Dissertations / Theses on the topic 'Anticancer'

Las toxicidades limitantes de dosis fuero una neutropenia febril grado 4 (en el brazo de docetaxel) y una neutropenia grado 3 en el brazo de gemcitabina. La combinación de carlumab no tuvo un impacto El cáncer es una enfermedad altamente frecuente y con alta mortalidad. El desarrollo de fármacos contra el cáncer se ha caracterizado por su ineficiencia, con una de las tasas de aprobación de fármacos más baja entre las diferentes especialidades médicas. El principal motivo de esta baja tasa de éxito es la falta de eficacia de los nuevos fármacos que entran al desarrollo clínico. Se han planteado diferentes estrategias para mejorar la eficiencia del desarrollo de fármacos, incluyendo la combinación de fármacos antitumorales, el desarrollo en paralelo de biomarcadores y la optimización del diseño de los ensayos clínicos usando modelización basada en farmacocinética y farmacodinamia Esta tesis es un compendio de dos artículos que evalúan estrategias para optimizar el desarrollo de fármacos mediante la combinación de agentes antitumorales. El primer proyecto es la evaluación preclínica en xenoinjertos derivados de pacientes (PDX) la combinación de inhibidores de PI3K-mTOR con diferentes agentes antitumorales y el segundo es el ensayo clínico fase I evaluando carlumab, un anticuerpo anti CCL2, en combinación con diferentes quimioterapias en pacientes con tumores sólidos avanzados. Proyecto 1: se seleccionaron tres modelos de PDX con deficiencia en PTEN: un PDX de cáncer de mama triple negativo (TNBC), otro de carcinoma de ovario de bajo grado KRAS G12R mutado y otro de adenocarcinoma de pulmón con mutaciones en KRAS G12C y TP53 R181P. En estos modelos se evaluaron dos inhibidores de PI3K-mTOR—PF-04691502 and PF-05212384— en combinación con cisplatino, paclitaxel o dacomitinib. La adición de los inhibidores de PI3K-mTOR a cisplatino o paclitaxel aumentó la actividad de la quimioterapia en los modelos de TNBC y LGSOC; sin embargo, no se objetivó este efecto en modelo de adenocarcinoma de pulmón con mutación de KRAS y TP53. Se objetivó modulación farmacodinámica de pAKT y pS6 en los grupos tratados con inhibidores de PI3K-mTOR. Nuestra investigación sugiere que añadir un inhibidor de PI3K-mTOR puede aumentar el efecto inhibitorio sobre el crecimiento de la quimioterapia en modelos PDX con deficiencia en PTEN. Sin embargo, este beneficio no se observó en el modelo de adenocarcinoma KRAS y TP53 mutado. En el futuro se deberá ahondar en el papel de la pérdida de PTEN en la actividad de estas combinaciones. Proyecto 2: se trata de un ensayo clínico fase Ib evaluando carlumab, un anticuerpo monoclonal contra CCL-2, en combinación con cuatro regímenes de quimioterapia (docetaxel, gemcitabina, carboplatino + paclitaxel y doxorrubicina liposomal pegilda (PLD). En este estudio participaron 53 pacientes en los que o bien los agentes quimioterápicos eran parte del tratamiento convencional o no tenían otras opciones de tratamiento convencional: docetaxel (n=15), gemcitabina (n=12), carboplatino + paclitaxel (n=12) y PLD (n=14). Las toxicidades limitantes de dosis incluyeron una neutropenia febril grado 4 (en el brazo de docetaxel) y una neutropenia grado 3 (en el brazo de gemcitabina). De acuerdo a los brazos de tratamiento, las toxicidades grado 3 o mayores más frecuentes fueron: neutropenia (6/15) y neutropenia febril (4/15) en el brazo de docetaxel, neutropenia (2/12) en el brazo de gemcitabina, neutropenia (4/12), trombocitopenia (4/12) y anemia (2/12) en el brazo de carboplatino-paclitaxel y anemia (3/14) y mucositis (2/14) en el brazo de PLD. Se objetivo una respuesta parcial y 18 estabilizaciones de la enfermedad (38%). La adición de carlumab no tuvo cambios relevantes en el perfil farmacocinético de ninguna de las quimioterapias evaluadas. Los niveles de CCL2 libres descendieron inmediatamente tras el tratamiento con carlumab, pero aumentaron con las administraciones posteriores, sugiriendo que carlumab secuestraba CCL2 de manera temporal. No se objetivaron anticuerpos anti-droga que justificasen dicho efecto. No se objetivaron cambios en las células tumorales circulantes ni en las células circulantes endoteliales. En 3 de 19 pacientes evaluables se objetivó una reducción del 30% en los niveles de N-telopeptido de colágeno tipo I en orina (uNTx). Carlumab es seguro administrado a dosis de 10 o 15 mg/kg en combinación con quimioterapia convencional y tiene buena tolerancia. Sin embargo, no se alcanza una inhibición sostenida de CCL2, ni se han objetivado un número de respuestas significativas.
Cancer is a highly frequent disease associated to high mortality. Drug development in Oncology has shown to be inefficient, having one of the lowest success rate of drugs entering in phase I trials that finally achieves marketed authorization. The main reason for this high failure rate is lack of efficacy. Different strategies have been adopted to improved anti-cancer drug development with the aim of improving patient care. This strategies include the combinatorial use of agents, biomarker co-development, and optimization of clinical trial design with the use of pharmacokinetic-pharmacodynamic modeling. This thesis is presented as compendium of work integrating two projects; the first project preclinically evaluates the combination of two PI3K-mTOR inhibitors and chemotherapy or the pan-HER inhibitor dacomitinib in patient derived xenografts. The second project evaluates de monoclonal antibody anti-CCL2 carlumab in patient derived xenografts. Project 1: Three PDXs were selected for their lack of PTEN expression by immunohistochemistry: a triple-negative breast cancer (TNBC), a KRAS G12R low-grade serous ovarian cancer (LGSOC), and KRAS G12C and TP53 R181P lung adenocarcinoma (LADC). Two dual PI3K-mTOR inhibitors were evaluated—PF-04691502 and PF-05212384—in combination with cisplatin, paclitaxel, or dacomitinib. The addition of PI3K-mTOR inhibitors to cisplatin or paclitaxel increased the activity of chemotherapy in the TNBC and LGSOC models; whereas no added activity was observed in the LADC model. Pharmacodynamic modulation of pS6 and pAKT was observed in the group treated with PI3K-mTOR inhibitor. Our research suggests that the addition of a PI3K-mTOR inhibitor may enhance tumor growth inhibition when compared to chemotherapy alone in PTEN-deficient PDXs. However, this benefit was absent in the KRAS and TP53 mutant LADC model. The role of PTEN deficiency in the antitumor activity of these combinations should be further investigated in the clinic. Project 2 is a first-in-human phase 1b study of carlumab with one of four chemotherapy regimens (docetaxel, gemcitabine, paclitaxel+carboplatin, and pegylated liposomal doxorubicin HCl [PLD]). Fifty-three patients with advanced solid tumors for which ≥1 of these regimens was considered standard of care or for whom no other treatment options existed participated in the study: docetaxel (n=15), gemcitabine (n=12), paclitaxel or carboplatin (n=12), or PLD (n=14). Dose-limiting toxicities included one grade 4 febrile neutropenia (docetaxel arm) and one grade 3 neutropenia (gemcitabine arm). The most common drug-related grade ≥3 adverse events were docetaxel arm—neutropenia (6/15) and febrile neutropenia (4/15); gemcitabine arm—neutropenia (2/12); paclitaxel+carboplatin arm—neutropenia, thrombocytopenia (4/12 each), and anemia (2/12); and PLD arm—anemia (3/14) and stomatitis (2/14). One partial response and 18 (38 %) stable disease responses were observed. Combination treatment with carlumab had no clinically relevant pharmacokinetic effect on any of the chemotherapeutic agents tested. Free CCL2 declined immediately post-treatment with carlumab but increased with further chemotherapy administrations in all arms, suggesting that carlumab could sequester CCL2 for only a short time. Neither antibodies against carlumab nor consistent changes in circulating tumor cells (CTCs) or circulating endothelial cells (CECs) enumeration were observed. Three of 19 evaluable patients showed a 30 % decrease from baseline urinary cross-linked N-telopeptide of type I collagen (uNTx). Carlumab could be safely administered at 10 or 15 mg/kg in combination with standard-of-care chemotherapy and was well-tolerated, although no long-term suppression of serumCCL2 or significant tumor responses were observed.

Drawbacks associated with anticancer chemotherapeutic cisplatin include tumour drug resistance, non-effectiveness against all tumours and lack of tumour-specificity resulting in severe side-effects (e.g. nausea, hair loss and kidney toxicity). The use of other metals such as transition metals rutheniumandosmium, may address the problems associated with platinum drugs and have received increased interest over the years. In this thesis the biological activity and aqueous solution chemistry of half-sandwichosmium (II) compounds of the type [(arene)OsII(X)(YZ)] n+ is explored. Chelating ligands containing nitrogen or nitrogen and oxygen donor atoms (N, NandN, O-chelatingligands) are investigated. It is shown that the chelating ligand has a large effect on the aqueous reactivity of the complexes. The introduction of functional groups on the chelate allowed for the ‘fine-tuning’ of the aqueous reactivity and nucleobase binding of the complexes. Also the nature of the coordinating arene was found to have an important effect on their biological activity. This could be rationalised by increased hydrophobicity with more extended arenes such as biphenylandtetrahydroanthracene, resulting in increased cellular uptake and increased cytotoxicity. Conjugating cell penetrating peptides to the complexes resulted in improved biological properties and opened a new way for functionalisation of the compounds. Several compounds reported in this thesis exhibit promising activity in the ovarian, colon and lung cancer cell lines and some could overcome cisplatin resistance in ovarian cisplatin resistant cell lines. Initial studies revealed cell death via apoptosis and the possible involvement of mitochondria in the apoptotic pathway. These results point to a novel pathway of activation for these complexes which is advantageous for addressing chemoresistance and effectiveness to oher types of cancers. This work shows that the biological properties of these compounds can be tuned by choice of ligands and also provides initial evidence for a novel pathway of activation.

