Combining galantamine and memantine in multitargeted, new chemical entities potentially useful in Alzheimer's disease

Article abstract of DOI:10.1021/jm3009458

Herein we report on a novel series of multitargeted compounds obtained by linking together galantamine and memantine. The compounds were designed by taking advantage of the crystal structures of acetylcholinesterase (AChE) in complex with galantamine derivatives. Sixteen novel derivatives were synthesized, using spacers of different lengths and chemical composition. The molecules were then tested as inhibitors of AChE and as binders of the N-methyl-D-aspartate (NMDA) receptor (NMDAR). Some of the new compounds were nanomolar inhibitors of AChE and showed micromolar affinities for NMDAR. All compounds were also tested for selectivity toward NMDAR containing the 2B subunit (NR2B). Some of the new derivatives showed a micromolar affinity for NR2B. Finally, selected compounds were tested using a cell-based assay to measure their neuroprotective activity. Three of them showed a remarkable neuroprotective profile, inhibiting the NMDA-induced neurotoxicity at subnanomolar concentrations (e.g., 5, named memagal, IC₅₀ = 0.28 nM).

Full text of DOI:10.1021/jm3009458

Article
pubs.acs.org/jmc
Combining Galantamine and Memantine in Multitargeted, New
Chemical Entities Potentially Useful in Alzheimer’s Disease
Elena Simoni,†,‡ Simona Daniele,†,§ Giovanni Bottegoni,† Daniela Pizzirani,† Maria L. Trincavelli,§
Luca Goldoni,† Glauco Tarozzo,† Angelo Reggiani,† Claudia Martini,§ Daniele Piomelli,†,⊥
Carlo Melchiorre,‡ Michela Rosini,*,‡ and Andrea Cavalli*,†,‡
†Drug Discovery and Development, Istituto Italiano di Tecnologia, via Morego 30, 16163 Genova, Italy
‡Department of Pharmaceutical Sciences, Alma Mater Studiorum − Bologna University, via Belmeloro 6, 40126 Bologna, Italy
§Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, via Bonanno 6, 56126 Pisa, Italy
⊥Departments of Pharmacology and Biological Chemistry, University of California, Irvine 92697-4621, United States
S
* Supporting Information
ABSTRACT: Herein we report on a novel series of multitargeted
compounds obtained by linking together galantamine and memantine. The
compounds were designed by taking advantage of the crystal structures of
acetylcholinesterase (AChE) in complex with galantamine derivatives.
Sixteen novel derivatives were synthesized, using spacers of different
lengths and chemical composition. The molecules were then tested as
inhibitors of AChE and as binders of the N-methyl-D-aspartate (NMDA)
receptor (NMDAR). Some of the new compounds were nanomolar
inhibitors of AChE and showed micromolar affinities for NMDAR. All
compounds were also tested for selectivity toward NMDAR containing the
2B subunit (NR2B). Some of the new derivatives showed a micromolar
affinity for NR2B. Finally, selected compounds were tested using a cell-
based assay to measure their neuroprotective activity. Three of them
showed a remarkable neuroprotective profile, inhibiting the NMDA-
induced neurotoxicity at subnanomolar concentrations (e.g., 5, named memagal, IC50 = 0.28 nM).
with 2.5,6 The rationale for this approach is that the NMDAR
antagonist will stop or delay neurodegeneration, while the
AChE inhibitor will improve memory and cognition by
stimulating the surviving neurons.
INTRODUCTION
■
Alzheimer’s disease (AD) is a progressive and fatal brain
disorder that causes memory loss, steady deterioration of
cognition, and dementia. Over 30 million people worldwide are
afflicted with the disease. It is estimated that, by 2050, there
The usefulness of NMDAR antagonists in AD patients has
been intensively debated over the years. It has long been known
could be more than 100 million AD patients worldwide.1 AD
that NMDAR activation can be either beneficial, promoting
dramatically affects the quality of life of the sufferers and their
neuronal survival, or detrimental, causing neuronal death.7 The
families, and despite massive investments, there are few, if any,
current consensus is that these opposite effects depend on
effective treatments for AD. The AD pathogenesis involves a
receptor location: activation of postsynaptic NMDARs is
complex interplay of genetic and biochemical factors, including
beneficial, whereas activation of extrasynaptic NMDARs
an increased production of β-amyloid peptide (amyloid
promotes neuronal death. This suggests that the latter receptor
hypothesis) and an increased phosphorylation of the micro-
population should be the target for novel molecules.7 In this
tubule-associated tau protein (tau hypothesis).2
respect, although 2 is far from being optimal, it is the only
AD progression is associated with a significant disruption of
NMDAR antagonist in clinical use. This is probably because of
several neurotransmitter systems, including the cholinergic,
its preferential action on the extrasynaptic NMDARs.8
GABAergic, adrenergic, serotoninergic, and glutamatergic
systems.3 Alterations of cholinergic and glutamatergic systems
Unlike other AChE inhibitor drugs, 1 has a postulated dual
mechanism of action: in addition to inhibiting AChE, 1 has
shown a facilitating effect on the nicotinic receptor-mediated
have been extensively investigated,4 leading to four acetylcho-
linesterase (AChE) inhibitors, donepezil, galantamine (1),
rivastigmine, and tacrine (now discontinued) and one non-
competitive N-methyl-D-aspartate (NMDA) receptor
transmission via allosteric modulation of the α-subunit of
nicotinic receptors.9 In particular, 1 can enhance synaptic
(NMDAR) antagonist, memantine (2, see Figure 1), which
have been approved for use in AD patients. The current
standard of care for AD is a combination of an AChE inhibitor
Received: July 4, 2012
© XXXX American Chemical Society
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Figure 1. (A) Dual-target design strategy, according to Bottegoni et al.20 In this study, we used the strategy of designing and synthesizing chimera
derivatives of (B) galantamine (1) and memantine (2). Suitable spacers of different length and chemical composition were used, affording 16 new
chemical entities, 3−18 (see Table 2).
NMDAR activity10,11 by activating α7 nicotinic receptors
located on presynaptic glutamatergic neurons.12 Working
together, 1 and 2 may thus improve neurophysiological
responses in AD patients,13 and their combination might
offer a promising therapeutic strategy for AD treatment. 1 can
provide the memory-enhancing activity of any AChE inhibitor,
but it can also complement the effect of 2.14
an unexpected ability to interact with the NMDAR containing
the 2B subunit of the receptor (hereafter referred to as NR2B),
as they were able to displace ifenprodil, a selective NR2B
ligand. It has been postulated that NR2B could be the major
component of the extrasynaptic NMDAR population. There-
fore, the neuroprotective profile of selected molecules was
examined using SHSY-5Y cells.
A possible alternative to drug combinations is to create
molecules that can hit multiple targets; these are known as
multitarget-directed ligands (MTDLs).15 MTDLs are gaining
increasing attention from the drug discovery community.
Recent review articles have pointed out that the strategy of
targeting two or more proteins with a single compound can
provide therapeutic effects superior to those of a selective
drug.15−18 In addition, MTDLs can offer a number of potential
benefits over cocktails or multicomponent drugs. These include
the following: (i) reduced uncertainty in clinical development
because predicting the pharmacokinetics of a single compound
is much easier than with a drug cocktail; (ii) improved efficacy
due to the synergistic effect of simultaneously inhibiting
multiple targets; (iii) improved safety by decreasing the side
effects related to the load of a drug cocktail with a reduced risk
of drug−drug interactions; (iv) simplified therapeutic regimen
and improved compliance, which is particularly important for
elderly AD patients and their caregivers.19
In the present study, we combined in single new chemical
entities the pharmacophoric moieties of 1 and 2 using our dual-
target design strategy20 schematically reported in Figure 1. The
new chimera compounds were initially investigated by docking
simulations, taking advantage of the crystallographic structure
of AChE in complex with a bivalent derivative of 1.21 The
spacers were designed to allow the new compounds to properly
dock into the AChE gorge and to simultaneously contact both
sites of the enzyme. In particular, different polymethylene
spacers and heteroatoms were chosen (3−8 and 12−15).
Moreover, we sought to evaluate the role of the adamantane
amine function through the design of amide (16−18) and N-
methyl (9−11) derivatives. In fact, the protonable nitrogen of 2
could establish cation−π interaction with the peripheral anionic
site of AChE, thus increasing the affinity for this target. The
molecules were then synthesized and tested against AChE and
NMDAR. Interestingly, some of the new compounds showed
RESULTS AND DISCUSSION
■
In this study, we investigated the feasibility of designing and
synthesizing dual-target compounds, which were able to inhibit
the AChE enzyme and to bind to the NMDAR. Other studies
have previously sought to obtain dual AChE/NMDAR
compounds.22,23 However, for the first time, we combined in
single new chemical entities two marketed drugs, 1 and 2 (see
Figure 1 for the design strategy). Docking simulations
demonstrated that the amine group of 1 was a suitable point
to functionalize this fragment, which would afford compounds
3−18. These compounds were then synthesized and bio-
logically characterized.
Computational Design. Docking simulations were carried
out to identify the proper length of the methylene linker
connecting 1 to 2. These simulations were performed on a
model structure of AChE obtained by imposing, on the crystal
structure of the human enzyme, the conformation adopted by
its Torpedo californica orthologue in complex with a bivalent
derivative of 1 (see the Experimental Section for further
details). Table 1 reports the results of the docking simulations.
Five to six methylenes were the minimal distance that allowed
the new hybrid compounds to efficiently contact both sites (the
internal and the peripheral anionic sites) of the AChE gorge
without excessive strain on the linker. Interestingly, compounds
with a linker length of six or seven units were characterized by
an optimal balance between binding score and log P. Moreover,
compounds with a six- or seven-methylene spacer displayed the
most suitable distance between the basic adamantane amine
and the indole ring of Trp286 to form a cation−π interaction. It
is worth emphasizing that, due to its electronic nature, this
specific interaction was not captured by the scoring function.
Figure 2 reports the binding mode of 6 at the AChE gorge.
The galantamine part of the compound matched almost
perfectly the crystallographic conformation, establishing a
B
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9−11 was accomplished by alkylation of 19 to furnish 21−23,
and the following activation of the alcohol group was achieved
without isolation of the intermediates, which were directly
reacted with 20 (Scheme 1). Amides 16−18 were easily
prepared by a two-step procedure starting from 2 hydrochloride
and the corresponding acylating agents. The resulting
intermediates 51−53 were reacted with 20 to furnish 16−18
(Scheme 1). The introduction of heteroatom chains in 12−14
was carried out by selectively activating the appropriate
dialcohol 54−56 (Scheme 2). The key intermediates 63−65
were prepared according to the procedure adopted for 38−44
and condensed with 20, affording 66−68. Then HCl-promoted
deprotection of the amino group provided final compounds
12−14. Because the above-described strategy failed for 15, a
convergent synthesis was performed by condensing 38 with 71,
which was derived from nucleophilic substitution between 20
and 69. Finally, HCl-mediated removal of the Boc group
furnished the target compound 15 (Scheme 3).
