University of Macau / Institute of Chinese Medical Sciences

by Cheung Yiu-ming. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Includes bibliographies. / ACKNOWLEDGMENT / ABSTRACT / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- REVIEW OF GAS-LIQUID CHROMATOGRAPHY --- p.1 / Chapter 1.2 --- APPLICATION OF GAS-LIQUID CHROMATOGRAPHY IN DRUG ANALYSIS --- p.8 / Chapter 1.3 --- AIMS OF THE PRESENT WORK --- p.9 / REFERENCES --- p.11 / Chapter CHAPTER 2 --- GAS-LIQUID CHROMATOGRAPHIC DETERMINATION OF ACTIVE INGREDIENTS IN COUGH-COLD SYRUP FORMULATIONS --- p.12 / Chapter 2.1 --- INTRODUCTION --- p.12 / Chapter 2.2 --- EXPERIMENTAL --- p.16 / Chapter 2.3 --- RESULTS AND DISCUSSION --- p.22 / Chapter 2.4 --- CONCLUSION --- p.73 / REFERENCES --- p.74 / Chapter CHAPTER 3 --- "GAS-LIQUID CHROMATOGRAPHIC DETERMINATION OF ATROPINE SULFATE/HYOSCYAMINE SULFATE, HOMATROPINE HYDROBROMIDE AND HYOSCINE HYDROBROMIDE IN PHARMACEUTICAL PREPARATIONS" --- p.75 / Chapter 3.1 --- INTRODUCTION --- p.75 / Chapter 3.2 --- EXPERIMENTAL --- p.79 / Chapter 3.3 --- RESULTS AND DISCUSSION --- p.85 / Chapter 3.4 --- CONCLUSION --- p.126 / REFERENCES --- p.127 / APPENDIX --- p.128 / Chapter A.1 --- INTRODUCTION --- p.128 / Chapter A.2 --- EXPERIMENTAL --- p.129 / Chapter A.3 --- RESULTS AND DISCUSSION --- p.132 / Chapter A.4 --- CONCLUSION --- p.147 / REFERENCE --- p.147 / LEGENDS FOR FIGURES / LEGENDS FOR TABLES

Considering the importance of nitrogen-rich heterocycles in drug discovery, a novel strategy towards heterocycle synthesis was envisioned using cyanamide chemistry. Synthesis which involve mild conditions, avoids multi-step sequence and non-toxic reagents are desirable for generation of large combinatorial libraries of drug molecules. We envisaged that the NCN linkage of the cyanamide as well as the concomitant use of the nucleo-and electrophilic centres of the cyanamide could provide a novel synthetic route towards nitrogen heterocycles. The first part (Ch-2) constitute the bulk of the thesis and it focuses on the generation of cyanamide ion and its cyclisative capture with a 1,3-dipole – nitrile oxide in situ. The cycloadduct -1,2,4-oxadiazol-5(4H)-imine was obtained in good yields, which was further transformed into pharmacologically important cores like oxadiazolone and amidines. A library of the different heterocyclic cores was generated, which tolerated a wide variety of functional groups in good to excellent yields. In the second part (Ch-3), we developed a novel protocol for the synthesis of 1,2,4-triazol-3-imine via a formal 1,3-dipolar cycloaddition of in situ generated nitrile imines and cyanamide ion. Further hydrolysis furnished with 1,2,4-triazol-3-one, which is an important core from medicinal chemistry point of view. The concomitant generation and reaction of two reactive species- 1,3-dipoles and cyanamide ion was achieved in a single pot in situ to provide a route towards novel and pharmaceutically important heterocyclic cores. The present work provides a platform for the development of cyanamide derivatives as a ‘single-reagent—diverse-scaffolds’ strategy for time efficient library delivery of structurally diverse molecules.