Half-sandwich complexes of ruthenium, iridium, and more recently osmium, have shown promise as anticancer agents. Many of these ‘piano-stool’ complexes appear to target the redox balance in cells. Separately, similar complexes have been investigated for the catalysis of hydrogenation reactions, with many examples achieving high turnover frequencies and enantioselectivities. This thesis is concerned with achieving in cell catalysis to increase drug potency and generate selectivity for cancer cells. A series of eighteen Os(II) and Ir(III) complexes, of the type [M(ηx-arene)(diamine)] (Os-arene: p-cymene, biphenyl, or m-terphenyl; Ir-arene: Cp*, CpxPh, or CpxBip), were synthesised and fully characterised. The structures were derived from a Ru(II) transfer hydrogenation catalyst [Ru(η6-p-cymene)(TsDPEN)], TsDPEN = N-tosyl-diphenyl-ethylenediamine. The complexes were isolated as 16-electron amido catalysts, which were highly stable in solution and upon storage, unlike their 16-electron Ru(II) counterparts, and were highly active for asymmetric transfer hydrogenation of ketones. Os complexes afforded enantiomerically-pure alcohols with high conversion and enantioselectivity (> 99%) at rates exceeding those of the existing Ru catalyst. Two Os and Ir complexes were explored for the conversion of NADH to its oxidised form (NAD+) under physiologically-relevant conditions. Antiproliferative activities determined in 14 human cell lines correlated with experimentally-determined hydrophobicities. Typically, Os catalysts were found to be more active than their Ir counterparts, though were internalised by cancer cells to a lesser degree, suggestive of a more potent in-cell mechanism of action. Structural modifications identified an apparent inert site of substitution on the sulfonamide substituent. Furthermore, their potency towards cancer cells was increased in combination with L-buthionine sulfoximine, an inhibitor of glutathione synthesis. Acute in vivo toxicities were determined in zebrafish, and all compounds investigated exhibited lower toxicities than the Pt anticancer drug, cisplatin. The complexes were shown to generate reactive oxygen species (ROS) in cancer cells, and similarly generated ROS in zebrafish. Transfer hydrogenation catalysis was explored under physiologically-relevant conditions using sodium formate as a biologically-compatible hydride source. Osmium complexes catalysed the reduction of pyruvate, a key metabolite in cells, to either L-lactate or D-lactate, selectively (ca. 83% ee). Upon co-administration of the catalyst and sodium formate, cancer cell proliferation was decreased by up to 13× (relative to cells treated with the catalyst alone), while no sodium formate effect was determined in non-cancerous cells. Importantly, the treatment of cells with a particular enantiomer of the Os catalyst and sodium formate facilitated the in cell reduction of pyruvate to D-lactate, providing, to the best of my knowledge, the first example of a synthetic catalyst carrying out asymmetric transfer hydrogenation chemistry in cells.

The 2-picoline (2-methylpyridine) complex, cis-[PtCl₂(NH₃)(2-pic)] (AMD473), is promising new generation platinum antitumour agent currently in clinical trials and highly active cisplatin resistant cell-lines. The antitumour activity of trans platinum complexes has attracted renewed interest since it has been shown that some trans compounds, in particular those possessing planar amine ligands, are anticancer-active. Therefore, three trans isomers, trans-[PtCl₂(NH­₃)(2-pic)] (1), trans-[PtCl₂(NH₃)(3-pic)] (2) and trans-[PtCl₂(NH₃)(4-pic)] (3), were synthesised and characterised. The crystal structure of 1 shows steric hindrance induced by the 2-methyl group towards an axial approach to Pt, while its 3-pic (2) and 4-pic (3) analogues are less sterically hindered. Notable however, is that in the solid state complex 1 is less sterically-hindered than its cis isomer. ¹⁵N-labelling of complexes 1-3 allowed both the hydrolysis rates and pK_a values of the complexes to be determined using 2D[¹H, ¹⁵N] NMR spectroscopy. Adducts of cis- and trans-(PtCl₂(NH₃)(2-pic)] with neutral 9-ethylguanine (9-EtGH) and anionic (N1-deprotonated) 9-ethylguanine (9-EtG) were prepared and their structures determined by X-ray crystallography. Platinum is coordinated at the guanine N7 position with a head-to-tail arrangement of the bases in all cases. Two of the complexes exhibited intermolecular triple hydrogen bonding between neutral and deprotonated guanine ligands. In addition, adducts of cis- and trans-[PtCl­₂(NH₃)(2-pic)] with guanosine and 2’-deoxyguanosine were prepared and characterised in solution by NMR spectroscopy and ESI mass spectrometry. The complexes cis-[Pt(NH₃)(2-pic)(Guo)₂]²⁺, and cis- and trans-(Pt(NH₃)(2-pic)(2’-dGuo)₂]²⁺ were assigned as head-to-tail conformations, on the basis of their NOE cross-peaks. The reaction of cis-[Pt(¹⁵NH₃)(2-pic)(OH₂)₂]²⁺ and guanosine (Guo) was followed by 2D [¹H, ¹⁵N] NMR spectroscopy and was found to proceed through two mono(guanosine) intermediate species to yield the dominant product cis-[Pt(¹⁵NH₃)(2-pic)(Guo)₂]²⁺. Initial guanosine substitution trans to 2-picoline was faster than substitution cis to 2-picoline due to steric hindrance, but the rates of the second guanosine substitution were similar. The binding of ¹⁵N-labelled-1 to a self-complementary DNA duplex, d(TATGGTACCATA)₂, was investigated using 1D ¹H and 2D [¹H, ¹⁵H] NMR spectroscopy. The first aquation step appeared to be the rate-limiting step in the formation of the monofunctional adducts. Several DNA products were observed but could not be identified unambiguously. The rate constants for reactions between ¹⁵N-laabelled 1 and guanosine 5’-monophosphate (5’-GMP) were determined via 2D NMR studies, and compared to those previously reported for cis-[PtCl₂(NH₃)(2-pic)].

Cisplatin has been used to treat various types of cancers for over 30 years, however, a number of serious side-effects of cisplatin have stimulated the quest for other metal-based anticancer agents. Iridium complexes are generally thought to be too inert to possess high reactivity, and therefore, there are only a few previous reports of the antitumour activity of iridium complexes. In this thesis a wide range of organometallic IrIII cyclopentadienyl complexes of the type [(η5-Cpx)Ir(XY)Cl]0/+ (where Cpx = pentamethylcyclopentadienyl (Cp*), tetramethyl(phenyl)cyclopentadienyl (Cpxph) or tetramethyl(biphenyl)cyclopentadienyl (Cpxbiph), XY = N,N-, N,O- or C N-chelating ligand) has been synthesised and characterised. All the complexes hydrolyse rapidly in aqueous solution. Complexes with N,N-chelating ligands readily form adducts with 9-ethylguanine but not 9-ethyladenine; C N- or N,O-chelated complexes bind to both purines. Guanine residues are preferential binding sites for 1,10-phenanthroline complexes on plasmid DNA. Replacement of the neutral N,N-bound chelating ligand by the negatively-charged C,N-bound analogues can improve biological activity. In addition, cytotoxic potency towards A2780 human ovarian cancer cells increases with phenyl substitution on Cp*: Cpxbiph > Cpxph > Cp*. This can be rationalised by increased hydrophobicity with more extended phenyl ring, resulting in increased cellular uptake and increased intercalative ability. Notably, several complexes exhibited submicromolar anticancer activity. The interconversion of 1,4-NADH and NAD+ through hydride-transfer reactions in the presence of cyclopentadienyl IrIII aqua complexes was studied. It is shown that the IrIII aqua complexes not only converts NAD+ to 1,4-NADH using formate as the hydride source, but can also catalyse the reverse reaction with hydride donation from 1,4-NADH to a iridium centre, recovered by protonation of bound hydride with generation of H2. This work demonstrates how the aqueous chemistry, nucleobase binding and anticancer activity of the IrIII cyclopentadienyl complexes can be controlled and fine-tuned by the modification of the chelating and cyclopentadienyl ligands. The results suggest that this new class of organometalic Ir(III) complexes is well suited for development as anticancer agents.

The platinum-based chemotherapy drugs cisplatin, carboplatin, and oxaliplatin remain, despite their long-term use, as integral components in the treatment of more than 25 different human cancers. As such, shortages in their supply can have serious health and societal impacts on both the outcome and welfare of patients and on the healthcare systems as a whole. As all three drugs are no longer under patent protection, they are supplied in Australia, the U.S. and the U.K. by between four and 17 different pharmaceutical companies, which reduces the risk of drug shortages. Determining the number and impact of platinum drug shortages in various regions of the world is difficult because legislation to monitor shortages has only been passed recently. All three drugs have suffered from shortages since 2017 with the most common shortage being due to discontinuation of the drug by the company. Other causes include production disruptions, changes in customer demand, problems in supply such as transport and storages, and other reasons. The median duration of drug shortage is 22 days (shortest and longest supply shortages are 3 and 79 days, respectively). Shortages appear to be rare in developed western countries and western European countries, but more common in eastern European countries where platinum drugs are never available or are available only half of the time. This project highlights the lack of information available on platinum drug shortages and the end to further examine platinum drug shortages in regions that are more likely to be impacted, such as Africa, south-east Asia, central and southern America, and the Middle East.

