Pivaloyloxymethyl Research Articles

Enolase Inhibitors as Early Lead Therapeutics against Trypanosoma brucei.

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, serving as the lone source of ATP production for the bloodstream form (BSF) parasite in the glucose-rich environment of the host blood. Recently, phosphonate inhibitors of human enolase (ENO), the enzyme responsible for the interconversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP) in glycolysis or PEP to 2-PG in gluconeogenesis, have been developed for the treatment of glioblastoma multiforme (GBM). Here, we have tested these agents against T. brucei ENO (TbENO) and found the compounds to be potent enzyme inhibitors and trypanocides. For example, (1-hydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (deoxy-SF2312) was a potent enzyme inhibitor (IC50 value of 0.60 ± 0.23 µM), while a six-membered ring-bearing phosphonate, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX), was less potent (IC50 value of 2.1 ± 1.1 µM). An analog with a larger seven-membered ring, (1-hydroxy-2-oxoazepan-3-yl) phosphonic acid (HEPTA), was not active. Molecular docking simulations revealed that deoxy-SF2312 and HEX had binding affinities of -6.8 and -7.5 kcal/mol, respectively, while the larger HEPTA did not bind as well, with a binding of affinity of -4.8 kcal/mol. None of these compounds were toxic to BSF parasites; however, modification of enzyme-active phosphonates through the addition of pivaloyloxymethyl (POM) groups improved activity against T. brucei, with POM-modified (1,5-dihydroxy-2-oxopyrrolidin-3-yl) phosphonic acid (POMSF) and POMHEX having EC50 values of 0.45 ± 0.10 and 0.61 ± 0.08 µM, respectively. These findings suggest that HEX is a promising lead against T. brucei and that further development of prodrug HEX analogs is warranted.

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

Acid‐Stable Nucleobase Protection for a Strongly Pairing Pyridone C‐Nucleoside Suitable for Solid‐Phase Synthesis of Oligonucleotides

AbstractOligonucleotides are indispensable tools in diagnostics, therapeutic applications and molecular biology. The low base pairing strength of thymine with adenine complicates their use. EthynylpyridoneC‐nucleosides are analogs of thymidine that pair more strongly and with improved base selectivity, and sequences containing these analogs show improved target affinity and selectivity, but their routine use is hampered by diminished yields of solid‐phase syntheses with the known building blocks. A partial loss of base protecting groups during the acidic deblocking step of chain extension cycles was identified as the cause of lower yields. Here we report the synthesis of an improved phosphoramidite building block featuring a pivaloyloxymethyl (POM) base protecting group. This building block gives oligonucleotides containing the strongly pairing ethynylmethylpyridoneC‐nucleoside in high yield and purity via solid‐phase synthesis.

Phosphonates and Phosphonate Prodrugs in Medicinal Chemistry: Past Successes and Future Prospects.

Compounds with a phosphonate group, i.e., –P(O)(OH)2 group attached directly to the molecule via a P-C bond serve as suitable non-hydrolyzable phosphate mimics in various biomedical applications. In principle, they often inhibit enzymes utilizing various phosphates as substrates. In this review we focus mainly on biologically active phosphonates that originated from our institute (Institute of Organic Chemistry and Biochemistry in Prague); i.e., acyclic nucleoside phosphonates (ANPs, e.g., adefovir, tenofovir, and cidofovir) and derivatives of non-nucleoside phosphonates such as 2-(phosphonomethyl) pentanedioic acid (2-PMPA). Principal strategies of their syntheses and modifications to prodrugs is reported. Besides clinically used ANP antivirals, a special attention is paid to new biologically active molecules with respect to emerging infections and arising resistance of many pathogens against standard treatments. These new structures include 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidines or so-called “open-ring” derivatives, acyclic nucleoside phosphonates with 5-azacytosine as a base moiety, side-chain fluorinated ANPs, aza/deazapurine ANPs. When transformed into an appropriate prodrug by derivatizing their charged functionalities, all these compounds show promising potential to become drug candidates for the treatment of viral infections. ANP prodrugs with suitable pharmacokinetics include amino acid phosphoramidates, pivaloyloxymethyl (POM) and isopropoxycarbonyloxymethyl (POC) esters, alkyl and alkoxyalkyl esters, salicylic esters, (methyl-2-oxo-1,3-dioxol-4-yl) methyl (ODOL) esters and peptidomimetic prodrugs. We also focus on the story of cytostatics related to 9-[2-(phosphonomethoxy)ethyl]guanine and its prodrugs which eventually led to development of the veterinary drug rabacfosadine. Various new ANP structures are also currently investigated as antiparasitics, especially antimalarial agents e.g., guanine and hypoxanthine derivatives with 2-(phosphonoethoxy)ethyl moiety, their thia-analogues and N-branched derivatives. In addition to ANPs and their analogs, we also describe prodrugs of 2-(phosphonomethyl)pentanedioic acid (2-PMPA), a potent inhibitor of the enzyme glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA). Glutamate carboxypeptidase II inhibitors, including 2-PMPA have been found efficacious in various preclinical models of neurological disorders which are caused by glutamatergic excitotoxicity. Unfortunately its highly polar character and hence low bioavailability severely limits its potential for clinical use. To overcome this problem, various prodrug strategies have been used to mask carboxylates and/or phosphonate functionalities with pivaloyloxymethyl, POC, ODOL and alkyl esters. Chemistry and biological characterization led to identification of prodrugs with 44–80 fold greater oral bioavailability (tetra-ODOL-2-PMPA).

