Pyrimidine Nucleosides Research Articles

Introduction of pseudo-base benzimidazole derivatives into nucleosides via base exchange by a nucleoside metabolic enzyme

In alternate organic synthesis, biocatalysis using enzymes provides a more stereoselective and cost-effective approach. Synthesis of unnatural nucleosides by nucleoside base exchange reactions using nucleoside-metabolizing enzymes has previously shown that the 5-position recognition of pyrimidine bases on nucleoside substrates is loose and can be used to introduce functional molecules into pyrimidine nucleosides. Here we explored the incorporation of purine pseudo bases into nucleosides by the base exchange reaction of pyrimidine nucleoside phosphorylase (PyNP), demonstrating that an imidazole five-membered ring is an essential structure for the reaction. In the case of benzimidazole, the base exchange proceeded to give the deoxyribose form in 96 % yield, and the ribose form in 23 % yield. The reaction also proceeded with 1H-imidazo[4,5-b]phenazine, a benzimidazole analogue with an additional ring, although the yield of nucleoside was only 31 %. Docking simulations between 1H and imidazo[4,5-b]phenazine nucleoside and the active site of PyNP (PDB 1BRW) supported our observation that 1H-imidazo[4,5-b]phenazine can be used as a substrate by PyNP. Thus, the enzymatic substitution reaction using PyNP can be used to incorporate many purine pseudo bases and benzimidazole derivatives with various functional groups into nucleoside structures, which have potential utility as diagnostic or therapeutic agents.

Biocoordination reactions in copper(II) ions and phosphocholine systems including pyrimidine nucleosides and nucleotides

The complexation reactions of phosphocholine and pyrimidine nucleosides as well as nucleotides with copper(II) ions were studied in the water system. Using potentiometric methods and computer calculations, the stability constants of the species were determined. Using spectroscopic methods such as UV-vis, EPR, 13C NMR, 31P NMR, FT–IR and CD, the coordination mode was established for complexes created in pH range 2.5–11.0. These studies will lead to a better understanding the role of copper(II) ions in living organisms and explain the interactions between them and the studied bioligands. The differences and similarities between nucleosides and nucleotides in the studied systems were also described, which testify to the significant influence of phosphate groups on the processes of metal ion complexation and interactions between ligands.

ATRT-09. COMBINATION OF THE PI3K INHIBITOR PAXALISIB WITH THE NUCLEOSIDE ANALOG GEMCITABINE ACTIVATES THE INTEGRATED STRESS RESPONSE AND EXTENDS SURVIVAL IN ORTHOTOPIC MODELS OF ATYPICAL TERATOID/RHABDOID TUMORS

Abstract Relapsed atypical teratoid/rhabdoid tumors (AT/RT) are a deadly disease without a known cure. The Pacific Pediatric Neuro-Oncology Consortium (PNOC) is looking for effective combination therapies for an upcoming clinical trial to better treat relapsed AT/RT. We have previously identified that AT/RT are dependent on activation of mTORC1 and mTORC2 for growth and survival. Paxalisib is a highly brain penetrant PI3k inhibitor which acts upstream of mTOR to simultaneously inhibit mTORC1/2. In murine orthotopic xenograft models of AT/RT, Paxalisib significantly extends survival as a single agent therapy (CHLA-06: 40 to 54 days, p=0.001; BT12: 21 to 35 days, p=0.02). However, to improve the durability of this single agent therapy, we evaluated the efficacy of rational therapeutic partners. Analysis of RNAseq following mTOR inhibition (mTORi) showed an upregulation of genes associated with the integrated stress response (ISR) (ATF4, CHOP, PPP1R15A; t-test p<0.05). Gemcitabine is a pyrimidine nucleoside prodrug, which inhibits DNA synthesis and induces DNA damage, also leading to ISR activation. While the ISR can be protective, intense or prolonged activation leads to cell death. We therefore hypothesized that Paxalisib would combine with Gemcitabine to over-activate the ISR and drive AT/RT cell death. Through protein analyses, we demonstrate that combination therapy further activates the ISR compared to DMSO control (Western Blot: phospho-eIF2α, ATF4, and CHOP). Combination therapy also increases apoptosis (Western blot cPARP, MUSE Annexin V Assay) and synergizes to decrease AT/RT cell growth (SynergyFinder BLISS score CHLA-06: 16.8, BT12: 14.3, CHLA-266: 13.9). In mice bearing CHLA-06 orthotopic tumors, Paxalisib/Gemcitabine combination therapy slows tumor growth (Bioluminescent imaging) and extends median survival from 22 to 82.5 days (p<0.0001) compared to vehicle control treated mice. We aim to translate these results in this upcoming PNOC clinical trial to treat relapsed AT/RT.

