We designed and synthesized a series of water-soluble prodrugs of the HIV-1 protease inhibitor KNI-727 (1), which is a sparingly water-soluble drug with a water solubility of 5.5 mug/mL. These prodrugs, which contain a water-soluble auxiliary with two tandem-linked units, i.e., a self-cleavable spacer and a solubilizing moiety with an ionized amino function, exhibited a marked increase in water solubility (>10(4)-fold) compared with the parent drug 1. The mechanism of conversion to the parent drug 1 is not enzymatic but through a chemical cleavage at the spacer via an intramolecular cyclization-elimination reaction through an imide formation under physiological conditions. To diversify the conversion time for the parent drug regeneration, chemical modification of the auxiliary was carried out focusing on the introduction of cyclic tertiary amines, which can modify the basicity and/or conformational flexibility of the terminal amino function at the solubilizing moiety, and the change in bond length, which can attenuate the five-membered ring intermediate formation in the cleavage. These newly synthesized water-soluble prodrugs exhibited a practical water solubility with values greater than 50 mg/mL and enabled the constant regeneration of the parent drug 1 with diversified conversion times ranging from 4 min to 34 h as t(1/2) values under physiological conditions. All the water-soluble prodrugs tested regenerated the parent drug 1 in vivo as well as in vitro. A clear increase in the gastrointestinal absorption was observed in prodrugs 8, 12, and 13 with bioavailability (BA) values of 23%, 26%, and 29%, respectively. These BA values were 1.5-1.9-fold higher than that in the administration of the parent drug 1 alone. Other prodrugs showed only a similar or decreased BA compared to the parent drug 1. From these results, we found that not only a high water solubility but also an appropriate conversion time of the prodrug with a relatively narrow limit of around 35 min via intraduodenal administration was necessary for significant improvement of the gastrointestinal absorption in water-soluble prodrugs based on the spontaneous chemical cleavage. This is the first successful water-soluble prodrug that suggests an increased BA value greater than the parent drug in HIV-1 protease inhibitors and is the first study to show the importance of optimal conversion time in water-soluble prodrugs. Consequently, a water-soluble strategy that can control the conversion time would be extensively applicable to improve the gastrointestinal absorption of sparingly water-soluble drugs. The present information is an intriguing discovery and is one of the key factors that will contribute to the future design of practical water-soluble prodrugs.