The design and synthesis of a new tumor-selective fluoropyrimidine carbamate, capecitabine

To identify an orally available fluoropyrimidine having efficacy and safety profiles greatly improved over those of parenteral 5-fluorouracil (5-FU: 1), we designed a 5-FU prodrug that would pass intact through the intestinal mucisa and be sequentially converted to 5-FU by enzymes that are highly expressed in the human liver and then in tumors. Among various N4-substituted 5'-deoxy-5-fluorocytidine derivatives, a series of N4-alkoxycarbonyl derivatives were hydrolyzed to 5'-deoxy-5-fluorocytidine (5'-DFCR: 8) specifically by carboxylesterase, which exists preferentially in the liver in humans and monkeys. Particularly, derivatives having an N4-alkoxylcarbonyl moiety with a C4-C6 alkyl chain were the most susceptible to the human carboxylesterase. Those were then converted to 5'-deoxy-5-fluorouridine (5'-DFUR: 4) by cytidine deaminase highly expressed in the liver and solid tumors and finally to 5-FU by thymidine phosphorylase (dThdPase) preferentially located in tumors. When administered orally to monkeys, a derivative having the N4-alkoxylcarbonyl moiety with a C5 alkyl chain (capecitabine: 6) The highest AUC and Cmax for plasma 5'-DFUR. In tests with various human cancer xenograft models, capecitabine was more efficacious at wider dose ranges than either 5-FU or 5'-DFUR and was significantly less toxic to the intestinal tract than the others in monkeys.     

Specific incorporation of 5-fluorocytidine into Escherichia coli RNA

RNAs isolated from Escherichia coli B grown in the presence of 5-fluorouracil have high levels of the analog replacing uridine and uridine-derived modified nucleosides. Cytidine has also been shown to be replaced in these RNAs by 5-fluorocytidine, a metabolic product of 5-fluorouracil, but to a considerably lesser extent. When 5-fluorocytidine is added to cultured of E. coli B little 5-fluorocytidine (0.20 mol%) is incorporated into cellular RNAs because of the active cytosine/cytidine deaminase activities. Addition of the cytidine deaminase inhibitor tetrahydrouridine (70 micrograms/ml) increases 5-fluorocytidine incorporation to about 3 mol% in tRNAs, but does not eliminate 5-fluorouridine incorporation. E. coli mutants lacking cytosine/cytidine deaminase activities are able to more than double the extent of 5-fluorocytidine incorporation into their transfer and ribosomal RNAs, replacing cytidine with no detectable 5-fluorouridine incorporation. Levels of 5-methyluridine, pseudouridine and dihydrouridine in tRNAs are not affected. These fluorocytidine-containing tRNAs show amino acid-accepting activities similar to control tRNAs. Fluorocytidine was found to be quite susceptible to deamination under alkaline conditions. Its conversion to primarily 5-fluorouridine follows pseudo-first-order reaction kinetics with a half-life of 10 h in 0.3 M KOH at 37 degrees C. This instability in alkali probably explains why 5-fluorocytidine was not found earlier in RNAs isolated from cells treated with 5-fluorouridine, since most early RNA hydrolyses were carried out in alkali. It may also explain the mild mutagenic properties observed in some systems following 5-fluorouridine treatment. Initial 19F-NMR measurements in fluorocytidine-containing tRNAs indicate that this modified tRNA may be useful in future structural studies of tRNAs and in probing tRNA-protein complexes.     

Solid-phase synthesis of 5'-deoxy-5'-amino-clitocine analogues

Several 6-substituted-amino-5'-deoxy-5'-amino-clitocine analogues were synthesized in a parallel fashion in solid phase. The desired scaffold was generated by coupling 2,3-O-bis-(t-butyldimethylsilyl)-5-N-(monomethoxytrityl-polystyrene-resin)-1,5-diamino-5-deoxy-beta-D-ribofuranose and 4, 6-dichloro-5-nitropyrimidine. The scaffold was then reacted with a variety of amines to generate a small library of 14 analogues of 5'-deoxy-5'-amino-clitocine following a protocol developed earlier.     

