Identification of novel SIRT1 activators endowed with cardioprotective profile

Article abstract of DOI:10.1016/j.ejps.2021.105930

Drugs targeting epigenetic mechanisms are attracting the attention of scientists since it was observed that the modulation of this post-translational apparatus, could help to identify innovative therapeutic strategies. Among the epigenetic druggable targe

Full text of DOI:10.1016/j.ejps.2021.105930

European Journal of Pharmaceutical Sciences 165 (2021) 105930
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Identification of novel SIRT1 activators endowed with
cardioprotective profile
Lorenzo Flori1, Giovanni Petrarolo1, Simone Brogi*, Concettina La Motta*, Lara Testai*,
Vincenzo Calderone
Department of Pharmacy, University of Pisa, Via Bonanno, 6, I-56126 Pisa, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Drugs targeting epigenetic mechanisms are attracting the attention of scientists since it was observed that the
modulation of this post-translational apparatus, could help to identify innovative therapeutic strategies. Among
the epigenetic druggable targets, the positive modulation of SIRT1 has also been related to significant car-
dioprotective effects. Unfortunately, actual SIRT1 activators (natural products and synthetic molecules) suffer
from several drawbacks, particularly poor pharmacokinetic profiles. Accordingly, in this article we present the
development of an integrated screening platform aimed at identifying novel SIRT1 activators with favorable
drug-like features as cardioprotective agents. Encompassing several competencies (in silico, medicinal chemistry,
and pharmacology), we describe a multidisciplinary approach for rapidly identifying SIRT1 activators and their
preliminary pharmacological characterization. In the first step, we virtually screened an in-house chemical li-
brary comprising synthetic molecules inspired by nature, against SIRT1 enzyme. To this end, we combined
molecular docking-based approach with the estimation of relative ligand binding energy, using the crystal
structure of SIRT1 enzyme in complex with resveratrol. Eleven computational hits were identified, synthesized
and tested against the isolated enzyme for validating the in silico strategy. Among the tested molecules, five of
them behave as SIRT1 enzyme activators. Due to the superior response in activating the enzyme and its favorable
calculated physico-chemical properties, compound 8 was further characterized in ex vivo studies on isolated and
perfused rat hearts submitted to ischemia/reperfusion (I/R) period. The pharmacological profile of compound 8,
suggests that this molecule represents a prototypic SIRT1 activator with satisfactory drug-like profile, paving the
way for developing novel epigenetic cardioprotective agents.
SIRT1 activators
Epigenetics
Cardioprotection
Computer-aided drug discovery
Organic synthesis
Pyrido[1,2-a]pyrimidin-4-ones
Imidazo[1,2-a]pyridines
1. Introduction
HDAC7, HDAC9, and IIb: HDAC6, HDAC10); class IV: HDAC11) (Gre-
goretti et al., 2004). Dysregulation of fine-tuning deacetylase cellular
Nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase
and/or ADP-ribosylase enzymes, namely, sirtuins (SIRTs, silent infor-
mation regulator genes) belong to the protein superfamily of histone
deacetylase (HDAC) (Rine et al., 1979; Park and Kim, 2020). This latter
modulates the epigenetic cellular machinery, playing a crucial role in
gene regulation, being able to interact with several histone and
non-histone substrates (Martinez-Redondo and Vaquero, 2013; Galli-
nari et al., 2007). This family is currently divided into four classes and
eighteen members. Along with the mentioned class III SIRTs (SIRT1–7)
(Frye, 2000), HDACs are classified on the basis of domains organization,
functions, cellular localization, and sequence homology to yeast-related
proteins (HDAC1-11, class I: HDAC1-3, HDAC8; class II (IIa: HDAC4,5,
activity has been observed in several human diseases, including cancers,
cardiovascular diseases, metabolic syndromes, and neurodegenerative
disorders (Haberland et al., 2009; Carafa et al., 2012; Haigis and Sin-
clair, 2010; Houtkooper et al., 2012). Recently, altered expression of
HDAC family members has been observed in numerous rare diseases
(Brindisi et al., 2019; Landucci et al., 2018). In this scenario, the mod-
ulation of deacetylase activity by inhibitors and activators has been
proposed as a valuable approach for developing innovative therapeutics
for treating the mentioned diseases (Brindisi et al., 2018; Dokmanovic
et al., 2007; Kumar and Chauhan, 2016; Carafa et al., 2016).
Among the human SIRTs, SIRT1 has been the most investigated
isoform. This enzyme is implicated in several biological processes
* Corresponding authors.
E-mail addresses: simone.brogi@unipi.it (S. Brogi), concettina.lamotta@unipi.it (C. La Motta), lara.testai@unipi.it (L. Testai).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejps.2021.105930
Received 6 May 2021; Received in revised form 16 June 2021; Accepted 3 July 2021
Available online 12 July 2021
0928-0987/© 2021 Elsevier B.V. All rights reserved.

L. Flori et al.
European Journal of Pharmaceutical Sciences 165 (2021) 105930
(Nakagawa and Guarente, 2011), being able to deacetylate numerous
non-histone targets (Haigis and Guarente, 2006; North et al., 2003).
According to numerous transcription factors behaving as substrate of
SIRT1, this enzyme has emerged as a crucial interplay in many physio-
logical and pathological conditions. In fact, it has been demonstrated
that SIRT1 regulation is directly involved in cellular senescence (Ober-
doerffer et al., 2008), activating DNA repair machinery (Wang et al.,
2008), genome integrity (Bosch-Presegue and Vaquero, 2015), cardio-
vascular disorders (Potente et al., 2007; Winnik et al., 2015), cancer
(Liu et al., 2009), environmental and cellular stresses (Caito et al., 2010;
Schug et al., 2010), age-related pathologies (Ng et al., 2015), renal and
metabolic diseases (Banks et al., 2008; Kitada et al., 2013), viral infec-
tion and transactivation (Budayeva et al., 2016), female fertility (Tatone
et al., 2015), inflammation (Chung and Joe, 2014), and neurodegener-
ative disorders (Kim et al., 2007; Gan and Mucke, 2008).
Due to the implication of SIRT1 in regulating several pathways
linked to human diseases described above, small-molecules able to
negatively and positively affect the activity of SIRT1 are emerging as
interesting pharmacological tools in many disorders (Kumar and
Chauhan, 2016). Accordingly, while SIRT1 inhibitors can represent the
starting point for developing effective drugs against cancers and in-
flammatory diseases (Kumar and Chauhan, 2016; Hu et al., 2014), SIRT1
Fig. 1. Natural and synthetic compounds acting as SIRT1 activators.
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L. Flori et al.
European Journal of Pharmaceutical Sciences 165 (2021) 105930
activators are pivotal for developing innovative therapeutics against
cardiovascular disorders, aging, age-related and neurodegenerative
diseases and metabolic syndrome (Hubbard and Sinclair, 2014;
Chaudhary and Pfluger, 2009; Li et al., 2020; Donmez and Outeiro,
2013; Chong et al., 2012).
small-molecules have been identified as SIRT1 activators through
computational and medicinal chemistry approaches (Scisciola et al.,
2020; An et al., 2019; Pulla et al., 2014). Among them, small-molecules
acting as SIRT1 activators (e.g. SRT1460, SRT2104, SRT2183, and
SRT3025, Fig. 1) developed by Sirtris Pharmaceuticals (GlaxoSmithK-
line) advanced into clinical studies, with SRT2104 involved in several
clinical trials. Unfortunately, these potential drug candidates showed in
general poor and variable pharmacokinetics upon oral administration.
Several attempts aimed at reducing pharmacokinetic inconsistency have
also been described, for example, by employing different formulations of
the compound SRT2104, although without any appreciable results (Dai
et al., 2018). In view of that, the search for SIRT1 activators with
satisfactory drug-like profile is still a challenging task.
