Subtly Modulating Glycogen Synthase Kinase 3 β: Allosteric Inhibitor Development and Their Potential for the Treatment of Chronic Diseases

Article abstract of DOI:10.1021/acs.jmedchem.7b00395

Glycogen synthase kinase 3 β (GSK-3β) is a central target in several unmet diseases. To increase the specificity of GSK-3β inhibitors in chronic treatments, we developed small molecules allowing subtle modulation of GSK-3β activity. Design synthesis, structure-Activity relationships, and binding mode of quinoline-3-carbohydrazide derivatives as allosteric modulators of GSK-3β are presented here. Furthermore, we show how allosteric binders may overcome the β-catenin side effects associated with strong GSK-3β inhibition. The therapeutic potential of some of these modulators has been tested in human samples from patients with congenital myotonic dystrophy type 1 (CDM1) and spinal muscular atrophy (SMA) patients. We found that compound 53 improves delayed myogenesis in CDM1 myoblasts, while compounds 1 and 53 have neuroprotective properties in SMA-derived cells. These findings suggest that the allosteric modulators of GSK-3β may be used for future development of drugs for DM1, SMA, and other chronic diseases where GSK-3β inhibition exhibits therapeutic effects.

Full text of DOI:10.1021/acs.jmedchem.7b00395

Article
Subtly modulating Glycogen Synthase Kinase 3 #: allosteric inhibitors
development and their potential for the treatment of chronic diseases
Valle Palomo, Daniel I. Pérez, Carlos Roca, Cara Anderson, Natalia Rodriguez-Muela,
Concepción Pérez, Jose A Morales-Garcia, Julio A Reyes, Nuria E. Campillo, Ana
M. Pérez-Castillo, Lee L Rubin, Lubov T. Timchenko, Carmen Gil, and Ana Martínez
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 26 May 2017
Downloaded from http://pubs.acs.org on May 26, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Subtly modulating Glycogen Synthase Kinase 3 β: allosteric
inhibitors development and their potential for the treatment of
chronic diseases
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a,#
a,#
a
b
Valle Palomo, Daniel I. Perez, Carlos Roca, Cara Anderson, Natalia Rodríguez-
c
d
e,f
d
Muela, Concepción Perez, Jose A. Morales-Garcia, Julio A. Reyes, Nuria E.
a
e,f
c
b
a
Campillo, Ana M. Perez-Castillo, Lee L. Rubin, Lubov Timchenko, Carmen Gil, and
a,
Ana Martinez, *
a
IPSBB Unit, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040
Madrid, Spain
b
Department of Pediatrics, Division of Neurology, Cincinnati Children's Hospital,
Cincinnati, OH 45219, USA
c
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge,
Massachusetts, USA. Harvard Stem Cell Institute, Harvard University, Cambridge,
Massachusetts, USA
d
Instituto de Quimica Médica-CSIC, Juan del Cierva 3, 28006 Madrid, Spain
e
Instituto de Investigaciones Biomedicas-CSIC, Arturo Duperier 4, Madrid, Spain
f
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas
(
CIBERNED), Madrid, Spain
#
these authors contribute equally to this work;
*
to whom correspondence should be addressed
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Abstract
Glycogen synthase kinase 3 β (GSK-3β) is a central target in several unmet diseases.
To increase the specificity of GSK-3β inhibitors in chronic treatments we are
developing small molecules allowing subtle modulation of GSK-3β activity. Design
synthesis, structure-activity relationships and binding mode of quinoline-3-
carbohydrazide derivatives as allosteric modulators of GSK-3β are here presented.
Furthermore, we show how allosteric binders may overcome the β-catenin side effects
associated to strong GSK-3β inhibition. The therapeutic potential of some of these
modulators has been tested in human samples from patients with congenital myotonic
dystrophy type 1 (CDM1) and spinal muscular atrophy (SMA) patients. We found that
compound 53 improves delayed myogenesis in CDM1 myoblasts, while compounds 1
and 53 have neuroprotective properties in SMA-derived cells. These findings suggest
that the allosteric modulators of GSK-3β may be used for future development of drugs
for DM1, SMA and other chronic diseases where GSK-3β inhibition exhibits therapeutic
effects.
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Keywords: glycogen synthase kinase 3 inhibitors, allosteric modulators, myotonic
dystrophy, β-catenin, spinal muscular atrophy
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Introduction
Glycogen synthase kinase 3 β (GSK-3β) is a constitutively active multi-functional
serine/threonine kinase involved in different physiological pathways such as
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metabolism, cell cycle, development and neuroprotection. It is essential for life, playing
2
a crucial role in the development and childhood stages. Simultaneously, GSK-3β has
been implicated in various diseases such as diabetes, inflammation, cancer,
amyotrophic lateral sclerosis (ALS), Alzheimer's and Parkinson’s diseases, bipolar
3-6
disorder and many other unmet severe pathologies. An increased expression and
7
activity of GSK-3β has been reported in type II diabetes and obese animal models, in
skeletal muscle of patients with neuromuscular disease, myotonic dystrophy type 1
8
(
DM1), and even more important, in brains of Alzheimer’s patients and spinal cord
9
-10
from ALS patients.
Consequently, GSK-3β inhibitors have demonstrated to be
1
1
powerful drugs in animal models and emerge as promising effective therapies for
1
2
these severe diseases.
13
However, these compounds rarely reach clinical trials, and only tideglusib is currently
in clinical development. Tideglusib, an ATP non-competitive GSK-3β inhibitor, is safe
14-15
for human treatments.
Clinical trial data indicate signs of efficacy such as dose
1
6
dependent decreased brain atrophy or reduction of phospho-tau and BACE-1 levels
17
in cerebrospinal fluid from Alzheimer’s patients. These data, in addition, supported
the target engagement of tideglusib and its ability to cross the human blood brain
barrier.
The involvement of GSK-3β in various essential molecular pathways, one of them
1
8
being the oncogenic β-catenin signaling, suggests that the inhibitors of GSK-3β might
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cause side effects and may be one of the reasons for the poor clinical translation.
However, recent data has shown that only a selective subtle modulation of the enzyme
is needed to provide a safe GSK-3β homeostasis recovery effect without interfering in
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other cellular signaling. This great challenge may be achieved considering allosteric
ligands. Today, allosterism is recognized as an inherent property of all dynamic
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proteins of biological relevance. In fact, allosteric inhibitors bring a novel mechanism
of GSK-3β modulation, and to date very few allosteric modulators of GSK-3β have
been described. In addition to substrate competitive GSK-3 inhibitors such as some
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specific peptides, including L803-mts, and 5-iminothiadiazoles, only the natural
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sesquiterpene palinurin and the quinoline-3-carbohydrazide 1 (VP0.7) have been
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described (Figure 1).
Here, Figure 1
Compound 1 is a small heterocyclic molecule which was postulated to bind to GSK-3β
25
between Arg209 and Thr235 in the described pocket 7. In this work we describe a
facile synthesis for 1 and expand the methodology for the preparation of related
molecules in order to explore the structure activity relationships of this novel family of
allosteric compounds. These studies are supported by refined geometry and docking
studies that reinforce the hypothesis of the binding to an allosteric site and open new
possibilities to specific drug design. Preliminary studies focused in following β-catenin
localization after treatments with different GSK-3β inhibitors, are performed. Finally, in
regard to the potential translation of this type of inhibitor into the clinic we report the
benefits of some of these allosteric inhibitors of GSK-3β in two muscular chronic
diseases: congenital myotonic dystrophy (CDM1) and spinal muscular atrophy (SMA).
Using human myoblasts from patients with CDM1 we have shown the correction of
delayed myogenesis after the treatment with these compounds. Moreover, the survival
of motor neurons derived from SMA patients’ induced pluripotent stem cells are
increased with allosteric GSK-3β modulator treatment.
Results and Discussion
Chemistry
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Considering our previous allosteric modulator 1 discovered by virtual screening of our
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in-house chemical library on GSK-3β as a prototype, here we proposed the
development of a feasible synthesis for this heterocyclic compound together with some
structural modifications that would allow us to build a structure-activity relationship and
confirm the allosteric modulation. The 3-carboxamide-quinoline scaffold is maintained
in all the synthetized compounds while different substituents that may modulate the
steric volume, electrostatic potential and hydrogen bond capabilities of the final
compounds are varied to decipher the key interactions with the allosteric pocket in
order to confirm this kind of enzymatic inhibition (Figure 2).
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Here, Figure 2
General synthesis of these quinoline derivatives was accomplished by a convergent
26
synthesis previously described
using 4-hydroxy-3-ethoxycarbonyl-2-oxo-dihydro-
quinolines 13-25 and different substituted amines or hydrazines (Scheme 1).
Here, Scheme 1
The synthesis of the dihydroquinolines starts from the corresponding isatoic anhydride
that is alkylated in presence of sodium hydride and the halogenated alkyl chain to
2
introduce the substituent R on the heterocyclic nitrogen (compounds 2-12). These
alkylated anhydrides, some of them from commercial suppliers, are treated with diethyl
malonate and sodium hydride to obtain the quinolines 13-25. Lastly, these quinolines
can be transformed into different amides or hydrazides. Thus, compounds 13-25 were
subsequently treated with different hydrazides in solution to achieve the final
compounds 26-62. Additionally, three compounds were prepared without the
carbohydrazide moiety, which were synthesized from the corresponding quinoline
carboxylate. This carboxylate was treated with amine or hydrazide in different solvents
at reflux temperature to obtain the amides 63 and 64 and the hydrazide 65.
