Design, Synthesis And Selection Of Benzamides As Inhibitors Of Human Cyclic Nucleotide Phosphodiesterase 4B2(hPDE4B2)

Human cyclic nucleotide phosphodiesterase 4(hPDE4) specificly hydrolyzed cyclic nucleotide phosphodiester, and its inhibitors have pharmacological activities of anti-inflammatory and anti-depression. Rolipram was first discovered as PDE4 inhibitor, but only used as a research reagent due to its serious side effects. Rolipram analogues contain skelecton of 3-(cyclopentyloxy)-4-methoxy-benzamide without chirality. Such compounds are easy to synthesize. Optimizing the amino moieties and 3, 4-substituents of 3-(cyclopentyloxy)-4-methoxy-benzamide are the clues to design new PDE4 inhibitors.First,based on inhibition potencies of some isovanillic amides at nanomole levels on pig aorta phosphodiesterase-4, some isovanillic amides of aromatic amines were selected and synthesized by alkylation, oxidation and amidation. Oxidation was optimized by using the hydrogen peroxide and metal catalysts to increase the rate and yield; Isovanillic acid was directly applied for amidation of aromatic amines by the dehydration under HBTU rather than DCC, to increase the reaction rate and yield and simplify purification process. The structures of candidate inhibitiors determined by 1H-NMR and the purity was higher than 88% by HPLC.Full-length human cyclic nucleotide phosphodiesterase 4B2(h PDE4B2) with N-terminal His-SUMO tag was recombinant expressed from Escherichia coli and purified by Ni2+-NTA. The inhibition potencies of these compounds for hPDE4B2 were determined as IC50 and thus Ki, by malachite green assay of inorganic phosphate released by calf intestinal alkaline phosphatase. Compareison with the results for pig aorta PDE4 enzyme as target enzyme found different activity structure relationships of these compounds. Among them, the inhibition constants of isovanillic amide of aniline is nanomolar.Accordingly, new isovanillic amides with amine moieties bearing small and flexible rings were designed and synthesized. Screen with hPDE4B2 gave Ki varing from micromoles to sub-micromoles that were less potent than phenyl isovanillic amide.The high-throughput-screen(HTS) of inhibitors against PDE4 by malachite green assay of inorganic phosphate required the PDE4 bearing specific activity higher than 8 U/g, but challenge in the fusion expression of the full-length human cyclic nucleotide phosphodiesterase 4B2(SF-hPDE4B2) promotes the artificial truncate(152~528 residue, ST-hPDE4B2) as the alternative target for HTS of inhibitors, if the hits can be reliably recognized by the two tagerts. After induced expression and affinity purification, ST-hPDE4B2 showed about 30-fold higher apparent specific activity and 100-fold higher activity yield than SF-hPDE4B2, but both preparations contained many 6His-tagged smaller polypeptides detected with anti-6His antibody. Of some inhibitors, Ki to SF-hPDE4B2 were consistently smaller than those to ST-hPDE4B2, but a dual-logarithm model quantitatively described their monotonic and close association. From Ki against ST-hPDE4B2, Ki from 0.010 to 8.5 μM against SF-hPDE4B2 was predicted by the dual-logarithm model with deviations below 2-fold. Therefore, based on spectrophotometric assay of PDE activity and the prediction with the dual-logarithm model, ST-hPDE4B2 was a suitable alternative target for HTS of hPDE4B2 inhibitors.