Discovery of novel 2,3,5-trisubstituted pyridine analogs as potent inhibitors of IL-1β via modulation of the p38 MAPK signaling pathway

Article abstract of DOI:10.1016/j.ejmech.2021.113620

Interleukin-1β is a central mediator of innate immune responses and inflammation. It plays a key role in a wide variety of pathologies, ranging from autoinflammatory diseases to metabolic syndrome and malignant tumors. It is well established that its inhibition results in a rapid and sustained reduction in disease severity, underlining the importance of having a repertoire of drugs of this class. At present, there are only three interleukin-1β blockers approved in the clinic. All of them are biologics, requiring parenteral administration and resulting in expensive treatments. In an exercise to identify small molecule allosteric inhibitors of MAP kinases, we discovered a series of compounds that block IL-1β release produced as a consequence of a stimulus involved in triggering an inflammatory response. The present study reports the hit-to-lead optimization process that permitted the identification of the compound 13b (AIK3-305) an orally available, potent and selective inhibitor of IL-1β. Furthermore, the study also reports the results of an in vivo efficacy study of 13b in a LPS endotoxic shock model in male BALB/c mice, where IL-1β inhibition is monitored in different tissues.

Full text of DOI:10.1016/j.ejmech.2021.113620

European Journal of Medicinal Chemistry 223 (2021) 113620
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel 2,3,5-trisubstituted pyridine analogs as potent
inhibitors of IL-1
b
via modulation of the p38 MAPK signaling pathway
,
Esther Carrasco a, Patricia Gomez-Gutierrez a c, Pedro M. Campos a, Miguel Vega a,
,
*
Angel Messeguer b, Juan J. Perez c
a Allinky Biopharma, Campus de Cantoblanco, Faraday 7, 28049, Madrid, Spain
b IQAC CSIC, Institute of Advanced Chemistry of Catalonia, Dept. Biological Chemistry, Jordi Girona 18-26, 08034, Barcelona, Spain
c Dept. of Chemical Engineering, Universitat Politecnica de Catalunya, 08028, Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Interleukin-1b is a central mediator of innate immune responses and inflammation. It plays a key role in
Received 12 February 2021
Received in revised form
8 April 2021
Accepted 1 June 2021
Available online 12 June 2021
a wide variety of pathologies, ranging from autoinflammatory diseases to metabolic syndrome and
malignant tumors. It is well established that its inhibition results in a rapid and sustained reduction in
disease severity, underlining the importance of having a repertoire of drugs of this class. At present, there
are only three interleukin-1
parenteral administration and resulting in expensive treatments. In an exercise to identify small mole-
cule allosteric inhibitors of MAP kinases, we discovered a series of compounds that block IL-1 release
b blockers approved in the clinic. All of them are biologics, requiring
b
Keywords:
Small molecule IL-1b blockers
MAP kinase Allosteric inhibitors
Hit-to-lead optimization
p38a MAPK allosteric Inhibitors
produced as a consequence of a stimulus involved in triggering an inflammatory response. The present
study reports the hit-to-lead optimization process that permitted the identification of the compound 13b
(AIK3-305) an orally available, potent and selective inhibitor of IL-1
the results of an in vivo efficacy study of 13b in a LPS endotoxic shock model in male BALB/c mice, where
IL-1 inhibition is monitored in different tissues.
b. Furthermore, the study also reports
Anti-inflammatory agents
b
© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-
NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
inflammatory diseases pathogenesis [6]. Accordingly, IL-1b inhibi-
tion has been focus of attention for the development of novel
therapeutic agents, with three biologics presently available for
clinical use. These compounds demonstrated that monotherapy
blocking IL-1R activity results in a rapid and sustained reduction in
disease severity [7,8]. However, despite the success of these drugs,
they exhibit drawbacks including the potential for development of
resistance, the requirement of parenteral administration, the lack of
efficacy against skeletal lesions and their high cost. These features
encourage the development of novel therapeutic agents that use
different mechanisms of action [9].
Interleukin-1b (IL-1b) is a central mediator of innate immune
responses and inflammation, released by diverse hematopoietic
cells in response to infection, injury or tissue stress [1e3]. Its action
is mediated through the IL-1 receptor (IL-1R1) to defend the host
against pathogens, inducing the expression of adhesion factors on
endothelial cells, resulting in immune cells migration to the site of
infection and provoking inflammation. IL-1
only produced on demand, requiring caspase-1 for its processing
[4]. Interestingly, IL-1R is also activated by the IL-1 isoform that
b Action is systemic and
a
elicits the same pattern of responses but in different cell types,
being an important driver of sterile inflammation following tissue
The mitogen-activated protein kinase (MAPK) p38
in the production of Pro-IL-1 through the p38
signaling pathway [10], so that its inhibition is expected to inhibit
IL-1 release. In addition, IL-1 release is also modulated by a post-
transcriptional mechanism through the p38 /MK2 pathway [11].
This underlines the key role played by p38 MAPKs in IL-1 pro-
a
participates
b
a/MK3/CREB
injury and cell damage. IL-1
a action is local, being its precursor
constitutively present in a variety of cell types, largely membrane
bound stored [5]. Dysfunctional monocyte-macrophage cells
b
b
a
induce excessive IL-1
b
production that is associated with auto-
a
b
duction, making it an attractive target for therapeutic intervention
for the treatment of auto-inflammatory diseases. Most of the small
molecule kinase inhibitors reported in the literature are ATP
competitive inhibitors [12,13]. They can be classified as type I and II,
* Corresponding author.
E-mail address: juan.jesus.perez@upc.edu (J.J. Perez).
https://doi.org/10.1016/j.ejmech.2021.113620
0223-5234/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
depending on the kinase conformation they bind to, either DFG-in
or DFG-out, respectively [14]. However, these inhibitors typically
exhibit a non-selective kinase profile due to the high conservation
of the ATP site across the kinome. In addition to competitive in-
hibitors, allosteric inhibitors have also been described. These
compounds exploit binding sites and regulatory mechanisms that
are unique to a particular kinase. Thus, type III inhibitors are mol-
MAPK as shown in Fig. 1, suggests that one of the nitrogen atoms of
the pyrazine ring is not necessary for binding. Accordingly, the
substitution of the pyrazine ring by a pyridine moiety was tested
with a successful retention of activity. Then, substitutions of the
oxadiazole by other five-member rings with a lower number of
hydrogen acceptors were tried with poor success. Thus for example,
the inhibitory efficacy of compound 3 (Scheme 1) drops the activity
ecules that bind to sites located in the proximity of the
a
C-helix and
to the low micromolar (IC50 ¼ 2.2
mM). In a further step, we
considered breaking the planarity of the chemical structure of these
compounds that may also contribute to enhance cohesion forces in
the ATP site, whereas type IV inhibitors are molecules that bind to
sites distant from the ATP binding site [15]. These two types of
inhibitors represent attractive compounds, since they exploit dif-
ferential mechanisms of kinase regulation with higher chances to
be selective and consequently, better suited for therapeutic inter-
vention [16]. Correspondingly, we aimed at discovering a series of
the solid form through
p-p stacking interactions. Accordingly, we
proceeded to replace the [1,2,5]oxadiazolo[3,4-b]pyrazine scaffold
and designed a series of pyridines with linear substituents in po-
sition 5 that hold a proton acceptor center in order to fulfil the
pharmacophore point P3. After a thorough investigation, we
identified the 3-(N,N-dimethylsulfonamido)pyridine as a suitable
core that was subsequently subject of an optimization process. The
results of such a process are reported in the present work.
small molecules, targeting the A-loop regulatory site of p38
a
MAPKs recently reported by us [17,18]. As a result of the hit-to-lead
optimization process, we disclose in the present work a series of
novel 2,3,5-trisubstituted pyridine analogs that act as potent in-
hibitors of the IL-1
The A-loop regulatory site in addition to be a transient pocket of
p38 MAPK it is also an anchoring point for its substrate MK2.
Analysis of the crystallographic structure of the p38 /MK2 heter-
b
release.
