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

Article abstract of DOI:10.1021/jm400944k

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 data

Full text of DOI:10.1021/jm400944k

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Article
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
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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Page 1 of 41
Journal of Medicinal Chemistry
3ꢀaroylmethyleneꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀones as
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potent Nrf2/ARE inducers in human cancer cells and AOMꢀDSS treated mice
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Meiꢀyang Xi,b,d Jianꢀmin Jia,b,d Haoꢀpeng Sun,a,b,d,* Zhongꢀying Sun,c Jieꢀwei Jiang,b,d Yaꢀjing Wang,c
Minꢀye Zhang,b,d Junꢀfeng Zhu,b,d Liꢀli Xu,b,d Zhengꢀyu Jiang,b,d Xin Xue,b,d Ming Ye,c Xi Yang,c Yuan
Gao,c Lei Tao,c Xiaoꢀke Guo,b,d Xiaoꢀli Xu,b,d Qingꢀlong Guo,c Xiaoꢀjin Zhang,b,d Rong Hu,c,* Qiꢀdong
You a,b,*
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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, Chiꢀ
na
c Department of Physiology, School of Pharmacy, China Pharmaceutical University, Nanjing, China
d Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
ABSTRACT
Nrf2ꢀmediated activation of ARE regulates expression of cytoprotective enzymes against oxidative stress, inflammaꢀ
tion and carcinogenesis. We have discovered a novel structure (1) as an ARE inducer via luciferase reporter assay to
screen the inꢀhouse database of our laboratory. The potency of 1 was evaluated by the expression of NQOꢀ1, HOꢀ1 and
nuclear translocation of Nrf2 in HCT116 cells. In vivo potency of 1 was studied using AOMꢀDSS models, showing that
the development of colorectal adenomas was significantly inhibited. Administration with 1 lowered the expression of
ILꢀ6, ILꢀ1β 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 identiꢀ
fied. To our knowledge, this is a potent ARE activator. Besides, its novel structure makes it promising for further optiꢀ
mization.
INTRODUCTION
Nuclear factor erythroid 2 p45ꢀrelated factor 2 (Nrf2), is a basicꢀleucine zipper (bZIP) transcription factor which conꢀ
tains a Cap ‘n’ Collar (CNC) structure.1ꢀ4 In quiescent condition, Nrf2 is sequestered in cytoplasm by Kelchꢀlike ECHꢀ
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associated protein 1 (Keap1), a 69ꢀkDa cytosolic protein usually existing as a dimer, targeting Nrf2 for ubiquitination
and proteosomal degradation. However, the binding conformation of Keap1ꢀNrf2 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
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shown to activate Nrf2 pathway.7 Nrf2ꢀregulated enzymes such as glutathione Sꢀtransferase (GST), NAD(P)H:quinone
8, 9 Molecules that
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oxidoreductaseꢀ1(NQOꢀ1) and heme oxygenaseꢀ1 (HOꢀ1)contain an ARE in their promoter region
.
activate Nrf2 are considered as potential antioxidant, antiꢀinflammatory 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 reꢀ
ported 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 chemoꢀ
preventive potency against inflammationꢀassociated carcinogenesis.23ꢀ25 In the models, AOM initially causes oxidative
and DNA damage, which is then promoted by DSS. DSS induces inflammation in the colon and promotes AOMꢀ
induced 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 chemoꢀ
preventive compounds including curcumin and nonsteroidal antiꢀinflammatory drugs are not only effective in animal
models, but also promising in the onꢀgoing clinical trials.29ꢀ31 Considering the importance of chemoprevention in the
treatment of cancer, novel inducers targeting Nrf2/ARE pathway are urgently needed and attract the attention of medicꢀ
inal chemists throughout the world 32ꢀ34
.
Here we report a series of 41 compounds with 3ꢀ(2ꢀoxoꢀ2ꢀphenylethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀ
a]isoquinolinꢀ4(11bH)ꢀone scaffold. Among these compounds, 1 was firstly identified from the screening of inꢀhouse
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. Structureꢀactivity 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 chemꢀ
opreventive agents.
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RESULTS
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Synthetic chemistry. A class of heterocyclic Compounds 1ꢀ41 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 1ꢀ41 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 1ꢀ41a.
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a Reagents and conditions: (a) chloracetyl chloride, NaHCO3, DCM, 0ꢀ10 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, iꢀPrOH/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 Nrf2ꢀregulated ARE, HOꢀ1, NQOꢀ1 and facilitate nuclear
translocation of Nrf2. In an effort to discover new Nrf2/ARE activators, we performed a cellꢀbased luciferase reporter
screening for compounds that induced intracellular ARE activities in HepG2ꢀAREꢀC8 cells. Compound 1 showed a
concentrationꢀdependent pattern for ARE inductivity. It significantly increased the level of ARE to 18.9ꢀfold at 5 ꢁM
compared to control (Figure 1A).In comparison, tertꢀbutylhydroquinone (tBHQ) showed 5.2ꢀfold induction at 80 ꢁM,
and SFN showed 5.5ꢀfold induction at 5 ꢁM (Table 2). As a result, 1 was far more efficacious than aboveꢀmentioned
positive control in their abilities to upregulate the intracellular ARE acticities. In addition, 1 induced HOꢀ1 and NQOꢀ
1 expression by measuring protein levels in human colorectal cancer cells (HCT116) in a timeꢀ and concentrationꢀ
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dependent manner (Figure 1B and 1C). A timeꢀresponse study with 1 demonstrated that nuclear translocation of Nrf2
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began within 1 h and maximized at 2 h (Figure 1D). It showed that expression level of HOꢀ1 and NQOꢀ1 was delayed
and maximized at 8 h and 24h, respectively.42ꢀ44
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Figure 1. 1 Induced expression of ARE, cytoprotective enzymes and nuclear translocation of Nrf2. (A) Concentrationꢀdependent induction
of 1 on the ARE activation. HepG2ꢀC8ꢀARE 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 ꢁM),
timeꢀcourse analysis indicated that 1 maximized HOꢀ1, NQOꢀ1 at 8 h and 24h separately. (C) After treatment with 1 at designated time (8
h for HOꢀ1, 24 h for NQOꢀ1), cell lysates were prepared from HCT116 cells and subjected to Western blot analysis for HOꢀ1, NQOꢀ1 and
βꢀactin, which showed concentrationꢀdependent. (D) Effects of 1on nuclear translocation of Nrf2. At indicated times after treatment with
1(10 ꢁM), nuclear fractions and cytoplasm extracts were prepared from HCT116 cells and subjected to Western blot analysis for Nrf2,
histone and βꢀactin. HOꢀ1, NQOꢀ1 and cytoplasmic Nrf2 expression used βꢀactin for normalization. Nuclear Nrf2 used histone for normalꢀ
ization. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant difference from the nonꢀtreated group.
