Protection-Free, Two-step Synthesis of C5-C Functionalized Pyrimidine Nucleosides.

A simple, reliable, and efficient method for the gram-scale chemical synthesis of pyrimidine nucleosides functionalized with C5-carboxyl, nitrile, ester, amide, or amidine, starting from unprotected uridine and cytidine, is described. The protocol involves the synthesis of 5-trifluoromethyluridine and 5-trifluoromethylcytidine with Langlois reagent (CF3 SO2 Na) in the presence of tert-butyl hydroperoxide and subsequent transformation of the CF3 group to the C5-C 'carbon substituents' under alkaline conditions. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of 5-trifluoromethyluridine (5-CF3 U) and 5-trifluoromethylcytidine (5-CF3 C) Basic Protocol 2: Conversion of 5-CF3 U and 5-CF3 C to several C5-substituted ribonucleosides.

Design and synthesis of novel 5-alkynyl pyrimidine nucleosides derivatives: Influence of C-6-substituent on antituberculosis activity.

We herein report new 5-substituted uridine derivatives as potent inhibitors of mycobacteria - causative agents of tuberculosis. A series of new 5-alkynyl-substituted uridine derivatives were synthesised via palladium-catalysed Sonogashira cross-coupling reaction of 5-iodo-6-methylpyrimidine base with terminal acetylenes with good yields in DMF at room temperature. It was found that methyl group in C-6 position of pyrimidine ring had no impact on yields of target compounds. All obtained compounds were evaluated for their antimycobacterial activity against Mycobacetrium bovis and Mycobacterium tuberculosis at concentrations of 1-100 µg/ml using MABA test. Synthesized nucleosides showed high antimycobacterial activity against M. bovis and M. Tuberculosis. The MIC50 values of 11 and 13 were similar or close to that of the reference drug rifampicin.

Dissecting the Nucleoside Antibiotics as Universal Translation Inhibitors.

Without question, natural products have provided the lion share of leads, if not drugs themselves, for the treatment of bacterial infections. The bacterial arms race, fueled by selection and survival pressures has delivered a natural arsenal of small molecules targeting the most essential of life processes. Antibiotics that target these critical intracellular processes face the formidable defense of both penetrating a bacterial cell membrane and avoiding efflux to exert their effect. These challenges are especially effective in Gram-negative (Gram-(-)) bacteria, which have a double membrane structure and efficient efflux systems from the combination of outer-membrane porins and inner membrane proton pumps. In this landscape of offense and defense, our clinically used antibiotics have only successfully targeted three intracellular processes for therapeutic intervention in Gram-(-) bacteria: dihydrofolate biosynthesis, transcription, and translation. Not surprisingly, such critical survival machinery is a popular target for bacterial warfare, and eight of our 14 classes of commonly used antibiotics target translation with the bacterial ribosome remaining one the most vetted targets for antimicrobial therapy. On the plus side, its anionic character attracts cationic inhibitors, which are generally more capable of penetrating the bacterial cell wall, and clinical resistance rates are usually manageable as mutation of such a highly evolved machine is difficult. On the down side, this highly evolved machine renders it difficult to inhibit selectively, and the inhibition of prokaryotic translation versus both eukaryotic cellular and mitochondrial translation is critical for clinical development and minimization of undesired toxicities.A class of natural products known as the "nucleoside antibiotics" have historically been recognized as universal inhibitors of the ribosome and can inhibit translation in prokaryotes, eukaryotes, and archaea. While they have served an essential role in dissecting the biochemical underpinnings of the enzymatic functions of the ribosome, they have not proven therapeutically useful as they target the highly conserved rRNA in the P-site and are toxic to mammalian cells. In this Account, we describe our studies on the natural product amicetin, a nucleoside antibiotic that we have demonstrated to break the rule of being a universal translation inhibitor. While the cytosine of amicetin mimics C75 of the 3'-CCA tail of the P-site tRNA akin to other nucleoside antibiotics, we advance a hypothesis that amicetin's unique interaction with the ribosomal protein uL16 exploits an untapped mechanism for selectively targeting the bacterial ribosome. A complex molecule comprised of a nucleoside, carbohydrates and amino acids, amicetin is also chemically unstable. Our initial attempts to stabilize and simplify this scaffold are presented with the ultimate goal of rebuilding the compound with improved penetrance to bacterial cells. If successful, this scaffold would demonstrate a path forward for a new class of antibiotics capable of selectively targeting the ribosomal P-site.

