The synthesis and biological evaluation of sanguinarine derivatives as anti-non-small cell lung cancer agents

Article abstract of DOI:10.1039/c9md00494g

A novel series of sanguinarine (SA) derivatives were synthesized and evaluated as anti-non-small cell lung cancer (NSCLC) agents. The compounds inhibited A549 and H1975 NSCLC cells with IC₅₀ values of 0.96->30 ΜM and 0.79->30 ΜM, respectively. Compounds 8d-8j exhibited low micromolar inhibitory activity and indicated that the C6-position of SA was tolerated to be substituted by hydrophilic groups and CN. Further investigation of their mechanism of action showed that compound 8h induced apoptosis of A549 and H1975 cells by inhibiting the Akt signaling pathway and elevating the reactive oxygen species (ROS). This study provided a strategy for developing new anti-cancer agents.

Full text of DOI:10.1039/c9md00494g

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RESEARCH ARTICLE
The synthesis and biological evaluation of
sanguinarine derivatives as anti-non-small cell
lung cancer agents†
Cite this: DOI: 10.1039/
c9md00494g
Liang Jiang,‡ Xiaolu Wang,‡ Yuting Wang, Fang Xu, Zhang Zhang,*
Ke Ding * and Xiaoyun Lu
*
A novel series of sanguinarine (SA) derivatives were synthesized and evaluated as anti-non-small cell lung
cancer (NSCLC) agents. The compounds inhibited A549 and H1975 NSCLC cells with IC50 values of 0.96 –
>30 μM and 0.79 – >30 μM, respectively. Compounds 8d–8j exhibited low micromolar inhibitory activity
and indicated that the C6-position of SA was tolerated to be substituted by hydrophilic groups and CN.
Further investigation of their mechanism of action showed that compound 8h induced apoptosis of A549
and H1975 cells by inhibiting the Akt signaling pathway and elevating the reactive oxygen species (ROS).
This study provided a strategy for developing new anti-cancer agents.
Received 20th October 2019,
Accepted 19th January 2020
DOI: 10.1039/c9md00494g
rsc.li/medchem
Non-small-cell lung cancer (NSCLC) is one of the common
aggressive malignant tumors, accounting for ∼85% of human
lung cancer, which is a disease that remains the leading
can effectively overcome epidermal growth factor receptor
T790M
threonine 790 to methionine 790 (EGFR
) mutation resis-
tance, one of the primary mechanisms for NSCLC resistance
in clinical, with the mechanism showing elevated reactive ox-
ygen species (ROS) and redox imbalance between NADPH oxi-
1
cause of cancer-related mortality worldwide. The reported
overall 5-year survival rate for NSCLC (all stages combined) is
roughly 17% and the 5-year survival rate in stage IV is only
1
4
dase isoform 3 (NOX3) and methionine reductase A (MsrA).
2
about 2%. The treatment of NSCLC has evolved over the past
However, the pharmacokinetic properties of SA are not so
good with low blood concentration and poor metabolic insta-
two decades, resulting in different therapeutic options, in-
3
4
15
cluding chemotherapy, tyrosine kinase inhibitors, monoclo-
bility. It has been found that SA can be metabolized into
5
6
nal antibodies, and some combinations. Remarkable ad-
vances in targeted small-molecule kinase inhibitors have led
to great improvements in the survival rates and quality of life
of NSCLC patients. However, resistance to targeted agents has
about seven metabolites by pig liver microsomes through oxi-
1
6
dation, reduction, demethoxylation, etc. One of the main
metabolites, dihydrosanguinarine (DHSA, 2), was more stable
1
6
than SA, thus suggesting that the optimization of the
7
+
inevitably emerged during the treatment. Therefore, new
CN bond may improve the metabolic stability of SA. Some
+
drugs and new strategies for the effective treatment of drug-
resistant NSCLC are urgently needed to be developed.
SA derivatives by reducing the CN bond or modifying the
5- or 6-position of SA have been reported and their antifungal
1
1,17
Sanguinarine (SA, 1) (Fig. 1), a quaternary benzo[c]
phenanthridine alkaloid, is mainly found in plant families
and antibacterial activities were explored,
while no anti-
cancer activity was reported. This encouraged us to further
investigate SA derivatives with the aim of discovering new
anti-NSCLC agents.
8
Papaveraceae, Amaranthus and Rutaceae. Because SA has a
unique structure, it has been widely studied by scientists. It
9
,10
was found that SA and its analogues have anticancer,
bacterial,
anti-
In this study, we designed and synthesized a new series of
1
1,12
13
+
anti-inflammatory activities, etc. For example,
SA derivatives by modifying the CN bond of SA (Fig. 2). All
Xu et al. found that SA induced the death of HeLa cervical
cancer cells with an IC50 value of 2.43 μM by reducing the ex-
pression of BCL-2 and NF-κB leading to the increased expres-
of the synthesized compounds were evaluated for their
9
sion of Bax protein. Recently, Leung et al. reported that SA
International Cooperative Laboratory of Traditional Chinese Medicine
Modernization and Innovative Drug Development of Chinese Ministry of Education
(MOE), School of Pharmacy, Jinan University, 601 Huangpu Avenue West,
Guangzhou 510632, China. E-mail: luxy2016@jnu.edu.cn
†
‡
Electronic supplementary information (ESI) available. See DOI: 10.1039/c9md00494g
L. J and X. W contributed equally to this work.
Fig.