Effect of administration with 1 on development of AOMꢀDSS 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 AOMꢀDSS group had 90 % colorectal tumor incidence (percentage of mice with colon adenomas), mulꢀ
tiplicity (number of adenomas) of 2.60 ± 0.68, an average adenomas size of 2.70 ± 0.62 mm and adenoma burden of
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7.02 ± 1.01. In contrast, with the treatment of 1, the above mentioned parameters was reduced to 50 % (tumor inciꢀ
dence), 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.
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Table1. Overview of the AOMꢀDSS models.a
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WBC
Group (No. of
mice examined)
Lengh of colon
(cm)
NR (%)
LR (%)
(10^9/L）
5.33±1.34
5.67±1.68
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AOMꢀDSS(20)
5.62±0.69
4.59±0.57
12.73±1.65
12.39±1.43
87.75±2.16
88.15±2.03
AOMꢀDSS+1(18)
a Data are mean ± SD. White Blood Cell (WBC), Neutrophils Ratio (NR), Lymphocyte Ratio (LR).
Figure 2. Observation of AOMꢀDSS models. (A) Representative pictures of colon from the AOMꢀDSS group (top panel) and AOMꢀ
DSS+1 group (bottom panel) in C57BL/6 female mice. (B) Body weight of the AOMꢀDSS group and AOMꢀDSS+1 group. (C) and (D)
Administration with 1 decreased incidence, multiplicity and size of colorectal tumors in an AOMꢀDSSꢀinduced 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 AOMꢀDSS group.
Adenoma pathology findings of AOMꢀDSS mice treated with 1. AOMꢀDSS treatment caused extensive high
grade dysplasia and formed adenomas in colorectal tissue. However, treatment with 1 decreased the developed dysplaꢀ
sia in AOMꢀDSS 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 AOMꢀDSS mice. Treatment with 1 improved dysplasia. Bars inserted indicate magnification (50 ꢁm). (ILꢀ
1β, ILꢀ6) Immunohistochemistry staining of ILꢀ6 and ILꢀ1β in indicated treatment. 1 suppressed inflammation factor in the AOM/DSS
model. Note the lower ILꢀ6 and ILꢀ1β staining in the colorectal crypts of animals treated with AOMꢀDSS+1 compared with AOMꢀDSS
alone. Bars inserted indicate magnification (50 ꢁm). (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 ꢁm).
The inhibitory effect of 1 on the over expression of ILꢀ6 and ILꢀ1β in AOMꢀDSS mouse models. We evaluated
inflammatory inhibition by ILꢀ6 and ILꢀ1β immunostaining after the treatment of 1 in AOMꢀDSS models. There was an
increased expression of inflammatory cytokines ILꢀ6 and ILꢀ1β in the group treated with AOMꢀDSS, 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 3ILꢀ1β, ILꢀ6 and Figure 4).
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Journal of Medicinal Chemistry
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Figure 4. Quantification of ILꢀ1β, ILꢀ6 and Nrf2. *p < 0.05, statistically significant difference from the AOMꢀDSS group.
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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 AOMꢀDSS 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 AOMꢀDSS+1 group, a significantly
increased Nrf2 translocation into the nuclei was observed in surrounding tissues compared to the AOMꢀDSS group.
This phenomenon indicated that 1 promoted Nrf2 activation to decrease the damage and slow down the development of
inflammationꢀassociated cancer in chronic inflammatory environment (Figure 3 Nrf2 and Figure 4). 45ꢀ48
Preliminary SAR. A series of analogs with different groups in both phenyl rings were tested at 0.01, 0.1, 1, 5, 10
ꢁM for their ability to induce ARE activity in HepG2ꢀAREꢀC8 cells (12 h treatments) (Table2). The optimal compound
was 17 with a 33ꢀfold 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 HOꢀ1, NQOꢀ1 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
aboveꢀmentioned compound, with metaꢀF substitution, was significantly more effective than 1 in ARE activity. It is
suggested that the bulk of ꢀF is similar with hydrogen (H). Moreover, electronꢀwithdrawing 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 sixꢀbond separation between carbonyl and ꢀF was crucial for the inducꢀ
tion activity. Also, the IC50 value showed that derivatives were not cytotoxic in administrated concentration (The
IC50value of most compounds is more than 100 ꢁM). When substituted with ꢀCF3 (39), 3,4ꢀF (10)and3,5ꢀF (40), the
activity decreased. It is considered that monoꢀfluoro introduction on ring B is enough. Similarly, with chloro (ꢀCl),
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methoxyl (ꢀOCH3) and nitro (ꢀNO2) substitution (3, 9 and 12) at the meta position, the activity was obviously predomiꢀ
nant compared to other positional substituted analogs (2, 4, 5, 18, 6, 16). With ꢀOCH3 and ꢀNO2 substitution, the simiꢀ
lar 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) subꢀ
stitution (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. Interestꢀ
ingly, 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 substiꢀ
tuted groups. When substituted with 3ꢀOCH3 and 2ꢀOCH3, the inductivity became lower. In general, 2ꢀOCH3 and 3ꢀ
OCH3 substitution were pessimistic compared to 4ꢀOCH3 substitution, which indicated substituted position was imꢀ
portant. However, the introduction of 3,4ꢀOCH3 significantly abolished the activity, indicating that introduction of diꢀ
methoxy completely destroyed a certain mode of action of target binding with this scaffold. It is thought that the subꢀ
stituent is small and electronꢀdonating property is nonessential. So the ꢀF effect on ring B was not the only key deterꢀ
minant 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 >25ꢀfold induction of ARE at 10 ꢁM can serve as ideal lead compounds for further
optimization.
