Exploring Basic Tail Modifications of Coumarin-Based Dual Acetylcholinesterase-Monoamine Oxidase B Inhibitors: Identification of Water-Soluble, Brain-Permeant Neuroprotective Multitarget Agents

Article abstract of DOI:10.1021/acs.jmedchem.6b00562

Aiming at modulating two key enzymatic targets for Alzheimer's disease (AD), i.e., acetylcholinesterase (AChE) and monoamine oxidase B (MAO B), a series of multitarget ligands was properly designed by linking the 3,4-dimethylcoumarin scaffold to 1,3- and 1,4-substituted piperidine moieties, thus modulating the basicity to improve the hydrophilic/lipophilic balance. After in vitro enzymatic inhibition assays, multipotent inhibitors showing potencies in the nanomolar and in the low micromolar range for hMAO B and eeAChE, respectively, were prioritized and evaluated in human SH-SY5Y cell-based models for their cytotoxicity and neuroprotective effect against oxidative toxins (H₂O₂, rotenone, and oligomycin-A). The present study led to the identification of a promising multitarget hit compound (5b) exhibiting high hMAO B inhibitory activity (IC₅₀ = 30 nM) and good MAO B/A selectivity (selectivity index, SI = 94) along with a micromolar eeAChE inhibition (IC₅₀ = 1.03 μM). Moreover, 5b behaves as a water-soluble, brain-permeant neuroprotective agent against oxidative insults without interacting with P-gp efflux system.

Full text of DOI:10.1021/acs.jmedchem.6b00562

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Article
Exploring Basic Tail Modifications of Coumarin-based Dual
Acetylcholinesterase-Monoamine Oxidase B Inhibitors: Identification
of Water-soluble, Brain-permeant Neuroprotective Multitarget Agents
Leonardo Pisani, Roberta Farina, Marco Catto, Rosamaria Iacobazzi, Orazio
Nicolotti, Saverio Cellamare, Giuseppe Felice Mangiatordi, Nunzio Denora, Ramón
Soto-Otero, Lydia Siragusa, Cosimo Damiano Altomare, and Angelo Carotti
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00562 • Publication Date (Web): 27 Jun 2016
Downloaded from http://pubs.acs.org on June 28, 2016
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Journal of Medicinal Chemistry
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Exploring Basic Tail Modifications of Coumarinꢀbased
Dual AcetylcholinesteraseꢀMonoamine Oxidase B
Inhibitors: Identification of Waterꢀsoluble, Brainꢀ
permeant Neuroprotective Multitarget Agents
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a
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,a
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a
Leonardo Pisani, Roberta Farina, Marco Catto,* Rosa Maria Iacobazzi, Orazio Nicolotti, Saverio
a
a
a
b
c
Cellamare, Giuseppe Felice Mangiatordi, Nunzio Denora, Ramon SotoꢀOtero, Lydia Siragusa,
a
a
Cosimo Damiano Altomare, Angelo Carotti
a
Dipartimento di Farmacia ꢀ Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”, via E.
Orabona, 4, Iꢀ70125 Bari, Italy
b
Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad de Santiago de
Compostela, San Francisco I, Eꢀ15782, Santiago de Compostela, Spain
c
Molecular Discovery Limited 215 Marsh Road, Pinner, Middlesex, London HA5 5NE, UK
RECEIVED DATE
KEYWORDS: monoamine oxidase inhibitors; acetylcholinesterase inhibitors; multitarget ligand
design; Alzheimer’s disease; neuroprotective agents; neurodegenerative diseases; coumarin derivatives.
Abstract
Aiming at modulating two key enzymatic targets for Alzheimer’s disease (AD), i.e.,
acetylcholinesterase (AChE) and monoamine oxidase B (MAO B), a series of multitarget ligands was
properly designed by linking the 3,4ꢀdimethylcoumarin scaffold to 1,3ꢀ and 1,4ꢀsubstituted piperidine
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moieties, thus modulating the basicity to improve the hydrophilic/lipophilic balance. After in vitro
enzymatic inhibition assays, multipotent inhibitors showing potencies in the nanomolar and in the low
micromolar range for hMAO B and eeAChE, respectively, were prioritized and evaluated in human SHꢀ
SY5Y cellꢀbased models for their cytotoxicity and neuroprotective effect against oxidative toxins
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(
H O , rotenone and oligomycinꢀA). The present study led to the identification of a promising
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multitarget hit compound (5b) exhibiting high hMAO B inhibitory activity (IC50 = 30 nM) and good
MAO B/A selectivity (Selectivity Index, SI = 94) along with a micromolar eeAChE inhibition (IC =
50
1.03 µM). Moreover, 5b behaves as a waterꢀsoluble, brainꢀpermeant neuroprotective agent against
oxidative insults without interacting with Pꢀgp efflux system.
Introduction
The incidence and socioꢀeconomic costs of neurodegenerative diseases (NDs) are constantly growing
as a consequence of the increased life expectancy and ageing population. Among NDs, a major role is
played by Alzheimer’s disease (AD), representing the most common cause of dementia. As population
ages, the devastating impact of AD increases worldwide, being estimated that more than 100 million
1
individuals will suffer from AD by 2050. Alzheimer’s patients experience an irreversible cognitive
decline, associated with severe memory, attention and learning deficits impairing daily life activities. In
the last decades, massive investments in both academic and private settings, even though lower than in
other healthcare programs (e.g., anticancer therapies), have been devoted to the discovery of novel
diagnostic and therapeutic tools against AD. Unfortunately, the road to effectively treat AD with both
2
small molecules and immunotherapies addressing amyloid as well as tau hypotheses has been paved
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4
with failures even in lateꢀstage clinical trials, and diseaseꢀmodifying drugs are missing. The
disheartening attrition rate has commonly been ascribed to the multifactorial etiopathogenesis of AD
5
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exhibiting several neuronal aberrations spanning from proteostasis, metal unbalance, oxidative stress
7
to mitochondrial dysfunctions, ultimately leading to disruption of cholinergic transmission in
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hippocampus and frontal cortex. It is still a matter of debate whether researchers are addressing the
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wrong targets and/or the disease models are not appropriate.
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So far, with the exception of memantine (approved in 2003), the restoration of basal
neurotransmitter acetylcholine (ACh) levels through acetylcholinesterase (AChE) inhibitors (Chart 1;
donepezil, galantamine and rivastigmine) is the only approved, albeit palliative, therapeutic strategy in
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mild forms of AD. In more recent years, the research shifted to the more promising multitarget
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strategy, rooted on the principle that a combination of actions may be beneficial for multifactorial
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pathologies including AD by hitting two or more relevant targets with the same molecular entity.
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In many antiꢀAlzheimer multitargeting programs, chelation of biometals, agonism to 5ꢀHT4
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receptors, antagonism to 5ꢀHT1A receptors, radical scavenging, release of vasodilating NO
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radical
have been considered valuable biochemical activities that could synergistically improve the
therapeutic potential of AChE inhibition often remaining the core feature of multipotent agents.
AChE (EC 3.1.1.7) is a serine hydrolase responsible for the deacetylation of ACh in both central and
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peripheral nervous system. A 20 Å narrow tunnel, chiefly lined by aromatic residues, separates a
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catalytic anionic site (CAS), close to the catalytic triad, from another anionic subsite (peripheral
anionic site, PAS) that binds the cationic heads of gorgeꢀspanning dual binding site (DBS) inhibitors
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and acts as chaperoneꢀlike motif during the formation of Aβ oligomers. Another ChE isoform
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(
butyrylcholinesterase, BChE, EC 3.1.1.8) has the same biological function and its inhibitors may also
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have impact in the therapy of AD since its activity increases in advanced AD forms. To alleviate
oxidative stress conditions of degenerating neurons, the inhibition of MAO (amineꢀoxygen
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oxidoreductase; EC 1.4.3.4) activity might be addressed. This flavoenzyme catalyzes the degradation
of endogenous and xenobiotic amines (including many neurotransmitters as catecholamines and
serotonin) and contributes to increase reactive oxygen species (ROS) level through its catalytic cycle by
producing the corresponding aldehyde metabolite and hydrogen peroxide as the end products. The two
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known isoforms, termed MAO A and MAO B,
differ in amino acid sequences, tissue distribution
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and selectivity for substrates and inhibitors. As far as the role of MAO in AD is concerned, the potential
application of MAO inhibitors in therapy needs to address a crucial selectivity issue. In fact, strong
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dietary restrictions are required to avoid unwanted side effects associated to the blockade of
peripheral MAO A by nonꢀselective inhibitors. Moreover, MAO B predominates in brain tissue and its
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activity increased in the elderly, especially in glial cells.
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As a part of our ongoing research on multiꢀfunctional ligands against NDs,
we recently devoted
our efforts to the identification of coumarinꢀbased AChEꢀMAO B inhibitors with improved drugꢀlike
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properties. Among privileged heterocycles, the coumarin core has been extensively decorated by
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40ꢀ42
several research groups addressing the discovery of novel selective MAO
and AChE
inhibitors
43
as well as to build new molecular scaffolds with dual AChEꢀMAO activity against NDs, in most cases
4
4,45
through a conjugative approach.
Over the past decade, our contribution to this field highlighted the
important role of substituents at position 4 and 7 of coumarin in tuning MAO B activity and
4
6ꢀ50
selectivity.
Aiming at obtaining multipotent compounds with good overall pharmacokinetic
properties, we herein focused on modifications of the basic head of the side chain at C7 and of the
spacer connecting the two key pharmacophore features (namely, the basic head and the coumarin core)
in order to improve aqueous solubility while maintaining a dual AChEꢀMAO B inhibitory activity.
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Starting from a potent MTDL (MultiꢀTarget Directed Ligand), the new molecular framework was built
as depicted in Figure 1. The lipophilic phenyl ring was removed from the spacer and the basic nitrogen
3
was enclosed in a flexible piperidine cycle, thus modulating the pK of Nꢀsp and the aqueous solubility
a
at physiological pH. By approaching this ringꢀclosing strategy around the basic nitrogen, we conceived
a series of 2Hꢀchromenꢀ2ꢀones bearing a donepezilꢀinspired benzylpiperidine moiety that may
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efficiently interact with AChE binding pocket. 3,4ꢀDimethylcoumarin was exploited as a molecular
fragment to enhance MAO B affinity since this scaffold proved to be efficiently accommodated in the
lipophilic enzymatic pocket that faces the FAD coenzyme and is unable to lodge sterically hindered
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groups.
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The designed compounds were tested in vitro to evaluate their ability to inhibit both ChEs and
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MAOs. The ability of novel compounds to permeate bloodꢀbrain barrier (BBB) was preliminarily
calculated in silico by using Volsurf+ package (Molecular Discovery, London, UK) to compute LogBB,
a Molecular Interaction Field (MIF) based parameter useful to estimate the CNS distribution of small
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molecules. The most promising compounds fished out from both the enzymatic screening and in silico
BBBꢀpermeation prediction, were prioritized and studied in human SHꢀSY5Y neuroblastoma cell lines
for their cytotoxicity and potential neuroprotective effects against oxidative insults (hydrogen peroxide,
rotenone and oligomycin A). Moreover, compounds endowed with the highest neuroprotective effects
were evaluated in vitro for their ability to cross BBB in a MadinꢀDarby Canine Kidney (MDCKIIꢀ
MDR1) cell model and to interact with glycoproteinꢀP (Pꢀgp) mediated transport. The
hydrophilic/lipophilic balance of selected compounds was investigated through wellꢀestablished
turbidimetric determination of aqueous solubility and RPꢀHPLC lipophilicity measurements.
