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Christopher Mcguigan - One of the best experts on this subject based on the ideXlab platform.

  • Symmetrical Diamidates as a Class of Phosphate Prodrugs to Deliver the 5′‐Monophosphate Forms of Anticancer Nucleoside Analogues
    ChemMedChem, 2018
    Co-Authors: Magdalena Slusarczyk, Gonczy Blanka, Valentina Ferrari, Michaela Serpi, Jan Balzarini, Christopher Mcguigan
    Abstract:

    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.

  • Phosphorodiamidates as a Promising New Phosphate Prodrug Motif for Antiviral Drug Discovery: Application to Anti-HCV Agents
    2016
    Co-Authors: Christopher Mcguigan, Karolina Madela, Mohamed Aljarah, Claire Bourdin, Maria Arrica, Emma Barrett, Alexander Kolykhalov, Blair Bleiman, Sarah Jones, Dawn K Bryant
    Abstract:

    We herein report Phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven Phosphorodiamidates are reported of two 6-O-alkyl 2′-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric Phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 μM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodrug method may have broad application outside of HCV and antivirals as it offers many of the advantages of phosphoramidate ProTides but without the chirality issues present in most cases

  • Design, synthesis and biological evaluation of phosphorodiamidate prodrugs of antiviral and anticancer nucleosides
    European journal of medicinal chemistry, 2013
    Co-Authors: Christopher Mcguigan, Karolina Madela, Claire Bourdin, Marco Derudas, Nadège Hamon, Karen Hinsinger, Sahar Kandil, Silvia Meneghesso, Fabrizio Pertusati, Michaela Serpi
    Abstract:

    We herein report the application of the phosphorodiamidate phosphate prodrug approach to a series of thirteen nucleoside analogs with antiviral or anticancer activity. Twenty-five symmetrical Phosphorodiamidates were synthesized, bearing esterified l-Alanine (and in one case d-Alanine) in the prodrug moiety, each as single stereoisomer. The presence of an achiral phosphorus represents a potential advantage over the phosphoramidate ProTide approach, where diastereoisomeric mixtures are routinely obtained, and different biological profiles may be expected from the diastereoisomers. Optimization of the synthetic pathway allowed us to identify two general methods depending on the particular nucleoside analogs. All the compounds were biologically evaluated in antiviral and anticancer assays and several showed improvement of activity compared to their parent nucleosides, as in the case of ddA, d4T, abacavir and acyclovir against HIV-1 and/or HIV-2. The biological results were supported by metabolism studies with carboxypeptidase Y monitored by 31P NMR to investigate their bioactivation. This work further validates the phosphorodiamidate approach as a monophosphate prodrug motif with broad application in the antiviral and anticancer fields.

  • Phosphorodiamidates as a promising new phosphate prodrug motif for antiviral drug discovery application to anti hcv agents
    Journal of Medicinal Chemistry, 2011
    Co-Authors: Christopher Mcguigan, Karolina Madela, Mohamed Aljarah, Claire Bourdin, Maria Arrica, Emma Barrett, Sarah Louise Jones, Alexander Kolykhalov, Blair Bleiman, Dawn K Bryant
    Abstract:

    We herein report Phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven Phosphorodiamidates are reported of two 6-O-alkyl 2′-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric Phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 μM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodr...

  • Phosphorodiamidates as a promising new phosphate prodrug motif for antiviral drug discovery application to anti hcv agents
    Journal of Medicinal Chemistry, 2011
    Co-Authors: Christopher Mcguigan, Karolina Madela, Mohamed Aljarah, Claire Bourdin, Maria Arrica, Emma Barrett, Sarah Louise Jones, Alexander Kolykhalov, Blair Bleiman, Dawn K Bryant
    Abstract:

    We herein report Phosphorodiamidates as a significant new phosphate prodrug motif. Sixty-seven Phosphorodiamidates are reported of two 6-O-alkyl 2'-C-methyl guanosines, with significant variation in the diamidate structure. Both symmetrical and asymmetric Phosphorodiamidates are reported, derived from various esterified amino acids, both d and l, and also from various simple amines. All of the compounds were evaluated versus hepatitis C virus in replicon assay, and nanomolar activity levels were observed. Many compounds were noncytotoxic at 100 μM, leading to high antiviral selectivities. The agents are stable in acidic, neutral, and moderately basic media and in selected biological media but show efficient processing by carboxypeptidases and efficiently yield the free nucleoside monophosphate in cells. On the basis of in vitro data, eight leads were selected for additional in vivo evaluation, with the intent of selecting one candidate for progression toward clinical studies. This phosphorodiamidate prodrug method may have broad application outside of HCV and antivirals as it offers many of the advantages of phosphoramidate ProTides but without the chirality issues present in most cases.

