Design, synthesis and biological characterization of a new class of osteogenic (1H)-quinolone derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.05.062

Smoothened (Smo) is the signal transducer of the Hedgehog (Hh) pathway and its stimulation is considered a potential powerful tool in regenerative medicine to treat severe tissue injuries. Starting from GSA-10, a recently reported Hh activator acting on Smo, we have designed and synthesized a new class of quinolone-based compounds. Modification and decoration of three different portions of the original scaffold led to compounds able to induce differentiation of multipotent mesenchymal cells into osteoblasts. The submicromolar activity of several of these new quinolones (0.4–0.9?μM) is comparable to or better than that of SAG and purmorphamine, two reference Smo agonists. Structure-activity relationships allow identification of several molecular determinants important for the activity of these compounds.

Full text of DOI:10.1016/j.ejmech.2016.05.062

Accepted Manuscript
Design, synthesis and biological characterization of a new class of osteogenic (1H)-
quinolone derivatives
Fabrizio Manetti, Elena Petricci, Annalisa Gabrielli, Andrè Mann, Hélène Faure,
Tatiana Gorojankina, Laurent Brasseur, Lucile Hoch, Martial Ruat, Maurizio Taddei
PII:
S0223-5234(16)30470-6
10.1016/j.ejmech.2016.05.062
EJMECH 8654
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2016
Revised Date: 26 May 2016
Accepted Date: 27 May 2016
Please cite this article as: F. Manetti, E. Petricci, A. Gabrielli, A. Mann, H. Faure, T. Gorojankina, L.
Brasseur, L. Hoch, M. Ruat, M. Taddei, Design, synthesis and biological characterization of a new class
of osteogenic (1H)-quinolone derivatives, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.05.062.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Design, synthesis and biological characterization of a new
class of osteogenic (1H)-quinolone derivatives
Fabrizio Manetti,§ Elena Petricci,*,§ Annalisa Gabrielli,§ Andrè Mann,¤ Hélène Faure,† Tatiana
Gorojankina,† Laurent Brasseur,† Lucile Hoch,† Martial Ruat,† and Maurizio Taddei§
Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, via Aldo Moro 2, I-53100, Siena, Italy;
¤Laboratoire d’Innovation Thérapeutique CNRS, UMR-7200, Université de Strasbourg, F-67401 Illkirch, France;
†CNRS, UMR-9197, Neuroscience Paris-Saclay Institute, 1 Avenue de la Terrasse, F-91198, Gif-sur-Yvette, France.
*
KEYWORDS: Stem cells, Smoothened agonists, structure-activity relationships, osteogenic activity, heterocycle,
synthesis, pharmacophore, Hedgehog signaling pathway
ABSTRACT: Smoothened (Smo) is the signal transducer of the Hedgehog (Hh) pathway and its
stimulation is considered a potential powerful tool in regenerative medicine to treat severe tissue
injuries. Starting from GSA-10, a recently reported Hh activator acting on Smo, we have designed
and synthesized a new class of quinolone-based compounds. Modification and decoration of three
different portions of the original scaffold led to compounds able to induce differentiation of
multipotent mesenchymal cells into osteoblasts. The submicromolar activity of several of these
new quinolones (0.4-0.9 µM) is comparable to or better than that of SAG and purmorphamine,
two reference Smo agonists. Structure-activity relationships allow identification of several
molecular determinants important for the activity of these compounds.

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Introduction
The Hedgehog (Hh) pathway is comprised of several components which orchestrate various
aspects of embryogenesis, development, and tissue remodeling, with critical functions during
embryonic, post-natal development and adult life.[1] In the absence of Hh ligands (Sonic, Indian,
and Desert Hedgehog), Patched (Ptc) inhibits the function of Smoothened (Smo) resulting in
phosphorylation and processing of glioblastoma (Gli) transcription factors into repressor, thus
preventing their target genes expression. On the contrary, when Hh ligands are released into the
extracellular environment, Smo repression is stopped and an intracellular signalling cascade is
started. As a consequence, glioblastoma (Gli) transcription factors are activated in order to
regulate the expression of Hh target genes. However, besides this canonical Hh signalling pathway,
several non-canonical pathways, independent of Gli but involving Smo, have been recently
reported.[2]
The pivotal role of Hh signalling in stem cells,[3][4] during development of many tissues and
organs,[5][6] and in remodelling, repair,[7] and regeneration of adult tissues,[8][9][10] prompted
to search small molecules that modulate the Hh pathway. While Hh inhibition has found
application in anticancer therapy,[11][12][13][14][2] the activation of the Hh pathway may
promote tissue regeneration and repair after severe injury in numerous organs. As an example,
coronary vessel density is increased and coronary function is improved by Hh agonists after
myocardial infarction[15] and in the case of ischemic heart disease.[16] On the other hand,
stimulation of the Hh cascade induces insulin production in pancreatic cells,[10][9] and affects
bone marrow.[17] In the adult brain, Hh signaling is implicated in neural stem cell
maintenance[18] and has been shown to positively regulate remyelination.[1][19]
Small molecules that act as positive modulators of Smo are able to promote or improve human
embryonic stem cell differentiation[3][20] and have been proposed as therapeutic tools for
stimulating tissue functions or restoring repair mechanisms that are compromised by disease. Smo
agonists have been also used to facilitate tissue and organ repair, and for implantation of
engineered tissues.[21] Of particular importance is the osteoinductive activity of Smo agonists
which are able to increase bone mass, thus representing attractive molecules in the field of
osteopenia, osteoporosis and bone diseases.[17]
Scheme 1: Positive modulators of Smo

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Hh agonists act also as an effective tool for bone formation after forearm amputation in
mouse,[22] and promote proliferation and survival of cortical/hippocampal cells, without affecting
their differentiation or inducing apoptosis.[5] The ability to modulate neurogenesis and promote
survival of neural cells could represent an attractive therapeutic tool for replacement of dead or
damaged neural cells in the case of nerve damage or neurodegenerative diseases (i.e., Parkinson’s
disease).[6][23][24] [18] Smo agonists could finally have a potential utility in regenerative
medicine as well as a tool for studying stem cell proliferation mechanisms.[25] The recent
evidence that Smo agonists are implicated in insulin-independent glucose uptake in muscle and
brown fat tissue through activating a non-canonical AMPK (5’ adenosine monophosphate-
activated protein kinase) axis, raised novel interests for Smo agonists for obesity and diabetes.[26]
Despite this wide range of potential applications, a very limited number of Smo agonists have
been reported so far, without definitive experimental evidence of their mechanism of action at
the molecular level. Purmorphamine (Scheme 1) was first identified as a potent osteogenic agent
in rodent cells.[27][28] However, opposite results showed that this compound may inhibit[29] or
enhance[30] osteoblast differentiation from human bone marrow mesenchymal cells. Other
applications of purmorphamine in tissue engineering were also recently proposed.[31][32] SAG, a
chloro benzothiophene derivative with a 30 nM EC50 value on the Gli-Luc assay is another example
of a well studied Smo agonist.[33][34] A complete series of SAG derivatives have been
described[35][36] and the difluoromethoxy analogue (Scheme 1) showed an activity improved by
two orders of magnitude (0.3 nM).[37] Several glucocorticoids[38][25] and oxysterols[39][40]
(Scheme 1) have been also described as potential Smo agonists, able to directly interact with Smo
in an allosteric site distinct from the one responsible for cyclopamine and purmorphamine
activities.
In this context, within a drug discovery program aimed at identifying new small molecules that
stimulate Hh signaling pathway by direct activation of Smo, we have built a pharmacophoric
model on the basis of already known Smo agonists. This model was then applied for an in silico
virtual screening of commercial compound databases and quinolone GSA-10 (1 in Scheme 1) was
identified and characterized as a positive modulator of Smo.[41] Compound 1 induces osteogenic
differentiation of multipotent mesenchymal C3H10T1/2 progenitor cells stimulating the alkaline
phosphatase (AP) response more than 16-fold above the basal level, with an EC50 value (1.2 µM,
Table 1) comparable to that of purmorphamine (0.8 µM), although lower than SAG (0.13 µM)
Results and discussion
Following a classical hit optimization routine, the influence of different substituents on the activity
of this compound was studied. For this purpose, the synthesis of analogues of compound 1 was
planned by introducing chemical diversity at three portions of the molecule, corresponding to the
N1 chain (R1), the ester moiety (R2), and the condensed phenyl ring (R3) (See Scheme 1 and Table
1).
The oxo-quinoline ring was synthesized by two different approaches depending on the target
products. For the introduction of different substituents on N-1 position, commercially available N-
alkylated anilines 1a-g were treated with triethyl methane tricarboxylate under microwave (MW)
dielectric heating at 225 °C for 30 min obtaining expected products 2a-g in good yields (Scheme 2).
The proposed synthetic approach failed when applied to anilines having different substituents on
the aromatic ring, as the first cyclisation to the 3 carboxy-quinolone intermediate did not occur. In
this case, desired derivatives 2h-k were synthesized starting from functionalized ethyl 2-
nitrobenzoates 4h-k that were first reduced to give the corresponding anthranilic derivatives. The
N-propylation was carried out by reductive amination with propanal (5h-k) followed by a further