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Chemistry
Scheme 1 illustrates the synthetic pathway to piperidine derivatives 4aꢀm and (±)ꢀ5aꢀi reported in
Table 1. The preparation of compounds bearing a methylene spacer started from the activation of Bocꢀ
protected 3ꢀ and 4ꢀpiperidinemethanol 1bꢀc as mesylate esters (1dꢀe) that underwent SN with 3,4ꢀ
dimethylꢀ7ꢀhydroxycoumarin in DMF at 70 °C by using cesium carbonate as the base and triethylamine
to buffer the reaction mixture, thus obtaining intermediates 2bꢀc. Coumarins lacking the methylene
linker were synthesized starting from the Mitsunobu etherification to couple 1a with 3,4ꢀdimethylꢀ7ꢀ
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hydroxycoumarin under buffered conditions through triethylamine, thus yielding 2a. The removal of
the carbamate protecting group of 2aꢀc in acidic conditions by TFA unmasked the piperidine
intermediates 3aꢀc that underwent final alkylation under microwaveꢀassisted nucleophilic substitution
conditions (compounds 4bꢀh, 4jꢀm, (±)ꢀ5aꢀi) in acetonitrile with the appropriate benzyl bromide or
methylation at room temperature (4a, 4i and (±)ꢀ5a). NaBH(OAc) mediated the reductive amination of
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c and commercially available 3,4ꢀdimethoxybenzaldehyde to prepare coumarin (±)ꢀ5i. A sequential
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twoꢀsteps alkylation protocol under microwave irradiation afforded the rigid 1,2,3,4ꢀ
tetrahydroisoquinoline derivatives 7aꢀb (Scheme 2). Initially, 3,4ꢀdimethylꢀ7ꢀhydroxycoumarin was
reacted with 1,3ꢀ or 1,4ꢀdibromoalkane, thus furnishing intermediate bromides 6aꢀb. Under the same
reaction conditions, the suitable 1,2,3,4ꢀtetrahydroisoquinoline was alkylated with 6aꢀb yielding the
desired compounds 7aꢀb.
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Biological assays
All compounds were tested in vitro for their inhibitory activities on human MAOs (hMAOs), electric
eel AChE (eeAChE) and equine serum BChE (esBChE) enzymes. For hMAOs inhibition assay the
protocol was carried out with a fluorescenceꢀbased method using kynuramine as a nonꢀselective
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substrate of hMAO A and hMAO B.
As for ChEs, the wellꢀknown Ellman’s spectrophotometric
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test was used to determine IC s for both isoforms. The inhibition data are reported in Table 1 as IC
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(µM) or, for poorly active compounds, as percentage of inhibition at 10 µM. The kinetic behavior of
compound (±)ꢀ5b for the inhibition of eeAChE was investigated and is illustrated in Figure 2 by means
of LineweaverꢀBurk diagram where the reciprocals of enzyme activity (eeAChE) vs. reciprocals of
substrate (Sꢀacetylthiocholine) concentration in the presence of different concentrations (0ꢀ8 µM) of
inhibitor have been reported.
Human neuroblastoma SHꢀSY5Y cell lines were used to evaluate the cytotoxic effect of compounds
4jꢀk, (±)ꢀ5aꢀe and (±)ꢀ5i through the 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide
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(MTT) viability assay (Figure 3). By using the same cellular models, the ability of selected
compounds to protect neurons against three different toxic insults (hydrogen peroxide, oligomycinꢀA
and rotenone) was studied (Figure 4) and compared to donepezil taken as a reference antiꢀAlzheimer’s
drug. Data concerning cytotoxicity and neuroprotection assays are expressed as percentage of viability
referred to control experiment as illustrated in Figures 3 and 4.
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As previously reported, cellꢀbased transport studies were aimed at monitoring the BBB permeating
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potential of compounds (±)ꢀ5aꢀb and (±)ꢀ5d displaying the highest neuroprotective activity.
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Bidirectional transport studies were carried out by measuring apical to basolateral (APꢀBL) and
basolateral to apical (BLꢀAP) apparent permeability (P ) in MDCK cells. After retroviral transfection
app
with the human MDR1 cDNA (MDCKIIꢀMDR1), these cells highly express Pꢀgp thus alerting
compounds likely to be efflux pumps substrates. Papp (in units of cm/sec) and efflux ratio (ER) were
calculated and summarized in Table 2.
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Results and discussion
It is worth underlining that MAO B selectivity of the multipotent ligands reported herein was a
pursued feature to avoid wellꢀknown side effects raising from the inhibition of peripheral MAO A
isoform. On the other hand, isoform selectivity was not considered a crucial issue in the case of ChEs.
In fact, in advanced AD increasing body of evidence supports the importance of BChE whose activity
increases as the disease progresses. Both ChEs are capable of catalyzing the hydrolysis of ACh.
25
Therefore, BChE inhibition can be a viable strategy to counteract the cholinergic depletion too.
Keeping the 3,4ꢀdimethylcoumarin as the common structural feature, the main modifications regarded
the flexibility and geometry of the linker tethering the coumarin backbone at position 7 to the basic head
and the alkylation of the piperidine nitrogen.
Looking at the inhibition data for piperidineꢀbearing coumarins reported in Table 1, as expected from
the presence of the Nꢀbenzylpiperidine moiety mimicking donepezil, all compounds showed from good
to high affinity towards AChE (IC s ≤ 10 ꢁM) along with good selectivity values over BChE. AChE
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inhibitory potencies shift was not remarkably influenced by the structural modification herein reported
and IC s for AChE laid in the range 0.68 µM (4i) – 9.57 µM (4l). In contrast, a wider activity window
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could be observed for MAO B (0.030 µM for (±)ꢀ5b < IC s < 2.49 µM for 4g) suggesting a more
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pronounced effect of the substitution pattern on the MAO affinity.
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All hybrids displayed from moderate to high affinity towards hMAO B with submicromolar IC s but
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for 4b and 4g. All compounds inhibited selectively MAO B, with Selectivity Index (SI, IC MAO A /
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IC MAO B) ranging from 2 (4eꢀf) to 133 ((±)ꢀ5h).
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Furthermore, MAO B affinity was affected by the flexibility of the linker and the piperidine
substitution site (3 vs. 4) more than AChE affinity. In fact, higher MAO B inhibitory potencies could be
fished out from compounds 4iꢀm and (±)ꢀ5aꢀi where a methylene spacer tethers the basic head to the
planar coumarin. In absence of the spacer, increasing the size and lipophilicity of the substituent on
piperidine nitrogen from methyl (4a) to nꢀbutyl (4b) and benzyl group (4c) decreased MAO B affinity
while improving AChE affinity. The opposite trend was observed in presence of the methylene linker
connecting the coumarin to the 3ꢀ and 4ꢀsubstituted piperidinyl ring when moving from methyl to
benzyl (4i vs. 4j, (±)ꢀ5a vs. (±)ꢀ5b). Homologation of the terminal chain from benzyl (4c) to phenethyl
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4g) group influenced negatively the affinities towards both MAOs and reduced AChE affinity, too. The
introduction of a carbonyl group (4h) produced a 4ꢀfold MAO B affinity improvement with respect to
4c whereas AChE inhibitory potency diminished.
The substitution position at the piperidine cycle strongly influenced MAO B affinity. As a matter of
fact, three derivatives out of the 4ꢀsubstituted piperidine series (4aꢀm) showed an IC50 < 0.250 ꢁM
whereas all 3ꢀsubstituted piperidines (±)ꢀ5aꢀi showed MAO B inhibitory potencies < 0.250 ꢁM. The
presence of 3ꢀsubstituted piperidines was preferred from MAO B with respect to 4ꢀpiperidinyl moieties
((±)ꢀ5a > 4a and 4b; (±)ꢀ5b > 4c and 4j; (±)ꢀ5c > 4d and 4k; (±)ꢀ5f > 4e and 4l; (±)ꢀ5h > 4f and 4m),
irrespective of the nitrogen substituents. Looking at the basic head, the introduction of halogens (3’ꢀCl,
3
’ꢀBr, 4’ꢀF, 4’ꢀCl) as well as electronꢀwithdrawing (4’ꢀSO CH and 4’ꢀCN) and electronꢀdonating
2 3
groups (3’,4’ꢀdiOMe) on the phenyl ring exhibited a detrimental effect regards to MAO B affinity and
drastically reduced MAO B/A selectivity. Metaꢀsubstitution represented the preferred position for
chlorine in the binding to MAO B enzymatic cleft, as inferred by comparing 4d vs. 4e, 4k vs. 4l, (±)ꢀ5c
vs. (±)ꢀ5f. In para position a cyano group was preferred to chloro (4e < 4f, 4l < 4m, (±)ꢀ5f < (±)ꢀ5h) in
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both MAO B and AChE. Sulfonyl derivative (±)ꢀ5g displayed the highest MAO A affinity along with a
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low IC towards MAO B.
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To assess the AChE inhibition mechanism of donepezilꢀlike Nꢀbenzylꢀpiperidines the kinetic
behaviour of compound (±)ꢀ5b was investigated. As shown in Figure 2, LineweaverꢀBurk plot indicated
a mixedꢀtype inhibition with a K equal to 1.37 ± 0.05 µM, thus suggesting a partial occupancy of PAS
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and potential Aβ antiꢀaggregating properties.
HPLC separations were undertaken to check a possible influence of chirality in the binding
interactions of this series, and racemic mixtures of 5b and 5d were resolved as prototypes on a
CHIRALPAK IA column yielding enantiomeric excess > 99% (see Supporting Information for details).
Regarding hMAO B affinity, an eudismic ratio equal to 8 was observed for both racemates. Eutomers
(+)ꢀ5b and (+)ꢀ5d exhibited outstanding hMAO B affinities (IC50 = 23 and 26 nM, respectively).
Interestingly, the most potent enantiomer (+)ꢀ5b showed also an outstanding MAO B over MAO A
selectivity (SI = 167), much greater than the other eutomer (+)ꢀ5d (SI = 21). Both mixtures displayed a
lower activity ratio (≤ 2) towards hMAO A, being (+)ꢀ5b and (−)ꢀ5d the distomers. A lower and
inverted eudismic ratio was found towards eeAChE for both pairs of enantiomers.
To overcome the undesirable drawback of chirality, 1,2,3,4ꢀtetrahydroisoquinoline derivatives 7aꢀb
were designed as rigid achiral analogues of the most potent hit compound ((±)ꢀ5b). In vitro evaluation
proved a sharp drop of MAO B affinity albeit the potency was still in the nanomolar range.
Interestingly, compounds 7aꢀb showed low micromolar affinities towards both ChEs, with 7a being the
most potent BChE inhibitor of the whole series with a submicromolar IC = 0.95 µM. This result could
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be ascribed to the flatness and wideness of BChE enzymatic cleft that could better accommodate rigid
and sterically hindered basic groups.