Increased renal elimination of endogenous and synthetic pyrimidine nucleosides in concentrative nucleoside transporter 1 deficient mice

Concentrative nucleoside transporters (CNTs) are active nucleoside influx systems, but their in vivo roles are poorly defined. By generating CNT1 knockout (KO) mice, here we identify a role of CNT1 in the renal reabsorption of nucleosides. Deletion of CNT1 in mice increases the urinary excretion of endogenous pyrimidine nucleosides with compensatory alterations in purine nucleoside metabolism. In addition, CNT1 KO mice exhibits high urinary excretion of the nucleoside analog gemcitabine (dFdC), which results in poor tumor growth control in CNT1 KO mice harboring syngeneic pancreatic tumors. Interestingly, increasing the dFdC dose to attain an area under the concentration-time curve level equivalent to that achieved by wild-type (WT) mice rescues antitumor efficacy. The findings provide new insights into how CNT1 regulates reabsorption of endogenous and synthetic nucleosides in murine kidneys and suggest that the functional status of CNTs may account for the optimal action of pyrimidine nucleoside analog therapeutics in humans.

Chemical Diversification of Carbocyclic Fluorinated Pyrimidine Nucleosides: Introducing 2′-Arabino Analogues and Ring Unsaturation

AbstractAnalogues of the canonical nucleosides have a longstanding presence and proven capability within medicinal chemistry and drug-discovery research. Herein, we report chemical diversification of carbocyclic pyrimidine nucleosides containing CF2 and CHF in place of the furanose oxygen to introduce ring unsaturation and 2′-epimers. Utilizing gram-scale access to 6′-(R)-monofluoro- and 6′-gem-difluorouridine, we explore the provision of 2′,3′-didehydro-2′,3′-dideoxy, and 1′,2′-didehydro-2′-deoxy analogues, alongside the first example of a 6′-(R)-fluoro arabino-carbauridine. Key stereochemistries and the presence of unsaturation are confirmed using X-ray crystallography and NMR, and an indicative conformational preference for a monofluoro 2′,3′-didehydro-2′,3′-dideoxy system is presented. This synthetic blueprint offers a potential to explore biological activity for these hitherto unavailable materials, including a direct comparison to established nucleoside analogue drugs.

Preparation of bicyclo[1.1.1]pentane-derived nucleoside analogues

Nucleoside analogues are prevalent in drug design and call for more diversified structures. Bicyclo[1.1.1]pentane (BCP) structure has recently found wide applications in drug discovery. However, incorporation of BCP fragment into nucleoside analogues is hitherto unknown. Thus, from readily available BCP-containing building blocks, six new compounds, including pyrimidine nucleoside analogues, purine nucleoside analogues, and C-nucleoside analogues were prepared in 1–4 steps, generally with good yields.

Determination of Gemcitabine and Sorafenib in Spiked Human Plasma Using Multivariate Model Update Chemometric Methods.

Gemcitabine (GEM), a pyrimidine nucleoside, has been used as a first-line treatment in non-small-cell lung cancer (NSCLC). Sorafenib (SOR), a nonselective multi-kinase inhibitor, is used as a chemotherapeutic agent in different types of cancers including NSCLC in preclinical studies. Co-administration of GEM and SOR was found to be effective and well-tolerated in the treatment of NSCLC. The aim of the present work is to determine the studied drugs in spiked human plasma simultaneously through resolving the overlapping spectra and removing the interference of the plasma matrix. Two updated chemometric models were developed using UV absorbance of the drugs, which named principal component regression (PCR) and partial least-squares (PLS) for determination of GEM and SOR in the ranges of 5-25 and 2-22 µg/mL, respectively. Validation of the two updated models has been achieved in accordance with US Food and Drug Administration (FDA) guidelines, and the results were satisfactory. The two methods had the advantages of high predictive ability of the studied drugs with high precision and accuracy. Moreover, there was no significant difference obtained when statistical comparison was done between the developed and reported methods, showing good validity of the suggested methods. The two updated models have the advantages of being rapid, accurate, sensitive, and cost-effective for the determination of GEM and SOR in quality control laboratories without any need for initial separation procedures. Two updated chemometric methods, PCR and PLS, were developed for the estimation of GEM and SOR in spiked human plasma using their UV absorbance data.

