Development of prohibitin ligands against osteoporosis

Article abstract of DOI:10.1016/j.ejmech.2020.112961

Current therapeutic approaches to osteoporosis display some potential adverse effects and a limited efficacy on non-vertebral fracture reduction. Some sulfonylamidines targeting the scaffold proteins prohibitins-1 and 2 (PHB1/2) have been showed to inhibit the formation of osteoclasts in charge of bone resorption. Herein, we report the development of a second generation of anti-osteoclastic PHB ligands. The most potent compound, IN45, showed 88% inhibition at the low concentration of 5 μM, indicates that it might serve as a basis for the development of new antiosteoporotic drugs.

Full text of DOI:10.1016/j.ejmech.2020.112961

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Development of prohibitin ligands against osteoporosis
Redouane Tabti, François Lamoureux, Céline Charrier, Benjamin Ory, Dominique
Heymann, Embarek Bentouhami, Laurent Désaubry
PII:
S0223-5234(20)30933-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112961
EJMECH 112961
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 September 2020
Revised Date: 17 October 2020
Accepted Date: 17 October 2020
Please cite this article as: R. Tabti, F. Lamoureux, C. Charrier, B. Ory, D. Heymann, E. Bentouhami,
L. Désaubry, Development of prohibitin ligands against osteoporosis, European Journal of Medicinal
Chemistry, https://doi.org/10.1016/j.ejmech.2020.112961.
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Graphical Abstract

Development of prohibitin ligands against osteoporosis
Redouane Tabti,1,2 François Lamoureux,3 Céline Charrier,3 Benjamin Ory,3 Dominique
Heymann,4,5,6 Embarek Bentouhami,2 and Laurent Désaubry1,7
1
Laboratory of Regenerative Nanomedicine (RNM), INSERM U 1260, CRBS, Rue Eugène
Boeckel, 67000, Strasbourg, France.
2 LCIMN Laboratory, Faculty of Technology, University Ferhat Abbas, Sétif, Algeria
3
INSERM, UMR 1238, Nantes, France; Université de Nantes, Nantes Atlantique Universités,
Laboratoire « Sarcome osseux et remodelage des tissus osseux calcifiés », Faculté de
Médecine, Nantes, France.
4
Institut de Cancérologie de l'Ouest, site René Gauducheau, Boulevard Professeur Jacques
Monod, Saint-Herblain, France.
5 Université de Nantes, Faculty of Medicine, Nantes, France.
6. University of Sheffield, Dept of Oncology and Metabolism, Sheffield, UK.
7
Sino-French Joint Lab of Food Nutrition/Safety and Medicinal Chemistry, College of
Biotechnology, Tianjin University of Science and Technology, Tianjin, China
Correspondence to: Laurent Désaubry, email: Désaubry@unistra.fr
Keywords: osteoclastogenesis; prohibitins; SAR; osteoporosis; STAT3.
ABSTRACT: Current therapeutic approaches to osteoporosis display some potential adverse
effects and a limited efficacy on non-vertebral fracture reduction. Some sulfonylamidines
targeting the scaffold proteins prohibitins-1 and 2 (PHB1/2) have been showed to inhibit the
formation of osteoclasts in charge of bone resorption. Herein, we report the development of a
second generation of anti-osteoclastic PHB ligands. The most potent compound, IN45, showed
88 % inhibition at the low concentration of 5
development of new antiosteoporotic drugs.
μM, indicates that it might serve as a basis for the
1

