New imidazotetrazinones have been synthesised that possess features in their structures to release aziridinium ions upon ring opening. Unstable 2-aminoethylisocyanates were required in this preparation, which were synthesized with BOC-protection of the amino group to counteract the reactivity of the amine towards the isocyanate group in the case of aliphatic amines; in contrast, anilinoethylisocyanates were synthesized unprotected. Substituents with a range of electron-withdrawing and electron-releasing properties were introduced at the p-position of the aniline ring. A 13C-labelled study confirmed the release of the aziridinium ion by these imidazotetrazinones in neutral pH buffer solution. Furthermore the kinetics of the hydrolysis in neutral aqueous solution of some these new tetrazines were similar to temozolomide, in addition to useful acid stability. Other imidazotetrazinones were synthesised for the purpose of releasing alcohols and phenols. Their synthesis was performed with a one-carbon linker between the imidazotetrazinone 3-position and the alcohols or phenols to be released. The release of alcohol and phenol through the hydrolysis of the intermediate diazonium ions to the unstable hemiacetals that decomposed to the alcohol and phenol was confirmed by 1H NMR. The kinetics of the hydrolysis of these tetrazines in neutral aqueous solution showed a faster reaction rate compared with temozolomide (t1/2 = 0.53 and 0.36 h compared with temozolomide 1.4 h). / Full text was made available at the end of the embargo period, 1st Feb 2016.

