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1.
Bioorg Med Chem Lett ; 27(18): 4323-4330, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28835346

ABSTRACT

Herein we describe the discovery of IDX21437 35b, a novel RPd-aminoacid-based phosphoramidate prodrug of 2'-α-chloro-2'-ß-C-methyluridine monophosphate. Its corresponding triphosphate 6 is a potent inhibitor of the HCV NS5B RNA-dependent RNA polymerase (RdRp). Despite showing very weak activity in the in vitro Huh-7 cell based HCV replicon assay, 35b demonstrated high levels of active triphosphate 6 in mouse liver and human hepatocytes. A biochemical study revealed that the metabolism of 35b was mainly attributed to carboxyesterase 1 (CES1), an enzyme which is underexpressed in HCV Huh-7-derived replicon cells. Furthermore, due to its metabolic activation, 35b was efficiently processed in liver cells compared to other cell types, including human cardiomyocytes. The selected RP diastereoisomeric configuration of 35b was assigned by X-ray structural determination. 35b is currently in Phase II clinical trials for the treatment of HCV infection.


Subject(s)
Antiviral Agents/pharmacology , DNA-Directed RNA Polymerases/antagonists & inhibitors , Drug Discovery , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , Uridine Monophosphate/analogs & derivatives , Uridine/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , DNA-Directed RNA Polymerases/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Hepacivirus/enzymology , Hepatocytes/drug effects , Hepatocytes/virology , Humans , Liver/drug effects , Liver/virology , Mice , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity Relationship , Uridine/chemical synthesis , Uridine/chemistry , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/chemistry , Uridine Monophosphate/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
2.
J Med Chem ; 60(14): 6098-6118, 2017 07 27.
Article in English | MEDLINE | ID: mdl-28654257

ABSTRACT

The increase in the number of bacterial strains resistant to known antibiotics is alarming. In this study we report the synthesis of novel compounds termed Lipophosphonoxins II (LPPO II). We show that LPPO II display excellent activities against Gram-positive and -negative bacteria, including pathogens and multiresistant strains. We describe their mechanism of action-plasmatic membrane pore-forming activity selective for bacteria. Importantly, LPPO II neither damage nor cross the eukaryotic plasmatic membrane at their bactericidal concentrations. Further, we demonstrate LPPO II have low propensity for resistance development, likely due to their rapid membrane-targeting mode of action. Finally, we reveal that LPPO II are not toxic to either eukaryotic cells or model animals when administered orally or topically. Collectively, these results suggest that LPPO II are highly promising compounds for development into pharmaceuticals.


Subject(s)
Anti-Bacterial Agents/chemistry , Uridine Monophosphate/analogs & derivatives , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Apoptosis/drug effects , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , Cell Survival/drug effects , Drug Design , Drug Resistance, Multiple, Bacterial , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Humans , Lipid Bilayers/chemistry , Male , Mice, Inbred ICR , Microbial Sensitivity Tests , Phospholipids/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyrazoles/pharmacology , Rabbits , Skin Irritancy Tests , Stereoisomerism , Structure-Activity Relationship , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/chemistry , Uridine Monophosphate/pharmacology
3.
Handb Exp Pharmacol ; 238: 307-337, 2017.
Article in English | MEDLINE | ID: mdl-27896476

ABSTRACT

After decades of intensive research on adenosine-3',5'-cyclic monophosphate (cAMP)- and guanosine-3',5'-cyclic monophosphate (cGMP)-related second messenger systems, also the noncanonical congeners cyclic cytidine-3',5'-monophosphate (cCMP) and cyclic uridine-3',5'-monophosphate (cUMP) gained more and more interest. Until the late 1980s, only a small number of cCMP and cUMP analogs with sometimes undefined purities had been described. Moreover, most of these compounds had been rather synthesized as precursors of antitumor and antiviral nucleoside-5'-monophosphates and hence had not been tested for any second messenger activity. Along with the recurring interest in cCMP- and cUMP-related signaling in the early 2000s, it became evident that well-characterized small molecule analogs with reliable purities would serve as highly valuable tools for the evaluation of a putative second messenger role of cyclic pyrimidine nucleotides. Meanwhile, for this purpose new cCMP and cUMP derivatives have been developed, and already known analogs have been resynthesized and highly purified. This chapter summarizes early medicinal chemistry work on cCMP and cUMP and analogs thereof, followed by a description of recent synthetic developments and an outlook on potential future directions.


