Synthesis and structure-activity relationship study of novel 3-heteroarylcoumarins based on pyridazine scaffold as selective MAO-B inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.07.045

Compounds of hybrid structure pyridazine-coumarin were discovered as potent, selective and reversible inhibitors of monoamine oxidase B (MAO-B). These compounds were synthesized in good yield following a multistep approach based on Knoevenagel reaction and using as key intermediate pyridazinone 16, which was obtained from maleic anhydride and furan. Compounds 9b and 9d are the most active compounds of these series, with IC₅₀ values in the sub-micromolar range, and lack of cytotoxic effects. Theoretical calculation of ADME properties also suggested a good pharmacokinetic profile for both compounds. Docking simulations provided insights into enzyme inhibitor interactions and allowed us to rationalize the observed structure-activity relationships (SARs).

Full text of DOI:10.1016/j.ejmech.2017.07.045

Accepted Manuscript
Synthesis and structure-activity relationship study of novel 3-heteroarylcoumarins
based on pyridazine scaffold as selective MAO-B inhibitors
María Carmen Costas-Lago, Pedro Besada, Fernanda Rodríguez-Enríquez, Dolores
Viña, Santiago Vilar, Eugenio Uriarte, Fernanda Borges, Carmen Terán
PII:
S0223-5234(17)30567-6
10.1016/j.ejmech.2017.07.045
EJMECH 9608
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 June 2017
Revised Date: 19 July 2017
Accepted Date: 21 July 2017
Please cite this article as: Marí.Carmen. Costas-Lago, P. Besada, F. Rodríguez-Enríquez, D. Viña,
S. Vilar, E. Uriarte, F. Borges, C. Terán, Synthesis and structure-activity relationship study of novel 3-
heteroarylcoumarins based on pyridazine scaffold as selective MAO-B inhibitors, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.07.045.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis and structure-activity relationship study of novel 3-heteroarylcoumarins
based on pyridazine scaffold as selective MAO-B inhibitors
María Carmen Costas-Lago, Pedro Besada, Fernanda Rodríguez-Enríquez, Dolores
Viña, Santiago Vilar, Eugenio Uriarte, Fernanda Borges, Carmen Terán*
Novel pyridazine-coumarin hybrids were described as potent, selective and reversible
MAOI-B, being analogues 9b and 9d, both substituted with a bromine atom in the
pyridazinyl fragment the most promising compounds.

ACCEPTED MANUSCRIPT
Title: Synthesis and structure-activity relationship study of novel 3-
heteroarylcoumarins based on pyridazine scaffold as selective MAO-B
inhibitors
María Carmen Costas-Lagoa, Pedro Besadaa, Fernanda Rodríguez-Enríquezb, Dolores
Viñab, Santiago Vilarc, Eugenio Uriartec,d, Fernanda Borgese, Carmen Terána *
a
Departamento de Química Orgánica and Instituto de Investigación Sanitaria Galicia
Sur (IISGS), Universidade de Vigo, 36310 Vigo, Spain
b Centro de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS)
Universidade de Santiago de Compostela,15782 Santiago de Compostela, Spain
c Departamento de Química Orgánica, Facultad de Farmacia, Universidade de Santiago
de Compostela, 15782 Santiago de Compostela, Spain.
d
Instituto de Ciencias Químicas Aplicadas, Universidad Autónoma de Chile, 7500912
Santiago, Chile.
e CIQUP/Departamento de Química e Bioquímica, Faculdade de Ciencias, Universidade
do Porto, 4169-007 Porto, Portugal
* Corresponding author.
Phone: 34 986 812276
Fax: 34 986 812262
E.mail: mcteran@uvigo.es

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ABSTRACT
Compounds of hybrid structure pyridazine-coumarin were discovered as potent,
selective and reversible inhibitors of monoamine oxidase B (MAO-B). These
compounds were synthesized in good yield following a multistep approach based on
Knoevenagel reaction and using as key intermediate pyridazinone 16, which was
obtained from maleic anhydride and furan. Compounds 9b and 9d are the most active
compounds of these series, with IC50 values in the sub-micromolar range, and lack of
cytotoxic effects. Theoretical calculation of ADME properties also suggested a good
pharmacokinetic profile for both compounds.
Docking simulations provided insights into enzyme inhibitor interactions and allowed
us to rationalize the observed structure-activity relationships (SARs).
Keywords: Coumarin, Pyridazine, MAO-B, Neurodegenerative Disorders, Molecular
modeling, ADME

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1. Introduction
Monoamine oxidase (MAO) is a FAD-dependent oxidoreductase located in the
mitochondrial outer membrane of glia, neurons and other mammalian tissue cells, such
as liver, kidney, intestinal epithelium, or glandular cells [1]. Two isoenzymes of MAO
have been identified and characterized, the MAO-A and MAO-B, both have about 70%
sequence identity, showing differences not only in their tissue distribution but also in
substrate and inhibitor specificity [2]. MAO-A is the most ubiquitous MAO isoform,
playing an important role in deactivation of neurotransmitter, dietary and xenobiotic
amines, such as norepinephrine, serotonin, dopamine and tyramine, while MAO-B is the
predominant isoform in brain, where it helps to metabolize dopamine and also β-
phenylethylamine, an endogenous amine that promotes dopamine release and inhibits its
neuronal reuptake, without affecting the serotonin or norepinephrine metabolism [3].
These properties determine the clinical importance of both isoforms as targets for drug
design, such as antidepressant (MAO-A) or neuroprotective (MAO-B) agents.
The MAO-catalyzed deamination reaction produces ammonia, the corresponding
aldehyde and hydrogen peroxide, a reactive oxygen species (ROS) which normally is
inactivated by glutathione peroxidase; however, it can be also converted by metal ions,
such as the ferrous ion Fe+2, into the highly reactive hydroxyl radical causing
neurotoxicity [4].
The MAO-B levels in the human species increases during aging and the oxidative stress
induced by MAO in brain is a potential risk factor for neuronal damage and death in
aging and age related neurodegenerative disorders, among them Parkinson´s (PD) and
Alzheimer´s disease (AD) [2].
Nowadays, selective inhibition of MAO-B is considered as a good therapeutic option
for PD in order to reduce the metabolic degradation of dopamine. Thus, selegiline (1,
Figure 1) and rasagiline (2), two irreversible MAO-B inhibitors (MAOI-B), are
commonly used for symptomatic treatment of PD [5]. Another MAOI-B, safinamide
(3), was recently approved in the European Union (EU) for treatment of PD [6].
Safinamide has both dopaminergic (via reversible MAO-B inhibition) and non-
dopaminergic properties, acting also as modulator of glutamate release through the
inhibition of voltage-gated sodium and calcium channels [6].

