New pyridazinone-4-carboxamides as new cannabinoid receptor type-2 inverse agonists: Synthesis, pharmacological data and molecular docking

Article abstract of DOI:10.1016/j.ejmech.2017.01.002

In the last few years, cannabinoid type-2 receptor (CB₂R) selective ligands have shown a great potential as novel therapeutic drugs in several diseases. With the aim of discovering new selective cannabinoid ligands, a series of pyridazinone-4-c

Full text of DOI:10.1016/j.ejmech.2017.01.002

Accepted Manuscript
New pyridazinone-4-carboxamides as new cannabinoid receptor type-2 inverse
agonists: Synthesis, pharmacological data and molecular docking
Giulio Ragusa, María Gómez-Cañas, Paula Morales, Carmen Rodríguez-Cueto,
María R. Pazos, Battistina Asproni, Elena Cichero, Paola Fossa, Gerard A. Pinna,
Nadine Jagerovic, Javier Fernández-Ruiz, Gabriele Murineddu
PII:
S0223-5234(17)30002-8
10.1016/j.ejmech.2017.01.002
EJMECH 9153
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 October 2016
Revised Date: 4 December 2016
Accepted Date: 1 January 2017
Please cite this article as: G. Ragusa, M. Gómez-Cañas, P. Morales, C. Rodríguez-Cueto, M.R.
Pazos, B. Asproni, E. Cichero, P. Fossa, G.A. Pinna, N. Jagerovic, J. Fernández-Ruiz, G. Murineddu,
New pyridazinone-4-carboxamides as new cannabinoid receptor type-2 inverse agonists: Synthesis,
pharmacological data and molecular docking, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.01.002.
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ACCEPTED MANUSCRIPT
GRAFICAL ABSTRACT

ACCEPTED MANUSCRIPT
New pyridazinone-4-carboxamides as new cannabinoid receptor type-2 inverse
agonists: synthesis, pharmacological data and molecular docking.
Giulio Ragusa , María Gómez-Cañas , Paula Moralese, Carmen Rodríguez-Cueto , María R. Pazos
a
b-d
b-d
b-
d
a
f
f
a
e
,
Battistina Asproni , Elena Cichero , Paola Fossa , Gerard A. Pinna , Nadine Jagerovic , Javier
b-d
a,*
Fernández-Ruiz , Gabriele Murineddu
a) Department of Chemistry and Pharmacy, University of Sassari, via F. Muroni 23/A, 07100 Sassari,
Italy.
b) Instituto Universitario de Investigación en Neuroquímica, Departamento de Bioquímica y Biología
Molecular, Facultad de Medicina, Universidad Complutense, 28040-Madrid, Spain.
c) Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED),
Madrid, Spain.
d) Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain.
e) Instituto de Química Médica, CSIC, Calle Juan de la Cierva, 3, 28006, Madrid, Spain.
f) Department of Pharmacy, University of Genoa, Viale Benedetto XV n. 3, 16132, Genoa, Italy.
*This author serve as corresponding author:
Gabriele Murineddu: Phone: +39079228740; Fax: +39079228720; E-mail: muri@uniss.it
1

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ABSTRACT
In the last few years, cannabinoid type-2 receptor (CB R) selective ligands have shown a great
2
potential as novel therapeutic drugs in several diseases. With the aim of discovering new selective
cannabinoid ligands, a series of pyridazinone-4-carboxamides was designed and synthesized, and the
new derivatives tested for their affinity toward the hCB R and hCB R. The 6-(4-chloro-3-
1
2
methylphenyl)-2-(4-fluorobenzyl)-N-(cis-4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-
carboxamide (9) displayed high CB -affinity (K CB = 2.0 ± 0.81 nM) and a notable selectivity
2
i
2
(
K CB /K CB > 2000). In addition, 9 and other active new synthesized entities have demonstrated to
i 1 i 2
3
5
behave as CB R inverse agonists in [ S]-GTPγS binding assay. ADME predictions of the newly
2
synthesized CB R ligands suggest a favourable pharmacokinetic profile. Docking studies disclosed the
2
specific pattern of interactions of these derivatives. Our results support that pyridazinone-4-
carboxamides represent a new promising scaffold for the development of potent and selective CB R
2
ligands.
Keywords: scaffold hopping, synthesis, cannabinoid receptors, CB antagonism, ADME model, docking studies
2
2

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1. Introduction
At the beginning of 1960, with the increase in recreational use of marijuana, the scientific
community started to study the pharmacological mechanisms underlying the psychotropic action of
cannabis plant. The identification of the main psychotropic component of Cannabis sativa, the (−)-
9
9
trans-ꢀ -tetrahydrocannabinol (ꢀ -THC) [1], and the discovery of its molecular targets [2,3], the so-
called cannabinoid receptors, along with their respective endocannabinoids and the enzymes
responsible for their regulation [4-6], led to the identification of a complex intercellular communicating
system, named endocannabinoid system involved in numerous physiological and physiopathological
processes in the body.
Among the two types of G-protein coupled receptors (GPCRs) [7], cannabinoid receptors type-1
(CB Rs) and type-2 (CB Rs), the former are expressed predominantly in the central nervous system
1 2
(CNS), in which they are implicated in the control of many neurobiological processes, including
nociception, control of movement, emesis, learning and cognitive function [8], whereas the latter are
expressed mainly in cells of the immune system such as monocytes, macrophages, B-cells, and T-cells,
in which they modulate cytokine release and immune cell migration [9]. Nevertheless, further
investigations revealed that CB R are also expressed in many other types of cells and tissues, including
2
activated microglia and infiltrated macrophages in the brain [10], vascular endothelial cells,
cardiomyocytes, and bone cells, as well as tissues of the gastrointestinal tract [11]. In fact, CB R and
1
CB R are often co-expressed in the same peripheral tissues and cells [12], and even they may form
2
heteromers enabling synergies at their receptor signalling [13].
As CB R is probably involved in most of side effects of cannabinoid treatments in the CNS
1
[
14], researchers focused their attention to the development of CB R selective ligands, either as
2
agonists or as antagonist/inverse agonists. These ligands would lack of undesirable psychoactive side-
3

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effects due to the limited distribution at the CNS of CB R, although such side-effects could appear in
2
the immune system (e.g. immunosuppression). The preclinical evidences accumulated in the last years
support that CB R selective ligands could be used for the treatment of various pathologies including
2
some CNS-related disorders [15,16]. Activation of CB R has shown to be an interesting approach for
2
pain, inflammation, arthritis, neuroprotection, addictions, and cancer. However, none of the CB2
agonists tested in clinical phases has reached the market yet, probably due to their lack of efficacy [17].
Among the CB ligands developed by academic and pharmaceutical researchers, only few showed
2
inverse agonism/antaginism activity at CB R. Thus, it is interesting to discover new CB antagonist
2
2
scaffolds.
Recently, we have synthesized a series of pyrrole-analogues of the robust CB R antagonist
2
SR144528 typified by derivative 1 (Fig. 1), which showed high CB R affinity (K = 5.7 ± 0.7 nM) and
2
i
selectivity (K CB /K CB = 258) [18]. The same derivative 1 was also reported by Reggio’s group
i
1
i
2
(
K CB = 15.3 nM) as a tool for molecular modelling studies focused on the hydrogen bonding,
i 2
hydrophobic, and aromatic stacking interactions in the CB R binding pocket of the inactive state of the
2
receptor [19].
INSERT FIG. 1
The structure-activity relationship (SAR) studies on CB R pyrazole- and pyrrole-based ligands
2
developed in our group [18,20-24] allowed us to determine the key features to CB R affinity: a pivotal
2
aromatic ring containing one or more nitrogen atoms bearing a N-benzyl group, a carboxamide moiety
and a lipophilic aromatic ring. Keeping in mind these pharmacophoric elements and pushed by our
interest in pyridazinone derivatives [25,26], we postulated that the introduction of those
pharmacophoric elements in the dihydropyridazinone core might provide CB R ligands endowed of
2
receptor selectivity and biological activity.
4

