Cinnamic anilides as new mitochondrial permeability transition pore inhibitors endowed with ischemia-reperfusion injury protective effect in vivo

Article abstract of DOI:10.1021/jm500547c

In this account, we report the development of a series of substituted cinnamic anilides that represents a novel class of mitochondrial permeability transition pore (mPTP) inhibitors. Initial class expansion led to the establishment of the basic structural requirements for activity and to the identification of derivatives with inhibitory potency higher than that of the standard inhibitor cyclosporine-A (CsA). These compounds can inhibit mPTP opening in response to several stimuli including calcium overload, oxidative stress, and thiol cross-linkers. The activity of the cinnamic anilide mPTP inhibitors turned out to be additive with that of CsA, suggesting for these inhibitors a molecular target different from cyclophylin-D. In vitro and in vivo data are presented for (E)-3-(4-fluoro-3-hydroxy-phenyl)-N-naphthalen-1-yl- acrylamide 22, one of the most interesting compounds in this series, able to attenuate opening of the mPTP and limit reperfusion injury in a rabbit model of acute myocardial infarction.

Full text of DOI:10.1021/jm500547c

Article
pubs.acs.org/jmc
Cinnamic Anilides as New Mitochondrial Permeability Transition
Pore Inhibitors Endowed with Ischemia-Reperfusion Injury Protective
Effect in Vivo
Daniele Fancelli,*,†,○ Agnese Abate,‡,◆ Raffaella Amici,†,○ Paolo Bernardi,⊥ Marco Ballarini,†,§
Anna Cappa,‡,○ Giacomo Carenzi,‡,¶ Andrea Colombo,∇,+ Cristina Contursi,†,▲ Fabio Di Lisa,#
Giulio Dondio,∇, □ Stefania Gagliardi,∇,◇ Eva Milanesi,† Saverio Minucci,§,∥ Gilles Pain,†,△
Pier Giuseppe Pelicci,§ Alessandra Saccani,†,■ Mariangela Storto,†,§ Florian Thaler,†,○ Mario Varasi,‡,○
Manuela Villa,†,○ and Simon Plyte†,●
†Genextra Group, Congenia s.r.l., Via Adamello 16, 20139 Milan, Italy
‡Genextra Group, DAC s.r.l., , Via Adamello 16, 20139 Milan, Italy
§Department of Experimental Oncology, European Institute of Oncology IEO, Via Adamello 16, 20139 Milan, Italy
∥Department of Biosciences, University of Milan, 20100 Milan, Italy
⊥Department of Biomedical Sciences, University of Padua, Via Ugo Bassi 58/B, 35121 Padua, Italy
#Department of Biomedical Sciences, University of Padua, Viale G. Colombo 3, 35131 Padua, Italy
∇NiKem Research s.r.l., Via Zambeletti 25, 20021 Baranzate, MI, Italy
S
* Supporting Information
ABSTRACT: In this account, we report the development of a series
of substituted cinnamic anilides that represents a novel class of
mitochondrial permeability transition pore (mPTP) inhibitors. Initial
class expansion led to the establishment of the basic structural
requirements for activity and to the identification of derivatives with
inhibitory potency higher than that of the standard inhibitor
cyclosporine-A (CsA). These compounds can inhibit mPTP opening
in response to several stimuli including calcium overload, oxidative
stress, and thiol cross-linkers. The activity of the cinnamic anilide
mPTP inhibitors turned out to be additive with that of CsA, suggesting
for these inhibitors a molecular target different from cyclophylin-D. In
vitro and in vivo data are presented for (E)-3-(4-fluoro-3-hydroxy-
phenyl)-N-naphthalen-1-yl-acrylamide 22, one of the most interesting
compounds in this series, able to attenuate opening of the mPTP and
limit reperfusion injury in a rabbit model of acute myocardial infarction.
formation by dimers of the ATP synthase.4 Much of our
understanding of the functioning of the mPTP comes from
INTRODUCTION
■
The mitochondrial permeability transition (mPT) is a
phenomenon whereby the inner mitochondrial membrane
becomes permeable to solutes. This induces cessation of
studies using the immunosuppressive agent 47 (cyclosporine A,
CsA)6 (Figure 1), which in the late 1980s was discovered to
inhibit the mPTP, as well as from cyclophilin D knockout
mitochondrial respiration and energy production, mitochon-
mice.7 Opening of the mPTP can be induced by calcium (Ca2+)
drial swelling and outer membrane rupture, release of apoptotic
overload and reactive oxygen species (ROS). These conditions
factors, and cell death leading to organ damage. The mPT is
are common to several diseases including reperfusion injury
mediated by opening of a nonselective pore within the
(angioplasty after a heart attack, transplantation surgery, and
mitochondria: the mitochondrial permeability transition pore
stroke), neurological diseases such as amyloid lateral sclerosis
(mPTP). Even after intensive investigation, the exact molecular
nature of the mPTP is still not known.1 Several proteins have
been implicated in facilitating the mPTP: adenine nucleotide
transporter, phosphate carrier protein, cyclophilin D, and
and Alzheimer’s disease, traumatic brain injury, dystrophies,
and myopathies.8−11 In essence, prolonged opening of the
mPTP can have catastrophic effects on organ function in many
recently the F1Fo ATP synthase complex, but no single protein
has been unambiguously demonstrated to be the pore-forming
Received: March 13, 2014
Published: June 11, 2014
unit.2−5 However, strong evidence now points to pore
© 2014 American Chemical Society
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Figure 1. Chemical structures of cinnamic anilides (I) and known mPTP inhibitors.
diverse diseases. In particular, opening of the mPTP is
considered to be a key to the immediate tissue damage caused
by reperfusion of ischemic tissue.12 In the case of acute
myocardial infarction (AMI), the reperfusion of ischemic tissue
causes cardiomyocyte damage and contributes to final infarct
size. Studies in animal models suggest that lethal reperfusion
injury (LRI) may contribute up to 50% of the final infarct
size.12,13 Indeed, this form of myocardial injury may in part
explain why, even after optimal myocardial reperfusion, the
death rate is still approximately 10% and the incidence of
cardiac failure remains almost 20%.14 Currently, there are no
registered therapies for the treatment of LRI.15,16 Hence,
inhibition of opening of the mPTP upon tissue reperfusion
should attenuate lethal reperfusion injury and reduce final
infarct size, thus permitting the full realization of the benefits of
reperfusion strategies in AMI. Consistent with this hypothesis,
Piot et al. demonstrated in a proof-of-concept study that
administration of CsA to patients with ST-elevated myocardial
infarction 5 min prior to PCI significantly reduced troponin I
and creatinine kinase release, indicative of reduced cardiac
damage.17 Larger clinical studies are currently underway to
confirm this initial observation. Despite its usefulness as an
investigational tool, the use of CsA in therapy could be severely
limited by several factors, such as its multiple pharmacological
activities, well documented adverse effects,18,19 and risk of
cardiovascular side effects induced by the cremophor
formulation.20,21
administered to mice undergoing myocardial infarction rescued
cardiac function compared to sham operated mice.23 The
cholesterol-oxime 50 (TRO40303) (Figure 1) specifically binds
to the cholesterol site of the mitochondrial translocator protein
(18 kDa)26 (TSPO), which is potentially involved in mPTP
regulation.27 Compound 50 demonstrated cardioprotective
properties in preclinical models26 and is currently being
evaluated for reduction of reperfusion injury in acute ST-
elevation myocardial infarction patients in a phase II proof-of-
concept clinical study.28
Because of the extremely high therapeutic potential of mPTP
inhibition and the paucity and limitations of known inhibitors,
we embarked on a program aimed at identifying and developing
novel, selective, nonpeptide inhibitors of the mPTP. In this
article, we describe the synthesis, the structure−activity
relationships (SAR) for inhibition of mPTP opening induced
by calcium overload, and a preliminary biological character-
ization of cinnamic anilides (I), a novel series of potent
inhibitors of mPTP.
The parent compound of this series (3), the 3-chlorophenyl
anilide of caffeic acid, was originated by a high-throughput
screen (data not shown) carried out to identify compounds
able to inhibit the swelling induced by calcium overload in
mitochondrial preparations from rat liver. Subsequent class
expansion led to the establishment of the basic structural
requirements for activity and to the identification of derivatives
with inhibitory potency higher than that of the standard
inhibitor CsA. These compounds can inhibit mPTP opening in
response to several stimuli including calcium overload, oxidative
stress, and cross-linkers. Further, in vitro and in vivo data are
presented for the representative compound (E)-3-(4-fluoro-3-
hydroxy-phenyl)-N-naphthalen-1-yl-acrylamide (22) that show
the potential of these inhibitors to attenuate opening of the
mPTP and their ability to limit reperfusion injury in a rabbit
model of acute myocardial infarction.
