Design, synthesis and apoptosis-related antiproliferative activities of chelidonine derivatives

Article abstract of DOI:10.1016/j.bmcl.2019.126913

To get chelidonine derivatives with enhanced antiproliferative activity and selectivity, a series of nitric oxide donating derivatives (10a-f and 11a-j) were designed, synthesized and biologically evaluated. Compared with chelidonine, these compounds exhibited lower IC₅₀ values against human hepatoma cells HepG2, breast cancer cells MCF-7, colon cancer cells HCT-116, as well as leukemia cells K562. Compound 11j displayed the strongest antiproliferative activity with IC₅₀ values of 3.91, 6.90, 4.36 and 1.12 μM against the above four cells, respectively. Nevertheless, it showed an IC₅₀ value >40 μM against human peripheral blood mononuclear cells (PBMCs), which demonstrated high selectivity between normal and cancer blood cells. In further mechanism studies, 11j showed the capability to induce K562 cells apoptosis, S phase cell cycle arrest and mitochondrial membrane potential disorder. Besides, 11j was found to be effective in promoting the expression of proapoptotic protein Bad and suppressing the expression of anti-apoptotic proteins Bcl-xL, catalase, survivin, claspin and clusterin.

Full text of DOI:10.1016/j.bmcl.2019.126913

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and apoptosis-related antiproliferative activities of
chelidonine derivatives
⁎
Xueyan Huanga,1, Keguang Chengb,1, Lilin Liua, Xu Hua, Xiang Gaoa, Haonan Lia, Fanxing Xuc,
,
⁎
⁎
Zhanlin Lia, Huiming Huaa, , Dahong Lia,
a Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
103 Wenhua Road, Shenyang 110016, PR China
b State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, and School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai
Raod, Guilin 541004, PR China
c Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
To get chelidonine derivatives with enhanced antiproliferative activity and selectivity, a series of nitric oxide
donating derivatives (10a-f and 11a-j) were designed, synthesized and biologically evaluated. Compared with
chelidonine, these compounds exhibited lower IC50 values against human hepatoma cells HepG2, breast cancer
cells MCF-7, colon cancer cells HCT-116, as well as leukemia cells K562. Compound 11j displayed the strongest
antiproliferative activity with IC50 values of 3.91, 6.90, 4.36 and 1.12 μM against the above four cells, re-
spectively. Nevertheless, it showed an IC50 value > 40 μM against human peripheral blood mononuclear cells
(PBMCs), which demonstrated high selectivity between normal and cancer blood cells. In further mechanism
studies, 11j showed the capability to induce K562 cells apoptosis, S phase cell cycle arrest and mitochondrial
membrane potential disorder. Besides, 11j was found to be effective in promoting the expression of proapoptotic
protein Bad and suppressing the expression of anti-apoptotic proteins Bcl-xL, catalase, survivin, claspin and
clusterin.