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In the present study, we report the synthesis of the new pyridazinone derivatives 2-19, 39
Table 1), preliminary pharmacological data from receptor binding and functionality assays, and
(
docking studies.
2. Result and discussion
2.1. Chemistry
The 3-oxo-6-phenyl-2,3-dihydropyridazines 2−19 were obtained following the synthetic route
depicted in scheme 1. The condensation of the bromide 21 or 22 with diethyl malonate in presence of
sodium hydride, gave the corresponding diester 23 and 24 [27], which reacted with hydrazine
monohydrate in ethanol to give the dihydropyridazinones 25 and 26 [27]. Their bromine-assisted
oxidation in acetic acid provided compounds 27 and 28 [27]. Heating pyridazinones with the
appropriate benzyl halide in presence of K CO gave the N-alkylated pyridazinones 29−31. Compared
2
3
to conventional heating, ultrasonic irradiation led to an improved yield when pentylchloride was used
instead of benzyl halides obtaining derivatives 32-33. Then, hydrolysis of esters 29−33 afforded the
carboxylic acids 34-38 that were treated with EDC and HOBt followed by the appropriate amines to
give the desired compounds 2-19.
INSERT SCHEME. 1
The tricyclic derivative 39 was synthesized according to the synthetic pathway illustrated in
scheme 2. Commercial 1-indanone 40 was treated under heating with diethylketomalonate to obtain the
diester 41 [28], that reacted with hydrazine dihydrochloride in ethanol to form the ring system 42 [28].
Then, N-alkylation of 43 under ultrasound irradiation with 4-fluorobenzylbromide in the presence of
K CO gave 43. Saponification of 43 afforded the acid 44, whose activation with EDC and HOBt,
2
3
followed by reaction with cycloheptylamine yielded the desired tricyclic pyridazinone 3.
5

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INSERT SCHEME. 2
2
.2. CB R and CB R affinities
1 2
With the exception of compounds 3, 4 and 14, which proved to be insoluble under the test
conditions, the affinity and selectivity of all the new pyridazinone-4-carboxamides for cannabinoid
3
receptors were determined by radioligand competition binding assay. Their ability to displace [ H]CP,
5
5-940 from human cannabinoid CB R or CB R genes transfected into HEK293 EBNA cells were
1 2
evaluated. They were first evaluated into a preliminary screening at a concentration of 4 µM. Only
3
those compounds able to displace the [ H]CP,55-940 to CB R or CB R more than 70% in such
1
2
preliminary screening were subjected to a secondary evaluation consisting in a complete dose-response
curve to determine the inhibition constants (K values). The CB R and CB R K values of the new
i
2
1
i
synthesized compounds are reported in Table 1. For comparison, the K values of the reference
i
cannabinoid ligands HU-308 (selective for the CB R) and WIN55,212-2 (it binds CB R but also CB R)
2
2
1
in our receptor binding analysis procedure have been also included in Table 1.
INSERT TABLE. 1
Firstly, we evaluated the effect of the modulation of the C4 carboxamide moiety on the 6-(4-
chloro-3-methylphenyl)-2-(4-methylbenzyl)-3-oxo-2,3-dihydropyridazine scaffold: among the three
selected derivatives 2-4, only compound 2, bearing a cyclohexyl group, could be tested due to
insolubility problems. The CB R binding data of 2 showed moderate CB R affinity (K = 240
± 190
2
2
i
nM). With the aim to improve the solubility of these templates, we planned the synthesis of three
analogues of 2-4 with a 4-F-benzyl group in N position, compounds 5-7. The modification of the N -
2
2
benzyl group in the dihydropiridazinone ring led to an improve of CB R affinity in the carboxamides
2
bearing a cyclohexyl (5), cycloheptyl (6) and 1-adamantyl (7) group with K values of 4.7
±
2.1 nM, 4.9
i
6

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±
2.6 nM and 3.4
±
3.0 nM, respectively, and a good selectivity (K CB /K CB > 851, 816 and 1176,
i
1
i
2
respectively). Encouraged by these results, we synthesized compound 8, bearing a fenchyl group in the
amide portion, which showed an increase in CB R affinity (K = 2.6 0.47 nM) with higher CB R
±
2
i
2
selectivity (K CB /K CB > 1538) compared to 7. Then, we modulated the carboxamide portion by
i
1
i
2
introduction of simplified cyclic group as cis-4-methyl-cyclohexyl (9), its trans isomer (10) and
ethylmorpholine derivative (11), leading to the best compound of this new series of derivatives,
carboxamide 9, endowed with the high CB R affinity (K = 2.0
±
0.81 nM) and selectivity
2
i
(
K CB /K CB > 2000) in the whole series, whereas the ethylmorpholine group (11) was unfavourable
i
1
i
2
for the receptor affinity.
In light of the results obtained with these modifications in the N and C4 positions in
2
compounds 5-10, it was of interest to further determine the influence of the elimination of 4-Cl,3-CH3
substituents in the C6 phenyl ring in the 3-oxo-2,3-dihydropyridazine scaffold. The selectivity for
CB R drastically changed with this modification. All derivatives (12-16) showed mixed CB R and
2
1
CB R affinities, greater for the CB R, always in the low-medium nanomolar range (<100 nM), and
2
2
lower for the CB R, always in the submicromolar range (1000-100 nM).
1
N -Alkyl substitution of the pyridazine ring was also analysed. Interestingly, the resulting
2
compounds (17−19) maintained high affinity for the CB R. However, they lost CB R selectivity
2
2
showing moderate affinity for CB R, in particular compound 17 with a K in the nM range (K CB =7.6
1
i
i
2
±
4.0 nM, K CB = 25 ± 8.6 nM).
i 1
Also in this series, as already noted in previously described [18], the constraining of the flexible
-(4-chloro-3-methylphenyl)-2-(4-methylbenzyl)-3-oxo-2,3-dihydropyridazine pharmacophore
drastically changed the binding behaviour, resulting the 3-oxo-3,5-dihydro-2H-indeno[1,2-c]pyridazine
6
3
9 with not affinity for the CBRs (K > 4000 nM). Therefore, the presence of the methylene bridge,
i
7

ACCEPTED MANUSCRIPT
modifying pharmacophoric conformation of the pyridazine ring, was unfavourable particularly for
CB R binding.
2
2
.3. CB R/CB R-stimulated GTPγS binding
1 2
Six compounds (5, 8, 9, 15, 17 and 18) were selected for further characterization based on their affinity
for the CB R. We investigated their functional activity at this receptor, and also of the reference
2
compound SR144528, by conducting GTPγS binding assays that demonstrated that, as happens with
SR144528, the six compounds behaved as inverse agonists of the CB R with IC and Emax values
2
50
indicated in Fig. 2. The mean curve of one of these compounds (5) at the GTPγS binding bioassay,
together with the curve corresponding to SR144528, is shown in Fig. 2. The functional potency (IC )
50
of all the tested compounds, except 17, was much better than SR144528 (Fig. 2).
INSERT FIG. 2
2.4. ADME properties in silico
ADME properties of the newly synthesized CB R ligands and of well-known cannabinoids such
2
as SR144528 and HU308 have been calculated in silico by using a set of 34 physicochemical
descriptors implemented in QikProp (Maestro Software). In-silico ADME approaches can be used to
help bias drug discovery processes at an early stage, in a lower cost and time manner [30].
The predicted data for the novel pyridazinone derivatives indicated that Lipinski and Jorgensen
pharmacokinetics rules are followed [31,32]. As displayed in Table 2, the prediction of blood-brain
barrier (BBB) partition coefficient, human oral absorption, bioavailability, and gut-blood barrier
permeability suggests a favourable drug-like profile for these new compounds, at least similar to the
8

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reference CB ligands SR144528 and HU308. Like most cannabinoids, pyridazinones are lipophilic and
2
they show aqueous solubility values that fall outside the optimum range of values for 95% of known
drugs [33,34]. Even though all of these compounds are hydrophobic, curiously, only compounds 3, 4
and, 14 could not be tested in binding assays due to solubility deficiency. This could be due to the
crystalline form in which these compounds solidified.
The only compound that is predicted to have a better biodisponibility than the reference
cannabinoids SR144528 and HU308 with improved aqueous solubility and decreased unspecific human
serum binding is the ethylmorpholine analogue (11). However, this compound (11) showed moderate
affinity for CB (K = 311±63 nM). Finally, the novel compounds 2-10, 12-19 and 39 have similar
2
i
ADME predicted data than most of cannabinoids. Therefore, selection of the best candidate (9) for
future studies was realized based on binding and functionality data. The next step for compound 9 to be
considered as suitable drug-like cannabinoid will be the acquisition of experimental ADME data.
INSERT TABLE. 2
2.5. Molecular docking
In this work, molecular docking studies were performed in order to rationalize the
pharmacological data describing the affinity and selectivity profile of the newly synthesized CB R
2
inverse agonists. The computational studies were developed relying on a a ligand-based homology
model of the human CB R we previously built, taking into account the molecular structure of the
2
SR144528 and of related analogues [35].
Briefly, the derived pattern of contacts between the inverse agonists and the biological target
proved to include H-bonds at least with S165 for weaker compounds, or both the T114 and S165
9

