Structure-Based Design and Optimization of Multitarget-Directed 2 H -Chromen-2-one Derivatives as Potent Inhibitors of Monoamine Oxidase B and Cholinesterases

Article abstract of DOI:10.1021/acs.jmedchem.5b00599

The multifactorial nature of Alzheimer's disease calls for the development of multitarget agents addressing key pathogenic processes. To this end, by following a docking-assisted hybridization strategy, a number of aminocoumarins were designed, prepared, and tested as monoamine oxidases (MAOs) and acetyl- and butyryl-cholinesterase (AChE and BChE) inhibitors. Highly flexible N-benzyl-N-alkyloxy coumarins 2-12 showed good inhibitory activities at MAO-B, AChE, and BChE but low selectivity. More rigid inhibitors, bearing meta- and para-xylyl linkers, displayed good inhibitory activities and high MAO-B selectivity. Compounds 21, 24, 37, and 39, the last two featuring an improved hydrophilic/lipophilic balance, exhibited excellent activity profiles with nanomolar inhibitory potency toward hMAO-B, high hMAO-B over hMAO-A selectivity and submicromolar potency at hAChE. Cell-based assays of BBB permeation, neurotoxicity, and neuroprotection supported the potential of compound 37 as a BBB-permeant neuroprotective agent against H₂O₂-induced oxidative stress with poor interaction as P-gp substrate and very low cytotoxicity.

Full text of DOI:10.1021/acs.jmedchem.5b00599

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Article
Structure-Based Design and Optimization of Multitarget-
Directed 2H-Chromen-2-one Derivatives as Potent
Inhibitors of Monoamine Oxidase B and Cholinesterases
Roberta Farina, Leonardo Pisani, Marco Catto, Orazio Nicolotti, Domenico Gadaleta, Nunzio
Denora, Ramón Soto-Otero, Estefanía Méndez-Álvarez, Carolina S. Passos, Giovanni
Muncipinto, Cosimo D. Altomare, Alessandra Nurisso, Pierre Alain Carrupt, and Angelo Carotti
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 24 Jun 2015
Downloaded from http://pubs.acs.org on June 25, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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StructureꢀBased Design and Optimization of
MultitargetꢀDirected 2HꢀChromenꢀ2ꢀone Derivatives
as Potent Inhibitors of Monoamine Oxidase B and
Cholinesterases.
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a
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Roberta Farina, Leonardo Pisani, Marco Catto,* Orazio Nicolotti, Domenico Gadaleta,
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Nunzio Denora, Ramon Soto-Otero, Estefania Mendez-Alvarez, Carolina S. Passos, Giovanni
a,
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Muncipinto, † Cosimo D. Altomare, Alessandra Nurisso, Pierre-Alain Carrupt, Angelo
,a
Carotti*
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Dipartimento di Farmacia − Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”,
Via E. Orabona, 4, Iꢀ70125 Bari, Italy
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Grupo de Neuroquimica, Departamento de Bioquimica y Biologia Molecular, Facultad de
Medicina, Universidad de Santiago de Compostela, San Francisco I, Eꢀ15782, Santiago de
Compostela, Spain
c
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ernest
Ansermet, 30, CHꢀ1211, Geneva 4, Switzerland
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KEYWORDS: monoamine oxidase inhibitors; acetylcholinesterase inhibitors; multiꢀtarget ligand
design; Alzheimer’s disease; Parkinson’s disease; neurodegenerative diseases; coumarin
derivatives.
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ABSTRACT
The multifactorial nature of Alzheimer’s disease calls for the development of multitarget agents
addressing key pathogenic processes. To this end, by following a dockingꢀassisted hybridization
strategy, a number of aminocoumarins were designed, prepared and tested as monoamine
oxidases (MAOs), acetylꢀ and butyrylꢀcholinesterase (AChE and BChE) inhibitors. Highly
flexible NꢀbenzylꢀNꢀalkyloxy coumarins 2ꢀ12 showed good inhibitory activities at MAOꢀB,
AChE and BChE, but low selectivity. More rigid inhibitors, bearing metaꢀ and paraꢀxylyl
linkers, displayed good inhibitory activities and high MAOꢀB selectivity. Compounds 21, 24, 31,
37 and 39, the last two featuring an improved hydrophilic/lipophilic balance, exhibited excellent
activity profiles with nanomolar inhibitory potency toward hMAOꢀB, high hMAOꢀB over
hMAOꢀA selectivity and subꢀmicromolar potency at hAChE. Cellꢀbased assays of BBBꢀ
permeation, neurotoxicity and neuroprotection supported the potential of compound 37 as a
BBBꢀpermeant neuroprotective agent against H O ꢀinduced oxidative stress with poor
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interaction as Pꢀgp substrate and very low cytotoxicity.
INTRODUCTION
Neurodegenerative diseases (NDs) are widely investigated pathologies, because of the low
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efficacy of current therapies and severe functional impairments for daily life activities resulting
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in high familiar, social and financial costs of patient care. Despite the huge efforts in private and
public research settings, most clinical trials of potential drug candidates for NDs, and for
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Alzheimer’s disease (AD) in particular, failed. As a result, valuable diseaseꢀmodifying
therapies for NDs are still missing.
AD and Parkinson’s disease (PD) are the most widespread and severe NDs. AD is the result of
a progressive loss of neurons in basal forebrain regions, associated to abnormal accumulation of
beta amyloid protein (Aβ) in neuronal plaques and hyperꢀphosphorylated tau protein in
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neurofibrillary tangles. Neuronal degeneration in AD is triggered and maintained by low
molecular weight Aβ oligomers and by reactive oxygen species (ROS), produced by oxidative
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degradation of neurotransmitters and xenobiotics. The brain regions mostly affected by neuronal
loss are essentially made of cholinergic neurons, so that restoring physiological acetylcholine
levels has been considered a viable therapy in AD, as claimed by the soꢀcalled cholinergic
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hypothesis. To date, drugs approved for AD therapy are the cholinesterase (ChE) inhibitors
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,10
rivastigmine, galantamine and donepezil (Chart 1). The NMDA partial antagonist memantine
has also been approved for the symptomatic treatment of AD for its contrasting effects over
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glutamate excitotoxicity.
Acetylcholinesterase (AChE, EC 3.1.1.7), the key enzyme targeted in the palliative therapy of
AD, is present in both central and peripheral nervous system and in muscular motor plaques, and
is responsible for the enzymatic cleavage of neurotransmitter acetylcholine (ACh). The other
ChE, butyrylcholinesterase (BChE, EC 3.1.1.8), present in brain and peripheral tissues, but
prevalently in serum, is upꢀregulated in advanced AD and may play a role in the maintenance
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and progression of the disease.
The catalytic cleavage of ACh involves a tight cooperation of three amino acids, the soꢀcalled
catalytic triad (SerꢀHisꢀGlu) and aromatic amino acid residues responsible for cationꢀπ
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interactions. The catalytic bottom cleft represents one of the binding sites for substrates and
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inhibitors, along with the peripheral anionic binding site constituted by a larger region lined
chiefly by aromatic amino acids. This structural arrangement accounts for the strong binding
interactions observed for inhibitors such as decamethonium and donepezil, able to interact with
both catalytic (CAS) and peripheral (PAS) anionic binding sites of AChE and, therefore, called
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dual binding site (DBS) inhibitors. As binding interactions at the PAS reduces the aggregation
of Aβ peptide(s) leading to amyloid oligomers DBS inhibitors are endowed with dual inhibitory
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activity on AChE and Aβ aggregation.
The availability of Xꢀray crystallographic coordinates of many AChEꢀinhibitor complexes has
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allowed the identification of key interactions for high ligand binding affinity and has enabled
the targetꢀbased design of potent and selective AChE inhibitors (AChEIs). In this context, potent
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AChE inhibition by coumarin derivatives,
homoꢀ and heteroꢀbisꢀquaternary ammonium
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salts,
2ꢀquinolones and other heterocyclic compounds
have been reported by our group.
