Rational design of central selective acetylcholinesterase inhibitors by means of a "bio-oxidisable prodrug" strategy

Article abstract of DOI:10.1039/b903041g

This work deals with the design of a "bio-oxidisable prodrug" strategy for the development of new central selective acetylcholinesterase inhibitors. This prodrug approach is expected to reduce peripheral anticholinesterase activity responsible for various

Full text of DOI:10.1039/b903041g

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Rational design of central selective acetylcholinesterase inhibitors by means
of a “bio-oxidisable prodrug” strategy†
Pierre Bohn,a,b Nicolas Le Fur,a Guillaume Hagues,b Jean Costentin,b Nicolas Torquet,b Cyril Papamicae¨l,a
Francis Marsaisa and Vincent Levacher*a
Received 13th February 2009, Accepted 8th April 2009
First published as an Advance Article on the web 18th May 2009
DOI: 10.1039/b903041g
This work deals with the design of a “bio-oxidisable prodrug” strategy for the development of new
central selective acetylcholinesterase inhibitors. This prodrug approach is expected to reduce peripheral
anticholinesterase activity responsible for various side effects observed with presently marketed AChE
inhibitors. The design of these new AChE inhibitors in quinoline series is roughly based on cyclic
analogues of rivastigmine. The key activation step of the prodrug involves an oxidation of an
N-alkyl-1,4-dihydroquinoline 1 to the corresponding quinolinium salt 2 unmasking the positive charge
required for binding to the catalytic anionic site of the enzyme. The synthesis of a set of
1,4-dihydroquinolines 1 and their corresponding quinolinium salts 2 is presented. An in vitro biological
evaluation revealed that while all reduced forms 1 were unable to exhibit any anticholinesterase activity
(IC50 > 106 nM), most of the quinolinium salts 2 displayed high AChE inhibitory activity (IC50 ranging
from 6 mM to 7 nM). These preliminary in vitro assays validate the use of these cyclic analogues of
rivastigmine in quinoline series as appealing chemical tools for further in vivo development of this
“bio-oxidisable prodrug” approach.
the sole clinically effective method for a palliative treatment of mild
to moderate AD.4
Introduction
Over the past decade, several AChE inhibitors such as tacrine,5
donepezil,6 rivastigmine7 and more recently galanthamine8 have
been launched on the market. Although all AChE inhibitors
employed in AD therapy exhibit a preferential action in the central
nervous system (CNS), the manifestation of peripheral activity
in the course of the treatment causes serious adverse effects on
peripheral organs and seriously limits the therapeutic potential of
these cholinesterase inhibitors. Therefore, the design of central
selective AChE inhibitors free from adverse peripheral effects
remains a major challenging therapeutic goal.
The strategy disclosed in this work brings into play a “bio-
oxidisable prodrug” which, after having crossed the blood-brain
barrier (BBB), is expected to be converted to the parent drug in the
CNS, via a redox-activation process.9 Our aim is to report herein
the design, synthesis and preliminary in vitro biological evaluation
of these new potential central selective AChE inhibitors.
Alzheimer’s disease (AD), the most common form of dementia
affecting elderly people, is a complex irreversible neurological
affection clinically characterized by a progressive loss of memory
and impairment of cognitive functions. While 1% to 3% of the
population aged under 65 years are affected, more than 30% of
people over 85 years of age are affected by this neurodegenerative
disorder. Given the ageing of the population, AD is becoming
one of the most important healthcare problems in developed
countries.1 Although the origin of AD is still under debate,
amyloid plaques and neurofibrillary tangles observed in post-
mortem brain studies in neurocortex and hippocampus regions are
held responsible for the devastating clinical effects of the disease.2
Another important and significant aspect of neurodegeneration
in the brain of AD patients is the loss of the basal forebrain cholin-
ergic system, thought to play a crucial role in producing cognitive
impairments and memory deficiency. Therefore, enhancement of
the central cholinergic transmission has been considered as a
promising approach for the symptomatic treatment of AD. Among
the diverse strategies explored,3 acetylcholinesterase (AChE) in-
hibitors have been extensively studied. This approach has provided
Results and discussion
“Bio-oxidisable prodrug” design of cyclic rivastigmine analogues
aLaboratoire de Chimie Organique Fine et He´te´rocyclique, UMR 6014,
IRCOF, CNRS, Universite´ et INSA de Rouen, B.P. 08, F-76131, Mont-
Saint-Aignan Cedex, France. E-mail: vincent.levacher@insa-rouen.fr;
Fax: +33(0)235522962
The design of this “bio-oxidisable prodrug” approach is closely
connected with the action mechanism of AChE inhibitors.
