Synthesis and biological evaluation of pyridazinone derivatives as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2018.01.035

Cyclic nucleotide phosphodiesterase type 4 (PDE4), that controls intracellular level of cyclic nucleotide cAMP, has aroused scientific attention as a suitable target for anti-inflammatory therapy in respiratory diseases. Here we describe the development of two families of pyridazinone derivatives as potential PDE4 inhibitors and their evaluation as anti-inflammatory agents. Among these derivatives, 4,5-dihydropyridazinone representatives possess promising activity, selectivity towards PDE4 isoenzymes and are able to reduce IL-8 production by human primary polymorphonuclear cells.

Full text of DOI:10.1016/j.ejmech.2018.01.035

Accepted Manuscript
Synthesis and biological evaluation of pyridazinone derivatives as potential anti-
inflammatory agents
Chantal Barberot, Aurélie Moniot, Ingrid Allart-Simon, Laurette Malleret, Tatiana
Yegorova, Marie Laronze-Cochard, Abderrazzaq Bentaher, Maurice Médebielle,
Jean-Philippe Bouillon, Eric Hénon, Janos Sapi, Frédéric Velard, Stéphane Gérard
PII:
S0223-5234(18)30048-5
10.1016/j.ejmech.2018.01.035
EJMECH 10112
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 October 2017
Revised Date: 10 January 2018
Accepted Date: 11 January 2018
Please cite this article as: C. Barberot, Auré. Moniot, I. Allart-Simon, L. Malleret, T. Yegorova, M.
Laronze-Cochard, A. Bentaher, M. Médebielle, J.-P. Bouillon, E. Hénon, J. Sapi, Fréé. Velard, Sté.
Gérard, Synthesis and biological evaluation of pyridazinone derivatives as potential anti-inflammatory
agents, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.035.
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ACCEPTED MANUSCRIPT
H
N
N
O
MeO
N
3c
H
PDE4
IL-8

ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of Pyridazinone derivatives
as Potential Anti-inflammatory Agents
a
b
a
c
Chantal Barberot, Aurélie Moniot, Ingrid Allart-Simon, Laurette Malleret, Tatiana
d
a
c
e
Yegorova, Marie Laronze-Cochard, Abderrazzaq Bentaher, Maurice Médebielle, Jean-
d
a
a
b
*a
Philippe Bouillon, Eric Hénon, Janos Sapi, Frédéric Velard and Stéphane Gérard
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, 51096
Reims, France.
b) Université de Reims-Champagne-Ardenne, EA 4691 Biomatériaux & Inflammation en site OSseux
(
BIOS), UFR Pharmacie and UFR Odontologie, 51 rue Cognacq-Jay, 51096 Reims, France
c) Centre International de Recherche en Infectiologie (CIRI), EA7426, Faculté de Médecine Lyon-Sud,
65 chemin du Grand Revoyet, 69921 Oullins, France
1
d) Normandie Univ, INSA Rouen, UNIROUEN, CNRS, COBRA (UMR 6014), 76000 Rouen, France
e) Univ Lyon, Université Lyon 1, CNRS, INSA, CPE-Lyon, ICBMS, UMR 5246, 43 bd du 11 Novembre
1
918, 69622 Villeurbanne, France
Abstract : Cyclic nucleotide phosphodiesterase type 4 (PDE4), that controls intracellular
level of cyclic nucleotide cAMP, has aroused scientific attention as a suitable target for anti-
inflammatory therapy in respiratory diseases. Here we describe the development of two
families of pyridazinone derivatives as potential PDE4 inhibitors and their evaluation as anti-
inflammatory agents. Among these derivatives, 4,5-dihydropyridazinone representatives
possess promising activity, selectivity towards PDE4 isoenzymes and are able to reduce IL-8
production by human primary polymorphonuclear cells.
Keywords : Pyridazinone - phosphodiesterase inhibitors - anti-inflammatory - PDE4
Introduction
The chemistry of pyridazinones has been an interesting field of study since decades
and this six membered ring has then become a scaffold of choice for the development of
potential drug-candidates [1]. Thus these nitrogen-rich heterocyclic derivatives have been
known to exhibit many pharmacological actions against ulcer [2] or cardiovascular diseases
[
3] or as anti-proliferative agents [4,5]. Development of new pyridazinone-based analgesic
and anti-inflammatory derivatives acting as selective COX-2 inhibitors was recently described
as well as the design of pyrazolo pyrimidopyridazinones for the treatment of erectile
dysfunction [6,7]. An identified therapeutic application of such scaffold is its anti-
inflammatory activity by targeting phosphodiesterases and our interest in this heterocyclic
system stems from the pyridazinone type PDE3/PDE4 dual inhibitor, Zardaverine (Scheme 1),
1

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which reached clinical development for the treatment of chronic obstructive pulmonary
disease (COPD) [8].
In recent years, phosphodiesterases, a superfamily of eleven intracellular enzymes,
responsible for the hydrolysis of cyclic adenosine and/or guanosine monophosphate
(intracellular second messengers facilitating the action of hormones and inflammatory
mediators) have emerged as new therapeutic targets with special attention given to
phosphodiesterase type-4 (PDE4). From a biological point of view, PDE4 is the predominant
isoenzyme found in inflammatory and immunomodulatory cells and is overexpressed in many
airway cells such as epithelial and immune cells including neutrophils, T-cells, and
macrophages [9]. 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 [10]. More recently
PDE4 inhibition also demonstrated positive effects against aberrant immune response diseases
such as atopic dermatisis, rheumatoid arthritis or psoriasis or in the modulation of metabolic
disorders such as obesity or type 2 diabetes [11]. Although a large number of PDE4 inhibitors
have been clinically evaluated, the use of first-generation 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) [12,13]. Additionally,
Barreiro et al. also showed the assessment of N-acyl hydrazones (possessing bioisosteric
relationship with the pyridazinone ring) as conformationally constrained PDE4 inhibitors to
limit the inflammatory response observed in pulmonary diseases [14] while biphenyl
pyridazinone derivatives were recently developed by Gracia et al. as inhaled PDE4 inhibitors
[
15].
In continuation of our efforts to develop selective inhibitors of PDE4 for the treatment
of respiratory diseases possessing fewer side effects than marketed compounds [16], we
describe herein the synthesis of the first representatives of two new classes of pyridazinone
derivatives (structures A and B, scheme 1) and their evaluation as potential anti-inflammatory
agents. As dialkoxyphenyl containing molecules have shown to be selective inhibitors of
PDE4 [17], probably by formation of hydrogen bonds with a glutamine residue at the back of
the catalytic site [18], we chose to introduce alkoxy or catechol moieties on the pyridazinone
scaffold of compounds of A family. Simultaneously the design of B family was based on the
work of Pieretti and Dal Piaz [19,20]. They developed constrained pyridazinones fused with
heterocyclic nucleus (thiophene or isoxazole) as possible anti-inflammatory agents. In
2

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2
SO Me
O
O
Cl
OEt
N
O
NH
N
O
O
O
F
O
O
OMe
F
N NH
Cl
OCHF2
Zardaverine
Roflumilast
Apremilast
O
O
R3
Het
R3
N
N
N
N
1
R O
R2
A
B
Scheme 1: Structures of Zardaverine, approved drugs Roflumilast and Apremilast and the two targeted
families
complementary manner Timmerman focused his work on dihydropyridazinones possessing
cis-fused cyclohexane ring as PDE4 inhibitors [21]. So, in our derivatives, we would like to
introduce a degree of conformational freedom between pyridazinone scaffold and heterocyclic
moiety in order to study if these constrained conformations can influence potency and
selectivity over PDE4. Preparation of the targeted compounds was efficiently achieved by
using two flexible stepwise procedures.
Results and discussions
Chemistry
First representatives of pyridazinone derivatives of A family were obtained by
condensation of hydrazines onto 5-arylidenetetronates intermediates prepared from the
corresponding methyl or benzyl tetronates by our aldolisation/deshydratation one pot
sequence (Scheme 2) [22]. This approach furnished pyridazinones bearing 5-alkoxy
substituent 1a-f in moderate to good yields (Table 1).
3