Robert E. Ireland - One of the best experts on this subject based on the ideXlab platform.

  • Organic Syntheses - Reductive Cleavage of Vinyl Phosphorodiamidates: 17β‐tert‐Butoxy‐Gα‐androst‐2‐ene
    Organic Syntheses, 2003
    Co-Authors: Robert E. Ireland, Thomas H. O'neil, Glen L. Tolman
    Abstract:

    Reductive cleavage of vinyl Phosphorodiamidates: 17β-tert-butoxy-Gα-androst-2-ene intermediate: vinyl phosphorodiamidate intermediate: 1.61 g (4.6 mmol) of 17β-tert-butoxy-5α-androstan-3-one reactant: N,N,N′,N′-Tetramethyldiamidophosphorochloridate, 5.83 mL (0.038 mol) product: 17β-tert-butoxy-5α-androst-2-ene Keywords: addition, to CC; alkylation, O-alkylation; deoxygenation; phosphonation; reduction, CX  CH; reduction, miscellaneous; assay methods, for butyllithium, with diphenylacetic acid; tert-butyl alcohol; diisopropylamine; dimethylamine; lithium diisopropylamide (LDA), preparation of; phosphoric acid, anhydrous; tetrahydro-1,3-dimethyl-2(1h)-pyrimidinone (DMPU); tetramethylethylenediamine; hexamethylphosphoric triamide (HMPA), toxicity

  • reductive cleavage of vinyl Phosphorodiamidates 17β tert butoxy gα androst 2 ene
    Organic Syntheses, 2003
    Co-Authors: Robert E. Ireland, Thomas H Oneil, Glen L. Tolman
    Abstract:

    Reductive cleavage of vinyl Phosphorodiamidates: 17β-tert-butoxy-Gα-androst-2-ene intermediate: vinyl phosphorodiamidate intermediate: 1.61 g (4.6 mmol) of 17β-tert-butoxy-5α-androstan-3-one reactant: N,N,N′,N′-Tetramethyldiamidophosphorochloridate, 5.83 mL (0.038 mol) product: 17β-tert-butoxy-5α-androst-2-ene Keywords: addition, to CC; alkylation, O-alkylation; deoxygenation; phosphonation; reduction, CX  CH; reduction, miscellaneous; assay methods, for butyllithium, with diphenylacetic acid; tert-butyl alcohol; diisopropylamine; dimethylamine; lithium diisopropylamide (LDA), preparation of; phosphoric acid, anhydrous; tetrahydro-1,3-dimethyl-2(1h)-pyrimidinone (DMPU); tetramethylethylenediamine; hexamethylphosphoric triamide (HMPA), toxicity

Glen L. Tolman - One of the best experts on this subject based on the ideXlab platform.

  • Organic Syntheses - Reductive Cleavage of Vinyl Phosphorodiamidates: 17β‐tert‐Butoxy‐Gα‐androst‐2‐ene
    Organic Syntheses, 2003
    Co-Authors: Robert E. Ireland, Thomas H. O'neil, Glen L. Tolman
    Abstract:

    Reductive cleavage of vinyl Phosphorodiamidates: 17β-tert-butoxy-Gα-androst-2-ene intermediate: vinyl phosphorodiamidate intermediate: 1.61 g (4.6 mmol) of 17β-tert-butoxy-5α-androstan-3-one reactant: N,N,N′,N′-Tetramethyldiamidophosphorochloridate, 5.83 mL (0.038 mol) product: 17β-tert-butoxy-5α-androst-2-ene Keywords: addition, to CC; alkylation, O-alkylation; deoxygenation; phosphonation; reduction, CX  CH; reduction, miscellaneous; assay methods, for butyllithium, with diphenylacetic acid; tert-butyl alcohol; diisopropylamine; dimethylamine; lithium diisopropylamide (LDA), preparation of; phosphoric acid, anhydrous; tetrahydro-1,3-dimethyl-2(1h)-pyrimidinone (DMPU); tetramethylethylenediamine; hexamethylphosphoric triamide (HMPA), toxicity

