The hydrazine moiety in the synthesis of modified nucleosides and nucleotides.

Synthetic nucleoside mimics are re-emerging as crucial contenders for antiviral and anticancer medications. While, Ribavirin stands out for its unique antiviral properties, predominantly associated with its distinctive triazole heterocycle as a nucleobase, the exploration of alternative nitrogen-based aromatic heterocycles hold great promises for the discovery of novel bioactive nucleoside mimics. Although nucleoside derivatives synthesised from hydrazine-ribose units have been in development for many decades, they have been little evaluated biologically and even less for their antiviral properties. With the aim of taking a closer look at these under-explored derivatives and investigating their synthetic pathways, this concise review provides an overview of the molecular design, the chemical synthesis, and the biological activity, when available, of these nucleoside analogues. Overall, the entire body of work already done motivates further exploration of theses analogues and encourages us of formulating structurally novel nucleoside drug candidates featuring innovative mode of action.

Probing the Use of Triphenyl Phosphonium Cation for Mitochondrial Nucleoside Delivery.

Herein, we report the design, the synthesis, and the study of novel triphenyl phosphonium-based nucleoside conjugates. 2'-Deoxycytidine was chosen as nucleosidic cargo, as it allows the introduction of fluorescein on the exocyclic amine of the nucleobase and grafting of the vector was envisaged through the formation of a biolabile ester bond with the hydroxyl function at the 5'-position. Compound 3 was identified as a potential nucleoside prodrug, showing ability to be internalized efficiently into cells and to be co-localized with mitochondria.

Purine containing carbonucleoside phosphonate analogues as novel chemotype for Plasmodium falciparum Inhibition.

The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and ß-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration.

Second-Generation CD73 Inhibitors Based on a 4,6-Biaryl-2-thiopyridine Scaffold.

Various series of 4,6-biaryl-2-thiopyridine derivatives were synthesized and evaluated as potential ecto-5'-nucleotidase (CD73) inhibitors. Two synthetic routes were explored and the coupling of 4,6-disubstituted 3-cyano-2-chloro-pyridines with selected thiols allowed us to explore the structural diversity. Somehow divergent results were obtained in biological assays on CD73 inhibition using either the purified recombinant protein or cell-based assays, highlighting the difficulty to target protein-protein interface on proteins existing as soluble and membrane-bound forms. Among the 18 new derivatives obtained, three derivatives incorporating morpholino substituents on the 4,6-biaryl-2-thiopyridine core were shown to be able to reverse the adenosine-mediated immune suppression on human T cells. The higher blockade efficiency was observed for 2-((3-cyano-4,6-bis(4-morpholinophenyl)pyridin-2-yl)thio)-N-(isoxazol-3-yl)acetamide (with total reversion at 100 µM) and methyl 2-((3-cyano-4,6-bis(4-morpholinophenyl)pyridin-2-yl)thio)acetate (with partial reversion at 10 µM). Thus, this series of compounds illustrates a new chemotype of CD73 allosteric inhibitors.

Prodrugs of Nucleoside 5'-Monophosphate Analogues: Overview of the Recent Literature Concerning their Synthesis and Applications.

Nucleoside analogues are widely used as anti-infectious and antitumoral agents. However, their clinical use may face limitations associated with their physicochemical properties, pharmacokinetic parameters, and/or their peculiar mechanisms of action. Indeed, once inside the cells, nucleoside analogues require to be metabolized into their corresponding (poly-)phosphorylated derivatives, mediated by cellular and/or viral kinases, in order to interfere with nucleic acid biosynthesis. Within this activation process, the first-phosphorylation step is often the limiting one and to overcome this limitation, numerous prodrug approaches have been proposed. Herein, we will focus on recent literature data (from 2015 and onwards) related to new prodrug strategies, the development of original synthetic approaches and novel applications of nucleotide prodrugs (namely pronucleotides) leading to the intracellular delivery of 5'-monophosphate nucleoside analogues.

Mononucleoside phosphorodithiolates as mononucleotide prodrugs.

