Discovery of a novel class of potent coumarin monoamine oxidase B inhibitors: Development and biopharmacological profiling of 7-[(3-chlorobenzyl) oxy]-4-[(methylamino)methyl]-2H-chromen-2-one methanesulfonate (NW-1772) as a highly potent, selective, reversible, and orally active monoamine oxidase B inhibitor

Article abstract of DOI:10.1021/jm9010127

In an effort to discover novel selective monoamine oxidase (MAO) B inhibitors with favorable physicochemical and pharmacokinetic profiles, 7-[(m-halogeno)benzyloxy]coumarins bearing properly selected polar substituents at position 4 were designed, synthesized, and evaluated as MAO inhibitors. Several compounds with MAO-B inhibitory activity in the nanomolar range and excellent MAO-B selectivity (selectivity index SI > 400) were identified. Structure-affinity relationships and docking simulations provided valuable insights into the enzyme-inhibitor binding interactions at position 4, which has been poorly explored. Furthermore, computational and experimental studies led to the identification and biopharmacological characterization of 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)-methyl]-2H-chromen-2-one methanesulfonate 22b (NW-1772) as an in vitro and in vivo potent and selective MAO-B inhibitor, with rapid blood-brain barrier penetration, short-acting and reversible inhibitory activity, slight inhibition of selected cytochrome P450s, and low in vitro toxicity. On the basis of this preliminary preclinical profile, inhibitor 22b might be viewed as a promising clinical candidate for the treatment of neurodegenerative diseases.

Full text of DOI:10.1021/jm9010127

J. Med. Chem. 2009, 52, 6685–6706 6685
DOI: 10.1021/jm9010127
Discovery of a Novel Class of Potent Coumarin Monoamine Oxidase B Inhibitors: Development and
Biopharmacological Profiling of 7-[(3-Chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-2-one
Methanesulfonate (NW-1772) as a Highly Potent, Selective, Reversible, and Orally Active Monoamine
Oxidase B Inhibitor
Leonardo Pisani,†,^ Giovanni Muncipinto,†,^ Teresa Fabiola Miscioscia,† Orazio Nicolotti,† Francesco Leonetti,†
Marco Catto,† Carla Caccia,§ Patricia Salvati,§ Ramon Soto-Otero,‡ Estefania Mendez-Alvarez,‡ Celine Passeleu, and
Angelo Carotti*,†
†Dipartimento Farmacochimico, Universitaꢀ degli Studi di Bari, Via Orabona 4, 70125-Bari, Italy, §Newron Pharmaceuticals Spa, Bresso (MI),
Italy, ‡Grupo de Neuroquꢁımica, Departamento de Bioquꢁımica y Biologꢁıa Molecular, Facultad de Medicina, Universidad de Santiago de
Compostela, Santiago de Compostela, Spain, and School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Switzerland.
^ L.P. and G.M. contributed equally to this work.
Received July 9, 2009
In an effort to discover novel selective monoamine oxidase (MAO) B inhibitors with favorable
physicochemical and pharmacokinetic profiles, 7-[(m-halogeno)benzyloxy]coumarins bearing properly
selected polar substituents at position 4 were designed, synthesized, and evaluated as MAO inhibitors.
Several compounds with MAO-B inhibitory activity in the nanomolar range and excellent MAO-B
selectivity (selectivity index SI > 400) were identified. Structure-affinity relationships and docking
simulations provided valuable insights into the enzyme-inhibitor binding interactions at position 4,
which has been poorly explored. Furthermore, computational and experimental studies led to the
identification and biopharmacological characterization of 7-[(3-chlorobenzyl)oxy]-4-[(methylamino)-
methyl]-2H-chromen-2-one methanesulfonate 22b (NW-1772) as an in vitro and in vivo potent and
selective MAO-B inhibitor, with rapid blood-brain barrier penetration, short-acting and reversible
inhibitory activity, slight inhibition of selected cytochrome P450s, and low in vitro toxicity. On the basis
of this preliminary preclinical profile, inhibitor 22b might be viewed as a promising clinical candidate for
the treatment of neurodegenerative diseases.
Introduction
interventions in neurological disorders. The lack of selective
inhibition, irreversible mechanism of action, severe side ef-
fects, e.g., hepatotoxicity and life-threatening hypertensive
crisis, associated with the first-generation of antidepressant
MAO-A inhibitors have stimulated further research aimed to
the discovery of novel, less toxic drugs.12,13 Several selective
MAO-A inhibitors14 acting as antidepressants (i.e., moclo-
bemide,15 brofaromine, clorgyline, and toloxatone; Chart 1),
and selective MAO-B inhibitors acting as anti-Parkinson
agents (i.e., lazabemide, selegiline, safinamide, and rasagiline;
Chart 1)16-18 have been discovered. Notably, rasagiline,
approved by the U.S. Food and Drug Administration
(FDA) as an anti-Parkinson drug, has been recently shown
to be the first neuroprotective disease modifying drug for
Parkinson’s disease (PD).19 In addition, selegiline has been
recently approved as transdermal once-a-day patch formula-
tion to treat major depressive disorders. The high doses of
selegiline used in such a formulation may be responsible for
MAO-A inhibition and, consequently, for the antidepressant
effect.20
Monoamine oxidase (MAO,a EC 1.4.3.4, amine-oxygen
oxidoreductase) is a membrane-bound flavoenzyme respon-
sible for the oxidative deamination of xenobiotic amines1 and
monoamine neurotransmitters such as serotonin (5-HT),
norepinephrine (NE), and dopamine (DA).2 Two distinct
enzymatic isoforms, named MAO-A and MAO-B, have been
identified; they differ in amino acid sequence, three-dimen-
sional structure, organ and tissue distribution, substrate
specificity, and sensitivity to inhibitors.3-9 Both enzymes
oxidatively deaminate DA, whereas MAO-A preferentially
deaminates 5-HT, epinephrine, and NE and is selectively
inhibited by clorgyline. MAO-B preferentially metabolizes
benzylamine and β-phenethylamine (PEA) and is selectively
inhibited by selegiline and rasagiline (Chart 1).10,11 The
involvement of MAO in the metabolism of key neurotrans-
mitters has made it an attractive target for pharmacological
*To whom correspondence should be addressed. Phone: þ39-080-
5442782. Fax: þ39-080-5442230. E-mail: carotti@farmchim.uniba.it.
a Abbreviations: AD, Alzheimer’s disease; FAD, flavin adenine
dinucleotide; HDM, hexadecane membrane; MAO, monoamine oxi-
dase; MAO-A, monoamine oxidase A; MAO-B, monoamine oxidase B;
PAMPA, parallel artificial membrane permeability assay; PD, Parkin-
son’s disease; PDB, Protein Data Bank; PLS-DA, partial least-squares
discriminant analysis; PSA, polar surface area; SAFIR, structure-affi-
nity relationships; SSR, structure-selectivity relationships.
The MAO-B isoform is predominant in the human brain,21
and its level increases significantly in aging-related neurode-
generative diseases (NDs).22-24 As a consequence, neurotrans-
mitters levels are lowered and oxidative stress25-27 may be
induced by highly reactive hydroxyl radicals formed in the
reaction between hydrogen peroxide, produced during amine
r
2009 American Chemical Society
Published on Web 10/07/2009
pubs.acs.org/jmc

6686 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
Chart 1. Chemical Structures of Nonselective and Selective
MAO-A (A) and MAO-B (B) Inhibitors
oxidation, and iron ion (Fenton’s reaction).28 Selective MAO-B
inhibitors have therefore attracted new interest also as neuro-
protective agents in the elderly and as potential drugs in the
therapy of Alzheimer’s disease (AD).29,30 Indeed a pronounced
neuroprotective effect of selegiline and rasagiline has been
claimed31,32 but clinical trials of selegiline in combination with
antioxidants or acetylcholinesterase inhibitors and alone in AD
have shown contrasting results.33-35
Although significant advances in the design of selective
MAO inhibitors (MAOIs) have been achieved in the past
2 decades, a veritable breakthrough in the field occurred only
a few years ago with the publication of the X-ray crystal
structures of human MAO-B (hMAO-B)5,36 and, to a lesser
extent, rat MAO-A (rMAO-A).8 The high-resolution 3D
structure of hMAO-B6 and, more recently, of hMAO-A37
bound to selective, reversible, and irreversible inhibitors finally
paved the way to the structure-based design of new and
selective modulators of MAO activity. The wealth of informa-
tion on structure and recently published methods for a medium
throughput screening of MAOIs on cloned hMAOs38,39 might
be particularly helpful for a more efficient and faster discovery
of new MAOIs.
As a part of our ongoing research in this field,40-47 herein we
report the design, synthesis, and biochemical evaluation of a
new series of coumarins that maintained the potent and selective
MAO-B inhibition found for this class of compounds48-50 but
that exhibited more appropriate physicochemical and pharma-
cokinetic properties for clinical applications as novel therapeu-
tics of neurological disorders. In fact, most of the potent and
selective MAO-B coumarin inhibitors studied so far have
generally displayed too high lipophilicity and poor aqueous
solubility, and this might strongly limit their investigations even
at the level of experimental preclinical profiling. Guided by the
wealth of information on structure-affinity and structure-
selectivity relationships (SAFIRs and SSRs, respectively) deve-
loped for coumarinic MAO inhibitors,45-47,51-53 we designed a
series of novel MAO-B inhibitors, maintaining the benzyloxy
and m-fluorobenzyloxy or m-chlorobenzyloxy substituents at
position 7 and introducing adequate polar moieties, that is,
cyano, amido, amidoamino, and aliphatic amino groups, at
position 4. These limited, albeit crucial, structural modifica-
tions, if tolerated, would have enabled us to identify new
MAO-B inhibitors with an improved pharmacokinetic profile
and a higher druggability.
Scheme 1. Synthesis of 7-Hydroxycoumarin Intermediates
1a-da
a Reagents and conditions: (method A) H2SO4 conc (cat.), 120 ꢀC, 1 h;
(method B) H2SO4 conc (large excess), 0 ꢀC, 2 h; (method C) BF3 diethyl
3
etherate, 90 ꢀC, 24 h; (i) POCl3, reflux, 4 h.
Chart 2. Starting 4-Substituted-7-hydroxycoumarin Derivatives
1a,b,d,e and 5
The results of this investigation, which led to the discovery
of an in vitro and in vivo very potent, reversible, and selective
MAO-B inhibitor exhibiting appropriate pharmacologic
Scheme 2. Synthesis of Compound 3 and Aminoacidic Coumarin Derivatives 4a-h (Table 1)a
a Reagents and conditions: (i) R3C6H4CH2Br, K2CO3, EtOH, reflux, 2 h; (ii) piperonyl alcohol, DIAD, PPh3, dry THF, room temperature, 18 h;
(iii) R2NHCHR1CONH2 HCl, DIEA, dry DMF, 80 ꢀC, 5 h.
3

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6687
Scheme 3. Synthesis of Amidoaminocoumarin Derivatives 8a-c (Table 2)a
a Reagents and conditions: (i) NH2(CH2)nNHBoc, DCC, HOBt, dry DMF, room temperature, 5 h; (ii) R3C6H4CH2Br, dry K2CO3, absolute EtOH,
reflux, 0.5 h; (iii) TFA, CH2Cl2, room temperature, 20 min.
Scheme 4. Solid-Phase Synthesis of Primary Amides 11a-d and 13a,b (Table 2)a
a Reagents and conditions: (i) DIC, HOBt, dry DMF, room temperature, 15 h; (ii) DIEA, R3C6H4CH2Br or 17, dry DMF, 70 ꢀC, 2.5 h; (iii) KHMDS,
CH3I, dry DMF, room temperature, 4 h; (iv) CH3ONa, dry THF, room temperature, 4 h; (v) TFA/H2O/TES, 9.5/0.25/0.25, room temperature, 1 h.
features to be progressed to clinical trials, will be presented
and discussed in this paper, along with new SAFIRs, SSRs,
and docking studies that provided a consistent picture of the
main binding interactions modulating the MAO inhibitory
potency and MAO-B isoform selectivity of this new class of
coumarins. The data discussed in the present paper have been
anticipated in part in a patent48 and at the ACS-EFMC
meeting of medicinal chemistry.54
Chemistry
The synthesis of the novel coumarin derivatives was per-
formed according to the reaction pathways illustrated in

6688 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
Schemes 1-10. The starting 7-hydroxycoumarin intermedi-
ates 1a,b,d,e and 5 depicted in Chart 2 were obtained through
the well-known von Pechmann reaction55 with slight modifi-
cations56,57 depending on the stability and reactivity of the
β-dicarbonyl reagent used (Scheme 1).
Scheme 5. Synthesis of Benzoate 17a
Chlorination of derivative 1c in refluxing phosphorus oxy-
chlorideyielded regioselectively coumarin1d. Thebenzylation
of 1b,d,e with the appropriate benzyl bromide furnished target
compound 3 and intermediate chlorides 2a-e (Scheme 2).
Intermediate piperonyl chloride 2f was prepared through a
Mitsunobu reaction58 starting from 7-hydroxycoumarin 1b
and piperonyl alcohol in the presence of diisopropyl azodi-
carboxylate (DIAD) and triphenylphospine (PPh3). Chlorides
2a-f were reacted with suitable, commercially available (S)-
R-aminoamides, affording the target coumarins 4a-h
(Scheme 2).
DCC-HOBt mediated coupling of commercially available
acid 5 and mono-Boc-protected hydrazine or ethylenediamine
represented the first step for the synthesis of compounds 8a-c
(Scheme 3). The resulting amides 6a,b were benzylated at the
7-OH and then reacted with TFA in CH2Cl2 to remove the
Boc protecting group. The selective benzylation of the
7-OH was problematic because of competitive alkylations at
the acidic methylenic group at position 4 and at the amidic
nitrogen of low sterically hindered secondary amides. The
elimination of these undesired side reactions was accom-
plished by applying a solid-phase protocol to prepare primary
amides 11a-d and the corresponding C-methylated analogues
13a,b (Scheme 4). 7-Hydroxycoumarinacetic acid 5 was
loaded onto a Rink amide, followed by a benzylation reaction
that proceeded cleanly, without the formation of byproduct,
yielding the derivatized resins 10a-c. Regioselective methyla-
tion of the 4-CH2 group was obtained using a strong and bulky
base (potassium hexamethyldisilazane, KHMDS) and methyl
iodide to afford resins 12a,b. The target amides 11a-d and
a Reagents and conditions: (i) TEA, dry THF, room temperature, 0.5
h; (ii) NBS, AIBN, CCl4, reflux, 1 h.
Scheme 6. Synthesis of 7-Pyridylmethoxycoumarin Derivatives
19a,b (Table 2)a
a Reagents and conditions: (i) aq NH3, sealed tube, 90 ꢀC, 60 h; (ii) 3- or
4-pyridinylmethanol, PBu3, ADDP, dry THF, room temperature, 18 h.
Scheme 7. Synthesis of Amidocoumarin Derivatives 21a-g (Table 2)a
a Reagents and conditions: (i) ADDP, PPh3, R3C6H4CH2OH, dry THF, room temperature, 18 h; (ii) R1R2NH, dry THF, 90 ꢀC, sealed tube, 24-60 h.
Scheme 8. Synthesis of Aminocoumarin Derivatives 22a-k and 24a,b (Table 3)a
a Reagents and conditions: (i) R1R2NH, dry THF, 50 ꢀC, 5 h; (ii) EtNH2 70% water solution, room temperature, 3 h; (iii) isopropylamine, reflux, 2 h;
(iv) N-methylbenzylamine or benzylamine, K2CO3, EtOH, reflux, 2-5 h; (v) sodium azide, EtOH, reflux, 2 h; (vi) SnCl2, MeOH, room temperature, 3 h.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6689
Scheme 9. Synthesis of 4-Aminoethylcoumarin Derivative 26 (Table 3)a
a Reagents and conditions: (i) TFAA, dry pyridine and dioxane, 0 ꢀC to room temperature, 10 min; (ii) NaBH4/CoCl2, MeOH, room temperature, 1 h.
Scheme 10. Synthesis of 4-Aminoethylcoumarin Derivatives 30a,b (Table 3)a
a Reagents and conditions: (i) BH3 THF complex, dry THF, room temperature, 6 h; (ii) R3C6H4CH2Br, K2CO3, EtOH, reflux, 45 min; (iii) CBr4,
3
PPh3, dry CH2Cl2, room temperature, 1 h; (iv) R1R2NH, K2CO3, KI, dry THF, 50 ꢀC, 15 h.
13a,b were obtained upon cleavage from the resin with a TFA/
H2O mixture containing triethylsilane as proton scavenger.
The preparation of amide 11d required different additional
synthetic steps consisting of the removal of the benzoyl
protecting group with sodium methoxide (from resin 10d)
and of the separated synthesis of benzyl bromide 17
(Scheme 5) that began with the benzoylation of m-cresol
followed by the bromination of ester 16 with NBS and AIBN.
The ammonolysis of ester 1a with aqueous NH3 in a sealed
tube afforded amide 18 in excellent yields. The introduction
of 3- and 4-pyridinylmethoxy moiety at position 7 (com-
pounds 19a,b; Scheme 6) was performed following a standard
Mitsunobu protocol with tributylphosphine (PBu3) and
1,10-(azodicarbonyl)dipiperidine (ADDP), thus avoiding the
use of relatively unstable pyridylmethyl bromides.
many years ago.61 The reduction of nitrile 25b with sodium
borohydride and CoCl2 afforded the desired primary amine 26.
An alternative synthetic pathway (Scheme 10) was followed to
access secondary and tertiary amines.
The carboxylic group of compound 5 was selectively
reduced by the BH3-THF complex, and the resulting dihy-
droxylated derivative 27 was benzylated at the phenolic OH
in the presence of potassium carbonate and a catalytic
amount of KI in refluxing ethanol. The alcohols 28a,b under-
went an Appel-type mild bromination with CBr4 and PPh3.
The subsequent nucleophilic displacement of the bromide
anion with the appropriate amine yielded the final amines
30a,b.
Physicochemical Assays
As described in Scheme 7, preliminary benzylation reaction
of 7-hydroxycoumarin 1a under Mitsunobu conditions af-
forded the esters 20a,b that were reacted with a suitable
aliphatic amine to yield secondary and tertiary amides 21a-g.
AsoutlinedinScheme 8, the substituted amines22a-k were
easily obtained by nucleophilic substitution of chlorides 2c-f
with the appropriate amines. Primary amines 24a,b were
synthesized from azides 23a,b obtained by refluxing chlorides
2c,d with sodium azide in ethanol, as illustrated in Scheme 8.
In this case, despite the tedious and potentially toxic purifica-
tion procedure, SnCl2 proved to be the most effective method
for reducing azides compared to the traditional Staudinger
reduction with PPh3/H2O,59 Sn(II)/thiophenol/triethyla-
mine60 reducing system, or catalytic hydrogenation.
The elongation of the aliphatic bridge linking the aminic
moiety at position 4 to the coumarin ring was pursued accord-
ing to the reaction pathway illustrated in Scheme 9. Amides
11a,b were converted into the corresponding nitriles 25a,b
upon treatment with trifluoroacetic anhydride in pyridine
according to an efficient method developed by some of us
log P, pKa, and Aqueous Solubility Measurements. The pKa
and octanol-water partition coefficient were measured by
potentiometric methods62 with a Sirius GLpKa instrument.
Aqueous solubility was determined with the DMSO-buffer
dilution turbidimetric method63 according to a reported
procedure.64
PAMPA Permeation Assay. Selected MAO inhibitors
were analyzed using PAMPA (parallel artificial membrane
permeability assay), a method recently developed for
the rapid determination of passive transport.65 In PAMPA,
an artificial liquid membrane is used to separate two
compartments, one containing a buffer solution of com-
pounds to be tested (defined as the donor compartment)
and the other an initial fresh buffer solution (defined as the
acceptor compartment) assembled in a “sandwich-like”
configuration. The permeation of tested compounds
through the artificial membrane is determined after a fixed
incubation time by disassembling the “sandwich” and
measuring sample concentrations in the donor and acceptor
compartments.