1. Introduction
Aging accelerates bone loss and skeletal fragility, leading to morbidity and mortality in both
men and women [1]. A continual dynamic self-regeneration process in bone called remodeling is
a coordinated cycle of resorption of old bone by osteoclasts followed by the deposition of new
bone by osteoblasts in response to mechanical loadings and micro damages. With aging this
balance shifts in a negative direction, favoring greater bone resorption and less bone formation,
affecting the musculoskeletal system and ultimately resulting in osteoporosis, thus increasing
fracture risk. In Europe, fractures caused by osteoporosis (usually hip or spinal) affect 1 in 2
women and 1 in 5 men aged more than 50 years old [2]. While numerous anti-catabolic (e.g.
bisphosphonates, anti-RANKL) and pro-anabolic (e.g. PTH fragments) drugs have been
developed in the recent past, most of them reduce bone resorption and increase bone mineral
density by artificially suppressing the physiological bone remodeling process, leading to a new
formed bone prone to breakage [3]. In addition current treatments of osteoporosis display some
potential adverse effects and a limited efficacy on non-vertebral fracture reduction. Due to these
limitations and the ageing of the population, the discovery of new anti-osteoporotic agents is a
matter of public health.
Osteoclasts are generated by the fusion of mononuclear progenitors of bone marrow
monocyte / macrophage lineage that resorb bone in the presence of the receptor activator of NF-
κ
B ligand (RANKL) [4]. Indeed, binding of RANKL to its cell-surface receptor, RANK, activates
several signaling pathways leading notably to the activation of the mitogen-activated protein
(MAP) kinases p38 and ERK, the nuclear factor B (NF- B), and the nuclear factor of activated T
κ
κ
cells c1 (NFATc1), which are critical to regulate osteoclastogenesis and maintain the survival of
mature osteoclasts. NFATc1 regulates in particular the expression of the tartrate-resistant acid
phosphatase (TRAP) and cathepsin K, which are involved in the cell fusion of osteoclast
progenitors and in the bone resorptive activity of osteoclasts.
In addition to RANKL, several humoral factors, such as interleukin-6 (IL-6), can also
promote osteoclastogenesis through a regulation of the transcription factor STAT3 [5]. Indeed, a
basal level of STAT3 phosphorylated at serine-727 associated with an absence of
phosphorylation on tyrosine-705 is required for osteoclast differentiation and STAT3 inhibitors
abolish RANKL-induced osteoclastogenesis. Importantly, STAT3 has been shown to be
regulated by PHBs in several cell types [6-8].
2

To identify a novel class of antiosteoporotic agents, Chang et al. screened a library of
sulfonylamidines for their in vitro ability to block pre-osteoclast differentiation into osteoclasts,
which mediate the removal of bone cells [9]. The sulfonylamidine 1 (Figure 1) was found to be a
particularly potent inhibitor of osteoclast differentiation with an IC50 of 3.3
μM. Using affinity
chromatography, prohibitin-1 (PHB1) was identified as the molecular target of these compounds
[10]. PHB1 and its homolog PHB2 are ubiquitous scaffold proteins that coordinate many
signaling pathways to regulate all aspects of cell functions, including metabolism, apoptosis, cell
migration, division, and differentiation [11]. As such, PHBs are involved in the etiology of
cancers, in inflammatory, cardiovascular, and neurodegenerative diseases, diabetes, obesity,
and osteoporosis. The various functions and intracellular localization of PHBs are regulated by a
wide array of post-translational modifications.
Figure 1. Structure of anti-osteoclastic sulfonylamidine 1 [10].
Since the identification of PHB1 as the molecular target of anti-osteoporotic
sulfonylamidines, PHB1 has been demonstrated to inhibit the formation of mature osteoclasts
[12], but the engagement of PHB1 in this activity of 1 has not been established yet, even though
1 has shown to block the PHB-dependent entry of Chikungunya virus into microglial cells [13].
Yet, some key events in the mechanism of action of these compounds have been clarified [14].
Osteoclast differentiation is initiated by the binding of RANK ligand (RANKL) to its receptor
RANK, which activates TRAF6 and consecutively, IKK and ERK, leading to the activation of the
transcription factors NF-
dependently inhibit the RANKL-induced activation of ERK, and both the translocation to the
nucleus and the transcriptional activity of NF- B. In addition, they repressed mRNA expression
κB, c-Fos and NFATc1. Sulfonylamidines were shown to dose-
κ
levels of c-Fos and NFATc1 induced by RANKL, indicating that sulfonylamidines mitigate
RANKL-induced signaling and transcription factors required for osteoclast differentiation.
Sulfonylamidines also repressed the expression of the matrix metalloproteinase 9 (MMP-9) and
the kinase c-Src that both promote osteoclastic bone resorption.
Hitherto, no further study on sulfonylamidines as PHB ligands has been reported, possibly
3