Development of High-Performance Pyrimidine Nucleoside and Oligonucleotide Diarylethene Photoswitches.

Nucleosidic and oligonucleotidic diarylethenes (DAEs) are an emerging class of photochromes with high application potential. However, their further development is hampered by the poor understanding of how the chemical structure modulates the photochromic properties. Here we synthesized 26 systematically varied deoxyuridine- and deoxycytidine-derived DAEs and analyzed reaction quantum yields, composition of the photostationary states, thermal and photochemical stability, and reversibility. This analysis identified two high-performance photoswitches with near-quantitative, fully reversible back-and-forth switching and no detectable thermal or photochemical deterioration. When incorporated into an oligonucleotide with the sequence of a promotor, the nucleotides maintained their photochromism and allowed the modulation of the transcription activity of T7 RNA polymerase with an up to 2.4-fold turn-off factor, demonstrating the potential for optochemical control of biological processes.

Convergent Assembly of Highly Oxygenated Natural Products Enabled by Intermolecular Radical Reactions.

Natural products with a high ratio of sp3-hybridized atoms and oxygen-substituted stereogenic centers represent privileged structures for the development of pharmaceuticals and chemical probes. The multiple oxygen functionalities of these natural products endow their potent and selective biological activities, although they significantly heighten the challenge of their chemical assemblies. We focused on developing efficient strategies for the total syntheses of this biologically and chemically important class of molecules. A convergent strategy is more advantageous than a linear strategy for designing a shorter synthetic route because a convergent strategy enables direct coupling of functionalized fragments whereas a linear strategy involves stepwise construction of a molecule from its terminus. Radical reactions are preferred over polar reactions for the coupling of heavily functionalized and sp3-rich fragments, as they allow for C(sp3)-C(sp3) coupling without damaging diverse polar functional groups. With these considerations in mind, we designed radical-based convergent strategies for assembling highly oxygenated natural products. Here we summarize the concise total syntheses of asimicin (1, antibiotic activity), 1-hydroxytaxinine (2, cytotoxicity), polyoxins (3, antifungal activity), and hikizimycin (4, anthelmintic activity) as representative examples. Retrosynthetic disconnection at the central part of these molecules produces highly substituted α-alkoxy radicals as synthons. In the synthetic direction, the α-alkoxy radicals were generated from the corresponding α-alkoxyacyl tellurides in a unified fashion, and then utilized for four distinct coupling reactions. Formation of the Et radical from Et3B and O2 homolytically cleaves the C-Te bond of α-alkoxyacyl telluride, and the facile expulsion of carbon monoxide from the acyl radical leads to the α-alkoxy radical. Dimerization of the stabilized α-alkoxy radical resulted in the core structure of 1 with 10 contiguous stereocenters. The coupling adduct was derivatized to 1 through the attachment of two different carbon chains (17 steps as the longest linear sequence). Alternatively, intermolecular addition reactions of the α-alkoxy radicals to electron-deficient C═C, C═N, and C═O bonds, followed by Et3B-mediated radical termination, led to the core structures of 2, 3, and 4, respectively. Intermolecular coupling between the α-alkoxy radical and the cyclohexenone derivative and intramolecular pinacol coupling gave rise to the 6/8/6-fused ring system of 2, which was transformed to 2 (26 steps). The two amino acid moieties of 3 were prepared by combining the α-alkoxy radical and the oxime and were then condensed to complete the synthesis of 3 (11 steps). Furthermore, a combination of α-alkoxyacyl telluride and Et3B/O2 realized a novel addition reaction of α-alkoxy radicals to aldehydes. This method was incorporated in the construction of the core 4-amino-5-deoxyundecose with 10 contiguous stereocenters, which was fabricated with two appendage structures to deliver 4. The four total syntheses described here demonstrate the versatility and robustness of intermolecular radical reactions. These syntheses will also provide new insights for retrosynthetic analyses in the field of organic chemistry and streamline synthetic routes to various bioactive natural products with multiple oxygen functionalities.

