Synthesis of Piperlongumine Analogues and Discovery of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Activators as Potential Neuroprotective Agents

Article abstract of DOI:10.1021/acs.jmedchem.5b00410

The cellular antioxidant system plays key roles in blocking or retarding the pathogenesis of adult neurodegenerative disorders as elevated oxidative stress has been implicated in the pathophysiology of such diseases. Molecules with the ability in enhancing the antioxidant defense thus are promising candidates as neuroprotective agents. We reported herein the synthesis of piperlongumine analogues and evaluation of their cytoprotection against hydrogen peroxide- and 6-hydroxydopamine-induced neuronal cell oxidative damage in the neuron-like PC12 cells. The structure-activity relationship was delineated after the cytotoxicity and protection screening. Two compounds (4 and 5) displayed low cytotoxicity and confer potent protection of PC12 cells from the oxidative injury via upregulation of a panel of cellular antioxidant molecules. Genetically silencing the transcription factor Nrf2, a master regulator of the cellular stress responses, suppresses the cytoprotection, indicating the critical involvement of Nrf2 for the cellular action of compounds 4 and 5 in PC12 cells.

Full text of DOI:10.1021/acs.jmedchem.5b00410

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Article
Synthesis of Piperlongumine Analogues and Discovery
of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)
Activators as Potential Neuroprotective Agents
Shoujiao Peng, Baoxin Zhang, Xianke Meng, Juan Yao, and Jianguo Fang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00410 • Publication Date (Web): 16 Jun 2015
Downloaded from http://pubs.acs.org on June 18, 2015
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Synthesis of Piperlongumine Analogues and Discovery of Nuclear Factor
Erythroid 2-Related Factor 2 (Nrf2) Activators as Potential Neuroprotective
Agents
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Shoujiao Peng#, Baoxin Zhang#, Xianke Meng, Juan Yao, Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical
Engineering, Lanzhou University, Lanzhou 730000, China
# These authors contribute equally to this work.
*Corresponding author, Eꢀmail: fangjg@lzu.edu.cn (J. Fang); Fax: +86 931 8915557.
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ABSTRACT
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The cellular antioxidant system plays key roles in blocking or retarding the pathogenesis of adult
neurodegenerative disorders as elevated oxidative stress has been implicated in the pathophysiology
of such diseases. Molecules with the ability in enhancing the antioxidant defense thus are promising
candidates as neuroprotective agents. We reported herein the synthesis of piperlongumine analogues
and evaluation of their cytoprotection against hydrogen peroxideꢀ and 6ꢀhydroxydopamineꢀinduced
neuronal cell oxidative damage in the neuronꢀlike PC12 cells. The structureꢀactivity relationship
was delineated after the cytotoxicity and protection screening. Two compounds (4 and 5) displayed
low cytotoxicity and confer potent protection of PC12 cells from the oxidative injury via
upregulation of a panel of cellular antioxidant molecules. Genetically silencing the transcription
factor Nrf2, a master regulator of the cellular stress responses, suppresses the cytoprotection,
indicating the critical involvement of Nrf2 for the cellular action of compounds 4 and 5 in PC12
cells.
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INTRODUCTION
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A small part of oxygen is continuously metabolized to reactive oxygen species (ROS) in aerobic
cells under physiological conditions. The production of ROS and their synchronous elimination by
an array of cellular antioxidant mechanisms are fundamental in maintaining the cellular redox
homeostasis, which is pivotal for the regulation of diverse cellular functions, such as differentiation,
proliferation, and death, via the activation or inactivation of transcription factors, metabolic
enzymes, and membrane channels and so on.1ꢀ3 Oxidative stress, arising from an increase of the
ROS production and/or a simultaneous impairment of the cellular antioxidant capacity, causes
deleterious effects and oxidative damage to various biomolecules, such as lipids, proteins, and
nucleic acids, and ultimately leads to many pathophysiological conditions. Neurodegenerative
disorders share many pathological features, such as accumulation of aberrant protein aggregates,
microglial activation and mitochondrial dysfunction, all of which are associated with the elevated
oxidative stress.4 As neuronal cells are particular vulnerable to oxidative stress and have limited
replenishment during entire lifespan, increasing lines of evidence have suggested oxidative stress as
one of pathogenic causes in the neuropathology of adult neurodegenerative disorders such as
Alzheimer's disease and Parkinson's disease.4, 5 Supplementation of antioxidants or stimulation of
cellular endogenous antioxidant defense system could efficiently block or retard the process of such
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diseases.6, Compared to the supplementation of exogenous antioxidants, the induction of
endogenous antioxidant defense is preferred as such action is more sustainable and regulated at
transcriptional level.
The Nrf2 (nuclear factor erythroid 2ꢀrelated factor 2) transcription factor plays a critical role in
cellular stress response and its malfunction is involved in a number of disease processes.8, 9 In
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unstressed cells, Nrf2 forms a complex with the inhibitory protein Kelchꢀlike ECHꢀassociated
protein 1 (Keap1), which anchors Nrf2 in cytosol and directs it to ubiquitinꢀmediated proteasome
degradation.10 The association of Keap1 with Nrf2 relies on the active sulfhydryl group of cysteine
residue(s) in the protein Keap1. Stimulants, such as electrophiles and oxidants, that could modify
such cysteine residue(s) cause the dissociation of Nrf2 from the Keap1, hence increasing Nrf2
stability and further facilitating Nrf2 to translocate into nucleus, where Nrf2 binds to a common
DNA sequence called antioxidant response elements (ARE) to initiate the transcription of a panel of
cytoprotective genes (phase II genes).9 As a global regulator of cellular antioxidant responses, Nrf2
controls a majority of antioxidants pathways, including the production of cellular universal reducing
equivalent NADPH, the synthesis, utilization and regeneration of glutathione (GSH), the expression
of thioredoxin system, and the synthesis of heme oxygenaseꢀ1 (HOꢀ1) and NAD(P)H quinone
oxidoreductase 1 (NQO1). In this sense, activation of Nrf2ꢀARE pathway is a promising therapeutic
approach in treatment of neurodegenerative disorders,11, 12 and thus the past years have witnessed
the expanding interests in identifying and developing naturally occurring or synthetic small
molecule activators of Nrf2ꢀARE pathway as potential neuroprotective agents.6, 13ꢀ16
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Natural products and their synthetic derivatives are invaluable fountain for therapeutic agents,
and have driven pharmaceutical discovery over the past century. Piperlongumine (PL, Fig. 1) is a
naturally occurring alkaloid from the plant Long pepper (Piper longum), which has long been used
as a fold medicine as well as spicy food additive. PL has been documented having multiple
pharmacological activity,17 such as antiꢀplatelet aggregation,18 antiꢀinflammation19 and
cardiovascular protection.20 The discovery that PL induces cell death preferentially in cancer cells
via elevation of cellular ROS levels turns the page for considering treatment of cancers with PL.21
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Since then, diverse analogues of PL have been prepared to find novel compounds with improved
properties.22ꢀ25 Although increasing attention has been devoted to the biological functions of PL and
its synthetic analogues, there is no study on the neuroprotection of PL. We reported herein the
synthesis and evaluation of PL and its analogues as potential neuroprotective agents against
oxidative stressꢀmediated cell damage in a neuronꢀlike cell line, PC12 cells. Most of the PL
analogues display remarkable protection of PC12 cells against hydrogen peroxideꢀ or
6ꢀhydroxydopamine (6ꢀOHDA)ꢀinduced cell injury. Two compounds (4 and 5) were chosen for the
followꢀup studies as they possess lower cytotoxicity and better protection. Hydrogen peroxide or
6ꢀOHDA induces ROS accumulation and apoptosis in PC12 cells, which could be efficiently
suppressed by the pretreatment of the cells with 4 or 5. Mechanistic studies reveal that both
compounds are potent Nrf2 activators to promote the nuclear translocation of Nrf2, which facilitates
the expression of a series of Nrf2ꢀdriven antioxidant molecules, including HOꢀ1, NQO1, GSH,
thioredoxin 1 (Trx1) and thioredoxin reductase 1 (TrxR1). In addition, knockdown of Nrf2 by
shRNA transfection abolishes the cytoprotection, demonstrating that activation Nrf2 is the
molecular mechanism underlying the cellular action of compounds 4 and 5 in PC12 cells.
