Phosphoramidate Prodrugs Research Articles

A Phosphoramidate Prodrug Platform: One-Pot Amine Functionalization of Kinase Inhibitors with Oligoethylene Glycol for Improved Water-Solubility.

Small molecular kinase inhibitors play a key role in modern cancer therapy. Protein kinases are essential mediators in the growth and progression of cancerous tumors, rendering involved kinases an increasingly important target for therapy. However, kinase inhibitors are almost insoluble in water because of their hydrophobic aromatic nature, often lowering their availability and pharmacological efficacy. Direct drug functionalization with polar groups represents a simple strategy to improve the drug solubility, availability, and performance. Here, we present a strategy to functionalize secondary amines with oligoethylene glycol (OEG) phosphate using a one-pot synthesis in three exemplary kinase inhibiting drugs Ceritinib, Crizotinib, and Palbociclib. These OEG-prodrug conjugates demonstrate superior solubility in water compared to the native drugs, with the solubility increasing up to 190-fold. The kinase inhibition potential is only slightly decreased for the conjugates compared to the native drugs. We further show pH dependent hydrolysis of the OEG-prodrugs which releases the native drug. We observe a slow release at pH 3, while the conjugates remain stable over 96 h under physiological conditions (pH 7.4). Using confocal microscopy, we verify improved cell uptake of the drug-OEG conjugates into the cytoplasm of HeLa cells, further supporting our universal solubility approach.

Discovery of GS-7682, a Novel 4'-Cyano-Modified C-Nucleoside Prodrug with Broad Activity against Pneumo- and Picornaviruses and Efficacy in RSV-Infected African Green Monkeys.

Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 (1), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C-nucleoside GS-646089 (2) with broad antiviral activity against RSV (EC50 = 3-46 nM), human metapneumovirus (EC50 = 210 nM), human rhinovirus (EC50 = 54-61 nM), and enterovirus (EC50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [(S)-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo. 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.

Mechanism and spectrum of inhibition of a 4′-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRp) of prototypic respiratory viruses. GS-646939 is the active 5′-triphosphate (TP) metabolite of a 4ʹ-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 (EV-71) incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase (h-mtRNAP) does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain-terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4ʹ-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1′-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1ʹ-cyano and 4ʹ-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.

Fingolimod phosphoramidate prodrugs: Synthesis, photophysical characterisation and lipid bilayer interaction of fluorescent tagged Prodrug

FTY720 (Fingolimod, Gilenya, 1), a structural analog of sphingosine (2) was the first orally administered drug approved for the treatment of multiple sclerosis (MS). However, the phosphate derivative of Fingolimod, namely Fingolimod-1-phosphate (FTY720-1P, 3) is the biologically active molecule in MS as well as in various lysosomal storage diseases such as mucolipidosis IV and Nieman-Pick. In all cases, Fingolimod requires to be phosphorylated, resembling the natural sphingosine phosphate (4), to accomplish its therapeutic/biological effects. Here, we report the synthesis of a small library of prodrugs that could provide the efficient delivery of 3, the bioactive species. Moreover, to gain insight about the biological behaviour of these novel candidates within human body such as cell and brain blood barrier penetration we have prepared and spectroscopically characterised a fluorescent tagged version of one of the new prodrugs. The behavior of our prodrugs with respect to biological membranes was evaluated by studying the interaction between the fluorescent prodrug 18 and DOPC/DPPC liposome models.

Development of a Novel DNA Mono-alkylator Platform for Antibody-Drug Conjugates.

Although microtubule inhibitors (MTI) remain a therapeutically valuable payload option for antibody-drug conjugates (ADC), some cancers do not respond to MTI-based ADCs. Efforts to fill this therapeutic gap have led to a recent expansion of the ADC payload "toolbox" to include payloads with novel mechanisms of action such as topoisomerase inhibition and DNA cross-linking. We present here the development of a novel DNA mono-alkylator ADC platform that exhibits sustained tumor growth suppression at single doses in MTI-resistant tumors and is well tolerated in the rat upon repeat dosing. A phosphoramidate prodrug of the payload enables low ADC aggregation even at drug-to-antibody ratios of 5:1 while still delivering a bystander-capable payload that is effective in multidrug resistant (MDR)-overexpressing cell lines. The platform was comparable in xenograft studies to the clinical benchmark DNA mono-alkylator ADC platform DGN459 but with a significantly better tolerability profile in rats. Thus, the activity and tolerability profile of this new platform make it a viable option for the development of ADCs.

A ProTide of AZT Shows Activity Against Human Papillomaviruses.

