Donepezil-Based Central Acetylcholinesterase Inhibitors by Means of a "bio-Oxidizable" Prodrug Strategy: Design, Synthesis, and in Vitro Biological Evaluation

Article abstract of DOI:10.1021/acs.jmedchem.7b00702

With the aim of reducing side effects of acetylcholinesterase inhibitors (AChEIs) during symptomatic treatment of Alzheimer's disease, we report herein a new class of donepezil-based "bio-oxidizable" prodrugs 1 designed to be converted into dual binding site AChEIs 2. While most of indanone-derived N-benzylpyridinium salts 2 revealed to be highly potent dual binding site hAChEIs (IC₅₀ up to 3 nM), outperforming the standard drug donepezil (IC₅₀ = 11 nM), most of the corresponding 1,4-dihydropyridines 1 were found to be inactive. Promisingly, whereas the selected prodrug 1r showed good permeability in the PAMPA-BBB model and high in vitro antioxidant activity, its conversion to AChEI 2r could be easily achieved under mild conditions when incubated in various oxidizing media. Lastly, both compounds 1r and 2r did not show genotoxicity in vitro and displayed high LD₅₀ values in mice, making this prodrug 1r/drug 2r couple a good candidate for further in vivo biological experiments.

Full text of DOI:10.1021/acs.jmedchem.7b00702

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Article
Donepezil-based Central Acetylcholinesterase Inhibitors
by means of a “Bio-oxidizable” Prodrug Strategy:
Design, Synthesis and in vitro Biological Evaluation.
Ludovic Peauger, Rabah Azzouz, Vincent Gembus, Mihaela-Liliana Tintas, Jana
Sopková-de Oliveira Santos, Pierre Bohn, Cyril Papamicaël, and Vincent Levacher
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017
Downloaded from http://pubs.acs.org on June 14, 2017
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Donepezilꢀbased Central Acetylcholinesterase
Inhibitors by means of a “Bioꢀoxidizable” Prodrug
Strategy: Design, Synthesis and in vitro Biological
Evaluation.
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‡
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Ludovic Peauger, Rabah Azzouz, Vincent Gembus,* MihaelaꢀLiliana Ţînţaꢁ, Jana Sopkováꢀ
§
║
†
,†
de Oliveira Santos, Pierre Bohn, Cyril Papamicaël, and Vincent Levacher*
†
Normandie Univ, COBRA, UMR 6014 et FR 3038; Univ Rouen; INSA Rouen; CNRS, IRCOF,
1
rue Tesnière, 76821 Mont Saint Aignan Cedex, France
‡
§
VFP Therapies, 15 rue François Couperin, 76000 Rouen, France
Centre d’Etudes et de Recherche sur le Médicament de Normandie, UFR des Sciences
Pharmaceutiques; Université de Caen, 1 rue Vaubénard, 14032 Caen Cedex, France
║
Department of Nuclear Medicine, Henri Becquerel Cancer Center and Rouen University
Hospital & QuantIF LITIS (Equipe d’Accueil (EA) 4108ꢀFederation Recherche (FR) National
Center for Scientific Research (CNRS) 3638), Faculty of Medicine, University of Rouen, Rouen,
7
6821, France.
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ABSTRACT
With the aim of reducing side effects of acetylcholinesterase inhibitors (AChEIs) during
symptomatic treatment of Alzheimer’s disease, we report herein a new class of donepezilꢀbased
“bioꢀoxidizable” prodrugs 1 designed to be converted into dual binding site AChEIs 2. While
most of indanoneꢀderived Nꢀbenzylpyridinium salts 2 revealed to be highly potent dual binding
site hAChEIs (IC50 up to 3 nM), outperforming the standard drug donepezil (IC50 = 11 nM),
most of the corresponding 1,4ꢀdihydropyridines 1 were found to be inactive. Promisingly,
whereas the selected prodrug 1r showed good permeability in the PAMPAꢀBBB model and high
in vitro antioxidant activity, its conversion to AChEI 2r could be easily achieved under mild
conditions when incubated in various oxidizing media. Lastly, both compounds 1r and 2r did not
show genotoxicity in vitro and displayed high LD50 values in mice, making this prodrug 1r/drug
2r couple a good candidate for further in vivo biological experiments.
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INTRODUCTION
Alzheimer’s disease (AD), the most common type of dementia affecting elderly people, is a
complex irreversible neurological affection clinically characterized by a progressive loss of
cognitive abilities associated with the death of central neuronal cells. Given the ageing of the
population over the past decades, AD is becoming one of the main public health issues we have
to face in developed countries. While one in nine people age 65 years and older is concerned,
approximately oneꢀthird of people over 85 years of age are affected by this neurodegenerative
1
disorder. Although the origin of AD is still controversial, amyloid plaques and neurofibrillary
tangles are pathological hallmarks, which are held accountable for the neuronal cell death
observed in neurocortex and hippocampus regions during postꢀmortem brain examinations and
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for the devastating clinical effects of AD. Mounting evidence indicates that oxidative stress is an
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important factor contributing to the initiation and progression of AD.
Another hallmark of AD is the dysfunction and loss of the basal forebrain cholinergic system,
thought to be at the roots of cognitive impairments and deficits in memory. Consequently,
improvement of the central cholinergic transmission emerged as a promising approach for the
symptomatic treatment of AD. Among the different strategies explored to boost acetylcholine
levels in brain regions involved in memory, acetylcholinesterase inhibitors (AChEIs) have been
extensively investigated providing the sole palliative treatment of mild to moderate AD approved
by the FDA. To date, three AChEIs are launched on the market; namely galantamine,
rivastigmine and donepezil. Although these AChEIs show a marked preferential central
cholinergic activity, severe peripheral cholinergic side effects restrict their therapeutic potential
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and lead in the most cases to treatment discontinuation in advanced stages of AD. While the
development of preventive and curative treatments of AD remains the ultimate goal, the search
of central selective AChEIs devoid of adverse effects is still a challenging research topic in the
years to come to improve cholinergic treatment of AD.
All these three marketed AChEIs possess a basic nitrogen which is protonated at physiological
pH. The resulting temporary positive charge interacts within the active site of the enzyme and
contributes significantly to the overall affinity of AChE for these ligands. This acidꢀbase
equilibrium can also be held responsible for both peripheral and central cholinergic activity of
these AChEIs by allowing their neutral form to cross the bloodꢀbrain barrier (BBB).
With the aim to relieve side effects arising from peripheral cholinergic activation, we
previously reported on the development of “bioꢀoxidizable” prodrugs derived from cyclic
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rivastigmine analogues. This firstꢀgeneration “bioꢀoxidizable” prodrugs of AChEIs relies on the
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masking of the positive charge that is intended to bind with W86 and to guide the carbamate
moiety into a proper position so that carbamylation of Ser200 can occur to promote the pseudoꢀ
irreversible inhibition of AChE. A first set of in vivo and ex vivo experiments in mice provided
proofꢀofꢀconcept for central redoxꢀactivation of the prodrug A (IC > 1 mM), brain delivery of
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the drug B (IC =20 nM) and central cholinergic activation. Although peripheral redoxꢀactivation
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of prodrug A cannot be ruled out, only limited peripheral cholinergic activation was observed
probably because of the permanent positive charge in drug B that favors its rapid elimination
from the circulation of mice.
Figure 1. a) The three marketed AChEis rivastigmine, galantamine and donepezil b) Firstꢀ
generation “bioꢀoxidizable” prodrugs of AChEIs derived from rivastigmine.
More recently, some experimental evidence suggest that central AChE would also play nonꢀ
cholinergic functions in the development of AD. Indeed, its presence in senile plaques give
strong support that central AChE is involved in the aggregation and deposition of βꢀamyloid
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peptide (Aβ). More specifically, it has been suggested that the peripheral anionic site (PAS) of
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AChE would catalyze βꢀamyloid peptide (Aβ) aggregation.
This finding has triggered a
renewed interest in the development of new dual AChEIs able to interact with both the catalytic
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anionic site (CAS) and PAS of AChE.
Such dual AChEIs are expected not only to alleviate
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the symptoms but also to slow down the progression of AD.
Among the three marketed
AChEIs, only donepezil displays a dualꢀbinding mode of action (Figure 2a).