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acylation with ethyl malonyl chloride in DCM giving the key intermediates 6h-k. Cyclisation with
NaH in DMF gave expected 3 carboxy-quinoline derivatives 2h-k in good to excellent yields.
Scheme 2. Preparation of quinolones 2a-k. 1a,2a R1=-C6H13; 1b,2b R1=-C5H11, 1c,2c R1=-C4H9; 1d,2d R1=-C3H7;
1e,2e R1=Me; 1f,2f R1=Allyl; 1g,2g R1 = Bn, 4h, 5h,6h R2 = 6-F; 4i, 5i, 6i R2= 6-Cl; 4j, 5j, 6j R2 = 6 Me; 4k, 5k, 6k R3 =
6-OMe, 7-OMe.
Amidation of derivatives 2a-k with commercially available or in house prepared anilines 7a-i was
run again under MW irradiation at 250 °C in toluene for 30-60 min, depending on the substrate.
Expected derivatives 8-24 and 26 were obtained in good to excellent yields (Scheme 3). For the
synthesis of the acid derivative 25, t-butyl ester of 9 was removed by treatment with TFA.
Scheme 3. Preparation of quinolones 1, 8-26. 7a R3=4-COOBu; 7b R3=4-COOPr; 7c R3=4-COOt-Bu; 7d R3=4-
CONHPr; 7e R3=3-COOPr; 7f R3=3-COOEt; 7g R3=3-Me, 5-COOPr; 7h R3=3-Cl, 4-COOPr; 7i R3=2-CF3, 3-COOPr.
With the aim to improve the chemical diversity of this small library of compounds, commercially
available derivative 28-42 were also bought and evaluated for their activity as Smo agonists.
The new compounds were tested for their ability to induce osteoblast differentiation in
mesenchymal C3H10T1/2 cells by evaluating the AP response. Biological results (summarized in
Table 1) allowed us to deduce several considerations on the relationships between structural
changes and compounds activity. Variations on the length and bulkiness of the propyl ester chain
somewhat changed activity. In particular, while a butyl group (8) did not affect activity significantly
(EC50 = 1.5 versus 1.2 µM found for reference compound GSA-10) the shorter ethyl (as in 28) and
the bulkier i-propyl or t-butyl substituents (as in 9 and 29, respectively) caused a 2-5-fold
reduction of activity. The benzyl derivative 30 retained an activity (2.0 µM) comparable to that of
8. Transformation of the ester moiety into the corresponding acidic and amide group led to
opposite changes in activity. In fact, while the acidic derivative 25 was two-fold better than 1 (EC50
= 0.6 µM, Table 1, Figure 2A), compounds bearing a primary (31) or secondary (10 and 32) amide
moiety were inactive or significantly less active with respect to the corresponding ester analogues
(compare 10 and 32 to 1). Moving the R3 substituent from position 4 to 3 or 5 generates inactive
compounds (21-24 and 26-27). The introduction of different heterocycles in place of the “right
side” phenyl group or the absence of a carbonyl on this ring (compounds 38-42) gave almost

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inactive products. In a similar way, replacement of the ester chain with an ether group produced
an inactive compound (33). These data suggested that the propyl ester group should be
maintained. Accordingly, keeping fixed the propyl ester terminus, the N-alkyl chain (R1) was
shortened, leading to a pentyl and a butyl moiety that showed the optimal length for a
submicromolar activity (0.7 and 0.8 µM found for 11 and 12, respectively). In addition, while a
propyl chain (as in 13) retained an activity two-fold lower than that of 1, a further length reduction
to a methyl group led to an inactive compound (14). Moreover, the allyl derivative 15 showed an
activity (3.4 µM) comparable to that of the propyl analogue 13, while 16, bearing a benzyl moiety,
was inactive. Structural rigidification as in 34 gave an interesting activity (EC50 = 0.8 µM).
Variations of the propyl ester chain of 11 into a butyl (35) or ethyl (36) group did not change
activity (EC50 = 0.7 vs 0.6 and 0.9 µM, respectively). Among these compounds, 11 and 35 showed
an activity two-fold better than that of 1. Transformation of the pentyl chain of 36 into a benzyl
group led to an inactive compound (37), thus confirming that a benzyl moiety at the heterocyclic
nitrogen atom is detrimental for agonist effect, as already found for the propyl analogue 16.
Finally, to further enlarge SAR considerations, the condensed phenyl ring of 13 was also decorated
with various small substituents. Introduction of a F, Cl, and Me group at position 6 (as in 17, 18,
and 19, respectively, Table 1) did not influence significantly activity (from 1.2 to 4.9 µM).
Interestingly, the 6,7-dimethoxy substitution of 20 resulted in a submicromolar activity (0.36 µM),
three-fold better than that of the reference compounds 1 and purmorphamine and comparable
with SAG (Table 1, Figure 2A).
These results are in agreement with the orientation of the new series of compounds within the
pharmacophoric model previously built for Smo agonists (Figure 1).[41] In fact, conformational
freedom of the Cphenyl-Ccarbonyl bond does allow both the oxygen atoms of the ester moiety in 1 and
20 to correspond to the hydrogen bond acceptor feature of the pharmacophore. The same ability
is expected for both the oxygen atoms of the carboxyl terminus of 25 (Figure 1). On the contrary,
formation of hydrogen bonds involving the amide NH as the donor group could be the driving
force for the orientation of the amide of 10, 31 and 32 within the receptor binding site, possibly
avoiding the carbonyl moiety to have any additional profitable contact as acceptor group, as
predicted by the pharmacophore. This could explain the general lack of activity for amide
compounds in comparison to ester analogues. Moreover, the ether oxygen of 33 is too far from
the region where the hydrogen bond acceptor group should be located, thus accounting for the
inactivity of this compound. On the other hand, alkyl chains of different length (from propyl to
hexyl) at the quinolone nitrogen result in active compounds. In agreement with experimental
data, two hydrophobic features in correspondence of C2-C3 and C6 of a linear alkyl moiety suggest
a hexyl chain as optimal for activity, even if shorter chains of 3-4 carbon atoms should be
profitable for activity.
Figure 1. Schematic representation of 25 superposed to the six-feature pharmacophoric model for Smo agonists
comprised of four hydrophobic regions (blue) and two hydrogen bond acceptor groups (green). Hydrophobic

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features are filled by the condensed phenyl ring, by the central phenyl ring, and by the C2-C3 and C6 carbon
atoms of the hexyl chain at N1. Hydrogen bonds acceptors are represented by the carbonyl group at position 2
of the quinolone core and by the carboxylic function.

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Table 1. Structure and osteoinductive activity of quinolones 8-42 in C3H10T1/2 cell differentiation.
R1
R2
R3
EC50 (μM)a
Stim.b
SAG
0.13 ± 0.01 75 ± 14
Purmorph.
0.8 ± 0.1
78 ± 12
1
8
9
-C6H13
-C6H13
-C6H13
-C6H13
-C5H11
-C4H9
-C3H7
Me
4-COOC3H7
4-COOC4H9
4-COOtBu
1.2 ± 0.1
1.5 ± 0.4
3.1 ± 0.5
ND
100
88 ± 6
69 ± 9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
4-CONHC3H7
4-COOC3H7
4-COOC3H7
4-COOC3H7
4-COOC3H7
4-COOC3H7
4-COOC3H7
4-COOC3H7
4-COOC3H7
4-COOC3H7
4-COOC3H7
3-COOC3H7
3-Me, 5-COOC3H7 ND
3-COOtBu
3-COOEt
4-COOH
3-Cl, 4-COOC3H7
1.6 ± 0.5
0.70 ± 0.01 138 ± 12
0.8 ± 0.2
2.6 ± 0.9
ND
3.4 ± 0.6
ND
2.4 ± 0.1
4.9 ± 0.4
1.2 ± 0.8
135 ± 10
105 ± 11
1.0 ± 0.4
22 ± 6
7 ± 2
98 ± 4
Allyl
Bn
-C3H7 6-F
-C3H7 6-Cl
-C3H7 6-Me
-C3H7 6,7-diMeO
-C6H13
-C6H13
-C6H13
-C6H13
-C6H13
-C6H13
-C6H13
-C6H13
-C6H13
-C6H13
-C6H13
-C6H13
-C6H13
41 ± 2
85 ± 3
0.36 ± 0.04 138 ± 10
ND
2 ± 1
3.0 ± 0.1
1.3 ± 0.3
1.7 ± 0.1
137 ± 11
11 ± 3
1.4 ± 0.3
62 ± 13
33 ± 3
ND
ND
0.6 ± 0.1
ND
2-CF3, 3-COOC3H7 ND
4-CO2Et
3.6 ± 0.7
4-COOCHMe2
4-COOBn
4-CONH2
4-CONHBu
4-OEt
6.5 ± 0.5
2.0 ± 0.2
ND
5.8 ± 1.6
ND
0.8 ± 0.3
0.6 ± 0.1
0.9 ± 0.1
47 ± 19
1.0 ± 0.3
92 ± 2
1± 1
38 ± 17
123 ± 16
76 ± 1
-C5H11
-C5H9
4- COOC4H9
4-CO2Et

