Thesis advisor: Larry W. McLaughlin / Hydration in the minor groove of B-form DNA influences the stability and conformation of duplex DNA. An analogue nucleoside that attempts to mimic the N3-hydrated adenine was designed and synthesized. Obtaining the modified adenine was a significant synthetic effort. The attempted incorporation of the new analogue in a DNA strand yielded inconclusive results, and further tests are ongoing. The incorporation of the new modified purine in a DNA duplex may be useful for a better understanding of the hydration of B-form DNA. / Thesis (MS) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

No description available.

The incidence of cancer worldwide has increased over the years, and gastrointestinal cancers (G.I.) are amongst the most common forms of cancer. Nevertheless, there is still no curative treatments for this group of tumors. Nucleoside analogues are widely used in cancer treatment. The prevailing compounds are Gemcitabine (used for pancreatic cancer and other carcinomas), 5-Fluorouracil (used in breast, colon, and other cancers), Cytarabine and Clofarabine (used in leukemias). Gemcitabine, the current standard of care for various forms of solid tumors, has a limited efficacy against pancreatic cancer. The objective of this project was the development of effective drugs against pancreatic cancer. We focused on a novel class of nucleoside analogues designed to bypass the most common cellular road blocks and resistance mechanisms. After an extensive screen for cell killing activity, two lead molecules were exclusively studied: LCB2151 and LCB2132. These two molecules showed high efficacy in killing human cancer cells from three different human G.I. cell lines: BxPC3 and Capan-2, two pancreatic cell lines representative of K-Ras positive and negative tumors, as well as the liver cell line HepG2. LCB2151 showed high efficacy in killing Gemcitabine-resistant cancer cells, and a low toxicity in normal cells. Interestingly, results show that these prodrugs can efficiently bypass key resistance mechanisms developed by cancer cells. The results obtained in this project are promising and could pave the way for a more effective treatment of pancreatic cancer.

Les analogues de nucléosides représentent une famille thérapeutique très largement utilisée en cancérologie dans le traitement des hémopathies malignes et autres cancers. Leur implication sur les variations des pools des nucléotides endogènes a été largement décrite et a constitué le point de départ de ce travail. C’est dans le but d’étudier ces variations que nous avons développé une méthode de dosage sensible et spécifique des ribonucléotides et désoxyribonucléotides physiologiques intracellulaires. Dans la première partie de ce mémoire nous avons réalisé une revue de la bibliographie sur les méthodes de dosage décrites dans la littérature puis nous avons détaillé le métabolisme des nucléotides endogènes et de leurs analogues. Dans la deuxième partie, nous présentons la validation de notre méthode de dosage et l’application à l’étude des variations des pools endogènes de ribonucléotides et désoxyribonucléotides dans différents modèles cellulaires : lignées résistantes versus lignées parentales et lignées traitées versus lignées non traitées. Les résultats obtenus sur les cellules montrent que notre méthode de dosage permet d’étudier l’impact des analogues nucleosidiques sur les pools endogènes de nucléotides mais aussi les dosages des dérivés phosphates de ces nucléosides / Nucleoside analogues constitute family of drugs widely used in cancer therapeutic for the treatment of malignancy hemopathies and several type of cancer. Numerous studies described their action on the variation of endogen nucleotide pools. This constitutes the point of starting of the present work. A sensible and specific assay for the quantification of intracellular ribonucleotides and désoxyribonucleotides was developed. In the first part, a review concerning the analytical methods published is presented. We also focused on the metabolism of endogen nucleotides and their analogues. In the second part, our method for the quantification of endogen nucleotides and désoxynucleotides is presented from different cellular models: resistant cell lines versus wild type lines, treated cells with analogues versus untreated cells. Results obtain from the cells lines studied show that our analytical method allows to study the impact of nucleoside analogues on endogenous nucleotides, but also the quantification of the phosphate derivatives of these analogues