In particular, in the last decade, numerous compounds from natural
sources and synthetic molecules have been identified as SIRT1 activa-
tors. Among them, one of the most studied is resveratrol (Fig. 1). This
natural polyphenol has been characterized as SIRT1 activator by directly
binding to the enzyme (Cao et al., 2015). This binding produces an
activation of the enzyme that is relevant in several animal models of
disorders (cardiovascular and neurodegenerative) (Ma et al., 2017; Ma
et al., 2019). Along with resveratrol and its metabolite piceatannol
(Karaman Mayack et al., 2020), other natural compounds such as
quercetin, fisetin, vitexin, orientin, naringenin, curcumin, trans-(-
In this framework, pursuing our interest in SIRT1 activation related
to cardioprotection, and using a multidisciplinary approach, we suc-
ceeded in identifying novel SIRT1 activators as cardioprotective agents
by combining in silico techniques, organic synthesis, in vitro evaluation
and ex vivo studies. Starting from a structure-based approach based on
molecular docking and relative ligand binding energy, and taking into
account the mechanism of action of resveratrol (Cao et al., 2015), we
computationally screened a focused in-house library of synthetic mole-
cules inspired by natural compounds including both known (La Motta
et al., 2007) and original compounds to find compounds sharing a
ꢀ )-ε-viniferin, ginsenoside Rc and berberine were found to behave as
SIRT1 activators (Fig. 1) (Karaman Mayack et al., 2020; Iside et al.,
2020; Wang et al., 2016; Testai et al., 2020; Huang et al., 2021). Un-
fortunately, the low bioavailability and rapid metabolism of natural
products can preclude their effective clinical use. Today, new formula-
tions of these compounds, especially made for resveratrol and curcumin,
are under investigation to improve their pharmacokinetic profile (de
Vries et al., 2018). Along with natural products, different
Table 1
Final hits and their computational parameters derived from in silico studies.
Cpd
GlideScore (kcal/mol)
ΔGbind (kcal/mol)
SASAa
QPlogPb
2.43
QPlogSc
QPPCacod
2512
QPPMDCKe
1339
QPlogHERGf
%HOAg
100
1, GP14
#site1 ꢀ 6.32
#site2 ꢀ 6.43
#site3 ꢀ 6.78
#site1 ꢀ 7.91
#site2 ꢀ 6.45
#site3 ꢀ 6.22
#site1 ꢀ 7.32
#site2 ꢀ 6.18
#site3 ꢀ 6.94
#site1 ꢀ 8.49
#site2 ꢀ 6.41
#site3 ꢀ 6.29
#site1 ꢀ 8.14
#site2 ꢀ 7.36
#site3 ꢀ 7.19
#site1 ꢀ 8.62
#site2 ꢀ 6.31
#site3 ꢀ 7.88
#site1 ꢀ 7.09
#site2 ꢀ 6.22
#site3 ꢀ 6.41
#site1 ꢀ 8.56
#site2 ꢀ 6.91
#site3 ꢀ 8.95
#site1 ꢀ 7.91
#site2 ꢀ 6.81
#site3 ꢀ 6.47
#site1 ꢀ 8.44
#site2 ꢀ 6.99
#site3 ꢀ 6.83
#site1 ꢀ 7.00
#site2 ꢀ 6.86
#site3 ꢀ 6.92
#site1 ꢀ 7.12
#site2 ꢀ 7.16
#site3 ꢀ 7.21
#site1 ꢀ 81.6
#site2 ꢀ 63.1
#site3 ꢀ 73.5
#site1 ꢀ 86.3
#site2 ꢀ 65.1
#site3 ꢀ 72.6
#site1 ꢀ 86.8
#site2 ꢀ 65.7
#site3 ꢀ 79.5
#site1 ꢀ 94.7
#site2 ꢀ 69.1
#site3 ꢀ 75.7
#site1 ꢀ 88.3
#site2 ꢀ 73.7
#site3 ꢀ 74.2
#site1 ꢀ 86.5
#site2 ꢀ 72.9
#site3 ꢀ 81.6
#site1 ꢀ 84.2
#site2 ꢀ 66.8
#site3 ꢀ 75.6
#site1 ꢀ 106.3
#site2 ꢀ 75.7
#site3 ꢀ 85.2
#site1 ꢀ 93.8
#site2 ꢀ 71.4
#site3 ꢀ 75.2
#site1 ꢀ 107.4
#site2 ꢀ 72.7
#site3 ꢀ 75.9
#site1 ꢀ 86.3
#site2 ꢀ 75.7
#site3 ꢀ 85.1
#site1 ꢀ 89.2
#site2 ꢀ 64.7
#site3 ꢀ 81.4
459
ꢀ 2.65
ꢀ 5.46
2, GP36
3, GP35
4, GP42
5, GP32
6, GP37
7, GP46
8, GP44
9, GP30
10, GP27d
11, GP41
Rsvh
590
661
589
565
588
665
590
688
635
583
482
5.23
5.39
5.22
4.39
2.57
5.35
2.82
6.027
4.30
5.27
2.00
ꢀ 5.91
ꢀ 6.13
ꢀ 5.88
ꢀ 5.42
ꢀ 4.66
ꢀ 6.2
6867
6868
6866
2083
2423
6889
2910
6881
2432
8383
279
3970
3971
3969
1093
2198
3971
1830
3989
1301
4925
123
ꢀ 6.67
ꢀ 6.46
ꢀ 6.65
ꢀ 6.63
ꢀ 6.40
ꢀ 6.49
ꢀ 6.38
ꢀ 7.16
ꢀ 7.08
ꢀ 6.56
ꢀ 5.33
100
100
100
89
85
100
90
ꢀ 4.67
ꢀ 6.36
ꢀ 5.74
ꢀ 5.77
ꢀ 2.81
91
88
100
71
a
SASA predicted the total solvent accessible surface (range or recommended value for 95% of known drugs 300 - 1000).
QPlogP predicted octanol/water partition coefficient (range or recommended value for 95% of known drugs ꢀ 2 - 6.5).
QPlogS predicted aqueous solubility in mol/dm3(range or recommended value for 95% of known drugs ꢀ 6.5–0.5).
QPPCaco predicted apparent Caco-2 cell permeability in nm/sec (range or recommended value for 95% of known drugs >500 great).
b
c
d
e
f
QPPMDCK predicted apparent MDCK cell permeability in nm/sec (range or recommended value for 95% of known drugs >500 great).
QPlogHERG predicted IC50 values for blockage of HERG K+ channels (range or recommended value for 95% of known drugs below ꢀ 5).
g
h%HOA predicted human oral absorption on 0 to 100% scale (range or recommended value for 95% of known drugs >80% high). Range or recommended values
are reported in QikProp user manual.
h
Resveratrol.
3

L. Flori et al.
European Journal of Pharmaceutical Sciences 165 (2021) 105930
similar mechanism of SIRT1 allosteric activation. The identified in silico
hits were further screened on the bases of their drug-like profile, to select
molecules with potential SIRT1 activator profile endowed with favor-
able physico-chemical properties. The final hits obtained from this
computational protocol were then synthesized in appropriate amounts
for further characterization. In fact, to validate the observed in silico
activities, compounds were investigated in vitro for their capability to
activate SIRT1 enzyme. Moreover, a compound showing the most potent
SIRT1-activating profile, namely, 5-(2-(4-hydroxyphenyl)imidazo[1,
2-a]pyridin-6-yl)benzene-1,3-diol (8, GP44; Table 1), was assessed for
its potential as cardioprotective agent on isolated and perfused rat hearts
submitted to ischemia/reperfusion (I/R) period. Following this
screening platform, it has been possible to rapidly identify SIRT1 acti-
vators with significant cardioprotective profile.
(highlighted as #site1, #site2, and #site3 in Fig. 2A), as determined by
experimental studies (Cao et al., 2015). Moreover, the experimentally
solved complex SIRT1/resveratrol was acquired with a specific peptide
system
(7-amino-4-methylcoumarin
AMC-containing
peptide),
mimicking the p53-interacting residues. This peptide is positioned be-
tween NTD and catalytic domain (CD) interfaces (Fig. 2A). The identi-
fied binding sites diversely contribute in activating SIRT1 enzyme. In
fact, mutagenesis studies indicated that the interactions with #site1 and
#site2 were found necessary for activating SIRT1, whereas #site3 is
demarcated as “accessory site,” displaying a lesser impact regarding
enzyme activation (Cao et al., 2015). Accordingly, two resveratrol
molecules facilitate the interaction among the AMC-peptide and the
SIRT1-NTD and are extremely relevant for promoting a strong binding
between the peptide and SIRT1 stimulating the enzyme activity.