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37
38
39
40
41
42
Bn
4-CO2Et
ND
ND
ND
ND
ND
ND
4 ± 1
5 ± 3
3 ± 1
3 ± 2
3 ± 1
5 ± 3
-C5H11
-C5H11
2-OH, 4-NO2
4-NO2
aDifferentiation of the mesenchymal pluripotent C3H10T1/2 cells into osteoblasts was measured
using an AP assay. bMaximal stimulation (Stim.) relative to 1 (100 %) obtained at 10 µM, except
forSAG (3 µM). All data are expressed as mean ± S.E.M. (n ≥ 3). ND: not determined.
Among the most potent osteogenic compounds belonging to the class of quinolone derivatives,
the most active compounds 20 and 25 were further profiled (in comparison with already known 1)
to dissect their biological activity at the molecular level and to fully understand their involvement
in the Hh signaling pathway.
A
B
20
ShRNAControl
ShRNASmo
25
20
15
10
5
SAG
**
1
20
25
**
**
15
10
5
**
0
0
0
-8
-7
-6
-5
Log[Drug], M
C
D
_
_
25
25
20
15
10
5
Fsk
Fsk
IBMX
***
***
***
+
+ IBMX
***
20
15
10
5
***
***
***
***
0
0
Figure 2. Quinolones 1, 20 and 25 mediates differentiation of the pluripotent C3H10T1/2 mesenchymal cells. (A)
1, 20, 25 and the Smo agonist SAG stimulate differentiation of C3H10T1/2 cells into osteoblasts as measured by
alkaline phosphatase (AP) activity. (B-D) Effects of Smo knockdown (B), Forskolin (Fsk, C) and
isobutylmethylxanthine (IBMX, D) treatment on quinolones 1 (1 µM), 20 (0.3 µM), 25 (1 µM, B; 0.3 µM, C-D), and
SAG (0.3 µM) activities. (B) Smo silencing prevents the differentiation effects of 1, 20, 25 and SAG. (C, D) 1, 20,
25 and SAG display opposite regulation by cAMP modulators. [41]Treated C3H10T1/2 cells were previously
infected with Smo shRNA or control shRNA. The AP activity was then measured. Data are expressed as fold
change of basal AP activity and are the mean ± S.E.M. (n=4) of 4-6 independent experiments. **P ≤ 0.01, ***P ≤
0.001 compared with control.

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Figure 3. Quinolones 1, 20 and 25 do not stimulate canonical Hh responses. (A-C) Effect on Gli-luciferase
reporter activity in Shh-light2 cells was measured at increasing concentrations of SAG, 1 (A), 20 (B) or 25 (C). (D-
F) Effect on rat cerebellar granule cell precursors proliferation. [3H]Thymidine incorporation is increased in
response to SAG but not to 1 (D), 20 (E) or 25 (F). (A-F) Data are the mean ± S.E.M. of quadruplicates from a
representative experiment over 3 independent experiments and are expressed as fold change, compared with
cells treated with vehicle alone. (G) Effect on Smo translocation to the primary cilium. Representative
immunofluorescent images of NIH3T3 cells treated overnight with vehicle (Veh), 1 (10 µM), 20 (10 µM), 25 (10
µM) or SAG (1 µM). Cells were stained with antibodies against endogenous Smo (green)[42] and acetylated (Ac)
tubulin (red). Nuclei were stained with DAPI (blue). (H) The percentage of Smo-positive cilia was calculated for
each drug in three independent fields. Data are the mean ± S.E.M. (n=3) of 3 independent experiments. 1, 20
and 25 do not induce Smo trafficking to the cilium. ***P ≤ 0.001, compared with Veh.
Figure 4. Quinolones 1, 20 and 25 inhibit SAG-induced Gli-luciferase activity. (A-C) Gli-luciferase reporter activity
in Shh-light2 cells was measured at increasing concentrations of SAG alone or in presence of 1 µM of 1 (A), 20 (B)
or 25 (C). Data are the mean ± S.E.M. of quadruplicates of a representative experiment over 3 independent
experiments and are expressed as percentage of the maximal response induced by SAG. SAG maximal response
was reduced by 31, 54 and 26 % in presence of 1, 20 and 25, respectively.

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Compounds 1, 20, 25, like SAG, activated C3H10T1/2 differentiation in a dose dependent manner.
An increased maximal activity for 20 and 25 compared to 1 was observed (Figure 2A, Table 1). This
activity is dependent on the Smo receptor since Smo silencing using specific shRNAs reduces
significantly their maximal stimulation by 61 ± 8%, 51 ± 4%, 55 ± 6 for 1, 20 and 25 compared to 92
± 3% for SAG (Figure 2B). The data is consistent with the involvement of Smo in the action of these
molecules. Increases in concentration of the second messenger cAMP result from the stimulation
of adenylate cyclase which catalyzes the conversion of ATP into cAMP, or from the inhibition of
phosphodiesterase which dephosphorylates cAMP into AMP. These increases may lead to the
activation of protein kinase A (PKA), which in turn phosphorylates several key effectors. During
development, increases of cAMP have been shown to block canonical Hh signaling in various
tissues.[1, 14, 21] Thus, to evaluate the effects of cAMP during cell differentiation, we applied
forskolin and IBMX that stimulate cAMP production by activating adenylate cyclase or inhibiting its
degradation by phosphodiesterase, respectively. These treatments resulted in the inhibition of
SAG-induced C3H10T1/2 differentiation while potentiating the differentiation induced by 1, 20
and 25 by two to four fold (Figure 2C-D). This is in agreement with a negative regulation of the Hh
canonical signaling pathway by the cAMP/PKA transduction pathways for SAG and is reminiscent
of a noncanonical activity for the quinolone compounds.
We further explored this possibility through multiple cell based assays routinely used for Hh
evaluation.[41] We first used a NIH3T3 cell line stably transfected with a Gli-dependent firefly
luciferase reporter (Shh-light2 cells)[33] and observed that, contrary to SAG, compounds 1, 20 and
25 were not able to stimulate the Gli transcription factors (Figure 3A-C). It has been demonstrated
that cerebellar granule cell precursors (GCPs) proliferate in response to Hh activation.[43]
However, when we measured the ability of compounds 1, 20 and 25 to stimulate GCPs
proliferation in primary culture, as measured by [3H]thymidine incorporation, none of them could
mimic the stimulatory effect observed with SAG (Figure 3D-F). We also observed that the
quinolone compounds were not able to induce the translocation of Smo to the primary cilium, a
response associated to the Hh canonical pathway (Figure 3G). The percentage of Smo positive cilia
in the presence of 1, 20 and 25 was not significantly different from the vehicle treated ones. On
the opposite, SAG promoted the receptor translocation in more than 60% of the cilia (Figure 3H)
as expected.[44] We also evaluated the transcription of genes likely regulated by Hh signaling such
as Gli1-3, Ptc1 and Smo using quantitative RT-PCR (qPCR) at 48 h treatment. We observed that Gli1
(7.8-fold) and Ptc1 (5.3-fold) were up-regulated upon SAG treatment while we noticed a significant
decrease of Gli1 (2-fold) and Ptc1 (3-fold) transcripts level after 1, 20 and 25 treatments
(Supplemental Table S1). All these data indicated that quinolinone compounds, although acting
through Smo, do not use the Hh canonical pathway to stimulate differentiation.
Since 1, 20 and 25 failed to induce Gli-luciferase reporter activity, and even inhibited Gli1
transcription as measured by qPCR, we wondered if they could negatively regulate this response.
Thus, response to SAG was evaluated in Shh-light2 cells in the presence of 1 µM of 1, 20 and 25 or
vehicle. We observed a marked reduction of the maximal response (25% to 55%) without
significant reduction of SAG EC50 (Figure 4). These data indicated that 1, 20 and 25 behaved as
noncompetitive antagonists of this response.
Smo belongs to the class F of GPCRs and displays high homology with Frizzled receptors
responsible for Wnt signaling.[2] The Wnt pathway was also reported to stimulate C3H10T1/2
osteoblastic differentiation.[45] To exclude that our compounds could stimulate this pathway, we
used a TCF-LEF luciferase reporter[41] and demonstrated that neither 1, 20 or 25 could stimulate
this response while transfection by the Wnt3a control plasmid induced a robust stimulation of
more than 15-fold compared to basal level (Supplemental Figure S1).

