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3-aroylmethylene-2,3,6,7-tetrahydro-1H-pyrazino[2,1a]isoquinolin-4(11bH)-ones as potent Nrf2/ARE inducers in human cancer cells and AOM-DSS treated mice Meiyang Xi, Zhongying Sun, Haopeng Sun, Jiewei Jiang, Yajing Wang, Mingye Zhang, Junfeng Zhu, Jianmin Jia, Lili Xu, Zhengyu Jiang, Xin Xue, Ming Ye, Xi Yang, Yuan Gao, Lei Tao, Xiaoke Guo, Xiaoli Xu, Qinglong Guo, Xiaojin Zhang, Rong Hu, and Qidong You J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 22 Sep 2013 Downloaded from http://pubs.acs.org on September 22, 2013

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Journal of Medicinal Chemistry

3-aroylmethylene-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-ones as potent Nrf2/ARE inducers in human cancer cells and AOM-DSS treated mice

Mei-yang Xi,b,d Jian-min Jia,b,d Hao-peng Sun,a,b,d,* Zhong-ying Sun,c Jie-wei Jiang,b,d Ya-jing Wang,c Min-ye Zhang,b,d Jun-feng Zhu,b,d Li-li Xu,b,d Zheng-yu Jiang,b,d Xin Xue,b,d Ming Ye,c Xi Yang,c Yuan Gao,c Lei Tao,c Xiao-ke Guo,b,d Xiao-li Xu,b,d Qing-long Guo,c Xiao-jin Zhang,b,d Rong Hu,c,* Qi-dong You a,b,*

a

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China

b

Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China

c

Department of Physiology, School of Pharmacy, China Pharmaceutical University, Nanjing, China

d

Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China

ABSTRACT Nrf2-mediated activation of ARE regulates expression of cytoprotective enzymes against oxidative stress, inflammation and carcinogenesis. We have discovered a novel structure (1) as an ARE inducer via luciferase reporter assay to screen the in-house database of our laboratory. The potency of 1 was evaluated by the expression of NQO-1, HO-1 and nuclear translocation of Nrf2 in HCT116 cells. In vivo potency of 1 was studied using AOM-DSS models, showing that the development of colorectal adenomas was significantly inhibited. Administration with 1 lowered the expression of IL-6, IL-1β and promoted Nrf2 nuclear translocation. These results indicated that 1 is a potent Nrf2/ARE activator, both in vitro and in vivo. 41 derivatives were synthesized for SAR study, and a more potent compound 17 was identified. To our knowledge, this is a potent ARE activator. Besides, its novel structure makes it promising for further optimization.

INTRODUCTION Nuclear factor erythroid 2 p45-related factor 2 (Nrf2), is a basic-leucine zipper (bZIP) transcription factor which contains a Cap ‘n’ Collar (CNC) structure.1-4 In quiescent condition, Nrf2 is sequestered in cytoplasm by Kelch-like ECHACS Paragon Plus Environment

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associated protein 1 (Keap1), a 69-kDa cytosolic protein usually existing as a dimer, targeting Nrf2 for ubiquitination and proteosomal degradation. However, the binding conformation of Keap1-Nrf2 complex is changed in response to centain stress.5, 6 The degradation process of Nrf2 is then prohibited, inducing the accumulation of newly synthesized Nrf2 and its subsequent translocation to the nuclei. In addition to Keap1, several activated protein kinases have been shown to activate Nrf2 pathway.7 Nrf2-regulated enzymes such as glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase-1(NQO-1) and heme oxygenase-1 (HO-1)contain an ARE in their promoter region.8, 9 Molecules that activate Nrf2 are considered as potential antioxidant, anti-inflammatory or cancer chemopreventive agents. At present, there are several classes of antioxidant response element (ARE)-regulated inducers reported. Most of them are either isolated from dietary food or synthetic small molecules, such as curcumin,10, 11 caffeic acid phenethyl ester (CAPE),12, 13 resveratrol,14, 15 genistein,16, 17 chalcone18, 19 and sulforaphane (SFN).20, 21 These compounds are reported to exert chemopreventive activities in human cancer cell lines and mouse models. For example, SFN, a known Nrf2 activator, was reported to have influence on the expression of phase II enzymes and on inflammation in Azoxymethane (AOM)-dextran sodium sulfate (DSS) mouse models.22 These models are used to evaluate the chemopreventive potency against inflammation-associated carcinogenesis.23-25 In the models, AOM initially causes oxidative and DNA damage, which is then promoted by DSS. DSS induces inflammation in the colon and promotes AOMinduced colorectal cancer.26, 27 Herein, targeting the Nrf2/ARE path way may not only treat colorectal inflammation but also the frequently occurred subsequent development of colorectal cancer (CRC).28 Studies have shown that chemopreventive compounds including curcumin and nonsteroidal anti-inflammatory drugs are not only effective in animal models, but also promising in the on-going clinical trials.29-31 Considering the importance of chemoprevention in the treatment of cancer, novel inducers targeting Nrf2/ARE pathway are urgently needed and attract the attention of medicinal chemists throughout the world.32-34 Here we report a series of 41 compounds with 3-(2-oxo-2-phenylethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1a]isoquinolin-4(11bH)-one scaffold. Among these compounds, 1 was firstly identified from the screening of in-house database using luciferase reporter assay. It showed potent activity not only in vitro but also in vivo. Thus 1 was selected as a hit for further optimization. Other 40 derivatives were designed, synthesized and evaluated their ability to increase intracellular ARE activity. Structure-activity relationship (SAR) studies indicated a more promising compound 17 for further analysis. To the best of our knowledge, these compounds with the novel structure are firstly confirmed as Nrf2/ARE inducers, exhibiting good activity and serving as ideal lead compounds for the development of novel chemopreventive agents.

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RESULTS Synthetic chemistry. A class of heterocyclic Compounds 1-41 was synthesized using the reaction sequence illustrated in Scheme 1. Accordingly, 47 was furnished via acylated,35 alkylated, cyclizated,36 reducted37 and deprotected38 from 42. At the same time, Claisen condensation of substituted acetophenone 48 with diethyl oxalate gave the respective 49.39,40 Subsequent reaction of 49 with 47 in EtOH/H2O at reflux afforded the corresponding end products 1-41 in good yield.41 All derivatives were characterized by 1H NMR, 13C NMR, IR, HRMS and HPLC. To the best of our knowledge, most compounds have never been reported before, whereas compounds 1, 4, 5, 7, 9, 13 and 15 are available as via the SciFinder search; however, there is no data about the activity of these compounds on human cancer cells and mice. Scheme 1. Synthetic procedure of 1-41a.

a

Reagents and conditions: (a) chloracetyl chloride, NaHCO3, DCM, 0-10 oC, 2 h ; (b) phthalimide potassium, DMF, 90 oC, 12 h; (c) P2O5,

CH3CN, reflux, 2 h; (d) NaBH4, CH3OH, 25 oC, 3 h; (e) NaBH4, i-PrOH/H2O (6:1), AcOH, 80 oC, 2 h; (f) diethyl oxalate, NaOMe, MeOH, reflux, 4 h; (g) NaHCO3, EtOH, H2O, AcOH, reflux, 1 h.

Potency of compound 1 to activate the expression of Nrf2-regulated ARE, HO-1, NQO-1 and facilitate nuclear translocation of Nrf2. In an effort to discover new Nrf2/ARE activators, we performed a cell-based luciferase reporter screening for compounds that induced intracellular ARE activities in HepG2-ARE-C8 cells. Compound 1 showed a concentration-dependent pattern for ARE inductivity. It significantly increased the level of ARE to 18.9-fold at 5 µM compared to control (Figure 1A).In comparison, tert-butylhydroquinone (tBHQ) showed 5.2-fold induction at 80 µM, and SFN showed 5.5-fold induction at 5 µM (Table 2). As a result, 1 was far more efficacious than above-mentioned positive control in their abilities to upregulate the intracellular ARE acticities. In addition, 1 induced HO-1 and NQO1 expression by measuring protein levels in human colorectal cancer cells (HCT116) in a time- and concentrationACS Paragon Plus Environment

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dependent manner (Figure 1B and 1C). A time-response study with 1 demonstrated that nuclear translocation of Nrf2 began within 1 h and maximized at 2 h (Figure 1D). It showed that expression level of HO-1 and NQO-1 was delayed and maximized at 8 h and 24h, respectively.42-44

Figure 1. 1 Induced expression of ARE, cytoprotective enzymes and nuclear translocation of Nrf2. (A) Concentration-dependent induction of 1 on the ARE activation. HepG2-C8-ARE cells were treated for 12 h at indicated concentrations followed by luciferase assays. ARE inductivity was shown as a ratio to the DMSO control. Data are expressed as mean ± SD (n = 3). (B) At indicated concentration (10 µM), time-course analysis indicated that 1 maximized HO-1, NQO-1 at 8 h and 24h separately. (C) After treatment with 1 at designated time (8 h for HO-1, 24 h for NQO-1), cell lysates were prepared from HCT116 cells and subjected to Western blot analysis for HO-1, NQO-1 and β-actin, which showed concentration-dependent. (D) Effects of 1on nuclear translocation of Nrf2. At indicated times after treatment with 1(10 µM), nuclear fractions and cytoplasm extracts were prepared from HCT116 cells and subjected to Western blot analysis for Nrf2, histone and β-actin. HO-1, NQO-1 and cytoplasmic Nrf2 expression used β-actin for normalization. Nuclear Nrf2 used histone for normalization. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant difference from the non-treated group.

Effect of administration with 1 on development of AOM-DSS induced colorectal adenoma in mice. Neither changes in body weight (Figure 2B) nor noticeable signs of toxicity (Table 1) of mice were observed in all groups up to 130 days. The AOM-DSS group had 90 % colorectal tumor incidence (percentage of mice with colon adenomas), multiplicity (number of adenomas) of 2.60 ± 0.68, an average adenomas size of 2.70 ± 0.62 mm and adenoma burden of ACS Paragon Plus Environment

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7.02 ± 1.01. In contrast, with the treatment of 1, the above mentioned parameters was reduced to 50 % (tumor incidence), 0.78 ± 0.42 (multiplicity), 2.20 ± 0.37 mm (average size) and 1.71 ± 0.50 (burden) (Figure 2A, 2C and 2D), showing apparent inhibitory effect in the colorectal tumor development. Table1. Overview of the AOM-DSS models.a Group (No. of mice examined) AOM-DSS(20) AOM-DSS+1(18) a

Lengh of colon (cm) 5.62±0.69 4.59±0.57

WBC (10^9/L） 5.33±1.34 5.67±1.68

NR (%)

LR (%)

12.73±1.65 12.39±1.43

87.75±2.16 88.15±2.03

Data are mean ± SD. White Blood Cell (WBC), Neutrophils Ratio (NR), Lymphocyte Ratio (LR).

Figure 2. Observation of AOM-DSS models. (A) Representative pictures of colon from the AOM-DSS group (top panel) and AOMDSS+1 group (bottom panel) in C57BL/6 female mice. (B) Body weight of the AOM-DSS group and AOM-DSS+1 group. (C) and (D) Administration with 1 decreased incidence, multiplicity and size of colorectal tumors in an AOM-DSS-induced mouse model. The data are expressed as the mean ± SD (n=20 or 18). *p < 0.05, **p < 0.01, statistically significant difference from the AOM-DSS group.

Adenoma pathology findings of AOM-DSS mice treated with 1. AOM-DSS treatment caused extensive high grade dysplasia and formed adenomas in colorectal tissue. However, treatment with 1 decreased the developed dysplasia in AOM-DSS treated mice. A better tissue organization was observed (Figure 3H & E staining).