1 The chemical structures of sanguinarine (1) and
dihydrosanguinarine (2).
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Scheme
conditions: (g) NaBH
3–64% or CH ONa, MeOH, 6 h, reflux, pH 10, 51%; (i) CH
°C → rt, 10 h, 29%; (j) CsF, TMSCN, CH CN, rt, 59%; (k) KOH, HCl,
FeIJCN) , H O, 3 h, 90 °C, 43%.
2
Chemical synthesis of
, MeOH, rt, 30 min, 36%; (h) amines, CH
MgBr, THF,
2
and 8d–8m. Reagents and
Fig. 2 Designed strategy for the target compounds.
4
3
CN, rt,
3
0
K
3
3
3
in vitro anticancer activities against A549 and H1975 NSCLC
cells, and a representative compound was used to explore the
mechanism of action (MOA) for anticancer activity.
3
6
2
All newly synthesized compounds were evaluated for their
The synthetic route of compounds 8a–8c is described in
1
8,19
anti-proliferative activities against A549 and H1975 NSCLC
cells using the MTT method (Table 1). The SA was used as
the control drug. The results showed that DHSA and 8a–8c al-
most totally lost their activities with IC50 values >30 μM. It
Scheme 1.
Alkylation of the starting material
naphthalene-2,3-diol (3) gave naphthoij2,3-d]ij1,3]dioxole (4),
followed by the iridium-catalyzed C–H borylation and Suzuki
coupling reaction obtaining intermediate 6. Treatment of 6
with hydroxylamine hydrochloride and then acylation with
p-trifluoromethylbenzoyl chloride provided acyl oxime (7). Ul-
timately, the alkaloid norsanguinarine (8a) was obtained by
the cyclization of 7 under established conditions. Further re-
duction of 8a in the presence of CH COOH or CH CH COOH
+
was indicated that the CN bond was critical for the anti-
cancer activity. Meanwhile the 6-position hydrophilic group
substituted SA derivatives (8d–8j) exhibited potent anti-
proliferative activities with IC50 values of 1.25–4.35 μM and
0.94–3.13 μM against A549 and H1975, respectively, which
3
3
2
was equivalent to that of SA. The results show that the 6-posi-
tion of SA can be replaced by hydrophilic groups, such as
aminoethyl (8d), aminopropyl (8e), aminoisopropyl (8f), cyclo-
pentylamino (8g), morpholinyl (8h) and methoxy (8j). Impor-
tantly, the CN substituted compound (8l) exhibited the most
potent anti-proliferative activity with IC50 values of 0.96 and
obtained 8b and 8c, respectively.
The synthetic route of compounds 8d–8m is described in
1
1,20
Scheme 2.
starting material to synthesize DHSA and 8d–8m. In detail,
reduction of 1 with NaBH gave DHSA. Compounds 8d–8j
were synthesized by nucleophilic addition of 1 with various
amines or CH ONa in a one-pot reaction. Treatment of 1 with
Grignard reagent CH BrMg, cyanide reagent (CH SiCN and
Sanguinarine chloride (1) was used as the
4
0.79 μM against A549 and H1975, respectively. However, the
3
CH and CO substituted derivatives (8k and 8m) totally lost
3
3
3 3
)
their activities (Table 1).
oxidizing agent K Fe (CN) gave the desired product 8k, 8l
3
6
and 8m, respectively.
Table 1 The in vitro anticancer activities of target compounds against
A549 and H1975 cells
Cellular inhibition (μM)
Compound R1
R2
H
A549
H1975
2
8
8
8
8
8
8
8
8
8
8
CH
—
3
>30
>30
>30
>30
19.14 ± 0.85
>30
>30
a
b
c
d
e
f
g
h
i
C
C
2
H
H
5
H
H
3
7
>30
CH
CH
CH
CH
CH
CH
CH
3
NHCH
NHIJCH
3 2
NHCHIJCH )
Cyclopentylamino 1.97 ± 0.22 1.57 ± 0.22
Morpholinyl
Benzylamino
OCH
CH3
CN
2
CH
3
1.25 ± 0.15 0.94 ± 0.01
1.48 ± 0.25 1.00 ± 0.32
1.46 ± 0.20 0.96 ± 0.29
3
3
3
3
3
3
2 2
) CH
3
Scheme 1 Chemical synthesis of 8a–8c. Reagents and conditions: (a)
CH Br , K CO , DMF, 100 °C, 10 h, 58%; (b) bisIJpinacolato)diboron,
IrIJCOD)OMe] , 4,4′-di-tert-butyl-2,2′-bipyridine, cyclohexane, 24 h, rt
reflux, 15 h, 31%; (c) 5-bromobenzoijd]ij1,3]dioxole-4-carbaldehyde,
CO , PdCl IJPPh , H O, (CH OMe) , 80 °C, 4 h, 77.1%; (d) C N,
NOH–HCl, MeOH, 8 h, rt; Et N, 4-(trifluoromethyl)-benzoyl chloride,
CH Cl , 0 °C; 10 min, 0 °C, 49.3%; (e) lrIJppy) , DMF, rt, 30 h, 86.3%; (f)
for 8b: NaBH , AcOH, rt, 30 min, 28.7%; for 8c: NaBH , CH CH COOH,
2
2
2
3
[
2
1.79 ± 0.60 1.16 ± 0.21
4.35 ± 0.36 3.13 ± 0.84
1.59 ± 0.27 1.17 ± 0.45
>30
0.96 ± 0.07 0.79 ± 0.04
>30 >30
1.59 ± 0.33 1.32 ± 0.36
→
K
2
3
2
3
)
2
2
2
2
5 5
H
j
3
H
2
3
8k
8l
8m
1
CH3
CH3
CH3
—
>30
2
2
3
4
4
3
2
O
rt, 30 min, 30%.