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Scheme 2: Synthesis of 5-alkoxy-pyridazinones
1
2
3
Entry
Compound
R
R
R
Yield (%)
1
2
3
4
5
6
1a
1b
1c
1d
1e
1f
Me
Me
Bn
Bn
Bn
Bn
Ph
H
Me
H
52
41
27
50
88
59
Ph
Ph
Ph
Me
H
3,4-(MeO) C H
2
6
3
3,4-(MeO) C H
Me
2
6
3
Table 1: Isolated yields of 5-alkoxy-pyridazinones
We then turn our attention to the preparation of compounds of B family. Theses
pyridazinone derivatives were obtained by a three-step sequence starting from levulinic acid
(Scheme 3) [23]. After preparation of the corresponding α,β-unsaturated levulinate [24],
regioselective introduction of the heterocyclic system was carried out by Friedel-Crafts type
reaction leading to intermediate compounds 2a-h [25]. Both aliphatic and aromatic
heterocycles were used. As shown in the Table 2, formation of the corresponding adduct
proceeded in about 48h in moderate to good yields. Different indoles as well as benzotriazole
could be used in this condensation. Moreover, the use of C-3 substituted indole offered the
possibility to obtain condensation in the less reactive 2 position (entry 4). Addition product of
morpholine and N-methylpiperazine could also be obtained in satisfactory yields while dimer
adduct of levulinate was the major product of the reaction with piperazine (entry 7).
Subsequent condensation of intermediates 2a-h with hydrazine or methylhydrazine led to the
formation of the desired 4,5-dihydropyridazinones 3a-h functionalized by a heterocyclic
moiety in position 4 with good yields.
H
Het
O
O
1) EtOH, H SO
2 4 O
R3
Het
O
Het
R NHNH
EtOH or
AcOH
2
) Br , CHCl3
2
N
N
CO H
CO Et
2
2
CO Et
2
3
3
) Et N
PTSA (cat)
Toluene, ∆
2
3
2
a-h
3a-h
Scheme 3: Synthesis of 4,5-dihydropyridazinones bearing heterocycle in position 4
4

ACCEPTED MANUSCRIPT
3
Compound
Compound
(Yield (%))
Entry
Heterocycle (Het)
R
a
a
(
Yield (%))
H
3a (61 %)
1
2a (53 %)
N
H
Me
3i (81 %)
2
2b (56 %)
2c (55 %)
H
H
3b (53 %)
3c (76 %)
N
MeO
3
4
N
H
2d (69 %)
2e (65 %)
H
H
3d (54 %)
3e (15 %)
N
H
N
N
N
5
6
7
H
3f (45 %)
3g (48 %)
N
O
2f (76 %)
Me
b
N
2g (38 %)
2h (57 %)
H
H
/
HN
N
N
8
3h (85 %)
Table 2: Synthesis of 4,5-dihydropyridazinones bearing heterocycle in position 4 [note: a) yield of
isolated product; b) dimer product with traces of monomer (see experimental section)]
PDE4 inhibition
All synthesized compounds were then screened for their potential inhibitory activity
on PDE4 (Table 3). The evaluation approach, based on a "non radioactive" assay recently
developed in our laboratory [16a], could be directly applied for the selection of privileged
pyridazinone derivatives during "hit detection" step. Results were expressed as inhibition
percentage of PDE4 at an initial concentration of 500 µM. For the most potent hits,
preliminary PDE selectivity (PDE1 isoform) and evaluated IC are also collected in Table 3.
5
0
Preliminary results of in-vitro evaluations performed on the two series of molecules
showed better activity of B family members rather than 5-alkoxy-pyridazinones A although
5

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introduction of catechol moiety slightly increased potency of this pyridazinone scaffold
(entries
a,b
a,b
b,c
Entry
Compound
PDE4 (%) PDE1 (%)
IC50
(µM)
1
2
3
4
5
6
7
8
9
1a
43
53
39
46
67
61
87
59
91
89
21
43
94
38
53
nd
nd
nd
nd
nd
nd
23
nd
9
nd
nd
nd
nd
nd
nd
32
nd
23
nd
nd
nd
35
nd
nd
2
1b
1c
1d
1e
1f
3a
3b
3c
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
3d
22
nd
31
34
nd
nd
3e
3f
3g
3h
3i
d
d
Zardaverine
IBMX
74
28
25
92
d
d
38
Table 3: Biochemical evaluation of pyridazinones as PDEs inhibitors [note: a) inhibition at initial
concentration of 500 µM; b) mean of two or three experiments, each performed in triplicate, protocols
are given in the experimental section; c) IC50 on PDE4 evaluated by non-linear regression; d)
inhibition at 40 µM; nd: not determined]
5
and 6). Indeed, most of 4,5-dihydropyridazinones bearing heterocycle in position 4 possess
significant inhibitory activity (IC50 = 20-40 µM for the best compounds) and promising
selectivity profile for PDE4 (vs PDE1) compared to reference substances: Zardaverine (IC =
5
0
2
µM and 25% PDE1 inhibition at 40 µM in our conditions) and non-specific PDE inhibitor 3-
isobutyl-1-methylxanthine (IBMX).
First in-vitro results obtained on the B family showed that all of 4,5-
dihydropyridazinones bearing indole moiety on the 4-position of the heterocyclic scaffold
exhibited quite similar activities and selectivities with interesting percentages of PDE4
inhibition. Our attention turned to the N-substituent of the indole and pyridazinone rings. It
was determined that these hydrogen bond donor functions were optimal for the affinity for
PDE4 in so far as N-methyl derivatives 3b and 3i appeared about 1.5-fold less potent than the
6

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corresponding analogue 3a. In this family, replacing indole by benzotriazole drastically
reduced the affinity for PDE4 receptors, thus 3e was found to be practically inactive at
screening concentration. Interestingly, contrary to our initial expectation, decreasing
hydrophobic character of the heterocyclic moiety (3g vs 3c) slightly modified the inhibitory
effect but might have an impact on selectivity.
Comparison of these different pharmacological data showed that the more active
pyridazinone is the compound 3c functionalized with 5-methoxyindole moiety possessing
9
1% of inhibition of PDE4 at 500 µM and IC50 about 20 µM. However this compound
remained about 10-fold less active than our reference compound Zardaverine but displayed a
better selectivity towards PDE1. This compound 3c, and its analog 3a, were selected for
further investigations.
Molecular modeling
After screening and hit-detection a challenging task is to address the binding mode of
our ligands. The protein structure was prepared from the available PDB structure 1MKD
(PDE4D isoform, see computational details section) and molecular docking simulations were
performed on the most active compounds. 3D representations of interactions regions are
reported for two of them on Figures 1 and 2. As pointed out by a previous study [26],
browsing the first ranked poses of all compounds shows two key features: (1) hydrogen
bonding (HB) with Gln466 and (2) van der Waals (vdW) interactions on both sides of the
ligand fitting an oblong cavity formed by residues Ile433, Phe437 and Phe469. This is in line
with the X-ray pose of the Zardevarine in PDE4 [27]. Accordingly, in our 4,5-
dihydropyridazinone 3a (pose not reported here), the planar indole moiety is more appropriate
than the pyridazinone motif to fit in the hydrophobic pocket, as observed in the top-ranked
0
-1
pose obtained (∆ G = -8.7 kcal.mol ). However, in that case, no HB can be observed between
r
Gln466 and the indole part (HB-donor). Similarly (see Figure 1a), the best pose obtained for
0
-1
3
c (∆ G = -8.7 kcal.mol ) also puts the planar indole group in the oblong hydrophobic pocket
r
of PDE4. But the presence of the methoxy group in 3c allows for additional interactions,
either vdW contacts with residues Trp429 and Asn418 (Figure 1b, pose 1), or even HB with
Gln466 (Figure 1c, pose 2). In that case, the methyl group can also interact with Met454 as
clearly revealed by the corresponding weak-interaction iso-surfaces. Although the Vina scores
for 3a and 3c best poses are identical, the additional conformational possibilities provided by
7

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the methoxy group could explain the better affinity observed for 3c. On the other hand, the
pyridazinone motif interacts with Met370 and can also establish HB with His257 as illustrated
on Figure 1c. It is worth noting that the ligand doesn’t interact directly with the two metal
ions but rather with the coordinated water molecules. On Figure 1a, the atoms are colored
according to their contribution to the interaction (NCI/IGM analysis [28], a new methodology
derived from NCI). This analysis clearly shows that the indole moiety primarily contributes to
the interactions in this complex. Finally, a pose is presented on Figure 1d where the
-1
pyridazinone moiety fills the oblong hydrophobic pocket (-7.6 kcal.mol , pose 5). Obviously
it is not a favorable situation because the strong HB interaction with the key residue Gln466 is
lost in that case.
Figure 1. Analysis of non-covalent interactions (NCI) between PDE4 and 3c, using the NCI/IGM
method derived from the NCI tool; poses obtained with software Autodock-Vina [28]; (a) atoms of the
complex colored according to their contribution to the interaction iso-surfaces described in panel b; a
grey-to-red color scheme is used in the range 0 to 2.89, with grey: no contribution to the NCI and red:
8