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RESULTS AND DISCUSSION
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Chemical synthesis. PL and its analogues were synthesized by a convergent strategy that
entails coupling commercially available or synthetically accessible lactams and carboxylic acid
chlorides (Scheme 1) according to the published procedures.22, 23, 25ꢀ27 All compounds were fully
characterized by 1H, 13C NMR and MS, and their structures were illustrated in Fig. 1. The purity of
PL and its synthetic analogues was determined by the HPLC analysis, and was greater than 95%.
Initial screening. The cytotoxicity of all compounds towards the PC12 cells and L02 cells (an
immortal hepatic cell line) were determined by the MTT assay. As shown in Fig. 2A, PL, among all
the tested compounds, displays the highest toxicity towards PC 12 cells. Compounds 9-18 also give
significant toxicity to the cells at high concentration (100 ꢀM), while compounds 2-8, and 19 only
show marginal effect on the growth of the cells. The cytotoxicity of the compounds follows a
similar trend in the L02 cells (Fig. 2B): PL~9-14>15-18>2-8, 19. Except PL, there is no apparent
toxicity of the tested compounds towards the PC12 cells at 20 ꢀM. Next, we determined the
protection of compounds 2-19 against the H2O2ꢀ and 6ꢀOHDAꢀinduced PC12 cell damage, two
wellꢀestablished cellular models of neurodegenerative disorders.13, 14, 28 All the tested compounds,
except 7, are capable of relieving the cell injury. Notably, 4 and 5 give the better protection (Fig. 3).
Protection of PC12 cells from H2O2- and 6-OHDA-induced damage by 4 and 5. As
compounds 4 and 5 display lower cytotoxicity (Fig. 2) and better protection (Fig. 3) in the initial
screening, we then selected these two compounds for the followꢀup studies. As shown in Fig. 4A,
PC12 cells treated with H2O2 only showed about half cell death compared with the control group.
However, if the cells were pretreated with 4 or 5 (10 or 20 ꢀM) for 24 h followed by H2O2 insult,
the population of viable cells increased remarkably in doseꢀdependent manners. Compounds 4 and 5
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(20 ꢀM) increased the cell viability to ~75% and ~80%, respectively. 6ꢀOHDA is a potent
neurotoxin, and widely used to generate an experimental model of Parkinson's disease.
6ꢀOHDAꢀmediated neurotoxicity is engendered, at least in part, by its ability to generate ROS.29 As
shown in Fig. 4B, 6ꢀOHDA reduced cell viability to about 50 % of the control. Similarly, addition
of 4 or 5 also markedly promoted cell viability to ~75 % (Fig. 4B). Lactate dehydrogenase (LDH) is
a soluble cytosolic enzyme that is released into the culture medium following loss of membrane
integrity resulting from cell damage. To confirm the cytoprotection of compounds 4 and 5, we
further determined the content of LDH leakage after H2O2 or 6ꢀOHDA insult. As shown in Fig. 4C
& D, H2O2 and 6ꢀOHDA cause ~2.2ꢀfold and ~1.8ꢀfold increase of LDH release, respectively.
Pretreatment of PC12 cells with 4 or 5 significantly reduces the leakage of LDH. Taken together,
compounds 4 and 5 at nonꢀtoxic concentration can significantly protect PC12 cells from H2O2ꢀ or
6ꢀOHDAꢀinduced cell injury.
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Alleviation of H2O2- and 6-OHDA-induced PC12 cell apoptosis. Nuclear chromatin
condensation and cytosolic caspases activation are hallmarks of cell apoptosis. The morphologic
changes of the nuclei were revealed by the Hoechst staining assay. Hoechst dyes are a family of
blue fluorescent molecules used to stain DNA. Hoechst 33342 is a popular cellꢀpermeant dye that
emits strong blue fluorescence when bound to double strand DNA, and is widely applied to
distinguish condensed pycnotic nuclei in apoptotic cells.13, 14, 30ꢀ33 As shown in Fig. 5A & 5B, both
H2O2 and 6ꢀOHDA elicit apoptosis in PC12 cells evidenced by the appearance of the characteristic
apoptotic nuclei as highly fluorescent, condensed bodies (indicated by arrows), while no apparent
apoptotic nuclei were observed in the control cells. Pretreatment of the cells with nonꢀtoxic
concentrations of 4 or 5 remarkably decreases the population of apoptotic nuclei. Next, we
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measured the caspaseꢀ3 activity in PC12 cells. Both H2O2 and 6ꢀOHDA activate the cellular
caspaseꢀ3 (Fig. 5C & 5D). Again, compounds 4 and 5 efficiently alleviate the extent of caspaseꢀ3
activation in a concentration–dependent manner. Collectively, these results indicate that compounds
4 and 5 could assuage the H2O2ꢀ and 6ꢀOHDAꢀinduced PC12 cell apoptosis.
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Prevention of ROS accumulation in PC12 cells. 2’,7’ꢀdichlorfluorescein diacetate
(DCFHꢀDA) is a cellꢀpermeable, nonꢀfluorescent dye. After diffusing into cells, DCFHꢀDA is
deacetylated by cellular esterases to nonꢀfluorescent 2’,7’ꢀdichlorodihydrofluorescin, which is
rapidly converted to highly fluorescent 2’,7’ꢀdichlorofluorescein upon reacting with ROS.
Stimulation of the cells with H2O2 or 6ꢀOHDA leads to appearance of bright green fluorescence,
indicating the burst of ROS in the cells. The control cells display negligible ROS level as they
remain nonꢀfluorescent (Fig. 6A & 6B). Pretreatment of the cells with 4 or 5 remarkably and
doseꢀdependently reduces the ROS accumulation induced by either H2O2 or 6ꢀOHDA. Oxidative
stress, arising from the overproduction of ROS beyond the cellular antioxidant capacity to remove
them, has been implicated in a common pathway of neurotoxicity in a wide variety of acute and
chronic neurologic disorders. Prevention of ROS accumulation in neuronal cells by compounds 4
and 5 might account for their cytoprotection against oxidative injury.
Induction of antioxidant genes expression. To further study the role(s) of 4 and 5 in
releasing the ROS stress imposed by H2O2 or 6ꢀOHDA in PC12 cells (Fig. 6), we then determined
their direct free radical scavenging activity. Compounds 4 and 5 are incapable of intercepting either
2,2ꢀdiphenylꢀ1ꢀpicrylhydrazyl (DPPH) or 2,2′ꢀazinobisꢀ(3ꢀethylbenzothiazolineꢀ6ꢀsulfonic acid)
(ABTS) free radicals (Fig. S1 in the Supporting Information), indicating that 4 and 5 cannot act as
direct radical scavengers. This may be understood by that the free radicalꢀscavenging antioxidants
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generally contain active functionalities, such as phenoxy group or/and sulfhydryl group, which
easily donates electrons to free radicals via a hydrogen abstraction reaction or electron transfer
reaction. However, compounds 4 and 5 don’t have such active groups, and thus they cannot
scavenge free radicals directly via chemical ways. In support of this, 19, the demethylated
derivative of 5, efficiently neutralizes both radicals (Fig. S1 in the Supporting Information).
Analysis of the structure of 4 and 5 reveals that they belong to the chalcone family, which bears α,
βꢀunsaturated ketone structure. Due to the electronꢀdeficient character of the double bond, this
moiety is renowned as Michael acceptor, which is a core structure of many reported small molecule
ARE inducers.15 In analogy to the known ARE activators, we thus hypothesized that 4 and 5 might
activate the cellular ARE response. We determined the expression of Nrf2ꢀdriven
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antioxidant/detoxifying genes (HOꢀ1, Trx1, TrxR1, NQO1, GCLC and GCLM) at 0, 3, 6 and 12 h
after treatment with 4 or 5 (20 ꢁM) in PC12 cells (Fig. 7). Significant induction of all the tested
genes was observed at 6 h. Among all the genes, Trx1 and TrxR1 are most notable as they were
induced even at 3 h, and the induction of both genes is sustainable even after 12 h. Our data
demonstrate that treatment of the PC12 cells with 4 and 5 effectively promotes the transcription of
Nrf2ꢀdriven antioxidant/detoxifying genes.