The C-5 modified pyrimidine analogues are well-known anticancer and antiviral drugs which underscore further development of novel probes to study their physical, chemical, and biological properties. In my dissertation the syntheses and properties of (β-halo)vinyl sulfone and/or (β-keto)sulfone analogues of C-5 modified pyrimidine have been discussed. In the first part of the dissertion, the synthesis of 5-(β-halo)vinyl sulfones either by transition metal-catalyzed or iodine-mediated halosulfonylation reaction of 5-acetylene pyrimidine nucleosides have been explored. The novel (β-chloro/bromo/iodo)vinyl sulfones efficiently undergo addition-elimination reaction with different nucleophiles such as thiols, amines, amino acid, peptides to provide (β-substituted)vinyl sulfone analogues. The rate of these substitution reactions depends on the nature of halogen atom presents at the β-position and increases with the order of I ≥ Br > Cl. (β-chloro/bromo/iodo)vinyl sulfones possess exclusively E stereochemistry while their β-substitued analogues possess either E (for β–thio analogues) or Z (for β–amino analogue) stereochemistry. It has been observed that the vinylic proton of (β-chloro) or (β-amino)sulfone analogue undergoes exchanges with deuterium in polar protic deutorated solvents. The antiproliferative activities of those analogues have been explored and was found that protected 5-(E)-(1-chloro-2-tosylvinyl)-2'-deoxyuridine inhibited the growth of L1210, CEM and HeLa cells in lower micromolar range. In the second part of the dissertation the syntheses and reactivities of 5-(β-keto) sulfone of pyrimidine nucleosides were investigated. Thus, 5-(β-halovinyl)sulfone of uracil and cytosine nucleosides have been efficiently converted into corresponding 5-(β-keto) sulfone analogues by displacement of halogen with ammonia followed by acid-catalyzed hydrolysis of the resulting (β-amino)sulfone analogues. A number of electrophiles were trapped at the acidic α-carbon of the 5-(β-keto)sulfones by treatment with electrophiles such as methyl, benzyl, or allyl halide in the presence of base. The 5-(α-iodo-β-keto)sulfone analogues of uracil nucleosides have been tested as an alternative substrates to probe the incorporation of nucleophiles at α-carbon. In the third part of the dissertation, the synthesis of 5'-phosphates of 5-(β-chloro) and 5-(β-keto) sulfones of 2'-deoxyuridine and their polymerase-catalyzed incorporation into DNA were evaluated. Thus, 5'-O-phosphorylated analogues have been efficiently incorporated into the DNA by human DNA repair polymerase (pol β) or bacterial polymerase (pol I).

Two classes of C5 azido-modified pyrimidine nucleosides were synthesized and explored as radiosensitizers. The 5-azidomethyl-2'-deoxyuridine (AmdU) was prepared from thymidine and converted to its cytosine counterpart (AmdC). The 5-(1-azidovinyl) modified 2'-deoxyuridine (AvdU) and 2'-deoxycytidine (AvdC) were prepared employing regioselective Ag-catalyzed hydroazidation of 5-ethynyl pyrimidine substrates with TMSN3. AmdU and AmdC were converted to 5'-triphosphates AmdUTP and AmdCTP, and incorporated into DNA-fragments via polymerase-catalyzed reaction during DNA replication and base excision repair. Radiation-mediated prehydrated electrons formed in homogeneous aqueous glassy (7.5 M LiCl) systems in the absence of oxygen at 77 K led to site-specific formation of π-type aminyl radicals (RNH•) from AmdU, AmdC, AvdU, and AvdC. The ESR spectral studies and DFT calculations showed RNH• undergo facile conversion to thermodynamically more stable σ-type iminyl radicals, R=N•. For AmdU, conversion of RNH• to R=N• was bimolecular involving α-azidoalkyl radical as intermediate; however, for AvdU, RNH• tautomerized to R=N•. Our work provides the first evidence for the formation of RNH• attached to C5 position of azidopyrimidine nucleoside and its facile conversion to R=N• under reductive environment. These aminyl and iminyl radicals can generate DNA damage via oxidative pathways. The azido-nucleosides were successfully applied as radiosensitizers in EMT6 cancer cells in both hypoxic and normoxic conditions. To explore the generation and reactivity of 2'‑deoxyguanosin-N2-yl radical (dG(N2-H)•) postulated to generate from guanine moiety towards •OH, 2-azido-2'-deoxyinosine (2-N3dI) was prepared by conversion of 2-amino group in protected dG into 2-azido via diazotization with tert-butyl nitrite followed by displacement with azide and deprotection. The investigation of dG(N2-H)• generated from 2-N3dI and its subsequent reactions using ESR will be discussed. Cycloaddition between 5-ethynylpyrimidine or 8-ethynylpurine nucleosides and TMSN3 in the presence of Ag2CO3, CuI, or CuSO4/sodium ascorbate provided N-unsubstituted 1,2,3-triazol-4-yl analogues of the parental DNA bases (i.e. 5-TrzdU, 5‑TrzdC, 8-TrzdA, and 8-TrzdG). These novel triazolyl nucleosides showed excellent fluorescent properties: 8-TrzdA exhibits the highest quantum yield (ΦF) of 44% while 8‑TrzdG had ΦF of 9%. The 5-TrzdU and 5-TrzdC showed a large Stokes shift of ~110 nm. The application of these fluorescent nucleosides to cell imaging and DNA modifications will also be discussed.