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Figure 3. General histological observation of stained tissues. (H & E) Adenomas and dysplasia stained by H & E. Con represents normal tissue. Adenomas developed in AOM-DSS mice. Treatment with 1 improved dysplasia. Bars inserted indicate magnification (50 µm). (IL1β, IL-6) Immunohistochemistry staining of IL-6 and IL-1β in indicated treatment. 1 suppressed inflammation factor in the AOM/DSS model. Note the lower IL-6 and IL-1β staining in the colorectal crypts of animals treated with AOM-DSS+1 compared with AOM-DSS alone. Bars inserted indicate magnification (50 µm). (Nrf2) Immunofluorescence staining. Cotreatment with 1 increased the expression of Nrf2 in colorectal tissue. In each individual sample, Nrf2 and nuclei was labeled with FITC and DAPI, respectively. Bars inserted indicate magnification (50 µm).

The inhibitory effect of 1 on the over expression of IL-6 and IL-1β in AOM-DSS mouse models. We evaluated inflammatory inhibition by IL-6 and IL-1β immunostaining after the treatment of 1 in AOM-DSS models. There was an

increased expression of inflammatory cytokines IL-6 and IL-1β in the group treated with AOM-DSS, compared to the control group. After the treatment of 1, the decreased expression of these two factors was observed, indicating that 1 was able to inhibit the colorectal inflammation (Figure 3IL-1β, IL-6 and Figure 4). ACS Paragon Plus Environment

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Figure 4. Quantification of IL-1β, IL-6 and Nrf2. *p < 0.05, statistically significant difference from the AOM-DSS group.

In vivo potency of indentified hit 1 to activate Nrf2 in mouse models. We examined the nuclear translocation of Nrf2 through immunofluorescence. The Nrf2 staining differed significantly among the three groups. In the control group, Nrf2 was kept in a normal level. However, in both AOM-DSS treated groups, overexpressed Nrf2 translocated into nuclei enormously in tumor tissues independent on administration with 1. It was disadvantageous and revealed the “dark” side of Nrf2. The details were discussed in the following discussion. In the AOM-DSS+1 group, a significantly increased Nrf2 translocation into the nuclei was observed in surrounding tissues compared to the AOM-DSS group. This phenomenon indicated that 1 promoted Nrf2 activation to decrease the damage and slow down the development of inflammation-associated cancer in chronic inflammatory environment (Figure 3 Nrf2 and Figure 4). 45-48 Preliminary SAR. A series of analogs with different groups in both phenyl rings were tested at 0.01, 0.1, 1, 5, 10 µM for their ability to induce ARE activity in HepG2-ARE-C8 cells (12 h treatments) (Table2). The optimal compound was 17 with a 33-fold increase at maximum concentration in ARE activity as compared to control followed by 1, 24, 8, 12 and 3 (Figure 5). Moreover, 17 activated the expression of HO-1, NQO-1 and facilitated nuclear translocation of Nrf2 (Figure 6), which was in a very similar manner with 1. Induction of Nrf2 with 17 was not statistically different from that of 1. Because 17 was identified later, most of above mechanistic works have been focused on the original hit 1.On ring B, the importance of the fluoro (-F) can be clearly seen compared to other substituted derivatives. The above-mentioned compound, with meta-F substitution, was significantly more effective than 1 in ARE activity. It is suggested that the bulk of -F is similar with hydrogen (H). Moreover, electron-withdrawing effect of fluoro is more advantageous to enhance activity. The position of -F substitution was crucial for the inductivity of these compounds. In general, the derivatives with -F substitution at meta position were the most active compounds (17), followed by ortho (8) and para (7) substitution. This showed that six-bond separation between carbonyl and -F was crucial for the induction activity. Also, the IC50 value showed that derivatives were not cytotoxic in administrated concentration (The IC50value of most compounds is more than 100 µM). When substituted with -CF3 (39), 3,4-F (10)and3,5-F (40), the activity decreased. It is considered that mono-fluoro introduction on ring B is enough. Similarly, with chloro (-Cl), ACS Paragon Plus Environment

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methoxyl (-OCH3) and nitro (-NO2) substitution (3, 9 and 12) at the meta position, the activity was obviously predominant compared to other positional substituted analogs (2, 4, 5, 18, 6, 16). With -OCH3 and -NO2 substitution, the similar activity at respective position indicated that the electrostatic state of the substituted group was not responsible for mediating the ARE activity. At para position, the decreased activity with methyl (-CH3), -OCH3 and phenyl (-ph) substitution (13, 5, 14) due to inappropriate large bulk of a substituent. It was thought that a substituent small in size was acceptable. The binding pattern of a compound with a target would be changed if a larger group is introduced. Interestingly, analogs with -OCH3 introduction on ring A were less active possibly owing to steric hindrance and inappropriate electrical characteristic. The higher activity of 24, 21 also showed the importance of -F group compared to other substituted groups. When substituted with 3-OCH3 and 2-OCH3, the inductivity became lower. In general, 2-OCH3 and 3OCH3 substitution were pessimistic compared to 4-OCH3 substitution, which indicated substituted position was important. However, the introduction of 3,4-OCH3 significantly abolished the activity, indicating that introduction of dimethoxy completely destroyed a certain mode of action of target binding with this scaffold. It is thought that the substituent is small and electron-donating property is nonessential. So the -F effect on ring B was not the only key determinant for the higher activity. Therefore, the electrophile with smaller bulk introduction or no substitution on ring A and B probably showed acceptable activities. A brief description of SAR was presented in Figure 7. On the basis of these data, derivatives that showed >25-fold induction of ARE at 10 µM can serve as ideal lead compounds for further optimization. Table 2. Induction of ARE activity in HepG2-ARE-C8 cells and EC50 value measured in HCT116 cells.

R

Compound /Vehicle Relative Luciferase Units (µM) a

Compd. ID

R1

EC50 (µM)

1

H

2

H

3

H

3-Cl

4

H

4-Cl

1.30±0.42

2.33±0.87

2.61±0.56

5

H

4-OCH3

1.15±0.07

2.30±1.55

2.74±1.21

6

H

4-NO2

1.20±0.14

2.34±0.56

1.76±0.34

2.43±1.16

3.81±0.98

>100

7

H

4-F

2.58±1.39

4.39±0.62

8.00±1.27

12.31±1.56

14.01±2.84

>100

8

H

2-F

2.50±0.71

3.64±1.74

8.85±1.80

16.51±0.71

16.92±0.40

>100

9

H

3-OCH3

1.48±0.54

3.69±1.86

5.68±1.97

8.65±1.07

10.04±0.06

>100

10

H

3,4-F

2.12±0.64

2.25±1.09

3.23±1.48

5.47±1.94

6.38±1.80

>100

11

H

2,4-Cl

1.68±0.17

1.10±0.28

1.42±0.62

1.78±0.04

2.49±1.53

>100

0.01

0.1

1

5

10

H

2.00±0.21

5.44±1.21

9.51±0.14

18.87±0.11

26.87±0.03

>100

2-Cl

1.19±0.14

2.19±1.09

2.77±1.07

5.46±1.13

6.04±1.36

>100

1.94±0.08

3.77±1.28

10.67±0.39

13.98±1.93

16.50±1.63

>100

3.57±1.62

4.00±1.41

>100

3.30±1.84

5.07±1.51

>100


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Table 1. Continued 12

H

3-NO2

3.28±1.81

3.52±1.14

7.22±1.90

14.50±0.14

18.54±2.18

>100

13

H

4-CH3

2.01±0.01

3.30±1.25

3.25±1.90

4.42±2.09

5.22±2.46

>100

14

H

4-ph

1.62±0.54

1.67±0.79

1.41±1.10

1.69±0.77

2.09±0.12

>100

15

H

3,4-OCH3

1.52±0.30

1.77±0.69

1.93±1.48

2.11±1.32

2.15±1.34

93.02±7.12

16

H

2-NO2

1.28±0.06

1.29±0.40

1.30±0.52

2.41±1.22

2.80±1.10

>100

17

H

3-F

9.99±3.10

29.06±0.49

33.00±0.49

>100

H

2-OCH3

2.18±0.58 0.96±0.38

3.89±0.69

18

1.68±1.14

1.51±0.11

1.78±0.27

1.99±0.85

>100

19

10-OCH3

H

1.35±0.64

1.93±0.30

3.50±0.86

10.43±1.12

12.99±1.35

>100

20

10-OCH3

3-Cl

2.26±1.19

2.28±0.40

4.53±0.78

6.49±1.34

7.28±1.85

>100

21

10-OCH3

2-F

1.44±0.04

2.75±1.39

3.44±0.09

9.84±1.36

12.44±2.55

>100

1.75±0.40

3.08±1.32

22

10-OCH3

3-NO2

1.45±0.49

>100

10-OCH3

3-OCH3

1.21±0.01

1.24±0.02

1.59±0.44

>100

24

10-OCH3

3-F

1.08±0.45 1.31±0.29

5.76±1.85 1.36±0.01

6.50±2.45

23

3.93±0.53

7.23±2.77

16.93±2.54

20.38±2.18

>100

25

10-OCH3

4-F

1.29±0.41

1.35±0.35

1.74±0.14

1.82±0.32

2.48±0.14

87.95±6.05

26

9,10-OCH3

H

0.86±0.25

0.85±0.40

1.35±0.14

1.46±0.25

1.67±.71

>100

1.47±0.66

1.07±0.21

1.15±0.29

1.44±0.49

>100

0.80±0.16

0.95±0.14

0.97±0.13

1.10±0.15

75.85±8.11

27

9,10-OCH3

3-Cl

1.41±0.58

28

9,10-OCH3

2-F

1.07±0.27

29

9,10-OCH3

3-NO2

1.31±0.11

1.08±0.11

1.03±0.04

1.44±0.45

2.51±0.56

49.61±2.53

30

9,10-OCH3

3-OCH3

1.45±0.49

1.18±0.24

0.95±0.33

1.11±0.24

1.06±0.03

>100

31

9,10-OCH3

3-F

2.31±0.38

0.93±0.27

1.05±0.36

1.68±0.10

1.09±0.13

92.00±8.34

32

9,10-OCH3 8-OCH3

4-F

1.25±0.61

1.42±0.35

2.70±0.43

1.82±0.53

1.25±0.46

81.54±6.31

33

H

1.00±0.40

3.53±0.44

3.57±0.80

6.89±0.51

6.93±0.73

58.54±5.61

34

8-OCH3

3-F

0.79±0.05

1.43±0.28

4.02±1.48

4.85±1.07

3.70±1.11

>100

35

8-OCH3

2-F

1.93±1.02

2.83±0.49

2.98±1.38

5.89±2.36

4.26±0.11

>100

36

9-OCH3

H

0.65±0.13

1.70±0.44

3.80±1.23

8.18±1.90

5.36±1.49

>100

37

9-OCH3

3-F

1.83±0.03

5.19±0.10

3.96±1.90

5.65±2.18

6.66±1.20

>100

38

9-OCH3

2-F

2.58±1.33

1.45±0.64

3.73±2.48

5.18±3.10

7.70±3.48

>100

39

H

3-CF3

2.22±0.20

1.70±0.21

1.76±0.09

3.14±0.18

3.25±0.75

>100

40

H

3,5-F

1.34±0.49

2.92±1.25

4.75±1.71

5.22±0.38

7.62±2.28

58.86±6.48

41

H

3-Br

3.10±0.21

3.40±1.49

8.97±2.61

11.99±0.04

15.73±5.49

59.12±3.63

SFN tBHQ

1µM

5µM

10µM

12.5µM

20µM

2.7±1.30

5.52±0.23

8.35±0.96

11.05±1.56

18.10±2.45

5µM

10µM

20µM

40µM

80µM

19.08±1.54

91.53±4.12 1.04±0.21 1.50±0.11 2.06±0.25 3.24±0.32 5.21±0.10 a Values shown are mean±SD (n = 3, 12 h of treatment). Cells were exposed to compounds tested for their ability to increase levels of relative luciferase units.

Figure 5. The fold of Nrf2-dependent ARE-regulated activity compared to control. *p < 0.05, **p < 0.01, ***p < 0.001, statistically sig-


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nificant difference from the non-treated group.

