Tetrazanbigen Derivatives as Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) Partial Agonists: Design, Synthesis, Structure-Activity Relationship, and Anticancer Activities

Article abstract of DOI:10.1021/acs.jmedchem.0c01512

Tetrazanbigen (TNBG) is a novel sterol isoquinoline derivative with poor water solubility and moderate inhibitory effects on human cancer cell lines via lipoapoptosis induction. Herein, we developed a series of novel TNBG analogues with improved water sol

Full text of DOI:10.1021/acs.jmedchem.0c01512

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Tetrazanbigen Derivatives as Peroxisome Proliferator-Activated
Receptor Gamma (PPARγ) Partial Agonists: Design, Synthesis,
Structure−Activity Relationship, and Anticancer Activities
Linling Gan,# Zongjie Gan,# Yanrong Dan, Yaowei Li, Peiming Zhang, Shanwen Chen, Zaijun Ye,
Tao Pan, Chunmei Wan, Xuelian Hu, and Yu Yu*
Cite This: J. Med. Chem. 2021, 64, 1018−1036
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ABSTRACT: Tetrazanbigen (TNBG) is a novel sterol isoquinoline derivative with poor water solubility and moderate inhibitory
effects on human cancer cell lines via lipoapoptosis induction. Herein, we developed a series of novel TNBG analogues with
improved water solubility and antiproliferative activities. The CCK-8 assay enabled us to identify a novel compound, 14g, which
strongly inhibited HepG2 and A549 cell growth with IC50 values of 0.54 and 0.47 μM, respectively. The anticancer effects might be
explained by the partial activation and upregulation of PPARγ expression, as indicated by the transactivation assay and western
blotting evaluation. Furthermore, the in vitro antiproliferative activity was verified in an in vivo xenograft model in which 14g strongly
reduced tumor growth at a dose of 10 mg/kg. In line with these positive observations, 14g exhibited an excellent water solubility of
31.4 mg/mL, which was more than 1000-fold higher than that of TNBG (4 μg/mL). Together, these results suggest that 14g is a
promising anticancer therapeutic that deserves further investigation.
specificity in action and show unfavorable toxicity profiles,
such as the weight loss and decreased food intake, which
prevents them from advancing to clinical use.17
INTRODUCTION
■
Lipid metabolism is involved in many cellular processes,
including cell growth, proliferation, differentiation, survival,
and membrane homeostasis, and it plays an essential role in
cancer metabolism.1,2 Compared with normal cells, most
cancer cells exhibit dysregulated lipid metabolism, which
provides cancer cells with macromolecular building blocks,
signaling lipid molecules, posttranslational modifications of
proteins, and energy supply to support rapid cell proliferation
and survival.3,4 Therefore, targeting the modulation of lipid
metabolism in cancer cells is believed to be a promising
strategy for cancer treatment.5−10
The reduction of de novo lipogenesis in cancer cells is an
emerging strategy to combat cancer.11,12 To this end, several
inhibitors and/or drugs that target a variety of enzymes
involved in de novo lipogenesis have been produced, including
acetyl CoA carboxylase inhibitors (ND-654),13 fatty acid
synthase inhibitors (Orlistat and TVB-2640),14,15 and
carnitine palmitoyltransferase 1 inhibitor (Etomoxir)16
(Figure 1). However, these lipogenesis inhibitors usually lack
In addition to the advanced development of lipogenesis
inhibitors, lipoapoptosis induction in tumor cells is another
attractive strategy for cancer treatment.18,19 Excess lipid
accumulation affects fundamental cellular processes by
disturbing the utilization of lipids in cancer cells,20 and
unlimited accumulation of lipid droplets would also occupy
most of the space in the cytoplasm and interfere with the
functions of other organelles, leading to lipoapoptosis of
differentiated cancer cells.21−23
Received: August 30, 2020
Published: January 11, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c01512
© 2021 American Chemical Society
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Figure 1. Chemical structures of lipogenesis inhibitors (ND-645, Orlistat, TVB-2640, and Etomoxir) and lipid accumulation inducers
(tetrazanbigen (TNBG) and TNBG-5602).
a
Scheme 1. Synthesis Routes for Compounds 8a−8o
a
Reagents and conditions: (a) ClCH2COCl, K2CO3, CH2Cl2, 0−10 °C; (b) potassium phthalimide, DMF, reflux; (c) P2O5, dimethylbenzene, 140
°C; (d) (CH3COO)3BHNa, CH3OH/CH2Cl2, r.t.; (e) N2H4·H2O, CH3OH, reflux; (f) HCl, C2H5OH, 0 °C; (g) DMF, K2CO3, 2,3-
dichloroquinoxalines (7), 110 °C; (h) 40% HBr reflux; (i) (dppf) PdCl2, K2CO3, dioxane/H2O, 120 °C, microwave.
Peroxisome proliferator-activated receptor gamma (PPARγ),
a member of the nuclear receptor of the superfamily of ligand-
inducible transcription factors,24 has been recognized as a
transcriptional regulator of fat-specific gene expression25 and
plays an important role in the control of lipid uptake, transport,
storage, and disposal in cell types with active lipid
metabolism.26,27 Recently, it has been revealed that some
compounds inhibit the growth and induce differentiation of
malignant cells due to the PPARγ-mediated excessive
accumulation of lipid droplets.28 Similarly, our group
previously reported a novel isoquinoline derivative, TNBG
(Figure 1), which exhibits good inhibitory potencies on many
human cancer cell lines and shows no cross-resistance with
many other antitumor drugs in vitro.29−31 Specifically, TNBG-
induced lipid accumulation in cancer cells, which may be
associated with PPARγ.29 However, the solubility of TNBG
did not meet the criteria as a good candidate compound.
Therefore, we herein report the design, synthesis, and
biological evaluation of novel and potent TNBG analogues
with improved solubility.
RESULTS AND DISCUSSION
■
Rational Design. Introducing hydrophilic substituents into
a core ring is a conventional and effective method for
increasing the water solubility of lead compounds, which has
been widely used in the process of discovering candidates.32,33
For example, irinotecan,34 benotican,35 and topotecan36 were
obtained by introducing dimethylamino, isopropylamino, and
1,4′-bipiperidine groups at the 7, 9, or 10 positions of
camptothecin, respectively, and they all showed a dramatic
increase in water solubility compared to that of the lead
compound.37 Based on the same design strategy, modification
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a
Scheme 2. Synthesis Routes of Compounds 9a−9e
a
Reagents and conditions: (a) DMF, NaH, various chlorinated aliphatic chains, 50 °C.
a
Scheme 3. Synthesis Routes of Compounds 11a−11d, 12a, and 12b
a
Reagents and conditions: (a) LiOH, CH3OH/H2O, 85 °C; (b) DMF, EDCI, HOBT, DIEA, r.t.; (c) LiAlH4, THF, r.t.
of TNBG by incorporating a flexible linker bearing an N,N-
dimethyl moiety into the nucleus led to the discovery of
TNBG-5602 (Figure 1), which demonstrated enhanced
antiproliferative activity and water solubility compared to
TNBG.30,31 Encouraged by this positive outcome, a series of
hydrophilic linkers containing hydroxyl groups, carboxyl
groups, morpholine, piperidine, and piperazine with different
carbon lengths were introduced into the A, C, or E rings of
TNBG with the aim of further improving the antitumor
activity and water solubility of TNBG-5602 in this study.
Moreover, different substituents, including electron-donating
or electron-withdrawing groups, were introduced to the A and
E rings to systemically investigate the structure−activity
relationship (SAR) and pharmacodynamic properties.
Chemistry. All target compounds were prepared according
to the routes outlined in Schemes 1−4. As shown in Scheme 1,
substituted phenethylamines 1a−1e were reacted with
chloroacetyl chloride in CH2Cl2 to afford amides 2a−2e,
which were refluxed with potassium phthalimide in DMF to
give 3a−3e. Compounds 3a−3e underwent a subsequent
cyclization reaction in the presence of phosphorus pentoxide,
affording 4a−4e. Then, the reduction of 4a−4e using sodium
triacetoxyborohydride in CH3OH/CH2Cl2 solution at room
temperature generated 5a−5e, which were subjected to
ammonification and acidification reactions to yield important
intermediates 6a−6e. After treating 6a−6e with 2,3-dichlor-
oquinoxalines38,39 and K2CO3 in DMF, TNBG (8g) and its
analogues 8a−8f and 8h−8o were prepared via the ring
closure reaction. Finally, condensation of TNBG with different
alkyl halides produced target compounds 9a−9e and is shown
in Scheme 2.
In Scheme 3, hydrolyzation of methylacetate 8j produced
carboxylic acid 10, followed by acylation with amines to give
11a−11d. Reduction of the carbonyl group in 11a and 11b
generated target compounds 12a and 12b.
Furthermore, to understand the contribution of the N,N-
dimethylamide substituents on both rings C and E or rings A
and C of TNBG, compounds 13a−13h and 14a−14g were
synthesized. As shown in Scheme 4, TNBG analogues 8b, 8e,
and 8 l were alkylated with various chlorinated aliphatic chains
in dimethylformamide to afford target compounds 13a−13h
and 14a−14g.
Structure−Activity Relationship. All the newly synthe-
sized compounds 8−14 were evaluated for their antiprolifer-
ative activities on human hepatoma cell lines (HepG2 and
QGY-7701), a human colon cancer cell line (LOVO), and a
human lung adenocarcinoma epithelial cell line (A549).
TNBG and the anticancer drugs, Sorafenib and Erlotinib,
were used as positive control drugs in standard CCK-8 assays.
All results of the antiproliferative activities are described in
Table 1.
The present study found that the incorporation of a methoxy
group or hydroxy group (8d or 8e) into TNBG slightly
improved antiproliferative activities against the tested cell lines
compared to TNBG, and the introduction of Cl (8i), F (8f), or
Br (8m) groups into TNBG resulted in decreased inhibitory
potencies, indicating that halogen groups are inferior to this
series of compounds for exerting biological activity. In
addition, the replacement of the Br group in 8m by pyrazole
rings generated 8n and 8o; however, no improvement in
antiproliferative activity was observed.
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a
Scheme 4. Synthesis Routes of TNBG Analogues 13a−13h and 14a−14g
a
Reagents and conditions: (a) DMF, NaH, various chlorinated aliphatic chains, 50 °C. Compounds 14a, 14e, 14f, and 14g were obtained as
hydrochloride salt.
Moreover, according to our previous results, nitrogen atom-
containing alkyl chains in ring C of TNBG-5602 are favorable
for activity improvement, thus various aliphatic chains,
including morpholine, piperidine, and piperazine, with differ-
ent carbon lengths were introduced. Unsurprisingly, the
resulting compounds (9a−9e) demonstrated a 2- to 3-fold
improvement in antiproliferative activities compared to
TNBG. Although the anticancer activities of the carboxyl
analogues (10) and their aminated analogues (11a−11d) were
less potent than TNBG, their reductive compounds 12a and
12b inhibited the proliferation of the tested cancer cells as
potently as TNBG.
Encouraged by the good activities produced by 9a−9e,
compounds containing two side chains were synthesized to
investigate their effects on cytotoxicity potencies. Fortunately,
with the introduction of two alkyl chains into the structure, the
antiproliferative activities were greatly enhanced (13a−13c,
13f−13h, and 14d−14g), except compounds 13d, 13e, and
14a−14b. In particular, 14d and 14g, which had three-carbon
linkers, were identified as the most effective anticancer agents
against HepG2 and A549 cells, and these compounds were
more potent than TNBG and TNBG-5602 by 10- to 30-fold
with IC50 values of 0.42 and 0.54 μM against HepG2 cells and
0.33 and 0.47 μM against A549 cells, respectively. Surprisingly,
the water solubility of 14g was 31.4 mg/mL, which was more
than 1000-fold higher than that of TNBG (4 μg/mL) in a pH
7.4 buffer solution.
Compound 14g Induces Cell Cytoplasmic Vacuola-
tion and Inhibits Cell Colony Formation. Due to the
potent and favorable water-soluble candidates among these
newly synthesized compounds, 14g was selected as a candidate
compound for further testing. First, A549 cells were treated
with 14g (1.6 and 2.0 μM) or the TNBG control (10 μM) for
8, 12, 24, and 48 h, and the morphological changes were then
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Table 1. Biological Activity and Solubility of Target Compounds 8−14
a
b
a
b
IC50 (μM)
solubility
IC50 (μM)
solubility
QGY-
7701
QGY-
7701
comp.
HepG2
>30
>30
LOVO
>30
>30
18.64
3.30
6.044
0.49
A549
>30
>30
2.05
0.38
4.95
0.49
(μg/mL)
comp.