Nonimmunosuppressive cyclosporin A analogues such as the
antiviral agents 48 (NIM811) and 49 (Debio 025) (Figure 1)
retain cyclophilin D inhibitory activity and consequently inhibit
mPTP, but their potency is restricted by the limits of the
regulatory role of cyclophilin D in mPTP function and its level
of expression.22,23 Compound 49 was demonstrated to protect
mouse dystrophic cells against mitochondria-mediated death
and showed efficacy in mouse models of collagen VI24 and
Duchenne muscular dystrophy,25 suggesting that therapies
targeting the mPTP may be helpful to patients. In addition, 49
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a
Scheme 1
a
Reagents and conditions: coupling method A, ArNH2, DCC, THF, reflux; coupling method B, (i) SOCl2, THF, 50 °C, (ii) ArNH2, NEt3, THF, 0
°C to rt; coupling method C, (i) (COCl)2, DCM, reflux, (ii) ArNH2, NEt3, DCM, 0 °C to rt; coupling method D, ArNH2, EDC, HOBt, DCM, 0 °C
to rt; coupling method E, (i) SOCl2, reflux, (ii) ArNH2, pyridine, 85 °C.
by direct coupling of the Weinreb amide of 2-(3-hydroxy-4-
methoxyphenyl)cyclopropanecarboxylic acid (36) were un-
successful, thus the amide was hydrolyzed to the corresponding
carboxylic acid, activated with EDC and HOBt, and finally
coupled with 3-chloroaniline 2a (Scheme 4).
N-3-Acetoxy-4-methoxyphenyl-N′-3-Cl-benzoylthiourea 40,
obtained by the reaction of the 3-Cl-aniline 2a and 3-acetyl-
4-methoxy benzoyl isothiocyanate 39, was converted into the
amino-triazole derivative 41 by condensation with hydrazine
and a concomitant loss of the acetyl protecting group (Scheme
5).
CHEMISTRY
■
Most of the cinnamic amides 3−34 were directly obtained by
standard coupling reactions (Scheme 1) carried out on the
commercially available carboxylic acids 1a−m and anilines 2a−
t. Specifically, the carboxylic acids were either activated with
SOCl2 (method B) or (COCl)2 (method C) to form their acyl
chlorides and then coupled with the anilines or the acids were
directly reacted with the anilines in the presence of DCC
(method A) or EDC/HOBt (method D). In some cases, the
phenolic moiety of the 3-hydroxy-4-methoxy-cinnamic acid 1d
was first protected as an acetate (Scheme 2).
The 1,3,4-oxadiazole analogue 44 was prepared in two steps
by coupling 3-chlorobenzoic acid hydrazide 42 and 3-acetyl-4-
methoxy-cinnamic acid 1n followed by alkaline hydrolysis of
Compounds 10, 14, and 18 were obtained by reduction of
the corresponding nitro derivatives 10a, 14a, and 18a,
respectively (Scheme 3). Attempts to prepare compound 37
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a
Scheme 2
a
Reagents and conditions: (a) (i) NaH, THF, 0 °C, (ii) Ac2O reflux; (b) (i) SOCl2, THF, reflux, (ii) ArNHR3, DIPEA, THF, reflux; (c) HCl,
MeOH, THF, rt; (d) (i) NaOHaq MeOH, reflux, (ii) HClaq.
a
the acetate protecting group (Scheme 6). Finally, the
phenylpropanoic analogue 46 was obtained by coupling the
corresponding 3-hydroxy-4-methoxy-phenylpropanoic acid 45
that was activated with SOCl2 and then treated with 3-chloro-
aniline 2a.
Scheme 3
SAR
■
The mPTP inhibitory activity of cinnamic anilides 3−34 was
initially evaluated by measuring their ability to protect murine
mitochondria from mPTP opening induced by the exposure to
increasing concentrations of Ca2+ (calcium retention capacity
assay (CRC)). This method allows measuring of the propensity
of mitochondria to open the mPTP after calcium uptake. In the
presence of extra-mitochondrial calcium, isolated mitochondria
take up calcium into the matrix via the calcium uniporter.
Continued addition of extra-mitochondrial calcium and
subsequent uptake leads to the calcium-induced opening of
the mPTP, mitochondrial depolarization, and release of stored
calcium. The total amount of calcium that can be retained until
calcium-induced mPTP opening occurs is termed the calcium
retention capacity.29 An example of the calcium retention
a
Reagents and conditions: (a) SnCl2·2H2O, EtOH, EtOAc, reflux,
1.5−7 h.
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a
Scheme 4. Synthesis of Racemic trans-Cyclopropane Analogue
a
Reagents and conditions: (a) Me-NH-OMe·HCl, EDC, HOBt, DCM, rt, 16 h; (b) (i) NaH, Me3SO+I−, DMSO, rt, 16 h, (ii) HClaq; (c) (i) tBuOK,
H2O, Et2O, rt, 20 h, (ii) 3-ClPhNH2, EDC, HOBt, DCM, rt, 36 h.
a
Scheme 5
a
Reagents and conditions: (a) (i) SOCl2, DMF, THF, reflux, 2 h, (ii) KSCN, MeCN, reflux, 2 h; (b) 3-Cl-aniline, MeCN, 0.5 h rt; (c) NH2NH2,
CHCl3, reflux, 2 h.
a
Scheme 6
a
Reagents and conditions: (a) POCl3, 1,4-dioxane, reflux, 1 h; (b) 4N HCl, 1,4-dioxane, MeOH, rt.
capacity assay using the well-characterized mPTP inhibitor CsA
is shown in Figure 2.
Measuring the increase of calcium needed to induce the
mPTP in mitochondria treated with mPTP inhibitor vs
untreated mitochondria (Ca2+ overloading, μM) is a convenient
and sensitive assay for assessing the ability of the compounds to
inhibit mPTP opening. The ratio between the amount of
calcium required to trigger mPT in the presence of the
compound (CRCi) with respect to that required to induce
mPT in the absence of the compound (CRCo) is a measure of
the inhibitory effect of the compound on the mPTP.
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Table 1. N-(3-Chlorophenyl)cinnamic Anilides
a
b
compd
R1
R2
CRCi/CRC0 (%)
SD
CsA
3
224
155
136
87
12
12
12
8.9
14
11
6.2
10
8.6
8.1
8.4
11
10
16
OH
OH
OH
H
4
5
H
OH
OMe
OH
OMe
H
6
OH
186
93
7
OMe
OMe
F
8
75
9
90
Figure 2. Calcium retention capacity assay. CaCl2 (1 μL of 2 mM
stock solution) was added every minute to mouse liver mitochondria
(200 μL of a 0.5 mg/mL suspension) in the presence of the
fluorescent Ca2+ indicator Calcium Green 5N. Calcium uptake was
followed by measuring extra-mitochondrial calcium green fluorescence
until mPTP opening was achieved. CsA (1 μM) was added to the
mitochondria immediately prior to the start of the experiment.
10
11
12
13
14
23
NH2
SO2NHMe
H
94
H
91
−NHCHCH−
87
OH
OH
OH
Me
NH2
F
133
102
175
a
Increase of the calcium retention capacity of mouse liver
b
mitochondria after incubation with 1 μM inhibitor. n = 3.
All the cinnamic anilides here described were tested in the
CRC assay in a dose escalation series at 0.1, 0.5, 1, and 5 μM.
Because no significant differences were observed in the potency
ranking of the compounds tested at different concentrations
(see Supporting Information for the complete result data set),
only the CRC values measured at 1 μM inhibitor concentration
are reported in the following SAR tables.
that of the reference standard CsA and was used for the very
initial characterization of the mPTP inhibitory properties of the
class. Thus, we decided to fix the right-hand side of compound
6 with the 3-OH-4-OMe moiety while exploring different
central linkers and anilines.
The impact of the central linker on the activity of the
compounds was explored starting from analogue 6, but all the
modifications introduced in this region such as N-methylation
(34), double bond reduction (46), cyclopropanation (37), or
cyclization (41, 44) completely abolished activity (Table 2).
Finally, the effect of different anilines was investigated (Table
3, 15−33). For this purpose, the 3-hydroxy-4-methoxy-
cinnamic moiety was kept fixed. Far from being exhaustive,
this initial exploration suggests that, while the chlorine present
in the initial hit turned out to be the best among the residues
In the absence of structural information on the binding mode
of these cinnamic anilides to their target mPTP component, we
first performed a limited SAR investigation to identify the
structural elements of the hit compound 3 that are essential for
mPTP inhibition.
The structure of 3 was divided into three regions, namely the
cinnamic phenyl ring, the central linker, and the aniline aryl
ring (Figure 3), and systematic variations were carried out in
each region.
Table 2. Modification of the Central Linker
Figure 3. Regions of the structure of the hit compound 3 subjected to
separate SAR investigation.
For this purpose and as the first attempt, the influence of the
vicinal hydroxy moieties at the cinnamic phenyl ring was
investigated (Table 1). The comparison of the activities of
compounds 4 vs 5 and 6 vs 7 immediately suggests a crucial
role for the hydroxyl group at position 3. This was further
confirmed by the complete inactivity of compounds 9−12,
wherein the 3-hydroxyl is replaced by potential bioisosters such
as fluorine, amino, or sulfonamido groups. In contrast,
replacement of the hydroxyl at position 4 with different
moieties (compounds 4, 6, 13, 14, and 23) was better tolerated
and the modifications variously modulated the potency. The
most active example in this series resulted to be the 3-OH-4-
OMe derivative 6, which demonstrated potency comparable to
a
Increase of the calcium retention capacity of mouse liver
b
mitochondria after incubation with 1 μM inhibitor. n = 3.
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well as the replacement of the methoxy moiety with fluorine
(22). In the latter case, the aim was to reduce lipophilicity and
potential metabolic liabilities of the molecule. While the
heterobicyclic derivatives were less active, the fluorine analogue
22 showed the highest potency in the series and was selected as
a class representative compound for a deeper biological
characterization.
