Novel Ligustrazine-Based Analogs of ... - ACS Publications

Article Cite This: J. Med. Chem. 2018, 61, 1821−1832

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Novel Ligustrazine-Based Analogs of Piperlongumine Potently Suppress Proliferation and Metastasis of Colorectal Cancer Cells in Vitro and in Vivo Yu Zou,†,‡,∥,# Di Zhao,†,§,# Chang Yan,†,‡ Yanpeng Ji,†,‡ Jin Liu,†,‡ Jinyi Xu,† Yisheng Lai,†,‡ Jide Tian,⊥ Yihua Zhang,*,†,‡ and Zhangjian Huang*,†,‡ †

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical University, Nanjing 210009, P. R. China § Clinical Pharmacokinetics Laboratory, Department of Clinical Pharmacy, School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing 211198, P. R. China ∥ Department of Pharmacy, College of Medicine, Wuhan University of Science and Technology, Wuhan, Hubei Province 430065, P. R. China ⊥ Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California 90095, United States ‡

S Supporting Information *

ABSTRACT: Piperlongumine 1 increases reactive oxygen species (ROS) levels and preferably induces cancer cell apoptosis by triggering different pathways. However, the poor solubility of 1 limits its intensive investigation and clinical application. Ligustrazine possesses a water-soluble pyrazine skeleton and can inhibit proliferation and metastasis of cancer cells. We synthesized compound 3 by replacement of the trimethoxyphenyl of 1 with ligustrazine moiety and further introduced 2-Cl, -Br, and -I to 3 for synthesis of 4−6, respectively. Compound 4 possessed 14-fold greater aqueous solubility than 1 and increased ROS levels in colorectal cancer HCT-116 cells. Additionally, 4 preferably inhibited proliferation, migration, invasion, and heteroadhesion of HCT-116 cells. Treatment with 4 suppressed tumor growth and lung metastasis in vivo and prolonged the survival of tumor-bearing mice. Furthermore, 4 mitigated TGF-β1-induced epithelial-mesenchymal transition and Wnt/β-catenin activation by inhibiting the Akt and GSK-3β phosphorylation in HCT-116 cells. Collectively, 4 displayed significant antiproliferation and antimetastasis activities, superior to 1.

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INTRODUCTION Accumulating evidence reveals that increased levels of reactive oxygen species (ROS) by elevating ROS production or reducing ROS-scavenging capacity is often associated with promoting cancer cell growth.1 Moderate levels of oxidative stress can enhance proliferation, angiogenesis, and metastasis of cancer cells.2 In contrast, high levels of ROS can irreversibly damage DNA and lipids and induce cancer cell apoptosis.3 In addition, some therapeutic agents and high levels of oxidative stress can synergistically damage cancer cells.4,5 Therefore, manipulation of ROS levels is an important strategy to selectively kill cancer cells. Actually, there have been many efforts to increase the levels of ROS specifically in cancer cells, which is known as “oxidation therapy”.6−8 One strategy for the oxidation therapy is to directly deliver ROS-promoting agents, such as piperlongumine (1, Figure 1),9 arsenic trioxide (As2O3),10 or glucose oxidase11 to tumor tissues. Piperlongumine 1, an alkaloid in the fruit of long pepper, is a naturally occurring small molecule product with multiple © 2018 American Chemical Society

Figure 1. Structure-based hybridization and scaffold hopping of novel piperlongumine-ligustrazine hybrids 3−6.

pharmacological activities.12−16 Recent studies have documented that it selectively increases ROS in tumor cells, inhibits Received: July 26, 2017 Published: February 9, 2018 1821

DOI: 10.1021/acs.jmedchem.7b01096 J. Med. Chem. 2018, 61, 1821−1832

Journal of Medicinal Chemistry

Article

Scheme 1. The Synthetic Route of (A) Compounds 3−6 and (B) 17, 19, and 21a

a For compounds 3−6: Reagents and conditions: a. NaOH, H2O, 1-2 h, 96%; b. 30% H2O2, AcOH, 70 °C, 8 h, 85%; c. Ac2O, reflux for 2 h, 87%; d. 20% NaOH, 90%; e. IBX, DMSO, rt, 0.5 h, 92%; f. Wittig−Hornor reaction: toluene, NaH, triethyl phosphonoacetate, rt, under dark, 8−10 h, 76%; g. KOH, 16 h, 89%; h. anhydrous DCM, TEA, pivaloyl chloride, one drop of DMF; then n-butyllithium, 17, 19, or 21 for 3 (65%), 4 (61%), or 5 (59%). i. iodine, Py, THF, rt, overnight for 6, 74%; j. (1) anhydrous DCM, pivaloyl chloride, one drop of DMF; (2) anhydrous THF, TEA, nbutyllithium and 14 for 13, 67%. For compounds 17, 19 and 21: Reagents and conditions: a. toluene, TEA, TMSCl, 0 °C, 4 h; b. THF, phenylselenyl chloride, LDA, −50 °C; c. THF, H2O2, 77% (a−c); d. PCl5, chloroform, rt; e. Li2CO3, LiCl, anhydrous DMF, 130 °C, 7 h, 75% (d,e); f. (i) PCl5, DCM, 0 °C; (ii) ZnI, Br2, rt, overnight; g. Li2CO3, LiCl, anhydrous DMF, 130 °C, 7 h, 68% (f,g).

cardiovascular and cerebrovascular diseases.28 Recently, 2 has been reported to inhibit the proliferation and metastasis of tumor cells, increase intracellular ROS accumulation, and induce cancer cell apoptosis.29,30 Accordingly, we hypothesized that hybrids of 1 with 2 via replacement of the trimethoxyphenyl of 1 by ligustrazine moiety and with or without introduction of a 2-halogen could produce a hitherto unknown class of analogs of 1 possessing better solubility and more potent antiproliferative and antimetastasis activities against cancer cells. To test the hypothesis, we synthesized novel hybrids 3−6 (Figure 1) and investigated their bioactivity in vitro and in vivo and the potential mechanisms.

proliferation, invasion, and migration, and induces apoptosis of the cells.17−19 However, the poor solubility of 1 limits its intensive investigation and clinical application.20,21 Although a great number of derivatives and analogs of 1 have been synthesized in order to get a more promising anticancer drug,22−26 the solubility currently remains challenging. Additionally, the derivatives and analogs of 1 with both antiproliferative and antimetastatic activities have so far not been documented. In this regard, the development of new anticancer analogs of 1 with improved water solubility as well as potent antiproliferative and antimetastatic effects should be of great importance. Our previous study indicated that replacing the trimethoxyphenyl of 1 by a pyrazine or pyridine ring remarkably improved aqueous solubility and displayed stronger anticancer activity in vitro.27 In addition, the 2-halogenated 1 reportedly showed more potent in vitro anticancer activity than 1 due to its higher reactivity of C2−C3 Michael acceptor.26,27 It is notable that ligustrazine (2) with a water-soluble pyrazine skeleton is a Chinese traditional medicine for treatment of