Subject(s)
Cyclic CMP/chemical synthesis , Nucleotides, Cyclic/chemical synthesis , Prodrugs/chemical synthesis , Uridine Monophosphate/chemical synthesis , Animals , Crystallization , Cyclic CMP/analogs & derivatives , Cyclic CMP/metabolism , Cyclic CMP/pharmacology , Humans , Molecular Structure , Nucleotides, Cyclic/metabolism , Nucleotides, Cyclic/pharmacology , Permeability , Prodrugs/metabolism , Prodrugs/pharmacology , Uridine Monophosphate/metabolism , Uridine Monophosphate/pharmacology
4.
J Org Chem ; 78(17): 8320-9, 2013 Sep 06.
Article in English | MEDLINE | ID: mdl-23895237

ABSTRACT

Nucleoside-(5'→P) methylenebisphosphonodithioate analogues are bioisosteres of natural nucleotides. The potential therapeutic applications of these analogues are limited by their relative instability. With a view toward improving their chemical and metabolic stability as well as their affinity toward zinc ions, we developed a novel nucleotide scaffold, nucleoside-5'-tetrathiobisphosphonate. We synthesized P1-(uridine/adenosine-5')-methylenebisphosphonodithioate, 2 and 3, and P1,P2-di(uridine/adenosine-5')-methylenebisphosphonodithioate, 4 and 5. Using (1)H and (31)P NMR-monitored Zn(2+)/Mg(2+) titrations, we found that 5 coordinated Zn(2+) by both N7 nitrogen atoms and both dithiophosphonate moieties, whereas 3 coordinated Zn(2+) by an N7 nitrogen atom and Pß. Both 3 and 5 did not coordinate Mg(2+) ions. (31)P NMR-monitored kinetic studies showed that 3 was more stable at pD 1.5 than 5, with t(1/2) of 44 versus 9 h, respectively, and at pD 11 both showed no degradation for at least 2 weeks. However, 5 was more stable than 3 under an air-oxidizing atmosphere, with t1/2 of at least 3 days versus 14 h, respectively. Analogues 3 and 5 were highly stable to NPP1,3 and NTPDase1,2,3,8 hydrolysis (0-7%). However, they were found to be poor ectonucleotidase inhibitors. Although 3 and 5 did not prove to be effective inhibitors of zinc-containing NPP1/3, which is involved in the pathology of osteoarthritis and diabetes, they may be promising zinc chelators for the treatment of other health disorders involving an excess of zinc ions.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Diphosphonates/chemistry , Nucleosides/chemistry , Nucleosides/chemical synthesis , Organothiophosphorus Compounds/chemistry , Uridine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemical synthesis , Adenosine Monophosphate/chemistry , Diphosphonates/chemical synthesis , Molecular Structure , Organothiophosphorus Compounds/chemical synthesis , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/chemistry
5.
J Med Chem ; 55(6): 2649-71, 2012 Mar 22.
Article in English | MEDLINE | ID: mdl-22339166

ABSTRACT

Targeted molecular radiotherapy opens unprecedented opportunities to eradicate cancer cells with minimal irradiation of normal tissues. Described in this study are radioactive cyclosaligenyl monophosphates designed to deliver lethal doses of radiation to cancer cells. These compounds can be radiolabeled with SPECT- and PET-compatible radionuclides as well as radionuclides suitable for Auger electron therapies. This characteristic provides an avenue for the personalized and comprehensive treatment strategy that comprises diagnostic imaging to identify sites of disease, followed by the targeted molecular radiotherapy based on the imaging results. The developed radiosynthetic methods produce no-carrier-added products with high radiochemical yield and purity. The interaction of these compounds with their target, butyrylcholinesterase, depends on the stereochemistry around the P atom. IC(50) values are in the nanomolar range. In vitro studies indicate that radiation doses delivered to the cell nucleus are sufficient to kill cells of several difficult to treat malignancies including glioblastoma and ovarian and colorectal cancers.


