Discovery of a novel family of FKBP12 “reshapers” and their use as calcium modulators in skeletal muscle under nitro-oxidative stress

Article abstract of DOI:10.1016/j.ejmech.2021.113160

The hypothesis of rescuing FKBP12/RyR1 interaction and intracellular calcium homeostasis through molecular “reshaping” of FKBP12 was investigated. To this end, novel 4-arylthioalkyl-1-carboxyalkyl-1,2,3-triazoles were designed and synthesized, and their e

Full text of DOI:10.1016/j.ejmech.2021.113160

European Journal of Medicinal Chemistry 213 (2021) 113160
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of a novel family of FKBP12 “reshapers” and their use as
calcium modulators in skeletal muscle under nitro-oxidative stress
,
*
a
a
a
a
Jesus M. Aizpurua a , Jose I. Miranda , Aitziber Irastorza , Endika Torres , Maite Eceiza ,
ꢀ
a
b
c, d
ꢀ
Maialen Sagartzazu-Aizpurua , Pablo Ferron , Garazi Aldanondo
,
Haizpea Lasa-Fernandez
e, Pablo Marco-Moreno c d, Naroa Dadie e,
c
,
d,
,
ꢀ
c
,
d,
, d, e, **
e, Ainara Vallejo-Illarramendi c
ꢀ
Adolfo Lopez de Munain
Joxe Mari Korta R&D Center, Departamento de Química Organica-I, Universidad Del País Vasco UPV/EHU, Avda. Tolosa-72, 20018, San Sebastian, Spain
a
ꢀ
ꢀ
b
c
ꢀ
Miramoon Pharma S.L., Avda Tolosa-72, 20018, San Sebastian, Spain
ꢀ
ꢀ
Instituto de Investigacion Sanitaria Biodonostia, Grupo de Enfermedades Neuromusculares, Paseo Dr Begiristain s/n, 20014, San Sebastian, Spain
d CIBERNED, Instituto de Salud Carlos III, 28031, Madrid, Spain
e Grupo de Neurosciencias, Departamentos de Pediatría y Neurociencias, Universidad Del País Vasco UPV/EHU, Hospital Donostia, Paseo Dr Begiristain S/n,
ꢀ
20014, San Sebastian, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The hypothesis of rescuing FKBP12/RyR1 interaction and intracellular calcium homeostasis through
molecular “reshaping” of FKBP12 was investigated. To this end, novel 4-arylthioalkyl-1-carboxyalkyl-
1,2,3-triazoles were designed and synthesized, and their efficacy was tested in human myotubes. A li-
brary of 17 compounds (10a-n) designed to dock the FKBP12/RyR1 hot-spot interface contact residues,
Received 6 October 2020
Received in revised form
23 December 2020
Accepted 4 January 2021
Available online 14 January 2021
was readily prepared from free
a-amino acids and arylthioalkynes using CuAAC “click” protocols
amenable to one-pot transformations in high overall yields and total configurational integrity. To model
nitro-oxidative stress, human myotubes were treated with the peroxynitrite donor SIN1, and evidence
was found that some triazoles 10 were able to normalize calcium levels, as well as FKBP12/RyR1 inter-
action. For example, compound 10 b at 150 nM rescued 46% of FKBP12/RyR1 interaction and up to 70% of
resting cytosolic calcium levels in human myotubes under nitro-oxidative stress. All compounds 10
analyzed showed target engagement to FKBP12 and low levels of cytotoxicity in vitro. Compounds 10b,
10c, 10h, and 10iR were identified as potential therapeutic candidates to protect myotubes in muscle
disorders with underlying nitro-oxidative stress, FKBP12/RyR1 dysfunction and calcium dysregulation.
© 2021 Elsevier Masson SAS. All rights reserved.
Keywords:
Ryanodine receptor
Triazoles
Calcium channel
1. Introduction1
conversion of a still not precisely identified XxxePro segment in
the FKBP-binding domain of ryanodine receptor RyR1 [2]. This
Intrinsically slow secondary amide rotations in proline peptides
can be catalyzed by rotamerase enzymes to elicit a reversible pro-
tein backbone switch from the open (trans-) to the closed (cis-)
state to activate transmembrane ion channels [1]. In particular
FKBP12, also known as Calstabin1, is a peptidyl-prolyl isomerase
(PPIase) expressed in the cytosolic region of skeletal muscle, among
other tissues, and is thought to catalyze the trans/cis inter-
channel regulates the periodical efflux of calcium ions from the
sarcoplasmic reticulum (SR) to the actin-myosin contraction sys-
tem and is critical in excitation-contraction coupling (EC). RyR1
malfunction, generally caused by congenital mutations, may lead to
“leaky” calcium channels that are related to severe muscle condi-
tions [3e5]. In addition, the FKBP12/RyR1 interaction can also be
altered or compromised by post-translational chemical modifica-
tions of RyR1 residues placed close to the switching XxxePro
segment. For example, S-nitrosylation of RyR1 cysteine residues
caused by the aberrant activity of nitric oxide synthase (nNOS)
enzyme [6], exhausting exercise, or aging, is known to interfere
with the proper closing of ryanodine receptor channels, and results
in calcium ion leakage in skeletal muscles [7].
* Corresponding author.
** Corresponding author. Grupo de Neurociencias. Departamento de Pediatría,
ꢀ
UPV/EHU, Paseo Dr Begiristain s/n, 20014, San Sebastian, Spain.
E-mail addresses: jesusmaria.aizpurua@ehu.eus (J.M. Aizpurua), ainara.vallejo@
ehu.eus (A. Vallejo-Illarramendi).
https://doi.org/10.1016/j.ejmech.2021.113160
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
Two structures of mammalian RyR1 channels bound to FKBP12
in both open and closed conformations have been recently eluci-
dated at a near-atomic resolution [8,9]. These structures have
provided an unprecedentedly detailed explanation for the long-
range allosteric regulation of calcium ion conductance of tetra-
meric RyR1 channels following a complex cascade of synchronized
conformational changes [10,11]. Despite these remarkable ad-
vances, the molecular clues behind the rotamerase activity of
FKBP12 on RyR1 remain obscure. The generally accepted mecha-
nism for the rotamerase action of FKBP12 involves the stabilization
group was formed by the polar Asp-37 and Arg-42 residues
participating in a pocket of electrostatic interactions, and the sec-
ond one comprised the Tyr-82 and Ile-90 residues engaged in hy-
drophobic interactions. The counterpart RyR1 interface amino acids
include the Asp-1690 residue and the segment Pro-1780, Cys-1781,
Phe-1782, Val-1783 (Fig. 1A).
After identifying the hot-spot cluster, several triazole structures
10 were designed as potential ligands to simultaneously fit the ionic
and the hydrophobic pockets of the FKBP12/RyR1 complex (Fig. 1B).
Recently, 1H-1,2,3-triazoles have proved to be suitable scaffolds for
small drug development [22]. Furthermore, such compounds
would be easily accessible from the clicking of substituted thio-
of
a
twisted prolyl amide transition state with
a
key
C
a
XxxeCOProeNProeCaPro dihedral angle of about 90ꢀ in the iso-
merizable protein [12]. FKBP12 is estimated to accelerate the trans/
cis transformation by 104 fold compared to the uncatalyzed isom-
erization [13,14].
phenol propargyl sulfides and a-azido azides (see below, Scheme
Rapamycin and immunosuppressant FK506 are the most effi-
cient known inhibitors of FKBP12 and both contain in their struc-
tures a central L
-pipecolic acid residue strained to 98ꢀ and 95ꢀ
respectively by rigid macrocyclic structures. This motif has inspired
the synthesis of many low molecular weight FKBP12 ligands aimed
to elicit effective immunosuppression [15]. However, rapamycin,
FK506 and most of the L-pipecolic acid peptide surrogates displace
FKBP12 from RyR1 channels, resulting in an increased calcium
leakage through RyR1 from the SR into the cytosol [16]. On the
other hand, different studies have demonstrated the beneficial ef-
fects of RyR1 channel stabilizers on animal models of muscular
dystrophy. For instance, benzothiazepines JTV519/K201 [17], S107
[18] and S48168/ARM210 [19], also known as “rycals”, have
reportedly improved in vivo muscle function and exercise perfor-
mance in the mdx Duchenne muscular dystrophy mouse model.
Such compounds decrease the Ca2þ leakage from the SR by pre-
venting FKBP12 depletion from RyR1 channels. However, the
mechanistic details of their activity are poorly understood [20].
