Electrophilic Triterpenoid Enones: A Comparative Thiol-Trapping and Bioactivity Study

Article abstract of DOI:10.1021/acs.jnatprod.7b00271

Bardoxolone methyl (1) is the quintessential member of triterpenoid cyanoacrylates, an emerging class of bioactive compounds capable of transient covalent binding to thiols. The mechanistic basis for this unusual "pulsed reactivity" profile and the mode of its biological translation are unknown. To provide clues on these issues, a series of Δ¹-dehydrooleanolates bearing an electron-withdrawing group at C-2 (7a-m) were prepared from oleanolic acid (3a) and comparatively investigated in terms of reactivity with thiols and bioactivity against a series of electrophile-sensitive transcription factors (Nrf2, NF-κB, STAT3). The emerging picture suggests that the triterpenoid scaffold sharply decreases the reactivity of the enone system by steric encumbrance and that only strongly electrophilic and sterically undemanding substituents such as a cyanide or a carboxylate group can re-establish Michael reactivity, albeit in a transient way for the cyanide group. In general, a substantial dissection between the thiol-trapping ability and the modulation of biological end-points sensitive to thiol alkylation was observed, highlighting the role of shape complementarity for the activity of triterpenoid thia-Michael acceptors.

Full text of DOI:10.1021/acs.jnatprod.7b00271

Article
pubs.acs.org/jnp
Electrophilic Triterpenoid Enones: A Comparative Thiol-Trapping and
Bioactivity Study
Danilo Del Prete,† Orazio Taglialatela-Scafati,‡ Alberto Minassi,† Carmina Sirignano,‡ Cristina Cruz,§
Maria L. Bellido,§ Eduardo Munoz,*,# and Giovanni Appendino*,†
̃
†Dipartimento di Scienze del Farmaco, Universita
̀
del Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy
di Napoli Federico II, Via Montesano 49, 80131 Napoli, Italy
§VivaCell Biotechnology Espana, Parque Científico Tecnolog
ico de Cordoba, 14014 Cordoba, Spain
doba, Reina Sofía University Hospital, Department of Cell Biology, Physiology and
doba, Avenida Menendez Pidal s/n, 14004 Cordoba, Spain
‡Dipartimento di Farmacia, Universita
̀
́
́
́
̃
#Maimonides Biomedical Research Institute of Cor
Immunology, University of Cor
́
́
́
́
S
* Supporting Information
ABSTRACT: Bardoxolone methyl (1) is the quintessential
member of triterpenoid cyanoacrylates, an emerging class of
bioactive compounds capable of transient covalent binding to
thiols. The mechanistic basis for this unusual “pulsed
reactivity” profile and the mode of its biological translation
are unknown. To provide clues on these issues, a series of Δ1-
dehydrooleanolates bearing an electron-withdrawing group at
C-2 (7a−m) were prepared from oleanolic acid (3a) and
comparatively investigated in terms of reactivity with thiols
and bioactivity against a series of electrophile-sensitive
transcription factors (Nrf2, NF-κB, STAT3). The emerging
picture suggests that the triterpenoid scaffold sharply decreases the reactivity of the enone system by steric encumbrance and that
only strongly electrophilic and sterically undemanding substituents such as a cyanide or a carboxylate group can re-establish
Michael reactivity, albeit in a transient way for the cyanide group. In general, a substantial dissection between the thiol-trapping
ability and the modulation of biological end-points sensitive to thiol alkylation was observed, highlighting the role of shape
complementarity for the activity of triterpenoid thia-Michael acceptors.
nucleophilic species is “virtual”, only detectable by spectro-
scopic measurements (NMR, UV) and not backed up by actual
isolation of the adducts.2 Since cyanoenones are strongly
electrophilic agents and the parent system (2-cyano-2-cyclo-
hexenone, 2) forms stable adducts with thiols (vide infra), the
transient nature of the reaction when the electrophilic system is
embedded into a triterpenoid scaffold is seemingly related to a
Gestalt (shape) effect, as suggested by the presence of overall
five substituents on the three tetrahedral carbons of the
cyclohexenone A-ring and the steric size7 of the C-1 sulfur
substituent in the adduct. To shed light on this issue and
identify alternative groups capable of inducing Michael
reactivity in ring A triterpenoid enones, we have investigated
the reaction of thiols with a series of 2-substituted Δ1-oleanane
triterpenoids related to bardoxolone methyl using the cyste-
amine assay, an NMR method to study thia-Michael reactivity
based on the reversal of transient addition upon a solvent
switch between DMSO and CHCl3.8 General acid−base
catalysis from the adjacent amino group renders the thiol
group of cysteamine a good mimic of a cysteine residue in a
ardoxolone methyl (CDDO-Me, RTA402, 1) is the
1,2
Bquintessential member of triterpenoid cyanoenones,
an
interesting class of bioactive compounds that has revitalized
interest in pentacyclic triterpenoids, a major group of plant
phytochemicals.3 After failing a phase-3 study for chronic
kidney disease because of cardiovascular side-effects,4 bardox-
olone methyl is currently undergoing phase-2 clinical studies
for the management of pulmonary arterial hypertension, a
severe condition difficult to manage with existing drugs.4 The
mechanism of action of triterpenoid cyanoenones is seemingly
related to their capacity to transiently trap reactive cysteine
residues,2 with the triterpenoid scaffold providing shape
complementarity and the cyanoenone moiety covalently
reinforcing this interaction.1,2 This dock-and-lock mechanism
is well precedented within bioactive compounds, underlying,
for instance, the mechanism of action of penicillin and
aspirin.5,6 On the other hand, although all thia-Michael
reactions are in principle reversible, the interaction of
triterpenoid cyanoenones with thiols is transient and expected
to translate into a pulsed, rather than permanent target
modulation, not unlike the one associated with noncovalent
ligands.2,6 Thus, triterpenoid cyanoenones do not form stable
and isolable adducts with thiols, and their reaction with
Received: March 28, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
Article
protein functional site,9 resulting in a rapid addition in dipolar
solvents such as DMSO. On the other hand, virtually no
reaction occurs in apolar solvents such as CHCl3, where the
zwitterionic form of cysteamine and the delicate network of
hydrogen bonding required by the organo-catalytic mechanism
do not benefit from solvent stabilization.8 To evaluate if the
reactivity data from the cysteamine assay could be translated in
terms of bioactivity modulation, we have complemented this
chemical end-point with biological assays on thiol-sensitive
important medicinal chemistry targets such as the transcription
factors Nrf2, NF-κB, and STAT3.1
lower potency than the triterpenoid scaffold of bardoxolone
methyl (1), which carries a C-12 carbonyl and Δ9(11)
-
unsaturation.2 Nevertheless, the cyanoacrylates from the two
series (1 and 7a) show the same profile of thia-Michael
reaction,2 and comparative, rather than absolute, bioactivity
data were relevant for this study.