Upregulation of the antioxidant defense system in PC12 cells. As a series of antioxidant
genes has been upꢀregulated in PC12 cells after compounds 4 or 5 stimulation (Fig. 7), we then
examined the corresponding gene products. The amount of GSH was quantified by the enzymatic
assay as described in the Experimental section. The expression and function of antioxidant enzymes
were determined by Western blots and activity assays, respectively. As shown in Fig. 8A, D, G, & J,
compounds 4 and 5 remarkably elevates GSH level and antioxidant enzymes activity (NQO1, Trx
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and TrxR). In the H2O2 or 6ꢀOHDA injury model (Fig. 8B, C, E, F, H, I, K & L), the amount of
GSH, and the activity of NQO1, Trx and TrxR were significantly impaired. Pretreatment of the cells
with 4 or 5 could rescue both the GSH amount and the enzymes activity. The expression patterns of
HOꢀ1, NQO1, Trx1 and TrxR1 were illustrated in Fig. 8M, and all the proteins were upregulated,
consistent with the elevation of their activity. We further confirmed the induction of TrxR activity in
live PC12 cells by our recently developed probe, TRFSꢀgreen.34 The increment of the green
fluorescence indicated the boost of the TrxR activity (Fig. 8N).
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Promotion of Nrf2 nuclear localization. Induction of transcription of antioxidant genes via
the Nrf2ꢀdependent cytoprotective pathway requires translocation of Nrf2 from cytosol to nucleus.
We therefore examined whether 4 and 5 could promote the Nrf2 to accumulate in nuclei. After
isolating the nuclei from the cells, the total Nrf2, the cytosolic Nrf2, and the nuclear Nrf2 were
determined by immunoblots. Upon 4 or 5 treatment, the immunoblotting results revealed that the
total Nrf2 expression was slightly upregulated (Fig. 9, bottom panel). Notably, the cytosolic Nrf2
decreased gradually (Fig. 9, middle panel). Coincidently, the amount of Nrf2 in nuclei increased
(Fig. 9, top panel), indicating the translocation of Nrf2 from the cytosol to the nuclei. The presence
of Nrf2 in nuclei would facilitate its binding to ARE, and thus subsequently initiates the
transcription process to induce the antioxidant genes expression.
Requirement of Nrf2 for the cytoprotection of 4 and 5. We further asked the role of Nrf2
for the cellular protection of 4 and 5 in response to oxidative insults. For this purpose, we
transfected the cells with the shRNA plasmid specifically targeting the Nrf2 to generate stable
transformants of PC12 cells silencing the expression of Nrf2 (PC12ꢀshNrf2 cells). As a control,
PC12 cells were also transfected with a nonꢀtargeting shRNA to afford the control cells
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(PC12ꢀshNT cells). After confirming the knockdown efficiency of Nrf2 by Western blots (Fig. 10A),
we evaluated the effect of 4 and 5 against oxidative challenge toward different cells. As shown in
Fig. 10B & C, compounds 4 and 5 display the similarly protective pattern in PC12ꢀshNT cells as
that observed in wild type PC12 cells. However, this protection is almost completely suppressed in
PC12ꢀshNrf2 cells, demonstrating the definite involvement of Nrf2 for the cytoprotection of 4 and 5
in PC12 cells.
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Medicinal chemical properties of compounds 4 and 5. The medicinal chemical properties of
a molecule could predict whether it is capable of passing the bloodꢀbrain barrier (BBB). The general
criteria for a compound to cross the BBB are as the follows:35 the molecular weight (MW) is less
than 450, cLogP is between 0 and 5, and the polar surface area (PSA) is less than 70 Å2. The values
of cLogP, PSA and MW of compounds 4 and 5 were summarized in Table 1. By determination of
the partition coefficient between nꢀoctanol and water, the cLogP values were obtained. The positive
control oꢀcresol gives a value of 1.95, consistent with the data in the reference (1.98±0.05).36 Thus,
the medicinal chemical properties of compounds 4 and 5 suggest they have the ability to penetrate
the BBB for a potential CNS drug.
ROS are physiological metabolites from numerous cellular processes in all aerobes.1, 37 Low
levels of ROS are signaling molecules to promote proliferation and survival pathways.38 However,
high levels of ROS exert an oxidative stress on the cell that damages various cellular components,
such as lipids, proteins and DNA, and ultimately cause cell senescence or death.38, 39 Common
potential targets and putative mechanisms of neurodegenerative diseases have been elegantly
summarized in the recent Perspective.6 Although the molecular mechanism underlying the
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pathogenesis of neurodegeneration is still in debate, increasing evidence has supported that
oxidative stress is a causal, or at least an ancillary, factor in the progressive degeneration of a subset
of neurons, which is the pathologic hallmark of adultꢀonset neurodegenerative disorders.7, 40 The
incapability of the adult human brain to regenerate and the limited success of the surgical operation
to replace the lost or damaged neurons have led to intense efforts to explore the pharmacological
management aiming to suppress the oxidative stress, which is expected eventually leading to
retardation or blockage of the neurodegenerative progression. As a master regulator of the cellular
antioxidant responses, the transcription factor Nrf2 has appeared as an emerging drug target to
counteract the oxidative stressꢀmediated pathogenic processes.12, 15 The observation that genetic
activation of Nrf2 signaling efficiently ameliorates neurodegenerative phenotypes in a fly model of
Parkinson’s disease further supports the therapeutic potential of Nrf2.41
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Preceding studies have indicated that PL and its analogues have a number of pharmacological
functions.17ꢀ21, 42 However, the neuroprotective function of PL has not been investigated prior to this
report. Molecules with orthoꢀdiphenoxyl groups or/and Michael acceptor units appear to potential
Nrf2 activators to induce various endogenous antioxidant and detoxifying molecules.13, 14, 43 PL
contains two Michael acceptor units (highlighted as M1 and M2 in Fig. 1), suggesting that PL might
be promising for cytoprotection. However, the high cytotoxicity of PL hinders its application as a
cytoprotective agent.21 As our continued interests in discovering and developing novel redox active
small molecules as potential therapeutic or diagnostic agents,30, 32, 34, 44ꢀ47 we described the synthesis
of PL and its analogues, and the discovery of two potent compounds (4 and 5) as potential
neuroprotective agents to rescue the H2O2ꢀ or 6ꢀOHDAꢀinduced PC12 cell damage. Compared to
the cytotoxicity data of 4 (a compound lacking the M2 moiety), PL shows greater toxic to both L02
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cells and PC12 cells. These results are consistent with the previous report that M2 is critical for the
cytotoxicity of PL.25 As the goal of this study is to seek potential neuroprotective agents, a set of PL
analogues without the M2 were prepared to minimize the cytotoxicity of the compounds. Based on
the initial cytotoxicity and protection screening results (Figures 2 & 3), the preliminary
structureꢀactivity relationship (SAR) could be drawn: 1) The Michael acceptor within the lactam
ring (M2) is responsible for the cytotoxicity as PL shows higher cytotoxicity than 4, while the M1 is
required for the cytoprotection as all analogues except N7 are capable of rescuing the cells from
oxidative damage; 2) Introducing the electronꢀwithdrawing groups (EWG) to the benzene ring, such
as 9-15, generally increase the cytotoxicity, while introducing the electronꢀdonating groups, such as
2-6 and 8, decreases the cytotoxicity; 3) The ring size of the lactam appears no apparent effect on
the protective potency or cytotoxicity as the compounds 2, 4 and 8, and the compounds 16-18
exhibit indiscriminate activity. The SAR disclosed here will guide the further modification of PL
with improved biological functions.
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Our results demonstrated that compounds 4 and 5 displayed potent cytoprotection against
H2O2ꢀ or 6ꢀOHDAꢀinduced neuronal cell damage in PC12 cells via the induction of phase II
antioxidant/detoxifying enzymes. Treatment of PC12 cells with 4 or 5 at nontoxic concentrations
promotes nuclear translocation of the transcription factor Nrf2, and subsequently upregulates a set
of Nrf2ꢀdriven genes as well as the corresponding gene products, including the antioxidant enzymes
(HOꢀ1, NQO1, Trx1, and TrxR1) and small peptide antioxidant GSH. The elevation of cellular
endogenous antioxidant system prevents the accumulation of ROS, and thus confers protection
against oxidative insults to the cells. Knockdown of Nrf2 expression almost completely deprives the
protection, supporting the physiological significance of targeting Nrf2 by 4 and 5 in PC12 cells. The
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clarification of the cellular target and the action mechanism would provide information for the
further development of PL analogues as potential therapeutic drugs. One major challenge for most
neuroprotective agents in vivo is their ability to pass through the bloodꢀbrain barrier (BBB). The
recent study disclosed that PL could cross the BBB after oral administration.48 The small size
(PSA<70 and MW<450), nonꢀpolar character (cLogP<5) and similar structure with PL would favor
both compounds to penetrate the BBB. It is much like that 4 and 5 has the ability to pass through
the BBB in vivo.
