Nine-Step Stereoselective Synthesis of Islatravir from Deoxyribose.

A stereoselective nine-step synthesis of the potent HIV nucleoside reverse transcriptase translocation inhibitor (NRTTI) islatravir (EfdA, MK-8591) from 2-deoxyribose is described. Key findings include a diastereodivergent addition of an acetylide nucleophile to an enolizable ketone, a chemoselective ozonolysis of a terminal olefin and a biocatalytic glycosylation cascade that uses a unique strategy of byproduct precipitation to drive an otherwise-reversible transformation forward.

Synthesis of nucleotide analogues, EFdA, EdA and EdAP, and the effect of EdAP on hepatitis B virus replication.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) and 4'-ethynyl-2'-deoxyadenosine (EdA) are nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the replication of the influenza A virus. The common structural feature of these compounds is the ethynyl group at the 4'-position. In this study, these nucleoside analogues were prepared by a common synthetic strategy starting from the known 1,2-di-O-acetyl-D-ribofuranose. Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus (HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.

Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts.

Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2'-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs.

Design, Synthesis, and Biological Evaluation of EdAP, a 4'-Ethynyl-2'-Deoxyadenosine 5'-Monophosphate Analog, as a Potent Influenza a Inhibitor.

Influenza A viruses leading to infectious respiratory diseases cause seasonal epidemics and sometimes periodic global pandemics. Viral polymerase is an attractive target in inhibiting viral replication, and 4'-ethynyladenosine, which has been reported as a highly potent anti-human immunodeficiency virus (HIV) nucleoside derivative, can work as an anti-influenza agent. Herein, we designed and synthesized a 4'-ethynyl-2'-deoxyadenosine 5'-monophosphate analog called EdAP (5). EdAP exhibited potent inhibition against influenza virus multiplication in Madin-Darby canine kidney (MDCK) cells transfected with human α2-6-sialyltransferase (SIAT1) cDNA and did not show any toxicity toward the cells. Surprisingly, this DNA-type nucleic acid analog (5) inhibited the multiplication of influenza A virus, although influenza virus is an RNA virus that does not generate DNA.

Homopurine RP-stereodefined phosphorothioate analogs of DNA with hampered Watson-Crick base pairings form Hoogsteen paired parallel duplexes with (2'-OMe)-RNAs.

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of 5'-O-DMT-N6-methyl-deoxyadenosine and 5'-O-DMT-N2,N2-dimethyl-O6-diphenylcarbamoyl-deoxyguanosine (OTP-NY, NY = DMT-m6dA or DMT-m,m2dGDPC) were synthesized, resolved onto pure P-diastereomers, and used in P-stereocontrolled synthesis of dinucleoside 3',5,-phosphorothioates NXPST (NX = m6dA or m,m2dG), in which the absolute configuration of the stereogenic phosphorus atom was established enzymatically. Diastereomerically pure OTP-NY and standard OTP-N (N = DMT-dABz or DMT-dGBz,DPC) were used in the synthesis of chimeric RP-stereodefined phosphorothioate oligomers ((RP-PS)-DN(NX)A) with hampered Watson-Crick base pairings. It was found that the m6dA units slightly reduce the thermodynamic stability of antiparallel duplexes formed with RNA and (2'-OMe)-RNA matrices, whereas m,m2dG units prevent their formation. The m6dA units stabilize (by up to 4.5 °C per modified unit) the parallel duplexes formed by (RP-PS)-DN(NX)A with Hoogsteen-paired (2'-OMe)-RNA templates compared to the analogous reference duplex containing only unmodified nucleobases. In contrast, the m,m2dG units destabilize such duplexes by up to 3 °C per modified unit. Both units prevent the formation of the corresponding parallel triplexes.

Prebiotic phosphorylation of 2-thiouridine provides either nucleotides or DNA building blocks via photoreduction.

