In order to discover novel

In order to discover novel small-molecule compounds with the activity of GLUT4 translocation, we conducted a cell-based phenotype screening in L6-GLUT4-myc myoblasts by measuring appearance of GLUT4 on the plasma membrane (GLUT4 translocation activity). Evaluation of total 183,400 compounds gave several series of compounds, which were further validated with functional glucose uptake assays using the L6 cells and isolated mouse skeletal muscle. Consequently, we could successfully find compound , which can activate GLUT4 translocation and also uptake in 2DG dose-dependently. As far as we know, there are only a few reports with showing the functional small molecules activate GLUT4 translocation, e.g. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a potential AMP-dependent protein kinase (AMPK) activator. Furthermore, those compounds are deficient and do not have enough potentials for the use in clinical practice. Encouraged by these results, we carried out derivatization of hit compound by dividing the scaffold into three parts (A-, B-, and C-part) as shown in . Test compounds in and were synthesized from commercially available dichloropyridazine compounds () as shown in . The installation of the lower aniline part (C-part) toward dichloro pyridazine was implemented by the treatment of aniline in the alcoholic solvent to give in good yield. The Suzuki-Miyaura cross-coupling reaction of with the appropriate boronic Proteinase K reagents by the presence of a Pd catalyst afforded compound , and then the ester moiety was converted to amide by saponification with NaOH and subsequent amination in the presence of a coupling reagent. The synthetic route of heteroaryl pyridazine compounds in was illustrated in . Starting from asymmetric dichloropyridazine compounds , the two regio isomers ( and ) that were produced could be separated with column chromatography and each isomer ( or ) was converted to the test compound ( or ) along with the similar route in . Although the route in successfully yielded the test compounds shown in , , this procedure included tangled steps for the transformation of C-part. To improve the above method, we tried to prepare chloropyridazine compound bearing the upper benzene part (A-part), and the synthetic route was presented in . Commercially available compound was converted to triflate with TfO and -PrNEt in CHCl. Treatment of 2-methoxybenzaldehyde () with TMSCN and EtN in THF followed by exposure to in the presence of KHMDS afforded in 74% in two steps. Cyclization of with hydrazine in EtOH afforded in 79%. Compound was chlorinated by the treatment of POCl to give the key intermediate in 72%. Using this intermediate , test compounds could be easily obtained by the direct installation of various anilines in a similar manner in . Thienopyridazine compound in was synthesized in the following manner (). Lithiation of thiophene with -BuLi followed by exposure to 2-methoxybenzene Weinreb-amide afforded in good yield. Cyclization and chlorination in the same manner as the cyclopentyl compound gave compound . Transformation of the methoxy moiety to another ether was implemented by demethylation and alkylation, where demethylation of with BBr in CHCl afforded phenol , and the subsequent appropriate alkylation in the presence of NaH or CsCO in DMF gave compound in good yield. Finally test compound was obtained by the previous described methods. To obtain SAR information, A- and B-part of hit compound were explored first. shows the results of the introduction onto the benzene ring of A-part. All the activities of the tested compounds (-) were calculated by specific values against the non-substituted compound at 30 μM. In general, the introduction of a substituent at the 2-position on the benzene ring of A-part was found to be more favorable than at other positions. That is, the mothoxy moiety at the 2-position onto the upper benzene ring exhibited better results ( vs. , ), and the methyl group reflected the same trend ( vs. ). As for the 2-position, the methoxy moiety was superior to simple alkyl moieties, such as methyl and ethyl ( vs. , ), suggesting that the interaction between oxygen atom of A-part and nitrogen atom of B-part plays somehow important role regarding the GLUT4 activities.