Targeting the Nrf2/Amyloid-Beta Liaison in Alzheimer's Disease: A Rational Approach

Article abstract of DOI:10.1021/acschemneuro.7b00100

Amyloid is a prominent feature of Alzheimer's disease (AD). Yet, a linear linkage between amyloid-β peptide (Aβ) and the disease onset and progression has recently been questioned. In this context, the crucial partnership between Aβ and Nrf2 pathways is a

Full text of DOI:10.1021/acschemneuro.7b00100

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Article
Targeting the Nrf2/amyloid-beta liaison in
Alzheimer’s disease: a rational approach
Elena Simoni, Melania M Serafini, Roberta Caporaso, Chiara Marchetti, Marco
Racchi, Anna Minarini, Manuela Bartolini, Cristina Lanni, and Michela Rosini
ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 19 Apr 2017
Downloaded from http://pubs.acs.org on April 20, 2017
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Targeting the Nrf2/amyloidꢀbeta liaison in Alzheimer’s
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disease: a rational approach
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Elena Simoni,1,ɸ Melania M. Serafini,2,3,ɸ Roberta Caporaso,1 Chiara Marchetti,1 Marco Racchi,2
Anna Minarini,1 Manuela Bartolini,1 Cristina Lanni,2 Michela Rosini*1
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1Department of Pharmacy and Biotechnology, Alma Mater StudiorumꢀUniversity of Bologna, Via
Belmeloro 6, 40126 Bologna, Italy
2Department of Drug Sciences (Pharmacology Section), V.le Taramelli 14, 27100 Pavia, Italy
3Scuola Universitaria Superiore IUSS Pavia, P.zza Vittoria, 15, 27100 Pavia, Italy
Keywords:
Alzheimer's disease, amyloid aggregation, Nrf2 pathway, oxidative stress, electrophilic
compounds.
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Amyloid is a prominent feature of Alzheimer’s disease (AD). Yet, a linear linkage between
amyloidꢀβ peptide (Aβ) and the disease onset and progression has recently been questioned. In this
context, the crucial partnership between Aβ and Nrf2 pathways is acquiring paramount importance,
offering prospects for deciphering the Aβꢀcentered disease network. Here, we report on a new class
of antiꢀaggregating agents rationally designed to simultaneously activate transcriptionꢀbased
antioxidant responses, whose lead 1 showed interesting properties in a preliminary investigation.
Relying on the requirements of Aβ recognition, we identified the catechol derivative 12. In SHꢀ
SY5Y neuroblastoma cells, 12 combined remarkable free radical scavenger properties to the ability
to trigger the Nrf2 pathway and induce the Nrf2ꢀdependent defensive gene NQO1 by means of
electrophilic activation of the transcriptional response. Moreover, 12 prevented the formation of
cytotoxic stable oligomeric intermediates, being significantly more effective, and per se less toxic,
than prototype 1. More importantly, as different chemical features were exploited to regulate Nrf2
and Aβ activities, the two pathways could be tuned independently. These findings point to
compound 12 and its derivatives as promising tools for investigating the therapeutic potential of the
Nrf2/Aβ cellular network, laying foundation for generating new drug leads to confront AD.
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INTRODUCTION
Alzheimer’s disease (AD) is the most common form of neurodegenerative dementia.1 As for major
chronic diseases, its phenotype reflects several pathological processes that interact in a complex
network.2 Despite considerable efforts, the underlying disease mechanisms and their connections
are still poorly understood, and it remains unclear whether they participate in neuronal degeneration
with causative roles or they merely represent the telltale remains of earlier pathogenic events.
The process of amyloidogenesis is thought to be an important driver of AD. The amyloidꢀβ peptide
(Aβ) becomes harmful when Aβ monomers combine in various aggregates to form oligomers and
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fibrils.3 Aβ aggregates emerge as manifestations and mediators of a variety of neurobiological
events, including inflammatory responses, tau phosphorylation impairment, mitochondrial
dysfunction and oxidative damage.4 Thus, focusing on Aβ alone has probably guided to a simplistic
linear disease model, which is now appearing inadequate to represent the complexity of the
disease.5 In this context, the simultaneous observation of multiple components is increasingly being
perceived as a more adequate way for addressing Aβ pluralism of causes and effects.6ꢀ9 In this
scenario, oxidative stress seems to play an important role, interconnecting diverse ADꢀrelated
phenomena.10 Indeed, the proꢀoxidant environment induced by Aβ in AD pathology is a
consolidated concept.11 At the same time, oxidative stress is known to trigger the amyloidogenic
pathway and promote Aβ toxicity in a vicious circle.12 This crucial partnership involves the nuclear
factor (erythroidꢀderived 2)ꢀlike 2 (Nrf2) transcriptional pathway, an intrinsic mechanism of
defense that, under neuropathological conditions, reduces oxidative stress and inflammation by
promoting the transcription of cytoprotective genes, including NAD(P)H:quinone reductase
(NQO1), heme oxygenaseꢀ1 (HOꢀ1), and glutathione Sꢀtransferase (GST) to name a few.13, 14 The
activation of this master regulator occurs by disrupting interaction and binding of Nrf2 to Kelchꢀlike
ECHꢀassociated protein 1 (Keap 1), a cytosolic Nrf2 repressor that acts as a sensor of
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oxidative/electrophilic stress.15 Notwithstanding the extensive oxidative damage characterizing AD,
levels of some Nrf2ꢀinduced gene products are reduced in AD patients, suggesting disruption of the
transcriptional pathway.16 In addition, recent studies have demonstrated activation of the Keap1ꢀ
Nrf2 system to be essential for counteracting Aβꢀinduced toxicity while genetic ablation of Nrf2
worsens amyloid deposition and neuroinflammation in a mouse model of AD.17
On these premises, we believe that the identification of pharmacologic tools able to modulate
Aβ and/or Nrf2 pathways might contribute to the comprehension of this crucial crossꢀtalk, offering
a powerful key for interpreting the mechanistic connection between the process of protein
aggregation and tissue degeneration. We have preliminary reported on a set of three molecules as
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versatile tools for investigating the molecular mechanisms potentially involved in chronic Aβ
damage.18 In particular, we identified the catechol derivative 1, which joins a remarkable antiꢀ
aggregating ability to oxidant properties. Interestingly, the biological profile of compound 1 was
strategically tuned by the hydroxyl substituents on the aromatic moiety, offering a peculiar “on–off”
pattern of control of the antiꢀaggregating efficacy that has contributed to shed light on the
interconnection between the overproduction of radical species and Aβ.18
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On this basis and in the pursuit of more effective molecules, we performed systematic modifications
of 1, by focusing on the aryl substitution pattern, the thioester function, and the aliphatic skeleton
(compounds 2ꢀ13, Figure 1).
Figure 1. Drug design strategy of compounds 2ꢀ13.
In this paper, we delineate the general structure−activity relationships (SAR) of 2−12 by
investigating antioxidant and antiꢀaggregating properties. The efficacy in inhibiting fibrilization of
Aβ42, the most amyloidogenic isoform of Aβ, was first studied in vitro by a fluorescenceꢀbased
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assay. Compounds were then assayed in human SHꢀSY5Y neuroblastoma cells to explore their
ability to contrast oxidative stress and to exert neuroprotective effect against Aβ42ꢀinduced toxicity.
To draw connections between the structural requirements involved in inhibition of amyloid
aggregation and transcriptionꢀbased antioxidant responses, selected compounds were studied as
Nrf2 inducers in human SHꢀSY5Y neuroblastoma cells, and the ability of promoting the
endogenous upꢀregulation of the Nrf2ꢀdependent defensive gene NQO1 was also assessed.
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RESULTS AND DISCUSSION
Syntheses of the thioester derivatives 2ꢀ4 were accomplished by one pot reaction with minor
modifications of literature procedure referred to caffeate esters.19 As reported in Scheme 1 (method
A), this procedure allowed Meldrum’s acid monoꢀthioesterification with allyl sulphide to give the
nonꢀisolable intermediate, which was then readily condensed with the appropriate aldehyde
affording cinnamic derivatives 2ꢀ4 with moderate to good yields.
