Discovery of 3,3′-pyrrolidinyl-spirooxindoles as cardioprotectant prohibitin ligands

Article abstract of DOI:10.1016/j.ejmech.2019.111859

The scaffold proteins prohibitins-1 and 2 (PHB1/2) play many important roles in coordinating many cell signaling pathways and represent emerging targets in cardiology and oncology. We previously reported that a family of natural products derivatives, flavaglines, binds to PHB1/2 to exert cardioprotectant and anti-cancer effects. However, flavaglines also target the initiation factor of translation eIF4A, which doesn't contribute to cardioprotection and may even induce some adverse effects. Herein, we report the development of a convenient and robust synthesis of the new PHB2 ligand 2′-phenylpyrrolidinyl-spirooxindole, and its analogues. We discovered that these compounds displays cardioprotective effect against doxorubicin mediated cardiotoxicity and uncovered the structural requirement for this activity. We identified in particular some analogues that are more cardioprotectant than flavaglines. Pull-down experiments demonstrated that these compounds bind not only to PHB2 but also PHB1. These novel PHB ligands may provide the basis for the development of new drugs candidates to protect the heart against the adverse effects of anticancer treatments.

Full text of DOI:10.1016/j.ejmech.2019.111859

Journal Pre-proof
Discovery of 3,3′-pyrrolidinyl-spirooxindoles as cardioprotectant prohibitin ligands
Sabria Elderwish, Anaïs Audebrand, Canan G. Nebigil, Laurent Désaubry
PII:
S0223-5234(19)31011-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.111859
EJMECH 111859
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 September 2019
Revised Date: 4 November 2019
Accepted Date: 5 November 2019
Please cite this article as: S. Elderwish, Anaï. Audebrand, C.G. Nebigil, L. Désaubry, Discovery of
3,3′-pyrrolidinyl-spirooxindoles as cardioprotectant prohibitin ligands, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111859.
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Graphical Abstract

Discovery of 3,3’-pyrrolidinyl-spirooxindoles as cardioprotectant
prohibitin ligands
Sabria Elderwish,a Anaïs Audebrand,b Canan G. Nebigil,b and Laurent Désaubry,a
aLaboratory of Medicinal Chemistry and Cardio-oncology, CNRS, 4 rue Blaise Pascal, 67081 Strasbourg,
France
bLaboratory of Medicinal Chemistry and Cardio-oncology, CNRS, Ecole Supérieure de Biotechnologie de
Strasbourg, Illkirch, France
Correspondence to: Laurent Désaubry, email: Désaubry@unistra.fr
Keywords: oxindoles; cardiotoxicity; prohibitins; STAT3; doxorubicin
ABSTRACT: The scaffold proteins prohibitins-1 and 2 (PHB1/2) play many important roles in
coordinating many cell signaling pathways and represent emerging targets in cardiology and oncology.
We previously reported that a family of natural products derivatives, flavaglines, binds to PHB1/2 to
exert cardioprotectant and anti-cancer effects. However, flavaglines also target the initiation factor of
translation eIF4A, which doesn’t contribute to cardioprotection and may even induce some adverse
effects. Herein, we report the development of a convenient and robust synthesis of the new PHB2
ligand 2'-phenylpyrrolidinyl-spirooxindole, and its analogs. We discovered that these compounds
displays cardioprotective effect against doxorubicin mediated cardiotoxicity and uncovered the
structural requirement for this activity. We identified in particular some analogues that are more
cardioprotectant than flavaglines. Pull-down experiments demonstrated that these compounds bind
not only to PHB2 but also PHB1. These novel PHB ligands may provide the basis for the development
of new drugs candidates to protect the heart against the adverse effects of anticancer treatments.
1