The synthesis and in vitro anti-HIV activity of a novel series of pronucleotides are reported. These prodrugs were characterized by a phosphorodithiolate structure, incorporating two O-pivaloyl-2-oxyethyl substituents as biolabile phosphate protections. The compounds were obtained following an original one-pot three-step procedure, involving the formation of a phosphorodithioite intermediate which is in situ oxidized. In vitro, comparative anti-HIV evaluations demonstrate that such original prodrugs are able to allow the efficient intracellular release of the corresponding 5'-mononucleotide. The pronucleotide of 2',3'-dideoxyadenosine (ddA) 3 exhibited a very potent antiretroviral effect with 50% effective concentration (EC50) values in nanomolar concentration range in various cell lines. In primary monocytes/macrophages, this derivative was 500 times more potent in inhibiting HIV replication (EC50 0.23 pM) than ddA and the selectivity index of the prodrug is fifty times higher than the one of the parent nucleoside.

Cytotoxic and Antitumoral Activity of N-(9H-purin-6-yl) Benzamide Derivatives and Related Water-soluble Prodrugs.

BACKGROUND: The development of small molecules as cancer treatments is still of both interest and importance. OBJECTIVE: Having synthesized and identified the initial cytotoxic activity of a series of chemically related N-(9H-purin-6-yl) benzamide derivatives, we continued their evaluation on cancer cell models. We also synthesized water-soluble prodrugs of the main compound and performed in vivo experiments. METHOD: We used organic chemistry to obtain compounds of interest and prodrugs. The biological evaluation included MTT assays, synergy experiments, proliferation assays by CFSE, cell cycle distribution and in vivo antitumoral activity. RESULTS: Our results show activities on cancer cell lines ranging from 3-39 µM for the best compounds, with both induction of apoptosis and decrease in cell proliferation. Two compounds evaluated in vivo showed weak antitumoral activity. In addition, the lead compound and its prodrug had a synergistic activity with the nucleoside analogue fludarabine in vitro and in vivo. CONCLUSION: Our work allowed us to gain better knowledge on the activity of N-(9H-purin-6-yl) benzamide derivatives and showed new examples of water-soluble prodrugs. More research is warranted to decipher the molecular mechanisms of the molecules.

An original pronucleotide strategy for the simultaneous delivery of two bioactive drugs.

The synthesis and in vitro anti-HIV activity of a novel series of phosphoramidate pronucleotides including a S-pivaloyl-2-thioethyl (tBuSATE) group as biolabile phosphate protecting group are reported. Such constructs, obtained through different phosphorus chemistries, are characterized by the association of two different anti-HIV nucleoside analogues linked to the phosphorus atom respectively by the sugar residue and the exocyclic amino function of the nucleobase. In vitro, comparative anti-HIV evaluation demonstrates that such original prodrugs are able to allow the efficient intracellular combination release of a 5'-mononucleotide as well as another nucleoside analogue. In human T4-lymphoblastoid cells, the pronucleotide 1 shows remarkable antiviral activity with an EC50 in the nanomolar range (0.6 ηM) and without additional cytotoxicity. In addition, these two pronucleotide models exhibit higher selectivity index than the equimolar mixture of their constitutive nucleoside analogues opening the way to further studies with regard to the current use of drug combinations.

4-Substituted-1,2,3-triazolo nucleotide analogues as CD73 inhibitors, their synthesis, in vitro screening, kinetic and in silico studies.

Three series of nucleotide analogues were synthesized and evaluated as potential CD73 inhibitors. Nucleobase replacement consisted in connecting the appropriate aromatic or purine residues through a triazole moiety that is generated from 1,3-dipolar cycloaddition. The first series is related to 4-substituted-1,2,3-triazolo-ß-hydroxyphosphonate ribonucleosides. Additional analogues were also obtained, in which the phosphonate group was replaced by a bisphosphonate pattern (P-C-P-C, series 2) or the ribose moiety was removed leading to acyclic derivatives (series 3). The ß-hydroxyphosphonylphosphonate ribonucleosides (series 2) were found to be potent inhibitors of CD73 using both purified recombinant protein and cell-based assays. Two compounds (2a and 2b) that contained a bis(trifluoromethyl)phenyl or a naphthyl substituents proved to be the most potent inhibitors, with IC50 values of 4.8 ± 0.8 µM and 0.86 ± 0.2 µM, compared to the standard AOPCP (IC50 value of 3.8 ± 0.9 µM), and were able to reverse the adenosine-mediated immune suppression on human T cells. This series of compounds illustrates a new type of CD73 inhibitors.