Chelidonine
Nitric oxide
Antiproliferative
Apoptosis
Chelidonium majus L. is known as a commonly-used Chinese med-
icinal herb for cough inhibition, analgesia and detoxification.1 In recent
studies, the crude extract of C. majus was observed to inhibit tumor cells
from migration and induce cell cycle arrest and apoptosis.2,3 Alkaloids
are the active components of C. majus.4 Chelidonine (1), a benzophe-
nanthridine alkaloid, is the major alkaloid in C. majus with the cap-
ability of performing various biological activities,3 including
antitumor,5–8 anti-inflammatory,9–11 antibacterial,12,13 analgesic,14 in-
secticide15 and spasmolytic16,17 effects. Chelidonine inhibits various
cancers, including leukemia,18 liver cancer,19 breast cancer,20 lung
cancer18 and so on. Its antitumor mechanisms have been investigated
extensively. Chelidonine induces human T-leukemia cells apoptosis via
a mitochondrial death pathway by slightly releasing cytochrome c in
12 h and causes intensive DNA damage in 24 h.21,22 It also prompts cells
mitotic arrest and modulates tyrosine kinase activity.23 In gastric cancer
SGC-7901 cells, chelidonine signally down-regulates the expression of
Cdk1 and cyclinB1, up-regulates the protein p-Cdk1 (Thr14) and in-
duces the G2/M phase cell cycle arrest.6 Chelidonine not only inhibits
the formation of integrin-linked kinase, PINCH and α-parvin complex,
but also reduces migration and invasion by inhibiting tubulin poly-
merisation.24,25 In addition, chelidonine down-regulates hTERT.20 As
confirmed by Mahmoud Zaki El-Readi et al., chelidonine overcomes
multidrug resistance (MDR) in cancer cells through interaction with
ABC-transporters, CYP3A4 and GST, induction of apoptosis and cyto-
toxic effects.26 Apart from these, chelidonine attenuates eosinophilic
airway inflammation by suppressing IL-4 and eotaxin-2 expression in
asthmatic mice.10 It also exerts inhibitory effect on human neutrophil
elastase activity.11
Abbreviations: PBMC, peripheral blood mononuclear cell; MDR, multidrug resistance; NO, nitric oxide; Et3N, triethylamine; DMAP, 4-dimethylaminopyridine; EDC-
HCl, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; DCM, dichloromethane; MeOH, methanol; THF, tetrahydrofuran; 5-Fu, 5-fluorouracil; JC-1,
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol caebocyanine; PBS, phosphate buffered solution; NMR, nuclear magnetic resonance; HR(ESI)MS, high resolu-
tion electrospray ionization mass spectrometry; m/z, mass-to-charge ratio; OR, optical rotation
⁎ Corresponding authors.
E-mail addresses: fanxing0011@163.com (F. Xu), huimhua@163.com (H. Hua), lidahong0203@163.com (D. Li).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2019.126913
Received 21 November 2019; Received in revised form 11 December 2019; Accepted 16 December 2019
0960-894X/©2019ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:XueyanHuang,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2019.126913

X. Huang, et al.
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Scheme 1. (A) Synthesis of NO donors 7a-f. Reagents and conditions: (a) ClCH2COOH, NaOH (aq), 140 ℃, 2 h; (b) 30% H2O2, rt, 3 h; (c) fuming HNO3, AcOH, 90 ℃,
4 h; (d) hydramine, NaH, 0 ℃, 5 h; (e) anhydride, anhydrous, pyridine, rt, 3 h. (B) Synthesis of NO donors 9a-j. Reagents and conditions: (f) corresponding dihydric
alcohol, THF, 30% NaOH, 0 ℃, 4–8 h; (g) corresponding anhydride, Et3N, DMAP, DCM, rt, 3–4 h. (C) Synthesis of chelidonine derivatives 10a-f and 11a-j. Reagents
and conditions: (h) 7a-f or 9a-j, EDC-HCl, DMAP, DCM, rt, 10–12 h.
2

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Table 1
Antiproliferative activities of chelidonine and its 16 NO-releasing derivatives against four human cancer and two normal cell lines.