In the therapy of PD, a key target enzyme is represented by monoamine oxidase (MAO;
amineꢀoxygen oxidoreductase; EC 1.4.3.4), a FADꢀdependent enzyme, responsible for oxidative
deamination of amine neurotransmitters, including dopamine that is depleted in PD, and
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exogenous amines.
Two isoforms of MAO, namely MAOꢀA and MAOꢀB, have been
characterized in terms of amino acid sequence, tissue distribution, and selectivity towards
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substrates and inhibitors.
Selective MAOꢀA inhibitors (MAOꢀAIs: e.g., clorgyline and
moclobemide) are used in the treatment of depression, while selective MAOꢀBIs, i.e., rasagiline
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and selegiline, are employed as adjuvant or alternative drugs to LꢀDOPA in PD therapy.
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The resolution of the Xꢀray crystal structures of both human MAOꢀA
and MAOꢀB
bound to several inhibitors has newly spurred the research in the field of MAO inhibition, given
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to its therapeutic potential in neurological disorders, including AD where selective MAOꢀBIs
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may play a role.
Growing evidence in the last few years has outlined the multifactorial etiopathogenesis of AD,
PD and other NDs. Actually, neurodegeneration is a complex pathological event resulting from
the imbalance and deregulation of multiple biochemical pathways, ultimately depending on
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transcriptional and epigenetic modulations and on environmental factors. As a consequence,
holistic, multifaceted pathologies are currently tackled by a polypharmacological approach based
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on multiꢀtargeted therapy.
The paradigm “one drugꢀone target” has nowadays evolved into
a more challenging “one drugꢀmore targets” approach, provided that a good balance among
potencies and efficacy towards selected targets, and optimal ADMEꢀT properties, can be
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achieved.
MULTITARGETꢀLIGAND DESIGN: RATIONALE AND METHODS
The experience achieved by our group in the field of ligandꢀ and targetꢀbased design of potent,
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selective and reversible MAO
and ChE inhibitors,
prompted us to further explore the
challenging field of multiꢀtargeted ligand design, by addressing compounds with dual MAO and
AChE inhibition. The earliest application of this approach by us was reported nearly 15 years
ago, when we discovered coumarin derivatives endowed with good and moderate inhibition
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against MAOꢀB and AChE, respectively. Additional data have been later presented in an
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international meeting.
Since then, many authors have described the potential therapeutic application of multimodal
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MAOꢀIs displaying additional activities, such as AChE inhibition,
metal ion chelation,
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antioxidant
and neuroprotective activities. Further studies suggested the combination of
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MAO and ChE inhibition in the same molecule as a promising strategy in the treatment of AD.
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Starting from our previous findings we designed, synthesized and tested a new series of
suitably substituted coumarin derivatives with the aim of discovering multipotent compounds
with different bioactivity profiles towards MAOꢀB and AChE. While MAOꢀB selectivity was an
important goal of our study to avoid unwanted side effects arising from the intestinal MAOꢀA
inhibition and the consequent hypertensive effect coming from tyramineꢀrich food (tyramine is
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indeed metabolized by intestinal MAOꢀA), the lack of AChE over BChE selectivity was not
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deemed as important due to a likely pathogenic role of BChE in advanced AD.
The molecular framework of our new multiꢀtarget ligands was built following a hybridization
strategy. Starting from known ligands of the two enzymes, simple pharmacophore motifs were
selected and joined in a unique molecular entity. As molecular flexibility plays a key role in
accommodating ligands inside the AChE narrow gorge, a small series of hybrids was designed
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by connecting the coumarin core of 7ꢀ(3ꢀchlorobenzyloxy)ꢀ3ꢀmethylcoumarin (MC 1095) to
the Nꢀbenzylaminomethyl group characterizing many AChE inhibitors (e.g., donepezil) through
a flexible spacer (Figure 1). The 2Hꢀchromenꢀ2ꢀone ring of such compound was chosen as the
moiety able to efficiently fit the MAOꢀB enzymatic cleft by facing the isoalloxazine ring of
FAD. In the additional series of more rigid hybrids, to improve AChE inhibitory potency of 7ꢀ
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benzyloxycoumarins we approached a “designing in” strategy
by adding in a suitable
position of the coumarin ring a protonatable basic moiety that might bind CAS through πꢀcation
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interactions (Figure 2). Taking into account that, as recently reported,
the steric hindrance of
substituents placed at position 4 of the coumarin ring exerts a negative impact on MAO affinity,
the basic head was anchored to the 7ꢀbenzyloxy substituent. This rational hybrid design was
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supported by prospective docking studies based on Xꢀray crystal structures of the two target
enzymes. As suggested by docking calculations, the protonatable head may establish πꢀcation
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interactions at the CAS of AChE, while the coumarin moiety may interact with PAS. Such a
pose resembled that of donepezil in the PDB complexes (entries: 1EVE and 4EY7). As for the
binding to MAOꢀB, docking studies revealed that the coumarin nucleus can be accommodated in
the catalytic region in proximity of FAD thus matching the pose experimentally observed in the
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Xꢀray crystal structure of MAOꢀB selective coumarin inhibitor NWꢀ1772 (Chart 1). The spacer
tethering the two main pharmacophore features, namely the coumarin nucleus and the
protonatable amine moiety, was examined also in terms of a reduced flexibility, by synthesizing
the metaꢀ and paraꢀxylyl derivatives listed in Table 2, to determine the optimal distance between
the two key binding moieties in both target enzymes.
Docking studies suggested that both polymethylene and xylyl linkers overlaid, at least in part,
with the 7ꢀbenzyloxy group of NWꢀ1772 that faces the MAOꢀB entrance cavity (acting as B/A
structural determinants for selectivity) and keeps the basic moiety close to the aromatic region
more proximal to the solvent.
The newly synthesized coumarin derivatives 2ꢀ40, 42, 43 were tested in vitro for their
inhibition of rat MAOꢀA and MAOꢀB (rMAOs), electric eel AChE (eeAChE) and equine serum
BChE (esBChE). The quinolone isoster 45 was also synthesized and tested, taking into account
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our previous findings on AChE inhibitors, with the aim of retaining activity towards the target
enzymes, while possibly improving the pharmacokinetics properties (i.e., lowering lipophilicity
and increasing metabolic stability). The most potent inhibitors at rMAOꢀB and eeAChE were
also assayed on the human isoforms of the two MAOs and ChEs. The inhibition data of nonꢀ
human and human enzymes are reported in Tables 1ꢀ3 and 4, respectively. Finally, compound
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7, that showed outstanding activities on human enzymes together with wellꢀbalanced
solubility/lipophilicity properties, was also investigated as potential CNSꢀpermeant
neuroprotective agent in vitro.
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CHEMISTRY
Compounds 2ꢀ40 were prepared from bromoꢀcoumarin intermediates 1aꢀk through
microwaveꢀassisted reaction with appropriate benzylamines, in the presence of anhydrous
potassium carbonate and a catalytic amount of potassium iodide, in anhydrous acetonitrile.
Bromides 1aꢀk were, in turn, prepared from 7ꢀhydroxycoumarin derivatives and commercial
dibromides, as depicted in Scheme 1. Synthesis of analogs 42 and 43 (Scheme 2) started from the
reduction of 4ꢀ(bromomethyl)phenylacetic acid with BH ·SMe complex to obtain alcohol 41a
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that was then coupled with 7ꢀhydroxycoumarin. CBr /PPh ꢀmediated bromination of 41b
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afforded 41c that underwent a final microwaveꢀassisted nucleophilic substitution with the
appropriate benzylamine yielding the desired coumarins 42ꢀ43. As illustrated in Scheme 3, 7ꢀ
hydroxyꢀ2ꢀquinolone 44b was prepared from the condensation of transꢀcinnamic acid chloride
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with mꢀanisidine followed by an intramolecular FriedelꢀCrafts acylation/dearylation reaction in
refluxing chlorobenzene in the presence of aluminum chloride as Lewis acid. Sequential
regioselective alkylation of phenolicꢀOH with α,α’ꢀdibromoꢀpꢀxylene and reaction of
intermediate 44c with benzylmethylamine in the usual conditions gave final compound 45.