6690 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
Chart 3. General Structures of Novel 4,7-Disubstituted Coumarin MAOIs (Tables 1-4)
Table 1. MAO Inhibitory Activity of (S)-Aminoamidocoumarin
Derivatives 4a-h
CYP3A4) was measured in distinct assays by using specific
substrates that become fluorescent upon CYP-promoted meta-
bolism (Gentest Kit assay, BD Biosciences, Bedford, MA).
In Vitro Cell Viability Assay. The SHSY-5Y continuous
cell line from a human neuroblastoma was used and cell
viability measured by the colorimetric MTS assay.69
compd
n
R1
CH3
CH3
R2
R3 MAO-Aa MAO-Ba
SIb
4a
4b
4c
4d
4e
4f
0
0
0
0
0
1
1
1
H
H
H
Cl
H
Cl
Cl
H
H
F
100
0.68
0.21
16.1
9.60
2.40
1.80
0.38
0.85
147
35%
99.8
5%
-CH2CH2CH2-
-CH2CH2CH2-
CH2OH
CH3
6
H
>100
22.7
35
>42
13
Computational and Molecular Modeling Studies
H
Building and optimization of small molecules and MAO
homology modeling were performed with Sybyl (Tripos, St.
Louis, MO) and Modeller (DBSPC, University of California;
San Francisco, San Francisco, CA) software, respectively. Dock-
ing simulations were carried using the GOLD program
(Cambridge Crystallographic Data Centre, Cambridge, U.K.).
Polar surface area (PSA), a molecular descriptor largely used
to roughly estimate oral bioavailability of potential drugs,70 and
LogBBB, a parameter related to the drug capability to cross the
blood brain barrier (BBB)71 were calculated by using Volsurfþ
(Molecular Discovery, Perugia, Italy).
4g
4h
-CH2CH2CH2-
-CH2CH2CH2-
92
>117
>100
a IC50 (μM) or % inhibition at 10 μM. b SI = [IC50 MAO-A
(μM)]/[IC50 MAO-B (μM)].
The HDM-PAMPA (hexadecane membrane assay target-
ing GI track) assay developed by Wohnsland and Faller,65
making use of an artificial liquid membrane composed of
hexadecane supported on polycarbonate filters for the predic-
tion of passive transcellular permeability, was used as a low
cost and efficient alternative to the Caco-2 cell culture model.66
Results and Discussion
Biological Assays
4-Methyl-7-(m-chlorobenzyloxy)coumarin 3 was designed
and synthesized as the compound bearing the simplest hydro-
phobic substituent at position 4. It was considered as an
appropriate reference compound to which the biochemical,
physicochemical, and pharmacokinetic properties of the
newly designed 4,7-disubstituted coumarins, bearing polar
groups at position 4, were to be compared.
Compound 3 exhibited high in vitro MAO-B affinity and a
very low affinity to MAO-A (IC50 of 0.007 and 5.2 μM,
respectively). Its estimated lipophilicity and aqueous solubi-
lity indicated a high octanol-water partition coefficient
(P; log P = 4.73, from Bioloom-BioByte Corp., Claremont,
CA) and a poor aqueous solubility (S = 2.63 ꢀ 10-5 M,
from ACD, version 12, Advanced Chemistry Development,
Toronto, Canada).
In Vitro MAO Inhibition. Rat brain mitochondria were
used as the source for the two MAO isoforms. MAO enzy-
matic activities were assessed with a radioenzymatic assay
using 14C-serotonin (5-HT) and 14C-phenylethylamine
(PEA) as selective radiosubstrates for MAO-A and MAO-
B, respectively, according to a well consolidated procedure.67
For compound 22b the MAO-B radioenzymatic assay was
performed also in human platelet rich plasma (PRP).68
In Vitro MAO B Reversibility Inhibition Studies. The
reversible nature of MAO-B inhibition was assessed by
evaluating the enzymatic activity upon different incubation
time experiments. Time-dependent association kinetics were
measured as the IC50 values after 0 and 30 min enzyme-
inhibitor incubation in the assay medium.
Ex Vivo MAO Inhibition. Mice were treated with the test
compound at different oral and intraperitoneal concentra-
tions and sacrificed at different time intervals. The brains
were removed, and crude homogenates were prepared in
0.1 M phosphate buffer, pH 7.40. Ex vivo MAO-A and
MAO-B enzymatic activities were assessed according to the
radioenzymatic assay described above.
In Vitro Human Recombinant Cytocrome P450 Isoform
Assay. Inhibition of six key cytochrome P450 isoforms
(CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and
The chemical structures of the newly designed series of
4,7-disubstituted coumarins are shown in Chart 3, whereas
theirMAO-A and MAO-B affinitiesand selectivity indices are
listed in Tables 1-4.
Inhibition data of both MAO isoforms are reported as
IC50 (μM) or as the percentage of inhibition at the indicated
concentration for low-active inhibitors. Selectivity was
expressed as selectivity index (SI), which is the ratio of the
IC50 of MAO-A to the IC50 of MAO-B. For an immediate and
more efficient analysis of SAFIRs and SSRs, inhibition data

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6691
Table 2. MAO Inhibitory Activity of Amidocoumarin Derivatives
8a-c, 11a-d, 13a,b, 19a,b, and 21a-g
compd R1
R2
R4 R3
X
Y
MAO-Aa MAO-Ba SIb
8a
8b
H
H
H
H
H
H
H
H
H
NH2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH CH 1.44
0.040 36
0.090 28
>10 nd
Cl CH CH 2.50
Cl CH CH >100
8c
(CH2)2NH2
11a
11b
11c
11d
19a
19b
H
H
H
H
H
H
H
CH CH 8.0
Cl CH CH 26
CH CH 70
OH CH CH 4.27
0.20
0.030 867
40
F
0.050 1400
1.0
4
H
H
H
CH N
CH 26.5
CH CH 2.80
21.0
1.30
2.0
16
13
N
21a Me H
21b Me H
21c Bu H
21d Bn H
21e Me Me
21f Me Me
21g Bu Me
0.015 187
0.024 21
>10 nd
Cl CH CH 0.50
Cl CH CH >100
Cl CH CH >100
5.0
0.40
>20
58
H
CH CH 23.1
Cl CH CH >100
Cl CH CH >100
0.040 >2400
>10 nd
Figure 1. Square plot of rMAO affinity and selectivity. The differ-
ent classes of coumarin inhibitors in Tables 1-4 and reference
compound 3 are indicated with red, blue, green, orange, and black
circles, respectively. Bottom-right corner contains potent and highly
selective rMAO-B inhibitors. The most potent and selective MAO-
B inhibitors are highlighted by black labels along with the only
nonselective inhibitor 25a.
13ac
13bc
H
H
H
H
Me H CH CH 6.26
Me F CH CH 2.0
0.10
0.10
63
20
a IC50 (μM). b SI = [IC50 MAO-A (μM)]/[IC50 MAO-B (μM)].
c Tested as racemate.
which is a potent and selective MAO-B inhibitor, was plotted
with an estimated pIC50 of MAO-A equal to 3.50.
Table 3. MAO Inhibitory Activity of Aminocoumarin Derivatives
22a-k, 24a,b, 26, and 30a,b
compd
n
R1
R2
R3 MAO-Aa MAO-Ba
SIb
Compounds with identical affinities at both isoenzymes lie
on the bisector (y = x) of the graph, whereas highly selective
MAO-B inhibitors lie well below the bisector, the distance of
their pIC50 values from the bisector being a direct measure of
their degree of selectivity. A line traced at two pIC50 unit
distance below the bisector enables the straightforward loca-
tion of inhibitors with a selectivity index-affinity ratio (SI)
higher than 100 (i.e., ΔpIC50 > 2).
At a first glance, the plot indicates that very potent and
selective MAO-B inhibitors were discovered and that no
apparent relationship exists between MAO-A and MAO-B
inhibitory affinities. Only the cyano derivative 25a lies on the
bisector, exhibiting nearly equal affinity to both enzymes.
MAO-A affinity was generallyvery low, and onlyina very few
cases, i.e., for the two cyanomethyl derivatives 25a,b and the
N-methlycarboxamidomethyl derivative 21b, did affinities
reach the submicromolar range.
Inhibitors endowed with both high MAO-B affinity and
selectivity (i.e., both high pIC50 and SI values) are located at
the bottom of the right-hand corner of the plot and belong
mostly to the aminic and the amidic series (Tables 2 and 3,
respectively). The most interesting compounds in terms of
MAO-B affinity and selectivity, e.g., amides 22a-d and
amines 11b,c, are highlighted in the plot.
A comparison of the MAO-B inhibition of 4-CH2X-7-
(m-chlorobenzyloxy)coumarins (Tables 1-4) revealed the
following order of potency 24b g 25b > 11b > 8b > 4b >
26, suggesting that the simple cyano, primary amino, and
amido groups (X = CN, NH2, and CONH2, respectively) are
highly preferred polar substituents at position 4 for an effi-
cient binding to MAO-B.
22a
22bc
22cc
22d
22ec
22fc
22g
22h
22i
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
CH3
H
H
Cl
F
15.4
5.94
13.5
5.40
22.6
91.0
>100
100
0.028
0.013
0.018
0.020
0.10
3.80
3.60
1.80
1.12
0.51
8.0
550
CH3
CH3
CH3
CH2CH3
CH(CH3)2
Bn
H
457
750
270
226
24
H
H
e
H
F
H
F
H
CH3
Cl
H
>28
56
CH3
CH3
CH3
CH3
H
CH3 Cl
50.8
>100
>100
4.40
2.0
45
22j
CH3
Bn
H
F
>196
>13
210
133
127
247
56
22k
24a
24b
26d
Cl
H
Cl
Cl
Cl
H
0.021
0.015
0.25
0.30
0.46
H
H
H
H
31.8
74.0
25.9
30a
30b
CH3
CH3
H
CH3
a IC50 (μM). b SI = [IC50 MAO-A (μM)]/[IC50 MAO-B (μM)].
=
e
c Tested as mesylate. d Tested as hydrochloride. Rmeta-para
-OCH2O-.
Table 4. MAO Inhibitory Activity of 4-Cyanomethylcoumarin Deri-
vatives 25a,b
compd
R3
MAO-Aa
MAO-Ba
SIb
25a
25b
H
Cl
0.20
0.47
0.23
0.016
0.9
29
a IC50 (μM). b SI = [IC50 MAO-A (μM)]/[IC50 MAO-B (μM)].
are presented also in Figure 1 as a plot of pIC50 of MAO-B
(x-axis) versus pIC50 of MAO-A (y-axis) using the same scale
and rangefor bothaxes(square plot). For immediatelocation,
the different classes of coumarin inhibitors, i.e., aminoamides
(Table 1), carboxyhydrazides and amides (Table 2), amines
(Table 3), nitriles (Table 4), and the reference compound 3 are
indicated in the plot with differently colored circles. Unfortu-
nately, the low MAO-A affinity, often associated with a
limited solubility in the aqueous assay medium, did not permit
the measurement of IC50 (IC50 > 100 μM) for some com-
pounds, and therefore, they are not shown in the plot. To
avoid the loss of an important data point, compound 21f,
A similar comparison of the MAO-B affinity of differently
meta-substituted benzyloxy derivatives revealed that inhibi-
tors bearing m-chloro and -fluoro substituents were generally
more active than the corresponding benzyl-unsubstituted par-
ent compounds. Moreover, the replacement of the 7-benzyloxy
group with the 3- and 4-pyridylmethoxy isosteric substituents
led to a significant decrease of affinity (compare 11a vs 19a and
19b). These results are in full agreement with our previous

6692 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
findings and confirm that the lipophilicity of the substituent at
position 7 plays a crucial role in selective and efficient binding
to MAO-B.45,46,72
The most salient results emerging from a careful evaluation
of the MAO-B affinity and selectivity variation within the
different classes of coumarin derivatives reported in
Tables 1-3 can be summarized as follows.
A dramatic reduction of MAO-B affinity, in comparison
with reference compound 3, was observed for all the synthe-
sized alanyl-, prolin-, and serin-amide derivatives 4 (Table 1)
bearing the R-aminoamido moiety directly linked to the
coumarin ring (i.e., 4a-e) or bridged to it through a methy-
lenic unit (i.e., 4f-h). The less active MAO-B inhibitors were
the more rigid prolinamide derivatives 4c,d, whereas alanina-
mide derivatives 4a,b and the more conformationally flexible
prolinamide derivatives 4g,h exhibited submicromolar MAO-
B affinities. The SIs ranged from 6 to 147, and no clear SSR
emerged from their analysis.
derivatives (Table 2). The finding that the most hindered
N,N-dialkylamine (22k) and N,N-dialkylcarboxamide (21g)
derivatives yielded the lowest MAO-B affinity (IC50 of 8
and>10 μM) of the entire series of the two classes of examined
inhibitors in Tables 2 and 3 might lend support to this
hypothesis.
The short elongation of the aliphatic chain at position 4 of
7-m-chlorobenzyloxy derivatives 24b and 22b afforded com-
pounds 26 and 30a, respectively, exhibiting significantly lower
MAO-B affinities.
Molecular Modeling Studies. The SAFIRs and SSRs dis-
cussed previously provided useful information on the main
molecular determinants of the inhibitor potency and selec-
tivity; however, a clear picture of the main interactions
taking place at the binding sites of the two rat isoenzymes
was still missing. A modeling study was therefore carried out
through homology building and docking simulations for the
purpose of (i) gaining insights on the nature and spatial
location of the key interactions of the 4-substituents mod-
ulating the MAO-B affinity, (ii) explaining the excellent
MAO-B selectivity observed for some classes of examined
coumarins, and (iii) confirming, complementing, and better
interpreting the results of previous studies conducted by our
group on different sets of coumarin derivatives.45,46
Analysis of crystal structures of hMAO-B in complex with
several inhibitors6,36,56 revealed that the binding site consists
of two cavities, the “substrate cavity” located near the FAD
cofactor and the “entrance cavity”, connected to the protein
surface. The two cavities are separated by residues Tyr326,
Ile199, Leu171 and Phe168, with Ile199 and Tyr326 behaving
as gate-keeper residues. However, our attention was also
drawn by other, apparently less important, structural fea-
tures, i.e., the cocrystallized water molecules embedded in
the reported X-ray complexes of hMAO-B. It is known that
water molecules may become trapped in the protein crystal
structure and, in addition, a number of them may contribute
to the stabilization of the protein structure or of the pro-
tein-ligand complex through the formation of a network of
hydrogen bonds bridging the ligand to specific binding site
residues.73,74 These structural water molecules are often con-
served in crystallographic complexes of different ligands with
the same macromolecular ligate and may play an important
role in docking simulations and virtual screening.75,76 There-
fore, the detection of water molecules assuming a structural
role in ligand binding is an important prerequisite to improve
binding mode prediction of new ligands and the hit selection
rate in docking virtual screening.77,78 Accordingly, in a recent
study of hMAO-B crystal structure (PDB code 1ojc),46 we
designated as structural water those molecules intercepting
the energetic hot spots mapped on a grid through a water
molecule probe available in GRID.79 A minimum of four
water molecules were assumed to be critical in the ligand
binding with MAO-B. To our satisfaction, our findings were
recently largely confirmed in a study based on the structural
sampling of water molecules occurring in different PDB
crystals released for hMAO-B.39 Starting with these observa-
tions, we carried out an accurate analysis of the hMAO-B
complex (PDB code 2v60) that included 11 water molecules
within a sphere of 6 A around the cocrystallized inhibitor, the
4-formyl-7-m-chlorobenzyloxycoumarin (4-FCBC). Cam-
paigns of molecular docking simulations were then conducted
to select among the 11 water molecules those influencing
inhibitor binding. Such a selection was done comparing the
accuracy of docking simulations in successfully reproducing
Nanomolar MAO-B inhibitory potencies were shown by 4-
carboxyhydrazidomethylcoumarin derivatives 8a,b and by
the 4-carboxamidomethylcoumarin, 4-N-methylcarboxami-
domethylcoumarin, and 4-N,N-dimethylcarboxamidomethyl-
coumarin derivatives 11b,c, 21a,b, and 21f, respectively.
MAO-B affinity of 7-m-chlorobenzyloxy derivatives re-
mained high and nearly constant going from carboxamido
(11b) to N-methylcarboxamido (21b) and to N,N-dimethyl-
carboxamido (21f) coumarin derivatives but dramatically
diminished as the size of the N-alkyl substituent(s) increased
as in compounds 21c,d,g. As previously anticipated, the
isosteric substitution of the 7-benzyloxy group with the
3- and 4-pyridylmethoxy groups led to a significant decrease
of affinity (compare 19a,b vs 11a). C-Methylated 7-m-chloro-
benzyloxy and 7-m-fluorobenzyloxy racemates 13a,b dis-
played high and equal affinities to MAO-B. Compound 8c,
an N-aminoethyl derivative of the 4-carboxamidomethylcou-
marin 11b, was found completely inactive at both MAO
isoforms. Potent and highly selective MAO-B inhibitors were
found in the class of 4-carboxamidomethylcoumarins in
Table 2. The 7-m-halogenobenzyloxy derivatives 11b,c
and 21f exhibited IC50/SI values equal to 0.030/867, 0.050/
1400, and 0.040/2400, respectively. Very surprisingly, the N-
methylcarboxamide 21b was endowed with the highest MAO-
A affinity of the entire series of tested inhibitors. Conversely,
its close N,N-dimethyl analogue 21f was completely inactive
at the same enzyme isoform (IC50>100 μM). This striking
difference was quite unexpected and very difficult to interpret.
Affinity data of 4-aminomethyl- and 4-aminoethylcoumar-
ins (Table3) indicatedthatthe most potent MAO-Binhibitors
belong to this class of compounds and that high SIs are also
associated with many of them. In fact, compounds 22a-d can
be seen in the region of highly potent and selective inhibitors
(i.e., in the lower right-hand corner of the plot in Figure 1).
The 4-N-methylaminomethyl derivatives 22b and 22c, bear-
ing at position 7 a m-halogenosubstituted benzyloxy group,
and the 4-aminomethyl-7-(m-clorobenzyloxy)coumarin deri-
vative 24b exhibited the highest MAO-B inhibitory potencies
(IC50 of 13, 18, and 15 nM, respectively) and high SIs (457, 750,
and 133, respectively). The presence of N-alkyl groups larger
than the methyl group decreased the affinity (compare 22c vs
22e and 22f, and 22b vs 22g). Similarly, N,N-dimethyl deriva-
tives were less potent than the corresponding N-methyl analo-
gues (compare 22a vs 22h, 22b vs 22i, and 22c vs 22j). A steric
effect can be hypothesized around the position 4 as previously
observed for the N-alkyl- and the N,N-dialkylcarboxamido