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Altogether, these results suggest that this new class of quinolone derivatives acts with a different
mechanism with respect to SAG. The two classes of compounds could act independently on
different Smo receptor sites presumably located at the level of the transmembrane domain of the
Smo receptor.[46] These data corroborate those reported by Fleury et al. demonstrating that
inhibition of adipocyte differentiation of 3T3-L1 cells by SAG and GSA-10 are mediated through
different signalling pathways: GSA-10 effects being mediated by a Smo/AMPK axis and associated
to inhibition of Gli1. [47]
Conclusion
In summary, a hit optimization routine led to the design and synthesis of several quinolones
derivatives with a 2-3-fold improved osteoinductive activity compared to 1. The submicromolar
activity of several new quinolones (0.4-0.9 µM) was comparable to or better than that of
purmorphamine (0.8 µM) and slightly lower than that of SAG (0.13 µM). A SAR analysis suggested
that: 1) short linear alkyl chains up to four carbon atoms on the ester moiety are profitable for
activity, while bulkier substituents are detrimental. Replacement of the ester moiety with the
corresponding acidic function was profitable, while an amide or ether group caused loss of
activity; 2) linear N1 alkyl chains from three to six carbon atoms are tolerated; 3) a dimethoxy
substitution was the sole decoration on the condensed phenyl ring with a profitable influence on
activity.
The new compounds could be considered as novel osteogenic agents for multipotent
mesenchymal cells and therefore may be of therapeutic interest in bone-related diseases.
However, a biological profile significantly different from that of the known Smo agonist SAG
emerged, although our study provided strong evidence for the direct interaction between our
products and Smo. We hypothesized that SAG and the quinolone family have different binding
sites on Smo and mediate osteoblast differentiation with a different mechanism of action.
Experimental
Chemistry
All reagents were used as purchased from commercial suppliers without further purification. The reactions
were carried out in oven dried or flamed vessels. Solvents were dried and purified by conventional
methods prior use. Toluene was distilled from sodium. Flash column chromatography was performed with
Merck silica gel 60, 0.040-0.063 mm (230-400 mesh). Merck aluminium backed plates pre-coated with silica
gel 60 (UV254) were used for analytical and preparative thin layer chromatography and were visualized by
staining with a KMnO4 solution. NMR spectra were recorded at 25 °C and 400 MHz for 1H and 100 MHz for
13C. The solvent is specified for each spectrum. Splitting patterns are designated as s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br, broad. Chemical shifts (δ) are given in ppm relative to the resonance of
their respective residual solvent peak. High and low resolution mass spectroscopy analyses were recorded
by electrospray ionization. Melting points were determined in open capillary tubes and are uncorrected.
HPLC/MS conditions: ES ionisation after passage through a C-18, 35x5 mm, 3 μ column, elution: mixture A
(99.9 % water, 0.1 % HCOOH); mixture B (99.9 % acetonitrile, 0.1 % HCOOH): 0–6.0 min, 95 % mixture A;
6.0–9.0 min 95– 0 % mixture A; 9.0–15 min 0–95 % mixture A, flow 1.0 mL/min, T = 40 °C. Reactions carried
out under MW dielectric heating were performed with a microwave oven (Discover from CEM) under
monomode irradiation in a 10 mL sealed vial. The internal temperature was monitored through an internal
IR sensor and the maximal internal pressure monitored and maintained under the value of 300 psi.
Commercially available compounds 28-42 were purchased from different vendors. The purity of all the
compounds tested was checked by HPLC/MS analysis resulting always higher than 95%.

ACCEPTED MANUSCRIPT
General procedure for the synthesis of 4-hydroxy-quinolones 2a-g. Ethyl 1-hexyl-1,2-dihydro-4-hydroxy-2-
oxo-quinoline-3-carboxylate (2a).
A mixture of N-hexylaniline (0.88 g, 5 mmol) and triethyl methanetricarboxylate 3 (3.48 g, 15 mmol) was
irradiated with microwaves at the fixed power of 250 Watt (max temp 250 °C) for 20 minutes in open vessel
conditions. The crude mixture was directly purified by flash chromatography (PE/AcOEt 4:1) giving
compound 2a as a yellow oil. (0.856 g, 54 % yield). 1H NMR: (200 MHz, CDCl3): δ 14.13 (bs, 1H), 8.10 (d, 1H,
J = 7.8 Hz), 7.58 (t, 1H, J = 8.12 Hz), 7.28-7.09 (m, 2H), 4.49 (q, 2H, J = 7.2 Hz), 4.12 (t, 4H, J = 7.6), 1.42-0.98
(m, 8H), 0.63 (t, 3H, J = 7.2 Hz). ES-MS: 318 [M+H]+, 340 [M+Na]+, 356 [M+K]+, 657 [2M+Na]+.
1
Ethyl 1-pentyl-1,2-dihydro-4-hydroxy-2-oxo-quinoline-3-carboxylate (2b). Yield: 60%. H NMR: (200 MHz,
CDCl3): δ 13.88 (bs, 1H), 7.79 (d, 1H, J = 7.8 Hz), 7.33 (t, 1H, J = 7.3 Hz), 6.96-6.84 (m, 2H), 4.20 (q, 2H, J =
7.2 Hz), 3.87 (t, 4H, J = 7.6 Hz), 1.41-0.99 (m, 6H), 0.63 (t, 3H, J = 7.2 Hz). ES-MS: 304 [M+H]+, 326 [M+Na]+,
342 [M+K]+.
1
Ethyl 1-butyl-1,2-dihydro-4-hydroxy-2-oxo-quinoline-3-carboxylate (2c). Yield: 55%. H NMR: (200 MHz,
CDCl3): δ 13.91 (bs, 1H), 7.79 (d, 1H, J = 7.8 Hz), 7.33 (t, 1H, J = 7.3 Hz), 6.96-6.84 (m, 2H), 4.22 (q, 2H, J = 7.2
Hz), 3.87 (t, 4H, J = 7.6 Hz), 1.40-1.21 (m, 4H), 0.63 (t, 3H, J = 7.2 Hz). ES-MS: 290 [M+H]+, 312 [M+Na]+, 328
[M+K]+.
1
Ethyl 1-propyl-1,2-dihydro-4-hydroxy-2-oxo-quinoline-3-carboxylate (2d). Yield: 53%. H NMR: (200 MHz,
CDCl3): δ 12.54 (bs, 1H), 8.24 (d, 1H, J = 7.92 Hz), 7.71-7.65 (m, 1H), 7.40-7.14 (m, 2H), 4.31-4.08 (m, 4H),
1.83-1.72 (m, 2H), 1.27-1.20 (m, 3H), 1.14-0.96 (m, 3H). ES-MS: 297 [M+Na]+.
1
Ethyl 1-methyl-1,2-dihydro-4-hydroxy-2-oxo-quinoline-3-carboxylate (2e). Yield: 35%. H NMR: (200 MHz,
CDCl3): δ 12.64 (bs,1H), 7.30-7.26 (m, 4H), 4.35 (q, 2H, J = 7.3 Hz), 3.62 (s, 3H), 1.19 (t, 3H, J = 7.3 Hz). ES-
MS: 270 [M+Na]+.
Ethyl 1-allyl-1,2-dihydro-4-hydroxy-2-oxo-quinoline-3-carboxylate (2f). Yield: 77%. 1H NMR (400 MHz,
CDCl3): δ 14.25 (bs, 1H), 8.15 (d, 2H, J = 8.4 Hz), 7.60 (t, 1H, J = 7.2 Hz), 7.24-7.19 (m, 2H), 5.95-5.85 (m, 1H),
5.18-5.06 (m, 2H), 4.94-4.96 (m, 2H), 4.47 (q, 2H, J = 6.4 Hz), 1.37 (t, 3H, J = 6.4 Hz). 13C NMR (100 MHz,
CDCl3): δ 172.27, 171.47, 140.3, 133.84, 133.74, 131.49 , 125.29, 121.42, 116.58, 114.27, 61.91, 43.99,
13.74. ES-MS: 296 [M+Na]+.
Ethyl 1-benzyl-1,2-dihydro-4-hydroxy-2-oxo-quinoline-3-carboxylate (2g). 1H NMR: (400 MHz, CDCl3): δ
14.32 (bs, 1H), 8.17 (d, 2H, J = 7.6 Hz), 7.50 (t, 2H, J = 7.6 Hz), 7.16-7.27 (m, 5H), 5.48 (s, 2H), 4.51 (q, 2H, J =
7.6 Hz), 1.46 (t, 3H, J = 7.6 Hz). ES-MS: 262 [M+Na]+. Yield: 49%.
General procedure for the synthesis of 4-hydroxy-quinolones 2h-k. Ethyl 1-propyl-1,2-dihydro-4-
hydroxy-6-fluoro-2-oxo-quinoline-3-carboxylate (2h) To a suspension of NaH (1 mmol) in dry DMF
(10 mL), a solution of ethyl 2-[(2-ethoxycarbonylacetyl)-N-hexylamino]-5-fluorobenzoate 6h (0.381
g, 1 mmol) in dry DMF (20 mL) was added at 0 °C. The reaction mixture was stirred at room
temperature under N2 for 20 min. The mixture was diluted with AcOEt (50 mL) and washed with
H2O (3 x 30 mL), and Brine (30 mL). The organic layers were dried over dry Na2SO4, filtered and
evaporated under reduced pressure giving compound 6h (145 mg, 46% yield) product after flash
1
chromatography with PE/toluene/AcOEt 1:2:1. H NMR: (400 MHz, CDCl3): δ 14.16 (bs, 1H), 7.83-
7.79 (m, 1H), 7.40-7.35 (m, 1H), 7.25-7.18 (m, 1H), 4.48 (q, 2H, J = 7.2 Hz), 4.13 (t, 2H, J = 7.6 Hz),
1.71 (q, 2H, J = 7.2 Hz), 1.45 (t, 3H, J = 6.8 Hz), 1.01 (t, 3H, J = 7.2 Hz). 13C NMR: (100 MHz, CDCl3): δ
170.0, 165.0, 163.8, 158.4, 130.7, 124.8, 117.2, 114.9, 111.4, 102.9, 61.02, 44.09, 20.82, 14.29,
11.27. ES/MS: 316 [M+Na]+.