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RESULTS AND DISCUSSION Chemistry. The synthetic routes of target compounds 3−6 are depicted in Scheme 1. 2-Hydroxymethyl-3,5,6-trimethylpyrazine 9 was prepared by the Boekelheide reaction starting from hydrochloride of 2 via a four-step reaction sequence as described previously.31 The compound 9 was oxidized by 21822



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alone or combination of 1 with 2. The most active compound 4 exhibited a strong cytotoxicity against human colorectal cancer HCT-116 cells with an IC50 of 0.30 ± 0.03 μM, which was 27fold stronger than 1 (IC50 = 8.13 ± 0.51 μM). Notably, compounds 1 and 3−6 more strongly inhibited the proliferation of HCT-116 and K562 cells than that of U87MG and A549 cells. Probably, the former cells are more sensitive to the ROS elevation induced by the tested compounds than the latter ones. Interestingly, compound 13 without C2−C3 double bond failed to inhibit the proliferation of cancer cells tested, suggesting that the C2−C3 double bond may be essential for the inhibitory activity of piperlongumineligustrazine hybrids. Further tests demonstrated that 4 was also powerful against other colorectal cancer cells with IC50 values ranging from 0.54 to 1.21 μM, which were much less than that of 1 (Table 3). In contrast, 4 showed much lower inhibitory activity against nontumor colon CCD-841 cells (IC50 = 51.55 μM), suggesting that 4 may selectively inhibit the proliferation of colorectal cancer cells. Thus, we selected it for further investigations. Comparison of the Chemical Reactivity of the Two Olefins in 4. It has been reported that the C2−C3 olefin is more reactive than the C7−C8 olefin in compound 122 and that the C2−C3 olefin on the ring A is more reactive than C9−C11 olefin on the ring C in 2-cyano-3,12-dioxooleana-1,9(11)-dien28-oic acid methyl ester (CDDO-Me).33 Structurally, compound 4 possesses two Michael acceptors, that is, C2−C3 and C7−C8 olefins, that may influence its actions on cells. Therefore, it was of interest to examine the chemical reactivity of C2−C3 and C7−C8 olefins in 4 on a representative thiolcontaining nucleophile, methyl thioglycolate. It could be seen from Figure S1, the proton signal and integral value of C3−H (designated as Hc) in 4 at δ 7.37 ppm was time dependently decreased, and two new proton signals at δ 3.60 and 5.08 ppm appeared, with the integral value gradually increased, which was closely related to C3−H. Thus, in combination of other relative variations in the 1H NMR spectra (Figure S1, Tables S1 and S2), two protons could be unambiguously assigned as Hi and Hj of compound 22 (Scheme S1), respectively. Interestingly, no addition reaction was observed at C8, even in the presence of 3.0 equiv of methyl thioglycolate (Figure S2, Tables S3 and S4). And this selective reaction on C2−C3 olefin is consistent with the previous reports on compound 1 and CDDO-Me.22,33 Pharmacokinetics of 4 in Rats. The pharmacokinetics of 4 was evaluated in rats in comparison with 1. Compound 1 or 4 at 20 mg/kg was intravenously administered to rats (n = 3), and blood samples were taken from suborbital vein at 0.033, 0.083, 0.25, 0.5, 0.75, 1, 2, 4 h, and analyzed by LC-MS-MS. The corresponding blood concentration−time profiles of 1 and 4 were shown in Figure S5 and pharmacokinetic parameters in Table S6. AUC0−∞ (area under concentration−time curve from time zero to infinite), t1/2 (half-life), and Cmax (maximum plasma concentration) of 1 and 4 were 273.0 ± 40.1 ng·h/mL, 2.87 ± 0.22 h, and 563.8 ± 129.6 ng/mL as well as 70.2 ± 16.9 ng·h/mL, 2.17 ± 0.95 h, and 44.3 ± 4.8 ng/mL, respectively. The CL (clearance rates) and Vss (distribution volumes) of 1 and 4 were 0.074 ± 0.012 mL/h/kg and 0.31 ± 0.07 mL/kg as well as 0.297 ± 0.077 mL/h/kg and 0.903 ± 0.4 mL/kg, respectively. Considering the fact that the 2-halogenated derivatives or analogs of 1 display higher reactivity of C2− C3Michael acceptor than 1,26,27 it would be expected that 4 would possess a faster metabolic behavior relative to 1, leading to the lower values of AUC0‑∞, t1/2, and Cmax as well as higher

iodoxybenzoic acid (IBX) to give aldehyde 10, which underwent Wittig−Horner reaction to furnish ester 11. Hydrolysis of 11 led to free acid 12, which was converted to acyl chloride, without isolation and purification followed by reacting with corresponding 5,6-dihydropyridin-2(1H)-one 17 as well as with 6-substituted-5,6-dihydro-pyridin- 2(1H)-ones 19 and 21 to provide compounds 3−5, respectively. Compound 6 was obtained by iodination at position 2 of 3. Compound 13, an analogue of 3 without C2−C3 double bond, was synthesized by treatment of 12 with the commercially available piperidin-2-one 14 for biological evaluation. As key intermediates, 17, 19, and 21 were prepared via twoor three-step reactions from 14 (Scheme 1B).23 Compound 17 was obtained by treatment of 14 with trimethyl chlorosilane (TMSCl), phenylselenyl chloride, and hydrogen peroxide, successively. Treatment of 14 with PCl5 yielded 2,2dichloropiperidin-2-one 18, followed by dehydrochlorination in the presence of Li2CO3 to offer 6-chloro-5,6-dihydropyridin2(1H)-one 19. 6-Bromo-5,6-dihydro-pyridin- 2(1H)-one 21 was similarly prepared as 19. The structures of all hybrids were fully characterized by 1H NMR, 13C NMR, MS, and HRMS, and the 7,8-olefinic bond in the hybrids was identified as E configuration. All target compounds with a purity of >95%, determined by highperformance liquid chromatography (HPLC) analysis, were used for subsequent experiments. The solubility of compounds 1 and 3−6 was determined in both pure water and phosphate buffer (50 mM, pH 7.4) at 20 °C by HPLC (Table 1).32 It was found that all hybrids were Table 1. Solubility (μg/mL) of Compounds 1 and 3−6 compd

1

3

4

5

6

in water in PBS (pH 7.4)