Whereas donepezil and galanthamine are competitive inhibitors,
rivastigmine displays a pseudo-irreversible inhibition involving
the carbamylation of the serine hydroxyl group located at the
“esterasic site” of the enzyme. Both classes of these inhibitors
share in common a tertiary amine which is known to play a central
role in the mechanism of AChE inhibition. At physiological pH,
the protonation of this amine results in the formation of a positive
bLaboratoire de Neuropharmacologie Expe´rimentale associe´ au CNRS
(FRE-2735). Faculte´ de Me´decine et de pharmacie, Universite´ de Rouen,
F-76000, France
† Electronic supplementary information (ESI) available: Analytical data
and purification conditions of 11a–g, 1a–g, 2a–g, 1H and 13C NMR spectra
of 3, 4, 6–9, 10b–f, 11a–f, 1a–d,f, 2a–f, preparation of AChE and detailed
procedure for AChE inhibition assay. See DOI: 10.1039/b903041g
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charge which binds to the “catalytic anionic site” of the enzyme
(Fig. 1). This binding element, although not sufficient, is essential
in the design of these inhibitors. It is also conceptually important
to note that while the charged bioactive form cannot cross the
BBB by passive diffusion, both central and peripheral cholinergic
activities commonly observed with these inhibitors stem from the
existence of an acid–base equilibrium by permitting the neutral
inactive form to cross this physiological barrier.
“locked-in” into the brain is expected to display a central selective
AChE inhibitory activity, thus exerting ameliorating effects on
cholinergic deficits without showing excessive peripheral effects.
This ideal scenario may be compromised if peripheral oxidation of
the 1,4-dihydroquinoline 1 takes place. Indeed, redox activation of
the prodrug 1 closely related to the conversion of the NAD(P)H ꢀ
NAD(P)+ coenzyme system may arise easily everywhere in the
body. However, due to its high hydrophilicity, the resulting
quinolinium salt 2 which might be formed prematurely in the
periphery is expected to be easily eliminated from the peripheral
circulation by the kidneys and bile. The presence of an electron
withdrawing group (EWG) at C-3 is essential to the stabilization
of the enamine function in prodrug 1. This stabilizing element may
be helpful to find the delicate balance between stabilization of the
prodrug 1 at the periphery and its activation in the CNS. Although
1,4-dihydropyridine-type compounds have been widely developed
by Bodor and Prokai for targeting neuroactive compounds to the
brain,11 the use of this redox chemical delivery system to design
new prodrug strategies has remained almost unexplored (Fig. 2).12
Preparation of 1,4-dihydroquinolines 1a–g and quinolinium
salts 2a–g
Fig. 1 AChE inhibitors clinically used for the symptomatic treatment of
AD. (A) Schematic representation of the active site of AChE showing the
“anionic” and “esterasic” sites of the enzyme.
These cyclic rivastigmine analogues can be described as consisting
of a quinoline scaffold incorporating a carbamate moiety and an
electron withdrawing group at C-3. To optimize both interactions
at “esterasic” and “anionic” sites of the enzyme, we first investi-
gated the relative position between the carbamate and positive
charge of the quinolinium salt 2 by installing the carbamate
successively at C-5 and C-7 positions of the quinoline ring. The
preparation of both 5- and 7-hydroxyquinoline intermediates 3
and 4 is depicted in Scheme 1. The requisite 5-hydroxyquinoline
3 was obtained in a three-step sequence from N-Boc-3-methoxy
aniline in a 65% overall yield. The reaction sequence started
with the ortho-lithiation of N-Boc-3-methoxy aniline under the
reaction conditions previously reported by Woggon.13 Trapping
the resulting lithiated species at -78 ◦C with DMF afforded the 2-
aminobenzaldehyde 5 in 90% isolated yield. The latter compound
was reacted with methyl trans-3-methoxyacrylate, according to a
modified Friedla¨nder reaction, to yield the desired quinoline 6
in 80% yield. Both O-demethylation and ester hydrolysis steps
were achieved in a one-pot procedure in refluxing aqueous HBr,
furnishing quinoline 3 in 80% yield. Using the above conditions,
7-hydroxyquinoline 4 was straightforwardly prepared in 73% yield
in a single step from the known 3-cyano-7-methoxyquinoline.14
On the basis of these mechanistic considerations, a prodrug ap-
proach was envisaged to design central selective AChE inhibitors
by temporarily masking the positive charge at the periphery. To
reach this goal, we focused our attention on cyclic rivastigmine
analogues 1 as appealing prodrug candidates (Fig. 2). Although
1,4-dihydroquinoline carbamates 1 are structurally closely related
to rivastigmine, these analogues mainly differ in that they possess
a non-protonable enamine nitrogen atom at physiological pH and
consequently cannot bind to the “anionic site” of the enzyme.
After gaining access to the brain through the BBB by passive
diffusion, oxidation of the 1,4-dihydroquinoline 1 would generate
the corresponding quaternary quinolinium salt 2,10 allowing the
latter to interact with the “anionic site” by unmasking the positive
charge. The resulting permanently charged quinolinium salt 2 can
no longer re-cross the BBB, in contrast to most other previously
reported AChE inhibitors for which both neutral and charged
forms are in equilibrium at physiological pH. This ionic species
Scheme 1 Preparation of 5- and 7-hydroxyquinolines 3 and 4. Reagents
and conditions: (a) t-BuLi/Et2O/-10 ◦C/3h then DMF/-78 ◦C to rt
over 18h; (b) methyl trans-methoxyacrylate/HCl 3M/MeOH/reflux/3h;
(c) aqueous HBr (48%)/reflux/24h for 3, 12h for 4.
Fig. 2 Design of a “bio-oxidisable prodrug” 1 for central selective AChE
inhibitory activity (EWG = electron withdrawing group).
