A novel series of 6-substituted 3-(pyrrolidin-1-ylmethyl)chromen-2-ones as selective monoamine oxidase (MAO) A inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.11.035

A series of 6-substituted 3-(pyrrolidin-1-ylmethyl)chromen-2-ones (coumarins) have been synthesized and their inhibitory activity to human monoamine oxidase A (MAO A) and B (MAO B) determined. Incorporation of a basic amino function in the C3 position together with substitution at the C6 position produced novel coumarin compounds with selectivity for the MAO A subtype. Substitution in the C6 position with small hydrophilic groups such as hydroxy (19, IC₅₀ = 1.46 μM) or amino (18, IC₅₀ = 3.77 μM) gave the most potent and selective compounds for MAO A. These compounds also showed excellent aqueous solubility properties. Compound 18 [6-amino-3- (pyrrolidin-1-ylmethyl)chromen-2-one] administrated in vivo induced in rat brain a neurotransmitter metabolite profile typical of MAO A inhibition: decreased 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) but increased 3-methoxytyramine (3-MT) levels.

Full text of DOI:10.1016/j.ejmech.2013.11.035

European Journal of Medicinal Chemistry 73 (2014) 177e186
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A novel series of 6-substituted 3-(pyrrolidin-1-ylmethyl)chromen-2-
ones as selective monoamine oxidase (MAO) A inhibitors
1
,
Cecilia Mattsson , Peder Svensson 2, Clas Sonesson 2
*
Medicinal Chemistry, NeuroSearch Sweden AB, Biotech Center, Arvid Wallgrens Backe 20, SE-413 46 Gothenburg, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 6-substituted 3-(pyrrolidin-1-ylmethyl)chromen-2-ones (coumarins) have been synthesized
and their inhibitory activity to human monoamine oxidase A (MAO A) and B (MAO B) determined.
Incorporation of a basic amino function in the C3 position together with substitution at the C6 position
produced novel coumarin compounds with selectivity for the MAO A subtype. Substitution in the C6
Received 23 June 2013
Received in revised form
26 November 2013
Accepted 27 November 2013
Available online 15 December 2013
position with small hydrophilic groups such as hydroxy (19, IC50 ¼ 1.46
m
M) or amino (18, IC50 ¼ 3.77
mM)
gave the most potent and selective compounds for MAO A. These compounds also showed excellent
aqueous solubility properties. Compound 18 [6-amino-3-(pyrrolidin-1-ylmethyl)chromen-2-one]
administrated in vivo induced in rat brain a neurotransmitter metabolite profile typical of MAO A inhi-
bition: decreased 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) but
increased 3-methoxytyramine (3-MT) levels.
Keywords:
Monoamine oxidase A inhibitor
Dopamine D2 antagonist
Coumarin
Ó 2013 Elsevier Masson SAS. All rights reserved.
DOPAC
5-HIAA
Molecular modeling
1. Introduction
led to important contributions in the therapy of several neuro-
psychiatric and neurological disorders [4].
Monoamine oxidase (MAO) is a flavoenzyme located intracel-
lularly at the outer mitochondrial membrane responsible for the
oxidative deamination of dietary amines, monoamine neurotrans-
mitters and hormones. The main function of MAOs is to terminate
the actions of neurotransmitter amines in the brain and peripheral
tissues and also to protect the brain from a variety of trace amines
The older MAOIs (e.g. iproniazid) were unselective and irre-
versible and had broad side effect profiles and dietary restrictions
due to “the cheese reaction”, a severe hypertensive crisis upon
consumption of food containing large quantities of tyramine. [5].
Newer reversible inhibitors of MAO A (RIMA) are easily displaced
by ingested tyramine in the gut and thus do not cause the “the
cheese reaction” and no dietary restrictions are needed. Inhibitors
of MAO B are mainly associated with symptomatic treatment of
Parkinson’s disease [6,7]. However, the age-related increase in MAO
B activity, as well as the neuroprotective effects of MAO B inhibitors,
have been considered as a rational to use MAO B inhibitors in
Alzheimer’s disease [8] (e.g. safinamide [9], selegiline [R-(-)-dep-
renyl] [10] and rasagiline [11]). MAO A on the other hand is found in
catecholaminergic neurons and is responsible for the metabolism
of the major neurotransmitters 5-HT, NE and DA, offering a multi
neurotransmitter strategy for the treatment of depression. The
development of reversible inhibitors of MAO A (RIMAs) with better
tolerability profile has sparked a renewed interest for this category
of compounds, particularly in view of their efficacy in treatment
resistant depression (e.g. moclobemide [12,13], brofaromine [12],
toloxatone [14], amifuraline [15] and CX157 [16]) [17].
such as benzylamine, b-phenylethylamine and tyramine [1]. The
MAO mediated degradation of the monoamines serotonin (5-HT),
norepinephrine (NE) and dopamine (DA) in the brain is essential for
a correct function of synaptic neurotransmission. Two distinct
types of MAOs were discovered by Johnston and co workers in the
1970s [2,3], namely MAO A and MAO B. They share 70% amino acid
sequence homology and have different organ and tissue distribu-
tion, substrate specificity and sensitivity to inhibitors and therefore
it appears that the two subtypes have diverse physiological func-
tions. The development of selective MAO inhibitors has therefore
* Corresponding author. Tel.: þ46 (0)709 518355.
E-mail addresses: cecilia.m.mattsson@gmail.com (C. Mattsson), peder.
svensson@astrazeneca.com (P. Svensson), clas.sonesson@irlab.se (C. Sonesson).
1
Current affiliation: AstraZeneca R&D Mölndal, CVGI IMed, SE-431 83 Mölndal,
The coumarins are one of the structural scaffolds that have
shown MAO inhibitory activity and in recent years the knowledge
of how to develop selective MAO B ligands within this class has
Sweden.