12b
HepG2
5.67
1.06
0.75
0.19
0.65
0.08
0.95
0.10
13.40
1.32
10.97
1.38
0.82
0.09
2.89
0.62
0.88
0.11
12.38
1.21
6.44
0.82
3.95
0.45
0.42
0.14
0.74
0.11
LOVO A549
(μg/mL)
c
8a
8b
8c
>30
>30
8.02
0.77
6.18
1.13
NT
6.72 8.71
12.94
2.03
1.60
0.11
0.53
0.09
0.68
0.07
NT
1.58
2.01
0.76
0.70
0.11
1.11
0.08
NT
1.31
0.98
0.13
0.62
0.12
0.73
0.06
10.40
0.98
NT
NT
13a
13b
13c
13d
13e
13f
<5000
<5000
42.84
NT
7.14
1.26
5.59
0.23
8d
8e
8f
NT
NT
NT
NT
15.4
1.2
14.95
2.87
10.68
1.42
>30
5.05
1.02
12.15
1.72
8.21
0.77
18.68
0.95
11.46
1.19
16.42
0.18
7.62
1.45
9.86
1.16
4.12
0.56
4.23
0.37
5.17
0.41
22.23
3.90
9.7 2.0 8.3 1.0 14.2
2.1
NT
15.24
1.83
21.08
0.76
>30
7.56
1.24
24.91
4.20
5.03
0.87
16.33
2.62
19.07
1.07
13.6
1.96
4.67
0.46
8.74
1.36
5.33
1.26
4.43
0.33
2.92
0.69
24.44
3.19
18.14
3.27
27.53
1.37
25.57
2.35
>30
10.23
1.83
15.07
0.94
>30
5.03
0.69
9.15
1.04
>30
12.02
2.09
9.19
2.12
10.05
2.08
23.68
4.49
>30
NT
12.40
1.02
NT
NT
8h
0.72
0.06
0.66
0.07
0.67
0.22
79.74
384.61
<5000
NT
8i
8j
NT
NT
13g
13h
14a
14b
14c
14d
14e
14f
NT
NT
6.63
1.28
0.16
1.23
0.23
8.36
0.92
1.79
0.16
5.21
0.21
0.33
0.01
0.23
0.01
1.21
0.01
NT
NT
2.27
8k
8l
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
<100
NT
NT
NT
NT
NT
0.89
3.14
0.58
9.58
1.10
0.40
0.09
0.63
0.06
2.91
0.32
0.84
0.12
8.92
1.10
0.23
1.60
0.19
2.96
0.41
0.31
0.032
0.39
0.02
1.25
0.045
1.09
0.06
6.95
0.89
7.58
0.93
8m
8n
>30
NT
22.36
0.40
25.15
0.92
8.40
0.30
20.98
4.46
8.88
0.39
2.99
0.56
1.84
0.09
20.22
1.27
16.16
2.17
10.03
1.34
11.98
1.09
19.20
1.89
NT
8o
25.17
0.16
2.47
0.34
8.52
098
5.20
0.69
3.54
0.76
2.71
0.65
25.38
2.16
17.12
1.90
19.77
1.42
25.21
1.12
17.65
2.12
14.47
1.09
101.38
< 5000
< 5000
9a
9b
1.05
0.17
0.54
0.06
7.81
0.35
9c
d
14g
TNBG
0.47
0.04
9.65
0.56
31421.79
4
9d
9e
TNBG5602 8.79
0.91
Sorafenib
NT
NT
152.95
NT
10
5.06
0.89
10.23
1.01
14.57
1.34
6.83
0.80
11.27
1.08
NT
11a
11b
11c
11d
12a
17.68
1.95
8.65
1.99
12.10
2.62
18.53
1.02
Erlotinib
8.61
0.89
20.86
1.12
NT
13.02
2.33
14.59
2.89
12.75
1.74
10.62
1.80
a
IC50 values were calculated from three independent experiments
using the CCK-8 assay after 72 h of treatment. The values were
reported as the mean SD. The aqueous solubility was measured at
pH 7.4 by a thermodynamic solubility assay. NT indicates not tested.
b
c
d
Tested as hydrochloride salt.
6.48
1.17
14.05
2.56
monitored by a microscope. As shown in Figure 2, extensive
cytoplasmic vacuolation was observed in A549 cells treated
with 14g for 24 or 48 h at both concentrations. Given that the
paraptosis, a form of nonapoptotic cell death characterized by
cytoplasmic vacuolation was involved in the induction of cell
death.40 We hypothesized that the paraptosis induced by 14g
may contribute to the cell death.
In addition, the colony formation of A549 and HepG2 cells
was repressed by compound 14g. As shown in Figure 3, TNBG
partially inhibited A549 cell colony formation at 3.0 μM, while
14g completely prevented colony formation at 0.3 μM. Even at
0.2 μM, 14g reduced most of the colony formation of A549
and HepG2 cells, suggesting that 14g is much more potent
than TNBG. Furthermore, A549 and HepG2 cells incubated
with 14g formed fewer colonies in a dose-dependent manner
than TNBG treatment cells. These results demonstrated that
14g has stronger anticancer activity than TNBG, which was in
agreement with the cytotoxicity assay.
Compound 14g Induces Cell Cycle Arrest and
Apoptosis. To characterize the mode of cell death induced
by compound 14g, we next evaluated the effect of 14g on cell
cycle distribution using a propidium iodide (PI) staining kit.
As shown in Figure 4, 14g led to an accumulation of A549 cells
in the S phase with percentages of 18.56% (0.9 μM), 18.23%
(1.2 μM), and 25.58% (1.5 μM), accompanied by decreases in
the G1 phase. Moreover, a similar trend was also observed in
HepG2 cells, indicating that 14g mildly induces S-phase arrest
in both cell lines.
Subsequently, the apoptosis-inducing ability of compound
14g was examined by the Annexin V-FITC/PI FACS assay in
A549 and HepG2. As shown in Figure 5, the apoptosis ratios of
compound 14g at 0.9, 1.2, and 1.5 μM in A549 cells were 8.87,
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with 14g and stained with Oil Red O to examine its effect on
lipid accumulation. As shown in Figure 6, red lipid droplet
accumulation in the cytoplasm of cancer cells was observed
after treatment with 14g, which was in line with that of TNBG-
5602, as evidenced by Oil Red O staining of the cellular lipid.
Compound 14g Shows a Weak Agonist Activity on
PPARγ. Previous evidence showed that PPARγ plays an
important role in regulating lipid metabolism, and activation of
PPARγ is associated with inhibition of malignant cell
proliferation.41−45 Thus, we evaluated the agonistic activity
of 14g on PPARγ at 1 and 10 μM in luciferase reporter assays,
utilizing PPARγ full agonist rosiglitazone (RSG) and partial
agonist decanoic acid (DA) as positive controls.46,47 Figure 7A
demonstrates that 14g increased PPARγ reporter expression by
2.4-fold at 10 μM, much lower than that of RSG. The dose−
response curves in Figure 7B showed that 14g (EC50 = 0.9
μM) has a weak agonistic activity on the transactivation assay
of PPARγ, which is similar to DA (EC50 = 7.6 μM).
Furthermore, the binding affinity of 14g to PPARγ was also
determined using the LanthaScreen TR-FRET assays. The
calculated inhibition constant (Ki) values of 14g, DA, and RSG
were 8.9 μM, 22 μM, and 17 nM, respectively (Figure 8).
These results support that 14g is a partial agonist of PPARγ.
T0070907 PPARγ Antagonist Partially Reverses the
Antiproliferative Effect of 14g on A549 Cells. To further
explore the relationship between the antiproliferative activity of
14g and PPARγ, a combinational inhibitory test of PPARγ
agonist/antagonist with 14g was conducted. The CCK-8
results revealed that the PPARγ agonist, RSG, substantially
enhanced the activity of 14g, while the PPARγ antagonist,
T0070907, partially reversed the antiproliferative effect of 14g
on A549 cells (Figure 9A). Moreover, the crystal violet assay
results in A549 cells were consistent with the above
experimental results (Figure 9B). All the aforementioned
Figure 2. Growth of A549 cells was monitored by microscopy after
treatment with TNBG at 10 μM or 14g at 1.6 and 2.0 μM for 0, 8, 12,
24, and 48 h.
10.97, and 52.3%, respectively, suggesting the induction of
apoptosis in A549 cells by 14g followed a dose-dependent
manner. For TBNG at 20 μM and Erlortinib at 8 μM, the
percentages were 4.04 and 19.41%, suggesting that the
apoptosis-inducing ability of 14g in A549 was stronger than
TNBG and Erlortinib. It was worth noting that 1.5 μM of 14g
could induce over 25% apoptosis in HepG2 cells, which is
similar to that of 14g in A549 cells.
Compound 14g Induces Lipid Accumulation. Our
previous data demonstrated that TNBG and its analogue
TNBG-5602 could induce lipid accumulation in cancer
cells.29−31 Therefore, A549 and HepG2 cells were treated
Figure 3. Colony formation of A549 and HepG2 cells after treatment with 14g and TNBG. A549 and HepG2 cells (400/well) were cultured with
varying concentrations of 14g at 0.1, 0.2, and 0.3 μM or TNBG at 1, 2 and 3 μM for 3 days. Cells were then imaged after an additional 12 days of
drug-free medium incubation. Bar chart representation of the colonies formed after treatment with 14g and TNBG. Data are shown as the mean
SD of three independent experiments. **P < 0.01 and ***P < 0.005 vs the control group.
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Figure 4. Compound 14g induces cell cycle arrest in A549 and HepG2 cells. Both cell lines were treated with 14g at 0.9, 1.2, and 1.5 μM for 48 h.
Erlotinib and Sorafenib were used as positive controls. Cells were then fixed in ice-cold 70% ethanol at 4 °C and stained with propidium iodide dye
(50 μg/mL), and the cell cycle was evaluated via flow cytometry.
Figure 5. Flow cytometric analysis of A549 and HepG2 cell apoptoses. Both cell lines were treated with 14g, TBNG, Erlotinib, or Sorafenib for 48
h and then stained with a FITC Annexin V/PI kit. Cells in the upper right quadrant indicate PI-positive/Annexin V-positive, late apoptotic, or
necrotic cells, and cells in the lower right quadrant indicate early apoptotic cells.
results suggested that the antiproliferative effects of 14g on
A549 cells might be mediated by PPARγ.
14g bound to PPARγ in a similar mode to GSK1997132B
(Figure 10A−D). 14g formed hydrogen-bond interactions with
Arg288, Ser289, and Tyr327, similar to GSK1997132B, while
TNBG is missing with these polar interactions with Ser289 and
Tyr327, which rationalized that the introduction of double
chains into TNBG may enhance the affinity of 14g to PPARγ.
We also propose that the lack of H-bonding interactions in this
area, particularly with Tyr473 positioned on the AF-2 helix, is
potentially responsible for the partial agonist character of the
series, in a similar manner to GSK1997132B.48
Binding Models of 14g with PPARγ. The binding modes
of 14g and TNBG with PPARγ were predicted by the software
SYBYL-X 2.0. First, we docked a novel centrally penetrant
benzimidazole PPARγ partial agonist GSK1997132B back to
the crystal of PPARγ (PDB code: 3S9S); the pose in our
predicted binding model is well overlapped with the pose in
crystal, with the root-mean-square deviation (RMSD) value is
0.6, indicating our docking assay is credible (see in the
Supporting Information). Then, compound TNBG and 14g
were docked to the PPARγ. As shown in Figure 10, TNBG and
Compound 14g Upregulates PPARγ Protein Expres-
sion in Cancer Cells. According to reported literature,
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Figure 6. Optical microscope images (200×) of A549 and HepG2 cells after treatment with 14g for 72 h and staining with Oil Red O.
Figure 7. Luciferase reporter assay of PPARγ activation in Cos-7 cells. The luciferase activity was normalized against Renilla luciferase units. Fold
activation was calculated against DMSO with no ligand treatment. (A) Fold activation of 14g, DA, and RSG was observed at 1 and 10 μM, and fold
activation of TNBG was observed at 10 μM. Values represent the mean SD (n = 3), (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). (B) Dose−
response curve of 14g. The concentration used ranged from 0.01 nM to 10 μM. Concentration is represented as a log10 scale.
Figure 8. Characterization of the binding of compound 14g to PPARγ. The LanthaScreen TR-FRET assay (Invitrogen) was used to measure the
ligand binding affinities. LBD (0.5 nM GST-PPARγ) was incubated with 5 nM fluormone and terbium-coated GST antibodies. The elevated
concentrations of DA, 14g, and RSG competed the interaction, resulting in a decrease in the FRET ratio. Values represent the mean SD (n = 2)
(*, P < 0.05). (A) Competition by 10 and 100 μM of DA, 14g, and RSG. (B) Dose−response competition curves for 14g, DA, and RSG,
concentration ranged from 0.01 nM to 125 μM. Concentration is represented on a log10 scale.
upregulation of PPARγ is associated with the inhibition of
malignant cell growth.49−52 To elucidate the mechanistic
pathway of PPARγ, we next investigated the effects of 14g on
the protein expression related to PPARγ in A549 and HepG2
cells. As shown in Figure 11, 14g upregulated PPARγ
expression levels in a concentration-dependent manner in
both tested cell lines, suggesting that the anticancer effect of
14g may be associated with the increased level of PPARγ.
Moreover, increasing evidence has suggested that the full
PPARγ agonist, rosiglitazone, or partial PPARγ agonists,
troglitazone and efatutazone, exert their antiproliferative effects
by activating the expression of the tumor suppressor gene,
phosphate and tension homology deleted on chromosome ten
(PTEN).51−53 Consistent with previous literature studies, the
expression of PTEN increased after 14g treatment indicated by
the western blot analysis. Moreover, 14g inhibited AKT
phosphorylation in A549 and HepG2 cells in a dose-dependent
manner with an almost complete abolishment at 1.8 μM in
both tested cells. These results indicated that the anticancer
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Figure 9. Effects of PPARγ on the anticancer activity of 14g in A549 lung cancer cells. (A) CCK-8 assay indicates the effect of a PPARγ agonist
(RSG) and PPARγ antagonist (T0070907) on the antiproliferative effect of 14g on A549 cells after 72 h (*, P < 0.05; **, P < 0.01; and ***, P <
0.001). Values represent the mean SD (n = 3). (B) Cell viability was measured with a crystal violet assay in A549 cells.