  • reductive cleavage of vinyl Phosphorodiamidates 17β tert butoxy gα androst 2 ene
    Organic Syntheses, 2003
    Co-Authors: Robert E. Ireland, Thomas H Oneil, Glen L. Tolman
    Abstract:

    Reductive cleavage of vinyl Phosphorodiamidates: 17β-tert-butoxy-Gα-androst-2-ene intermediate: vinyl phosphorodiamidate intermediate: 1.61 g (4.6 mmol) of 17β-tert-butoxy-5α-androstan-3-one reactant: N,N,N′,N′-Tetramethyldiamidophosphorochloridate, 5.83 mL (0.038 mol) product: 17β-tert-butoxy-5α-androst-2-ene Keywords: addition, to CC; alkylation, O-alkylation; deoxygenation; phosphonation; reduction, CX  CH; reduction, miscellaneous; assay methods, for butyllithium, with diphenylacetic acid; tert-butyl alcohol; diisopropylamine; dimethylamine; lithium diisopropylamide (LDA), preparation of; phosphoric acid, anhydrous; tetrahydro-1,3-dimethyl-2(1h)-pyrimidinone (DMPU); tetramethylethylenediamine; hexamethylphosphoric triamide (HMPA), toxicity

Chul-hoon Kwon - One of the best experts on this subject based on the ideXlab platform.

  • 1,2-Benzisoxazole Phosphorodiamidates as novel anticancer prodrugs requiring bioreductive activation
    2015
    Co-Authors: Monish Jain, Chul-hoon Kwon
    Abstract:

    Several 1,2-benzisoxazole Phosphorodiamidates have been designed as prodrugs of phosphora-mide mustard requiring bioreductive activation. Enzymatic reduction of 1,2-benziosoxazole moiety is expected to result in the formation of imine intermediate due to the cleavage of the N-O bond. The imine should then be spontaneously hydrolyzed to a ketone metabolite, thereby facilitating base-catalyzed â-elimination of cytotoxic phosphoramide mustard. As expected, the proposed prodrugs 4, 9, and 12 were at least 3-5-fold more potent cytotoxins than control compounds 5 and 15, which lack in the phosphoramide mustard group. Upon incubation with phenobarb-induced rat liver S-9 fraction, compounds 4, 9, and 12 underwent extensive NADPH-dependent metabolism with concomitant generation of alkylating activity under both hypoxic and oxic conditions. Corresponding ketone metabolites were detected for 9 and 15. NADPH-dependent bioreduction of 15 to its ketone metabolite 16 was located in the microsomal fraction and inhibited by SKF-525A and pCMBA. Compared with phenobarb-induced rat liver microsomal fraction, incubation of 15 with rat or human P450 reductase microsomes showed moderate generation of 16. Microsomal cytochrome P450 and/or P450 reductase appear to be involved in the reductive metabolism of 1,2-benzisoxazole moiety under hypoxic as well as oxic conditions

  • 1 2 benzisoxazole Phosphorodiamidates as novel anticancer prodrugs requiring bioreductive activation
    Journal of Medicinal Chemistry, 2003
    Co-Authors: Monish Jain, Chul-hoon Kwon
    Abstract:

    Several 1,2-benzisoxazole Phosphorodiamidates have been designed as prodrugs of phosphoramide mustard requiring bioreductive activation. Enzymatic reduction of 1,2-benziosoxazole moiety is expected to result in the formation of imine intermediate due to the cleavage of the N−O bond. The imine should then be spontaneously hydrolyzed to a ketone metabolite, thereby facilitating base-catalyzed β-elimination of cytotoxic phosphoramide mustard. As expected, the proposed prodrugs 4, 9, and 12 were at least 3−5-fold more potent cytotoxins than control compounds 5 and 15, which lack in the phosphoramide mustard group. Upon incubation with phenobarb-induced rat liver S-9 fraction, compounds 4, 9, and 12 underwent extensive NADPH-dependent metabolism with concomitant generation of alkylating activity under both hypoxic and oxic conditions. Corresponding ketone metabolites were detected for 9 and 15. NADPH-dependent bioreduction of 15 to its ketone metabolite 16 was located in the microsomal fraction and inhibited b...