Starting from methyl dehydrooleanolate (3b),10 the Δ1-
dehydro derivative 7b was obtained by dehydrogenation with
DDQ (Scheme 2). This enone was separately reacted with
Scheme 2. Synthesis of the 2-Substituted Oleanolylenones
a
7b−k from 3b
RESULTS AND DISCUSSION
■
Oleanolic acid (3a) was used as a triterpenoid scaffold for this
study. After conversion to its more soluble methyl ester and
oxidation to 3b, it was subjected to the classic four-step
sequence for the synthesis of triterpenoid cyanoenones
(formylation, reaction with hydroxylamine, iosoxazole fragmen-
tation, and dehydrogenation with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone, DDQ),10 eventually affording 7a (Scheme 1). In
terms of cyanoacrylate-associated bioactivity, the ring C
functionalization of oleanolic acid (3a) leads to biologically
a
Conditions: (a) Br2, HBr, HOAc, 35°C, 68%; (b) I2, DMAP, Pyr,
CHCl3, 90 °C, 79%; (c) Pd(PPh3)4, NaOMe, p-cyanophenylboronic
acid, toluene, 80 °C, 10%; (d) Pd(PPh3)4, NaOMe, phenylboronic
acid, toluene, 80 °C, quant.; (e) Pd(PPh3)4, NaOMe, p-methox-
yphenylboronic acid, toluene, 80 °C, quant.; (f) Pd(PPh3)4, TEA, CuI,
phenylacetylene, EtOAc, 60 °C, 73%; (g) ethyl acrylate, Pd(OAc)2,
TPP, TEA,CH3CN, 80 °C, quant.; (h) DDQ, toluene, quant.; (i)
EDC, DMAP, MeOH, 98%.
a
Scheme 1. Synthesis of the Oleanolylcyanoenone 7a
bromine and iodine to afford, by an addition−elimination
mechanism, the corresponding 2-halo derivatives 7c and 7d,
respectively. The 2-iodoenone (7d) served as a good substrate
for palladium-mediated coupling. Suzuki reaction with p-
cyanophenylboronic acid afforded the phenylogous cyanoenone
7e, and the 2-phenyl- and 2-(p-methoxyphenyl)enones 7f and
7g were similarly prepared from the corresponding boronic
acids. Sonogashira coupling with 2-phenylacetylene yielded the
acetylenic enone 7h, while Heck coupling with ethyl acrylate
afforded the cross-conjugated keto-ester 7i.
Alternatively, methyl dehydrooleanolate (3b) was carboxy-
lated with the Stiles reagent (methyl magnesium carbonate)11
to afford an unstable β-ketoacid, which was immediately
dehydrogenated with DDQ12 to afford the stable enone 7j and
subsequently esterified to the methyl ester 7k. The 2-formyl
derivative 7l was prepared via 4 by DDQ-mediated
a
Conditions: (a) HCOOMe, NaOMe, benzene, 95%; (b) NH2OH·
HCl, aq EtOH; (c) NaOMe, Et2O, MeOH (85% from 5); (d) DDQ,
benzene, 60%.
B
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dehydrogenation, while the enol ester 7m was prepared from
chromic oxidation of maslinic acid (3c) followed by acetylation.
To investigate their reactivity with thiols, the 2-substituted
triterpenoid enones were then subjected to the cysteamine
assay.8 This NMR-based assay capitalizes on solvent-related
differences in the reactivity of cysteamine with electron-poor
double bonds to identify transient acceptors. Since a rapid
reaction takes place in DMSO-d6 but no reaction occurs in
CDCl3, dilution of the DMSO-d6 reaction mixture with CDCl3
will regenerate the starting olefin in the case of a reversible
addition, but will leave the adduct unscathed with an
irreversible addition.8 2-Cyclohexenone gave a nontransient
addition with cysteamine, but no reaction occurred in 7b,
where this structural element is part of the triterpenoid
framework. Also 2-cyano-2-cyclohexenone (2) gave a non-
transient addition with cysteamine, but the corresponding
triterpenoid analogue 7a reacted only transiently. These
observations suggest that the bulky triterpenoid scaffold
interferes with the thia-Michael addition, completely deactivat-
ing the parent enone 7b and downgrading the reactivity of the
cyanoenone 7a to the realm of transiency. No reactivity was
also observed in the phenylogous cyanoenone 7e, in the two
other 2-phenylsubstituted enones 7f and 7g, and in the 2-
phenylethynyl derivative 7h. The 2-acryloyl derivative 7i
reacted with cysteamine in a nontransient way, but at the
acryloyl double bond and not at the Δ1-double bond of the
triterpenoid scaffold. An interesting behavior was observed with
the 2-halosubstituted enones, since their thia-Michael adducts
underwent reductive dehalogenation to the Michael-unreactive
unsubstituted Δ1-enone 7b. The 2-halo derivatives did not react
with the simple odorless thiol 1-dodecanethiol, suggesting the
involvement of the amino group of cysteamine, possibly via the
mechanism outlined in Scheme 3. Finally, the 2-formyl enone
by running competition experiments, we could establish that
the 2-carboxylate 7j was more reactive than the cyanoenone 7a,
which showed similar reactivity in the assay to the 2-haloenones
7b and 7c, while the acrylate 7i was significantly less reactive.
The high reactivity of the 2-carboxylate 7j might be related to a
facilitation of the addition by a network of hydrogen bondings,
as depicted in Scheme 4. The role of general acid catalysis in
the reaction is consistent with the observation that methylation
of the carboxylate quenched the Michael reactivity, as shown by
7k.
Scheme 4. General Acid Catalysis in the Addition of
Cysteamine to the 2-Carboxyenone Moiety of 7j
The Δ1-enones 7a−l were comparatively evaluated with
bardoxolone methyl (1) for their capacity to activate the
transcription factor Nrf2, a major target of this compound,1,2
and for the inhibition of two further transcription factors
sensitive to thiol trapping (NF-κB and STAT3) (Table 1). The
Table 1. Biological Activities of Oleanolic Acid Derivatives
on Specific Targets
IC50
efficacy NRF2 (IRA potency EC50
STAT3
IC50 NF-
κB (μM)
a
compound
coefficient)
Nrf2 (μM)
(μM)
1
40.94
0.06
2.38
1.20
3a
3b
4
−(>50)
−(>25)
−(>25)
−(>25)
−(>25)
1.097
−(>50)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>50)
−(>25)
−(>25)
−(>25)
−(>25)
15.27
Scheme 3. Possible Mechanism for the Cysteamine-Induced
Dehalogenation of 7c (X = Br) and 7d (X = I)
5
6
7a
7b
7c
7d
7e
7f
7g
7h
7i
1.69
−(>25)
11.48
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
7.69
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
−(>25)
b
b
7l
ND
ND
7m
7j
−(>25)
2.45
−(>25)
25.74
−(>25)
−(>25)
−(>25)
0.41
7k
−(>25)
−(>25)
a
The IRA (intrinsic relative activity) was calculated relative to tert-
7l reacted with cysteamine to give, in a nontransient way, a
mixture of mono- and bis-thiol adducts, in accordance with
previous observations of α,β-unsaturated aldehydes,8 while the
enol acetate 7m behaved as an acylating reagent for the amino
group of cysteamine, affording the starting enolyzed α-
dicarbonyl (diosphenol).
