Effect of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil on mitochondrial functions in HepG2 cells.

The effects of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil (D-FEAU) on mitochondrial functions were examined in HepG2 cells. D-FEAU between 0.1 and 10 microM had no apparent inhibitory effect on cell proliferation for 2-week period; however, D-FEAU caused a decrease in mitochondrial DNA (mtDNA) content in a dose-dependent manner with an IC50 value of 2.7 microM. A 20.9% of increase in lactic acid production was observed after the cells were incubated with 10 microM of D-FEAU for 4 days without substantial effect being detected at 0.1 and 1 microM. In addition, no significant changes on mitochondrial morphology were observed in the cells treated with 10 microM of D-FEAU for 14 days under the electron microscope.     

9-(2-Deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine: a metabolically stable cytotoxic analogue of 2'-deoxyguanosine

The synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine (1b) from 1,3-di-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranose (2a) and 2,6-dichloropurine in six steps using an enzymatic deamination as the last step is reported. The target compound was found to be stable to purine nucleoside phosphorylase cleavage and was cytotoxic in two cell lines, one a T-cell line. Incubation of L1210 cells with 1b results in an inhibition of DNA synthesis as judged by the reduced incorporation of labeled thymidine into DNA, while RNA and protein syntheses were unaffected.     

Nucleosides. 146. 1-Methyl-5-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil, the C-nucleoside isostere of the potent antiviral agent 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (FMAU). Studies directed toward the synthesis of 2'-deoxy-2'-substituted arabino nucleosides. 6

The synthesis of 5-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1-methyluracil (1, C-FMAU), an isostere of the potent antiviral and antitumor nucleoside 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (2'-fluoro-5-methyl-ara-U or FMAU), was achieved. Pseudouridine (2) was converted into 4,5'-anhydro-3'-O-acetyl-2'-O-triflylpseudouridine (4), which was treated with tris(dimethylamino)sulfur (1+) difluorotrimethylsilicate (TASF) to give 4,5'-anhydro-5-(3-O-acetyl-2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-1- methyluracil (5b) in 40% yield. Acid hydrolysis of the 4,5'-anhydro linkage of 5b with Dowex 50 (H+) afforded C-FMAU. The inhibitory activity of C-FMAU against HSV-1 and HSV-2 was about 10-fold less than that of FMAU in tissue culture. This compound, however, did not show significant activity in mice inoculated with HSV-1 or HSV-2.     

Evaluation of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil in a rabbit model of herpetic keratitis.

The nucleoside analog 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- ethyluracil (FEAU) was tested in a rabbit model of acute herpetic keratitis and its effectiveness compared with that of acyclovir (ACV). FEAU or ACV was applied topically 3 times daily, beginning 3 days post-HSV-1 inoculation and continued for a period of 7 days. FEAU at a concentration of 1% (w/v) or 3% ACV resulted in significant lessening of the severity of corneal lesions, conjunctivitis, iritis, and corneal clouding at 24 to 48 h after beginning chemotherapy. No toxic reaction was observed in any rabbit eyes treated with either FEAU or ACV. The duration of virus shedding into tear film and colonization of the trigeminal ganglia, however, were not reduced by either FEAU or ACV treatment begun 3 days post-inoculation. Fifty percent effective dose (ED50) of FEAU determinations performed on isolates from tear film and on the virus inoculum in secondary rabbit kidney cultures yielded a range of 4.6-7 microM, with two in vitro resistant isolates having ED50S of greater than or equal to 1500 microM of FEAU. Fifty percent cell growth inhibition for FEAU was 3000 microM at 72 h.     

Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine.

