Fine molecular tuning at position 4 of 2H-chromen-2-one derivatives in the search of potent and selective monoamine oxidase B inhibitors

European Journal of Medicinal Chemistry 70 (2013) 723e739

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European Journal of Medicinal Chemistry

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Original article

Fine molecular tuning at position 4 of 2H-chromen-2-one derivatives

in the search of potent and selective monoamine oxidase B inhibitors

Leonardo Pisani ^a, Marco Catto ^a, Orazio Nicolotti ^a, Giancarlo Grossi ^b, Mario Di Braccio ^b,

Ramon Soto-Otero ^c, Estefania Mendez-Alvarez ^c, Angela Stefanachi ^a,

,

Domenico Gadaleta ^a, Angelo Carotti ^a

*

^aDipartimento di Farmacia e Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, via E. Orabona 4, I-70125 Bari, Italy

^bDipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, viale Benedetto XV 3, I-16132 Genova, Italy

^cGrupo de Neuroquimica, Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad de Santiago de Compostela, San Francisco I

E-15782, Santiago de Compostela, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:

The effects on the inhibition potencies of monoamine oxidase isoforms A (MAO-A) and B (MAO-B)

depending upon changes in the physicochemical properties (size, shape, H-bonding, lipophilicity, etc.) of

substituents at the C4 position of 2H-chromen-2-one derivatives were extensively investigated, and the

results signiﬁcantly added to our knowledge on this class of MAO inhibitors. All the 67 examined

compounds showed high MAO-B selectivity, some of them achieving potency in the low nanomolar

range. In particular, the 7-metachlorobenzyloxy-4-oxyacetamido-2H-chromen-2-one (entry 62) showed

single digit nanomolar MAO-B potency (IC₅₀¼ 3.1 nM) and high selectivity over the MAO-A isoform

(selectivity ratio ¼ 7244). The great variety of the investigated substituents at C4 of the 2H-chromen-2-

one nucleus, combined with binding models generated from docking studies carried out on selected

compounds, allowed us to shed light on the main molecular requirements for potent and selective MAO-

B inhibition, highlighting the dominant role of the steric effects. Interestingly, many of the designed

substituents could be metabolically related to each other (e.g., CH₃/CH₂OH/CHO/COOH; NH₂/NHCH₃,

NHAc), and therefore the results obtained may help in predicting the in vivo activity of some putative

metabolites of lead MAO-B inhibitors.

Received 26 June 2013

Received in revised form

11 September 2013

Accepted 14 September 2013

Available online 17 October 2013

Keywords:

Monoamine oxidase inhibitors

4-Substituted-7-benzyloxy-2H-chromene-

2-ones

Molecular docking

Selective MAO-B inhibitors

Parkinson’s disease

Alzheimer’s disease

Neurodegenerative diseases

1. Introduction

hypotheses have been formulated and, among others, single elec-

tron transfer and polar nucleophilic mechanisms appear as the

Monoamine oxidases (MAOs, E.C. 1.4.3.4, amine-oxygen oxido-

reductase) are responsible for the catalytic oxidative deamination

of endogenous and exogenous arylalkylamines to the correspond-

ing iminium salts that subsequently are non-enzymatically hy-

drolyzed to the ﬁnal aldehyde products [1]. The catalytic cycle

employs molecular oxygen as the electron acceptor and produces

lower molecular weight amines, or ammonia (from primary

amines), and the hydrogen peroxide that triggers the formation of

reactive oxygen species (ROS) as harmful by-products. Even though

over the years detailed studies have been dedicated to MAOs

structure and function, the mechanistic features of the oxidative

pathway catalyzed by MAOs are still under debate. Different

most plausible ones being sustained by more robust experimental

evidence [2].

MAO substrates include dietary amines (e.g., tyramine), mono-

amine neurotransmitters (e.g., serotonin, histamine and catechol-

amines, such as epinephrine, norepinephrine and dopamine) and

the Parkinson-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine (MPTP). After the seminal paper describing

two different MAO isoforms in 1968, termed MAO-A and -B [3],

great research efforts have been devoted to the understanding of

their main structural and functional differences, often related to

species diversity. Encoded by two different genes on X chromo-

some, the two isoenzymes show nearly 70% sequence identity and

share close biological functions [4]. Flavin adenine dinucleotide

(FAD), covalently linked through a thioether bond to a cysteine

residue of the MAO protein backbone, serves as redox prosthetic

cofactor in the oxidative deamination process. The highly hydro-

* Corresponding author. Tel.: þ39 080 5442782; fax: þ39 080 5442230.

E-mail

addresses:

angelo.carotti@uniba.it,

carotti_angelo@hotmail.com

phobic C-terminal a-helix anchors each isoenzymes to the outer

(A. Carotti).

http://dx.doi.org/10.1016/j.ejmech.2013.09.034

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L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

mitochondrial membrane with a characteristic dimeric fashion [5],

which made their isolation and puriﬁcation troublesome. The

identiﬁcation and optimization of efﬁcient high-throughput

expression and puriﬁcation systems [6e8] paved the way for the

preparation of protein-inhibitors co-crystals. The X-ray determi-

nation at high resolution of MAO complexed with both reversible

and irreversible inhibitors enlightened the main molecular de-

terminants governing the binding interactions in the active site and

allowed the identiﬁcation of the key three-dimensional structural

differences between the two isoenzymes [9]. The MAO enzymatic

active site is a lipophilic cage, largely ﬁlled by aromatic amino acids,

surrounding the isoalloxazine moiety of the FAD cofactor. The

diverse shape and structure of this region in MAO-A and MAO-B

result in the different selectivity/afﬁnity for substrates and in-

hibitors [4]. Depending on the bound substrate/inhibitor, MAO-B

active site can exhibit a bipartite cavity so that the ligand has to

negotiate a smaller entrance room before entering the substrate

cavity where FAD is accommodated. As an alternative, these two

subsites may be merged because of conformational changes of a

ﬂexible residue (Ile199) occurring upon binding of speciﬁc ligands

spanning both cavities (e.g., 1,4-diphenyl-2-butene and trans,trans-

farnesol) [10]. This bipartite protein motif is absent in MAO-A,

where the “gate-keeper” Ile199 residue is replaced by a Phe208

(numbering are referred to human enzymes) and the substrate/

inhibitor has to gain access to a unique and wide hydrophobic

pocket containing FAD [11,12].

Most mammalian peripheral tissues express both MAO iso-

forms, with the exception of platelets which contain just MAO-B

[13]. High levels of MAO-A have been found in placenta and in-

testine. Marked differences in brain distribution have been

described in humans through positron emission tomography (PET)

experiments [14]. The prevalence of MAO-A in brain areas under

catecholamine control (e.g., substantia nigra and locus coeruleus)

has been documented. MAO-B has been mainly localized in astro-

cytes, basal ganglia, dorsal raphe nucleus and bloodebrain barrier

microvessels [15]. In neurons and glial cells, the catalytic activity of

MAOs exerts the function of protecting from exogenous amines and

terminating the nerve impulse signalling.

The ability of MAOs to control the levels of neurotransmitters in

central nervous system (CNS) attracted the interest of many re-

searchers involved in the discovery of new drugs targeting brain

disorders [16,17]. Broad consensus about the clinical relevance of

MAO inhibition raised when abnormalities in central MAO-A and -B

enzymatic activity have been associated to behavioural distur-

bances and Parkinson’s disease (PD), respectively [18]. Propargyl-

amines (e.g., clorgyline and selegiline), hydrazine derivatives (e.g.,

phenelzine) and arylcyclopropylamines (e.g., tranylcypromine)

have been described as mechanism-based irreversible inhibitors

(Chart 1). After initial ﬁtting to MAOs enzymatic site, these in-

hibitors are transformed into suicide inhibitors, as a consequence of

the catalytic activity itself, that inactivate the enzyme by forming

covalent bonds to isoalloxazine N(5) or C(4a) atoms [9]. The for-

mation of FAD adducts is allowed by the unusual bent conformation

of the tricyclic isoalloxazine skeleton. Aryl oxazolidinones (e.g.,

toloxatone), brofaromine and moclobemide belong to the class of

MAO reversible inhibitors, which can be further classiﬁed as

competitive or slow, tight-binding inhibitors [19].

Although abundance of MAOs in liver, placenta, intestine and

lung [20] exceeds by far brain expression, little is known about their

physiological role in non-cerebral districts. Moreover, the activity of

MAO inhibitors (MAO-Is) in peripheral tissues has been largely

associated with the toxicity of the earliest non-selective and irre-

versible inhibitors. Hypertensive crises have been ascribed to the

potentiation of sympathomimetic amines such as tyramine (mainly

present in cheese and red wine and hence triggering the so called

“cheese-effect”) that is not scavenged by MAOs after inhibitors

administration. Hepatotoxic reactions have been related to the in-

hibition of CYP450s activity. In addition to the above-mentioned,

unwanted and severe side-effects, stringent dietary restrictions

and harmful drugedrug interactions explain the reasons why some

MAO inhibitors have been discontinued in the clinical practice. In

order to overcome the toxicity issue, the research on MAO-Is has

been focused on solving chieﬂy the selectivity problem. In this way,

moclobemide (a reversible and selective MAO-A inhibitor) [21] and

a transdermal formulation system of selegiline [22] (irreversible

and selective MAO-B inhibitor) were discovered and marketed

H

N

NH₂

Phenelzine (NS)

Tranylcypromine (NS)

Cl

HN

O

N

Cl

Rasagiline (B)

Clorgyline (A)

Selegiline (B)

H

N

OH

O

N

O

N

H

O

Cl

O

Br

Brofaromine (A)

Toloxatone (A)

Moclobemide (A)

Chart 1. Chemical structures of non-selective and selective reversible and irreversible inhibitors of monoamine oxidases A and B.

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

725

against depressive disturbances. On the other side, selective MAO-

BIs (e.g., rasagiline) [23] have been adopted in the treatment of PD

as dopamine-sparing agents. Recent trials [24] suggested a poten-

tial disease-modifying effects of rasagiline in early PD as an alter-

native to long-term levodopa treatment that is associated to

disabling motor impairments (e.g., motor ﬂuctuations and

dyskynesia).

bearing at position 4 mainly polar substituents, including ionized

carboxylic, enolic and amino groups. The large variety of inspected

substituent/molecular properties allowed the derivation of sound

SAR while docking simulations provided easily interpretable

binding modes and enabled the detection of the main molecular

requirements for potent and selective MAO-B inhibition. In this

regard, outstanding in vivo data have been collected in the past for

derivative 13f that can be taken as the reference compound for its

potent and selective MAO-B inhibition both in vitro and in vivo

[39,40]. The great modulation of lipophilicity, water solubility and

metabolic stability of the designed molecules should guarantee a

successful inhibitor selection for oral administration and speciﬁc

delivery and high activity at the CNS. Interestingly, many of the

designed substituents/molecules can be seen as metabolically-

related and this would prospect a reliable prediction of their

pharmacokinetic proﬁle and in vivo activity. Eventually the original

insights from the present work will help the design of new, in vivo-

active, multitarget-directed ligands having the selective MAO-B

inhibition as the core biological activity [30].

Initially conceived as therapeutics to treat depressive distur-

bances and PD, and after a brief decline linked to the clear-cut

unwanted toxic effects, a renewed interest towards MAO in-

hibitors is ﬂourishing nowadays. Recent studies unravelled a novel

role for MAO-A [25] and MAO-B [26] in the pathogenesis and

progression of heart failure because of MAO-driven ROS produc-

tion, enhanced norepinephrine catabolism and impaired aldehyde

metabolism. Moreover, enabling the diminishment of ROS pro-

duction, MAO-Is have been proposed as neuroprotectants against

many debilitating neurodegenerative diseases (NDs) linked to

ageing and oxidative stress [27]. The multi-factorial nature of these

disorders claims for a paradigmatic shift in the drug discovery

process that should be mainly oriented to multi-target ligands able

to modulate multiple and differently altered biochemical pathways

in a synergic manner [28,29]. Multi-potent compounds showing

MAO inhibition as the core activity might exhibit high therapeutic

potential against multifaceted brain disorders such as Alzheimer’s

disease (AD), PD, Huntington’s disease (HD), and amyotrophic

lateral sclerosis (ALS) [30].

2. Chemistry

The synthetic routes to 4-substituted coumarin derivatives 2ae

d, 3aep, 4a, 5a, 6aef, 7e9, 10aed, 11aed, 13aen, 14e18 and 20e22

are illustrated in Schemes 1e6.

Different heterocyclic scaffolds have been explored to design

selective and reversible MAO-A/-B inhibitors. Among them, 2H-

chromene-2-ones, that is coumarins, have been investigated as

potent and selective MAO-Is [31] because their well-consolidated

chemistry enables an easy access to mono- and poly-substituted

derivatives with a great molecular diversity. The most explored

position has been position 7 where appropriate substituents may

confer to the molecule high afﬁnity and, more so, selectivity to-

wards either one of the two MAO enzymatic isoforms [32]. Inter-

estingly, similar results came from a recent study of benzofuran

analogues carrying substituents at position 6 that can be consid-

ered as topologically equivalent to position 7 of coumarin [33]. 3-

Substituted coumarins have also been studied as moderately to

highly potent and selective MAO-B inhibitors, by the groups of

Chimenti [34,35] and Uriarte [36,37]. The position 4 has been also

explored by considering chieﬂy hydrophobic [38], and more occa-

sionally, hydrophilic substituents [39]. As substituents at position 4

1-(2,4-Dihydroxyphenyl)ethanone was treated with proper

benzyl halides to give the corresponding benzyloxyderivatives

1aed (anhydrous K₂CO₃, dry 2-butanone at reﬂux) whose cyclo-

condensation with diethyl carbonate in the presence of Na

(Dowtherm A, 160 ^ꢀC) afforded the sodium salt of 4-

hydroxycoumarin derivatives 2aed [41], from which 4-

hydroxycoumarins were obtained by treatment with aqueous

hydrochloric acid. 4-Alkoxyderivatives 3aep were obtained in

satisfactory yields by treating the sodium salts of appropriate 4-

hydroxycoumarins 2aed, with proper alkyl halides in DMF at

room temperature (Scheme 1).

As shown in Scheme 2, the reaction of 7-substituted-4-

hydroxycoumarins 2c,d with ethyl bromoacetate in reﬂuxing 2-

butanone in the presence of anhydrous K₂CO₃afforded ethyl 4-

oxyacetates 4a,b, which in turn were transformed into the corre-

sponding acids 5a,b through saponiﬁcation with KOH in ethanol.

The reaction of acids 5a,b with an excess of SOCl₂in reﬂuxing

CH₂Cl₂gave the corresponding acyl chlorides, which were treated

with excess 30% aqueous ammonia or with an excess of suitable

amines in CH₂Cl₂solution yielding amides 6a,b or 6cef (Scheme 2).