Competition experiments where equimolar amounts of two
substrates were reacted with a substoichiometric amount of
cysteamine were carried out to establish a reactivity scale
between the compounds positive in the cysteamine assay. Since
all reactions were complete within the time frame of the assay
(ca. 5 min), a leveling effect was observed. On the other hand,
b
butylhydroquinone (TBHQ) (see Experimental Section). Toxic at
concentrations above 5 and 10 μM in the cell lines used in the assay.
function of these regulatory proteins is critically dependent on
the presence of a free cysteine that works as a veritable on/off
switch. For instance, inactive Nrf2 is retained in the cytoplasm
by Keap-1, which contains two critical cysteine moieties. Upon
interaction with reactive molecules, Keap-1 is degraded and
Nrf2 is translocated to the nucleus, where it activates Nrf2-
dependent genes.13 The NF-κB pathway is critically dependent
on the upstream kinase IKKβ, which hosts a loop-exposed
C
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cytsteine (Cys-179) sensitive to electrophilic compounds that
inhibit its kinase activity and the activation of the NF-κB
pathway.14 Finally, STAT3 also contains critical cysteine units,
whose alkylation prevents binding to DNA.15 The assays were
done on cultured cells transfected with the appropriate protein
gene [human keratynocytes (HaCaT-ARE-Luc) for Nrf2,
mouse fibroblasts (NIH-3T3-KBF-Luc) for NF-κB, and
human cervix carcinoma (HeLa-STGAT-3-Luc) for STAT3]
using luciferase-based protocols (see Experimental Section for
further details). The cyanoenone 7a showed potency at low
estimated “residence time” of ca. 14 min (Scheme 5).18 This
iterative and pulsed cycle of activation and inhibition is
Scheme 5. Transient Covalent Trapping of the Serine
Residue of Lactamase (ROH) by Avibactam
micromolar concentration in the activation of Nrf2 (EC50
1.10 μM), more than 1 order of magnitude lower, however,
than bardoxolone methyl, an ultrapotent activator (EC50
=
=
0.060 μM).2 Within the compounds investigated, only the 2-
bromoenone 7c and the 2-carboxyenone 7j were active in
assays of Nrf2 activation. Overall, a substantial dissection
between thiol trapping and activation of Nrf2 was observed in
terms of thiophilicy and mode of addition of the substrates.
Thus, both the 2-bromoenone 7c and the 2-iodoenone 7d
could covalently modify thiol groups, but only the former was
active, while the 2-carboxyenone 7j and its vinylogous ester 7i
were both nontransient Michael acceptors, but only 7j could
activate Nrf2. All other substrates were unable to react in vitro
with thiol groups and to modulate Nrf2 activity. Regarding NF-
κB inhibition, only bardoxolone methyl (1) was significantly
active (IC50 = 2.38 μM), and this compound was also the most
fundamentally distinct from the kinetically irreversible profile of
the other inhibitors9 and might be involved in the superior
clinical activity of avibactam compared to the other lactamase
inhibitors.19
The reversibility of action of avibactam has been related to
the lower strain, and therefore easier regeneration, of its
imidazolinone-active moiety compared to the β-lactam moiety
of the other inhibitors (Scheme 5).18 The data on triterpenoid
cyanoenones suggest that the reversibility of their thia-Michael
addition is mainly due to an increased steric congestion on the
triterpenoid A-ring. Interestingly, replacement of the 2-cyano
group with a 2-halo group changes the reactivity mode, overall
resulting in the reductive dehalogenation of the triterpenoid
probe and the irreversible covalent modification of the
nucleophilic partner. This peculiar reactivity profile is worth
investigating also in a different molecular framework aimed at
the modulation of macromolecules according to a “lock and
dock” strategy.20
Finally, by highlighting the critical relevance of shape
complementarity and pulse reactivity for bioactivity, our
observations on triterpenoid cyanoacrylates emphasize the
difficulty of sorting out compounds into good leads and PAINS
(pan-assay interference compounds) based solely on test tube
data on reactivity and tendency to aggregate,21 which can both
be critical for binding to biological macromolecules and elicit
activity of clinical relevance.20,21
potent inhibitor of γ-IFN-induced STAT3 activation (IC50
=
1.20 μM), although also the cyanoenone 7a and the 2-
formylenone 7l showed activity in this assay (IC50 = 15.27 and
7.69 μM, respectively). Collectively, the bioactivity data support
the view that, within electrophilic triterpenoids, shape
complementarity has a critical role for the biological translation
of covalent reactivity, with thiol-trapping capacity being
necessary but not sufficient for bioactivity against a specific
target. This is in accordance with the observation that
bardoxolone methyl (1) is at least one order of magnitude
more potent than its ring A analogue 7a for the modulation of
the three thiol-trapping-sensitive end-points investigated.
Oleanolic acid (3a) is the archetypal TGR5 dietary agonist,16
and the activity of compounds 7a−m and the intermediates for
their synthesis (3b,c−6) was also investigated for the activation
of this end-point. While confirming the activity of oleanolic acid
in the assay (EC50 = 18.90 μM), our data also evidenced the
detrimental effect of changes on ring A and the C-28
carboxylate, since all compounds, including maslinic acid
(3b), were inactive in the assay. Similar results have been
reported in the betulinic acid series.17
Cysteine is one of the least abundant protein residues, and,
due to a unique combination of metal affinity, redox properties,
and reactivity with electrophiles, it is often located at functional
sites, acting as a veritable sensor of the local environment.9 The
selective manipulation of these “active” cysteine residues has
therefore a profound effect on the homeostatic response,
although its phenotypical translation is difficult to predict, as is
the distinction between its pulsed rather than continuous
modulation. Reversible covalent modification is not limited to
thia-Michael acceptors, but has been reported also for the β-
lactamases inhibitor avibactam (8), a hydroxy-trapping agent.18
Unlike the adducts from irreversible inhibitors of these serine
proteases (clavulanic acid, tazobactam, sulbactam), which are
cleaved by hydrolytic turnover and deactivated, the β-
lactamase-avibactam adduct (9) undergoes spontaneous
lactamization, with regeneration of the inhibitor after an
EXPERIMENTAL SECTION
■
General Experimental Procedures. 1H (500 MHz) and 13C
(125 MHz) NMR spectra were measured on a Varian INOVA
spectrometer. Chemical shifts were referenced to the residual solvent
signal (CDCl3: δH = 7.26, δC = 77.0; DMSO-d6: δH = 2.50).
Homonuclear 1H connectivities were determined by the COSY
experiment. One-bond heteronuclear 1H−13C connectivities were
determined with the HSQC experiment. Two- and three-bond
1H−13C connectivities were determined by 2D HMBC experiments
optimized for 2,3J = 9 Hz. Spectra were obtained on an Avatar 370 FT-
IR Thermo Nicolet. Low- and high-resolution ESIMS spectra were
obtained on an LTQ OrbitrapXL (Thermo Scientific) mass
spectrometer. Silica gel 60 (63−200 mesh) used for gravity column
chromatography (GCC) was purchased from Merck. Reactions were
monitored by TLC on Merck 60 F254 silica gel (0.25 mm) plates and
Macherey-Nagel ALUGRAM neutral alumina (0.20 mm) plates that
were visualized by UV inspection (254 and 365 nm) and/or staining
with 5% H2SO4 in EtOH and heating. Organic phases were dried with
Na2SO4 before evaporation. Chemical reagents and solvents were from
Aldrich. Oleanolic acid and maslinic acid were provided by Vivacell
Spain. Petroleum ether with a boiling point of 40−60 °C was used.