Starting from this mechanism of action, we developed a docking
protocol able to evaluate the affinity of a molecule into the three distinct
binding sites of SIRT1. Accordingly, we used this methodology to eval-
uate the affinity of the molecules, contained in the collection, for these
binding sites. To identify the most promising molecules we retained only
the ones showing a favorable affinity for each site comparable or better
than that found for resveratrol (Table 1). The affinity for the mentioned
binding sites for each molecule was evaluated considering the docking
score combined with the relative ligand binding energy (ΔGbind) (full
details of the computational protocol are reported in Materials and
Methods section). The calculation allowed us to identify compounds
showing a docking score for the three selected binding sites comparable
or better to that found for resveratrol. In particular, regarding #site1 we
found a docking score ranging from ꢀ 6.32 kcal/mol for compound 1
(GP14) to ꢀ 8.62 kcal/mol for compound 6 (GP37), while the ΔGbind
ranging from ꢀ 81.6 kcal/mol for compound 1 to ꢀ 107.4 kcal/mol for
compound 10 (GP27d). Considering the #site2 we found a docking score
ranging from ꢀ 6.18 kcal/mol for compound 3 (GP35) to ꢀ 7.36 kcal/mol
for compound 5 (GP32), while the ΔGbind ranging from ꢀ 63.1 kcal/mol
2. Results and discussion
2.1. In silico screening of potential SIRT1 activators
In the first step of our integrated screening platform, we performed a
virtual screening of a chemical library to rapidly identify potential
SIRT1 activators. To this end, we combined molecular docking-based
approach with the estimation of relative ligand binding energy, using
the crystal structure of SIRT1 enzyme in complex with resveratrol as
previously described by us (Testai et al., 2020). In this study, we used an
in-house library of synthetic molecules designed by taking inspiration
from natural compounds, including constrained analogs of resveratrol.
On the basis of their chemical structures, we hypothesized that these
compounds might share the same mechanism of SIRT1 activation
described for the natural derivatives.
Briefly, resveratrol activates SIRT1 by targeting a specific region of
the enzyme located in its N-terminal domain (NTD) (Fig. 2A). Specif-
ically, resveratrol binds NTD interacting with three distinct binding sites
Fig. 2. (A) Cristal structure of SIRT1 (gray cartoon representation) in complex with resveratrol (sticks representation) (PDB ID 5BTR); the different binding sites of
resveratrol, namely, #site1, #site2 and #site3 are represented in cyan, magenta, and orange, respectively; (B) binding mode of compound 8 (GP44, yellow sticks)
into SIRT1 with particular focus on #site1, #site2 (C) and #site3 (D). The p53-AMC-peptide is reported as green sticks. Residues in the binding sites are illustrated by
lines while hydrogen bonds are indicated as gray dotted lines. Pictures were produced employing PyMOL software (The PyMOL Molecular Graphics System, v1.8;
¨
Schrodinger, LLC, New York, 2015).
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L. Flori et al.
European Journal of Pharmaceutical Sciences 165 (2021) 105930
for compound 1 to ꢀ 75.7 kcal/mol for compounds 8 (GP44) and 11
(GP41). Furthermore, for the #site3 our calculation highlighted a
docking score ranging from ꢀ 6.22 kcal/mol for compound 2 (GP36) to
ꢀ 8.95 kcal/mol for compound 8, while the ΔGbind ranging from ꢀ 72.6
kcal/mol for compound 2 to ꢀ 85.2 kcal/mol for compound 8.
SIRT1, as highlighted by docking scores and relative ligand binding
energies (compound 8: #site1, GlideScore = ꢀ 8.56 kcal/mol, and
ΔGbind = ꢀ 106.3 kcal/mol; #site2, GlideScore = ꢀ 6.91 kcal/mol, and
ΔGbind = ꢀ 75.7 kcal/mol; #site3, GlideScore = ꢀ 8.95 kcal/mol, and
ΔGbind = ꢀ 85.2 kcal/mol. Resveratrol: #site1, GlideScore = ꢀ 7.12 kcal/
mol, and ΔGbind = ꢀ 89.2 kcal/mol; #site2, GlideScore = ꢀ 7.16 kcal/
mol, and ΔGbind = ꢀ 64.7 kcal/mol; #site3, GlideScore = ꢀ 7.21 kcal/
mol, and ΔGbind = ꢀ 81.4 kcal/mol) (Table 1). In particular, compound 8
into the #site1 establishes a H-bond with N226 and a relevant network
of hydrophobic interactions with the residues in the binding site and
with the p53-AMC-peptide. Regarding the #site2, compound 8 can
target the sidechain of N226 and p53-AMC-peptide by establishing two
H-bonds, in a similar fashion as resveratrol. Notably, in addition to the
mentioned H-bonds, compound 8 establishes a polar contact with the
backbone of E214. Hydrophobic contacts were observed and in partic-
The second step of our computational protocol consisted of evalu-
ating the drug-like profile of compounds with favorable affinity as found
by molecular docking calculation. From this assessment, we selected
only molecules having a drug-like profile more favorable than the one
displayed by resveratrol. In fact, the identified compounds showed
satisfactory LogP and solubility (LogS) along with a good capacity to
cross membranes, as suggested by the predicted values regarding the
models of permeability (Caco-2 and MDCK cells). In fact, these values
are abundantly over 500, indicating a strong ability to cross biological
barriers (Table 1). Moreover, the potential SIRT1 activators were pre-
dicted to be devoid of cardiotoxicity. The assessment of oral absorption
showed a favorable predicted profile for the identified compounds with
respect to resveratrol. Furthermore, this assessment highlighted the
scarce pharmacokinetic profile for resveratrol as largely reported in
literature. Additionally, the evaluation of the physico-chemical features
showed that the screened compounds are not pan-assay interference
compounds (PAINS), although the output highlighted a low risk warning
for compounds containing phenol and catechol moiety. In conclusion,
our computational approaches provided eleven potential SIRT1 activa-
tors with favorable drug-like profile (Table 1).
ular we noted a π-π stacking with the aromatic moiety of p53-AMC-
peptide. Finally, when docked into #site3, compound 8 can target,
with its catechol moiety, D292 and D298 by two H-bonds. Also, a H-
bond with the sidechain of E208 was observed (Fig. 2). This pattern of
interaction within SIRT1-binding sites accounted for a significant
interaction with the enzyme, sharing a similar mechanism found for
resveratrol. Accordingly, due to this similarity, compound 8 is identified
as one of the most promising SIRT1 activators.
Lastly, to validate the computational approach, the identified po-
tential SIRT1 activators (Fig. 3) were synthesized and submitted to a
biological evaluation.
Accordingly, applying the described in silico protocol, we identified
drug-like compounds with computational scores comparable or even
better with respect to the scores found for resveratrol. In Fig. 2 is re-
ported the docking output of compound 8, that is one of the most
promising screened compounds. Briefly, based on the computational
output, this compound can strongly target all three binding sites on
2.2. Synthesis of selected hit compounds
Compound 1, 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one (Fig. 3)
was synthesized as previously described (La Motta et al., 2007) by
Fig. 3. Chemical structures of potential SIRT1 activators.
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L. Flori et al.
European Journal of Pharmaceutical Sciences 165 (2021) 105930
condensing 2-aminopyridine 12 with ethyl 3-oxo-3-phenylpropanoate,
in the presence of polyphosphoric acid (PPA) at 100 ◦C (Scheme 1).
Derivatives 2–6 (Fig. 3), characterized by an imidazo[1,2-a]pyridine
central core, were obtained as outlined in Scheme 2. Reaction of 2-
amino-5‑bromo pyridine 13 with the suitably substituted 2‑bromo-1-
phenylethan-1-one, in the presence of potassium carbonate, achieved
the corresponding 2-phenyl-imidazo[1,2-a]pyridines 14a-c, which were
then coupled with differently substituted-phenylboronic acids under
Suzuky-Miyaura conditions (Blakemore, 2016) to obtain the desired
activators 2–4. Treatment of the methoxy derivatives 2 and 3 with BBr3
in a dichloromethane solution gave the corresponding hydroxy‑sub-
stituted compounds 5 and 6.
the range from 1 to 300 µM, exhibited a perfect concentration-response
curve (Fig. 5), demonstrating to be an activator of SIRT1. Accordingly,
compound 8 was further investigated in ex vivo experiments, to evaluate
its cardioprotective properties.