In the search of novel central selective AChEIs exhibiting a dualꢀbinding mode of action, we
report herein a donepezilꢀbased “bioꢀoxidizable” prodrug strategy (Figure 2b). This study
includes (1) the rational design of this secondꢀgeneration “bioꢀoxidizable” prodrugs of AChEIs;
(
2) the synthesis of indanoneꢀderived Nꢀbenzylpyridinium salts 2 as potential new AChEIs and
(3) their in vitro biological evaluation [AChE and butyrylcholinesterase (BChE) inhibitory
activity, propidium iodide displacement, antiꢀamyloid aggregation activity]; (4) a preliminary in
vitro evaluation of selected “bioꢀoxidizable” prodrug 1 /drug 2 couples (hAChE inhibitory
activity, kinetic studies, PAMPAꢀBBB permeability, initial assessment of the oxidative
activation step of the prodrug 1r in various media, antioxidant properties of prodrug 1r,
genotoxicity, human neuroblastoma cell viability, docking studies) as well as an evaluation of
their acute toxicity in mice.
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Figure 2. a) Dualꢀbinding mode of donepezil with rhAChE active site b) Design of selective
central AChEꢀdonepezilꢀbased inhibitors 2 by means of a bioꢀoxidizable prodrug strategy.
RESULTS AND DISCUSSION
Rational design of a donepezil-based “bio-oxidizable” prodrug system. All the three
marketed AChEIs possess a basic nitrogen which is protonated at physiological pH. This
resulting temporary positive charge interacts within the active site of the enzyme and contributes
significantly to the overall affinity of AChE for these ligands. This acidꢀbase equilibrium can
also be held responsible for both peripheral and central cholinergic activity of these AChEIs by
allowing their neutral form to cross the bloodꢀbrain barrier (BBB). As far as donepezil is
concerned, Xꢀray crystallography and docking studies established that donepezil has a dualꢀ
binding mode of action. While the benzyl piperidine residue interacts with the CAS (stacking
against W86 in rhAChE) and the indanone moiety binds to the PAS (stacking with W286 in
rhAChE), the protonated piperidine nitrogen makes additional cationꢀπ interactions with
aromatic residues at the active site gorge (waterꢀmediated hydrogen bond between piperidine
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nitrogen and Y341 and Y377 of rhAChE).
The rational design of this prodrug approach is
based on the masking of the positive charge at the nitrogen, by replacing the piperidine moiety of
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donepezil by a 1,4ꢀdihydropyridine ring. Accordingly, the 1,4ꢀdihydropyridine nitrogen of the
prodrug 1, not basic enough to be protonated at physiological pH, is expected to lead to a
significant decrease in AChE inhibitory activity, while being able to cross the BBB as a result of
their good lipophilicity. Once in the brain, a redoxꢀactivation step mediated by oxidoreductases
would convert the 1,4ꢀdihydropyridine prodrug 1 into the corresponding pyridinium drug 2. The
presence of a permanent positive charge within the pyridinium drug 2 is expected not only to
restore AChE inhibitory activity but also to entail a “lockedꢀin” effect in the brain, thus
preventing peripheral cholinergic adverse effects, while enabling prolonged duration of AChEIs
action in brain tissue. At this stage, it is worth noting that an electronꢀwithdrawing group (EWG)
at Cꢀ3 position of the pyridine moiety constitutes a key element in the design of the prodrug. It is
intended to tune the redox properties of the 1,4ꢀdihydropyridine 1/pyridinium 2 couple so that to
provide a prodrug stable enough at the periphery, and which once in the brain can be easily
oxidized to exert a central AChE inhibitory activity. Skillful structural variations at the EWG
should help to strike a good balance between stability and redoxꢀactivation ability of the prodrug.
Although 1,4ꢀdihydropyridineꢀtype compounds have been extensively exploited by N. Bodor and
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L. Prokai as redox targetor to transport and release neuroactive drugs into the brain,
their use
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in the design of “bioꢀoxidizable” prodrug strategies has scarcely been explored.
Last but not
least, the pertinence of this approach was also supported by a survey of the literature which
revealed that several Nꢀbenzyl pyridinium derivatives closely related to the target compounds 2
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have shown to exhibit appealing AChE inhibitory activities.
Chemistry. In order to conduct a preliminary SAR analysis of this new class of Nꢀbenzyl
pyridinium quaternary donepezil analogues, a set of target compounds 2 were prepared by
varying the nature of the withdrawing group (EWG) at Cꢀ3 position of the pyridine ring, as well
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as by using differently substituted indanones and benzyl groups (Scheme 1). Thus, indanones 3a-
f were reacted with 4ꢀpyridinecarboxaldehydes 4,5 in the presence of PTSA under DeanꢀStark
conditions to afford the corresponding α,βꢀunsaturated indanones 6,7a-f in moderate to good
yields (26ꢀ79%). Standard catalytic hydrogenation of 6,7a-f was performed in the presence of
platinum on carbon to afford the doubleꢀbond reduction products 8,9a-f in good to excellent
yields (59ꢀ99%). Further functionalizations of 3ꢀbromopyridine derivatives 9a-f allowed to
install various EWGs at Cꢀ3 position of the pyridine ring. To this end, a Pdꢀcatalyzed crossꢀ
coupling reaction between 3ꢀbromopyridine derivatives 9a-f and 1ꢀ(ethoxyvinyl)tri(nꢀ
butyl)stannane furnished the desired 3ꢀacetylpyridine derivatives 10a-f in good yields (60ꢀ
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87%). Alternatively, starting from 9a and 2,4,6ꢀtrichlorophenyl formate, a Pdꢀcatalyzed crossꢀ
coupling reaction afforded the corresponding reactive ester 11 in 55% yield. The later was
subsequently reacted with various heteronucleophiles to give rise to variously 3ꢀsubtituted
pyridines 12-14 (75ꢀ99% yields). Thereafter, the dehydration reaction of pyridineꢀ3ꢀcarboxamide
derivative 14 conducted with MSTFA and CuCl led to the desired pyridineꢀ3ꢀcarbonitrile
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derivative 15 in 83% yield. Indanones 8, 10a-f, 12-15 thus obtained were easily transformed into
the corresponding indanoneꢀderived Nꢀbenzylpyridinium salts 2a-r (63ꢀ99% yields) by
quaternization in the presence of the appropriate aryl bromide.
a
Scheme 1. Synthesis of novel indanoneꢀderived Nꢀbenzylpyridinium salts 2
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a
Reagents and conditions: (i) PTSA, toluene, DeanꢀStark (26ꢀ79%); (ii) H , Pt/C, EtOH, 20°C
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(59ꢀ99%); (iii) For 10a-f from 9a-f: Pd(dba) , PPh , 1ꢀ(ethoxyvinyl)tri(nꢀbutyl)stannane then
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HCl, reflux (60ꢀ87%); (iv) For 11 from 9a: Xantphos, Pd(OAc) , 2,4,6ꢀtrichlorophenyl formate,
toluene, Schlenk tube, 110°C, overnight (55%); (v) MeOH, NEt , Schlenk tube, 70°C, 20h for 12
(75%) or MeNH , NEt , THF, Schlenk tube, 70°C, 24h for 13 (99%) or NH in dioxane, Schlenk
tube, 70°C, 20h for 14 (78%); (vi) CuCl , MSTFA, toluene (83%); (vii) ArCH Br, CH Cl or
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ACN, reflux, 12h (63ꢀ99%).
In vitro Inhibition Studies of hAChE / eqBChE and SARs. In addition to AChE, recent
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studies revealed that BChE may also have a crucial role in the progression of AD.
