Investigating alkyl nitrates as nitric oxide releasing precursors of multitarget acetylcholinesterase-monoamine oxidase B inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.10.016

Herein we envisaged the possibility of exploiting alkyl nitrates as precursors of alcohol-bearing dual inhibitors targeting acetylcholinesterase (AChE) and monoamine oxidase B (MAO B), key enzymes in neurodegenerative syndromes such as Alzheimer's disease (AD), through biotransformation unmasking an alcoholic function upon nitric oxide (NO) release. The cooperation to neuroprotection of low fluxes of NO and target enzymes’ inhibition by the alcohol metabolites might return a multitargeting effect. The in vitro screening towards ChEs and MAOs of a collection of 21 primary alcohols disclosed a subset of dual inhibitors, among which three diverse chemotypes were selected to study the corresponding nitrates. Nitrate 14 proved to be a brain permeant, potent AChE-MAO B inhibitor by itself. Moreover, it protected human SH-SY5Y lines against rotenone and hydrogen peroxide with a poor inherent cytotoxicity and showed a slow conversion profile to its alcohol metabolite 9d that still behaved as bimodal and neuroprotective molecule.

Full text of DOI:10.1016/j.ejmech.2018.10.016

Accepted Manuscript
Investigating alkyl nitrates as nitric oxide releasing precursors of multitarget
acetylcholinesterase-monoamine oxidase B inhibitors
Leonardo Pisani, Rosa Maria Iacobazzi, Marco Catto, Mariagrazia Rullo, Roberta
Farina, Nunzio Denora, Saverio Cellamare, Cosimo Damiano Altomare
PII:
S0223-5234(18)30878-X
10.1016/j.ejmech.2018.10.016
EJMECH 10802
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 July 2018
Revised Date: 4 October 2018
Accepted Date: 8 October 2018
Please cite this article as: L. Pisani, R.M. Iacobazzi, M. Catto, M. Rullo, R. Farina, N. Denora, S.
Cellamare, C.D. Altomare, Investigating alkyl nitrates as nitric oxide releasing precursors of multitarget
acetylcholinesterase-monoamine oxidase B inhibitors, European Journal of Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.ejmech.2018.10.016.
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ACCEPTED MANUSCRIPT
Investigating alkyl nitrates as nitric oxide releasing precursors of multitarget
acetylcholinesterase-monoamine oxidase B inhibitors
b
a
a
Leonardo Pisani *,a, Rosa Maria Iacobazzi , Marco Catto , Mariagrazia Rullo , Roberta
Farina a, Nunzio Denora a, Saverio Cellamare a, Cosimo Damiano Altomare a
a
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”,
via E. Orabona 4, 70125 Bari, Italy
b Istituto Tumori IRCCS Giovanni Paolo II, viale O. Flacco 65, 70124 Bari, Italy
KEYWORDS
Acetylcholinesterase; monoamine oxidase B; nitric oxide donor; multitargeting agents.
ABBREVIATIONS
ACh, acetylcholine; AChE, acetylcholinesterase; AD, Alzheimer’s disease; BBB, blood brain
barrier; BChE, butyrylcholinesterase; DIEA, N,N-diisopropylethylamine; DMEM, Dulbecco’s
modified eagle medium; ER, efflux ratio; FBS, fetal bovine serum; FD4, fluorescein
isothiocyanate-dextran; GSH, glutathione; MAO, monoamine oxidase; MDCK, Madin-Darby
Canine Kidney; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; ND,
neurodegenerative disease; NMDA, N-methyl-D-aspartate; NO, nitric oxide; Papp, apparent
permeability; Papp AP, apparent permeability apical-to-basal; Papp BL, apparent permeability
basal-to-apical; PBS, phosphate buffered saline; P-gp, P-glycoprotein; ROS, reactive oxygen
species; RP-HPLC, reversed-phase high-performance liquid chromatography; SAR, structure-
activity relationships; TEER, trans-epithelial electrical resistance; TLC, thin layer
chromatography.
ABSTRACT
Herein we envisaged the possibility of exploiting alkyl nitrates as precursors of alcohol-bearing
dual inhibitors targeting acetylcholinesterase (AChE) and monoamine oxidase B (MAO B), key
enzymes in neurodegenerative syndromes such as Alzheimer’s disease (AD), through
biotransformation unmasking an alcoholic function upon nitric oxide (NO) release. The
cooperation to neuroprotection of low fluxes of NO and target enzymes’ inhibition by the alcohol
metabolites might return a multitargeting effect. The in vitro screening towards ChEs and MAOs
of a collection of 21 primary alcohols disclosed a subset of dual inhibitors, among which three
diverse chemotypes were selected to study the corresponding nitrates. Nitrate 14 proved to be a
brain permeant, potent AChE-MAO B inhibitor by itself. Moreover, it protected human SH-
1

ACCEPTED MANUSCRIPT
SY5Y lines against rotenone and hydrogen peroxide with a poor inherent cytotoxicity and
showed a slow conversion profile to its alcohol metabolite 9d that still behaved as bimodal and
neuroprotective molecule.
1. Introduction
Alzheimer’s disease (AD) is a devastating and ultimately fatal pathology [1] accounting for
most of dementia cases worldwide. Giving that ageing is the main risk factor, the incidence of
AD is expected to constantly mirror the increased life-expectancy. In addition, the cure for AD
represents a health priority because of the lack of effective therapies.[2,3] Cognitive, motor and
psychiatric deficits that impact daily-life activities appear as a consequence of dysfunctions in
cerebral cortex areas mainly under cholinergic control.[4,5] Therefore, drugs able to restore
appropriate neurotransmitter (namely acetylcholine, ACh) levels funded their rationale in the so-
called cholinergic theory addressing the inhibition of ACh catabolic enzyme
(acetylcholinesterase, AChE).[6] Albeit merely palliative,[7] AChE inhibitors are still the
mainstay of AD treatment.[8] On the other side, the more recent amyloid hypothesis was related
to the formation of senile plaques constituted by beta-amyloid (Aβ) peptide fibrils in AD
brains.[9] Aβ deposits and neurofibrillary tangles (NFTs), formed by hyperphosphorylated tau
protein, are the main hallmarks that led to the discovery of the most-validated AD
biomarkers,[10] whereas drug research programs addressing both mechanisms have failed so far
in offering novel therapies.[11]
Different abnormalities in neurons have been proved in AD brains, which include oxidative
stress conditions [12] and reactive oxygen species (ROS) overloading,[13] protein misfolding
and deposition, unbalance in biometal concentrations,[14] loss of synaptic function,
inflammatory processes and many others,[1] thus triggering the identification of several
therapeutic options including insulin resistance [15] and lipid transport,[16] too. However, none
of the above-mentioned events has been clearly associated to the onset or to the relentless
progression of the disease and, more importantly, all efforts have not resulted in new drugs on
the market so far. In recent years, the multifactorial aetiology inspired the so-called multitarget
strategy rooted on the idea that exploiting cooperative activities by multipotent molecules [17]
could produce a more effective disease modifying action.[18,19] As a matter of fact, the latest
innovative progression has been scored by a combination of two active ingredients each acting
with a different mechanism,[20] namely memantine (NMDA antagonist) and donepezil (AChE
inhibitor).
In this context, dual-targeting compounds inhibiting AChE and monoamine oxidases (MAOs)
have been reported by several authors.[21,22,23,24,25] AChE inhibition has been maintained as
the core activity to reduce central ACh deficits, whereas the inhibition of MAOs could
potentially interfere with oxidative stress. In fact, MAO catalytic cycle produces hydrogen
peroxide and its activity increases with ageing in both normal brains and in activated plaque-
related astrocytes in AD.[26] As a follow-up of our recent contribution to the field of dual
AChE-MAO B inhibitors,[27,28]we devoted our efforts to add a third synergic activity to
bimodal compounds that is the release of nitric oxide (NO). NO is a gaseous, endogenous, and
rapidly diffusing messenger able to exert dose-dependent effects into the CNS.[29] High fluxes
are cytotoxic since NO can react with superoxide anion, thus increasing peroxynitrite levels and
consequent protein aberration upon nitration. In contrast, NO can work as neuroprotectant at low
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levels through S-nitrosylation of up-stream caspase-3 and activation of pro-survival pathways
related to soluble guanylate cyclase (sGC). Up to date, the most explored therapeutic effect of
NO is smooth muscle relaxation and, to this scope, several nitric oxide donors have been
developed over the years.[30] More recently, nitrate hybrids have gained consensus in several
therapeutic approaches, spanning from inflammation to anticancer therapies.[31,32] In most
cases, they add a vasodilating effect to the core activity through a labile pendant moiety or a NO
donor group. Since the metabolism of organic alkyl nitrates can release an alcoholic
function,[33] our idea was to explore whether selected alkyl nitrates can serve as precursors of
alcohol bearing dual AChE-MAO B inhibitors upon nitric oxide release, thus working as
multipotent small-molecule agents.
Organic nitrates lack a common degradation pathway in eukaryotes, and then a case-by-case
study on different chemotypes is needed. On this basis, we initially developed a small collection
of primary alcohols that were screened towards the target enzymes, namely acetyl- and
butyrylcholinesterase (AChE and BChE) and monoamine oxidases (MAO A and B). The most
promising inhibitors were then shortlisted to focused diverse samples and the corresponding
nitrates were prepared and studied. The Griess assay along with the HPLC studies of nitrates
stability in different experimental conditions (phosphate buffer at physiological pH with or
without active thiols, namely cysteine and glutathione; human serum; cytosolic fraction from
neuroblastoma cells) were undertaken to assess NO release and alcohol formation profile.
Moreover, a preliminary investigation addressing cytotoxicity and neuroprotective action against
oxidative insults (rotenone and H2O2) was carried out in human SH-SY5Y neuroblastoma cells.
A cell-based model employing MDCKII-MDR1 cell lines was used to estimate the ability to
cross blood-brain barrier (BBB) by the nitrates eligible for the desired alcohol metabolite release.
2. Design of alcohol probes
Diverse alcohol-based dual inhibitors were designed by combining three molecular bricks
(Figure 1), each carrying a potential pharmacodynamic feature, through different connection
patterns: i) a basic and protonatable head was used to bind AChE anionic subsites;[34,35,36] ii) a
planar coumarin scaffold to fit the MAO aromatic cage;[37,38] iii) a primary alcohol group as
the structural element unmasked from the parent nitrate group after in vivo NO release. The
decoration of the heterocyclic framework and the focused structural variations herein explored
started from our previous studies highlighting two classes of dual AChE-MAO B coumarin-
based inhibitors differing for the substitution at C7 (benzyloxy [27] and 1-piperidin-3(4)-yl-
methoxy groups [28]) and for the multitarget profile. Taking advantage of previous extensive
structure-activity relationships (SAR) developed for 7-benzyloxycoumarins, hindered
substituents were avoided at position 4 [38] where a hydroxymethyl group greatly enhanced the
dual activity. Unexplored positions 3 and 4 of 7-{piperidin-4-yl]methoxy}-2H-chromen-2-ones
have been concerned by addressing the effect of methyl groups whereas spacer and alcohol chain
lengths were investigated at position 7. The design aimed at probing primary alkyl and benzyl
alcohol groups in both compound classes to allow the selection of different alcohol/nitrate
chemotypes.
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Figure 1. Combination of molecular bricks to generate a collection of multipotent alcohols.
3. Methods
3.1 Chemistry
Scheme 1. Synthesis of compounds 3a-b and 6a-ca
N
Cl
a)
O
O
O
O
O
O
HO
HO
2a
3a
b)
N
Cl
N
b)
a)
HO
O
O
O
HO
O
O
HO
O
O
1
2b
3b
R1
R1
O
R1
O
R2
R2
O
R2
O
b)
a) or c)
R3
O
O
O
O
(Br)Cl
N
R3
HO
4a-b
5a-c
6a-c
a
Reagents and conditions: a) for 2b, 3a, 6a-b: 2.0 N solution of N,N-dimethylamine in THF,
room temperature, dry THF, 6 h; b) suitable benzyl chloride or bromide, K2CO3, dry acetonitrile,
reflux, 1-8 h; c) for 6c: 4-piperidinemethanol, DIEA, dry acetone, room temperature, overnight.
As shown in Scheme 1, the synthesis of alcohols 3a-b and 6a-c was accomplished by the
benzylation of the suitable 7-hydroxycoumarin (1, 2b or 4a-b) followed by nucleophilic
substitution with N,N-dimethylamine or 4-piperidinemethanol in THF. Scheme 2 illustrates the
preparation of piperidine-bearing compounds that started from the common intermediates 8a-g
obtained upon removal of Boc-protecting group from 7a-g in acidic environment by TFA.
Subsequent alkylation with the appropriate benzyl halides or ω-chloro(bromo)-1-alcohols in
refluxing acetonitrile gave access to N-benzylpiperidines 9a-i and N-alkylpiperidines 12a-f,
respectively. Unprotected piperidine 8a was reacted with 3-(chloromethyl)phenol in the presence
of DIEA to afford phenol 10 that underwent final alkylation with 3-bromopropan-1-ol thus
yielding derivative 11.
4