1.63 0.32

42.63 328.16

22.84 74.62

20.17 67.28

14.50 49.85

more soluble than 1. Among them, solubility of 3 (42.63 μg/ mL) and 4 (22.84 μg/mL) was approximately 26- and 14-fold greater than 1 (1.63 μg/mL), respectively. The improvement of the aqueous solubility is probably attributable to the ligustrazine moiety, which is more water soluble than thrimethoxyphenyl ring in 1. Biological Evaluations. Assessment of in Vitro Antiproliferative Activity. Compounds 3−6 were tested for their antiproliferative activity against human glioma U87MG, colorectal cancer HCT-116, lung cancer A549, and leukemic K562 by MTT assay. As shown in Table 2, all hybrids showed a more potent activity in inhibiting the proliferation of cancer cells than 1 Table 2. IC50 (μM) of 3−6 against Human Cancer Cellsa compd

U87MG

HCT-116

A549

K562

3 4 5 6 13 1 1+2

8.73 ± 0.40 3.43 ± 0.31 4.28 ± 0.87 7.26 ± 0.83 >50.00 17.34 ± 0.23 16.15 ± 0.81

1.67 ± 0.30 0.30 ± 0.03 0.46 ± 0.03 0.76 ± 0.05 >50.00 8.13 ± 0.51 8.17 ± 0.26

4.86 ± 0.21 2.21 ± 1.19 2.48 ± 0.65 3.19 ± 0.48 >50.00 15.28 ± 0.19 15.22 ± 1.20

1.13 ± 0.01 0.25 ± 0.01 0.48 ± 0.02 0.64 ± 0.01 >50.00 5.05 ± 0.02 5.09 ± 0.10

a

Cells were treated with indicated compounds in triplicate for 72 h, and the cell viability was determined using MTT assay, respectively. 1823



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Table 3. IC50 (μM) of 4 against Colorectal Cancer Cells and Nontumor Colon Cellsa

a

cell lines

HT-29

SW620

HCT-8

HCT-116

CCD-841

1 4

2.65 ± 0.17 0.54 ± 0.04

4.62 ± 0.22 0.87 ± 0.06

4.10 ± 0.20 1.21 ± 0.08

8.13 ± 0.11 0.30 ± 0.03

44.32 ± 1.77 51.55 ± 2.08

Cells were treated with indicated compounds in triplicate for 72 h, and the cell viability was determined using MTT assay, respectively.

Figure 2. Compound 4 selectively promotes the accumulation of intracellular ROS in colorectal cancer cells. HCT-116 and CCD-841 cells were treated in triplicate with 1 and 4 at 1, 5, and 10 μM for 1, 2, and 3 h, respectively. The intracellular ROS were stained with DCFH-DA and measured by a fluorescent spectrometry and microscope. Data are representative images or expressed as the means of individual groups of cells (relative to DMSO control) from three separate experiments. (A−C) The levels of ROS in HCT-116 and CCD-841 cells (relative to DMSO control). (D−I) Fluorescence images of HCT-116 cells (D−F), fluorescent images of CCD-841 cells (G−I), and both types of cells were treated with, or without, 1 and 4 at 10 μM for 3 h, respectively.

values of CL and Vss of 4 as compared to those of 1. In addition, the stability of 4 in HCT-116 cell lysis, simulated gastric fluid (pH 1.4), and intestinal fluid (pH 6.8) was investigated. As shown in Figures S6−S8, 4 bearing a basic ligustrazine moiety was much more stable in simulated gastric fluid than in HCT-116 cell lysis and intestinal fluid. Compound 4 Selectively Promotes More ROS Accumulation than 1 in HCT-116 Cells. It is known that the inhibition of glutathione S-transferase P1 (GSTP1) activity by 1 disturbs glutathione (GSH) balance, leading to intracellular ROS accumulation in cancer cells.34 Accordingly, we examined whether and how 4 impacted ROS levels in HCT-116 and CCD-841 cells. HCT-116 and CCD-841 cells were treated in triplicate with 1 and 4 at 1, 5, and 10 μM for 1, 2, and 3 h, respectively. The levels of intracellular ROS were detected with 2′,7′-dichlorodihydrofluoresceine diacetate (DCFH-DA) by using a fluorescent microplate reader to measure the fluorescent signals. As shown in Figure 2A−C, treatment with 4 rapidly and significantly increased the levels of intracellular ROS in HCT-116 cells, while the same treatment only increased moderate levels of ROS in CCD-841 cells. The selectivity of 4 was obviously greater than that of 1, indicating that 4 more preferably promoted intracellular ROS accumulation relative to 1 in the cancer cells. Fluorescence microscopy analysis indicated that treatment with 1 and 4 at 10 μM for 3 h resulted in green fluorescent positive HCT-116 and CCD-841 cells (Figure 2D−I). The

images (Figure 2E,F) further supported that 4 more selectively increased the levels of ROS in the cancer cells than 1. Importantly, pretreatment with GSH not only reduced the ROS levels (data not shown) but also diminished the inhibition of 4 on the proliferation of HCT-116 cells from 82.9% to 36.1% (Figure 3). A similar result was observed in the case of

Figure 3. Pretreatment with GSH diminishes the inhibition of 4 or 1 on the proliferation of HCT-116 cells. HCT-116 cells were pretreated in the presence or absence of GSH (10 mM) for 1 h, then washed, and treated with 4 (1.52 μM) or 1 (8.0 μM) for 72 h. The viability of cells was determined by MTT, and inhibitory rates were calculated. Data are expressed as the mean ± SD of individual groups of cells from three independent experiments. ***P < 0.001. 1824



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with 4 significantly reduced the volume and size of the colorectal tumors in mice. Moreover, there was no obvious abnormality in the liver, kidney, lung, and heart in terms of the size and morphology in both controls and 4-treated mice except for a slight, but not significant, reduction in body weight as compared with each other (Figure 4 and Table S5). Compound 4 Mitigates the TGF-β1-Mediated Migration and Invasion of HCT-116 Cells. To verify that the hybrid 4 could inhibit cancer metastasis, a series of migration and invasion assays were conducted in vitro. Transforming growth factor-β1 (TGF-β1) can stimulate tumor cell migration and invasion.35,36 Accordingly, we first examined the cytotoxic effects of TGF-β1 (10 ng/mL) or combination of TGF-β1 (10 ng/mL) with different concentrations (2, 10, or 50 nM) of 4 on HCT-116 cells by MTT assay using 1 and oxaliplatin as controls. The results indicated that treatment with TGF-β1 or treatment in combination of TGF-β1 with either 4 or 1 at three concentrations for 24 and 48 h did not affect the viability of HCT-116 cells, although treatment with 4 at 50 nM for 72 h reduced the viability of HCT-116 cells independent of TGF-β1 treatment (Figure S3). Accordingly, the antimigration and antiinvasion effects of 4 were further determined by transwell migration (Figure 5A,D), invasion (Figure 5B,E), and lateral migration (Figure 5C,F) assays for maximum incubation time for 48 h. It was found that treatment with TGF-β1 significantly promoted migration and invasion of HCT-116 cells, while treatment with 4 minimized the TGF-β1-stimulated migration and invasion in a dose- and time-dependent manner. The antimigration and anti-invasion effects of 4 were significantly stronger than that of 1 and slightly potent or similar to that of oxaliplatin in HCT-116 cells. Therefore, compound 4 had a potent activity against the migration and invasion of HCT-116 cells in vitro. Compound 4 Inhibits Adhesion of HCT-116 Cells to HUVECs. Adhesion of cancer cells, especially for circulating tumor cells (CTCs), to vascular endothelium is crucial for