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6693
Figure 2. Docking poses of inhibitors 22b and 22k (rendered in yellow and cyan stick models, respectively) into rMAO-B binding site. Amino
acid residues and FAD cofactor are represented in thin line form, colored according to the atom code (C atoms in green and yellow for amino
acid residues and cofactor, respectively). Structural water molecules are represented as red balls. Hydrogen bonds are drawn as dark-blue
dashed lines.
the binding mode of the cocrystallized 4-FCBC into hMAO-B
binding site. As in previous studies, GOLD software was used
in all the simulations. The best combination of water mole-
cules, returning the lowest rmsd value of 0.578 A with a
docking score of 59.46 kJ/mol, included eight water molecules
labeled as w1055, w1159, w1166, w1171, w1206, w1224,
w1309, and w1351 according to the numbering reported in
the cited hMAO-B X-ray complex. Given that our MAO
inhibition data were determined on rat brain mitochondria
assay, wefirstdeveloped a 3Dhomologymodel ofrat MAO-B
for which no crystallographic data were available, starting
from the rMAO-B sequence and the Cartesian coordinates of
the PDB 2v60 complex. A proper fitting was also set for
reconstructing the position in rMAO-B of both the FAD
cofactor and the eight designated water molecules coming
from the hMAO-B analysis. Confidence in the developed
homology model was ensured by the high primary structure
similarity (88%) and more so by the high degree of residue
conservation into the binding site of rat and human enzymes.
The cocrystallized 4-FCBC ligand of the 2v60 PDB structure
did not exhibit appreciable binding differences with the
hMAO-B when docked into the rMAO-B model. Only
slight geometrical differences(rmsd = 0.855 A) were observed
without any remarkable change in the binding topology. In
light of these encouraging results, the chemical scaffold of the
best ranked solution of the coumarin inhibitor 4-FCBC
docked into the rMAO-B was used to physically constrain
the docking simulations of selected inhibitors. The constraint
was limited to the 7-benzyloxy substituents that were found to
always assume a preferred binding conformation into the
hydrophobic binding site composed of Val316, Pro102, Tyr
326, Ile198, and Ile199 side chains. A visual inspection of
Figures 2 and 3 revealed that the selected and docked inhibi-
tors 22b and 22k, and 22c and 22f, respectively, adopted a
convergent binding mode with the coumarin ring facing the
FAD moiety and the lactonic carbonyl generally involved in a
hydrogen bond with the hydroxyl group of Tyr398. In addi-
tion, it was found that while amides or amines with small N-
alkyl substituents at position 4 of the coumarin ring were well
accommodated into the protein binding site and the proto-
nated nitrogen atoms formed hydrogen bonds with water
molecules w1351, w1166, and w1159 affording high Gold-
Score values (59.86, 55.30, 53.35, and 65.70 kJ/mol, for 11b,
22b, 22c, and 24b, respectively), the presence of bulkier N-
alkyl groups such as isopropyl (22f), n-butyl (21c), or benzyl
(22k) decreased the capability of establishing valuable binding
interactions with specific amino acid side chains and water mole-
cules into the protein binding site, leading to significantly lower
GoldScores values (45.38, 46.56, and 44.44 kJ/mol, respectively).
Although docking score energy was mainly related to
hydrophobic interactions that should be increasingly rele-
vant for larger groups, in our analysis no significant differ-
ence occurred when comparing van der Waals terms of
coumarins substituted at position 4 with small (22b and
22c) and large (22k and 22f) substituents. A major propensity
to form hydrogen bonds was instead observed in 4-substi-
tuted coumarins bearing small (22b and 22c) rather than
large (22k and 22f) substituents as denoted by the values of
the external hydrogen bond term. As unequivocally shown in
Figures 2 and 3, inhibitors with large groups at position 4
adopted folded conformations, and this may distort the
correct geometry for a strong hydrogen bond interaction

6694 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
Figure 3. Docking poses of inhibitors 22c and 22f (rendered in
light-blue and white stick models, respectively) into rMAO-B bind-
ing site. Amino acid residues and FAD cofactor are represented in
thin line form, colored according to the atom code (C atoms in green
and yellow for amino acid residues and cofactor, respectively).
Structural water molecules are represented as red balls. Hydrogen
bonds are drawn as dark-blue dashed lines.
Figure 4. Top-score docking pose of inhibitor 11c, rendered in
yellow stick model, into rMAO-A binding site. Amino acid residues
and FAD cofactor are represented in thin line form, colored
according to the atom code (C atoms in blue and cyan for amino
acid residues and cofactor, respectively). Structural water molecules
are represented as red balls. Hydrogen bonds are drawn as dark-
blue dashed lines. The different amino acids with respect to rMAO-
B residues are labeled in magenta according to the numbering of
rMAO-B.
between protonated nitrogens and surrounding water mole-
cules. In addition, the analysis of GoldScore values attrib-
uted to each of the 10 top-scored poses per compound
revealed a narrower standard deviation for both 22b and
22c (approximately (4%) compared to both 22k and 22f
(approximately (6.5%). Finally, the energy gap between top
and bottom scored docking solutions of 22b and 22c com-
pared to 22k and 22f increased from about 13 to 29 kJ/mol.
Taken together, all the above observations clearly indicated
that large groups at position 4 can be accommodated only at
the expense of high-energy inhibitor conformations unable
to engage optimal ligand-protein interactions.
mode was observed in both rMAO-A and rMAO-B. More-
over, in rMAO-A, a remarkable lowering of docking scores
was generally found. For instance, for inhibitor 11c a Gold-
Score of 19.92 kJ/mol was measured. The presence of diverse
residues forming the entrance cavity of the rMAO-A com-
pared to rMAO-B (i.e., Phe199Ile, Ala102Pro, Cys314Thr,
Ile316Val and Ile326Tyr) is supposed to determine a differ-
ent orientation of 11c. As illustrated in Figure 4, a number of
favorable hydrogen bonds and hydrophobic interactions
was lost in rMAO-A compared to rMAO-B.
Binding, Physicochemical, and Biopharmacological Profil-
ing of 22b Mesylate. Experimental MAO-B affinity and
selectivity, as well as the estimated promising lipophilicity
and aqueous solubility of the 7-(m-chlorobenzyloxy)-
coumarin derivative 22b, prompted a deeper in silico and
experimental investigation of its physicochemical and bio-
pharmacological profiles. Indeed, the X-ray crystallographic
structure of the complex of 22b with human MAO-B has
been solved and published by the Mattevi group and our
group 2 years ago56 and the main inhibitor-enzyme binding
interactions have been identified and discussed therein.
As for the physicochemical characterization of 22b, aqu-
eous solubility (S), pKa, octanol-water partition coefficient
(P), permeability coefficient (Ps, for passive oral absorption),
blood-brain barrier permeability coefficient (LogBBB), and
PSA, all relevant physicochemical parameters for drug dis-
solution, absorption, and distribution were experimentally
or computationally determined.
Interestingly, 4-substituted 7-(m-halogenobenzyloxy)cou-
marins exhibited generally higher docking scores compared
to the corresponding unsubstituted benzyloxy derivatives,
according to the experimental inhibition data.
Further analyses were carried out to investigate the rea-
sons behind the pronounced selective inhibition of MAO-B
exhibited by most of the coumarins analyzed. Given that
only a low-resolution rMAO-A complex was available and
water molecules were even missing (PDB code 1o5w), our
investigation was focused on the hMAO-A/harmine com-
plex (PDB code 2z5x). Rat and human MAO-A exhibited
strong structural resemblance with an rmsd equal to 0.438 A,
a sequence similarity of 87%, and a binding site with
identical amino acid residues. Interestingly, 7 out of the 8
selected water molecules for hMAO-B (w1055, w1159,
w1166, w1171, w1206, w1309, and w1351) were also pre-
served in the hMAO-A binding site. These water molecules
were therefore transferred into the rMAO-A crystal struc-
ture before running molecular docking simulations on a
number of highly selective MAO-B inhibitors (i.e., 11c,
11b, 22a, and 22c). Because no MAO-A X-ray complex with
coumarin ligands was available, the docking simulations
were conducted without constraints. Not surprisingly, as
shown in Figure 4, a diverse and somehow divergent binding
Aqueous solubility of 22b, assessed by the DMSO-buffer
dilution turbidimetric method63 at pH 7.4 and 3.0 was
52 μg/mL and 2.82 mg/mL, respectively, whereas pKa and
log P determined by potentiometric titration using the Sirius
GLpKa instrument (Sirius Analytical Instruments Ltd.,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6695
Figure 5. (a) Discriminant PLS t1-t2 analysis score plot for the BBB permeation model. BBBþ and BBB(/BBB- compounds are represented
as dark and light solid gray circles respectively. Predicted compound 22b is indicated by the solid white diamond. (b) Experimental vs predicted
BBB values for model compound along with projection for the predicted 22b.
Table 5. Permeability Data from HDM-PAMPA Assay
CA(t)/CD(0) b (%)
0.582, PC = 3). As shown in Figure 5, the PLS t1-t2 score plot
compd
R
a (%)
log Pe
c
disclosed a good discrimination between the BBBþ on one side
and BBB( and BBB- compounds on the other side. It was, in
fact, immediately evident that the great majority of compounds
was properly assigned to the correct class. On the basis of these
solid statistics, the ability of 22b to cross the BBB was evaluated
with much confidence. To our satisfaction, 22b, rendered as
solid white diamond, was centered in the BBBþ region where
CNS drugs are localized (Figure 5). It can therefore be assumed
that 22b is endowed with physicochemical properties that are
typical of CNS drugs and that its choice for a further experi-
mental biological characterization was appropriate.
22b
3
10 ( 3
72 ( 1
33.8 ( 0.3
3.7 ( 0.3
-3.48 ( 0.01
-4.13 ( 0.10
a R = % of retention through the artificial membrane. b CA(t)/CD(0) =
% of compound that reaches the acceptor compartment. c Pe = effective
permeability coefficient expressed in cm/s.
Forest Row, East Sussex, U.K.) were 7.18 ( 0.05 and 2.49 (
0.03, respectively. PSA, another computational parameter
used along with the rule of five of Lipinski to roughly
estimate oral drug absorption,66 was 49.8 A2, a value com-
patible with good biovailability and BBB permeation.71
Passive oral absorption of 22b, was also evaluated experi-
mentally by using the parallel artificial membrane perme-
ability assay (PAMPA).65,80 Experimental values of the
retention through the artificial membrane (R, %), the quan-
tity of compound reaching the acceptor compartment
(CA(t)/CD(0), %), and the effective permeability coefficient
(Pe) of 22b and 3 obtained using HDM-PAMPA are shown
in Table 5. Both compounds presented a relatively high
permeability coefficient through the hexadecane membrane.
However, 3 was mostly retained in the membrane because of
its notable affinity for hexadecane (as expected owing to its
high log P predicted equal to 4.73 by Bioloom, Biobyte
Corp., Claremont, CA). In contrast 22b largely reached the
acceptor compartment and was slightly retained in the
artificial membrane.
Compared to the reference compound 3, the aqueous
solubility, lipophilicity, membrane and BBB permeability
(log Pe and LogBBB, respectively), and the PSA of 22b
suggested a favorable profile for a CNS in vivo activity
and prompted its in-depth biopharmacological profiling.
MAO-B inhibitory affinity of 22b was therefore measured
also in human PRP with a radioenzymatic assay using
[14C]PEA, a selective substrate already used in rat mitochon-
dria. An IC50 of 3 nM, very close to that determined in the rat
mitochondria assay (13 nM), was measured.
The reversible nature of MAO-B inhibition by 22b was
assessed in time-dependent inhibition experiments, by mea-
suring the IC50 after 0 and 30 min of inhibitor-enzyme
incubation (PRP as enzyme source). No significant differ-
ence between the IC50 with or without incubation was
observed (6 vs 3 nM, respectively), as is typically detected
with reversible inhibitors.
The ability of 22b to cross the BBB was assessed in silico
through the estimation of the distribution parameter LogBBB
from MIF-based Volsurfþ descriptors.81 A LogBBB value of
0.386 was calculated for 22b, a figure that was indicative of a
good BBB permeation. Indeed, LogBBB values lower than
-0.5 denote very poor brain permeation whereas values
greater than 0.5 indicate high brain permeation.82 To lend
further support to the potentially good BBB permeation
ability of 22b, a knowledge-based approach was applied to
a database containing a number of related, but chemically
diverse, compounds exhibiting high, moderate, and poor
BBB permeation (coded as BBBþ, BBB(, and BBB-,
respectively). After calculation of MIF-based Volsurfþ
descriptors for the entire data set (245 compounds), y-response
categorized variable was set to 1 for BBBþ compounds and
to -1 for both BBB( and BBB- compounds. Partial least
square discriminant analysis (PLS-DA)83 was then carried out
to develop a reliable BBB permeation model having confident
statistics (r2 = 0.708, q2 = 0.659, SDEC = 0.538, SDEP =
Subsequently, 22b was also tested in ex vivo experiments as
a selective MAO-B inhibitor. In C57Bl mice, 1 h after an oral
or intraperitoneal dose of 22b (3 mg/kg), brain MAO-B
activity was almost completely inhibited while MAO-A
was not affected (Figure 6).
In time-course experiments, following a 0.5 mg/kg oral dose
of 22b, MAO-B enzymatic activity was significantly inhibited
at 30 min, 1 h, and 2 h (84%, 86%, and 90%, respectively),
indicating a very rapid inhibition onset. Good MAO-B in-
hibition was maintained up to 8 h (nearly 60%), and full
MAO-B activity was recovered after 16 h. During this time-
course, MAO-A activity remained unchanged (Figure 7),
suggesting that compound 22b may be a tight binding inhi-
bitor as lazabemide and safinamide. In a dose-response
experiment, the ED50 for brain MAO-B inhibition, calculated
1 h after dose administration, was 0.24 mg/kg po (Figure 8).
To evaluate potential drug-drug interaction effects of
22b, its inhibitory-induction activity on the selected series of

