Student Number : 0011001D - PhD thesis - School of Chemistry - Faculty of Science / The subject of this thesis is the development of a potentially simple, general and economical synthetic protocol for the potent antimalarial alkaloid febrifugine (1) and its analogues. In Chapter 1, the interesting history of 1, which includes a description of several reported total syntheses of 1, is discussed. Natural products derived from 1, as well as promising synthetic derivatives that display good antimalarial activity, are also discussed. The structure-activity relationships determined to date for 1 and its derivatives are presented in order to substantiate the need for the development of new synthetic strategies towards derivatives of 1 and its 3″-unsubstituted analogue, (±)- deoxyfebrifugine (14). A brief overview of the general strategies used in the Organic Chemistry Group at the University of the Witwatersrand for the synthesis of alkaloids is given in Chapter 2. The Eschenmoser sulfide contraction reaction between a thiolactam and an α- bromocarbonyl compound results in the formation of a vinylogous amide (or “enaminone”) product, which can be further manipulated to produce commonly encountered alkaloidal molecular skeletons. The chosen approach to 1 is based on reaction between the pivotal bromide 3-(3-bromo-2-oxopropyl)quinazolin-4(3H)-one (105) and suitable 3-substituted piperidine-2-thiones. A series of model studies, described in Chapter 3, was performed in order to test the feasibility of the synthetic strategy. These studies resulted in a new preparation of the key bromide 105 and a new and efficient synthesis of (±)-deoxyfebrifugine (14). Enaminone analogues derived from five-, seven-, eight-, nine- and thirteen-membered thiolactams were also prepared for comparison. An interesting difference in the sensitivity of the five- and six-membered model cyclic vinylogous amides towards reducing agents was observed. Whereas three piperidine analogues of 14 could be prepared in high yields by the chemoselective hydrogenation of six-membered cyclic vinylogous amide precursors, the five-membered analogues either reacted nonselectively under various standard hydrogenation conditions, or resisted reduction entirely. An extension of the approach towards the synthesis of a 3″-amino analogue of febrifugine (1) from L-ornithine is discussed in Chapter 4. Several 3-aminated piperidin-2-ones and thiones were prepared, but the subsequent enaminones were stable and characterizable only when the piperidinylidene ring nitrogen was alkylated. However, chemoselective reduction of the enaminone C=C bond in 3-{(E)-3-[(3S)-3- (tert-butoxycarbonylamino)-1-(4-methoxybenzyl)piperidin-2-ylidene]-2-oxopropyl}- quinazolin-4(3H)-one (221), an interesting 3-acylamino dehydro analogue of 1, did not give the desired azafebrifugine, but instead yielded a 5,6,7,8-tetrahydro-1Hpyrido[ 3,2-c][1,2]oxazine derivative. Several approaches to febrifugine (1) itself based on the successful model studies are discussed in Chapter 5. Initially, stereoselective α-bromination and subsequent substitution by oxygen of piperidin-2-ones derived from the chiral auxiliary (S)- phenylglycinol was explored. Unexpected racemization occurred at the α-position of the lactam during this route. A second approach to 3-hydroxypiperidin-2-one from Larginine was also problematic. Finally, the utility of α-hydroxylation methodology (including Davis methodology) on N-substituted piperidin-2-ones was explored. This route yielded a range of 3-oxygenated lactams and thiolactams, many of which displayed optical activity. The crystal structures of several 3-substituted thiolactams were determined and compared. However, attempts to apply the sulfide contraction procedure to these precursors were unsuccessful. Some investigations designed to explore the structural differences between vinylogous amides derived from the quinazoline-containing bromide 105 and thiolactams of different ring sizes are discussed in Chapter 6. Single crystal X-ray diffraction and NMR spectral data are compared for this series of compounds, the results revealing that the enaminone group in the five-membered ring derivative 3- [(3Z)-2-oxo-3-(pyrrolidin-2-ylidene)propyl]quinazolin-4(3H)-one 155 possesses a significantly different electronic distribution to the other analogues in the series.