Chemistry of NH2OH and its related species in the ISM

ABSTRACT Hydroxylamine (NH2OH) is a key precursor of both the formation of amino acids and the concurrent synthesis of pyrimidine and purine nucleosides. Recently, NH2OH was detected towards the quiescent molecular cloud G+0.693-0.027, which is located in the Galactic Centre. In this study, we construct the chemical network for NH2OH and its related species via quantum chemical calculations, and simulate their chemistry using the astrochemical code Nautilus in isothermal models and shock models. According to the disagreement distance formula and molecular abundance ratios, we acquire the best-fitting isothermal model T18 and the best-fitting C-shock model S2, respectively. Of these two models, S2 is more suitable for G+0.693-0.027 due to its reproduction of abundances of all considered molecules. From the model, we found that NH2OH and CH3NH2 are mainly formed on grain surfaces, while HNCN and CH2NH, which are the post-shock tracers, are produced by both gas-phase and grain-surface reactions. Our chemical modelling also agrees with the observed abundances or upper limits of NH2OH and its related species towards IRAS 16293-2422 B and Sgr B2(N). Moreover, we predict that NH2OH and the undiscovered species H2NO and HNOH are of high abundance, thus are detectable in cold clouds and pre-stellar cores, and in G+0.693-0.027, respectively.

Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases

Enzymes from thermophilic organisms are interesting biocatalysts for a wide variety of applications in organic synthesis, biotechnology, and molecular biology. Next to an increased stability at elevated temperatures, they were described to show a wider substrate spectrum than their mesophilic counterparts. To identify thermostable biocatalysts for the synthesis of nucleotide analogs, we performed a database search on the carbohydrate and nucleotide metabolism of Thermotoga maritima. After expression and purification of 13 enzyme candidates involved in nucleotide synthesis, these enzymes were screened for their substrate scope. We found that the synthesis of 2'-deoxynucleoside 5'-monophosphates (dNMPs) and uridine 5'-monophosphate from nucleosides was catalyzed by the already known wide-spectrum thymidine kinase and the ribokinase. In contrast, no NMP-forming activity was detected for adenosine-specific kinase, uridine kinase, or nucleotidase. The NMP kinases (NMPKs) and the pyruvate-phosphate-dikinase of T.maritima exhibited a rather specific substrate spectrum for the phosphorylation of NMPs, while pyruvate kinase, acetate kinase, and three of the NMPKs showed a broad substrate scope with (2'-deoxy)nucleoside 5'-diphosphates as substrates. Based on these promising results, TmNMPKs were applied in enzymatic cascade reactions for nucleoside 5'-triphosphate synthesis using four modified pyrimidine nucleosides and four purine NMPs as substrates, and we determined that base- and sugar-modified substrates were accepted. In summary, besides the already reported TmTK, NMPKs of T.maritima were identified to be interesting enzyme candidates for the enzymatic production of modified nucleotides.

Chemoenzymatic synthesis of bridged homolyxofuranosyl pyrimidine nucleosides: Bicyclic AZT analogues

A greener chemo-enzymatic methodology has been developed for the synthesis of conformationally restricted diastereomeric homolyxofuranosyl pyrimidines (AZT analogue), i.e., (5′R)-3′-azido-3′-deoxy-2′-O,5′-C-bridged-β-d-homolyxofuranosyl-uracil and thymine starting from inexpensive diacetone-d-glucofuranose in 18% and 21% overall yields, respectively. In one of the key steps in multistep synthesis of bicyclic AZT analogues, the primary hydroxyl group of 3′-azido-3′-deoxy-β-d-glucofuranosyl pyrimidines has been acetylated using Novozyme® 435 in THF in 92% and 97% yields, respectively. The monoacetylated nucleoside was converted to desired bicyclic AZT analogue in two steps in an overall yield of 82% and 83%, respectively.