The khat plant, catha edulis, has been abused for some time in the Middle East and the African horn for its short-term stimulant effects. However, it was not until 1975 when cathinone, β-ketoamphetamine, was identified as the major stimulant component of khat. Structural analogues of cathinone, synthetic cathinones, are new psychoactive substances available on the clandestine market of numerous countries including the USA. Abuse of these new illicit stimulants is a worldwide growing health concern which necessitates the investigation of the pharmacological properties of these new drugs of abuse. The abuse liabilities of these compounds seem to be related to the three major monoamine transporters (MATs): the dopamine, norepinephrine, and serotonin transporters (DAT, NET, and SERT, respectively). Synthetic cathinones act as either releasing agents by stimulating the release of the presynaptic neuronal content of neurotransmitters, or as reuptake inhibitors by inhibiting normal physiological reuptake of neurotransmitters from the synaptic cleft. Bupropion (DAT/NET reuptake inhibitor) is clinically prescribed for the treatment of depression and smoking cessation, whereas its closely related cousin, cathinone (DAT/NET releasing agent), is a drug of abuse. Deconstruction of bupropion (i.e., a stepwise conversion – or structural transition – of bupropion to cathinone) and investigation of the actions of the deconstructed analogues at the three major MATs showed that the steric bulk at the terminal amine controls the molecular mechanisms of these compounds at MATs (i.e. reuptake inhibition versus substrate-induced release). This study also concluded that bupropion is abused, because it is a cathinone derivative. Methcathinone (MCAT), N-methylcathinone, (DAT/NET releasing agent) is a recreational street drug and a US Schedule I substance; however, new MCAT analogues are continually appearing on the clandestine market to circumvent prosecution under the Controlled Substance Analog Enforcement Act. We investigated the actions and structure-activity relationships of a series of 3-substituted MCAT analogues at MATs and their quantitative structure-activity relationships to determine the physicochemical properties of the 3-position substituents important for the releasing actions of these compounds. This study indicated that the steric bulk of the 3-position substituents controls the selectivity of these compounds at MATs.

Atherosclerotic disease is an almost universal phenomenon and increases in severity and frequency with increasing age. Atherosclerosis may contribute to several disorders including bursting of an artery, blockage of an artery, or induction of arterial clotting. The culmination of these diseases is usually premature since at the time of death the unaffected organs are in reasonably satisfactory condition and could have operated for several more years. Among the many factors which act in concert to produce the disease, serum cholesterol levels play a central role. It has been suggested that the lowering of cholesterol levels will provide an effective means of treatment and prophylaxis of atherosclerosis. The purpose of this investigation was to rationally design, synthesize, and evaluate agents to lower serum cholesterol levels by the inhibition of cholesterol biosynthesis at the site of the reduction of β-hydroxy-β-methylglutaryl coenzyme A (HMG CoA) to mevalonic acid. This reaction, mediated by HMG CoA reductase, was chosen as the inhibition target because it is the first irreversible reaction, the rate limiting step for the pathway, and the site of physiological regulation of cholesterol biosynthesis. Rat liver HMG CoA reductase provided a convenient test system for these agents. Using the previously reported compound, 1-(4-biphenylyl)-n-pentyl hydrogen succinate as parent inhibitor, the present study accomplished two goals: first, a contribution toward elucidation of reversible binding sites for these inhibitors and second, probing of the suggested nonpolar n-pentyl binding area of the enzyme by introduction of a functional group capable of alkylating the enzyme and providing irreversible inhibition. The first objective was approached by replacing the ester of the parent inhibitor with an amide functional group. The resulting glutarimide exhibited inhibition comparable to that of the parent inhibitor. This may be taken as evidence that isosteric replacement of the parent ester group with the amide N-H did not seriously alter the ability of the inhibitor to bind to the enzyme and that an additional binding site in this region is not available. The data also support the suggestion that the ester group of the parent agent is not necessary for binding. A similar inhibition study was made possible by synthesis of 1-(4-biphenylyl)-n-pentyl hydrogen 3-methyl-3-methoxyglutarate. With respect to the corresponding 3-methyl-3-hydroxyglutarate this compound showed an eleven fold decrease of activity. This considerable activity loss indicates that the 3-methoxy group interferes with reversible binding of inhibitor to the enzyme. The inhibition data indicate that the 3-hydroxy group of the 3-methyl-3-hydroxy compound contributes to reversible binding by participating as a hydrogen donor in hydrogen bonding with the enzyme. The major portion of this investigation was designed to probe a region of the enzyme which is nonpolar and binds the n-alkyl moiety of the parent inhibitor. The purpose was to determine the feasibility of incorporating a functional group into this region of the inhibitor which could act as an acceptor for an enzymic nucleophile located in proximity to the reversible binding area. If successful, this could provide irreversible inhibition of the enzyme. A series of compounds was synthesized which bore a terminal alkenyl group two to four carbon atoms removed from the ester moiety. Testing showed that, with respect to the parent inhibitor, no appreciable loss of binding took place. Similarly, a series of epoxyalkyl esters was prepared, the epoxide group being the portion of the inhibitor susceptible to nucleophilic attack. Reversible binding of these compounds was found to be equal to that of the parent inhibitor and it was therefore concluded that the enzyme does accommodate this alkylating group with no loss of reversible binding. This provided the necessary preliminary work upon which subsequent irreversible binding studies will be based.