All the compounds here prepared were unequivocally characterized using analytical
1
13
and spectroscopic data ( H- and C-NMR), which are collected in the experimental and
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supplementary parts and when possible for some previously described compounds,
melting points were compared (see experimental section).
GSK-3β inhibition assays
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The GSK-3β enzymatic inhibition of all the compounds here prepared was analyzed in
our laboratories using human recombinant enzyme and a luminescence method (see
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experimental section). All the compounds were tested initially at a fixed concentration
of 10 µM and only in the cases where the compound’s inhibition was more than 50% of
the total activity the dose response curve was calculated. Data collected in tables 1 and
2 are the mean of two different experiments. Our results show that most of the 3-
carboxamide-quinolines here prepared are GSK-3β inhibitors with IC50 values in the
low micromolar range, very similar to that of the first compound discovered 1.
Here Table 1 and Table 2
The IC50 values obtained here for the new synthetized compounds are rather similar, in
the same order of magnitude, pointing to a flat structure-activity relationship which is in
agreement with an allosteric modulation. However, the lack of activity of some
compounds revealed important structural requirements for the enzymatic inhibition.
Thus, the carbonyl group at N’-position of the hydrazide is crucial for the inhibition,
leading to inactive compounds 63-65 when omitted from the structure. Moreover, a
long lipophilic tail of 7 or 11 carbon atoms joined to the carboxamide moiety is also a
prerequisite for active compounds. In fact, when it is substituted by phenyl, alkyl,
benzyl or dimethylene heteroaryl fragments (compounds 31, 32, 33 and 34, 36), the
biological activity is lost. Other changes in the nature of the substituent attached to the
heterocyclic nitrogen atom are rather insensitive for GSK-3β inhibition, resulting in
compounds with similar enzymatic potency. Finally, introduction of halogen atoms in
positions 6 or 7 of the quinoline scaffold produces a slight increase in IC50 value
(
derivatives 53 and 59 versus 1, for example).
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To corroborate the kind of inhibition of these new compounds we selected derivatives
53 and 59 with IC50 values lower than the reference compound 1 for different kinetic
studies. We determined the competition with ATP or with the substrate used in the
enzymatic reaction
synthase was used as substrate. The first set of studies were done varying both ATP
from 1 to 50 µM) and quinoline concentrations. Double-reciprocal plotting of the data is
. The small peptide GS-2 similar to skeletal muscle glycogen
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(
depicted in figure 3. The intercept of the plot in the vertical axis (1/V) rises when the
quinoline concentration increases (from 2.5 to 5 µM), whereas the intercept in the
horizontal axis (1/[ATP]) does not change, meaning that, while the enzyme maximal
rate (Vmax) decreases in the presence of the inhibitor, the Michaelis-Menten constant
(
Km) remains unaltered. These results would suggest that derivatives 53 and 59 act as
non-competitive inhibitors of ATP binding, because an increase in the ATP
concentration (from 1 to 50 µM) does not interfere with enzymatic inhibition.
Here, Figure 3
Finally, kinetic experiments were also performed varying the concentrations of both
GS-2 (from 12.5 to 100 µM) and quinoline inhibitors 53 and 59 (2.5 and 5 µM), while
the ATP concentration was kept constant (1 µM). Double-reciprocal plotting of the data
(
Figure 4), suggests that these compounds act as non-competitive inhibitors of GS-2
2
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binding. These results are similar to that observed for compound 1 and confirm that
the new 3-carbohydrazide-quinolines here described are also allosteric inhibitors of
GSK-3β, binding to a different pocket to the ones that ATP and substrate bind to.
Here, Figure 4
One of the main challenges to overcome when a protein kinase is modulated
allosterically is selectivity over other protein kinases. To check that fact, we have tested
a couple of compounds, 53 and 59, together with the first prototype, quinoline 1, on a
panel of 50 different kinases. Kinase profiling plots are shown in figure 5, showing a
consistent and robust selectivity compatible with an ATP non-competitive inhibition.
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These results not only support the selective profile of the allosteric modulators but also
rule out the possibility to be pan assay interference compounds.
Here, Figure 5
Molecular modeling studies
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All these experimental results prompted us to perform a computational molecular
docking study to understand the ligand-protein interaction in detail and the allosteric
mechanism of action of these heterocyclic derivatives.
The quinoline compounds studied here have at least 3 or 4 protons that can be moved
between 6-7 different positions marked by nitrogen and oxygen atoms present in the
heterocycle and the substituent attached in position 3 leading to different tautomeric
forms (Figure 2). Thus, a preliminary computational study was performed to determine
the different tautomeric micro species using the Marvin Sketch software and compound
1 as prototype of this heterocyclic family. Results showed that the more stable
tautomeric forms were those with a hydroxyl group in positions 2 or 4 of the quinoline
heterocycle, forms A and B, respectively (Figure 6).
Moreover, using the same software, the pKa profile for A and B tautomeric forms of
compound 1, were calculated. Deprotonated micro species distributions at different pH
values are depicted in Figure 6. At physiological pH, the tautomeric form A will have
two micro species coexisting, one with the protonated hydroxyl group (micro specie A)
and another deprotonated (micro specie A1). However, the high acidic nature of the 4-
hydroxyl group in tautomer B, leads to a unique anionic form at physiological
environment (micro specie B1) (Figure 6).
Here, Figure 6
Ab initio geometry optimizations and frequency calculations were performed for both
anionic species (A1 and B1) showing a difference in Gibbs free energy, between them
-1
of 0.818 kcal.mol (Figure S1). Both optimized structures maintain the intramolecular
H-bond between the hydroxyl group of the quinoline ring and the amine of
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carbohydrazide, being the tautomer with the hydroxyl moiety in position 4 (anionic form
B1) slightly more favored energetically.
Docking experiments were carried out in order to understand the allosteric mechanism
of action of 3-carboxamide-quinolines and the differences in the behavior of active and
inactive compounds. These studies were performed using AutoDock software (see
experimental section), centering the grid in the allosteric cavity previously identified as
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pocket 7, but considering the favorable protonation state at physiological conditions.
The calculated docking scores together with the predicted Ki values were used to
evaluate the binding mode of these allosteric modulators.
The results obtained showed that these GSK-3β inhibitors need to maintain three
different key interactions simultaneously in the allosteric site for enzymatic inhibition.
The first one faced the quinoline moiety with Arg209 located in the activation loop of
this protein kinase. The nature of this interaction is different for the compounds studied,
thus electrostatic, cation-π or hydrogen bond types are found. The second one, is a
crucial hydrogen bond formed between the carbohydrazide group of the inhibitor and
Ser236; and the last one is produced by hydrophobic interactions between the aliphatic
chain of the inhibitor with the hydrophobic region of the pocket (Leu169, Arg328,
Thr330 and Pro331) (Figure 7). These three different interactions seem to be essential
for enzyme inhibition.
Here, Figure 7
The non-existence of some of the three key interactions above mentioned is translated
in a decrease or loss of the activity. As an example, inactive compounds 32, 63 and 64
were analyzed. Compounds 32 and 63 bind to the cavity by interactions with Arg209,
although these are not present with Ser236 neither the hydrophobic region. These facts
explain the low affinity of these quinolines and their lack of biological activity (see
Figure S2). Other interesting example is compound 65 which interacts with Arg209 and
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Ser236, but the less hydrophobic nature of the chain could prevent its binding to the
allosteric site (Figure S2).
Among active compounds the docking study has allowed us to rationalize the
relationship between chemical structure and biological activity. Regarding the
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substituent R , different halogen atoms in positions 6 and 7 of the quinoline ring,
docking results show that the substituent is exposed to solvent without any interaction
with target residues. This fact is reflected in the slight variation of IC50 values of the
halogen derivatives 1, 53 and 59.
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Considering R , the substituent attached to the heterocyclic nitrogen atom of the
quinoline ring, small moieties such as H, Me and Et allow the ionic interaction between
the 4-hydroxyl moiety and Arg209 (Figure 7). In the case of bigger substituents such as
isoprenyl, CH -cyclopropyl and benzyl groups (compounds 39, 40, 41, 42, 61, 62), the
2
steric hindrance makes the quinoline ring to flip, allowing the formation of an hydrogen
bond between the hydroxyl attached to the quinoline and Asn213’s backbone –NH (see
Figure S3). This alternative binding mode favors H-bonds between the carbonyl group
2
of the quinoline ring and Arg209 and in addition hydrophobic interactions among R
substituents and the Arg209’s side chain (Figure S3). In the case of aromatic
substituents (Compounds 42, 61 and 62), cation-π interaction between the benzyl
group and Arg209 has been found, which would explain that in spite of their different
binding mode, the interactions with the Arg209 are still maintained and therefore the
inhibitory activity is maintained (Figure S3).
3
Finally, docking studies show that bigger aliphatic chains in R (C11
H23) score better
than the smaller ones (C H ) in agreement with the experimental data (See Table 1).
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Hydrophobic interactions between aliphatic chain and Leu169, Val240, Arg328, Thr330
and Pro331 residues appear to be very important to improve the ligand affinity by the
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allosteric pocket, whereas compounds with the shorter C
7
H15 chain are less efficient
(Figure S4).