Figure 10. Proposed binding models of 14g and TNBG with PPARγ. (A) Crystal structure of a partial agonist GSK1997132B with PPARγ (PDB
code: 3S9S). (B/C) Hydrogen-bond interactions of TNBG/14g with PPARγ. (D/E/F) Hydrophobic interaction of the hydrophobic residues in
PPARγ with GSK1997132B/TNBG/14g. The graphics of 3D views were drawn by PyMOL, and the amino acids involved in hydrophobic
interaction were shown as stick (gray), while the S289, Y328, and R288 were highlighted as red.
effect of 14g may be exerted through partial activation of
PPARγ and upregulation of PTEN.
inflammatory cells were observed, indicating that 14g may
have toxic effects on the respiratory system.
To verify the in vivo anticancer effects of 14g, an A549
xenograft model in nude mice was established. Nude mice
were administered 10 mg/kg 14g or physiological saline
solution (vehicle group) once daily via intraperitoneal injection
for 18 days. The results demonstrated that 14g significantly
inhibited tumor growth (Figure 13A). Tumor samples were
harvested at the end of the animal study, weighed, and imaged.
As shown in Figure 13C,D, the tumor weight and volume were
reduced by 14g treatment. In addition, 14g decreased A549
xenograft tumor growth by approximately 62% (P < 0.01),
while there was no remarkable decline in the body weight
(Figure 13B).
Compound 14g Reduces Tumor Growth in vivo.
Encouraged by the inhibitory effects of 14g on several human
cancer cell lines in vitro, the in vivo acute toxicity of the
compound was determined. It was found that when the
intraperitoneal injection dose of 14g was increased to 125 mg/
kg, hypersomnia, reduced activity, gait instability, convulsions,
and other symptoms occurred (Table 2). After a decent
interval, some of the mice recovered gradually, while others did
not tolerate or even died. According to the mortality, the
median lethal dose 50% (LD50) value was calculated with a
value of 127.2 mg/kg. At the end of the animal study, the main
tissues of the mice, including the heart, liver, kidney, and lung,
were harvested for ex vivo investigation. As shown in Figure 12,
the H&E staining results showed no pathological changes in
the collected tissues, except in the lung tissues at doses of 145
and 200 mg/kg in which hemorrhage and infiltration of
CONCLUSIONS
■
The present study described the development of novel TNBG
analogues with improved potency and water solubility based
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Figure 11. Western blot analysis of the effects of 14g on A549 and HepG2 cancer cells. Both cell lines were exposed to 14g at various
concentrations for 72 h, and cells were then lysed and processed for western blot assays.
Table 2. Experimental Results of Acute Toxicity of
Intraperitoneal Injection of 14g in Mice
dose
mice
numbers
death of the mortality
LD50
(mg/kg)
groups (mg/kg)
mice
(rate)
1
2
3
4
5
6
0
10
10
10
10
10
10
0
0
6
8
8
10
0
0
60
80
80
100
127.2 9.8
106
125
145
170
200
on our previous results. The CCK-8 assay enabled us to
identify a novel compound, 14g, which inhibited cancer cell
proliferation with IC50 values of 0.54 and 0.47 μM against
HepG2 and A549 cells, respectively. The in vivo acute toxicity
study revealed that the LD50 value of 14g was 127.2 mg/kg. In
xenograft models, compound 14g strongly reduced tumor
growth at a dose of 10 mg/kg. In addition, the transactivation
and binding affinity assays revealed that 14g exhibited weak
agonistic activities and binding affinity to PPARγ and the
PPARγ antagonist, T0070907, partially reversed the anti-
Figure 12. H&E (40 ×) staining of tumor tissues (heart, liver, kidney,
and lung) after treatment with vehicle and 14g.
proliferative effect of 14g on A549 cells. Moreover, western
blotting analyses demonstrated that 14g upregulated the
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Figure 13. Compound 14g inhibited tumor growth in vivo. A549 cell xenograft mouse models were treated daily with 10 mg/kg 14g or vehicle
control for 18 consecutive days. Tumor volume changes (A) and body weight (B) were recorded twice a week. Tumor tissues of each sample were
harvested, weighed (C), and photographed (D).
ethyl acetate (50 mL) was added, and then the organic phase was
washed with water (3 × 50 mL), dried over MgSO4, and
concentrated. The crude products were purified by column
chromatography, affording 8a (240 mg, 76% yield) as yellow solid.
mp 193−195 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.87 (d, J = 2.8
Hz, 1H), 7.44 (d, J = 7.3 Hz, 1H), 7.36 (d, J = 7.4 Hz, 1H), 7.21−
7.15 (m, 3H), 7.02 (s, 1H), 6.84 (d, J = 8.3 Hz, 1H), 5.03−5.01 (m,
1H), 4.77 (d, J = 8.2 Hz, 1H), 4.04 (d, J = 11.6 Hz, 1H), 3.77 (s, 3H),
3.29 (t, J = 10.9 Hz, 1H), 3.05−3.01 (m, 1H), 2.91−2.81 (m, 2H).
13C NMR (151 MHz, DMSO-d6) δ 158.32, 144.24, 143.52, 137.67,
expression of PPARγ and PTEN and inhibited AKT
phosphorylation in A549 and HepG2 cells, which may trigger
apoptosis leading to cancer cell death. Therefore, we
concluded that 14g is a partial PPARγ agonist and the
anticancer activity of 14g may be due to the partial activation
and upregulation of PPARγ. Collectively, our data demon-
strated that 14g is a promising lead for the development of
potent anticancer agents.
137.15, 135.21, 130.34, 127.27, 125.40, 124.56, 124.46, 123.88,
113.77, 111.11, 55.68, 53.49, 45.76, 39.99, 27.86. HRMS (ESI): calcd
for C19H19ON4 [M + H]+ 319.1553, found 319.1554.
EXPERIMENTAL SECTION
■
Chemistry. All building blocks were purchased from commercial
sources and used as received; all solvents and reagents were
analytically pure. Reactions were monitored by thin-layer chromatog-
raphy (TLC) and carried out on precoated plates HSGF-254
(Qingdao Haiyang Chemicals Co., Ltd., China), which could be
visualized under UV light at 254 nm. Column chromatography was
4b,5,14,15-Tetrahydro-6H-isoquinolino[2′,1′:1,6]pyrazino-
[2,3-b] Quinoxalin-3-ol (8b). Compound 8a (1.59 g, 5 mmol) and
20 mL of 40% HBr solution were mixed and refluxed for 6 h, then
cooled to room temperature. Removal of the solvent was performed,
ethyl acetate (50 mL) was added, the organic phase was washed with
saturated sodium bicarbonate water (3 × 50 mL) to adjust the pH to
7−8, and then the organic phase was dried over MgSO4 and
concentrated. The crude products were purified by column
chromatography, affording 8b (1.14 g, 75% yield) as yellow solid.
1
performed on silica gel (300−400 mesh). All H NMR spectra were
obtained on a BRUKER AVANCE 400 or 600 MHz instrument
(Bruker company, Germany) with CDCl3 or DMSO-d6 as solvents at
ambient temperature and reported in ppm downfield from TMS (0
ppm). All 13C NMR spectra were obtained with proton decoupling on
a BRUKER AVANCE 400 MHz (100 MHz) or 600 MHz (151 MHz)
instrument and reported in ppm relative to CDCl3 (77.16 ppm).
Coupling constants were reported as Hz with multiplicity denoted as s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br
(broad). High-resolution mass spectra (HRMS) were obtained on an
Aligent 6210 system. The melting point was recorded on a WRS-2A
digital melting point apparatus. The purity of some synthesized target
compounds exhibited purities of no less than 95% as determined by
HPLC, which equipped with reverse-phase chromatography through a
Lichrospher C18 column (4.6 mm × 250 mm, 5 μm). The samples
(20 μL) were injected, using MeOH/H2O (80/20, v/v) as the mobile
phase. The flow rate was 1 mL/min, and the absorbance at 330 nm
was monitored.
1
mp 267−270 °C. H NMR (600 MHz, DMSO-d6) δ 9.35 (s, 1H),
7.79 (s, 1H), 7.45−7.43 (m, 1H), 7.36−7.35 (m, 1H), 7.20−7.16 (m,
2H), 7.03 (d, J = 8.3 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 6.69−6.67
(m, 1H), 5.00−4.96 (m, 1H), 4.74−4.72 (m, 1H), 3.89−3.86 (m,
1H), 3.32−3.29 (m, 1H), 3.05−3.00 (m, 1H), 2.87−2.82 (m, 1H),
2.78−2.76 (m, 1H). 13C NMR (151 MHz, DMSO-d6) δ 156.23,
144.17, 143.56, 137.67, 137.18, 135.02, 130.28, 125.48, 125.39,
124.53, 124.49, 123.87, 114.90, 112.31, 53.30, 45.79, 39.98, 27.79.
HRMS (ESI): calcd for C18H17N4O [M + H]+ 305.1397, found
305.1398.
2-Methoxy-4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b] Quinoxaline (8c). The product was
obtained as yellow solid. Yield 57%. mp 228−230 °C. 1H NMR (600
MHz, DMSO-d6) δ 7.83 (d, J = 3.5 Hz, 1H), 7.45−7.43 (m, 1H),
7.36−7.34 (m, 2H), 7.21−7.17 (m, 2H), 6.84 (d, J = 8.5 Hz, 1H),
6.81 (s, 1H), 5.00−4.97 (dd, J = 12.7, 2.8 Hz, 1H), 4.75−4.73 (dd, J
= 9.6, 2.5 Hz, 1H), 3.98−3.95 (m, 1H), 3.75 (s, 3H), 3.26 (t, J = 10.8
Hz, 1H), 3.10−3.05 (m, 1H). 2.98−2.93 (m, 1H), 2.86 (d, J = 15.9
Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 158.44, 144.23, 143.57,
137.69, 137.17, 136.83, 127.29, 126.23, 125.40, 124.55, 124.49,
123.86, 113.76, 113.27, 55.54, 52.98, 45.85, 39.99, 28.89. HRMS
(ESI): calcd for C19H19ON4 [M + H]+ 319.1553, found 319.1558.
Syntheses of Compounds 2−6. Compounds 2−6 were
prepared according to our previous reported literature.54−56
3-Methoxy-4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b] Quinoxaline (8a). Compound 6a (1.0
mmol, 264 mg) and compound 7 (1.0 mmol, 200 mg) were dissolved
in 25 mL of DMF. Then, potassium carbonate (4.0 mmol, 550 mg)
was added; the mixture was heated to 110 °C for 24 h and then
cooled to room temperature. Removal of the solvent was performed,
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1-Chloro-4-methoxy-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b] Quinoxaline (8d). The
product was obtained as yellow solid. Yield 62%. mp 245−248 °C. 1H
NMR (600 MHz, CDCl3) δ 7.60 (d, J = 5.2 Hz, 1H), 7.44 (s, 1H),
7.31−7.26 (m, 3H), 7.08 (s, 1H), 6.75 (d, J = 8.3 Hz, 1H), 5.26 (d, J
= 9.2 Hz, 1H), 4.92 (d, J = 8.4 Hz, 1H), 4.35 (d, J = 9.9 Hz, 1H), 3.86
(s, 3H), 3.21−3.15 (m, 2H), 2.95 (t, J = 11.8 Hz, 1H), 2.80 (t, J =
11.5 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 155.38, 143.97,
143.35, 137.43, 136.89, 135.52, 128.35, 126.04, 125.52, 124.64,
124.31, 124.14, 123.58, 109.49, 55.58, 51.39, 44.60, 38.21, 27.63.
HRMS (ESI): calcd for C19H18ON4Cl [M + H]+ 353.1164, found
353.1167.
5.01−4.99 (m, 1H), 4.91−4.84 (m, 1H), 4.04−4.01 (m, 1H), 3.86 (s,
3H), 3.33−3.29 (m, 1H), 3.13−3.09 (m, 1H), 3.01−2.97 (m, 1H),
2.91 (d, J = 15.8 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 166.93,
145.46, 144.12, 141.51, 136.89, 135.32, 133.89, 129.40, 127.41,
126.97, 126.90, 126.18, 124.72, 124.43, 124.39, 53.01, 52.37, 45.64,
39.98, 28.57. HRMS (ESI): calcd for C20H19N4O2 [M + H]+
347.1503, found 347.1505.
9-Methoxy-4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b] Quinoxaline (8k). The product was
obtained as yellow solid. Yield 69%. mp 212−215 °C. 1H NMR (600
MHz, CDCl3) δ 7.52 (d, J = 8.8 Hz, 1H), 7.27−7.23 (m, 4H), 6.96−
6.93 (m, 2H), 5.96 (s, 1H), 5.16−5.13 (m, 1H), 4.81 (dd, J = 10.1,
3.0 Hz, 1H), 3.99 (dd, J = 11.4, 3.5 Hz, 1H), 3.86 (s, 3H), 3.52 (t, J =
10.7 Hz, 1H), 3.14−3.07 (m, 2H), 2.93−2.89 (m, 1H). 13C NMR
(151 MHz, CDCl3) δ 157.40, 143.71, 142.01, 137.75, 135.48, 133.21,
132.32, 129.40, 127.19, 126.52, 126.41, 125.31, 114.86, 105.24, 55.47,
53.57, 46.87, 39.23, 29.02. HRMS (ESI): calcd for C18H19ON4 [M +
H]+ 319.1553, found 319.1555.
