Design, Synthesis And Biological Evaluation Of Novel N-(1,3-dioxoisoindolin-5-yl) Benzamides As Chemical Uncoupler

Mitochondria as the center of the cell metabolic and signal transduction networks exhibit various physiological functions. The mitochondria research has become one of the hotpots for new drug research and development of drugs targeting. The chemical uncoupler as direct regulator of mitochondrial function may provide new opportunities for the treatment of diabetes and other metabolic diseases and anticancer drugs found. However, the structure-activity relationship (SAR) between the chemical uncoupler and mitochondrial membrane potential (MMP) has rarely been reported until recently.Based on high-throughput screening (HTS), the Hit compound LGH00277 was gained as mitochondrial uncoupler. In this paper, the compound AUK1001 was synthesized as the novel scaffold of N-(1,3-dioxoisoindolin) benzamides according to the principle of isosteric. In order to further research the SAR of this uncoupler on MMP, the modification and optimization of the scaffold, preliminary mechanism research and function tests were carried out.On the basis of the structural modification of AUK1001, about 170 novel compounds were designed and synthesized, and the structures of the compounds were characterized by NMR, HRMS spectra. To confirmed the structure of the series of compounds, AUK1057 was selected for the single-crystal X-ray diffraction analysis.MMP tests on L6 myotube were set as the preliminary evaluation method for uncoupling activity. The modification showed that the substituent changes on the ring A significantly affect the activity of reducing MMP. The change on the electric effect of substituent group, the volume size, and substituted position of ring A regulated compounds to reduce MMP. The substitution with electron-withdrawing group improved the activity of compounds reducing MMP, and the strong electron-donating group and the block group significantly weaken the ability of reducing MMP; The m-chlorine and p-methyl substituted compounds showed strong activity on the reduction of MMP, while other position of chloro-substituted or methyl-substituted compounds showed low activity; The presence of the hydroxy-substituted compounds substantially increase the ability of reducing MMP, a low concentration of o-hydroxy-substituted compound showed good activity of reducing MMP and dose-dependent; Instead of benzene, thiophene also showed good activity of reducing MMP.In the discussion of SAR in Area D, the length of N-alkyl chain affected the activity of the compounds on reducing MMP. With N-alkyl chain length of pentyl or hexyl, the compunds showed the maximum decrease of the MMP. The decease of MMP was mild for the compunds with N-butyl or N-dodecyl chain. However, the activity showed no difference regardless of the length of N-alkyl chain for the inactive compounds on MMP. Hence, Area D may help provide the lipophilic property and as the assitance efficiency group. In addition, the introduction of a branched carboxyl group on N-alkyl chain may modulate the hydrophilic lipophilic balance, and have the structural characteristic of amino acids.Preliminary structural modification of Area B showed that the compounds with sulfonamide group, instead of amide, connecting regions A and C weakened the ability of decrease MMP, but if combined the modification of regions A and D, some good results may be obtained with obvious decreasing MMP which indicated that the sulfonamide group is useful for further modification.Structural modification of Area C has not got satisfied results till now, it needs further work.On the basis of the activity evaluation and preliminary SAR analysis on N-(1,3-dioxoisoindolin)benzamides analogues, part of the compounds with good activity have been selected for the futher tests.In the experiment of glucose consumption (GC) in L6 myotube of rats, a series of compounds can significantly effect glucose consumption, such as AUK1001, AUK2029 which are much better than the classic uncoupler DNP, and even better than the positive control, berberine.The further oxygen consumption (OC) tests not only confirmed that the compounds can effect the respiratory chain, but also further clarified the fact that MMP decreasing function of the analogues of N-(1,3-dioxoisoindolin) benzamides was uncoupling mechanism. The results also showed that the compounds with high GC effect and low membrane potential could highly promote oxygen consumption under the same concentration.121 compounds were selected for antitumor test on MGC-803 cell. The results showed that 22 compounds have inhibitory activity to some extent, and the best compound with IC50 2.06μM.The cytotoxic evaluation of all the compounds were testes in the L6 myoblast cells by the SRB analysis method.Basis on the above works, some novel chemical uncouplers and their SAR for regulating MMP have been explored, uncoupler mechanism has also been clarified. Futhermore, GC tests in L6 myotube and the growth inhibition tests on MGC-803 tumor cell have been prelimilarily studied which provides some information for the further research.