Neurodegenerative diseases are a class of conditions that lead to progressive atrophy of different parts of the central nervous system (CNS). These diseases lead to devastating clinical outcomes to patients and give rise to an enormous socio-economical burden on society.1 One commonality among some of the most well-known neurodegenerative disorders, e.g. Alzheimer’s disease (AD), Parkinson’s disease (PD), and multiple sclerosis (MS), is neuroinflammation.2-4 Neuroinflammation stems from interactions of the innate immune system with toxins and insults to the central nervous system. In the case of irremovable or chronic insults and toxins, this leads to chronic damaging inflammation that hastens neuronal degeneration and exacerbates disease pathology.5,6 Recently, inflammasomes of the innate immune system have been indicated in playing essential roles in the observed inflammatory responses. The most studied inflammasome is the nod-like receptor pyrin containing 3 (NLRP3) inflammasome.7–9 Recently our research group has successfully developed sulfonamide-based small molecule inhibitors of the NLRP3 inflammasome, such as JC-21 and JC-171, as potential therapeutics for AD and MS. Our studies established that JC-21 is a selective inhibitor of the NLRP3 inflammasome.10,11 Structural modifications led to the development of JC-171 with improved pharmacokinetic properties. More importantly, our studies demonstrated the in vivo activity of JC-171 to effectively ameliorate the experimental autoimmune encephalomyelitis (EAE), a mouse model of MS.12 Our data also strongly suggested that inhibitors based on this chemical scaffold may directly target the NLRP3 inflammasome.10–12 In this dissertation, we conducted biophysical, biochemical, and modeling studies to further elucidate the mechanistic information of these compounds as inhibitors of the NLRP3 inflammasome. In order to conduct further mechanistic studies, the NLRP3 protein was produced via transfection of HEK 293 cells with a modified plasmid of full-length human NLRP3 protein.13 Furthermore, LC-MS studies were conducted to confirm the blood-brain barrier penetration (BBB) of JC-171. Our studies established that JC-171 directly binds to the NLRP3 protein. The results also suggested that JC-171 may bind to the NACHT domain of NLRP3 while in a site that is distinct from the ATP binding site. This notion is supported by the fact that our compounds do not interfere with the ATPase activity of NLRP3. Docking studies of JC-171 to the homology model of the NACHT domain of NLRP3 also supported this assertion by showing the interaction of JC-171 with residues that are not overlapping with the ATP binding pocket. BBB penetration studies in combination with LC-MS analysis confirmed that JC-171 shows better BBB penetration when compared to MCC950. Collectively, our results strongly support that our compounds function as NLRP3 inflammasome inhibitors by directly binding to the NLRP3 protein, a novel and distinct mechanism of action when compared to the known inhibitors that target the NLRP3 inflammasome pathway. These results strongly encourage further development of such inhibitors as potential therapeutics for neurodegenerative diseases.