Cysteamine Assay. In an NMR tube, an exact amount of substrate
(ca. 5 mg) was dissolved in 500 μL of dry DMSO-d6, and the 1H NMR
spectrum was recorded. Two equivalents of cysteamine was then
added, and the spectrum was immediately recorded, with acquisition
D
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finishing within 5 min from the addition. The reaction was typically
monitored by observing the disappearance of the deshielded (>7 ppm)
signal of H-1. To test the reversibility of the addition, the sample was
diluted 1:10 with CDCl3, and the spectrum was recorded again.
Reversion was evaluated by comparing this spectrum with an original
spectrum of the product under investigation in CDCl3. All competition
assays were carried out by reacting a ca. equimolar solution of a pair of
Michael acceptors with a substoichiometric amount (60−80% of the
theoretical amount) of cysteamine and registering the spectrum within
5 min from the addition.
Methyl 3-Dehydrooleanoate (3b). To a solution of oleanolic acid
(3a, 6.00 g, 13.13 mmol) in dimethylformamide (100 mL) were slowly
added K2CO3 (3.61 g, 26.27 mmol; 2 molar equiv) and iodomethane
(817 μL, 13.13 mmol; 1 molar equiv) under stirring at 0 °C (ice bath).
The reaction mixture was stirred at room temperature for 24 h and
then worked up by dilution with brine and extraction with EtOAc. The
organic phase was dried, filtered, and evaporated, affording a white
powder (6.00 g, 97%) of methyl oleanolate. The latter (6.00 g, 12.75
mmol) was dissolved in acetone (100 mL), and Jones reagent was
added dropwise at 0 °C. The reaction mixture was stirred for 1 h, until
its color had turned from orange to persistent green, and was then
worked up by dilution with brine, addition of a few drops of EtOH,
and extraction with EtOAc. The organic phase was dried, filtered, and
evaporated, affording a semicrystalline residue that was purified by
GCC on silica gel (petroleum ether/EtOAc, 9:1, → petroleum ether/
EtOAc, 8:2, as eluent) to yield 3b (6.0 g) as a white powder.10
Methyl 2-Formyl-3-dehydrooleanoate (4). To a stirred solution of
3b (250 mg, 0.426 mmol) in benzene (6 mL) were sequentially added
NaOMe (138 mg, 2.5 mmol; 6 molar equiv) and ethyl formate (796
μL, 3.41 mmol; 8 molar equiv) at room temperature. After 1 h, the
reaction was worked up by dilution with 5% HCl and extraction with
petroleum ether/Et2O, 2:1. The organic phase was dried, filtered, and
evaporated to give a white crystalline product (250 mg, quantitative
yield).22
ions): m/z 514 [M + Na]+, 1005 [2 M + Na]+; HR-ESIMS m/z
514.3299, calcd for C32H45NNaO3 514.3297.
Methyl 1,3-Bisdehydrooleanolate (7b). To a solution of 3b (2.0 g,
4.27 mmol) in THF (100 mL) was added DDQ (970 mg, 4.27 mmol;
1 molar equiv) at room temperature After stirring at 60 °C for 24 h,
the reaction was worked up by dilution with brine and extraction with
EtOAc. After drying and evaporation, the residue was purified by GCC
on silica gel (petroleum ether/EtOAc, 9:1, as eluent) to give 7b (white
solid, 1.79 g, 90%): IR νmax (KBr) 1726, 1672, 1463, 1379, 1262, 1199,
1125 cm−1; 1H NMR (CDCl3, 500 MHz) δH 7.03 (1H, d, J = 10.1 Hz,
H-1), 5.80 (1H, d, J = 10.1 Hz, H-2), 5.36 (1H, t, J = 3.5 Hz, H-12),
3.64 (3H, s, 28-OMe), 2.90 (1H, dd, J = 14.0, 4.0 Hz, H-18), 2.10−
1.10 (series of multiplets), 1.16 (3H, s, H-25), 1.15 (3H, s, H-24), 1.15
(3H, s, H-27), 1.09 (3H, s, H-23), 0.94 (3H, s, H-30), 0.91 (3H, s, H-
29), 0.82 (3H, s, H-26); 13C NMR (CDCl3) δC 205.5 (C-3), 178.4 (C-
28), 159.3 (C-1), 144.5 (C-13), 125.2 (C-2), 121.9 (C-12), 53.6 (C-
5), 51.8 (OCH3-28), 47.0 (C-17), 45.9 (C-19), 44.7 (C-4), 42.2 (C-
14), 42.0 (C-18), 41.7 (C-9), 40.3 (C-10), 39.7 (C-8), 34.1 (C-21),
33.3 (C-7), 32.7 (C-22), 32.5 (C-20), 30.9 (C-24), 28.0 (C-15), 27.9
(C-29), 26.0 (C-27), 23.8 (C-30), 23.5 (C-11), 23.2 (C-16), 21.8 (C-
23), 19.1, (C-6) 18.8 (C-26), 17.5 (C-25); ESIMS (positive ions) m/z
489 [M + Na]+, 955 [2 M + Na]+; HR-ESIMS m/z 514.3341, calcd for
C31H46NaO3 489.3345.
Methyl 2-Bromo-1,3-bisdehydrooleanolate (7c). To a solution of
7b (50 mg, 0.107 mmol) in HOAc (2 mL) were added dropwise HBr
(33%, 9 μL, 0.047 mmol, 0.44 equiv) and bromine (14 μL, 0.257
mmol, 2.4 equiv). After stirring at 35 °C for 3 h, the reaction was
worked up by dilution with 20% aqueous sodium thiosulfate (20 mL)
and extracted with EtOAc. The combined organic phases were washed
with saturated NaHCO3 (2 × 10 mL) and brine (1 × 10 mL) and
dried. After evaporation, the residue was purified by GCC (petroleum
ether/EtOAc, 99:1, as eluant) to give 7c (40 mg, 68%) as a yellowish
powder: IR νmax (KBr) 1783, 1724, 1685, 1460, 1432, 1386, 1312,
1
1259 cm−1; H NMR (CDCl3, 500 MHz) δH 7.41 (1H, s, H-1), 5.29
(1H, t, J = 3.5 Hz, H-12), 3.68 (3H, s, 28-OMe), 2.66−1.17 (series of
multiplets), 1.38 (3H, s, H-27), 1.22 (3H, s, H-24), 1.21 (3H, s, H-25),
1.15 (3H, s, H-23), 1.01 (3H, s, H-30), 0.96 (3H, s, H-29), 0.86 (3H,
s, H-26); 13C NMR (CDCl3, 125 MHz) δC 196.6 (C-3), 176.8 (C-28),
157.5 (C-1), 122.3 (C-2), 55.0, 52.4, 52.1, 49.5, 47.4, 46.5, 46.1 (C-
19), 43.0, 42.9, 42.8, 41.0, 40.5 (C-8), 40.2, 37.2, 35.9, 35.2, 33.5, 32.8,
31.6, 30.9 (C-20), 28.4 (C-24), 25.7, 23.1 (C-16), 22.0 (C-23), 19.0
(C-6), 18.4 (C-26), 17.7 (C-25); ESIMS (positive ions) m/z 567 and
569 [M + Na]+ in 1:1 ratio; HR-ESIMS m/z 567.2443, calcd for
C31H4579BrNaO3 567.2450.