In order to provide a reasonable hypothesis for the allosteric mechanism of inhibition of
this quinoline heterocyclic family, a structural comparative study was performed
between the apo form of GSK-3β (1PYX) and the complexes with compound 1 and
compound 63, after molecular dynamics experiments. Trajectories for each ligand-
GSK-3β complexes and GSK-3β without ligand were calculated along 25 ns.
Simulations for all systems maintain the stability along the trajectories. Compound 1 is
able to restrict the movement of the protein backbone, while compound 63 is not able
to restrict this movement (Figure S5), which is in correspondence with the lack of
activity of this last compound. More in detail, the comparative analysis of the root-
mean-square fluctuations (RMSF) for each residue between the trajectory for apo-
GSK-3β and GSK-3β complexed with compound 1 shows the movement of different
residues present in the allosteric pocket, such as Arg209 (Figure S6). The major
changes are produced in the activation loop region (residues 200-226) inducing an
important rearrangement of the residues of the catalytic site. Due to the electrostatic
interactions of 1 with Arg209 there is a decrease in the flexibility of the activation loop.
The main change is the drastic reduction of the distance between Glu211 and Lys205
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(
Figure 8 and S7). The interaction of Arg209 with compound 1 does not allow the
movement of Glu211, stimulating a reorientation of Glu211 to Lys205 favoring the
formation of a strong hydrogen bond. This new bond with Lys205, one of the different
basic residues involved in the catalytic recognizing site network (Asn213, Arg180 and
Arg96) produces different changes of more than 0.5 Å. These conformational changes
restrict the movement of GSK-3β activation loop, modifying the substrate binding.
Here, Figure 8
Effects of different GSK-3β inhibitors on β-catenin localization
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Levels of β-catenin, a multifunctional protein with pivotal role in embryonic patterning,
organogenesis and adult homeostasis, are normally kept low by a phosphorylation
event mediated by GSK-3 (α- and β-isoforms), which targets β-catenin for ubiquitylation
28
and proteosomal degradation. When this phosphorylation event is blocked, β-catenin
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is stabilized and accumulated in the cytosol. Stabilized β-catenin translocates to the
2
9
nucleus, where it co-activates transcription of different oncogenes. The great
challenge for a small molecule targeting GSK-3 (α- or β-) is to reduce its aberrant
activity found in several diseases without altering its role on β-catenin signaling.
30-32
By using different techniques (X-ray, molecular dynamics or NMR)
several allosteric
sites that may allow the recognition of one substrate or other, have been described in
GSK-3β. Moreover, the structural basis for the recruitment of GSK-3β to the Axin-APC
33
scaffold crucial for β-catenin phosphorylation was described, and structural biology
studies have shown that it is possible to target the three-dimensional structure of GSK-
3
β to select for specific substrate-bound forms of the enzyme, enabling selective
3
4
targeting of one pathway controlled by GSK-3β.
Considering this background, we decided to explore if the use of several inhibitors of
GSK-3β that bind in different enzymatic sites may alter or not β-catenin localization.
We selected two widely used ATP-competitive inhibitors, 66
(
SB216763)35 and 67
36
(
alsterpaullone)
and two allosteric modulators here described, specifically
3
7
compounds 1 and 53. In addition we also included 68 (TDZD-8), a well-known ATP
non-competitive inhibitor that modulates the enzyme by covalent modification of
Cys199. We have used two different human cell lines of glioblastoma and
neuroblastoma, LN-18 and SH-SY5Y, respectively, and treated them during long term
(72 h) with the different GSK-3β inhibitors. Localization of β-catenin was analyzed by
immunohistochemistry. Our results show that whereas in the cell cultures treated with
the two ATP-competitive inhibitors, β-catenin is translocated to the nucleus, but in the
three cases where the inhibitor targets the enzyme allosterically, localization of β-
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catenin remains in the cytosol (Figure 9). We here corroborate that allosteric
modulators of GSK-3β not only induce a conformational change in the active site by
modification of GSK-3β activation loop flexibility, but also point to overcoming an
important challenge in GSK-3β targeting: reducing the aberrant GSK-3β activity found
in several pathologies while not promoting oncogenesis through aberrant β-catenin
signaling. Based on these results, this kind of inhibitors should be of great benefit in
chronic diseases treatments.
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Here, Figure 9
Effect of GSK-3β allosteric modulator on chronic diseases
Until now, few data have been obtained from the clinic regarding the safety and the
efficacy of GSK-3β inhibitors in patients and some issues remain unclear. However, it
is important to consider that lithium, a weak inhibitor of GSK-3β, is the standard
pharmacological therapy for chronic bipolar disorder treatment which may discard
38
mechanism-based toxicities associated with GSK-3β inhibition. Moreover, tideglusib,
a TDZD-related compound that targets GSK-3β in an ATP non-competitive manner,
has no safety concerns in patients of chronic diseases such as progressive
supranuclear palsy and Alzheimer’s disease after long-term treatments of 12 and 6
14-15
months, respectively.
Until now, the quinoline allosteric modulators reported here have not reached clinical
trials, but compound 1 has shown safety and great efficacy in preclinical models of
39
40
multiple sclerosis and fragile X syndrome, pointing to a potential use in the chronic
treatment of such neurological diseases.
Among the unmet diseases where GSK-3β recently emerges as a potential
pharmacological target are some rare neuromuscular degenerative diseases. We
recently found that the levels of active GSK-3β are increased in skeletal muscle
8
,41
biopsies from patients with myotonic dystrophy type 1 (DM1).
The inhibitors of GSK-
3β, lithium and 68 correct the levels of GSK-3β and the GSK-3β-cyclin D3 pathway in a
LR
DM1 mouse model (HSA mice) significantly reducing myotonia, muscle weakness
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and myopathy.
Furthermore, although lithium did not improve the neuromuscular
42
phenotype in a mouse model of SMA, maleimide-based GSK-3β inhibitors prolonged
4
3
the median survival time of these mice.
Given the specific effect of the allosteric modulators here described on GSK-3β activity,
we tested if these modulators could be of therapeutic interest for GSK-3β associated
neuromuscular diseases, mainly DM1 and SMA.
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To examine if the allosteric modulators have a beneficial effect in DM1, we determined
whether compound 53 improves delayed myogenesis in primary myoblasts from
skeletal muscle of patients with congenital DM1 (CDM1). Figure 10 shows that un-
treated CDM1 myoblasts fail to fuse and form myotubes in cell culture dish. However,
treatment of CDM1 myoblasts during differentiation with the compound 53 improved
differentiation of CDM1 myoblasts in a dose-dependent manner. Moreover, an analysis
of protein extracts of the different cultures showed that desmin, a differentiation marker,
is present in healthy myotubes and is deficient in the CDM1 ones. Treatment with the
GSK-3β inhibitor, quinoline 53, corrects expression levels of desmin. These data
suggest the therapeutic potential of the allosteric GSK-3β modulator 53 for the future
therapy of CDM1.
Here, Figure 10
Furthermore, we have used human motor neurons (MNs) derived from induced
pluripotent stem cells (iPSCs) obtained from a healthy subject (1016A) and SMA
patients affected by different disease severities – a severe type II (I-51C) line and a
44
very severe type I (I-38G) line – that have been previously described, to evaluate
their survival with or without different GSK-3β inhibitors. We have selected two
allosteric modulators described here, compounds 1 and 53, and 67, a well-known GSK-
3
β ATP-competitive inhibitor, and treated the MN cultures from 7 days. Importantly,
both 1 and 53 compounds produced a dose-dependent MN survival increase in type I
SMA MN cultures (Figure 11). Given that the rate of MN death is notably higher in the
most severe type I than in the type II and healthy control cultures, a higher rescue was
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also expected. The results also showed that these compounds do not display toxicity,
even at the highest concentrations tested, whereas 67 was toxic at concentrations
higher than 1.25 µM and produced some basal toxicity in the healthy control MNs.
These data demonstrate that these non-ATP competitive GSK-3β inhibitors prevent the
death of iPSC-derived SMA MNs in chronic treatments (7 days).
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Here, Figure 11
Conclusions
GSK-3β represents an important drug target for many severe and unmet human
45
diseases. Here we describe a new family of selective allosteric modulators of GSK-3β
and provide a careful structural study of their binding mode. The compounds reported
here are able to modify the flexibility of the GSK-3β activation loop which not only
induces a smooth conformational change in the active site, decreasing the activity, but
also points to overcoming an important challenge in GSK-3β targeting: not interfering in
the β-catenin signaling.
We have shown in this paper that modulation of GSK-3β activity by the allosteric
quinoline 53 in myoblasts from skeletal muscle of patients with congenital DM1 shows
that this compound clearly improves CDM1 myogenesis. Moreover, we have shown
that allosteric modulation of GSK-3β with quinolines 1 and 53 promotes the survival of
SMA motor neurons. Thus, modulation of GSK-3β activity by these allosteric quinoline
derivatives opens a new avenue for future chronic treatments of GSK-3β associated
diseases, including CDM1 and SMA.
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Experimental section
Chemistry
Substrates were either purchased from commercial sources or used without further
purification. Melting points were determined with a Mettler Toledo MP70 apparatus and
are uncorrected. Flash column chromatography was carried out at medium pressure
using silica gel (E. Merck, grade 60, particle size 0.040−0.063 mm, 230−240 mesh
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0
ASTM) with the indicated solvent as eluent. Compounds were detected with UV light
1
(
254 nm). H NMR spectra were obtained on a Bruker AVANCE-300 spectrometer
working at 300 MHz and on the Varian INNOVA-300 operating at 300 MHz. Typical
spectral parameters: spectral width 10 ppm, pulse width 9 ꢀs (57°), data size 32 K.