Table 1. Outcomes of Docking Simulations Carried Out with
Galantamine−Memantine Chimeras Containing Spacers of
Different Length
cation−π
c
linker docking score
ligand
distance
(Å)
a
b
compd length (score units) log P efficiency
LELP
3
4
5
6
7
8
4
5
6
7
8
9
−10.00
−13.38
−20.15
−22.03
−23.15
−18.72
3.98
4.46
4.94
5.42
5.90
6.38
0.27
0.35
0.52
0.55
0.56
0.44
14.74
12.67
9.57
4.89
4.91
3.75
3.40
4.43
4.76
9.84
10.46
14.33
a
Ligand efficiency is expressed as Docking Score/no. of non-hydrogen
b
c
atoms. Ligand efficiency lipophilicity. Distance between the
protonated adamantan-1-amine and the center of the indole ring of
Trp286.
Biological Assays. All the compounds were tested to assess
their abilities to inhibit AChE activity and bind to NMDAR.
The AChE-inhibiting activities were measured using the classic
method of Ellman.25 Binding to NMDAR and NR2B-
containing NMDAR was assessed using the assay based on
the displacement of [3H]MK-801 and [3H]ifenprodil, respec-
tively. In addition, the inhibition of the NMDA-mediated
neurotoxicity (500 μM of NMDA for 6 h) was assessed using
the SHSY-5Y neuroblastoma cell line.
Multitargeted Structure−Activity Relationships. All
the compounds were initially tested to assess their ability to
inhibit the two selected targets, AChE and NMDAR. Table 2
summarizes the biological data generated for compounds 3−18.
As reported above, 1 could be suitably modified at the amine
function, because substituents at this position could fit into the
AChE gorge and contact the peripheral anionic site of the
enzyme, identified by the Trp286 residue (Figure 2). It has
been extensively reported in the literature that compounds,
which can simultaneously interact with both sites of AChE
(internal and peripheral anionic sites), are expected to potently
inhibit the enzyme.26−32 According to these observations, we
have discovered quite potent AChE inhibitors. In fact, 5−8,
which were able to interact with both sites of AChE (see Figure
2 and Supporting Information), were nanomolar inhibitors of
this enzyme. In addition, the introduction of a methyl group on
the nitrogen of 2 further improved the AChE-inhibiting
potency, with 9 and 11 being the most potent AChE inhibitors
of the present series (IC50 = 1.03 nM and 0.52 nM,
respectively). Conversely, replacing methylenes of the spacer
with heteroatoms (12−15) had a slight detrimental effect on
the AChE-inhibiting potency (docking models of 12−18 in
complex with AChE are reported in the Supporting
Information). In addition, we also observed that “switching
off” the charge on the moiety of 2, by replacing the methylene
close to the nitrogen with a carbonyl group, greatly affected the
AChE-inhibiting profile. Docking simulations showed that the
binding mode of the present series of compounds was quite
well conserved (compare Figure 2 and the binding modes
reported in Supporting Information), and therefore removal of
the charge prevented 16−18 from properly interacting with
Trp286 by means of a cation−π interaction, thus decreasing
their affinity for the enzyme. Furthermore, reducing the chain
length to four methylenes, as in compound 3, prevented it from
properly reaching Trp286 and significantly decreased its affinity
(IC50 = 695.9 nM) when compared to 4−8. All these data
Figure 2. Bound conformation of 6 at the inner and peripheral anionic
binding site of human AChE. The inhibitor is displayed in green, while
the protein key residues are reported in gray and labeled explicitly. An
orange mesh highlights the boundaries of the binding pocket.
hydrophobic interaction with the indole ring of Trp86 and
donating a hydrogen bond to the side chain of Glu202. The
linker was lodged in a narrow cavity described by Asp74,
Tyr124, Phe297, Tyr337, Phe338, and Tyr341. The protonated
nitrogen of the adamantane amine formed a cation−π
interaction with the indole of Trp286. The adamatane moiety
pointed toward the bulk of the solvent and established
hydrophobic interaction with Val294, the backbone of
Phe295, and the backbone of Gly342.
Chemical Syntheses. 3−18 were obtained following the
synthetic approaches depicted in Schemes 1−3. Due to the
easier chemical accessibility and availability of 2 with respect to
desmethyl galantamine (20), the synthesis of 3−14 and 16−18
began by generating derivatives of 2, 21−23, 39−44, 51−53,
and 63−65 as key intermediates, which were then connected to
20 (Schemes 1 and 2). The alkylation of 2 as hydrochloride salt
with the appropriate bromo alcohol under basic conditions gave
24−30. Then the amino group was Boc-protected, achieving
31−37, which were treated with tosyl chloride to afford the
easily substitutable tosyl-activated alcohols 38−44. 38 was
employed for the synthesis of 15 (Scheme 3), whereas 39−44
were subsequently reacted with 20, providing compounds 45−
50. 3−8 were obtained in good yield after carbamate
deprotection under mild acidic conditions to avoid degradation
of the desired compounds (Scheme 1). 9−11 were synthesized
starting from N-methylmemantine (19), which, in turn, was
prepared through reductive methylation of the primary amine
of 2 with aqueous formaldehyde and zinc dust by means of
slight modification of a literature procedure.24 The synthesis of
C
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a
Scheme 1. Synthesis of 3−11 and 16−18
a
Reagents and conditions: (a) CH2O, MeCOOH, Zn, H2O/dioxane, rt; (b) HO(CH2)nBr, K2CO3, DMF, 80 °C; (c) Boc2O, Na2CO3, THF/H2O,
rt; (d) TsCl, Et3N, DMAP, CH2Cl2, 0 °C to rt; (e) TsCl, Et3N, DMAP, CH2Cl2, 0 °C to rt followed by 20, Et3N, MeCN, 80 °C; (f) Et3N, MeCN,
80 °C; (g) 4 M HCl in dioxane, 0 °C to rt; (h) ClCO(CH2)nCl, MeCN, K2CO3, rt.
a
a
Scheme 2. Synthesis of 12−14
Scheme 3. Synthesis of 15
a
a
Reagents and conditions: (a) Et3N, MeCN, 80 °C; (b) 4 M HCl in
dioxane, 0 °C to rt.
Reagents and conditions: Y = O(CH2)2O; O; S; (a) K2CO3, DMF,
80 °C; (b) Boc2O, Na2CO3, THF/H2O, rt; (c) TsCl, Et3N, DMAP,
CH2Cl2, 0 °C to rt; (d) 20, Et3N, MeCN, 80 °C; (e) 4 M HCl in
dioxane, 0 °C to rt.
As for NMDAR, some of the new compounds were active in
the micromolar range (see Table 2), but none of the new
derivatives was remarkably potent against this receptor. The
confirmed the binding mode reported in Figure 2 for the
present series of chimeras.
D
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Table 2. Biological Activities of 3−18 against Rat AChE, NMDAR, NR2B, and the NMDA-Induced Neurotoxicity in
Neuroblastoma Cell Lines
NMDAR (NR2B) [3H]
b
AChE, IC50
NMDAR [3H] MK-801
ifenprodil binding assay ,
SHSY-5Ycell viability assay,
c
c
a
d
d
compd
n
R
Y
(nM)
binding assay , Ki (μM)
Ki (μM)
IC50 (nM) vs 500 μM NMDA
3
4
4
H
−
−
−
−
−
−
−
−
−
695.9 28.4
4.31 0.25
1.16 0.03
1.79 0.06
5.36 0.77
2.32 0.50
1.03 0.18
1.33 0.12
0.52 0.05
80.9 12.8
3920 690
131.1 29.5
2.32 0.43
>100
5
H
>10
40.9 5.6
4.6 0.7
10.95 3.44
2.9 1.0
>100
5 (memagal)
6
H
4.6 0.6
0.28 0.05
0.21 0.02
6
7
H
10.0 2.1
7
8
H
4.99 0.20
8
9
H
4.63 1.54
9
6
CH3
CH3
CH3
−
−
−
−
−
−
−
>10
5.25 1.6
>100
10
7
>10
11
8
>10
>100
12
−
−
−
−
3
O(CH2)2O
>10
>100
>10 000
13
O
S
>10
>100
14
>10
>100
15
CH2NHCH2 167.8 24.3
5.41 1.11
3.32 0.09
>100
16
−
−
−
55.7 10.3
369 7.6
>10
17
4
>10
>100
e
18
5
11.3 1.6
2550 690
33.87 4.73% at 10 μM
9.76 4.48
>100
0.37 0.04
747 67
718 63
1 (galantamine)
2 (memantine)
>10
8.50% 1.30 1.16 0.07
at 10 μM
>100
ifenprodil
MK801
3.08 0.76
0.029 0.0003
>100
0.73 0.04
7.91 0.45
Kd = 4.0 nM
a
b
Displacement of specific [3H]MK-801 binding in rat cortex membranes. Displacement of specific [3H]ifenprodil binding in rat cortex membranes.
c
A nonlinear multipurpose curve-fitting program, Prism (GraphPad Software Inc., San Diego, CA), was used for data analysis of rat AChE activity
and cell viability; the IC50 value represents the compound concentration able to give 50% inhibition of rat AChE activity or NMDA-induced toxicity.
d
Data are reported as the means SEM of three or four different experiments (performed in duplicate). The Ki values are means SEM derived
e
from an iterative curve-fitting procedure (Prism program, GraphPad, San Diego, CA). Percentage of inhibition is reported for compound 18.
affinity for this receptor appeared to be mainly driven by 2,
while the galantamine structure was not detrimental and did
not cause any significant drop in affinity. In the MK-801
binding assay, the most potent derivative of the present series
was 3, showing a Ki value of 2.34 μM. In this case, the chain
length did not affect the affinity against NMDAR (compare the
potencies of 3−8), while switching off the charge affected the
affinities for this receptor (see compounds 16−18), in
agreement with previous observations.33 The introduction of
a methyl group on the nitrogen of 2 (9−11) had a detrimental
effect on binding to NMDAR. Replacing methylenes of the
spacer with heteroatoms (12−15) had a slight detrimental
effect on the affinity for NMDAR. The only exception was 15,
where a methylene was replaced by an NH group. 15 showed a
Ki value of 5.41 μM, comparable to that of the most potent
derivative of the present series (3, Ki = 2.34 μM). As for 15, we
can hypothesize that a further protonable nitrogen, present on
its spacer, can positively contribute to the recognition between
the ligand and the channel that physiologically controls the
permeation of bivalent positive cations.
activities against NMDAR, call for a subsequent campaign of
optimization to properly equalize the profile of 3−18 against
the two targets. However, it should also be noted that, for in
vivo efficacy, it is not strictly necessary that the two targets are
modulated to the same extent. In the field of multitarget drug
discovery, the issue of affinity-balancing is particularly
challenging for the following two reasons: (i) increasing the
affinity against the first target can decrease affinity against the
second, and vice versa; (ii) there is the possibility that the two
targets should be engaged to different extents (i.e., diverse
target engagement profile) to provide optimal in vivo activity
and efficacy. The latter aspect should be carefully considered
when selecting two (or more) targets at the very early stages of
a multitarget drug discovery program.