In order to seek additional evidence on the nature of the key
amino acids involved in the molecular mechanism of the FKBP12/
RyR1 interaction and the deleterious effect caused by the S-nitro-
sylation of putative cysteine residues, we decided to conduct a
combined computational and experimental study. Our objective
was to modify such protein-protein interaction using selective li-
gands of FKBP12 that cause a “reshaping” of its surface and,
therefore, a potential restoration of the peptidyl-prolyl isomerase
activity of FKBP12 when RyR1 is hyper-nitrosylated. Herein we
report the design and synthesis of a novel family of 1,2,3-triazole-
based reshapers of FKBP12 and their biological activity as stabi-
lizers of FKBP12/RyR1 interaction and modulators of cytosolic cal-
cium concentration in human myotubes submitted to chemical
nitro-oxidative stress.
2. Results and discussion
2.1. Design
The crystal structure of the FKBP12/RyR1 tetramer complex
(Protein Data Bank code 3J8H) [9] was used as the starting point to
design triazole ligands suitable to modify the protein-protein
interaction (PPI). A successful modulator ligand should dock the
critical interaction surface areas by binding together several amino
acid hot-spots. The identification of such key residues was con-
ducted computationally using the virtual alanine scanning
approach, which provides interaction energy differences for indi-
vidual residues upon simulated mutation to alanine.
After subjecting the FKBP12/RyR1 complex to virtual alanine
scanning by using the DrugScore PPI server [21] and setting the cut-
off value at DDG > 1.5 kcal molꢁ1, we identified two groups of
FKBP12 residues involved in the protein-protein interaction. One
Fig. 1. (A) FKBP12/RyR1 interaction (PDB ID: 3J8H) [9]. Hot-spot residues
DDG > 1.5 kcal molꢁ1) obtained after virtual alanine scanning are shown in blue for
(
FKBP12, and in red for RyR1. Electrostatic and hydrophobic contact pockets are high-
lighted in purple and orange, respectively. (B) Triazoles studied in this work (general
structure 10) designed to incorporate polar (purple) and hydrophobic (orange) frag-
ments. (C) Docking of ligand 10b (in green) to the FKBP12/RyR1 complex. For a detailed
2D interaction diagram of 10b, see Supplementary Material (S3).
2

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
Scheme 1. Synthesis of triazoles 10a-n.
1). To check this hypothesis, we conducted a computational dock-
ing study using the Autodock FR [23] software package. Proteins
and ligands were prepared using the AutoDock Tools 1.5.7 software
following standard protocols. The selection of the docking area and
mobile side-chains in the FKBP12/RyR1 was achieved using the
AutoGrid FR software. This area was limited to the residues Tyr-26,
Phe-36, Asp-37, Phe-46, Phe-48, Glu-54, Trp-59, Phe-99 (in FKBP12)
and Phe-1782, Val-1783 (in RyR1). All these residues surround the
key interaction of amino acids Arg-42 (in FKBP12) and Pro-1780 (in
RyR1), which are placed in the center of a characteristic groove-like
structure. The RyR1 protein docking surface was limited to the
1777e1783 residues comprising a peptide loop interacting with the
FKBP12 groove. Each ligand was docked 50 times to obtain average
interaction energies.
As exemplified in Fig.1C for ligand 10b, such triazoles repeatedly
docked the FKBP12/RyR1 complex hot-spot interface with typical
docking energies in the range of 12e14 kcal molꢁ1 showing a
uniform spatial disposition. In particular, compound 10b was found
to form two hydrogen bonds with the Tyr-26 and His-87 residues of
FKBP12 and a weak interaction with the Cys-1781 residue of RyR1
(see Supplementary Material (S3). Considering that this ligand
contains the minimum essential groups for activity, we chose it as a
representative model of compounds 10 to conduct target engage-
ment experiments with FKBP12 complexes (see below). Further-
more, we hypothesized that triazole ligands 10 could modulate the
long-range allosteric effect elicited by FKBP12 on RyR1 channels.
intermediate
a
-azido acids using imidazole-1-sulfonylazide
hydrogenosulfate 11, followed by CuAAC reaction with the corre-
sponding alkynes (Method B) [25]. It is worth mentioning that
triflyl azide proved to be an unsuitable N-diazotation reagent to
conduct the last transformations because, after the workup,
inseparable mixtures of carboxylic triazoles and triflamide were
obtained. The chiral integrity of compounds 10h-n was assessed by
analytical HPLC using a chiral stationary phase (Supplementary
Material S32eS42).
2.3. Pharmacology
First, we aimed to verify the target engagement of triazole li-
gands 10 to FKBP12. To do this, we used a well-characterized pro-
tein fragment complementation assay where the rapamycin-
inducible FKBP/FRB interaction produces quantitative lumines-
cence, due to the association of luciferase fragments fused to
FKBP12 and FRB proteins. Indeed, this assay has been previously
used to determine binding kinetics underlying drug action [26]. In
this system, FKBP12 binds rapamycin with high affinity and then
the FKBP-rapamycin complex binds FRB to form a ternary complex
[27]. The addition of rapamycin to cells co-expressing FKBP12 and
FRB fusion proteins resulted in an instantaneous increase of lumi-
nescence of around 2 orders of magnitude (Fig. 2B). The observed
potency of rapamycin to induce protein dimerization (EC50 value)
was 36.39 nM (Fig. 2C), which is in agreement with previously
published data [27]. As expected, neither FK506 nor triazole 10b
were able to induce FKBP12/FRB dimerization, even at high con-
2.2. Chemistry
centrations (100 mM and 10 mM respectively), since none of them
For the preparation of triazoles 10 (Scheme 1) we developed
several one-pot protocols starting from alkynes 1e7 which, in turn,
were easily obtained in 56e99% yields from propargyl bromide or
homopropargyl alcohol mesylate by a nucleophilic substitution
reaction with several thiophenols under basic conditions.
Copper(I)-catalyzed CuAAC reaction of arylthioalkyl alkynes 1e7
present the rapamycin site that binds to FRB (Fig. 2A, blue residues).
We next wanted to evaluate the effect of FK506 and triazoles 10
on the FKBP/rapamycin/FRB ternary complex. Since this protein
fragment complementation assay is reportedly reversible and
robust for more than 1 h [27], we designed a competition assay
where FK506 or triazoles 10 were added after rapamycin induction
of FKBP/FRB dimerization (Fig. 2D for schematics). Hence, both
FK506 and triazoles 10 would compete with rapamycin for FKBP12
binding pocket. Indeed, we found that these compounds displace
with suitable a-azido acid derivatives [24] was performed in two
complementary ways. To synthesize achiral triazoles 10a-f (Method
A), methyl bromoacetate 8 was transformed with sodium azide into
methyl azidoacetate and was “clicked” in situ with alkynes 1e6
rapamycin from FKBP12 with a potency (IC50) of 38.29 mM and
affording
the
corresponding
intermediate
1-
3.32 mM, respectively (Fig. 2E). We next wanted to determine
whether other triazoles 10 also bind to FKBP12. To do so, we per-
formed a competitive binding assay with all different triazole 10
compounds. In this assay, we used a supramaximal concentration
methoxycarbonymethyl-1,2,3-triazole esters. Subsequent saponifi-
cation of the CO2Me group with lithium hydroxide provided the
free carboxylic acids in good to excellent overall yields (Table 1,
entries 1e6). The hindered triazole 10g (entry 7) was prepared
of rapamycin (1.2 mM) and the effect of triazole compounds (3 mM)
similarly starting from methyl
triazoles 10h-n (Table 1, entries 8e17) were synthesized from free
-amino acids upon Cu(II)-catalyzed diazotation to the requisite
a
-bromoisobutyrate. Finally, chiral
were determined subsequently (Fig. 2F). All tested compounds 10
showed target engagement to FKBP12, as shown by the decay
observed in the rapamycin-induced FKBP12/FRB dimerization,
a
3

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
Table 1
Preparation of triazole ligands 10a-n.
Entry
Alkyne
R1
R2
R3
n
Config.(*)
Product
Method
Yield (%)a
1
2
3
4
5
6
7
8
1
2
3
4
5
6
3
3
3
3
3
3
3
7
7
7
4
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Bn
Bn
iBu
iBu
iPr
iPr
Bn
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
e
10a
10b
10c
10d
10e
10f
10g
10hR
10hS
10iR
10iS
10jR
10jS
10kR
10mR
10mS
10nS
A
A
A
A
Ab
A
Ac
B
B
B
B
B
80
74
88
67
68
91
72
76
73
80
75
69
65
63
73
86
63
3-MeO
4-MeO
3-Cl
e
e
e
4-Me
e
2,3,5,6-F4
3-MeO
3-MeO
3-MeO
3-MeO
3-MeO
3-MeO
3-MeO
3-F3C
e
e
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(R)
(S)
(S)
9
10
11
12
13
14
15
16
17
B
B
B
B
3-F3C
3-F3C
3-Cl
4-HOC6H4CH2
4-HOC6H4CH2
BocHN(CH2)4
B
a
Overall yields of pure isolated products.