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Scheme 2. Synthesis of compounds 9a-i, 11, and 12a-fa
a
Reagents and conditions: a) TFA, dry CH2Cl2, room temperature, overnight; b) [3- or [4-
(chloromethyl)-phenyl]methanol, K2CO3, KI (cat.), dry acetonitrile, reflux, 6 h (for 9a-h) or 2-
[4-(bromomethyl)phenyl]ethanol, DIEA, dry acetone, room temperature, 15 h (for 9i); c) 3-
(chloromethyl)phenol, DIEA, dry acetone, room temperature, 7 h; d) 3-bromopropan-1-ol (for
11, 12a-b, 12d-f) or 4-chlorobutan-1-ol (for 12c), K2CO3, KI, dry acetonitrile (for 12a-f) or dry
acetone (for 11), reflux, 4-8 h.
To prepare benzyl nitrate 14 (Scheme 3), intermediate 8a was alkylated with α,α’-dichloro-m-
xylene in acetone before transformation into 14 in the presence of silver nitrate in acetonitrile
under reflux conditions. Conversely, nitrate esters 15 and 16 were prepared through a
nucleophilic substitution employing 8a or 10 and 3-bromopropyl nitrate,[39] respectively.
Scheme 3. Synthesis of nitrates 14-16a
a
Reagents and conditions: a) α,α’-dichloro-m-xylene, DIEA, dry acetone, room temperature,
15 h; b) AgNO3, dry acetonitrile, reflux, 6 h; c) 3-bromopropyl nitrate, K2CO3, dry acetonitrile,
room temperature (24 h, for 15) or reflux (4 h, for 16).
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3.2 Biological Assays
Alcohol-bearing derivatives 3a-b, 6a-c, 9a-i, 11, 12a-f and nitrates 14-16 were tested in vitro
as inhibitors of human MAOs (hMAOs) and ChEs (eeAChE and esBChE) by applying a
fluorescence-based protocol [27] and the well-established Ellman’s spectrophotometric
assay,[40] respectively, to determine IC50s on both isoforms. Human ChEs isoforms were
applied on selected samples. The inhibition data are reported in Tables 1-4 as IC50 (µM) or as
percentage of inhibition at 10 µM for the less active compounds.
NO release and alcohol release profiles were estimated to advance nitrates towards biological
studies addressing cytotoxicity and neuroprotection. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay [41] was used to measure the percentage of viable
human neuroblastoma SH-SY5Y cells referred to control experiment and determine both the
cytotoxic effect of compounds 9d, 12a, 14-15 (Table 5) and their protective effect when co-
incubated with two oxidative insults (hydrogen peroxide and rotenone) as illustrated in Figure 8.
Neuroprotection effects were compared to that of donepezil as standard marketed anti-
Alzheimer’s drug.
As previously reported,[27] cell-based bidirectional transport studies were aimed at estimating
the potential BBB permeation of compounds 9d, 12a, 14-15, by measuring apical to basolateral
(AP) and basolateral to apical (BL) apparent permeability (Papp) in Madin-Darby Canine Kidney
(MDCK) cells. Papp (in units of cm/s) and efflux ratio (ER) were calculated and listed in Table 5.
4. Results and discussion
4.1 SAR and diverse alcohol probes identification
For the sake of convenience in SAR discussion, all the compounds tested on enzymatic targets
(MAOs and ChEs) were grouped into three small congeneric subsets. Concerning MAOs, B/A
selectivity was desirable to avoid cheese-effect drawbacks.[42,43] On the other side, several
lines of evidence indicated the activity of BChE within AD brains as an attractive feature
[44,45,46] thus the lack of ChEs isoform selectivity was not considered a major drawback.
Looking at the benzyloxycoumarins 3a-b and 6a-c, the best results towards MAO B were
obtained by installing the primary alcoholic function at the position 4 of the heterocyclic core
and the basic nitrogen on the 7-benzyloxy group. The smallest basic chain was tolerated at both
meta- and para-position of the phenyl ring, giving rise to selective MAO B inhibitors resulting
much more potent than the “flipped” analogues where the basic and alcohol groups linkage
positions are inverted (6a vs. 3a, IC50 = 0.078 and 0.63 µM, respectively; 6b vs. 3b, IC50 = 0.054
and 1.1 µM, respectively). Unfortunately, an opposite trend was observed for AChE affinity (3a
> 6a, 3b > 6b), even so in the micromolar range. BChE affinity was quite low and improved by
branching the benzyloxy group with a bulkier alcohol-bearing protonatable head (6c).
Within N-benzylpiperidines (9a-i, 11), 1,4-piperidine ramification slightly enhanced MAO B
inhibition unrelated to m- or p-phenyl substitution (9c > 9a and 9d > 9b) without affecting AChE
affinity to a great extent. A molecule belonging to this subset (9c) showed the highest potency
towards MAO B of the whole series with IC50 equal to 0.029 µM along with a good B/A
selectivity (SI = 48). An interesting effect could be ascribed to the methyl groups at C3 and C4
of the coumarin ring. Indeed, both groups were essential for dual activity and 4-methyl scored
better binding interactions than 3-methyl towards both MAO B and AChE (9c > 9e > 9g; 9d > 9f
6

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> 9h). The homologation of the alcohol chain from 9c to 9i worsened mainly MAO B targeting
activity. It is worth noting that a longer terminal chain (11) at position meta of the phenyl ring
was able to revert selectivity (SI = 0.70 and 0.10 for MAO B/A and AChE/BChE, respectively)
leading to a potent dual MAO A (IC50 = 0.55 µM) and BChE inhibitor (IC50 = 0.18 µM).
Table 1. MAOs and ChEs inhibition data for alcohols 3a-b, 6a-c
R2
R1
R3
O
O
O
IC50 (µM) or % inhibition at 10 µ
Ma
compd
R1
R2
R3
MAO Ab
23±1.0%
21±3.2%
MAO Bb
0.63±0.12
1.1± 0.11
AChEc
1.3±0.020
4.4±1.0
BChEd
H
H
H
H
CH2N(CH3)2
CH2N(CH3)2
CH2OH
p-CH2OH
m-CH2OH
11±1.6
3a
3b
6a
6b
35±2.2%
43±1.1%
p-CH2N(CH3)2 20±3.4%
0.078±0.013
4.5±0.52
CH2OH
m-
42±2.9% 0.054±0.0050
5.9±0.61
2.9±0.43
36±1.4%
CH2N(CH3)2
CH3
CH3
m-[4-(CH2OH) 23±1.3%
0.32±0.12
1.0±0.040
6c
piperidin-1-yl]
20±3.0% 0.018±0.0030
safinamide
clorgyline
0.0038±
2.5±0.43
0.00050
0.024±0.0040
2.1±0.22
donepezil
a Values are the means of three independent experiments. b Human recombinant MAOs. c AChE
from electric eel. d BChE from equine serum.
7

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Table 2. MAOs and ChEs inhibition data for alcohols 9a-i, 11
IC50 (µM) or % inhibition at 10 µ
Ma
MAO Ab
MAO Bb
AChEc
BChEd
compd
R1
R2
X
R3
CH3
CH3
CH3
CH3
p-CH2OH
m-CH2OH
1.1±0.20
2.2±0.10
0.076±0.010
0.11±0.015
1.2±0.020
8.6±0.050
2.7±0.28
9a
9b
0.90±0.080
CH3
CH3
H
CH3
CH3
CH3
CH3
H
p-CH2OH
m-CH2OH
p-CH2OH
m-CH2OH
p-CH2OH
m-CH2OH
1.4±0.30
1.9±0.060
11±1.5
0.029±0.0080
0.076±0.0080
0.60±0.064
0.48±0.081
3.1±0.047
1.0±0.070
1.0±0.040
1.6±0.33
1.6 ±0.11
3.0±0.30
3.2±0.37
1.2±0.22
1.8±0.32
8.8±1.4
9c
9d
9e
9f
1.3±0.12
45±3.4%
1.9±0.21
8.1±0.34
0.91±0.19
41±7.3%
H
3.5±0.040
43±5.2%
3.9±0.81
CH3
CH3
CH3
CH3
9g
9h
9i
H
1.1±0.034
CH3
CH3
p-CH2CH2OH 1.7±0.17
m-O(CH2)3OH 0.55±0.038
0.12±0.020
0.78±0.050
0.18±0.020
11
a
b
c
Values are the means of three independent experiments. Human recombinant MAOs.
AChE from electric eel. d BChE from equine serum.
8

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Table 3. MAOs and ChEs inhibition data for alcohols 12a-f
R
X
n
m
IC50 (µM) or % inhibition at 10 µ
Ma
compd
MAO Ab
44±5.2%
26±4.4%
30±4.1%
35±3.2%
41±3.0%
MAO Bb
0.48±0.050
48±1.1%
3.6±0.24
31±3.1%
1.5±0.13
AChEc
BChEd
CH3
H
1
1
1
2
1
1
1
2
1
1
1.3±0.040
38±2.3%
0.50±0.020
0.72±0.097
1.8±0.060
30±3.1%
26±2.4%
8.3±1.2
12a
12b
12c
12d
12e
CH3
CH3
CH3
39±4.4%
26±3.2%
H
1
1
14±3.3%
42±1.0%
3.5±0.82
1.7±0.76
12f
a
b
c
Values are the means of three independent experiments. Human recombinant MAOs.
AChE from electric eel. d BChE from equine serum.
The third series includes N-alkylpiperidines 12a-f, where the structural exploration addressed
methyl groups on the coumarin ring, piperidine ramification pattern and linker length. As
previously noticed, the 3,4-dimethyl substitution on the heterocyclic nucleus is a key player for
the multitargeting activity (i.e., MAO B and AChE), as can be inferred by comparing 12a vs.
12b and 12e vs. 12f. 1,4-Piperidine substitution pattern performed a more efficient fit within
MAO B enzymatic cleft than 1,3-ramification (12a > 12e, 12b > 12f). Interestingly, the length of
the spacer connecting coumarin to piperidine ring proved to be crucial for MAO B affinity since
its homologation drastically dropped down the potency of 12d compared to 12a (IC50 > 10 µM
and IC50 = 0.48 µM, respectively), while increasing AChE affinity (12d, IC50 = 0.72 µM; 12a,
IC50 = 1.3 µM). A similar effect resulted from the alkyl chain elongation within the terminal N-
substituent, therefore butanol-derivative 12c showed a detrimental MAO B affinity (IC50 = 3.6
µM) along with an improved AChE inhibition (IC50 = 0.50 µM) compared to propanol-derivative
12a.
After clustering the alcohols in three subsets (Figure S1 in Supporting Information: benzyl
alcohols, alkyl alcohols bearing a N-alkyl basic head, alkyl alcohols bearing a N-benzyl basic
head), we selected one sample from each group (9d, 11, 12a) to study diverse possible
chemotypes: i) benzyl nitrate 14 (from 9d); ii) N-propyl nitrate 15 (from 12a); iii) phenoxypropyl
nitrate 16 (from 11). Within the most potent dual benzyl alcohols showing nanomolar MAO B
inhibition potency along with low micromolar AChE affinity (3a-b, 6a-b, 9a-h), 9d was
preferred to the equipotent, yet chiral, analogue 9a and to para-substituted congener 9c in order
to circumvent solubility issues, more likely detectable with symmetric compounds.[28] Nitrate
15, deriving from N-propyl alcohol 12a, was chosen to probe compounds with both alkyl
alcohols and alkyl basic nitrogen (12a-f) since it displayed the best multitarget profile. The study
9

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encompassed also phenoxyalkyl nitrate 16, showing an interesting pan-inhibitor profile related to
alcohol 11. The corresponding nitrates were synthesized according to Scheme 3, tested in vitro
against target enzymes, and their stability and NO-release, as well as the alcohol formation
profile, were investigated in different experimental conditions.
4.2 In vitro assays for nitrates: NO-release and target enzymes’ inhibition
The colorimetric quantitation of the azo-dye formed in the Griess assay was employed to
measure NO release.[47] As inferred from Figure 2, all compounds behaved as NO-donors and a
time-dependent release was performed in all cases with the benzyl chemotype 14 providing the
highest nitrite yields.
In vitro biological evaluation towards human target enzymes was extended to nitrates 14-16.
Data are reported in Table 4 compared to alcohols, which showed that the nitrate group was
well-tolerated by both hMAO B and hAChE. More importantly, it enhanced MAO B affinity in
all cases compared to the alcohol product (14 > 9d, 15 > 12a, 16 > 11) and originated potent
inhibitors in the nanomolar range. Non-homogeneous clues could be gathered about the other
targets, where the presence of nitrate promoted different affinity performances.
-
Figure 2. NO release from nitrates 14-16 in the colorimetric Griess assay. The levels of NO2 ion
(µm/mL) produced by compounds 14-16 (150 µM) were measured at different incubation time
(30, 60, 90, 120, 180 minutes) with phosphate buffer (50 mM, pH = 7.40) containing cysteine (5
mM), before the addition of the Griess reagent. Calibration curve was derived from sodium
nitrite standard solutions. Values are the mean ± SEM.
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Table 4. hMAOs and hChEs inhibition data for alcohols 9d, 12a, 11, and corresponding nitrates
14-16
compd
R
IC50 (
µ
M) or % inhibition at 10
µ
Ma
AChEc
MAO Ab
MAO Bb
BChEd
14
9d
0.23±0.0080
0.051±0.0050
1.3±0.27
1.4±0.21
n.t. e
O2NO
1.9±0.060
0.076±0.0080
n.t. e
HO
O2NO
HO
36±2.2%
44±5.2%
0.25±0.068
0.48±0.050
0.86±0.040
0.89±0.12
< 5%
< 5%
15
12a
16
1.2±0.12
0.55±0.038
0.78±0.050
0.48±0.041
0.84±0.11
0.80±0.14
O2NO
HO
O
11
0.55±0.038
0.74±0.013
O
a
b
c
Values are the means of three independent experiments. Human recombinant MAOs.
Human recombinant AChE. d BChE from human serum. e n.t.= not tested.
4.3 RP-HPLC study of nitrate-alcohol conversion profiles
In vivo nitrate decomposition can occur via hydrolysis or reductive denitration. However, no
specific enzymatic pathway is available in eukaryotes because of the absence of specific
hydrolases acting on nitrates. Importantly, hydrolytic by-products include chemical species
different from the putative alcohol that might originate from elimination (α-H, β-H) or
nucleophilic substitution reactions (e.g., alkene, aldehyde), which could in principle decrease
alcohol release rate and yield.
The RP-HPLC method enabled to check the parent nitrates stability and their conversion rate
to the alcohol metabolites in phosphate buffer at pH 7.40 and in human serum (Figure 3). At
physiological pH (Figure 3A), about 7% of 9d was retrieved from 14 and trace alcohol 12a from
15 after 24 h incubation, suggesting that non-enzymatic hydrolysis at physiological pH does not
play any role in the biotransformation of these nitrates into the alcohol product. Levels of alcohol
11 (data not shown) under the limit of detection were formed from 16. In human serum, all three
candidates exhibited a degradation profile that followed pseudo-first order kinetics and displayed
an outstanding stability (t1/2 = 23 h, 36.8 h, 458 h for 15, 14 and 16 respectively) without
releasing appreciable amounts of alcohols. In fact, a conversion yield to alcohol lower than 5%
was retrieved in all cases after 48 h.
11

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Figure 3. Diagram of hydrolytic conversion to alcohol (A) and human serum stability plots (B)
for nitrates 14-16. Time-dependent formation of alcohols 9d (orange plot) and 12a (red plot)
from 14 and 15, respectively, was measured under hydrolytic conditions (A) in phosphate buffer
(50 mM, pH 7.40, 0.20 M KCl) at 37 °C. Time-dependent disappearance of nitrates 14-16 was
studied in human serum (B) at 37 °C. Compounds 14-16 were incubated at initial concentration
equal to 100 µM and concentration at various time points was determined by RP-HPLC. Data
points represent means ± SD of three independent measurements.
Apart from non-enzymatic degradation, we considered intriguing to investigate the reactivity
towards reductive denitration pathways. This type of cleavage might be catalyzed in vivo by
hemoproteins (iron-promoted) and active thiols (cytosolic and microsomal glutathione-S-
transferases, GST, and glutathione, GSH), in both cases releasing NO and alcohol by-
product.[33]
Figure 4. Release profiles of alcohols 9d (orange diagram) and 12a (red diagram) from nitrates
14 and 15, respectively, in the presence of thiols. Time-dependent formation of alcohols 9d
(orange) and 12a (red) from nitrates 14 and 15, respectively, was determined in phosphate buffer
(50 mM, pH 7.40, 0.20 M KCl) containing 1 mM cysteine (○) or 1 mM glutathione (●) at 37 °C
as determined by RP-HPLC. Nitrates 14-15 were incubated at initial concentration equal to 100
µM. Data are means ± SD of three independent assays.
As a preliminary examination, the susceptibility towards reductive cleavage promoted by
active thiols was studied by adding an excess of cysteine or GSH to degassed phosphate buffer
and by applying a 24 h co-incubation window in order to limit background disulfide formation.
By monitoring the nitrate disappearance and the formation of the alcohol product, a RP-HPLC
12