Extension of furopyrimidine nucleosides with 5-alkynyl substituent: Synthesis, high fluorescence, and antiviral effect in the absence of free ribose hydroxyl groups.

A novel methodology to access alkynyl nucleoside analogues is elaborated. Highly fluorescent 5-alkynylfuropyrimidines were synthesized (97-46%) and their antiviral properties investigated in vitro. Regiochemistry of the functionalization was achieved with the aid of 5-endo-dig electrophilic halocyclization of acetyl 5-p-tolyl- or 5-p-pentylphenyl-2'-deoxyuridine. Structure of one of the resulting nucleosides, 6-p-tolyl-5-iodo-2'-deoxyribofuranosyl-furo[2,3-d]pyrimidin-2-one, was confirmed by X-ray crystallography, and its conformation was compared to related nucleosides. Diverse alkynyl substituents were introduced at the heterobicyclic base C-5 position via Sonogashira coupling of 5-iodo-2'-deoxyribofuranosyl-furo[2,3-d]pyrimidin-2-ones. The resulting compounds had fluorescence emissions of 452-481 nm. High quantum yields of 0.53-0.60 were observed for 9-ethynyl-9-fluorenol and propargyl alcohol/methyl ether-modified furopyrimidines. These modified nucleosides, designed in the form of ribose acetyl esters, are potential tools for fluorescent tagging, studying nucleoside metabolism, 2'-deoxyribonucleoside kinase activity, and antiviral activity. Antiviral assays against a broad spectrum of DNA and RNA viruses showed that in human embryonic lung (HEL) cell cultures some of the compounds posess antiviral activity (EC50 1.3-13.2 µM) against varicella-zoster virus (VZV). The alkynyl furopyrimidine with two p-pentylphenyl substituents emerged as the best compound with reasonable and selective anti-VZV activity, confirming p-pentylphenyl potency as a pharmacophore.

Selective prebiotic formation of RNA pyrimidine and DNA purine nucleosides.

The nature of the first genetic polymer is the subject of major debate1. Although the 'RNA world' theory suggests that RNA was the first replicable information carrier of the prebiotic era-that is, prior to the dawn of life2,3-other evidence implies that life may have started with a heterogeneous nucleic acid genetic system that included both RNA and DNA4. Such a theory streamlines the eventual 'genetic takeover' of homogeneous DNA from RNA as the principal information-storage molecule, but requires a selective abiotic synthesis of both RNA and DNA building blocks in the same local primordial geochemical scenario. Here we demonstrate a high-yielding, completely stereo-, regio- and furanosyl-selective prebiotic synthesis of the purine deoxyribonucleosides: deoxyadenosine and deoxyinosine. Our synthesis uses key intermediates in the prebiotic synthesis of the canonical pyrimidine ribonucleosides (cytidine and uridine), and we show that, once generated, the pyrimidines persist throughout the synthesis of the purine deoxyribonucleosides, leading to a mixture of deoxyadenosine, deoxyinosine, cytidine and uridine. These results support the notion that purine deoxyribonucleosides and pyrimidine ribonucleosides may have coexisted before the emergence of life5.

Chemo-enzymatic route to bridged homolyxofuranosyl-pyrimidines.