To assess the brainꢀpermeating capability of the novel multipotent molecules reported herein,
Volsurf+ was employed to compute LogBB, a parameter expressing the logarithmic ratio between the
concentration of a drug in brain and blood. More specifically, compounds showing logBB value greater
than 0.5 readily penetrate into CNS and are classified as BBB+. LogBB values lower than ꢀ0.5 disclose
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very poor brain permeation (BBBꢀ compounds) whereas values higher than ꢀ0.5 and lower than 0.5
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indicate moderate permeation (BBB± compounds). Calculations suggested that most compounds in
Table 1 should rapidly be distributed into the brain (BBB+ compounds). LogBB of derivatives bearing
polar substituents on the phenyl ring (a cyanoꢀgroup for 4f, 4m and (±)ꢀ5h or a dimethoxyꢀgroup for
(±)ꢀ5i) and the ketoꢀderivative 4h suggested moderate permeation. The lowest logBB value (0.011) was
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returned by (±)ꢀ5g with a highly hydrophilic substituent on the basic head (ꢀSO CH group). The high
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confidence of Volsurfꢀbased predictions was assessed through the projection of the studied compounds
in the chemical space represented by its applicability domain, determined according to the Hotelling's Tꢀ
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Squared approach (see Supporting Information).
Cellꢀbased assays: cytotoxicity, neuroprotection and brainꢀpermeation
By combining data from enzymatic screening with in silico calculations, a number of hits was
shortlisted and selected for further assays in cellꢀbased models in order to evaluate their cytotoxic and
neuroprotective effects and their ability to permeate BBB without interacting with efflux pumps such as
Pꢀgp. Regarding the inhibitory properties, the following selection criteria were applied: i) IC towards
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MAO B < 150 nM; ii) IC towards AChE < 2 µM. With respect to brain permeation predictions, logBB
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threshold was set equal to 0.450. Seven coumarin derivatives (4j, 4k, (±)ꢀ5a, (±)ꢀ5b, (±)ꢀ5c, (±)ꢀ5d and
(±)ꢀ5e) met all the selection criteria and were submitted to preliminary in vitro investigations in human
neuroblastoma SHꢀSY5Y cell lines. Compound (±)ꢀ5i was also included in the study in order to
investigate the effect of the orthoꢀdimethoxyphenyl moiety.
Since in vitro enzymatic assays for both enantiomers of 5b and 5d provided AChE inhibition
activities close to racemate and the differences between enantiomers and racemate for MAO B were
reasonably low (ratio of IC s < 6 for both isomers in comparison to racemate 5b and 5d), we deemed
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unnecessary to perform the cellꢀbased studies on single enantiomers at this early stage investigations.
As shown in Figure 3, most compounds displayed negligible cytotoxicity up to 50 µM after 24 h
incubations. Some derivatives produced a reasonable cellular damage only at highest concentrations. In
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particular, the concentrations responsible for 50% inhibition of cell growth (IC s) for compounds (±)ꢀ
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a, (±)ꢀ5b, (±)ꢀ5c and (±)ꢀ5i were equal to 30 ± 0.01 µM, 44 ± 0.02 µM, 13 ± 0.01 µM and 10 ± 0.03
µM, respectively.
The overproduction of harmful radicals, above all reactive oxygenated species (ROS), and the
unbalance in detoxification systems produces severe oxidative stress conditions in neurons affected by
AD. Nucleic acids, proteins and lipids become loaded with aberrant alterations that, in turn, could
trigger ADꢀrelated neurotoxicity and indeed the reduction of oxidative stress has been claimed as a
viable strategy to slow down the progression of the disease. Therefore, we tested also the ability of
selected multitarget molecules to protect SHꢀSY5Y cells against oxidative injuries. The cytoprotective
effect was determined by measuring cell viability after incubation with a radical initiator (hydrogen
peroxide, H O ) and two mitochondrial poisons (rotenone and oligomycinꢀA), both capable of arresting
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respiratory chain and energy production (Figure 4). Rotenone induces cellular damage by interfering
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with the activity of complex I of the respiratory chain whereas oligomycinꢀA binds to F part of H ꢀ
o
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ATPꢀsynthase and exerts its proꢀapoptotic effect by blocking ATP formation. Compounds under
investigation were incubated at two concentrations (1 and 10 µM) and untreated cells were used as
control. As depicted in Figure 4a, compounds (±)ꢀ5aꢀe markedly protected SHꢀSY5Y cells against H O
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even at the lowest concentration (1 µM). A low increase of cell viability in the presence of oligomycinꢀ
A was produced by 4j (at 1 and 10 µM), 4k (at 1 µM), (±)ꢀ5c (at 10 µM) and (±)ꢀ5i (at 1 and 10 µM).
Moreover, derivative (±)ꢀ5a was not able to counteract cellular damage induced by oligomycinꢀA,
whereas it exerted a moderate neuroprotective activity against rotenone at 1 and 10 µM. A significant
increase of viable cells was obtained when rotenone was coꢀincubated with (±)ꢀ5e at 1 µM. Derivative
(±)ꢀ5b and (±)ꢀ5d remarkably increased cell viability in the presence of both mitochondrial toxins used
in this assay (rotenone and oligomycinꢀA) at 1 and 10 µM and exerted a cytoprotective activity by far
superior to that of donepezil. Taken together these results highlighted dual inhibitors (±)ꢀ5b and (±)ꢀ5d
as the most promising neuroprotective agents of the series, as they proved to be effective against all the
insults employed in SHꢀ SY5Y cellꢀbased experimental models.
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To endorse the potential of both compounds as promising multipotent anti AD leads, in vitro transport
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studies were undertaken. As for CNSꢀacting drugs, a critical issue is represented by the ability to cross
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BBB acting as a highly lipophilic boundary. Compounds permeate BBB mainly by passive diffusion
mechanism and several efflux systems prevent the entrance of xenobiotics into CNS. Since the extrusion
activity is essentially governed by Pꢀgp, the possible behavior of a hit as a Pꢀgp substrate should be
assessed in the early stage of drug discovery along with its brain permeation properties. MDCK cells
were used as a model to examine the behavior of selected compounds in crossing BBB and eventually
interacting with extrusion pumps. When retrovirally transfected with the human MDR1 cDNA
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(
MDCKIIꢀMDR1), these cell lines highly express Pꢀgp (MDR1) and represent a robust BBB mimicking
in vitro model. Furthermore, permeation analyses were undertaken also with (±)ꢀ5a, being the most
potent neuroprotective agent against H O at 10 µM and moderately effective against rotenone at both
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tested concentrations (1 and 10 µM). In contrast with the computed LogBB, lipophilic derivative (±)ꢀ5d
was not able to permeate the monolayer (see Table 2), probably because of quite high membrane
retention as deduced from low permeation rate in both directions, i.e., apicalꢀtoꢀbasolateral (APꢀBL) and
basolateralꢀtoꢀapical (BLꢀAP). Moreover, an ER > 2 (ER = P , BLꢀAP / P , APꢀBL) can be taken as
app
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a figure of undesirable interactions with Pꢀgp. On the other hand, as shown in Table 2 compounds (±)ꢀ
b and (±)ꢀ5a confirmed rapid permeation and low ER comparable to donepezil used as reference CNSꢀ
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active drug, thus envisaging good brain distribution and poor interactions as Pꢀgp substrates.
Aqueous solubility and lipophilicity
In the present work, the aim of optimizing the aqueous solubility at pH 7.4 of a coumarinꢀbased hit
recently reported by us (7ꢀ(4ꢀ(NꢀBenzylꢀNꢀmethylaminomethyl)benzyloxy)ꢀ4ꢀ(hydroxymethyl)ꢀ2Hꢀ
34
chromenꢀ2ꢀone hydrochloride I; Figure 1) while maintaining a dual AChEꢀMAO B inhibitory activity
was pursued by introducing focused structural modification on the protonatable head and on the linker
at position 7 of the coumarin core. Kinetic aqueous solubility at pH 7.4 was experimentally determined
for compounds I, 4j, 5a and 5b by applying a turbidimetric method.
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As showed in Table 3, compound I returned the lowest value of water solubility (log S= ꢀ4.42) and
the Nꢀmethylpiperidinꢀ3ꢀyl derivative 5a exhibited the highest solubility (logS = ꢀ3.67). Compared to I,
the piperidinꢀ4ꢀyl derivative 4j showed only a modest increase of solubility (logS = ꢀ4.25), whereas the
isomer piperidinꢀ3ꢀyl derivative 5b proved to be 3ꢀfold more soluble (119 µM, logS = ꢀ3.92).
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The observed increase in water solubility at pH 7.4 of 4j, 5a and 5b over I, and the small but
significant differences among them as well, could be reasonably explained taking into account the pKa
shift (i.e., the degree of amine protonation) in the examined molecules and their hydrophobicity.
Regarding the basicity of the amino moiety at C(7) of the coumarin core, the ACD/labs (ver. 6.00)
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however within the optimal range 7.5ꢀ10.5 proposed for CNS drugs). The lipophilicity of the
molecules in neutral form was assessed by calculation (BioꢀLoom 1.6, Biobyte Corp., Claremont,
U.S.A.), whereas a relative lipophilicity scale of the compounds, predominantly in the protonated form,
was experimentally determined by a reversed phase (RP) HPLC method. Zorbax EclypseꢀC18 column
was used as the stationary phase and mixtures of ammonium acetate buffer (20 mM, pH 5.00) and
methanol were used as the mobile phases to measure chromatographic capacity factor (logk’) as a
lipophilicity index.
Looking at the data in Table 3, interesting structureꢀproperty relations could be derived. Quite
obviously, the most waterꢀsoluble is 5a, that is the most hydrophilic one (i.e., the lowest logk’ and
cLogP values) and fully protonated at pH 7.4. Both Nꢀbenzylpiperidinyl derivatives 4j and 5b, despite
their higher lipophilicity (as supported by the cLogP and logk’ values), are more soluble in water than I,
most likely due to the different protonation degree of the amino group which, based on the estimated
pK s, should be higher in 4j and 5b than in I. Albeit showing similar lipophilicity and very close
a
basicity, the piperidinꢀ3ꢀyl derivative 5b proved to be more than 2ꢀfold more soluble in water that the
isomer piperidinꢀ4ꢀyl compound 4j. This result may be related on one hand to the disruption of
molecular symmetry elements in 5b with respect to 4j, which could be an entropic factor favouring
solubility in water, whereas on the other hand the proximity of the polar groups in 5b, which are closer
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than in 4j, could explain the slightly better partitioning of 5b in apolar media, as cLogP and logk’ values
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account for.
Conclusions
The multitarget approach has been extensively exploited by several research groups as a promising
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6ꢀ69
therapeutic option to face neurodegenerative disorders.
In the case of AD, in the last decades
inhibition of enzymatic activities of MAOs and ChEs has been pursued to identifying novel therapeutic
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agents with a potential diseaseꢀmodifying effect.
A seminal discovery in the field is represented by
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ladostigil, a dual AChEꢀMAO inhibitor, that has been recently announced to finish ad interim phase
IIb and to enter phase III clinical trials by Avraham Pharmaceuticals for the treatment of mild cognitive
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impairment. Looking at the literature, common drawbacks of multitarget dual inhibitors hitting MAOs
and ChEs are given by low MAO B over MAO A selectivity and, at least, one violation of Lipinski’s
Rule of Five (in most cases MW > 500 and/or cLogP > 5), the latter representing a compromising
feature for good oral bioavailability. Peripheral inhibition of MAO A implies safety issues arising from
75
the soꢀcalled cheese effect. Over the years, we devoted attention to the development of coumarinꢀ
based dual AChEꢀMAO B inhibitors. To this end, our design strategy was aimed at improving the
physicochemical properties for drug bioavailability (aqueous solubility, above all) of a previously
34
described multiꢀpotent hit compound while maintaining a dual AChEꢀMAO B inhibitory activity.
