Discovery of New Monocarbonyl Ligustrazine-Curcumin Hybrids for Intervention of Drug-Sensitive and Drug-Resistant Lung Cancer

Article abstract of DOI:10.1021/acs.jmedchem.5b01203

The elevation of oxidative stress preferentially in cancer cells by inhibiting thioredoxin reductase (TrxR) and/or enhancing reactive oxygen species (ROS) production has emerged as an effective strategy for selectively targeting cancer cells. In this study, we designed and synthesized 21 ligustrazine-curcumin hybrids (10a-u). Biological evaluation indicated that the most active compound 10d significantly inhibited the proliferation of drug-sensitive (A549, SPC-A-1, LTEP-G-2) and drug-resistant (A549/DDP) lung cancer cells but had little effect on nontumor lung epithelial-like cells (HBE). Furthermore, 10d suppressed the TrxR/Trx system and promoted intracellular ROS accumulation and cancer cell apoptosis. Additionally, 10d inhibited the NF-κB, AKT, and ERK signaling, P-gp-mediated efflux of rhodamine 123, P-gp ATPase activity, and P-gp expression in A549/DDP cells. Finally, 10d repressed the growth of implanted human drug-resistant lung cancer in mice. Together, 10d acts a novel TrxR inhibitor and may be a promising candidate for intervention of lung cancer.

Full text of DOI:10.1021/acs.jmedchem.5b01203

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Article
Discovery of New Monocarbonyl Ligustrazine-Curcumin Hybrids
for Intervention of Drug-Sensitive and Drug-Resistant Lung Cancer
Yong Ai, Bin Zhu, Caiping Ren, Fenghua Kang, Jinlong Li, Zhangjian
Huang, Yisheng Lai, Sixun Peng, Ke Ding, Jide Tian, and Yihua Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01203 • Publication Date (Web): 18 Feb 2016
Downloaded from http://pubs.acs.org on February 19, 2016
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Discovery of New Monocarbonyl Ligustrazine-Curcumin Hybrids
for Intervention of Drug-Sensitive and Drug-Resistant Lung
Cancer
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Yong Ai,‡a,b Bin Zhu,‡c Caiping Ren,*c Fenghua Kang,a,b Jinlong Li,c Zhangjian
Huang,*a,b Yisheng Lai,a,b Sixun Peng,a,b Ke Ding,d Jide Tian,e and Yihua Zhang*a,b
aState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing
210009, PR China
bJiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China
Pharmaceutical University, Nanjing 210009, PR China
cCancer Research Institute, Collaborative Innovation Center for Cancer Medicine, Key
Laboratory for Carcinogenesis of Chinese Ministry of Health, School of Basic Medical
Sciences, Central South University, Changsha 410078, PR China
dKey Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou
Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530,
PR China
eDepartment of Molecular and Medical Pharmacology, University of California, Los
Angeles, California 90095, United States
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ABSTRACT The elevation of oxidative stress preferentially in cancer cells by inhibiting
thioredoxin reductase (TrxR) and/or enhancing reactive oxygen species (ROS) production
has emerged as an effective strategy for selectively targeting cancer cells. In this study,
we designed and synthesized twenty-one ligustrazine-curcumin hybrids (10a-u).
Biological evaluation indicated that the most active compound 10d significantly inhibited
the proliferation of drug-sensitive (A549, SPC-A-1, LTEP-G-2) and drug-resistant
(A549/DDP) lung cancer cells but had little effect on non-tumor lung epithelial-like cells
(HBE). Furthermore, 10d suppressed the TrxR/Trx system and promoted intracellular
ROS accumulation and cancer cell apoptosis. Additionally, 10d inhibited the NF-κB,
AKT and ERK signaling, P-gp-mediated efflux of rhodamine 123, P-gp ATPase activity
and P-gp expression in A549/DDP cells. Finally, 10d repressed the growth of implanted
human drug-resistant lung cancer in mice. Together, 10d acts a novel TrxR inhibitor and
may be a promising candidate for intervention of lung cancer.
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INTRODUCTION
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Cancer cells are usually exposed to a moderate level of reactive oxygen species (ROS),
primarily due to their active metabolism in response to oncogenic signals.1 In fact, cancer
cells take advantage of this moderate oxidative stress for several important processes
such as proliferation, angiogenesis, and metastasis.2 However, high levels of ROS
irreversibly damage DNA and lipids, and ultimately cause cancer cell apoptosis.3
Recently, pharmacological elevation of intracellular ROS has emerged as an effective
strategy for selectively targeting cancer cells. While an exogenous ROS insult is tolerable
to normal cells it may exceed thethreshold cancer cells can endure and lead to selective
cytotoxicity against cancer cells.4,5 Actually, there have been increasing efforts to
increase the levels of ROS specifically in cancer cells for ‘oxidation therapy’.6-8 One
strategy for oxidation therapy is to directly deliver ROS promoting agents such as
ligustrazine (1, Figure 1),9 arsenic trioxide (As2O3)10,11 and glucose oxidase to tumor
tissues.12,13
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Figure 1. Chemical structures of ligustrazine, curcumin and SCAs.