ACCEPTED MANUSCRIPT
Ethyl 1-propyl-1,2-dihydro-4-hydroxy-6-chloro-2-oxo-quinoline-3-carboxylate (2i). Yield: 40%.
1
Eluent for chromatography: PE/AcOEt 3:1. H NMR: (400 MHz, CDCl3): δ 7.23 (d, 1H, J = 8.2), 7.17
(d, 1H, J = 8.2), 6.99 (s, 1H), 3.97 (q, 2H, J = 6.8 Hz), 3.57 (t, 2H, J = 7.2 Hz), 1.36 (q, 2H, J = 7.2 Hz),
1.04 (t, 3H, J = 7.2 Hz), 0.74 (t, 3H, J = 6.8 Hz). ES/MS: 332 [M+Na]+.
Ethyl 1-propyl-1,2-dihydro-4-hydroxy-6-methyl-2-oxo-quinoline-3-carboxylate (2j). Yield: 64%.
1
Eluent for chromatography: CH2Cl2/MeOH 95:5. H NMR: (400 MHz, CDCl3): δ 14.02 (bs, 1H), 7.76
(s, 1H), 7.29 (d, 1H, J = 8.2 Hz), 6.99 (d, 1H, J = 8.2 Hz), 4.37 (q, 2H, J = 7.2 Hz), 3.99 (t, 2H, J = 7.8
Hz), 2.26 (s, 3H), 1.59 (q, 2H, J = 8 Hz), 1.35 (t, 3H, J = 7.8 Hz), 0.89 (t, 3H, J = 7.2 Hz). 13C NMR: (100
MHz, CDCl3): δ 172, 171,26, 158,98, 138.57, 135.43, 131.1, 125,08, 114.66, 113.93, 97.65, 62.06,
43.65, 20.70, 20.45, 14.17, 11.22. ES/MS: 312 [M+Na]+.
Ethyl 1-propyl-1,2-dihydro-4-hydroxy-6,7-dimethoxy-2-oxo-quinoline-3-carboxylate (2k). Yield:
80%. Eluent for Chromatography: CH2Cl2/MeOH 95:5. 1H NMR: (400 MHz, CDCl3): δ 16.38 (bs, 1H),
14.18 (bs, 1H), 7.45 (s, 1H), 6.62 (s, 1H), 4.43 (q, 2H, J = 7.2 Hz), 4.11 (t, 2H, J = 7.6 Hz), 3.94 (s, 3H),
3.91 (s, 3H), 1.71 (q, 2H, J = 7.6 Hz), 1.43 (t, 3H, J = 7.2 Hz), 0.99 (t, 3H, J = 7.2 Hz). 13C NMR: (400
MHz, CDCl3): δ 173.08, 170.8, 159.53, 155.25, 145.07, 137.29, 107.71, 105.61, 96.74, 96.36, 62.10,
56.23, 56.14, 44.05, 20.84, 14.28, 11.46. ES/MS: 358 [M+Na]+.
General procedure for the synthesis of 4-hydroxy quinolones 1 and 8-27 Propyl 4-(1-hexyl-4-
hydroxy-2-oxo-1,2-dihydroquinolin-3-carboxamido)benzoate (GSA-10, 1) A mixture of 2a (0.63 g 2
mmol) and propyl 4-aminobenzoate (0.394 g, 2.2 mmol) in toluene (4 mL) was submitted to MW
irradiation at 250 Watt (max temperature set 250 °C) for 1 h (two times of 30 min each) in open
vessel conditions. After cooling, the crude mixture was purified by flash chromatography eluting
1
with PE/AcOEt: 6/1 to give 1 (0.630 g, 70% yield). H NMR: (400 MHz, CDCl3): δ 16.30 (bs, 1H),
15.71 (bs, 1H), 8.24 (d, 1H, J = 8.0 Hz), 7.85 (d, 2H, J = 8.4 Hz), 7.77 (t, 1H, J = 8.4 Hz), 7.44-7.24 (m,
2H), 6.52 (d, 2H, J = 8.8 Hz), 4.27 (t, 2H, J = 7.6 Hz), 4.19 (t, 2H, J = 6.4 Hz), 2.85-2.80 (m, 2H), 1.76-
1.71 (m, 2H), 1.45-1.23 (m, 6H), 0.98 (t, 3H, J = 7.6 Hz), 0.89 (t, 3H, J = 6.8 Hz). 13C NMR: (100 MHz,
CDCl3): δ 197.23, 173.05, 170.67, 164.25, 139.13, 134.52, 130.97, 125.94, 123.06, 72.54, 65.95,
42.32, 29.71, 26.1, 21.95, 13.53, 10.11. ES-MS: 473 [M+Na]+. LC/MS: RT 5.29 min; 473 [M+Na]+.
Purity: 98%. Anal calcd for C26H30N2O5 C 69.31, H 6.71, N 6.22, O 17.76; found C 69.29, H 6.68, N
6.25. %.
1
Butyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-carboxamido)benzoate (8). Yield: 75%. H
NMR: (400 MHz, CDCl3): δ 16.30 (bs, 1H), 15.71 (bs, 1H), 8.24 (d, 1H, J = 8.0 Hz), 7.85 (d, 2H, J = 8.4
Hz), 7.77 (t, 1H, J = 8.4 Hz), 7.44-7.24 (m, 2H), 6.52 (d, 2H, J = 8.8 Hz), 4.27 (t, 2H, J = 7.6 Hz), 4.19
(t, 2H, J = 6.4 Hz), 2.85-2.80 (m, 2H), 1.79-1.71 (m, 2H), 1.49-1.23 (m, 8H), 0.98 (t, 3H, J = 7.6 Hz),
13
0.89 (t, 3H, J = 6.8 Hz). C NMR: (100 MHz, CDCl3): δ 197.23, 173.05, 170.67, 164.25, 139.13,
134.52, 130.97, 125.94, 123.06, 72.54, 65.95, 42.32, 30.82, 29.71, 26.1, 21.95, 19.12, 13.53, 10.11.
ES-MS: 487 [M+Na]+. LC/MS: RT 5.32 min; 487 [M+Na]+. Purity: 97%. Anal calcd for C27H30N32O5 C,
69.81; H, 6.94; N, 6.03; O, 17.22; found C 69.79, H 6.95, N 6.05.
tert-Butyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-carboxamido)benzoate (9). Yield: 65%.
1H NMR: (400 MHz, CDCl3): δ 16.37 (bs, 1H), 15.82 (bs, 1H), 8.24 (d, 1H, J = 8.0 Hz), 7.87 (d, 2H, J =
8.4 Hz), 7.77 (t, 1H, J = 8.4 Hz), 7.44-7.24 (m, 2H), 6.52 (d, 2H, J = 8.8 Hz), 4.27 (t, 2H, J = 7.6 Hz),
13
2.85-2.80 (m, 2H), 1.57 (s, H), 1.49-1.23 (m, 8H), 0.89 (t, 3H, J = 6.8 Hz). C NMR: (100 MHz,
CDCl3): δ 197.23, 173.05, 170.67, 164.25, 139.13, 134.52, 130.97, 125.94, 123.06, 82.22, 72.54,
42.32, 30.82, 29.71, 29.01, 26.13, 19.12, 13.53. ES-MS: 487 [M+Na]+. LC/MS: RT 5.33 min; 487