Table 3. 3-Hydroxycinnamic Anilides
BIOLOGY
■
Typically, opening of the mPTP in isolated mitochondria can
be achieved by challenging the mitochondria with high Ca2+
concentrations and can be assayed spectrophotometrically by
monitoring the decrease of absorbance at 540 nm (A540),
which indicates mitochondrial swelling as a result of solute
influx into the mitochondrial matrix via the open mPTP.
In addition, the mPTP opening can be induced by
challenging isolated mitochondria with oxidizing agents (e.g.,
diamide or menadione) or uncouplers of electron transport
(e.g., trifluorocarbonylcyanide phenylhydrazone (FCCP)). In a
preliminary assessment of the mechanism of action of this novel
class of inhibitors, compound 6 was tested in this swelling assay
for its ability to prevent mPTP opening in response to the
different stimuli mentioned above in isolated mouse liver
mitochondria. The results reported in Figure 4 show that
cinnamic anilides prevent mPTP opening induced by stimuli
independent of changes in calcium flux, thus suggesting that
they behave as genuine inhibitors of the mPTP and not of
calcium homeostasis (Figure 4B−D).
Because the cinnamide skeleton is present in diverse
compounds with a wide range of pharmacological activ-
ities,30−34 we profiled our initial lead molecule in a Cerep
diversity profile. Thus, compound 6 was tested at a
concentration of 10 μM versus a panel of 69 receptors and
ion channels and 14 enzymes (see complete list in the
Supporting Information). Affinity for each target is measured
by displacement of a radiolabeled ligand specific for each
particular receptor or enzyme. The results, expressed in terms
of percentage of displaced ligand, confirmed a notably clean
profile for 6: in fact, for just 1 (norepinephrine transporter) out
of 83 assessed targets was found a displacement higher than
50%. These initial results, positive in terms of mechanism of
action and selectivity, encouraged us in further developing this
class.
After the initial round of class expansion, compound 22 was
selected for wider biological characterization. Prompted by the
presence of an α,β-unsaturated amide group in the structure,
the electrophilic behavior of compound 22 was first examined.
The compound was incubated at a test concentration of 100
μM with glutathione (100 μM) or N-acetyl-cysteine (100 μM)
at 25 °C. HPLC analysis carried out after 24 h incubation
showed quantitative recovery of 22 without detection of any
thiol adducts (see Supporting Information).
A more extensive assessment of the potency in the CRC
assay was then carried out in comparison to the standard CsA.
The CRC of freshly prepared mouse liver mitochondria was
determined for 22 and CsA in a dose escalation series from 0.1
to 5.0 μM (Figure 5). A clear dose response was observed with
22 and the compound enabled mitochondria to withstand very
high levels of Ca2+, significantly superior to CsA. In addition,
the CRC of CsA reached a plateau around 0.5 μM of CsA and
did not increase at higher doses. This is probably due the
titration of cyclophillin D, its primary mitochondrial target.
a
Increase of the calcium retention capacity of mouse liver
b
mitochondria after incubation with 1 μM inhibitor. n = 3.
explored at position 3 of the phenyl ring (6, 15−19, 24), this
region of the scaffold tolerates a wide range of modifications. In
fact, phenyl substitution at positions 2 and 4 (20, 21), phenyl
disubstitution (25−27), and replacement of the phenyl with
bicyclic moieties (28−33) led to compounds with maintained
or improved potency.
In summary, the above findings allowed us to identify the
phenol at position 3 of the cinnamic phenyl ring and the
acrylamido linker as the structural determinants for the activity
of this novel class of mPTP inhibitors, while modifications in
the aniline region of the scaffold as well as position 4 of the
cinnamic phenyl showed to be well tolerated and thus usable
for the optimization of potency and properties.
Searching for a lead compound more potent than the initial
lead 6, we drew our attention to the high potency showed by
the naphthyl derivative 30 and investigated the effect of the
introduction of heteroatoms in the bicyclic ring (31−33) as
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Figure 4. The cinnamic anilides inhibit opening of the mPTP in isolated mitochondria in response to various stimuli. Compound 6 (1 μM) inhibits
swelling of mouse liver mitochondria exposed to (A) calcium overload (CaCl2 150 μM), (B) uncoupling of electron transport chain (FCCP 50 nM),
(C,D) oxidative stress (menadione 100 μM and diamide 300 μM, respectively).
CsA in combination with our inhibitors. Figure 6 shows that
there is a clear additivity between CsA and 22. The protective
effect of the combination is significantly higher than the
maximum effect obtainable with CsA alone, which further
confirms the hypothesis that the cinnamic anilides do not target
cyclophilin D.
Figure 5. CRC of mouse liver mitochondria treated with increasing
concentrations of 22 or CsA.
The observation that the CRC of CsA reached a plateau at
relatively low concentrations while the cinnamic anilides
continued to increase the CRC was the first indication that
the molecular target of this new class might not be cyclophilin
D. To further investigate this point, we measured the CRC of
Figure 6. CRC of mouse liver mitochondria treated with combination
of 22 and CsA; the results are the mean of three experiments
standard deviation.
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The potential use of mPTP inhibitors in preventing LRI after
PCI requires that the inhibitor can reach the cardiac
mitochondria very rapidly due to the fact that the mPTP
opens within the first few minutes of reperfusion. To this end,
we assessed the CRC of cardiac mitochondria after whole
hearts had been perfused with the compound for just 2 min.
Freshly prepared beating mouse hearts, immobilized on a
Langendorff reperfusion apparatus, were perfused with
compound 22 at a 5 μM concentration or the corresponding
amount of DMSO for 2 min. Hearts were then flushed with
buffer, the mitochondria prepared, and the CRC measured
(Figure 7A).
respect to the amount of tissue exposed to the ischemic insult.
To correlate the pharmacodynamics response with plasma
concentration of 22, a satellite group of animals (n = 5) was
treated with the same dose of 22 and the test item quantified in
plasma up to 240 min.
The data in Figure 8 clearly show that 22, when given just
prior to organ reperfusion, is cardioprotective in this animal
Figure 8. Compound 22 is cardioprotective in a rabbit model of acute
myocardial infarction. Rabbits were subjected to left anterior
descending (LAD) coronary artery occlusion for 30 min followed by
4 h of reperfusion. Compound 22 (5 mg/kg in 20% DMSO, 40%
PEG400) and CsA (10 mg/kg as Sandimmune) were administered by
iv bolus 5 min prior to reperfusion. Infarct size (IS) and area at risk
(AAR) were determined by measuring TTC and Evans blue-stained
heart slices. Vehicle treated group AAR/IS = 57.3%, SD = 18.1 n = 8;
compound 22 treated group AAR/IS = 30.9%, SD = 7.8 (n = 8) p <
0.01; CsA treated group group AAR/IS = 32.6%, SD = 14.1 (n = 8) p
< 0.01.
Figure 7. (A) Calcium retention capacity of mitochondria isolated
from mouse hearts after perfusion with compound 22 (Langendorff
apparatus) at 5 μM for 2 min. Data expressed as absolute quantity of
calcium retained. Vehicle (DMSO) treated group CRC = 80 μM, SD =
6.8, n = 6; treated group CRC = 125 μM, SD = 6.0 (n = 6); p < 0.01.
(B) Calcium retention capacity of mitochondria isolated from mouse
hearts after 5 min from iv administration of compound 22 (15 mg/kg).
Data expressed as absolute quantity of calcium retained. Vehicle
(DMSO) treated group CRC = 69.8 μM, SD = 15.7, n = 16; treated
group CRC = 108.4 μM, SD = 20.2 (n = 6); p < 0.01.
model of acute myocardial infarction and can reduce infarct size
by almost 50%. Compound 22 showed a plasma concentration
after 5 min of 4.8 μM that reduced to 0.02 μM after 240 min
from the iv injection. The compound was cleared very rapidly
in rabbit plasma, showing a t1/2 of about 30 min. However, as
expected for an agent targeting I/R injury, this short-termed
plasma exposure was able to induce a significant pharmacody-
namic effect in this model of acute myocardial infarction.
Further, the reduction in infarct size was similar to that seen
with CsA, which works primarily by attenuating the mPTP,
thus confirming the validity of targeting the mPTP.
Finally, to exclude potential cardiovascular side effects that
could hamper the development of these compounds as novel
therapeutics to treat acute ischemia-reperfusion injury,
compound 22 was tested in vitro on the cardiac hERG channel
and in vivo in a standard rat telemetry study. Compound 22 did
not show any significant binding to hERG up to the
concentration of 30 μM, while no significant alterations of
any of the monitored parameters (body temperature, heart rate,
systolic and diastolic blood pressure) were observed in the rat
safety study up to a dose of 30 mg/kg given iv, which
correspond to plasma levels well above those observed in the
efficacy experiments (see Supporting Information).
Mitochondria from hearts perfused with 22 showed a
significantly higher CRC than those treated with the vehicle
(DMSO). These data clearly show that 22 can reach the cardiac
mitochondria in whole organs in as little as 2 min.
Further, similar CRC experiments performed ex vivo on
cardiac mitochondria prepared 5 min after iv injection of 22
(15 mg/kg) into the tail vein of mice confirmed that the
compound, after systemic administration, is able to reach
rapidly cardiac mitochondria and exert its protecting effect
(Figure 7B).
On the basis of these results, we decided to assess the efficacy
of 22 in a rabbit model of acute myocardial infarction.