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-1
largest relative contribution to the NCI; (b) top-ranked pose: -8.7 kcal.mol ; (c) second pose: -7.8
-1
-1
inter
kcal.mol ; (d) fifth pose: -7.6 kcal.mol ; every iso-surface has been obtained using δg =0.007 a.u.
and is colored according to the BGR scheme over the signed electron density range -0.05 < sign(λ )ρ
2
<
0.05 a.u.
In addition we would like to understand the pharmacological data obtained for 4,5-
dihydropyridazinone 3g. This complementary work indicates a different positioning of this
-1
compound (Fig 2). Here, the best pose (-6.6 kcal.mol , pose 1) displays the non-planar
pyridazinone motif filling the hydrophobic pocket. The interaction iso-surfaces reveal a
substantial HB interaction with Gln466. However, according to the atomic analysis of the
interactions (Figure 2a), the oxygen and nitrogen atoms of the heterocyclic moiety hardly
contribute to the interactions. The ligand affinity and lower selectivity observed for this
species could be explained by this less effective binding mode. Finally, it can be noted that
our compounds are too small to reach an additional hydrophobic channel present in the active
site (at the bottom right of Figures 1a and 2a). This theoretical study establishes a first
interesting basis for compound comparison and points out key features for efficient binding.
Further investigations (molecular dynamics for instance) might provide better insight in the
observed affinities and selectivities by introducing the protein flexibility into the simulations.
However, theoretical computations are nonetheless limited and cannot capture the overall
complex biological response.
Figure 2. Analysis of non-covalent interactions (NCI) between PDE4 and 3g, using the NCI/IGM
method derived from the NCI tool; poses obtained with software Autodock-Vina; (a) atoms of the
complex colored according to their contribution to the interaction iso-surfaces described in panel b; a
grey-to-red color scheme is used in the range 0 to 2.89, with grey: no contribution to the NCI and red:
-1
largest relative contribution to the NCI; (b) top-ranked pose: -6.6 kcal.mol ; iso-surfaces have been
9

ACCEPTED MANUSCRIPT
inter
obtained using δg
=0.007 a.u. and are colored according to the BGR scheme over the electron
density range -0.05 < sign(λ )ρ < 0.05 a.u.
2
Biology
The two selected compounds 3a and 3c were then evaluated for their in vitro cellular
activity in the blockade of chemokine production. We first evaluated the toxicity of PDE4
inhibitors in pro-inflammatory environment by measuring LDH release by human
polymorphonuclear leukocytes (PMNs) (Figure 3). Test compounds were dissolved in culture
media containing 0.5% DMSO with no evidence of lack of solubility. After four hours of
culture in presence of lipopolysaccharide (LPS), PMNs supernatants showed a slight but
significant increase (20% increase of median value) in LDH activity which was not retrieved
in presence of PDE4 inhibitors as compared to the control condition. Such a tolerance toward
PDE4 inhibitors was already suggested in studies demonstrating their anti-inflammatory
potential against LPS-induced immune cells activation [29].
0
,4
,35
,3
,25
,2
,15
,1
0
0
0
0
*
0
0
0
,05
0
Figure 3: Cell death evaluation by measurement of LDH activity in human PMNs culture supernatants
st
th
(
n=10). Red bar represents median value. Black bars represent 1 and 9 decile and limits of
st
rd
rectangles represent 1 and 3 quartile. * p < 0.05 when compared with non-stimulated (control) cells.
1
0

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We then measured the inhibition of IL-8 production in the PMNs to give a translation
between the in vitro enzymatic potency and cell-based assay. IL-8 production by PMNs in
response to pro-inflammatory molecules is a very well described phenomenon. Here we
confirmed in our model that LPS is able to induce an increased IL-8 secretion by human
primary PMNs (3 fold increase of median value). Of interest, we highlighted that PDE4
inhibitors 3a and 3c were able to statistically reduce this IL-8 production in 7 and 9 of 10
donors respectively (10% and 15% reduction of median value) whereas Zardaverine failed to
demonstrate such an effect (Figure 4).
#
4
3
3
2
2
1
1
00
50
00
50
00
50
00
*
*
*
#
*
5
0
0
Figure 4: ELISA measurements of IL-8 concentrations in unexposed or LPS-exposed cell supernatants
st
th
after 4 h. Red bar represents median value. Black bars represent 1 and 9 decile and limits of
st
rd
rectangles represent 1 and 3 quartile. n=10 independent experiments. * p < 0.05 when compared
with nonstimulated (control) cells. # p < 0.05 when compared with LPS-stimulated cells. n=10
independent experiments.
As IL-8 is both a potent activator and also a powerful chemoattractant for PMNs, we
next assessed the effect of anti-PDE4 compounds on PMNs migratory abilities. Our data
indicated that rhIL-8 induced an increased number of migrating PMNs as compared to
spontaneous migration in basal conditions (Figure 5). PMNs ability to follow chemotactic
gradient of IL-8 was impaired with all anti PDE4 compounds. In addition, when cells were
co-incubated with Zardaverine the chemotaxis is statistically reduced versus rhIL-8. In the
1
1

ACCEPTED MANUSCRIPT
same way, compound 3a tended to decrease the migrating cell number (median value
decreased by 50%) but failed to reach statistical threshold. These data are consistent with
previous studies highlighting neutrophils activation and chemotaxis impairments after PDE4
inhibition independently of the initial stimulus [30,31].
6
Figure 5: Boyden chamber chemotaxis assay. PMNs were seeded at 10 per mL in the upper wells and
-1
allowed to migrate for 45 min against rhIL-8 200 ng.mL (lower wells). Migrated cells through the
st
membrane were counted after staining. Red bar represents median value. Black bars represent 1 and
th
st
rd
9
decile and limits of rectangles represent 1 and 3 quartile. Shown are representative photographs
of MGG staining. Scale bar = 25 µm. Objective x40, Zeiss Axiovert 200M. n=8 independent
experiments. * p < 0.05 when compared with basal migrating cells. # p < 0.05 when compared with
rhIL-8-chemoattracted cells.
Conclusion
In conclusion, two new families of molecules possessing pyridazinone scaffold were
obtained in few steps from 5-arylidenetetronates or levulinic acid derivative and were
evaluated for their potential anti-inflammatory activity. After screening and hit-detection a
theoretical study (NCI/IGM method) helped us to rationalize the binding mode of our ligands.
Among the prepared compounds, 4,5-dihydropyridazinones 3a and 3c bearing an heterocyclic
moiety (indole and 5’-methoxyindole, respectively) on the 4-position exhibited PDE4
selectivity vs PDE1 and interesting activity (IC50 ~ 20 and 30 µM, respectively). Of
1
2

ACCEPTED MANUSCRIPT
physiologic relevance, identified hits 3a and 3c possess neither cytotoxic effect nor abnormal
pro-inflammatory role on polymorphonuclear leukocytes cells as judged by LDH cytotoxicity
assay and we highlighted that PDE4 inhibitors 3c was able to reduce IL-8 production. Other
structural modifications are currently under investigation to enhance the activity and modulate
physico-chemical properties of this class of compounds.
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 CPER project is acknowledged. C.B. thanks the Regional Council of Champagne–
Ardenne (France) for a Ph.D. fellowship. A.M. Ph.D. fellowship is 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. Thanks to ANR-11-EMMA-028-03-03 and “Fonds Agir pour les Maladies
Chroniques” for research support to A.B.
1
3