Since our efforts were directed to improve the pharmacological
profile of this series of compounds preserving their capacity to
a
inhibit IL-1
b
release, optimization process was carried out using a
a
in vitro IL-1
b inhibition assay as end-point.
odimer (PDB ID: 2OZA) [19] permitted to define a pharmacophore
of the interaction between the segment 264e269 of MK2 bound to
the p38a A-loop regulatory site that was used to undertake a virtual
2. Results and discussion
screening study using the MOE lead-like database [20]. Fruit of this
study, we identified UPC-K-005 (1, Scheme 1), a hit that exhibits a
non-competitive weak kinase inhibitory profile and binder to the
A-loop regulatory site as supported by site-directed mutagenesis
studies [17]. In order to get a deeper insight into the functional role
of this novel site, compound 1 was subsequently used as starting
point in a molecular modeling guided study that led to the dis-
covery of a series of disubstituted [1,2,5]oxadiazolo[3,4-b]pyrazine
compounds [21]. These compounds were key to demonstrate that it
2.1. Lead optimization
As described above, starting from 2 (Scheme 1) and guided by its
prospective bound conformation to p38a MAPK (Fig. 1), we iden-
tified the N,N-dimethylsulfonamide substituent at C-5 of the pyri-
dine ring as that suitable for the synthesis of 2,3,5-trisubstituted
pyridine derivatives that preserving its inhibitory properties,
exhibited a better pharmacological profile. Specifically, several
substitutions in positions 2 and 3 were investigated guided by the
results from our previous study on the [1,2,5]oxadiazolo[3,4-b]
pyrazine analogs. First, the substituent in position 3 was optimized
maintaining the 4-(40-chlorophenoxy)piperidinyl moiety in posi-
tion 2, according to the results of our previous study [21]. Among
the diverse substitutions explored, Table 1 lists a selection of
diverse compounds synthesized along with their biological activity,
is possible to modulate p38
regulatory site, exhibiting a potent downstream inhibitory effect on
the IL-1 release in human monocyte-derived macrophages.
a MAPK activity through the A-loop
b
Among this series of molecules, compound 2 (Scheme 1) was one of
the most potent disclosed, with IC50 ¼ 28 nM. Unfortunately, this
series of compounds exhibit low solubility, which underlined the
need of exploring structural modifications to improve their phar-
macological profile. We hypothesized that the low solubility of the
disubstituted [1,2,5]oxadiazolo[3,4-b]pyrazine compounds was
due to specific structural features that enhance cohesion forces in
the solid form, making dissolution energetically more costly [22].
Bearing this hypothesis in mind, we designed different molec-
ular scaffolds with less hydrogen bond acceptors/donors preserving
ligand-receptor interactions. Specifically, inspection of the pro-
measured by their capacity to inhibit IL-1
b release in human
monocyte-derived macrophages. Inspection of Table 1 shows that
the 2,3-dihydrobenzofuran substitution renders good efficacy to
compounds directly attached to the pyridine ring or via either an
ether or amine bridge (11a, 12a, 13a). Moreover, substitutions on
the dihydrobenzofuran bicycle do not affect much the activity of
the compounds (12b, 12c). Unfortunately, these dihydrobenzofuran
analogs exhibit an inhibitory action on diverse forms of the cyto-
chrome P450 family and consequently, were disregarded.
spective bound conformation of compound 2 (Scheme 1) into p38
a
Scheme 1. Chemical structures of diverse initial p38a MAPK inhibitors.
2

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
Scheme 2. Synthesis of compound 3.
2.3. Synthesis of N,N-dimethylsulfonamidopyridine analogs
The diverse N,N-dimethylsulfonamidopyridine derivatives
were synthesized from the 5,6-dichloro-N,N-dimethylpyridine-3-
sulfonamide, following the procedure described in Scheme 3.
Initially, treatment of 8 with iodomethane in the presence of
potassium carbonate afforded the dimethylated derivative 9 in
95% yield [25]. Intermediate compounds 10a-f were prepared in
good to excellent yields by a procedure analog as that described
above for compound 7 but using compound 9 as starting mate-
rial. Compounds 11a and 11b were prepared in moderate yields
by condensation of 10a or 10b with the corresponding arylbor-
onic acid in the presence of Pd(PPh3)4 [26].
For the preparation of compounds 12a,12b and 12e, a mixture of
the corresponding intermediate 10a, the appropriate aniline and
BrettPhos-precatalyst (method A in the experimental section), was
allowed to react in the presence of LiHMDS in a sealed screw-cap
test-tube [27]. The expected adducts were isolated in 6e23%
yields. Alternatively (method B in the experimental part), by
introducing some modifications to the conditions reported by
Buchwald for the case of deactivated aromatic amines [28], reaction
of 10a with the corresponding aniline in the presence of potassium
carbonate under microwave activation yielded 12d and 12f in 70%
and 54% yields, respectively. The case of compound 12c required
additional manipulations since the aniline contained a primary
alcohol as additional functional group (Scheme 4). Thus, this
starting material should be protected by silylation to give 14 [29],
which was condensed with 10a under the conditions of method B
to yield the protected adduct 15. Further deprotection afforded
pure alcohol 12c in 8% overall yield [30].
Finally, following the general methodology reported by Rarig
et al. [31], compounds 13 were synthesized in low to moderate
yields by reaction of the corresponding intermediate 10 with the
appropriate phenol in the presence of cesium carbonate and so-
dium hydride under microwave activation. Once again, for the case
of primary alcohol adduct 13c it was needed to prepare the corre-
sponding phenol reagent bearing a protected primary alcohol
group as a silylated ether (Scheme 5). Thus, conversion of 4-
hydroxyphenylacetic acid into its methyl ester [32] followed to
reduction to the primary alcohol [33] and further protection [30]
gave the expected reagent for the condensation with 10a to give
directly deprotected adduct 13c.
Fig. 1. Prospective bound structure of compound 2 (orange) to the A-loop regulatory
site in p38a MAPK. Circles represent the positions of the pharmacophore in a color
code: light blue are proton-accepting centers whereas green represents hydrophobic/
aromatic centers.
Replacement of the dihydrofuran ring by an ether or a ketone at C-4
of the phenyl ring were detrimental for the efficacy, except in the
case of an alkyl ether substituent when the ring was connected via
an oxygen bridge to the pyridine moiety (13b). This compound
exhibits an inhibitory capacity of cytochrome P450 < 30
mM.
Accordingly, the 40-ethoxyphenoxy substituent was selected for
exhibiting the best results in terms of activity and solubility for
position 3.
2.2. Chemical synthesis of the compounds
Compound 3 as well as the different analogs listed in Tables 1
and
2
were synthesized from the corresponding 2,3-
dichloropyridine derivatives through Suzuki cross-coupling re-
actions or nucleophilic substitution reactions with the corre-
sponding amines or phenols, using the protocols shown in Schemes
2 and 3.
Scheme 2 displays the synthesis of compound 3. The diverse
analogs bearing a bicycle scaffold fused to the pyridine ring were
synthesized from the reaction of commercially available 2,3-
dichloropyridine derivative 6 with 5-(40-chloro phenoxy)piperi-
dine hydrochloride in the presence of triethylamine under micro-
wave irradiation to give intermediate 7 [23] and subsequent
treatment with 5-amino-2,3-dihydrobenzo[b]furan in the presence
of BrettPhos-precatalyst under the usual conditions of the Buch-
wald replacement of chloroaryl derivatives by aromatic amines
[24], to yield compound 3 in 55% overall yield.
2.4. In vitro studies
In vitro studies show that 13b (Scheme 6) is a potent selective
inhibitor of IL-1b secretion. The dose-response inhibition curve of
IL-1 secretion in human monocyte-derived macrophages (Fig. 2a)
b
shows that the compound elicits a potent inhibitory action with a
IC50 ¼ 28 nM. Dexamethaxone dose-response curve was also
measured in the same conditions, as positive control. As can be
seen, 13b being more potent, exhibits the same efficacy as dex-
amethaxone on the inhibition of IL-1
b release. Moreover, the
3

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
Table 1
Table 2
Selected list of 2,3,5-trisubstituted pyridine derivatives used for the optimization of
the substituent at C-2. Their inhibitory capacity of IL-1 release in human monocyte-
Selected list of 2,3,5-trisubstituted pyridine derivatives used for the optimization of
the substituent at C-3. Their IL-1
b
release inhibitory capacity in human monocyte-
b
derived macrophages is also listed. Individual IC50 were computed using 10 points
at different concentrations (N ¼ 2). Standard error of the means (Z-score) is also
reported.
derived macrophages is also listed. Individual IC50 were computed using 10 points at
different concentrations (N ¼ 2). Standard error of the means (Z-score) is also re-
ported.