6696 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
Figure 8. Ex vivo brain MAO activity after oral administration of
22b (0.05, 0.1, 0.25, 0.5 mg/kg) in C57Bl mice: dose-response
experiment. Mice (n = 6 per group) were treated with the test
compound at different doses (0.05 - 0.5 mg/kg/po) and sacrificed
after 1 h. The brains were removed, crude homogenates prepared,
and MAO enzyme activities assessed with a radioenzymatic assay.
Data were expressed as the mean ( SEM and were analyzed by
ANOVA followed by Dunnett’s test.
Figure 6. Ex vivo brain MAO activity after oral (po) and intraper-
itoneal administration (ip) of 22b (3 mg/kg) in C57Bl mice. Mice (n =
6 per group) were treated (po or ip) with the test compound (3 mg/kg)
and sacrificedafter 1 h. The brainswereremoved, crude homogenates
were prepared, and MAO enzyme activities were assessed with a
radioenzymatic assay. Data were expressed as the mean ( SEM and
were analyzed by ANOVA followed by Dunnett’s test.
Figure 7. Ex vivo brain MAO activity after oral administration of
22b (0.5 mg/kg) in C57Bl mice: time course experiment. Mice (n =
6 per group) were treated (po) with the test compound, and at
different time intervals (0.5, 1, 2, 4, 8, and 16 h) they were sacrificed.
The brains were removed, crude homogenates prepared and MAO
enzyme activities assessed with a radioenzymatic assay. Data were
expressed as the mean ( SEM and were analyzed by ANOVA
followed by Dunnett’s test.
Figure 9. Effect of 22b on SH-SY5Y cell viability.
parameters relevant for drug activity in vivo led us to select
compound 22b for an in-depth biopharmacological profiling.
In vitro radioenzymatic assays in rat brain mitochondria and in
human PRP showed that 22b was a strong MAO-B inhibitor
(IC50 of 13 and 3 nM, respectively) endowed with a high MAO-
B selectivity (SI = 453, in rat brain mitochondria). The MAO-
B inhibition of 22b was not time-dependent as it would be for
reversible inhibitors. In ex vivo experiments, 22b showed an
excellent BBB penetrating capacity when administered both
recombinant human CYP450s was evaluated. Data revealed
that 22b inhibited the CYP450 isoforms at high micromolar
concentrations with IC50 values between 5 and 50 μM. These
data were quite promising, since compounds exhibiting
IC50 > 5-10μM canbe consideredat lowpotentialfor drug-
drug interaction because of the inhibition of CYP450 activity.
Finally, the in vitro effect of 22b on SH-SY5Y cell viability
was measured. As shown in Figure 9, incubation of cells for
24 h in a serumless medium containing 22b did not induce
any cellular damage up to 10 μM with IC50 = 29.7 μM, thus
exhibiting very little in vitro toxicity.
parenterally and orally and behaved as a very potent (ED50
=
0.24 mg/kg po), reversible, and short-acting (full recovery of
the enzymatic activity at 16 h) MAO-B inhibitor with no
significant effect on MAO-A. Moreover, compound 22b
exhibited no CYP isoform liabilities and was devoided of
cytotoxic effects.
Because of its excellent in vitro and in vivo MAO-B
inhibitory activity and appropriate pharmacokinetic proper-
ties, compound 22b may be considered as a promising clinical
candidate for the therapy of neurodegenerative diseases and,
analogous to selegeline,20 for major depressive disorders.
Finally, we anticipate that compound 22b also exhibits an
interesting, albeit low, inhibitory activity toward acetylcholi-
nesterase, an enzyme targeted by most of the anti-Alzheimer’s
drugs currently used in the therapy of this continuously
growing neurodegenerative disease.84 An improvement of
AChE inhibitory activity, while maintaining a sufficiently
strong MAO-B inhibitory potency, is an important goal of
Conclusions
A new class of higly potent and selective coumarin MAO-B
inhibitors with an excellent biopharmacological profile was
identified. SAFIRs and SSRs and modeling studies provided a
clear picture of the main interactions taking place at position 4
of the newly designed 4,7-disubstituted coumarin derivatives
and deepened our understanding of the structural requirements
for high MAO-B affinity and selectivity. The determination or
computational estimation of a number of physicochemical

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6697
our ongoing research addressing the preparation of multi-
potent ligands for the therapy of neurological disorders.54,85
J = 8.8 Hz, 1H), 10.89 (s, 1H, dis. with D2O). IR cm-1 (KBr):
1706, 1624, 1272, 841.
General Procedure for the Synthesis of Chlorides 2a-e. An
amount of 4.6 mmol of 1b or 1d was dissolved in 45 mL of hot
ethanol. Then potassium carbonate (0.63 g, 4.6 mmol) and
5.5 mmol of the appropriate benzyl bromide (0.65 mL of benzyl
bromide in the case of 2a and 2c, 0.72 mL of 3-chlorobenzyl
bromide in the case of 2b and 2d, 0.68 mL of 3-fluorobenzyl
bromide in the case of 2e) were added. The mixture was refluxed
for 2 h and then cooled, and the inorganic residue was filtered
off. The solvent was evaporated and the resulting solid was
treated with diethyl ether and filtered, giving the desired pro-
ducts as off-white solids.
7-(Benzyloxy)-4-chloro-2H-chromen-2-one (2a). Yield: 55%.
1H NMR (300 MHz, CDCl3) δ: 5.15 (s, 2H), 6.43 (s, 1H), 6.89 (d,
J = 2.2 Hz, 1H), 6.99 (dd, J1 = 2.2 Hz, J2 = 9.1 Hz, 1H),
7.34-7.44 (m, 5H), 7.75 (d, J = 9.1 Hz, 1H). IR cm-1 (KBr):
1735, 1359, 1126, 833.
4-Chloro-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one (2b).
Yield: 68%. 1H NMR (300 MHz, CDCl3) δ: 5.12 (s, 2H),
6.45 (s, 1H), 6.87 (d, J = 2.5 Hz, 1H), 6.99 (dd, J1 = 2.5 Hz,
J2 = 9.1 Hz, 1H), 7.30-7.38 (m, 3H), 7.44 (s, 1H), 7.78 (d, J =
9.1 Hz, 1H). IR cm-1 (KBr): 1721, 1365, 1275, 865, 838.
7-(Benzyloxy)-4-(chloromethyl)-2H-chromen-2-one (2c). Yield:
69%. 1H NMR (300 MHz, CDCl3) δ: 4.62 (s, 2H), 5.14 (s, 2H),
6.40 (s, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.97 (dd, J1 = 2.5 Hz, J2 =
8.8 Hz, 1H), 7.34-7.45 (m, 5H), 7.57 (d, J = 8.8 Hz, 1H).
IR cm-1 (KBr): 1732, 1615, 1396, 1264, 1199, 1057, 1008.
7-[(3-Chlorobenzyl)oxy]-4-(chloromethyl)-2H-chromen-2-one
(2d). Yield: 78%. 1H NMR (300 MHz, CDCl3) δ: 4.62 (s, 2H),
5.11 (s, 2H), 6.41 (s, 1H), 6.88 (d, J = 2.5 Hz, 1H), 6.96 (dd, J1 =
2.5 Hz, J2 = 8.8 Hz, 1H), 7.27-7.37 (m, 3H), 7.43 (br s, 1H),
7.58 (d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 1699, 1611.
4-(Chloromethyl)-7-[(3-fluorobenzyl)oxy]-2H-chromen-2-one
(2e). Yield: 73%. 1H NMR (300 MHz, CDCl3) δ: 4.62 (s, 2H),
5.14 (s, 2H), 6.41 (s, 1H), 6.89 (d, J = 2.5 Hz, 1H), 6.97 (dd,
J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.01-7.07 (m, 1H), 7.13-7.25
(m, 2H), 7.34-7.41 (m, 1H), 7.58 (d, J = 8.8 Hz, 1H). IR cm-1
(KBr): 1718, 1611.
Experimental Section
Chemistry. Starting materials and reagents (including 1e and
5), the resin for solid phase synthesis and analytical grade
solvents were from commercial sources. Melting points were
determined by the capillary method on a Stuart Scientific SMP3
electrothermal apparatus and are uncorrected. Elemental ana-
lyses were performed on the Euroea 3000 analyzer only on the
final compounds tested as MAO inhibitors. The measured
values for C, H, and N agreed to within (0.40% of the
theoretical values. The purity of the intermediates, checked by
1H NMR and HPLC, was always better than 95%. IR spectra
were recorded using potassium bromide disks on a Perkin-Elmer
Spectrum One FT-IR spectrophotometer; only the most signi-
ficant IR absorption bands are reported. 1H NMR spectra were
recorded in the specified deuterated solvent at 300 MHz on a
Varian Mercury 300 instrument. Chemical shifts are expressed
in parts per million (δ) relative to the solvent signal and the
coupling constants J in hertz (Hz). The following abbreviations
were used: s (singlet), d (doublet), t (triplet), q (quadruplet), dd
(double doublet), m (multiplet), br (broad signal); signals due to
OH or NH protons were located by deuterium exchange with
D2O. Chromatographic separations were performed on silica
gel (230-400 mesh, Merck) by using the flash methodology.
Ethyl (7-Hydroxy-2-oxo-2H-chromen-4-yl)acetate (1a). Resor-
cinol (2.2 g, 20 mmol), diethyl 1,3-acetonedicarboxylate (4.0 mL,
22 mmol), and few drops of 96% sulfuric acid were stirred at
120 ꢀC for 1 h. The oily residue obtained was crystallized from
1
absolute ethanol, yielding a red solid (2.48 g, 50% yield). H
NMR (300 MHz, DMSO-d6) δ: 1.16 (t, J = 7.1 Hz, 3H), 3.91 (s,
2H), 4.09 (q, J = 7.1 Hz, 2H), 6.21 (s, 1H), 6.71 (d, J = 2.3 Hz,
1H), 6.78 (dd, J1 = 2.3 Hz, J2 = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz,
1H), 10.55 (br s, 1H). IR cm-1 (KBr): 3230, 1713, 1682, 1205,
1026, 849.
4-(Chloromethyl)-7-hydroxy-2H-chromen-2-one (1b). Resor-
cinol (10 g, 91 mmol) was dissolved in 100 mL of 96% sulfuric
acid at 0 ꢀC. Then ethyl 4-chloroacetoacetate (10 mL, 74 mmol)
was slowly added and the mixture was stirred at 0-5 ꢀC for 2 h.
The reaction mixture was then poured onto ice-water (1000 g)
and the solid was filtered and washed with water, yielding the
7-(1,3-Benzodioxol-5-ylmethoxy)-4-(chloromethyl)-2H-chromen-
2-one (2f). An amount of 1.0 mmol (0.21 g) of 1b was dissolved in
10 mL of anhydrous THF. Piperonyl alcohol (0.46 g, 3.0 mmol),
DIAD (0.59 mL, 3.0 mmol), and PPh3 (0.79 g, 3.0 mmol) were
added, and the mixture was stirred at room temperature for 18 h.
The solvent was removed under vacuum, and the resulting crude oil
was purified by column chromatography using as eluent a mixture
1
desired coumarin as white solid (12.2 g, 78% yield). H NMR
(300 MHz, acetone-d6) δ: 4.92 (s, 2H), 6.40 (s, 1H), 6.80 (d,
J = 2.5 Hz, 1H), 6.91 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.73 (d,
J = 8.8 Hz, 1H), 9.50 (s, 1H, dis. with D2O). IR cm-1 (KBr):
3283, 1686, 1235, 1136, 850.
4,7-Dihydroxy-2H-chromen-2-one (1c). Resorcinol (8.8 g,
80 mmol), malonic acid (25 g, 240 mmol), and 40 mL of boron
1
of chloroform/petroleum ether, 1/1 (v/v). Yield: 76%. H NMR
(300 MHz, CDCl3) δ: 4.62 (s, 2H), 5.03 (s, 2H), 5.98 (s, 2H), 6.40 (s,
1H), 6.79-6.84 (m, 2H), 6.87-6.91 (m, 2H), 6.93-6.96 (m, 1H),
7.55-7.58 (m, 1H). IR cm-1 (KBr): 1731, 1614.
trifluoride-diethyl etherate complex (BF3 Et2O) were mixed at
7-[(3-Chlorobenzyl)oxy]-4-methyl-2H-chromen-2-one (3). An
amount of 0.81 g (4.6 mmol) of commercially available 1e was
dissolved in 45 mL of hot ethanol. Then potassium carbonate
(0.63 g, 4.6 mmol) and 3-chlorobenzyl bromide (0.72 mL, 5.5
mmol) were added. The mixture was refluxed for 2 h and then
cooled. The inorganic residue was filtered off and the product
crystallized from the resulting solution. Yield: 88%. Mp: 148-9
3
90 ꢀC for 24 h under magnetic stirring. The crude mixture was
poured onto crushed ice (300 g), and the resulting precipitate
was filtered, washed with diethyl ether (50 mL), and then
purified by column chromatography using as eluent the mixture
CHCl3/CH3OH, 9.5/0.5 (v/v). Yield: 23%. 1H NMR (300 MHz,
DMSO-d6) δ: 5.36 (s, 1H), 6.64 (d, J = 2.2 Hz, 1H), 6.75 (dd,
J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 10.51 (s,
1H, dis. in D2O), 12.23 (s, 1H, dis. in D2O). IR cm-1 (KBr):
1662, 1635, 1098, 802.
1
ꢀC. H NMR (300 MHz, CDCl3) δ: 2.40 (d, J = 1.1 Hz, 3H),
5.10 (s, 2H), 6.15 (d, J = 1.1 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H),
6.93 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.28-7.37 (m, 3H), 7.44
(s, 1H), 7.51 (d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 1713, 1614.
Anal. (C17H13ClO3) C, H.
4-Chloro-7-hydroxy-2H-chromen-2-one (1d). An amount of
2.0 g (11 mmol) of 1c was refluxed in 12 mL of POCl3 for 4 h. The
mixture was poured onto crushed ice (100 g), and the aqueous
layer was extracted with ethyl acetate (3 ꢀ 40 mL). The organic
phases were collected and dried over sodium sulfate, and the
solvent was evaporated under reduced pressure. The resulting
red oil was purified by flash chromatography (eluent n-hexane/
ethyl acetate, 8/2 v/v), furnishing a red solid (995 mg, 46%
yield). 1H NMR (300 MHz, DMSO-d6) δ: 6.60 (s, 1H), 6.76 (d,
J = 2.2 Hz, 1H), 6.88 (dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 7.69 (d,
General Procedure for the Synthesis of Coumarin Amines
4a-h. An amount of 0.50 mmol of the appropriate chloro
derivative 2a-e was dissolved in 4.0 mL of dry DMF. Then
0.26 mL (1.5 mmol) of DIEA and 0.75 mmol of the appropriate
amino acid (for compounds 4a,b,f, 0.093 g of L-alaninamide
hydrochloride; for compound 4c,d,g,h, 0.086 g of L-prolinamide;
for compound 4e, 0.11 g of L-serinamide hydrochloride) were
added, and the mixture was stirred at 80 ꢀC for 5 h. The solvent