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6] J. Singh, D. Sharma, R. Bansal, Pyridazinone: An attractive lead for anti-inflammatory
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Zhang, Phosphodiesterase-4 as a therapeutic target, Drug Discov. Today 10 (2005) 1503-
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A. Martinez, C. Gil, cAMP-specific phosphodiesterase inhibitors: promising drugs for
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A.M. Mulhall, C.A. Droege, N.E. Ernst, R.J. Panos, M.A. Zafar, Phosphodiesterase 4
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13] H. Abdulrahim, S. Thistleton, A.O. Adebajo, T. Shaw, C. Edwards, A. Wells,
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15] J. Gracia, M. A. Buil, J. Castro, P. Eichhorn, M. Ferrer, A. Gavalda, B. Hernandez, V.
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Andres, J. Cabedo, D. Vilella, E. Calama, C. Carcasona, M. Miralpeix, Biphenyl pyridazinone
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3-(cyclopentyloxy)-4-methoxyphenyl]hexahydro-3H-pyrrolizin-3-one via reductive domino
[
transformations of 3-β-carbomethoxyethyl-substituted six-membered cyclic nitronates, J. Org.
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18] Q. Huai, H. Wang, Y. Sun, H.-Y. Kim, Y. Liu, H. Ke, Three-dimensional structures of
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1
6

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259-6269.
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23] For review on the chemistry of levulinic acid, see: B.V. Timokhin, V.A. Baransky, G.D.
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AlgoGen: a tool coupling a linear scaling-quantum method with a genetic algorithm for
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Ther. 18 (2005) 93-101.
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8

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Experimental section
Chemistry
General
Solvents : Solvents were dried and purified by standard literature methods prior to use.
Reagents : Reagents were bought from Acros Organics and Sigma-Aldrich and were used without
further purification.
Instrumentation : Reactions were monitored on silicagel plates (Silicagel 60F254 from Merck) and
column chromatography was carried out on silica gel 60 (70-200 µm).
1
13
Proton and Carbon Nuclear Magnetic Resonance spectra ( H NMR C NMR) were recorded on a
Bruker DPX 300 apparatus at 300 and 75 MHz respectively, in CDCl . Chemical shifts are reported in
3
parts per million (ppm) downfield from tetramethylsilane (TMS) as internal standard followed by
multiplicity in which s, bs, d, t, q, dd, dt, td, tt, m are respectively singlet, broad singlet, doublet,
triplet, quadruplet, dedoubled doublet, dedoubled triplet, detripled doublet, detripled triplet and
multiplet (or unwell-resolved signals). Coupling constants J are quoted in Hertz (Hz). Bidimensional
NMRs (COSY, HSQC and HMBC) were used to assign signals.
Infrared spectra (IR) (thin film or KBr pastille) were measured on a Perkin-Elmer SPECTRUM
-1
BX/RX Fourier transform spectrometer. Principal absorption bands are given in cm .
Electronic Impact (EI), Chemical Ionisation (CI), ElectroSpray Ionisation (ESI) Mass Spectra (MS)
and High Resolution Mass Spectra (HRMS) were either recordedc with an ESI-Q-TOF mass
spectrometer from Waters or with a GCT CA 170 Micromass Waters apparatus. The (+)ESI Mass
Spectra were performed in a positive mode with CH OH as solvent. The masses are measured in
3
Dalton (Da). 5-Arylidenetetronate precursors were prepared by in situ aldolisation/dehydration
sequence according to reference 18.
General procedure for the formation of the 5-alkoxy-pyridazinone 1a-f:
To a solution of furan-2(5H)-one (1 eq.) in methanol (10 mL/1 mmol) is added hydrazine (from 2 to 5
eq.). The mixture is stirred at reflux during 12 hours. After cooling, the pyridazinone usually
precipitates. The solid is then filtered and washed with a little amount of cold methanol to afford the
desired compound. The mother liquor is then evaporated under reduced pressure and the resulting
residue is recrystallized in hot MeOH or diethyl ether (for compounds 1a,c,e) or is chromatographed
on silica gel (eluent: petroleum ether/AcOEt or CH Cl /AcOEt, for compounds 1b,d,f). Overall yields
2
2
were calculated on the combined fractions.
1
9

ACCEPTED MANUSCRIPT
5
-Methoxy-6-benzylpyridazin-3(2H)-one 1a. According to the general procedure, and starting from
1
00 mg of (Z)-4-methoxy-5-benzylidenefuran-2(5H)-one and 80 µL of hydrazine (35 % w/t in water),
pure compound 1a is obtained after filtration followed by recrystallisation in methanol as a white
powder. Yield: 55 mg (52 %).
1
Mp: 125-127 °C. H NMR (300 MHz, CD OD) δ (ppm) = 3.84 (s, 3 H), 3.95 (s, 2 H), 6.22 (s, 1 H),
3
1
3
7
.1-7.3 (m, 5 H). C NMR (75 MHz, CD OD) δ (ppm) = 35.5, 56.0, 103.2, 126.3, 128.3 (2 C), 128.7
3
-1
(
2 C), 137.6, 141.6, 159.6, 162.6. IR (KBr) ν (cm ) = 3200, 2943, 2866, 1677, 1625, 1589, 1451,
+
+
+
+
+
1
245. MS (ESI ) m/z 455 [2M+Na] , 433 [2M+H] , 239 [M+Na] , 217 [M+H] .
5
-Methoxy-6-benzyl-2-methylpyridazin-3(2H)-one 1b. According to the general procedure, and
starting from 110 mg of (Z)-4-methoxy-5-benzylidenefuran-2(5H)-one and 153 µL of methyl-
hydrazine, pure compound 1b is obtained after chromatography on silica gel (eluent: petroleum
ether/AcOEt, 2/8) as a white powder. Yield: 51 mg (41 %).
1
Mp: 239-241 °C. H NMR (300 MHz, CD OD) δ (ppm) = 3.71 (s, 3 H), 3.84 (s, 3 H), 3.96 (s, 2 H),
3
-
1
6
1
.26 (s, 1 H), 7.2-7.4 (m, 5 H). IR (KBr) ν (cm ) = 3027, 2942, 1650, 1591, 1455, 1404, 1336, 1243,
+
225. HRMS (ESI; [M+Na] ) calculated for C H N O Na 253.0953, found 253.0950.
1
3
14
2
2
5
-Benzyloxy-6-benzylpyridazin-3(2H)-one 1c. According to the general procedure, and starting from
6
0 mg of (Z)-4-benzyloxy-5-benzylidenefuran-2(5H)-one and 34 µL of hydrazine (35 % w/t in water),
pure compound 1c is obtained after filtration followed by recrystallisation in methanol as a white
powder. Yield: 17 mg (27 %).
1
Mp: 221-223 °C. H NMR (300 MHz, DMSO-d ) δ (ppm) = 3.88 (s, 2 H), 5.10 (s, 2 H), 6.25 (s, 1 H),
6
1
3
7
.1-7.5 (m, 10 H), 12.5 (bs, 1 H). H). C NMR (75 MHz, DMSO-d6) δ (ppm) = 35.9, 69.7, 104.3,
1
26.3, 127.6 (2C), 128.2, 128.3 (2C), 128.4 (2C), 128.7 (2C), 135.2, 137.6, 141.7, 158.4, 162.6. IR
-
1
(
[
KBr) ν (cm ) = 3200, 3061, 2966, 2854, 1753, 1627, 1590, 1555, 1451, 1422, 1256. HRMS (ESI;
+
M+H] ) calculated for C H N O 293.1290, found 293.1284.
1
8
17
2
2
5
-Benzyloxy-6-benzyl-2-methylpyridazin-3(2H)-one 1d. According to the general procedure, and
starting from 150 mg of (Z)-4-benzyloxy-5-benzylidenefuran-2(5H)-one and 152 µL of
methylhydrazine, pure compound 1d is obtained after chromatography on silica gel (eluent: petroleum
ether/AcOEt, 2/8) as a white powder. Yield: 83 mg (50 %).
1
Mp: 161-163 °C. H NMR (300 MHz, CD OD) δ (ppm) = 3.73 (s, 3 H), 3.98 (s, 2 H), 5.08 (s, 2 H),
3
1
3
6
.31 (s, 1 H), 7.1-7.5 (m, 10 H). C NMR (75 MHz, DMSO-d6) δ (ppm) = 36.0, 38.6, 69.9, 104.1,
2
0