Figure 6. 17 Induces expression of cytoprotective enzymes and nuclear translocation of Nrf2. (A) At indicated concentration (10 µM), cell lysates were prepared fromHCT116 cells and subjected to Western blot for time-course analysis. (B) Effects of 17on nuclear translocation of Nrf2. At indicated times after treatment with 17 (10 µM), nuclear fractions and cytoplasm extracts were prepared from HCT116 cells and subjected to Western blot analysis for Nrf2, histone and β-actin. HO-1, NQO-1 and cytoplasmic Nrf2 expression used β-actin for normalization. Nuclear Nrf2 used histone for normalization. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant difference from the non-treated group.

-OCH3 substitution is less active. 3,4-OCH3 substitution abolishs the ARE inductivity. A smaller bulk O N substitution is reasonable. N H O

we bet ion t. rat be s epe is d s -F on nd 6-b =O a -C .

en

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The electrostatic state is not responsible for higher activity. Inductivity decreases with increased bulk introduction. o- and p-sustitution are (H) F less active. m-F (H) substitution is the most active.

Figure 7. SAR of compounds 1-41.

DISCUSSION Dietary administration with chemopreventive agents could inhibit oncogenesis in inflammation-associated cancer mouse models. Inflammatory environment may promote some signaling pathways to be aberrantly activated, and increasing targets required in cancer promotion were then further addressed.49-51 Here we used AOM-DSS models to investigate thepreventive effects of 1 on oncogenesis and its potential application for inhibiting colorectal cancer develACS Paragon Plus Environment

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opment, although the target for 1 remains to be further investigated. Our results indicated that upregulated Nrf2 may provide a mechanistic link between inflammation and cancer. It is generally believed that boosting the Nrf2-dependent response to counteract environmental stress is a promising strategy for cancer prevention. Also, compounds able to induce Nrf2/ARE expression may have beneficial effects in a number of diseases.52-54 We hope that upregulated Nrf2 can prevent from cancer. On the other hand, overexpressed Nrf2 is detected in many cancer tissues as mentioned in Figure 4, giving cancer cells a chance to survive and develop. Nrf2 and its downstream genes are upregulated in cancer cells, leading acquired resistance to therapies.55, 56 This phenomenon reveals the “dark” side of Nrf2.57 Fortunately, Compound 1 did not increase transactivation of Nrf2 in tumor tissues to cause the aggravation of the “dark” side. In this condition, Nrf2 should be inhibited rather than induction. It is considered to be necessary to inhibit the Nrf2 during cancer therapy. Thus, Nrf2 inducer should be administrated in a right timing. Although a dual role of Nrf2 exists, it is optimistic to discover new Nrf2 activators for chemoprevention. In this study, we report a series of potent Nrf2/ARE activators with novel 3-(2-oxo-2-phenylethylidene)-2,3,6,7-tetrahydro1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one structure, which were never reported before. The compounds were efficacious not only in vitro but also in vivo. Most derivatives showed outstanding ability to induce the intracellular ARE activity compared to previously reported activators, such as a kavalactone derivatives,34 quinolinederivatives (AI-1),7 resveratrol, genistein. The ARE enhanced effect was also significantly higher than those of tBHQ and SFN, wellknown positive control of Nrf2/ARE. It is worthy of being mentioned that terpenoids which are known to activate Nrf2 at nanomolar concentrations. The compound CDDO-IM, discrepancy exists whether a response mechanism is Cys151 dependent.58 Also, modification in Cys273 and Cys288 sites is expected to ablate Nrf2 degradation. The two corresponding mechanisms are disruption of the Keap1-Cul3 interaction and a conformational change in Keap1 to change the Keap1-Nrf2 interaction. It is suggested that electrophiles prefer some specific cysteine residues in Keap1 and exhibit a particular mode of cysteine modification. Also, it is speculated that a binding pocket exists in Keap1 and it contains above important cysteines to covalently bind with the target molecule. As the pocket is spatially small, introduction of large groups such as dimethoxy on ring A or phenyl on ring B may cause unsuitable molecular interactions.. Furthermore, triterpenoid seems to share the similar tricyclic structure with 1 that may represent an opening of the basic pentacyclic framework to simplify the scaffold. Based on similarity, we will study relationships aiming at structure modification and activity improvement in the future. Another common and interesting phenomenon is that they induce HO-1 much more responsive than NQO-1. Maybe it is due to cell types. Induction of different proteins in the same tissues or induction of the same protein in different tissues is different. Besides, it is also detected in mRNA tranACS Paragon Plus Environment

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script levels. 43,59 To the best of our knowledge, these compounds are potent Nrf2/ARE activators, and they are worthy of further structural optimization so as to develop novel chemopreventive agents.

CONCLUSIONS We have identified compound 1 with a novel structure as an ARE inducer via screening an in-house database using the luciferase reporter assay. The inductivity was excellent compared to previously reported compounds. Human cancer cell-based and AOM-DSS mouse models were used to demonstrate that 1 was a potent activator of Nrf2/ARE signaling pathway. Furthermore, 1 significantly suppressed the development of inflammation-associated cancer in colon. In summary, we demonstrated the role of 1 in the Nrf2/ARE signaling pathway both in vitro and in vivo. According to these findings, we made a structural modification and SAR investigation, furthermore discovered a promising compound 17 with the most potency to enhance the ARE levels. This compound can be ideal lead for further optimization.

EXPERIMENTAL SECTION Synthesis chemistry. All reagents were purchased from commercial sources. Organic solutions were concentrated in a rotary evaporator (BüchiRotavapor) below 55 °C under reduced pressure. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silica gel plates (GF254) and visualized under UV light. Melting points were determined with a Melt-Temp II apparatus. IR spectra were recorded on a Nicolet iS10 Avatar FT-IR spectrometer using KBr film. The 1H NMR and 13C NMR spectra were measured on a Bruker AV-300 instrument using deuterated solvents with tetramethylsilane (TMS) as internal standard. ESI-mass and high resolution mass spectra (HRMS) were recorded on a Water Q-Tof micro mass spectrometer. The purity (≥ 95%) of the compounds is verified by the HPLC study performed on Agilent C18 (4.6×150 mm, 3.5 µm) column using a mixture of solvent methanol/water or acetonitrile/water at the flow rate of 0.5 mL/min and peak detection at 254 nm under UV. 41 compounds were synthesized as follows: In an ice-bath, chloracetyl chloride (4.69 mL, 61.9 mmol) was drop by drop added into a solution of 42 (41.3 mmol) and NaHCO3 (5.19 g, 61.9 mmol) in CH2Cl2 (60 mL). The mixture was reacted at 10 oC for 2 h, washed by 1 M HCl (50 mL) and saturated NaCl solution (50 mL), dried and recrystallized from methanol and water (3 : 2). Yield 94.698.5 %. 43 (39.06 mmol) and phthalimide potassium (10.85 g, 58.6 mmol) was added into a solution of DMF (50 mL), and kept 90 oC for 12 h. After cooling, the mixture was poured to water (200 mL). The precipitate was filtered and dried. Yield 94.1-98.1 %. ACS Paragon Plus Environment

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P2O5 (18.78 g, 132.3 mmol) and 44 (36.7 mmol) was added into a solution of refluxed CH3CN (200 mL), and another P2O5(12.50 g, 88.1 mmol) was added 5 min later. After 2 h, remove CH3CN and add water (300 mL). The mixture was extracted with ethyl acetate (2 × 150 mL), adjusted pH to 9 with NH3·H2O, filtered and recrystallized from THF. Yield 82.0-84.1 %. 45 (30.1 mmol) was dissolved in CH3OH (300 mL), then NaBH4 (1.71 g, 45.2 mmol) was added in small portions over 20 min. After 2 h, add water (2 mL) and filtrate was concentrated under vacuum. Then dissolve it in dichloromethane (200 mL), remove inorganic salt, reduce to dryness and crystallized from ethyl acetate. Yield 74.1-75.3%. NaBH4 (4.64 g, 122.7 mmol) was added into a solution of 46 (22.3 mmol) in isopropanol : H2O (6 : 1, 280 mL) in room temperature. Acetic acid was added slowly until the evolution of gas ceased. The mixture was heated at 80 oC for 2 h. Then adjust to alkaline with 2 M NaOH solution, extract with CHCl3 (3 × 50 mL), dry, add concentrated HCl (4 mL), and recrystallized from ethanol. Yield 73.6-75.2 %. A solution of diethyl oxalate (3.25 mL, 24.0 mmol) and 48 (12.0 mmol) in CH3OH (20 mL) was added dropwise to a solution of CH3ONa in CH3OH (5.2 mL of 25% w/v, 24.0 mmol), and the reaction was allowed to proceed at reflux for 4 h. After cooling to r.t., the mixture was poured into water (100 mL), acidified with HCl (2 mL of 37% w/v). The precipitate was filtered to afford the respective product 49. Yield 60.1-97.9%. A solution of NaHCO3 (1.7 mmol) in water (0.85 mL) was added to a solution of the 47 (0.85 mmol) in ethanol (0.85 mL) and slightly heated until the evolution of gas ceased. A solution of the 49 (0.85 mmol) in ethanol (0.85 mL) and glacial AcOH (0.425 mL) was then added and refluxed for 1 h. After cooling, the precipitated solid was filtered off and recrystallized from ethanol. Yield 44.2-85.6 %. 1 (5 g) was synthesized on a large scale for dietary administration of AOM-DSS mice. 3-(2-oxo-2-phenylethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (1). Yield 25.6 %; Yellow solid; m.p. 203-204 oC; 1HNMR (300 MHz, DMSO) δ 10.84 (d, J = 4.68 Hz, 1H), 7.89-7.87 (m, 2H),7.86-7.45 (m, 3H), 7.33-7.24 (m, 4H), 6.58 (s, 1H), 5.11 (q, J1= 8.49 Hz, J2= 3.85 Hz, 1H); 4.63 (q, J1= 7.77 Hz, J2= 3.46 Hz,1H), 4.01 (m, 1H), 3.25 (t, J = 12.75 Hz, 1H), 3.00-2.86 (m, 3H); 13C NMR (75 MHz, DMSO) δ 188.85, 158.62, 150.16,139.33, 134.88, 132.41, 131.41, 128.84, 128.56, 128.56, 127.09, 126.76, 126.76, 125.65, 125.94, 89.78, 53.40, 45.29, 38.44, 28.36; IR (cm-1, KBr film): 3134(-NH), 1663(-CON), 1612(-CO); HRMS (ESI): calcd. for C20H19N2O2 [M + H]+ 319.1441, found 319.1431; HPLC (80% methanol in water): tR = 6.28 min, 99.24 %. 3-(2-(2-chlorophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (2). Yield 23.2 %; Yellow solid; m.p.>210 oC; 1HNMR (300 MHz, DMSO) δ 10.65 (d, J = 4.81 Hz, 1H), 7.48-7.41 (m, 4H),7.307.24 (m, 4H),6.10 (s, 1H), 5.14 (q, J1 = 8.25 Hz, J2= 3.90 Hz, 1H); 4.59 (q, J1 = 9.00 Hz, J2 = 2.34 Hz, 1H), 4.01 (m, ACS Paragon Plus Environment