Compound
X
R
IC50
Z-score
0.8
Compound
X
Y
X1
X2
R
IC50
Z-score
11a
e
27 nM
13b
13d
13e
13f
13g
13h
C
N
C
N
C
C
O
C
O
C
O
O
C
C
C
C
N
C
C
C
N
N
N
N
Cl
Cl
Cl
Cl
Cl
28 nM
>10
4 mM
0.6
0.7
0.9
0.7
0.9
0.7
m
M
>10
1.1
337 nM
m
M
M
11b
12a
e
>10 mM
m
NH
29 nM
0.8
Moreover, while our compound elicits a strong inhibition of IL-1
secretion, it also exerts an increase of TNF- secretion, possibly to
compensate IL-1 inhibition, due to the cross-talk between MAPKs
[34]. Finally, the expected increase of the anti-inflammatory cyto-
b
12b
12c
12d
NH
NH
NH
121 nM
12.8 nM
0.8
0.8
a
b
kine IL-10 secretion could be due to its modulatory effect on TNF-
a
increased secretion.
Surface Plasmon Resonance (SPR) was utilized to characterize
the biophysical interactions between 13b and p38 MAPK. SPR
a
>10
>10
m
m
M
M
Binding sensograms measured at five different concentrations
(Fig. 3a) show a steady state achieved within 400 s. Inspection of
Fig. 3a suggests that while the association binding rate exhibits an
average value (kon ¼ 2.08 ꢀ 104 Mꢁ1sꢁ1), the dissociation process is
slow (koff ¼ 2.48 ꢀ 10ꢁ4sꢁ1). Steady-state affinity analysis (Fig. 3b)
indicates that 13b has a KD of 12 nM, which is consistent with the
12e
12f
NH
NH
IC50
macrophages.
¼
28 nM determined on human monocyte-derived
>10
m
M
2.5. Pharmacokinetic studies
Fig. 4 shows the plasma concentration of 13b vs time after a
single oral administration of 65 mg/kg, dissolved in PEG400/Kolli-
phor HS 15 (70/30) v/v to 8 weeks old female Wistar rats. Inspec-
tion of Fig. 4 shows that the compound is absorbed slowly after a
single dose oral administration with a Tmax of 1.0 h. After reaching
13a
O
28 nM
28 nM
0.7
13b
13c
O
O
0.6
0.8
T
max, the compound shows a two-compartmental elimination with
first-order elimination kinetics in the terminal phase. According to
this model, the compound exhibits a long distribution half-life time
of 2.1 h with an elimination half-life time of 9 h.
>10 mM
2.6. In vivo efficacy of 13b in IL-1b secretion inhibition
The efficacy of 13b in the inhibition of IL-1
tested in vivo. Specifically, we tested the efficacy of the compound
to inhibit IL-1 secretion in a LPS endotoxic shock model in male
BALB/c mice. Levels of IL-1 were measured in plasma as well as
levels of IL- m-RNA expression in diverse peripheral tissues
including brain, liver and lungs (Fig. 5). Fig. 5a shows the concen-
tration of IL-1 in plasma. As can be seen, levels of IL-1 secreted
b secretion was also
compound selectively inhibits IL-1b release, as shown in an assay of
a panel of cytokines on lipopolysaccharide (LPS) stimulated human
monocyte-derived macrophages assay (Fig. 2b). Specifically, orange
bars represent the concentration of diverse cytokines in the lysate
of human monocyte-derived macrophages after a LPS stimulation,
normalized to 100%, whereas grey bars represent the correspond-
b
b
b
b
b
ing secretion in the presence of 13b at 1
mM. As shown, compound
increased 3-fold after the LPS challenge and 13b was capable to
reduce these levels in a dose-response manner. Similarly, Fig. 5bed
13b exhibits a selective inhibition profile towards IL-1
b
secretion.
4

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
Scheme 3. General synthesis for N,N-dimethylsulfonamidopyridine derivatives 11e13.
Scheme 4. Synthesis of compound 12c.
shows the increase of IL-1
sues, respectively, as well as the inhibitory efficacy of 13b in
reducing these levels in a dose-response manner.
b
expression in brain, liver and lung tis-
biologics, MABp1 is a human monoclonal antibody specific for
human IL-1 that is presently in phase III clinical trials [38].
a
Up to our knowledge, there are no small molecule blockers of IL-
1 available in the market. A small molecule IL-1 secretion blocker
bears advantages in regard to biologics including oral administra-
tion, adequate half-life and effect duration, and the expected cost/
effect. There are few small molecule IL-1 blockers in preclinical or
phase I clinical trials recently disclosed. Thus for example, CDD-450
IL-1R inhibition has demonstrated efficacy in a sustained
reduction in disease severity for an ample spectrum of IL-1 medi-
ated pathologies including autoinflammatory, autoimmune, infec-
tious, ischemic diseases, metabolic syndrome and malignant
tumors [7,8]. Currently there are three biologics IL-1 inhibitors
available in the clinic including anakinra, a recombinant human
intrinsic IL-1 receptor antagonist [35], rilonacept, a soluble decoy
receptor [36] and canakinumab, a specific human monoclonal IgG1
is a small molecule IL-1 blocker acting as inhibitor of the p38a/MK2
pathway [39]. In addition, there are also diverse NLRP3 inflam-
masome inhibitors like MCC950 or IFM-2427 [40,41]. Related to
them, belnacasan (VX-765) is an oral potent and selective inhibitor
of NLRP3-activated caspase-1 [42]. Both types of compounds pre-
antibody targeting IL-1
and IL-1 , whereas canakinumab is a monoclonal antibody against
IL-1 . These drugs have been shown effective in treating a number
of IL-1 mediated diseases [7]. To complete the repertoire of
b [37]. Anakinra and rilonacept inhibit IL-1a
b
b
vent maturation of IL-1
b and IL-18. Our results point to 13b as a
promising small molecule candidate for the treatment IL-1
b
5

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
Scheme 5. Preparation of the protected primary alcohol as silyl ether used in the synthesis of 13c.
associated with increased levels of IL-1b in brain like Alzheimer's
disease [45]. Finally, in the Chronic Obstructive Pulmonary Disease
a progressive decline of lung function and airway remodeling are
due to chronic inflammatory responses, where IL-1
b plays an
important role [46].
3. Conclusions
Scheme 6. Chemical structure of lead compound 13b (AIK3-305).
In the search for small molecule inhibitors of IL-1 secretion, we
recently discovered an allosteric MAPK modulator site, named the
A-loop allosteric site and disclosed a hit binder. We carried out a
hit-to-lead optimization process guided by the prospective bound
conformation of the hit that led us to series of disubstituted [1,2,5]
oxadiazolo[3,4-b]pyrazine derivatives that were potent and selec-
dysregulated diseases. The fact that it does not inhibit IL-1
(Fig. 2b) could be considered an advantage, since IL-1 blockade
should preserve antimicrobial responses intact to prevent a risk of
opportunistic infections [43]. Moreover, demonstration of IL-1
reduction in diverse tissues (Fig. 5), suggests that 13b could be
useful for the treatment of diverse organ-specific diseases with an
a
b
tive inhibitors of IL-1b release. Nevertheless, they were discarded
due to their low solubility. A subsequent optimization process led
us to a series of 2,3,5-trisubstituted pyridine analogs described in
the present report, that preserve a potent and selective inhibition of
important inflammatory component. Thus, for example, an IL-1
b
inhibitor is expected to be useful in the treatment of Nonalcoholic
Steatohepatitis (NASH), a disease characterized by fat accumulation
and inflammation in the liver [44]. Similarly, the compound could
also be useful in the treatment of neurodegenerative diseases
IL-1
The pharmacological profile of the lead compound 13b was
analyzed. The compound exhibits a potent inhibitory effect of IL-1
b release with improved pharmacological properties.
b
Fig. 2. a) Dose-response curve of the efficacy of 13b (solid dots) and dexamethaxone (empty dots) on the release inhibition of IL-1b in human monocyte-derived macrophages
(N ¼ 2). b) Inhibitory action of 13b on a panel of diverse inflammatory cytokines. Orange bars represent the secretion of diverse cytokines (normalized to 100%) in human monocyte-
derived macrophages after a LPS stimulation, whereas grey bars represent the corresponding secretion in the presence of 13b at 1 mM.
6

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
Fig. 3. Surface Plasmon Resonance sensograms and saturation curve corresponding to the titration of 13b on p38
a
MAPK immobilized on a sensor chip. a) Sensograms obtained by
using different concentrations of ligand are displayed in different colors. b) Binding curves were fitted to a steady-state affinity model to get KD value of 12 n.
Taken together, present results point to 13b as a promising small
molecule candidate for the treatment IL-1b dysregulated diseases.
We are currently testing the compound in diverse animal models.