ACCEPTED MANUSCRIPT
1
26.4, 127.5 (2C), 128.1, 128.3 (2C), 128.4 (2C), 128.5 (2C), 135.1, 137.5, 141.3, 157.9, 161.1. IR
-1
+
(
KBr) ν (cm ) = 3025, 2933, 1657, 1593, 1436, 1375, 1338, 1245. HRMS (ESI; [M+H] ) calculated
for C H N O 307.1447, found 307.1450.
1
9
19
2
2
5
-(Benzyloxy)-6-(3,4-dimethoxybenzyl)pyridazin-3(2H)-one 1e. According to the general procedure,
and starting from 33 mg of (Z)-4-benzyloxy-5-(3,4-dimethoxybenzylidene)furan-2(5H)-one and 18 µL
of hydrazine (35 % w/t in water), pure compound 1e is obtained after filtration followed by
recrystallisation in diethyl ether as a beige powder. Yield: 30 mg (88 %).
1
Mp: 166-168 °C. H NMR (300 MHz, CDCl ) δ (ppm) = 3.69 (s, 3 H), 3.86 (s, 3 H), 3.88 (s, 2 H),
3
4
.97 (s, 2 H), 6.17 (s, 1 H), 6.68-6.78 (m, 3 H), 7.21-7.24 (m, 2 H), 7.36-7.38 (m, 3 H), 10.47 (bs, 1
1
3
H). C NMR (75 MHz, CDCl ) δ (ppm) = 36.5, 55.8, 56.0, 70.7, 104.3, 111.2, 112.4, 121.1, 127.9 (2
3
+
.
C), 128.8 (3 C), 129.7, 134.3, 143.8, 147.8, 148.8, 159.7, 164.5. MS (EI) m/z 352 [M ], 261, 230, 227,
+.
9
1. HRMS (EI; [M ]) calculated for C H N O 352.1423, found 352.1429.
20 20 2 4
5
-(Benzyloxy)-6-(3,4-dimethoxybenzyl)-2-methylpyridazin-3(2H)-one 1f. According to the general
procedure, and starting from 15 mg of (Z)-4-benzyloxy-5-(3,4-dimethoxybenzylidene)furan-2(5H)-one
and 14 µL of methylhydrazine, pure compound 1f is obtained after flash column chromatography
(
dichloromethane/ethyl acetate, from 8/2 to 7/3) as a yellow solid. Yield: 10 mg (59 %).
1
Mp: 140-142 °C. H NMR (300 MHz, CDCl ) δ (ppm) = 3.68 (s, 3 H), 3.74 (s, 3 H), 3.85 (s, 3 H),
3
3
.89 (s, 2 H), 4.95 (s, 2 H), 6.22 (s, 1 H), 6.69-6.78 (m, 3 H), 7.18-7.21 (m, 2 H), 7.34-7.36 (m, 3 H).
1
3
C NMR (75 MHz, CDCl ) δ (ppm) = 36.6, 39.5, 55.8, 56.0, 70.7, 104.4, 111.2, 112.4, 121.0, 127.8
3
+.
(
2 C), 128.8, 128.9 (2 C), 130.0, 134.5, 142.6, 147.8, 148.9, 158.7, 162.5. MS (EI) m/z 366 [M ], 275,
+.
2
44, 227, 105, 91. HRMS (EI; [M ]) calculated for C H N O 366.1580, found 366.1586.
21 22 2 4
Typical procedure for the formation of ethyl 4-oxopent-2-enoate:
Ethyl 4-oxopent-2-enoate. Levulinic acid (1 eq) is dissolved in ethanol. Catalytic amount of sulfuric
acid (0.1 eq) is added to the solution. The mixture is then stirred and headed at reflux. After 16 h, the
reaction is stopped and the solvent is evaporated under reduced pressure to yield the ethyl levulinate in
quantitative yield. Then bromine (1 eq) is dropwise added (during a period of 1 hour) to a cold (0°C)
solution of ethyl levulinate (1 eq) in chloroform. After 30 minutes of stirring at 0°C, triethylamine (3
eq) is then dropwise added during 1 hour. The mixture is stirred for 1h30 after which water is added to
the solution. The crude mixture is then extracted by dichloromethane and the combined organic phases
are washed successively with aqueous solutions of hydrochloric acid (10 %) and saturated ammonium
chloride, dried over anhydrous MgSO and concentrated under reduced pressure. The crude mixture is
4
2
1

ACCEPTED MANUSCRIPT
purified by flash chromatography over silica gel using dichloromethane as eluent to yield 47 % of a
1
yellow oil. H NMR (300 MHz, CDCl ) δ (ppm) = 1.33 (t, J= 7.1 Hz, 3 H), 2.37 (s, 3 H), 4.28 (q, J=
3
1
3
7
.1 Hz, 2 H), 6.65 (d, J= 16.1 Hz, 1 H), 7.02 (d, J= 16.1 Hz, 1 H). C NMR (75 MHz, CDCl ) δ
3
-1
(
ppm) = 14.2, 27.6, 60.4, 135.3, 139.7, 165.7, 196.8. IR (KBr) ν (cm ) = 3052, 2982, 1721, 1701,
+
+
1
422, 1291, 1263, 1157. MS (CI) m/z 143 [M+H] , 129. HRMS (CI; [M+H] ) calculated for C H O
7 11 3
1
43.0751, found 143.0708.
General procedure for Friedel-Crafts alkylation:
Ethyl 4-oxopenten-2-oate (1 eq) is dissolved in toluene. One equivalent of heterocyclic compound is
added to the solution with a catalytic amount of para-toluenesulfonic acid (PTSA, 0.1 eq). The
reaction is stirred and heated at reflux. After about 48 hours (monitoring by thin layer
chromatography), the reaction is stopped and the solvent is evaporated under reduced pressure. The
crude mixture is then purified by column chromatography and sometimes cristallized in diethyl ether.
Ethyl 2-(1H-indol-3-yl)-4-oxo-pentanoate 2a. According to the general procedure for Friedel-Crafts
alkylation, starting from 147 mg of ethyl 4-oxopenten-2-oate and 120 mg of indole, the title compound
is isolated after column chromatography (cyclohexane/AcOEt=7/3) followed by recrystallization in
diethyl ether as a beige powder. Yield: 139 mg (53 %).
1
Mp: 116-118 °C. H NMR (300 MHz, CDCl ) δ (ppm) = 1.18 (t, J= 7.1 Hz, 3 H), 2.17 (s, 3 H), 2.83
3
(
dd, J= 17.9, 4.4 Hz, 1 H), 3.51 (dd, J= 18.0, 10.2 Hz, 1 H), 4.01-4.22 (m, 2 H), 4.39 (dd, J= 10.2, 4.5
Hz, 1 H), 7.05 (d, J= 2.7 Hz, 1 H), 7.10-7.22 (m, 2 H), 7.34 (d, J= 8.1 Hz, 1 H), 7.71 (d, J= 7.8 Hz, 1
1
3
H), 8.25 (bs, 1 H). C NMR (75 MHz, CDCl ) δ (ppm) = 14.2, 30.1, 38.0, 46.3, 61.2, 111.5, 113.0,
3
-1
1
19.3, 119.8, 122.2, 122.4, 126.2, 136.4, 173.9, 207.1. IR (KBr) ν (cm ) = 3405, 2919, 1721, 1708,
+
.
1
457, 1413, 1361, 1336, 1323, 1258, 1232, 1173, 1090. MS (EI) m/z 259 [M ], 213, 186, 144, 115.
+
.
HRMS (EI; [M ]) calculated for C H NO 259.1208, found 259.1212.
1
5
17
3
Ethyl 2-(1H-methyl-indol-3-yl)-4-oxo-pentanoate 2b. According to the general procedure for Friedel-
Crafts alkylation with 157 mg of ethyl 4-oxopenten-2-oate and 133 mg of 1-methylindole, the mixture
is heated at reflux during 65 h. The title compound is isolated after column chromatography
(
cyclohexane/ ethyl acetate =8/2) as a brown lacquer. Yield: 153 mg (56 %).
1
H NMR (300 MHz, CDCl ) δ (ppm) = 1.20 (t, J= 7.1 Hz, 3 H), 2.18 (s, 3 H), 2.83 (dd, J= 18.0, 4.5
3
Hz, 1 H), 3.49 (dd, J= 18.0, 10.2 Hz, 1 H), 3.74 (s, 3 H), 4.02 (q, J= 7.1 Hz , 2 H), 4.37 (dd, J= 10.2,
1
3
4
.5 Hz, 1 H), 6.96 (s, 1 H), 7.10-7.31 (m, 3 H), 7.69 (d, J= 7.9 Hz, 1 H). C NMR (75 MHz, CDCl ) δ
3
(
ppm) = 14.2, 30.1, 32.9, 37.8, 46.6, 61.1, 109.5, 111.4, 119.4, 119.5, 122.0, 126.6, 126.8, 137.1,
2
2