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Table 1 Functionalization of the quinoline scaffold: carbamylation,a
The functionalization of this quinoline scaffold was then
investigated (Scheme 2). We first undertook to install various
electron withdrawing groups at C-3 to find a good compromise
between stability of the prodrugs 1 and AChE inhibitory activity of
the related quinolinium salts 2. To this end, quinoline esters 10a–c
were readily prepared in 60–70% yields by esterification of the cor-
responding quinoline carboxylic acids 3 and 4. Quinoline amide
10f was easily obtained in 60% yield, according to a literature
procedure,15 by reacting ester 10c with morpholine hydrochloride
in the presence of AlMe3. Attempts to use this former one-
step procedure during the preparation of quinoline amides 10d,e
failed. To thwart this failure, a protection/deprotection sequence
of the phenol function of 3 was therefore investigated. The so-
prepared O-benzylated quinoline 7 was subsequently converted
into quinoline amides 8 and 9 in 75 and 80% yields respectively
by conventional activation methods of the carboxylic acid with
thionyl chloride or oxalyl chloride. The O-debenzylation step,
conducted under hydrogen atmosphere, was accompanied by a
partial reduction of the quinoline ring which could be easily re-
oxidized by means of air bubbling to finally afford quinolines 10d
and 10e in 95 and 74% yields respectively.
quaternizationb and reductionc steps
Entry
Carbamylationa
Quaternizationb
Reductionc
1
2
3
4
5
6
7
11a: 90%
11b: 78%
11c: 85%
11d: 65%
11e: 55%
11f: 18%
11g: 94%
2a: >95%
2b: >95%
2c: >95%
2d: >95%
2e: >95%
2f: >95%
2g: >95%
1a: 47%
1b: 46%
1c: 47% (67%)d
1d: 53%
1e: 60%
1f: 46%
1g: 62%
a Ethyl isocyanate or dimethylcarbamoyl chloride/THF/NaH/12h/
reflux. b TfOMe/CH2Cl2/rt/2h. c Na2S2O4/Na2CO3/CH2Cl2/H2O/rt/2h.
d N-Benzyl-1,4-dihydronicotinamide/CH2Cl2/rt/1h.
1,4-dihydroquinolines 1 have to be reactive enough to undergo an
in vivo oxidation, these prodrug candidates should also display
a satisfactory chemical stability for further in vivo development.
From this point of view, we were pleased to notice that all 1,4-
dihydroquinolines 1a–g could be handled without any special cau-
tion and stored for several weeks without significant degradation.
In vitro biological evaluation (AChE inhibitory activity)
Scheme 2 Functionalization of the quinoline scaffold: screening of
various electron withdrawing groups (EWGs) at C-3. Reagents and con-
ditions: (a) EtOH/SOCl2/reflux/12h; (b) 4/MeOH/H2SO4/reflux/24h;
(c) morpholine hydrochloride/AlMe3/toluene/rt/3h; (d) 3/BnBr/
K2CO3/DMF/65 ◦C/36h; (e) SOCl2/reflux/1h then CH2Cl2/Me2NH/
reflux/12h; (f) (COCl)2/few drops of DMF/CH2Cl2/1h/rt then
CH2Cl2/methylamine (2M in THF)/reflux 12h; (g) EtOH/5% Pd/C/1atm
of H2/rt/3h then bubbling of air through the solution.
AChE inhibitory activity of these newly designed compounds was
determined by Ellman’s colorimetric assay18 on human AChE
using donepezil as a reference compound. Whereas quinolinium
salt 2a bearing the carbamate moiety at C-7 revealed a moderate
AChE inhibitory activity (IC50 = 0.5 mM, Table 2 entry 2),
quinolinium salt 2b proved to be much more potent (IC50 = 7 nM,
Table 2 entry 3), designating C-5 as an appropriate attachment
position to reach optimal inhibition. For comparison, donepezil,
the most potent marketed AChE inhibitor, showed an IC50 value
7 times higher than that obtained for compound 2b (Table 2 entry
1). Surprisingly, quinolinium salt 2c is about 15-fold less potent
than 2b (Table 2 entries 3, 4), indicating that inhibitory activity is
noticeably affected by slight modification of the ester group at C-3.
We then focused our attention on the influence of the stabilizing
group at C-3 on the AChE inhibitory activity, by replacing ethyl
ester by different amides. With the exception of quinolinium salt
2f, which exhibited a rather modest inhibition (IC50 = 0.86 mM,
Table 2 entry 7), both quinolinium salts 2d,e showed high AChE
inhibitory activity (IC50 = 29 nM and 15 nM, Entries 5, 6). Lastly,
the nature of the carbamate was briefly investigated by replacing
N,N -dimethylcarbamate by N-ethylcarbamate. It turned out that
The last steps include the carbamylation of quinolines 10a–
f, their quaternization and the regioselective reduction of the
corresponding quinoliniun salts 2 into 1,4-dihydroquinolines 1
(Table 1). The carbamylation of quinolines 10a–f occurred with
fair to good yields, with only the exception of quinoline 10f which
afforded 11f in only 18% isolated yield (Table 1, entry 6). In all
cases, quinolinium salts 2a–g were obtained in almost quantitative
yields under mild conditions using methyl trifluorosulfonate
as methylating agent. The desired 1,4-dihydroquinolines 1a–g
were obtained in moderate yields (46–62%) by regioselective
reduction of 2a–g in the presence of sodium dithionite under basic
conditions.16 Alternatively, quinolinium salt 2c could be regiose-
lectively reduced by means of N-benzyl-1,4-dihydronicotinamide
(BNAH) in somewhat better yield (Table 1, entry 3).17 Although
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Table 2 In vitro inhibition of AChE by 1,4-dihydroquinoline 1a–g and
quinolinium salts 2a–g
Experimental
General methods
Melting points (◦C) were determined with a WME Ko¨fler
apparatus. IR spectra were recorded on a PERKIN ELMER
IRFT 1650 spectrometer. Liquids were applied as a film be-
tween KBr windows and solids were dispersed in KBr tablets.