Analytical and spectroscopic data of tested compounds 2ꢀ40, 42, 43, 45 are reported in Table 6
and Supporting Information.
BIOLOGICAL ASSAYS
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All compounds were tested for their inhibitory activities on rat MAOs (rMAOs), electric eel
AChE (eeAChE) and equine serum BChE (esBChE) enzymes. For MAO inhibition test, the
protocol using mitochondrial rMAOꢀA and ꢀB obtained from rat brain homogenates was used as
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previously described.
As for eeAChE and esBChE, the wellꢀknown Ellman’s
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spectrophotometric test was used to determine both IC s and inhibition kinetics. Results are
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reported in Tables 1ꢀ3 as IC (ꢀM) or, for poorly active compounds, as percentage of inhibition
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at 10 ꢀM. Inhibition kinetics plots are depicted in Figure 3.
Compounds 2, 4, 5, 11, 12, 21ꢀ24, 29, 31ꢀ34, 37, 39, 40 and 45 were also tested on human
isoenzymes of MAOs (hMAOs) and/or ChEs (hChEs). While hChEs were tested through the
Ellman’s method, the assays for hMAOs were carried out with a fluorescenceꢀbased method
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using kynuramine as a nonꢀselective substrate of hMAOꢀA and hMAOꢀB. Results of inhibition
tests on hMAOs and hChEs are reported in Table 4.
Apical to basolateral (APꢀBL) and basolateral to apical (BLꢀAP) apparent permeability (Papp)
of compound 37 was measured using MadinꢀDarby Canine Kidney (MDCK) cells, retrovirally
transfected with the human MDR1 cDNA (MDCKIIꢀMDR1). Papp and efflux ratio (ER) were
calculated and reported in Table 5.
Cytotoxicity of compound 37 was evaluated in human neuroblastoma cell line SHꢀSY5Y
through the 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenylꢀtetrazolium bromide (MTT) viability
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assay (Figure 8). The same cellꢀbased method was used to for preliminarily investigate the
cytoprotective effects of compound 37 against cell death induced by H O (Figure 9).
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RESULTS AND DISCUSSION
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6
For the sake of clarity, structureꢀactivity and structureꢀselectivity relationships (SAR and SSR,
respectively) will be discussed first separately for the different classes of compounds reported in
Tables 1ꢀ3, then a comparison will be made among the inhibition data from rMAOs and eeAChE
with the corresponding data from hMAOs, hAChE and to a lesser extent hBChE (Table 4).
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SARs and SSRs of N-benzyl-N-alkyloxy coumarin derivatives (Table 1)
Compounds 2ꢀ12 were designed to investigate the effects on inhibition potency and selectivity
at the target enzymes of the length of the polymethylene linker and of the N-substitution at the
NHꢀbenzyl moiety with methyl and ethyl groups. A few substituents were also introduced on the
benzyl ring of compound 6, bearing a pentamethylene linker, to extend the knowledge of SARs
and SSRs.
The inhibition data in Table 1 showed that, with a few exceptions, the designed compounds
display from lowꢀ to subꢀmicromolar potencies against rMAOꢀB and eeAChE, whereas potencies
against rMAOꢀA and esBChE were slightly lower. As the result, promising dual MAOꢀB and
AChE inhibitors were discovered with limited MAOꢀB over MAOꢀA and AChE over BChE
selectivity.
Interestingly, compounds 3, 5, 6 and 12, characterized by linkers of different length, displayed
subꢀmicromolar affinities at both rMAOꢀB and eeAChE with a limited selectivity over rMAOꢀA
and esBChE, respectively. The gradual elongation of the linker in the N-Me derivative 2,
affording compounds 4, 6 and 11 provided a consistent increase of potency towards eeAChE
(from 1.3 ꢀΜ of 2 to 0.095 ꢁM of 11) and esBChE (from 7.8 ꢀΜ of 2 to 0.67 ꢀΜ of 11)
whereas an opposite effect was observed with rMAOꢀB (from 0.33 ꢀΜ of 2 to 1.7 ꢀΜ of 11).
Therefore, in this series of compounds, the goal of optimizing both MAOꢀB and AChE activities
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could not be achieved by varying the length of the linker. However, moving from the Nꢀmethyl
derivative 11 to the N-ethyl homologue 12 good activities at the three target enzymes, rMAOꢀB,
eeAChE and esBChE, were obtained but along with a very low MAOꢀB over MAOꢀA selectivity
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(
SI = 3; SI is the selectivity index calculated as the ratio IC MAOꢀA/IC MAOꢀB). Compound
50 50
11 was endowed with the highest eeAChE inhibitory potency within the whole examined series
IC = 0.095 ꢀΜ) and, surprisingly, the highest rMAOꢀA inhibitory potency (IC = 0.51 ꢀΜ)
(
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50
leading to a reversal of rMAO selectivity (SI = 0.3). Kinetics of eeAChE inhibition resulted in a
mixedꢀtype mechanism, as expected from putative dual binding site inhibitors (Figure 3A), with
K equal to 0.080 ± 0.002 ꢀΜ.
i
Notably, compounds with the longest linker 11 and 12 displayed the highest inhibitory potency
on rMAOꢀA (IC50 = 0.51 and 2.1 ꢀΜ, respectively) and ChEs (IC50 = 0.095 and 0.32
ꢀΜ, respectively, at AChE, and 0.67 and 0.49 ꢀΜ, respectively, at BChE). No clear relationship
emerged from the comparison of inhibition potency of Nꢀmethyl and N-ethyl derivatives.
The introduction in compound 6 of a cyano group in position meta and para (7 and 9,
respectively) and of a chlorine at position meta (comp. 8) did diminish the inhibitory potency
towards all the tested enzymes except for compound 8 at both ChEs.
SARs and SSRs of meta- and para-N,N-dialkylaminomethyl-7-benzyloxycoumarin derivatives
(Table 2).
Compounds 13ꢀ40 were designed to investigate the effects on inhibition potency and
selectivity at the three target enzymes of the N,Nꢀdialkylaminomethyl substituents at the meta
and para positions of the 7ꢀbenzyloxy moiety and of substituents at position 3 and 4 of the
6
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coumarin ring, having in mind our previous findings,
which highlighted the steric
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requirements for this region in binding MAOꢀB enzymatic cleft. A number of substituents were
also introduced on the Nꢀbenzyl ring in R to extend the study of SARs and SSRs.
4
It is worth to underline that all the examined compounds shared a metaꢀ or paraꢀxylyl linker
joining the oxygen at position 7 of coumarin with the basic N,Nꢀdialkylamino substituents. Both
xylyl linkers have a lower conformational flexibility compared to the polymethylene linkers of
compounds 2ꢀ12 and this might result in a higher (iso)enzyme selectivity. Indeed, this was the
case for rMAOs, as most compounds achieved micromolar to subꢀmicromolar rMAOꢀB
inhibitory potency whereas an inhibition lower than 20% at 10 ꢀM was generally displayed at
rMAOꢀA. The same effect on selectivity was not observed for the ChEs even though most
compounds appeared slightly more AChEꢀselective. Inhibition potency on eeAChE reached the
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subꢀmicromolar level in eleven cases, the most potent inhibitor being compound 24 (IC50
.10 ꢀM), while on esBChE only six compounds exhibited a subꢀmicromolar activity, with
compound 19 showing the highest potency (IC = 0.24 ꢀM).