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protocol was applied to estimate the stability of parent nitrates in the presence of free thiols and
to predict the possible transformation from nitrate to primary alcohol metabolites along with
inherent NO release in vivo. As for N-benzyl derivative 14, GSH boosted the conversion from
parent nitrate and showed a higher conversion rate compared to cysteine (Figure 4). An opposite
scale and an attenuated reactivity were observed in the case of the N-propyl chemotype 15 that
behaved as a quite poor donor with low alcohol formation as illustrated by red plots in Figure 4.
Figure 5. Conversion rates of N-benzyl nitrate 14 (A) and N-propyl nitrate 15 (B) in different
conditions. Time-dependent formation of alcohols 9d (A) and 12a (B) from nitrates 14 (100 µM)
and 15 (100 µM), respectively, was determined in phosphate buffer (50 mM, pH 7.40, 0.20 M
KCl) containing 1 mM cysteine (○) or 1 mM glutathione (●) and compared to thiol-free
phosphate buffer (blue) at 37 °C as determined by RP-HPLC. Data are means ± SD of three
independent assays.
As a trend, plots in Figure 5 indicate potentiating effects of thiols enabling higher alcohol
yields and rates from 14 and 15 compared to hydrolysis in phosphate buffer at pH 7.40 (blue
lines).
Figure 6. Decomposition rate of nitrate 16 in different conditions. Time-dependent
disappearance of compound 16 (100 µM) was measured at 37 °C in phosphate buffer (50 mM,
pH 7.40, 0.20 M KCl) with 1 mM cysteine (○, green) or 1 mM glutathione (●, green) and
without thiols (●, blue). The ratio between concentrations at various time points (C) and the
initial concentration at t0 (C0) was determined by RP-HPLC. Data points represent means ± SD
of three independent measurements.
In the case of the phenoxypropyl chemotype 16, no appreciable level of alcohol 11 was
detected up to 24 h with and without thiols. However, this low release could not be ascribed to
13

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the nitrate stability in the experimental conditions. As can be inferred from the graph in Figure 6,
the disappearance of parent nitrate 16 proceeded rapidly, consuming more than 50% after 24 h in
all experiments. Therefore, unwanted degradation by-products were formed, which ruled out the
desired biotransformation into alcohol 11 and this sample was excluded from subsequent assays.
Figure 7. Alcohol formation profiles from nitrates 14-15 in cellular environment. Time-
dependent formation of alcohols 9d (orange) and 12a (red) from nitrates 14 and 15, respectively,
both incubated (50 µM) at 37 °C in cytosolic fractions of human SH-SY5Y neuroblastoma cells,
as determined by RP-HPLC. Data represent triplicates’ means ± SD.
We further probed the behaviour in a cellular environment of the two chemotypes (14 and 15)
enabling thiol-mediated alcohol release (Figure 7). The stability and conversion profiles were
assessed in the freshly prepared cytosolic fractions of human neuroblastoma cell lines. Both
nitrates disclosed a retard-release profile and 15 exhibited higher alcohol yield than 14 (52% and
71% for 15, 31% and 34% for 14 after 24 h and 48 h, respectively). The slow conversion rate
may prefigure low NO fluxes in vivo, which could shift the bioactivity balance towards
neuroprotection.
4.4 Cell-based studies
The ability of compounds to protect neurons from oxidative damage is a critical feature for
anti-AD candidates. In the human neuroblastoma SH-SY5Y cell-based protocol,[48] only the
nitrates enabling alcohol release (14 and 15) were co-incubated at different concentrations (2.5,
0.5 and 0.1 µM) with two pro-oxidant toxins (rotenone at 20 µM and hydrogen peroxide at 200
µM). Cell viability was measured through MTT-assay.[41] Untreated cells were used as control
and donepezil was taken as the reference compound. For comparative purposes, the respective
alcohol metabolites 9d and 12a were engaged in the assays.
As shown in Figure 8, all tested compounds (parent nitrates 14, 15 and alcohols 9d, 12a)
showed a dose-dependent inverse correlation with neuroprotection against cytotoxic insults, and
the percentage of viable cells increased to a greater extent with the lowest concentration (0.1
µM). Interestingly, comparable figures of neuroprotection were obtained for nitrate 14 and
donepezil with the exception of the highest concentration (2.5 µM) against rotenone when
viability increase with donepezil was by far superior. In all experiments with nitrate ester 14, the
percentage of viable cells relative to control was higher or quite close to its putative metabolite
9d. In particular, a greater viability gain than 9d was observed when 14 was co-incubated at 0.5
and 0.1 µM concentrations with rotenone and at 2.5 µM concentration in the presence of
hydrogen peroxide. An opposite trend was returned by N-propyl chemotypes (15 and 12a), thus
alcohol 12a protected SH-SY5Y neurons from both toxins more efficiently or closely to NO-
donor 15 at all doses.
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In addition, the same cell line was used to assess the cytotoxicity of compounds 9d, 12a, 14
and 15 at concentrations ranging from 100 to 0.01 µM. As indicated by data in Table 5, 9d and
the respective parent nitrate 14 produced a low cellular damage (IC50 = 42.9 and 35.5 µM,
respectively) whereas both 12a and 15 were much less cytotoxic (IC50 > 100 µM).
Figure 8. Neuroprotective action on human neuroblastoma SH-SY5Y cells of nitrates 14-15 and
alcohols 9d and 12a with rotenone (20 µM) or H2O2 (200 µM) after incubation at different
concentrations (2.5, 0.5 or 0.1 µM). Cell proliferation was assessed by MTT assay. Data are
expressed as percentage of viable cells (referred to untreated cells as control) and shown as mean
± SD (n = 3). Donepezil was used as a reference anti-AD marketed drug. Statistical significance
was calculated using a two-way analysis of variance (ANOVA) followed by the Bonferroni post
hoc tests (GraphPad Prism vers. 5); **p < 0.01 , *** p < 0.001.
Table 5. Cytotoxicity and Bidirectional Transport across MDCKII-MDR1 Cells of Nitrates 14-
15 and Alcohols 9d, 12a
Cytotoxicitya
36±2.2 µM
43±1.2 µM
> 100 µM
> 100 µM
n.d.
Papp AP (cm/sec × 10-5)b Papp BL (cm/sec × 10-5)c ERd
compd
1.24±0.113
1.98±0.191
0.496±0.610
0.992±0.984
1.49±0.323
0.874±0.640
1.65±0.772
2.15±0.322
1.57±0.574
1.49±0.290
1.37±0.781
1.03±0.210
1.33
1.09
3.17
1.50
0.92
1.18
14
9d
15
12a
diazepam
FD4
n.d.
a
Cytotoxicity of compounds tested at concentrations in the range 0.1−100 µM in human
neuroblastoma SH-SY5Y cell lines for 24 h. Data are reported as IC50 values, relative to
b
untreated cells (control). Data represent means ± SD (n = 3). Papp AP is the apparent
c
permeability of apical-to-basal transport expressed in cm/sec × 10-5. Papp BL is the apparent
d
permeability of basal-to-apical transport expressed in cm/sec × 10-5. Efflux ratio (ER) was
calculated using the following equation: ER = Papp BL / Papp AP.
15

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To endorse the potential as CNS drugs, an in vitro blood-brain barrier penetration model was
employed to study the bidirectional transport across MDCKII-MDR1 cells and predict CNS
permeation.[49] Moreover, this protocol provides a reliable evaluation of the chance that
xenobiotics are pumped out by P-glycoprotein (P-gp), which prevents central bioactivity. Both
alcohols (9d, 12a) and nitrate 14 might penetrate into CNS through passive diffusion with
permeability comparable to diazepam and without interactions with efflux system (ER < 2).
Unfortunately, N-propyl compound 15 seems to suffer from P-gp liability, which could hamper
its further development.
5. Conclusions
In this study, we aimed at finding multitargeting organic nitrates that can serve as precursors of
alcohol bearing dual AChE-MAO B inhibitors after biotransformation that can be non-
enzymatically promoted by active thiols (e.g., glutathione). We initially focused our attention to
the development of the active metabolites, namely alcohol-bearing dual AChE-MAO B
inhibitors. Then, by applying a structural diversity filter, we fished three samples out the most
potent alcohols (9d, 12a, 11) to study the corresponding nitrates. In the RP-HPLC study, the
three nitrates displayed outstanding stability at physiological pH and in human serum, without
releasing the alcohol product. All of them proved to be potent in vitro MAO B inhibitors,
stronger than the putative alcohol metabolites. In the presence of bioactive thiols, two different
NO donors (14-15) might work as co-drugs of alcohol-based bimodal AChE-MAO B inhibitors.
In particular, we identified a brain permeant and serum stable nitrate (i.e., the N-benzyl
chemotype 14) able to perform a slow NO release and to yield good amount of the suitable
primary alcohol (9d) in the presence of GSH as well as in cytosolic preparations of SH-SY5Y
human neuroblastoma cells. Therefore, compound 14 should deserve further attention since it
behaved as a multipotent molecule by itself (MAO B IC50 = 0.051 µM, AChE IC50 = 1.6 µM)
and as a multitargeting precursor for a still active dual inhibitor (namely the alcohol metabolite
9d, IC50 = 0.076 and 1.0 µM towards MAO B and AChE, respectively) upon NO release. Both
parent NO-donor and the related alcohol exhibited significant neuroprotective activities against
the pro-oxidative insults rotenone and H2O2 without producing cytotoxic effect in human
neuroblastoma cell lines.
6. Experimental section
6.1 Chemistry
Starting materials, reagents, and analytical grade solvents were purchased from Sigma-Aldrich
(Europe). The purity of all the intermediates, checked by HPLC, was always better than 95%. All
the newly prepared and tested compounds showed purity higher than 95% (elemental analysis).
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. Column chromatography was performed using Merck silica gel
60 (0.063-0.200 mm, 70-230 mesh). 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 flame-dried and then cooled under a stream
of dry argon before the use. Nuclear magnetic resonance spectra were recorded on a Varian
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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 D2O. The attribution of 1H
NMR signals was supported by comparison with gHSQC experiments carried out for selected
samples (9b, 9c, 11, 12e) as reported in Supporting Information. HRMS experiments were
performed with a dual electrospray interface (ESI) and a quadrupole time-of-flight mass
spectrometer (Q-TOF, Agilent 6530 Series Accurate-Mass Quadrupole Time-of-Flight LC/MS,
Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, Italy). Full-scan mass spectra were
recorded in the mass/charge (m/z) range 50-3000 Da. Melting points for solid final compounds
were determined by the capillary method on a Stuart Scientific SMP3 electrothermal apparatus
and are uncorrected. The following intermediates have been already described in the literature:
tert-butyl
4-{[(3,4-dimethyl-2-oxo-2H-chromen-7-yl)oxy]methyl}piperidine-1-carboxylate
(7a),[28] 3,4-dimethyl-7-(piperidin-4-ylmethoxy)-2H-chromen-2-one (8a),[28] tert-butyl 3-
{[(3,4-dimethyl-2-oxo-2H-chromen-7-yl)oxy]methyl}piperidine-1-carboxylate (7f),[28] 3,4-
dimethyl-7-(piperidin-3-ylmethoxy)-2H-chromen-2-one
(8f),[28]
7-hydroxy-4-methyl-
coumarin,[50] 7-hydroxy-3-methyl-coumarin,[51] 7-hydroxy-3,4-dimethyl-coumarin (4b),[52]
tert-butyl
{[(methylsulfonyl)oxy]methyl}piperidine-1-carboxylate.[28]
6.1.1 4-(Chloromethyl)-7-{[4-(hydroxymethyl)benzyl]oxy}-2H-chromen-2-one (2a)
4-{[(methylsulfonyl)oxy]methyl}piperidine-1-carboxylate,[28]
tert-butyl
3-
.
4-
Chloromethyl-7-hydroxycoumarin [53] (0.21 g, 1.0 mmol) was suspended in dry acetonitrile (8
mL) before adding [4-(chloromethyl)-phenyl]methanol (0.78 g, 5.0 mmol) and potassium
carbonate (0.14 g, 1.0 mmol). The reaction mixture was refluxed for 8 h and then the solid
residue was filtered off. The solution was concentrated under rotary evaporation and purified
through column chromatography (gradient eluent: methanol in dichloromethane 0%→5%). Pale
1
yellow solid; yield: 0.26 g, 80%. H NMR (500 MHz, Acetone-d6) δ: 4.65 (s, 2H), 4.93 (s, 2H),
5.27 (s, 2H), 6.45 (s, 1H), 7.02 (d, J = 2.4 Hz, 1H), 7.06 (dd, J = 8.8, 2.4 Hz, 1H), 7.41 (d, J = 8.3
Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H), 7.79 (d, J = 8.8 Hz, 1H), OH not detected.
6.1.2 4-[(Dimethylamino)methyl]-7-{[4-(hydroxymethyl)benzyl]oxy}-2H-chromen-2-one (3a)
.
Intermediate 2a (0.20 g, 0.60 mmol) was dissolved in dry THF (4 mL) and commercially
available 2.0 N solution of N,N-dimethylamine in THF (0.90 mL, 1.8 mmol) was added. After
stirring at room temperature for 6 h, the solvent and excess amine were evaporated under
reduced pressure and the crude residue was purified through column chromatography (gradient
eluent: methanol in dichloromethane 0%→5%). Pale yellow solid; yield: 0.18 g, 87%; mp: 93-5
°C. 1H NMR (500 MHz, Acetone-d6) δ: 2.29 (s, 6H, N(CH3)2), 3.58 (s, 2H, CH2N), 4.65 (s, 2H,
CH2OH), 5.25 (s, 2H, CH2OAr), 6.25 (s, 1H, 3-Hcoum), 6.95 (d, J = 2.5 Hz, 1H, 8-Hcoum), 6.97
(dd, J = 8.8, 2.5 Hz, 1H, 6-Hcoum), 7.40 (d, J = 8.3 Hz, 2H, HAr), 7.47 (d, J = 8.3 Hz, 2H, HAr),
7.90 (d, J = 8.8 Hz, 1H, 5-Hcoum), OH not detected. Anal. (C20H21NO4) calcd. % C, 70.78; H,
6.24; N, 4.13. Found % C, 70.82; H, 6.31; N, 4.04. HRMS (Q-TOF) calcd. for C20H21NO4
[M+H]+ m/z 340.1543, found 340.1541; [M+Na]+ m/z 362.1363, found 362.1360.
6.1.3 4-[(Dimethylamino)methyl]-7-{[3-(hydroxymethyl)benzyl]oxy}-2H-chromen-2-one (3b)
.
[3-(Chloromethyl)-phenyl]methanol [54] (0.23 g, 1.5 mmol) and potassium carbonate (0.12 g,
0.90 mmol) were added to a stirred suspension of 4-[(dimethylamino)methyl]-7-hydroxy-2H-
chromen-2-one (2b) [55] (0.13 g, 0.60 mmol) in dry acetonitrile (5 mL). After refluxing for 1 h,
17