ACCEPTED MANUSCRIPT
residues for the most potent analogues, as recently reported in literature [36]. In particular, the 4-
chloro-3-methyl-phenyl and the benzyl group of SR144528 were engaged in Van der Waals
interactions and π−π stacking with two hydrophobic crevices, delimited by L195, Y190, W194 and
I110, L169, respectively. In this way, the fenchyl substituent of SR144528 was oriented toward L160,
V164, F197 and F202.
Interestingly, the relevance of this kind of contacts was also confirmed by docking studies we
recently performed within a series of in house potent inverse agonists bearing a tricyclic pyrazole
carboxamide core [37].
In this context, the newly synthesized derivatives here proposed are characterized by a
comparable binding mode, moving the benzyl moiety toward Y190 and W194 whereas the phenyl ring
is engaged in Van der Waals contacts with L181. Moreover, the carboxamide function is stabilized by
H-bonds contacts with the key residues T114 and S165 (Fig. 3).
INSERT FIG. 3
In particular, as shown in Fig. 4, the cis-methyl-cyclohexyl carboxamide portion of compound
15 is able to mimic the monoterpene moiety and the pyrazole ring of the refererence compound
SR144528, whereas the phenyl ring and the pyridazinone core partially play the same role of the
SR144528 4-chloro-3-methyl-phenyl substituent. On the other hand, the benzyl group of the new
inverse agonist is engaged in π−π stacking with Y190 and W194, moving quite far from the area
occupied by the related benzyl of SR144528.
INSERT FIG. 4
Interestingly, all the inverse agonists here disclosed show a similar affinity profile toward the
hCB R (K = 2.0−7.4 nM) whereas they are characterized by a different selectivity trend over the CB R.
2
i
1
10

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In details, those compounds bearing a disubstitued phenyl ring rather than the related analogues bearing
an unsubstituted phenyl ring are more promising selectivity profile to CB R over CB R.
2
1
Concerning this issue, with the aim at exploring any pattern of contacts that could turn in CB R
1
binding, we also performed docking studies involving the CB R. This kind of analysis was applied
1
starting from the hCB R homology model we previously described [38], further refined taking into
1
account the X-ray data of the human β -adrenoreceptor [39].
2
Several computational studies we already carried out within a number of different series of
CB R antagonists allowed us to reveal that three different receptor sub cavities would be necessary
1
involved in receptor-ligand interaction [40]. In particular, the first one revolves around the residues
K192 and S383, which are involved in H-bonds with the most potent antagonists, whilst the second and
third cavities are placed in the area including F278, I280, M363 and F200, F289, Y292, W356,
respectively.
Interestingly, according to our calculations only those compounds bearing an unsubstituted
phenyl ring linked to the pyridazinone core are able to occupy the three aforementioned cavities. As
shown in Fig. 5, the carboxamide moiety of compound 15 displays H-bond with the key residue K192,
moving the Q group toward F170 and I175. In addition, the benzyl group and the phenyl moiety
display hydrophobic contacts with F200, F289 and F278, I280, V285 and M363, respectively.
INSERT FIG. 5
On the contrary, the related substituted analogues do not fit the CB R binding site. Indeed, the
1
3-methyl, 4-chloro derivatives are expected to clash with the I280, V285 and M363 amino acids.
On this basis, the final computational data prove to be in good agreement with the
pharmacological results providing a further validation of the overall method.
11

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3. Conclusions
Using a scaffold hopping approach on pyrazole CB R ligands, we designed novel 3-oxo-6-
2
phenyl-2,3-dihydropyridazine-4-carboxamides as a new interesting scaffold for the development of
potent CB R ligands. SAR studies derived from compound 2 led us to synthesize the best derivative of
2
this series, compound 9 with nanomolar affinity and high selectivity for the CB R. The functionality at
2
3
5
CB R determined by an in vitro assay based on [ S]-GTPγS binding showed that compound 9 and
2
other representative compounds behave as CB R inverse agonists as evidenced by docking studies.
2
Pyridazinone 9 presents the highest potency at this receptor with an IC value of 20.7 nM. Therefore, it
5
0
can be concluded that compound 9 has better affinity and selectivity than other frequently-used inverse
agonists for this receptor such as AM630 [41] or JTE-907 [42] and similar affinity to SR144528 but
resulting to be more selective than this reference compound [43,44]. In-silico ADME calculations
indicate that these novel compounds can be considered as suitable drug-like cannabinoid candidates.
Then, compound 9 and related derivatives may serve as experimental tools for investigating CB R-
2
mediated effects in cellular and tissue models as well as in in vivo studies. They may also have interest
for a further therapeutic development aimed at selectively inhibiting the CB R-mediated activity in
2
certain pathologies, e.g. immunodeficiency, bone disorders, cerebral malaria, in which an excess of
CB R-dependent endocannabinoid activity has been associated with the progression of the disease
2
and/or with the occurrence of specific symptoms [45].
4. Experimental section
4.1. General procedures
12

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All reactions involving air or moisture-sensitive compounds were performed under nitrogen
atmosphere. Solvents and reagents were obtained from commercial suppliers and were used without
further purification. The starting ketones 40 and 20, bromoketone 22 and amines for the synthesis of
final compounds were purchased from Sigma-Aldrich®: fenchylamine [46] was synthesized according
to the literature procedure. Ultrasound irradiation experiments were carried out in Bandelin® Sonorex
ultrasonic bath RK-100H. Flash column chromatography was performed automatically on Flash-master
(Biotage®) with pre-packed Biotage® SNAP silica gel cartridges or manually on silica gel (Kieselgel
60, 0.040−0.063 mm, Merck®). Thin layer chromatography (TLC) was performed with Polygram SIL
N-HR/HV254 pre-coated plastic sheets (0.2 mm) on aluminum sheets (Kieselgel 60 F254, Merck®).
Melting points were obtained on a Köfler melting point apparatus and are uncorrected. IR spectra were
recorded as nujol mulls on NaCl plates with a Jasco FT/IR 460 plus spectrophotometer and are
-1
expressed in ν (cm ). NMR experiments were run on a Bruker Avance III Nanoboy 400 system
1
13
(
400.13 MHz for H, and 100.62 MHz for C). Spectra were acquired using deuterated chloroform
1
(
chloroform-d) or deuterated dimethylsulfoxide (DMSO-d ) as solvents. Chemical shifts (
δ
) for H- and
6
1
3
C-NMR spectra are reported in parts per million (ppm) using the residual non-deuterated solvent
1
13
resonance as the internal standard (for chloroform-d: 7.26 ppm, H and 77.16 ppm, C; for DMSO-d :
6
1
13
2
.50 ppm, H, 39.52 ppm, C). Data are reported as follows: chemical shift (sorted in descending
order), multiplicity (s for singlet, br s for broad singlet, d for doublet, dd for double doublet, t for
triplet, q for quadruplet, m for multiplet), integration and coupling constants (J) in Hertz (Hz). LC/MS
analyses were run on an Agilent 1100 LC/MSD system consisting of a single quadrupole detector
(SQD) mass spectrometer (MS) equipped with an electrospray ionization (ESI) interface and a
photodiode array (PDA) detector; PDA range was 120−550 nm. ESI in positive mode was applied;
mobile phases: (A) ACN in H O (8:2). Analyses were performed with a flow rate of 0.9 mL/min; and a
2
13

ACCEPTED MANUSCRIPT
troom temperature All final compounds displayed ≥ 95% purity as determined by elemental analysis on
a Perkin-Elmer 240-B analyser, for C, H, and N.
4.1.1. 2-Bromo-1-(4-chloro-3-methylphenyl)ethan-1-one (21)
Bromine (3,02 mL, 59,3 mmol, 1 eq) was added dropwise to a stirred solution of commercial
ketone 20 (10 g, 59,3 mmol, 1 eq) in glacial AcOH (65 mL), the resulting solution was stirred at room
temperature for 8 h. Then ice-water was poured into reaction flask and the resulting precipitate was
filtered off, washed with H O, and air-dried to give 21 as a white solid (14,6 g, 95%). R =0,71
2
f
1
(
petroleum ether/EtOAc 9:1); mp 60-61 °C. H NMR (400 MHz, Chloroform-d) δ 7.61 (s, 1H), 7.49 (d,
J = 8.2 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 4.56 (s, 2H), 2.40 (s, 3H).
4.1.2. General Procedure for the preparation of ketodiester derivatives 23 and 24 [27]
Diethylmalonate (1.83 mL, 12 mmol, 1.2 eq) was treated with NaH in mineral oil (0.480 g, 20
mmol, 2 eq) in anhydrous THF (25 mL) under N flow at 0 °C for 30 min. Then a solution of
2
bromoketone 21 or 22 (10 mmol, 1eq) in anhydrous THF (7 mL) was added dropwise and the resulting
mixture was stirred at room temperature until complete conversion on starting materials (TLC). Then
the solution diluted with EtOAc was poured into a separatory funnel, and washed with saturated NH Cl
4
solution and brine. The organic solution was dried (Na SO ) and the solvent removed under reduced
2
4
pressure. The resulting residue was purified by column flash chromatography using the appropriate
eluents.
4.1.3. Diethyl 2-(2-(4-chloro-3-methylphenyl)-2-oxoethyl)malonate (23)
Mixture of 21, diethylmalonate and NaH was stirred at room temperature for 3 h. Crude of
reaction was purified by flash chromatography (petroleum ether/EtOAc 95:5) to afford 23 as a light
1
yellow oil (1,79 g, 55%), R = 0.43 (petroleum ether/EtOAc 9:1); H NMR (400 MHz, Chloroform-d) δ
f
14

