Design, Synthesis, and Antitumor Activity of Olmutinib Derivatives Containing Acrylamide Moiety

Two series of olmutinib derivatives containing an acrylamide moiety were designed and synthesized, and their IC50 values against cancer cell lines (A549, H1975, NCI-H460, LO2, and MCF-7) were evaluated. Most of the compounds exhibited moderate cytotoxic activity against the five cancer cell lines. The most promising compound, H10, showed not only excellent activity against EGFR kinase but also positive biological activity against PI3K kinase. The structure–activity relationship (SAR) suggested that the introduction of dimethylamine scaffolds with smaller spatial structures was more favorable for antitumor activity. Additionally, the substitution of different acrylamide side chains had different effects on the activity of compounds. Generally, compounds H7 and H10 were confirmed as promising antitumor agents.


Introduction
Recent studies have shown that the mutation rate and overexpression rate of epidermal growth factor receptor (EGFR; a transmembrane protein) of patients with non-small-cell lung cancer (NSCLC) are as high as 75% in vivo [1,2]. EGFR is associated with various growth processes of cancer, including proliferation, differentiation, migration, apoptosis, and angiogenesis [3]. Therefore, EGFR has garnered considerable attention as an antitumor drug target, giving rise to numerous investigations of EGFR kinase inhibitors to disrupt and inhibit the proliferation and growth of tumor cells [4]. The first-generation EGFR inhibitor gefitinib (1) (Figure 1) and the second-generation EGFR inhibitor afatinib (2) have good inhibitory effects on the EGFR L858R mutation and the EGFR T790M mutation, respectively [5][6][7]. However, the first-and second-generation inhibitors have poor kinase selectivity between the EGFR T790M mutant and the wild type. Moreover, their clinical efficacy is limited. The third-generation EGFR inhibitor olmutinib [8,9] (3) was developed by Hanmei Pharmaceutical Company, and is an irreversible inhibitor for the treatment of patients with locally advanced or EGFR T790M -mutant NSCLC ( Figure 2) [10]. The IC 50 values of 3 against HCC827 (EGFR dell19 ), H1975 (EGFR L858R/T790M ), and A549 (EGFR WT ) cells were 9.2 nM, 10 nM, and 225 nM, respectively. This indicated that 3 had strong selectivity for the EGFR mutation. However, 3 had toxic side effects such as palmoplantar keratoderma and diarrhea. Therefore, the goal of this study to obtain new olmutinib derivatives which can overcome the side effects of skeleton migration.
To guide our modification, a molecular docking simulation of 3 and EGFR protein was performed. The thienopyrimidine structure of 3 was inserted into the larger hydrophobic band of the protein and could form hydrogen bonds with MET-793 in the hinged region, as shown in Figure 2A,B. Therefore, we transformed the thiophene pyrimidine structure into thiophene and pyrimidine to explore the antiproliferative activity of the target compounds when fully occupying the protein cavity. At the same time, inspired by WZ4002

Chemistry
According to the structure-based drug design (SBDD) strategy, we designed and synthesized two series of olmutinib derivatives as EGFR inhibitors containing an acrylamide moiety. The synthetic routes of target compounds H1-H16 are outlined in Schemes 1 and 2.  To design of the second series of target compounds (Figure 4), we retained the thiophene and pyrimidine core and introduced the triazine structure of phosphatidylinositol-3-kinase (PI3K) inhibitor ZSTK474 (5) while at the same time, the structures of thiophene and pyrimidine were transformed into triazine and pyrimidine [12,13]. The introduction of the 1,3,5-triazine ring increased the polarity of the molecule to better form key hydrogen bonds with MET-793 [14]. The oxygen of the morpholine ring of GDC-0941 (6) can form hydrogen bonds with the VAL-851 of the PI3K hinge region. Moreover, the introduction of the morpholine ring enhanced the mTOR-inhibitory activity of the compound; thus, the morpholine ring group was preserved [15][16][17]. Michael receptors were introduced in the solvent region to explore the interaction with the surrounding amino acids. Based on this design strategy, we completed the synthesis of the second series of compounds. We expected to obtain an ideal inhibitor with a better EGFR-inhibitory activity and to explore whether the target compound has an inhibitory effect on PI3K following the modifications described above.
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Chemistry
According to the structure-based drug design (SBDD) strategy, we designed and synthesized two series of olmutinib derivatives as EGFR inhibitors containing an acrylamide moiety. The synthetic routes of target compounds H1-H16 are outlined in Schemes 1 and 2.

