Tanshinone-IIA-Based Analogs of Imidazole Alkaloid Act as Potent

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Tanshinone-IIA-Based Analogs of Imidazole Alkaloid Act as Potent Inhibitors to Block Breast Cancer Invasion and Metastasis in vivo Qiong Wu, Kangdi Zheng, Xiaoting Huang, Li Li, and Wenjie Mei J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01018 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 6, 2018

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Journal of Medicinal Chemistry

Tanshinone-IIA-Based Analogs of Imidazole Alkaloid Act as Potent Inhibitors to Block Breast Cancer Invasion and Metastasis in vivo Qiong Wu,†,‡ Kangdi Zheng,† Xiaoting Huang, † Li Li,† Wenjie Mei*,†,§ †

School of Pharmacy, Guangdong Pharmaceutical University, Gaungzhou, 510006, China. ‡

Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou 510632, China

§

Guangzhou key laboratory of construction and application of new drug screening

model systems, Guangdong Pharmaceutical University, Guangzhou 510006, China

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ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Tanshinone-IIA (Tan-IIA), a primary active component extracted from commonly used Chinese herbal, Salvia miltiorrhiza (Danshen), is considered as a potential inhibitor against tumor-cell proliferation. However, the potential application of Tan-IIA is hindered by its poor water solubility and low bioavailability. In this work, an imidazole moiety was linked to the skeleton of Tan-IIA to enhance its antitumor activity. A series of Tan-IIA-based analogs TA01-TA12 were synthesized, and their inhibitory activities against the migration and invasion of MDA-MB-231 cells were investigated. All compounds, particularly TA12, markedly inhibited the proliferation, migration and invasion of MDA-MB-231cells. TA12 also prominently blocked cancer-cell metastasis in blood vessels and surrounding tissues in zebrafish xenograft model. Further studies showed that the mechanisms may involve S-phase arrest pathway, which was probably caused by inducing reactive oxygen species production and activating DNA damage. These results indicated that the Tan-IIA-based analogs of imidazole derivates can act as potent anti-metastasis agents.

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INTRODUCTION Tanshinone-IIA (Tan-IIA) is a major active component extracted from a commonly used Chinese herbal, Salvia miltiorrhiza (Danshen), that is responsible for the antiangiogenic, antioxidant stress, anti-inflammatory, antiplatelet aggregation, and antitumor activities.1, 2 A number of studies have shown that Tan-IIA significantly inhibits the growth, migration, invasion, and metastasis of various cancer cells by inducing cell cycle arrest and apoptosis.3-5 However, further preclinical or clinical development of Tan-IIA as a new antitumor therapeutic agent has been dampened by its weak potency, extremely low aqueous solubility, and poor bioavailability.6 Therefore, continued research on Tan-IIA analogs is currently focused on structural optimization to promote both the antitumor effect and the drug-like properties.7, 8 A limited number of structural modifications have been researched, including the introduction of some moieties (long-chain alkanes, N-heterocycle, or oxygenous groups) that help to increase water solubility.9-11 Most of these analogs still suffer from modest in vitro antitumor efficacy. Imidazole and related azole moiety, which are well-known significant pharmacophores in various clinical drugs, generally increase the antitumor potency of drugs.12, 13 For example, metronidazole-amino-acidum-natrium, which contains two imidazole rings in the molecular structure, exhibits significant radiosensitization for head and neck cancer.14 Another commonly used antitumor drug for fadrozole (Benzonitrile,4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridi) contains one imidazole ring, which is a clinically active aromatase inhibitor with a low incidence of side effects during breast cancer treatment.15 Moreover, temozolomide is tolerated and is emerging as a feasible first-line choice to treat patients with glioblastoma multiforme,16 and dacarbazine is one of the most important effective drugs in treating 3



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melanoma in clinic.17 Although certain im The antitumor idazole derivates have been reported with potential applications in medicinal chemistry, the introduction of imidazole into Tan-IIA has not been investigated as a potential anticancer agent. We therefore designed a series of Tan-IIA-based analogs through introducing imidazole moiety to increase aqueous solubility and molecular stability that may improve physicochemical property and modify the middle B ring to reduce lipophilicity and anchor an electron-rich aromatic ring on the N-moiety that helps to enhance antitumor activity. We described the synthesis and the enhanced potency of this new series of Tan-IIA analogs to block metastasis breast cancer in vitro and in vivo.

Tanshinone-IIA Based Imidazole

Tanshinone-IIA TA01 R1=-CF3

R2=-H

R3=-H

TA02 R1=-H R2=-CF3

R3=-H

TA03 R1=-H R2=-H R3=-CF3

TA04 R1=-OCH3 R2=-H

R3=-H

TA05 R1=-H R2=- OCH3 R3=-H

TA06 R1=-H R2=-H R3=- OCH3

TA07 R1=-NO2

R2=-H

R3=-H

TA08 R1=-H R2=-NO2

R3=-H

TA09 R1=-H R2=-H R3=- NO2

TA10 R1=-OH

R2=-H

R3=-H

TA11 R1=-H R2=-OH

R3=-H

TA12 R1=-H R2=-H R3=-OH

Scheme 1. Synthetic route of Tan-IIA-based analogs of imidazole derivatives RESULTS Synthesis and Characterization Tan-IIA, as a naturally active substance, can be chemically modified easily, which promotes the intermolecular reductive cyclization of 1,2-diketone and aldehyde groups in basic media (NH4Ac/HAc) and provides a convenient method for synthesizing imidazole derivates. Herein, a series of novel Tan-IIA derivates 4


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(TA01–TA12) was prepared from Tan-IIA and benzaldehyde modified with different substituent groups at 100 °C under microwave irradiation for 20 min in a Pyrex vessel with a high yield range of 85% to 95%, which was markedly higher than that in conventional synthesis methods (Scheme 1). Moreover, three crystallites were obtained through dissolvent crystallization to clarify the typical spatial structure of this class of compounds by single-crystal X-ray structural analysis. As shown in Figure 1, the crystal structures of TA04, TA09, and TA10 showed that this type of Tan-IIA derivative is a special compound that contains macrocyclic aromatic conjugation system in the skeleton, in which two heteroaromatic structures (furan and imidazole rings) might be significant active centers. The selected bond distances and angles are listed in Tables S1 and S2 of the Supporting Information. TA04, as a typical example, represented an aromatic conjugation system with in-plane aromaticity constructed by A, B, C, E, and F rings and a twisted chair structure displayed by D ring.18 Two other compounds (TA09 and TA10) seemed to have reached the same structural features independently. However, for E ring, the imidazole ring is a typical transition state, in which the H atom can transfer freely from one N to another N atom according to the changes in surrounding chemical environment. For TA04 and TA10, H atom was in the N1 and N2, respectively, owing to the presence of -OCH3 and -OH in the ortho-position in F ring, in which the H atom of N2 of TA04 can form H bond with adjacent O atom and N2 of TA10 can form H bond with the adjacent H atom of -OH group by intramolecular interactions.19 For TA09, H atom movably sited in N1 position. The spatial structure of Tan-IIA-based imidazole derivates might help us to analyze the structure–activity relationship in the growth inhibition of tumor cells.

