-Azido-2 0 -deoxy-2 0 -methylcytidine (14) is a potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase, displaying an EC50 value of 1.2 lM and showing moderate in vivo bioavailability in rat (F = 14%). Here we describe the synthesis and biological evaluation of 4 0 -azido-2 0 -deoxy-2 0 -methylcytidine and prodrug derivatives thereof.

4'-Azido-2'-deoxy-2'-methylcytidine (14) is a potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase, displaying an EC(50) value of 1.2 μM and showing moderate in vivo bioavailability in rat (F=14%). Here we describe the synthesis and biological evaluation of 4'-azido-2'-deoxy-2'-methylcytidine and prodrug derivatives thereof.

A set of low molecular weight compounds containing a hydroxyethylamine (HEA) core structure with different prime side alkyl substituted 4,5,6,7-tetrahydrobenzazoles and one 4,5,6,7-tetrahydropyridinoazole was synthesized. Striking differences were observed on potencies in the BACE-1 enzymatic and cellular assays depending on the nature of the heteroatoms in the bicyclic ring, from the low active compound 4 to inhibitor 6, displaying BACE-1 IC50 values of 44 nM (enzyme assay) and 65 nM (cell-based assay).

There is a growing interest in markers for cartilage degradation in synovial joints because of their potential diagnostic and prognostic value. Therefore, the aim of this study was to identify valuable degradation markers for temporomandibular joint (TMJ) osteoarthritis (OA) by comparing the relative concentrations of carboxyterminal telopeptides type I and II (CTX-I and II), cartilage oligomeric matrix protein (COMP), and prostaglandin E2 (PGE2) in the synovial fluid (SF) of TMJs with OA with those of healthy symptom-free TMJs.In this cross-sectional case-control study, participants were recruited from the University Medical Center Groningen (Groningen, the Netherlands). Cases were defined as patients with TMJ OA, and control patients had symptom-free TMJs. The outcome variables were the relative concentrations of CTX-I, CTX-II, COMP, and PGE2 in osteoarthritic TMJ SF compared with symptom-free joints. An independent-samples Mann-Whitney U test was used to compare the relative concentrations.Thirty cases (9 male, 21 female; mean age, 40.1 yr; standard deviation, 15.3 yr) and 10 controls (5 male, 5 female; mean age, 30.3 yr; standard deviation, 10.8 yr) were studied. No significant differences in relative concentrations of CTX-I (P = .548), CTX-II (P = .842), COMP (P = .140), and PGE2 (P = .450) were found between the groups. Unexpected low relative concentrations of CTX-I and high relative concentrations of CTX-II were observed.Assumed changes in the SF concentration of CTX-I, CTX-II, COMP, and PGE2 in TMJ OA seem to occur proportionally. Furthermore, the unexpected large contribution of CTX-II suggests that this marker may be useful to quantify cartilage degradation in TMJ OA.

Novel NS3/4A protease inhibitors comprising quinazoline derivatives as P2 substituent were synthesized. High potency inhibitors displaying advantageous PK properties have been obtained through the optimization of quinazoline P2 substituents in three series exhibiting macrocyclic P2 cyclopentane dicarboxylic acid and P2 proline urea motifs. For the quinazoline moiety it was found that 8-methyl substitution in the P2 cyclopentane dicarboxylic acid series improved on the metabolic stability in human liver microsomes. By comparison, the proline urea series displayed advantageous Caco-2 permeability over the cyclopentane series. Pharmacokinetic properties in vivo were assessed in rat on selected compounds, where excellent exposure and liver-to-plasma ratios were demonstrated for a member of the 14-membered quinazoline substituted P2 proline urea series.

A novel series of P3-truncated macrocyclic HCV NS3/4A protease inhibitors containing a P2 proline-urea or carbamate scaffold was synthesized. Very potent inhibitors were obtained through the optimization of the macrocycle size, urea and proline substitution, and bioisosteric replacement of the P1 carboxylic acid moiety. Variation of the lipophilicity by introduction of small lipophilic substituents resulted in improved PK profiles, ultimately leading to compound 13Bh, an extremely potent (K(i)=0.1 nM, EC(50)=4.5 nM) and selective (CC(50) (Huh-7 cells)>50 microM) inhibitor, displaying an excellent PK profile in rats characterized by an oral bioavailability of 54% and a high liver exposure after oral administration.

C-CAM is a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family. Two co-expressed isoforms, C-CAM-L and C-CAM-S, are known, having different cytoplasmic domains both of which can be phosphorylated in vivo. Here we have characterized the PKC-mediated phosphorylation of the short cytoplasmic domain isoform, C-CAM-S. Phorbol myristyl acetate induced phosphorylation of C-CAM-S in transfected CHO cells. Using synthetic peptides and Edman degradation we identified Ser449 as the PKC-phosphorylated amino acid residue. Binding experiments with modified peptides indicated that this phosphorylation decreases the ability of the cytoplasmic domain of C-CAM-S to bind calmodulin.

The cell adhesion molecule C-CAM belongs to the immunoglobulin superfamily and is expressed in epithelia, vessel endothelia, and hematopoietic cells. Differential splicing gives rise to different isoforms, of which the major two are C-CAM1 and C-CAM2, which both have four Ig-like domains in their extracellular portions, but differ in their cytoplasmic domains. Two different allelic variants of C-CAM, namedaandb,occur in the rat. The adhesive binding mechanism(s) of C-CAM is not known in detail. Evidence for both homophilic and heterophilic binding has been presented, and different species and splice variants of C-CAM have shown differences in temperature and cation dependence when expressed in different cell types. Here, we have analyzed the binding mechanism of rat C-CAM2athat was expressed in CHO cells. In this system C-CAM2a-mediated adhesion was calcium- and temperature-independent. C-CAM2a-transfected cells did not adhere to nontransfected cells, demonstrating that the binding was homophilic. Cells transfected with C-CAM2ain which the N-terminal Ig-domain (D1) was deleted did not aggregate, and cells with intact C-CAM2acould not bind to these cells. This was in contrast to cells that were transfected with C-CAM2ain which the fourth Ig-like domain (D4) had been deleted; they both aggregated and bound to cells with intact C-CAM2a.Thus, C-CAM2amediates intercellular adhesion of CHO cells by a homophilic mechanism, in which the D1 domain binds reciprocally to a D1 domain on an opposed C-CAM molecule.

C-CAM is a Ca2+-independent rat cell adhesion molecule belonging to the CEA gene family of the immunoglobulin superfamily. Two major isoforms that differ in the length of their cytoplasmic domains exist. In previous studies it has been reported that only the long isoform (C-CAM1) but not the short isoform (C-CAM2) can mediate adhesion. However, in the mouse, isoforms with both long and short cytoplasmic domains have been reported to have adhesive activity. In order to analyze this apparent conflict we transfected C-CAM1 or C-CAM2 into CHO Pro5 cells and examined their adhesive phenotype in an aggregation assay. We found that in this cellular system both C-CAM1 and C-CAM2 could mediate cell-cell adhesion in a Ca2+-independent and temperature-independent way. The results suggest that the cellular environment is important for the activity of C-CAM isoforms.