Thesis (MTech (Chemistry))--Cape Peninsula University of Technology, 2009 / N3-substituted pyrimidine nucleoside derivatives containing either an iodovinyl moiety or an allyl group, i.e. [121]-N3-(3-iodoprop-2-en- l -yl)thymidine, (1~-711 and e21]-N3-(prop-2-enl- yl)-5-iodo-Z'-deoxyuridine, (1~_10, were synthesised and preliminarily evaluated by determining their uptake into CHO-Kl cells. Compound e~-711 was designed to be a delivery vector of 121: into the DNA of the cells, while (1~_10 was designed to serve as a control. Compound (1231]-711 was also intended to have a higher metabolic radiochemical stability than 5-iodo-Z'-deoxyuridine ([123I]_IUdR) for the therapeutic use in cancer. The synthesis of the N3-substituted intermediate precursors 411, 5 and 9 was achieved by Nalkylation of suitably' protected thymidine and 5-iodo-Z'-deoxywidine analogues, respectively. The intmediate precursors for radiolabelling, 411 and 11, were obtained by incorporating a trialkylstannyl group at the respective labelling positions prior to radioidination. (1~1-711 was recovered from (1~_6G after acid-hydrolysis of the protecting groups and 10 was obtained from direct oxidative iodination of11. The radiochemical yields ranged from 73% to 91% at the end ofthe synthesis and radiochemical purities were in excess of99"10 after HPLC purification. The cell-uptakes ofthe radiotracers were carried out and assessed by a direct comparison with the gold standard e23I]-IUdR, which is known to bephosphorylated and incorporated into the DNA of cells during the cells S-phase. The cell-uptake results of (1~_711 and (1~-10 were roughly 4% and 3% relative to [123I]-IUdR, respectively. The poor cell-uptake of (1~_10 suggested thatthe uptake into the cells is not influenced by the position of the iodine atom in the molecule, but most probably by the availability of the N3-position in its non-substituted form. As a result of its poor incorporation into cells, it was concluded that the synthesised radiotracer (1231]_711 would be a poor candidate for use in the eradication ofmalignant cells.

本文描述了碳環核苷的發展過程，同時也描述了將不同環融合在五元碳環上的方法來對碳環核苷的構象進行鎖定。 / 以D-核糖為起始原料，經過12 步反應並以分子內的Diels-Alder 反應 (IMDA)為關鍵步驟合成出了關鍵的中間體192，它的總產率為27%。通過對中間體192中的環己烯的結構進行修飾，經過3-4 步反應成功合成出了7 個具有雙環[4.3.0]壬烷結構的碳環核苷（185-191），它們的構型也通過其X 光單晶結構圖來進行確定。[附圖] / 以一價銅催化端炔和疊氮化物的Huisgen 環加成反應為關鍵步驟，成功地合成出了12 個具有雙環[4.3.0]壬烷結構的碳環三唑核苷。[附圖] / 同時，我們也合成12 個具有雙環[4.3.0]壬烷結構的碳環三唑核苷羧酸。[附圖] / 另外，我們也合成出了两個在三唑環上含有羧酸的碳環三唑核苷（298 和 299）。[附圖] / 最後，我們還合成出了16 個含有大基團叔丁基羧酸酯的碳環三唑核苷(260-263, 273-276, 286-289 和294-297)。[附圖] / In this thesis, a review regarding the development of carbobicyclic nucleosides and conformationally locked carbobicyclic nucleosides by fusing different rings onto the five-member ring was presented. / The key intermediate 192 was synthesized from D-ribose in 12 steps with 27% overall yield, using an Intramolecular Diels-Alder reaction (IMDA) as the key step. By modification of the cyclohexene ring, seven carbobicyclic adenosine analogues 185-191 with a bicyclo[4.3.0]nonane framework were prepared successfully from intermediate 192 in 3 to 4 steps and their conformations were examined by X-ray crystallography [with diagram]. / Twelve carbobicyclic ribavirin analogues 232-239 and 250-253 with a bicyclo[4.3.0]nonane framework were synthesized successfully by using a copper catalyzed azide-alkyne cycloaddition (Huigsen reaction) as the key step [with diagram]. / Another twelve ribavirin analogues bearing a carboxylate group (264-267, 277-280 and 290-293) with a bicyclo[4.3.0]nonane framework were also obtained [with diagram]. / Furthermore, two more ribavirin analogues bearing a carboxylic acid in triazole (298 and 299) with a bicyclo[4.3.0]nonane framework were obtained. [with diagram] / Finally, twelve ribavirin analogues bearing a tert-butyl carboxylate ester (260-263, 273-276, 286-289 and 290-293) with a bicyclo[4.3.0]nonane framework were also obtained [with diagram]. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Fanglin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2015. / Includes bibliographical references (leaves 175-187). / Abstracts also in Chinese.

