Supplementary MaterialsTable_1. of 1 1.301 M. In HCC cells, compound 50 suppressed cell proliferation in a MARK4-dependent manner. Moreover, compound 50 could sensitize the anticancer Brazilin function of paclitaxel against HCC cells, providing a new therapeutic approach for HCC and enlarging the potential application of paclitaxel in cancer treatment. Dialogue and Outcomes The catalytic site of Tag4 identifies its substrate MAP4, leading to the phosphorylation of MAP4 to improve microtubule dynamics, can be a key theme for Tag4 function (Trinczek et al., 2004). Lately, the crystal Brazilin framework of Tag4 in complicated using its inhibitor (PDB Identification: 5ES1) have already been disclosed (Shape 1A), facilating the finding of small-molecule Tag4 inhibitors (Sack et al., 2016). Therefore, we planned to determine the molecular docking model predicated on this crystal framework, and carry out computer-aided virtual testing of TargetMol and self-built substance collection via Lipinski’s filtering and Yellow metal molecular docking in Finding Studio room v3.1 software program; The hit substances will become synthesized and posted to natural evaluation to acquire promising lead substances (Shape 1B). Open up in another window Shape 1 Computer-aided digital screening of potential MARK4 inhibitors. (A) Crystal structure of MARK4 catalytic domain in complex with pyrazolopyrimidine inhibitor (PDB ID: 5ES1). (B) Workflow for computer-aided screening of MARK4 inhibitor. (C) Selected hit compound with coumarin moiety after Lipinski’s filtering and GOLD molecular docking. (D) Biological evaluation of hit coumarin via kinase assay (left) using MARK4 as enzyme and cell viability assay (right) in HepG2 cells. Small molecules after Lipinski’s filtering in a library containing 5,972 compounds were screened through GOLD molecular docking. Higher GoldScore.Fitness value implies higher potential affinity between protein and small molecules. Among the hit compounds, 3-arylcoumarin 6,8-dichloro-3-(3-methoxyphenyl)-2H-chromen-2-one had favorable drug-likeness and GOLDScore (Figure 1C). This coumarin was then submitted to kinase assay and cell viability assay to evaluate its biological activity. The results suggested 6,8-dichloro-3-(3-methoxyphenyl)-2H-chromen-2-one inhibited MARK4 activity with an IC50 value of 7.804 M and suppressed the Brazilin cell viability of HepG2 cells with an IC50 value of 15.92 M (Figure 1D). Thus, we speculated that coumarin derivatives were favorable to inhibit MARK4 function. To verify this speculation, a series of coumarin derivatives, including 3-acry-, 3-aryl- 4-alkyl-, or 4-aryl coumarins and 3-arylthiocoumarins, were designed and synthesized in vision with structural and electronic features. Starting from substituted salicylaldehydes, coumarins 1C10 were successfully prepared via the Perkin reaction (Scheme 1). 3-Arylcoumarins 11C46 were synthesized from salicylaldehyde derivatives and phenylacetic acid derivatives through the Perkin condensation followed by acid-promoted hydrolysis if necessary, which were described in our previous work (Scheme 1) (Pu et al., 2014a). Moreover, salicylaldehydes and reactive methylene compounds were utilized as substrates in the presence of L-proline via the Knoevenagel reaction (Karade et al., 2008), 3-acrycoumarin 47C54 were afforded with high yields (Scheme 2). Open in a separate window Scheme 1 Synthesis of coumarins 1C46 via Perkin reaction. Open in a separate window Scheme 2 Synthesis of coumarins 47C54 via Knoevenagel reaction. To prepare 4-methyl or 4-phenyl coumarins, we adapted Pechmann reaction-based strategy (Smitha and Sanjeeva Reddy, 2004). By using phenol derivatives and reactive methylene compounds as substrates, zircomiun tetrachloride as the mediator, compound 55C57 were synthesized with acceptable yields (Scheme 3). 56 and 57 were subsequently transformed into 58C61 via alkylation (Scheme 3). Similarly, 4-benzyloxy- or 4-methoxylcoumarins (63, 64) were obtained Cd86 from commercially-available compound 62 through benzylation and methylation, respectively (Scheme 4). In addition, following a two-step strategy (Meth-Cohn and Tarnowski, 1978), we also synthesized thiocoumarin 65C68 with moderate overall yields (Scheme 5). Together, through multiple synthetic strategies, sixty-eight coumarin derivatives were prepared as candidates for the investigation of potential MARK4 Brazilin inhibitors. Open in a separate window Scheme 3 Synthesis of coumarins 55C61 based on the Pechmann reaction. Open in a separate window Scheme 4 Preparation of 63 and 64 Brazilin from alkylation of 62. Open in a separate window Structure 5 Synthesis of thiocoumarin 65C68. With.