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The Michael acceptor moieties, notorious for the electrophilic property which gives them
promiscuous reactivity towards various electronꢀrich biomolecules, were generally excluded in the
traditional drug design process. However, accumulating evidence has supported that Michael
acceptors are promising pharmacophores for cytoprotective drugs as molecules bearing such
functionalities are effective activators of Keap1ꢀNrf2ꢀARE pathway.28, 49, 50 The recently
FDAꢀapproved drug dimethyl fumarate (BGꢀ12, a Michael acceptor) for multiple sclerosis
consolidates this concept.51 We previously reported that several natural products bearing α,
βꢀunsaturated ketone structure (a Michael acceptor moiety), such as xanthohumol and
6ꢀdehydrogingerdione,13, 14 are potent Nrf2 activators to afford neuroprotection in cellular models.
Compounds 7, a compound similar to 4 but lacking the Michael acceptor unit, has little protection,
indicating the importance of the α, βꢀunsaturated amide structure for the cellular action of PL
analogues. In analogy to the activation of Nrf2 by xanthohumol13 and 6ꢀdehydrogingerdione,14 we
reasoned that bind of the compounds via their Michael acceptor moiety to the cysteine residue(s) in
the inhibitory protein Keap1 might be the molecular basis for the activation of Nrf2.
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CONCLUSIONS
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In summary, a series of PL analogues has been synthesized, and two PL analogues, i. e., 4 and 5,
have been identified as potent Nrf2 activators with minimal cytotoxicity. Compounds 4 and 5
promote Nrf2 nuclear translocation, and hence facilitate transcription of a variety of cytoprotective
genes, conferring protection of PC12 cells against oxidative insults. The clarification of the cellular
target, the action mechanism and the structural determents for the activity of 4 and 5 would guide
the further development of PL analogues as potential therapeutic drugs.
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EXPERIMENTAL PROCEDURES
Materials.
Dulbecco's
modified
Eagle’s
medium
(DMEM),
NꢀacetylꢀAspꢀGluꢀValꢀAspꢀpꢀnitroanilide (AcꢀDEVDꢀpNA), dimethyl sulfoxide (DMSO), yeast
glutathione reductase (GR), Hoechst 33342, DCFHꢀDA, 5,5’ꢀdithiobisꢀ2ꢀnitrobenzoic acid (DTNB),
AAPH, DPPH, 2,6ꢀdichlorophenolꢀindophenol (DCPIP), NADH, 3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,
5ꢀdiphenyltetrazolium bromide (MTT), 2,2’ꢀazinobis(3ꢀethylbenzthiazolineꢀ6ꢀ sulfonic acid
(ABTS), 2, 2ꢀdiphenylꢀ1ꢀpicrylhydrazyl (DPPH), penicillin and streptomycin were obtained from
SigmaꢀAldrich (St. Louis, USA). NADPH was obtained from Roche (Mannheim, Germany). Fetal
bovine serum (FBS) was obtained from HyClone. 6ꢀOHDA, hydrogen peroxide, antibodies against
TrxR1 and Nrf2 were from Santa Cruz Biotechnology. Antibodies against HOꢀ1, NQO1 and Trx1
were from Sangon Biotech (Shanghai, China). The shRNA plasmids targeting coding regions of the
rat Nrf2 gene (shNrf2) and the control nonꢀtargeting shRNA (shNT) were purchased from Gene
Pharma Co, Ltd (Shanghai, China). GeneTran III transfection reagent was obtained from Biomiga
(CA, USA). Bovine serum albumin (BSA), phenylmethanesulfonyl fluoride (PMSF), sodium
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orthovanadate (Na3VO4), and antiꢀlamin and antiꢀactin antibodies were from Beyotime (Nantong,
China). The recombinant rat TrxR1 is a gift from Prof. Arne Holmgren at Karolinska Institute. The
recombinant E. coli Trx was prepared according to our published procedures.31 All other reagents
were of analytical grade.
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Compounds Purity Analysis. PL and its analogues were analyzed by HPLC to determine their
purity. The analyses were performed on Waters 1525 2998 series HPLC system (Cꢀ18 column, Sun
Fire, 5 µm, 4.6 mm×150 mm) at room temperature. The HPLC chromatograms of compounds 1ꢀ19
were included in the Supporting Information (Fig. S2ꢀS6). All the tested compounds were dissolved
in methanol, and 10 µL of the sample was loaded onto the column. Methanol and water were used
as mobile phase, and the flow rate was set at 1.0 mL/min. The maximal absorbance at the range of
210ꢀ400 nm was used as the detection wavelength. The purity of all the tested compounds
(compounds 1ꢀ19) is >95%, which meets the purity requirement by the Journal.
Chemical Synthesis. The detailed starting materials and synthetic intermediates in the Scheme
1 were summarized in Table S1 in the Supporting Information.
Synthesis of substituted cinnamic acids (Step a in Scheme 1).27, 52, 53 Malonic acid (0.3 g, 2.86
mmol) was added to the stirred solution of the corresponding aldehyde (1.3 mmol) in pyridine (15
mL) and piperidine (1.5 mL). The mixture was heated to reflux for 8 h. The reaction mixture was
neutralized with hydrochloric acid in an ice bath. The white precipitates were filtered and washed
with cold water. The crude products were recrystallized from aqueous ethanol to afford the
corresponding acids.
Synthesis of substituted cinnamic acid chlorides (Step b in Scheme 1).54 To a solution of acids
(1.0 mmol) in dry CH2Cl2 was added oxalyl chloride (5.0 equiv.) and catalytic amount of DMF
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(0.01 equiv.). The reaction mixture was stirred at room temperature for 5 h before the solvent was
removed. The residue was dried under high vacuum, and used to the next step without any further
purification.
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Synthesis of PL and its analogues (Step c in Scheme 1).25, 26 To a solution of acid chloride (1.0
equiv.) in CH2Cl2 were added triethylamine (TEA, 3.0 equiv.) and lactam (1.2 equiv.). The reaction
mixture was stirred at room temperature overnight before it was quenched with saturated aqueous
NH4Cl, and extracted with CH2Cl2. The combined organic phases were washed with brine and dried
over MgSO4. After filtration and concentration, the residue was purified by flash chromatography to
provide the desired amides.
Synthesis of 7 (Step d in Scheme 1). To a mixture of 100 mg of Pd/C (10 %) in 10 ml of ethyl
acetate under a hydrogen atmosphere was added 4 (1.0 mmol). The reaction was kept at room
temperature for 12 h. After the reaction completing, the Pd/C was removed by filtration, and the
filtrate was concentrated and purified by silica gel column chromatography to afford 7.
(E)-1-(3-(3,4,5-Trimethoxyphenyl)acryloyl)-5,6-dihydropyridin-2(1H)-one (1).25 Yield: 25%.
1H NMR (400 MHz, CDCl3) δ: 7.72 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.41 (d, J = 15.6 Hz, 1H,
ArꢀCH=CHꢀ), 6.80 (s, 2H, ArH2), 6.58 (m, 1H, ꢀCH=CHꢀ), 6.13 (d, J = 11.6 Hz, 1H, ꢀCH=CHꢀ),
4.02 (t, 2H, ꢀCH2ꢀ), 3.90 (s, 6H, 2 ꢀOCH3), 3.88 (s, 3H, ꢀOCH3), 2.44(m, 2H, ꢀCH2ꢀ); 13C NMR
(100 MHz, CDCl3) δ: 171.4, 167.6, 153.2, 144.1, 142.1, 139.9, 130.5, 127.9, 120.1, 105.45, 60.9,
56.1, 41.2, 26.1, 25.4; ESIꢀMS(m/z): [M+H]+ 318.2; mp: 156ꢀ158 oC; purity: 95.21% (MeOH/H2O
= 70/30, Rt= 4.964 min).