Infection by human papillomaviruses (HPV) can cause warts and tumors. So far, no small molecule antiviral has been approved for the treatment of infections with this DNA virus, although preclinical studies show activity for nucleosidic compounds, such as 9-(2-phosphonylmethoxy)ethylguanine (PMEG) or cidofovir. This prompted us to test new prodrug versions of the nucleoside analog 3'-azido-2',3'-dideoxythymidine (AZT), known to be active against reverse transcriptases and approved for the treatment of HIV. Here we report the synthesis of an ethylbutyl alaninyl ester phosphosphoramidate prodrug of AZT, dubbed AZAEB, and its activity against HPV, a target not known to be sensitive to AZT. A methyl ester derivative was found to be inactive against this and three other DNA viruses, while the phosphoramidate prodrug AZAEB showed a modest inhibitory effect against HPV types 6, 11, 18 and 31. Our results open up new avenues of study for the treatment of diseases caused by members of the papillomaviridae family.

Cytotoxicity of phosphoramidate, bis-amidate and cycloSal prodrug metabolites against tumour and normal cells.

Phosphonate and phosphate prodrugs are integral to enhancing drug permeability, but the potential toxicity of their metabolites requires careful consideration. This study evaluates the impact of widely used phosphoramidate, bis-amidate, and cycloSal phosph(on)ate prodrug metabolites on BxPC3 pancreatic cancer cells, GL261-Luc glioblastoma cells, and primary cultured mouse astrocytes. 1-Naphthol and 2-naphthol demonstrated the greatest toxicity. Notably, 2-naphthol exhibited an ED50 of 21 μM on BxPC3 cells, surpassing 1-naphthol with an ED50 of 82 μM. Real-time xCELLigence experiments revealed notable activity for both metabolites at a low concentration of 16 μM. On primary cultured mouse astrocyte cells, all prodrugs exhibited reduced viability at 128 to 256 μM after only 4 hours of exposure. A cell-type-dependent sensitivity to phosph(on)ate prodrug metabolites was evident, with normal cells showing greater susceptibility than corresponding tumour cells. The results suggest it is essential to consider the potential cytotoxicity of phosph(on)ate prodrugs in the drug design and evaluation process.

Modified Synthesis and Isolation of an Advanced Intermediate of Remdesivir

Abstract: Synthesis and isolation of an advanced intermediate (S)-2-Ethylbutyl 2-(((S)-(4- nitrophenoxy) (phenoxy) phosphoryl) amino) propanoate (1b), which is being used for the manufacture of the prodrug diastereoisomer 1d called Remdesivir have been carried out in high yield with efficient stereoselectivity. The isolated advanced intermediate 1b was a diastereoselective nucleoside phosphoramidate prodrug used as an antiviral agent having a mixture of two (SS) and (SR) diastereomers with stereocenter at phosphorus, which was purified by converting into a more stable diastereoselective isomer (SS) by simple physical fractional crystallization process, resulting in an improved yield of ~45%. The recrystallization has been afforded diastereomerically in 99% pure (SS)-isomer 1b. The 1H NMR characterization data confirm the (SS)-isomer (1b). The developed process holds significant potential for large-scale reactions relatively with commercially available low-cost solvents and co-solvents, resulting in an alternative cheaper process.

Recent advances in drug substance development – prodrug strategies for enhancing the bioavailability and potency of antiviral nucleosides

Bioavailability is a prerequisite for drug activity. In vivo bioavailability (intestinal permeability), linked to drug substance solubility and drug product dissolution, became the basis of Gordon L. Amidon’s Biopharmaceutical Classification System. One method of improving the drug substance’s bioavailability is to modify its structure chemically, leading to increased lipophilicity and the ability to penetrate the phospholipid bilayer of the cell membrane. These modifications, known as prodrug strategies, involve derivatizing the drug substance by introducing substituents that reduce the hydrophilicity of the molecule. The present mini-review outlines the examples of Christopher McGuigan’s prodrug strategies used to obtain antiviral nucleosides with enhanced bioavailability and activity. These strategies primarily involve forming and optimizing the structure of esters and amino acid esters, phosphoramidates, octadecyl phosphates, and bis-pivaloxymethyl phosphates. The review discusses the optimization of the phosphoramidate prodrug moiety of the SARS-CoV-2 antiviral nucleoside remdesivir in detail. It presents the resulting improvement in bioavailability and antiviral activity. Moreover, it focuses on the modern prodrug strategy as one of the major recent advances in drug substance development. This strategy effectively optimized physicochemical properties and improved the functional activity of the existing drug substances and drug substance candidates for the first time.