Unfortunately, this convenient method was not effective to access the phenolic derivatives 5ꢀ13.
They were therefore synthesized by means of an alternative procedure, which minimized sideꢀ
reactions and purification efforts (Scheme 1, method B). TBDMSꢀprotected alcohols 14ꢀ19
underwent coupling reaction with the appropriate allyl or propyl derivative with DCC in presence of
DMAP, to give intermediates 20ꢀ28. Treatment of 20ꢀ28 with TBAF effected desilylation to give
1
the final compounds 5ꢀ13. HNMR spectra show that compounds 2ꢀ8 and 11, featuring a carbonꢀ
carbon double bond between the catechol ring and the carbonyl function, have an E configuration as
indicated by the large spin coupling constants (around 16 Hz) of αꢀH and βꢀH on double bonds.
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Scheme 1
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Cell toxicity assay.
In order to define the range of concentration to be used in cellular experimental settings, the
cytotoxicity of compounds 2ꢀ13 was assessed in SHꢀSY5Y human neuroblastoma cells, in
comparison with 1. Cells were exposed to the compounds at concentrations ranging from 1 to 12.5
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M for 24 h and cell viability was evaluated by MTT assay. As shown in figure 2, all the
compounds were well tolerated (reduction of cell viability of about 10%) at a concentration up to 5
M, resulting significantly less toxic than prototype 1, that at this concentration determined a slight
decrease (about 20%) of cell viability.
ꢀ
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Figure 2. Cellular toxicity of compounds 2ꢀ12 on human neuroblastoma SHꢀSY5Y cells. The cell toxicity
profiles of reference compound 1 and the nonꢀ(pro)electrophilic derivative 13 are also shown. Cells were
treated with 1 µM, 2.5 µM, 5 µM, 7.5 µM, 10 µM and 12.5 µM of each compound for 24 h. Cell viability
was assessed by MTT assay. Data are expressed as percentage of cell viability versus CTR; * p<0.05,
**p<0.01, ***p<0.001 versus CTR; Dunnett’s multiple comparison test.
Inhibition of Aβ42 selfꢀaggregation.
In our previous study, we identified catechol derivative 1 as a good inhibitor of Aβ42 selfꢀ
aggregation.18 Its inhibitory effect was studied by a thioflavin T (ThT)ꢀbased fluorometric assay,
giving an IC50 of 12.5 ± 0.9 ꢀM, and confirmed by a mass spectrometry assay,20 which allowed to
detect and quantitate the monomeric form of Aβ42. Interestingly, the antiꢀaggregating profile of 1
seemed to be strictly related to the catechol moiety, as a complete loss of efficacy was observed
following single removal of the mꢀ or pꢀhydroxyl function.
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Table 1. Inhibition of Aβ42 selfꢀaggregation by
compounds 1ꢀ12
Compd
Inhibition of Aβ42 selfꢀaggregation [a]
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% inhibition (± SEM)
[I] = 50 µM
IC50 µM (± SEM)
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> 90
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< 10
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< 10
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< 10
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> 90
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> 90
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> 90
[a] Inhibition of Aβ42 50 µM selfꢀaggregation by [I] = 50 ꢀM.
The Aβ42/inhibitor ratio was equal to 1/1. For compounds
showing a % inhibition higher than 50% when screened at 50
ꢀM the IC50 value was determined. Values are the mean of
two independent experiments each performed in duplicate.
nd stands for not determined. SEM = standard error of the
mean.
Herein, systematic modifications of the aromatic substitution pattern corroborated the importance of
the catechol group in inhibiting amyloid aggregation21 as, in compounds 2ꢀ6, the removal or
masking into a methoxyꢀ or ethoxyꢀfunction of one or both the hydroxyl substituents of 1 resulted
in a complete loss of antiꢀaggregating efficacy (% inhibition at 50 ꢀM <10%, Table 1). Following
this observation, catecholꢀbased compounds were studied to assess the role of the thioester function.
Replacement of this moiety with an ester or an amide, affording compounds 7 and 8, respectively,
resulted in a gradual decrease in the ability of limiting fibril formation (ꢀCONHꢀ<ꢀCOOꢀ<ꢀCOSꢀ,
Table 1). Based on these results, we can argue that the catechol motif is essential but not per se
sufficient to guarantee antiꢀaggregating efficacy, indicating the thioester moiety as a second
requisite of relevance in this respect. Thus, we focused on the chemical link between the aboveꢀ
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mentioned key features of 1. For compound 9, where saturation of the cinnamoyl double bond
avoids conjugation of the catechol moiety and the thioester function, a slight increase in activity is
observed with respect to prototype 1 (IC50 values equal to 8.72 and 12.5 ꢀM for 9 and 1,
respectively), suggesting that no electronic influence between the two groups is required.
Conversely, when the conjugation persists but the distance is shortened, as in 10, a significant drop
in activity is detected (from % inhibition >90 to 49.1 at 50 ꢀM), revealing the importance of the
relative position of the catechol group and the thioester side chain in amyloid recognition.
Interestingly, the antiꢀaggregating effect of most active compounds 1 and 9 was further improved
by replacing the terminal allyl moiety with an alkyl function, affording 11 (IC50 = 3.99 ꢀM) and 12
(IC50 = 3.80 ꢀM), respectively. This modification, in addition to potentiating prototype’s efficacy,
opens perspectives for further functionalization in this position as a promising multitarget drug
discovery strategy.22
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Protective effect of 12 on Aβ42ꢀinduced toxicity in SHꢀSY5Y neuroblastoma cells.
Based on the promising data obtained in the in vitro assessment of the antiꢀaggregating properties of
the new derivatives, we studied whether the most active compound 12 could also exert
neuroprotective effect against Aβ42ꢀinduced toxicity in SHꢀSY5Y human neuroblastoma cells.
Incubation of SHꢀSY5Y cells with 10 ꢁM Aβ42 resulted in a reduction of about 40% of cell viability
(as determined by MTT assay), which can be ascribed to oligomeric species formation.23
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Figure 3. Effect of 1 and 12 on Aβ42ꢀmediated toxicity in neuroblastoma cells. SHꢀSY5Y cells were coꢀ
incubated for 24 h with 5 µM and 10 µM compound 1 or with 5 µM compound 12 in presence of 10 ꢁM
Aβ42. Cell viability was determined by MTT assay. Data are expressed as percentage of cell viability versus
CTR; ***p<0.001 versus CTR, $ p<0.05 versus Aβ42; Dunnett’s multiple comparison test.
A strong protective effect was observed for compound 12 which, at 5 µM, almost completely
prevented the Aβ42ꢀinduced cell death, showing to be more effective than 1 that, in the same assay,
was not able to counteract Aβ42 toxicity up to 10 µM concentration (Figure 3). These data are in
agreement with the inhibitory potency (as IC50 values) determined by ThTꢀbased assay, which
showed a 3.3ꢀfold higher antiꢀaggregating activity for 12 compared to 1 (Table 1). A good
correlation between data obtained by ThTꢀ and cellꢀbased assays was also previously demonstrated
by others.24
Protective effect towards H2O2ꢀinduced damage.
To determine the potential interest of compounds 2ꢀ12 as antioxidants, we evaluated their scavenger
ability when coꢀincubated with 300 µM H2O2, using prototype 1 as comparison.
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Figure 4. Compounds 1ꢀ12 reverse ROS formation induced by H2O2ꢀinduced oxidative stress in SHꢀSY5Y
neuroblastoma cells. Cells were loaded with 25 mM DCFHꢀDA for 45 min. DCFHꢀDA was removed by
centrifugation, cells were resuspended in PBS into a black 96ꢀwells plate and exposed to 5 µM concentration
of compounds 1ꢀ12 and 300 µM H2O2. ROS levels were determined from 0 to 270 min using a fluorescence
microplate reader. Fluorescence intensity for all compounds is significant at any time from 30 to 270 min
with p<0.001 versus H2O2. Dunnett’s multiple comparison test.