There is now compelling evidence that NR2B-containing
NMDAR could preferentially contribute to pathologies linked
to glutamate overexcitation.35−37 Therefore, we turned our
attention to NR2B, testing all the compounds using the
ifenprodil binding assay (see the Experimental Section for
details). While most of the compounds were unable to displace
ifenprodil (see Table 2), some of the new derivatives
(compounds 5−7, 9, 15, and 18) showed Ki values in the
micromolar range. A comment is required here on the SAR of
the present series of compounds against NR2B-containing
NMDAR. In fact, we noticed that slight structural modifications
In summary of the profiles against AChE and NMDAR, some
of the new derivatives were nanomolar inhibitors of AChE, and
micromolar binders of NMDAR. A comment is required here
on the affinity-balancing issue.34 The nanomolar profiles of the
new compounds against AChE, along with the micromolar
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caused a drop in affinity for NR2B-containing NMDAR. To
rationalize such a behavior, we carried out docking simulations
using the crystal structure of NR2B in complex with ifenprodil
(see Experimental Section for details). In the Supporting
Information, we report the docking model of NR2B in complex
with 5. The compound fitted quite well into the infenprodil
binding pocket, establishing relevant hydrophobic and H-bond
interactions (for a detailed description of all interactions, see
the Supporting Information). The proposed binding mode
could explain the good affinity of 5 for NR2B (Ki = 4.6 μM), as
well as the ability of the compound to displace ifenprodil as
experimentally observed. However, the SARs for this receptor
subunit remained puzzling and difficult to be properly
rationalized on the basis of our docking model. Further studies
are required to properly generate meaningful SARs for the
present series of derivatives against NR2B-containing NMDAR.
These encouraging data prompted us to test some
representative compounds using a cell-based assay to assess
their neuroprotective profile against NMDA-mediated neuro-
toxicity. SHSY-5Y cells were treated with 500 μM NMDA for 6
h in the absence or presence of different concentrations of
selected compounds, to generate the dose−response curves of
inhibition of the NMDA-induced neurotoxicity. Compounds 5,
6, and 18 were remarkably potent in these experimental
conditions, as they were able to inhibit NMDA-induced
neurotoxicity with subnanomolar potencies. In particular, 6
showed an IC50 value of 0.21 nM, while 5 (which we named
memagal) and 18 showed IC50 values of 0.28 nM and 0.37 nM,
respectively. As negative control, we tested 12 (NR2B Ki > 100
μM), which started to inhibit NMDA neurotoxicity at 10 μM.
Other compounds could not be tested up to 10 μM, because
they were cytotoxic at this concentration. Under the same
conditions, we also tested 2, which affected cell viability at a
much higher concentration (718 nM; see Table 2). These cell-
based data were extremely promising and not fully explained by
the binding potencies (in the micromolar range) of these
compounds against NR2B. Interestingly, while a nanomolar
antagonist of NMDAR unable to bind to NR2B (i.e., MK-801;
see Table 2) inhibited the NMDA-induced toxicity at
nanomolar concentration (IC50 = 7.91 nM), a selective
nanomolar ligand of NR2B (ifenprodil) exerted the same effect
at a subnanomolar concentration (IC50 = 0.73 nM). Similarly,
we observed that our compounds, which displaced both MK-
801 and ifenprodil at micromolar concentrations, were able to
inhibit NMDA-induced neurotoxicity in the subnanomolar
range. A possible explanation for this behavior could be that
nonselective binders, able to displace both MK-801 and
ifenprodil, could result in potent neuroprotective agents, due
to possible synergistic and/or additive effects.
multitargeted compounds obtained by chemically combining
1 and 2, two marketed drugs for the treatment of AD. Our
molecules turned out to be remarkably active against AChE
with the most potent derivative showing an IC50 value of 0.52
nM. For NMDAR, we obtained micromolar binders. The most
potent derivative (3) showed a Ki of 2.32 μM against NMDAR.
However, it was slightly more potent against AChE (IC50
=
695.9 nM). This series of molecules has further highlighted the
inherent difficulty of balancing the affinity profiles against
diverse targets, when discovering and developing MTDLs. The
molecules were then tested against NR2B-containing NMDAR,
using the ifenprodil binding assay. A few compounds were
active in the micromolar range, with 7 being the most potent
binder of NR2B (Ki = 2.9 μM). These molecules are the first
ever derivatives reported to show a dual AChE/NR2B profile.
They could be promising tools for investigating the
simultaneous modulation of these two targets in AD. Finally,
in light of the NR2B profile, selected derivatives were tested
using cellular assays, to investigate the neuroprotective profile
after insult by NMDA. While most of the derivatives were
inactive or cytotoxic at micromolar concentration, compounds
5, 6, and 18 turned out to be subnanomolar inhibitors of the
NMDA-mediated neurotoxicity. The high potencies of these
molecules as neuroprotective agents might lie in their ability to
nonselectively modulate NMDARs. This intriguing biological
profile deserves further investigation as it could open up new
avenues for generating leads and drug candidates endowed with
optimal anticholinesterase and neuroprotective profiles against
AD.
EXPERIMENTAL SECTION
■
Chemical Synthesis. Chemistry. Chemical reagents were
purchased from Sigma Aldrich, Fluka (Italy), and TCI-Europe.
N-Desmethylgalantamine was purchased from Synfine research.
Solvents were RP grade. Dichloromethane was distilled from
calcium chloride. Dry dimethylformamide and triethylamine
were used as supplied. Nuclear magnetic resonance spectra
(NMR) were recorded at 400 MHz on Varian VXR 400
spectrometers and reported in parts per million. All final
compounds 3−18 are >95% pure by UPLC-MS analyses. The
analyses were performed on a Waters ACQUITY UPLC-MS
instrument consisting of an SQD single quadropole mass
spectrometer equipped with an electrospray ionization interface
and a photodiode array detector. The analyses were performed
on an ACQUITY UPLC BEH C18 column (50 × 2.1 mm ID,
particle size 1.7 μm) with a VanGuard BEH C18 precolumn (5
× 2.1 mm ID, particle size 1.7 μm). The mobile phases were 10
mM NH4OAc at pH 5 adjusted with AcOH (A) and 10 mM
NH4OAc in MeCN−H2O (95:5) at pH 5 (B). Linear Gradient:
0−0.2 min, 10% B; 0.2−6.2 min, 10−90% B; 6.2−6.3 min, 90−
100% B; 6.3−7 min, 100% B; at a flow rate of 0.5 mL/min.
Electrospray ionization in positive and negative mode was used
in the mass scan range 100−500 Da. Optical rotations were
measured on a Rudolf Research Analytical Autopol II
Automatic polarimeter using a sodium lamp (589 nm) as the
light source; concentrations expressed in g/100 mL using
CHCl3 as a solvent and a 1 dm cell. Column chromatography
purifications were performed under “flash conditions” using
Sigma Aldrich silica gel grade 9385, 230−400 mesh. TLC were
performed on 0.20 mm silica gel 60 F254 plates (Merck,
Germany), which were visualized by exposure to ultraviolet
light and potassium permanganate stain. Reactions involving
generation or consumption of amine were visualized by using
CONCLUSIONS
■
In AD, there is still a need for drugs that simultaneously
stimulate the cholinergic system while suppressing glutamate
neurotoxicity, providing molecules endowed with neuro-
protective profiles. In light of the recent failures of compounds
that block targets belonging to the amyloid and the tau
cascades, there is interest in broadening efficacy of
anticholinesterase drugs, by contrasting the progressive neuro-
degeneration responsible for their decline in efficacy. The
neuroprotective profile, achieved through modulating
NMDARs, can lengthen the time in which anticholinesterase
drugs can provide beneficial effects on memory and cognition.
With this in mind, we designed and synthesized novel
F
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Journal of Medicinal Chemistry
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bromocresol green spray (0.04% in EtOH made blue by
NaOH) following heating of the plate. Compounds were
named following IUPAC rules as applied by Beilstein-Institute
AutoNom (version 2.1), a PC integrated software package for
systematic names in organic chemistry.
General Procedure for the Synthesis of 45−50 and 66−
68. A mixture of the appropriate activated alcohol (1.2 equiv),
20 (1 equiv) and Et3N (2 equiv) in MeCN (0.1 M) was stirred
at 80 °C for 48 h in a pressure tube. After evaporation of the
solvent, the residue was purified by flash chromatography (see
Supporting Information for details).
General Procedure for the Synthesis of 3−8 and 12−14.
HCl (4 M) in dioxane (1 mL) was carefully added to the
appropriate Boc derivative (1 equiv) at 0 °C. The resulting
solution was stirred at room temperature for 2 h. After removal
of the solvent,, the obtained residue was purified by flash
chromatography.
N,3,5-Trimethyladamantan-1-amine (19). Glacial acetic
acid (0.382 mL, 6.66 mmol), Zn dust (0.436 g, 6.66 mmol),
and 35% aqueous formaldehyde (0.833 mL, 16.65 mmol) were
sequentially added to a solution of 2 hydrochloride (0.720 g,
3.33 mmol) in dioxane/H2O (1:1). The mixture was stirred at
30 °C for 2 days. Then, saturated NH4Cl aqueous solution was
added (5 mL), and the resulting mixture extracted with CH2Cl2
(3 × 10 mL). The combined organic layers were then washed
with brine and dried over Na2SO4. The crude product was
purified by flash chromatography (CH2Cl2/MeOH/33%
aqueous NH3 9:1:1.8) to achieve 19 as a white solid: 0.480 g
(75%). 1H NMR (CDCl3, 400 MHz) δ 0.76 (s, 6H), 1.04−1.05
(AB m, 2H), 1.15−1.23 (m, 8H), 1.40−1.41 (m, 2H), 2.05−
2.07 (m, 1H), 2.26 (s, 3H), 2.75 (br s, exchangeable with D2O,
1H).
General Procedure for the Synthesis of 54−56. p-TsCl (1.2
equiv) was added dropwise to a mixture of the appropriate diol
(1 equiv), Et3N (1 equiv), and DMAP (catalytic amount) in
dry CH2Cl2 (0.01 M) under N2 atm, with stirring at 0 °C. The
stirring was continued at room temperature for 4 h. The
reaction was quenched with saturated NH4Cl aqueous solution,
and the whole was extracted with AcOEt. The extract was
washed with brine and dried over Na2SO4, and, following
solvent evaporation, the crude material was purified by flash
chromatography (see Supporting Information for details).