Breakthroughs in the study of the origin of life have demonstrated how some of the building blocks essential to biology could have been formed under various primordial scenarios, and could therefore have contributed to the chemical evolution of life. Missing building blocks are then sometimes inferred to be products of primitive biosynthesis, which can stretch the limits of plausibility. Here, we demonstrate the synthesis of 2'-deoxy-2-thiouridine, and subsequently 2'-deoxyadenosine and 2-deoxyribose, under prebiotic conditions. 2'-Deoxy-2-thiouridine is produced by photoreduction of 2,2'-anhydro-2-thiouridine, which is in turn formed by phosphorylation of 2-thiouridine-an intermediate of prebiotic RNA synthesis. 2'-Deoxy-2-thiouridine is an effective deoxyribosylating agent and may have functioned as such in either abiotic or proto-enzyme-catalysed pathways to DNA, as demonstrated by its conversion to 2'-deoxyadenosine by reaction with adenine, and 2-deoxyribose by hydrolysis. An alternative prebiotic phosphorylation of 2-thiouridine leads to the formation of its 5'-phosphate, showing that hypotheses in which 2-thiouridine was a key component of early RNA sequences are within the bounds of synthetic credibility.

Synthesis of New Potential Lipophilic Co-Drugs of 2-Chloro-2'-deoxyadenosine (Cladribine, 2-CdA, Mavenclad®, Leustatin®) and 6-Azauridine (z6 U) with Valproic Acid.

2-Chloro-2'-deoxyadenosine (cladribine, 1) was acylated with valproic acid (2) under various reaction conditions yielding 2-chloro-2'-deoxy-3',5'-O-divalproyladenosine (3) as well as the 3'-O- and 5'-O-monovalproylated derivatives, 2-chloro-2'-deoxy-3'-O-valproyladenosine (4) and 2-chloro-2'-deoxy-5'-O-valproyladenosine (5), as new co-drugs. In addition, 6-azauridine-2',3'-O-(ethyl levulinate) (8) was valproylated at the 5'-OH group (→9). All products were characterized by 1 H- and 13 C-NMR spectroscopy and ESI mass spectrometry. The structure of the by-product 6 (N-cyclohexyl-N-(cyclohexylcarbamoyl)-2-propylpentanamide), formed upon valproylation of cladribine in the presence of N,N-dimethylaminopyridine and dicyclohexylcarbodiimide, was analyzed by X-ray crystallography. Cladribine as well as its valproylated co-drugs were tested upon their cancerostatic/cancerotoxic activity in human astrocytoma/oligodendroglioma GOS-3 cells, in rat malignant neuro ectodermal BT4Ca cells, as well as in phorbol-12-myristate 13-acetate (PMA)-differentiated human THP-1 macrophages. The most important result of these experiments is the finding that only the 3'-O-valproylated derivative 4 exhibits a significant antitumor activity while the 5'-O- as well as the 3',5'-O-divalproylated cladribine derivatives 3 and 5 proved to be inactive.

Cordycepin reduces weight through regulating gut microbiota in high-fat diet-induced obese rats.

BACKGROUND: An increasing number of studies have shown that obesity is the key etiological agent of cardiovascular diseases, nonalcoholic fatty liver disease, type 2 diabetes and several kinds of cancer and that gut microbiota change was one of the reasons suffering from obesity. At present, the gut microbiota has gained increased attention as a potential energy metabolism organ. Our recent study reported that cordycepin, a major bioactive component separated from Cordyceps militaris, prevented body weight gain in mice fed a high-fat diet directly acting to adipocytes, however, the effect of cordycepin regulating gut microbiota keeps unknown. METHODS: In this research, we synthesized cordycepin (3-deoxyadenosine) by chemical methods and verified that cordycepin reduces body weight gain and fat accumulation around the epididymis and the kidneys of rats fed a high-fat diet. Furthermore, we used high-throughput sequencing on a MiSeq Illumina platform to test the species of intestinal bacteria in high-fat-diet-induced obese rats. RESULTS: We found that cordycepin modifies the relative abundance of intestinal bacteria in high-fat-diet-induced obese rats. However, cordycepin did not alter the variety of bacteria in the intestine. Cordycepin treatment dramatically reversed the relative abundance of two dominant bacterial phyla (Bacteroidetes and Firmicutes) in the high-fat-diet-induced obese rats, resulting in abundance similar to that of the chow diet group. CONCLUSION: Our study suggests that cordycepin can reduce body weight and microbiome done by cordycepin seems be a result among its mechanisms of obesity reduction.