Platinum complexes, e.g. cisplatin, oxaliplatin, carboplatin, have been used for near 40 years in clinic as potent anticancer drugs. However, these drugs can cause severe side effects, and hence there is an urgent need to develop other metal based antiproliferative agents. For example, organometallic Ru II/III complexes possess potent anticancer activity but low cytotoxicity towards normal cells, which makes them promising alternatives to currently used anticancer drugs. In this thesis, a series of neutral pseudo-octahedral Ru II-sulfonamidoethylenediamine complexes [(η6-arene)Ru(N,N’)X] where η6-arene is p-cym, biph and benzene, N,N’ is ethylenediamine chelating ligands with sulfonyl substituents (e.g. Ts or Nb) on one terminal N and various functional groups (e.g. Me, Me2, Et, benzyl, 4-fluorobenzyl or naphthalen-2-ylmethyl) on the other terminal N were synthesized and fully characterized, including X-ray crystal structures. These complexes catalyse the reduction of NAD+ regioselectively to 1, 4-NADH using sodium formate as hydride source under biologically relevant conditions. The catalytic efficiency depends markedly on the steric and electronic effects of the N-substituent, with turnover frequencies (TOFs) increasing with the enhancement of bulkiness and electron withdrawing of the substituents, achieving a highest TOF of 12.9 h−1 for complex 10 [(η6-biph)Os(TsEnBz)Cl]. These complexes exhibited antiproliferative activity against A2780 human ovarian cancer cells. Co-administration with sodium formate (2 mM) increased their potency significantly towards A2780 cells. Substituted Ru II sulfonylethylenediamine complexes can also interact rapidly with glutathione (GSH) and N-acetyl-L-cysteine (NAC) to form S-bridged [(η6-arene)2Ru2(GS)3]2+ or [(η6-arene)2Ru2(NAC)3]+ dimers, and the presence of GSH can effectively hamper the catalytic reduction of NAD+ to NADH. Next, a series of neutral organometallic tethered [Ru(η6-benzene-N-R-ethylenediamine)Cl] complexes was synthesized and characterized, where R = methylsulfonyl (Ms), toluenesulfonyl (Ts), trifluorobenzenesulfonyl (Tf) and 4-nitrobenzenesulfonyl (Nb), including their X-ray crystal structures. In general, these complexes also exhibited potent catalytic activity in the transfer hydrogenation of NAD+ to NADH with formate as hydride donor (310 K, pH 7), but with moderate antiproliferative activity towards human ovarian, lung, liver and breast cancer cell lines. Tethered Ru II complexes showed preferential binding to 9-ethylguanine (9-EG) over adenosine 5’-monophosphate (5’-AMP). However, DNA appears not to be the target, as little binding of complex 17 [Ru(η6-benzene-N-Ts-ethylenediamine)Cl] to ct-DNA or bacterial plasmid DNA was observed. Also, the tethered complexes bind rapidly to GSH, which might again hamper the transfer hydrogenation reactions in cells. Interestingly, these tethered Ru II complexes can induce a dose-dependent G1 cell cycle arrest and high level of reactive oxygen species (ROS) generation, which is likely to contribute to their antiproliferative activity. Diseases caused by bacterial infections, especially by multidrug-resistance bacteria, are the major cause of deaths worldwide. Traditional clinical drugs cannot cope with the rapid rise of drug resistance. In this thesis, a new class of organometallic antimicrobial complexes of the type [(arene/Cpx)Ir(Big)Z]Z (where arene is para-cymene or biphenyl, Cpx = Cp* (tetramethylcyclopentadienyl), Cp*ph or Cp*biph, Big = biguanide ligands and functional sulfonyl substituted biguanide ligands, Z = Cl, Br and I) were synthesized and characterized by NMR, ESI-MS, elemental analysis and X-ray crystallography. These complexes not only have promising antibacterial activity against Gram-negative bacteria and excellent potency against Gram-positive bacteria, but also exhibit high antifungal potency towards C. albicans and C. neoformans. Most of the complexes have low cytotoxicity towards mammalian cells (HEK-293 human red blood cells and HaCaT keratinocyte cells), indicating a high selectivity. These Ir III complexes have a high stability in both medium even at high temperature (315 K). A mutant generation study suggests that S. aureus exhibits a low tendency to generate mutants in response to these complexes. Ir biguanide complexes 27 [(η5-CpXbiph)Ir(PhBig)]Cl, 30 [(η5-CpXbiph)Ir(TolBig)Cl]Cl and 33 [(η5-CpXbiph)Ir(TsTolBig)Cl] exhibited synergy with clinical drug vancomycin when co-administered in vancomycin-resistant Enterococci (VRE), with the MIC 256× lower at sub-MIC complex concentration. These complexes also exhibited potent anti-biofilm activity against biofilms generated by S. aureus. Potent antimicrobial activity against various microbes might provide an alternative pathway to treat drug resistant nosocomial pathogens.

Photoactive platinum compounds have the potential to reduce some of the debilitating side-effects associated with conventional chemotherapeutics, such as cisplatin. Stable, inert platinum(IV) compounds which are reduced to active platinum(II) species only upon irradiation, could provide a site-specific treatment. The Pt^IV azide complexes, cis, trans, cis-[Pt(N₃)₂(OH)₂(NH₃)₂] and cis, trans-[Pt(en)(N₃)₂, have previously been shown to be stable in the dark but reduced to Pt^II upon irradiation. The synthesis and characterisation of new platinum azide compounds, designed to improve important properties such as solubility and wavelength of absorbance are described here. Complexes which have azide ligands in a trans position were synthesised, the general formula is trans, trans, trans-[Pt(N₃)₂(OH)₂(NH₃)R] where R is NH₃, pyridine, methylamine, ethylamine, thiazole, 2-picoline, 3-picoline, 4-picoline or cyclohexylamine. Several Pt^IV diazido compounds containing chelating aromatic ligands, such as 2,2’-bipyridine and 1,10-phenanthroline were also prepared. Many of the novel compounds synthesised were characterised by X-ray structure determination. The complexes with trans azides generally showed improved water solubility as well as a shift of the main absorbance band towards the visible region, compared to their cis analogues. A transcription mapping study of a fragment of pSP73KB plasmid DNA treated with cis, trans-[Pt(en)(N₃)₂(OH)₂] and visible light, has shown that platination mainly occurs at consecutive guanine bases. The major binding sites were similar to those of cisplatin. No platination was seen in an identical sample which was not irradiated.

The interest in the development of anticancer metal complexes for cancer therapy is growing spurred by the encouraging successful stories of platinum drugs. Osmium arene chlorido complexes had been found to show anticancer activity in vitro. In this thesis, the osmium arene iodido complexes were mainly explored and investigated. It is found that iodido OsII arene complexes with a general structure: [Os(η6- arene)(XY)I]PF6 (XY = p-hydroxy or p-dimethylamino phenylazopyridine, arene = p-cymene or biphenyl) are potently cytotoxic at nanomolar concentrations toward a panel of human cancer cell lines. In contrast to the chlorido osmium arene anticancer complexes, the iodido complexes are stable and inert toward aquation. More than thirty half sandwich azopyridine OsII arene complexes [Os(η6- arene)(azopyridine)X]+ (where X is chloride or iodide, the arene is p-cymene or biphenyl and the pyridine ring of azopyridine ligand bearing a variety of substituents) were synthesized and characterized. A preliminary structure activity relationships (SARs) were built up based on the anticancer activity towards A2780 human ovarian cancer cell line. In general, the introduction of an electronwithdrawing group (e.g. F, Cl, Br or I) at specific positions on the pyridine ring significantly increases cytotoxic activity and aqueous solubility. Changing the arene from p-cymene to biphenyl or the monodentate ligand (X) from chloride to iodide resulted in a significant increase in the anticancer activity. Studies in A2780 human ovarian cancer cells suggested that cellular uptake and targeting to cellular organelles play important roles in determining the anticancer activity. According to the 60 cancer cell lines screening results from National Cancer Institute (NCI), the anticancer activity achieved by the most potent OsII arene azopyridine complex is 100 times more than cisplatin; 1000 times activity was found in some cell lines. The mechanism of action may involve the inhibition of tubulin polymerization. One iodido osmium complex was selected for anticancer efficiency evaluation in vivo: [Os(η6-p-cym)(Azpy-NMe2)I]PF6 (FY026). This complex delayed the growth of HCT116 human colon cancer xenografts in mice, with negligible toxicity. It is the first example of in vivo antitumour activity for an organometallic osmium arene complex. Its activity appears to involve redox mechanisms. Its potency towards A2780 ovarian and A549 lung cancer cells is increased significantly when used in combination with L-buthionine-sulfoximine (L-BSO) indicating that L-BSO can be a good candidate for combination therapy treatment with iodido osmium complexes. Further study on the bioisosteres of FY026 was carried out by changing the azo bond (N=N) to imine bond (CH=N). Sixteen osmium arene iminopyridine complexes were synthesized, well characterized and showed good anticancer activity. Different structure-activity relationships comparing iminopyridine complexes with azopyridine complexes were identified which suggested a different anticancer mechanism. In contrast to FY026, [Os(η6-p-cym)(Impy- NMe2)I]PF6 (6) and [Os(η6-p-cym)(Impy-NMe2)Cl]PF6 (14) were found to undergo hydrolysis and the binding was observed between their hydrolyzed product (14A) and 9-ethylguanine. Moreover, a hydride transfer from NADH to form an osmium hydride intermediate which is involved in a catalytic process resulting in the formation of NAD+ was discovered. This process might be involved in the anticancer mechanism of action. A dual mechanism of action was proposed based in the interaction of these compounds with DNA nucleobase and catalytic oxidation of NADH.

This thesis is concerned with the design of ruthenium(II) arene anticancer complexes where the chelating ligand contains an azo-imine group (­N=N-C=N) which binds to the ruthenium to form a five-membered chelate ring. Several mononuclear complexes containing the chelating 2-phenylazopyridine ligand or derivatives thereof were found to be moderately cytotoxic towards A2780 human ovarian and A549 human lung cancer cells (IC₅₀ 18-88 μM). These complexes were found to hydrolase (Ru-C1 → Ru-OH₂) slowly in aqueous solution and arene loss was a competing reaction. X-ray crystal structures revealed that the arene ligand is not as tightly bound to the ruthenium as in the case when the chelating ligand is en. Synthesis of the corresponding dinuclear analogues, where two 2-phenylazopyridine ligands were joined together via a linker, did not improve the cytotoxicity. Unlike their mononuclear analogues, the complexes appeared to undergo electrochemical reduction with radical formation in aqueous solution. An EPR spectrum of the mono-reduced anion revealed an entirely ligand based radical. Surprisingly, replacement of Cl^- by I^- in the mononuclear complexes led to compounds that were highly cytotoxic towards both A2780 and A549 cancer cells (IC₅₀ 1-5 μM). Interestingly these complexes were resistant to hydrolysis in aqueous solution suggesting that this class of compounds had a novel mechanism of cytotoxic action not involving activation by hydrolysis. The redox chemistry of these complexes proved to be central to their observed cytotoxicity. The complexes were rapidly reduced by the biological reductant ascorbate, catalytically oxidised the tripeptide glutathione and generated reactive oxygen species (radicals) inside A549 cancer cells. These reactive oxygen species were shown to be involved in cell death. These iodide complexes were also found to be cytotoxic towards non-cancerous WI38 human lung cells (IC₅₀ 1-6 μM) and attempts were made to target one complex specifically to cancer cells, through conjugation to transferrin.