Absorption bands are given in cm-1. NMR spectra were recorded
Entry Carbamate position R1
1
R2
EWG
IC50 (nM)
1
on a BRUKER AVANCE-300 (300 MHz) spectrometer: H at
300 MHz, 13C at 75 MHz and 19F at 282.5 MHz using CDCl3
or DMSO-d6 as solvents and with the residual solvent signals
as internal standards unless otherwise indicated. The following
abbreviations are used to describe peak patterns: s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).
Elemental analyses were carried out at the University of Rouen
(Microanalytical Service Laboratory) on a CARLO ERBA 1160.
Measurement accuracy is around 0.4% on carbon. Mass spectra
(EI, CI, FAB) were recorded on aJEOL JMS AX-500. Analytical
thin layer chromatography (TLC) was performed on silica gel
plates (Kieselgel 60F254) from Merck laboratory or on alumina
gel plates [DC-Alufolien Aluminiumoxid 150F254 neutral (Typ
T)] from Merck laboratory. Flash chromatography on silica gel
was carried out using silica (70–230 mesh ASTM) from Merck
laboratory. Flash chromatography on alumina was carried out
with aluminium oxide, activated, neutral, 50–200 micron from
Acros Organics. Anhydrous dioxane, THF and Et2O were distilled
from sodium-benzophenone ketyl. Toluene, Et3N, DMF, CH2Cl2
and CH3CN were distilled over CaH2 before use.
Donepezil: 50
1a: >106
2a: 500
2
3
4
5
6
7
7
5
5
5
5
5
Me Me COOEt
Me Me COOEt
Me Me COOMe
1b: >106
2b: 7
1c: >106
2c: 110
Me Me CONHMe 1d: >106
2d: 29
Me Me CONMe2
1e: >106
2e: 15
Me Me
1f: >106
2f: 860
8
5
Et
H
COOMe
1g: >106
2g: 6000
the resulting quinoliniun salt 2g is 60-fold less potent than 2c
(Table 2 entry 8).
More importantly, whereas a large number of quinolium salts 2
displayed modest to excellent AChE inhibitory activities (IC50
=
5-Hydroxyquinoline-3-carboxylic acid (3). A solution of 6
(7.3 g, 33.6 mmol) in an aqueous solution of HBr (150 mL, 48%)
was heated under reflux for 24 hours. After cooling the reaction
mixture to room temperature, the pH was adjusted to 2 with 2M
aqueous NaOH. After filtration of the insoluble matter, the pH
was adjusted between 5 and 6, and the resulting precipitate was
filtered off and dried at 70 ◦C to afford 3 (5.25 g, 80%) as a brown
solid. Mp >260 ◦C. 1H NMR (DMSO-d6) d 10.91 (s, 1H), 9.25 (s,
1H), 9.04 (s, 1H), 7.70 (dd, J = 7.9 Hz and 8.3 Hz, 1H), 7.53 (d,
J = 8.5 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H). 13C NMR (DMSO-d6)
d 166.7 (C), 154.8 (C), 150.3 (C), 150.1 (CH), 133.5 (CH), 133.1
(CH), 122.4 (C), 119.3 (CH), 118.5 (C), 109.8 (CH). Found: C,
63.2; H, 3.85; N, 7.4. Calc. for C10H7NO3: C, 63.49; H, 3.73; N,
7.40%.
6 mM–7 nM), all corresponding reduced forms 1a–g proved to be
completely inactive up to the limit of solubility (IC50 >106 nM,
Table 2 entries 2–8). These preliminary in vitro assays strongly
support our working hypothesis that while the positive charge in
quinolinium salts 2 gives rise to a critical binding element at the
“anionic site” of the enzyme, this positive charge is masked in the
reduced forms 1 thanks to the low basicity of the enamine nitrogen
atom which remains non-protonated at physiological pH. With
such acetylcholinesterase inhibitors 2 and their corresponding
inactive reduced forms 1 in hand, we have at our disposal
promising chemical tools for further in vivo biological evaluation
of this “bio-oxidisable prodrug” strategy.
Conclusion
7-Hydroxy 3-quinolinecarboxylic acid (4). A solution of 3-
cyano-7-methoxyquinoline15 (20 g, 110 mmol) in an aqueous so-
lution of HBr (300 mL, 48%) was stirred under reflux for 12 hours.