Synthesis of mizoribine prodrugs and their in vivo evaluation as immunosuppressive agents

Mizoribine is a well-known immunosuppressive drug, based on a nucleoside scaffold, that targets inosine-monophosphate dehydrogenase (IMPDH). In an effort to increase its in vivo efficacy, three different types of prodrugs (a phosphoramidate prodrug, a lipophilic ester derivative and an amino acid conjugate) were prepared. Screening of these prodrugs in a rapid whole blood assay revealed that the two ester-based mizoribine prodrugs potently inhibited interleukin 2 secretion. Moreover, these prodrugs were able to prolong graft survival, when evaluated in a mouse model of cardiac allograft transplantation. Strikingly, a combination therapy of these mizoribine prodrugs with tacrolimus had a synergistic in vivo effect.

Discovery of GS-5245 (Obeldesivir), an Oral Prodrug of Nucleoside GS-441524 That Exhibits Antiviral Efficacy in SARS-CoV-2-Infected African Green Monkeys.

Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 (2) into lung cells, thereby forming the bioactive triphosphate 2-NTP. 2-NTP, an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP. Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP, leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2, which supports development of 3 as a promising COVID-19 treatment.

The Many Faces of Histidine Triad Nucleotide Binding Protein 1 (HINT1).

The histidine triad nucleotide binding protein 1 (HINT1) is a nucleoside phosphoramidase that has garnered interest due to its widespread expression and participation in a broad range of biological processes. Herein, we discuss the role of HINT1 as a regulator of several CNS functions, tumor suppressor, and mast cell activator via its interactions with multiple G-protein-coupled receptors and transcription factors. Importantly, altered HINT1 expression and mutation are connected to the progression of multiple disease states, including several neuropsychiatric disorders, peripheral neuropathy, and tumorigenesis. Additionally, due to its involvement in the activation of several clinically used phosphoramidate prodrugs, tremendous efforts have been made to better understand the interactions behind nucleoside binding and phosphoramidate hydrolysis by HINT1. We detail the substrate specificity and catalytic mechanism of HINT1 hydrolysis, while highlighting the structural biology behind these efforts. The aim of this review is to summarize the multitude of biological and pharmacological functions in which HINT1 participates while addressing the areas of need for future research.

In vivo pharmacodynamic study of contezolid acefosamil, a prodrug of contezolid for oral and intravenous administration.

Contezolid acefosamil is a novel O-acyl phosphoramidate prodrug of contezolid. In the current study, we aimed to systemically evaluate the efficacy of contezolid acefosamil against infections caused by multiple Gram-positive pathogens, and compare the efficacy of the prodrug by oral and intravenous administrations. The in vivo pharmacodynamic efficacy of contezolid acefosamil was evaluated in mouse models of systemic (with five S. aureus, three S. pneumoniae and two S. pyogenes bacterial isolates) and thigh (with two S. aureus isolates) infections using linezolid as the reference agent. In both models, contezolid acefosamil administrated either orally or intravenously, demonstrated high antibacterial efficacy similar to linezolid, and the antibacterial efficacy of oral and intravenous contezolid acefosamil were comparable. The high aqueous solubility and great efficacy of contezolid acefosamil support its clinical development as an injectable and oral antibiotic suitable for serious Gram-positive infections.

Derivatives of 3′‐Azidothymidine with 6‐Cyanopyridone as Base or as Phosphoramidate Ester and their Antiretroviral Activity

AbstractStrongly pairing ethynylpyridone C‐nucleosides are attractive surrogates for thymidine in oligonucleotides. Exploratory work on the antiviral activity of 3′‐azidothymidine (AZT) derivatives with ethynylpyridone as base had identified strong lipophilicity as a limiting factor. Two strategies are being pursued to overcome this issue. In order to make the base more polar, the ethynyl group has been replaced with a cyano group, leading to a cyanopyridone C‐nucleoside, whose eleven‐step synthesis is reported here, together with the synthesis of a 3′‐azido‐2′,3′‐dideoxynucleoside derivative. The base pairing with adenine in a DNA duplex was studied by UV melting analysis of a self‐complementary hexamer containing the 6‐cyano‐2′‐deoxynucleoside instead of thymidine. A melting point increase of 2 °C compared to the unmodified control was found. The other strategy employs a phosphoramidate prodrug design with less lipophilic amino acid esters. Here, anti‐HIV test of the alaninyl and prolinyl methyl esters of AZT gave promising results in cell culture experiments, increasing the selectivity index up to 5.8‐fold for the IIIB strain and up to 5‐fold for the ROD strain of the virus, as compared to the parent nucleoside. These findings help to design the next generation of pyridone C‐nucleosides with potential applications as antivirals.

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.

Discovery of a 2'-Fluoro,2'-Bromouridine Phosphoramidate Prodrug Exhibiting Anti-Yellow Fever Virus Activity in Culture and in Mice.