No description available.

Trypanosoma brucei is a causative agent of African sleeping sickness. It is an extracellular parasite which circulates in the blood, lymph and eventually invades the central nervous system. There is a great need for new medicines against the disease and specific properties of nucleoside kinases in the pathogen can be exploited as targets for chemotherapy.  T. brucei contains a gene where two thymidine kinase sequences are fused into a single open reading frame. These types of tandem thymidine kinases were found only in different types of parasites, which made us to believe that it might be beneficial for them. Each thymidine kinase sequence in these tandem enzymes are here referred to as a domain. By cloning and expressing each domain from T. brucei separately, we found that domain 1 was inactive and domain 2 was as active as the full-length enzyme. T. brucei thymidine kinase phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine and to some extent purine nucleosides like deoxyinosine and deoxyguanosine. Human thymidine kinase increases the affinity to its substrates when it forms oligomers. Similarly, the T. brucei two thymidine kinase sequences, which can be viewed as a pseudodimer, had a higher affinity to its substrates than domain 2 alone.  T. brucei lacks de novo purine biosynthesis and it is therefore dependent on salvaging the required purine nucleotides for RNA and DNA synthesis from the host. Purine salvage is considered as a target for drug development. It has been shown that in the presence of deoxyadenosine in the growth medium, the parasites accumulate high levels of dATP and the extensive phosphorylation of deoxyadenosine leads to depleted ATP pools. Initially, we wondered if deoxyadenosine could be used as a drug against T. brucei. However, we found that T. brucei is partially protected against deoxyadenosine because it was cleaved by the enzyme methylthioadenosine phosphorylase (MTAP) to adenine and ribose-1-phosphate. At higher concentration of deoxyadenosine, 3 the formed adenine was not efficiently salvaged into ATP and started to inhibit MTAP instead. The deoxyadenosine was then instead phosphorylated by adenosine kinase leading to accumulation of dATP. The MTAP reaction makes deoxyadenosine itself useless as a drug and instead we focused on finding analogues of deoxyadenosine or adenosine that were cleavage-resistant and at the same time good substrates of T. brucei adenosine kinase. Our best hit was then 9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine (FANA-A). An additional advantage of FANA-A as a drug was that it was taken up by the P1 nucleoside transporter family, which makes it useful also against multidrug resistant parasites that often have lost the P2 transporter function and take up their purines solely by the P1 transporter. In parallel with our study of nucleoside metabolism in T. brucei, we also have a collaboration project where we screen essential oils from plants which are used in traditional medicine. If the essential oils are active against the trypanosomes, we further analyze the different components in the oils to identify new drugs against African sleeping sickness. One such compound identified from the plant Smyrnium olusatrum is isofuranodiene, which inhibited T. brucei proliferation with an IC50 value of 3 μM.

Dans le premier chapitre de cette thèse, nous nous sommes intéressés aux virus de l'immunodéficience humaine et des hépatites B et C ainsi qu'aux thérapies utilisées dans le traitement de ces affections. Nous avons introduit l'importance des nucléosides fluorés, et nous avons donné quelques exemples de nucléosides fluorés utilisés en chimiothérapie antivirale et antitumorale. Dans le second chapitre, nous avons présenté une synthèse rapide de 2',3'-didésoxy-3'-fluoro-beta-D-thréo-nucléosides portant les bases pyrimidiques naturelles et substituées en position N3 par un groupement nitro ou amino. Les composés obtenus ont été évaluées contre divers virus à ADN et ARN (y compris le VIH) dans des expériences de culture cellulaire. Dans le troisième chapitre, nous nous somme intéressés à la synthèse de différents 2',3'-didésoxy-2'-fluoro-3'-(N-hydroxyimino), (N-methoxyimino) and (hydroxyl-amino) nucléosides en série pyrimidine. Les composés obtenus ont été évaluées contre divers virus à ADN et ARN dans des expériences de culture cellulaire. / In the first chapter, we presented the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV), as well as the therapies used to treat these diseases. In a second part, we discussed about the importance of the incorporation of fluorine atom into nucleoside analogues, and in a third part of this chapter, we presented the recent literature sources of the synthesis and biological activity of fluorinated nucleosides. In the second chapter, we designed and synthesized a series of 2',3'-dideoxy-3'-fluoro-threo-pyrimidine nucleosides by direct and rapid methodology and evaluated them for their inhibitory effects on a number of RNA and DNA viruses in cell culture experiments. None of these nucleoside derivatives showed any antiretroviral activity nor cytotoxicity. In the third chapter of this manuscript, we synthesized a new series of 2',3'-dideoxy-2'-fluoro-3'-(N-hydroxyimino),(N-methoxyimino) and (hydroxylamino)pyrim idine nucleosides and also evaluated for their inhibitory effects on a number of RNA and DNA viruses, without finding any activity or cytotoxicity.

