Design, synthesis and biological evaluation of aryloxy thiophosphoramidate triesters of anticancer nucleoside analogues.

Aryloxy phosphoroamidate triesters, known as ProTides, are a class of prodrugs developed to enhance the physicochemical and pharmacological properties of therapeutic nucleosides. This approach has been extensively investigated in the antiviral and anticancer areas leading to three prodrugs on the market and several others in clinical stage. In this article we have prepared the PS analogues of three ProTides that have reached the clinic as anticancer agents. These novel PS ProTides were tested for their capacity in enzymatic activation and for their cytotoxic properties against a panel of solid and liquid tumor cell lines. As expected, the replacement of the PO with a PS bond led to increased metabolic stability albeit concomitant to a decrease in potency. Surprisingly, the intermediate formed after the first activation step of a thiophosphoramidate with carboxypeptidase Y is not the expected PS aminoacyl product but the corresponding PO aminoacyl compound.

Synthesis, molecular docking and antibacterial activity of an oxadiazole-based lipoteichoic acid inhibitor and its metabolites.

Amongst drug resistant Gram-positive bacteria, Staphylococcus aureus is a pathogen of great concern as it is the leading cause of life-threatening nosocomial and community acquired infections which are often associated with implanted medical devices. The biosynthesis of lipotheicoic acid (LTA) by S. aureus has been recognized as a promising antibacterial target, owing its critical role in the growth and survival of Gram-positive bacteria. Here we report for the first time the chemical synthesis and characterisation of an oxadiazole based compound (1771), previously described as an inhibitor of LTA biosynthesis by targeting Lta synthase enzyme (LtaS). To investigate its controversial mode of action, we also performed molecular docking studies, which indicated that 1771 behaves as a competitive inhibitor against LtaS. We also synthesised and evaluated the antimicrobial activity of 1771 metabolites which we have identified from its decomposition in mouse serum, proving that the biological activity was caused by intact 1771.

The nucleotide prodrug CERC-913 improves mtDNA content in primary hepatocytes from DGUOK-deficient rats.

Loss-of-function mutations in the deoxyguanosine kinase (DGUOK) gene result in a mitochondrial DNA (mtDNA) depletion syndrome. DGUOK plays an important role in converting deoxyribonucleosides to deoxyribonucleoside monophosphates via the salvage pathway for mtDNA synthesis. DGUOK deficiency manifests predominantly in the liver; the most common cause of death is liver failure within the first year of life and no therapeutic options are currently available. in vitro supplementation with deoxyguanosine or deoxyguanosine monophosphate (dGMP) were reported to rescue mtDNA depletion in DGUOK-deficient, patient-derived fibroblasts and myoblasts. CERC-913, a novel ProTide prodrug of dGMP, was designed to bypass defective DGUOK while improving permeability and stability relative to nucleoside monophosphates. To evaluate CERC-913 for its ability to rescue mtDNA depletion, we developed a primary hepatocyte culture model using liver tissue from DGUOK-deficient rats. DGUOK knockout rat hepatocyte cultures exhibit severely reduced mtDNA copy number (~10%) relative to wild type by qPCR and mtDNA content remains stable for up to 8 days in culture. CERC-913 increased mtDNA content in DGUOK-deficient hepatocytes up to 2.4-fold after 4 days of treatment in a dose-dependent fashion, which was significantly more effective than dGMP at similar concentrations. These early results suggest primary hepatocyte culture is a useful model for the study of mtDNA depletion syndromes and that CERC-913 treatment can improve mtDNA content in this model.

Drug repurposing: phosphate prodrugs of anticancer and antiviral FDA-approved nucleosides as novel antimicrobials.