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) is an antiviral phosphonate nucleotide analogue that displays activity against a range of herpesviruses. Anion exchange high performance liquid chromatography analysis of the 60% methanol extract from [14C]HPMPC-treated cells reveals the formation of three major metabolites. Two of these were identified as phosphorylated forms of HPMPC, HPMPC phosphate, and HPMPC diphosphate, by liberation of HPMPC upon acid digestion and coelution with synthetic standards on high performance liquid chromatography. The third metabolite, which is resistant to alkaline phosphatase cleavage but sensitive to phosphodiesterase, is proposed to be an HPMPC phosphate adduct. In herpes simplex virus-1-infected cells the same three metabolites are detected, at concentrations comparable to those in uninfected cells. When HPMPC is removed from the medium, the concentrations of the metabolites in cells decrease slowly, with half-lives of approximately 6, 17, and 48 hr for HPMPC phosphate, HPMPC diphosphate, and the HPMPC phosphate adduct, respectively. HPMPC diphosphate inhibits herpes simplex virus-1 and -2 DNA polymerases with a lower Ki than that for DNA polymerase alpha, and enzyme inhibition is competitive in each case. The formation and the persistence of HPMPC phosphates in cells and the selective inhibition of viral DNA polymerases by HPMPC diphosphate can explain why cells pretreated with HPMPC remain refractory to viral infection even long after HPMPC is removed from the medium.     

Fluoro carbocyclic nucleosides: synthesis and antiviral activity of 2'- and 6'-fluoro carbocyclic pyrimidine nucleosides including carbocyclic 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil and carbocyclic 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil

The racemic carbocyclic 2'-fluoroarabinosyl pyrimidine nucleosides 8, 9 (C-FIAU), 12, and 13 (C-FMAU) and the 2'-fluororibosyl pyrimidine nucleosides 17, 20, and 21 were prepared from their respective protected 2'-fluoro amino diols 5 and 14. The carbocyclic 2'-2'-difluorothymidine analogue 27 was obtained from the protected difluoro amino diol 24 which was prepared from the ketone 23 and (diethylamino)sulfur trifluoride (DAST). The chiral carbocyclic 2'-deoxy-6'-fluorouridines 33, 34, 38, and 39 were synthesized from the protected 6'-fluoro amino diols 30 and 36, which were prepared by reduction of the azides 28 and 35. C-FMAU (13) and C-FIAU (9) were active in vitro against HSV-1 with ID50 values of 4.4 and 11 micrograms/mL, respectively, but they were inactive against HSV-2. The cytidine analogues 12 and 20 displayed modest activity in vitro against HSV-1 and HSV-2 but were inactive against human influenza A virus.     

Nucleosides. 129. Synthesis of antiviral nucleosides: 5-alkenyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracils

Synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracils containing a vinyl (4a), 2-halovinyl (4b-d), or ethyl substituent at C-5 was achieved. These nucleosides were found to be about a log order less active than 2'-fluoro-5-iodo-ara-C (FIAC) against HSV-1, but they are much less cytotoxic against normal human lymphocytes than FIAC. Nucleosides 4a and 4e showed good activity against HSV-1 (ED50 = 0.16 and 0.24 microM, respectively) and HSV-2 (ED50 = 0.69 and 0.65 microM) with very little cytotoxicity (ID50 greater than 100 microM).     

Nucleosides. 136. Synthesis and antiviral effects of several 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-alkyluracils. Some structure-activity relationships

In order to study structure-activity relationships between antiherpetic activity and the size of the C-5 alkyl substituents of 2'-fluoro-ara-U derivatives, six new nucleosides (1c-h) were synthesized. The 5-allyl analogue 1c was prepared by a Pd(II)-catalyzed reaction of 5-(chloromercuri)-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil with allyl chloride. Partial hydrogenation of 1c afforded the 5-n-propyl derivative 1d (FPAU). Nucleosides 1e-h were obtained by condensation of 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinosyl bromide with the corresponding 5-substituted uracils. Preliminary in vitro data show that, as the alkyl side chain is increased by one carbon unit, the antiherpetic potency is decreased by approximately 1 log order. The cytotoxicity also diminishes as the size of the 5-substituent is increased. FPAU exerts good activity against HSV-1 and HSV-2. FiPAU still shows good therapeutic indices, whereas the higher alkyl analogues are essentially inactive.     

Time-dependent action of 5-propyl deoxyuridine as antiherpes simplex virus type 1 and type 2 agents.