Subject(s)
Neoplasms/radiotherapy , Radiopharmaceuticals/chemical synthesis , Thymidine Monophosphate/analogs & derivatives , Thymidine Monophosphate/chemical synthesis , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemical synthesis , Butyrylcholinesterase/metabolism , Cell Line, Tumor , Cell Survival , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/pharmacology , Colorectal Neoplasms , Drug Screening Assays, Antitumor , Female , Glioblastoma , Humans , Hydrolysis , Iodine Radioisotopes , Molecular Targeted Therapy , Neoplasms/diagnostic imaging , Neoplasms/enzymology , Ovarian Neoplasms , Radionuclide Imaging , Radiopharmaceuticals/pharmacology , Stereoisomerism , Structure-Activity Relationship , Thymidine Monophosphate/pharmacology , Uridine Monophosphate/pharmacology
6.
Nucleosides Nucleotides Nucleic Acids ; 30(11): 886-96, 2011 Nov.
Article in English | MEDLINE | ID: mdl-22060553

ABSTRACT

In order to support bioanalytical LC/MS method development and plasma sample analysis in preclinical and clinical studies of the anti-hepatitis C-virus nucleotides, PSI-7977 and PSI-352938, the corresponding stable isotope labeled forms were prepared. These labeled compounds were prepared by addition reaction of the freshly prepared Grignard reagent (13)CD(3)MgI to the corresponding 2 '-ketone nucleosides followed by fluorination of the resulting carbinol with DAST. As expected, these 2 '-C-(trideuterated-(13)C-methyl) nucleotide prodrugs showed similar anti-HCV activity to that of the corresponding unlabeled ones.


Subject(s)
Antiviral Agents/chemistry , Cyclic P-Oxides/chemistry , Hepacivirus/drug effects , Nucleosides/chemistry , Prodrugs/chemistry , Uridine Monophosphate/analogs & derivatives , Antiviral Agents/chemical synthesis , Antiviral Agents/pharmacology , Cyclic P-Oxides/chemical synthesis , Cyclic P-Oxides/pharmacology , Halogenation , Hepatitis C/drug therapy , Humans , Isotope Labeling/methods , Nucleosides/chemical synthesis , Nucleosides/pharmacology , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Sofosbuvir , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/chemistry , Uridine Monophosphate/pharmacology
7.
J Org Chem ; 76(20): 8311-9, 2011 Oct 21.
Article in English | MEDLINE | ID: mdl-21916475

ABSTRACT

Prodrugs of therapeutic nucleoside monophosphates masked as phosphoramidate derivatives have become an increasingly important class of antiviral drugs in pharmaceutical research for delivering nucleotides in vitro and in vivo. Conventionally, phosphoramidate derivatives are prepared as a mixture of two diastereomers. We report a class of stable phosphoramidating reagents containing an amino acid ester and two phenolic groups, one unsubstituted and the other with electron-withdrawing substituents. The reagents can be isolated as single diastereomers and reacted with the 5'-hydroxyl group of nucleosides through selective nucleophilic displacement of the substituted phenol to prepare single diastereomer phosphoramidate products. This method has been used to prepare the HCV clinical candidate PSI-7977 in high yield and high diastereomeric purity.


Subject(s)
Amides/chemistry , Antiviral Agents/chemical synthesis , Chemistry, Pharmaceutical/methods , DNA-Directed RNA Polymerases/antagonists & inhibitors , Hepacivirus/drug effects , Hepatitis C/drug therapy , Phosphoric Acids/chemistry , Uridine Monophosphate/analogs & derivatives , Viral Proteins/antagonists & inhibitors , Amino Acids/chemistry , Antiviral Agents/pharmacology , Chromatography, High Pressure Liquid , DNA-Directed RNA Polymerases/metabolism , Electrons , Esters/chemistry , Hepacivirus/enzymology , Hepatitis C/virology , Humans , Magnetic Resonance Spectroscopy , Nucleosides/chemistry , Nucleotides/chemistry , Phenols/chemistry , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Sofosbuvir , Stereoisomerism , Structure-Activity Relationship , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/pharmacology , Viral Proteins/metabolism
8.
J Med Chem ; 53(19): 7202-18, 2010 Oct 14.
Article in English | MEDLINE | ID: mdl-20845908

