Design, synthesis, and anticancer properties of isocorydine derivatives

Bioorganic & Medicinal Chemistry 25 (2017) 6542–6553

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Design, synthesis, and anticancer properties of isocorydine derivatives Qian Yan a,c,d, Ruxia Li a,b,d, Aiyi Xin a,c, Yin Han a, Yanxia Zhang a, Junxi Liu a,⇑, Wenguang Li b, Duolong Di a a CAS Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (CAS), Lanzhou 730000, PR China b Gansu Key Laboratory of Preclinical Studies for New Drugs, Institute of Pharmacology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, PR China c University of Chinese Academy of Sciences, Beijing 10049, PR China

a r t i c l e

i n f o

Article history: Received 14 September 2017 Revised 13 October 2017 Accepted 20 October 2017 Available online 21 October 2017 Keywords: Isocorydine Aporphine alkaloid Structure modification Anticancer activity Hepatocellular carcinoma

a b s t r a c t Isocorydine (ICD), an aporphine alkaloid, is widely distributed in nature. Its ability to target side population (SP) cells found in human hepatocellular carcinoma (HCC) makes it and its derivative 8-amino-isocorydine (NICD) promising chemotherapeutic agents for the treatment of HCC. To improve the anticancer activity of isocorydine derivatives, twenty derivatives of NICD were designed and synthesized through chemical structure modifications of the aromatic amino group at C-8. The anti-proliferative activities of all synthesized compounds against human hepatocellular (HepG2), cervical (HeLa), and gastric (MGC-803) cancer cell lines were evaluated using an MTT assay. The results showed that all the synthetic compounds had some tumor cell growth inhibitory activity. The compound COM33 (24) was the most active with IC50 values under 10 lM (IC50 for HepG2 = 7.51 mM; IC50 for HeLa = 6.32 lM). FICD (12) and COM33 (24) were selected for further investigation of their in vitro and in vivo activities due to their relatively good antiproliferative properties. These two compounds significantly downregulated the expression of four key proteins (C-Myc, b-Catenin, CylinD1, and Ki67) in HepG2 cells. The tumor inhibition rate of COM33 (24) in vivo was 73.8% after a dose 100 mg/kg via intraperitoneal injection and the combined inhibition rate of COM33 (24) (50 mg/kg) with sorafenib (50 mg/kg) was 66.5%. The results indicated that these isocorydine derivatives could potentially be used as targeted chemotherapy agents or could be further developed in combination with conventional chemotherapy drugs to target cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT), the main therapeutic targets in HCC. Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Aporphines are a group of tetrahydroisoquinoline alkaloids which possessed a lots of markedly biological activities, such as central nervous system activity, antitumor activity, et al.1,2 Isocorydine (1), approved as an antispasmodic analgesic by the China Food and Drug Administration (CFDA), is a typical natural aporphine alkaloid and is widely distributed in many plants, including plants of the papaveraceae, menispermanceae, and annonaceae families. It has antispasmodic, analgesia, antimalarial, antiarrhythmic, and antitumor properties along with other pharmacological effects.3–6 Recent studies have shown that isocorydine not only inhibits cell proliferation in hepatocellular carcinoma cell (HCC) lines by inducing G2/M cell cycle arrest and apoptosis, but also targets drugresistant cellular side populations (or cancer stem cells, CSCs) by inducing PDCD4-related apoptosis. Furthermore, isocorydine could ⇑ Corresponding author. d

E-mail address: [email protected] (J. Liu). Co-first author: These authors contributed equally to this work.

https://doi.org/10.1016/j.bmc.2017.10.027 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.

selectively reduce the size and weight of side population cellinduced tumor masses in nude mice, which suggested that isocorydine is a potential therapeutic drug for targeting side population cancer cells in hepatocellular carcinoma.7,8 CSCs possess selfrenewal properties and are chemoresistant to the majority of anticancer agents, which is a major challenge to clinical chemotherapy. Isocorydine could not only significantly reduce the percentage of CD133+- and EpCAM-expressing cells, but also suppress the ability of primary liver carcinoma PLC/PRF/5 CD133+ cells to form hepatospheres and tumor-like spheres in vitro. To improve the anticancer activity of ICD, 8-amino-isocorydine (NICD, 6) was prepared through chemical structure modifications.9 A study on its antitumor activity and the mechanism of action has shown that NICD could inhibit HCC growth, particularly of the CD133+ subpopulation, and render HCC more sensitive to sorafenib treatment.10 In particular, NICD inhibited the expression of IGF2BP3 in HCCs, which could enrich the CD133+ CSC subpopulation in HCCs. CD133+ overexpression could then induce the overexpression of drug resistance-related genes such as ABCB1 and ABCG2. NICD could also induce G2/M cycle arrest in HCCs via DNA damage 45

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alpha (GADD45A) and the p21 pathway.11 NICD also could inhibit HCC cell migration and invasion partly by downregulating E2F1/ ITGA1 expression.12 NICD oxidizes easily in water due to its p-aminophenol substructure, which limits it as an effective antitumor agent.9 In addition, more effective and selective antitumor agents are urgently required, especially for the treatment of HCC. Sorafenib (Fig. 1) is the only successful multi-target tyrosine kinase inhibitor currently available for the treatment of advanced liver cancer that is ineffective or intolerant to standard therapy.13 Completed research in our group supported that the S absolute configuration of the hydrogen atom at C-6a is a key factor in the anticancer activity of aporphine alkaloids and the different substituent at C-8 position can significantly affect the anticancer activities of isocorydine derivatives.9–12 So amide- and urea-type derivatives of NICD were designed and synthesized. Following the chemical structure design philosophy of sorafenib and CHMFL-KIT-8140,13–15 NICD was considered as the lead compound, and the C-8 amino group and the C-11 phenolic hydroxyl group were chemically combined with the pharmacophore of above mentioned anticancer agents. The anti-proliferative activities of all synthesized compounds against human hepatocellular (HepG2), cervical (HeLa) and gastric (MGC-803) cancer cell lines were evaluated via MTT assays. FICD (12) and COM33 (24) were selected from the obtained compounds because of their better anti-proliferative activity, especially COM33, and their in vitro and in vivo activities were investigated. Emerging research data have indicated that epithelial-to-mesenchymal transition (EMT) and CSCs may be related to a high risk for recurrence and poor prognosis.16 Recent studies in HCCs have shown that the EMT phenotype and CSC biology are intricately linked and patients manifesting both CSC and EMT phenotypes are unresponsive to standard chemotherapies and had low progression-free survival rates.16–19 Therefore, the oncogene proteins Cmyc, cell cycle-related protein Cyclin D1, cell proliferation related protein Ki67, the main EMT marker vementin, and the cellular differentiation-related signaling pathway Wnt/b-catenin were selected to investigate the anticancer mechanism via western blot analysis. The results indicated that NICD derivatives could target CSCs and EMT, which are the main therapeutic targets in HCC. COM33 showed potential as a target chemotherapy agent to be used in combination with conventional chemotherapy to target CSCs and EMT. 2. Results and discussion 2.1. Chemistry To obtain enough aporphine alkaloids, isocorydine (1), corydine (2), corytuberine (3), and isocorytuberine (4) were selectively isolated from Dicranostigma leptopodum (Maxim.) Fedde and Stephania yunnanensis Lo via column chromatography with silica gel in our laboratory (Fig. 2).20,21 Isocorydione and NICD were prepared from isocorydine according to the method reported in the literature.9 Nuclear magnetic resonance (NMR), High-

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Fig. 2. Structures of compounds 1–6.

resolution electrospray ionization mass spectrometry (HR-ESI-MS) and X-ray crystallography (Fig. 3) were performed to confirm four chemical structures.22–24 To synthesize enough NICD derivatives with an aromatic amino functional group at C-8, three strategies were applied. A nucleophilic substitution reaction between the aromatic amino group of NICD and an aromatic carboxylic acid/aroyl chloride was the first applied method, which produced compounds 7–11 with synthetic yields of 53%–81% (Scheme 1). The second strategy was a nucleophilic addition reaction of aromatic isocyanate with the aromatic amino group of NICD, which formed the key urea functional group of the HCC target drugs, such as sorafenib and regorafenib (Scheme 2). Compounds 12–21 were prepared with synthetic yields of 3%–72%. The aromatic isocyanates have different electrophilic reactivities for the different substituents at -R1, -R2 and -R3. Consequently, the nucleophilic addition reaction could occur at the C-11 hydroxy group or at the C-8 amino group in NICD. The key urea functional group could also be synthesized through a symmetrical nucleophilic additional-condensational reaction between triphosgene and two different kinds of aromatic amino groups (Scheme 3), which was performed to obtain compounds 23 and 27. The key intermediates 22 and 26 were prepared via a nucleophilic substitution reaction between Boc-protected-4-aminopiperidine and 2-chloro-5- nitrobenzotrifluoride, which was achieved in two steps with 66% yield. Next, under acidic conditions, the protectional Boc group was removed to afford compounds 24 and 28. Subsequently, amide bond formation with acyl chloride led to the formation of compounds 25 and 29. The structures of these target compounds were characterized by 1H NMR, 13C NMR and high-resolution mass spectra (HR-ESI-MS). 2.2. Biological activity 2.2.1. In vitro anticancer activity The anti-proliferative activities of all obtained compounds (1–2 9) were evaluated against human hepatocellular (HepG2), cervical (HeLa), and gastric (MGC-803) cancer cell lines via MTT assays with sorafenib as a positive control. The IC50 values obtained are

Fig. 1. Structures of sorafenib and CHMFL-KIT-8140.