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As the N,Nꢀdimethylaminomethyl derivative 13, the lead compound of the metaꢀxylyl series,
showed very low inhibitory effect on rMAOꢀA and eeAChE (IC = 10 ꢀM and 38% inhibition at
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0 ꢀM, respectively) and moderate activity on rMAOꢀB and esBChE (IC50 = 3.2 ꢀM and
.3 ꢀM, respectively), its N,Nꢀdiethylamino 14 and NꢀmethylꢀNꢀbenzylamino 15 congeners were
5
synthesized. Improved potencies were definitely gained with compound 15 that showed an
interesting profile of inhibition with low micromolar potencies rMAOꢀB, eeAChE and esBChE
(
IC = 2.0, 2.3 and 1.0 ꢀM, respectively) and very weak activity at rMAOꢀA (8% inhibition at
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10 ꢀM). The introduction of cyano, methoxy and chloro substituents at the meta position of the
phenyl ring of the Nꢀbenzylamino moiety, leading to compounds 16, 17 and 18, respectively,
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resulted in lower inhibitory potencies on rMAOꢀB and eeAChE whereas a 3ꢀfold increased
inhibition was observed for the chloro derivative 18 on esBChE (IC = 0.31 ꢀΜ vs. 1.0 ꢀΜ).
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The para-N,Nꢀdimethylaminoꢀ and N,Nꢀdiethylaminoꢀmethyl derivatives 19 and 20 displayed
lower activity against rMAOꢀA, similar activity against rMAOꢀB, and higher activity against
esBChE when compared to the corresponding metaꢀsubstituted isomers 13 and 14. Better results
were obtained with the para Nꢀmethyl, Nꢀbenzylaminomethyl derivative 21 that achieved subꢀ
micromolar potency towards rMAOꢀB and eeAChE (IC = 0.85 and 0.75 ꢀΜ, respectively), and
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a significantly lower activity against rMAOꢀA and esBChE (7% at 10 ꢀΜ and IC = 11 ꢀΜ,
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respectively).
Methyl substituents were introduced at the positions 3 and 4 of the coumarin ring to give the
monomethyl derivatives 22 and 23 and the dimethyl derivative 24. Compared to the lead
compound 21, the rMAOꢀB activity decreased while a significant increase of the inhibitory
potency on eeAChE (IC = 0.26, 0.18 and 0.10 ꢀΜ) and esBChE (IC = 1.1, 1.0 and 0.69
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ꢀΜ) was observed for compounds 22, 23 and 24, respectively. It is worth noting that these very
simple structural changes led to the most potent eeAChE inhibitors of the whole series of
compounds and, even more interestingly, for compound 24 an impressive combination of high
inhibitory activities towards rMAOꢀB, eeAChE and esBChE and high rMAOꢀBꢀselectivity was
revealed (IC = 1.2, 0.10 and 0.69 ꢀΜ at rMAOꢀB, eeAChE and esBChE, respectively, and 0%
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inhibition at 10 ꢀΜ at rMAOꢀA). As for open chain derivative 11, also compound 24 displayed
mixedꢀmode kinetics for eeAChE inhibition, with K equal to 0.22 ± 0.03 ꢁM (Figure 3B).
i
As done for lead compound 15, the isomeric compound 21 was modified by introducing
cyano, methoxy and chloro substituents at position meta, and the cyano group at position para as
well, in the aromatic ring of the Nꢀbenzylamino moiety. Metaꢀsubstituted derivatives 25ꢀ27
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proved to be less potent inhibitors than the lead compound 21 on rMAOꢀB. Metaꢀchloro
derivative 27 maintained, however, an attractive inhibition profile (12% inhibition at 10 ꢀΜ at
rMAOꢀA; IC50 = 2.7, 0.59 and 1.2 ꢀΜ, at rΜΑΟ−Β, eeΑChE and esBChE, respectively).
Therefore, compound 27 was slightly modified by introducing methyl substituents at the
positions 3 and 4 to afford the monomethyl derivatives 28 and 29 and the dimethyl derivative 30.
Compared to 27, rMAOꢀB, rMAOꢀA and esBChE activities considerably decreased, while the
potency towards AChE was maintained, or even improved in particular for the 4ꢀ
methylderivative 29 (IC = 0.12 vs. 0.59 ꢀΜ). Compared to 21, 4ꢀcyanobenzylamino derivative
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1 displayed lower activities towards rMAOꢀA, eeAChE and esBChE and a 3ꢀfold increase of
rMAOꢀB activity. The introduction in compound 31 of methyl substituents at the positions 3 and
, to give the monomethyl derivatives 32 and 33, and the dimethyl derivative 34, generally
4
diminished the activity on almost all enzymatic targets.
The final structural modifications of lead compound 21 were aimed at reducing molecular
lipophilicity while maintaining good inhibitory activities on the target enzymes. One approach
aimed at the modulation of the pK of the basic head, through the elongation of an arm of the
b
linker that allowed a higher distance between the two electron withdrawing phenyl rings from the
basic nitrogen, resulting in lower pK and logD. This structural modification increased the
b
distance between the two key binding moieties of 21, that is the coumarin ring and the N-benzyl
group, giving rise to the more flexible Nꢀbenzyl, Nꢀphenethyl analog 42 (Table 3). In addition,
Nꢀdemethylated derivative 43 was synthesized to test the effect of having a more hydrophilic
basic moiety endowed with HBꢀdonor ability. Compared to compound 21, the homologue 42
showed a similar activity towards eeAChE (IC = 0.85 vs. 0.75 ꢀΜ) and rΜΑΟꢀΑ (12% vs.
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%), a much higher activity at esBChE (IC = 0.52 vs. 11 ꢀΜ), and a decreased activity at
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rMAOꢀB (IC = 4.1 vs. 0.85 ꢀΜ). To our surprise, the Nꢀdemethylated congener 43 reversed
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MAOꢀselectivity, being the activity on rMAOꢀA higher than on rMAOꢀB (IC50
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1.7 ꢀΜ and 45% inhibition at 10 ꢀΜ, respectively). The inhibition potency on eeAChE
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worsened (IC50 = 4.7 vs. 0.85 ꢀM), while conversely the potency against esBChE remained
nearly unchanged (IC = 0.64 vs. 0.52 ꢀΜ). An additional less lipophilic analog of 21, namely
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45 in Table 3, was prepared through bioisosteric replacement of the 2Hꢀchromenꢀ2ꢀone with the
2
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ꢀquinolone as successfully done in the past for a dual binding site AChE inhibitor. Actually,
the activity towards both cholinesterases improved (IC = 0.49 vs. 0.75 ꢀΜ and 1.7 vs. 11 ꢀΜ at
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eeAChE and esBChE, respectively) and the low activity on rMAOꢀA was maintained but,
unexpectedly, the activity on rMAOꢀB dramatically decreased (29% inhibition at 10 ꢀΜ vs. IC50
= 0.85 ꢀΜ).
As the homologation of the basic head and the isosteric replacement yielded unsatisfying
results, compound 21 was modified by introducing polar groups on the coumarin core. The
insertion of a polar cyano group on the coumarin ring at position 3 (comp. 35) was highly
detrimental for rMAOꢀB activity (41% at 10 ꢀΜ vs. IC50 = 0.85 ꢀΜ) while a 3ꢀ and 2ꢀfold
potency increase was observed against eeAChE (IC = 0.20 vs. 0.75 ꢀΜ) and esBChE (IC =
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5.5 vs. 11 ꢀΜ), respectively. Better results on rMAOꢀB but much worse toward both ChEs came
from the introduction of a second polar substituent, a paraꢀcyano group on the Nꢀbenzyl ring
(comp. 36).