Patrick L Iversen - One of the best experts on this subject based on the ideXlab platform.

  • A Single Phosphorodiamidate Morpholino Oligomer Targeting VP24 Protects Rhesus Monkeys against Lethal Ebola Virus Infection
    mBio, 2015
    Co-Authors: Travis K Warren, Lisa S Welch, Patrick L Iversen, Jay Wells, Chris A. Whitehouse, Alison E. Heald, Jay S. Charleston, Pete Sazani, St. Patrick Reid, Sina Bavari
    Abstract:

    ABSTRACT Ebola viruses (EBOV) cause severe disease in humans and nonhuman primates with high mortality rates and continue to emerge in new geographic locations, including several countries in West Africa, the site of a large ongoing outbreak. Phosphorodiamidate morpholino oligomers (PMOs) are synthetic antisense molecules that are able to target mRNAs in a sequence-specific fashion and suppress translation through steric hindrance. We previously showed that the use of PMOs targeting a combination of VP35 and VP24 protected rhesus monkeys from lethal EBOV infection. Surprisingly, the present study revealed that a PMO plus compound targeting VP24 alone was sufficient to confer protection from lethal EBOV infection but that a PMO plus targeting VP35 alone resulted in no protection. This study further substantiates recent data demonstrating that VP24 may be a key virulence factor encoded by EBOV and suggests that VP24 is a promising target for the development of effective anti-EBOV countermeasures. IMPORTANCE Several West African countries are currently being ravaged by an outbreak of Ebola virus (EBOV) that has become a major epidemic affecting not only these African countries but also Europe and the United States. A better understanding of the mechanism of virulence of EBOV is important for the development of effective treatments, as no licensed treatments or vaccines for EBOV disease are currently available. This study of phosphorodiamidate morpholino oligomers (PMOs) targeting the mRNAs of two different EBOV proteins, alone and in combination, demonstrated that targeting a single protein was effective at conferring a significant survival benefit in an EBOV lethal primate model. Future development of PMOs with efficacy against EBOV will be simplified if only one PMO is required instead of a combination, particularly in terms of regulatory approval.

  • discovery and early development of avi 7537 and avi 7288 for the treatment of ebola virus and marburg virus infections
    Viruses, 2012
    Co-Authors: Patrick L Iversen, Travis K Warren, Dan V Mourich, Lisa S Welch, Jay Wells, Rekha G Panchal, Nicole L. Garza, Sina Bavari
    Abstract:

    There are no currently approved treatments for filovirus infections. In this study we report the discovery process which led to the development of antisense Phosphorodiamidate Morpholino Oligomers (PMOs) AVI-6002 (composed of AVI-7357 and AVI-7539) and AVI-6003 (composed of AVI-7287 and AVI-7288) targeting Ebola virus and Marburg virus respectively. The discovery process involved identification of optimal transcript binding sites for PMO based RNA-therapeutics followed by screening for effective viral gene target in mouse and guinea pig models utilizing adapted viral isolates. An evolution of chemical modifications were tested, beginning with simple Phosphorodiamidate Morpholino Oligomers (PMO) transitioning to cell penetrating peptide conjugated PMOs (PPMO) and ending with PMOplus containing a limited number of positively charged linkages in the PMO structure. The initial lead compounds were combinations of two agents targeting separate genes. In the final analysis, a single agent for treatment of each virus was selected, AVI-7537 targeting the VP24 gene of Ebola virus and AVI-7288 targeting NP of Marburg virus, and are now progressing into late stage clinical development as the optimal therapeutic candidates.