In this work, trans-diazido Pt(IV) complexes with general formula [Pt(N3)2(OH)(OCOR)(pyr)2] (where OCOR is a carboxylate axial ligand) and [Pt(N3)2(OH)2(L1)(L2)] (where L1 and L2 are aromatic N-heterocyclic ligands) have been synthesised and characterised. The chemical and photochemical properties of these complexes, as well as their photobiological behaviour, have been studied in order to check their potential as photoactivatable anticancer drugs. Four trans-diazido Pt(IV) complexes with general formula trans, trans, trans- [Pt(N3)2(OH)(OCOR)(pyr)2] (OCOR= succinate, 4-oxo-4-propoxybutanoate, Nmethylisatoate and succinate-(RGD)f peptide ligands) have been obtained by axial derivatisation of one hydroxido ligand from trans, trans, trans- [Pt(N3)2(OH)2(pyr)2]. The crystal structures of three axially-derivatised complexes have been determined by X-ray diffraction. Photoirradiation studies have shown an improved photoactivity of the carboxylate versus the dihydroxido complexes at the longer wavelengths. Release of the axial ligands was observed in the studied complexes. This fact is especially relevant in the case of the Pt(IV)-(cRGD)f complex, where the RGD was incorporated as a tumour cell targeting moiety. DFT-TDDFT calculations performed on the complex trans, trans, trans- [Pt(N3)2(OH)(Succ)(pyr)2] showed dissociative transitions at longer wavelength, which could explain the photolability observed in these carboxylate derivatives. Studies of photoactivation of the diazido Pt(IV) complexes in the presence of 5’- GMP indicate the formation of a mono-GMP Pt(II) adduct as main photoproduct, therefore DNA could be considered a potential target site for these anticancer compounds. Additionally, EPR studies showed that azidyl radical release was observed when complexes bearing the succinate and 4-oxo-4-propoxybutanoate ligands were irradiated with green light. No such result was obtained for the dihydroxo precursor showing that these complexes could be phototoxic with longer wavelength light activation. Seven trans-diazido Pt(IV) complexes, trans, trans, trans- [Pt(N3)2(OH)2(L1)(L2)] (where L1 and L2 are pyridine, 2-picoline, 3-picoline, 4- picoline, thiazole or 1-methylimidazole ligands), have been obtained by oxidation of the corresponding trans-[Pt(N3)2(L1)(L2)] precursor. The X-ray crystal structures have been determined for four Pt(IV) diazido complexes from this family of compounds. Photoirradiation studies indicate that the incorporation of a sterically demanding ligand, e.g. trans, trans, trans-[Pt(N3)2(OH)2(2-pic)(pyr)], greatly enhances the photoactivity in these complexes. DFT-TDDFT calculations are in agreement with these results, since higher intensity transitions were observed for such complex at longer wavelength. Phototoxicity studies carried out on A2780, A2780cis and OE19 cell lines with the trans, trans, trans-[Pt(N3)2(OH)2(pyridine)(n-picoline)] family concluded that steric hindrance close to the platinum centre does not favour phototoxicity. Most of the complexes were equally potent in cisplatin resistance against the ovarian cancer cell line (A2780cis), except [Pt(N3)2(OH)2(3-pic)2] and [Pt(N3)2(OH)2(4-pic)2] which exhibited some cross resistance. All of the complexes tested in both OE19 and A2780 cell lines have shown less sensitivity to OE19 than to A2780. Studies in S. pombe yeast strains (WT and ΔRad3) with trans, trans, trans-[Pt(N3)2(OH)2(pyr)2] suggest that DNA is potentially an important target for this type of compounds, although other targets are not excluded. Furthermore, live-cell confocal microscopy was performed on A2780 cells treated with the complex trans, trans, trans-[Pt(N3)2(OH)2(pyr)2] and irradiated with a low dose of blue light. The cell death, monitored by propidium iodide uptake, was captured occurring 2 h 30 min post activation.

Modifications of the potent anticancer agent taxol were carried out in order to gain an understanding of the chemical reactivity of the drug and the factors which contribute to its biological activity. The C-2' and/or the C-7 hydroxyl groups of taxol were substituted with acetyl, ßalanyl, silyl, succinyl, trichloroethyloxycarbonyl or carbonate linked dibenzylidene protected glucosyl groups. The C-7 position was selectively epimerized under free radical conditions and a 2'-epiacetyltaxol was produced via base catalysed epimerization. The C-2' amide became nucleophilic in the presence of base and could attack a C-2; acyl substituent. The C-13 ester side chain was selectively reduced by borohydride. The 7 position of taxol was selectively oxidized by Jones reagent and longer reaction times also oxidized the 2' position. The D rings of the 7 oxotaxols were readily opened via beta elimination; hydrogenation of the double bond in the enone of the D seco products produced a product in which the C ring was opened. The D ring was also susceptible to electrophilic attack. Reaction of taxol with triethyloxonium tetrafluoroborate or acetyl chloride/HC1 produced D seco taxols. C-7 deoxygenation was not achieved due to steric hindrance at C-7 and the instability of taxol under free radical conditions. Biological testing of modified taxols showed that substitution of the C-2' hydroxyl removed biological activity but that C-2' acyl groups were readily removed vivo. The water soluble 2'-βalanyItaxol possessed in vivo activity equal to that of taxol. Substitution of the C-7 hydroxyl did not inhibit the ability of a taxol derivative to polymerize tubulin but did decrease in vivo activity; epimerization of C-7 decreased in vivo activity slightly. A 2'-oxotaxol was found to be less active than, but still comparable to, its nonoxidized analogue. All taxol derivatives having a 7-oxo group and/or not possessing a D ring lost almost all biological activity.
Ph. D.

The immune system plays a critical role in disease prevention via collaboration between the adaptive and innate immune responses. Due to a variety of immune cell subpopulations with different functions including opposing roles, the tumour microenvironment provides opportunities as a therapeutic target for anticancer drug development. Recently the essential oils and their extracts from plants have been gaining attention in cancer research, due to their antioxidant and anti-inflammatory activities making them good candidates as sources of novel anticancer compounds. The essential oil of Melaleuca Alternifolia and its derived products have been previously reported to cause tumour regression, accompanied by an accumulation of neutrophils at treatment sites in various studies. Therefore, the present study was aimed at investigating the immunological responses induced by intratumoural injection of Melaleuca Alternifolia Concentrate (MAC) into subcutaneous tumours of the female transgenic FVB/N c-neu spontaneous murine model of breast cancer, particularly focusing on the role of neutrophils. Following emerging evidence demonstrating the anticancer activity of neutrophils under different stimulants, the present study investigated the immune responses induced by directly injecting MAC into solid tumours of transgenic FVB/N c-neu mice. 4 % v/v MAC was injected directly into the subcutaneous tumours of the female mice and significantly slowed growth of tumours with no observable side effects. The results showed increased levels of neutrophils purified from the treated tumours as compared to vehicle control and untreated control tumours. The results were consistent with previous studies which reported an increased influx of neutrophils into the tumours following MAC injections. The present study further characterised the neutrophils isolated from treated tumours, and the results showed a phenotypic diversity of neutrophils isolated from tumours mostly containing a hypersegmented nucleus constituting the bulk (62.76 %) of the cytotoxic high-density neutrophils. Emerging evidence has indicated the phenotypic diversity and heterogeneity of neutrophils in cancer. Furthermore, this study evaluated the cytotoxicity of the neutrophils isolated from treated tumours, ex vivo. Neutrophils isolated from tumours treated with 4 % v/v MAC showed cytotoxicity towards NeuTL murine breast cancer cells in vitro and effective killing by neutrophils at effector-to-target ratios from10:1 down to 1.25:1 (p<0.001). In summary, the present study found that 4 % v/v MAC impairs the growth of tumours and with no observable toxicity in mice. Also, neutrophils activated in response to treatment of FVB/N tumours with MAC are cytotoxic for NeuTL murine breast cancer cells and are characterised by a hypersegmented nuclear morphology. At present, literature on specific anti-tumorigenic roles played by TANs in the tumour microenvironment is limited. Thus, data from this study contributes towards identification of specific roles and mechanisms underlying the chemoattraction of TANs into the tumour microenvironment and more importantly in response to MAC intervention.
Thesis (Masters)
Master of Medical Research (MMedRes)
School of Medical Science
Griffith Health
Full Text

Several members of the schweinfurthin family are potent and selective inhibitors of cancer cell growth in the NCI 60-cell cancer screen. The schweinfurthins display unique activity in this screen which suggests that these compounds have a previously unexploited target. This activity encourages development of the schweinfurthins as anti-cancer agents; however, the scarcity of the natural products has hindered biological evaluation. For this reason, a program was initiated to synthesize the natural products and analogues thereof. These efforts have made 5 natural product schweinfurthins and over 60 analogues available for biological evaluation. Studies presented herein evaluate the biological activity, intracellular localization, and potential protein interactions of schweinfurthins analogues. To investigate the potent and differential effects of these compounds, a two-cell line screen was developed incorporating a sensitive cell (SF-295) and insensitive cell line (A549). The structure-function data obtained from this screen identified a schweinfurthin site amenable to modification. Based on this finding, fluorescent schweinfurthins were designed and synthesized. These compounds retain schweinfurthin-like potency and activity which is critical for their study. The intracellular localization of these schweinfurthins was investigated using fluorescence microscopy. These studies identified the lysosomes and peri-nuclear organelles as potential sites of schweinfurthin activity. Additionally, we describe the attachment of a fluorescent schweinfurthin to a solid support, which was utilized to identify protein-schweinfurthin interactions. This approach identified vimentin as a potential target of schweinfurthin activity.

