Design, Synthesis, Biological Evaluation and Silico Prediction of Novel Sinomenine Derivatives

Sinomenine is a morphinan alkaloid with a variety of biological activities. Its derivatives have shown significant cytotoxic activity against different cancer cell lines in many studies. In this study, two series of sinomenine derivatives were designed and synthesized by modifying the active positions C1 and C4 on the A ring of sinomenine. Twenty-three compounds were synthesized and characterized by spectroscopy (IR, 1H-NMR, 13C-NMR, and HRMS). They were further evaluated for their cytotoxic activity against five cancer cell lines, MCF-7, Hela, HepG2, SW480 and A549, and a normal cell line, Hek293, using MTT and CCK8 methods. The chlorine-containing compounds exhibited significant cytotoxic activity compared to the nucleus structure of sinomenine. Furthermore, we searched for cancer-related core targets and verified their interaction with derivatives through molecular docking. The chlorine-containing compounds 5g, 5i, 5j, 6a, 6d, 6e, and 6g exhibited the best against four core targets AKT1, EGFR, HARS and KARS. The molecular docking results were consistent with the cytotoxic results. Overall, results indicate that chlorine-containing derivatives might be a promising lead for the development of new anticancer agents.


Introduction
Natural products, also known as secondary metabolites, are organic compounds with biological functions and activities synthesized by microorganisms or plants in their cells. They play an irreplaceable role and have broad application prospects in pharmaceutical, agricultural, chemical industries and other fields [1,2]. In fact, 6.1% of the total 1394 small molecule drugs developed from 1981 to 2019 were natural compounds, and 27.5% were developed by structural optimization. Furthermore, small molecule cancer drugs developed between 1940 and 2019 are directly or indirectly derived from natural products [3].
Alkaloids are nitrogen-containing organic compounds widely found in plants, animals and microorganisms. Most alkaloids have complex structures with basic nitrogencontaining heterocycles. A few alkaloids are organic amine alkaloids in which nitrogen atoms are absent in the ring structure. Thus far, around 12,000 alkaloids have been isolated from nature [4]. Based on their source or chemical structure, the alkaloids are divided into about 60 categories, including tropine, isoquinoline, indole alkaloids, terpenoids and In previous work, structural modification of sinomenine was carried out in the active groups of its four rings [9]. The A ring is a benzene ring where C3 and C4 are substituted by methoxy and hydroxyl groups. Structural modification on the A ring is often present on the active reaction sites C1 and C4, and sometimes on C2 or C3, but it is relatively rare [16]. Different sinomenine derivatives were reported in the previous studies as having good anti-inflammatory, immunosuppressive and neuroprotective activities compared to sinomenine [17]. In this study, we designed and synthesized two new series of sinomine derivatives in a quest to obtain potent compounds.
Many scholars have reported the structural modification of the sinomenine A ring. Due to the good application effect of piperidine and tetrahydropyrrole in medicine, we took sinomenine as the leading compound and introduced piperidine methylene or tetrahydropyrrole methylene at C1 by Mannich reaction to obtain 1-piperidine methylated sinomenine (5a-5k) and 1-tetrahydropyrrole methylated sinomenine (6a-6l) [18]. As the phenolic hydroxyl group of C4 is easy to oxidize and decompose and may be a factor leading to allergic reactions in vivo, it is necessary to modify the structure to protect the phenolic hydroxyl group. Esters are important intermediates of organic synthesis. The phenol hydroxyl ester can not only increase liposolubility, enhance substance activity and change metabolic characteristics, but it can also slow down the oxidation rate of compounds and greatly reduce their allergic reactions. Therefore, a series of 1,4-bisubstituted sinomenine derivatives were synthesized by the reaction of phenylacetyl chloride with 1-tetrahydropyrrole methylated sinomenine. All structures of the products were determined by IR, 1 H-NMR, 13 C-NMR, and HRMS. Furthermore, the cytotoxicity of the synthesized compounds was assessed. Compounds 5a-5k were tested against three cancer cell lines, MCF-7, Hela, and HepG2, using the MTT method. Compounds 6a-6l were tested against four cancer cell lines, MCF-7, Hela, SW480, and A549, and a normal cell line Hek293 with the CCK8 method. Finally, all In previous work, structural modification of sinomenine was carried out in the active groups of its four rings [9]. The A ring is a benzene ring where C 3 and C 4 are substituted by methoxy and hydroxyl groups. Structural modification on the A ring is often present on the active reaction sites C 1 and C 4 , and sometimes on C 2 or C 3 , but it is relatively rare [16]. Different sinomenine derivatives were reported in the previous studies as having good anti-inflammatory, immunosuppressive and neuroprotective activities compared to sinomenine [17]. In this study, we designed and synthesized two new series of sinomine derivatives in a quest to obtain potent compounds.
Many scholars have reported the structural modification of the sinomenine A ring. Due to the good application effect of piperidine and tetrahydropyrrole in medicine, we took sinomenine as the leading compound and introduced piperidine methylene or tetrahydropyrrole methylene at C 1 by Mannich reaction to obtain 1-piperidine methylated sinomenine (5a-5k) and 1-tetrahydropyrrole methylated sinomenine (6a-6l) [18]. As the phenolic hydroxyl group of C 4 is easy to oxidize and decompose and may be a factor leading to allergic reactions in vivo, it is necessary to modify the structure to protect the phenolic hydroxyl group. Esters are important intermediates of organic synthesis. The phenol hydroxyl ester can not only increase liposolubility, enhance substance activity and change metabolic characteristics, but it can also slow down the oxidation rate of compounds and greatly reduce their allergic reactions. Therefore, a series of 1,4-bisubstituted sinomenine derivatives were synthesized by the reaction of phenylacetyl chloride with 1-tetrahydropyrrole methylated sinomenine. All structures of the products were determined by IR, 1 H-NMR, 13 C-NMR, and HRMS.
Furthermore, the cytotoxicity of the synthesized compounds was assessed. Compounds 5a-5k were tested against three cancer cell lines, MCF-7, Hela, and HepG2, using the MTT method. Compounds 6a-6l were tested against four cancer cell lines, MCF-7, Hela, SW480, and A549, and a normal cell line Hek293 with the CCK8 method. Finally, all the synthesized compounds underwent network pharmacology and molecular-docking studies in order to forecast the possible targets of the most active compounds.

