Molecular-docking-guided design, palladium-catalyzed synthesis and anticancer activity of paclitaxel-benzoxazoles hybrids

A series of new paclitaxel-benzoxazoles hybrids were designed based on both the molecular docking mode of beta-tubulin with paclitaxel derivatives (7a and 7g), and the activity-structure relationship of C-13 side chain in paclitaxel. Palladium-catalyzed direct Csp2–H arylation of benzoxazoles with different aryl-bromides was used as the key synthetic strategy for the aryl-benzoxazoles moieties in the hybrids. Twenty-six newly synthesized hybrids were screened for their antiproliferative activity against human cancer cell lines such as human breast cancer cells (MDA-MB-231) and liver hepatocellular cells (HepG2) by the MTT assay and results were compared with paclitaxel. Interestingly, most hybrids (7a–7e, 7i, 7k, 7l, 7A, 7B, 7D and 7E) showed significantly active against both cell lines at concentration of 50 µM, which indicated that the hybrid strategy is effective to get structural simplified paclitaxel analogues with high anti-tumor activity.


Results and discussion
Molecular docking study. It was believed that the simplified paclitaxel analogues with better antitumor activities could accelerate the polymerization of tubulin and stabilize the resultant microtubules to apoptosis through cell-signaling cascade either. In our continued work on finding paclitaxel analogues with better antitumor activities, the docking studies of a series of paclitaxel-nitrogen containing heterocyclic moiety hybrids with beta-tubulin were carried out. To our delight, the paclitaxel-benzoxazoles hybrids 7a and 7g showed good binding affinity with beta-tubulin. -Cdocker Interaction Energy values of compounds 7a and 7g with beta-tubulin were 52.9245 and 54.6571 kcal/mol, respectively, along with -Cdocker Energy values of them were 38.4268 and 38.2471 kcal/mol, respectively.
An overlay of the structures for compounds 7a, 7g and positive control paclitaxel was shown in Fig. 2. These three molecules embedded in 6I2I cavity and the binding region of compounds 7a and 7g was consistent with that of paclitaxel. The oxygen atom of the ester carbonyl in paclitaxel established the hydrogen bond interaction
According to the desirable effect of above synthetics on the 2 cancer cell lines, the compounds with antiproliferative activity over 70% at 50 µM were tested for their half maximal inhibitory concentration (IC 50 ) (Fig. 5). The potential inhibitory activities expressed as IC 50 values for all compounds were shown in Tables 2 and 3. It is regrettable that the IC 50 value of all hybrids both against MDA-MB-231 and HepG2 were equally much higher than those of paclitaxel (IC 50 values of 0.32 ± 0.08 and 0.78 ± 0.09 μM, respectively). According to the results, it is supposed that the antiproliferative activities of synthesized hybrids had dose-dependent effect with concentration. Nonetheless, IC 50 values of compound 7A against MDA-MB-231 cells was 21.7 ± 0.8 µM, better than that of

Conclusion
In summary, twenty-six new paclitaxel-benzoxazoles hybrids bearing only a C-13 side chain of paclitaxel were designed on both the SAR of paclitaxel and the molecular docking study. The newly hybrids were rapidly prepared through five simple reactions. The key synthetic strategy for the aryl-benzoxazoles moieties in the hybrids was palladium-catalyzed direct Csp 2 -H arylation of benzoxazoles with different aryl-bromides. At the concentration of 50 µM, most compounds showed moderate to good antiproliferative activity against human breast cancer cells (MDA-MB-231) and liver hepatocellular cells (HepG2). Some of the compounds (7a-7e, 7i, 7k, 7l, 7A, 7B, 7D and 7E) at this concentration exhibited stronger activity against the two cell lines than paclitaxel. Unfortunately, the IC 50 value of potential inhibitor were higher than those of paclitaxel. It is supposed that synthesized hybrids exhibited antiproliferative activities in a dose-dependent manner. Among of them, the optimal compound 7a showed the best therapeutic potential to inhibit HepG2 cell growth with the IC 50 values of 17.6 ± 0.8 µM. Owing to the remarkable cytotoxicity, 7a needs an in-depth investigation to improve in terms of reducing dose-dependent effect, which might assist in the development of anticancer agents in the future. The results suggests that the use of baccatin-free hybrid component might be an effective strategy to establish paclitaxel-based hybrid library to find lead compounds against cancer.

Experimental
General experimental procedures. 1 H NMR spectra were recorded on a Bruker AV 400 or 600 nuclear magnetic resonance instrument (400 or 600 MHz). Chemical shifts were recorded in ppm relative to tetramethylsilane as the internal standard. 13 C NMR data were collected on a Bruker AV 400 or 600 nuclear magnetic resonance instrument (100 or 150 MHz) with complete proton decoupling. Chemical shifts are reported in ppm with tetramethylsilane as the internal standard. HRESIMS were determined using a Waters Acquity UPLC/Xevo G2-S QTof mass spectrometer. Thin-layer chromatography silica gel GF254 plates and silica gel G (200-400 mesh) for column chromatography were purchased from Qingdao Ocean Chemical Plant (Qingdao, People's Republic of China). Unless otherwise specified, the reagents and solvents used in this work were all commercially available analytical or chemical grades, and used directly without any purification.

Molecular docking study.
The crystallographic structures of tubulin (PDB ID: 6I2I) was chosen as the template for the modeling study of compounds. The pdb file about the crystal structure of refined 13pf Hela Cell tubulin microtubule (6I2I.pdb) was obtained from the Protein Date Bank at http:// www. rcsb. org/. The structures of compound were drawn by ChemBioDraw software and converted to mol file. The ligands and bound water were removed from the protein and the polar hydrogen was added. The energy minimized structures of tubulin (6I2I.pdb) and the ligand were generated by using prepare protein module and prepare ligand module, respectively. The whole tubulin complex was defined as a receptor. The molecular docking procedure was performed by using CDOCKER protocol for receptor-ligand interactions section of Discovery Studio Client software. Then, the lowest energy configuration of docking molecule was docked into 20 different binding sites of the prepared protein molecule using the standard parameters of Discovery Studio throughout the simulation. The preferred     Table 3. Antiproliferative activities of selected paclitaxel-benzoxazoles hybrids against HepG2.

Percentage of antiproliferation against HepG2
IC 50 (µM) 3.125 µM 6 7.52-7.27 (7H, m), 7.24-7.23 (4H, m), 7.11 (2H, t, J = 7.6 Hz), 6.85 (2H, d, J = 7.8 Hz), 6.80 6.87 (2H, d, J = 8. 6 Hz), 6.78 (1H, dd, J = 7.2, 1.6 Hz), 6.39 (1H, s), 5.42 (1H, s), 4.62 (1H, d, J = 4.2 Hz), 4.59-4.40 (4H, m), 3.94 (3H, s), 3.75 (3H, s), 1 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.