Synthesis and biological evaluation of β-ionone oriented proapoptosis agents by enhancing the ROS generation

Article abstract of DOI:10.1016/j.bioorg.2020.104273

β-ionone, a cyclic terpenoid compound present in many fruits, has been showed a broad spectrum of biological activities. In this paper, we synthesized a panel of β-ionone derivatives and tested their anti-proliferation activity on cancer cell by the MTT a

Full text of DOI:10.1016/j.bioorg.2020.104273

BioorganicChemistry104(2020)104273
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of β-ionone oriented proapoptosis
agents by enhancing the ROS generation
Jie Yang⁎, Wen-Wen Mu, Yu-Xin Cao, Guo-Yun Liu⁎
School of Pharmacy, Liaocheng University, 1 Hunan Street, Liaocheng, Shandong 252000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
β-ionone, a cyclic terpenoid compound present in many fruits, has been showed a broad spectrum of biological
activities. In this paper, we synthesized a panel of β-ionone derivatives and tested their anti-proliferation activity
on cancer cell by the MTT assay. The results showed that most of the β-ionone derivatives were more active than
β-ionone and curcumin. Particularly, the β-ionone derivatives (1a, 1d and 1g) with ortho-substituents on the
aromatic ring exhibited much stronger cytotoxicity than their corresponding meta- and para-substituted com-
pounds. Importantly, the cytotoxicity of the β-ionone derivatives (1a, 1d and 1g) were relationship with their
reactive oxygen species (ROS)-generation abilities, which could lead to the redox imbalance, lipid peroxidation,
the loss of mitochondrial membrane potential (MMP), the activation of Bax and Caspase 3, followed by cell
apoptosis. This work suggest that the “ortho effect”, the ROS-generation ability and drawing fluorine atom into
drugs may play a potent role in enhancing the anticancer activity of β-ionone derivatives.
Ionone
Curcumin analog
Reactive oxygen species
Apoptosis
1. Introduction
induce apoptosis in cancer cell through p53-dependent mitochondrial
signaling pathway [11]. Inspired by the anticancer property of mono-
The electrophilic natural products, characterized with Michael ac-
ceptor units, have attracted much attention in medicinal chemistry,
particularly, in the field of cancer chemoprevention and chemotherapy
[1]. The Michael acceptor untis of the electrophilic natrual products
could be attacked by the nuclephilic centers of proteins, DNA and
glutathione, resulting in a covalent compound with a wide variety of
biological functions [2]. α,β-unsaturated ketones, widespread existed in
curcumin, β-ionone, chalcone and cinnamic acid derivatives, were a
typical Michael acceptor and could remarkebaly enhance the pharma-
cological properties [3]. Curcumin, as one of the simplest phenolic
compound found in turmeric, contains two α,β-unsaturated ketones
units and has been used for the purpose of cancer prevention and
treatment [4]. In the last few decades, extensive efforts have been de-
voted to seek new curcumin analogs for the sake of enhancing its sta-
bility. Mon-carbonyl curcumin, the representative curcumin analogs,
showed more stability and potent anticancer activity in vitro than cur-
cumin [5,6].
carbonyl curcumins and β-ionone, a series of β-ionone oriented mono-
carbonyl curcumin were synthesized by Aldol condension (Scheme 1).
Thus, we focus on the structure-activity relationships of these com-
pounds underlying cytotoxic potential on tumor cell lines and probe
into the anticancer mechanism against A549 cells.
2. Results and discussion
2.1. Synthesis
The synthetic routs of β-ionone derivatives 1a-m are depicted in
Scheme 1. The desired compounds were synthesized by aldol con-
densation reaction between β-ionone and respective benzaldehyde in
the presence of 20% NaOH aqueous using ethanol as solvent. The title
compounds were obtained with medium yields after purification by
column chromatography. All compounds were characterized by 1H
NMR, 13C NMR and HRMS.
β-ionone, a cyclic terpenoid compound present in many fruits, ve-
getables and grains, has been showed a broad spectrum of physiological
and pharmacological activities, including anti-inflammatory [7], an-
ticancer [8], antibacterial [9] and tryosine kinase inhibitor [10]. Of
particular interest is the anticancer activity of β-ionone, which could
2.2. Cytotoxicity and SAR
All the title compounds were initially tested the cytotoxicity against
lung cancer A549 and ovarian cancer SK-OV3 cells, and the results were
⁎ Corresponding author.