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Figure 1 comes about here
In addition, several studies point out the neuroprotective role of MAOI-B in order to
prevent the progression of PD and other neurodegenerative diseases, including AD
[4,7]. In fact, the interest in selective MAOI-B has increased in the last few years [8],
due to not only their therapeutic potential for this kind of disorders but also other
pathologies in which the hyperactivity of this isoenzyme plays a crucial role [8a]. The
research in this field has resulted in a significant number of compounds based on great
variety of nitrogen and/or oxygen-containing cores [8a-b,9,10], among them it is
noteworthy the coumarin scaffold [8a,10c-e]. Different substitution patterns at the
coumarin ring have been studied in order to develop potent and selective MAOI-B.
Substitution at 3 position with an acyl function, such as carboxamide (compound 4) or
carbohydrazide (compound 5), or an aryl group (compound 6), are some of the most
interesting [11] (Figure 2). For instance, 3-phenylcoumarins induce strong activity and
selectivity against human MAO-B (hMAO-B), showing outstanding differences with
the kind of substituents and their position at the phenyl rings [11c-d,12]. A small group
(methyl or methoxy) at C6 of the coumarin core seems to be important for both effects
and methoxy, methyl and bromine were the preferred substituents at the 3-phenyl ring.
In addition, replacement of the phenyl group at C3 by some heterocyclic cores, like
thiophene or indole, provided new hMAOI-B (compounds 7 and 8, Figure 2). However,
these compounds inhibited the hMAO-B activity in the sub-micromolar range only
when a methoxy group was located at C6 or C7 of the coumarin nucleus [13].
Figure 2 comes about here
In this work we describe three new series of 3-heteroarylcoumarins showing at C3 a
halogen (bromine, chlorine) or methoxy substituted pyridazine ring and a methyl or
methoxy group at C6, C7 or C8 of the coumarin core (compounds 9-11, Figure 2).
These novel 3-heteroaryl analogues, which combine in their structure two privileged
scaffolds for drug design [14], coumarin and piridazine [15], were proposed with the
aim to analyse the effect of bioisosteric substitution benzene/pyridazine on MAO-B
affinity and selectivity.
2. Results and discussion

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2.1. Chemistry
The target compounds 9-11 were synthesized following the multi-step strategy shown in
Schemes 1 and 2. This approach was based on Knoevenagel reaction of pyridazinone 16
with the appropriate o-hydroxybenzaldehyde.
The key synthon 16 was obtained in four steps from maleic anhydride and furan and via
the enol-lactone 15 [16], such as was displayed in Scheme 1. Although Wittig reaction
between stabilized phosphoranes and cyclic anhydrides supposes a direct approach to
get enol-lactones unsatisfactory results were obtained when this methodology was
applied to maleic anhydride [16]. Accordingly, the alkene group of maleic anhydride
was protected through a Diels-Alder reaction with furan providing the furan-maleic
anhydride cycloadduct 14, which subsequently undergoes a sequence of Wittig and retro
Diels-Alder reactions to give the (2E)-ethyl 2-(5-oxofuran-2(5H)-ylidene)acetate 15 in
high global yield (80% three steps). Finally, the intermediate 15, by reaction with
hydrazine in ethanol, was converted into pyridazinone 16 in moderate yield (52%).
Scheme 1 comes about here
Once compound 16 was obtained, the different coumarin cores were constructed by
condensing heteroaralkyl ester 16 with several methyl or methoxy substituted o-
hydroxybenzaldehydes (17a-f) (Figure 3). The reaction was performed in isopropyl
alcohol at reflux for 5 h in the presence of piperidine giving rise to the intermediates
18a-f of hybrid structure coumarin-pyridazinone in good yields (69-91%).
Figure 3 comes about here
Finally, compounds 18 were converted to the bromides 9a-f by refluxing with POBr3 in
toluene (42-63% yield). Likewise, heating of compounds 18 with an excess of POCl3
led to the chlorides 10a-f (59-70% yield). Chloro derivatives 10 were subsequently
treated with a 25% wt solution of NaOCH3 in methanol at reflux to afford the methoxy
analogues 11a-f (65-81% yield) (Scheme 2).
Scheme 2 comes about here

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2.2. Monoamine oxidase inhibition studies
The biological study of newly synthesized compounds 9-11 on hMAO activity was
carried out by measuring their effects on the production of hydrogen peroxide (H2O2)
from p-tyramine, a common substrate for hMAO-A and hMAO-B, using the two
microsomal MAO isoforms, which were obtained from insect cells (BTI-TN-5B1-4)
infected with recombinant baculovirus containing cDNA inserts for hMAO-A or
hMAO-B, and the Amplex Red MAO assay kit, following a previously described
procedure [12,13a]. The target compounds and reference inhibitors (selegiline and
iproniazide) were unable to react directly with the Amplex Red reagent, which indicates
that these drugs do not interfere with the measurements. In addition, the control activity
of hMAO-A and hMAO-B was performed using p-tyramine as the common substrate
and adjusting the enzyme concentrations to catalyze the oxidation to p-
hydroxyphenylacetaldehyde at a rate of 165±2 pmol/min (n = 20).
The results of the hMAO-A and hMAO-B inhibition studies for compounds 9a-f, 10a-f
and 11a-f, as well as the MAO-B selectivity index (SI hMAO-B = [IC50(hMAO-
A)]/[IC50(hMAO-B)]), were displayed on Table 1. Enzymatic studies revealed that
seventeen of eighteen tested compounds are selective inhibitors of hMAO-B at
micromolar or sub-micromolar concentrations.
Table 1 comes about here
We observed that a halogen atom (chlorine or bromine) at C6 of the pyridazine ring
seems to be better for the activity than a methoxy group, in particular if it was combined
with a small substituent (methoxy or methyl) at C6 or C7 of the coumarin core. In
addition, a methyl group at C8 seems to be tolerated; however, a methoxy group at this
position decreases the MAO-B activity and selectivity in all cases.
Two of the most active compounds of these series are the pyridazinylcoumarins 9b and
9d, with IC50 values against hMAO-B of 0.75 and 0.56 µM, respectively. Both
compounds are substituted in the 3-pyridazinyl ring with a bromine atom, although they
differ in the kind and position of substituent on the coumarin core. Thus, compound 9b
is a C7 methyl derivative while compound 9d contains a methoxy fragment at C6.

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2.3. Reversibility studies of MAO inhibition
Propargylamines, such as selegiline and rasagiline, are irreversible inhibitors of hMAO-
B, since when they bind to the enzyme active centre via a covalent bond between the N-
propargyl fragment and FAD. However, inhibitors lacking propargyl or any other
reactive group could behave as reversible agents, such as safinamide or isatin.
Reversible MAO-B inhibitors, because of their minor pharmacological side effects, can
be considered more promising than irreversible ones [17]. Accordingly, reversibility
experiments were performed to evaluate the type of inhibition for compounds 9b and
9d, the most active compounds in the series (Table 1). An effective dilution method was
used and selegiline (irreversible inhibitor) and isatin (reversible inhibitor) were taken as
standard drugs [18]. Compounds 9b and 9d resulted to be reversible MAO-B inhibitors
(Table 2). However, their reversibility degree is lower than that described for isatin.
Table 2 comes about here
2.3. Molecular docking study
We carried out molecular docking simulations with Glide [19] in the hMAO-B to
determine the hypothetical binding mode for the compounds and study the key
interactions with the enzyme. The crystallized structure selected to run the docking
calculations was 2V60 (PDB code) [20] with the hMAO-B co-crystallized with a
coumarin (c17) in the pocket. Details of the docking protocol and validation can be
found in Experimental Protocols section and in previous studies described by our group
[12b,21].
The novel compounds positioned the coumarin scaffold toward the FAD cofactor and
the pyridazine ring toward the hydrophobic entrance cavity. Figure 4a shows the
binding mode extracted from docking for compound 9d, the most potent compound in
these series. For comparative purposes, co-crystallized coumarin c17 is also shown in
Figure 4a. In compound 9d the α-benzopyrone system orientates the oxygen atoms
towards the bottom of the pocket, so that the carbonyl oxygen establishes a hydrogen
bond with the residue Cys172 (see Figure 4b). In addition, important π-π stacking
interactions between the benzopyrone moiety and phenyl ring of the residue Tyr326
stabilize the complex. We have found similar poses for compounds 9e and 9f with