(E)-1-(3-(3,4,5-Trimethoxyphenyl)acryloyl)pyrrolidin-2-one (2).55 Yield: 45%. 1H NMR (400
MHz, CDCl3) δ: 7.87 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.78 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ),
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6.84 (s, 2H, ArH2), 3.96 (t, 2H, NꢀCH2ꢀ),3.91 (s, 6H, 2 ꢀOCH3), 3.89 (s, 3H, ꢀOCH3), 2.69 (t, 2H,
ꢀCOCH2ꢀ), 2.13 (m, 2H, ꢀCH2ꢀ); 13C NMR (100 MHz, CDCl3) δ:175.8, 174.6, 166.2, 153.4, 152.7,
145.6, 140.2, 130.4, 118.2, 105.6, 60.9, 56.2, 45.9, 34.0, 17.2; ESIꢀMS(m/z): [M+H]+ 306.3; mp:
152ꢀ154 oC; purity: 98.49% (MeOH/H2O = 65/35, Rt= 4.335 min).
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(E)-1-(3-(3,4-Dimethoxyphenyl)acryloyl)pyrrolidin-2-one (3).56 Yield: 44%. 1H NMR (400
MHz, CDCl3) δ: 7.86 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.81 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ),
7.21 (dd, J = 8.4, 2.0 Hz, 1H, ArH), 7.14 (d, J = 1.6 Hz, 1H, ArH), 6.89 (d, J = 8.4 Hz, 1H, ArH)
3.94 (s, 6H, 2 ꢀOCH3), 3.93 (t, 2H, NꢀCH2ꢀ), 2.68 (t, 2H, ꢀCOCH2ꢀ), 2.12 (m, 2H, ꢀCH2ꢀ); 13C NMR
(100 MHz, CDCl3) δ: 175.7, 166.4, 151.2, 149.1, 145.6, 127.9, 123.1, 116.6, 110.9, 109.9, 55.9,
55.8, 45.9, 34.0, 17.1; ESIꢀMS(m/z): [M+H]+ 275.8; mp: 128ꢀ130 oC; purity: 95.95% (MeOH/H2O
= 65/35, Rt= 3.962 min).
(E)-1-(3-(3,4,5-Trimethoxyphenyl)acryloyl)piperidin-2-one (4).25 Yield: 40%. 1H NMR (400
MHz, CDCl3) δ: 7.65 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.37 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ),
6.78 (s, 2H, ArH2), 3.88 (s, 6H, 2 ꢀOCH3), 3.87 (s, 3H, ꢀOCH3), 3.81 (t, 2H, NꢀCH2ꢀ), 2.63 (t, 2H,
ꢀCOCH2ꢀ), 1.90(m, 4H, ꢀCH2CH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 173.9, 169.6, 153.3, 143.4,
139.7, 130.6, 121.3, 105.4, 60.9, 56.1, 44.6, 34.9, 22.5, 20.6; ESIꢀMS(m/z): [M+H]+ 320.2; mp:
109ꢀ110 oC; purity: 99.88% (MeOH/H2O = 65/35, Rt=4.941min).
(E)-1-(3-(4-Methoxyphenyl)acryloyl)pyrrolidin-2-one (5).57 Yield: 55%. 1H NMR (400 MHz,
CDCl3) δ: 7.84 (d, J = 16.0 Hz, 1H, ArꢀCH=CHꢀ), 7.80 (d, J = 16.0 Hz, 1H, ArꢀCH=CHꢀ), 7.57 (d, J
= 8.8 Hz, 2H, ArH2), 690 (d, J = 8.8 Hz, 2H, ArH2), 3.92 (t, 2H, NꢀCH2ꢀ), 3.82 (s, 3H, ꢀOCH3), 2.65
(t, 2H, ꢀCOCH2ꢀ), 2.08 (m, 2H, ꢀCH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 175.6, 166.5, 161.4, 145.2,
130.2, 127.6, 116.4, 114.2, 55.3, 45.8, 34.0, 17.1; ESIꢀMS(m/z): [M+H]+ 245.8; mp: 110ꢀ112 oC;
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purity: 98.36% (MeOH/H2O = 65/35, Rt= 5.744 min).
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(E)-1-(3-(4-Methoxyphenyl)acryloyl)piperidin-2-one (6). Yield: 43%. 1H NMR (400 MHz,
CDCl3) δ: 7.72 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.54 (d, J = 8.8 Hz, 2H, ArH2), 7.38 (d, J = 15.6
Hz, 1H, ArꢀCH=CHꢀ), 6.90 (d, J = 8.4 Hz, 2H, ArH2) , 3.84 (s, 3H, ꢀOCH3), 3.82 (t, 2H, NꢀCH2ꢀ),
2.62 (t, 2H, ꢀCOCH2ꢀ), 1.90 (m, 4H, ꢀCH2CH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 173.8, 169.8,
161.1, 143.1, 129.9, 127.8, 119.6, 114.1, 55.3, 44.5, 34.9, 22.5, 20.5; ESIꢀMS(m/z): [M+Na]+ 282.0;
mp: 70ꢀ71 oC; purity: 98.40% (MeOH/H2O = 70/30, Rt= 4.895 min).
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1-(3-(3,4,5-Trimethoxyphenyl)propanoyl)piperidin-2-one (7).25 Yield: 20%. 1H NMR (400
MHz, CDCl3) δ: 6.46 (s, 2H, ArH2), 3.84 (s, 6H, 2 ꢀOCH3), 3.81 (s, 3H, ꢀOCH3), 3.72 (t, 2H,
NꢀCH2ꢀ), 3.24 (t, 2H, ArCH2ꢀ), 2.93 (t, 2H, ꢀCH2COꢀ), 2.55 (t, 2H, ꢀCH2COꢀ),1.82 (t, 4H,
ꢀCH2CH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 176.0, 173.4, 137.0, 136.2, 105.4, 60.8, 56.0, 44.0,
41.3, 34.8, 31.5, 22.4, 20.2; ESIꢀMS(m/z): [M+H]+ 322.3; mp: 75ꢀ76 oC; purity: 96.03%
(MeOH/H2O = 65/35, Rt= 4.582 min).
(E)-1-(3-(3,4,5-Trimethoxyphenyl)acryloyl)azepan-2-one (8). Yield:.40%. 1H NMR (400 MHz,
CDCl3) δ: 7.65 (d, J = 15.2 Hz, 1H, , ArꢀCH=CHꢀ), 7.35 (d, J = 15.6 Hz, 1H, , ArꢀCH=CHꢀ), 6.79 (s,
2H, ArH2), 3.99 (t, 2H, NꢀCH2ꢀ),3.89 (s, 6H, 2ꢀOCH3), 3.88 (s, 3H, OCH3), 2.27 (t, 2H, ꢀCOCH2ꢀ),
1.84 (m, 6H, ꢀCH2CH2CH2ꢀ); 13C NMR (100 MHz, CDCl3) δ:178.1, 168.8, 153.2, 143.5, 139.9,
130.7, 121.2, 105.4, 60.9, 56.1, 43.8, 39.6, 29.2, 28.6, 23.7; ESIꢀMS(m/z): [M+H]+ 334.2; mp:
92ꢀ93 oC; purity: 96.61% (MeOH/H2O = 65/35, Rt= 6.753 min).
(E)-1-(3-(4-(Trifluoromethyl)phenyl)acryloyl)pyrrolidin-2-one (9).58 Yield: 25%. 1H NMR
(400 MHz, CDCl3) δ: 8.00 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.82 (d, J = 15.6 Hz, 1H,
ArꢀCH=CHꢀ), 7.71 (d, J = 8.0 Hz, 2H, ArH2), 7.64 (d, J = 8.0 Hz, 2H, ArH2), 3.94 (t, 2H, NꢀCH2ꢀ),
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2.67 (t, 2H, ꢀCOCH2ꢀ), 2.10 (m, 2H, ꢀCH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 176.0, 166.0, 143.5,
138.4, 132.1, 131.7, 128.8, 126.0, 125.9, 122.7, 121.7, 46.0, 34.1, 17.4; ESIꢀMS (m/z): [M + H+]
283.8; mp: 126ꢀ128 oC; purity: 98.90% (MeOH/H2O = 70/30, Rt= 7.635 min).