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23 22 O1

3 2 4 25

A

O1 3 2 4 25

1

C 5 6

26

D 7

9

8

24 21 20

N1

E

19 18

13 14

N2

F

O2

15

13 14 N2

18

19

17

F 15

16

17

16

27

24

E

11 12 10

B

TA04

21 20 N1

B

7

A

11 12 10

6D 26

23 22

C

5

1

O1 1 3 2

O3

4

N3

25

6

5

26

7

TA09

A B

24 21 20 N1

11 12 10 N2

D

O2

9 8

C

23 22

8

E

13

14

F

9

TA10

O2 15

19 18 17

16

Figure 1. Ball-and-stick diagrams of the Tan-IIA-based analogs of imidazole derivatives TA04, TA09, and TA10. For clarity, the solvent molecules and the counteranions were omitted. CCDC No: 1045309 (TA04); 1519705 (TA09); 1831456 (TA10). Structure–activity Relationship of Tan-IIA–imidazole Derivatives The antiproliferative activities of synthetic Tan-IIA-based imidazole derivatives (TA01–TA12) against various human cancer-cell lines were evaluated by MTT assay. The inhibitory activities (IC50) of these synthetic compounds against the growth of various human tumor cells, including MDA-MB-231 highly metastatic human breast cancer cells line, HepG2 human liver carcinoma cell line, and HeLa human cervical cancer-cell line, after 72-h treatment are listed in Table 1. All of these compounds exhibited certain inhibition to various tumor cells after 72-h treatment, especially TA12 displayed a better antitumor activity than other compounds. The inhibitory activity (IC50) of TA12 against breast cancer MDA-MB-231 cells and normal liver L02 cells were approximately 13.2 μM and 49.1, 6


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μM, respectively, which was much better than that of Tan-IIA (115.9 μM and 78.5 μM) and the tolerable level of DOX (8.53 μM and 3.6μM). These results showed that the introduction of imidazole ring in Tan-IIA derivatives can effectively enhance the growth inhibition of tumor cells via exhibiting certain sensitivity to highly metastatic MDA-MB-231 cells and low toxicity to normal L02 cells. Table 1. IC50 (μM) of TA01−12 against Human Cancer Cells a Comp.

MDA-MB-231

HepG2

HeLa

A549

L02

TA01

23.8±7.5

38.4±6.4

61.0±11.7

25.6±3.1

93.9±16.5

TA02

52.7±6.4

43.8±7.9

68.8±12.3

31.4±2.8

54.5±8.5

TA03

44.3±13.2

25.2±8.7

65.3±11.4

48.8±4.5

30.7±11.3

TA04

110.2±15.7

125.7±18. 7

>300

45.9±5.6

86.8±12.7

TA05

52.3±6.8

28.4±7.4

67.8±15.5

40.1±7.3

>300

TA06

21.1±6.1

13.9±4.2

62.0±12.8

35.3±6.2

84.0±15.1

TA07

23.6±6.3

24.8±5.8

114.9±21.4 202.8±12.7

TA08

48.8±11.6

33.9±9.2

>300

63.7±10.5

>300

TA09

30.7±6.7

31.0±7.7

157.3±24.3

24.9±8.3

>300

TA10

20.2±6.1

26.3±8.5

84.3±16.2

4.9±3.2

51.9±8.3

TA11

25.5±7.3

37.1±5.7

52.6±8.1

18.4±5.1

32.9±12.7

TA12

13.2±3.9

21.2±7.2

39.8±7.3

6.9±2.4

49.1±7.4

Tan-II A

115.9±25.4

65.7±12.7 193.2±29.4

53.2±13.4

78.5±6.3

DOX

8.53 ± 2.9

4.3 ± 1.4

3.4±2.1

3.6±1.2

8.66 ± 2.9

>300

a

Cells were treated with indicated compounds to repeat three times in 72 h, and the cell viability was determined by MTT assay. The position, electric effect, and steric hindrance of substituted groups often play key roles in the biological activities of drugs. Unlike the IC50 value for different substituted positions of compounds to MDA-MB-231 cells, a complex relationship and significant regularity indicated that the ortho- and para-substituted compounds of 7



the electron-donating group in the terminal benzene ring exhibited higher growth suppression than meta-position, especially para-position was the most active one.20 For example, with -OCH3 and -OH modification, the para-substituted TA06 and TA12 exerted minimum IC50 values (21.1 and 13.2 μM, respectively) compared with ortho- (the IC50 values of TA04 and TA10 were 110.2 and 20.2μM, respectively) and meta-position (the IC50 values of TA05 and TA11 were 50.2 and 25.5 μM, respectively). Although for the compounds of the electron-withdrawing group, orthoand para-substituted compounds were still better than meta-position, the best one was ortho-substituted compound. With -CF3 and -NO2 modification, the ortho-substituted compounds TA01 and TA07 exhibited better antitumor activities (with IC50 of 23.8 and 23.6 μM, respectively) than meta- (the IC50 values of TA02 and TA08 were 110.2 and 20.2 μM, respectively) and para-position (the IC50 values of TA03 and TA09 where 44.3 and 30.7 μM, respectively). Comprehensive analysis of these results indicated that the compounds modified by the electron-donating group in the para-position might exert excellent antitumor activities, which provided guiding significance for optimizing Tanshinone IIA derivatives.21 On the basis of its promising in vitro activity, TA12 was selected as an early lead for a preliminary evaluation in the further study. As shown in Figure S37A, with increasing TA12, some cell membrane surface became rough and uneven with large and numerous particles. The cell membrane structure was incomplete, and a number of cells were shrinking to globe.22 The treatment of MDA-MB-231 cells with complex TA12 significantly decreased the cell viability in a dose-dependent manner, which kept a considerable level with DOX. Migration and Invasion of MDA-MB-231 Cells Inhibited by TA12 Metastasis is the main cause of morbidity and mortality in patients with breast 8


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cancer. Research indicating that the direct actor of tumor cells migration and invasion is invadopodia, which are actin-rich protrusions that localize proteolytic activity to areas of the cell in contact with extracellular matrix (ECM) and release matrix metalloproteinase to degrade the ECM.23 The inhibitory effect of TA12 on the migration and invasion of MDA-MB-231 cells was further evaluated by wound-healing and FITC-gelatin assays. Figure 3A shows an obvious decrease in the distance of wound closure when treated without drugs for 72 h, but a notable inhibition of wound closure was observed with the addition of TA12. When treatment with TA12 for 3 μM was applied, the wound-healing rate was less than half of that in the control group. With increasing TA12 for 6 μM, the wound-healing rate further reduced lower than that treated with 3 μM, which suggested that the inhibition of TA12 against migration had a positive dosage-dependent manner.24 Moreover, the inhibitory effect of TA12 on the invasion of MDA-MB-231 cells was studied by FITC-gelatin invasion assay. Highly invasive cells with invadopodia generally can release matrix metalloproteinases (MMPs) to degrade the FITC-gelatin.25 Consequently, the number of dark holes (nonfluorescent areas) in the FITC-gelatin showed the invasion capability of tumor cells, in which the high number of dark holes implied great invasiveness of the tumor cells. Figure 3C depicts that when treatment without drugs was implemented, numerous black holes can be found in the FITC-gelatin, which implied that MDA-MB-231 exerted high invasive capability.26 After TA12 for 3 μM was applied, the number of black holes decreased notably. With the further increase in TA12 concentration for 6 μM, the invasive capability of MDA-MB-231 cells was markedly inhibited, and no black holes were observed in FITC-gelatin.27 These results suggested that this type of Tan-IIA–imidazole derivatives can effectively inhibit the migration and invasion of 9