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methoxy groups at C7 and C8 position of the coumarin scaffold, respectively (see
Figure 4c). However, both compounds (9e and 9f) have shown lower hMAO-B
inhibitory activity compared to compound 9d. Binding modes found for these
compounds showed the coumarin ring slightly shifted towards the FAD region and did
not yield the hydrogen bond with the residue Cys172.
Figure 4 comes about here
We also calculated the interaction between the residues in the hMAO-B pocket and
compounds 9d, 9e and 9f (see Figure 4d). The residue interaction is calculated as the
sum of the terms of Coulomb, van der Waals and hydrogen bonding. According to these
calculations, there are some important residues in ligand recognition, such as Phe168,
Leu171, Cys172, Ile198, Ile199 and Tyr326. The three analyzed compounds showed a
similar interaction profile, although compound 9d yielded a higher contribution with
residue Cys172 compared to compounds 9e and 9f. Moreover, methoxy substituent in
the described poses for compounds 9e and 9f would cause the disruption of the H-
bonding network established by two water molecules in the crystallized hMAO-B
pocket (HOH1309 and HOH1351). This displacement of water molecules could have
effect on binding thermodynamics (see Figure 5a showing the superposition of ligand 9f
and two co-crystallized water molecules in the pocket).
Compounds 9a, 9b and 9c with a methyl substituent at C6, C7 and C8 position of the
benzopyrone moiety, respectively, showed also good binding affinities. Their binding
modes are in agreement with the poses described for 9d. In addition, a hydrogen bond
with the residue Cys172 is also preserved in compound 9b. However, pose described for
compound 9c did not yield any hydrogen bond and the coumarin ring is slightly shifted
toward the FAD region (see Figure 5b). This fact could contribute in the reduction of
hMAO-B inhibitory activity shown by 9c compared to 9b. Residue interaction scores
for these last three compounds are provided in Figure S1 of the Supporting Information.
We have also calculated the hydrophobic/hydrophilic surface generated by the hMAO-B
(see Figure 5c). This surface shows favored hydrophobic and hydrophilic regions in the
pocket that can help to explain the ligand binding modes. The bromine atom in
compound 9b is placed in the hydrophobic region, in the entrance cavity. Likewise,
placement of the C7-methyl substituent shows a good accommodation inside the
hydrophobic surface, which can contribute to its activity (IC50=0.75µM). As described

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previously, compounds 9b and 9c placed the methyl substituent in a region where the
crystal structure contains a water molecule (HOH1351) that must be displaced for the
described binding modes to occur (see Figure S2 of the Supporting Information). The
shift of water molecules could positively or negatively influence the ligand binding.
This water molecule participates in the H-bond network with other waters although it
does not establish H-bonds with the protein. Moreover, the cited water molecule is not
present in the ligand-protein interaction described in alternative hMAO-B crystal
structures, such as 1OJ9, 2BK3 or 2V5Z [22].
On the other hand, compounds with chlorine and methoxy substituents at position C6 of
the pyridazine ring showed a similar binding mode as described previously (see Figure
S3 of the Supporting Information). In the same manner as bromine in compound 9d,
chlorine atom and methoxy group are placed in the hydrophobic region of the active
centre (see Figure 5c with the hMAO-B hydrophobic/hydrophilic surface). The lower
hydrophobic nature showed by chlorine atom [23] could be a key factor to explain the
slight reduction in hMAO-B affinity (IC50 values are 0.75 and 2.51 µM for compounds
9b and 10b respectively).
Figure 5 comes about here
In addition, binding modes described for the new 3-pyridazinylcoumarins are in
agreement with previous results shown by molecular docking studies performed for 3-
phenylcoumarins, as well as with the fact that a small and hydrophobic substituent in
the 3-phenyl ring yielded good results in hMAO-B inhibition and selectivity [12a-b,
20]. Our results corroborated the ability of the compounds to interact with the residue
Ile199. This fact could modify the disposition of the residue Ile199 and preserve an
open conformation in order to make both entrance and substrate cavities join together,
which is determinant for the selective MAO-B inhibition when the molecule is suitable
of binding to the whole active site [24]. Moreover, isoenzyme A and B present in the
pocket some differences in residue composition that can affect selectivity. Additionally,
the presence of different water molecules in their pockets can have an important impact
in ligand binding and complex formation [12a, 25].
2.5. Cytotoxicity studies

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The cytotoxic effects of two concentrations (20 and 10 μM) of compounds 9-11 were
evaluated by using the human neuroblastome cell line SH-SY5Y because of their
similarity to dopaminergic neurons. Compounds were incubated at 20 μM or 10 μM
concentration and maintained for 24h period in cell culture. Afterward, the percentage
of cell viability was measured as MTT reduction [26].
As depicted in Figure 6, only compound 11f (at 20 µM concentration) significantly
decreased the viability of SH-SY5Y cells (Fig 6a). However for a lower concentration
(10 µM), all the compounds resulted well tolerated by the cells (Fig 6b).
Considering the IC50 values obtained for MAOI-B activity, these results justify the
interest of these compounds.
Figure 6 comes about here
2.6 Prediction of ADME properties and BBB permeability
Theoretical calculations were also performed using Molispiration property program [27]
to predict some physicochemical and ADME (absortion, distribution, metabolism and
excretion) parameters of the target compounds and compare with reference inhibitors.
The values of predicted parameters, including lipophilicity, expressed as log P,
molecular weight (MW), topological polar surface area (TPSA), number of hydrogen
acceptors (nON), number of hydrogen bond donors (nOHNH) and molecular volume,
are presented in Table 3. According to these data, all designed compounds (9-11) follow
the Lipinski’s rule without causing any violation [28], which supposes important
information about the potential drug-likeness of these new 3-pyridazinylcoumarins. In
addition, ability of compounds 9-11 to cross the blood-brain barrier (BBB, +/-) was also
predicted by using a CBligand-BBB predictor program [29]. This program uses two
different algorithms AdaBoost and SVM combined with four different fingerprints,
providing predictor scores higher than 0 if the analyzed compound can pass the BBB
(BBB+). As it was detailed in Table 3, all compounds were predicted as BBB (+),
which is essential for MAO inhibition in brain.
Table 3 comes about here
3. Conclusions

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Eighteen 3-pyridazinylcoumarins were synthesized in good yield following a multistep
approach based on Knoevenagel reaction and using as key intermediate pyridazinone
16, which was obtained from maleic anhydride and furan. The novel compounds of
hybrid structure coumarin-pyridazine, whose design was based on bioisosteric
replacement benzene/pyridazine, display potent, selective and reversible effects on
MAO-B activity in the micromolar or submicromolar range, in which are devoid of
cytotoxic activity, being compounds 9b (IC50 = 0.75 µM) and 9d (IC50 = 0.56 µM), both
substituted with a bromine atom in the 3-pyridazinyl ring and a methyl or methoxy
group at C7 or C6, respectively, the most active compounds of these series, displaying
good theoretical ADME properties and BBB penetration.
Molecular docking yielded binding modes for the compounds with the coumarin
nucleus oriented toward the FAD and the pyridazine ring toward the hydrophobic
entrance cavity of the hMAO-B. Our simulations led us to explain the MAOI-B
selectivity and rationalize the structure-activity relationships (SARs) for the studied
series and provided important insights for the rational drug design of new derivatives. In
addition, SARs suggested that, although the bioisosteric substitution benzene-pyridazine
decreases the MAOI-B activity and selectivity, both effects seem to be attenuated by the
inclusion in the pyridazine core of groups with adequate steric and hydrophobic
parameters. All these results proved the potential of this chemical family as a promising
lead for the hMAO-B activity.
4. Experimental Protocols
4.1. General methods and materials
All starting materials and common laboratory chemicals were purchased from
commercial sources and used without further purification. All solvents were distilled
and dried according to standard procedures. Melting points were determined in capillary
1
tubes in a Stuart Scientific apparatus. H and 13C NMR spectra were recorded on a
Bruker ARX400 instrument, using TMS as internal standard [chemical shifts (δ) in
ppm, J in Hz]. The assignment of the signals was performed by COSY, DEPT, HSQC
experiments. High resolution mass spectra were recorded using a Bruker microTOF
focus spectrometer. Silica gel (Merck 60, 230–400 mesh) was used for flash