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(E)-1-(3-(4-Bromophenyl)acryloyl)pyrrolidin-2-one (10).58 Yield: 20%. 1H NMR (400 MHz,
CDCl3) δ: 7.92 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.75 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.52 (d, J
= 8.8 Hz, 2H, ArH2), 7.47 (d, J = 8.8 Hz, 2H, ArH2), 3.92 (t, 2H, NꢀCH2ꢀ), 2.66 (t, 2H, ꢀCOCH2ꢀ),
2.09 (m, 2H, ꢀCH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 176.0, 166.3, 144.2, 134.0, 132.3, 130.1,
124.8, 119.9, 46.1, 34.2, 17.4; EIꢀMS m/z (%): 293 (M+, 36), 211 (91), 209 (100), 183 (26), 102
(75), 84 (27); mp: 112ꢀ113 oC; purity: 99.23% (MeOH/H2O = 65/35, Rt= 11.508 min).
(E)-1-(3-(4-(Trifluoromethyl)phenyl)acryloyl)piperidin-2-one (11). Yield: 40%. 1H NMR (400
MHz, DMSOꢀd6) δ: 7.84 (d, J = 8.0 Hz, 2H, ArH2), 7.78 (d, J = 8.0 Hz, 2H, ArH2), 7.56 (d, J = 15.6
Hz, 1H, ArꢀCH=CHꢀ), 7.41 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 3.68 (t, 2H, NꢀCH2ꢀ), 2.53 (t, 2H,
ꢀCOCH2ꢀ), 1.80(m, 4H, ꢀCH2CH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 174.2, 169.5, 141.1, 138.8,
131.8, 131.4, 128.6, 126.0, 125.9, 124.9, 122.8, 45.0, 35.2, 22.8, 20.9; ESIꢀMS (m/z): [M + H+]
298.7; mp: 86ꢀ87 oC; purity: 99.68% (MeOH/H2O = 65/35, Rt= 14.103 min).
(E)-1-(3-(4-Nitrophenyl)acryloyl)piperidin-2-one (12). Yield: 35%. 1H NMR (400 MHz,
DMSOꢀd6) δ: 8.26 (d, J = 8.8 Hz, 2H, ArH2), 7.89 (d, J = 8.8 Hz, 2H, ArH2), 7.58 (d, J = 16.0 Hz,
1H, ArꢀCH=CHꢀ), 7.44 (d, J = 16.0 Hz, 1H, ArꢀCH=CHꢀ), 3.68 (t, 2H, NꢀCH2ꢀ), 2.54 (t, 2H,
ꢀCOCH2ꢀ), 1.80 (m, 4H, ꢀCH2CH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 174.2, 169.0, 148.3, 141.6,
139.4, 128.8, 126.5, 124.2, 44.9, 35.0, 22.6, 20.7; EIꢀMS m/z (%): 274 (M+, 100), 246 (41), 176
(95), 130 (80), 102(34); mp: 146ꢀ148 oC; purity: 95.87% (MeOH/H2O = 70/30, Rt= 4.579 min).
(E)-1-(3-(4-Bromophenyl)acryloyl)piperidin-2-one (13). Yield: 30%. 1H NMR (400 MHz,
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DMSOꢀd6) δ: 7.94 (d, J = 16.0 Hz, 1H, ArꢀCH=CHꢀ), 7.83 (d, J = 16.0 Hz, 1H, ArꢀCH=CHꢀ), 7.61
(d, 2H, ArH2), 7.38 (d, 2H, ArH2), 3.92 (t, 2H, NꢀCH2ꢀ), 2.65 (t, 2H, ꢀCOCH2ꢀ), 2.06 (m, 4H,
ꢀCH2CH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 174.1, 169.6, 141.8, 134.2, 132.1, 129.8, 124.3, 122.9,
44.8, 35.1, 22.7, 20.8; ESIꢀMS (m/z): [M + Na+] 330.0; mp: 114ꢀ116 oC; purity: 98.98%
(MeOH/H2O = 70/30, Rt= 8.771 min).
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(E)-1-(3-(4-(Trifluoromethyl)phenyl)acryloyl)azepan-2-one (14). Yield: 25%. 1H NMR (400
MHz, DMSOꢀd6) δ: 7.85 (d, J = 8.4 Hz, 2H, ArH2), 7.78 (d, J = 8.4 Hz, 2H, ArH2), 7.55 (d, J=16.0,
1H, ArꢀCH=CHꢀ), 7.39 (d, J=16.0, 1H, ArꢀCH=CHꢀ), 3.92 (t, 2H, NꢀCH2ꢀ), 2.76 (t, 2H, ꢀCOCH2ꢀ),
1.72ꢀ1.62 (m, 6H, ꢀCH2CH2CH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 178.5, 168.7, 141.1, 138.8,
131.2, 128.5, 125.9, 125.9, 124.8, 44.0, 39.8, 29.5, 28.8, 23.9; ESIꢀMS (m/z): [M + H+] 312.5; mp:
89ꢀ90 oC; purity: 99.75% (MeOH/H2O = 70/30, Rt= 12.299 min).
(E)-1-(3-(4-Nitrophenyl)acryloyl)azepan-2-one (15). Yield: 25%. 1H NMR (400 MHz,
DMSOꢀd6) δ: 8.26 (d, J = 8.8 Hz, 2H, ArH2), 7.90 (d, J = 8.8 Hz, 1H, ArH2), 7.57 (d, J=16.0, 1H,
ArꢀCH=CHꢀ), 7.43 (d, J=16.0, 1H, ArꢀCH=CHꢀ), 3.93 (t, 2H, NꢀCH2ꢀ), 2.76 (t, 2H, ꢀCOCH2ꢀ),
1.72ꢀ1.65 (m, 6H, ꢀCH2CH2CH2ꢀ); 13C NMR (100 MHz, CDCl3) δ: 178.5, 168.4, 148.4, 141.6,
139.6, 129.6, 126.6, 124.3, 123.7, 44.1, 39.7, 29.4, 28.8, 23.9; EIꢀMS m/z (%): 288 (M+, 100), 260
(15), 176 (56), 130 (47), 102 (20); mp: 82ꢀ84 oC; purity: 99.91% (MeOH/H2O = 70/30, Rt= 6.102
min).
1-Cinnamoylpyrrolidin-2-one (16).59 Yield: 43%. 1H NMR (400 MHz, CDCl3) δ: 7.94 (d, J =
16.0 Hz, 1H, ArꢀCH=CHꢀ), 7.83 (d, J = 16.0 Hz, 1H, ArꢀCH=CHꢀ), 7.61 (d, 2H, ArH2), 7.38 (m, 3H,
ArH3), 3.92 (t, 2H, NꢀCH2ꢀ), 2.65 (t, 2H, ꢀCOCH2ꢀ), 2.06 (m, 2H, ꢀCH2ꢀ); 13C NMR (100 MHz,
CDCl3) δ: 175.6, 166.2, 145.4, 134.8, 130.3, 128.7, 128.4, 118.9, 45. 8, 33.9, 17.1; ESIꢀMS (m/z):
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[M + H+] 215.8; mp: 98ꢀ99 oC; purity: 99.97% (MeOH/H2O = 65/35, Rt= 5.687 min).
1-Cinnamoylpiperidin-2-one (17).59 Yield: 45%. 1H NMR (400 MHz, CDCl3) δ: 7.72 (d, J =
15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.58 (d, 2H, ArH2), 7.45 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.35 (m, 3H,
ArH3), 3.81 (t, 2H, NꢀCH2ꢀ), 2.62 (t, 2H, ꢀCOCH2ꢀ), 1.90 (m, 4H, ꢀCH2CH2ꢀ); 13C NMR (100 MHz,
CDCl3) δ: 173.8, 169.6, 143.0, 135.0, 129. 9, 128.7, 128.2, 122.0, 44.5, 34.8, 22.4, 20.6; ESIꢀMS
(m/z): [M + H+] 229.9; mp: 57ꢀ58 oC; purity: 96.56% (MeOH/H2O = 65/35, Rt= 6.629 min).
1-Cinnamoylazepan-2-one (18). Yield: 37%. 1H NMR (400 MHz, CDCl3) δ: 7.70 (d, J = 16.0
Hz, 1H, ArꢀCH=CHꢀ), 7.57 (d, 2H, ArH2), 7.40 (d, J = 16.0 Hz, 1H, ArꢀCH=CHꢀ), 7.36 (m, 3H,
ArH3), 3.97 (m, 2H, NꢀCH2ꢀ), 2.77 (m,, 2H, ꢀCOCH2ꢀ), 1.81 (m, 6H, ꢀCH2CH2CH2ꢀ); 13C NMR
(100 MHz, CDCl3) δ: 178.2, 169.0, 143.2, 135.2, 130.0, 128.8, 128.3, 122.1, 43.8, 39.6, 29.3, 28.7,
23.8; ESIꢀMS (m/z): [M + Na+] 266.0; mp: 70ꢀ71 oC; purity: 99.17% (MeOH/H2O = 65/35, Rt=
9.557 min).