1. Introduction
Prohibitins-1 and 2 (PHB1/2) are scaffold proteins found in every eukaryotic cells that control a
wide range of physiological processes through direct interactions with more than one hundred proteins
[1]. The various functions and the intracellular localization of PHBs are regulated by many post-
translational modifications that modulate their interaction with their protein partners.
Several classes of PHB ligands that display different a wide array of pharmacological activities
have been identified [1]. This variety of biological activities probably stems from the multitude of post-
translational modifications of the PHBs targeted by these drugs. The most studied PHB ligands are
natural compounds called flavaglines, such as FL3 (1, Figure 1) that induce both cardioprotectant and
anticancer effects [1-12]. In addition to PHBs, flavaglines also inhibit the initiation factor of protein
synthesis eIF4a, which may account for some adverse effects [13]. Thus, to develop new
cardioprotectant PHB ligands that do not inhibit eIF4a is highly desirable. Such drugs would be also very
useful to explore the role of PHBs in many physiological and pathophysiological settings.
Recently, Sen and coll. reported a new PHB2 ligand, the spiro-oxindole 2 (Figure 1), that is weakly
cytotoxic in MCF-7 breast cancer cells (EC50: 3.5 μM) [14]. Unfortunately whether this cytotoxicity was
mediated by PHB2 was not examined. Moreover, the stereochemistry of this compound (which could
correspond to 2a or 2b) was not reported. We noticed that spiro-oxindole 2a displays some structural
features with the pharmacophore of flavaglines 1. Indeed, the two aryl moieties of 2a perfectly overlap
with those of flavaglines. The replacement of the ether of flavaglines by a methylene abolishes the
pharmacological activities, indicating that a dipole is necessary in this area. The lactam of 2a may provide
an even stronger dipolar interaction with PHB2, which could compensate for the absence of the
dimethoxyaryl moiety, the tertiary hydroxyl and the dipole (hydroxy, ketone or imine) adjacent to it.
Figure 1. Structure of flavagline FL3 (1), spiro-oxindole 2a and 2b and overlay of 1 with 2a.
2

As part of our ongoing medicinal chemistry on new PHB ligands [2-12, 15, 16], we aimed at
comparing the effects of our previous PHB ligands to those of 2a/b and explore the structural
requirement for its effects on PHB signaling.
2. Results
2.1. Synthesis of 3,3’-pyrrolidinyl-spirooxindoles
The reported synthesis of 2 involves a one pot Pictet-Spengler condensation of tryptamine 3 to
benzaldehyde combined to N-bromosuccinimide-induced ring contraction catalyzed by trifluoroacetic
acid (Scheme 1) [14]. This straightforward synthesis occurred with a 94% yield. Unfortunately, the
stereochemistry of the obtained adducts was not disclosed. It is also unclear whether one or two
diastereoisomers were produced.
Scheme 1. Reported one-pot Pictet Spengler-Oxidative ring contraction to furnish the oxindole 2 [14] and
current observation of the formation of the dihydro-β-carboline 7.
Our attempts to reproduce the disclosed synthesis of 2 [14] did not to deliver the expected
compound. Indeed, we only observed the formation of the dihydro-β-carboline 7, probably through
intermediates 5 and 6 (Scheme 1), which is in line with previous reports [17, 18]. We then explored
several routes and eventually found that protection of the secondary amine of tetrahydro-β-carboline
with a 2-nitrobenzyl group followed by a NBS-induced oxidative rearrangement affords the protected
spiro-oxindole 9, which was irradiated by UV [19] to afford an inseparable 71:29 mixture of the desired
3

secondary amines 2a and 2b (Scheme 2). The structure of 2a/2b was confirmed by key HMBC
correlations from the protons of the pyrrolidine moiety and the C-2 and C-3 carbons (Figure 2).
1
Surprisingly the H NMR spectrum of this product did not correspond to the reported one [14]. Indeed,
the protons of the pyrrolidine moiety Ha and Hb show evident upfield shifts when compared to the
reported ones. In addition, the benzylic protons Hc of 2a and 2b are upfield and downfield of the
reported one, and the chemical shifts of the aromatic protons extend from 6.50 to 7.48 ppm (vs 7.01 to
7.50 for the reported ones).
Figure 2. Key HMBC correlations of 2a/2b.
This approach was also used to prepare the N-methyl derivatives 11 via the methylation of
oxindole 10 with methyl iodide.
4