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Ligustrazine is a Chinese medicine for treatment of cardiovascular and cerebrovascular
diseases.14 Recently, ligustrazine has been shown to increase intracellular ROS
accumulation, and to induce cancer cell apoptosis.9,15,16 More importantly, ligustrazine is
a reversal agent against ATP-binding cassette (ABC) transporter-mediated multidrug
resistance (MDR).17,18 Unfortunately, ligustrazine has a short half-life and low
bioavailability in vivo and is required for frequent treatments with a high dose, which
results in the drug-accumulation related toxicity.19 Besides, ligustrazine is usually used in
combination with an anticancer drug to circumvent MDR, which may lead to
pharmacokinetic interactions of the drugs and to increase adverse effects of the anticancer
therapy. Thus, modification of ligustrazine is needed to improve its therapeutic potency.
‘Oxidation therapy’ is to disrupt the redox balance in cancer cells by suppressing the
antioxidant systems.6,20-22 The thioredoxin system, one of the key antioxidant systems is
composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, and regulates
numerous cellular signal pathways involved in cell survival and proliferation.23-25 TrxR is
the only known physiological enzyme to catalyze the reduction of oxidized Trx.
Importantly, the thioredoxin system is often overexpressed in many tumors,26,27
associated with chemoresistance of cancers.28 Recently, accumulating data support that
TrxR is a promising target for development of novel anticancer agents, and the efficacy
of this redox-modulating method has been demonstrated in models of drug-resistant
cancers.29 In the past years, a great endeavor has been witnessed in discovering and
developing a variety of TrxR-targeting small molecules, including curcumin (2, Figure 1),
cinnamaldehydes, acylfulvenes, flavonoids, etc., many of which have been combined
with an anticancer drug as a potential therapy against drug-sensitive and drug-resistant
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cancers.30-37
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Curcumin, a yellow spice extracted from the Curcuma longa, has been identified as a
TrxR inhibitor with anticancer activity.38-41 To improve its potency, many synthetic
curcumin analogs (SCAs) have been prepared, including EF24 (3), EF31 (4), UBS109 (5)
(Figure 1),42 6, and others.43,44 Among them, those monocarbonyl curcumin analogs
display strong anticancer activity by inhibiting cancer cell proliferation and inducing
cancer cell apoptosis.45-50
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Given that use of an ROS promoting agent together with an inhibitor of ROS
scavenging can amplify oxidative stress and be effective for treatment of advanced solid
tumors,21,51 we hypothesized that new hybrids of ligustrazine scaffold with curcumin
moiety might not only elevate ROS production but also diminish the antioxidant defense
systems, leading to ROS accumulation preferentially in cancer cells and to subsequent
ROS-mediated cytotoxicity against cancer cells. Accordingly, we synthesized hybrids
10a-u by substituting one of the two aromatic rings of curcumin analogs with ligustrazine
via a Claisen-Schmidt condensation reaction, and evaluated their bioactivity in vitro and
in vivo.
RESULTS AND DISCUSSION
Chemistry. The synthetic routes to compounds 10a-u are depicted in Scheme 1.
2-Hydroxymethyl-3,5,6-trimethylpyrazine 7 was prepared by the Boekelheide reaction
starting from ligustrazine 1 as described previously.52 The generated 7 underwent a
2-iodoxybenzoic acid (IBX) oxidation giving the aldehyde group-containing intermediate
8, which was directly reacted with the aryl-substituted enones 9a-u, prepared from
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corresponding aldehydes 11a-u,53 in the presence of borontrifluoride-etherate (BF3·Et2O)
via a Claisen-Schmidt condensation reaction to get the target compounds 10a-u. The
purity of all hybrids was greater than 95% and determined by HPLC analysis. The
structures of all hybrids were fully characterized and identified as E,E configuration.
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Scheme 1. Synthetic Routes of Ligustrazine-Curcumin Hybrids 10a-u.a
aReagents and conditions: (a) 30% H2O2, acetic acid, 70 °C, 8 h.; (b) acetic anhydride,
reflux, 2 h; (c) 20% NaOH; (d) IBX, DMSO, room temperature, 0.5 h; (e) Morpholinium
trifluoroacetate, acetone, 60 °C, sealing tube reaction for 24 h; (f) BF3·Et2O, 1,4-dioxane,
reflux, 4-6 h.
BIOLOGICAL EVALUATION
Evaluation of Antiproliferative Activity. Given that compounds 10a-u we designed
may effectively target TrxR expressed in cancer cells, we first examined the levels of
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TrxR expression in drug-sensitive human lung cancer A549, drug-resistant human lung
cancer A549/DDP cells and human non-tumor bronchial epithelial HBE cells by Western
blot assay. We observed that the levels of TrxR expression in A549 cells were
significantly higher than that in HBE cells, but significantly lower than that in A549/DDP
cells (P < 0.001). Hence, TrxR was up-regulated in lung cancer cells, particularly in
drug-resistant A549/DDP cells.
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Figure 2. TrxR Expression in HBE, A549 and A549/DDP cells. HBE, A549 and
A549/DDP cells were harvested and lyzed. The relative levels of TrxR to control β-actin
in HBE, A549 and A549/DDP cell lysates were determined by Western blot. Data are
representative of three independent experiments. ***P < 0.001.
Next, the antiproliferative activity of compounds 10a-u against A549 and A549/DDP
cells was initially screened by the MTT assay using cisplatin (DDP) as a positive control.