compound 1 (Figure 3), which is reportedly a ROS-promoting agent.9 These results suggest that 4 may inhibit the proliferation of colorectal cancer cells by selective promotion of ROS accumulation, at least in part, through disturbing the GSH balance. Compound 4 Inhibits the Growth of HCT-116 Xenograft Tumors in Mice. To investigate the safety profile of the hybrids, the acute toxicity of 4 was determined in ICR mice at doses of 50, 100, 150, 200, 300, and 400 mg/kg (ip, n = 10 per group) for 14 days. As shown in Table 4, treatment with 4 at the lowest Table 4. Acute Toxicity of 4 in Mice dose (mg/kg)

no. of mice

total mortality

survival (%)

LD50a (mg/kg)

400 300 200 150 100 50

10 10 10 10 10 10

10 9 7 6 2 0

0 10 30 40 80 100

147.54

a

The 95% confidence limits: 114.88−181.16 mg/kg.

dose (50 mg/kg) did not cause any death in mice. However, treatment with 4 at 400 mg/kg killed all the mice. Finally, the median lethal dose (LD50) value of 4 was calculated to be 147.54 mg/kg. To evaluate in vivo anticolon cancer activity of 4, BALB/c nude mice were inoculated subcutaneously with colorectal cancer HCT-116 cells. After the establishment of solid tumors, the mice were randomized and treated intraperitoneally with oxaliplatin (5 mg/kg), 1 (5 mg/kg), or 4 (2.5 or 5 mg/kg) daily for 21 consecutive days (Figure 4), respectively. As shown in Figure 4 and Table S5, treatment with 4 at 5 mg/kg inhibited the growth of the colon cancer cells in the mice by 71.4% (w/ w), which was more potent than 1 (60.6%) and positive control oxaliplatin (65.3%) at the same does. In addition, treatment

Figure 4. Treatment with 4 inhibits the growth of HCT-116 cells in vivo. Nude mice were inoculated with HCT-116 cells, and after establishment of solid tumors, the mice were treated intraperitoneally with vehicle, oxaliplatin (5 mg/kg), 1 (5 mg/kg), or 4 (2.5 and 5 mg/kg) daily. The tumor volumes and body weights of individual groups of mice were monitored, and the tumor sizes and weights were measured at the end of experiment. Data are expressed as the mean ± SD of individual groups of mice (n = 8 per group). (A) Image of all tumors. (B) The body weights of mice. (C) The volumes of tumors. ***P < 0.001 vs control at the last measurement. (D) The weights of tumors. ***P < 0.001 vs control, #P < 0.05. 1825



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Figure 5. Effects of the tested compounds on transwell migration (A, D), invasion (B, E), and lateral migration (C, F) of HCT-116 cells. (A) The HCT-116 cells were seeded on chambers and incubated with the compounds for 48 h. Cells that migrated through the chambers were stained with crystal violet, and representative images were captured. (B) The HCT-116 cells were seeded on chambers and incubated with the indicated factors for 48 h. Cells that migrated through the matrigel-coated chambers were stained with crystal violet, and representative images were captured. (C) The HCT-116 cells were seeded on 48-well plates. After 24 or 48 h incubation with the indicated factors, representative images of the wound were captured. (D) The cells that migrated through the chambers were counted from three independent experiments. (E) The cells that migrated through the matrigel-coated chambers were counted from three independent experiments. (F) The rate of lateral migration is presented. Experiment was confirmed for three independent times. All the data in (D−F) were expressed as the means ± SD of each group of cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs respective TGF-β group; #P < 0.05, ###P < 0.001.

Figure 6. Compound 4 inhibits adhesion of HCT-116 cells to HUVECs induced by IL-1β. HUVEC monolayers were pretreated with IL-1β (1 ng/ mL) for 4 h and then were co-incubated with Rhodamine 123-labeled HCT-116 cells with or without 4 and 1 at indicated concentrations for 1 h. The adhered HCT-116 cells to HUVECs were captured, and the percentages of adhered HCT-116 cells were determined. Data are representative images or expressed as the mean ± SD of each group of cells from three separate experiments. (A) Fluorescent images of HCT-116 cells (green) adhered to the HUVECs. (B) Quantitative analysis of the percentages of adhered HCT-116 cells. ** P < 0.01, ***P < 0.001 vs IL-1β group; #P < 0.05, ###P < 0.001.

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Figure 7. Compound 4 inhibits the lung metastasis of colorectal cancer in mice. HCT-116 cells in the logarithmic growth phase were prepared in 5 × 106/mL cell suspension with serum-free medium under sterile conditions and injected with 0.2 mL into the tail vein of nude mice. The mice were randomized and treated intraperitoneally with 4, 1, or oxaliplatin three times a week for 6 weeks. The body weight of mice was recorded every week after injection, and all the nude mice were sacrificed after 40 days. At the end of experiment, the lung metastatic tumors of colon cancers were examined by histology. Data are representative images or expressed as the mean ± SD of individual groups of mice. (A) The body weights. (B) The survival rates (n = 6). Four groups where rates were treated with 4 at 2 or 4 mg/kg, 1 at 4 mg/kg, and oxaliplatin at 4 mg/kg have 100% survival rate. (C) Representative images of lung metastatic tumors (n = 3, scale bar: 100 μm). (D) The number of lung metastatic nodules in mice (n = 4−6 per group). *P < 0.05, **P < 0.01 vs the control group; #P < 0.05, ##P < 0.01.