Scheme 2. First approach for the synthesis of oxindoles 2a/b and 12a/b.
To circumvent the use of the 2-nitrobenzyl as a protecting group, we developed an alternative
approach for the synthesis of spiro-oxindoles 2a and 2b in 4 steps with an overall yield of 83% (average
yield: 95%) with only two steps of purification by chromatography (Scheme 3). Tryptamine 3 was most
efficiently protected by ethyl trifluoroacetate and oxidized into oxindole by HCl in DMSO [20]. Removal
of the trifluoroacetamide protecting group and condensation with benzaldehyde in the presence of
triethylamine afforded the expected adducts 2a and 2b as a 71:29 inseparable mixture of 2
diastereomers in 92% yield (Scheme 3). A modification of this approach afforded the brominated
analogues 17a/b. Bromination of 13 and hydrolysis in strong acidic medium afforded the amino-oxindole
16 that underwent a Mannich condensation with benzaldehyde to provide the expected adducts 17a/b
with a 12% yield.
5

Scheme 3. Second approach for the synthesis of oxindoles 2a and 2b and synthesis of the brominated
derivative 16a/b.
To explore the structural requirements of pyrrolidinyl-spirooxindoles for their pharmacological
effects, we introduced several substituents on 2a/b. A Pictet-Spengler condensation of tryptophan
methyl ester 18 with benzaldehyde followed by a N-bromosuccinimide-induced ring contraction afforded
a mixture of 4 diastereoisomers 20a-d that could be separated (Scheme 4). We also synthesized a series
of N-alkylated derivatives 22 using the same approach as before.
6

Scheme 4. Synthesis of oxindole derivatives 20 and 22.
To verify that 3,3’-pyrrolidinyl-spirooxindoles do bind to PHBs, we conjugated these compounds
to beads of Affigel-10 through a linker anchored at N1 or N1’ via the same strategies as above using
iodoazide 23 [21] as an alkylating agents (Scheme 5). Azides 25 and 29 were conveniently reduced by
1,3-propanedithiol [22] into amines 26 and 30.
7

Scheme 5. Synthesis of immobilized ligands 27 and 31.
2.2. Synthesis of flavaglines-spirooxindoles hybrids
To enhance the similarity between spirooxindoles and flavaglines, we considered to introduce a
hydroxyl and an amide to mimic the cognate hydroxyl and dipolar function (hydroxyl, enamine or imine)
that are critical to flavaglines activity (Figure 3).
Figure 3. Spiro-oxindole 2a, pharmacophore of flavaglines 32, hybrid PHB ligands 33 and 34 and
substructure searched in SciFinder 35.
8

Surprisingly, a substructure search for the motif 35 in SciFinder identified only 3 compounds [23-
25], raising the possibility that the preparation of this type of compounds might present some
difficulties. Indeed, several routes were explored for the synthesis of 16a and 16b and we finally used the
recently developed one-pot thiol-Michael /Mannich/ lactamization reaction [26] to construct the
functionalized spiro-γ-lactam oxindoles 38a/b as key intermediates (Scheme 6). The starting oxindoles
37a/b were conveniently prepared from isatins 36a/b by a Horner–Wadsworth–Emmons olefination.
Successive condensation of 37a with p-toluenethiol, benzaldehyde, NH4OAc and a catalytic amount of
piperidine gave a mixture of diastereoisomeric adducts 38a (21%) and 39a (5%). Gratefully, the N-methyl
oxindole 37b gave better yields (46% of the desired isomer 38b and 28% of diastereomer 39b).
Scheme 6. Synthesis of spiro-γ-hydroxylactam oxindoles 33 and 34.
With 38a/b in hand, we pushed forward toward the completion of the hydroxylated spiro-γ-
lactam oxindoles 33 and 34. To replace the thioether moiety by a hydroxyl, we envisioned to generate an
α-ketoamide by a Pummerer rearrangement that could be reduced into an α- hydroxyamide. Thioethers
38 were oxidized to sulfoxides 40 using mCPBA. Initial attempts to perform a Pummerer-type reaction
using HCl in DMSO, TBABr and PIDA, Ac2O, or AcBr did not deliver the expected compound. Finally, we
found that a treatment with TFAA in presence of triethylamine delivered a mixture of α-acyloxy sulfides
9