Hydrogen sulfide (H2S) has been recognized as a biological signaling molecule for over twenty years now. Since these important findings emerged, many collaborative projects among chemists, biologists, and clinicians have demonstrated the physiological roles and potential therapeutic benefits of exogenous H2S delivery. As our understanding of the active roles H2S plays in biological systems has increased, so has the desire to investigate other related sulfur species (i.e. persulfides, R–SSH) for their physiological interactions with H2S and potential therapeutic efficacy. This recent interest in persulfides has stimulated a flurry of research in the field and created a new set of scientific problems to solve and opportunities to improve our understanding of persulfides in a biological context. With this surge of interest in persulfides, chemists set out to synthesize and characterize a variety of stimuli-responsive compounds that release persulfides under specific, biologically relevant conditions. In order to better understand persulfide reactivity and biological activity, and provide several prodrug platforms that respond to a variety of stimuli, this dissertation describes four persulfide-releasing prodrug systems, a pyrene-based fluorescent probe that measures H2S release in the presence of thiols, and efforts toward a peptide-based system for the release of H2S from a peptide thioacid (C(O)SH). The first four systems described utilize the well-known 1,6-benzyl elimination reaction (sometimes called self-immolation) to trigger release of a persulfide from a small molecule, polymeric, or peptide-based prodrug platform. Importantly, the first self-immolative small molecule persulfide prodrug (termed BDP-NAC) was designed to respond to reactive oxygen species (ROS). Specifically, BDP-NAC utilized a para-positioned boronic acid pinacol ester functionality to selectively react with H2O2, yielding N-acetylcysteine persulfide (NAC-SSH) and p-hydroxybenzyl alcohol as a byproduct. BDP-NAC showed trigger specificity towards H2O2, as determined by the use of a structurally analogous fluorescent probe (termed BDP-fluor). The prodrug also exhibited antioxidant properties in vitro, and served as the first example in the literature of a self-immolative persulfide donor. The second group of donors, self-immolative small molecule and peptide-based persulfide prodrugs (termed SOPD-Pep and SOPD-NAC), were designed to be responsive to superoxide (O2∙–), the primary precursor to all other ROS. In this work, the advantages of attaching small molecule persulfide donors to peptides were explored. In vitro experiments showed that SOPD-Pep mitigated toxicity induced by phorbol 12-myristate 13-acetate (PMA) more effectively than its small molecule counterpart SOPD-NAC and several common H2S donors. It is proposed that peptide scaffolds offer increased cellular uptake due to their nanoscale size, allowing for better antioxidant activity, as confirmed by fluorescence microscopy. The third section of this dissertation compares an esterase-responsive small molecule to an analogous polymeric persulfide releasing prodrug (termed EDP-NAC and polyEDP-NAC) and their abilities to decrease oxidative stress in response to immediate (H2O2) and sustained (5-fluorouracil, 5-FU) forms of ROS. Persulfide release half-lives were characterized using 1H NMR spectroscopy and showed over one order of magnitude difference between EDP-NAC and polyEDP-NAC. In vitro evaluation of the donors showed polyEDP-NAC was better suited to combat sustained production of ROS induced by 5-FU, whereas EDP-NAC was better suited to combat immediately available ROS from H2O2. These discrepancies in antioxidant activity between the two donors were deemed to be a result of their different persulfide release half-lives, indicating that scientists must take these factors into consideration when designing R–SSH prodrugs for specific disease indications. The fourth donor, NDP-NAC, responded to the bacteria-specific enzyme nitroreductase to release its persulfide payload. NDP-NAC elicited gastroprotective effects in mice that were not observed in animals treated with control compounds incapable of persulfide release or in animals treated with Na2S. NDP-NAC induced these effects by the upregulation of beneficial small and medium chain fatty acids and through increasing growth of Turicibacter sanguinis, a beneficial gut bacterium. It also decreased the populations of Synergistales bacteria, opportunistic pathogens implicated in gastrointestinal infections. Lastly, two appendices are provided in this dissertation that briefly describe the synthesis of a pyrene-based H2S sensor and efforts toward a readily accessible peptide-based thioacids as H2S donors. / Doctor of Philosophy / Hydrogen sulfide (H2S), produced naturally in hydrothermal vents and as a byproduct of industrial processes, has historically been known for its potent smell and toxicity. However, the recent discovery of H2S as a naturally-produced signaling molecule (termed gasotransmitter) in mammals has changed the way scientists view this malodorous gas. Our understanding of the biological roles and production of H2S is still growing, and recent research has suggested various links between changes in H2S concentrations in the body and a variety of disease states, including Alzheimer's, cardiovascular disease, and inflammation. Because of this link between various diseases and alterations in natural H2S production, collaborative efforts among chemists, biologists, and pharmacologists have demonstrated the usefulness of therapeutics that contain H2S-donating moieties, in an effort to alleviate these disease conditions. Persulfides (R-SSH), biological signaling molecules related to H2S, have emerged as critical species in sulfur signaling because of the similar observed antioxidative effects compared to H2S. This dissertation focuses on the synthesis and characterization of several compounds that release persulfides in response to specific stimuli (called persulfide donors). The first donor system described here releases persulfides in response to hydrogen peroxide (H2O2), a major cellular oxidant, and reduces oxidative stress in response to H2O2. The second donor system responds to superoxide (O2∙–), a precursor oxidant to H2O2 in cells, to release persulfides. Specifically, two variants of these donors, a small molecule and a peptide-based donor, exhibited antioxidant activity in response to O2∙–, but to varying degrees based on differences in cellular uptake of small molecules and self-assembled peptide nanostructures. The third donor system compares persulfide release from a small molecule and polymeric scaffold, both of which release persulfides in response to esterase enzymes. A large persulfide release half-life range was observed between the two donor systems, and antioxidant activity in response to H2O2 also varied based on the source and timescale of oxidant (H2O2 versus 5-fluorouracil). The fourth section of this dissertation focuses on a persulfide donor that responds to the bacterial enzyme nitroreductase. This donor increased levels of beneficial bacteria and short and medium chain fatty acids in murine models, while simultaneously decreasing levels of a niche subset of harmful bacteria. Taken together, these persulfide donor systems exhibit the strong reducing ability of persulfides in a biological context, showcasing the potential for therapeutic efficacy and avenues for more advanced donors to be synthesized in the future.

Parkinson's disease (PD) is a progressive neurodegenerative disorder of unknown cause that afflicts about 1.5 million Americans. The characteristic feature of PD is a deficiency of dopamine in the terminals of nigrostriatal neurons. Two enzyme systems, the neuronal form of nitric oxide synthase (nNOS) and monoamine oxidase B (MAO-B), have been linked to neurodegenerative pathways leading to PD. Several MAO-B and nNOS inhibitors have been evaluated for their neuroprotective properties in the mouse model of neurodegeneration which employs the parkinsonian inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). One such compound is 7-nitroindazole (7-NI), a compound which is reported to inhibit both enzymes. This thesis focuses on the synthesis and biological evaluation of a potential prodrug form of 7-NI and related indazolyl containing compounds which are designed to release the active drugs following a metabolic bioactivation process. These studies have led to a detailed description of the nucleophilic aromatic substitution reactions between 4-chloro-1-methylpyridinium iodide and the indazolyl reactants that were employed as the initial step in the synthesis of the target compounds. The MAO-B substrate and inhibition properties of these "prodrugs" as well as the parent indazolyl compounds were examined. The results are discussed in relation to a previously developed active site model of MAO-B. / Master of Science