E-mail addresses: yangjie1110@163.com (J. Yang), guoyunliu@126.com (G.-Y. Liu).
https://doi.org/10.1016/j.bioorg.2020.104273
Received 28 April 2020; Received in revised form 3 September 2020; Accepted 7 September 2020
Availableonline11September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

J. Yang, et al.
BioorganicChemistry104(2020)104273
Scheme 1. Molecular structures and synthetic routs of ionone-oriented analogs.
Table 1
Cytotoxicity of β-ionone and its analogs against SK-OV3 and A549 cells.
Comps.
IC50 (µM)
Comps.
IC50 (µM)
SK-OV3
A549
19.3
SK-OV3
57.8
A549
40.9
23.4
1.4
0.6
2.2
3.6
48.9
40.6
24.2
1.1
2.5
1.7
19.9
18.8
37.5
1.4
1.2
1.4
26.0
89.4
56.8
1.6
6.9
1.9
37.8
39.6
60.9
2.7
3.4
4.7
34.0
42.2
1.2
1.8
36.5
65.5
2.3
1.1
47.6
58.1
3.2
2.5
32.2
53.2
0.7
1.1
49.9
79.6
4.0
6.2
20.6
46.3
0.4
1.8
> 200
> 200
aThe IC50 value is the concentration of a compound tested to cause 50% inhibition of cell viability after 48 h of treatment, and is expressed as the mean
three determinations.
SD for
summarized in Table 1. The compounds with substitution at ortho- of
the aromatic ring exhibited higher cytotoxicity than the corresponding
meta- and para-substituted compounds in SK-OV3 cells. The results were
in accordance with the “ortho effect” [12], which had been reported in
our previous report and could enhance the cytotoxicity of mono-car-
bonyl curcumin analogs [5]. The “ortho effect” in the cases of A549
cells showed some effect, but not very obvious. The compound 1g and
1j (with trifluoromethyl or hydroxyl at ortho position) showed much
stronger cytotoxicity than the corresponding meta- and para-tri-
fluoromethyl (1h and 1i) or hydroxyl (1k and 1l) compounds. The
“ortho effect” of fluorine or methoxyl substituted compounds showed
no abvious in the case of A549 cells. This reason may be relationship
with the overexpression of thioredoxin (Trx) and thioredoxin reductase
(TrxR) in cancer cells [13,14]. The thioredoxin system, comprising Trx,
TrxR and NADPH, is a potent thiol redox system and critical for cell
survival and growth [15]. Additionally, drugs, containing Michael ac-
ceptor, can irreversibly inhibit the TrxR, and the modified enzyme
could trigger ROS generation and then lead cancer cell apoptosis [12].
Therefore, “ortho effect” showed no obvious in the cases of A549 may
be due to the overexpression of Trx and TrxR of A549 cells was not
consistent with that of SK-OV3 cells.
Compounds, containing Michael acceptor, could trigger ROS gen-
eration and then lead cancer cell apoptosis through irreversibly in-
hibiting the TrxR [12]. Therefore, we subjected β-ionone analogs (1a,
1b, 1c and 1d) to TrxR1 (PDBID 2J3N) in docking. As shown in Fig. 1A,
the Michael acceptor group of all the four β-ionone analogs were close
to the C-terminal Cys498 and the distance was about 3 Å. This suggest
that the β-ionone analogs have the potential to interact with the C-
terminal Cys-SH of mammalian TrxR. In the other hand, ortho-fluorine
atom of 1a may be formed a hydrophobicity interaction with the me-
thyl of Leu409, and ortho-methoxyl of 1d may be formed hydrophilic
interaction with the hydroxyl of Trp407. These interactions may be
contribute to the inhibition of TrxR by 1a and 1d and enhancing the
cytotoxicity of compounds with ortho-substituents. Further, the mole-
cules with F or CF3 substituent on the aromatic ring exerted more po-
tent cytotoxicity than that with OH or OMe substituent. This result may
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J. Yang, et al.
BioorganicChemistry104(2020)104273
Fig. 1. (A) β-ionone analogs (1a, 1b, 1c and 1d) in the binding site of human TrxR1. (B) Stability assessment on curcumin (50 μM) and β-ionone analogs (1a (50 μM),
1d (50 μM) and 1g (50 μM)) in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal calf serum at 25 °C by monitoring the decrease in their maximum
absorbance.
be due to the fluorine atom, which could increase the lipophilicity and
biological activity of the drugs [16]. It is obvious from the results that
most of the β-ionone derivatives were more active than that of the
leading curcumin and β-ionone. This result was consistent with the
more stability of the compounds (1a, 1d and 1g) than curcumin in
RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal calf
serum (Fig. 1B). We also found that most of the β-ionone derivatives
showed stronger anti-proliferation activity on A549 cells than that on
SK-OV3 cells. Therefore, compounds 1a, 1d and 1g were selected for
further exploration of the anti-cancer mechanisms against A549 cells.
cells. These results were in accordance with the cytotoxicity and sta-
bility of curcumin and β-ionone derivatives.