Synthesis
Two series of sinomenine derivatives were synthesized as shown in Scheme 1 and Table 1. A total of 23 new 1,4-disubstituted sinomenine derivatives (5a-5k and 6a-6l) were obtained by the introduction of piperidine and tetrahydropyrrole methylene (at 1-position, step a) using the Mannich reaction, and then the substitution of the phenylacetyl group by an acylation reaction was performed (at 4-OH, step b). the synthesized compounds underwent network pharmacology and molecular-docking studies in order to forecast the possible targets of the most active compounds.

Synthesis
Two series of sinomenine derivatives were synthesized as shown in Scheme 1 and Table 1. A total of 23 new 1,4-disubstituted sinomenine derivatives (5a-5k and 6a-6l) were obtained by the introduction of piperidine and tetrahydropyrrole methylene (at 1-position, step a) using the Mannich reaction, and then the substitution of the phenylacetyl group by an acylation reaction was performed (at 4-OH, step b). Scheme 1. Synthetic scheme of compounds 2-6 (R' = Compound 4, the red gradient circle represents the ester substitution R.)

Anticancer Activity of the Synthesized Compounds 5a-5k
The in vitro growth inhibitory activity of the sinomenine derivatives (5a-5k) was evaluated against different three cell lines, MCF-7, Hela, and HepG2, using sinomenine as the reference compound kept under the same conditions in an MTT assay [19]. The inhibition ratios of synthetic compounds were determined at concentrations of 2, 20 and 200 µM. The obtained data in triplicate for each concentration were plotted, as shown in Figure 2.
The results indicated that all the compounds had significant activity against the cancer cell lines. The synthesized compounds had a structure and dose-dependent relationship. At a low drug concentration of 2 µM, all of the tested compounds showed more toxicity towards the three cancer cells lines with inhibition ratios in the lower to medium percentage range for Hela and HepG2 cell lines, or higher inhibition ratios for MCF-7 cell lines. At a moderate drug concentration of 20 µM, the compounds 5i and 5j had significant effects on MCF-7, Hela, and HepG2 cell lines with the inhibition ratios of 54.13 and 63.35%, 94.43 and 80.22%, and 90.69 and 89.57%, respectively. Moreover, the compound 5g showed better anticancer activity against HepG2 cell lines with inhibition ratios reaching more than 53.2%. Other tested compounds showed moderate to weak inhibition against both cancer cell lines. At a high drug concentration of 200 µM, the inhibition ratios of all synthetic compounds, except 5c, against three cell lines exceeded 50%. In conclusion, the presence of two chlorine atoms in the R group was critical for the observed cytotoxicity activities.