From homopropargyl mesylate.
From methyl a-bromoisobutyrate (see experimental section).
b
c
which was in the range of 20e100%.
FKBP12/RyR1 interaction in non-stressed control myotubes (see
Supplementary Material, Fig. S1).
In order to prove the mechanism of action and efficacy of novel
FKBP12/RyR1 interaction stabilizers, we need specific in vitro
models that recapitulate disease phenotypes with the involvement
of nitro-oxidative stress (e.g. Duchenne muscular dystrophy). In
this regard, the peroxynitrite donor SIN1 [28] (3-morpholino-
sydoniminium chloride) can be used to induce post-translational S-
nitrosylation of RyR1 in vitro and generate cellular models of
FKBP12 depletion that are useful to test drug candidates targeting
these processes. Aerobic decomposition of SIN1 produces super-
oxide anion (Oꢂ2e) and nitric oxide (NOꢂ), which react to form per-
oxynitrite anion and induce the S-nitrosylation of cysteine residues,
leading to alterations in intracellular calcium homeostasis [29]. On
the other hand, in situ Proximity Ligation Assay (PLA) is a technique
that allows direct detection of protein interactions and modifica-
tions with high specificity and sensitivity. It can readily detect,
localize and quantify two interacting proteins located within
30e40 nm in unmodified cells and tissues [30]. This is achieved by
using species-specific secondary antibodies linked to complemen-
tary oligonucleotides that hybridize in close proximity. Upon sub-
Our results indicate that both compounds, 10b and S107, show
efficacy in the rescue of FKBP12 depletion from RyR1 complexes
that occurs in human myotubes under nitro-oxidative stress.
Indeed, at 150 nM, 10b and S107 compounds showed similar re-
covery of FKBP12/RyR1 interaction, corresponding to 46% and 56%
recovery scores, respectively. Furthermore, our data confirm that
human myotubes stressed with SIN1 mimic the FKBP12 depletion
from RyR channels that result in “leaky” RyR1 channels, which is
reportedly caused, among other things, by RyR1 S-nitrosylation.
We then carried out further in vitro experiments for direct
determination of the effect of these ligands on intracellular calcium
levels. Human myoblasts were differentiated into myotubes and
pretreated with the benzothiazepine S107 and triazole ligands 10a-
n at 150 nM. Then, myotubes were subjected to nitro-oxidative
stress with 5 mM SIN1 and after 6 h, calcium levels were deter-
mined using the ratiometric fluorochrome Fura-2AM, which mea-
sures cytosolic calcium concentration in living cells (see Fig. 4A for
schematics).
sequent addition of
a
ligase,
a
polymerase, and
a
labeled
In agreement with previous reports [17], a submicromolar
concentration of S107 partially rescued the higher cytosolic calcium
ion levels observed in SIN1-stressed human myotubes. In this line,
several triazole ligands 10 displayed a similar regulating effect on
the S-nitrosylated FKBP12/RyR1 complex. For example, achiral
complementary oligonucleotide, distinct bright spots are obtained,
which can be imaged and quantified by fluorescence microscopy.
Taking this background into account, we surmised the convenience
of applying the in situ PLA technique in human myotubes to eval-
uate the effect of the S-nitrosylation stress caused by SIN1 on
FKBP12/RyR1 co-localization (see Experimental for details).
As shown in Fig. 3, the FKBP12/RyR1 interaction was specifically
quantified by in situ PLA. In healthy human myotubes, SIN1 treat-
ment induced a 60% reduction of FKBP12/RyR1 co-localization
compounds 10a-10g, derived from azidoglycine and
a-azidoiso-
butyric acid, showed recovery scores up to 90% when the aromatic
ring was substituted by electron-donating R1 groups (e. g. MeO).
Electron-withdrawing groups showed much less activity (e. g. 10d).
This last trend persisted in chiral ligands 10k-n derived from
phenylalanine, tyrosine and lysine. An exception of such tendency
was the tetrafluorinated compound 10f. However, such apparent
bias change could be more related to the highly hydrophobic nature
of the 2,3,5,6-tetraflurophenyl moiety than to the electron-
withdrawing effect of fluoro groups. The effect of chirality was
also checked by comparing the enantiomer pairs of ligands 10h-j
and 10m. Interestingly, compared to S enantiomers, we observed
higher calcium-modulating activities of R enantiomers in all in-
stances, excepting the phenylalanine-derived triazoles 10hR and
10hS, which showed similar activity.
(39.87
(100
8.46%) compared to non-stressed myotubes
9.80%; P < 0.05). This reduction was similar to the one
observed in myotubes treated with rapamycin (46.63
2.96%),
which upon binding to FKBP12 [31] depletes FKPB12 from RyR1
complexes [15]. Pre-treatment of myotubes with the RyR1 stabi-
lizer S107 resulted in a partial recovery of FKBP12/RyR1 interaction,
as shown by an 84% increase in FKBP12/RyR1 co-localization
compared to non-treated myotubes stressed with SIN1 (from
39.87
treatment resulted in a significant increase of FKBP12/RyR1 co-
localization in SIN1-stressed myotubes (from 39.87 8.46% to
67.45 9.55%; P < 0.05). As expected, neither S107 nor 10b altered
8.46 to 73.30
6.26%; P < 0.05). Similarly, 10b pre-
Finally, we evaluated the in vitro cytotoxicity profile of selected
triazoles 10 in human myoblasts by using the lactate
4

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
Fig. 2. Target engagement of triazoles 10 was evaluated in HEK293 cells using a luminescence protein complementation assay, by measuring FKBP12-FRB dimerization induced with
rapamycin. (A) Chemical structures of rapamycin, FK506, and 10b compounds showing FKBP12 binding groups in red and FRB binding residues in blue. (B) Monitorization of
temporal FKBP12/FRB dimerization in HEK293 cells expressing FKBP12 and FRB fused to luciferase fragments. Cells were treated with 100 nM rapamycin (green line), 100 mM FK506
(black line) or 10 mM 10b compound (red line). RLU, relative light units. (C) Dose-dependent effect of rapamycin on FKBP12-FRB dimerization. In our system, maximal activity was
reached with 300 nM rapamycin. (D) Schematics of competition assays. (E) Dose-dependent inhibition of FK506 and 10b of FKBP12-Rapamycin-FRB interaction induced with
100 nM rapamycin. IC50 values were 38.29
m
M for FK506 and 3.32 mM for 10b. Data are expressed as mean SEM of n ¼ 3e6 independent experiments performed in triplicate. (F)
Target engagement was demonstrated in triazoles 10 (3 mM) where FKBP12/FRB dimerization was induced with 1.2 mM rapamycin. Data are expressed as mean SEM of three
measurements. Lower activity values indicate higher inhibition potency.
dehydrogenase release method [32]. All the triazole ligands
checked were essentially nontoxic up to millimolar concentration,
while S107 was myotoxic at 1 mM concentration (see Supple-
mentary Material, Fig. S2).
ProePro-Cys(NO)-Phe-Val) of the ryanodine receptor RyR1, which
compromises the normal peptidyl-prolyl isomerase (PPI) activity of
FKBP12 and the calcium conductance of the RyR1 channel. A
combination of computational modeling (virtual alanine mutation)
and docking studies based on such a model has allowed the iden-
tification of polar and hydrophobic hot-spot residues at the FKBP12
protein and the design of a novel family of triazole ligands 10.
Target engagement of several 10 compounds to FKBP12 has been
confirmed experimentally using a competitive ligand binding assay
with rapamycin. Furthermore, the activity of 10b compound to
enhance the binding of FKBP12 to nitrosylated RyR1 calcium
channels has been confirmed experimentally in vitro by using in situ
PLA analysis in healthy human myotubes stressed with the
3. Conclusions
In this study, we propose a detailed FKBP12/RyR1 interaction
mechanism that accounts for the RyR leakage of calcium ion in
skeletal muscle fibers under nitro-oxidative stress, that may occur
during aging, extreme fatigue, or in several diseases such as
Duchenne muscular dystrophy. The model highlights the role of the
S-nitrosylated Cys 1781 residue in the peptide segment (Phe-Ser-
5

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
Fig. 3. Triazole 10b rescues FKPB12/RyR1 co-localization in human myotubes under nitro-oxidative stress. (A) Representative in situ proximity ligation assay (PLA) images showing
FKBP12/RyR1 co-localization (red) and MyHC double staining (green) in non-stressed myotubes (control), as well as in myotubes stressed with 5 mM SIN1 for 30 min (SIN1).