Such compound (i.e., derivative I in ref. 34, Figure 1) showed outstanding in vitro inhibitory potencies
against hAChE and hMAO B in the nanomolar range along with a limited aqueous solubility at pH 7.4
(38 µM).
Structureꢀactivity relationships (SARs) of multipotent compounds described herein shed light on the
most relevant structural features modulating hMAO B affinity: i) the flexibility of the spacer linking the
coumarin core to the basic tail at position 7 and ii) the substituents and the branching position on the
piperidinyl ring (1,3 vs. 1,4). AChE affinity was influenced by these structural modifications to a lesser
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extent. Interestingly, the presence of piperidinyl fragment along with its substitution pattern markedly
influenced aqueous solubility.
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The present study allowed the development of a novel multiꢀfunctional agent (±)ꢀ5b with a good
balance of biochemical activities and improved aqueous solubility, definitively deserving further
attention. In vitro enzymatic assays highlighted an outstanding hMAO B affinity (IC = 30 nM) along
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with pronounced MAO B/A selectivity (SI = 94) and low micromolar eeAChE affinity (IC50 = 1.03
µM). In cellꢀbased assays, (±)ꢀ5b produced low cytotoxic damage (IC50 = 44 ± 0.02 µM) after 24 h
incubation and increased cell viability of human neuroblastoma lines incubated with some toxic insults
(
H O , rotenone and oligomycinꢀA). Interestingly, its neuroprotective activity against the oxidative
2 2
stress insults produced by hydrogen peroxide, oligomycinꢀA and rotenone was superior to that of
donepezil, used as a reference antiꢀAD drug. Bidirectional transport studies on MDCKIIꢀMDR1 model
denoted a rapid BBB permeation without suffering from likely Pꢀgp interactions thus suggesting good
brain permeation and CNS distribution. Moreover, coumarin (±)ꢀ5b does not violate Lipinski’s rule of
five (MW = 377.48, HBꢀdonor = 0ꢀ1 depending on the protonation state, HBꢀacceptor = 2ꢀ3 depending
on the protonation form, cLogP (ChemAxon) = 4.57). The presence of the basic piperidinyl moiety does
34
increase water solubility of 5b at pH 7.4 (3ꢀfold increase compared to I ). Taken together, these
findings highlighted the potential of this class of compounds and particularly of (±)ꢀ5b as multitarget
antiꢀAD neurotherapeutic, deserving further pharmacological investigations to prove its ability to
prevent the onset, hamper the progression or reverse the neurodegenerative process in animal models.
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Experimental section
Chemistry
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Starting materials, reagents, intermediate 1a and analytical grade solvents were purchased from
1
SigmaꢀAldrich (Europe). The purity of all the intermediates, checked by H NMR and HPLC was
always better than 95%. All the newly prepared and tested compounds showed HPLC purity higher than
0
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8%. Column chromatography was performed using Merck silica gel 60 (0.063ꢀ0.200 mm, 70ꢀ230
mesh). Flash chromatographic separations were performed on Biotage SP1 purification system using
flash cartridges prepacked with KPꢀSil 32ꢀ63 ꢁm, 60 Å silica. All reactions were routinely checked by
TLC using Merck Kieselgel 60 F254 aluminum plates and visualized by UV light or iodine. Regarding
the reaction requiring the use of dry solvents, the glassware was flameꢀdried and then cooled under a
stream of dry argon before the use. Optical rotation was measured on a PerkinꢀElmer 241 polarimeter
with a Na lamp (589 nm). Nuclear magnetic resonance spectra were recorded on a Varian Mercury 300
instrument (at 300 MHz) or on a Agilent Technologies 500 apparatus (at 500 MHz) 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), dd (doublet of doublet), t (triplet), q
(
quadruplet), qn (quintuplet), m (multiplet), br s (broad signal); signals due to OH and NH protons were
located by deuterium exchange with D O. Chiral HPLC separations and enantiomeric excess
2
determinations were carried out on CHIRALPAK IA (Chiral Technologies Europe, 25 cm x 0.46 cm I.
D., 5 ꢁm size particles) built on a Analytic Agilent 1260 Infinity Multidetector system equipped with
1
200 Series UVꢀDiode Array in isocratic conditions. Elemental analyses were performed on the EuroEA
3000 analyzer only on the final compounds tested as MAOs and ChEs inhibitors. The measured values
for C, H, and N agreed to within ± 0.40% of the theoretical values. Melting points were determined by
the capillary method on a Stuart Scientific SMP3 electrothermal apparatus and are uncorrected.
General procedure for the synthesis of tertꢀbutyl 3ꢀ and 4ꢀhydroxymethyl piperidineꢀ1ꢀ
carboxylate (1bꢀc). To a suspension of the suitable commercially available 3ꢀ and 4ꢀ
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Page 17 of 59
Journal of Medicinal Chemistry
hydroxymethylpiperidine (8.6 g, 75 mmol) in a mixture of acetonitrile (110 mL) and saturated aq.
sodium hydrogen carbonate solution (35 mL), diꢀtertꢀbutyl dicarbonate (21 g, 94 mmol) was added in
portions while cooling to 0 °C. The reaction mixture was then kept to room temperature and left under
magnetic stirring for 18 h. Brine (300 mL) was added and the aqueous layer was extracted with ethyl
acetate (3 x 150 ml). The organic phases were collected, dried over sodium sulphate, and evaporated to
dryness to give the desired product that was used without further purification.
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tertꢀButyl 4ꢀ(hydroxymethyl)piperidineꢀ1ꢀcarboxylate (1b). Yield: 88%. H NMR (300 MHz,
CDCl ) δ: 1.06−1.19 (m, 2H), 1.44 (s, 9H), 1.60−1.74 (m, 4H), 2.69 (t, J = 12.7 Hz, 2H), 3.48 (t, J = 5.8
3
Hz, 2H), 4.09−4.13 (m, 2H, 1H dis. with D O).
2
1
tertꢀButyl 3ꢀ(hydroxymethyl)piperidineꢀ1ꢀcarboxylate (1c). Yield: 94%. H NMR (500 MHz,
DMSOꢀd ) δ: 1.01−1.10 (m, 1H), 1.21−1.47 (m, 3H), 1.37 (s, 9H), 1.52−1.68 (m, 2H), 2.63−2.72 (m,
6
1H), 3.11−3.19 (m, 1H), 3.21−3.28 (m, 1H), 3.73−3.81 (m, 1H), 3.87−3.96 (m, 1H), 4.48 (t, J = 8.8 Hz,
1
H, dis. with D O).
2
General procedure for the synthesis of tertꢀbutyl 4ꢀ{[(methylsulfonyl)oxy]methyl}piperidineꢀ1ꢀ
carboxylate (1d) and tertꢀbutyl 3ꢀ{[(methylsulfonyl)oxy]methyl}piperidineꢀ1ꢀcarboxylate (1e). The
suitable Bocꢀprotected piperidine 1bꢀc (7.5 g, 35 mmol) was dissolved in CH Cl (100 mL) before the
2
2
addition of triethylamine (20 mL, 140 mmol). The mixture was cooled to 0 °C with an external ice bath
and methanesulfonyl chloride (3.0 mL, 39 mmol) was added dropwise. After warming at room
temperature, the reaction was kept under magnetic stirring for 3 h. The mixture was diluted with CH Cl
2
2
(200 mL) and washed with sat. aq. Na CO (3 x 200 mL). The organic phase was dried over Na SO .
2
3
2
4
Evaporation of the solvent yielded the desired product in high purity.
1
tertꢀButyl 4ꢀ{[(methylsulfonyl)oxy]methyl}piperidineꢀ1ꢀcarboxylate (1d). Yield: 86%. H NMR
(
500 MHz, DMSOꢀd ) δ: 1.03−1.11 (m, 2H), 1.38 (s, 9H), 1.61−1.65 (m, 2H), 1.82−1.88 (m, 1H),
6
2
.62−2.75 (br s, 2H), 3.15 (s, 3H), 3.90−3.97 (m, 2H), 4.04 (d, J = 6.4 Hz, 2H).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 59
1
tertꢀButyl 3ꢀ{[(methylsulfonyl)oxy]methyl}piperidineꢀ1ꢀcarboxylate (1e). Yield: 85%. H NMR
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(
500 MHz, DMSOꢀd ) δ: 1.20−1.26 (m, 1H), 1.28−1.35 (m, 1H), 1.38 (s, 9H), 1.56−1.62 (m, 1H),
6
1.69−1.74 (m, 1H), 1.76−1.82 (m, 1H), 2.78−2.84 (m, 2H), 3.16 (s, 3H), 3.67−3.72 (br s, 2H),
4
.01−4.10 (m, 2H).
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tertꢀButyl
4ꢀ((3,4ꢀdimethylꢀ2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy)piperidineꢀ1ꢀcarboxylate
(2a).
Triethylamine (17 mL, 120 mmol), commercially available 1a (8.1 g, 40 mmol), and 1,1'ꢀ
azodicarbonyl)ꢀdipiperidine (20 g, 80 mmol) were added to a suspension of 3,4ꢀdimethylꢀ7ꢀ
hydroxycoumarin (15 g, 80 mmol) in dry CH Cl (250 mL). The reaction mixture was kept to 0 °C and
(
2
2
triphenylphosphine (21 g, 80 mmol), previously dissolved in CH Cl (100 mL), was added dropwise.
2
2
The reaction was kept to room temperature and left under magnetic stirring overnight. The solvent was
evaporated under reduced pressure and the resulting crude solid was purified through flash
1
chromatography (gradient eluent: ethyl acetate in nꢀhexane 20%→60%). Yield: 60%. H NMR (300
MHz, CDCl ) δ: 1.47 (s, 9H), 1.71−1.82 (m, 2H), 1.92−1.98 (m, 2H), 2.18 (s, 3H), 2.37 (s, 3H),
3
3.31−3.39 (m, 2H), 3.66−3.74 (m, 2H), 4.48−4.55 (m, 1H), 6.80 (d, J = 2.2 Hz, 1H), 6.84 (dd, J = 2.2
1
Hz, J = 8.8 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H).
2
General procedure for the synthesis of tertꢀbutyl 3ꢀ or 4ꢀ(((3,4ꢀdimethylꢀ2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀ
yl)oxy)methyl)piperidineꢀ1ꢀcarboxylate (2bꢀc). The appropriate mesylate ester 1dꢀe (8.2 g, 28 mmol)
was dissolved in dry DMF (50 mL) followed by the addition of triethylamine (7.8 g, 56 mmol), cesium
carbonate (9.1 g, 28 mmol) and 3,4ꢀdimethylꢀ7ꢀhydroxycoumarin (4.5 g, 28 mmol). After heating at 70
°C for 72 h, the mixture was poured onto crushed ice (500 g). The precipitate was collected and
thoroughly washed with water thus furnishing the desired derivative 2bꢀc.
tertꢀbutyl 4ꢀ{[(3,4ꢀdimethylꢀ2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy]methyl}piperidineꢀ1ꢀcarboxylate (2b).