Die Schlafkrankheit hat ihren Schrecken seit den Zeiten Robert Kochs und Paul Ehrlichs nicht verloren. Die zielgerichtete Entwicklung neuer Medikamente ist für die Menschen in den Endemiegebieten damals wie heute von elementarer Bedeutung. Die Naphtylisochinolin-Alkaloide stellen eine neue chemische Substanzklasse dar, die gute Kandidaten für die Entwicklung neuer Medikamente enthält. Mit GBAP 94 im speziellen liegt eine Substanz vor, die gute Startvorrausetzungen hierfür mitbringt. Diese sind eine sehr gute Wirksamkeit gegen Trypanosomen, gepaart mit einer hohen Selektivität durch einen sehr wahrscheinlich relativ spezifisch anti-trypanosomalen Wirkmechanismus. Die verwendeten Naphtylisochinolin-Alkaloide GBAP 94 und GBAP 146 wurden nach unterschiedlichen Gesichtspunkten ausgewählt. GBAP 94 wurde aufgrund seiner guten antitrypanosomalen Wirkung und seiner hohen Selektivität für Trypanosomen ausgewählt. Die IC50 liegt mit 0,383 µmol/l im Vergleich zu den aktuell verwendeten Medikamenten sehr niedrig. Die Selektivitätsindices (IC50 Trypanosoma brucei brucei / IC50 Makrophagen J774.1) mit 85,6 und (IC50 Try-panosoma brucei brucei / IC50 Leishmania major) mit 15,1 liegen in einem sehr günstigen Bereich. GBAP 146 wurde hauptsächlich wegen seiner guten Fluoreszenz-Eigenschaften ausgewählt. Die antitrypanosomale Aktivität ist mit einer IC50 von 0,289 µmol/l zwar sehr gut, eine große Selektivität ist aber nicht gegeben. Die beiden Alkaloide waren aufgrund ihrer Eigenfluoreszenz gut fluoreszenz-mikroskopisch in den Parasiten zu detektieren. Nach 10 min war in den ersten Trypanosomen die Anreicherung der Wirkstoffe erkennbar. Nach 30 min war bei fast allen Parasiten eine Färbung erkennbar. Die Wirkstoffe reicherten sich zunächst in mehreren kleinen Vakuolen an. Bei längeren Inkubationszeiten zeigte sich eine fast homogene Verteilung innerhalb des kompletten Parasiten. Durch-gängig ausgespart blieb eine vakuolische Struktur. Diese entwickelte oder vergrößerte sich im Verlauf der Inkubationszeit im vorderen Drittel des Parasiten, etwa im Bereich des Kinetoplasten. Diese Vakuole konnte auch lichtmikroskopisch in der Giemsa-Färbung nachgewiesen werden. Der Anteil der veränderten Trypanosomen lag bei diesen Untersuchungen nach 1 h bei 25,4%, stieg bis zum Zeitpunkt 2 h auf 46,6% und stabilisierte sich nach 4 h bei 44,8%. Die vakuolische Struktur führte durch ihre Vergrößerung zur zunehmenden Verplumpung der Trypanosomen bis zu einer kugelförmigen Zellform mit geisselartig-wirkender Flagelle. Aufgrund der veränderten Form wurden die Zellorganellen verdrängt. Dies konnte durch die Fluoreszenzmarkierung des Mitochondriums mit Rodamine B Hexylester und der sauren Kompartimente, besonders des Lysosoms, mit LysoTracker® gezeigt werden. Die Vakuolisierung von Trypanosomen im Zusammenhang mit Apoptose ist bekannt. Die neu entstehende Vakuole konnte weder mit LysoTracker® green, noch mit dem endosomalen Farbstoff FM 4-64 angefärbt werden. Damit können eine lysosomale und eine endosomale Herkunft der Vakuole ausgeschlossen werden. Eine genaue Klärung der Genese der Vakuole steht noch aus. In den Untersuchungen mit Annexin V und Propidium-Jodid im FACS® konnte gezeigt werden, dass die Wirkung der NIQs sehr wahrscheinlich Apoptose induziert. Annexin V ist auch bei Trypanosomen als Marker für Apoptose etabliert. Zudem zeigte sich ein Anstieg der Anzahl