The reaction mixture was then cooled to room temperature and
neutralized by adding an aqueous solution of KOH (20%). The
resulting precipitate was filtered and dried under vacuum to afford
4 (15.2 g, 73%) as a brown powder. Mp >260 ◦C. 1H NMR
(DMSO-d6) d 10.68 (s, 1 H), 9.20 (d, J = 2 Hz, 1 H), 8.84 (d, J =
2 Hz, 1 H), 8.06 (d, J = 9 Hz, 1 H), 7.34 (s, 1 H), 7.29 (d, J = 9 Hz,
1 H). HRMS calc. for (M+) C10H7NO3: 189.0426. Found: 189.0423.
A series of new carbamylated quinolinium salts 2, structurally
related to cyclic analogues of rivastigmine, exhibited high AChE
inhibitory activity with IC50 values in the nanomolar range.
Interestingly, the corresponding reduced forms 1 proved to be
inactive in inhibiting AChE. These results pave the way for the
development of a novel “bio-oxidisable prodrug” by means of
such redox systems with the aim to reduce undesirable peripheral
activity reported with marketed AChE inhibitors. At this stage,
the key to success in this “bio-oxidisable prodrug” strategy lies
in the preferential redox activation of prodrug 1 in the CNS,
versus periphery. An in vivo study of this critical activation step
will be reported in due course by investigating the central and
peripheral cholinergic activity profiles in mice after administering
the prodrug.
N-(2-Formyl-3-methoxyphenyl)-2,2-dimethylpropanamide (5)14.
To a solution of N-(3-methoxyphenyl)-2,2-dimethylpropanamide
(1 g, 4.48 mmol) in Et2O (20 mL) was added ◦dropwise t-BuLi
(7.04 mL, 9.86 mmol, 1.4 M in hexane) at -15 C. This mixture
was stirred for 3h at -10 ◦C. A solution of DMF in Et2O (5 mL)
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was added dropwise at -78 ◦C. The resulting mixture was stirred
overnight at room temperature. The reaction was quenched by
addition of a saturated aqueous solution of NH4Cl (20 mL). After
phase separation, the aqueous layer was extracted with CH2Cl2,
and the combined organic layers were washed with saturated
aqueous NaCl and dried (MgSO4). Flash chromatography (eluent:
CH2Cl2) provided pure compound 5 (1.0 g, 90%) as a pale yellow
solid. 1H NMR (CDCl3) d 10.88 (s, 1H), 10.35 (s, 1H), 8.00 (d, J =
7.9 Hz, 1H), 7.43 (t, J = 7.9 Hz, 1H), 6.44 (d, J = 7.9 Hz, 1H), 3.83
(s, 3H), 1.44 (s, 9H). IR nmax (KBr): 3265, 1728, 1403, 1148 cm-1.
HRMS calc. for (M+) C13H17NO4: 251.1158. Found: 251.1162.
0 ◦C under N2 atmosphere. The reaction mixture was then heated
under reflux overnight and evaporated. Flash chromatography on
silica gel of the residue (eluent: CH2Cl2 then CH2Cl2/i-PrOH: 9/1)
provided pure compound 8 (412 mg, 75%) as a pale yellow solid.
1H NMR (CDCl3) d 8.95 (s, 1H), 8.72 (s, 1H), 7.60–7.75 (m, 2H),
7.30–7.55 (m, 5H), 6.97 (d, J = 7.5 Hz, 1H), 5.23 (s, 2H), 3.16
(s, 3H), 3.04 (s, 3H). Found: C, 74.45; H, 5.9; N, 9.0. Calc. for
C19H18N2O2: C, 74.49; H, 5.92; N, 9.14%.
5-(Benzyloxy)-3-(N-methylcarboxamido)quinoline (9). To
a
suspension of compound 7 (1 g, 3.6 mmol) in dry CH2Cl2 (50 mL)
were added 3 drops of dry DMF. The solution was stirred under N2
at 0 ◦C for a few minutes before adding dropwise oxalyl chloride
(2.2 mL, 25.2 mmol). The reaction mixture was stirred for 1 hour
at room temperature and then evaporated under reduced pressure.
The residue was dissolved in dry CH2Cl2 (30 mL) and this solution
was added dropwise to a solution of methylamine (9 mL, 18 mmol,
2M in THF) in dry CH2Cl2 (50 mL) pre-cooled to 0 ◦C. The
reaction mixture was then heated under reflux overnight. After
cooling to room temperature, water (50 mL) was added. After
phase separation, the aqueous layer was extracted with CH2Cl2
(3 ¥ 50 mL). The combined organic layers were dried (MgSO4)
and filtered. The solvent was removed under reduced pressure and
the residue was purified by chromatography on silica gel (eluent:
EtOAc) to afford compound 9 (849 mg, 80%) as an orange solid.