General Procedure for the Synthesis of 21−30 and 57−
59. In a pressure tube (Sigma Aldrich, ACE pressure tube) the
appropriate bromo alcohol or the activated alcohol 54−56 (1.2
equiv) was added to a vigorously stirred solution of 19 (1
equiv) or 2 hydrochloride (1 equiv) and K2CO3 (2.5 equiv) in
DMF (0.2 M). The reaction mixture was stirred at 80 °C for 48
h. After evaporation of the solvent, the residue was taken up
with water and extracted with CH2Cl2 (3 × 10 mL). The
collected organic layers were concentrated, and the crude was
purified by flash chromatography (see Supporting Information
for details).
General Procedure for the Synthesis of 31−37 and 60−
62. The appropriate secondary amine (1 equiv) was dissolved
in THF/H2O (2:1) and treated with Na2CO3 (2.5 equiv) and
di-tert-butyl dicarbonate (1.5 equiv). The resulting suspension
was stirred at room temperature overnight. The solvent was
then removed in vacuo, yielding a residue, which was dissolved
in water and extracted with AcOEt (3 × 15 mL). The extracts
were combined, washed with brine, dried over Na2SO4,
concentrated, and purified by flash chromatography (see
Supporting Information for details).
General Procedure for the Synthesis of 38−44 and 63−
65. p-TsCl (1 equiv) was added to a mixture of the appropriate
alcohol (1 equiv), Et3N (2 equiv), and DMAP (catalytic
amount) in dry CH2Cl2 (0.1 M) under N2 atmosphere, with
stirring at 0 °C. The stirring was continued at room
temperature for 3.5 h. After completion, the reaction was
quenched with saturated NH4Cl aqueous solution and
extracted with AcOEt (3 × 10 mL). Combined extracts were
washed with brine, dried over Na2SO4, and evaporated. The
tosylate derivative was then purified by flash chromatography
(see Supporting Information for details).
(4aS,6R,8aS)-11-(4-((3,5-Dimethyladamantan-1-yl)-
amino)butyl)-3-methoxy-5,6,9,10,11,12-hexahydro-4aH-
benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (3). 3 was synthe-
sized from 45 (0.066 g, 0.11 mmol). Elution with CH2Cl2/
CH3OH/33% aqueous ammonia (9:1:0.1) afforded 3 as a waxy
1
solid: 0.050 g (91%). H NMR (CDCl3, 400 MHz) δ 0.84 (s,
6H), 1.08−1.16 (AB m, 2H), 1.25−1.37 (m, 9 H), 1.52−1.61
(m, 6H), 1.98−2.09 (m, 2H), 2.13−2.14 (m, 1H), 2.48−2.58
(m, 2H), 2.61−2.71 (m, 3H), 3.19−3.33 (2H), 3.82 (AB d, J =
15.2, 1H), 3.83 (s, 3H), 4.10−4.13 (m+AB d, J = 15.2 Hz, 2H),
4.60 (br m, 1H), 6.00 (dd, J1 = 10.4, Hz, J2 = 4.8 Hz, 1H), 6.07
(d, J = 10.4 Hz, 1H), 6.63 (d, J = 8.0, 1H), 6.66 (d, J = 8.0, 1H).
13C NMR (CDCl3, 100 MHz) δ 25.27, 27.91, 29.67, 29.92,
30.06, 30.16, 32.37, 33.01, 40.28, 42.70, 47.69, 48.32, 50.65,
51.46, 51.81, 55.85, 57.62, 62.02, 88.71, 111.19, 122.14, 126.75,
127.70, 128.80, 133.13, 144.17, 145.83. [α]25D = −57.4 (c 0.07,
CHCl3). UPLC/MS: purity 97%, MS (ESI+): m/z 507.4 [M +
H]+.
(4aS,6R,8aS)-11-(5-((3,5-Dimethyladamantan-1-yl)-
amino)pentyl)-3-methoxy-5,6,9,10,11,12-hexahydro-4aH-
benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (4). 4 was synthe-
sized from 46 (0.170 g, 0.27 mmol). Elution with CH2Cl2/
CH3OH/33% aqueous ammonia (9:1:0.1) afforded 4 as a waxy
1
solid: 0.095 g (66%). H NMR (CDCl3, 400 MHz) δ 0.84 (s,
6H), 1.11−1.21 (AB m, 2H), 1.26−1.35 (m, 11 H), 1.46−1.52
(m, 6H), 1.98−2.03 (m, 2H), 2.13−2.14 (m, 1H), 2.42−2.50
(m, 2H), 2.59 (t, J = 7.6, 2H), 2.66−2.71 (m, 1H), 3.16−3.17
(1H), 3.33−3.34 (m, 1H), 3.79 (AB d, J = 15.2, 1H), 3.83 (s,
3H), 4.10−4.13 (m+AB d, J = 15.2 Hz, 2H), 4.60 (br m, 1H),
6.00 (dd, J1=10.4, Hz, J2 = 4.8 Hz, 1H), 6.08 (d, J = 10.0 Hz,
1H), 6.60 (d, J = 8.4, 1H), 6.65 (d, J = 8.4, 1H). 13C NMR
(CDCl3, 100 MHz) δ 25.14, 27.25, 29.94, 30.17, 30.23, 32.37,
32.94, 40.43, 42.85, 48.41, 50.79, 51.52, 55.88, 57.76, 62.07,
88.71, 111.13, 121.97, 126.96, 127.53, 129.52, 133.14, 144.03,
144.77. [α]25 = −63.2 (c 0.05, CHCl3). UPLC/MS: purity
D
97%, MS (ESI+): m/z 521.6 [M + H]+.
(4aS,6R,8aS)-11-(6-((3,5-Dimethyladamantan-1-yl)-
amino)hexyl)-3-methoxy-5,6,9,10,11,12-hexahydro-4aH-
benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (5). 5 was synthe-
sized from 47 (0.160 g, 0.25 mmol). Elution with CH2Cl2/
CH3OH/33% aqueous ammonia (9:1:0.1) afforded 5 as a waxy
1
solid: 0.075 g (56%). H NMR (CDCl3, 400 MHz) δ 0.84 (s,
6H), 1.09−1.15 (AB m, 2H), 1.18−1.37 (m, 11H), 1.42−1.54
(m, 8H), 1.98−2.04 (m, 2H), 2.13−2.14 (m, 1H), 2.42−2.51
(m, 2H), 2.60 (t, J = 7.6, 2H), 2.66−2.71 (m, 1H), 3.16−3.17
(1H), 3.31−3.33 (m, 1H), 3.79 (AB d, J = 16.0, 1H), 3.83 (s,
3H), 4.10−4.13 (m+AB d, J = 16.0 Hz, 2H), 4.60 (br m, 1H),
6.00 (dd, J1 = 10.4, Hz, J2 = 4.8 Hz, 1H), 6.08 (d, J = 10.0 Hz,
1H), 6.60 (d, J = 8.4, 1H), 6.65 (d, J = 8.4, 1H). 13C NMR
(CDCl3, 100 MHz) δ 22.66, 26.67, 27.21, 27.38, 29.67, 29.94,
30.14, 30.21, 31.89, 32.38, 32,95, 33.66, 40.45, 42.80, 47.95,
G
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Journal of Medicinal Chemistry
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48.41, 50.74, 51.50, 53.66, 55.85, 55.88, 57.77, 62.07, 88.70,
111.11, 121.97, 126.97, 127.51, 129.56, 133.15, 144.00. 145.76.
[α]25D = −34.9 (c 0.10, CHCl3). UPLC/MS: purity 100%, MS
(ESI+): m/z 535.6 [M + H]+.
CH2Cl2/CH3OH/33% aqueous ammonia (9:1:0.05) afforded
12 as a waxy solid: 0.060 g (88%). H NMR (CDCl3, 400
1
MHz) δ 0.84 (s, 6H), 1.08−1.16 (AB m, 2H), 1.26−1.37 (m,
8H), 1.51−1.54 (m, 3H), 1.98−2.09 (m, 2H), 2.15 (br m, 1H),
2.66−2.70 (m, 1H), 2.74 (t, J = 5.8 Hz, 2H), 2.83 (t, J = 4.8 Hz,
2H), 3.21−3.25 (m, 1H), 3.38−3.44 (m, 1H), 3.54−3.67 (m,
8H), 3.83 (s, 3H), 3.88 (d, J = 15.2 Hz, 1H), 4.13−4.15 (m,
1H), 4.18 (d, J = 15.2 Hz, 1H), 4.60 (br m, 1H), 6.00 (dd, J1 =
10.4, J2 = 5.2 Hz, 1H), 6.08 (d, J = 10.4 Hz, 1H), 6.61 (d, J =
(4aS,6R,8aS)-11-(7-((3,5-Dimethyladamantan-1-yl)-
amino)heptyl)-3-methoxy-5,6,9,10,11,12-hexahydro-4aH-
benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (6). 6 was synthe-
sized from 48 (0.130 g, 0.20 mmol). Elution with CH2Cl2/
1
CH3OH (9:1) afforded 6 as a waxy solid: 0.070 g (64%). H
13
NMR (CDCl3, 400 MHz) δ 0.84 (s, 6H), 1.08−1.15 (AB m,
2H), 1.23−1.34 (m, 15H), 1.44−1.51 (m, 6H), 1.94−2.07 (m,
2H), 2.13 (br m, 1H), 2.40−2.52 (m, 2H), 2.57 (t, J = 7.2 Hz,
2H), 2.66−2.69 (m, 1H), 3.14−3.18 (1H), 3.31−3.37 (m, 1H),
3.79 (AB d, J = 15.2, 1H), 3.83 (s, 3H), 4.09−4.15 (m+AB d, J
= 15.2 Hz, 2H), 4.60 (br m, 1H), 5.99 (dd, J1 = 10.4, Hz, J2 =
4.8 Hz, 1H), 6.08 (d, J = 10.0 Hz, 1H), 6.61 (d, J = 8.4, 1H),
6.65 (d, J = 8.4, 1H). 13C NMR (CDCl3, 100 MHz) δ 27.35,
27.50, 27.55, 27.72, 29.40, 29.91, 30.05, 30.16, 32.14, 32.72,
33.19, 38.29, 40.36, 42.46, 45.58, 48.64, 50.41, 51.74, 56.11,
57.25, 58.02, 62.31, 88.94, 111.37, 122.21, 127.21, 127.77,
8.0 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H). C NMR(CDCl3, 100
MHz) δ 29.92, 30.12, 30.18, 32.38, 32.91, 40.05, 40.29, 42.77,
47.95, 48.40, 50.72, 51.83, 55.87, 57.89, 62.03, 68.99, 70.04,
70.16, 70.27, 88.68, 111.17, 122.21, 126.80, 127.63, 129.22,
133.14, 144.12, 145.82. [α]25 = −45.4 (c 0.11, CHCl3).
D
UPLC/MS: purity 99%, MS (ESI+): m/z 567.5 [M + 1]+.