Guided by docking simulations and a 3DꢀQSAR model recently developed from a large series
6
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of 4,7ꢀdisubstituted coumarins, a different, more polar substituent, that is the hydroxymethyl
group, was introduced at position 4 of the coumarin core. Very satisfactorily inhibitor 37 showed
good inhibitory activities with the three target enzymes (IC = 0.41, 0.42 and 1.1 ꢀΜ at rMAOꢀ
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B, eeAChE and esBChE, respectively) and no activity on rMAOꢀA (0% inhibition at 10 ꢀΜ). As
for compounds 11 and 24, also 37 showed a mixedꢀtype inhibition mode (Figure 3C), with a Ki
equal to 0.10 ± 0.01 ꢁΜ. The Nꢀdemethylated derivative of 37, that is comp. 38, was also
prepared and tested. Its inhibitory potencies and selectivity profile (IC50 = 0.53, 0.44 and
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0.57 ꢀΜ at rMAOꢀB, eeAChE and esBChE, respectively and 19% inhibition at rMAOꢀA at 10
ꢀΜ) were very good and compared well with those of the Nꢀmethyl analog.
45
As previous SAR/3DꢀQSAR and current docking studies (data not shown) suggested
favorable interactions of a halogen atom at the meta position of a benzyl ring at AChE and
MAOꢀB binding sites, the metaꢀchloro derivative 39 was synthesized and tested. Its excellent
inhibitory activities on the three target enzymes (IC = 0.24, 0.25 and 0.63 ꢀΜ for rMAOꢀB,
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eeAChE and esBChE, respectively, and 0% inhibition toward rMAOꢀA at 10 ꢀΜ) proved the
goodness of our molecular design. Indeed, compound 39 attained the pursued combination of
strong and wellꢀbalanced inhibitory potencies and an excellent rMAOꢀB over rMAOꢀA
selectivity.
The introduction of a para-cyano substituent in the Nꢀbenzyl ring of 37 led to 40 which
showed an exquisite rMAOꢀB activity and selectivity (rMAOꢀB: IC = 0.035 ꢀΜ; rMAOꢀA: 0%
50
inhibition at 10 ꢀΜ), but also a dramatic drop of activity at eeAChE (IC50 = 6.3 ꢀΜ) and
esBChE (23% inhibition at 10 ꢀΜ), indeed resulting as the most potent MAOꢀB inhibitor of the
whole set of compounds herein investigated.
Inhibitory activities on human enzymes
The good biological profiles of many inhibitors towards MAO and ChE enzymes, prompted us
to extend our inhibition assays to the corresponding human enzymes. On the basis of our
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previous studies, significant changes of inhibitory activities were expected for hMAOs
(
generally an improvement, especially at hMAOꢀB) whereas more limited differences were
awaited for hAChE inhibition. However, to gain further support to these general predictions and
select compounds with high activity on hMAOꢀB and hAChE, a prospective docking study was
performed on the series of inhibitors listed in Table 4. Inhibition activities on human enzymes
were not assessed for compounds exhibiting low potency against the corresponding rMAO and
eeAChE enzymes.
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Prospective docking simulations were carried out using GOLD Suite v5.2 on the Xꢀray
crystal structures of AChE and MAOꢀB available from the PDB. For AChE studies, the Xꢀray
crystal structures of Torpedo californica AChE (TcAChE) (PDB entry: 1EVE) and human
recombinant AChE (hAChE) (PDB entry: 4EY7), both complexed with donepezil, were used.
The careful inspection of the two crystal structures revealed that PAS and CAS binding sites of
AChE are at about 18 Å distance (measured between the Cα carbon atoms of Trp86 and Trp286
of hAChE). Distances between the two putative binding moieties close to such a value, might
ensure efficient interactions at both PAS and CAS. On this basis, we conceived predictive
docking studies aimed at forecasting the optimal length of the polymethylene spacer joining the
pharmacophore features ensuring strong AChE binding. In this respect, the in silico studies were
focused on two compounds (2 and 11 of Table 1) bridging the protonatable basic tail and the
coumarin head with a short (trimethylene) or long (hexamethylene) linear linker, respectively.
Interestingly, docking simulations returned similar poses for the two compounds, which were
both able to engage steady πꢀπ stacking interactions with the aromatic residues of Trp86 and
Trp286. The compound bearing the longer linear linker showed a higher score (ꢀ106.2 kJ/mol for
hAChE and ꢀ96.3 kJ/mol for TcAChE) compared to that bearing the shorter linker (score: ꢀ98.2
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kJ/mol for hAChE and ꢀ88.6 kJ/mol for TcAChE). For clarity, a zoomed in view of topꢀscored
solutions is shown in Figure 4. Compounds 2 and 11 actually proved effective in inhibiting
AChE, in good agreement with the docking scores.
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For MAOꢀB studies, the Xꢀray crystal structure of hMAOꢀB (PDB entry: 2V60) and a
homology model for the rMAOꢀB were used. We performed a number of exploratory docking
runs to assess whether the decoration of the well known 7ꢀbenzyloxycoumarin with a
protonatable amino tail (mimicking the basic head of donepezil) could be suitable in biasing also
MAOꢀB. To this end, we docked three probe compounds (23, 32, and 33 in Table 2) into the
binding site of MAOꢀB, and observed that the ad-hoc incorporated benzylamine fragment was
located in a peripheral region surrounded by Tyr112, His115 (mutated to Tyr115 in rMAOꢀB)
and Asn116. As shown in Figure 5, the residue at position 115 is critical in establishing πꢀπ
stacking interactions with the terminal benzyl ring of 32, although the scores at hMAOꢀB were
consistently higher than those at rMAOꢀB. These interspecies differences were even more
pronounced in enzyme inhibition potency of 32, showing two log units higher activity at hMAOꢀ
B compared to rMAOꢀB (IC = 0.017 vs. 1.7 ꢀΜ). We hypothesized that such scoring and
5
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biological differences in hMAOꢀB and rMAOꢀB can rely on a likely greater strength of the πꢀπ
stacking interactions engaged by the imidazole ring of His115 compared to that of the phenyl
7
5
ring of Tyr115.
The potential benefit of introducing a cyano group at the para position of the benzylamine
was also challenged by docking compounds 23 and 33. Actually, we did not observe any
appreciable difference in the scores; however, the presence of the cyano group increased the
rMAOꢀB inhibition approximately of 10 folds (23, IC = 4.1 ꢀM vs. 33, IC = 0.41 ꢀM). We
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tentatively explained this experimental result by visually inspecting the docking poses of 23 and
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3. As shown in Figure 6, the cyano group could form HBs (even waterꢀmediated) with Asn116
or Tyr112.
For the sake of completeness, additional modeling studies were conducted on 37, one of the
most potent multiꢀtarget inhibitors of the series. 37 was first docked into both hAChE and
TcAChE to assess the binding interactions resulting from the substitution of an aliphatic linker
with an aromatic one and of the 4ꢀH atom with a more polar 4ꢀhydroxymethyl group. Beyond
hydrophobic interactions already described for inhibitors 2 and 11 bearing an aliphatic linker,
inhibitor 37 was engaged in a πꢀπ interaction involving the paraꢀxylyl linker and the aromatic
10
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17
side chain of Tyr341 (hAChE numbering referred to PDB entry 4EY7). In addition, a HB
occurred between the 4ꢀhydroxymethyl group and Ser293 of hAChE as shown in Figure 7A. The
higher score of 37 towards hAChE (ꢀ109.38 kJ/mol) compared to 2 and 11 may be therefore
ascribed to both πꢀπ and HB interactions (Figure 7A).
6
8,69
As reported in previous studies,
the introduction of polar groups at position 4 of the
coumarin scaffold improved physicochemical relevant properties (e.g., aqueous solubility and
lipophilicity) while maintaining good MAOꢀB inhibitory potency and selectivity. Thus inhibitor
3
7 was docked also on hMAOꢀB resulting in a binding pose similar to that of 23, 32 and 33 but
with a higher score (ꢀ95.95 kJ/mol). Most likely the high hMAOꢀB inhibitory potency arose from
45,49,68
the formation of multiple HBs between the hydroxyl group and structural water molecules
as illustrated in Figure 7B.