ACCEPTED MANUSCRIPT
7
.61 (s, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 4.28 – 4.19 (m, 4H), 4.09 – 4.01 (m,
H), 3.63 (d, J = 7.1 Hz, 2H), 2.40 (s, 3H), 1.34 – 1.22 (m, 9H).
1
4.1.4. Diethyl 2-(2-oxo-2-phenylethyl)malonate (24) [27]
A mixture of 22, diethylmalonate and NaH was stirred at room temperature for 1 h. Crude of
reaction was purified by flash chromatography (petroleum ether/EtOAc 9:1) to afford 24 [27] as a light
1
yellow oil (1,67 g, 60%), R = 0.48 (petroleum ether/EtOAc 8:2); H NMR (400 MHz, Chloroform-d) δ
f
7
.99 (d, J = 7.7 Hz, 2H), 7.58 (t, J = 7.3 Hz, 1H), 7.47 (t, J = 7.4 Hz, 2H), 4.29 – 4.19 (m, 4H), 4.09 –
.03 (m, 1H), 3.63 (d, J = 7.1 Hz, 2H), 1.34 – 1.22 (m, 9H).
4
4.1.5. General procedure for the preparation of dihidropyridazinone derivatives 25 and 26 [27]
A solution of diketone 23 or 24 [27] (10 mmol, 1 eq) and hydrazine monohydrate 64-65% (0,48
mL, 10 mmol, 1 eq) in absolute ethanol (50 mL), was heated to reflux and strirred for 24 h. Then the
solution was allowed to stand at room temperature and the resulting precipitate was isolated by
filtration in vacuum and purified by column flash chromatography using the appropriate eluents.
4.1.6. Ethyl 5-(4-chloro-3-methylphenyl)-2-oxo-1,2,3,4-tetrahydropyridine-3-carboxylate (25)
From diketone 23 was obtained a solid which purified by flash chromatography (petroleum
ether/EtOAc 7:3) gave 25 as a pale yellow solid (1,01 g, 35%). R = 0.32 (petroleum ether/EtOAc 7:3);
f
1
mp 164-165 °C; H NMR (400 MHz, Chloroform-d) δ 8.90 (s, 1H, NH, exch with D O), 7.61 (s, 1H),
2
7
.49 (d, J = 8.2 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 4.25 (q, J = 7.0 Hz, 2H), 3.60 (t, J = 7.6 Hz, 1H),
.41 (dd, J = 16.9, 8.2 Hz, 1H), 3.07 (dd, J = 17.0, 6.9 Hz, 1H), 2.41 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H).
3
4.1.7. Ethyl 3-oxo-6-phenyl-2,3,4,5-tetrahydropyridazine-4-carboxylate (26) [27]
From diketone 24 was obtained a solid which purified by flash chromatography (petroleum
ether/EtOAc 1:1) gave 26 [27] as a white solid (1,2 g, 50%). R = 0.32 (petroleum ether/EtOAc 1:1);
f
15

ACCEPTED MANUSCRIPT
2
6 1
mp 156-158 °C (158 °C); H NMR (400 MHz, Chloroform-d) δ 8.68 (s, 1H, NH, exch with D O),
2
7.81 – 7.67 (m, 2H), 7.50 – 7.37 (m, 3H), 4.26 (q, J = 7.1 Hz, 2H), 3.61 (t, J = 7.7 Hz, 1H), 3.45 (dd, J
=
16.9, 8.4 Hz, 1H), 3.12 (dd, J = 16.9, 7.0 Hz, 1H), 1.28 (t, J = 7.1 Hz, 3H).
4.1.8. General procedure for the preparation of pyridazinones 27 and 28 [27]
To a solution of dihydropyridazinone 25 or 26 [27] (10 mmol, 1 eq) in glacial AcOH (65 mL)
was added dropwise a solution of bromine (1 mL, 20 eq) in glacial AcOH (15 mL). The resulting
mixture was stirred at room temperature for 6 h. Then the solution was poured into ice-water, and
aqueous solution was extracted in a separatory funnel with EtOAc. Afterward the collected organic
phases were washed with H O, saturated NaHCO solution and brine. The organic solution dried
2
3
(
Na SO ) and concentrated under reduced pressure yielding a residue which was purified by column
2 4
flash chromatography using the appropriate eluents.
4.1.9. Ethyl 6-(4-chloro-3-methylphenyl)-3-oxo-2,3-dihydropyridazine-4-carboxylate (27)
General procedure for the preparation of pyridazinones was used to oxidize 25. Mixture of
reaction was purified by flash chromatography (petroleum ether/EtOAc 4:6) to afford 27 as a pale
1
yellow solid (1.57 g, 55%). R = 0.33 (petroleum ether/EtOAc 1:1); mp 153-154 °C; H NMR (400
f
MHz, Chloroform-d) δ 8.27 (s, 1H), 7.70 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H),
4.47 (q, J = 7.1 Hz, 2H), 2.45 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H).
4.1.10. Ethyl 3-oxo-6-phenyl-2,3-dihydropyridazine-4-carboxylate (28) [27]
General procedure for the preparation of pyridazinones was used to oxidize 26. Mixture of
reaction was purified by flash chromatography (petroleum ether/EtOAc 1:1) to afford 28 [27] as a pale
2
6 1
yellow solid (0.87 g, 36%). R = 0.38 (petroleum ether/EtOAc 1:1); mp 153-154 °C (150 °C);
H
f
16

ACCEPTED MANUSCRIPT
NMR (400 MHz, Chloroform-d) δ 8.32 (s, 1H), 7.82 (d, J = 7.3 Hz, 2H), 7.53 – 7.45 (m, 3H), 4.47 (q,
J = 7.1 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H).
4.1.11. General procedures for the preparation of N-alkyl pyridazinone derivatives 29-33
Method A. K CO (8.5 mmol, 2.5 eq) and appropriate benzyl-halide (6.8 mmol, 2 eq) were
2
3
added to a solution of pyridazinones 27 or 28 (3.4 mmol, 1 eq) in anhydrous DMF (17 mL). The
resulting mixture was stirred at 60 °C until complete reaction of starting materials (TLC). Then after
cooling, H O was added into a reaction flask, and the aqueous solution was extracted with EtOAc in a
2
separatory funnel. The collected organic phases were washed with H O, LiCl 5% solution and brine,
2
then was dried (Na SO ) and concentrated under reduced pressure. The obtained residues were purified
2
4
by column flash chromatography using the appropriate eluents.
Method B. A solution of potassium carbonate (10.2 mmol, 3 eq), pentyl chloride (10.2 mmol, 3
eq) and pyridazinones 27 or 28 (3.4 mmol, 1 eq) in anhydrous DMF (15 mL) was exposed to
ultrasound irradiation for 2 h at room temperature. Then the mixture of reaction was poured into ice-
water, filtered off, and extracted with EtOAc in a separatory funnel. The collected organic phases were
washed with H O, LiCl 5% solution and brine, then dried (Na SO ) and concentrated under reduced
2
2
4
pressure. No further purification was required.
4.1.12.
Ethyl
6-(4-chloro-3-methylphenyl)-2-(4-methylbenzyl)-3-oxo-2,3-dihydropyridazine-4-
carboxylate (29)
Method A was used to convert pyridazinone 27 and methylbenzylchloride into the title product.
Mixture of reaction was stirred at 60 °C for 4 h and purification by flash chromatography (petroleum
1
ether/EtOAc 8:2) yielded 29 as a yellow oil (1.21 g, 89.6%), R = 0.36 (petroleum ether/EtOAc 7:3); H
f
NMR (400 MHz, Chloroform-d) δ 8.15 (s, 1H), 7.65 (s, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.47 – 7.36 (m,
3H), 7.14 (d, J = 7.8 Hz, 2H), 5.40 (s, 2H), 4.43 (q, J = 7.1 Hz, 2H), 2.45 (s, 3H), 2.32 (s, 3H), 1.40 (t,
17