As shown in Table 1, compounds 10a-l and 10q displayed potent inhibitory activity
against both A549 (IC50 = 1.16-4.78 ꢀM) and A549/DDP (IC50 = 0.60-5.20 ꢀM) cell
viability, and their anti-proliferative activity was more potent than DDP (IC50 = 8.10 and
45.14 ꢀM) their individual moieties, 1 (IC50 > 200 and 200 ꢀM) and 2 (IC50 = 38.12 and
32.39 ꢀM), and even the combination of 1 and 2 (IC50 = 24.33 and 25.17 ꢀM). It was
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obvious that R substitutes at benzene ring of the hybrids (Table 1) may be crucial for their
anticancer activity. In general, the compounds with an electron-donating group such as
methoxy, hydroxy or methyl at benzene ring (10a-l and 10q) usually showed more potent
inhibitory activity than those bearing an electron-withdrawing group such as halogen,
trifluoromethyl, nitro or benzene (10n-p and 10r-u).
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Subsequently, the five potent compounds (10d, 10e, and 10i-k) were further
investigated for their antitumor efficacy by MTT (Table 2). We found that all of the five
compounds displayed more potent activity (IC50 = 2.34-6.32 ꢀM) than DDP (IC50
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14.18-15.51 ꢀM) against human lung cancer SPC-A-1 and LTEP-G-2 cells that were
similar to A549 cells with high levels of TrxR expression. While DDP had similar
inhibitory activity against both lung cancer and non-tumor HBE cells, 10d exhibited a
7~35-fold less antiproliferative activity against HBE cells (IC50 = 21.34 ꢀM), suggesting
that 10d may have antiproliferative activity selectively against cancer cells.
Table 1. The antiproliferative activity of target compounds (10a-u) against human lung
cancer A549 and A549/DDP cells.
Compd
R substituents
IC50 (ꢀM)a
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2
OH
H
3
H
4
H
5
A549
A549/DDP
3.90 0.14b
3.70 0.21b
4.79 0.77b
0.60 0.11b
2.19 0.15b
4.74 0.41b
3.44 0.24b
3.61 0.77b
2.44 0.24b
2.85 0.55b
1.00 0.13b
H
H
H
H
H
H
H
H
H
H
4.78 0.35b
4.30 0.32b
4.01 0.53b
2.19 0.14b
2.85 0.22b
3.20 0.69b
3.30 0.12b
3.50 0.89b
3.79 0.22b
3.07 1.55b
1.60 0.11b
10a
10b
10c
10d
10e
10f
10g
10h
10i
OH
H
H
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H
OH
OH
H
H
OCH3
OCH3
H
OH
OCH3
H
H
OCH3
H
H
H
OCH3
OCH3
OCH3
H
OCH3
H
OCH3
H
10j
10k
OCH3
OCH3 OCH3
H
H
4.50 0.54b
5.20 0.97b
10l
H
H
H
H
H
F
H
H
H
H
H
H
H
12.31 1.11
5.90 0.47
15.8 1.23
11.7 1.09
4.3 1.29b
17.33 0.54
6.70 0.14
11.88 0.66b
7.44 0.59b
7.49 0.67b
6.90 0.53b
3.99 0.33b
6.39 0.21b
6.69 0.11b
10m
10n
10o
10p
10q
10r
10s
H
H
Cl
H
H
CF3
CH3
H
H
H
NO2
H
H
NO2
H
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H
H
NO2
H
H
9.60 0.97
7.10 0.74
7.90 0.17b
10t
11.80 0.91b
10u
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> 200
> 200
1
2
38.12 2.87
24.33 2.77
8.10 0.97
32.39 3.14
25.17 2.89
45.14 2.11
1+2
DDP
aCells were treated in triplicate with tested compounds for 72 h and the cell viability was
determined using MTT assay. bP < 0.01 vs the DDP.
Table 2. The antiproliferative activity of selected compounds against human lung cancer
SPC-A-1, LTEP-G-2 and non-tumor HBE cells.
IC50 (ꢀM)a
Compd
SPC-A-1
LTEP-G-2
2.88 0.13b
5.01 0.34b
4.36 0.23b
5.91 0.45b
2.34 0.13b
14.18 1.24
HBE
3.12 0.41b
5.71 0.47b
5.42 0.38b
6.32 0.59b
5.35 0.44b
15.51 1.23
21.34 3.76b
17.56 1.55b
16.23 2.68b
20.76 1.55b
14.35 1.37
10.37 1.54
10d
10e
10i
10j
10k
DDP
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aCells were treated in triplicate with tested compounds for 72 h and the cell viability was
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Inhibition of TrxR by selected compounds in both cell-free and cellular assay. Since
10d, 10e, and 10i-k displayed the promising antiproliferative activity against lung cancer
cells, we tested their TrxR inhibitory activity in purified TrxR enzyme and in A549 and
A549/DDP cells. Firstly, the TrxR inhibitory activity of 10d, 10e, and 10i-k in purified
TrxR enzyme was determined with DTNB assay.37 As shown in Table 3, 10d, 10e, and
10i-k exhibited more potent TrxR inhibitory activity (IC50 = 2.95-7.63 ꢀM) than
curcumin 2 (IC50 = 41.42 3.21 ꢀM), while ligustrazine 1 showed little TrxR inhibitory
activity at 200 ꢀM. Next, we determined the TrxR inhibitory activity of 10d, 10e, and
10i-k in cells. Briefly, A549 and A549/DDP cells were treated with different
concentrations of each compound for 24 h. The activity of TrxR in different groups of
cells was determined using a Thioredoxin Reductase Assay Kit (Cayman).54 As shown in
Table 3, all of the five compounds exerted potent TrxR inhibitory activity (IC50
=
1.22-4.73 ꢀM). As expected, 10d showed the highest TrxR inhibitory activity, which was
~32-fold more potent than curcumin 2 (IC50s = 40.56, 39.42 ꢀM), suggesting that 10d
may be a novel TrxR inhibitor for further investigation.