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Inhibition data in Table 4 fulfilled our expectations and docking calculations. As far as the
inhibitory activities on hMAOꢀB are concerned, a consistent increase of activity, from 2.5ꢀfold
for compound 2 up to 121ꢀfold for 34, was recorded whereas the activities on hMAOꢀA
remained low or were slightly incremented, from 2.7ꢀfold for compound 11 to 18ꢀfold for
compound 12. As the result, the hMAOꢀB over hMAOꢀA selectivity strongly raised. Actually,
the SIs measured for compounds 37, 39 and 40 were 2210, 842 and 910, respectively. Since for
many other inhibitors activities on hMAOꢀA remained very low, the IC50 values were not
measured and the hMAO selectivity could not be exactly assessed. Nevertheless, taking into
account the low percentage of inhibition of hMAOꢀA at 10 ꢀM concentration, high hMAOꢀB
over hMAOꢀA selectivity can be confidently foreseen also for inhibitors 21, 31 and 32.
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It is worth noting that all the coumarin derivatives listed in Table 4 attained subꢀmicromolar
inhibitory on hMAOꢀB. Coumarin derivatives 37 and 40, bearing the 4ꢀCH OH group, were the
2
most potent inhibitors within the whole molecular series examined with IC values in the low
50
nanomolar range (10 and 5.7 nM, respectively). Interestingly, some similarities and striking
differences emerged by comparing inhibitory activities at rat and human MAO enzymes. For
instance, 4ꢀCH OHꢀbearing coumarin derivatives 37, 39 and 40 resulted the most active MAOꢀB
2
inhibitors at both the human and rat enzymes, with compound 40 showing the highest activities
(IC = 0.0057 and 0.035 ꢀΜ, respectively). Noteworthy, the inverted selectivity observed on
5
0
rMAOs for compound 11, bearing the longest polymethylene linker, was maintained also on
hMAOs.
As anticipated in the introduction, the selective inhibition of either AChE or BChE was not a
primary aim of our work. Nonetheless a number of representative compounds, that is 5, 11, 21,
24, 37 and 39, were tested also on hBChE (Table 4). Despite an 89.4 % of amino acid sequence
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identity, the human and equine enzymes displayed significant differences in the catalytic site and
even more in the peripheral sequence. Notably, Gly277, Pro285, Phe398 in human mutated to
Asp, Leu and Ile, respectively, in equine. The amino acid numbering refers to hBChE as reported
in PDB. Therefore different inhibition potencies at the two enzymes may be expected and this
was indeed observed for compounds 24, 39, 21 and 37 (46% and 14% at 10 ꢀM and IC = 0.89
10
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5
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and 9.3 ꢀM versus IC = 0.69, 0.63, 11 and 1.1 ꢀM respectively). In contrast, compounds 5 and
5
0
11 displayed subꢀmicromolar inhibition potencies on hBChE close to the ones observed on
esBChE.
Appealing inhibition profiles resulted for compounds 5, 37 and 39. The first displayed subꢀ
micromolar potencies at all the four tested human enzymes, along with a significant, but limited,
selectivity for hMAOꢀB (SI = 13.5) and no selectivity for either one of the two ChEs. The latter
two showed high hMAOꢀB inhibitory potencies and selectivities (IC = 10 and 24 nM, and SI =
50
1580 and 542, respectively) and also a good hAChE inhibitory potency and selectivity (IC =
50
0.12 and 0.33 ꢀM , and SI = 77.5 and >>30, respectively).
Taking into account inhibition potencies, selectivity data and physicochemical parameters,
inhibitor 37 was selected for a preliminary evaluation of its ability to cross bloodꢀbrain barrier
(BBB) and its cytotoxicity and cytoprotective properties.
BBB PERMEABILITY AND TOXICITY PROFILING OF COMPOUND 37
MDCKꢀMDR1 cell lines are known to express Pꢀglycoprotein (Pꢀgp), which plays an
important role in the efflux transport of drugs from brain to blood. Thus, we were particularly
interested to see if compound 37 was able to permeate by passive diffusion the BBB and to
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interact with Pꢀgp as well. To this purpose, transport studies were performed on MDCKIIꢀMDR1
cells which are characterized by high Pꢀgp expression, and represent a wellꢀestablished in vitro
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model mimicking the BBB.
Transport studies were conducted in both apicalꢀtoꢀbasal (APꢀ
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BL) and basalꢀtoꢀapical (BLꢀAP) directions and the results were reported in Table 5. Fluorescein
isothiocyanateꢀdextran (FD4) and diazepam were used as paracellular and transcellular markers,
respectively, of cell monolayers integrity and as internal controls to verify tight junction integrity
during the assay; the results for controls were within the expected values. Both apparent
ꢀ5
ꢀ1
ꢀ5
ꢀ1
permeabilities (APꢀBL: 1.91·10 cm·sec ; BLꢀAP: 3.38·10 cm·sec ) were comparable with
those of diazepam, thus supporting a good BBB penetration. The efflux ratio (ER) equal to 1.77
detected for compound 37 disclosed no significant differences in Papp values between APꢀtoꢀBL
and BLꢀtoꢀAP direction. Since a value of ER greater than 2 indicates that a test compound is
likely to be a substrate for Pꢀgp transport, the measured value suggested that compound should
be able to permeate the monolayer without significant interactions with such efflux system.
The effects of compound 37 on the viability of human neuroblastoma cell line SHꢀSY5Y were
studied, using donepezil as reference compound. As shown in Figure 8, compound 37, like
donepezil, was not cytotoxic at the tested concentrations ranging from 0.1 to 50 ꢁM after 24, 48
and 72 h of incubation. The neuroprotective capacity of 37 against oxidative stress was also
evaluated using the same cell line and hydrogen peroxide (H O ) for the generation of exogenous
2
2
free radicals. Cells were incubated with 37 at two nonꢀtoxic concentrations (1 and 10 ꢀM) for 24
h; then, H O (60 ꢀM) was added and the cells maintained for further 24 h. Cell death was
2
2
determined using the MTT assay. As shown in Figure 9, compound 37 at 10 µM concentration
prevented the H O ꢀinduced cell death (ca. 25%) with a statistically significant effect (P < 0.01).
2
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CONCLUSIONS
The most salient features emerging from the SARs and SSRs of the two classes of compounds
examined in this paper can be summarized as follows. Highly flexible inhibitors of the Nꢀbenzyl,
Nꢀalkyloxy coumarin series in Table 1 showed good inhibitory activities at the target enzymes,
when bearing a pentaꢀ or hexamethylene linkers, but low selectivity (see comps. 6, 11 and 12 in
Table 1). The inhibitors bearing a more rigid xylyl linker (Table 2), displayed good inhibitory
potencies and high rMAOꢀB over rMAOꢀA selectivity ratios. Compound 21, a more rigid analog
of 6, exhibited good activities and selectivity towards rMAOꢀB and eeAChE. Metaꢀchlorobenzyl
analogs (i.e., 27, 35 and 39) showed an improved inhibition at eeAChE and esBChE but a worse
inhibition at rMAOꢀB, whereas in contrast the paraꢀcyanobenzyl analogues 31, 33 and 36
showed better potencies toward rMAOꢀB and decreased potencies toward eeAChE. The more
polar and hydrophilic 4ꢀhydroxymethyl derivatives 37ꢀ39 displayed the most interesting activity
profiles with low submicromolar activity at the three target enzymes rMAOꢀB, eeAChE and
esBChE, and low or no activity at rMAOꢀA.