New Zealand white rabbits were subjected to 30 min of left
anterior descending (LAD) coronary artery ligation followed by
4 h of reperfusion. Animals were then sacrificed, and the hearts
were analyzed to determine the area that had been ischemic
(area at risk, AAR) during the ligation and the area that was
necrotic (infarct size, IS) at the end of the reperfusion period.
Animals were given either vehicle, 22, or CsA via iv bolus
injection 5 min prior to unligation of the LAD coronary artery.
The ratio of the area at risk to infarct area (AAR/IS) is a
measure of the extent of the final damage to the heart with
Taken together, these experiments strongly support the
notion that administration of our cinnamic anilide mPTP
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ide 3 (270 mg 48%) as a white powder. 1H NMR (400 MHz, DMSO-
d6) δ 10.27 (bs, 1H), 9.38 (bs, 2H), 7.96 (m, 1H), 7.54 (m, 1H), 7.46
(d, J = 15.6 Hz, 1H), 7.38 (m, 1H), 7.14 (m, 1H), 7.05 (m, 1H), 6.96
(m, 1H), 6.82 (m, 1H), 6.54 (d, J = 15.6 Hz, 1H). m/z (ES+), (M +
H)+ = 290.
inhibitors during acute myocardial infarction and just prior to
reperfusion therapy should prevent mPTP opening, attenuate
lethal reperfusion injury, and reduce infarct size. Indeed, a drug
candidate from this chemical series is currently undergoing
clinical evaluation for the eventual treatment of lethal
reperfusion injury in the setting of acute myocardial infarction.
(E)-N-(3-Chlorophenyl)-3-(3-hydroxyphenyl)-prop-2-enam-
ide (4). A solution of 3-hydroxycinnamic acid 1b (1.0 g, 6.1 mmol)
and thionyl chloride (0.53 mL, 7.32 mmol) in dry THF (15 mL) was
stirred at 55 °C for 3 h. Then a further aliquot of thionyl chloride (0.1
mL, 1.38 mmol) was added, and the mixture was stirred at reflux
temperature for additional 1.5 h. After cooling to about 5 °C, a
solution of 3-chloroaniline 2a (0.65 mL, 6.1 mmol) and triethylamine
(3.4 mL, 24.4 mmol) in dry THF (5 mL) was added dropwise. After
stirring at rt for 16 h, the reaction mixture was diluted with DCM and
washed with water, 0.5N aqueous hydrochloric acid, and brine.
Organic layer was dried over sodium sulfate, concentrated under
reduced pressure, and purified by flash silica chromatography
(petroleum ether/EtOAc 45:55) to afford (E)-N-(3-chlorophenyl)-3-
(3-hydroxyphenyl)-prop-2-enamide 4 (671 mg, 40%) as a beige solid.
1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.64 (s, 1H), 7.93
CONCLUSIONS
■
We identified a series of novel mPTP inhibitors based on the
cinnamic anilido scaffold that are characterized by simplicity in
structure, low molecular weight, and mechanism of action
different from cyclophilin D inhibition.
On the basis of initial SAR studies, we identified the
following structural requirements to gain mPTP inhibitory
activity: (i) a phenol moiety at position 3 of the cinnamic
phenyl ring, (ii) an unsubstituted acrylamido linker joining the
cinnamic phenyl ring and the anilinic aryl moiety.
The high potential of cinnamic anilides is exemplified by the
rapid identification of compound 22, which demonstrated
potency equal or higher than the gold standard, CsA, in a series
of in vitro and in vivo experiments.
(m, 1H), 7.53−7.49 (m, 2H), 7.37 (m, 1H), 7.25 (m, 1H), 7.13 (m,
1H), 7.05 (m, 1H), 6.70 (m, 1H), 6.83 (m, 1H), 6.72 (d, J = 15.6 Hz,
1H). m/z (ES+), (M + H)+ = 274.
(E)- 3-(3-Aminophenyl)-N-(3-chlorophenyl)-prop-2-enamide
(10). SnCl2·2H2O (530 mg, 2.35 mmol) was added to a solution of
(E)-3-(3-nitrophenyl)-N-(3-chlorophenyl)-prop-2-enamide 10a (142
mg, 0.47 mmol) in EtOH/EtOAc 1/1 (6 mL). After stirring at reflux
for 7 h, the reaction mixture was cooled to rt and poured onto ice/
water; pH was adjusted to 8 with NaHCO3 (satd aq), and the resulting
aqueous solution was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over sodium sulfate, and
concentrated under reduced pressure to afford (E)-3-(3-amino-
phenyl)-N-(3-chlorophenyl)-prop-2-enamide 10 (88 mg, 69%) as an
Taken together, all these findings highlight that this series of
cinnamic anilides offer the possibility of studying the biology of
the mPTP and the therapeutic potential of mPTP inhibition
using potent, low molecular weight inhibitors.
EXPERIMENTAL SECTION
■
Reagents and solvents used, unless stated otherwise, were of
commercially available reagent grade quality and were used without
further purification. Flash chromatography purifications were
performed on Merck silica gel 60 (0.04−0.063 mm). Nuclear magnetic
resonance spectra (1H NMR) were recorded on a Bruker 400 MHz
spectrometer at 300 K and are referenced in ppm (δ) relative to TMS.
Coupling constants (J) are expressed in hertz (Hz). HPLC−MS
experiments were performed either on an Acquity UPLC apparatus,
equipped with a diode array and a Micromass SQD single quadruple
(Waters) or on an Agilent 1100, equipped with a diode array and a
Bruker ion−trap Esquire 3000+. Purity was monitored at 254 nm, and
the purities of the compounds used for biological tests were found to
be at least 95% with the exception of 10 (90%) and 11 (90%) (see
Supporting Information).
1
orange solid. H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H), 7.94
(m, 1H), 7.52 (m, 1H), 7.44 (d, J = 15.67 Hz, 1H), 7.36 (m, 1H),
7.14−7.07 (m, 2H), 6.78−6.75 (m, 2H), 6.66 (d, J = 15.67 Hz, 1H),
6.61 (m, 1H), 5.25 (s, 2H). m/z (ES+), (M + H)+ = 273.
(E)-N-(3-Chlorophenyl)-3-[3-[(methylsulfonyl)amino]-
phenyl]-prop-2-enamide (11). A solution of 3-[(methylsulfonyl)-
amino]cinnamic acid 1i (329 mg, 1.36 mmol), oxalyl chloride (0.35
mL, 4.1 mmol), and catalytic dry DMF in dry DCM (15 mL) was
stirred at reflux for 1.5 h. After cooling to rt, solvent and excess oxalyl
chloride were evaporated under reduced pressure and the residue was
taken up in dry toluene and concentrated. A solution of 3-
chloroaniline 2a (0.144 mL, 1.36 mmol) and triethylamine (0.284
mL, 2.04 mmol) in dry DCM (8 mL) was added dropwise at 0 °C to
the raw acyl chloride in toluene. After stirring at rt for 16 h, the
reaction mixture was diluted with DCM and washed with water, 0.5N
aqueous hydrochloric acid, NaHCO3 (satd aq), and brine. Organic
layer was dried over sodium sulfate, concentrated under reduced
pressure, and purified by flash silica chromatography (DCM/acetone
97:3) and trituration with diisopropyl ether/methanol to afford (E)-N-
(3-chlorophenyl)-3-[3-[(methylsulfonyl)amino]phenyl]-prop-2-enam-
ide 11 (50 mg, 14%) as a beige solid. 1H NMR (400 MHz, DMSO-d6)
δ 11.5 (s, 1H), 9.92 (s, 1H), 7.94 (m,bs, 1H), 7.60−7.34 (m, 6H),
7.25−7.12 (m, 2H), 6.77 (d, J = 15.65 Hz, 1H), 3.35 (s, 3H). m/z (ES
+), (M + H)+ = 351.
3-Acetoxy-4-hydroxycinnamic Acid (1n). NaH (60%; 4.5 g,
113.29 mmol) was added portionwise to a solution of 3-hydroxy-4-
methoxycinnamic acid (10 g, 51.5 mmol) in dry THF (220 mL) while
stirring at 0 °C under a nitrogen atmosphere. Stirring was continued at
0 °C for about 40 min, then acetic anhydride (7.3 mL, 77.25 mmol)
was added dropwise at 0 °C. The reaction mixture was heated at reflux
for about 3 h, further acetic anhydride (1.46 mL, 15.4 mmol) was
added, and heating continued for additional 6 h. After cooling to rt,
solvents were evaporated; the residue was taken up in 2 N HCl and
water, extracted with EtOAc. The combined organic layers were
washed with water and brine, dried over sodium sulfate, and
concentrated to dryness. The resulting colorless powder was triturated
in EtOAc, filtered, and dried under vacuum at 40 °C to afford 3-
acetoxy-4-hydroxycinnamic acid 1n (10 g, 82%) as a colorless powder.