While the
level of AChE decrease dramatically in the advanced stage of AD, that of BChE is maintained
constant or, better still, may be increased in brain regions involved in cognitive functions. These
observations lead one to believe that inhibition of both AChE and BChE may contribute to
improve current symptomatic treatments of AD. Therefore, both AChE and BChE inhibitory
activities of the newly prepared racemic indanoneꢀderived Nꢀbenzylpyridinium salts 2a-r was
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evaluated by means of Ellman’s spectrophotometric method using AChE from human
erythrocytes and BChE from equine serum. Donepezil and tacrine were used as reference
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standards (Table 1). In pyridinium salt 2a, the substitution pattern of both indanone and benzyl
moieties being the same as that of donepezil, this compound was prepared as reference for
comparison purpose with donepezil. To our delight, this first pyridinium 2a displayed an
interesting twoꢀdigit nanomolar hAChE inhibitory activity (IC =36 nM) comparable to that of
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donepezil (IC =11 nM). We then undertook to study the influence of the EWG at Cꢀ3 of the
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pyridine scaffold on hAChE inhibitory activity. This stabilizing functional group, essential to
ensure good stability of the 1,4ꢀdihydropyridine prodrugs 1, should nevertheless not disrupt the
inhibitory activity of the corresponding pyridinium drugs 2. This is all the more interesting as, to
the best of our knowledge, all SAR investigations regarding donepezil mainly focused on both
ends of the molecule (namely indanone and benzyl moieties located at the PAS and CAS
respectively), leaving aside the central part of the donepezil which interacts with the active mid
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gorge.
As depicted in Table 1, a set of indanoneꢀderivated Nꢀbenzylpyridinium salts 2 bearing at Cꢀ3
position a nitrile (2b), an acetyl (2c-l), an ester (2o,p) and a primary carboxamide (2q,r) were
evaluated on hAChE. Most of them turned out to be highly potent hAChEIs, exhibiting oneꢀ to
twoꢀdigit nanomolar IC values ranging from 2.9 to 91 nM, except for targets 2l and 2q whose
5
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IC values dropped to 262 and 678 nM respectively. Of all EWGs surveyed, only secondary
5
0
carboxamides (2m,n) showed no inhibition on hAChE (IC > 10ꢀM), ruling out the use of this
50
class of EWGs as stabilizing element in prodrugs 1. We then focused our interest on the
influence of the substitution pattern at the benzyl moiety by comparing the hAChE inhibitory
2
3
activities of compounds 2c-g (EWG=COMe; R =R =OMe). In this series, the benzyl appendage
is differently substituted at the orthoꢀ or metaꢀposition by a methyl group or a chlorine atom.
Whereas the unsubstituted benzyl derivative 2c already showed a remarkable hAChE inhibitory
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activity (IC =18 nM), the substituted benzyl derivatives 2d-g prove to be slightly more potent
5
0
(
IC = 3ꢀ14 nM), 3ꢀchlorobenzyl derivative 2g offering the best result of this series (IC =3 nM).
50 50
Lastly, we also briefly looked at the substitution pattern of the indanone ring by comparing the
10
11
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23
24
25
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
performance in inhibiting hAChE of a series of compounds 2d,h-l (EWG=COMe; Ar=2ꢀMePh)
2
3
2
3
differently substituted at the indanone moiety (R = H, OMe, Me; R = H, OMe, Me, Cl; R = R =
18
OCH O). In short, IC values followed the same trend as donepezil; namely compound 2d
2
50
bearing a 5,6ꢀdimethoxy indanone ring revealed to be the most potent (IC = 8 nM). All other
5
0
compounds 2h-k displayed a good but lower twoꢀdigit nanomolar potency against hAChE (IC =
5
0
2
7ꢀ91 nM), while a much lower inhibition was recorded with compound 2l (IC =262 nM)
50
highlighting the deleterious effect of the electron withdrawing chlorine atom at the Cꢀ5 position.
Although donepezil is known to be selective for AChE and to display low affinity for BChE
18
(
IC = 3.3 ꢀM), the inhibitory activity of a large selection of our Nꢀbenzylpyridinium
5
0
analogues 2d-m,o,q,r towards eqBChE was evaluated (Table 1). Unsurprisingly, the majority of
compounds 2 exhibited a low inhibitory activity against eqBChE in the same micromolar range
as that measured for donepezil. Interestingly, compound 2j stands out with a satisfactory IC50
value of 262 nM on eqBChE together with a high hAChE inhibitory activity (IC =27 nM).
5
0
Table 1. Inhibition of AChE from Human Erythrocytes (hAChE) and Propidium Iodide
Displacement (EeAChE) by the Target Compounds
IC50 (nM) ± SD
2
3
a
b
Selectivity
for AChE
Propidium iodide
displacement (%)
compd
EWG
H
R
R
Ar
Ph
hAChE
eqBChE
c
d
e
e
2
a
OCH
3
OCH
3
36.5±4
nd
ꢀ
nd
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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
e
e
2
b
CN
OCH
OCH
OCH
OCH
OCH
OCH
OCH
H
3
3
3
3
3
3
2
OCH
OCH
OCH
OCH
OCH
OCH
ꢀ
3
3
3
3
3
3
Ph
2.92±0.1
18±1.5
8.8±0.2
14±1.6
18±3
nd
nd
ꢀ
ꢀ
25±2
26±3
14±1
19±1
19±1
16±1
19±2
23±3
18±2
20±2
20±3
2c
COCH
COCH
COCH
COCH
COCH
COCH
COCH
COCH
COCH
COCH
3
3
3
3
3
3
3
3
3
3
Ph
2
2
2
d
e
f
2ꢀMePh
3ꢀMePh
2ꢀClPh
3ꢀClPh
2ꢀMePh
2ꢀMePh
2ꢀMePh
2ꢀMePh
2ꢀMePh
Ph
5930±440
5990±510
1915±145
3340±180
1615±45
2990±240
262±15
674
427
106
1021
26.9
133
6.95
41.9
13.9
ꢀ
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
2
3
4
5
6
7
8
9
0
2g
3.27±0.1
60±3
2
2
2
h
i
O
OCH
30±1
3
j
CH
H
3
CH
CH
Cl
3
3
27.7±3.7
91±9
2k
3815±375
3630±90
4765±425
2
2
2
l
H
262±17
>10ꢀM
>10ꢀM
25.6±4.7
5.8±0.2
678±42
41±20
e
m
n
CONHCH
CONHCH
3
3
OCH
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
3
OCH
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
3
nd
e
e
2ꢀMePh
Ph
nd
ꢀ
nd
2o
COOCH
3
3
3055±65
1792±170
>10ꢀM
119
309
ꢀ
16±0
e
2
2
2
p
q
r
COOCH
2ꢀMePh
2ꢀMePh
3ꢀClPh
nd
CONH
CONH
16±1
20±4
17±1
2
ꢀ
2
>10ꢀM
1
8
17
donepezil
6.4±0.4 or 11
3365
306
55
tacrine
45.1±7
3.2±0.2
a
The 50% inhibitory concentration (means ± SD of three expermiments) of AChE from human
erythrocytes. The 50% inhibitory concentration (means ± SD of three experiments) of BChE
from equine serum. Selectivity for AChE = IC (eqBChE)/IC (hAChE). Propidium iodide
displacement conducted on EeAChE by the tested compound and donepezil as control at 1.0ꢀM.
b
c
d
5
0
50
e
Not determined.
Inhibition of the Peripheral Anionic Site (PAS), kinetic studies and anti-amyloid
aggregation activity. The affinity of AChEIs 2 for the PAS of AChE was examined at 1.0ꢀM
5
6
concentration using the method of Taylor et al. which implements displacement studies with
propidium iodide, a known PASꢀspecific ligand of AChE. The results obtained in Table 1
showed that most of the tested drugs 2 were slightly more efficient to displace propidium (18ꢀ
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2
6%) than donepezil (17%), with the exception of 2d,o which appeared to be somewhat less
potent (12ꢀ16%). To gain further insight into the mechanism of AChE enzyme inhibition and to
ascertain the dualꢀbinding site character of this new class of AChEIs 2, an enzyme kinetic study
was performed on selected drug 2r using hAChE. As explained bellow, the better solubility of
the corresponding prodrug 1r prompted us to pursue our in vitro biological investigation with 2r.
The LineweaverꢀBurk double reciprocal plots were constructed with a range of substrate
concentrations as depicted in Fig. 3. Analysis of the plots revealed that by increasing the
concentrations of 2r, an increase in slope occurred (reflecting a reduction of Vmax) whereas Km
remains unchanged. These two findings are consistent with a nonꢀcompetitive inhibition of
AChE, thus supporting the dual binding character of 2r that binds, in all likelihood, to both CAS
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5
7
and PAS of the enzyme. A thioflavin Tꢀbased fluorometric assay was accomplished with the
dual binding site inhibitor 2r to ascertain whether inhibition of the PAS may prevent Aβ peptide
aggregation. Gratifyingly, 2r inhibits AChEꢀinduced Aβ1–40 aggregation by 41±2 % at the
concentration of 100 µM, compared to 75±2% with propidium iodide as reference standard
under the same conditions. In addition, although 2r exhibits moderate inhibition of selfꢀinduced
Aβ1−42 aggregation (25%±3) at 10 μM concentration, it is worth noting that donepezil displays a
58
lower antiꢀaggregation activity (13%) at the same concentration.