Anticancer Activity of the Synthesized Compounds 5a-5k
The in vitro growth inhibitory activity of the sinomenine derivatives (5a-5k) evaluated against different three cell lines, MCF-7, Hela, and HepG2, using sinome as the reference compound kept under the same conditions in an MTT assay [19]. inhibition ratios of synthetic compounds were determined at concentrations of 2, 20 200 μM. The obtained data in triplicate for each concentration were plotted, as show Figure 2.
The results indicated that all the compounds had significant activity against the cer cell lines. The synthesized compounds had a structure and dose-dependent rela ship. At a low drug concentration of 2 μM, all of the tested compounds showed m toxicity towards the three cancer cells lines with inhibition ratios in the lower to med percentage range for Hela and HepG2 cell lines, or higher inhibition ratios for MCF-7 lines. At a moderate drug concentration of 20 μM, the compounds 5i and 5j had signifi effects on MCF-7, Hela, and HepG2 cell lines with the inhibition ratios of 54.13 and 63. 94.43 and 80.22%, and 90.69 and 89.57%, respectively. Moreover, the compound showed better anticancer activity against HepG2 cell lines with inhibition ratios reac more than 53.2%. Other tested compounds showed moderate to weak inhibition aga both cancer cell lines. At a high drug concentration of 200 μM, the inhibition ratios o synthetic compounds, except 5c, against three cell lines exceeded 50%. In conclusion presence of two chlorine atoms in the R group was critical for the observed cytotox activities.

Anticancer Activity of the Synthesized Compounds 5a-5k
The in vitro growth inhibitory activity of the sinomenine derivatives (5a-5k) evaluated against different three cell lines, MCF-7, Hela, and HepG2, using sinome as the reference compound kept under the same conditions in an MTT assay [19]. inhibition ratios of synthetic compounds were determined at concentrations of 2, 20 200 μM. The obtained data in triplicate for each concentration were plotted, as show Figure 2.
The results indicated that all the compounds had significant activity against the cer cell lines. The synthesized compounds had a structure and dose-dependent rela ship. At a low drug concentration of 2 μM, all of the tested compounds showed m toxicity towards the three cancer cells lines with inhibition ratios in the lower to med percentage range for Hela and HepG2 cell lines, or higher inhibition ratios for MCF-7 lines. At a moderate drug concentration of 20 μM, the compounds 5i and 5j had signifi effects on MCF-7, Hela, and HepG2 cell lines with the inhibition ratios of 54.13 and 63.3 94.43 and 80.22%, and 90.69 and 89.57%, respectively. Moreover, the compound showed better anticancer activity against HepG2 cell lines with inhibition ratios reac more than 53.2%. Other tested compounds showed moderate to weak inhibition aga both cancer cell lines. At a high drug concentration of 200 μM, the inhibition ratios o synthetic compounds, except 5c, against three cell lines exceeded 50%. In conclusion presence of two chlorine atoms in the R group was critical for the observed cytotox activities. 19

Anticancer Activity of the Synthesized Compounds 5a-5k
The in vitro growth inhibitory activity of the sinomenine derivatives (5a-5k) evaluated against different three cell lines, MCF-7, Hela, and HepG2, using sinome as the reference compound kept under the same conditions in an MTT assay [19]. inhibition ratios of synthetic compounds were determined at concentrations of 2, 20 200 μM. The obtained data in triplicate for each concentration were plotted, as show Figure 2.
The results indicated that all the compounds had significant activity against the cer cell lines. The synthesized compounds had a structure and dose-dependent rela ship. At a low drug concentration of 2 μM, all of the tested compounds showed m toxicity towards the three cancer cells lines with inhibition ratios in the lower to med percentage range for Hela and HepG2 cell lines, or higher inhibition ratios for MCF-7 lines. At a moderate drug concentration of 20 μM, the compounds 5i and 5j had signifi effects on MCF-7, Hela, and HepG2 cell lines with the inhibition ratios of 54.13 and 63.3 94.43 and 80.22%, and 90.69 and 89.57%, respectively. Moreover, the compound showed better anticancer activity against HepG2 cell lines with inhibition ratios reac more than 53.2%. Other tested compounds showed moderate to weak inhibition aga both cancer cell lines. At a high drug concentration of 200 μM, the inhibition ratios o synthetic compounds, except 5c, against three cell lines exceeded 50%. In conclusion presence of two chlorine atoms in the R group was critical for the observed cytotox activities.

