Synthesis of monomeric derivatives to probe memoquin's bivalent interactions

Article abstract of DOI:10.1021/jm200691d

Eight monomeric congeners, related to the multitarget lead candidate memoquin, were prepared and evaluated at multiple targets to determine their profile against Alzheimer's disease. 2-4 bind to AChE with similar low nanomolar affinities and function as effective inhibitors of amyloid aggregation. The most potent monovalent ligand 2 also inhibits BACE-1 in vitro and APP metabolism in primary chicken telencephalic neurons. (Figure presented)

Full text of DOI:10.1021/jm200691d

Article
pubs.acs.org/jmc
Synthesis of Monomeric Derivatives To Probe Memoquin’s Bivalent
Interactions
Maria Laura Bolognesi,*,† GianPaolo Chiriano,‡ Manuela Bartolini,† Francesca Mancini,†
Giovanni Bottegoni,§ Valentina Maestri,† Stefan Czvitkovich,∥ Manfred Windisch,∥ Andrea Cavalli,†,§
Anna Minarini,† Michela Rosini,† Vincenzo Tumiatti,† Vincenza Andrisano,† and Carlo Melchiorre†
†Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
‡Statistical and Biological Physics Sector, SISSA-ISAS, Via Bonomea 265, 34136 Trieste, Italy
§Department of Drug Discovery and Development, Italian Institute of Technology, Via Morego 30, 16163 Genova, Italy
∥JSW Lifesciences GmbH, Parkring 12, 8074 Grambach, Austria
S
* Supporting Information
ABSTRACT: Eight monomeric congeners, related to the multitarget
lead candidate memoquin, were prepared and evaluated at multiple
targets to determine their profile against Alzheimer’s disease. 2−4 bind to
AChE with similar low nanomolar affinities and function as effective
inhibitors of amyloid aggregation. The most potent monovalent ligand 2
also inhibits BACE-1 in vitro and APP metabolism in primary chicken
telencephalic neurons.
that simultaneously bind the catalytic and the peripheral
anionic (PAS) sites of AChE.14−18
INTRODUCTION
■
Memoquin (1) was rationally designed to create a new
chemical entity (NCE) with a polypharmacological profile
against Alzheimer’s disease (AD).1,2 An in vitro and in vivo
characterization revealed its multifunctional mechanism of
action and its interaction with three molecular targets involved
in AD pathology, namely, acetylcholinesterase (AChE), β-
amyloid (Aβ), and β-secretase (BACE-1).3 1 is thus the
successful product of one of the first AD multitarget drug
discovery efforts. This strategy is emerging as an alternative way
to develop effective anti-AD drugs.4−7 Although the single-
target approach remains the main strategy in big pharmaceut-
ical companies, the ongoing failure of current candidates is
convincing the pharmaceutical community that the desired
outcome may be provided more effectively by a drug that
modulates multiple targets.8 From a medicinal chemistry point
of view, 1 is a bivalent ligand, with a symmetrical structure
composed of two 2-methoxybenzyldiamino moieties connected
by a benzoquinone spacer. Several examples of linking two
pharmacophoric units via spacers of different length and
flexibility were reported by Portoghese in the field of opioids.9
In AD too, the bivalent ligand strategy has received attention
over the past decade.10−12 This was mostly motivated by the
peculiar topology of a classical AD target, the enzyme AChE,
which has two recognition sites sharing common molecular
features.13 Consequently, improved potency is shown by drugs
The multitarget approach could be considered an evolution
of the bivalent ligands concept. This is because combining
structural elements from two ligands is the simplest way of
incorporating activity at two targets into a single molecule.19
The rationale for using the bivalent ligand approach in AD also
stems from the possibility that dimeric structures may be
capable of bridging independent recognition sites on other
validated targets (such as Aβ and BACE-1), resulting in a
binding interaction that is thermodynamically more favorable
than the monovalent binding of two molecules. In principle,
this would be particularly advantageous in view of the
complexity of the recognition mechanism of protein−protein
interactions in amyloidosis.20 As a matter of fact, several
amyloid binding compounds share a common bivalent structure
and bivalent “molecular tweezers” have been envisaged as the
next generation of ligands.21 Another positive feature of anti-
AD bivalent ligands is that because of their high hindrance, they
can efficiently fit the extended substrate binding site of BACE-
1.22 However, their high molecular weight (MW) has negative
consequences for the pharmacokinetic profile.