Abstract 5490: Combination of the PI3k inhibitor Paxalisib with the nucleoside analog Gemcitabine over-activates the integrated stress response and induces atypical teratoid/rhabdoid tumor cell death

Abstract We have previously identified high activation of both mTORC1 and mTORC2 in the aggressive infantile brain tumor, atypical teratoid/rhabdoid tumor (AT/RT). Paxalisib is a highly brain penetrant PI3k inhibitor which acts upstream of mTOR to simultaneously inhibit mTORC1/2. Paxalisib is well-tolerated in ongoing pediatric clinical trials. In murine orthotopic xenograft models of AT/RT, Paxalisib significantly extended survival (CHLA-06: 40 to 54 days, p=0.001; BT12: 21 to 35 days, p=0.02). However, to improve the durability of this single agent therapy, we evaluated the efficacy of rational therapeutic partners. Analysis of RNAseq following mTOR inhibition (mTORi) of 4 cell lines representative of AT/RT showed an upregulation of genes associated with the integrated stress response (ISR), (ATF4: CHLA-06, t-test p<0.05; CHOP: CHLA-02, CHLA-06 t-test p<0.001). The activation of the ISR is further suggested through induction of PPP1R15A, a gene whose activation is closely associated with stress events and leads to downstream cell growth arrest and apoptosis (BT37 t-test p<0.001, CHLA-02, CHLA-06, CHLA-266, t-test p<0.01). Gemcitabine is a pyrimidine nucleoside prodrug, which inhibits DNA synthesis and induces DNA damage. As a result, Gemcitabine also induces phosphorylation of eIF2α and the activation of the ISR. While the ISR can be protective, intense or prolonged activation leads to cell death. We therefore hypothesized that Paxalisib would combine with Gemcitabine to hyper-activate the ISR and drive AT/RT cell death. Through Western blot analysis, we demonstrate that combination therapy increases phospho-eIF2α, ATF4, and CHOP compared to DMSO control. Furthermore, we demonstrate that combination therapy increases apoptosis (Western blot cPARP, MUSE Annexin V Assay) and synergizes to decrease AT/RT cell growth (SynergyFinder BLISS score CHLA-06: 16.8, BT12: 14.3, CHLA-266: 13.9, BT37: 11.4, CHLA-05: 10.4). Ongoing treatment of mice bearing CHLA06 orthotopic tumors demonstrate that combination therapy further extends median survival. Our studies suggest that pharmacologically activating the ISR may be an effective strategy to target AT/RT and combination of Paxalisib and Gemcitabine may be an effective treatment to help extend survival in this deadly disease. Citation Format: Tyler Findlay, Kristen Malebranche, Anupa Geethadevi, Eric Raabe, Charles Eberhart, Jeffrey Rubens. Combination of the PI3k inhibitor Paxalisib with the nucleoside analog Gemcitabine over-activates the integrated stress response and induces atypical teratoid/rhabdoid tumor cell death. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5490.

A Unifying Concept for the Prebiotic Formation of RNA Pyrimidine Nucleosides

AbstractThe question of how nucleosides might have formed as essential precursor molecules on the early Earth is one of the many challenges associated with the origin of life. In this context, the prebiotic synthesis of pyrimidine nucleosides is controversially discussed. For the pyrimidines, two at first glance contradictory prebiotically plausible reaction pathways have been proposed, based on either oxazole or isoxazole chemistry. This study shows that these two reaction sequences can be merged under prebiotically reasonable conditions, suggesting that both pathways could have co‐existed and possibly interacted. The key precursor 3‐aminoisoxazole was found to react with the key intermediate of the oxazole route (ribo‐2‐(methylthio)oxazoline), to form a ribo‐isoxazole‐oxazoline hybrid structure, which collapses upon reductive N−O bond cleavage to give the nucleoside cytidine. The data suggest that different, interacting prebiotically plausible chemical pathways may have created the key molecules of life on the early Earth.

Synthesis of 1,2,3‐Triazole‐Linked Hexopyranosylpyrimidine Nucleosides and Their Application as Hepatitis B Viral DNA, HBsAg and HBeAg Suppressants

AbstractA series of novel 1,2,3‐triazole‐linked hexopyranosyl mono/double‐headed pyrimidine nucleosides from D‐glucose was synthesized by Cu(I)‐mediated [3+2] cycloaddition reactions (CuAAC) of mono/diazido 2,6‐anhydro‐glucoheptitol and propynylated pyrimidines in good yields. It was observed that all the four synthesized pyrimidine nucleosides were found to be non‐cytotoxic upto 500 μM concentration. The 1,2,3‐triazole‐linked hexopyranosyl mono/double‐headed pyrimidine nucleosides have shown significant suppression of Hepatitis B surface antigen (HBsAg) and Hepatitis B e‐antigen (HBeAg). Amongst all the synthesized compounds, nucleoside dimer 1‐[1‐(6′‐deoxy‐6′‐(4‐(1‐methyluracil)‐1,2,3‐triazol‐1‐yl)‐β‐D‐glucopyranosyl methyl)‐1,2,3‐triazol‐4‐yl]methyl‐uracil was found to be more effective against HBeAg. In the suppression of viral DNA level, all the four synthesized nucleoside analogues were comparable to known drug Entecavir (ETV) with IC50 value in the range of 8.39 μM–18.58 μM and are two folds significant as compared to control.