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120
Control
2r 1nM
1
00
2
2
r 10nM
80
r 100nM
6
0
2r 1000nM
0
1
2
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8
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0
40
2
0
0
-
5
5
15
25
-
20
-1
-1
ATC mM
Figure 3. Kinetics study on the mechanism of hAChE inhibition by 2r. Lineweaver−Burk
reciprocal plots of the AChE initial velocity at increasing substrate concentrations (0,04ꢀ0,2 mM)
in the absence and presence of 2r (1ꢀ1000 nM) are represented. ATC = acetylthiocholine; V =
initial velocity rate.
In vitro evaluation of the donepezil-based “bio-oxidizable” prodrug system. After having
demonstrated the potential of indanoneꢀderived Nꢀbenzylpyridinium salts 2 to act as highly
potent dual binding site AChEIs, it remained to be established whether the corresponding 1,4ꢀ
dihydropyridines 1 are inactive or much less active against AChE to be considered as “bioꢀ
oxidizable” prodrug candidates. To this end, regioselective reduction of selected indanoneꢀ
derived Nꢀbenzylpyridinium salts 2d,h-l,q,r afforded the corresponding 1,4ꢀdihydropyridines
1
d,h-l,q,r as a diastereoisomeric mixture which were subsequently evaluated for their inhibitory
activity against hAChE (Table 2, 35ꢀ59% yields). Pleasingly, most of the dihydropyridines 1
proved to be inactive against AChE (IC >1 ꢀM), with the exception of 1d,j which revealed a
5
0
weak activity (IC = 428nM and 653 nM respectively), however much lower than that of the
5
0
parent pyridinium salts 2d,j (IC = 8.8 nM and 27.7 nM respectively).
5
0
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Table 2. Inhibition of AChE from Human Erythrocytes (hAChE) by Selected Prodrugs 1
R1
R1
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18
19
20
21
22
23
24
25
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31
32
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34
35
36
37
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41
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O
O
R2
R2
EWG BNAH/CH Cl
EWG
2
2
or
N
N
sodium dithionite
dioxane/water
2
1
Ar
Ar
IC50
(nM)±SD
1
2
compd EWG
R
R
Ar
a
hAChE
1
1
d
h
COCH
COCH
COCH
COCH
COCH
COCH
3
3
3
3
3
3
OCH
OCH
H
3
OCH
3
3
2ꢀMePh
2ꢀMePh
2ꢀMePh
2ꢀMePh
2ꢀMePh
2ꢀMePh
2ꢀMePh
3ꢀClPh
428±42
>1ꢀM
>1ꢀM
653±39
>1ꢀM
>1ꢀM
>1ꢀM
>1ꢀM
O
ꢀ
2
1i
OCH
1
1
1
1
j
CH
H
3
CH
CH
Cl
3
3
k
l
H
q
CONH
2
2
OCH
3
3
OCH
3
3
1r
CONH
OCH
OCH
A crucial task of the “bioꢀoxidizable” prodrug 1 consists in crossing the BBB to deliver the
active drug 2 into the brain after a subsequent redoxꢀactivation step. The aptitude of 1,4ꢀ
dihydropyridines 1h,i,r and their corresponding pyridinium salts 2h,i,r to cross the BBB by
passive diffusion were then evaluated using an in vitro parallel artificial membrane permeability
ꢀ6
assay of bloodꢀbrain barrier (PAMPAꢀBBB). Permeability values P (10 cm/s) for the prodrug
e
1
/drug 2 systems tested at 100 ꢀM in a buffer pH 7.4, were measured to be 14.3±1.5/0±0
(
1h/2h), 14.8±0.6/0.3±0.2 (1i/2i), 25.6±2.3/0.15±0.07 (1r/2r). On the basis of these results, it
can be reasonably concluded that 1,4ꢀdihydropyridines 1h,i,r are lipophilic enough to reach the
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2
2
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2
2
2
2
2
2
2
3
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5
5
6
brain, as their P values are well above the threshold predicting high BBB permeability (P >
e
e
5.2). As might be expected, the corresponding quaternary pyridinium salts 2h,i,r would not
penetrate the BBB by passive diffusion suggesting that once in the brain, AChEIs 2 will be
subjected to a “lockedꢀin” effect to exhibit a central selective AChE inhibition. For this scenario
to be successful, prodrugs 1 should be stable enough to ensure that any early conversion to the
parent AChEIs 2 occurs at the periphery, while being able to undergo rapid oxidation once in the
brain to thwart competing metabolisms and to trap the permanently charged AChEIs 2 within the
0
1
2
3
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0
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0
1
2
3
4
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9
0
brain tissue. To gain insights into this activation step of prodrugs 1, the rate and the nature of the
+
oxidation was investigated by incubation in various oxidative media (AgNO , NAD and
3
riboflavin solutions, mice brain homogenate). For solubility reasons, we decided to focus our
study on the 1,4ꢀdihydropyridine/pyridinium salt couple 1r/2r as “bioꢀoxidizable” prodrug
system. First, we evaluated the in vitro stability of prodrug 1r in PBS and in human plasma at
37°C (Fig. 4). After 3 hours, only traces of oxidation product 2r could be detected without any
other degradation product, allowing us to foresee a good stability of 1r at the periphery. In the
presence of AgNO , a significant amount of oxidation product 2r was detected after 3 hours.
3
Although a chemical oxidation route can occur, an enzymatic activation step of the prodrug 1r
cannot be ruled out, all the more so as oxidation of dihydropyridines by NADH dehydrogenase
5
9
has already been reported. A relevant observation that argues in favor of this hypothesis is the
+
marked acceleration in the rate of oxidation of prodrug 1r when using a 2% NAD or a 2%
+
riboflavin solution. It is worth noting that NAD and riboflavin act respectively as coenzyme and
prosthetic group precursor of NADH dehydrogenase. Finally yet importantly, whereas incubation
of prodrug 1r conducted in 10% fresh mice brain homogenate led to the oxidation product 2r
(35%), the use of mice brain homogenate stored beforehand for 3 months at ꢀ20°C failed to
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furnish 2r. This timeꢀdependent loss of enzyme activity is the hallmark of NADH hydrogenase
and therefore consistent with the involvement of this enzymatic system in the oxidation of
prodrug 1r (Figure 3).
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60
7
7.90%
8
0%
60%
39.6%
4
2
0%
0%
35.0%
2
8.0%
2.9%
.6%
3.0%
1
0
%
Figure 4. Activation of the “bioꢀoxidizable” prodrug 1r in various oxidative media.
Another appealing aspect of our “bioꢀoxidizable” prodrug approach arises from the possibility
for 1,4ꢀdihydropyridines 1 to exhibit radical scavenging and antioxidant properties during its
conversion to the corresponding AChEIs 2. The antioxidant activity of prodrug 1r was therefore
6
0ꢀ62
assessed using DPPH (2,2ꢀdiphenylꢀ1ꢀpicrylhydrazyl) free radical scavenging method,
one of
the most frequently used assay for evaluating in vitro antioxidant activity by measuring the
ability of tested compounds to act as free radical scavengers or hydrogen donors. From this
assay, it was observed that prodrug 1r exhibits a noticeable scavenging activity (EC = 90 ꢀM)
5
0
whereas donepezil does not show any activity. Furthermore, a set of NMR experiments clearly
.
demonstrated that prodrug 1r reacts with the stable radical DPPH to produce the desired AChEI
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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2
r as the main oxidation product (see supporting information). These findings suggest that
central conversion of prodrug 1r to AChEI 2r may possibly lead to additional antioxidant
properties with potential protective effects against oxidative stress.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
1
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0
1
2
3
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0
1
2
3
4
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0
Genotoxicity tests are now recommended at earlier stages of drug discovery to reduce the
failure probability in advanced stage of development. Therefore, the genotoxicity of compounds
1
r and 2r was evaluated by the Ames fluctuation test from Toxem (France). The genotoxic
activity was determined using Salmonella thyphimurium TA98 and TAMix (mixture of six base
pair mutant strains TA7001ꢀTA7006) with and without metabolic activation. Satisfactorily,
compounds 1r and 2r did not induce significant number of revertant colonies both in presence
and in absence of metabolic activation.