2.4. Anti-ischemic activity of compound 8 in an ex vivo model of I/R
The functional efficacy of compound 8 was investigated in a heart
isolated and perfused on Langendorff apparatus submitted to I/R pro-
tocol, which represents a well-known model to reproduce myocardial
damage induced by ischemic events. In our experimental conditions, an
I/R episode produced marked damage to the isolated hearts of vehicle-
treated rats. At this regard, a marked decrease of the functional pa-
rameters of myocardial contractile function (RPP) was observed during
the reperfusion time and at the end the value of RPP was 29 ± 3%,
compared to the pre-ischemic period (Fig. 6A). Accordingly, in vehicle-
treated hearts a wide and fast ischemic contracture was observed (MIC
= 85 ± 18% and Thc = 18 ± 1 min, Fig. 6B-C). The damage induced by
the I/R event was also visible through morphometric analysis, and a size
of ischemic area equal to 42 ± 2% was measured using formazan salt
(Fig. 6D). Finally, the dosage of lactate dehydrogenase (LDH) levels in
the perfusate, a well-known biochemical marker of myocardial injury,
confirmed the compromise of cardiac function. LDH was abundantly
released in the perfusate during the first part of reperfusion, according to
the literature (Breschi et al., 2006) and the total amount at the end of
reperfusion was 86 ± 9 U/g (Fig. 6E).
The imidazopyridines 7 and 8 (Fig. 3) were synthesized according to
the alternative synthetic sequence described in Scheme 3. A coupling
reaction between 2-amino-5-bromopyridine 13 and 3,5-dimethoxyphe-
nylboronic acid, under the standard conditions referenced above, gave
the key intermediate 5-(3,5-dimethoxyphenyl)pyridin-2-amine 15,
which was cyclized to the imidazo[1,2-a]pyridine 7 by reaction with
2‑bromo-1-(4-methoxyphenyl)ethan-1-one. The following ether cleav-
age, performed using BBr3 in a dichloromethane solution, afforded the
hydroxy‑substituted activator 8.
Compounds 9 and 10 (Fig. 3), bearing a 7-styryl fragment, were
obtained as illustrated in Scheme 4. Cyclization of the commercially
accessible 4-bromopyridin-2-amine 16 to 7‑bromo-2-(4-methox-
yphenyl)imidazo[1,2-a]pyridine 17, achieved upon reaction with
2‑bromo-1-(4‑methoxy)phenylethan-1-one, and the subsequent
coupling of 17 with 1‑methoxy-4-vinylbenzene under the Heck condi-
tions (Heck, 2005), afforded the di‑methoxy derivative 9, which was
converted into the corresponding di‑hydroxy-substituted product 10 by
reaction with BBr3 in a dichloromethane solution.
Very interestingly, treatment with compound
8 (50 mg/kg)
improved functional as well as morphometric and biochemical markers.
Indeed, at the end of the reperfusion period the RPP value was signifi-
cantly increased (53 ± 7%) and the typical ischemic contracture was
later and reduced if compared to vehicle-treatment (MIC = 48 ± 17%
and Thc = 24 ± 1 min, Figs. 6A-C). The reduced impairment induced by
I/R on the functional parameters of isolated hearts from compound 8-
treated animals was consistent with a significantly reduced degree of
damage (Ai/ALV% = 19 ± 5%, Fig. 6D) and with the almost-halved
amount of LDH released during the reperfusion period (40 ± 1 U/g,
Fig. 6E). Taken together, these results unequivocally demonstrate the
cardioprotective activity of compound 8 against I/R injury.
Derivative 11 (Fig. 3), characterized by a pendant phenyl ring in
position 8 of the imidazopyridine core, was obtained by a Suzuky-
Miyaura coupling on the 8‑bromo-2-(3-methoxyphenyl)imidazo[1,2-a]
pyridine intermediate 18, obtained in turn by cyclization of commercial
3-bromopyridin-2-amine 19 with 2‑bromo-1-(3‑methoxy)phenylethan-
1-one in basic conditions (Scheme 5).
2.3. In vitro evaluation of potential SIRT1 activators
3. Conclusions
The potential SIRT1 activators, identified by the computational
approach, were tested using a SIRT1-specific enzymatic assay at a con-
centration of 100 µM, to compare their activity with the reference
compound, resveratrol (Rsv, tested, according to literature, at 100 µM,
Fig. 4). Among the tested compounds, 4 (GP42), 6 (GP37), 8 (GP44), 9
(GP30), and 11 (GP41) showed significant stimulation of SIRT1 enzyme.
Conversely, compounds 1 (GP14) and 5 (GP32) were unable to influence
the activity of SIRT1 enzyme. Compounds 2 (GP36), 3 (GP35), 7 (GP46)
and 10 (GP27d) were discontinued from the test due to the inconsistency
of the results, precluding the reproducibility of the behavior of the
compounds as SIRT1 activators.
In summary, in this work we described the development of an inte-
grated screening platform, enclosing computational, medicinal chemis-
try, and pharmacological competencies, for the identification of SIRT1
activators and their preliminary pharmacological validation as car-
dioprotective agents. Starting from an in-house library containing syn-
thetic molecules inspired by nature, including both bioisosters of natural
flavonoids and rigidified and constrained analogs of resveratrol, we
developed a computational screening protocol employing the crystal
structure of SIRT1 enzyme and the mentioned library, to identify a small
set of potential SIRT1 activators. To validate our computational
approach, the retrieved eleven compounds were synthesized in appro-
priate amount and submitted to in vitro studies for evaluating their ca-
pacity to activate SIRT1 enzyme. Among the tested compounds, five of
them were found able to activate SIRT1 enzyme and one of them
(compound 8, GP44) was selected for further characterization due to the
superior response in activating the enzyme and its favorable calculated
drug-like profile. Compound 8 was employed in ex vivo studies for a
preliminary evaluation of its pharmacological profile.
In summary, based on the in vitro test on isolated enzyme, our
screening protocol had a success rate of roughly 45% (hit rate) in
identifying compounds behaving as SIRT1 activators, as five of eleven
compounds showed significant activation of the enzyme.
Regarding the compounds endowed with stimulating properties,
only compound 8 showed comparable potency to resveratrol, reaching
at 100 µM an enzyme activity of 79 ± 1%. Furthermore, compound 8, in
This study clearly confirms a close link between the activation of
SIRT1 and cardioprotection, identifying the SIRT1 activator compound
8 as the most promising cardioprotective agent of the screened com-
pounds. This pharmacological tool paves the way for developing inno-
vative cardioprotective agents able to target this epigenetic drug target.
Scheme 1. Synthesis of 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one 1. Re-
agents and conditions: (i) ethyl 3-oxo-3-phenylpropanoate, PPA, Δ.
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European Journal of Pharmaceutical Sciences 165 (2021) 105930
Scheme 2. Synthesis of imidazo[1,2-a]pyridine derivatives 2–6. Reagents and conditions: (i) 2-Bromo-1-(substituted)phenylethan-1-one, NaHCO3, Δ; (ii)
substituted-phenylboronic acid, Pd(OAc)2, PPh3, Na2CO3, Δ; (iii) BBr3, CH2Cl2.
Scheme 3. Synthesis of imidazo[1,2-a]pyridine derivatives 7 and 8. Reagents and conditions: (i) 3,5-dimethoxyphenylboronic acid, Pd(OAc)2, PPh3, Na2CO3, Δ; (ii)
2‑bromo-1-(4‑methoxy)phenylethan-1-one, NaHCO3, Δ; iii) BBr3, CH2Cl2.
Scheme 4. Synthesis of imidazo[1,2-a]pyridine derivatives 9 and 10. Reagents and conditions: (i) 2‑bromo-1-(4-methoxyphenyl)ethan-1-one, Δ; (ii) 1‑methoxy-4-
vinylbenzene, Pd(OAc)2, PPh3, Et3N, DMF, Δ; (iii) BBr3, CH2Cl2.