because the moderate activity of these compounds. Thus we aimed to develop more active
analogues to explore the therapeutic potential of these PHB ligands in models of osteoporosis.
2. Results
2.1. Chemical synthesis
A first approach to prepare sulfonylamidines relies on the condensation of amides 2 with
tosylisocyanate following King’s method (Scheme 1) [15].
Scheme 1. Synthesis of sulfonylamidines 3a-e by condensation of amides with tosylisocyanate.
To restrain the conformational flexibility of these PHB ligands, we synthesized the cyclic
sulfonylamidines from valerolactam (4a) and caprolactam (4b) (Scheme 2). N-alkylation of these
compounds with isopropyl iodide occurred in low yield. Condensation with tosylisocyanate
conveniently furnished 6a/b [15]. Deprotonation with LDA and reaction with n-iodobutane
following Magnus’ procedure [16] afforded the monoadducts 7a/b. Unfortunately, this reaction
did not proceed with cyclohexyl bromide or iodide. To overcome this limitation we explored
several routes and finally developed an original synthesis of substituted caprolactams (Scheme
3).
Scheme 2. Synthesis of sulfonylamidines 6a/b and 7a/b.
4

Our synthesis commenced with the reductive condensation of aldehyde 8 with Meldrum
acid 9 in the presence of formic acid and trimethylamine to furnish the monoadduct 10 (Scheme
3). A second alkylation with 1,4-diodobutane delivered the adduct 11 in 51% yield. A
condensation of the latter with isopropylamine gave the secondary amine 12 in 78% yield. With
this advanced intermediate in hand, we proceeded to its cyclization via the formation of ketene
intermediate 13. Original attempts to perform this reaction at reflux in DMF afforded the
expected caprolactam 14 in only 10% yield. Using microwave conditions improved the yield up
to 62%. As far as we know, it is the first time that a caprolactam is synthesized by this type of
strategy. Completion of the synthesis was accomplished by a condensation with tosylisocyanate
in a 27% yield.
Scheme 3. Synthesis of sulfonylamidine 15.
To restrict the conformational freedom of 3a, we synthesized the alkene 19 using a
Wadsworth–Emmons reaction and a condensation of the obtained amide with tosylisocyanate
(Scheme 4).
Scheme 4. Synthesis of alkene 19.
Sulfonylamidines 23a-
γ
were synthesized in a straightforward manner using a Cu-
5

catalyzed three-component coupling of sulfonyl azides 20, alkynes 21, and amines 22 (Scheme
5) [17].
Scheme 5. Synthesis of sulfonylamidines 23a-γ.
We examined the isosteric replacement of the tosylamidine moiety synthesizing the tosylguanidines
27 and 30 (Scheme 6). Aza-isostere 27 was prepared in 3 steps starting from the addition of amine
25 to cyclohexylisothiocyanate 24, followed by S-methylation and condensation with tosylamide [18].
The synthesis of the cyclic tosylguanidine 30 started by the protection of cyclohexylamine 28 as a
nosylamide, which was alkylated by 1,4-diiodobutane, condensed with isopropylamine and
deprotected. The obtained diamine 29 reacted with N-tosyldichloromethanimine [19] to afford the
cyclic tosylguanidine 30.
6

Scheme 6. Synthesis of the tosylguanidines 27 and 30.
2.2. Inhibition of osteoclastogenesis
We functionally examined whether our new sulfonylamidines inhibit osteoclastogenesis.
Osteoclast differentiation was induced from human CD14+ precursors cultured in the presence of M-
CSF and RANKL and the formation of multinucleated cells was followed by TRAP staining (Table 1)
[20]. We confirmed that 1 indeed blocked osteoclast differentiation (54 % inhibition at 10 µM).
Importantly, replacement of the n-pentyl by a cyclohexylmethyl 3a greatly inhibited osteoclast
differentiation.
The replacement by a cyclohexyl (3b) was unfavorable. Restraining the conformational
flexibility through the inclusion of the alkyl chain in a 6-membered ring was slightly detrimental (7a),
but switching to a 7 membered ring (7b) restored a good inhibition of osteoclast differentiation.
Deleting the n-butyl substituents on these two latter compounds (6a and 6b) abolished the activity.
The replacement of the cyclohexylmethyl of 3a by a cyclopentylmethyl (3c) did not modify the
activity. Exchanging the 2,6-dimethylpiperidine moiety by a decahydroquinoline (3d) was somewhat
unfavorable. Introduction of an unsaturation between the cyclohexyl and the sulfoamidine moiety led
to an inactive compound (19).
7