Synthesis and Antiviral Evaluation of Carbocyclic Nucleoside Analogs of Nucleoside Reverse Transcriptase Translocation Inhibitor MK-8591 (4'-Ethynyl-2-fluoro-2'-deoxyadenosine).

MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine) is a novel nucleoside analog that displays a differentiated mechanism of action as a nucleoside reverse transcriptase translocation inhibitor (NRTTI) compared to approved NRTIs. Herein, we describe our recent efforts to explore the impact of structural changes to the properties of MK-8591 through the synthesis and antiviral evaluation of carbocyclic derivatives. Synthesized analogs were evaluated for their antiviral activity, and the corresponding triphosphates were synthesized and evaluated in a biochemical assay. 4'-Ethynyl-G derivative (±)-29 displayed a promising IC50 of 33 nM in a hPBMC cell-based antiviral assay, and its triphosphate (TP), (±)-29-TP, displayed an IC50 of 324 nM in a biochemical RT-polymerase assay. Improved TP anabolite delivery resulting in improved in vitro potency was achieved by preparing the corresponding phosphoramidate prodrug of single enantiomer 29b, with 6-ethoxy G derivative 34b displaying a significantly improved IC50 of 3.0 nM, paving the way for new directions for this novel class of nucleoside analogs.

Enzymatic Fluorination of Biotin and Tetrazine Conjugates for Pretargeting Approaches to Positron Emission Tomography Imaging.

The use of radiolabelled antibodies and antibody-derived recombinant constructs has shown promise for both imaging and therapeutic use. In this context, the biotin-avidin/streptavidin pairing, along with the inverse-electron-demand Diels-Alder (iEDDA) reaction, have found application in pretargeting approaches for positron emission tomography (PET). This study reports the fluorinase-mediated transhalogenation [5'-chloro-5'-deoxyadenosine (ClDA) substrates to 5'-fluoro-5'-deoxyadenosine (FDA) products] of two antibody pretargeting tools, a FDA-PEG-tetrazine and a [18 F]FDA-PEG-biotin, and each is assessed either for its compatibility towards iEDDA ligation to trans-cyclooctene or for its affinity to avidin. A protocol to avoid radiolytically promoted oxidation of biotin during the synthesis of [18 F]FDA-PEG-biotin was developed. The study adds to the repertoire of conjugates for use in fluorinase-catalysed radiosynthesis for PET and shows that the fluorinase will accept a wide range of ClDA substrates tethered at C-2 of the adenine ring with a PEGylated cargo. The method is exceptional because the nucleophilic reaction with [18 F]fluoride takes place in water at neutral pH and at ambient temperature.

Synthesis and evaluation of analogs of 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (MDL 73811, or AbeAdo) - An inhibitor of S-adenosylmethionine decarboxylase with antitrypanosomal activity.

We describe our efforts to improve the pharmacokinetic properties of a mechanism-based suicide inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), essential for the survival of the eukaryotic parasite Trypanosoma brucei responsible for Human African Trypanosomiasis (HAT). The lead compound, 5'-(((Z)-4-amino-2-butenyl)methylamino)-5'-deoxyadenosine (1, also known as MDL 73811, or AbeAdo), has curative efficacy at a low dosage in a hemolymphatic model of HAT but displayed no demonstrable effect in a mouse model of the CNS stage of HAT due to poor blood-brain barrier permeation. Therefore, we prepared and evaluated an extensive set of analogs with modifications in the aminobutenyl side chain, the 5'-amine, the ribose, and the purine fragments. Although we gained valuable structure-activity insights from this comprehensive dataset, we did not gain traction on improving the prospects for CNS penetration while retaining the potent antiparasitic activity and metabolic stability of the lead compound 1.

Development of Cordycepin Formulations for Preclinical and Clinical Studies.