Figure 8. Treatment with 4 reverses the TGF-β1-induced EMT process in HCT-116 cells. HCT-116 cells were treated with, or without, TGF-β1 (10 ng/mL) for 48 h in the presence or absence of 1 or 4 at the indicated concentrations. The relative levels of E-cadherin, Vimentin, MMP-9, Snail, and Twist to the control β-actin expression were determined by Western blotting. Data are representative images or expressed as the mean ± SD of each group of cells from three independent experiments. (A) Western blot analysis. The levels of (B) E-cadherin, (C) Vimentin, (D) MMP-9, (E) Snail, and (F) Twist. *p < 0.05, **p < 0.01 vs TGF-β group; #P < 0.05, ##P < 0.01.

invasion and metastasis of cancers.35,36 Therefore, we tested the impact of 1 and 4 on adhesion of HCT-116 cells to human umbilical vein endothelial cells (HUVECs).35 Monolayers of HUVECs were pretreated with, or without, interleukin 1β (IL1β, 1 ng/mL) for 4 h and were co-incubated with fluorescence labeled HCT-116 in the presence or absence of 1 (50 nM) and 4 (2, 10, 50 nM) for 1 h. After being washed, the percentages of HCT-116 cells that had adhered to the HUVECs were determined (Figure 6). The results indicated that while treatment with IL-1β significantly enhanced the adhesion of

HCT-116 cells to HUVECs, treatment with 4 markedly decreased the percentages of adhered HCT-116 to HUVECs in a dose-dependent manner, which was much more potent than 1 at the same dose. Compound 4 Suppresses in Vivo Lung Metastasis of Colorectal Cancer. We further examined whether treatment with 4 could modulate the metastasis of colorectal cancer. We established a lung metastasis model of human colon cancer HCT-116 cells in BALB/c nude mice to examine its in vivo anticolon cancer metastatic activity. The mice were intra1827



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Figure 9. Treatment with 4 inhibits the AKT and Wnt/β-catenin signaling induced by TGF-β1 in HCT-116 cells. HCT-116 cells were treated with, or without, TGF-β1 (10 ng/mL) for 48 h in the presence or absence of 1 or 4 at the indicated concentrations. The relative levels of Akt and GSK-3β phosphorylation and cytoplasm and nuclear β-catenin expression in HCT-116 cells were determined by Western blot assays. Data are representative images or expressed as the mean ± SD of each group of cells from three independent experiments. (A) Western blot analysis. (B) The levels of AKT and GSK-3β phosphorylation. (C) Western blot analysis of cytoplasm and nuclear β-catenin expression. (D) The levels of cytoplasm and nuclear βcatenin expression. *p < 0.05, **p < 0.01 vs TGF-β group; #P < 0.05, ##P < 0.01.

effect of treatment with 4 on the TGF-β1-induced EMT process in HCT-116 cells. As shown in Figure 8, treatment with TGF-β1 (10 ng/mL) for 48 h significantly reduced the levels of E-cadherin expression (p < 0.01), but increased the levels of vimentin, MMP-9, Snail, and Twist expression (P < 0.01), demonstrating that TGF-β1 promoted the EMT process in HCT-116 cells (Figure 8). Treatment with 4 significantly mitigated the TGFβ1-reduced E-cadherin expression and TGF-β1-enhanced vimentin, MMP-9, Snail, and Twist expression in HCT-116 cells. Its effects were dose dependent and significantly stronger than that of 1 (p < 0.01). Hence, treatment with 4 reversed the TGF-β1-induced EMT process in HCT-116 cells. Treatment with 4 Inhibits the AKT and Wnt/β-Catenin Signaling Induced by TGF-β1 in HCT-116 Cells. TGF-β1 can bind to its receptor to activate the Smad signaling, which crosstalks with the PI3K and Wnt/β-catenin signaling to promote metastasis of cancer.42,43 The Wnt/β-catenin activation was negatively regulated by GSK-3β. Finally, we determined the impact of treatment with 4 on the TGF-β1-regulated activation of the PI3K and Wnt/β-catenin signaling in HCT-116 cells by Western blot assays. As shown in Figure 9A,B, although treatment with TGF-β1 did not alter the relative levels of AKT and GSK-3β expression, the treatment significantly increased the relative levels of AKT Ser473 and GSK-3β S9 phosphorylation in HCT-116 cells. In contrast, treatment with 4 significantly mitigated the TGF-β1-enhanced AKT and GSK-3β phosphorylation in a dose-dependent manner and the regulatory effects of treatment with 4 on TGF-β1-enhanced AKT and GSK-3β phosphorylation were significantly stronger than that of 1 (p < 0.01). Given that up-regulated GSK-3β phosphorylation should promote its degradation, which will enhance the Wnt/β-catenin activation, we further tested the impact of treatment with 4 on the TGF-β1-regulated β-catenin nuclear translocation in HCT-116 cells. As shown in Figure 9 C,D, treatment with TGF-β1 reduced the relative levels of cytoplasm β-catenin, but elevated the levels of nuclear βcatenin, a hallmark of promoting nuclear translocation of β-

peritoneally administrated 4 at 1, 2, or 4 mg/kg. Compound 1 and oxaliplatin (4 mg/kg) were employed as positive controls. The test proceeded three times a week for six consecutive weeks. Their body weights and survival in individual groups of mice were monitored. At the end of the experiment, the lung metastatic tumors of colon cancers were examined by histology (Figure 7). In comparison with the control mice, treatment with 4, like treatment with 1 or oxaliplatin, prevented the loss of body weights in tumor-bearing mice, and treatment with 4 at 4 mg/kg increased the body weights of tumor-bearing mice (Figure 7A). Similarly, treatment with 4 at 2 or 4 mg/kg, like treatment with 1 or oxaliplatin, prevented the death of tumorbearing mice (Figure 7B). In addition, treatment with 4 appeared to reduce the size of lung metastatic tumors and significantly decreased the numbers of lung metastatic nodules (Figure 7C,D). The therapeutic effect of 4 was significantly stronger than that of 1 and oxaliplatin. Collectively, treatment with 4 significantly inhibited the lung metastasis of colorectal cancers. Compound 4 Reverses the TGF-β1-Induced Process of Epithelial-Mesenchymal Transition in HCT-116 Cells. The process of epithelial-mesenchymal transition (EMT) is important for tumor metastasis, and cancer cells undergoing the EMT process usually have the following characteristics:37−40 (i) reduction or loss of adhesion molecule Ecadherin; (ii) up-regulation expression of vimentin, one of the main mesenchymal cell markers and transcription factors, Snail and Twist; (iii) variation in the compositions of extracellular matrix, which facilitate tumor invasion and metastasis; and accompanied by changes in the multiple signal pathways. The upregulated Snail can promote the expression of matrix metalloproteinase (MMP), but repress E-cadherin expression, while up-regulated Twist expression not only enhances Ncadherin expression but also reduces E-cadherin expression as well as activates the PI3K/AKT and Wnt/β-catenin signaling.37 Moreover, TGF-β1 can enhance the EMT process and promote migration and invasion of cancers.41 Accordingly, we tested the 1828