ACCEPTED MANUSCRIPT
-
1
1
73.9, 207.0. IR (KBr) ν (cm ) = 3426, 2925, 1721, 1609, 1542, 1470, 1369, 1328, 1155, 1015. MS
+
.
+.
(
EI) m/z 273 [M ], 227, 225, 216, 200, 158, 156. HRMS (EI; [M ]) calculated for C H NO
16 19 3
2
73.1365, found 273.1368.
Ethyl 2-(5-methoxy-1H-indol-3-yl)-4-oxo-pentanoate 2c. According to the general procedure for
Friedel-Crafts alkylation using 154 mg of ethyl 4-oxopenten-2-oate and 151 mg of 5-methoxyindole,
the title compound is isolated after column chromatography (cyclohexane/ ethyl acetate =7/3) as beige
crystals. Yield: 198 mg (55 %).
1
Mp: 137-139 °C. H NMR (300 MHz, CDCl ) δ (ppm) = 1.21 (t, J= 7.2 Hz, 3 H), 2.20 (s, 3 H), 2.83
3
(
dd, J= 18.0, 4.4 Hz, 1 H), 3.50 (dd, J= 18.0, 10.4 Hz, 1 H), 3.87 (s, 3 H), 4.02-4.24 (m, 2 H), 4.35
dd, J= 10.4, 4.4 Hz, 1 H), 6.87 (dd, J= 9.0, 2.4 Hz, 1 H), 7.06 (d, J= 2.4 Hz, 1 H), 7.16 (d, J= 2.4 Hz,
(
1
3
1
H), 7.24 (d, J= 8.7 Hz, 1 H), 8.01 (bs, 1 H). C NMR (75 MHz, CDCl ) δ (ppm) = 14.2, 30.1, 37.9,
3
-
4
6.1, 56.0, 61.1, 100.9, 112.2, 112.5, 112.8, 122.8, 126.6, 131.4, 154.2, 173.9, 207.2. IR (KBr) ν (cm
1
)
= 3333, 2925, 1718, 1708, 1620, 1581, 1483, 1457, 1364, 1302, 1266, 1199, 1170, 1023. MS (EI)
+
.
+.
m/z 289 [M ], 243, 216, 174, 159, 130, 115. HRMS (EI; [M ]) calculated for C H NO 289.1314,
1
6
19
4
found 289.1309.
Ethyl 2-(3-methyl-1H-indol-2-yl)-4-oxo-pentanoate 2d. According to the general procedure for
Friedel-Crafts alkylation starting from 142 mg of ethyl 4-oxopenten-2-oate and 136 mg of 3-
methylindole, the title compound is isolated after column chromatography (cyclohexane/ ethyl acetate
=
6/4) as a brown solid . Yield: 188 mg (69 %).
1
Mp: 115-117 °C. H NMR (300 MHz, CDCl ) δ (ppm) = 1.15 (t, J= 7.2 Hz, 3 H), 2.16 (s, 3 H), 2.45
3
(
s, 3 H), 2.75 (dd, J= 18.0, 5.0 Hz, 1 H), 3.63 (dd, J= 18.0, 9.3 Hz, 1 H), 3.97-4.21 (m, 2 H), 4.37 (dd,
J= 9.3, 5.0 Hz, 1 H), 7.04-7.14 (m, 2 H), 7.25-7.28 (m, 2 H), 7.59 (dd, J= 7.8, 1.5 Hz, 1 H), 7.83 (bs, 1
13
H). C NMR (75 MHz, CDCl ) δ (ppm) = 12.1, 14.3, 30.4, 37.4, 45.3, 61.1, 108.5, 110.5, 118.8,
3
-
1
1
1
19.7, 121.3, 127.2, 132.4, 135.3, 173.8, 207.2. IR (KBr) ν (cm ) = 2971, 2919, 1721, 1702, 1454,
+
.
392, 1369, 1346, 1320, 1248, 1214, 1160, 1088. MS (EI) m/z 273 [M ], 227, 216, 200, 158, 130.
+
.
HRMS (EI; [M ]) calculated for C H NO 273.1365, found 273.1367.
1
6
19
3
Ethyl 2-(benzotriazol-1-yl)-4-oxo-pentanoate 2e. According to the general procedure for Friedel-
Crafts alkylation starting from 148 mg of ethyl 4-oxopenten-2-oate and 125 mg of 1,2,3-benzotriazole,
the title compound is isolated after column chromatography (cyclohexane/ ethyl acetate =7/3) as a
brown oil. Yield: 164 mg (65 %).
2
3

ACCEPTED MANUSCRIPT
1
H NMR (300 MHz, CDCl ) δ (ppm) = 1.15 (t, J= 7.1 Hz, 3 H), 2.26 (s, 3 H), 3.65 (dd, J= 18.2, 7.4
3
Hz, 1 H), 3.79 (dd, J= 18.0, 6.0 Hz, 1 H), 4.12-4.23 (m, 2 H), 5.98 (dd, J= 7.4, 6.2 Hz, 1 H), 7.37-7.42
1
3
(
m, 1 H), 7.50-7.56 (m, 1 H), 7.63 (d, J= 8.4 Hz, 1 H), 8.07 (d, J= 8.4 Hz, 1 H). C NMR (75 MHz,
CDCl ) δ (ppm) = 14.0, 30.1, 44.1, 56.1, 62.7, 109.7, 120.1, 124.4, 128.0, 133.4, 145.8, 168.0, 204.0.
3
-1
IR (KBr) ν (cm ) = 3054, 2982, 2925, 1746, 1718, 1615, 1591, 1491, 1449, 1405, 1367, 1269, 1204,
+.
+.
1
163, 1104, 1015. MS (EI) m/z 261 [M ], 204, 188, 160, 127, 118, 97, 91. HRMS (EI; [M ])
calculated for C H N O 261.1113, found 261.1115.
1
3
15
3
3
Ethyl 2-morpholinyl-4-oxopentanoate 2f. According to the general procedure for Friedel-Crafts
alkylation starting from 284 mg of ethyl 4-oxopenten-2-oate and 174 mg of morpholine, the title
compound is isolated after column chromatography (dichloromethane/methanol=9.5/0.5) as a yellow
oil. Yield: 348 mg (76 %).
1
H NMR (300 MHz, CDCl ) δ (ppm) = 1.22 (t, J= 7.1 Hz, 3 H), 2.12 (s, 3 H), 2.47-2.54 (m, 2 H),
3
1
3
2
.64-2.75 (m, 3 H), 2.97 (dd, J=17.1, 9.2 Hz, 1 H), 3.65-3.75 (m, 5 H), 4.16-4.25 (m, 2 H). C NMR
(
75 MHz, CDCl ) δ (ppm) = 14.4, 30.2, 42.9, 50.2 (2 C), 60.7, 63.0, 67.3 (2 C), 170.6, 206.2. IR
3
-1
+.
(
KBr) ν (cm ) = 3602, 3411, 2961, 2853, 1724, 1713, 1362, 1166, 1111. MS (EI) m/z 229 [M ], 184,
+
.
1
56, 127, 114. HRMS (EI; [M ]) calculated for C H NO 229.1314, found 229.1317.
11 19 4
Diethyl 2,2'-(piperazine-1,4-diyl)bis(4-oxopentanoate) 2'g. According to the general procedure for
Friedel-Crafts alkylation with 284 mg of ethyl 4-oxopenten-2-oate and 172 mg of piperazine, the
compound (yellow oil) isolated after column chromatography (dichloromethane/methanol=9.5/0.5) is
the corresponding dimere adduct (Yield: 38%).
1
H NMR (300 MHz, CDCl ) δ (ppm) = 1.33 (t, J= 7.1 Hz, 6 H), 2.18 (s, 6 H), 2.48 (t, J= 7.8 Hz, 2 H),
3
2
4
.63-2.73 (m, 10 H), 3.08 (dd, J=17.1, 9.3 Hz, 2 H), 3.73 (dd, J= 9.2, 5.0 Hz, 2 H), 4.18 (q, J= 7.1 Hz,
+
.
+.
H). MS (EI) m/z 370 [M ], 327, 297, 239, 155, 127, 97. HRMS (EI; [M ]) calculated for
C H N O 370.2104, found 370.2111.
18
30
2
6
Ethyl 4-oxo-2-(4-methylpiperazin-1-yl)-pentanoate 2h. According to the general procedure for
Friedel-Crafts alkylation starting from 207 mg of ethyl 4-oxopenten-2-oate and 160 µL of 1-
methylpiperazine, the compound is obtained after column chromatography (dichloromethane/
methanol=9/1) as a yellow oil. Yield: 202 mg (57 %).
1
H NMR (300 MHz, CDCl ) δ (ppm) = 1.22 (t, J= 7.1 Hz, 3 H), 2.11 (s, 3 H), 2.23 (s, 3 H), 2.32-2.51
3
(
(
m, 6 H), 2.60 (dd, J= 17.0, 5.3 Hz, 1 H), 2.66-2.73 (m, 2 H), 2.99 (dd, J= 17.1, 9.3 Hz, 1 H), 3.69
1
3
dd, J= 9.3, 5.1 Hz, 1 H), 4.11 (q, J= 7.2 Hz, 2 H). C NMR (75 MHz, CDCl ) δ (ppm) = 14.4, 29.7,
3
2
4