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Table 2. Induction of ARE activity in HepG2ꢀAREꢀC8 cells and EC50 value measured in HCT116 cells.
Compound /Vehicle Relative Luciferase Units (ꢁM) a
Compd. ID
R1
R
EC50 (ꢁM)
0.01
2.00±0.21 5.44±1.21 9.51±0.14 18.87±0.11 26.87±0.03
1.19±0.14 2.19±1.09 2.77±1.07 5.46±1.13 6.04±1.36
1.94±0.08 3.77±1.28 10.67±0.39 13.98±1.93 16.50±1.63
0.1
1
5
10
H
H
H
H
H
H
H
H
H
H
H
H
2ꢀCl
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
1
2
3ꢀCl
3
4ꢀCl
1.30±0.42 2.33±0.87 2.61±0.56
1.15±0.07 2.30±1.55 2.74±1.21
1.20±0.14 2.34±0.56 1.76±0.34
3.57±1.62
3.30±1.84
2.43±1.16
4.00±1.41
5.07±1.51
3.81±0.98
4
4ꢀOCH3
4ꢀNO2
4ꢀF
5
6
2.58±1.39 4.39±0.62 8.00±1.27 12.31±1.56 14.01±2.84
2.50±0.71 3.64±1.74 8.85±1.80 16.51±0.71 16.92±0.40
7
2ꢀF
8
3ꢀOCH3
3,4ꢀF
2,4ꢀCl
1.48±0.54 3.69±1.86 5.68±1.97
2.12±0.64 2.25±1.09 3.23±1.48
1.68±0.17 1.10±0.28 1.42±0.62
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9
5.47±1.94
1.78±0.04
6.38±1.80
2.49±1.53
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Table 1. Continued
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H
3ꢀNO2
4ꢀCH3
4ꢀph
3,4ꢀOCH3
2ꢀNO2
3ꢀF
3.28±1.81 3.52±1.14 7.22±1.90 14.50±0.14 18.54±2.18
>100
>100
>100
12
H
2.01±0.01 3.30±1.25 3.25±1.90
1.62±0.54 1.67±0.79 1.41±1.10
1.52±0.30 1.77±0.69 1.93±1.48
1.28±0.06 1.29±0.40 1.30±0.52
4.42±2.09
1.69±0.77
2.11±1.32
2.41±1.22
5.22±2.46
2.09±0.12
13
H
14
5
6
7
8
H
2.15±1.34 93.02±7.12
15
H
2.80±1.10
>100
>100
>100
>100
>100
>100
>100
>100
>100
16
H
2.18±0.58 3.89±0.69 9.99±3.10 29.06±0.49 33.00±0.49
17
H
0.96±0.38
1.35±0.64
2.26±1.19
1.44±0.04
1.45±0.49
2ꢀOCH3
H
1.68±1.14 1.51±0.11
1.78±0.27
1.99±0.85
18
9
10
11
12
13
14
15
16
17
18
19
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
10ꢀOCH3
10ꢀOCH3
10ꢀOCH3
10ꢀOCH3
10ꢀOCH3
10ꢀOCH3
10ꢀOCH3
9,10ꢀOCH3
9,10ꢀOCH3
9,10ꢀOCH3
9,10ꢀOCH3
9,10ꢀOCH3
9,10ꢀOCH3
1.93±0.30 3.50±0.86 10.43±1.12 12.99±1.35
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
3ꢀCl
2ꢀF
2.28±0.40 4.53±0.78
2.75±1.39 3.44±0.09
1.75±0.40 3.08±1.32
6.49±1.34
7.28±1.85
9.84±1.36 12.44±2.55
3ꢀNO2
3ꢀOCH3
3ꢀF
5.76±1.85
1.36±0.01
6.50±2.45
1.59±0.44
1.08±0.45 1.21±0.01 1.24±0.02
1.31±0.29
1.29±0.41
0.86±0.25
1.41±0.58
1.07±0.27
1.31±0.11
1.45±0.49
2.31±0.38
3.93±0.53 7.23±2.77 16.93±2.54 20.38±2.18
4ꢀF
1.35±0.35 1.74±0.14
0.85±0.40 1.35±0.14
1.47±0.66 1.07±0.21
0.80±0.16 0.95±0.14
1.08±0.11 1.03±0.04
1.18±0.24 0.95±0.33
0.93±0.27 1.05±0.36
1.82±0.32
1.46±0.25
1.15±0.29
0.97±0.13
1.44±0.45
1.11±0.24
1.68±0.10
1.82±0.53
6.89±0.51
4.85±1.07
5.89±2.36
8.18±1.90
5.65±2.18
5.18±3.10
3.14±0.18
5.22±0.38
2.48±0.14 87.95±6.05
H
1.67±.71
>100
>100
3ꢀCl
2ꢀF
1.44±0.49
1.10±0.15 75.85±8.11
2.51±0.56 49.61±2.53
3ꢀNO2
3ꢀOCH3
3ꢀF
1.06±0.03
>100
1.09±0.13 92.00±8.34
1.25±0.46 81.54±6.31
6.93±0.73 58.54±5.61
9,10ꢀOCH3
8ꢀOCH3
4ꢀF
1.25±0.61 1.42±0.35 2.70±0.43
1.00±0.40 3.53±0.44 3.57±0.80
0.79±0.05 1.43±0.28 4.02±1.48
1.93±1.02 2.83±0.49 2.98±1.38
0.65±0.13 1.70±0.44 3.80±1.23
1.83±0.03 5.19±0.10 3.96±1.90
2.58±1.33 1.45±0.64 3.73±2.48
2.22±0.20 1.70±0.21 1.76±0.09
1.34±0.49 2.92±1.25 4.75±1.71
H
8ꢀOCH3
8ꢀOCH3
9ꢀOCH3
9ꢀOCH3
9ꢀOCH3
H
3ꢀF
3.70±1.11
4.26±0.11
5.36±1.49
6.66±1.20
7.70±3.48
3.25±0.75
>100
>100
>100
>100
>100
>100
2ꢀF
H
3ꢀF
2ꢀF
3ꢀCF3
3,5ꢀF
3ꢀBr
H
7.62±2.28 58.86±6.48
H
3.10±0.21 3.40±1.49 8.97±2.61 11.99±0.04 15.73±5.49 59.12±3.63
1ꢁM
5ꢁM
10ꢁM
12.5ꢁM
20ꢁM
SFN
19.08±1.54
2.7±1.30 5.52±0.23 8.35±0.96 11.05±1.56 18.10±2.45
5ꢁM
10ꢁM
20ꢁM
40ꢁM
80ꢁM
tBHQ
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 relaꢀ
tive luciferase units.