The most effective period of 5-propyl deoxyuridine action was 3–6 hr post-infection in both herpes simplex virus type 1- and type 2-infected HeLa BU cells. This antiviral activity could not be reversed by removing the drug from the culture medium. A correlation exists between inhibition of virus growth and inhibition of induction of virus specific DNase and DNA polymerase. No inhibition of induction of virus specific thymidine kinase was observed when virus replication was inhibited by 5-propyl deoxyuridine.     

(2S)-1-(arylacetyl)-2-(aminomethyl)piperidine derivatives: novel, highly selective kappa opioid analgesics.

This paper describes the synthesis and structure-activity relationships as kappa opioid analgesics of a novel class of 1-(arylacetyl)-2-(aminomethyl)piperidine derivatives. The active conformation of the pharmacophore, with a torsional angle (N1C2C7N8) of 60 degrees, was defined with computational studies and 1H NMR. A quantitative structure-activity relationship study of the arylacetic moiety substitution indicated that the presence of an electron-withdrawing and lipophilic substituent in para and/or meta positions is required for good analgesic activity and kappa affinity. The lead compounds (2S)-1-[(3,4-dichlorophenyl)acetyl]-2-(pyrrolidin-1-ylmethyl )piperidine hydrochloride and (2S)-1-[4-(trifluoromethyl)phenyl]acetyl]-2-(pyrrolidin-1-ylmet hyl) piperidine hydrochloride are the most kappa/mu selective (respectively 6500:1 and 4100:1) and among the most potent (Ki kappa 0.24 and 0.57 nM, respectively) kappa ligands identified so far. In the mouse tail flick model of antinociception, compound 14 (ED50 = 0.05 mg/kg sc) was 25 times more potent than morphine and 16 times more potent than the standard kappa ligand U-50488.     

A radioimmunoassay for the new antiviral agent 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil.

A specific and sensitive radioimmunoassay (RIA) for the measurement of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) in biological fluids has been developed. The assay has a range of 2.5-1,000 ng/ml and 10-1,000 ng/ml for serum and urine, respectively, and has the sensitivity to detect 2.5 and 25 ng/ml of BV-araU in serum and urine, respectively. A satisfactory zero binding and sensitivity were obtained after an overnight incubation at 4 degrees C. Separation of the antibody-bound and free ligand was achieved by employing polyethylene glycol-goat anti-rabbit gamma globulin separant. A quantitative recovery of the exogenous analyte was obtained at all concentrations of BV-araU tested. The assay is specific for the parent drug and is not affected by the presence of its metabolite, BV-U (bromovinyl uracil) or serum components (nucleotides, nucleosides, or sugars). Intraassay coefficients of variation were 3.1-4.4% and 2.5-4.2% for serum and urine controls, respectively. Interassay variability was < 8.6% for all serum and urine controls. Linear regression analysis showed that the correlation between RIA and high-pressure liquid chromatography was excellent (r = 0.997). The ascending dosage studies have been analyzed by the BV-araU RIA, and results indicate that the values of area under the serum concentration-time curve increased proportionally with the administered dose of BV-araU up to 80 mg. Cumulative urinary excretion data showed that approximately 50% of unchanged BV-araU was excreted in the urine within 24 h.     

Nucleosides. 133. Synthesis of 5-alkenyl-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)cytosines and related pyrimidine nucleosides as potential antiviral agents

The synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)cytosines with a halovinyl or vinyl substituent at C-5 was accomplished from the corresponding 5-iodo (FIAC, 1) and/or 5-chloromercuri nucleoside analogues with use of Li2PdCl4- and Pd(OAc)2-mediated coupling reactions. Thiation of the benzoylated derivative of the 5-ethyluracil nucleoside 3 followed by S-methylation and then ammonolysis provided 5-ethyl-2'-fluoro-ara-C. 5-Ethynyl-2'-fluoro-ara-C (19a) and 5-ethynyl-2'-fluoro-ara-U (19b) were also obtained from the persilylated 5-iodo nucleosides 1 and 16, respectively, by PdII/CuI catalyzed coupling with (trimethylsilyl)acetylene. With use of selective sugar deprotection of the initial coupling products with H2O/Me2SO, the corresponding 5-[2-(trimethylsilyl)ethynyl] derivatives 18a and 18b could be isolated. Most of the new compounds showed activity in vitro against both HSV-1 and HSV-2, as did the known corresponding 5-alkenyluracil nucleosides synthesized earlier. The 5-vinylcytosine and -uracil nucleosides 10 and 24, respectively, were highly effective against HSV-1 (ED90 = 0.40 and 0.043 microM, respectively) and HSV-2 (ED90 = 0.59 and 0.56 microM, respectively). Unlike BVDU, the 2'-fluoroarabinosyl derivatives of 5-(halovinyl)cytosine and -uracil showed activity against both types of herpes simplex virus. The therapeutic indices of these compounds are in some cases superior to those of 2'-fluoro-5-methyl-ara-U (FMAU, 2). Moderate antileukemic activity was observed in vitro for the 5-alkynyl and 5-vinyl compounds. The competition of these compounds with thymidine for viral-induced thymidine kinases was also studied.     