De par leur grande diversité chimique et leur implication dans de nombreux mécanismes biologiques, les molécules phosphorées font l'objet de nombreuses recherches scientifiques. Depuis près de 30 ans, plusieurs composés phosphorés ont été développés et utilisés pour leurs propriétés médicinales ou phytosanitaires. Les dérivés de type phosphinates ou phosphonates se sont révélés être des composés de choix, grâce à leur grande stabilité chimique et enzymatique. Par ailleurs, les sucres et leurs analogues ont largement démontré leur potentiel biologique en raison de leur implication dans de nombreux processus biologiques. Dans ce contexte, il a été envisagé de développer des analogues de sucres phosphorés possédant un atome de phosphore endocyclique. Il s'agit de structures totalement inédites obtenues à partir de précurseurs phosphorés simples et par des réactions de type : P-alkylation, Pudovik, ouverture d'époxyde, cyclisation par transacétalisation ou transestérification. Grâce à des synthèses en 4 à 7 étapes, deux familles de phosphinosucres ont été préparées ainsi qu'une famille de phosphonosucres en tant qu'analogues de sucre. En parallèle, la synthèse d'analogues de nucléosides phosphorés a également été étudiée par introduction de nucléobases sur les analogues de sucres phosphorés précédemment cités. Ces travaux ont mis en jeu des réactions issues à la fois de la chimie de nucléosides mais également de la chimie du phosphore, permettant d'accéder à des analogues de nucléosides totalement inédits dont l'activité antivirale a pu être évaluée. Enfin, une nouvelle famille de phosphonates nucléosidiques acycliques a été synthétisée en 6 ou 7 étapes en série pyrimidique. Deux prodrogues dérivées de la cytosine et de l'uracile ont également été préparées avec des groupements enzymolabiles de type Bis-(S-acyl-2-thioéthyle). L'ensemble des analogues de nucléotides synthétisés ont été évalués pour leurs propriétés antivirales contre les virus de l'hépatite C et du SIDA. Ainsi, à travers ce projet, trois grandes familles de nouvelles molécules phosphorées ont été développées : les hétérocycles phosphorés oxygénés en tant qu'analogues de sucres, des analogues de nucléosides phosphorés, une nouvelle classe de phosphonates nucléosidiques acycliques. / According to their wide chemical diversity and their implication in many biological processes, phosphorus compounds are intensively studied by organic chemists. Since 30 years, many phosphorus molecules have been developed and used for their biological properties in medicine or agrochemistry. Chemically and enzymatically stable compounds, phosphonates and phosphoninates are potential derivatives for drug design. Besides, sugars are an important biological family involved in numerous biological pathways which have widely revealed a high therapeutic potential. In this context, the first objective of these works was to develop sugar analogues with an endocyclic phosphorus atom (phosphasugars) to synthesize unpublished families of phosphinosugars and phosphonosugars. Their synthesis were carried out in 4 to 7 steps, using as key reactions : P-alkylation, Pudovik, epoxyde ring-opening reaction, cyclisation by transacetalisation or transesterification. Moreover, these phosphasugars were extended to the synthesis of new nucleoside analogues by introducing nucleobase on phosphasugar moiety. The antiviral activity of these new compounds was evaluated. Finally, a new class of acyclic nucleoside phosphonates was prepared in pyrimidinyl series. Different nucleotide analogues and prodrugs were synthesized in 6-7 steps with uracil, thymine and cytosine and evaluated against HCV and HIV.