2.4. The redox imbalance induced by β-ionone derivatives in A549 cells
An accumulation of ROS, as powerful signaling molecules involved
in the regulation of biological processes [17], usually resulted in the
redox imbalance, which could be evaluated by the ratio of GSH/GSSG.
As our expectation, we started to test whether β-ionone derivatives
could result in the redox imbalance of A549 cells. As shown in Fig. 3,
we found that the three of β-ionone derivatives (1a, 1d and 1g) sharply
decreased the ratio of GSH/GSSG in a dose-dependent fashion. More
important, 1a exhibited the most severe redox imbalance in A549 cells.
In particular, A549 cells treated with 60 µM 1a displayed about 4-fold
decrease in the ratio of GSH/GSSG compared to the control. The ac-
tivity sequence of 1a, 1d and 1g was in line with their ROS-generating
ability. These results also indicated that the sustained flux of ROS fre-
quently brought out redox imbalance.
2.3. β-ionone derivatives induced-ROS generation in A549 cells
ROS, which have been linked to numerous biological processes and
disease conditions, is a potent mediator of cytotoxicity [17]. Therefore,
we detected the ROS generation induced by β-ionone derivatives (1a,
1d and 1g) and curcumin in A549 cells with 2′,7′-dichlorofluorescein
diacetate (DCFH-DA). As shown in Fig. 2, 1a, 1d and 1g promoted an
extensive increase in the ROS levels in a dose-dependent manner.
Especially, β-ionone derivatives (1a, 1d and 1g) markedly induced
much higher ROS production than curcumin. Notablely, compound 1a,
having the strongest anti-proliferation activity against A549 cells, ex-
hibited the most potent ROS-generating ability and induced about 5-
fold increase in ROS levels at 60 µM as compared to untreated control
2.5. The lipid peroxidation induced by β-ionone derivatives in A549 cells
Plasma membrane, playing a critical role in cellular signaling
transduction, can be vulnerably attacked by the ROS and resulting in
lipid peroxidation [18]. Furthermore, the amount of malondialdehyde
Fig. 3. The changes of GSH/GSSG ratios in A549 cells after treatment with 1a,
1d and 1g at the indicated concentrations for 6 h. Each experiment was per-
formed in triplicate.
Fig. 2. The ROS generation induced by1a, 1d and 1g at the indicated con-
centrations against A549 cells. Each experiment was performed in triplicate.
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J. Yang, et al.
BioorganicChemistry104(2020)104273
2.7. The apoptosis-inducting activity of β-ionone derivatives in A549 cells
Given that the disruption of MMP is a catastrophic event during in
apoptosis. Therefore, we determined the effects of β-ionone derivatives
(1a, 1d and 1g) on cells apoptosis by flow cytometry. As illustrated in
Fig. 6, the three of β-ionone derivatives were more potent inducer of
apoptosis than the leading curcumin, and exhibited perfect dose-de-
pendent manner. Specially, treatment with 60 µM 1a for 24 h resulted
in 51.8% late apoptotic cells. These results suggest that the apoptosis as
well the cytotoxicity, the loss of MMP, lipid peroxidation and redox
imbalance were in relation with the ROS production triggered by β-
ionone derivatives (1a, 1d and 1g) in A549 cells.
2.8. β-ionone derivatives activated Bax and Caspase 3 expression in A549
cells
Next, we test the proteins expression of Bax and Caspase 3, the well-
known apoptosis-regulating proteins, which exhibited a major role in
the apoptosis of cancer cells [20]. As a result, compounds 1a and 1g
could significantly increase the expression level of Bax and Caspase 3
compared with the control group in A549 cells (Fig. 7). Particularly, the
expression level of Bax and Caspase 3 proteins promoted by 1g was
more than that of 1a and 1d. This result suggested that the β-ionone
derivatives-induced apoptosis was mediated through the activation of
Bax and Caspase 3.
Fig. 4. MDA concentrations of A549 cells was determined after treatment with
1a, 1d and 1g at the indicated concentrations for 15 h. Values are expressed as
MDA equivalents (pmol)/ mg protein. Each experiment was performed in tri-
plicate.
(MDA), the biomarker of lipid peroxidation, was measured by using
thiobarbituric acid-reactive substance. As illustrated in Fig. 4, the for-
mation of MDA was dramatic increase in a dose-dependent manner
after treatment with 1a, 1d and 1g at the indicated concentrations.