5′-CMP and 5′-UMP alleviate dexamethasone-induced muscular atrophy in C2C12 myotubes

Atrogin-1 and muscle RING finger 1 (MuRF1) are ubiquitin ligases specifically expressed during skeletal muscle atrophy and mediate muscle protein degradation. In contrast, PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), which is a master regulator of mitochondrial biosynthesis, protects skeletal muscle from atrophy. Pyrimidine nucleoside 5′-monophosphates, such as cytidine 5′-monophosphate (5′-CMP) and uridine 5′-monophosphate (5′-UMP), induce PGC-1α expression and promote myotube formation in mouse C2C12 cells. In this study, we determined the effect of 5′-CMP and 5′-UMP on muscular atrophy in C2C12 myotube cells. 5′-UMP decreased Atrogin-1 and MuRF1 mRNA levels that were upregulated by dexamethasone treatment. 5′-CMP and 5′-UMP ameliorated dexamethasone-mediated atrophy in C2C12 myotubes. Furthermore, the combination of 5′-CMP and 5′-UMP further alleviated dexamethasone-mediated atrophy. In addition, cytidine and uridine, the precursors of 5′-CMP and 5′-UMP, markedly ameliorated dexamethasone-mediated atrophy. Considering nucleotide metabolism and absorption, the active metabolites underlying the observed effects of 5′-CMP and 5′-UMP appear to be cytidine and uridine. Our results indicate that 5′-CMP alleviates muscle atrophy by activating PGC-1α and differentiation, and 5′-UMP alleviates muscle atrophy by suppressing the activation of the myolytic system, whereas the combined use of both enhances the muscle atrophy inhibitory effect. 5′-CMP and 5′-UMP may be an effective and safe treatment for muscular atrophy.

Why 6-Iodouridine Cannot Be Used as a Radiosensitizer of DNA Damage? Computational and Experimental Studies

Previous density functional theory (DFT) studies on 6-brominated pyrimidine nucleosides suggest that 6-iodo-2'-deoxyuridine (6IdU) should act as a better radiosensitizer than its 5-iodosubstituted 2'-deoxyuridine analogue. In this work, we show that 6IdU is unstable in an aqueous solution. Indeed, a complete disappearance of the 6IdU signal was observed during its isolation by reversed-phase high-performance liquid chromatography (RP-HPLC). As indicated by the thermodynamic characteristics for the SN1-type hydrolysis of 6IdU obtained at the CAM-B3LYP/DGDZVP++ level and the polarizable continuum model (PCM) of water, 6-iodouracil (6IU) was already released quantitatively at ambient temperatures. The simulation of the hydrolysis kinetics demonstrated that a thermodynamic equilibrium was reached within seconds for the title compound. To assess the reliability of the calculations carried out, we synthesized 6-iodouridine (6IUrd), which was, unlike 6IdU, sufficiently stable in an aqueous solution at room temperature. The activation barrier for the N-glycosidic bond dissociation in 6IUrd was estimated experimentally using an Arrhenius plot. The stabilities in water calculated for 6IdU, 6IUrd, and 5-iodo-2'-deoxyuridine (5IdU) could be explained by the electronic and steric effects of the 2'-hydroxy group present in the ribose moiety. Our studies highlight the issue of the hydrolytic stability of potentially radiosensitizing nucleotides which, besides having favorable dissociative electron attachment (DEA) characteristics, must be stable in water to have any practical application.

