Despite of its known toxicity and potential to cause cancer, arsenic has been proven to be a very important tool for the treatment of various refractory neoplasms. One of the promising arsenic-containing chemotherapeutic agents in clinical trials is Darinaparsin (dimethylarsinous glutathione, DMAIII(GS)). In order to understand its toxicity and therapeutic efficacy, the metabolism of Darinaparsin in human cancer cells was evaluated. With the aim of detecting all potential intermediates and final products of the biotransformation of Darinaparsin and other arsenicals, an analytical method employing high performance liquid chromatography inductively coupled mass spectrometry (HPLC-ICP-MS) was developed. This method was shown to be capable of separating and detecting fourteen human arsenic metabolites in one chromatographic run. The developed analytical technique was used to evaluate the metabolism of Darinaparsin in human cancer cells. The major metabolites of Darinaparsin were identified as dimethylarsinic acid (DMAV), DMAIII(GS), and dimethylarsinothioyl glutathione (DMMTAV(GS)). Moreover, the method was employed to study the conditions and mechanisms of formation of thiol-containing arsenic metabolites from DMAIII(GS) and DMAV as the mechanisms of formation of these important As species were unknown. The arsenic sulfur compounds studied included but were not limited to the newly discovered human arsenic metabolite DMMTAV(GS) and the unusually highly toxic dimethylmonothioarsinic acid (DMMTAV). It was found that these species may form from hydrogen sulfide produced in enzymatic reactions or by utilizing the sulfur present in protein persulfides. Possible pathways of thiolated arsenical formation were proposed and supporting data for their existence provided. In addition to known mechanism of arsenic toxicity such as protein-binding and reactive oxygen formation, it was proposed that the utilization of thiols from protein persulfides during the formation of thiolated arsenicals may be an additional mechanism of toxicity. The toxicities of DMAV(GS), DMMTAV, and DMMTAV(GS) were evaluated in cancer cells, and the ability of these cells to take the compounds up were compared. When assessing the toxicity by exposing multiple myeloma cells to arsenicals externally, DMMTAV(GS) was much less toxic than DMAIII(GS) and DMMTAV, probably as a result of its very limited uptake (less than 10% and 16% of DMAIII(GS) and DMMTAV respectively).

Pahayokolides A-D are cytotoxic cyclic polypeptides produced by the freshwater cyanobacterium Lyngbya sp. strain 15-2 that possess an unusual β-amino acid, 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configuration of pahayokolides A-D was determined using advanced Marfey’s method. It was also confirmed that a pendant N-acetyl-N-methyl leucine moiety in pahayokolide A was absent in pahayokolides B and pahayokolides C-D were conformers of pahayokolide A. Feeding experiments indicated that the biosynthesis of the Athmu sidechain arises from leucine or α-ketoisovalerate, however could not be further extended by three rounds of condensation with malonate units. Putative four peptide and one unique polyketide synthetases in Lyngbya sp. strain 15-2 were identified by using a PCR method and degenerate primers derived from conserved core sequences of known NRPSs and PKSs. Identification of one unique KS domain conflicted with the logic rule that the long side chain of Athmu was assembled by three rounds of ketide extensions if PKSs were involved. A gene cluster (pah) encoding a peptide synthetase putatively producing pahayokolide was cloned, partially sequenced and characterized. Seven modules of the non-ribosomal peptide synthetase (NRPS) were identified. Ten additional opening reading frames (ORFs) were found, responsible for peptide resistance, transport and degradation. Although the predicted substrate specificities of NRPS agreed with the structure of pahayokolide A partially, the disagreement could be explained. However, no PKS gene was found in the pah gene cluster.

In the 1960s, Rosenberg and his colleagues confirmed the anti-cancer activity of cisplatin. Although cisplatin was capable of killing testicular cancer cells there were also serious side effects. It was necessary to find alternate ways of overcoming side effects, and soon many researchers have discovered novel platinum compounds that show similar reactivity. Recently, replacing one chloride group to a heterocyclic amine group showed significant cytotoxicity with a different binding activity than cisplatin. Previously in our lab, [Pt(Me5dien)(NO3)]+ and [Pt(Et2dien)Cl]+ have been synthesized and reacted with NAcetyl- L-methionine (N-AcMet) and Guanosine 5’-monophosphate (5’-GMP) showed unusual reactivity. Unlike most previously studied platinum triamine compounds, Me5dien compound was reacting faster with 5’-GMP than N-AcMet, due to the bulkiness of the triamine ligand. When both N-AcMet and 5’-GMP were reacted with Et2dien, 5’- GMP displaced one amine group of the triamine ligand and replaced that spot to form a bis-adducts, when the pH was kept below 4. Here a new novel platinum compound has been synthesized with a seven-membered chelate ring triamine ligand, Chloro[2-(4- methyl-1,4-diazepan-1-yl)ethanamine]platinum(II) chloride ([Pt(L)Cl]+). The unusual binding activity of [Pt(L)Cl]+ showed a unique pair of products under 1H NMR, 195Pt NMR and LC/MS spectrometry.