Chemistry
According to the structure-based drug design (SBDD) strategy, we designed and synthesized two series of olmutinib derivatives as EGFR inhibitors containing an acrylamide moiety. The synthetic routes of target compounds H1-H16 are outlined in Schemes 1 and 2.
As shown in Scheme 1, we used commercially available 2,4,6-trichloropyrimidine (7) and thiophen-2-ylboronic acid to obtain 11a-11b through cyclization, chlorination, and nucleophilic substitution reactions. Compounds 11a-11b reacted with different amide side chains to give the target compounds H1-H8. As shown in Scheme 2, we used 1,3,5triazine as the starting material to obtain 18a-18b with different amines via six steps of substitution, reduction, and chlorination. Finally, 18a-18b reacted with different amide side chains to give the target compounds H9-H16. The structural information of target compounds was confirmed by 1 H-NMR, 13 C-NMR, and TOF MS (ES+), the results of which were consistent with the structures depicted.

Biological Discussion
Four human tumor cell lines (A549, H1975, NCI-H460, and MCF-7) and human normal cell line LO2 were selected to evaluate the antiproliferative activity of all target compounds in vitro. Olmutinib was used as a positive control. Results are summarized in Table 1, where the values are the average of at least three independent experiments. Compared with the lead compound olmutinib, most target compounds were less toxic to the normal cell line LO2, which indicates that the target compounds had a selective inhibitory effect on cancer cells. After the introduction of electron-withdrawing cyanide, the antiproliferative activities of compounds H1-H6 that were substituted with cyano groups showed moderate inhibitory activity against all the cell lines. However, the antiproliferative activities of the compounds that were substituted with the anisidine side chain performed better than the cyano group chain. This indicates that the introduction of the electron-withdrawing units could not increase the antiproliferative activity of the compounds. Obviously, compound H7 showed the greatest inhibitory activities against A549 and H1975 cancer cell lines, with IC 50 values of 4.37 ± 0.50 µM and 4.59 ± 0.46 µM, respectively, which were similar to the reference compound of olmutinib. Table 1 shows that the introduction of the 1,3,5-triazine ring and morpholine ring significantly enhanced the antiproliferation activity of the compounds. At the same time, we found that the antiproliferative activities of the compounds that were substituted with dimethylamine groups, were better than those that were substituted with diethylamine groups. Therefore, we speculate that the inner cavity area of the hydrophobic region is limited and only can accommodate molecules with smaller structures. From the docking results ( Figure 5B), it was found that the dimethylamine group of compound H10 penetrated into the interior of the protein (4zau) and completely occupied the space, which suggests that the introduction of a larger group than dimethylamine will not increase the activity of these compounds. The optimal compound H10 showed excellent antiproliferative activity against A549 and MCF-7 cancer cell lines, with IC 50 values of 3.36 ± 1.59 µM and 13.05 ± 1.36 µM, respectively, which were superior to the drug of reference. The selectivity of compound H10 to A549 cells was 29.76 times than to LO2 cells, and about 5 times that of the lead compound olmutinib.  Table 1 shows that the introduction of the 1,3,5-triazine ring and morpholine ring significantly enhanced the antiproliferation activity of the compounds. At the same time, we found that the antiproliferative activities of the compounds that were substituted with dimethylamine groups, were better than those that were substituted with diethylamine groups. Therefore, we speculate that the inner cavity area of the hydrophobic region is limited and only can accommodate molecules with smaller structures. From the docking results ( Figure 5B), it was found that the dimethylamine group of compound H10 penetrated into the interior of the protein (4zau) and completely occupied the space, which suggests that the introduction of a larger group than dimethylamine will not increase the activity of these compounds. The optimal compound H10 showed excellent antiproliferative activity against A549 and MCF-7 cancer cell lines, with IC50 values of 3.36 ± 1.59 μM and 13.05 ± 1.36 μM, respectively, which were superior to the drug of reference. The selectivity of compound H10 to A549 cells was 29.76 times than to LO2 cells, and about 5 times that of the lead compound olmutinib.  