ABSTRACT
A series of novel 3,4-diarylpyrazolines was synthesized and evaluated in cannabinoid (hCB(1) and hCB(2)) receptor assays. The 3,4-diarylpyrazolines elicited potent in vitro CB(1) antagonistic activities and in general exhibited high CB(1) vs CB(2) receptor subtype selectivities. Some key representatives showed potent pharmacological in vivo activities after oral dosing in both a CB agonist-induced blood pressure model and a CB agonist-induced hypothermia model. Chiral separation of racemic 67, followed by crystallization and an X-ray diffraction study, elucidated the absolute configuration of the eutomer 80 (SLV319) at its C(4) position as 4S. Bioanalytical studies revealed a high CNS-plasma ratio for the development candidate 80. Molecular modeling studies showed a relatively close three-dimensional structural overlap between 80 and the known CB(1) receptor antagonist rimonabant (SR141716A). Further analysis of the X-ray diffraction data of 80 revealed the presence of an intramolecular hydrogen bond that was confirmed by computational methods. Computational models and X-ray diffraction data indicated a different intramolecular hydrogen bonding pattern in the in vivo inactive compound 6. In addition, X-ray diffraction studies of 6 revealed a tighter intermolecular packing than 80, which also may contribute to its poorer absorption in vivo. Replacement of the amidine -NH(2) moiety with a -NHCH(3) group proved to be the key change for gaining oral biovailability in this series of compounds leading to the identification of 80.
Subject(s)
Pyrazoles/chemical synthesis , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Sulfonamides/chemical synthesis , Administration, Oral , Animals , Arachidonic Acid/metabolism , Binding, Competitive , Biological Availability , CHO Cells , Cricetinae , Crystallography, X-Ray , Fever/chemically induced , Fever/physiopathology , Humans , Hypotension/chemically induced , Hypotension/physiopathology , Male , Mice , Models, Molecular , Molecular Conformation , Pyrazoles/chemistry , Pyrazoles/pharmacology , Radioligand Assay , Rats , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
Successful application of antisense oligonucleotides (ODNs) in cell biology and therapy will depend on the ease of design, efficiency of (intra)cellular delivery, ODN stability, and target specificity. Equally essential is a detailed understanding of the mechanism of antisense action. To address these issues, we employed phosphorothioate ODNs directed against specific regions of the mRNA of the serotonin 5HT1A receptor, governed by sequence and structure. We demonstrate that rather than various intracellular factors, the gene sequence per se primarily determines the antisense effect, since 5HT1a autoreceptors expressed in RN46A cells, postsynaptic receptors expressed in SN48 cells, and receptors overexpressed in LLP-K1 cells are all efficiently downregulated following ODN delivery via a cationic lipid delivery system. The data also reveal that the delivery system as such is a relevant parameter in ODN delivery. Antisense ODNs bound extensively to the RNA matrix in the cell nuclei, thereby interacting with target mRNA and causing its subsequent degradation. Antisense delivery effectively diminished the mRNA pool, thus resulting in downregulation of newly synthesized 5HT1A proteins, without the appearance of truncated protein fragments. In conjunction with the selected mRNA target sequences of the ODNs, the latter data indicated that effective degradation rather than a steric blockage of the mRNA impedes protein expression. The specificity of the antisense approach, as described in this study, is reflected by the effective functional downregulation of the 5-HT1A receptor.