Particularly, 60 µM 1a caused a 1-fold increase of the amount of MDA
compared to the control in A549 cells.
3. Conclusions
In summary, we synthesized a panel of β-ionone derivatives through
Aldol condension and evaluated their cytotoxicity against A549 and SK-
OV3 cells. The results illustrated that 1a, 1d and 1g, with ortho-sub-
stituents on the aromatic ring, showed much stronger anti-proliferation
activity than other β-ionone derivatives. Mechanism investigation
hinted that the cytotoxity of β-ionone derivatives were originated from
their ROS-generation abilities, which subsequently resulted in the redox
imbalance, lipid peroxidation, the collapse of the MMP, the activation
of apoptosis-regulating proteins (Bax and Caspase 3) and untimately
caused cell apoptosis (Scheme 2). This work revealed that the “ortho
effect”, promoting ROS-generation and the introduction of fluorine
atom into drugs may be potent strategies to improve anticancer activity
of β-ionone derivatives.
2.6. The loss of mitochondrial membrane potential (MMP) induced by β-
ionone derivatives in A549 cells
Mitochondrial, playing a potent role in the regulation of cell sur-
vival, can be damaged by the excess ROS [19]. We next determined the
loss of mitochondrial membrane potential using rhodamine 123 by flow
cytometry. As shown in Fig. 5, we found that the three of the β-ionone
derivatives were more active than 60 µM curcumin, and lead to a re-
markable decrease in the loss of MMP in a dose-dependent fashion.
Especially, 60 µM 1a brought about an 80% reduction of MMP in A549
cells relative to the control. The abilities of the three β-ionone deriva-
tives (1a, 1d and 1g) and curcumin were in accordance with their anti-
proliferation activities.
4. Materials and methods
4.1. Materials
Roswell Park Memorial Institute (RPMI)-1640 was from Hyclone. 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
rhodamine 123, 2´,7´-dichlorofluorescein diacetate, reduced L-glu-
tathione (GSH), oxidized -glutathione (GSSG), 2-vinylpyridine (97%)
L
and thiobarbituric acid were purchased from Beyotime. Annexin V-
FITC/PI apoptosis detection kit was from BD Biosciences. RIPA buffer,
50*Cooktail proteinase inhibitor, GAPDH, the primary antibodies and
HRP-labeled secondary antibodies were obtained from Servicebio.
Substituted benzaldehyde, β-ionone and curcumin were from
EnergyChemical. All other chemicals were of the highest quality
available.
4.2. Synthesis of the β-ionone oriented mono-carbonyl curcumin analogs
4.2.1. General procedure for the synthesis of 1a-1i
The mono-carbonyl curcumin analogs were synthesized according
to the published procedure [21]. Briefly, the aqueous NaOH (20%, w/v,
6 mL) was added dropwise to the mixture, containing β-ionone
(5 mmol) and various subsstituted benzaldehyde (5 mmol) in ethanol
(15 mL), at 0 °C. After completion of the reaction, distilled water
Fig. 5. The loss of mitochondrial membrane potential induced by 1a, 1d and
1g at the indicated concentrations in A549 cells for 12 h. Each experiment was
performed in triplicate.
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J. Yang, et al.
BioorganicChemistry104(2020)104273
Fig. 6. Flow cytometric analysis for apoptotic induction of A549 cells treated with curcumin and β-ionone derivatives (1a, 1d and 1g) for 24 h. Each experiment was
performed in triplicate.
6.96 (d, J = 16.0 Hz, 1H), 6.44 (d, J = 16.0 Hz, 1H), 2.09 (t,
J = 8.0 Hz, 2H), 1.83 (s, 3H), 1.62-–1.65 (m, 2H), 1.49–1.52 (m, 2H),
1.12 (s, 6H); 13C NMR (100 MHz, CDCl3), δ 188.8, 164.2, 161.8, 143.5,
141.2, 141.1, 137.3, 137.2, 136.5, 130.5, 130.4, 129.4, 126.7, 124.4,
124.3, 117.2, 117.0, 114.4, 114.2, 39.8, 34.2, 33.7, 28.9, 21.9, 18.9.
HRMS m/z (ES+): [M+H]+ 299.1825 (theor 299.1811).
(1E,4E)-1-(4-fluorine-phenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-yl)
penta-1,4-dien-3-one (1c): Yield 40.8%, yellow solid, 1H NMR
Fig. 7. Effects of β-ionone derivatives (1a, 1d and 1g) on the activation of Bax
and Caspase 3 in A549 cells for 18 h.