4. Experimental section
4.1. Chemistry
All reagents were obtained from commercial sources and used
without further purification. Reactions were controlled by TLC
(silica 60 F254, Merck, hexane-ethyl acetate mixtures) or by
analytical RP-HPLC with a Hewlett Packard Series 110 (UV detector
1315A) modular system using a reverse phase Kromasil 100C8
(15 ꢀ 0.46 cm, 5
mm) column, CH3CNeH2O mixtures containing
0.1% TFA (gradient method from 20% to 100% CH3CN in 20 min) at
1 mL/min were used as mobile phase and monitoring wavelength
was set at 220 nm. Product purification was performed by column
chromatography (silicagel 0.035e0.070 mm, 60 Å, hexane-ethyl
acetate mixtures) or by using a Isolera Biotage system (reverse
phase C18 column, CH3CNeH2O mixtures containing 0.1% TFA as
mobile phase). High resolution mass spectra were obtained by a LCT
premier HPLC/MS UPLC-TOF (Waters) with an electrospray ioni-
zation detector. 1H NMR and 13C NMR spectra were recorded in
CDCl3 by an Agilent Unity 400 spectrometer with an indirect
Fig. 4. Kinetics of 13b. Evolution of the concentration of 13b in plasma vs time after a
single 65 mg/kg dose by oral administration to 8 weeks old female Wistar rats (N ¼ 9).
secretion on monocyte-induced macrophages with an IC50 ¼ 28 nM.
Analysis of the inhibitory profile on a range of cytokines shows that
the compound does not interfere expression levels of IL-1
6, IL-8 or GM-CSF. In contrast, levels of TNF- are increased about a
20%, probably due to a compensatory effect of IL-1 inhibition due to
the cross-talk among MAPKs. In addition, levels of IL10 are also
increased to compensate the increase of TNF- secretion. Moreover,
in order to study its mode of action, SPR was utilized to characterize
the biophysical interactions between 13b and p38 MAPK. The re-
a
, IL-2, IL-
detection probe, and chemical shifts are given in d units referred to
a
TMS. Mass and NMR spectra profiles of final compounds are
depicted described in the supplementary material. Chloro[2-
(dicyclohexylphosphino)-3,6-dimethoxy-20,40,60-triisopropyl-1,10-
biphenyl][2-(2-aminoethyl)phenyl]palladium(II) (“BrettPhos pre-
catalyst”) was purchased from Aldrich (718750) and activated as
described [24].
b
a
a
sults of the experiment showa KD of 12 nM, which is consistent with
the IC50 value determined on human monocyte-derived macro-
phages. DMPK studies show that compound can be administered
orally and exhibits a long distribution half-life of 2.1 h with an
elimination half-life of 9 h.
Synthesis of furo[3,4b]pyridin-5(7H)-one derivatives (Scheme 2).
Synthesis
of
the
intermediate
3-chloro-2-(4-[4-
chlorophenoxy]piperidin
1 yl)furo [3,4-b]pyridin-5(7H)-one
(7) [23]
A mixture of commercially available 2,3-dichloro-5He7H-furo
[3,4-b]pyridin-5-one (6, 1 eq.), 4-(4-chlorophenoxy)piperidine hy-
drochloride (1.2 eq.) and TEA (2.2 eq.) in EtOH (4.8 mL/mmol), was
heated first at 80 ꢂC for 5 min and then at 120 ꢂC for 30 min under
microwave irradiation (200 W) (HPLC control). Then the solvent
was evaporated and ethyl acetate was added, the organic mixture
was washed twice with water and the combined organic layers
were dried with MgSO4. The crude product 7 was used in the next
13b was also tested in vivo in a LPS endotoxic shock model in
male BALB/c mice. Levels of IL-1
diverse peripheral tissues including brain, liver and lungs. Levels of
IL-1 secreted in plasma are shown to be reduced in a dose-
response manner. Similarly, the increase of IL-1 expression in
b were measured in plasma and in
b
b
brain, liver and lung tissues produced by the LPS insult were
reduced in a dose-response manner.
7

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
Fig. 5. Levels of IL-1
b
in plasma and diverse organs after a LPS challenge and the inhibitory effect of AIK3 305. a) Levels of IL-1
b in plasma; b) Average fold increase of IL-1b mRNA
expression in brain; average fold increase mRNA IL-1
b
expression in liver; average fold increase mRNA IL-1 expression in lung (***p < 0.0001; **p < 0.001; *p < 0.05).
b
step without purification. Light brown solid, 99% yield. 1H NMR
(CDCl3, 400 MHz)
(d, J ¼ 8 Hz, 2H), 5.14 (s, 2H), 4.58e4.54 (m, 1H), 3.87e3.81 (m, 2H),
3.62e3.56 (m, 2H), 2.15e2.09 (m, 2H), 2.00e1.97 (m, 2H).
2-(4-[4-Chlorophenoxy]piperidin-1-yl)-3-([2,3-
acetate 2:1), to isolate 3 as a brown amorphous solid, 56% yield, 98%
purity by HPLC. 1H NMR (CDCl3, 400 MHz)
(ppm) 7.55 (s, 1H), 7.25
d
(ppm) 7.97 (s, 1H), 7.26 (d, J ¼ 8 MHz, 2H), 6.88
d
(d, J ¼ 12 Hz, 2H), 7.04 (s, 1H), 6.88 (dd, J ¼ 8 Hz, J ¼ 4 Hz, 2H), 6.79
(d, J ¼ 8 Hz, 1H), 5.18 (s, 2H), 4.62 (t, J ¼ 8 Hz, 2H), 4.64e4.56 (m,
1H), 3.71e3.67 (m, 2H), 3.38 (m, 2H), 3.23 (t, J ¼ 8 Hz, 2H), 2.36 (m,
2H), 2.08e2.06 (m, 2H). HRMS (ESI) calcd for C26H25ClN3O4
[Mþ1]þ: 478.1534, found: 478.1501.
dihydrobenzofuran-5-yl]amino)furo [3,4b]pyridin-5 (7H)-one
(3) [24]
A mixture of 7 (1 eq.), 5-amino-2,3-dihydrobenzo[b]furan (1.0
eq.) and K2CO3 (2.5 eq.) in 1,4-dioxane (4.8 mL/mmol) in the
presence of BrettPhos-precatalyst (0.005 eq.), was heated at 110 ꢂC
until reaction was completed (1 h). The solvent was evaporated,
ethyl acetate was added and the resulting mixture was washed
with water. Then the organic layer was dried and the solvent was
removed under reduced pressure. The crude reaction residue was
purified by preparative chromatography (silica, hexane/ethyl
General procedure for the synthesis of compounds 11e13
(Scheme 3).
Synthesis of the intermediate 5,6-dichloro-3-(N,N-dime-
thylsulfonamido)pyridine (9) [25]
To a suspension of commercially available 5,6-dichloro-3-
sulfonamidopyridine (8, (1 eq.) and K2CO3 (2 eq.) in anhydrous
DMF (1 mL/eq.), iodomethane (2 eq.) in DMF (5 ml/mmol) was
added and the mixture was stirred at room temperature until
8

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
reaction was completed (15 h, TLC control). Then the solvent was
evaporated and ethyl acetate was added. The mixture was washed
twice with water and the organic layer was dried with MgSO4. After
evaporation of the solvent, the resulting crude product 9 was pure
enough to be used in the next step (95% yield). 1H NMR (CDCl3,
A solution of 10 (1 eq.), the corresponding boronic acid (1.1 eq.),
K2CO3 (1.4 eq.) and Pd(PhPh3)4 (0.1 eq.) in toluene (5 mL/mmol),
was heated at 150 ꢂC until reaction was completed (15 h, HPLC
control). Then the catalyst was filtered through a Celite plug and
solvent was evaporated. The crude reaction mixture was purified by
Isolera Biotage system (C18, acetonitrile/water).
400 MHz)
(s, 6H).
d
(ppm) 8.66 (d, J ¼ 4 Hz, 1H), 8.12 (d, J ¼ 4 Hz, 1H), 2.81
6-(4-[40-Chlorophenoxy]piperidinyl)-5-(2,3-
General procedure for the synthesis of intermediate compounds
10 (Scheme 3).
dihydrobenzofuran-5-yl)-3-(N,N-dimethylsulfonamido)pyridine
(11a).
Intermediate compounds 10 were prepared analogously as
described above for compound 7 but using compound 9 as starting
material.
The title compound was prepared from 10a and (2,3-
dihydrobenzofuran-5-yl)boronic acid. Compound 11a was isolated
as a white foam, 34% yield, 98% purity (HPLC, 254 nm). 1H NMR
5-Chloro-6-(4-[4-chlorophenoxy]piperidin-1-yl)-3-(N,N-dime-
thylsulfonamido) pyridine (10a).