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Fig. 3. X-ray crystal structures of compounds 1–4.

Scheme 1. Synthesis of compounds 7–11. Reagents and reaction conditions: (a) carboxylic acid, HBTU, DIPEA, DMF, rt, 8 h.

Scheme 2. Synthesis of compounds 12–21. Reagents and reaction conditions: (a) Phenyl isocyanate, CH2Cl2, 0 °C, 1 h.

summarized in Table 1. Natural aporphine alkaloids (such as compounds 1–4) had poor inhibitory activities with IC50 values above 100 lM, consistent with the literature.25 In the chemical structure of compounds 1–4, the methoxyl and hydroxyl groups although have different substituted positions, such as C-1, C-2, C-10, or C11, their antitumor activity was limited which means the natural aporphine alkaloids have weakly antitumor activity. So it was necessary to chemical modifications about natural aporphine alkaloids in order to improve their activities and effective useage. All synthesized compounds (5–2 9) showed significant improvements in anti-proliferative activity with IC50 values in micromolar levels (9.12–75.37 lM) over all cell lines. FICD (12) and COM33 (24) showed better anti-proliferative activity against three cancer cell

lines, especially COM33. Their IC50 values less than 10 lM was superior to those of sorafenib, which indicated that the structure modification of NICD was effective. Pharmacophores of sorafenib and CHMFL-KIT-8140, such as the urea, CF3, and 4-aminomenthyl-piperdine groups, were introduced into these test compounds to increase their biological activities. The results showed that a urea group at C-8 was superior to an amino group, such as in compounds 7–11 where the antitumor activity was less than that of compound 12. An aromatic amine substitution of the hydroxyl group at C-11 also increased the cytotoxic activity, such as in compound 14 (IC50: HepG2, 16.15 mM) and compound 17 (IC50: HepG2, 17.25 lM). The activity of the 4-aminomenthyl-substituted compound (24, IC50: HepG2, 7.51 mM) was superior to that of the

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Scheme 3. Synthesis of compounds 22–29. Reagents and reaction conditions: (a) K2CO3, DMF, 100 °C, 8 h; (b) H2, 10% Pd/C, MeOH, rt, 3 h; (c) triphosgene, Et3N, DMAP, DCM, 0 °C to rt, 4 h; (d) 4 M HCl in ethyl acetate, rt, 2 h; (e) propionyl chloride, Et3N, DMF, 0 °C, 0.5 h.

Table 1 The antiproliferative activities (IC50: lM) of compound 1–29 and sorafenib against human cancer cell lines in vitro. Compounds

1 2 3 4 5 6 (NICD) 7 8 9 10 11 12 (FICD) 13 14 15 16 17 18 19 20 21 23 24 (COM33) 25 27 28 29 Sorafenib

IC50 (mM) HepG2

Hela

MGC-803

>200 >200 >200 153.9 40.02 51.12 21.53 63.37 30.19 69.75 75.37 17.16 28.36 16.15 53.17 36.74 17.25 31.35 45.86 44.11 26.67 18.94 7.51 20.69 25.91 28.61 28.52 15.00

>200 >200 >200 109.1 31.34 44.10 16.55 74.49 29.56 49.58 62.11 26.52 21.73 19.10 46.64 36.94 27.05 43.89 48.99 47.67 26.83 22.12 6.32 32.52 26.72 33.74 33.37 12.01

>200 >200 >200 124.1 49.57 62.08 21.89 37.86 27.20 34.66 56.63 16.64 30.02 25.62 47.79 35.17 26.90 22.83 57.27 39.32 25.08 23.18 11.14 20.41 28.74 32.30 32.48 19.92

propionyl-protected compound (25, IC50: HepG2, 20.69 mM), although the amino group needs to be protected to improve the pharmacokinetics in compound CHMFL-KIT-8140.14 Thus, five nitrogen atoms introduced into the chemical structure of compound 24 (COM33) increased the hydrogen bond, hydrophilic, and Van der Walls interactions between the compound and target proteins or drug metabolic enzymes in the cancer cell, which was useful to increase the antitumor activity of NICD derivatives. 2.2.2. The synergistic effect of FICD (12) and sorafenib for HepG2 growth inhibition The anti-proliferative activity of FICD (12) against U251, HeLa, MGC-803, SMMC-7721, HepG2, and MAD-MB-231 cells lines was evaluated (Fig. 4A). During the concentration ranged from 5 lg/

mL to 40 lg/mL, the growth inhibitory activities of FICD (12) against six human cancer cells were all dose and time dependent. Of the six cancer cell lines, FICD exhibited the best anticancer activity in HepG2. Thus, HepG2 was selected to evaluate the detailed anti-cancer mechanism exhibited by FICD. As shown in Fig. 4B, FICD had a dose- and time-dependent inhibitory effect on the proliferation of HepG2. The inhibition rate increased when the incubation time was prolonged, whereas the inhibition rate gradually increased with the concentration of FICD. As sorafenib is a common, clinically used anti-HCC agent, we analyzed the effect of low-dose FICD (2.5 lg/mL, 5.0 lg/mL, and 10.0 lg/mL) combined with different doses of sorafenib in HepG2 cells to determine whether these compounds act synergistically. MTT assays performed 24 h after treatment showed that the HepG2 cells were more sensitive to this combination than to sorafenib treatment alone, which indicated that a low dose of FICD enhances the cytotoxic effect of sorafenib on HCC cells in vitro (Table 2, Fig. 4C–E). As shown in Fig. 4E, at a FICD concentration of 10.0 lg/mL, the growth inhibitory activity of the combination against HepG2 significantly increased (P < .01) compared to that of sorafenib at 2.0 lg/mL, 4.0 lg/mL, and 6.0 lg/mL. The values of the combination index (CI) were all less than 0.9, and the synergistic multiple achieved was 2.04. 2.2.3. Cell cycle analysis of HepG2 with FICD treatment FICD (12) The cell cycle distribution of HepG2 under treatment with different concentrations of FICD for 48 h was also evaluated. Untreated cells were used as a negative control and HepG2 cells were treated with FICD (10.0 lg/mL), sorafenib (4.0 lg/mL), and the combination (sorafenib 4.0 lg/mL + FICD 10.0 lg/mL) for 48 h. After the cells were harvested, the cell cycle phases were examined via flow cytometry. As shown in Fig. 5A, the sorafenib group showed a significant increase in the number of cells in the G0/G1 phase, whereas the number of cells in the S phase decreased significantly. In the FICD group, the number of cells in the G2/M phase increased significantly, and the number of cells in the G0/G1 phase decreased. In the combination group, the number of cells in the G0/G1 phase decreased compared to that of the sorafenib group which means that FICD and sorafenib have a completely different mechanism of pharmacological action. 2.2.4. FICD (12) induces apoptosis in HepG2 cells As shown in Fig. 5B, FICD induced apoptosis (both early and late) in HepG2 cells after treatment with FICD (5.0 lg/mL or 10.0 lg/mL), sorafenib (4.0 lg/mL), or the combination (FICD: 10.0

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Fig. 4. Antiproliferative activities of FICD (12) and FICD/sorafenib combination against human cancer cells in vitro. A: The growth inhibitory activity of FICD against six human cancer cells is dose dependent with concentrations ranging from 5.0 lg/mL to 40.0 lg/mL. B: FICD has a dose- and time-dependent inhibitory effect on the proliferation of HepG2. C (FICD: 2.5 mg/mL + sorafenib), D (FICD: 5.0 mg/mL + sorafenib), and E (FICD: 10.0 mg/mL + sorafenib) support the hypothesis that FICD has a synergistic effect with sorafenib. (*P < .01, **P < .01).