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1H), 3.26 (t, J = 12.43 Hz, 1H), 2.50-2.47 (m, 3H); 13C NMR (75 MHz, DMSO) δ 189.57, 158.26, 149.83, 140.79, 134.88, 132.33, 130.87, 130.01, 128.99, 128.89, 128.84, 127.27, 127.10, 126.67, 125.91, 93.83, 53.30, 45.33, 38.46, 28.35; IR (cm-1, KBr film): 3131(-NH), 1667(-CON), 1608(-CO); HRMS (ESI): calcd. for C20H18ClN2O2 [M + H]+ 353.1051, found 353.1054; HPLC (80% methanol in water): tR = 5.98 min, 95.89%. 3-(2-(3-chlorophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (3). Yield 22.1 %; Yellow solid; m.p. 183-185 oC; 1HNMR (300 MHz, DMSO) δ 10.89 (d, J = 4.59 Hz, 1H), 7.85-7.83 (m, 2H), 7.62-7.58 (m, 1Н), 7.52 (t, J = 5.97 Hz, 1H), 7.30-7.25 (m, 4H), 6.54 (s, 1H), 5.12(q, J = 12.03 Hz, 1H), 4.63 (q, J = 11.34 Hz, 1H), 4.05 (m, 1H), 3.31 (t, J = 10.84 Hz, 1H), 2.98-2.87 (m, 3H); 13C NMR (75 MHz, DMSO) δ 186.88, 158.39, 150.73, 141.29, 134.86, 133.55, 132.32, 131.08, 130.58, 128.84, 127.10, 126.66, 126.37, 125.91, 125.42, 89.56, 53.32, 45.33, 38.47, 28.35; IR (cm-1, KBr film): 3132 (-NH), 1666 (-CON), 1619 (-CO); HRMS (ESI): calcd. for C20H18ClN2O2 [M + H]+ 353.1051, found 353.1056; HPLC (90% methanol in water): tR = 5.124 min, 98.05%. 3-(2-(4-chlorophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (4). Yield 23.0 %; Yellow solid; m.p. 210-211 oC; 1HNMR (300 MHz, DMSO) δ 10.86 (d, J = 4.83 Hz, 1H), 7.89 (d, J = 8.61 Hz, 2H), 7.53 (d, J = 8.58 Hz, 2H), 7.30-7.24 (m, 4H),6.54 (s, 1H), 5.09 (m, appar q, 1H); 4.60 (m, appar q, 1H), 4.02 (m, 1H), 3.25 (t, J = 13.43 Hz, 1H), 2.93-2.86 (m, 3H); 13CNMR (75 MHz, DMSO) δ 187.33, 158.48, 150.51, 137.99, 136.19, 134.87, 132.36, 128.84, 128.84, 128.67, 128.67, 128.62, 127.10, 126.65, 125.92, 89.53, 53.35, 45.31, 38.46, 28.35; IR (cm-1, KBr film): 3132(-NH), 1658(-CON), 1608(-CO); HRMS (ESI): calcd. for C20H18ClN2O2 [M + H]+ 353.1051, found 353.1055; HPLC (90% methanol in water): tR = 5.18 min, 97.70%. 3-(2-(4-methoxyphenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (5). Yield 21.0 %; Yellow solid; m.p. 187-188 oC; 1HNMR (300 MHz, DMSO) δ 10.72 (d, J = 4.53 Hz, 1H), 7.85 (d, J = 8.79 Hz, 2H), 7.32-7.24 (m, 4H),7.00 (d, J = 8.82 Hz, 2H), 6.54 (s, 1H), 5.08 (q, J = 8.13 Hz, 1H); 4.61 (m, appar q, 1H), 4.00 (m, 1H), 3.81 (s, 3H), 3.22 (t, J = 11.16 Hz, 1H), 2.93-2.85 (m, 3H); 13CNMR (75 MHz, DMSO) δ 188.03, 161.94, 158.79, 149.60, 134.89, 132.50, 132.04, 128.82, 128.82, 128.82, 127.06, 126.63, 125.92, 113.78, 113.78, 89.65, 55.34, 53.50, 45.26, 38.44, 28.38; IR (cm-1, KBr film): 3132(-NH), 1658(-CON), 1600(-CO); HRMS (ESI): calcd. for C21H21N2O3 [M + H]+ 349.1547, found 349.1547; HPLC (80% methanol in water): tR = 5.91 min, 96.42%. 3-(2-(4-nitrophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (6). Yield 20.2 %; Yellow solid; m.p.>210 oC; 1HNMR (300 MHz, DMSO) δ 11.03 (d, J = 4.95 Hz, 1H), 8.30 (d, J = 8.91 Hz, 2H), 8.10 (d, J = 8.91 Hz, 2H), 7.32-7.25 (m, 4H),6.60 (s, 1H), 5.12 (q, J1 = 8.16 Hz,J2 = 3.78 Hz, 1H); 4.62 (q, J1 = 8.67 Hz,J2 = 2.85 Hz, 1H), 4.04 (m, 1H), 3.30 (t, J = 12.57 Hz, 1H), 2.98-2.87 (m, 3H); 13CNMR (75 MHz, DMSO) δ 186.32, 158.24, 151.26, 148.89, 144.49, 134.87, 132.26, 128.86, 128.12, 128.12, 127.14, 126.69, 125.93, 123.82, ACS Paragon Plus Environment

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123.82, 89.94, 53.24, 45.37, 38.50, 28.33; IR (cm-1, KBr film): 3128(-NH), 1670(-CON), 1603 (-CO); HRMS (ESI): calcd. for C20H18N3O4 [M + H]+ 364.1292, found 364.1291; HPLC (80% methanol in water): tR = 6.87 min,97.16%. 3-(2-(4-fluorophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (7). Yield 24.0 %; Yellow solid; m.p. 210-212 oC; 1HNMR (300 MHz, DMSO) δ 10.82 (d, J = 4.77 Hz, 1H), 7.97-7.92 (m, 2H),7.32-7.10 (m, 6H), 6.54 (s, 1H), 5.10 (q, J1= 8.79 Hz, J2= 3.60 Hz, 1H); 4.62 (q, J = 9.33 Hz, 1H), 4.01 (m, 1H), 3.21 (t, J = 12.36 Hz, 1H), 3.02-2.73 (m, 3H); 13CNMR (75 MHz, DMSO) δ 187.40, 164.02 (d, J = 247.58 Hz), 158.55, 150.30, 135.90 (d, J = 2.68 Hz), 134.88, 132.39, 129.44 (d, J = 9.15 Hz), 129.44 (d, J = 9.15 Hz), 128.84, 127.09, 126.65, 125.92, 115.43 (d, J = 21.53 Hz), 115.43 (d, J = 21.53 Hz), 89.53, 53.39, 45.29, 38.46, 28.36; IR (cm-1, KBr film): 3132(-NH), 1661 (-CON), 1610(-CO); HRMS (ESI): calcd. for C20H18FN2O2 [M + H]+ 337.1347, found 337.1347; HPLC (80% methanol in water): tR = 6.88 min, 96.60%. 3-(2-(2-fluorophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (8). Yield 27.0 %; Yellow solid; m.p. 199-201 oC; 1HNMR (300 MHz, DMSO) δ 10.84 (d, J = 4.95 Hz, 1H), 7.73 (m, appar t, 1H),7.56-7.51 (m, 1H),7.33-7.25 (m, 6H),7.30-7.24 (m, 4H),6.42 (s, 1H), 5.11 (m, appar q, 1H); 4.60 (m, appar q, 1H), 4.02 (m, 1H), 3.28 (t, J = 12.66 Hz, 1H), 2.94-2.87 (m, 3H); 13CNMR (75 MHz, DMSO) δ 185.83, 159.91 (d, J = 223.95 Hz), 158.08, 150.20, 134.86, 132.82 (d, J = 8.85 Hz), 132.33, 129.91 (d, J = 2.78 Hz), 128.84, 128.09 (d, J = 13.35 Hz), 127.09, 126.65, 125.90, 124.61 (d, J = 3.38 Hz), 116.38 (d, J = 23.25 Hz), 94.11 (d, J = 8.10 Hz), 53.31, 45.34, 38.45, 28.35; IR (cm-1, KBr film): 3135(-NH), 1667(-CON), 1609(-CO); HRMS (ESI): calcd. for C21H21N2O3 [M + H]+ 349.1547, found 349.1549; HPLC (80% methanol in water): tR = 5.80 min, 99.26%. 3-(2-(3-methoxyphenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (9). Yield 26.4 %; Yellow solid; m.p. 159-161 oC; 1HNMR (300 MHz, DMSO) δ 10.83 (d, J = 4.31 Hz, 1H), 7.47-7.24 (m, 7H),7.12-7.11 (m, appar q, 1H), 6.54 (s, 1H), 5.10 (m, apparq,1H); 4.61 (m, apparq, 1H), 4.01 (m, 1H), 3.80 (s, 3H), 3.25 (t, J = 12.61 Hz, 1H), 2.93-2.86 (m, 3H); 13CNMR (75 MHz, DMSO) δ 188.55, 159.42, 158.60, 150.19, 140.92, 134.88, 132.40, 129.70, 128.84, 127.09, 126.65, 125.92, 119.16, 117.17, 111.68, 89.95, 55.16, 53.40, 45.30, 38.45, 28.36; IR (cm-1, KBr film): 3132(-NH), 1661(-CON), 1609(-CO); HRMS (ESI): calcd. for C21H21N2O3 [M + H]+ 349.1547, found 349.1549; HPLC (80% methanol in water): tR = 6.67 min, 99.38%. 3-(2-(3,4-difluorophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (10). Yield 16.4 %; Yellow solid; m.p. 182-183 oC; 1HNMR (300 MHz, DMSO) δ 10.89 (d, J = 3.57 Hz, 1H), 7.90-7.77 (m, 2H),7.58-7.49 (m, appar q, 1H), 7.31-7.26 (m, 4H),6.54 (s, 1H), 5.11 (q, J1= 8.58 Hz, J2= 3.27 Hz, 1H); 4.64 (d, J = 11.19 Hz, 1H), 4.03 (m, 1H), 3.28 (t, J = 13.08 Hz, 1H), 3.02-2.87 (m, 3H); 13CNMR (75 MHz, DMSO) δ 185.91, 158.37, 152.26 (d, J = 155.93 Hz), 150.76, 148.94 (d, J = 174.15 Hz), 134.87, 132.32, 128.83, 127.10, 126.65, 125.91, ACS Paragon Plus Environment

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124.16 (d, J = 7.20 Hz), 124.07, 117.65 (d, J = 17.33 Hz), 115.84 (d, J = 17.10 Hz), 89.33, 56.18, 45.33, 38.46, 28.35; IR (cm-1, KBr film): 3126(-NH), 1660(-CON), 1608(-CO); HRMS (ESI): calcd. for C20H17F2N2O2 [M + H]+ 355.1253, found 355.1255; HPLC (80% methanol in water): tR = 8.01 min, 96.00%. 3-(2-(2,4-dichlorophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (11). Yield 28.5 %; Yellow solid; m.p. 209-210 oC; 1HNMR (300 MHz, DMSO) δ 10.70 (d, J = 4.83 Hz, 1H), 7.68 (m, appar d, 1H), 7.51 (m, appar t, 2Н), 7.32-7.25 (m, 4H), 6.11 (s, 1H), 5.13(q, J1= 8.31 Hz, J2= 3.72 Hz, 1H), 4.60 (m, appar q, 1H), 4.03 (m, 1H), 3.24 (t, J = 12.75 Hz, 1H), 2.97-2.87 (m, 3H); 13CNMR (75 MHz, DMSO) δ 188.74, 158.13, 150.13, 139.39, 134.84, 134.58, 132.26, 130.71, 130.35, 129.51, 128.84, 127.53, 127.12, 126.67, 125.92, 93.53, 53.24, 45.33, 38.46, 28.32; IR (cm-1, KBr film): 3132(-NH), 1661(-CON), 1609 (-CO); HRMS (ESI): calcd. for C20H17Cl2N2O2 [M + H]+ 387.0662, found 387.0666; HPLC (90% methanol in water): tR = 5.13 min, 98.12%. 3-(2-(3-nitrophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (12). Yield 27.1 %; Yellow solid; m.p.>210 oC; 1HNMR (300 MHz, DMSO) δ 10.98 (d, J = 4.47 Hz, 1H), 8.58 (m, appar t, 1H), 8.40-8.32 (m, 2Н), 7.79 (t, J = 7.95 Hz, 1H), 7.32-7.26 (m, 4H), 6.62 (s, 1H), 5.13(q, J1= 8.49 Hz, J2= 3.96 Hz, 1H), 4.64 (q, J = 11.43 Hz, 1H), 4.08 (m, 1H), 3.33 (t, J = 12.87 Hz, 1H), 3.00-2.88 (m, 3H); 13CNMR (75 MHz, DMSO) δ 185.76, 158.28, 151.23, 148.10, 140.57, 134.86, 133.00, 132.27, 130.47, 128.85, 127.14, 126.68, 125.94, 125.72, 121.10, 89.30, 53.26, 45.37, 38.52, 28.33; IR (cm-1, KBr film): 3127(-NH), 1664(-CON), 1605 (-CO); HRMS (ESI): calcd. for C20H18N3O4 [M + H]+ 364.1292, found 364.1297; HPLC (80% methanol in water): tR = 6.69 min, 97.88 %. 3-(2-oxo-2-(p-tolyl)ethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (13). Yield 22.8 %; Yellow solid; m.p. 205-206 oC; 1HNMR (300 MHz, DMSO) δ 10.80 (m, appar d, 1H), 7.80-7.77 (m, 2H),7.30-7.26 (m, 6H), 6.56 (s, 1H), 5.10 (q, J1 = 8.85 Hz, J2 = 3.45 Hz, 1H); 4.62 (d, J = 10.89 Hz, 1H), 4.01 (m, 1H), 3.31 (t, J = 12.84 Hz, 1H), 3.01-2.86 (m, 3H), 2.35 (s, 3H); 13CNMR (75 MHz, DMSO) δ 188.71, 158.71, 149.90, 141.46, 136.76, 134.89, 132.46, 129.11, 129.11, 128.84, 127.07, 126.86, 126.86, 126.64, 125.92, 89.79, 53.45, 45.27, 38.45, 28.38, 20.98; IR (cm-1, KBr film): 3131(-NH), 1665(-CON), 1607(-CO); HRMS (ESI): calcd. for C21H21N2O2 [M + H]+ 333.1598, found 333.1598; HPLC (80% methanol in water): tR = 7.86 min, 98.56%. 3-(2-([1,1'-biphenyl]-4-yl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (14). Yield 23.0 %; Yellow solid; m.p.>210 oC; 1HNMR (300 MHz, DMSO) δ 10.90 (d, J = 4.74 Hz, 1H), 7.99 (d, J = 7.50 Hz, 2H), 7.80 (d, J = 7.74 Hz, 2H), 7.73 (d, J = 7.71 Hz, 2H), 7.50 (t, J = 7.50 Hz, 2H), 7.42 (d, J = 7.38 Hz, 1H), 7.337.27 (m, 4H), 6.64 (s, 1H), 5.10 (d, J = 9.27 Hz, 1H); 4.62 (d, J = 10.80 Hz, 1H), 4.04 (m, 1H), 3.28 (t, J = 13.47 Hz, 1H), 3.00-2.88 (m, 3H); 13CNMR (75 MHz, DMSO) δ 188.29, 158.64, 150.17, 142.94, 139.22, 138.18, 134.89, 132.43, 129.01, 129.01, 128.85, 128.04, 127.49, 127.49, 127.09, 126.82, 126.82, 126.82, 126.82, 126.66, 125.93, 89.86, 53.42, ACS Paragon Plus Environment