1-Chloro-4b,5,14,15-tetrahydro-6H-isoquinolino[2′,1′:1,6]-
pyrazino[2,3-b] Quinoxalin-4-ol (8e). Compound 8e was
obtained according to the protocol of preparing of 8b. The product
1
was obtained as white solid. Yield 65%. mp 249−252 °C. H NMR
(600 MHz, DMSO) δ 10.67 (s, 1H), 9.77 (s, 1H), 7.63−7.58 (m,
2H), 7.38−7.17 (m, 3H), 6.95 (d, J = 6.5 Hz, 1H), 4.99 (s, 1H), 4.45
(d, J = 10.9 Hz, 1H), 3.91−3.40 (m, 1H), 3.21 (t, J = 11.3 Hz, 1H),
3.08 (d, J = 15.9 Hz, 1H), 2.99 (t, J = 12.2 Hz, 1H), 2.73 (t, J = 11.8
Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 154.11, 151.85, 144.00,
140.81, 134.58, 131.44, 129.38, 128.81, 126.24, 126.09, 123.07,
122.88, 121.00, 115.25, 50.17, 44.03, 36.14, 26.95. HRMS (ESI):
calcd for calcd for C18H16ClN4O [M + H]+ 339.1007, found
339.1009.
4b,5,14,15-Tetrahydro-6H-isoquinolino[2′,1′:1,6]pyrazino-
[2,3-b]quinoxalin-9-ol (8l). To a stirred solution of compound 8k
(0.5 mmol, 160 mg) was added 40% aqueous HBr (4.5 mmol equiv of
substrate) in a single lot. The resulting reaction mixture was heated at
100 °C, and the progress of reaction was monitored by TLC. After
completion of reaction, the reaction mixture was cooled to room
temperature and quenched by adding water (25 mL). The resulting
reaction mass was extracted with 3 × 30 mL of ethyl acetate. The
ethyl acetate layer was washed twice with 20 mL of water, dried over
anhydrous Na2SO4, and evaporated under reduced pressure. The
crude product was purified by silica gel column chromatography using
ethyl acetate/hexane system, affording 8l (120 mg, 81% yield) as
3-Fluoro-4b,5,14,15-tetrahydro-6H-isoquinolino[2′,1′:1,6]-
pyrazino[2,3-b] Quinoxaline (8f). The product was obtained as
yellow solid. Yield 48%. mp 248−250 °C. 1H NMR (600 MHz,
CDCl3) δ 7.62−7.59 (m, 1H), 7.50−7.47 (m, 1H), 7.33−7.28 (m,
2H), 7.22−7.19 (m, 1H), 6.99−6.93 (m, 2H), 5.25−5.22 (m, 1H),
4.83 (dd, J = 10.1, 3.0 Hz, 1H), 3.96 (dd, J = 11.5, 3.6 Hz, 1H), 3.55−
3.51 (m, 1H), 3.15−3.10 (m, 1H), 3.07−3.01 (m, 1H), 2.89 (d, J =
15.6 Hz, 1H). 13C NMR (151 MHz, CDCl3) δ 161.43, 143.11,
137.49, 136.40, 134.84, 131.10, 130.93, 130.88, 125.61, 125.11,
124.88, 124.30, 114.58, 112.06, 53.52, 46.42, 39.42, 28.29. HRMS
(ESI): calcd for C18H16N4F [M + H]+ 307.1354, found 307.1356.
9-Methyl-4b,5,14,15-tetrahydro-6H-isoquinolino[2′,1′:1,6]-
pyrazino[2,3-b] Quinoxaline (8h). Compound 2,3-dichloro-6-
methylquinoxaline (1.0 mmol, 226 mg) and compound 6f (1.0
mmol, 162 mg) were dissolved in 25 mL of DMF. Then potassium
carbonate (4.0 mmol, 550 mg) was added; the mixture was heated to
110 °C for 24 h and then cooled to room temperature. Removal of
the solvent was performed, ethyl acetate (50 mL) was added, and then
the organic phase was washed with water (3 × 50 mL), dried over
MgSO4, and concentrated. The crude products were purified by
column chromatography, affording 8h (130 mg, 43% yield) as yellow
1
yellow solid. mp 271−273 °C. H NMR (600 MHz, DMSO-d6) δ
10.08 (s, 1H), 9.78 (s, 1H), 7.48−7.46 (m, 2H), 7.30 (d, J = 8.0 Hz,
3H), 6.98 (s, 1H), 6.92 (d, J = 8.8 Hz, 1H), 4.93 (d, J = 8.8 Hz, 1H),
4.82 (d, J = 11.7 Hz, 1H), 4.21 (d, J = 12.1 Hz, 1H), 3.45 (t, J = 11.7
Hz, 1H), 3.12 (t, J = 10.6 Hz, 1H), 3.02−2.93 (m, 2H). 13C NMR
(151 MHz, DMSO-d6) δ 156.52, 152.46, 141.95, 140.50, 135.23,
135.02, 132.65, 129.52, 127.75, 127.10, 126.22, 116.14, 113.98,
102.26, 51.97, 46.16, 45.82, 39.81, 28.28. HRMS (ESI): calcd for
C18H17N4O [M + H]+ 305.1397, found 305.1399.
9-Bromo-4b,5,14,15-tetrahydro-6H-isoquinolino[2′,1′:1,6]-
pyrazino[2,3-b] Quinoxaline (8m). The product was obtained as
yellow solid. Yield 59%. mp 214−215 °C. 1H NMR (400 MHz,
CDCl3): δ 7.61 (d, J = 2 Hz, 1H), 7.46−7.44 (d, J = 8.4 Hz, 1H),
7.37−7.34 (dd, J = 2, 2 Hz, 1H), 7.29−7.23 (m, 4H), 5.80 (s, 1H),
5.21−5.17 (m, 1H), 4.88−4.85 (m, 1H), 4.01−3.97 (dd, J = 3.2, 3.2
Hz, 1H), 3.54−3.48 (m, 1H), 3.18−3.08 (m, 2H), 3.06−2.90 (m,
1H); 13C NMR (100 MHz, CDCl3): δ 143.70, 143.30, 137.93,
136.58, 135.32, 132.85, 129.41, 127.70, 127.37, 126.95, 126.76,
126.69, 125.31, 117.53, 53.60, 46.64, 39.3,5 28.95; HRMS (ESI):
calcd for C18H15BrN4 [M + H]+ 367.0553, found 367.0557.
9-(1H-Pyrazol-3-yl)-4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino [2,3-b]Quinoxaline (8n). To a 5 mL of
dioxane/water (v/v = 5/1) were added compound 8m (73 mg, 0.2
mmol), 1-methylpyrazole-4-boronic acid pinacol ester (65 mg, 0.3
mmol), K2CO3 (56 mg, 0.4 mmol), and (dppf)PdCl2 (75 mg, 0.1
mmol); the mixture was heated at 120 °C for 20 min under a nitrogen
atmosphere. After completion of the reaction, ethyl acetate (50 mL)
was added, the organic phase was washed with saturated sodium
bicarbonate water (3 × 50 mL); the organic phase was dried over
MgSO4 and then concentrated. The crude products were purified by
column chromatography, affording 8n. Yield, 52%. mp 112−114 °C.
1H NMR (400 MHz, DMSO-d6) 13.04 (d, J = 176.3 Hz, 1H), 7.87−
1
solid. mp 158−160 °C. H NMR (600 MHz, CDCl3) δ 7.50 (d, J =
8.2 Hz, 1H), 7.26−7.22 (m, 5H), 7.12 (d, J = 8.0 Hz, 1H), 6.41 (s,
1H), 5.21−5.17 (m, 1H), 4.86−4.82 (m, 1H), 3.99−3.96 (m, 1H),
3.51 (t, J = 10.8 Hz, 1H), 3.15−3.06 (m, 2H), 2.90 (d, J = 13.1 Hz,
1H), 2.44 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 143.66, 142.95,
136.72, 135.54, 135.49, 134.68, 133.27, 129.39, 127.21, 126.57,
126.28, 125.36, 125.21, 124.06, 53.64, 46.76, 39.28, 29.06, 21.49.
HRMS (ESI): calcd for C19H19N4 [M + H]+ 303.1604, found
303.1605.
9-Chloro-4b,5,14,15-tetrahydro-6H-isoquinolino[2′,1′:1,6]-
pyrazino[2,3-b] Quinoxaline (8i). The product was obtained as
yellow solid. Yield 30%. mp 224−227 °C. 1H NMR (600 MHz,
CDCl3) δ 7.51 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 2.3 Hz, 1H), 7.29−
7.22 (m, 5H), 6.13 (s, 1H), 5.20−5.17 (m, 1H), 4.88−4.85 (m, 1H),
4.01−3.98 (m, 1H), 3.52 (d, J = 10.9 Hz, 1H), 3.17−3.06 (m, 2H),
2.98−2.90 (m, 1H). 13C NMR (151 MHz, CDCl3) δ 143.60, 143.20,
136.76, 136.03, 135.26, 132.71, 129.92, 129.43, 127.40, 126.72,
126.52, 125.33, 125.17, 123.37, 53.49, 46.63, 39.35, 28.92. HRMS
(ESI): calcd for C18H16N4Cl [M + H]+ 323.1058, found 323.1059.
Methyl 4b,5,14,15-Tetrahydro-6H-isoquinolino[2′,1′:1,6]-
pyrazino[2,3-b]quinoxaline-10-carboxylate (8j). The product
7.68 (s, 3H), 7.55−7.44 (m, 3H), 7.30−7.25 (m, 3H), 6.73 (s, 1H),
5.01 (dd, J = 10.3, 2.5 Hz, 1H), 4.84 (d, J = 7.9 Hz, 1H), 4.02−3.99
(m, 1H), 3.32−3.16 (m, 1H), 3.17−3.07 (m, 1H), 3.03−2.88 (m,
2H); 13C NMR (100 MHz, DMSO-d6): δ 144.53, 143.44, 137.83,
136.66, 135.55, 135.42, 134.26, 134.20, 129.40, 127.36, 126.87,
126.17, 125.55, 123.18, 121.59, 120.55, 102.45, 53.36, 45.74, 39.59,
28.66; HRMS (ESI): calcd for C21H19N6 [M + H]+ 355.1666, found
355.1668.
1
was obtained as yellow solid. Yield 34%. mp 216−218 °C. H NMR
(600 MHz, DMSO-d6) δ 8.35 (d, J = 4.0 Hz, 1H), 8.03 (d, J = 1.9 Hz,
1H), 7.75−7.72 (m, 1H), 7.49−7.39 (m, 2H), 7.29−7.25 (m, 3H),
1029
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Article
9-(1-Methyl-1H-pyrazol-4-yl)-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxaline (8o). The
product was obtained as yellow solid. Yield 51%. mp 118−120 °C.
1H NMR (400 MHz, CDCl3): δ 7.85 (s, 1H), 7.72 (s, 1H), 7.71 (s,
1H), 7.67 (s, 1H), 7.48−7.41 (m, 2H), 7.28−7.23 (m, 4H), 5.68 (s,
1H), 5.25−5.20 (m, 1H), 4.90−4.88 (m, 1H), 4.02−3.98 (m, 1H),
3.96 (s, 3H), 3.55−3.49 (t, J = 16.2 Hz, 1H), 3.21−3.07 (m, 2H),
2.95−2.91 (d, J = 22.6 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ
143.54, 143.03, 138.00, 136.88, 135.51, 135.42, 133.09, 129.39,
129.20, 127.30, 126.85, 126.63, 125.32, 124.85, 123.42, 122.94,
121.46, 53.79, 46.74, 39.37, 39.10, 29.02; HRMS (ESI): calcd for
C22H21N6 [M + H] + 369.1822, found 369.1824.
4-(3-(4b,5,14,15-Tetrahydro-6H-isoquinolino[2′,1′:1,6]-
pyrazino[2,3-b]quinoxalin-6-yl)propyl)morpholine (9a).
TNBG (1.0 mmol, 288 mg) and 4-(3-chloropropyl)morpholine (2
mmol, 326 mg) were dissolved in 25 mL of DMF. Then, 60% sodium
hydride (4 mmol, 160 mg) was added; the mixture was heated to 50
°C for 12 h and then cooled to room temperature. Removal of the
solvent was performed, ethyl acetate (50 mL) was added, and then the
organic phase was washed with water (3 × 50 mL), dried over
MgSO4, and concentrated. The crude products were purified by
column chromatography, affording 9a (182 mg, 44% yield) as yellow
solid. mp 124−127 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.44 (d, J
= 7.4 Hz, 2H), 7.37 (d, J = 7.86 Hz, 1H), 7.29−7.16 (m, 5H), 4.99−
4.97 (m, 1H), 4.83 (d, J = 9.5 Hz, 1H), 4.10−4.07 (m, 1H), 3.89−
3.84 (m, 1H), 3.59 (m, 4H), 3.45 (t, J = 10.9 Hz, 1H), 3.38 (s, 1H),
3.09−3.05 (m, 1H), 2.99−2.94 (m, 1H), 2.87 (d, J = 15.8 Hz, 1H),
2.33−2.35 (m, 6H), 1.85−1.82 (m, 2H). 13C NMR (151 MHz,
DMSO) δ 143.53, 143.19, 137.51, 136.63, 135.43, 133.93, 129.48,
127.44, 126.81, 126.10, 125.26, 124.94, 124.66, 123.97, 66.66, 56.09,
53.69, 52.68, 51.79, 46.56, 39.59, 28.52, 23.14. HRMS (ESI): calcd
for C25H30ON5 [M + H]+ 416.2445, found 416.2449.
4-(2-(4b,5,14,15-Tetrahydro-6H-isoquinolino[2′,1′:1,6]-
pyrazino[2,3-b]quinoxalin-6-yl)ethyl)morpholine (9b). The
product was obtained as yellow solid. Yield 51%. mp 125−128 °C.