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(E)-4-(3-oxo-3-(2-oxopyrrolidin-1-yl)prop-1-en-1-yl)phenyl acetate(19a). Yield: 56%; 1H
NMR (400 MHz, CDCl3) δ: 7.90 (d, J = 15.6 Hz, 1H, ArꢀCH=CHꢀ), 7.80 (d, J = 15.6 Hz, 1H,
ArꢀCH=CHꢀ), 7.64 (d, J = 8.4 Hz, 2H, ArH2), 7.12 (d, J = 8.4 Hz, 2H, ArH2), 3.91 (t, 2H, NꢀCH2ꢀ),
2.65 (t, 2H, ꢀCOCH2ꢀ), 2.31 (s, 3H, ꢀCOCH3), 2.07 (m, 2H, ꢀCH2ꢀ); 13C NMR (100 MHz, CDCl3) δ:
175.8, 169.2, 166.2, 152.2, 144.4, 132.7, 129.7, 122.1, 119.3, 45.9, 34.0, 21.2, 17.3; ESIꢀMS (m/z):
[M + H+] 274.8; mp: 154ꢀ155 oC.
Synthesis of B8 (Step e in Scheme 1). To a solution of pꢀcoumaric acid (B8a, 10 mmol, 1.64g)
in 4 mL acetic anhydride was added DMAP (0.08 equiv.) and catalytic amount of pyridine (0.01
equiv.). The mixture was heated to reflux for 4 h before it was washed with saturated aqueous
NaHCO3, and extracted with ethyl acetate. The combined organic phases were dried over MgSO4.
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After filtration and concentration, the residue was purified by flash chromatography to obtain the
desired product. Yield: 92%.
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Synthesis of 19 (Step f in Scheme 1). To a solution of amide (0.37 mmol, 100 mg) in dry
CH2Cl2 (10 mL) was added pyrollidine (1 mL). The reaction mixture was stirred for 15 min at room
temperature. The reaction was quenched with HCl (20 mL, 1 M) and extracted with ethyl acetate.
The combined organic phases were dried over MgSO4. After filtration and concentration, the
residue was purified by flash chromatography to obtain the desired product.
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(E)-1-(3-(4-hydroxyphenyl)acryloyl)pyrrolidin-2-one(19).59 Yield: 66%; 1H NMR (400 MHz,
DMSOꢀd6) δ: 10.04(s, 1H, ArꢀOH), 7.69(d, J = 16.0 Hz, 1H, ArꢀCH=CHꢀ), 7.64(d, J = 16.0 Hz, 1H,
ArꢀCH=CHꢀ), 7.49(d, J = 7.2 Hz, 2H, ArH2), 7.64(d, J = 7.2 Hz, 2H, ArH2), 3.76(t, J = 7.2 Hz, 2H,
NꢀCH2ꢀ), 2.57(t, J = 8.2 Hz, 2H, ꢀCOCH2ꢀ), 1.96(m, 2H, ꢀCH2ꢀ); 13C NMR (100 MHz, DMSOꢀd6) δ:
175.8, 165.5, 159.9, 144.3, 130.1, 125.6, 115.9, 115. 6, 45.5, 33.4, 16.6; mp: 201ꢀ202 oC; ESIꢀMS
(m/z): [M + H+] 232.0. purity: 96.58% (MeOH/H2O = 48/52, Rt = 6.773 min).
Biological Studies. Cell cultures. PC12 cells (rat adrenal pheochromocytoma cell line) and
L02 cells (the normal liver cell line) were obtained from Shanghai Institute of Biochemistry and
Cell Biology, Chinese Academy of Sciences and were cultured in DMEM supplemented with 10%
FBS, 2 mM glutamine, and 100 units ml−1 penicillin/streptomycin and maintained in a humidified
atmosphere of 5% CO2 at 37 °C.
MTT assay and Lactate dehydrogenase (LDH) release assay. PC12 cells (1×104 cells/well)
were seeded in 96ꢀwell plates for 1 day followed by incubation with the drugs for 24 h. The
cytotoxicity of drugs was determined by the MTT assay according to our published procedure.32, 33
For the H2O2 or 6ꢀOHDA injury model, PC12 cells (1×104 cells/well) were plated in a 96ꢀwell plate
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and allowed to adhere for 1 day, and then treated with the drugs for 24 h. After replacing with fresh
medium containing 500 ꢁM H2O2 or 200 ꢁM 6ꢀOHDA for 12 h, the cell viability was determined
by the MTT assay. For the LDH release assay, the cells (2×105 cells/well) were plated in 12ꢀwell
plates. On the following day, the cells were exposed to various concentrations of 4 or 5 for 24 h,
followed by addition of 500 ꢁM H2O2 or 200 ꢁM 6ꢀOHDA for 12 h. The leakage of LDH from the
cultured cells was quantified by measuring LDH activity in the culture medium according to our
published procedure.13, 14
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Hoechst 33342 staining. PC12 cells (2×105 cells/well) were seeded into 12ꢀwell plates. On the
following day, the cells were treated with 4 or 5 for another 24 h followed by replacing with the
fresh medium containing 500 ꢁM H2O2 or 200 ꢁM 6ꢀOHDA for 5 h. Hoechst 33342 was
subsequently added to a final concentration of 5 ꢁg/mL to stain the nuclei. The cells were visualized
and photographed under a Leica inverted fluorescent microscopy. Cells displayed condensed and
highly fluorescent nuclei, a characteristic morphology of cells undergoing apoptosis, were counted
as apoptotic cells.
Measurement of caspase-3 activity. For determination of the caspaseꢀ3 activity, PC12 cells
(1×106 cells/well) were seeded in 60ꢀmm dishes for 24 h, and then treated with different
concentrations of 4 or 5 for another 24 h. After replacing with the fresh medium containing 500 ꢁM
H2O2 or 200 ꢁM 6ꢀOHDA and continuing culture for 24 h, the cells were lysed with RIPA buffer (2
mM EDTA, 0.5% deoxycholate, 150 mM NaCl, 1% TritonXꢀ100, 0.1% SDS, 1 mM Na3VO4, and 1
mM PMSF in 50 mM Tris–HCl, pH 7.5). The protein content was quantified by the Bradford
procedure using BSA as a standard. The caspaseꢀ3 activity in the lysate was determined by a
colorimetric assay using AcꢀDEVDꢀpNA as a substrate.30, 31 The activity of caspaseꢀ3 in the
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drugꢀtreated cells was expressed as the percentage of the control cells.
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Determination of intracellular ROS.32, 33 For assaying the intracellular accumulation of ROS,
PC12 cells (2×105cells/well) were seeded into 12ꢀwell plates. After 24 h, the cells were treated with
4 or 5 for another 24 h followed by replacing with the fresh medium containing 500 ꢁM H2O2 or
200 ꢁM 6ꢀOHDA for 5 h. After removing the medium, DCFHꢀDA (10 ꢁM) in fresh FBSꢀfree
medium was added, and continued incubation for 30 min at 37 °C in dark. The cells were visualized
and photographed under fluorescent microscopy. The appearance of green fluorescence indicates
the accumulation of ROS in cells.
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Assessment of intracellular thiols. PC12 cells were plated in 60ꢀmm dishes at a concentration
of 8×105 cells/well and allowed to adhere for 24 h, then were treated with 4 or 5 (0ꢀ20 µM) for 24 h
followed treated with H2O2 or 6ꢀOHDA for another 24 h in the absence of drugs. The cells were
collected, washed twice with PBS, and lysed with RIPA buffer for 30 min on ice. The protein
content was quantified by the Bradford procedure using BSA as a standard. Total thiol levels were
determined by DTNBꢀtitration.31ꢀ33, 60 Briefly, 10 µL of cell lysate was added to cuvettes containing
90 ꢁL of 1 mM DTNB in 6 M guanidine hydrochloride, pH 8.0. After keeping the mixture for 5 min
at room temperature, the absorbance was read at 412 nm. Thiol levels were calculated by a
calibration curve using GSH as the standard.