Table 2 The synergistic effect (IC50: lg/mL) and synergistic multiple of FICD (12) combined with sorafenib against HepG2 growth inhibition. Treatment group

IC50 (Synergistic multiple)

Treatment group

IC50 (Synergistic multiple)

Sorafenib (lg/mL) S + 10.00 lg/mL FICD

12.40 ± 0.044 5.44 (2.04)

S + 5.00 lg/mL FICD S + 2.50 lg/mL FICD

8.97 (1.25) 11.20 (0.91)

Notes: S means sorafenib.

lg/mL + sorafenib: 4.0 lg/mL) for 24 h. Apoptosis in HepG2 cells increasedgraduallyinadose-dependentmanner.Theapoptosisratios of FICD (low and high doses), sorafenib, and the combination were found to be 8.94%, 11.89%, 28.86%, and 63.67%, respectively. These resultsshowedthatFICD,especiallythecombinationofFICDwithsorafenib, effectively induced apoptosis in HepG2 cells. 2.2.5. FICD (12) and COM33 (24) prevent HCC proliferation through induced differentiation and reverse the epithelial-tomesenchymal transition by regulating related protein expression The onco-inducing protein C-myc, cell cycle-related protein Cyclin D1, cell proliferation-related protein Ki67, EMT marker vementin, and cell differentiation-related signaling proteins Wnt/ b-catenin were selected to investigate the underlying anticancer mechanism of FICD and COM33 via western blot analysis. The results indicated that the proteins levels of C-myc, b-catenin, Cyclin D1, and Ki67 decreased in a dose-dependent manner after 48 h of treatment with FICD, COM33, or sorafenib. The results were significantly different between the treatment groups and the control group, as shown in Fig. 6A–B. Compared with other groups, the combination group (FICD or COM33 with sorafenib) showed clear a decrease in the levels of four typical proteins (C-myc, b-catenin, Cyclin D1 and Ki67) which are closely related to highly aggressive tumor invasiveness, intrahepatic spread, and extrahepatic metastasis, which occurs in HCC. This supports the hypothesis that isoco-

rydine derivatives have a synergistic effect with sorafenib, a multikinase inhibitor that has shown efficacy against a wide variety of HCC subtypes. In contrast, the expression of the mesenchymal marker vementin increased after treatment with either sorafenib or NICD derivatives. This was a disappointing result with respect to the classic targeted therapy, as it meant that the induction of EMT in HCC resulted in the expression of CSC markers or the creation of CSCs, which leads to self-renewal, drug resistance, enhanced invasiveness, and increased migratory potential. Fortunately, as shown in Fig. 6A, the combination of sorafenib with FICD markedly suppressed the increased expression of vementin. The combination of sorafenib with COM33, however, had limited effects. Overall, COM33 showed stronger antitumor activity than FICD. NICD derivatives could suppress the expression of the oncogene protein C-myc, induce apoptosis and cell cycle arrest, restrain HCC proliferation, and reverse EMT. The anticancer mechanism is closely related to the Wnt/b-Catenin signaling pathway. The detailed mechanism should be explored in the future studies. 2.2.6. In vivo anticancer activity Tumor growth inhibition was evaluated in tumor-bearing mice after treatment with various dosages of FICD and COM33. The results are detailed in Tables 3 and 4, Fig.7, S Table 1, and S Fig. 1. The inhibitory rate and tumor weight of the treatment group were significantly different from those of the blank control group

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Fig. 5. The effect of FICD (12) and FICD/sorafenib on cell cycle distribution and apoptosis in HepG2 cells. A: The cell cycle distribution of HepG2 cells treated with FICD (12) for 48 h via flow cytometric analysis. Control group, FICD (10.0 lg/mL), sorafenib (4.0 lg/mL), and FICD (10.0 lg/mL) and sorafenib (4.0 lg/mL). B: Apoptosis in HepG2 cells incubated with FICD (12) or sorafenib for 24 h via flow cytometric analysis. Control group (PBS), FICD (5.0 lg/mL), FICD (10.0 lg/mL), sorafenib (4.0 lg/mL), and FICD (10.0 lg/mL) combined with sorafenib (4.0 lg/mL).

Fig. 6. Western blot analysis of b-Catenin, C-Myc, vimentin, Ki-67, and Cyclin D1 after treatment of HepG2 cells with FICD (12) (A), COM33 (24) (B), and their combination with sorafenib. Notes: b-actin antibody was used as the reference control. A: Sorafenib (4.0 lg/mL), FICD (5.0 lg/mL), FICD (10.0 lg/mL), FICD (20.0 lg/mL), and the combination (FICD: 10.0 lg/mL + Sorafenib: 4.0 lg/mL). B: Sorafenib (4.0 lg/mL), COM33 (2.5 lg/mL), COM33 (5.0 lg/mL), COM33 (10.0 lg/mL), and the combination (COM33: 5.0 lg/mL + Sorafenib: 4.0 lg/mL).

Table 3 Inhibitory effect of FICD (12) and the combination with Sorafenib on H22-bearing mice. Treatment group

Mice number

Dose (mg/kg/d)

Tumor weight (g, Mean ± SD)

Inhibition ratio (%)

Control FICD FICD FICD FICD+Sorafenib FICD+Sorafenib FICD+Sorafenib Sorafeinb

10 10 10 10 10 10 10 10

– 150 100 50 50 + 150 50 + 100 50 + 50 50

2.62 ± 0.92 1.29 ± 0.74** 1.43 ± 0.61** 1.60 ± 0.65** 0.77 ± 0.36** 0.92 ± 0.36** 0.97 ± 0.47** 1.16 ± 0.54**

– 50.8 45.6 39.1 70.6 65.1 63.1 55.8

VS Control group, **P < .01.

(physiological saline). When the dosage increased, the FICD inhibitory rates increased significantly. The tumor inhibition rates exceeded 40%, and the results were statistically significant (Fig. 7A/a–b). Combination with sorafenib significantly increased the inhibitory effect of FICD, indicating that FICD and sorafenib are synergistic, such that the inhibitory rate of the high combination dosage group (50 mg/kg sorafenib + 150 mg/kg FICD) exceeded 70.6%. The synergistic effect of FICD with sorafenib was

supported by the results of in vitro experiments. To investigate the oral absorption and bioavailability of FICD, the pharmacological properties, and the choice of administrational method, a tumor growth inhibition experiment with Kunming mice was performed. The results are showed in S Table 1 and S Fig. 1. There was no statistically significant difference between the different modes of administration (ip vs ig), indicating that FICD could be absorpted under ig administration of FICD.

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Table 4 Inhibitory effect of COM33 (24) on H22-bearing mice. Treatment group

Mice number

Dose (mg/kg/d)

Tumor weight (g, Mean ± SD)

Inhibition ratio (%)

Control COM33 COM33 Combination Sorafeinb

10 10 10 10 10

– 100 50 50 + 50 50

1.89 ± 0.49 0.49 ± 0.35** 1.37 ± 0.61* 0.63 ± 0.49** 0.82 ± 0.66**

– 73.8 27.2 66.5 56.5

VS Control group *p < .05, **P < .01.

Fig. 7. Inhibitory effect of FICD (12) (A), COM33 (24) (B), and their combinations with sorafenib on murine hepatoma H22-bearing mice. (*P < .01, **P < .01).

The tumor inhibition rate of sorafenib was approximately 56.5% according to three batches of different experiments, which indicated that the murine H22-bearing mice could be used to evaluate the targeted therapeutic effect of NICD derivatives and sorafenib. The inhibitory effect of COM33 is detailed in Table 4 and Fig. 7B. The inhibitory rate of high-dose COM33 (100 mg/kg/d) on tumor growth was 73.8%, and its tumor inhibition rate was significantly higher than that of sorafenib (56.5%), (Fig. 7B/b). The effect of this compound on the body weight of mice was small, which indicated that COM33 had better tumor inhibition properties in vivo. The combination also increased the inhibitory effect of COM33, which indicated that COM33 and sorafenib act synergistically. The inhibitory rate of the combination dosage group (50 mg/kg sorafenib + 50 mg/kg COM33) exceeded 66.5%.

compounds, FICD (12) and COM33 (24) were selected for further anticancer research using in vitro and in vivo experiments. The results suggested that structural modifications could significantly improve the anticancer activity of isocorydine. Identifying the mechanisms by which EMT-transformed CSCs initiate relapse could facilitate the development of new or enhanced personalized therapeutic regimens. The association of EMT and CSC may also form the basis for identifying novel targeting agents to improve clinical outcomes in HCC patients. The results of this study and previous investigations about isocorydine and NICD indicated that NICD derivatives could target CSCs and EMT, which are the main therapeutic targets in HCC. COM33 could be a potential targeted chemotherapy drug or be further developed into a combination with conventional chemotherapy drugs to target CSCs and EMT.