Synthesis of 5'-deoxy-5'-nucleosideacetic acid derivatives

The synthesis of several new 5'-deoxy-5'-nucleosideacetic acid derivatives by the reactions of alkoxycarbonylmethylene triphenylphosphoranes with nucleoside 5'-aldehydes is described.     

Alterations in the maturation and structure of ribosomal precursor RNA in Novikoff hepatoma cells induced by 5-fluorocytidine

The effects of 5-fluorocytidine on ribosomal RNA maturation and structure in Novikoff hepatoma cells were investigated. Like other nucleic acid base analogues that are incorporated into RNA, this compound inhibits maturation of the 45S ribosomal RNA precursor. The 45S RNA precursor produced in the presence of 5-fluorocytidine has an abnormal electrophoretic mobility compared with that of the control precursor under nondenaturing conditions, but the two have identical mobilities under denaturing conditions. Under the conditions of these experiments, 5-fluorocytidine inhibited cellular protein synthesis only slightly, whereas equimolar concentrations of 5-azacytidine resulted in nearly 75% inhibition of this process. Despite this difference in the effects of the two analogues as well as the greater chemical lability of the 5-azacytidine, their effects on ribosomal RNA maturation are identical.     

Synthesis of 2'-deoxy-2'-fluoroguanyl-(3',5')-guanosine

The protected analogue of 2-amnio-6-chloropurine arabinoside (3b) was subjected to reaction with diethylaminosulfur trifluoride (DAST) and subsequently treated with NaOAc in Ac2O/AcOH to give N2, O3', O5'-triacetyl-2'-deoxy-2'-fluoroguanosine (5a). After deacetylation of the sugar moiety and protection of 5'-OH by a 4,4'-dimethoxytrityl group, this nucleoside component was converted to 2'-deoxy-2'-fluoroguanyl-(3',5')-guanosine (6c, GfpG).     

Liquid chromatography-mass spectrometry method for the analysis of the anti-cancer agent capecitabine and its nucleoside metabolites in human plasma

A reversed-phase high-performance liquid chromatography method with electrospray ionization and mass spectral detection is described for the determination of capecitabine, 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine in human plasma with 5-chloro-2'-deoxyuridine as the internal standard. An on-line sample clean-up procedure allows dilution of the plasma sample with the initial mobile phase. The linear dynamic range is 0.0500-10.0 microgram/ml for capecitabine, and 0.0500-25.0 microgram/ml for the metabolites, 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine, respectively. This method has been used to analyze plasma samples from patients receiving capecitabine in combination with oxaliplatin.     

A novel hyperthermostable 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus

We report herein the first molecular characterization of 5'-deoxy-5'-methylthio-adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII). The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low Km (0.7 microm) for 5'-deoxy-5'-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 degrees C and extremely thermostable with an apparent Tm of 112 degrees C that increases in the presence of substrates. The enzyme is characterized by high kinetic stability and remarkable SDS resistance and is also resistant to guanidinium chloride-induced unfolding with a transition midpoint of 3.3 m after 22-h incubation. Limited proteolysis experiments indicated that the only one proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. Moreover, the binding of 5'-deoxy-5'-methylthioadenosine induces a conformational transition that protected the enzyme against protease inactivation. By site-directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutants C259S/C261S and C262S show thermophilicity and thermostability features significantly lower than those of the wild-type enzyme. In order to get insight into the physiological role of SsMTAPII a comparative kinetic analysis with the homologous 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was carried out. Finally, the alignment of the protein sequence of SsMTAPII with those of SsMTAP and human 5'-deoxy-5'-methylthioadenosine phosphorylase (hMTAP) shows several key residue changes that may account why SsMTAPII, unlike hMTAP, is able to recognize adenosine as substrate.     