2
Current affiliation: Integrative Research Laboratories Sweden AB, Biotech Cen-
ter, Arvid Wallgrens Backe 20, SE-413 46 Gothenburg, Sweden.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.035

178
C. Mattsson et al. / European Journal of Medicinal Chemistry 73 (2014) 177e186
4
3
6
7
1 Esuprone (A)
2 (A)
3 LU 53439 (B)
R3 = -COOH, -COCl, -COOEt, -COPh,
-CONH2, -CONHNH2, -Ph, -Me
5 (A)
6 (A)
4 (B)
7 (A)
8a (A/B)
8b (inactive A/B)
9 (B)
Fig. 1. Reversible MAO A (A) and MAO B (B) inhibitors in the coumarin class.
emerged. On the other hand only a few publications can be found
describing MAO A selective coumarins [18]. In 1994, Rendenbach-
Muller et al. [19] discovered the importance of the C7 position in
a similar series with a polar acyl group at position C3 with different
substituents at position C5eC8 of the coumarin nucleus, the mon-
osubstituted C6 analogs were found to yield low potent to inactive
simple
7-substitued
3,4-dimethylcoumarines
(3,4-
compounds e.g. 8b (MAO A IC50 ¼ 40.2
mM, MAO B inactive).
dimethylchromen-2-one) for the selectivity between the MAO A
and B isoforms. A sulfonic ester linkage yielded MAO A selective
inhibitors (1, esuprone, Fig. 1) and Gnerre et al. [20] identified the
potent and selective MAO A ligand 2 (IC50 ¼ 10.7 nM, Fig. 1), based
on the same motif as 1. However, a phenolic linker with a spaced
heteroaromatic group was found to be preferred for MAO B selec-
tivity (3, LU 53439, Fig. 1), while the corresponding intermediate 7-
hydroxy-3,4-dimethylcoumarin was found to be inactive for both
MAO A and MAO B. In addition, both compounds 1 and 3 were
found to bind reversible to the enzyme [19]. Further studies on 7-
substituted coumarins (4, Fig. 1) provided knowledge about a
wide range of different groups (carboxylic acid, carboxylic ester,
acyl, phenyl, benzyloxy, etc.) that are tolerated in position C3 with
retained selectivity for MAO B [20e27]. In addition, simultaneous
substitution at C3 and C4 with methyl were shown to improve MAO
B inhibition, as did 3,4-annelation with 5- and 6-membered alkyl
rings. However, Santana et al. [28] and Abdelhafez et al. [29,30] also
demonstrated that within the C3-, C4- and C7-substituted cou-
marins, potent and selective MAO A inhibitors can be developed
(i.e. 5, 6 and 7, Fig. 1). This in turn indicates that the selection of
substituent(s), especially in C7 position, is crucial for the outcome
of potency and selectivity for MAO A and B.
Furthermore, Matos et al. [27] reported on a study where they have
instead introduced lipophilic groups at position C3 and C6 such as a
phenyl and methyl group e.g. compound 9 (Fig. 1), yielding a potent
MAO B inhibitor (IC50 ¼ 0.8 nM) without any MAO A activity.
In our search for novel compounds active on the dopaminergic
system in the brain, we applied scaffold hopping from the known
partial DA type 2 agonist (D2), 3-[(benzylamino)methyl]-2,3-
dihydro-1,4-benzodioxin-6-ol (10, Fig. 2) [32] to the 3-(amino-
methyl)chromen-2-one (coumarin) scaffold (11, Fig. 2). We main-
tained a spaced basic amino function in C3 position and focused on
different functionalities in C6 position (e.g. electron withdrawing
and donating groups). The new compounds were screened in vivo
for effects on the dopaminergic, adrenergic, and serotonergic sys-
tem in the rat brain (i.e. striatum) and the aim was to identify
compounds with a DA D2 antagonistic property. However, the 3-
(aminomethyl)chromen-2-one substituted ligands showed
a
different profile in vivo than expected and when we further
analyzed the results we concluded that these new compounds
turned out to be MAO A inhibitors.
Based on the generic structure 11 (Fig. 2) and different sub-
stituents (R1, R2 and R6) listed in Table 1 (12e22), we hereby report
a novel series of selective MAO A ligands. The new compounds were
tested for in vitro activity for human MAO A and B and selected
compounds were also screened for their selectivity against off-
targets (i.e. monoaminergic, serotonergic receptors and their
transporters). In vivo data (i.e. effects on 3,4-dihydroxyphenylacetic
acid [DOPAC], 3-methoxytyramine [3-MT] and 5-hydroxyindo
leacetic acid [5-HIAA] in rat brain, Fig. 1S) is also reported for a
number of compounds which further support the in vitro finding.
Among the existing publications on coumarins as MAO in-
hibitors only a few have reported the effect of substitution at the C6
position. Chimenti et al. [31] reported on a series of C6-substituted
compounds with a polar acyl group at the C3 position, most of these
derivatives turned out to be potent MAO B inhibitors with low
selectivity over MAO A e.g. the nitro analog 8a (pIC50 ¼ 7.17 and 7.71
for MAOA and B, respectively). However, Secci et al. [25] reported on

C. Mattsson et al. / European Journal of Medicinal Chemistry 73 (2014) 177e186
179
10
11
R1, R2 = -H, -Et; -H, -nPr; -(CH2)4-
R6 = -H, -OH, -OMe, -OnPr, -OnBu,
-OCF3 , -NH2, -NO2, -CF3
Fig. 2. Design strategy “scaffold hopping” from the dopamine agonist 1,4-benzodioxan core (10) to coumarin core (11, generic structure).