Figure 5. The fold of Nrf2ꢀdependent AREꢀregulated activity compared to control. *p < 0.05, **p < 0.01, ***p < 0.001, statistically sigꢀ
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nificant difference from the nonꢀtreated group.
1
2
3
4
5
6
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8
9
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14
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17
18
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Figure 6. 17 Induces expression of cytoprotective enzymes and nuclear translocation of Nrf2. (A) At indicated concentration (10 ꢁM), cell
lysates were prepared fromHCT116 cells and subjected to Western blot for timeꢀcourse analysis. (B) Effects of 17on nuclear translocation
of Nrf2. At indicated times after treatment with 17 (10 ꢁM), nuclear fractions and cytoplasm extracts were prepared from HCT116 cells
and subjected to Western blot analysis for Nrf2, histone and βꢀactin. HOꢀ1, NQOꢀ1 and cytoplasmic Nrf2 expression used βꢀactin for norꢀ
malization. Nuclear Nrf2 used histone for normalization
nonꢀtreated group.
. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant difference from the
ꢀOCH3 substitution is less active.
3,4ꢀOCH3 substitution abolishs the
ARE inductivity.
A smaller bulk
substitution is
reasonable.
N
O
The electrostatic state is
not responsible for higher
activity.
6
ꢀ
b
N
ꢀ
C
o
.
n
H
=
O
Inductivity decreases
with increased bulk
introduction.
d
s
e
O
a
p
n
e
d
r
ꢀ
a
e
F
t
i
o
i
oꢀ and pꢀsustitution are
F less active.
mꢀF (H) substitution is
the most active.
s
n
b
b
(H)
e
s
t
t
w
.
e
e
n
Figure 7. SAR of compounds 1ꢀ41.
DISCUSSION
Dietary administration with chemopreventive agents could inhibit oncogenesis in inflammationꢀassociated cancer
mouse models. Inflammatory environment may promote some signaling pathways to be aberrantly activated, and inꢀ
creasing targets required in cancer promotion were then further addressed.49ꢀ51 Here we used AOMꢀDSS models to inꢀ
vestigate thepreventive effects of 1 on oncogenesis and its potential application for inhibiting colorectal cancer develꢀ
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Journal of Medicinal Chemistry
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.
1
2
3
4
5
6
7
8
9
It is generally believed that boosting the Nrf2ꢀdependent 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.52ꢀ54 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 pheꢀ
nomenon 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.
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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 3ꢀ(2ꢀoxoꢀ2ꢀphenylethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ
1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone structure, which were never reported before. The compounds were efficaꢀ
cious 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 (AIꢀ1),7
resveratrol, genistein. The ARE enhanced effect was also significantly higher than those of tBHQ and SFN, wellꢀ
known positive control of Nrf2/ARE. It is worthy of being mentioned that terpenoids which are known to activate Nrf2
at nanomolar concentrations. The compound CDDOꢀIM, 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 correꢀ
sponding mechanisms are disruption of the Keap1ꢀCul3 interaction and a conformational change in Keap1 to change
the Keap1ꢀNrf2 interaction. It is suggested that electrophiles prefer some specific cysteine residues in Keap1 and exꢀ
hibit a particular mode of cysteine modification. Also, it is speculated that a binding pocket exists in Keap1 and it conꢀ
tains above important cysteines to covalently bind with the target molecule. As the pocket is spatially small, introducꢀ
tion 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 strucꢀ
ture modification and activity improvement in the future. Another common and interesting phenomenon is that they
induce HOꢀ1 much more responsive than NQOꢀ1. 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 tranꢀ
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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.
1
2
3
4
5
6
7
CONCLUSIONS
8
9
We have identified compound 1 with a novel structure as an ARE inducer via screening an inꢀhouse database using the
luciferase reporter assay. The inductivity was excellent compared to previously reported compounds. Human cancer
cellꢀbased and AOMꢀDSS mouse models were used to demonstrate that 1 was a potent activator of Nrf2/ARE signaling
pathway. Furthermore, 1 significantly suppressed the development of inflammationꢀassociated 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 comꢀ
pound 17 with the most potency to enhance the ARE levels. This compound can be ideal lead for further optimization.
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60
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 thinꢀlayer
chromatography (TLC) on 0.25 mm silica gel plates (GF254) and visualized under UV light. Melting points were deꢀ
termined with a MeltꢀTemp II apparatus. IR spectra were recorded on a Nicolet iS10 Avatar FTꢀIR spectrometer using
1
KBr film. The H NMR and 13C NMR spectra were measured on a Bruker AVꢀ300 instrument using deuterated solꢀ
vents with tetramethylsilane (TMS) as internal standard. ESIꢀmass and high resolution mass spectra (HRMS) were recꢀ
orded on a Water QꢀTof 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 ꢁm) column using a mixture of solvent methanol/water or acetoniꢀ
trile/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 iceꢀbath, 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 94.6ꢀ
98.5 %.