MDA-MB-231 cells. 0h

24 h

48 h

72 h

B

6 μM

3 μM

Control

A

Rhodamine-phalloidin

Merge

Overlay

300

Fluorescence intensity

FITC-Gelatin

Control

C

Rhodamine-phalloidin FITC-Gelatin

250 200 150 100 50 0

5

10

15

20

25

30

35

40

Distance (Pixel)

3 μM

Fluorescence intensity

300


250 200 150 100 50 0

0

5

10 15 20 25 30 35 40 45 50

Distance (Pixel) Fluorescence intensity

300

6 μM

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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250 200 150 100 50 0

0

5

10 15 20 25 30 35 40 45 50

Distance (Pixel)

Figure 2. Inhibition of migration and invasion of MDA-MB-231 cells by TA12 in vitro. (A) The wound-healing assay was used to evaluate the migration of MDA-MB-231 cells after treatment with TA12 (0, 3, and 6 μM) and DMEM without FBS. Cells were wounded and monitored with a microscope every 24 h. The migration was determined by the rate of cells filling the scratched area. (B) Wound-healing rate of MDA-MB-231 cells induced by TA12. (C) The invasion of MDA-MB-231 cells was blocked by TA12 (0, 3, and 6 μM). The whole view of many MDA-MB-231 cells, the number of black holes observed without TA12, and those treated with TA12. Cell cytoskeletons were dyed by rhodamine-conjugated phalloidin. The dark area in the FITC-gelatin was identified as the position degraded by 10


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MDA-MB-231 cells. All data were obtained from three independent experiments and presented as the means ± SD. *P < 0.05 vs untreated control. In vivo Activity of TA12 on the Metastasis of MDA-MB-231 Cells in Zebrafish. Some researches suggested that xenotransplantation of human tumor cells in zebrafish has been an important major model utilized by cancer biologists for many years.28 Due to extensive advantage of high reproductive ability, superior imaging qualities and little immunorejection,29 combined with their cost effectiveness makes zebrafish attractive for developing potent cancer research tool to study the mechanism of drug action in vivo. MDA-MB-231 breast cancer cells xenografts in zebrafish embryos, were used to evaluate the suppression of TA12 on the proliferation and metastasis of MDA-MB-231 cells in zebrafish for studying the inhibitory activity of TA12 in vivo. Here, with a transgenic zebrafish (fil1:EGFP), a breast cancer-bearing model was established, in which the blood vessels were labeled green fluorescence, and the red fluorescently labeled MDA-MB-231 cells were serially transplanted in limiting dilutions to identify the tumor cells near the subintestinal vessel (SIV) of the zebrafish.30 Figure 5A shows that unlike the result after 0 h when tumor cells were microinjected into zebrafish embryos, after 48 h, the MDA-MB-231 cells in the control group markedly enhanced the sprout of angiogenesis and widely spread away from the primary site. Some tumor cells even migrated in the blood vessel of the zebrafish tail.31 When the time was increased to 96 h, a number of MDA-MB-231 cells invaded neighboring tissues and spread to the zebrafish head. When treated with TA12 (5 μM), the MDA-MB-231-cell xenografts lowly induced the sprout of angiogenesis and markedly blocked cell migration and invasion. Furthermore, the MDA-MB-231-cell xenografts treated with TA12 showed reduced fluorescence 11



intensities and decreased tumor area compared with the control group, which indicated that TA12 can inhibit the proliferation of MDA-MB-231 cells in zebrafish xenografts.32 TA12 effectively inhibited the proliferation and metastasis of MDA-MB-231 cells in zebrafish xenografts, which can be developed as a potential candidate to inhibit the metastasis of breast cancer.

12


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Figure 3. Inhibitory effect of TA12 on the proliferation and metastasis of MDA-MB-231 cells in zebrafish xenografts model. (A) Metastasis of MDA-MB-231 cells xenografts zebrafish without and with TA12. Dil-labeled MDA-MB-231 cells (red) were microinjected into zebrafish embryos, and treatment with TA12 (5 μM) for 96 h. After 48 h, the proliferation and metastasis of the xenografts of MDA-MB-231 cells were imaged under a fluorescence microscope. (B) Quantification of the fluorescent area of the tumor xenografts, representing total MDA-MB-231 cells in zebrafish (n=10/group). (C) Fluorescence intensity of the tumor xenografts in trunk (n=10/group). (D) Fluorescence intensity of the tumor xenografts in tail (n=10/group). All data were obtained from at 3 independent experiments and presented as the means ± SD. *P < 0.05 vs untreated control. Here, the inhibition of TA12 to angiogenic activity was further evaluated by using transgenic Tg (fli1:EGFP) zebrafish, in which the vascular endothelial cells were labeled with green fluorescent protein. The SIVs started to develop in the zebrafish until 48 hpf. Therefore, 24 hpf zebrafish embryos were selected, dechorionated, and incubated with 2 and 4 μM of TA12 for 48 h. The effects of TA12 on the changes in SIVs were detected at 72 hpf and observed under a fluorescence microscope. Figure 6A illustrates that unlike the control group, the angiogenesis treated with different concentrations of TA12 (2 and 4 μM) showed a minimal damage and an abnormal development, and an inconsiderable distinction was observed in other parts.33 For the positive control PTK787, a classic inhibitor to angiogenesis, the formation of angiogenesis was blocked obviously.32 These results indicated that TA12 exhibited minimal inhibition to normal angiogenesis in the SIVs of the zebrafish. TA12 can greatly suppress the sprouts of angiogenesis induced by tumor cells but rarely blocked the growth of normal angiogenesis. Moreover, The in vivo toxicity assessments of 13



TA12 were conducted on developing zebrafish embryos, which suggested that 3 had low toxicity to zebrafish embryos, and such toxicity could extend the developmental period (Figure S54). The metastasis of MDA-MB-231 was thus blocked not through inhibiting angiogenesis in vivo.