Synthesis of Aminomethylene-gem-bisphosphonates Containing an Aziridine Motif: Studies of the Reaction Scope and Insight into the Mechanism.

A broad range of N-carbamoylaziridines were obtained and then treated by the diethyl phosphonate anion to afford α-methylene-gem-bisphosphonate aziridines. Study of the reaction's scope and additional experiments indicates that the transformation proceeds via a new mechanism involving the chelation of lithium ion. This last step is crucial for the reaction to occur and disfavors the aziridine ring-opening. A phosphonate-phosphate rearrangement from a α-hydroxybisphosphonate aziridine intermediate is also proposed for the first time. This reaction provides a simple and convenient method for the synthesis of a highly functionalized phosphonylated aziridine motif.

2-(Substituted amino)-8-azachromones from 4,6-Diaryl-2-pyridones: A Synthetic Strategy toward Compounds of Broad Structural Diversity.

3-Acetoacetyl-4,6-diaryl-2-pyridones are synthesized in three steps from chalcones and then condense with carbon disulfide to afford 8-azachromones containing a methylthio group at C2. This leaving group offers an entry point for the insertion of more complex moieties via nucleophilic substitution. For this purpose, N-nucleophiles are explored according to their positions in the Mayr's nucleophilicity scale (N parameter), and three main classes are distinguished depending on whether the substitution takes place from their neutral forms, from their deprotonated anionic forms, or under nucleophilic catalysis. A broad range of primary and secondary amines may be inserted by this method, including enantiomerically pure amino acids, enabling us to explore structural diversity.

ß-Hydroxy- and ß-Aminophosphonate Acyclonucleosides as Potent Inhibitors of Plasmodium falciparum Growth.

Malaria is an infectious disease caused by a parasite of the genus Plasmodium, and the emergence of parasites resistant to all current antimalarial drugs highlights the urgency of having new classes of molecules. We developed an effective method for the synthesis of a series of ß-modified acyclonucleoside phosphonate (ANP) derivatives, using commercially available and inexpensive materials (i.e., aspartic acid and purine heterocycles). Their biological evaluation in cell culture experiments and SAR revealed that the compounds' effectiveness depends on the presence of a hydroxyl group, the chain length (four carbons), and the nature of the nucleobase (guanine). The most active derivative inhibits the growth of Plasmodium falciparum in vitro in the nanomolar range (IC50 = 74 nM) with high selectivity index (SI > 1350). This compound also showed remarkable in vivo activity in P. berghei-infected mice (ED50 ∼ 0.5 mg/kg) when administered by the ip route and is, although less efficient, still active via the oral route. It is the first ANP derivative with such potent antimalarial activity and therefore has considerable potential for development as a new antimalarial drug.

Synthetic Strategies for Dinucleotides Synthesis.

Dinucleoside 5',5'-polyphosphates (DNPs) are endogenous substances that play important intra- and extracellular roles in various biological processes, such as cell proliferation, regulation of enzymes, neurotransmission, platelet disaggregation and modulation of vascular tone. Various methodologies have been developed over the past fifty years to access these compounds, involving enzymatic processes or chemical procedures based either on P(III) or P(V) chemistry. Both solution-phase and solid-support strategies have been developed and are reported here. Recently, green chemistry approaches have emerged, offering attracting alternatives. This review outlines the main synthetic pathways for the preparation of dinucleoside 5',5'-polyphosphates, focusing on pharmacologically relevant compounds, and highlighting recent advances.