No / Purpose: Combretastatin A4 phosphate (CA4P) and its structural analog, combretastatin A1 phosphate (CA1P), are soluble prodrugs capable of interacting with tubulin and causing rapid vascular shutdown within tumors. CA4P has completed Phase I clinical trials, but recent preclinical studies have shown that CA1P displays a greater antitumor effect than the combretastatin A4 (CA4) analog at equal doses. The aim of this study, therefore, is to compare pharmacokinetics and metabolism of the two compounds to determine whether pharmacokinetics plays a role in their differential activity. Experimental Design: NMRI mice bearing MAC29 tumors received injection with either CA4P or CA1P at a therapeutic dose of 150 mg/kg-1 , and profiles of both compounds and their metabolites analyzed by a sensitive and specific liquid chromatography/mass spectroscopy method. Results: The metabolic profile of both compounds is complex, with up to 14 metabolites being detected for combretastatin A1 (CA1) in the plasma. Many of these metabolites have been identified by liquid chromatography/mass spectroscopy. Initial studies, however, focused on the active components CA4 and CA1, where plasma and tumor areas under the curve were 18.4 and 60.1 microgram/h/ml-1 for CA4, and 10.4 and 13.1 microgram/h/ml-1 for CA1, respectively. In vitro metabolic comparisons of the two compounds strongly suggest that CA1 is metabolized to a more reactive species than the CA4. Conclusions: Although in vitro studies suggest that variable rates of tumor-specific prodrug dephosphorylation may explain these differences in pharmacokinetics profiles, the improved antitumor activity and altered pharmacokinetic profile of CA1 may be due to the formation of a more reactive metabolite.

Yes / Silane-substituted imidazotetrazines 1,2 were investigated for their activity as anticancer prodrugs related to temozolomide (TMZ). The TMS-derivative 1 showed an activity profile against TMZ susceptible and resistant cell lines very similar to TMZ; in contrast, the SEM-derivative 2 showed activity irrespective of MGMT expression or MMR deficiency (Table). Probing the prodrug activation mechanism by NMR kinetic studies determined that the TMS compound 1 follows a reaction pathway and time-course very similar to temozolomide. 1H-NMR spectra of the reaction mixture showed considerable incorporation of deuterium into the final alkylation products of the reaction (methanol and methyl phosphate) as had previously been shown for temozolomide (Wheelhouse, R.T., et al. Chem. Commun. 1993, 15, 1177–1178). The SEM-derivative 2 reacted more rapidly than TMZ or TMS-derivative 1. Somewhat surprisingly, the silane remained intact throughout the experiment and the observed reaction was the hydrolysis of the imidazo-tetrazine to ultimately release formaldehyde hydrate and 2-TMS-ethanol. In conclusion, TMS-derivative 1 is a diazomethane precursor with prodrug activation mechanism, kinetics and anti-cancer activity in vitro similar to TMZ. In contrast, the SEM derivative 2 was more rapidly hydrolysed, a precursor of 2-TMS-ethanol and had activity in vitro different from TMZ. 2-TMS-ethanol was previously reported as a non-toxic compound in mice (Voronkov, M.G., et al. Dokl. Akad. Nauk SSSR 1976, 229, 1011–1013) and is known as a substrate for alcohol dehydrogenase (Zong, M.-H., et al. Appl. Microbiol. Biotechnol. 1991, 36, 40–43) and as a modest inhibitor of acetylcholinesterase (Aberman, A., et al. Biochim. Biophys. Acta 1984, 791, 278–280; Cohen, S.G., et al. J. Med. Chem. 1985, 28, 1309–1313).