There is extensive literature on in vivo studies with cordycepin, but these studies were generally conducted without validation of the various formulations, especially in terms of the solubility of cordycepin in the dosing vehicles used. Cordycepin is a promising drug candidate in multiple therapeutic areas, and there is a growing interest in studies aimed at assessing the pharmacological activity of this compound in relevant animal disease models. It is likely that many reported in vivo studies used formulations in which cordycepin was incompletely soluble. This can potentially confound the interpretation of pharmacokinetics and efficacy results. Furthermore, the presence of particles in intravenously administered suspension can cause adverse effects and should be avoided. Here, we present the results from our development of simple and readily applicable formulations of cordycepin based on quantitative solubility assessment. Homogeneous solutions of cordycepin were prepared in phosphate-buffered saline (PBS) at different pH levels, suitable as formulations for both intravenously and oral administration. For the purpose of high-dose oral administration, we also developed propylene glycol (PPG)-based vehicles in which cordycepin is completely soluble. The stability of the newly developed formulations was also assessed, as well as the feasibility of their sterilisation by filtration. Additionally, an HPLC-UV method for the determination of cordycepin in the formulations, which may also be useful for other purposes, was developed and validated. Our study could provide useful information for improvement of future preclinical and clinical studies involving cordycepin.

Facile Access to Bromonucleosides Using Sodium Monobromoisocyanurate (SMBI).

Bromonucleosides constitute a significant class of molecules and are well known for their biological activity. 5-Bromouridine, 5-bromo-2'-deoxyuridine, 5-bromouridine-5'-triphosphate, and nucleotides containing 5-bromouridine have been tested and used for numerous biological studies. 8-Bromopurine nucleosides have been used as essential precursors for the synthesis of nucleosides with fluorescent properties. This unit describes protocols for the synthesis of bromonucleosides using sodium monobromoisocyanurate (SMBI) in a straightforward way. Reactions are carried out at room temperature, and aqueous solvent mixtures are used to dissolve the nucleosides. Sodium azide is used as catalyst for the bromination of pyrimidine nucleosides, and no catalyst is necessary for the bromination of purine nucleosides. Unprotected 2'-deoxy pyrimidine and 2'-deoxy purine nucleosides are treated with SMBI to afford C-5 bromo pyrimidine and C-8 bromo purine nucleosides, respectively. This methodology has been found to be efficient for the synthesis of bromonucleosides on gram scale with consistently high yields. © 2017 by John Wiley & Sons, Inc.

Aminyl Radical Generation via Tandem Norrish Type I Photocleavage, ß-Fragmentation: Independent Generation and Reactivity of the 2'-Deoxyadenosin- N6-yl Radical.

Formal hydrogen atom abstraction from the nitrogen-hydrogen bonds in purine nucleosides produces reactive intermediates that are important in nucleic acid oxidation. Herein we describe an approach for the independent generation of the purine radical resulting from hydrogen atom abstraction from the N6-amine of 2'-deoxyadenosine (dA•). The method involves sequential Norrish Type I photocleavage of a ketone (7b) and ß-fragmentation of the initially formed alkyl radical (8b) to form dA• and acetone. The formation of dA• was followed by laser flash photolysis, which yields a transient with λmax ≈ 340 nm and a broader weaker absorption centered at ∼560 nm. This transient grows in at ≥2 × 105 s-1; however, computations and reactivity data suggest that ß-fragmentation occurs much faster, implying the consumption of dA• as it is formed. Continuous photolysis of 7b in the presence of ferrous ion or thiophenol produces good yields of dA, whereas less reactive thiols afford lower yields presumably due to a polarity mismatch. This tandem photochemical, ß-fragmentation method promises to be useful for site-specific production of dA• in nucleic acid oligomers and/or polymers and also for the production of aminyl radicals, in general.

Enantioselective Synthesis of 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) via Enzymatic Desymmetrization.

