Asiatic acid also has an extra hydroxyl group at position 2. However the same substitution and this feature greatly improved the inhibitory activity. In fact, among the 15 commercial compounds tested, maslinic acid was the best hABHD12 inhibitor having an IC50 value of 1.3 mM. The oleanane series further confirmed our findings that dimethyl at position 4 in combination with a carboxyl group at position 17 were important features for hABHD12 inhibition. Finally, we tested four triterpenoids, 2-cyano-3,12dioxo-oleana-1,9-dien-28-oic acid, CDDO methyl ester, celastrol, and the established MAGL inhibitor pristimerin. All four derivatives failed to show any inhibition of hABHD12 and the findings with pristimerin are in agreement with those in the study by King et al. where pristimerin was tested against different endocannabinoid targets. Poor inhibitory PCI-32765 activity of triterpenoids 12�C15 allowed us to conclude that triterpene backbone was crucial for the hABHD12 inhibitor activity. As betulinic acid, ursolic acid and oleanolic acid had only minor differences in their inhibitory activities, neither the size of the ring E nor its substituents have a role in hABHD12 inhibition. In order to establish additional structural features that are critical for hABHD12 inhibition, we chose a series of previously reported derivatives of betulinic acid for further evaluation. Importance of the carboxyl group at position 17 was further verified by testing an aldehyde 16 which only weakly inhibited hABHD12 at 10 mM concentration. When comparing two similar aldehydes, the inhibition was enhanced to moderate level when hydroxyl substituent at position 3 was replaced to carbonyl, i.e. a plain hydrogen bond accepting group. An amide bond as well as an insertion of an ester or ether similarly decreased inhibitor activity. When carboxyl group was replaced with an oximino group, modest inhibitory activity was observed. Inhibitory activity of the oxime 24 was retained by replacing hydroxyl group at position 3 with another oximino group. When carboxyl group at position 17 was retained and an oximino group was added at position 3, decreased inhibitory activity was observed. However, it was interesting that compound 19 was able to fully inhibit the enzyme whereas maximum inhibition of the compound 24 was only 61%. The effect of the modifications on the ring A on hABHD12 inhibitor activity are presented in the Figures 3�C4 and Table S3. As shown in the case of maslinic acid, an additional hydroxyl group at the position 2 ABT-199 resulted in good inhibition. We synthesized the corresponding betulinic acid derivative 32 and observed that the activity of this compound was similar to that of the parent betulinic acid. Additional heterocyclic ring system attached to the ring A generally gave good inhibition. For example, when hydroxyl groups at positions 2 and 3 were protected as an acetonide, modest inhibitory activity was observed. Replacement of a ring A with a lactam ring resulted in modest inhibitory activity, however, the lactam ring also decreased selectivity, as compound 35 also inhibited MAGL. Introduction of a pyridine or a pyrazine ring or an indole ring revealed an important structural feature. The position of a nitrogen atom 
in the pyridine ring turned out to be important for the inhibitory activity as the compound 41 showed improved activity over the compound 40. Activity was further improved by replacing the pyridine ring with an indole ring or a pyrazole. In fact, compound 33 was the most potent compound of the entire series having an IC50 value of 0.9 mM. As evidenced by the total loss of the inhibitory activity in the case of the indole-fused allobetulin derivative 35, the carboxyl group at position 17 was still needed for inhibitory activity.