A

B

Control

2 μM

4 μM

PTK787

Figure 4. Inhibitory effect of TA12 on the angiogenesis of transgenic zebrafish (fil1:EGFP). PTK787 was used as a positive control. Zebrafish (48 hpf old) treatment with different concentration of TA12 (0, 2 and 4 μM ) for 48 (n=10/group) . All data were obtained from three independent experiments and presented as the means ± SD. *P < 0.05 vs untreated control. The effect on the inhibition of focal adhesions and stress fibers in MDA-MB-231 cells was determined by TA12 immunofluorescence experiments using a laser scanning confocal light microscope. Stress fibers, the leading actors in the cell migration process, are usually composed of bundles of approximately 10 to 30 actin filaments. Focal adhesions, such as integrin and proteoglycan-mediated adhesion links to the actin cytoskeleton, regulate cell migration.34 The focal adhesions were observed by paxillin because characterization of the protein revealed that paxillin was localized at discrete structures of focal adhesions, which were sites of close cellular contact with the underlying extra-cellular matrix. Invadopodia are actin-rich protrusions located in 14


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the areas of focal adhesions in contact with the ECM.35 The possible mechanism of antimetastasis by TA12 in vivo was studied. Fig. 5 depicts a number of focal adhesions around the edge of MDA-MB-231 cells without TA12 treatment. However, after being treated with TA12, the number of paxillins in MDA-MB-231 cells decreased significantly, and the cellular morphology changed. The stress fibers were also reduced, as represented by the F-actin with red fluorescence after being treated with TA12. The expression of focal adhesion kinase, a key protein to point the position of invadopodia, downregulated markedly. By contrast, the expression of GSK3β was upregulated. Inhibition of releasing MMPs to suppress the invasion of tumor cells consequently occurred.26 The results indicated that TA12 can inhibit the migration and invasion of MDA-MB-231 cells through suppressing focal adhesions and stress fibers.

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Figure 5. (A) The distribution of focal adhesion proteins (green) and stress fibers (red) of MDA-MB-231 cells induced by TA12. MDA-MB-231 cells were treated with TA12 (0, 5 and 10 μM) for 24 h. Green: Paxillin. Red: F-actin. (B) The number of paxillins on the cell membrane margin. (C) Regulation of invasion related protein expression of FAK, GSK3β and MMP9 at the protein level. MDA-MB-231 cells were treated with TA12 (0, 5, 10 and 15 μM) for 72 h. All data were obtained from three independent experiments and presented as the means ± SD. *P < 0.05 vs untreated control. Growth Inhibition of TA12 by Joint Action of S-Phase Arrest and Apoptosis Flow cytometry was performed to explore the possible mechanism of TA12 in 16


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inhibiting the growth of MDA-MB-231 cells. As shown in Figures 6A and 6B, the exposure of MDA-MB-231 cells to TA12 (0, 5, 10, and 15 μM) for 24 h triggered a significant increase in the number of cells under S-phase arrest from 42.60% to 54.51%, accompanied by a decrease in the percentage of cells at G1 phase from 49.4% to 35.37%.37 Upon the addition of TA12, certain degree of induction of cell apoptosis was observed.38 The obvious upregulation of p21 and downregulation of cyclin A (the key proteins in the S-phase of the cell cycle) and the small upregulation of caspase 3, cleaved-caspase 3 and poly (ADP-ribose) polymerase (the major proteins in the apoptosis),39 upon the increase in TA12 indicated that the growth inhibition induced by TA12 was mainly caused by the S-phase arrest and slight joint action of apoptosis.

Figure 6. S-phase arrest (A) and (B) apoptosis of MDA-MB-231 cells induced by TA12. (C) Regulation of the S-phase arrest related protein expression of cyclin A, p21 by TA12. (D) Regulation of the apoptosis related protein expression of caspase 3 17



and PARP at the protein level. MDA-MB-231 cells were treated with TA12 (0, 5, 10 and 15 μM) for 72 h. All data were obtained from three independent experiments and presented as the means ± SD. *P < 0.05 vs untreated control.

Figure 7. (A) DNA damage induced by TA12 as examined by Comet assay. (B) Effects of TA12 on the expression level γH2A X. Cells were treated with TA12 (0, 10 and 20 μM) for 24 h and the length of tail reflects DNA damage in the cells. (C) 18


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ROSUP and TA12 (5, 10 15 and 20 μM) induced ROS accumulation in MDA-MB-231 cells. Cells were pretreatment with DCFH-DA (10 μM) in FBS-free DMEM at 37 oC for 30 min. (D) DNA damage related protein expression of γH2AX, p53 and AKT induced by TA12. Statistical analysis of the percentage of γH2AX foci in MDA-MB-231 cells from three independent experiments and presented as the means ± SD. *P < 0.05 vs untreated control. The results of flow cytometry assay demonstrated that TA12 inhibited the proliferation of MDA-MB-231 through induced S-phase arrest, which is a stage of DNA synthesis.40 Subsequently, we detected the induction of DSBs in MDA-MB-231 cells by neutral comet assays and confocal immunofluorescence assays, stained with DSB biomarker γH2AX.41 Figure 8A shows that TA12 significantly increased the generation of endogenous DSBs in MDA-MB-231 cells, as indicated by the statistical increase in olive tail moment.42 The percentages of γH2AX focal positive cells were obviously increased in MDA-MB-231 cells treated with different concentrations of TA12 (0, 10, and 20 μM). These results indicated that TA12 mainly induced the S-phase arrest of MDA-MB-231 cells through DNA damage, which led to endogenous DSBs. Reactive oxygen species (ROS) plays an important role in the induction of DNA damage.43 We speculated the possibility that overproduced ROS induced by TA12 entered into nucleus to enhance ROS-mediated DNA damage. Fig. 7C demonstrates that like positive ROSUP, TA12 treatments triggered a time- and dose-dependent increase in ROS generation. Abundant intracellular ROS might cause DNA damage and

activate

down-streamed

signaling

pathway.20

Therefore,

combined

treatment-induced DNA damage was also detected using DNA damage markers, p53 and γH2AX. Fig. 7D illustrates that treatment with TA12 triggered DNA damage, as 19



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confirmed by the upregulated level of p53 and γH2AX, which led to the inactivated AKT signal pathway.44 These results provided compelling evidence that TA12 inhibited the invasion and metastasis through inducing S-phase arrest in MDA-MB-231 cells by triggering ROS overproduction, which activated DNA damage-mediated p53 inactivated AKT pathway. DISCUSSION In the last decades, a number of studies suggested that

mainly suppress tumor

growth through S phase arrest, and also might via apoptosis induction.45,46 Moreover, some researchers studied the structure modification of Tanshinone IIA and their structure-activity relationship. Bi et al introduce some substituent groups such as halogen and functional benzene ring into the C ring of Tanshinone IIA, it is found that the polarity of products contrasted with DIIA was increase and some synthetic products exhibited good vasodilative activity.47,48 However, in this study, we designed and synthesized a class of Tan-IIA derivates by introduced the imidazole ring into B ring and increased the planarity by the terminal benzene ring, which the introduction of imidazole ring slightly improved the water solubility and enhanced the anti-tumor activity against diverse tumor cells, especially to triple-negative breast cancer cells. Further studies shown that this class of compounds uptake into cell mainly rose the ROS level and induce DNA damage to activate AKT signal pathway induced S-pahse arrest and inhibited the metastasis of breast cancer cells. Comprehensive analysis suggested that TA12 can effectively induce S-phase arrest and markedly block the migration and invasion of MDA-MB-231 cells in vitro and in vivo. However, the mechanism is still unclear and is being studied further. When TA12 uptake in cells, the ROS level were increased rapidly and induce DNA damage of MDA-MB-231 cells, which further activate the up-regulation of p53. Considering 20


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AKT signal pathway take an important part in influence diverse cellular processes, including cell proliferation, proliferation, cell cycle, apoptosis and metastasis, which is decreased with the increased of p53. When the AKT expression level increased, the key regulating protein of cell cycle of p21 and cyclin A were up-regulated and down-regulated obviously, resulting in the S-phase arrest of tumor cells. Meanwhile, ROS activation induce the down-regulation of FAK, which further promote the up-regulation of the expression of GSK3β leads to the decreased release of MMPs to block the metastasis of tumor cells.