compound
IC50 (μM)a
HepG2
MCF-7
HCT-116
K562
L-02
PBMC
10a
10b
10c
11.36
16.83
19.43
26.44
10.68
0.83
1.20
1.27
1.73
0.62
7.94
13.59
8.37
14.58
5.29
4.92
3.68
4.78
7.94
8.92
5.41
7.34
6.81
13.24
6.55
6.90
> 40
26.65
0.55
1.04
0.51
1.17
11.54
17.26
16.45
> 40
7.25
0.59
1.38
0.33
3.17
9.80
6.94
12.13
3.18
2.68
3.08
2.91
7.61
4.52
3.78
3.73
6.14
9.59
2.60
1.12
17.65
3.94
0.14
0.46
0.31
27.68
36.48
> 40
> 40
19.94
23.45
14.56
19.71
> 40
23.93
28.19
20.32
> 40
> 40
16.52
6.04
1.33
1.74
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
> 40
10d
10e
0.34
0.14
0.22
0.20
0.35
0.44
0.30
0.31
0.52
0.40
0.48
0.30
0.19
0.18
0.23
0.39
0.23
0.22
0.26
0.52
0.62
0.17
0.11
0.97
1.38
1.05
0.80
10f
7.59
0.45
12.17
15.73
8.27
0.46
0.94
11a
11b
11c
10.54
6.39
0.72
0.24
0.43
9.45
0.59
0.37
16.56
8.68
1.87
11d
11e
8.65
0.65
1.22
1.46
1.08
14.39
13.27
19.58
> 40
6.55
1.02
23.54
12.88
19.14
> 40
8.47
1.39
0.68
0.52
11f
0.88
1.36
11g
11h
11i
0.83
0.30
0.21
0.28
0.35
0.53
1.24
11j
3.91
4.36
0.29
0.37
0.50
chelidonine
5-Fu
> 40
32.57
> 40
6.86
1.56
0.17
> 40
> 40
1.98
1.92
a
IC50: concentration of the tested compound that inhibits 50% of cell growth by the MTT assay, the cells were incubated for 72 h. The values are expressed as
standard deviations of three independent experiments.
averages
cancer cells.35,36 The combination of NO donors with natural products,
such as terpenoids, alkaloids and flavonoids, is conducive to improving
antitumor activity and reducing toxicity.37–39 Among NO-donors, fur-
azan has a relative stable structure and inhibits tumor proliferation by
the release of NO.40
35
30
25
20
15
10
5
10a
11e
10c
11g
10e
11j
10b
11b
10d
11d
10f
11a
11i
11h
In this work, chelidonine was combined with a series of furazan-
type NO donors to obtain 16 NO-donating derivatives, which were
screened for their antiproliferative activities. Further mechanism study
of 11j, concerning apoptosis related properties, were also investigated.
Chelidonine was isolated from the aerial parts of C. majus L. and its
structure was confirmed via a combination of 1H NMR, 13C NMR and
optical rotation. The configuration of C-11 of chelidonine was con-
firmed by comparing the optical rotation with the literature.41 The
tested optical rotation of chelidonine was [α] + 135 (c 0.12, MeOH)
which was fitted with [α] + 112 (c 0.65, MeOH) in the literature.
Thiophenol (2) was reacted with chloroacetic acid in the presence of
sodium hydroxide to generate phenylthio acetic acid 3. In the presence
of 30% H2O2, 4 was obtained from the oxidation of 3. Further reaction
with fuming nitric acid at 90 °C generated 5. 5 was reacted with ami-
noalcohol, in THF to derive 6a-c. 7a-f were obtained by further reaction
with anhydride and pyridine (Scheme 1A). In addition, 5 was reacted
with propanediol, butanediol, hexanediol, diethylene glycol or 2-bu-
tyne-1,4-diol in THF containing 30% NaOH to generate 8a-e, then
treated with anhydride, triethylamine (Et3N) and 4-dimethylamino-
pyridine (DMAP) to derive 9a-j (Scheme 1B). In addition, 7a-f or 9a-j
were reacted with chelidonine in the presence of DMAP and EDC-HCl in
DCM, and then they were purified by silica gel column chromatography
(DCM:MeOH = 200:1) to get the target compounds 10a-f and 11a-j
(Scheme 1C).
0
15
30
45
60
90
Time (min)
120
150
180
210
Fig. 1. NO-releasing ability of chelidonine derivatives. The values are expressed
as averages of three independent experiments.
Structural modification of nature products is considered as a major
approach of drug discovery. For example, the well-known anticancer
drugs 10-hydroxycamptothecin, topotecan, belotecan and lurtotecan
are all classed as derivatives of the alkaloid camptothecin. They are
used to treat colon cancer, lung cancer and ovarian cancer in clinic.27
Chelidonine possesses antitumor activities and is potential to be an anti-
neoplastic precursor. It is reported in the literature, the alkaloids in C.
majus L. show detectable hepatotoxicity.28 In order to enhance the
antiproliferative activity of chelidonine and improve the selectivity
between cancer and normal cells, chelidonine is structurally modified
in this study.