Description of signals: s = singlet, bs = broad singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, dd = doublet of doublet, ddd = doublet of dd, dt = doublet of
13
triplets.
C NMR experiments were carried out on the Bruker AVANCE-300
spectrometer operating at 75 MHz. The acquisition parameters: spectral width 16 kHz,
acquisition time 0.99 s, pulse width 9 ꢀs (57°), data size 32 K. J values are reported in
hertz. Elemental analyses were performed by the analytical department at CENQUIOR
(
CSIC), are reported in the supplementary information and the results obtained were
within ±0.4% of the theoretical values. HPLC analyses were performed on an Alliance
Waters 2690 equipment with a UV detector photodiode array Waters 2996 with MS
detector MicromassZQ (Waters) positive electrospray, using the Sunfire column C18,
3.5 ꢀm (50 mm × 4.6 mm) and acetonitrile and MilliQ water (with 0.1% formic acid) as
mobile phase. The standard gradient consisted of a20 min run (unless otherwise
stated) from 10% to 100% acetonitrile gradient at a flow rate of 1.0 mL/min. All the final
compounds have a purity >95% tested by HPLC
General synthesis procedure compounds 2-12
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Following the procedure described by Ukrainteset al. , the corresponding isatoic
anhydride (1 equiv.) was dissolved in dry DMF (15 mL) and the sodium hydride (1.5
equiv.) was added slowly at 0 ºC. After 30 min, the corresponding halogenated
compound (1.5-3 equiv.) was added and the reaction mixture was stirred at room
temperature overnight. Dichloromethane (15 mL) and water (15 mL) were added. The
organic phases were washed with brine (15 mL), dried over magnesium sulphate,
filtered and the solvent evaporated. The obtained solid was recrystallized in
dichloromethane/hexane to afford the desired compound.
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N-Isobutylisatoic anhydride (2). Reagents: isatoic anhydride (1 g, 6.1 mmol); sodium
hydride (0.23 g, 9.2 mmol); isobutyl iodide (3.4 g, 18 mmol). White solid (600 mg,
4
6%). M.p. = 85-86 ºC. HPLC: Purity > 99%, R.t. = 8.6 min. ESI MS (m/z): 220 [M +
+
1
H] . H NMR (300 MHz, DMSO-d ) δ (ppm): 8.10 (dd, J = 1.7, 7.9 Hz, 1H), 7.68 (ddd, J
6
=
1.6, 7.3, 8.4 Hz, 1H), 7.25 – 7.18 (m, 1H), 7.10 (d, J = 8.5 Hz, 1H) 3.93 (d, J = 7.6
13
Hz, 2H), 2.25-2.10 (m,1H), 1.03 (d, J = 6.7 Hz, 6H); C NMR (75 MHz, CDCl ) δ (ppm):
3
1
62.4, 158.7, 148.3, 141.8, 137.2, 131.1, 124.0, 114.4, 112.0, 51.7, 26.9, 20.1, 19.9.
Anal. (C12 ).
H13NO
3
N-Isoprenylisatoic anhydride (3). Reagents: isatoic anhydride (1 g, 6.1 mmol);
sodium hydride (0.23 g, 9.2 mmol); isoprenyl bromide (1.9 g, 12 mmol). White solid
46
(
950 mg, 67%). M.p. = 119-120 ºC (lit. 119 ºC).
N-Cyclopropylmethylisatoic anhydride (4). Reagents: isatoic anhydride (0.8 g, 4.9
mmol); sodium hydride (0.14 g, 5.9 mmol); cyclopropyl methyl bromide (3.4 g, 18
4
7
mmol). Brown solid (530 mg, 50%). M.p. = 122-123 ºC (lit. 118-121 ºC).
N-Benzylisatoic anhydride (5). Reagents: isatoic anhydride (1 g, 6.1 mmol); sodium
hydride (0.22 g, 9.2 mmol); benzyl bromide (1.4 g, 12.3 mmol). White solid (200 mg,
4
7
15%). M.p. = 139-140 ºC (lit. 140-141 ºC).
5
-Chloro-N-methylisatoic anhydride (6). Reagents: 5-chloroisatoic anhydride (0.8 g,
3.9 mmol); sodium hydride (0.14 g, 5.9 mmol); methyl iodide (1.6 g, 11.4 mmol). White
47
solid (500 mg, 60%). M.p. = 194-195 ºC (lit. 192-195 ºC).
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5-Chloro-N-ethylisatoic anhydride (7). Reagents: 5-chloroisatoic anhydride (0.5 g,
2.5 mmol); sodium hydride (0.09 g, 3.8 mmol); ethyl iodide (0.6 g, 3.8 mmol). White
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solid (400 mg, 70%). M.p. = 145-146 ºC (lit. 143-145 ºC).
5-Bromo-N-methylisatoic anhydride (8). Reagents: 5-bromoisatoic anhydride (1 g,
4.1 mmol); sodium hydride (0.15 g, 6.2 mmol); methyl iodide (0.9 g, 6.2 mmol). White
0
1
2
3
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8
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solid (690 mg, 66%). M.p. = 201-202 ºC (lit. 204ºC).
5-Bromo-N-ethylisatoic anhydride (9). Reagents: 5-bromoisatoic anhydride (2 g, 8.2
mmol); sodium hydride (0.3 g, 12.5 mmol); ethyl iodide (1.9 g, 12.5 mmol). White solid
(
1.4 g, 63%). M.p. = 177-178 ºC. HPLC: Purity >97%, R.t. = 8.4 min. ESI MS (m/z): 272
+
+
1
[
M+2H] , 270 [M + H] . H NMR (300 MHz, CDCl
3
) δ (ppm): 8.21 (d, J = 2.4 Hz, 1H),
7.78 (dd, J = 2.4, 8.9 Hz, 1H), 7.02 (d, J = 8.9 Hz, 1H), 4.05 (q, J = 7.2 Hz, 2H), 1.31 (t,
13
J = 7.2 Hz, 3H); C NMR (75 MHz, CDCl ) δ (ppm): 157.3, 146.9, 140.0, 139.0, 133.2,
3
1
4
4
16.6, 115.5, 113.4, 40.3, 12.0. Anal. (C10
H
8
BrNO ).
3
-Chloro-N-methylisatoic anhydride (10). Reagents: 4-chloroisatoic anhydride (0.9 g,
.5 mmol); sodium hydride (0.12 g, 5.4 mmol); methyl iodide (1.9 g, 13.7 mmol). Yellow
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solid (400 mg, 42%). M.p. = 215-216 ºC (lit. 216-218 ºC).
4-Chloro-N-ethylisatoic anhydride (11). Reagents: 4-chloroisatoic anhydride (0.9 g,
4.5 mmol); sodium hydride (0.12 g, 5.0 mmol); ethyl iodide (1.3 g, 8.3 mmol). White
solid (650 mg, 63%). M.p. = 156-157 ºC. HPLC: Purity > 97%, R.t. = 8.1 min. ESI MS
+
+ 1
(
m/z): 228 [M+2H] , 226 [M + H] . H NMR (300 MHz, DMSO-d
6
) δ (ppm): 8.00 (d, J =
8
2
1
.4 Hz, 1H), 7.64 (d, J = 1.7 Hz, 1H), 7.37 (dd, J = 1.7, 8.4 Hz, 1H), 4.06 (q, J = 7.0 Hz,
1
3
H), 1.20 (t, J = 7.0 Hz, 3H); C NMR (75 MHz, DMSO-d
6
) δ (ppm): 158.8, 147.6,
ClNO ).
42.8, 142.5, 131.8, 124.1, 114.9, 111.4, 40.2, 12.3. Anal. (C10
H
8
3
N-Benzyl-4-chloroisatoic anhydride (12). Reagents: 4-chloroisatoic anhydride (1.0 g,
.0 mmol); sodium hydride (0.19 g, 7.8 mmol); benzyl bromide (1.7 g, 10.1 mmol).
5
4
7
White solid (650 mg, 63%). M.p. = 167-168 ºC (lit. 165-170 ºC).
General synthesis procedure compounds 13-25
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Following the procedure described by Coppola et al. diethylmalonate was added
dropwise to a sodium hydride solution in dry DMF (15 mL) under argon atmosphere.
The reaction was stirred for 30 min at room temperature. Then the mixture was added
to a round bottom flask containing the corresponding isatoic anhydride derivative
dissolved in DMF (15 mL) under argon atmosphere. The mixture was heated at 50 ºC
during 5 hours. To the mixture dichlorometane (30 mL), water (50 mL) and HCl (1N)
were added to reach a pH=5. The organic layer was isolated and washed with brine
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(
30 mL). Then the organic phase was dried over dry magnesium sulfate, filtered and
the solvent was evaporated. The solid was recrystallized in dichloromethane /hexane to
obtain the desired compound.
Ethyl
4-hydroxy-1-isobutyl-2-oxo-1,2-dihydroquinoline-3-carboxylate
(13).