Scheme 2. Diagram of the S-phase arrest and metastasis inhibiton of MDA-MB-231 cells induced by TA12 through promoting ROS-activated DNA damage by regulating AKT signal pathway mediated p53. CONCLUSIONS A class of Tan-IIA-based imidazole analogs was prepared by microwave-assisted synthesis technology. This class of compounds exerted selectively potent inhibition 21



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against highly invasive breast cancer cells, especially TA12, which exhibited an excellent inhibitory activity against the proliferation, migration, and invasion of MDA-MB-231 breast cancer cells. The structure–activity relationship analysis of these results suggested that the compounds modified by the electron-donating group in the para-position might exert an excellent antitumor activity, which provided guiding significance for further optimization of Tan-IIA derivatives. Our mechanistic study result showed that TA12 acted on S-phase arrest through ROS-mediated DNA damage activation. Further evaluation of in vivo activity indicated that TA12 effectively inhibited the proliferation and metastasis of MDA-MB-231 cells in zebrafish xenografts at a low concentration. In summary, our studies provided evidence that Tan-IIA-based imidazole analogs can be developed as potential treatment agents to delay or prevent the metastasis of breast cancer. EXPERIMENTAL SECTION Chemicals. Tanshinone IIA All reagents and solvents were purchased commercially and used without further purification unless specially noted. Tanshinone-IIA-based analogs of imidazole alkaloid were synthesized by using Anton Paar Monowave 300 microwave reactor. ESI-MS spectra were obtained in methanol on Agilent 1100 ESI-MS system operating at room temperature. The 1H NMR and

13

C NMR spectra

were recorded on a dimethyl-d6 sulfoxide (DMSO-d6) solution on a Bruker Avance Ⅲ 500 spectrometer operating at room temperature. All the synthetic Tanshinone -IIA-based analogs were confirmed with ≥95% purity by using HPLC (Agilent 1290 Infinity II) analysis in the eluate (acetonitrile: water =1:1). The X-ray intensity data were collected on an X-ray diffractometer equipped with a graphite-monochromated

22


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1,6,6-trimethyl-11-(2-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-furo[2', 3':1,2]phenanthro[3,4-d]imidazole (TA01) A mixture of tanshinone IIA (100.0 mg, 0.34 mmol), 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol), ammonium acetate (4 g, 51.9 mmol) and glacial acetic acid᧤15 mL) was heated at 100 °C for 20 min under microwave irradiation. Then, 20 ml of water was added and the pH value was adjusted to 7.0 at room temperature. The solution was filtered and dried in vacuum to obtain brown precipitate.22 After filtration and evaporation of the solvent, the residue was purified using silica gel flash column chromatography with a mixed solution of petroleum ether and ethyl acetate as the eluent. TA01 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 447.5 ([M-H+]-). 1H NMR (500 MHz, DMSO) δ 13.22 (s, 1H), 8.10 (d, J = 8.6 Hz, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.94 – 7.83 (m, 3H), 7.77 (t, J = 7.5 Hz, 1H), 7.62 (d, J = 8.6 Hz, 1H), 3.83 (t, J = 6.1 Hz, 2H), 2.54 (s, 3H), 1.90 (dd, J = 28.5, 9.6 Hz, 2H), 1.72 (d, J = 5.5 Hz, 2H), 1.37 (d, J = 12.2 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 150.11 (s), 146.08 (s), 143.95 (s), 142.61 (s), 137.37 (s), 133.99 (s), 133.48 (s), 132.05 (s), 130.98 (s), 129.57 (s), 128.07 (s), 126.35 (s), 125.14 (s), 124.11 (s), 119.06 (s), 118.34 (s), 116.55 (s), 113.09 (s), 39.80 (s), 35.64 (s), 33.27 (s), 32.03 (s), 27.70 (s), 20.86 (s), 10.80 (s). 1,6,6-trimethyl-11-(3-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-furo[2', 3':1,2]phenanthro[3,4-d]imidazole (TA02) TA02 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 3-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol). TA02 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 449.1 ([M+H+]+). 1H NMR (500 MHz, 23



DMSO) δ 12.94 (s, 1H), 8.67 (s, 1H), 8.64 – 8.60 (m, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.89 (dd, J = 6.6, 1.3 Hz, 1H), 7.83 – 7.80 (m, 2H), 7.62 (d, J = 8.7 Hz, 1H), 3.93 (t, J = 6.3 Hz, 2H), 2.60 (dd, J = 6.0, 1.3 Hz, 3H), 2.01 – 1.90 (m, 2H), 1.81 – 1.70 (m, 2H), 1.36 (s, 6H). 13C NMR (126 MHz, DMSO) δ 150.44 (s), 146.81 (s), 144.15 (s), 142.62 (s), 137.76 (s), 134.16 (s), 133.09 (s), 131.84 (s), 131.24 (s), 131.04 (s), 127.16 (s), 126.49 (s), 125.40 (s), 124.58 (s), 123.91 (s), 119.12 (s), 118.58 (s), 117.58 (s), 116.62 (s), 113.08 (s), 39.79 (s), 35.67 (s), 33.28 (s), 32.13 (s), 20.98 (s), 11.16 (s). 1,6,6-trimethyl-11-(4-(trifluoromethyl)phenyl)-7,8,9,10-tetrahydro-6H-furo[2', 3':1,2]phenanthro[3,4-d]imidazole (TA03) TA03 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 4-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol). TA03 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 449.3 ([M+H+]+). 1H NMR (500 MHz, DMSO) δ 13.11 (s, 1H), 8.64 (d, J = 8.1 Hz, 2H), 8.18 (d, J = 8.6 Hz, 1H), 8.05 (d, J = 8.3 Hz, 2H), 8.01 (d, J = 1.3 Hz, 1H), 7.73 (d, J = 8.6 Hz, 1H), 4.05 (t, J = 6.3 Hz, 2H), 2.70 (d, J = 1.3 Hz, 3H), 2.11 – 1.99 (m, 2H), 1.90 – 1.77 (m, 2H), 1.48 (s, 6H). 13

C NMR (126 MHz, DMSO) δ 150.53 (s), 146.84 (s), 144.21 (s), 142.67 (s), 137.93

(s), 135.92 (s), 134.19 (s), 128.33 (s), 127.31 (s), 127.00 (s), 126.78 (s), 125.46 (s), 124.66 (s), 123.93 (s), 119.15 (s), 118.63 (s), 116.67 (s), 113.06 (s), 39.80 (s), 35.68 (s), 33.28 (s), 32.16 (s), 20.98 (s), 11.09 (s). 1,6,6-trimethyl-11-(2-methoxyphenyl)-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]ph enanthro[3,4-d]imidazole. (TA04) TA04 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 24