(4aS,6R,8aS)-11-(2-(2-((3,5-Dimethyladamantan-1-yl)-
amino)ethoxy)ethyl)-3-methoxy-5,6,9,10,11,12-hexahydro-
4aH-benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (13). 13 was
synthesized from 67 (0.115 g, 0.18 mmol). Elution with
CH2Cl2/CH3OH/33% aqueous ammonia (9:1:0.05) afforded
129.74, 133.39, 144.25, 146.01. [α]25 = −64.1 (c 0.11,
13 as a waxy solid: 0.085 g (88%). H NMR (CDCl3, 400
1
D
CHCl3). UPLC/MS: purity 100%, MS (ESI+): m/z 549.6 [M +
MHz) δ 0.87 (s, 6H), 1.11−1.01 (AB m, 2H), 1.24−1.33 (m,
4H), 1.36−1.45 (AB m, 4H), 1.54−1.62 (m, 3H), 1.98−2.11
(m, 2H), 2.20 (br m, 1H), 2.66−2.71 (m, 1H), 2.76 (t, J = 5.2
Hz, 2H), 2.90 (t, J = 4.8 Hz, 2H), 3.34−3.39 (m, 2H), 3.53−
3.62 (m, 2H), 3.71−3.72 (m, 2H), 3.83 (s, 3H), 3.95 (d, J =
15.2 Hz, 1H), 4.14−4.17 (m+d, J = 15.2 Hz, 2H), 4.61 (br m,
1H), 6.00−6.08 (m, 2H), 6.65−6.67 (m, 2H).13 C NMR
(CDCl3, 100 MHz) δ 30.06, 32.58, 40.34, 42.55, 48.35, 50.50,
51.54, 55.87, 57.30, 62.00, 88.70, 111.27, 122.38, 126.52,
H]+.
(4aS,6R,8aS)-11-(8-((3,5-Dimethyladamantan-1-yl)-
amino)octyl)-3-methoxy-5,6,9,10,11,12-hexahydro-4aH-
benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (7). 7 was synthe-
sized from 49 (0.160 g, 0.24 mmol). Elution with CH2Cl2/
1
CH3OH (9:1) afforded 7 as a waxy solid: 0.093 g (68%). H
NMR (CDCl3, 400 MHz) δ 0.86 (s, 6H), 1.14−1.15 (AB m,
2H), 1.22−1.37 (m, 13H), 1.43−1.53 (m, 7H), 1.64−1.68 (m,
4H), 1.98−2.05 (m, 2H), 2.16−2.17 (m, 1H), 2.43−2.49 (m,
2H), 2.66−2.70 (m, 2H), 3.16−3.17 (m, 1H), 3.29−3.35 (m,
1H), 3.79 (AB d, J = 15.2, 1H), 3.83 (s, 3H), 4.10−4.15 (m
+AB d, J = 15.2 Hz, 2H), 4.61 (br m, 1H), 6.00 (dd, J1 = 10.4,
Hz, J2= 4.8 Hz, 1H), 6.08 (d, J = 10.0 Hz, 1H), 6.61 (d, J = 8.4,
1H), 6.66 (d, J = 8.4, 1H). 13C NMR(CDCl3, 100 MHz) δ
27.27, 27.30, 27.40, 29.25, 29.51, 29.66, 29.94, 30.06, 32.42,
32.97, 40.18, 42.48, 46.21, 50.43, 51.46, 55.84, 55.90, 58.60,
62.07, 88.74, 111.12, 121.97, 126.97, 127.53, 129.51, 133.15,
127.86, 133.10, 144.28, 145.83. [α]25 = −65.3 (c 0.10,
D
CHCl3). UPLC/MS: purity 95%, MS (ESI+): m/z 523.4 [M +
H]+.
(4aS,6R,8aS)-11-(2-((2-((3,5-Dimethyladamantan-1-yl)-
amino)ethyl)thio)ethyl)-3-methoxy-5,6,9,10,11,12-hexahy-
dro-4aH-benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (14). 14
was synthesized from 68 (0.075 g, 0.12 mmol). Elution with
CH2Cl2/CH3OH/33% aqueous ammonia (9:1:0.05) afforded
1
14 as a waxy solid: 0.055 g (88%). H NMR (CDCl3, 400
144.01, 145.77, 177.21. [α]25 = −63.5 (c 0.12, CHCl3).
MHz) δ 0.82 (s, 6H), 1.04−1.13 (AB m, 2H), 1.19−1.27 (m,
6H), 1.43 (br s, 2H), 1.48−1.51 (m, 1H), 1.98−2.04 (m, 2H),
2.09−2−11(m, 1H), 2.59−2.75 (m, 8H), 3.15 (m, 1H), 3.18
(m, 1H), 3.36−3.42 (m, 1H), 3.60−3.64 (m, 1H), 3.72−3.76
(m, 1H), 3.81 (s, 3H), 3.82 (d, J = 15.2 Hz, 1H), 4.10−4.17 (m
+d, J = 15.2 Hz, 2H), 4.58 (br m, 1H), 5.99 (dd, J1 = 10.0, J2 =
4.4 Hz, 1H), 6.05 (d, J = 10.0 Hz, 1H), 6.58 (d, J = 8.4 Hz,
1H), 6.63 (d, J = 8.4 Hz, 1H). 13 C NMR (CDCl3, 100 MHz) δ
29.67, 29.86, 29.93, 30.25, 30.30, 32.37, 33.00, 33.68, 39.65,
41.27, 42.85, 42.97, 48.41, 49.00, 49.04, 50.91, 51.70, 52.25,
55.89, 57.38, 62.03, 71.11, 72.29, 88.69, 111.17, 121.94, 126.74,
127.69, 129.15, 133.07, 144.20, 145.90. [α]25D = −68.7 (c 0.09,
CHCl3) UPLC/MS: purity 97%, MS (ESI+): m/z 539 [M +
H]+.
General Procedure for the Synthesis of 9−11. p-TsCl (1
equiv) was added to a mixture of the appropriate alcohol (1
equiv), Et3N (2 equiv), and DMAP (catalytic amount) in dry
CH2Cl2 (0.1 M) under N2 atmosphere, with stirring at 0 °C.
The stirring was continued at room temperature for 3.5 h. After
completion, the reaction was quenched with saturated NH4Cl
aqueous solution and extracted with AcOEt (3 × 10 mL).
Combined extracts were washed with brine, dried over Na2SO4,
and evaporated to give a residue that was transferred into a
pressure tube. 20 (0.5 equiv) and Et3N (2 equiv) in MeCN
D
UPLC/MS: purity 100%, MS (ESI+): m/z 563.4 [M + H]+.
(4aS,6R,8aS)-11-(9-((3,5-Dimethyladamantan-1-yl)-
amino)nonyl)-3-methoxy-5,6,9,10,11,12-hexahydro-4aH-
benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (8). 8 was synthe-
sized from 50 (0.095 g, 0.14 mmol). Elution with CH2Cl2/
1
CH3OH (9:1) afforded 8 as a waxy solid: 0.060 (75%). H
NMR (CDCl3, 400 MHz) δ 0.85 (s, 6H), 1.11−1.14 (AB m,
2H), 1.26−1.59 (m, 25 H), 1.98−2.09 (m, 2H), 2.16−2.17 (m,
1H), 2.40−2.53 (m, 2H), 2.60−2.71 (m, 3H), 3.15−3.18 (m,
1H), 3.21−3.37 (m, 1H), 3.79 (AB d, J = 15.2, 1H), 3.83 (s,
3H), 4.10−4.14 (m+AB d, J = 15.2 Hz, 2H), 4.61 (br m, 1H),
6.00 (dd, J1= 10.4 Hz, J2= 4.8 Hz, 1H), 6.10 (d, J = 10.0 Hz,
1H), 6.61 (d, J = 8.4, 1H), 6.65 (d, J = 8.4, 1H). 13C NMR
(CDCl3, 100 MHz) δ 27.35, 27.45, 29.40, 29.48, 29.52, 29.95,
30.12, 30.19, 32.40, 32.98, 40.56, 42.76, 48.41, 50.70, 51.50,
55.87, 57.82, 62.07, 88.71, 111.10, 121.95, 126.98, 127.51,
129.62, 133.15, 143.10, 145.77. [α]25 = −57.2 (c 0.10,
D
CHCl3). UPLC/MS: purity 99%, MS (ESI+): m/z 577.6 [M +
1]+.
(4aS,6R,8aS)-11-(2-(2-(2-((3,5-Dimethyladamantan-1-yl)-
amino)ethoxy)ethoxy)ethyl)-3-methoxy-5,6,9,10,11,12-hexa-
hydro-4aH-benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (12). 12
was synthesized from 66 (0.080 g, 0.12 mmol). Elution with
H
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Journal of Medicinal Chemistry
Article
were added, and the resulting mixture was stirred at 80 °C for
48 h. After evaporation of the solvent, the residue was purified
by flash chromatography.
dropwise the appropriate acylating agent. The reaction was
stirred at room temperature for 4 h. After evaporation of the
solvent, the crude was purified by flash chromatography (see
Supporting Information for details).
(4aS,6R,8aS)-11-(6-((3,5-Dimethyladamantan-1-yl)-
(methyl)amino)hexyl)-3-methoxy-5,6,9,10,11,12-hexahydro-
4aH-benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (9). 9 was syn-
thesized from 21 (0.210 g, 0.72 mmol). Elution with CH2Cl2/
CH3OH/33% aqueous ammonia (9:1:0.15) afforded 9 as a
waxy solid: 0.085 g (43%). 1H NMR (CDCl3, 400 MHz) δ 0.87
(s, 6H), 1.08−1.17 (AB m, 2H), 1.24−1.33 (m, 9H), 1.38−1.52
(m, 7H), 1.60−1.65 (m, 3H), 1.98−2.07 (m, 2H), 2.19 (br m,
1H), 2.35 (s, 3H), 2.41−2.53 (m, 4H), 2.67−2.71 (m, 1H),
3.11−3.18 (m, 1H), 3.31−3.38 (m, 1H), 3.80 (AB d, J = 16 Hz,
1H) 3.83 (s, 3H), 4.10−4.14 (m+AB d, J = 16 Hz, 2H), 4.61
(br m, 1H), 6.00 (dd, J1= 10.0 Hz, J2= 4.8 Hz, 1H), 6.09 (d, J =
10.0 Hz, 1H), 6.61 (d, J = 8.4 Hz, 1H), 6.66 (d, J = 8.4 Hz,
1H). 13C NMR (CDCl3, 100 MHz) δ 24.36, 27.20, 27.38,
27.51, 28.63, 29.94, 30.14, 30.38, 32.48, 32.91, 33.64, 36.45,
41.26, 42.07, 42.67, 44.02, 47.15, 48.41, 49.69, 50.60, 51.48,
55.88, 55.94, 57.78, 62.07, 88.71, 111.13, 121.99, 126.98,
127.52, 129.52, 133.14, 144.01, 145.76. [α]25D = −45.8 (c 0.11,
CHCl3). UPLC/MS: purity 95%, MS (ESI+): m/z 549.5 [M +
H]+.