43 (39.06 mmol) and phthalimide potassium (10.85 g, 58.6 mmol) was added into a solution of DMF (50 mL), and
o
kept 90 C for 12 h. After cooling, the mixture was poured to water (200 mL). The precipitate was filtered and dried.
Yield 94.1ꢀ98.1 %.
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Journal of Medicinal Chemistry
P2O5 (18.78 g, 132.3 mmol) and 44 (36.7 mmol) was added into a solution of refluxed CH3CN (200 mL), and anothꢀ
er 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 NH3ꢂH2O, filtered and recrystallized from THF.
Yield 82.0ꢀ84.1 %.
1
2
3
4
5
6
7
8
9
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 dichloroꢀ
methane (200 mL), remove inorganic salt, reduce to dryness and crystallized from ethyl acetate. Yield 74.1ꢀ75.3%.
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 73.6ꢀ75.2 %.
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49
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59
60
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 preꢀ
cipitate was filtered to afford the respective product 49. Yield 60.1ꢀ97.9%.
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 44.2ꢀ85.6 %.
1 (5 g) was synthesized on a large scale for dietary administration of AOMꢀDSS mice.
3ꢀ(2ꢀoxoꢀ2ꢀphenylethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (1). Yield 25.6 %; Yelꢀ
low solid; m.p. 203ꢀ204 oC; 1HNMR (300 MHz, DMSO) δ 10.84 (d, J = 4.68 Hz, 1H), 7.89ꢀ7.87 (m, 2H),7.86ꢀ7.45 (m,
3H), 7.33ꢀ7.24 (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), 3.00ꢀ2.86 (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 (cmꢀ1, 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 %.
3ꢀ(2ꢀ(2ꢀchlorophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(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), 7.48ꢀ7.41 (m, 4H),7.30ꢀ
7.24 (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,
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1H), 3.26 (t, J = 12.43 Hz, 1H), 2.50ꢀ2.47 (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 (cmꢀ1, 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%.
1
2
3
4
5
6
7
8
9
3ꢀ(2ꢀ(3ꢀchlorophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (3). Yield
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13
14
15
16
17
18
19
20
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23
24
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26
27
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29
30
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43
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48
49
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59
60
22.1 %; Yellow solid; m.p. 183ꢀ185 oC; 1HNMR (300 MHz, DMSO) δ 10.89 (d, J = 4.59 Hz, 1H), 7.85ꢀ7.83 (m, 2H),
7.62ꢀ7.58 (m, 1Н), 7.52 (t, J = 5.97 Hz, 1H), 7.30ꢀ7.25 (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), 2.98ꢀ2.87 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(4ꢀchlorophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (4). Yield
23.0 %; Yellow solid; m.p. 210ꢀ211 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), 7.30ꢀ7.24 (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), 2.93ꢀ2.86 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(4ꢀmethoxyphenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (5). Yield
21.0 %; Yellow solid; m.p. 187ꢀ188 oC; 1HNMR (300 MHz, DMSO) δ 10.72 (d, J = 4.53 Hz, 1H), 7.85 (d, J = 8.79 Hz,
2H), 7.32ꢀ7.24 (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), 2.93ꢀ2.85 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(4ꢀnitrophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(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), 7.32ꢀ7.25 (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), 2.98ꢀ2.87 (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,
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Journal of Medicinal Chemistry
123.82, 89.94, 53.24, 45.37, 38.50, 28.33; IR (cmꢀ1, 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%.
3ꢀ(2ꢀ(4ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (7). Yield
24.0 %; Yellow solid; m.p. 210ꢀ212 oC; 1HNMR (300 MHz, DMSO) δ 10.82 (d, J = 4.77 Hz, 1H), 7.97ꢀ7.92 (m,
2H),7.32ꢀ7.10 (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), 3.02ꢀ2.73 (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 (cmꢀ1, 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%.
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3ꢀ(2ꢀ(2ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (8). Yield
27.0 %; Yellow solid; m.p. 199ꢀ201 oC; 1HNMR (300 MHz, DMSO) δ 10.84 (d, J = 4.95 Hz, 1H), 7.73 (m, appar t,
1H),7.56ꢀ7.51 (m, 1H),7.33ꢀ7.25 (m, 6H),7.30ꢀ7.24 (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), 2.94ꢀ2.87 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(3ꢀmethoxyphenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (9). Yield
26.4 %; Yellow solid; m.p. 159ꢀ161 oC; 1HNMR (300 MHz, DMSO) δ 10.83 (d, J = 4.31 Hz, 1H), 7.47ꢀ7.24 (m,
7H),7.12ꢀ7.11 (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), 2.93ꢀ2.86 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(3,4ꢀdifluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (10). Yield
16.4 %; Yellow solid; m.p. 182ꢀ183 oC; 1HNMR (300 MHz, DMSO) δ 10.89 (d, J = 3.57 Hz, 1H), 7.90ꢀ7.77 (m,
2H),7.58ꢀ7.49 (m, appar q, 1H), 7.31ꢀ7.26 (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), 3.02ꢀ2.87 (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,
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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 (cmꢀ1, 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%.
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Yield 28.5 %; Yellow solid; m.p. 209ꢀ210 oC; 1HNMR (300 MHz, DMSO) δ 10.70 (d, J = 4.83 Hz, 1H), 7.68 (m, apꢀ
par d, 1H), 7.51 (m, appar t, 2Н), 7.32ꢀ7.25 (m, 4H), 6.11 (s, 1H), 5.13(q, J1= 8.31 Hz, J2= 3.72 Hz, 1H), 4.60 (m, apꢀ
par q, 1H), 4.03 (m, 1H), 3.24 (t, J = 12.75 Hz, 1H), 2.97ꢀ2.87 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(3ꢀnitrophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(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),
8.40ꢀ8.32 (m, 2Н), 7.79 (t, J = 7.95 Hz, 1H), 7.32ꢀ7.26 (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), 3.00ꢀ2.88 (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 (cmꢀ1, 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 %.