As a first generation of compounds, the nucleosides adenosine 8, cytidine 11, guanosine 9, inosine 116 and uridine 12, as well as the sugar ᴅ-(-)-ribose 100, were transformed into the corresponding 5’-O-(tert-butyldiphenylsilyl)- and 5’-O-(4,4’-dimethoxytrityl)-derivatives. These were subsequently protected as acetyl, benzoyl and allyl derivatives at various positions on the molecules, to give a range of twenty five unique compounds for biological testing. The nucleoside and corresponding ᴅ-(-)-ribose derivatives were evaluated for their antibacterial activity against two Gram-positive (Staphylococcus aureus ATCC 25923 and Bacillus cereus DL5) and two Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922), for their anti-HIV activity against strain HLTVIIIB as well as for their anticancer properties, by evaluating inhibition of cell proliferation in two adherent (HT-29 and Caco-2) and three suspension (HL-60, Jurkat and K-562) cell lines. From these screens, and based on the 2,3,5-triphenyltetrazolium chloride (TTC) assay, it was found that 5’-O-(tert-butyldiphenylsilyl)uridine 107, 5’-O-(tert-butyldiphenylsilyl)-1'-O-methoxy-ᴅ-(-)-ribose 102 and tert-butyldiphenylsilyl alcohol 145 exhibited antimicrobial activity towards only the Gram-positive bacteria when compared to the ciprofloxacin 153 control. None of the compounds tested showed any antiviral activity when assayed against HIV; however, all compounds indicated some form of toxicity to the uninfected cells. Subsequent cell proliferation studies indicated pronounced activity against both the adherent and suspension cancer cell lines for 5’-O-(tert-butyldiphenylsilyl)uridine 107, 5’-O-(tert-butyldiphenylsilyl)cytidine 134, tert-butyldiphenylsilyl alcohol 145, 5’-O-(4,4’-dimethoxytrityl)uridine 126 and 4,4’-dimethoxytrityl alcohol 147. Our initial screen indicated that ᴅ-(-)-ribose derivatives do not show any significant general biological activity; whereas (tert-butyldiphenylsilyl)-protected nucleoside derivatives and the corresponding tert-butyldiphenylsilyl alcohol control are intrinsically more bio-active. From the data reported for the anti-bacterial and the cell proliferation studies, we concluded that the nucleoside showing the most promising results was uridine 12 and our mini structure- activity study on the uridine derivatives found that the best position for performing modifications to the nucleoside was at the 5'-OH position on the sugar ring. As such, this would become the initial focus for the synthesis of the second generation compounds. The second generation compounds included a series of ten uridine 12 and five 5-methyluridine 233 derivatives which were protected on the primary alcohol with a range of different silicon-containing protecting groups. At the same time, we used a general procedure to synthesize a series of fourteen silanols for use as control compounds. The uridine 12, 5-methyluridine 233 and corresponding silanol derivatives were screened for their antibacterial activity against the same two Gram-positive and two Gram-negative bacteria as above, as well as for their anticancer properties, by evaluating inhibition of cell proliferation in a series of six adherent cell lines (five human: Hs683, MCF-7, PC-3, SKMEL-28, U373, and one murine: B16F10) cell lines. The data obtained for our TTC assay showed that converting the base in 1-[(6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,-2,4]trioxadisilocin-8-yl]-pyrimidine-2,4(1H,-3H)dione 234 to the 5-methyl derivative 254 caused a corresponding loss in antibacterial activity for the compound, whereas oxidising the secondary alcohol on the 2'-position of the sugar ring to give compound 239 caused a corresponding increase in antibacterial activity. As such, we concluded that 1-[(6aR,8R,9aR)-2,2,4,4-tetraisopropyl-9-oxotetrahydro-6H-furo[3,2-f][1,3,5,-2,4]trioxadisilo-cin-8-yl]pyrimidine-2,4(1H,3H)dione 239 was the compound with the best antibacterial activity out all of the first and second generations of nucleoside derivatives assayed. The results obtained in the TTC assay, were supported by our scanning electron (SEM) and confocal scanning electron (CSLM) microscopy studies. Interestingly, the