OBJECTIVES: Following a drug repurposing approach, we aimed to investigate and compare the antibacterial and antibiofilm activities of different classes of phosphate prodrugs (HepDirect, cycloSal, SATE and mix SATE) of antiviral and anticancer FDA-approved nucleoside drugs [zidovudine (AZT), floxouridine (FUDR) and gemcitabine (GEM)] against a variety of pathogenic Gram-positive and -negative bacteria. METHODS: Ten prodrugs were synthesized and screened for antibacterial activity against seven Gram-negative and two Gram-positive isolates fully susceptible to traditional antibiotics, alongside six Gram-negative and five Gram-positive isolates with resistance mechanisms. Their ability to prevent and eradicate biofilms of different bacterial pathogens in relation to planktonic growth inhibition was also evaluated, together with their effect on proliferation, viability and apoptosis of different eukaryotic cells. RESULTS: The prodrugs showed decreased antibacterial activity compared with the parent nucleosides. cycloSal-GEM-monophosphate (MP) prodrugs 20a and 20b were the most active agents against Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus) and retained their activity against antibiotic-resistant isolates. cycloSal-FUDR-MP 21a partially retained good activity against the Gram-positive bacteria E. faecalis, Enterococcus faecium and S. aureus. Most of the prodrugs tested displayed very potent preventive antibiofilm specific activity, but not curative. In terms of cytotoxicity, AZT prodrugs did not affect apoptosis or cell viability at the highest concentration tested, and only weak effects on apoptosis and/or cell viability were observed for GEM and FUDR prodrugs. CONCLUSIONS: Among the different prodrug approaches, the cycloSal prodrugs appeared the most effective. In particular, cycloSal (17a) and mix SATE (26) AZT prodrugs combine the lowest cytotoxicity with high and broad antibacterial and antibiofilm activity against Gram-negative bacteria.

λ5-Phosphorus-Containing α-Diazo Compounds: A Valuable Tool for Accessing Phosphorus-Functionalized Molecules.

The compounds characterized by the presence of a λ5-phosphorus functionality at the α-position with respect to the diazo moiety, here referred to as λ5-phosphorus-containing α-diazo compounds (PCDCs), represent a vast class of extremely versatile reagents in organic chemistry and are particularly useful in the preparation of phosphonate- and phosphinoxide-functionalized molecules. Indeed, thanks to the high reactivity of the diazo moiety, PCDCs can be induced to undergo a wide variety of chemical transformations. Among them are carbon-hydrogen, as well as heteroatom-hydrogen insertion reactions, cyclopropanation, ylide formation, Wolff rearrangement, and cycloaddition reactions. PCDCs can be easily prepared from readily accessible precursors by a variety of different methods, such as diazotization, Bamford-Stevens-type elimination, and diazo transfer reactions. This evidence along with their relative stability and manageability make them appealing tools in organic synthesis. This Review aims to demonstrate the ongoing utility of PCDCs in the modern preparation of different classes of phosphorus-containing compounds, phosphonates, in particular. Furthermore, to address the lack of precedent collective papers, this Review also summarizes the methods for PCDCs preparation.

Expedient synthesis and biological evaluation of alkenyl acyclic nucleoside phosphonate prodrugs.

The importance of phosphonoamidate prodrugs (ProTides) of acyclic nucleoside phosphonate (ANPs) is highlighted by the approval of Tenofovir Alafenamide Fumarate for the treatment of HIV and HBV infections. In the present paper we are reporting an expedient, one-pot, two-steps synthesis of allyl phosphonoamidates and diamidates that offers a time saving strategy when compared to literature methods. The use of these substrates in the cross metathesis reactions with alkenyl functionalised thymine and uracil nucleobases is reported. ANPs prodrugs synthesized via this methodology were evaluated for their antiviral activities against DNA and RNA viruses. It is anticipated that the use of 5,6,7,8-tetrahydro-1-napthyl as aryloxy moiety is capable to confer antiviral activity among a series of otherwise inactive uracil ProTides.

Synthesis and biological evaluation of phosphoramidate prodrugs of two analogues of 2-deoxy-d-ribose-1-phosphate directed to the discovery of two carbasugars as new potential anti-HIV leads.

2-Deoxy-α-d-ribose-1-phosphate is of great interest as it is involved in the biosynthesis and/or catabolic degradation of several nucleoside analogues of biological and therapeutic relevance. However due to the lack of a stabilising group at its 2-position, it is difficult to synthesize stable prodrugs of this compound. In order to overcome this lack of stability, the synthesis of carbasugar analogues of 2-deoxyribose-1-phosphate was envisioned. Herein the preparation of a series of prodrugs of two carbocyclic analogues of 2-deoxyribose-1-phosphate using the phosphoramidate ProTide technology, along with their biological evaluation against HIV and cancer cell proliferation, is reported.