Synthesis of 2'-O,5'-C-bridged-ß-d-homolyxofuranosyl nucleosides U and T have been achieved starting from diacetone-d-glucose in overall yields 55.7 and 57.1%, respectively. Quantitative regioselective monoacetylation of the lone primary hydroxyl group in trihydroxy nucleoside intermediate, i.e. 3'-O-benzyl-ß-d-glucofuranosyl nucleosides mediated by Novozyme®-435 has been utilized as the key step in the synthesis of homolyxofuranosyl nucleosides. The structure of the synthesized 2'-O,5'-C-bridged-ß-d-homolyxofuranosyl uracil and -thymine has been established on the basis of their spectral (IR, 1H, 13C NMR and HRMS) data analysis and the structure of earlier nucleoside was confirmed by its X-rays diffraction analysis which revealed that these 2'-O,5'-C-bridged homo-nucleosides are locked into S-type sugar puckering.

Design, Synthesis, and Biological Evaluation of Novel C5-Modified Pyrimidine Ribofuranonucleosides as Potential Antitumor or/and Antiviral Agents.

BACKGROUND: Nucleoside analogues are well-known antitumor, antiviral, and chemotherapeutic agents. Alterations on both their sugar and the heterocyclic parts may lead to significant changes in the spectrum of their biological activity and the degree of selective toxicity, as well as in their physicochemical properties. METHODS: C5-arylalkynyl-ß-D-ribofuranonucleosides 3-6, 3΄-deoxy 12-15, 3΄-deoxy-3΄-C-methyl- ß-D-ribofurananucleosides 18-21 and 2΄-deoxy-ß-D-ribofuranonucleosides 23-26 of uracil, were synthesized using a one-step Sonogashira reaction under microwave irradiation and subsequent deprotection. RESULTS: All newly synthesized nucleosides were tested for their antitumor or antiviral activity. Moderate cytostatic activity against cervix carcinoma (HeLa), murine leukemia (L1210) and human lymphocyte (CEM) tumor cell lines was displayed by the protected 3΄-deoxy derivatives 12b,12c,12d, and the 3΄-deoxy-3΄-methyl 18a,18b,18c. The antiviral evaluation revealed appreciable activity against Coxsackie virus B4, Respiratory syncytial virus, Yellow Fever Virus and Human Coronavirus (229E) for the 3΄-deoxy compounds 12b,14, and the 3΄-deoxy-3΄-methyl 18a,18c,18d, accompanied by low cytotoxicity. CONCLUSION: This report describes the total and facile synthesis of modified furanononucleosides of uracil, with alterations on both the sugar and the heterocyclic portions. Compounds 12b,14 and 18a,c,d showed noticeable antiviral activity against a series of RNA viruses and merit further biological and structural optimization investigations.

Synthesis and anti-CVB3 activity of 4-amino acid derivative substituted pyrimidine nucleoside analogues.

Seven novel 4-amino acid derivative substituted pyrimidine nucleoside analogues were designed, synthesized, and tested for their anti-CVB3 activity. Initial biological studies indicated that among these 4-amino acid derivative substituted pyrimidine nucleoside analogues, 4-N-(2'-amino-glutaric acid-1'-methylester)-1-(2'- deoxy-2'-ß-fluoro-4'-azido)-furanosyl-cytosine 2 exhibited the most potent anti-CVB activity (IC50 = 9.3 µM). The cytotoxicity of these compounds has also been assessed. The toxicity of compound 2 was similar to that of ribavirin.

Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides.

Theories about the origin of life require chemical pathways that allow formation of life's key building blocks under prebiotically plausible conditions. Complex molecules like RNA must have originated from small molecules whose reactivity was guided by physico-chemical processes. RNA is constructed from purine and pyrimidine nucleosides, both of which are required for accurate information transfer, and thus Darwinian evolution. Separate pathways to purines and pyrimidines have been reported, but their concurrent syntheses remain a challenge. We report the synthesis of the pyrimidine nucleosides from small molecules and ribose, driven solely by wet-dry cycles. In the presence of phosphate-containing minerals, 5'-mono- and diphosphates also form selectively in one-pot reactions. The pathway is compatible with purine synthesis, allowing the concurrent formation of all Watson-Crick bases.