Isoxazole 5. To a solution of 4 (250 mg, 0.50 mmol) in H2O/
EtOH (10:1, 15 mL) was added hydroxylamine hydrocloride (313 mg,
4.512 mmol, 9 molar equiv) at room temperature and under
continuous stirring. The mixture was stirred at room temperature
for 1 h and then worked up by dilution with brine and extraction with
EtOAc. The organic phase was dried with Na2SO4 and filtered, and the
solvent evaporated to give 5 as a white powder (250 mg, ca.
quantitative).23
Base-Induced Fragmentation of 5. To a cooled (ice bath) and
stirred 5% solution of 5 (300 mg, 0.61 mmol) in Et2O/MeOH (2:1, 30
mL) was added NaOMe (328 mg, 6.08 mmol; 10 molar equiv). After
stirring at room temperature for 1 h, the reaction was worked up by
dilution with HCl 5% and extraction with Et2O/CH2Cl2 (2:1). The
organic phase was dried, filtered, and evaporated to afford 6 as a white
powder (260 mg, 87%).24
Methyl 2-Cyano-1,3-bisdehydrooleanoate (7a). To a solution of 6
(100 mg, 0.20 mmol) in dry benzene (4 mL) was added DDQ (92 mg,
0.40 mmol, 2 molar equiv). After stirring at room temperature for 30
min, the reaction was worked up by dilution with brine, extracted with
EtOAc, dried, and evaporated. The residue was purified by GCC on
silica gel (petroleum ether/EtOAc, 9:1, → petroleum ether/EtOAc,
7:3, as eluent) to give 70 mg of 7a (70%) as a white powder: IR νmax
(KBr) 3528, 3281, 1723, 1684, 1455, 1386, 1362, 1262, 1191 cm−1; 1H
NMR (CDCl3, 500 MHz) δH 7.74 (1H, s, H-1), 5.36 (1H, t, J = 3.5
Hz, H-12), 3.64 (3H, s, 28-OMe), 2.91 (1H, dd, J = 14.0, 4.0 Hz, H-
18), 2.11 (1H, dd, J = 8.9, 3.5 Hz, H-11), 2.01−1.09 (series of
multiplets), 1.22 (3H, s, H-25), 1.21 (3H, s, H-24), 1.15 (3H, s, H-27),
1.14 (3H, s, H-23), 0.94 (3H, s, H-30), 0.92 (3H, s, H-29), 0.83 (3H,
s, H-26); 13C NMR (CDCl3, 125 MHz) δC 198.3 (C-3), 178.3 (C-28),
170.2 (C-1), 144.8 (C-13), 121.1 (C-12), 115.2 (CN-2), 114.0 (C-2),
52.8 (C-5), 51.8 (OCH3-28), 46.9 (C-17), 45.8 (C-19), 45.1 (C-4),
42.3 (C-14), 41.7 (C-18), 41.3 (C-9), 40.8 (C-10), 40.5 (C-8), 34.0
(C-21), 32.4 (C-7), 32.3 (C-22), 30.9 (C-20), 27.9 (C-24), 27.8 (C-
15), 27.0 (C-29), 26.0 (C-27), 23.8 (C-30), 23.4 (C-11), 23.1 (C-16),
21.8 (C-23), 18.9 (C-6), 18.1 (C-26), 17.6 (C-25); ESIMS (positive
Methyl 2-Iodo-1,3-bisdehydrooleanolate (7d). To a solution of 7b
(100 mg, 0.21 mmol) were added pyridine/CHCl3 (1:1 v/v, 2 mL), 4-
dimethylaminopyridine (DMAP, 6 mg, 0.043 mmol, 0.20 molar
equiv), and I2 (163 mg, 0.64 mmol, 3.0 molar equiv). After heating to
90 °C under N2 and in the dark for 24 h, the reaction was worked up
by dilution with EtOAc (50 mL). and sequential washing with 20%
sodium thiosulfate (10 mL), 1 N HCl (3 × 10 mL), saturated
NaHCO3 (10 mL), and brine (10 mL). After drying and evaporation,
7d was obtained as a pale yellow powder (100 mg, 79%): IR νmax
1
(KBr) 1727, 1680, 1462, 1384, 1321, 1261, 1195, 1164 cm−1; H
NMR (CDCl3, 500 MHz) δH 7.81 (1H, s, H-1), 5.36 (1H, t, J = 3.5
Hz, H-12), 3.64 (3H, s, 28-OMe), 2.91 (1H, dd, J = 14.0, 4.0 Hz, H-
18), 2.20−1.09 (series of multiplets), 1.19 (3H, s, H-25), 1.18 (3H, s,
H-24), 1.16 (3H, s, H-27), 1.15 (3H, s, H-23), 0.94 (3H, s, H-30),
0.91 (3H, s, H-29), 0.81 (3H, s, H-26); 13C NMR (CDCl3, 125 MHz)
δC 198.1 (C-3), 178.2 (C-28), 167.6 (C-1), 144.4 (C-13), 121.4 (C-
12), 101.5 (C-2), 53.2 (C-5), 51.6 (OCH3-28), 46.9 (C-17), 45.8 (C-
19), 45.5 (C-4), 42.1 (C-14), 41.7 (C-18), 41.5 (C-9), 41.3 (C-10),
40.1 (C-8), 34.2 (C-21), 32.7 (C-7), 32.2 (C-22), 30.7 (C-20), 28.9
(C-24), 27.6 (C-15), 27.0 (C-29), 25.8 (C-27), 23.6 (C-30), 23.4 (C-
11), 23.0 (C-16), 22.1 (C-23), 18.5 (C-6), 18.1 (C-26), 17.3 (C-25);
ESIMS (positive ions) m/z 615 [M + Na]+, 1207 [2 M + Na]+; HR-
ESIMS m/z 615.2307, calcd for C31H45INaO3 615.2311.
Methyl 2-(4-Cyanophenyl)-1,3-dehydrooleanolate (7e). To a
stirred solution of 7d (200 mg, 0.34 mmol) in toluene (8 mL) were
added NaOMe (18 mg, 0.337 mmol, 1 molar equiv), p-
E
DOI: 10.1021/acs.jnatprod.7b00271
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Journal of Natural Products
Article
cyanophenylboronic acid (50 mg, 0.34 mmol, 1 molar equiv), and a
catalytic amount of Pd(PPh3)4. After stirring under N2 at 80 °C for 24
h, the mixture was worked up by dilution with brine and extraction
with EtOAc. After drying and evaporation, the residue was purified by
GCC on neutral alumina (petroleum ether/EtOAc, 99:1 → 9:1, as
eluant) to give 20 mg (10%) of 7e as a white powder: IR νmax (KBr)
1726, 1672, 1604, 1511, 1462, 1388, 1361, 1247, 1178 cm−1; 1H NMR
(CDCl3, 500 MHz) δH 7.61 (2H, d, J = 8.2 Hz, H-3′/5′), 7.41 (2H, d,
J = 8.2 Hz, H-2′/6′), 7.16 (1H, s, H-1), 5.37 (1H, t, J = 3.5 Hz, H-12),
3.64 (3H, s, 28-OMe), 2.91 (1H, dd, J = 14.0, 4.0 Hz, H-18), 2.20−
1.09 (series of multiplets), 1.21 (3H, s, H-25), 1.21 (3H, s, H-24), 1.21
(3H, s, H-27), 1.17 (3H, s, H-23), 0.94 (3H, s, H-30), 0.90 (3H, s, H-
29), 0.86 (3H, s, H-26); 13C NMR (75 MHz, CDCl3) δ 200.3, 178.2,
144.2, 141.3, 140.1, 138.1, 132.3, 127.6, 123.2, 118.6, 111.8, 56.0, 48.3,
47.3, 46.6, 42.3, 41.9, 40.0, 39.4, 39.1, 37.7, 35.8, 34.6, 33.8, 33.3, 33.1,
31.4, 28.8, 28.3, 27.9, 26.6, 24.3, 24.1, 23.7, 19.0, 17.6; ESIMS (positive
ions) m/z 590 [M + Na]+; HR-ESIMS m/z 590.3615, calcd for
C38H49NNaO3 590.3610.