We also investigated the cytotoxicity profile of pyridinium compound 2r on human
neuroblastoma SHꢀSY5Y cell lines using a CalceinꢀAM assay. As a control, the neuroblastoma
cells were also treated with donepezil and the known neurotoxin 1ꢀmethylꢀ4ꢀphenylpyridinium
6
3
(MPP+). Cells were treated with compound 2r, donepezil and MPP+ at various concentrations
(0.1 to 1 mM) for 24h prior to measuring cell viability. In contrast to MPP+ and donepezil for
which a doseꢀrelated effect was clearly observed, we are pleased to note that compound 2r did
not induced toxicity in all tested doses on the SHꢀSY5Y cells (see supporting information).
Docking Study of 1r and 2r with hAChE. To gain insight into binding interactions of AChEI
in the hydrolytic active site, we carried out molecular docking studies of 1r and 2r compounds.
2
8
These studies were performed into a human AChE structure (PDB ED: 4EY7) and during the
docking a water molecule interacting with protonated piperidine ring of donepezil was conserved
(residue number 931) and the protonation schema proposed by proPKA software was applied on
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AChE (Glu202 protonate, His447 protonate at δ position…). (R)ꢀ and (S)ꢀenantiomers of 2r and
(R,R)ꢀ and (S,S)ꢀenantiomers of 1r were docked independently in the active site using the GOLD
program and applying ChemPLP scoring function. Both enantiomers took a similar position
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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60
2
8
close to donepezil in the crystal structure of hAChE/donepezil complex (Figure 5C). The
visualization of AChE binding site surface showed two cavities having the possibility to
accommodate the EWG group, the first in W86 proximity and the second one in His447
proximity (Figure 4D). The docking placed the EWG group of 1r and 2r systematically in the
His447 cavity. The docking results suggest that, as donepezil, the indanone moiety of 1r and 2r
are situated in W286 proximity of the Peripheric binding site (PAS) well positioned for πꢀ
stacking. Nꢀbenzyl group stacks above W86 of anionic hole and the indanone carbonyl group
establishes an Hꢀbond with Phe295 backbone NH. The cyclic nitrogen atom of both ligands takes
a position suitable for an electrostatic interaction with the water molecule in the proximity of
Tyr337 and Tyr341. According to the docking results, all key interactions are preserved by 1r
and 2r. However, 1r and 2r exhibit very different inhibition activities. As the unique difference
between both compounds lies in the presence of the pyridinium ion in 2r, we assumed that the
positively charged nitrogen atom is necessary to display high AChE inhibition activity. Finally,
we were also very concerned about the difference on hAChE inhibition observed between the
secondary amide group in 2m,n and ester function in 2o,p. So, docking studies were also
performed on 2n and 2p. Interestingly, the ester EWG in 2p for which the oxygen atom from the
OMe group is in front of His447 HN (see supporting information) can give interactions and is
δ
then in agreement with the measured inhibition activities. On the contrary, the NH part of the
secondary amide group of 2n was placed in front of HN of His447 which is not suitable for an
δ
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interaction. This result is thus consistent with the observed ineffectiveness of 2n on the hAChE
inhibition (>10ꢀM).
W286
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B
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F295
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HOH931
H447
Y341
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C
D
Figure 5. Dihydropyridine (S,S)ꢀ1r (A) and pyridinium (S)ꢀ2r (B) positioned in AChE binding
sites using the docking studies, compared to the donepezil position from the Xꢀray structure (C)
as well as the binding cavity surface (D). The compound and the selected side chains of the
binding site residues are in stick and the protein in ribbon representation. This figure was made
with PYMOL (DeLano Scientific, 2002, San Carlo, USA).
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In vivo toxicity evaluation of the donepezil-based prodrug system. Finally, preliminary
toxicological experiments were accomplished to provide a first insight in the toxicological
profiles of the selected compounds 1r and 2r. Donepezil and rivastigmine were used as reference
drugs. The median lethal dose (LD ) is a helpful pointer of the substance’s acute toxicity. The
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LD dose was established after intraperitoneal injection in female CDꢀ1 IGS mice 20ꢀ22g
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(Charles River Laboratories France) followed by an observation period of 48 hours. The LD50
values in mg/kg were determined to be as follows: for 1r (>50 solubility limit), 2r (70±10),
donepezil (22.5±1.5) and rivastigmine (6.2±0.6). These results point out a low acute toxicity of
the prodrug/drug system 1r/2r with particular high LD values compared with reference drugs.
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The potential toxicity of compound 1r was then evaluated following once daily intraperitoneal
administration (10 mg/kg) in mice for 14 days. At this dose level, the compound 1r did not
induce mortality. A slight freezing was observed with only one animal. This sign appeared
within 30 minutes to 1h after treatment the first 3 days and was not observed thereafter until the
end of the treatment. No other clinical signs were observed during the treatment period.
Compared to control group, a slight weight loss was observed in 4/5 animals during the first
three days after the start of treatment with 1r but was discontinued until the day 14. After
necroscopy, principal organs (brain, heart, kidney, liver, spleen…) were submitted to a
macroscopic and microscopic examination. At the studied dose, compound 1r did not induce
changes on the controlled organs. To ensure that no accumulation of 2r in the brain occurs after
repeated daily administration, ex vivo analysis of brain tissue after 24h and 14 days were
performed (by LC MSMS) and no significant quantities of 2r were detected.
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CONCLUSION
Although the search of curative treatments for AD has long been, and is still a priority,
improvement of currently prescribed symptomatic treatments remains an important field of
research. These symptomatic treatments, which mainly rely on AChEIs to restore the cholinergic
balance in brain of AD patients in order to preserve cognitive functions, are often associated with
serious side effects attributable to peripheral cholinergic stimulation. With the ultimate goal to
prevent these deleterious adverse effects, we reported herein a “bioꢀoxidizable” prodrug
approach by taking a cue from Bodor’s redox chemical delivery system to develop new central
AChEIs based on the standard drug donepezil. Thus, a set of indanoneꢀderived Nꢀ
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benzylpyridinium salts 2 and their corresponding “bioꢀoxidizable” prodrug 1,4ꢀdihydropyridines
1
were prepared and evaluated in vitro. Our study provides in vitro proofꢀofꢀconcept of this “bioꢀ
oxidizable” prodrug strategy by validating the main features required; namely (1) high inhibitory
activity of indanoneꢀderived Nꢀbenzylpyridinium salts 2 towards AChE; (2) dual binding site
mode of interactions by binding to both the CAS and PAS of AChE leading to antiꢀamyloid
aggregation activity; (3) any relevant inhibitory activity of the corresponding indanoneꢀderived
Nꢀbenzyl 1,4ꢀdihydropyridines 1 against AChE; (4) good prediction of BBB penetration for
prodrugs 1 and brain trapping of the corresponding permanently charged AChEIs 2; (5) good
stability of the prodrug 1r in human plasma and smooth conversion back to the parent drug 2r
under various mild oxidizing conditions, not to mention promising antioxidant properties of
prodrug 1r. Finally, both compounds 1r and 2r did not exhibit genotoxicity from Ames tests and
showed relatively low acute toxicity in mice, offering the prospect of further in vivo biological
developments.
EXPERIMENTAL SECTION
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Chemistry. All commercial reagents were used without further purification. The solvents were
dried with appropriate desiccants and distilled prior to use or were obtained anhydrous from
commercial suppliers. Silica gel (60, 230–400 mesh or 70–230 mesh) was used for column
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chromatography. Reactions were monitored by thin layer chromatography on silica gel precoated
1
aluminium plates. UV light at 254 nm or KMnO stains were used to visualize TLC plates. H,
4
1
3
C NMR spectra were recorded using a spectrometer operating at 300 and 75 MHz respectively.