CSLM study suggests that the synthetic compound 239 is bacteriocidal and is inactivating cells, not simply inhibiting their growth. From the inhibition of cell proliferation assay performed on the fifty combined first and second generation derivatives and their corresponding controls, we found that the six most active compounds (5'-O-(tert-butyldiphenylsilyl)adenosine 142, 5'-O-(tert-butyldiphenyl-silyl)cytidine 134, 5'-O-(tert-butyldiphenylsilyl)uridine 107, 2',3'-O-diacetyl-5'-O-(tert-butyl-diphenylsilyl)uridine 123, 2',3'-O-diacetyl-5'-O-(4,4'-dimethoxytrityl)uridine 127 and 3-benzoyl-1-[(6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,-5,2,4]trioxa-disilocin-8-yl]pyrimidine-2,4(1H,3H)-dione 235) had mean IC50 values of approximately 24-28 μM.

It has recently been shown that the binding site of SAHase, an enzyme critical in the replication mechanism of viruses, is quite flexible and exhibits a large difference between the "open" and "closed" conformations, thus presenting an obstacle towards design efforts. As a possible solution to this dilemma, we have strategically designed and synthesized a series of structurally innovative nucleosides possessing a heteroaromatic purine ring split into its two components (for example, an imidazole and pyrimidine ring), thereby conferring additional degrees of conformational freedom and torsional flexibility to the ligand. As a result, these molecular "chameleons" can adapt to the environment of the flexible binding site in order to maximize and complement structural interactions, without losing the integrity of the crucial contacts involved in the enzyme's mechanism of action. The synthesis of several proximal analogues is presented herein.

Nucleic acids are ubiquitous in nature and modified nucleosides are present in a wide range of anti-viral, anti-cancer drugs and antibiotics. Although a variety of naturally occurring nucleoside analogues exist, few include modifications to the ribose or deoxyribose ring. Intriguingly, the uridyl peptide antibiotics (UPAs), such as pacidamycin, contain a biosynthetically unique 3'-deoxyuridine which resembles synthetic anti-retrovirals. Elucidation of the biosynthesis of this structuraly unique nucleoside motif suggests a degree of substrate flexibility, making it a highly attractive prospect for biosynthetic approaches to nucleoside modification. In order to fully exploit the biotransformative potential, a detailed mechanistic understanding of the individual enzymes involved in the biosynthesis of the nucleoside moiety, and especially the enzyme employed at the installation of the 3'-deoxy modification, is required. Chapter 1, the introduction the thesis, discusses the importance of nucleosides for Chemistry and Biology. The section describes the biosynthesis of the nucleoside antibiotics and reviews the recent advances relating to the synthesis and biosynthesis of 3'-deoxy-nucleosides. The Chapter proceeds to describes the biosynthesis of deoxy-sugars, deoxy-nucleosides and nucleotides, reviewing the most common dehydratase mechanisms in addition to examining unusual dehydratases involved in carbohydrate metabolism. Chapter 2, the study of Pac13, the uridine-5'-aldehyde dehydratase of the pacidamyicin nucleoside cluster, is reported. Through detailed functional, structural and kinetic analysis of the wild-type enzyme as well a series of mutants, Chapter 2 provides insight into the mechanism emplyed by this unusual enzyme. Chapter 3 describes the structural and functional analysis of Pac11, the flavin-dependent oxidoreductase of the nucleoside biosynthetic cluster, while Chapter 4 revolves around Pac5, the PLP-dependent aminotransferase. In Chapter 5, the chemical synthesis of fluorinated nucleosides, as probes for exploring the enzymes' mechanism is investigated. Chapter 7 reports the experimental procedures for the research described in this document. The work described in this thesis broadens the understanding of the biosynthesis of deoxy-nucleosides and constitutes the first structural and mechanistic study of the biosynthesis of the biosynthesis of the valuable yet, synthetically challenging 3'-deoxy nucleosides.