Reagents: N-isobutylisatoic anhydride (1) (0.3 g, 1.3 mmol); sodium hydride (0.138 g,
5
.7 mmol); diethyl malonate (1.1 g, 6.8 mmol). Yellow oil (150 mg, 38%). HPLC: Purity
+
1
>
95%, R.t. = 10.0 min. ESI MS (m/z): 290 [M + H] . H NMR (300 MHz, DMSO-d ) δ
6
(
ppm): 13.02 (s, 1H), 8.06 (dd, J = 1.55, 8.3 Hz, 1H), 7.72 (ddd, J = 1.60, 7.0, 8.7 Hz,
1
H), 7.55 (d, J = 8.6 Hz, 1H), 7.29 (ddd, J = 0.86, 7.0, 7.9 Hz, 1H), 4.33 (q, J = 8.6 Hz,
2
H), 4.07 (d, J = 7.4 Hz, 2H), 2.10 (sept, J = 7.0 Hz, 1H), 1.31 (t, J = 7.4 Hz, 3H), 0.89
13
(
d, J = 7.4 Hz, 6H); C NMR (75 MHz, DMSO-d ) δ (ppm): 169.2, 165.5, 158.9, 140.0,
6
1
33.6, 124.6, 121.7, 115.3, 114.5, 101.0, 61.4, 47.5, 26.6, 19.8, 13.9. Anal.
).
4-hydroxy-1-isoprenyl-2-oxo-1,2-dihydroquinoline-3-carboxylate
(
C
16
H19NO
4
Ethyl
(14).
Reagents: N-isopropenylisatoic anhydride (2) (0.3 g, 1.3 mmol); sodium hydride (0.065
g, 1.3 mmol); diethyl malonate (1.0 g, 6.5 mmol). White solid (80 mg, 21%). M.p. = 123-
+
1
1
24 ºC. HPLC: Purity > 96%, R.t. = 10.2 min. ESI MS (m/z): 302 [M + H] . H NMR
300 MHz, DMSO-d ) δ (ppm): 14.21 (s, 1H), 8.18 (dd, J = 1.6, 8.3 Hz, 1H), 7.65 (td, J
1.6, 7.1, 7.9 Hz, 1H), 7.24 (m, 2H), 5.10 (m, 1H), 4.88 (d, J = 6.0 Hz, 2H), 4.51 (q, J =
(
6
=
1
3
7.1 Hz, 2H), 1.80 (s, 3H), 1.63 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H); C NMR (75 MHz,
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CDCl
3
) δ (ppm): 172.9, 171.8, 159.5, 140.9, 135.8, 134.3, 125.9, 121.8, 119.7, 115.2,
114.7, 98.0, 62.5, 40.9, 25.7, 18.5, 14.4. Anal. (C13
H13NO
3
).
Ethyl 1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate
(16).
Reagents: N-benzylisatoic anhydride (4) (0.85 g, 3.4 mmol); sodium hydride (0.18 g,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.37 mmol); diethyl malonate (2.7 g, 16.8 mmol). White solid (100 mg, 9%), M.p. = 117-
+
1
118 ºC. HPLC: Purity > 99%, R. t. = 9.9 min. ESI MS (m/z): 324 [M + H] . H NMR (300
MHz, CDCl ) δ (ppm): 14.29 (s, 1H), 8.13 (dd, J = 1.5, 8.0 Hz, 1H), 7.48-7.43 (m, 1H),
.23-7.18 (m, 2H), 7.15-7.11 (m, 5H), 5.44 (s, 2H), 4.45 (q, J = 7.1 Hz, 2H), 1.42 (t, J =
3
7
7
1
13
.1 Hz, 3H); C NMR (75 MHz, CDCl ) δ (ppm): 171.7, 171.0, 158.7, 139.8, 135.4,
3
33.3, 127.7, 126.2, 125.4, 124.7, 121.0, 113.9, 96.8, 61.4, 44.6, 13.21. Anal.
(
C
19
H
4
17NO ).
Ethyl 6-chloro-4-hydroxy-2-oxo-1,2-dihydroquiniline-3-carboxylate (17). Reagents:
5-chloroisatoicanhydride (1.0 g, 5.0 mmol); sodium hydride (0.25 g, 10.4 mmol); diethyl
50
malonate (4.0 g, 25 mmol). White solid (0.06 g, 9%). M.p. = 251-ºC (lit. 250 ºC
decomp).
Ethyl 6-chloro-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (18).
Reagents: 5-chloro-N-methylisatoicanhydride (5) (0.8 g, 3.5 mmol); sodium hydride
(
0.18 g, 7.1 mmol); diethyl malonate (2.8 g, 18 mmol). White solid (300 mg, 30%). M.p.
49
=
136-137 ºC (lit. 132-135 ºC).
Ethyl 6-chloro-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (19).
Reagents: 5-chloro-N-ethylisatoicanhydride (6) (0.8 g, 3.5 mmol); sodium hydride (0.18
g, 7.1 mmol); diethyl malonate (2.8 g, 18 mmol). White solid (500 mg, 48%). M.p. =
+
140-141 ºC. HPLC: Purity > 99%, R.t. = 9.9 min. ESI MS (m/z): 298 [M+2H] , 296 [M +
+
1
H] . H NMR (300 MHz, CDCl
3
) δ (ppm): 14.14 (s, 1H), 8.09 (d, J = 2.5 Hz, 1H), 7.54
(
dd, J = 2.5, 9.1 Hz 1H), 7.19 (d, J = 2.0 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 4.21 (q, J =
1
3
7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H), 1.25 (t, J = 7.1 Hz, 3H); C NMR (75 MHz, CDCl3)
δ (ppm): 171.4, 169.5, 157.7, 137.8, 133.3, 126.4, 124.2, 115.1, 114.5, 97.5, 61.2,
36.4,14.3, 13.2. Anal. (C14 ).
H
14ClNO
4
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Ethyl 6-bromo-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (21).
Reagents: 5-bromo-N-methylisatoicanhydride (7) (0.6 g, 2.3 mmol); sodium hydride
(0.12 g, 4.8 mmol); diethyl malonate (1.8 g, 11.5 mmol). White solid (0.3 g, 40%). M.p.
= 139-140 ºC. HPLC: Purity > 96%, R.t. = 9.6 min. ESI MS (m/z): 330 [M+2H], 328 [M +
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
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
1
H] . H NMR (300 MHz, CDCl
3
) δ (ppm): 14.14 (s, 1H), 8.28 (d, J = 2.4 Hz, 1H), 7.74
(dd, J = 2.4, 9.0 Hz, 1H), 7.18 (d, J = 9.0 Hz, 1H), 4.50 (q, J = 7.1 Hz, 2H), 3.62 (s, 3H),
13
1.48 (t, J =7.1 Hz, 3H); C NMR (75 MHz, CDCl
3
) δ (ppm): 172.5, 170.6, 159.3, 140.3,
).
1
37.2, 128.2, 116.5, 116.0, 115.0, 98.7, 62.7, 29.4, 14.3. Anal. (C13
H
12BrNO
4
Ethyl 6-bromo-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (22).
Reagents: 5-bromo-N-ethylisatoicanhydride (8) (0.5 g, 1.8 mmol); sodium hydride (0.09
g, 3.7 mmol); diethyl malonate (1.5 g, 4.25 mmol). White solid (0.3 g, 48%). M.p. = 129-
+
1
30 ºC. HPLC: Purity > 99%, R.t. = 10.6 min. ESI MS (m/z): 342 [M + 2H] , 340 [M+H].
1
H NMR (300 MHz, CDCl
3
) δ (ppm): 14.14 (s, 1H), 8.23 (d, J = 2.4 Hz, 1H), 7.67 (dd, J
=
2
2.4, 9.0 Hz 1H), 7.13 (d, J = 9.0 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 4.20 (q, J = 7.1 Hz,
13
H), 1.42 (t, J = 0.5,7.1 Hz, 3H), 1.24 (t, J = 1.5,7.2 Hz, 3H); C NMR (75 MHz, CDCl3)
δ (ppm): 172.4, 170.4, 158.7, 139.2, 137.0, 128.7, 116.6, 115.7, 114.6, 98.5, 62.5,
7.4, 14.2, 12.7. Anal. (C14 ).
3
H14BrNO
4
Ethyl 7-chloro-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (23).
Reagents: 4-chloro-N-methylisatoicanhydride (9) (0.4 g, 2.1 mmol); sodium hydride
(
0.1 g, 4.2 mmol); diethyl malonate (1.7 g, 10.2 mmol). White solid (280 mg, 49%). M.p.
+
=
+
7
3
1
153-154 ºC. HPLC: Purity > 99%, R.t. = 5.1 min. ESI MS (m/z): 284 [M+2H] , 282 [M
+
1
H] . H NMR (300 MHz, DMSO-d
6
) δ (ppm): 12.95 (s, 1H), 8.02 (d, J = 8.6 Hz, 1H),
.59 (d, J = 1.8 Hz, 1H), 7.33 (dd, J = 1.9, 8.7 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 3.52 (s,
1
3
H), 1.30 (t, J = 7.1 Hz, 3H); C NMR (75 MHz, DMSO-d
59.2, 141.7, 138.9, 126.7, 122.5, 115.1, 113.7, 102.3, 61.8, 49.1, 29.4, 14.4. Anal.
).
6
) δ (ppm): 168.9, 164.6,
(
C
13
H
12ClNO
4
Ethyl 7-chloro-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (24).
Reagents: 4-chloro-N-ethylisatoic anhydride (10) (0.6 g, 2.6 mmol); sodium hydride
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6
(0.1 g, 4.6 mmol); diethyl malonate (1.9 g, 11.5 mmol). White solid (460 mg, 62%). M.p.