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chromatography (FC). Analytical TLC was performed on plates precoated with silica
gel (Merck 60 F254, 0.25 mm). Synthesis of (2E)-ethyl 2-(5-oxofuran-2(5H)-
ylidene)acetate 15, which was performed adapting experimental protocols previously
described [16], as well as its analytic and spectroscopic data were described in the
supporting information.
4.2. Ethyl 2-(6-oxo-1,6-dihydropyridazin-3-yl)acetate (16)
To a solution of compound 15 (6.03 g, 35.9 mmol) in ethanol (25 mL) was added
hydrazine monohydrate (2.6 mL, 53.8 mmol) and the reaction mixture was stirred under
reflux for 6 h. The solvent was removed and the residue was purified by column
chromatography on silica gel (1% MeOH/EtOAc) to afford 16 (3.4 g, 52%) as a white
solid. Rf = 0.3 (5% MeOH/EtOAc); m.p. = 108 - 109 oC; 1H NMR (CDCl3): δ = 10.92
(s, 1H, NH), 7.31 (d, 1H, J = 9.8 Hz, H4), 6.95 (d, 1H, J = 9.8 Hz, H5), 4.20 (q, 2H, J =
7.1 Hz, CH2CH3), 3.64 (s, 2H, CH2), 1.28 (t, 3H, J = 7.1 Hz, CH3); 13C NMR (CDCl3):
δ = 169.6 (CO2Et), 162.1 (C6), 142.4 (C3), 134.6 (C4), 129.9 (C5), 61.5 (CH2CH3),
40.0 (CH2), 14.1 (CH3); HRMS (ESI): m/z [M+H]+ calcd for C8H11N2O3, 183.07642;
found 183.07604.
4.3. General procedure for the synthesis of compounds 18a-f
To a solution of the pyridazinone 16 (0.90 mmol) in isopropyl alcohol (8 mL) was
added the corresponding o-hydroxybenzaldehyde 17a-f (1.62 mmol) and piperidine
(0.99 mmol). The reaction mixture was stirred at reflux for 5 h and the resulting
precipitate was filtered, washed with diethyl ether and dried under vacuum to afford the
desired compound.
4.3.1. 6-Methyl-3-(6-oxo-1,6-dihydropyridazin-3-yl)coumarin (18a)
The title compound was prepared using 2-hydroxy-5-methylbenzaldehyde (17a). White
o
1
solid; yield 76%; Rf = 0.3 (EtOAc); m.p. = 299 - 300 C; H NMR (DMSO-d6): δ =
13.35 (s, 1H, NH), 8.30 (s,1H, H4), 7.85 (d, 1H, J = 9.9 Hz, H4’), 7.61 (d, 1H, J = 1.8
Hz, H5), 7.44 (dd, 1H, J = 8.4, 1.8 Hz, H7), 7.32 (d, 1H, J = 8.4 Hz, H8), 6.92 (d, 1H, J
13
= 9.9 Hz, H5’), 2.33 (s, 3H, CH3); C NMR (DMSO-d6): δ = 160.4 (CO), 159.5 (CO),
151.6 (C8a), 141.6 (C4), 141.0 (C3’), 134.2 (C6), 133.7 (C7), 133.6 (C4’), 128.9, 128.7,
122.3 (C3), 118.7 (C4a), 115.9 (C8), 20.3 (CH3); HRMS (ESI): m/z [M+H]+ calcd for
C14H11N2O3, 255.07642; found 255.07642.

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4.3.2. 7-Methyl-3-(6-oxo-1,6-dihydropyridazin-3-yl)coumarin (18b)
The title compound was prepared using 2-hydroxy-4-methylbenzaldehyde (17b). White
o
1
solid; yield 84%; Rf = 0.2 (EtOAc); m.p. = 300 - 301 C; H NMR (DMSO-d6): δ =
13.36 (s, 1H, NH), 8.36 (s, 1H, H4), 7.89 (d, 1H, J = 9.9 Hz, H4’), 7.74 (d, 1H, J = 7.9
Hz, H5), 7.28 (s, 1H, H8), 7.21 (d, 1H, J = 7.9 Hz, H6), 6.95 (d, 1H, J = 9.9 Hz, H5’),
2.42 (s, 3H, CH3); 13C NMR (DMSO-d6): δ = 160.4 (CO), 159.5 (CO), 153.6 (C8a),
143.8 (C7), 141.7 (C4), 141.0 (C3’), 133.6 (C4’), 129.0 (C5), 128.7 (C5’), 126.0 (C6),
121.2 (C3), 116.6 (C4a), 116.1 (C8), 21.4 (CH3); HRMS (ESI): m/z [M+H]+ calcd for
C14H11N2O3, 255.07642; found 255.07657.
4.3.3. 8-Methyl-3-(6-oxo-1,6-dihydropyridazin-3-yl)coumarin (18c)
The title compound was prepared using 2-hydroxy-3-methylbenzaldehyde (17c). White
o
1
solid; yield 77%; Rf = 0.2 (EtOAc); m.p. = 285 - 286 C; H NMR (DMSO-d6): δ =
13.38 (s, 1H, NH), 8.41 (s, 1H, H4), 7.92 (d, 1H, J = 9.9 Hz, H4’), 7.71 (d, 1H, J = 7.5
Hz, H5), 7.55 (d, 1H, J = 7.5 Hz, H7), 7.31 (t, 1H, J = 7.5 Hz, H6), 6.98 (d, 1H, J = 9.9
Hz, H5’), 2.41 (s, 3H, CH3); 13C NMR (DMSO-d6): δ = 160.4 (CO), 159.4 (CO), 151.7
(C8a), 142.0 (C4), 140.9 (C3’), 133.8, 133.6, 128.8 (C5’), 127.1 (C5), 125.0 (C8), 124.5
(C6), 122.1 (C3), 118.7 (C4a), 14.9 (CH3); HRMS (ESI): m/z [M+H]+ calcd for
C14H11N2O3, 255.07642; found 255.07702.
4.3.4. 6-Methoxy-3-(6-oxo-1,6-dihydropyridazin-3-yl)coumarin (18d)
The title compound was prepared using 2-hydroxy-5-methoxylbenzaldehyde (17d).
White solid; yield 91%; Rf = 0.2 (EtOAc); m.p. = 278 - 279 oC; 1H NMR (DMSO-d6): δ
= 13.38 (s, 1H, NH), 8.37 (s, 1H, H4), 7.91 (d, 1H, J = 9.9 Hz, H4’), 7.44 (d, 1H, J =
2.9 Hz, H5), 7.41 (d, 1H, J = 9.1 Hz, H8), 7.25 (dd, 1H, J = 9.1, 2.9 Hz, H7), 6.96 (d,
13
1H, J = 9.9 Hz, H5’), 3.80 (s, 3H, CH3); C NMR (DMSO-d6): δ = 160.4 (CO), 159.5
(CO), 155.8 (C6), 147.9 (C8a), 141.5 (C4), 140.9 (C3’), 133.6 (C4’), 128.7 (C5’), 122.6
(C3), 120.3 (C7), 119.5 (C4a), 117.2 (C8), 111.3 (C5), 55.8 (CH3); HRMS (ESI): m/z
[M+H]+ calcd for C14H11N2O4, 271.07133; found 271.07120.
4.3.5. 7-Methoxy-3-(6-oxo-1,6-dihydropyridazin-3-yl)coumarin (18e)
The title compound was prepared using 2-hydroxy-4-methoxylbenzaldehyde (17e).
White solid; yield 69%; Rf = 0.2 (EtOAc); m.p. = 301 – 302 oC; 1H NMR (DMSO-d6): δ