Anticancer Activity of the Synthesized Compounds 6a-6l
Cytotoxicity screens were performed to assess the in vitro anticancer activity o synthesized sinomenine derivatives 6a-6l on the various cancer cell lines MCF-7, H SW480, and A549, as well as on Hek293 as a normal cell line, at doses of 2.5 and 25 The obtained inhibition ratios data were used to construct a heat map ( Figure 3, Ta S1-S2) in order to select compounds that exert cancer cell line-specific cytotoxicity. T the IC50 values of the promising compound were determined. Cisplatin was used as a erence standard. The antiproliferative activity was based on the evaluation of the per viability using the CCK8 assay, which is a rapid and sensitive method for determinin number of viable cells in culture. In the presence of electronic coupling reagents, W can be reduced by a dehydrogenase in the mitochondria to produce a highly water-sol orange-yellow formazan product. Its color is proportional to the proliferation of the n ber of viable cells and is related to cytotoxicity. Using a microplate reader to measur OD value can indirectly reflect the number of living cells at 450 nm wavelength [20].
Based on the heat map, 6a and 6c-6g exhibited a minimum of 50% inhibition r on at least one cancer cell line and had no or a very mild effect on normal cell line understand structure-function relationships, these selected compounds were subject subsequent analyses. All six compounds (6a and 6c-6g) were further examined to d mine their IC50 concentrations on the previously mentioned cell lines, and their effi was compared to the reference drug cisplatin. For this, six compounds and cisplatin evaluated on Hela, MCF-7, SW480, and A549, and on noncancerous Hek293 cell lin various concentrations for 60 h. IC50 values were calculated ( Table 2).
In agreement with the primary cytotoxicity screen, the obtained IC50 concentra clearly indicated which compounds were selectively effective on one or more cance lines with IC50 values less than 20 μM. In compounds 6a, 6d, 6e and 6g, with a chlo atom attached to the R group, significant anticancer activities were observed compar 6f. The derivative 6d, being the superior compound, displayed an IC50 of 5.73, 8.20, 6.08 μM against MCF-7, Hela, and SW480, respectively. Concerning the cytotoxici compound 6e, significant IC50 values were observed for MCF-7 and Hela cell lines (6e = 14.86, 13.28, and 16.57 μM for MCF-7, Hela, and Hek293, respectively), indicating 6e more selective toxicity toward cancer cells than normal cells.

Anticancer Activity of the Synthesized Compounds 6a-6l
Cytotoxicity screens were performed to assess the in vitro anticancer activity of the synthesized sinomenine derivatives 6a-6l on the various cancer cell lines MCF-7, Hela, SW480, and A549, as well as on Hek293 as a normal cell line, at doses of 2.5 and 25 µM. The obtained inhibition ratios data were used to construct a heat map (Figure 3, Tables S1 and S2) in order to select compounds that exert cancer cell line-specific cytotoxicity. Then, the IC 50 values of the promising compound were determined. Cisplatin was used as a reference standard. The antiproliferative activity was based on the evaluation of the percent viability using the CCK8 assay, which is a rapid and sensitive method for determining the number of viable cells in culture. In the presence of electronic coupling reagents, WST-8 can be reduced by a dehydrogenase in the mitochondria to produce a highly water-soluble orangeyellow formazan product. Its color is proportional to the proliferation of the number of viable cells and is related to cytotoxicity. Using a microplate reader to measure the OD value can indirectly reflect the number of living cells at 450 nm wavelength [20].
Based on the heat map, 6a and 6c-6g exhibited a minimum of 50% inhibition ratios on at least one cancer cell line and had no or a very mild effect on normal cell lines. To understand structure-function relationships, these selected compounds were subjected to subsequent analyses. All six compounds (6a and 6c-6g) were further examined to determine their IC 50 concentrations on the previously mentioned cell lines, and their efficacy was compared to the reference drug cisplatin. For this, six compounds and cisplatin were evaluated on Hela, MCF-7, SW480, and A549, and on noncancerous Hek293 cell lines at various concentrations for 60 h. IC 50 values were calculated ( Table 2).
In agreement with the primary cytotoxicity screen, the obtained IC 50 concentrations clearly indicated which compounds were selectively effective on one or more cancer cell lines with IC 50 values less than 20 µM. In compounds 6a, 6d, 6e and 6g, with a chlorine atom attached to the R group, significant anticancer activities were observed compared to 6f. The derivative 6d, being the superior compound, displayed an IC 50 of 5.73, 8.20, and 6.08 µM against MCF-7, Hela, and SW480, respectively. Concerning the cytotoxicity of compound 6e, significant IC 50 values were observed for MCF-7 and Hela cell lines (6e, IC 50 = 14.86, 13.28, and 16.57 µM for MCF-7, Hela, and Hek293, respectively), indicating 6e had more selective toxicity toward cancer cells than normal cells.