The title compound was prepared from 9 and commercially
available 4-(4-chlorophenoxy)piperidine hydrochloride. Interme-
diate compound 10a (91% yield) was used without purification. 1H
(CDCl3, 400 MHz)
d
(ppm) 8.52 (d, J ¼ 4 Hz, 1H), 7.69 (d, J ¼ 4 Hz,
1H), 7.33 (s, 1H), 7.28 (d, J ¼ 8 Hz, 1H), 7.21 (d, J ¼ 8 Hz, 2H), 6.85 (d,
J ¼ 8 Hz, 1H), 6.82 (d, J ¼ 8 Hz, 2H), 4.64 (t, J ¼ 8 Hz, 2H), 4.42e4.38
(m, 1H), 3.61e3.56 (m, 2H), 3.27 (t, J ¼ 8 Hz, 2H), 3.21e3.15 (m, 2H),
2.76 (s, 6H), 1.91e1.89 (m, 2H), 1.75e1.71 (m, 2H). HRMS (ESI) calcd
for C26H29ClN3O4S [Mþ1]þ: 514.1567, found: 514.1581.
NMR (CDCl3, 400 MHz)
d (ppm) 8.49 (s, 1H), 7.88 (s, 1H), 7.26 (d,
J ¼ 8 Hz, 2H), 6.88 (d, J ¼ 8 Hz, 2H), 4.55e4.53 (m, 1H), 3.84e3.79
(m, 2H), 3.56e3.50 (m, 2H), 2.76 (s, 6H), 2.11e2.08 (m, 2H),
1.98e1.96 (m, 2H).
5-Chloro-6-(4-[4-chlorobenzyl]piperazin-1-yl)-3-(N,N-dime-
thylsulfonamido) pyridine (10b).
6-(4-[40-Chlorophenoxy]piperidinyl)-5-(40-propionylphenyl)-3-
(N,N-dimethyl sulfonamido)pyridine (11b).
The title compound was prepared from compound 10a and (4-
propionylphenyl)boronic acid. Compound 11b was isolated as a
white foam, 40% yield, 100% purity (HPLC, 254 nm). 1H NMR (CDCl3,
The title compound was prepared from compound 9 and
commercially available 1-(4-chlorobenzyl)piperazine and it was
used without purification (87% yield). 1H NMR (CDCl3, 400 MHz)
400 MHz)
d
(ppm) 8.57 (d, J ¼ 4 Hz, 1H), 8.05 (d, J ¼ 8 Hz, 2H), 7.73
(d, J ¼ 4 Hz,1H), 7.61 (d, J ¼ 8 Hz, 2H), 7.20 (d, J ¼ 12 Hz, 2H), 6.79 (d,
J ¼ 12 Hz, 2H), 4.41e4.37 (m, 1H), 3.56e3.49 (m, 2H), 3.21e3.15 (m,
2H), 3.03 (c, J ¼ 8 Hz, 2H), 2.76 (s, 6H), 1.91e1.86 (m, 2H), 1.74e1.69
(m, 2H), 1.25 (t, J ¼ 8 Hz, 2H). HRMS (ESI) calcd for C27H31ClN3O4S
[Mþ1]þ: 528.1724, found: 528.1691.
d
(ppm) 8.47 (d, J ¼ 4 Hz, 1H), 7.86 (d, J ¼ 4 Hz, 1H), 7.33 (m, 4H),
3.66 (m, 6H), 2.75 (s, 6H), 2.65 (m, 4H).
5-Chloro-6-(4-[(6-chloropyridin-3-yl)oxy]piperidin-1-yl)-3-
(N,N-dimethyl sulfonamido)pyridine (10c).
The title compound was prepared from compound 9 and
commercially 2-chloro-5-(piperidin-4-yloxy)pyridine dihydro-
chloride and it was used without purification (99% yield). 1H NMR
General Procedure for the synthesis of compounds 12
(Scheme 3, Method A) [27]
A screw-cap test-tube, equipped with a magnetic stir bar, was
charged with BrettPhos-precatalyst (4 mol %), the corresponding
aniline (1 eq.) and intermediate compound 10 (1 eq.). The vial was
sealed with a teflon screw-cap, evacuated and backfilled with ni-
trogen; this procedure was repeated two additional times. Then,
the LiHMDS (1 M in THF, 2.5 eq.) was added. The reaction mixture
was stirred at 40 ꢂC until reaction was completed (2.5 h, HPLC
control). The solution was allowed to cool to room temperature,
quenched by the addition of aqueous, saturated NH4Cl solution
(5 mL) and then diluted with ethyl acetate. The organic phase was
separated and the aqueous phase was extracted one more time
with ethyl acetate. The combined organic layers were washed with
brine and dried over MgSO4. The solvent was removed under
reduced pressure and the residue was purified by Isolera Biotage
System (C18, acetonitrile/water).
(CDCl3, 400 MHz)
d
(ppm) 8.49 (s, 1H), 8.09 (d, J ¼ 4 Hz, 1H), 7.88 (d,
J ¼ 4 Hz, 1H), 7.24 (m, 2H), 4.60e4.57 (m, 1H), 3.83e3.78 (m, 2H),
3.56e3.50 (m, 2H), 2.75 (s, 6H), 2.13e2.11 (m, 2H), 1.99e1.98 (m,
2H).
5-Chloro-6-(4-[(6-chloropyridin-3-yl)methyl]piperazin-1-yl)-
3-(N,N-dimethyl sulfonamido)pyridine (10d).
The title compound was prepared from compound 9 and
commercially available 1-[(6-chloropyridin-3-yl)methyl]pipera-
zine and it was used without purification (95% yield). 1H NMR
(CDCl3, 400 MHz) d (ppm) 8.47 (s, 1H), 8.30 (s, 1H), 8.10 (s, 1H), 7.30
(d, J ¼ 12 Hz, 1H), 7.10 (d, J ¼ 12 Hz, 1H), 3.62 (m, 2H), 3.55 (m, 4H),
2.75 (s, 6H), 2.62 (m, 2H), 2.51 (m, 2H).
5-Chloro-6-(4-[(6-chloropyridazin-3-yl)oxy]piperidin-1-yl)-3-
(N,N-dimethyl sulfonamido)pyridine (10e).
6-(4-[40-Chlorophenoxy]piperidinyl)-5-([2,3-
The title compound was prepared from compound 9 and
commercially available 3-chloro-6-(piperidin-4-yloxy)pyridazine
and it was used be used without purification (78% yield). 1H NMR
dihydrobenzofuran-5-yl]amino)-3-(N,N-dimethylsulfonamido)pyr-
idine (12a).
The title compound was prepared from compound 10a and
commercially available 5-amino-2,3-dihydrobenzo[b]furan. Com-
pound 12a was isolated as a brown solid, 6% yield, 97% purity (HPLC,
(CDCl3, 400 MHz)
d
(ppm) 8.46 (d, J ¼ 4 Hz, 1H), 7.90 (d, J ¼ 4 Hz,
1H), 7.02 (d, J ¼ 12 Hz, 1H), 6.98 (d, J ¼ 12 Hz, 1H), 5.48e5.39 (m,
1H), 4.02e3.91 (m, 2H), 3.49e3.48 (m, 2H), 2.70 (s, 6H), 2.31e2.25
(m, 2H), 2.12e1.90 (m, 2H).
254 nm). 1H NMR (CDCl3, 400 MHz)
d (ppm) 8.33 (s, 1H), 7.26e7.21
(m, 3H), 7.02 (s, 1H), 6.95 (d, J ¼ 12 Hz, 1H), 6.84e6.78 (m, 3H), 5.87
(s, 1H), 4.62 (t, J ¼ 8 Hz, 2H), 4.47 (m, 1H), 3.79e3.72 (m, 2H), 3.44
(m, 2H), 3.23 (t, J ¼ 8 Hz, 2H), 2.79 (s, 6H), 1.98e1.93 (m, 2H), 1.81
(m, 2H). HRMS (ESI) calcd for C26H30ClN4O4S [Mþ1]þ: 529.1676,
found: 529.1677.
5-([1-(30-Chloro-5-[N,N-dimethylsulfamoyl]pyridin-2-yl)piper-
idin-4-yl]oxy) picolinamide (10f).
The title compound was prepared from compound 9 and
commercially
carboxamide and it was used in the next step without purifica-
tion (93% yield). 1H NMR (CDCl3, 400 MHz)
(ppm) 8.50 (s,1H), 8.39
available
5-(piperidin-4-yloxy)pyridine-2-
5-(Benzo[d]
[1,3]dioxol-5-ylamino)-6-(4-[4-chlorophenoxy]
d
piperidinyl)-3-(N,N-sulfonamido)pyridine (12b).