1H NMR (600 MHz, DMSO-d6) δ 7.47−7.44 (m, 2H), 7.40−7.39
(m, 1H), 7.31−7.24 (m, 3H), 7.21−7.17 (m, 2H), 5.00−4.98 (m,
1H), 4.83 (d, J = 7.56 Hz, 1H), 4.19−4.16 (m, 1H), 4.03−3.98 (m,
1H), 3.75−3.71 (m, 1H), 3.53−3.50 (m, 5H), 3.36 (s, 2H), 3.10−
3.06 (m, 1H), 2.99−2.96 (m, 1H), 2.89 (d, J = 15.7 Hz, 1H), 2.63 (t,
J = 5.2 Hz, 2H), 2.48 (s, 2H). 13C NMR (151 MHz, DMSO) δ
143.52, 143.08, 137.43, 136.63, 135.43, 133.91, 129.49, 127.44,
126.83, 126.07, 125.26, 124.98, 124.66, 124.00, 66.76, 55.42, 53.95,
52.72, 52.06, 44.89, 39.57, 28.49. HRMS (ESI): calcd for C24H28ON5
[M + H]+ 402.2288, found 402.2290.
6-(3-(Piperidin-1-yl)propyl)-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxaline (9c). The
product was obtained as yellow solid. Yield 39%. mp 105−108 °C.
1H NMR (600 MHz, DMSO-d6) δ 7.47−7.43 (m, 2H), 7.39−7.37
(m, 1H), 7.30−7.24 (m, 3H), 7.22−7.16 (m, 2H), 5.01−4.97 (m,
1H), 4.88−4.85 (m, 1H), 4.14−4.10 (dd, J = 6, 6 Hz, 1H), 3.88−3.83
(m, 1H), 3.65−3.60 (m, 1H), 3.48 (t, J = 11.0 Hz, 1H), 3.11−3.06
(m, 1H), 3.00−2.95 (m, 1H), 2.91−2.88 (m, 1H), 2.52−2.49 (m,
1H), 2.34−2.30 (m, 5H), 1.88−1.81 (m, 2H), 1.48−1.52 (m, 4H),
1.38 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 143.53, 143.20,
137.52, 136.61, 135.46, 133.96, 129.49, 127.44, 126.81, 126.08,
125.25, 124.95, 124.63, 123.95, 56.39, 54.41, 52.69, 51.83, 46.64,
39.59, 28.54, 26.04, 24.62, 23.60. HRMS (ESI): calcd for C26H32N5
[M + H]+ 414.2652, found 414.2656.
124.65, 123.95, 55.66, 54.71, 52.72, 52.16, 45.34, 28.49, 26.16, 24.47.
HRMS (ESI): calcd for C25H30N5 [M + H]+ 400.2496, found
400.2499.
6-(3-(4-Methylpiperazin-1-yl)propyl)-4b,5,14,15-tetrahy-
dro-6H-isoquinolino[2′,1′:1,6]pyrazino [2,3-b]quinoxaline
(9e). The product was obtained as yellow solid. Yield 51%. mp
1
140−142 °C. H NMR (600 MHz, CDCl3) δ 7.57−7.55 (m, 1H),
7.51−7.49 (m, 1H), 7.29−7.22 (m, 6H), 5.20−5.18 (m, 1H), 4.82−
4.80 (m, 1H), 3.92−3.88 (m, 2H), 3.74−3.69 (m, 1H), 3.53 (t, J =
11.0 Hz, 1H), 3.12−3.06 (m, 2H), 2.93−2.87 (m, 1H), 2.52−2.42
(m, 10H), 2.32 (s, 3H), 2.00−1.89 (m, 2H). 13C NMR (151 MHz,
DMSO-d6) δ 138.55, 138.20, 132.60, 131.97, 130.70, 128.37, 124.69,
122.51, 121.81, 120.60, 120.51, 120.21, 119.90, 119.42, 50.68, 49.52,
48.24, 47.99, 47.31, 41.67, 40.56, 34.55, 24.19, 18.80. HRMS (ESI):
calcd for C26H33N6 [M + H]+ 429.2761, found 429.2765.
4b,5,14,15-Tetrahydro-6H-isoquinolino[2′,1′:1,6]pyrazino-
[2,3-b]quinoxaline-10-carboxylic Acid (10). Compound 8j (11.6
mmol, 4.089 g) was dissolved in CH3OH/H2O (v/v = 4/1); then
lithium hydroxide (47.6 mmol, 2.010 g) was added. The mixture was
stirred at room temperature for 4 h. Removal of the solvent was
performed, and cool water (200 mL) was added, adjusted the pH to
4−5, and then filtered it to obtain a light yellow solid 10 (3.553 g,
1
92% yield) as yellow solid. mp 268−270 °C. H NMR (600 MHz,
DMSO-d6) δ 10.12 (s, 1H), 8.07 (s, 1H), 7.85 (d, J = 8.6 Hz, 1H),
7.67 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 6.5 Hz, 1H), 7.31−7.28 (m,
3H), 5.07−5.01 (m, 1H), 4.95−4.93 (m, 1H), 4.26−4.23 (m, 1H),
3.44 (t, J = 11.8 Hz, 1H), 3.22−3.17 (m, 1H), 3.04−2.94 (m, 2H).
13C NMR (151 MHz, DMSO-d6) δ 167.24, 144.26, 141.80, 134.97,
133.96, 132.48, 129.46, 128.11, 127.79, 127.16, 126.64, 126.58,
126.41, 126.22, 118.63, 52.17, 45.89, 39.52, 28.24. HRMS (ESI):
calcd for C19H17N4O2 [M + H]+ 333.1346, found 333.1349.
N,N-Dimethyl-4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b]quinoxaline-10-carboxamide (11a).
Compound 10 (3 mmol, 1000 mg) was dissolved in DMF (15
mL); then EDCl (3 mmol, 630 mg), HOBT (3.3 mmol, 450 mg),
DIEA (15.5 mmol, 2000 mg), and dimethylamine hydrochloride (10
mmol, 720 mg) were added. The mixture was stirred for 2 h. After
completion of the reaction, removal of the solvent was performed,
ethyl acetate (50 mL) was added, and then the organic phase was
washed with water (3 × 50 mL), dried over MgSO4, and
concentrated. The crude products were purified by column
chromatography, affording 11a (450 mg, 41% yield) as yellow solid.
mp 221−224 °C. 1H NMR (600 MHz, DMSO-d6) δ 8.07 (d, J = 3.8
Hz, 1H), 7.45−7.43 (m, 2H), 7.37 (d, J = 8.2 Hz, 1H), 7.27−7.22 (m,
4H), 5.03−4.99 (m, 1H), 4.84 (dd, J = 10.0, 3.3 Hz, 1H), 4.03−4.00
(m, 1H), 3.61−3.59 (m, 1H), 3.33−3.30 (m, 1H), 3.12−3.08 (m,
1H), 2.99 (s, 6H), 2.90 (d, J = 15.9 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6) δ 170.79, 144.79, 143.98, 138.37, 136.28, 135.36, 134.04,
131.52, 129.39, 127.37, 126.88, 126.17, 124.19, 124.13, 123.66, 53.21,
45.68, 39.57, 28.61, 25.60. HRMS (ESI): calcd for C21H22ON5 [M +
H]+ 360.1819, found 360.1821.
N,N-Diethyl-4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b]quinoxaline-10-carboxamide (11b).
The product was obtained as yellow solid. Yield 45%. mp 153−155
°C. 1H NMR (600 MHz, CDCl3) δ 7.62 (d, J = 1.6 Hz, 1H), 7.47 (d,
J = 8.2 Hz, 1H), 7.31−7.24 (m, 5H), 6.36 (s, 1H), 5.22−5.19 (m,
1H), 4.88 (dd, J = 10.1, 3.0 Hz, 1H), 4.01 (dd, J = 11.6, 3.6 Hz, 1H),
3.54−3.51 (m, 3H), 3.35 (s, 2H), 3.18−3.07 (m, 2H), 2.92 (d, J =
15.4 Hz, 1H), 1.25 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 171.43,
143.84, 143.64, 136.93, 136.77, 135.29, 133.27, 132.82, 129.40,
127.36, 126.69, 125.35, 124.22, 123.58, 123.25, 53.50, 46.59, 39.36,
29.71, 28.93, 14.31. HRMS (ESI): calcd for C23H26ON5 [M + H]+
388.2132, found 388.2135.
6-(2-(Piperidin-1-yl)ethyl)-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxaline (9d). The
product was obtained as yellow solid. Yield 56%. mp 122−124 °C.
1H NMR (600 MHz, DMSO-d6) δ 7.45 (t, J = 6.3 Hz, 2H), 7.39 (d, J
= 7.4 Hz, 1H), 7.30−7.16 (m, 5H), 5.00−4.97 (m, 1H), 4.81 (d, J =
9.4 Hz, 1H), 4.17−4.15 (m, 1H), 3.99−3.95 (m, 1H), 3.73−3.68 (m,
1H), 3.51 (t, J = 10.9 Hz, 2H), 3.09−3.05 (m, 1H), 3.01−2.95 (m,
1H), 2.88 (d, J = 15.7 Hz, 1H), 2.59−2.54 (m, 2H), 2.51 (s, 1H),
2.42 (s, 3H), 1.44 (t, J = 4.8 Hz, 4H), 1.35 (d, J = 2.3 Hz, 2H). 13C
NMR (151 MHz, DMSO-d6) δ 143.53, 143.07, 137.49, 136.61,
135.43, 133.95, 129.50, 127.44, 126.82, 126.04, 125.24, 124.98,
Morpholino(4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-10-yl)methanone (11c).
The product was obtained as yellow solid. Yield 38%. mp 257−259
°C. 1H NMR (600 MHz, DMSO-d6) δ 8.09 (d, J = 2.2 Hz, 1H), 7.45
(s, 2H), 7.38 (d, J = 8.2 Hz, 1H), 7.27−7.23 (m, 4H), 5.01 (d, J =
10.6 Hz, 1H), 4.84 (d, J = 8.8 Hz, 1H), 4.01 (d, J = 11.6 Hz, 1H),
3.61−3.54 (m, 8H), 3.33−3.30 (m, 1H), 3.11 (t, J = 11.9 Hz, 1H),
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Article
3.01−2.96 (m, 1H), 2.91 (d, J = 15.8 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6) δ 169.84, 144.83, 144.01, 138.58, 136.36, 135.35, 134.02,
130.55, 129.39, 127.39, 126.89, 126.19, 124.39, 124.16, 123.66, 66.63,
53.19, 45.66, 39.56, 28.61. HRMS (ESI): calcd for C23H24O2N5 [M +
H]+ 402.1925, found 402.1927.
(4-Methylpiperazin-1-yl)(4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-10-yl) Meth-
anone (11d). The product was obtained as yellow solid. Yield 44%.
mp 236−239 °C. 1H NMR (600 MHz, CDCl3) δ 7.63 (d, J = 1.8 Hz,
1H), 7.47 (d, J = 8.3 Hz, 1H), 7.35−7.33 (m, 1H), 7.30−7.23 (m,
4H), 6.27 (s, 1H), 5.23−5.20 (m, 1H), 4.89−4.87 (dd, J = 10.2, 3.0
Hz, 1H), 4.01−3.99 (m, 1H), 3.84 (brs, 2H), 3.58 (brs, 1H), 3.54−
3.51 (m, 2H), 3.18−3.07 (m 2H), 2.92 (d, J = 15.5 Hz, 1H), 2.51−
2.41 (m, 4H), 2.34 (s, 3H). 13C NMR (151 MHz, CDCl3) δ 170.59,
144.25, 143.74, 137.75, 136.89, 135.26, 132.84, 131.42, 129.38,
127.35, 126.68, 125.35, 124.45, 123.79, 53.49, 46.56, 46.06, 39.35,
29.69, 28.93, 22.85. HRMS (ESI): calcd for C24H27 O2N6 [M + H]+
415.2241, found 415.2245.
39.57, 27.69. HRMS (ESI): calcd for C26H35N6O [M + H]+ 447.2867,
found 447.2872.
3-(3-(3-(Dimethylamino)propoxy)-4b,5,14,15-tetrahydro-
6H-isoquinolino[2′,1′:1,6]pyrazino [2,3-b]quinoxalin-6-yl)-
N,N-dimethylpropan-1-amine (13b). The product was obtained
1
as white solid. Yield 35%. mp 110−112 °C. H NMR (600 MHz,
DMSO-d6) δ 7.44−7.39 (m, 2H), 7.21−7.16 (m, 2H), 7.14 (d, J = 8.4
Hz, 1H), 7.03 (d, J = 2.0 Hz, 1H), 6.84 (dd, J = 8.3, 2.3 Hz, 1H),
5.00−4.98 (m, 1H), 4.80 (dd, J = 9.7, 2.6 Hz, 1H), 4.15 (dd, J = 11.8,
3.5 Hz, 1H), 4.04−3.97 (m, 2H), 3.88−3.83 (m, 1H), 3.63−3.44 (m,
6H), 3.03−2.99 (m, 1H), 2.91−2.85 (m, 1H), 2.80 (d, J = 15.6 Hz,
1H), 2.35−2.32 (m, 4H), 2.19 (d, J = 24.0 Hz, 12H). 13C NMR (151
MHz, DMSO-d6) δ 157.70, 143.53, 143.15, 137.51, 136.63, 134.91,
130.40, 127.22, 125.24, 124.96, 124.60, 123.94, 114.09, 111.83, 66.35,
57.00, 56.16, 52.84, 51.81, 46.56, 45.61, 45.47, 39.57, 27.71, 27.37,
24.20. HRMS (ESI): calcd for C28H39N6O [M + H]+ 475.3180, found
475.3178.
2-(1-Chloro-4-(2-(dimethylamino)ethoxy)-4b,5,14,15-tetra-
hydro-6H-isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-6-
yl)-N,N-dimethylethan-1-amine (13c). The product was obtained
N,N-Dimethyl-1-(4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-10-yl) Methanamine
(12a). To a mixture of lithium aluminum hydride (1.0 mmol, 37
mg) and tetrahydrofuran (20 mL) was added compound 11a (0.5
mmol, 180 mg); the reaction was stirred at room temperature for 2 h.