(400 MHz, (CDCl3), δ 7.61 (d, J = 16.0 Hz, 1H), 7.56 (dd, J = 8.0 Hz,
2H), 7.49 (d, J = 16.0 Hz, 1H), 7.07 (t, J = 8.0 Hz, 2H), 6.90 (d,
J = 16.0 Hz, 1H), 6.44 (d, J = 16.0 Hz, 1H), 2.09 (t, J = 8.0 Hz, 2H),
1.83 (s, 3H), 1.63–1.66 (m, 2H), 1.49–1.52 (m, 2H), 1.11 (s, 6H); 13C
NMR (100 MHz, CDCl3), δ 189.0, 165.1, 162.6, 143.3, 141.4, 137.0,
136.5, 130.2, 130.1, 129.4, 125.3, 116.2, 115.9, 39.8, 34.2, 33.7, 28.9,
21.9, 18.9. HRMS m/z (ES+): [M+H]+ 299.1826 (theor 299.1811).
(1E,4E)-1-(2-methoxy-phenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-
yl)penta-1,4-dien-3-one (1d): Yield 35.2%, yellow oil, 1H NMR
(400 MHz, (CDCl3), δ 7.98 (d, J = 16.0 Hz, 1H), 7.58 (d, J = 8.0 Hz,
1H), 7.46 (d, J = 16.0 Hz, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.05 (d,
J = 16.0 Hz, 1H), 6.96 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H),
6.48 (d, J = 16.0 Hz, 1H), 3.90 (s, 3H), 2.08 (t, J = 8.0 Hz, 2H), 1.82
(s, 3H), 1.63–1.66 (m, 2H), 1.49–1.52 (m, 2H), 1.11 (s, 6H); 13C NMR
(100 MHz, CDCl3), δ 189.9, 158.5, 142.8, 138.2, 136.6, 136.0, 131.5,
129.7, 128.8, 126.4, 123.9, 120.7, 111.1, 55.5, 39.8, 34.2, 33.6, 28.9,
21.9, 18.9. HRMS m/z (ES+): [M+H]+ 311.2025 (theor 311.2011).
(1E,4E)-1-(3-methoxy-phenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-
yl)penta-1,4-dien-3-one (1e): Yield 32.4%, yellow solid, 1H NMR
(400 MHz, (CDCl3), δ 7.61 (d, J = 16.0 Hz, 1H), 7.49 (d, J = 16.0 Hz,
1H), 7.30 (t, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H),7.11 (s, 1H),
(20 mL) was addeed, the mixture was neutralized with 10% hydro-
chloric acid solution and extracted with EtOAc. Then, the organic sol-
vent was removed and the residue was purified by a silica gel column.
Their structures were confirmed by 1H, 13C NMR and HRMS spectro-
scopy.
(1E,4E)-1-(2-fluorine-phenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-yl)
penta-1,4-dien-3-one (1a)
:
Yield 31.7%, yellow solid, 1H NMR
(400 MHz, (CDCl3), δ 7.76 (d, J = 16.0 Hz, 1H), 7.58 (t, J = 8.0 Hz,
1H), 7.50 (d, J = 16.0 Hz, 1H), 7.33–7.39 (m, 1H), 7.16 (t, J = 8.0 Hz,
1H), 7.09 (t, J = 8.0 Hz, 1H), 7.07 (d, J = 16.0 Hz, 1H), 6.47 (d,
J = 16.0 Hz, 1H), 2.09 (t, J = 8.0 Hz, 2H), 1.83 (s, 3H), 1.61–1.66 (m,
2H), 1.49–1.52 (m, 2H), 1.12 (s, 6H); 13C NMR (100 MHz, CDCl3), δ
189.3, 162.8, 160.3, 143.5, 137.0, 136.5, 135.3, 135.2, 131.7, 131.6,
129.4, 129.3, 128.0, 124.5, 124.4, 116.3, 116.1, 39.8, 34.2, 33.7, 28.9,
21.9, 18.9. HRMS m/z (ES+): [M+H]+ 299.1821 (theor 299.1811).
(1E,4E)-1-(3-fluorine-phenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-yl)
penta-1,4-dien-3-one (1b): Yield 36.4%, yellow solid, 1H NMR
(400 MHz, (CDCl3), δ 7.59 (d, J = 16.0 Hz, 1H), 7.50 (d, J = 16.0 Hz,
1H), 7.35–7.38 (m, 2H), 7.27 (d, J = 8.0 Hz, 1H), 7.08–7.10 (m, 1H),
5

J. Yang, et al.
BioorganicChemistry104(2020)104273
Scheme 2. β-ionone derivatives induce A549 apoptosis via enhancing ROS generation.
