four human cancer cell lines and the non-cancerous MDCK cell line. In this series, the lipophilicity correlates directly with the cytotoxic activity against cancer cells. The second family of compounds concerns cationic gold(I) bis(NHC) complexes containing aromatic amino-side arms. The in vitro cytotoxicity of these complexes and proligands on the representative PC-3 prostate and T24 bladder cancer cell lines has been evaluated. All these complexes show higher Log P values (lipophilicity) than the first series of complexes, and in line with this higher cytotoxicity, nevertheless too high lipophilicity can also lead to lower selectivity. In order to develop a drug candidate with optimized activity and selectivity, we designed and synthesized the third family of cationic gold(I) bis(NHC) complexes. The Log P values of this series were between the first series and the second series. The lower cytotoxicity towards non-cancerous NIH3T3 cells was found for this series of complexes whereas they also displayed less activities towards cancer cells than the second series. The mechanistic studies on two gold complexes by monitoring the cellular uptake showed the rapid cellular accumulation of the intact gold bis(NHC) and a good bioavailability, in good agreement with the antiproliferative activity of these two complexes. Moreover, both complexes inhibit TrxR, a common target for gold(I) complexes. The cell death induced by these two complexes was ROS-dependent. Besides anticancer activities, we also tested gold(I) mono-NHC complexes for other biomedical applications in parasite disease Leishmaniasis. They were screened in vitro against both promastigote and axenic amastigote forms of L. infantum. Moreover, their cytotoxicity was evaluated on the murine J774A.1 macrophages in order to determine their selectivity of action. Another topic of this thesis concerns iridium(III)-NHC complexes. Three families of theranostic iridium(III)-NHC complexes were prepared and characterized. The in vitro cytotoxicity of all the complexes against PC-3 prostate, T24 bladder cancer cells and non-cancerous NIH3T3 cells was evaluated. Moreover, all complexes are theranostic agents, and the confocal microscopy experiments of one complex showed that it can be quickly and effectively taken up into PC-3 cells and specifically localize into mitochondria. Interestingly, these complexes can act as efficient photosensitizers. The cytotoxicity of these complexes was increased substantially upon 365 nm light irradiation, which suggested the high potential to be mitochondria-targeting theranostic anticancer agents for photodynamic therapy.

Cancer has become a major health problem due to its high rate of morbidity and mortality. Severe side effects associated with most of the available anticancer drugs and the development of the drug resistant cells are the major hurdles limiting their application and therapeutic success. Much attention has thus been focused on natural compounds with minimal or no toxicity in humans and having capacity to suppress proliferation and/or induce apoptosis in cancer cells. Therefore, it is imperative to discover and develop new anticancer drugs. Endophytic fungi though underexploited have remained a rich source of structurally novel and biologically active secondary metabolites. That they are a prolific resource for new compounds in drug discovery is evident from a plethora of reported research findings of the recent past. Those of endophytic fungi especially isolated from medicinal plants with known therapeutic value have especially caught the attention of several research groups worldwide for the production of host associated or novel lead molecules. In this backdrop, the focus of my research work has been centered on exploring the endophytic fungal population associated with Catharanthus roseus in search of production of novel anticancer compounds. Twenty endophytic fungi were isolated from different parts of Catharanthus roseus plant. The ethyl acetate (EA) extracts of three week grown liquid cultures of the individual endophytes were assessed in vitro for their cytotoxic activities in HeLa and HepG2 cancer cells using MTT assay. Of all the isolates thus screened, the EA extract from an isolate identified as Chaetomium nigricolor exhibited significant cytotoxicity in the tested cell lines with a better activity profile against HeLa cells. To understand if the liquid culture filtrate or the mycelium harbored the active cytotoxic principle, the C. nigricolor culture filtrate and mycelia was extracted separately using organic solvents with varying polarity namely ethyl acetate, dichloromethane, chloroform and hexane. These extracts were further tested for cytotoxicity induction in HeLa and MCF-7 cells by MTT assay. The ethyl acetate culture filtrate and mycelial extracts showed best cytotoxic activity on HeLa cells compared to other