br Results and discussion A
Results and discussion A diverse set of compounds were synthesized to improve the PK and physicochemical properties of 2. Compounds' agonist effects on hGPR40 were assessed by calcium mobilization assay using hGPR40-HEK293 cell line which stably expressed human GPR40. Preliminary explorations focused on the linker of 2 (Table 1). Lilly reported that the methylene between the phenyl and the piperidyl group was metabolically susceptible to N-dealkylation, so we replaced the methylene with a carbonyl moiety to block the metabolic site [23,48] (Fig. 2). In addition, the carbonyl replacement increased tPSA and reduced LogP, which would be expected to reduce the CNS penetration. Direct replacement with the carbonyl in the linker gave 13a. 13a was less potent but possessed lower LogP (5.9) and higher tPSA (67) than 2. Based on the structure of another compound from Lilly, LY2922083, we produced 13b with a thiophene ring in the linker. 13b displayed comparable activity to that of 13a. Further liver microsomal stability and Caco-2 permeability assays (Table 2) showed that, 13a and 13b retained the permeability of 2. Introduction of the carbonyl group gave a slight increase in metabolic stability in mouse liver microsomes. As 13b was more preferred for better metabolic stability on both human and mouse liver microsomes than 2 and 13a, 5-carbonylthiophene moiety was chosen as linker for the subsequent compounds. In the optimization of both the stability and activity, we modified the indene ring in the tail to improve stability because the nonaromatic double bond of indene could be susceptible to oxidative metabolism in liver microsomes . First, we opened the five-member ring of indene in two ways (Fig. 2) and introduced a nitrogen dopamine-2 receptor antagonist to the piperidyl group, affording 13c and 13d with 4-phenylpiperazine and 4-benzylpiperazine as the tail, respectively. 4-Phenylpiperazine (13c) was more favorable and displayed a 2.2-fold increase in potency than 13d. As expected, 13c revealed promoted stability in human and mouse liver microsomes. Furthermore, conversion of the benzene ring into a methyl of cyclohexyl group gave 13e and 13f respectively. These two compounds had either markedly decreased potency or complete loss of activity, suggesting that the aromatic ring was critical for maintaining the agonistic activity. Also, we replaced the benzene ring with pyridyl rings (13g, 13h, 13i), giving lower LogP and higher tPSA. Alteration of the benzene ring into 2-pyridyl (13i) was well tolerated, while 3-pyridyl (13h) and 4-pyridyl (13g) replacement resulted in more than 16-fold and 70-fold loss in potency compared with 13c respectively, demonstrating that moving the nitrogen atom to different positions greatly impacted the activity. In view of these outcomes, 13c was selected for further exploration. The SAR of 13c was then investigated according to three parts of the molecule, the 4-phenylpiperazine tail, the central linker, and the β-propynyl substituted 3-benzenepropanoic acid head. We first investigated the influence of various substituents para to the phenyl of the tail considering that the para-position was potentially the major site of oxidative metabolism for the phenylpiperazine moiety  (Table 3). Among these substituents, 13k with a tert-butyl group showed the greatest loss of potency (5.8-fold), while the smaller methyl group (13j) had a 3-fold decrease in potency only. This indicated that a bulky substituent at the para position of the phenyl group was deleterious to activity. Neither an electron-donating methoxy group (13l) nor a strong electron-withdrawing nitro group (13m) was beneficial for activity. A comparison of 13l and 13m with 13k suggested that the size of R2 contributed to the major loss of potency. Similar outcomes were observed with 13n and 13o, that the compound with the bulkier chlorine group (13n) was less potent than that with the smaller fluorine group (13o), and that 13o had higher potency than 13c. These SAR findings led to our speculation that small substituents at the para position of the phenyl would be favorable.