6.96 (d, J = 16.0 Hz, 1H), 6.93 (t, J = 8.0 Hz, 1H), 6.46 (d,
J = 16.0 Hz, 1H), 3.85 (s, 3H), 2.09 (t, J = 8.0 Hz, 2H), 1.83 (s, 3H),
1.61–1.64 (m, 2H), 1.49–1.52 (m, 2H), 1.11 (s, 6H); 13C NMR
(100 MHz, CDCl3), δ 189.1, 159.9, 143.2, 142.6, 136.8, 136.5, 136.3,
129.9, 129.4, 126.0, 121.0, 116.1, 113.1, 55.3, 39.8, 34.2, 33.7, 28.9,
21.9, 18.9. HRMS m/z (ES+): [M+H]+ 311.2024 (theor 311.2011).
(1E,4E)-1-(4-methoxy-phenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-
yl)penta-1,4-dien-3-one (1f): Yield 40.5%, yellow oil, 1H NMR
(400 MHz, (CDCl3), δ 7.62 (d, J = 16.0 Hz, 1H), 7.54 (d, J = 8.0 Hz,
2H), 7.46 (d, J = 16.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 2H), 6.86 (d,
J = 16.0 Hz, 1H), 6.44 (d, J = 16.0 Hz, 1H), 3.85 (s, 3H), 2.08 (t,
J = 8.0 Hz, 2H), 1.82 (s, 3H), 1.63–1.66 (m, 2H), 1.49–1.51 (m, 2H),
1.11 (s, 6H); 13C NMR (100 MHz, CDCl3), δ 189.1, 161.4, 142.6, 142.5,
136.6, 136.2, 130.0, 129.7, 127.6, 123.6, 114.4, 55.4, 39.8, 34.2, 33.7,
28.9, 21.9, 18.9. HRMS m/z (ES+): [M+H]+ 311.2026 (theor
311.2011).
NMR (100 MHz, CDCl3), δ 188.7, 143.8, 140.6, 138.4, 137.5, 136.5,
129.3, 128.3, 127.7, 125.9, 125.8, 39.8, 34.2, 33.8, 28.9, 21.9, 18.9.
HRMS m/z (ES+): [M+H]+ 349.1807 (theor 349.1779).
(1E,4E)-1-(2-hydroxy-phenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-yl)
penta-1,4-dien-3-one (1j): Yield 26.3%, yellow solid, 1H NMR
(400 MHz, (CDCl3), δ 8.07 (d, J = 16.0 Hz, 1H), 7.75 (s, 1H), 7.55 (d,
J = 16.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H),
7.22 (d, J = 16.0 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.90 (t, J = 8.0 Hz,
1H), 6.54 (d, J = 16.0 Hz, 1H), 6.38 (s, 1H), 2.09 (t, J = 8.0 Hz, 2H),
1.84 (s, 3H), 1.63–1.65 (m, 2H), 1.49–1.52 (m, 2H), 1.12 (s, 6H); 13C
NMR (100 MHz, CDCl3), δ 191.1, 156.3, 143.6, 139.8, 137.2, 129.1,
126.1, 122.1, 120.4, 116.8, 39.8, 34.2, 33.8, 28.9, 21.9, 18.9. HRMS m/
z (ES+): [M+H]+ 297.1869 (theor 297.1855).
(1E,4E)-1-(3-hydroxy-phenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-yl)
penta-1,4-dien-3-one (1 k): Yield 28.8%, yellow oil, 1H NMR (400 MHz,
(CDCl3), δ 7.62 (d, J = 16.0 Hz, 1H), 7.53 (d, J = 16.0 Hz, 1H), 7.25 (t,
J = 8.0 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.14 (s, 1H), 6.97 (d,
J = 16.0 Hz, 1H),6.91 (d, J = 16.0 Hz, 1H), 6.47 (d, J = 16.0 Hz, 1H),
6.38 (s, 1H), 2.09 (t, J = 8.0 Hz, 2H), 1.83 (s, 3H), 1.62–1.65 (m, 2H),
1.48–1.51 (m, 2H), 1.11 (s, 6H); 13C NMR (100 MHz, CDCl3), δ 189.9,
156.6, 143.9, 143.3, 137.8, 136.5, 136.2, 130.1, 129.1, 125.7, 120.6,
117.9, 115.1, 39.8, 34.2, 33.8, 28.9, 21.9, 18.9. HRMS m/z (ES+): [M
+H]+ 297.1870 (theor 297.1855).