extracts; therefore, the mycelial and culture filtrate ethyl acetate extracts of C. nigricolor were pooled and was used for further work. Hexane culture filtrate extract of C. nigricolor showed best antiproliferative activity against MCF-7 cells. Thus, the compounds with anticancer potential from both ethyl acetate pooled extracts and hexane culture filtrate extract of C. nigricolor were purified using bioassay guided isolation method. This finally resulted in the isolation of two different anticancer compounds, one each from EA pooled extract and Hexane culture filtrate extracts of C. nigricolor named as compound 1 and compound 2, respectively. Based on various analyses including mass spectrometry (MS and MS-MS) and UV-visible, Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, the compounds 1 and 2 were identified as 1, 2 Bis (diethylamino) ethene-1, 2 diol (BDED) and Nonacos-4-enoic acid (NA), respectively An improved understanding of the cellular responses to chemotherapeutic agents in cancer cells and the underlying molecular mechanisms gains particular relevance in the efforts to improve the clinical outcome of chemotherapeutic agents. Arresting the growth of cancer cells in one way or the other and induction of apoptosis in a drug specific manner is the hallmark of almost all the clinically used anticancer drugs. Towards this end, evaluation of the cytotoxic effects of the newly identified compounds on various human cancer cells was carried out. BDED was examined for in vitro cytotoxicity of against a panel of five human cancer cell lines namely HeLa, A-431, A549, COLO 205, and MCF-7. Among the cell lines screened, HeLa cells were most vulnerable to BDED treatment with an IC50 value of 27 μM. This cytotoxicity was later identified as resulting from apoptosis induction by BDED which was scored by the characteristic events of membrane externalization of phosphatidylserine, cytoplasmic shrinkage and chromatin condensation which were observed in a large majority of the BDED-treated cells. Further detailed studies were performed to delineate the cellular mechanisms of BDED-induced apoptosis in HeLa cells. Analysis of cell cycle progression by propidium iodide (PI) staining revealed BDED-induced cell cycle arrest in the G1 phase of cell cycle. The results indicated that BDED induced a significant ROS generation and a significant loss of mitochondrial membrane potential which were abrogated upon pretreatment of the cells with an antioxidant, N-acetyl cysteine (NAC). These observations suggested the probable involvement of the initial events of BDED-induced ROS production in causing cell death via mitochondria-based intrinsic pathway of apoptosis. In addition, the results from the expression profile of pro- and anti- apoptotic proteins by western blot analysis in the BDED-treated HeLa cells further corroborated this hypothesis. These included an abnormally deregulated cellular abundance of BAX 1 and BCL 2 proteins, elevated levels of APAF-1, and activated cleaved species of procaspases 9 and 3. In addition, a pronounced abundance of cleaved PARP protein was observed in these experiments. Thus, the results suggest that BDED induced apoptosis in HeLa cells via ROS mediated mitochondrial dependent pathway. In parallel, the in vitro cytotoxic effects of NA was also studied, the second antiproliferative compound identified from hexane extracts of C. nigricolor using a panel of four human cancer cell lines - A-431, A549, COLO 205, and MCF 7. NA was thus found to be most potent against MCF 7 (breast cancer) cells. PI staining-based viability assays and microscopic observations showed a dose dependent cytotoxicity of NA on MCF 7 cells. Our data reveled an IC 50 of 40 μM for NA The NA-induced apoptosis was confirmed by flow cytometric detection of membrane externalization of phosphatidylserine using Annexin V FITC/PI dual staining. NA was also observed to induce cytotoxicity best at 72 h this could be attributed to the fact that it is a derivative of a Nonocosane-(a naturally prevalent molecule found in several vegetables). Similar to our results from BDED-treated HeLa cells, ROS generation and loss of mitochondrial membrane potential which were abrogated on pretreatment with NAC, were also observed in NA-treated MCF-7cells. Similar to several other fatty acids, NA was also observed to induce cell cycle arrest in the G1 phase of cell cycle. Further, apoptotic signature of an altered expression of anti-apoptotic BCL-2 and pro-apoptotic BAX, APAF-1 and procaspase 9 and 3 and a cleaved PARP were observed in NA-treated MCF 7 cells. In conclusion, the results indicate that BDED and NA have cytotoxic and apoptotic effects on HeLa (cervical cancer) and MCF 7 (breast cancer) cell lines, respectively. This leaves open further avenues to evaluate their potential application as anticancer agents for treatment of human cervical and breast cancers.