1H NMR (500 MHz, DMSO-d6) δ 12.27 (s, 1H), 7.58−7.55 (m, 1H),
(E)-N-(3-Chlorophenyl)-3-(1H-indol-6-yl)-prop-2-enamide
(12). EDC hydrochloride (245 mg, 1.28 mmol) and HOBt (173 mg,
1.28 mmol) were added to a solution of (E)-3-(1H-indol-6-yl)-prop-2-
enoic acid 1j (120 mg, 0.64 mmol) in dry DCM (6 mL) while cooling
at 0 °C. The mixture was allowed to warm to room temperature and
stirred for 45 min. 3-Chloroaniline 2a (0.082 mL, 0.77 mmol) was
added, and the mixture was stirred at room temperature for 4 h, then
at reflux for 7 h. After cooling, the mixture was diluted with DCM,
washed with NaHCO3 (satd aq), dried over sodium sulfate, filtered,
and concentrated. The residue was purified by flash silica
chromatography (elution gradient: DCM/MeOH 100:0.5) to afford
(E)-N-(3-chlorophenyl)- 3-(1H-indol-6-yl)-prop-2-enamide 12 (82
7.54−7.49 (m, 2H), 7.15 (m, 1H), 6.40 (d, J = 16.1 Hz, 1H), 3.81 (s,
3H), 2.26 (s, 3H). m/z (ES+), (M + Na)+ = 259.
(E)-N-(3-Chlorophenyl)-3-(3,4-dihydroxyphenyl)-prop-2-en-
amide (3). 3-Chloroaniline 2a (23.6 μL, 2.22 mmol) and N,N′-
dicyclohexylcarbodiimide (504 mg, 2.44 mmol) were added to a
solution of caffeic acid 1a (400 mg, 2.22 mmol) in THF (10 mL), and
the resulting mixture was stirred at reflux for 7 h. After cooling to
room temperature, the solid residue was filtered off and the remaining
solution was evaporated. The residue was purified by flash silica
chromatography (elution gradient: 20−50% EtOAc in n-hexane) to
afford (E)-N-(3-chlorophenyl)-3-(3,4-dihydroxyphenyl)-prop-2-enam-
1
mg, 43%) as a pale-yellow powder. H NMR (400 MHz, DMSO-d6)
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δ 11.35 (s, 1H), 10.30 (s, 1H), 7.95 (t, J = 2, 1H), 7.70 (d, J = 15.6,
1H), 7.64 (s, 1H), 7.59 (d, J = 8.4, 1H), 7.54−7.52 (m, 1H), 7.46 (t, J
= 2.6, 1H), 7.36 (t, J = 8, 1H), 7.30 (dd, J = 8.4, J = 1.2, 1H), 7.11 (dd,
J = 8, J = 1.2, 1H), 6.75 (d, J = 15.6, 1H), 6.47 (m, 1H). m/z (ES+),
(2M + Na)+ = 615.
(5 mL, 1.67 mmol), at room temperature for about 1.5 h. Solvents
were evaporated, and the residue was taken up in MeOH and
concentrated (twice), then triturated with diethyl ether and filtered to
give (E)-3-(3-hydroxy-4-methoxyphenyl)-N-indan-1-yl-acrylamide 28
1
(237 mg, 100%) as a green powder. H NMR (400 MHz, DMSO-d6)
(E)-N-(3-Chlorophenyl)-3-(4-amino-3-hydroxyphenyl)-prop-
2-enamide (14). SnCl2·2H2O (562 mg, 2.5 mmol) was added to a
solution of (E)-N-(3-chlorophenyl)-3-(3-hydroxy-4-nitrophenyl)-
prop-2-enamide 14a (159 mg, 0.5 mmol) in EtOH (6 mL). The
mixture was stirred at reflux for about 1.5 h, cooled to room
temperature, and concentrated. The residue was taken up with
NaHCO3 (satd aq) and Rochelle’s salt solution and extracted with
EtOAc. The combined organic layers were washed with brine, dried
over sodium sulfate, and concentrated under reduced pressure to
afford (E)-N-(3-chlorophenyl)-3-(4-amino-3-hydroxyphenyl)-prop-2-
enamide 14 (89 mg, 62%) as a yellow powder. 1H NMR (400
MHz, DMSO-d6) δ 10.16 (s, 1H), 9.34 (s, 1H), 7.93 (t, J = 1.6 Hz,
1H), 7.50 (m, 1H), 7.34 (m, 2H), 7.08 (m, 1H), 6.92 (s, 1H), 6.87 (m,
1H), 6.59 (d, J = 8.0 Hz, 1H), 6.38 (t, J = 15.6 Hz, 1H), 5.15 (s, 2H).
m/z (ES+), (M + H)+ = 289.
(E)-N-(3-Aminophenyl)-3-(3-hydroxy-4-methoxyphenyl)-
prop-2-enamide (18). A suspension of (E)-3-(3-hydroxy-4-methox-
yphenyl)-N-(3-nitrophenyl)-prop-2-enamide 18a (314 mg, 1.00
mmol) and SnCl2·2H2O (1.128 g, 5.00 mmol) in EtOH (20 mL)
was heated at reflux for 1.5 h. The resulting solution was cooled to
room temperature and concentrated. The residue was taken up with
NaHCO3 (satd aq) and Rochelle’s salt solution and extracted with
EtOAc. The combined organic layers were dried over sodium sulfate,
filtered, and concentrated. Purification by flash silica chromatography
(DCM/MeOH 98:2) afforded (E)-N-(3-aminophenyl)-3-(3-hydroxy-
4-methoxyphenyl)-prop-2-enamide 18 (110 mg, 39%) as an off-white
powder. 1H NMR (400 MHz, DMSO-d6) δ 9.75 (s, 1H), 9.19 (s, 1H),
7.38 (d, J = 15.6 Hz, 1H), 7.02−6.90 (m, 5H), 6.79 (d, J = 8.0 Hz,
1H), 6.60 (d, J = 15.6 Hz, 1H), 6.26 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H),
5.05 (s, 2H), 3.81 (s, 3H). m/z (ES+), (2M + Na)+ = 591.
( E)- N- ( 3 - C a r b o x a m i d o p h e n yl ) - 3 - ( 3 - a c e t o xy - 4 -
methoxyphenyl)prop-2-enamide (24a). A solution of 3-acetoxy-4-
methoxycinnamic acid 1n (0.45 g, 1.9 mmol), thionyl chloride (0.14
mL, 2.66 mmol) ,and 3 drops of DMF in dry THF (10 mL) was
stirred at reflux for 2 h. Then a further aliquot of thionyl chloride (0.05
mL, 0.76 mmol) was added, and the mixture was stirred at reflux
temperature for additional 2 h. After cooling to about 5 °C, a solution
of 3-aminobenzamide 2j (0.259 g, 1.9 mmol) and triethylamine (0.53
mL, 3.8 mmol) in dry DCM (5 mL) was added dropwise. After stirring
at rt overnight, THF was removed under vacuum and the residue was
triturated in DCM to give a first aliquot of the target cinnamic anilide
24a as a beige solid (185 mg). After filtration, the solution was washed
with 1N aqueous hydrochloric acid and NaHCO3 (satd aq), dried over
sodium sulfate, and evaporated to afford a second aliquot of 24a. The
two solids were mixed and triturated in DCM to yield (E)-N-(3-
carboxamidophenyl)-3-(3-acetoxy-4-methoxyphenyl)prop-2-enamide
24a (671 mg, 79%). m/z (ES+), (M + Na)+ = 377.
(E)-N-(3-Carboxamidophenyl)-3-(3-hydroxy-4-
methoxyphenyl)prop-2-enamide (24). A suspension of (E)-N-(3-
carboxamidophenyl)-3-(3-acetoxy-4-methoxyphenyl)prop-2-enamide
24a (535 mg, 1.5 mmol) and NaOH (50% in water) (0.16 mL, 3
mmol) in MeOH (5 mL) was stirred at reflux for 1 h. After cooling to
room temperature, MeOH was evaporated, the reaction mixture was
diluted with water, and pH was adjusted to 6 with 2 N HCl. The
resulting precipitate was filtered off, washed with water, and dried
under vacuum to afford (E)-N-(3-carboxamidophenyl)-3-(3-hydroxy-
4-methoxyphenyl)prop-2-enamide 24 (252 mg, 54%) as a white solid.
1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 9.23 (s, 1H), 8.10 (s,
δ 8.39 (m, 1H), 7.35 (d, J = 15.6 Hz, 1H), 7.27−7.17 (m, 4H), 6.98−
6.93 (m, 3H), 6.44 (d, J = 16.0 Hz, 1H), 5.39 (q, J = 7.6 Hz, 1H), 3.79
(s, 3H), 2.99−2.92 (m, 1H), 2.86−2.78 (m, 1H), 2.47−2.39 (m, 1H),
1.86−1.80 (m, 1H). m/z (ES+), (2M + Na)+ = 641.
(E)-3-(3-Hydroxy-4-methoxy-phenyl)-N-(2-naphthyl)-prop-2-
enamide (29). K2CO3 (61.4 mg, 0.44 mmol) was added to a solution
of (E)-3-(3-acetoxy-4-methoxyphenyl)-N-(2-naphthyl)-prop-2-enam-
ide 29a (80 mg, 0.22 mmol) in MeOH:THF:water 10:1:1 (4.8 mL).
After stirring for 2 h at rt, solvents were evaporated, the residue was
partitioned between EtOAc, and water and the aqueous phase was
extracted with EtOAc. The combined organic layers were dried over
sodium sulfate, filtered, and concentrated to dryness to afford (E)-3-
(3-hydroxy-4-methoxyphenyl)-N-(2-naphthyl)-prop-2-enamide 29 (70
1
mg, 100%) as a yellow powder. H NMR (500 MHz, DMSO-d6) δ
10.39 (s, 1H), 9.02−8.97 (m, 1H), 8.41 (s, 1H), 7.91−7.77 (m, 3H),
7.72−7.64 (m, 1H), 7.43−7.42 (m, 1H), 7.50−7.32 (m, 3H), 7.09−
7.03 (m, 1H), 7.00−6.88 (m, 2H), 6.66 (d, J = 15.7 Hz, 1H), 3.80 (s,
3H). m/z (ES+), (M + H)+ = 318.