For 1, we could verify absorption through oral administration
and access to the central nervous system.23 Nevertheless, if we
Received: May 31, 2011
Published: November 7, 2011
© 2011 American Chemical Society
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Chart 1. Design Strategy for Monovalent Ligands 2−9
a
Scheme 1
a
Reagents and conditions: (a) EtOH, 4 h, reflux; (b) CH2Cl2, 3 h, air, room temp; (c) MnO2, dry Et2O, 4 h, room temp; (d) MeOH, 4 h, air, room
temp; (e) TFA, CHCl3, 5 h, room temp.
strictly reason in terms of Lipinski’s rule,24 1 violates the MW
parameter, being out of range (632 vs 500). On this basis, we
sought to generate analogues of 1 with a reduced MW, yet
maintaining its promising multitarget profile.
methoxybenzoquinone (5). Conversely, in 6 and 7 a structural
simplification has been performed to investigate the role of the
ethyl- and 2-methoxybenzyl substituents on the basic nitrogen
of 2. Moreover, we reduced the flexibility at the polymethylene
chain of 2 and 5 because conformationally restricted analogues
8 and 9 might show improved potency. Notably, all compounds
displayed a MW below the 500 Da cutoff (see Chart 1 for
design strategy).
In previous studies, we verified that 1,4-Michael addition or
substitution reactions would allow us to effectively incorporate
a diamine chain into the 2-position of a quinone.1,2,28 In the
cases of 2 and 3, we used a substitution reaction, which also led
to the unambiguous synthesis of 3. Thus, treating 2-
methoxynaphtalene-1,4-dione or 6-methoxyquinoline-5,8-
dione (13)29 with diamine 101,2 gave target compounds in
RESULTS AND DISCUSSION
■
To this end, we prepared and then evaluated at multiple targets
(AChE, Aβ, BACE-1) eight congeners (2−9), which we
formally obtained by cutting the dimeric structure of 1 in two
halves. In the resulting monomeric compounds 2−5, one of the
two 2-methoxybenzyldiamino chains of 1 is always preserved,
with the ending fragments being a quinone, as a privileged
motif for modulating protein−protein interactions.25−27 The
selected quinones were a napthoquinone (2), a quinolinoqui-
none (3), a 2,3-dimethylbenzoquinone (4), and a 2-
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a
Scheme 2
a
Reagents and conditions: (a) K2CO3, KI, DMF, 4 h, reflux; (b) dry THF, (CH3COO)3BHNa, CH3COOH, N2, overnight, room temp; (c) TFA,
dry CH2Cl2, 5 h, room temp; (d) MeOH, 5 h, air, room temp.
a
Scheme 3
a
Reagents and conditions: (a) NaBH3CN, KOH, MeOH, 3 h, room temp; (b) CHCl3, 72 h, reflux.