ABSTRACT

Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the development of HCV therapies. ß-d-2'-Deoxy-2'-α-fluoro-2'-ß-C-methyl nucleosides are selective inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic. Phosphoramidate prodrugs of the 5'-phosphate derivative of the ß-d-2'-deoxy-2'-α-fluoro-2'-ß-C-methyluridine nucleoside were prepared and showed significant potency in the HCV subgenomic replicon assay (<1 µM) and produced high levels of triphosphate 6 in primary hepatocytes and in the livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric mixture 14 was crystallized, and an X-ray structure was determined establishing the phosphoramidate stereochemistry as Sp, thus correlating for the first time the stereochemistry of a phosphoramidate prodrug with biological activity. 51 (PSI-7977) was selected as a clinical development candidate.


Subject(s)
Antiviral Agents/chemical synthesis , Hepacivirus/drug effects , Prodrugs/chemical synthesis , Uridine Monophosphate/analogs & derivatives , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Cell Line , Crystallography, X-Ray , Dogs , Drug Resistance, Viral , Esters , Hepacivirus/genetics , Hepatocytes/metabolism , Humans , In Vitro Techniques , Liver/metabolism , Macaca fascicularis , Mutation , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Rats , Replicon , Sofosbuvir , Stereoisomerism , Structure-Activity Relationship , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/pharmacokinetics , Uridine Monophosphate/pharmacology , Viral Nonstructural Proteins/genetics
9.
J Med Chem ; 51(3): 439-48, 2008 Feb 14.
Article in English | MEDLINE | ID: mdl-18189347

ABSTRACT

Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5'-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5'-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6- N-methylamino, and 6- N, N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5'-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6- N-Methylamino and 6- N, N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.


Subject(s)
Antimalarials/chemical synthesis , Orotidine-5'-Phosphate Decarboxylase/antagonists & inhibitors , Plasmodium/drug effects , Uridine/analogs & derivatives , Uridine/chemical synthesis , Animals , Antimalarials/pharmacology , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Models, Molecular , Plasmodium/enzymology , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Plasmodium vivax/drug effects , Structure-Activity Relationship , Uridine/pharmacology , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/pharmacology
10.
J Antibiot (Tokyo) ; 60(7): 407-35, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17721001

ABSTRACT

The design and synthesis of novel 15-membered 11-azalides and 16-membered 11,12-diazalide starting from 16-membered macrolides are reported. A mobile linear dialdehyde was isolated via a cyclic tetraol which was prepared by osmium oxidation of a conjugated diene. One-pot macrocyclization of this dialdehyde with an amine or a diamine afforded corresponding 15-membered azalides or 11,12-diazalide. Fundamental SAR studies of 15-membered 11-azalides disclosed their potentiality as a lead molecule for further chemical modifications. For environmental preservation, sustainable chemistry for synthesis of these azalides is also discussed.


Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Aza Compounds/chemical synthesis , Azithromycin/analogs & derivatives , Macrolides/chemical synthesis , Macrolides/pharmacology , Anti-Bacterial Agents/chemistry , Aza Compounds/pharmacology , Azithromycin/chemistry , Azithromycin/pharmacology , Bacteria/drug effects , Humans , Kitasamycin/chemical synthesis , Kitasamycin/pharmacology , Macrocyclic Compounds , Macrolides/chemistry , Microbial Sensitivity Tests , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/pharmacology
11.
J Med Chem ; 50(5): 915-21, 2007 Mar 08.
Article in English | MEDLINE | ID: mdl-17290979

ABSTRACT

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.