ProTides of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide as potential anti-tubercular and anti-viral agents.

The flavin-dependent thymidylate synthase X (ThyX), rare in eukaryotes and completely absent in humans, is crucial in the metabolism of thymidine (a DNA precursor) in many microorganisms including several human pathogens. Conserved in mycobacteria, including Mycobacterium leprae, and Mycobacterium tuberculosis, it represents a prospective anti-mycobacterial therapeutic target. In a M. tuberculosis ThyX-enzyme inhibition assay, N-(3-(5-(2'-deoxyuridine-5'-phosphate))prop-2-ynyl)octanamide was reported to be the most potent and selective 5-substituted 2'-deoxyuridine monophosphate analogue. In this study, we masked the two charges at the phosphate moiety of this compound using our ProTide technology in order to increase its lipophilicity and then allow permeation through the complex mycobacterial cell wall. A series of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide phosphoroamidates were chemically synthesized and their biological activity as potential anti-tuberculars was evaluated. In addition to mycobacteria, several DNA viruses depend on ThyX for their DNA biosynthesis, thus these prodrugs were also screened for their antiviral properties.

Improved Antibacterial Activity of 1,3,4-Oxadiazole-Based Compounds That Restrict Staphylococcus aureus Growth Independent of LtaS Function.

The lipoteichoic acid (LTA) biosynthesis pathway has emerged as a promising antimicrobial therapeutic target. Previous studies identified the 1,3,4 oxadiazole compound 1771 as an LTA inhibitor with activity against Gram-positive pathogens. We have succeeded in making six 1771 derivatives and, through subsequent hit validation, identified the incorporation of a pentafluorosulfanyl substituent as central in enhancing activity. Our newly described derivative, compound 13, showed a 16- to 32-fold increase in activity compared to 1771 when tested against a cohort of multidrug-resistant Staphylococcus aureus strains while simultaneously exhibiting an improved toxicity profile against mammalian cells. Molecular techniques were employed in which the assumed target, lipoteichoic acid synthase (LtaS), was both deleted and overexpressed. Neither deletion nor overexpression of LtaS altered 1771 or compound 13 susceptibility; however, overexpression of LtaS increased the MIC of Congo red, a previously identified LtaS inhibitor. These data were further supported by comparing the docking poses of 1771 and derivatives in the LtaS active site, which indicated the possibility of an additional target(s). Finally, we show that both 1771 and compound 13 have activity that is independent of LtaS, extending to cover Gram-negative species if the outer membrane is first permeabilized, challenging the classification that these compounds are strict LtaS inhibitors.

Structure of cyclic nucleoside phosphonate ester prodrugs: an inquiry.

The configuration at phosphorus in cyclic (S)-HPMPC (1, cidofovir) and (S)-HPMPA (2) phenyl ester (5 and 6, respectively) diastereomers ((R(p))-5, (R(p))-6, (S(p))-6) was determined by X-ray crystallography and correlated to their (1)H and (31)P NMR spectra in solution. (R(p))-5 and (R(p))-6 have chair conformations with the nucleobase substituent equatorial and the P-OPh axial. Perhaps surprisingly, (S(p))-6 is (a, a) in the crystal and exists largely as an equilibrium of (a, a)/(e, e) conformers in chloroform or acetonitrile.

Synthesis and Characterization of NUC-7738, an Aryloxy Phosphoramidate of 3'-Deoxyadenosine, as a Potential Anticancer Agent.

3'-Deoxyadenosine (3'-dA, Cordycepin, 1) is a nucleoside analogue with anticancer properties, but its clinical development has been hampered due to its deactivation by adenosine deaminase (ADA) and poor cellular uptake due to low expression of the human equilibrative transporter (hENT1). Here, we describe the synthesis and characterization of NUC-7738 (7a), a 5'-aryloxy phosphoramidate prodrug of 3'-dA. We show in vitro evidence that 7a is an effective anticancer drug in a panel of solid and hematological cancer cell lines, showing its preferential cytotoxic effects on leukemic stem cells. We found that unlike 3'-dA, the activity of 7a was independent of hENT1 and kinase activity. Furthermore, it was resistant to ADA metabolic deactivation. Consistent with these findings, 7a showed increased levels of intracellular 3'-deoxyadenosine triphosphate (3'-dATP), the active metabolite. Mechanistically, levels of intracellular 3'-dATP were strongly associated with in vitro potency. NUC-7738 is now in Phase II, dose-escalation study in patients with advanced solid tumors.