41 and α-ketoamides 42, which were stereoselectively reduced in situ by sodium borohydride to deliver
the expected α-hydroxylactams 33 and 34.
2.2. Biological results
Cardioprotectant activity
We first examined whether spiro-oxindole 2a/b protects cardiomyocytes against the apoptosis
induced by doxorubicin, similarly to other PHB ligands such as flavaglines [2, 6]. Indeed, anthracyclines,
such as doxorubicin, remain the cornerstone of chemotherapies for a various cancers, even if their use is
limited by significant dose-dependent cardiotoxicity, which is caused, at least partially, by the
accumulation of reactive oxygen species (ROS) in cardiomyocytes [27]. Previous reports from our lab
showed that flavaglines, such as FL3 (1, Figure 1) protect cardiomyocytes in vitro and in vivo against the
apoptosis induced by doxorubicin due to their action on PHBs leading to the phosphorylation of STAT3
[2, 8].
The cardioprotective activity of 2a/b was first determined in H9c2 cardiomyocytes that
represents a well-established in vitro model of doxorubicin-induced cardiotoxicity (Figure 4A).
Preincubation of H9c2 cardiomyocytes for 10h with different concentrations 2a/b significantly promoted
cardioprotection against acute doxorubicin-induced apoptosis with a maximal effect at 1 nM.
Accordingly, 2a/b was also found to suppress the production of ROS by 26 ± 0.1 % at 1 nM (Figure 4B).
Figure 4. Cardioprotectant activity of spiro-oxindole 2a/b. H9c2 cardiomyocytes were pretreated with
2a/b (1 nM-10 µM) or its vehicle for 10 h and then treated with doxorubicin (DOX) (1 μM) for additional
14 h (acute model of cardiotoxicity). A) Pro-survival effect of 2a/b. Viability levels were measured by
cQuant (n =3-4). B) Inhibition of doxorubicin-induced ROS production by 2a/b. ROS production was
detected by DCFDA (n=3-4). **P<0.05 vs vehicle;
*P<0.05 vs DOX.
10

Considering that 2a/b displayed optimal activities at 0.1 and 0.3 nM, its derivatives were
examined at these concentrations for their effects on survival and the level of ROS in H9c2
cardiomyocytes treated by doxorubicin (Table 1).
Table 1. Cardioprotectant activity of spiro-oxindoles. H9c2 cardiomyocytes were pretreated by FL3, 2a/b,
its analogues (0.3 or 1 nM) or their vehicle for 10 h and then treated with doxorubicin (DOX) (1 μM) for
additional 14 h (acute model of cardiotoxicity). A) Pro-survival effect of 2a/b. Viability levels were
measured by cQuant (n =3-4). B) Inhibition of doxorubicin-induced ROS production by 2a/b. ROS
production was detected by DCFDA (n=3-4).
Sub-
Increase in survival (%)
Decrease in ROS (%)
Entry Compound
Structural featurea
structure
0.3 nM
0
1nM
0
0.3 nM
0
1nM
0
1
2
3
4
5
Vehicle
1 (FL3)
2a/b
3.5±2.9
7.7±0.2
0.3±0.2
1.1±0.2
9.7±0.2
9.5±0.2
12.5±0.1
0.0±0.1
3.2±0.1
15.9±0.4
A
A
A
11.3±0.3
2.6±0.4
6.5±0.1
18.8±0.1
3.2±0.1
6.5±0.1
12a/b
17a/b
R2 = Me
R1 = Br
R4 = COOMe
(2'R,3R,5'R)
R4 = COOMe
(2'S,3S,5'R)
R3 = Me
6
7
20a
20b
B
B
4.0±0.1
2.9±0.2
6.4±0.1
7.1±0.2
3.1±0.2
2.9±0.2
9.4±0.1
2.7±0.2
9
22a
22b
22c
22d
22e
22f
B
B
B
B
B
B
5.4±0.4
6.0±0.4
1.3±0.3
0.9±0.3
2.6±2.6
1.4±5.5
11
7.6±0.3
7.4±0.2
0.8±0.3
5.7±0.1
0.2±0.2
0.2±0.2
9.4±0.1
9.4±0.1
3.1±0.2
6.2±0.2
0.0±0.1
0.0±0.2
15.6±0.1
12.5±0.1
6.2±0.1
10
11
12
13
14
R3 = Et
R3 = n-Pr
R3 = n-Bu
12.5±0.2
12.5±0.2
9.4±0.2
R3 = cyclopentyl
R3 = CH2CONMe2