ACCEPTED MANUSCRIPT
[M+Na]+. Purity: 98%. Anal calcd for C27H30N32O5 C, 69.81; H, 6.94; N, 6.03; O, 17.22; found C
69.80, H 6.95, N 6.01.
1-Hexyl-4-hydroxy-2-oxo-N-(4-(propylcarbamoyl)phenyl)-1,2-dihydroquinoline-3-carboxamide (10)
1
Yield: 53%. H NMR (CDCl3, 400 MHz) δ (ppm): 8.25 (d, J = 8 Hz, 2H), 7.77 (s, J = 8, 1H), 7.70 (t, J =
7.6 2H), 7.36 (d, J = 8 Hz, 1H), 7.31 (t, J = 7.6 Hz, 2H), 4.26 (t, J = 8 Hz, 3H), 3.41 (q, J1 = 7.6 Hz, J2 =
6.4 Hz 4H), 1.53 (s, 2H), 1.33 (d, J = 3.6 Hz, 6H), 1.00 (t, J = 6.8 Hz 3H), 0.89 (t, J = 6.8 Hz, 3H).13C
NMR (CDCl3, 100 MHz): δ (ppm): 172.11, 19.67, 127.83 125.83, 122.52, 120.73, 114.40, 77.63,
77.20, 77.00, 76.36, 73.05, 42.57, 41.76, 31.53, 29.69, 27.65, 26.68, 22.97, 22.61, 14.01. ES-MS:
450 [M+H]+, 472 [M+Na]+. M.p.: 205-210 °C. LC/MS: RT 4.98 min; 472 [M+Na]+. Purity: 98%. Anal
calcd for C26H31N3O4 C, 69.47; H, 6.95; N, 9.35; O, 14.24; found C 69.44, H 6.98, N 9.30.
Propyl 4-(1-pentyl-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-carboxamido)benzoate (11). Yield: 67%.
1H NMR: (400 MHz, CDCl3): δ 16.30 (bs, 1H), 15.71 (bs, 1H), 8.24 (d, 1H, J = 8.0 Hz), 7.85 (d, 2H, J =
8.4 Hz), 7.77 (t, 1H, J = 8.4 Hz), 7.44-7.24 (m, 2H), 6.52 (d, 2H, J = 8.8 Hz), 4.27 (t, 2H, J = 7.6 Hz),
4.19 (t, 2H, J = 6.4 Hz), 2.83-2.79 (m, 2H), 1.76-1.71 (m, 2H), 1.45-1.27 (m, 4H), 1.01 (t, 3H, J = 7.6
Hz), 0.91 (t, 3H, J = 6.8 Hz). 13C NMR: (100 MHz, CDCl3): δ 197.23, 173.05, 170.67, 164.25, 139.13,
134.52, 130.97, 125.94, 123.06, 72.54, 65.95, 42.32, 29.71, 26.1, 21.95, 13.53, 10.11. ES-MS: 459
[M+Na]+. LC/MS: RT 5.75 min; 459 [M+Na]+. Purity: 98%. Anal calcd for C25H28N2O5 C, H, N: calc. C
68.79, H 6.47, N 6.42, O 18.33; found C 68.82, H 6.45, N 6.45.
Propyl 4-(1-butyl-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-carboxamido)benzoate (12). Yield: 52%.
1H NMR: (400 MHz, CDCl3): δ 16.22 (bs, 1H), 15.38 (bs, 1H), 8.27 (d, 1H, J = 8.0 Hz), 7.85 (d, 2H, J =
8.4 Hz), 7.72 (t, 1H, J = 8.4 Hz), 7.44-7.19 (m, 2H), 6.48 (d, 2H, J = 8.8 Hz), 4.27 (t, 2H, J = 7.6 Hz),
4.19 (t, 2H, J = 6.4 Hz), 2.83-2.61 (m, 2H), 1.70-1.45 (m, 4H), 1.01 (t, 3H, J = 7.6 Hz), 0.91 (t, 3H, J =
13
6.8 Hz). C NMR: (100 MHz, CDCl3): δ 197.23, 173.02, 170.67, 164.27, 139.13, 134.52, 130.97,
125.94, 123.06, 72.54, 65.90, 42.37, 29.31, 21.80, 13.13, 10.11. ES-MS: 445 [M+Na]+. LC/MS: RT
5.68 min; 445 [M+Na]+. Purity: 97%. Anal calcd for C24H26N2O5 C, H, N: calc. C 68.23, H 6.20, N 6.63,
O 18.94; found C 68.19, H 6.18, N 6.60.
Propyl 4-(1-propyl-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-carboxamido)benzoate (13). Yield: 42%.
1H NMR: (400 MHz, CDCl3): δ 16.36 (bs, 1H), 12.83 (bs, 1H), 8.25 (d, 1H, J = 8.4 Hz), 8.04 (d, 2H. J =
8.4 Hz), 7.77 (d, 2H, J = 8.4 Hz), 7.76-7.67 (m, 1H), 7.37-7.24 (m, 2H), 4.25 (q, 4H, J = 6.4 Hz), 1.78
13
(q, 4H, J = 7.2 Hz), 1.05 (q, 6H, J = 7.2 Hz). C NMR: (100 MHz, CDCl3): δ 133.71, 130.27, 125.42,
119.94, 114, 76.57, 66.02, 43.55, 21.10, 10.86. LC/MS: RT 5.71 min; 409 [M+H]+; 431 [M+Na]+.
Purity: 96%. Anal calcd for C23H24N2O5 C, H, N: C 67.63, H 5.92, N 6.86, O 19.59; found C 67.60, H
5.94, N 6.84.
Propyl 4-(1-methyl-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-carboxamido)benzoate (14) Yield: 42%.
1H NMR: (400 MHz, CDCl3): δ 16.52 (bs, 1H), 12.81 (bs, 1H), 8.24 (d, 1H, J = 8 Hz), 7.85 (d, 2H, J =
8.4 Hz), 7.76 (d, 2H, J = 8.4 Hz), 7.71 (t, 1H, J = 8 Hz), 7.38 (d, 1H, J = 7.6 Hz), 7.32 (t, 1H, J = 8 Hz),
4.26 (t, 2H, J = 6.4 Hz), 3.71 (s, 3H), 1.78 (q, 2H, J = 7.2 Hz), 1.02 (t, 3H, J = 7.2 Hz). 13C NMR: (100
MHz, CDCl3): δ 173.23, 164.25, 141.32, 133.81, 130.30, 125.28, 123.06, 119.89, 113.94, 66.03,
31.51, 22.26, 13.67. ES-MS: 403 [M+Na]+. LC/MS: RT 5.62 min; 481 [M+H]+. Purity: 98%. Anal calcd
for C21H20N2O5 C, H, N: C 66.31, H 5.30, N 7.36, O 21.03; found C 66.29, H 5.31, N 7.32.
1
Propyl 4-(1-allyl-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-carboxamido)benzoate (15). Yield: 46% H
NMR: (400 MHz, CDCl3): δ 16.45 (bs, 1H), 12.72 (bs, 1H), 8.25 (d, 1H, J = 8 Hz), 8.03 (d, 2H, J = 8.4