2. Chemistry
Compound 16 (Scheme 2), which is substituted with a 6-
trifluoromethyl group, was synthesized using a modified version
of the Baylis-Hillman methodology. In this case, using 2-
tetrahydropyranyl (THP) as protecting group instead of a benzyl
group was warranted, as the reactivity of the trifluoromethyl group
is greatly enhanced in p-trifluoromethyl substituted phenols and
thus benzylation of the starting material [2-hydroxy-5-(tri-
fluoromethyl)benzaldehyde] under basic conditions leads to 1,6-
elimination of hydrogen fluoride [38,39]. Using an acid labile pro-
tecting group, such as THP, solved this problem and 2-
The different 6-substituted coumarin derivatives 12e14, 17, 18
and 20 were prepared by the Baylis-Hillman methodology,
described by Kaye et al. [33], with minor modifications. The
different salicylaldehydes (23aee, Scheme 1) were benzylated
under standard conditions using potassium carbonate as base (48e
97%, 24aee). 23a was made from 4-butoxyphenol with a magne-
sium mediated ortho specific formylation in excellent yield (98%)
[34]. The benzylated derivatives (24aee, Scheme 1) were mixed
with methyl acrylate, 1,4-diazabicyclo[2.2.2]octane (DABCO) and
deuterated chloroform and stirred at room temperature for 1e7
weeks yielding Baylis-Hillman products in good yield (73e97%,
25aee) [33]. When the salicylaldehyde was substituted with elec-
tron withdrawing groups (24b, 24c) the reaction accelerated (1e2
weeks) and this has also been reported by others [35e37]. The
conjugate addition was made with: ethylamine, propylamine and
pyrrolidine using methanol as solvent, and producing excellent
conversion (80e100%, 26aef). Debenzylation by hydrogenolysis
achieved the ring opened hydroxyl derivatives (27aef) which after
filtration was stirred over night at ambient temperature, yielding
spontaneous cyclization to coumarins 12e14, 17, 18 and 20 (21e
62%, in some cases base catalyzed with potassium carbonate). In
the nitro substituted 26c concomitant reduction of the nitro func-
tionality to the corresponding aniline was observed (27c).
tetrahydropyran-2-yloxy-5-(trifluoromethyl)benzaldehyde
(29,
Scheme 2) was synthesized according to Geneste et al. [40] by
directed ortho-lithiation of the THP protected 4-(trifluoromethyl)
phenol. Compound 29 was further reacted with methyl acrylate
and DABCO to yield the Baylis-Hillman product 30 with full con-
version after five days (rate enhancement). Conjugate addition to
31 (Scheme 2) with pyrrolidine as base and deprotection under
acidic conditions yielded the ring opened hydroxyl derivative 32
(Scheme 2). Correction of pH to basic conditions (triethylamine)
and concomitant heating (microwave irradiation) gave ring closure
to 16 in 40% yield (Scheme 2).
A few compounds were made by transformation or addition
of functional groups at the C6-position of the aromatic ring
(Scheme 3). Compound 14 was selectively nitrated at C6 in 45%
yield (15) [41,42]. Furthermore, compound 20 with a 6-substituted
methoxy group was demethylated with hydrobromic acid, pro-
ducing 19 in 63% yield (as a hydrobromic salt). Attempts to further
alkylate the 6-hydroxy compound (19) were made, the most
favorable conditions were found to be large excess of triethylamine
and benzyl bromide with refluxing acetonitrile as solvent (41%, 22).
The coumarins seems to be sensitive for nucleophile attack (C2,
C4 and the exocyclic methylene carbon), as well as a competing
quaternization reaction at the basic amine and this could explain
the low yield and many byproducts seen when alkylating the
coumarin with 1-iodopropane (9%, 21) [43].
Table 1
Monoamine oxidase inhibitory activity of compounds 12e22.a,b
Compound
R1, R2
R6
MAO B
IC50 M)
MAO A
IC50 M)
SIc
(m
(m
3. Biological assays
12
13
14
15
16
17
18
19
eH, enPr
eH, eEt
e(CH2)4e eH
e(CH2)4e eNO2
e(CH2)4e eCF3
e(CH2)4e eOCF3
e(CH2)4e eNH2
e(CH2)4e eOH
e(CH2)4e eOMe
e(CH2)4e eOnPr
e(CH2)4e eOBn
eH
62.3 ꢀ 5.35
17.1 ꢀ 2.19
1.95 ꢀ 0.38
6.32 ꢀ 0.78
>100
8.46 ꢀ 3.36
4.24 ꢀ 0.61
3.77 ꢀ 0.65
1.46 ꢀ 0.47
4.48 ꢀ 0.50
2.16 ꢀ 0.16
>100
3.6
3.7
eOnBu 7.34 ꢀ 1.06
3.1. In vitro pharmacology
>100
>100
>100
>100
>100
>100
>100
>15.8
e
All the coumarins described in this report (12e22, Table 1) were
evaluated for their ability to inhibit the A and B isoforms of hMAO
(Table 1). Kynuramine, a MAO A and MAO B mixed substrate, served
as substrate for inhibition studies with both enzymes. The deter-
mination of MAO catalytic rates in the presence of compounds 12e
22 was accomplished by measuring the concentration of 4-
hydroxyquinoline, the MAO catalyzed oxidation product of kynur-
amine, via LC-MS/MS [Cyprotex Discovery Ltd (Macclesfield, UK),
see Supplementary material] [44]. The corresponding IC50 values
and the MAO A selectivity [expressed as IC50 (MAO B)/IC50 (MAO A)]
are reported in Table 1. A subset of the synthesized compounds (12,
15e18 and 20) were tested for their ability to inhibit DA D2 receptor
functional response using human HEK cells with D2-Gqi5 clone in
>11.8
>23.5
>26.5
>68
>22.3
10.3
e
20
21
22
22.3 ꢀ 4.77
>100
Tranylcypromine
e
e
0.25 ꢀ 0.02
0.24 ꢀ 0.01
1
Abbreviations: Kynuramine, 3-(2-aminophenyl)-3-oxopropanamine; SI, selectivity
index; SE, standard error.
a
Inhibitory activity to human MAO A and MAO B with kynuramine as substrate
[44].
b
All values are expressed as the mean ꢀ SE of duplicate determinations.
c
The selectivity index is the selectivity for MAO A isoform and is given as the
ratios of IC50 (MAO B)/IC50 (MAO A).