(E)-N-Benzoxazol-4-yl-3-(3-acetoxy-4-methoxyphenyl)prop-
2-enamide (31a). A solution of 3-acetoxy-4-hydroxycinnamic acid 1n
(236 mg, 1 mmol) and thionyl chloride (0.09 mL, 1.2 mmol) in dry
THF (5 mL) was stirred at 55 °C for 3 h. Then a further aliquot of
thionyl chloride (0.07 mL, 1 mmol) was added, and the mixture was
stirred at reflux for additional 2 h. After cooling to rt, solvents were
evaporated, and dry THF (3 mL) was added, followed by a solution of
4-amino-benzoxazole 2q (168 mg, 1.25 mmol) and triethylamine (0.42
mL, 3 mmol) in dry THF (2 mL). After stirring at rt for 2 h, solvent
was evaporated, and the residue was taken up in EtOAc and washed
with water, NaHCO3 (satd aq), and brine. The organic layer was dried
over sodium sulfate, filtered, and evaporated. The residue was
triturated in acetone and then DCM to afford (E)-N-benzoxazol-4-
yl-3-(3-acetoxy-4-methoxyphenyl)prop-2-enamide 31a (66 mg, 19%)
1
as a light-yellow powder. H NMR (500 MHz, DMSO-d6) δ 2.28 (s,
3H) 3.82 (s, 3H) 7.11−8.34 (m, 8H) 8.79 (s, 1H) 10.26 (s, 1H). m/z
(ES+), (M + H)+ = 353.
(E)-3-(3-Hydroxy-4-methoxyphenyl)-N-(1-methyl-1H-inda-
zol-4-yl)prop-2-enamide hydrochloride (32). A solution of 3-
acetoxy-4-hydroxycinnamic acid 1n (236 mg, 1 mmol) and thionyl
chloride (0.08 mL, 1.1 mmol) in dry THF (5 mL) was stirred at 55 °C
for 2 h. Then a further aliquot of thionyl chloride (0.03 mL, 0.4 mmol)
was added, and the mixture was stirred at reflux for additional 2 h.
After cooling to rt, a solution of 4-amino-1-methyl-indazole 2r (147
mg, 1 mmol) and triethylamine (0.42 mL, 3 mmol) in dry THF (2
mL) was added dropwise. After stirring at rt for 22 h, solvent was
evaporated, and the residue was taken up in EtOAc and washed with
water, NaHCO3 (satd aq), and brine. The organic layer was dried over
sodium sulfate, concentrated under reduced pressure, and purified by
flash silica chromatography (DCM/EtOAc 90:10), followed by
trituration with diethyl ether. The resulting solid (30 mg, 0.08
mmol) was suspended in 3 N HCl in MeOH (2 mL) and stirred at rt
for 2 h. Solvents were evaporated, and the residue was taken up in
MeOH and concentrated (twice) and finally triturated in diethyl ether
to afford (E)-3-(3-hydroxy-4-methoxyphenyl)-N-(1-methyl-1H-inda-
zol-4-yl)prop-2-enamide hydrochloride 32 (20 mg, 6%) as a beige
1
powder. H NMR (400 MHz, DMSO-d6) δ 10.10 (s, 1H), 9.24 (s,
1H), 8.31 (s, 1H), 7.88 (m, 1H), 7.50 (d, J = 15.6 Hz, 1H), 7.37−7.32
(m, 2H), 7.09−7.06 (m, 2H), 6.99 (d, J = 8.0 Hz, 1H), 6.86 (d, J =
15.6 Hz, 1H), 4.03 (s, 3H), 3.82 (s, 3H). m/z (ES+), (M + H)+ = 324.
(E)-3-(3-Hydroxy-4-methoxyphenyl)-N-methoxy-N-methyl-
prop-2-enamide (35). N,O-Dimethylhydroxylamine hydrochloride
(1.2 g, 12.4 mmol), EDC hydrochloride (2.38 g, 12.4 mmol), and
HOBt (837 mg, 6.2 mmol) were added to a suspension of 3-hydroxy-
4-methoxycinnamic acid 1d (2 g, 10.3 mmol) in DCM (10 mL). After
stirring at room temperature for 16 h, the reaction mixture was diluted
with DCM and washed with water. Purification by flash silica
1H), 7.92−7.87 (m, 2H), 7.54 (d, J = 7.6 Hz, 1H), 7.45 (d, J = 15.6
Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.32 (s, 1H), 7.05−7.03 (m, 2H),
6.97 (d, J = 8.4 Hz, 1H), 6.60 (d, J = 15.6 Hz, 1H), 3.81 (s, 3H). m/z
(ES+), (2M + H)+ = 625.
(E)-3-(3-Hydroxy-4-methoxyphenyl)-N-indan-1-yl-acryla-
mide (28). (E)-3-(3-Acetoxy-4-methoxyphenyl)-N-indan-1-yl-acryla-
mide 28a (267 mg, 0.76 mmol) was treated with 3 N HCl in MeOH
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chromatography (n-hexane/EtOAc 1:1) afforded (E)-3-(3-hydroxy-4-
methoxyphenyl)-N-methoxy-N-methyl-prop-2-enamide 35 (607 mg,
25%) as an off-white powder. 1H NMR (400 MHz, CDCl3) δ 7.66 (d,
1H, J = 15.7), 7.23 (d, 1H, J = 2.1), 7.07 (dd, 1H, J = 8.3, 2.1), 6.91 (d,
1H, J = 15.7), 6.86 (d, 1H, J = 8.3), 3.94 (s, 3H), 3.78 (s, 3H), 3.32 (s,
3H). m/z (ES+), (M + H)+ = 238.
purified by flash silica chromatography (elution gradient: 2−5%
MeOH in DCM) to afford 5-[5-(3-chloroanilino)-4H-1,2,4-triazol-3-
yl]-2-methoxyphenol 41 (81 mg, 32%) as a white powder. H NMR
(400 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.28 (br s, 1H), 7.76 (t, J = 2.05
Hz, 1H), 7.45 (dd, J = 8.22, 2.05 Hz, 1H), 7.36−7.42 (m, 2H), 7.24 (t,
J = 8.07 Hz, 1H), 7.06 (d, J = 9.10 Hz, 1H), 6.83 (dd, J = 7.63, 1.47
Hz, 1H), 3.83 (s, 3H). m/z (ES+), (M + H)+ = 317.
1
trans-rac-N-Methoxy-N-methyl-2-(3-hydroxy-4-
methoxyphenyl)cyclopropanecarboxamide (36). DMSO (7
mL) was added dropwise to a mixture of NaH (274 mg, 6.88
mmol) and trimethylsulfoxonium iodide (1.5 g, 6.88 mmol). After
stirring at room temperature for 30 min, a solution of (E)-3-(3-
hydroxy-4-methoxyphenyl)-N-methoxy-N-methyl-prop-2-enamide 35
(407 mg, 1.72 mmol) in DMSO (3 mL) was added, and the resulting
mixture was stirred overnight. The reaction mixture was poured into
water, acidified to pH 3 with 2 N HCl, and extracted with diethyl
ether. The combined organic layers were dried over sodium sulfate and
concentrated under reduced pressure. Purification by flash silica
chromatography afforded trans-rac-N-methoxy-N-methyl-2-(3-hy-
droxy-4-methoxyphenyl)cyclopropane-carboxamide 36 (331 mg,
77%) as an off-white powder. 1H NMR (400 MHz, CDCl3) δ
6.79−6.69 (m, 3H), 3.89 (s, 3H), 3.72 (s, 3H), 3.25 (s, 3H), 2.48−
2.41 (m, 1H), 2.36 (m, 1H), 1.62−1.58 (m, 2H), 1.28−1.25 (m, 1H) .
m/z (ES+), (M + H)+ = 252.
5-[(E)-2-[5-(3-Chlorophenyl)-1,3,4-oxadiazol-2-yl]ethenyl]-2-
methoxyphenyl acetate (43). A mixture of 3-acetoxy-4-hydrox-
ycinnamic acid 1n (210 mg, 0.9 mmol), 3-chlorobenzoic acid
hydrazide 42 (157.5 mg, 0.9 mmol), and POCl3 (2.5 mL) was heated
at reflux for 1 h. After cooling to rt, the reaction mixture was poured
onto ice/water and extracted with EtOAc. The combined organic
layers were washed with NaHCO3 (satd aq) and brine, dried over
sodium sulfate, filtered, and concentrated to dryness. Purification by
flash silica chromatography (n-hexane/EtOAc 80:20) afforded 5-[(E)-
2-[5-(3-chlorophenyl)-1,3,4-oxadiazol-2-yl]ethenyl]-2-methoxyphenyl
acetate 43 (100 mg, 30%) as a colorless powder. 1H NMR (500 MHz,
DMSO-d6) δ 8.11 (m, 1H), 8.08−8.05 (m, 1H), 7.77 (d, J = 16.4 Hz,
1H), 7.75−7.65 (m,4H), 7.28 (d, J = 16.4 Hz, 1H), 7.22 (m, 1H), 3.84
(s, 3H), 2.30 (s, 3H). m/z (ES+), (M + H)+ = 371.