3ꢀ(2ꢀoxoꢀ2ꢀ(pꢀtolyl)ethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (13). Yield 22.8 %;
Yellow solid; m.p. 205ꢀ206 oC; 1HNMR (300 MHz, DMSO) δ 10.80 (m, appar d, 1H), 7.80ꢀ7.77 (m, 2H),7.30ꢀ7.26 (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), 3.01ꢀ2.86 (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 (cmꢀ1, 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%.
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3ꢀ(2ꢀ([1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(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), 7.33ꢀ
7.27 (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), 3.00ꢀ2.88 (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,
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Journal of Medicinal Chemistry
45.31, 38.48, 28.38; IR (cmꢀ1, 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%.
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Yield 24.6 %; Yellow solid; m.p. 173ꢀ175 oC; 1HNMR (300 MHz, DMSO) δ 10.73 (m, appar d, 1H), 7.53 (m, appar d,
1H), 7.43 (s, 1Н), 7.32ꢀ7.26 (m, 4H), 7.05ꢀ7.03 (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), 2.94ꢀ2.87 (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 (cmꢀ1, 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 %.
3ꢀ(2ꢀ(2ꢀnitrophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (16). Yield
25.5 %; Yellow solid; m.p. 194ꢀ195 oC; 1HNMR (300 MHz, DMSO) δ 10.62 (d, J = 4.41 Hz, 1H), 7.93ꢀ7.91 (m, 1H),
7.79ꢀ7.66 (m, 3Н), 7.30ꢀ7.26 (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), 3.24ꢀ2.87 (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 (cmꢀ1, 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 %.
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21.2 %; Yellow solid; m.p. 199ꢀ200 oC; 1HNMR (300 MHz, DMSO) δ 10.91 (s, 1H), 7.74 (m, appar t, 1H), 7.62ꢀ7.51
(m, 2Н), 7.39 (t, J = 8.13 Hz, 1H), 7.31ꢀ7.27 (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), 2.99ꢀ2.87 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(2ꢀmethoxyphenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(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), 7.51ꢀ7.40 (m, 2H),
7.31ꢀ7.24 (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), 2.95ꢀ2.85 (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 (cmꢀ1, KBr film): 3131(ꢀNH), 1661 (ꢀCON), 1609 (ꢀCO);
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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%.
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24.4 %; Yellow solid; m.p. 188ꢀ190 oC; 1HNMR (300 MHz, DMSO) δ 10.85 (d, J = 4.80 Hz, 1H), 7.87 (t, J = 6.48 Hz,
2H), 7.53ꢀ7.45 (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), 2.93ꢀ2.77 (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 (cmꢀ1, 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%.
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(20). Yield 26.8 %; Yellow solid; m.p. 176ꢀ177 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), 2.95ꢀ2.79
(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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(2ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ10ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone
(21). Yield 23.4 %; Yellow solid; m.p. 178ꢀ179 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),7.32ꢀ7.25 (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), 3.26ꢀ2.77 (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 (cmꢀ1, 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 %.
3ꢀ(2ꢀ(3ꢀnitrophenyl)ꢀ2ꢀoxoethylidene)ꢀ10ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(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),
8.38ꢀ8.32 (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,
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Journal of Medicinal Chemistry
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), 2.93ꢀ2.79 (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 (cmꢀ1, 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%.
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3ꢀ(2ꢀ(3ꢀmethoxyphenyl)ꢀ2ꢀoxoethylidene)ꢀ10ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone
(23). Yield 17.9 %; Yellow solid; m.p. 115ꢀ116 oC; 1HNMR (300 MHz, DMSO) δ 10.83 (d, J = 4.62 Hz, 1H), 7.47ꢀ
7.35 (m, 3H), 7.17ꢀ7.08 (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), 2.96ꢀ2.79 (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 (cmꢀ1, 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 %.
3ꢀ(2ꢀ(3ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ10ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone
(24). Yield 20.1 %; Yellow solid; m.p. 163ꢀ164 oC; 1HNMR (300 MHz, DMSO) δ 10.89 (d, J = 4.74 Hz, 1H), 7.72 (d,
J = 8.07 Hz, 1H),7.63ꢀ7.50 (m, 2H), 7.40ꢀ7.35 (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), 2.97ꢀ2.79 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(4ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ10ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone
(25). Yield 22.8 %; Yellow solid; m.p. 195ꢀ196 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), 2.93ꢀ2.77 (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 (cmꢀ1, KBr film): 3132(ꢀ
NH), 1663(ꢀCON), 1620(ꢀCO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.1489; HPLC (80%
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methanol in water): tR = 7.34 min, 96.97%.
3ꢀ(2ꢀoxoꢀ2ꢀphenylethylidene)ꢀ9,10ꢀdimethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (26).
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Yield 25.6 %; Yellow solid; m.p. 172ꢀ173 oC; 1HNMR (300 MHz, DMSO) δ 10.85 (s, 1H), 7.89ꢀ7.86 (m, appar t, 2H),
7.54ꢀ7.48 (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), 3.14ꢀ2.76 (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 (cmꢀ1, 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%.
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one (27). Yield 25.7 %; Yellow solid; m.p. 176ꢀ177 oC; 1HNMR (300 MHz, DMSO) δ 10.91 (s, 1H), 7.84 (s, 2H),
7.62ꢀ7.52 (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), 2.90ꢀ2.78 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(2ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ9,10ꢀdimethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀ
one (28). Yield 16.5 %; Yellow solid; m.p. 176ꢀ177 oC; 1HNMR (300 MHz, DMSO) δ 10.84 (d, J = 4,23 Hz, 1H), 7.72
(t, J = 7.68 Hz, 1H), 7.54ꢀ7.52 (m, 1H), 7.31ꢀ7.24 (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), 2.88ꢀ2.76 (m, 3H);
13CNMR (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 (cmꢀ1,
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%.