ACCEPTED MANUSCRIPT
13
J = 7.1 Hz, 3H). C NMR (101 MHz, Chloroform-d) δ 163.72 (C), 156.37 (C), 142.60 (C), 137.95 (C),
36.86 (C), 136.00 (C), 132.76 (C), 132.67 (C), 131.68 (CH), 130.36 (C), 129.69 (CH), 129.30 (CH
x2), 129.13 (CH x2), 128.16 (CH), 124.48 (CH), 62.32 (CH ), 56.17 (CH ), 21.17 (CH ), 20.26 (CH ),
1
2
2
3
3
14.22 (CH3).
4.1.13. Ethyl
6-(4-chloro-3-methylphenyl)-2-(4-fluorobenzyl)-3-oxo-2,3-dihydropyridazine-4-
carboxylate (30)
Method A was used to convert pyridazinone 27 and 4-fluorobenzyl bromide into the title
product. Mixture of reaction was stirred at 60 °C for 3 h and purification by flash chromatography
(
petroleum ether/EtOAc 8:2) yielded 30 as a light yellow solid (1.2 g, 87%), R = 0.47 (petroleum
f
1
ether/EtOAc 7:3); mp 124-125 °C; H NMR (400 MHz, Chloroform-d) δ 8.16 (s, 1H), 7.65 (s, 1H),
7.60 – 7.48 (m, 3H), 7.43 (d, J = 8.3 Hz, 1H), 7.02 (t, J = 8.6 Hz, 2H), 5.40 (s, 2H), 4.43 (q, J = 7.1 Hz,
1
3
2
H), 2.46 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H); C NMR (101 MHz, Chloroform-d) δ 163.87 (C), 163.56
(
(
(
C), 161.42 (C), 156.35 (C), 142.87 (C), 136.94 (C), 136.15 (C), 132.54 (C), 131.87 (CH), 131.09
CH), 131.01 (CH), 130.43 (C), 129.74 (CH), 128.16 (CH), 124.48 (CH), 115.64 (CH), 115.43
CH), 62.39 (CH ), 55.72 (CH ), 20.27 (CH ), 14.22 (CH ).
2
2
3
3
4.1.14. Ethyl 2-(4-fluorobenzyl)-3-oxo-6-phenyl-2,3-dihydropyridazine-4-carboxylate (31)
Method A was used to convert pyridazinone 28 and 4-fluorobenzyl bromide into the title
product. Mixture of reaction was stirred at 60 °C for 2 h and purification by flash chromatography
(
petroleum ether/EtOAc 7:3) yielded 31 as a brown solid (1.12 g, 93%), R = 0.40 (petroleum
f
1
ether/EtOAc 7:3); mp 113-115 °C; H NMR (400 MHz, Chloroform-d) δ 8.21 (s, 1H), 7.80 (d, J = 7.0
Hz, 2H), 7.57 – 7.44 (m, 5H), 7.01 (t, J = 8.7 Hz, 2H), 5.41 (s, 2H), 4.43 (q, J = 7.1 Hz, 2H), 1.41 (t, J
1
3
=
7.1 Hz, 3H); C NMR (101 MHz, Chloroform-d) δ 163.86 (C), 163.59 (C), 161.41 (C), 156.46 (C),
18

ACCEPTED MANUSCRIPT
1
43.74 (C), 134.08 (C), 132.09, 131.50 (C), 131.13 (CH), 131.04 (CH), 130.37 (CH), 129.75 (CH),
29.07 (CH x2), 125.82 (CH x2) ,115.62 (CH), 115.40 (CH), 62.32 (CH ), 55.65 (CH ), 14.22 (CH ).
1
2
2
3
4.1.15. Ethyl 6-(4-chloro-3-methylphenyl)-3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxylate (32)
Using method B from pyridazinone 27 and pentylchloride was obtained 32 as a yellow oil (1.0
1
g, 85%), R = 0.36 (petroleum ether/EtOAc 8:2); H NMR (400 MHz, Chloroform-d) δ 8.16 (s, 1H),
f
7.67 (s, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 4.45 (q, J = 7.1 Hz, 2H), 4.29 (t, J = 7.4
1
3
Hz, 2H), 2.46 (s, 3H), 1.95 – 1.82 (m, 2H), 1.49 – 1.34 (m, 7H), 0.91 (t, J = 6.2 Hz, 3H); C NMR
101 MHz, Chloroform-d) δ 163.94 (C), 156.54 (C), 142.41 (C), 136.88 (C), 135.92 (C), 132.79 (C),
31.41 (CH), 129.97 (C), 129.68 (CH), 128.11 (CH), 124.41 (CH), 62.32 (CH ), 53.02 (CH ), 28.79
(
1
2
2
(
CH ), 28.10 (CH ), 20.24 (CH ), 14.22 (CH ), 13.95 (CH ).
2 2 3 3 3
4.1.16. Ethyl 3-oxo-2-pentyl-6-phenyl-2,3-dihydropyridazine-4-carboxylate (33)
Using method B from pyridazinone 28 and pentylchloride was obtained 33 as a yellow oil (0.92
1
g, 88%), R = 0.31 (petroleum ether/EtOAc 8:2); H NMR (400 MHz, Chloroform-d) δ 8.21 (s, 1H),
f
7
1
.81 (d, J = 7.6 Hz, 2H), 7.47 (q, J = 9.1, 7.8 Hz, 3H), 4.44 (q, J = 7.1 Hz, 2H), 4.30 (t, J = 7.4 Hz, 2H),
1
3
.96 – 1.83 (m, 2H), 1.47 – 1.30 (m, 7H), 0.91 (t, J = 6.4 Hz, 3H). C NMR (101 MHz, Chloroform-d)
δ 163.97 (C), 156.65 (C), 143.29 (C), 134.35 (C), 131.64 (CH), 129.93 (C), 129.54 (CH), 129.03 (CH
x2), 125.76 (CH x2), 62.24 (CH ), 52.98 (CH ), 28.80 (CH ), 28.09 (CH ), 22.32 (CH ), 14.23 (CH ),
2
2
2
2
2
3
1
3.95 (CH3).
.1.17. General procedure for the preparation of pyridazinone carboxilic acid derivatives 34-38
A solution of NaOH 2 M (50 mL) was added to a stirred solution of pyridazinone ester 29-33 (3
mmol) in EtOH (50 mL). The resulting mixture was heated to reflux and stirred for 8 h. Then the
4
19

ACCEPTED MANUSCRIPT
solution was cooled at room temperature and acidified with HCl 6N .The resulting precipitate was
filtered under vacuum, washed with H O and air-dried to yield the analytically pure acid.
2
4.1.18.
6-(4-Chloro-3-methylphenyl)-2-(4-methylbenzyl)-3-oxo-2,3-dihydropyridazine-4-carboxylic
acid (34)
Hydrolysis of ester 29 allowed to obtain acid 34 as a white solid (0.80 g, 73%), R = 0.49
f
1
(
CHCl /MeOH 9:1); mp 233-235 °C; H NMR (400 MHz, DMSO-d ) δ 7.90 (s, 2H), 7.72 (d, J = 8.3
3
6
Hz, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 7.4 Hz, 2H), 7.15 (d, J = 7.2 Hz, 2H), 5.29 (s, 2H), 2.39
1
3
(
(
(
s, 3H), 2.27 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 165.09 (C), 157.76 (C), 142.96 (C), 138.69
6
C), 136.81 (C), 136.07 (C), 134.35 (C), 133.58 (C), 133.23 (C), 129.33 (CH), 128.99 (CH x2), 128.44
CH), 128.07 (CH x2), 126.08 (CH), 124.85 (CH), 54.68 (CH ), 20.66 (CH ), 19.57 (CH ).
2
3
3
4.1.19. 6-(4-Chloro-3-methylphenyl)-2-(4-fluorobenzyl)-3-oxo-2,3-dihydropyridazine-4-carboxylic acid
(35).
Hydrolysis of ester 30 allowed to obtain acid 35 as a white solid (0.95 g, 85%), R = 0.52
f
1
(
CHCl /MeOH 9:1); mp 209-210 °C; H NMR (400 MHz, Chloroform-d) δ 8.62 (s, 1H), 7.70 (s, 1H),
3
1
3
7
.65 – 7.45 (m, 3H), 7.07 (t, J = 8.6 Hz, 2H), 5.48 (s, 2H), 2.47 (s, 3H); C NMR (101 MHz,
Chloroform-d) δ 164.18 (C), 163.03 (C), 161.44 (C), 146.08 (C), 141.56 (C), 138.37 (C), 137.33 (C),
33.36 (CH), 131.58 (CH), 131.17 (CH), 131.08 (C), 130.04 (CH), 128.49 (CH), 126.45 (C), 124.78
CH), 115.81 (CH), 56.19 (CH ), 20.28 (CH ).
1
(
2
3
4.1.20. 2-(4-Fluorobenzyl)-3-oxo-6-phenyl-2,3-dihydropyridazine-4-carboxylic acid (36)
Hydrolysis of ester 31 allowed to obtain acid 36 as a pale yellow solid (0.93 g, 94.5%), R =
f
1
0
.20 (CHCl /MeOH 9:1); mp 151-152 °C; H NMR (400 MHz, Chloroform-d) δ 8.66 (s, 1H), 7.85 (d, J
3
1
3
=
4.6 Hz, 2H), 7.64 – 7.38 (m, 5H), 7.06 (t, J = 8.4 Hz, 2H), 5.49 (s, 2H); C NMR (101 MHz,
20