4. Materials and methods
4.1. Computational details
4.1.1. Database and protein preparation
The in-house library of chemical compounds was built using tools
¨
available in Maestro software (Maestro release 2018, Schrodinger, LLC,
Scheme 5. Synthesis of 2-(3-methoxyphenyl)ꢀ 8-phenylimidazo[1,2-a]pyri-
dine 11. Reagents and conditions: (i) 2‑bromo-1-(3-methoxyphenyl)ethan-1-
one, Δ; (ii) 1‑methoxy-4-vinylbenzene, Pd(OAc)2, PPh3, Δ.
New York, NY, 2018). This library, for a total of 54 compounds, includes
both pyrido[1,2-a]pyrimidine derivatives, developed as bioisosters of
flavonoids, and imidazo[1,2-a]pyridine compounds, designed as
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L. Flori et al.
European Journal of Pharmaceutical Sciences 165 (2021) 105930
loops. To optimize the hydrogen bond network, His-tautomers and
ionization states were predicted, 180◦ rotations of the terminal angle of
Asn, Gln, and His-residues were assigned, and hydrogen atoms of the
hydroxyl and thiol groups were sampled. Finally, a restrained minimi-
zation was performed using the Impact Refinement (impref) module,
employing OPLS3 force field to optimize the geometry and minimize the
energy of the protein. The minimization was terminated when the en-
ergy converged, or the RMSD reached a maximum cutoff of 0.30 Å.
4.1.2. Virtual screening
The library, prepared as described, was screened against SIRT1
¨
enzyme using Glide software (Glide release 2018, Schrodinger, LLC,
New York, NY, 2018) with XP-scoring function as previously performed
by us (Testai et al., 2020). Due to the mechanism of activation described
for resveratrol and highlighted by the crystal structure in which
resveratrol is bound to SIRT1 in three binding sites; we prepared the
energy grids for docking, using the default value of the protein
atom-scaling factor (1.0 Å), with a cubic box centered on crystallized
resveratrol molecules. In this way, we prepared a specific grid for each
binding site found for resveratrol. After the generation of the grids, the
library of compounds was docked into SIRT1, considering the three
binding sites of resveratrol. The docked poses considered for the
post-docking minimization step were 1000, evaluating the Glide XP
docking score. Notably, the XP technique was capable of correctly ac-
commodating the resveratrol into three distinct binding sites
(Figure S1). In particular, the calculation of the root-mean-square de-
viation (RMSD), performed using the tool available in Maestro suite,
showed a lower value between the crystallized structure and the docked
pose of resveratrol for each selected binding site (#site1, RMSD 0.148 Å;
#site2, RMSD 0.143 Å; #site3, RMSD 0.151 Å). This procedure is a
well-established technique for validating the docking protocol employed
in virtual screening campaigns (Mascarenhas et al., 2020; Santos et al.,
2021; Leao et al., 2020; Araujo et al., 2020; Neto et al., 2020).
Fig. 4. Stimulation of SIRT1 enzyme promoted by the selected compounds.
SIRT1 activity is expressed as % vs the effect observed with Rsv 100 µM. The
vertical bars symbolize the standard errors (n = 6).
For improving the quality of the screening, we also evaluated the
ligand binding energies from the complexes derived by the docking
calculation. For this purpose, Prime/MM-GBSA method available in
Fig. 5. Concentration-response curve of SIRT1 enzyme activation mediated by
compound 8. SIRT1 activity is expressed as % vs the effect observed with Rsv
100 µM. The vertical bars symbolize the standard errors (n = 6). Asterisks show
a statistically significant difference from the value observed in the hearts of
vehicle-treated animals (*P < 0.05; **P < 0.01; ***P < 0.005).
¨
Prime software (Prime release 2018, Schrodinger, LLC, New York, NY,
2018). This technique computes the variation between the free and the
complex state of both the ligand and enzyme after energy minimization
(Brogi et al., 2018; Frydenvang et al., 2020).
4.1.2. Evaluation of drug-like profile
rigidified and constrained analogs of the natural product resveratrol. All
structures, generated in the previous step, were minimized employing
The drug-like profile was assessed in silico employing QikProp
¨
application (QikProp release 2018, Schrodinger, LLC, New York, NY,
¨
MacroModel program (MacroModel release 2018, Schrodinger, LLC,
2018). Pan Assay Interference Compounds (PAINS) assessment was
executed using FAFDrugs4 web-server (Lagorce et al., 2015) (https:
//fafdrugs4.rpbs.univ-paris-diderot.fr/ access date February 2021) as
previously reported (Zaccagnini et al., 2017). In particular, regarding
the calculation performed by QikProp, QPPCaco (model for the
gut-blood barrier) predicts apparent Caco-2 cell permeability in nm/sec,
while QPPMDCK (model for the for the blood brain barrier) predicts
apparent MDCK cell permeability in nm/sec. Remarkably, QikProp
predictions are for non-active transport.
New York, NY, 2018) adopting the force field OPLS3 (Jorgensen et al.,
1996). For simulating the solvent effects the GB/SA model was
employed, selecting ‘‘no cutoff’’ for non-bonded interactions. The PRCG
technique (5000 maximum iterations and threshold for gradient
convergence = 0.001) was employed. The resulting structures were
¨
treated by LigPrep software (LigPrep release 2018, Schrodinger, LLC,
New York, NY, 2018) for identifying the most probable ionization state
at cellular pH value (7.4 ± 0.5).
The three-dimensional structure of the human SIRT1 enzyme was
obtained from the Protein Data Bank (PDB ID 5BTR (Cao et al., 2015);
crystal structure of SIRT1 in complex with resveratrol and an
AMC-containing peptide) and imported into Maestro suite 2018 and
prepared by means of protein preparation wizard protocol for obtaining
a suitable starting structure for further computational experiments
(Brindisi et al., 2018; Di Capua et al., 2016). Using this protocol, we
performed a series of computational steps to: (1) add hydrogens, (2)
optimize the orientation of hydroxyl groups, Asn, and Gln, and the
protonation state of His, and (3) perform a constrained minimization
refinement using the impref utility. In particular, at first, the protein was
preprocessed by adding all hydrogen atoms to structure, assigning bond
orders, creating disulfide bonds and filling missing side chains and
4.2. Chemistry
¨
Melting points were determined using a Reichert Kofler hot-stage
apparatus and are uncorrected. Routine 1H NMR and 13C NMR spectra
were recorded in DMSO‑d6 solution on a Bruker 400 spectrometer
operating at 400 MHz. Evaporation was done in vacuo (rotary evapo-
rator). Analytical TLCs were conducted on Merck 0.2 mm precoated
silica gel aluminum sheets (60 F-254). Purity of compounds was deter-
mined by HPLC analysis, using a Shimadzu LC-20AD liquid chromato-
graph (PDA, 250ꢀ 500 nm) and a Luna C18 column (250 mm × 4.6 mm,
5
μ
m, Phenomenex), with a gradient of either water and acetonitrile
(compounds 1–5,9,11) or water, acetonitrile and acetic acid
8

L. Flori et al.
European Journal of Pharmaceutical Sciences 165 (2021) 105930
Fig. 6. The histograms represent the functional,
biochemical and morphological changes induced by
treatment of the animals with compound 8 or with
vehicle before I/R episode. (A) Changes in RPP% at the
end of reperfusion; (B) Changes in the maximal
ischemic contraction (MIC) expressed as % vs pre-
ischemic inotropism; (C) Changes in the time of half-
ischemic contracture (Thc) expressed as min; (D)
Changes in LDH amount released in the reperfusion
period (U/g, E) Changes in the percentage of ischemic
area vs left ventricle area (Ai/ALV%). The vertical bars
symbolize the standard errors (n = 6). Asterisks show a
statistically significant difference from the value
observed in the hearts of vehicle-treated animals (*P <
0.05; **P < 0.01).