Double prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents

A series of eleven double prodrug derivatives of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. A pivaloyloxymethyl (POM) phosphonate prodrug modification was combined with various prodrug derivatisations of the hydroxamate moiety. The majority of compounds showed activity comparable with or inferior to fosmidomycin against P. falciparum. N-benzyl substituted carbamate prodrug 6f was the most active antimalarial analog with an IC50 value of 0.64 µM. Contrary to fosmidomycin and parent POM-prodrug 5, 2-nitrofuran and 2-nitrothiophene prodrugs 6i and 6j displayed promising antitubercular activities.

Facile Synthesis, Geometry, and 2'-Substituent-Dependent in Vivo Activity of 5'-(E)- and 5'-(Z)-Vinylphosphonate-Modified siRNA Conjugates.

(E)-Vinylphosphonate ((E)-VP), a metabolically stable phosphate mimic at the 5'-end of the antisense strand, enhances the in vivo potency of siRNA. Here we describe a straightforward synthetic approach to incorporate a nucleotide carrying a vinylphosphonate (VP) moiety at the 5'-end of oligonucleotides under standard solid-phase synthesis and deprotection conditions by utilizing pivaloyloxymethyl (POM) protected VP-nucleoside phosphoramidites. The POM protection enhances scope and scalability of 5'-VP-modified oligonucleotides and, in a broader sense, the synthesis of oligonucleotides modified with phosphonate moieties. Trivalent N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) comprising (E)-geometrical isomer of VP showed improved RISC loading with robust RNAi-mediated gene silencing in mice compared to the corresponding (Z)-isomer despite similar tissue accumulation. We also obtained structural insights into why bulkier 2'-ribosugar substitutions such as 2'-O-[2-(methylamino)-2-oxoethyl] are well tolerated only when combined with 5'-(E)-VP.