Simultaneous decrease in deamination of 5'-adenylic acid and 5'-deoxy-5'-S-isobutylthioadenosine in chick-embryo fibroblasts infected by Rous sarcoma virus

The rate of deamination of 5'-deoxy-5'-S-isobutylthioadenosine [(iBuS5'Ado] in chick embryo fibroblasts was substantially reduced after their infection and morphological transformation by Rous sarcoma virus. Concomitant with the reduction in rate of (iBuS)5'Ado deamination there was a decrease in adenosine deaminase and 5'-adenylic acid deaminase activities. The drop of these activities was related to infection and not to the expression of the src gene. (iBuS)5'Ado was deaminated by at least three enzymes or isoenzymes whose apparent molecular weights have been estimated to be 295000, 121000 and 37000 respectively. Two of these enzymes have been characterized as 5'-adenylic acid deaminase and the heavy form of adenosine deaminase, respectively.     

Mechanism of inactivation of S-adenosyl-L-homocysteine hydrolase by 5'-deoxy-5'-S-allenylthioadenosine and 5'-deoxy-5'-S-propynylthioadenosine

5'-Deoxy-5'-S-allenylthioadenosine 1 and 5'-deoxy-5'-S-propnylthioadenosine 2, derived from adenosine, were prepared. 1 and 2 caused irreversible inactivation of AdoHcy hydrolase. ESI mass spectra analysis of the inactivated enzyme demonstrated that 1 and 2 were type II "mechanism-based" inhibitors.     

Design, synthesis and biological evaluation of 2'-deoxy-2',2'-difluoro-5-halouridine phosphoramidate ProTides

We report the synthesis of a series of novel 2'-deoxy-2',2'-difluoro-5-halouridines and their corresponding phosphoramidate ProTides. All compounds were evaluated for antiviral activity and for cellular toxicity. Interestingly, 2'-deoxy-2',2'-difluoro-5-iodo- and -5-bromo-uridines showed selective activity against feline herpes virus replication in cell culture due to a specific recognition (activation) by the virus-encoded thymidine kinase.     

5'-deoxy-5'-thioanalogs of adenosine and inosine 5'-monophosphate: studies with 5'-nucleotidase and alkaline phosphatase

The interaction of 5'-deoxy-5'-thioadenosine 5'-monophosphate (A(S)MP) and 5'-deoxy-5'-thioinosine 5'-monophosphate (I(S)MP) with snake venom, 5'nucleotidase, and calf intestinal mucosa alkaline phosphatase has been characterized. The substrates, A(S)MP and I(S)MP, are analogs of adenosine 5'-monophosphate and inosine 5'-monophosphate in which sulfur replaces oxygen as the bridge between the 5'-carbon of the ribose and the phosphorous. The P-S bond of both A(S)MP and I(S)MP was hydrolyzed by alkaline phosphatase producing the corresponding thionucleoside as a reaction product. The Km for A(S)MP was 270 microM and the V for alkaline phosphatase was 110 nmol/min/mg (8% of the V for AMP), whereas the corresponding values for I(S)MP were 300 microM and 530 nmol/min/mg protein, respectively. In contrast, 5'-nucleotidase did not catalyze hydrolysis of either A(S)MP or I(S)MP. A(S)MP and I(S)MP were competitive inhibitors of the 5'-nucleotidase hydrolysis of AMP and IMP, respectively, with Ki values of 975 and 13 microM. Decreasing the pH of the reaction from 8.1 to 7.1 lowered the Ki for I(S)MP by 100-fold, to a value of 0.15 microM.     

Rapid microwave-assisted fluorination yielding novel 5'-deoxy-5'-fluorouridine derivatives

The preparation of (18)F-labeled ligands for positron emission tomography (PET) and the subsequent imaging have to be completed within a half-life of the neutron-deficient isotope ((18)F=110 min). In this paper, we report a rapid fluorination approach to obtain 5'-deoxy-5'-fluoro-substituted uracil nucleoside analogues. Nucleophilic substitution at the 5'-position of the nucleosides was achieved within 45 min providing excellent yields of 75-92% by application of microwaves.     