General Procedure for the Synthesis of 16−18. A mixture
of the chloride derivative (1.2 equiv), 20 (1 equiv), Et3N (2
equiv), and KI (catalytic amount) in MeCN (0.1 M) was stirred
at 80 °C for 48 h in a pressure tube. After evaporation of the
solvent, the residue was purified by flash chromatography.
N-(3,5-Dimethyladamantan-1-yl)-4-((4aS,6R,8aS)-6-hy-
droxy-3-methoxy-5,6,9,10-tetrahydro-4aH-benzo[2,3]-
benzofuro[4,3-cd]azepin-11(12H)-yl)butanamide (16). 16
was synthesized from 51 (0.070 g, 0.25 mmol). Elution with
CH2Cl2/CH3OH/33% aqueous ammonia (9:1:0.02) afforded
1
16 as a waxy solid: 0.070 g (65%). H NMR (CDCl3, 400
MHz) δ 0.83 (s, 6H), 1.13−1.15 (AB m, 2H), 1.26−1.38 (AB
m, 4H), 1.61 (br m, 4H), 1.72−1.79 (m, 4H), 1.98−2.04 (m,
2H), 2.11−2.14 (m, 3H), 2.47−2.55 (m, 3H), 2.65−2.70 (m,
1H), 3.11−3.18 (m, 1H), 3.31−3.38 (m, 1H), 3.77 (AB d, J =
16 Hz, 1H) 3.83 (s, 3H), 4.09−4.14 (m+AB d, J = 16 Hz, 2H),
4.60 (br m, 1H), 5.57 (br s,1H), 6.00 (dd, J1= 10.0 Hz, J2= 4.8
Hz, 1H), 6.09 (d, J = 10.0 Hz, 1H), 6.61 (d, J = 8.4 Hz, 1H),
6.66 (d, J = 8.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ
22.92, 30.05, 32.30, 33.04, 35.24, 40.17, 42.63, 47.60, 48.33,
50.56, 51.52, 53.25, 58.81, 55.89, 55.96, 57.60, 61.98, 88.68,
111.24, 122.04, 126.82, 127.65, 129.01, 133.10, 144.11, 145.82,
(4aS,6R,8aS)-11-(7-((3,5-Dimethyladamantan-1-yl)-
(methyl)amino)heptyl)-3-methoxy-5,6,9,10,11,12-hexahy-
dro-4aH-benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (10). 10
was synthesized from 22 (0.190 g, 0.62 mmol). Elution with
CH2Cl2/CH3OH/33% aqueous ammonia (9:1:0.15) afforded
172.01. [α]25 = −60.1 (c 0.11, CHCl3). UPLC/MS: purity
D
96%, MS (ESI+): m/z 521.4 [M + H]+.
1
10 as a waxy solid: 0.084 g (49%). H NMR (CDCl3, 400
N-(3,5-Dimethyladamantan-1-yl)-5-((4aS,6R,8aS)-6-hy-
droxy-3-methoxy-5,6,9,10-tetrahydro-4aH-benzo[2,3]-
benzofuro[4,3-cd]azepin-11(12H)-yl)pentanamide (17). 17
was synthesized from 52 (0.070 g, 0.24 mmol). Elution with
CH2Cl2/CH3OH/33% aqueous ammonia (9:1:0.02) afforded
MHz) δ 0.82 (s, 6H), 1.03−1.08 (AB m, 2H), 1.22−1.36 (m,
14H), 1.40−1.49 (m, 5H), 1.52−1.55 (m, 2H), 1.95−2.00 (m,
2H), 2.12−2.18 (m, 1H), 2.25 (s, 3H), 2.37−2.48 (m, 4H),
2.67−2.67 (m, 1H), 3.11−3.15 (m, 1H), 3.28−3.31 (m, 1H),
3.78 (AB d, J = 16 Hz, 1H) 3.83 (s, 3H), 4.07−4.11 (m+AB d, J
= 16 Hz, 2H), 4.57 (br m, 1H), 5.97 (dd, J1= 10.0 Hz, J2= 4.8
Hz, 1H), 6.09 (d, J = 9.6 Hz, 1H), 6.61 (d, J = 8.4 Hz, 1H),
1
17 as a waxy solid: 0.070 g (67%). H NMR (CDCl3, 400
MHz) δ 0.83 (s, 6H), 1.11−1.19 (AB m, 2H), 1.24−1.47 (AB
m, 4H), (m, 4H), 1.49−1.60 (m, 5H), 1.62−1.80 (br m, 2H),
1.98−2.09 (m, 2H), 2.11−2.14 (m, 3H), 2.44−2.54 (m, 2H),
2.65−2.70 (m, 1H), 3.13−3.17 (m, 1H), 3.31−3.37 (m, 1H),
(AB d, J = 15.6 Hz, 1H) 3.83 (s, 3H), 4.09−4.14 (m+AB d, J =
15.6 Hz, 2H), 4.60 (br m, 1H), 5.12 (br s, 1H), 6.00 (dd, J1=
9.6 Hz, J2= 5.2 Hz, 1H), 6.08 (d, J = 10.0 Hz, 1H), 6.60 (d, J =
8.0 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H). 13C NMR (CDCl3, 100
MHz) δ 23.51, 26.87, 30.12, 32.34, 32.99, 37.50, 40.21, 42.63,
47.65, 48.38, 50.57, 51.60, 53.34, 55.92, 57.67, 62.05, 88.72,
111.18, 121.96, 126.94, 127.57, 129.38, 133.15, 144.07, 145.81,
13
6.66 (d, J = 8.4 Hz, 1H). C NMR (CDCl3, 100 MHz) δ
27.32, 27.54, 29.49, 29.65, 29.94, 30.18, 30.50, 32.33, 32.93,
33.73, 36.69, 42.88, 44.33, 48.40, 49.64, 50.79, 51.48, 55.86,
57.76, 62.07, 88.71, 111.11, 121.97, 126.99, 127.51, 129.53,
133.15, 144.00, 145.75. [α]25 = −50.4 (c 0.11, CHCl3).
D
UPLC/MS: purity 98%, MS (ESI+): m/z 563.5 [M + H]+.
(4aS,6R,8aS)-11-(8-((3,5-Dimethyladamantan-1-yl)-
(methyl)amino)octyl)-3-methoxy-5,6,9,10,11,12-hexahydro-
4aH-benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (11). 11 was
synthesized from 23 (0.160 g, 0.50 mmol). Elution with
CH2Cl2/CH3OH/33% aqueous ammonia (9:1:0.15) afforded
171.97. [α]25 = −57.8 (c 0.11, CHCl3). UPLC/MS: purity
D
100%, MS (ESI+): m/z 535.4 [M + H]+.
1
11 as a waxy solid: 0.075 g (52%). H NMR (CDCl3, 400
N-(3,5-Dimethyladamantan-1-yl)-6-((4aS,6R,8aS)-6-hy-
droxy-3-methoxy-5,6,9,10-tetrahydro-4aH-benzo[2,3]-
benzofuro[4,3-cd]azepin-11(12H)-yl)hexanamide (18). 18
was synthesized from 53 (0.070 g, 0.224 mmol). Elution with
CH2Cl2/CH3OH/33% aqueous ammonia (9:1:0.02) afforded
MHz) δ 0.81 (s, 6H), 1.01−1.13 (AB m, 2H), 1.25−1.31 (m,
16H), 1.38−1.46 (m, 5H), 1.47−1.50 (m, 2H), 1.94−2.02 (m,
2H), 2.10−2.11 (m, 1H), 2.19 (s, 3H), 2.31−2.38 (m, 4H),
2.40−2.77 (m, 1H), 3.11−3.15 (m, 1H), 3.28−3.32 (m, 1H),
3.78 (AB d, J = 16 Hz, 1H) 3.83 (s, 3H), 4.07−4.11 (m+AB d, J
= 16 Hz, 2H), 4.58 (br m, 1H), 5.97 (dd, J1= 10.0 Hz, J2= 5.2
Hz, 1H), 6.07 (d, J = 10.0 Hz, 1H), 6.58 (d, J = 8.4 Hz, 1H),
1
18 as a waxy solid: 0.080 g (78%). H NMR (CDCl3, 400
MHz) δ 0.83 (s, 6H), 1.07−1.14 (AB m, 2H), 1.18−1.38 (AB
m, 4H), 1.48−1.66 (m, 10H), 1.81 (br m, 2H), 1.96−2.06 (m,
4H), 2.12 br (m, 1H), 2.41−2.54 (m, 3H), 2.41−2.54 (m, 1H),
3.14−3.18 (m, 1H), 3.27−3.37 (m, 1H), 3.77 (AB d, J = 16 Hz,
1H) 3.83 (s, 3H), 4.09−4.14 (m+AB d, J = 16 Hz, 2H), 4.60
(br m, 1H), 5.23 (br s, 1H), 6.00 (dd, J1= 10.0 Hz, J2= 4.8 Hz,
1H), 6.09 (d, J = 10.0 Hz, 1H), 6.61 (d, J = 8.4 Hz, 1H), 6.66
(d, J = 8.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ 25.54,
26.84, 27.08, 29.63, 30.04, 32.30, 32.85, 37.52, 40.15, 42.62,
47.58, 48.35, 50.55, 51.43, 53.30, 55.81, 55.87, 57.65, 62.01,
13
6.63 (d, J = 8.4 Hz, 1H). C NMR (CDCl3, 100 MHz) δ
27.45, 29.54, 29.95, 30.24, 30.62, 32.23, 32.98, 33.90, 37.02,
43.08, 44.74, 48.41, 49.70, 50.99, 51.51, 55.87, 57.81, 62.08.
[α]25 = −48.6 (c 0.11, CHCl3). UPLC/MS: purity 95%, MS
D
(ESI+): m/z 577.5 [M + H]+.
General Procedure for the Synthesis of 51−53. To a
vigorously stirred solution of 2 hydrochloride (0.300 g, 1.40
mmol) and K2CO3 (0.480 g, 3.50 mmol) in MeCN was added
I
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Journal of Medicinal Chemistry
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61.99, 88.67, 111.18, 121.98, 126.91, 127.53, 129.25, 133.09,
afforded 15 as a waxy solid: 0.012 g (41%). 1H NMR
(CDCl3, 400 MHz) δ 0.84 (s, 6H), 1.09−1.17 (AB m, 2H),
1.17−1.39 (m, 8H), 1.48−1.57 (m, 4H), 1.67−1.68 (m, 2H),
1.79−1.80 (m, 2H), 1.97−2.08 (m, 2H), 2.16 (br m, 1H),
2.49−2.58 (m, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.76−2.83 (m,
4H), 2.90 (br m, exchangeable with D2O, 1H), 3.13−3−16 (m,
1H), 3.31−3.34 (m, 1H), 3.77−3.81 (s+AB d, J = 15.2 Hz,
4H), 4.09−4.13 (m+AB d, J = 15.2 Hz, 2H), 4.58 (br m, 1H),
5.97 (dd, J1 = 10.2 Hz, J2 = 4.3 Hz, 1H), 6.05 (d, J = 10.4 Hz,
144.01, 145.74, 172.17. [α]25 = −60.8 (c 0.11, CHCl3).