Quinones attract the attention of chemists and biologists because of their unique structure and biological properties. Cancer is a leading cause of death worldwide, killing millions of people every year. Several antitumour agents, frequently employed in the treatment of different forms of cancer, contain in their structure the quinone motif, discovered to have marked cytotoxic effects. Chapter 1 presents an overview of the unique properties of quinones, with particular emphasis on their anticancer activity. Bioactivation of quinones by cellular reductive enzymes generates reactive oxygen species and/or alkylating species responsible for the cytotoxic effects of these compounds. Chapter 2 discusses the synthetic approaches to the novel quinone-containing natural product (+ )-terreusinone, recently isolated from the marine algicolous fungus Aspergillus terreus. Although the total synthesis of the natural product could not be accomplished, an innovative and efficient symmetric strategy, with a double Claisen rearrangement as the key step, led to the formation of the dipyrrolobenzoquinone core of ( + )-terreusinone. Chapter 3 describes the design, the synthesis and the biological evaluation of novel 2-unsubstituted indolequinones based on the structure of the indolequinones, previously synthesised in our group, that showed growth inhibitory activity against human pancreatic cancer cells in vitro and in vivo. Human thioredoxin reductase 1, a selenium-containing enzyme, was identified as the molecular target of these indolequinones. All the novel indolequinones made were tested for their anticancer activity in vitro against human pancreatic cancer and some of them were found very potent compounds at inducing inhibition of growth in human pancreatic cancer cells, confirming the potential use of these indolequinone-based compounds as a therapeutic approach to pancreatic cancer.

Our studies on the synthesis and biological evaluation of novel anticancer drugs consist of three research areas; namely, synthesis of Mitogen Activated Protein (MAP) kinase inhibitors, Checkpoint (Chk1) inhibitors and nordihydroguaiaretic acid (NDGA) analogues. The first research area involved synthesis of MAP kinase inhibitors. MAP kinases are a family of serine I and threonine II kinases which can act together to generate a process of phosphorylation events within the cell signalling pathway leading eventually to cell division. The compounds made in this project were specifically designed to target the stress related kinases, a MAP kinase pathway which controls the expression of genes involved in cell proliferation. The stress related kinases are known to have serine or threonine joined to a proline III residue. In an attempt to prepare selective inhibitors of stress related kinases, compounds of types IV and V were deigned in which a conformationally restricted serine analogue is joined to L-proline via an amide link in one of two possible ways. Examples of these two sets of compounds were synthesised and those that were tested by Professor David Gillespie at the Beatson Institute for Cancer Research, Glasgow were shown not to be inhibitors of these kinases. (Fig. 1144A) The second research area concentrated on the checkpoint signalling pathway. Components in the DNA damage checkpoint signalling pathway such as ChK1 could be potential targets for chemical intervention. Caffeine VI and pentoxifylline VII have been shown to sensitise p53-deficient tumour cells to killing by DNA damage. We envisaged that the xanthine derivatives, caffeine VI and pentoxifylline VII might also disrupt the G2 checkpoint by preventing activation of Chk1. To test his hypothesis, a range of xanthine derivatives shown below were prepared by alkylation of theophylline VIII or theobromine IX. (Fig. 1144B) The biological evaluation of these xanthine derivatives by Professor Gillespie revealed that three of these compounds, X, XI and XII, suppressed G2/M arrest very effectively. All three active compounds possess a long aliphatic chain that provides a large degree of flexibility to the structures. The long aliphatic chains could bind to a hydrophobic pocket in the enzyme’s active site that might confer selectivity on the compounds. (Fig. 1144C) The third area, synthesis of NDGA analogues, was the major part of the synthetic work. NDGA XIII is known to be a selective inhibitor of lipoxygenase and blocks small cell lung cancer growth in vitro and in vivo. In addition to its lipoxygenase activity, NDGA was demonstrated to inhibit c-kit, a tyrosine kinase that has been observed preferentially in SCLC. The main drawbacks to the use of NDGA in cancer treatment are its poor solubility and moderate potency. Therefore chemical modifications are required to provide better compounds for clinical use. Preliminary work in our group was performed by McDonald and Macleod. They synthesised a range of analogues of NDGA which were tested for their activity in vitro by Professor Michael Seckl at the Medical Oncology Department of Hammersmith Hospital, London. Improved potency over NDGA for new analogues with 4-6 atoms between the two aromatic rings was observed. Furthermore introduction of an amide linkage between the two aromatic residues resulted in NDGA analogues which are more active than NDGA. Based on these preliminary results, the structural modifications proposed for this project focused on three areas. The main programme of research was drug solubilisation of new analogues which have higher potency than NDGA for in vivo work. The second area of study sought to introduce position variations of the amide linkage between the two aromatic residues. The third area of work involved modification of the substituents on the two aromatic rings. (Fig. 1144D) A range of NDGA analogues was successfully synthesised and evaluated for anticancer activity in vitro. Compounds XIV and XV were confirmed as lead compounds which are ten times more active than NDGA. Compound XIV was successfully transformed into a water soluble form XVI which is now available for in vivo work. In addition NDGA was converted into a water soluble form which was more potent than NDGA in vitro. Moreover a NDGA analogue XVII with no free hydroxy groups was found to be as active as NDGA, which was an unexpected discovery. (Fig. 1144E)

In this thesis the biological activity and aqueous solution chemistry of half-sandwich Os¹¹ arene complexes of the type [(η⁶-arene)Os(XY)C1] is explored, and it is demonstrated that these properties can be tuned by careful choice of XY chelating ligand (N,N-, O,O- and N,O-chelates) to achieve cancer cytotoxicity comparable to carboplatin. The osmium complexes containing N,N-chelates hydrolyse more slowly than their ruthenium analogues and the pK_a of the resulting water is more acidic. Efforts to increase the rates of hydrolysis and the resulting pK_a led to replacement of the neutral N,N-chelating ligand by an anionic O,O-chelate. This was successful in that hydrolysis is more rapid and the pK_a of the coordinated water has increased by ca 0.8 units. However, these complexes are deactivated by formation of the inert and thermodynamically stable hydroxo-bridged dimers. Attempts to tune the stability of complexes containing XY = O,O-chelate, by replacing the 6-membered O,O-chelate with 5-membered analogues, was partially successful for the development of active complexes, but was unsuccessful in preventing hydroxo-bridged dimer formation. Within the class of N,N- and N,O-chelated complexes the choice of donor group is important. Replacing amine N-donor groups with the Π-acceptor pyridine, reduced both the rate of hydrolysis and pK_a or coordinated water, and increased the overall stability of the complex. This was especially the case for complexes containing N,O-chelates, which displayed aqueous chemistry in between that of the parent compounds containing neutral N,N-or anionic O,O-chelates. Within this group of osmium arene complexes, [(η⁶-arene)Os(N,O)C1], active cytotoxic complexes were obtained, and the first X-ray crystal structures of osmium bound to either G or A nucleobases is reported. This work shows that a wide range of reactivity can be obtained for complexes of the form [(η⁶-arene)Os(XY)C1]ⁿ⁺ by careful choice of the XY chelating ligand, and this knowledge has allowed complexes with cancer cell cytotoxicity to be designed.

Bien que les agents anticancéreux à base de platine soient bien établis en clinique, plusieurs paramètres restent à optimiser, notamment leur toxicité et les phénomènes de résistances. La combinaison platine-carbènes N-hétérocycliques (NHCs) a montré des résultats prometteurs dans la lutte antitumorale. Ainsi, afin de développer des agents anticancéreux innovants, ce travail résume les modifications structurales étudiées dans le but d’augmenter la cytotoxicité tout en réduisant les effets secondaires des complexes. Une stratégie développée ici porte sur l’introduction de pnictogènes par échange de ligand pour observer un effet synergétique. Autrement, les efforts se sont concentrés sur les complexes de NHC-platine(IV) de leur synthèse à l’étude de leur stabilité, activité anticancéreuse et mécanisme d’action. Finalement, la combinaison des NHC-Pt à des nanotransporteurs a été étudiée afin d’améliorer leur biocompatibilité et sélectivité
Although platinum-based anticancer drugs are well established, several shortcomings have raised concerns, namely their toxicity and resistance mechanisms. Therefore, improved anticancer drugs are strongly awaited to substitute drugs currently used in clinics. Remarkably, the combination of N-Heterocyclic Carbenes (NHCs) to platinum has recently demonstrated very promising results as anticancer agents. In the aim to access novel drugs, this work emphasizes several structural modifications to improve the cytotoxicity and lower side effects. One strategy developed herein focus on the introduction of pnictogens by ligand exchange to access a synergistic effect. Otherwise, efforts mainly focused on NHC-platinum(IV) complexes from their synthesis to stability investigation and anticancer activities and mechanism of action. Finally, the combination of NHC-Pt drugs to nanodelivery devices has been investigated in order to improve both their biocompatibility and selectivity toward cancer cells