Treatment with S107 and 10b protective compounds at 150 nM concentration partially rescues SIN1-mediated depletion of FKBP12 from RyR1 complexes. In control myotubes,
treatment with 15 mM rapamycin for 30 min induces a similar FKBP12 depletion from RyR1 complexes as the one observed with SIN1. Scale bar: 25 mm. (B) Quantification of in situ
proximity ligation assay (PLA) images showed that both rapamycin and SIN1 (left panel) significantly reduced FKBP12/RyR1 interaction by 53% and 60%, respectively. *P < 0.0001 vs.
control myotubes. Right panel, reduced FKBP12/RyR1 co-localization in SIN1-stressed myotubes was significantly rescued by pretreatment with S107 or 10b, with recovery scores
(RS) of 56% and 46%, respectively. Data are represented as the percentage of co-localization, where the average of non-treated control myotubes was taken as 100%. Data are
expressed as the mean SEM of n ¼ 11e15 fields from 3 to 4 independent cultures with at least 8 myotubes per field. Statistical significance was determined using one-way ANOVA
followed by Dunnett’s test.
peroxynitrite-donor SIN1. Finally, we found that some ligands 10
can rescue up to 100% of the normal cytosolic calcium levels in
SIN1-stressed human myotube cultures, while showing a lack of
substantial toxicity, suggesting that they could minimize the
pathogenic effects of nitro-oxidative stress on skeletal muscles.
According to these results, the novel triazole ligands described
here, and in particular 10b-c, 10h or 10iR, may be useful as thera-
peutic candidates for the treatment of muscular diseases with
compromised calcium homeostasis and/or reduced FKBP12/RyRl
affinity due to nitro-oxidative stress. Further investigation of the
in vivo activity of compounds 10 in cellular and animal models of
Duchenne muscular dystrophy is underway in our laboratories and
the results will be reported in due course.
sources (Merck, Acros Organics, Alfa Aesar, Fluka and Scharlab) and
were used without further purification unless stated otherwise.
Purification of reaction products was carried out by flash chroma-
tography using silica gel 60 (230e400 mesh). Analytical thin layer
chromatography was performed on 0.25 mm silica gel 60-F plates
and visualization was accomplished with UV light (
l
¼ 254 nm) or
KMnO4 as a TLC stain. 1H NMR spectra were recorded on Bruker
Avance spectrometers operated at 500 and 400 MHz. 13C NMR
spectra were recorded at 125 and 101 MHz. The chemical shifts are
reported as
internal standards: for CDCl3 dH (7.26 ppm) and
CD3OD dH (3.31 ppm) and C (49.0 ppm). Analytical HPLC was
d
values (ppm) relative to residual deuterated solvent as
dC (77.16 ppm); for
d
performed on a Waters 1525 Binary HPLC pump (dual wavelength
detector), using a Daicel Chiralpak IC 50 column. The mobile phase
was iPrOH (% 0.1 TFA)/Hex 20:80 with a flow rate of 1 mL/min
monitored by UV detection at 210 nm. High-resolution mass
spectra were performed by SGIker and were acquired on a time of
flight (TOF) mass spectrometer (SYNAPT G2 HDMS from Waters,
Milford, MA, USA) equipped with an electrospray source in positive
mode (ESIþ). Melting points were measured with a Büchi SMP-20
4. Experimental section
4.1. Chemistry
4.1.1. General
All reagents and solvents were obtained from commercial
6

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
Fig. 4. Triazoles 10a-n rescue cytosolic calcium levels in SIN1-stressed human myotubes. (A) Schematics of the experimental protocol. (B) Representative pseudocolored images of
human myotubes loaded with Fura-2AM fluorochrome showing the F340/380 fluorescence ratio recordings. Scale bar: 50 m. (C) Quantification of intracellular calcium levels in
m
non-stressed myotubes (Ctl) and myotubes stressed with 5 mM SIN1. All compounds were tested at 150 nM concentration. Data are expressed as mean SD. Each point in the bar
chart represents an independent myotube culture, with a minimal analyzed area of 0.325 mm2, corresponding to at least 30 myotubes. Statistical significance was determined using
one-way ANOVA followed by Dunnett’s test, where all groups were compared to the SIN1-stressed group. #P ¼ 0.05; ##P ¼ 0.03; ###P ¼ 0.01; ###P ¼ 0.0006.
melting point apparatus and are uncorrected. Infrared spectra were
recorded on a Bruker Alpha P. Optical rotations were measured on a
Jasco P-200 polarimeter using a sodium lamp (589 nm, D line) at
25 0.2 ꢀC and concentrations are reported in g/mL units.
evaporated at reduced pressure to afford a crude product, which
was used without further purification in subsequent reactions.
Analytically pure samples were obtained by flash column chro-
matography (silica gel; eluent: hexanes, then MeOH/CH2Cl2 5:95).
4.1.2.1. Phenyl propargyl sulfide (1) [33]. The general procedure was
4.1.2. General procedure for the synthesis of alkynes 1e7
followed starting from thiophenol (10.0 mmol, 1.38 mL). Oil. Yield
A suspension of the corresponding thiophenol (10 mmol) and
anhydrous K2CO3 (21 mmol, 2.90 g) in MeCN (35 mL) was stirred at
room temperature for 5 min. Propargyl bromide (15 mmol,1.67 mL;
80% soln in toluene) was added dropwise and the resulting mixture
was heated at 50 ꢀC for 3 h. Quenching with H2O (10 mL) and
evaporation of organic volatiles under reduced pressure provided a
solution, which was extracted with CH2Cl2 (2 ꢃ 10 mL). The organic
extract was washed with brine (10 mL), dried (Na2SO4) and
0.82 g (56%). 1H NMR (400 MHz, CDCl3)
d
7.49 (dt, J ¼ 8.1, 0.9 Hz,
2H), 7.40e7.34 (m, 2H), 7.32e7.27 (m, 1H), 2.27 (t, J ¼ 2.6 Hz, 1H).
13C NMR (101 MHz, CDCl3)
d 135.2, 130.2, 129.2, 127.2, 80.1, 71.8,
22.8. IR (cmꢁ1): 3289, 1480, 1438, 737, 688, 637.
4.1.2.2. 3-Methoxyphenyl propargyl sulfide (2) [34]. The general
procedure was followed starting from 3-methoxybenzenethiol
7

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
(10.0 mmol, 1.22 mL). Orange liquid. Yield 1.66 g (93%). 1H NMR
(5 mL: 1 mL), CuOAc (0.05 mmol, 6 mg), NaOAc (3 mmol, 246 mg)
and sodium ascorbate (0.5 mmol, 99 mg) and the mixture was
stirred at 30 ꢀC overnight. Methanol was evaporated in vacuo, aq
20% ammonia was added (0.25 mL) and the solution was extracted
with EtOAc (3 ꢃ 10 mL). The organic extract was washed with a
solution sat aq of NH4Cl (5 mL), dried (Na2SO4) and evaporated at
reduced pressure. The crude product was purified by column
chromatography (silica gel; eluent: EtOAc:Hex mixture) to give the
corresponding intermediate triazole methyl ester. To a solution of
this compound in THF/H2O (4 mL: 4 mL) was added LiOH$H2O
(2.00 mmol, 84 mg) and the mixture was stirred at room temper-
ature for 3 h. The organic solvent was evaporated in vacuo, the
resulting aqueous solution was acidified with 1 M HCl and it was
extracted with EtOAc (2 ꢃ 10 mL). The combined organic phase was
dried (MgSO4) and evaporated at reduced pressure. The crude
products were purified by crystallization in a mixture of EtOAc:
hexanes.
(500 MHz, CDCl3):
d
7.48 (d, J ¼ 8.9 Hz, 2H), 6.87 (d, J ¼ 8.8 Hz, 2H),
3.81 (s, 3H), 3.49 (d, J ¼ 2.6 Hz, 2H), 2.22 (s, 1H). IR (cmꢁ1): 3286,
1588, 1574, 1245, 1228, 1036, 638.
4.1.2.3. 4-Methoxyphenyl propargyl sulfide (3) [35]. The general
procedure was followed starting from 4-methoxybenzenethiol
(10.0 mmol, 1.22 mL). Orange solid. Yield 1.95 g (99%). 1H NMR
(500 MHz, CDCl3): d 7.30e7.22 (m, 1H), 7.07e6.99 (m, 2H), 6.81 (dd,
J ¼ 7.6, 2.4 Hz, 1H), 3.83 (s, 3H), 3.65 (s, 2H), 2.27 (t, J ¼ 2.6 Hz, 1H).
IR (cmꢁ1): 3287, 1590, 1492, 1242, 1026, 823, 636.