ACCEPTED MANUSCRIPT
= 13.33 (s, 1H, NH), 8.35 (s, 1H, H4), 7.89 (d, 1H, J = 9.9 Hz, H4’), 7.78 (d, 1H, J =
8.7 Hz, H5), 7.05 (d, 1H, J = 2.3 Hz, H8), 6.99 (dd, 1H, J = 8.7, 2.3 Hz, H6), 6.94 (d,
13
1H, J = 9.9 Hz, H5’), 3.87 (s, 3H, CH3); C NMR (DMSO-d6): δ = 163.2 (C7), 160.4
(CO), 159.6 (CO), 155.5 (C8a), 141.9 (C4), 141.1 (C3’), 133.6 (C4’), 130.5 (C5), 128.7
(C5’), 118.6 (C3), 113.1 (C6), 112.6 (C4a), 100.4 (C8), 56.1 (CH3); HRMS (ESI): m/z
[M+H]+ calcd for C14H11N2O4, 271.07133; found 271.07168.
4.3.6. 8-Methoxy-3-(6-oxo-1,6-dihydropyridazin-3-yl)coumarin (18f)
The title compound was prepared using 2-hydroxy-3-methoxylbenzaldehyde (17f).
White solid; yield 87%; Rf = 0.2 (EtOAc); m.p. = 307 - 308 oC; 1H NMR (DMSO-d6): δ
= 13.38 (s, 1H, NH), 8.38 (s, 1H, H4), 7.91 (d, 1H, J = 9.9 Hz, H4’), 7.41 (dd, 1H, J =
7.1, 2.1 Hz, H5), 7.38 - 7.27 (m, 2H, H6, H7), 6.96 (d, 1H, J = 9.9 Hz, H5’), 3.92 (s,
3H, CH3); 13C NMR (DMSO-d6): δ = 160.4 (CO), 159.1 (CO), 146.3 (C8), 142.7 (C8a),
141.8 (C4), 140.8 (C3’), 133.6 (C4’), 128.7 (C5’), 124.9 (C6), 122.5 (C3), 120.4 (C5),
119.5 (C4a), 114.8 (C7), 56.2 (CH3); HRMS (ESI): m/z [M+H]+ calcd for C14H11N2O4,
271.07133; found 271.07043.
4.4. General procedure for the synthesis of compounds 9a-f
To a solution of compound 18a-f (0.08 mmol) in toluene (4 mL) was added phosporous
oxybromide (2.80 mmol) and the reaction mixture was stirred at reflux for 8 h. After the
solvent was removed, H2O (5 mL) was added and the resulting precipitate was filtered,
washed with diethyl ether and dried under vacuum to afford the proper compound.
4.4.1. 3-(6-Bromopyridazin-3-yl)-6-methylcoumarin (9a)
o
1
Brown solid; yield 42%; Rf = 0.5 (50% EtOAc/hexane); m.p. = 201 - 202 C; H NMR
(CDCl3): δ = 8.95 (s, 1H, H4), 8.48 (d, 1H, J = 9.1 Hz, H4’), 7.73 (d, 1H, J = 9.1 Hz,
H5’), 7.50 - 7.42 (m, 2H, H5, H7), 7.31 (d, 1H, J = 8.4 Hz, H8), 2.45 (s, 3H, CH3); 13C
NMR (CDCl3): δ = 160.3 (C2), 154.7 (C3’), 152.7 (C8a), 147.7 (C6’), 144.2 (C4),
135.1 (C6), 134.7 (C7), 131.4 (C5’), 129.3, 129.0, 121.5 (C3), 119.0 (C4a), 116.5 (C8),
21.0 (CH3); HRMS (ESI): m/z [M+H]+ calcd for C14H10BrN2O2, 316.99202; found
316.99154.
4.4.2. 3-(6-Bromopyridazin-3-yl)-7-methylcoumarin (9b)

ACCEPTED MANUSCRIPT
o
1
Brown solid; yield 52%; Rf = 0.5 (50% EtOAc/hexane); m.p. = 211 - 212 C; H NMR
(CDCl3): δ = 8.97 (s, 1H, H4), 8.48 (d, 1H, J = 9.1 Hz, H4’), 7.72 (d, 1H, J = 9.1 Hz,
H5’), 7.58 (d, 1H, J = 7.9 Hz, H5), 7.22 (s, 1H, H8), 7.19 (d, 1H, J = 7.9 Hz, H6), 2.51
13
(s, 3H, CH3); C NMR (CDCl3): δ = 160.3 (C2), 154.7 (C3’, C8a), 147.5 (C6’), 145.3
(C7), 144.2 (C4), 131.4 (C5’), 129.3 (C5), 128.9 (C4’), 126.6 (C6), 120.5 (C3), 116.9
(C4a, C8), 22.2 (CH3); HRMS (ESI): m/z [M+H]+ calcd for C14H10BrN2O2, 316.99202;
found 316.99207.
4.4.3. 3-(6-Bromopyridazin-3-yl)-8-methylcoumarin (9c)
o
1
Brown solid; yield 57%; Rf = 0.6 (50% EtOAc/hexane); m.p. = 199 - 200 C; H NMR
(CDCl3): δ = 9.01 (s, 1H, H4), 8.51 (d, 1H, J = 9.1 Hz, H4’), 7.75 (d, 1H, J = 9.1 Hz,
H5’), 7.55 (d, 1H, J = 7.6 Hz, H5), 7.49 (d, 1H, J = 7.6 Hz, H7), 7.27 (t, 1H, J = 7.6 Hz,
13
H6), 2.52 (s, 3H, CH3); C NMR (CDCl3): δ = 160.2 (C2), 154.6 (C3’), 153.0 (C8a),
147.6 (C6’), 144.8 (C4), 134.9 (C7), 131.6 (C5’), 129.2 (C4’), 127.5 (C5), 126.3 (C8),
124.9 (C6), 121.0 (C3), 119.0 (C4a), 15.5 (CH3); HRMS (ESI): m/z [M+H]+ calcd for
C14H10BrN2O2, 316.99202; found 316.99278.
4.4.4. 3-(6-Bromopyridazin-3-yl)-6-methoxycoumarin (9d)
o
1
Brown solid; yield 53%; Rf = 0.5 (50% EtOAc/hexane); m.p. = 203 - 204 C; H NMR
(CDCl3): δ = 8.96 (s, 1H, H4), 8.50 (d, 1H, J = 9.1 Hz, H4’), 7.74 (d, 1H, J = 9.1 Hz,
H5’), 7.33 (d, 1H, J = 9.1 Hz, H8), 7.21 (dd, 1H, J = 9.1, 2.9 Hz, H7), 7.10 (d, 1H, J =
13
2.9 Hz, H5), 3.88 (s, 3H, CH3); C NMR (CDCl3): δ = 160.2 (C2), 156.6 (C6), 154.5
(C3’), 149.1 (C8a), 147.7 (C6’), 144.1 (C4), 131.5 (C5’), 129.1 (C4’), 121.9 (C7), 121.7
(C3), 119.5 (C4a), 117.8 (C8), 110.7 (C5), 56.1 (CH3); HRMS (ESI): m/z [M+H]+ calcd
for C14H10BrN2O3, 332.98693; found 332.98756.
4.4.5. 3-(6-Bromopyridazin-3-yl)-7-methoxycoumarin (9e)
o
1
Brown solid; yield 63%; Rf = 0.5 (50% EtOAc/hexane); m.p. = 222 - 223 C; H NMR
(CDCl3): δ = 8.97 (s, 1H, H4), 8.48 (d, 1H, J = 9.1 Hz, H4’), 7.71 (d, 1H, J = 9.1 Hz,
H5’), 7.60 (d, 1H, J = 8.7 Hz, H5), 6.93 (dd, 1H, J = 8.7, 2.4 Hz, H6), 6.87 (d, 1H, J =
13
2.4 Hz, H8), 3.92 (s, 3H, CH3); C NMR (CDCl3): δ = 164.5 (C7), 160.3 (C2), 156.7
(C8a), 154.8 (C3’), 147.2 (C6’), 144.3 (C4), 131.4 (C5’), 130.8 (C5), 128.7 (C4’), 117.8
(C3), 113.8 (C6), 113.0 (C4a), 100.5 (C8), 56.1 (CH3); HRMS (ESI): m/z [M+H]+ calcd
for C14H10BrN2O3, 332.98693; found 332.98794.