ROS scavenging effects were evaluated in SHꢀSY5Y cells by using the fluorescent probe dichloroꢀ
dihydroꢀfluorescein diacetate (DCFHꢀDA) as a specific marker for quantitative intracellular ROS
formation. In comparison to untreated cells (dashed line, Figure 4), the intracellular DCFHꢀ
fluorescence intensity in H2O2ꢀtreated cells significantly increased (dotted line, Figure 4).
Treatment with all compounds markedly suppressed H2O2ꢀinduced intracellular ROS production,
albeit to a different extent. In particular, catecholꢀbased derivatives 1 and 7ꢀ12 emerged as the most
potent antioxidants as, at any time tested, they were able to keep ROS levels below those observed
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for control (Figure 4). This strong antioxidant activity was particularly evident for compounds
where conjugation between the catechol moiety and the carbonyl function (1, 7, 8, 10 and 11)
occurs, while compounds 9 and 12, lacking the cinnamoyl double bond, were slightly less effective.
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Activation of Nrf2 pathway in neuroblastoma cells.
Activation of the Keap1/Nrf2 pathway and the consequent induction of phase 2 antioxidant genes
trigger an elaborate network of protective mechanisms against oxidative damage.25 When exposed
to oxidative insults, Keap1 undergoes conformational changes as a result of oxidation at specific
cysteine residues. This disrupts Nrf2 binding, promotes Nrf2 translocation into the nucleus and the
activation of transcriptionꢀmediated protective responses.26, 27 Interestingly, the Keap1ꢀNrf2
interaction can also be disrupted by small molecules,28 most of which have electrophilic
properties.29 Thus, the opportunity for tuning the inducible antioxidant response is offered by Keap1
cysteine residues, which covalently conjugate the electrophilic inducers.30 Based on these
observations, electrophiles and proelectrophiles from synthetic or natural sources have attracted
interest from a broad range of researchers in the drug discovery community.31 In particular,
proelectrophile compounds, which include hydroquinone cores of terpenoids and flavonoids, are
only converted to their active electrophilic forms in response to pathological oxidation, offering
prospects of minimal potential side effects.32
Herein, based on the (pro)electrophilic features of compounds 2ꢀ12, we selected a number of
catecholꢀbased derivatives to be studied as Nrf2 inducers. First, by acting as the “on” switch for
antiꢀaggregating activity, the catechol group represented a prerequisite for exploring the
amyloid/Nrf2 cellular network. Secondly, catechols, which become active orthoꢀquinones on
oxidation, prospect benefits of proelectrophiles, which should provide neuroprotection in oxidative
conditions.33 In addition, catecholꢀbearing compounds 1 and 7ꢀ12 can count on more favorable
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scavenger abilities (as highlighted by their ability to reverse H2O2ꢀinduced ROS formation), which
can significantly contribute to the overall antioxidant profile of the new molecules.
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Thus, catechol derivatives 1 and 7ꢀ12 were investigated in SHꢀSY5Y neuroblastoma cell line to
verify whether they may affect the Nrf2 pathway. To note, some of the selected compounds also
presented an electrophilic α,βꢀunsaturated carbonyl group (Michael acceptor functionality), which
may represent an additional source for Nrf2 activation. To discriminate the individual contribution
of the two (pro)electrophilic features, compound 5, where the Michael acceptor is not associated to
the catechol moiety, and a new compound (13) lacking both electrophilic functionalities (purposely
synthesized) were tested for comparison. Nrf2 protein levels were evaluated by western
immunoblotting in SHꢀSY5Y cells after treatment for 24 h with compounds 1, 5 and 7ꢀ13 at 5 ꢁM
concentration. Interestingly, all compounds with the exception of 13 increased Nrf2 levels when
compared to control (Fig. 5a), suggesting that Nrf2 modulation can be driven by both the catechol
function and the α,βꢀunsaturated carbonyl group, while the other structural features seemed to have
modest relevance in this respect. In particular, differently from what observed for the antiꢀ
aggregating activity, the thioester group is not a key feature for inducing Nrf2 activation, as
demonstrated by the strong efficacy elicited by ester derivative 7. The lack of efficacy observed for
compound 13 suggests that nucleophilic addition of Keap1 cysteine residues to (pro)electrophilic
portions of the molecule may represent the initiating event of the transcriptional process. Thus, we
focused on the activation of Nrfꢀ2 signalling by analyzing its translocation into nucleus and its
ability to induce NQO1, a prototypical cytoprotective Nrf2ꢀtarget gene related to cellular stress
response. In particular, we selected compounds 1, 5, 7, 9 and 12, which increased significantly the
protein levels of Nrf2. The selected compounds carry alternatively or simultaneously the two
(pro)electrophilic features responsible for Nrf2 induction. Compound 13 was also tested as negative
control. All compounds, with the only exception of 13, induced remarkable Nrf2 nuclear
translocation, with catechol derivatives 1, 7, 9 and 12 being slightly more effective than 5, lacking
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the catechol moiety (Figure 5b). Moreover, when analyzing the induction of NQO1, all the
compounds but 13 increased NQO1 levels, with the same trend of activity detected for Nrf2
activation and translocation to the nucleus (Figure 5c). Noteworthy, differently from what recently
observed for other multiꢀelectrophile compounds,34 the combined presence of the two
(pro)electrophilic features, as in 1, did not result in a synergistic efficacy (compare activity of 1
with that of 9 and 12, which only carry the catechol group). This can be possibly ascribed to the
conjugation, occurring in 1, between the two features, that consequently may not behave as separate
entities.
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Figure 5. Activation of Nrf2ꢀmediated phase II detoxification pathway. a) Total cellular extracts of SHꢀ
SY5Y cells treated for 24 h with 5 µM concentration of compounds 1, 5, 7ꢀ13 were analyzed for Nrf2
expression by western blot. Anti tubulin was used as protein loading control. Results are shown as ratio
Nrf2/tubulin (% of CTR) ± SEM. Dunnett’s multiple comparison test. b) Nuclear cellular extracts of SHꢀ
SY5Y cells were treated for 3 h with compounds 1, 5, 7, 9, 12, 13 at 5 µM concentration and homogenized to
obtain nuclear fraction. Nrf2 expression was determined by western blot. Anti lamin AꢀC was used as protein
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loading control. Results are shown as ratio Nrf2/lamin AꢀC (% of CTR) ± SEM. **p<0.01 and ***p<0.001
versus CTR; Dunnett’s multiple comparison test. c) Total cellular extracts of SHꢀSY5Y cells treated for 24 h
with 5 µM concentration of compounds 1, 5, 7, 9, 12, 13 were analyzed for NQO1 expression by western
blot. Antiꢀactin was used as protein loading control. Results are shown as ratio NQO1/actin (% of CTR) ±
SEM. *p<0.05, **p<0.01 and ***p<0.001 versus CTR; Dunnett’s multiple comparison test.
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Electrophilic features may account for potential toxicity issues associated with offꢀtarget
interactions.34 When studied in SHꢀSY5Y cells the nonꢀ(pro)electrophilic compound 13 was not
toxic in the whole range of tested concentrations (cell viability > 90%, Figure 2). Noteworthy, the
behavior at 5 ꢀM (concentration of interest for biological activity) of (pro)electrophilic compounds
2ꢀ12 was not significantly dissimilar from that of 13.
Conclusion
Advances in Aβꢀcentered drug discovery for AD suffer the lack of a molecular mechanistic theory
of Aβ causative role. Thus, a deeper comprehension of the intertwined correlation between Aβ and
oxidative damage might contribute to the understanding and treatment of the disease. The interest
towards this critical partnership is reinforced by the emerging evidence of an aberrant regulation of
the Nrf2ꢀmediated antioxidant response in AD, with Aβ contributing to the dysfunctional activation
of the transcriptional pathway.