Subject(s)
Antimalarials/chemical synthesis , Orotidine-5'-Phosphate Decarboxylase/antagonists & inhibitors , Uridine Monophosphate/analogs & derivatives , Uridine/analogs & derivatives , Animals , Antimalarials/chemistry , Antimalarials/pharmacology , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Mass Spectrometry , Methanobacterium/enzymology , Models, Molecular , Orotidine-5'-Phosphate Decarboxylase/chemistry , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Plasmodium falciparum/isolation & purification , Stereoisomerism , Structure-Activity Relationship , Uridine/chemical synthesis , Uridine/chemistry , Uridine/pharmacology , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/chemistry , Uridine Monophosphate/pharmacology
12.
J Med Chem ; 49(24): 7076-87, 2006 Nov 30.
Article in English | MEDLINE | ID: mdl-17125260

ABSTRACT

A series of UTP, UDP, and UMP derivatives and analogues were synthesized and evaluated at the human pyrimidinergic P2Y receptor subtypes P2Y2, P2Y4, and P2Y6 stably expressed in 1321N1 astrocytoma cells. Substituents at N3 of UTP were poorly tolerated by P2Y2 and P2Y4 receptors. In contrast, a large phenacyl substituent at N3 of UDP was well tolerated by the P2Y6 receptor, yielding a potent and selective P2Y6 receptor agonist (3-phenacyl-UDP, EC50=70 nM, >500-fold selective). The most potent and selective P2Y2 receptor agonist of the present series was 2-thio-UTP (EC50=50 nM, >or=30-fold selective vs P2Y4 and P2Y6). All modifications at the uracil base of UTP led to a decrease in potency at the P2Y4 receptor. A beta,gamma-dichloromethylene modification in the triphosphate chain of 5-bromo-UTP was tolerated by all three receptor subtypes, thus opening up a new strategy to obtain ectonucleotide diphosphohydrolase- and phosphatase-resistant P2Y2, P2Y4, and P2Y6 receptor agonists.


Subject(s)
Purinergic P2 Receptor Agonists , Uracil Nucleotides/chemical synthesis , Bridged Bicyclo Compounds/chemical synthesis , Bridged Bicyclo Compounds/pharmacology , Cell Line, Tumor , Humans , Inositol Phosphates/biosynthesis , Purines/chemical synthesis , Purines/pharmacology , Receptors, Purinergic P2 , Receptors, Purinergic P2Y2 , Structure-Activity Relationship , Uracil Nucleotides/pharmacology , Uridine Diphosphate/analogs & derivatives , Uridine Diphosphate/chemical synthesis , Uridine Diphosphate/pharmacology , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/pharmacology , Uridine Triphosphate/analogs & derivatives , Uridine Triphosphate/chemical synthesis , Uridine Triphosphate/pharmacology
13.
J Med Chem ; 49(16): 4937-45, 2006 Aug 10.
Article in English | MEDLINE | ID: mdl-16884305

ABSTRACT

Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.


Subject(s)
Orotidine-5'-Phosphate Decarboxylase/antagonists & inhibitors , Orotidine-5'-Phosphate Decarboxylase/chemistry , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemical synthesis , Calorimetry , Computer Simulation , Drug Design , Kinetics , Models, Molecular , Thermodynamics , Uridine Monophosphate/chemistry
14.
Adv Space Res ; 30(6): 1525-31, 2002.
Article in English | MEDLINE | ID: mdl-12575717

ABSTRACT

Modern studies now favor the fact that extraterrestrial organic molecules served as an important source of biological important substances on the primitive Earth. It is presumed that these space-made organic molecules could be transported safely to the Earth surface being associated with mineral grains. It is important to test whether nucleotides synthesized in Earth orbit could be protected by lunar surface regolite. The phosphorylation of adenosine, uridine and thymidine has been studied with respect of their further transformations and degradation in presence of mineral bed. After retrieval, HPLC analysis is used to identify all the mononucleotides of certain nucleosides. It has been shown, that exposure of the investigated nucleosides as dry films in space conditions in the presence of Lunar soil increases the yield of synthesized nucleotides in 1.1-3.0 times as compared with the exposure of the same samples in absence of Lunar soil. To identify and evaluate the principal source of energy in open space responsible for nucleotide synthesis reaction laboratory experiments were performed. It has been shown, that vacuum ultra violet (VUV 145 nm) radiation promotes nucleotide synthesis more effectively than ultra violet (UV 254 nm) while the presence of Lunar soil increases reaction yield in 1.5-2.0 times. Formation of 5'-mononucleotides seemed to be the most effective reaction both in flight and in laboratory experiments. Protective action of lunar soil on synthesized nucleotides against UV radiation has been shown in open Space conditions.