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More interesting results came from the assays of selected inhibitors on human MAOs and
AChE. In comparison with nonꢀhuman enzymes, a significant increase of inhibitory activities
was observed for hMAOs, more pronounced on hMAOꢀB, with a consequent increase of the
selectivity index. More limited variations, generally a decrease of inhibitory potency, were
instead observed with hAChE and hBChE. The most interesting inhibitors, e.g. 5, 12, 21, and 37ꢀ
3
9, showed excellent activity profiles with low nanomolar inhibitory potency on hMAOꢀB, high
MAOꢀB over MAOꢀA selectivity and subꢀmicromolar potency on hAChE. Kinetic inhibition
data and docking studies on selected compounds suggested a mixedꢀtype mechanism of
inhibition and binding interactions at both the CAS and PAS of AChE. Therefore our compounds
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do behave as dual binding site inhibitors and have the potential to block another pathological
7
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mechanism of AD, that is the AChEꢀpromoted Aꢀbeta aggregation taking place at the PAS.
Ultimately, our hybridization strategy proved successful in designing and optimizing novel
coumarinꢀcontaining compounds targeting MAOꢀB and ChEs, two key enzymes involved in AD,
PD and other neurodegenerative diseases, with wellꢀbalanced inhibition potencies. Activity,
selectivity and physicochemical properties were improved compared to other ligands targeting
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the same enzymes described so far.
Moreover, toxicity, neuroprotection and transport data, as
preliminarily assessed using cellꢀbased models, suggested that the 4ꢀhydroxymethyl coumarin
derivative 37, which resulted the most promising inhibitor, is devoid of significant neurotoxicity,
shows moderate neuroprotective effects against H O ꢀinduced cell death, as well as a good BBB
2
2
permeability profile with limited Pꢀgp affinity.
In conclusion, some of the multipotent inhibitors reported herein, and compound 37 in
particular, may be considered promising leads for further preꢀclinical studies in cognitive and
neurodegenerative disease models.
EXPERIMENTAL SECTION
Chemistry.
Starting materials, reagents, and analytical grade solvents were purchased from SigmaꢀAldrich
Europe. All reactions were routinely checked by TLC using Merck Kieselgel 60 F254 aluminum
plates and visualized by UV light. Microwave reactions were performed in a Milestone
MicroSynth apparatus, setting temperature and hold times, fixing maximum irradiation power to
500 W and heating ramp times to 2 minutes. The purity of all the intermediates was checked by
1
H NMR and ESIꢀMS. ESIꢀMS analyses were performed on an Agilent 1100 LC–MSD trap
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system VL. Flash chromatographic separations were performed on a Biotage SP1 purification
system using flash cartridges prepacked with KPꢀSil 32−63 ꢁm, 60 Å silica. Elemental analyses
were performed on a EuroEA 3000 analyzer only on the final compounds and are reported in
Supporting Information. The measured values for C, H, and N agreed to within ±0.40% of the
theoretical values. Melting points (MP) were taken on a Gallenkamp MFB 595010M apparatus
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(open capillary method) and are uncorrected. Nuclear magnetic resonance (NMR) spectra were
recorded at 300 MHz on a Varian Mercury 300 instrument at ambient temperature in the
specified deuterated solvent. Chemical shifts (δ) are quoted in parts per million (ppm) and are
referenced to the residual solvent peak. The coupling constants J are given in hertz (Hz). The
following abbreviations were used: s (singlet), d (doublet), t (triplet), dd (doublet of doublet), m
(multiplet), brs (broad signal). Signals due to NH/OH protons were located by deuterium
45
exchange with D O. Nonꢀcommercial 7ꢀhydroxyꢀ3ꢀmethylcoumarin,
7ꢀhydroxyꢀ3,4ꢀ
2
4
4
80
35
dimethylcoumarin, 7ꢀhydroxyꢀ3ꢀcyanocoumarin, 7ꢀhydroxyꢀ4ꢀ(hydroxymethyl)coumarin,
were prepared as referenced.
Analytical data of final compounds 2ꢀ40, 42, 43, 45 are reported in Table 6. Analytical details
and spectroscopic data of all the intermediates 1aꢀk and final compounds are available as
Supporting Information.
General procedure for the synthesis of 7ꢀ(ωꢀbromoalkyloxy)ꢀ3,4ꢀdimethylcoumarins 1aꢀ
d, 7ꢀ(3ꢀ(bromomethyl)benzyloxy)coumarin 1e and 7ꢀ(4ꢀ(bromomethyl)benzyloxy)ꢀ3,4ꢀ
substituted coumarins 1fꢀk.
A Pyrex vessel was charged with a magnetic stirring bar, and then the appropriate 7ꢀ
hydroxycoumarin derivative (5.0 mmol) and potassium carbonate (0.70 g, 5.0 mmol) were
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suspended in dry acetonitrile (20 mL). The suitable commercially available dibromoꢀderivative
(1,ωꢀdibromoalkane or α,α’ꢀdibromoꢀmꢀxylene or α,α’ꢀdibromoꢀpꢀxylene, 25 mmol) was added.
The reactor was placed in a microwave apparatus and irradiated at 130 °C for 30 min. After
cooling to room temperature, the solid residue was filtered and washed with dichloromethane.
The solution was concentrated to dryness and the resulting crude was purified through flash
chromatography (gradient eluent, different mixtures of ethyl acetate in nꢀhexane).
0
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2ꢀ(4ꢀ(Bromomethyl)phenyl)ethanol (41a). (4ꢀBromomethyl)phenylacetic acid (0.60 g, 2.6
mmol) was dissolved under magnetic stirring with 8 mL of anhydrous THF in a flameꢀdried
roundꢀbottomed flask kept to 0 °C. Borane·dimethyl sulfide (0.40 mL, 3.9 mmol) was then
added dropwise and the mixture carefully cooled to room temperature and then left for additional
2
h. Water was cautiously added and the organic solvent was evaporated. Aqueous layer was
extracted with ethyl acetate; the organic layers were collected, dried over sodium sulfate and
1
evaporated to dryness to give the title product. Yield: 87%. H NMR (DMSOꢀd ) δ: 2.69 (t, J =
6
7
.1 Hz, 2H), 3.57 (t, J = 7.1 Hz, 1H, exch. D O), 3.79ꢀ3.87 (m, 2H), 4.66 (s, 2H), 7.18 (d, J = 7.7
2
Hz, 2H), 7.33 (d, J = 7.7 Hz, 2H).
7ꢀ{[4ꢀ(2ꢀHydroxyethyl)benzyl]oxy}ꢀ2Hꢀchromenꢀ2ꢀone (41b). A Pyrex vessel was charged
with a magnetic stirring bar, and then 7ꢀhydroxycoumarin (0.32 g, 2.0 mmol) and potassium
carbonate (0.28 g, 2.0 mmol) were suspended in dry acetone (12 mL). Bromide 41a (0.43 g, 2.0
mmol) was added and the reactor was placed in a microwave apparatus and irradiated at 130 °C
for 30 min. After cooling to room temperature, the solid residue was filtered and washed with
dichloromethane. The solution was concentrated to dryness and the resulting oil was purified
through flash chromatography (gradient eluent, methanol in dichloromethane 0%→10%). Yield:
1
7
7%. H NMR (DMSOꢀd ) δ: 2.70 (t, J = 7.1 Hz, 2H), 3.56ꢀ3.61 (m, 2H), 4.59ꢀ4.63 (m, 1H,
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exch. D O), 5.15 (s, 2H), 6.27 (d, J = 9.6 Hz, 1H), 6.98ꢀ7.06 (m, 2H), 7.35 (d, J = 8.0 Hz, 2H),
2
7.22 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.5 Hz, 1H), 7.97 (d, J = 9.6 Hz, 1H).