Abbreviations used for peak multiplicities are s: singlet, d: doublet, t: triplet, q: quadruplet dd =
doublet of doublet, br = broad and m: multiplet. Coupling constants J are in Hz and chemical
1
shifts are given in ppm and calibrated with DMSOꢀd or CDCl (residual solvent signals). H
6
3
13
NMR spectra obtained in CDCl were referenced to 7.26 ppm. C NMR spectra obtained in
3
CDCl were referenced to 77.16 ppm and in DMSOꢀd were referenced to 39.52 ppm. Elemental
3
6
analyses were performed by the microanalysis service of the University of Rouen and were
recorded with a Thermo Scientific FLASH 2000 analyzer.
General procedure A for synthesis of compounds 1d, 1h, 1i, 1j, 1k, 1l. To a solution of
pyridinium salt 2 (0.2 mmol) in CH Cl (6 mL) was added Nꢀbenzylꢀ1,4ꢀdihydronicotinamide
2
2
(BNAH) (0.2 mmol, 1 equiv) at room temperature under Argon. The resulting solution was
stirred in darkness until completion of the reaction (TLC). The reaction mixture was then washed
with H O (2x) and brine, dried over MgSO , filtered and concentrated under reduced pressure.
2
4
Flash chromatography on silica gel of the crude residue afforded compounds 1 as a
diastereomeric mixture.
2ꢀ[[3ꢀAcetylꢀ1ꢀ(oꢀtolylmethyl)ꢀ4Hꢀpyridinꢀ4ꢀyl]methyl]ꢀ5,6ꢀdimethoxyꢀindanꢀ1ꢀone (1d). The
title compound 1d was prepared according to the general procedure A with compound 2d (76.2
mg, 0.15 mmol) and BNAH (32.1 mg, 1 equiv) as reactants. Rf = 0.50/ 0.59 (100% EtOAc). Pale
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brown solid. (Yield 32.3 mg, 50%). H NMR (300 MHz, CDCl , δ): 1.35ꢀ1.46 (m, 0.55H), 1.48ꢀ
3
1.60 (m, 0.45H), 1.88ꢀ2.09 (m, 1H), 2.12 (s, 1.65H), 2.18 (s, 1.35H), 2.29 (s, 3H), 2.63ꢀ2.85 (m,
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.45H), 3.07 (dd, 0.55H, J = 17.1 Hz, J = 3.3 Hz), 3.17ꢀ3.35 (m, 1H), 3.58ꢀ3.66 (m, 0.55H),
.69ꢀ3.79 (m, 0.45H), 3.88 (s, 3H), 3.94ꢀ3.95 (m, 3H), 4.44 (s, 2H), 4.95 (dd, 0.45H, J = 7.8 Hz,
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J = 5.4 Hz), 5.12 (dd, 0.55H, J = 7.8 Hz, J = 5.1 Hz), 5.89ꢀ5.96 (m, 1H), 6.84 (s, 0.45H), 6.88 (s,
13
0
.55H), 7.06ꢀ7.28 (m, 6H). C NMR (75 MHz, CDCl , δ): 19.2, 24.5, 24.6, 29.2, 29.4, 32.6,
3
33.8, 40.5, 41.4, 44.1, 45.2, 56.0, 56.1, 104.2, 107.4, 107.6, 108.3, 110.0, 113.1, 113.8, 126.6,
1
27.6, 128.2, 129.4, 130.9, 134.4, 136.1, 142.7, 143.3, 149.3, 149.7, 149.8, 155.3, 155.4, 194.9,
1
95.5, 208.4, 208.5. Anal. Calcd. for C H NO : C, 75.15; H, 6.77; N, 3.25. Found: C, 75.37; H,
2
7
29
4
6.89; N, 3.21.
6
ꢀ[[3ꢀAcetylꢀ1ꢀ(oꢀtolylmethyl)ꢀ4Hꢀpyridinꢀ4ꢀyl]methyl]ꢀ5,6ꢀ
dihydrocyclopenta[f][1,3]benzodioxolꢀ7ꢀone (1h). The title compound 1h was prepared
according to the general procedure A with compound 2h (100 mg, 0.20 mmol) and BNAH (43
mg, 1 equiv) as reactants. Pale brown solid. (Yield 32.1 mg, 38%). Rf = 0.46/ 0.61 (100%
1
EtOAc). H NMR (300 MHz, CDCl , δ): 1.34ꢀ1.59 (m, 0.54H), 1.86ꢀ2.00 (m, 0.46H), 2.13 (s,
3
1
.6H), 2.18 (s, 1.4H), 2.31 (2s, 3H), 2.64ꢀ2.86 (m, 1.46H), 3.03 (dd, 0.54H, J = 17.1 Hz, J = 3.3
Hz), 3.15ꢀ3.32 (m, 1H), 3.56ꢀ3.65 (m, 0.54H), 3.70ꢀ3.79 (m, 0.46H), 4.43 (s, 2H), 4.96 (dd,
0
.44H, J = 7.5 Hz, J = 5.6 Hz), 5.10 (dd, 0.56H, J = 7.5 Hz, J = 5.1 Hz), 5.91ꢀ5.96 (m, 1H), 6.04
13
(
brs, 2H), 6.79 (s, 0.46H), 6.82 (s, 0.54H), 7.05ꢀ7.28 (m, 6H). C NMR (75 MHz, CDCl , δ):
3
19.2, 19.3, 24.5, 24.6, 29.2, 29.3, 32.8, 34.1, 40.5, 41.1, 44.3, 45.3, 56.0, 102.1, 102.3, 105.7,
1
05.9, 108.2, 109.9, 113.1, 113.8, 126.6, 126.7, 127.6, 127.7, 128.2, 130.9, 131.2, 134.4, 136.1,
+
1
42.7, 143.3, 148.1, 151.8, 151.9, 154.2, 154.3, 194.9, 195.4, 207.5, 207.6. HRMS (ESI ) m/z
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16.1844 [M+H] , calcd for C H NO 416.1862. Anal. Calcd. for C H NO : C, 75.16; H,
26 26 4 26 25 4
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.06; N, 3.37. Found: C, 75.31; H, 6.14; N, 3.41.
ꢀ[[3ꢀAcetylꢀ1ꢀ(oꢀtolylmethyl)ꢀ4Hꢀpyridinꢀ4ꢀyl]methyl]ꢀ5ꢀmethoxyꢀindanꢀ1ꢀone (1i). The title
2
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compound 1i was prepared according to the general procedure A with compound 2i (100 mg,
0.21 mmol) and BNAH (45 mg, 1 equiv) as reactants. Yellow solid. (Yield 34.5 mg, 41%). Rf =
1
0
0
1
3
.52/0.60 (100% EtOAc). H NMR (300 MHz, CDCl , δ): 1.38ꢀ1.59 (m, 0.54H), 1.83ꢀ2.03 (m,
3
.46H), 2.12 (s, 1.6H), 2.17 (s, 1.4H), 2.29 (2s, 3H), 2.61ꢀ2.88 (m, 1.46H), 3.12 (dd, 0.54H, J =
7.4 Hz, J = 3.6 Hz), 3.19ꢀ3.39 (m, 1H), 3.56ꢀ3.66 (m, 0.54H), 3.72ꢀ3.88 (m, 0.46H), 3.86 (s,
H), 4.43 (s, 2H), 4.95 (dd, 0.44H, J = 7.8 Hz, J = 5.7 Hz), 5.10 (dd, 0.56H, J = 7.8 Hz, J = 5.4
1
3
Hz), 5.89ꢀ5.94 (m, 1H), 6.84ꢀ6.88 (m, 2H), 7.07ꢀ7.28 (m, 5H), 7.61ꢀ7.65 (m, 1H). C NMR (75
MHz, CDCl , δ): 19.2, 24.5, 24.6, 29.2, 29.3, 32.9, 34.2, 40.4, 41.2, 44.0, 45.0, 55.6, 56.0, 108.3,
3
1
09.6, 109.7, 109.9, 113.1, 113.8, 115.2, 115.4, 125.4, 126.6, 126.7, 127.6, 127.7, 128.2, 130.0,
30.1, 134.4, 136.0, 142.8, 143.3, 157.4, 157.5, 165.2, 165.3, 194.9, 195.5, 207.8, 207.9. HRMS
1
+
+
(
ESI ) m/z 402.2053 [M+H] , calcd for C H NO 402.2069. Anal. Calcd. for C H NO : C,
26 28 3 26 27 3
77.78; H, 6.78; N, 3.48. Found: C, 77.9; H, 6.85; N, 3.45.