180
C. Mattsson et al. / European Journal of Medicinal Chemistry 73 (2014) 177e186
a
b
23a-e
24a-e
c
d
e
25a-e
26a-f
27a-f
cmpd
R6
R1, R2
f or g
23a-28a -OnBu
23b-28b -OCF3
23c-26c -NO2
27c-28c -NH2
23d-28d -OMe
23e-28e -H
-H, -Et
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-H, -nPr
-(CH2)4-
28a-f
12-14, 17, 18, 20
26f-28f
-H
Scheme 1. Ring synthesis of 6-substituted coumarin derivatives 12e14, 17, 18 and 20. Reagents and conditions: (a) 1. magnesium methoxide 6e10% in methanol, 2. para-
formaldehyde, toluene, 3. 10% hydrochloric acid; (b) benzyl bromide, K2CO3, acetonitrile, 80 ꢁC; (c) DABCO, CDCl3, rt, 1e7 weeks; (d) NR1R2: ethylamine, propylamine or pyrrolidine,
methanol, rt; (e) H2, Pd/C, methanol, rt; (f) methanol, rt; (g) K2CO3, methanol, rt; Abbreviations: DABCO, 1,4-diazabicyclo[2.2.2]octane.
the presence of DA reported by Dyhring et al. [45] (Table 2S,
Supplementary material). Two compounds (16, 18) were further
evaluated for their selectivity in competition binding assays by
CEREP (Poitiers, France) for affinity to the serotonin type 1A re-
ceptor (5-HT1A) [46], serotonin type 2A receptor (5-HT2A) [47],
sacrificed after 1 h. The post-mortem levels of 3-MT, DOPAC, and 5-
HIAA in striatum were quantified using the method described in
the Supplementary material. The results for compounds 12, 14e16
and 18e20 are shown in Table 2.
adrenergic type 1 receptor (a1) [48], adrenergic type 2 receptor (a2
)
3.3. Microdialysis
[49] and histaminergic type 1 receptor (H1) [50], as well as the
dopamine transporter protein (DAT) [51], serotonergic transporter
protein (SERT) [52] and adrenergic transporter protein (NAT) [53]
(Table 3).
Microdialysis is an invasive sampling technique that makes it
possible of continuous measurement of neurotransmitters (DA, 5-
HT) and their metabolites (DOPAC, 5-HIAA and 3-MT) concentra-
tions in the extracellular fluid in rat brain (in vivo). This is made by
insertion of a microdialysis probe in the region of interest in living
animals. Compound 18 was administrated subcutaneously to freely
moving rats and the levels of DOPAC and 3-MT was measured
in vivo by HPLC/EC system in striatum during 2 h (Fig. 4). The
method used is described in Supplementary material and by Wa-
ters et al. [54].
3.2. In vivo pharmacology
A selection of the synthesized compounds in Table 1 and the
known MAO A and MAO B inhibitors, moclobemide (MAO A),
selegiline (high dose MAO A/B) and tranylcypromine (MAO A/B)
were administrated subcutaneously to freely moving rats that were
a
b
29
30
31
c
d
32
16
Scheme 2. Ring synthesis of 3-(pyrrolidin-1-ylmethyl)-6-(trifluoromethyl)chromen-2-one (16). Reagents and conditions: (a) DABCO, CDCl3, rt, 5 days; (b) pyrrolidine, methanol, rt;
(c) conc. hydrochloric acid, methanol, rt; (d) triethylamine, methanol, microwave irradiation 100 ꢁC. Compound 29 was synthesized according to Geneste et al. [40]. Abbreviations:
DABCO, 1,4-diazabicyclo[2.2.2]octane.

C. Mattsson et al. / European Journal of Medicinal Chemistry 73 (2014) 177e186
181
a
b or c
20
19
21 R = -nPr
22 R = -Bn
d
14
15
Scheme 3. Functional group modifications on coumarin core to 15, 19, 21 and 22. Reagents and conditions: (a) 48% hydrobromic acid, 110 ꢁC; (b) benzyl bromide, triethylamine,
acetonitrile, 70 ꢁC; (c) 1-iodopropane, K2CO3, N,N-dimethylformamide, 80 ꢁC; (d) conc. H2SO4/HNO3, H2O, rt.
3.4. Molecular modeling
explained but we speculate that the planar geometry of the
coumarin ring forces the basic amino group into a position that is
not optimal for the affinity to DA D2 receptors (compared to the
geometry for the benzodioxane ring in 10) [61]. However, the new
molecules were instead found to be MAO inhibitors with selectivity
for MAO A. From Table 1, which shows the MAO inhibition data for
all new compounds, it can be concluded that most of them display
weak to moderate inhibitory activity at MAO A (range IC50 1.46e
The modeling presented in this paper was performed in the
Schrödinger 2012.04 suite of modeling tools [55]. The X-ray crystal
complex of MAO A with the reversible inhibitor harmine (resolution
ꢀ
2.17 A, PDB entry 2Z5Y [56]) was processed with the standard
protein preparation protocol [55] including generating appropriate
protonation states for the ligand, co-factor, and protein, optimizing
the hydrogen bond network and orientation of Asn, Glu and His
residue, and a restrained force field minimization using OPLS 2005
[57]. Ligand docking was performed using GLIDE, with standard
settings for receptor grid generation and partial charges and
docking procedure [58,59]. Ligands were prepared for docking us-
ing LigPrep [60] with standard settings for generation of proton-
ation and tautomer states using OPLS 2005 force field.
17.1
mM), but with a clear selectivity towards MAO A. For the
unsubstituted coumarin rings 12 and 14 the switch from the sec-
ondary amine (n-propylamine, 12) to the tertiary pyrrolidine ring
(14) improved the potency and selectivity for MAO A. The intro-
duction of functional groups at position C6 led to various effects on
both MAO A and B. Small electron donating groups such as amino
(18) and hydroxyl (19) had a positive influence on the inhibitory
activity at MAO A, with the hydroxyl compound (19) being the most
potent within this series (IC50 ¼ 1.46
mM) and with high selectivity
towards MAO B (SI > 68). Adding small alkyl groups to the hydroxy
group slightly reduced the activity at MAO A; methoxy (20,
4. Results and discussion
In the search for novel compounds with effects on DA D2 re-
ceptors in the rat brain, the scaffold jumping from the 1,4-
benzodioxan core to the coumarin ring led to a profile distinctly
different from what would be expected. The compounds 12, 15e18
and 20 were found to be completely abolished of any functional
activity at the DA D2 receptors (IC50 > 16,000 nM, Table 2S). This
effect was found to be independent on which substituent was used
in the coumarin C6 position or if the basic amino group was a
secondary or tertiary amine (i.e. n-propylamine or pyrrolidine ring).