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45.31, 38.48, 28.38; IR (cm-1, KBr film): 3132(-NH), 1666(-CON), 1605(-CO); HRMS (ESI): calcd. for C26H23N2O2 [M + H]+ 395.1754, found 395.1788; HPLC (100% methanol): tR = 4.03 min, 95.22%. 3-(2-(3,4-dimethoxyphenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (15). Yield 24.6 %; Yellow solid; m.p. 173-175 oC; 1HNMR (300 MHz, DMSO) δ 10.73 (m, appar d, 1H), 7.53 (m, appar d, 1H), 7.43 (s, 1Н), 7.32-7.26 (m, 4H), 7.05-7.03 (m, appar q, 1H), 6.56 (s, 1H), 5.09 (m, appar q, 1H), 4.63 (m, appar q, 1H), 4.01 (m, 1H), 3.82 (s, 6H), 3.24 (t, J = 12.54 Hz, 1H), 2.94-2.87 (m, 3H); 13CNMR (75 MHz, DMSO) δ 188.04, 158.79, 151.78, 149,54, 148.56, 134.88, 132.48, 132.18, 128.82, 127.06, 126.62, 125.92, 120.63, 111.01, 109.71, 89.76, 55.59, 55.40, 53.49, 45.27, 38.42, 28.37; IR (cm-1, KBr film): 3128(-NH), 1664(-CON), 1612(-CO); HRMS (ESI): calcd. for C22H23N2O4 [M + H]+ 379.1653, found 379.1652; HPLC (80% methanol in water): tR = 5.55 min, 99.43 %. 3-(2-(2-nitrophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (16). Yield 25.5 %; Yellow solid; m.p. 194-195 oC; 1HNMR (300 MHz, DMSO) δ 10.62 (d, J = 4.41 Hz, 1H), 7.93-7.91 (m, 1H), 7.79-7.66 (m, 3Н), 7.30-7.26 (m, 4H), 6.16 (s, 1H), 5.15(q, J1= 8.10 Hz, J2= 3.18 Hz, 1H), 4.61 (d, J = 9.09 Hz, 1H), 4.01 (m, 1H), 3.28 (t, J = 12.51 Hz, 1H), 3.24-2.87 (m, 3H); 13C NMR (75 MHz, DMSO) δ 187.72, 158.03, 150.75, 148.02, 135.88, 134.85, 132.91, 132.23, 130.93, 128.84, 128.47, 127.12, 126.67, 125.92, 123.94, 91.57, 53.20, 45.35, 38.47, 28.33; IR (cm-1, KBr film): 3127(-NH), 1662(-CON), 1605 (-CO); HRMS (ESI): calcd. for C20H18N3O4 [M + H]+ 364.1292, found 364.132; HPLC (80% methanol in water): tR = 4.65 min, 99.10 %. 3-(2-(3-fluorophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (17). Yield 21.2 %; Yellow solid; m.p. 199-200 oC; 1HNMR (300 MHz, DMSO) δ 10.91 (s, 1H), 7.74 (m, appar t, 1H), 7.62-7.51 (m, 2Н), 7.39 (t, J = 8.13 Hz, 1H), 7.31-7.27 (m, 4H), 6.55 (s, 1H), 5.12(d, J = 12.03 Hz, 1H), 4.63 (d, J = 9.72 Hz, 1H), 4.03 (m, 1H), 3.28 (t, J = 12.84 Hz, 1H), 2.99-2.87 (m, 3H); 13C NMR (75 MHz, DMSO) δ 187.60, 162.53 (d, J = 214.13 Hz), 158.43, 150.68, 141.78, 134.87, 132.34, 130.71 (d, J = 7.88 Hz), 128.84, 127.11, 126.66, 125.92, 122.92, 118.18 (d, J = 20.70 Hz), 113.16 (d, J = 22.05 Hz), 89.65, 53.33, 45.33, 38.46, 28.35; IR (cm-1, KBr film): 3133(-NH), 1658(-CON), 1614 (-CO); HRMS (ESI): calcd. for C20H18FN2O2 [M + H]+ 337.1347, found 337.1372; HPLC (80% methanol in water): tR = 6.94 min, 99.60%. 3-(2-(2-methoxyphenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (18). Yield 25.2 %; Yellow solid; m.p.>210 oC; 1HNMR (300 MHz, DMSO) δ 10.65 (d, J = 4.41 Hz, 1H), 7.51-7.40 (m, 2H), 7.31-7.24 (m, 4H),7.09 (d, J = 8.28 Hz, 1H), 6.97 (t, J = 6.60 Hz, 1H), 6.46 (s, 1H), 5.08 (q, J1= 8.19 Hz, J2= 3.45 Hz, 1H), 4.63 (m, appar q, 1H), 3.97 (m, 1H), 3.82 (s, 3H), 3.22 (m, appar t, 1H), 2.95-2.85 (m, 3H); 13C NMR (75 MHz, DMSO) δ 190.01, 158.77, 156.92, 148.79, 134.89, 132.50, 131.76, 130.41, 129.24, 128.83, 127.05, 126.63, 125.92, 120.32, 112.13, 95.64, 55.62, 53.47, 45.29, 38.37, 28.38; IR (cm-1, KBr film): 3131(-NH), 1661 (-CON), 1609 (-CO); ACS Paragon Plus Environment

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HRMS (ESI): calcd. for C21H21N2O3 [M + H]+ 349.1547, found 349.1544; HPLC (80% methanol in water): tR = 5.56 min, 98.06%. 3-(2-oxo-2-phenylethylidene)-10-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (19). Yield 24.4 %; Yellow solid; m.p. 188-190 oC; 1HNMR (300 MHz, DMSO) δ 10.85 (d, J = 4.80 Hz, 1H), 7.87 (t, J = 6.48 Hz, 2H), 7.53-7.45 (m, 3H), 7.16 (d, J = 8.37 Hz, 1H), 6.90 (s, 1H), 6.85 (d, J = 8.34 Hz, 1H),6.58 (s, 1H), 5.05 (q, J1= 8.67 Hz, J2= 3.51 Hz, 1H), 4.63 (q, J1= 8.91 Hz, J2= 3.24 Hz, 1H), 4.04 (m, 1H), 3.74 (s, 3H), 3.24 (t, J = 12.54 Hz, 1H), 2.93-2.77 (m, 3H); 13CNMR (75 MHz, DMSO) δ 188.92, 158.59, 158.01, 150.22, 139.34, 133.42, 131.39, 129.83, 128.54, 128.54, 126.76, 126.72, 126.72, 113.21, 111.17, 89.84, 55.20, 53.56, 45.32, 38.70, 27.57; IR (cm-1, KBr film): 3132(-NH), 1664(-CON), 1619 (-CO); HRMS (ESI): calcd. for C21H21N2O3 [M + H]+ 349.1547, found 349.1549; HPLC (80% methanol in water): tR = 6.72 min, 99.25%. 3-(2-(3-chlorophenyl)-2-oxoethylidene)-10-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (20). Yield 26.8 %; Yellow solid; m.p. 176-177 oC; 1HNMR (300 MHz, DMSO) δ 10.89 (s, 1H), 7.83 (s, 2H), 7.59 (t, J = 7.29 Hz, 1H), 7.52 (t, J = 7.86 Hz, 1H), 7.16 (d, J = 8.25 Hz, 1H), 6.90 (s, 1H), 6.85 (d, J = 8.22 Hz, 1H),6.53 (s, 1H), 5.05 (d, J = 11.82 Hz, 1H), 4.63 (d, J = 11.55 Hz, 1H), 4.06 (m, 1H), 3.74 (s, 3H), 3.24 (t, J = 12.00 Hz, 1H), 2.95-2.79 (m, 3H); 13CNMR (75 MHz, DMSO) δ 186.93, 158.36, 158.01, 150.80, 141.27, 133.55, 133.32, 131.08, 130.58, 129.83, 126.70, 126.36, 125.42, 113.23, 111.17, 89.56, 55.20, 53.46, 45.35, 38.71, 27.54; IR (cm-1, KBr film): 3132(NH), 1662(-CON), 1617 (-CO); HRMS (ESI): calcd. for C21H20ClN2O3 [M + H]+ 383.116, found 383.1157; HPLC (90% methanol in water): tR = 5.32 min, 98.84%. 3-(2-(2-fluorophenyl)-2-oxoethylidene)-10-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (21). Yield 23.4 %; Yellow solid; m.p. 178-179 oC; 1HNMR (300 MHz, DMSO) δ 10.83 (s,1H), 7.71 (t, J = 7.47 Hz, 1H),7.52 (t, J = 6.78 Hz, 1H),7.32-7.25 (m, 2H), 7.16 (d, J = 8.25 Hz, 1H), 6.89 (s, 1H), 6.85 (d, J = 8.28 Hz, 1H), 6.41 (s, 1H), 5.05 (q, J1= 8.28 Hz, J2= 3.60 Hz, 1H), 4.62(q, J1= 9.60 Hz, J2= 2.67 Hz, 1H), 4.03 (m, 1H), 3.73 (s, 3H), 3.29 (t, J = 11.34 Hz, 1H), 3.26-2.77 (m, 3H); 13CNMR (75 MHz, DMSO) δ 185.86, 159.89 (d, J = 226.13 Hz), 158.08, 158.01, 150.27, 133.34, 132.83 (d, J = 8.63 Hz), 129.94, 129.83 (d, J = 4.65 Hz), 128.07 (d, J = 12.68 Hz), 126.71, 124.61(d, J = 3.45 Hz), 116.37 (d, J = 23.25 Hz), 113.20, 111.17, 94.12 (d, J = 8.70 Hz), 55.19, 53.46, 45.37, 38.70, 27.54; IR (cm-1, KBr film): 3128(-NH), 1662(-CON), 1613(-CO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.1452; HPLC (80% methanol in water): tR = 6.13 min, 99.39 %. 3-(2-(3-nitrophenyl)-2-oxoethylidene)-10-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (22). Yield 25.8 %; Yellow solid; m.p.>210 oC; 1HNMR (300 MHz, DMSO) δ 10.98 (d, J = 4.02 Hz, 1H), 8.57 (s, 1H), 8.38-8.32 (m, 2H), 7.78 (t, J = 7.98 Hz, 1H),7.17 (d, J = 8.49 Hz, 1H), 6.91 (s, 1H), 6.85 (d, J = 8.52 Hz, 1H), 6.61 (s, ACS Paragon Plus Environment