An overview of ProTide technology and its implications to drug discovery.

Introduction: The ProTide technology is a phosphate (or phosphonate) prodrug method devised to deliver nucleoside monophosphate (or monophosphonate) intracellularly bypassing the key challenges of antiviral and anticancer nucleoside analogs. Three new antiviral drugs, exploiting this technology, have been approved by the FDA while others are in clinical studies as anticancer agents.Areas covered: The authors describe the origin and development of this technology and its incredible success in transforming the drug discovery of antiviral and anticancer nucleoside analogues. As evidence, discussion on the antiviral ProTides on the market, and those currently in clinical development are included. The authors focus on how the proven capacity of this technology to generate new drug candidates has stimulated its application to non-nucleoside-based molecules.Expert opinion: The ProTide approach has been extremely successful in delivering blockbuster antiviral medicines and it seems highly promising in oncology. Its application to non-nucleoside-based small molecules is recently emerging and proving effective in other therapeutic areas. However, investigations to explain the lack of activity of certain ProTide series and comprehensive structure activity relationship studies to identify the appropriate phosphoramidate motifs depending on the parent molecule are in our opinion mandatory for the future development of these compounds.

Single Diastereomers of the Clinical Anticancer ProTide Agents NUC-1031 and NUC-3373 Preferentially Target Cancer Stem Cells In Vitro.

A 3'-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

The Novel Nucleoside Analogue ProTide NUC-7738 Overcomes Cancer Resistance Mechanisms In Vitro and in a First-In-Human Phase I Clinical Trial.

PURPOSE: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a preactivated nucleoside released. 3'-Deoxyadenosine (3'-dA) is a naturally occurring adenosine analogue with established anticancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells, and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3'-dA was chemically modified to create the novel ProTide NUC-7738. EXPERIMENTAL DESIGN: We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in patients with cancer. RESULTS: We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3'-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide-binding protein 1. PK and tumor samples obtained from the ongoing first-in-human phase I clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective proapoptotic agent in cancer cells with effects on the NF-κB pathway. CONCLUSIONS: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and supports its further clinical evaluation as a novel cancer treatment within the growing pantheon of anticancer ProTides.

Serine side chain-linked peptidomimetic conjugates of cyclic HPMPC and HPMPA: synthesis and interaction with hPEPT1.

Cidofovir (HPMPC), a broad spectrum antiviral agent, cannot be administered orally due to ionization of its phosphonic acid group at physiological pH. One prodrug approach involves conversion to the cyclic form (cHPMPC, 1) and esterification by the side chain hydroxyl group of a peptidomimetic serine. Transport studies in a rat model have shown enhanced levels of total cidofovir species in the plasma after oral dosing with L-Val-L-Ser-OMe cHPMPC, 2a. To explore the possibility that 2a and its three L/D stereoisomers 2b-d undergo active transport mediated by the peptide-specific intestinal transporter PEPT1, we performed radiotracer uptake and electrophysiology experiments applying the two-electrode voltage clamp technique in Xenopus laevis oocytes overexpressing human PEPT1 (hPEPT1, SLC15A1). 2a-d did not induce inward currents, indicating that they are not transported, but the stereoisomers with an L-configuration at the N-terminal valine (2a and 2b) potently inhibited transport of the hPEPT1 substrate glycylsarcosine (Gly-Sar). A "reversed" dipeptide conjugate, L-Ser-L-Ala-OiPr cHPMPC (4), also did not exhibit detectable transport, but completely abolished the Gly-Sar signal, suggesting that affinity of the transporter for these prodrugs is not impaired by a proximate linkage to the drug in the N-terminal amino acid of the dipeptide. Single amino acid conjugates of cHPMPC (3a and 3b) or cHPMPA (5, 6a and 6b) were not transported and only weakly inhibited Gly-Sar transport. The known hPEPT1 prodrug substrate valacyclovir (7) and its L-Val-L-Val dipeptide analogue (8) were used to verify coupled transport by the oocyte model. The results indicate that the previously observed enhanced oral bioavailability of 2a relative to the parent drug is unlikely to be due to active transport by hPEPT1. Syntheses of the novel compounds 2b-d and 3-6 are described, including a convenient solid-phase method to prepare 5, 6a and 6b.