equiv), and Pd(PPh3)4 (29 mg, 0.025 mmol, 0.1 molar equiv). After
refluxing for 12 h, the reaction was worked up by dilution with brine
and extraction with EtOAc. The organic phase was dried and
evaporated, and the residue was purified by column chromatography
on alumina (petroleum ether/EtOAc, 9:1, as eluant) to give 105 mg
(73%) of 7h as a white powder: IR νmax (KBr) 1726, 1676, 1560, 1459,
1363, 1260, 1164, 1124 cm−1; 1H NMR (CDCl3, 500 MHz) δH 7.50−
7.35 (phenyl protons), 7.27 (1H, s, H-1), 5.37 (1H, t, J = 3.5 Hz, H-
12), 3.64 (3H, s, 28-OMe), 2.91 (1H, dd, J = 14.0, 4.0 Hz, H-18),
2.20−1.09 (series of multiplets), 1.21 (3H, s, H-25), 1.20 (3H, s, H-
24), 1.16 (3H, s, H-27), 1.16 (3H, s, H-23), 0.94 (3H, s, H-30), 0.91
(3H, s, H-29), 0.84 (3H, s, H-26); 13C NMR (CDCl3, 125 MHz) δC
201.5 (C-3), 178.2 (C-28), 162.5 (C-1), 144.3 (C-13), 137.3 (C-2),
131.8 (Ph), 128.3 (Ph), 128.2 (Ph), 121.6 (C-12), 120.9 (Ph), 91.3
(C-2′), 85.0 (C-1′), 52.8 (C-5), 51.6 (OCH3-28), 46.8 (C-17), 45.7
(C-19), 44.9 (C-4), 42.1 (C-14), 41.6 (C-18), 41.5 (C-9), 40.1 (C-
10), 39.8 (C-8), 33.9 (C-21), 33.1 (C-7), 32.3 (C-22), 32.1 (C-20),
30.7 (C-24), 28.4 (C-15), 27.6 (C-29), 25.8 (C-27), 23.6 (C-30), 23.4
(C-11), 23.0 (C-16), 21.8 (C-23), 19.0 (C-6), 18.5 (C-26), 17.4 (C-
25); ESIMS (positive ions) m/z 589 [M + Na]+, 1155 [2 M + Na]+;
HR-ESIMS m/z 589.3657, calcd for C39H50NaO3 589.3658.
Methyl 2-Phenyl-1,3-bisdehydrooleanolate (7f). To a stirred
solution of 7d (50 mg, 0.084 mmol) in toluene (4 mL) were added
NaOMe (4 mg, 0.084 mmol, 1 equiv), phenylboronic acid (11 mg,
0.084 mmol, 1 equiv), and catalytic Pd(PPh3)4. After stirring at 80 °C
for 2 h, the reaction was worked up by dilution with brine and
extraction with EtOAc. After drying and evaporation, the residue was
purified by GCC on silica gel (petroleum ether/EtOAc, 9:1, as eluant)
to give 50 mg (95%) of 7d as a white powder: IR νmax (KBr) 1726,
Methyl (E)-2-(2-Ethoxycarbonylethenyl)-1,3-bisdehydrooleano-
late (7i). To a stirred solution of 7d (20 mg, 0.034 mmol, 1 molar
equiv) in CH3CN (10 mL) were sequenatially added ethyl acrylate (17
μL, 0.17 mmol, 5 molar equiv), TEA (7 μL, 0.051 mmol, 1.5 molar
equiv), Pd(OAc)2 (0.76 mg, 0.0030 mmol, 0.1 molar equiv), and Ph3P
(1 mg, 0.003 mmol, 0.1 molar equiv). After stirring at 80 °C for 2 h,
the reaction was worked up by dilution with brine and extraction with
EtOAc. After drying and evaporation, the residue was purified by GCC
(petroleum ether/EtOAc, 9:1, as eluant) to give 20 mg (95%) of 7i as
a white powder: IR (KBr) νmax 1720, 1679, 1629, 1462, 1303, 1261,
1
1671, 1459, 1362, 1302, 1195, 1163 cm−1; H NMR (CDCl3, 500
MHz) δH 7.34−7.29 (5H, phenyl signals), 7.10 (1H, s, H-1), 5.36 (1H,
t, J = 3.5 Hz, H-12), 3.64 (3H, s, 28-OMe), 2.90 (1H, dd, J = 14.0, 4.0
Hz, H-18), 2.20−1.09 (series of multiplets), 1.21 (3H, s, H-25), 1.21
(3H, s, H-24), 1.20 (3H, s, H-23), 1.17 (3H, s, H-27), 0.94 (3H, s, H-
30), 0.90 (3H, s, H-29), 0.85 (3H, s, H-26); 13C NMR (CDCl3, 125
MHz) δC 204.2 (C-3), 178.2 (C-28), 155.5, 144.3 (C-13), 137.2,
136.2, 128.3 (C-2′/6′), 128.1 (C-3′/5′), 127.5 (C-4′), 121.8 (C-12),
52.8 (C-5), 51.6 (OCH3-28), 46.8 (C-17), 45.6 (C-19), 45.2 (C-4),
42.1 (C-14), 42.0 (C-18), 41.6 (C-9), 40.0 (C-10), 38.9 (C-8), 33.9
(C-21), 33.1 (C-7), 32.3 (C-22), 32.4 (C-20), 30.7 (C-24), 28.7 (C-
15), 27.7 (C-29), 25.8 (C-27), 23.6 (C-30), 23.5 (C-11),23.0 (C-16),
21.6 (C-23), 19.3 (C-6), 18.8 (C-26), 17.3 (C-25); ESIMS (positive
ions) m/z 565 [M + Na]+, 1107 [2 M + Na]+; HR-ESIMS m/z
565.3657, calcd for C37H50NaO3 565.3658.