Subject(s)
Evolution, Chemical , Nucleotides/chemical synthesis , Soil/analysis , Space Flight , Ultraviolet Rays , Adenosine Monophosphate/chemical synthesis , Earth, Planet , Exobiology , Extraterrestrial Environment , Moon , Thymidine Monophosphate/chemical synthesis , Uridine Monophosphate/chemical synthesis
15.
J Med Chem ; 44(25): 4475-80, 2001 Dec 06.
Article in English | MEDLINE | ID: mdl-11728193

ABSTRACT

A novel approach to the intracellular delivery of nucleotides using phosphoramidate-based prodrugs is described. Specifically, we have developed phosphoramidate prodrugs of the anticancer nucleotide 5-fluoro-2'-deoxyuridine-5'monophosphate (FdUMP). These phosphoramidate prodrugs contain an ester group that undergoes intracellular activation liberating phosphoramidate anion, which undergoes spontaneous cyclization and P-N bond cleavage to yield the nucleoside monophosphate quantitatively. In vitro evaluation of 5-fluoro-2'-deoxyuridine phosphoramidate prodrugs 2a and 3b against L1210 mouse leukemia cells show potent inhibition of cell growth (IC(50) 0.5-3 nM). Cell-based thymidylate synthase inhibition studies show that, in contrast to FUdR, the nitrofuran compound 2a is of comparable potency in wild type vs thymidine kinase deficient LM cells. This result indicates that the activation of this novel prodrug occurs via the proposed mechanism of intracellular delivery. However, naphthoquinone 3b has an IC(50) value for thymidylate synthase inhibition that is comparable to FUdR in thymidine kinase deficient cells. Further studies revealed that 3b rapidly decomposes to the nucleotide in cell culture medium, suggesting that the naphthoquinone analogue is not sufficiently stable to function as a nucleotide prodrug.


Subject(s)
Antineoplastic Agents/chemical synthesis , Fluorodeoxyuridylate/analogs & derivatives , Fluorodeoxyuridylate/chemical synthesis , Prodrugs/chemical synthesis , Uridine Monophosphate/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Division/drug effects , Cell Line , Drug Screening Assays, Antitumor , Enzyme Inhibitors/pharmacology , Floxuridine/pharmacology , Fluorodeoxyuridylate/chemistry , Fluorodeoxyuridylate/pharmacology , Magnetic Resonance Spectroscopy , Mice , Prodrugs/chemistry , Prodrugs/pharmacology , Thymidine Kinase/deficiency , Thymidylate Synthase/antagonists & inhibitors , Tumor Cells, Cultured , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemistry , Uridine Monophosphate/pharmacology
16.
Article in English | MEDLINE | ID: mdl-11562946

ABSTRACT

A synthesis of 6-formyluridine 5'-monophosphate (6-formylUMP) in 6 steps starting from uridine is described. This approach should be applicable to the preparation of other O5'-phosphorylated 6-formylUrds such as 6-formylUDP and 6-formylUTP.


Subject(s)
Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemical synthesis
17.
Biochemistry ; 40(21): 6227-32, 2001 May 29.
Article in English | MEDLINE | ID: mdl-11371183