(1E,4E)-1-(2-trifluoromethy-phenyl)-5-(2,6,6-trimethylcyclohex-1-
en-1-yl)penta-1,4-dien-3-one (1 g): Yield 38.7%, yellow oil, 1H NMR
(400 MHz, (CDCl3), δ 7.98 (d, J = 16.0 Hz, 1H), 7.77 (d, J = 8.0 Hz,
1H), 7.71 (d, J = 8.0 Hz, 1H), 7.57 (t, J = 8.0 Hz, 1H), 7.50 (d,
J = 16.0 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 6.89 (d, J = 16.0 Hz, 1H),
6.50 (d, J = 16.0 Hz, 1H), 2.09 (t, J = 8.0 Hz, 2H), 1.84 (s, 3H),
1.61–1.66 (m, 2H), 1.49–1.52 (m, 2H), 1.12 (s, 6H); 13C NMR
(100 MHz, CDCl3), δ 189.1, 143.8, 138.0, 137.4, 136.5, 132.1, 130.2,
130.0, 129.7, 129.5, 128.6, 127.8, 126.2, 39.8, 34.2, 33.8, 28.8, 21.8,
18.9. HRMS m/z (ES+): [M+H]+ 349.1808 (theor 349.1779).
(1E,4E)-1-(3-trifluoromethy-phenyl)-5-(2,6,6-trimethylcyclohex-1-
en-1-yl)penta-1,4-dien-3-one (1 h): Yield 39.7%, yellow solid, 1H NMR
(400 MHz, (CDCl3), δ 7.83 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.65 (d,
J = 16.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H),
7.51 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 16.0 Hz, 1H), 6.47 (d,
J = 16.0 Hz, 1H), 2.10 (t, J = 8.0 Hz, 2H), 1.84 (s, 3H), 1.61–1.66 (m,
2H), 1.50–1.52 (m, 2H), 1.13 (s, 6H); 13C NMR (100 MHz, CDCl3), δ
188.6, 143.7, 140.6, 137.4, 136.5, 135.8, 131.3, 129.4, 129.2, 127.3,
126.6, 126.5, 124.6, 39.9, 34.2, 33.8, 28.9, 21.9, 18.9. HRMS m/z
(ES+): [M+H]+ 349.1801 (theor 349.1779).
(1E,4E)-1-(4-hydroxy-phenyl)-5-(2,6,6-trimethylcyclohex-1-en-1-yl)
penta-1,4-dien-3-one (1 l): Yield 30.5%, yellow oil, 1H NMR (400 MHz,
(CDCl3), δ 7.65 (d, J = 16.0 Hz, 1H), 7.53 (d, J = 16.0 Hz, 1H), 7.53 (s,
1H), 7.47 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 16.0 Hz, 1H),6.49 (d,
J = 16.0 Hz, 1H), 2.08 (t, J = 8.0 Hz, 2H), 1.82 (s, 3H), 1.62–1.65 (m,
2H), 1.48–1.51 (m, 2H), 1.11 (s, 6H); 13C NMR (100 MHz, CDCl3), δ
190.3, 159.1, 144.0, 143.6, 137.6, 136.5, 130.5, 129.2, 126.8, 122.9,
116.2, 39.8, 34.2, 33.8, 28.9, 21.9, 18.9. HRMS m/z (ES+): [M+H]+
297.1867 (theor 297.1855).
(1E,4E)-1-phenyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl)penta-1,4-
dien-3-one (1 m): Yield 26.9%, yellow solid, 1H NMR (400 MHz,
(CDCl3), δ 7.65 (d, J = 16.0 Hz, 1H), 7.58 (dd, J = 8.0, 4.0 Hz, 2H),
7.49 (d, J = 16.0 Hz, 1H), 7.39–7.40 (m, 3H), 6.98 (d, J = 16.0 Hz,
1H), 6.46 (d, J = 16.0 Hz, 1H), 2.09 (t, J = 8.0 Hz, 2H), 1.83 (s, 3H),
1.63–1.64 (m, 2H), 1.49–1.52 (m, 2H), 1.12 (s, 6H); 13C NMR
(100 MHz, CDCl3), δ 189.2, 143.2, 142.8, 136.8, 136.5, 134.9, 130.3,
129.5, 128.9, 128.3, 125.6, 39.8, 34.2, 33.7, 28.9, 21.9, 18.9. HRMS m/
z (ES+): [M+H]+ 281.1921 (theor 281.1905).