Abstract A39: Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer

Abstract Glycolysis inhibition is an active area of investigation for the treatment of cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions solely reliant on ENO2, rendering them exquisitely sensitive to enolase inhibitors Collateral Lethality. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We recently reported that a structural analogue of PhAH, the natural phosphonate antibiotic SF2312, is a high potency inhibitor of Enolase. While more potent than PhAH, SF2312 remains poorly cell permeable. Here, we generated a Pivaloyloxymethyl (POM) ester pro-drug derivative of SF2312, termed POMSF, which increased the potency in cell based systems by ~50-fold. POMSF is selectively active against ENO1-deleted glioma cells in culture at ~19 nM, versus μM for SF2312. However, POMSF displayed poor aqueous stability. A derivative of POMSF, termed POMHEX, showed greater stability and its active form, HEX, showed 4-fold preference for ENO1 over ENO2. Labeled 13C-glucose tracing shows that POMHEX inhibits glycolysis at the Enolase step in all cell lines tested, but with ~100-fold greater potency in ENO1-deleted lines. POMHEX selectively killed ENO1-deleted glioma cells with an IC50 <30nM, whilst non-deleted cells could readily tolerate μM levels of inhibitor. As such, POMHEX was selected for in vivo experiments. Using an orthotopic intracranial xenografted model where tumor growth and response to therapy are monitored by MRI, we show that POMHEX is capable of eradicating intracranial ENO1-deleted tumors, with mice remaining recurrence-free even after treatment discontinuation. Taken together, these results reinforce that glycolysis is a viable target and provide in vivo proof-of-principal for the concept of using passenger deletions as targetable vulnerabilities in personalized cancer therapy. Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Federica Pisaneschi, Paul Leonard, David Maxwell, Zhenghong Peng, Todd Link, Lee IV R. Gilbert, Ananth Bosajou, Duoli Sun, Joe Marszalek, Yuting Sun, John S. McMurray, Pijus K. Mandal, Maria E. Di Francesco, Barbara Czako, Alan Wang, William Bornmann, Ronald A. DePinho, Florian Muller. Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A39.

Lipophilic 2'-O-Acetal Ester RNAs: Synthesis, Thermal Duplex Stability, Nuclease Resistance, Cellular Uptake, and siRNA Activity after Spontaneous Naked Delivery.

The in vivo application of siRNA depends on its cellular uptake and intracellular release, and this is an unsatisfactorily resolved technical hurdle in medicinal applications. Promising concepts directed towards providing efficient cellular and intracellular delivery include lipophilic chemical modification of siRNA. Here we describe chemistry for the production of modified siRNAs designed to display improved transmembrane transport into human cells while preserving the potency of the RNAi-based inhibitors. We report the synthesis and the biochemical and biophysical characteristics of 2'-O-phenylisobutyryloxymethyl (PiBuOM)-modified siRNAs and their impact on biological activity. In the case of spontaneous cellular uptake of naked PiBuOM-modified siRNA, we observed increased target suppression in human cells relative to unmodified or pivaloyloxymethyl (PivOM)-modified siRNA. We provide evidence of improved spontaneous cellular uptake of naked PiBuOM-modified siRNA and of substantial target suppression in human cells in serum-containing medium.

Discovery of Orally Available Prodrugs of the Glutamate Carboxypeptidase II (GCPII) Inhibitor 2-Phosphonomethylpentanedioic Acid (2-PMPA).

2-Phosphonomethylpentanedioic acid (1, 2-PMPA) is a potent inhibitor of glutamate carboxypeptidase II which has demonstrated robust neuroprotective efficacy in many neurological disease models. However, 1 is highly polar containing a phosphonate and two carboxylates, severely limiting its oral bioavailability. We strategized to mask the polar groups via a prodrug approach, increasing the likelihood of passive oral absorption. Our initial strategy was to cover the phosphonate with hydrophobic moieties such as pivaloyloxymethyl (POM) and isopropyloxycarbonyloxymethyl (POC) while keeping the α- and γ-carboxylates unsubstituted. This attempt was unsuccessful due to the chemical instability of the bis-POC/POM derivatives. Addition of α,γ-diesters and α-monoesters enhanced chemical stability and provided excellent oral exposure in mice, but these mixed esters were too stable in vivo, resulting in minimal release of 1. By introducing POC groups on both the phosphonate and α-carboxylate, we synthesized Tris-POC-2-PMPA (21b), which afforded excellent release of 1 following oral administration in both mice and dog.

Mono-anionic phosphopeptides produced by unexpected histidine alkylation exhibit high Plk1 polo-box domain-binding affinities and enhanced antiproliferative effects in HeLa cells.

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides provide starting points for developing PBD-directed inhibitors, to date the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising, in part, from the di-anionic nature of the phosphoryl group or its mimetics. In our current article we report the unanticipated on-resin N(τ)-alkylation of histidine residues already bearing a N(π)- alkyl group. This resulted in cationic imidazolium-containing pThr peptides, several of which exhibit single-digit nanomolar PBD-binding affinities in extracellular assays and improved antimitotic efficacies in intact cells. We enhanced the cellular efficacies of these peptides further by applying bio-reversible pivaloyloxymethyl (POM) phosphoryl protection. New structural insights presented in our current study, including the potential utility of intramolecular charge masking, may be useful for the further development of PBD-binding peptides and peptide mimetics.