  • Bacterial resistance to antisense peptide phosphorodiamidate morpholino oligomers.
    Antimicrobial agents and chemotherapy, 2012
    Co-Authors: Susan E. Puckett, Patrick L Iversen, Kaleb A. Reese, Georgi M. Mitev, Valerie M. Mullen, Rudd C. Johnson, Kyle R. Pomraning, Brett L. Mellbye, Lucas D. Tilley, Michael Freitag
    Abstract:

    Peptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)(3)RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is β-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF)(3)RXB-AcpP was 2.5 μM (14 μg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)(3)RXB-AcpP was 4 × 10(-7) mutations/cell division. A spontaneous (RFF)(3)RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)(3)RXB-AcpP was 40 μM (224 μg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)(3)RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)(3)RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)(3)RXB-AcpP. Deletion of sbmA caused resistance to (RFF)(3)RXB-AcpP. We conclude that resistance to (RFF)(3)RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)(3)R-XB PPMOs may be transported across the plasma membrane by SbmA.

  • Cellular Uptake of Neutral Phosphorodiamidate Morpholino Oligomers
    Current pharmaceutical biotechnology, 2009
    Co-Authors: Patrick L Iversen, Katherine M. Aird, Michael M. Morse, Gayathri R. Devi
    Abstract:

    Phosphorodiamidate morpholino oligomers (PMO), which have a neutral chemistry, are extensively being used as tools for selective inhibition of gene expression in cell culture models and are currently in human clinical trials. Unlike phosphorothioates (PS ODN) and other charged oligonucleotides, little is known about the uptake characteristics of neutral oligomers. The purpose of this study was to understand the kinetics of PMO transport in cells and correlate with antisense activity. In contrast to primary cells and some transformed cell lines which were uptake permissive, established cancer cell lines showed very poor uptake with an occasional diffuse intracellular pattern. Differential PMO uptake was also observed in immune cells, with dendritic cells and monocytes showing highest uptake compared to T and B cells. In addition, PMO localization was observed to be heterogeneous within a population of uptake permissive cells. Unassisted PMO delivery targeting specific genes was correlated with functional antisense efficacy in experiments showing correction of pre-mRNA missplicing and inhibition of target enzyme activity in cells in culture. PMO internalization in uptakepermissive cells was identified to be specific, saturable, and energy-dependent, suggesting a receptor mediated uptake mechanism. Understanding PMO transport should facilitate the design of more effective synthetic antisense oligomers as therapeutic agents.

  • Inhibition of Intracellular Growth of Salmonella enterica Serovar Typhimurium in Tissue Culture by Antisense Peptide-Phosphorodiamidate Morpholino Oligomer
    Antimicrobial agents and chemotherapy, 2009
    Co-Authors: Georgi M. Mitev, Patrick L Iversen, Brett L. Mellbye, Bruce L. Geller
    Abstract:

    Two types of phosphorodiamidate morpholino oligomers (PMOs) were tested for inhibition of growth of Salmonella enterica serovar Typhimurium. Both PMOs have the same 11-base sequence that is antisense to the region near the start codon of acpP, which is essential for lipid biosynthesis and viability. To the 3 end of each is attached the membrane-penetrating peptide (RXR)4XB (R, X, and B indicate arginine, 6-aminohexanoic acid, and -alanine, respectively). One peptide-PMO (AcpP PPMO) has no charge on the PMO moiety. The second PPMO has three cations (piperazine) attached to the phosphorodiamidate linkages (3Pip-AcpP PPMO). A scrambled-sequence PPMO (Scr PPMO) was synthesized for each type of PMO. The MICs of AcpP PPMO, 3Pip-AcpP PPMO, and either one of the Scr PPMOs were 1.25 M( 7g/ml), 0.156 M (0.94 g/ml), and >160 M (>900 g/ml), respectively. 3Pip-AcpP PPMO at 1.25 or 2.5 M significantly reduced the growth rates of pure cultures, whereas AcpP PPMO or either Scr PPMO had no effect. However, the viable cell count was significantly reduced at either concentration of 3Pip-AcpP PPMO or AcpP PPMO, but not with either Scr PPMO. In other experiments, macrophages were infected intracellularly with S. enterica and treated with 3 M3 Pip-AcpP PPMO. Intracellular bacteria were reduced >99% with 3Pip-AcpP PPMO, whereas intracellular bacteria increased 3 orders of magnitude in untreated or Scr PPMO-treated cultures. We conclude that either AcpP PPMO or 3Pip-AcpP PPMO inhibited growth of S. enterica in pure culture and that 3Pip-AcpP PPMO reduced intracellular viability of S. enterica in macrophages.