apoptotischer Trypanosomen mit Periode von 6 h – 8 h. Diese Dauer entspricht ungefähr der Dauer des trypanosomalen Zellzyklus. Ein Eingriff der NIQs in den Zellzyklus ist somit sehr wahrscheinlich. Eine Hemmung von Teilen des Zellzyklus ist als Auslöser für Apoptose bekannt. Über die genaue Zielstruktur der NIQs kann allerdings nur spekuliert werden. Die apotose-induzierende Wirkung anderer Alkaloide auf Trypanosomen ist inzwischen nachgewiesen. Ein weiteres Indiz ist, dass die Ergebnisse von Ponte-Sucre mit den NIQs bei Leishmanien ebenfalls in Richtung Apoptose weisen. / The trypanosomiasis is still an emerging problem in sub-Saharan Africa. Due to the limitations of the currently used drugs and emerging drug resistance, there is an urgent need for the target-oriented development of novel therapies. Naturally occurring naphthylisoquinoline alkaloids (NIQs), axially chiral acetogenic products derived from tropical plants, have been investigated for their activity against Trypanosoma brucei brucei TC 221. The NIQ N-(3'-Methoxyphenyl)-6,8-dimethoxy-1,3-dimethylisochinoliniumtetrafluoroborate seems to be quite specific antitrypanosomal agent. This compound shows a low IC50-value of 0.383 µmol/l against Trypanosoma brucei brucei TC 221 in comparison to the current drugs. For controls another NIQ, N-(4'-N'-Dansylaminophenyl)-6,8-dimethoxy-1,3-dimethylisochinoliniumtrifluoro-acetate, eflornithine an amphotericin B, witch is described to induce apoptosis in trypanosomes, were used. Both NIQ could be detected directly because of their self-fluorescence in the fluorescence-microscopy. After 10 min an accumulation in the first parasites could be detected. After 30 min almost all parasites show the compounds. After an initial accumulation in small vesicles the NIQ spread homogeneous over nearly the whole parasite. Only a vacuole was spared. This structure developed or increased during incubation time. It was located in the front part of the parasite near the kinetoplast. This vacuole could also be detected in light-microscopy of Giemsa-stained parasites. The fraction of the affected trypanosomes was after 1 h 25.4% and increased up to 46.6% after 2 h and stayed almost in this level (44.8% after 4 h). The increase of the vacuole induced a dumpier up to spherical shape. The organelles were displaced. This could be shown by fluorescence-labelled mitochondria, stained with rodamine-B-hexylester, and the acidic compartments, especially the lysosome stained with LysoTracker®. The vacuolisation of trypanosoma brucei is described during apoptosis. The staining of the developing vacuole wasn’t possible neither with LysoTracker® nor with the endosomal staining FM 4-64®. A lysosomal or endosomal origin of this vacuole could be excluded. The genesis of this vacuole needs further investigation. In the FACS®-investigations with annexin V and propidium-iodide staining we got strong hints that the NIQs induce apoptosis. Annexin V is established as a marker for apoptosis in trypanosome. We found an increase of apoptotic parasites in a 6 h – 8 h period. This is also the time for the trypanosomal cell cycle. NIQs seem to interfere with the cell cycle. This is descried from various authors as a trigger for apoptosis. The target structure is however still unknown. Results of other groups indicate an apoptosis-inducing effect of alkaloids in trypanosoma or leishmania.