Recent pharmaceutical developments have investigated using supramolecular nanoparticles in order to increase the bioavailability and solubility of drugs delivered in various methods. Modification of the carbohydrate cyclodextrin increases the ability to encapsulate hydrophobic pharmaceutical molecules by forming a carrier with a hydrophobic core and hydrophilic exterior. Guest molecules are commonly added to these inclusion complexes in order to add stability and further increase targeting abilities of the carriers. One such guest molecule is adamantine combined with a poly(ethylene glycol) chain. Vesicles are formed by hydrating a thin film of amphiphilic cyclodextrin and guest molecules in buffer solution that mimics physiological conditions. The solution is subject to freeze-thaw cycles and extrusion, and the complexes are separated out via size exclusion chromatography. Dynamic Light Scattering instrumentation is used to observe the particle size distribution. Cargo release can be observed in fluorescent dye-loaded vesicles by addition of a membrane-cleaving agent under a fluorimeter instrument. Future work involving this drug delivery technology includes synthesizing a chemically sensitive guest that will cleave in the presence of an intra-cellular anti-oxidant, and finally observing the uptake of these vesicles into live cells and testing the delivery of cargo in vitro under physiological conditions.

This study aims to prepare theranostic nanoparticles (NPs) that are expected to increase cancer diagnostics and therapeutic efficacy. We prepared the NPs constituting carbon dots (CDs) as an imaging agent, folic acid as a targeting agent, doxorubicin (DOX), or gemcitabine (GEM) as chemotherapy agents. The NPs include noncovalent FA-CDs-DOX, covalent CDs-FA-DOX, and covalent FA-CDs-GEM. Through ultraviolet-visible spectroscopy, fluorescence spectroscopy, and Fourier transform-infrared spectroscopy, the fabrication of these NPs was confirmed. It was discovered that the high drug loading efficiency is the noncovalent series while the high drug loading capacity is the covalent series The in-vitro pH-dependent drug release data indicate the NPs release more drugs at around pH 5.0 than at pH 7.4. The NPs sizes are between 2-5 nm. The Cell viability was investigated using the Alamar Blue assay and the three NPs complexes exhibited strong therapeutic efficacy against MDA-MB-468 breast cancer cells as compared with CDs-drug.

D-γ-tocotrienol (G-T3) chiefly present in palm oil has stolen the spotlight as a promising radiation protective agent for the treatment of acute radiation syndrome (ARS). Although G-T3 is a promising counteracting agent discovered, the separation and purification from its matrix are painstaking. These have limited its characterization, derivatization, and biomedical application. In this study, we developed chromatographic and distillation methods to enhance the purity and synthesis of G-T3 derivatives. Exactly 8% ethyl acetate (EA) in hexanes used in TLC resulted in right spots separation. Two gradient column chromatographic methods were examined using solvent mixtures of EA/hexanes and EA/petroleum ether (PE), respectively. We verified that a gradient elution with EA in PE led to the maximum purity (≥95%) based on the NMR and GC-MS outcomes. Also, G-T3 was stable in an emulsion to some extent, and some soluble G-T3 derivatives were synthesized, and their structure was confirmed

Man has always searched for greater understanding of himself and his environment. As a result, much scientific investigation has been directed towards the understanding of the life process. In recent years, such scientific inquiry has become increasingly focused on the fundamental biochemical events taking place at the cellular, subcellular, and molecular levels. In the course of investigation into the nature of living things, research has often necessitated the development of new techniques and investigational tools to serve as experimental systems. Very often such new methods have made possible the discovery of previously unknown processes. One very interesting and useful investigational tool which has been developed is the method by which cells and tissues are maintained in vitro.

As a part of the overall program in the Grandy laboratory at Oregon Health & Science University (OHSU), studying the underlying chemical biology of methamphetamine (Meth) addiction, this dissertation reports on the development of six new thyronamine analogs which were synthesized and assayed against trace amine associated receptor 1 (TAAR1), giving preliminary results consistent with the analogs being inverse agonists. Due to highly variable TAAR1 expression levels in the assays, based on inter-assay response to control Meth stimulation as well as other possible factors, kinetic models were developed to qualitatively explain the assay results. The models set approximate limits on the analogs' binding and disassociation rates relative to those of Meth. Analysis of the assays also provides more evidence of TAAR1's basal activity. Based on the models, the conversion rate of ligand-free inactive TAAR1 to ligand-free active TAAR1 is less than 6% of the binding rate of Meth to TAAR1. The models also suggest that the inverse agonists bind to the inactive ligand-free form of TAAR1 between 10 and 100 times faster than Meth binds to the inactive ligand-free form of TAAR1. Three of the new analogs, G5-110s8, G5-112s5, and G5-114s5, bind to the ligand-free active form of TAAR1 faster than they bind to the inactive ligand-free form of TAAR1. The models do not suggest an upper limit on the binding rate of those 3 analogs to the ligand-free active form of TAAR1. A control assay lacking TAAR1 revealed an electrophysiological off-target effect caused by G5-109s8. Also, a novel synthetic route was developed for ET-92, the lead compound for this project, which reduced the number of synthetic steps from 14 to 5 and improved the overall yield from 15.3% to 18.3% (77.4 mg) with the hope that further improvements in yield are possible.

