Table 4.

Inhibition of human sEH by urea derivatives substituted with oxyoxalamide function

No.	R	R1	R2	R3	Human sEH IC50 a (nM)	Solubilityb (μM)
27c					16	20
28		H	H	CH3	6.6	312
29		H	H		19	39
30		H	CH3	CH3	5.0	312
31		CH3	CH3	CH3	1.2	625
32		H	H		19	78
AUDAd					3.2	63
IK950d					14	625

^aTest compounds prepared in DMSO was reacted with human sEH (1 nM) for 10 min in 25 mM Bis–Tris/HCl buffer (202 μL; pH 7.0) at 30 °C. The fluorescent substrate (CMNPC; [S] = 5 μM) was then introduced to the incubation mixture. Inhibition potency against the human sEH was determined by measuring the appearance of the 6-methoxy-2-naphthaldehyde with an excitation wavelength of 330 nm and an emission wavelength of 465 nm for 10 min on a fluorometer. Results are averages of three separate measurements. See the Supplementary data for the detailed procedures.

^bWater solubility was determined by adding a variety of concentrations of a test compound prepared in DMSO to 0.1 M sodium phosphate buffer (pH 7.4) in a final ratio of 5:95 (v/v). The turbidity of the water solution was measured at 650 nm to determine solubility in water. Results are the average of triplicate determinations.

^cUrea inhibitor with no substitution by oxyoxalamide function, which was synthesized by the reaction of 1-adamantyl isocyanate with benzyl amine in DMF in 100% yield.¹³

^dPotent urea-based inhibitors.^13,14 IK950 with improved water solubility was developed based on the structure of AUDA. IK950 was used as a control compound for the measurement of water solubility in this study.