4.1.2.4. 3-Chlorophenyl propargyl sulfide (4). The general procedure
was followed starting from 3-clorobenzenethiol (4.68 mmol,
0.54 mL). Yellowish liquid. Yield 0.70 g (82%). 1H NMR (400 MHz,
CDCl3):
d
7.43 (t, J ¼ 1.9 Hz, 1H), 7.31 (dt, J ¼ 7.5, 1.7 Hz, 1H),
7.28e7.24 (m,1H), 7.24e7.20 (m,1H), 3.62 (d, J ¼ 2.6 Hz, 2H), 2.26 (t,
J ¼ 2.6 Hz, 1H). 13C NMR (101 MHz, CDCl3):
d 137.1, 134.7, 130.0,
129.3, 127.7, 127.0, 79.3, 72.1, 22.3. IR (cmꢁ1): 3295, 1576, 1460, 1407,
772, 636. MS (ESI-) m/z (%) 180 (M ꢁ H). HRMS calculated for
(C9H6SCl): 180.9879, found 180.9886.
4.1.3.1. 1-Carboxymethyl-4-(phenylthiomethyl)-1H-1,2,3-triazole
(10a). The general procedure A was followed starting from phenyl
propargyl sulfide (1) (1.0 mmol, 148 mg). The intermediate methyl
ester [1H NMR (400 MHz, CDCl3)
d
7.49 (s, 1H), 7.37e7.30 (m, 2H),
7.28e7.24 (m, 2H), 7.23e7.13 (m, 1H), 5.09 (s, 2H), 4.23 (s, 2H), 3.75
(s, 3H). 13C NMR (101 MHz, CDCl3)
166.5, 145.2, 135.2, 129.4, 128.8,
4.1.2.5. 2,3,5,6-Tetrafluorophenyl propargyl sulfide (6). The general
procedure
was
followed
starting
from
2,3,5,6-
d
tetrafluorobenzenethiol (10 mmol, 1.19 mL). Yellowish liquid.
126.3, 123.4, 52.8, 50.5, 28.6] was submitted to saponification to
Yield 2.18 g (99%). 1H NMR (400 MHz, CDCl3)
d
7.19e7.07 (m, 1H),
afford the title product. Yield: 80% (198 mg). White solid. Mp:
3.68 (d, J ¼ 2.6 Hz, 2H), 2.22 (t, J ¼ 2.6 Hz, 1H). 13C NMR (101 MHz,
189e191 ꢀC. 1H NMR (400 MHz, CD3OD)
d 7.80 (s, 1H), 7.41e7.35 (m,
CDCl3)
d
148.2, 147.1, 145.8, 144.6, 113.6, 106.8, 78.0, 72.6, 22.5. IR
2H), 7.34e7.27 (m, 2H), 7.26e7.19 (m, 1H), 5.23 (s, 2H), 4.24 (s, 2H).
IR (cmꢁ1): 2413, 1879, 1705, 1232, 690. HRMS calculated for
(C11H11N3O2S): 249.0572, found 249.0570.
(cmꢁ1): 3306, 1485, 1437, 1240, 1174, 915, 712, 642. IR (cmꢁ1): 3306,
1485, 915, 642.
4.1.2.6. 3-Trifluoromethylphenyl propargyl sulfide (7). The general
procedure was followed starting from 3-trifluorobenzenethiol
(1 mmol, 1.24 mL). Oil. Yield: 1.10 g (61%). 1H NMR (400 MHz,
4.1.3.2. 1-Carboxymethyl-4-(phenylthiomethyl)-1H-1,2,3-triazole
(10b). The general procedure A was followed starting from 3-
methoxyphenyl propargyl sulfide (2) (1.0 mmol, 175 mg). The in-
CDCl3)
d
7.73 (s,1H), 7.67e7.62 (m,1H), 7.50 (dt, J ¼ 15.5, 7.7 Hz, 2H),
termediate methyl ester [1H NMR (500 MHz, CDCl3)
d 7.51 (s, 1H),
3.72e3.64 (m, 2H), 2.29 (dd, J ¼ 3.2, 2.2 Hz, 1H). 13C NMR (101 MHz,
7.18 (t, J ¼ 8.0 Hz, 1H), 6.95e6.86 (m, 2H), 6.77e6.69 (m, 1H), 5.11 (s,
CDCl3)
d
136.8, 133.1, 129.6, 126.7, 126.6, 123.9, 123.8, 79.3, 72.4,
2H), 4.26 (s, 2H), 3.78 (s, 3H), 3.77 (s, 3H). 13C NMR (126 MHz,
22.5. IR (cmꢁ1): 3306, 1320, 1164, 1120, 1070, 792, 694, 648.
CDCl3) d 166.7, 160.0, 145.7, 136.8, 129.9, 123.6, 121.5, 114.6, 112.5,
55.4, 53.1, 50.8, 28.7] was submitted to saponification to provide
4.1.2.7. 4-Methylphenyl 3-butyn-1-yl sulfide (5). Triethylamine
(22.5 mmol, 3.1 mL) and methanesulfonyl chloride (22.5 mmol,
1.73 mL) were successively added to a solution of 3-butyn-1-ol
(15 mmol, 1.13 mL) in anhydrous CH2Cl2 (90 mL) and the mixture
was stirred at room temperature overnight. Upon completion (TLC),
the organic solution was washed with sat aq NH4Cl (30 mL), dried
over MgSO4 and evaporated under reduced pressure, to afford
crude 3-butyn-1-yl mesylate, which was used in the next step
without further purification. 4-Methylbenzenethiol (18.6 mmol,
2.3 g), 3-butyn-1-yl mesylate and anhydrous K2CO3 (30 mmol,
4.14 g) were suspended in MeCN (60 mL) and the reaction mixture
was heated at 80 ꢀC overnight. After evaporation of the solvents
under reduced pressure, the crude product was purified by column
chromatography (silica gel; eluent: CH2Cl2/MeOH, 95:5). Yield:
the title compound. Yield: 210 mg (74%). White solid. Mp
118e119 ꢀC. 1H NMR (500 MHz, CD3OD):
d
7.80 (s, 1H), 7.18 (t,
J ¼ 8.0 Hz, 1H), 6.96e6.86 (m, 2H), 6.79e6.70 (m, 1H), 5.19 (s, 2H),
4.23 (s, 2H), 3.75 (s, 3H). 13C NMR (126 MHz, CD3OD):
169.7, 161.4,
d
146.1, 138.0, 130.9, 125.9, 123.1, 116.1, 113.6, 55.7, 51.7, 29.3. IR
(cmꢁ1): 2999, 2971, 1707, 1229. MS (ESIþ) m/z (%) 280 (M þ H).
HRMS calculated for (C12H14N3O4S): 280.0756, found 280.0753.
4.1.3.3. 1-Carboxymethyl-4-[4-(methoxy)phenylthiomethyl]-1H-
1,2,3-triazole (10c). The general procedure A was followed starting
from 4-methoxyphenyl propargyl sulfide (3) (1.0 mmol, 175 mg).
The intermediate methyl ester [1H NMR (500 MHz, CDCl3)
d
7.38 (s,
1H), 7.33e7.28 (m, 2H), 6.85e6.76 (m, 2H), 5.09 (s, 2H), 4.12 (s, 2H),
3.78 (s, 3H), 3.77 (s, 3H). 13C NMR (126 MHz, CDCl3)
166.7, 159.4,
d
1.50 g (53%). 1H NMR (400 MHz, CDCl3)
d
7.36e7.29 (m, 2H), 7.15 (d,
145.8, 134.0, 125.4, 123.5, 114.7, 55.4, 53.1, 50.8, 31.0] was submitted
J ¼ 7.9 Hz, 2H), 3.05 (t, J ¼ 7.5 Hz, 2H), 2.49 (td, J ¼ 7.5, 2.7 Hz, 2H),
to saponification to afford the title product. Yield: 88% (250 g).
2.36 (s, 3H), 2.06 (t, J ¼ 2.6 Hz, 1H). 13C NMR (101 MHz, CDCl3)
White solid. Mp 168e170 ꢀC. 1H NMR (500 MHz, CD3OD):
d
7.66 (s,
d
136.8, 131.2, 131.0, 129.8, 82.3, 69.5, 33.5, 21.0, 19.3. IR (cmꢁ1):
1H), 7.28 (d, J ¼ 8.8 Hz, 2H), 6.84 (d, J ¼ 8.8 Hz, 2H), 5.19 (s, 2H), 4.07
3292, 1492, 802, 632, 490.
(s, 2H), 3.76 (s, 3H). 13C NMR (126 MHz, CD3OD):
d 169.7,161.1,146.3,
135.6, 126.3, 125.9, 115.7, 55.8, 51.6, 31.5. IR (cmꢁ1): 2923, 2848,
1730, 1223, 1188. HRMS calculated for (C12H14N3O3S): 280.0756,
found 280.0760.