3. Conclusion 4. Experimental In summary, four structurally similar natural isocorydine analogs were extracted from plants, and more than twenty isocorydine derivatives were prepared through chemical structure modification. Through the preliminary antitumor screening of the obtained

1 H NMR and 13C NMR spectral data were recorded in CDCl3, acetone d6, CD3OD, or DMSO d6 using Bruker AVANCE HD 400 NMR spectrometers. High-resolution accurate mass spectra

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(HR-ESI-MS) of all final target compounds were obtained on a Bruker Micromass time flight mass spectrometer equipped with an electrospray ionization (ESI) detector. X-ray crystallographic data were collected on a Bruker D8 SMART APEX II. Column chromatography was performed on silica gel (200–300 mesh, Qingdao Puke Parting Materials Co., Ltd., Qingdao, China). TLC was also carried out on silica gel GF254 (Qingdao Marine Chemical Ltd., Qingdao, China). Other chemical solvents and reagents used in synthesis were of analytical grade and provided by Aladdin Industrial Corporation and used without further purification, unless noted specifically. 4.1. General procedure for the preparation of compounds 1–6, 7–11, 12–21, and 22–29 Isocorydine (1), corydine (2), corytuberine (3), and isocorytuberine (4) were extracted from the plants D. leptopodum (Maxim) Fedde and S. yunnanensis Lo which were identified by Professor Zhigang Ma (Lanzhou University), and voucher specimens of both plants were deposited at Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, China. The synthesis of compounds 5 and 6 from isocorydine and the identification of compound 1, 5 and 6 have been reported previously.9 Compound 2: 1H NMR (400 MHz, CDCl3) d 6.95 (1H, s, H-3), 3.00 (2H, m, H-4), 2.62 (2H, m, H-5), 3.69 (1H, dd, J = 16. 4, 3. 6 Hz, H-6a), 3.16 (1H, m, H-7a), 3.11 (1H, m, H-7b), 6.88 (1H, d, J = 8.0 Hz, H-8), 6.98 (1H, d, J = 8.0 Hz, H-9), 3.15 (3H, s, N-CH3), 3.64 (3H, s, 2-OCH3), 3.79 (3H, s, 10-OCH3), 3.85 (3H, s, 11-OCH3). 13C NMR (101 MHz, CDCl3) d 143.7 (C-1), 122.9 (C-1a), 124.7 (C-1b), 152.4 (C-2), 111.6 (C-3), 126.2 (C-3a), 25.5 (C-4), 51.2 (C-5), 62.0 (C-6a), 31.7 (C-7), 126.9 (C-7a), 118.8 (C-8), 111.4 (C-9), 144.0 (C-10), 148.7 (C-11), 118.9 (C-11a), 61.6 (2-OCH3), 55.9 (10-OCH3), 61.2 (11-OCH3), 43.4 (N-CH3). Compound 3: 1H NMR (400 MHz, DMSO d6) d 6.92 (1H, s, H-3), 3.40 (2H, m, H-4), 3.08 (2H, m, H-5), 4.08 (1H, m, H-6a), 3.71 (1H, m, H-7a), 2.97 (1H, m, H-7b), 6.94 (1H, d, J = 8.2 Hz, H-8), 7.00 (1H, d, J = 8.2 Hz, H-9), 3.17 (3H, s, N-CH3), 3.83 (3H, s, 2-OCH3), 3.84 (3H, s, 10-OCH3). 13C NMR (101 MHz, DMSO d6) d 141.8 (C-1), 120.5 (C-1a), 127.0 (C-1b), 149.7 (C-2), 111.1 (C-3), 122.0 (C-3a), 25.9 (C-4), 51.8 (C-5), 62.2 (C-6a), 32.2 (C-7), 122.8 (C-7a), 120.1 (C-8), 111.8 (C-9), 149.1 (C-10), 142.7 (C-11), 120.2 (C-11a), 56.5 (2-OCH3), 56.3 (10-OCH3), 49.1 (N-CH3). Compound 4: 1H NMR (400 MHz, DMSO d6) d 6.68 (1H, s, H-3), 3.14 (2H, m, H-4), 3.05 (2H, m, H-5), 2.67 (1H, m, H-6a), 2.50 (1H, m, H-7a), 2.30 (1H, m, H-7b), 6.68 (1H, d, J = 8.0 Hz, H-8), 6.80 (1H, d, J = 8.0 Hz, H-9), 2.60 (3H, s, N-CH3), 3.74 (3H, s, 2-OCH3), 3.74 (3H, s, 11-OCH3). 13C NMR (101 MHz, DMSO d6) d 142.9 (C-1), 120.9 (C-1a), 128.5 (C-1b), 148.5 (C-2), 109.7 (C-3), 126.0 (C-3a), 27.2 (C-4), 51.9 (C-5), 62.3 (C-6a), 34.1 (C-7), 120.5 (C-7a), 121.4 (C-8), 110.4 (C-9), 144.2 (C-10), 148.6 (C-11), 121.3 (C-11a), 55.4 (2-OCH3), 55.6 (11-OCH3), 42.3 (N-CH3). Synthesis of compounds 7–11. To a solution of carboxylic acid (1.17 mmol), HBTU (0.4495 g, 1.18 mmol), and DIPEA (0.5 mL, 2.21 mmol) in anhydrous DMF (20 mL), a solution of NICD (0.4186 g 1.17 mmol) in anhydrous DMF (10 mL) was added at 44 °C. After the mixture was stirred for 6 h, it was diluted with CH2Cl2 (100 mL) and extracted with water (250 mL
3). The organic phase was dried with Na2SO4 and concentrated under reduced pressure. The residue was purified via column chromatography on silica gel with ethyl acetate/ methanol (V:V = 8:1) to afford the compound as dark green solids. Compound 7: yield 53.2%, 1H NMR (400 MHz, CDCl3) d 7.71(dd, J = 8.0, 2.0, 1H), 7.58 (dd, J = 8.0 2.0Hz, 1H), 7.52 (s, 1H), 7.34 (dd, J = 2.0, 2.0, Hz, 1H), 6.68 (s, 1H), 3.88 (s, 3H), 3.87 (s, 3H), 3.82 (s,

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1H), 3.70 (s, 3H), 3.48 (s, 1H), 3.42 (d, J = 12.0 Hz 1H), 3.20–2.88 (m, 2H), 2.68 (d, J = 16.0 Hz, 1H), 2.50 (s, 3H), 2.15 (t, J = 12.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 164.1, 152.7, 151.6, 149.6, 145.0, 142.4, 138.0, 137.6, 132.2, 131.8, 131.8, 126.4, 125.2, 124.8, 123.2, 122.9, 122.3, 117.1, 112.5, 111.6, 62.2, 61.4, 56.2, 55.9, 52.5, 43.9, 29.7, 28.6. HRMS (ESI, m/z): calculated for C28H27ClF3N2O5 [M+H]+ 563.1555, found 563.1541. Compound 8: yield, 81.5%, 1H NMR (400 MHz, acetone d6) d 7.75 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 16.0 Hz, 1H), 7.61 (dd, J = 8.0, 2.0 Hz, 1H), 7.52 (dd, J = 8.0, 2.0 Hz, 1H), 7.38 (m, 1H), 7.29 (s, 1H), 7.22 (m, 1H), 7.04 (d, J = 16.0 Hz, 1H), 6.79 (s, 1H), 4.06 (dd, J = 16.0, 8.0 Hz, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.66 (s, 3H), 3.42 (dd, J = 16.0, 2.0 Hz, 1H), 3.36–3.24 (m, 2H), 2.82 (m, 2H), 2.71 (s, 3H), 2.42 (t, J = 12.0 Hz, 1H). 13C NMR (101 MHz, acetone d6) d 164.4, 152.0, 148.9, 143.3, 142.9, 142.3, 140.6, 135.2, 129.6, 128.9, 128.6, 127.9, 127.8, 127.0, 125.1, 123.6, 121.9, 118.5, 111.4, 110.4, 110.1, 62.1, 61.3, 59.7, 55.7, 55.4, 52.1, 41.8, 27.2. HRMS (ESI, m/z): calculated for C29H31N2O5 [M+H]+ 487.2227, found 487.2219. Compound 9: yield, 60.1%, 1H NMR (400 MHz, CDCl3) d 8.46 (d, J = 8.0 Hz, 1H), 8.36 (d, J = 7.8.0 Hz, 1H), 8.16 (dd, J = 7.8, 0.8 Hz, 1H), 7.77 (s, 1H), 7.44–7.39 (d, J = 8.0 Hz, 1H), 7.36 (s, 1H), 7.28 (s, 1H), 7.12 (dd, J = 8.0, 4.0 Hz, 1H), 6.72 (s, 1H), 4.34 (s, H-N), 3.94 (s, 3H), 3.91 (s, 3H), 3.71 (s, 3H), 3.27 (dd, J = 12.0, 4.0 Hz, 1H), 3.23–3.14 (m, 1H), 3.05 (m, 1H), 2.92 (m, 1H), 2.71 (m, 2H), 2.50 (s, 3H), 2.23 (t, J = 12.0 Hz, 1H), 2.05 (s, 1H), 1.26 (t, J = 8.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 161.8, 160.7, 160.6, 158.3, 151.4, 149.1, 145.6, 142.6, 142.4, 134.6, 133.7, 132.2, 131.5, 129.7, 129.5, 128.7, 128.3, 127.2, 125.7, 125.0, 123.7, 121.5, 120.6, 116.8, 116.5, 111.4, 109.0, 62.2, 60.4, 56.2, 55.9, 52.5, 43.6, 37.8, 29.7, 29.0. HRMS (ESI, m/z): calculated for C36H34FN4O6 [M+H]+ 637.2457, found 637.2445. Compound 10: yield, 69.1%, 1H NMR (400 MHz, CDCl3) d 7.48 (m, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.96 (s, 1H), 6.64 (s, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 3.65 (s, 3H), 3.33 (d, J = 12.0 Hz, 1H), 3.18–3.04 (m, 1H), 2.70 (m, 1H), 2.37 (s, 3H), 2.36 (m, 2H), 2.07 (m, 2H). 13C NMR (101 MHz, CDCl3) d 164.4, 152.0, 148.9, 143.3, 142.9, 142.3, 140.6, 135.2, 129.6, 128.9, 128.6, 128.0, 127.8, 127.01, 125.13, 123.6, 121.9, 118.5, 62.1, 61.3, 55.7, 55.4, 52.1, 41.8, 29.0, 27.3. HRMS (ESI, m/z): calculated for C26H28N3O5 [M+H]+ 462.2023, found 462.2016. Compound 11: yield, 56.1%, 1H NMR (400 MHz, CDCl3) d 7.75 (dd, J = 8.0, 2.0 Hz, 1H), 7.75 (7.8, 8.0 1H), 7.68 (s, 1H), 7.52 (d, J = 7.8, 8.0 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.79 (s, 1H), 4.14 (dd, J = 14.0, 7.2 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.72 (s, 3H), 3.54 (d, J = 13.8 Hz, 1H), 3.33–3.21 (m, 1H), 3.12 (m, 1H), 3.00 (m, 1H), 2.75 (m, 1H), 2.58 (s, 3H), 2.06 (m, 1H). 13C NMR (101 MHz, CDCl3) d 154.1, 151.6, 149.9, 142.5, 135.3, 129.5, 128.8, 127.7, 125.4, 125.1, 125.1, 123.4, 122.3, 121.1, 120.6, 120.5, 111.6, 111.1, 110.9, 62.5, 62.2, 56.3, 55.9, 52.6, 43.5, 29.7, 29.6. HRMS (ESI, m/ z): calcd for C27H30N3O5 [M+H]+ 476.1947, found 476.2186. Synthesis of compounds 12–21. To a solution of NICD (0.7915 g, 2.23 mmol) in anhydrous CH2Cl2 (50 mL), a solution of phenyl isocyanate (2.33 mmol) in anhydrous CH2Cl2 (50 mL) was added at 0 °C under Nitrogen. After the mixture was stirred for 1 h, it was extracted with 250 mL of water three times. The organic phase was dried with Na2SO4 and concentrated under reduced pressure. The residue was purified through column chromatography on silica gel with petroleum ether/ethyl acetate/methanol (V:V:V = 6:2:1) to afford the compound as a brown solid. Compound 12 (FICD): yield, 72.3%, 1H NMR (400 MHz, CDCl3) d 7.71 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 6.99 (s, 1H), 6.70 (s, 1H), 3.89 (s, 6H), 3.71 (s, 3H), 3.41 (dd, J = 13.2, 1.2 Hz, 1H), 3.19 (t, J = 14.0 Hz, 1H), 3.04–2.98 (m, 1H), 2.83 (m, 1H), 2.70 (m, 1H), 2.47 (s, 3H), 2.15 (t, J = 14.0 Hz, 1H), 1.91 (s, 1H). 13C NMR (101 MHz, CDCl3) d 154.1, 152.7, 151.5, 149.6, 144.9, 142.4, 138.0, 137.6, 132.2, 131.8, 131.8, 126.4, 125.2, 124.8, 123.2, 122.8, 122.3,