This thesis is concerned with the design of half-sandwich iridium(III) complexes of the type [(η5-Cp*)Ir(2-(Rˈ-phenyl)pyridine-R)X]0/+ (Cp* = pentamethylcyclopentadienyl), X = Cl- or pyridine derivatives) as anticancer agents, with particular focus on the effects that functionality in the chelating and monodentate ligands has on their chemical and biological properties. A set of phenyliminopyridyl (ImPy) complexes of the type [(η5-Cpx)Ir(ImPy)Cl]PF6 (Cpx = Cp*, tetramethyl(phenyl)-cyclopentadienyl (CpxPh) or tetramethyl(biphenyl)- cyclopentadienyl (CpxBiPh) was also synthesised and their solution chemistry and potential applications as antibacterial and anticancer agents investigated. Electron donating (-CH3, -OH, -CH2OH and –OCH3) or electron withdrawing (-F, -CF3 -CHO and –NO2) groups were introduced to various positions on the 2-PhPy ligand, giving rise to seventeen complexes [(η5-Cp*)Ir(2-(Rˈ- phenyl)pyridine-R)Cl]. Three X-ray crystal structures were determined, showing the expected pseudo-octahedral configuration. The functional groups have a profound effect on the resulting anticancer activities against a range of cell lines. Some complexes showed activity against A2780 human ovarian cancer cells comparable to cisplatin, and similar activity to [(η5-CpxPh)Ir(2-phenyl)pyridine)Cl]. The complexes all show similar extents of hydrolysis. The complexes preferentially bind to the model nucleobase 9-EtG over 9-MeA, and show the ability to catalytically oxidise the coenzyme NADH to NAD+. Contrasting anticancer activities were found for structural isomers. The hydrophobicity is related to substituent type and position on the ligand. The more hydrophobic complexes accumulated in A2780 cells to a greater extent. The extent of accumulation appeared to correlate with the potency of the complexes. The most potent complex [(η5-Cp*)Ir(2-(2ˈ-methylphenyl)pyridine)Cl] 13 (IC50 = 1.18 μM against A2780 cells) was modified via replacement of the chlorido monodentate ligand with pyridine derivatives (18 = pyridine (Py), 19 = 4- dimethylaminopyridine (Py-NMe2), 20 = 4-trifluoromethylpyridine (Py-CF3). The X-ray crystal structure was determined for 19. These pyridine complexes show less monodentate ligand release in aqueous solution and less binding to 9-EtG than parent complex 13. Complexes 13 and 19 show mild catalytic activity towards the oxidation of NADH to NAD+. Reactivity towards glutathione (GSH) decreased only in the case of 19 when compared with the parent complex 13. The reaction products include [(η5-Cp*)Ir(2-(2ˈ-methylphenyl)pyridine)(S-O)G] (sulfenate) and [(η5- Cp*)Ir(2-(2ˈ-methylphenyl)pyridine)(S-O2)G] (sulfinate) which are reliant on the presence of O2. The antiproliferative activity against A2780 cells increased upon enhancement of stability at the monodentate site (19 > 18 > 20 ~ 13) when activity was measured with no cell recovery time, with 19 exhibiting nanomolar activity (IC50 = 650 nM). All of the complexes induced high levels of total reactive oxygen species (ROS). Apoptotic cell death after 24 h recovery time was only observed for the pyridyl complexes. The ability to functionalise 2-PhPy complexes via Schiff base formation was examined using the aldehyde-containing complex [(η5-Cp*)Ir(2-phenyl-5- pyridinecarboxaldehyde)Cl] 6, and new conjugates were synthesised by conjugation to primary amines. Reactions with lysine-containing peptides were analysed by ultrahigh resolution mass spectrometry (UHR-MS) techniques which showed formation of new iridium-peptide conjugates via Schiff base formation with the free amino group of lysine. The imine bond in a complex bearing the fluorescent dansyl moiety (for fluorescence microscopy) was reduced using the hydride source Et3SiH, which appears to involve the metal centre through proposed formation of an Ir-H species. Eighteen [(η5-Cpx)Ir(ImPy)Cl]PF6 complexes with various ImPy ligands were synthesised and characterised, and six X-ray crystal structures were reported. They were found to exhibit more complex aqueous chemistry than the 2-PhPy complexes, but exhibit minimal hydrolysis at biologically-relevant concentrations of chloride, remaining predominantly as the chlorido species. The hydrophobicity of the complexes increased upon extension of the Cpx capping ligand: CpxBiPh > CpxPh > Cp*. The complexes show minimal activity against Gram-negative E. coli but CpxBiPh complexes show good activity (MIC = 8 – 15 μM) against Gram-positive S. aureus bacteria. The antibacterial activity is generally dependent on the hydrophobicity and extension of the Cpx capping ligand. Disruption of the bacterial cell membrane appears to be involved in the mechanism of action. The antiproliferative activity against A2780 cells follows the trend CpxBiPh > CpxPh > Cp*, where the complex [(η5-CpBiPh)Ir(ImPy-NMe2)Cl]PF6 31 exhibits an IC50 value against A2780 cells of 640 nM.

The importance of Fe(III)-chelation in the biological activity of the anticancer pharmaceutical Razoxane has been investigated. Since Razoxane is known to be hydrolysed in vivo to form a potential complexing agent, the observed biological effect o(Razoxane (ICRF 159) and two less active homologues (ICRF 154 and ICRF 192) were compared with the properties of their hydrolysis products; ICRF 198 (N ,N'-dicarboxamidomethy 1-N ,N'-dicarboxymethyl-1,2-diaminopropane, ICRF 175 (N,N'-dicarboxamidomethyl-N,N'-dicarboxymethyl-1,2-diamino-ethane) and ICRF 226 (N,N'-dicarboxarnidomethyl-N,N'-dicarboxymethyl-1,2-diaminobutane), respectively. The equilibrium constants for the binding of these compounds with Fe(III) were determined by cyclic voltammetry and potentiometric titration. In addition, equilibrium constants of ICRF 175 and 198 binding with Zn(II) and of citrate binding with Fe(III) were determined. Relevant equilibrium constants for the binding of the proton with these complexing agents were also obtained. These constants were incorporated into a computer simulation model of blood plasma, permitting calculation of the extent of chelation of these agents in vivo. Differences were observed in the ability of the compounds to complex Fe(III) under physiological conditions. Nevertheless, it was concluded that the anticancer activity of Razoxane is not directly related to Fe(III) chelation. The rates of reaction of superoxide (O-2) with the Zn(II), Cu(II) and Fe(III) complexes of EDTA, DTPA, ICRF 175, I 98 and 226 were subsequently measured by pulse radiolysis. Significantly, ICRF 198-Fe(III) was found to react relatively rapidly with superoxide. In addition, the computer simulations indicate that the presence of ICRF 198 would restrict the formation of iron complexes of the anticancer pharmaceutical Adriamycin, these complexes being implicated in its cardiotoxicity. The ability to compete in vivo for iron and to react with superoxide thus appear to be important aspects of the mechanism of action of Razoxane to protect against the clinically-important side-effects of Adriamycin administration.

Carbendazim (methyl-2-benzimidazolecarbamate), a well known anti-fungal agent that may have significant anti-cancer activity, is a poorly water-soluble ampholyte. Unfortunately its projected oral dose up to hundred mg per day is far greater than its water solubility of 6 μg/ml. The overall aim of this research was to conduct preformulation studies and develop therapeutically viable oral and parenteral formulations. The solubility of carbendazim was altered by using both solute and solvent modification approaches. The solvent modification was carried out by investigating the solubilization of carbendazim by pH in combination with cosolvents, surfactants or complexants. At pH 7 the total drug solubility is 6.11 ± 0.45 μg/ml which increases by 1 to 7 fold with cosolvent, surfactant or complexant. However, at pH 2 the solubility increases by 400 times and at pH 1.3 over 3000 times. Both cosolvents and non-ionic surfactants have a negligible effect on the total drug solubility at pH 2, whereas the total drug solubility increases by combining pH 2 with anionic surfactants or complexants. The solute modification was accomplished by preparing different salts of carbendazim. In all six different salts, viz., hydrochloride dihydrate, phosphate, hemisulfate hydrate, mesylate, besylate, and tosylate were prepared. Their structures were determined using single crystal x-ray diffraction. The developability attributes (i.e. hygroscopicity, thermal behavior, aqueous solubility, solid state stability, and dissolution rate) of these compounds were evaluated. The dissolution studies showed that all the salts had better dissolution rate than the free base, the mesylate salt been the best. Based on the preformulation studies, two formulations were chosen for oral dosing in mice. The proposed oral formulation of 9.4 mg/ml carbendazim at pH 1 in phosphate buffer and 20% SBEβCD phosphate buffer, respectively, did not precipitate on in vitro static serial dilution with water and seven up. However, the formulation containing only phosphate buffer showed activity in mice by significantly reducing the tumor growth in B-16 melanoma injected mice. Also, it gave active blood levels, which were comparable to IV dosing.

The Antibody-Drug Conjugates (ADCs) is one of the novel therapeutic strategies with a great potential in many medicinal treatments. ADCs are composed of a monoclonal antibody (mAb) connected to a drug (payload) through a properly designed linker. The mAb is responsible for the selective transport of the drug into target cells expressing the specific antigen; in this way, the off-target toxicity of the drug can be avoided. The linker plays an important role due to its dual features: stability in blood and ability to release the payload into the target cells thanks to its degradation. In this thesis, several different aspects of the ADC development are explored. Different ADCs charged with SMO inhibitors as payload are investigated for the treatment of melanoma, and novel multifunctional linkers with trigger groups are designed for the drug release in different environments in a very elegant and controlled way. Besides the work on bioconjugates aforementioned, micellar catalysis is also investigated to perform catalytic transformations in water media avoiding the use of organic solvents. Micellar catalysis is applied in the presence of a proper rhodium salt to develop a suitable hydroformylation process with very good results.

Metal-based antitumor drugs play a relevant role in antiblastic chemotherapy. Cisplatin is regarded as one of the most effective drugs, even if severe toxicities and drug resistance phenomena limit its clinical use. Therefore, in recent years there has been a rapid expansion in research and development of novel metal-based anticancer drugs to improve clinical effectiveness, to reduce general toxicity and to broaden the spectrum of activity. The variety of metal ion functions in biology has stimulated the development of new metallodrugs other than Pt drugs with the aim to obtain compounds acting via alternative mechanisms of action. This work has been dedicated to the investigation of the behaviour, at cellular, subcellular and molecular level of copper(I) and gold(I) phosphine complexes. Concerning gold(I) complexes, we highlighted their ability of interacting with one of the most important system involved in redox cellular homeostasis, thus creating the pathophysiological conditions stimulating mitochondria to undergo the way of apoptosis. Acting as potent inhibitors of the selenoenzyme thioredoxin reductase, gold complexes determine an alteration of the redox state of the cell leading to an increased production of hydrogen peroxide and oxidation of the components of the thioredoxin system, therefore creating the conditions for augmented apoptosis. Differently, phosphine copper complexes were found to activate an atypical programmed cell death pathway. Indeed, they inhibit tumor cell growth through paraptosis, an alternative non-apoptotic cell death mechanism characterized by a massive cytoplasmatic vacuolization and a consequent ER stress. This different way to induce a programmed cell death appeared particular interesting as these copper complexes may be utilized in inducing cell death even in tumors resistant to apoptosis-inducting therapies.
I complessi metallici ricoprono un ruolo rilevante nella terapia anticancro. Il cisplatino è riconosciuto come uno dei farmaci più attivi, anche se sia la sua tossicità che lo sviluppo di resistenza limitano fortemente il suo impiego clinico. Negli ultimi anni, quindi, la ricerca si è indirizzata verso lo sviluppo di nuovi complessi metallici come potenziali agenti antitumorali, al fine di ottenere composti che presentino maggior efficacia e posseggano una minore tossicità ed un più ampio spettro d’azione. La varietà di funzioni biologiche svolte dagli ioni metallici ha notevolmente stimolato in particolare lo sviluppo di complessi metallici contenenti metalli diversi dal platino, allo scopo di ottenere composti che agiscano attraverso un meccanismo d’azione diverso. In tale ambito, questo lavoro è stato dedicato allo studio gli effetti a livello cellulare, subcellulare e molecolare indotti da complessi fosfinici di oro(I) e rame(I). Per quanto concerne i complessi fosfinici di oro(I), è stato messo in luce la loro capacità di interagire a livello il selenoenzima tioredossina reduttasi, creando condizioni fisiopatologiche in grado di stimolare la via mitocondriale dell’apoptosi in cellule tumorali. I complessi di oro(I), agendo come potenti e selettivi inibitori della tioredossina reduttasi determinano una alterazione dello stato redox cellulare che a sua volta porta all’aumento della produzione cellulare di perossido di idrogeno oltre che all’ossidazione dei componenti del sistema tioredossinico. Questo sbilancio dell’omeostasi cellulare determina induzione della morte cellulare per apoptosi. Diversamente, l’attività antitumorale indotta dai complessi di rame(I) è stata correlata alla loro capacità di indurre una morte cellulare programmata atipica. Infatti, essi inibiscono la crescita cellulare tumorale mediante induzione di paraptosi, un meccanismo di morte cellulare caratterizzato dalla massiccia formazione di vacuoli e da un conseguente stress del reticolo endoplasmatico. L’induzione di morte cellulare mediante un meccanismo diverso da quello dell’apoptosi appare particolarmente interessante poiché questi complessi di rame(I) potrebbero essere impiegati nel trattamento di cellule tumorali che hanno sviluppato numerosi meccanismi al fine di eludere l’induzione di apoptosi.