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2-methoxybenzaldehyde (107.0 mg, 0.51 mmol)). TA04 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 411.2 ([M+H+]+). 1H NMR (600 MHz, CDCl3) δ 11.77 (s, 1H), 8.66 (d, J = 6.7 Hz, 2H), 8.19 (t, J = 12.3 Hz, 2H), 7.56 (d, J = 8.9 Hz, 2H), 7.41 – 7.29 (m, 2H), 7.25 (s, 1H), 7.15 (t, J = 7.5 Hz, 2H), 7.01 (d, J = 8.3 Hz, 2H), 4.07 (d, J = 12.4 Hz, 3H), 3.47 (t, J = 6.2 Hz, 2H), 2.73 (s, 2H), 1.43 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 156.02 (s), 149.13 (s), 147.00 (s), 142.48 (s), 140.45 (s), 136.00 (s), 129.99 (s), 129.23 (s), 128.89 (s), 125.30 (s), 123.79 (s), 121.91 (s), 119.09 (s), 118.54 (s), 117.97 (s), , 111.33 (s), 77.26 (s), 77.05 (s), 76.84 (s), 56.18 (s), 38.29 (s), 34.38 (s), 31.89 (s), 29.92 (s), 19.89 (s), 9.71 (s). 1,6,6-trimethyl-11-(3-methoxyphenyl)-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]ph enanthro[3,4-d]imidazole (TA05). TA05 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 3-methoxybenzaldehyde (107.0 mg, 0.51 mmol). TA05 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 411.3 ([M+H+]+). 1H NMR (500 MHz, DMSO) δ 12.78 (s, 1H), 8.06 (t, J = 10.5 Hz, 1H), 7.92 (s, 1H), 7.89 (t, J = 5.2 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.48 (s, 1H), 7.12 – 6.98 (m, 1H), 3.95 (t, J = 5.8 Hz, 2H), 3.89 (s, 3H), 2.60 (s, 3H), 1.93 (d, J = 19.6 Hz, 2H), 1.83 – 1.68 (m, 2H), 1.37 (s, 6H).

13

C NMR (126 MHz, DMSO) δ 160.89 (s), 150.21 (s), 148.29 (s),

143.97 (s), 142.54 (s), 137.68 (s), 134.13 (s), 133.43 (s), 131.72 (s), 131.20 (s), 126.84 (s), 125.19 (s), 123.95 (s), 120.46 (s), 119.08 (s), 116.63 (s), 115.08 (s), 114.02 (s), 113.15 (s), 56.69 (s), 39.85 (s), 35.67 (s), 33.30 (s), 32.18 (s), 21.00 (s), 11.13 (s).

25



1,6,6-trimethyl-11-(4-methoxyphenyl)-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]ph enanthro[3,4-d]imidazole (TA06). TA06 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 4-methoxybenzaldehyde (107.0 mg, 0.51 mmol). TA06 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 411.2 ([M+H+]+). 1H NMR (500 MHz, DMSO) δ 12.66 (s, 1H), 8.25 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 1.3 Hz, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.13 (d, J = 8.6 Hz, 2H), 4.02 – 3.88 (m, 2H), 3.85 (s, 3H), 2.59 (s, 3H), 1.97 (d, J = 18.5 Hz, 2H), 1.76 (d, J = 5.4 Hz, 2H), 1.37 (s, 6H). 13C NMR (126 MHz, DMSO) δ 161.26 (s), 149.93 (s), 148.63 (s), 143.75 (s), 142.46 (s), 137.63 (s), 134.66 (s), 134.05 (s), 129.46 (s), 126.58 (s), 124.99 (s), 123.90 (s), 119.04 (s), 118.25 (s), 116.54 (s), 115.41 (s), 113.18 (s), 56.67 (s), 39.89 (s), 35.65 (s), 33.32 (s), 32.19 (s), 21.01 (s), 11.11 (s). 1,6,6-trimethyl-11-(2-nitrophenyl)-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]phena nthro[3,4-d]imidazole (TA07). TA07 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 2-nitrobenzaldehyde (78.6 mg, 0.51 mmol). TA07 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 426.3 ([M+H+]+). 1H NMR (500 MHz, DMSO) δ 13.28 (s, 1H), 8.18 (dd, J = 7.8, 1.2 Hz, 1H), 8.09 (d, J = 8.6 Hz, 1H), 8.03 – 7.98 (m, 1H), 7.91 (t, J = 5.1 Hz, 1H), 7.89 – 7.85 (m, 1H), 7.75 – 7.71 (m, 1H), 7.63 (d, J = 8.7 Hz, 1H), 3.67 (t, J = 6.3 Hz, 2H), 2.56 (d, J = 1.2 Hz, 3H), 1.95 – 1.82 (m, 2H), 1.80 – 1.60 (m, 2H), 1.35 (s, 6H). 13C NMR (126 MHz, DMSO) δ 150.62 (s), 150.38 (s), 144.17 (s), 143.83 (s), 142.72 (s), 137.63 (s), 134.07 (s), 133.31 (s), 132.23 (s), 131.36 (s),

26


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126.68 (s), 125.41 (s), 125.29 (s), 123.87 (s), 119.07 (s), 118.45 (s), 116.58 (s), 113.06 (s), 39.76 (s), 35.65 (s), 33.24 (s), 31.72 (s), 20.93 (s), 10.93 (s). 1,6,6-trimethyl-11-(3-nitrophenyl)-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]phena nthro[3,4-d]imidazole (TA08). TA08 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 3-nitrobenzaldehyde (78.6 mg, 0.51 mmol). TA08 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 426.4 ([M+H+]+). 1H NMR (500 MHz, DMSO) δ 12.99 (s, 1H), 9.13 (s, 1H), 8.71 (d, J = 7.4 Hz, 1H), 8.24 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.88 (s, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.62 (t, J = 10.4 Hz, 1H), 3.91 (s, 2H), 2.55 (s, 3H), 1.97 (d, J = 16.4 Hz, 2H), 1.84 – 1.61 (m, 2H), 1.37 (s, 6H). 13C NMR (126 MHz, DMSO) δ 150.53 (s), 149.62 (s), 146.09 (s), 144.16 (s), 142.60 (s), 137.75 (s), 134.16 (s), 134.02 (s), 133.61 (s), 131.55 (s), 127.27 (s), 125.43 (s), 124.44 (s), 123.87 (s), 121.82 (s), 119.11 (s), 118.62 (s), 116.62 (s), 113.01 (s), 39.77 (s), 35.66 (s), 33.27 (s), 32.11 (s), 20.99 (s), 11.17 (s). 1,6,6-trimethyl-11-(4-nitrophenyl)-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]phena nthro[3,4-d]imidazole (TA09). TA09 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 4-nitrobenzaldehyde (78.6 mg, 0.51 mmol). TA09 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 425.3 ([M-H+]-).