ꢀ[[3ꢀAcetylꢀ1ꢀ(oꢀtolylmethyl)ꢀ4Hꢀpyridinꢀ4ꢀyl]methyl]ꢀ5,6ꢀdimethylꢀindanꢀ1ꢀone (1j). The
2
title compound 1j was prepared according to the general procedure A with compound 2j (62.0
mg, 0.13 mmol) and BNAH (33 mg, 1 equiv) as reactants. Yellow solid. (Yield 20.0 mg, 38%).
1
Rf = 0.7/0.78 (100% EtOAc). H NMR (300 MHz, CDCl , δ): 1.37ꢀ1.61 (m, 0.55H), 1.89ꢀ1.99
3
(m, 0.45H), 2.12 (s, 1.6H), 2.18 (s, 1.4H), 2.25ꢀ2.36 (m, 9H), 2.61ꢀ2.86 (m, 1.45H), 3.05 (dd,
0
.55H, J = 17.1 Hz, J = 3.3 Hz), 3.17ꢀ3.37 (m, 1H), 3.59ꢀ3.66 (m, 0.55H), 3.72ꢀ.379 (m, 0.45H),
.44 (s, 2H), 4.95 (dd, 0.45H, J = 7.5 Hz, J = 5.4 Hz), 5.10 (dd, 0.55H, J = 7.5 Hz, J = 5.1 Hz),
4
1
3
5.89ꢀ5.95 (m, 1H), 7.07 (s, 0.55H), 7.12ꢀ7.45 (m, 5.45H), 7.50 (s, 1H). C NMR (75 MHz,
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CDCl , δ): 19.2, 19.3, 19.8, 20.8, 24.5, 24.6, 29.3, 29.4, 32.4, 33.7, 40.4, 41.0, 44.0, 45.1, 56.0,
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108.2, 109.9, 113.2, 113.9, 124.1, 126.6, 127.3, 127.5, 127.6, 128.2, 130.9, 134.4, 134.9, 136.1,
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42.7, 143.2, 144.7, 144.8, 152.5, 152.6, 194.9, 195.4, 209.5, 209.6. HRMS (ESI ) m/z 400.2263
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[
M+H] , calcd for C H NO 400.2277. Anal. Calcd. for C H NO : C, 81.17; H, 7.32; N, 3.51.
2
7
30
2
27 29
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Found: C, 81.38; H, 7.35; N, 3.54.
ꢀ[[3ꢀAcetylꢀ1ꢀ(oꢀtolylmethyl)ꢀ4Hꢀpyridinꢀ4ꢀyl]methyl]ꢀ5ꢀmethylꢀindanꢀ1ꢀone (1k). The title
compound 1k was prepared according to the general procedure A with compound 2k (150 mg,
2
0.32 mmol) and BNAH (69 mg, 1 equiv) as reactants. Pale yellow solid. (Yield 43.5 mg, 35%).
1
Rf = 0.69/ 0.74 (100% EtOAc). H NMR (300 MHz, CDCl , δ): 1.38ꢀ1.58 (m, 0.56H), 1.89ꢀ1.96
3
(m, 0.44H), 2.12 (s, 1.6H), 2.17 (s, 1.4H), 2.30 (s, 3H), 2.41 (s, 3H), 2.62ꢀ2.71 (m, 0.44H), 2.72ꢀ
2
4
1
2
.88 (m, 1H), 3.05ꢀ3.45 (m, 1.56H), 3.56ꢀ3.66 (m, 0.56H), 3.72ꢀ3.76 (m, 0.44H), 4.44 (s, 2H),
.95 (dd, 0.44H, J = 7.8 Hz, J = 5.7 Hz), 5.10 (dd, 0.56H, J = 7.5 Hz, J = 5.1 Hz), 5.90ꢀ5.94 (m,
1
3
H), 7.07ꢀ7.28 (m, 7H), 7.58ꢀ7.61 (m, 1H). C NMR (75 MHz, CDCl , δ): 19.2, 19.3, 22.1,
3
4.5, 24.6, 29.2, 29.3, 32.7, 34.0, 40.3, 41.0, 44.0, 45.0, 56.0, 108.2, 109.9, 113.2, 113.8, 123.6,
126.6, 126.7, 126.9, 127.1, 127.6, 128.2, 128.5, 130.9, 134.4, 134.5, 136.1, 142.7, 143.3, 145.7,
+
+
1
45.8, 154.9, 155.0, 194.9, 195.4, 209.3, 209.4. HRMS (ESI ) m/z 386.2108 [M+H] , calcd for
C H NO 386.2120. Anal. Calcd. for C H NO : C, 81.01; H, 7.06; N, 3.63. Found: C, 81.22;
26 28
2
26 27
2
H, 7.15; N, 3.62.
ꢀ[[3ꢀAcetylꢀ1ꢀ(oꢀtolylmethyl)ꢀ4Hꢀpyridinꢀ4ꢀyl]methyl]ꢀ5ꢀchloroꢀindanꢀ1ꢀone (1l). The title
2
compound 1l was prepared according to the general procedure A with compound 2l (42 mg, 0.08
mmol) and BNAH (19 mg, 1 equiv) as reactants. Yellow solid. (Yield 17.5 mg, 50%). Rf =
1
0
.60/0.71 (100% EtOAc). H NMR (300 MHz, CDCl , δ): 1.38ꢀ1.59 (m, 1.54H), 1.88ꢀ2.01 (m,
3
1.46H), 2.13 (s, 1.6H), 2.17 (s, 1.4H), 2.29 (s, 3H), 2.64ꢀ2.75 (m, 1.46H), 3.12 (dd, 0.54H, J =
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7.7 Hz, J = 3.9 Hz), 3.20ꢀ3.43 (m, 1H), 3.58ꢀ3.66 (m, 0.54H), 3.71ꢀ3.79 (m, 0.46H), 4.44 (s,
H), 4.94 (dd, 0.44H, J = 7.5 Hz, J = 5.4 Hz), 5.08 (dd, 0.56H, J = 7.8 Hz, J = 5.1 Hz), 5.89ꢀ5.97
2
1
3
(m, 1H), 7.08ꢀ7.28 (m, 5H), 7.41ꢀ7.44 (m, 1H), 7.61ꢀ7.66 (m, 1H). C NMR (75 MHz, CDCl ,
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δ): 19.2, 19.3, 24.5, 24.6, 29.1, 29.2, 29.8, 32.7, 33.9, 40.2, 40.9, 44.1, 45.1, 56.0, 108.1, 113.0,
109.9, 113.7, 124.9, 126.6, 126.7, 126.8, 127.6, 127.0, 127.6, 127.7, 128.0, 128.1, 128.3, 128.4,
130.9, 134.3, 135.2, 136.1, 141.0, 141.2, 142.8, 143.3, 155.8, 155.9, 194.9, 195.5, 208.2, 208.3.
+
+
HRMS (ESI ) m/z 406.1568 [M+H] , calcd for C H ClNO 406.1574. Anal. Calcd. for
25 25
2
C H ClNO : C, 73.97; H, 5.96; N, 3.45. Found: C, 74.21; H, 5.99; N, 3.46.
25
24
2
General procedure B for synthesis of compounds 1q and 1r.To a solution of pyridinium salt
2 (0.25 mmol) in a mixture of dioxane/water (8 mL, 1/1) was added potassium carbonate (1.5
mmol, 6 equiv) and sodium dithionite (1.5 mmol, 6 equiv) at room temperature. The resulting
mixture was stirred at 50°C until completion of the reaction (TLC). After cooling at room
temperature and adding glacial acetic acid to reach pH 6 and phase separation, the aqueous layer
was extracted with EtOAc (3x). The combined organic layers were washed with water and brine,
dried (MgSO ) and concentrated under vacuum. The crude residue was purified by flash
4
chromatography on silica gel to afford compounds 1 as a diastereomeric mixture.