Mp 60 ◦C (degrad.) 1H NMR (CDCl3) d 9.22 (d, J = 2.1 Hz, 1H),
8.90 (d, J = 2.1 Hz, 1H), 7.50–7.67 (m, 2H), 7.26–7.45 (m, 5H),
7.16 (br, 1H), 6.85 (d, J = 7.2 Hz, 1H), 5.13 (s, 2H), 2.97 (d, J =
4.7 Hz, 3H). 13C NMR (CDCl3) d 166.7 (C), 154.7 (C), 149.8 (C),
149.0 (CH), 136.1 (C), 131.3 (CH), 130.1 (CH), 128.7 (CH), 128.3
(CH), 127.6 (CH), 126.3 (C), 121.3 (CH), 119.5 (C), 106.3 (CH),
70.6 (CH2), 26.9 (CH3). IR nmax/cm-1 (KBr) 1640, 1265, 1091, 813.
Found: C, 73.8; H, 5.5; N, 9.45. Calc for C18H16N2O2: C, 73.95; H,
5.52; N, 9.58%.
Methyl 5-methoxyquinoline-3-carboxylate (6). To a solution of
5 (7.8 g, 31 mmol) and methyl trans-3-methoxyacrylate (7.4 mL,
68.4 mmol) dissolved in 150 mL of methanol was slowly added
3M aqueous hydrochloric acid (100 mL). The resulting mixture
was stirred under reflux for 3 hours. The reaction mixture was
then cooled to room temperature and neutralized by adding
Na2CO3. The aqueous solution was extracted with CH2Cl2 (4 ¥
150 mL). The combined organic layers were dried (MgSO4),
filtered and evaporated under vacuum. The crude product was
then filtered on silica gel (EtOAc:cyclohexane, 50:50) to afford
6 (5.6 g, 80%) as a yellow powder. Mp 102 ◦C. 1H NMR
(CDCl3) d 9.38 (s, 1H), 9.18 (s, 1H), 7.68 (m, 2H), 6.85 (m,
1H), 3.99 (s, 3H), 3.98 (s, 3H). 13C NMR (CDCl3) d 166.1
(C), 156.1 (C), 150.6 (C), 150.5 (CH), 133.9 (CH), 132.3 (CH),
122.0 (C), 121.4 (CH), 119.5 (C), 105.1 (CH), 55.9 (CH3), 52.5
(CH3). IR nmax/cm-1 (KBr) 1728, 1281, 1110, 815. Found: C,
66.2; H, 5.05; N, 6.4. Calc. for C12H11NO3: C, 66.35; H, 5.10;
N, 6.45%.
5-(Benzyloxy)quinoline-3-carboxylic acid (7). To a solution of
compound 3 (0.1 g, 0.53 mmol) in dry DMF (5 mL) was added
benzyl bromide (132 mL, 1.11 mmol) and finely powdered K2CO3
◦
(183 mg, 1.3 mmol). This mixture was stirred at 65 C under N2
for 36 hours. The reaction was worked up by pouring the solution
into water (10 mL) and EtOAc (10 mL). After phase separation,
the aqueous layer was extracted with EtOAc (3 ¥ 10 mL). The
organic layers were combined, washed with water and brine, and
dried (MgSO4). Filtration and evaporation under vacuum gave
0.14 g of a viscous brown oil which was treated with ethanolic
KOH (150 mg, 2.66 mmol in 10 mL of ethanol) and heated under
reflux for 3 hours. After evaporation of ethanol, the product was
dissolved in 5 mL of water and washed with diethyl ether (2 ¥
10 mL). The aqueous layer was neutralized with 3 N aqueous HCl.
The acid was filtered and dried under vacuum to afford compound
7 (93 mg, 63%) as a brown powder. 1H NMR (DMSO-d6) d 9.30
(s, 1H), 9.04 (s, 1H), 7.83 (dd, J = 8.5 and 7.9 Hz, 1H), 7.68 (d,
J = 8.5 Hz, 1H), 7.56 (d, J = 7.0 Hz, 2H), 7.35–7.48 (m, 3H),
7.27 (d, J = 7.7 Hz, 1H), 5.37 (s, 2H). 13C NMR (DMSO-d6) d
166.8 (C), 154.5 (C), 150.6 (CH), 149.7 (C), 136.6 (C), 132.0 (CH),
131.9 (CH), 128.7 (CH), 128.2 (CH), 127.9 (CH), 124.7 (C), 121.0
(CH), 119.0 (C), 107.0 (CH), 70.1 (CH2). HRMS calc. for (M+)
C17H13NO3: 279.0895. Found: 279.0889.