ACCEPTED MANUSCRIPT
+.
3
1
0.3, 43.0 (2 C), 45.7, 48.2, 55.3 (2 C), 60.7, 62.7, 170.7, 206.4. MS (EI) m/z 242 [M ], 199, 169,
+
.
27, 100. HRMS (EI; [M ]) calculated for C H N O 242.1630, found 242.1633.
1
2
22
2
3
General procedure for the formation of the 4,5-dihydropyridazin-3(2H)-one 3a-h:
Method a) Hydrazine (1.5 eq) is added to a solution of precursor (1 eq) in ethanol (0.2 M). The
mixture is stirred at room temperature and the reaction is monitored by TLC. After total conversion,
evaporation under reduced pressure provided the corresponding 4,5-dihydropyridazinone which could
be purified by silica gel column chromatography.
Method b) Hydrazine (12.5 eq) is added to a solution of precursor (1 eq) in acetic acid (0.15 M). The
mixture is stirred at reflux and the reaction is monitored by TLC. After total conversion, evaporation
under reduced pressure provided the corresponding 4,5-dihydropyridazinone which could be purified
by silica gel column chromatography.
4
-(1H-Indol-3-yl)-6-methyl-4,5-dihydropyridazin-3(2H)-one 3a. According to the general procedure
for the formation of the dihydropyridazinone cycle (method a)) starting from 70 mg of ethyl 2-(3-1H-
indole)-4-oxo-pentanoate 2a and 24 µL of hydrazine hydrate. The desired compound is obtained after
flash column chromatography (dichloromethane/methanol=9.6/0.4) followed by a recrystallization in
dichloromethane, as a brown powder. Yield: 42 mg (61 %).
1
Mp: 244-246 °C. H NMR (300 MHz, DMSO-d6) δ (ppm) = 2.00 (s, 3 H), 2.71-2.87 (m, 2 H), 3.83
(
dt, J= 7.4, 1.1 Hz, 1 H), 6.97 (dt, J= 7.1, 1.1 Hz, 1 H), 7.05-7.10 (m, 2 H), 7.34 (d, J= 8.1 Hz, 1 H),
1
3
7
.61 (d, J= 7.8 Hz, 1 H), 10.51 (bs, 1 H), 10.95 (bs, 1 H). C NMR (75 MHz, DMSO-d6) δ (ppm) =
-
2
2.8, 32.4, 33.8, 111.3, 111.4, 118.5, 119.3, 121.2, 122.3, 126.4, 136.3, 151.9, 167.4. IR (KBr) ν (cm
1
+.
)
= 3366, 2914, 1667, 1455, 1416, 1374, 1330, 1248, 1222, 1173, 1101, 1013. MS (EI) m/z 227 [M ],
+
.
1
98, 157, 143, 129, 115. HRMS (EI; [M ]) calculated for C H N O 227.1059, found 227.1057.
13 13 3
6
-Methyl-4-(1-methyl-1H-indol-3-yl)-4,5-dihydropyridazin-3(2H)-one 3b. According to the general
procedure for the formation of the dihydropyridazinone cycle (method a)) starting from 55 mg of ethyl
-(1H-methylindol-3-yl)-4-oxo-pentanoate 2b and 18 µL of hydrazine hydrate. The desired compound
2
is obtained after flash column chromatography (dichloromethane/ethyl acetate=8/2 to 7/3) as a beige
lacquer. Yield: 26 mg (53 %).
1
H NMR (300 MHz, CD OD) δ (ppm) = 2.03 (s, 3 H), 2.87 (ddd, J= 21.3, 16.8, 7.2, Hz, 2 H), 3.75 (s,
3
3
H), 3.97 (dt, J= 7.2, 0.6 Hz, 1 H), 6.99 (s, 1 H), 7.05 (dt, J= 6.9, 0.9 Hz, 1 H), 7.18 (dt, J= 7.2, 1.1
1
3
Hz, 1 H), 7.34 (d, J= 8.1 Hz, 1 H), 7.61 (d, J= 8.1 Hz, 1 H). C NMR (75 MHz, CDCl ) δ (ppm) =
3
2
3.4, 32.9, 33.8, 34.4, 109.7, 110.2, 119.2, 119.6, 122.2, 126.4, 126.7, 137.3, 153.5, 168.3. MS (EI)
2
5

ACCEPTED MANUSCRIPT
+.
+.
m/z 241 [M ], 212, 171, 157, 143, 115. HRMS (EI; [M ]) calculated for C H N O 241.1215, found
1
4
15
3
2
41.1217.
4
-(5-Methoxy-1H-indol-3-yl)-6-methyl-4,5-dihydropyridazin-3(2H)-one 3c. According to the general
procedure for the formation of the dihydropyridazinone cycle (method a)) starting from 80 mg of ethyl
-(5-methoxy-1H-indol-3-yl)-4-oxo-pentanoate 2c and 22 µL of hydrazine hydrate. The desired
2
compound is obtained after flash column chromatography (dichloromethane/methanol=9.6/0.4)
followed by a recrystallization in dichloromethane as a beige powder. Yield: 55 mg (76 %)
1
Mp: 225-227 °C. H NMR (300 MHz, CD OD) δ (ppm) = 2.05 (s, 3 H), 2.81-2.97 (m, 2 H), 3.82 (s, 3
3
H), 3.96 (dt, J= 6.9, 0.6 Hz, 1 H), 6.77 (dd, J= 8.7, 2.4 Hz, 1 H), 7.02 (s, 1 H), 7.12 (d, J= 2.4 Hz, 1
1
3
H), 7.23 (dd, J= 8.7, 0.6 Hz, 1 H). C NMR (75 MHz, DMSO-d6) δ (ppm) = 22.9, 32.3, 33.7, 55.3,
+.
1
01.2, 111.0, 111.3, 112.0, 122.9, 126.8, 131.4, 152.0, 153.0, 167.4. MS (EI) m/z 257 [M ], 187, 159.
+
.
HRMS (EI; [M ]) calculated for C H N O 257.1164, found 257.1164.
1
4
15
3
2
6
-Methyl-4-(3-methyl-1H-indol-2-yl)-4,5-dihydropyridazin-3(2H)-one 3d. According to the general
procedure for the formation of the dihydropyridazinone cycle (method a)) starting from 80 mg of ethyl
-(3-methyl-1H-indol-2-yl)-4-oxo-pentanoate 2d and 23 µL of hydrazine hydrate. The desired
2
compound is obtained after flash column chromatography (dichloromethane/ethyl acetate=5/5)
followed by a recrystallization in dichloromethane as a white powder. Yield: 38 mg (54 %).
1
Mp: 225-227 °C. H NMR (300 MHz, CD OD) δ (ppm) = 2.04 (s, 3 H), 2.36 (s, 3 H), 2.71 (dd, J=
3
1
8
7.4, 7.8 Hz, 1 H), 2.89 (ddd, J= 17.1, 12.3, 0.9 Hz, 1 H), 4.01 (dd, J= 12.3, 7.5 Hz, 1 H), 6.93 (dt, J=
13
.1, 1.5 Hz, 1 H), 7.00 (dt, J= 7.2, 1.5 Hz, 1 H), 7.24 (d, J= 7.8 Hz, 1 H), 7.34 (d, J= 7.8 Hz, 1 H). C
NMR (75 MHz, DMSO-d6) δ (ppm) = 11.5, 22.6, 33.0, 33.2, 107.5, 110.5, 118.0, 118.1, 119.9, 126.8,
+.
+.
1
32.8, 135.3, 152.5, 168.1. MS (EI) m/z 241 [M ], 171, 143, 130, 115. HRMS (EI; [M ) calculated
for C H N O 241.1215, found 241.1214.
1
4
15
3
4
-(1H-Benzo[d][1,2,3]triazol-1-yl)-6-methyl-4,5-dihydropyridazin-3(2H)-one 3e. According to the
general procedure for the formation of the dihydropyridazinone cycle (method a)) starting from 93 mg
of ethyl 2-(benzotriazol-1-yl)-4-oxo-pentanoate 2e and 50 µL of hydrazine (35 % wt in water). The
desired compound is obtained after column chromatography (dichloromethane/ethyl acetate = 7/3 to
5
/5) as a yellow oil. Yield: 12 mg (15 %).
1
H NMR (300 MHz, DMSO-d6) δ (ppm) = 2.08 (s, 3 H), 3.14 (dd, J= 16.8, 7.2 Hz, 1 H), 3.50 (dd, J=
6.8, 13.2 Hz, 1 H), 6.05 (dd, J= 13.2, 7.2 Hz, 1 H), 7.43 (ddd, J= 8.4, 6.9, 0.9 Hz, 1 H), 7.57 (ddd, J=
1
1
3
8
.4, 6.9, 0.9 Hz, 1 H), 7.85 (d, J= 8.4 Hz, 1 H), 8.08 (d, J= 8.4 Hz, 1 H), 11.05 (s, 1 H). C NMR (75
2
6