On these bases, in the pursuit of more effective antiꢀaggregating and antioxidant properties, we
herein expanded our previous study on the catechol derivative 1 through systematic modifications
of its structure. We also deepened insight the antioxidant profile of the compounds by investigating
their ability to trigger the Nrf2 pathway in terms of upꢀregulation of Nrf2 expression, translocation
into the nucleus and induction of the Nrf2ꢀdependent defensive gene NQO1. Interestingly, in SHꢀ
SY5Y neuroblastoma cells, all compounds tested exhibited remarkable free radical scavenging
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properties and protected against oxidative stress by means of electrophilic activation of Nrf2ꢀ
mediated response. This multimodal behavior was accompanied by a significant reduction of the
cytotoxicity with respect to 1. Relying on the requirements of Aβ recognition, which was driven by
the catechol function and the thioester group, we identified compound 12. It joined the aboveꢀ
mentioned antioxidant effects to a marked ability of preventing the formation of cytotoxic stable
oligomeric intermediates, being significantly more effective than prototype 1. Most importantly, as
different chemical features were exploited to regulate Nrf2 and Aβ activities, we could finely and
separately tune the two pathways. These findings point to compound 12 and its derivatives as
powerful tools for investigating the therapeutic potential of the Nrf2/Aβ cellular network, paving
the way for the generation of new drug leads to confront AD.
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EXPERIMENTAL SECTION
Chemistry. General Chemical methods. Chemical reagents were purchased from Sigma Aldrich,
Fluka and Lancaster (Italy). The course of the reactions was observed by TLC on 0.20 mm silica gel
60 F254 plates (Merck, Germany), then visualized with an UV lamp. Nuclear magnetic resonance
spectra (NMR) were recorded at 400 MHz for 1H and 100 MHz for 13C on Varian VXR 400
spectrometer. Chemical shifts are reported in parts per millions (ppm) relative to tetramethylsilane
(TMS), and spin multiplicities are given as s (singlet), br s (broad singlet), d (doublet), t (triplet), q
(quartet), or m (multiplet). Direct infusion ESIꢀMS mass spectra were recorded on a Waters ZQ
4000 apparatus. Final compounds 1−13 were >95% pure as determined by HPLC analyses. The
analyses were performed under reversedꢀphase conditions on a Phenomenex Jupiter C18 (150x4.6
mm I.D.) column, using a binary mixture of H2O/acetonitrile (60/40, v/v for 2, 5, 6; 30/70, v/v for
3,4; 65/35, v/v for 7, 10, 11; 70/30, v/v for 8; 50/50, v/v for 9, 12, 13) as the mobile phase, UV
detection at λ = 302 nm (for 2ꢀ8, 10) or 254 nm (for 9, 12, 13) and a flow rate of 0.7 mL/min.
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Analyses were performed on a liquid chromatograph model PUꢀ1585 UV equipped with a 20 µL
loop valve (Jasco Europe, Italy).
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General procedure for the target compounds 2ꢀ4. To a solution of Meldrum’s acid (1 equiv,
114.13 mg, 4.05 mmol) in toluene (8 mL) was slowly added 2ꢀpropeneꢀ1ꢀthiol (1 equiv, 300 mg,
4.05 mmol). The mixture was refluxed for 7 h. After the formation of the intermediate, the reaction
was cooled to room temperature followed by sequential addition of the appropriate aldehyde (0.4
equiv), pyridine (400 ꢀL) and piperidine (40 ꢀL). The stirring continued at room temperature 4
days. Following evaporation of the solvent, the residue was purified by column chromatography on
silica gel to yield the desired cinnamic derivatives 2ꢀ4.
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Sꢀallyl (E)ꢀ3ꢀphenylpropꢀ2ꢀenethioate (2). 2 was synthesized from benzaldehyde (170 mg, 1.6
mmol). Elution with petroleum ether/ethyl acetate (9.7:0.3) afforded 2 as a waxy solid: 108 mg
(33%). 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J=15.6 Hz, 1H), 7.47ꢀ7.44 (m, 2H), 7.32ꢀ7.29 (m,
3H), 6.67 (d, J=15.6 Hz, 1H), 5.89ꢀ5.79 (m, 1H), 5.26 (dd, 1J=16.8 Hz, 2J=1.2 Hz, 1H), 5.10 (d,
J=10 Hz, 1H), 3.64 (d, J=6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 190.04, 140.63, 134.06,
133.19, 130.58, 128.94 (2 C), 128.43 (2 C), 124.75, 117.99, 31.80. MS [ESI+] m/z 227 [M+Na]+.
Sꢀallyl (E)ꢀ3ꢀ(3,4ꢀdimethoxyphenyl)propꢀ2ꢀenethioate (3). 3 was synthesized from 3,4ꢀ
dimethoxybenzaldehyde (266 mg, 1.6 mmol). Elution with petroleum ether/ethyl acetate (7:3)
afforded 3 as a white solid: 220 mg (52%), m.p.= 123 °C. 1H NMR (400 MHz, CDCl3) δ 7.57 (d,
J=16 Hz, 1H), 7.13 (dd, 1J=8 Hz, 2J=2 Hz, 1H), 7.05 (d, J=2Hz, 1H), 6.86 (d, J=8 Hz, 1H), 6.59 (d,
J=16 Hz, 1H), 5.91ꢀ5.84 (m, 1H), 5.29 (d, J=19.6, 1H), 5.13 (d, J=11.6, 1H), 3.92 (s, 6H), 3.67 (d,
J=8 Hz, 2H). 13CꢀNMR (100 MHz, CDCl3) δ 188.88, 151.44, 149.25, 140.77, 133.21, 126.95,
123.26, 122.58, 117.86, 111.04, 109.74, 55.98 (2 C), 31.71. MS [ESI+] m/z 265 [M+1]+.
Sꢀallyl (E)ꢀ3ꢀ(3,4ꢀdiethoxyphenyl)propꢀ2ꢀenethioate (4). 4 was synthesized from 3,4ꢀ
diethoxybenzaldehyde (310 mg, 1.6 mmol). Cristallization from ethanol gave 4 as a white solid:
220 mg (47%), m.p. =145 °C; 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J=15.6 Hz, 1H), 7.09ꢀ7.05
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(m, 2H), 6.83 (d, J=8 Hz, 1H), 6.57 (d, J=15.6 Hz, 1H), 5.89ꢀ5.83 (m, 1H), 5.27 (d, J=17.2 Hz, 1H),
5.11 (d, J=10.4 Hz, 1H), 4.13ꢀ4.08 (m, 4H), 3.66 (d, J=6.8 Hz, 2H), 1.47ꢀ1.44 (m, 6H). 13CꢀNMR
(100 MHz, CDCl3) δ 188.76, 151.28, 148.79, 140.90, 133.27, 126.75, 123.21, 122.36, 117.78,
112.63, 112.0, 64.58 (2 C), 31.67, 14.74 (2 C). MS [ESI+] m/z 315 [M+Na]+.
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General procedure for the intermediates 14ꢀ19. To a solution of the appropriate commercially
available acid (1 equiv) in dry DMF (5 mL) were added TBDMSꢀCl (2ꢀ3 equiv) and imidazole (5
equiv) under nitrogen atmosphere. After leaving the reaction to room temperature overnight, the
mixture was concentrated to dryness, and the residue purified by column chromatography on silica
gel to yield the desired intermediates 14ꢀ19. Experimental data of known compounds 14 and 16ꢀ19
were in agreement with the literature.