Thesis (S.M. in Inorganic chemistry)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012.
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references (p. 38-40).
Nucleotide excision repair, a DNA repair mechanism, is the major repair pathway responsible for removal of platinum-based anticancer drugs. In this study, 146 bp duplexes were prepared containing either a site-specific cisdiammineplatinum( Il)-DNA intrastrand d(GpG) cross-link or a cisdiamminephenanthridinechloroplatinum( Il)-DNA dG adduct. Comparison of the repair efficiencies of the two adducts reveals that the diamminephenanthridinechloroplatinum(lI)-DNA dG lesion eludes the nucleotide excision repair pathway better than diammineplatinum(lI)-DNA intrastrand d(GpG) cross-link. A factor that may be relevant to the difference is the influence of platination on DNA-mediated charge transfer. Atomic force microscopy is a method by which we can explore the possibility that phenanthriplatin influences charge transfer properties of DNA. Long DNA duplexes site-specifically modified with cisplatin or phenthanriplatin were prepared for AFM studies.
by Meiyi Li.
S.M.in Inorganic chemistry

Thesis (Ph. D.)--University of Maryland, College Park, 2005.
Thesis research directed by: Chemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (March 5, 2007) Vita. Includes bibliographical references.

The World Health Organization has named the resistance of microbes to known antimicrobial drugs as an increasingly serious threat to global public health. Isolates of the ESKAPE pathogens (E. faecium, S. aureus, K. pneumonia, A. baumanii, P. aeruginosa, and Enterobacter species) are responsible for many nosocomial infections each year that require complicated, and therefore expensive, medical treatment, often leading to death in immune-compromised patients. Over the past 50 years, (fluoro-)quinolone antimicrobial agents have been widely used in the clinic as broad-spectrum antibiotics, but lately growing resistance against this drug class has been reported. Combining metal ions with known organic small-molecule drugs is one strategy to overcome such developed resistances. Previously, the antimicrobial properties of copper(II) and gallium(III) had been investigated, leading to Greek mythology comparisons for their mechanism of action: Cu²⁺ as the ”Achilles Heel”, Ga³⁺ as the ”Trojan Horse” subterfuge for Fe³⁺. In this thesis, gallium(III) and copper(II) coordination complexes of (fluoro-)quinolone antimicrobial agents, and derivatives thereof, were synthesized in an attempt to combine the antimicrobial potency of Cu²⁺ and Ga³⁺ with that of the quinolone antimicrobial agents in one molecule. The antimicrobial susceptibility of these coordination complexes was evaluated against five isolates of the ESKAPE pathogens; combinational effects between the metals and the quinolone ligands were not observed. While the combination of metal ions with small, organic drug molecules may lead to novel potent metallodrugs, the interaction of metal ions with drugs in vivo is often associated with toxic side-effects of medical treatment, for which the iron(III)-mediated cumulative-dose cardiotoxicity of doxorubicin is one example. Vosaroxin is a first-in-class anticancer quinolone derivative in clinical trials. Unlike the anthracycline anticancer drug doxorubicin, vosaroxin is minimally metabolized in vivo. Spectrophotometric titrations and further studies of the isolated tris(vosaroxino)iron(III) and -gallium(III) complexes supported a strong coordination of the metal ion suggesting that vosaroxin treatment may not result in cardiotoxicity.
Science, Faculty of
Chemistry, Department of
Graduate

The mechanism of resistance of tumor cells to chemotherapeutic agents is explored using probabilistic methods where it is assumed that resistant cells arise spontaneously with a defined frequency. The resistance process is embedded in a discrete time Markov branching process which models the growth of the tumor and contains three seperate cell types: stem, transitional and end cells. Using the asymptotic properties of such models it is shown that the proportion of each type of cell converge to constants almost surely. It is shown that the parameters relating to stem cell behaviour determine the asymptotic behaviour of the system. It is argued that for biologically likely parameter values, cure of the tumor will occur if, and only if, all stem cells are eliminated. A model is developed for the acquisition of resistance by stem cells to a single drug. Probability generating functions are derived which describe the behaviour of the process after an arbitrary sequence of drug treatments. The probability of cure, defined as the probability of ultimate extinction of the stem cell compartment, is characterised as the central quantity reflecting the success of therapeutic intervention. Expressions for this function are derived for a number of experimental situations. The effects of variation in the parameter values are examined. The model is extended to the case where two anticancer drugs are available and formulae for the probability of cure are developed. The problem of therapeutic scheduling is examined and under situations where drugs are of "equal" effectiveness, but may not be given together, it is shown that the mean number of tumor cells is minimised by sequential alternation of the drugs. The models are applied to data collected on the L1210 leukemia treated by the drugs Cyclophosphamide and Arabinosylcytosine. In both cases the analysis of the data provide evidence that resistant cells arise spontaneously with a frequency of approximately 10⁻⁷ per division. When applied to human breast cancer, the model indicates that neoadjuvant therapy is unlikely to greatly influence the likelihood that the patient will die from the growth of drug-resistant cells.
Science, Faculty of
Statistics, Department of
Graduate

This dissertation reports the synthesis and characterization of nanoscale coordination polymers (NCPs) for anticancer drug delivery. Nanoparticles have been explored in order to address the limitations of small molecule chemotherapeutics. NCPs have been investigated as drug delivery vehicles as they can exhibit the same beneficial properties as the bulk metal-organic frameworks as well as interesting characteristics that are unique to nanomaterials.

Gd-MTX (MTX = methotrexate) NCPs with a MTX loading of 71.6 wt% were synthesized and stabilized by encapsulation within a lipid bilayer containing anisamide (AA), a small molecule that targets sigma receptors which are overexpressed in many cancer tissues. Functionalization with AA allows for targeted delivery and controlled release to cancer cells, as shown by enhanced efficacy against leukemia cells. The NCPs were doped with Ru(bpy)₃²⁺ (bpy = 2,2'-bipyridine), and this formulation was utilized as an optical imaging agent by confocal microscopy.

NCPs containing the chemotherapeutic pemetrexed (PMX) were synthesized using different binding metals. Zr-based materials could not be stabilized by encapsulation with a lipid bilayer, and Gd-based materials showed that PMX had degraded during synthesis. However, Hf-based NCPs containing 19.7 wt% PMX were stabilized by a lipid coating and showed in vitro efficacy against non-small cell lung cancer (NSCLC) cell lines. Enhanced efficacy was observed for formulations containing AA.

Additionally, NCP formulations containing the cisplatin prodrug disuccinatocisplatin were prepared; one of these formulations could be stabilized by encapsulation within a lipid layer. Coating with a lipid layer doped with AA rendered this formulation an active targeting agent. The resulting formulation proved more potent than free cisplatin in NSCLC cell lines. Improved NCP uptake was demonstrated by confocal microscopy and competitive binding assays.

Finally, a Pt(IV) oxaliplatin prodrug was synthesized and incorporated in different NCPs using various binding metals. A moderate drug loading of 44.9 wt% was determined for Zr-based NCPs. This drug loading, along with a diameter less than 200 nm, make these particles promising candidates for further stabilization via lipid encapsulation.

The concept of bioreductive prodrug chemotherapy is introduced in chapter 1. Tumour cell hypoxia is a significant factor in limiting tumour growth control with conventional radiotherapy and some chemotherapeutic agents. Following therapy these cells can repopulate and cause a relapse of the cancer. On the other hand, hypoxia is unique to tumours, and is therefore potentially exploitable. Bioreductive prodrugs are compounds in which a oxygen inhibited redox-based bioactivation step triggers a reaction leading to a lethal intermediate. The concept of bioreductive DNA alkylators and DNA topoisomerase 11 inhibitors is discussed. The synthesis of model thiazolylindole compounds based on the natural product BE \0988 are discussed in chapter 2. Two strategies were employed for the construction of the thiazolylindoles: the Hantzsch reaction; and nucleophilic substitution on 2-bromothiazole by an indolyl anion. The synthesis of thiazolylindolequinone compounds are discussed in chapter 3. The quinone C(5) position of the thiazolylindolequinone analogues was elaborated to provide a series of cyclic and acyclic C(5)-amino derivatives. Synthetic strategies towards the synthesis of indole-2-carboxylates are discussed in chapter 4. The Moody-Rees and Cadogan-Sundberg reactions were employed to provide a synthesis of the useful highly substituted indole [154]. The Brederek imidazole reaction (i.e., the reaction of a amidine and a-halo ketone) is discussed in chapter 5. Application of the Brederek reaction was employed towards the construction of the bisindole imidazole natural compounds, the nortopsentins. The biological properties of the compounds of the compounds synthesised are discussed in chapter 6. The compounds were tested for DNA topoisomerase 11 inhibitory activity and cytotoxicity under a hypoxic environment.

