Preparation of the pyridyl ether
Preparation of the 2-pyridyl ether analogs (–) was accomplished by alkylation of alcohol with 2-bromopyridines in the presence of NaH to afford the desired pyridyl ether analogs in low to moderate yield. Preparation of the 3-pyridyl analog was carried out by conversion of alcohol to the tosylate, followed by displacement with 3-hydroxypyridine in the presence of NaH to afford . As previously reported, it was found that there was little difference in the potency of enantiomers at the chiral center in the R substituent in this pyrazinone chemotype; hence, it was decided to synthesize only -enantiomers for this investigation. Preparation of carbamate analogs containing a difluoromethoxypyridyl group in place of the methoxypyridyl group was carried out in a similar fashion whereby methyl ether was treated with BBr followed by alkylation with an isocyanate in the presence of sodium hydride to furnish – (). The CRF receptor binding affinities for the described compounds are shown in . Compounds were tested in a CRF receptor binding titration assay using rat frontal pkc inhibitor homogenate, in which inhibition of specific binding of [I]Tyr-ovine CRF by our test compounds was measured to determine their receptor binding affinities, as measured by the IC. The SAR of the five ester containing analogs showed that the phenyl substituted ester was similar in potency to methyl ether and slightly more potent than methyl ester . Replacement of the phenyl group with a 2-pyridyl group () resulted in a nearly 50-fold reduction in potency relative to , while replacement of the phenyl group with either a 3-pyridyl () or 4-pyridyl () group was better tolerated and resulted in only a 3–4-fold decrease in potency. Analysis of the SAR for the alkyl and aryl carbamate analogs showed that aryl carbamates (–) were similar in potency to the methyl ether and phenyl ester and were slightly more potent than the alkyl carbamate analogs (–), although it was found that increasing the length of the alkyl group resulted in a trend to improved binding affinity. Small substituents on the phenyl ring of the aryl carbamates did not have a significant effect on the binding affinity (–). Similar to the ester containing compounds, the 3-pyridyl analog was found to be less potent (7-fold) than the corresponding phenyl carbamate . The SAR of a series of aryl ether analogs was analyzed and compared with the methyl ether . A decrease in potency of approximately 60-fold was observed when the methoxy group in compound was replaced with a hydroxyl group (compound ). In contrast to the ester containing analog , 2-pyridyl ether was similar in potency to the methyl ether analog and was 6-fold more potent than the corresponding 3-pyridyl analog (). Incorporation of either a 4-bromo or 5-trifluoromethyl substituent on the pyridyl ring resulted in no significant change in potency (compare and versus ), whereas incorporation of a cyano substituent at either the 4- or 5-position on the pyridyl ring resulted in a 6-fold decrease in potency (compare and versus ). It was previously reported that the potency and the metabolic stability in the pyrazinone series of CRF antagonists can be improved by replacing the lower methoxypyridyl group with a difluoromethoxypyridyl group (compound ). Two carbamate analogs were synthesized with the lower difluoromethoxypyridyl group (–). Consistent with previous findings, introduction of the difluoromethoxypyridyl substituent resulted in an improvement in potency compared to the corresponding methoxypyridyl analogs (compare with and with ). Incubation of , , , and () with human and rat liver microsomes showed that compounds with the lower difluoromethoxypyridyl group had improved metabolic stability in both human and rat liver microsomes compared to the corresponding analogs with the lower methoxypyridyl group. The metabolic stability for the phenyl carbamate () and the 3-pyridyl carbamate () analogs were each improved over their counterparts ( and , respectively).