Abstract P552: Mendelian Randomization Analysis of Metabolites Associated With Severe Obesity in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL)

Obesity remains a global public health burden, with 4.7 million premature deaths globally attributed to obesity. Severe obesity (SevO: defined as body mass index (BMI)≥40) is a major risk factor for other comorbidities, including heart disease and Type 2 Diabetes, which disproportionately impact historically marginalized populations, including Hispanic/Latinos. Based on BRFSS data, 24.5% of US Hispanic/Latino adults are projected to have severe obesity by 2030. However, the etiology of the underlying metabolic dysfunction remains unknown. To address this gap, we identified metabolites associated with SevO in genotyped participants aged ≥20 with 25 < BMI ≥ 40 and metabolic data in the Hispanic Community Health Study/Study of Latinos (HCHS/SOL). We investigated cross-sectional associations between Blom-transformed metabolites and baseline SevO, adjusting for age, study center, background group, smoking status, and principal components of ancestry, stratified by sex (SUGEN), and meta-analyzed (SAS 9.4). For the top 20 metabolites, we extracted publicly available HCHS/SOL metabolite GWAS (mGWAS) summary statistics to derive metaboQTLs, and summary statistics from a multi-population meta-analysis of SevO (N>70,000) and implemented forward MR analysis using the MendelianRandomization R package (v0.3.0), which provides various robust causal estimation methods for summary data. Anthropometry and data for 640 known Metabolon metabolites were available for 551 females (mean age: 43.3 years, 27% SevO) and 371 males (mean age 43.4 years, 15% SevO). We identified Bonferroni-corrected significant SevO associations (p<0.05/640) for 224 metabolites in the meta-analysis. Of the top 20 metabolites associated with SevO, we excluded six with no genome-wide significant SNPs in the mGWAS. For the remaining 14, we included independent SNPs with p<1E-6 as instrumental variables in the MR for each metabolite. After excluding SNPs ±500kb from known BMI loci, two metabolites showed causal associations with SevO, cytidine (penalized robust MR-Egger: ß (SE) =0.121 (0.051), p=0.018) and indoleproprionate (penalized weighted median-MR: ß (SE) =-0.138 (0.043), p=0.001). Cytidine is a pyrimidine nucleoside consisting of D-ribose and cytosine, which is a precursor of cytidine triphosphate required in the one-carbon metabolism pathway to convert phosphatidylcholine (PC) to phosphatidylethanolamine (PE). PC biosynthesis is higher in adipose tissue macrophages in obese mice and humans. Indoleproprionate is a microbial metabolite of tryptophan produced by gut bacteria. Indoleproprionate levels have been shown to be associated with higher microbiome diversity and lower incidence of T2D. Our work points to future efforts to validate findings in other cohorts, including reverse MR to further elucidate the causal relationship between metabolites and severe obesity.

Design, synthesis, and evaluation of 4'-phosphonomethoxy pyrimidine ribonucleosides as potential anti-influenza agents.

Influenza viruses belong to the Orthomyxoviridae family and cause acute respiratory distress in humans. The developed drug resistance towardexisting drugs and the emergence of viral mutants that can escape vaccines mandate the search for novel antiviral drugs. Herein, the synthesis of epimeric 4'-methyl-4'-phosphonomethoxy [4'-C-Me-4'-C-(O-CH2 P═O)] pyrimidine ribonucleosides, their phosphonothioate [4'-C-Me-4'-C-(O-CH2 P═S)] derivatives, and their evaluation against an RNA viral panel are described. Selective formation of the α- l-lyxo epimer, [4'-C-(α)-Me-4'-C-(β)-(O-CH2 -P(═O)(OEt)2 )] over the β- d-ribo epimer [4'-C-(β)-Me-4'-C-(α)-(O-CH2 -P(═O)(OEt)2 )] was explained by DFT equilibrium geometry optimizations studies. Pyrimidine nucleosides having the [4'-C-(α)-Me-4'-C-(β)-(O-CH2 -P(═O)(OEt)2 )] framework showed specific activity against influenza A virus. Significant anti-influenza virus A (H1N1 California/07/2009 isolate) was observed with the 4'-C-(α)-Me-4'-C-(β)-O-CH2 -P(═O)(OEt)2 -uridine derivative 1 (EC50 = 4.56 mM, SI50 > 56), 4-ethoxy-2-oxo-1(2H)-pyrimidin-1-yl derivative 3 (EC50 = 5.44 mM, SI50 > 43) and the cytidine derivative 2 (EC50 = 0.81 mM, SI50 > 13), respectively. The corresponding thiophosphonates 4'-C-(α)-Me-4'-C-(β)-(O-CH2 -P( S)(OEt)2 ) and thionopyrimidine nucleosides were devoid of any antiviral activity. This study shows that the 4'-C-(α)-Me-4'-(β)-O-CH2 -P(═O)(OEt)2 ribonucleoside can be further optimized to provide potent antiviral agents.