ACCEPTED MANUSCRIPT
MHz, DMSO-d6) δ (ppm) = 22.7, 31.2, 52.5, 111.1, 119.2, 124.2, 127.5, 133.6, 145.1, 152.5, 162.7.
+.
+.
MS (EI) m/z 229 [M ], 159, 119, 110. HRMS (EI; [M ) calculated for C H N O 229.0964, found
1
1
11
5
2
29.0968.
6
-Methyl-4-morpholino-4,5-dihydropyridazin-3(2H)-one 3f. According to the general procedure for
the formation of the dihydropyridazinone cycle (method a)) starting from 150 mg of ethyl 2-
morpholino-4-oxopentanoate 2f and 92 µL of hydrazine (35 % wt in water). The desired compound is
obtained as a yellow oil. Yield: 58 mg (45 %).
1
H NMR (300 MHz, CD OD) δ (ppm) = 2.04 (s, 3 H), 2.40-2.48 (m, 1 H), 2.60-2.72 (m, 4 H), 3.15-
3
1
3
3
6
1
.29 (m, 1 H), 3.66-3.71 (m, 5 H). C NMR (75 MHz, CDCl ) δ (ppm) = 22.9, 29.6, 49.5 (2 C), 58.3,
3
-1
7.0 (2 C), 153.0, 165.3. IR (KBr) ν (cm ) = 3421, 2858, 1656, 1649, 1602, 1551, 1432, 1380, 1334,
+
.
+.
135, 1106. MS (EI) m/z 197 [M ], 112, 111, 110, 87, 86, 82, 81, 57, 53. HRMS (EI; [M ])
calculated for C H N O 197.1164, found 197.1171.
9
15
3
2
2
,6-Dimethyl-4-morpholino-4,5-dihydropyridazin-3(2H)-one 3g. According to the general procedure
for the formation of the dihydropyridazinone cycle (method a)) starting from 150 mg of ethyl 2-
morpholino-4oxopentanoate 2f and 53 µL of methylhydrazine. The desired compound is obtained after
flash column chromatography (dichloromethane/methanol = 9.8/0.2) as a yellow oil. Yield: 65 mg (48
%
).
1
H NMR (300 MHz, CDCl ) δ (ppm) = 2.08 (s, 3 H), 2.62-2.64 (m, 6 H), 3.22 (t, J= 7.2 Hz, 1 H), 3.33
3
13
(
s, 3 H), 3.69-3.72 (m, 4 H). C NMR (75 MHz, CDCl ) δ (ppm) = 23.3, 30.4, 36.2, 50.0 (2 C), 59.0,
3
+
.
+.
6
7.2 (2 C), 152.9, 163.5. MS (EI) m/z 211 [M ], 124, 96, 87. HRMS (EI; [M ]) calculated for
C H N O 211.1321, found 211.1321.
10
17
3
2
6
-Methyl-4-(4-methylpiperazin-1-yl)-4,5-dihydropyridazin-3(2H)-one 3h. According to the general
procedure for the formation of the dihydropyridazinone cycle (method a)) starting from 500 mg of
ethyl 4-oxo-2-(4-methylpiperazin-1-yl)-pentanoate 2h and 280 µL of hydrazine (35 % wt in water).
The desired compound is obtained after flash column chromatography (dichloromethane/methanol
gradient=9.8/0.2 to 7/3) as a yellow lacquer. Yield: 369 mg (85 %).
1
H NMR (300 MHz, CDCl ) δ (ppm) = 2.06 (s, 3 H), 2.30 (s, 3 H), 2.43-2.55 (m, 4 H), 2.62-2.72 (m,
3
1
3
6
4
H), 3.34 (t, J= 7.5 Hz, 1 H), 8.47 (bs, 1 H). C NMR (75 MHz, CD OD) δ (ppm) = 22.7, 30.0, 45.9,
3
+.
+.
8.7 (2 C), 56.0 (2 C), 59.1, 156.2, 167.5. MS (EI) m/z 210 [M ], 110, 99. HRMS (EI; [M )
calculated for C H N O 210.1481, found 210.1488.
1
0
18
4
2
7

ACCEPTED MANUSCRIPT
4
-(1H-Indol-3-yl)-2,6-methyl-4,5-dihydropyridazin-3(2H)-one 3i. According to the general procedure
for the formation of the dihydropyridazinone cycle (method a)) starting from 100 mg of ethyl 2-(3-1H-
indole)-4oxo-pentanoate 2a and 31 µL of methylhydrazine. The desired compound is obtained after
flash column chromatography (dichloromethane/methanol gradient=9.9/0.1 to 97/3) as a yellow
lacquer. Yield: 76 mg (81 %).
1
H NMR (300 MHz, CDCl ) δ (ppm) = 2.03 (s, 3 H), 2.71-2.86 (m, 2 H), 2.41 (s, 3 H), 3.97 (t, J= 7.1
3
Hz, 1 H), 6.72 (dd, J= 2.4, 0.2 Hz, 1 H), 7.07-7.18 (m, 2 H), 7.24 (dd, J= 7.5, 1.5 Hz, 1 H), 7.61 (d, J=
1
3
7
1
2
2
.8 Hz, 1 H), 8.56 (bs, 1 H). C NMR (75 MHz, CDCl ) δ (ppm) = 23.5, 33.7, 34.8, 36.6, 111.6,
3
-1
11.8, 119.0, 119.7, 121.8, 122.2, 126.3, 136.5, 153.7, 166.7. IR (KBr) ν (cm ) = à faire. MS (EI) m/z
+.
+.
41 [M ], 212, 183, 157, 143, 129, 115. HRMS (EI; [M ]) calculated for C H N O 241.1215, found
1
4
15
3
41.1221.
Phosphodiesterase assay
PDE IV activity was monitored by using a PDE IV assay kit (Enzo) adapted to human
recombinant phosphodiesterase isoform PDE IV expressed in E. coli. PDE I come from
bovine brain and is available in PDE assay kit from Enzo. The basis for the assay is the
cleavage of cAMP by the cyclic nucleotide phophodiesterase. The 5’-nucleotide released is
further cleaved by 5’-nucleotidase into the nucleoside and phosphate which is quantified
®
using BIOMOLGREEN reagent. The assay mixture (50 µL/well) contained in the assay
buffer (10mM Tris-HCl, pH 7.4), PDE incubating with cAMP, 5’-nucleotidase with or
without inhibitor (compounds or control) at 30°C for 60 min. The reaction was stopped by
®
addition of 100 µL of BIOMOLGREEN reagent and the plate was incubated for another 30
min to allow color to develop before reading OD on a microplate reader.
Test compounds were dissolved in DMSO with a final concentration (2%) which did not
significantly affect PDE activity. A non-specific PDE inhibitor, 3-isobutyl-1-methylxanthine
(
IBMX) and Zardaverine as reference, were included as a test control. The initial screening on
PDE IV is conducted at 500 µM. For the most potent compounds, the inhibition study on PDE
IV activity included five concentrations of the drug. The IC50 values were evaluated by
nonlinear regression and represent the mean value of two or three independent determinations,
each performed in triplicate.
2
8