(compounds 6,8,10) and a flow rate of 1.0 mL/min. All the compounds
showed percent purity values ≥ 95%. Pyridin-2-amine, 3-bromopyridin-
2-amine, 4-bromopyridin-2-amine, 5-bromopyridin-2-amine, ethyl 3-
oxo-3-phenylpropanoate, 2‑bromo-1-phenylethan-1-one 2‑bromo-1-(3-
methoxyphenyl)ethan-1-one, 2‑bromo-1-(4-methoxyphenyl)ethan-1-
one, 1‑methoxy-4-vinyl benzene, and suitably substituted-
phenylboronic acids used to obtain the target compounds were from
Aldrich and Activate Scientific.
7.653–7.609 (m, 4H); 7.444 (t, J = 6.84 Hz, 1H); 7.342 (d, J = 6.35 Hz,
2H); 7.047 (d, J = 7.32 Hz, 2H), 3.985 (s, 3H). 13C NMR (δ, ppm):
159.56; 145.14; 144.42; 134.27; 129.37; 129.20; 128.15; 126.03;
125.52; 123.63; 116.96; 115.03; 109.92.
4.2.3. 6-(3,4-dimethoxyphenyl)ꢀ 2-(4-methoxyphenyl)imidazo[1,2-a]
pyridine (3)
M.p. 141–144 ◦C. Cryst. Solvent: Acetonitrile. Yield: 49%. 1H NMR
(δ, ppm): 8.805 (s, 1H); 8.259 (s, 1H); 7.906 (d, J = 8.79 Hz, 2H);
7.624–7.588 (m, 3H); 7.284–7.218 (m, 2H); 7.034 (t, J = 8.54 Hz, 3H);
3.865 (s, 3H); 3.796 (s, 6H). 13C NMR (δ, ppm): 159.52; 149.67; 149.12;
145.37; 144.45; 129.83; 127.34; 126.99; 125.53; 125.26; 123.63;
119.16; 116.64; 114.62; 112.74; 110.74; 108.75; 56.12; 55.60.
2-Phenyl-4H-pyrido[1,2-a]pyrimidin-4-one 1 (La Motta et al., 2007),
6-Bromo-2-phenylimidazo[1,2-a]pyridine 14a (Quattrini et al., 2020),
6‑bromo-2-(4-methoxyphenyl)imidazo[1,2-a]pyridine 14b (Quattrini
et al., 2020), 6‑bromo-2-(3-methoxyphenyl)imidazo[1,2-a]pyridine 14c
(Quattrini et al., 2020), 8‑bromo-2-(3-methoxyphenyl)imidazo[1,
2-a]-pyridine 18 (Quattrini et al., 2020) were synthesized following the
described procedures.
4.2.4. 2-(3-methoxyphenyl)ꢀ 6-phenylimidazo[1,2-a]pyridine (4)
M.p. 139–141 ◦C. Cryst. Solvent: Acetonitrile. Yield: 39%. 1H NMR
(δ, ppm): 8.877 (s, 1H); 8.433 (s, 1H); 7.741 (d, J = 7.24 Hz, 2H); 7.690
(d, J = 9.36 Hz, 1H); 7.618 (dd, J1 = 9.36 Hz, J2 = 1.76 Hz, 1H);
7.565–7.570 (m, 2H); 7.522 (t, J = 7.44 Hz, 2H); 7.418–7.403 (m, 1H);
7.370 (t, J = 8.20 Hz, 1H); 6.912 (dd, J1 = 7.91 Hz, J2 = 1.76 Hz, 1H);
3.845 (s, 3H). 13C NMR (δ, ppm): 160.13; 145.25; 144.54; 137.12;
135.72; 130.29; 129.58; 128.20; 127.00; 125.72; 125.46; 124.45;
118.46; 117.13; 114.11; 111.11; 110.32; 55.56.
4.2.1. General procedure for the synthesis of 2,6-(Substituted)
diphenylimidazo[1,2-a]pyridines (2–4)
A solution of the appropriate 6-bromoimidazo[1,2-a]pyridine de-
rivative 14a-c (1.00 mmol), Pd(OAc)2 (0.10 mmol) and PPh3 (0.20
mmol) in toluene was added with the suitable phenylboronic acid (1.50
mmol), dissolved in ethanol, and 2 mL of Na2CO3 2 M. The resulting
mixture was refluxed under stirring until the disappearance of the
starting material (TLC analysis). After cooling, the crude obtained were
evaporated to dryness and the residue was extracted (water/ethyl ace-
tate) and purified by column chromatography (silica gel, eluting system
ethyl acetate/petroleum ether). The pure product was recrystallized
from the suitable solvent then characterized by the physico-chemical
and spectroscopic data.
4.2.5. General procedure for the synthesis of 4-(2-phenylimidazo[1,2-a]
pyridin-6-yl)phenol (5) and 4-(2-(4-methoxyphenyl)imidazo[1,2-a]
pyridin-6-yl)benzene-1,2-diol (6)
A solution of the appropriate methoxy derivative, 2 and 3 (1.00
mmol), in CH2Cl2 anhydrous was cooled at ꢀ 10 ◦C, then added with
BBr3 (1.50 mmol) and left under stirring at room temperature until the
disappearance of the starting material (TLC analysis). The resulting
suspension was then carefully poured into crushed ice and the solid
precipitated was collected by filtration, recrystallized from the suitable
4.2.2. 6-(4-methoxyphenyl)ꢀ 2-phenylimidazo[1,2-a]pyridine (2)
M.p. 157–161 ◦C. Cryst. Solvent: Acetonitrile. Yield: 70%. 1H NMR
(δ, ppm): 8.765 (s, 1H); 8.369 (s, 1H); 7.944 (d, J = 7.81 Hz, 2H);
9

L. Flori et al.
European Journal of Pharmaceutical Sciences 165 (2021) 105930
solvent and characterized by the physico-chemical and spectroscopic
data.
= 8.80 Hz, J2 = 2.80 Hz, 1H); 6.717 (d, J = 2.00 Hz, 2H); 6.530 (d, J =
8.80 Hz, 1H); 6.433 (t, J = 4.40 Hz, 1H); 6.106 (s, 2H, exc.); 3.812 (s,
6H).
4.2.6. 4-(2-phenylimidazo[1,2-a]pyridin-6-yl)phenol (5)
M.p. > 230 ◦C. Cryst. Solvent: Methanol. Yield: 44%. 1H NMR (δ,
ppm): 9.896 (s, 1H, exc.); 9.161 (s, 1H); 8.766 (s, 1H); 8.230 (d, J = 10.0
Hz, 1H); 7.992 (d, J = 8.40 Hz, 3H); 7.653–7.541 (m, 5H); 6.962 (d, J =
8.40 Hz, 2H). 13C NMR (δ, ppm): 158.85; 139.56; 136.27; 133.10;
130.86; 130.42; 129.98; 128.74; 127.00; 126.67; 125.60; 125.16;
116.66; 112.47; 111.75.
4.2.11. Synthesis of 7‑bromo-2-(4-methoxyphenyl)imidazo[1,2-a]pyridine
(17)
A mixture of 4-bromopyridin-2-amine 16 (1.00 mmol) and 2‑bromo-
1-(4-methoxyphenyl)ethan-1-one (1.00 mmol) was heated at 80 ◦C for 8
h, until the disappearance of the starting material (TLC analysis). The
crude obtained were purified through recrystallization from methanol
and characterized with physico-chemical and spectroscopic data. M.p.
290–295 ◦C. Yield: 77%. 1H NMR (δ, ppm): 8.75 (d, 1H); 8.433 (s, 1H);
4.2.7. 4-(2-(4-hydroxyphenyl)imidazo[1,2-a]pyridin-6-yl)benzene-1,2-
diol (6)
7.991 (s, 1H); 7.845 (d, J = 8.8 Hz, 2H); 7.344 (dd, J1 = 2.00 Hz, J2
1.60 Hz, 1H); 7.074 (d, J = 8.8 Hz, 2H); 3.806 (s, 3H).
=
M.p. > 230 ◦C. Cryst. Solvent: 2-propanol. Yield: 23%. 1H NMR (δ,
ppm): 10.164 (s, 1H, exc.); 9.469 (s, 1H, exc.); 9.259 (s, 1H, exc.); 9.029
(s, 1H); 8.526 (s, 1H); 8.106 (d, J = 9.60 Hz, 1H); 7.905 (d, J = 9.20 Hz,
1H); 7.793 (d, J = 8.80 Hz, 2H); 7.143 (d, J = 2.40 Hz, 1H); 7.072 (dd,
J1 = 8.00 Hz, J2 = 2.20 Hz, 1H); 6.987 (d, J = 8.40 Hz, 2H); 6.916 (d, J
= 8.40 Hz, 1H). 13C NMR (δ, ppm): 159.91; 146.96; 146.56; 139.33;
130.47; 129.92; 128.36; 126.29; 124.93; 118.65; 116.79; 116.73;
114.61; 112.27; 110.02.