Ethyl 7-hydroxyquinoline-3-carboxylate (10a). To a solution of
compound 4 (15 g, 79 mmol) in EtOH (500 ml) was added dropwise
SOCl2 (40 mL). The resulting mixture was stirred under reflux for
12 hours. After adding water (200 mL), the pH was adjusted to
7 with 20% aqueous Na2CO3. The mixture was extracted with
CH2Cl2 (3 ¥ 200 mL). The combined organic layers were dried
(MgSO4), filtered and evaporated to afford compound 10a (11 g,
◦
1
64%) as a pale brown powder. Mp 182 C. H NMR (CDCl3) d
9.24 (d, J = 2 Hz, 1H), 8.70 (d, J = 2 Hz, 1H), 7.66 (d, J = 9 Hz,
1H), 7.36 (s, 1H), 7.02 (dd, J = 9 and 2 Hz, 1H), 4.48 (q, J = 7 Hz,
2 H), 1.48 (t, J = 7 Hz, 3H). HRMS calc. for (M+) C12H11NO3 :
+
217.0739. Found: 217.0728.
Ethyl 5-hydroxyquinoline-3-carboxylate (10b). To a solution of
compound 3 (15 g, 79 mmol) in EtOH (500 ml) was added dropwise
SOCl2 (40 mL). The resulting mixture was stirred under reflux for
12 hours. After adding water (200 mL), the pH was adjusted to
7 with 20% aqueous Na2CO3. The mixture was extracted with
CH2Cl2 (3 ¥ 200 mL). The combined organic layers were dried
(MgSO4), filtered and evaporated to afford compound 10b (10.5 g,
60%) as a yellow solid. Mp 240 ◦C (degrad.) 1H NMR (DMSO-d6)
11.01 (s, 1H), 9.26 (d, J = 2.3 Hz, 1H), 9.06 (d, J = 1.9 Hz, 1H),
7.73 (dd, J = 8.1 and 8.1 Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.04
(d, J = 7.7 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 1.38 (t, J = 7.2 Hz,
5-(Benzyloxy)-3-(N,N-dimethylcarboxamido)quinoline (8).
A
solution of compound 7 (0.5 g, 1.79 mmol) in thionyl chloride
(15 mL) was heated under reflux for 1h. After evaporation of
thionyl chloride, the residue was dissolved in dry CH2Cl2 (15 mL)
and dimethylamine (4.5 mL, 8.96 mmol, 2M in THF) was added at
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3H). IR nmax/cm-1 (KBr) 1713, 1377, 1258, 1111. Found: C, 66.05;
H, 5.5; N, 6.5. Calc. for C12H11NO3: C, 66.35; H, 5.10; N, 6.45%.
(270 mL, 3.2 mmol). The reaction mixture was stirred at room
temperature for 3h. The reaction was quenched with a saturated
aqueous solution of NH4Cl, washed with water, and the aqueous
solution was extracted with CH2Cl2 (2 ¥ 200 mL). The combined
organic layers were dried over MgSO4, filtered and evaporated.
The residue was washed with heptane to provide pure compound
Methyl 5-hydroxyquinoline-3-carboxylate (10c). To a suspen-
sion of 3 (3.5 g, 17.3 mmol) in methanol (250 mL) was added
concentrated H2SO4 (1.5 mL). The reaction mixture was heated
under reflux for 24 hours and then evaporated to half volume
before adding 100 mL of water. After neutralization with 2M
aqueous NaOH, the mixture was extracted with EtOAc (4 ¥
200 mL). The combined organic layers were washed with brine
(100 mL), dried (MgSO4), filtered and evaporated under vacuum.
Flash chromatography on silica gel (eluent: CH2Cl2:EtOAc 50:50)
provided 10c (2.54 g, 70%) as a yellow powder. Mp: 200 ◦C
(degrad.) 1H NMR (DMSO-d6) d 11.04 (s, 1H), 9.24 (s, 1H), 9.05
(s, 1H), 7.71 (dd, J = 8.1 and 8.1 Hz, 1H), 7.53 (d, J = 8.3 Hz,
1H), 7.02 (d, J = 7.7 Hz, 1H), 3.93 (s, 3H). 13C NMR (DMSO-d6)
d 165.3 (C), 154.6 (C), 150.2 (C), 149.3 (CH), 133.1 (CH), 133.0
(CH), 121.0 (C), 119.1 (CH), 118.1 (C), 109.6 (CH), 52.4 (CH3).
IR nmax/cm-1 (KBr) 1721, 1277, 1115. Found: C, 65.12; H, 4.55;
N, 6.85. Calc. for C11H9NO3: C, 65.02; H, 4.46; N, 6.89%.
1
10f (240 mg, 60%) as a brown powder. H NMR (CDCl3) d 8.91
(d, J = 2.1 Hz, 1H), 8.76 (d, J = 1.9 Hz, 1H), 7.57 (d, J =
8.5 Hz, 1H), 7.48 (dd, J = 7.9 and 8.3 Hz, 1H), 6.90 (d, J =
7.5 Hz, 1H), 3.40–4.00 (m, 8H). 13C NMR (CDCl3) d 168.7 (C),
154.0 (C), 148.6 (C), 148.0 (CH), 132.1 (CH), 131.9 (CH), 126.2
(C), 119.3 (C), 119.2 (CH), 110.3 (CH), 66.9 (CH2). Found: C,
65.2; H, 5.6; N, 10.75. Calc. for C14H14N2O3: C, 65.11; H, 5.46; N,
10.85%.
General procedure for the carbamylation of 10a–f (procedure A).