PPI Interaction Network Construction
The gene was uploaded to the String database (https://string-db.org/, version 11.0b, October 17, 2020) for the analysis of PPI; the species were limited to Homo sapiens and the minimum interaction threshold was set to "medium confidence" 0.4. The network construction used Cytoscape software (https://cytoscape.org/, release 3.8.0, April 15, 2020). We calculated the degree of each node through the CytoHubba plug-in and selected the top 10 targets as potential targets for cancer diseases according to their associated degree. A total of 16 targets playing an important role in cancer diseases were obtained (Table 3). Subsequently, the PPI network was constructed for the top ten targets of each cancer disease and visual analysis ( Figure 5). The genes AKT1, TP53, EGFR, MYC and PTEN were determined as high-frequency genes that may have a potential anticancer role.

PPI Interaction Network Construction
The gene was uploaded to the String database (https://string-db.org/, version 11.0b, accessed on 17 October 2020) for the analysis of PPI; the species were limited to Homo sapiens and the minimum interaction threshold was set to "medium confidence" 0.4. The network construction used Cytoscape software (https://cytoscape.org/, release 3.8.0, accessed on 15 April 2020). We calculated the degree of each node through the CytoHubba plug-in and selected the top 10 targets as potential targets for cancer diseases according to their associated degree. A total of 16 targets playing an important role in cancer diseases were obtained (Table 3). Subsequently, the PPI network was constructed for the top ten targets of each cancer disease and visual analysis ( Figure 5). The genes AKT1, TP53, EGFR, MYC and PTEN were determined as high-frequency genes that may have a potential anticancer role.

GO and KEGG Enrichment Analysis
The GO and KEGG enrichment analysis of the top 10 targets related to five cancer diseases were analyzed with the DAVID gene annotation tool. GO analysis was applied to enrich and functionally interpret differentially expressed key candidate genes (KCGs) at the molecular and cellular levels. The result of the KCGs GO was decomposed into three subontologies viz. Biological Process (BP), Cellular Component (CC) and Molecular Function (MF) as shown in Figure 6. For BP, the KCGs were mainly enriched in positive regulation of transcription from RNA polymerase II promoter, negative regulation of the apoptotic process, positive regulation of protein phosphorylation and positive regulation of cell proliferation. The KCGs in MF mainly participate in protein binding. In addition, most of the KCGs were localized to regions called the cytoplasm, plasma membrane, cytosol and the nucleus of the CC.