Dehydration of the 4-oxyacetamide 6b in reﬂuxing POCl₃afforded

the corresponding nitrile 7 (Scheme 2).

play a pivotal role in determining

a productive binding of

coumarin-based MAO-B inhibitors, in the present work a rational

and systematic exploitation of physicochemical and spatial domain

of that region was carried out with the design, synthesis and bio-

logical evaluation of a large series of 2H-chromen-2-one derivatives

OR²

O

OH

O

a)

b)

c)

HO

OH

R¹O

OH

R¹O

O

R¹O

O

3a-p

2a-d

1a-d

R¹= 3'-ClBn; R²= n-C₃H₇

R¹= Bn; R²= CH₃

R¹= Bn; R²= C₂H₅

R¹= Bn; R²= n-C₃H₇

3i

3j

¹= Bn

3a

3b

3c

3d

3e

3f

1a, 2a

R

R¹= 3'-BrBn; R²= CH₃

¹= 3'-FBn

1b, 2b

1c, 2c

1d, 2d

¹= 3'-BrBn; R²= C₂H₅

R¹= 3'-ClBn

R¹= 3'-BrBn

3k

3l

R

¹= 3'-FBn; R²= CH₃

¹= 3'-FBn; R²= C₂H₅

R

¹= 3'-BrBn; R²= n-C₃H₇

R¹= 3'-ClBn; R²= i-C₃H₇

R¹= 3'-ClBn; R²= CH₂OCH₃

R¹= 3'-ClBn; R²= CH₂SCH₃

3m

3n

3o

R¹= 3'-FBn; R²= n-C₃H₇

3g R¹= 3'-ClBn; R²= CH₃

R¹= 3'-ClBn; R²= C₂H₅

3p R¹= 3'-ClBn; R²= CH₂COCH₃

3h

Scheme 1. Synthesis of 4-hydroxy- and 4-alkyloxy-coumarin derivatives 2aed and 3aep. Reagents and conditions: a) appropriate benzyl halide, anhydrous K₂CO₃, 2-butanone,

reﬂux, 24 h; b) i): diethyl carbonate, Na, Dowtherm A, 160 ^ꢀC, 1 h; ii): aq. HCl; c) (from sodium salt of 2aed) appropriate alkyl halide, dry DMF, room temperature, 1.5 h.

726

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

O

OH

O

OH

O

b)

a)

R¹O

O

R¹O

O

R¹O

O

2c, 4a, 5a

R

¹= 3'-ClBn

2d, 4b, 5b R¹= 3'-BrBn

2c-d

4a-b

5a-b

c)

R²

¹= 3'-BrBn; R²= NH₂

O

6a

R

N

R¹= 3'-ClBn; R²= NH₂

O

d)

(from 6b)

6b

6c R¹= 3'-ClBn; R²= NHCH₃

6d

R¹O

O

R¹O

O

R¹= 3'-ClBn; R²= N(CH₃)₂

R₁= 3'-ClBn; R²= piperidin-1-yl

6e

R₁= 3'-ClBn; R²= morpholin-4-yl

7

R¹= 3'-ClBn

6a-f

6f

Scheme 2. Synthesis of coumarin derivatives 4a,b, 5a,b, 6aef and 7. Reagents and conditions: a) ethyl bromoacetate, anhydrous K₂CO₃, 2-butanone, reﬂux, 3 h; b) KOH, ethanol,

room temperature, 1 h; c) i: SOCl₂, CH₂Cl₂, reﬂux, 2 h; ii: 30% aq. NH₃or appropriate amine in CH₂Cl₂, room temperature, 30 min; d) from 6b: POCl₃(excess), reﬂux, 2 h.

7-[(3-Chlorobenzyl)oxy]-4-hydroxycoumarin 2c was treated

with excess triethylamine in methanol to give a solution of the

corresponding triethylammonium salt which, after removal of

solvents, was reﬂuxed with excess POCl₃giving 4-chloroderivative

8 (Scheme 3). The reaction of compound 8 with a large excess of

phenol afforded the 4-phenoxy derivative 9. 4-Aminoderivative 10a

was conveniently obtained by the reaction of 4-hydroxyderivative

2c with excess ammonium acetate in DMSO at 120 ^ꢀC for 3 h

(Scheme 3). The acylation of 10a in the presence of acetic anhydride

under microwave irradiation originated the corresponding amide

10b. Carbamate derivative 10c and urea 10d were obtained by

treating 10a with ethyl chloroformate or ethyl isocyanate,

respectively. By reacting 4-chloroderivative 8 with an ethanolic

solution of methylamine in a sealed vessel at 60 ^ꢀC enamine 11a

was obtained. By following the same synthetic strategy, nucleo-

philic substitutions on 4-chloroderivative 8 with ethylamine hy-

drochloride (in the presence of triethylamine) or isopropylamine

were performed in reﬂuxing CH₂Cl₂furnishing 11b,c, whereas the

synthesis of the anilino derivative 11d required higher temperature

(140 ^ꢀC), as illustrated in Scheme 3.

4-Substituted-7-hydroxycoumarins 12a (from commercial

source), 12b,c (prepared through a von Pechmann reaction) [39,42]

or 12d [43] underwent benzylation reactions under different con-

ditions with the suitable benzyl bromides affording compounds

OH

Cl

O

a)

b)

R¹O

O

R¹O

O

R¹O

O

2c

R¹= 3'-ClBn

R

¹= 3'-ClBn

8

R

¹= 3'-ClBn

9

e)

c)

NH₂

NHR²

R¹O

O

R¹O

R¹= 3'-ClBn

O

10a

R¹= 3'-ClBn

11a

11b

R

²= CH₃

²= C₂H₅

R

R²= i-C₃H₇

R²= Ph

11c R¹= 3'-ClBn

11d

¹= 3'-ClBn

d)

R

O

HN

R³

O

10b

10c

10d

R

¹= 3'-ClBn

R³= CH₃

R³= OC₂H₅

R³= NHC₂H₅

R¹O

O

Scheme 3. Synthesis of coumarin derivatives 8, 9, 10aed and 11aed. Reagents and conditions: a) i): triethylamine, MeOH, room temperature; ii): POCl₃, reﬂux, 3 h; b) phenol,

anhydrous K₂CO₃, 2-butanone, reﬂux, 2 h; c) ammonium acetate, DMSO, 120 ^ꢀC, 3 h; d) for 10b: Ac₂O, 150 ^ꢀC, MW, 30 min; for 10c: ethyl chloroformate, K₂CO₃, reﬂux, 10 h; for 10d:

ethyl isocyanate, 60 ^ꢀC, 120 h; e) for 11a: methylamine 60% wt. in EtOH, 60 ^ꢀC, 7 h; for 11b: ethylamine hydrochloride, triethylamine, EtOH, reﬂux, 2 h; for 11c: isopropylamine,

CH₂Cl₂, reﬂux, 5 h; for 11d: aniline, DIEA, 140 ^ꢀC, 48 h.

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

727

HO

CH₂R¹

a)

e)

R²O

O

HO

O

R²O

O

13a R¹= H

13b R¹= CH₃

R²= 3'-ClBn

12a R¹= H

14

R²= 3'-ClBn

12b R¹= CH₃

12c R¹= Cl

12d R¹= OH

R²= 3'-ClBn

13c R¹= Cl

13d R¹= OH

f)

b)

R¹= NH₂

13e

O

c)

R¹= NHCH₃

R²= 3'-ClBn

13f

¹= N(CH₃)₂

R

²= 3'-ClBn

13g

13h

R

R²O

O

c)

¹= NHnC₃H₇

R²= 3'-ClBn

R

¹= NHnC₄H₉

R²= 3'-ClBn

R

²= 3'-ClBn

13i

13j

15

R¹= pyrrolidin-1-yl

R¹= morpholin-4-yl

R

²= 3'-ClBn

R²= 3'-ClBn

²= 3'-BrBn

13k

13l

R

¹= Cl

R

d)

13mR¹= OH

¹= NHCH₃

R²= 3'-BrBn

c)

R

13n

Scheme 4. Synthesis of coumarin derivatives 13aen, 14 and 15. Reagents and conditions: a) for 13a: see Ref. [39]; for 13b: 12b, sodium ethoxide, 3⁰-chlorobenzyl bromide, DMF,

100 ^ꢀC, 1 h; for 13c,l: 12c, Cs₂CO₃, suitable benzyl bromide, DMF, room temperature, 24 h; for 13d: 12d, Cs₂CO₃, 3⁰-chlorobenzyl bromide, DMF, room temperature, 48 h; b) see

Ref. [39]: i) sodium azide, EtOH, reﬂux, 2 h; ii) SnCl₂, MeOH, room temperature, 3 h; c) appropriate amine, THF, room temperature, 3 h; d) water, reﬂux, 48 h; e) from 13b: SeO₂,

xylene, 140 ^ꢀC, 4 h; f) PCC, dry CH₂Cl₂, room temperature, 2 h.

13aec and 13l (Scheme 4). Aqueous hydrolysis of chloride 13l

yielded the primary alcohol 13m. Chlorides 13c and 13l were

reacted with suitable amines in THF yielding compounds 13eek

and 13n. The oxidation of the 4-ethylderivative 13b with SeO₂in

reﬂuxing xylene afforded in moderate yield (43%) 7-[(3-

chlorobenzyl)oxy]-4-(1-hydroxyethyl)-2H-1-benzopyran-2-one

14. PCC-mediated oxidation of alcohol 14 afforded ketone 15

(Scheme 4). In the same way, aldehyde 16 was obtained starting

from primary alcohol 13d, as shown in Scheme 5. Compound 16

was then converted into the corresponding oxime 17, by reaction

with NH₂OH$HCl in the presence of stoichiometric amount of

AcONa$3H₂O. By reﬂuxing oxime 17 in Ac₂O, dehydration to 4-

cyanoderivative 18 was performed.

Ethyl ester 20 was obtained through a reaction between 3-

chlorobenzyl bromide and phenol 19 [33] in the presence of

caesium carbonate (Scheme 6). Ester 20 was in turn hydrolyzed

with KOH to carboxylic acid 21 which was then transformed into

the intermediate acyl chloride with SOCl₂, that reacted with

concentrated aq. NH₃to afford primary amide 22.

Compounds 23 and 24 were obtained through benzylation of

the

suitable

commercially

available

4-substituted-7-

hydroxycoumarins as described in the Experimental section.

The synthetic procedures leading to compounds 13a,13eeg, and

25e28 (corresponding to entries 23e26 in Table 1) have been

already reported elsewhere [39].

OH

N

OH

N

O

H

c)

a)

b)

R²O

O

R²O

O

R²O

O

R²O

O

13d R²= 3'-ClBn

R

²= 3'-ClBn

18 R²= 3'-ClBn

16

R

²= 3'-ClBn

17

ꢀ

Scheme 5. Synthesis of coumarin derivatives 16e18. Reagents and conditions: a) PCC, sodium acetate, 4 A mol. sieves, dry CH₂Cl₂, room temperature, 12 h; b) hydroxylamine

hydrochloride, sodium acetate, EtOH/H₂O, reﬂux, 30 min; c) Ac₂O, reﬂux, 15 h.

O

OH

O

NH₂

O

a)

b)

c)

R¹O

O

R¹O

20

O

HO

O

R¹O

O

19

R

¹= 3'-ClBn

21

R

¹= 3'-ClBn

22 R¹= 3'-ClBn

Scheme 6. Synthesis of coumarin derivatives 20e22. Reagents and conditions: a) 3⁰-chlorobenzyl bromide, Cs₂CO₃, DMF, room temperature, 24 h; b) KOH, ethanol, reﬂux, 15 min; c)

i) SOCl₂, CH₂Cl₂, reﬂux, 2 h; ii) aq. 30% NH₃, room temperature, 6 h.

728

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

Table 1 (continued)

Table 1

Chemical structure and MAO inhibition data of coumarin derivatives (entries 1e67).

Entry^aCompd^bR₁R₂

MAO-A^cMAO-B^cSI^d

R₂

62

63

64

65

66

67

6b

6a

6c

6d

6e

6f

Cl

OCH₂CONH₂

4.62

5.05

8.48

8.05

7.47

6.30

4.41

4.89

3.86

3.00

nd

Br OCH₂CONH₂

R₁

Cl

OCH₂CONHMe

11%

10%

9.0%

3.2%

O

OCH₂CON(Me)₂

OCH₂CO-1⁰-piperidinyl

OCH₂CO-4⁰-morpholinyl

nd

a

Entry numbers used in the section “Results and discussion”.

Entry^aCompd^bR₁R₂

MAO-A^cMAO-B^cSI^d

b

Compound numbers used for the section “Chemistry” and “Experimental

section”.

Class I

c

MAO-A and MAO-B inhibitory activities are expressed as pIC₅₀or as percentage

1

2

3

4

5

6

7

23

Cl

H

Me

Et

CF₃

Cl

CH₂Cl

40%

5.28

7.77

8.13

7.54

6.06

7.68

7.36

6.68

nd

2.85

nd

of inhibition at 10

mM. Values are the mean of two or three independent

13a

13b

24

8

13c

13l

experiments.

48%

16%

d

SI, is the selectivity index expressed as pIC₅₀MAO-BepIC₅₀MAO-A (DpIC₅₀);

nd

nd ¼ not determined.

5.05

21%

28%

2.63

nd

e

Estimated value from the % of inhibition at 10 mM concentration (see text).

Br CH₂Cl

Class II

8

9

2a

2b

2c

H

F

Cl

OH

2%

0%

6%

0%

5.38

14%

0%

4.4%

14%

23%

48%

5.04

5.68

6.32

6.31

7.32

7.28

7.40

4.00^e

6.38

6.63

6.99

6.66

nd

1.94

nd

3. Biological assays

10

11

12

13

14

15

16

17

18

19

Class III

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

Class IV

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

Class V

52

53

54

55

56

57

58

Class VI

59

60

61

The inhibition of monoamine oxidases A and B activity was

measured in vitro by using crude rat brain mitochondrial homog-

enates through a spectrophotometric method [44] based on the

monitoring of the oxidation rate of the non-selective non-ﬂuores-

cent MAO substrate kynuramine to 4-hydroxyquinoline. The per-

centage of MAO inhibition was determined for all the examined

compounds at 10 mM concentration and IC₅₀data were measured

only for compounds showing an inhibition greater than 50%. In a

few cases the IC₅₀was measured also for compounds with low

2d

10a

16

15

21

20

22

18

17

Br OH

Cl

NH₂

CHO

COCH₃

COOH

COOEt

CONH₂

CN

CH]NOH

13d

13m

14

25

26

Cl

CH₂OH

4.99

49%

22%

6.33

4.59

6.30

0%

5.70

5.23

8.28

7.85

7.11

7.80

7.52

7.62

7.40

7.82

7.89

7.96

6.69

6.35

5.95

5.77

5.64

3.29

nd

1.47

2.94

1.32

nd

2.12

2.66

nd

activity (MAO-I < 50% at 10 mM) when deemed necessary for a

Br CH₂OH

quantitative evaluation of the structure-activity and/or structuree

selectivity relationships (SAR and SSR respectively). Inhibitory po-

tency was expressed as pIC₅₀and the selectivity index (SI) as the

Cl

CH(OH)CH₃

CH₂CN

CH₂CONH₂

CH₂CONHMe

CH₂CON(Me)₂

CH₂NH₂

27

28

difference pIC₅₀MAO-BepIC₅₀MAO-A (DpIC₅₀). pIC₅₀s, SIs and % of

MAO inhibition are reported in Table 1 along with the chemical

structures of the examined 4,7-disubstituted coumarins. To avoid

the loss of important structural information in the derivation of

sound SAR, for the low active acid (entries 15 and 60 in Table 1) and

ureido (entry 58) MAO-B inhibitors, estimated pIC₅₀values of 4.0

and 4.5 were used, respectively.

13e

13f

13n

13h

13i

13g

13j

13k

CH₂NHMe

Br CH₂NHMe

40%

Cl

CH₂NHⁿPr

CH₂NHnBu

CH₂N(Me)₂

CH₂-1⁰-pyrrolidinyl

CH₂-4⁰-morpholinyl

3.2%

1.1%

5.7%

16%

11%

nd

4. Molecular modelling studies

3a

3d

3g

3j

3b

3e

3h

3k

3c

3f

3i

3l

3m

3n

3o

7

H

F

Cl

OMe

22%

49%

4.93

27%

0%

25%

2%

2.5%

0%

12%

5.3%

0%

3%

23%

8.6%

17%

0%

7.00

7.44

8.11

8.24

6.12

6.58

6.94

6.90

6.24

6.39

7.21

7.13

4.61

7.00

6.50

7.34

5.39

nd

3.18

nd

The need to clarify at a 3D level some important molecular as-

pects of previously discussed SAR, prompted us to undertake a

number of docking simulations to better assess, at the MAO-B

binding site, the effect on binding of some examined substituents

at position 4 of coumarin. The steric effect was analyzed in terms of

both posing and scoring by investigating the nature of the binding

interactions along with an analysis of the enzymatic room available

at position 4 and the subtle conformational rearrangements expe-

rienced by the 4-substituents. Besides the steric effect, docking

models permitted to clearly spot enzymatic regions where the

occurrence of HBD interactions can enhance the MAO-B afﬁnity.