6698 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
was evaporated, and the resulting crude was purified by column
chromatography (eluent CHCl3/CH3OH, 9.5/0.5 v/v).
N2-[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]alaninamide (4a).
Yield: 77%. Mp: 247-9 ꢀC. H NMR (300 MHz, DMSO-d6)
diamine (for 6b). The mixture was then stirred at room tem-
perature for 5 h.
tert-Butyl 2-[(7-Hydroxy-2-oxo-2H-chromen-4-yl)acetyl]hydra-
zinecarboxylate (6a). Yield: 98%. 1H NMR (300 MHz, DMSO-
d6) δ: 1.40 (s, 9H), 3.60 (s, 2H), 6.22 (s, 1H), 6.70 (d, J = 2.4 Hz,
1H), 6.77 (dd, J1 = 2.4 Hz, J2 = 8.7 Hz, 1H), 7.61 (d, J = 8.7 Hz,
1H), 8.85 (s, 1H), 9.93 (s, 1H), 10.57 (s, 1H). IR cm-1 (KBr): 3302,
3206, 1727, 1705, 1683, 1607.
1
δ: 1.42 (d, J = 6.9 Hz, 3H), 3.97-4.01 (m, 1H), 4.88 (s, 1H), 5.19
(s, 2H), 6.94 (d, J = 2.2 Hz, 1H), 6.99 (dd, J1 = 2.2 Hz, J2 =
9.1 Hz, 1H), 7.30-7.47 (m, 6H), 7.17 (s, 1H), 7.61 (s, 1H), 8.12
(d, J = 9.1 Hz, 1H). IR cm-1 (KBr): 3325, 1668, 1609. Anal.
(C19H18N2O4) C, H, N.
tert-Butyl 2-{[(7-Hydroxy-2-oxo-2H-chromen-4-yl)acetyl]-
amino}ethylcarbamate (6b). Yield: 65%. H NMR (300 MHz,
N2-{7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}alanin-
1
1
amide (4b). Yield: 82%. Mp: 237-9 ꢀC. H NMR (300 MHz,
DMSO-d6) δ: 1.35 (s, 9H), 2.96-3.05 (m, 4H), 3.60 (s, 2H), 6.14 (s,
1H), 6.69-6.78 (m, 3H), 7.57 (d, J = 8.8 Hz, 1H), 8.19 (br s, 1H),
10.54 (br s, 1H). IR cm-1 (KBr): 3360, 3335, 1695, 1680, 1656.
Synthesis of Amides 7a-c. The appropriate intermediate 6a,b
(1.5 mmol) was dissolved in absolute ethanol (10 mL) and
K2CO3 (0.21 g, 1.5 mmol), and the suitable benzyl bromide
(1.5 mmol) was added to the solution. The mixture was refluxed
for 30 min. The solid was filtered off and the solution cooled at
room temperature. The solvent was evaporated to give a solid
that was purified by column chromatography (eluent CHCl3/
MeOH, 9.5/0.5 v/v).
DMSO-d6) δ: 1.42 (d, J = 6.9 Hz, 3H), 3.97-4.01 (m, 1H), 4.88
(s, 1H), 5.21 (s, 2H), 6.95 (d, J = 2.2 Hz, 1H), 7.00 (dd, J1 =
2.2 Hz, J2 = 9.1 Hz, 1H), 7.17 (s, 1H), 7.39-7.44 (m, 4H), 7.54
(s, 1H), 7.62 (s, 1H), 8.13 (d, J = 9.1 Hz, 1H). IR cm-1 (KBr):
3437, 3180, 1674, 1613. Anal. (C19H17ClN2O4) C, H, N.
1-[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]prolinamide (4c).
Yield: 59%. Mp: 203-5 ꢀC. 1H NMR (300 MHz, DMSO-d6) δ:
1.82-1.99 (m, 3H), 2.26-2.30 (m, 1H), 3.78-3.79 (m, 1H),
3.92-4.00 (m, 1H), 4.29-4.34 (m, 1H), 4.91 (s, 1H), 5.20 (s, 2H),
6.92 (dd, J1 = 2.8 Hz, J2 = 9.1 Hz, 1H), 6.97 (d, J = 2.8 Hz, 1H),
7.21 (s, 1H), 7.31-7.47 (m, 5H), 7.67 (s, 1H), 7.95 (d, J = 9.1 Hz,
1H). IR cm-1 (KBr): 3475, 3195, 1691, 1669, 1615. Anal.
(C21H20N2O4) C, H, N.
tert-Butyl 2-{[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]acetyl}-
hydrazinecarboxylate (7a). Yield: 30%. H NMR (300 MHz,
1
DMSO-d6) δ: 1.37 (s, 9H), 3.68 (s, 2H), 5.22 (s, 2H), 6.30 (s, 1H),
7.01 (dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 7.08 (d, J = 2.2 Hz, 1H),
7.30-7.46 (m, 5H), 7.70 (d, J = 8.8 Hz, 1H), 8.85 (s, 1H), 9.94 (s,
1H). IR cm-1 (KBr): 3371, 3262, 1754, 1670.
tert-Butyl 2-{[7-(3-Chlorobenzyl)oxy)-2-oxo-2H-chromen-4-yl]-
acetyl}hydrazinecarboxylate (7b). Yield: 56%. 1H NMR (300
MHz, DMSO-d6) δ: 1.37 (s, 9H), 3.68 (s, 2H), 5.22 (s, 2H), 6.30
(s, 1H), 7.01 (dd, J1 = 2.2 Hz, J2 = 8.8 Hz, 1H), 7.08 (d, J =
2.2 Hz, 1H), 7.39-7.40 (m, 3H), 7.51 (s, 1H), 7.70 (d, J = 8.8 Hz,
1H), 8.85 (s, 1H), 9.94 (s, 1H). IR cm-1 (KBr): 3366, 3259, 1755,
1683.
tert-Butyl 2-[({7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}-
acetyl)amino]ethylcarbamate (7c). Yield: 95%. 1H NMR (300
MHz, DMSO-d6) δ: 1.35 (s, 9H), 2.94-2.98 (m, 2H), 3.03-3.07
(m, 2H), 3.64 (s, 2H), 5.23 (s, 2H), 6.23 (s, 1H), 6.81 (br s, 1H), 7.03
(dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H),
7.39-7.41 (m, 3H), 7.53 (s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 8.21 (br s,
1H). IR cm-1 (KBr): 3356, 3290, 1729, 1685.
Synthesis of Amides 8a-c. The appropriate intermediate
7a-c (0.060 mmol) was dissolved in 1.0 mL of a 1/1 v/v mixture
of CH2Cl2/CF3COOH and the solution stirred at room temp-
erature for 20 min. The solvent was evaporated under vacuum
and the oily residue obtained was treated with ether to give a
precipitate that was filtered and crystallized from ethanol (8a,b)
or chloroform/n-hexane (8c).
1-{7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}prolina-
1
mide (4d). Yield: 73%. Mp: 222-3 ꢀC. H NMR (300 MHz,
DMSO-d6) δ: 1.82-1.99 (m, 3H), 2.26-2.27 (m, 1H), 3.73-3.78
(m, 1H), 3.94-3.97 (m, 1H), 4.29-4.32 (m, 1H), 4.91 (s, 1H), 5.22
(s, 2H), 6.93 (dd, J1 = 2.8 Hz, J2 = 9.1 Hz, 1H), 6.97 (d, J =
2.8 Hz, 1H), 7.19 (s, 1H), 7.40-7.44 (m, 3H), 7.53 (s, 1H), 7.66 (s,
1H), 7.95 (d, J = 9.1 Hz, 1H). IR cm-1 (KBr): 3478, 3193, 1688,
1669, 1615. Anal. (C21H19ClN2O4) C, H, N.
N2-{7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}serina-
mide (4e). Yield: 48%. Mp: 244 ꢀC (dec). 1H NMR (300 MHz,
DMSO-d6) δ: 3.75-3.80 (m, 2H), 3.96-4.02 (m, 1H), 4.94 (d,
J = 1.9 Hz, 1H), 5.05-5.09 (m, 1H), 5.22 (s, 2H), 6.96 (d, J =
2.5 Hz, 1H), 7.02 (dd, J1 = 2.5 Hz, J2 = 9.1 Hz, 1H), 7.23 (d,
J = 6.9 Hz, 1H), 7.26 (s, 1H), 7.38-7.43 (m, 3H), 7.54 (s, 1H),
7.64 (s, 1H), 8.08 (d, J = 9.1 Hz, 1H). IR cm-1 (KBr): 3368,
3275, 3188, 1658, 1618. Anal. (C19H17ClN2O5) C, H, N.
N2-{[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]methyl}alanina-
1
mide (4f). Yield: 63%. Mp: 151-3 ꢀC. H NMR (300 MHz,
DMSO-d6) δ: 1.23 (d, J = 6.6 Hz, 3H), 3.23-3.33 (m, 1H),
3.98-4.01 (m, 2H), 5.21 (s, 2H), 6.37 (s, 1H), 7.01 (dd, J1 =
2.5 Hz, J2 = 9.1 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.13 (br s, 1H),
7.33-7.47 (m, 6H), 7.73 (d, J = 9.1 Hz, 1H), 1NH not detectable.
IR cm-1 (KBr): 3203, 1705, 1639, 1614. Anal. (C20H20N2O4) C,
H, N.
1-{[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]methyl}prolinamide
(4g). Yield: 74%. Mp: 67-9 ꢀC. 1H NMR (300 MHz, DMSO-d6)
δ: 1.72-1.78 (m, 3H), 2.02-2.06 (m, 1H), 2.25-2.30 (m, 1H),
3.02-3.10 (m, 2H), 3.63 (d, J = 15.4 Hz, 1H), 3.93 (d, J =
15.4 Hz, 1H), 5.21 (s, 2H), 6.44 (s, 1H), 6.98 (dd, J1 = 2.2 Hz, J2
= 8.8 Hz, 1H), 7.06 (d, J = 2.2 Hz, 1H), 7.20 (s, 2H), 7.30-7.47
(m, 5H), 7.93 (d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 3328, 3194,
1723, 1677, 1611. Anal. (C22H22N2O4) C, H, N.
2-[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]acetohydrazide (8a).
Yield: 93%. Mp: 164 ꢀC (dec). 1H NMR (300 MHz, DMSO-d6)
δ: 3.60 (s, 2H), 4.29 (br s, 2H, dis. with D2O), 5.22 (s, 2H), 6.23 (s,
1H), 7.02 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz,
1H), 7.30-7.47 (m, 5H), 7.70 (d, J = 8.8 Hz, 1H), 9.31 (s, 1H, dis.
with D2O). IR cm-1 (KBr): 3303, 1716, 1644, 1615. Anal.
(C18H16N2O4) C, H, N.
2-[7-(3-Chlorobenzyloxy)-2-oxo-2H-chromen-4-yl]acetohydrazide
(8b). Yield: 86%. Mp 174-6 ꢀC. 1H NMR (300 MHz, DMSO-d6)
δ: 3.61 (s, 2H), 4.43 (br s, 2H, dis. with D2O), 5.24 (s, 2H), 6.24 (s,
1H), 7.03-7.06 (m, 1H), 7.07 (br s, 1H), 7.41-7.42 (m, 3H), 7.53 (s,
1H), 7.71 (d, J = 8.8 Hz, 1H), 9.34 (s, 1H, dis. with D2O). IR cm-1
(KBr): 3309, 1719, 1646. Anal. (C18H15ClN2O4) C, H, N.
N-(2-Aminoethyl)-2-{7-[(3-chlorobenzyl)oxy]-2-oxo-2H-chro-
men-4-yl}acetamide (8c). Yield: 83%. Mp: 144-5 ꢀC. 1H NMR
(300 MHz, DMSO-d6) δ: 2.81-2.83 (m, 2H), 3.26-3.28 (m, 2H),
3.69 (s, 2H), 5.23 (s, 2H), 6.25 (s, 1H), 7.03 (dd, J1 = 2.5 Hz,
J2 = 8.8 Hz, 1H), 7.09 (d, J = 2.5 Hz, 1H), 7.39-7.42 (m, 3H),
7.53 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.74 (br s, 2H, dis. with
D2O), 8.33 (br s, 1H). IR cm-1 (KBr): 3356, 3290, 1700, 1683.
Anal. (C20H19ClN2O4) C, H, N.
1-({7-[(3-Fluorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}methyl)-
1
prolinamide (4h). Yield: 61%. Mp: 162-4 ꢀC. H NMR (300
MHz, DMSO-d6) δ: 1.72 (br s, 3H), 1.98-2.08 (m, 1H),
2.25-2.33 (m, 1H), 3.01-3.10 (m, 2H), 3.63 (d, J = 15.4 Hz,
1H), 3.93 (d, J = 15.4 Hz, 1H), 5.23 (s, 2H), 6.44 (s, 1H),
6.97-7.05 (m, 3H), 7.13-7.31 (m, 4H), 7.40-7.47 (m, 1H), 7.95
(d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 3193, 1739, 1649, 1613.
Anal. (C22H21FN2O4) C, H, N.
Synthesis of Amides 6a,b. The commercially available
7-hydroxycoumarin-4-acetic acid 5 (0.44 g, 2.0 mmol) and
HOBt (0.63 g, 4.0 mmol) were dissolved in 12 mL of anhydrous
DMF. DCC (0.8 g, 4.0 mmol) was added followed by an amount
of 4.0 mmol of tert-butyl carbazate (for 6a) or N-Boc-ethylene-

Article
Solid-Phase Synthesis of Amides 11a-d and 13a,b. Resin 9.
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6699
2-{7-[(3-Hydroxybenzyl)oxy]-2-oxo-2H-chromen-4-yl}acetamide
(11d). Purified by column chromatography (eluent: CH2Cl2/
MeOH, 8.5/1.5 v/v). Yield: 30%. Mp: 190-1 ꢀC (dec). 1H NMR
(300 MHz, DMSO-d6) δ: 3.62 (s, 2H), 5.13 (s, 2H), 6.23 (s, 1H),
6.68-6.70 (m, 1H), 6.81-6.85 (m, 2H), 6.99-7.18 (m, 4H),
7.63-7.66 (m, 2H), 9.51 (s, 1H). IR cm-1 (KBr): 3373, 3198,
1698, 1672, 1607. Anal. (C18H15NO5) C, H, N.
An amount of 0.060 mmol (0.10 g) of Rink amide AM resin was
suspended in 2.0 mL of anhydrous DMF and shaken for 20 min.
The swelled resin was suspended in 2.0 mL of a 20% solution of
piperidine in anhydrous DMF and shaken for 30 min, filtered,
and washed with anhydrous DMF (3 ꢀ 3.0 mL). The depro-
tected resin was suspended in 2 mL of anhydrous DMF, and
7-hydroxycoumarin-4-acetic acid 5 (0.070 g, 0.32 mmol), hydro-
xybenzotriazole (0.049 g, 0.32 mmol), and N,N-diisopropylcar-
bodiimide (0.050 mL, 0.32 mmol) were added. The mixture was
shaken overnight. The resin was filtered and washed with DMF
(3 ꢀ 2.0 mL) and THF (3 ꢀ 2.0 mL).
2-[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]propanamide (13a).
Crystallized from ethanol. Yield: 34%. Mp: 196 ꢀC (dec),
1
204-6 ꢀC. H NMR (300 MHz, DMSO-d6) δ: 1.38 (d, J =
7.2 Hz, 3H), 3.99 (q, J = 7.2 Hz, 1H), 5.21 (s, 2H), 6.19 (s, 1H),
7.04 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.08 (d, J = 2.5 Hz, 1H),
7.14 (s, 1H), 7.32-7.46 (m, 5H), 7.59 (s, 1H), 7.76 (d, J = 8.8 Hz,
1H). IR cm-1 (KBr): 3398, 1716, 1667, 1618. Anal. (C19H17-
NO4) C, H, N.
Resins 10a-d. An amount of 0.20 mmol (0.30 g) of resin-
bound 9 was suspended in 4.0 mL of anhydrous DMF and
shaken for 20 min. The swelled resin was suspended in 3.0 mL of
anhydrous DMF. Then 1.9 mmol of the appropriate benzyl
bromide (benzyl bromide for 10a, 3-chlorobenzyl bromide
for 10b, 3-fluorobenzyl bromide for 10c, and 17 for 10d) and
0.34 mL (1.9 mmol) of DIEA were added. The shaking was
continued at 70 ꢀC for 2.5 h. The mixture was cooled at room
temperature, the resin was filtered, washed with anhydrous
DMF (3 ꢀ 3.0 mL), and the coupling was repeated in the same
conditions. The mixture was filtered, and the resin was washed
with DMF (3 ꢀ 3.0 mL), THF (3 ꢀ 3.0 mL), and dichloro-
methane (3 ꢀ 3.0 mL).
2-{7-[(3-Fluorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}propana-
mide (13b). Crystallized from ethanol. Yield: 24%. Mp:
1
168-9 ꢀC. H NMR (300 MHz, DMSO-d6) δ: 1.38 (d, J =
6.9 Hz, 3H), 3.99 (q, J = 6.9 Hz, 1H), 5.24 (s, 2H), 6.20 (s, 1H),
7.03-7.19 (m, 4H), 7.28-7.30 (m, 2H), 7.44 (dd, J1 = 7.4 Hz,
J2 = 7.7 Hz, 1H), 7.59 (s, 1H), 7.77 (d, J = 8.5 Hz, 1H). IR cm-1
(KBr): 3422, 1723, 1655, 1618. Anal. (C19H16FNO4) C, H, N.
3-Methylphenyl Benzoate(16). An amount of 1.0mL (10 mmol)
of m-cresol 14 and 7.0 mL (50 mmol) of anhydrous triethylamine
were dissolved in 10 mL of anhydrous THF. Then 1.2 mL
(10 mmol) of benzoyl chloride 15, previously dissolved in 10 mL
of anhydrous THF, were added dropwise at 0 ꢀC. The mixture was
stirred at room temperature for 30 min. The obtained precipitate
was filtered and the solvent was evaporated under vacuum to
give an oil that was purified by flash chromatography (eluent
CHCl3/n-hexane, 1/1 v/v). Yield: 98%. 1H NMR (300 MHz,
CDCl3) δ: 2.18 (s, 3H), 7.15-7.18 (m, 1H), 7.26-7.30 (m, 2H),
7.39-7.48 (m, 1H), 7.53-7.55 (m, 2H), 7.63-7.68 (m, 1H),
8.19-8.22 (m, 2H). IR cm-1 (KBr): 1733, 1268, 1244.
Resin 10e. An amount of 0.17 mmol of swelled resin 10d was
suspended in 2.0 mL of anhydrous THF, and 0.13 mL of
saturated solution in methanol of sodium methoxide was added.
The mixture was shaken for 4 h, filtered, and washed with THF/
H2O, 1/1 v/v (3 ꢀ 2.0 mL), THF/HCl 2.0 N, 1/1 v/v (3 ꢀ 2.0 mL),
THF/H2O, 1/1 v/v (3 ꢀ 2.0 mL), THF (3 ꢀ 2.0 mL), and CH2Cl2
(3 ꢀ 2.0 mL).
Resins 12a,b. An amount of 0.18 mmol of the resin 10a (for
12a) or 10c (for 12b) was suspended in3.0mLofanhydrousDMF
and shaken for 20 min. The swelledresin was suspended in 2.0 mL
of anhydrous DMF, and an amount of 1.1 mL (0.55 mmol) of a
0.5 M solution of KHMDS in toluene was added. The mixture
was shaken for 10 min. The resin was filtered, washed with
anhydrous DMF, resuspended in 2.0 mL of anhydrous DMF,
and methyl iodide (0.23 mL, 3.7 mmol) was added. The resin was
shaken for 4 h, filtered, and washed with DMF (3 ꢀ 2.0 mL),
THF (3 ꢀ 2.0 mL), and CH2Cl2 (3 ꢀ 2.0 mL).
3-(Bromomethyl)phenyl Benzoate (17). The intermediate 16
(1.3 g, 6.0 mmol), N-bromosuccinimide (1.3 g, 7.2 mmol), and
2,20-azobis(2-methylpropionitrile) were dissolved in 6.0 mL of
carbon tetrachloride. The solution was refluxed for 1 h, filtered,
and added to n-hexane to give a precipitate that was filtered and
washed with methanol. Yield: 53%. 1H NMR (300 MHz,
CDCl3) δ: 4.51 (s, 2H), 7.16-7.18 (m, 1H), 7.25-7.32 (m,
2H), 7.39-7.46 (m, 1H), 7.52-7.55 (m, 2H), 7.63-7.68 (m,
1H), 8.19-8.22 (m, 2H). IR cm-1 (KBr): 1728, 1270, 1240.
2-[7-(Hydroxy)-2-oxo-2H-chromen-4-yl]acetamide (18). In a
glass vessel 0.80 g (3.2 mmol) of 1a was added to 8.0 mL
(16 mmol) of a 2.0 M solution of ammonia in methanol. The
ampule was sealed and placed in an oven at 90 ꢀC for 60 h. The
solution was evaporated under vacuum, and the solid obtained
was crystallized from ethanol. Yield: 50%. 1H NMR (300 MHz,
DMSO-d6) δ: 3.64 (s, 2H), 6.25 (s, 1H), 7.05 (dd, J1 = 2.4 Hz,
J2 = 8.8 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H), 7.17 (s, 1H), 7.63 (s,
1H), 7.68 (d, J = 8.7 Hz, 1H), OH not detectable. IR cm-1
(KBr): 3447, 3341, 3148, 1712, 1623.
Synthesis of Pyridyl Compounds 19a,b. Amounts of 0.11 g
(0.50 mmol) of 18, 1.5 mmol of 3-(hydroxymethyl)pyridine
(0.15 mL, in the case of 19a) or 4-(hydroxymethyl)pyridine
(0.16 g, in the case of 19b), 0.38 g (1.5 mmol) of 1,10-(azodi-
carbonyl)dipiperidine (ADDP), and 0.38 mL (1.5 mmol) of
tributylphosphine were dissolved in 5.0 mL of anhydrous
THF, and the mixture was stirred at room temperature for
18 h. The obtained precipitate was filtered, washed with chloro-
form, and then crystallized from ethanol.
General Procedure for the Cleavage of Amides 11a-d and 13a,
b. The functionalized resin 12a,b or 10a-c or 10e was suspended
in 3.0 mL of a 95% CF3COOH, 2.5% H2O, and 2.5% triethyl-
silane solution and shaken for 1 h. The resin was filtered and
washed with the same solution (3 ꢀ 3.0 mL) and the solvent was
evaporated under vacuum to give an oil that was treated with
toluene, yielding a solid that was crystallized from ethanol or
purified by column chromatography.
2-[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]acetamide
(11a).
Crystallized from ethanol. Yield: 53%. Mp: 218-221 ꢀC (dec).
1H NMR (300 MHz, DMSO-d6) δ: 3.63 (s, 2H), 5.21 (s, 2H),
6.24 (s, 1H), 7.02-7.08 (m, 2H), 7.17 (s, 1H), 7.30-7.47 (m, 5H),
7.65-7.68 (m, 2H). IR cm-1 (KBr): 3395, 3305, 1713, 1661,
1612. Anal. (C18H15NO4) C, H, N.
2-{7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}acetamide
(11b). Purified by column chromatography (eluent: CHCl3/
MeOH, 9.5/0.5 v/v). Yield: 30%. Mp: 185-6 ꢀC. 1H NMR
(300 MHz, DMSO-d6) δ: 3.64 (s, 2H), 5.24 (s, 2H), 6.25 (s, 1H),
7.03-7.09 (m, 2H), 7.17 (s, 1H), 7.38-7.44 (m, 3H), 7.54 (s, 1H),
7.63 (s, 1H), 7.68 (d, J = 8.7 Hz, 1H). IR cm-1 (KBr): 3447, 3341,
1733, 1693, 1618. Anal. (C18H14ClNO4) C, H, N.
2-[2-Oxo-7-(pyridin-3-ylmethoxy)-2H-chromen-4-yl]acetamide
(19a). Yield: 40%. Mp: 212 ꢀC (dec). 1H NMR (300 MHz,
DMSO-d6) δ: 3.63 (s, 2H), 5.26 (s, 2H), 6.25 (s, 1H), 7.05
(dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.11 (d, J = 2.5 Hz, 1H),
7.17(br s, 1H), 7.40-7.44 (m, 1H), 7.64 (brs, 1H), 7.67 (d, J = 8.8
Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 8.54 (d, J = 4.7 Hz, 1H), 8.68
(s, 1H). IR cm-1 (KBr): 1714, 1667, 1615. Anal. (C17H14N2O4)
C, H, N.
2-{7-[(3-Fluorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}acetamide
(11c). Purified by column chromatography (eluent: CHCl3/MeOH,
1
9.5/0.5 v/v). Yield: 44%. Mp: 177-8 ꢀC. H NMR (300 MHz,
DMSO-d6) δ: 3.63 (s, 2H), 5.24 (s, 2H), 6.24 (s, 1H), 7.03-7.07 (m,
2H), 7.13-7.17 (m, 2H), 7.29 (d, J = 7.7 Hz, 2H), 7.40-7.47 (m,
1H), 7.64 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 3415,
1717, 1667, 1614. Anal. (C18H14FNO4) C, H, N.