Table 1. Inhibition of osteoclastogenesis by the 1st series of sulfonylamidines.
Cpd Structure Inhibition % Cpd Structure
1µM 10 µM
Inhibition %
1µM 10 µM
1
6a
20.9 ± 12.1 99.8 ± 1.0
35.0 ± 13.3 97.9 ± 2.4
27.9 ± 4.06 6.1 ± 4.1
3a
3b
7a
7b
6b
3c
3d
19
27.1 ± 9.5
39.3 ± 6.1
22.8 ± 4.3
31.9 ± 4.1
12.1 ± 5.4
24.1 ± 9.8
80.6 ± 8.1
90.1± 2.7
32.7 ± 11.4 91.5 ± 6.1
33.1 ± 0.5
78.9 ± 7.8
25.5 ± 19.2 23.3 ± 10.8
Bone marrow-derived CD14+ preosteoclasts were treated by sulfonylamidines with 100 ng/mL RANKL
and 25 ng/mL M-CSF for 10 days. TRAP staining was then performed. Nuclei greater than 3 were
counted as osteoclasts. The percentage of inhibition is an average corresponding to the number of
osteoclasts in each treated condition compared to corresponding positive control, which represents the
100 % value.
The following compounds have been tested with different lots of human monocytes which may have
various sensitivity to the RANK ligand (RANKL). To compare all of these results, the compound 3a
was used as the reference compound.
In accordance with 7b activity, the rigidification of 3a through a 7-memberd ring (15) was beneficial
(Supplementary table S1). The isosteric replacement of the tosylamidine moiety by a tosylguanidine
(27 and 30) suppressed the activity. In contrast to what we observed with 3d, the introduction of a
decahydroquinoline (3e) was highly detrimental, indicating that the substituent effects of the alkyl and
tosylamidine moieties are highly interdependent
Next, we pursued our SAR investigation by changing the nature of the arylsulfonyl group (Table 2
and supplementary table S2). Replacement of the 4-tolyl by a phenyl (23a), a trifluoromethyl (23b) or
a 5-(dimethylamino)naphthalenyl (23i) was well tolerated, but replacement by a 2-pyridyl, 3-pyridyl,
dimethylamino or camphorsulfonyl was unfavorable at the dose of 10 µM (23c, 23d, 23e, 23g, Figure
2). This deleterious effect was more pronounced with an isopropyl or a methyl or (23f and 23h). 23c
and 23d displayed a biphasic effect: at 1 µM they promoted osteoclastogenesis, but at 10 µM they
8

displayed the opposite effect. This increase in the number of osteoclasts can be explained by a
transient release of pro-osteoclastogenic agents in the culture medium.
Table 2. Inhibition of osteoclastogenesis by the 3rd series of sulfonylamidines.
Cpd
Structure
Inhibition %
1 µM
Cpd Structure
Inhibition %
1 µM
10 µM
10 µM
3a
23d
23e
23f
14.4 ± 13.9
97.6 ± 2.1
-23.6 ± 33.9
11.8 ± 16.5
1.6 ± 8.96
-1.5 ± 10.4
56.1 ± 39.7
23a
23b
23c
3.3 ± 26.41
-1.1 ± 22.9
-20.2 ± 35.6
97.9 ± 0.5
96.1 ± 3.9
44.9± 49.1
68.4 ± 5.5
12.6 ± 28.1
57.5 ± 29.8
23g
9

Figure 2. Representative pictures of CD14+ monocytes treated (positive control) or not with
RANKL (negative control), and +/- treated with 23c or 23d at indicated doses. Cells were then
stained with TRAP staining kit to reveal multinucleated cells (osteoclasts).
The replacement of the tosyl by a mesyl (23h) or dansyl (23i) were respectively very and
slightly detrimental (Supplementary table S2). Gratefully, the replacement of 2,6-
dimethylpiperidine by an N-isopropylcyclohexylamine (23j) maintained the anti-osteoclastogenic
efficacy. At the doses of 1 M and 10 M, 23j suppressed osteoclastogenesis by 10% and 99%
respectively.
In the next set of experiments, 3a was found to promote osteoclastogenesis at 1 M,
confirming that the observed effect is highly dependent of the monocyte donors. Gratefully, the
introduction of a N-isopropylcyclopentylamine (23k) significantly enhanced the anti-osteoclastogenic
efficacy (Table S3). Curiously the transition to a 2,6-dimethylpyrrolidine 23l rendered the compound
10