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(E)-1-(3-(3,5,6-Trimethylpyrazin-2-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (3). Yellow powder; yield 53%; mp 115−118 °C. 1H NMR (300 MHz, CDCl3), δ (ppm): 2.41−2.45 (m, 2H, CH2), 2.48 (s, 6H, 2 × CH3), 2.59 (s, 3H, CH3), 4.01 (t, J = 6.0 Hz, 2H, NCH2), 6.03 (d, J = 4.5 Hz, 1H, CHCH), 6.90−6.96 (m, 1H, CHCH), 7.80− 7.91 (m, 2H, CHCH); 13C NMR (75 MHz, CDCl3): δ 20.9, 21.9, 22.2, 26.5, 42.3, 126.2, 138.4, 143.2, 149.3, 150.1, 152.6, 161.4, 169.0; ESI-MS: 294.1 [M + Na]+; HRMS calculated for C15H17N3O2Na [M + Na]+ 294.1218, found 294.1219, ppm error 0.3. (E)-3-Chloro-1-(3-(3,5,6-trimethylpyrazin-2-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (4). Yellow powder; yield 56%; mp 111−114 °C. 1H NMR (300 MHz, CDCl3), δ (ppm): 2.49 (s, 6H, 2 × CH3), 2.54−2.56 (m, 2H, NCH2CH2), 2.59 (s, 3H, CH3), 4.06 (t, J = 6.0 Hz, 2H, NCH2CH2), 7.06 (t, J = 4.5 Hz, 1H, COClCCH), 7.82−7.95 (m, 2H, CHCH); 13C NMR (75 MHz, CDCl3): δ 21.0, 21.9, 22.2, 25.5, 42.0, 126.2, 128.3, 138.4, 141.3, 143.2, 149.3, 150.1, 152.6, 161.4, 168.8; ESI-MS: 328.1 [M + Na] + ; HRMS calculated for C15H16N3O2ClNa [M + Na]+ 328.0829, found 328.0835, ppm error 1.8. Elemental analysis, calculated: C, 58.92; H, 5.27; N, 13.74. Found: C, 58.60; H, 5.41; N, 13.44. (E)-3-Bromo-1-(3-(3,5,6-trimethylpyrazin-2-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one (5). Yellow powder; yield 43%; mp 109−113 °C. 1H NMR (300 MHz, CDCl3), δ (ppm): 2.44−2.59 (m, 11H, NCH2CH2 and 3 × CH3), 4.06 (t, J = 7.5 Hz, 2H, NCH2CH2), 7.06 (t, J = 4.5 Hz, 1H, COClCCH), 7.83−7.93 (m, 2H, CHCH); 13C NMR (75 MHz, CDCl3): δ 21.0, 21.9, 22.2, 26.9, 42.1, 113.2, 126.3, 138.3, 143.3, 147.3, 149.3, 150.1, 152.6, 161.3, 169.0; ESI-MS: 372.0 [M + Na]+; HRMS calculated for C15H16N3O2BrNa [M + Na]+ 372.0368, found 372.0371, ppm error 0.8. Synthesis of (E)-3-Iodo-1-(3-(3,5,6-trimethylpyrazin-2-yl)acryloyl)-5,6- dihydropyridin-2(1H)-one (6). Iodine (104 mg) and 25 (50 mg) were added to a mixed solution of pyridine and tetrahydrofuran (V(Py)/V(THF) = 1:1). The reaction solution was stirred overnight at room temperature, and then saturated aqueous ammonium chloride solution (16 mL) and suitable amount of sodium sulfite aqueous solution were added successively. The obtained mixture was extracted with chloroform, the aqueous phase was washed with ethyl acetate (20 mL × 3), and the organic phase was combined, dried over magnesium sulfate, and filtered. The filtrate was concentrated in vacuum to give the crude product, which was purified by column chromatography (PE/EA = 3:1) to give 6. Yellow powder; yield 47%; mp 108−113 °C. 1H NMR (300 MHz, CDCl3), δ (ppm): 2.45−2.51 (m, 8H, NCH2CH2 and 2 × CH3), 2.59 (s, 3H, CH3), 4.07 (t, J = 7.5 Hz, 2H, NCH2), 7.65 (t, J = 4.5 Hz, 1H, CCH), 7.84 (s, 2H, CHCH); 13C NMR (75 MHz, CDCl3): δ 21.0, 21.9, 22.2, 28.7, 42.3, 96.6, 126.4, 138.2, 143.3, 149.3, 150.1, 152.6, 154.7, 161.3, 169.1; ESI-MS: 420.0 [M + Na]+; HRMS calculated for C15H16N3O2INa [M + Na]+ 420.0185, found 420.0191, ppm error 1.4. Synthesis of 3,5,6-trimethylpyrazine-2-carbaldehyde (10). Compound 9 (1.5 g, 10 mmol) was dissolved in DMSO (15 mL) and cooled to 0 °C. IBX (2.8 g, 11 mmol) was slowly added, and the reaction solution was stirred for 0.5 h at room temperature. Then the reaction mixture was diluted with saturated sodium bicarbonate (NaHCO3) aqueous solution and extracted with EtOAc. The organic layer was washed with brine, dried with anhydrous sodium sulfate, filtered, and evaporated in vacuum. The crude product was purified by column chromatography (PE/EtOAc = 10:1−1:1) to give the title compound 10 as a yellow powder in 87% yield; mp 90−92 °C. 1H NMR (300 MHz, CDCl3): δ 2.61 (s, 6H, 2 × CH3), 2.80 (s, 3H, CH3), 10.15 (s, 1H, CHO) ppm. 13C NMR (75 M Hz, CDCl3): δ 20.9, 21.8, 141.4, 149.5, 151.0, 154.9, 193.8. MS (ESI): 151 [M + H]+. Synthesis of (E)-Ethyl 3-(3,5,6-trimethylpyrazin-2-yl)acrylate (11). Compound 10 (710 mg, 4.6 mmol) was dissolved in toluene (70 mL) in a flask which was wrapped with tin foil, and sodium hydride (200 mg) and triethyl phosphonoacetate (940 μL) were added, respectively. Then the reaction solution was stirred at room temperature under dark until TLC analysis showed complete conversion. The reaction mixture was extracted with EtOAc, and the combined organic phase was washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuum. The crude product was

catenin in HCT-116 cells. However, treatment with 4 significantly minimized the TGF-β1-decreased cytoplasm βcatenin and TGF-β1-increased nuclear β-catenin in HCT-116 cells. The regulatory effect of treatment with 4 was significantly stronger than that of 1. Therefore, treatment with 4 inhibited the AKT and Wnt/β-catenin signaling induced by TGF-β1 in HCT-116 cells.