4.2.12. Synthesis of (E)ꢀ 2-(4-methoxyphenyl)ꢀ 7-(4-methoxystyryl)
imidazo[1,2-a]pyridine (9)
A mixture of 7‑bromo-2-(4-methoxyphenyl)imidazo[1,2-a]pyridine
17, (10.0 mmol), PPh3 (0.70 mmol) and Pd(OAc)2 (0.30 mmol) was
added with 1‑methoxy-4-vinylbenzene (20.0 mmol), Et3N (20.0 mmol)
and DMF, then heated under stirring at 100 ◦C for 18 h until the
disappearance of the starting material (TLC analysis). After cooling, the
crude obtained were evaporated to dryness, washed with water, purified
through recrystallization from methanol and characterized with
physico-chemical and spectroscopic data. M.p. 267–270 ◦C. Yield: 48%.
1H NMR (δ, ppm): 8.454 (d, J = 7.20 Hz, 1H); 8.251 (s, 1H); 7.897 (d, J
= 8.80 Hz, 2H); 7.588 (d, J = 8.40 Hz, 3H); 7.330 (d, J = 16.40 Hz, 1H);
7.256 (d, J = 7.20 Hz, 1H); 7.174 (d, J = 16.40 Hz, 1H); 7.029–6.977 (m,
4H); 3.806 (d, J = 3.20 Hz, 6H). 13C NMR (δ, ppm): 159.81; 159.70;
145.66; 145.35; 145.07; 132.36; 130.55; 129.77; 128.59; 127.46;
127.38; 115.90; 114.75; 114.67; 114.40; 109.01; 108.93; 55.62.
4.2.8. Synthesis of 6-(3,5-dimethoxyphenyl)ꢀ 2-(4-methoxyphenyl)
imidazo[1,2-a]pyridine (7)
A mixture of 5-(3,5-dimethoxyphenyl)pyridin-2-amine 15 (1.00
mmol), 2‑bromo-1-(4-methoxyphenyl)ethan-1-one (1.00 mmol) and
NaHCO3 (1.20 mmol) was refluxed under stirring at 100 ◦C until the
disappearance of the starting material (TLC analysis). After cooling, the
crude obtained were evaporated to dryness; water was then added to the
residue and the desired heterocycle, 7, separated as a white solid, was
collected by filtration, purified by recrystallization from ethanol, and
characterized with physico-chemical and spectroscopic data. M.p.
140–142 ◦C. Yield: 34%. 1H NMR (δ, ppm): 8.879 (s, 1H); 8.272 (s, 1H);
7.910 (d, J = 8.80 Hz, 2H); 7.597 (t, J = 3.60 Hz, 2H); 7.015 (d, J = 8.80
Hz, 2H); 6.860 (d, J = 2.00 Hz, 2H); 6.530 (t, J = 4.40 Hz, 1H); 3.824 (s,
6H); 3.798 (s, 3H).
4.2.13. Synthesis of (E)ꢀ 4-(2-(2-(4-hydroxyphenyl)imidazo[1,2-a]
pyridin-7-yl)vinyl)phenol (10)
A solution of the methoxy derivative 9 (1.00 mmol) in CH2Cl2
anhydrous was cooled at ꢀ 10 ◦C and added with BBr3 (2.00 mmol). The
resulting mixture was then left under stirring at room temperature until
the disappearance of the starting material (TLC analysis). At the end, it
was carefully poured into crushed ice and the solid precipitated was
collected by filtration, recrystallized from methanol and characterized
by the physico-chemical and spectroscopic data. M.p. > 230 ◦C. Yield:
28%. 1H NMR (δ, ppm): 10.122 (s,1H, exc.); 9.89 (s, 1H, exc.); 8.741 (d,
J = 5.60 Hz, 1H); 8.50 (s, 1H); 7.779–7.729 (m, 4H); 7.652–7.548 (m,
3H); 7.314 (d, J = 15.20 Hz, 1H); 6.971 (d, J = 8.40 Hz, 2H); 6.847 (d, J
= 8.40 Hz, 2H). 13C NMR (δ, ppm): 159.25; 158.91; 145.66; 145.35;
144.87; 132.77; 130.55; 129.08; 128.15; 127.73; 125.84; 116.82;
116.68; 115.90; 114.40; 109.01; 108.93.
4.2.9. Synthesis of 5-(2-(4-hydroxyphenyl)imidazo[1,2-a]pyridin-6-yl)
benzene-1,3-diol (8)
A solution of 6-(3,5-dimethoxyphenyl)ꢀ 2-(4-methoxyphenyl)imi-
dazo[1,2-a]pyridine 7 (1.00 mmol) in anhydrous CH2Cl2 was cooled at
ꢀ 10 ◦C and added with BBr3 (3.20 mmol). The resulting mixture was
then left under stirring at room temperature until the disappearance of
the starting material (TLC analysis). At the end, it was carefully poured
into crushed ice and the solid precipitated was collected by filtration,
recrystallized from iPrOH and characterized by the physico-chemical
and spectroscopic data. M.p. 259–262 ◦C. Yield: 35%. 1H NMR (δ,
ppm): 10.086 (s, 1H, exc.); 9.588 (s, 2H, exc.); 9.043 (s, 1H); 8.507 (s,
1H); 8.028 (d, J = 8.00 Hz, 1H); 7.889 (d, J = 9.20 Hz, 1H); 7.791 (d, J
= 8.80 Hz, 2H); 6.978 (d, J = 8.80 Hz, 2H); 6.575 (d, J = 2.40 Hz, 2H);
6.365 (s, 1H). 13C NMR (δ, ppm): 159.93; 159.68; 139.78; 137.14;
132.44; 130.39; 128.39; 125.97; 116.74; 112.41; 110.12; 105.57;
103.37.
4.2.14. Synthesis of 2-(3-methoxyphenyl)ꢀ 8-phenylimidazo[1,2-a]
pyridine (11)
A solution of 8‑bromo-2-(3-methoxyphenyl)imidazo[1,2-a]pyridine
18 (1.00 mmol), Pd(OAc)2 (0.10 mmol) and PPh3 (0.20 mmol) in
toluene was added with phenyl boronic acid (1.50 mmol), dissolved in
ethanol, and 2 mL of Na2CO3 2 M. The resulting mixture was refluxed
under stirring until the disappearance of the starting material (TLC
analysis). After cooling, the crude obtained were evaporated to dryness
and the residue was extracted (water/ethyl acetate) and purified by
column chromatography (silica gel, eluting system ethyl acetate/pe-
troleum ether) obtaining an oily product. The pure product was then
characterized by the physico-chemical and spectroscopic data. Yield:
49%. 1H NMR (δ, ppm): 8.544 (dd, J1 = 6.70 Hz, J2 = 1.00 Hz, 1H);
8.522 (s, 1H); 8.220 (d, J = 7.20 Hz, 2H); 7.560–7.508 (m, 4H); 7.498
(dd, J1 = 7.10 Hz, J2 = 1.10 Hz, 1H); 7.460–7.412 (m,1H); 7.377 (t, J =
4.2.10. Synthesis of 5-(3,5-dimethoxyphenyl)pyridin-2-amine (15)
A solution of 5-bromopyridin-2-amine 13 (1.00 mmol), Pd(OAc)2
(0.10 mmol) and PPh3 (0.20 mmol) in ethanol and water (3:1) was
added with (3,5-dimethoxyphenyl)boronic acid (1.50 mmol) and
Na2CO3 (1.5 mmol). The resulting mixture was refluxed under stirring at
110 ◦C until the disappearance of the starting material (TLC analysis).