(d, J ¼ 8 Hz, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.79 (s, 1H), 7.00 (m, 1H),
5.58 (s, 1H), 4.82 (m, 1H), 3.86e3.80 (m, 2H), 3.57e3.51 (m, 2H),
2.76 (s, 6H), 2.20e2.15 (m, 2H), 2.05e1.98 (m, 2H).
General procedure for the synthesis of compounds 11
(Scheme 3) [26].
The title compound was prepared from compound 10a and
commercially available 3,4-(methylenedioxy)aniline. Compound
12b was isolated as a brown solid, 23% yield, 98% purity (HPLC,
254 nm). 1H NMR (CDCl3, 400 MHz)
d
(ppm) 8.34 (s, 1H), 7.98 (s,
1H), 7.23 (d, J ¼ 8 Hz, 2H), 6.85e6.80 (m, 3H), 6.71 (s, 1H), 6.65 (dd,
9

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
J ¼ 4 Hz, J ¼ 12 Hz, 1H), 6.00 (s, 2H), 5.96 (s, 1H), 4.49e4.46 (m, 1H),
3.80e3.74 (m, 2H), 3.49e3.43 (m, 2H), 2.78 (s, 6H), 2.01e1.93 (m,
2H), 1.84e1.76 (m, 2H). HRMS (ESI) calcd for C25H28ClN4O5S
[Mþ1]þ: 531.1469, found: 531.1470.
3.21e3.17 (m, 2H), 2.69 (s, 6H), 2.18e2.15 (m, 2H), 2.01e1.94 (m,
2H). HRMS (ESI) calcd for C27H30ClN4O5S [Mþ1]þ: 557.1625, found:
557.1622.
6-(4-[40-Chlorophenoxy]piperidinyl)-5-([40-ethoxyphenyl]
amino)-3-(N,N-dimethyl sulfonamide)pyridine (12d).
The title compound was prepared from compound 10a and
commercially available 4-ethoxyaniline. Compound 12d was iso-
lated as a brown foam, 70% yield, 99% purity (HPLC, 254 nm). 1H
6-(4-[4-Chlorophenoxy]piperidinyl)-5-([40-propoxyphenyl]
amino)-3-(N,N-dimethyl sulfonamido)pyridine (12e).
The title compound was prepared from compound 10a and
commercially available 4-chloropropoxy aniline. Compound 12e
was isolated as a brown solid, 22% yield, 95% purity (HPLC,
NMR (CDCl3, 400 MHz)
d (ppm) 8.16 (s, 1H), 7.43 (s, 1H), 7.26e7.23
254 nm).1H NMR (CDCl3, 400 MHz)
d
(ppm) 8.35 (s,1H), 8.00 (s,1H),
(m, 3H), 7.09e7.06 (m, 2H), 6.93e6.87 (m, 3H), 4.5 (m, 1H), 4.05 (c,
J ¼ 8 Hz, 2H), 3.56 (m, 2H), 3.19 (m, 2H), 2.70 (s, 6H), 2.17 (m, 2H),
1.99 (m, 2H), 1.43 (t, J ¼ 8 Hz, 3H), 1.39 (s, 1H). HRMS (ESI) calcd for
7.23 (d, J ¼ 8 Hz, 2H) 7.12 (d, J ¼ 8 Hz, 2H), 6.92 (dd, J ¼ 4 Hz, J ¼ 8 Hz,
2H), 6.84 (dd, J ¼ 4 Hz, J ¼ 8 Hz, 2H), 5.93 (s, 1H), 4.49e4.45 (m, 1H),
3.94 (t, J ¼ 8 Hz, 2H), 3.79e3.73 (m, 2H), 3.47e3.41 (m, 2H), 2.80 (s,
6H), 1.98e1.93 (m, 2H), 1.86e1.77 (m, 4H), 1.06 (t, J ¼ 8 Hz, 3H).
HRMS (ESI) calcd for C27H34ClN4O4S [Mþ1]þ: 545.1989, found:
545.1973.
C
27H32ClN4O4S [Mþ1]þ: 543.1833, found: 531.1840.
6-(4-[40-Chlorophenoxy]piperidinyl)-5-([40-propionylphenyl]
amino)-3-(N,N-dimethylsulfonamido)pyridine (12f).
The title compound was prepared from compound 10a and
commercially available 4-aminopropiophenone. Compound 12f
was isolated as a brown oil, 54% yield, 92% purity (HPLC, 254 nm).
General Procedure for the Synthesis of Compounds 12
(Scheme 3, Method B) [28].
A mixture of 10a (1 eq.), the corresponding aniline (1.0 eq.),
BrettPhos-precatalyst (0.005 eq.) and K2CO3 (2.5 eq.) in tert-BuOH
(4.8 mL/mmol) was heated at 130 ꢂC under microwave irradiation
(200 W) for 1 h (HPLC control). Solvent was evaporated and ethyl
acetate was added. Then the reaction mixture was washed with
water, the organic layers were dried and the solvent was concen-
trated. The crude reaction mixture was purified by Isolera Biotage
system (C18, acetonitrile/water).
1H NMR (CDCl3, 400 MHz)
d
(ppm)
d
8.35 (d, J ¼ 4 Hz, 1H), 7.96 (d,
J ¼ 8 Hz, 2H), 7.88 (bs, 1H), 7.23 (d, J ¼ 12Hz, 2H), 7.05 (d, J ¼ 8 Hz,
2H), 6.85 (d, J ¼ 12 Hz, 2H), 4.50e4.48 (m, 1H), 3.58e3.54 (m, 2H),
3.25e3.21 (m, 2H), 2.96 (c, J ¼ 8 Hz, 2H), 2.76 (s, 6H), 2.11 (m, 2H),
1.94 (m, 2H), 1.23 (t, J ¼ 8 Hz, 3H). HRMS (ESI) calcd for
C
27H32ClN4O4S [Mþ1]þ: 543.1833, found: 543.1835.
General Procedure for the synthesis of compounds 13
(Scheme 3) [31].
The corresponding phenol (1.1 eq.) and Cs2CO3 (1.2 eq.) were
taken in DMF (1.16 mL/mmol) and treated with neat sodium hy-
dride (1.1 eq.). After hydrogen evolution ceased, intermediate
compound 10 (1 eq.) was added and the reaction was stirred at
150 ꢂC under microwave irradiation (200 W) for 2 h. The solvent
was removed under vacuum and the residue diluted with ethyl
acetate (6 mL/mmol) and water (6 mL/mmol). The aqueous layer
was extracted with ethyl acetate three times. The combined organic
layers were washed with 2 N NaOH, dried and concentrated under
vacuum. The residue was purified by Isolera Biotage system (C18,
acetonitrile-water) and/or preparative chromatography (silica,
hexane-ethyl acetate 4:1).
4.2. 6-(4-[40-Chlorophenoxy]piperidinyl)-5-([2’-(hydroxymethyl)
benzofuran-5-yl] amino)-3-(N,N-dimethylsulfonamido)pyridine
(12c) (Scheme 4)
4.2.1. Preparation of the silylated intermediate 14 [29]
2-Hydroxymethyl-5-aminobenzofuran (1.0 equiv.) and imid-
azole (3 equiv.) were dissolved in dichloromethane (0.35 mL/mmol)
and the solution was cooled to 0 ꢂC. Then triisopropylsilyl chloride
TIPSCl (1.1 equiv.) was added and the solution was allowed to warm
to room temperature and stirred for 12 h. The solution was washed
with water, the organic layer was dried over MgSO4 and the filtrate
was concentrated under reduced pressure. The crude reaction
mixture was purified by reverse phase chromatography (C18, ACN-
H2O, 5e100% ACN), to yield 14 a colorless oil (94% yield). 1H NMR
6-(4-[40-Chlorophenoxy]piperidinyl)-5-([2,3-
dihydrobenzofuran-5-yl]oxy)-3-(N,N-dimethylsulfonamido)pyri-
dine (13a).
(CDCl3, 400 MHz)
d
(ppm) 7.23 (d, J ¼ 12 Hz, 1H), 6.85e6.83 (m, 1H),
The title compound was prepared from 10a and commercially
available 2,3-dihydro-5-hydroxybenzo[b]furan. Compound 13a
was isolated as a colorless oil, 14% yield, 95% purity (HPLC, 254 nm).