The removal of the tetrahydrofuran was performed, ethyl acetate (50
mL) was added, and then the organic phase was washed with water (3
× 50 mL), dried over MgSO4, and concentrated. The crude products
were purified by column chromatography, affording 12a (160 mg,
1
as white solid. Yield 41%. mp 121−123 °C. H NMR (600 MHz,
CDCl3) δ 7.57−7.52 (m, 2H), 7.30−7.23 (m, 3H), 6.76 (d, J = 8.7
Hz, 1H), 5.21 (dd, J = 12.8, 4.0 Hz, 1H), 4.90 (d, J = 9.0 Hz, 1H),
4.34 (dd, J = 11.1, 2.5 Hz, 1H), 4.18−4.05 (m, 3H), 3.71−3.66 (m,
1H), 3.28 (t, J = 10.5 Hz, 1H), 3.14 (d, J = 16.4 Hz, 1H), 2.95−2.91
(m, 1H), 2.84−2.78 (m, 2H), 2.73−2.67 (m, 2H), 2.63−2.61 (m,
1H), 2.35 (d, J = 10.6 Hz, 12H). 13C NMR (151 MHz, CDCl3) δ
154.54, 143.23, 142.88, 137.40, 136.75, 135.72, 128.32, 126.18,
125.12, 125.04, 124.51, 124.05, 123.67, 110.21, 66.58, 58.32, 55.99,
51.02, 50.79, 46.21, 46.04, 45.58, 38.11, 29.73, 27.59. HRMS (ESI):
calcd for C26H34ON6Cl [M + H]+ 481.2477, found 481.2481.
4-(2-(3-(2-Morpholinoethoxy)-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-6-yl)ethyl)-
morpholine (13d). The product was obtained as white solid. Yield
1
90% yield) as yellow solid. mp 145−147 °C. H NMR (600 MHz,
DMSO-d6) δ 7.80 (d, J = 3.3 Hz, 1H), 7.43 (d, J = 6.9 Hz, 1H), 7.35
(s, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.26−7.23 (m, 3H), 7.15 (d, J = 8.2
Hz, 1H), 5.00 (dd, J = 12.6, 2.6 Hz, 1H), 4.80 (d, J = 7.3 Hz, 1H),
3.99 (dd, J = 7.9, 3.7 Hz, 1H), 3.45 (s, 2H), 3.30 (t, J = 10.8 Hz, 1H),
3.10−3.05 (m, 1H), 3.00−2.95 (m, 1H), 2.88 (d, J = 15.8 Hz, 1H),
2.17 (s, 6H). 13C NMR (151 MHz, DMSO-d6) δ 144.10, 143.56,
136.79, 136.73, 135.42, 134.30, 134.24, 129.39, 127.32, 126.84,
126.14, 125.64, 125.49, 124.20, 63.82, 53.37, 45.73, 45.33, 39.58,
28.66. HRMS (ESI): calcd for C21H24N5 [M + H]+ 346.2026, found
346.2030.
1
65%. mp 46−48 °C. H NMR (600 MHz, DMSO-d6) δ 7.44−7.43
(m, 1H), 7.39−7.38 (m, 1H), 7.21−7.16 (m, 2H), 7.15 (d, J = 8.4 Hz,
1H), 7.04 (d, J = 2.4 Hz, 1H), 6.86 (dd, J = 8.4, 2.4 Hz, 1H), 4.99−
4.96 (m, 1H), 4.78 (dd, J = 9.9, 3.0 Hz, 1H), 4.22 (dd, J = 11.9, 3.6
Hz, 1H), 4.14−4.07 (m, 2H), 4.05−4.01 (m, 1H), 3.73−3.69 (m,
1H), 3.60−3.49 (m, 8H), 3.05−3.00 (m, 1H), 2.92−2.89 (m, 1H),
2.81 (d, J = 15.7 Hz, 1H), 2.70 (t, J = 5.8 Hz, 2H), 2.63 (t, J = 6.6 Hz,
2H), 2.50 (dd, J = 3.6, 1.8 Hz, 4H), 2.48 (s, 5H). 13C NMR (151
MHz, DMSO-d6) δ 157.49, 143.53, 143.11, 137.42, 136.62, 134.95,
130.44, 127.36, 125.23, 124.96, 124.65, 123.99, 114.13, 111.90, 66.78,
66.66, 65.91, 57.55, 55.42, 54.13, 53.96, 52.88, 51.97, 44.89, 40.52,
27.67. HRMS (ESI): calcd for C30H39O3N6 [M + H]+ 531.3078,
found 531.3060.
N-Ethyl-N-((4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-10-yl)methyl) Ethan-
amine (12b). The product was obtained as yellow solid. Yield
1
94%. mp 171−173 °C. H NMR (600 MHz, DMSO-d6) δ 7.84 (s,
1H), 7.43−7.39 (m, 2H), 7.32 (d, J = 8.2 Hz, 1H), 7.27−7.22 (m,
4H), 5.01−4.98 (m, 1H), 4.80 (dd, J = 9.7, 3.4 Hz, 1H), 3.99 (dd, J =
11.3, 2.7 Hz, 1H), 3.70 (s, 2H), 3.31−3.27 (m, 1H), 3.10−3.05 (m,
1H), 3.00−2.94 (m, 1H), 2.89−2.85 (m, 1H), 2.57 (s, 4H), 1.04 (t, J
= 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) δ 144.30, 144.13,
143.58, 137.37, 136.83, 135.40, 134.21, 129.38, 127.32, 126.84,
126.12, 125.82, 125.14, 124.23, 56.86, 53.36, 46.40, 45.74, 39.57,
28.65, 11.52. HRMS (ESI): calcd for C23H28N5 [M + H]+ 374.2339,
found 374.2341.
4-(3-(3-(3-Morpholinopropoxy)-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-6-yl)propyl)-
morpholine (13e). The product was obtained as white solid. Yield
1
74%. mp 44−46 °C. H NMR (600 MHz, DMSO-d6) δ 7.43 (d, J =
1.7 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.21−7.17 (m, 2H), 7.02 (s,
1H), 7.14 (d, J = 8.4 Hz, 1H), 6.85 (dd, J = 8.3, 2.2 Hz, 1H), 5.00−
4.97 (m, 1H), 4.81−4.79 (m, 1H), 4.14 (dd, J = 11.8, 3.5 Hz, 1H),
4.05−3.99 (m, 2H), 3.92−3.87 (m, 1H), 3.60−3.57 (m, 8H), 3.49−
3.45 (m, 2H), 3.04−3.00 (m, 1H), 2.91−2.85 (m, 1H), 2.81 (d, J =
15.5 Hz, 1H), 2.44−2.42 (m, 2H), 2.44−2.42 (m, 2H), 2.37 (d, J =
4.1 Hz, 8H), 1.90−1.83 (m, 4H). 13C NMR (151 MHz, DMSO-d6) δ
157.67, 143.53, 143.19, 137.49, 136.62, 134.95, 130.42, 127.26,
125.24, 124.91, 124.63, 123.95, 114.01, 111.97, 66.70, 66.67, 66.37,
56.12, 55.43, 53.88, 53.73, 52.83, 51.76, 46.54, 39.56, 27.71, 26.43,
23.20. HRMS (ESI): calcd for C32H43N6O3 [M + H]+ 559.3391,
found 559.3388.
2-(3-(2-(Dimethylamino)ethoxy)-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-6-yl)-N,N-di-
methylethan-1-amine (13a). Compound 8b (0.5 mmol, 152 mg)
was dissolved in DMF (10 mL); then sodium hydride was added. The
mixture was stirred at room temperature for 0.5 h; then 2-chloroo-
N,N-dimethylethan-1-amine hydrochloride (2 mmol, 287 mg) was
added. Removal of the solvent was performed, ethyl acetate (50 mL)
was added, and then the organic phase was washed with water (3 × 50
mL), dried over MgSO4, and concentrated. The crude products were
purified by column chromatography, affording 13a (70 mg, 33%
yield) as white solid. mp 95−97 °C. 1H NMR (600 MHz, DMSO-d6)
δ 7.44 (d, J = 7.3 Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 7.22−7.12 (m,
3H), 7.06 (s, 1H), 6.86 (d, J = 8.4 Hz, 1H), 5.00−4.98 (m, 1H), 4.77
(d, J = 8.9 Hz, 1H), 4.21−4.19 (m, 1H), 4.13−4.03 (m, 3H), 3.63−
3.60 (m, 1H), 3.50 (t, J = 10.9 Hz, 1H), 3.01 (t, J = 10.7 Hz, 1H),
2.91−2.80 (m, 2H), 2.64−2.54 (m, 4H), 2.22 (d, J = 7.1 Hz, 12H).
13C NMR (151 MHz, DMSO-d6) δ 157.54, 143.53, 143.09, 137.45,
3-(2-(Piperidin-1-yl)ethoxy)-6-(2-(piperidin-1-yl)ethyl)-
4b,5,14,15-tetrahydro-6H-isoquinolino[2′,1′:1,6]pyrazino[2,3-
b]quinoxaline (13f). The product was obtained as white solid. Yield
40%. mp 143−145 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.44−7.43
(m, 1H), 7.39−7.38 (m, 1H), 7.20−7.15 (m, 2H), 7.12 (d, J = 8.4 Hz,
1H), 7.01 (d, J = 2.3 Hz, 1H), 6.84 (dd, J = 8.4, 2.4 Hz, 1H), 5.00−
4.97 (m, 1H), 4.74 (dd, J = 9.9, 2.8 Hz, 1H), 4.21 (dd, J = 11.9, 3.5
Hz, 1H), 4.10−4.02 (m, 2H), 3.99−3.95 (m, 1H), 3.74−3.69 (m,
136.60, 134.91, 130.42, 127.26, 125.23, 124.98, 124.64, 123.96,
114.03, 111.98, 66.35, 58.26, 56.20, 52.82, 51.88, 46.06, 45.89, 45.73,
1031
https://dx.doi.org/10.1021/acs.jmedchem.0c01512
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1H), 3.50−3.46 (m, 1H), 3.40 (bs, 4H), 3.02−2.98 (m, 1H), 2.90−
2.85 (m, 1H), 2.79 (d, J = 15.6 Hz, 1H), 2.65 (t, J = 5.97 Hz, 4H),
2.60−2.58 (m, 4H), 2.43 (s, 8H), 1.52−1.45 (m, 8H), 1.37 (d, J = 5.3
Hz, 4H) ppm. 13C NMR (151 MHz, DMSO) δ 157.53, 143.52,
143.06, 137.49, 136.62, 134.88, 130.42, 127.25, 125.23, 124.96,
124.60, 123.91, 114.24, 111.65, 66.13, 57.89, 55.75, 54.91, 54.85,
54.70, 52.89, 52.18, 45.23, 39.57, 27.70, 26.05, 24.40 ppm. HRMS
(ESI, m/z): calcd for C32H43N6O [M + H]+ 527.3493, found
527.3495.
3.63−3.58 (m, 9H), 3.48 (t, J = 10.9 Hz, 1H), 3.35 (s, 2H), 3.04−
2.94 (m, 2H), 2.87 (d, J = 15.2 Hz, 1H), 2.44−2.34 (m, 10H), 1.91−
1.80 (m, 4H). 13C NMR (151 MHz, DMSO-d6) δ 156.44, 143.42,
142.25, 138.47, 135.44, 134.01, 131.07, 129.49, 127.39, 126.75,
126.12, 126.08, 114.26, 106.62, 66.69, 66.33, 56.08, 55.38, 53.86,
53.74, 52.65, 52.01, 46.49, 28.53, 26.41, 23.23. HRMS (ESI): calcd
for C32H43O3N6 [M + H]+ 559.3391, found 559.3397.
9-(2-(Piperidin-1-yl)ethoxy)-6-(2-(piperidin-1-yl)ethyl)-
4b,5,14,15-tetrahydro-6H-isoquinolino[2′,1′:1,6]pyrazino[2,3-
b]quinoxaline (14c). The product was obtained as yellow solid.
Yield 63%. mp 125−128 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.46
(d, J = 6.8 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.25 (s, 3H), 6.85−6.80
(m, 2H), 4.91 (d, J = 10.2 Hz, 1H), 4.80 (d, J = 8.2 Hz, 1H), 4.12 (d,
J = 9.9 Hz, 1H), 4.03 (s, 1H), 3.86 (s, 1H), 3.62 (s, 1H), 3.51−3.47
(m, 1H), 3.04−2.98 (m, 2H), 2.88 (d, J = 15.3 Hz, 1H), 2.36 (d, J =
37.1 Hz, 11H), 1.88 (s, 4H), 1.51−1.38 (m, 10H). 13C NMR (151
MHz, DMSO-d6) δ 156.02, 155.80, 143.44, 142.26, 138.51, 135.46,
134.05, 131.03, 129.51, 125.84, 114.25, 108.88, 106.69, 66.50, 55.48,
54.62, 52.90, 52.43, 43.12, 26.12, 24.21. HRMS (ESI): calcd for
C32H43N6O [M + H]+ 527.3493, found 527.3495.
9-(3-(Piperidin-1-yl)propoxy)-6-(3-(piperidin-1-yl)propyl)-
4b,5,14,15-tetrahydro-6H-isoquinolino[2′,1′:1,6]pyrazino[2,3-
b]quinoxaline (14d). The product was obtained as yellow solid.