An Eco-Friendly Technique: Solvent-Free Microwave Synthesis and Docking Studies of Some New Pyridine Nucleosides and Their Pharmacological Significance.

Two series of novel 5-arylazo-3-cyano-2-(2″,3″,4″,6″-tetra-O-acetyl-ß-d-galacto pyranosyloxy) pyridines and 3-cyano-2-(2″,3″,4″,6″-tetra-O-acetyl-ß-d-galactopyranosyloxy) pyridines were synthesized in high yields utilizing a microwave-assisted synthesis tool guided by the principles of green chemistry. The chemical structures of the new substances were confirmed on the basis of their elemental analysis and spectroscopic data (FT-IR, 1D, 2D-NMR). Activity against different bacterial strains was studied. The anticancer potential of the new compounds is also discussed. Molecular docking was used as a tool in this research work to get better insight into the possible interactions, affinities, and expected modes of binding of the most promising derivatives of the potential chemotherapeutic target (DHFR).

Synthesis of a Protected keto-Lysidine Analogue via Improved Preparation of Arabino-isoCytosine Nucleosides.

Anhydrouridines react with aliphatic amines to give N-alkyl isocytosines, but reported procedures often demand very long reaction times and can be low yielding, with narrow scope. A modified procedure for such reactions has been developed, using microwave irradiation, significantly reducing reaction time and allowing facile access to a diverse range of novel nucleosides on the gram scale. The method has been used to prepare a precursor to a novel analogue of lysidine, a naturally occurring iminonucleoside found in tRNA.

An Expeditious Total Synthesis of 5'-Deoxy-toyocamycin and 5'-Deoxysangivamycin.

In present paper, an expeditious total synthesis of naturally occurring 5'-deoxytoyocamycin and 5'-deoxysangivamycin was accomplished. Because of the introduction of a benzoyl group at N-6 of 4-amino-5-cyano-6-bromo-pyrrolo[2,3-d]pyrimidine, a Vorbrüggen glycosylation with 1,2,3-tri-O-acetyl-5-deoxy-ß-D-ribofuranose afforded a completely regioselective N-9 glycosylation product, which is unambiguously confirmed by X-ray diffraction analysis. All of the involved intermediates were well characterized by various spectra.

Fluorescent 5-Pyrimidine and 8-Purine Nucleosides Modified with an N-Unsubstituted 1,2,3-Triazol-4-yl Moiety.

The Cu(I)- or Ag(I)-catalyzed cycloaddition between 8-ethynyladenine or guanine nucleosides and TMSN3 gave 8-(1- H-1,2,3-triazol-4-yl) nucleosides in good yields. On the other hand, reactions of 5-ethynyluracil or cytosine nucleosides with TMSN3 led to the chemoselective formation of triazoles via Cu(I)-catalyzed cycloaddition or vinyl azides via Ag(I)-catalyzed hydroazidation. These nucleosides with a minimalistic triazolyl modification showed excellent fluorescent properties with 8-(1- H-1,2,3-triazol-4-yl)-2'-deoxyadenosine (8-TrzdA), exhibiting a quantum yield of 44%. The 8-TrzdA 5'-triphosphate was incorporated into duplex DNA containing a one-nucleotide gap by DNA polymerase ß.

Electron-Mediated Aminyl and Iminyl Radicals from C5 Azido-Modified Pyrimidine Nucleosides Augment Radiation Damage to Cancer Cells.