5-[(E)-2-[5-(3-Chlorophenyl)-1,3,4-oxadiazol-2-yl]ethenyl]-2-
methoxyphenol (44). A solution of 5-[(E)-2-[5-(3-chlorophenyl)-
1,3,4-oxadiazol-2-yl]ethenyl]-2-methoxyphenyl acetate 43 (100 mg,
0.27 mmol) in MeOH (2 mL) was treated with 4 N HCl in 1,4-
dioxane (1.1 mL, 0.27 mmol) at rt for 6 h. Solvents were evaporated
and the residue taken up in diethyl ether and filtered. Purification by
flash silica chromatography (elution gradient: 10−25% acetone in n-
hexane) afforded 5-[(E)-2-[5-(3-chlorophenyl)-1,3,4-oxadiazol-2-yl]-
ethenyl]-2-methoxyphenol 44 (20 mg, 22%) as a colorless powder. 1H
NMR (500 MHz, DMSO-d6) δ 9.21 (s, 1H), 8.13 (m, 1H), 8.07 (m,
1H), 7.74−7.64 (m, 3H), 7.22 (m, 2H), 7.09 (d, J = 16.4 Hz, 1H),
7.01 (m, 1H), 3.83 (s, 3H). m/z (ES+), (M + H)+ = 329.
tra ns-r ac -N-(3-Chlorophenyl)-2-(3-hydroxy-4-
methoxyphenyl)cyclopropanecarboxamide (37). To a suspen-
sion of trans-rac-N-methoxy-N-methyl-2-(3-hydroxy-4-
methoxyphenyl)cyclopropane-carboxamide 36 (330 mg, 1.31 mmol)
in diethyl ether (8 mL) was added t-BuOK (884 mg, 7.88 mmol)
followed by water (47 mL). The reaction mixture was stirred for 20 h,
then poured onto crushed ice and extracted twice with diethyl ether.
The aqueous phase was cooled to 0 °C and acidified to pH 1 with 1 N
HCl. Extraction with diethyl ether afforded raw trans-rac-2-(3-hydroxy-
4-methoxyphenyl)cyclopropanecarboxylic acid as a yellow oil that was
used in the next step without any further purification. 2-(3-Hydroxy-4-
methoxyphenyl)cyclopropanecarboxylic (130 mg, 0.62 mmol), 3-
chloroaniline 2a (86 μL, 0.81 mmol), and a catalytic amount of HOBt
were added to a suspension of EDC hydrochloride (144 mg, 0.75
mmol) in DCM (5 mL). The resulting mixture was stirred at room
temperature for 36 h, poured into water, and the organic phase was
washed twice with water, dried over sodium sulfate, filtered, and
concentrated. Purification by flash silica chromatography (n-hexane/
EtOAc 3:1) afforded trans-rac-N-(3-chlorophenyl)-2-(3-hydroxy-4-
methoxyphenyl)cyclopropanecarboxamide 37 (57 mg, 29%) as an
N-(3-Chlorophenyl)-3-(3-hydroxy-4-methoxy-phenyl)-
propanamide (46). A solution of 3-(3-hydroxy-4-methoxy-phenyl)-
propanoic acid (0.5 g, 2.57 mmol), thionyl chloride (0.2 mL, 2.83
mmol), and 3 drops of DMF in dry THF (15 mL) was stirred at reflux
for 2 h. Then a further aliquot of thionyl chloride (50 μL, 0.69 mmol)
was added, and the mixture was stirred at reflux temperature for
additional 2 h. After cooling to about 5 °C, a solution of 3-Cl-aniline
(0.27 mL, 2.57 mmol) and triethylamine (0.71 mL, 5.14 mmol) in dry
DCM (5 mL) was added dropwise. After stirring at rt for 16 h, the
reaction mixture was concentrated under vacuum, diluted with DCM,
and washed with water, 0.5 N aqueous hydrochloric acid, and brine.
Organic layer was dried over sodium sulfate, concentrated under
reduced pressure, and purified by flash silica chromatography (hexane/
EtOAc 6:4) to afford N-(3-chlorophenyl)-3-(3-hydroxy-4-methoxy-
1
off-white powder. H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H),
8.90 (s, 1H), 7.83 (t, 1H, J = 2 Hz), 7.44−7.43 (m, 1H), 7.32 (m, 1H,
J = 8.1 Hz), 7.10−7.07 (m, 1H), 6.83 (d, 1H, J = 8.1), 6.60−6.56 (m,
2H), 3.73 (s, 3H), 2.25 (m,1H), 1.95 (m, 1H), 1.43 (m, 1H), 1.24 (m,
1H). m/z (ES+), (M + H)+ = 318.
1
phenyl)propanamide 46 (430 mg, 55%) as an off-white powder. H
N-[(3-Chlorophenyl)carbamothioyl]-3-acetoxy-4-methoxy-
benzamide (40). A solution of 3-acetoxy-4-methoxybenzoic acid 38
(0.5 g, 2.38 mmol), thionyl chloride (0.19 mL, 2.62 mmol), and 3
drops of DMF in dry THF (10 mL) was stirred at reflux for 2 h. The
mixture was then concentrated in vacuo, treated twice with toluene,
and dried. The crude was taken up with CH3CN (5 mL), KSCN (231
mg, 2.38 mmol) was added, and the resulting mixture was heated at
reflux for additional 2 h. After cooling to room temperature, KCl was
filtered off, 3-chloroaniline (225 μL, 2.14 mmoL) in CH3CN (2 mL)
was added, and the solution was stirried for 0.5 h at rt. Filtration of the
resulting solid afforded N-[(3-chlorophenyl)carbamothioyl]-3-acetoxy-
NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.79 (s, 1H), 7.81 (t, J =
2.0 Hz, 1H), 7.43−7.40 (m, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.09−7.06
(m, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.66 (d, J = 2.0 Hz, 1H), 6.60 (dd, J
= 8.4 Hz, J = 2.0 Hz, 1H), 3.71 (s, 3H), 2.76 (t, J = 8.0 Hz, 2H), 2.55
(t, J = 8.0 Hz, 2H). m/z (ES+), (2M + Na)+ = 633.
Induction of mPTP Opening in Isolated Mitochondria.
Mitochondria were freshly prepared from C57/bl6 male mouse livers
as previously described.35 Briefly, each mouse liver was excised after
cervical dislocation and placed in ice-cold “mitochondria buffer” (250
mM sucrose, 10 mM Tris-HCl, 0.1 mM EGTA, pH 7.4). Liver was
rinsed 3−4 times with ice-cold mitochondria buffer, minced with
scissors, and passed through a 7 mL Dounce homogenizer (Wheaton)
using three strokes with a “loose” pestle in ice. The homogenate was
diluted to 50 mL and centrifuged at 900g for 10 min at 4 °C (Beckman
Avanti J-25 refrigerated). The supernatant was carefully decanted and
centrifuged at 7000g for 10 min at 4 °C. The mitochondrial pellet was
carefully washed with 2 mL of ice-cold mitochondria buffer, diluted to
50 mL, and spun at 7000g for 10 min at 4 °C. The resulting
mitochondrial pellet was resuspended in 500 μL of mitochondria
buffer and stored in ice. Protein concentration was determined using
the Biuret assay.
1
4-methoxybenzamide 40 (704 mg, 87%) as a white powder. H NMR
(300 MHz, DMSO-d6) δ 12.58 (br s, 1H), 11.49 (br s, 1H), 8.00 (dd, J
= 8.6, 2.2 Hz, 1H), 7.95 (s, 1H), 7.84 (d, J = 2.3 Hz, 1H), 7.65−7.53
(m, 1H), 7.45 (t, J = 7.9, 1H), 7.33 (ddd, J = 7.9, 2.0, 0.9 Hz, 1H), 7.29
(d, J = 8.8 Hz, 1H), 3.89 (s, 3H), 2.30 (s, 3H). m/z (ES+), (M + H)+
= 379.
5-[5-(3-Chloroanilino)-4H-1,2,4-triazol-3-yl]-2-methoxyphe-
nol (41). A solution of N-[(3-chlorophenyl)carbamothioyl]-3-acetoxy-
4-methoxybenzamide 40 (0.3 g, 0.79 mmol) and hydrazine hydrate
(0.19 μL, 3.95 mmol) in CHCl3 (8 mL) was stirred at reflux for 2 h.
After cooling to room temperature, the resulting solid was filtered and
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Isolation of Mitochondria from Mouse Heart. Hearts were
minced with scissors (into very small pieces) in 3 mL of mitochondria
buffer (250 mM Sucrose, 10 mM Tris-HCl, 0.1 mM EGTA, 0.1% BSA;
pH 7.4) and homogenized with a 1 mL Dounce homogenizer
(Wheaton) using four strokes of a “loose” pestle and two strokes of a
“tight” pestle. The homogenates were centrifuged in 1.5 mL tubes at
3100 rpm (Heraeus Biofuge Fresco centrifuge) for 10 min at 4 °C.
The supernatant was then centrifuged at 8700 rpm for 10 min, and the
resulting pellet was washed with 1 mL of mitochondria buffer (without
BSA) followed by another centrifugation at 8700 rpm for 10 min.
Pellet was then resuspended in CRC buffer (see CRC assay
description).