Synthesis of (E)-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine and related analogues: potent and unusually selective antiviral activity of (E)-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine against herpes simplex virus type 1

Syntheses of (E)-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine (TFPe-dUrd) (1), 5-(3,3,3-trifluoro-1-propyl)-2'-deoxyuridine (11), 5-(3,3,3-trifluoro-1-methoxy-1-propyl)-2'-deoxyuridine (8), and 5-(3,3,3-trifluoro-1-hydroxy-1-propyl)-2'-deoxyuridine (10) from 5-chloromercuri-2'-deoxyuridine are described. The antiviral activity of TFPe-dUrd was determined in cell culture against herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and vaccinia virus and compared concurrently with 5-(1-propenyl)-2'-deoxyuridine, 5-(2-bromovinyl)-2'-deoxyuridine, 5-iodo-2'-deoxyuridine, and 5-(trifluoromethyl)-2'-deoxyuridine. TFPe-dUrd demonstrated a potent and unusually selective activity against HSV-1, with a 2-log reduction in virus yield at 0.03 micrograms/mL (0.09 microM); L-1210 cell growth was inhibited by 50% only at 290 micrograms/mL. Isopycnic centrifugation of 32P-labeled DNA indicated that if 0.5 or 2 microM TFPe-dUrd was present for 0-6 h postinfection, viral DNA synthesis was reduced by ca. 50 and 85%, respectively; concomitantly, a new DNA band appeared at lower density than normal cellular or viral DNA.     

2-(Arylalkylamino)adenosin-5'-uronamides: a new class of highly selective adenosine A2 receptor ligands

The synthesis and receptor-binding profiles at adenosine receptor subtypes for a series of 2-(arylalkylamino)-adenosin-5'-uronamides is described. Halogenated 2-phenethylamino analogues such as 3e show greater than 200-fold selectivity for the A2 receptor subtype on the basis of rat brain receptor binding. The general structure-activity relationship of this series of compounds is discussed both in terms of potency at A2 receptors as well as receptor subtype selectivity. It is possible to introduce a hydrophilic carboxyalkyl substituent to this series such as in CGS 21680A (3h) and still retain good potency and selectivity for A2 receptors. In addition, functional data in a perfused working rat heart model shows that these compounds possess full agonist properties at A2 receptors with 3h having a greater than 1500-fold separation between A2 (coronary vasodilatory) and A1 (negative chronotropic) receptor mediated events.     

N-(cycloalkylamino)acyl-2-aminothiazole inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4- piperidinecarboxamide (BMS-387032), a highly efficacious and selective antitumor agent

N-Acyl-2-aminothiazoles with nonaromatic acyl side chains containing a basic amine were found to be potent, selective inhibitors of CDK2/cycE which exhibit antitumor activity in mice. In particular, compound 21 [N-[5-[[[5-(1,1-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide, BMS-387032], has been identified as an ATP-competitive and CDK2-selective inhibitor which has been selected to enter Phase 1 human clinical trials as an antitumor agent. In a cell-free enzyme assay, 21 showed a CDK2/cycE IC(50) = 48 nM and was 10- and 20-fold selective over CDK1/cycB and CDK4/cycD, respectively. It was also highly selective over a panel of 12 unrelated kinases. Antiproliferative activity was established in an A2780 cellular cytotoxicity assay in which 21 showed an IC(50) = 95 nM. Metabolism and pharmacokinetic studies showed that 21 exhibited a plasma half-life of 5-7 h in three species and moderately low protein binding in both mouse (69%) and human (63%) serum. Dosed orally to mouse, rat, and dog, 21 showed 100%, 31%, and 28% bioavailability, respectively. As an antitumor agent in mice, 21 administered at its maximum-tolerated dose exhibited a clearly superior efficacy profile when compared to flavopiridol in both an ip/ip P388 murine tumor model and in a s.c./i.p. A2780 human ovarian carcinoma xenograft model.     