1
Yield: 85%. H NMR (300 MHz, CDCl ) δ: 1.24−1.33 (m, 2H), 1.46 (s, 9H), 1.77−1.86 (m, 2H),
3
1
.93−2.03 (m, 1H), 2.18 (s, 3H), 2.37 (s, 3H), 2.75 (t, J = 12.4 Hz, 2H), 3.84−3.86 (m, 2H), 4.16−4.18
(
m, 2H), 6.77 (d, J = 2.5 Hz, 1H), 6.83 (dd, J = 2.5 Hz, J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H).
1
2
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Page 19 of 59
Journal of Medicinal Chemistry
tertꢀbutyl 3ꢀ{[(3,4ꢀdimethylꢀ2ꢀoxoꢀ2Hꢀchromenꢀ7ꢀyl)oxy]methyl}piperidineꢀ1ꢀcarboxylate (2c).
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9
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1
Yield: 89%. H NMR (300 MHz, DMSOꢀd ) δ: 1.33 (s, 9H), 1.26−1.35 (m, 2H), 1.59−1.66 (m, 1H),
6
1.71−1.83 (m, 1H), 1.85−1.91 (m, 1H), 2.00 (s, 3H), 2.35 (s, 3H), 2.80−2.95 (m, 2H), 3.88−3.97 (m,
4
H), 6.91−6.95 (m, 2H), 7.68 (d, J = 8.8 Hz, 1H).
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General procedure for the synthesis of 3,4ꢀdimethylꢀ7ꢀ(piperidinꢀ4ꢀyloxy)ꢀ2Hꢀchromenꢀ2ꢀone
(3a) and 3,4ꢀdimethylꢀ7ꢀ(piperidinꢀ3ꢀ and 4ꢀylmethoxy)ꢀ2Hꢀchromenꢀ2ꢀone (3bꢀc). To a solution of
a (7.5 g, 20 mmol) or 2bꢀc (7.7 g, 20 mmol) in CH Cl (40 mL), trifluoroacetic acid (40 mL) was
2
2
2
added dropwise while cooling to 0 °C. After 15 minutes, the reaction mixture was kept to room
temperature and left under magnetic stirring for 1 h. The solvents and excess trifluoroacetic acid were
removed under reduced pressure. The resulting oil was diluted with ethyl acetate (100 mL) and washed
with Na CO (3 x 30 mL). The organic phase was dried over Na SO and concentrated to dryness, thus
2
3
2
4
obtaining the unprotected piperidines as white solids.
1
3
,4ꢀDimethylꢀ7ꢀ(piperidinꢀ4ꢀyloxy)ꢀ2Hꢀchromenꢀ2ꢀone (3a). Yield: 83%. H NMR (500 MHz,
DMSOꢀd ) δ: 1.77−1.84 (m, 2H), 2.06 (s, 3H), 2.08−2.15 (m, 2H), 2.35 (s, 3H), 3.06−3.12 (m, 2H),
6
3
.22−3.31 (m, 2H), 4.73−4.78 (m, 1H), 6.96−6.98 (m, 1H), 7.07−7.08 (m, 1H), 7.68−7.70 (m, 1H), NH
not detected.
1
3
,4ꢀDimethylꢀ7ꢀ(piperidinꢀ4ꢀylmethoxy)ꢀ2Hꢀchromenꢀ2ꢀone (3b). Yield: 89%. H NMR (300
MHz, DMSOꢀd ) δ: 1.38−1.51 (m, 2H), 1.86−1.95 (m, 2H), 2.03−2.11 (m, 5H), 2.35 (s, 3H), 2.84−2.98
6
(
m, 2H), 3.25−3.34 (m, 2H), 3.95−3.97 (m, 2H), 6.91 (d, J = 2.5 Hz, 1H), 6.94 (dd, J = 2.5 Hz, J = 8.8
1 2
Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H).
1
3,4ꢀDimethylꢀ7ꢀ(piperidinꢀ3ꢀylmethoxy)ꢀ2Hꢀchromenꢀ2ꢀone (3c). Yield: 92%. H NMR (300 MHz,
DMSOꢀd ) δ: 1.21−1.38 (m, 2H), 1.58−1.64 (m, 1H), 1.80−1.84 (m, 2H), 2.05 (s, 3H), 2.19 (br s, 1H),
6
2
.34 (s, 3H), 2.73−2.81 (m, 2H), 3.22−3.37 (m, 2H), 3.92−4.05 (m, 2H), 6.91−6.95 (m, 2H), 7.69 (d, J =
8
.3 Hz, 1H).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 59
General procedure for the synthesis of final compounds 4a, 4i and 5a. The appropriate coumarin
aꢀc (0.50 mmol) was suspended in dry acetone (5 mL) before the addition of potassium carbonate
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(0.069 g, 0.50 mmol) and methyl iodide (0.031 mL, 0.50 mmol). The mixture was stirred at room
temperature for 6 h. The inorganic residue was then filtered off and the resulting solution was
concentrated to dryness under rotary evaporation. The desired products were purified as described
below.
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,4ꢀDimethylꢀ7ꢀ[(Nꢀmethylpiperidinꢀ4ꢀyl)oxy]ꢀ2Hꢀchromenꢀ2ꢀone (4a). Purification procedure: the
crude was treated with THF and the insoluble residue was filtered off. Evaporation of the solvent under
rotary evaporation and crystallization from nꢀhexane furnished the desired product. Yield: 59%. Mp: 95ꢀ
1
6
°C (nꢀhexane). H NMR (300 MHz, CDCl ) δ: 1.88−1.96 (m, 2H), 2.10−2.15 (m, 2H), 2.18 (s, 3H),
3
2.37 (s, 3H), 2.38 (s, 3H), 2.40−2.50 (m, 2H), 2.75−2.80 (m, 2H), 4.41 (br s, 1H), 6.80 (d, J = 2.5 Hz,
1
7
H), 6.84 (dd, J = 2.5 Hz, J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H). Anal. (C H NO ) calcd. % C,
1 2 17 21 3
1.06; H, 7.37; N, 4.87. Found % C, 71.39; H, 7.23; N, 5.02.
3
,4ꢀDimethylꢀ7ꢀ[(Nꢀmethylpiperidinꢀ4ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone
hydrochloride
(4i).
Purification procedure: the crude was treated with HCl 1.25 N in ethanol, collecting the precipitate.
1
Yield: 65%. Mp > 250 °C. H NMR (300 MHz, DMSOꢀd ) δ: 1.57−1.65 (m, 2H), 1.91−1.96 (m, 3H),
6
2.05 (s, 3H), 2.35 (s, 3H), 2.68 (s, 3H), 2.89−2.99 (m, 2H), 3.36−3.42 (m, 2H), 3.94 (d, J = 6.0 Hz, 2H),
6
.91−6.97 (m, 2H), 7.69 (d, J = 9.0 Hz, 1H), 10.40 (br s, 1H, dis. with D O). Anal. (C H NO ·HCl)
2 18 23 3
calcd. % C, 63.99; H, 7.16; N, 4.15. Found % C, 64.31; H, 7.26; N, 4.20.
(±)ꢀ3,4ꢀDimethylꢀ7ꢀ[(Nꢀmethylpiperidinꢀ3ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone hydrochloride (5a).
Purification procedure: the crude was treated with HCl 1.25 N in ethanol yielding a precipitate that was
1
collected and washed with ethanol. Yield: 60%. Mp: 150ꢀ2 °C. H NMR (300 MHz, DMSOꢀd ) δ:
6
1.19−1.30 (m, 1H), 1.70−1.89 (m, 4H), 2.06 (s, 3H), 2.35 (s, 3H), 2.76 (s, 3H), 3.25−3.54 (m, 4H),
3
.89−4.08 (m, 2H), 6.91−6.97 (m, 2H), 7.70 (d, J = 9.0 Hz, 1H), 9.91 (br s, 1H, dis. with D O). Anal.
2
(
C H NO ꢂHCl) calcd. % C, 63.99; H, 7.16; N, 4.15. Found % C, 63.89; H, 7.21; N, 3.93.
18 23 3
ACS Paragon Plus Environment

Page 21 of 59
Journal of Medicinal Chemistry
General procedure for the synthesis of final compounds 4bꢀh, 4jꢀm and (±)ꢀ5bꢀh. A Pyrex vessel
was charged with a magnetic stirring and a Weflon bar, and then the appropriate 3,4ꢀdimethylꢀ7ꢀ
(piperidinꢀ3ꢀ and 4ꢀyloxy)ꢀ2Hꢀchromenꢀ2ꢀone 3aꢀb, or 3,4ꢀdimethylꢀ7ꢀ(piperidinꢀ3ꢀ and 4ꢀylmethoxy)ꢀ
1
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Hꢀchromenꢀ2ꢀone 3cꢀd derivative (0.50 mmol) and potassium carbonate (1.0 mmol) were suspended in
acetone (4.0 mL). The suitable commercially available butyl chloride (0.50 mmol, for 4b) or substituted
benzyl bromide (0.5 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 off after
thorough washing with CH Cl . The solution was concentrated to dryness, and the resulting crude was
0
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purified as detailed below. Compounds 4jꢀm, (±)ꢀ5bꢀh, 7aꢀb were obtained as hydrochloride salts as
described later.
3,4ꢀDimethylꢀ7ꢀ[(Nꢀbutylpiperidinꢀ4ꢀyl)oxy]ꢀ2Hꢀchromenꢀ2ꢀone (4b). Isolation procedure: column
chromatography (eluent: methanol in chloroform 5%) followed by crystallization from hot ethanol.
1
Yield: 55%. Mp: 86ꢀ7 °C (ethanol). H NMR (300 MHz, CDCl ) δ: 0.98 (t, J = 7.3 Hz, 3H), 1.39−1.46
3
(m, 2H), 1.91−1.98 (m, 2H), 2.18−2.23 (m, 2H), 2.20 (s, 3H), 2.38 (s, 3H), 2.76−2.83 (m, 2H),
2
.93−3.04 (m, 2H), 3.06−3.12 (m, 2H), 3.42−3.46 (m, 2H), 4.76−4.79 (m, 1H), 6.81−6.85 (m, 2H), 7.54
(
d, J = 8.5 Hz, 1H). Anal. (C H NO ) calcd. % C, 72.92; H, 8.26; N, 4.25. Found % C, 73.34; H, 8.10;
2
0
27
3
N, 3.97.
3
,4ꢀDimethylꢀ7ꢀ[(Nꢀbenzylpiperidinꢀ4ꢀyl)oxy]ꢀ2Hꢀchromenꢀ2ꢀone (4c). Isolation procedure:
1
crystallization from hot ethanol. Yield: 82%. Mp: 111ꢀ2 °C (ethanol). H NMR (300 MHz, CDCl ) δ:
3
1
.89−2.07 (m, 4H), 2.19 (s, 3H), 2.32−2.38 (m, 5H), 2.77−2.85 (m, 2H), 3.51−3.57 (m, 2H), 4.37−4.45
(
m, 1H), 6.77ꢀ6.90 (m, 2H), 7.24−7.34 (m, 5H), 7.49 (d, J = 8.5 Hz, 1H). Anal. (C H NO ) calcd. %
2
3
25
3
C, 76.00; H, 6.93; N, 3.85. Found % C, 76.01; H, 6.89; N, 4.01.
3
,4ꢀDimethylꢀ7ꢀ[(Nꢀ(3ꢀchlorobenzyl)piperidinꢀ4ꢀyl)oxy]ꢀ2Hꢀchromenꢀ2ꢀone
(4d).