ACCEPTED MANUSCRIPT
Hz), 7,75 (d, 2H, J = 7.6 Hz), 7.66 (t, 1H, J = 8), 7.32-7,24 (m, 2H), 6.00-5.94 (m, 1H), 5.24 (d, 1H, J =
8 Hz), 5.08 (d, 1H, J = 10.8 Hz), 4.94 (d, 2H, J = 2.8 Hz), 4.25 (t, 2H, J = 6.8 Hz), 1.80-1.75 (m, 2H),
13
1.01 (t, 3H, J = 8.4 Hz). C NMR: (100 MHz, CDCl3): δ 172.11, 161.18, 140.96, 133.74, 130.92,
130.27, 125.98, 125.28, 122.38, 119.91, 116.73, 114.60, 66.03, 44.19, 21.71, 10.1. ES-MS: 407
[M+H]+, 429 [M+Na]+. Mp : 132-135 °C. LC/MS: RT 5.88 min; 457 [M+H]+, 479 [M+Na]+. Purity: 97%.
Anal calcd for C23H22N2O5 C, H, N: C 67.97, H 5.46, N 6.89, O 19.68; found C 67.99, H 5.44, N 6.86.
Propyl 4-(1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinolin-3-carboxamido)benzoate (16). Yield: 40%.
1H NMR: (400 MHz, CDCl3): δ 16.51 (bs, 1H), 12.74 (bs,1H), 8.23 (d, 1H, J = 8 Hz), 8.03 (d, 2H, J = 8.4
Hz), 7.75 (d, 2H, J = 8.4 Hz), 7.52 (t, 1H, J = 8 Hz), 7.32-7.17 (m, 7H), 5.54 (s, 2H), 4.25 (t, 2H, J = 6.8
Hz), 1.80-1.77 (m, 2H), 1.02 (t, 3H, J = 8 Hz). 13C NMR: (100 MHz, CDCl3): δ 172.26, 169.23, 162.55,
140.93, 139.07, 135.37, 133.81, 130.26, 128.58, 125.97, 125.23, 119.91, 114.76, 96.57, 66.03,
45.50, 21.74, 10.02. LC/MS: RT 5.87 min; 457 [M+H]+, 479 [M+Na]+. Purity: 99%. Anal calcd for
C27H24N2O5 C, H, N: C 71.04, H 5.30, N 6.14, O 17.52; found C 71.05, H 5.34, N 6.11.
Propyl
4-(1-propyl-4-hydroxy-N-methyl-2-oxo-1,2-dihydroquinolin-3-carboxamido)-6-fluoro-
1
benzoate (17). Yield: 40%. H NMR: (200 MHz, CDCl3): δ 8.04 (d, 2H, J=8.6 Hz), 7.89 (dd, 1H, J1 =
2.8 Hz, J2 = 8.6 Hz, 1H), 7.76 (d, 2H, J = 8.6 Hz), 7.42 (m, 1H), 4.23 (q, 2H, J = 6.6 Hz), 1.78 (q, 4H, J =
7.6 Hz), 1.04 (t, 6H, J = 7.6 Hz). LC/MS: RT 5.23 min; 427 [M+H]+. Purity: 98%. Anal calcd for
C23H23FN2O5 C, H, N: calc. C 64.78, H 5.44, F 4.46, N 6.57, O 18.76; found C 64.80, H 5.41, N 6.53.
Propyl
4-(1-propyl-4-hydroxy-N-methyl-2-oxo-1,2-dihydroquinolin-3-carboxamido)-6-
chlorobenzoate (18). Yield: 36%. 1H NMR: (400 MHz, CDCl3): δ 16.44 (bs, 1 H), 12.73 (bs, 1H), 8.21
(d, 1H, J = 8.6 Hz), 8.04 (d, 2H, J = 8.4 Hz), 7.76 (d, 2H, J=8.4 Hz), 7.62 (q, 1H, J = 8.6 Hz), 7.29 (t, 1H,
13
J = 9.2 Hz), 4.27-4.19 (m, 4H), 1.82-1.71 (m, 4H), 1.39-1.34 (m, 6H). C NMR: (100 MHz, CDCl3): δ
167.2, 165.9, 163.8, 163.0, 141.9, 133.73, 130.29, 124.70, 124.70, 119.98, 115.60, 66.07, 43.73,
21.71, 20.54, 10.82, 10.09. LC/MS: RT 5.35 min; 443 [M+H]+. Purity: 98%. Anal calcd for
C23H23ClN2O5 C, H, N: C 62.37, H 5.23, Cl 8.00, N 6.33, O 18.06; found C 62.40, H 5.25, N 6.34.
Propyl
4-(1-propyl-4-hydroxy-N-methyl-2-oxo-1,2-dihydroquinolin-3-carboxamido)-6-
1
methylbenzoate (19). Yield: 55%. H NMR: (400 MHz, CDCl3): δ 16.29 (bs, 1H), 12.86 (bs, 1H), 8.01
(d, 2H, J = 8.2 Hz), 7.74 (d, 2H, J = 8.2 Hz), (d, 1H, J = 7.4 Hz), 7.22 (d, 2H, J = 8.2 Hz), 4.26-4.17 (m,
4H), 2.38 (s, 3H), 1.80-1.72 (m, 2H), 1.25-0.98 (m, 6H). LC/MS: RT 6.05 min; 446 [M+Na]+. Purity:
97%. Anal (C24H26N2O5) C, H, N: calc. C 68.23, H 6.20, N 6.63, O 18.94; found C 68.25, H 6.19, N
6.66.
Propyl
4-(1-propyl-4-hydroxy-N-methyl-2-oxo-1,2-dihydroquinolin-3-carboxamido)-6,7-
1
dimethoxylbenzoate (20). Yield: 48%. H NMR: (400 MHz, CDCl3): δ 16.28 (bs, 1H), 12.87 (bs, 1H),
8.02 (d, 2H, J = 8.4 Hz), 7.76 (d, 2H, J = 8.8 Hz), 7.55 (s, 1H), 6.72 (s, 1H), 4.24 (q, 4H, J = 6.4 Hz),
4.00 (s, 3H), 3.96 (s, 3H), 1.82-1.75 (m, 4 H), 1.5-0.92 (m, 6H). C NMR: (100 MHz, CDCl3): δ
13
171.19, 170.00, 166.25, 162.49, 155,14, 145.74, 141,64, 135,66, 130,68, 126.19, 125.70, 120.30,
109.07, 105.33, 96.92, 95.82, 77.33, 77.01, 76.69, 66.44, 56.25, 44.18, 20.17, 22.70, 21.01, 11.44,
10.54. LC/MS: RT 5.95 min; 469 [M+H]+, 491 [M+Na]+, 959 [2M+Na]+. Purity: 98%. Anal calcd for
C25H28N2O7 C, H, N: C 64.09, H 6.02, N 5.98, O 23.91; found C 64.12, H 6.00, N 5.95.
Propyl 3-(1-hexyl-4-hydroxy-2-oxo-1,2dihydroquinolin-3-carboxamido)-benzoate (21). Yield: 52% 1H
NMR (CDCl3, 400 MHz): 16.21 (bs, 1H), 13.12 (bs, 1H), 8.28 (t, 1H, J = 6.8 Hz), 7.98 (d, 1H, J = 6.8
Hz), 7.82 (d, 1H, J = 8Hz), 7.69-6.57 (m, 2H), 7.42 (t, 1H, J = 8 Hz), 7.35-7.24 (m, 2H), 4.38 (q, 2H, J =