1

H NMR (500 MHz, DMSO) δ 13.03 (s,

1H), 8.53 – 8.49 (m, 2H), 8.39 – 8.33 (m, 2H), 8.05 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 1.3 Hz, 1H), 7.63 (d, J = 8.6 Hz, 1H), 3.91 (t, J = 6.3 Hz, 2H), 2.57 (d, J = 1.3 Hz, 3H), 2.02 – 1.86 (m, 2H), 1.83 – 1.68 (m, 2H), 1.37 (s, 6H). 27


13

C NMR (126 MHz,


DMSO) δ 150.79 (s), 148.23 (s), 146.10 (s), 144.36 (s), 142.72 (s), 138.33 (s), 137.85 (s), 134.29 (s), 128.36 (s), 127.74 (s), 125.62 (s), 125.36 (s), 123.87 (s), 119.17 (s), 118.77 (s), 116.72 (s), 112.95 (s), 39.76 (s), 35.68 (s), 33.26 (s), 32.13 (s), 20.96 (s), 11.07 (s). 1,6,6-trimethyl-11-(2-hydroxyl)-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]phenant hro[3,4-d]imidazole (TA10). TA10 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 2-hydroxybenzaldehyde (63.6 mg, 0.51 mmol). TA10 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 397.1 ([M+H]+). 1H NMR (500 MHz, DMSO) δ 13.67 (s, 1H), 12.96 (s, 1H), 8.37 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 8.7 Hz, 1H), 7.92 (s, 1H), 7.74 (d, J = 8.1 Hz, 1H), 7.69 – 7.58 (m, 1H), 7.41 – 7.27 (m, 1H), 7.04 (dd, J = 7.8, 4.1 Hz, 1H), 3.73 (t, J = 5.9 Hz, 2H), 2.60 (s, 3H), 2.03 – 1.89 (m, 2H), 1.72 – 1.64 (m,2H), 1.37 (s, 6H). 13C NMR (126 MHz, DMSO) δ 161.76 (s), 158.74 (s), 150.53 (s), 148.71 (s), 143.60 (s), 134.72 (s), 132.89 (s), 130.03 (s), 128.00 (s), 125.75 (s), 125.31 (s), 121.33 (s), 121.16 (s), 120.76 (s), 119.37 (s), 118.25 (s), 116.65 (s), 114.47 (s), 112.89 (s), 38.72 (s), 35.65 (s), 32.80 (s), 30.81 (s), 20.07 (s), 9.86 (s). 1,6,6-trimethyl-11-(3-hydroxyl)-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]phenant hro[3,4-d]imidazole (TA11). TA11 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 3-hydroxybenzaldehyde (63.6 mg, 0.51 mmol). TA11 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water. ESI-MS (in CH3COOCH2CH3, m/z): 397.3 ([M+H]+). 1H NMR (500 MHz, DMSO) δ 12.76 (s, 1H), 9.67 (s, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 1.3 Hz, 1H), 7.77 – 7.72 (m, 28


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1H), 7.60 (d, J = 8.7 Hz, 1H), 7.39 – 7.33 (m, 1H), 6.90 – 6.82 (m, 1H), 3.94 (t, J = 6.2 Hz, 2H), 2.58 (d, J = 1.2 Hz, 3H), 2.04 – 1.86 (m, 2H), 1.74 (dt, J = 33.8, 15.3 Hz, 2H), 1.36 (s, 6H). 13C NMR (126 MHz, DMSO) δ 159.59 (s), 150.79 (s), 149.35 (s), 144.55 (s), 143.17 (s), 138.32 (s), 134.73 (s), 134.08 (s), 131.65 (s), 127.44 (s), 125.78 (s), 124.61 (s), 119.74 (s), 119.58 (s), 119.02 (s), 117.96 (s), 117.31 (s), 115.51 (s), 113.82 (s), 40.52 (s), 36.33 (s), 33.97 (s), 32.86 (s), 21.67 (s), 11.75 (s). 1,6,6-trimethyl-11-(4-hydroxyl)-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]phenant hro[3,4-d]imidazole (TA12). TA12 was prepared using the method described above, but with 2-(trifluoromethyl)benzaldehyde (90.6 mg, 0.51 mmol) replaced by 4-hydroxybenzaldehyde (63.6 mg, 0.51 mmol). TA12 easily dissolve in chloroform, ethyl acetate and can slightly dissolve in ethyl alcohol and water.ESI-MS (in CH3COOCH2CH3, m/z): 397.3 ([M+H]+). 1H NMR (500 MHz, DMSO) δ 13.37 (s, 1H), 10.62 (s, 1H), 8.95 – 8.91 (m, 2H), 8.86 (d, J = 8.6 Hz, 1H), 8.67 (d, J = 1.3 Hz, 1H), 8.39 (d, J = 8.7 Hz, 1H), 7.76 – 7.73 (m, 2H), 4.74 (t, J = 6.4 Hz, 2H), 3.38 (d, J = 1.3 Hz, 3H), 2.80 – 2.66 (m, 2H), 2.58 – 2.52 (m, 2H), 2.17 (s, 6H). 13C NMR (126 MHz, DMSO) δ 185.28 (s), 177.81 (s), 163.27 (s), 161.11 (s), 151.29 (s), 145.11 (s), 143.88 (s), 139.05 (s), 136.23 (s), 131.05 (s), 129.52 (s), 127.91 (s), 124.80 (s), 122.68 (s), 119.63 (s), 118.22 (s), 114.64 (s), 40.21 (s), 37.13 (s), 34.29 (s), 32.31 (s), 21.55 (s), 11.36 (s). Cell culture. Human cancer cell lines, including human breast cancer MDA-MB-231 cells, human hepatocarcinoma HepG2 cells, human cervical cancer HeLa cells, human lung cancer A549 cells and human normal liver L02 cells, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) media 29



supplemented with fetal bovine serum (10%), penicillin (100 units/mL), and streptomycin (50 units/mL) at 37 °C in a CO2 incubator (95% relative humidity, 5% CO2). MTT assay. Cell viability was investigated by measuring the ability of cells to transform 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a purple formazan dye. Cells were seeded in 96-well tissue culture plates (5×103 cells per well) for 24 h. The cells were then treatment with the tested compounds at different concentrations for 72h. After incubation, 20 μL per well of MTT solution (5 mg/mL in phosphate buffered saline, PBS) was added, followed by incubation for a further 5 h. The medium was aspirated and replaced with 150 μL/well of DMSO to dissolve the formazan salt formed. The color intensity, which reflects the cell growth condition, was measured at 570 nm using a microplate spectrophotometer (SpectroAmaxTM250, BioTek Instruments, Inc., Winooski, VT, USA). Wound-Healing Assay. Cells were seeded in six-well tissue culture plates, which were respectively marked on the back (1 × 105 cells per well) until the monolayer cells covered more than 80% of the bottom of the culture plate. A line was then scratched on the culture using a tip (200 μL pipet) orthogonal to the mark on the plate. Subsequently, these cells were incubated with the test compounds at different concentrations (0, 3 and 6 μM) for 72 h. Migrating cells were observed in the same visual field every 12 h for 3 days.49 Using Slidebook and Excel software, the average migration rate and the end-to-end distance of cell trajectory were calculated based on 10 fields of view for each cell type. Fluorescein Isothiocyanate (FITC)-Conjugated Gelatin Invasion Assay. The FITC-gelatin invasion assay was performed according to the manufacturer’s 30