Unraveling the structural and functional differences between purine nucleoside phosphorylase and 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Purine nucleoside metabolism in the archaeon Pyrococcus furiosus is catalyzed by purine nucleoside phosphorylase (PfPNP) and 5'-deoxy-5'-methylthioadenosine phosphorylase (PfMTAP). These enzymes, characterized by 50% amino acid sequence identity, show non-common features of thermophilicity and thermostability and are stabilized by intramolecular disulfide bonds. PfPNP is highly specific for 6-oxopurine nucleosides while PfMTAP is characterized by a broad substrate specificity with 6-aminopurine nucleosides as preferred substrates. Amino acid sequence comparison clearly shows that the hypothetical active sites of PfPNP and PfMTAP are almost identical and that, in analogy with human 5'-deoxy-5'-methylthioadenosine phosphorylase and human purine nucleoside phosphorylase, residue changes at level of the same crucial positions could be responsible for the switch of substrate specificity. To validate this hypothesis we changed the putative active site of PfPNP by site-directed mutagenesis. Substrate specificity and catalytic efficiency of PfPNP mutants were then analyzed by kinetic studies and compared with the wild-type enzyme. We carried out the molecular modeling of PfPNP and PfMTAP to obtain a picture of the overall enzyme structure and to identify structural features as well as interactions playing critical roles in thermostability. Finally, we utilized the structural models of mutant enzyme-substrate complex to rationalize the functional effects of the mutations.     

Appearance of atypical 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in spgp knockout mice

Bile formation and its canalicular secretion are essential functions of the mammalian liver. The sister-of-p-glycoprotein (spgp) gene was shown to encode the canalicular bile salt export protein, and mutations in spgp gene were identified as the cause of progressive familial intrahepatic cholestasis type 2. However, target inactivation of spgp gene in mice results in nonprogressive but persistent cholestasis and causes the secretion of unexpectedly large amounts of unknown tetrahydroxylated bile acid in the bile. The present study confirms the identity of this tetrahydroxylated bile acid as 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid. The data further show that in serum, liver, and urine of the spgp knockout mice, there is a significant increase in the concentration of total bile salts containing a large amount of tetrahydroxy-5 beta-cholan-24-oic acid. The increase in total bile acids was associated with up-regulation of the mRNA of cholesterol 7 alpha-hydroxylase in male mice only. It is suggested that the lower severity of the cholestasis in the spgp knockout mice may be due to the synthesis of 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid, which neutralizes in part the toxic effect of bile acids accumulated in the liver.     

The crystal structure of 5'-deoxy-5'-methylthioadenosine phosphorylase II from Sulfolobus solfataricus, a thermophilic enzyme stabilized by intramolecular disulfide bonds

The crystal structure of Sulfolobus solfataricus 5'-deoxy-5'-methylthioadenosine phosphorylase II (SsMTAPII) in complex with 5'-deoxy-5'-methylthioadenosine (MTA) and sulfate was determined to 1.45A resolution. The hexameric structure of SsMTAPII is a dimer-of-trimers with one active site per monomer. The oligomeric assembly of the trimer and the monomer topology of SsMTAPII are almost identical with trimeric human 5'-deoxy-5'-methylthioadenosine phosphorylase (hMTAP). SsMTAPII is the first reported hexameric member in the trimeric class of purine nucleoside phosphorylase (PNP) from Archaea. Unlike hMTAP, which is highly specific for MTA, SsMTAPII also accepts adenosine as a substrate. The residues at the active sites of SsMTAPII and hMTAP are almost identical. The broad substrate specificity of SsMTAPII may be due to the flexibility of the C-terminal loop. SsMTAPII is extremely thermoactive and thermostable. The three-dimensional structure of SsMTAPII suggests that the unique dimer-of-trimers quaternary structure, a CXC motif at the C terminus, and two pairs of intrasubunit disulfide bridges may play an important role in its thermal stability.     