6700 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
2-[2-Oxo-7-(pyridin-4-ylmethoxy)-2H-chromen-4-yl]acetamide
(19b). Yield: 71%. Mp: 193-4 ꢀC. 1H NMR (300 MHz, DMSO-
d6) δ: 3.63 (s, 2H), 5.30 (s, 2H), 6.25 (s, 1H), 7.04-7.07 (m, 2H),
7.16 (br s, 1H), 7.43 (d, J = 5.6 Hz, 2H), 7.63 (br s, 1H), 7.68 (d,
J = 9.6 Hz, 1H), 8.57 (d, J = 5.6 Hz, 2H). IR cm-1 (KBr): 3319,
1663, 1627. Anal. (C17H14N2O4) C, H, N.
Synthesis of Esters 20a,b. Amounts of 0.12 g (0.50 mmol) of
1a, 1.5 mmol of the suitable benzyl alcohol, 0.38 g (1.5 mmol) of
ADDP, and 0.39 g (1.5 mmol) of triphenylphosphine were
dissolved in 5.0 mL of anhydrous THF, and the mixture was
stirred at room temperature for 18 h. The precipitate was filtered
off, the solvent evaporated under vacuum and the oily residue
purified by flash chromatography (eluent CHCl3).
2-{7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}-N,N-di-
methylacetamide (21f). Purified by column chromatography
(eluent: AcOEt). Yield: 62%. Mp: 159-160 ꢀC. 1H NMR
(300 MHz, CDCl3) δ: 3.02 (s, 3H), 3.10 (s, 3H), 3.79 (s, 2H),
5.10 (s, 2H), 6.14 (s, 1H), 6.87 (d, J = 2.5 Hz, 1H), 6.92 (dd,
J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.29-7.34 (m, 3H), 7.43 (br s,
1H), 7.51 (d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 1717, 1644, 1617.
Anal. (C20H18ClNO4) C, H, N.
N-Butyl-2-{7-[(3-chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}-
N-methylacetamide (21g). Purified by column chromatography
(eluent: CHCl3/MeOH, 9.7/0.3 v/v). Yield: 25%. 1H NMR
(300 MHz, CDCl3) δ: 0.90-1.03 (m, 3H), 1.25-1.67 (m, 4H),
2.98 (s, 3H) 3.06 (s, 3H), 3.32 (t, J = 7.4 Hz, 1H), 3.41 (t, J =
7.4 Hz, 1H) 3.78 (s, 2H), 5.10 (s, 2H), 6.15 (s, 1H), 6.86 (s, 1H),
6.91 (d, J = 8.8 Hz, 1H), 7.26-7.36 (m, 3H), 7.42 (br s, 1H), 7.49
(d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 2958, 2872, 1714, 1639,
1609. Anal. (C23H24ClNO4) C, H, N.
General Procedure for the Preparation of Amines 22a,d,h-j
and Free Amines of Mesylates 22b,c. The appropriate intermedi-
ate 2c-f (0.17 g, 0.50 mmol) and a 2.0 M solution of
N-methylamine or N,N-dimethylamine in THF (5.0 mL,
10 mmol) were stirred at 50 ꢀC under argon for 5 h. The mixture
was then cooled to room temperature, and the inorganic pre-
cipitate was filtered off. The solvent was then evaporated, and
the resulting solid was purified by column chromatography
using AcOEt as eluent.
7-(Benzyloxy)-4-[(methylamino)methyl]-2H-chromen-2-one
(22a). Yield: 44%. 1H NMR (300 MHz, DMSO-d6) δ: 2.67 (s,
3H), 4.42 (s, 2H), 5.24 (s, 2H), 6.45 (s, 1H), 7.09 (dd, J1 =
2.2 Hz, J2 = 8.8 Hz, 1H), 7.13 (d, J = 2.2 Hz, 1H), 7.33-7.47
(m, 5H), 7.76 (d, J = 8.8 Hz, 1H), NH not detectable. IR cm-1
(KBr): 1700, 1611. Anal. (C18H17NO3) C, H, N.
Ethyl [7-(Benzyl)oxy-2-oxo-2H-chromen-4-yl]acetate (20a).
1
Yield: 61%. H NMR (300 MHz, DMSO-d6) δ: 1.16 (t, J =
7.1 Hz, 3H), 3.64 (s, 2H), 4.09 (q, J = 7.1 Hz, 2H), 5.24 (s, 2H),
6.25 (s, 1H), 7.03-7.09 (m, 2H), 7.33-7.46 (m, 5H), 7.68 (d, J =
8.7 Hz, 1H). IR cm-1 (KBr): 1732, 1709, 1615.
Ethyl {7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}acetate
1
(20b). Yield: 48%. H NMR (300 MHz, DMSO-d6) δ: 1.16 (t,
J = 7.1 Hz, 3H), 3.64 (s, 2H), 4.09 (q, J = 7.1 Hz, 2H), 5.24 (s,
2H), 6.25 (s, 1H), 7.03-7.09 (m, 2H), 7.26-7.35 (m, 3H), 7.54 (s,
1H), 7.68 (d, J = 8.7 Hz, 1H). IR cm-1 (KBr): 1736, 1712, 1615.
General Procedure for the Synthesis of Amides 21a-g. An
amount of 2.0 mmol of a suitable ester 20a,b was placed in a
glass vessel together with 20 mmol of the appropriate amine in a
2.0 N THF solution, commercially available or freshly prepared
from the parent liquid amine. The vessel was sealed and placed
in an oven at 90 ꢀC for 24-60 h, until the starting ester
was consumed as indicated by the TLC control. The solvent
was then evaporated under vacuum, and the oily residue was
purified by column chromatography or crystallized from abso-
lute ethanol.
2-[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]-N-methylacetamide
(21a). Crystallized from ethanol. Yield: 50%. Mp: 235-6 ꢀC. 1H
NMR (300 MHz, DMSO-d6) δ: 2.57 (d, J = 4.4 Hz, 3H), 3.64 (s,
2H), 5.22 (s, 2H), 6.22 (s, 1H), 7.02 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz,
1H), 7.06 (d, J = 2.5 Hz, 1H), 7.30-7.47 (m, 5H), 7.66 (d, J =
8.8 Hz, 1H), 8.07 (br s, 1H). IR cm-1 (KBr): 3303, 1716, 1635,
1614. Anal. (C19H17NO4) C, H, N.
7-(1,3-Benzodioxol-5-ylmethoxy)-4-[(methylamino)methyl]-2H-
1
chromen-2-one (22d). Yield: 40%. Mp: 168-170 ꢀC. H NMR
(300 MHz, DMSO-d6) δ: 2.33 (s, 3H), 3.81 (s, 2H), 5.09 (s, 2H),
6.01 (s, 2H), 6.28 (s, 1H), 6.89-6.99 (m, 3H), 7.02-7.04 (m, 2H),
7.72 (d, J = 8.8 Hz, 1H), NH not detectable. IR cm-1 (KBr):
1705, 1614. Anal. (C19H17NO5) C, H, N.
7-(Benzyloxy)-4-[(dimethylamino)methyl]-2H-chromen-2-one
(22h). Yield: 61%. 1H NMR (300 MHz, CDCl3) δ: 2.34 (s, 6H),
3.54 (s, 2H), 5.13 (s, 2H), 6.32 (s, 1H), 6.88 (d, J = 2.5 Hz, 1H),
6.93 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.33-7.45 (m, 5H),
7.77 (d, J = 8.8 Hz, 1H). IR (cm-1): 1723, 1611, 1392. Anal.
(C19H19NO3) C, H, N.
7-[(3-Chlorobenzyl)oxy]-4-[(dimethylamino)methyl]-2H-chro-
men-2-one (22i). Yield:71%. Mp:78-80 ꢀC. 1HNMR(300MHz,
CDCl3) δ: 2.33 (s, 6H), 3.53 (s, 2H), 5.10 (s, 2H), 6.33 (s, 1H), 6.86
(d, J = 2.5 Hz, 1H), 6.92 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H),
7.28-7.34 (m, 3H), 7.43 (s, 1H), 7.78 (d, J = 8.8 Hz, 1H). IR cm-1
(KBr): 1708, 1614, 1385. Anal. (C19H18ClNO3) C, H, N.
4-[(Dimethylamino)methyl]-7-[(3-fluorobenzyl)oxy]-2H-chro-
men-2-one (22j). Yield: 74%. Mp: 84-6 ꢀC. 1H NMR (300 MHz,
CDCl3) δ: 2.32 (s, 6H), 3.51 (s, 2H), 5.12 (s, 2H), 6.31 (s, 1H),
6.85 (d, J = 2.5 Hz, 1H), 6.91 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H),
7.00-7.06 (m, 1H), 7.13-7.20 (m, 2H), 7.33-7.40 (m, 1H), 7.79
(d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 1704, 1615. Anal. (C19H18-
FNO3) C, H, N.
2-{7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}-N-methyl-
acetamide (21b). Purified by column chromatography (eluent:
CHCl3/MeOH, 9.5/0.5 v/v). Yield: 50%. Mp: 174-5 ꢀC. 1H
NMR (300 MHz, DMSO-d6) δ: 2.56 (s, 3H), 3.64 (s, 2H), 5.23
(s, 2H), 6.23 (s, 1H), 7.01-7.08 (m, 2H), 7.39-7.42 (m, 3H), 7.53
(s, 1H), 7.67 (d, J = 8.8 Hz, 1H), 8.07 (br s, 1H). IR cm-1 (KBr):
3294, 1718, 1640, 1614. Anal. (C19H16ClNO4) C, H, N.
N-Butyl-2-{7-[(3-chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}-
acetamide (21c). Crystallized from ethanol. Yield: 15%. Mp:
1
112-3 ꢀC. H NMR (300 MHz, CDCl3) δ: 0.87 (t, J = 7.2 Hz,
3H), 1.21-1.31 (m, 2H), 1.38-1.50 (m, 2H), 3.23 (q, J = 6.7 Hz,
2H), 3.64 (s, 2H), 5.10 (s, 2H), 5.51 (br s, 1H), 6.23 (s, 1H), 6.87 (d,
J= 2.5 Hz, 1H), 6.91 (dd, J1 =2.5Hz,J2 = 8.8 Hz, 1H), 7.30-7.34
(m, 3H), 7.43 (br s, 1H), 7.60 (d, J = 8.8 Hz, 1H). IR cm-1 (KBr):
3287, 1718, 1638, 1613. Anal. (C22H22ClNO4) C, H, N.
N-Benzyl-2-{7-[(3-chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}-
acetamide (21d). Crystallized from ethanol. Yield: 25%. Mp:
1
170-1 ꢀC. H NMR (300 MHz, CDCl3) δ: 3.69 (s, 2H), 4.42
7-[(3-Chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-
2-one (Free Amine of 22b). Yield: 18%. H NMR (300 MHz,
1
(d, J = 5.8 Hz, 2H), 5.10 (s, 2H), 5.90 (br s, 1H), 6.22 (s, 1H), 6.86
(d, J = 2.5 Hz, 1H), 6.92 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H),
7.15-7.18 (m, 2H), 7.26-7.37 (m, 6H), 7.43 (br s, 1H), 7.60 (d,
J = 8.8 Hz, 1H). IR cm-1 (KBr): 3280, 1727, 1639, 1619. Anal.
(C25H20ClNO4) C, H, N.
CDCl3) δ: 2.54 (s, 3H), 3.90 (s, 2H), 5.10 (s, 2H), 6.38 (s, 1H), 6.86 (d,
J= 2.8 Hz, 1H), 6.92 (dd, J1 =2.8Hz, J2 = 8.8 Hz, 1H), 7.31-7.34
(m, 3H), 7.43 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H), NH not detectable.
IR cm-1 (KBr): 1710, 1610. Anal. (C18H16ClNO3) C, H, N.
7-[(3-Fluorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-
2-one (Free Amine of 22c). Yield: 22%. Mp: 115-7 ꢀC. 1H NMR
(300 MHz, DMSO-d6) δ: 2.32 (s, 3H), 3.81 (s, 2H), 5.23 (s, 2H),
6.29 (s, 1H), 7.00 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.05 (d,
J = 2.5 Hz, 1H), 7.12-7.19 (m, 1H), 7.28-7.31 (m, 2H),
7.40-7.47 (m, 1H), 7.73 (d, J = 8.8 Hz, 1H), NH not detectable.
IR cm-1 (KBr): 1707, 1611. Anal. (C18H16FNO3) C, H, N.
2-[7-(Benzyloxy)-2-oxo-2H-chromen-4-yl]-N,N-dimethylaceta-
mide (21e). Crystallizedfromethanol.Yield:55%. Mp: 162-3 ꢀC.
1H NMR (300 MHz, DMSO-d6) δ: 2.83 (s, 3H), 3.07 (s, 3H), 3.93
(s, 2H), 5.21 (s, 2H), 6.15 (s, 1H), 6.99 (dd, J1 = 2.5 Hz, J2 =
8.8 Hz, 1H), 7.06 (d, J = 2.5 Hz, 1H), 7.30-7.47 (m, 5H), 7.55 (d,
J = 8.8 Hz, 1H). IR cm-1 (KBr): 1724, 1648, 1611. Anal.
(C20H19NO4) C, H, N.