ACCEPTED MANUSCRIPT
Chloroform-d) δ 164.16 (C), 163.17 (C), 161.48 (C), 146.93 (C), 133.54 (CH), 133.18 (C), 131.19
(
CH), 131.10 (CH), 130.60 (CH), 130.24 (C), 129.31 (CH x2), 126.55 (C), 126.17 (CH x2), 116.00
CH), 115.78 (CH), 56.11 (CH2).
(
4.1.21. 6-(4-Chloro-3-methylphenyl)-3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxylic acid (37)
Hydrolysis of ester 32 allowed to obtain acid 37 as a pink solid (0.95 g, 94%), R = 0.59 (CHCl /MeOH
f
3
1
9
:1); mp 128-129 °C; H NMR (400 MHz, Chloroform-d) δ 8.64 (s, 1H), 7.72 (s, 1H), 7.63 (d, J = 8.0
Hz, 1H), 7.47 (d, J = 7.8 Hz, 1H), 4.37 (t, J = 7.6 Hz, 2H), 2.47 (s, 3H), 1.93 (s, 2H), 1.41-1.33 (m,
1
3
4
1
5
H), 0.93 (t, J = 6.2 Hz, 3H). C NMR (101 MHz Chloroform-d) δ 163.43 (C), 161.68 (C), 145.74 (C),
37.26 (C), 136.97 (C), 132.88 (CH), 131.78 (C), 129.98 (CH), 128.47 (CH), 125.92 (C), 124.75 (CH),
3.40 (CH ), 28.63 (CH ), 28.11 (CH ), 22.23 (CH ), 20.26 (CH ), 13.90 (CH ).
2
2
2
2
3
3
4.1.22. 3-Oxo-2-pentyl-6-phenyl-2,3-dihydropyridazine-4-carboxylic acid (38)
Hydrolysis of ester 33 allowed to obtain acid 38 as a pink solid (0.71 g, 82%), R = 0.57
f
1
(
CHCl /MeOH 9:1); mp 89-91 °C; H NMR (400 MHz, Chloroform-d) δ 8.68 (s, 1H), 7.87 (s, 2H),
3
1
3
7
.51 (s, 3H), 4.39 (t, J = 7.2 Hz, 2H), 1.94 (s, 2H), 1.41-1.33 (m, 4H), 0.93 (t, J = 6.2 Hz, 3H). C
NMR (101 MHz, Chloroform-d) δ 163.49 (C), 161.75 (C), 146.63 (C), 133.37 (C), 133.08 (CH),
130.45 (CH), 129.27 (CH x2), 126.12 (CH x2), 125.87 (C), 53.35 (CH ), 28.63 (CH ), 28.09 (CH ),
2 2 2
2
2.24 (CH ), 13.89 (CH ).
2
3
4.1.23. Diethyl 2-hydroxy-2-(1-oxo-2,3-dihydro-1H-inden-2-yl)malonate (41) [28]
A mixture of 1-indanone 40 (2 g, 15.1 mmol, 1 eq) and diethylketomalonate (2.76 mL, 18.1
mmol, 1.2 eq) was heated at 100 °C overnight. After cooling at room temperature, the residue was
purified by flash chromatography (petroleum ether/EtOAc 8:2) to obtain 41 as a pale yellow solid (4.3
1
g, 93%), R = 0.32 (petroleum ether/EtOAc 8:2); mp 75-77 °C (79-80 °C) [28]; H NMR (400 MHz,
f
21

ACCEPTED MANUSCRIPT
Chloroform-d) δ 7.71 (d, J = 7.6 Hz, 1H), 7.59 (t, J = 7.4 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.35 (t, J =
7
1
.4 Hz, 1H), 4.50 – 4.37 (m, 2H), 4.29 (dd, J = 10.9, 7.1 Hz, 2H), 3.79 – 3.69 (m, 1H), 3.43 (dd, J =
7.2, 8.3 Hz, 1H), 3.20 (dd, J = 17.2, 4.8 Hz, 1H), 1.36 (t, J = 7.1 Hz, 3H), 1.31 (t, J = 7.1 Hz, 3H).
4.1.24. Ethyl 3-oxo-3,5-dihydro-2H-indeno[1,2-c]pyridazine-4-carboxylate (42) [28]
A solution of diester 41 (4.3 g, 14 mmol, 1 eq) and hydrazine dihydrochloride (1.46 g, 14 mmol,
1
eq) in absolute ethanol (85 mL) was heated to reflux and stirred for 24 h. After cooling at room
temperature the solvent was removed under reduced pressure, and the residue was diluted with
saturated KHCO solution and H O, then the aqueous solution was extracted in a separatory funnel
3
2
with CH Cl . The collected organic phases dried (Na SO ) and concentrated under reduced pressure
2
2
2
4
gave a residue which purified by flash chromatography (petroleum ether/EtOAc 4:6) yelded 42 as a
1
white solid (1.5 g, 40%), R = 0.39 (petroleum ether/EtOAc 4:6); mp 228-230 °C; H NMR (400 MHz,
f
Chloroform-d) δ 11.17 (br s, 1H, NH, exch with D O), 7.86 (d, J = 6.9 Hz, 1H), 7.66 – 7.33 (m, 3H),
2
1
3
4
1
1
.49 (q, J = 7.1 Hz, 2H), 4.18 (s, 2H), 1.45 (t, J = 7.1 Hz, 3H); C NMR (101 MHz, Chloroform-d) δ
63.32 (C), 158.11 (C), 150.89 (C), 150.45 (C), 142.67 (C), 135.21 (C), 130.66 (CH), 128.13 (CH),
25.83 (C), 125.40 (CH), 121.18 (CH), 62.02 (CH ), 35.32 (CH ), 14.31 (CH ).
2
2
3
4.1.25. Ethyl 2-(4-fluorobenzyl)-3-oxo-3,5-dihydro-2H-indeno[1,2-c]pyridazine-4-carboxylate (43)
A mixture of pyridazinone 42 (0.80 g, 3.12 mmol, 1 eq), potassium carbonate (1.3 g, 9.36
mmol, 3 eq) and 4-fluorobenzyl bromide (1.16 mL, 9.36 mmol, 3 eq) in anhydrous DMF (9 mL) was
exposed to ultrasound irradiation for 4 h at 30 °C. Then the suspension was poured into a separatory
funnel, diluted with EtOAc and washed with H O and LiCl 5% solution (x5). The organic solution
2
dried (Na SO ) and concentrated under reduced pressure gave 0.7 g of a yellow oil which was used for
2
4
further reaction without purification.
.1.26. 2-(4-Fluorobenzyl)-3-oxo-3,5-dihydro-2H-indeno[1,2-c]pyridazine-4-carboxylic acid (44)
4
22

ACCEPTED MANUSCRIPT
To a solution of ester 43 (0.7 g, 1.9 mmol) in EtOH (30 ml) was added a solution of NaOH 2 M
(30 mL) and the resulting mixture was refluxed for 6 h. After cooling at room temperature the solution
was acidified with HCl 6N. The precipitate was filtered, washed with H O and air-dried to yield the
2
analytically pure acid 44 as a brown solid (0.3 g, 46%), R = 0.36 (CHCl /MeOH 98:2); mp 80-82 °C;
f
3
1
H NMR (400 MHz, Chloroform-d) δ 7.69 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.48 (d, J = 7.6
Hz, 1H), 7.37 (t, J = 7.4 Hz, 1H), 7.00 – 6.88 (m, 4H), 5.12 (s, 2H), 4.14 (d, J = 13.1 Hz, 1H), 3.26 (d,
J = 13.1 Hz, 1H).
4.1.27. General procedure for the synthesis of carboxamides 2-19,39
A mixture of HOBt (0.15 g, 1.05 mmol, 1.5 eq), EDC (0.27 g, 1,4 mmol, 2 eq) and acids 34-38
and 44 (0,7 mmol, 1 eq) in CH Cl (6 mL) was stirred at room temperature for 30 min. Afterward a
2
2
solution of appropriate amine in CH Cl (6 mL) was added dropwise at 0 °C. The resulting mixture was
2
2
allowed to stand at room temperature, and then stirred for 12-24 h. The solution was then poured into a
separatory funnel and H O was added. The aqueous phase was separated and extracted with CH Cl
2.
2
2
The collected organic phases were washed with H O and brine, dried (Na SO ) and concentrated under
2
2
4
reduced pressure. The analytically pure product was isolated by column flash chromatography
purification using the appropriate eluents.
4
.1.28. 6-(4-Chloro-3-methylphenyl)-N-cyclohexyl-2-(4-methylbenzyl)-3-oxo-2,3-dihydropyridazine-4-
carboxamide (2)
General procedure for the synthesis of carboxamides was used to convert acid 34 and
cyclohexylamine into the title product. The mixture of reaction was stirred for 12 h, then the crude of
reaction was purified by flash chromatography (petroleum ether/EtOAc 8:2) to afford 2 (91 mg, 29%)
as a white solid. R = 0.24 (petroleum ether/EtOAc 95:5); mp 133-135 °C; IR 1677 (C=O), 3260 (NH);
f
1
H NMR (400 MHz, Chloroform-d) δ 9.60 (d, J = 8.1 Hz, 1H, NH, exch with D O), 8.64 (s, 1H), 7.73
2
23