ACCEPTED MANUSCRIPT
the residue was filtered and washed with dichloromethane. The solution was concentrated to
dryness and purified through column chromatography (gradient eluent: methanol in
dichloromethane 0%→5%). Colorless oil; yield: 0.15 g, 76%. 1H NMR (500 MHz, DMSO-d6) δ:
2.21 (s, 6H, N(CH3)2), 3.53 (s, 2H, CH2N), 4.49 (d, J = 5.4 Hz, 2H, CH2OH), 5.19-5.21 (m, 3H,
1H dis. with D2O, CH2OAr + OH), 6.25 (s, 1H, 3-Hcoum), 6.99 (dd, J = 8.8, 2.5 Hz, 1H, 6-Hcoum),
7.05 (d, J = 2.5 Hz, 1H, 8-Hcoum), 7.26-7.33 (m, 3H, 4-HAr + 5-HAr + 6-HAr), 7.41 (s, 1H, 2-HAr),
7.84 (d, J = 8.8 Hz, 1H, 5-Hcoum). Anal. (C20H21NO4) calcd. % C, 70.78; H, 6.24; N, 4.13. Found
% C, 70.94; H, 6.31; N, 3.99. HRMS (Q-TOF) calcd. for C20H21NO4 [M+H]+ m/z 340.1543,
found 340.1540; [M+Na]+ m/z 362.1363, found 362.1363.
6.1.4 7-{[3-(Chloromethyl)benzyl]oxy}-4-(hydroxymethyl)-2H-chromen-2-one (5b)
.
7-
Hydroxy-4-(hydroxymethyl)-2H-chromen-2-one [56] (4a, 0.38 g, 2.0 mmol) was refluxed in dry
acetonitrile (15 mL) in the presence of potassium carbonate (0.41 g, 3.0 mmol) and α,α’-
dichloro-m-xylene (1.8 g, 10 mmol) for 5 h. After cooling to room temperature, the mixture was
filtered and the solid was thoroughly washed with chloroform. The solution was concentrated to
dryness and the crude residue was washed several times with n-hexane/diethyl ether 2/1 v/v
mixture until removal of excess starting halide, thus yielding the desired 5b. Off-white solid;
1
yield: 0.58 g, 88%. H NMR (300 MHz, DMSO-d6) δ: 4.71 (s, 2H), 4.77 (s, 2H), 5.22 (s, 2H),
5.60 (s, 1H, dis. with D2O), 6.28 (s, 1H), 6.99 (dd, J = 8.8, 2.4 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H),
7.33-7.46 (m, 3H), 7.54 (s, 1H), 7.61 (d, J = 8.8 Hz, 1H).
6.1.5 7-{[3-(Chloromethyl)benzyl]oxy}-3,4-dimethyl-2H-chromen-2-one (5c). 7-Hydroxy-3,4-
dimethyl-2H-chromen-2-one [28] (4b, 0.38 g, 2.0 mmol) was refluxed in dry acetonitrile (15
mL) in the presence of potassium carbonate (0.41 g, 3.0 mmol) and α,α’-dichloro-m-xylene (1.8
g, 10 mmol) for 8 h. After cooling to room temperature, the solvent was removed under rotary
evaporation and the residue was purified through column chromatography (gradient eluent: ethyl
1
acetate in n-hexane 10%→30%). White solid; yield: 0.43 g, 65%. H NMR (500 MHz, DMSO-
d6) δ: 2.06 (s, 3H), 2.35 (s, 3H), 4.77 (s, 2H), 5.21 (s, 2H), 7.01 (dd, J = 8.8, 2.4 Hz, 1H), 7.04 (d,
J = 2.4 Hz, 1H), 7.39-7.45 (m, 3H), 7.53 (s, 1H), 7.70 (d, J = 8.8 Hz, 1H).
6.1.6 General procedure for the synthesis of 7-({4-[(dimethylamino)methyl]benzyl}oxy)-4-
(hydroxymethyl)-2H-chromen-2-one (6a) and 7-({3-[(dimethylamino)methyl]benzyl}oxy)-4-
(hydroxymethyl)-2H-chromen-2-one (6b). The appropriate intermediate 4-(hydroxymethyl)-7-(4-
(bromomethyl)benzyloxy)coumarin (5a) [28] or 5b (0.50 mmol) was dissolved in dry THF (4
mL) and 2.0 N solution of N,N-dimethylamine in THF (0.75 mL, 1.5 mmol) was added
dropwise. After stirring at room temperature for 6 h, the reaction mixture was concentrated under
vacuum and purified through column chromatography (gradient eluent: methanol in
dichloromethane 0%→10%).
6.1.6.1
7-({4-[(Dimethylamino)methyl]benzyl}oxy)-4-(hydroxymethyl)-2H-chromen-2-one
(6a). Prepared from 5a (0.19 g, 0.50 mmol) and 2.0 N solution of N,N-dimethylamine in THF
1
(0.75 mL, 1.5 mmol). Pale yellow oil; yield: 0.15 g, 91%. H NMR (500 MHz, DMSO-d6) δ:
2.36 (s, 6H, N(CH3)2), 3.80 (s, 2H, CH2N), 4.70 (d, J = 5.4 Hz, 2H, CH2OH), 5.21 (s, 2H,
CH2OAr), 5.60 (t, J = 5.4 Hz, 1H, dis. with D2O, OH), 6.28 (s, 1H, 3-Hcoum), 6.99 (dd, J = 8.8,
2.4 Hz, 1H, 6-Hcoum), 7.07 (d, J = 2.4 Hz, 1H, 8-Hcoum), 7.38 (d, J = 7.8 Hz, 2H, HAr), 7.46 (d, J =
7.8 Hz, 2H, HAr), 7.60 (d, J = 8.8 Hz, 1H, 5-Hcoum). Anal. (C20H21NO4) calcd. % C, 70.78; H,
6.24; N, 4.13. Found % C, 70.91; H, 6.36; N, 4.07. HRMS (Q-TOF) calcd. for C20H21NO4
[M+H]+ m/z 340.1543, found 340.1542; [M+Na]+ m/z 362.1363, found 362.1365.
6.1.6.2
7-({3-[(Dimethylamino)methyl]benzyl}oxy)-4-(hydroxymethyl)-2H-chromen-2-one
(6b). Prepared from 5b (0.17 g, 0.50 mmol) and 2.0 N solution of N,N-dimethylamine in THF
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(0.75 mL, 1.5 mmol). Colorless oil; yield: 0.095 g, 56%. 1H NMR (500 MHz, DMSO-d6) δ: 2.12
(s, 6H, N(CH3)2), 3.37 (s, 2H, CH2N), 4.70 (d, J = 5.4 Hz, 2H, CH2OH), 5.19 (s, 2H, CH2OAr),
5.60 (t, J = 5.4 Hz, 1H, dis. with D2O, OH), 6.28 (s, 1H, 3-Hcoum), 6.99 (dd, J = 8.8, 2.5 Hz, 1H,
6-Hcoum), 7.06 (d, J = 2.5 Hz, 1H, 8-Hcoum), 7.22-7.24 (m, 1H, HAr), 7.32-7.33 (m, 2H, HAr), 7.37
(s, 1H, 2-HAr), 7.59 (d, J = 8.8 Hz, 1H, 5-Hcoum). Anal. (C20H21NO4) calcd. % C, 70.78; H, 6.24;
N, 4.13. Found % C, 70.86; H, 6.31; N, 4.08. HRMS (Q-TOF) calcd. for C20H21NO4 [M+H]+ m/z
340.1543, found 340.1541; [M+Na]+ m/z 362.1363, found 362.1360.
6.1.7 7-[(3-{[4-(Hydroxymethyl)piperidin-1-yl]methyl}benzyl)oxy]-3,4-dimethyl-2H-chromen-
2-one (6c). Compound 5c (0.16 g, 0.50 mmol) was dissolved in dry acetone (4 mL) and stirred
with 4-piperidinemethanol (0.069 g, 0.60 mmol) in the presence of DIEA (0.13 mL, 0.75 mmol)
at room temperature overnight. The solvent was removed under rotary evaporation and the crude
oil was purified through column chromatography (gradient eluent: methanol in dichloromethane
1
0%→10%). Dark yellow oil; yield: 0.14 g, 67%. H NMR (300 MHz, CDCl3) δ: 1.54-1.68 (m,
3H, 3-Ha(piper) + 4-H(piper)), 1.76-1.91 (m, 2H, 3-Hb(piper)), 2.16 (s, 3H, 3-CH3), 2.35 (s, 3H, 4-
CH3), 2.38-2.48 (m, 2H, 2-Ha(piper)), 3.15-3.33 (m, 2H, 2-Hb(piper)), 3.49 (d, J = 4.2 Hz, 2H,
CH2OH), 3.92 (s, 2H, ArCH2N), 5.12 (s, 2H, ArCH2OCoum), 6.81 (d, J = 2.4 Hz, 1H, 8-Hcoum),
6.91 (dd, J = 8.9, 2.4 Hz, 1H, 6-Hcoum), 7.32-7.44 (m, 3H, 4-HAr + 5-HAr + 6-HAr), 7.47-7.51 (m,
2H, 2-HAr + 5-Hcoum), OH not detected. Anal. (C25H29NO4) calcd. % C, 73.68; H, 7.17; N, 3.44.
Found % C, 73.85; H, 7.28; N, 3.31. HRMS (Q-TOF) calcd. for C25H29NO4 [M+H]+ m/z
408.2169, found 408.2166; [M+Na]+ m/z 430.1989, found 430.1987.
6.1.8 General procedure for the synthesis of substituted tert-butyl 4-{[(2-oxo-2H-chromen-7-
yl)oxy]alkyl}piperidine-1-carboxylate (7b-e) and tert-outyl 3-{[(2-oxo-2H-chromen-7-
yl)oxy]methyl}piperidine-1-carboxylate (7g). By following a previously reported method,[28] a
mixture of the appropriate 7-hydroxycoumarin (7.0 mmol), the suitable mesylate ester (6.7
mmol), triethylamine (2.8 mL, 20 mmol) and cesium carbonate (2.3 g, 7.0 mmol) were heated in
dry DMF (15 mL) at 70 °C for 72 h. Then the mixture was poured onto crushed ice. The filtrate
was collected and washed with abundant water yielding the desired Boc-protected intermediates
7b-e, 7g that were deprotected without further purification.
6.1.8.1 tert-Butyl 4-{[(4-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl}piperidine-1-carboxylate
(7b). Prepared from 7-hydroxy-4-methyl-coumarin (1.2 g, 7.0 mmol) and tert-butyl 4-
{[(methylsulfonyl)oxy]methyl}piperidine-1-carboxylate (2.0 g, 6.7 mmol). White solid; yield:
1
2.3 g, 93%. H NMR (300 MHz, CDCl3) δ: 1.22-1.37 (m, 2H), 1.46 (s, 9H), 1.63-1.85 (m, 3H),
2.39 (s, 3H), 2.62-2.78 (m, 2H), 3.85-3.87 (m, 2H), 4.10-4.18 (m, 2H), 6.13 (s, 1H), 6.79 (d, J =
2.5 Hz, 1H), 6.84 (dd, J = 8.8, 2.5 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H).
6.1.8.2 tert-Butyl 4-{[(3-methyl-2-oxo-2H-chromen-7-yl)oxy]methyl}piperidine-1-carboxylate
(7c). Prepared from 7-hydroxy-3-methyl-coumarin (1.2 g, 7.0 mmol) and tert-butyl 4-
{[(methylsulfonyl)oxy]methyl}piperidine-1-carboxylate (2.0 g, 6.7 mmol). White solid; yield:
1
2.2 g, 86%. H NMR (300 MHz, DMSO-d6) δ: 1.24-1.31 (m, 2H), 1.60 (s, 9H), 1.70-1.83 (m,
3H), 2.03 (s, 3H), 2.64-2.81 (m, 2H), 3.90-3.95 (m, 4H), 6.86-6.97 (m, 2H), 7.50 (d, J = 8.2 Hz,
1H), 7.79 (s, 1H).
6.1.8.3 tert-Butyl 4-{[(2-oxo-2H-chromen-7-yl)oxy]methyl}piperidine-1-carboxylate (7d)
.
Prepared from commercially available 7-hydroxy-coumarin (1.1 g, 7.0 mmol) and tert-butyl 4-
{[(methylsulfonyl)oxy]methyl}piperidine-1-carboxylate (2.0 g, 6.7 mmol). Pale yellow solid;
yield: 2.2 g, 91%. 1H NMR (500 MHz, Acetone-d6) δ: 1.23-1.31 (m, 3H), 1.44 (s, 9H), 1.82-1.85
(m, 2H), 2.81-2.92 (m, 2H), 4.01 (d, J = 6.4 Hz, 2H), 4.12-4.18 (m, 2H), 6.21 (d, J = 9.3 Hz, 1H),
19