Targeting GNE Myopathy: A Dual Prodrug Approach for the Delivery of N-Acetylmannosamine 6-Phosphate.

ProTides comprise an important class of prodrugs currently marketed and developed as antiviral and anticancer therapies. The ProTide technology employs phosphate masking groups capable of providing more favorable druglike properties and an intracellular activation mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein, we describe the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac3ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac3ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. Prodrug 12b showed rapid activation in a carboxylesterase (CPY) enzymatic assay and favorable ADME properties, while also being more effective than ManNAc at increasing sialic acid levels in GNE-deficient cell lines. These results provide a potential platform to address substrate deficiencies in GNE myopathy and other CDGs.

Preparation of Pyrimidine Alkenyl Acyclic Nucleoside Phosphonoamidates.

This synthetic protocol describes two strategies for the preparation of pyrimidine alkenyl acyclic nucleoside phosphonoamidates (ANPs), including linear and trisubstituted alkenyl derivatives. For the first procedure, a bis-trimethylsilyl ester of the parent alkenyl ANPs is the key intermediate that reacts with the desired amino acid ester and aryl alcohol. For the second procedure, an allyl phosphonoamidate bearing the ProTide promoieties is the key synthon employed as olefin partner for a cross-metathesis reaction with an alkylated nucleobase. © 2018 by John Wiley & Sons, Inc.

Phosphoramidates and phosphonamidates (ProTides) with antiviral activity.

Following the first report on the nucleoside phosphoramidate (ProTide) prodrug approach in 1990 by Chris McGuigan, the extensive investigation of ProTide technology has begun in many laboratories. Designed with aim to overcome limitations and the key resistance mechanisms associated with nucleoside analogues used in the clinic (poor cellular uptake, poor conversion to the 5'-monophosphate form), the ProTide approach has been successfully applied to a vast number of nucleoside analogues with antiviral and anticancer activity. ProTides consist of a 5'-nucleoside monophosphate in which the two hydroxyl groups are masked with an amino acid ester and an aryloxy component which once in the cell is enzymatically metabolized to deliver free 5'-monophosphate, which is further transformed to the active 5'-triphosphate form of the nucleoside analogue. In this review, the seminal contribution of Chris McGuigan's research to this field is presented. His technology proved to be extremely successful in drug discovery and has led to two Food and Drug Administration-approved antiviral agents.

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, molecular modeling studies, and preliminary pharmacological characterization of all possible 2-(2'-sulfonocyclopropyl)glycine stereoisomers as conformationally constrained L-homocysteic acid analogs.

Bioisosteric replacements of the distal acidic group of L-glutamic acid (L-Glu, 1) and conformational constraining of its carbon skeleton, have been widely exploited to discover competitive modulators of glutamate receptors. Noteworthy, L-homocysteic acid (L-HCA, 18), a neurotransmitter belonging to the class of excitatory sulfur-containing amino acids, may be considered an endogenous occurring bioisoster of L-Glu (1). L-HCA (18) has been reported to mediate signaling between glial cells and postsynaptic neurons through the activation of glutamate receptors and others hitherto not well-characterized receptors. As a continuation of our work in the preparation of conformationally constrained glutamate analogs, we report the synthesis and the preliminary pharmacological characterization at iGluRs and mGluRs of all eight stereoisomers of 2-(2'-sulfonocyclopropyl)glycine (SCGs, 8-15). Among the reported compounds, S-SCG-4 (15) showed to be a potent and relatively selective AMPA ligand. Docking experiments coupled to molecular electrostatic potential calculations allowed insight into the molecular basis of the activity of this compound to be gained. The library of SCGs (8-15), while providing a novel source of modulators of the glutamate receptors, represents a valuable chemical tool to better characterize L-HCA pathways in the CNS.