The thesis is divided in to five chapters. Chapter I: Synthesis of New thiolevomannosan derivatives In this chapter, a general and efficient method for the synthesis of conformationally locked thiosugars has been discussed. An unprecedented synthesis of a novel thioorthoester and its synthetic utility in glycosylation has been demonstrated. Chapter II: Studies on -Mannosidase Inhibitors The synthesis and evaluation of novel, conformationally locked glycomimic, thiolevomannosan and its analogs sulfoxide and sulfone starting from readily available D-mannose is discussed in this chapter. This is the first report of thiosugar derivatives with enhanced potency compared to kifunensine. Docking and biochemical studies have been discussed. Chapter III: Studies on Novel Cyclic Tetraselenides of Mannose In this chapter, the syntheses and structural properties of novel cyclic tetraselenides starting from mannose have been discussed. These tetraselenides are the first of their kind where all four selenium atoms are arranged in a cyclic manner as the backbone of mannose. X-ray structures have been reported for the tetraselenides. Chapter IV: Novel Chalcogenides of Uridine and Thymidine: Synthesis and Applications An efficient and simple method to synthesise disulfides and diselenides of thymidine and uridine derivatives has been demonstrated in this chapter. The utility of these disulfides in various ring opening reactions as well in Michael addition reactions has been demonstrated. Chapter V: Studies on New, Potent Urease Inhibitors In this chapter, a facile one-pot synthesis of thio and selenourea derivatives under mild conditions by the reaction of amines and commercially available Viehe’s iminium salt in the presence of benzyltriethylammonium tetrathiomolybdate as sulfur transfer reagent and tetraethylammonium tetraselenotungstate as selenium transfer reagent has been discussed. A few of the urea derivatives have shown potent inhibitor activity in the nanomolar range for jackbean urease.

The thesis entitled ‘Studies of NIS Mediated Cyclopropane Ring Opening Reactions in Carbohydrate Chemistry’ is divided into four chapters. Chapter 1: Section 1: Efficient Synthesis of Fused Perhydrofuro[2,3-b]pyrans (and furans) by Ring Opening of 1,2-Cyclopropanated Sugar Derivatives. In this section a general and efficient methodology for the synthesis of carbohydrate derived perhydrofuro[2,3-b]pyrans (and furans) from the corresponding 1,2-cyclopropane carboxylates has been discussed. A wide range of linear-fused perhydrofuro[2,3-b]pyran or furan ring systems are encountered in a number of biologically active natural products. A few approaches are available for the construction of this kind of fused motifs which involve harsh reaction conditions and lengthy reaction sequence. The methodology utilizes the potential ability of cyclopropanated sugars to undergo N-iodosuccinimide (NIS) mediated electrophilic ring opening assisted by the pyran ring oxygen followed by intramolecular trapping of oxonium intermediate to generate the furan ring system. Cyclopropantion of tribenzyl glucal using methyl diazoacetate and catalytic amount of dirhodiumtetracetate furnished corresponding exo-1,2-cyclopropane carboxylate exclusively. To generate a nucleophile, cyclopropane carboxylate ester was reduced to the corresponding alcohol which upon treatment with NIS in CH3CN underwent ring opening followed by intramolecular ring closure to give the corresponding perhydro[2,3-b]furopyran along with an oxidized product. After various modifications we found that using CH2Cl2 as a solvent gave the expected perhydrofuropyran as the sole product in good yield (Scheme I). The stereochemistry of the product was established on the basis of 1H-1H NOESY experiment. There are many natural products that contain the perhydrofuro[2,3-b]furyl glycal core such as clerodin, jodrelline B and caryoptin, which show insect anti-feedant properties. With this in mind, the methodology has been successfully extended to the cyclopropanated tetrahydrofuran derivatives resulting in the synthesis of furofuryl glycal moiety (Scheme II). Scheme II Chapter 1: Section 2: Synthesis of Carbohydrate Derived