R3=
15
22g
B
0.3±0.3
0.3±0.2
0.0±0.1
6.2±0.1
16
17
18
19
22h
22i
33
B
B
C
C
R3 = allyl
4.6±0.2
6.7±0.8
3.0±0.2
1.9±0.1
11.6±0.3
10.7±0.2
5.80.1
12.7±0.3
14.3±1.9
6.25±0.2
3.2±0.1
20.6±1.8
20.5±1.6
21.9±0.2
2.1±1.8
R3 = i-Pr
34
R2 = Me
2.9±0.6
a: only the substituents different from a proton are indicated.
While the introduction of a methyl on N1 (12a/b), a bromine on C6 (17a/b) or a methyl
carboxylate on 5’ (20a/b) were detrimental, the introduction of a methyl (22a) or an ethyl (22b) on the
amine was well tolerated, but bulkier substituents, such as n-propyl (22c-g) was less effective. Gratefully,
the introduction of an allyl (22h) an isopropyl (22i) slightly enhanced the cardioprotectant activity. This
observation also suggests that in the mixture 2a/b the cardioprotectant activity is carried by 2a, which
has the same configuration as 22h and 22i. These two compounds being particularly active, we
compared their cardioprotectant effect at several concentrations to that of flavagline FL3 (Figure 5). At
subnanomolar concentration 22i was more cardioprotectant than FL3, but at 10 and 30 nM FL3 was
slightly more efficient than 22i.
Figure 5. Cardioprotectant activity of FL3, 22h and 22i. A) H9c2 cardiomyocytes were pretreated with
FL3, 22h or 22i (0.1-30 nM) for 10 h and then treated with doxorubicin (DOX) (1 μM) for additional 14 h
(acute model of cardiotoxicity). Viability levels were measured by cQuant and indicated as % of increase
12

in survival over DOX (n =3-4). B) % of Inhibition of DOX-induced ROS production detected by DCFDA (n=3-
4).
Replacement of the pyrrolidine moiety by a 3-hydroxy-5-aryl-pyrrolidin-2-one that displays some
commune features with both flavaglines and spiro-oxindole 2a was disappointing: 33 and 34 exhibited
cardioprotectant activities lower than that of 2a/b or the flavagline FL3, suggesting that 3,3’-pyrrolidinyl-
spirooxindoles and flavaglines don’t have the same binding mode with PHBs [28].
Binding to PHBs and activation of STAT3
To verify that spiro-oxindole do bind to PHB2 and possibly to PHB1, we performed a pull-down
experiment with immobilized ligands 27 and 31. Interestingly, Western blot analysis showed that these
ligands interact with both PHB1 and PHB2 (Figure 6A). No unspecific binding was observed with Affi-Gel
10 beads containing only the linker as a negative control. Similarly to FL3 [6], 22i induced a
phosphorylation of the pro-survival factor STAT3 indicating that both compounds share a same
mechanism of action involving PHBs (Figure 6B).
Figure 6. Binding to PHBs and STAT3 activation. (A) Pull-down assay results demonstrating an interaction
with PHB1 and PHB2. Western blot analysis of proteins pulled down from the spiro-oxindole-coupled
13