To a solution of hydroxyquinoline 10 (1 mmol) in dry THF (50 mL)
was added portionwise NaH (1.1 mmol) and the mixture was
stirred at room temperature for one hour. Dimethylcarbamoyl
chloride or ethyl isocyanate (1.1 mmol) was added dropwise
and the resulting mixture was heated under reflux overnight. A
saturated aqueous solution of NH4Cl (30 mL) was added and after
phase separation the aqueous phase was extracted with CH2Cl2
(2 ¥ 100 mL). The combined organic phases were washed with
brine, dried over MgSO4 and concentrated under vacuum. The
residue was purified by flash chromatography to afford the desired
5-Hydroxy-3-(N-methylcarboxamido)quinoline (10d). A solu-
tion of compound 9 (391 mg, 1.34 mmol) in ethanol (50 mL)
was stirred under H2 atmosphere for 3 hours at room temperature
in the presence of 5% Pd/C (276 mg, 0.13 mmol). The catalyst
was then removed by filtration and ethanol was evaporated under
1
reduced pressure. The H NMR analysis of the crude product
1
showed partial reduction of the quinoline ring. The mixture was
dissolved in ethanol and air was bubbled through the solution
until complete re-oxidation of the product. After evaporation of
solvent, compound 10d was obtained (263 mg, 95%) as a brownish
powder. Mp 200 ◦C (degrad.) 1H NMR (CDCl3) d 10.82 (s, 1H),
9.22 (d, J = 2.3 Hz, 1H), 8.97 (d, J = 2.1 Hz, 1H), 8.83 (br, 1H),
7.64 (dd, J = 8.3 and 7.9 Hz, 1H), 7.50 (d, J = 8.3, 1H), 6.99 (d,
J = 7.5 Hz, 1H), 2.84 (d, J = 4.5 Hz, 3H). IR nmax/cm-1 (KBr)
1622, 1320, 1279, 812. Found: C, 65.4; H, 4.9; N, 13.7. Calc. for
C11H10N2O2: C, 65.34; H, 4.98; N, 13.85%.
carbamylated quinolines 11a–g. All spectroscopic data, H and
13C NMR spectra are provided in the Electronic Supplementary
Information (ESI†).
General procedure for the quaternisation of 11a–g (procedure B).
To a stirred solution of carbamylated quinoline 11a–g (1 mmol)
in CH2Cl2 (10 mL) was added methyl trifluoromethanesulfonate
(1.2 mmol) at room temperature. The resulting mixture was stirred
for 2h at room temperature after which CH2Cl2 was evaporated
in vacuo to afford the quinolinium salts 2a–g. All spectroscopic
1
data, H and 13C NMR spectra are provided in the Electronic
5-Hydroxy-3-(N,N-dimethylcarboxamido)quinoline (10e).
A
Supplementary Information (ESI†).
solution of compound 8 (275 mg, 0.90 mmol) in ethanol (40 mL)
was stirred under H2 atmosphere for 3 hours at room temperature
in the presence of 5% Pd/C (191 mg, 0.09 mmol). The catalyst
was then filtered and ethanol was evaporated under reduced
General procedure for the reduction of 2a–g (procedure C).
To a mixture of CH2Cl2 (6 mL) and water (6 mL) was added
quinolinium salt 2 (0.22 mmol) and the resulting solution was vig-
orously stirred under nitrogen (water and CH2Cl2 were degassed
by nitrogen bubbling before use). The following procedure was
repeated twice: a solution of sodium dithionite (1mL; 190 mg/mL)
and a solution of sodium carbonate (1 mL, 70 mg/mL) were
added. The resultant biphasic solution was vigorously stirred at
room temperature for 1 hour. After adding glacial acetic acid to
reach pH 6 and phase separation, the aqueous layer was extracted
with CH2Cl2 (2 ¥ 5 mL). The combined organic layers were washed
with brine, dried (MgSO4) and concentrated under vacuum to
afford 1,4-dihydroquinolines 1a–g. All spectroscopic data, 1H and
13C NMR spectra are provided in the Electronic Supplementary
Information (ESI†).
1
pressure. The H NMR analysis of the crude product showed
partial reduction of the quinoline ring. The mixture was dissolved
in ethanol and air was bubbled through the solution until complete
re-oxidation of the product. After evaporation of solvent and
purification by flash chromatography on silica gel, compound
1
10e was obtained (144 mg, 74%) as a brown powder. H NMR
(DMSO-d6) d 10.88 (s, 1H), 8.98 (d, J = 2.0 Hz, 1H), 8.63 (d, J =
2.0 Hz, 1H), 7.72 (dd, J = 8.1 and 7.9 Hz, 1H), 7.57 (d, J = 8.3 Hz,
1H), 7.08 (d, J = 7.7 Hz, 1H), 3.10 (br, 6H). 13C NMR (DMSO-d6)
d 168.2 (C), 154.3 (C), 148.7 (CH), 148.0 (C), 132.0 (CH), 130.5
(CH), 128.2 (C), 118.7 (CH), 118.6 (C), 109.8 (CH), 36.0 (CH3),
39.6 (CH3). Found: C, 66.5; H, 5.5; N, 12.9. Calc. for C12H12N2O2:
C, 66.65; H, 5.59; N, 12.96%.
4-(5-Hydroxyquinolin-3-yl-carbonyl)morpholine (10f). To
a
In vitro AChE inhibition assay. Inhibitory activity against
AChE was evaluated according to Ellman’s method.18 Full ex-
perimental details are provided in the Electronic Supplementary
Information (ESI†).
suspension of compound 10c (300 mg, 1.48 mmol) in toluene
(60 mL) pre-cooled to 0 ◦C were added AlMe3 (1.6 mL,
3.2 mmol, 2M in heptane) and freshly distilled morpholine
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Acknowledgements
We thank the CNRS, the re´gion Haute-Normandie and the
CRIHAN for financial and technical support.
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