Isolation
1
procedure: crystallization from hot ethanol. Yield: 84%. Mp: 106ꢀ7 °C (ethanol). H NMR (300 MHz,
CDCl ) δ: 1.83−1.91 (m, 2H), 2.02−2.07 (m, 2H), 2.18 (s, 3H), 2.34−2.40 (m, 5H), 2.70−2.77 (m, 2H),
3
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Page 22 of 59
3
.51−3.56 (m, 2H), 4.37−4.43 (m, 1H), 6.80 (d, J = 2.5 Hz, 1H), 6.83 (dd, J = 2.5 Hz, J = 8.5 Hz, 1H),
1 2
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
.23−7.29 (m, 3H), 7.37 (s, 1H), 7.49 (d, J = 8.5 Hz, 1H). Anal. (C H ClNO ) calcd. % C, 69.43; H,
23 24 3
6.08; N, 3.52. Found % C, 69.70; H, 6.06; N, 3.56.
3
,4ꢀDimethylꢀ7ꢀ[(Nꢀ(4ꢀchlorobenzyl)piperidinꢀ4ꢀyl)oxy]ꢀ2Hꢀchromenꢀ2ꢀone
(4e).
Isolation
1
procedure: crystallization from hot ethanol. Yield: 87%. Mp: 140ꢀ1 °C (ethanol). H NMR (300 MHz,
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
CDCl ) δ: 1.83−1.89 (m, 2H), 1.98−2.05 (m, 2H), 2.18 (s, 3H), 2.27−2.33 (m, 2H), 2.36 (s, 3H),
3
2
.70−2.77 (m, 2H), 3.52 (s, 2H), 4.36−4.41 (m, 1H), 6.79 (d, J = 2.5 Hz, 1H), 6.83 (dd, J = 2.5 Hz, J =
1 2
8.5 Hz, 1H), 7.28−7.33 (m, 4H), 7.48 (d, J = 8.5 Hz, 1H). Anal. (C H ClNO ) calcd. % C, 69.43; H,
2
3
24
3
6
.08; N, 3.52. Found % C, 69.43; H, 6.13; N, 3.85.
ꢀ[(4ꢀ((3,4ꢀDimethylꢀ2Hꢀ2ꢀoxochromenꢀ7ꢀyl)oxy)piperidinꢀ1ꢀyl)methyl]benzonitrile
Isolation procedure: crystallization from hot ethanol. Yield: 71%. Mp: 120ꢀ1 °C (ethanol). H NMR
300 MHz, CDCl ) δ: 1.79−2.00 (m, 4H), 2.18 (s, 3H), 2.35−2.38 (m, 5H), 2.78−2.82 (m, 2H),
4
(4f).
1
(
3
3
.50−3.56 (m, 2H), 4.41 (s, 1H), 6.79 (d, J = 2.5 Hz, 1H), 6.82 (dd, J = 2.5 Hz, J = 8.8 Hz, 1H),
1 2
7.15−7.33 (m, 4H), 7.49 (d, J = 8.8 Hz, 1H). Anal. (C H N O ) calcd. % C, 74.21; H, 6.23; N, 7.21.
2
4
24
2
3
Found % C, 74.74; H, 6.19; N, 7.21.
,4ꢀDimethylꢀ7ꢀ[(Nꢀ(2ꢀphenylethyl)piperidinꢀ4ꢀyl)oxy]ꢀ2Hꢀchromenꢀ2ꢀone
3
(4g).
Isolation
1
procedure: crystallization from hot ethanol. Yield: 87%. Mp: 107ꢀ8 °C (ethanol). H NMR (300 MHz,
CDCl ) δ: 1.93−2.01 (m, 2H), 2.17−2.20 (m, 4H), 2.35−2.39 (m, 4H), 2.72−2.95 (m, 8H), 4.45−4.51 (m,
3
1H), 6.81 (d, J = 2.5 Hz, 1H), 6.84 (dd, J = 2.5 Hz, J = 8.5 Hz, 1H), 7.22−7.33 (m, 5H), 7.50 (d, J =
1
2
8
.5 Hz, 1H). Anal. (C H NO ) calcd. % C, 76.36; H, 7.21; N, 3.71. Found % C, 76.53; H, 7.14; N,
24 27 3
3
.97.
3,4ꢀDimethylꢀ7ꢀ[(Nꢀ(2ꢀphenylꢀ2ꢀoxoethyl)piperidinꢀ4ꢀyl)oxy]ꢀ2Hꢀchromenꢀ2ꢀone (4h). Isolation
1
procedure: crystallization from hot ethanol. Yield: 90%. Mp: 94ꢀ6 °C (dec.) from ethanol. H NMR (300
MHz, CDCl ) δ: 1.63−1.72 (m, 2H), 2.05−2.16 (m, 2H), 2.19 (s, 3H), 2.34−2.41 (m, 5H), 2.96−3.02 (m,
3
2
H), 4.19−4.27 (m, 2H), 4.57−4.63 (m, 1H), 6.85 (d, J = 2.5 Hz, 1H), 6.89 (dd, J = 2.5 Hz, J = 8.8 Hz,
1 2
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Journal of Medicinal Chemistry
1
H), 7.51 (t, J = 7.1 Hz, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.64 (t, J = 7.1 Hz, 1H), 7.98 (d, J = 7.1 Hz, 2H).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Anal. (C H NO ) calcd. % C, 73.64; H, 6.44; N, 3.58. Found % C, 74.15; H, 6.38; N, 3.85.
2
4
25
4
3,4ꢀDimethylꢀ7ꢀ[(Nꢀbenzylpiperidinꢀ4ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone
hydrochloride
(4j).
Isolation procedure: column chromatography (eluent: ethyl acetate in chloroform 50%). The compound
was transformed into the corresponding hydrochloride salt by dissolving the solid free base in the
minimum volume of 1,4ꢀdioxane before adding HCl 4.0 N in 1,4ꢀdioxane. The resulting precipitate was
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
1
collected by filtration and washed with dry dioxane, yielding 4j. Yield: 60%. Mp: 215ꢀ6 °C (dec.). H
NMR (300 MHz, DMSOꢀd ) δ: 1.87−1.95 (m, 2H), 2.06 (s, 3H), 2.22−2.29 (m, 3H), 2.35 (s, 3H),
6
2
.79−2.84 (m, 2H), 3.13−3.20 (m, 2H), 3.91−4.03 (m, 2H), 4.31−4.35 (m, 2H), 6.88−6.96 (m, 2H),
7
.46−7.58 (m, 5H), 7.69 (d, J = 9.1 Hz, 1H), 9.68 (s, 1H, dis. with D O). Anal. (C H NO ·HCl) calcd.
2
24 27
3
% C, 69.64; H, 6.82; N, 3.38. Found % C, 70.09; H, 6.55; N, 3.40.
3
,4ꢀDimethylꢀ7ꢀ[(Nꢀ(3ꢀchlorobenzyl)piperidinꢀ4ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone hydrochloride
(4k). Isolation procedure: the crude was suspended in 1,4ꢀdioxane and the insoluble residue discarded.
HCl 4.0 N in 1,4ꢀdioxane was added to the solution, yielding a white precipitate that was filtered and
1
crystallized from hot ethanol. Yield: 99%. Mp: 246ꢀ8 °C (ethanol). H NMR (300 MHz, DMSOꢀd6) δ:
1
2
2
.55−1.66 (m, 2H), 1.90−1.97 (m, 3H), 2.06 (s, 3H), 2.35 (s, 3H), 2.89−3.00 (m, 2H), 3.35−3.42 (m,
H), 3.94 (d, J = 6.0 Hz, 2H), 4.26−4.30 (m, 2H), 6.91 (dd, J = 2.5 Hz, J = 8.8 Hz, 1H), 6.95 (d, J =
1
2
.5 Hz, 1H), 7.48−7.55 (m, 3H), 7.66−7.71 (m, 2H), 10.16 (s, 1H, dis. with D O). Anal.
2
(C H ClNO ·HCl) calcd. % C, 64.29; H, 6.07; N, 3.12. Found % C, 64.68; H, 5.88; N, 2.83.
2
4
26
3
3
,4ꢀDimethylꢀ7ꢀ[(Nꢀ(4ꢀchlorobenzyl)piperidinꢀ4ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone hydrochloride
(4l). Isolation procedure: the crude was suspended in 1,4ꢀdioxane and the insoluble residue was
discarded after filtration. HCl 4.0 N in 1,4ꢀdioxane was added to the solution, yielding a white
1
precipitate that was filtered and crystallized from hot ethanol. Yield: 77%. Mp > 250 °C (ethanol). H
NMR (300 MHz, DMSOꢀd ) δ: 1.57−1.65 (m, 2H), 1.92−1.97 (m, 3H), 2.05 (s, 3H), 2.35 (s, 3H),
6
2
.91−2.99 (m, 2H), 3.38−3.45 (m, 2H), 3.94 (d, J = 6.0 Hz, 2H), 4.25−4.28 (m, 2H), 6.91 (dd, J = 2.5
1
Hz, J = 8.5 Hz, 1H), 6.95 (d, J = 2.5 Hz, 1H), 7.53 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.67
2
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Journal of Medicinal Chemistry
Page 24 of 59
(
d, J = 8.5 Hz, 1H), 10.12 (s, 1H, dis. with D O). Anal. (C H ClNO ·HCl) calcd. % C, 64.29; H, 6.07;
2 24 26 3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N, 3.12. Found % C, 64.70; H, 6.00; N, 3.05.
4ꢀ[(4ꢀ((3,4ꢀDimethylꢀ2Hꢀ2ꢀoxochromenꢀ7ꢀyl)oxymethyl)piperidinꢀ1ꢀyl)methyl]benzonitrile
hydrochloride (4m). Isolation procedure: the crude was suspended in 1,4ꢀdioxane and the insoluble
residue was filtered off. HCl 4.0 N in 1,4ꢀdioxane was added to the solution, yielding a white precipitate
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
1
that was filtered and crystallized from hot ethanol. Yield: 93%. Mp > 250 °C (ethanol). H NMR (300
MHz, DMSOꢀd ) δ: 1.64−1.79 (m, 2H), 1.88−1.97 (m, 3H), 2.05 (s, 3H), 2.34 (s, 3H), 2.94 (q, J = 12.0
6
Hz, 2H), 3.36−3.43 (m, 2H), 3.93 (d, J = 6.3 Hz, 2H), 4.32−4.37 (m, 2H), 6.90−6.99 (m, 2H), 7.68 (d, J
=
8.5 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.4 Hz, 2H), 11.17 (s, 1H, dis. with D O). Anal.
2
(C H N O ·HCl) calcd. % C, 68.41; H, 6.20; N, 6.38. Found % C, 68.86; H, 6.03; N, 6.17.
25 26 2 3
(±)ꢀ3,4ꢀDimethylꢀ7ꢀ[(Nꢀbenzylpiperidinꢀ3ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone hydrochloride (5b).