Synthesis of Substituted 5'-Aminoadenosine Derivatives and Evaluation of Their Inhibitory Potential toward CD73.

Derivatives of 5'-aminoadenosine containing methyl carboxylate, methyl phosphonate, gem-bisphosphonate, bis(methylphosphonate), and α-carboxylmethylphosphonate or phosphonoacetate moieties were synthesized from key intermediate 5'-aminonucleoside. These nucleotide analogues were envisaged as 5'-mono- or diphosphate nucleoside mimics. All compounds were evaluated for CD73 inhibition in a cell-based assay (MDA-MB-231) and toward the purified recombinant protein. Most of them failed to reach significant inhibition of AMP hydrolysis by CD73 at 100 µm. Among the new compounds, the most interesting candidates, 5 (5'-deoxy-5'-N-phosphonomethyladenosine) and 7 (5'-deoxy-5'-N-(ethoxyphosphorylacetate)adenosine), inhibited recombinant CD73 by 36 and 46 % and cellular CD73 by 61 and 45 % at 100 µm, respectively. Molecular modeling partially explains this lack of activity, as the initially predicted docking scores had been encouraging, especially for compound 9.

Plasmodium Purine Metabolism and Its Inhibition by Nucleoside and Nucleotide Analogues.

Malaria still affects around 200 million people and is responsible for more than 400,000 deaths per year, mostly children in subequatorial areas. This disease is caused by parasites of the Plasmodium genus. Only a few WHO-recommended treatments are available to prevent or cure plasmodial infections, but genetic mutations in the causal parasites have led to onset of resistance against all commercial antimalarial drugs. New drugs and targets are being investigated to cope with this emerging problem, including enzymes belonging to the main metabolic pathways, while nucleoside and nucleotide analogues are also a promising class of potential drugs. This review highlights the main metabolic pathways targeted for the development of potential antiplasmodial therapies based on nucleos(t)ide analogues, as well as the different series of purine-containing nucleoside and nucleotide derivatives designed to inhibit Plasmodium falciparum purine metabolism.

Lead optimization and biological evaluation of fragment-based cN-II inhibitors.

The development of cytosolic 5'-nucleotidase II (cN-II) inhibitors is essential to validate cN-II as a potential target for the reversion of resistance to cytotoxic nucleoside analogues. We previously reported a fragment-based approach combined with molecular modelling, herein, the selected hit-fragments were used again in another computational approach based on the Ilib-diverse (a software enabling to build virtual molecule libraries through fragment based de novo design) program to generate a focused library of potential inhibitors. A molecular scaffold related to a previously identified compound was selected and led to a novel series of compounds. Ten out of nineteen derivatives showed 50-75% inhibition on the purified recombinant protein at 200 µM and among them three derivatives (12, 13 and 18) exhibited Ki in the sub-millimolar range (0.84, 2.4 and 0.58 mM, respectively). Despite their only modest potency, the cN-II inhibitors showed synergistic effects when used in combination with cytotoxic purine nucleoside analogues on cancer cells. Therefore, these derivatives represent a family of non-nucleos(t)idic cN-II inhibitors with potential usefulness to overcome cancer drug resistance especially in hematological malignancies in which cN-II activity has been described as an important parameter.

Straightforward Ball-Milling Access to Dinucleoside 5',5'-Polyphosphates via Phosphorimidazolide Intermediates.

A solvent-assisted mechanochemical approach to access symmetrical and mixed dinucleoside 5,5'-polyphosphates is reported. Under ball-milling conditions, nucleoside 5'-monophosphates were quantitatively activated using 1,1'-carbonyldiimidazole, forming their phosphorimidazolide derivatives. The addition of a nucleoside 5'-mono-, di- or triphosphate directly led to the formation of the corresponding dinucleotides. Benefits of the reported one-pot method include the use of unprotected nucleotides in their sodium or acid form, activation by the eco-friendly 1,1'-carbonyldiimidazole, non-dry conditions, short reaction time, high conversion rates, and easy setup and purification. This work offers new perspectives for the synthesis of nucleotide conjugates and analogues, combining the phosphorimidazolide approach and milling conditions.