In the present research, nicotine alkaloid production by Nicotiana tabacum (tobacco) hairy roots and tropane alkaloid production by Hyoscyamus niger hairy roots were investigated. The first objective of this research was to improve the oxygen mass transfer in hairy root cultures with microbubbles. Oxygen was shown as a critical nutrient for the growth of tobacco and H. niger hairy roots. In a 1-liter fermentor, microbubble dispersion improved the oxygen mass transfer, tobacco hairy root growth, and nicotine production in the medium. In a novel ground-joint column bioreactor, microbubbles enhanced the oxygen mass transfer and the growth of H. niger hairy roots. The second objective of this research was to enhance the release of alkaloids from the hairy roots into the culture medium. In a l-liter fermentor, nicotine concentration in medium was improved by adjusting the medium pH to 6. Unlike the nicotine alkaloid, hyoscyamine concentration in medium was not detectable at medium pH 6, whereas hyoscyamine in medium increased to 42 mg l-1 at medium pH 3. Similar to the hyoscyamine, scopolamine in medium increased from 0.1 to 11 mg l-1 when the medium pH was adjusted from 6 to 3. The release of alkaloids into culture medium provides opportunities to isolate a high-value alkaloid directly from the culture fluid, and reduces the cost of product recovery.

We have successfully developed an efficient approach to 3,4-disubstituted £\,£]-unsaturated £_-lactams from glutarimide, and proved to be applicable for the synthesis of hydrocinchonine and hydrocinchonidine.

Marine natural products biogenetically derived from the pyrrole-imidazole oroidin (i) display fascinating structural diversity and exhibit a wide range of significant biological activities. Hence, they have attracted great interest from the synthetic community leading to the development of new synthetic methodologies and providing an admirable set of strategies and tactics to assemble the most complex representatives of this class of alkaloids. In this work, a synthesis of the potent antitumor dibromophakellstatin (ii) was devised featuring a highly diastereoselective acylation of the C2-symmetric prolyl-prolyl anhydride (iii), an intramolecular Mitsunobu reaction installing the C6 aminal and a tandem Hofmann rearrangement/cyclization that delivered the targeted imidazolidinone of the phakellstatins. Both enantiomers of phakellstatin were prepared starting from either D- or L- proline. Building on several findings made in the course of the synthesis of phakellstatin, namely the differential reactivity of the aminal centers (C6 and C9) and the formation of an oxidized phakellstatin, a second generation approach towards these deceitfully simple alkaloids was initiated. The second generation synthetic strategy towards phakellstatin (iv) and phakellin (v) utilizes a novel variation of Du Bois' C-H insertion involving a urea or a guanidine. This approach appears more amenable to the annulation of the phakellin substructure for the final stages of the synthesis of the potent immunosuppressant palau'amine (vi).

Piperine, the major alkaloid derived from black pepper corns, has played an important role in traditional medicine worldwide. Current research has demonstrated piperine to have several anti-inflammatory properties, however, little is known concerning the effect of piperine on B cells. Spleen-derived murine B cells were cultured in the presence or absence of piperine during T-dependent or T-independent activation. Piperine reversibly inhibited B cell proliferation in a dose-dependent manner. This was due to a G0/1-phase cell cycle arrest, and was associated with a reduction in phospho-ERK, phospho-AKT, and Cyclin D1, D2, and D3. Piperine also inhibited antibody and cytokine production. Furthermore, piperine treatment diminished B cell-mediated antigen presentation determined by measuring OT-II transgenic T cell proliferation in response to OVA, which was attributed to the decreased MHC-II ad co-stimulatory molecule expression observed. This in vitro study shows that piperine has potent immuno-suppressive effects on B cell activation and effector function.