The reason for the lack of affinity to the DA D2 receptors is not easily
IC50 ¼ 4.48
m
M) and trifluoromethoxy group (17, IC50 ¼ 4.24
mM)
while the introduction of a bulky benzyloxy group (22) abolished
all activity at both MAO A and B. However, intermediate size alkoxy
groups such as n-propoxy (21, IC50 ¼ 2.16
mM) and n-butoxy group
(13, IC50 ¼ 1.95
m
M) was very well tolerated at MAO A, but the
activity at MAO B was also enhanced and the selectivity towards
MAO B was therefore reduced (SI ¼ 4e10). The introduction of
electron withdrawing groups at position C6 was also investigated
and the trifluoromethyl group (16) had an IC50 of 8.46 mM at MAO A
Table 2
Ex vivo brain monoaminergic biochemistry in rats given various doses of compounds 12, 14e16, 18e20 and reference compounds.
Compound
R1, R2
R6
Dose (
m
mol/kg)
DOPACa,b % of control
3-MTa,b % of control
5-HIAAa,b % of control
12
14
15
16
18
19
20
eH, enPr
e(CH2)4e
e(CH2)4e
e(CH2)4e
e(CH2)4e
e(CH2)4e
e(CH2)4e
e
eH
eH
100
100
100
100
86
80
100
37
70*
58
105
77
45*
47*
65*
18*
20*
36*
89
164*
75
116
106
118
104
72*
86*
90
80*
87
96
eNO2
eCF3
eNH2
eOH
eOMe
e
88
170*
126
129
398*
257*
146*
Moclobemide
Tranylcypromine
Selegiline
e
e
0.14
53
e
e
*P-values <0.05 using student’s t-test.
Abbreviations: DOPAC, 3,4-dihydroxyphenylacetic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 3-MT, 5-methoxytryptamine; SEM, standard error of the mean.
a
Post mortem neurochemistry (subcutaneous injection) analysis of striatal DOPAC, 3-MT, and 5-HIAA levels compared with saline control (n ¼ 4).
b
ꢀSEM are reported in supplementary material (Table 1S).

182
C. Mattsson et al. / European Journal of Medicinal Chemistry 73 (2014) 177e186
Table 3
In vitro selectivity data for a selection of serotonergic, adrenergic and histaminergic receptors as well as for dopamine, serotonin and norepinephrine transporters for com-
pounds 16 and 18.a
DATa (%)
SERTa (%)
NATa (%)
a
a
a
a
Cmpd
5-HT1A (%)
5-HT2A (%)
a1 (%)
a2a (%)
H1 (%)
16
18
8
0
20
ꢂ4
4
20
2
4
13
8
0
ꢂ4
1
ꢂ2
4b
1b
Abbreviations: 5-HT, serotonin; a1, adrenergic type 1 receptor; a2, adrenergic type 2 receptor; H1, histamine type 1 receptor; NAT, norepinephrine transporter protein; SERT,
serotonin transporter protein; DAT, dopamine transporter protein.
a
Inhibition of control specific binding at 1 m
M reported with [3H]8-OH-DPAT as ligand for h5-HT1A (agonist), [3H]ketanserin as ligand for h5-HT2A (antagonist), [3H]prazosin
as ligand for rat a1 (non selective, antagonist), [3H]RX 821002 as ligand for rat a2 (non selective, antagonist), histamine as ligand for hH1 (antagonist), [3H]BTCP as ligand for
hDAT (antagonist), [3H]imipramine as ligand for hSERT (antagonist) and [3H]nisoxetine as ligand for hNAT (antagonist).
b
Inhibition of control specific binding at 10 mM reported.
while the nitro (15) was found to be inactive at both MAO A and
MAO B. To give some further explanation to the SAR findings in this
new series of MAO A ligands, the new compounds were subjected
to molecular modeling, based on the high resolution X-ray crystal
complex of MAO A with the reversible inhibitor harmine (resolution
mode docking. In the extra precision mode only one binding mode
of 19 (Fig. 3) and two similar modes of 18 were obtained. The top
ranked binding mode of 18 is shown in Fig. 2S (Supplementary
material). The binding mode of 19 revealed that the protonated
amino group (C3) makes a hydrogen bond interaction to the amide
group (oxygen) of the Gln-215 side chain. Moreover, a hydrogen
bond between the 6-hydroxy group of 19 and the backbone
carbonyl of Phe-208 was also observed. The Phe-208 residue is one
of two amino acids residues (i.e. Phe-208, Ile-335) in the active site
of MAO A responsible for substrate/inhibitor selectivity over MAO B
[56,62,63]. By examining Fig. 3 it can be concluded that the inactive
6-benzyloxy analog (22) could not adopt the same binding mode to
MAO A as 18 (Fig. 2S) and 19 (Fig. 3 and Fig. 3S in Supplementary
material) since there is not enough space for such group in C6-
position. The binding mode found (18, 19) would not permit the
phenyl ring to point towards the entrance of the cavity; instead the
phenyl would clash into the protein. The opening would in any case
be too narrow to accommodate a benzyl group. However, smaller
more flexible alkyls like n-propyl (21) are more likely to fit within
the opening and possibly enhance binding through hydrophobic
interactions with Phe-208, Leu-97 and Val-210. In addition, other
reversible MAO A inhibitors (i.e. other structural series) with a basic
nitrogen have been found to make a hydrogen bond between the
protonated amine and the amide group (oxygen) of the Gln-215
ꢀ
2.17 A, PDB entry 2Z5Y [56]). The X-ray crystal structure of this
entry was selected in spite of the presence of a single mutation
(G110A) in the sequence of the protein. This mutation introduces
only a minor local distortion of the backbone, and the introduced
methyl (alanine) is pointing away from the active site (with a dis-
ꢀ
tance to harmine of >10 A). However, one of the reasons for
selecting this structure is that 2Z5Y has higher resolution than the
structure of the wild type (PDB entry 2Z5X). Another reason is that
2Z5Y contains five water molecules in the active site instead of
seven (2Z5X), and all water molecules are at identical positions to
the corresponding waters in the wild type 2Z5X. The two missing
waters may, therefore, be regarded as more easily replaced by li-
gands, and thus one could argue that the active site in 2Z5Y is
slightly more suitable for docking. Flexible ligand docking using
GLIDE was performed for the two most potent and selective ligands
6-amino (18) and 6-hydroxy (19), together with the 6-benzoloxy
inactive analog (22). Similar types of binding modes were identi-
fied for 18 and 19. However, GLIDE failed to produce a binding
mode for 22, neither when applying standard nor extra precision
Fig. 3. Binding pose of compound 19 (thick bonds with green carbon atoms) in the active site of human MAO A (PDB: 2Z5Y) viewed alongside the active site. Part of FAD is shown in
thick bonds with white carbon atoms. The molecular surface of the active site is shown as a transparent shape. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

C. Mattsson et al. / European Journal of Medicinal Chemistry 73 (2014) 177e186
183
800
700
600
500
400
300
200
100
0
3MT
DOPAC
-60
-40
-20
0
20
40
60
80
100
120
140
160
180
time (min)
Fig. 4. Striatal levels of the dopaminergic metabolites 3-MT and DOPAC, measured by microdialysis in freely moving rats (n ¼ 1e2) and expressed as percentage of saline control,
after administration of 18 (43 mol/kg) at 0 min. Abbreviations: DOPAC, 3,4-dihydroxyphenylacetic acid; 3-MT, 3-methoxytyramine.