6.65 (dd, J ¼ 4 Hz, J ¼ 12 Hz), 6.49 (s, 1H), 4.85 (d, J ¼ 4 Hz, 2H), 1.11
(d, J ¼ 8 Hz, 18H).
Step A: The title compound was prepared from 10a and 14
following the general procedure for compound 12 (method B). The
purified product 15 (28% yield) was used in the next step without
1H NMR (CDCl3, 400 MHz)
d (ppm) 8.31 (s, 1H), 7.25e7.22 (m, 3H),
6.87e6.82 (m, 3H), 6.76e6.68 (m, 2H), 4.62 (t, J ¼ 8 Hz, 2H),
4.51e4.48 (m, 1H), 4.01e3.98 (m, 2H), 3.65e3.61 (m, 2H), 3.21 (t,
J ¼ 8 Hz, 2H), 2.67 (s, 6H), 2.03e2.01 (m, 2H), 1.89e1.84 (m, 2H).
HRMS (ESI) calcd for C26H29ClN3O5S [Mþ1]þ: 530.1516, found:
530.1550.
further purification. 1H NMR (CDCl3, 400 MHz)
d (ppm) 8.19 (d,
J ¼ 4 Hz, 1H), 7.51 (s, 1H), 7.44 (d, J ¼ 8 Hz, 1H), 7.31 (d, J ¼ 4 Hz, 1H),
7.26 (d, J ¼ 12 Hz, 2H), 7.04 (dd, J ¼ 4 Hz, J ¼ 8 Hz, 1H), 6.89 (d,
J ¼ 12 Hz, 2H), 6.61 (s, 1H), 4.90 (s, 2H), 4.51 (m, 1H), 3.57 (m, 2H),
3.20 (m, 2H), 2.70 (s, 6H), 2.18 (m, 2H), 2.01 (m, 2H), 1.12 (d, J ¼ 8 Hz,
18H).
6-(4-[40-Chlorophenoxy]piperidinyl)-5-(40-ethoxyphenoxy)-3-
(N,N-dimethyl sulfonamido)pyridine (13b, AIK3-305).
The title compound was prepared from compound 10a and
commercially available 4-ethoxyphenol. Compound 13b was iso-
lated as a brown solid, 41% yield, 96% purity (HPLC, 254 nm). 1H
Step B [30]: To a solution of the above TIPS-protected product 15
(1 eq.) in anhydrous THF (7 mL/mmol), tetrabutylammonium
fluoride (TBAF) 1.0 M in THF (1.5 eq.) was added. After 1.5 h (HPLC
control), ethyl acetate was added and the mixture was washed with
water. Then the organic layer was dried over MgSO4 and concen-
trated under reduced pressure. The crude reaction mixture was
purified by preparative chromatography (silica, hexane-ethyl ace-
tate 2:1), to isolate 12c as a white foam, 27% yield, 99% purity (HPLC,
NMR (CDCl3, 400 MHz)
d (ppm) 8.32 (s, 1H), 7.25e7.22 (m, 3H),
6.87e6.83 (m, 6H), 4.49e4.46 (m, 1H), 4.04 (c, J ¼ 8 Hz, 2H),
4.04e3.95 (m, 2H), 3.65e3.58 (m, 2H), 2.66 (s, 6H), 1.99e1.97 (m,
2H), 1.87e1.81 (m, 2H), 1.42 (t, J ¼ 8 Hz, 3H). 13C NMR (101 MHz,
CDCl3) d (ppm) 155.93, 155.84,154.62, 148.89,142.51, 141.95,129.59,
126.01, 124.60, 122.19, 119.52, 117.62, 115.97, 72.81, 64.12, 44.55,
38.01, 30.80, 14.99. HRMS (ESI) calcd for C26H31ClN3O5S [Mþ1]þ:
532.1673, found: 532.1680.
254 nm). 1H NMR (CDCl3, 400 MHz)
7.49e7.45 (m, 2H), 7.32 (d, J ¼ 2 Hz, 1H), 7.25 (d, J ¼ 8 Hz, 2H), 7.06
(dd, J ¼ 2 Hz, J ¼ 8 Hz, 1H), 6.88 (d, J ¼ 8 Hz, 2H), 6.64 (d, J ¼ 2 Hz,
1H), 6.02 (s, 1H), 4.80 (s, 2H), 4.52e4.48 (m, 1H), 3.58e3.54 (m, 2H),
d
(ppm)
d
8.20 (d, J ¼ 4 Hz, 1H)
6-(4-[40-Chlorophenoxy]piperidinyl)-5-(4’-[200-hydroxyethoxy]
phenoxy)-3-(N,N-dimethylsulfonamido)pyridine (13c).
10

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
Synthesis of 4-(2-[(tert-butyldimethylsilyl)oxy]ethyl)phenol
(Scheme 5).
6-(4’-[(600-Chloropyridin-3-yl)methyl]piperazinyl)-5-(40-ethox-
yphenoxy)-3-(N,N-dimethylulfonamido)pyridine (13f).
Step A [32]: To a solution of the commercially available 2-(40-
hydroxyphenyl)acetic acid (1 eq.) in MeOH (1 mL/mmol), few drops
of concentrated H2SO4 were added and the resulting mixture was
heated under reflux until reaction was completed (2 h, TLC control).
Then solvent was evaporated, ethyl acetate was added and the
organic solution was washed with NaHCO3. Then organic layer was
dried with MgSO4 and the solvent was evaporated to give the
corresponding methyl ester in 89% yield.
The title compound was prepared from compound 10d and
commercially available 4-ethoxyphenol. Compound 13f was iso-
lated as a yellow solid, 15% yield, 100% purity (HPLC, 254 nm). 1H
NMR (CDCl3, 400 MHz) (two rotamers)
d (ppm) 8.47 (s, 1H),
8.32e8.30 (m, 2H), 8.10 (s, 1H), 7.86 (s, 1H), 7.30 (d, J ¼ 12 Hz, 1H),
7.22 (d, J ¼ 4 Hz, 2H), 7.06 (d, J ¼ 12 Hz, 2H), 6.91 (d, J ¼ 12 Hz, 2H),
6.86e6.84 (m, 5H), 4.06e3.99 (m, 4H), 3.74 (m, 5H), 3.62 (m, 3H),
3.55e3.51 (m, 4H), 2.74 (s, 6H), 2.65 (s, 6H), 2.62 (m, 5H), 2.51 (m,
5H), 1.44e1.40 (m, 6H). HRMS (ESI) calcd for C25H31ClN5O4S
[Mþ1]þ: 532.1785, found: 532.1780.
Step B [33]: A solution of methyl 2-(40-hydroxyphenyl)acetate (1
eq.) in THF (25 mL/mmol) was added over a suspension of lithium
tetrahydroaluminate (1.1 eq.) in THF and the resulting mixture was
stirred for 15 h at room temperature. Then water (340 mL) and 4 N
NaOH solution (340 ml) were added cautiously and the mixture
was filtered. The filtrate was concentrated under vacuum to give
the crude alcohol which was used in the next step without purifi-
cation (96% yield). (Described product).
Step C [30]: To a solution of commercially available 4-(2-
hydroxyethoxy)-phenol (4.2 g, 27 mmol) in anhydrous DMF
(30 mL) was added imidazole (2.1 g, 31 mmol) at 0 ꢂC and tert-
butyldimethylchlorosilane (4 g, 27 mmol), and the reaction mixture
was stirred at 0 ꢂC for 1 h. Then the mixture was partitioned be-
tween ethyl acetate and water, the organic layer was separated and
the aqueous layer was extracted with ethyl acetate. The combined
organic layers were washed with water and brine, dried over
MgSO4 and concentrated. The residue was purified by column
chromatography (ethyl acetate:hexane 1:4) to give 4-[2-(tert-
butyldimethylsilanyloxy)-ethoxy]-phenol as a colorless oil (142 mg,
52% yield). (Described product).
6-(4’-[(600-Chloropyridazin-3-yl)oxy]piperidinyl)-5-(40-ethox-
yphenoxy)-3-(N,N-dimethylsulfonamido)pyridine (13g).
The title compound was prepared from compound 10e and
commercially available 4-ethoxyphenol. Compound 13g was iso-
lated as a white solid, 10% yield, 90% purity (HPLC, 254 nm). 1H NMR
(CDCl3, 400 MHz)
d
(ppm) 8.48 (d, J ¼ 4 Hz, 1H), 7.86 (d, J ¼ 4 Hz,
1H), 7.15e7.09 (m, 3H), 6.99 (d, J ¼ 12 Hz,1H), 6.91 (d, J ¼ 12 Hz, 2H),
5.44e5.40 (m, 1H), 4.02 (c, J ¼ 8 Hz, 2H), 3.95e3.89 (m, 2H),
3.42e3.35 (m, 2H), 2.74 (s, 6H), 2.26e2.21 (m, 2H), 2.00e1.92 (m,
2H), 1.41 (t, J ¼ 8 Hz, 3H). HRMS (ESI) calcd for C24H29ClN5O5S
[Mþ1]þ: 534.1578, found: 534.1589.
5-([1-(5-[N,N-Dimethylsulfamoyl]-3-(4-ethoxyphenoxy)pyr-
idin-2-yl)piperidin-4-yl]oxy)picolinamide (13h).
The title compound was prepared from compound 10f and
commercially available 4-ethoxyphenol. Compounds 13h was iso-
lated as a light brown solid, 30% yield, 100% purity (HPLC, 254 nm).
1H NMR (CDCl3, 400 MHz)
d
(ppm) 8.37 (d, J ¼ 4 Hz, 1H), 8.33e8.32
(d, J ¼ 2 Hz, 1H), 7.88 (bs, 1H), 7.73 (d, J ¼ 2 Hz, 1H), 6.93 (dd,
J ¼ 2 Hz, J ¼ 8 Hz, 1H), 6.88 (s, 4H), 5.66 (bs, 1H), 4.77e4.73 (m, 1H),
4.05e3.98 (m, 4H), 3.68e3.62 (m, 2H), 2.68 (s, 6H), 2.10e2.04 (m,
2H), 1.92e1.85 (m, 2H), 1.43 (t, J ¼ 8 Hz, 3H). HRMS (ESI) calcd for
4.3. Compound 13c
The title compound was prepared from 10a and the above
protected phenol following the general procedure for compounds
13. Directly TBDMS-deprotected product 13c was isolated as a
colorless oil, 7% yield (after several purifications by Isolera Biotage
system (C18, acetonitrile-water) and preparative TLC (silica,
hexane-ethyl acetate), 96% purity (HPLC, 254 nm). 1H NMR (CDCl3,
C
26H32N5O6S [Mþ1]þ: 542.2073, found: 542.2085.
4.4. In vitro studies
Monocyte-derived macrophages differentiated from a Periph-
eral Blood Mononuclear Cell (PBMC) pool were isolated from hu-
man whole blood by Histopaque density gradient centrifugation
(Sigma-Aldrich, St. Louis, MO, USA). The freshly isolated PBMCs
were seeded at 2 ꢀ 105 numbers/well in a 96-well plate and
monocytes were differentiated into macrophages by treatment
with 200 ng/mL of recombinant human M-CSF (4 mg/mL stock)
(Biolegend, San Diego, CA, USA). After incubation at 37 ꢂC in a CO2
incubator for 6 days, monocyte-derived macrophages were dosed
with 13b at different concentrations, starting at a top concentration
400 MHz)
d
(ppm) 8.33 (s, 1H), 7.27 (s, 1H), 7.23 (d, J ¼ 8 Hz, 2H),
6.93e6.83 (m, 6H), 4.50e4.47 (m, 1H), 4.08 (t, J ¼ 4 Hz, 2H),
4.02e3.96 (m, 4H), 3.69e3.63 (m, 2H), 2.68 (s, 6H), 2.04e1.98 (m,
2H), 1.89e1.83 (m, 2H). HRMS (ESI) calcd for C26H31ClN3O6S
[Mþ1]þ: 548.1622, found: 548.1582.
6-(4-[40-Chlorobenzyl]piperazinyl)-5-(40-ethoxyphenoxy)-3-
(N,N-dimethyl sulfonamido)pyridine (13d).
The title compound was prepared from compound 10b and
commercially available 4-ethoxyphenol. Compound 13d was iso-
lated as a yellow oil, 10% yield, 100% purity (HPLC, 254 nm). 1H NMR
of 10 mM with a half log step down dilution for 10 points. After
incubation at 37 ꢂC in a CO2 incubator for 2 h, PBMCs were stim-
ulated with 100 ng/mL of LPS (1 mg/mL stock) (Sigma-Aldrich, St.
Louis, MO, USA) and incubated in the same conditions for 6 h. Cell
supernatant was subsequently collected and measured by ELISA
(CDCl3, 400 MHz)
d
(ppm) 8.30 (d, J ¼ 4Hz, 1H), 7.28e7.26 (m, 4H),
7.22 (d, J ¼ 4 Hz, 1H), 6.86 (m, 4H), 4.02 (c, J ¼ 4 Hz, 2H), 3.75 (m,
4H), 3.50 (m, 2H), 2.65 (s, 6H), 2.51 (m, 4H), 1.42 (t, J ¼ 4 Hz, 3H).
HRMS (ESI) calcd for C26H32ClN4O4S [Mþ1]þ: 531.1833, found:
531.1830.
technique using the LEGEND MAX human IL-1
b ELISA kit (Bio-
legend, San Diego, CA, USA) with a SpectraMax microplate reader
set at 450 nm.
6-(4’-[(600-Chloropyridin-3-yl)oxy]piperidinyl)-5-(40-ethox-
yphenoxy)-3-(N,N-dimethylsulfonamido)pyridine (13e).
The title compound was prepared from compound 10c and
commercially available 4-ethoxyphenol. Compound 13e was iso-
lated as a white solid, 24% yield, 97% purity (HPLC, 254 nm). 1H NMR
For the cytokine panel the same protocol was followed.
Monocyte-derived macrophages were dosed or not with 13b at
1 m
M. After incubation at 37 ꢂC in a CO2 incubator for 2 h, PBMCs
were stimulated with 100 ng/mL of LPS (100 ng/mL stock) (Sigma-
Aldrich, St. Louis, MO, USA) and incubated in the same conditions
(CDCl3, 400 MHz)
d
(ppm) 8.32 (d, J ¼ 4 Hz, 1H), 8.07 (d, J ¼ 4 Hz,
1H), 7.25 (d, J ¼ 2 Hz, 1H), 7.22 (s, 1H), 7.22 (d, J ¼ 2 Hz, 1H), 6.87 (s,
4H), 4.54e4.51 (m, 1H), 4.04e3.95 (m, 4H), 3.66e3.59 (m, 2H), 2.66
(s, 6H), 2.06e2.00 (m, 2H), 1.87e1.83 (m, 2H), 1.42 (t, J ¼ 8 Hz, 3H).
HRMS (ESI) calcd for C25H30ClN4O5S [Mþ1]þ: 533.1625, found:
533.1638.
for 6 h. Twelve different cytokines including IL-1
a
, IL-1b, IL-2, IL-4,
IL-6, IL-8, IL-10, IL-12, IL-17A, TNF , GM-CSF and INF-
a
g
were tested
in the cell supernatant and measured by ELISA technique, using the
ELISAMulti-Analyte ELISArray Kit (QIAGEN, Hilden, Germany) with
a SpectraMax microplate reader set at 450 nm.
11

E. Carrasco, P. Gomez-Gutierrez, P.M. Campos et al.
European Journal of Medicinal Chemistry 223 (2021) 113620
4.5. Surface Plasmon Resonance imaging (SPRi)
and liver tissue samples were harvested and submitted for IL-1
mRNA analysis. Levels of mRNA were measured by RT PCR.
b
Diverse concentrations of p38 MAPK dissolved in water were
manually printed onto the bare gold-coated (thickness 47 nm)
PlexArray Nanocapture Sensor Chip (Plexera Bioscience, Seattle,
WA, USA) at 40% humidity. Each concentration was printed in
Declaration of competing interest
The authors declare the following financial interests/personal
relationships which may be considered as potential competing
interests: EC, PG-G, PMC and MG are Allinky Biopharma employees.
replicate with each spot containing 0.2 mL of protein solution. The
chip was incubated in 80% humidity at 4 ꢂC overnight and rinsed
with 10 ꢀ PBST for 10 min, 1 ꢀ PBST for 10 min and deionized water
twice for 10 min. Subsequently, the chip was blocked with 5% (w/v)
non-fat milk in water overnight and washed with 10 ꢀ PBST for
10 min, 1 ꢀ PBST for 10 min and deionized water twice for 10 min,
before being dried under a stream of nitrogen prior to use. SPRi
Measurements were performed with PlexAray HT (Plexera Biosci-
ence, Seattle, WA, USA), where collimated light (660 nm) passing
through the coupling prism, is reflected off the SPR-active gold
surface and received by a charge-coupled device camera. Buffers
and samples were injected by a non-pulsatile piston pump into the
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113620.
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13