ACCEPTED MANUSCRIPT
(s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.37 (d, J = 7.7 Hz, 2H), 7.17 (d, J = 7.7 Hz,
2
2
1
1
4
H), 5.44 (s, 2H), 4.03 – 3.91 (m, 1H), 2.45 (s, 3H), 2.33 (s, 3H), 2.02 – 1.91 (m, 2H), 1.83 – 1.70 (m,
1
3
H), 1.59 (d, J = 15.2 Hz, 2H), 1.49 – 1.21 (m, 4H). C NMR (101 MHz, Chloroform-d) δ 160.64 (C),
59.74 (C), 144.40 (C), 138.08 (C), 136.85 (C), 136.16 (C), 132.70 (C), 132.48 (C), 131.31 (CH),
29.96 (C), 129.70 (CH), 129.41 (CH x2), 128.67 (CH x2), 128.43 (CH), 124.70 (CH), 56.19 (CH2),
8.60 (CH), 32.68 (CH x2), 25.61 (CH ), 24.69 (CH x2), 21.19 (CH ), 20.23 (CH ); MS (ESI):
2
2
2
3
3
+
C H ClN O requires m/z 449.1, found 450.1 [M + 1] ; Anal. calcd for C H ClN O : C, 69.40; H,
26
28
3
2
26 28
3
2
6.27; N, 9.34; Found: C, 69.42; H, 6.23; N, 9.36.
4.1.29. 6-(4-Chloro-3-methylphenyl)-N-cycloheptyl-2-(4-methylbenzyl)-3-oxo-2,3-dihydropyridazine-4-
carboxamide (3)
General procedure for the synthesis of carboxamides was used to convert acid 34 and
cycloheptylamine into the title product. The mixture of reaction was stirred for 16 h, then the crude of
reaction was purified by flash chromatography (petroleum ether/EtOAc 8:2) to afford 3 (78 mg, 24%)
as a pale yellow solid. R = 0.22 (petroleum ether/EtOAc 95:5); IR 1682 (C=O), 3257 (NH); mp 110-
f
1
1
1
12 °C; H NMR (400 MHz, Chloroform-d) δ 9.65 (d, J = 8.0 Hz, 1H, NH, exch with D O), 8.64 (s,
2
H), 7.72 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.37 (d, J = 7.7 Hz, 2H), 7.16 (d, J
=
7.7 Hz, 2H), 5.43 (s, 2H), 4.27 – 4.04 (m, 1H), 2.45 (s, 3H), 2.33 (s, 3H), 2.09 – 1.91 (m, 2H), 1.75 –
1
3
1
1
.49 (m, 10H); C NMR (101 MHz, Chloroform-d) δ 160.41 (C), 159.73 (C), 144.38 (C), 138.07 (C),
36.85 (C), 136.15 (C), 132.70 (C), 132.49 (C), 131.26 (CH), 129.96 (C), 129.71 (CH), 129.40 (CH
x2), 128.68 (CH x2), 128.42 (CH), 124.68 (CH), 56.15 (CH ), 50.91 (CH), 34.72 (CH x2), 28.07 (CH
2
2
2
x2), 24.16 (CH x2), 21.19 (CH ), 20.23 (CH ); MS (ESI): C H ClN O requires m/z 463.2, found
2
3
3
27 30
3
2
+
4
64.4 [M + 1] ; Anal. calcd for C H ClN O : C, 69.89; H, 6.52; N, 9.06; Found: C, 69.86; H, 6.54;
27 30 3 2
N, 9.03.
24

ACCEPTED MANUSCRIPT
4.1.30.
N-(Adamantan-1-yl)-6-(4-chloro-3-methylphenyl)-2-(4-methylbenzyl)-3-oxo-2,3-
dihydropyridazine-4-carboxamide (4)
General procedure for the synthesis of carboxamides was used to convert acid 34 and 1-
adamantylamine into the title product. The mixture of reaction was stirred for 18 h, then the crude of
reaction was purified by flash chromatography (petroleum ether/EtOAc 9:1) to afford 4 (84 mg, 24%)
as a white solid. R = 0.39 (petroleum ether/EtOAc 95:5); IR 1680 (C=O), 3264 (NH); mp 169-170 °C;
f
1
H NMR (400 MHz, Chloroform-d) δ 9.50 (br s, 1H, NH, exch with D O), 8.63 (s, 1H), 7.72 (s, 1H),
2
7
5
.63 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.36 (d, J = 7.7 Hz, 2H), 7.16 (d, J = 7.6 Hz, 2H),
13
.43 (s, 2H), 2.43 (s, 3H), 2.33 (s, 3H), 2.23 – 2.04 (m, 9H), 1.83 – 1.55 (m, 6H); C NMR (101 MHz,
Chloroform-d) δ 159.13 (C), 158.83 (C), 143.32 (C), 137.01 (C), 135.80 (C), 135.09 (C), 131.71 (C),
1
1
1
31.49 (C), 129.90 (CH), 129.77 (C), 128.68 (CH), 128.36 (CH x2), 127.66 (CH x2), 127.36 (CH),
23.58 (CH), 55.11 (CH ), 51.35 (C), 40.30 (CH x3), 35.40 (CH x3), 28.40 (CH x3), 20.17 (CH ),
2
2
2
3
+
9.19 (CH ); MS (ESI): C H ClN O requires m/z 501.2, found 502.2 [M + 1] ; Anal. calcd for
3
30 32
3
2
C H ClN O : C, 71.77; H, 6.42; N, 8.37; Found: C, 71.80; H, 6.41; N, 8.35.
30
32
3
2
4
4
.1.31. 6-(4-Chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-(cyclohexyl)-3-oxo-2,3-dihydropyridazine-
-carboxamide (5)
General procedure for the synthesis of carboxamides was used to convert acid 35 and
cyclohexylamine into the title product. The mixture of reaction was stirred for 18 h, then the crude of
reaction was purified by flash chromatography (petroleum ether/EtOAc 8:2) to afford 5 (91 mg, 29%)
as a white solid. R = 0.72 (petroleum ether/EtOAc 8:2); mp 151-152 °C; IR 1684 (C=O), 3259 (NH);
f
1
H NMR (400 MHz, Chloroform-d) δ 9.56 (d, J = 8.1 Hz, 1H, NH, exch with D O), 8.65 (s, 1H), 7.72
2
(d, J = 2.3 Hz, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.52 – 7.38 (m, 3H), 7.04 (t, J = 8.6 Hz, 2H), 5.43 (s, 2H),
13
4
.04 – 3.91 (m, 1H), 2.45 (s, 3H), 2.01 – 1.92 (m, 2H), 1.79 – 1.59 (m, 3H), 1.51 – 1.21 (m, 5H). C
25

ACCEPTED MANUSCRIPT
NMR (101 MHz, Chloroform-d) δ 163.88 (C), 161.43 (C), 160.51 (C), 159.70 (C), 144.62 (C), 136.91
(C), 136.28 (C), 132.56 (C), 131.47 (CH), 130.72 (CH), 130.64 (CH), 130.06 (C), 129.74 (CH), 128.41
(
CH), 124.69 (CH), 115.76 (CH), 115.55 (CH), 55.64 (CH ), 48.60 (CH) 32.67 (CH x 2), 25.60 (CH ),
2
2
2
+
2
4.66 (CH x2), 20.23 (CH ); MS (ESI): C H ClFN O requires m/z 453.2, found 454.4 [M + 1] ;
2 3 25 25 3 2
Anal. calcd for C H ClFN O : C, 66.15; H, 5.55; N, 9.26; Found: C, 66.19; H, 5.54; N, 9.22.
2
5
25
3
2
4
4
.1.32. 6-(4-Chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-(cycloheptyl)-3-oxo-2,3-dihydropyridazine-
-carboxamide (6)
General procedure for the synthesis of carboxamides was used to convert acid 35 and
cycloheptylamine into the title product. The mixture of reaction was stirred for 18 h, then the crude of
reaction was purified by flash chromatography (petroleum ether/EtOAc 8:2) to afford 6 (144 mg, 30%)
as a white solid. R = 0.70 (petroleum ether/EtOAc 8:2); mp 140-141 °C; IR 1680 (C=O), 3270 (NH);
f
1
H NMR (400 MHz, Chloroform-d) δ 9.61 (d, J = 8.0 Hz, 1H, NH, exch with D O), 8.65 (s, 1H), 7.71
2
(s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.52 – 7.41 (m, 3H), 7.04 (t, J = 8.6 Hz, 2H), 5.43 (s, 2H), 4.22 – 4.07
1
3
(
m, 1H), 2.45 (s, 3H), 2.08 – 1.92 (m, 2H), 1.74 – 1.50 (m, 10H); C NMR (101 MHz, Chloroform-d)
δ 163.89 (C), 161.43 (C), 160.29 (C), 159.71 (C), 144.62 (C), 136.92 (C), 136.29 (C), 132.57 (C),
31.44 (CH), 130.73 (CH), 130.65 (CH), 130.08 (C), 129.75 (CH), 128.41 (CH), 124.67 (CH), 115.76
CH), 115.55 (CH), 55.61 (CH ), 50.91 (CH), 34.71 (CH x2), 28.07 (CH x2), 24.14 (CH x2), 20.24
1
(
(
2
2
2
2
+
CH ); MS (ESI): C H ClFN O requires m/z 467.2, found 468.3 [M + 1] ; Anal. calcd for
3
26 27
3
2
C H ClFN O : C, 66.73; H, 5.82; N, 8.98; Found: C, 66.75; H, 5.81; N, 8.95.
26
27
3
2
4.1.33.
N-(Adamantan-1-yl)-6-(4-chloro-3-methylphenyl)-2-(4-fluorobenzyl)-3-oxo-2,3-
dihydropyridazine-4-carboxamide (7)
General procedure for the synthesis of carboxamides was used to convert acid 35 and 1-
adamantylamine into the title product. The mixture of reaction was stirred for 18 h, then the crude of
26