Furthermore, we determined the impact of 10d treatment on the expression of TrxR
and Trx in A549 and A549/DDP cells by Western blot. As shown in Figure 3, 10d
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significantly reduced the relative levels of TrxR and Trx to GAPDH expression in A549
and A549/DDP cells. Hence, the decrease in the levels of TrxR and Trx expression by
10d may contribute to its inhibition on TrxR and anticancer activity in lung cancer cells.
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Table 3. Inhibition of compounds on TrxR activity in cell-free and cellular assays.
In cellular assay IC50 (ꢀM)b
In cell-free assay
Compd
IC50 (ꢀM)a
A549
A549/DDP
1.22 0.13c
4.64 0.34c
4.22 0.31c
4.26 0.44c
1.98 0.14c
> 200
2.95 0.17c
5.30 0.22c
7.09 0.35c
7.63 0.13c
3.06 0.21c
> 200
1.49 0.11c
4.73 0.37c
4.17 0.24c
4.12 0.2 c
2.36 0.21c
> 200
10d
10e
10i
10j
10k
1
41.42 3.21
40.56 3.67
39.42 2.11
2
aThe TrxR inhibition activities in cell-free assay were measured by DTNB assay as
b
described in the Experimental Section. The TrxR inhibitory activity of selected
compounds in A549 and A549/DDP cells using a Thioredoxin Reductase Assay Kit
(Cayman). cP < 0.05 vs the ligustrazine 1 and curcumin 2.
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Figure 3. Effect of 10d on the relative levels of TrxR and Trx expression in A549 and
A549/DDP cells. Cells were treated with 10d or vehicle for 24 h and the relative levels of
TrxR and Trx to control GAPDH expression were determined by Western blot. Data are
representative of three independent experiments. *P < 0.05, **P < 0.01 vs the control.
Effect of 10d on the ROS Accumulation in cells. The Trx system is crucial for the
intracellular redox balance to prevent excess ROS accumulation.34,54,55 The inhibition of
TrxR and Trx may disturb the redox balance, leading to intracellular ROS accumulation
in cancer cells. Accordingly, we examined the impact of 10d on ROS levels in A549 and
A549/DDP cells. A549 and A549/DDP cells were treated in triplicate with various
concentrations of 10d, 1, and 2 for 60 min. The cells were collected longitudinally and
stained with dihydroethidium (DHE, Beyotime). The levels of intracellular ROS were
determined by measuring the fluorescent signals using a fluorescence microplate reader.
As shown in Figure 4A and B, treatment with 10d rapidly and significantly increased the
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levels of intracellular ROS in both A549 and A549/DDP cells. More importantly,
treatment with 10d significantly reduced the viability of A549 and A549/DDP cells
(Figure 4C), which was partially or completely abrogated by pre-treatment with 10 mM
N-acetyl-L-cysteine (NAC) or glutathione (GSH), respectively. These data indicated that
10d promoted ROS accumulation that was cytotoxic to lung cancer cells.
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Figure 4. Treatment with 10d induces the accumulation of toxic ROS in A549 (A) and
A549/DDP (B) cells. (C) Pre-treatment with 10 mM GSH or NAC for 1 h demolishes the
cytotoxicity of 10d against A549 and A549/DDP cells. Data are representative of three
independent experiments. **P < 0.01, ***P < 0.001 vs the control. #P < 0.05 vs the 10d
group.
Induction of A549 and A549/DDP cell apoptosis. To determine whether the inhibitory
effects of 10d on lung cancer cellular proliferation are accompanied by enhanced cancer
cell apoptosis, Annexin V-FITC and propidium iodide (PI) staining were carried out, and
the percentages of apoptotic cells were tested using flow cytometry assay. A549 and
A549/DDP cells were incubated with different concentrations of vehicle, 10d,
ligustrazine 1, or curcumin 2 for 24 h. We observed that treatment with 10d induced
apoptosis in both A549 and A549/DDP cells (Figure 5), which were significantly
stronger than that of 1 and 2.
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Figure 5. Apoptosis of A549 (A, C) and A549/DDP (B, D) cells treated with 10d,
ligustrazine 1 and curcumin 2 for 24 h. Values are the means SD from at least three
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independent experiments. **P < 0.01, ***P < 0.001 vs the ligustrazine 1 and curcumin 2
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Furthermore, Western blot assay revealed that treatment with 10d dramatically
decreased the levels of anti-apoptotic Bcl-2, but increased the levels of pro-apoptotic Bax
expression in A549 and A549/DDP cells (Figure 6A). Quantitative analysis revealed that
treatment with 10d significantly increased the ratios of Bax to Bcl-2 (Figure 6B). Caspase
activation is a critical event in the initiation and execution of apoptosis in cells.
Treatment with 10d also significantly increased the relative levels of cleaved caspase 3
and poly(ADP-ribose)polymerase (PARP) in A549 and A549/DDP cells.
