Synthesis of a focused library (FL) is an efficient method to develop novel compounds regulating functions of specific enzymes. Compounds in a FL are composed of a common core structure with different building blocks. Herein, our design and synthesis of FLs based on selective inhibitors of dual-specificity protein phosphatases (DSPs) is summarized. A first generation FL having an acidic core structure extracted from a natural product, RK-682, does not contain a highly selective inhibitor for DSPs, and showed very weak activity at the cellular level, possibly due to poor cell membrane permeability. Upon building the second FL, the property of the core structure was modified from acidic to neutral. Construction of a second-generation FL (RE derivatives) having the enamine derivative of 3-acyltetronic acid as the core structure resulted in dramatic improvement of cell membrane permeability and inhibitory selectivity. As a result, VHR-selective RE12 and CDC25A/B-selective RE44 were discovered. Replacement of the side chain in RE12 afforded RE176, which showed more potent anti-proliferative activity against HeLa cells. Core structure modification from acidic to neutral also changed the mode of action of inhibitors. RE derivatives showed a non-competitive inhibition profile and interacts with a pocket adjacent to the active site of CDC25s.