ACCEPTED MANUSCRIPT
4.4.6. 3-(6-Bromopyridazin-3-yl)-8-methoxycoumarin (9f)
o
1
Brown solid; yield 53%; Rf = 0.5 (50% EtOAc/hexane); m.p. = 198 - 199 C; H NMR
(CDCl3): δ = 8.99 (s, 1H, H4), 8.50 (d, 1H, J = 9.1 Hz, H4’), 7.73 (d, 1H, J = 9.1 Hz,
H5’), 7.34 - 7.26 (m, 2H, H5, H6), 7.18 (dd, 1H, J = 7.3, 2.4 Hz, H7), 4.01 (s, 3H, CH3);
13C NMR (CDCl3): δ = 159.5 (C2), 154.5 (C3’), 147.7 (C6’), 147.2 (C8), 144.4 (C8a),
144.2 (C4), 131.6 (C5’), 129.1 (C4’), 125.1 (C6), 121.8 (C3), 120.9 (C5), 119.8 (C4a),
115.1 (C7), 56.5 (CH3); HRMS (ESI): m/z [M+H]+ calcd for C14H10BrN2O3, 332.98693;
found 332.98617.
4.5. General procedure for the synthesis of compounds 10a-f
Compound 18a-f (0.36 mmol) was treated with an excess of phosphorus oxychloride (2
mL). The reaction mixture was stirred overnight at reflux, then carefully poured onto ice
(≈1 mL), and finally rendered alkaline with a 25% wt. aqueous solution of NH3. The
resulting precipitate was filtered, washed with water and dried under vacuum to afford
the proper compound.
4.5.1. 3-(6-Chloropyridazin-3-yl)-6-methylcoumarin (10a)
o
1
Brown solid; yield 67%; Rf = 0.6 (50% EtOAc/hexane); m.p. = 187 - 188 C; H NMR
(CDCl3): δ = 8.95 (s, 1H, H4), 8.59 (d, 1H, J = 9.1 Hz, H4’), 7.59 (d, 1H, J = 9.1 Hz,
H5’), 7.51 - 7.40 (m, 2H, H5, H7), 7.31 (d, 1H, J = 8.4 Hz, H8), 2.45 (s, 3H, CH3); 13C
NMR (CDCl3): δ = 160.3 (C2), 156.3 (C6’), 154.4 (C3’), 152.8 (C8a), 144.2 (C4),
135.1 (C6), 134.7 (C7), 129.3, 129.2, 128.1 (C5’), 121.5 (C3), 119.0 (C4a), 116.5 (C8),
21.0 (CH3); HRMS (ESI): m/z [M+H]+ calcd for C14H10ClN2O2, 273.04253; found
273.04322.
4.5.2. 3-(6-Chloropyridazin-3-yl)-7-methylcoumarin (10b)
Brown solid; yield 70%; Rf = 0.6 (50% EtOAc/hexane); m.p. = 217. - 218 oC; 1H NMR
(CDCl3): δ = 8.97 (s, 1H, H4), 8.58 (d, 1H, J = 9.1 Hz, H4’), 7.60 - 7.56 (m, 2H, H5’,
H5), 7.24 - 7.13 (m, 2H, H6, H8), 2.51 (s, 3H, CH3); 13C NMR (CDCl3): δ = 160.4 (C2),
156.1 (C6’), 154.7, 154.5, 145.3 (C7), 144.2 (C4), 129.3, 129.2, 128.0 (C5’), 126.6
(C6), 120.4 (C3), 116.9 (C8), 116.8 (C4a), 22.2 (CH3); HRMS (ESI): m/z [M+H]+ calcd
for C14H10ClN2O2, 273.04253; found 273.04294.

ACCEPTED MANUSCRIPT
4.5.3. 3-(6-Chloropyridazin-3-yl)-8-methylcoumarin (10c)
o
1
Brown solid; yield 59%; Rf = 0.6 (50% EtOAc/hexane); m.p. = 197 - 198 C; H NMR
(CDCl3): δ = 8.99 (s, 1H, H4), 8.59 (d, 1H, J = 9.1 Hz, H4’), 7.60 (d, 1H, J = 9.1 Hz,
H5’), 7.54 (d, 1H, J = 7.6 Hz, H5), 7.49 (d, 1H, J = 7.6 Hz, H7), 7.27 (t, 1H, J = 7.6 Hz,
13
H6), 2.52 (s, 3H, CH3); C NMR (CDCl3): δ = 160.3 (C2), 156.3 (C6’), 154.5 (C3’),
152.9 (C8a), 144.6 (C4), 134.8 (C7), 129.3 (C4’), 128.1 (C5’), 127.4 (C5), 126.3 (C8),
124.8 (C6), 121.2 (C3), 119.0 (C4a), 15.6 (CH3); HRMS (ESI): m/z [M+H]+ calcd for
C14H10ClN2O2, 273.04253; found 273.04334.
4.5.4. 3-(6-chloropyridazin-3-yl)-6-methoxycoumarin (10d)
o
1
Brown solid; yield 61%; Rf = 0.4 (50% EtOAc/hexane); m.p. = 224 - 225 C; H NMR
(CDCl3): δ = 8.97 (s, 1H, H4), 8.60 (d, 1H, J = 9.1 Hz, H4’), 7.60 (d, 1H, J = 9.1 Hz,
H5’), 7.35 (d, 1H, J = 9.1 Hz, H8), 7.23 (dd, 1H, J = 9.1, 2.9 Hz, H7), 7.10 (d, 1H, J =
13
2.9 Hz, H5), 3.89 (s, 3H, CH3); C NMR (CDCl3): δ = 160.2 (C2), 156.6 (C6), 156.3
(C6’), 154.4 (C3’), 149.1 (C8a), 144.0 (C4), 129.3 (C4’), 128.1 (C5’), 121.9 (C3), 121.8
(C7), 119.5 (C4a), 117.9 (C8), 110.8 (C5), 56.1 (CH3); HRMS (ESI): m/z [M+H]+ calcd
for C14H10ClN2O3, 289.03745; found 289.03729.
4.4.5. 3-(6-Chloropyridazin-3-yl)-7-methoxycoumarin (10e)
o
1
Brown solid; yield 63%; Rf = 0.5 (50% EtOAc/hexane); m.p. = 243- 244 C; H NMR
(CDCl3): δ = 8.98 (s, 1H, H4), 8.58 (d, 1H, J = 9.1 Hz, H4’), 7.60 (d, 1H, J = 8.7 Hz,
H5), 7.57 (d, 1H, J = 9.1 Hz, H5’), 6.94 (dd, 1H, J = 8.7, 2.4 Hz, H6), 6.89 (d, 1H, J =
13
2.4 Hz, H8), 3.93 (s, 3H, CH3); C NMR (CDCl3): δ = 164.4 (C7), 160.4 (C2), 156.6,
155.9, 154.6 (C3’), 144.3 (C4), 130.7 (C5), 129.0 (C4’), 128.0 (C5’), 117.8 (C3), 113.8
(C6), 113.0 (C4a), 100.5 (C8), 56.1 (CH3); HRMS (ESI): m/z [M+H]+ calcd for
C14H10ClN2O3, 289.03745; found 289.03678.
4.4.6. 3-(6-Chloropyridazin-3-yl)-8-methoxycoumarin (10f)
o
1
Brown solid; yield 65%; Rf = 0.4 (50% EtOAc/hexane); m.p. = 230 - 231 C; H NMR
(CDCl3): δ = 8.98 (s, 1H, H4), 8.61 (d, 1H, J = 9.1 Hz, H4’), 7.60 (d, 1H, J = 9.1 Hz,
H5’), 7.34 - 7.23 (m, 2H, H5, H6), 7.18 (dd, J = 7.3, 2.1 Hz, H7), 4.01 (s, 3H, CH3); 13C
NMR (CDCl3): δ = 159.6 (C2), 156.3 (C6’), 154.3 (C3’), 147.1 (C8), 144.4 (C4), 144.1
(C8a), 129.4 (C4’), 128.1 (C5’), 125.1 (C6), 121.8 (C3), 120.8 (C5), 119.8 (C4a), 115.0