Figure 6. Effect of 10d on the relative levels of Bcl-2, Bax, PARP, and Caspase 3
expression in A549 and A549/DDP cells. Cells were treated with 10d (A549, 2.1 ꢀM or
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4.2 ꢀM; A549/DDP, 0.6 ꢀM or 1.2 ꢀM) or vehicle for 24 h and the relative protein levels
of Bcl-2, Bax, PARP, and Caspase 3 to β-actin were determined using Western blot
assays. Data are representative of three independent experiments. *P < 0.05, **P < 0.01,
***P < 0.001 vs the control.
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Effects of 10d on the NF-κB, AKT, and ERK Signaling. Recent studies indicate that
high levels of ROS in cancer cells directly inhibit the NF-κB, AKT and ERK
activation,55-57 and aberrant activation of NF-κB, AKT and ERK are associated with the
proliferation and drug resistance of tumor cells.58-63 To further understand the molecular
mechanisms underlying the activity of 10d, we investigated the regulatory effects of 10d
on the NF-κB, AKT, and ERK signaling in A549 and A549/DDP cells. The cells were
treated with various concentrations of 10d. The levels of NF-κB, AKT, and ERK
expression and phosphorylation were determined using Western blotting. Although
treatment with 10d did not alter the levels of NF-κB and ERK expression, it significantly
suppressed the phosphorylations of NF-κB, AKT and ERK in A549 and A549/DDP cells
(Figure 7). These results suggest that 10d may inhibit spontaneous activation of the
NF-κB, AKT, and ERK pathways, contributing to its antitumor activity in lung cancer
cells. Nevertheless, the precise mechanisms underlying the action of 10d remain to be
further investigated.
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Figure 7. Effect of 10d on the NF-κB, AKT and ERK signaling in A549 and A549/DDP
cells. Cells were treated with 10d (A549, 2.1 ꢀM or 4.2 ꢀM; A549/DDP, 0.6 ꢀM or 1.2
ꢀM) or vehicle for 24 h and the relative levels of NF-κB, AKT and ERK expression and
phosphorylation were determined using Western blot assays. Data are representative of
three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs the control.
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Effect of 10d on P-gp in A549/DDP cells. Previous studies have shown that
over-expression of P-glycoprotein (P-gp) in cancer cells can export anticancer drugs out
and cause consequent drug ineffectiveness,64,65 which is a leading factor for tumor
MDR.66-71 Thus, to gain more insights into the role of 10d in suppressing A549/DDP cell
proliferation, we examined the effect of 10d on P-gp in A549/DDP cells.
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We first determined the levels of P-gp expression in A549 and A549/DDP cells using
Western blot assay. As shown in Figure 8A, high levels of P-gp were expressed in
A549/DDP cells while only little was detected in A549 cells. Next, the impact of 10d on
the levels of P-gp expression was determined by Western blot and reverse
transcription-PCR (RT-PCR). Treatment with 10d significantly decreased the relative
levels of P-gp mRNA transcripts and protein expression in A549/DDP cells (Figure 8B
and C).
In addition, the effect of 10d on P-gp-mediated efflux of rhodamine 123 (Rh123) in
A549/DDP cells was examined (Figure 8D). Treatment with different concentrations
(0.03-1.2 ꢀM) of 10d increased the levels of intracellular Rh123 accumulation in
A549/DDP cells and the effect of 10d at 1.2 ꢀM was stronger than that of the positive
control verapamil (VRP). Collectively, these results suggest that 10d had potent
antiproliferative activity against A549/DDP cells by inhibiting P-gp expression in
drug-resistant human lung cancer cells.
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Figure 8. (A) Expression profiles of P-gp in cells. (B) Effect of 10d on the protein
expression of P-gp in A549/DDP cells. Cells were treated with 10d (0.6 or 1.2 ꢀM) or
vehicle for 24 h and the relative levels of P-gp to β-actin expression were determined by
Western blot assay. (C) The relative levels of P-gp mRNA transcripts in A549/DDP cells.
The cells were treated with compounds for 24 h. The relative levels of P-gp to GAPDH
mRNA transcripts were determined by RT-PCR and expressed as fold change of the
control (in the presence of 0.1% DMSO). Data are representative of three independent
experiments. **P < 0.01 vs the DMSO group. (D) The accumulation of rhodamine 123 in
A549/DDP cells was analyzed by flow cytometry.
Effect of 10d on P-gp ATPase activity. The efflux of drug by P-gp is dependent on ATP,
which is derived from ATP hydrolysis by the ATPase, and the activity of P-gp ATPase is
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closely related to the transport capacity of P-gp. To further understand its action, we
examined the impact of 10d on the activity of P-gp ATPase in A549/DDP cells using
Pgp-GloTM assay system (Promega, USA), according to a previously described
method.72,73 We isolated the crude membranes from A549/DDP cells and treated in
triplicate with 10d (40 µM), 200 µM Sodium vanadate (Na3VO4, a negative control),
VRP (the inhibitor of P-gp) or vehicle in the presence of MgATP (5 mM, 37 °C, 40 min).
The RLU (relative light unit) representing the ATP consumed by P-gp ATPase was
measured using a luminescent detector. The RLU values in the VRP-treated membrane
samples were significantly lower than that in the vehicle-treated controls (Table 4). In
contrast, treatment with 10d, similar to that of negative control Na3VO4, significantly
increased the values of RLU in A549/DDP cell membranes. Given that the values of
RLU were negatively correlated with the activities of ATPase in the samples, these data
indicated that VRP stimulated the ATPase activity while 10d inhibited the ATPase
activity.
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Table 4. Effect of 10d on P-gp ATPase activity.