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1H), 5.07 (q, J1= 8.49 Hz, J2= 3.39 Hz, 1H), 4.64(d, J = 12.27 Hz, 1H), 4.06 (m, 1H), 3.74 (s, 3H), 3.30 (m, appar t, 1H), 2.93-2.79 (m, 3H); 13CNMR (75 MHz, DMSO) δ 185.80, 158.25, 158.02, 151.30, 148.11, 140.54, 133.27, 133.00, 130.44, 129.86, 126.71, 125.73, 121.12, 113.27, 111.18, 89.32, 55.22, 53.41, 45.40, 38.70, 27.53; IR (cm-1, KBr film): 3132(-NH), 1663(-CON), 1609(-CO); HRMS (ESI): calcd. for C21H20N3O5 [M + H]+ 394.1397, found 394.1428; HPLC (80% methanol in water): tR = 6.73 min, 95.01%. 3-(2-(3-methoxyphenyl)-2-oxoethylidene)-10-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (23). Yield 17.9 %; Yellow solid; m.p. 115-116 oC; 1HNMR (300 MHz, DMSO) δ 10.83 (d, J = 4.62 Hz, 1H), 7.477.35 (m, 3H), 7.17-7.08 (m, 2H), 6.90 (s, 1H), 6.85 (d, J = 8.28 Hz, 1H), 6.54 (s, 1H), 5.06 (q, J1= 8.79 Hz, J2= 3.00 Hz, 1H), 4.64(d, J = 12.33 Hz, 1H), 4.05 (m, 1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.24 (t, J = 12.96 Hz, 1H), 2.96-2.79 (m, 3H); 13CNMR (75 MHz, DMSO) δ 188.59, 159.42, 158.57, 158.01, 150.24, 140.91, 133.39, 129.83, 129.68, 126.71, 119.17, 117.19, 113.22, 111.66, 111.14, 89.98, 55.19, 55.16, 53.55, 45.32, 38.69, 27.56; IR (cm-1, KBr film): 3132(NH), 1663(-CON), 1609 (-CO); HRMS (ESI): calcd. for C22H23N2O4 [M + H]+ 379.1652, found 379.1677; HPLC (80% methanol in water): tR = 6.50 min, 97.24 %. 3-(2-(3-fluorophenyl)-2-oxoethylidene)-10-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (24). Yield 20.1 %; Yellow solid; m.p. 163-164 oC; 1HNMR (300 MHz, DMSO) δ 10.89 (d, J = 4.74 Hz, 1H), 7.72 (d, J = 8.07 Hz, 1H),7.63-7.50 (m, 2H), 7.40-7.35 (m, 1H), 7.15 (d, J = 8.22 Hz, 1H), 6.90 (s, 1H), 6.85 (d, J = 8.34 Hz, 1H), 6.54 (s, 1H), 5.06 (q, J1= 8.79 Hz, J2= 3.12 Hz, 1H), 4.63(d, J = 12.33 Hz, 1H), 4.06 (m, 1H), 3.76 (s, 3H), 3.26 (t, J = 11.58 Hz, 1H), 2.97-2.79 (m, 3H); 13CNMR (75 MHz, DMSO) δ 187.09, 162.32 (d, J = 243.08 Hz), 158.39, 158.00, 150.73, 141.79 (d, J = 6.00 Hz), 133.33, 130.69 (d, J = 7.80 Hz), 129.83, 126.71, 122.88 (d, J = 2.55 Hz), 118.18 (d, J = 21.00 Hz), 113.21, 113.17 (d, J = 21.98 Hz), 111.17, 89.67, 55.19, 53.47, 45.36, 38.70, 27.54; IR (cm-1, KBr film): 3132(-NH), 1663(-CON), 1621(-CO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.1483; HPLC (80% methanol in water): tR = 6.76 min, 98.91%. 3-(2-(4-fluorophenyl)-2-oxoethylidene)-10-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (25). Yield 22.8 %; Yellow solid; m.p. 195-196 oC; 1HNMR (300 MHz, DMSO) δ 10.82 (d, J = 4.20 Hz, 1H), 7.92 (m,2H), 7.29 (t, J = 6.15 Hz, 2H), 7.15 (d, J = 8.28 Hz, 1H), 6.90 (s, 1H), 6.85 (d, J = 8.37 Hz, 1H), 6.54 (s, 1H), 5.06 (q, J1= 8.16 Hz, J2= 3.87 Hz, 1H), 4.62(q, J1= 6.15 Hz, J2= 3.24 Hz, 1H), 4.04 (m, 1H), 3.74 (s, 3H), 3.24 (t, J = 12.99 Hz, 1H), 2.93-2.77 (m, 3H); 13CNMR (75 MHz, DMSO) δ 187.46, 164.03 (d, J = 247.58 Hz), 158.52, 158.01, 150.36, 135.89 (d, J = 2.63 Hz), 133.39, 129.83, 129.45 (d, J = 9.00 Hz), 129.45 (d, J = 9.00 Hz), 126.72, 115.43 (d, J = 21.45 Hz), 115.43 (d, J = 21.45 Hz), 113.21, 111.17, 89.56, 55.20, 53.53, 45.31, 38.71, 27.55; IR (cm-1, KBr film): 3132(NH), 1663(-CON), 1620(-CO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.1489; HPLC (80% ACS Paragon Plus Environment

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methanol in water): tR = 7.34 min, 96.97%. 3-(2-oxo-2-phenylethylidene)-9,10-dimethoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (26). Yield 25.6 %; Yellow solid; m.p. 172-173 oC; 1HNMR (300 MHz, DMSO) δ 10.85 (s, 1H), 7.89-7.86 (m, appar t, 2H), 7.54-7.48 (m, 3H), 6.89 (s, 1H), 6.82 (s, 1H), 6.56 (s, 1H), 4.98 (q, J1= 8.97 Hz, J2= 3.33 Hz, 1H), 4.68(d, J1= 9.18 Hz, J2= 2.46 Hz, 1H), 4.05 (m, 1H), 3.74 (s, 6H), 3.22 (t, J = 12.51 Hz, 1H), 3.14-2.76 (m, 3H); 13C NMR (75 MHz, DMSO) δ 188.90, 158.62, 150.32, 147.92, 147.71, 139.33, 131.39, 128.54, 128.54, 126.97, 126.76, 126.76, 123.90, 111.99, 109.61, 89.82, 55.76, 55.49, 53.30, 45.58, 38.45, 27.88; IR (cm-1, KBr film): 3132(-NH), 1664(-CON), 1612(CO); HRMS (ESI): calcd. for C22H23N2O4 [M + H]+ 379.1652, found 379.1684; HPLC (80% methanol in water): tR = 5.46 min, 99.97%. 3-(2-(3-chlorophenyl)-2-oxoethylidene)-9,10-dimethoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)one (27). Yield 25.7 %; Yellow solid; m.p. 176-177 oC; 1HNMR (300 MHz, DMSO) δ 10.91 (s, 1H), 7.84 (s, 2H), 7.62-7.52 (m, 2H), 6.91 (s, 1H), 6.82 (s, 1H), 6.54 (s, 1H), 5.01 (d, J = 11.76 Hz, 1H), 4.66(d, J = 11.76 Hz, 1H), 4.07 (m, 1H), 3.75 (s, 6H), 3.21 (t, J = 12.45 Hz, 1H), 2.90-2.78 (m, 3H); 13C NMR (75 MHz, DMSO) δ 186.90, 158.40, 150.90, 147.92, 147.71, 141.26, 133.54, 131.08, 130.59, 126.94, 126.37, 125.42, 123.79, 111.99, 109.61, 89.54, 55.76, 55.49, 53.20, 45.62, 38.47, 27.85; IR (cm-1, KBr film): 3133(-NH), 1664(-CON), 1617(-CO); HRMS (ESI): calcd. for C22H22ClN2O4 [M + H]+413.1263, found 413.1304; HPLC (80% methanol in water): tR = 7.57 min, 98.37%. 3-(2-(2-fluorophenyl)-2-oxoethylidene)-9,10-dimethoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)one (28). Yield 16.5 %; Yellow solid; m.p. 176-177 oC; 1HNMR (300 MHz, DMSO) δ 10.84 (d, J = 4,23 Hz, 1H), 7.72 (t, J = 7.68 Hz, 1H), 7.54-7.52 (m, 1H), 7.31-7.24 (m, 2H), 6.90 (s, 1H), 6.81 (s, 1H), 6.41 (s, 1H), 4.99 (q, J1= 9.00 Hz, J2= 2.76Hz, 1H), 4.66(d, J = 11.82 Hz, 1H), 4.06 (m, 1H), 3.74 (s, 6H), 3.18 (t, J = 12.57 Hz, 1H), 2.88-2.76 (m, 3H); 13

CNMR (75 MHz, DMSO) δ 185.85 (d, J = 2.93 Hz), 159.91 (d, J = 222.45 Hz), 158.08, 150.38, 147.94, 147.73,

132.84 (d, J = 9.15 Hz), 129.92 (d, J = 2.93 Hz), 128.06 (d, J = 13.05 Hz), 126.98, 124.61 (d, J = 3.23 Hz), 123.84, 116.37 (d, J = 23.33 Hz), 112.03, 109.64, 94.11 (d, J = 9.08 Hz), 55.78, 55.51, 53.21, 45.63, 38.46, 27.86; IR (cm-1, KBr film): 3131(-NH), 1663(-CON), 1621 (-CO); HRMS (ESI): calcd. for C22H22FN2O4 [M + H]+ 397.1558, found 397.1586; HPLC (80% methanol in water): tR = 5.05 min, 99.84%. 3-(2-(3-nitrophenyl)-2-oxoethylidene)-9,10-dimethoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (29). Yield 20.4 %; Yellow solid; m.p.>210 oC; 1HNMR (300 MHz, DMSO) δ 11.00 (s, 1H), 8.58 (s, 1H), 8.36 (t, J = 9.15 Hz, 2H), 7.79 (t, J = 8.07 Hz, 1H), 6.92 (s, 1H), 6.83 (s, 1H), 6.61 (s, 1H), 5.03 (d, J = 12.63 Hz, 1H), 4.66(d, J = 11.19 Hz, 1H), 4.09 (m, 1H), 3.75 (s, 6H), 3.24 (t, J = 12.18 Hz, 1H), 2.91-2.79 (m, 3H); 13CNMR (75 MHz, DMSO) δ 185.78, 158.28, 151.40, 148.10, 147.95, 147.73, 140.53, 133.00, 130.44, 126.97, 125.73, 123.75, 121.12, 112.03, ACS Paragon Plus Environment

