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.

Kinase-independent phosphoramidate S1P1 receptor agonist benzyl ether derivatives.

Previously published S1P receptor modulator benzyl ether derivatives have shown potential as being viable therapeutics for the treatment of neurodegenerative diseases, however, two of the most S1P1-selective compounds are reported as being poorly phosphorylated by kinases in vivo. Phosphoramidates of BED compounds (2a, 2b) were synthesised with the aim of producing kinase-independent S1P receptor modulators. Carboxypeptidase, human serum and cell lysate processing experiments were conducted. ProTide BED analogues were found to have an acceptable level of stability in acidic and basic conditions and in vitro metabolic processing experiments showed that they are processed to the desired pharmacologically active monophosphate. The research describes the development of an entirely novel family of therapeutic agents.

Rational design and synthesis of novel anti-prostate cancer agents bearing a 3,5-bis-trifluoromethylphenyl moiety.

Prostate cancer is a major cause of male death worldwide and the identification of new and improved treatments is constantly required. Among the available options, different non-steroidal androgen receptor (AR) antagonists are approved also to treat castration-resistant forms. Most of these drugs show limited application due to the development of resistant mutants of their biological target. Following docking-based studies on a homology model for the AR open antagonist conformation, a series of novel 3,5-bis-trifluoromethylphenyl compounds was designed with the aim to improve the antiproliferative activity of anti-androgen drugs bicalutamide and enzalutamide. The new structural modifications might impede the receptor to adopt its closed agonist conformation also in the presence of adaptive mutations. Among the novel compounds synthesised, several displayed significantly improved in vitro activity in comparison with the parent structures, with IC50 values in the low micromolar range against four different prostate cancer cell lines (LNCaP, VCaP, DU-145, 22Rv1). Selected hits demonstrated full AR antagonistic behaviour and promising candidates for further development were identified.

Synthesis of an apionucleoside family and discovery of a prodrug with anti-HIV activity.

We report the synthesis of a family of D- and L-furano-D-apionucleosides, their 3'-deoxy, as well as their 2',3'-dideoxy analogues with thymine and adenine nucleobases. Single carbon homologation of 1,2-O-isopropylidene-D-glycero-tetrafuranos-3-ulose (15) and optimized glycosylation conditions involving microwave irradiation were key to the successful synthesis of the target compounds. While all target nucleosides failed to show significant antiviral activity, we demonstrated that the triphosphate of 2',3'-deoxy-D-apio-D-furanoadenosine (1), in contrast to that of its D-apio-L-furanose epimer 2, was readily incorporated into a DNA template by HIV reverse transcriptase to act as a DNA chain terminator. This led us to convert adenine derivative 1 into two phosphoramidate prodrugs. ProTide 9b was found active against HIV-1 and HIV-2 (EC50 = 0.5-1.5 µM), indicating that the lack of activity of the parent nucleoside, and possibly also other members of the D-apio-D-furanose nucleoside family must be sought in the inefficient cellular conversion to the monophosphate.

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.

Poly-beta-amino-ester licofelone conjugates development for osteoarthritis treatment.

Disease-modifying osteoarthritis drugs (DMOADs) are a new therapeutic class for osteoarthritis (OA) prevention or inhibition of the disease development. Unfortunately, none of the DMOADs have been clinically approved due to their poor therapeutic performances in clinical trials. The joint environment has played a role in this process by limiting the amount of drug effectively delivered as well as the time that the drug stays within the joint space. The current study aimed to improve the delivery of the DMOADs into cartilage tissue by increasing uptake and retention time of the DMOADs within the tissue. Licofelone was used a model DMOAD due to its significant therapeutic effect against OA progression as shown in the recent phase III clinical trial. For this purpose licofelone was covalently conjugated to the two different A16 and A87 poly-beta-amino-ester (PBAEs) polymers taking advantage of their hydrolysable, cytocompatible, and cationic nature. We have shown cartilage uptake of the licofelone-PBAE conjugates increased 18 times and retention in tissues was prolonged by 37 times compared to the equivalent dose of the free licofelone. Additionally, these licofelone conjugates showed no detrimental effect on the chondrocyte viability. In conclusion, the cationic A87 and A16 PBAE polymers increased the amount of licofelone within the cartilage, which could potentially enhance the therapeutic effect and pharmacokinetic performance of this drug and other DMOADs clinically.

Synthesis of 2-Acetamido-1,3,4-Tri-O-Acetyl-2-Deoxy-D-Mannopyranose -6-Phosphate Prodrugs as Potential Therapeutic Agents.