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CONCLUSIONS Elevation in ROS production preferentially or selectively in cancer cells has emerged as an effective strategy for anticancer therapy. In this study, we designed, synthesized, and biologically evaluated the novel hybrids 3−6. The most active compound 4 had a 14-fold greater aqueous solubility than 1 and selectively enhanced the ROS levels in colorectal cancer cells. In view of that 4 structurally bears two Michael acceptors, that is, C2−C3 and C7−C8 olefins, that may influence its actions on cells, we examined and compared their chemical activity. We found that treatment of 4 with one or three equivalents of methyl thioglycolate in DMSO-d6 provided the product of conjugate addition at C3, and no addition was observed at C8 by 1H NMR technique. This selective action on C2−C3 olefin is consistent with the previous report on 1. Functionally, 4 preferably inhibited the proliferation, migration, invasion, and heteroadhesion of colorectal cancer cells. In addition, 4 substantially suppressed the growth and long metastasis of implanted colon cancer as well as prevented cancer-related death in mice. Moreover, 4 mitigated TGF-β1promoted EMT process and Wnt/β-catenin signaling by inhibiting the Akt and GSK-3β phosphorylation in HCT-116 cells. The biological activity of 4 was superior to 1. Together, our findings suggest that 4 may be a promising candidate for intervention of colorectal cancers.

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EXPERIMENTAL SECTION

Chemistry. General Methods. Nuclear magnetic resonance (NMR) spectra were obtained from a Bruker Avance 300 (1H, 300 MHz; 13C, 75 MHz) or 500 (1H, 500 MHz; 13C, 125 MHz) spectrometer at 300 K using TMS as an internal standard. Mass spectrometry (MS) spectra were recorded on a Mariner mass spectrometer. Melting points (mp) were measured by a Mel-TEMP II apparatus and uncorrected. TLC was performed on silica gel GF/ UV 254, and column chromatography was conducted by silica gel (200−300 mesh). The purities of target compounds were characterized by HPLC analysis (Shimadzu DGU-20A3R) and HRMS (Agilent Technologies LC/MSD TOF). The target compounds 3−6 with a purity of >95% were used for subsequent experiments. The synthesis and chemical characterization of compounds 7−9 and 15− 21 are shown in the Supporting Information. General Procedure for the Synthesis of 3−5. Compound 12 (4 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), and triethylamine (0.6 mL) was slowly added and cooled to −20 °C, then pivaloyl chloride (0.6 mL) was added to the reaction solution, which was stirred for about 45 min to prepare the corresponding acyl chloride. Next, the compound 17, 19, or 21 in dry THF was added to the n-butyllithium solution in another flask and stirred for 20 min. Then the acyl chloride solution was dropwise added to the nbutyllithium reaction solution, stirred for about 40 min, and monitored by TLC. After the reaction was completed, a saturated aqueous solution of ammonium chloride (10 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine (30 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuum. The crude product was purified by column chromatography (PE/EA = 3:1) to afford 3−5. 1829



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[(Wcontrol − Wtreated)/Wcontrol] × 100%. Wtreated and Wcontrol were the average tumor weights of the treated and control mice, respectively. The tumor diameters were measured with calipers, and the tumor volume was calculated by the formula V (mm3) = d2 × D/2, where D is the largest diameter and d the smallest diameter. Cell Migration, Invasion, and Lateral Migration Assay. Cell Migration Assay. HCT-116 cells (1 × 105 cells/well) were cultured in the upper chambers of 24-well transwell plates (8 μm pore, Corning Costar), and the bottom chambers were added in triplicate with medium alone or containing TGF-β1 (10 ng/mL) alone or combined with 1 (50 nM) or 4 (2, 10, 50 nM) or oxaliplatin (50 nM) for 48 h. The cells on the upper surface of the membrane were removed, and the migrated cells on the bottom surface of the membranes were stained with crystal violet, followed by photoimaging. The cells in 10 fields of each well were counted in a blinded manner. Cell Invasion Assay. HCT-116 cells (2.5 × 105 cells/well) were seeded on matrigel-coated chambers. 1 (50 nM) or 4 (2, 10, 50 nM) or oxaliplatin (50 nM) was treated on the lower surface for 48 h. Cells that migrated through the matrigel-coated chambers were stained with crystal violet. Representative images were captured, and the cells were counted from three independent experiments. For the detailed steps, refer to the cell migration experiment. Lateral Migration Assay. HCT-116 cells (2.5 × 105 cells/well) were cultured in 48-well plates until 95−100% of confluency. The monolayer of cells was wounded using a plastic tip, and after being measured for the wounded areas, the cells were cultured in triplicate in medium alone, medium containing TGF-β1 (10 ng/mL) alone, or combined with 1 (50 nM) or 4 (2, 10, 50 nM) or oxaliplatin (50 nM) for 48 h, followed by photoimaging. The healed areas in individual groups of cells were calculated. Adhesion Assay. The impact of treatment with 4 on adhesion of HCT-116 cells to HUVECs was determined by a fluorescence-based analysis. Briefly, HCT-116 cells were labeled with Rhodamine 123. HUVECs were cultured in 24-well plates up to monolayer and pretreated with, or without, IL-1β (1 ng/mL) for 4 h, followed by washing with medium. The Rhodamine 123-labeled HCT-116 cells were added onto the monolayer of HUVECs in medium containing vehicle or 1 or 4 at the indicated concentrations at 37 °C for 1 h. The nonadherent HCT-116 cells were washed with warm medium, and the remaining adherent HCT-116 cells were examined under a fluorescent microscope. Anticolon Cancer Lung Metastases Test in Vivo. All animal experimental protocols were approved by the Animal Research and Care Committee of China Pharmaceutical University. Male BALB/c nude mice at 3−5 weeks of age were obtained from Silaike Experiment Animal, Shanghai, China and housed in a specific pathogen-free facility with free access to autoclaved food and water. To determine the effect of treatment with 4 on the lung metastasis of colon cancer, BALB/c nude mice were injected intravenously with 1 × 107 HCT-116 cells in their tail veins and randomized. The groups of mice were injected intraperitoneally with vehicle alone, 1 (4 mg/kg), or 4 (1, 2, or 4 mg/ kg) three times per week for 7 weeks. Their body weights were measured weekly. At the end of the experiment, their lungs were dissected, and the lung sections (4 μm) were stained with H&E. The numbers and areas of metastatic nodules in three sections of each mouse were examined in a blinded manner. Western Blotting. HCT-116 cells (1 × 106 cells/well) were cultured in six-well plates overnight and treated in triplicate with vehicle DMSO (0.1%, v/v) alone, 1, or 4 at the indicated concentrations for 24 h. The cells were harvested and lysed in a lysis buffer [50 mM Tris, pH 7.4, 1 mM MgCl2, 100 mM NaCl, 2.5 mM EDTA, 0.5% Triton X-100, 1 mM phenylmethanesulfonylfluoride (PMSF), 2.5 mM Na3VO4, 0.5% NP-40, pepstatin A, leupeptin, and 5 g/mL of aprotinin]. After being centrifuged, the concentrations of total proteins in the cell lysates were determined by bicinchonininc acid assay. The cell lysates (20 μg/lane) were separated by SDSpolyacrylamide gel electrophoresis (7.5% gel) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with skim dry milk (5%) in Tris-buffered saline containing 0.05% Tween 20 and incubated with primary antibodies at 4 °C