4.1.3. General procedure for the preparation of achiral triazoles
10a-g (method A)
A mixture of methyl bromoacetate (1.2 mmol, 113 mL) and so-
dium azide (1.3 mmol, 84 mg) in MeOH (0.5 mL) and water (0.3 mL)
was stirred at 50 ꢀC for 5 h. To this solution was added successively
the corresponding alkyne 1e6 (1.0 mmol), dissolved in MeOH/H2O
4.1.3.4. 1-Carboxymethyl-4-[3-(chloro)phenylthiomethyl]-1H-1,2,3-
triazole (10d). The general procedure A was followed starting from
3-chlorophenyl propargyl sulfide (4) (1.0 mmol, 178 mg). The
8

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
intermediate methyl ester [1H NMR (400 MHz, CDCl3)
d
7.52 (s, 1H),
1.78 (s, 6H). 13C NMR (101 MHz, CD3OD):
d 174.2, 161.3, 145.3, 137.9,
7.31 (s, 1H), 7.22e7.13 (m, 3H), 5.12 (s, 2H), 4.26 (s, 2H), 3.79 (s, 3H).
130.8,123.4,116.5,113.7, 65.9, 55.7, 29.5, 25.9. IR (cmꢁ1): 2931,1589,
1573, 1283, 1229, 1178, 1030. MS (ESIþ) m/z (%) 308 (M þ H). HRMS
calculated for (C14H18N3O3S): 308.1069, found 308.1074.
13C NMR (101 MHz, CDCl3)
d
166.7, 145.2, 137.7, 134.8, 130.2, 129.0,
127.4, 126.7, 123.6, 53.2, 50.9, 28.7] was submitted to saponification
to afford the title product. Yield: 67% (0.38 g). White solid. Mp
146e148 ꢀC. 1H NMR (500 MHz, CDCl3):
d
7.86 (s, 1H), 7.38 (s, 1H),
4.1.4. General procedure for the preparation of chiral triazoles 10h-
n (method B)
7.27 (d, J ¼ 6.8 Hz, 2H), 7.21 (d, J ¼ 1.9 Hz, 1H), 5.22 (s, 2H), 4.28 (s,
2H). 13C NMR (126 MHz, CD3OD):
d
168.4, 144.2, 137.9, 134.4, 130.0,
The corresponding a-amino acid (1.0 mmol) and potassium
128.8, 127.6, 126.3, 124.7, 50.4, 27.7. IR (cmꢁ1): 2928, 2850, 1731,
1224, 1184. MS (ESIþ) m/z (%) 284 (M þ H). HRMS calculated for
(C11H10ClN3O2S): 284.0182, found 284.0190.
carbonate (2.0 mmol, 276 mg) were dissolved in a mixture of water
(5 mL) and methanol (5 mL). Then, CuSO4$5H2O (0.01 mmol,
2.5 mg) and 1-(azidosulfonyl)imidazolium hydrogen sulfate 11
(1.1 mmol, 275 mg) were added. The reaction mixture was stirred at
4.1.3.5. 1-Carboxymethyl-4-[2-(4-methylphenylthio)ethyl]-1H-1,2,3-
triazole (10e). The general procedure A was followed starting from
4-methylphenyl 3-butyn-1-yl sulfide (5) (1.0 mmol, 176 mg). The
room temperature for 5 h to give a solution of the corresponding a-
azido acid to which were added successively the corresponding
alkyne (2), (4) or (7) (1.2 mmol) dissolved in methanol (1 mL),
CuOAc (0.05 mmol, 6 mg), NaOAc (5 mmol, 410 mg) and sodium
ascorbate (0.5 mmol, 100 mg). After stirring the mixture overnight
at 30 ꢀC, the organic solvent was evaporated in vacuo, pH was
adjusted to 10 with a saturated aqueous solution of ammonia and
the aqueous phase was washed with CH2Cl2 (2 ꢃ 10 mL). The
aqueous phase was acidified with 2 M HCl, extracted with EtOAc
(2 ꢃ 15 mL) and the extract was dried (MgSO4) and evaporated at
reduced pressure to give the product.
intermediate methyl ester [1H NMR (400 MHz, CDCl3)
d 7.53 (s, 1H),
7.35e7.26 (m, 2H), 7.13 (d, J ¼ 7.8 Hz, 1H), 5.16 (s, 2H), 3.82 (s, 3H),
3.23 (t, J ¼ 7.3 Hz, 2H), 3.06 (t, J ¼ 7.4 Hz, 2H), 2.34 (s, 3H). 13C NMR
(101 MHz, CDCl3)
d 167.1, 146.8, 136.8, 132.0, 130.8, 130.0, 123.0,
53.3, 50.9, 34.2, 26.0, 21.2] was submitted to saponification to afford
the title product. Yield: 188 mg (68%). White solid. 1H NMR
(400 MHz, CD3OD):
d
7.84 (s, 1H), 7.31 (d, J ¼ 8.2 Hz, 1H), 7.16 (d,
J ¼ 7.9 Hz, 2H), 5.23 (s, 3H), 3.21 (dd, J ¼ 8.0, 6.9 Hz, 2H), 2.99 (t,
J ¼ 7.4 Hz, 3H), 2.34 (s, 3H). 13C NMR (101 MHz, CD3OD):
d 169.8,
147.2, 137.7, 133.3, 131.7, 130.8,125.3, 51.6, 34.7, 26.6, 21.0. IR (cmꢁ1):
2917, 2500, 2109, 1724, 1215, 803, 486. HRMS calculated for
(C13H15N3O2S): 277.0885, found 277.0881.
4.1.4.1. (R)-1-(1-Carboxy-2-phenylethyl)-4-[3-(methoxy)phenyl-
thiomethyl]-1H-1,2,3-triazole (10hR). The general procedure B was
followed starting from D-phenylalanine (1 mmol, 165 mg) and 3-
methoxyphenyl propargyl sulfide (2) (1.2 mmol, 213 mg) to give
4.1.3.6. 1-Carboxymethyl-4-[2,3,5,6-(tetrafluoro)phenylthiomethyl]-
1H-1,2,3-triazole (10f). The general procedure A was followed
starting from 2,3,5,6-tetrafluorophenyl propargyl sulfide (6)
(1.00 mmol, 220 mg). The intermediate methyl ester [1H NMR
the product. Yield: 76% (280 mg). White solid. Mp 85e86 ꢀC. 1H
NMR (500 MHz, CDCl3):
d 10.11 (s, 1H), 7.49 (s, 1H), 7.14 (d,
J ¼ 7.4 Hz, 4H), 6.89 (d, J ¼ 6.6 Hz, 2H), 6.82 (d, J ¼ 6.7 Hz, 2H), 6.72
(d, J ¼ 7.4 Hz, 1H), 5.53 (dd, J ¼ 9.4, 4.8 Hz, 1H), 4.31e4.08 (m, 2H),
3.70 (s, 3H), 3.52 (dd, J ¼ 14.3, 4.6 Hz, 1H), 3.36 (dd, J ¼ 14.1, 9.8 Hz,
(400 MHz, CDCl3) d 7.64 (s, 1H), 7.16e7.01 (m, 1H), 5.16 (s, 2H), 4.29
(s, 2H), 3.83 (s, 3H)] was submitted to saponification to afford the
1H). 13C NMR (126 Hz, CD3OD):
d 171.1, 161.4, 145.9, 138.0, 137.2,
title product. Yield: 292 mg (91%). White solid. M.p: 205e207 ꢀC. 1H
130.9, 129.9, 129.6, 128.1, 124.8, 122.9, 115.9, 113.5, 65.8, 55.7, 39.0,
29.1. IR (cmꢁ1): 2930, 1727, 1246, 1230. MS (ESIþ) m/z (%) 384
(M þ H). HRMS (ESIþ, m/z) calculated for C19H20N3O3S: 370.1225;
NMR (500 MHz, CD3OD):
d
7.88 (s, 1H), 7.43 (tt, J ¼ 10.0, 7.3 Hz, 1H),
168.2, 147.0
5.23 (s, 2H), 4.29 (s, 2H). 13C NMR (126 MHz, CD3OD):
d
1
2
1
(dt, JCF ¼ 252.0 Hz, JCF ¼ 12.6 Hz), 145.8 (dt, JCF ¼ 243.0 Hz,
found: 370.1239. [
a
] ¼ þ16.50 (c 0.98, CH2Cl2).
2JCF ¼ 16.4 Hz), 143.7, 124.6, 107.0, 106.8, 106.7, 50.2, 27.9. 19F NMR
(471 MHz, CD3OD):
d
ꢁ135.73 (4JFF ¼ 9.4 Hz) ꢁ140.56 (4JFF ¼ 9.3 Hz).