4ꢀ[(5,6ꢀDimethoxyꢀ1ꢀoxoꢀindanꢀ2ꢀyl)methyl]ꢀ1ꢀ(oꢀtolylmethyl)ꢀ4Hꢀpyridineꢀ3ꢀcarboxamide
(1q). The title compound 1q was prepared according to the general procedure B with compound
2q (100.0 mg, 0.196 mmol) as reactant. Pale yellow solid. (Yield 50.0 mg, 59%). Rf = 0.48
1
(
EtOAc/MeOH = 98:2). H NMR (300 MHz, CDCl , δ): 1.65ꢀ1.67 (m, 0.45H), 1.85ꢀ1.96 (m,
3
0.55H), 2.27ꢀ2.28 (m, 3H), 2.63ꢀ2.68 (m, 1.45H), 2.69ꢀ2.72 (m, 0.45H), 2.97ꢀ3.03 (m, 0.55H),
3.23ꢀ3.31 (m, 1.55H), 3.63ꢀ3.65 (m, 0.45H), 3.82ꢀ3.95 (m, 6.45H), 4.37ꢀ4.43 (m, 2H), 4.82 (dd,
0.55H, J = 7.5 Hz, J = 5.7 Hz), 5.00 (dd, 0.45H, J = 7.8 Hz, J = 5.1 Hz), 5.90 (d, 2H, J = 7.8 Hz),
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.84 (s, 1H), 7.12ꢀ7.35 (m, 7H). C NMR (75 MHz, CDCl , δ): 19.3, 29.5, 31.4, 34.2, 34.5,
3
41.8, 42.5, 42.9, 43.8, 55.8, 56.0, 56.2, 56.4, 103.2, 103.6, 104.2, 104.3, 105.9, 106.3, 107.4,
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26.4, 128.0, 128.8, 128.9, 129.1, 129.2, 130.7, 135.0, 136.2, 139.8, 140.3, 149.3, 149.6, 156.0,
0
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0
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0
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0
+
+
1
70.3, 171.0, 209.4, 209.7. HRMS (ESI ) m/z 433.2115 [M+H] , calcd for
C H N O 433.2127. Anal. Calcd. for C H N O : C, 72.20; H, 6.53; N, 6.48. Found: C,
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2
4
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4
7
2.41; H, 6.57; N, 6.51.
1
ꢀ[(3ꢀChlorophenyl)methyl]ꢀ4ꢀ[(5,6ꢀdimethoxyꢀ1ꢀoxoꢀindanꢀ2ꢀyl)methyl]ꢀ4Hꢀpyridineꢀ3ꢀ
carboxamide (1r). The title compound 1r was prepared according to the general procedure B
with compound 2r (265.9 mg, 0.5 mmol). Pale brown solid. (Yield 110.1 mg, 48%). Rf = 0.46
1
(EtOAc/MeOH = 98:2). H NMR (300 MHz, CDCl , δ): 1.61ꢀ1.69 (m, 0.45H), 1.85ꢀ1.93 (m,
3
0
.55H), 2.53ꢀ2.69 (m, 1H), 2.73ꢀ2.85 (m, 0.55H), 2.91ꢀ2.99 (m, 0.45H), 3.19ꢀ3.27 (m, 1H), 3.57ꢀ
3
.63 (m, 0.45H), 3.84ꢀ3.93 (m, 6.55H), 4.33ꢀ4.38 (m, 2H), 4.80 (dd, 0.55H, J = 7.5 Hz, J = 5.7
Hz), 4.98 (dd, 0.45H, J = 7.8 Hz, J = 5.1 Hz), 5.87ꢀ5.92 (m, 1H), 5.99 (br s, 1H), 6.20 (br s, 1H),
13
6
.82 (s, 1H), 7.05ꢀ7.35 (m, 7H). C NMR (75 MHz, CDCl , δ): 29.3, 31.2, 34.2, 34.4, 41.5,
3
42.2, 42.8, 43.7, 56.2, 56.3, 57.1, 57.2, 103.8, 104.2, 104.3, 104.4, 106.3, 106.9, 107.4, 125.3,
1
27.2, 128.1, 128.7, 129.8, 129.0, 129.1, 130.3, 134.8, 139.3, 139.6, 139.9, 149.3, 149.6, 155.8,
+
+
1
56.0, 170.0, 170.7, 209.3, 209.6. HRMS (ESI ) m/z 453.1588 [M+H] , calcd for C H ClN O
25 26 2 4
4
53.1581. Anal. Calcd. for C H ClN O : C, 66.29; H, 5.56; N, 6.18. Found: C, 66.41; H, 5.62;
25 25 2 4
N, 6.20.
General procedure C for synthesis of compounds 2a-r. Compounds 8, 10a-e, 12-15 (0.2
mmol) were dissolved in dichloromethane (2 mL). Benzyl bromide derivative (1.2 to 2.0 equiv)
was then added and the solution was heated under reflux for 12 h in a sealed tube. After
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concentration under reduced pressure, the solid was triturated in diethyl ether, filtered and
washed (3x) with diethyl ether to afford the compounds 2a-r.
(±)ꢀ2ꢀ[(1ꢀBenzylpyridinꢀ1ꢀiumꢀ4ꢀyl)methyl]ꢀ5,6ꢀdimethoxyꢀindanꢀ1ꢀone bromide (2a). The title
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compound 2a was prepared according to the general procedure C with compound 8 (48 mg, 0.17
mmol) and benzyl bromide (41 µL, 0.34 mmol) as reactants. Pale brown solid solid. (Yield 61.3
1
mg, 90%). H NMR (300 MHz, CDCl , δ): 2.75 (dd, 1H, J = 17.1 Hz, J = 3.3 Hz), 3.05ꢀ3.14 (m,
3
2H), 3.30ꢀ3.41 (m, 2H), 3.89 (s, 3H), 3.96 (s, 3H), 6.17 (s, 2H), 6.87 (s, 1H), 7.11 (s, 1H), 7.40ꢀ
1
3
7
.42 (m, 3H), 7.61ꢀ7.64 (m, 2H), 7.92 (d, 2H, J = 6.6 Hz), 9.23 (d, 1H, J = 6.6 Hz). C NMR (75
MHz, CDCl , δ): 32.2, 37.2, 47.1, 56.2, 56.6, 63.8, 104.3, 107.7, 128.3, 128.7, 129.8, 129.9,
3
130.3, 132.3, 144.0, 148.5, 149.9, 156.3, 161.2, 204.2. Anal. Calcd. for C H BrNO : C, 63.44;
2
4
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H, 5.32; N, 3.08. Found: C, 63.39; H, 5.24; N, 3.15.
±)ꢀ1ꢀBenzylꢀ4ꢀ[(5,6ꢀdimethoxyꢀ1ꢀoxoꢀindanꢀ2ꢀyl)methyl]pyridinꢀ1ꢀiumꢀ3ꢀcarbonitrile
(
bromide (2b). The title compound 2b was prepared according to the general procedure C with
compound 15 (100.0 mg, 0.32 mmol) and benzyl bromide (66 µL, 0.55 mmol). Pale yellow
1
solid. (Yield 74.2 mg, 95%). H NMR (300 MHz, DMSOꢀd , δ): 2.81ꢀ2.85 (m, 1H), 3.23ꢀ3.25
6
(m, 3H), 3.42ꢀ3.48 (m, 1H), 3.8 (s, 3H), 3.87 (s, 3H), 5.87 (s, 2H), 7.09 (s, 1H), 7.13z (s, 1H),
7
1
1
.46ꢀ7.48 (m, 3H), 7.60ꢀ7.62 (m, 2H), 8.41 (d, 1H, J = 6.5Hz), 9.38 (d, 1H, J = 6.5Hz), 9.98 (s,
1
3
H). C NMR (75 MHz, DMSOꢀd , δ): 31.7, 35.5, 46.2, 55.7, 56.1, 63.4, 104.1, 108.2, 113.1,
6
14.2, 127.6, 129.0, 129.1, 129.3, 129.6, 133.62, 146.8, 148.6, 149.4, 155.8, 163.8, 203.6.
+
+
HRMS (ESI ) m/z 399.1707 [M] , calcd for C H N O 399.1709. Anal. Calcd. for
2
5
23
2
3
C H BrN O : C, 62.64; H, 4.84; N, 5.84. Found: C, 62.71; H, 4.85; N, 5.87.
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3
(±)ꢀ2ꢀ[(3ꢀAcetylꢀ1ꢀbenzylꢀpyridinꢀ1ꢀiumꢀ4ꢀyl)methyl]ꢀ5,6ꢀdimethoxyꢀindanꢀ1ꢀone bromide
(2c). The title compound 2c was prepared according to the general procedure C with compound
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