Sugar phosphates are emerging as potential therapeutic candidates for certain diseases. However, their high polarity makes them poorly absorbed by the body and their penetration inside the cell is even more difficult without a proper transporter. Amino sugar phosphates (n-amino-n-deoxy-sugars, carbohydrates in which a hydroxyl group has been replaced with an amine group), such as N-acetyl-D-mannosamine (ManNac)-6-phosphate have shown potential as a treatment for a muscular disease called GNE myopathy caused by a deficiency in the production of sialic acid. However, its high polarity leads to poor absorption and consequent high dosage in humans, causing unwanted side effects. Herein, we describe the application of phosphoramidate prodrug 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 the gene "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. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 2-Acetamido-1,3,4-tri-O-acetyl-2-deoxy-D-mannopyranose. Basic Protocol 2: Preparation of 3-acetamido-6-((((((S)-1-ethoxy-4-methyl-1-oxo-pentan-2-yl) amino) (phenoxy)phosphoryl) oxy) methyl) tetrahydro-2H-pyran-2,4,5-triyl triacetate (5). Support Protocol: Preparation of ethyl (chloro(phenoxy)phosphoryl)-l-leucinate.

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.

A new series of bicalutamide, enzalutamide and enobosarm derivatives carrying pentafluorosulfanyl (SF5) and pentafluoroethyl (C2F5) substituents: Improved antiproliferative agents against prostate cancer.

SAR studies on bicalutamide, enobosarm and enzalutamide analogues, functionalised with polyfluorinated groups, is presented. Among the novel bicalutamide and enobosarm derivatives synthesised, several displayed significantly improved in vitro anticancer activity, with IC50 values in the low micromolar range against four different prostate cancer cell lines (LNCaP, VCaP, DU-145 and 22Rv1), showing up to 48-fold increase in comparison with the parent structures. In particular, SF5 enobosarm analogues were found to be most potent compounds, full AR antagonists and with favourable ADME properties. The most promising compound (48a) was evaluated for its in vivo efficacy in PC xenograft mouse model (22Rv1) with results comparable to the standard-of-care docetaxel.

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.

Phosphonoamidate prodrugs of C5-substituted pyrimidine acyclic nucleosides for antiviral therapy.

Acyclic nucleoside phosphonates (ANPs) are nowadays one of the key drugs in the treatment of DNA virus and retrovirus infections. In this work, we report the synthesis and antiviral evaluation of phosphonoamidate and diamidates prodrugs of C5-pyrimidine acyclic nucleosides derivatives functionalized with but-2-enyl- chain. In the phosphonoamidate series, the most active compound 15, showed sub-micromolar activity against varicella zoster virus (VZV) (EC50 = 0.09-0.5 µM) and µM activity against human cytomegalovirus (HCMV) and herpes simplex virus (HSV). Separation of single diastereoisomers for compound 14, showed that 14b had better anti-herpesvirus activity and no cytotoxicity compared to the diastereoisomeric mixture 14. Very interestingly, phosphonodiamidate 21 showed anti-herpesvirus activity with excellent activity against wild type and thymidine kinase-deficient (TK-) VZV strains (EC50 = 0.47 and 0.2 µM, respectively) and HCMV (EC50 = 3.5-7.2 µM) without any cytotoxicity (CC50 > 100).

Synthetic Approaches for the Preparation of Phosphoramidate Prodrugs of 2'-Deoxypseudoisocytidine.

A synthetic procedure for the preparation of phosphoramidate prodrugs of C-nucleosides is reported. Different phosphorochloridates were reacted with 3'-O-protected N-acetyl-2'-deoxypseudoisocytidine or 3'-O-protected 2'-deoxypseudoisocytidine, followed by acidic hydrolysis of the protecting group. In the presence of the N-acetyl moiety, the enolisable keto group of the nucleobase was able to react (like the 5'-OH) with the phosphorochloridates to give bisphosphorylated derivatives. Epimerisation (ß to α) occurred if the amino group of the nucleobase was unprotected. These side reactions demonstrate the peculiar behaviour of C-nucleosides compared to their nucleoside analogues. It was demonstrated that the first enzymatic activation step for this new class of prodrugs can be mediated by carboxypeptidase and that it follows the same pathway and rate reported for ProTides of more conventional nucleoside analogues. These new phosphoramidate derivatives deserve further investigation for their therapeutic potential as anti-cancer agents.

Rational design and synthesis of novel phenylsulfonyl-benzamides as anti-prostate cancer agents.

Prostate cancer is a major cause of male death worldwide and the identification of new efficient treatments is constantly needed. Different non-steroidal androgen receptor antagonists are approved also in the case of castration-resistant cancer forms. Using a rational approach and molecular modelling studies to modify the structure of antiandrogen drug bicalutamide, a new series of phenylsulfonyl-benzamide derivatives was designed and synthesised. Their antiproliferative activities were evaluated in four different human prostate cancer cell lines and several new compounds showed significantly improved IC50 values in the low µM range. The cytotoxicity profile was also evaluated for the novel structures in the HEK293 cell line.