ACCEPTED MANUSCRIPT
6.89 (d, J = 2.4 Hz, 1H), 6.94 (dd, J = 8.8, 2.4 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 9.3
Hz, 1H).
6.1.8.4 tert-Butyl 4-{2-[(methylsulfonyl)oxy]ethyl}piperidine-1-carboxylate. Commercially
available N-Boc-4-piperidineethanol (2.3 g, 10 mmol) was dissolved in CH2Cl2 (30 mL) before
the addition of triethylamine (5.7 mL, 40 mmol). The mixture was cooled to 0 °C with an
external ice bath, and methanesulfonyl chloride (0.9 mL, 11 mmol) was added dropwise. After
warming at room temperature, the reaction was kept under magnetic stirring for 3 h. The mixture
was diluted with CH2Cl2 (70 mL) and washed with satd. aq. Na2CO3 (3 × 80 mL). The organic
phase was dried over Na2SO4 and the evaporation of the solvent under reduced pressure yielded
the desired product that was used without further purification. Pale yellow solid; yield: 2.4 g,
1
77%. H NMR (500 MHz, DMSO-d6) δ: 0.91-1.02 (m, 2H), 1.36 (s, 9H), 1.58-1.63 (m, 5H),
2.60-2.66 (m, 2H), 3.14 (s, 3H), 3.86-3.90 (m, 2H), 4.21 (t, J = 6.4 Hz, 2H).
6.1.8.5
tert-Butyl
4-{2-[(3,4-dimethyl-2-oxo-2H-chromen-7-yl)oxy]ethyl}piperidine-1-
carboxylate (7e). Prepared from 7-hydroxy-3,4-dimethyl-coumarin (1.3 g, 7.0 mmol) and tert-
butyl 4-{2-[(methylsulfonyl)oxy]ethyl}piperidine-1-carboxylate (2.0 g, 6.7 mmol). Off-white
1
solid; yield: 2.1 g, 77%. H NMR (300 MHz, DMSO-d6) δ: 1.01-1.11 (m, 2H), 1.37 (s, 9H),
1.64-1.71 (m, 5H), 2.05 (s, 3H), 2.34 (s, 3H), 2.68-2.71 (m, 2H), 3.87-3.93 (m, 2H), 4.09 (t, J =
5.9 Hz, 2H), 6.89-6.93 (m, 2H), 7.67 (d, J = 8.8 Hz, 1H).
6.1.8.6 tert-Butyl 3-{[(2-oxo-2H-chromen-7-yl)oxy]methyl}piperidine-1-carboxylate (7g)
.
Prepared from 7-hydroxy-coumarin (1.1 g, 7.0 mmol) and tert-butyl 3-
{[(methylsulfonyl)oxy]methyl}piperidine-1-carboxylate (2.0 g, 6.7 mmol). White solid; yield:
1
2.0 g, 81%. H NMR (500 MHz, Acetone-d6) δ: 1.41-1.49 (m, 11H), 1.68-1.72 (m, 1H), 1.92-
1.95 (m, 1H), 2.00-2.03 (m, 1H), 2.67-2.72 (m, 1H), 2.92-2.97 (m, 1H), 3.80-3.91 (m, 2H), 4.01-
4.08 (m, 2H), 6.21 (d, J = 9.3 Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 6.95 (dd, J = 8.8, 2.4 Hz, 1H),
7.58 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 9.3 Hz, 1H).
6.1.9 General procedure for the synthesis of substituted 7-(piperidin-4-ylalkoxy)-2H-chromen-
2-ones (8b-e) and 7-(piperidin-3-ylmethoxy)-2H-chromen-2-one (8g). The suitable coumarin 7b-
e, 7g (5.0 mmol) was dissolved in dry CH2Cl2 (10 mL) followed by the dropwise addition of
trifluoroacetic acid (3.0 mL) at 0 °C through an external ice bath. The reaction mixture was
stirred at room temperature overnight and the solvents were removed under rotary evaporation.
The crude oil residue was diluted with dichloromethane (50 mL) and washed with Na2CO3 (3 ×
20 mL). The organic phase was dried over Na2SO4 and concentrated to dryness, thus obtaining a
solid residue that was purified through column chromatography (5% methanol in
dichloromethane) giving the desired coumarin 8b-e, 8g as solids.
6.1.9.1 4-Methyl-7-(piperidin-4-ylmethoxy)-2H-chromen-2-one (8b). Prepared from 7b (1.9 g,
5.0 mmol). Off-white solid; yield: 1.2 g, 91%. 1H NMR (300 MHz,CDCl3) δ: 1.40-1.51 (m, 2H),
1.82-1.96 (m, 3H), 2.05-2.12 (m, 2H), 2.40 (s, 3H), 2.90-2.98 (m, 2H), 3.89-3.94 (m, 2H), 6.13
(s, 1H), 6.79-6.86 (m, 2H), 7.50 (d, J = 8.8 Hz, 1H), NH not detected.
6.1.9.2 3-Methyl-7-(piperidin-4-ylmethoxy)-2H-chromen-2-one (8c). Prepared from 7c (1.9 g,
1
5.0 mmol). White solid; yield: 0.96 g, 70%. H NMR (300 MHz, DMSO-d6) δ: 1.21-1.28 (m,
2H), 1.71-1.77 (m, 2H), 1.89-1.93 (m, 1H), 2.04 (s, 3H), 2.59-2.64 (m, 2H), 3.06-3.09 (m, 2H),
3.90 (d, J = 6.4 Hz, 2H), 6.90 (dd, J = 8.8, 2.5 Hz, 1H), 6.95 (d, J = 2.5 Hz, 1H), 7.51 (d, J = 8.8
Hz, 1H), 7.79 (s, 1H), NH not detected.
6.1.9.3 7-(Piperidin-4-ylmethoxy)-2H-chromen-2-one (8d). Prepared from 7d (1.8 g, 5.0
1
mmol). Brown solid; yield: 1.1 g, 88%. H NMR (500 MHz, DMSO-d6) δ: 1.10-1.22 (m, 2H),
1.64-1.69 (m, 2H), 1.79-1.88 (m, 1H), 2.41-2.46 (m, 2H), 2.90-2.95 (m, 2H), 3.88 (d, J = 6.4 Hz,
20

ACCEPTED MANUSCRIPT
2H), 6.26 (d, J = 9.3 Hz, 1H), 6.92 (dd, J = 8.8, 2.4 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 7.59 (d, J =
8.8 Hz, 1H), 7.97 (d, J = 9.3 Hz, 1H), NH not detected.
6.1.9.4 3,4-Dimethyl-7-(2-piperidin-4-ylethoxy)-2H-chromen-2-one (8e). Prepared from 7e
1
(2.0 g, 5.0 mmol). White solid; yield: 1.1 g, 73%. H NMR (500 MHz, DMSO-d6) δ: 1.03-1.10
(m, 2H), 1.51-1.57 (m, 2H), 1.61-1.64 (m, 3H), 2.04 (s, 3H), 2.33 (s, 3H), 2.41-2.45 (m, 2H),
2.88-2.91 (m, 2H), 4.07 (t, J = 6.4 Hz, 2H), 6.89-6.91 (m, 2H), 7.64 (d, J = 8.8 Hz, 1H). NH not
detected.
6.1.9.5 7-(Piperidin-3-ylmethoxy)-2H-chromen-2-one (8g). Prepared from 7g (1.8 g, 5.0
1
mmol). Pale yellow solid; yield: 1.3 g, 99%. H NMR (500 MHz, DMSO-d6) δ: 1.32-1.35 (m,
1H), 1.61-1.64 (m, 1H), 1.82-1.84 (m, 2H), 2.17-2.19 (m, 1H), 2.75-2.81 (m, 2H), 3.24-3.29 (m,
1H), 3.36-3.40 (m, 1H), 3.95-3.99 (m, 1H), 4.04-4.07 (m, 1H), 6.30 (d, J = 9.3 Hz, 1H), 6.96 (dd,
J = 8.8, 2.4 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.99 (d, J = 9.3 Hz, 1H),
NH not detected.
6.1.10 General procedure for the synthesis of substituted 7-({1-[benzyl]piperidin-3-
yl}methoxy)-2H-chromen-2-ones (9a-b) and 7-({1-[benzyl]piperidin-4-yl}methoxy)-2H-chromen-
2-ones (9c-h). The appropriate compound 8a-c or 8f (0.50 mmol) was suspended in dry
acetonitrile (4 mL) followed by the addition of potassium carbonate (0.14 g, 1.0 mmol), 3- or [4-
(chloromethyl)-phenyl]methanol [54] (0.094 g, 0.60 mmol) and a catalytic amount of potassium
iodide. After refluxing for 6 h, the solvent was removed under rotary evaporation. The resulting
crude was suspended in dichloromethane and the inorganic residue was filtered-off. The solution
was concentrated to dryness and purified as indicated below to obtain final coumarins 9a-h as
white solids.
6.1.10.1 7-({1-[4-(Hydroxymethyl)benzyl]piperidin-3-yl}methoxy)-3,4-dimethyl-2H-chromen-
2-one (9a). [57] Prepared from 8f (0.14 g, 0.50 mmol) and [4-(chloromethyl)-phenyl]methanol
(0.094 g, 0.60 mmol). Purified through column chromatography (gradient eluent: methanol in
1
dichloromethane from 0% to 5%). White solid; yield: 0.13 g, 64%; mp: 52-5 °C. H NMR (300
MHz, Acetone-d6) δ: 1.17-1.28 (m, 1H, 4-Ha(piper)), 1.50-1.61 (m, 1H, 5-Ha(piper)), 1.62-1.75 (m,
1H, 5-Hb(piper)), 1.80-1.87 (m, 1H, 4-Hb(piper)), 1.97-2.17 (m, 3H, 2-Ha(piper) + 6-Ha(piper) + 3-
H(piper)), 2.11 (s, 3H, 3-CH3), 2.39 (s, 3H, 4-CH3), 2.68-2.71 (m, 1H, 6-Hb(piper)), 2.83-2.94 (m,
1H, 2-Hb(piper)), 3.44 (d, J = 13.4 Hz, 1H, ArCHaN), 3.51 (d, J = 13.4 Hz, 1H, ArCHbN), 4.00 (d, J
= 6.4 Hz, 2H, CH2OAr), 4.59 (s, 2H, CH2OH), 6.82 (d, J = 2.3 Hz, 1H, 8-Hcoum), 6.88 (dd, J =
8.8, 2.3 Hz, 1H, 6-Hcoum), 7.27-7.30 (s, 4H, HAr), 7.65 (d, J = 8.8 Hz, 1H, 5-Hcoum), OH not
detected. Anal. (C25H29NO4) calcd. % C, 73.68; H, 7.17; N, 3.44. Found % C, 73.88; H, 7.21; N,
3.33. HRMS (Q-TOF) calcd. for C25H29NO4 [M+H]+ m/z 408.2169, found 408.2161; [M+Na]+
m/z 430.1989, found 430.1991.
6.1.10.2 7-({1-[3-(Hydroxymethyl)benzyl]piperidin-3-yl}methoxy)-3,4-dimethyl-2H-chromen-
2-one (9b). [57] Prepared from 8f (0.14 g, 0.50 mmol) and [3-(chloromethyl)-phenyl]methanol
(0.094 g, 0.60 mmol). Purified through crystallization from hot ethanol 95%. White solid; yield:
1
0.18 g, 86%; mp: 118-120 °C. H NMR (500 MHz, DMSO-d6) δ: 1.07-1.14 (m, 1H, 4-Ha(piper)),
1.43-1.50 (m, 1H, 5-Ha(piper)), 1.60-1.64 (m, 1H, 5-Hb(piper)), 1.72-1.78 (m, 1H, 4-Hb(piper)), 1.86-
2.03 (m, 3H, 2-Ha(piper) + 6-Ha(piper) + 3-H(piper)), 2.05 (s, 3H, 3-CH3), 2.34 (s, 3H, 4-CH3), 2.62-
2.66 (m, 1H, 6-Hb(piper)), 2.82-2.85 (m, 1H, 2-Hb(piper)), 3.40 (d, J = 13.2 Hz, 1H, ArCHaN), 3.43
(d, J = 13.2 Hz, 1H, ArCHbN), 3.92 (d, J = 6.4 Hz, 2H, CH2OAr), 4.44 (d, J = 5.4 Hz, 2H,
CH2OH), 5.12 (t, J = 5.4 Hz, 1H, dis. with D2O, OH), 6.87-6.89 (m, 2H, 6-Hcoum + 8-Hcoum), 7.12
(d, J = 7.5 Hz, 1H, HAr), 7.16 (d, J = 7.5 Hz, 1H, HAr), 7.20-7.22 (m, 2H, HAr), 7.65 (d, J = 8.8
Hz, 1H, 5-Hcoum). Anal. (C25H29NO4) calcd. % C, 73.68; H, 7.17; N, 3.44. Found % C, 73.75; H,
21