Design and synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the preparation of peptide prodrugs containing phosphothreonine in fully protected form.

The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.

RETRACTED: Peptide-Based Inhibitors of Plk1 Polo-box Domain Containing Mono-anionic Phosphothreonine Esters and Their Pivaloyloxymethyl Prodrugs

Binding of polo-like kinase 1 (Plk1) polo-box domains (PBDs) to phosphothreonine (pThr)/phosphoserine (pSer)-containing sequences is critical for the proper function of Plk1. Although high-affinity synthetic pThr-containing peptides may be used to disrupt PBD function, the efficacy of such peptides in whole cell assays has been poor. This potentially reflects limited cell membrane permeability arising in part from the di-anionic nature of the phosphoryl group. We report five-mer peptides containing mono-anionic pThr phosphoryl esters that exhibit single-digit nanomolar PBD binding affinities in extracellular assays and improved antimitotic efficacies in whole cell assays. The cellular efficacies of these peptides have been further enhanced by the application of bio-reversible pivaloyloxymethyl (POM) phosphoryl protection to a pThr-containing polypeptide. Our findings may redefine structural parameters for the development of PBD-binding peptides and peptide mimetics.

The biolabile 2′-O-pivaloyloxymethyl modification in an RNA helix: an NMR solution structure

The pivaloyloxymethyl (PivOM) group is a biolabile 2'-O-ribose protection that is under development in a prodrug-based approach for siRNA applications. Besides an expected cellular uptake, nucleic acid sequences carrying PivOM showed also increased nuclease resistance and, in most cases, an affinity for complementary RNA. The r(CGCU*ACGC)dT:r(GCGUAGCG)dT model duplex containing a single modified residue (U*) was synthesized and its solution structure was determined by NMR. The duplex showed a maintained A-RNA helix. In U*, both 2'-O-acetal ester side chain and ring pucker presented a notable rigid conformation. The PivOM moiety was oriented with the carbonyl group turned outside the minor groove and with trans, -ac and -ac torsion angles around the C2'-O2', O2'-CA and CA-OB1 bonds respectively. Gauche effects and dipolar interactions between the PivOM and the backbone appeared to be the predominant factors influencing the PivOM conformation and the orientation of the two supplementary H acceptors suggested that hydration could also play a role in the duplex stability.

Synthesis of novel tetrazole C5-linked C0- and C2-ribonucleoside phosphoramidites using MePOM and POM groups for probing RNA catalysis

Novel C5-linked C0- and C2-tetrazole ribonucleoside phosphoramidites were designed and synthesized via tetrazole C-nucleosides. Pivaloyloxymethyl (POM) and methyl-substituted POM (MePOM) groups were introduced as N-protecting groups in the tetrazole ring that can be readily removed under mild basic conditions. The phosphoramidites were successfully incorporated into the VS ribozyme substrate and hence providing a chemogenetic approach to determine which nucleobases of ribozymes function as the acid or base, in the studies of ribozyme general acid and base catalysis.

Synthesis and Preliminary Evaluation of pro-RNA 2′-O-Masked with Biolabile Pivaloyloxymethyl Groups in an RNA Interference Assay

The cellular delivery of bioactive nucleic acid-based drugs such as small interfering RNA (siRNA) represents a major technical hurdle for their pharmaceutical application. Prodrug-like approaches provide an attractive concept to address the delivery problem. With the aim to prepare RNA-based prodrugs bearing biolabile protections which facilitate cellular uptake and are prone to be removed enzymatically inside cells in order to release functional RNA, we synthesized pro-RNA totally or partially masked in 2'-OH position with pivaloyloxymethyl (PivOM) groups. A suitable strategy has been developed to synthesize and to purify base-sensitive mixed 2'-OH/2'-O-PivOM oligoribonucleotides, and to include them in siRNA. In this strategy, the fluoride labile [(triisopropylsilyl)oxy]-benzyloxycarbonyl group (tboc) as nucleobase protection (for A and C), the TBS group as 2'-OH protection and the Q-linker to solid-support were compatible with the PivOM groups masking some 2'-OH. We have taken advantage of the specific stability of the PivOM group to apply selected acidic, basic, and fluoride ions treatment for the deprotection and release of pro-RNA. This kind of pro-siRNA was studied in a human cell culture-based RNAi assay and preliminary promising data are discussed.