pro-osteoclastogenic at 1 µM and cytotoxic at 10 µM. Switching to an azepane 23m had a
deleterious effect, but an azocane 23n was well tolerated. The tetrahydropyran derivative 23o was
less active than its larger, less polar, sulfur isostere 23p. Cycloheptanic derivatives 23q had an
activity at least equal to that of 3a.
The cyclooctanic derivative 23r was less active than 3a at 1 μM, but it was more active at 10
μM (Supplementary table S4). Interestingly, this compound was found to display some cytotoxicity.
The introduction of chlorine on the arylsulfonyl group improved the activity, especially in position
ortho (23s). The introduction of an isopropyl in position para (23w) also increased the anti-
osteoclastogenic activity.
Next, we replaced the 2,6-dimethylpiperidino moiety in 23v by various hydrophobic amines, and
we observed that the N-isopropylcyclooctanamine (23γ) increased slightly the anti-
osteoclastogenic activity (Table 3). Interestingly this compound, called IN45, was found to be
cytotoxic at 10 µM.
Table 3. Inhibition of osteoclastogenesis by the 7th series of sulfonylamidines.
Inhibition %
5 µM
Inhibition %
1µM 5 µM 10 µM
1 µM
10 µM
3a
-9 ± 31 72 ± 13 99 ± 2
23α
23β
23γ
-10± 25 53 ±10 93 ± 4
-6 ± 18 74± 4 99 ± 2
23v
23x
23y
23z
0 ± 32
62 ± 9
98 ± 2
-86± 26 26 ± 12 99 ± 1
5 ± 16
88 ± 8 100 ± 0
-30± 20 49 ± 4
88 ± 7
-14 ± 5 28 ± 20 39 ± 21
2.3. Effects of sulfonylamidine 3a on cell signaling
Having probes with inhibition of osteoclastogenesis in hand; we next examined the molecular
11

mechanisms of 3a on key signaling pathways that regulate the differentiation of CD14+ monocytes in
osteoclasts.
p38 and ERK are two MAP kinases involved in osteoclastic differentiation that have been shown
to be activated by other PHB ligands in several cell types [21-26]. We observed that 3a did not
modulate the expression or phosphorylation of these 2 kinases at protein level after IL-6
stimulation. However, 3a inhibited IL6-induced STAT3 phosphorylation at serine-727 (Figure 3),
which is involved in osteoclast differentiation by regulating the transcription of NFATc1 [5, 27].
Figure 3. Effects of compound 3a on the activation of STAT3, P38 and ERK in CD14+
monocytes. Human CD14+ monocytes were treated with 25 ng/ml for 3 days. At day 3, human
CD14+ monocytes were treated with vehicle or 10 µM 3a for 4h without FBS and M-CSF,
followed by IL-6 stimulation (25 ng/mL) for 15 min. Western blot analysis was performed on cell
lysate to determine the phosphorylation level of STAT3 (at Ser-727 and Tyr-705), P38 and ERK.
Sulfonylamidines 1 and 3a were found to decrease the mRNA expression of the matrix-
degrading enzyme cathepsin K, the tartrate-resistant acid phosphatase (TRAP) and NFATc1,
indicating that these compounds inhibit osteoclastogenesis through inhibition of RANKL-induced
NFATc1 expression (Figure 4). This inhibition was more pronounced with 3a than with reference
compound 1.
12

Figure 4. Effects of compounds 1 and 3a on mRNA expression of cathepsin K, TRAP and
NFATc1. At day 3, Human CD14+ monocytes were treated with RANKL with or without 1 or 3a (1
or 10 µM) for 4 days. Total RNA was isolated and analyzed by RT-qPCR. Each assay was
performed in triplicate. A. Cathepsin K mRNA expression. B. TRAP mRNA expression. C.
NFATc1 mRNA expression.
4. Conclusion
With the aging of the population, the need to improve the treatment of osteoporosis will
become more and more preeminent. Therefore, new drugs targeting novel therapeutic agents
are in great need. Although PHBs are involved in the regulation of osteoclostagenesis, these
signaling proteins are not yet established therapeutic targets against osteoporosis. Since the
discovery of anticlastogenic sulfonylamidines targeting PHBs in 2010, no medicinal chemistry
project aimed at improving these compounds has been reported.
13