7ꢀ(4ꢀ(2ꢀBromoethyl)benzyloxy)ꢀ2Hꢀchromenꢀ2ꢀone (41c). Intermediate 41b (0.40 g, 1.4
10
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mmol) was dissolved in 5.0 mL of anhydrous dichloromethane under stirring with carbon
tetrabromide (0.49 g, 1.5 mmol). To this mixture, a solution of triphenylphosphine (0.43 g, 1.6
mmol) in 5.0 mL of anhydrous dichloromethane was dropped at 0 °C and the resulting solution
was kept at room temperature for 4 h. Evaporation of the solvent and purification by flash
chromatography (gradient eluent, ethyl acetate in nꢀhexane 0%→60%) afforded bromide 41c.
1
Yield: 77% yield. H NMR (DMSOꢀd ) δ: 3.12 (t, J = 7.1 Hz, 2H), 3.72 (t, J = 7.1 Hz, 2H), 5.17
6
(
s, 2H), 6.28 (d, J = 9.6 Hz, 1H), 7.00 (dd, J = 8.5 Hz, J = 2.5 Hz, 1H), 7.06 (d, J = 2.5 Hz, 1H),
1 2
7.29 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.5 Hz, 1H), 7.97 (d, J = 9.6 Hz,
H).
2E)ꢀNꢀ(3ꢀMethoxyphenyl)ꢀ3ꢀphenylacrylamide (44a). To a suspension of transꢀcinnamic
1
(
acid (0.89 g, 6.0 mmol) in dry dichloromethane (5.0 mL), thionyl chloride (5.0 mL) was added.
The resulting reaction mixture was refluxed for 6 h and then concentrated to dryness. The oily
residue was dissolved in dry dichloromethane (20 mL) and mꢀanisidine (0.67 mL, 6.0 mmol) was
slowly added. After refluxing for 4 h, the solvent was evaporated under vacuum and the resulting
1
crude was crystallized from hot ethanol. Yield: 70%. H NMR (DMSOꢀd ) δ: 3.73 (s, 3H), 6.61ꢀ
6
6.66 (m, 1H), 6.81 (d, J = 15.7 Hz, 1H), 7.18ꢀ7.25 (m, 2H), 7.36ꢀ7.46 (m, 4H), 7.53ꢀ7.62 (m,
3
H), 10.17 (s, 1H, exch, D O).
2
7
0
7
ꢀHydroxyꢀ2ꢀquinolinone (44b). Phenylacrylamide 44a (0.63 g, 2.5 mmol) was dissolved in
chlorobenzene (12 mL) in a flameꢀdried roundꢀbottomed flask. AlCl (1.3 g, 10 mmol) was
3
added portionwise while cooling to 0 °C. The reaction mixture was refluxed for 8 h, then cooled
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and poured into crushed ice. The resulting precipitate was washed with chloroform followed by
1
diethyl ether, thus yielding the desired intermediate. Yield: 67%. H NMR (DMSOꢀd ) δ: 6.19
6
(
d, J = 9.3 Hz, 1H), 6.59ꢀ6.71 (m, 2H), 7.42 (d, J = 8.3 Hz, 1H), 7.72 (d, J = 9.3 Hz, 1H), 10.08
s, 1H, exch. D O), 11.48 (s, 1H, exch. D O).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2
2
7ꢀ(4ꢀ(Bromomethyl)benzyloxy)ꢀ2Hꢀquinolinꢀ2ꢀone (44c). In a Pyrex vessel charged with a
magnetic and a Weflon stirring bar, phenol 44b (0.24 g, 1.5 mmol) and potassium carbonate
0.21 g, 1.5 mmol) were suspended in dry acetone (10 mL). α,α’ꢀDibromoꢀpꢀxylene (1.2 g, 4.5
(
mmol) was added and the reactor was placed in a microwave apparatus and irradiated at 130 °C
for 30 min. After cooling to room temperature, the solid residue was filteredꢀoff and washed with
dichloromethane. The solution was concentrated to dryness and the resulting crude solid was
purified through flash chromatography (gradient eluent, methanol in dichloromethane
1
0
%→10%). Yield: 49%. H NMR (DMSOꢀd ) δ: 4.71 (s, 2H), 5.13 (s, 2H), 6.28 (d, J = 9.3 Hz,
6
1
H), 6.84ꢀ6.85 (m, 2H), 7.40ꢀ7.57 (m, 5H), 7.78 (d, J = 9.3 Hz, 1H), 11.60 (brs, 1H, exch. D O).
2
General procedure for the synthesis of final compounds 2ꢀ40, 42, 43, 45
Appropriate bromide 1aꢀk, 41c or 44c (0.50 mmol) were suspended under magnetic stirring in
4.0 mL of anhydrous acetonitrile in a Pyrex microwave reactor in the presence of potassium
carbonate (0.64 g, 0.50 mmol) and a catalytic amount of potassium iodide (for compounds 2ꢀ12
8
1,82
and 42ꢀ43). Benzylamine (2.5 mmol), suitable substituted N-methylbenzylamine
(0.75 mmol)
or N-ethylbenzylamine (0.75 mmol) was added. The vessel was placed in a microwave apparatus
and heated at 130 °C for 30 min. After cooling to room temperature, the reaction mixture was
poured into iceꢀcold water (50 g) and extracted with dichloromethane (3 x 20 mL). The organic
fractions were collected, dried over anhydrous sodium sulfate and evaporated to dryness, to give
a residue that was crystallized from an appropriate solvent (as indicated in the Supporting
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Page 29 of 71
Journal of Medicinal Chemistry
1
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Information) or purified by flash chromatography (gradient eluent: different mixtures of
methanol in dichloromethane or ethyl acetate in nꢀhexane). Compounds 2, 3, 7ꢀ10 and 12 were
crystallized as hydrochlorides by treatment with HCl 1.25 N in ethanol. Compounds 5, 14, 16ꢀ
10
11
12
13
14
15
16
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1
8, 22ꢀ37, 39ꢀ40, 42, 43 and 45 were transformed into the corresponding hydrochloride salts by
treating the crude oil with HCl 4.0 N in 1,4ꢀdioxane (commercially available) or by dissolving
the solid crude in the minimum volume of 1,4ꢀdioxane before adding HCl 4.0 N in 1,4ꢀdioxane.
Rat and Human Monoamine Oxidases Inhibition Assays.
rMAO inhibitory activity of compounds in Tables 1ꢀ3 was assessed using a continuous
83
spectrophotometric assay, monitoring the rate of oxidation of the nonselective nonfluorescent
MAO substrate kynuramine to 4ꢀhydroxyquinoline. MAOꢀA and MAOꢀB activities in rat
mitochondrial preparations were assayed using as the controls the selective and irreversible
inhibitors clorgyline (250 nM) and (−)ꢀLꢀdeprenyl (250 nM), respectively. IC50 values were
determined by nonlinear regression of MAO inhibition vs. −log of the concentration plots, using
the program Origin, version 6.0 (Microcal Software Inc., Northampton, MA).
Human monoamine oxidase inhibition assays were carried out with a fluorescence based
7
2
method, also using kynuramine as nonꢀselective substrate of MAOꢀA and MAOꢀB. Briefly,
reactions were performed in triplicate in black, flatꢀbottomed polystyrene 96ꢀwell microtiter
plates (FluoroNunc/LumiNund, MaxiSorpTM surface, NUNC, Roskild, Denmark) containing
potassium phosphate buffer (158 ꢁL), an aqueous stock solution of kynuramine 0.5 mM (final
kynuramine concentration corresponding to 50 ꢁM), and DMSO solution of inhibitor in final
ꢀ4
ꢀ11
concentrations ranging from 10 to 10 M. Samples were incubated at 37 °C, and then diluted
human recombinant MAOꢀA and MAOꢀB (Supersomes; BD Gentest, Woburn, MA) were
delivered to obtain final protein concentrations of 0.009 mg/mL and 0.015 mg/mL, respectively.
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