The syntheses and biological activities of a number of benzamide derivatives, designed from rolipram, which are selective inhibitors of cyclic AMP-specific phosphodiesterase (PDE IV), are described. The effects of changes to the alkoxy groups, amide linkage, and benzamide N-phenyl ring on the inhibition of the cytosolic PDE IV from pig aorta have been investigated. As a result, some highly potent and selective PDE IV inhibitors have been identified. The most potent compounds have been further evaluated for their inhibitory potencies against PDE IV obtained from and superoxide O2- generation from guinea pig eosinophils in vitro. Selected compounds have also been examined for their activities in inhibiting histamine-induced bronchospasm in anaesthetized guinea pigs. 3-(Cyclopentyloxy)-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide (15j) showed exceptional potency in all tests and may have therapeutic potential in the treatment of asthma.

We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte cyclic AMP‐specific phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2‐induced cyclic AMP accumulation and lipopolysaccharide (LPS)‐induced TNFα production and TNFα mRNA expression. PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP‐inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT‐PCR) of human monocyte poly (A+) mRNA revealed amplified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed. RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 ± 0.6 nM, n = 3). (±)‐Rolipram (IC50: 313 ± 6.7 nM, n = 3) was at least 200 fold less potent than RP 73401. R‐(−)−rolipram was approximately 3 fold more potent than S‐(+)‐rolipram against cytosolic PDE4. RP 73401 (IC50: 9.2 ± 2.1 nM, n = 6) was over 50 fold more potent than (±)‐rolipram (IC50: 503 ± 134 nM, n = 6)) in potentiating PGE2‐induced cyclic AMP accumulation. R‐(−)−rolipram (IC50: 289 ± 121 nM, n = 5) was 4.7 fold more potent than its S‐(+)‐enantiomer (IC50: 1356 ± 314 nM, n = 5). A strong and highly‐significant, linear correlation (r = 0.95, P < 0.01, n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte cyclic AMP accumulation. A poorer, though still significant, linear correlation (r = 0.67, P < 0.01, n = 13) was observed between the potencies of the same compounds in potentiating PGE2‐induced monocyte cyclic AMP accumulation and their abilities to displace [3H]‐rolipram binding to brain membranes. RP 73401 (IC50: 6.9 ± 3.3 nM, n = 5) was 71 fold more potent than (±)‐rolipram (IC50: 490 ± 260 nM, n = 4) in inhibiting LPS‐induced TNFα release from monocytes. R‐(−)−rolipram (IC50: 397 ± 178 nM, n = 3) was 5.2‐fold more potent than its S‐(+)‐ enantiomer (IC50: 2067 ± 659 nM, n = 3). As with cyclic AMP, accumulation a closer, linear correlation existed between the potency of structurally distinct compounds in suppressing TNFα with PDE4 inhibition (r = 0.93, P < 0.01, n = 13) than with displacement of [3H]‐rolipram binding (r = 0.65, P < 0.01, n = 13). RP 73401 (IC50: 2 nM) was 180 fold more potent than rolipram (IC50: 360 nM) in suppressing LPS (10 ng ml−1)‐induced TNFα mRNA. The results demonstrate that RP 73401 is a very potent inhibitor of TNFα release from human monocytes suggesting that it may have therapeutic potential in the many pathological conditions associated with over‐production of this pro‐inflammatory cytokine. Furthermore, PDE inhibitor actions on functional responses are better correlated with inhibition of PDE4 catalytic activity than displacement of [3H]‐rolipram from its high‐affinity binding site, suggesting that the native PDE4 in human monocytes ex...

1. We have investigated the inhibitory potency of RP 73401, a novel, highly selective and potent inhibitor of cyclic AMP-specific phosphodiesterase (PDE IV), against partially-purified PDE isoenzymes from smooth muscle and the particulate PDE IV from guinea-pig eosinophils. The inhibitory effects of RP 73401 on the generation of superoxide (.O2-), major basic protein (MBP) and eosinophil cationic protein (ECP) from guinea-pig eosinophils have also been studied. 2. RP 73401 potently inhibited partially-purified cyclic AMP-specific phosphodiesterase (PDE IV) from pig aortic smooth muscle (IC50 = 1.2 nM); it was similarly potent against the particulate PDE IV from guinea-pig peritoneal eosinophils (IC50 = 0.7 nM). It displayed at least a 19000 fold selectivity for PDE IV compared to its potencies against other PDE isoenzymes. Rolipram was approximately 2600 fold less potent than RP 73401 against pig aortic smooth muscle PDE IV (IC50 = 3162 nM) and about 250 times less potent against eosinophil PDE IV (IC50 = 186 nM). 3. Solubilization of the eosinophil particulate PDE IV increased the potency of rolipram 10 fold but did not markedly affect the potency of RP 73401. A similar (10 fold) increase in the PDE IV inhibitory potency of rolipram, but not RP 73401, was observed when eosinophil membranes were exposed to vanadate/glutathione complex (V/GSH). 4. Reverse transcription polymerase chain reaction (RT-PCR), using primer pairs designed against specific sequences in four distinct rat PDE IV subtype cDNA clones (PDE IVA-D), showed only mRNA for PDE IVD in guinea-pig eosinophils. PDE IVD was also the predominant subtype expressed in pig aortic smooth muscle cells. 5. RP 73401 (Kiapp = 0.4 nM) was 4 fold more potent than (+/-)-rolipram (Kiapp = 1.7 nM) in displacing[3H]-(+/-)-rolipram from guinea-pig brain membranes.6. In intact eosinophils, RP 73401 potentiated isoprenaline-induced cyclic AMP accumulation(EC50 = 79 nM). RP 73401 also inhibited leukotriene B4-induced generation of *02- (IC50 = 25 nM), and the release of major basic protein (ICo = 115 nM) and eosinophil cationic protein (IC50 = 7 nM). Rolipram was 3-14 times less potent than RP 73401.7. Thus RP 73401 is a very potent and selective PDE IV inhibitor which suppresses eosinophil function suggesting that it may be a useful agent for the treatment of inflammatory diseases such as asthma. The greatly different inhibitory potencies of rolipram against PDE IV from smooth muscle and eosinophils(in contrast to the invariable effects of RP 73401) are unlikely to be attributable to diverse PDE IV subtypes but suggest distinct interactions of the two inhibitors with the enzyme.

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