Discovery of (7R)-14-cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl) amino}-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic acid (MK-3281), a potent and orally bioavailable finger-loop inhibitor of the hepatitis C virus NS5B polymerase.

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery efforts. Here, we report on the further development of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33 (MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in viremia in vivo using the chimeric mouse model of HCV infection.

Cationic peptides that increase the thermal stabilities of 2'-O-MeRNA/RNA duplexes but do not affect DNA/DNA melting.

Several different cationic nonapeptides have been synthesized and investigated with respect to how they can influence the thermal melting of 2'-O-methylRNA/RNA and DNA/DNA duplexes. Each peptide has a C-terminal L-phenylalanine unit and is otherwise uniformly composed of a sequence of a specific basic D-amino acid that in most cases will be largely charged at neutral pH. These N-terminal octamer stretches are composed variously of the amino acids D-lysine, D-diaminobutyric acid (D-Dab), D-diaminopropionic acid (D-Dap), or D-histidine. None of the peptides substantially affected the thermal melting of DNA/DNA duplexes, which was in sharp contrast with their effects on 2'-O-methylRNA/RNA duplexes. In particular, the peptides based on diaminopropionic and diaminobutyric acid units had strong positive effects on the melting temperatures of the 2'-O-methylRNA duplexes (up to 16 °C higher with 1 equivalent of peptide) at pH 7, whereas at pH 6 the effect was even more drastic (ΔT(m) up to +25 °C). The shorter R groups of the Dap and Dab groups appear to have a better length than lysine for enhancement of the thermal melting of the 2'-O-methylRNA/RNA duplex, an effect that is more pronounced at lower pH but substantial even at pH 7, although the Dap derivative is not likely to be fully protonated. The dramatic difference between the influence, or lack thereof, on the 2'-O-methylRNA/RNA and the DNA/DNA thermal meltings suggest that, although electrostatic interactions probably play a role, there is another major and structurally dependent component influencing the properties of the duplexes. This is also seen in the observation that the oligo-Dap and oligo-Dab peptides give greater melting point enhancements than both the lysine peptide (with a longer side chain) and a ß-linked Dap peptide with a shorter side chain and a longer backbone.

A method for solid-phase synthesis of oligonucleotide 5'-peptide-conjugates using acid-labile alpha-amino protections.

We describe the development of a solid-phase technique for the synthesis of 5'-peptide-oligonucleotide conjugates (POCs) with a uniform protection strategy for the nucleic acid and the peptide fragments. On the alpha-amino function, the amino acid building blocks were protected with the 2-(biphenyl-4-yl)propan-2-yloxycarbonyl (Bpoc) group. This protection is removed during the stepwise peptide elongation by the same acidic conditions used for removal of the dimethoxytrityl (DMT) group used in the oligonucleotide assembly (3% trichloroacetic acid, 2 min). The 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz) group was also tested. With this somewhat more stable group, a prolonged contact with the acid (at least 16 min) was required for accomplishing complete alpha-amino deprotection, which resulted in some degree of depurination of the acid-sensitive DNA chain. Base-labile acyl protections were adopted for the side-chains of histidine, lysine, and the nucleobase amino functions. These were all removed in the final deblocking step by ammonolysis. This uniform protection scheme for the peptide and the oligonucleotide enabled the total stepwise synthesis of model conjugates in the 3' --> N direction with high efficiency and purity.

Facile determination of the protecting group location of Nim-protected histidine derivatives by 1H-15N heteronuclear correlation NMR.

The positioning of the imidazole protecting group of several histidine derivatives was determined by means of (1)H-(15)N heteronuclear multiple-bond correlation NMR experiments. The cross-peak originated from the three-bond correlation between the histidine side-chain H(beta) and the imidazole N(pi) was used for the identification of the N(pi) signal in the (15)N spectrum. Therefore, based on the fact that the signal of the substituted imidazole nitrogen appears always at lower chemical shift (delta) than the unsubstituted one, the position of the blocking group could easily be inferred. The obtained data confirmed previous findings that were accomplished with other less generally applicable spectroscopic or crystallographic techniques.