GO and KEGG Enrichment Analysis
The GO and KEGG enrichment analysis of the top 10 targets related to five cancer diseases were analyzed with the DAVID gene annotation tool. GO analysis was applied to enrich and functionally interpret differentially expressed key candidate genes (KCGs) at the molecular and cellular levels. The result of the KCGs GO was decomposed into three subontologies viz. Biological Process (BP), Cellular Component (CC) and Molecular Function (MF) as shown in Figure 6. For BP, the KCGs were mainly enriched in positive regulation of transcription from RNA polymerase II promoter, negative regulation of the apoptotic process, positive regulation of protein phosphorylation and positive regulation of cell proliferation. The KCGs in MF mainly participate in protein binding. In addition, most of the KCGs were localized to regions called the cytoplasm, plasma membrane, cytosol and the nucleus of the CC.
KEGG pathway databases contain vital information for systematic pathway enrichment analysis of gene functions. Significantly enriched pathways as therapeutic targets in cancer, including the PI3K/Akt signaling pathway, MAPK signaling pathway, ErbB signaling pathway, p53 signaling pathway and FoxO signaling pathway, were identified. The top 30 integrated KEGG pathways of each disease are shown in Figure 7.  KEGG pathway databases contain vital information for systematic pathway enrichment analysis of gene functions. Significantly enriched pathways as therapeutic targets in cancer, including the PI3K/Akt signaling pathway, MAPK signaling pathway, ErbB signaling pathway, p53 signaling pathway and FoxO signaling pathway, were identified. The top 30 integrated KEGG pathways of each disease are shown in Figure 7.

Molecular Docking
Molecular docking was carried out to elucidate the binding modes of the 24 compounds to the 16 targets (AKT1, CCND1, CDH1, EGF, EGFR, ERBB2, GAPDH, HRAS,  IL6, INS, KRAS, MYC, PTEN, STAT3, TP53 and VEGFA) for which crystal structures were known. Interestingly, most of the compounds showed a relatively much higher binding affinity against targets such as AKT1 (PDB ID: 4EJN), KRAS (PDB ID: 4LYH), HRAS (PDB ID: 121P) and EGFR (PDB ID: 1M17) than the other 10 targets (Figures 8 and 9). For example, 6a interacted with AKT1 with a docking score of −155.153 kcal/mol, and 6d interacted with HRAS with a docking score of −133.267, which should be deemed as potent binding. The docking scores of the best-ranked molecules against the selected targets are shown in Tables S3 and S4 and Figure S59.

Molecular Docking
Molecular docking was carried out to elucidate the binding modes of the 24 compounds to the 16 targets (AKT1, CCND1, CDH1, EGF, EGFR, ERBB2, GAPDH, HRAS, IL6,  INS, KRAS, MYC, PTEN, STAT3, TP53 and VEGFA) for which crystal structures were known. Interestingly, most of the compounds showed a relatively much higher binding affinity against targets such as AKT1 (PDB ID: 4EJN), KRAS (PDB ID: 4LYH), HRAS (PDB ID: 121P) and EGFR (PDB ID: 1M17) than the other 10 targets (Figures 8 and 9). For example, 6a interacted with AKT1 with a docking score of −155.153 kcal/mol, and 6d interacted with HRAS with a docking score of −133.267, which should be deemed as potent binding. The docking scores of the best-ranked molecules against the selected targets are shown in Tables S3-S4 and Figure S59.   Molecular docking was carried out to elucidate the binding modes of the 24 compounds to the 16 targets (AKT1, CCND1, CDH1, EGF, EGFR, ERBB2, GAPDH, HRAS, IL6, INS, KRAS, MYC, PTEN, STAT3, TP53 and VEGFA) for which crystal structures were known. Interestingly, most of the compounds showed a relatively much higher binding affinity against targets such as AKT1 (PDB ID: 4EJN), KRAS (PDB ID: 4LYH), HRAS (PDB ID: 121P) and EGFR (PDB ID: 1M17) than the other 10 targets (Figures 8 and 9). For example, 6a interacted with AKT1 with a docking score of −155.153 kcal/mol, and 6d interacted with HRAS with a docking score of −133.267, which should be deemed as potent binding. The docking scores of the best-ranked molecules against the selected targets are shown in Tables S3-S4 and Figure S59.  . The total energy of molecular docking between compounds 6a-6l and 13 potential targets. Figure 9. The total energy of molecular docking between compounds 6a-6l and 13 potential targets.
As shown in Figures 10 and 11 and Tables S5-S8, a stable composite structure was formed between the compound and the protein through hydrophobic interaction, hydrogen bonding, halogen bonding, salt bridges and π stacking, thereby affecting the structure and function of the protein and causing cancer cell death. For example, 6e bound to the cavity of 121P (HRAS) and interacted with eight amino acid residues. The interaction between 6e and six amino acid residues (ALA18, PHE28, TYR32, LYS117, ALA146 and LYS147) was hydrophobic. Moreover, 6e interacted with ASP30 and LYS117 through hydrogen bonds, with GLY15A through halogen bonds, with LYS117 through salt bridges and with PHE28 through π stacking interaction. and function of the protein and causing cancer cell death. For example, 6e bound to the cavity of 121P (HRAS) and interacted with eight amino acid residues. The interaction between 6e and six amino acid residues (ALA18, PHE28, TYR32, LYS117, ALA146 and LYS147) was hydrophobic. Moreover, 6e interacted with ASP30 and LYS117 through hydrogen bonds, with GLY15A through halogen bonds, with LYS117 through salt bridges and with PHE28 through π stacking interaction.