4.1.4.2. (S)-1-(1-Carboxy-2-phenylethyl)-4-[3-(methoxy)phenyl-
thiomethyl]-1H-1,2,3-triazole (10hS). The general procedure B was
followed starting from L-phenylalanine (1 mmol, 165 mg) and 3-
IR (cmꢁ1): 2435, 1869, 1710, 1486, 1234, 911, 710. HRMS calculated
for (C11H7F4N3O2S): 321.0195, found 321.0195.
methoxyphenyl propargyl sulfide (2) (1.2 mmol, 213 mg) to give
4.1.3.7. 1-(1-Carboxy-1-methylethyl)-4-[3-(methoxy)phenyl-
the product. Yield: 73% (270 mg). White solid. Mp 81e83 ꢀC. NMR,
thiomethyl]-1H-1,2,3-triazole (10g). A solution of methyl
a
-bro-
IR and HRMS data were identical to example 10hR. [
1.12, CH2Cl2).
a
] ¼ ꢁ23.31 (c
moisobutyrate (6.6 mmol, 854
m
L) and sodium azide (19.8 mmol,
1.3 g) in DMSO (10 mL) was stirred at 50 ꢀC for 2 h. The reaction
mixture was quenched with sat aq NaCl (20 mL), extracted with
CH2Cl2 (2 ꢃ 10 mL) and the organic phase was dried over MgSO4, to
4.1.4.3. (R)-1-(1-Carboxy-3-methylbutyl)-4-[3-(methoxy)phenyl-
thiomethyl]-1H-1,2,3-triazole (10iR). The general procedure B was
followed starting from D-leucine (1.1 mmol, 144 mg) and 3-
give methyl
a-azidoisobutyrate. To a solution of the crude ester in
MeOH/H2O (9/3.5 mL) was added 3-methoxyphenyl propargyl
sulfide (5.5 mmol, 980 mg), CuOAc (0.28 mmol, 34 mg), NaOAc
(16.5 mmol, 1.4 g) and sodium ascorbate (2.75 mmol, 545 mg) and
the mixture was stirred at 30 ꢀC overnight. The reaction mixture
was worked up and purified according to Method A to provide the
corresponding intermediate ester [1H NMR (400 MHz, CD3OD):
methoxyphenyl propargyl sulfide (2) (1.3 mmol, 231 mg) to give
the product. Yield: 80% (301 mg). White solid. Mp: 102e104 ꢀC. 1H
NMR (500 MHz, CDCl3):
d
11.88 (s,1H), 7.54 (s, 1H), 7.12 (t, J ¼ 7.9 Hz,
1H), 6.95e6.77 (m, 2H), 6.75e6.62 (m,1H), 5.38 (dd, J ¼ 10.7, 5.0 Hz,
1H), 4.35e4.15 (m, 2H), 3.70 (s, 3H), 2.33e1.75 (m, 2H), 1.17 (dq,
J ¼ 13.2, 6.5 Hz, 1H), 0.88 (d, J ¼ 6.5 Hz, 3H), 0.82 (d, J ¼ 6.5 Hz, 3H).
d
7.77 (s, 1H), 7.09 (t, J ¼ 7.8 Hz, 1H), 6.90e6.73 (m, 2H), 6.67 (d,
13C NMR (126 Hz, CDCl3):
d 171.2, 159.8, 144.7, 136.0, 129.8, 122.6,
J ¼ 7.7 Hz, 1H), 4.12 (s, 2H), 3.65 (s, 3H), 1.78 (s, 6H). 13C NMR
122.4, 115.6, 112.9, 61.8, 55.3, 41.2, 28.5, 24.7, 22.7, 21.1. IR (cmꢁ1):
2958, 2513, 1726, 1588, 1478, 1229, 1037, 773, 685. MS (ESIþ) m/z (%)
336 (M þ H). HRMS (ESIþ, m/z) calculated for C16H22N3O3S:
(101 MHz, CD3OD):
d 174.2, 161.3, 145.3, 137.9, 130.8, 123.4, 116.5,
113.7, 65.9, 55.7, 29.5, 25.9]. This compound was submitted to
saponification with lithium hydroxide monohydrate (2.00 mmol,
84 mg) in THF/H2O (4 mL/4 mL) and worked up to provide the title
compound. Yield: 72% (138 mg). Brown solid: Mp 119e121 ꢀC. 1H
336.1382; found: 336.1389. [
a] ¼ ꢁ12.06 (c 0.95, CH2Cl2).
4.1.4.4. (S)-1-(1-Carboxy-3-methylbutyl)-4-[3-(methoxy)phenyl-
NMR (400 MHz, CD3OD):
6.90e6.73 (m, 2H), 6.67 (d, J ¼ 7.7 Hz, 1H), 4.12 (s, 2H), 3.65 (s, 3H),
d
7.77 (s, 1H), 7.09 (t, J ¼ 7.8 Hz, 1H),
thiomethyl]-1H-1,2,3-triazole (10iS). The general procedure B was
followed starting from
L-leucine (1.1 mmol, 197 mg) and 3-
9

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
24
methoxyphenyl propargyl sulfide (2) (1.8 mmol, 320 mg) to give
the product. Yield: 75% (360 mg). NMR, IR and HRMS data were
[
a
]
¼ ꢁ32.1.0ꢀ (c. 1.21 g/100 mL, MeOH).
D
identical to example 10iR. [
a
] ¼ þ9.31 (c 1.11, CH2Cl2).
4.1.4.10. (S)-1-[5-tert-Butoxycarbonylamino-1-carboxypentyl]-4-[3-
(chloro)phenylthiomethyl]-1H-1,2,3-triazole (10nS). The general
4.1.4.5. (R)-1-(1-Carboxy-3-methylpropyl)-4-[3-(methoxy)phenyl-
procedure B was followed starting from N-Boc-L-lysine (1 mmol,
thiomethyl]-1H-1,2,3-triazole (10jR). The general procedure B was
246 mg) and 3-chlorophenyl propargyl sulfide (4) (1.2 mmol,
followed starting from
D-valine (1 mmol, 117 mg) and 3-
219 mg) to give the product. Yield: 63% (286 mg). Oil. 1H NMR
methoxyphenyl propargyl sulfide (2) (1.2 mmol, 213 mg) to give
(400 MHz, CD3CN)
d
7.76 (s, 1H), 7.43 (q, J ¼ 1.3 Hz, 1H), 7.32e7.28
the product. Yield: 69% (220 mg). White solid. Mp 120e123 ꢀC. 1H
(m, 2H), 7.25 (qt, J ¼ 4.8, 2.2 Hz, 1H), 5.29 (dd, J ¼ 10.4, 5.0 Hz, 1H),
NMR (500 MHz, CD3OD):
d
7.81 (s, 1H), 7.16 (t, J ¼ 7.9 Hz, 1H),
4.30 (s, 2H), 2.97 (qd, J ¼ 6.8, 2.9 Hz, 2H), 2.30e2.07 (m, 2H), 1.41 (s,
24
7.03e6.82 (m, 2H), 6.82e6.66 (m, 1H), 5.06 (d, J ¼ 8.1 Hz, 1H), 4.22
(s, 2H), 3.73 (s, 3H), 2.46 (dt, J ¼ 13.5, 6.7 Hz, 1H), 0.95 (d, J ¼ 6.7 Hz,
11H), 1.03 (tt, J ¼ 8.7, 4.4 Hz, 2H). [
a
]
¼ ꢁ9.8ꢀ (c. 0.95 g/100 mL,
D
MeOH). IR (cmꢁ1): 2976, 1684, 1180, 1152, 778, 607. HRMS calcu-
lated for (C20H27ClN4O4S): 454.1442, found 454.1458.
3H), 0.74 (d, J ¼ 6.7 Hz, 3H). 13C NMR (126 Hz, CD3OD):
d 170.7,
159.9, 144.6, 136.1, 129.9, 123.0, 122.8, 115.8, 113.0, 69.3, 55.4, 32.2,
28.7, 19.3, 18.3. IR (cmꢁ1): 2966, 2461, 1907, 1591, 1573, 1479, 1281,
1228, 1206, 1037, 783, 721, 687. MS (ESIþ) m/z (%) 336 (M þ H).
HRMS (ESIþ, m/z) calculated for C15H20N3O3S: 332.1227; found:
4.2. Biological evaluation
4.2.1. Cell cultures
332.1225. [
a
] ¼ ꢁ6.68 (c 1.01, CH2Cl2).
LHCN-M2 and 8220 immortalized human myoblasts were
kindly provided by Dr. Vincent Mouly (Myology Institute, Paris).