(E)ꢀ3ꢀ(4ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀ3ꢀethoxyphenyl)acrylic acid (15). 15 was synthesized
from (E)ꢀ3ꢀ(3ꢀethoxyꢀ4ꢀhydroxyphenyl)acrylic acid (500 mg, 2.40 mmol). Elution with petroleum
ether/ethyl acetate/methanol (5:4.5:0.5) afforded 15 as a waxy solid: 325 mg (42%); 1H NMR (400
MHz, CDCl3) δ 7.70 (d, J=15.6 Hz, 1H), 7.03 (s, 1H), 7.02 (d, J=4.0 Hz, 1H), 6.84 (d, J=4.0 Hz,
1H), 6.30 (d, J=15.6 Hz, 1H), 4.04 (q, J=7.2 Hz, 2H), 1.45 (t, J=6.8 Hz, 3H), 0.99 (s, 9H), 0.17 (s,
6H). 13C NMR (100 MHz, CDCl3) 171.57, 152.21, 148.89, 147.65, 126.62, 122.91, 121.31, 114.01,
111.09, 64.93, 31.76, 25.61 (3 C), 14.73, ꢀ4.61 (2 C).
General procedure for the intermediates 20ꢀ28. To an iceꢀcooled solution of the appropriate
protected acid (14ꢀ19) (1 equiv) in dry CH2Cl2 (4 mL) was added DCC (1.1 equiv), and DMAP
(cat.). The reaction mixture was stirred for 10 min, followed by addition of the appropriate
nucleophile (3 equiv). Stirring was then continued at room temperature overnight, and the reaction
worked up by filtration and evaporation. The crude was purified by chromatography on silica gel.
Sꢀallyl (E)ꢀ3ꢀ(4ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀ3ꢀmethoxyphenyl)propꢀ2ꢀenethioate (20). 20 was
synthesized from 1435 (410 mg, 1.33 mmol) and 2ꢀpropeneꢀ1ꢀthiol. Elution with petroleum
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ether/ethyl acetate (9.4:0.6) afforded 20 as a waxy solid: 330 mg (68%); H NMR (400 MHz,
CDCl3) δ 7.48 (d, J=15.6 Hz, 1H), 6.98ꢀ6.95 (m, 2H), 6.76 (d, J=8 Hz, 1H), 6.50 (d, J=15.6 Hz,
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1H), 5.80ꢀ5.76 (m, 1H), 5.21 (d, J=18 Hz, 1H), 5.05 (d, J=10 Hz, 1H), 3.76 (s, 3H), 3.59 (d, J=6.80
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Hz, 2H), 0.91 (s, 9H), 0.09 (s, 6H). C NMR (100 MHz, CDCl3) 174.87, 151.23, 147.94, 140.97,
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133.25, 127.84, 122.81, 122.66, 121.14, 117.84, 111.00, 55.44, 31.70, 25.63 (3 C), 18.46, ꢀ4.61 (2
C).
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Sꢀallyl (E)ꢀ3ꢀ(4ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀ3ꢀethoxyphenyl)propꢀ2ꢀenethioate (21). 21 was
synthesized from 15 (200 mg, 0.62 mmol) and 2ꢀpropeneꢀ1ꢀthiol. Elution with petroleum
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ether/ethyl acetate (9.5:0.5) afforded 21 as a waxy solid: 130 mg (55%); H NMR (400 MHz,
CDCl3) δ 7.50 (d, J=16 Hz, 1H), 7.00ꢀ6.89 (m, 2H), 6.79 (d, J=8 Hz, 1H), 6.52 (d, J=16 Hz, 1H),
5.86ꢀ5.77 (m, 1H), 5.22 (d, J=17.2 Hz, 1H), 5.10 (d, J=10 Hz, 1H), 4.01 (q, J=6.4 Hz, 2H), 3.62 (d,
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J=6.8 Hz, 2H), 1.41 (t, J=6.4 Hz, 3H), 0.96 (s, 9H), 0.03 (s, 6H). C NMR (100 MHz, CDCl3) δ
189.04, 146.34, 145.99, 140.85, 132.27, 125.39, 124.69, 122.15, 117.64, 114.96, 111.02, 64.93,
31.90, 24.93 (3 C), 18.66, 14.35, ꢀ4.31 (2 C).
Allyl (E)ꢀ3ꢀ(3,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)phenyl)acrylate (22). 22 was synthesized from
1635 (190 mg, 0.465 mmol) and allyl alcohol. Elution with petroleum ether/ethyl acetate (9.5:0.5)
afforded 22 as a waxy solid: 120 mg (57%); 1H NMR (400 MHz, CDCl3) δ 7.57 (d, J=16 Hz, 1H),
6.99 (s, 1H), 6.98 (d, J=8 Hz, 1H) 6.80 (d, J=8 Hz, 1H) 6.23 (d, J=16 Hz, 1H), 6.00ꢀ5.98 (m, 1H),
5.34 (d, J=16 Hz, 1H), 5.24 (d, J=11 Hz, 1H), 4.67 (d, J=6 Hz, 2H), 0.97 (s, 9H), 0.96 (s, 9H), 0.19
(s, 6H), 0.18 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 169.8, 146.93, 146.04, 143.71, 133.03, 126.06,
122.03, 118.64, 115.29, 114.85, 114.00, 30.07, 26.63 (6 C), 18.56 (2 C), ꢀ4.21 (4 C).
(E)ꢀNꢀallylꢀ3ꢀ(3,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)phenyl)acrylamide (23). 23 was synthesized
from 1635 (400 mg, 0.978 mmol) and allylamine. Elution with petroleum ether/ethyl acetate (8:2)
afforded 23 as a waxy solid: 100 mg (23%); 1H NMR (400 MHz, CDCl3) δ 7.49 (d, J=15.6 Hz, 1H),
6.98 (s, 1H), 6.96 (d, J=8 Hz, 1H), 6.77 (d, J=8 Hz, 1H), 6.21 (d, J=15.6 Hz, 1H), 5.91ꢀ5.80 (m,
1H), 5.81 (br s, 1H), 5.21 (d, J=17.2 Hz, 1H), 5.13 (d, J=11.6 Hz, 1H), 3.99 (d, J=5.6 Hz, 2H), 0.97
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(s, 9H), 0.96 (s, 9H), 0.19 (s, 6H), 0.18 (s, 6H). C NMR (100 MHz, CDCl3) δ 166.95, 147.31,
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145.20, 141.17, 133.93, 127.02, 121.55, 117.63, 115.34, 114.80, 113.52, 41.20, 25.63 (6 C), 18.76
(2 C), ꢀ4.11 (4 C).
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ꢀallyl 3ꢀ(3,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)phenyl)propanethioate (24). 24 was synthesized
from 1736 (300 mg, 0.73 mmol) and 2ꢀpropeneꢀ1ꢀthiol. Elution with petroleum ether/ethyl acetate
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(9.7:0.3) afforded 24 as a waxy solid: 200 mg (59%); H NMR (400 MHz, CDCl3) δ 6.71(d, J=8
Hz, 1H), 6.62ꢀ6.57 (m, 2H), 5.78ꢀ5.73 (m, 1H), 5.20 (dd, J=16.8 Hz, 2J=1.2 Hz, 1H), 5.07 (d, J=8
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Hz, 1H), 3.51 (d, J=8 Hz, 2H), 2.84ꢀ2.77 (m, 4H), 0.97 (s, 18H), 0.17 (s, 12H). 13CꢀNMR (100
MHz, CDCl3) δ 198.09, 146.78 (2 C), 145.42, 133.22, 121.34, 121.24, 121.10, 117.99, 45.79, 31.09,
30.86, 26.09 (6 C), 18.57 (2 C), ꢀ3.96 (4 C).
S
ꢀallyl 3,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)benzothioate (25). 25 was synthesized from 1837 (330
mg, 0.862 mmol) and 2ꢀpropeneꢀ1ꢀthiol. Elution with petroleum ether/ethyl acetate (8:2) afforded
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25 as a waxy solid: 130 mg (34%); H NMR (400 MHz, CDCl3) δ 7.51ꢀ7.47 (m, 2H), 6.84 (d, J=8
Hz, 1H), 5.88 (m, 1H), 5.30 (d, J=17.2 Hz, 1H), 5.13 (d, J=10 Hz, 1H), 3.69 (d, J=6.8 Hz, 2H), 0.99
(s, 18H), 0.22 (s, 12H). 13C NMR (100 MHz, CDCl3) δ 190.30, 151.34, 146.49, 134.01, 130.94,
120.42, 117.82, 115.96, 114.76, 32.28, 25.72 (6 C), 18.57 (2 C), ꢀ4.06 (4 C).