ACCEPTED MANUSCRIPT
8 Hz), 4.25 (q, 2H, J = 6.4 Hz), 1.80-1.71 (m, 2H), 1.47-1.33 (m, 6H), 1.09 (t, 3H, J = 6.8 Hz), 0.92 (t,
3H, J = 7.3 Hz). 13C NMR (CDCl3, 100 MHz) δ: 172.09, 134.01, 125.80, 122.45, 114.37, 42.55, 31.55,
27.67, 26.70, 22.62, 14.01, 10.52. EI-MS: 450 [M+H]+, 473 [M+Na]+. M.p.: 103-108 °C LC/MS: RT
5.32 min; 473 [M+Na]+. Purity: 98%. Anal calcd for C26H30N2O5 C, H, N: C 69.31, H 6.71, N 6.22, O
17.76; found C 69.28, H 6.67, N 6.20.
Propyl 3-methyl-5-(1-hexyl-4-hydroxy-2-oxo-1,2dihydroquinolin-3-carboxamido)-benzoate (22).
Yield: 48%. 1H NMR (CDCl3, 200 MHz): δ 17.01 (bs, 1H), 12.45 (bs, 1H), 8.30 (d, 1H, J = 4.6 Hz), 8.22
(d, 1H, J = 3.8 Hz), 7.89 (s, 1H), 7.67 (t, 2H, J = 4 Hz), 7.34-7.24 (m, 2H), 4.34 (q, 2H, J = 6.8 Hz), 4.25
13
(t, 2H, J = 6.5 Hz), 2.47 (s, 3H), 1.80-1.68 (m, 2H), 1.59-1.32 (m, 8H), 1.10-0.80 (m, 6H). C NMR
(CDCl3, 50 MHz): δ 172.05, 169.71, 140.12, 134.02, 131.67, 128.23, 125.77, 122.39, 121.33, 114.37,
66.38, 42.30, 31.50, 27.56, 26.59, 22.60, 22.13, 18.47, 14.01, 10.53.EI-MS: 465 [M+H]+, 487
[M+Na]+. M.p.: 120-122 °C. LC/MS: RT 5.98 min; 465 [M+H]+, 487 [M+Na]+. Purity: 99%. Anal calcd
for C27H32N2O5 C, H, N: C 69.81, H 6.94, N 6.03, O 17.22; found C 69.84, H 6.95, N 6.02.
tert-Butyl 3-(1-hexyl-4-hydroxy-2-oxo-1,2dihydroquinolin-3-carboxamido)-benzoate (23). Yield:
1
32%. H NMR (CDCl3, 4OO MHz): δ 16.45 (bs, 1H), 12.39 (bs, 1H), 8.80 (d, 1H, J= 6.8 Hz), 7.91 (d,
1H, J = 7.6 Hz), 7.75 (d, 1H J = 7.6 Hz), 7.66 (t, 1H, J = 7.6 Hz), 7.39 (t, 1H, J = 8 Hz), 7.30 (d, 1H, J =
1.2 Hz), 7.26 (dd, 2H, J1 = 8 Hz, J2 = 7.6 Hz,), 4.24 (t, 2H, J = 6.4 Hz), 1.58-1.33 (m, 6H), 1.23 (s, 9H),
13
0.89 (t, 3H, J = 6.8 Hz). C NMR (CDCl3, 100 MHz): δ 172.04, 169.63, 165.33, 165.33, 162.51,
138.20, 137.31, 133.98, 132.94, 128.82, 125.76, 125.67, 125.17, 122.41,122.14, 116.30, 114.35,
97.03, 81.24, 42.52, 31.54, 30.89, 29.70, 28.22, 27.66, 26.69, 22.62, 14.02. ES-MS: 465 [M+H]+, 487
[M+Na]+. M.p.: 120-125 °C. LC/MS: RT 5.72 min; 465 [M+H]+, 487 [M+Na]+. Purity: 97%. Anal calcd
for C27H32N2O5 C, H, N: C 69.81, H 6.94, N 6.03, O 17.22; found C 69.83, H 6.97, N 6.00.
1
Ethyl 3-(1-hexyl-4-hydroxy-2-oxo-1,2dihydroquinolin-3-carboxamido)-benzoate (24) Yield: 58%. H
NMR (CDCl3, 400 MHz): δ 16.39 (bs, 1H), 12.57 (bs, 1H), 8.28 (d, 1H, J = 8 Hz), 7.98 (d, 1H, J =8 Hz),
7.82 (d, 1H, J = 8 Hz), 7.68 (t, 1H, J = 8 Hz,), 7.42 (t, 1H, J = 8 Hz), 7.35-7.27 (m, 2H), 4.38 (q, 2H, J =
8 Hz), 4.25 (t, 2H, J = 8 Hz,), 1.77-1.69 (m, 4H), 1.56-1.35 (m, 7H), 0.92 (t, 3H, J = 8 Hz,).13C NMR
(CDCl3, 100 MHz): δ 169.65, 139.22, 134.02, 131.39, 129.02, 125.80, 125.49, 122.46, 122,16,
114.38, 97.05, 61.14, 42.56, 31.56, 27.67, 26.70, 22.62, 14.38, 14.02. EI-MS: 437 [M+H]+, 459
[M+Na]+. M.p.: 70-73 °C. LC/MS: RT 5.07 min; 437 [M+H]+, 459 [M+Na]+. Purity: 97%. Anal calcd for
C25H28N2O5 C, H, N: C 68.79, H 6.47, N 6.42, O 18.33; found C 68.82, H 6.45, N 6.41.
Propyl 2-chloro-5-(1-hexyl-4-hydroxy-2-oxo-1,2dihydroquinolin-3-carboxamido)-benzoate (26).
Yield: 46%. 1H NMR (CDCl3, 400 MHz): δ 16.21 (bs, 1H), 13.12 (bs, 1H), 8.23 (d, 1H, J = 8 Hz), 7.85 (t,
1H, J = 12 Hz), 7.67 (t, 1H, J = 7.64 Hz), 7.58 (d, 2H, J = 8 Hz), 7.34-7.24 (m, 2H), 4.23 (q, 4H, J = 7.2
13
Hz), 1.82-1.69 (m, 2H), 1.58-1.30 (m, 6H), 1.03 (t, 3H, J = 6.8 Hz,), 0.89 (t, 3H, J = 7.6 Hz). C NMR
(CDCl3, 100 MHz): δ 172.10, 162.47, 141.10, 139.22 134.93, 134.27, 132.50, 125.83, 125.34,
122.72, 118.41, 114.42, 97.00, 67.00, 42.63, 31.53, 27.63, 26.68, 22.61, 22.05, 14.02, 10.62. EI-MS:
485 [M+H]+, 507 [M+Na]+. M.p.: 110-113 °C. LC/MS: RT 4.38 min; 485 [M+H]+, 507 [M+Na]+. Purity:
99%. Anal calcd for C26H29ClN2O5 C, H, N: C 64.39, H 6.03, Cl 7.31, N 5.78, O 16.50; found C 64.38, H
6.05, N 5.75.
Propyl 3-trifluoromethyl-4-(1-hexyl-4-hydroxy-2-oxo-1,2dihydroquinolin-3-carboxamido)-benzoate
(27). Yield: 56%. 1H NMR (CDCl3, 200 MHz): δ 16.21 (bs, 1H), 12.31 (bs, 1H), 8.47 (s, Hz, 1H), 8.46 (s,
1H), 8.39 (d, 1H, J= 4.4 Hz), 8.33 (s, 1H), 8.20 (d, 1H, J = 4.4 Hz), 7.68 (t, 1H, J = 4.6 Hz), 7.34-724 (m,
1H), 4.39-4.25 (m, 4H), 1.82-1.87 (m, 2H), 1.46-1.18 (m, 2H), 1.05-1.0 (m, 6H), 0.90-0.87 (m, 3H),

ACCEPTED MANUSCRIPT
0.86-0.83 (m, 3H).13C NMR (CDCl3, 50 MHz): δ 172.09, 170.31, 165.09, 162.40, 139.46, 138.67,
134.30, 133.56, 127.86, 127.76, 126.57, 125.82, 125.28, 124.87, 122.44, 115.98, 114.45, 97.05,
83.97, 82.97, 81.95, 81.03, 67.72, 67.28, 66.95, 42.25, 31.49, 31.49, 29.68, 27.52, 26.54, 22.58,
22.07, 13.98, 10.46, 10.41. EI-MS: 519 [M+H]+. M.p.: 85-89 °C. LC/MS: RT 4.23 min; 519 [M+H]+.
Purity: 98%. Anal calcd for C27H29F3N2O5 C, H, N: C 62.54, H 5.64, F 10.99, N 5.40, O 15.43; found C
62.50, H 5.65, N 5.37.
4-(1-Hexyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido)benzoic acid (25). Compound 9
(46 mg, 0.1 mmol) was dissolved in dry CH2Cl2 (10 mL). Trifluoroacetic acid (114 μL, 1.5 mmol) was
added and the reaction mixture was stirred at r.t. for 3 h. Pure 25 ( 37 mg, 92%) was obtained
after evaporation under reduced pressure . An analytical sample was purified by flash
chromatography (eluent EtOAc). 1H NMR: (400 MHz, CD3OD): δ 16.37 (bs, 1H), 15.82 (bs, 1H), 8.24
(d, 1H, J = 8.0 Hz), 7.87 (d, 2H, J = 8.4 Hz), 7.77 (t, 1H, J = 8.4 Hz), 7.44-7.24 (m, 2H), 6.52 (d, 2H, J =
13
8.8 Hz), 4.27 (t, 2H, J = 7.6 Hz), 2.85-2.80 (m, 2H), 1.49-1.23 (m, 8H), 0.89 (t, 3H, J = 6.8 Hz). C
NMR: (100 MHz, CD3OD): δ 197.21, 173.05, 170.67, 169.23, 139.13, 134.58, 130.92, 125.99,
123.06, 72.53, 42.302, 30.85, 29.71, 26.11, 19.13, 13.49. ES-MS: 407 [M-H]-. LC/MS: RT 3.98 min;
407 [M-H]-. Purity: 98%. Anal calcd for (C23H24N2O5) C, 67.63; H, 5.92; N, 6.86; O, 19.58; found C
67.58, H 5.90, N 6.87.
Acknowledgments
PRIN-2009RMW3Z5_006 (M.T.) is acknowledged for a partial financial support to M.T., La Ligue
contre le Cancer (comité des Yvelines et de l’Essonne) and the Association pour la Recherche sur le
Cancer (ARC) for supporting M.R. and La ligue contre le Cancer and the Fondation pour la
Recherche Médicale (FRM; FDT20140930825) for a doctoral fellowship to L. H. Andreina Ramirez is
acknowledged for expert technical support.
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ACCEPTED MANUSCRIPT
Highlights
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Desing and synthesis of new analogues of GSA-10
Test of the synthesized compounds as Smo agonists
Identification of compounds showing 3 fold improved activity with respect to GSA-10
SAR analysis