Article
Mesylates 22b,c were prepared from the corresponding free
amines following the general procedure reported below.
General Procedure for the Preparation of Methanesulfonate
Salts. The suitable 4-aminoalkyl-7-benzyloxycoumarin deriva-
tive (1.1 mmol) was dissolved in dry THF (6.0 mL), and
methanesulfonic acid (0.080 mL, 1.2 mmol) was slowly added.
The resulting white solid was filtered, washed with dry THF,
and crystallized from absolute ethanol.
7-[(3-Chlorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-
2-one Methanesulfonate (22b). Mp: 213-5 ꢀC. 1H NMR (DMSO-
d6) δ: 2.31 (s, 3H), 2.71 (s, 3H), 4.44 (s, 2H), 5.27 (s, 2H), 6.41 (s,
1H), 7.10 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.14 (d, J = 2.5 Hz,
1H), 7.37-7.44 (m, 3H), 7.54 (s, 1H), 7.77 (d, J = 8.8 Hz, 1H),
9.01 (s, 2H, dis. with D2O). IR cm-1 (KBr): 1716, 1614. Anal.
(C19H20ClNO6S) C, H, N.
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6701
detectable. IR cm-1 (KBr): 3298, 1696, 1610. Anal. (C24H20-
ClNO3) C, H, N.
4-{[Benzyl(methyl)amino]methyl}-7-[(3-chlorobenzyl)oxy]-2H-
chromen-2-one (22k). Yield: 46%. Mp: 107-8 ꢀC. 1H NMR
(300 MHz, DMSO-d6) δ: 2.13 (s, 3H), 3.58 (s, 2H), 3.67 (s, 2H),
5.23 (s, 2H), 6.35 (s, 1H), 7.02 (dd, J1 = 1.9 Hz, J2 = 8.8 Hz, 1H),
7.05 (d, J = 1.9 Hz, 1H), 7.20-7.39 (m, 5H), 7.41-7.44 (m, 3H),
7.53(s, 1H), 7.85 (d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 1708, 1619.
Anal. (C25H22ClNO3) C, H, N.
Synthesis of Azides 23a,b. The appropriate chlorides 2c,d
(1.7 mmol) and NaN3 (0.44 g, 6.8 mmol) were refluxed in
absolute ethanol (17 mL) for 2 h. The mixture was cooled to
room temperature, and the solid residue was filtered off. The
solvent was then evaporated and the resulting oil was purified by
column chromatography using n-hexane/AcOEt, 8/2 (v/v), as
eluent, yielding a yellow solid.
7-[(3-Fluorobenzyl)oxy]-4-[(methylamino)methyl]-2H-chromen-
2-one Methanesulfonate (22c). Mp 215-6 ꢀC. 1H NMR (300
MHz, DMSO-d6) δ: 2.28 (s, 3H), 2.70 (s, 3H), 4.43 (s, 2H), 5.27
(s, 2H), 6.40 (s, 1H), 7.10 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.15
(d, J = 2.5 Hz, 1H), 7.16 (m, 1H), 7.28-7.31 (m, 2H), 7.41-7.45
(m, 1H), 7.76 (d, J = 8.8 Hz, 1H), 8.96 (s, 2H, dis. with D2O).
IR cm-1 (KBr): 1717, 1615. Anal. (C19H20FNO6S) C, H, N.
4-[(Ethylamino)methyl]-7-[(3-fluorobenzyl)oxy]-2H-chromen-
2-one Methanesulfonate (22e). Intermediate 2e (0.25 g, 0.80 mmol)
and the commercially available 70% solution of ethylamine in
water (3.0 mL) were stirred at room temperature under argon
for 3 h. The mixture was then poured into ice-water, and a
precipitated side product was filtered off. The solvent was evapo-
rated to dryness and the resulting solid was purified by column
chromatography using CHCl3/n-hexane/AcOEt, 7/2/1 (v/v/v) as
eluent, yielding a red oil that was further transformed into the
corresponding mesylate salt according to the procedure described
above. Yield: 35%. Mp: 242-4 ꢀC. 1H NMR (300 MHz, DMSO-
d6) δ: 1.25 (t, J = 7.2 Hz, 3H), 2.28 (s, 3H), 3.11-3.12 (m, 2H),
4.42-4.46 (m, 2H), 5.27 (s, 2H), 6.45 (s, 1H), 7.11 (dd, J1 =
2.5 Hz, J2 = 8.8 Hz, 1H), 7.15 (d, J = 2.5 Hz, 1H), 7.17-7.20 (m,
1H), 7.29-7.32 (m, 2H), 7.41-7.48 (m, 1H), 7.77 (d, J = 8.8 Hz,
1H), 8.88(s, 2H, dis.withD2O). IRcm-1 (KBr):1714, 1613. Anal.
(C20H22FNO6S) C, H, N.
7-[(3-Fluorobenzyl)oxy]-4-[(isopropylamino)methyl]-2H-chro-
men-2-one Methanesulfonate (22f). Intermediate 2e (0.25 g,
0.80 mmol) and the commercially available isopropylamine
(2.7 mL, 31 mmol) were refluxed for 2 h. The solution was
evaporated to dryness and the resulting oil was purified by
column chromatography using CHCl3/n-hexane/AcOEt 7/2/1
(v/v/v) as eluent, yielding a yellow oil that was further trans-
formed into the corresponding mesylate derivative according to
the procedure described above. Yield: 42%. Mp: 196-8 ꢀC. 1H
NMR (300 MHz, DMSO-d6) δ: 1.33 (d, J = 6.5 Hz, 6H), 2.29 (s,
3H), 3.47 (m, 1H), 4.43-4.47 (m, 2H), 5.29 (s, 2H), 6.47 (s, 1H),
7.12 (dd, J1 = 2.4 Hz, J2 = 8.9 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H),
7.21 (m, 1H), 7.30-7.33 (m, 2H), 7.42-7.49 (m, 1H), 7.81 (d,
J = 8.9 Hz, 1H), 8.82 (s, 2H, dis. with D2O). IR cm-1 (KBr):
1713, 1612. Anal. (C21H24FNO6S) C, H, N.
4-(Azidomethyl)-7-benzyloxy-2H-chromen-2-one (23a). Yield:
45%. 1H NMR (300 MHz, CDCl3) δ: 4.51 (s, 2H), 5.14 (s, 2H),
6.36 (s, 1H), 6.93 (br s, 1H), 6.96-6.97 (br s, 1H), 7.34-7.46
(m, 6H). IR cm-1 (KBr): 2116, 1714, 1614.
4-(Azidomethyl)-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one
(23b). Yield: 43%. 1H NMR (300 MHz, CDCl3) δ: 4.51 (s, 2H),
5.11 (s, 2H), 6.38 (s, 1H), 6.90 (d, J = 2.5 Hz, 1H), 6.94 (dd,
J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.30-7.35 (m, 3H), 7.43-7.47
(m, 2H). IR cm-1 (KBr): 2108, 1703, 1611.
Synthesis of Primary Amines 24a,b. To a clear solution of
SnCl2 dihydrate (0.66 g, 3.5 mmol) in dry methanol (5.0 mL) was
added over 1 h the appropriate 4-(azidomethyl)-7-(benzyloxy)-
2H-chromen-2-one 23a,b (0.40 mmol) in small portions. The
mixture was then stirred at room temperature for 3 h. The
solvent was then evaporated, the residue was poured into cold
water, the pH was made strongly basic with 3 N NaOH, and then
the resulting aqueous solution was extracted with AcOEt. The
organic layers were collected, washed with brine, dried over
Na2SO4, and evaporated in vacuum. The resulting solid was
further purified by column chromatography using CHCl3/
CH3OH, 9.7/0.3 (v/v), as eluent, yielding the desired primary
amines as white solids.
4-(Aminomethyl)-7-(benzyloxy)-2H-chromen-2-one
(24a).
Yield: 36%. Mp: 135-7 ꢀC (dec). 1H NMR (300 MHz,
DMSO-d6) δ: 3.90 (s, 2H), 5.21 (s, 2H), 6.38 (s, 1H), 6.99 (dd,
J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H),
7.30-7.47 (m, 5H), 7.67 (d, J = 8.8 Hz, 1H), 2 NHs not
detectable. IR cm-1 (KBr): 3391, 3320, 1703, 1611. Anal.
(C17H15NO3) C, H, N.
4-(Aminomethyl)-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one
(24b). Yield: 39%. Mp: 166-7 ꢀC. 1H NMR (300 MHz,
DMSO-d6) δ: 3.90 (s, 2H), 5.23 (s, 2H), 6.39 (s, 1H), 7.00 (dd,
J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H),
7.39-7.48 (m, 3H), 7.53 (s, 1H), 7.69 (d, J = 8.8 Hz, 1H), 2 NHs
not detectable. IR cm-1 (KBr): 3393, 1711, 1612. Anal.
(C17H14ClNO3) C, H, N.
Synthesis of Nitriles 25a,b. An amount of 0.38 mmol of 11a or
11b and 0.061 mL (0.76 mmol) of anhydrous pyridine were
dissolved in 4.0 mL of anhydrous dioxane. The mixture
was cooled at 0 ꢀC with an external ice bath, and trifluoroacetic
anhydride (0.068 mL, 0.48 mmol) was added dropwise. The
clear solution was allowed to reach room temperature and after
10 min was poured into ice. The aqueous solution was extracted
with chloroform and the organic phase was dried over anhy-
drous Na2SO4, filtered, and evaporated under vacuum to give a
white solid crystallized from ethanol.
Synthesis of Amines 22g and 22k. 7-[(3-Chlorobenzyl)oxy]-
4-(chloromethyl)-2H-chromen-2-one 2d (0.40 g, 1.2 mmol),
K2CO3 (0.17 g, 1.2 mmol), and benzylamine (0.66 mL, 6.0 mmol)
or N-benzyl-N-methylamine (0.15 mL, 1.2 mmol) were stirred in
refluxing absolute ethanol (10 mL) for 2-5 h, until the disappear-
ance of the 2d spot as indicated by the TLC control. The reaction
mixture was cooled to room temperature, the inorganic solid
residue was filtered off, the solvent was evaporated, and the
resulting oil was purified by column chromatography using
CHCl3/n-hexane/AcOEt 7/2/1 (v/v/v) as eluent, giving a solid,
which was crystallized from absolute ethanol yielding a yellow solid.
4-[(Benzylamino)methyl]-7-[(3-chlorobenzyl)oxy]-2H-chromen-
(7-Benzyloxy-2-oxo-2H-chromen-4-yl)acetonitrile (25a). Yield:
86%. Mp: 178-9 ꢀC. 1H NMR (300 MHz, DMSO-d6) δ: 4.37 (s,
2H), 5.23 (s, 2H), 6.32 (s, 1H), 7.09 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz,
1H), 7.13 (d, J = 2.5 Hz, 1H), 7.30-7.46 (m, 5H), 7.66 (d, J =
8.8 Hz, 1H). IR cm-1 (KBr): 2938, 1714, 1612, 1395, 1283. Anal.
(C18H13NO3) C, H, N.
1
2-one (22g). Yield: 18%. Mp: 133-5 ꢀC. H NMR (300 MHz,
CDCl3) δ: 3.93 (s, 2H), 3.94 (s, 2H), 5.09 (s, 2H), 6.49 (s, 1H), 6.87
(dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H),
7.27-7.39 (m, 8H), 7.43 (s, 1H), 7.54 (d, J = 8.8 Hz, 1H), NH not
{7-[(3-Chlorobenzyl)oxy]-2-oxo-2H-chromen-4-yl}acetonitrile
(25b). Yield: 97%. Mp: 180-2 ꢀC. 1H NMR (300 MHz,

6702 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
DMSO-d6) δ: 4.37 (s, 2H), 5.25 (s, 2H), 6.33 (s, 1H), 7.09 (d, J =
2.5 Hz, 1H), 7.12-7.14 (m, 1H), 7.37-7.44 (m, 3H), 7.54 (s, 1H),
7.67 (d, J = 8.8 Hz, 1H). IR cm-1 (KBr): 2936, 1716, 1613, 1396,
1268. Anal. (C18H12ClNO3) C, H, N.
and the oily residue was purified by flash chromatography
(eluent CHCl3/n-hexane, 8/2 v/v, for 29a and CHCl3/n-hexane,
7/3 v/v, for 29b) to give white solids.
7-(Benzyloxy)-4-(2-bromoethyl)-2H-chromen-2-one (29a).
Yield: 82%. 1H NMR (300 MHz, DMSO-d6) δ: 3.34 (t, J = 6.8
Hz, 2H), 3.82 (t, J = 6.8 Hz, 2H), 5.21 (s, 2H), 6.27 (s, 1H), 7.02
(dd, J1 = 2.2 Hz, J2 = 9.0 Hz, 1H), 7.08 (d, J = 2.2 Hz, 1H),
7.30-7.47 (m, 5H), 7.75 (d, J = 9.0 Hz, 1H). IR cm-1 (KBr):
1727, 1609.
4-(2-Bromoethyl)-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one
(29b). Yield: 59%. 1H NMR (300 MHz, CDCl3) δ: 3.30 (t, J =
7.2 Hz, 2H), 3.64 (t, J = 7.2 Hz, 2H), 5.11 (s, 2H), 6.20 (s, 1H),
6.89 (d, J = 2.5 Hz, 1H), 6.95 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H),
7.32-7.33 (m, 3H), 7.44 (s, 1H), 7.50 (d, J = 8.8 Hz, 1H). IR
cm-1 (KBr): 1718, 1610.
Synthesis of Amines 30a,b. An amount of 0.51 mmol of the
suitable bromide 29a,b was added to 3.8 mL (7.6 mmol) of a
2.0 M solution of methylamine (for 30a) or dimethylamine
(for 30b) in THF, followed by anhydrous K2CO3 (0.070 g,
0.51 mmol) and KI (0.009 g, 0.051 mmol). The mixture was
stirred at 50 ꢀC overnight. The precipitate was filtered off and
the solvent was evaporated under vacuum to give an oily residue
that was purified by chromatography (eluent CHCl3/MeOH,
9/1 v/v) and crystallized from ethanol.
4-(2-Aminoethyl)-7-[(3-chlorobenzyl)oxy]-2H-chromen-2-one
Hydrochloride (26). Amounts of 0.033 g (0.10 mmol) of 25b and
0.048 g (0.20 mmol) of CoCl2 H2O were suspended in 2.0 mL of
3
methanol. An amount of 0.038 g (1.0 mmol) of sodium bor-
ohydride was added to the suspension, and the reaction mixture
was stirred at room temperature for 1 h. Then 1.0 mL of 2.0 N
HCl was added and the organic solvent was evaporated under
vacuum. The acid solution was cooled at 0 ꢀC, and an amount
of 5.0 mL of a solution of ammonia 30% in water was added.
The basic solution was extracted with ethyl acetate and the
organic phase dried over anhydrous Na2SO4, filtered, and
evaporated to dryness, yielding a yellow solid which was
dissolved in 2.0 mL of chloroform, and 1.0 mL of 3 N HCl
was added. A precipitate was obtained and filtered, corre-
sponding to the hydrochloric salt of the desired product. Yield:
20%. Mp: 113 ꢀC (dec), 176-8 ꢀC. 1H NMR (300 MHz,
DMSO-d6) δ: 3.08 (br, 4H), 5.26 (s, 2H), 6.27 (s, 1H), 7.07
(dd, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 7.11 (d, J = 2.5 Hz, 1H),
7.42-7.44 (m, 3H), 7.54 (s, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.96
(br s, 3H, dis. with D2O). IR cm-1 (KBr): 3056, 1723, 1615.
Anal. (C18H17Cl2NO3) C, H, N.
7-[(3-Chlorobenzyl)oxy]-4-[2-(methylamino)ethyl]-2H-chromen-
1
7-Hydroxy-4-(2-hydroxyethyl)-2H-chromen-2-one (27). An
(4.3 mmol) of commercially available
2-one (30a). Yield: 29%. Mp: 72 ꢀC (dec), 137-9 ꢀC. H NMR
amount of 0.94
g
(300 MHz, DMSO-d6) δ: 2.34 (s, 3H), 2.84-2.92 (m, 4H), 5.24 (s,
2H), 6.19 (s, 1H), 7.04 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 7.08 (d,
J = 2.5 Hz, 1H), 7.42-7.44 (m, 3H), 7.54 (s, 1H), 7.76 (d, J =
8.8 Hz, 1H), NH not detectable. IR cm-1 (KBr): 1719, 1609. Anal.
(C19H18ClNO3) C, H, N.
7-hydroxycoumarin-4-acetic acid 5 was dissolved in 25 mL of
anhydrous tetrahydrofuran. The mixture was cooled at 0 ꢀC with
an external ice bath, and an amount of 13 mL (13 mmol) of a 1.0 M
solution of borane in THF was added dropwise. The mixture was
allowed to reach room temperature and stirred for 6 h. The mixture
was cooled at 0 ꢀC, and an amount of 20 mL of methanol was added.
The solvent was evaporated under vacuum and the residue obtained
was dissolved in ethyl acetate, washed with water, dried over
anhydrous Na2SO4, filtered, and evaporated to give a solid that
was purified by flash chromatography (eluent CHCl3/MeOH, 9/1
v/v) to give a white solid of satisfactory purity. Yield: 54%. 1HNMR
(300 MHz, DMSO-d6) δ: 2.86 (t, J = 6.3 Hz, 2H), 3.65-3.71 (m,
2H), 4.80 (t, J = 5.2 Hz, 1H), 6.09 (s, 1H), 6.70 (d, J = 2.2 Hz, 1H),
6.78 (dd, J1 = 2.2 Hz, J2 = 8.7 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H),
10.54 (s, 1H). IR cm-1 (KBr): 1686, 1611.
Synthesis of Alcohols 28a,b. The intermediate 27 (0.21 g,
1.0 mmol) was dissolved in 10.0 mL of absolute ethanol, and
K2CO3 (0.14 g, 1.0 mmol) and the corresponding benzyl bro-
mide (2.0 mmol) were added to the solution, and the mixture was
refluxed for 45 min. The precipitate was filtered off, and the
organic solution was evaporated under vacuum. The oily resi-
due was purified by flash chromatography (eluent CHCl3/
MeOH, 9.5/0.5 v/v) to give a white solid.
7-(Benzyloxy)-4-[2-(dimethylamino)ethyl]-2H-chromen-2-one
(30b). Yield: 20%. Mp: 163-4 ꢀC. 1H NMR (300 MHz, DMSO-
d6) δ: 2.84 (s, 6H), 3.17-3.25 (m, 2H), 3.32-3.40 (m, 2H), 5.23
(s, 2H), 6.28 (s, 1H), 7.06 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H),
7.11 (d, J = 2.5 Hz, 1H), 7.31-7.48 (m, 5H), 7.84 (d, J = 8.8 Hz,
1H). IR cm-1 (KBr): 1721, 1617. Anal. (C20H21NO3) C, H, N.
pKa and log P Determination of 22b. pKa and octanol-water
partition coefficient of compound 22b were determined by
potentiometric titration using the Sirius GLpKa instrument
implemented by the software RefinementPro (Sirius Analitical
Ltd., Forest Row, East Sussex, U.K.). For pKa determination, a
5 ꢀ 10-4 M concentration of the sample in a 0.15 M KCl
solution was brought to pH 1.8 through the addition of 0.5 M
HCl and then titrated up to pH 12.2 with carbonate-free 0.5 M
KOH at a temperature of 25 ( 0.5 ꢀC, under a slow argon flow to
avoid CO2 absorption at high pHs. An analogous measurement
has been performed in the absence of compound, for the
standardization of the instrument (blank curve). Detailed
instrumental procedures can be found elsewhere.86,87
7-(Benzyloxy)-4-(2-hydroxyethyl)-2H-chromen-2-one (28a).
Yield: 53%. 1H NMR (300 MHz, DMSO-d6) δ: 2.89 (t,
J = 6.3 Hz, 2H), 3.66-3.72 (m, 2H), 4.80 (t, J = 5.5 Hz, 1H),
5.21 (s, 2H), 6.17 (s, 1H), 7.01 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz,
1H), 7.06 (d, J = 2.5 Hz, 1H), 7.30-7.47 (m, 5H), 7.73 (d, J =
8.8 Hz, 1H). IR cm-1 (KBr): 1697, 1614.
The curves of sample and blank titrations were elaborated
through a computerized mathematical algorithm with the soft-
ware RefinementPro to get the differential curve of Bjerrum,
that shows the number of free protons from the compound
dissociation (n) versus pH. These calculations indicate a pKa of
7.18 ( 0.05, from three independent measurements.
7-[(3-Chlorobenzyl)oxy]-4-(2-hydroxyethyl)-2H-chromen-2-one
(28b). Yield: 67%. 1H NMR(300MHz, DMSO-d6) δ: 2.89 (t, J =
6.3 Hz, 2H), 3.69 (t, J = 6.3 Hz, 2H), 4.78 (br s, 1H), 5.23 (s, 2H),
6.18 (s, 1H), 7.02 (dd, J1 = 2.5 Hz, J2 = 8.6 Hz, 1H), 7.06 (d, J =
2.5 Hz, 1H), 7.41-7.43 (m, 3H), 7.53 (s, 1H), 7.74 (d, J = 8.6 Hz,
1H). IR cm-1 (KBr): 3068, 1718, 1610.
Synthesis of Bromides 29a,b. Amounts of 1.0 mmol of 28a
(0.30 g, for 29a) or 28b (0.33 g, for 29b) and 0.73 g (2.2 mmol) of
carbon tetrabromide were dissolved in 10 mL of anhydrous
dichloromethane. The mixture was cooled at 0 ꢀC with an
external ice bath, and triphenylphosphine (0.53 g, 2.0 mmol)
dissolved in 2.0 mL of anhydrous dichloromethane, was added
dropwise. The mixture was allowed to reach room temperature
and stirred for 1 h. The solvent was evaporated under vacuum
The experimental log P values was determined through a
potentiometric titration in a biphasic system constituted by a
solution of 0.15 M KCl saturated with 1-octanol.
In a two-phases system, the dissociation of acids or bases is
reduced because of the partitioning. This involves a shift of the
Bjerrum plot, toward the right for acids and the left for bases.
The new value of dissociation constant measured is an apparent
pKa (poKa).
The entity of pKa shift allows the determination of the
experimental value of log P through the following equations.
For monoprotic acid, it is
10poKa -pK
a
P ¼
-1
r