An enantioselective synthesis of the potent anti-HIV nucleoside EFdA is presented. Key features of stereocontrol include construction of the fully substituted 4'-carbon via a biocatalytic desymmetrization of 2-hydroxy-2-((triisopropylsilyl)ethynyl)propane-1,3-diyl diacetate and a Noyori-type asymmetric transfer hydrogenation to control the stereochemistry of the 3'-hydroxyl bearing carbon. The discovery of a selective crystallization of an N-silyl nucleoside intermediate enabled isolation of the desired ß-anomer from the glycosylation step.

C-C Bond Formation: Synthesis of C5 Substituted Pyrimidine and C8 Substituted Purine Nucleosides Using Water Soluble Pd-imidate Complex.

The synthesis of a highly efficient, water soluble [Pd(Sacc)2 (TPA)2 ] complex for C-C bond formation is described. Additionally, application of the [Pd(Sacc)2 (TPA)2 ] complex for Suzuki-Miyaura arylation of all four nucleosides (5-iodo-2'-deoxyuridine [5-IdU], 5-iodo-2'-deoxycytidine [5-IdC], 8-bromo-2'-deoxyadenosine, and 8-bromo-2'-deoxyguanosine) with various aryl/heteroaryl boronic acids in plain water under milder conditions is demonstrated. © 2016 by John Wiley & Sons, Inc.

Nucleoside-Based Diarylethene Photoswitches: Synthesis and Photochromic Properties.

Diarylethene photoswitches based on the natural nucleoside deoxyadenosine were designed and synthesized. In aqueous solution, some of them exhibited good photochromic properties, including clear changes in color upon irradiation at 365 nm, red-shifts of the absorption wavelength, with good fatigue resistance, thermal stability, conversion efficiency, and base-pairing properties.

Design and synthesis of environmentally sensitive fluorescent 2-naphthylethynylated 2'-deoxyadenosines: Detection of target DNA via changes in fluorescence intensity and wavelength.

Various C2-naphthylethynylated 2'-deoxyadenosines were synthesized as environmentally sensitive fluorescent (ESF) nucleosides and their photophysical properties were examined. Among the ESF nucleosides synthesized, four exhibited strong solvatochromicity, two of which were incorporated into oligodeoxynucleotides (ODNs). These ODN probes were able to detect target DNA through distinct changes in fluorescence intensity and wavelength and acted as effective reporter probes.

Synthesis of 8-aza-3,7-dideaza-2'-deoxyadenosines possessing a new adenosine skeleton as an environmentally sensitive fluorescent nucleoside for monitoring the DNA minor groove.

8-Aza-3,7-dideaza-2'-deoxyadenosine 1 and its C3-naphthylethynylated derivative (3n7z)A (2) comprising a 8-aza-3,7-dideazapurine (pyrazolo[4,3-c]pyridine) skeleton were synthesized for the first time. In particular, nucleoside (3n7z)A (2) exhibited environmentally sensitive intramolecular charge transfer (ICT) emission because of electron transition in the coplanar conformer formed by nucleobase and naphthalene moieties. Its incorporation into oligodeoxynucleotide (ODN) probes enable a clear identification of a perfectly matched thymine (T) in the complementary strand by a distinct change in the emission wavelength. In addition, the fluorescence emission of the duplexes containing a cytosine/guanine (C/G) base pair flanking (3n7z)A (2) was strongly quenched by guanine only when the opposite base of the modified nucleoside was mismatched, enhancing its base identification ability. Thus, ODN probes containing (3n7z)A (2) acted as effective reporter probes for homogeneous single nucleotide polymorphism (SNP) typing.

Synthesis of EFdA via a diastereoselective aldol reaction of a protected 3-keto furanose.

An efficient enantioselective total synthesis of EFdA, a remarkably potent anti-HIV nucleoside analogue with various favorable pharmacological profiles, has been achieved in 37% overall yield from diacetone-D-glucose by a 14-step sequence that features a highly diastereoselective installation of the tetrasubstituted stereogenic center at the C4' position, direct oxidative cleavage of an acetonide-protected diol derivative to an aldehyde, and one-pot 2'-deoxygenation of a ribonucleoside intermediate.