ACCEPTED MANUSCRIPT
(C7), 56.5 (CH3); HRMS (ESI): m/z [M+H]+ calcd for C14H10ClN2O3, 289.03745; found
289.03759.
4.5. General procedure for the synthesis of compounds 11a-f
To a solution of compound 10a-f (0.10 mmol) in methanol (5 mL) was added a 25% wt.
solution of NaOCH3 in methanol (0.15 mmol) and the reaction mixture was stirred
under reflux overnight. After the solvent was removed, the residue was purified by
column chromatography on silica gel (20% EtOAc/hexane for 11a-c; 30%
EtOAc/hexane for 11d-f) to afford the desired compound.
4.5.1. 3-(6-Methoxypyridazin-3-yl)-6-methylcoumarin (11a)
o
1
White solid; yield 68%; Rf = 0.3 (2:1 hexane/EtOAc); m.p. = 195 - 196 C; H NMR
(CDCl3): δ = 8.78 (s, 1H, H4), 8.43 (d, 1H, J = 9.3 Hz, H4’), 7.43 - 7.37 (m, 2H, H5,
H7), 7.29 (d, 1H, J = 8.3 Hz, H8), 7.05 (d, 1H, J = 9.3 Hz, H5’), 4.19 (s, 3H, CH3O),
13
2.44 (s, 3H, CH3); C NMR (CDCl3): δ = 164.8 (C6’), 160.7 (C2), 152.4 (C8a), 151.3
(C3’), 142.5 (C4), 134.8 (C6), 133.8 (C7), 130.2 (C4’), 128.8 (C5), 122.8 (C3), 119.3
(C4a), 116.8 (C5’), 116.4 (C8), 55.1 (CH3O), 21.0 (CH3); HRMS (ESI): m/z [M+H]+
calcd for C15H13N2O3, 269.09207; found 269.09201.
4.5.2. 3-(6-Methoxypyridazin-3-yl)-7-methylcoumarin (11b)
o
1
White solid; yield 65%; Rf = 0.3 (2:1 hexane/EtOAc); m.p. = 231 - 232 C; H NMR
(CDCl3): δ = 8.80 (s, 1H, H4), 8.42 (d, 1H, J = 9.3 Hz, H4’), 7.52 (d, 1H, J = 7.9 Hz,
H5), 7.20 (s, 1H, H8), 7.15 (d, 1H, J = 7.9 Hz, H6), 7.05 (d, 1H, J = 9.3 Hz, H5’), 4.19
(s, 3H, CH3O), 2.49 (s, 3H, CH3); 13C NMR (CDCl3): δ = 164.7 (C6’), 160.7 (C2),
154.4 (C8a), 151.3 (C3’), 144.3 (C7), 142.5 (C4), 130.2 (C4’), 128.9 (C5), 126.3 (C6),
121.7 (C3), 117.2 (C4a), 116.8, 116.8, 55.0 (CH3O), 22.2 (CH3); HRMS (ESI): m/z
[M+H]+ calcd for C15H13N2O3, 269.09207; found 269.09201.
4.5.3. 3-(6-Methoxypyridazin-3-yl)-8-methylcoumarin (11c)
o
1
White solid; yield 81%; Rf = 0.3 (2:1 hexane/EtOAc); m.p. = 169 - 170 C; H NMR
(CDCl3): δ = 8.82 (s, 1H, H4), 8.44 (d, 1H, J = 9.3 Hz, H4’), 7.48 (d, 1H, J = 7.5 Hz,
H5), 7.44 (d, 1H, J = 7.5 Hz, H7), 7.23 (t, 1H, J = 7.5 Hz, H6), 7.06 (d, 1H, J = 9.3 Hz,
H5’), 4.19 (s, 3H, CH3O), 2.51 (s, 3H, CH3); 13C NMR (CDCl3): δ = 164.8 (C6’), 160.7
(C2), 152.7 (C8a), 151.2 (C3’), 142.9 (C4), 134.0 (C7), 130.2 (C4’), 126.9 (C5), 126.2

ACCEPTED MANUSCRIPT
(C8), 124.6 (C6), 122.6 (C3), 119.3 (C4a), 116.9 (C5’), 55.1 (CH3O), 15.6 (CH3);
HRMS (ESI): m/z [M+H]+ calcd for C15H13N2O3, 269.09207; found 269.09170.
4.5.4. 6-Methoxy-3-(6-methoxypyridazin-3-yl)coumarin (11d)
o
1
White solid; yield 68%; Rf = 0.2 (2:1 hexane/EtOAc); m.p. = 179 - 180 C; H NMR
(CDCl3): δ = 8.77 (s, 1H, H4), 8.43 (d, 1H, J = 9.3 Hz, H4’), 7.33 (d, 1H, J = 9.1 Hz,
H8), 7.17 (dd, J = 9.1, 2.9 Hz, H7), 7.07 - 7.02 (m, 2H, H5’, H5), 4.19 (s, 3H, 6’-
13
OCH3), 3.87 (s, 3H, 6-OCH3); C NMR (CDCl3): δ = 164.8 (C6’), 160.6 (C2), 156.5
(C6), 151.2 (C3’), 148.8 (C8a), 142.3 (C4), 130.3 (C4’), 123.2 (C7), 120.7 (C3), 119.9
(C4a), 117.7 (C8), 116.8 (C5’), 110.7 (C5), 56.0 (6-OCH3), 55.1 (6’-OCH3); HRMS
(ESI): m/z [M+H]+ calcd for C15H13N2O4, 285.08698; found 285.08658.
4.5.5. 7-Methoxy-3-(6-methoxypyridazin-3-yl)coumarin (11e)
o
1
White solid; yield 66%; Rf = 0.2 (2:1 hexane/EtOAc); m.p. = 224 - 225 C; H NMR
(CDCl3): δ = 8.79 (s, 1H, H4), 8.41 (d, 1H, J = 9.3 Hz, H4’), 7.53 (d, 1H, J = 8.6 Hz,
H5), 7.03 (d, 1H, J = 9.3 Hz, H5’), 6.90 (dd, 1H, J = 8.6, 2.3 Hz, H6), 6.86 (d, 1H, J =
2.3 Hz, H8), 4.17 (s, 3H, 6’-OCH3), 3.90 (s, 3H, 7-OCH3); 13C NMR (CDCl3): δ =
164.6 (C6’), 163.7 (C7), 160.8 (C2), 156.3 (C8a), 151.4 (C3’), 142.6 (C4), 130.2 (C5),
130.0 (C4’), 119.3 (C3), 116.8 (C5’), 113.5 (C6), 113.3 (C4a), 100.5 (C8), 56.0 (7-
OCH3), 55.0 (6’-OCH3); HRMS (ESI): m/z [M+H]+ calcd for C15H13N2O4, 285.08698;
found 285.08693.
4.5.6. 8-Methoxy-3-(6-methoxypyridazin-3-yl)coumarin (11f)
o
1
White solid; yield 66%; Rf = 0.2 (2:1 hexane/EtOAc); m.p. = 232 - 233 C; H NMR
(CDCl3): δ = 8.82 (s, 1H, H4), 8.45 (d, 1H, J = 9.3 Hz, H4’), 7.32 - 7.19 (m, 2H, H5,
H6), 7.14 (dd, 1H, J = 7.8, 1.6 Hz, H7), 7.05 (d, 1H, J = 9.3 Hz, H5’), 4.19 (s, 3H, 6’-
13
OCH3), 4.00 (s, 3H, 8-OCH3); C NMR (CDCl3): δ = 164.7 (C6’), 159.7 (C2), 150.9
(C3’), 147.0 (C8), 143.8 (C8a), 142.6 (C4), 130.2 (C4’), 124.7 (C6), 122.9 (C3), 120.4
(C5), 120.0 (C4a), 116.9 (C5’), 114.2 (C7), 56.3 (8-OCH3), 55.0 (6’-OCH3); HRMS
(ESI): m/z [M+H]+ calcd for C15H13N2O4, 285.08698; found 285.08644.
4.6. Determination of MAO isoforms activity