Two classes of azido-modified pyrimidine nucleosides were synthesized as potential radiosensitizers; one class is 5-azidomethyl-2'-deoxyuridine (AmdU) and cytidine (AmdC), while the second class is 5-(1-azidovinyl)-2'-deoxyuridine (AvdU) and cytidine (AvdC). The addition of radiation-produced electrons to C5-azido nucleosides leads to the formation of π-aminyl radicals followed by facile conversion to σ-iminyl radicals either via a bimolecular reaction involving intermediate α-azidoalkyl radicals in AmdU/AmdC or by tautomerization in AvdU/AvdC. AmdU demonstrates effective radiosensitization in EMT6 tumor cells.

Symmetrical Diamidates as a Class of Phosphate Prodrugs to Deliver the 5'-Monophosphate Forms of Anticancer Nucleoside Analogues.

The application of phosphorodiamidate technology to pyrimidine and purine nucleosides with anticancer activity to potentially overcome the resistance mechanisms associated with parent nucleosides is reported. Sixteen symmetrical phosphorodiamidates were prepared from the natural amino acids l-alanine and glycine. All the compounds were evaluated for their cytotoxic activity against a wide panel of solid and leukaemic tumour cell lines. In addition, a carboxypeptidase Y assay was performed on a representative phosphorodiamidate in order to reveal the putative bioactivation pathway for the reported phosphorodiamidate-type prodrugs.

Synthesis of Cyclic and Acyclic Pyrimidine Nucleosides Analogues with Anticipated Antiviral Activity.

A convenient method for preparation of cyclic and acyclic nucleosides was achieved by alkylation of 6-(2,4-dichlorophenoxymethyl) pyrimidine-2,4-dione (1) with a variety of acyclic and cyclic activated sugar analogues, namely (2-acetoxyethoxy)methyl acetate (3), 2-(acetoxymethoxy)propane-1,3-diyl dibenzoate (4), benzyloxymethyl acetate (5), 2-acetoxy-5-(benzoyloxymethyl)tetrahydrofuran-3,4-diyl dibenzoate (12), 5-chloro-2-((4-chlorobenzoyloxy)methyl) tetrahydrofuran-3-yl 4-chlorobenzoate (13) and 2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (14), respectively. Deprotection of the synthesized nucleosides was achieved by using methanolic ammonia. The structures of the newly synthesized nucleoside analogues were fully characterized by analytical methods (mass spectrometry, 1H NMR, 13C NMR, and elemental analysis).

Abiotic synthesis of purine and pyrimidine ribonucleosides in aqueous microdroplets.

Aqueous microdroplets (<1.3 µm in diameter on average) containing 15 mM d-ribose, 15 mM phosphoric acid, and 5 mM of a nucleobase (uracil, adenine, cytosine, or hypoxanthine) are electrosprayed from a capillary at +5 kV into a mass spectrometer at room temperature and 1 atm pressure with 3 mM divalent magnesium ion (Mg2+) as a catalyst. Mass spectra show the formation of ribonucleosides that comprise a four-letter alphabet of RNA with a yield of 2.5% of uridine (U), 2.5% of adenosine (A), 0.7% of cytidine (C), and 1.7% of inosine (I) during the flight time of ∼50 µs. In the case of uridine, no catalyst is required. An aqueous solution containing guanine cannot be generated under the same conditions given the extreme insolubility of guanine in water. However, inosine can base pair with cytidine and thus substitute for guanosine. Thus, a full set of ribonucleosides to generate the purine-pyrimidine base pairs A-U and I-C are spontaneously generated in aqueous microdroplets under similar mild conditions.

Synthesis of unique spirocyclic orthoester-type derivatives of isothiazolo[4,3-d]pyrimidine nucleosides.

A set of unique nucleoside analogs, containing 'spirocyclic orthoester-type' scaffolds, were synthesized from a common isothiazolo[4,3-d]pyrimidine-riboside precursor. The key reaction, using 1,2-di-heteroatomic nucleophiles (e.g., 1,2-ethandithiol) and BF3•OEt2, converts an exocyclic imine into the spirocyclic analogs. The novel structural scaffold is confirmed through the use of one- and two-dimensional 1H and 13C NMR experiments.