Yield 42%. mp 121−123 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.46
(d, J = 7.3 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.30−7.24 (m, 3H),
6.85 (d, J = 2.6 Hz, 1H), 6.81 (dd, J = 8.8, 2.7 Hz, 1H), 4.93−4.90
(m, 1H), 4.80 (dd, J = 9.8, 2.8 Hz, 1H), 4.12 (dd, J = 11.8, 3.4 Hz,
1H), 4.03 (t, J = 6.5 Hz, 2H), 3.88−3.83 (m, 1H), 3.64−3.59 (m,
1H), 3.48 (t, J = 10.9 Hz, 1H), 3.05−2.95 (m, 2H), 2.89 (d, J = 15.2
Hz, 1H), 2.40−2.31 (m, 12H), 1.90−1.79 (m, 4H), 1.52−1.49 (m,
8H), 1.39 (s, 4H). 13C NMR (151 MHz, DMSO-d6) δ 156.46,
143.44, 142.26, 138.51, 135.46, 134.05, 131.03, 129.51, 127.39,
126.75, 126.10, 126.07, 114.25, 106.68, 66.52, 56.41, 55.66, 54.62,
54.47, 52.66, 52.05, 46.61, 39.57, 28.54, 26.84, 26.11, 24.72, 24.66,
23.69. HPLC purity: 99.10%, tR = 15.7 min. HRMS (ESI): calcd for
C34H47ON6 [M + H]+ 555.3806, found 555.3809.
3-(3-(Piperidin-1-yl)propoxy)-6-(3-(piperidin-1-yl)propyl)-
4b,5,14,15-tetrahydro-6H-isoquinolino[2′,1′:1,6]pyrazino[2,3-
b]quinoxaline (13g). The product was obtained as white solid.
1
Yield 69%. mp 101−103 °C. H NMR (600 MHz, CDCl3) δ 7.56−
7.52 (m, 2H), 7.25−7.22 (m, 2H), 7.12 (d, J = 8.4 Hz, 1H), 6.83−
6.81 (m, 1H), 6.78−6.77 (m, 1H), 5.18−5.16 (m, 1H), 4.74 (dd, J =
10.2, 3.0 Hz, 1H), 4.02 (t, J = 6.4 Hz, 2H), 3.88−3.85 (m, 2H), 3.76−
3.72 (m, 1H), 3.51 (t, J = 10.9 Hz, 1H), 3.07−2.96 (m, 2H), 2.82 (d,
J = 15.1 Hz, 1H), 2.50−2.38 (m, 12H), 2.21 (t, J = 7.3 Hz, 4H),
2.02−1.95 (m, 3H), 1.94−1.89 (m, 1H). 13C NMR (151 MHz,
CDCl3) δ 157.69, 143.33, 142.96, 137.45, 136.65, 134.16, 130.24,
127.41, 125.14, 125.03, 124.55, 123.98, 113.57, 111.47, 66.76, 56.60,
56.01, 54.67, 54.64, 53.06, 52.70, 46.74, 39.53, 29.70, 28.13, 26.84,
25.93, 24.42, 24.41, 24.05. HRMS (ESI): calcd for C30H39N6O3 [M +
H]+ 555.3806, found 555.3829.
3-(3-(4-Methylpiperazin-1-yl)propoxy)-6-(3-(4-methylpiper-
azin-1-yl)propyl)-4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b]quinoxaline (13h). The product was
1
obtained as white solid. Yield 69%. mp 88−90 °C. H NMR (600
MHz, DMSO-d6) δ 7.43 (dd, J = 7.7, 1.7 Hz, 1H), 7.36 (dd, J = 7.8,
1.6 Hz, 1H), 7.21−7.15 (m, 2H), 7.13 (d, J = 8.4 Hz, 1H), 7.00 (d, J
= 2.3 Hz, 1H), 6.83 (dd, J = 8.4, 2.4 Hz, 1H), 5.00−4.97 (m, 1H),
4.80−4.78 (m, 1H), 4.12 (dd, J = 11.8, 3.6 Hz, 1H), 4.02−3.98 (m,
2H), 3.88−3.83 (m, 1H), 3.62−3.58 (m, 1H), 3.46 (dd, J = 11.6, 10.3
Hz, 2H), 3.04−3.01 (m, 1H), 2.90−2.85 (m, 1H), 2.80 (d, J = 15.6
Hz, 1H), 2.51−2.50 (m, 2H), 2.43−2.40 (m, 4H), 2.38−2.31 (m,
13H), 2.14 (d, J = 1.6 Hz, 6H), 1.89−1.80 (m, 4H). 13C NMR (151
MHz, DMSO-d6) δ 157.69, 143.52, 143.18, 137.52, 136.62, 134.94,
130.41, 127.23, 125.24, 124.91, 124.60, 123.91, 113.98, 111.95, 66.42,
55.63, 55.29, 55.24, 54.98, 53.24, 53.06, 52.82, 51.86, 46.60, 46.26,
46.22, 40.43, 27.72, 26.81, 23.58. HRMS (ESI): calcd for C34H49N8O
[M + H]+ 585.4024, found 585.4005.
9-(3-(4-Methylpiperazin-1-yl)propoxy)-6-(3-(4-methylpiper-
azin-1-yl)propyl)-4b,5,14,15-tetrahydro-6H-isoquinolino-
[2′,1′:1,6]pyrazino[2,3-b]quinoxaline Dihydrochloride (14e).
The product was obtained as yellow solid. Yield 30%. mp 230−231
1
°C. H NMR (600 MHz, DMSO-d6) δ 12.14 (s, 3H), 7.67 (s, 1H),
7.55 (d, J = 7.0 Hz, 1H), 7.33−7.30 (s, 4H), 6.99 (dd, J = 8.9, 2.5 Hz,
1H), 5.03 (d, J = 8.2 Hz, 1H), 4.93 (dd, J = 8.6, 3.4 Hz, 1H), 4.29 (d,
J = 9.0 Hz, 1H), 4.14 (t, J = 5.5 Hz, 2H), 4.07−4.00 (m, 1H), 3.85−
3.69 (m, 14H), 3.62−3.59 (m, 2H), 3.55−3.42 (m, 6H), 3.23 (s, 1H),
3.06−3.22 (m, 1H), 2.96 (d, J = 16.1 Hz, 1H) 2.83 (s, 6H), 2.28−
2.53 (m, 4H). 13C NMR (151 MHz, DMSO-d6) δ 156.37, 144.45,
142.24, 138.69, 135.43, 134.29, 131.67, 129.42, 127.31, 126.81,
126.23, 126.15, 113.90, 106.55, 66.38, 56.41, 55.21, 54.97, 53.25,
53.16, 47.71, 46.17, 45.93, 39.57, 34.55, 28.66, 26.79, 24.23, 18.80.
HRMS (ESI): calcd for C34H48N8O [M + H]+ 585.4024, Found
585.4022.
2-(9-(2-(Dimethylamino)ethoxy)-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-6-yl)-N,N-di-
methylethan-1-amine Dihydrochloride (14f). The product was
obtained as yellow solid. Yield 50%. mp 259−262 °C. 1H NMR (600
MHz, DMSO-d6) δ 11.12 (s, 1H), 10.66 (s, 1H), 7.83 (s, 1H), 7.62
(d, J = 7.4 Hz, 1H), 7.41−7.36 (m, 3H), 7.19 (s, 1H), 7.09 (dd, J =
8.9, 2.4 Hz, 1H), 5.21 (d, J = 8.5 Hz, 1H), 5.02 (dd, J = 9.1, 3.7 Hz,
1H), 4.51 (t, J = 4.9 Hz, 2H), 4.37 (dd, J = 11.7, 3.3 Hz, 1H), 4.28−
4.26 (m, 1H), 4.17−4.15 (m, 1H), 3.68−3.54 (m, 5H), 3.13−2.90
(m, 15H). 13C NMR (151 MHz, DMSO) δ 155.73, 143.99, 135.07,
133.12, 129.33, 127.75, 127.21, 126.97, 126.24, 123.16, 114.88,
107.65, 63.16, 55.63, 53.90, 43.17, 43.06, 39.57, 28.31. HRMS (ESI):
calcd for C26H35ON6 [M + H]+ 447.2867, found 447.2869.
4-(2-(9-(2-Morpholinoethoxy)-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-6-yl)ethyl)-
morpholine Dihydrochloride (14a). Compound 8l (1.65 mmol,
500 mg) was dissolved in DMF (10 mL); then sodium hydride (16.5
mmol, 650 mg) was added. The mixture was stirred at room
temperature for 0.5 h; then 4-(2-chloroethyl)morpholine hydro-
chloride (6.59 mmol, 1.2 g) was added. Removal of the solvent was
performed, then ethyl acetate (50 mL) was added, the organic phase
was washed with water (3 × 50 mL), dried over MgSO4, and
concentrated. The crude products were purified by column
chromatography affording a clear oil, which was treated with saturated
hydrogen chloride in dioxane under an ice bath for 2 h to give 14a
(249 mg, 28% yield) as yellow solid. mp 269−271 °C. 1H NMR (600
MHz, DMSO-d6) δ 11.86 (s, 1H), 11.07 (s, 1H), 7.78 (s, 1H), 7.54
(d, J = 7.4 Hz, 1H), 7.35−7.30 (m, 3H), 7.11 (s, 1H), 7.02 (dd, J =
8.9, 2.6 Hz, 1H), 5.16 (d, J = 8.5 Hz, 1H), 4.95 (dd, J = 8.7, 3.8 Hz,
1H), 4.78−4.08 (m, 8H), 4.06−3.72 (m, 8H), 3.66 (t, J = 11.1 Hz,
2H), 3.58−3.50 (m, 5H), 3.30−3.21 (m, 4H), 3.07−2.95 (m, 2H).
13C NMR (151 MHz, DMSO-d6) δ 155.78, 144.16, 141.24, 136.33,
135.05, 133.09, 130.12, 129.31, 127.78, 127.01, 126.24, 126.18,
114.98, 107.47, 63.59, 63.44, 63.11, 55.25, 53.52, 53.23, 52.14, 50.73,
42.59, 40.52, 39.56, 28.26. HRMS (ESI): calcd for C30H39O3N6 [M +
H]+ 531.3078, found 531.3084.
4-(3-(9-(3-Morpholinopropoxy)-4b,5,14,15-tetrahydro-6H-
isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-6-yl)propyl)-
morpholine (14b). The product was obtained as yellow solid. Yield
55%. mp 264−266 °C. 1H NMR (600 MHz, DMSO-d6) δ 7.45 (d, J =
7.4 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.29−7.23 (m, 3H), 6.83−6.80
(m, 2H), 4.92−4.90 (m, 1H), 4.78 (dd, J = 9.8, 2.8 Hz, 1H), 4.11 (dd,
J = 11.8, 3.4 Hz, 1H), 4.03 (t, J = 6.4 Hz, 2H), 3.91−3.86 (m, 1H),
3-(9-(3-(Dimethylamino)propoxy)-4b,5,14,15-tetrahydro-
6H-isoquinolino[2′,1′:1,6]pyrazino[2,3-b]quinoxalin-6-yl)-N,N-
dimethylpropan-1-amine Dihydrochloride (14g). The product
1
was obtained as yellow solid. Yield 66%. mp 253−256 °C. H NMR
(600 MHz, DMSO-d6, D2O) δ 7.58 (d, J = 8.8 Hz, 1H), 7.47 (d, J =
6.5 Hz, 1H), 7.30 (d, J = 9.4 Hz, 3H), 7.21 (s, 1H), 6.99 (d, J = 8.7
1032
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J. Med. Chem. 2021, 64, 1018−1036

Journal of Medicinal Chemistry
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Article
Hz, 1H), 4.99 (d, J = 9.4 Hz, 1H), 4.72 (d, J = 9.5 Hz, 1H), 4.21 (d, J
= 11.8 Hz, 1H), 4.10 (t, J = 5.6 Hz, 2H), 3.93−3.90 (m, 1H), 3.85−
3.76 (m, 1H), 3.60 (t, J = 11.3 Hz, 1H), 3.28−3.22 (m, 3H), 3.17 (t, J
= 7.4 Hz, 2H), 3.05−2.91 (m, 2H), 2.79 (d, J = 14.5 Hz, 12H), 2.16−
2.11 (m, 4H). 13C NMR (151 MHz, DMSO-d6, D2O) δ 156.50,
143.02, 141.66, 135.11, 132.95, 129.43, 127.76, 126.98, 126.28,
114.88, 65.71, 54.43, 42.51, 42.47, 42.39, 29.49, 28.30, 24.32, 21.59.
HPLC purity: 98.60%, tR = 13.7 min. HRMS (ESI): calcd for
C28H39ON6 [M + H]+ 475.3180, found 475.3184.
Biological Evaluation. Cells and Materials. The human
hepatoma cancer cell lines QGY-7701 and HepG2, lung cancer cell
line A549, and human colon cancer cell line LOVO were obtained
from the Cell Bank of the Shanghai Institute of Biochemistry and Cell
Biology (Chinese Academy of Sciences, China). All the cells were
cultured with DMEM containing 10% fetal bovine serum (FBS), 100
U/mL penicillin and 100 μg/mL streptomycin. The cells were
cultured at 37 °C and 5% CO2. All primary antibodies in this study
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Oil red O dye was purchased from Yuanye Biotechnology
(Shanghai, China). Cell counting kit-8 (CCK-8) was purchased from
Yeasen Biotech Co., Ltd. (Shanghai, China). Rosiglitazone (RSG) was
purchased from Sigma-Aldrich (St. Louis, MO, USA). T0070907 was
purchased from MedChemExpress China.