Br OMe

H

F

OEt

Cl

Br OEt

H

F

OⁿPr

Cl

Br OⁿPr

Cl

OⁱPr

OCH₂OMe

OCH₂SMe

OCH₂CN

OPh

9

5. Results and discussion

11a

11b

11c

11d

10b

10c

10d

Cl

NHMe

5.39

5.03

0%

6.55

8.06

7.55

5.11

5.01

7.41

6.23

4.50^e

2.67

2.53

nd

0.86

nd

NHEt

NHⁱPr

A preliminary search to identify the best anchoring substituent

at position 7 for high potency and selectivity towards MAO-B, was

carried out on the basis of previous SARs and X-ray data from a

molecular complex of entry 13 with human MAO-B. Since previous

SARs suggested a halogen atom at meta-position as the best sub-

stituents in the 7-benzyloxy group, F, Cl and Br were selected as

suitable binding elements. To keep the overall molecular lip-

ophilicity at an acceptable level, the inhibitory potency of 7-

NHPh

NHCOCH₃

NHCOOEt

NHCONHEt

21%

0%

nd

3p

5a

4a

Cl

OCH₂COCH₃

OCH₂COOH

OCH₂COOEt

22%

0%

7.57

4.50^e

5.74

nd

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

729

benzyloxy derivatives bearing in the meta positions F, Cl and Br

substituents was ﬁrstly measured on coumarins carrying at posi-

tion 4 substituents with increasing lipophilicity, that is OH (entries

8e11), OMe (entries 35e38), OEt (entries 39e42), and OⁿPr (entries

43e46). Comparative analysis of the resulting inhibition data (see

infra) suggested the 3-chloro atom on the benzyloxy moiety as the

ideal substituent ensuring both comparable potency and lower

lipophilicity than the corresponding 3-bromo substituted

analogues.

The 3-chorobenzyloxy substituent was therefore kept con-

stant in the design of all the other 4-substituted coumarins re-

ported in Table 1. Since in previous studies the substituent effects

on MAO afﬁnity and selectivity at 4-, and to lesser extent, 3-

position, have been mainly assessed with hydrophobic sub-

stituents, in the present work polar substituents with different

conformational, hydrophobic, electronic and steric properties

were investigated. Bulky substituents carrying aliphatic or

cycloaliphatic moieties were introduced at position 4 to better

assess the steric effects.

that no apparent relationship exists between MAO-A and MAO-B

inhibitory potency. MAO-A afﬁnity was generally very low and

only in a very few cases a sub-micromolar IC₅₀value was observed

(i.e., entries 23, 25 and 56). The most potent MAO-B inhibitors

(pIC₅₀ꢁ 7.80) were entries 2, 20, 21, 23, 27e29, 37, 38, 52, 62 and 63

which were distributed nearly over all the classes of compound in

Table 1 but prevalently within class III (see Fig. 1). Entry 62, that is

the 4-oxyacetamido-7-metachlorobenzyloxy coumarin, resulted

the most potent and selective MAO-B inhibitor of the whole series

of examined compounds, exhibiting outstanding pIC₅₀and SI

values equal to 8.48 and 3.86, respectively.

Anionic carboxylic acid derivatives (entries 15 and 60) displayed

almost no MAO inhibition. Interestingly, some inhibitory activity

towards MAO-B was recovered in the corresponding ethyl esters

(entries 16 and 61, respectively).

4-OH coumarin derivatives, which at physiological pH are fully

ionized, exhibited low MAO-B inhibition for the 3⁰-unsubstituted-

and 3⁰-ﬂuoro-benzyloxy derivatives (entries 8 and 9, respectively)

whereas a higher and nearly identical, sub-micromolar inhibitory

activity at MAO-B was displayed by the 3⁰-chloro- and 3⁰-bromo-

benzyloxy congeners (entries 10 and 11, respectively). As foresee-

able, the substitution of the highly polar anionic 4-OH group with

linear alkoxy groups afforded a series of potent and selective MAO-

B inhibitors. These MAO-B inhibition data prompted us to choose

the 3⁰-chlorobenzyloxy substituent at position 7 of the coumarin

ring as the most efﬁcient anchoring moiety to the MAO-B binding

site while introducing a large variety of substituents at position 4 to

thoroughly exploit the physicochemical and spatial binding domain

of that region.

Possible metabolic transformations of one substituent/molecule

to another through demethylation, deacylation, hydrolysis and

oxidation reactions were also considered in the molecular design to

prospect the in vivo behaviour of lead inhibitors.

5.1. SAR and SSR

For a better analysis and discussion of the SARs and SSRs, 7-

(meta-halogenobenzyloxy)-4-substituted coumarins in Table

1

were divided in six different classes as follows: Class I,

comprising lead compound (entry 1) and compounds bearing small

hydrophobic substituents at position 4 (entries 2e7); Class II,

containing small polar groups directly linked at the C-4 (entries 8e

19); Class III, containing 4-CH₂R groups (entries 20e34) where R is

a polar substituent; Class IV, containing 4-alkoxy groups and a 4-

phenoxy group (entries 35e50 and 51, respectively); Class V,

comprising 4-NHR and 4-NHCOR groups (entries 52e55 and 56e

58, respectively) and Class VI, collecting compounds with 4-

OCH₂COR groups (entries 59e67) where R is a polar substituent.

For a rapid visual inspection of MAO-B afﬁnity distribution over the

different classes, pIC₅₀values were plotted as a histogram in Fig. 1.

The limited availability of MAO-A pIC₅₀makes useless a similar

representation for MAO-A inhibition.

The following discussion of the SAR within the classes of in-

hibitors IeVI was limited to MAO-B inhibition because MAO-A

pIC₅₀were available only for a limited number of compounds.

5.2. Inhibitors of class I

MAO-B Inhibitory potency of compounds of class I appeared to

be mainly modulated by steric effects. Taft Es steric parameters are

linearly related to the inhibitory potency (data not shown), being

the unsubstituted entry 1 an underpredicted outlier.

The most potent inhibitors are the 4-Me and 4-Cl coumarin

derivatives (entries 2 and 5, respectively). As it will be seen later

also for other classes of compounds reported herein, substituent

steric effects seemed to be the most relevant modulators of MAO-B

inhibitory potency.

At a ﬁrst glance, inhibition data in Fig. 1 and Table 1 indicate that

very potent and selective MAO-B inhibitors were discovered and

Fig. 1. pIC₅₀s values distribution within the different classes of coumarin MAO-B inhibitors. Highly potent inhibitors (pIC₅₀ꢁ 7.80) are highlighted with black bars.

730

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

5.3. Inhibitors of class II

bearing a 7-(3⁰-chlorobenzyloxy) group 37, 41, 45 and 47e50. The

4-OCH₂CN derivative (entry 50, pIC₅₀¼ 7.34) was a strong outlier

and was discarded from the regression analysis as it showed a very

high activity despite a steric hindrance higher than its OEt analogue

(entry 41). It may be speculated that the CN group might be

involved in the formation of a HB reinforcing the inhibitor binding

to MAO-B.

The fully ionized 4-enol (entries 8e11) and 4-carboxy (entry 15)

coumarin derivatives exhibited a very low inhibitory potency. As

anticipated in the general discussion, the afﬁnity of the 4-OH de-

rivatives increased with the increase of the lipophilic character of

the meta substituent of the benzyloxy group. Small hydrophilic

substituents at position 4 displayed a good inhibitory potency (see

entries 12e14 and 18) whereas compound 16, bearing the larger

and more lipophilic carbethoxy substituent, was the less active

inhibitor among the unionized compounds of this series.

5.6. Inhibitors of class V

Despite the limited number of 4-NHR and 4-NHCOR derivatives

prepared and tested, also for this class of inhibitors a clear steric

effect of the R substituent was detected being the compounds with

bulkier substituents (i.e., entries 54, 55, 57 and 58) the least active

of the series. It is worth noting that the 4-NHAc derivative (entry

56) displayed the highest MAO-A inhibitory potency (pIC₅₀¼ 6.55

and, consequently, a very low selectivity index, SI ¼ 0.86).

5.4. Inhibitors of class III

R groups of the 4-CH₂R moiety are very polar and mainly rep-

resented by diversely substituted amino groups (see entries 27e

34). Very small and polar R groups (i.e., OH, CN, NH₂, NHCH₃,

CONH₂, and CONHCH₃) characterize compounds with a very high

MAO-B inhibitory potency. However a comparable activity was also

observed for the corresponding 4-CH₂Cl (entry 6) and this sug-

gested that in the “R region” small, highly polar or lipophilic groups

can be easily accommodated. As described in the modelling section,

different interactions could take place and contribute to the bind-

ing afﬁnity.

It is worth noting that position 4 did not tolerate bulky groups as

can be inferred comparing the afﬁnities of entry 27 bearing an N-

methyl amino group with the corresponding N-monoalkyl

substituted congeners (entries 28, 30 and 31) and the N,N-disub-

stituted congeners (entries 32e34) bearing alkyl and cyclolakyl

groups of increasing size. Actually entries 33 and 34 bearing the

bulky pyrrolidine and morpholine rings are the least potent MAO-B

inhibitors within this class of derivatives with pIC₅₀values equal to

5.77 and 5.64, respectively.

5.7. Inhibitors of class VI

Also for this class of compounds, that is 4-OCH₂COR substituted

derivatives, steric hindrance of the R group plays a key role in the

modulation of MAO-B inhibitory potency. Actually, entries 59 and

62e64, bearing small R groups, exhibited the highest MAO-B

inhibitory activity whereas compounds carrying bulkier groups

(e.g., the ethyl ester 61 and the N,N-dimethyl-, piperidine- and

morpholine-amides 65e67) showed a very low inhibitory potency.

Also for these compounds the decrease of activity seemed to be

directly correlated to the steric hindrance of the R group being

more pronounced for the bulkiest piperidine and morpholine

groups present in entries 66 and 67 (pIC₅₀¼ 4.41 and 4.89,

respectively). As already mentioned, the anionic 4-oxyacetic acid

derivative 60 showed no afﬁnity for both MAO isoforms. Remark-

ably, the most potent and selective MAO-B inhibitor, that is the 7-

(3⁰-chlorobenzyloxy)-4-oxyacetamido coumarin 62 (pIC₅₀¼ 8.48,

SI ¼ 3.86) was found in this class of inhibitors. Its corresponding 3⁰-

bromobenzyloxy analogue 63 displayed a slightly lower inhibitory

potency (pIC₅₀¼ 8.05) and a signiﬁcantly lower selectivity index

(SI ¼ 3.00) thus conﬁrming the correctness of our selection of the

3⁰-chlorobenzyloxy substituent a the position 7 of the coumarin

ring as the ideal binding moiety for a high MAO-B inhibitory po-

tency and MAO-B over MAO-A selectivity.

Previous analysis and discussion of SARs pointed out the

importance of steric effects at position 4 and lipophilic effects at the

meta position of the 7-benzyloxy group, providing signiﬁcant in-

sights on the pivotal physicochemical interactions taking place at

the MAO-B binding site. However, the lack of suitable electronic,

lipophilic and hydrogen bonding parameters for most of the

explored 4-substituents precluded the derivation of a more general,

comprehensive and informative QSAR model. Consequently, to

detect and locate at the three-dimensional level possible additional

interactions driving the ligand binding at position 4, inhibition data

of some selected inhibitors were modelled through docking

simulations.

5.5. Inhibitors of class IV

Among 4-alkoxy coumarins (entries 35e49), methoxy de-

rivatives (entries 35e38) exhibited the highest activity being the 3⁰-

chloro- and 3⁰-bromobenzyloxy congeners two of the most potent

and selective MAO-B inhibitors of the whole series of studied

compounds (i.e., pIC₅₀¼ 8.24 and 8.11; SI ¼ 3.18 and SI > 3.24,

respectively). In general, in the three different series of 4-alkoxy

coumarins, that is 4-methoxy, 4-ethoxy and 4-n-propoxy, the

following rank of activity was observed 3⁰-ClOBn ꢁ 3⁰-BrOBn > 3⁰-

FOBn > 3⁰-unsubstituted OBn. These ﬁndings were slightly sur-

prising because a signiﬁcant increase of activity was expected from

the interaction of the more lipophilic 3-BrOBn derivatives,

compared to the corresponding 3⁰-ClOBn derivatives, in the lipo-

philic pocket of the MAO-B binding site, well delineated by X-ray

crystallography of the complexes of entry 13, saﬁnamide and

coumarin NW-1772 with MAO-B [43]. MAO-B inhibitory potency of

4-alkoxy coumarins bearing a 7-(3⁰-chlorobenzyloxy) group was

ranked as follows: 4-OMe > 4-OⁿPr > 4-OEt >> 4-OⁱPr and this was

again indicative of a strong steric effect at position 4. Actually, the

inhibitory potency of the 4-isopropoxy derivative, entry 47, was

3141-fold lower than that of the 4-methoxy analogue (entry 37).

The isosteric substitution of one methylene group of the n-propoxy

(entry 45) with one oxygen (entry 48) or one sulphur atom (entry

49) led to a decrease of activity, especially for compound 49 which

showed a 5-fold decrease of potency (pIC₅₀¼ 6.50 vs 7.21). To

explain these data as well as the very low activity observed for the

4-phenoxy derivative (entry 51, pIC₅₀¼ 5.39) again a steric effect

could be advocated. Interestingly a good linear relationship (data

6. Docking simulations

Docking simulations aimed to explain mainly the drop of afﬁnity

caused by the introduction of bulky substituents at position 4 of the

coumarin ring (e.g., entry 20 vs 66) strictly related to the limited

steric accessibility of the MAO-B binding counterpart. A number of

derivatives, of which some have at position 4 branched or cyclic

bulky groups (i.e., entries 6, 20, 33, 34, 51, 54, 62 and 66), were

selected and their binding interactions evaluated through docking

studies. The GOLD 5.1 [46] program was used to carry out docking

not shown) can be found between inhibitory potency and the

n

steric parameter of Charton [45] for the 4-alkoxy coumarins

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

731

simulations, since in several studies it yielded better performances

compared to other similar programs. Given that our MAO-B inhi-

bition data were determined on rat brain mitochondria homoge-

nates, docking simulations were run on homology model of rat

MAO-B isoform, for which no crystallographic data were avail-

able. As reported in our previous papers [33,39], eight structural

water molecules were explicitly taken into account in the docking

screens, and a conformational ﬂexibility was allowed to two critical

residues, Q206 and Y398, through the exploration of a rotamer li-

brary of GOLD software.

the morpholinyl (entry 34, pIC₅₀¼ 5.64) and pyrrolidinyl (entry

33, pIC₅₀¼ 5.77) rings had a clear negative impact on the docking

score lowered to 56.14 kJ/mol and 66.87 kJ/mol, respectively. The

docking poses (data not shown) conﬁrmed the hypothesized

conformational rearrangements visited by those cyclic

substituents.

7. Conclusions

Small sized hydrophobic and polar substituents, including the

aliphatic charged amino groups, were well tolerated at the position

4 of 7-benzyloxy coumarins. Compounds bearing highly polar and

anionic 4-substituents were instead disallowed. The systematic and

ﬁnely-tuned exploration of position 4 allowed a very precise

detection of the physicochemical features of the admitted sub-

stituents. In particular docking simulations helped to detect and

locate at the 3D level the main steric, hydrophobic, and HB in-

teractions underlying the selective MAO-B inhibition potency. The

prevalent role of steric interactions was clearly pointed out along

with the important role of hydrophobic interactions, chieﬂy of the

7-meta-substituted benzyloxy groups, previously observed for

coumarins [47] and condensed diazine derivatives [48].