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6703
For monoprotic basic, it is
compartments; 0.1 M phosphate buffer solution (pH 6.8) was
used).
10pK
a -poKa
P ¼
-1
After 5 h, the sandwich was disassembled and both donor and
acceptor compartments were transferred to a UV quartz plate
(Hellma GmbH & Co., Mullheim, Germany). UV absorption
was measured with a PowerWave (Bio-Tek Instruments, Inc.,
Winooski, VT), and the reading was performed at λmax of the
compounds.
The very low concentration (in both donor and acceptor
compartments) of 3 renders the UV detection ineffective, and
therefore, the detection was performed using HPLC Merck
Hitachi apparatus with a Discovery RP amide C16 (20 mm ꢀ
4 mm i.d., 5 μm) (Supelco, Bellefonte, PA).
To assess membrane stability, electrical resistance measure-
ments were conducted on the filter plate at the end of the
incubation time, using an electrometer system especially de-
signed for PAMPA assays (EVOMX and MULTI96, World
Precision Instruments, Sarasota, FL).
Molecular Modeling. Molecular Building and Optimization.
The coumarin inhibitors were built from the Sybyl (version 8.2)
fragment libraries starting from the reference ligand (4-FCBC)
cocrystallized with hMAO-B (PDB code 2v60). Geometrical
optimization and charge calculation were made by means of a
quantum mechanical method with the PM3 Hamiltonian. The
starting conformation for docking of the 7-(m-halogeno)-
benzyloxy substituents was recovered from the X-ray crystal-
lographic structures of 4-FCBC and safinamide bound to
hMAO-B.56
r
The log P value of 22b coming from these calculations was
2.48 ( 0.03 (from three separated experiments).
Aqueous Solubility Assay. Compound 22b was dissolved in
DMSO at a concentration of 2 and 180 mg/mL. The appropriate
solution was added in portions (2 μL at a time) to 1 mL of a 50
mM Tris-HCl, pH 7.4, or of a 50 mM acetate buffer, pH 3.0,
solution at room temperature. Typically a total of 10 additions
were made so that the final volume of DMSO was well below
5%. The appearance of the precipitate was detected by an
absorbance increase due to light scattering by particulate ma-
terial, in a dedicated diode array UV spectrometer (GE Health-
care, Uppsala, Sweden). Increased UV absorbance was
measured in the 600-820 nm range. In its simplest implementa-
tion, the precipitation point (i.e., the upper aqueous solubility
limit) was calculated from a bilinear curve fit in a plot of the
absorbance (y axis) versus μL of DMSO (x axis). The solubility
was 52 ( 5 μg /mL and 2.82 ( 0.35 mg/mL at pH 7.4 and pH 3.0,
respectively, as the mean ( SEM of three independent assays.
HDM-PAMPA Permeability Assay. Equation 1 below was
used to calculate the effective permeability coefficient (Pe):
"
#
ꢀ
ꢁ
ꢀ
ꢁ
2:303VD
VA
VAþVD CAðtÞ
Pe ¼ -
log 1 -
Aðt -τLAG
Þ
VAþVD
VDð1 -RÞ CDð0Þ
(1Þ
Homology Modeling. The 3D model of the rMAO-B (entry
name of AOFB_RAT; primary accession number of P19643 at
the ExPASy proteomics server) was developed starting from the
X-ray structure of the hMAO-B (PDB code 2v60) complexed
with the 4-FCBC ligand. The two MAO-B from different species
presented highly close amino acid sequences (520aa vs 520aa,
88% of sequence similarity) and almost identical amino acid
residues in the binding site where the only difference was the
substitution of Ile316 in human by Val316 in rat. The three-
dimensional model of rMAO-B was constructed through
homology modeling within Modeller (version 9.2). Among the
five best solutions derived from Modeller, the one provided with
the lowest value of the Modeller objective scoring function was
selected for the subsequent docking simulations. The stereo-
chemical quality of this model, as well as the overall residue-by-
residue geometry, was controlled with Procheck (version 3.5.4).
By selection of a resolution value equal to 2.0 A, the Ramachan-
dran plot returned 99.5% of residues in the core regions that
represent the most favorable and allowed combinations of j and
ψ angle values while only the remaining 0.5% of residues (Lys52
and Ala346) was located in disallowed regions. As expected,
the fitting of the backbone R-carbons of theoretical (rMAO-B)
and experimental (hMAO-B) models gave a very low devia-
tion (rms 0.096 A), indicating a substantial conservation of
their spatial position. By Modeller modeling of all the heteroa-
toms, it was possible to rebuild the coordinates of FAD cofactor
and the eight structural water molecules coming from hMAO-B
2v60.
where A the accessible filter area (cm2) (i.e., filter area multiplied
by filter porosity), t is incubation time (s), VA and VD are
respectively the volumes in the acceptor and the donor wells
(cm3), CA(t) is the concentration of the compound (mol cm-3) in
the acceptor well at time t, and CD(0) is the concentration of the
compound (mol cm-3) in the donor well at time 0. R is the
retention factor, calculated by eq 2 and defined as the mole
fraction that is lost in the membrane and in the microplates (i.e.,
filters and plate materials):
ꢀ
ꢁꢀ
ꢁ
CDðtÞ
CDð0Þ
VA
CAðtÞ
R ¼ 1 -
-
(2Þ
VD CDð0Þ
τLAG is the steady-state time (s), i.e., the time needed for the
permeant’s concentration gradient to become stabilized. Math-
ematically τLAG is the time at which Fick’s second law has
transformed into the limiting situation of Fick’s first law.
Steady-state times (τLAG) to saturate the membranes in PAMPA
are short relative to the total permeation time (∼20 min with
unstirred plates), and for this reason they are usually neglected.
Permeation experiments were carried out in polycarbonate
(PC) 96-well microtiter filter plates (Millipore, Bellerica, MA).
Filter specifications of PC filter plates were 3 μm pore size, 10
μm thickness, and 12.5% porosity (the manufacturer specifica-
tions were 5-20% porosity range; thus, the average porosity
value was taken in this work). Each well was coated with 0.75 μL
of hexadecane. Because of the high viscosity of hexadecane
(HDC), hexane was used as a solvent (15 μL of 5% (v/v) HDC
in hexane was dispensed in each well), and after the application
of the membrane, the resulting filter plates were placed under a
hood for 20 min to completely evaporate the hexane. Subse-
quently, the donor plate was placed upon a Teflon acceptor
plate (MSSACCEPTOR, Millipore, Bellerica, MA) which had
been prefilled with 280 μL of buffer containing 5% DMSO. The
system was finally hydrated with 280 μL of tested compounds in
buffer containing 5% DMSO, and the resulting sandwich was
incubated for 5 h at room temperature under constant shaking
(75 rpm). Reference compound concentrations were chosen
according to their solubility (i.e., 2.5 mM for 22b and 617 μM
for 3). Each compound was measured in quadruplicate, and
iso-pH conditions were used (same pH in donor and acceptor
The seven conserved water molecules in hMAO-A crystal
structure (PDB code 2z5x) were transferred into the binding site
of rMAO-A by using Sybyl.
Docking Simulations. GOLD (version 2.4), a genetic algorithm-
based software, was used for a 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). Para-
meters used in the fitness function (hydrogen bond energies, atom
radii and polarizabilities, torsion potentials, hydrogen bond
directionalities, and so forth) were taken from the GOLD para-
meter file.

6704 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Pisani et al.
In the present study, the 3D coordinates of hMAO-B (PDB
code 2v60), rat and human MAO-A (PDB codes 1o5w and 2z5x,
respectively) were retrieved from the Protein Data Bank. The
protein preparation for docking study was performed in Sybyl:
the cocrystallized ligands were removed; the correct atom type
and the bond orders were assigned to FAD cofactor; by use
of the Biopolymer tool, hydrogen atoms were added. The
structure model of rMAO-B from Modeller was prepared for
docking simulation applying the same protocol described
above. For each coumarin inhibitor, 10 conformations were
generated in a sphere of a 12 A radius centered on phenolic
oxygen atom of Tyr435 and Tyr444 in rMAO-B and rMAO-A,
respectively. In our docking runs, the molecular scaffold of the
best ranked solution of the 2v60 reference ligand docked into the
rMAO-B was set as physical constraint to favor the occurrence
of the known binding mode of coumarin inhibitors.
Biological Assays. In Vitro MAO-A and MAO-B Inhibition.
MAO Enzyme Source and Membrane Preparations (Crude Rat
Brain Mitochondrial Fraction). Male Wistar rats (Harlan, Italy,
150-175 g) were sacrificed under light anesthesia, and brains
were rapidly removed and homogenized in 10 volumes of ice-
cold 0.32 M sucrose buffer containing 0.1 M EDTA, pH 7.40.
The crude homogenate was centrifuged at 2220 rpm for 10 min
and the supernatant recovered. The pellet was homogenized and
centrifuged again, and the two supernatants were pooled and
centrifuged at 9250 rpm for 10 min at 4 ꢀC. The pellet was
resuspended in fresh buffer and centrifuged at 11250 rpm for
10 min at 4 ꢀC. The resulting pellet was stored at -80 ꢀC until use.
Human Platelet Rich Plasma (PRP) Preparation. The human
venous blood (3.8% sodium citrate as anticoagulant) was im-
mediately centrifuged at 125g for 15 min at 4 ꢀC. The super-
natant was removed and stored in ice. The pellet was again
centrifuged at 600g for 5 min at 4 ꢀC. The two PRP supernatants
were pooled and stored at -80 ꢀC until assay.
In Vitro MAO-A and MAO-B Inhibition Assays in Rat Brain
Mitochondria. The enzyme activities were assessed with a radio-
enzymatic assay using the selective substrates 14C-serotonin
(5-HT) and 14C-phenylethylamine (PEA) for MAO-A and
MAO-B, respectively. The mitochondrial pellet (500 μg protein)
was resuspended in 0.1 M phosphate buffer, pH 7.40, and
500 μL was added to 50 μL of the test compound or buffer for
30 min at 37 ꢀC (preincubation). Then the substrate (50 μL) was
added. The incubation was carried out for 30 min at 37 ꢀC
([14C]5-HT, 5 μM) or for 10 min at 37 ꢀC ([14C]PEA, 0.5 μM).
The reaction was stopped by adding 0.2 mL of 37% HCl or
0.2 M perchloric acid. After centrifugation, the deaminated
metabolites were extracted with 3 mL of diethyl ether (5-HT)
or toluene (PEA) and the radioactive organic phase was mea-
sured by liquid scintillation spectrometry at 90% efficiency.
Radioactivity in the eluate indicates the production of neutral
and acidic metabolites formed as a result of MAO activity. The
enzymatic activity was expressed as nanomoles of substrate
transformed per milligram of protein per minute. The activity
of MAO in the sample was expressed as a percentage of control
activity in the absence of inhibitors after subtraction of appro-
priate blank values.
InVitro MAO-B Inhibition Assay inPRP Preparation. MAO-B
enzyme activity was assessed with a radioenzymatic assay using
[14C]PEA as selective substrates (3). The PRP was diluted 1:5 in
0.1 M phosphate buffer, pH 7.40, and 500 μL (in duplicate) was
added to 50 μL of buffer and to 50 μL of substrate ([14C]PEA,
0.5 μM). The incubation was carried out for 10 min at 37 ꢀC.
The reaction was stopped by adding 0.2 mL of perchloric acid.
After centrifugation, the acidic metabolites were extracted by
3.5 mL of toluene and an amount of 3 mL of the radioactive
organic phase was added to 10 mL of scintillation cocktail Insta-
Fluor and measured by liquid scintillation spectrometry at 90%
efficiency.
dependent experiments. Time-dependent inhibition kinetics
were measured as IC50 values after 0 or 30 min of enzyme-
inhibitor preincubation. The absence of a significant difference
between IC50 with or without preincubation was considered as
indicative of reversible inhibition.
Ex Vivo MAO-A and MAO-B Inhibition. C57Bl mice were
treated with the test compound at different concentrations
(0.5 to 20 mg/kg po and ip), and at different time intervals
(0.5, 1, 2, 4, 8, 16, and 24 h) they were sacrificed. The brains were
removed, and crude homogenates were prepared in 0.1 M
phosphate buffer pH 7.40. The enzyme inhibition were per-
formed with a radioenzymatic assay as described above.
In Vitro Human Recombinant Cytocrome P450 Isoform Assay.
Inhibition of the six most important P450 isoforms (CYP1A2,
CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) was
measured in specific assays using suitable substrates that
become fluorescent upon CYP metabolism (Gentest Kit assay,
BD Biosciences, Bedford, MA). Compounds were tested in a
96-well plate containing incubation/NADPH regenerating
buffer. Specific human recombinant isoenzymes (supersomes)
and substrates were added and incubated at 37 ꢀC. The specific
substrates were the following: 3-cyano-7-ethoxycoumarin
(CYP2C19 and CYP1A2), 7-methoxy-4-trifluoromethylcou-
marin (CYP2C9), 3-[2-(N,N-diethyl-N-methylamonium)ethyl]-
7-methoxy-4-methylcoumarin (CYP2D6), 7,8-benzoquinoline
(CYP3A4), 7-methoxy-4-phenylcoumarin (2E1). The plates
were read on a Victo3vr plate reader (Perkin-Elmer, Wellesley,
MA) at the appropriate emission/excitation wavelengths.
Known inhibitors from literature for each isoenzyme were tested
in every assay as positive control.
In Vitro Cell Viability Assay. The SHSY-5Y continuous cell
line from a human neuroblastoma (Istituto Zooprofilattico,
Brescia, Italy) was chosen from the present study. Cells, showing
a neuronal-like morphology and a growth adherent to the
plastic culture surface, were routinely maintained in Dulbecco’s
modified essential medium (DMEM) supplemented with 10%
of heat inactivated FBS plus 1% nonessential amino acid, 2 mM
glutamine, and 100 U/mL penicillin þ 100 μg/mL streptomycin
and were grown at 37 ꢀC in an atmosphere of 95% air and 5%
CO2. Culture medium was replaced every 24 h. Growth con-
stituents unless otherwise stated were purchased from Life
Technologies. Cells, routinely split 1:5, were transplanted by
trypsin-EDTA solution (0.05-0.02% in PBS) and plated
according to experimental needing.
The experimental protocol assay was as follows: at time 0 cells
were seeded at 6.25 ꢀ 104/cm2 in 96-well cell culture plates in
complete growth medium, and after 72 h at subconfluent phase,
growth medium was removed and neurobasal medium (with
2 mM glutamine and 100 U/mL penicillin þ 100 μg/mL strep-
tomycin, without serum) was added 30 min before the addition
of compound 22b. The assay proceeded with a 24 h incubation at
37 ꢀC in serumless neurobasal medium (200 μL/well) with or
without 22b at 1, 3, 10, 30, and 100 μM. At the end of incubation,
cell viability was assessed by a colorimetric assay by using
the MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]) kit by
Promega (Promega Corp., Madison, WI).
Data Elaboration and Statistical Analysis. Enzyme inhibition
curves were obtained from at least seven different concentrations,
each in duplicate (from 10-10 to 10-4 M), and the IC50 values
with confidence intervals were determined using nonlinear
regression analysis (GraphPad Prism). Data were expressed
as the mean ( SEM and were analyzed by ANOVA followed
by Dunnett’s test. Generally, SEM values were lower than 15%
of the calculated mean.
Acknowledgment. We thank Dr. Sophie Martel and Prof.
Pierre-Alain Carrupt from the School of Pharmaceutical
Sciences, University of Geneva;University of Lausanne,
In Vitro MAO-B Inhibition Reversibility Studies. The reversi-
bility of MAO-B inhibition was assessed performing time-

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6705
Switzerland, for helpful advice on the HDM-PAMPA assay
and Dr. Piero Melloni from Newron Pharmaceuticals, Bresso,
Italy, for helpful discussions and support in the execution of
the present work. The experimental determinations of pKa
and log P by Filomena Fiorella, Dipartimento Farmacochi-
mico, University of Bari, Italy, and of the aqueous solubility
by Jose Manuel Brea Floriani, Department of Pharmacology,
University of Santiago de Compostela, Spain, are kindly
acknowledged. The Spanish authors thank the Ministerio de
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