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109.63, 89.31, 55.77, 55.51, 53.16, 45.67, 38.51, 27.85; IR (cm-1, KBr film): 3132(-NH), 1669(-CON), 1616(-CO); HRMS (ESI): calcd. for C22H22N3O6 [M + H]+424.1503, found 424.1545;HPLC (80% methanol in water): tR = 5.72 min, 97.67%. 3-(2-(3-methoxyphenyl)-2-oxoethylidene)-9,10-dimethoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)one (30). Yield 17.6 %; Yellow solid; m.p. 173-175 oC; 1HNMR (300 MHz, DMSO) δ 10.84 (d, J = 4.71 Hz, 1H), 7.46-7.35 (m, 3H), 7.11 (t, J = 5.34 Hz, 1H), 6.91 (s, 1H), 6.81 (s, 1H), 6.54 (s, 1H), 5.00 (q, J1= 8.19 Hz, J2= 3.72 Hz, 1H), 4.65(d, J = 11.70 Hz, 1H), 4.05 (m, 1H), 3.80 (s, 3H), 3.74 (s, 6H), 3.18 (t, J = 12.57 Hz, 1H), 2.89-2.76 (m, 3H); 13

CNMR (75 MHz, DMSO) δ 188.56, 159.41, 158.59, 150.35, 147.91, 147.71, 149.90, 129.69, 126.95, 123.89, 119.16,

117.22, 112.00, 111.62, 109.63, 89.94, 55.77, 55.50, 55.17, 53.29, 45.58, 38.45, 27.87; IR (cm-1, KBr film): 3134(-NH), 1662(-CON), 1616(-CO); HRMS (ESI): calcd. for C23H25N2O5 [M + H]+409.1758, found 409.1792;HPLC (80% methanol in water): tR = 5.70min, 99.12 %. 3-(2-(3-fluorophenyl)-2-oxoethylidene)-9,10-dimethoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)one (31). Yield 20.1 %; Yellow solid; m.p. 200-201 oC; 1HNMR (300 MHz, DMSO) δ 10.90 (d, J = 4.26 Hz, 1H), 7.72 (d, J = 7.53 Hz, 1H), 7.61-7.49 (m, 2H), 7.37 (t, J = 8.19 Hz, 1H), 6.91 (s, 1H), 6.81 (s, 1H), 6.54 (s, 1H), 5.02 (q, J1= 10.68 Hz, J2= 1.47 Hz, 1H), 4.65(d, J = 11.43 Hz, 1H), 4.05 (m, 1H), 3.73 (s, 6H), 3.18 (t, J = 12.33 Hz, 1H), 2.932.72 (m, 3H); 13CNMR (75 MHz, DMSO) δ 187.11, 162.47 (d, J = 220.88 Hz), 158.44, 150.85, 147.94, 147.73, 141.85, 130.72 (d, J = 7.65 Hz), 126.98, 123.84, 122.87, 118.16 (d, J = 24.45 Hz), 113.16 (d, J = 22.28 Hz), 112.04, 109.66, 89.65, 55.79, 55.52, 53.22, 45.62, 38.48, 27.87; IR (cm-1, KBr film): 3131(-NH), 1672(-CON), 1613(-CO); HRMS (ESI): calcd. for C22H22FN2O4 [M + H]+ 397.1558, found 397.1592; HPLC (80% methanol in water): tR = 5.91 min, 97.64%. 3-(2-(4-fluorophenyl)-2-oxoethylidene)-9,10-dimethoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)one (32). Yield 20.0 %; Yellow solid; m.p. 210-211 oC; 1HNMR (300 MHz, DMSO) δ 10.83 (d, J = 4.29 Hz, 1H), 7.97-7.494 (m, 2H), 7.28 (t, J = 8.37 Hz, 2H), 6.91 (s, 1H), 6.81 (s, 1H), 6.54 (s, 1H), 5.00 (d, J = 9.45 Hz, 1H), 4.66(d, J = 11.94 Hz, 1H), 4.04 (m, 1H), 3.75 (s, 6H), 3.18 (t, J = 12.69 Hz, 1H), 2.90-2.76 (m, 3H); 13CNMR (75 MHz, DMSO) δ 187.43, 163.94 (d, J = 242.03 Hz), 158.56, 150.47, 147.92, 147.72, 135.87, 129.45 (d, J = 8.70 Hz), 129.45 (d, J = 8.70 Hz), 126.21, 123.89, 115.42 (d, J = 21.60 Hz), 115.42 (d, J = 21.60 Hz), 112.05, 109.66, 89.54, 55.79, 55.52, 53.28, 45.58, 38.47, 27.45; IR (cm-1, KBr film): 3131(-NH), 1672(-CON), 1616(-CO); HRMS (ESI): calcd. for C22H22FN2O4 [M + H]+ 397.1558, found 397.16; HPLC (80% methanol in water): tR = 5.87 min, 96.50 %. 3-(2-oxo-2-phenylethylidene)-8-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (33). Yield 24.3 %; Yellow solid; m.p. 207-208 oC; 1HNMR (300 MHz, DMSO) δ 10.82 (d, J = 4.56 Hz, 1H), 7.90-7.87 (m, appar ACS Paragon Plus Environment

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d, 2H),7.54-7.47 (m, 3H), 7.27 (t, J = 7.95 Hz, 1H), 6.93-6.90 (m, 2H), 6.59 (s, 1H), 5.10 ( q, J1 = 9.36 Hz, J2 = 2.82 Hz, 1H), 4.71 (q, J1 = 7.92 Hz, J2 = 4.44 Hz, 1H), 4.02 (m, 1H), 3.81 (s, 3H), 3.22 (t, J = 12.60 Hz, 1H), 2.99-2.83 (m, 2H), 2.54 (m, 1H); 13CNMR (75 MHz, DMSO) δ 188.85, 158.61, 156.36, 150.04, 139.34, 133.58, 131.39, 128.55, 128.55, 127.36, 126.75, 126.75, 123.29, 117.81, 108.74, 89.79, 55.40, 53.21, 45.36, 37.79, 22.16; IR (cm-1, KBr film): 3132(-NH), 1661(-CON), 1611(-CO); HRMS (ESI): calcd. for C21H21N2O3 [M + H]+ 349.1547, found 349.1552; HPLC (80% methanol in water): tR = 7.24 min, 98.81 %. 3-(2-(3-fluorophenyl)-2-oxoethylidene)-8-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (34). Yield 24.8 %; Yellow solid; m.p.>210 oC; 1HNMR (300 MHz, DMSO) δ 10.88 (d, J = 4.62 Hz, 1H), 7.40 (d, J = 7.65 Hz, 1H),7.62-7.53 (m, appardt, 2H), 7.39-7.38 (m, appar t, 1H), 7.27 (t, J = 8.01 Hz, 1H), 6.91 (d, J = 8.25 Hz, 2H), 6.56 (s, 1H), 5.09 (q, J1= 8.46 Hz, J2= 3.63 Hz, 1H), 4.70 (m, appar q, 1H), 4.05 (m, 1H), 3.81 (s, 3H), 3.27 (t, J = 13.29 Hz, 1H), 2.99-2.86 (m, 2H), 2.50 (m, appar t, 1H); 13CNMR (75 MHz, DMSO) δ 187.07, 162.32 (d, J = 243.53 Hz), 158.42, 156.36, 150.57, 142.20, 133.50, 130.72 (d, J = 7.65 Hz), 127.38, 123.29, 122.87, 118.32, 117.80, 113.15 (d, J = 22.28 Hz), 108.77, 89.63, 55.41, 53.13, 45.39, 37.81, 22.14; IR (cm-1, KBr film): 3131(-NH), 1661(-CON), 1610(-CO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.1459; HPLC (80% methanol in water): tR = 8.05 min, 95.55 %. 3-(2-(2-fluorophenyl)-2-oxoethylidene)-8-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (35). Yield 25.0 %; Yellow solid; m.p. 172-173 oC; 1HNMR (300 MHz, DMSO) δ 10.82 (d, J = 3.93 Hz, 1H), 7.75 (t, J = 7.68 Hz, 1H),7.59-7.52 (m, 1H), 7.34-7.26 (m, 3H), 6.90 (m, appar t, 2H), 6.44 (s, 1H), 5.11 (d, J = 11.82 Hz, 1H), 4.68 (q, J1= 8.76 Hz, J2= 3.45 Hz, 1H), 4.02 (m, 1H), 3.82 (s, 3H), 3.24 (t, J = 12.72 Hz, 1H), 2.99-2.83 (m, 3H); 13

CNMR (75 MHz, DMSO) δ 185.82, 159.89 (d, J = 224.33 Hz), 158.07, 156.36, 150.09, 133.50, 132.83 (d, J = 9.30

Hz), 129.90 (d, J = 2.93 Hz), 128.01, 127.37, 124.61 (d, J = 3.53 Hz), 123.28, 117.78, 116.37 (d, J = 23.18 Hz), 108.76, 94.08 (d, J = 8.48 Hz), 55.40, 53.11, 45.40, 37.80, 22.14; IR (cm-1, KBr film): 3132(-NH), 1668(-CON), 1607(-CO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.1492; HPLC (80% methanol in water): tR = 6.55 min, 99.51%. 3-(2-oxo-2-phenylethylidene)-9-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (36). Yield 21.3 %; Yellow solid; m.p. 173-174 oC; 1HNMR (300 MHz, DMSO) δ 10.85 (d, J = 4.59 Hz, 1H), 7.89 (m, appar t, 2H),7.55-7.47 (m, 3H), 7.24 (d, J = 8.43 Hz, 1H), 6.87 (d, J = 8.58 Hz, 1H), 6.84 (s, 1H),6.59 (s, 1H), 5.05 (q, J1= 8.19 Hz, J2= 3.63 Hz, 1H), 4.62(d, J = 12.42 Hz, 1H), 4.01 (m, 1H), 3.76 (s, 3H), 3.23 (t, J = 12.39 Hz, 1H), 3.02-2.86 (m, 3H); 13CNMR (75 MHz, DMSO) δ 188.86, 158.65, 158.14, 150.21, 139.37, 136.33, 131.37, 128.54, 128.54, 127.06, 126.75, 126.75, 124.39, 113.33, 113.08, 89.79, 55.10, 53.07, 45.44, 38.45, 28.60;IR (cm-1, KBr film): 3132 (-NH), ACS Paragon Plus Environment