Effect of 5'-deoxy-5'-S-isobutyladenosine on hematopoietic stem cells

Unlike known cytostatics, 5'-deoxy-5'-S-isobutyladenosine (SIBA) does not inhibit proliferative activity of hemopoietic stem cells of intact bone marrow. On the contrary, it raises CFUs number of 15-28% in vitro and in vivo. SIBA inhibits by 40% the 3',5'-cAMP-induced increase in CFUs proliferation.     

Amino substituted derivatives of 5'-amino-5'-deoxy-5'-noraristeromycin

The potent antiviral potential of 5'-amino-5'-deoxy-5'-noraristeromycin (2) is limited by associated toxicity. To seek derivatives of 2 that circumvent this undesirable property, three amino substituted derivatives (acetyl, 3; formyl, 4; and methyl, 5) of 2 have been prepared in 4-7 steps from the same intermediate, (1S,4R)-4-(6-chloropurin-9-yl)cyclopent-2-en-1-ol (6). Key steps involved an improved Pd(0)-catalyzed allylic azidation and a novel Pd(0)-catalyzed allylic amidation. The three target compounds were evaluated against a large number of viruses and found to be inactive except for a very weak effect of 5 on human cytomegalovirus, varicella zoster virus, and Epstein-Barr virus. There was also no noteworthy cytotoxicity associated with the new derivatives. Thus, these results indicate variation of the cyclopentyl amine of 2 does not offer a means to improve upon its antiviral potential.     

Three-dimensional structure of a hyperthermophilic 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus

The structure of 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) has been determined alone, as ternary complexes with sulfate plus substrates 5'-deoxy-5'-methylthioadenosine, adenosine, or guanosine, or with the noncleavable substrate analog Formycin B and as binary complexes with phosphate or sulfate alone. The structure of unliganded SsMTAP was refined at 2.5-A resolution and the structures of the complexes were refined at resolutions ranging from 1.6 to 2.0 A. SsMTAP is unusual both for its broad substrate specificity and for its extreme thermal stability. The hexameric structure of SsMTAP is similar to that of purine-nucleoside phosphorylase (PNP) from Escherichia coli, however, only SsMTAP accepts 5'-deoxy-5'-methylthioadenosine as a substrate. The active site of SsMTAP is similar to that of E. coli PNP with 13 of 18 nearest residues being identical. The main differences are at Thr(89), which corresponds to serine in E. coli PNP, and Glu(163), which corresponds to proline in E. coli PNP. In addition, a water molecule is found near the purine N-7 position in the guanosine complex of SsMTAP. Thr(89) is near the 5'-position of the nucleoside and may account for the ability of SsMTAP to accept either hydrophobic or hydrophilic substituents in that position. Unlike E. coli PNP, the structures of SsMTAP reveal a substrate-induced conformational change involving Glu(163). This residue is located at the interface between subunits and swings in toward the active site upon nucleoside binding. The high-resolution structures of SsMTAP suggest that the transition state is stabilized in different ways for 6-amino versus 6-oxo substrates. SsMTAP has optimal activity at 120 degrees C and retains full activity after 2 h at 100 degrees C. Examination of the three-dimensional structure of SsMTAP suggests that unlike most thermophilic enzymes, disulfide linkages play a key in role in its thermal stability.     

Chemical stability of 2'-deoxy-5-methylisocytidine during oligodeoxynucleotide synthesis and deprotection

Previous studies have encountered difficulties with degradation of some isocytidine derivatives during solid-phase synthesis and deprotection of oligonucleotides. Here we investigate the degradation of a commonly used derivative, 2'-deoxy-5-methylisocytidine, during oligodeoxynucleotide synthesis and deprotection. A small, but detectable amount of hydrolytic deamination occurred at ca. 0.5% of 2'-deoxy-5-methylisocytidine residues using routine synthesis and deprotection conditions. Depyrimidination, or cleavage of the glycosylic bond, occurred to a far lesser extent during alkaline deprotection than previously suggested. In contrast to model studies of nucleoside monomers, significant depyrimidination was not observed, even at extended incubation times.