D
UPLC/MS: purity 100%, MS (ESI+): m/z 549.5 [M + H]+.
3-((tert-Butoxycarbonyl)amino)propyl 4-Methylbenzene-
sulfonate (69). 69 was synthesized starting from 3-amino-
propanol according to a literature procedure.38 1H NMR
(CDCl3, 400 MHz) δ 1.35 (s, 9H), 1.75−1.81 (m, 2H), 2.38,
(s, 3H), 3.09 (br m, 2H), 4.02 (t, J = 6.0 Hz, 2H), 4.70 (br m,
1H), 7.30 (d, J = 8.0 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H).
tert-Butyl (3-((4aS,6R,8aS)-6-Hydroxy-3-methoxy-5,6,9,10-
tetrahydro-4aH-benzo[2,3]benzofuro[4,3-cd]azepin-11-
(12H)-yl)propyl)carbamate (70). In a pressure tube a mixture
of 69 (0.101 g, 0.31 mmol), 20 (0.070 mg, 0.26 mmol), and
Et3N (0.07 mL, 0.73 mmol) in CH3CN (3 mL) was stirred at
80 °C for 48 h. After evaporation of the solvent, the residue was
purified by flash chromatography. Elution with CH2Cl2/
CH3OH (9:1) afforded 70 as a waxy solid: 0.080 g (73%).
1H NMR (CDCl3, 400 MHz) δ 1.40 (s, 9H), 1.47−1.50 (AB m,
1H), 1.55−1.65 (m, 2H), 1.94−2.04 (m, 2H), 2.46−2.56 (m,
2H), 2.63−2.68 (AB m, 1H), 3.11−3.14 (m, 3H), 3.28−3.35
(m, 1H), (AB d, J = 15.2 Hz, 1H), 3.81 (s, 3H), 4.07−4.11 (m
+AB d, J = 15.2 Hz, 2H), 4.57 (br m, 1H), 4.99 (br m, 1H),
5.97 (dd, J1 = 10.2 Hz, J2 = 4.3 Hz, 1H), 6.06 (d, J = 10.4 Hz,
1H), 6.57 (d, J = 8.4, 1H), 6.62 (d, J = 8.0, 1H).
13
1H), 6.60 (d, J = 8.4, 1H), 6.63 (d, J = 8.0, 1H). C NMR
(CDCl3, 100 MHz) δ 22.66, 26.86, 29.33, 29.63, 29.67, 29.92,
29.96, 30.07, 31.90, 32.42, 32.93, 39.79, 40.33, 42.58, 47.32,
47.83, 48.40, 49.35, 50.54, 51.76, 55.88, 57.51, 62.03, 88.71,
111.17, 122.02, 126.81, 127.66, 129.13, 133.13, 144.14, 115.82.
[α]25 = −40.3 (c 0.09, CHCl3). UPLC/MS: purity 95%, MS
D
(ESI+): m/z 550 [M + H]+.
Biological Evaluation. Materials. [3H]Ifenprodil (40 Ci/
mmol) and [3H]MK-801 (27.5 Ci/mmol) were obtained from
Perkin-Elmer Life Science Inc. (Boston, MA). Memantine and
galantamine were obtained from Tocris (Bristol, U.K.). Other
compounds and reagents were purchased from Sigma-Aldrich
(Milan, Italy).
[3H]MK-801 Binding Assay. Crude synaptic membranes
were prepared from the cerebral cortex of Sprague−Dawley rats
using a modified method from Marvizon
̀
et al.39 The cerebral
(4aS,6R,8aS)-11-(3-Aminopropyl)-3-methoxy-
5,6,9,10,11,12-hexahydro-4aH-benzo[2,3]benzofuro[4,3-cd]-
azepin-6-ol (71). HCl (4 M) in dioxane (1 mL) was carefully
added to 70 (0.080 g, 0.186 mmol) at 0 °C, and the solution
was stirred at room temperature for 2 h. After removal of the
solvent, the obtained residue was purified by flash chromatog-
raphy. Elution with CH2Cl2/CH3OH/33% aqueous ammonia
(9:1:0.15) afforded 71 as a waxy solid: 0.046 g (75%). 1H NMR
(CDCl3, 400 MHz) δ 1.47−1.50 (AB m, 1H), 1.61−1.72 (m,
2H), 1.94−2.04 (m, 2H), 2.52−2.60 (m, 2H), 2.62−2.66 (AB
m, 1H), 2.82 (br m, 2H), 3.14−3.36 (m+br m exchangeable
with D2O, 4H), 3.80 (s, 3H), 3.81 (AB d, J = 15.2 Hz, 1H),
4.07−4.11 (m+AB d, J = 15.2 Hz, 2H), 4.56 (br m, 1H), 5.96
(dd, J1 = 10.2 Hz, J2 = 4.3 Hz, 1H), 6.06 (d, J = 10.4 Hz, 1H),
6.58 (d, J = 8.4, 1H), 6.60 (d, J = 8.0, 1H).
tert-Butyl (3,5-Dimethyladamantan-1-yl)(3-((3-
((4aS,6R,8aS)-6-hydroxy-3-methoxy-5,6,9,10-tetrahydro-
4aH-benzo[2,3]benzofuro[4,3-cd]azepin-11(12H)-yl)propyl)-
amino)propyl)carbamate (72). In a pressure tube a mixture of
71 (0.046 g, 0.139 mmol), 38 (0.068 mg, 0.14 mmol), and
Et3N (0.039 mL, 0.278 mmol) in MeCN (2 mL) was stirred at
80 °C for 24 h. Elution with CH2Cl2/CH3OH/33% aqueous
ammonia (9:1:0.04) afforded 72 as a waxy solid: 0.035 g (39%).
1H NMR (CDCl3, 400 MHz) δ 0.82 (s, 6H), 1.10−1.12 (AB m,
2H), 1.23−1.27 (m, 12H), 1.47 (s, 9H), 1.61−1.68 (m, 4H),
1.79−1.83 (m, 2H), 1.89−2.04 (m, 2H), 2.16 (br m, 1H),
2.53−2.54 (m, 1H), 2.80−2.83 (m, 2H), 2.92−2.94 (m, 1H),
3.03−3.09 (m, 2H), 3.28−3.49 (m, 3H), 3.81 (s, 3H), 3.99 (AB
d, J = 15.2 Hz, 1H), 4.15 (AB d, J = 15.2 Hz, 1H), 4.58 (br m,
1H), 5.98−5.99 (m, 2H), 6.60 (d, J = 8.4, 1H), 6.63 (d, J = 8.0,
1H).
cortex was removed and homogenized in 10 vol. (v/w) of ice-
cold 0.32 M sucrose solution. The homogenate was centrifuged
at 1000g for 10 min and the resulting supernatant centrifuged at
10 000g for 20 min. The pellets were washed (resuspended,
homogenized, and centrifuged at 48 000g for 20 min) twice in
50 vol of Tris−HEPES buffer (Tris 4.5 mM, HEPES 5 mM, pH
7.4) containing EDTA 1 mM and twice in Tris−HEPES buffer
without EDTA. The pellets were stored at −80 °C for at least
18 h.
Before the binding assay, the membrane pellets were washed
three more times with Tris−HEPES buffer (Tris 4.5 mM,
HEPES 5 mM, pH 7.4) to remove the endogenous amino acids.
In the binding assay, [3H]MK-801 (final concentration 3 nM),
50 μL of membrane preparation (40−50 μg protein), and 10
μL of compound were mixed at 25 °C in the presence of L-
glutamate (10 μM), glycine 50 μL (10 μM). Tris−HEPES
buffer (Tris 4.5, HEPES 5 mM, pH 7.4) was added to a final
volume of 0.5 mL. Following incubation for 2 h at 25 °C,
binding was terminated by filtration using Whatman GF/B
filters and a Brandel M-48 Cell Harvester. Radioactivity was
measured using a PerkinElmer liquid scintillation counter.
Nonspecific binding was determined in the presence of
unlabeled 100 μM (1)-MK-801. The dissociation constant
(Kd) of [3H]MK-801 in rat cortex membranes was 4.0 nM. For
compound activity determination, aliquots of membrane pellets
were incubated with different ligand concentrations in the
presence of 3 nM [3H]MK-801 for 2 h at 25 °C. Samples were
then filtered, and the radioactivity was counted.
[3H]Ifenprodil Binding Assay. Rat cortex membranes were
prepared as previously reported.40 The frontal cortex
membranes from adult male Sprague−Dawley rats were pooled
and homogenized in 10 volumes of ice-cold 0.32 M sucrose/50
mM Tris/acetate buffer (pH 7.4) (homogenizing buffer) in a
Teflon pestle homogenizer. The homogenate was centrifuged at
1000g for 10 min at 4 °C, and the supernatant was collected.
The pellet was dissolved in a small volume of homogenizing
buffer and centrifuged again at 1000g for 10 min at 4 °C. The
supernatant was collected together with the first supernatant
(4aS,6R,8aS)-11-(3-((3-((3,5-Dimethyladamantan-1-yl)-
amino)propyl)amino)propyl)-3-methoxy-5,6,9,10,11,12-hex-
ahydro-4aH-benzo[2,3]benzofuro[4,3-cd]azepin-6-ol (15).
HCl (4 M) in dioxane (1 mL) was carefully added to 72
(0.035 g, 0.05 mmol) at 0 °C, and the solution was stirred at
room temperature for 2 h. After removal of the solvent, the
obtained residue was purified by flash chromatography. Elution
with CH2Cl2/CH3OH/33% aqueous ammonia (9:1:0.20)
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and centrifuged at 17 000g for 20 min at 4 °C. The pellet (P2)
was homogenized in 20 volumes of ice-cold 50 mM Tris/
acetate buffer, pH 7.0 and centrifuged at 50 000g for 30 min at
4 °C. This last centrifugation step was repeated three times.
The final pellet was suspended in ice-cold 50 mM Tris/acetate
buffer, pH 7.0, and aliquots of 0.5 mL were stored at −80 °C.
The protein content was measured by the method described by
Lowry et al.41
SEM of AChE activity, set to 100%. IC50 values were
determined graphically from log concentration−inhibition
curves (GraphPad Prism 4.03 software, GraphPad Software
Inc.).
Cell Culture, Pharmacological Treatments, and Viability
Assay. SHSY-5Y cells were maintained in DMEM/F12 medium
supplemented with 15% fetal bovine serum, 1% nonessential
amino acids, 2 mM L-glutamine, and penicillin/streptomycin in
a humidified atmosphere (5% CO2) at 37 °C.