+
= 141-142 ºC. HPLC: Purity > 99%, R.t. = 9.9 min. ESI MS (m/z): 298 [M+2H] , 296 [M
+
1
+ H] . H NMR (300 MHz, DMSO-d
6
) δ (ppm): 13.00 (s, 1H), 8.03 (d, J = 8.6 Hz, 1H),
7.64 (d, J = 1.8 Hz, 1H), 7.33 (dd, J = 1.9, 8.6 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 4.19
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
(q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H); C NMR (75 MHz,
DMSO-d ) δ (ppm): 169.0, 164.8, 158.6, 140.7, 139.0, 127.0, 122.3, 114.6, 113.9,
6
102.0, 61.8, 37.1,14.4, 13.0. Anal. (C14
H
14ClNO ).
4
Ethyl 1-benzyl-7-chloro-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (25).
Reagents: N-benzyl-4-chloroisatoic anhydride (11) (0.5 g, 1.9 mmol); sodium hydride
(
0.09 g, 3.9 mmol); diethyl malonate (1.6 g, 9.6 mmol). White solid (900 mg, 94%). M.p.
+
=
172-173 ºC. HPLC: Purity > 99%, R.t. = 10.8 min. ESI MS (m/z): 360 [M+2H] , 358
+
1
[
M + H] . H NMR (300 MHz, DMSO-d ) δ (ppm): 13.17 (s, 1H), 8.07 (d, J = 8.6 Hz,
6
1H), 7.43 (d, J = 1.8 Hz, 1H), 7.35-7.32 (m, 3H), 7.26-7.23 (m, 1H), 7.18 (dd, J = 1.3,
13
8
.6 Hz, 2H), 5.47 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H); C NMR
(
75 MHz, DMSO-d ) δ (ppm): 168.5, 164.8, 158.9, 140.6, 138.7, 136.5, 128.7, 127.1,
6
126.6, 126.4, 122.3, 114.9, 113.8, 101.6, 61.5, 44.5, 13.9. Anal. (C19
H
16ClNO ).
4
General procedure for the synthesis of compounds 1 and 26-62
51
Following the procedure described by Jonsson et al., the corresponding ethyl
carboxylates were mixed with the corresponding hydrazide and the mixture is heated in
DMF at 160 °C for 3 min. The mixture was cooled down to room temperature, MeOH (5
mL) was added and a white precipitate was formed. The precipitate was filtered and
washed with MeOH to afford the products.
N’-Dodecanoyl-1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
(
1). Reagents: ethyl 1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.17
g, 0.67 mmol), dodecanoylhydrazide (0.14 g, 0.67 mmol). White solid (142 mg, 91%).
M.p. = 133-134 ºC. HPLC (10 min run): Purity > 99%, R.t. = 3.82 min. ESI MS (m/z):
1
4
30 [M+H]. H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.22 (s, 1H), 12.05 (d, J = 3.7 Hz,
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1H), 10.80 (d, J = 3.7 Hz, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 7.1 Hz, 1H), 7.68 (d,
J = 8.6 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 4.31 (d, J = 6.7 Hz, 2H), 2.24 (m, 2H), 1.55 (m,
1
3
2H), 1.23 (m, 19H), 0.84 (m, 3H). C NMR (75 MHz, DMSO-d
170.3, 168.0, 139.6, 135.3, 125.4, 123.2, 115.8, 96.4, 37.6, 33.7, 31.9, 29.6, 29.6, 29.4,
29.2, 25.7, 22.7, 14.5, 13.4. Anal. (C24 ).
N’-Dodecanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
6
) δ (ppm): 161.5, 171.4,
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
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
35
N
3
O
4
(26).
Reagents: ethyl 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.030 g, 0.15
mmol), dodecanoylhydrazide (0.030 g, 0.15 mmol). White solid (34 mg, 98%). M.p. =
256-257 ºC. HPLC (10 min run): Purity > 99%, R.t. = 2.19 min. ESI MS (m/z): 402
1
[
M+H]. H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.11 (s, 1H), 11.98 (s, 1H), 11.92 (s,
1
1
1
1
2
4
H), 10.63 (s, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.39 (d, J = 8.3 Hz,
H), 7.31 (t, J = 7.6 Hz, 1H), 2.23 (t, J = 7.2 Hz, 2H), 1.57 (d, J = 6.6 Hz, 2H), 1.25 (s,
13
6H), 0.85 (t, J = 6.2 Hz); C NMR (75 MHz, DMSO-d ) δ (ppm): 162.4, 172.2, 170.0,
6
67.8, 139.2, 134.6, 124.3, 122.9, 116.3, 114.5, 99.4, 33.3, 31.7, 31.0, 29.4, 29.1, 28.9,
5.3, 22.5, 14.3. Anal. (C H N O ).
2
2
31
3
4
-Hydroxy-N’-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
(27).
Reagents: ethyl 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.050 g, 0.2
mmol), octanoylhydrazide (0.030 g, 0.2 mmol). White solid (48 mg, 83%). M.p. = 254-
+
1
2
55 ºC. HPLC (10 min run): Purity 95%, R.t. = 2.14 min. ESI MS (m/z): 346 [M+H] . H
NMR (300 MHz, DMSO-d ) δ (ppm): 16.10 (s, 1H), 11.97 (s, 2H), 10.67 (s, 1H), 7.99
d, J = 8.1 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.49-6.98 (m, 2H), 2.23 (t, J = 7.2 Hz, 2H),
6
(
1
3
1
.55 (s, 2H), 1.27 (s, 8H), 0.86 (d, J = 6.6 Hz, 3H); C NMR (75 MHz, DMSO-d
ppm): 162.8, 172.5, 170.0, 167.9, 139.2, 134.4, 124.4, 122. 9, 116.3, 96.1, 33.3, 31.5,
8.9, 28.8, 25.3, 22.4, 14.3. Anal. (C18 ).
N’-Dodecanoyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
28). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate
6
) δ
(
2
H
23
N
3
O
4
(
(0.15 g, 0.61 mmol), dodecanoylhydrazide (0.13 g, 0.61 mmol). White solid (137 mg,
93%). M.p. = 219-220 ºC. HPLC (10 min run): Purity > 99%, R.t. = 3.02 min. ESI MS
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4
4
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5
5
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5
5
5
6
1
(m/z): 416 [M+H]. H NMR (300 MHz, DMSO-d ) δ (ppm): 16.15 (s, 1H), 11.91 (s, 1H),
6
10.38 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.85 (ddd, J = 8.7, 7.1, 1.6 Hz, 1H), 7.66 (d, J =
8.6 Hz, 1H), 7.49–7.25 (m, 1H), 3.69 (s, 3H), 2.28 (t, J = 7.3 Hz, 2H), 1.64 (s, 2H),
1
3
1.64-1.11 (m, 16H), 0.89 (t, J = 6.7 Hz, 3H). C NMR (75 MHz, CDCl ) δ (ppm): 161.9,
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
171.1, 169.1, 166.6, 140.1, 134.1, 125.4, 122.5, 115.6, 114.4, 96.3, 34.4, 31.9, 29.6,
29.4, 29.3, 29.2, 25.4, 22.7, 14.1. Anal. (C H N O ).
2
3
33
3
4
4-Hydroxy-1-methyl-N’-octanoil-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
(
29). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate
(
0.30 g, 1.2 mmol), octanoylhydrazide (0.19 g, 1.2 mmol). White solid (330 mg, 99%).
M.p. = 157-158 ºC. HPLC (10 min run): Purity > 99%, R.t. = 5.53 min. ESI MS (m/z):
1
3
1
8
1
60 [M+H]). H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.24 (s, 1H), 11.99 (d, J = 3.5 Hz,
H), 10.75 (d, J = 3.4 Hz, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.92-7.74 (m, 1H), 7.64 (d, J =
.7 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 3.64 (t, J = 4.9 Hz, 3H), 2.22 (t, J = 7.3 Hz, 2H),
13
.79-1.34 (m, 2H), 1.25 (s, 8H), 0.85 (t, J=6.1 Hz, 3H). C NMR (75 MHz, DMSO-d
6
) δ
(
ppm): 161.5, 171.0, 169.9, 167.6, 140.2, 134.9, 124.8, 123.1, 115.9, 115.1, 96.1, 33.3,
3
1.5, 29.6, 28.9, 28.7, 25.3, 22.4, 14.3. Anal. (C19
-Hydroxy-1-methyl-N’-palmitoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-
carboxylate(0.30 g, 1.2 mmol), palmitoylhydrazide (0.33 mg, 1.2 mmol). White solid
H
25
N
3
O
4
).
4
(
30).
(
532 mg, 94%). M.p. = 149-150 ºC. HPLC (10 min run): Purity 95%, R.t. = 7.24 min.
1
ESI MS (m/z): 472 [M+H]. H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.25 (s, 1H), 11.99
(
s, 1H), 10.76 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.83 (m, 1H), 7.64 (d, J = 8.3 Hz, 1H),
7
.39 (m, 1H), 3.63 (m, J = 2.7 Hz, 3H), 2.21 (m, 2H), 1.52 (s, 2H), 1.20 (s, 24H), 0.81
).
N’-Benzoyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
31). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate
(0.50 g, 2.02 mmol), phenylhydrazide (0.27 g, 2.02 mmol). White solid (585 mg, 86%).