S
ꢀpropyl (E)ꢀ3ꢀ(3,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)phenyl)propꢀ2ꢀenethioate (26). 26 was
synthesized from 1635 (480 mg, 1.174 mmol) and 1ꢀpropanethiol. Elution with petroleum
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ether/ethyl acetate (9.8:0.2) afforded 26 as a waxy solid: 430 mg (78%); H NMR (400 MHz,
CDCl3) δ 7.49 (d, J=16 Hz, 1H), 7.05ꢀ7.01 (m, 2H), 6.82 (d, J=8.4 Hz, 1H), 6.52 (d, J=16 Hz, 1H),
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2.99 (t, J=7.2 Hz, 2H), 1.70ꢀ1.64 (m, 2H), 1.03ꢀ0.97 (m, 18H + 3H), 0.21 (s, 12H). C NMR (100
MHz, CDCl3) δ 190.70, 145.84, 144.52, 141.95, 126.95, 122.98, 121.65, 115.54, 114.83, 32.14,
24.08, 25.52 (6 C), 18.53 (2 C), 13.32, ꢀ4.16 (4 C).
S
ꢀpropyl 3ꢀ(3,4ꢀbis((tertꢀbutyldimethylsilyl)oxy)phenyl)propanethioate (27). 27 was synthesized
from 1736 (300 mg, 0.73 mmol) and 1ꢀpropanethiol. Elution with petroleum ether/ethyl acetate
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(9.7:0.3) afforded 27 as a waxy solid: 342 mg (44%); H NMR (400 MHz, CDCl3) δ 6.72 (d, J= 8
Hz, 1H), 6.64 (s, 1H), 6.61 (d, J=8 Hz, 1H), 2.86ꢀ2.79 (m, 6H), 1.60ꢀ1.55 (m, 2H), 0.99ꢀ0.93 (m,
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18H+3H), 0.18 (s, 12H). C NMR (100 MHz, CDCl3) δ 198.86, 146.74, 145.35, 133.35, 121.33,
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121.25, 121.06, 45.91, 30.94, 30.87, 26.07 (6 C), 23.07, 18.54 (2 C), 13.45, ꢀ3.98 (4 C).
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Sꢀpropyl 3ꢀ(4ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀ3ꢀmethoxyphenyl)propanethioate (28). 28 was
synthesized from 1938 (110 mg, 0.35mmol) and 1ꢀpropanethiol. Elution with petroleum ether/ethyl
acetate (9.5:0.5) afforded 28 as a pale oil: 70 mg (54%); 1H NMR (400MHz, CDCl3) δ 6.75 (d, J=8
Hz, 1H), 6.67 (d, J=2 Hz, 1H), 6.63 (dd,1J=8 Hz,2J=2 Hz, 1H), 3.78 (s, 3H), 2.91ꢀ2.86 (m, 2H),
2.85ꢀ2.82 (m, 4H), 1.62ꢀ1.56 (m, 2H), 0.99 (s, 9H), 0.97ꢀ0.94 (m, 3H), 0.14 (s, 6H).13C NMR
(100MHz, CDCl3) δ 198.96, 150.90, 143.55, 143.55, 133.69, 120.90, 112.54, 55.57, 45.95, 31.38,
30.88, 25.83 (3 C), 23.06, 18.53, 13.39, ꢀ4.56 (2 C).
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General procedure for the synthesis of 5ꢀ13. To a solution of the appropriate organosilane
intermediate 20ꢀ28 (1 equiv) in THF (5 mL) was added TBAF (4 equiv) and stirring was continued
at room temperature. After 20ꢀ30 min, the reaction was quenched by addition of saturated aqueous
NH4Cl solution; the aqueous phase was extracted with EtOAc (3 x 10 mL), and the combined
organic layer was dried over Na2SO4. Following evaporation of the solvent, the residue was purified
by column chromatography on silica gel.
Sꢀallyl (E)ꢀ3ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)propꢀ2ꢀenethioate (5). 5 was synthesized from 20
(330 mg, 0.91mmol). Elution with petroleum ether/ethyl acetate (8.5:1.5) afforded 5 as a waxy
solid: 140 mg (62%); 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J=15.6 Hz, 1H), 7.08 (d, J=8 Hz, 1H),
7.01 (s, 1H), 6.90 (d, J=8 Hz, 1H), 6.56 (d, J=15.6 Hz, 1H), 5.90 (br s, 1H), 5.87ꢀ5.80 (m, 1H), 5.27
13
(d, J=16.8 Hz, 1H), 5.11 (d, J=10 Hz, 1H), 3.92 (s, 3H), 3.65 (d, J=7.6 Hz, 2H). C NMR (100
MHz, CDCl3) δ 188.91, 148.31, 146.79, 140.95, 133.23, 126.57, 123.66, 122.35, 117.86, 114.81,
109.49, 55.98, 31.71. MS [ESIꢀ] m/z 249 [Mꢀ1]ꢀ.
Sꢀallyl (E)ꢀ3ꢀ(3ꢀethoxyꢀ4ꢀhydroxyphenyl)propꢀ2ꢀenethioate (6). 6 was synthesized from 21 (120
mg, 0.317 mmol). Elution with petroleum ether/ethyl acetate (8.5:1.5) afforded 6 as a waxy solid:
50 mg (60%); 1H NMR (400 MHz, CDCl3) δ 7.52 (d, J=15.6 Hz, 1H), 7.06 (d, J=8 Hz, 1H), 6.99 (s,
1H), 6.90 (d, J=8 Hz, 1H), 6.54 (d, J=15.6 Hz, 1H), 5.90 (br s, 1H), 5.89ꢀ5.84 (m, 1H), 5.26 (d,
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J=16.8 Hz, 1H), 5.10 (d, J=9.6 Hz, 1H), 4.13 (q, J=7.2 Hz, 2H), 3.64 (d, J=7.2 Hz, 2H), 1.45 (t,
J=7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 188.94, 148.44, 146.09, 141.05, 133.25, 126.49,
123.49, 122.25, 117.84, 114.76, 110.42, 64.63, 31.70, 14.75. MS [ESI+] m/z 265 [M+1]+; [ESIꢀ] m/z
263 [Mꢀ1]ꢀ.
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Allyl (E)ꢀ3ꢀ(3,4ꢀdihydroxyphenyl)acrylate (7).19 7 was synthesized from 22 (120 mg, 0.267
mmol). Elution with petroleum ether/ethyl acetate (5:5) afforded 7 as a waxy solid: 43 mg (73%);
1H NMR (400 MHz, CDCl3) δ 7.59 (d, J=16 Hz, 1H), 7.09 (s, 1H), 6.98 (d, J=8 Hz, 1H), 6.86 (d,
J=8 Hz, 1H), 6.26 (d, J=16 Hz, 1H), 5.97ꢀ5.93 (m, 1H), 5.35(d, J=17.2 Hz, 1H), 5.26(d, J=10.4 Hz,
1H), 4.70 (d, J=5.6 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 167.8, 146.63, 145.65, 143.91, 132.03,
127.26, 122.53, 118.44, 115.49, 114.97, 114.40, 29.67. MS [ESI+] m/z 221 [M+1]+; [ESIꢀ] m/z
219 [Mꢀ1]ꢀ.