Yellow fever virus (YFV) is a potentially lethal, zoonotic, blood-borne flavivirus transmitted to humans and non-human primates by mosquitoes. Owing to multiple deadly epidemics, the WHO classifies YFV as a "high impact, high threat disease" with resurgent epidemic potential. At present, there are no approved antiviral therapies to combat YFV infection. Herein we report on 2'-halogen-modified nucleoside analogs as potential anti-YFV agents. Of 11 compounds evaluated, three showed great promise with low toxicity, high intracellular metabolism into the active nucleoside triphosphate form, and sub-micromolar anti-YFV activity. Notably, we investigated a 2'-fluoro,2'-bromouridine phosphate prodrug (C9), a known anti-HCV agent with good stability in human blood and favorable metabolism. Predictive modeling revealed that C9 could readily bind the active site of the YFV RdRp, conferring its anti-YFV activity. C9 displayed potent anti-YFV activity in primary human macrophages, 3D hepatocyte spheroids, and in mice. In an A129 murine model, shortly after infection, C9 significantly reduced YFV replication and protected against YFV-induced liver inflammation and pathology with no adverse effects. Collectively, this work identifies a potent new anti-YFV agent with strong therapeutic promise.

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 O-Benzyl Phosphonamidate Prodrug of Tenofovir for the Treatment of Hepatitis B Virus Infection

A series of new O-(substituted benzyl) phosphoramidate prodrugs of tenofovir for the treatment of hepatitis B virus (HBV) infections have been designed and synthesized. An investigation of structure-activity relationships revealed that the compound bearing an o-methylbenzyl group (1a) has the most potent in vitro anti-HBV activity. This prodrug (1a) was well-tolerated in KM mice via intragastric administration at a dosage of up to 1.5 g/kg. In DHBV-infected ducks, prodrug 1a displayed a good inhibitory effect on the viral DNA replication in both the serum and the liver in a time- and dose-dependent manner and did not cause any necrosis, hemorrhage, or inflammatory response in the animal livers. Further investigation demonstrated that prodrug 1a achieved a higher exposure of the bioactive metabolite (tenofovir diphosphate, TFV-DP) in the liver, the target organ for the treatment of HBV infection, than tenofovir alafenamide fumarate (TAF) did at an equimolar dose.

Discovery of Antibacterial Contezolid Acefosamil: Innovative O-Acyl Phosphoramidate Prodrug for IV and Oral Therapies.

New oral antibiotic contezolid (CZD) is effective against Gram-positive infections but unsuitable for intravenous (IV) administration due to its modest solubility. To address the medical need for an IV form of CZD, its isoxazol-3-yl phosphoramidate derivatives have been explored, and contezolid acefosamil (CZA, 8), the first representative of a novel O-acyl phosphoramidate prodrug class, has been identified. CZA exhibits high aqueous solubility (>200 mg/mL) and good hydrolytic stability at media pH suitable for IV administration. CZA rapidly converts into the active drug CZD in vivo. In a pharmacokinetic (PK) rat model, the exposure of active drug CZD after IV administration of the prodrug CZA was similar to or higher than that from the IV administration of CZD. The prodrug CZA is bioequivalent to or better than CZD in several preclinical infection models. CZA is likewise active upon its oral administration. To date, CZA has been evaluated in Phase 1 and Phase 2 clinical trials in the USA. It is advancing into further clinical studies including step-down therapy with in-hospital intravenous CZA administration followed by outpatient oral CZD treatment.

Development and validation of an LC-MS/MS method for simultaneous determination of remdesivir and its hydrolyzed metabolite and nucleoside, and its application in a pharmacokinetic study of normal and diabetic nephropathy mice.

Remdesivir (RDV), a phosphoramidate prodrug, has broad-spectrum antiviral activity. It is the first antiviral drug approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19. Remdesivir is rapidly metabolized in the body to produce derivatives: alanine metabolite (RM-442) and RDV C-nucleoside (RN). Here, the phosphatase inhibitor PhosSTOP and carboxylesterase inhibitor 5,5'-dithiobis-2-nitrobenzoic acid were used to improve stability of RDV in mouse blood. We developed a rapid and sensitive LC-MS/MS method to simultaneously quantify RDV, RM-442 and RN in mouse blood. Chromatographic separation was achieved by gradient elution on an Acquity HSS T3 column. The run time was 3.2min. The linearity ranges of the analytes were 0.5-1,000 ng/ml for RDV and 5-10,000 ng/ml for both RM-442 and RN. The method had an acceptable precision (RSD < 8.4% for RDV, RSD < 10.7% for RM-442 and RSD < 7.2% for RN) and accuracy (91.0-106.3% for RDV, 92.5-98.6% for RM-442 and 87.5-98.4% for RN). This method was successfully applied to analyze RDV, RM-442 and RN in the blood of normal and diabetic nephropathy DBA/2J mice after intravenous injection of RDV at 20 mg/kg. The area under the concentration-time curve of RN between the normal and diabetic nephropathy mice showed a significant difference (P < 0.01).