BACKGROUND: Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. RESULTS: Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized 13C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. CONCLUSIONS: Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

Fluorinated systems are defined by unique properties that offer advantages in drug delivery, material synthesis and industrial applications. In comparison to their hydrocarbon counterparts, the design of fluorinated solutes for tailored applications is limited by the inability to predict the effect of fluorination on phase behavior. This work examines and interprets the influence of fluorination on the phase behavior of fluorinated solutes and surfactants, with emphasis on their impact on vesicle bilayers. Thermodynamic partitioning of functionalized series of fluorinated and hydrocarbon nicotinate prodrugs fashioned to promote solubility in a fluorocarbon solvent (perfluorooctyl bromide; PFOB) is measured. Predictive approaches are also employed to describe partitioning of these nicotinates between immiscible phases relevant to drug delivery. The findings reveal no strong correlation of the partitioning trends with biological markers of cytotoxicity and prodrug uptake for PFOB mediated delivery. However, partitioning in model membranes (liposomes), which, increases with the hydrophobicity of the perhydrocarbon nicotinates, suggests incorporation in a cellular matrix is chain length dependent. The impact of incorporating fluorinated surfactants in catanionic vesicles, which form spontaneously in dilute aqueous solutions and serve as potential substitutes to conventional meta-stable liposome-based vesicles, is studied. Much larger isotropic vesicle regions are observed in the phase map of the partially fluorinated catanionic surfactant pair, cetylpyridinium bromide/ sodium perfluorooctanoate (CPB/SPFO) than in fully fluorinated HFDPC (1,1,2,2,-tetrahydroperfluorododecyl pyridinium chloride )/SPFO. Fluorescence probing of the vesicle bilayers suggest more fluid bilayers in CPB/SPFO than in HFDPC/SPFO due to better chain packing in the fully fluorinated bilayer. However, the vesicle region is expanded in more asymmetric fluorinated bilayers of HFDPC/SPFH (sodium perfluorohexanoate). The increased chain asymmetry in HFDPC/SPFH results in reduced packing density and more fluid bilayers than in HFDPC/SPFO. The robustness of CPB/SPFO and HFDPC/SPFO vesicles is demonstrated in the synthesis of silica hollow spheres by templating and the retention of encapsulated solutes. Higher colloidal stability of the silica spheres is achieved in HFDPC/SPFO relative to CPB/SPFO due to the barrier effect of the fluorinated bilayer. Similarly, higher solute retention in HFDPC/SPFO is observed. The modulation of phase behavior with fluorination offers opportunities in tunable applications of fluorinated bilayers.

The present study was divided between two different drug delivery goals, each involving naltrexone (NTX) or its active metabolite, 6-β-naltrexol (NTXOL). First, amino acid esters of NTX and NTXOL were prepared in order to test their candidacy for microneedle-enhanced transdermal delivery. Second, a 3-O-(-)-cytisine-naltrexone (CYT-NTX) codrug was prepared for screening as a potential oral delivery form of NTX and (-)-cytisine (CYT). The amino acid prodrugs were intended for the treatment of alcohol abuse, while the codrug was designed as a single agent for the treatment of alcoholism and tobacco-dependency co-morbidities. One hypothesis of this work was that prodrugs of NTX or NTXOL can be designed that possess superior skin transport properties through microneedle-treated skin compared to parent NTX or NTXOL. Nine amino acid ester prodrugs were prepared, and only three 6-O amino acid ester prodrugs of NTXOL were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. 6-O-β-Ala-NTXOL, the lead compound, exhibited the most rapid bioconversion to NTXOL in human plasma (t1/2 = 2.2 ± 0.1 h); however, this in vitro stability value indicates that the prodrug may require hepatic enzyme-mediated hydrolysis for sufficiently rapid bioconversion to NTXOL in vivo. A second hypothesis of this work was that a CYT-NTX codrug could be designed with appropriate stability characteristics for oral delivery. CYT-NTX was found to be stable over the time course of 24 h in buffer systems of pH 1.5, 5.0, 7.4 and 9.0, and in 80% rat plasma, 80% human plasma, simulated gastric fluid and simulated intestinal fluid. Six (3 rats/group) Sprague-Dawley male rats were dosed i.v. with 1 mg/kg CYT-NTX codrug, or 10 mg/kg, p.o. Oral administration of a 10 mg/kg dose of CYT-NTX codrug resulted in rapid absorption and distribution (5 min) of CYT-NTX codrug, and NTX was released from codrug with a peak plasma concentration of 6.8 ± 0.9 nmol/L reached within 65 minutes. Plasma CYT was not detected; however, NTX delivery was achieved with a fraction absorbed value of 13%. Thus, CYT-NTX may hold promise as a potential oral codrug for further optimization and development.