Affi-Gel beads 27 and 31 and negative control (NC) coupled to Affi-Gel10 beads (NC). (B) Phosphorylation
of STAT3 in H9c2 cardiomyocytes exposed to 22i (10 nM) relative to actin levels determined by
immunoblotting of whole-cell lysates.
3. Discussion
3,3’-Pyrrolidinyl-spirooxindoles have attracted intense interest for their diverse pharmacological
activities [29-33]. Sen and coll. disclosed a straightforward synthesis of 3,3’-pyrrolidinyl-spirooxindole 3
and found that it binds to PHB2 [14]. In our hand this reported synthesis of PHB2 ligand 3 could not be
reproduced. We thus developed two robust syntheses of this compound and founds that it exists as a
1
71:29 mixture of two regioisomers 2a and 2b in equilibrium, which have an H and 13C NMR spectra
different from the reported ones [34]. We also prepared a series of 17 analogues of 2a, including spiro-γ-
hydroxylactam oxindoles 16a and 16b, which required the development of an original strategy based on
a one-pot thiol-Michael/Mannich/lactamization reaction followed by a 3-step conversion of a thioether
into a hydroxyl.
Several families of PHBs ligands have been described, and up to now only flavaglines, such as FL3
(1), have been shown to display cardioprotective activities [1]. Other classes of compounds, such as
sulfonylamidines, Mel9 or fluorizoline are inactive or even toxic to cardiomyocytes (unreported results)
[3, 35, 36]. This study is the first one to uncover a second family of cardioprotective PHB ligands. We
performed the first hit to lead optimization of 2a/b and demonstrated that the introduction of an
isobutyl or an allyl on the pyrrolidine nitrogen promotes the cardioprotection. Such compounds present
the advantage of not being a mixture of diastereomers in equilibrium like 2a/b.
Our in vitro data established that a pretreatment with 3,3’-pyrrolidinyl-spirooxindoles
significantly increased viability and reduced ROS accumulation against doxorubicin-injured H9c2
cardiomyocytes. We showed that 3,3’-pyrrolidinyl-spirooxindoles bind both PHB1 and PHB2 and induce
the phosphorylation of STAT3 to promote the survival of H9c2 cardiomyocytes against doxorubicin
induced cardiotoxicity. PHBs have been reported to promote the survival of many noncancerous cell
types, including cardiomyocytes [37-48]. STAT3 phosphorylation [49] and overexpression [50] have also
been shown to protect cardiomyocytes from doxorubicin toxicity. In mitochondria, STAT3 inhibits the
14

opening of mitochondrial permeability transition pores (MPTP) [51] and modulates the electron
transport chain [52]. STAT3 has also been shown to regulate ROS generation and mitochondrial
respiration by interacting with mitochondrial complex I and complex II [53,54]. In addition, recent
findings indicate that STAT3 directly interact with the mitochondrial calcium uniporter (MCU) to alleviate
calcium overload [55], which is a key event in doxorubicin-induced cardiotoxicity [56].
Previously, we showed that the flavagline FL3 binds to PHBs to induce the phosphorylation of
STAT3 and its translocation to mitochondria to trigger a cardioprotective pathway [44]. As far as we
know, 3,3’-pyrrolidinyl-spirooxindoles, have not been reported to activate STAT3 or to protect
cardiomyocytes against doxorubicin toxicity.
4. Conclusion
We uncovered a new class of PHB ligand that will provide the basis for the development of new
medicines to protect cancer patients from the cardiac adverse effects of chemotherapies. These
compounds can also be very valuable to explore PHB signaling in various disease-relevant contexts.
5. Material and methods
5.1. Synthetic Procedures.
Detailed synthetic methods can be found in SI Materials and Methods.
5.2. Cell viability
H9c2 cardiomyocytes was purchased from American Type Culture Collection (ATCC) (Rockville,
MD, USA). Viability of H9c2 cardiomyocytes was evaluated using CyQUANT-NF proliferation kit
(Invitrogen) as previously described [57]. Briefly, H9c2 cardiomyocytes were seeded at 10,000/well, in 96
wells plate in their completed medium overnight before the experiment. Cells were pretreated with the
compounds in 1% fetal calf serum (FCS) medium for 10 h and 1 µmol/l of DOX for additional 14h. Then
cell viability was determined on the supplier’s protocol. Relative cell viability was then assessed, using a
96-well microplate reader (Thermo fisher) with absorbance set at 490 nm.
5.3. ROS assays
ROS accumulation was measured in the H9c2 cardiomyocytes using 2’, 7’-
dichlorodihydrofluorescein diacetate (DCFDA) ROS detection assay kit (Abcam) according to
15