Interestingly, many substituents were designed as

metabolically-related moieties (e.g., 4-CH₃, 4-CH₂OH, 4-CHO and 4-

COOH; 4-N(CH₃)₂, 4-NHCH₃, 4-NH₂and 4NHAc; 4-XCH₂CON(CH₃)₂

and 4-XCH₂CONHCH₃; 4-OCH₃and 4-OH) and this may help the

reliable prediction of the activity of putative metabolites of the

most interesting parent inhibitors.

The large variety of substituents at position 4 characterizing

highly potent and selective MAO-B inhibitors may enable the se-

lection of the most appropriate compounds for safe and site-

selective delivery (e.g., at CNS), for a long- or short-lasting action

(high or low metabolic stability) and/or for the eventual prepara-

tion of suitable pro-drugs (e.g., from amine 13e and alcohol 13d).

The main outcomes from our SAR and docking studies might

help the design of new and selective MAO-B inhibitors based on

heterocyclic scaffold different from coumarins (scaffold hopping)

[33] and eventually multimodal drugs addressing other biochem-

ical targets relevant for NDs diseases along MAO-B. Indeed, long-

term studies are going on in our laboratories to address NDs

Results from our docking screen pointed out the steric hin-

drance of the 4-substituents as a clear molecular determinant

inﬂuencing the MAO-B inhibitory potency. In this respect, the

structural water molecules might play a role also in forcing

highly branched or bulky substituents to folded conformations at

the expense not only of their energetic content but also of their

chance of establishing favourable hydrogen bonds with ordered

water molecules. Satisfactorily, a good agreement was observed

between experimental inhibitory activities and docking scoring

values. As shown in Fig. 2a, one of the most active compound

(entry 20, pIC₅₀¼ 8.28), bearing the small hydroxymethyl group

at position 4, was well accommodated in the MAO-B binding site

and was involved in a stabilizing HB network including at least

three structural water molecules as shown in Fig. 2. A docking

score as high as 79.85 kJ/mol was calculated. Replacing the hy-

droxyl with the chlorine atom led to a slight decrease of activity

(entry 6, pIC₅₀¼ 7.36) likely due to the loss of HB interactions

yielding to a lower docking score (76.61 kJ/mol). On the other

hand, the ﬂexibility of the larger polar group at position 4 of the

most active inhibitor (entry 62, pIC₅₀¼ 8.48) ensured the

occurrence of an HB with a speciﬁc amino acid residues resulting

in a high docking score value (74.76 kJ/mol). As anticipated, the

introduction of bulkier groups had the effect of strongly lowering

the docking score. For instance, the consistent loss of activity of

piperidine derivative (entry 66, pIC₅₀¼ 4.41) was likely due to

the need of rearranging in a high-energy folded conformation

yielding a score as low as 34.79 kJ/mol despite, as shown in

Fig. 2b, the displacement of Y398 from its native position to

reduce possible steric clashes. Although less pronounced, a

similar behaviour was experienced by other cyclic part of 4-

substituents in the search of larger room into the MAO-B bind-

ing site. The relevant drop of activity due to the introduction of

through

a multitarget-directed-ligand approach [49]. Some

encouraging preliminary results have been reported [50] and

Fig. 2. Top-ranked docking pose of entries 20 (a) (pIC₅₀¼ 8.28) and 66 (b) (pIC₅₀¼ 4.41) into rMAO-B binding site. Relevant amino acid side chains, FAD cofactor, and inhibitors are

represented as stick models coloured according to the following atom code: C atoms in white, yellow, and cyan for amino acid side chains, cofactor and inhibitor, respectively.

Ordered water molecules are represented as red balls. The protein binding site is rendered as a light blue surface. (a) The black dashed lines indicate the HB network established by

entry 20 with ordered water molecules. (b) The orange wireframe and stick model of Y398 indicate its native and rotated conformations following docking optimization of the

piperidinyl inhibitor 6e (entry 66). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

732

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

original insights from the present work will help the design of new,

in vivo-active, multitarget-directed ligands having the selective

MAO-B inhibition as the core biological activity [30].

6.47 (d, J ¼ 2.4 Hz, 1H), 6.52 (dd, J₁¼ 2.4 Hz, J₂¼ 8.8 Hz, 1H), 6.99e

7.07 (m, 1H), 7.11e7.21 (m, 1H), 7.32e7.40 (m, 2H), 7.65 (d,

J ¼ 8.8 Hz, 1H), 12.73 (s, 1H, dis. with D₂O).

8. Experimental section

8.1.1.2. 1-{4-[(3-Chlorobenzyl)oxy]-2-hydroxyphenyl}ethanone (1c).

White solid; yield: 56%; mp: 127e8 ^ꢀC (diisopropyl ether). ¹H NMR

8.1. Chemistry

(200 MHz, CDCl₃)

d

: 2.59 (s, 3H), 5.05 (s, 2H), 6.50 (d, J ¼ 2.4 Hz, 1H),

6.54 (dd, J₁¼ 2.4 Hz, J₂¼ 8.8 Hz, 1H), 7.22e7.40 (m, 3H), 7.48 (s, 1H),

Starting materials, reagents and analytical grade solvents were

purchased from SigmaeAldrich (Europe). The purity of all the in-

termediates, checked by ¹H NMR and HPLC was always better than

95%. Column chromatography was performed using Carlo Erba

silica gel (0.05e0.20 mm) or Carlo Erba neutral aluminium oxide

(Brockmann activity I). Flash chromatographic separations were

7.68 (d, J ¼ 8.8 Hz, 1H), 12.77 (s, 1H, dis. with D₂O).

8.1.1.3. 1-{4-[(3-Bromobenzyl)oxy]-2-hydroxyphenyl}ethanone (1d).

White solid; yield: 94%; mp: 141e2 ^ꢀC (diisopropyl ether). ¹H NMR

(200 MHz, CDCl₃)

d

: 2.57 (s, 3H), 5.06 (s, 2H), 6.47 (d, J ¼ 2.4 Hz, 1H),

6.51 (dd, J₁¼ 2.4 Hz, J₂¼ 8.8 Hz, 1H), 7.23e7.36 (m, 2H), 7.48 (m,

performed on Biotage SP1 puriﬁcation system using ﬂash cartridges

prepacked with KP-Sil 32e63

1H), 7.58 (s, 1H), 7.65 (d, J ¼ 8.8 Hz, 1H), 12.73 (s, 1H, dis. with D₂O).

ꢀ

m

m, 60 A silica. All reactions were

routinely checked by TLC using Merck Kieselgel 60 F₂₅₄aluminium

plates and visualized by UV light or iodine. Regarding the reaction

requiring the use of dry solvents, the glassware was ﬂame-dried

and then cooled under a stream of dry argon before the use. Mi-

crowave reactions were performed in a Milestone MicroSynth

apparatus, setting temperature and hold times, ﬁxing maximum

irradiation power to 500 W and heating ramp times to 2 min.

Elemental analyses were performed on the Carlo Erba Elemental

Analyzer Model EA 1110 or EuroEA 3000 analyzer only on the ﬁnal

compounds tested as MAOs inhibitors. The measured values for C,

H, and N agreed to within ꢂ0.40% of the theoretical values. Nuclear

magnetic resonance spectra were recorded on a Varian Mercury

300 instrument (at 300 MHz) or on a Varian Gemini 200 instru-

ment (at 200 MHz) at ambient temperature in the speciﬁed

8.1.2. General procedure for the synthesis of 7-substituted-4-

hydroxy-2H-chromen-2-ones 2bed

A mixture of the proper 4-benzyloxy-2-hydroxyacetophenone

1bed (10 mmol), sodium (0.58 g, 25 mmol), diethyl carbonate

(3.5 g, 30 mmol) and Dowtherm A (10 mL) was heated at 160 ^ꢀC

with stirring. Within a few minutes, after the sodium melting, a

sudden reaction occurred with gas evolution and formation of an

abundant whitish precipitate. Xylene (30 mL) was added in order

to dilute the mixture and allow a better stirring, then the

mixture was further heated at 160 ^ꢀC for 1 h. After cooling,

diethyl ether (30 mL) was added and the solid (nearly pure

compound 2bed as sodium salts) was collected by ﬁltration,

washed with abundant diethyl ether and dried. Because in the

subsequent synthetic step (O-alkylation) compounds 2bed were

used as sodium salts, a major amount of the crude reaction

product was stored just as was obtained, whereas a minor

amount was dissolved in water (50 mL) and the alkaline aqueous

solution was acidiﬁed with 6 N aqueous HCl so that compounds

2bed separated out as amorphous solids which were collected

by ﬁltration, washed with a little water, dried and crystallized

from the proper solvent.

deuterated solvent. Chemical shifts (d) are quoted in parts per

million (ppm) and are referenced to the residual solvent peak

(when registered at 300 MHz) or using (CH₃)₄Si as an internal

reference (

d

¼ 0, when recorded at 200 MHz). The coupling con-

stants J are given in Hertz (Hz). The following abbreviations were

used: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q

(quadruplet), quint (quintet), sext (sextet), m (multiplet), br (broad

signal); signals due to OH and NH protons were located by deute-

rium exchange with D₂O. Melting points were determined by the

capillary method on a Stuart Scientiﬁc SMP3 electrothermal

apparatus or using a Fisher-Johns apparatus and are uncorrected.

Synthesis, analytic and spectroscopic data of compounds 1a [41], 2a

[41], 12b [42], 12c [39], 12d [43], 13a [39], 13eeg [39], 19 [33] and

25e28 (corresponding to entries 23e26 in Table 1) have been

already reported in the literature. Coumarin 12a was from com-

mercial source.

8.1.2.1. 7-[(3-Fluorobenzyl)oxy]-4-hydroxy-2H-chromen-2-one (2b).

White crystals; yield: 66%; mp: 294e5 ^ꢀC dec. (ethanol). ¹H NMR

(300 MHz, DMSO-d₆)

d

: 5.22 (s, 2H), 5.43 (s, 1H), 6.98 (d, J ¼ 2.5 Hz,

1H), 7.02 (dd, J₁¼ 2.5 Hz, J₂¼ 8.5 Hz, 1H), 7.16e7.20 (m, 1H), 7.28e

7.31 (m, 2H), 7.40e7.48 (m, 1H), 7.71 (d, J ¼ 8.5 Hz, 1H), 12.36 (s, 1H,

dis. with D₂O). Anal. (C₁₆H₁₁FO₄) C, H.

8.1.2.2. 7-[(3-Chlorobenzyl)oxy]-4-hydroxy-2H-chromen-2-one (2c).

White crystals; yield: 85%; mp: 261e3 ^ꢀC dec. (acetone). ¹H NMR

8.1.1. General procedure for the synthesis of substituted

acetophenones 1bed

(300 MHz, DMSO-d₆)

d

: 5.22 (s, 2H), 5.42 (s, 1H), 6.98 (d, J ¼ 2.2 Hz,

1H), 7.02 (dd, J₁¼ 2.2 Hz, J₂¼ 8.4 Hz, 1H), 7.39e7.44 (m, 3H), 7.53 (s,

1H), 7.71 (d, J ¼ 8.4 Hz, 1H), 12.35 (s, 1H, dis. with D₂O). Anal.

(C₁₆H₁₁ClO₄) C, H.

A

mixture of 1-(2,4-dihydroxyphenyl)ethanone (3.0 g,

20 mmol), 20 mmol of the proper benzyl halide, 14.5 mmol (2.0 g)

of anhydrous K₂CO₃and 2-butanone (100 mL) was heated at reﬂux

for 24 h with stirring. The mixture was then poured into cold water

(500 mL) and exhaustively extracted with a mixture ethyl acetatee

diethyl ether (1:1, v/v). The residue obtained from the combined

extracts (washed with water and dried over anhydrous Na₂SO₄)

was concentrated under reduced pressure and then puriﬁed

through column chromatography on a silica gel column eluting

with dichloromethaneepetroleum ether (1:1, v/v), to recover pure

compounds 1bed as white solids which were then crystallized

from a suitable solvent.

8.1.2.3. 7-[(3-Bromobenzyl)oxy]-4-hydroxy-2H-chromen-2-one (2d).

White crystals; yield: 83%; mp: 291e2 ^ꢀC dec. (ethanol/acetone). ¹H

NMR (200 MHz, DMSO-d₆) d: 5.24 (s, 2H), 5.47 (s,1H), 6.95e7.06 (m,

2H), 7.32e7.62 (m, 3H), 7.70 (s, 1H), 7.74 (d, J ¼ 8.8 Hz, 1H), 12.15e

12.80 (br s, 1H, dis. with D₂O). Anal. (C₁₆H₁₁BrO₄) C, H.

8.1.3. General procedure for the synthesis of 7-(benzyloxy)-4-

alkoxy-2H-chromen-2-ones 3aec and 7-[(3-halobenzyl)oxy]-4-

alkoxy-2H-chromen-2-ones 3dep

A large excess (1.0 mL) of the proper alkyl halide was added to

the solution of the sodium salt of compounds 2aed (1.0 mmol) in

25 mL of dry DMF and the mixture was stirred at room temperature

for 1.5 h, then poured into cold water. The resulting emulsion was

8.1.1.1. 1-{4-[(3-Fluorobenzyl)oxy]-2-hydroxyphenyl}ethanone (1b).

White solid; yield: 47%; mp: 108e9 ^ꢀC (diisopropyl ether/petro-

leum ether). ¹H NMR (200 MHz, CDCl₃)

d: 2.56 (s, 3H), 5.09 (s, 2H),

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

733

exhaustively extracted with the mixture diethyl ethereethyl ace-

tate (1:1, v/v). The combined extracts (dried over anhydrous Na₂SO₄

and evaporated to dryness at reduced pressure) afforded a thick oil

which was chromatographed on a silica gel column eluting with

CH₂Cl₂. The fraction collected, after removal of solvents, gave pure

compounds 3aep as white solids which were then crystallized

from proper solvents.

8.1.3.10. 7-[(3-Bromobenzyl)oxy]-4-methoxy-2H-chromen-2-one

(3j). White crystals; yield: 64%; mp: 140e1 ^ꢀC (CH₂Cl₂/petroleum

ether). ¹H NMR (200 MHz, CDCl₃)

d: 4.00 (s, 3H), 5.12 (s, 2H), 5.61 (s,

1H), 6.82e6.98 (m, 2H), 7.23e7.42 (m, 2H), 7.52 (m, 1H), 7.63 (s, 1H),

7.74 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₇H₁₃BrO₄) C, H.

8.1.3.11. 7-[(3-Bromobenzyl)oxy]-4-ethoxy-2H-chromen-2-one (3k).

White crystals; yield: 64%; mp: 98.5e100 ^ꢀC (diisopropyl ether). ¹H

8.1.3.1. 7-(Benzyloxy)-4-methoxy-2H-chromen-2-one

White crystals; yield: 60%; mp: 141e3 ^ꢀC (ethyl acetate/petroleum

ether). ¹H NMR (200 MHz, CDCl₃)

: 3.96 (s, 3H), 5.11 (s, 2H), 5.56 (s,

(3a).

NMR (300 MHz, DMSO-d₆)

d

: 1.38 (t, J ¼ 7.2 Hz, 3H), 4.23 (q,

J ¼ 7.2 Hz, 2H), 5.21 (s, 2H), 5.72 (s, 1H), 6.99e7.07 (m, 2H), 7.33e

7.39 (m, 1H), 7.45e7.48 (m, 1H), 7.53e7.55 (m, 1H), 7.67e7.69 (m,

1H), 7.70 (d, J ¼ 1.9 Hz, 1H). Anal. (C₁₈H₁₅BrO₄) C, H.

d

1H), 6.85 (d, J ¼ 2.4 Hz, 1H), 6.90 (dd, J₁¼ 2.4 Hz, J₂¼ 8.8 Hz, 1H),

7.31e7.46 (m, 5H), 7.69 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₇H₁₄O₄) C, H.