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117.1, 112.5, 111.6, 62.2, 61.4, 56.2, 55.9, 52.5, 43.9, 29.7, 28.6. HRMS (ESI, m/z): calcd for C28H28ClF3N3O5 [M+H]+ 578.1651, found 578.1670. Compound 13: yield, 46.9%, 1H NMR (400 MHz, CDCl3) d 7.60 (s, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.24 (s, 1H), 7.19 (d, J = 8.0 Hz, 1H), 6.75 (s, 1H), 3.89 (s, 6H), 3.70 (s, 3H), 3.43 (d, J = 14.0 Hz, 1H), 3.22 (m, 1H), 3.08 (m, 1H), 2.83 (m, 1H), 2.74 (m, 1H), 2.52 (s, 3H), 2.18 (t, J = 13.6 Hz, 1H), 2.06 (d, J = 3.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 154.0, 151.6, 149.7, 142.5, 135.3, 129.5, 128.8, 127.7, 125.4, 125.1, 125.0, 123.4, 122.3, 121.1, 120.6, 120.5, 111.6, 111.0, 110.9, 62.5, 62.2, 56.2, 55.9, 52.6, 43.5, 29.7, 26.9. HRMS (ESI, m/z): calculated for C27H28Cl2N3O5 [M+H]+ 544.1401, found 544.1386. Compound 14: yield, 30.7%, 1H NMR (400 MHz, CDCl3) d 7.44 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.28 (s, 1H), 7.12 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 6.61 (s, 1H), 3.87–3.80 (m, 3H), 3.68 (brs, 6H), 3.08 (s, 2H), 3.05 (d, J = 2.4 Hz, 1H), 2.99 (s, 1H), 2.79 (d, J = 14.0 Hz, 2H), 2.47 (s, 3H), 2.35 (s, 3H), 2.29 (s, 3H), 1.99 (s, 1H). 13C NMR (101 MHz, CDCl3) d 153.4 (2
C), 151.0, 150.1, 145.1, 136.8, 134.8, 133.9, 133.6, 132.8, 131.3, 129.6 (2
C), 129.0 (2
C), 127.8, 126.2, 126.2, 123.3, 120.2, 118.7 (2
C), 112.4, 107.7, 62.2, 61.2, 56.0, 55.5, 52.6, 43.9, 28.7, 26.9, 20.8, 20.7. HRMS (ESI, m/z): calculated for C36H38N4NaO6 [M+Na]+ 645.2684, found 645.2645. Compound 15: yield, 43.9%; 1H NMR (400 MHz, CDCl3) d 7.16 (d, J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 6.96 (s, 1H), 6.69 (s, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 3.65 (s, 3H), 3.33 (d, J = 16.0 Hz, 1H), 3.18–3.04 (m, 1H), 2.95 (dd, J = 11.2, 5.6 Hz, 1H), 2.72 (d, J = 8.0, 1H), 2.63 (d, J = 8.0, 1H), 2.37 (s, 3H), 2.36 (s, 3H), 2.07 (d, J = 8.0 Hz, 2H). 13C NMR (101 MHz, CDCl3) d 154.7, 151.4, 149.4, 142.9, 142.3, 136.1, 132.5, 129.8, 128.7, 128.3 (2
C), 125.7, 125.4, 121.0, 120.6 (2
C), 111.5, 110.5, 62.3, 62.2, 60.4, 56.2, 55.9, 52.5, 43.5, 29.7, 28.9, 16.8. HRMS (ESI, m/z): calculated for C28H32N3O5S [M+H]+ 522.2057, found 522.2066. Compound 16: yield, 3.9%, 1H NMR (400 MHz, CDCl3) d 8.32 (d, J = 8.0 Hz, 1H), 7.28 (s, 1H), 7.25 (dd, J = 8.0, 2.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.95 (dd, J = 8.0, 2.0 Hz, 1H), 6.75 (s, 1H), 4.14 (dd, J = 16.0, 8.0 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H), 3.72 (s, 3H), 3.54 (d, J = 12.0 Hz, 1H), 3.33–3.21 (m, 1H), 3.12 (d, J = 12.0 Hz, 1H), 3.00 (brs, 1H), 2.75 (dd, J = 16.0, 2.0 Hz, 1H), 2.58 (s, 3H), 2.30 (t, J = 16.0 Hz, 1H), 2.06 (d, J = 3.8 Hz, 1H), 1.35–1.21 (m, 2H). 13C NMR (101 MHz, CDCl3) d 154.1, 151.6, 149.9, 142.5, 135.3, 129.5, 128.8, 127.7, 125.4, 125.1, 125.0, 123.4, 122.3, 121.1, 120.6, 120.5, 111.6, 111.0, 110.9, 62.5, 62.2, 56.2, 55.9, 52.6, 43.5, 29.7, 29.6. HRMS (ESI, m/z): calculated for C27H29ClN3O5 [M+H]+ 510.1790, found 510.1790. Compound 17: yield, 43.0%, 1H NMR (400 MHz, CDCl3) d 7.65 (s, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.28 (s, 1H), 7.25–7.10 (m, 4H), 7.01 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.68 (s, 1H), 3.93 (s, 1H), 3.85 (s, 3H), 3.67 (brs, 6H), 3.19 (d, J = 12.0 Hz, 1H), 3.03 (m, 1H), 2.86 (d, J = 12.0 Hz, 2H), 2.52 (s, 3H), 2.27–2.09 (m, 2H), 2.07 (s, 1H). 13C NMR (101 MHz, CDCl3) d 152.9, 152.4, 151.4, 150.5, 145.8, 135.9, 134.4, 132.6, 129.3, 129.1, 128.8, 127.8, 127.6, 127.3, 126.9, 124.2, 123.4, 123.2, 122.9, 122.8, 122.5, 120.9, 120.7, 113.4, 107.7, 62.3, 61.2, 60.4, 56.3, 55.9, 52.7, 43.8, 28.6, 28.5. HRMS (ESI, m/z): calculated for C34H32Cl2N4NaO6 [M+Na]+ 685.1591, found 685.1588. Compound 18: yield, 61.6%, 1H NMR (400 MHz, CDCl3) d 7.28 (s, 1H), 7.21 (m, 5H), 7.05 (m, 2H), 6.94 (m, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.58 (s, 1H), 3.85 (s, 3H), 3.61 (m, 2H), 3.31–2.82 (m, 2H), 2.28 (s, 6H), 2.26 (m, 2H), 2.18 (s, 3H), 1.28 (s, 1H). 13C NMR (101 MHz, CDCl3) d 153.2, 152.4, 151.4, 150.5, 145.4, 139.1, 138.8, 138.3, 129.0, 128.8, 128.5, 127.8, 127.6, 126.1, 125.0, 124.1, 123.3, 123.0, 122.9, 122.8, 122.5, 120.5, 120.3, 119.2, 117.0, 116.7, 115.8, 107.7, 62.2, 61.3, 55.8, 55.6, 52.6, 43.8, 29.7, 28.6. HRMS (ESI, m/z): calculated for C36H39N4O6 [M+H]+ 623.2864, found 623.2848.