m
side chain (Pettersson et al. [64], Gallardo-Godoy et al. [65] and La
Regina et al. [66]). The coumarins are usually docked within the
MAO A cavity with the coumarin core in the same mode as we
found, with the heterocyclic ring closest to FAD cofactor. However,
no reports of MAO A selective coumarins with a basic 3-amino side
chain have been investigated on this core before. Recently Abdel-
hafez et al. [29,30] reported of potent MAO A selective coumarins
on a C7 structural motif e.g. 7, and was found by molecular
modeling to have four different hydrogen bonding interactions
within MAO A cavity (two each to Tyr-444 and Asn-181), and
further claimed that Ile-335 and Phe-208 and the mono vs. dipar-
tite cavity of MAO A respective MAO B are responsible for the
selectivity seen over MAO B [29,30].
The in vitro findings were further supported by in vivo studies in
rat brain (Table 2). The most potent and selective MAO A com-
pounds in our series (14, 18e20) administrated in vivo induced a
neurotransmitter metabolite profile typical of MAO A inhibition:
decreased DOPAC and increased 3-MT levels (Table 2). The major
metabolite of 5-HT, 5-HIAA was only slightly altered by the most
potent ligands, 6-amino (18) and 6-hydroxy (19). These data are in
line with previously reported in vivo profiles for MAO A inhibitors
such as moclobemide or a high dose of selegiline (non selective)
[64,67]. One of the most potent ligand within this series, compound
18, was further investigated by a microdialysis experiment in freely
moving rats. A large increase in 3-MTalong with a slight decrease of
DOPAC was detected in striatum (Fig. 4), further supporting the
inhibitory effect on MAO A [64,68]. In a test panel for off target
affinity for various receptors and protein transporters which may
have an impact on the in vivo profile, compound 18 did not show
any significant affinity (Table 3) and we therefore conclude that the
in vivo effects seen of 18 is most likely attributed to the inhibition of
MAO A. We have not measured whether the new compounds (i.e.18
and 19) are reversible or irreversible MAO A inhibitors. However,
due to the fact that these compounds share the chemical motif
known for coumarins which are classified as reversible MAO in-
hibitors (plus that they lack reactive functional groups), it is most
likely that these new coumarins also are reversible MAO A in-
hibitors [31,69].
One of the major drawbacks with the coumarins developed so
far, are that the chemical properties such as low aqueous solubility
and weak metabolic stability hampers further development of
clinical candidates [18]. Therefore a search for new coumarins with
improved pharmacokinetic properties and better aqueous solubil-
ity is ongoing. Recently, Pisani et al. [69] reported the discovery of a
new potent and selective reversible MAO B inhibitor with improved
pharmacokinetic and toxicity properties (NW-1772, 23, Fig. 5)
[69,70]. Introduction of the polar amino group seems to be in favor
of previously developed MAO B coumarin inhibitors in terms of
these properties. The new coumarins (12, 14e16 and 18e20) were
prepared as hydrochloric salts (except 19 which is a hydrobromic
salt) and dissolved in saline solutions at room temperature and
they were all found to be easily dissolved in concentrations up to
5e7 mg/mL (the highest amount tested). For compounds 15, 16, 18,
and 20 the temperature was raised to approx 40 ꢁC to speed up the
dissolution. Furthermore, compounds 12, 14, 17 and 18 were tested
(conc. 5
mM) for enzymatic stability in the presence of rat liver
microsomes (Table 3S, Supplementary material) and it was found
that substitution at the C6 position greatly affected this stability.
Compounds 12, 14 and 17 were not metabolically stable (<5%
remaining compound after 60 min) while compound 18 had
significantly higher stability (80% remaining compound after
60 min).
5. Conclusion
A new series of MAO A selective inhibitors have been identified
within the coumarin scaffold, with a basic amino function at the C3
position together with small functional groups at the C6 position.
These new compounds have, as expected, high aqueous solubility
(>5 mg/mL in saline solution) and the metabolic stability was
found to be dependent on the substitution at the C6 position. The
23
Fig. 5. Reversible monoamine oxidase B methylamino spaced coumarin inhibitor.

184
C. Mattsson et al. / European Journal of Medicinal Chemistry 73 (2014) 177e186
most potent and selective coumarins are the 6-subsituted 3-(pyr-
rolidin-1-ylmethyl)chromen-2-ones with small substituents such
as eNH2, eOH, eOMe and eOCF3 in the C6 position. Among them
the 6-amino (18) and 6-hydroxy (19) compounds were the most
6.1.2. General method for “BayliseHillman products” methyl 2-[(2-
benzyloxyphenyl)-hydroxy-methyl]prop-2-enoates
derivatives (25aee)
A
mixture of 2-benzyloxybenzaldehyde (24aee) (1 equiv,
potent inhibitors of MAO A (IC50 3.77 and 1.46
m
M, respectively),
5 mmol), methyl acrylate (1 equiv, 5 mmol) and DABCO (0.52 equiv,
2.63 mmol) in CDCl3 (0.25 mL) was stirred for 1e7 weeks. The
mixture was concentrated in vacuo to give an oily residue, which
was purified by flash chromatography using isooctaneeethyl ace-
tateemethanol in appropriate ratios to give the title compounds
(25aee).
while the corresponding benzyloxy (22) was found to be inactive.