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Fig. 3 8h induces the G0/G1 cell cycle arrest in A549 and H1975 cells.
(
A) PI-stained A549 and H1975 cells were subjected to flow cytometry
Fig. 5 8h induces the ROS production in A549 and H1975 cells. (A)
The cells were treated with 8h for 20 minutes and then collected and
incubated with a fluorescent probe for 30 minutes for flow cytometry.
for the detection of cell cycle distribution. (B) Quantification of cell cy-
cle distribution. The cells were treated with 8h for 24 h and then col-
lected for flow cytometry.
(B) Quantification of ROS production.
In order to study the anticancer mechanism of the com-
pounds, the representative compound 8h was selected for
their MOA study. We firstly determined whether 8h affected
the cell cycle of NSCLC cells. The results suggested that 8h
can induce the G0/G1 phase cell cycle arrest of A549 and
H1975 cells in a dose-dependent manner, with the percent of
the cells in the G0/G1 phase increasing from 63.11% to
respectively. Consistently, 8h increased the levels of cleaved-
poly ADP-ribose polymerase (PARP), cleaved-caspase-3, and
cleaved-caspase-9 by western blotting analysis (Fig. 4B). As
Akt signaling plays a critical role in cell proliferation and sur-
21
vival, we further investigated whether 8h can induce the ap-
optosis of NSCLC through Akt signaling. The results (Fig. 4B)
indicated that 8h significantly decreased both the basal level
and phosphorylation level of Akt, suggesting that the com-
pound inhibits cell proliferation and induces cell apoptosis
via the inhibition of the Akt signaling pathway.
7
1.21% in A549 cells and 62.86% to 77.70% in H1975 cells,
respectively (Fig. 3).
Further Annexin V/7-AAD double staining and flow
cytometric analysis were performed to examine the apoptotic
effect of 8h on A549 and H1975 cells. The results (Fig. 4A)
showed that 8h significantly induced apoptosis of NSCLC
cells, with the apoptotic rate increasing from 6.20% to
To further examine whether oxidative stress was involved in
14
8
8
h-induced cell apoptosis, we detected the ROS production in
h-treated A549 and H1975 cells using the oxidation-sensitive
fluorescent probe DCFH-DA. The results showed that 8h in-
duces the ROS production in A549 and H1975 cells in a dose-
dependent manner (Fig. 5), indicating that oxidative stress
plays an important role in mediating 8h-induced cell apoptosis.
In conclusion, a novel series of SA derivatives were synthe-
sized and inhibited A549 and H1975 NSCLC cells with IC50
values of 0.96 – >30 μM and 0.79 – >30 μM, respectively. The
SARs studies indicated that the C6-position of SA was toler-
ated to be substituted by small volume of hydrophilic groups
and CN. Further biological evaluation illustrated that 8h can
exert an antineoplastic effect in A549 and H1975 cells via the
induction of ROS production and the inhibition of the Akt
signaling pathway. Collectively, this study may provide a
strategy to develop new anticancer agents.
9
7.64% in A549 cells and 11.13% to 93.61% in H1975 cells,
Conflicts of interest
There are no conflicts to declare.
Fig. 4 8h induces cell apoptosis and decreases the level of AKT/p-
AKT in A549 and H1975 cells. (A) The cells were treated with 8h for 48
h. Apoptosis rate was quantified through the Annexin V-FITC/7-AAD
assay. (B) The cells were treated with 8h for 24 h, the expression levels
of Akt, p-Akt, PARP, caspase 3, cleaved-caspase-3, caspase 9, cleaved-
caspase-9 were detected by western blot analysis, GAPDH was used as
a loading control. p-Akt: phosphorylated Akt.
Acknowledgements
The authors appreciate the financial support from the
National Natural Science Foundation of China (81874285,
81922062 and 81973158), the Guangdong International
Science and Technology Cooperation Project (2018A050506043),
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