Compound 19: yield, 9.1%, 1H NMR (400 MHz, CDCl3) d 7.51 (s, 3H), 7.28 (s, 2H), 6.73 (s, 1H), 3.92 (s, 6H), 3.73 (s, 3H), 3.47 (d, J = 16.0 Hz, 1H), 3.24 (m, 1H), 3.09 (m, 1H), 2.93 (m, 1H), 2.75 (d, J = 16.0Hz, 1H), 2.53 (s, 3H), 2.29–2.15 (m, 1H), 1.27 (s, 1H). 13C NMR (101 MHz, CDCl3) d 154.0, 151.8, 151.7, 149.8, 142.6, 141.8, 136.8, 136.6, 129.5, 126.2, 126.2, 125.3, 124.8, 122.8, 121.0, 120.5, 120.4, 118.6 (2
C), 111.6, 110.5, 62.6, 62.2, 56.3, 55.9, 52.5, 43.2, 29.7, 29.6. HRMS (ESI, m/z): calculated for C28H29F3N3O5 [M+H]+ 544.2054, found 544.2050. Compound 20: yield, 44.5%, 1H NMR (400 MHz, CDCl3) d 7.65– 7.56 (m, 5H), 7.37 (m, 3H), 7.28 (s, 1H), 6.62 (s, 1H), 3.90–3.78 (m, 1H), 3.64–3.49 (s, 6H), 3.04–2.96 (s, 3H), 2.82–2.72 (m, 2H), 2.51 (s, 2H), 2.28–2.19 (m, 1H), 2.13–2.08 (m, 1H). 13C NMR (101 MHz, CDCl3) d 152.7 (2
C), 151.2, 150.2, 145.0, 144.2, 142.3, 140.3, 133.5, 132.7, 126.5 (2
C), 125.9 (2
C), 125.6 (2
C), 125.3, 124.4, 124.1, 123.0, 122.9, 122.7, 119.5 (2
C), 117.9 (2
C), 112.4, 107.5, 62.1, 61.3, 55.8, 55.7, 52.5, 43.8, 29.7, 28.5. HRMS (ESI, m/z): calculated for C36H33F6N4O6 [M+H]+ 731.2299, found 731.2286. Compound 21: yield, 64.5%, 1H NMR (400 MHz, acetone d6) d 7.63–7.50 (m, 5H), 7.08–7.03 (m, 4H), 6.80 (s, 1H), 3.91 (s, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.68 (d, J = 4.0 Hz, 1H), 3.45 (s, 3H), 3.36 (dd, J = 14.0, 2.0 Hz, 1H), 3.05–2.98 (m, 1H), 2.79 (m, 1H), 2.68 (m, 1H), 2.50 (s, 3H), 1.29 (s, 1H). 13C NMR (101 MHz, acetoned6) d 151.8, 151.3, 150.1, 148.0, 145.1, 140.3, 135.8, 134.3, 133.2, 132.5, 129.2, 127.9, 125.8, 124.4, 121.9, 121.3, 120.5, 124.3, 120.1, 118.9, 115.2, 115.1, 115.0, 114.9, 112.6, 107.5, 62.9, 60.5, 55.6, 55.6, 52.7, 43.3, 30.1, 29.8. HRMS (ESI, m/z): calculated for C34H33F2N4O6 [M+H]+ 631.2363, found 631.2354. Synthesis of compound 22. To a solution of 2-chloro-5nitrobenzotrifluoride (2.5363 g, 11.27 mmol) and K2CO3 (4.2203 g, 30.58 mmol) in DMF (60 mL), tert-butyl (piperidin-4-ylmethyl) carbamate (2.4127 g, 11.27 mmol) was added. The mixture was stirred at 100 °C for 8 h. The solvent was removed under vacuum after it was allowed to cool down to 25 °C. The residue was dissolved in ethyl acetate (100 mL), washed with water (250 mL
3) and brine (250 mL
3), dried with Na2SO4, and then concentrated to afford the crude nitrobenzene product as a light yellow solid. This was used in the following steps without further purification. To a solution of the crude nitrobenzene in methanol (50 mL), 10% Pd/C (2.0 g, 20% w/w) was added at 25 °C. The reaction mixture was stirred under hydrogen in a high-pressure reactor (0.3 MPa) for 3 h and then filtered through busher funnel. The filtrate was concentrated to obtain the residue, which was purified through column chromatography on silica gel with ethyl acetate/methanol (V:V = 8:1) to afford compound 22 as a light brown solid. Yield, 65.4% in two steps, 1H NMR (400 MHz, CDCl3) d 7.17 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.81 (dd, J = 8.0, 2.0 Hz, 1H), 4.63 (s, H-N, 1H), 3.94 (brs, H-N, 1H), 3.06 (t, J = 12.0 Hz, 2H), 2.95 (dd, J = 12.0, 2.0 Hz, 2H), 2.63 (t, J = 12.0 Hz, 2H), 1.70 (dd, J = 12.0, 2.0 Hz, 2H), 1.45 (s, 9H), 1.38 (dd, J = 12.0, 4.0 Hz, 2H). 1.23 (m, 1H) 13C NMR (101 MHz, CDCl3) d 156.1, 144.5, 142.9, 125.6, 125.3, 120.8 118.8, 113.3, 79.1, 54.1 (2
C), 46.4, 36.2, 30.5 (2
C), 29.8 (3
C). HRMS (ESI, m/z): calculated for C18H27F3N3O2 [M +H]+ 374.2050, found 374.2038. Synthesis of compound 23. To a solution of triphosgene (0.7537 g, 2.54 mmol) in anhydrous CH2Cl2 (30 mL), a solution of NICD (2.0870 g, 5.86 mmol), Et3N (1 mL, 7.30 mmol), and DMAP (0.0753 g, 0.62 mmol) in anhydrous CH2Cl2 (30 mL) was added at 0 °C under Nitrogen. After stirring for 2 h, a solution of 22 (2.1862 g, 5.86 mmol), Et3N (1 mL, 7.30 mmol), and DMAP (0.0757 g, 0.62 mmol) in anhydrous CH2Cl2 (30 mL) was added to the above-mentioned reaction mixture at 0 °C under nitrogen. The mixture was warmed to room temperature and stirred for 2 h. The reaction mixture was washed with water (250 mL
3) and brine (250 mL
3), dried with Na2SO4, filtered, and concentrated