(E)ꢀN
ꢀallylꢀ3ꢀ(3,4ꢀdihydroxyphenyl)acrylamide (8).39 8 was synthesized from 23 (100 mg, 0.224
mmol). Elution with CHCl3/MeOH (9.7:0.3) afforded 8 as a waxy solid: 40 mg (81%);1H NMR
(400 MHz, CD3OD) δ 7.39 (d, J=15.6 Hz, 1H), 6.99 (s, 1H), 6.89 (d, J=8 Hz, 1H), 6.74 (d, J=8 Hz,
1H), 6.37 (d, J=15.6 Hz, 1H), 5.91ꢀ5.82 (m, 1H), 5.18(d, J=17.2 Hz, 1H), 5.11 (d, J=13.2 Hz, 1H),
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3.89 (d, J=7.2 Hz, 2H). C NMR (100 MHz, CD3OD) δ 167.75, 147.41, 145.30, 141.14, 134.03,
126.82, 120.75, 116.63, 115.04, 114.90, 113.68, 41.50. MS [ESI+] m/z 220 [M+1]+; [ESIꢀ] m/z
218 [Mꢀ1]ꢀ.
Sꢀallyl 3ꢀ(3,4ꢀdihydroxyphenyl)propanethioate (9). 9 was synthesized from 24 (130 mg, 0.28
mmol). Elution with petroleum ether/ethyl acetate (5.5:4.5) afforded 9 as a waxy oil: 45 mg (67%);
1H NMR (400 MHz, CDCl3) δ 6.77 (d, J=8 Hz, 1H), 6.69 (s, 1H), 6.61 (d, J=8 Hz, 1H), 5.84ꢀ5.73
(m, 1H), 5.22 (d, J=17.2 Hz, 1H), 5.10 (d, J=10.8 Hz, 1H), 3.53 (d, J=7.2 Hz, 2H), 2.89ꢀ2.80 (m,
13
4H). C NMR (100 MHz, CDCl3) δ 199.25, 143.71, 142.18, 132.95 (2 C), 120.77, 118.18, 115.56,
115.50, 45.68, 31.96, 30.91. MS [ESI+] m/z 261 [M+Na]+.
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Sꢀallyl 3,4ꢀdihydroxybenzothioate (10). 10 was synthesized from 25 (130 mg, 0.296 mmol).
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Elution with dichloromethane/methanol (9.8:0.2) afforded 10 as a dark oil: 15 mg (20%); H NMR
(400 MHz, CD3OD) δ 7.41ꢀ7.38 (m, 2H), 6.81 (d, J=8.4 Hz, 1H), 5.91ꢀ5.82 (m, 1H), 5.27 (d,
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J=16.8 Hz, 1H), 5.09 (d, J=9.6 Hz, 1H), 3.66 (d, J=6.8 Hz, 2H). C NMR (100 MHz, CD3OD) δ
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191.30, 152.35, 146.49, 135.01, 130.24, 121.53, 117.92, 115.92, 114.96, 32.38. MS [ESI+] m/z
209 [M+1]+.
Sꢀpropyl (E)ꢀ3ꢀ(3,4ꢀdihydroxyphenyl)propꢀ2ꢀenethioate (11). 11 was synthesized from 26 (430
mg, 0.921 mmol). Elution with dichloromethane/methanol (9.6:0.4) afforded 11 as a pale oil: 63 mg
(30%); 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J=15.6 Hz, 1H), 7.10 (s, 1H), 7.02 (d, J=8 Hz, 1H),
6.88 (d, J=8 Hz, 1H), 6.56 (d, J=15.6 Hz, 1H), 2.98 (t, J=7.2 Hz, 2H), 1.69ꢀ1.64 (m, 2H), 1.00 (t,
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J=7.2 Hz, 3H). C NMR (100 MHz, CDCl3) δ 191.80, 146.88, 144.02, 140.92, 127.25, 122.98 (2
C), 115.74, 114.93, 31.13, 23.08, 13.52. MS [ESIꢀ] m/z 237 [Mꢀ1]ꢀ.
Sꢀpropyl 3ꢀ(3,4ꢀdihydroxyphenyl)propanethioate (12). 12 was synthesized from 27 (119 mg,
0.25 mmol). Elution with petroleum ether/ethyl acetate (5.5:4.5) afforded 12 as a waxy oil: 41 mg
(66%); 1H NMR (400 MHz, CDCl3) δ 6.76 (d, J=8.4 Hz, 1H), 6.68 (d, J=2 Hz 1H),6.59 (dd, 1J=8.4,
13
2J=2 Hz 1H), 2.86ꢀ2.81 (m, 6H), 1.58ꢀ1.57 (m, 2H), 0.94 (t, J=7.2 Hz, 3H). C NMR (100 MHz,
CDCl3) δ 200.23, 143.60, 142.07, 132.89, 120.61, 115.44, 115.39, 45.71, 30.92, 30.88, 22.82,
13.27. MS [ESI+] m/z 263 [M+Na]+.
Sꢀpropyl 3ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)propanethioate (13). 13 was synthesized from 28 (70
mg, 0.189 mmol). Elution with petroleum ether/ethyl acetate (5.5:4.5) afforded 13 as a pale oil: 45
1
mg (94%); H NMR (400 MHz, CDCl3) δ 6.83ꢀ6.81 (m, 1H), 6.68 (s, 1H), 6.66 (d, J=2 Hz, 1 H),
13
3.86 (s, 3H), 2.92ꢀ2.80 (m, 6H), 1.61ꢀ1.56 (m, 2 H), 0.95 (t, J=8 Hz, 3H). C NMR (100 MHz,
CDCl3) δ 199.14, 146.62, 144.23, 132.25, 121.10, 114.56, 111.14, 56.07, 46.15, 31,47, 30.10,
23.16, 13.51. MS [ESI+] (m/z) 277 [M+Na]+.
Sample preparation for Aβ42 selfꢀaggregation
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1,1,1,3,3,3ꢀHexafluoroꢀ2ꢀpropanol (HFIP)ꢀpretreated Aβ42 samples (Bachem AG, Switzerland)
were resolubilized with a CH3CN/0.3 mM Na2CO3/250 mM NaOH (48.4:48.4:3.2) mixture to have
a stable stock solution ([Aβ42]=500 ꢁM).20, 40 Tested inhibitors were dissolved in MeOH and diluted
in the assay buffer. Experiments were performed by incubating the peptide diluted in 10 mM
phosphate buffer (pH 8.0) containing 10 mM NaCl at 30°C (Thermomixer Comfort, Eppendorf,
Italy) for 24 h (final Aβ concentration=50 ꢁM) with and without inhibitor.
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Inhibition of Aβ42 selfꢀaggregation: ThT assay. Inhibition studies were performed by incubating
Aβ42 samples in the assay conditions reported above, with and without tested inhibitors. Inhibitors
were first screened at 50 ꢀM in a 1:1 ratio with Aβ42. To quantify amyloid fibril formation, the ThT
fluorescence method was used.41 After incubation, samples were diluted to a final volume of 2.0 mL
with 50 mM glycineꢀNaOH buffer (pH = 8.5) containing 1.5 ꢀM ThT. A 300ꢀsecondsꢀtime scan of
fluorescence intensity was carried out (λexc = 446 nm; λem = 490 nm), and values at plateau were
averaged after subtracting the background fluorescence of 1.5 ꢀM ThT solution. Blanks containing
inhibitor and ThT were also prepared and evaluated to account for quenching and fluorescence
properties. The fluorescence intensities were compared and the % inhibition was calculated. For
compounds 7, 9, 11 and 12, the IC50 value was also determined. To this aim four increasing
concentrations were tested. IC50 value was obtained from the % inhibition vs log[inhibitor] plot.
Reagents for cellular experiments. All culture media, supplements and Foetal Bovine Serum (FBS)
were obtained from Euroclone (Life Science Division, Milan, Italy). Electrophoresis reagents were
obtained from BioꢀRad (Hercules, CA, USA). All other reagents were of the highest grade available
and were purchased from Merck KGaA (Darmstadt, Germany) unless otherwise indicated. Aβ42
was solubilized in dimethyl sulfoxide (DMSO) at the concentration of 100 ꢁM and frozen in stock
aliquots. All the experiments performed with Aβ were made in 1% of serum. H2O2 was diluted to
working concentration (300 ꢁM) in phosphate buffer saline (PBS) at the moment of use.