In 1962, Owen, Dietz, and Camiener reported the isolation of a new antitumor antibiotic from the culture filtrate of Streptomyces sparsogenes. The structure of the crystalline antibiotic, named sparsomycin, remained elusive until 1970, when Wiley and MacKellar reported results of spectroscopic and degradation studies which elucidated the structure. In addition to the molecular structure, investigators have examined the mechanism of action, toxicity, and related analogues, striving to establish sparsomycin or a synthetic analogue's usefulness as an effective chemotherapeutic agent. The initial pharmacological evaluation of sparsomycin revealed it possessed activity against KB human epidermoid carcinoma cells, a variety of gram-negative and gram-positive bacteria, and fungi. This broad spectrum of activity prompted a closer examination of the biochemical mechanisms. These studies revealed sparsomycin interfered with protein synthesis by inhibiting peptide bond formation near the enzyme peptidyl transferase. Ottenheijm, Liskamp, and Tijhuis reported the first total synthesis of sparsomycin in 1979, which provided access to greater quantities of the material for investigational use. Sparsomycin was selected for use by cancer patients in phase I clinical trials, but was found to cause ocular toxicity which hindered its development as an antitumor agent. In an effort to reduce or eradicate the toxic effects while maintaining the antitumor activity, analogues of sparsomycin were prepared. Using the sparsomycin analogues which were synthesized, studies were performed to determine the effect alteration of key structural parameters had on the efficacy of the compounds. Previous investigators examined analogues which incorporated modifications of the uracil ring, the unique mono-oxodithioacetal moiety, and the stereochemical configuration of the chiral centers. Vince and Lee reported there was an apparent requirement for the Q-configuration at the asymmetric carbon atom. Overall, however, the small number of sparsomycin analogues prepared and evaluated limited the definitive statements concerning the functional groups required for antitumor activity. In order to expand and clarify the structure-activity relationships, three series of new sparsomycin analogues were prepared for this project. The compounds of Series I and II, distinguished by the inclusion or exclusion of a hydroxymethyl functional group, were designed to elucidate the effect on activity of replacing the mono-oxodithioacetal side chain of sparsomycin with 4-substituted benzyl groups. The Series III analogues, which excluded the hydroxymethyl functional group, featured a 4-substituted benzyl amide group in place of the mono-oxodithioacetal moiety of sparsomycin, and were designed to investigate the potential interaction of an amide oxygen in contrast to the sulfoxide oxygen of sparsomycin. The target compounds synthesized for this project were experimentally examined to quantitate their effects on [75Se]-selenomethionine incorporation as an 125 cell growth, indirect measurement of protein synthesis, and 5-125I-iodo- 2-deoxyuridine incorporation as an indirect measurement of DNA synthesis in bone marrow, P388 lymphocytic leukemia, and P815 mastocytoma cells. The results for the Series I and II analogues indicated the removal of the hydroxymethyl functional group as seen in sparsomycin affected activity to varying degrees depending upon the assay and the type of cells used. The results for the Series III compounds suggested the removal of the hydroxymethyl functional group and substitution of the mono-oxodithioacetal side chain of sparsomycin with a substituted benzyl amide moiety was not beneficial for activity. Finally, examination of the collective data revealed that the bromobenzyl-substituted analogues consistently imparted the greatest inhibitory activity, while the methoxybenzyl-substituted analogues displayed the least. The methyflnnzyl and the unsubstituted benzyl compounds were intermediate in inhibitory potency. The activity may correspond to the lipophilic and electronic characteristics of the substituents on the benzyl moiety of the analogues. It appears that the bromobenzyl-substituent of hydrophobic and electron withdrawing character is optimal for inhibitory activity, and conversely, the methoxybenzyl substituent of hydrophilic and electron donating character is least desirable.

Human immunodeficiency virus (HIV) and opioid abuse have been described as synergistic epidemics. Pharmacologically, it has been found that opioids have the capacity to enhance HIV infection and replication. Research has shown that activation of the mu-opioid receptor (MOR) elevates the expression of the HIV-1 entry co-receptor CXCR4 on T-lymphocytes in the peripheral nervous system, thus allowing for enhanced viral entry and invasion. Although the exact mechanism for opioid modulation of CXCR4 expression and subsequent exacerbation of HIV is unknown, several hypotheses exist. One hypothesis is that MOR and CXCR4 are functionally interacting through the formation of a heterodimer. This hypothesis is supported by studies substantiating the ability for MOR and CXCR4 to form heterodimers with other GPCRs, and the finding that MOR and CXCR4 were co-expressed in several central and peripheral regions including immune cells. To test this hypothesis, a series of bivalent ligands containing both a mu opioid receptor (MOR) antagonist and a CXCR4 antagonist pharmacophore was designed and synthesized to understand the pharmacological role of the putative CXCR4-MOR heterodimer in opioid exacerbated HIV progression. These bivalent ligands were evaluated for their binding and functional activities in radioligand binding, antibody binding, [35S]GTPγS, and calcium mobilization assays. In these assays, the bivalent ligands were shown to maintain binding and functional activities in both MOR and CXCR4 monoclonal cell lines. In addition, these bivalent ligands were evaluated for their ability to block HIV entry in a reverse transcriptase assay, and for their ability to inhibit morphine exacerbated HIV invasion in an LTR-luciferase assay. In these assays, the bivalent ligands were shown to inhibit HIV entry in a dose dependent manner. However, due to experimental limitations in our morphine exacerbated reporter system, the ability for the bivalent ligands to inhibit viral entry upon morphine co-exposure was not fully validated. Finally, molecular modeling approaches were utilized to visualize the putative binding modes of the bivalent ligands in a constructed MOR-CXCR4 heterodimer model. Overall, these studies have provided a solid basis for the utility of bivalent ligands in studying MOR-CXCR4 interactions and their involvement in opioid potentiated HIV progression. Further studies are ongoing to optimize the bivalent ligands construct and explore new analyses to evaluate their ability to block opioid modulation of the virus.