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under reduced pressure. The residue was purified using column chromatography on silica gel with dichloromethane/methanol (V: V = 20:1) to afford compound 23 as a brown solid. Yield, 58.7%, 1 H NMR (400 MHz, CDCl3) d 7.50 (d, J = 8.0 Hz, 1H), 7.31 (brs, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.96 (s, 1H), 6.64 (s, 1H), 4.83 (s, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.58 (s, 3H), 3.26 (d, J = 8.0 Hz, 1H), 2.99 (m, 3H), 2.91–2.74 (m, 4H), 2.62 (m, 3H), 2.48 (m, 2H), 2.33 (s, 3H), 2.07–1.95 (m, 1H), 1.62 (m, 2H), 1.42 (s, 9H), 1.36–1.21 (m, 2H). 13C NMR (101 MHz, CDCl3) d 156.4, 154.7, 151.2, 149.1, 148.1, 142.2, 135.5, 129.9, 128.9, 127.6, 127.3, 125.9, 125.4, 125.0, 124.6, 123.6, 122.3, 120.8, 118.1, 111.4, 110.2, 79.3, 63.2, 62.2, 62.0, 56.0, 55.8, 53.8, 52.5, 46.3, 43.5, 37.3, 36.2, 30.4, 29.7, 28.9, 28.4 (3
C). HRMS (ESI, m/z): calculated for C39H49F3N5O7 [M+H]+ 756.3579, found 756.3571. Synthesis of compound 24. Compound 23 (1.4590 g, 1.93 mmol) was dissolved in 4 N HCl in ethyl acetate (20 mL) at 25 °C. The resulting mixture was stirred for 2 h and then filtered to obtain compound 24 as an off-white solid. Yield, 91.6%, 1H NMR (400 MHz, CD3OD) d 8.03 (d, J = 2.0 Hz, 1H), 7.75 (dd, J = 8.0, 2.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.16 (s, 1H), 6.98 (s, 1H), 4.02 (dd, J = 12.0, 2.0 Hz, 2H), 3.91 (s, 3H), 3.90 (s, 3H), 3.81–3.75 (m, 2H), 3.71 (s, 3H), 3.58 (dd, J = 14.0, 2.0 Hz, 2H), 3.47 (m, 1H), 3.45 (s, 1H), 3.41 (d, J = 4.0 Hz, 1H), 3.18 (s, 4H), 3.08 (m, 2H), 2.95 (d, J = 8.0 Hz, 3H), 2.52 (t, J = 12.0 Hz, 1H), 2.01 (d, J = 12.0 Hz, 4H), 1.67 (d, J = 10.7 Hz, 2H). 13C NMR (101 MHz, CD3OD) d 156.2, 154.5, 150.3, 145.4, 143.3, 140.4, 138.2, 128.1, 127.6, 126.9, 126.6, 126.4, 125.7, 124.3, 123.7, 122.7, 120.7, 118.4, 112.6, 111.4, 63.8, 62.3, 56.8, 56.6, 56.2, 53.5, 47.0, 45.4, 42.4, 34.2, 30.1, 29.0, 28.9, 26.9. HRMS (ESI, m/z): calculated for C34H41F3N5O5 [M+H]+ 656.3054, found 656.3039. Synthesis of compound 25. To a solution of compound 24 (0.1903 g, 0.29 mmol) and Et3N (0.13 mL, 0.95 mmol) in anhydrous DMF (3 mL) at 0 °C under nitrogen, propionyl chloride (0.03 mL, 0.31 mmol) was added. After being stirred for 0.5 h, the reaction mixture was quenched with water, diluted with CH2Cl2 (10 mL), and washed with water (20 mL
3) and brine (20 mL
3). The organic phase was dried with Na2SO4, filtered, and concentrated under vacuum to obtain the residue, which was purified through column chromatography on silica gel with petroleum dichloromethane/methanol (V:V = 20:1) to afford the compound as a white solid. Yield, 48.6%. 1H NMR (400 MHz, DMSO d6) d 7.81 (dd, J = 8.0, 2.0 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.07 (s, 1H), 6.93 (s, 1H), 3.89 (s, 3H), 3.80 (s, 3H), 3.69 (s, 3H), 3.55 (m, 2H), 3.44 (dd, J = 14.0, 2.0 Hz, 2H), 2.65 (d, J = 3.8 Hz, 1H), 2.41 (s, 3H), 2.35 (s, 1H), 2.24 (d, J = 4.0 Hz, 1H), 2.12 (m, 4H), 2.08 (dd, J = 14.0, 2.0 Hz, 2H), 1.86 (t, J = 6.0 Hz, 3H), 1.67 (t, J = 14.0 Hz, 1H), 1.20 (m, 5H), 1.01 (t, J = 8.0, 2.0 Hz, 3H). 13C NMR (101 MHz, DMSO d6) d 173.3, 153.9, 151.5, 148.1, 147.1, 143.1, 141.4, 137.7, 129.4, 127.3, 126.6, 126.3, 125.8, 125.6, 125.3, 123.8, 123.1, 123.0, 120.4, 116.4, 111.9, 109.5, 62.9, 61.7, 56.26, 56.2, 54.1, 52.7, 44.5, 44.1, 40.6, 40.4, 40.2, 40.0, 39.8, 39.6, 39.4, 35.9, 30.8, 30.2, 29.0, 10.6. HRMS (ESI, m/z): calculated for C37H45F3N5O6 [M +H]+ 712.3322, found 712.3308. Compound 26 was prepared following the synthetic procedure for compound 22. Yield, 66.7% in two steps; 1H NMR (400 MHz, CDCl3) d 7.15 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.78 (dd, J = 8.0, 2.0 Hz, 1H), 4.54 (s, 1H, H-N), 2.90 (d, J = 12.0 Hz, 2H), 2.72 (t, J = 8.0 Hz, 2H), 1.95 (d, J = 12.0 Hz, 2H), 1.54 (ddd, J = 14.0, 12.0, 4.0 Hz, 3H), 1.46 (s, 9H). 13C NMR (101 MHz, CDCl3) d 155.3, 143.7, 143.5, 125.2, 122.5, 118.6, 113.0, 100.1, 79.2, 53.1, 47.7, 33.2, 28.4 (3
C). HRMS (ESI, m/z): calculated for C17H24F3N3NaO2 [M+H]+ 382.1713, found 382.1718. Compound 27 was prepared following the synthetic procedure for compound 23. Yield, 43.2%, 1H NMR (400 MHz, CDCl3) d 7.51 (s, 1H), 7.40 (dd, J = 8.0, 2.0 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.95 (s, 1H), 6.62 (s, 1H), 4.64 (s, 1H), 3.81 (s, 3H), 3.72 (s, 3H), 3.55

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(s, 3H), 3.33 (s, 1H), 3.23 (d, J = 12.0 Hz, 2H), 3.04 (s, 2H), 2.81 (m, 4H), 2.60 (m, 4H), 2.31 (s, 3H), 1.99 (t, J = 12.0 Hz, 1H), 1.86 (s, 2H), 1.41 (s, 9H). 13C NMR (101 MHz, CDCl3) d 155.5, 154.7, 151.2, 148.9, 147.4, 142.2, 135.9, 129.8, 128.8, 127.6, 127.3, 126.1, 125.4, 125.0, 124.5, 123.4, 122.3, 120.7, 117.9, 111.3, 110.1, 79.3, 62.1, 62.0, 55.9, 55.8, 52.7, 52.4, 50.2, 47.6, 43.4, 33.0, 29.6, 28.8, 28.4 (3
C). HRMS (ESI, m/z): calculated for C38H47F3N5O7 [M+H]+ 742.3422, found 742.3430. Compound 28 was prepared following the synthetic procedure for compound 24. Yield, 92.8%; 1H NMR (400 MHz, CD3OD) d 7.91 (d, J = 2.0 Hz, 1H), 7.63 (dd, J = 8.0, 2.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.14 (s, 1H), 6.97 (s, 1H), 4.00 (d, J = 12.0 Hz, 2H), 3.90 (s, 3H), 3.89 (s, 3H), 3.80–3.72 (m, 2H), 3.70 (s, 3H), 3.61–3.53 (m, 2H), 3.50–3.37 (m, 3H), 3.28–3.21 (m, 2H), 3.17 (s, 3H), 3.11– 2.97 (m, 4H), 2.87 (m, 2H), 2.51 (t, J = 12.0 Hz, 1H), 2.07 (m, 2H), 1.81 (m, 2H). 13C NMR (101 MHz, CD3OD) d 155.0, 153.2, 149.0, 146.0, 144.0, 141.8, 137.3, 126.9, 126.2, 125.2, 124.9, 122.8, 122.6, 122.3, 121.4, 119.3, 116.8, 111.2, 110.1, 68.1, 62.5, 60.9, 55.4, 55.2, 52.2, 51.8, 41.0, 30.5, 29.7, 27.6, 27.4, 25.6. HRMS (ESI, m/z): calculated for C33H39F3N5O5 [M+H]+ 642.2898, found 642.2891. Compound 29 was prepared following the synthetic procedure for compound 25. Yield, 48.3%;1H NMR (400 MHz, CD3OD) d 7.78 (d, J = 2.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.18 (s, 3H), 6.86 (s, 3H), 3.84 (s, 3H), 3.80 (s, 3H), 3.62 (s, 3H), 3.19 (d, J = 2.0 Hz, 1H), 3.01 (m, 4H), 2.86 (m, 2H), 2.63 (m, 3H), 2.41 (m, 2H), 2.35 (s, 3H), 2.10 (m, 2H), 1.85 (m, 1H), 1.67 (m, 1H), 1.24 (m, 1H), 1.01 (t, J = 8.0 Hz, 3H). 13C NMR (101 MHz, CD3OD) d 173.3, 153.9, 151.4, 147.1, 141.4, 137.7, 127.3, 127.0, 126.9, 126.3, 126.2, 126.3, 125.6, 123.8, 123.0, 120.4, 116.4, 111.9, 109.5, 79.6, 62.9, 61.7, 56.3, 56.2, 54.1, 52.7, 44.5, 44.1, 39.0, 35.9, 30.8, 30.2, 29.0, 10.6. HRMS (ESI, m/z): calculated for C36H43F3N5O6 [M +H]+ 698.3160, found 698.3158. 4.2. Biological evaluation 4.2.1. Materials All human cancer cell lines used in this study were provided by the Cell Bank of the Biochemistry and Cell Biology at the China Academy of Sciences (Shanghai, China). Murine hepatoma cell line H22 was obtained from the cell library of the Institute of Cancer Biology and Drug Discovery, Lanzhou University. All cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 g/mL of streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. Kunming male mice weighing 18.0–22.0 g were purchased from the Experimental Animal Center at Lanzhou University. The use and treatment of mice were in accordance with institutional guidelines for Laboratory Animal Care. 4.2.2. Cell growth inhibition assay The anticancer activities of all compounds were investigated in three human cancer cell lines (HepG2, HeLa, and MGC-803). The in vitro proliferative response of the cancer cells was estimated using an MTT dye assay. Cancer cells were maintained in RPMI 1640 and in DMEM medium, respectively. After counting the number of cells with a hemocytometer by using the 0.04% Trypan Blue dye exclusion technique, the cells were cultured in 96-well microplates (5
103 cells/100 mL in each well) with RPMI 1640 medium and incubated for 24 h at 37 °C in 5% CO2. Inhibitor solutions were prepared by serial dilution of 5 mM DMSO stock solution with the culture medium. The final amount of DMSO per well was maintained below 0.1% V/V. The cells were then treated with different concentrations of the test compounds, positive control sorafenib, and medium alone with or without 0.1% DMSO as negative controls for 48 h at 37 °C with 5% CO2. Next, 10 mL MTT solution diluted in