8.1.3.12. 7-[(3-Bromobenzyl)oxy]-4-propoxy-2H-chromen-2-one

8.1.3.2. 7-(Benzyloxy)-4-ethoxy-2H-chromen-2-one

crystals; yield: 57%; mp: 132e3 ^ꢀC (diisopropyl ether). ¹H NMR

(300 MHz, CDCl₃)

(3b). White

(3l). White crystals; yield: 51%; mp: 113e4 ^ꢀC (diisopropyl ether).

¹H NMR (300 MHz, CDCl₃)

d: 1.09 (t, J ¼ 7.4 Hz, 3H), 1.93 (sext,

d

: 1.52 (t, J ¼ 6.9 Hz, 3H), 4.18 (q, J ¼ 6.9 Hz, 2H),

J ¼ 7.4 Hz, 2H), 4.07 (t, J ¼ 7.4 Hz, 2H), 5.09 (s, 2H), 5.55 (s, 1H), 6.83

(d, J ¼ 1.9 Hz, 1H), 6.90 (dd, J₁¼ 1.9 Hz, J₂¼ 8.8 Hz, 1H), 7.29e7.37

(m, 2H), 7.47e7.49 (m, 1H), 7.60 (s, 1H), 7.73 (d, J ¼ 8.8 Hz, 1H). Anal.

(C₁₉H₁₇BrO₄) C, H.

5.12 (s, 2H), 5.54 (s, 1H), 6.86 (d, J ¼ 2.2 Hz, 1H), 6.91 (dd, J₁¼ 2.2 Hz,

J₂¼ 8.8 Hz, 1H), 7.33e7.45 (m, 5H), 7.73 (d, J ¼ 8.8 Hz, 1H). Anal.

(C₁₈H₁₆O₄) C, H.

8.1.3.3. 7-(Benzyloxy)-4-propoxy-2H-chromen-2-one

White crystals; yield: 48%; mp: 107e8 ^ꢀC (diisopropyl ether). ¹H

NMR (300 MHz, CDCl₃)

J ¼ 7.4 Hz, 2H), 4.06 (t, J ¼ 7.4 Hz, 2H), 5.12 (s, 2H), 5.54 (s, 1H), 6.86

(d, J ¼ 2.5 Hz, 1H), 6.91 (dd, J₁¼ 2.5 Hz, J₂¼ 8.8 Hz, 1H), 7.33e7.45

(m, 5H), 7.72 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₉H₁₈O₄) C, H.

(3c).

8.1.3.13. 7-[(3-Chlorobenzyl)oxy]-4-isopropoxy-2H-chromen-2-one

(3m). White crystals; yield: 44%; mp: 137e8 ^ꢀC (diisopropyl ether).

d

: 1.09 (t, J ¼ 7.4 Hz, 3H), 1.91 (sext,

¹H NMR (200 MHz, CDCl₃)

5.12 (s, 2H), 5.57 (s, 1H), 6.82e6.98 (m, 2H), 7.28e7.42 (m, 3H), 7.47

(s, 1H), 7.75 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₉H₁₇ClO₄) C, H.

d

: 1.48 (d, J ¼ 6.2 Hz, 6H), 4.73 (m, 1H),

8.1.3.14. 7-[(3-Chlorobenzyl)oxy]-4-(methoxymethoxy)-2H-chro-

8.1.3.4. 7-[(3-Fluorobenzyl)oxy]-4-methoxy-2H-chromen-2-one (3d).

White crystals; yield: 76%; mp: 142e3 ^ꢀC (diisopropyl ether). ¹H

men-2-one (3n). White crystals; yield: 69%; mp: 130e1 ^ꢀC (ethyl

acetate/petroleum ether). ¹H NMR (200 MHz, CDCl₃)

d: 3.57 (s, 3H),

NMR (200 MHz, CDCl₃)

6.82e7.48 (m, 6H), 7.74 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₇H₁₃FO₄) C, H.

d

: 4.00 (s, 3H), 5.15 (s, 2H), 5.60 (s, 1H),

5.13 (s, 2H), 5.37 (s, 2H), 5.81 (s, 1H), 6.82e6.98 (m, 2H), 7.26e7.42

(m, 3H), 7.47 (s, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₈H₁₅ClO₅) C, H.

8.1.3.5. 4-Ethoxy-7-[(3-ﬂuorobenzyl)oxy]-2H-chromen-2-one (3e).

White crystals; yield: 73%; mp: 123e5 ^ꢀC (diisopropyl ether/pe-

8.1.3.15. 7-[(3-Chlorobenzyl)oxy]-4-[(methylthio)methoxy]-2H-chro-

men-2-one (3o). White crystals; yield: 33%; mp: 137e9 ^ꢀC (ethyl

troleum ether). ¹H NMR (200 MHz, CDCl₃)

d

: 1.55 (t, J ¼ 7.0 Hz, 3H),

acetate/petroleum ether). ¹H NMR (200 MHz, CDCl₃)

d: 2.36 (s, 3H),

5.13 (s, 2H), 5.28 (s, 2H), 5.62 (s, 1H), 6.82e6.98 (m, 2H), 7.22e7.40

4.21 (q, J ¼ 7.0 Hz, 2H), 5.15 (s, 2H), 5.58 (s, 1H), 6.82e7.53 (m, 6H),

7.78 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₈H₁₅FO₄) C, H.

(m, 3H), 7.47 (s,1H), 7.77 (d, J ¼ 8.8 Hz,1H). Anal. (C₁₈H₁₅ClO₄S) C, H,

S.

8.1.3.6. 7-[(3-Fluorobenzyl)oxy]-4-propoxy-2H-chromen-2-one (3f).

White crystals; yield: 55%; mp: 99e100 ^ꢀC (diethyl ether). ¹H NMR

8.1.3.16. 7-[(3-Chlorobenzyl)oxy]-4-(2-oxopropoxy)-2H-chromen-2-

(300 MHz, CDCl₃)

d: 1.09 (t, J ¼ 7.4 Hz, 3H),1.92 (sext, J ¼ 7.4 Hz, 2H),

one (3p). White crystals; yield: 31%; mp: 210e2 ^ꢀC (ethyl acetate).

4.07 (t, J ¼ 7.4 Hz, 2H), 5.12 (s, 2H), 5.54 (s, 1H), 6.83 (d, J ¼ 2.2 Hz,

1H), 6.90 (dd, J₁¼ 2.2 Hz, J₂¼ 8.8 Hz, 1H), 7.00e7.07 (m, 1H), 7.13e

7.21 (m, 2H), 7.33e7.40 (m, 1H), 7.73 (d, J ¼ 8.8 Hz, 1H). Anal.

(C₁₉H₁₇FO₄) C, H.

¹H NMR (200 MHz, CDCl₃)

d: 2.35 (s, 3H), 4.74 (s, 2H), 5.14 (s, 2H),

5.44 (s, 1H), 6.83e7.01 (m, 2H), 7.26e7.42 (m, 3H), 7.47 (s, 1H), 7.83

(d, J ¼ 8.8 Hz, 1H). Anal. (C₁₉H₁₅ClO₅) C, H.

8.1.4. General procedure for the synthesis of ethyl ({7-[(3-

8.1.3.7. 7-[(3-Chlorobenzyl)oxy]-4-methoxy-2H-chromen-2-one

halobenzyl)oxy]-2-oxo-2H-chromen-4-yl}oxy)acetates 4a,b

A mixture of the 4-hydroxycoumarin 2c (1.0 g, 3.0 mmol) or 2d

(0.91 g, 3.0 mmol), ethyl bromoacetate (1.0 mL), anhydrous K₂CO₃

(1.0 g) and 2-butanone (25 mL) was heated at reﬂux for 3 h with

stirring. The reaction mixture was poured into cold water (300 mL)

and exhaustively extracted with ethyl acetateediethyl ether (1:1, v/

v). The combined extracts were washed with aqueous 1.0 N NaOH,

then with water and dried over anhydrous Na₂SO₄. After removal of

solvents, the oily residue was treated with a small amount of

diethyl ether and petroleum ether so that the nearly pure com-

pounds 4a,b separated out as whitish solids.

(3g). White crystals; yield: 92%; mp: 131e3 ^ꢀC (ethyl acetate/pe-

troleum ether). ¹H NMR (200 MHz, CDCl₃)

d: 4.00 (s, 3H), 5.12 (s,

2H), 5.60 (s, 1H), 6.82e6.98 (m, 2H), 7.30e7.40 (m, 3H), 7.46 (s, 1H),

7.74 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₇H₁₃ClO₄) C, H.

8.1.3.8. 7-[(3-Chlorobenzyl)oxy]-4-ethoxy-2H-chromen-2-one (3h).

White crystals; yield: 79%; mp: 128e130 ^ꢀC (diisopropyl ether). ¹H

NMR (200 MHz, CDCl₃)

2H), 5.12 (s, 2H), 5.57 (s, 1H), 6.82e6.98 (m, 2H), 7.28e7.43 (m, 3H),

7.47 (s, 1H), 7.76 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₈H₁₅ClO₄) C, H.

d

: 1.55 (t, J ¼ 7.0 Hz, 3H), 4.21 (q, J ¼ 7.0 Hz,

8.1.3.9. 7-[(3-Chlorobenzyl)oxy]-4-propoxy-2H-chromen-2-one (3i).

White crystals; yield: 55%; mp: 108e9 ^ꢀC (diisopropyl ether). ¹H

8.1.4.1. Ethyl ({7-[(3-chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}oxy)

acetate (4a). White crystals; yield: 56%; mp: 135e6 ^ꢀC (ethyl ace-

NMR (200 MHz, CDCl₃)

d: 1.12 (t, J ¼ 7.0 Hz, 3H),1.95 (m, 2H), 4.10 (t,

tate). ¹H NMR (200 MHz, CDCl₃)

d: 1.36 (t, J ¼ 7.0 Hz, 3H), 4.34 (q,

J ¼ 7.0 Hz, 2H), 5.12 (s, 2H), 5.58 (s, 1H), 6.82e6.98 (m, 2H), 7.25e

7.42 (m, 3H), 7.47 (s, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₉H₁₇ClO₄)

C, H.

J ¼ 7.0 Hz, 2H), 4.77 (s, 2H), 5.13 (s, 2H), 5.48 (s, 1H), 6.84e7.00 (m,

2H), 7.27e7.42 (m, 3H), 7.47 (s, 1H), 7.85 (d, J ¼ 8.8 Hz, 1H). Anal.

(C₂₀H₁₇ClO₆) C, H.

734

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

8.1.4.2. Ethyl

({7-[(3-bromobenzyl)oxy]-2-oxo-2H-chromen-4-yl}

1H), 7.52e7.55 (m, 2H), 7.68 (s, 2H, dis. with D₂O), 7.98 (d, J ¼ 8.8 Hz,

1H). Anal. (C₁₈H₁₄BrNO₅) C, H, N.

oxy)acetate (4b). White crystals; yield: 73%; mp: 125e6 ^ꢀC (ethyl

acetate/petroleum ether). ¹H NMR (300 MHz, DMSO-d₆)

d: 1.22 (t,

J ¼ 7.2 Hz, 3H), 4.19 (q, J ¼ 7.2 Hz, 2H), 5.04 (s, 2H), 5.22 (s, 2H), 5.76

(s, 1H), 7.05 (d, J ¼ 2.5 Hz, 1H), 7.08 (dd, J₁¼ 2.5 Hz, J₂¼ 8.5 Hz, 1H),

7.36 (t, J ¼ 7.7 Hz,1H), 7.47 (d, J ¼ 7.7 Hz,1H), 7.53e7.56 (m,1H), 7.68

(s, 1H), 7.73 (d, J ¼ 8.5 Hz, 1H). Anal. (C₂₀H₁₇BrO₆) C, H.

8.1.6.2. 2-({7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}oxy)

acetamide (6b). Whitish crystals; yield: 80%; mp: 283e4 ^ꢀC dec.

(acetone). ¹H NMR (200 MHz, DMSO-d₆)

d: 4.71 (s, 2H), 5.27 (s, 2H),

5.68 (s, 1H), 6.95e7.15 (m, 2H), 7.33e7.51 (m, 3H), 7.58 (s, 1H), 7.73

(s, 2H, dis. with D₂O), 8.00 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₈H₁₄ClNO₅) C,

H, N.

8.1.5. General procedure for the synthesis of ({7-[(3-halobenzyl)

oxy]-2-oxo-2H-chromen-4-yl}oxy)acetic acids 5a,b

Derivative 4a (0.43 g, 1.0 mmol) or 4b (0.39 g, 1.0 mmol) was

added to an ethanolic solution of solid KOH (0.11 g, 2.0 mmol).

Rapidly, the solution became yellow and a whitish solid (com-

pounds 5a,b as potassium salts) began to precipitate within few

minutes. The suspension was kept stirring at room temperature for

1 h, then it was diluted with diethyl ether, ﬁltered, washed with

diethyl ether and dried. The potassium salts collected were dis-

solved in water and the resulting solution was acidiﬁed with 6 N

HCl, so that the pure compound 5a or 5b separated out as a white

solids which were collected by ﬁltration, washed, with water, dried

and crystallized from acetone.

8.1.6.3. 2-({7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}oxy)-

N-methylacetamide (6c). Obtained by the reaction of acyl chloride

of 5a with methylamine. White crystals; yield: 94%; mp: 193e5 ^ꢀC

(CH₂Cl₂/ethyl acetate). ¹H NMR (200 MHz, DMSO-d₆)

d: 2.71 (d,

J ¼ 4.8 Hz, 3H), 4.74 (s, 2H), 5.27 (s, 2H), 5.71 (s, 1H), 7.01e7.16 (m,

2H), 7.37e7.55 (m, 3H), 7.58 (s, 1H), 8.02 (d, J ¼ 8.8 Hz, 1H), 8.16 (br

q, 1H, dis. with D₂O). Anal. (C₁₉H₁₆ClNO₅) C, H, N.

8.1.6.4. 2-({7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}oxy)-

N,N-dimethylacetamide (6d). Obtained by the reaction of acyl

chloride of 5a with dimethylamine. White crystals; yield: 85%; mp:

152e3 ^ꢀC (ethyl acetate). ¹H NMR (200 MHz, CDCl₃)

d: 3.05 (s, 3H),

8.1.5.1. ({7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}oxy)ace-

3.09 (s, 3H), 4.87 (s, 2H), 5.12 (s, 2H), 5.53 (s, 1H), 6.86 (s, 1H), 6.93

(d, J ¼ 8.8 Hz, 1H), 7.26e7.41 (m, 3H), 7.46 (s, 1H), 7.85 (d, J ¼ 8.8 Hz,

1H). Anal. (C₂₀H₁₈ClNO₅) C, H, N.

tic acid (5a). White crystals; yield: 76%; mp: 228e230 ^ꢀC (acetone).

¹H NMR (200 MHz, DMSO-d₆)

d: 4.90 (s, 2H), 5.26 (s, 2H), 5.69 (s,

1H), 7.01e7.16 (m, 2H), 7.37e7.53 (m, 3H), 7.58 (s, 1H), 7.78 (d,

J ¼ 8.8 Hz, 1H), OH not detected. Anal. (C₁₈H₁₃ClO₆) C, H.