Chemistry
Unless specified otherwise, all the materials were obtained from commercial suppliers and used without further purification. Thin layer chromatography (TLC) was performed using silica gel 60 F 254 and visualized using UV light. Column chromatography was performed with silica gel (mesh 300-400). 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer in CDCl 3 with Me 4 Si as an internal standard. Data were reported as follows: chemical shift in ppm (δ), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad and m = multiplet), coupling constant in Herts (Hz), and integration. IR spectra were recorded on an FT-IR spectrometer, and only major peaks are reported in cm −1 . Mass data were recorded by ESI on an FT mass spectrometer.

General Procedure for the Synthesis of Compounds 5a-5k
To a mixture of 1-(1-pyrrolidinylmethyl)-Sinomenine or 1-(1-piperidinylmethyl)-Sinomenine (1.0 mmol) and Et 3 N (2.5 mmol) in CH 2 Cl 2 (5 mL) was added acyl chloride (2.5 mmol) at 0 • C. The reaction mixture was then stirred at room temperature. After the completion of the reaction, sat. NaHCO 3 was added and then extracted with CH 2 Cl 2 . The organic layers were combined and dried with anhydrous Na 2 SO 4 and evaporated under reduced pressure. The mixture was evaporated under vacuum, and the residue was purified by flash chromatography with dichloromethane and methanol as the eluents to produce the pure product.

Cell Lines
The MCF-7 (human breast cancer cell lines), Hela (human cervical cancer cells lines), and HepG2 (human hepatocellular carcinoma cell lines) cells were used to measure the cytotoxicity of the 5a-5k series, which were provided by the Academy of Military Medical Sciences (Beijing, China). The cell lines were maintained on RPMI 1640 nutrient medium supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, 100 mg/mL streptomycin and 100 units/mL penicillin. The cells were grown at 37 • C in a humidified atmosphere with 5% CO 2 and were subcultured two to three times a week.
The MCF-7 (human breast cancer cell lines), Hela (human cervical cancer cell lines), SW480 (human colon adenocarcinoma cell lines), A549 (human lung adenocarcinoma cell lines) and nontoxic normal cells Hek293 (human embryonic kidney cell lines) cells were used to measure the cytotoxicity of the 6a-6l. They were obtained from the Beijing Normal University (Beijing, China). The test cell lines were cultured in DMEM, IMDM, F12 and DMEM nutrient medium containing 10% heat-inactivated fetal bovine serum, 100 mg/mL streptomycin and 100 units/mL penicillin in a humified, 5% (v/v) CO 2 atmosphere at 37 • C.

Anticancer Evaluation MTT Assay
The MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay with a slight modification was used to determine the inhibition effects of substrates 5a-5k. MCF-7, Hela and HepG2 were used for testing. Briefly, cells (5.0 × 10 3 cells per well) were seeded in 100 µL of the RPMI-1640 medium in 96-well plates for 24 h, treated with drugs and complete medium in wells up to 200 µL for 24 h. RPMI-1640 samples were employed as negative controls, and cisplatin as a positive control. Four quadruplicates of each concentration for all tested compounds were evaluated in three independent assays. For this, after removing the drug containing media 100 µL, 10 µL of MTT solution was added to wells and incubated for 4 h under similar conditions. After that, the supernatant from each well was removed and the formazan crystals formed by viable cells were dissolved with DMSO (100 µL/well). At the end of incubation, optical densities at 570 nm were measured using a plate reader (BIO-TEK, USA). Cell viability was calculated based on the measured absorbance relative to the absorbance of the cells exposed to the negative control, which represented 100% cell viability.