ACCEPTED MANUSCRIPT
reaction was purified by flash chromatography (petroleum ether/EtOAc 8:2) to afford 7 (110 mg, 30%)
as a white solid. R = 0.73 (petroleum ether/EtOAc 8:2); mp 130-132 °C; IR 1685 (C=O), 3262 (NH);
f
1
H NMR (400 MHz, Chloroform-d) δ 9.45 (br s, 1H, NH, exch with D O), 8.64 (s, 1H), 7.71 (s, 1H),
2
7.62 (d, J = 8.3 Hz, 1H), 7.52 – 7.40 (m, 3H), 7.04 (t, J = 8.3 Hz, 2H), 5.42 (s, 2H), 2.44 (s, 3H), 2.23 –
1
3
2
.08 (m, 9H), 1.83 – 1.67 (m, 6H); C NMR (101 MHz, Chloroform-d) δ 163.87 (C=O), 161.42
(
C=O), 160.01 (C), 159.83 (C), 144.59 (C), 136.90 (C), 136.25 (C), 132.60 (C), 131.11 (CH), 130.91
C), 130.72 (CH), 130.64 (CH), 129.75 (CH), 128.37 (CH), 124.59 (CH), 115.74 (CH), 115.53 (CH),
(
5
5.60 (CH ), 52.41 (C), 41.33(CH x3), 36.41(CH x3), 29.42(CH x3), 20.22(CH ); MS (ESI):
2 2 2 3
+
C H ClFN O requires m/z 505.2, found 506.2 [M + 1] ; Anal. calcd for C H ClFN O : C, 68.84;
29
29
3
2
29 29
3
2
H, 5.78; N, 8.30; Found: C, 68.88; H, 5.77; N, 8.31.
4
4
.1.34. 6-(4-Chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-((2S)-fenchyl)-3-oxo-2,3-dihydropyridazine-
-carboxamide (8)
General procedure for the synthesis of carboxamides was used to convert acid 35 and N-(1)-(S)-
fenchylamine into the title product. The mixture of reaction was stirred for 18 h, then the crude of
reaction was purified by flash chromatography (petroleum ether/EtOAc 8:2) to afford 8 (174 mg, 49%)
as a white solid. R = 0.23 (petroleum ether/EtOAc 95:5); mp 143-145 °C; IR 1683 (C=O), 3269 (NH);
f
1
H NMR (400 MHz, Chloroform-d) δ 9.83 (d, J = 9.2 Hz, 1H, NH, exch with D O), 8.66 (s, 1H), 7.70
2
(s, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.55 – 7.46 (m, 2H), 7.43 (d, J = 8.3 Hz, 1H), 7.05 (t, J = 8.6 Hz, 2H),
5.46 (s, 2H), 3.81 (d, J = 8.8 Hz, 1H), 2.44 (s, 3H), 1.85 – 1.76 (m, 2H), 1.76 – 1.69 (m, 1H), 1.63 –
13
1
.47 (m, 2H), 1.33 – 1.20 (m, 2H), 1.17 (s, 3H), 1.09 (s, 3H), 0.86 (s, 3H); C NMR (101 MHz,
Chloroform-d) δ 162.88 (C), 161.22 (C), 160.42 (C), 158.84 (C), 143.56 (C), 135.87 (C), 135.23 (C),
31.58 (C), 130.35 (CH), 129.92 (CH), 129.83 (CH), 128.85 (C), 128.73 (CH), 127.36 (CH), 123.61
CH), 114.70 (CH), 114.48 (CH), 63.86 (CH), 54.23 (CH ), 47.86 (C), 47.28 (CH), 41.72 (CH ), 38.78
1
(
2
2
27

ACCEPTED MANUSCRIPT
(
C), 29.96 (CH ), 26.65 (CH ), 25.02 (CH ), 20.58 (CH ), 19.20 (CH ), 18.77 (CH ); MS (ESI):
3
2
2
3
3
3
+
C H ClFN O requires m/z 507.2, found 508.3 [M + 1] ; Anal. calcd for C H ClFN O : C, 68.56;
29
31
3
2
29 31
3
2
H, 6.15; N, 8.27; Found: C, 68.54; H, 6.18; N, 8.23.
.1.35. 6-(4-Chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-(cis-4-methylcyclohexyl)-3-oxo-2,3-
4
dihydropyridazine-4-carboxamide (9) and 6-(4-Chloro-3-methylphenyl)-2-(4-fluorobenzyl)-N-(trans-4-
methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (10)
Compound 9 and 10 were obtained from acid 35 using the general procedure for the synthesis
of carboxamides. The mixture of reaction was stirred for 20 h, then cis/trans isomers were separated
by flash chromatography (petroleum ether/EtOAc 9:1). 9 (65 mg, 27 %) white solid; R = 0.62
f
1
(
petroleum ether/EtOAc 8:2); IR 1682 (C=O), 3271 (NH); mp 125-126 °C; H NMR (400 MHz,
Chloroform-d) δ 9.85 (d, J = 7.8 Hz, 1H, NH, exch with D O), 8.65 (s, 1H), 7.71 (s, 1H), 7.63 (d, J =
2
8
3
.4 Hz, 1H), 7.57 – 7.36 (m, 3H), 7.05 (t, J = 8.4 Hz, 2H), 5.45 (s, 2H), 4.28 – 4.16 (m, 1H), 2.45 (s,
1
3
H), 1.88 – 1.76 (m, 2H), 1.70 – 1.51 (m, 4H), 1.36 – 1.19 (m, 3H), 0.98 (d, J = 6.5 Hz, 3H); C NMR
(
101 MHz, Chloroform-d) δ 163.89 (C), 161.44 (C), 160.64 (C), 159.79 (C), 144.58 (C), 136.91 (C),
36.27 (C), 132.59 (C), 131.32 (CH), 130.82 (CH), 130.73 (CH), 130.10 (C), 129.75 (CH), 128.41
CH), 124.67 (CH), 115.74 (CH), 115.52 (CH), 55.42 (CH ), 45.93 (CH), 30.98 (CH), 30.08 (CH x 2),
1
(
2
2
29.45 (CH x 2), 21.49 (CH ), 20.23 (CH ); MS (ESI): C H ClFN O requires m/z 457.5, found
2 3 3 26 27 3 2
+
4
58.5 [M + 1] ; Anal. calcd for C H ClFN O : C, 66.73; H, 5.82; N, 8.98; Found: C, 66.70; H, 5.84;
2
6
27
3
2
N, 8.90. 10 (26 mg, 11 %) white solid; R = 0.72 (petroleum ether/EtOAc 8:2); IR 1680 (C=O), 3267
f
1
(
NH); mp 119-121 °C; H NMR (400 MHz, Chloroform-d) δ 9.46 (d, J = 8.1 Hz, 1H, NH, exch with
D O), 8.65 (s, 1H), 7.72 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.52 – 7.41 (m, 3H), 7.04 (t, J = 7.9 Hz, 2H),
2
5.43 (s, 2H), 3.87 (d, J = 10.5 Hz, 1H), 2.45 (s, 3H), 2.04 (d, J = 12.3 Hz, 2H), 1.75 (d, J = 13.3 Hz,
1
3
2
H), 1.43 – 1.22 (m, 3H), 1.18 – 1.04 (m, 2H), 0.91 (d, J = 6.5 Hz, 3H); C NMR (101 MHz,
28