There is an urgent need to find novel anticancer therapeutics with different mechanisms of action than platinum-containing drugs, particularly for patients who relapse after having been initially treated with a platinum-containing chemotherapy regimen. This chemoresistance phenomena, along with the serious side effects observed with cisplatin, have led research in Medicinal Inorganic Chemistry to using other precious metals for the design of novel anticancer therapeutics. This work reports on the synthesis and characterisation of a series of organometallic drug candidates based on ruthenium, osmium, rhodium, and iridium, followed by investigation of their cancer-inhibiting properties via in vitro and in vivo studies. The cytotoxicity of these complexes against various human cancer cell lines is presented, as well as preliminary studies on their possible modes of action, determined via gene expression studies, cell cycle and apoptosis analysis, reactive oxygen species detection and mitochondrial-membrane potential assays. In addition, to confirm the surprising absence of in vitro toxicity against normal cells exhibited by some compounds, studies on ex vivo/in vitro isolated human lymphocytes from healthy individuals, have been conducted. One lead molecule has been progressed to in vivo studies in mice and toxicity and efficacy were assessed with a series of assays including determination of the maximum tolerated dose and pharmacodynamic studies. Structural modifications of the lead molecule with water-soluble phosphines were subsequently undertaken, with the aim to improve the stability and solubility of the parent 16-electron specie, and evaluations of the biological activity of these novel complexes are presented.

During this thesis we tried to design, synthesise and analyse some novel devices for the selective delivery of peptides. These systems are based on the enzyme-activated anticancer prodrugs developed by Prof. Gesson in Poitiers and the peptide rotaxanes developed by Prof. Leigh in Edinburgh. The innovative rotaxanes we constructed are devised to protect and selectively release a peptide in response to an enzyme-specific stimulus for the targeted therapy of cancer. In Chapter 1 we tried to expose the main synthetic strategies aimed at improving the stability and permeation features of biologically active peptides. We examined some prodrug approaches and particularly the tumour-activated prodrugs (TAPs), largely investigated for use in anticancer chemotherapy. TAPs are generally three-part molecules composed of trigger, spacer and effector units. We also presented the original methodology developed by Prof. Leigh, namely the hydrogen bond-directed assembly of peptide rotaxanes, to protect a peptide thread from external environment. Finally we presented our project which consists of a combination of the peptide prodrug and rotaxane approaches. Therefore, based on the knowledge of both research groups we tried in Chapter 2 to develop some model systems in order to study the influence of the rotaxane architecture upon prodrug molecules. The first step towards such rotaxane-based peptide prodrugs relied on the efficient design of a spacer which has to be bulky enough to work as a stopper for the macrocycle. Much of the work presented in this chapter is based on the design and synthesis of such self-immolative units. We then explored the response of our model rotaxanes under the action of the activating enzyme. After this detailed study, in Chapter 3 we applied our concept to the biologically active peptide Met-enkephalin. In this chapter we presented a comparison between a rotaxane prodrug of Met-enkephalin and its non-interlocked derivative. Thus both compounds were successfully synthesised and evaluated to release the free peptide after enzymatic activation. The protective effect of encapsulating the peptide within a rotaxane assembly was also studied in human plasma and with different proteases. Finally, in Chapter 4, we introduced the construction of a rotaxane-based molecular machine programmed to synthesise a short peptide unit from the amino acids carried on its thread. We synthesised with success a one-station model rotaxane to study the catalyst effect of the macrocycle. Unfortunately this model machine proved not to work and current research is still ongoing to achieve such a synthetic device.

Compounds from the marine environment exhibit a wide variety of biological activities, and thus hold much promise as potential drugs. The halichondrins, isolated from the Kaikoura sponge Lissodendoryx sp. are no exception to this, demonstrating potent anticancer activity. Novel cytotoxic compounds have also been isolated from the Chatham Rise sponge Lamellomorpha strongylata. Knowledge of the cellular origins of such compounds is desirable, in order to establish if the sponge or associated micro-organisms are producing the compounds of interest. Siderophores are also important molecules, which are produced on demand by bacteria in order to obtain sufficient iron necessary for their growth. Knowledge of the biosynthesis of these compounds has potential for the control of undesirable bacteria, such as the anthrax-causing pathogen Bacillus anthracis. Cell separation studies have been carried out on Lamellomorpha strongylata, locating a swinholide in sponge-associated filamentous bacteria and theonellapeptolides in sponge-associated unicellular bacteria. A microscopic analysis of dissociated cells from Lissodendoryx sp. was also undertaken. The structures of four new halichondrins (3.13 - 3.16), isolated from Lissodendoryx sp., have been determined from spectral data. All of these compounds are very similar to known B series halichondrins, with differences occurring only beyond carbon 44. As biological activity has been shown to be derived from the portion of the molecule between carbons 1 and 35, they all retain good activity in the P388 assay as expected. A new siderophore, petrobactin sulfonate (4.2), was characterised, along with three cyclic imide siderophore derivatives (4.3 - 4.5). Petrobactin sulfonate is the first marine siderophore containing a sulfonated 3,4-dihydroxy aromatic ring. The structures were elucidated from spectral data, resulting in a revision of the NMR assignments of petrobactin.