After cooling, the crude obtained were evaporated to dryness and the
residue was washed with water and extracted with ethyl acetate, then
purified by column chromatography (silica gel, eluting system ethyl
acetate/petroleum ether) obtaining an oily product. The pure product
was then characterized by physico-chemical and spectroscopic data.
Yield: 82%. 1H NMR (δ, ppm): 8.278 (d, J = 2.00 Hz, 1H); 7.724 (dd, J1
7.80 Hz, 1H); 7.020 (t, J = 6.90 Hz, 1H); 6.917 (dd, J1 = 7.80 Hz, J2
=
2.60 Hz, 1H); 3.834 (s, 3H). 13C NMR (δ, ppm): 160.09; 144.61; 143.74;
136.60; 135.74; 130.28; 129.12; 128.77; 128.59; 126.58; 123.49;
10

L. Flori et al.
European Journal of Pharmaceutical Sciences 165 (2021) 105930
118.58; 113.61; 113.05; 111.66; 110.49; 55.55.
end of the ischemic period the hearts were re-perfused for 120 min and
RPP value was acquired at intervals of 10 min. Before ischemia and
during reperfusion, the perfusate was collected to evaluate the enzyme
LDH. In particular, LDH, a biochemical marker of ischemic damage, was
measured by spectrophotometric method in the perfusate collected in
the last 5 min of the pre-ischemic phase and then every 5 min during the
first 30 min of reperfusion, and every 10 min in the remaining period of
reperfusion. LDH was assessed by adding 27.6 mM pyruvate and 4.8 mM
NADH, and measuring the conversion of NADH to NAD+ at λ = 340 nm,
in kinetics for 2.5 min. The amount of released LDH has been expressed
in U/g in 120 min of reperfusion, resulting from the AUC analysis (area
under the curve of the LDH amount recorded) and related to 1 g of the
heart weight.
4.3. In vitro assay
SIRT1 activity was measured using a SIRT1 direct fluorescent
screening assay kit (Cayman, Ann Arbor, MI), following the manufac-
turer’s protocol. Before performing the test, the possible interferences of
the compounds under examination with the fluorophore and/or devel-
oper were evaluated according to manufacturer’s protocol. Compounds
with an interference % to the fluorophore and developer less than or
equal to 10% were considered suitable. Briefly, 25
μ
L of Assay Buffer, 5
μ
L of SIRT1 human recombinant appropriately reconstituted and diluted
and 5
μ
L of the test compounds suitably solubilized in DMSO (10ꢀ 2 M)
At the end of reperfusion, the hearts were removed from the appa-
ratus, dried, weighed and subsequently the left ventricle was isolated.
This was cut in slices of about 2 mm, which were immersed in a 1% w/v
solution of 2,3,5 triphenyltetrazolium chloride (TTC, Sigma-Aldrich)
dissolved in a phosphate buffer (pH = 7.4) for 20 min in the dark and
at 37 ◦C. TTC reacts with dehydrogenases present in intact and viable
cells, and oxidizes to formazan, a red and insoluble compound. Then,
slices were fixed in a water solution of formaldehyde 10% v/v. Subse-
quently, the ventricular slices were photographed and analyzed to
identify the necrotic areas (visible as a white or light pink color) and the
healthy areas (visible as a strong red due to the TTC reaction). It was
possible to calculate the ischemic area as a percentage of the total area of
the left ventricle, through photographs of the sections subjected to
planimetric analysis carried out 24 h after the reaction with the TTC (Ai/
and diluted in bidistilled water, were added to the three wells. 30
μ
L of
Assay Buffer and 5
examination (DMSO) were plated to three wells (background). 25
Assay Buffer, 5 L of SIRT1 and 5
were plated to three wells.
The final concentration in the wells of the tested compounds and
resveratrol was 100 M and 1% v/v for DMSO. Compound 8 was also
tested at 300 M, 30 M, 10 M, 3 M, 1 M concentrations. The fluo-
μ
L of solvent used to solubilize the compounds under
μL of
μ
μL of reference activator (resveratrol)
μ
μ
μ
μ
μ
μ
rescence intensity was monitored with EnSpire® (2300 Multilabel
Reader, PerkinElmer) by setting an excitation wavelength of 350 nm and
an emission wavelength of 450 nm. The fluorescence values referred to
the blank were subtracted, and the data were normalized considering
the vehicle fluorescence value as 0% activity and the value recorded
with the fluorescence emitted in the presence of resveratrol 100
100%.
μM as
A
LV).
4.4.1. Data analysis
4.3.1. Data analysis
All values are expressed as a mean ± standard error for six different
experiments.
All values are expressed as a mean ± standard error for three
different experiments in duplicate.
The effects of compound 8 on RPP, MIC, Thc, Ai/ALV and LDH were
statistically analyzed by Student’s t-test. P < 0.05 was considered
indicative of a significant difference.
The effects of SIRT1 activator compounds were statistically analyzed
by one-way ANOVA followed by Bonferroni post-test. P < 0.05 was
considered indicative of a significant difference.
4.4. Ex vivo studies
CRediT authorship contribution statement
All procedures were performed according to European (EEC Direc-
tive 2010/63) and Italian (D.L. March 4, 2014 n. 26) legislation (pro-
tocol number 45,972, 21/09/2016). Animals were housed in cages with
food and water ad libitum, and exposed to 12 h:12 h light/dark cycles.
Adult male Wistar rats, 300–350 g, were treated with an intraperi-
toneal injection of compound 8 (50 mg/kg) or vehicle (dimethyl sulf-
oxide; DMSO). The dosage of compound 8 was established according to
the dosage of resveratrol in studies in which cardioprotective effects
were evaluated (Testai et al., 2013, 2017, 2013; Calderone et al., 2010).
After 2 h, the rats were anaesthetised with sodium pentobarbital
(100 mg/kg, i.p.) and heparinised (100 U, i.p.) to prevent blood clotting.
Subsequently, the hearts were quickly excised, mounted on a Langen-
dorff apparatus and perfused at constant pressure (70–80 mmHg), as
previously published (Calderone et al., 2010, 2010; Testai et al., 2017,
2013, 2017). To check the functional parameters of the heart, a latex
balloon filled with bidistilled water at a pressure of 5–10 mmHg was
inserted into the left ventricle through the mitral valve and connected to
a pressure transducer (Bentley Trantec, mod 800, Ugo Basile, Comerio,
Italy) and in turn, to a data acquisition system (Byopac, California USA).
The functional parameter of rate pressure product (RPP) was calculated
as RPP = HR x LVDP, where HR was the heart rate value and LVDP was
the left ventricular developed pressure, continuously monitored by a
computerised Byopac system (Goleta, CA, USA). After 30 min of equil-
ibration, the hearts were submitted to 30 min of global ischemia. During
this period, a typical ischemic contracture was observed, thus the
amplitude of the maximal ischemic contracture (MIC) was expressed as a
percentage of pre-ischemic LVDP; while the time required to reach a
half-maximal ischemic contracture (Thc) was measured in min. At the
Lorenzo Flori: Investigation, Methodology, Formal analysis, Vali-
dation. Giovanni Petrarolo: Investigation, Methodology, Formal anal-
ysis, Validation. Simone Brogi: Conceptualization, Investigation,
Methodology, Data curation, Software, Formal analysis, Validation,
Writing – original draft, Writing – review & editing, Supervision. Con-
cettina La Motta: Conceptualization, Investigation, Methodology,
Formal analysis, Validation, Writing – review & editing, Supervision.
Lara Testai: Conceptualization, Investigation, Methodology, Formal
analysis, Validation, Writing – review & editing, Supervision. Vincenzo
Calderone: Data curation, Formal analysis, Validation, Writing – review
& editing.
Acknowledgments
`
This work is supported by the Universita di Pisa under the “PRA –
Progetti di Ricerca di Ateneo” (Institutional Research Grants) – Project
no PRA_2020_58 “Agenti innovativi e nanosistemi per target molecolari
nell’ambito dell’oncologia di precisione” to Simone Brogi.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.ejps.2021.105930.
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