8.1.6.5. 7-[(3-Chlorobenzyl)oxy]-4-(2-oxo-2-piperidin-1-ylethoxy)-

2H-chromen-2-one (6e). Obtained by the reaction of acyl chloride

of 5a with piperidine. White crystals; yield: 72%; mp: 146e7 ^ꢀC

8.1.5.2. ({7-[(3-Bromobenzyl)oxy]-2-oxo-2H-chromen-4-yl}oxy)ace-

tic acid (5b). White crystals; yield: 72%; mp: 231e3 ^ꢀC (acetone). ¹H

(ethyl acetate). ¹H NMR (200 MHz, CDCl₃)

d: 1.47e1.78 (m, 6H),

NMR (300 MHz, DMSO-d₆)

d

: 4.94 (s, 2H), 5.22 (s, 2H), 5.71 (s, 1H),

3.32e3.70 (m, 4H), 4.87 (s, 2H), 5.12 (s, 2H), 5.55 (s, 1H), 6.85 (d,

J ¼ 2.4 Hz, 1H), 6.93 (dd, J₁¼ 2.4 Hz, J₂¼ 8.8 Hz, 1H), 7.21e7.40 (m,

3H), 7.46 (s, 1H), 7.83 (d, J ¼ 8.8 Hz, 1H). Anal. (C₂₃H₂₂ClNO₅) C, H, N.

7.03e7.08 (m, 2H), 7.36 (t, J ¼ 7.7 Hz, 1H), 7.47 (d, J ¼ 7.7 Hz, 1H),

7.53e7.55 (m, 1H), 7.68 (s, 1H), 7.74 (d, J ¼ 8.5 Hz, 1H), OH not

detected. Anal. (C₁₈H₁₃BrO₆) C, H.

8.1.6.6. 7-[(3-Chlorobenzyl)oxy]-4-(2-morpholin-4-yl-2-oxoethoxy)-

2H-chromen-2-one (6f). Obtained by the reaction of acyl chloride of

5a with morpholine. White crystals; yield: 79%; mp: 161e3 ^ꢀC

8.1.6. General procedure for the synthesis of 2-({7-[(3-halobenzyl)

oxy]-2-oxo-2H-chromen-4-yl}oxy)-N-alkylacetamides 6aef

SOCl₂(20 mL) was added to the suspension of compound 5a

(0.40 g, 1.0 mmol) or 5b (0.36 g, 1.0 mmol) in CH₂Cl₂(25 mL)

and the mixture was reﬂuxed with stirring until a clear solution

was obtained (w2 h). The solvent and the excess of SOCl₂were

then removed in vacuo and the resulting residue (crude acyl

chloride of 5a or 5b) was cooled with an ice bath and treated

with an excess of proper amine. In the case of 6a,b, 15 mL of 30%

aqueous ammonia were directly added to crude acyl chlorides

and the resulting suspension was then stirred at room temper-

ature for 30 min, then ﬁltered to give the nearly pure com-

pounds 6a or 6b. In all other cases, the ice-cooled acyl chloride

of 5a was treated with 5 mL of suitable amine dissolved in

30 mL of CH₂Cl₂. The resulting solution was stirred at room

temperature for 30 min, then treated with aqueous 10% Na₂CO₃

(15 mL) and water (15 mL). The mixture was transferred in a

separatory funnel, the organic solution was collected and the

aqueous phase extracted twice with CH₂Cl₂. The combined ex-

tracts, after drying over anhydrous Na₂SO₄, were evaporated to

dryness under reduced pressure to give an oily residue which

was chromatographed on a silica gel column, eluting with the

mixture CH₂Cl₂/ethyl acetate (1:1, v/v). The collected eluate

afforded pure compounds 6cef as solids which were then

crystallized from the proper solvent.

(ethyl acetate/petroleum ether). ¹H NMR (200 MHz, CDCl₃)

d: 3.42e

3.90 (m, 8H), 4.89 (s, 2H), 5.12 (s, 2H), 5.58 (s,1H), 6.86 (d, J ¼ 2.4 Hz,

1H), 6.94 (dd, J₁¼ 2.4 Hz, J₂¼ 8.8 Hz, 1H), 7.21e7.40 (m, 3H), 7.46 (s,

1H), 7.80 (d, J ¼ 8.8 Hz, 1H). Anal. (C₂₂H₂₀ClNO₆) C, H, N.

8.1.7. ({7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}oxy)

acetonitrile (7)

Amide 6b (0.36 g, 1.0 mmol) was suspended in POCl₃(10 mL)

and the mixture was reﬂuxed for 2 h, with stirring. The resulting

solution was poured into ice-water and, after hydrolysis of excess

POCl₃, was exhaustively extracted with CH₂Cl₂. The combined ex-

tracts were dried over anhydrous Na₂SO₄and concentrated in

vacuo. Chromatographic separation on a silica gel column, by

eluting with the mixture CH₂Cl₂/ethyl acetate (1:1, v/v) gave pure

compound 7. White crystals; yield: 61%; mp: 157e8 ^ꢀC (ethyl ace-

tate). ¹H NMR (200 MHz, CDCl₃)

d: 4.96 (s, 2H), 5.14 (s, 2H), 5.69 (s,

1H), 6.89 (d, J ¼ 2.4 Hz, 1H), 6.97 (dd, J₁¼ 2.4 Hz, J₂¼ 8.8 Hz, 1H),

7.25e7.40 (m, 3H), 7.46 (s, 1H), 7.73 (d, J ¼ 8.8 Hz, 1H). Anal.

(C₁₈H₁₂ClNO₄) C, H, N.

8.1.8. 4-Chloro-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one (8)

Triethylamine (5 mL) was added to 7-[(3-chlorobenzyl)oxy]-4-

hydroxycoumarin 2c (0.91 g, 3.0 mmol) previously suspended in

methanol (5 mL). The resulting solution was evaporated to dryness

in vacuo to give a resinous residue to which POCl₃(5 mL) was

added. The mixture was heated at 130 ^ꢀC for 3 h, with stirring. The

dark solution obtained was poured into ice-water and, after hy-

drolysis of excess POCl₃, was exhaustively extracted with CH₂Cl₂.

8.1.6.1. 2-({7-[(3-Bromobenzyl)oxy]-2-oxo-2H-chromen-4-yl}oxy)

acetamide (6a). Whitish crystals; yield: 94%; mp: 277e9 ^ꢀC dec.

(ethanol). ¹H NMR (300 MHz, DMSO-d₆)

d

: 4.67 (s, 2H), 5.23 (s, 2H),

5.65 (s, 1H), 7.00e7.07 (m, 2H), 7.33e7.38 (m, 1H), 7.42e7.48 (m,

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

735

The combined extracts were dried over anhydrous Na₂SO₄,

concentrated in vacuo, and chromatographed on a silica gel col-

umn, eluting with CH₂Cl₂to give pure compound 8. White crystals;

yield: 66%; mp: 175e7 ^ꢀC (ethyl acetate). ¹H NMR (200 MHz, CDCl₃)

J₁¼ 2.4 Hz, J₂¼ 8.7 Hz, 1H), 6.98 (br s, 1H, dis. with D₂O), 7.28e7.37

(m, 3H), 7.43 (s, 1H), 7.65 (d, J ¼ 8.7 Hz, 1H). Anal. (C₁₉H₁₆ClNO₅) C,

H, N.

d

: 5.15 (s, 2H), 6.48 (s, 1H), 6.90 (d, J ¼ 2.4 Hz, 1H), 7.01 (dd,

8.1.13. N-{7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}-N⁰-

ethylurea (10d)

J₁¼ 2.4 Hz, J₂¼ 8.8 Hz, 1H), 7.23e7.40 (m, 3H), 7.46 (s, 1H), 7.80 (d,

J ¼ 8.8 Hz, 1H). Anal. (C₁₆H₁₀Cl₂O₃) C, H.

In a closed Pyrex vial equipped with a magnetic stirring bar, 10a

(0.30 mmol, 0.091 g) was suspended in ethyl isocyanate (2.0 mL).

The vessel was kept for 5 days in an oil bath at 60 ^ꢀC, then cooled at

room temperature and the mixture poured into crushed ice. The

resulting precipitate was ﬁltered and crystallized from ethanol.

White crystals; yield: 84%; mp: 227e8 ^ꢀC (ethanol). ¹H NMR

8.1.9. 7-[(3-Chlorobenzyl)oxy]-4-phenoxy-2H-chromen-2-one (9)

The mixture of 4-chlorocoumarin 8 (0.32 g, 1.0 mmol), phenol

(1.9 g, 20 mmol), anhydrous K₂CO₃(1.0 g, 7.2 mmol) and 2-

butanone (40 mL) was reﬂuxed for 2 h with stirring. The solvent

was then removed in vacuo and the residue was partitioned be-

tween H₂O and CH₂Cl₂. The organic layer was collected and the

aqueous phase was further extracted twice with CH₂Cl₂. The

combined extracts were washed with 0.5 N aqueous NaOH (to

remove the excess phenol), then with water and dried over Na₂SO₄.

After removal of solvent, pure compound 9 was obtained. White

crystals; yield: 74%; mp: 164e5 ^ꢀC (ethyl acetate/petroleum ether).

(300 MHz, DMSO-d₆)

d

: 1.16 (t, J ¼ 6.9 Hz, 3H), 3.81 (q, J ¼ 6.9 Hz,

2H), 5.26 (s, 2H), 6.63 (s, 1H), 7.03 (dd, J₁¼ 2.4 Hz, J₂¼ 9.0 Hz, 1H),

7.19 (d, J ¼ 2.4 Hz, 1H), 7.41e7.43 (m, 3H), 7.55 (s, 1H), 7.88 (d,

J ¼ 9.0 Hz, 1H), 2 NH not detected. Anal. (C₁₉H₁₇ClN₂O₄) C, H, N.

8.1.14. 7-[(3-Chlorobenzyl)oxy]-4-(methylamino)-2H-chromen-2-

one (11a)

¹H NMR (200 MHz, CDCl₃)

d: 5.16 (s, 2H), 5.31 (s, 1H), 6.91 (d,

4-Chlorocoumarin derivative 8 (0.24 g, 0.75 mmol) was sus-

pended in a solution of methylamine 60% wt. in ethanol (8.0 mL) and

heated at 60 ^ꢀC in a closed Pyrex vessel for 7 h, with stirring. After

cooling at room temperature, the solvent and the excess amine were

removed under reduced pressure and the resulting crude solid was

puriﬁed through ﬂash chromatography (gradient eluent, ethyl ace-

tate in n-hexane 40% / 100%) to give pure compound 11a. White

crystals; yield: 46%; mp: 205e7 ^ꢀC (diethyl ether). ¹H NMR

J ¼ 2.4 Hz, 1H), 7.03 (dd, J₁¼ 2.4 Hz, J₂¼ 8.8 Hz, 1H), 7.10e7.60 (m,

9H), 7.96 (d, J ¼ 8.8 Hz, 1H). Anal. (C₂₂H₁₅ClO₄) C, H.

8.1.10. 4-Amino-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one (10a)

The mixture of 4-hydroxycoumarin 2c (1.2 g, 4.0 mmol),

ammonium acetate (2.0 g, 26 mmol) and DMSO (10 mL) was heated

at 120 ^ꢀC for 3 h, with stirring. The resulting solution was poured

into cold water (w300 mL) and the amorphous solid that separated

out was recovered by ﬁltration, washed with water and dried. This

solid (mixture of starting compound 2c and 4-aminoderivative 10a)

was ﬁnely powdered in a mortar, then stirred in hot CH₂Cl₂for

15 min. The resulting suspension was ﬁltered to give a good re-

covery (0.30 g) of starting compound 2c. The ﬁltrate was concen-

trated in vacuo, then puriﬁed through a silica gel column, eluting

with the mixture CH₂Cl₂/ethyl acetate (1:1, v/v). The eluate

collected afforded pure compound 10a. White crystals; yield: 48%;

(300 MHz, DMSO-d₆)

d

: 2.81 (d, J ¼ 4.7 Hz, 3H), 4.94 (s, 1H), 5.20 (s,

2H), 6.95 (d, J ¼ 2.5 Hz, 1H), 6.99 (dd, J₁¼ 2.5 Hz, J₂¼ 8.8 Hz, 1H),

7.39e7.42 (m, 3H), 7.53 (s, 1H), 7.68 (br q, J ¼ 4.7 Hz, 1H, dis. with

D₂O). 7.86 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₇H₁₄ClNO₃) C, H, N.

8.1.15. 7-[(3-Chlorobenzyl)oxy]-4-(ethylamino)-2H-chromen-2-one

(11b)

The mixture of 4-chlorocoumarin derivative

8 (0.24 g,

0.75 mmol), ethylamine hydrochloride (0.61 g, 7.5 mmol) and

triethylamine (2.0 mL) in ethanol (25 mL) was reﬂuxed for 2 h, with

stirring. The mixture was then evaporated to dryness in vacuo and

the residue was partitioned between aqueous 5% NaHCO₃and

CH₂Cl₂. The organic layer was collected and the aqueous phase was

further extracted twice with CH₂Cl₂. The combined extracts were

dried over anhydrous Na₂SO₄, concentrated in vacuo, and chro-

matographed on a silica gel column, eluting with the mixture

CH₂Cl₂/ethyl acetate (1:1, v/v) to give pure compound 11b. White

crystals; yield: 73%; mp: 183e4 ^ꢀC (ethyl acetate/petroleum ether).

mp: 248e250 ^ꢀC (ethanol). ¹H NMR (200 MHz, DMSO-d₆)

d: 5.09 (s,

1H), 5.24 (s, 2H), 6.88e7.08 (m, 2H), 7.23e7.52 (m, 5H, 2 protons dis.

with D₂O), 7.57 (s, 1H), 7.92 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₆H₁₂ClNO₃)

C, H, N.

8.1.11. N-{7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}

acetamide (10b)

In a closed Pyrex vessel equipped with a magnetic stirring bar,

10a (0.30 mmol, 0.091 g) was suspended in acetic anhydride

(2.0 mL). The reaction was exposed to microwave irradiation

(150 ^ꢀC, 30 min), cooled to room temperature and poured into

crushed ice. The resulting precipitate was ﬁltered, thus yielding the

desired product that was further puriﬁed by crystallization from

hot ethanol. White crystals; yield: 85%; mp: 139e141 ^ꢀC dec.

¹H NMR (200 MHz, CDCl₃)

d

: 1.39 (t, J ¼ 7.2 Hz, 3H), 1.80e2.30 (br s,

1H, dis. with D₂O), 3.34 (q, J ¼ 7.2 Hz, 2H), 5.11 (s, 2H), 5.25 (s, 1H),

6.80e6.97 (m, 2H), 7.24e7.55 (m, 5H). Anal. (C₁₈H₁₆ClNO₃) C, H, N.

8.1.16. 7-[(3-Chlorobenzyl)oxy]-4-(isopropylamino)-2H-chromen-

2-one (11c)

(ethanol). ¹H NMR (300 MHz, DMSO-d₆)

d: 2.31 (s, 3H), 5.25 (s, 2H),

6.61 (s, 1H), 7.01 (dd, J₁¼1.7 Hz, J₂¼ 8.8 Hz, 1H), 7.07 (d, J ¼ 8.8 Hz,

1H), 7.19 (d, J ¼ 1.7 Hz, 1H), 7.41e7.43 (m, 3H), 7.55 (s, 1H), 9.95 (br s,

1H, dis. with D₂O). Anal. (C₁₈H₁₄ClNO₄) C, H, N.

The mixture of 4-chlorocoumarin derivative 8 (0.24 g,

0.75 mmol), isopropylamine (3.0 mL) and CH₂Cl₂(25 mL) was

reﬂuxed for 5 h, with stirring. The mixture was then diluted with

CH₂Cl₂, transferred in a separatory funnel and washed with

aqueous 5% NaHCO₃. The organic solution was collected, dried over

anhydrous Na₂SO₄, and evaporated to dryness in vacuo to give an

oily residue which was chromatographed on a silica gel column,

eluting ﬁrst with CH₂Cl₂to remove some impurities, then with the

mixture CH₂Cl₂/ethyl acetate (1:1, v/v). The eluate collected affor-

ded pure compound 11c. White crystals; yield: 47%; mp: 180e2 ^ꢀC

8.1.12. Ethyl {7-[(3-chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}

carbamate (10c)

Compound 10a (0.30 mmol, 0.091 g) and anhydrous potassium

carbonate (0.30 mmol, 0.041 g) were reﬂuxed for 10 h under

magnetic stirring in 2.0 mL of ethyl chloroformate. The reaction was

cooled at room temperature, poured into crushed ice and ﬁltered.