ACCEPTED MANUSCRIPT
7.18; N, 3.39. HRMS (Q-TOF) calcd. for C25H29NO4 [M+H]+ m/z 408.2169, found 408.2169;
[M+Na]+ m/z 430.1989, found 430.1988.
6.1.10.3 7-({1-[4-(Hydroxymethyl)benzyl]piperidin-4-yl}methoxy)-3,4-dimethyl-2H-chromen-
2-one (9c). Prepared from 8a (0.14 g, 0.50 mmol) and [4-(chloromethyl)-phenyl]methanol (0.094
g, 0.60 mmol). Purified through crystallization from hot ethanol 95%. White solid; yield: 0.13 g,
64%; mp: 157-9 °C. 1H NMR (500 MHz, DMSO-d6) δ: 1.24-1.33 (m, 2H, 3-Ha(piper)), 1.70-1.72
(m, 3H, 3-Hb(piper) + 4-H(piper)), 1.87-1.92 (m, 2H, 2-Ha(piper)), 2.04 (s, 3H, 3-CH3), 2.33 (s, 3H, 4-
CH3), 2.78-2.82 (m, 2H, 2-Hb(piper)), 3.40 (s, 2H, ArCH2N), 3.89 (d, J = 5.9 Hz, 2H, CH2OAr),
4.44 (d, J = 5.9 Hz, 2H, CH2OH), 5.11 (t, J = 5.9 Hz, 1H, dis. with D2O, OH), 6.90-6.93 (m, 2H,
6-Hcoum + 8-Hcoum), 7.21 (d, J = 8.3 Hz, 2H, HAr), 7.23 (d, J = 8.3 Hz, 2H, HAr), 7.65 (d, J = 9.2
Hz, 1H, 5-Hcoum). Anal. (C25H29NO4) calcd. % C, 73.68; H, 7.17; N, 3.44. Found % C, 73.95; H,
7.28; N, 3.49. HRMS (Q-TOF) calcd. for C25H29NO4 [M+H]+ m/z 408.2169, found 408.2165;
[M+Na]+ m/z 430.1989, found 430.1990.
6.1.10.4 7-({1-[3-(Hydroxymethyl)benzyl]piperidin-4-yl}methoxy)-3,4-dimethyl-2H-chromen-
2-one (9d). Prepared from 8a (0.14 g, 0.50 mmol) and [3-(chloromethyl)-phenyl]methanol (0.094
g, 0.60 mmol). Purified through column chromatography (gradient eluent: methanol in
1
dichloromethane from 0% to 5%). White solid; yield: 0.16 g, 80%; mp: 128-130 °C. H NMR
(500 MHz, DMSO-d6) δ: 1.26-1.33 (m, 2H, 3-Ha(piper)), 1.71-1.75 (m, 3H, 3-Hb(piper) + 4-H(piper)),
1.88-1.95 (m, 2H, 2-Ha(piper)), 2.05 (s, 3H, 3-CH3), 2.34 (s, 3H, 4-CH3), 2.80-2.83 (m, 2H, 2-
Hb(piper)), 3.44 (s, 2H, ArCH2N), 3.91 (d, J = 6.4 Hz, 2H, CH2OAr), 4.46 (d, J = 5.4 Hz, 2H,
CH2OH), 5.13 (t, J = 5.4 Hz, 1H, dis. with D2O, OH), 6.90-6.93 (m, 2H, 6-Hcoum + 8-Hcoum),
7.12-7.16 (m, 2H, HAr), 7.22-7.27 (m, 2H, HAr), 7.66 (d, J = 9.3 Hz, 1H, 5-Hcoum). Anal.
(C25H29NO4) calcd. % C, 73.68; H, 7.17; N, 3.44. Found % C, 73.79; H, 7.18; N, 3.40. HRMS
(Q-TOF) calcd. for C25H29NO4 [M+H]+ m/z 408.2169, found 408.2168; [M+Na]+ m/z 430.1989,
found 430.1988.
6.1.10.5
7-({1-[4-(Hydroxymethyl)benzyl]piperidin-4-yl}methoxy)-4-methyl-2H-chromen-2-
one (9e). Prepared from 8b (0.14 g, 0.50 mmol) and [4-(chloromethyl)-phenyl]methanol (0.094
g, 0.60 mmol). Purified through column chromatography (gradient eluent: methanol in
dichloromethane from 0% to 5%). White solid; yield: 0.10 g, 53%; mp: 103-5 °C. 1H NMR (500
MHz, DMSO-d6) δ: 1.26-1.33 (m, 2H, 3-Ha(piper)), 1.70-1.75 (m, 3H, 3-Hb(piper) + 4-H(piper)), 1.90-
1.96 (m, 2H, 2-Ha(piper)), 2.37 (s, 3H, 4-CH3), 2.79-2.83 (m, 2H, 2-Hb(piper)), 3.41 (s, 2H,
ArCH2N), 3.92 (d, J = 7.5 Hz, 2H, CH2OAr), 4.44 (d, J = 5.8 Hz, 2H, CH2OH), 5.09 (t, J = 5.8
Hz, 1H, dis. with D2O, OH), 6.18 (s, 1H, 3-Hcoum), 6.92-6.96 (m, 2H, 6-Hcoum + 8-Hcoum), 7.22-
7.26 (m, 4H, HAr), 7.65 (d, J = 9.5 Hz, 1H, 5-Hcoum). Anal. (C24H27NO4) calcd. % C, 73.26; H,
6.92; N, 3.56. Found % C, 73.44; H, 6.99; N, 3.45. HRMS (Q-TOF) calcd. for C24H27NO4
[M+H]+ m/z 394.2013, found 394.2008; [M+Na]+ m/z 416.1832, found 416.1831.
6.1.10.6
7-({1-[3-(Hydroxymethyl)benzyl]piperidin-4-yl}methoxy)-4-methyl-2H-chromen-2-
one (9f). Prepared from 8b (0.14 g, 0.50 mmol) and [3-(chloromethyl)-phenyl]methanol (0.094 g,
0.60 mmol). Purified through column chromatography (gradient eluent: methanol in
dichloromethane from 0% to 5%). White solid; yield: 0.12 g, 61%; mp: 146-8 °C. 1H NMR (500
MHz, DMSO-d6) δ: 1.26-1.33 (m, 2H, 3-Ha(piper)), 1.70-1.73 (m, 3H, 3-Hb(piper) + 4-H(piper)), 1.92-
1.97 (m, 2H, 2-Ha(piper)), 2.37 (s, 3H, 4-CH3), 2.80-2.83 (m, 2H, 2-Hb(piper)), 3.41 (s, 2H,
ArCH2N), 3.93 (d, J = 5.8 Hz, 2H, CH2OAr), 4.46 (d, J = 5.8 Hz, 2H, CH2OH), 5.12 (t, J = 5.8
Hz, 1H, dis. with D2O, OH), 6.18 (s, 1H, 3-Hcoum), 6.93-6.95 (m, 2H, 6-Hcoum + 8-Hcoum), 7.12-
7.17 (m, 2H, HAr), 7.21-7.26 (m, 2H, HAr), 7.65 (d, J = 7.1 Hz, 1H, 5-Hcoum). Anal. (C24H27NO4)
22

ACCEPTED MANUSCRIPT
calcd. % C, 73.26; H, 6.92; N, 3.56. Found % C, 73.41; H, 7.00; N, 3.47. HRMS (Q-TOF) calcd.
for C24H27NO4 [M+H]+ m/z 394.2013, found 394.2010; [M+Na]+ m/z 416.1832, found 416.1831.
6.1.10.7
7-({1-[4-(Hydroxymethyl)benzyl]piperidin-4-yl}methoxy)-3-methyl-2H-chromen-2-
one hydrochloride (9g). Prepared from 8c (0.14 g, 0.50 mmol) and [4-(chloromethyl)-
phenyl]methanol (0.094 g, 0.60 mmol). Purified through column chromatography (gradient
eluent: methanol in dichloromethane from 0% to 5%) and transformed into the hydrochloride salt
1
with 4.0 N solution of HCl in dioxane. Off-white solid; yield: 0.095 g, 44%; mp: 163-4 °C. H
NMR (free base, 500 MHz, DMSO-d6) δ: 1.24-1.32 (m, 2H, 3-Ha(piper)), 1.70-1.75 (m, 3H, 3-
Hb(piper) + 4-H(piper)), 1.90-1.94 (m, 2H, 2-Ha(piper)), 2.03 (s, 3H, 3-CH3), 2.79-2.81 (m, 2H, 2-
Hb(piper)), 3.41 (s, 2H, ArCH2N), 3.89 (d, J = 6.5 Hz, 2H, CH2OAr), 4.44 (d, J = 6.2 Hz, 2H,
CH2OH), 5.09 (t, J = 6.2 Hz, 1H, dis. with D2O, OH), 6.91 (dd, J = 8.8, 2.4 Hz, 1H, 6-Hcoum),
6.94 (d, J = 2.4 Hz, 1H, 8-Hcoum), 7.20-7.26 (m, 4H, HAr), 7.49 (d, J = 8.8 Hz, 1H, 5-Hcoum), 7.78
(s, 1H, 4-Hcoum). Anal. (C24H27NO4·HCl) calcd. % C, 67.05; H, 6.56; N, 3.26. Found % C, 67.28;
H, 6.68; N, 3.17. HRMS (Q-TOF) calcd. for C24H27NO4 [M+H]+ m/z 394.2013, found 394.2009;
[M+Na]+ m/z 416.1832, found 416.1829.
6.1.10.8
7-({1-[3-(Hydroxymethyl)benzyl]piperidin-4-yl}methoxy)-3-methyl-2H-chromen-2-
one hydrochloride (9h). Prepared from 8c (0.14 g, 0.50 mmol) and [3-(chloromethyl)-
phenyl]methanol (0.094 g, 0.60 mmol). Purified through column chromatography (gradient
eluent: methanol in dichloromethane from 0% to 5%) and transformed into the hydrochloride salt
with 4.0 N solution of HCl in dioxane. White solid; yield: 0.11 g, 50%; mp: 207-9 °C (dec.). 1H
NMR (300 MHz, DMSO-d6) δ: 1.40-1.54 (m, 2H, 3-Ha(piper)), 1.77-2.01 (m, 3H, 3-Hb(piper) + 4-
H(piper)), 2.03 (s, 3H, 3-CH3), 2.89-3.01 (m, 2H, 2-Ha(piper)), 3.35-3.40 (m, 2H, 2-Hb(piper)), 3.91-
3.94 (d, J = 6.4 Hz, 2H, CH2OAr), 4.27 (br d, 2H, ArCH2NH+), 4.51-4.54 (br d, 2H, CH2OH),
5.29 (br s, 1H, dis. with D2O, OH), 6.87-6.95 (m, 2H, 6-Hcoum + 8-Hcoum), 7.39-7.53 (m, 5H, HAr
+ 5-Hcoum), 7.79 (s, 1H, 4-Hcoum), 9.63 (s, 1H, dis. with D2O, NH+). Anal. (C24H27NO4·HCl)
calcd. % C, 67.05; H, 6.56; N, 3.26. Found % C, 67.35; H, 6.71; N, 3.08. HRMS (Q-TOF) calcd.
for C24H27NO4 [M+H]+ m/z 394.2013, found 394.2011; [M+Na]+ m/z 416.1832, found 416.1830.
6.1.11 7-({1-[4-(2-Hydroxyethyl)benzyl]piperidin-4-yl}methoxy)-3,4-dimethyl-2H-chromen-2-
one (9i). Intermediate 8a (0.17 g, 0.60 mmol) was suspended in dry acetone (5 mL) and then
DIEA (0.11 mL, 0.65 mmol) was added followed by 2-[4-(bromomethyl)phenyl]ethanol [27]
(0.11 g, 0.50 mmol). After stirring overnight at room temperature, the mixture was concentrated
to dryness and purified through column chromatography (gradient eluent: methanol in
1
dichloromethane from 0% to 3%). Brown solid; yield: 0.099 g, 47%; mp: 86-8 °C (dec.). H
NMR (300 MHz, DMSO-d6) δ: 1.13-1.33 (m, 2H, 3-Ha(piper)), 1.67-1.81 (m, 3H, 3-Hb(piper) + 4-
H(piper)), 1.83-1.97 (m, 2H, 2-Ha(piper)), 2.05 (s, 3H, 3-CH3), 2.34 (s, 3H, 4-CH3), 2.59-2.75 (m,
2H, ArCH2CH2OH), 2.81-2.94 (m, 2H, 2-Hb(piper)), 3.38 (br s, 2H, ArCH2N), 3.50-3.66 (m, 2H,
CH2OH), 3.93 (br s, 2H, CH2OAr), 4.62 (br s, 1H, dis. with D2O, OH), 6.86-6.99 (m, 2H, 6-
Hcoum + 8-Hcoum), 7.05-7.47 (m, 4H, HAr), 7.67 (d, J = 8.9 Hz, 1H, 5-Hcoum). Anal. (C26H31NO4)
calcd. % C, 74.08; H, 7.41; N, 3.32. Found % C, 74.38; H, 7.59; N, 3.19. HRMS (Q-TOF) calcd.
for C26H31NO4 [M+H]+ m/z 422.2326, found 422.2323; [M+Na]+ m/z 444.2145, found 444.2142.
6.1.12 7-{[1-(3-Hydroxybenzyl)piperidin-4-yl]methoxy}-3,4-dimethyl-2H-chromen-2-one (10)
.
Coumarin 8a (0.68 g, 2.4 mmol) was suspended in dry acetone (15 mL) followed by the addition
of DIEA (0.42 mL, 2.4 mmol) and 3-chloromethylphenol [54] (0.28 g, 2.0 mmol). After stirring
at room temperature for 7 h, the mixture was concentrated under rotary evaporation and purified
through column chromatography (gradient eluent methanol in dichloromethane from 0% to 5%)
1
furnishing the desired intermediate as an off-white solid. Yield: 0.52 g, 66%. H NMR (300
23

ACCEPTED MANUSCRIPT
MHz, DMSO-d6) δ: 1.21-1.31 (m, 2H), 1.71-1.74 (m, 3H), 1.81-1.92 (m, 2H), 2.05 (s, 3H), 2.34
(s, 3H), 2.69-2.80 (m, 2H), 3.37 (s, 2H), 3.91 (d, J = 5.3 Hz, 2H), 6.59-6.71 (m, 3H), 6.91-6.94
(m, 2H), 7.07 (t, J = 7.0 Hz, 1H), 7.66 (d, J = 9.9 Hz, 1H), 9.20 (s, 1H, dis. with D2O).
6.1.13
7-({1-[3-(3-Hydroxypropoxy)benzyl]piperidin-4-yl}methoxy)-3,4-dimethyl-2H-
chromen-2-one (11). Phenol 10 (0.20 g, 0.50 mmol) was refluxed with potassium carbonate (0.14
g, 1.0 mmol), 3-bromopropan-1-ol (0.043 mL, 0.50 mmol) and a catalytic amount of KI in dry
acetone (1.5 mL) for 8 h. The solvent was removed under vacuum and the resulting crude oil was
purified through column chromatography (eluent: 5% methanol in dichloromethane) yielding a
yellow solid. Yield: 0.12 g, 51%; mp: 74-6 °C. 1H NMR (500 MHz, DMSO-d6) δ: 1.25-1.33 (m,
2H, 3-Ha(piper)), 1.70-1.73 (m, 3H, 3-Hb(piper) + 4-H(piper)), 1.83 (qn, J = 6.4 Hz, 2H,
ArOCH2CH2CH2OH), 1.88-1.96 (m, 2H, 2-Ha(piper)), 2.05 (s, 3H, 3-CH3), 2.34 (s, 3H, 4-CH3),
2.77-2.85 (m, 2H, 2-Hb(piper)), 3.40 (s, 2H, ArCH2N), 3.53 (q, J = 6.4 Hz, 2H,
ArOCH2CH2CH2OH), 3.91 (d, J = 5.9 Hz, 2H, CH2O-Coum), 3.98 (t, J = 6.4 Hz, 2H,
ArOCH2CH2CH2OH), 4.50 (t, J = 6.4 Hz, 1H, dis. with D2O, OH), 6.76-6.79 (m, 1H, 4-HAr),
6.82-6.85 (m, 2H, 6-HAr + 2-HAr), 6.90-6.93 (m, 2H, 8-Hcoum + 6-Hcoum), 7.19 (t, J = 7.3 Hz, 1H,
5-HAr), 7.67 (d, J = 9.8 Hz, 1H, 5-Hcoum). Anal. (C27H33NO5) calcd. % C, 71.82; H, 7.37; N, 3.10.
Found % C, 72.09; H, 7.50; N, 3.01. HRMS (Q-TOF) calcd. for C27H33NO5 [M+H]+ m/z
452.2431, found 452.2426; [M+Na]+ m/z 474.2251, found 474.2252.
6.1.14 General procedure for the synthesis of substituted 7-{[1-(ω-hydroxyalkyl)piperidin-4-
yl]methoxy}-2H-chromen-2-ones (12a-d) and 7-{[1-(3-hydroxypropyl)piperidin-3-yl]methoxy}-
2H-chromen-2-ones (12e-f). The suitable unprotected piperidine 8a, 8d-g (0.50 mmol) was
suspended in dry acetonitrile (5 mL). Potassium carbonate (0.10 g, 0.75 mmol), 3-bromo-1-
propanol (0.090 mL, 1.0 mmol, for 12a-b and 12d-f) or 4-chlorobutan-1-ol (0.10 mL, 1.0 mmol,
for 12c), and a catalytic amount of KI were added and the mixture was refluxed for 4-6 h. The
solvent was evaporated under reduced pressure and the solid residue was filtered-off after
washing with CH2Cl2. The solution was concentrated to dryness, and the resulting crude product
was purified as detailed below.
6.1.14.1
7-{[1-(3-Hydroxypropyl)piperidin-4-yl]methoxy}-3,4-dimethyl-2H-chromen-2-one
(12a). Prepared from 8a (0.14 g, 0.50 mmol) and 3-bromo-1-propanol (0.090 mL, 1.0 mmol).
Purified through crystallization from hot ethanol. White solid; yield: 0.12 g, 68%; mp: 117-9 °C.
1H NMR (500 MHz, DMSO-d6) δ: 1.22-1.29 (m, 2H, 3-Ha(piper)), 1.55 (qn, J = 6.9 Hz, 2H,
NCH2CH2CH2OH), 1.71-1.76 (m, 3H, 3-Hb(piper) + 4-H(piper)), 1.80-1.86 (m, 2H, 2-Ha(piper)), 2.05
(s, 3H, 3-CH3), 2.30 (t, J = 6.9 Hz, 2H, NCH2CH2CH2OH), 2.34 (s, 3H, 4-CH3), 2.85-2.87 (m,
2H, 2-Hb(piper)), 3.41 (t, J = 6.9 Hz, 2H, CH2OH), 3.90 (d, J = 5.9 Hz, 2H, CH2OAr), 6.90-6.93
(m, 2H, 6-Hcoum + 8-Hcoum), 7.67 (d, J = 8.8 Hz, 1H, 5-Hcoum), OH not detected. Anal.
(C20H27NO4) calcd. % C, 69.54; H, 7.88; N, 4.05. Found % C, 69.69; H, 7.96; N, 3.98. HRMS
(Q-TOF) calcd. for C20H27NO4 [M+H]+ m/z 346.2013, found 346.2011; [M+Na]+ m/z 368.1832,
found 368.1830.
6.1.14.2 7-{[1-(3-Hydroxypropyl)piperidin-4-yl]methoxy}-2H-chromen-2-one (12b). Prepared
from 8d (0.13 g, 0.50 mmol) and 3-bromo-1-propanol (0.090 mL, 1.0 mmol). Purified through
column chromatography (gradient eluent: methanol in dichloromethane from 0% to 10%). White
solid; yield: 0.063 g, 40%; mp: 137-9 °C (dec.). 1H NMR (500 MHz, DMSO-d6) δ: 1.27-1.34 (m,
2H, 3-Ha(piper)), 1.63 (qn, J = 6.4 Hz, 2H, NCH2CH2CH2OH), 1.78-1.85 (m, 3H, 3-Hb(piper) + 4-
H(piper)), 2.13-2.27 (m, 2H, 2-Ha(piper)), 2.56 (br s, 2H, NCH2CH2CH2OH), 2.92-3.06 (m, 2H, 2-
Hb(piper)), 3.42 (t, J = 6.4 Hz, 2H, CH2OH), 3.93 (d, J = 5.9 Hz, 2H, CH2OAr), 6.26 (d, J = 9.5 Hz,
1H, 3-Hcoum), 6.93 (dd, J = 8.6, 2.4 Hz, 1H, 6-Hcoum), 6.94 (d, J = 2.4 Hz, 1H, 8-Hcoum), 7.60 (d, J
24