3ꢀ(2ꢀ(3ꢀnitrophenyl)ꢀ2ꢀoxoethylidene)ꢀ9,10ꢀdimethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(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), 2.91ꢀ2.79 (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,
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109.63, 89.31, 55.77, 55.51, 53.16, 45.67, 38.51, 27.85; IR (cmꢀ1, 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%.
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one (30). Yield 17.6 %; Yellow solid; m.p. 173ꢀ175 oC; 1HNMR (300 MHz, DMSO) δ 10.84 (d, J = 4.71 Hz, 1H),
7.46ꢀ7.35 (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), 2.89ꢀ2.76 (m, 3H);
13CNMR (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 (cmꢀ1, KBr film): 3134(ꢀNH),
1662(ꢀCON), 1616(ꢀCO); HRMS (ESI): calcd. for C23H25N2O5 [M + H]+409.1758, found 409.1792;HPLC (80% methꢀ
anol in water): tR = 5.70min, 99.12 %.
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one (31). Yield 20.1 %; Yellow solid; m.p. 200ꢀ201 oC; 1HNMR (300 MHz, DMSO) δ 10.90 (d, J = 4.26 Hz, 1H), 7.72
(d, J = 7.53 Hz, 1H), 7.61ꢀ7.49 (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), 2.93ꢀ
2.72 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(4ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ9,10ꢀdimethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀ
one (32). Yield 20.0 %; Yellow solid; m.p. 210ꢀ211 oC; 1HNMR (300 MHz, DMSO) δ 10.83 (d, J = 4.29 Hz, 1H),
7.97ꢀ7.494 (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), 2.90ꢀ2.76 (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 (cmꢀ1, 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 %.
3ꢀ(2ꢀoxoꢀ2ꢀphenylethylidene)ꢀ8ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (33). Yield
24.3 %; Yellow solid; m.p. 207ꢀ208 oC; 1HNMR (300 MHz, DMSO) δ 10.82 (d, J = 4.56 Hz, 1H), 7.90ꢀ7.87 (m, appar
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Page 22 of 41
d, 2H),7.54ꢀ7.47 (m, 3H), 7.27 (t, J = 7.95 Hz, 1H), 6.93ꢀ6.90 (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), 2.99ꢀ2.83 (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 (cmꢀ1, 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 %.
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3ꢀ(2ꢀ(3ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ8ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(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),7.62ꢀ7.53 (m, appardt, 2H), 7.39ꢀ7.38 (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), 2.99ꢀ2.86 (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 (cmꢀ1, KBr film): 3131(ꢀNH), 1661(ꢀCON),
1610(ꢀCO); HRMS (ESI): calcd. for C21H20FN2O3 [M + H]+ 367.1452, found 367.1459; HPLC (80% methanol in waꢀ
ter): tR = 8.05 min, 95.55 %.
3ꢀ(2ꢀ(2ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ8ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone
(35). Yield 25.0 %; Yellow solid; m.p. 172ꢀ173 oC; 1HNMR (300 MHz, DMSO) δ 10.82 (d, J = 3.93 Hz, 1H), 7.75 (t, J
= 7.68 Hz, 1H),7.59ꢀ7.52 (m, 1H), 7.34ꢀ7.26 (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), 2.99ꢀ2.83 (m, 3H);
13CNMR (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 (cmꢀ1, 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%.
3ꢀ(2ꢀoxoꢀ2ꢀphenylethylidene)ꢀ9ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (36). Yield
21.3 %; Yellow solid; m.p. 173ꢀ174 oC; 1HNMR (300 MHz, DMSO) δ 10.85 (d, J = 4.59 Hz, 1H), 7.89 (m, appar t,
2H),7.55ꢀ7.47 (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), 3.02ꢀ2.86 (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 (cmꢀ1, KBr film): 3132 (ꢀNH),
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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%.
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(37). Yield 22.3 %; Yellow solid; m.p. 166ꢀ167 oC; 1HNMR (300 MHz, DMSO) δ 10.89 (d, J = 4.86 Hz, 1H), 7.74 (d,
J = 7.77 Hz, 1H), 7.63ꢀ7.52 (m, 2H), 7.42ꢀ7.36 (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), 2.99ꢀ2.86 (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
(cmꢀ1, 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%.
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3ꢀ(2ꢀ(2ꢀfluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ9ꢀmethoxyꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone
(38). Yield 20.0 %; Yellow solid; m.p. 165ꢀ167 oC; 1HNMR (300 MHz, DMSO) δ 10.81 (d, J = 4.26 Hz, 1H), 7.75ꢀ
7.70 (m, 1H), 7.54ꢀ7.50 (m, 1H),7.32ꢀ7.24 (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), 2.96ꢀ2.83 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀoxoꢀ2ꢀ(4ꢀ(trifluoromethyl)phenyl)ethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(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), 8.20ꢀ8.04 (m,
2H), 7.91ꢀ 7.71 (m, 2H),7.31ꢀ7.25 (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), 3.04ꢀ2.89 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(3,5ꢀdifluorophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (40). Yield
26.5 %; Yellow solid; m.p. 176ꢀ177 oC; 1HNMR (300 MHz, DMSO) δ 10.95 (d, J = 5.01 Hz, 1H), 7.54ꢀ7.52 (m, appar
d, 2H), 7.44 (d, J = 8.97 Hz, 1H), 7.31ꢀ 7.27 (m, 4H), 6.52 (s, 1H), 5.12 (q, J1= 8.37 Hz, J2= 3.69 Hz, 1H), 4.53(q, J1=
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8.10 Hz, J2= 4.26 Hz, 1H), 4.06 (m, 1H), 3.30 (t, J = 12.21 Hz, 1H), 3.05ꢀ2.84 (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 (cmꢀ1, 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%.
3ꢀ(2ꢀ(3ꢀbromophenyl)ꢀ2ꢀoxoethylidene)ꢀ2,3,6,7ꢀtetrahydroꢀ1Hꢀpyrazino[2,1ꢀa]isoquinolinꢀ4(11bH)ꢀone (41). Yield
26.1 %; Yellow solid; m.p. 185ꢀ187 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), 7.32ꢀ 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), 3.05ꢀ2.89
(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 (cmꢀ1, 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 %.