Loline alkaloids (LA) are secondary metabolites produced by Epichloandamp;euml; (anamorph, Neotyphodium) grass endophytes. They are toxic and deterrent to a broad range of herbivorous insects but not to livestock. This protective bioactivity has spurred considerable research into the LA biosynthetic pathway. LOL, the gene cluster containing nine genes, is required for LA biosynthesis. The regulation of LOL genes during LA production in culture and in symbio is of interest. In this study, coordinate regulation between LOL gene expression and LA production level was investigated in both MM culture and symbiota. Results showed that expression of LOL genes in N. uncinatum MM culture were tightly correlated with each other (p andamp;lt; 0.0005), and all presented a significant temporal quadratic pattern during LA production. Gene expression started before LA were detectable, and increased while LA accumulated. The highest gene expression level was reached before the highest amounts of LA were detected, and gene expression level declined to a very low level after amounts of LA plateaued. Observations suggested that the hierarchical clusters based on the correlation coefficient could help to predict the roles of LOL genes in the LA pathway. In symbiota, coordinate coregulation of LOL gene expression with LA was found in E. festucae-meadow fescue inflorescences and stromata, whereby lower LOL gene expression corresponded with the lower LA level in stromata. In N. uncinatum (or N. siegelii)-meadow fescue vegetative tissues, dramatically higher LA levels were found in younger leaf tissue than in older leaf tissue, yet no evidence was found to relate this difference to LOL gene expression differences. Instead, substrate availability may regulate the LA level. In particular, asparagine was more than 10-fold higher in young leaf tissue than in old tissue, although proline was significantly lower in young tissue. Therefore, different regulatory mechanisms underlie LOL gene expression and LA production in different circumstances. The GUS activity of Pro-lolC2-GUS and Pro-lolA2-GUS in Neotyphodium species was almost undetectable in culture, though the activity could be detected in symbiota. The mRNA of GUS did not exhibit the same pattern as lolC2 or lolA2 in culture during LA production time course. A Pro-lolC2-cre transgene was expressed in complex medium, in which lolC2 mRNA was not detectable. These results suggest that proper regulation of LOL genes in culture or symbiota is dependent on the LOL cluster.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacognosy Candidate: Michaela Teplanská Supervisor: PharmDr. Daniela Hulcová, Ph.D. Title of diploma thesis: Derivatives of Amaryllidaceae alkaloid vittatine as potential drugs Haemanthamine type Amaryllidaceae alkaloids are characterized by interesting biological activity. This group also includes alkaloid vittatine with antitumor, antibacterial, antifungal and antimalarial effects. Although vittatine does not inhibit cholinesterases, its derivatives have shown promising activity against butyrylcholinesterase, which is one of the targets of potential drugs in the treatment of Alzheimer's disease. Another series of semisynthetic vittatine derivatives was prepared in order to examine their biological activity. Reactions with acyl chlorides gave 11 aromatic esters. Identification of the prepared substances was performed by ESI HRMS, NMR and optical rotation measurements. The derivatives were tested for inhibitory activity against human cholinesterases. The results show that the substances were not active against acetylcholinesterase, but almost all of them inhibited butyrylcholinesterase. The most active was 3-O-(6-chloro-2- fluoro-3-methylbenzoyl)vittatine with an IC50 value 0.29 ± 0.03 μM. According to the calculated value...

The role of the labile methyl groups of betaine in the transmethylation reactions in the barley plants was investigated using carbon-14 methyl labelled betaine. The N-methyl groups of N-methyl tyramine, hordenine and choline were found to arise from betaine. Betaine was also administered to castor bean seedlings and the alka­loid ricinine isolated. It was found that its N- and 0-mthyl groups did not arise from betaine methyl. The origin of the methylenedioxy and N-methyl groups of the alkaloid protopine was also investigated by the tracer technique. Carbon-14 methyl labelled L-methionine, carbon-14 labelled choline and carbon-14 labelled sodium formate were fed to Dicentra species and the extent and position of labelling of the protopine molecule determined by degradation / Thesis / Master of Science (MS)

<p> An investigation of flabelliformine, an alkaloid obtained from Lycopodium flabelliforme, led to the establishment of its structure. </p> <p> A study of the mass spectra of annotine (a minor alkaloid present in Lycopodium annotinum) and several of its derivatives in combination with degradative work gave support to a total structure proposed previously for the alkaloid.</p> <p> A synthetic approach to the Lycopodium alkaloids was considered. 1-Carbomethoxy-3-methyl-7-methoxybicyclo [3.3.1] non-3-en-9-one, a compound which appeared suitable as an intermediate in the synthesis, was prepared in low yield. The difficulty encountered in the purification of this compound, coupled with the low yield in which it was obtained, indicated the impracticality of this approach to a total synthesis of a Lycopodium alkaloid.</p> / Thesis / Doctor of Philosophy (PhD)