The precipitate was puriﬁed on column chromatography (ethyl

acetate/n-hexane 2:1 v/v as eluent). White crystals; yield: 46%. ¹H

(ethyl acetate/petroleum ether). ¹H NMR (300 MHz, DMSO-d₆)

d:

1.21 (d, J ¼ 6.1 Hz, 6H), 3.76 (m, 1H), 5.02 (s, 1H), 5.20 (s, 2H), 6.93e

6.99 (m, 2H), 7.13 (d, J ¼ 7.4 Hz, 1H, dis. with D₂O), 7.39e7.43 (m,

3H), 7.53 (s, 1H), 8.05 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₉H₁₈ClNO₃) C, H, N.

NMR (300 MHz, CDCl₃)

2H), 5.11 (s, 2H), 6.50 (s, 1H), 6.88 (d, J ¼ 2.4 Hz, 1H), 6.94 (dd,

d

: 1.41 (t, J ¼ 7.2 Hz, 3H), 4.40 (q, J ¼ 7.2 Hz,

736

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

8.1.17. 4-Anilino-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one (11d)

4-Chlorocoumarin derivative 8 (0.096 g, 0.30 mmol) was sus-

pended in aniline (1.0 mL) and N,N-diisopropylethylamine (0.11 mL,

0.60 mmol) was added. The mixture was heated at 140 ^ꢀC for 48 h

and then partitioned between brine (50 mL) and diethyl ether

(30 mL). The organic layer was washed with brine (3 ꢃ 20 mL) and

then dried over Na₂SO₄. After removal of the solvent under vacuum,

the mixture was puriﬁed by ﬂash chromatography (gradient eluent,

methanol in dichloromethane 0% / 5%) yielding the desired

product 11d. Brown powder; yield: 53%, mp: 221e3 ^ꢀC. ¹H NMR

3.0 mmol; morpholine for 13k: 0.26 mL, 3.0 mmol) was added. The

reaction mixture was stirred at room temperature under an Argon

atmosphere for 3 h. The excess amine and the solvent were removed

under reduced pressure. The resulting crude oil was puriﬁed by ﬂash

chromatography (gradient eluent, ethyl acetate in n-hexane

40% / 100% for 13hei or ethyl acetate in n-hexane 0% / 50% for

13jek) yielding the desired compounds. The isolated free amines

13hei, corresponding to the ﬂuorescent TLC spots with lower R_f,

were transformed in the corresponding hydrochloride salts by using

a commercially available 4.0 N solution of HCl in 1,4-dioxane.

(300 MHz, DMSO-d₆)

d

: 5.16 (s, 1H), 5.25 (s, 2H), 7.03 (d, J ¼ 2.5 Hz,

1H), 7.08 (dd, J₁¼ 2.5 Hz, J₂¼ 8.8 Hz,1H), 7.27 (t, J ¼ 7.7 Hz,1H), 7.34

(d, J ¼ 7.7 Hz, 2H), 7.41e7.49 (m, 5H), 7.55 (s, 1H), 8.15 (d, J ¼ 8.8 Hz,

1H), 9.23 (s, 1H, dis. with D₂O). Anal. (C₂₂H₁₆ClNO₃) C, H, N.

8.1.21.1. 7-[(3-Chlorobenzyl)oxy]-4-[(propylamino)methyl]-2H-chro-

men-2-one hydrochloride (13h). White solid; yield: 59%; mp: 241e

3

^ꢀC. ¹H NMR (300 MHz, DMSO-d₆)

d

: 0.92 (t, J ¼ 7.4 Hz, 3H), 1.69

(sext, J ¼ 7.4 Hz, 2H), 2.99 (t, J ¼ 7.4 Hz, 2H), 4.42 (s, 2H), 5.27 (s, 2H),

6.51 (s, 1H), 7.11 (dd, J₁¼ 2.4 Hz, J₂¼ 9.1 Hz, 1H), 7.15 (d, J ¼ 2.5 Hz,

1H), 7.40e7.44 (m, 3H), 7.54 (s, 1H), 7.78 (d, J ¼ 9.1 Hz, 1H), 9.06 (br

s, 2H, dis. with D₂O). Anal. (C₂₀H₂₀ClNO₃$HCl) C, H, N.

8.1.18. 7-[(3-Chlorobenzyl)oxy]-4-ethyl-2H-chromen-2-one (13b)

Intermediate 12b [42] (1.9 g, 10 mmol) was added to an ethanol

solution of sodium ethoxide (10 mmol, 0.23 g of sodium) and the

resulting solution was evaporated to dryness in vacuo. To the solid

residue DMF (30 mL) and 3-chlorobenzyl chloride (1.6 g, 10 mmol)

were added and the mixture was heated at 100 ^ꢀC for 1 h with

stirring. The resulting solution was poured into cold water (400 mL)

so that the nearly pure compound 13b separated out as a whitish

solid that was recovered by ﬁltration, washed with water and dried.

White solid; yield: 83%; mp: 130e1 ^ꢀC (ethyl acetate/petroleum

8.1.21.2. 4-[(Butylamino)methyl]-7-[(3-chlorobenzyl)oxy]-2H-chro-

men-2-one hydrochloride (13i). White solid; yield: 43%; mp: 217.6e

219.4 ^ꢀC. ¹H NMR (300 MHz, DMSO-d₆)

d

: 0.90 (t, J ¼ 7.2 Hz, 3H),

1.35 (sext, J ¼ 7.2 Hz, 2H), 1.64 (quint, J ¼ 7.2 Hz, 2H), 3.03 (t,

J ¼ 7.2 Hz, 2H), 4.42 (s, 2H), 5.27 (s, 2H), 6.50 (s, 1H), 7.10e7.15 (m,

2H), 7.39e7.45 (m, 3H), 7.54 (s, 1H), 7.78 (d, J ¼ 8.8 Hz, 1H), 9.02 (br

s, 2H, dis. with D₂O) Anal. (C₂₁H₂₂ClNO₃$HCl) C, H, N.

ether). ¹H NMR (300 MHz, CDCl₃)

d

: 1.32 (t, J ¼ 7.4 Hz, 3H), 2.78 (q,

J ¼ 7.4 Hz, 2H), 5.10 (s, 2H), 6.16 (s, 1H), 6.87 (d, J ¼ 2.5 Hz, 1H), 6.93

(dd, J₁¼ 2.5 Hz, J₂¼ 8.8 Hz, 1H), 7.28e7.35 (m, 3H), 7.44 (s, 1H), 7.55

(d, J ¼ 8.8 Hz, 1H). Anal. (C₁₈H₁₅ClO₃) C, H.

8.1.21.3. 7-[(3-Chlorobenzyl)oxy]-4-(pyrrolidin-1-ylmethyl)-2H-

chromen-2-one (13j). White solid; yield: 79%; mp: 120e121.7 ^ꢀC. ¹H

NMR (300 MHz, DMSO-d₆) d: 1.68e1.72 (m, 4H), 2.50e2.52 (m, 4H),

8.1.19. 7-[(3-Chlorobenzyl)oxy]-4-(chloromethyl)-2H-chromen-2-

one (13c)

3.74 (s, 2H), 5.23 (s, 2H), 6.28 (s, 1H), 7.01 (dd, J₁¼ 2.5 Hz,

J₂¼ 8.8 Hz, 1H), 7.06 (d, J ¼ 2.5 Hz, 1H), 7.39e7.44 (m, 3H), 7.53 (s,

1H), 7.83 (d, J ¼ 8.8 Hz, 1H). Anal. (C₂₁H₂₀ClNO₃) C, H, N.

4-Chloromethylcoumarin 12c (2.1 g, 10 mmol) was dissolved in

dry DMF (35 mL) and then Cs₂CO₃(3.9 g, 12 mmol) was added fol-

lowed by 3-chlorobenzyl bromide (1.3 mL, 10 mmol) after 15 min of

stirring. The mixture was stirred at room temperature for 24 h and

thenpoured onto crushed ice. The resulting solid wasﬁltered, washed

several times with hot water and crystallized from CHCl₃/n-hexane

yielding the desired product. Yield: 87%. Spectroscopic and analytical

data are in full agreement with those reported in the literature [39].

8.1.21.4. 7-[(3-Chlorobenzyl)oxy]-4-(morpholin-4-ylmethyl)-2H-

chromen-2-one (13k). White solid; yield: 88%; mp: 140.8e142.6. ¹H

NMR (300 MHz, DMSO-d₆) d: 2.43e2.46 (m, 4H), 3.55e3.58 (m, 4H),

3.63 (s, 2H), 5.23 (s, 2H), 6.31 (s, 1H), 7.02 (dd, J₁¼ 2.5 Hz,

J₂¼ 8.8 Hz, 1H), 7.06 (d, J ¼ 2.5 Hz, 1H), 7.37e7.44 (m, 3H), 7.53 (s,

1H), 7.86 (d, J ¼ 8.8 Hz, 1H). Anal. (C₂₁H₂₀ClNO₄) C, H, N.

8.1.20. 7-[(3-Chlorobenzyl)oxy]-4-(hydroxymethyl)-2H-chromen-

2-one (13d)

8.1.22. 7-[(3-Bromobenzyl)oxy]-4-(chloromethyl)-2H-chromen-2-

one (13l)

7-Hydroxy-4-(hydroxymethyl)-2H-chromen-2-one 12d [43]

(0.38 g, 2.0 mmol) was dissolved in dry DMF (8.0 mL) followed by

the addition of Cs₂CO₃(0.65 g, 2.0 mmol). After stirring for 30 min,

3-chlorobenzyl bromide (0.64 mL, 2.0 mmol) was added. The

resulting mixture was stirred at room temperature for 48 h and the

solvent was evaporated under reduced pressure. The resulting solid

was partitioned between brine (80 mL) and chloroform. After

extraction with chloroform (3 ꢃ 40 mL), the organic phases were

collected, dried over Na₂SO₄and concentrated under vacuum.

Crystallization of the obtained crude yellow solid from ethyl acetate

furnished the desired compound. Yellow crystals; yield: 64%; mp:

4-Chloromethylcoumarin 12c (0.42 g, 2.0 mmol) was dissolved

in dry DMF (10 mL) and then Cs₂CO₃(0.65 g, 2.0 mmol) was added

followed by 3-bromobenzyl bromide (0.75 g, 3.0 mmol) after

15 min of stirring. Stirring was continued at room temperature for

24 h and then reaction mixture was poured onto crushed ice. After

extraction with ethyl acetate (3 ꢃ 120 mL), the organic phases were

collected, dried over Na₂SO₄and concentrated under vacuum. Flash

chromatographic separation (gradient eluent, ethyl acetate in n-

hexane 0% / 50%) afforded pure 13l. Whitish crystals; yield: 61%;

mp: 126.5e128 ^ꢀC (diethyl ether). ¹H NMR (300 MHz, DMSO-d₆)

d:

4.98 (s, 2H), 5.24 (s, 2H), 6.49 (s, 1H), 7.09 (dd, J₁¼ 2.5 Hz,

J₂¼ 8.8 Hz, 1H), 7.12 (d, J ¼ 2.5 Hz, 1H), 7.34e7.39 (m, 1H), 7.46e7.48

(m, 1H), 7.53e7.55 (m, 1H), 7.68 (s, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H). Anal

(C₁₇H₁₂BrClO₃) C, H.

185e6 ^ꢀC (ethyl acetate). ¹H NMR (300 MHz, DMSO-d₆)

d: 4.71 (d,

J ¼ 3.3 Hz, 2H), 5.23 (s, 2H), 5.59 (t, J ¼ 3.3 Hz, 1H, dis. with D₂O),

6.29 (s, 1H), 7.00 (dd, J₁¼ 2.6 Hz, J₂¼ 8.8 Hz, 1H), 7.07 (d, J ¼ 2.6 Hz,

1H), 7.35e7.43 (m, 3H), 7.53 (s, 1H), 7.61 (d, J ¼ 8.8 Hz, 1H). Anal.

(C₁₇H₁₃ClO₄) C, H.

8.1.23. 7-[(3-Bromobenzyl)oxy]-4-(hydroxymethyl)-2H-chromen-

2-one (13m)

8.1.21. General procedure for the synthesis of amino-compounds

13hek

Coumarin 13c (0.17 g, 0.50 mmol) was dissolved in THF (5.0 mL)

and the suitable amine (n-propylamine for 13h: 0.82 mL, 10 mmol;

n-butylamine for 13i: 0.98 mL,10 mmol; pyrrolidine for 13j: 0.25 mL,

4-Chloromethylcoumarin 13l (0.11 g, 0.30 mmol) was reﬂuxed

in water (50 mL) for 48 h. After cooling at room temperature, the

reaction mixture was extracted with chloroform (3 ꢃ 20 mL). The

organic layers were collected, dried over Na₂SO₄and concentrated

to dryness. The resulting crude solid was puriﬁed by ﬂash

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

737

chromatography, thus obtaining the desired alcohol 13m. White

solid; yield: 46%; mp: 184.8e186.2 ^ꢀC (diethyl ether). ¹H NMR

added followed by sodium acetate (0.010 g, 0.12 mmol). While

cooling at 0 ^ꢀC, pyridinium chlorochromate (0.65 g, 3.0 mmol) was

added in 3 portions. The mixture was slowly brought back to room

temperature and then stirred for 12 h. The mixture was ﬁltered over

a silica pad and then washed with chloroform. The solution was

concentrated under reduced pressure and the resulting solid was

puriﬁed by ﬂash chromatography. Pure aldehyde 16 was obtained

as a yellow solid. Yield: 46%. Spectroscopic and analytic data are in

agreement with those reported in the literature [43].

(300 MHz, DMSO-d₆)

d

: 4.71 (d, J ¼ 5.5 Hz, 2H), 5.23 (s, 2H), 5.60 (t,

J ¼ 5.5 Hz, 1H, dis. with D₂O), 6.29 (s, 1H), 6.98e7.03 (m, 1H), 7.08e

7.09 (m, 1H), 7.33e7.39 (m, 1H), 7.45e7.48 (m, 1H), 7.52e7.55 (m,

1H), 7.61 (d, J ¼ 9.1 Hz, 1H), 7.67 (s, 1H). Anal (C₁₇H₁₃BrO₄) C, H.

8.1.24. 7-[(3-Bromobenzyl)oxy]-4-[(methylamino)methyl]-2H-

chromen-2-one hydrochloride (13n)

4-Chloromethylcoumarin 13l (0.11 g, 0.30 mmol) was dissolved in

THF (1.0 mL) and the commercially available 2.0 N solution of

methylamine in THF (3.0 mL, 6.0 mmol) was added. The reaction

mixture was stirred at room temperature for 3 h. After the removal of

the excess amine and the solvent under reduced pressure, the

resulting crude oil was puriﬁed by ﬂash chromatography (gradient

eluent, ethyl acetate in n-hexane 40% / 100%) and the isolated free

amine, corresponding to the ﬂuorescent TLC spots with lower R_f, was

transformed in the corresponding hydrochloride salts by using a

commercially available HCl 4.0 N solution in 1,4-dioxane. White

8.1.28. 7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromene-4-

carbaldehyde oxime (17)

According to a literature method [51], a mixture of hydroxyl-

amine hydrochloride (0.076 g, 1.1 mol) and AcONa$3H₂O (0.15 g,

1.1 mmol) previously dissolved in water (5 mL) was added to the

solution of aldehyde 16 (0.31 g, 1.0 mmol) in hot ethanol (100 mL).