Luminescence
Compound
Concentration (ꢀM)
(relative light units)a
706683 23554
Untreated
VRP
0
200
200
40
641490 17014*
790447 12182**
761592 14162*
Na3VO4
10d
aRelative light units (RLU) represent the levels of ATP in the samples, and are negatively
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correlated with activity of P-gp ATPase. Na3VO4, a negative control (not a substrate of
P-gp); Verapamil, a positive control (a substrate of P-gp). Data are representative of three
independent experiments. *P < 0.05, **P < 0.01 vs the untreated group.
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Antitumor Efficacy of 10d in Inhibiting the Growth of A549/DDP Xenograft Tumors
in Mice. To investigate the safety profile of the hybrids, the acute toxicity of 10d was
tested in ICR mice at doses of 200, 250, 300, 350, 400, and 450 mg/kg (ip, n = 10 per
group). As shown in Table 5, treatment with 10d at the lowest dose (200 mg/kg) only
killed one mouse at day 3 post treatment. However, treatment with 10d at 450 mg/kg
killed all the mice. Finally, the median lethal dose (LD50) value of 10d was calculated to
be 284.537 mg/kg.
Next, we tested the in vivo antitumor efficacy of 10d against A549/DDP xenografts.
After the solid tumors were established and reached 180-200 mm3, the mice were
randomized and treated intraperitoneally with indicated dose (20 mg/kg or 40 mg/kg) of
10d or the same volume of vehicle consisting of PBS/DMSO/cremophor-EL (8:1:1) daily
for 15 consecutive days. As shown in Figure 9, treatment with 20 or 40 mg/kg 10d
significantly reduced the growth of A549/DDP tumor (P < 0.01, P < 0.001 vs the control).
Importantly, the tumor weights (0.49 0.13 g) of mice treated with 10d at 40 mg/kg were
significantly reduced by 67 % as compared to the control (1.48 0.11 g, P < 0.01, Table
6). Besides, no mortality or significant weight loss was observed for any of 10d-treated
mice during the post-treatment period (Table 6). Together, these results indicated that 10d
had potent anticancer activity against the growth of implanted drug-resistant human lung
cancer cells in mice with little toxicity.
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Table 5. Acute toxicity of 10d in mice.
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a
Mouse mortality
1h 4h 3d 4-14d
Dose
(mg/kg)
450
No. of
mice
10
Total
Survival (%)
LD50
mortality
on day 14
(mg/kg)
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aThe 95% confidence limits: 248.892-316.481 mg/kg.
Figure 9. Growth curve of implanted A549/DDP xenografts in nude mice treated with ip
of vehicle alone or 10d (20 or 40 mg/kg). Data are presented as the mean SD (n = 8
nude mice per group). **P < 0.01, ***P < 0.001 vs the control.
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Table 6. Effects of 10d on the body and tumor weights in micea
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Body weight (g)
Dose
Inhibitory ratio
Group
Tumor weight (g)
(mg/kg)
(%, w/w)
day 1
day 15
Control
10d
-
19.3 1.55 21.0 1.28
18.3 1.85 18.6 1.75
19.9 1.64 19.6 1.33
1.48 0.11
0.94 0.21b
0.49 0.13b
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67
10d
aData are shown as the mean SD of body and tumor weights (n = 8). P < 0.01 vs the
b
control.
CONCLUSIONS
A new series of ligustrazine-curcumin hybrids (10a-u) were designed, synthesized and
biologically evaluated. We found that compound 10d displayed strong and selective
antiproliferative activity against both drug-sensitive A549, SPC-A-1, LTEP-G-2 and
drug-resistant A549/DDP cells. Furthermore, 10d suppressed TrxR expression and
activity, promoted intracellular ROS accumulation and lung cancer cell apoptosis, and its
anticancer activity was significantly attenuated by ROS scavengers. Accordingly, the
reduced TrxR expression and activity by 10d in cancer cells may reflect the consequence
of apoptosis due to reduced cell viability and energy depletion during apoptosis.
Additionally, 10d inhibited the NF-κB, AKT and ERK signaling, P-gp-mediated efflux of
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rhodamine 123, P-gp expression and ATPase activity in A549/DDP cells. Moreover, 10d
was relatively safe to mice and significantly inhibited the growth of implanted
drug-resistant lung tumor in mice. Together, these results indicate that 10d, a hybrid of
ligustrazine and curcumin moiety, has potent anticancer activity preferably against
drug-resistant lung cancer. Our findings may provide a proof of principle that the hybrids
like 10d as novel TrxR inhibitors may be promising candidates for the intervention of
drug-sensitive and drug-resistant lung cancer.
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EXPERIMENTAL SECTION
General Methods. The reagents commercially available were used without further
purification, unless noted specifically. The purity of 10a-u was determined using HPLC
(see the Supporting Information). Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker Avance 300 (1H, 300 MHz; C, 75 MHz) or 500 (1H, 500 MHz;
13
13C, 125 MHz) spectrometer at 300 K, using tetramethylsilane (TMS) as an internal
reference. Melting points (mp) were measured using a Mel-TEMP II apparatus and
uncorrected. ESI-mass and high-resolution mass spectra (HRMS) were recorded on a
Water Q-Tof micro mass spectrometer. Infrared (IR) spectra were recorded as KBr
pellets on a Nicolet Impact 410 instrument. 2-hydroxymethyl-3,5,6- trimethylpyrazine (7)
and intermediates 9a-u were prepared as described previously.52,53 9a-d and 9f-u were
reported in previous literatures,74-79 while 9e was an unknown compound and its chemical
characterization is shown in the Supporting Information.