These cells were generated by the Platform for Immortalization of
Human Cells (Myology Institute, Paris). Human myoblasts were
grown and differentiated as previously reported [36]. Experiments
were performed 7e9 days after first adding differentiation me-
dium, with highly mature myotubes.
4.1.4.6. (S)-1-(1-Carboxy-3-methylpropyl)-4-[3-(methoxy)phenyl-
thiomethyl]-1H-1,2,3-triazole (10jS). The general procedure B was
followed starting from
L-valine (1 mmol, 117 mg) and 3-
methoxyphenyl propargyl sulfide (2) (1.2 mmol, 259 mg) to give
the product. Yield: 65% (210 mg). White solid. Mp 119e121 ꢀC.
NMR, IR and HRMS data were identical to example 10jR. [
(c 1.06, CH2Cl2).
a] ¼ þ4.52
4.2.2. Competitive ligand binding assay
HEK293 cells were seeded onto white 96-well plates (Corning)
4.1.4.7. (R)-1-(1-Carboxy-2-phenylethyl)-4-[3-(trifluoromethyl)phe-
nylthiomethyl]-1H-1,2,3-triazole (10kR). The general procedure B
coated with poli-
in DMEM (Thermofisher) with 10% FBS for 6 h and transfected with
D
-lysine at 4 ꢃ 105 cells per well. Cells were grown
was followed starting from
D-phenylalanine (1 mmol, 165 mg) and
12.5 ng/mL of each FRB-lgBiT and FKBP-smBiT vectors (NanoBiT
3-trifluoromethylphenyl propargyl sulfide (7) (1.2 mmol, 259 mg)
Control Pair, Promega) using ViaFect. Kinetic measurements were
performed 20e24 h after transfection in CO2 Independent Medium
(Gibco, Thermofisher) with Nano-Glo® Live Cell Reagent (Prom-
ega), as described by the manufacturer. Luminescence was
measured at 37 ꢀC using the Glomax Discover Microplate Reader
(Promega). Rapamycin, FK506 and triazole compounds were
sequentially added and each effect was recorded for 15 min. To
establish the specific activity in competitive binding assays,
measured values of basal activity (BA) at 10 min, maximal activity
(MA) at 25 min, and compound activity (CA) at 40 min were
determined. Specific activity (SA) was calculated using the formula:
SA ¼ (MA-BA)/(CA-BA)*100. SA was normalized to the nondrug
(vehicle) measurements. IC50 and EC50 were calculated in
GraphPad Prism 8.0.1 by fitting the data to a four-parameter logistic
curve.
to give the product. Yield: 63% (258 mg) 81% (329 mg). Oil. 1H NMR
(500 MHz, CD3OD):
d 7.85 (s, 1H), 7.61 (s, 1H), 7.56e7.42 (m, 3H),
7.13 (dd, J ¼ 5.0, 2.0 Hz, 2H), 7.04e6.95 (m, 2H), 5.62 (dd, J ¼ 10.8,
4.7 Hz, 1H), 4.27 (s, 2H), 3.61 (dd, J ¼ 14.4, 4.7 Hz, 1H), 3.45 (dd,
J ¼ 14.3, 10.8 Hz, 1H). 13C NMR (126 MHz, CD3OD)
d 169.6, 143.8,
137.3, 135.7, 132.4,132.3, 130.9 (q, 2JCF ¼ 31.0 Hz), 129.4,128.5,128.2,
1
126.7, 125.4, 125.3, 123.9 (q, JCF ¼ 271.0 Hz), 123.6, 122.7, 122.6,
122.5, 64.3, 37.5, 27.3. 19F NMR (471 MHz, CD3OD):
d
ꢁ64.26. IR
(cmꢁ1): 1716, 1319, 1122, 692. HRMS (ESIþ, m/z) calculated for
24
C
19H17N3O2SF3: 408.0994; found: 408.1005. [
a
]
¼ þ16.8ꢀ (c.
D
0.98 g/100 mL, CH2Cl2).
4.1.4.8. (R)-1-[1-Carboxy-2-(4-hydroxyphenyl)ethyl]-4-[3-(tri-
fluoromethyl)phenylthiomethyl]-1H-1,2,3-triazole (1mR). The Gen-
eral Procedure B was followed starting from D-tyrosine (1 mmol,
181 mg) and 3-trifluoromethylphenyl propargyl sulfide (7)
4.2.3. In situ proximity ligation assay (PLA)
(1.2 mmol, 259 mg) to give the product. Yield: 73% (308 mg). Oil. 1H
In situ PLA assay was performed as previously described [37].
Briefly, paraformaldehyde-fixed myotubes were blocked and
incubated overnight with the following primary antibodies: anti-
RyR1 mouse mAb (1:200, Thermo Scientific), and anti-Calst1 rab-
bit pAb (1:100, Novus Biologicals). PLA assay was performed using
the Duolink in situ Orange kit (Sigma). Samples were incubated
with oligonucleotide strand conjugated secondary antibodies
(MINUS and PLUS PLA probes, Sigma). For counterstaining, samples
were incubated with FITC-conjugated Myosin Heavy Chain-CFS
mAb (1:50, R&D) for 30 min, and mounted with ProLong Gold
antifade reagent with DAPI (Life Technologies). Image quantifica-
tion was performed with ImageJ (NIH) and the “Batch spot analysis”
macro from the Henry Wellcome Laboratory (https://www.uea.ac.
uk/about/faculties-and-schools/faculty-of-science/facilities/bio-
imaging-platform/macros). For each image, the total number of
spots was normalized to the MyHC area. At least 4 images per
condition were analyzed with an average of 8e9 myotubes per
image.
NMR (400 MHz, CD3OD):
d 7.84 (s, 1H), 7.62 (s, 1H), 7.53e7.39 (m,
3H), 6.84e6.77 (m, 2H), 6.58 (d, J ¼ 8.5 Hz, 2H), 5.54 (dd, J ¼ 10.5,
4.7 Hz,1H), 4.25 (s, 2H), 3.49 (dd, J ¼ 14.4, 4.7 Hz,1H), 3.39e3.29 (m,
1H). 13C NMR (126 MHz, CD3OD)
d 167.7, 156.5, 135.7, 133.4, 131.0 (q,
2JCF ¼ 32.0 Hz), 129.8, 129.6, 129.5, 126.4, 125.3, 125.2, 123.8 (q,
1JCF ¼ 274.7 Hz), 123.5, 115.3, 65.9, 62.5, 36.4. 19F NMR (471 MHz,
CD3OD):
d
ꢁ64.01. IR (cmꢁ1): 2924 br, 1732, 1516, 1321, 1164, 1120,
1067, 792, 695. HRMS (ESIþ) m/z calculated for C19H17N3O3SF3:
24
424.0943; found: 424.0949. [
a
]
¼ þ28.7ꢀ (c. 1.14 g/100 mL,
D
MeOH).
4.1.4.9. (S)-1-[1-Carboxy-2-(4-hydroxyphenyl)ethyl]-4-[3-(tri-
fluorometil)phenylthiomethyl]-1H-1,2,3-triazole (10mS). The Gen-
eral Procedure B was followed starting from L-tyrosine (1 mmol,
181 mg) and 3-trifluoromethylphenyl propargyl sulfide (7)
(1.2 mmol, 259 mg) to give the product. Yield: 86% (318 mg). Oil.
NMR, IR and HRMS data were identical to example 10mR
10

J.M. Aizpurua, J.I. Miranda, A. Irastorza et al.
European Journal of Medicinal Chemistry 213 (2021) 113160
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Acknowledgments
This research was funded by the Basque Government (J.M.A.,
GIC-2015_IT-1033-16; J.M.A., ETORTEK-KK-2018/00108; A.V.-I.,
2016111091); Instituto de Salud Carlos III, co-funded by European
Regional Development Fund/European Social Fund, “Investing in
your future” (A.V.-I., PI17/00676; A.L.d.M., PI17/01841), Diputacion
Foral de Gipuzkoa (A.V.-I., 2019-00362-01-B); the University of the
Basque Country (A.V.-I., PPGA19/58, PPGA20/23), and Duchenne
Parent Project, Espana (A.V.-I.). A.S.-A., A.I., G.A. hold fellowships
from the Basque Government, H.L.-F. holds a PhD fellowship from
the UPV/EHU and A.V.-I. holds a Ramon y Cajal contract funded by
the Spanish Ministry of Economy and Competitiveness. The authors
thank Dr. Mouly and the Platform for Immortalization of Human
Cells (Myology Institute) for the human LHCN-M2 and 8220 cell
lines. The use of SGIker NMR facilities is acknowledged to the
University of the Basque Country UPV/EHU.
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