ABSTRACT

The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (ODCase) complexed with the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reveals the presence of a series of strong interactions between enzyme residues and functional groups of this ligand. Enzyme contacts with the phosphoribofuranosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contribute at least 16.6 kcal/mol of intrinsic binding free energy to the stabilization of the transition state for the reaction catalyzed by yeast ODCase. In addition to these enzyme-ligand contacts, active site residues contributed by both subunits of the dimeric enzyme are positioned to form hydrogen bonds with the 2'- and 3'-OH groups of the ligand's ribosyl moiety. These involve Thr-100 of one subunit and Asp-37 of the opposite subunit, respectively. To evaluate the contributions of these ribofuranosyl contacts to ground state and transition state stabilization, Thr-100 and Asp-37 were each mutated to alanine. Elimination of the enzyme's capacity to contact individual ribosyl OH groups reduced the k(cat)/K(m) value of the T100A enzyme by 60-fold and that of the D37A enzyme by 300-fold. Removal of the 2'-OH group from the substrate OMP decreased the binding affinity by less than a factor of 10, but decreased k(cat) by more that 2 orders of magnitude. Upon removal of the complementary hydroxymethyl group from the enzyme, little further reduction in k(cat)/K(m) for 2'-deoxyOMP was observed. To assess the contribution made by contacts involving both ribosyl hydroxyl groups at once, the ability of the D37A mutant enzyme to decarboxylate 2'-deoxyOMP was measured. The value of k(cat)/K(m) for this enzyme-substrate pair was 170 M(-1) s(-1), representing a decrease of more than 7.6 kcal/mol of binding free energy in the transition state. To the extent that electrostatic repulsion in the ground state can be tested by these simple alterations, the results do not lend obvious support to the view that electrostatic destabilization in the ground state enzyme-substrate complex plays a major role in catalysis.


Subject(s)
Orotidine-5'-Phosphate Decarboxylase/metabolism , Alanine/genetics , Aspartic Acid/genetics , Catalysis , Enzyme Stability/genetics , Kinetics , Orotidine-5'-Phosphate Decarboxylase/chemistry , Orotidine-5'-Phosphate Decarboxylase/genetics , Ribosemonophosphates/metabolism , Saccharomyces cerevisiae/enzymology , Substrate Specificity/genetics , Threonine/genetics , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/metabolism
18.
J Org Chem ; 65(20): 6515-24, 2000 Oct 06.
Article in English | MEDLINE | ID: mdl-11052096

ABSTRACT

This paper describes the design and synthesis of a conformationally rigid dimer building block Umpc3Um as a chiral center at the phosphate group with the S/N junction where c3 refers to a propylene bridge linked between the uracil 5-position and 5'-phosphate group of pUm. The extensive H1 NMR analysis of Umpc3Um suggests that the 5'-upstream Um has predominantly a C2'-endo conformation and the pc3Um moiety exists almost exclusively in a C3'-endo conformation. The absolute configuration of the diastereomers Umpc3Um(fast) (8a) and Umpc3Um(slow) (8b) was determined by CD spectroscopy as well as computer simulations. The oligonucleotides U4[Umpc3Um(fast)]U4 (13a) and U4[Umpc3Um(slow)]U4 (13b) incorporating 8a and 8b were synthesized by use of the phosphoramidite building blocks 11a and 11b, respectively. The Tm experiments of the duplexes formed between these modified oligomers and the complementary oligomers imply that the modified oligomer 13a having Umpc3Um(fast) has the Sp configuration at the chiral phosphoryl group.


Subject(s)
Nucleotides, Cyclic/chemical synthesis , Oligonucleotides/chemical synthesis , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemical synthesis , Circular Dichroism , DNA/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Nucleic Acid Conformation , Nucleotides, Cyclic/chemistry , Oligonucleotides/chemistry , Phosphorus/chemistry , RNA/chemistry , Stereoisomerism , Uridine Monophosphate/chemistry
19.
Biochemistry ; 39(9): 2261-8, 2000 Mar 07.
Article in English | MEDLINE | ID: mdl-10694392