ChemInform Abstract: Synthetic Studies of Imidazole C‐Nucleosides Toward Biofunctional Molecules

AbstractReview: 35 refs.

Chemical Synthesis of RNA with Base‐Labile 2′‐O‐(Pivaloyloxymethyl)‐Protected Ribonucleoside Phosphoramidites

The efficiency of chemical RNA synthesis has been radically improved by the use of pivaloyloxymethyl (PivOM) groups as 2'-protection, containing an acetal spacer that minimizes the steric effect of the ester group on the neighboring amidite during the coupling. However, the major benefit of the base-labile PivOM groups is their simple removal upon standard basic conditions applied to deprotect the RNA and release it from solid supports. Combined with standard acyl groups for nucleobases, cyanoethyl groups for phosphates, and base-cleavable linkers, PivOM groups make RNA deprotection as simple as DNA deprotection. Thus, no additional deprotection step with tedious desalting workup procedures is required, and RNA synthesis becomes as convenient and efficient as DNA synthesis.

Synthetic Studies of Imidazole C-Nucleosides toward Biofunctional Molecules

Synthetic studies of C4-linked imidazole C-nucleosides toward biofunctional molecules are described, in which the following items are covered. 1) Stereoselective synthesis of imidazole C- and pyrazole C-nucleosides via diazafulvene intermediates. 2) Synthesis of tetrahydrofuranylimidazoles using a PhSe group efficiently and its application to the new human histamine H(3) receptor (hH(3)R) agonist, imifuramine, and the first selective human histamine H(4) receptor (hH(4)R) agonist, OUP-16. 3) Synthesis of imidazole ribonucleoside phosphoramidite (Imz-PA) with pivaloyloxymethyl (POM) group for probing the catalytic mechanism of ribozymes. 4) Synthesis of a two-carbon-elongated homologue (Imz-C(2)-PA) with a combination of POM and 2-cyanoethyl groups. 5) Incorporation of C(2)-imidazole nucleoside into position 638 of VS ribozyme using Imz-C(2)-PA and catalytic activities of the thereby generated modified VS ribozyme (G638C(2)Imz).

Synthesis of Ester Prodrugs of 9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) as Anti-Poxvirus Agents

9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and its cyclic form were selected for further evaluation as potential drug candidates against poxvirus infections. To increase bioavailability of these compounds, synthesis of their structurally diverse ester prodrugs was carried out: alkoxyalkyl (hexadecyloxypropyl, octadecyloxyethyl, hexadecyloxyethyl), pivaloyloxymethyl (POM), 2,2,2-trifluoroethyl, butylsalicylyl, and prodrugs based on peptidomimetics. Most HPMPDAP prodrugs were synthesized in the form of monoesters as well as the corresponding cyclic phosphonate esters. The activity was evaluated not only against vaccinia virus but also against different herpes viruses. The most potent and active prodrugs against vaccinia virus were the alkoxyalkyl ester derivatives of HPMPDAP, with 50% effective concentrations 400-600-fold lower than those of the parent compound. Prodrugs based on peptidomimetics, the 2,2,2-trifluoroethyl, the POM, and the butylsalicylyl derivatives, were able to inhibit vaccinia virus replication at 50% effective concentrations that were equivalent or ∼10-fold lower than those observed for the parent compounds.

ChemInform Abstract: A Short-Step Synthesis of Orally Active Carbapenem Antibiotic CS-834.

An orally bioavailable carbapenem CS-834, which is a pivaloyloxymethyl (POM) ester-type prodrug and has (R)-5-oxopyrrolidin-3-ylthio moiety at the C-2 position of the 1 beta-methylcarbapenem skeleton, is currently under clinical trial. We accomplished a short-step synthesis of CS-834 by using phosphorus ylide from the intramolecular Wittig-type reaction in the key step for cyclization to the bicyclic carbapenem system. The POM ester group was found to be suitable for the cyclization conditions.