A tuberculose afeta mais de um bilhão de pessoas em todo o mundo. Desde a descoberta da rifampicina, em 1965, nenhum outro fármaco importante foi introduzido na terapêutica. A pirazinamida, um dos fármacos disponíveis na terapia da tuberculose, é atualmente considerada um bioprecursor do ácido pirazinóico, porque bactérias resistentes não expressam uma enzima, pirazinamidase, responsável pela conversão da pirazinamida no derivado ácido. Ésteres do ácido pirazinóico apresentam atividade antimicobacteriana, provavelmente por melhor penetração pela parede celular das micobactérias que o derivado ácido. Desta forma, podem ainda atuar em cepas resistentes por serem ativados por esterases. Algumas fluorquinolonas apresentam atividade antimicobacteriana, como o ciprofloxacino, ofloxacino e levofloxacino. O trabalho teve por objetivo obter formas latentes de ácido pirazinóico unindo-o a quinolonas com atividade antimicobacteriana, através de ligação éster, obtendo-se pró-fármacos recíprocos. Um dos compostos sintetizados apresentou atividade in vitro, com concentração inibitória mínima comparável ao ciprofloxacino, um dos fármacos mais ativos. / Tuberculosis affects over than one billion people around the world. Since the discovery of rifampin, in 1965, no one another important drug was introduced in therapeutics. Pyrazinamide, one of the drugs available in therapy of tuberculosis, is nowadays considered a bioprecursor of pyrazinoic acid, because resistant bacterias do not express an enzyme, pyrazinamidase, responsible by the conversion of pyrazinamide to pyrazinoic acid. Pyrazinoic acid esters exhibit antimycobacterial activity probably to better penetration through mycobacterial cell wall than the acid derivative. So, it could act in resistant strains because is activated by esterases. Some fluoroquinolones exhibit antimicobacterial activity, like ciprofloxacin, ofloxacin and levofloxacin. This work had as objective to obtain latent forms of pyrazinoic acid linking it to quinolones with antimycobacterial activity, through an ester bond, obtaining mutual prodrugs. One of the synthesized compounds showed activity in vitro, with minnimal inhibitory concentration comparable to ciprofloxacin, one of most active drugs.

Conventional treatments for prostate cancer have significant limitations making it difficult to control the disease. Cyclopropabenzindoles (CBI) are more biologically potent, stable and synthetically accessible analogues of cyclopropapyrroloindole (CPI) anti-tumour antibiotics, such as duocarmycin-SA and CC1065. A polymeric prodrug carrying a CBI drug attached to the polymeric backbone through a PSA cleavable linker peptide has two modes of selectivity: activation by PSA and the EPR effect. To synthesise a 5-amino-seco-CBI analogue, 2,4-dinitronaphthalen- 1-ol gave di-Boc-1-iodonaphthalene-2,4-diamine in five steps (triflation, SNAr displacement with iodide, reduction (loss of iodine), protection and restoration of the iodine. For the amino-seco-CBI, it was important to discriminate between N2 and N4. Acidic removal of the Boc-group(s) resulted in deiodination. NMR investigations showed an unexpected Wheland-like cationic intermediate. N3 of naphthalene-1,3-diamine was selectively trifluoroacetylated and N1 was masked with Boc. Electrophilic iodination gave an orthogonally protected 1-iodonaphthalene-2,4-diamine. Allylation at the trifluoroacetamide was followed by free radical cyclisation with TEMPO trap. Removal of the trifluoroacetyl group allowed coupling to 5-(2-(dimethylamino)ethoxy)-1H-indole-2-carboxylic acid. Reductive removal of 2,2,6,6-tetramethylpiperidine, substitution of the exposed hydroxy group with chloride and removal of the Boc-group gave the amino-seco-CBI drug, 5-amino-1-chloromethyl-3-(5-(2-dimethylaminoethoxy)indole-2-carbonyl)-2,3-dihydro-1H-benz[e]indole. A DNA-melting assay confirmed that it binds very strongly to dsDNA causing a 13 deg. C increase in melting temperature. The drug was a highly potent cytotoxin in vitro, with IC50 = 18 nM against LNCaP prostate cancer cells. The polymeric prodrug system involved the synthesis of the pentapeptide SSKLQ. The amide side chain of glutamine can be masked as the nitrile and this can be quantitatively hydrated to the γ-carboxamide of L-Gln with hydroperoxide. The pentapeptide was coupled to 4-methoxynaphthalen-1-amine and to poly(ethylene glycol) as a model polymeric prodrug system. Efficient release of the model drug from the polymeric prodrug by PSA will allow this polymeric prodrug system to be adopted for the synthesised amino-seco-CBI drug.