Mitochondrial Swelling Assay. For mitochondrial swelling
experiments, 100 μL of assay buffer (120 mM KCl, 1 mM H2KPO4,
10 mM MOPS, 20 μM EGTA, 5 mM glutamate, 2.5 mM malate, pH
7.4) containing mitochondrial solution at 1 mg/mL was placed in each
well of a 96-well plate. Then 100 μL of assay buffer containing 300 μM
CaCl2 was then added to the solution with a final concentration of 0.5
mg/mL for mitochondria and 150 μM CaCl2. Mitochondrial swelling
was then followed by measuring the change in absorbance at 540 nm
using a spectrophotometer (Spectramax Plus-384, Molecular Devices,
Sunnyvale, CA, USA), with a reading every 9 s/well for a total time of
10 min. Then 2 μL of the compounds at the desired concentrations
were added before the addition of the mitochondrial solution.
Mitochondrial Calcium Retention Capacity (CRC) Assay.29
Mitochondrial CRC was assessed fluorimetrically in the presence of
the fluorescent Ca2+ indicator Calcium Green 5N (Molecular Probes
C3737; ex-em 505−535 nm) using a temperature-controlled
PerkinElmer LS 55 spectrofluorimeter. Mitochondria (0.5 mg) were
diluted in 1 mL of CRC buffer (120 mM KCl, 1 mM H2KPO4, 10 mM
MOPS, 20 μM EGTA, 5 mM glutamate, 2.5 mM malate, pH 7.4)
containing 1 μM Calcium Green. CaCl2 (1 μL of 2 mM stock) was
added every minute to 200 μL of the mitochondrial suspension, and its
uptake was followed by measuring extra-mitochondrial calcium green
fluorescence (PerkinElmer spectrofluorimeter at 25 °C) until mPTP
opening was achieved. Compounds were added to the mitochondria
immediately prior to the start of the experiment. CsA was used as
positive control at a final concentration of 1 μM.
photographed again. The sections of the heart were evaluated for area
at risk (area of the heart perfused by the occluded artery) and infarct
size using the photographs taken during necropsy. The images
including the ruler were analyzed with ImageJ 1.32j software from the
National Institutes of Health (NIH) and evaluated by group pairwise
analysis (ANOVA).
hERG Binding Assay. Interaction with the hERG channel was
assessed by displacement of the radioligand [3H]-astemizole as
described by Chiu et al.36
Assessment of Cardiovascular Parameters in the Conscious
Rat. Cardiovascular parameters were measured in conscious female rat
following a single intravenous administration of compound 22 by
means of a telemetry system (Data Science International, St. Paul,
MN, USA). Animals used in this study had been instrumented about 1
week before the start of the study. Telemetry transmitters (model
TL11M2-C50-PXT) implanted in the abdominal cavity allowed
recording of blood pressure (via femoral artery) and body temper-
ature.
Prior to the start of the study, animals were checked for general
health status and for the functionality of the radio transmitters
(readable and normal blood pressure waveform).
Cardiovascular parameters were collected from at least 1 h before
treatment to about 24 h after treatment. The following parameters
were calculated from the recorded blood pressure and body
temperature waveforms: heart rate (bpm), systolic blood pressure
(mmHg), diastolic blood pressure (mmHg), average blood pressure
(mmHg), and body temperature (°C).
Plasma Exposure in the Rabbit. Blood samples were collected
from the jugular vein of New Zealand white rabbits (n = 5). Samples
were collected predose and at 5, 15, 30, 60, and 240 min post
reperfusion and placed in tubes containing K2EDTA as anticoagulant.
The blood samples were stored on an ice block until centrifuged. The
plasma was collected and temporarily stored on dry ice until stored
frozen at −50 to −90 °C until the analysis. Briefly, 100 μL of plasma
were added to a Sirocco filter plate (Waters) containing a mixture of
ACN (300 μL) and 25% H3PO4 (10 μL). The plate was shaken for 20
min and filtered under vacuum (15 mmHg) for 3 min. Sample analysis
was performed on an Acquity UPLC injecting 5 μL of the resulting
solution on a Acquity HSS T3 column (50 mm × 2.1 mm × 1.8 μm, T
= 40 °C; eluent, water, ACN, 0.1% HCOOH gradient from 2% B to
100% B in 1.3 min flow 0.45 mL/min), coupled with a sample
organizer and interfaced to a triple quadrupole Premiere XE (Waters,
Milford, MA). The mass spectrometer was operated using electrospray
interface (ESI) with a capillary voltage of 3.5 kV, cone voltage of 23 V,
collision energy of 17 V, extractor 5 V, source temperature of 120 °C,
desolvation gas flow of 800 L/h, and desolvation temperature of 480
°C. Collision energy was optimized for each compound. LC-MS/MS
analyses were carried out using a positive electrospray ionization
(ESI(+)) interface in MRM (multiple reaction monitoring) mode.
Plasma concentrations of compound 22 were extrapolated on an
eight-point calibration curve (2.5−1000 ng/mL). QC samples of the
test compound at three different concentrations (high, medium, and
low) were considered for acceptance of the analytical runs with an
accuracy within 15% except at the LLOQ of 2.5 ng/mL (lowest limit
of quantification) where 20% was accepted.
In Vivo Studies. All aspects of animal care, use, and welfare for all
animals used for in vivo studies were performed in strict compliance
with the U.S. Department of Agriculture’s (USDA) Animal Welfare
(Rabbit experiment) or with EU and Italian Guidelines for Laboratory
Animal Welfare (mouse and rat experiments).
Ex Vivo CRC. Mice hearts were excised, flushed with perfusion
buffer (118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 2.15 KH2PO4,
0.6 mM MgSO4, 1.69 mM CaCl2, 11.1 mM glucose), and connected to
a Langendorff reperfusion apparatus. Beating hearts were first perfused
with perfusion buffer for 2 min prior to treatment and then with
compound 22 at 5 μM concentration with 0.1% DMSO in the
perfusion buffer or DMSO (0.1% in perfusion buffer) for 2 min.
Hearts were then immediately placed in ice followed by mitochondria
preparation, and CRC evaluation was performed as described above.
In Vivo Ischemia Reperfusion Injury in Rabbits. New Zealand
white rabbits were subjected to left anterior descending coronary
artery ligation followed by reperfusion. Briefly, animals were
anesthetized (30 mg/kg sodium pentobarbitol iv), and a sternotomy
was performed to visualize the left anterior descending artery (LAD).
The LAD was temporarily ligated and maintained occluded for 30 min.
At 5 min prior to reperfusion, the animals were administered
compound 22 (5 mg/kg in 20% DMSO; 40% PEG400) or vehicle
via iv bolus. At the end of the 30 min occlusion, the knot was loosened
leaving the ligature in place, and the vessel was allowed to reperfuse for
4 h. At the end of the reperfusion period, each animal was injected
intravenously (iv) with 8−10 mL of 0.5% Fast Green. The animals
were euthanized, and the hearts were flushed with heparinized saline
until cleared of blood. The hearts were cut into four slices which were
photographed (cranial surface and caudal surface), along with a
calibrated measurement block. The slices were then incubated in a 1%
solution of triphenyltetrazolium chloride (TTC) in sodium phosphate
buffer (pH 7.4) for 20 min at 37 °C. The heart rings were weighed and
Pharmacokinetic parameters were calculated by a noncompartmen-
tal method using WinNonLin 5.1 software (Pharsight, Mountain View,
CA).
Plasma Exposure in the Rat. Blood samples were collected from
the retro-orbital plexus of WI(Glx/BRL/Han)IGS rats. Samples were
collected at 0.083, 0.332, 1, 2, 4, 7, and 24 h and placed in tubes
containing K2EDTA as anticoagulant. The blood samples were stored
on an ice block until centrifuged. The plasma was collected and
temporarily stored on dry ice until stored frozen at −50 to −90 °C
until the analysis. The same sample preparation and analytical method
developed for the rabbit (see above) was used to quantify compound
22 in the rat plasma.
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Article
glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid;
EtOAc, ethyl acetate; FCCP, trifluorocarbonylcyanide phenyl-
hydrazone; HOBt, 1-hydroxybenzotriazole; IS, infarct size;
LAD, left anterior descending; LRI, lethal reperfusion injury;
MOPS, 3-(N-morpholino)propanesulfonic acid; mPT, mito-
chondrial permeability transition; mPTP, mitochondrial per-
meability transition pore; PCI, percutaneous coronary inter-
vention; TTC, triphenyltetrazolium chloride
ASSOCIATED CONTENT
* Supporting Information
■
S
CRC of mouse liver mitochondria after incubation with 0.1, 0.5,
and 5 μM inhibitor; synthesis and characterization of
compounds 5−9, 13, 15−17, 19−23, 25−27, 30, 31, 33, 34,
10a, 14a, 18a, 25a, 26a, 27a−30a, 33a, and 34a; purity of key
1
compounds as determined by HPLC; H NMR and UPLC
traces of compounds 6 and 22; summary results of in vitro
selectivity assays (compound 6); reactivity assay of compound
22 with N-acetyl cysteine or glutathione; plasma levels of 22 in
the rat telemetry study. This material is available free of charge
via the Internet at http://pubs.acs.org.
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ABBREVIATIONS USED
■
Macia, C.; Raczka, F.; Sportouch, C.; Gahide, G.; Finet, G.; Andre-
́
AAR, area at risk; ACN, acetonitrile; CRC, calcium retention
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