Isolation procedure: column chromatography (eluent: ethyl acetate in chloroform 50%). The compound
was transformed into the corresponding hydrochloride salt by dissolving the solid free base in the
minimum volume of 1,4ꢀdioxane before adding HCl 4.0 N in 1,4ꢀdioxane. The resulting precipitate was
1
collected by filtration and washed with dry dioxane, yielding 5b. Yield: 62%. Mp: 215ꢀ6 °C (dec.). H
NMR (300 MHz, DMSOꢀd ) δ: 1.20−1.34 (m, 1H), 1.60−1.75 (m, 1H), 1.77−1.90 (m, 2H), 2.06 (s, 3H),
6
2
.20−2.27 (m, 1H), 2.35 (s, 3H), 2.72−2.86 (m, 2H), 3.30−3.49 (m, 2H), 3.90−4.07 (m, 2H), 4.28−4.33
(m, 2H), 6.90 (dd, J = 2.5 Hz, J = 8.8 Hz, 1H), 6.93 (d, J = 2.5 Hz, 1H), 7.42−7.57 (m, 5H), 7.69 (d, J
1
2
= 8.8 Hz, 1H), 10.12 (br s, 1H, dis. with D O). Anal. (C H NO ·HCl) calcd. % C, 69.64; H, 6.82; N,
2
24 27
3
3
.38. Found % C, 69.90; H, 6.81; N, 3.56.
+)ꢀ3,4ꢀDimethylꢀ7ꢀ[(Nꢀbenzylpiperidinꢀ3ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone
(
(5b).
HPLC
purification of (±)ꢀ5b on a CHIRALPAK IA, mobile phase: A = methanol, B = acetonitrile; isocratic
1
elution: 20% B; flow rate = 1 mL/min; λ = 320 nm; 100 ꢁL injection; k . Mp: 117ꢀ9 °C. H NMR (500
2
MHz, DMSOꢀd ) δ: 1.09−1.15 (m, 1H), 1.43−1.51 (m, 1H), 1.61−1.66 (m, 1H), 1.71−1.75 (m, 1H),
6
1
.86−2.02 (m, 3H), 2.05 (s, 3H), 2.34 (s, 3H), 2.62−2.67 (m, 1H), 2.81−2.85 (m, 1H), 3.41 (d, J = 13.7
Hz, 1H), 3.47 (d, J = 13.7 Hz, 1H), 3.92 (d, J = 6.4 Hz, 2H), 6.87−6.90 (m, 2H), 7.19−7.23 (m, 1H),
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Journal of Medicinal Chemistry
20
D
7
.25−7.30 (m, 4H), 7.65 (d, J = 8.8 Hz, 1H). [α] = +18.5° (c 0.13, MeOH). Anal. (C H NO ) calcd.
24 27
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
%
C, 76.36; H, 7.21; N, 3.71. Found % C, 76.50; H, 7.05; N, 3.46.
(−)ꢀ3,4ꢀDimethylꢀ7ꢀ[(Nꢀbenzylpiperidinꢀ3ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone
(5b).
HPLC
purification of (±)ꢀ5b on a CHIRALPAK IA, mobile phase: A = methanol, B = acetonitrile; isocratic
1
elution: 20% B; flow rate = 1 mL/min; λ = 320 nm; 100 ꢁL injection; k . Mp: 117ꢀ9 °C. H NMR (500
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
1
MHz, DMSOꢀd ) δ: 1.07−1.14 (m, 1H), 1.42−1.51 (m, 1H), 1.61−1.66 (m, 1H), 1.70−1.78 (m, 1H),
6
1
.84−2.02 (m, 3H), 2.05 (s, 3H), 2.34 (s, 3H), 2.62−2.67 (m, 1H), 2.81−2.85 (m, 1H), 3.42 (d, J = 13.7
Hz, 1H), 3.47 (d, J = 13.7 Hz, 1H), 3.91 (d, J = 6.4 Hz, 2H), 6.87−6.90 (m, 2H), 7.18−7.23 (m, 1H),
20
D
7
.26−7.30 (m, 4H), 7.64 (d, J = 8.8 Hz, 1H). [α] = −18.5° (c 0.13, MeOH). Anal. (C H NO ) calcd.
24 27
3
%
C, 76.36; H, 7.21; N, 3.71. Found % C, 76.80; H, 6.91; N, 3.55.
(±)ꢀ3,4ꢀDimethylꢀ7ꢀ[(Nꢀ(3ꢀchlorobenzyl)piperidinꢀ3ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone
hydrochloride (5c). Isolation procedure: the crude was suspended in 1,4ꢀdioxane and the insoluble
residue was filtered off. HCl 4.0 N in 1,4ꢀdioxane was added to the solution, yielding a white precipitate
1
that was filtered and crystallized from hot ethanol. Yield: 98%. MP 246ꢀ8 °C (dec.) from ethanol. H
NMR (300 MHz, DMSOꢀd ) δ: 1.21−1.35 (m, 1H), 1.77−1.85 (m, 3H), 2.05 (s, 3H), 2.34 (s, 3H),
6
2
.36−2.43 (m, 1H), 2.81 (qn, J = 11.5 Hz, 2H), 3.37−3.48 (m, 2H), 3.94−4.06 (m, 2H), 4.32 (br s, 2H),
6
.85−6.93 (m, 2H), 7.45−7.54 (m, 3H), 7.45−7.70 (m, 2H), 10.30 (br s, 1H, dis. with D O). Anal.
2
(C H ClNO ·HCl) calcd. % C, 64.29; H, 6.07; N, 3.12. Found % C, 63.82; H, 5.85; N, 2.93.
24 26 3
(±)ꢀ7ꢀ{[1ꢀ(3ꢀBromobenzyl)piperidinꢀ3ꢀyl]methoxy}ꢀ3,4ꢀdimethylꢀ2Hꢀchromenꢀ2ꢀone
hydrochloride (5d). Isolation procedure: flash chromatography (gradient eluent: ethyl acetate in nꢀ
hexane 20%→80%). The compound was transformed into the corresponding hydrochloride salt by
dissolving the solid free base in the minimum volume of 1,4ꢀdioxane before adding HCl 4.0 N in 1,4ꢀ
dioxane. The resulting precipitate was collected by filtration and washed with dry dioxane, yielding
1
racemic 5d. Yield: 87%. Mp: 231ꢀ2 °C. H NMR (free base, 300 MHz, DMSOꢀd ) δ: 1.05−1.18 (m,
6
1
H), 1.46−1.53 (m, 1H), 1.62−1.75 (m, 2H), 1.89−1.97 (m, 3H), 2.05 (s, 3H), 2.34 (s, 3H), 2.61ꢀ2.65
(m, 1H), 2.79−2.82 (m, 1H), 3.39−3.51 (m, 2H), 3.94 (d, J = 6.1 Hz, 2H), 6.86−6.89 (m, 2H), 7.21−7.29
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Journal of Medicinal Chemistry
Page 26 of 59
(
m, 2H), 7.40−7.45 (m, 2H), 7.65 (d, J = 9.6 Hz, 1H). Anal. (C H BrNO ·HCl) calcd. % C, 58.49; H,
24 26 3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
.52; N, 2.84. Found % C, 58.14; H, 5.48; N, 2.98.
(+)ꢀ7ꢀ{[1ꢀ(3ꢀBromobenzyl)piperidinꢀ3ꢀyl]methoxy}ꢀ3,4ꢀdimethylꢀ2Hꢀchromenꢀ2ꢀone (5d). HPLC
purification of (±)ꢀ5d on a CHIRALPAK IA, mobile phase: A = methanol, B = acetonitrile; isocratic
1
elution: 30% B; flow rate = 1 mL/min; λ = 320 nm; 100 ꢁL injection; k . Mp: 119ꢀ120 °C. H NMR
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
(300 MHz, DMSOꢀd ) δ: 1.03−1.18 (m, 1H), 1.46−1.53 (m, 1H), 1.64−1.75 (m, 2H), 1.87−1.97 (m,
6
3
H), 2.05 (s, 3H), 2.34 (s, 3H), 2.60−2.65 (m, 1H), 2.79−2.82 (m, 1H), 3.39−3.51 (m, 2H), 3.93 (d, J =
20
6.1 Hz, 2H), 6.86−6.89 (m, 2H), 7.22−7.29 (m, 2H), 7.40−7.45 (m, 2H), 7.65 (d, J = 9.6 Hz, 1H). [α]
D
=
+33.5° (c 0.08, MeOH). Anal. (C H BrNO ) calcd. % C, 63.16; H, 5.74; N, 3.07. Found % C, 63.50;
24 26 3
H, 5.58; N, 3.00.
(−)ꢀ7ꢀ{[1ꢀ(3ꢀBromobenzyl)piperidinꢀ3ꢀyl]methoxy}ꢀ3,4ꢀdimethylꢀ2Hꢀchromenꢀ2ꢀone (5d). HPLC
purification of (±)ꢀ5d on a CHIRALPAK IA, mobile phase: A = methanol, B = acetonitrile; isocratic
1
elution: 30% B; flow rate = 1 mL/min; λ = 320 nm; 100 ꢁL injection; k . Mp: 119ꢀ120 °C. H NMR
1
(300 MHz, DMSOꢀd ) δ: 1.05−1.18 (m, 1H), 1.46−1.53 (m, 1H), 1.62−1.75 (m, 2H), 1.89−1.97 (m,
6
3
H), 2.07 (s, 3H), 2.33 (s, 3H), 2.61−2.67 (m, 1H), 2.80−2.84 (m, 1H), 3.36−3.51 (m, 2H), 3.94 (d, J =
20
6
.1 Hz, 2H), 6.86−6.89 (m, 2H), 7.21−7.29 (m, 2H), 7.44−7.49 (m, 2H), 7.64 (d, J = 9.6 Hz, 1H). [α]
D
=
−33.5° (c 0.08, MeOH). Anal. (C H BrNO ) calcd. % C, 63.16; H, 5.74; N, 3.07. Found % C, 63.49;
24 26 3
H, 5.36; N, 2.96.
(±)ꢀ3,4ꢀDimethylꢀ7ꢀ[(Nꢀ(4ꢀfluorobenzyl)piperidinꢀ3ꢀyl)methoxy]ꢀ2H-chromenꢀ2ꢀone
hydrochloride (5e). Isolation procedure: the crude was suspended in 1,4ꢀdioxane and the insoluble
residue was filtered off. HCl 4.0 N in 1,4ꢀdioxane was added to the solution, yielding a white precipitate
1
that was filtered and crystallized from hot ethanol. Yield: 98%. Mp: 228ꢀ230 °C (dec.). H NMR (300
MHz, DMSOꢀd ) δ: 1.18−1.33 (m, 1H), 1.77−1.88 (m, 3H), 2.05 (s, 3H), 2.34 (s, 3H), 2.36−2.41 (m,
6
1
H), 2.78 (qn, J = 9.9 Hz, 2H), 3.40−3.46 (m, 2H), 3.90−4.05 (m, 2H), 4.27−4.32 (m, 2H), 6.89 (dd, J1
=
8.8 Hz, J = 2.5 Hz, 1H), 6.93 (d, J = 2.5 Hz, 1H), 7.26−7.32 (m, 2H), 7.59−7.64 (m, 2H), 7.68 (d, J =
2
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Journal of Medicinal Chemistry
8
3
.8 Hz, 1H), 10.34 (br s, 1H, dis. with D O). Anal. (C H FNO ·HCl) calcd. % C, 66.74; H, 6.30; N,
2 24 26 3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
.24. Found % C, 66.34; H, 6.22; N, 3.17.