Table 1 shows that the introduction of the 1,3,5-triazine ring and morpholine ring significantly enhanced the antiproliferation activity of the compounds. At the same time, we found that the antiproliferative activities of the compounds that were substituted with dimethylamine groups, were better than those that were substituted with diethylamine groups. Therefore, we speculate that the inner cavity area of the hydrophobic region is limited and only can accommodate molecules with smaller structures. From the docking results ( Figure 5B), it was found that the dimethylamine group of compound H10 penetrated into the interior of the protein (4zau) and completely occupied the space, which suggests that the introduction of a larger group than dimethylamine will not increase the activity of these compounds. The optimal compound H10 showed excellent antiproliferative activity against A549 and MCF-7 cancer cell lines, with IC50 values of 3.36 ± 1.59 μM and 13.05 ± 1.36 μM, respectively, which were superior to the drug of reference. The selectivity of compound H10 to A549 cells was 29.76 times than to LO2 cells, and about 5 times that of the lead compound olmutinib.  Table 1 shows that the introduction of the 1,3,5-triazine ring and morpholine ring significantly enhanced the antiproliferation activity of the compounds. At the same time we found that the antiproliferative activities of the compounds that were substituted with dimethylamine groups, were better than those that were substituted with diethylamine groups. Therefore, we speculate that the inner cavity area of the hydrophobic region is limited and only can accommodate molecules with smaller structures. From the docking results ( Figure 5B), it was found that the dimethylamine group of compound H10 pene trated into the interior of the protein (4zau) and completely occupied the space, which suggests that the introduction of a larger group than dimethylamine will not increase the activity of these compounds. The optimal compound H10 showed excellent antiprolifera tive activity against A549 and MCF-7 cancer cell lines, with IC50 values of 3.36 ± 1.59 μM and 13.05 ± 1.36 μM, respectively, which were superior to the drug of reference. The se lectivity of compound H10 to A549 cells was 29.76 times than to LO2 cells, and about 5 times that of the lead compound olmutinib.  Table 1 shows that the introduction of the 1,3,5-triazine ring and morpholine ring significantly enhanced the antiproliferation activity of the compounds. At the same time, we found that the antiproliferative activities of the compounds that were substituted with dimethylamine groups, were better than those that were substituted with diethylamine groups. Therefore, we speculate that the inner cavity area of the hydrophobic region is limited and only can accommodate molecules with smaller structures. From the docking results ( Figure 5B), it was found that the dimethylamine group of compound H10 penetrated into the interior of the protein (4zau) and completely occupied the space, which suggests that the introduction of a larger group than dimethylamine will not increase the activity of these compounds. The optimal compound H10 showed excellent antiproliferative activity against A549 and MCF-7 cancer cell lines, with IC50 values of 3.36 ± 1.59 μM and 13.05 ± 1.36 μM, respectively, which were superior to the drug of reference. The selectivity of compound H10 to A549 cells was 29.76 times than to LO2 cells, and about 5 times that of the lead compound olmutinib.  Table 1 shows that the introduction of the 1,3,5-triazine ring and morpholine ring significantly enhanced the antiproliferation activity of the compounds. At the same time we found that the antiproliferative activities of the compounds that were substituted with dimethylamine groups, were better than those that were substituted with diethylamine groups. Therefore, we speculate that the inner cavity area of the hydrophobic region is limited and only can accommodate molecules with smaller structures. From the docking results ( Figure 5B), it was found that the dimethylamine group of compound H10 pene trated into the interior of the protein (4zau) and completely occupied the space, which suggests that the introduction of a larger group than dimethylamine will not increase the activity of these compounds. The optimal compound H10 showed excellent antiprolifera tive activity against A549 and MCF-7 cancer cell lines, with IC50 values of 3.36 ± 1.59 μM and 13.05 ± 1.36 μM, respectively, which were superior to the drug of reference. The se lectivity of compound H10 to A549 cells was 29.76 times than to LO2 cells, and about 5 times that of the lead compound olmutinib. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib. These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib. These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib. These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib. These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib. These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib. These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K. Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K.