(
d, J = 3.0 Hz, 3H). Anal. (C27
H
41
N
3
O
4
(
2
6
M.p. = 138-139 ºC (lit. 137-139 ºC).
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8
9
N’-Acetyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (32).
Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.40 g,
1.62 mmol), methylhydrazide (0.12 g, 1.62 mmol). White solid (524 mg, 89%). M.p. =
252-253 ºC. HPLC (10 min run): Purity > 99%, R.t. = 3.83 min. ESI MS (m/z): 276
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
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
[M+H]. H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.21 (s, 1H), 11.99 (d, J = 4.1 Hz, 1H),
10.79 (d, J = 4.1 Hz, 1H), 8.08 (dd, J = 8.0, 1.3 Hz, 1H), 7.82 (t, J = 7.9 Hz, 1H), 7.63
1
3
(d, J = 8.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H) 3.63 (s, 3H), 1.96 (s, 3H). C NMR (75
MHz, DMSO-d ) δ (ppm): 161.5, 171.0, 167.5, 166.8, 140.2, 135.0, 124.8, 123.1,
15.9, 115.1, 96.0, 29.6, 20.7. Anal. (C H N O ).
6
1
13
13
3
4
4
-Hydroxy-1-methyl-2-oxo-N’-(2-phenylacetyl)-1,2-dihydroquinoline-3-
carbohydrazide (33). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-
-carboxylate (0.30 g, 1.2 mmol), benzylhydrazide (0.18 g, 1.2 mmol). White solid (273
3
mg, 80%). M.p. = 227-228 ºC. HPLC (10 min run): Purity > 99%, R.t. = 4.90 min. ESI
1
MS (m/z): 352 [M+H]. H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.13 (s, 1H), 12.07 (d,
J = 4.1 Hz, 1H), 11.13 (d, J = 4.2 Hz, 1H), 8.07 (dd, J = 8.0, 1.2 Hz, 1H), 7.81 (t, J = 7.8
13
Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.42-7.19 (m, 6H), 3.61 (s, 3H), 3.60 (s, 2H).
NMR (75 MHz, DMSO-d
29.1, 128.3, 126.6, 124.4, 122.7, 115.5, 114.6, 95.6, 39.7, 29.2. Anal. (C H N O ).
C
6
) δ (ppm): 161.1, 170.6, 167.3, 167.1, 139.8, 135.4, 134.6,
1
19
17
3
4
N’-2-(Indol-3-yl)propanoyl-4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-3-
carbohydrazide (34). Reagents: ethyl 4-hydroxy-1-methyl-2-oxo-1,2-dihydroquinoline-
-carboxylate (0.20 g, 0.81 mmol), 3-(indol-3-yl)-propanohydrazide (0.16 g, 0.81
3
mmol). White solid (0.90 mg, 45%). M.p. = 235-236 ºC. HPLC (10 min run): Purity 96%,
1
R.t. = 4.72 min. ESI MS (m/z): 405 [M+H]. H NMR (300 MHz, DMSO-d ) δ (ppm):
6
1
6.22 (s, 1H), 12.08 (d, J = 4.1 Hz, 1H), 10.92 (d, J = 4.1 Hz, 1H), 10.79 (s, 1H), 8.09
(
d, J = 7.9 Hz, 1H), 7.82 (t, J = 7.8 Hz, 1H), 7.63 (d, J = 8.6 Hz, 1H), 7.56 (d, J = 7.7
Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.16 (s, 1H), 7.06 (t, J = 7.4
Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 3.64 (s, 3H), 2.99 (t, J = 7.5 Hz, 2H), 2.63 (t, J = 7.6
1
3
Hz, 2H). C NMR (75 MHz, DMSO-d ) δ (ppm): 161.1, 170.6, 169.1, 166.9, 139.8,
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
136.2, 134.5, 126.9, 124.4, 122.7, 122.3, 120.9, 118.3, 118.1, 115.5, 114.7, 113.3,
111.3, 95.6, 33.8, 29.2, 20.7. Anal. (C22
H
20
N
4
4
O ).
1-Ethyl-4-hydroxy-N’-octanoil-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (35).
Reagents: ethyl 1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (0.10 g,
0.38 mmol), octanoylhydrazide (0.06 g, 0.38 mmol). White solid (100 mg, 99%). M.p. =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
146-147 ºC. HPLC (10 min run): Purity > 99%, R.t. = 5.80 min. ESI MS (m/z): 374
1
[M+H]. H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.24 (s, 1H), 12.03 (d, J = 3.9 Hz, 1H),
1
8
1
d
3
1
0.79 (d, J = 3.9 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.84 (t, J = 7.1 Hz, 1H), 7.70 (d, J =
.6 Hz, 1H), 7.40 (t, J = 7.3 Hz, 1H), 4.32 (q, J = 6.8 Hz, 2H), 2.24 (t, J = 7.1 Hz, 2H),
1
3
.56 (m, 2H), 1.33–1.21 (m, 11H), 0.86 (t, J = 5.9 Hz, 3H). C NMR (75 MHz, DMSO-
) δ (ppm): 160.7, 170.6, 169.5, 167.1, 138.7, 134.7, 124.6, 122.6, 115.2, 114.9, 95.6,
6.9, 32.8, 31.1, 28.4, 28.3, 25.0, 22.0, 13.9, 12.7. Anal. (C H N O ).
6
2
0
27
3
4
-Ethyl-N’-2-(indol-3-yl)propanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-
carbohydrazide (36). Reagents: ethyl 1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-
carboxylate (0.10 g, 0.38 mmol), 3-(indol-3-il)-propanohydrazide (0.08 g, 0.38 mmol).
White solid (65 mg, 72%). M.p. = 258-259 ºC. HPLC (10 min run): Purity 97%, R.t. =
1
3
.72 min. ESI MS (m/z): 419 [M+H]. H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.23 (s,
1H), 12.13 (d, J = 4.0 Hz, 1H), 10.96 (d, J = 3.9 Hz, 1H), 10.82 (s, 1H), 8.12 (d, J = 8.0
Hz, 1H), 7.84 (t, J = 7.8 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.57 (d, J = 7.7 Hz, 1H),
.53-7.29 (m, 2H), 7.17 (s, 1H), 7.08 (t, J = 7.1 Hz, 1H), 6.99 (t, J = 7.1 Hz, 1H), 4.33
q, J = 6.6 Hz, 2H), 3.01 (t, J = 7.5 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 1.24 (t, J = 6.9 Hz,
7
(
1
3
3
1
9
1
3
H). C NMR (75 MHz, DMSO-d
35.3, 127.8, 125.4, 123.3, 123.0, 121.6, 119.0, 118.9, 115.9, 115.8, 114.2, 112.0,
6.5, 37.6, 34.7, 21.4, 13.4. Anal. (C23 ).
6
) δ (ppm): 161.6, 171.4, 170.0, 167.8, 139.6, 137.0,
H
22
N
4
O
4
-Ethyl-N’-2-(4-hydroxyphenyl)propanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-
-carbohydrazide (37). Reagents: ethyl 1-ethyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-
3-carboxylate (0.10 g, 0.38 mmol), 3-(4-hydroxyphenyl)propanhydrazide (0.08 g, 0.38
mmol). White solid (70 mg, 73%). M.p. = 165-166 ºC. HPLC (10 min run): Purity 99%,
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R.t. = 4.45 min. ESI MS (m/z): 396 [M+H]. H NMR (300 MHz, DMSO-d
6
) δ (ppm):
16.21 (s, 1H), 12.09 (d, J = 4.2 Hz, 1H), 10.89 (d, J = 4.2 Hz, 1H), 9.18 (s, 1H), 8.12 (d,
J = 8.0 Hz, 1H), 7.84 (t, J = 7.1 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.40 (t, J = 7.5 Hz,
1H), 7.03 (d, J = 8.3 Hz, 2H), 6.68 (d, J = 8.4 Hz, 2H), 4.33 (q, J = 6.9 Hz, 2H), 2.77 (t,
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
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
3
J = 7.5 Hz, 2H), 2.50 (t, J = 7.5 Hz, 2H), 1.24 (t, J = 6.9 Hz, 3H). C NMR (75 MHz,
DMSO-d ) δ (ppm): 161.1, 171.0, 169.1, 167.3, 155.9, 139.2, 135.1, 131.3, 129.5,
6
125.1, 123.0, 115.6, 115.4, 115.3, 96.0, 37.3, 35.4, 30.3, 13.1. Anal. (C21
H
21
N
3
5
O ).
N’-Dodecanoyl-4-hydroxy-1-isobutyl-2-oxo-1,2-dihydroquinoline-3-
carbohydrazide (38). Reagents: ethyl carboxylate 12, (0.2 g, 0.66 mmol),
dodecanoylhydrazide (0.17 g, 0.78 mmol). White solid (50 mg, 21%). M.p. = 112-113
1
ºC. HPLC: Purity 97%, R.t. = 6.6 min. ESI MS (m/z): 458 [M+H]+. H NMR (300 MHz,
DMSO-d ) δ (ppm): 16.32 (s, 1H), 12.05 (d, J = 4.1 Hz, 1H), 10.79 (d, J = 4.1 Hz, 1H),
6
8
1
0
1
2
.12 (d, J = 1.6 Hz, 1H), 7.9-7.75 (m, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.35 (t, J = 7.6 Hz,
H), 4.18 (d, J = 7.5 Hz, 2H), 2.30-2.05 (m, 3H), 1.52-1.35 (m, 2H), 1.25-1.05 (m, 16H),
13
.95-0.80 (m, 9H); C NMR (75 MHz, DMSO-d ) δ (ppm): 170.6, 169.4, 167.0, 161.5,
6
39.4, 134.5, 124.6, 122.6, 115.9, 114.9, 95.5, 47.85, 31.3, 29.0, 28.9, 28.7, 28.6, 28.5,
6.7, 24.9, 22.0, 19.8, 13.9. Anal. (C26
H
39
N
3
4
O ).