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1664(-CON), 1607(-CO); HRMS (ESI): calcd. for C21H21N2O3 [M + H]+ 349.1547, found 349.1584; HPLC (80% methanol in water): tR = 6.51 min, 99.76%. 3-(2-(3-fluorophenyl)-2-oxoethylidene)-9-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (37). Yield 22.3 %; Yellow solid; m.p. 166-167 oC; 1HNMR (300 MHz, DMSO) δ 10.89 (d, J = 4.86 Hz, 1H), 7.74 (d, J = 7.77 Hz, 1H), 7.63-7.52 (m, 2H), 7.42-7.36 (m, 1H), 7.24 (d, J = 8.55 Hz, 1H), 6.87 (d, J = 8.58 Hz, 1H), 6.84 (s, 1H), 6.56 (s, 1H), 5.05 (q, J1= 8.31 Hz, J2= 3.54 Hz, 1H), 4.62(q, J1= 5.40 Hz, J2= 3.27 Hz, 1H), 4.01 (m, 1H), 3.76 (s, 3H), 3.24 (t, J = 12.48 Hz, 1H), 2.99-2.86 (m, 3H); 13CNMR (75 MHz, DMSO) δ 187.02, 162.32 (d, J = 243.08 Hz), 158.45, 158.15, 150.71, 141.82 (d, J = 6.23 Hz), 136.30, 130.67 (d, J = 7.80 Hz), 127.05, 124.28, 122.86 (d, J = 2.63 Hz), 118.14 (d, J = 21.08 Hz), 113.33, 113.18 (d, J = 17.78 Hz), 113.00, 89.63, 55.09, 52.99, 45.46, 38.46, 28.57; IR (cm-1, KBr film): 3133(-NH), 1664(-CON), 1609(-CO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.149; HPLC (80% methanol in water): tR = 7.17 min, 96.67%. 3-(2-(2-fluorophenyl)-2-oxoethylidene)-9-methoxy-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (38). Yield 20.0 %; Yellow solid; m.p. 165-167 oC; 1HNMR (300 MHz, DMSO) δ 10.81 (d, J = 4.26 Hz, 1H), 7.757.70 (m, 1H), 7.54-7.50 (m, 1H),7.32-7.24 (m, 2H), 7.22 (d, J = 8.67 Hz, 1H),6.85 (d, J = 8.46 Hz, 1H), 6.82 (s, 1H), 6.41 (s, 1H), 5.04 (q, J1= 8.67 Hz, J2= 3.48 Hz, 1H), 4.56 (q, J1= 5.22 Hz, J2= 3.03 Hz, 1H), 3.98 (m, 1H), 3.22 (t, J = 12.60 Hz, 1H), 2.96-2.83 (m, 3H); 13CNMR (75 MHz, DMSO) δ 185.82, 159.91 (d, J = 221.55 Hz), 158.14, 158.14, 150.25, 136.31, 132.80 (d, J = 9.45 Hz), 129.92, 128.09 (d, J = 14.03 Hz), 127.04, 124.61, 124.44 (d, J = 20.63 Hz), 116.36 (d, J = 23.40 Hz), 113.33, 113.07, 94.07 (d, J = 8.55 Hz), 55.09, 52.97, 45.48, 38.44, 28.57; IR (cm-1, KBr film): 3134(-NH), 1666(-CON), 1607 (-CO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.1465; HPLC (80% methanol in water): tR = 5.95 min, 99.96%. 3-(2-oxo-2-(4-(trifluoromethyl)phenyl)ethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (39). Yield 17.0 %; Yellow solid; m.p.>210 oC; 1HNMR (300 MHz, DMSO) δ 10.90 (d, J = 13.56 Hz, 1H), 8.20-8.04 (m, 2H), 7.91- 7.71 (m, 2H),7.31-7.25 (m, 4H), 6.59 (s, 1H), 5.10 (q, J1= 10.80 Hz, J2= 0.48 Hz, 1H), 4.53(m, appar q, 1H), 4.03 (m, 1H), 3.25 (m, 1H), 3.04-2.89 (m, 3H); 13CNMR (75 MHz, DMSO) δ 186.70, 158.36, 150.99, 140.09, 134.87, 132.31, 130.77, 130.00, 129.58, 128.85, 127.80, 127.75, 127.13, 126.68, 124.44 (d, J = 224.33 Hz), 122.90, 89.41, 53.30, 45.34, 38.50, 28.34; IR (cm-1, KBr film): 3132(-NH), 1665(-CON), 1620(-CO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.1459; HPLC (80% methanol in water): tR = 4.89 min, 96.16%. 3-(2-(3,5-difluorophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (40). Yield 26.5 %; Yellow solid; m.p. 176-177 oC; 1HNMR (300 MHz, DMSO) δ 10.95 (d, J = 5.01 Hz, 1H), 7.54-7.52 (m, appar d, 2H), 7.44 (d, J = 8.97 Hz, 1H), 7.31- 7.27 (m, 4H), 6.52 (s, 1H), 5.12 (q, J1= 8.37 Hz, J2= 3.69 Hz, 1H), 4.53(q, J1= ACS Paragon Plus Environment

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8.10 Hz, J2= 4.26 Hz, 1H), 4.06 (m, 1H), 3.30 (t, J = 12.21 Hz, 1H), 3.05-2.84 (m, 3H); 13CNMR (75 MHz, DMSO) δ 185.41, 162.57 (d, J = 245.85 Hz), 162.40 (d, J = 245.93 Hz), 158.24, 151.21, 142.94 (d, J = 7.58 Hz), 134.87, 132.27, 128.84, 127.12, 126.67, 125.92, 109.85 (d, J = 17.18 Hz), 109.75 (d, J = 17.18 Hz), 106.58 (t, J = 26.17 Hz), 89.44, 53.26, 45.37, 38.47, 28.34; IR (cm-1, KBr film): 3133(-NH), 1669(-CON), 1616(-CO); HRMS (ESI): calcd. for C20H17F2N2O2 [M + H]+ 355.1253, found 355.1252; HPLC (90% methanol in water): tR = 4.87 min, 97.25%. 3-(2-(3-bromophenyl)-2-oxoethylidene)-2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(11bH)-one (41). Yield 26.1 %; Yellow solid; m.p. 185-187 oC; 1HNMR (300 MHz, DMSO) δ 10.90 (d, J = 4.11 Hz, 1H), 7.99 (s, 1H), 7.90 (d, J = 7.86 Hz, 1H), 7.74 (d, J = 7.86 Hz, 1H), 7.47 (t, J = 7.80 Hz, 1H), 7.32- 7.27 (m, 4H), 6.55 (s, 1H), 5.11 (q, J1= 9.33 Hz, J2= 3.00 Hz, 1H), 4.53(q, J1= 9.72 Hz, J2= 5.61 Hz, 1H), 4.06 (m, 1H), 3.30 (t, J = 12.09 Hz, 1H), 3.05-2.89 (m, 3H); 13C NMR (75 MHz, DMSO) δ 186.80, 158.39, 150.74, 141.48, 134.87, 133.99, 132.32, 130.88, 129.27, 128.84, 127.11, 126.66, 125.92, 125.80, 122.07, 89.51, 53.31, 45.33, 38.48, 28.35; IR (cm-1, KBr film): 3132(-NH), 1666(-CON), 1619(-CO); HRMS (ESI): calcd. for C20H18BrN2O2 [M + H]+397.0546, found 397.0588; HPLC (90% methanol in water): tR = 5.37 min, 97.96 %. Biology. Cell culture conditions. HepG2 cells stably transfected with a luciferase reporter (HepG2-ARE-C8) were kindly provided by Professor Dr. A. N. Tony Kong (Rutgers University, Piscataway, NJ). Cells were maintained in modified RPMI-1640 medium (GiBco, Invitrogen Corp., USA) with 10% fetal bovine serum (FBS) (GiBco, Invitrogen Corp., USA) in a humidified atmosphere of 5% CO2 and 95% air at 37 ºC. HCT116 cells (Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured in McCoy's 5A (Sigma-Aldrich, USA) supplemented with 10% (v/v) FBS. MTT analysis. Cell viabilities were determined by using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide) assay. MTT was purchased from Sigma (St. Louis, MO). It was dissolved in phosphate buffered saline (PBS) to a concentration of 5 mg/mL as the stock solution and stored at -20 ºC. HCT116 cells were plated in 96-well plates, raised to a population of 8 × 103 cells/well and incubated overnight. The cells were exposed with different concentrations of test compounds for 24 h at 37 oC, 20 µL of MTT solution was added into 96-well plates and incubated for an additional 4 h. Then the solution was removed and 100 µL of DMSO was added into each well to dissolve formazan precipitate. The OD value which served as a measure of cell viability was determined at 570 nm by Elx800 Absorbance Microplate Reader (BioTek, Vermont, USA). EC50 = [1 – (OD test - OD blank)/ (OD control - OD blank)]

* 100 %.

ARE-luciferase activity assay. HepG2-ARE-C8 cells were plated in 96-well plates at a density of 4 × 104 cells/well and incubated overnight. The cells were exposed with different concentrations of test compounds, with tBHQ and SFN ACS Paragon Plus Environment

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serving as a positive control DMSO as a negative control and the luciferase cell culture lysis reagent as a blank. After 12 h of treatment, the medium was removed and 100 µL of cold PBS was added into each well. Then the cells were harvested in the luciferase cell culture lysis reagent. After centrifugation, 20 µL of the supernatant was used for determining the luciferase activity according to the protocol provided by the manufacturer (Promega, Madison, WI). The luciferase activity was measured by a luminoskan ascent (Thermo scientific, USA). The data were obtained in triplicates and expressed as fold induction over control. Western Blot. Anti-HO-1 (sc-136960) and -NQO1 (sc-271116) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin (AP0060) and -Nrf2 (BS1258) were purchased from Bioworlde (Bioworlde, USA). HCT116 Cells (5 × 106 cells/well) were washed once with ice-cold PBS and driven down with 2mL of EDTA. Cells were centrifuged at 2500 rpm and resuspended in 45 µL of lysis buffer, which was composed of 50 mMTris–HCl, 150 mMNaCl, NP-40, 1 mM EDTA, PMSF, NaF, Leu and DTT for 1 h. Then cells were centrifuged again at 12000 rpm for 20 min at 4 ºC.The supernatant was retained, and the protein concentration was determined by the BCA assay with Varioskan flash (Thermo, Waltham, MA) at 562 nm. Samples were stored at -80 ºC until use. Nuclear and cytosol (1 × 107 cells/well) were isolated using the nuclear-cytosolextraction kit (KeyGEN, NJ, China). Then the separate extracts were stored at -80 ºC until use. The extracts were separated by SDS–PAGE and then transferred onto PVDF membranes (Perkin Elmer, Northwalk, CT, USA). After blocking with 1% BSA for 2 h, membranes were incubated at 37℃ for 1h and then at 4 ºC overnight with a primary antibody. After that, they were reacted with a DyLight 800 labeled secondary antibody at 37 ºC for 1 h. The membranes were screened through the odyssey Infrared Imaging System (LI-COR, Lincoln, Nebraska, USA). AOM-DSS mouse models. Animal studies were conducted according to protocols approved by Institutional Animal Care and Use Committee of China Pharmaceutical University. All animals were appropriately used on a scientifically valid and ethical manner. AOM (Sigma-Aldrich, St. Louis) was dissolved in distilled water at 10 mg/mL as storage, and then diluted to a final concentration of 1 mg/mL in sterile isotonic saline. DSS (molecular weight at 36-50kDa) (MP Biomedicals, Morgan Irvine, CA) was dissolved at 2.5 % with distilled water. Female C57BL/6 mice (6-8 weeks old) were divided into 3 groups in random: control group, AOM-DSS group, and AOM-DSS+1 (50mg/kg/day, supplemented into the diet) group. At the second week, mice were injected intraperitioneally with AOM 10 mg/kg. DSS treatment were begun at the third week and continued for 3 cycles. Each cycle consisted of one week of 2.5% DSS in drinking water followed by two weeks normal water. Mice were weighted every two days and dietary administration of 1 was lasted until the end of the experiment. After 130 days, mice were sacrificed. The colon was fixed in10% buffered formalin (pH 7.4) for at least 24 h for further histopathological assessment, immunohistochemical and immunofluoresACS Paragon Plus Environment

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cence study. All the tissue slices were done by the Division of Pathology, Jiangsu Province Hospital, Nanjing, China. Histopathological evaluation. The colon was spread onto a plastic sheet, fixed with 10% neutral-buffered formalin for 48 h, and embedded in paraffin block. Sections of paraffin-embedded tissues were subjected to H&E (KeyGEN, NJ, China) staining for evaluating the severity of inflammation. Immunohistochemistry. Tissues were prepared from formalin-fixed, paraffin-embedded colon tissue. Stains against IL-1β and IL-6 (R & D system, USA) were performed according to the kit protocol (KeyGEN, NJ, China). Briefly, the slides were deparaffinized. Antigen unmasking was carried out by incubation in100 °C water bath in 10 mM sodium citrate buffer with 0.1% Tween 20 for 20 min. Slides were incubated with primary antibodies in PBS containing 5% BSA and 10% goat serum. Biotinylated secondary anti-rabbit antibodies were added and incubated at room temperature for 30 min. Streptavidin-HRP was added, and after 40 min the sections were stained with DAB substrate and counterstained with hematoxylin. Image Pro Plus 6.0 software (Media Cybernetics, Silver Spring, USA) was used for automatic counting of labeled inflammatory cytokines to quantify the experimental data. Immunofluorescence. Four-micron thick sections were washed in 10% PBS for 15 min, then incubated at 4 ºC overnight with Nrf2 primary antibodies (abcam, Cambridge, UK). After washed with PBS, tissues were incubated at 37 ºC for 1 h with FITC-labeled secondary sheep anti-rabbit IgG antibody (BOSTER, WH, China), then stained with fluorochrome dye DAPI (Santa Cruz Biotechnology, Santa Cruz, CA) to visualize the nuclei and observed under a laser scanning confocal microscope (OlympusFluoviewFV1000, Japan) with a peak excitation wave length of 570 nm and 340 nm. The method for quantification is the same as above-mentioned immunohistochemistry. Statistical analysis. All results are given as mean ± SD performed in parallel experiments for triplicate. P-values less than 0.05 (p

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