The assay was carried out in a total volume of 500 μL, in the
presence of about 10 nM [3H]ifenprodil in binding buffer
containing 50 μg of protein and 100 μM of trifluoperazine, to
block the non-NMDA receptor [3H]ifenprodil binding.
Unlabeled ifenprodil, 100 μM, was used to define nonspecific
binding. After 2 h of incubation at 25 °C in a water bath, bound
radioligand was separated from unbound by filtration through
presoaked (0.5% polyethyleneimine solution) presoaked glass
fiber filters (Whatman GF/B) under reduced pressure. The
filters were rinsed with 4 mL of cold binding buffer three times.
The filters were placed into individual 4 mL scintillation vials
and prepared for counting using conventional liquid spectros-
copy. The radioactivity was measured in a β-counter. The
dissociation constant (Kd) of [3H]ifenprodil in rat cortex
membranes was 28.0 nM. All compounds were routinely
dissolved in DMSO and diluted with assay buffer to the final
concentration with the final amount of DMSO never exceeding
2%. Percent inhibition values for specific radiolabeled ligand
binding results at 10−100 μM concentration are means SEM
of at least three determinations. At least five different
concentrations (spanning 3 orders of magnitude) were adjusted
appropriately for the IC50 of each compound examined. IC50
values were computer-generated using a nonlinear regression
formula (Graph-Pad, San Diego, CA) and were converted to Ki
values using the known Kd values of the radioligands in the
different tissues and using the Cheng−Prusoff equation.42 Ki
values were means SEM of at least three determinations.
Rat Acetylcholinesterase (AChE) Inhibition Assays. The
cholinesterase assay method of Ellman25 was used to determine
the in vitro cholinesterase activity. The activity was measured
by increase in absorbance at 412 nm due to the yellow color
produced from the reaction of acetylthiocholine iodide with the
dithiobisnitrobenzoate (DTNB) ion. AChE was prepared from
the brain of Wistar rats by homogenizing under a Teflon
blender for 10 min in 0.1 M KH2PO buffer, pH 8. A stock
solution of the enzyme in 0.1 M KH2PO buffer (pH 8),
containing 0.1% Triton X-100, was kept frozen. Acetylthiocho-
line iodide and DTNB were prepared daily using 0.1 M
KH2PO4 buffer (pH 7). The assay solution consisted of a 0.1 M
phosphate buffer, pH 8.0, with the addition of 340 μM DTNB,
0.02 unit/mL of rat AChE, and 550 μM of substrate
(acetylthiocholine iodide).
Cells were seeded in 96-well microplates (5000 cells/well);
on the next day, cells were incubated with 500 μM NMDA, in
the absence or presence of different compound concentrations.
At least six different concentrations, ranging from 0.01 nM to
100 μM, were tested for standard and newly synthesized
compounds. The compounds were added for 5 min prior to
addition of NMDA, to determine the inhibition of NMDA-
mediated cell toxicity.
Following incubation times, cell viability was determined
using the MTS assay according to the manufacturer’s
instructions. The dehydrogenase activity in active mitochondria
reduced 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) to the solu-
ble formazan product. The absorbance of formazan at 490 nM
was measured in a colorimetric assay with an automated plate
reader.
Within an experiment, each condition was assayed in
duplicate or triplicate and each experiment was performed at
least three times. The results were calculated by subtracting the
mean background from the values obtained from each test
condition and were expressed as the percentage of the control
(untreated cells). A nonlinear multipurpose curve-fitting
program, Prism (GraphPad Software Inc., San Diego, CA),
was used for data analysis of cell viability; the IC50 value
represents the compound concentration able to give 50% of
NMDA-induced toxicity. Student’s t test was used to evaluate
whether differences between the experimental groups and the
control were statistically significant.
Docking Simulations. Ligand docking simulations were
performed on a receptor model generated using ICM3.743
based on the crystal structure of the human AChE solved in
complex with fasciculin (PDB code: 1B41).44 Fasciculin was
removed from the complex. The protein atoms were assigned
the correct atom types according to a modified version of the
ECEPP/3 force field. Hydrogen atoms were added. Heavy side
chain atoms that were not solved in the crystal structure and
assigned occupancy equal to zero (Glu268: cg, cd, oe1, oe2;
Gln291: cd, oe1, ne2; Gln369: cd, oe1, ne2; Arg522: cz, nh1,
nh2) were added according to the topology of the ICM library
of standard residues. None of these side chain atoms turned out
to be involved in the definition of the binding pocket (see
below), being either far from the binding site or pointing
toward the bulk of the solvent.
Assay solutions were preincubated for 10 min at 37 °C,
followed by the addition of substrate. The inhibitory activity of
the newly synthesized compounds was assayed by adding
different compound concentrations to the reaction mixture
described above. All the inhibitors were dissolved in DMSO,
whose concentration never exceeded 1% in the final reaction
mixture. The rate of the increase in absorbance at 412 nm was
followed for 5 min at 37 °C with a PerkinElmer
spectrophotometer. Assays were carried out with a blank
containing all components except AChE to account for the
nonenzymatic reaction. The reaction rates were compared, and
the percent inhibition due to the presence of tested compounds
was calculated. Data are expressed as percentage of inhibition
The side chains of the residues lining the enzyme gorge had
to be optimized to allow relaxing of the conformation of
Tyr337 and nearby residues in such a way as to bind bulky
ligands. For this purpose, the structure of the human enzyme
was superimposed and tethered to the crystal structure of
TcAChE solved in complex with a dual derivative of 1 (PDB
code 1W4L)21 using the ICM built-in impose_conformation
macro. It appeared safe to adopt this procedure because of the
high level of conservation between the two orthologues.
According to BLAST, TcAChE and hAChE shared 58.9%
sequence identity45 and 75% homology.46 The superimposition
K
dx.doi.org/10.1021/jm3009458 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
procedure was based on an iterative algorithm47 that attempts
to find the ideal overlapping core between structures. Atom
equivalences were established on the fly according to sequence
alignment. On account of this approach, a minority of deviating
atoms did not affect the overall quality of the superimposition.
Then, the tethering procedure pulled hAChE atoms toward the
positions of corresponding atoms in TcAChE according to the
alignment and by means of harmonic restraints. Finally,
tautomeric states of histidines, the positions of asparagine and
glutamine side-chain amidic groups, and the side chains in
which heavy atoms were added were optimized through a
Monte Carlo procedure to improve the hydrogen bonding
pattern in the new conformation. Polar hydrogen atoms were
also optimized. Ligands were assigned the MMFF force field
atom types and charges.48
The boundaries of the binding pocket were defined by
selecting all the residues with at least one side-chain non-
hydrogen atom in the range of 5 Å from the crystallographic
position of a previously reported dual derivative of 1 (PDB
code 1W4L).21 When considering only the binding site
residues, sequence identity between hAChE and TcAChE was
86% and homology 94%. The backbone displacement in the
generated hAChE binding pocket conformation measured in
terms of RMSD with respect to the original crystal structure
was 0.7 Å (1.0 Å including side-chain heavy atoms). Tyr337
was the only amino acid that displayed a significant
conformational rearrangement. As expected, the dihedral angles
describing the position of the Tyr337 side chain in hAChE (χ1
−133, χ2 −34) ended up closely resembling those of Phe330 in
TcAChE (χ1 −137, χ2 −39).
Docking simulations were carried out by means of the Biased
Probability Monte Carlo stochastic optimizer as implemented
in ICM. The molecular conformation of the system was
described by means of internal coordinate variables. The
binding site residues were described by precalculated 0.5 Å
spacing potential grid maps, representing van der Waals
potentials for hydrogens and heavy atoms, electrostatics,
hydrophobicity, and hydrogen bonding, respectively. The van
der Waals interactions were described by a smoother form of
the 6−12 Lennard−Jones potential with the repulsive
contribution capped at a cutoff value of 4 kcal/mol. Poses
from Monte Carlo sampling were rescored by means of the
standard ICM empirical scoring function.
5 was docked into the crystal structure of the heterodimer
formed by the amino terminal domains of N1 and N2B
subunits of NMDA receptor solved in complex with
ifenprodil.49 The simulation was performed according to the
SCARE induced fit docking protocol that has been thoroughly
described by Bottegoni and colleagues,50 and it is only briefly
summarized here. SCARE implements the flexibility of the
receptor in the simulation generating different local versions of
the binding pocket. The protein atoms were assigned the
correct atom types according to a modified version of the
ECEPP/3 force field. Hydrogen atoms were added. Tautomeric
states of histidines, the positions of asparagine and glutamine
side-chain amidic groups, and polar hydrogen atoms were
optimized. The binding pocket boundaries were defined,
selecting the residues with at least one non-hydrogen atom
within 5 Å from the crystallographic position of ifenprodil.
Then, ifenprodil was removed from the structure. 5 was
assigned the MMFF force field atom types and charges.48
Twelve variants of the receptor binding pocket were generated,
systematically omitting pairs of proximal side chains. 5 was
docked at each variant according to the already described ICM
standard Monte Carlo docking protocol (see above). The best
scoring poses were collected and employed to seed
independent optimization runs on the receptor, after restoring
the original pocket composition and conformation. For each
optimized complex, the binding score was calculated. The
complex returning the best score was proposed as the putative
binding mode.
ASSOCIATED CONTENT
* Supporting Information
■
S
Docking models of AChE in complex with 5, 7, 8, and 12−18.
Docking model of NR2B-containing NMDAR in complex with
5. Effect of galantamine on NMDA-induced toxicity in SHSY-
5Y cells. Experimental details of chemical syntheses of
intermediates 21−68. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*(A.C.) E-mail: andrea.cavalli@iit.it, phone/fax: +39 010
71781530/710187; (M.R.) e-mail: michela.rosini@unibo.it,
phone/fax: +39 051 2099722/34.
Notes
The authors declare the following competing financial
interest(s): A patent protecting the class of compounds
disclosed in this paper was filed by the following authors:
Cavalli, A.; Rosini, M.; Simoni, E.; Reggiani, A.; Melchiorre, C.
ACKNOWLEDGMENTS
■
We thank Sine Mandrup Bertozzi for technical assistance. We
thank Grace Fox for editing and proofreading the manuscript.
Tiziano Bandiera is gratefully acknowledged for useful
discussions. C.M. and M.R. gratefully acknowledge MIUR for
the financial support (PRIN 2009ESXPT2_001).
ABBREVIATIONS USED
■
AChE, acetylcholinesterase; AD, Alzheimer’s disease; DTNB,
dithiobisnitrobenzoate; LELP, ligand efficiency lipophilicity;
MTDLs, multitarget-directed ligands; MTS, 3-(4,5-dimethylth-
iazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium; NMDA, N-methyl-D-aspartate; NMDAR, N-
methyl-D-aspartate receptor; NR2B, 2B subunit of the NMDA
receptor
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