Methyl 2-(4-Methoxyphenyl)-1,3-bisdehydrooleanolate (7g). To
a stirred solution of 7d (50 mg, 0.084 mmol) in toluene (4 mL) were
added NaOMe (4 mg, 0.084 mmol, 1 molar equiv), 4-methox-
yphenylboronic acid (13 mg, 084 mmol, 1 molar equiv), and catalytic
Pd(PPh3)4. After stirring at 80 °C for 2 h, the reaction was worked up
by dilution with brine and extraction with EtOAc. After drying and
evaporation, the residue was purified by GCC (petroleum ether/
EtOAc, 9:1, as eluant) to give 50 mg (95%) of 7g as a white powder:
IR νmax (KBr) 3307, 1726, 1672, 1604, 1511, 1462, 1388, 1361, 1247,
1178 cm−1; 1H NMR (CDCl3, 500 MHz) δH 7.24 (2H, d, J = 9.0 Hz,
H-2′/6′), 7.03 (1H, s, H-1), 6.86 (2H, d, J = 9.0 Hz, H-3′/5′), 5.37
(1H, t, J = 3.5 Hz, H-12), 3.81 (3H, s, 4′-OMe), 3.64 (3H, s, 28-
OMe), 2.90 (1H, dd, J = 14.0, 4.0 Hz, H-18), 2.20−1.09 (series of
multiplets), 1.20 (3H, s, H-25), 1.19 (3H, s, H-24), 1.18 (3H, s, H-23),
1.16 (3H, s, H-27), 0.94 (3H, s, H-30), 0.91 (3H, s, H-29), 0.85 (3H,
s, H-26); 13C NMR (CDCl3, 125 MHz) δC 204.8 (C-3), 178.6 (C-28),
159.3 (C-4′), 144.5 (C-13), 139.9 (C-2), 135.7 (C-1), 129.7 (C-2′/
6′), 129.6 (C-1′), 122.1 (C-12), 113.8 (C-3′/5′), 55.6 (OCH3-4′),
53.0 (C-5), 52.0 (OCH3-28), 47.1 (C-17), 45.8 (C-19), 45.4 (C-4),
42.3 (C-14), 42.2 (C-18), 41.8 (C-9), 40.3 (C-10), 39.1 (C-8), 33.4
(C-21), 33.3 (C-7), 32.6 (C-22), 32.4 (C-20), 31.0 (C-24), 28.9 (C-
15), 27.8 (C-29), 26.0 (C-27), 23.9 (C-30), 23.8 (C-11),23.7 (C-16),
21.8 (C-23), 19.6 (C-6), 19.1 (C-26), 17.5 (C-25); ESIMS (positive
ions) m/z 595 [M + Na]+, 1167 [2 M + Na]+; HR-ESIMS m/z
595.3767, calcd for C38H52NaO4 595.3763.
1
1164, 1031 cm−1; H NMR (CDCl3, 500 MHz) δH 7.28 (1H, d, J =
16.0 Hz, H-1′), 7.16 (1H, s, H-1), 6.51 (1H, d, J = 16 Hz, H-2′), 5.37
(1H, t, J = 3.5 Hz, H-12), 4.22 (2H, q, J = 14.2, 7.1 Hz, 3′-OCH2),
3.49 (3H, s, 28-OMe), 2.78 (1H, dd, J = 14.0, 4.0 Hz, H-18), 2.45−
1.10 (series of multiplets), 1.30 (3H, t, J = 7.1 Hz, 3′-OCH2CH3), 1.30
(3H, s, H-25), 1.18 (3H, s, H-24), 1.11 (3H, s, H-27), 1.08 (3H, s, H-
23), 1.01 (3H, s, H-30), 0.93 (3H, s, H-29), 0.92 (3H, s, H-26); 13C
NMR (CDCl3, 125 MHz) δC 200.3, 178.2, 162.5, 149.4, 143.1, 142.3,
123.6, 115.3, 88.6, 61.4, 56.0, 48.3, 47.3, 46.6, 42.3, 41.9, 40.0, 39.4,
39.1, 37.7, 35.8, 34.6, 33.8, 33.3, 33.1, 31.4, 28.8, 28.3, 27.9, 26.6, 24.3,
24.1, 23.7, 19.0, 17.6, 14.2; ESIMS (positive ions) m/z 587 [M + Na]+,
1151 [2 M + Na]+; HR-ESIMS m/z 587.3717, calcd for C36H52NaO5
587.3712.
Methyl 2-Carboxy-1,3-bisdehydrooleanolate (7j). Under a N2
atmosphere, a mixture of 3a (100 mg, 0.21 mmol) and a 2.0 M
solution of Stiles’s reagent (methyl magnesium carbonate, 8 mL, 16
mmol) was heated at 110 °C. After 1 h, the reaction was worked up by
dilution with 5% HCl and extraction with EtOAc. The organic phase
was washed with brine, dried, and evaporated. The residue, a white
powder, was dissolved in toluene (8 mL) and treated with DDQ (90
mg, 0.39 mmol, 2 molar equiv) under stirring at room temperature
After 1 h, the reaction was worked up by dilution with brine and
extraction with EtOAc. After drying and evaporation, the residue was
purified by GCC on silica gel (10 g, petroleum ether/EtOAc, 9:1, as
eluant) to afford 7j as a white powder (70 mg, 66% from 3a): IR νmax
(KBr) 1720, 1667, 1455, 1387, 1300, 1260, 1164 cm−1; for 1H and 13
C
NMR data, see ref 10; ESIMS (positive ions) m/z 533 [M + Na]+;
HR-ESIMS m/z 533.3239, calcd for C32H46NaO5 533.3243.
Methyl 2-Methoxycarbonyl-1,3-bisdehydrooleanolate (7k). To a
solution of 7j (100 mg, 0.19 mmol) in MeOH (8 mL) were added
ethylene dichloride (EDC) (38 mg, 0.195 mmol, 1 molar equiv) and
catalytic DMAP. After stirring at room temperature for 1 h, the
reaction was worked up by dilution with brine and extraction with
EtOAc. After washing with brine, drying, and evaporation, the residue
was purified by GCC (10 g silica gel, petroleum ether/EtOAc, 9:1, as
eluant) to give 100 mg (98%) of 7k as a white powder: IR νmax (KBr)
1730, 1661, 1730, 1661, 1455, 1387, 1300, 1260, 1164, 1098 cm−1; for
1H and 13C NMR data, see ref 10; ESIMS (positive ions) m/z 547 [M
Methyl 2-(2-Phenylethynyl)-1,3-bisdehydrooleanolate (7h). To a
stirred solution of 7d (150 mg, 0.25 mmol) in EtOAc (6 mL) were
added phenylacetylene (41 μL, 0.379 mmol, 1.5 molar equiv),
triethylamine (TEA) (500 μL), CuI (5 mg, 0.025 mmol, 0.1 molar
F
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+ Na]+, 1071 [2 M + Na]+; HR-ESIMS m/z 547.3333, calcd for
C33H48NaO5 587.3339.
For NF-κB inhibition the RLU (relative light units) was calculated, and
the results were expressed as percentage of inhibition of NF-κB activity
induced by TNFα (100% activation). For STAT3 inhibition the RLU
was calculated, and results were expressed as percentage of inhibition
of STAT3 activity induced by IFN-γ (100% activation). For Nrf2
activation the RLU was calculated, and the EC50 and IRA (intrinsic
relative activity) values were determined relative to 20 μM tert-
butylhydroquinone (TBHQ) using the following equation: IRA
coefficient = (EC50‑TBHQ × Emax)/(EC50 × Emax‑TBHQ), where EC50
and Emax denote EC50 and Emax of the agonist, and EC50‑TBHQ and
Emax‑TBHQ denote EC50 and Emax values of the standard agonist
TBHQ.26 The results represent the mean of at least five independent
experiments, and the SD was always lower than 15%.