In addition, molecular modeling results were in line with the SAR
found for the series. The in vitro findings were confirmed with
in vivo data, by measuring the changes in DOPAC, 3-MT and 5-HIAA
levels in rat brain (i.e. striatum). The most potent and selective MAO
A inhibitors within this series (18 and 19) gave the expected in vivo
MAO A profile (decrease in DOPAC and 5-HIAA levels with a
concomitant increase in 3-MT). This indicates that it is possible to
develop new selective MAO A inhibitors from this structural class
and that these compounds may have potential for the treatment of
depression and mood disorders.
6.1.3. General method for debenzylation and ring-closure to
coumarins (12e14, 17, 18, 20)
A mixture of 25aee (1 equiv) and amine (1.5 equiv) in methanol
was stirred over night at room temperature. Excess amine was
evaporated in vacuo to give an oily residue (26aef) which was re-
dissolved in methanol (10 mL). Pd/C (0.1 wt%) was added under
N2 atmosphere and the reaction mixture was hydrogenated under
H2 (50 psi) for 0.5e1 h and debenzylated product is achieved (27ae
f). The mixture was filtered through celite and washed with
methanol (20 mL). The methanolic solution was stirred over night
at room temperature, in some cases with addition of base K2CO3
(3 equiv) if the reaction stopped at “eOH intermediate” (28aef).
Filtration of K2CO3 and the solvent were removed in vacuo to give
an oily residue, which was purified by flash chromatography using
ethyl acetateemethanol in appropriate ratios as eluent to give the
title compounds (12e14, 17, 18, 20).
6. Experimental section
6.1. Chemistry general
1H and 13C NMR spectra were recorded in CD3OD, CDCl3, or
DMSO-d6 at 300 and 75 MHz, respectively, using a Varian XL 300
spectrometer (Varian, Darmstadt, Germany), or at 400 and
100 MHz, respectively, using a Mercury Plus 400 spectrometer
(Varian, Darmstadt, Germany). Chemical shifts are reported as
d
values (ppm) relative to an internal standard (tetramethylsilane).
Low-resolution mass spectra were recorded on a HP 5970A in-
strument (Agilent Technologies, Stockholm, Sweden) operating at
an ionization potential of 70 eV. The mass detector was interfaced
with a HP5700 gas chromatograph (Agilent Technologies, Stock-
holm, Sweden) equipped with a fused silica column (11 m, 0.22 mm
i.d.) coated with cross-linked SE-54 (film thickness 0.3 mm, He gas,
flow 40 cm/s). Electrospray ionization mass spectra were recorded
on Agilent 1200 series liquid chromatography/mass selective de-
tector (Agilent Technologies, Stockholm, Sweden). High resolution
mass spectrometer was recorded on MaXis, Q-TOF type instrument
(Bruker Daltonics Inc, Billerica, USA). The microwave heating was
performed in a Smith synthesizer single-mode microwave cavity
producing continuous irradiation at 2450 MHz (Personal Chemistry
AB, Uppsala, Sweden). For further instructions see Alterman et al.
[71] Elemental analyses were performed by MikroKemi AB
(Uppsala, Sweden). Melting points were determined with Büchi
545 instrument (Kebo Lab, Goteborg, Sweden) and are uncorrected.
For flash chromatography, silica gel 60 (0.040e0.063 mm, VWR, no.
109385) was used. The amine products were converted to the
corresponding salts by dissolving the free base in methanol or
ethanol and adding 1 equiv of ethanolic HCl solution. The solvent
was removed azeotropically with absolute ethanol in vacuo fol-
lowed by recrystallization from appropriate solvents. Purity of all
target compounds where assessed as greater than 95% by elemental
analysis (C, H, N) or HPLC/MS (one compound). Intermediates 24c
[35], 24d [72], 24e [33], 25e [33,35], and 29 [40] are known and
synthesized by published methods. Intermediate 25c [35] are
known but made by method reported within this publication.
6.1.4. 6-Butoxy-3-(ethylaminomethyl)chromen-2-one (13)
The intermediate methyl 3-(2-benzyloxy-5-butoxy-phenyl)-2-
(ethylaminomethyl)-3-hydroxy-propanoate (26a) was obtained in
100% yield ESIMS: m/z 416.0 (M þ H)þ. Ring closure afforded the
product in 45% yield by the general method described in Chapter
6.1.3. MS m/z (relative intensity, 70 eV) 275 (Mþ, 5), 246 (bp), 231
(20), 190 (27), 176 (24). ESIMS: m/z 276.0 (M þ H)þ. 1H NMR
(CD3OD, 400 MHz)
d
¼ 0.98 (t, J ¼ 7.42 Hz, 3H), 1.16 (t, J ¼ 7.22 Hz,
3H), 1.50 (qt, J ¼ 7.52, 7.27 Hz, 2H), 1.75 (dd, J ¼ 8.40, 6.44 Hz, 2H),
2.69 (q, J ¼ 7.29 Hz, 2H), 3.65 (s, 2H), 3.98 (t, J ¼ 6.44 Hz, 2H), 7.01e
7.15 (m, 2H), 7.21 (d, J ¼ 8.98 Hz, 1H), 7.80 (s, 1H). 13C NMR (CD3OD,
101 MHz)
d
¼ 14.10, 14.56, 20.18, 32.32, 44.02, 47.32, 69.32, 111.75,
118.12, 120.62, 120.89, 127.21, 141.70, 148.69, 157.19, 163.15. The
amine was converted to the HCl salt which was recrystallized in
ethanol/diethyl ether, mp 174e176 ꢁC. HRMS C16H21NO3 (M þ H)þ
calcd 276.1594, found 276.1594. Anal. (C16H21NO3$HCl) C, H, N.