CD73 inhibition by purine cytotoxic nucleoside analogue-based diphosphonates.

The ecto-5'-nucleotidase CD73 has emerged as an important drug target in oncoimmunology as well as in other diseases. We describe new ADP analogues as CD73 inhibitors based on the replacement of the adenosine moiety, in the reference inhibitor APCP, by purine nucleoside analogues. Compounds were assessed for CD73 inhibition both on purified recombinant protein and on CD73-expressing cancer cells. The clofarabine-containing compound (2) was shown to be more potent than APCP with IC50 values of 0.18 µM (vs. 3.8 µM) on purified protein and 0.24 µM (vs. 23.6 µM) on CD73 expressed on cells. This work gives additional insights into structure-activity relationship of substrate-analogues as CD73 inhibitors.

Synthesis and substrate properties towards HIV-1 reverse transcriptase of new diphosphate analogues of 9-[(2-phosphonomethoxy)ethyl]adenine.

Background The replacement of ß,γ-pyrophosphate by ß,γ-phosphonate moieties within the triphosphate chain of 5'-triphosphate nucleoside analogues was previously studied for various antiviral nucleoside analogues such as AZT and 2',3'-dideoxynucleosides. Thus, it has been shown that these chemical modifications could preserve, in some cases, the terminating substrate properties of the triphosphate analogue for HIV-RT. Herein, we aimed to study such 5'-triphosphate mimics based on the scaffold of the well-known antiviral agent 9-[(2-phosphonomethoxy)ethyl]adenine (PMEA, Adefovir). Methods Synthesis involved coupling of a morpholidate derivative of PMEA with appropriate pyrophosphoryl analogues. The relative efficiencies of incorporation of the studied diphosphate phosphonates were measured using subtype B WT HIV-1 RT in an in vitro susceptibility assay, in comparison to the parent nucleotide analogue (PMEApp). Results Searching for nucleoside 5'-triphosphate mimics, we have synthesized and studied a series of diphosphate analogues of PMEA bearing non hydrolysable bonds between the and phosphorus atoms. We also examined their relative inhibitory capacity towards HIV-1 reverse transcriptase in comparison to the parent nucleotide analogue (PMEApp). Only one of them appeared as a weak inhibitor (IC50 = 403.0 ± 75.5 µM) and proved to be less effective than PMEApp (IC50 = 6.4 ± 0.8 µM). Conclusion PMEA diphosphoryl derivatives were designed as potential substrates and/or inhibitors of various viral polymerases. These modifications dramatically affect their ability to inhibit HIV-RT.

Identification of allosteric inhibitors of the ecto-5'-nucleotidase (CD73) targeting the dimer interface.

The ecto-5'-nucleotidase CD73 plays an important role in the production of immune-suppressive adenosine in tumor micro-environment, and has become a validated drug target in oncology. Indeed, the anticancer immune response involves extracellular ATP to block cell proliferation through T-cell activation. However, in the tumor micro-environment, two extracellular membrane-bound enzymes (CD39 and CD73) are overexpressed and hydrolyze efficiently ATP into AMP then further into immune-suppressive adenosine. To circumvent the impact of CD73-generated adenosine, we applied an original bioinformatics approach to identify new allosteric inhibitors targeting the dimerization interface of CD73, which should impair the large dynamic motions required for its enzymatic function. Several hit compounds issued from virtual screening campaigns showed a potent inhibition of recombinant CD73 with inhibition constants in the low micromolar range and exhibited a non-competitive inhibition mode. The structure-activity relationships studies indicated that several amino acid residues (D366, H456, K471, Y484 and E543 for polar interactions and G453-454, I455, H456, L475, V542 and G544 for hydrophobic contacts) located at the dimerization interface are involved in the tight binding of hit compounds and likely contributed for their inhibitory activity. Overall, the gathered information will guide the upcoming lead optimization phase that may lead to potent and selective CD73 inhibitors, able to restore the anticancer immune response.