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Synthesis of 3,5,6-trimethylpyrazine-2-carbaldehyde (8) Compound 7 (1.5 g, 10 mmol)
was dissolved in DMSO (15 mL), and cooled to 0 °C. IBX (2.8 g, 11 mmol) was added
and the solution was stirred for 0.5 h at room temperature. Then, the reaction mixture was
diluted with saturated sodium bicarbonate (NaHCO3) aqueous solution and extracted with
EtOAc. The organic layer was washed with brine, dried with anhydrous sodium sulfate,
filtered and evaporated in vacuum. The crude product was purified by column
chromatography (PE/EtOAc = 10:1-1:1) to give the title compound 8 as a yellow powder
in 100 % yield. mp 90-92 °C; 1H NMR (300 M Hz, CDCl3): δ 2.61 (s, 6H, 2 × CH3), 2.80
(s, 3H, CH3), 10.15 (s, 1H, CHO) ppm; 13C NMR (75 M Hz, CDCl3): δ 20.9, 21.8, 141.4,
149.5, 151.0, 154.9, 193.8; MS (ESI): 151 [M+H]+.
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General Procedure for the Preparation of Compounds 10a-u. BF3·Et2O was added
(35.6 ꢀL, 0.13 mmol) to the stirred solution of compound 8 (100 mg, 0.26 mmol) and
various substituted intermediates 9a-u (0.28 mmol) in dry 1,4-dioxane at room
temperature under a nitrogen atmosphere. The reaction mixture was stirred and refluxed
for 4-6 h at 101 °C and cooled to room temperature. And then, the reaction mixture was
diluted with saturated NaHCO3 aqueous solution and extracted with EtOAc. The organic
layer was washed with brine, dried with anhydrous sodium sulfate, filtered and
evaporated in vacuum. The crude product was purified by column chromatography
(PE/EtOAc = 10:1-1:1) to yield the title compounds, respectively.
(1E,4E)-1-(2-Hydroxyphenyl)-5-(3,5,6-trimethylpyrazin-2-yl)penta-1,4-dien-3-one
(10a) Yellow powder; yield 78 %; mp 166-168 °C; IR (KBr, cm-1): 3339, 2914, 1645,
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1612, 1578; H NMR (300 M Hz, DMSO-d6): δ 2.48 (s, 3H, CH3), 2.50 (s, 3H, CH3),
2.60 (s, 3H, CH3), 6.85-6.96 (m, 2H, CH=CHCO, Ar-H), 7.28 (t, 1H, J = 7.5, 7.3 Hz,
Ar-H), 7.38 (d, 1H, J = 16.0 Hz, Ar-H), 7.51 (d, 1H, J = 15.3 Hz, Ar-H), 7.77-7.85 (m,
2H, CH=CHCO, CH=CHCO), 7.97 (d, 1H, J = 15.0 Hz, CH=CHCO), 10.33 (s, 1H, OH)
ppm; 13C NMR (75 M Hz, DMSO-d6): δ 20.4, 21.4, 21.6, 116.2, 119.4, 121.2, 124.6,
128.4, 129.8, 132.0, 136.3, 138.4, 142.2, 149.3, 149.6, 152.7, 157.2, 188.3; MS (ESI):
295 [M+H]+, 317 [M+Na]+; HRMS: calcd for C18H18N2O2Na [M+Na]+ 317.1266, found
317.1266.
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(1E,4E)-1-(3-Hydroxyphenyl)-5-(3,5,6-trimethylpyrazin-2-yl)penta-1,4-dien-3-one
(10b) Yellow powder; yield 66 %; mp 220-222 °C; IR (KBr, cm-1): 3339, 3009, 2924,
1
1670, 1621, 1592, 1574; H NMR (300 M Hz, DMSO-d6): δ 2.49 (s, 3H, CH3), 2.51 (s,
3H, CH3), 2.61 (s, 3H, CH3), 6.87 (d, 1H, J = 15.0 Hz, CH=CHCO), 7.13-7.41 (m, 4H, 4
× Ar-H), 7.52-7.66 (m, 2H, CH=CHCO, CH=CHCO), 7.85 (d, 1H, J = 15.0 Hz,
CH=CHCO), 9.66 (s, 1H, OH) ppm; 13C NMR (75 M Hz, DMSO-d6): δ 20.4, 21.4, 21.6,
115.1, 117.8, 119.7, 125.3, 129.8, 135.8, 136.8, 142.2, 149.4, 149.6, 152.8, 157.7, 188.3;
MS (ESI): 295 [M+H]+, 317 [M+Na]+; HRMS: calcd for C18H18N2O2Na [M+Na]+
317.1266, found 317.1266.