6.1.5. 5-Butoxy-2-hydroxy-benzaldehyde (23a)
4-Butoxyphenol (5.2 g, 31.28 mmol) was added to magnesium
methoxide (35 mL of 6e10 wt.% solution in methanol, 18.76 mmol)
and the mixture was heated to reflux. Approximately half the
amount of methanol was distilled off and toluene (40 mL) was
added to the residue. The azeotropic mixture of toluene and
methanol was removed by fractional distillation, until the tem-
perature of the reaction mixture rose to 95 ꢁC. A slurry of para-
formaldehyde powder (3.38 g, 112.6 mmol) in toluene (20 mL) was
added to the reaction mixture at 95 ꢁC with concurrent removal of
volatile materials by distillation for 1 h, after which the mixture
was cooled to room temperature and quenched with 10% hydro-
chloric acid and stirred over night at ambient temperature. Next
day the mixture was extracted with ethyl acetate (3 ꢃ 100 mL) and
the combined organic phases were dried (MgSO4) and concen-
trated in vacuo to give crude 23a (5.93 g, 98%): MS m/z (relative
intensity, 70 eV) 194 (Mþ, 25), 138 (bp), 137 (42), 120 (6) 92 (5). 1H
6.1.1. General method for benzylation of phenols (24aed)
Phenol (1 equiv) was dissolved in acetonitrile and then ben-
zylbromide (1.1 equiv) and K2CO3 (2 equiv) were added. The reac-
tion mixture was heated at 80 ꢁC for 1e2 h, cooled to ambient
temperature and K2CO3 was filtered off and subsequently washed
with 2 ꢃ 50 mL acetonitrile. The combined organic phases were
concentrated in vacuo and the residue was purified with flash
chromatography using isooctaneeethyl acetate in appropriate ra-
tios to give the title compounds (24aed).
NMR (CDCl3, 400 MHz)
d
¼ 0.98 (t, J ¼ 7.54 Hz, 3H), 1.39e1.59 (m,
2H),1.76 (dd, J ¼ 14.52, 6.80 Hz, 2H), 3.94 (t, J ¼ 6.43 Hz, 2H), 6.91 (d,
J ¼ 9.19 Hz, 1H), 6.99 (d, J ¼ 2.94 Hz, 1H), 7.14 (dd, J ¼ 9.19, 2.94 Hz,

C. Mattsson et al. / European Journal of Medicinal Chemistry 73 (2014) 177e186
185
1H), 9.83 (s, 1H), 10.64 (s, 1H). 13C NMR (CDCl3, 101 MHz)
19.15, 31.22, 68.59, 116.15, 118.55, 120.03, 125.78, 152.21, 155.87,
196.18.
d
¼ 13.77,
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6.1.6. 2-Benzyloxy-5-butoxy-benzaldehyde (24a)
The product was obtained in 68% yield by the general method
described under chapter 6.1.1. MS m/z (relative intensity, 70 eV) 284
(Mþ, 11), 192 (9), 137 (6), 136 (6), 91 (bp). 1H NMR (CDCl3, 400 MHz)
d
¼ 0.96 (t, J ¼ 7.41 Hz, 3H), 1.39e1.58 (m, 2H), 1.67e1.90 (m, 2H),
3.94 (t, J ¼ 6.63 Hz, 2H), 5.13 (s, 2H), 6.98 (d, J ¼ 8.97 Hz, 1H), 7.10
(dd, J ¼ 8.97, 3.12 Hz, 1H), 7.24e7.51 (m, 6H), 10.50 (s, 1H). 13C NMR
(CDCl3, 101 MHz)
d
¼ 13.78, 19.14, 31.21, 68.30, 71.23, 111.04, 115.02,
123.88, 125.48, 127.31, 128.18, 128.65, 136.28, 153.40, 155.65, 189.50.
6.1.7. Methyl 2-[(2-benzyloxy-5-butoxy-phenyl)-hydroxy-methyl]
prop-2-enoate (25a)
Reaction during 7 weeks yielded the product in 73% yield, by the
general method described under chapter 6.1.2. MS m/z (relative
intensity, 70 eV) 370 (Mþ,13), 262 (29),191 (55),113 (26) 91 (bp). 1H
NMR (CDCl3, 400 MHz)
d
¼ 0.96 (t, J ¼ 7.29 Hz, 3H), 1.39e1.56 (m,
2H), 1.67e1.82 (m, 2H), 3.35 (d, J ¼ 6.28 Hz, 1H), 3.73 (s, 3H), 3.91
(td, J ¼ 6.53, 1.76 Hz, 2H), 5.02 (s, 2H), 5.68 (t, J ¼ 1.26 Hz, 1H), 5.90
(d, J ¼ 6.03 Hz,1H), 6.28 (s, 1H), 6.76 (dd, J ¼ 8.8, 2.8 Hz, 1H), 6.85 (d,
J ¼ 8.8 Hz, 1H), 6.97 (d, J ¼ 3.2 Hz, 1H), 7.24e7.50 (m, 4H). 13C NMR
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(CDCl3, 101 MHz)
113.31, 114.17, 114.38, 126.04, 127.42, 127.98, 128.58, 130.74, 137.03,
141.30, 149.81, 153.54, 167.06.
d
¼ 13.91, 19.27, 31.45, 51.93, 68.26, 68.41, 70.97,
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Acknowledgments
We thank Theresa Andreasson, Elisabeth Ljung, Marianne
Thorngren, Kirsten Sönniksen, Boel Svanberg, Anna-Carin Jansson,
and Thérese Carlsson for their work with behavioral and neuro-
chemical experiments and analyses, Theresa Andreasson is thanked
for calculating in vivo and in vitro data for tested compounds, Tomas
A Jacobsen for kindly providing with high resolution MS data, Tino
Dyhring for kindly providing with FLIPR data and for reviewing the
manuscript, we thank Daniel Klamer and Fredrik Pettersson.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found
in the online version, at http://dx.doi.org/10.1016/j.ejmech.2013.
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Abbreviations
DOPAC 3,4-dihydroxyphenylacetic acid
5-HIAA 5-hydroxyindoleacetic acid
3-MT
3-methoxytyramine
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