Design and Synthesis of 2-(1-Alkylaminoalkyl)pyrazolo[1,5-a]pyrimidines as New Respiratory Syncytial Virus Fusion Protein Inhibitors.

Respiratory syncytial virus (RSV) is one of the most common causes of lower respiratory tract infections and a significant pathogen for both adults and children. Although two drugs have been approved for the treatment of RSV infections, the low therapeutic index of these drugs have led pharmaceutical companies to develop safe and effective small-molecule anti-RSV drugs. The pyrazolo[1,5-a]pyrimidine series of compounds containing a piperidine ring at the 2-position of the pyrazolo[1,5-a]pyrimidine scaffold are known as candidate RSV fusion (F) protein inhibitor drugs, such as presatovir and P3. The piperidine ring has been revealed to facilitate the formation of an appropriate dihedral angle between the pyrazolo[1,5-a]pyrimidine scaffold and the plane of the amide bond for exertion of anti-RSV activity. A molecular-dynamic study on newly designed compounds with an acyclic chain instead of the piperidine ring proposed and demonstrated a new series of pyrazolo[1,5-a]pyrimidine derivatives, such as 9c with a 1-methyaminopropyl moiety, showing similar dihedral angle distributions to those in presatovir. Compound 9c exhibited potent anti-RSV activity with an EC50 value of below 1 nM, which was similar to that of presatovir. A subsequent optimization study on the benzene ring of 9c led to the potent RSV F protein inhibitor 14f with an EC50 value of 0.15 nM. The possibility of improving the biological properties of anti-RSV agents by modification at the 7-position of pyrazolo[1,5-a]pyrimidine is also discussed.

Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora.

A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene (gpd) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.