This thesis describes the structure elucidation of drug metabolites in biological samples by the use of high performance liquid chromatography (HPLC) atmospheric pressure ionization (API) tandem mass spectrometry (MS/MS). Due to their different advantages, various mass analyzers have been used in the different experiments. The metabolism of clemastine, flutamide, and meloxicam were studied in vitro and/or in vivo in different species such as humans, dogs, and horses. Accurate mass measurements with the quadrupole-time of flight mass spectrometer and MSn data supplied by the ion trap instrument were useful in the structural investigation of the product ions of the drugs and their metabolites. Different scan modes of the triple quadrupole mass spectrometer resulted in great flexibility, selectivity, and sensitivity in the qualitative and semi-quantitative studies. Additionally, hydrogen/deuterium exchange and experiments with atmospheric pressure chemical ionization were conducted, and the fungus Cunninghamella elegans was utilized to produce amounts of drug metabolites sufficient for structural investigation. Six isomers of oxidized clemastine were detected and characterized in C. elegans incubations and their retention times and mass spectral data were compared to the metabolites detected in urine samples. Two of the metabolites were concluded to be diastereomeric N-oxides. In urine from horses treated with meloxicam, the peak of 5'-hydroxymethylmeloxicam resulted in much higher intensity than the parent drug or the other metabolites, and it was detectable for at least 14 days after the last dose in some of the horses. That is useful information in the development of analytical methods for the detection of prohibited use of meloxicam. A mercapturic acid conjugate of hydroxyflutamide was detected in urine from cancer patients, which indicated that a reactive metabolite was formed. This metabolite could be responsible for the adverse events reported for flutamide. The results from the four papers included in the thesis clearly demonstrate the usefulness and the flexibility of the HPLC-API-MS/MS technique.

Tuberculosis (TB) is a major infectious disease, killing about 2 million people annually throughout the world. Today's TB treatment is a lengthy procedure involving a combination of antibiotics. No new TB drug has been introduced onto the market in the past 40 years, and the emergence of multi- and extensively drug-resistant TB calls for new drugs. Finding new drug targets is important and one such target is the Mycobacterium tuberculosis enzyme glutamine synthetase (GS), which catalyses the formation of glutamine from glutamic acid. In this work, novel GS inhibitors and new Pd(0)-catalyzed methods have been developed. A microwave-enhanced Pd(0)-catalyzed α-arylation reaction was developed using water as solvent, and a phenylglycine scaffold was identified using structure-based design. A series of α-arylated phenylglycine derivates was produced at moderate to good yields. Some of these were biologically evaluated against GS. A novel scaffold, 3-amino-imidazo[1,2-a]pyridine, was identified by high-throughput screening directed towards GS. This type of compound could be easily produced via a Ugi-type, microwave-promoted multi-component reaction in 20 min. The scaffold was investigated by changing one substituent at a time, and in an experimental design where 8 factors were varied in the same design. Several potent inhibitors were identified; amongst them the most potent inhibitor to date (IC50 = 0.38 µM). Two discrete structure-activity relationships were established, and one of the inhibitors was co-crystallized. The first general aminocarbonylation of aryl chlorides and the first aminocarbonylation of alkenyl phosphates were developed. Alkenyl chlorides, bromides and triflates were investigated in the same transformation utilizing Mo(CO)6 as a solid carbon monoxide source. Two different Pd(0)-based catalytic systems were developed. A wide variety of aryl chlorides and amines could be transformed into the corresponding amides with good yields. The alkenyl substrates produced low to good yields.