CCK8 Assay
Cell viability was estimated using the CCK8 assay. Tumor cells (100 mL) viz. MCF-7, Hela, SW480, A549 and nontoxic normal cells Hek293 were seeded in medium containing Corning ® 96-well tissue culture plates at confluences of 20, 15, 20, 15, 40 and 13%, respectively, in Incucyte ZOOM. Fresh medium (100 µL) containing different concentrations of the test sample was added after 12 h of seeding. The microtiter plates were incubated at 37 • C in a humidified incubator with 5% CO 2 for 60 h. Triplicates from each concentration were used. Negative control cells were incubated without sample and cisplatin as a positive control. At the end of treatment, the numbers of viable cells were determined by the CCK8 test. In brief, the media was removed from the 96 well plates and replaced with 100 µL of fresh culture medium with 10% CCK8 solution added to each well including the untreated controls. The 96 well plates were then incubated at 37 • C and 5% CO2 for 1-4 h. Then, the absorbance was measured at 450 nm using a BMG LABTECH ® -POLAR star Omega microplate reader (Ortenberg, Germany) to determine the number of viable cells, and the percentage of viability was calculated as ((ODt-ODb/ODc-ODb)) × 100%, where ODt is the mean optical density of wells treated with the tested sample, ODc is the mean optical density of wells treated with DMSO and ODb is the mean optical density of PBS wells without cells. The relation between surviving cells and drug concentrations were plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each concentration [21,22].

Collection of Related Genes
Multiple disease-related gene databases viz. Online Mendelian Inheritance in Man (OMIM) [23], Human Gene Function and Network Analysis (CooLGeN) [24], The Comparative Toxicogenomics Database (CTD) [25] and GeneCards [26], were used to collect the cancer-associated genes. Either advanced search or custom filtering criteria were used for the gene retrieval. For Cool GeN, genes collected with all human genes were retrieved. For CTD, genes annotated with direct evidence and labeled as "M" (marker/mechanism) and/or "T" (therapeutic) were retrieved. For GeneCards, genes associated with "Protein Coding" were retrieved.

Pharmacology Network Analysis
STRING [27] was used to construct the protein-protein interaction (PPI) networks of the targets related to the five cancer diseases. Analysis and modularization were performed using Cytoscape. The MCODE algorithm [28] was used to determine highly interconnected regions in the PPI network. The degree cut off, node density cutoff, and node score cutoff were kept to 2, 0.1 and 0.2, respectively.

Enrichment Analysis of Five Cancer Diseases Targets
All targets of five cancer diseases-related cell lines were mapped into Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGGs) [29]. The GO functional annotations were carried for the biological process (BP), molecular function (MF) and cellular components (CC) terms [30].

Molecular Docking
Molecular docking was performed using iGEMDOCK (BioXGEM, Taiwan) [31]. iGEM-DOCK makes use of the genetic algorithm that selects the best solution from the population by calculating their fitness. Genetic algorithm is a general-purpose optimization and search technique which is based on the evolutionary process of natural selection. Genetic algorithm permits the individuals of a population to evolve under certain specific selection rules to a condition that maximizes the fitness function [32]. The PyMOL Molecular Graphics (Version 1.8.4.0, Schrödinger, LLC) was used for the visualizations and graphics generations [33]. To determine the interactions of all the docking complexes, the proteinligand interaction profiler (PLIP) was used to analyze the crystal structure of the available structural complexes [34].

Conclusions
In the current work, a series of sinomenine derivatives were synthesized with good yields. All the derivatives were tested for their cytotoxic activity against a variety of cancer cells in vitro and applied to a molecular docking study to investigate the potential molecular targets. The results show that compounds containing chlorine had significant anticancer activity. Furthermore, we obtained 16 core targets and some key signal pathways related to cancer diseases through comprehensive bioinformatics analysis. The molecular docking results showed that AKT1, EGFR, HARS and KARS could be considered as the most potential anticancer targets of sinomenine derivatives with high binding affinity. We found that these targets are important key genes in the PI3K/AKT and MAPK signaling pathways combined with the results of KEGG enrichment. Therefore, we boldly speculate that chlorine-containing sinomenine derivatives may cause the pathological death of cancer cells by regulating multiple genes in these pathways.
As the current amount of the compound is insufficient for more experimental verification, we intend to synthesize chlorine-substituted sinomenine derivatives with different substitutions to conduct a detailed structure-activity relationship (SAR) analysis. We will further explore the relationships between structure and activity combined with activity experiments in vitro.