Methyl 2-Formyl-1,3-dehydrooleanoate (7l). To a solution of 4
(200 mg, 0.40 mmol) in toluene (8 mL) was added DDQ (182 mg,
0.802 mmol, 2 molar equiv), and the reaction was stirred at room
temperature After 30 min, the reaction was worked up by dilution with
brine and extraction with EtOAc. Afte drying and evaporation, 200 mg
(quant.) of 7l was obtained as a white powder. The same reaction
could be obtained by treatment with PhSeCl (2 molar equiv) and
pyridine (6 molar equiv) in CH2Cl2: IR νmax (KBr) 1727, 1667, 1458,
1
1389, 1302, 1260, 1164, 1098 cm−1; H NMR (500 MHz, CDCl3) δ
10.01 (s, 1H), 7.79 (1H, s, H-1), 5.37 (1H, t, J = 3.5 Hz, H-12), 3.63
(s, 3H), 2.78 (1H, dd, J = 14.0, 4.0 Hz, H-18), 2.45−1.10 (series of
multiplets), 1.19 (3H, s, H-25), 1.17 (3H, s, H-24), 1.16 (3H, s, H-23),
1.14 (3H, s, H-27), 0.94 (3H, s, H-30), 0.91 (3H, s, H-29), 0.85 (3H,
s, H-26); 13C NMR (75 MHz, CDCl3) δ 203.7, 190.7, 178.3, 165.2,
144.5, 131.2, 121.6, 52.8, 51.8, 47.0, 45.8, 45.1, 42.3, 41.7, 41.3, 40.5,
39.8, 34.0, 33.3, 32.44, 32.38, 30.9, 28.2, 27.8, 26.0, 23.8, 23.5,
23.2,21.7, 19.2, 18.2, 17.6; ESIMS (positive ions) m/z 517 [M + Na]+,
1011 [2 M + Na]+; HR-ESIMS m/z 517.3296, calcd for C32H46NaO4
517.3294.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00271.
Selected MS and NMR spectra for the synthetic
compounds (PDF)
Methyl 2-Acetoxy-1,3-bisdehydrooleanolate (7m). To a stirred
and cooled (ice bath) solution of of maslinic acid (3c) (200 mg, 0.41
mmol) in acetone (5 mL) was added dropwise Jones’ reagent. After 1
h, the reaction was worked up by the addition of a few drops of EtOH
and then brine and subsequently extracted with EtOAc. After drying
and evaporation, the residue was purified by GCC on silica gel
(petroleum ether/EtOAc, 9:1, as eluent) to afford the enol tautomer of
methyl 2-oxo-3-dehydrooleanoate,20 which was dissolved in THF (10
mL) and treated with pyridine (500 μL) and Ac2O (27 μL 0.29 mmol,
1 molar equiv). After stirring at room temperature for 1 h, the reaction
was worked up by dilution with brine and extraction with EtOAc. After
drying and evaporation, the residue was purified by GCC on silica gel
(petroleum ether/EtOAc, 9:1, → petroleum ether/EtOAc, 9:1, as
eluent) to afford 7m as a white solid (80 mg, 52%): IR νmax (KBr)
AUTHOR INFORMATION
Corresponding Authors
■
*Tel: +34-957-213766. E-mail: e.munoz@uco.es (E. Munoz).
̃
*Tel: +39-0321-375744. Fax: +39-0321375744. E-mail:
giovanni.appendino@uniupo.it (G. Appendino).
ORCID
Giovanni Appendino: 0000-0002-4170-9919
Notes
The authors declare no competing financial interest.
1
1607, 1475, 1238, 998, 846, 774 cm−1; H NMR (CDCl3, 500 MHz)
δH 6.68 (1H, s, H-1), 5.34 (1H, t, J = 3.5 Hz, H-12), 3.63 (3H, s, 28-
OMe), 2.90 (1H, dd, J = 14.0, 4.0 Hz, H-18), 2.20 (3H, s, Ac), 2.05−
1.10 (series of multiplets), 1.23 (3H, s, H-25), 1.21 (3H, s, H-24), 1.15
(3H, s, H-27), 1.13 (3H, s, H-23), 0.93 (3H, s, H-30), 0.91 (3H, s, H-
29), 0.81 (3H, s, H-26); 13C NMR (CDCl3, 125 MHz) δC 197.9 (C-
3), 178.2 (C-28), 169.0 (C-1′), 144.4 (C-13), 144.0, 135.0, 121.5 (C-
12), 52.9 (C-5), 51.6 (OCH3-28), 46.8 (C-17), 46.7 (C-19), 45.7 (C-
4), 42.1 (C-14), 41.9 (C-18), 41.5 (C-9), 40.0 (C-10), 39.4 (C-8),
33.9 (C-21), 33.1 (C-7), 32.3 (C-22), 30.7 (C-20), 27.8 (C-24), 27.6
(C-15), 25.9 (C-29), 24.5 (C-27), 23.6 (C-30), 23.3 (C-11), 23.0 (C-
16), 21.5 (C-23), 20.4 (C-2′), 19.2 (C-6), 18.9 (C-26), 17.3 (C-25);
ESIMS (positive ions) m/z 547 [M + Na]+, 1071 [2 M + Na]+; HR-
ESIMS m/z 517.3402, calcd for C33H48NaO5 547.3399.
Cell Cultures. HaCaT-ARE-Luc (human keratinocytes), NIH-3T3-
KBF-Luc (mouse fibroblasts), and HeLa-STAT3-Luc (human cervix
carcinoma) cell lines were constructed in our lab and characterized as
previously described.25 All the cell lines were grown in supplemented
Dulbecco’s modified Eagle’s medium containing 10% fetal bovine
serum and 1% penicillin/streptomycin antibiotics, at 37 °C in a
humidified atmosphere of 5% CO2.
Luciferase Assays. For the anti-NF-κB activity NIH-3T3-KBF-
Luc cells were stimulated with TNFα (20 ng/mL) in the presence or
the absence of the compounds for 6 h. For the activation of the
antioxidant response element (ARE) that is activated by Nrf2
HaCaTARE-Luc cells were stimulated with the compounds for 6 h.
For the anti-STAT3 activity HeLa-STAT-3-Luc cells were stimulated
with γIFN (20 IU/mL) in the presence or the absence of the
compounds for 6 h. After treatment, the cells were washed twice in
phosphate-buffered saline and lysed in 25 mM Tris-phosphate pH 7.8,
8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 7% glycerol during
15 min at room temperature in a horizontal shaker. After
centrifugation, luciferase activity in the supernatant was measured
using a GloMax 96 microplate luminometer (Promega, Madison, WI,
USA) following the instructions of the luciferase assay kit (Promega).
ACKNOWLEDGMENTS
NMR spectra were recorded at the “Centro di Servizio
Interdipartimentale di Analisi Strumentale”, Universita
Napoli Federico II. The research leading to these results has
received funding to TriForC from the European Community’s
Seventh Framework Programme [FP7/2007-2013] under grant
agreement 613692, a Pipeline for the Discovery, Sustainable
Production and Commercial Utilization of Known and Novel
High-Value Triterpenes with New or Superior Biological
Activities.
■
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di
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