Cells Viability Assay. Cell viability upon the treatment of the
prepared compounds and control drug TNBG, Sorafenib, and
Erlotinib was tested by using the CCK-8 assay. Briefly, a suspension
of cells (3000/well for HepG2, LOVO, A549, and QGY-7701) was
seeded in 96-well plates and cultured for 24 h. Then, eight different
concentrations ranging from 30 to 0.20 μM (PBS buffer containing
1% DMSO) for the prepared compounds and 100−0.80 μM for
Sorafenib and Erlotinib were added into the 96-well plates in which
cells were incubated for 72 h. Then, 10 μL of CCK-8 solution was
added to each well of the plates and incubated at 37 °C for 2 h. The
absorbance of the each well was measured at 450 nm in a microplate
reader. At last, resultant OD450 nm values were expressed as IC50
values, which were the mean values derived from three independent
experiments.
Colony Formation Assay. A549 and HepG2 cells were seeded in
6-well plates with each well contained 400 cells in 2 mL of the
medium for 24 h. Then, 14g (0.1, 0.2 and 0.3 μM) and TNBG (1, 2,
and 3 μM) in the fresh medium were added into the plates to treat
cells for a continuous 3 days and then replaced by the drug-free
medium for every 3 days. Fifteen days after cell plating, cell colonies
were fixed in 95% ethanol for 15 min and stained by 0.1% Crystal
Violet (Sigma) and then dried for colonies counting. Three
independent experiments were conducted, each in triplicate.
Flow Cytometry Apoptosis Assay. A549 and HepG2 cells were
seeded at density of 5 × 105 cells/mL on each well of 6-well plates
and allowed to grow overnight. Then, they were treated with 14g (0.9,
1.2 and 1.5 μM), TNBG (10 and 20 μM) for A549, or 14g (0.9, 1.2
and 1.5 μM) for HepG2 for 72 h at 37 °C. The cells were then
trypsinized, repeatedly washed with cold PBS three times and
centrifuged at 1200 rpm for 5 min to pellet. Then, cells were
resuspended to ∼5 × 105 cells/mL in 1 × Annexin binding buffer
(100 mL); to which, 5 μL of Annexin V and 10 μL of PI were added
and allowed to bind for 15 min at room temperature. After the
incubation, 400 μL of binding buffer was added to each sample and
was gently mixed. The samples were analyzed immediately on a BD
Biosciences FACSCalibur flow cytometer.
BCA protein assay reagent (Beyotime, Jiangsu, China), followed by 8
min protein denaturation with SDS loading buffer at 100 °C. Proteins
(20 μg) were separated by sodium dodecyl sulfate-polyacrylamide
(SDS-PAGE) electrophoresis and transferred onto polyvinylidene
fluoride (PVDF) membranes, which were blocked with 5% fat-free
dry milk in 1 × Tris-buffered saline (TBS) containing 0.05% Tween
20 for 2 h at room temperature. Then, the membranes were incubated
with AKT, PTEN, and PPARγ antibodies at 4 °C for overnight,
followed by treatment with secondary horseradish-peroxidase-
conjugated anti-rabbitIgG for 2 h. Membranes were finally scanned
in a ChemiDoc MP Imaging System after 2 min incubation in a clarity
western ECL substrate.
Efficacy Study in the A549 Xenograft Model. The animal study
protocol was approved by The Animal Research Ethics Committee of
The Chongqing Medical University and carried out strictly under the
supervision and recommendation of the committee. Six-to-eight
weeks of nude mice were inoculated subcutaneously with 2 × 106
A549 cells suspended in serum-free DMEM in the right flank. Seven
days after inoculation, the tumor-bearing mice were randomly
assigned into individual groups and treated with 14g at a dose of
10 mg/kg daily for a continuous 18 days. The tumor volume of each
mouse was recorded twice a week. At the end of the experiment, mice
were humanely killed by CO2 inhalation, while tumor samples were
harvested, weighted, and photographed. For the acute toxicity of 14g,
the experiment was carried following the above mentioned. Mice were
treated with 14g at a dose of 106, 125, 145, 170, and 200 mg/kg daily
for 7 days. At the end of the animal study, main tissues of the mice
(heart, liver, kidney, and lung) were harvested to perform H&E
staining.
Solubility Measurement. The solubility measurement was
conducted as previous reported literature.57 Approximately 2 mg of
the TNBG, 13c, 13f, 13g, 14d, and 65 mg of 14g was added into a
syringeless filter (Whatman, Uniprep, 0.45 μm); then 2 mL of 50
mmol/L phosphate buffer pH 7.4 was added and the suspension was
shaken on an orbital shaker at 400 rpm for 6 h at 37 °C. Subsequently,
the suspension was shaken for a further 18 h. After completion of
incubation, the solid and liquid phases were separated by pushing
down the plunger of the syringeless filter. Immediately after the
filtration step, 200 μL of acetonitrile was added to the filtrate to avoid
precipitation from the saturated solution. The concentrations of the
tested compounds were determined by HPLC with UV detection.
Morphological Changes Detection. The A549 cells (5 × 105/
well) were seeded in 6-well plates for 24 h; then 14g at 1.6 and 2.0
μM and TNBG at 10 μM were added to individual well of the plates,
followed by extra 8, 12, 24, and 48 h incubation. The morphological
changes of the cells were imaged by a microscope at end of each time
incubation.
Oil Red O Staining. A549 and HepG2 cells were seeded in 24-well
plates (5 × 104 of cells/well) overnight. The cells were treated with
the 14g (0, 0.3, 0.6, and 0.9 μM) for 72 h. Then, the medium was
removed; cells were carefully washed with phosphate-buffered saline
(PBS) three times. The cells were fixed for at least 1 h with 10%
formalin at room temperature and rinsed with double distilled water.
The fixed cells were stained by complete immersion in the working
solution of Oil red O for 0.5 h. After the Oil Red O staining was
performed, the morphological changes in the formation and
accumulation of lipid droplets were observed by imaging with an
Olympus inverted microscope.
PPARγ Transactivation Assay of Compound 14g. Cell-based
transactivation assay: Cos-7 cells from ATCC were grown to 70%
confluence in DMEM containing 10% FBS. For assessing full-length
PPAR receptors, Cos-7 cells were transiently co-transfected with 100
ng of plasmid containing the luciferase gene under the control of three
tandem PPAR response elements (PPRE × 3 TK-luciferase) and 50
ng of full-length hPPARγ expression plasmid using lipofectamine 2000
(Invitrogen) along with 10 ng of Renilla luciferase expression plasmid
for standardization. 24 h after transfection, the cells were treated with
the RSG and compounds at the indicated concentration for 24 h.
Luciferase activity was determined with the reporter luciferase assay
kit (Promega) according to the manufacturer’s instructions using an
Flow Cytometry Cell Cycle Assay. A549 and HepG2 cells were
seeded in 6-well plates at a density of 5 × 105 cells/well and treated
with compound 14g (0.9, 1.2 and 1.5 μM) for 72 h at 37 °C. Then,
cells were harvested and fixed in 70% precooled ethanol. The cells
were then treated with RNase A, and stained by propidium iodide
dye. Finally, the suspended cells were analyzed with a flow cytometer
(Accuri C6, BD Biosciences), a minimum total of 10,000 events were
recorded. The data were analyzed with the FlowJo.
Western Blotting. After treating A549 and HepG2 cells with 14g
(0.9, 1.2, 1.5, and 1.8 μM) for 72 h, the total proteins were extracted
and their concentrations were balanced to the same level using the
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Article
Envision luminometer (PerkinElmer). Luciferase activity was
normalized to Renilla activity. Each condition was performed with n
≥ 3 for each experiment. As a control, the activity was measured in
the presence of a vehicle (DMSO).46,47
Zongjie Gan − Key Laboratory for Biochemistry and
Molecular Pharmacology of Chongqing, Department of
Medicinal Chemistry, School of Pharmacy, Chongqing
Medical University, Chongqing 400016, China
TR-FRET Binding Assay. The GST-PPARγ LBD was labeled with a
terbium-linked antibody and incubated with a fluorescent small
molecule pan-PPAR ligand (Fluormone Pan-PPAR Green, tracer).
Excitation of terbium fluorescence at 340 nm results in a time-
resolved fluorescence energy transfer (TR-FRET) to bound
Fluormone with an emission at 520 nm. Agonist binding was
determined as a decrease in FRET (the ratio of emission at 520 nm to
emission from terbium fluorescence at 495 nm. Notably, with regard
to compound 14g, the emission value at 520 nm should minus
fluorescent emission value at 520 nm of 14g excited by 340 nM
excitation) due to the displacement of the fluorescent tracer from the
ligand-binding domain upon agonist binding. The inhibition constant
(Ki) was calculated by applying the S6 Cheng−Prusoff equation as
following:
Yanrong Dan − Key Laboratory for Biochemistry and
Molecular Pharmacology of Chongqing, Department of
Medicinal Chemistry, School of Pharmacy, Chongqing
Medical University, Chongqing 400016, China
Yaowei Li − Key Laboratory for Biochemistry and Molecular
Pharmacology of Chongqing, Department of Medicinal
Chemistry, School of Pharmacy, Chongqing Medical
University, Chongqing 400016, China
Peiming Zhang − Key Laboratory for Biochemistry and
Molecular Pharmacology of Chongqing, Department of
Medicinal Chemistry, School of Pharmacy, Chongqing
Medical University, Chongqing 400016, China
Shanwen Chen − Key Laboratory for Biochemistry and
Molecular Pharmacology of Chongqing, Department of
Medicinal Chemistry, School of Pharmacy, Chongqing
Medical University, Chongqing 400016, China
Zaijun Ye − Key Laboratory for Biochemistry and Molecular
Pharmacology of Chongqing, Department of Medicinal
Chemistry, School of Pharmacy, Chongqing Medical
University, Chongqing 400016, China
Tao Pan − Key Laboratory for Biochemistry and Molecular
Pharmacology of Chongqing, Department of Medicinal
Chemistry, School of Pharmacy, Chongqing Medical
University, Chongqing 400016, China
Chunmei Wan − Key Laboratory for Biochemistry and
Molecular Pharmacology of Chongqing, Department of
Medicinal Chemistry, School of Pharmacy, Chongqing
Medical University, Chongqing 400016, China
Xuelian Hu − Key Laboratory for Biochemistry and Molecular
Pharmacology of Chongqing, Department of Medicinal
Chemistry, School of Pharmacy, Chongqing Medical
University, Chongqing 400016, China
IC50
Ki =
[tracer]
1 +
KD
where IC50 is the concentration of the competitor that produces 50%
displacement of the tracer, [tracer] is the concentration of Fluormone
Pan-PPAR Green used in the assay (5 nM), and KD is the binding
constant of Fluormone Pan-PPAR Green to PPAR γ-LBD. This KD
value has been determined to be 2.8 0.8 nM (average standard
deviation calculated from three separate experiments).46,47
Molecular Docking Study. Molecular docking was performed
using a Sybyl-X 2.0 software. The crystal structure of PPARγ protein
was downloaded from the Protein Data Bank (PDB ID: 3S9S). The
crystal structure of PPARγ was optimized with H added and charge
added by the AMBER7 FF99 method. The structure of small
molecules was subjected to the polar H adding and being energy
optimized with a tripos force field and charged optimized with the
Gasteiger−Huckel method. The graphics of 3D views were drawn by
PyMOL, and the amino acids involved in hydrophobic interaction
were shown as stick.
Statistical Analysis. Data are reported as mean
standard
deviation (SD). Statistical analysis was performed using SPSS
statistics software (Version 19.0) or GraphPad Prism version 6.0 for
Windows. Data were analyzed by performing one-way analysis of
variance (ANOVA) followed by post hoc Tukey’s test. *p < 0.05, **p
< 0.01, and ***p < 0.001 are considered as statistically significant.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01512
Author Contributions
#L.G. and Z.G. contributed equally to this study.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01512.
■
sı
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Ligands in PPARγ LBD, H NMR, 13C NMR, HRMS
1
■
This work was supported by a research grant from the National
Natural Science Foundation of China (grant nos. NSFC
30371632, 30772595, 30171070), and Chongqing Science and
Technology Commission (grant no. cstc2017jcyjAX0228), and
Scientific and Technological Research Program of Chongqing
Municipal Education Commission (grant nos. KJ1600201,
KJQN201900431). The authors thank Professor Wei Yi and
Dr. Lei Ma in Guangzhou Medical University for the support
on PPARγ transactivation assay and TR-FRET binding assay.
and purity analysis data and spectra (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
■
Yu Yu − Key Laboratory for Biochemistry and Molecular
Pharmacology of Chongqing, Department of Medicinal
Chemistry, School of Pharmacy, Chongqing Medical
University, Chongqing 400016, China; Phone: +86-23-
68485983; Email: yuyu3519@163.com
ABBREVIATIONS
■
Authors
CCK-8, Cell counting kit-8; DA, decanoic acid; FBS, fetal
bovine serum; LD50, lethal dose 50%; NT, not tested; PBS,
phosphate-buffered saline; PI, propidium iodide; PPARγ,
peroxisome proliferator-activated receptor gamma; PTEN,
phosphate and tension homology deleted on chromosome
ten; PVDF, polyvinylidene fluoride; RMSD, root-mean-square
Linling Gan − Key Laboratory for Biochemistry and Molecular
Pharmacology of Chongqing, Department of Medicinal
Chemistry, School of Pharmacy, Chongqing Medical
University, Chongqing 400016, China; orcid.org/0000-
0002-2012-9405
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deviation; RSG, rosiglitazone; SAR, structure−activity relation-
ship; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide;
TBS, tris-buffered saline; TLC, thin-layer chromatography;
TNBG, tetrazanbigen; TR-FRET, time-resolved fluorescence
energy transfer.
(20) Schaffer, J. E. Lipotoxicity: when tissues overeat. Curr. Opin.
Lipidol. 2003, 14, 281−287.
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