Here, a series of 50 new analogues was synthesized, and evaluated for their in vitro
osteoclastogenesis inhibitory activities. The most potent compound, IN45, showed 88 %
inhibition at a concentration as low as 5 μM. We have also contributed to better define the
mechanism of action of these compounds. Kim et al. had shown that these compounds bind to
PHB1 [10] and block ERK activation and NFATc1 expression [14]. Herein, we confirmed that
these compounds indeed inhibit the expression of NFATc1 and found that they also inhibit the
phosphorylation of the transcription factor STAT3 that is also necessary to osteoclast
differentiation [27]. Considering that PHB1 overexpression inhibits NFATc1 and blocks
osteoclast differentiation [12], and also that PHB1 regulate STAT3 signaling in several cell types,
these observations support an action of sulfonylamidines on PHB1 to inhibit osteoclastogenesis,
through a cascade of events that we are currently exploring. In addition, since sulfonylamidine 1
has been shown to block the entry of Chikungunya virus into microglial cells [13],
sulfonylamidines might also block also the PHB-dependent entry of other viruses, such as
chronic hepatitis C virus, dengue virus or enterovirus 71 (responsible of hand, foot and mouth
disease [28]. Considering that PHBs are expressed in every tissue, these compounds might also
be of interest to treat other types of diseases where modulation of PHB signaling is beneficial
[26].
Overall, our current study sets up a basis for the development of new antiosteoporotic
drugs with an original mechanism of action and also consolidate PHB1 as a novel therapeutic
target against osteoporosis.
5. Material and methods
5.1. Synthetic Procedures
Detailed synthetic methods can be found in SI Materials and Methods.
5.2. Selection of CD14+ cells
Blood sample from different human donors are first diluted with phosphate buffer to
obtain final 100ml and diluted samples are layered onto Ficoll solution in a centrifuge tube.
Human peripheral blood mononuclear cells (PBMCs) were then isolated by centrifugation over
Ficoll gradient for 25 minutes at 500g. Next, enrichment and purification of osteoclast precursors
(CD14+) allow the differentiation of high number of osteoclasts. CD14+ cells are magnetically
labeled with CD14 Microbeads and positively selected by MACS technology.
14

5.3. Osteoclast differentiation
To induce osteoclast formation, CD14+ cells are seeded at 45 x 104 cells/well in 96-well
plates in α-MEM (Invitrogen Life Technologies) supplemented with 10%FCS and 25 ng/ml hM-
CSF (hM-CSF; R&D Systems). From day 3 of the culture, medium is changed twice a week with
medium containing 10%FCS, 25 ng/ml hM-CSF and 100 ng/ml hRANKL +/- chemical
compounds. The formation of osteoclasts occurs after around 11 days and is confirmed by
Tartrate-Resistant Acid Phosphatase (TRAP) staining (Sigma, France).
5.4. Quantitative reverse transcription PCR
CD14+ cells were seeded in 96 wells plates. Three days after seeding, CD14+ cells were treated
with 25 ng/ml hM-CSF and 100ng/ml hRANKL +/-chemical compounds at the indicated doses for
4 days. Total RNA was extracted using the Macherey-Nagel Nucleospin RNA. Following a
reverse transcription on 1 µg of total RNA, cDNA was diluted to 10 ng/µL. A Real-time monitoring
of PCR amplification of cDNA was performed using DNA primers on CFX96 Detector System
(Bio-Rad) with SYBR PCR Master Mix (Bio-Rad). Target gene expression was normalized to
GAPDH level in respective samples as an internal standard, and the comparative cycle
threshold (Ct) method was used to calculate relative quantification of target mRNAs. Each assay
was performed in triplicate.
5.5. Western blot
Samples containing equal amounts of protein from lysates of cultured osteosarcoma cells
underwent electrophoresis on SDS-polyacrylamide gel and were transferred to PVDF filters
blocked in odyssey blocking buffer (LI-COR). Blots were probed overnight at 4°C with primary
antibodies. After incubation, the filters were washed in PBS containing 0.05% Tween. Filters
were then probed with a fluorescent antibody (Li-COR IRDye) diluted in the odyssey blocking
buffer. Antibody binding was visualized using the LI-COR odyssey Fc.
Acknowledgment
The authors are grateful to the French and Algerian Ministries for Foreign Affairs for a fellowship
to Redouane Tabti via the PHC Tassili scientific exchange program.
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Highlights
• There is an urgent need to improve antiosteoporotic treatments.
• Some sulfonylamidines target prohibitins to inhibit the formation of osteoclasts.
• We developed new sulfonylamidines with enhanced anti-osteoclastic activities.
• The most potent compound showed 88 % inhibition at the low concentration of 5
μM.

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