A series of 3-aminotetralins were synthesized as potential opioid antagonists. Each proposed target compound was based on a 3-(mono- or dialkylamino )-7 -(hydroxy or methoxy)-2, 2-dimethyl-1-tetralone parent structure. Three synthetic schemes were developed utilizing the common intermediate, ethyl3-benzylamino-2, 2-dimethyl-4-(4- methoxyphenyl)butyrate 3. In Scheme I, compound 3 was modified through a series of six steps to obtain 3-(N-methyl-N-cyclopropanecarboxamido )-7 -methoxy-2, 2-dimethyl- 1-hydroxy-1-phenyltetralin (9). To carry out further synthetic steps on the intermediate 9 required the reduction of the amide function, which proved to be problematic in terms of product isolation. Scheme II was a four-step procedure, which utilized the intermediate ethyl 3- amino-2, 2 dimethyl-4-(4-methoxyphenyl)butyrate (4), also utilized in Scheme I. Ester hydrolysis of the amino ester 4 produced the amino acid 12. Internal cyclization of 12 yielded the key intermediate, 3-amino-7 -methoxy-2, 2-dimethyl-1-tetralone (13). TheNalkylation step was carried out on 13 and this yielded the target compounds, 3-[N- ( cyclopropylmethyl)amino ]- and 3-[N, N-( dicyclopropylmethyl)amino ]-7 -methoxy-2, 2- dimethyl-1-tetralone (14, 15). Subsequently, compounds 14 and 15 were 0-demethylated to obtain the respective target compounds, 3-[N-(cyclopropylmethyl)amino]- and 3-[N, N-(dicyclopropylmethyl)amino ]-7-hydroxy-2, 2-dimethyl-1-tetralone (16, 17). Scheme III was an alternate synthetic route to obtain the target compounds 3-[Nmethyl- N-( cyclopropylmethyl)amino ]-2, 2-dimethyl-7-(hydroxy or methoxy)-1-hydroxy- 1-phenyltetralin (10, 11) without the amide reduction step required in Scheme I. The intermediate 3 was N-methylated to form the 3-N-methyl-N-benzylamino ester 18 by the Eschweiler-Clarke procedure. Compound 18 was converted through a series of four steps to obtain 3-[ N-methyl-N-( cyclopropylmethyl)amino ]-7 -methoxy-2, 2-dimethyl-1- tetralone (22), a target compound which was 0-demethylated to obtain compound 23, the 7-0H analog. The mono- and dialkylated 3-aminotetralins were synthesized and confirmed for purity and correct molecular formula by utilizing 1H NMR, 13C NMR, mass spectrometry, and elemental analysis. The target compounds 14, 15, 16, 17,22 and 23 were converted to their salts and are being analyzed for opioid-related activity in receptor binding assays.

A new enzyme activity assay has been developed for the target N8-acetylspermidine deacetylase, a not-well-studied but essential enzyme in the polyamine interconversion and reutilization pathway. The enzyme assay, based on mass spectrometric detection of a specific reaction product following sample introduction by flow injection, was shown to have a sensitivity of smaller than 1 micromolar and typical RSD of 3-10%. The linear range for analyte was from 1 μM to 100 μM, with R2 > 0.992. The new assay avoids the use of radio labels. Sample preparation is straightforward, and high specificity is provided by the selected reaction monitoring, SRM, using a triple quadrupole mass spectrometer. Acetylputrescine was used for the first time as the substrate for the assay of N8-acetylspermidine deacetylase in lieu of N8 -acetylspermidine. The crude enzyme extracted from rat liver had an apparent Km value of 80.6 μM for acetylputrescine and a Vmax of 1.1 nmol mg-1 min-1. Enzyme extracted from frozen rat liver was compared with that from fresh rat liver. Frozen rat liver extraction had similar kinetics parameters with the fresh preparation and had a specific activity of 0.8 nmol mg-1 min-1. N8-acetylspermidine deacetylase was partially purified by protein precipitation and gell filtration chromatography. Affinity chromatography was tentatively applied for further isolation of the enzyme, but was not yet successful.