LB Agar Miller australia As illustrated in among all synthes

As illustrated in , among all synthesized compounds, biphenyl derivatives , , , , and with substitutions at the 3-position possessed the best FAAH inhibitory capacity. These 5 compounds demonstrated a good activity against FAAH with IC values in a submicromolar range. Nevertheless, when replacing the phenyl ring appended to the isoxazole by a pyridinyl ring (–), a decrease of activity was observed. Indeed, these compounds manifested less than 25% FAAH inhibition. Moreover, compounds with a pyridinyl group (), a quinolinyl group () or a furanyl group () on the phenyl ring manifested slight inhibition activity against FAAH from 17% to 31% inhibition. When the substituent on the terminal phenyl ring is bearing at the 2-position (, 47% inhibition), a slight decrease of inhibitory activity was observed in comparison with the corresponding 3-substituted derivative (58% inhibition). The FAAH inhibitory capacity of the molecules was almost completely lost when the substituent is bearing at the 4-position. For instance, compounds , , , and were determined to exert less than 25% inhibition of FAAH. Additionally, biphenyl derivatives bearing a carbamoyl substituent either at 3-position (17) or at 4-position (18) were determined to show no obvious activity towards FAAH (<15%). Furthermore, the introduction of an additional methoxy group at the 4-position of LB Agar Miller australia led to a sharp decrease of inhibitory capacity from 58% to 14% (). Surprisingly, the introduction of a third methoxy group at the 5-position of the terminal phenyl ring of compound brought about an obvious recovery of inhibitory potency to 42% (). Moreover, the replacement of the methylene function appended to the terminal phenyl ring of compounds (69% inhibition) and (63% inhibition) by a carbonyl function led to a substantial attenuation of the inhibitory activity to 49% () and 44% (), respectively. Overall, a phenyl group is more favorable than other aromatic groups (such as pyridinyl, quinolinyl, and furanyl groups) for the design of FAAH inhibitors. Substituents at position 3 are more favorable than substituents at position 4. Specifically, acetyl, morpholinomethyl, cyano, methoxy, and 4-methyl-1-piperazinylmethyl substituents are preferred rather than a carbamoyl group. Notably, compound (IC=0.59μM) possessed similar FAAH inhibitory capacity in comparison with compound (IC=0.46μM), a previously identified potent FAAH inhibitor in our group. The replacement of the acetyl group of compound by a cyano group () brought about an impressive improvement of inhibitory potency against FAAH. Compound is approximately 2 times more potent against FAAH than compounds and . This compound manifests a moderate efficacy (maximum effect that a drug can produce, 64% inhibition at 10μM concentrations) but a good potency (amount required to produce a given effect, IC=0.24μM). It shows a similar inhibitory potency against FAAH than the positive control URB597 (IC=0.26μM), one of the most potent FAAH inhibitors to date. Cytotoxicity of these compounds was determined at 10μM using a cell proliferation assay on human colorectal adenocarcinoma cells HT29 and human embryonic kidney cells HEK293. This test is based on a colorimetric method, which measures the activity of cellular enzymes that reduce the tetrazolium dye (MTS, uncolored) to its insoluble formazan giving a purple color. This assay measures cellular metabolic activity via NADPH-dependent cellular oxidoreductase enzymes and reflects, under defined conditions, the number of viable cells. No cytotoxicity was observed for our new compounds on HT29 cells except for compounds with a piperazinyl moiety (, , , and ). These 4 compounds inhibited also proliferation of HEK293 cells. An investigation of the lipophilicity–activity relationships of compounds with substituents at the 3-position on the terminal phenyl ring was performed. Lipophilicity is an important property in drug discovery and the prediction of bioavailability. The logarithm of the octanol/water partition coefficient (Log) is widely used to assess the lipophilicity of molecules. In terms of different calculation rules, Log values can be calculated by atomic methods (such as Log and Log), which consider the contribution of each atom; fragmental methods (such as Log, ACD/Log and KowWIN), which consider the contribution of each small fragment in a molecule; or property-based methods (such as Log), which is based on the three-dimensional (3D) structures of the molecules and topological approaches. Actually, Log was identified to display more accurate prediction of Log in a wide range of 108 compounds, in comparison with ACD/Log and KowWIN, Hence, Log values of our compounds were calculated by Log method (). As represented in , our 5 most potent FAAH inhibitors (, , , , and ) possessed high Log values ranging from 5.5 to 6.6. Conversely, compounds with a low FAAH inhibitory capacity (, , , , and ) possessed Log values below 5.5. This result indicates that compounds with substituents at the 3-position of the terminal phenyl ring conferring higher Log values (such as acetyl, methoxy, and cyano group) showed better FAAH inhibition potency. Hence, the low inhibitory capacity of compound (Log=4.80) with a carbamoyl group might be attributed to its weak lipophilicity. The replacement of the methylene linker of compounds (Log=6.04) and (Log=6.60) with a carbonyl linker (, Log=4.92, , Log=5.48) and the replacement of the phenyl ring of compound (Log=5.73) by a pyridine ring (, Log=4.58) led to a substantial attenuation of the FAAH inhibitory activity probably because of the decrease of lipophilicity.