The antiproliferative activities of chelidonine derivatives 10a-f and
11a-j were tested against four human cancer cells (hepatoma HepG2,
breast cancer MCF-7, colon cancer HCT-116 and leukemia K562) and
two normal cells (hepatocytes L-02 and peripheral blood mononuclear
cells PBMC) in comparison with chelidonine (1). 5-Fu (5-fluorouracil)
was used as positive control (Table 1).
Nitric oxide (NO) represents a significant gaseous signaling mole-
cule that is applied to the treatment of cancer by inducing apoptosis,
increasing the sensitivity of tumor cells to drugs and attenuating cancer
cell metastasis.29–31 High levels of NO may exert anticancer activities
through upregulating p53 to stimulate apoptosis, degradating the anti-
apoptotic proteasomal molecule, increasing the mitochondrial perme-
ability and releasing cytochrome c, cell cycle arrest, cell necrosis, cell
necrosis and cytotoxicity.32–34 In recent years, the research conducted
into NO donating derivatives has been made a focus of attention. The
antitumor effects dramatically attenuate by NO scavenger or elimina-
tion of the NO-releasing capability which indicates that the NO pro-
duced by NO-donating derivatives contributes to its cytotoxicity against
As demonstrated by the results, a majority of the chelidonine deri-
vatives exhibited antiproliferative activities against these four human
tumor cells. All derivatives were indicated to perform significant cy-
totoxic activity with the IC50 values of 1.12–12.13 and 3.68–14.58 μM
against K562 and MCF-7 cell lines, respectively. Most of the derivatives
performed decent cytotoxic activity with the IC50 values of 4.36–23.54
3

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Fig. 2. Cell cycle analysis of different concentrations of 11j in K562 cells. K562 cells were treated with 11j (0, 0.55, 1.10 and 2.20 μM) for 72 h, then stained with PI.
A flow cytometer was used to analyze.
and 3.91–26.44 μM against HCT-116 and HepG2 cell lines, respectively.
For 10b, 10d and 10f with the same R2 of -(CH2)3-, generally in all four
tested cancer cells, 10b with R1 of ethylamine showed the weakest
antiproliferative potency while 10f with R1 of isopropyl moiety was the
strongest. 11f with R1 of -(CH2)6- moiety and R2 of -(CH2)3- showed the
weakest antiproliferative activity. None of the derivatives performed
antiproliferaitve activity against PBMCs with IC50s > 40 μM, which
showed selectivity between normal and cancer cells. Compounds 10b,
10e and 11d-f were discovered to possess no cytotoxicity against L-02,
which also showed selectivity. 11j42 with R1 of butyne moiety and R2 of
-(CH2)3- was the strongest one against the above four tumor cells with
IC50 values of 3.91, 6.90, 4.36 and 1.12 μM, respectively. Therefore, 11j
was selected for further mechanism study in K562 cell line.
cell cycle arrest in S phase in a concentration-dependent manner.
The morphological characteristics displayed by cells would manifest
a string of changes like cell shrinkage, chromatin accumulation and
nuclear membrane rupture when cell apoptosis occurs.44 In order to test
whether 11j is capable to induce apoptosis, K562 cells were treated
with varying concentrations of 11j (0.55, 1.10 and 2.20 μM) for 48 h
and subsequently stained with Hoechst 33258. The morphological
changes were observed by fluorescence microscopy. The results clearly
revealed (Fig. 3) that the morphology of cells in the control group
underwent no noticeable change, and the cells showed round homo-
geneous nuclei. Whereas different concentrations of 11j treated K562
cells showed significant morphological changes, including chromatin
aggregation, nuclear rupture and other signs of apoptosis. The results
showed that 11j was effective in facilitating apoptosis in K562 cells.