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Biology. Cell culture conditions. HepG2 cells stably transfected with a luciferase reporter (HepG2ꢀAREꢀC8) were
kindly provided by Professor Dr. A. N. Tony Kong (Rutgers University, Piscataway, NJ). Cells were maintained in
modified RPMIꢀ1640 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 Instiꢀ
tute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences)
were cultured in McCoy's 5A (SigmaꢀAldrich, USA) supplemented with 10% (v/v) FBS.
MTT analysis. Cell viabilities were determined by using MTT (3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazoliumbromide) 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 96ꢀwell 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 ꢁL of MTT solution was added into 96ꢀwell
plates and incubated for an additional 4 h. Then the solution was removed and 100 ꢁL 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 %.
AREꢀluciferase activity assay. HepG2ꢀAREꢀC8 cells were plated in 96ꢀwell 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
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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 ꢁL of cold PBS was added into each well. Then the cells were
harvested in the luciferase cell culture lysis reagent. After centrifugation, 20 ꢁL of the supernatant was used for deterꢀ
mining 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 tripliꢀ
cates and expressed as fold induction over control.
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Western Blot. AntiꢀHOꢀ1 (scꢀ136960) and ꢀNQO1 (scꢀ271116) antibodies were from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Antiꢀβꢀactin (AP0060) and ꢀNrf2 (BS1258) were purchased from Bioworlde (Bioworlde,
USA). HCT116 Cells (5 × 106 cells/well) were washed once with iceꢀcold PBS and driven down with 2mL of EDTA.
Cells were centrifuged at 2500 rpm and resuspended in 45 ꢁL of lysis buffer, which was composed of 50 mMTris–HCl,
150 mMNaCl, NPꢀ40, 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 nuclearꢀcytosolextraction kit (KeyGEN, NJ, China). Then the separate exꢀ
tracts 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 laꢀ
beled secondary antibody at 37 ºC for 1 h. The membranes were screened through the odyssey Infrared Imaging Sysꢀ
tem (LIꢀCOR, Lincoln, Nebraska, USA).
AOMꢀDSS 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 (SigmaꢀAldrich, 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 36ꢀ50kDa)
(MP Biomedicals, Morgan Irvine, CA) was dissolved at 2.5 % with distilled water. Female C57BL/6 mice (6ꢀ8 weeks
old) were divided into 3 groups in random: control group, AOMꢀDSS group, and AOMꢀDSS+1 (50mg/kg/day, supꢀ
plemented 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 immunofluoresꢀ
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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% neutralꢀbuffered formalin
for 48 h, and embedded in paraffin block. Sections of paraffinꢀembedded tissues were subjected to H&E (KeyGEN, NJ,
China) staining for evaluating the severity of inflammation.
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Immunohistochemistry. Tissues were prepared from formalinꢀfixed, paraffinꢀembedded colon tissue. Stains against
ILꢀ1β and ILꢀ6 (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 antiꢀrabbit antibodies were added and incubated at room temperaꢀ
ture for 30 min. StreptavidinꢀHRP was added, and after 40 min the sections were stained with DAB substrate and counꢀ
terstained with hematoxylin. Image Pro Plus 6.0 software (Media Cybernetics, Silver Spring, USA) was used for autoꢀ
matic counting of labeled inflammatory cytokines to quantify the experimental data.
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Immunofluorescence. Fourꢀmicron 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 FITCꢀlabeled secondary sheep antiꢀrabbit IgG antibody (BOSTER, WH, China), then stained with fluoꢀ
rochrome 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 aboveꢀmentioned immunohistochemistry.
Statistical analysis. All results are given as mean ± SD performed in parallel experiments for triplicate. Pꢀvalues
less than 0.05 (p <0.05) were considered statistically significant. They were undertaken using the GraphPad Prism
software (GraphPad Software Inc., Avenida, CA).
ACKNOWLEDGMENTS
This work was supported by the project 81230078 (key program), 81202463 (youth foundation), 91129732 and
81173087 of National Natural Science Foundation of China, Program of State Key Laboratory of Natural Medicines,
China Pharmaceutical University (No. JKGQ201103, No. ZJ11173), 2012AA020301 of 863 program, 2010ZX09401ꢀ
401 and 2009ZX09501ꢀ003 of the National Major Science and Technology Project of China (Innovation and Develꢀ
opment of New Drugs). The authors declare no other conflicts of interest.
ASSOCIATED CONTENT
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Supporting Information
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Spectral data for characterization.
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AUTHOR INFORMATION
Corresponding Author
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*H.S.: fax and phone, +86ꢀ25ꢀ83271216;eꢀmail, sunhaopeng@163.com. R.H.: fax and phone, +86ꢀ25ꢀ83271126; eꢀ
mail:michelhu@hotmail.com. Q.Y.: fax and phone, +86ꢀ25ꢀ83271351; eꢀmail, youqidong@gmail.com.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
AOMꢀDSS, azoxymethaneꢀ dextran sodium sulfate; Nrf2, Nuclear factor erythroid 2 p45ꢀrelated factor 2; ARE, antioxꢀ
idant response element; GST, glutathione Sꢀtransferase; NQOꢀ1, NAD(P)H:quinone oxidoreductaseꢀ1; HOꢀ1, heme
oxygenaseꢀ1; HCT116, human colorectal cancer cells; IL, interleukin; SAR, structureꢀactivity relationship; bZIP, basicꢀ
leucine zipper; CNC, Cap ‘n’ Collar; Keap1, Kelchꢀlike ECHꢀassociated protein 1; CAPE,caffeic acid phenethyl ester;
CRC, colorectal cancer; tBHQ, tertꢀbutylhydroquinone; SFN, Sulforaphane; WBC, White Blood Cell; NR, Neutrophils Raꢀ
tio;LR, Lymphocyte Ratio; H & E, hematoxylin&eosinstaining; MTT, 3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazoliumomide.
REFERENCES
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