Real-time reverse transcription-PCR (RT-PCR). PC12 cells (1×106 cells/well) were seeded in
60ꢀmm dishes for 1 day, and treated with 4 or 5 (20 ꢁM) for 0, 3, 6 and 12 h. Total RNA was
isolated from cells using the RNAiso plus (TaKaRa, Dalian, China) according to the manufacture’s
protocol and quantified through 260/280 absorbance. Reverse transcription was performed using
PrimescriptTM RT reagent kit according to the manufacture’s protocol (TaKaRa, Dalian, China).
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RTꢀPCR was performed on Mx3005PRTꢀPCR System (Agilent Technologies) using Power SYBR
Green PCR Master Mix. The glyceraldehydeꢀ3ꢀphosphate dehydrogenase (GAPDH) was used as an
internal control. Target gene expression was measured and normalized to the GAPDH expression
level. PCR primers specific to each gene are as follows: GAPDH: 5’ꢀcagtgccagcctcgtctcatꢀ3’ and
5’ꢀaggggccatccacagtcttcꢀ3’; HOꢀ1: 5’ꢀgccctggaagaggagatagagꢀ3’ and 5’ꢀtagtgctgtgtggctggtgtꢀ3’;
NQO1: 5’ꢀtcaccactctactttgctccaaꢀ3’ and 5’ꢀttttctgctcctcttgaacctcꢀ3’; Trx1:
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5’ꢀccttctttcattccctctgtgacꢀ3’ and 5’ꢀcccaaccttttgaccctttttatꢀ3’; TrxR1: 5’ꢀactgctcaatccacaaacagcꢀ3’
and 5’ꢀccacggtctctaagccaatagtꢀ3’; glutamate cysteine ligase catalytic subunit (GCLC):
5’ꢀcaaggacaagaacacaccatctꢀ3’ and 5’ꢀcagcactcaaagccataacaatꢀ3’; glutamate cysteine ligase modifier
subunit (GCLM): 5’ꢀggcacaggtaaaacccaatagtꢀ3’ and 5’ꢀttcaatgtcagggatgctttctꢀ3’. The GAPDH was
used as an internal control.
Measurement of total glutathione. After the drug treatment, the cells were collected and
resuspended in extraction buffer containing 0.1% TritonXꢀ100 and 0.6% sulfosalicyclic acid in 0.1
M PBS with 5 mM EDTA, pH 7.5 (KPE buffer). The suspension was sonicated on ice for 2ꢀ3 min
with vortexing every 30 s. After centrifugation at 3000 g for 4 min at 4 °C, the supernatant was
ready for the total GSH assay by enzymatic detection as described in the published protocols.33, 61
Determination of NQO1 activity. For measuring NQO1, the cells were lysed in RIPA buffer.
The total protein content was quantified by the Bradford procedure using BSA as a standard. The
NQO1 activity was determined spectrophotometrically by monitoring the reduction of the electron
acceptor DCPIP at 600 nm.13, 14, 62 The enzymatic reaction was initiated by the addition of cell lysate
(5 ꢁg of total protein) to the reaction mixture (20 mM TrisꢀHCl, pH 7.4, 200 ꢁM NADH, and 40
ꢁM DCPIP), and the decrease in absorbance at 600 nm was measured every 8 s for 2 min at room
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temperature in the presence or absence of 20 ꢁM dicoumarol, a specific inhibitor of NQO1. The
dicoumarolꢀinhibitable part of DCPIP's reduction was used to calculate the NQO1 activity. The
same amounts of DMSO were added to the control experiments and the activity was expressed as
the percentage of the control.
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Trx and TrxR activity assays. Measuring cellular Trx and TrxR activity were performed by
following our published protocols.30, 31, 63 Briefly, the cell extract containing 20 ꢁg of total proteins
was incubated in a final reaction volume of 50 ꢁL 100 mM TrisꢀHCl (pH 7.4) containing 0.3 mM
insulin, 660 ꢁM NADPH, 3 mM EDTA and 60 nM recombinant rat TrxR1 (for assaying the Trx
activity) or 4 ꢀM E. coli Trx (for assaying the TrxR activity) for 30 min at 37 °C. The reaction was
terminated by adding 200 ꢁL of 1 mM DTNB in 6 M guanidine hydrochloride, pH 8.0. A blank
sample, containing everything except Trx (for TrxR assay) or TrxR (for Trx assay), was treated in
the same manner. The absorbance at 412 nm was measured, and the blank value was subtracted
from the corresponding absorbance value of the sample. The same amounts of DMSO were added
to the control experiments and the activity was expressed as the percentage of the control.
Determination of TrxR activity by TRFS-green. TRFSꢀgreen, developed by our group, is a cell
membrane permeable dye, which specifically detects the activity of TrxR in the cultured cells.34
PC12 cells (2×105 cells/well) were seeded into 12ꢀwell plates. After 24 h, the cells were treated with
4 or 5 for 20 h. Then TRFSꢀgreen was added (the final concentration is 10 µM) and continued
incubation for 4 h at 37 °C in dark. The cells were visualized and photographed under fluorescent
microscopy. The intensity of the green fluorescence indicates the relative activity of TrxR in cells.
Preparation of different protein extracts for Western blots analysis. Different protein extracts
from PC12 cells, i. e., the whole cell protein extract, the cytosolic protein extract and the nuclear
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protein extract, were prepared according to our previously published procedures.13, 14 The target
proteins were determined by Western blots.
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Knockdown of Nrf2 expression by shRNA transfection. The shRNA (shNrf2ꢀ842) targeting rat
Nrf2 gene was used for Nrf2 knockdown experiments.13, 14 The shRNA with scrambled sequence
(shNT) was used as a control. Exponentially growing cells were transfected with different shRNAs
using GeneTran III transfection reagent according to the manufacture’s instruction. After 48 h of
transfection, the cells were maintained in DMEM containing 10% FBS, 2 mM glutamine, 100
units/ml penicillin/streptomycin at 37℃ in a humidified atmosphere of 5% CO2 and selected by
supplementation with 0.5 mg/ml of G418. Knockdown of the Nrf2 expression in the cells was
analyzed by Western blotting.
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Determination of cLogP and PSA. The cLogP values were obtained by determining their
partition coefficients between nꢀoctanol and water according to the published procedure and
oꢀcresol was used as a positive control.36, 64 The octanol–water partition coefficient was determined
as the follows. Approximately 100 mL of nꢀoctanol and 100 ml of distilled water were thoroughly
mixed in a 500 mL separatory funnel to get mutually saturated. To an Erlenmeyer flask containing
approximately 5 mg of tested compound in 10 ml of waterꢀsaturated octanol was added the same
volume of octanolꢀsaturated water. The flask was then shaked for 3 h at 25 oC to reach the
equilibrium. The two phases were separated by a separating funnel. Aliquots of water phase (100
ꢁL) and octanol phase (100 ꢁL) were transferred to cuvettes containing 1900 ꢁL of methanol, and
the concentrations of the compound in octanol phase (C1) and in water phase (C2) were quantified
by measuring the absorbance at 315 nm from an established calibration curve of the compound. The
cLogP value was calculated by the equation: cLogP=log(C1/C2). The PSA values were calculated
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Journal of Medicinal Chemistry
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on line via the website: www.molinspiration.com.
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Statistics. Data are presented as mean ± S. E. Statistical differences between two groups were
assessed by the Students t-test. Comparisons among multiple groups were performed using oneꢀway
analysis of variance (ANOVA), followed by a post hoc Scheffe test. P<0.05 was used as the
criterion for statistical significance.
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SUPPORTING INFORMATION
Supporting Information Available: The details of the synthetic intermediates, in vitro free radical
scavenging assay, HPLC chromatograms and NMR spectra of the final compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Eꢀmail: fangjg@lzu.edu.cn. Phone: +86 931ꢀ8912500.
Author Contributions
# These authors contribute equally to this work.
ACKNOWLEDGMENTS
The financial supports from Lanzhou University (the Fundamental Research Funds for the Central
Universities, lzujbkyꢀ2014ꢀ56) and Natural Science Foundation of Gansu Province (145RJZA225)
are greatly acknowledged. The authors also express appreciation to Prof. Kun Gao at the SKLAOC
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