ABSTRACT

Rat liver alpha(2-->6) sialyltransferase catalyzes the formation of a glycosidic bond between N-acetylneuraminic acid and the 6-hydroxyl group of a galactose residue at the nonreducing terminus of an oligosaccharide. This reaction has been investigated through the use of the novel sugar-nucleotide donor substrate UMP-NeuAc. A series of UMP-NeuAc radioisotopomers were prepared by chemical deamination of the corresponding CMP-NeuAc precursors. Kinetic isotope effects (KIEs) on V/K were measured using mixtures of radiolabeled UMP-NeuAc's as the donor substrate and N-acetyllactosamine as the acceptor. The secondary beta-(2)H KIE was 1.218 +/- 0.010, and the primary (14)C KIE was 1.030 +/- 0.010. A large inverse (3)H binding isotope effect of 0.944 +/- 0.010 was measured at the terminal carbon of the NeuAc glycerol side chain. These KIEs observed using UMP-NeuAc are much larger than those previously measured with CMP-NeuAc [Bruner, M., and Horenstein, B. A. (1998) Biochemistry 37, 289-297]. Solvent deuterium isotope effects of 1.3 and 2.6 on V/K and V(max) were observed with CMP-NeuAc as the donor, and it is revealing that these isotope effects vanished with use of the slow donor substrate UMP-NeuAc. Bell-shaped pH versus rate profiles were observed for V(max) (pK(a) values = 5.5, 9.0) and V/K(UMP)(-)(NeuAc) (pK(a)values = 6.2, 9.0). The results are considered in terms of a mechanism involving an isotopically sensitive conformational change which is independent of the glycosyl transfer step. The isotope effects reveal that the enzyme-bound transition state bears considerable charge on the N-acetylneuraminic acid residue, and this and other features of this mechanism provide new directions for sialyltransferase inhibitor design.


Subject(s)
N-Acetylneuraminic Acid/analogs & derivatives , N-Acetylneuraminic Acid/chemistry , Sialyltransferases/chemistry , Uridine Monophosphate/analogs & derivatives , Uridine Monophosphate/chemistry , Animals , Deuterium , Hydrogen-Ion Concentration , Kinetics , Liver/enzymology , N-Acetylneuraminic Acid/chemical synthesis , Rats , Solvents , Substrate Specificity , Uridine Monophosphate/chemical synthesis , beta-D-Galactoside alpha 2-6-Sialyltransferase
20.
Pharm Res ; 14(6): 706-12, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9210185

ABSTRACT

PURPOSE: This study is concerned with cellular delivery/generation of 2'-azido-2'-deoxyuridine and -deoxycytidine diphosphate (N3UDP or N3CDP), potent inhibitors of ribonucleotide reductase. It characterizes the phosphorylation steps involved in the conversion of 2'-azido-2'-deoxyuridine (N3Urd) and 2'-azido-2'-deoxycytidine (N3Cyd) to the corresponding diphosphates and explores a prodrug approach in cellular delivery of the inhibitor which circumvents the requirement of deoxynucleoside kinases. METHODS: Cell growth of CHO and 3T6 cells of known deoxycytidine kinase level was determined in the presence of N3Urd and N3Cyd. Activity of ribonucleotide reductase was determined in the presence of the azidonucleosides as well as their mono- or di-phosphates in a Tween 80-containing permeabilizing buffer. A prodrug of 5'-monophosphate of N3Urd was prepared and its biological activity was evaluated with CHO cells as well as with cells transfected with deoxycytidine kinase. RESULTS: N3Urd failed to inhibit the growth of both cell lines, while N3Cyd was active against 3T6 cells and moderately active against CHO cells. These results correlate with the deoxycytidine kinase levels found in the cells. Importance of the kinase was further established with the finding that the nucleoside analogs were inactive as reductase inhibitors in a permeabilized cell assay system while their mono- and di-phosphates were equally active. The prodrug was active in cell growth inhibition regardless of the deoxycytidine kinase level. CONCLUSIONS: The azidonucleosides become potent inhibitors of the reductase by two sequential phosphorylation steps. The present study indicates that the first step to monophosphate is rate-limiting, justifying a prodrug approach with the monophosphate.


Subject(s)
Antineoplastic Agents/pharmacology , Azides/pharmacology , Cytidine Diphosphate/analogs & derivatives , Deoxyuracil Nucleotides/pharmacology , Prodrugs/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Uridine Monophosphate/analogs & derivatives , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , CHO Cells , Cell Division/drug effects , Cell Line , Cricetinae , Cytidine Diphosphate/pharmacology , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine Kinase/metabolism , Deoxyuridine/analogs & derivatives , Deoxyuridine/pharmacology , Dideoxynucleotides , Hydrolysis , Permeability , Phosphorylation , Prodrugs/pharmacokinetics , Tumor Cells, Cultured , Uridine Monophosphate/chemical synthesis , Uridine Monophosphate/pharmacokinetics , Uridine Monophosphate/pharmacology
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