Table 1. Inhibitory Activity on Human AChE and BuChE, BACE-1, and Amyloid Aggregation by 2−9 and Reference
Compound 1
inhibition of Aβ aggregation
a
IC50 (nM)
(%)
inhibition of
b
c
d
compd
hAChE
hBuChE
BACE-1 (%)
AChE-induced self-induced
1
2
3
4
5
6
7
8
9
1.55 ± 0.11
9.73 ± 0.44
27.9 ± 1.6
29.0 ± 4.0
65.3 ± 2.2
144 ± 100
>80
87.1 ± 1.7
69.1 ± 3.2
41.3 ± 0.7
60.6 ± 0.2
nt
nt
nt
nt
nt
66.8 ± 4.4
27.3 ± 4.3
15.4 ± 6.7
30.2 ± 1.4
45.7 ± 3.4
29.5 ± 0.9
13.8 ± 4.1
21.4 ± 1.3
20.5 ± 1.0
1490 ± 100
2560 ± 170
314 ± 21
22800 ± 1600
3320 ± 260
294000 ± 25000
3160 ± 130
3580 ± 150
60.2 ± 1.6
na
12.8 ± 2.0
32.8 ± 2.4
na
1850 ± 30
64500 ± 2700
17200 ± 1000
24400 ± 1400
na
na
32.9 ± 1.0
a
Human recombinant AChE and BuChE from human serum were used. IC50 ± SEM values represent the concentration of inhibitor required to
b
decrease enzyme activity by 50% and are the mean of two independent measurements, each performed in duplicate. Inhibition of BACE-1. The
concentration of the tested inhibitor was 3 μM. Experimental conditions are as in ref 22. For 2 IC50 = 2.8 ± 0.1 μM. IC50 represents the
concentration of inhibitor required to decrease enzyme activity by 50% and is the mean of three independent measurements, each performed in
c
duplicate; na = not active. Inhibition of AChE-induced Aβ40 aggregation. The concentration of the tested inhibitor and Aβ40 was 100 and 230 μM,
respectively, whereas the Aβ40/AChE ratio was equal to 100:1. Values are the mean of two independent experiments each performed in duplicate; nt
d
= not tested. Inhibition of 50 μM Aβ42 self-aggregation when [I] = 10 μM was used. The Aβ42/inhibitor ratio was equal to 5:1. Values are the mean
of two independent experiments each performed in duplicate.
Figure 1. Low energy docking model of 2 (orange) into the active sites of AChE (A, residues displayed in light green) and BACE-1 (B, residues
displayed in light cyan).
good yields. Conversely, 4 was synthesized by reacting 10 with
quinone 14, obtained, in turn, by oxidation of 2,3-
dimethylbenzene-1,4-diol. Similarly, Michael addition of amines
112 and 12 afforded 6 and 15, respectively. Subsequent
deprotection of BOC protecting group of 15 led to final
compound 7 (Scheme 1). Michael addition was also used to
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synthesize 8, starting from naphthoquinone and 18 (Scheme
2). The amine 18 was derived by reductive amination between
the commercially available tert-butyl piperidin-4-ylcarbamate
and 16, obtained by a nucleophilic substitution reaction,
followed by BOC removal. Finally, to obtain 9, monosub-
stitution of 2,5-dimethoxy-1,4-benzoquinone with diamine 19,
synthesized through an efficient reductive amination protocol,
was carried out as reported for 530 (Scheme 3).
necessary for Aβ binding. Conversely, these data pinpoint to
the quinone core as an essential feature for potent aggregation
inhibition, in agreement with the well-documented inhibitory
capability of quinones toward Aβ assembly.25−27,36−38 Spacer
flexibility also seems to be important, with 9 showing halved
percent inhibition with respect to 5. When rigid 8 is compared
to flexible 2, the relatively lower reduction in potency (29% vs
22%, respectively) might be attributed to the concomitant
presence in 8 of an additional protonable nitrogen atom, which
could establish additional positive interactions.
To characterize the multitarget profiles of 2−9, their
inhibitory activity at human AChE and butyrylcholinesterase
(BuChE) (Table 1) was tested. Kinetic and molecular modeling
evidence has demonstrated that 1 is a dual binding
cholinesterases inhibitor, which accounts for its remarkable
nanomolar activity.1,2 Interestingly, all compounds, except 6−9
were effective inhibitors of AChE, with 2 being just 6 times less
potent than 1. This suggests that even the monovalent
structures of 2−5 could establish interactions with both sites
of the enzyme. However, the low activity displayed by 6 and 7
confirms the importance of an ethyl and a 2-methoxybenzyl
group as substituents on the terminal protonable nitrogen, as
verified for 1 derivatives.2,27 It is also plausible that the
constrained structures of 8 and 9 did not allow an optimal
docking into the AChE gorge. Figure 1A reports the bound
conformation of 2, resulting from docking simulations carried
out at the active site of hAChE (PDB code 1B41; see
Supporting Information). Here, we see that 2 was able to
interact with the catalytic region and at the same time to
protrude toward the solvent-exposed gorge entrance. The
following interactions between 2 and hAChE were observed:
(i) the ligand protonated nitrogen established a cation−π
interaction with the indole ring of W86 and the phenol ring of
Y337; (ii) the oxygen in position 1 of the quinone moiety
established an H-bond interaction with the backbone of F295;
(iii) the naphthalenedione moiety established a favorable π−π
stacking with the indole ring of W286 of the PAS. This last
finding was relevant in the context of previous reports that
linked inhibition of AChE-induced Aβ aggregation with a
binder’s ability to interact with the PAS of the enzyme.31,32
Indeed, the AChE-induced Aβ aggregation experiments,31,32
performed on the three most active AChEIs 2−4, are in
agreement with the proposed binding mode at AChE. Activities
in the AChE-induced aggregation and AChE inhibition were
highly correlated for 2, which was the most potent in both
assays. The IC50 for the inhibition of the AChE-induced Aβ
aggregation by 2 was also calculated, being 45 ± 6 μM. A
different pattern was found for 4, which is the less potent
against AChE, while showing an intermediate antiaggregation
activity. Concerning the physiological relevance of these in vitro
studies, it must be taken into account that the AChE
concentration in the induced Aβ aggregation assay is roughly
1000−3000 times higher than that in the cerebrospinal fluid of
AD patients.33 Consequently, inhibitory activity reported in
Table 1 refers to a normalized inhibitor concentration of 35−
100 nM, a concentration that is closely correlated with those
needed to exert the other activities.