ACCEPTED MANUSCRIPT
= 8.6 Hz, 1H, 5-Hcoum), 7.97 (d, J = 9.5 Hz, 1H, 4-Hcoum), OH not detected. Anal. (C18H23NO4)
calcd. % C, 68.12; H, 7.30; N, 4.41. Found % C, 68.33; H, 7.33; N, 4.35. HRMS (Q-TOF) calcd.
for C18H23NO4 [M+H]+ m/z 318.1700, found 318.1700; [M+Na]+ m/z 340.1519, found 340.1515.
6.1.14.3
7-((1-(4-Hydroxybutyl)piperidin-4-yl)methoxy)-3,4-dimethyl-2H-chromen-2-one
(12c). Prepared from 8a (0.14 g, 0.50 mmol) and 4-chloro-1-butanol (0.10 mL, 1.0 mmol).
Purified through column chromatography (gradient eluent: methanol in dichloromethane from
0% to 10%). White solid; yield: 0.072 g, 40%; mp: 125-7 °C. 1H NMR (300 MHz, DMSO-d6) δ:
1.21-1.28 (m, 2H, 3-Ha(piper)), 1.38-1.46 (m, 4H, NCH2CH2CH2CH2OH), 1.70-1.78 (m, 3H, 3-
Hb(piper) + 4-H(piper)), 1.80-1.92 (m, 2H, 2-Ha(piper)), 2.05 (s, 3H, 3-CH3), 2.21-2.29 (m, 2H,
NCH2CH2CH2CH2OH), 2.36 (s, 3H, 4-CH3), 2.85-2.88 (m, 2H, 2-Hb(piper)), 3.36-3.39 (m, 2H,
CH2OH), 3.89-3.91 (m, 2H, CH2OAr), 6.92-6.94 (m, 2H, 6-Hcoum + 8-Hcoum), 7.66 (d, J = 9.4 Hz,
1H, 5-Hcoum), OH not detected. Anal. (C21H29NO4) calcd. % C, 70.17; H, 8.13; N, 3.90. Found %
C, 70.44; H, 8.22; N, 3.84. HRMS (Q-TOF) calcd. for C21H29NO4 [M+H]+ m/z 360.2169, found
360.2165; [M+Na]+ m/z 382.1989, found 382.1991.
6.1.14.4
7-{2-[1-(3-Hydroxypropyl)piperidin-4-yl]ethoxy}-3,4-dimethyl-2H-chromen-2-one
(12d). Prepared from 8e (0.15 g, 0.50 mmol) and 3-bromo-1-propanol (0.090 mL, 1.0 mmol).
Purified through column chromatography (gradient eluent: methanol in dichloromethane from
1
0% to 10%). White solid; yield: 0.13 g, 71%; mp: 97-9 °C. H NMR (500 MHz, DMSO-d6) δ:
1.15-1.21 (m, 3H, 3-Ha(piper) + 4-H(piper)), 1.56 (qn, J = 6.4 Hz, 2H, NCH2CH2CH2OH), 1.63-1.70
(m, 4H, CH2CH2OAr + 3-Hb(piper)), 1.88-1.92 (m, 2H, 2-Ha(piper)), 2.05 (s, 3H, 3-CH3), 2.34 (s,
3H, 4-CH3), 2.32-2.36 (m, 2H, NCH2CH2CH2OH), 2.88 (br s, 2H, 2-Hb(piper)), 3.41 (t, J = 6.4 Hz,
2H, CH2OH), 4.08 (t, J = 6.9 Hz, 2H, CH2CH2OAr), 6.90-6.93 (m, 2H, 6-Hcoum + 8-Hcoum), 7.66
(d, J = 8.8 Hz, 1H, 5-Hcoum), OH not detected. Anal. (C21H29NO4) calcd. % C, 70.17; H, 8.13; N,
3.90. Found % C, 70.21; H, 8.05; N, 3.91. HRMS (Q-TOF) calcd. for C21H29NO4 [M+H]+ m/z
360.2169, found 360.2167; [M+Na]+ m/z 382.1989, found 382.1984.
6.1.14.5
7-{[1-(3-Hydroxypropyl)piperidin-3-yl]methoxy}-3,4-dimethyl-2H-chromen-2-one
(12e). [57] Prepared from 8f (0.14 g, 0.50 mmol) and 3-bromo-1-propanol (0.090 mL, 1.0
mmol). Purified through column chromatography (gradient eluent: methanol in dichloromethane
1
from 0% to 10%). Off-white solid; yield: 0.093 g, 54%; mp 104-7 °C. H NMR (500 MHz,
DMSO-d6) δ: 1.05-1.16 (m, 1H, 4-Ha(piper)), 1.39-1.53 (m, 1H, 5-Ha(piper)), 1.58 (qn, J = 6.3 Hz,
2H, NCH2CH2CH2OH), 1.62-1.69 (m, 1H, 5-Hb(piper)), 1.69-1.78 (m, 1H, 4-Hb(piper)), 1.85-1.95
(m, 1H, 2-Ha(piper)), 1.95-2.03 (m, 2H, 6-Ha(piper) + 3-H(piper)), 2.05 (s, 3H, 3-CH3), 2.36 (s, 3H, 4-
CH3), 2.38 (br s, 2H, NCH2CH2CH2OH), 2.73-2.81 (m, 1H, 6-Hb(piper)), 2.89-2.96 (m, 1H, 2-
Hb(piper)), 3.41 (t, J = 6.3 Hz, 2H, CH2OH), 3.89-3.98 (m, 2H, CH2OAr), 6.90-6.95 (m, 2H, 6-
Hcoum + 8-Hcoum), 7.66 (d, J = 9.5 Hz, 1H, 5-Hcoum), OH not detected. Anal. (C20H27NO4) calcd.
% C, 69.54; H, 7.88; N, 4.05. Found % C, 69.82; H, 7.95; N, 3.88. HRMS (Q-TOF) calcd. for
C20H27NO4 [M+H]+ m/z 346.2013, found 346.2011; [M+Na]+ m/z 368.1832, found 368.1834.
6.1.14.6 7-{[1-(3-Hydroxypropyl)piperidin-3-yl]methoxy}-2H-chromen-2-one (12f). Prepared
from 8g (0.13 g, 0.50 mmol) and 3-bromo-1-propanol (0.090 mL, 1.0 mmol). Purified through
1
crystallization from hot ethanol. Off-white solid; yield: 0.11 g, 69%; mp: 175-7 °C. H NMR
(500 MHz, DMSO-d6) δ: 1.08-1.12 (m, 1H, 4-Ha(piper)), 1.45-1.49 (m, 1H, 5-Ha(piper)), 1.55-1.57
(m, 2H, NCH2CH2CH2OH), 1.63-1.65 (m, 1H, 5-Hb(piper)), 1.71-1.73 (m, 1H, 4-Hb(piper)), 1.87-
1.98 (m, 3H, 2-Ha(piper) + 6-Ha(piper) + 3-H(piper)), 2.31-2.40 (m, 2H, NCH2CH2CH2OH), 2.72-2.74
(m, 1H, 6-Hb(piper)), 2.89-2.91 (m, 1H, 2-Hb(piper)), 3.40 (t, J = 6.4 Hz, 2H, CH2OH), 3.93-3.95 (m,
2H, CH2OAr), 6.26 (d, J = 9.3 Hz, 1H, 3-Hcoum), 6.93 (dd, J = 8.8, 2.4 Hz, 1H, 6-Hcoum), 6.96 (d,
J = 2.4 Hz, 1H, 8-Hcoum), 7.60 (d, J = 8.8 Hz, 1H, 5-Hcoum), 7.96 (d, J = 9.3 Hz, 1H, 4-Hcoum), OH
25

ACCEPTED MANUSCRIPT
not detected. Anal. (C18H23NO4) calcd. % C, 68.12; H, 7.30; N, 4.41. Found % C, 68.40; H, 7.36;
N, 4.29. HRMS (Q-TOF) calcd. for C18H23NO4 [M+H]+ m/z 318.1700, found 318.1696; [M+Na]+
m/z 340.1519, found 340.1518.
6.1.15 7-({1-[3-(Chloromethyl)benzyl]piperidin-4-yl}methoxy)-3,4-dimethyl-2H-chromen-2-
one (13). Coumarin 8a (0.28 g, 1.0 mmol) was suspended in dry acetone (8 mL) before the
addition of DIEA (0.17 mL, 1.0 mmol) and α,α′-dichloro-m-xylene (0.52 g, 3.0 mmol). The
mixture was stirred at room temperature overnight and then the solvent was removed under
vacuum. The resulting crude was purified through column chromatography (eluent: ethyl acetate
in dichloromethane 50%). Yellow solid; yield: 0.31 g, 73%. 1H NMR (300 MHz, CDCl3) δ: 1.24-
1.33 (m, 2H), 1.71-1.74 (m, 3H), 1.81-1.85 (m, 2H), 2.04 (s, 3H), 2.32 (s, 3H), 2.76-2.82 (m,
2H), 3.39 (s, 2H), 3.90-3.94 (m, 2H), 4.71 (s, 2H), 6.91-6.95 (m, 2H), 7.39-7.43 (m, 3H), 7.50 (s,
1H), 7.68 (d, J = 8.8 Hz, 1H).
6.1.16 3-[(4-{[(3,4-Dimethyl-2-oxo-2H-chromen-7-yl)oxy]methyl}piperidin-1-yl)methyl]benzyl
nitrate (14). Intermediate 13 (0.26 g, 0.60 mmol) was refluxed in dry acetonitrile (4 mL) in the
presence of AgNO3 (0.30 g, 1.8 mmol) for 6 h. After cooling, the inorganic residue was filtered
off. The solution was concentrated under rotary evaporation and purified through column
chromatography (gradient eluent: methanol in dichloromethane from 0% to 5%) yielding the
1
desired nitrate as a brown solid. Yield: 0.13 g, 48%. H NMR (500 MHz, CDCl3) δ: 1.43-1.60
(m, 2H, 3-Ha(piper)), 1.79-1.85 (m, 3H, 4-H(piper) + 3-Hb(piper)), 1.99-2.03 (m, 2H, 2-Ha(piper)), 2.18 (s,
3H, 3-CH3), 2.36 (s, 3H, 4-CH3), 2.91-2.97 (m, 2H, 2-Hb(piper)), 3.54 (br s, 2H, ArCH2N), 3.92 (d,
J = 5.9 Hz, 2H, CH2OAr), 5.47 (s, 2H, CH2ONO2), 6.73-6.81 (m, 2H, 6-Hcoum + 8-Hcoum), 7.48-
7.52 (m, 3H, 4-HAr + 5-HAr + 6-HAr), 7.70 (s, 1H, 2-HAr), 7.78 (d, J = 8.8 Hz, 1H, 5-Hcoum). Anal.
(C25H28N2O6) calcd. % C, 66.36; H, 6.24; N, 6.19. Found % C, 66.45; H, 6.38; N, 6.03. HRMS
(Q-TOF) calcd. for C25H28N2O6 [M+H]+ m/z 453.2020, found 453.2023.
6.1.17 General procedure for the synthesis of 3-(4-{[(3,4-dimethyl-2-oxo-2H-chromen-7-
yl)oxy]methyl}piperidin-1-yl)propyl nitrate (15) and 3-{3-[(4-{[(3,4-dimethyl-2-oxo-2H-
chromen-7-yl)oxy]methyl}piperidin-1-yl)methyl]phenoxy}propyl nitrate (16). The appropriate
piperidine 8a or 10 (0.60 mmol) was suspended in dry acetonitrile (5 mL) before adding K2CO3
(0.13 g, 0.90 mmol) and 3-bromopropyl nitrate [39] (0.17 g, 0.90 mmol). The mixture was stirred
at room temperature (24 h, for 15) or refluxed (4 h, for 16), and then concentrated to dryness.
The resulting crude was treated with chloroform and filtered. The solution was then purified
through column chromatography (gradient eluent: methanol in dichloromethane from 0% to 3%).
6.1.17.1
3-(4-{[(3,4-Dimethyl-2-oxo-2H-chromen-7-yl)oxy]methyl}piperidin-1-yl)propyl
nitrate (15). Prepared from 8a (0.17 g, 0.60 mmol) and 3-bromopropyl nitrate (0.17 g, 0.90
1
mmol). Brown solid; yield: 0.12 g, 53%. H NMR (500 MHz, CDCl3) δ: 1.40-1.44 (m, 2H, 3-
Ha(piper)), 1.81-1.84 (m, 3H, 4-H(piper) + 3-Hb(piper)), 1.95 (qn, J = 6.4 Hz, 2H,
NCH2CH2CH2ONO2), 2.00-2.05 (m, 2H, 2-Ha(piper)), 2.17 (s, 3H, 3-CH3), 2.37 (s, 3H, 4-CH3),
2.47 (t, J = 6.4 Hz, 2H, NCH2CH2CH2ONO2), 2.95-2.99 (m, 2H, 2-Hb(piper)), 3.85 (d, J = 5.9 Hz,
2H, CH2OAr), 4.56 (t, J = 6.4 Hz, 2H, CH2ONO2), 6.75 (d, J = 2.4 Hz, 1H, 8-Hcoum), 6.82 (dd, J
= 8.8, 2.4 Hz, 1H, 6-Hcoum), 7.50 (d. J = 8.8 Hz, 1H, 5-Hcoum). Anal. (C20H26N2O6) calcd. % C,
61.53; H, 6.71; N, 7.18. Found % C, 61.64; H, 6.80; N, 7.05. HRMS (Q-TOF) calcd. for
C20H26N2O6 [M+H]+ m/z 391.1864, found 391.1865; [M+Na]+ m/z 413.1683, found 413.1682.
6.1.17.2
3-{3-[(4-{[(3,4-Dimethyl-2-oxo-2H-chromen-7-yl)oxy]methyl}piperidin-1-
yl)methyl]phenoxy}propyl nitrate (16). Prepared from 10 (0.24 g, 0.60 mmol) and 3-
bromopropyl nitrate (0.17 g, 0.90 mmol). Brown solid; yield: 0.11 g, 36%. 1H NMR (500 MHz,
CDCl3) δ: 1.42-1.44 (m, 2H, 3-Ha(piper)), 1.79-1.82 (m, 3H, 3-Hb(piper) + 4-H(piper)), 1.98-2.02 (m,
26

