Selective inhibitors of cyclic nucleotide phosphodiesterase-5 (PDE5) have been used as drugs for treatment of male erectile dysfunction and pulmonary hypertension. An insight into the pharmacophores of PDE5 inhibitors is essential for development of second generation of PDE5 inhibitors, but has not been completely illustrated. Here we report the synthesis of a new class of the sildenafil derivatives and a crystal structure of the PDE5 catalytic domain in complex with 5-(2-ethoxy-5-(sulfamoyl)-3-thienyl)-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one (12). Inhibitor 12 induces conformational change of the H-loop (residues 660-683), which is different from any of the known PDE5 structures. The pyrazolopyrimidinone groups of 12 and sildenafil are well superimposed, but their sulfonamide groups show a positional difference of as much as 1.5A. The structure-activity analysis suggests that a small hydrophobic pocket and the H-loop of PDE5 are important for the inhibitor affinity, in addition to two common elements for binding of almost all the PDE inhibitors: the stack against the phenylalanine and the hydrogen bond with the invariant glutamine. However, the PDE5-12 structure does not provide a full explanation to affinity changes of the inhibitors. Thus alternatives such as conformational change of the M-loop are open and further structural study is required.