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instructions (Invitrogen). Briefly, coverslips (18 mm in diameter) were coated with 50 μg/mL poly-L-lysine for 20 min at room temperature, washed with phosphate buffered saline (PBS), fixed with 0.5% glutaraldehyde for 15 min, and rewashed with PBS three times.27 After washing, the coverslips were inverted on a drop of 0.2% FITC-conjugated gelatin in PBS containing 2.0% sucrose, incubated for 10 min at room temperature, washed with PBS thrice, quenched with sodium borohydride (5 mg/mL ) for 3 min, and finally incubated in 2 mL of complete medium for 2 h. Cells (2 × 105 per well) with different concentrations of TA12 (0, 3 and 6 μM) were plated onto the FITC-gelatin-coated coverslips and incubated at 37 °C for 24 h. The FITC-gelatin degradation status was evaluated and photographed with a laser confocal microscope. Flow cytometric analysis. Flow Cytometry Analysis. Cells were seeded in six-well tissue culture plates (1 × 105 cells per well), and the apoptosis rate and the cell cycle arrest were analyzed by flow cytometry as previously described. After incubating with different concentration of TA12 (0, 2.5, 5, 10, 20 and 40 μM) for 72 h, cells were trypsinized, washed with PBS, and fixed with 70% ethanol overnight at 4 °C. The fixed cells were washed with PBS and stained with propidium iodide (PI) for 15 min in the dark, and the cell cycle arrest was analyzed with an Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL, USA). Treated or untreated cells were trypsinized, washed with PBS, and costained with annexin-V and PI for 10 min, respectively.48 The apoptosis of cells was analyzed with an Epics XL-MCL flow cytometer (Beckman Coulter). Comet assay. Single-cell gel electrophoresis for detection of DNA damage was performed using the Comet assay reagent kit purchased from Trevigen according to 31



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the manufacturer’s instructions. DNA was stained with SYBR Green I (Trevigen) and visualized under a fluorescence microscope (Leica DMI8).50 Fifty cells per slide were selected randomly and their olive tail moments were determined using an image analysis system (ImagePro Plus). Immunofluorescence.

MDA-MB-231

cells

were

treated

with

different

concentrations of TA12 (0, 10 and 20μM). MDA-MB-231 cells in complete growth medium at 5 × 104 cells per mL were incubated for 24 h at 37 oC, unless otherwise stated. Cells were washed once in PBS, fixed, and permeabilized simultaneously using 4% paraformaldehyde with 1% Triton X-100 in PBS, quenched with 0.1 M glycine in PBS, and blocked overnight at 4 oC with 3% (wt/vol) BSA. Fixed cells were stained with primary antibodies as indicated.51 Cell morphology was observed using a laser confocal microscope. Western blot analysis. Total cellular proteins were extracted by incubating the cells in lysis buffer obtained from Cell Signaling Technology; protein concentrations were determined by BCA assay. Anti-body p21, AKT, GSK3β, MMP-9, FAK and PTEN were purchased from Abcam, Cell Signaling Technology and Proteintech. SDS-PAGE was carried out in 10% tricine gels, loading equal amounts of protein per lane, which was carried out as described previously. After electrophoresis, separated proteins were transferred to nitrocellulose membranes and blocked with 5% non-fat milk in TBST buffer for 1 h. After that, the membranes were incubated with primary antibodies at 1 : 1000 dilutions in 5% non-fat milk overnight at 4 1C, and then secondary antibodies were conjugated with horseradish peroxidase at 1 : 2000 dilution for 1 h at room temperature. Protein bands were visualized on LICOR Odyssey system. 32


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The establishment of zebra fish embryo breast cancer model. Firstly, the MDA-MB-231 cells were collected in free DMEM (without FBS) and labeled red fluorescence by DiI at a density of ~107 cells/mL. Then WPI microinjector was used to inject 10-50 nL suspended MDA-MB-231-DiI cells into the perivitelline space near the subintestinal vessels (SIVs) of the transgenic zebrafish (fil1:EGFP) embryos at 48 hpf to establish this zebrafish embryo breast cancer model.52 Juvenile zebra fish of breast cancer model (48 h old) were incubated in 6-well plates (10 fishes in every well) with 2 mL solutions without or with TA12 (0 and 5 μM) in aquaculture water. The effect of TA12 in breast cancer zebra fish were observed every 24 h with a fluorescence microscopy.53 The relevant ethical protocols used for the in vivo study for zebra fish were followed by the relevant laws. Statistical analysis. Results were expressed as the mean ± standard deviation. Data were analyzed using Graphpad Prism 7.0. One-way analysis of variance (ANOVA) was used to identify differences between group means, and statistical significance was assessed by t-test at a significance level of p < 0.05. ASSOCIATED CONTENT The file includes: -Supplementary figures: -Figure S1-S12. The ESI-MS spectra of TA01-TA12. -Figure S13-S24. The 1H NMR spectra of TA01-TA12. -Figure S25-S36. The 13C NMR spectra of TA01-TA12. -Figure S37-S48. HPLC purity data of TA01-TA12. -Figure S49. The inhibitory effect of TA12 against MDA-MB-231 cells.

33



-Figure S50. The graphs for the inhibitory rate of TA12 against MDA-MB-231, HepG2, Hela, A549 and L02 cells. -Figure S51. The real-time cell analysis of MDA-MB-231 cells migration. -Figure S52. The VEGF release of MDA-MB-231 cells induced by TA12. -Figure S53. The Zymography of MMPs in MDA-MB-231 cells treated with TA12. -Figure S54. Toxicity assessments of developing zebrafish embryos in vivo -Supplementary tables: -Table S1. Selected crystallographic data for TA04, TA09 and TA10. -Table S2. The mortality rate (%) of zebrafish embryo treated with different concentration of TA12. -Table S3. The survival rate (%) of zebrafish embryo treated with different concentration of TA12. -Table S4. The hatching rate (%) of zebrafish embryo treated with different concentration of TA12. Molecular formula strings are available AUTHOR INFORMATION Corresponding Author * For W. J. Mei: E-mail, [email protected] ACKNOWLEDGMENT The authors acknowledge the National Natural Science Foundation of China (Grant Nos. 81572926, 81703349). The China Postdoctoral Science Foundation (Grant No. 2017M610576). The Provincial Major Scientific Research Projects in Universities of Guangdong Province (Grant No. 2014KZDXM053), The Science and Technology Project of Guangdong Province (Grant No. 2014A020212312) 34


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Table of Contents graphic

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A series of Tanshinone IIA based imidazole analogs have been demonstrated to be potential low toxicity inhibitor against the proliferation, migration and invasion of MDA-MB-231 breast cancer cells in vitro and in vivo by DNA damage-mediated p53 inactivated AKT pathway. 247x114mm (300 x 300 DPI)


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Tanshinone-IIA-Based Analogs of Imidazole Alkaloid Act as Potent

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