To confirm the apoptosis-induced effects of 11j, annexin-V FITC/PI
binding tests were conducted. K562 cells were incubated with varying
concentrations of 11j (the same as cell cycle experiments) for 48 h,
previous to being stained with annexin-V FITC and PI. Then, the per-
centages of apoptotic cells were determined by conducting flow cyto-
metry analysis. As shown in Fig. 4, varying concentrations of 11j caused
the percentage of apoptosis to rise from 2.96% in the solvent group to
9.96%, 18.85% and 53.81%, respectively. Therefore, 11j induced
apoptosis in K562 cells in a dose-dependent manner.
The anti-neoplastic ability of NO was generally improved with the
concentration of NO.43 Therefore, to further investigate the association
between the NO-releasing ability of NO-donating chelidonine deriva-
tives and their antiproliferative activities, the target compounds were
subjected to the test of NO release ability (Fig. 1). As indicated, the
amounts of NO released from NO-donating derivatives increased
markedly from 90 min and continued this increasing over time. Espe-
cially, 11j exhibiting the strongest antiproliferative activity also
showed the highest NO release ability, the amount of NO release was
about 30 μM at 210 min, which suggested a certain association between
the NO release ability and the antiproliferative effects.
As mitochondrial membrane potential is closely associated with
apoptosis. The loss of mitochondrial membrane potential causes
changes of the permeability of mitochondrial membrane, which induces
cell apoptosis by releasing pro-apoptotic factors into cytosol.45,46 The
effects exerted by 11j on mitochondrial membrane potentials in K562
cell line were detected. Cells were co-cultured with varying con-
centrations (the same as cell cycle experiment) of 11j for a duration of
To validate the effect of 11j on K562 cell cycle, K562 cells were
subjected to the treatment with varying concentrations of 11j (0.55,
1.10 and 2.20 μM) for a 72 h spell. As indicated in Fig. 2, the G1, S and
G2/M phases of solvent control were 56.13%, 35.68% and 8.19%, re-
spectively. The proportion of cells in S phase increased from 35.68% to
41.89%, 52.04% and 59.72%, respectively. It suggested that 11j ex-
erted inhibitory effects on the proliferation of K562 cells by inducing
48
h prior to being stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-
4

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Fig. 3. Hoechst staining of 11j in K562 cells. After the treatment with 11j (0, 0.55, 1.10 and 2.20 μM) for 48 h, K562 cells were stained by Hoechst 33258 solution,
then detected by fluorescent microscope.
Fig. 4. Flow cytometry analysis of apoptosis induced by 11j in K562 cells. K562 cells were treated with 11j for 48 h and apoptosis was performed by flow cytometry
via an annexin V-FITC/PI binding assay.
5

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Fig. 5. Mitochondrial depolarization induced by 11j in K562 cells. K562 cells were treated with 11j and then stained with JC-1. The percentage of cells with
collapsed mitochondrial membrane potentials was monitored by flow cytometry.
tetraethylbenzimidazol caebocyanine (JC-1). The results (Fig. 5) de-
monstrated that 11j elevated the proportion of cells with mitochondrial
depolarization in a concentration-dependent manner, raising the per-
centage from 7.04% in the control group to 19.29%, 28.66% and
51.15%, respectively. So, 11j induced apoptosis by promoting the loss
of mitochondrial membrane potential in K562 cells.
K562 cell apoptosis via up-regulating the pro-apoptotic proteins and
down-regulating the anti-apoptotic proteins.