The resulting mixture was reﬂuxed for 30 min, then concentrated

in vacuo and poured into cold water (300 mL). The nearly pure

aldoxime 17 separated out as a whitish solid which was collected by

ﬁltration, washed with water and dried. Cream-coloured crystals;

quantitative yield; mp: 232e5 ^ꢀC (ethanol). ¹H NMR (300 MHz,

solid; yield: 66%; mp: 236e7 ^ꢀC. ¹H NMR (300 MHz, DMSO-d₆)

d:

2.68 (s, 3H), 4.42 (s, 2H), 5.26 (s, 2H), 6.46 (s,1H), 7.10 (dd, J₁¼ 2.5 Hz,

J₂¼ 8.8 Hz, 1H), 7.15 (d, J ¼ 2.5 Hz, 1H), 7.34e7.39 (m, 1H), 7.46e7.48

(m, 1H), 7.53e7.55 (m, 1H), 7.68 (s, 1H), 7.77 (d, J ¼ 8.8 Hz, 1H), 9.30

(br s, 2H, dis. with D₂O). Anal. (C₁₈H₁₆BrNO₃$HCl) C, H, N.

DMSO-d₆) d: 5.24 (s, 2H), 6.52 (s, 1H), 7.05e7.11 (m, 2H), 7.38e7.44

(m, 3H), 7.54 (s, 1H), 8.36 (d, J ¼ 9.2 Hz, 1H), 8.41 (s, 1H), 12.35 (s, 1H,

dis. with D₂O). Anal. (C₁₇H₁₂ClNO₄) C, H, N.

8.1.25. 7-[(3-Chlorobenzyl)oxy]-4-(1-hydroxyethyl)-2H-chromen-

2-one (14)

8.1.29. 7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromene-4-carbonitrile

(18)

The mixture of 13b (1.3 g, 4.0 mmol) and selenium dioxide

(0.67 g, 6.0 mmol) in xylene (30 mL) was heated at 140 ^ꢀC for 4 h,

with stirring. The mixture was ﬁltered to remove black selenium,

and the ﬁltrate was evaporated to dryness. The residue was dis-

solved in dichloromethane and chromatographed on a silica gel

column eluting with the same solvent to remove a mixture of

starting compound and 4-acetyl derivative by-product. The elution

was then pursued with the mixture dichloromethaneeethyl acetate

(1:1, v/v). The fraction collected, after removal of solvents, afforded

an oily residue, from which, after the addition of a small volume

diethyl ether, pure racemic compound 14 separated out as whitish

crystals. Yield: 43%; mp: 111e2 ^ꢀC (diisopropyl ether). ¹H NMR

Aldoxime 17 (0.26 g, 0.80 mmol) was suspended in acetic an-

hydride (10 mL) and the resulting mixture was then reﬂuxed

(140 ^ꢀC) with stirring overnight. The resulting solution was evap-

orated to dryness in vacuo and the solid residue was partitioned

between aqueous 5% NaHCO₃(100 mL) and dichloromethane

(100 mL); the aqueous phase was further extracted twice with

dichloromethane. The combined extracts were dried over anhy-

drous Na₂SO₄and evaporated to dryness, thus yielding the pure

nitrile 18. Whitish crystals; yield: 96%; mp: 180e1 ^ꢀC (ethyl acetate/

petroleum ether). ¹H NMR (300 MHz, DMSO-d₆)

d: 5.27 (s, 2H), 7.16

(dd, J₁¼ 2.5 Hz, J₂¼ 8.8 Hz, 1H), 7.19 (s, 1H), 7.21 (d, J ¼ 2.5 Hz, 1H),

7.39e7.44 (m, 3H), 7.54 (s, 1H), 7.66 (d, J ¼ 8.8 Hz, 1H). Anal.

(C₁₇H₁₀ClNO₃) C, H, N.

(300 MHz, DMSO-d₆)

d

: 1.37 (t, J ¼ 6.6 Hz, 3H), 5.07 (m, 1H), 5.23 (s,

2H), 5.58 (d, J ¼ 4.4 Hz, 1H. dis. with D₂O), 6.31 (s, 1H), 7.02 (dd,

J₁¼ 2.5 Hz, J₂¼ 8.8 Hz, 1H), 7.09 (d, J ¼ 2.5 Hz, 1H), 7.41e7.44 (m,

3H), 7.54 (s, 1H), 7.79 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₈H₁₅ClO₄) C, H.

8.1.30. Ethyl 7-[(3-chlorobenzyl)oxy]-2-oxo-2H-chromene-4-

carboxylate (20)

Ester 19³³(0.46 g, 2.0 mmol) was dissolved in dry DMF (8 mL)

and Cs₂CO₃(0.65 g, 2.0 mmol) was added. The mixture turned from

yellow to red in few minutes and then 3-chlorobenzyl bromide

(0.32 mL, 2.4 mmol) was added. After stirring at room temperature

for 24 h, the mixture was poured onto crushed ice and the resulting

solid was ﬁltered and washed with water. This precipitate was

treated with a small volume of diethyl ether and the resulting solid

was collected as the pure compound 20. Off-white solid; yield: 93%;

8.1.26. 4-Acetyl-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one (15)

A suspension of pyridinium chlorochromate (0.49 g, 2.3 mmol)

in 30 mL of CH₂Cl₂was slowly added to a solution of 14 (0.50 g,

1.5 mmol) in CH₂Cl₂(30 mL). This mixture was stirred at room

temperature for 2 h, then ﬁltered to remove a dark amorphous solid

which was washed with dichloromethane and discarded. The

organic phase was washed with abundant water in a separatory

funnel to remove the inorganic impurities, then dried over Na₂SO₄

and evaporated to dryness in vacuo. The residue was dissolved in

CH₂Cl₂and chromatographed on a silica gel column eluting with

the same solvent; the eluate collected, after removal of solvent,

afforded pure compound 15. Yellow crystals; yield: 46%; mp: 155e

mp: 133.5e135.6 ^ꢀC. ¹H NMR (300 MHz, DMSO-d₆)

d: 1.33 (t,

J ¼ 7.2 Hz, 3H), 4.35 (q, J ¼ 7.2 Hz, 2H), 5.25 (s, 2H), 6.69 (s, 1H), 7.08

(dd, J₁¼ 2.5 Hz, J₂¼ 8.8 Hz, 1H), 7.15 (d, J ¼ 2.5 Hz, 1H), 7.38e7.42

(m, 3H), 7.54 (s, 1H), 8.01 (d, J ¼ 8.8 Hz, 1H). Anal. (C₁₉H₁₅ClO₅) C, H.

6

^ꢀC (diisopropyl ether). ¹H NMR (300 MHz, DMSO-d₆)

d

: 2.61 (s,

8.1.31. 7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromene-4-carboxylic

acid (21)

3H), 5.24 (s, 2H), 6.82 (s, 1H), 7.04 (dd, J₁¼ 2.5 Hz, J₂¼ 8.8 Hz, 1H),

7.13 (d, J ¼ 2.5 Hz, 1H), 7.39e7.43 (m, 3H), 7.53 (s, 1H), 7.77 (d,

J ¼ 8.8 Hz, 1H). Anal. (C₁₈H₁₃ClO₄) C, H.

After complete dissolution of solid KOH (0.17 g, 3.0 mmol) in

boiling ethanol (7.5 mL), ester 20 (0.54 g, 1.5 mmol) was added and

the reﬂux was continued for 15 min. The mixture was slowly kept at

room temperature and the solvent was evaporated under reduced

pressure. The solid was diluted with water (50 mL) and the solution

was made acid (pH z 2) with 2.0 N HCl and then extracted with

ethyl acetate (3 ꢃ 30 mL). The organic layers were collected dried

8.1.27. 7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromene-4-

carbaldehyde (16)

Alcohol 13d (0.38 g, 1.2 mmol) was suspended in dichloro-

ꢀ

methane (10 mL) and 4 A powder molecular sieves (1.2 g) were

738

L. Pisani et al. / European Journal of Medicinal Chemistry 70 (2013) 723e739

over Na₂SO₄and concentrated to dryness yielding a yellow solid

that was washed with diethyl ether furnishing acid 21. Pale yellow

solid; mp: 159e161 ^ꢀC (dec.); yield: 89%. ¹H NMR (300 MHz, DMSO-

(250 nM), respectively. After a preincubation for 5 min with the

assayed compound dissolved in DMSO at a ﬁnal concentration of 5%

(v/v), kynuramine was added at a concentration equal to the cor-

d₆)

d

: 5.24 (s, 2H), 6.64 (s, 1H), 7.07 (dd, J₁¼ 2.5 Hz, J₂¼ 9.1 Hz, 1H),

responding K_Mvalue (90 mM for MAO-A and 60 mM for MAO-B).

7.13 (d, J ¼ 2.5 Hz, 1H), 7.39e7.44 (m, 3H), 7.54 (s, 1H), 8.08 (d,

J ¼ 9.1 Hz, 1H), OH not detected. Anal. (C₁₇H₁₁ClO₅) C, H.

Then the rate of formation of 4-hydroxyquinoline was monitored

at 314 nm for 5 min. Finally, IC₅₀values were determined by

nonlinear regression of MAO inhibition vs ꢄ log of the concentra-

tion plots, using the program Origin, version 6.0 (Microcal Software

Inc., Northampton, MA).

8.1.32. 7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromene-4-

carboxamide (22)

Derivative 21 (0.33 g, 1.0 mmol) was suspended in dry CH₂Cl₂

(6 mL) and thionyl chloride (2.0 mL) was added. The reaction

mixture was reﬂuxed for 2 h and then evaporated to dryness.

Concentrated aq. ammonia (5.0 mL) was added while cooling at 0 ^ꢀC.

The mixture was allowed to reach room temperature slowly and

then stirred for 6 h. The excess ammonia and water were removed in

vacuo. The resulting solid was washed several times with chloroform

and ﬁltered, giving pure amide 22. An additional amount of product

was obtained from chromatographic separation of the organic liquor

(eluent, 5% methanol in chloroform, v/v). Brown solid; yield: 52%;

8.3. Computational methods

The coumarin inhibitors were built from the LigPrep module

available in Maestro (vers. 9.2) starting from the reference

inhibitor (entry 13, 4-FCBC), that is the 4-formyl-7-

metachlorobenzyloxycoumarin, co-crystallized with hMAO-B

(pdb code: 2v60). As already reported [39], the spatial model of

the rMAO-B was constructed through homology modelling. Ac-

cording to a recent study of hMAO-B crystal structure, we

designated as ordered eight water molecules labelled as w1055,

w1159, w1166, w1171, w1206, w1224, w1309 and w1351 refer-

ring to the numbering reported in the cited hMAO-B X-ray

complex.

mp: >230 ^ꢀC. ¹H NMR (300 MHz, DMSO-d₆)

d: 5.24 (s, 2H), 6.36 (s,

1H), 7.05e7.08 (m, 1H), 7.11e7.13 (m, 1H), 7.38e7.42 (m, 3H), 7.54 (s,

1H), 7.72 (d, J ¼ 8.8 Hz,1H), 7.96 (s,1H, dis. with D₂O), 8.26 (s, 1H, dis.

with D₂O). Anal. (C₁₇H₁₂ClNO₄) C, H, N.

8.1.33. 7-[(3-Chlorobenzyl)oxy]-2H-chromen-2-one (23)

8.3.1. Docking simulations

Commercial 7-hydroxycoumarin (0.16 g, 1.0 mmol) and 3-

chlorobenzyl bromide (0.32 mL, 1.0 mmol) were reacted as

described for 13d. The workup rendered a crude that was puriﬁed

by crystallization. White crystals; yield: 64%; mp: 127e8 ^ꢀC

GOLD (vers. 5.1), a genetic algorithm-based software, was used

for the docking study selecting GoldScore as a ﬁtness function.

GoldScore is made up of four components that account for protein-

ligand binding energy: protein-ligand hydrogen bond energy

(external H-bond), protein-ligand van der Waals energy (external

vdw), ligand internal vdw energy (internal vdw), and ligand

torsional strain energy (internal torsion). Parameters used in the

ﬁtness function (hydrogen bond energies, atom radii and polariz-

abilities, torsion potentials, hydrogen bond directionalities, and so

forth) were taken from the GOLD parameter ﬁle. Docking runs on

MAO-B enzyme were performed through GOLD program selecting

GoldScore as a ﬁtness function. For each coumarin inhibitor, 10

(ethanol). ¹H NMR (300 MHz, CDCl₃)

d: 5.10 (s, 2H), 6.27 (d,

J ¼ 9.6 Hz, 1H), 6.87 (d, J ¼ 2.4 Hz, 1H), 6.91 (dd, J₁¼ 2.4 Hz,

J₂¼ 8.4 Hz, 1H), 7.32e7.34 (m, 3H), 7.39 (d, J ¼ 8.4 Hz, 1H), 7.44 (s,

1H), 7.64 (d, J ¼ 9.6 Hz, 1H). Anal. (C₁₆H₁₁ClO₃) C, H.

8.1.34. 7-[(3-Chlorobenzyl)oxy]-4-(triﬂuoromethyl)-2H-chromen-

2-one (24)

Commercially available 7-hydroxy-4-triﬂuoromethyl coumarin

(0.069 g, 0.30 mmol) was dissolved in dry DMF (2.0 mL) and then

60% NaH in mineral oil (0.012 g, 0.30 mmol) was added while

cooling at 0 ^ꢀC. The mixture was slowly warmed at room temper-

ature, then 3-chlorobenzyl bromide (0.040 mL, 0.30 mmol) was

added dropwise via syringe after 1 h and stirring was continued for

24 h at room temperature. After addition of crushed ice, the sus-

pension was treated with 2.0 N aq. solution of NaOH (5 mL). The

mixture was stirred for 1 h and the resulting precipitate was ﬁltered

and washed with water, yielding compound 24. White crystals;

yield: 84%, mp: 118.8e120.3 ^ꢀC (ethanol). ¹H NMR (300 MHz,

ꢀ

conformations were generated in a sphere of a 12 A radius centred

on phenolic oxygen atom of Y435. In our docking runs, the mo-

lecular scaffold of the best ranked solution of the 2v60 reference

ligand docked into the rMAO-B was set as physical constraint to

favour the occurrence of the known binding mode of mhalogeno-7-

benzyloxy substituents of coumarin inhibitors (together with a

distance constraint between the backbone oxygen atom if Ile164

and halogen atom of the inhibitor). Docking simulations towards

MAO-B were carried out by allowing torsions and ﬂexibility to Q206

and Y398. As already reported [39], eight ordered water molecules

were explicitly considered in all the docking runs.

DMSO-d₆)

d

: 5.27 (s, 2H), 6.85 (s, 1H), 7.13 (dd, J₁¼ 2.4 Hz,

J₂¼ 8.9 Hz, 1H), 7.23 (d, J ¼ 2.4 Hz, 1H), 7.40e7.45 (m, 3H), 7.54 (s,

1H), 7.61e7.64 (m, 1H). Anal. (C₁₇H₁₀ClF₃O₃) C, H.

Acknowledgements

8.2. Biological assays

The ﬁnancial support from MIUR, Italy (Grant PRIN

20085HR5JK_005), is kindly acknowledged.

MAO inhibitory activity of compounds in Table 1 was assessed

using a continuous spectrophotometric assay, monitoring the rate

of oxidation of the nonselective nonﬂuorescent MAO substrate

kynuramine to 4-hydroxyquinoline. Brieﬂy, male SpragueeDawley

rats (200e250 g) were sacriﬁced by decapitation. The brains were

immediately removed and washed in an ice-cold isotonic Na₂HPO₄/

KH₂PO₄buffer (pH 7.40) containing sucrose. A crude brain mito-

chondrial fraction was then prepared by differential centrifugation

[52] and stored at ꢄ40 ^ꢀC in an isotonic Na₂HPO₄/KH₂PO₄buffer

(pH 7.4) containing KCl. MAO-A and MAO-B activities in mito-

chondrial preparations (1 mg/mL) were assayed using as selective

and irreversible inhibitors clorgyline (250 nM) and (ꢄ)-L-deprenyl

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Article Doi

Fine molecular tuning at position 4 of 2H-chromen-2-one derivatives in the search of potent and selective monoamine oxidase B inhibitors

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