Fused Perhydrofuro/pyrano[2,3-b]-γ-butyrolactones. In this section a general and efficient methodology for the synthesis of carbohydrate derived perhydrofuro/pyrano[2,3-b]-γ-butyrolactones has been discussed. The fusion of the γ-butyrolactone onto a substituted tetrahydrofuran/pyran ring makes a distinctive class natural diterpenoids. Representative members of this family include the marine diterpenoids norrisilide and miniolutelide A. In this chapter we describe a neutral and general method for the construction of perhydrofuro/pyrano[2,3-b]-γ-butyrolactones by NIS mediated ring opening of carbohydrate derived 1,2-cyclopropane carboxylic acids (Scheme III). Scheme III The present strategy is complementary to the existing methods and it is useful since it incorporates an additional chiral center in the molecule under milder conditions, which can be used for further transformations. Chapter 2: Ring Opening of Activated Cyclopropanes with NIS/NaN3: One-pot Synthesis of C-1 Linked Pseudo Disaccharides. Ring opening reactions of activated cyclopropanes have been widely used in organic synthesis. But they are restricted to only selected nucleophiles such as alcohol/ water, as most of the ring opening reactions need acidic activation. This chapter deals with studies of reactivity of various activated cyclopropanes with NIS as a neutral activator and sodium azide as a source of nitrogen nucleophile (Scheme IV). Scheme IV We have clearly demonstrated not only the importance of the donor-acceptor feature in the cyclopropanes in the electrophilic ring opening reaction, but also the selectivity in its functionality. Scheme V This methodology has been successfully utilized in a one-pot synthesis of C-1 linked pseudo-disaccharides from carbohydrate derived 1,2-cyclopropane carboxylates (Scheme V). Chapter 3: Synthesis of Unnatural C-2 Amino Acid Nucleosides Using NIS Mediated Ring Opening of 1,2-Cyclopropane Carboxylated Sugar Derivatives. In this chapter, we have efficiently demonstrated the utility of NIS mediated regioselective ring opening of carbohydrate derived donor-acceptor cyclopropanes for the synthesis of C-2 amino acid nucleosides. This leads to a new class of analogs of peptidyl nucleosides (Scheme VI). Scheme VI One of the advantageous factors is the attachment of nucleobase as well as generation of amino acid precursor in the same reaction which avoids lengthy reaction sequence. We have also shown the synthetic utility of our methodology to pyrimidine based furanosyl C-2 amino acid nucleosides which are of interest, since polyoxins having similar structural core exhibit antifungal activity (Scheme VII). Chapter 4: Attempts Towards the Synthesis of Carbohydrate Derived Spiro-perhydrofuropyrans Using NIS Mediated Cyclopropane Ring Opening Reaction. In this chapter we present various attempts to synthesize spiro-perhydrofuropyran/furans by ring opening of spiro-cylopropane derivatives and attempts towards stereoselective synthesis of spiro-cyclopropane carboxylates. Spiroacetal can be synthesized from the corresponding exo-cyclopropyl methanol, which can be obtained from the corresponding exo- cyclopropane carboxylate. The cyclopropyl carboxylate can be obtained from an exo- vinyl ether. Cyclopropanation of carbohydrate derived exo-glycal failed to give any selectivity under a variety of reaction conditions (Scheme VIII). Carbohydrate derived C1-unsaturated ester on cyclopropanation reaction using standard conditions (Pd(OAc)2/CH2N2) was found to be inert. The reaction under Simmons-Smith cyclopropanation conditions also gave similar results. Reduction of the ester part of the molecule to the corresponding alcohol was found to be helpful in the Simmon-Smith cyclopropanation reaction (CH2I2, Et2Zn) to obtain the corresponding exo-cyclopropane, but disappointingly without any selectivity (Scheme IX). In order to get exo-cyclopropane carboxylate with high stereoselectivity, we decided to use one of the hydroxyl group present in the molecule, as a chiral auxiliary. All the established methods for the diazoester formation failed to attach diazo ester at C-4 position (Scheme X). Scheme X (For structural formula pl see the pdf file)