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The effects of the target compounds on enzymatic activity of hMAO isoforms were
evaluated by a fluorimetric method and following previously described protocols
[12,13a]. Briefly, 0.1 mL of sodium phosphate buffer (0.5M, pH 7.4) containing the test
drugs (novel compounds and reference inhibitors), in various concentrations, and
adequate amounts of recombinant hMAO-A or hMAO-B required, adjusted to obtain in
our experimental conditions the same reaction velocity that in the control group, that is
an oxidation rate of 165±2 pmol of p-tyramine/min (hMAO-A: 1.1 µg protein; specific
activity: 150 nmol of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg
protein; hMAO-B: 7.5 µg protein; specific activity: 22 nmol of p-tyramine
o
transformed/min/mg protein), were incubated for 15 min at 37 C in a flat-blackbottom
96-well microtestTM plate, placed in the dark fluorimeter chamber. After this incubation
period, the reaction was started by adding (final concentrations) 200 µM Amplex® Red
reagent, 1 U/mL horseradish peroxidase and 1 mM p-tyramine. The production of H2O2
o
and, consequently, of resorufin was quantified at 37 C in a multidetection microplate
fluorescence reader (FLX800TM, Bio-Tek® Instruments, Inc., Winooski, VT, USA)
based on the fluorescence generated (excitation, 545 nm, emission, 590 nm) over a 15
min period, in which the fluorescence increased linearly.
Control experiments were carried out simultaneously by replacing the test drugs (new
compounds and reference inhibitors) with appropriate dilutions of the vehicles. In
addition, the possible ability of the above test drugs to modify the fluorescence
generated in the reaction mixture due to non-enzymatic inhibition (e.g., for directly
reacting with Amplex® Red reagent) was determined by adding these drugs to solutions
containing only the Amplex® Red reagent in a sodium phosphate buffer.
The specific fluorescence emission (used to obtain the final results) was calculated after
subtraction of the background activity, which was determined from vials containing all
components except the hMAO isoforms, which were replaced by a sodium phosphate
buffer solution.
MAO activity of the test compounds and reference inhibitors is expressed as IC50 values
(concentration of each drug required to produce a 50% decrease on the control value
activity of the isoforms MAO). These values were calculated by using the Origin 5.0
software (Microcal Software Inc., Northampton, MA, USA), from the equations of the
lines obtained by linear regression (methods least squares) of the resulting points to
represent the log of the molar concentration of the test compound (x axis) versus the
percentage inhibition of the control MAO activity achieved with corresponding

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concentrations of each compound (y axis). This linear regression was performed by
using data obtained with 4–6 concentrations of each test compound capable of
inhibiting the control enzyme activity of the MAO isoenzymes by between 20 and 80%.
Also, the [IC50(MAO-A)]/[IC50(MAO-B)] ratio was calculated as an indicator of the rate
of selectivity in the inhibition of both isoforms.
4.7. Reversibility
To evaluate whether compounds 9b and 9d are reversible or irreversible hMAO-B
inhibitors, a dilution method was used [18]. A 100X concentration of the enzyme used
in the above described experiments was incubated with a concentration of inhibitor
equivalent to 10-fold the IC50 value. After 30 min, the mixture was diluted 100-fold into
reaction buffer containing Amplex® Red reagent, horseradish peroxidase and p-
tyramine and the reaction was monitored for 15 min, following the same procedure
previously described for MAO activity assay. Reversible inhibitors show linear progress
with a slope equal to ≈ 91% of the slope of the control sample, whereas irreversible
inhibition reaches only ≈ 9% of this slope. Control tests were carried out by pre-
incubating and diluting the enzyme in the absence of inhibitor.
4.8. Molecular docking
Schrödinger package [19] was used to carry out the docking calculations. The co-
crystallized hMAO-B structure (PDB code: 2V60) [20] bound to the coumarin 7-(3-
chlorobenzyloxy)-4-carboxaldehydecoumarin (c17) was selected for the docking. The
crystallized structure was pre-processed with the Protein Preparation Workflow
available in the modeling package. This process prepared the protein for the following
docking steps and included addition and optimization of hydrogens, cap termini
addition, optimization of protonation states of some residues, H-bond network
optimization, etc. Only a water molecule that establishes a hydrogen bond between the
co-crystallized coumarin and the protein is retained in the pocket for the docking
calculations. The ligands database was pre-processed with the LigPrep module [19].
This module generated different tautomers, protonation states (pH 7.0±2.0) and
optimized the structure of each ligand.

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A receptor grid was generated and centered in the co-crystallized c17 coumarin (outer
box=20 Å), with a van der Waals scaling factor of 1.0 and a partial charge cut-off of
0.25. Docking was carried out using the Standard Precision (SP) mode of Glide [19].
The final pose was selected using the energetic parameter Emodel. Final binding modes
were optimized along with the protein pocket using Prime MM-GBSA [19]. Docking
protocol was previously validated through the root mean square deviation (RMSD)
between the theoretical poses and the co-crystallized conformations of different ligands
[20,21]. As an example the RMSD for the co-crystallized coumarins in 2V60 and 2V61
[20] in the current study are 0.56 and 1.30 respectively.
4.9. Citotoxicity
SH-SY5Y, a twice-subcloned cell line derived from the SK-N-SH neuroblastoma cell
line (ATCC CRL-2266) which is used widely in experimental neurological studies such
as neurotoxicity and neuroprotection, were grown and kept in culture as described
elsewhere [30].
For experiments SH-SY5Y cells (1.106/mL) were seeded in each well of 96-well
culture plate. After overnight incubation, solution of compounds 9-11 in DMSO
(1%) was added to the cells.
Cells treated with DMSO 1% were used as negative control. After further incubation
for 24 h, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was
performed to measure cell viability [26].
Briefly, 10% of MTT solution (5 mg/ml in PBS) was added to each well. After
incubation for 2 h at 37 °C, MTT solution was removed and 100 µL of DMSO was
added to dissolve the crystals formed. Then, absorbance at 540 nm was read using a
microplate reader. The percentage cell viability was calculated as [Absorbance
(treatment)/Absorbance (negative control)] 100%.
Acknowledgments
Financial support from Universidade de Vigo, the Consellería de Cultura, Educación e
Ordenación Universitaria (CN2012/184, and EM2014/016 and Centro singular de
investigación de Galicia accreditación 2016‐2019, ED431G/05) and the European

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Regional Development Fund (ERDF), is gratefully acknowledged. M.C.C.-L. thanks the
Xunta de Galicia for her PhD fellowship.
Supplementary data
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Figure 1. Structure of reference hMAO-B inhibitors

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SO2CH3
O
O
O
NH2
N
H
N
H
OCH3
O
O
O
O
O
4
5
6
H
R
S
N
N
H3CO
6
7
N
R'
R = Br, Cl, OCH3
R' = CH3, OCH3
O
O
O
O
H3CO
O
O
8
9-11
7
8
Figure 2. Structure of several 3-substituted coumarins as MAO-B inhibitors (compounds 4-8)
and general structure of the novel analogues with pyridazine scaffold (compounds 9-11).

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Figure 3. o-Hydroxybenzaldehydes (17a-f) used in the target compounds synthesis.