Estimation of the activity of modified pyrimidine nucleoside derivatives on bacteria cells

The increase in prevalence of antimicrobial-resistant bacteria (ARB) is currently a serious threat, thus there is a need for new classes antimicrobial compounds to combat infections caused by these ARB. The growth inhibition ability of derivatives of the components of nucleic acids has been well-characterized but not for its antimicrobial characteristics. It was found that modified nucleosides arabinofuranosylcytosine (cytarabine, ara-C), [1-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)- 4-(1,2,4-triazol-1-yl)]uracil (TTU), and nucleotides cytarabine-5′-monophosphate (ara-CMP), and O2,2′-cyclocytidine-5′- monophosphate (cyclocytidine monophosphate, cyclo-CMP) were able to inhibit Escherichia coli, Sarcina lutea, Bacillus cereus, and Proteus mirabilis strains in a time and dose dependent manner via killing kinetics assay. It was demonstrated that studied modified pyrimidine nucleosides derivatives enhanced the production of intracellular reactive oxygen species (ROS) over time (validated via DCFA-DA probe assay). This study has revealed the mechanism of action of cytarabine, cyclocytidine monophosphate, and TTU as an antimicrobial agent for the first time, and has shown that these pyrimidine derivatives enhanced might be able to combat infections caused by E. coli, S. lutea, B. cereus, and P. mirabilis in the future.

Mechanism for dissociative hydrolysis of pyrimidine nucleoside d4N: Inversion vs retention

Employing the microhydration model that involves two to five explicit water molecules, two plausible dissociative hydrolysis pathways of 2′,3′-didehydro-2′,3′-dideoxyuridine (d4U), α-path with configuration-inversion and β-path with configuration-retention, have been investigated by M06-2X(CPCM)/6–31++G(d,p) method. Using this model, inclusion of three explicit water molecules (n = 3) is shown to be the smallest system that gives the minimal activation free energy for α-path and β-path. Our results suggest that the glycoside cleavage is the RDS, and α-path is more favorable kinetically than β-path. Whereas β-path with exergonic formation of β-dihydrofuran-like sugar with keto pyrimidine complex possesses thermodynamic preference over α-path, where the formation of α-dihydrofuran-like sugar with enol pyrimidine complex is endergonic. The free energy barriers of RDSs for d4U (25.8 kcal mol−1) and d4T (27.3 kcal mol−1) suggest that the glycosidic bond in d4T is more stable than that in d4U. The relative lower stability of d4U is probably an important factor for less antiviral activity of d4U. The small free energy barrier differences of ∼ 1 kcal mol−1 for β-path over α-path, and the reaction free energy differences of ∼ -12 kcal mol−1 for β-path lower than α-path in d4T and d4U suggest a competitive β-path in pyrimidine d4Ns. The higher free energy barriers of RDSs in ddU (27.6 kcal mol−1) and ddT (29.0 kcal mol−1) indicate that the saturated sugar moiety increases the stability of glycosidic bond in contrast to the unsaturated counter parts in d4U and d4T. NBO analysis also shows the kinetic preference of α-path over β-path. Our results provide an exploration for the less antiviral activity of d4U and the influence of saturated ribose on the glycosidic bond stability of pyrimidine d4Ns.

Cytidine deaminases catalyze the conversion of N(S,O)4-substituted pyrimidine nucleosides.

Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and 2'-deoxycytidine to uridine and 2'-deoxyuridine. Here, we report that prokaryotic homo-tetrameric CDAs catalyze the nucleophilic substitution at the fourth position of N4-acyl-cytidines, N4-alkyl-cytidines, and N4-alkyloxycarbonyl-cytidines, and S4-alkylthio-uridines and O4-alkyl-uridines, converting them to uridine and corresponding amide, amine, carbamate, thiol, or alcohol as leaving groups. The x-ray structure of a metagenomic CDA_F14 and the molecular modeling of the CDAs used in this study show a relationship between the bulkiness of a leaving group and the volume of the binding pocket, which is partly determined by the flexible β3α3 loop of CDAs. We propose that CDAs that are active toward a wide range of substrates participate in salvage and/or catabolism of variously modified pyrimidine nucleosides. This identified promiscuity of CDAs expands the knowledge about the cellular turnover of cytidine derivatives, including the pharmacokinetics of pyrimidine-based prodrugs.