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Cell cultures. Human neuroblastoma SHꢀSY5Y cell line from European Collection of Cell Cultures
(ECACC No. 94030304) were cultured in medium with equal amount of Eagle’s minimum essential
medium and Nutrient Mixture Ham’s Fꢀ12, supplemented with 10% foetal bovine serum, glutamine
(2mM), penicillin/streptomycin, nonꢀessential aminoacids at 37 °C in 5% CO2/95% air.
Cell viability. The mitochondrial dehydrogenase activity that reduces 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ
2,5ꢀdiphenylꢀtetrazolium bromide (MTT, Sigma, St Louis, MO, USA) was used to determine
cellular viability, in a quantitative colorimetric assay. At day 0 SHꢀSY5Y cells were plated at a
density of 2.5x104 viable cells per well in 96ꢀwell plates. After treatment, according to the
experimental setting, cells were exposed to an MTT solution in PBS (1 mg/mL). Following 4 h
incubation with MTT and treatment with SDS for 24 h, cell viability reduction was quantified by
using a Synergy HT multiꢀdetection microplate reader (BioꢀTek).
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Measurement of intracellular ROS. DCFHꢀDA (Merck KGaA, Darmstadt, Germany) was used to
estimate intracellular ROS. Two different experimental settings were performed. First, cells were
loaded with 25 ꢁM DCFHꢀDA for 45 min. After centrifugation DCFHꢀDA was removed and cells
were exposed to 5 µM of compounds 1ꢀ13 and 300 µM H2O2. Alternatively, cells (2 × 104
cells/well) were pretreated to compounds 1ꢀ13 (5 ꢁM) for 24 h and then loaded with 25 ꢁM DCFHꢀ
DA at 37°C for 45 min. DCFHꢀDA was removed after centrifuge and cells were resuspended in
PBS and then exposed to 300 ꢁM H2O2. The results were visualized using Synergy HT multiꢀ
detection microplate reader (BioTek) with excitation and emission wavelengths of 485 nm and 530
nm, respectively.
Immunodetection of Nrf2 and NQOꢀ1. Cell monolayers were washed twice with ice cold PBS,
lysed on the tissue culture dish by addition of iceꢀcold lysis buffer (50 mM Tris/HCl pH 7.4, 150
mM NaCl, 50 mM EDTA, 0.2 mM 4ꢀ(2ꢀaminoethyl) benzenesulfonyl fluoride hydrochloride
(AEBSF), 20 µg/mL leupeptin, 25 µg/mL aprotinin, 0,5 µg/mL pepstatin A and 1% Triton Xꢀ100)
and an aliquot was used for protein analysis with the Bradford assay, for protein quantification. Cell
lysates were diluited in sample buffer (62.5 mM Tris/HCl pH 6.8, 2% SDS, 10% glycerol, 50 mM
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dithiothreitol, 0.1% Bromophenol blue) and subjected to Western blot analysis. Proteins were
subjected to SDSꢀPAGE (10%) and then transferred onto PVDF membrane 0,45 µm (Merck KGaA
Darmstadt, Germany). The membrane was blocked for 1 h with 5% BSA in Trisꢀbuffered saline
containing 0.1% Tween 20 (TBST). Membranes were immunoblotted with the rabbit anti human
Nrf2 polyclonal antibody (at 1:2000 in 5% BSA) and the mouse antiꢀNQOꢀ1 monoclonal antibody
(1:1000 in 5% BSA) (Novus, Bioꢀtechne Minneapolis, USA). The detection was carried out by
incubation with horseradish peroxidase conjugated goat antiꢀrabbit IgG for Nrfꢀ2 or rabbit antiꢀ
mouse for NQOꢀ1 (1:5000 dilution in 5% BSA, from Merck KGaA Darmstadt, Germany) for 1 h.
The blots were then washed extensively and the proteins of interest were visualized using an
enhanced chemiluminescent method (Pierce, Rockford, IL, USA). Tubulin was also performed as a
normal control of proteins (Merck KGaA Darmstadt, Germany).
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Subcellular fractionation for Nrf2 nuclear translocation. 5×106 SHꢀSY5Y cells were seeded in
100mm2 dishes and treated for 3 h with 5 ꢀM compounds 1, 5, 7, 9, 11, 13; afterwards the medium
was removed, and cells were washed twice with iceꢀcold PBS. Cells were subsequently
homogenized 15 times using a glassꢀglass dounce homogenizer in 0.32 M sucrose buffered with 20
mM TrisꢀHCl (pH 7.4) containing 2 mM EDTA, 0,5 mM EGTA, 50 mM βꢀmercaptoethanol, and
20ꢀg/mL leupeptin, apotrinin and pepstatin. The homogenate was centrifuged at 300×g for 5 min to
obtain the nuclear fraction. An aliquot of the nuclear fraction was used for protein assay by the
Bradford method, whereas the remaining was boiled for 5 min after dilution with sample buffer and
subjected to polyacrylamide gel electrophoresis and immunoblotting as described.
Densitometry and statistics. All the experiments, unless specified, were performed at least three
times. Following acquisition of the Western blot image through an AGFA scanner and analysis by
means of the Image 1.47 program (Wayne Rasband, NIH, Research Services Branch, NIMH,
Bethesda, MD, USA), the relative densities of the bands were expressed as arbitrary units and
normalized to data obtained from control sample run under the same conditions. Data were
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analyzed by analysis of variance (ANOVA) followed when significant by an appropriate post hoc
comparison test as indicated in figure legend. The reported data are expressed as means ± SEM of at
least three independent experiments. A p value < 0.05 was considered statistically significant.
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AUTHOR INFORMATION
ɸ
The authors contributed equally to the work.
Corresponding Author
* Michela Rosini: michela.rosini@unibo.it, Phone/Fax: +39 051 2099722/34.
Author Contributions
M.R., C.L., E.S. participated in research design; M.R., C.L., M.B. and E.S. analysed results and
wrote the manuscript with input from all coauthors; M.R., C.L., M.B., A.M., M.R., E.S. conceived
the methodology, designed experiments and analysed data. E.S., R.C. and C.M. afforded synthesis
and characterization of compounds; M.B. ran experiments on amyloid aggregation; M.M.S.
performed cellular experiments.
Funding Sources
This work was supported by the University of Bologna (grants from the RFO).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
AD, Alzheimer’s disease; Aβ, amyloidꢀβ peptide; ANOVA, analysis of variance; DCFHꢀDA,
dichlorodihydrofluorescein diacetate; DCC, N,N'ꢀdicyclohexylcarbodiimide; DMAP, 4ꢀ
dimethylaminopyridine; DMF, N,Nꢀdimethylformamide; DMSO, dimethyl sulfoxide; ESIꢀMS,
electrospray ionisation mass spectrometry; FBS, Foetal Bovine Serum, GST, glutathione Sꢀ
transferase; HPLC, high performance liquid chromatography; HOꢀ1, heme oxygenaseꢀ1; Keap 1,
Kelchꢀlike ECHꢀassociated protein 1; MTT, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenylꢀtetrazolium
bromide; NMR, nuclear magnetic resonance; NQO1, NAD(P)H:quinone reductase; Nrf2, nuclear
factor (erythroidꢀderived 2)ꢀlike 2; PBS, phosphate buffer saline; ppm, parts per millions; SAR,
structureꢀactivity relationships; TBDMS, tertꢀbutyldimethylsilyl; TBAF, tetrabutylammonium
fluoride; TBDMSꢀCl, tertꢀbutyldimethylsilyl chloride; TBST, Trisꢀbuffered saline containing 0.1%
Tween 20; THF, tetrahydrofuran; TLC, thin layer chromatography; TMS, tetramethylsilane; ThT,
thioflavin T; UV, ultraviolet.
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