ACCEPTED MANUSCRIPT
2H, 2-Ha(piper)), 2.18 (s, 3H, 3-CH3), 2.20-2.22 (m, 2H, OCH2CH2CH2ONO2), 2.36 (s, 3H, 4-
CH3), 2.91-2.94 (m, 2H, 2-Hb(piper)), 3.48 (s, 2H, ArCH2N), 3.84-3.87 (m, 2H, CH2OCoum),
4.07-4.09 (m, 2H, CH2OAr), 4.67 (t, J = 6.4 Hz, 2H, CH2ONO2), 6.77-6.83 (m, 3H, 6-Hcoum + 8-
Hcoum + HAr), 6.89-6.92 (m, 2H, HAr), 7.20-7.24 (m, 1H, HAr), 7.47 (d, J = 8.8 Hz, 1H, 5-Hcoum).
Anal. (C27H32N2O7) calcd. % C, 65.31; H, 6.50; N, 5.64. Found % C, 65.60; H, 6.56; N, 5.52.
HRMS (Q-TOF) calcd. for C27H32N2O7 [M+H]+ m/z 497.2282, found 497.2281.
6.2 In vitro Inhibition Assays
Inhibitory activities were determined through the protocols described as follows by means of
nonlinear regressions of response/log(concentration) curve obtained with GraphPad Prism 5.0
software and are expressed as IC50 (µM) or as percentage of inhibition at 10 µM for less active
compounds. Results are the mean of three independent experiments.
6.2.1 Fluoroscence-based MAOs inhibition assays
As already reported,[27] human recombinant MAO A and MAO B (microsomes from
baculovirus infected insect cells; Sigma-Aldrich) were used to determine IC50s through a
fluorescence-based method, using kynuramine as non-selective MAO A and MAO B substrate.
Samples were pre-incubated 20 min at 37 °C before adding MAO solutions, then incubated for
additional 30 min. Fluorescence was recorded at excitation/emission wavelengths of 310/400 nm
(5 nm slit width for both excitation and emission) in a 96-well microplate fluorescence reader
(Tecan Infinite M1000 Pro). Experiments were performed in triplicates in black, round-bottomed
polystyrene 96-well microtiter plates (Greiner).
6.2.2 Direct kynuramine MAOs inhibition assay
Coumarins are well-known fluorescent molecules that may interfere with kynuramine-based
MAOs inhibition assay protocol here applied. In order to rule out artifacts arising from samples
fluorescent properties, an additional spectrophotometric in vitro protocol has been employed to
test four compounds as hMAO B inhibitors bearing prototypes of different fluorescent moieties
(3a:
4-[(dimethylamino)methyl]-7-hydroxy-2H-chromen-2-one;
6a:
7-hydroxy-4-
(hydroxymethyl)-2H-chromen-2-one; 9c; 7-hydroxy-3,4-dimethyl-2H-chromen-2-one, 12b; 7-
hydroxy-2H-chromen-2-one). Data have been reported in Table S2 (Supporting Information)
and are in agreement with the fluorescent method. In any case, this direct assay suffers from a
low method sensitivity due to the product (4-hydroxyquinoline) absorbance at 316 nm, ∆ε
=
12,000 M-1 · cm-1.
6.2.3 ChEs inhibition assay
Inhibition tests on ChEs were performed in vitro on AChE from electric eel (463 U/mg;
Sigma) or human recombinant AChE (2770 U/mg; Sigma), BChE from equine serum (13 U/mg;
Sigma) or BChE from human serum (50 U/mg; Sigma), by adapting the well-known
spectrophotometric Ellman’s method to a 96-well plate procedure.[58] Experiments were
performed in triplicates in transparent, flat-bottomed polystyrene 96-well microtiter plates.
6.3 Griess assay
Aliquots (150 µL) from 1 mM stock solutions in DMSO of nitrate under study were added to
DMSO (50 µL) and 800 µL of phosphate buffer (50 mM, pH = 7.40) containing 5 mM cysteine.
Compounds were incubated in the dark at 37 °C. At appropriate time intervals ranging from 30
to 180 minutes, 250 µL of Griess reagent (4.0 g of sulphanilamide, 0.20 g of N-
naphthylethylenediamine dihydrochloride, 10 mL of 85% phosphoric acid in distilled water to a
final volume of 100 mL) were added. After 30 minutes in the dark at room temperature,
absorbance was read at 540 nm using a Agilent 8453E UV-visible spectrophotometer equipped
-
with a cell changer. NO release (quantitated as NO2 , µg/mL) was measured from the calibration
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curve derived from standard sodium nitrite solutions (0.005, 0.01, 0.02, 0.05, 0.07, 0.10, 0.20,
and 0.40 µg/mL). Each compound was tested in triplicates.
6.4 Reversed-phase HPLC analysis
Following a protocol similar to studies already reported by us [59] to assess the hydrolytic
stability in buffer and in the presence of thiols, 150 µL of 1 mM stock solutions of compounds
14-16 in DMSO were added to 1.350 mL of aqueous buffer solution (50 mM phosphate buffer,
pH 7.40, 0.20 M KCl) or to 1.350 mL of buffer enriched with cysteine or glutathione (50 mM
phosphate buffer, pH 7.40, 0.20 M KCl, 1 mM thiol) thus incubating final compounds
concentration equal to 100 µM. Regarding serum stability experiments, a volume of 15 µL from
10 mM stock solutions of compounds 14-16 in DMSO was added to 1.485 mL of reconstituted
human serum preheated at 37 ± 0.5 °C (final compound concentrations 100 µM).
For the analysis in cytosolic fractions, each sample was prepared from five dishes of SH-SY5Y
cells, each containing 5.000.000 cells, that were collected and homogenized in 2 mL of
Dulbecco’s phosphate buffered saline (PBS), pH 7.4, at 4 °C with a IKA®Ultra-Turrax® T25
Digital Homogenizer (for 60 s). The homogenate was centrifuged at 5000 rpm for 10 min and the
surnatant was used for the assay. 15 µL of 5 mM stock solutions of compounds 14-15 in DMSO
were added to 1.485 mL of the cytosolic preparation thus incubating final compounds
concentration equal to 50 µM.
Samples were kept in the dark under gentle stirring (not in the case of human serum and
cytosolic fractions) and incubated at 37 ± 0.5 °C. At appropriate time intervals, aliquots (100 µL)
were diluted or deproteinized with methanol or acetonitrile (400 µL) before injection. In the case
of serum and cytosolic suspensions, after the addition of methanol the samples were vortexed for
1 minute and centrifuged at 3500 rpm for 5 minutes and the supernatant was filtered and
injected. Samples were analyzed by HPLC using a Kinetex-C18 (2.1 mm × 150 mm, with 2.6
µm size particles, 100 Å) on an Analytic Agilent 1260 Infinity multidetector system equipped
with a 1200 series UV-diode array detector. UV spectra were recorded at 320 nm. Analytes were
eluted in isocratic conditions by using mixtures of acetonitrile and ammonium formate buffer (20
mM, pH 5.00) as the mobile phase. The mobile phase was filtered through a Nylon-66 membrane
0.45 µm (Supelco, USA) before use. Injection volumes were 2 µL and the flow rate was 0.2
mL/min. Data were integrated and reported using OpenLAB software (Agilent Technologies).
Calibration lines for alcohols 9d, 11 and 12a were obtained by injecting 1 µL of different stock
solutions (1-200 µM in methanol containing 10% DMSO, v/v) after dilution with cold methanol
(100 µL to 500 µL) and measuring the peak area absorbance at 320 nm. Percentage of conversion
was calculated by using the following equation:
% conversion = (ct/cmax) x 100
where ct was the concentration measured at time t and cmax represents the maximal concentration
(equal to the initial concentration of 14-16 in the incubated samples at t0). Each kinetic
experiment was performed in triplicate. Linear and non-linear regression analyses were
performed with Prism 5.0. Data are the mean ± SD.
6.5 Cytotoxicity assays and neuroprotection against oxidative stress insults
Cytotoxicity assays were carried out against human neuroblastoma cell line SH-SY5Y as
previously described.[60] Cells were maintained at 37 °C in a humidified incubator containing
5% CO2 in DMEM (EuroClone) nutrient supplemented with 10% heat inactivated FBS
(EuroClone), 2 mM L-glutamine (EuroClone), 100 U/mL penicillin and 100 µg/mL streptomycin
(EuroClone). Cytotoxicity of compounds is expressed as IC50 values, the concentrations that
cause 50% growth inhibition. The results were determined using the 3-(4,5-dimethylthiazol-2-
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yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Cells were dispensed into 96-well microtiter
plates at a density of 5,000 cells/well. Following overnight incubation, cells were treated with the
compounds in the range concentration 0.01-100 µM. Then the plates were incubated at 37 °C for
24 h. An amount of 10 µL of MTT (0.5% w/v) was further added to each well and the plates
were incubated for an additional 2 h at 37 °C. Finally, the blue formazan crystals were dissolved
by addition of 100 µL of DMSO. Absorbance at 570 nm was determined using a Perkin Elmer
2030 multilabel reader Victor TM X3.
The oxidative stress in the same neuroblastoma lines was reproduced by incubating cells
rotenone (20 µM) as specific mitochondrial insult [61] or hydrogen peroxide (200 µM) as a non
selective reactive species.[62] To study the neuroprotective action of compounds 9d, 12a, 14, 15
and donepezil against the inhibition of cell proliferation induced by rotenone and H2O2, SH-
SY5Y cells were pre-incubated for 24 h with compounds at concentrations 0.1, 0.5 and 2.5 µM,
then the media was replaced with the media containing the combination of compounds plus the
insults and incubated for further 24 h.[48] Cell proliferation was assessed by MTT assay and data
are expressed as the percentage of viable cells referred to cells incubated without insults and
compounds (control). Each compound was tested in triplicate. Standard error of the mean (SD) is
given. Statistical significance was determined using a two-way analysis of variance (ANOVA)
followed by the Bonferroni post hoc tests (GraphPad Prism version 5) and was assigned to p <
0.01 (**) and p < 0.001 (***).
6.6 Bidirectional transport studies on MDCKII-MDR1 monolayers
The bi-directional transport studies were conducted as previously described by using Madin-
Darby Canine Kidney (MDCK) cells retrovirally transfected with the human MDR1 cDNA
(MDCKII-MDR1) as a blood-brain barrier penetration model.[63] The flux of fluorescein
isothiocyanate-dextran (FD4, Sigma-Aldrich, Italy) and diazepam was used to verify cell barrier
function and integrity. The analysis of compounds 9d, 12a, 14-15 was performed through
UV−visible spectroscopy using a PerkinElmer double-beam UV−visible spectrophotometer
Lambda Bio 20 (Milan, Italy), equipped with 10 mm path-length-matched quartz cells. Standard
calibration curves were prepared at maximum absorption wavelength of each compound using
PBS as solvent and were linear (r2 = 0.999) over the range of tested concentration (from 5 to 75
µM). The FD4 samples were analyzed with a Victor3 fluorimeter (Wallac Victor3, 1420
Multilabel Counter, PerkinElmer) at excitation and emission wavelengths of 485 and 535 nm,
respectively. Each compound was tested in triplicate, and the experiments were repeated three
times. Data are reported as the apparent permeability (Papp), calculated in units of cm/s.[28] The
efflux ratio (ER) was calculated using the following equation: ER = Papp, BL / Papp, AP where
Papp, AP is the apparent permeability of apical-to-basal transport expressed in cm/sec and Papp,
BL is the apparent permeability of basal-to-apical transport expressed in cm/sec. An ER greater
than 2 indicates that a test compound is likely to be a substrate for P-gp transport.
ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge.
Table of alcohol clusters 1-3; Table of inhibition data through kynuramine direct assay
(absorbance protocol) for compounds 3a, 6a, 9c and 12b; gHSQC spectra for compounds 9b, 9c,
11 and 12e (PDF)
29