(1E,4E)-1-(4-Hydroxyphenyl)-5-(3,5,6-trimethylpyrazin-2-yl)penta-1,4-dien-3-one
(10c) Yellow powder; yield 69 %; mp 249-251 °C; IR (KBr, cm-1): 3220, 3021, 2940,
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1660, 1617, 1598, 1552; H NMR (300 M Hz, DMSO-d6): δ 2.48 (s, 3H, CH3), 2.50 (s,
3H, CH3), 2.60 (s, 3H, CH3), 6.82 (d, 2H, J = 8.5 Hz, 2 × Ar-H), 7.22 (d, 1H, J = 15.8 Hz,
CH=CHCO), 7.51 (d, 1H, J = 15.3 Hz, CH=CHCO), 7.63-7.70 (m, 3H, CH=CHCO, 2 ×
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Ar-H), 7.80 (d, 1H, J = 15.3 Hz, CH=CHCO), 10.12 (s, 1H, OH) ppm; C NMR (75 M
Hz, DMSO-d6): δ 20.4, 21.4, 21.6, 115.8, 122.3, 125.6, 130.0, 130.9, 136.1, 142.3, 143.5,
149.3, 149.6, 152.6, 160.2, 187.9; MS (ESI): 295 [M+H]+, 317 [M+Na]+; HRMS: calcd
for C18H18N2O2Na [M+Na]+ 317.1266, found 317.1268.
(1E,4E)-1-(4-Hydroxy-3-methoxyphenyl)-5-(3,5,6-trimethylpyrazin-2-yl)penta-1,4-di
en-3-one (10d) Yellow powder; yield 63 %; mp 194-196 °C; IR (KBr, cm-1): 3398, 3062,
2921, 1671, 1646, 1621, 1574; 1H NMR (300 M Hz, CDCl3): δ 2.55 (s, 3H, CH3), 2.56 (s,
3H, CH3), 2.65 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 6.31 (s, 1H, OH), 6.88-6.96 (m, 2H,
CH=CHCO, Ar-H), 7.13-7.19 (m, 2H, 2 × Ar-H), 7.71-7.78 (m, 2H, CH=CHCO,
CH=CHCO), 7.88 (d, 1H, J = 15.0 Hz, CH=CHCO) ppm; 13C NMR (75 M Hz, CDCl3): δ
20.8, 21.8, 22.0, 56.0, 109.7, 115.0, 123.8, 124.5, 127.3, 129.0, 136.5, 143.2, 144.2, 147.0,
148.5, 149.7, 149.8, 152.6, 188.8; MS (ESI): 325 [M+H]+, 347 [M+Na]+; HRMS: calcd
for C19H20N2O3Na [M+Na]+ 347.1372, found 347.1383.
(1E,4E)-1-(2-Hydroxy-3-methoxyphenyl)-5-(3,5,6-trimethylpyrazin-2-yl)penta-1,4-di
en-3-one (10e) Yellow powder; yield 67 %; mp 177-179 °C; IR (KBr, cm-1): 3367, 3056,
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2922, 1658, 1618, 1574; H NMR (300 M Hz, CDCl3): δ 2.54 (s, 3H, CH3), 2.56 (s, 3H,
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CH3), 2.65 (s, 3H, CH3), 3.92 (s, 3H, OCH3), 6.37 (s, 1H, OH), 6.87-6.89 (m, 2H,
CH=CHCO, Ar-H), 7.17-7.27 (m, 2H, 2 × Ar-H), 7.75 (d, 1H, J = 15.1 Hz, CH=CHCO),
7.88 (d, 1H, J = 15.1 Hz, CH=CHCO), 8.04 (d, 1H, J = 16.2 Hz, CH=CHCO) ppm; 13C
NMR (75 M Hz, CDCl3): δ 20.8, 21.8, 22.0, 56.2, 112.0, 119.8, 121.1, 127.7, 129.3,
136.6, 138.8, 143.3, 145.8, 146.9, 149.6, 152.5, 189.6; MS (ESI): 325 [M+H]+; HRMS:
calcd for C19H20N2O3Na [M+Na]+ 347.1372, found 347.1381.
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(1E,4E)-1-(2-Methoxyphenyl)-5-(3,5,6-trimethylpyrazin-2-yl)penta-1,4-dien-3-one
(10f) Yellow powder; yield 57 %; mp 134-136 °C; IR (KBr, cm-1): 3067, 2914, 1645,
1591, 1572; 1H NMR (300 M Hz, CDCl3): δ 2.53 (s, 3H, CH3), 2.56 (s, 3H, CH3), 2.65 (s,
3H, CH3), 3.92 (s, 3H, OCH3), 6.92-7.01 (m, 2H, CH=CHCO, Ar-H), 7.11 (d, 1H, J =
16.2 Hz, Ar-H), 7.38 (t, 1H, J = 7.3 Hz, Ar-H), 7.59 (d, 1H, J = 6.9 Hz, Ar-H), 7.73 (d,
1H, J = 15.1 Hz, CH=CHCO), 7.87 (d, 1H, J = 15.1 Hz, CH=CHCO), 8.09 (d, 1H, J =
16.2 Hz, CH=CHCO) ppm; 13C NMR (75 M Hz, CDCl3): δ 20.3, 21.3, 21.5, 55.0, 110.7,
120.3, 123.2, 126.7, 128.5, 128.8, 131.4, 136.1, 138.8, 142.7, 149.1, 149.3, 152.0, 158.2,
189.0; MS (ESI): 331 [M+Na]+; HRMS: calcd for C19H20N2O2Na [M+Na]+ 331.1422,
found 331.1429.
(1E,4E)-1-(3-Methoxyphenyl)-5-(3,5,6-trimethylpyrazin-2-yl)penta-1,4-dien-3-one
(10g) Yellow powder; yield 73 %; mp 128-130 °C; IR (KBr, cm-1): 3050, 2918, 1670,
1620, 1575; 1H NMR (300 M Hz, CDCl3): δ 2.54 (s, 3H, CH3), 2.56 (s, 3H, CH3), 2.65 (s,
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