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culture medium (5 mg/mL) was added to each well and incubated for 4 h. The supernatants were carefully extracted, and formazan precipitates were dissolved in 100 mL of DMSO. Plates were then shaken vigorously (300 rpm) for 5 min. Finally, the difference in absorbance at 570 nm was measured using a Microplate Spectrophotometer (DNM-9602). Each assay was performed in triplicate. The inhibition percentage of test compounds with concentrations of 10–100 mg/mL was calculated as follows: (1  DODwells treated with compounds/DODwells treated with medium alone)
100. Next, IC50 values were calculated by nonlinear regression analysis using SPSS 19 (Version 6.0) software and expressed as mean values. The CI data were calculated by using the computer software from CompuSyn for drug combination (ComboSyn, Inc). 4.2.3. Cell cycle distribution detected via flow cytometry assay For cell cycle analysis, 2
105 cells were plated in a 6-well culture plates and allowed to grow for 24 h. The HepG2 cells were then incubated with 1 mM thymidine (Sigma-Aldrich) for 24 h to synchronize cells at the G1/S boundary. The HepG2 cells were treated with fresh media containing different concentrations of FICD, positive control sorafenib, and the combination of sorafenib and FICD for 24 h. After incubation, cells were harvested and washed twice with cold PBS, and fixed with cold 70% ethanol at 20 °C overnight. The cells were then treated with 100 mg/mL RNase A for 30 min at 37 °C after being washed twice with cold PBS, and finally stained with 1 mg/mL propidium iodide (PI) in the dark at 4 °C for 1 h. Cells were subsequently analyzed by flow cytometric analysis. Analyses were performed with the system software (Cell Quest; BD Biosciences). 4.2.4. Apoptosis analysis Apoptosis was examined via flow cytometry analysis of Annexin V/PI staining. HepG2 cells were seeded into each well of 96-well plates at a density of 1
105 cells/mL, and DMEM with 10% FBS was added to obtain a final volume of 2 mL. The plates were then incubated overnight and treated with different concentrations of FICD, positive control sorafenib, and the combination of sorafenib and FICD for 24 h. Briefly, cells were harvested and washed three times with ice-cold PBS, and then suspended in Annexin-binding buffer at a concentration of 5
105 cells/mL. Cells were incubated with 5 lL annexin V-FITC and 5 lL PI for 30 min at 25 °C in the dark. The cells were analyzed by system software (Cell Quest; BD Biosciences). 4.2.5. Western blot analysis Western blot analyses were performed as described previously.23 HepG2 cells were treated with different concentrations of FICD (12), COM33 (24), and the combination for 48 h. Following this, cells were harvested, centrifuged, and washed twice with icecold PBS. The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 20 min, the lysates were centrifuged at 12,000 rpm at 4 °C for 5 min. The protein concentration in the supernatant was analyzed using BCA protein assay reagents (Solarbio, China). Equal amounts of protein per line were separated by 12% SDS polyacrylamide gel electrophoresis and transferred to PVDF Hybond-P membranes (GE Healthcare). The membranes were incubated with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) buffer for 1 h. Next, the membranes were gently rotated overnight at 4 °C. The membranes were then incubated with primary antibodies against b-Catenin, C-myc, CyclinD1, Ki67, and Vimentin (Abcam, UK) overnight at 4 °C, followed by incubation with peroxidase-labeled secondary antibodies for 2 h. All membranes were washed with TBST three times for 15 min each, and the protein blots were analyzed with chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The X-ray films were developed with a developer and fixed by using a fixer solution.

4.2.6. Anticancer activity in vivo Model mice ascites (0.2 mL), including 1
107 H22 cells/mL, were injected into the right axilla of mice. From the second day after the implantation of cancer cells, tumor-bearing mice were grouped randomly as follows: the blank control group was treated with physiological saline (0.9%), and the positive control group was treated with sorafenib by intraperitoneal injection (ip) at a dose of 50 mg/kg. Medicated groups were treated by ip or intragastric administration (ig) with targeted compounds at 50 mg/kg, 100 mg/kg, and 150 mg/kg (except for COM33). Subsequently, mice were treated daily for the following 10 days. Twenty-four hours after the last administration, mice were killed, and their tumors were totally excised and accurately weighed. The anticancer activity of isocorydine derivatives in vivo is expressed as inhibitory rate (IR), which was calculated using the following formula:

IRð%Þ ¼ ð1  mean tumor weight of dose group=
mean tumor weight of control groupÞ
100%:

4.2.7. Statistical analysis All statistical analyses were performed using SPSS Version 9.0. Data were analyzed using one-way ANOVA. Student’s t-tests were used to analyze the significance of the differences between treated and control groups. All data are presented as mean values with their standard deviation (S.D), where p  .01 indicated statistical significance. Acknowledgment This study was supported by the National Natural Science Foundation of China (NSFC No. 21672225). A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.bmc.2017.10.027. References 1. Sandeep C, Shashikanth P, Onica L, et al. New aporphinoid 5-HT2A and a1A antagonists via structural manipulations of nantenine. Bioorg Med Chem. 2011;19:5861–5868. 2. Ye N, Wu Q, Zhu L, et al. Further SAR study on 11-O-substituted aporphine analogues: identification of highly potent dopamine D3 receptor ligands. Bioorg Med Chem. 2011;19:1999–2008. 3. Guinaudeau H, Leboeuf M, Cave A. Aporphinoid alkaloids. J Nat Prod. 1994;57:1033–1135. 4. Waud RA. The pharmacological action of the alkaloids of Fumaraceous plants I Isocorydine. J Pharmacol Exp Ther. 1934;50:100–107. 5. Sotnikova R, Kettmann V, Kostalova D, Taborska E. Relaxant properties of some aporphine alkaloids from Mahonia aquifolium. Method Find Exp Clin. 1997;19:589–597. 6. Wright CW, Marshall SJ, Russell PF. In vitro antiplasmodial, antiamoebic, and cytotoxic activities of some monomeric isoquinoline alkaloids. J Nat Prod. 2000;63:1638–1640. 7. Lu P, Sun H, Zhang L, et al. Isocorydine targets the drug-resistant cellular side population through PDCD4-related apoptosis in hepatocellular carcinoma. Mol Med. 2012;18:1136–1146. 8. Sun H, Hou H, Lu P, et al. Isocorydine inhibits cell proliferation in hepatocellular carcinoma cell lines by inducing G2/M cell cycle arrest and apoptosis. PLoS ONE. 2012;7:e36808. 9. Zhong M, Liu YJ, Liu JX, et al. Isocorydine derivatives and their anticancer activities. Molecules. 2014;19:12099–12115. 10. Li M, Zhang L, Ge C, et al. An isocorydine derivative (d-ICD) inhibits drug resistance by downregulating IGF2BP3 expression in hepatocellular carcinoma. Oncotarget. 2015;6:25149–25160. 11. Chen L, Tian H, Li M, et al. Derivate isocorydine inhibits cell proliferation in hepatocellular carcinoma cell lines by inducing G2/M cell cycle arrest and apoptosis. Tumour Biol. 2016;37:5951–5961. 12. Liu X, Tian H, Li H, et al. Derivate isocorydine (d-ICD) suppresses migration and invasion of hepatocellular carcinoma cell by downregulating ITGA1 expression. Int J Mol Sci. 2017;18:514.

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