Finally, poor inhibitory activity of the isoxazole 38 allowed us to conclude that a functional group with a hydrogen bond donor or acceptor at carbon 3 was crucial for ABHD12 inhibitory activity. In summary, the above-described SAR studies allowed us to identify four key determinants for hABHD12 inhibition potency and efficacy. These key features played an important role in building a pharmacophore model of ABHD12 that is described later in this chapter. Shape complementarity of the triterpene skeleton accompanied with four axial methyl substituents likely play an important role in inhibitor binding. Additional double bonds within the skeleton affect the overall planar shape of triterpene scaffold, leading to total lack of inhibitory activity. Carboxyl group at position 17 in triterpene core structure is of crucial importance, as basically any modification at this position reduced, or fully eliminated inhibitory activity. It is known from previous studies that the carboxyl group at this position is crucial also in many other biological Nutlin-3 structure targets. Small, hydrophobic substituents at the position 4 are required, 
as asiatic acid and hederagenin did not inhibit hABHD12. As summarized in Figure S3, hydrogen bond donor or acceptor attached to position 3 was a key feature required for high inhibitory potency. This was further illustrated by pyridine and pyrazine derivatives 40�C42. GDC-0941 compounds 41 and 42 have a nitrogen at this position and show moderate inhibitory activity. In contrast, no atom capable of hydrogen bonding is present in the compound 40, causing dramatic decrease of inhibitory activity. A good pair of compounds for comparison was 37 and 38. The pyrazole derivative 37 was one of the most potent compounds in the series. On the other hand, when an aromatic nitrogen was replaced by an oxygen, a complete loss of the inhibitory activity was observed. This was due to the fact that oxygen within an aromatic ring cannot form hydrogen bond. The most promising synthetic compounds in the series were compounds 33 and 34 which both have an indole ring attached to ring A, and thus, a nitrogen atom at this crucial hydrogen bonding position. The compound 34 has an electron donating methoxy group at the indole ring which may cause tighter interaction between indole nitrogen and amino acid residues of the enzyme. In addition, good inhibitory activity of this methoxy derivative also implies that there is additional space for bulkier substituents in this direction. Strength of a hydrogen bond may also explain why these compounds were equally potent in inhibiting hABHD12 activity but their maximal inhibition was significantly different. Similar trend in efficacy was observed with the compounds 41 and 42, i.e. additional nitrogen in pyrazine decreased efficacy. Collectively, the above data demonstrate the importance of a hydrogen bond donor at position 3. However, as betulinic acid that can act both as a hydrogen bond acceptor and a donor, and compound 18 that is a hydrogen bond acceptor, both showed good inhibitory activity, we were able to conclude that both hydrogen bond donor and acceptor are tolerated at this position. The key finding that both hydrogen bond donor and acceptor at position 3 are able to form good interactions with the enzyme led us to hypothesize that these interactions might involve a serine residue, possibly the catalytic serine of ABHD12, previously identified by site-directed mutagenesis. However, due to the reversible nature of triterpenoid inhibition, this hypothesis could not be experimentally tested. In conclusion, we report the discovery of the first phytocompounds and their synthetic analogues that inhibit human and mouse ABHD12. The studied compounds belong to the class of triterpenoids that are known to possess wide-ranging therapeutic effects.