Comparative metabolism of E-5-(2-bromovinyl)-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil in herpes simplex virus-infected cells

The antiviral activities and metabolic fates of E-5-(2-bromovinyl)-2'-deoxyuridine (BrVdUrd) and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BrVaraUra) were compared in a dThd kinase-deficient human fibroblast cell line, infected with parental strains of herpes simplex virus, and other strains expressing no viral dThd kinase activity. Metabolic experiments were performed at concentrations well above the ID50 for each compound because radiolabeled agents were not available. BrVaraUra and its nucleotides qualitatively displayed chromatographic and anabolic characteristics which closely paralleled those of BrVdUrd and its nucleotides. Monophosphorylation of both drugs was dependent upon the presence of viral dThd kinase activity except in the case of one dThd kinase-negative type 1 mutant (SC16R5C1) which retained BrVdUrd/BrVaraUra kinase activity. Intracellular uptake of either parent compound was absent during mock-infection and minimal in the cases of infection with mutants unable to phosphorylate the parent compound. Parental type 1 strains were able to induce diphosphorylation and triphosphorylation of both compounds to a similar, dose-dependent degree. Extracts of type 2-infected cells contained greater quantities of BrVdUrd and its monophosphate compared with BrVaraUra and its monophosphate, after identical drug exposure and infection conditions. As previously observed for BrVdUrd, diphosphorylated and triphosphorylated nucleotides of BrVaraUra were not detected after type 2 infection. BrVdUrd and BrVaraUra metabolic breakdown pathways differed, however, as evidenced by the formation of E-5-(2-bromovinyl)uracil (BrVUra). Unlike BrVdUrd, BrVaraUra formed no BrVUra in infected cells, suggesting that replacement of 2'-deoxyribose with arabinose makes the compound biologically more stable, presumably because of resistance to enzymatic breakdown by pyrimidine nucleoside phosphorylases. In this dThd kinase-negative cell line, BrVdUrd and BrVaraUra displayed qualitatively similar susceptibility profiles in that activities were type 1 selective and dThd kinase dependent. Antiviral activities against dThd kinase-positive type 1 strains were similar with both compounds. These data would suggest that BrVdUrd and BrVaraUra have identical type-specific dThd-dTMP kinase-dependent mechanisms of cellular uptake and phosphorylation, but that the latter is not subjected to phosphorolysis and resultant formation of an inactive metabolite. Furthermore, the absence of presence of phosphorolysis of the parent nucleoside does not apparently adversely affect in vitro antiviral activity.     

A selective human H(4)-receptor agonist: (-)-2-cyano-1-methyl-3-[(2R,5R)-5- [1H-imidazol-4(5)-yl]tetrahydrofuran-2-y] methylguanidine

A series of 16 compounds related to chiral 4(5)-(5-aminomethyltetrahydrofuran-2-yl)imidazoles (1) have been designed, synthesized, and examined in vitro by radioligand displacement studies and functional assays for both the human H(3)- and H(4)-receptors expressed in SK-N-MC cells. Among them, the (2S,5S)-isomer 1d of amino compounds showed approximately 300-fold higher selectivity at the H(3)-receptor than the H(4)-receptor. On the other hand, (2R,5S)- and (2R,5R)-cyanoguanidines 3b and 3c, in which the amino group of the compounds 1b and 1c was substituted by the cyanoguanidino moiety, bound to the H(4)-receptor with a pEC(50) value of 6.65 and 7.11, respectively, and had >40-fold selectivities over the H(3)-receptor. As such, 3b and 3c are the first selective H(4) receptor agonists.