The optimization of the lead compound was initiated and
The optimization of the lead compound () was initiated and the initial key SAR and the results of the structural modifications of lead compound are summarized in . Replacement of the carboxylic Derquantel receptor moiety with other functional groups led to loss of functional activity against EP4 receptor. Although the corresponding tetrazole exhibited slightly increased functional activity, the tetrazole analog was a substrate for efflux pumps and a strong inhibitor of CYP3A4. Shifting the carboxylic acid moiety from - to position showed loss of intrinsic activity. The benzoic acid moiety was not replaceable by nicotinic acid, cyclohexanecarboxylic acid, or 4-thiazolecarboxylic acid. Moreover, modifications of the amide moiety by exchanging the nitrogen with methylene, reduction of the amide carbonyl, or -methylation of the nitrogen also reduced intrinsic activity. These early SAR studies of the -(aminomethyl)benzoic acid moiety of lead compound showed that the carboxylic acid group at the -position was a key element for the EP4 antagonist activity. A limited class of substituents on the phenoxypyridine moiety of lead compound , maintained or enhanced the potencies when satisfied regiochemical requirements. The introduction of a substituent to the 4-position of pyridine ring caused loss of intrinsic activity. Additionally, it was found that the aliphaticoxy groups were not suitable surrogates for the phenoxy group on the 2-position of the pyridine of . Even though the aliphaticoxy substituted analogs retained intrinsic activity, their metabolic stabilities in HLM were decreased. In order to develop detailed SAR around lead compound for improving its profile, structural modifications were performed in three portions of lead compound : the benzylic position of aminomethylbenzoic acid moiety for Portion A, the central ring core with substituents for Portion B, and the substitution pattern of the phenoxy group for Portion C (). Furthermore, this strategy allowed us to easily construct the molecule in a concerted manner for effective SAR studies. We aimed to improve not only pharmacological properties, but also physiochemical properties such as MW<450 and cLogD<3, although the compounds with a MW over 450 and a cLogD over 3 were prepared for exploring SAR involving the selective EP4 antagonist. Our optimization studies on lead compound started by exploring portions A and B. The synthetic route to the target compounds from easily available materials is outlined in . Starting from -halo-substituted aromatic acids or , the corresponding methyl esters were formed, which were then converted to -phenoxy-substituted aromatic acids or with nucleophilic aromatic substitution (SNAr) reaction followed by alkali hydrolysis. Depending on the aromatic ring (A=CH or N) of starting materials or , the phenol was reacted under different conditions. The SNAr reaction of the benzoates derived from benzoic acids (A=CH) required more severe reaction conditions than that of the nicotinates derived from nicotinic acids (A=N). The target benzoic acids and were prepared by reaction of carboxylic acids or with -(methoxycarbonyl)benzylamine using EDCI/HOBt condensation conditions and subsequent hydrolysis of the methyl ester or -butyl ester intermediates or thereby obtained. The human EP4 binding and functional activities of representative compounds are presented in . Switching the aromatic ring in portion B from pyridine to benzene was tolerated ( vs ). Replacement of 5-fluoro substituent on the pyridine ring moiety of lead compound with 5-chloro substituent showed about 2-fold increase in EP4 binding and functional activity ( vs ). The 5-chlorobenzene analogs were almost equipotent to the corresponding 5-chloropyridine analogs ( vs ) although it is concerned that benzene has an evidently high cLogD value by 0.6 units relative to pyridine (3.21 vs 2.60). Substitution of hydrophilic functionality such as carboxamides, sulfones and sulfonamides on the benzene ring was also attempted, however none of them exhibited sufficient EP4 antagonistic activity at all (data not shown). 5-Methylpyridine and 6-methylpyridine analogs ( and ) displayed substantially decreased EP4 functional activity. Incorporation of a methyl group at the benzylic position in portion A led to a marked improvement in EP4 binding and functional activity in a stereochemical dependent manner (, , and ). The ()-methyl isomers were dramatically more active than the corresponding antipodes ( vs ). The geminal methyl groups or ethyl group at the same position decreased the binding affinity ( and ). Key compounds exhibiting EP4 antagonist activities were selected and tested to assess selectivity against the other EP receptor subtypes. These compounds did not show any significant binding to EP1 and EP3 at the highest concentration tested (20μM). Although some compounds were found to have weak binding affinity to EP2, most of compounds ( and ) showed high selectivity to EP4, including compounds with candidate credentials. In the Caco-2 assay, compound and exhibited sufficiently high permeability.