The work described in this thesis details the design, synthesis and evaluation of molecules intended as mechanistic probes to the enediyne anticancer antibiotics. In particular it examines the role of radical and ionic accepting groups in analogue systems related to the DNA cleaving agent Neocarzinostatin chromophore (NCS). The application of novel methodologies towards the synthesis of a number of key precursors and model systems is discussed. Initial studies concentrated on the versatile synthesis of a model system bearing dienediyne and styryl motifs, from 2-cyclopentenone. The role of cyclic amine bases in a critical Stille type cross-coupling was investigated, leading to an efficient and robust synthetic route. Attempts to selectively cyclopropanate, or epoxidise, the styryl olefin under a range of conditions however, failed to give the desired phenylcyclopropyl or phenyloxiranyl analogues. A novel application of the Kulinkovich reaction, to form an alkynylcyclopropanol is discussed. Attempts to synthesise a phenylcyclopropanol analogue via an advanced tris-silyl pre-cursor are also detailed. The successfulu se of Salaun's cyclopropanone-hemiacetatl o complete the synthesis of a novel analogue system bearing a cyclopropanol motif is detailed and discussed, with some mention of related synthetic endeavours. The analogue's reactivity to a range of nucleophiles, radicals and reaction conditions is evaluated, though no conclusive evidence for cycloaromatised products was found. Finally, approaches to the synthesis of a fully functionalised analogue system incorporating dienediyne, cyclopropanol and selectively protected trans-diol moieties (for possible coupling to DNA binding agents) are detailed. Although this synthesis incorporates some methodology previously reported, significant improvementsnotably the synthesis of a key pyrano-3-one directly from furfuryl alcohol using Nbromosuccinimide and aqueous sodium bicarbonate- make it noteworthy of inclusion

This thesis is concerned with the synthesis of small molecules that mimic tetramate- and pyrrolidinone-containing natural products, and the evaluation of their antibacterial and anticancer properties. Chapter 1 exposes the need for new antibiotics due to the emergence of resistance to current drugs, and it discusses the value of the natural product inspired approach for drug discovery. It also gives an overview of the structure, synthesis and biological properties of the natural products pramanicin and oxazolomycin, both of which contain a pyrrolidinone core. Chapter 2 describes an efficient synthetic route which allowed the preparation of a library of acyltetramates via Grignard addition to a Weinreb amide. This route was also used to prepare mimics of the acyl chain of pramanicin, comprising dienones and epoxy-enones of different lengths, and of the left-hand segment of oxazolomycin. In Chapter 3, reduction of the tetramate core of different closely-related systems was performed, and it was found that the diastereochemical outcome could be controlled by appropriate choice of substituents in the bicyclic system. Preparation of reduced acyltetramates was only successful via hydrogenation of the Weinreb amide and subsequent Grignard substitution. Chapter 4 describes the introduction of β-lactones to pyrrolidinones. Conditions for the selective formation of either spiro- or fused-β-lactones were found, which relied on the activation of the precursor β-hydroxyacid, accessed via N,O-acetal deprotection and ester saponification of the bicyclic systems. Further functionalisation of these analogues to mimic oxazolomycin was thwarted by the instability of some of the intermediates. Finally, the biological properties of the analogues are discussed in Chapter 5. The antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus was examined using the holeplate method, which showed that the aliphatic lipophilic tetramates were the most potent inhibitors, with selective Gram-positive activity. An anticancer high-content screen was also designed, but only weak activities of the synthesised compounds were observed against four cancer cell lines.

The research work described in this thesis concerns metal-based anticancer drugs with an emphasis on non-covalent DNA-binding supramolecular assemblies. The project involves the preparation of a series of mono- and bi-metallic ruthenium complexes with a primary focus on fluorescent dinuclear triple-stranded helicates with different structural topographies. Emphasis is then directed towards an investigation of the DNA binding characteristics of these molecules and an evaluation of their anticancer properties in human cancer cell lines. Attention is brought to the significance that the cylinder-building moieties and their structural characteristics have to these features. The studies also include an examination of the effects of chirality of the investigated supramolecular systems and the impact they have on molecular recognition. This is addressed via studies of the interaction of optical isomers of ruthenium triple-stranded helicates with DNA as a biomolecular target system and with Δ-TRISPHAT as a representative small chiral molecule.

Development of liposome formulation of an amphiphilic anticancer peptide using the ANTS/DPX leakage assay. The effects of lipid composition on the liposomes' resistance to an amphiphilic cyclic peptide c[KS.S.S.KWL W] were studied by the ANTS/DPX leakage assay. One or more unsaturated acyl chains in the phospholipids, small phospholipid headgroup size, the presence of cholesterol, and the presence of PEG-lipid were demonstrated as critical parameters to stabilize the liposome membrane. A liposome formulation of the peptide comprising POPE/POPC/cholesterol/C16 mPEG 2000 ceramide (20.8:31.2:40:8, mol%) was thereby developed with a peptide-encapsulation efficiency of 47.8%. The liposomal cyclic peptide exhibited dose-dependent toxicity to MCF7 human breast cancer cells and stability under incubation. Design, construction and in vitro characterization of a hydrazone-based convertible liposomal system for anticancer drug delivery. A novel PEG-lipid, PEG2ooo-Hz-DHG, with an acid-labile hydrazone linker between the PEG2ooo head group and the lipidic DHG moiety was synthesized. PEG2000-Hz-DHG was relatively stable at normal physiological pH 7.4, but hydrolyzed more quickly at tumor interstitium pH 6.5-7.0 and endosomal/lysosomal pH 5.0. A novel pH-sensitive "Convertible Liposome System" (CLS) was constructed comprising PEG2ooo-Hz-DHG, positively charged lipid DOTAP, and the zwitterionic phospholipid POPC (8:15:77, mol%). CLS converted from neutrally charged "stealth" liposome to positively charged liposome at tumor interstitual pH owing to the hydrolysis ofPEG2ooo-Hz-DHG. The doxorubicin-encapsulated CLS that had been pre-incubated at pH 6.5 for 30 h exhibited more intensive binding and higher toxicity to Bl6-Fl0 murine melanoma and MDA-MB-435S human breast cancer cells than doxorubicin encapsulated in pH-insensitive stealth liposome.

Traditionally, inoperable or metastatic cancers have been treated by causing massive DNA damage in order to induce self-destruction (apoptosis) of the rapidly multiplying cancer cells. Initially, this strategy works for many cancers, in particular those which express normal p53 tumor suppressor protein. However, most cancers eventually aquire mutations in either p53 or other signaling molecules and fail to initiate apoptosis in response to severe DNA damage. During this study three types of compounds were investigated for their DNA damaging and anticancer effects: a pair of novel metal containing compounds, a pair of natural products, and a known synthetic drug which had been used many years ago for completely different indication. It was shown that all stop the growth of cancer cells and that the latter two classes do not require functional p53 because they work equally well in cells with normal (wildtype), mutant or no p53. The two nickel complexes investigated in this dissertation, differ in their ability to cause DNA damage and cell death. The oxidized form of the nickel complex, [Ni(CR-2H)] 2+ causes DNA damage and cell death at a much lower concentration than its reduced counterpart [Ni(CR)] 2+ . The phenanthridine alkaloids, Sanguinarine and Chelerythine cause high levels of DNA strand breaks and extremely rapid apoptosis which is not due to DNA damage because the quick onset precludes extensive signaling. The effects of the phenanthridines were linked to production of large amounts of reactive oxygen species (ROS), in particular hydrogen peroxide (H 2 O 2 ). The importance of ROS for the action of anticancer drugs as well as antibiotics is increasingly being recognized. In addition we also investigated the thioxanthone Lucanthone or Miracil D (which was used for the treatment of parasitic worms more than 50 years ago). It causes DNA strand breaks and apoptosis. Apoptosis occurs on a timescale consistent with signaling. However, p53 does not seem to be involved and alternative mechanisms are being investigated. This work provides new directions for designing novel anticancer drugs that are not subject to the limitations of DNA damaging agents.

L'électrochimiothérapie est un traitement anticancéreux utilisé en routine en Europe dans près de 130 centres de traitement du cancer. Le taux de réponse objective atteint 85 % pour le traitment de tumeurs cutanées et sous-cutanées et des études sont en cours afin d'appliquer ce traitement à des tumeurs profondes. Au cours de ce doctorat, les méchanismes sous-jacents à cette excellente efficacité antitumorale ont été étudiés. Dans un premier temps, l'objectif a été d'évaluer la capacité de l'électrochimiothérapie à induire la mort des cellules souches cancéreuses, considérées comme les racines du cancer. Ensuite, les mécanismes immunologiques à l'origine du développement d'une immunité antitumorale mise en place par le traitement ont été investigués. Cependant, malgré le haut taux de réponse observé, l'électrochimiothérapie reste un traitement local qui n'induit pas de réponse antitumorale à distance, sur les tumeurs non-traitées. Afin de pallier à cette absence d'activité systémique, une collaboration a été mise en place avec une entreprise innovante de biotechnologies, INVECTYS, dans le but de développer une stratégie de vaccination à ADN ciblant la télomérase et basée sur l'électrogènetransfert. Il est attendu que la combinaison de cette immunothérapie avec un traitement local par électrochimiothérapie, détruise non seulement la tumeur primaire, dont les cellules souches cancéreuses, mais également les cellules cancéreuses circulantes et les métastases
Electrochemotherapy is an anticancer treatment used in routine in Europe in 130 cancer treatment centers. The objective response rate reaches 85 % for the treatment of cutaneous and subcutaneous tumors and studies are ongoing to spread the use of electrochemotherapy to deep-seated tumors. In the frame of this doctorate, the mechanisms underlying this excellent antitumor efficiency were investigated. First, the goal was to evaluate the ability of electrochemotherapy to induce the death of cancer stem cells, considered as the roots of cancer. Second, the immunological mechanisms responsible for the development of antitumor immune responses following the treatment were investigated. However, although a very high response rate is observed, electrochemotherapy remains a local treatment which does not induce antitumor responses in distant non-treated nodules. In order to circumvent this lack of systemic activity, a collaborative project was initiated with an innovating biotech company, INVECTYS, in order to develop a DNA vaccination strategy targeting the telomerase and based on electrogenetransfer. It is expected that the combination of this immunotherapy with a local treatment by electrochemotherapy could destroy not only the primary tumor, including cancer stem cells, but also circulating cancer cells and metastases