(1E,4E)-1-(4-trifluoromethy-phenyl)-5-(2,6,6-trimethylcyclohex-1-
en-1-yl)penta-1,4-dien-3-one (1i): Yield 42.6%, yellow solid, 1H NMR
(400 MHz, (CDCl3), δ 7.68 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 16.0 Hz,
2H), 7.64 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 16.0 Hz, 1H), 7.04 (d,
J = 16.0 Hz, 1H), 6.46 (d, J = 16.0 Hz, 1H), 2.10 (t, J = 8.0 Hz, 2H),
1.84 (s, 3H), 1.62–1.66 (m, 2H), 1.50–1.52 (m, 2H), 1.12 (s, 6H); 13C
6

J. Yang, et al.
BioorganicChemistry104(2020)104273
4.3. MTT assay
Cells were collected, washed three times with ice-cold phosphate-buf-
fered saline and lysed with ice-cold RIPA lysis buffer containing pro-
teinase inhibitors. Then, the equal amounts of protein were separated
on SDS-PAGE and blotted onto PVDF membranes. The membranes were
blocked with 5% skim milk and probed with the primary antibodies
before incubation with the corresponding HRP-labeled secondary an-
tibody. After washing, the blotted protein were detected with enhanced
chemiluminescence (ECL).
Human lung cancer cells (A549) and human ovarian cancer cells
(SK-OV3), which were from the Shanghai Institute of Biochemistry and
Cell Biology, Chinese Academy of Sciences, were grown and maintained
in RPMI-1640 medium at 37 °C in a humidified atmosphere with 5%
CO2. A549 (3 × 103/well) and SK-OV3 cells (5 × 103/well) were
plated in 96-well plates, then incubated with vehicle alone or the tested
compounds at the indicated concentrations for 48 h. Subsequently, the
MTT assay was done to evaluate the cytotoxic potential as reported
previously [22,23].
4.11. Molecular docking simulation
Crystal structure of human TrxR1 was downloaded from RCSB PDB
database (http://www.rcsb.org) with PDBID 2J3N. The protein was
prepared according to AutoDock 4.2 manual: The bound ligand was
extracted from the complexes, water molecules were removed, and
hydrogen and charge were automatically added. The ligands were en-
ergy minimized with MM2 force field using ChemBioOffice 2014
(http://www.cambridgesoft.com), and the prepared structures were
4.4. Stability assay
The stability of curcumin and ionone-oriented analogs, dissolving in
RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal calf
serum, was monitored at their maximun absorbance for 120 min at 10-
min intervals as described previously [24].
used as input files for docking. The grid box of 60 Ǻ size (x, y, z) with a
spacing of 0.375 Ǻ was created representing the active binding site
where contained the native ligand and all the important amino acids.
Finally, the best pose of Figure was showed by pyMol [28].
4.5. Cell apoptosis analysis
A549 (3 × 105) cells were incubated in 6-well plates and treated
with vehicle alone or the tested compounds at the indicated con-
centrations for 24 h. The cells were harvested, washed with PBS and
labeled with FITC Annexin-V and PI as described previously [24].
Subsequently, the samples were analyzed by using a flow cytometry.
4.12. Statistical analysis
The data was expressed as the mean
SD of at least three in-
dependent experiments. Significant differences (P < 0.05) between
4.6. Intracellular ROS measurement
the means of two groups are analyzed by Student’s t-test.
A549 (3 × 105) cells were seeded in 6-well plates and incubated
with the vehicle alone or the tested compounds at the indicated con-
centrations for 6 h. The cells were collected, washed with PBS and
stained with DCFH-DA at the dark. Then, the cells were washed with
PBS and resuspended in PBS followed by analyzing with a flow cyto-
metry.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
4.7. Measurement of GSH and GSSG levels
This work was supported by the National Natural Science
Foundation of China (Grant No. 81901420), Shandong Provincial
Natural Science Foundation (Grant No. ZR2018LH022), the Open
Project of Shandong Collaborative Innovation Centre for Antibody
Drugs (Grant No. CIC-AD1822 and CIC-AD1824).
A549 (3 × 105 cells/well) cells were cultured in 6-well plates and
incubated for 24 h. After treatment with the tested compounds at the
indicated concentrations for 6 h, the cells were harvested, washed with
PBS and lysed. Then, the intracellular GSH and GSSG content were
determined by the glutathione reductase-DTNB recycling [25].
Appendix A. Supplementary material
4.8. Determination of thiobarbituric acid-reactive substance (TBARS)
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104273.
A549 cells were cultured at 3 × 105 cells/well in 6-well plates and
incubated for 24 h. After incubation with the vehicle alone or the test
compounds for 18 h, the cells were harvested and lysed. Then, the
samples were tested by the protocol as previously described [26].
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