manufactures guidance. 1ꢀ×ꢀ104 cells were seeded in a clear bottom black 96-well plates and allowed for
attachment overnight in medium containing 10% FCS at 37°C in a 5% CO2 incubator. Cells were then
starved in the medium with 1% FCS for 10 h followed by treatment of the compounds for additional 14 h.
Cells were washed and loaded with 25 µM of DCFDA for 45 min at 37°C and exposed to DOX (15 µM) for
3h for the peak ROS accumulation as previously described [57]. At the end of treatment period,
fluorescence intensity was measured at Ex/Em, 485/535ꢀnm using Fluoskan AscentFluorescan Reader
(Thermo SCIENTIFIC).
5.4. Pull-down assay
Pull-down assay was performed as previously described [6]. H9c2 cardiomyocytes were lysed
using RIPA buffer (50 mM TrisHCl pH7.4, 150 mM NaCl, 2mM EDTA, 1% NP-40, 1.1% SDS) supplemented
with protease inhibitors (Halt protease inhibitor cocktail) and phosphatase inhibitors (Halt phosphatase
inhibitor cocktail) (all from Sigma). Cellular debris was removed by centrifugation at 13000 g for 10 mins
at 4°C. Cell lysate (500 μg) was incubated overnight with 40µl of spiro-oxindoles-coupled Affigel-10 beads
27 and 31 or negative control (NC) beads. Beads were washed three times with lysis buffer, bound
proteins were eluted by SDS-PAGE loading buffer and analyzed by western blot.
4.5. Western blot analysis
After the treatments, H9c2 cardiomyocytes were lysed in cold RIPA buffer (50mM TrisHCl pH7.4,
150mM NaCl, 2 mM EDTA, 1% NP-40, 1.1% SDS) and centrifuged 10 min at 14 000 rpm 4°C to obtain
protein extract. About 20 µg of protein per sample was loaded in SDS-PAGE electrophoresis and
transferred to Whatman PVDF membrane. Membranes were probed with antibody against
Phosphorylated-STAT3 (Ser724) (Cell Signaling, 1/1000), PHB1 (gift from Dr Valerie Bosch, DKFZ,
Heidelberg), PHB2 (Cell signaling, 1/1000) and β-Actin (Santa-Cruz, 1/5000). Membranes were treated
with peroxidase-conjugated secondary antibodies (Santa-Cruz, 1/10000) and immunoreactivity was
detected with an ECL Prime chemoluminescence detection kit (GE Healthcare) and quantified by Image J.
16

Statistical analysis
Data are expressed as mean ± standard error of the mean (SEM). Statistical analyses were
conducted by using GraphPad Prism software version 6.0 (GraphPad Software, La Jolla, California) for
unpaired student t test.
Acknowledgments
Financial support from Lybia's Ministry of Higher Education and Scientific Research for a
fellowship to S.E. is gratefully acknowledged. This work was also supported by a European ERA-NET grant
(ERA-CVD-JC2016, managed by Agence Nationale de la Recherche (ANR-16- ECVD-0005-01).
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Highlights
• A practical synthesis of 3,3’-pyrrolidinyl-spirooxindoles has been developed.
• 3,3’-pyrrolidinyl-spirooxindoles target the scaffold proteins prohibitins to display in vitro
cardioprotectant activities at nanomolar concentrations.
• The structural requirements for cardioprotection have been unraveled.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: