Full text of DOI:10.1039/d0md00423e

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Medicinal Chemistry
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RESEARCH ARTICLE
Pyridazinone derivatives as potential anti-
inflammatory agents: synthesis and biological
evaluation as PDE4 inhibitors†
Cite this: RSC Med. Chem., 2021, 12,
584
a
Ingrid Allart-Simon,‡a Aurélie Moniot,‡b Nicolo Bisi,a Miguel Ponce-Vargas,
c
a
Sandra Audonnet,
Marie Laronze-Cochard,a Janos Sapi,a Eric Hénon,
a
Frédéric Velardb and Stéphane Gérard
*
Cyclic nucleotide phosphodiesterase type 4 (PDE4), which controls the intracellular level of cyclic
adenosine monophosphate (cAMP), has aroused scientific attention as suitable target for anti-
a
Received 16th December 2020,
Accepted 10th February 2021
inflammatory therapy of respiratory diseases. This work describes the development and characterization of
pyridazinone derivatives bearing an indole moiety as potential PDE4 inhibitors and their evaluation as anti-
inflammatory agents. Among these derivatives, 4-(5-methoxy-1H-indol-3-yl)-6-methylpyridazin-3(2H)-one
possesses promising activity, and selectivity towards PDE4B isoenzymes and is able to regulate potent pro-
inflammatory cytokine and chemokine production by human primary macrophages.
DOI: 10.1039/d0md00423e
rsc.li/medchem
respiratory diseases. From a biological point of view, PDE4 is
the predominant isoenzyme found to be overexpressed in
Introduction
Chronic obstructive pulmonary disease (COPD) and asthma
are among the most common chronic diseases of the twenty-
first century. They are becoming a major global health
problem with high morbidity and mortality.1 Asthma is one
of the most common chronic conditions in children, while
more than 3 million people died from COPD worldwide in
airway cells such as epithelial and immune cells including
neutrophils, T-cells, and macrophages.4 By its inhibition, both
airway smooth muscle relaxation and inflammatory modulator
release can be managed; therefore, PDE4 may be considered
as
a valuable biological target for the treatment of
inflammatory or pulmonary diseases.5 More recently, PDE4
inhibition also demonstrated positive effects against aberrant
immune response diseases such as atopic dermatitis,
rheumatoid arthritis or psoriasis or in the modulation of
2015.2 These lung pathologies are characterized by
a
progressive airflow diminution accompanied by inflammatory
response. Even if combined inhaled corticosteroid and long-
acting beta agonist treatments play a central role in the
management of asthma and COPD, poor treatment adherence
and/or inhaler techniques have been reported.3
In the recent years, phosphodiesterases (PDEs),
a
superfamily of eleven intracellular enzymes, responsible for
the hydrolysis of cyclic adenosine and/or guanosine
monophosphate (ubiquitous intracellular second messengers),
have emerged as new therapeutic targets with special attention
given to phosphodiesterase type-4 (PDE4) for the treatment of
a Université de Reims Champagne-Ardenne, Institut de Chimie Moléculaire de
Reims (ICMR), UMR CNRS 7312, UFR Sciences, Moulin de la housse and UFR
Pharmacie, 51 rue Cognacq-Jay, F-51096 Reims, France.
E-mail: stephane.gerard@univ-reims.fr
b Université de Reims-Champagne-Ardenne, EA 4691 Biomatériaux & Inflammation
en site OSseux (BIOS), UFR Pharmacie and UFR Odontologie, 51 rue Cognacq-Jay,
F-51096 Reims, France
c Université de Reims-Champagne-Ardenne, URCACyt, UFR Pharmacie, 51 rue
Cognacq-Jay, F-51096 Reims, France
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00423e
Scheme 1 Structures and affinities of PDE4 inhibitors zardaverine and
‡ These authors equally contributed to this work.
approved drugs and the targeted family.
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metabolic disorders such as obesity or type 2 diabetes.6
Although a large number of PDE4 inhibitors have been
clinically evaluated, the use of first-generation compounds has
been hampered by cardiac and emetic mechanism-associated
side effects. Second-generation selective inhibitors like
roflumilast and apremilast were generally better-tolerated and
are currently approved for the treatment of COPD and plaque
psoriasis, respectively (Scheme 1).7,8
Macrophages are cells belonging to the innate immune
system. In several inflammatory diseases, macrophages
orchestrate the inflammatory response as in COPD.9 Actually, a
series of studies have evidenced an increase in macrophage
number in COPD patients.10–12 These cells are recruited from
blood flow in response to chemokine (CCL2, CXCL1)
gradients.13 Their activation leads to pro-inflammatory cytokine
release, among them TNF-α, IL-1β, IL-8 and IL-6, which are
pivotal to ensure the recruitment and activation of more
inflammatory cells like polynuclear neutrophils that become a
major cell population in COPD patient lungs.14 To avoid an
exacerbated inflammatory process, some therapeutics have
been tested to modulate macrophage activation: macrolides,
exhibiting both anti-microbial and anti-inflammatory
properties,15 and PDE4 inhibitors.16,17 For example, roflumilast
induces (at a dose of 5 mg kg−1 per day) reduction of
macrophages in the lung of mice exposed to tobacco smoke
and have demonstrated clinical benefits in COPD patients.18,19
In continuation of our efforts to develop selective
inhibitors of PDE4 for the treatment of respiratory diseases
possessing fewer side effects than marketed compounds,20
we have previously identified a hit compound possessing the
Scheme
moiety in position 4.
2
Synthesis of (dihydro)pyridazinones bearing an indole
(SAR) study by comparison with our preliminary hit. As
shown in Table 1, formation of the corresponding
compounds proceeded in moderate to good yields in about
36 h. Subsequent condensation of the intermediates 2a–f
with hydrazine derivatives led to the formation of the
corresponding 4,5-dihydropyridazinones 3a–f with good
yields. The desired pyridazinones 4a–f functionalized with an
indole moiety in position 4 were then obtained using
oxidative conditions (copper(II) chloride (method A) or
manganese dioxide (method B)) depending on the solubility
of the dihydropyridazinone derivatives.24
2. PDE4 inhibition
dihydropyridazinone scaffold and bearing
a heterocyclic
moiety.21 We have designed this hit by introducing a degree
of conformational freedom between the pyridazinone scaffold
and heterocyclic moiety in order to study if these
conformations can influence potency and selectivity over
PDE4. This hit possessed neither cytotoxic effects nor
abnormal pro-inflammatory roles in polymorphonuclear
leukocyte cells and we highlighted that this PDE4 inhibitor
was able to reduce IL-8 production. Based on this preliminary
data, we describe herein the synthesis of new pyridazinone
derivatives bearing an indole moiety in position 4 (structure
in Scheme 1), their evaluation as potential PDE4B-inhibitors
and their anti-inflammatory potential by regulating cytokine
and chemokine production by macrophages.
The effects of (dihydro)pyridazinones 3 and 4 on modulation
of the intracellular level of cyclic AMP were then examined
and compared to those of a non-specific PDE inhibitor
3-isobutyl-1-methylxanthine
(pyridazinone scaffold comparison) and
(IBMX),
zardaverine
therapeutic
a
reference, roflumilast. Taking into account the fact that drugs
that preferentially inhibit the PDE4B isoform over PDE4D
should retain many of their anti-inflammatory activity
benefits with fewer unwanted central nervous system side-
effects,25 all synthesized compounds were first screened for
their potential inhibitory activity on this subtype. The
evaluation approach, based on a “nonradioactive” assay
developed in our laboratory, could be directly applied for the
selection of privileged pyridazinone derivatives.21 Results
were expressed as inhibition percentage of PDE4B at an
initial screening concentration of 20 μM. First, in vitro results
(collected in Table 2) showed that all the pyridazinones,
bearing an indole moiety on the 4-position of the heterocyclic
scaffold, systematically exhibited greater percentages of
PDE4B inhibition (1.5–3 fold) compared to the corresponding
Results and discussion
1. Synthesis
Preparation of the targeted pyridazinone derivatives was
carried out through a flexible three-step sequence starting
from the α,β-unsaturated levulinate
1
(Scheme 2).22
Regioselective introduction of the indole moiety was carried
out by a Michael type reaction leading to intermediate
compounds 2a–f.23 Different indole derivatives (5-halogeno,
5-nitro and 5-methoxyindole) were used in this reaction in
order to initiate a preliminary structure–activity relationship
4,5-dihydropyridazinones (compounds
4
vs. 3). This
observation is probably due to the more planar character of
the pyridazinone scaffold (by comparison to a dihydro
structure) allowing better interactions with the relatively
closed and hydrophobic oblong pocket of the active site. Our
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Table 1 Synthesis of pyridazinone derivatives bearing an indole moiety in position 4
Entry
Heterocycle
Compound (yield)a
R2
Compound (yield)a
Oxidative methodb
Compound (yield)a
1
2
3
2a (61%)
H
Me
Bn
3aa (61%)
3ab (81%)
3ac (63%)
B
A
A
4aa (50%)
4ab (36%)
4ac (34%)
4
5
6
H
Me
Bn
3ba (76%)
3bb (25%)
3bc (41%)
B
B
B
4ba (60%)
4bb (35%)
4bc (46%)
2b (55%)
2c (39%)
7
8
H
Bn
3ca (77%)
3cc (44%)
B
B
4ca (90%)
4cc (50%)
9
10
2d (80%)
H
Bn
3da (81%)
3dc (43%)
B
B
4da (60%)
4dc (40%)
11
12
2e (33%)
2f (20%)
H
Bn
3ea (68%)
3ec (40%)
B
B
4ea (28%)
4ec (45%)
13
H
3fa (73%)
B
4fa (80%)
a
Yield of isolated product. b Method A: CuCl2/AcOH; method B: MnO2/THF.
Table 2 Biochemical evaluation of pyridazinone derivatives as PDE4B
inhibitors
to 2.5-fold less potent than their corresponding NH analogue.
Interestingly, contrary to our initial expectations, increasing
the hydrophobic character of the pyridazinone derivatives (R2
= Bn) slightly decreased the inhibitory effect but might have
an impact on selectivity. Concerning the substitution on
position 5′ of the indole moiety, modification of the nature of
the halogen could drastically reduce the affinity for PDE4
receptors (F > Br ∼ I); thus, iodo- and bromo-derivatives were
found to be practically inactive at screening concentration.
By comparison with 5′-fluoro derivative 4ea, compound 4fa
possessing a nitro function seems to interact more favorably
with PDE4 but remains about three times less active than
pyridazinone 4ba. Additional hydrogen-bond interaction with
key glutamine (Gln) could explain the observed increase in
inhibitory activity of the methoxy-functionalized compounds
(see the Molecular modeling section).21
In order to explore the potentiality of compound 4ba and
to reinforce the preliminary SAR results (vs. compounds 4aa
and 4bc), we submitted these pyridazinones to a selectivity
study versus PDE subtypes by comparison with references. We
selected PDEs which demonstrate different capabilities for
degrading cAMP/cGMP and regulating cell functions: PDE1 (a
Ca2+ and calmodulin dependent PDE), PDE10, an isoform
mainly expressed in the brain capable of hydrolyzing both
cAMP and cGMP (but with a higher affinity for cAMP than
cGMP), and PDE7, highly selective for cAMP isoenzymes.26
Based on previous studies suggesting that the emesis side-
a
b
Inhibition at an initial concentration of 20 μM. Mean of two or
three experiments, each performed in triplicate; protocols are given
in the ESI† experimental section. Percentage of inhibition range:
c
/
/
/>
.
attention turned to the N-substituent on the pyridazinone
ring (R2). It was confirmed that the hydrogen bond donor
function (R2 = H) was optimal for the PDE4B affinity in so far
as N-methyl derivatives 3ab, 4ab, 3bb and 4bb appeared up
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effects result from the inhibition of the PDE4D isoform and
that knocking out PDE4B but not PDE4A/PDE4D significantly
inhibited the synthesis of TNF-α in LPS-mediated monocyte
and macrophages, we also evaluated the capability of our
pyridazinones to modulate the three distinct PDE4 subtypes
(Fig. 1).27 The results of this study confirms the potency of
our scaffold enhanced by the selectivity over the PDE4
isoenzyme compared to PDE1, PDE10 and mostly PDE7. If, as
previously expected, the introduction of an N-benzyl substient
in our pyridazinone scaffold (compound 4bc) affects the
profile, clearly, the PDE4B selectivity index value is much
better for our pyridazinone 4ba than those of references.
Comparison of the different pharmacological data showed
that the more active pyridazinone is the compound 4ba,
functionalized with the 5-methoxyindole moiety, exhibiting
64% inhibition of PDE4B at 20 μM and IC50 = 251 18 nM
(log P 0.43). In our study, this compound is slightly less active
in vitro than therapeutic reference compound roflumilast
(75% of inhibition at 20 μM) but displays a better selectivity
profile towards the PDE4B isoform. This pyridazinone 4ba
was selected for further investigations.
But, previous authors have shown that the C-terminal control
region (CR3) present in the PDE4 isoforms can be exploited
to generate PDE4B-selective inhibitors.29,30 Actually, some
ligands cause the CR3 helix to close over the PDE4 active site,
thereby, locking the enzyme in an inactive conformation. In
that case, the PDE4B selectivity is due to a single amino acid
of the CR3 helix (Leu in PDE4B vs. Gln in PDE4D). This so-
called CR3 gating helix (GH) has been introduced in our
models during the docking simulations and its associated
residues will be referred hereafter with the subscript GH.
From the literature and from our previous work,21,31
although interactions vary from one inhibitor to another, the
two key features of PDE4–ligand binding are: i) hydrogen
bonding with Gln and ii) van der Waals interactions on both
sides of a ligand possessing flat structure and fitting an oblong
cavity, as illustrated in Fig. 2. Also, a shallow groove can be
taken advantage of, present at the foot of the active site, to
design new ligands. Upon binding the PDE4B active site, the
ligand 4ba fulfills the above-mentioned key criteria, and a good
complementarity shape is obtained (see Fig. 2 and 3). Its
interaction with the Gln615 residue through hydrogen-bonding
is limited in the reported pose. It should however be noticed
that we found many poses displaying this hydrogen-bond
between the methoxy group of 4ba and this Gln residue,
showing that the methoxy pattern plays a key role in the ligand
biorecognition by the PDE4B protein. The interactions between
4ba and the glutamine (Gln615) and phenylalanine (Phe586
and Phe618) residues of the protein are well recovered by the
IGM analysis as evidenced by the large associated isosurface
(Fig. 3 [upper line, central panel]). It should be noted that the
methyl group on the pyridazinone moiety exploits a small
hydrophobic cavity at the back of the active site. The
interactions between the methoxy group of 4ba and the leucine
(Leu674_GH) residue of the gating helix CR3, as well as those
involving the indole moiety of 4ba and the lysine (Lys677_GH)
and phenylalanine (Phe678_GH) groups, highlight the
relevance of the CR3 gating helix segment in the PDE4B–ligand
3. Molecular modeling
In a complementary way to the experimental investigations
and hit-detection,
a challenging task to explain the
preliminary SAR was to address the binding mode of our
ligands by means of theoretical tools. Here, molecular
docking simulations together with IGM analysis were carried
out to identify, characterize and quantify ligand–protein
interactions as well as atomic contributions to these
interactions (see computational details).28 The PDE4B and
PDE4D isoforms show a high structural similarity (98%)
making it extremely difficult to develop a selective inhibitor.
Fig. 1 PDE inhibition (% of inhibition) and PDE4B (vs. 4D)-selectivity
index values. [Inhibition at a concentration of 20 μM; mean of two or
three experiments, each performed in triplicate; protocols are given in
the ESI† experimental section].
Fig. 2 Presentation of the PDE4B active site complexed with ligand
4ba. The glutamine residue (Gln) plays a key role in the biomolecular
recognition, as well as hydrophobic residues on both sides of the
ligand; a hydrophobic groove exists at the foot of the active site.
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recognition. This is confirmed by a significant Δginter score of
1.44 a.u. (see Table S1 in the ESI†) for the CR3–4ba interaction.
According to the IGM atomic decomposition, the indole pattern
(42%) and pyridazinone moiety (42%) equally contribute to the
total interaction with the protein PDE4B, whilst the methoxy
group contribution amounts to 16% of the ligand–protein
PDE4B interaction.
In PDE4D–4ba, the key interactions are still found with
Gln (Gln535 in this isoform) and hydrophobic residues.
However, it is worth noting that the pyridazinone moiety can
no longer exploit the hydrophobic pocket in the back of the
active site. In this pose, the pyridazinone is twisted relative to
the indole heterocycle and is rather directed towards the
shallow groove and partially towards the CR3 helix. A Δginter
score of 1.03 a.u. (Table S1†), reflecting the interaction
between ligand 4ba and the PDE4D CR3 helix, is lower in
comparison with that of PDE4B–4ba (1.44 a.u.), suggesting a
reduced ability of the ligand to hold the active site closed in
this case. Removal of the methoxy group from 4ba (PDE4B–
4aa, see Fig. S1 in the ESI†) leads to a loss of the PDE4B
recognition through hydrogen-bonding to the Gln615 residue.
In other respects, the pose of 4aa–PDE4B looks very similar
to the pose of 4ba–PDE4B. The impact of the methoxy group
on the CR3–ligand interaction is demonstrated when the
4ba–CR3(PDE4B) and 4aa–CR3(PDE4D) Δginter scores are
compared (Table S1 in the ESI†). In the latter, a lower Δginter
(1.08 a.u.) relative to the former (1.44 a.u.) is found, revealing
the positive effect of the methoxy group incorporation in
terms of CR3 gating helix recognition. In PDE4B–4bc where a
benzyl substituent is present on the pyridazinone moiety, we
still observe the well-known interaction between the methoxy
group of 4bc and the glutamine (Gln615) as well as
interactions with hydrophobic residues (see Fig. S2 in the
ESI†). However, the methylpyridazinone moiety is no longer
directed towards the hydrophobic pocket in the back of the
active site but, once more, it is slightly twisted relative to the
indole heterocycle and oriented so that the benzyl group
points towards the shallow groove. Then, an additional
attractive
noncovalent
interaction
between
the
methylpyridazinone group and
a
methionine (Met519)
belonging the protein cavity is observed.
It is noteworthy that some unexpected results were
obtained in PDE4B–4ea with the 5′-fluorinated compound. As
in PDE4B–4aa (without the methoxy group on the indole), the
hydrogen-bonding recognition by Gln615 is lost (see
Fig. 3 [lower line, left panel]). Also, the pyridazinone moiety
is slightly twisted and pointing towards the shallow groove.
But, in return, the fluorine atom is directed towards the
Fig. 3 Molecular docking pose and IGM analysis of 4ba [upper line] and 4ea [lower line] inside the PDE4B-CR3 cavity (left panel). IGM 0.007 a.u.
δginter isosurface and a BGR color code in the range −0.05 < ρ sign (λ2) < 0.05 a.u. (center panel). IGM atomic decomposition with the ligand
colored according to the Δginter/At score using a BGryR color scale (right panel). Non-polar hydrogen atoms of the protein are not displayed for the
sake of clarity.
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gating helix CR3, favoring new interactions closing the active
site. After inspecting very carefully the pose of 4ea in the
PDE4B model (Fig. 3 [lower line, left panel]), there is no
indication of halogen bond formation between the fluorine
atom of 4ea and the gating helix residues. However, the IGM
analysis reveals attractive vdW contacts between this fluorine
atom and the lysine (Lys677_GH)/phenylalanine (Phe678_GH)
residues of the CR3 helix, as illustrated in Fig.
3
[central panel]. Although the overall CR3–4ea interaction
score is small (0.76 a.u.) compared to that of the other
ligands, it is worth noting that the single fluorine atom
interaction with the CR3 gating helix amounts to 4.6%.
4. Biology
The selected compound 4ba was then evaluated for in vitro
anti-inflammatory activity. We first evaluated the morphology
of the cells we used (Fig. 4). PBMCs cultured in the presence
of M-CSF are intended to become macrophages.32 SEM
analyses demonstrated that under basal conditions (DMSO,
Fig. 4), we obtained individual cells which are spherical,
exhibiting limited adhesion-related structures and few
vesicles at the cell membrane. After LPS stimulation, cells
tended to cluster together, increasing in size and exhibiting a
more ameboid shape. They also exhibited numerous
cytoplasmic extensions towards other cells, filopodia and
developed lamellipodia. A high number of vesicles have also
been observed and all these criteria are classical hallmarks of
activated macrophage morphology.33 When treated with
zardaverine, cells exhibited a morphology close to that
observed under control conditions whereas in the presence
of roflumilast, they were much more flattened, showing a
reduced vesicle number and highly intricate cytoplasms, but
were bigger in size. When treated with compound 4ba, cells
display an alternative phenotype with common features from
both zardaverine and roflumilast treatments.
In order to assess that morphological changes are not
attributable to cell suffering and death, we measured lactate
dehydrogenase (LDH) release in cell-stimulated supernatants.
As evidenced in Fig. 5, no statistical accumulation of LDH
was observed in the presence of any of the tested
compounds. We then measured the inhibition of pro-
inflammatory cytokine secretion in the macrophage culture
supernatants (Fig. 6). Here, we confirmed that in our hands
LPS was able to induce an increased secretion of IL-1β (30
fold increase of average value p = 0.008), TNF-α (6.25 fold
increase of average value p = 0.008), IL-6 (26.5 fold increase
of average value p = 0.008), and IL-8 (18.9 fold increase of
average value p = 0.008) by human primary macrophages
(Fig. 6A) as has been also evidenced and reviewed
elsewhere.32 We highlighted that zardaverine was unable to
limit such an increase for all the 4 cytokines studied
(Fig. 6B–E), while roflumilast only induced an average 10%
decrease in IL-8 measurement (p = 0.047, Fig. 6E) in LPS-
stimulated macrophage supernatants. Compound 4ba
demonstrated a clear dose effect on IL-1β concentration
Fig.
4
Representative scanning electron microscopy photographs
showing human primary macrophages after 24 h of stimulation with
DMSO alone (A) or with added LPS 10 ng mL−1 (B) and treated with
zardaverine 10 μM (C), roflumilast 10 μM (D) or compound 4ba 50 μM
(E). Magnification ×5000; scale bar = 1 μm. Inserts show representative
micrographs under the same conditions at magnification ×2000; scale
bar = 10 μm.
decrease that reached the statistical significance threshold
for concentrations of 20 μM and 50 μM (58% and 54%
reduction of average values, p = 0.023 and p = 0.039,
respectively, Fig. 6B). TNF-α concentration regulation by
compound 4ba followed a similar pattern but only the
highest concentration (50 μM) demonstrated a significant
47% decrease (p
=
0.008). Interestingly, the
5
μM
concentration failed to significantly reduce the LPS-induced
TNF-α production despite a 43% average decrease (p =
0.2422), but it was the only condition in which LPS
stimulation did not lead to
a significant increase as
compared to the control conditions (p = 0.219, Fig. 6C). The
IL-6 concentration was reduced by all the tested
concentrations of compound 4ba (average reduction of 21%,
28%, 23%, and 16% for 50 μM, 20 μM, 10 μM and 5 μM
respectively; p = 0.039, p = 0.008, p = 0.023 and p = 0.008,
Fig. 6D). IL-8 production by macrophages has been reduced
only when cells were treated by 50 μM and 20 μM (16% and
25% average reduction respectively, p = 0.039 and p = 0.008,
Fig. 6E). These results highlight the ability of our compound
4ba to regulate potent pro-inflammatory cytokine and
chemokine production by macrophages at
concentration of 20 μM. In addition, the anti-inflammatory
effects evidenced in this work are better than those
a
starting
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Fig. 5 Cell death evaluation by measurement of LDH activity in human
primary macrophage supernatants after 24 h treatment with zardaverine
(10 μM), roflumilast (10 μM) and compound 4ba (n = 8). The red bar
represents the median value. Black bars represent the 1st and 9th deciles
and the limits of rectangles represent the 1st and 3rd quartiles.
demonstrated by zardaverine or roflumilast, two reference
molecules for anti-PDE4 activity in inflammatory disease
models.34–36 On the one hand, as compared to the literature,
major discrepancies in the zardaverine and roflumilast
effects may come from the incubation time (4 h most of the
time) and cell source, which is very often lung-related and
from rodents in the literature, as compared to human whole
blood in our work.37 On the other hand, such an observation
is in line with recent data from our group highlighting in
human primary polymorphonuclear cells that zardaverine
exerted modest regulation of chemokine production
following LPS stimulation exhibiting thus a small impact on
cell recruitment in vitro.21
To assess the 4ba effect on monocyte/macrophage
recruitment in vivo, we used the murine air pouch model that
creates a delineated cavity inside which the 4ba effect was
tested. Initially dedicated to acute inflammation studies,38–39
this model has also been evidenced to allow the study of
monocyte/macrophage populations.40 In response to LPS
stimulation, we demonstrated an increase in monocyte and
macrophage recruitment in the air pouch exudate (1.9 and 1.7
fold increase of average values, p = 0.044 and p = 0.019
respectively, Fig. 7A and B). As compared to LPS or control
condition, Zardaverine failed to induce any modulation of
monocyte recruitment (Fig. 7A). Interestingly, zardaverine
exhibited a clear dose effect on macrophage recruitment with a
significant increase as compared to the control conditions
(average increase of 2 fold, p = 0.020) at a concentration of 2
μM, the absence of any variation at 20 μM, and a significant
reduction of macrophage recruitment as compared to LPS at
200 μM (26% average reduction, p = 0.006, Fig. 7B). Roflumilast
had no effect at 2 μM but increased monocyte recruitment at 20
μM and 200 μM as compared to the control conditions (3.5 and
2 fold increase of average values, p = 0.001 and p = 0.042
Fig.
6
ELISA measurements of IL-1β, TNF-α, IL-6 and IL-8
concentrations in unexposed or LPS-exposed cell supernatants after
24 h (A). IL-1β (B), TNF-α (C), IL-6 (D) and IL-8 (E) concentrations in
LPS-exposed cell supernatants after 24 h of treatment with zardaverine
(10 μM), roflumilast (10 μM) or compound 4ba. The red bar represents
the median value. Black bars represent the 1st and 9th deciles and the
limits of rectangles represent the 1st and 3rd quartiles.
n = 8
independent experiments. *p
stimulated (DMSO) cells. #p
stimulated cells.
<
<
0.05 when compared with non-
0.05 when compared with LPS-
respectively), and also as compared to LPS stimulation alone at
20 μM (average increase of 1.8 fold, p = 0.020). In contrast,
roflumilast exhibited no effect on macrophage recruitment
(Fig. 7B). In this in vivo model, our hit compound 4ba
statistically impacted monocyte recruitment at a concentration
of 200 μM for which it was the only compound able to reduce
the cell infiltrate in response to LPS (average reduction of 69%,
p = 0.002) and tended to decrease, at the same concentration,
macrophage recruitment after stimulation. Importantly, at a
concentration of 2 or 20 μM, compound 4ba also blocks both
the monocyte and macrophage LPS-recruitment (p > 0.05 vs.
control). These results highlighted a few significant effects of
anti-PDE4 molecules possessing a pyridazinone scaffold (vs.
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compound possesses neither a cytotoxic effect nor an abnormal
pro-inflammatory role and we highlighted that this PDE4
inhibitor 4ba was able to impact monocyte recruitment in vivo.
While other structural modifications are currently under
investigation to modulate the physico-chemical properties of
this class of compounds, our hit should be useful to investigate
the role of PDE4 in various pulmonary disease models.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Financial support from the French National Centre for Scientific
Research (CNRS), the Regional Council of Champagne-Ardenne
(France), the French Ministry of Higher Education and Research
(MESR) and the European Regional Development Fund (FEDER)
to the PlAneT and URCACyt CPER projects is acknowledged. A.
M.'s Ph.D. fellowship was co-funded by Reims-Metropole and
the European Union (Europe invests in Champagne-Ardenne
with the European Regional Development Fund). Thanks are
given to the Maison de la simulation de Champagne-Ardenne
(Reims, France) and the Centre Régional Informatique et
d'Applications Numériques de Normandie (CRIANN, Rouen,
France) for the provision of computational facilities. The
authors also thank the PICT-URCA platform (University of
Reims Champagne-Ardenne) for imaging core facilities.
Fig.
7 Quantification of monocytes (A) and macrophages (B) in
murine air-pouch exudate after 6 h of inflammation induction and
treatment with DMSO (control conditions), zardaverine at 2 μM, 20
μM, and 200 μM, roflumilast at
2 μM, 20 μM, and 200 μM or
compound 4ba at 2 μM, 20 μM and 200 μM. n = 8–15, *p < 0.05 vs.
control, #p < 0.05 vs. LPS.
Notes and references
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roflumilast) on monocyte/macrophage recruitment, but some
interesting trends in dose effect may be observed, especially for
monocytes.
Taking together our results, we can speculate that
pyridazinone scaffold-based derivatives and more specifically
our derivative 4ba are interesting molecules to limit
inflammatory processes, fighting against both immune cell
activation (via IL-1, TNF or IL-6) and also inflammatory cell
recruitment (via IL8) thanks to inflammatory mediator
regulated production.
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Conclusions
In conclusion, a new family of molecules possessing a
pyridazinone scaffold, bearing an indole moiety in position 4,
were obtained in a few steps from a levulinic acid derivative
and were evaluated for their potential anti-inflammatory
activity. After screening, hit-detection and docking/IGM
analysis to rationalize the binding mode of our ligands, we
have identified 4-(5-methoxy-1H-indol-3-yl)-6-methylpyridazin-
3(2H)-one 4ba to possess promising activity and selectivity
towards the PDE4B isoenzyme and be able to regulate potent
pro-inflammatory cytokine and chemokine production by
macrophages. Of physiological relevance is that our hit
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