purified by column chromatography (PE/EA = 8:1) to give a pale yellow solid (11) (650 mg, 62.1%). Mp 112−116 °C. 1H NMR (300 MHz, CDCl3): δ 1.21 (t, J = 7.5 Hz, 3H, CH3), 2.61 (s, 6H, 2 × CH3), 2.80 (s, 3H, CH3), 3.87−4.02 (m, 2H, CH2), 6.08 (d, J = 12.0 Hz, 1H, CHCH), 7.46 (d, J = 15.0 Hz, 1H, CHCH) ppm. MS (ESI): 221 [M + H]+ Synthesis of (E)-3-(3,5,6-Trimethylpyrazin-2-yl)acrylic acid (12). Compound 11 (650 mg) was dissolved in a mixed solution of tetrahydrofuran and water (V(THF)/V(H2O) = 2:1, 54 mL), then lithium hydroxide (283 mg) was slowly added, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC. Next, ethyl acetate (30 mL) was added to the reaction mixture, which was adjusted with 2N hydrochloric acid to pH = 4 to complete the reaction. The reaction mixture was extracted with EtOAc, and the combined organic phase was washed with saturated brine (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated in vacuum to give a pale yellow solid (530 mg, 93.9%). mp 120−125 °C. 1H NMR (300 MHz, CDCl3): 2.64 (s, 6H, 2 × CH3), 2.82 (s, 3H, CH3), 6.10 (d, J = 12.0 Hz, 1H, CHCH), 7.47 (d, J = 18.0 Hz, 1H, CH CH) ppm. MS (ESI): 215 [M + Na]+. (E)-1-(3-(3,5,6-Trimethylpyrazin-2-yl)acryloyl)-piperidin-2-one (13). The title compound was prepared by the reaction of compound 12 with 14 by using the same procedure for the synthesis of 3−5. Yellow powder; yield 66%; mp 115−118 °C. 1H NMR (300 MHz, CDCl3), δ (ppm): 1.84−1.90 (m, 4H, NCH2CH2CH2), 2.49−2.56 (m, 11H, 3 × CH3 and COCH2), 3.79 (t, J = 7.6 Hz, 2H, NCH2), 7.72− 7.82 (m, 2H, CHCH); 13C NMR (75 MHz, CDCl3): δ 20.9, 21.2, 21.8, 22.1, 26.1, 32.1, 42.0, 126.2, 127.0, 138.3, 143.2, 145.5, 149.2, 150.1, 152.6, 161.3, 168.9; ESI-MS: 294.1 [M + Na]+; HRMS calculated for C15H19N3O2Na [M + Na]+ 296.1375, found 296.1370, ppm error −1.7. Solubility Evaluation. Compounds 1 and 3−6 were added to pure water or phosphate buffer (50 mM, pH 7.4) at 20 °C. After shaking and centrifuging, the supernatant was taken to determine the concentration of compounds 1 and 3−6 in each solvent for calculation of the corresponding solubility. The samples (10 μL each) were analyzed by HPLC (Shimadzu DGU-20A3R) on Shimadzu-GL WondaSil C18-WR column. The mobile phase was acetonitrile-water (60/40, v/v) at a flow rate of 1.0 mL/min with the detection wavelength at 254 nm. This experiment was repeated in triplicates. MTT Assay. Human colon, colorectal, and nontumor colon epithelial cells (1 × 106 cells/well) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in 96-well plates overnight and treated with vehicle alone or the tested compounds at the indicated concentrations for 24, 48, or 72 h. During the last 4 h culture, the cells were exposed to 20 μL of MTT (5 mg/mL, in PBS). The generated formazan in individual wells was dissolved in DMSO (150 μL) and measured for the absorbance at 570 nm using a microplate reader. The inhibition rates were calculated by the formula of (1 − OD (experimental cells)/OD (control cells)) × 100%. Measurement of ROS Generation. The tested cells (1 × 106 cells/well) were treated in triplicate with vehicle DMSO (0.1%, v/v) alone, 1, or 4 at the indicated concentrations for 3 h, and the cells were stained with dihydroethidium (DHE, Beyotime). The levels of intracellular ROS were measured as the fluorescent signals using a fluorescence microplate reader (300 and 610 nm). In Vivo Antitumor Effects of 4. All animal experimental protocols were approved by the Administration Committee of Experimental Animals in Jiangsu Province and the Ethics Committee of China Pharmaceutical University. Male BALB/c nude mice 3−5 weeks of age (Silaike Experiment Animal Co.,Ltd., Shanghai, China) were inoculated subcutaneously with 1 × 107 HCT-116 cells. After the formation of a solid tumor with a volume of about 75 mm3, the tumorbearing mice were randomly divided into five groups with eight mice in each group. The groups with compound 4 treatment received two dosages (2.5 or 5 mg/kg) every day. Oxaliplatin (5 mg/kg) and 1 (5 mg/kg) were used as positive controls. At the end of the experiment, the mice were sacrificed, their tumors were dissected, and the tumor size and weight were measured. The tumor inhibitory ratio was calculated by the following formula: tumor inhibitory ratio (%) = 1830



Article

overnight. After being washed, the bound antibodies were detected with HRP-conjugated second antibodies (anti-β-actin antibody, anti-βcatenin, anti-p-Akt, anti-Akt antibody, anti-E-cadherin, anti-Vimentin, anti-MMP-9, anti-Snail, anti-Twist (Abcam, Cambridge, UK); antiGSK3β, anti-p-GSK3β (S9), anti-Lamin A (Bioworld, Minnesota, USA)) and visualized using the enhanced chemiluminescent reagents. The relative levels of target protein to the control or phosphorylated to expressed protein were determined by densitometric analysis using the ImageJ software. Statistical Analysis. Data are expressed as mean ± SD. The difference among the groups were analyzed by one-way ANOVA and post-hoc Tukey’s test using SPSS software. A P value of

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