N’-Dodecanoyl-4-hydroxy-1-isoprenyl-2-oxo-1,2-dihydroquinoline-3-
carbohydrazide (39). Reagents: ethyl carboxylate 13, (0.2 g, 0.66 mmol),
dodecanoylhydrazide (0.16 g, 0.73 mmol). White solid (125 mg, 40%). M.p. = 107-108
+
1
ºC. HPLC: Purity > 99%, R.t. = 4.9 min. ESI MS (m/z): 470 [M+2H] . H NMR (300
MHz, DMSO-d ) δ (ppm): 16.28 (s, 1H), 11.97 (d, J = 3.9 Hz, 1H), 10.77 (d, J = 3.9 Hz,
H), 8.11 (dd, J = 7.9, 1.5 Hz, 1H), 7.83 (td, J = 9.18, 7.2, 1.6 Hz, 1H), 7.51 (d, J = 8.6
6
1
Hz, 1H), 7.39 (t, J = 8.6 Hz, 1H), 5.51-5.05 (m, 1H), 4.95-4.85 (m, 2H), 2.23 (t, J = 7.3
Hz, 2H), 1.85 (s, 3H), 1.67 (s, 3H), 1.6-1.40 (m, 2H), 1.35-1.1 (m, 16H), 0.84 (t, J = 6.5
1
3
Hz, 3H); C NMR (75 MHz, DMSO-d ) δ (ppm): 170.7, 169.6, 167.3, 160.9, 139.0,
6
135.8, 134.7, 124.7, 122.7, 119.2, 115.5, 114.9, 95.7, 32.9, 31.3, 29.0, 28.9, 28.8, 28.7,
28.5, 25.3, 24.9, 22.1, 18.2, 13.9. Anal. (C27
H
39
N
3
O ).
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4-Hydroxy-1-isoprenyl-N’-octanoil-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
(40). Reagents: ethyl carboxylate 13 (0.2 g, 0.66 mmol), octanoylhydrazide (0.12 g,
0.73 mmol). White solid (125 mg, 22%). M.p. = 114-115 ºC. HPLC: Purity > 99%, R.t. =
1
13.7 min. ESI MS (m/z): 414 [M + H]+. H NMR (300 MHz, CDCl
3
) δ (ppm): 15.37 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1H), 12.38 (d, J = 5.3 Hz, 1H), 8.67-8.55 (m, 1H), 8.06 (d, J = 7.9 Hz, 1H), 7.57 (t, J =
7.9 Hz, 1H), 7.22-7.14 (m, 2H), 5.1-5.02 (m, 1H), 4.81-4.76 (m, 2H), 2.26 (t, J = 7.5 Hz,
1
3
2H), 1.80 (s, 3H), 1.7-1.52 (m, 5H), 1.38-1.1 (m, 8H), 0.79 (t, J = 6.4 Hz, 3H); C NMR
75 MHz, CDCl ) δ (ppm): 170.1, 169.6, 166.9, 161.7, 139.7, 136.5, 134.09, 125.1,
22.4, 119.1, 115.8, 114.9, 96.4, 40.9, 34.4, 31.8, 29.3, 29.1, 25.7, 26.6, 22.7, 18.5,
4.2. Anal. (C23 ).
-(Cyclopropylmethyl)-N’-dodecanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-
(
3
1
1
1
H
31
N
3
O
4
carbohydrazide (41). Following the general synthesis procedure for compounds 13-
5, ethyl 1-(cyclopropylmethyl)-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate
15) was synthesized and used here without further purification. Diethyl malonate (1.1
2
(
g, 6.8 mmol) was added dropwise to a sodium hydride (0.007 g, 2.9 mmol) solution in
dry DMF (15mL) under argon atmosphere. The reaction was stirred for 30 min at room
temperature. Then the mixture was added to a round bottom flask containing the N-
cyclopropylmethylisatoic anhydride 4 (0.3 g, 1.3 mmol) dissolved in DMF (15 mL) under
argon atmosphere. The mixture was heated at 50 ºC during 5 hours. To the mixture
dichloromethane (30 mL), water (50 mL) and HCL (1N) were added to reach pH=5.
The organic layer was isolated and washed with brine (30 mL). The organic phase was
dried over magnesium sulfate, filtered and the solvent was evaporated. The solid (0.13
g, 0.45 mmol) was used in the next step without further purification after reaction with
dodecanoylhydrazide (0.15 g, 0.68 mmol) to yield hydrazide 41. White solid (90 mg,
49%). M.p. = 121-122 ºC. HPLC: Purity > 99%, R.t. = 15.8 min. ESI MS (m/z): 456
1
[
M+H]+. H NMR (300 MHz, DMSO-d ) δ (ppm): 16.30 (s, 1H), 12.01 (d, J = 4.1 Hz,
6
1H), 10.76 (d, J = 4.1 Hz, 1H), 8.12 (d, J = 7.9 Hz, 1H), 7.80-7.61 (m, 2H), 7.35 (t, J =
7.6 Hz, 1H), 4.23 (d, J = 6.9 Hz, 2H), 2.23 (t, J = 7.6 Hz, 2H), 1.6-1.42 (m, 2H), 1.32-
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1
3
1.17 (m, 16H), 0.84 (t, J = 6.9 Hz, 3H), 0.52-0.42 (m, 5H); C NMR (75 MHz, DMSO-
) δ (ppm): 170.7, 169.5, 167.0, 161.3, 139.3, 134.6, 124.6, 122.7, 115.8, 114.9, 95.6,
45.2, 32.9, 31.3, 29.0, 28.9, 28.8, 28.7, 28.5, 25.0, 22.1, 13.9, 9.8, 3.7. Anal.
).
d
6
(C26
H
37
N
3
O
4
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
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1-Benzyl-N’-dodecanoyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
(42). Reagents: ethyl carboxylate 16, (0.04 g, 0.12 mmol), dodecanoylhydrazide (0.033
g, 0.15 mmol). White solid (45 mg, 75%). M.p. = 167-168 ºC. HPLC: Purity > 99%, R.t.
1
=
9.6 min. ESI MS (m/z): 516 [M+H]+. H NMR (300 MHz, DMSO-d
6
) δ (ppm): 15.63
(
s, 1H), 12.13 (d, J = 4.1 Hz, 1H), 8.25-8.08 (m, 2H), 7.3-7.1 (m, 7H), 5.46 (s, 2H),
1
3
2
.35-2.10 (m, 2H), 1.75-1.50 (m, 2H), 1.5-1.1 (m, 16H), 0.9-0.75 (m, 3H); C NMR (75
) δ (ppm): 170.5, 168.2, 165.8, 161.1, 138.7, 134.9, 133.2, 127.9,
26.4, 125.4, 121.6, 114.9, 114.2, 95.1, 44.9, 33.4, 30.9, 28.6, 28.4, 28.3, 28.2, 24.4,
1.7, 13.1. Anal. (C29 ).
MHz, DMSO-d
6
1
2
H N
37 3
O
4
N’-Dodecanoyl-6-fluoro-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
43). Reagents: ethyl 6-fluoro-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (20
(
mg, 0.07 mmol), dodecanoylhydrazide (17 mg, 0.07 mmol). White solid (15 mg, 93%).
M.p. = 178-179 ºC. HPLC (10 min run): Purity 95%, R.t. = 2.35 min. ESI MS (m/z): 419
+
1
[
M+H] . H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.16 (s, 1H), 12.11 (s, 1H), 11.94 (d,
J = 3.9 Hz, 1H), 10.72 (s, 1H), 7.72-7.56 (m, 2H), 7.42 (m, 1H), 2.21 (t, J = 7.3 Hz, 2H),
1.52 (m, 2H), 1.23 (s, 16H), 0.83 (t, J = 6.7 Hz, 3H). Anal. (C22
H30FN
3
O
4
).
6-Fluoro-4-hydroxy-N’-octanoyl-2-oxo-1,2-dihydroquinoline-3-carbohydrazide
(
44). Reagents: ethyl 6-fluoro-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (20
mg, 0.08 mmol), octanoylhydrazide (13 mg, 0.08 mmol). White solid (24 mg, 83%).
M.p. = 290-291 ºC. HPLC (10 min run): Purity 98%, R.t. = 2.37 min. ESI MS (m/z): 364
+
1
[
M+H] . H NMR (300 MHz, DMSO-d
6
) δ (ppm): 16.15 (s, 1H), 11.95 (d, J = 3.6 Hz,
1H), 12.13 (s, 1H), 10.75 (d, J = 3.7 Hz, 1H), 7.66 (m, 2H), 7.43 (m, 1H), 2.23 (t, J = 7.3
1
3
Hz, 2H), 1.55 (s, 2H), 1.40 (m, 8H), 0.86 (d, J = 6.6 Hz, 3H); C NMR (75 MHz,
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