(±)ꢀ3,4ꢀDimethylꢀ7ꢀ[(Nꢀ(4ꢀchlorobenzyl)piperidinꢀ3ꢀyl)methoxy]ꢀ2Hꢀchromenꢀ2ꢀone
hydrochloride (5f). Isolation procedure: the crude was suspended in 1,4ꢀdioxane and the insoluble
residue was filtered off. HCl 4.0 N in 1,4ꢀdioxane was added to the solution, yielding a white precipitate
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
1
that was filtered and crystallized from hot ethanol. Yield: 91%. Mp: 219ꢀ221 °C (dec.) from ethanol. H
NMR (300 MHz, DMSOꢀd ) δ: 1.18−1.34 (m, 1H), 1.76−1.88 (m, 3H), 2.05 (s, 3H), 2.35 (s, 3H),
6
2.36−2.41 (m, 1H), 2.71−2.86 (m, 2H), 3.39−3.46 (m, 2H), 3.90−4.05 (m, 2H), 4.29−4.31 (m, 2H), 6.88
(d, J = 2.5 Hz, 1H), 6.93 (dd, J = 2.5 Hz, J = 8.8 Hz, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz,
1 2
2
H), 7.68 (d, J = 8.8 Hz, 1H), 10.37 (br s, 1H, dis. with D O). Anal. (C H ClNO ·HCl) calcd. % C,
2 24 26 3
64.29; H, 6.07; N, 3.12. Found % C, 63.86; H, 5.97; N, 3.09.
±)ꢀ3,4ꢀDimethylꢀ7ꢀ[(Nꢀ(4ꢀ(methylsulfonyl)benzyl)piperidinꢀ3ꢀyl)methoxy]ꢀ2H-chromenꢀ2ꢀone
(
hydrochloride (5g). Isolation procedure: the crude was suspended in 1,4ꢀdioxane and the insoluble
residue was filtered off. HCl 4.0 N in 1,4ꢀdioxane was added to the solution, yielding a white precipitate
1
that was filtered and crystallized from hot ethanol. Yield: 99%. Mp: 172ꢀ4 °C (ethanol). H NMR (300
MHz, DMSOꢀd ) δ: 1.20−1.36 (m, 1H), 1.80−1.87 (m, 3H), 2.05 (s, 3H), 2.34 (s, 3H), 2.40−2.46 (m,
6
1
H), 2.76−2.87 (m, 2H), 3.24 (s, 3H), 3.28−3.48 (m, 2H), 3.91−4.03 (m, 2H), 4.40−4.45 (m, 2H), 6.89
(dd, J = 2.5 Hz, J = 8.8 Hz, 1H), 6.93 (d, J = 2.5 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.89 (d, J = 8.7 Hz,
1
2
2H), 8.00 (d, J = 8.7 Hz, 2H), 10.91 (br s, 1H, dis. with D O). Anal. (C H NO S·HCl) calcd. % C,
2
25 29
5
6
1.03; H, 6.15; N, 2.85. Found % C, 60.65; H, 5.84; N, 2.91.
±)ꢀ4ꢀ[(3ꢀ((3,4ꢀDimethylꢀ2Hꢀ2ꢀoxochromenꢀ7ꢀyl)oxymethyl)piperidinꢀ1ꢀyl)methyl]benzonitrile
(
hydrochloride (5h). Isolation procedure: the crude was suspended in 1,4ꢀdioxane and the insoluble
residue was filtered off. HCl 4.0 N in 1,4ꢀdioxane was added to the solution, yielding a white precipitate
1
that was filtered and crystallized from hot ethanol. Yield: 76%. Mp: 220ꢀ2 °C (ethanol). H NMR (300
MHz, DMSOꢀd ) δ: 1.26−1.30 (m, 1H), 1.80−1.85 (m, 3H), 2.06 (s, 3H), 2.35 (s, 3H), 2.39−2.43 (m,
6
1
H), 2.76−2.87 (m, 2H), 3.30−3.31 (m, 1H), 3.44−3.48 (m, 1H), 3.91−4.05 (m, 2H), 4.37−4.44 (m, 2H),
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Journal of Medicinal Chemistry
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6
.88−6.94 (m, 2H), 7.69 (d, J = 8.7 Hz, 1H), 7.77 (d, J = 7.8 Hz, 2H), 7.95 (d, J = 7.8 Hz, 2H), 10.30 (br
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
s, 1H, dis. with D O). Anal. (C H N O ·HCl) calcd. % C, 68.41; H, 6.20; N, 6.38. Found % C, 68.77;
2
25 26
2
3
H, 6.05; N, 6.17.
±)ꢀ7ꢀ((1ꢀ(3,4ꢀDimethoxybenzyl)piperidinꢀ3ꢀyl)methoxy)ꢀ3,4ꢀdimethylꢀ2Hꢀchromenꢀ2ꢀone (5i).
,4ꢀDimethylꢀ7ꢀ(piperidinꢀ3ꢀylmethoxy)ꢀ2Hꢀchromenꢀ2ꢀone (0.12 g, 0.40 mmol) was dissolved under
(
3
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
magnetic stirring with 1.3 mL of 1,2ꢀdichloroethane in a flameꢀdried roundꢀbottomed flask. 3,4ꢀ
dimethoxybenzaldehyde (0.66 g, 0.40 mmol) and sodium triacetoxyborohydride (0.12 g, 0.56 mmol)
were added and the reaction mixture was left under magnetic stirring, at room temperature and under
nitrogen atmosphere overnight. After the removal of the solvent, the solid crude was purified through
flash chromatography (gradient eluent: methanol in CH Cl 0%→10%). Yield: 45%. Mp: 62ꢀ4 °C
2
2
1
(dec.). H NMR (300 MHz, DMSOꢀd ) δ: 1.16−1.18 (m, 1H), 1.42−1.51 (m, 1H), 1.59−1.64 (m, 1H),
6
1
1
.69−1.77 (m, 1H), 1.83−1.99 (m, 3H), 2.05 (s, 3H), 2.34 (s, 3H), 2.60−2.67 (m, 1H), 2.77−2.83 (m,
H), 3.31 (d, J = 13.5 Hz, 1H), 3.40 (d, J = 13.5 Hz, 1H), 3.67 (s, 3H), 3.70 (s, 3H), 3.92−3.94 (m, 2H),
6.74−6.77 (m, 1H), 6.82−6.84 (m, 2H), 6.86−6.90 (m, 2H), 7.65 (d, J = 8.5 Hz, 1H). Anal. (C H NO )
2
6
31
5
calcd. % C, 71.37; H, 7.14; N, 3.20. Found % C, 71.56; H, 7.10; N, 3.09.
General procedure for the synthesis of 7ꢀ(4ꢀ and 3ꢀbromoalkyloxy)ꢀ3,4ꢀdimethylꢀ2Hꢀchromenꢀ2ꢀ
one 6aꢀb. A Pyrex vessel was charged with a magnetic stirring and Weflon bar and then 7ꢀhydroxyꢀ3,4ꢀ
dimethylꢀ2Hꢀchromenꢀ2ꢀone (0.57 g, 3.0 mmol) and anhydrous potassium carbonate (0.83 g, 6.0 mmol)
were suspended in acetone (10 mL). The suitable commercially available dibromoalkyl derivative (15
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 CH Cl . The
2
2
solution was concentrated to dryness, and the resulting crude was purified through flash
chromatography (gradient eluent as indicated below).
7
ꢀ(3ꢀBromopropoxy)ꢀ3,4ꢀdimethylꢀ2Hꢀchromenꢀ2ꢀone (6a). Purified by flash chromatography
1
(gradient eluent: ethyl acetate in nꢀhexane 0%→60%). Yield: 79%. H NMR (500 MHz, DMSOꢀd ) δ:
6
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Journal of Medicinal Chemistry
2
.07 (s, 3H), 2.27 (q, J = 6.4 Hz, 2H), 2.36 (s, 3H), 3.67 (t, J = 6.4 Hz, 2H), 4.18 (t, J = 6.4 Hz, 2H),
.95−6.98 (m, 2H), 7.70 (d, J = 8.8 Hz, 1H).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
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5
5
5
5
5
5
6
6
7ꢀ(4ꢀBromobutoxy)ꢀ3,4ꢀdimethylꢀ2Hꢀchromenꢀ2ꢀone (6b). Purified by flash chromatography
1
(gradient eluent: ethyl acetate in nꢀhexane 0%→50%). Yield: 88%. H NMR (500 MHz, DMSOꢀd ) δ:
6
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
1
.85 (qn, J = 6.4 Hz, 2H), 1.97 (qn, J = 6.4 Hz, 2H), 2.07 (s, 3H), 2.36 (s, 3H), 3.61 (t, J = 6.4 Hz, 2H),
4.10 (t, J = 6.4 Hz, 2H), 6.92−6.95 (m, 2H), 7.68 (d, J = 8.8 Hz, 1H).
General procedure for the synthesis of final compounds 7aꢀb. Appropriate bromide derivative 6aꢀ
b (0.50 mmol) was suspended under magnetic stirring in acetone (4 mL) in a Pyrex microwave reactor
in the presence of anhydrous potassium carbonate (0.21 g, 1.5 mmol) and catalytic amount of potassium
iodide. 1,2,3,4ꢀTetrahydroisoquinoline (0.080 g, 0.60 mmol) was added and the vessel was placed in a
microwave apparatus and heated at 130 °C for 45 min. After cooling to room temperature, the solid
residue was filtered off after washing with CH Cl . The solution was concentrated to dryness, and the
2
2
resulting crude was purified through flash chromatography (gradient eluent: ethyl acetate in nꢀhexane
0%→80%). The resulting solids were transformed into the corresponding hydrochlorides by dissolving
2
the base in the minimum amount of 1,4ꢀdioxane followed by the addition of HCl 4.0 N in 1,4ꢀdioxane.
The precipitate was collected after filtration and washed with dry 1,4ꢀdioxane under an Ar atmosphere.
7
ꢀ[3ꢀ(3,4ꢀDihydroisoquinolinꢀ2(1H)ꢀyl)propoxy]ꢀ3,4ꢀdimethylꢀ2Hꢀchromenꢀ2ꢀone hydrochloride
1
(7a). Yield: 74%. Mp > 250 °C. H NMR (free base, 300 MHz, DMSOꢀd ) δ: 1.98 (qn, J = 6.4 Hz, 2H),
6
2
.05 (s, 3H), 2.34 (s, 3H), 2.59 (t, J = 6.4 Hz, 2H), 2.65 (t, J = 5.8 Hz, 2H), 2.79 (t, J = 5.8 Hz, 2H), 3.55
(s, 2H), 4.13 (t, J = 6.4 Hz, 2H), 6.91−6.94 (m, 2H), 7.01−7.09 (m, 4H), 7.66 (d, J = 9.9 Hz, 1H). Anal.
(C H NO ·HCl) calcd. % C, 69.08; H, 6.55; N, 3.50. Found % C, 69.45; H, 6.58; N, 3.75.
2
3
25
3
7
ꢀ[4ꢀ(3,4ꢀDihydroisoquinolinꢀ2(1H)ꢀyl)butoxy]ꢀ3,4ꢀdimethylꢀ2Hꢀchromenꢀ2ꢀone hydrochloride
1
(7b). Yield: 72%. Mp: 206ꢀ8 °C. H NMR (free base, 500 MHz, DMSOꢀd ) δ: 1.67 (qn, J = 6.9 Hz, 2H),
6
1
.79 (qn, J = 6.9 Hz, 2H), 2.07 (s, 3H), 2.36 (s, 3H), 2.46−2.48 (m, 2H), 2.64 (t, J = 5.4 Hz, 2H), 2.79 (t,
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