General Information
Unless otherwise stated, all reagents used in the experiment were purchased at commercial analytical grade and used directly without further purification. Common solvents Compounds H7 and H10 had excellent antiproliferative activity and were further screened out for kinase inhibition testing. We evaluated compounds H7 and H10 with EGFR T790M/L858R kinases and PI3Kα kinases. As shown in Table 2, compounds H7 and H10 exhibited potent inhibition against EGFR T790M/L858R kinase. In particular, compound H10 showed better inhibitory activity against PI3Kα kinase than the control drug olmutinib. These data demonstrate that compound H10 is expected to be a dual inhibitor of EGFR and PI3K.

Molecular Docking Study
To explore the binding mode of the target compound (H10) with the active site of EGFR, molecular docking simulation was carried out using AutoDock 4.2 software. The docking tutorials and detailed explanations of the AutoDock basic methods we used can be found at the following address: http://autodock.scripps.edu/faqshelp/tutorial (accessed on 25 March 2021). According to the analysis results of the cells and kinases, we chose compound H10 as the example ligand; the structures of EGFR WT (PDB CODE: 4zau) and EGFR T790M (PDB CODE: 3ika) were selected as docking models. The combination of compound H10 with the EGFR T790M and PI3Kγ molecular active sites is shown in Figure 5. When compound H10 docked with the 3ika (Figure 5A), we observed that the dimethylamine group extended into the ATP hydrophobic pocket and formed hydrogen bonds with residues LYS-745, and the amino group formed hydrogen bonds with MET-793 residues. Figure 5C shows that the morpholine rings of compound H10 formed hydrogen bonds with the VAL-882 residues in 3L08. Among these, the acrylamide side chain of compound H10 formed hydrogen bonds with the LYS-833 residue in 3L08. This was in line with the combination model we predicted before. The abovementioned SAR (structure-activity relationship) analysis and molecular docking results indicate that compound H10 could be a potentially interesting anticancer agent.

General Information
Unless otherwise stated, all reagents used in the experiment were purchased at commercial analytical grade and used directly without further purification. Common solvents (ethanol, methanol, petroleum ether, ethyl acetate, dichloromethane 1,2-dimethoxyethane, etc.) were absolutely anhydrous. All reactions were monitored on a GF254 thin-layer chromatography plate (Laishan Penghan Plastic Industry Store, Yantai, Shandong, China), and spots were visualized at 254 nanometers or 365 nanometers with iodine or light. The target compound (20 mg) and 3.5 mL DMSO formed the corresponding compound solution, and the structure of the target compound was confirmed by 1 H-NMR and 13

EGFR and PI3Kα Kinase Assay
The potent compounds H7 and H10 were tested for their activities against EGFR T790M/L858R and PI3Kα enzyme using the Kinase-Glo Luminescent Kinase Assay, with olmutinib as a positive control. The specific operation was carried out according to our previous research [19,20].

Cytotoxicity Assay In Vitro
The in vitro cytotoxic activities of all compounds H1-H16 were evaluated with A549, H1975, LO2, and MCF-7 cell lines using the standard MTT assay, with olmutinib as a positive control [21].