In summary, in order to improve its antiproliferative activity against
Pro- and anti-apoptotic factors affect apoptosis through cellular
pathways. Mitochondria-dependent apoptotic pathways play important
roles in cell apoptosis.47,48 Bcl-2 family, including pro-apoptotic (Bax,
Bak, Bid and Bad) and anti-apoptotic (Bcl-2, Bcl-xL and Bcl-w) proteins,
induces apoptosis by influencing the permeability of mitochondria
membrane, causing cytochrome c released into the cytosol and acti-
vating caspase-3 and -9.49,50 Survivin is an anti-apoptotic factor, which
controls the activities of caspase-3, −7 and −9 to inhibit apop-
tosis.51–53 Claspin is a protein which is cleaved by caspase-7 during the
initiation of apoptosis and causes the dysfunction of claspin to induce
cell cycle arrest and repair subsequent damage. It regulates the activity
of Chk1 and arrest cell cycle at S phase.54–55 Therefore, down-regulated
the expression of claspin inhibits cancer cells survival. The relations of
these proteins and apoptosis are shown in Fig. 6. To figure out whether
the expression of pro- and anti-apoptotic factors were influenced by
11j, human apoptosis array was performed in K562 cells treated with
11j. The relative levels of 35 apoptosis-related proteins in K562 cells
were detected simultaneously (Fig. 7). The original image could be
found in the supporting information (Fig. S49). A comparison was
performed to determine the relative changes on apoptosis-related pro-
tein levels. As showed, 11j up-regulated the expression of proapoptotic
protein Bad while suppressing the expression of anti-apoptotic protein
Bcl-xL, survivin and claspin. Moreover, the expression of anti-apoptotic
factors catalase and clusterin were also reduced. Therefore, 11j induced
Fig. 6. The apoptosis pathways of apoptosis-related proteins influenced by 11j.
6

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Fig. 7. The apoptosis protein array analysis of 11j. Proteins were extracted from cells treated with 11j for 24 h and detected the chemiluminescent signals by a digital
imaging system.
tumor cells and reduce toxicity to normal cells, several furazan-type
NO-donating derivatives of chelidonine were designed and synthesized.
All the synthesized derivatives were tested for antiproliferative activ-
ities against four tumor cells HepG2, MCF-7, HCT-116 and K562, and
two normal cells. Among them, 11j exhibited good antiproliferative
activity with IC50 values of 3.91, 6.90, 4.36 and 1.12 μM, respectively.
NO-releasing ability results indicated that 11j also released the highest
amount of NO among the derivatives. In further mechanistic studies,
11j was discovered to induce cell cycle arrest at S phase, cause dis-
ruption of mitochondrial membrane potential and enhance apoptosis in
a dose-dependent manner. Subsequently, the results of human apop-
tosis array evidenced the effectiveness of 11j-induced apoptosis
through enhancing the expression of pro-apoptotic protein Bad and
suppressing the expression of anti-apoptotic proteins Bcl-xL, catalase,
survivin, claspin and clusterin in K562 cells.
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(d, 2H, J = 7.8 Hz, Ar-H), 7.89 (t, 1H, J = 7.4 Hz, Ar-H), 7.74 (t, 2H, J = 7.6 Hz, Ar-
H), 7.21 (d, 1H, J = 8.3 Hz, H-10), 7.10 (s, 1H, H-1), 6.75 (d, 1H, J = 8.3 Hz, H-9),
6.57 (s, 1H, H-4), 5.88-5.95 (m, 4H, 2 × -OCH2O-), 5.24 (s, 2H, -CH2-), 5.22 (m, 1H,
H-11), 4.84 (s, 2H, -CH2-), 4.11 (d, 1H, J = 3.8 Hz, H-4b), 3.60 (d, 1H, J = 17.0 Hz,
H-6), 3.53 (m, 1H, H-10b), 3.40 (d, 1H, J = 17.0 Hz, H-6), 2.79 (dd, 1H, J = 15.3, 4.
7 Hz, H-12), 2.69 (m, 1H, H-12), 2.45 (s, 3H, N-CH3), 2.41 (m, 4H, 2 × -CH2-), 1.80
(m, 2H, -CH2-); 13C NMR (DMSO-d6, 150 MHz) δ (ppm): 171.82, 171.79, 158.0,
146.43, 146.35, 144.8, 143.5, 136.9, 136.2, 130.0, 130.0, 129.0, 128.4, 128.4, 127.
0, 126.4, 121.2, 116.3, 110.7, 108.2, 107.4, 106.4, 100.9, 100.8, 84.1, 79.3, 71.7, 68.
5, 60.6, 59.0, 54.9, 45.0, 41.0, 32.4, 30.8, 29.6, 19.7; HR(ESI)MS m/z calcd for
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