As part of its multitarget profile, 1 inhibits BACE-1 quite
effectively.1 Therefore, preliminary studies were carried out to
assess whether the monomeric derivatives retained the ability to
inhibit BACE-1 in vitro. 2−9 were tested at 3 μM, and their
inhibition percentages are reported in Table 1 in comparison to
1 (80%). The most potent compounds were 2, 5, and 9, which
inhibited enzyme activity at 32−60%, whereas 3 and 6−8
showed no activity. For 2 we also determined an IC50 of 2.8
μM. To check for nonspecific effects, additional experiments
were performed on 1 and 2 with a different source of enzyme
and type of substrate and in the presence of a detergent (see
Supporting Information). In this second assay, 2 showed a
similar potency (IC50 = 3.0 μM) while 1 was less active (IC50
=
4.6 μM). The discrepancy of results between the two assays for
1 is likely to be a consequence of differences in the substrate,
protein, and assay buffer. Docking simulations were performed
in an attempt to provide a plausible binding mode compatible
with the BACE-1 inhibitory profile of 2. Figure 1B reports the
binding mode of 2 at BACE-1 proteasic site (PDB code
2QZL). The leading interactions that characterized the bound
complex were the following: (i) the proximal nitrogen of the
spacer established two H-bond interactions with the side chain
of catalytic D32 and with the carbonyl oxygen of G34
backbone; (ii) the oxygen in position 4 of naphthalenedione
moiety interacted via H-bond with the side chain of Y198; (iii)
the protonated nitrogen established an H-bond interaction with
the carbonyl oxygen of G230 backbone; (iv) the aliphatic chain
of the spacer was lodged in a hydrophobic subpocket described
by Y71, F108, I110, L30, I118, and W115; (v) the quinone
formed hydrophobic contacts with I226 and V332. In summary,
in the reported bound conformation, 2 contacted directly the
catalytic dyad and several subsites that have been previously
exploited to achieve inhibitory potency.39
To further substantiate the secretase inhibitory activity of 2,
5, and 9, we also tested whether they affect APP processing in a
cellular context. This study was carried out in primary chicken
telencephalon neurons to assess the effect on secretion of Aβ38,
Aβ40, and Aβ42.40 Thus, intrinsic cell toxicity of 2, 5, and 9 was
first evaluated, using 1 as reference compound. Treating
primary neurons for 24 h with 2 (0.01−50 μM) did not lead to
modified viability, whereas treatment with high concentrations
of 5 (50 μM), 1 (25 μM), and 9 (25 μM) significantly
abolished neuronal viability (see Supporting Information).
Notably, 1 exhibited a similar toxicity in the SH-SY5Y cell
line.27 These data are encouraging because they point to a
molecule-based toxicity rather than to a potential mechanism of
toxicity related to the quinone moiety. Concerning studies on
amyloid peptides production, 2 inhibited Aβ38, Aβ40, and Aβ42
secretion, with IC50 values of 19, 21, and 46 μM, respectively.
These values were corrected with mean neurons viability,
obtained in the MTT reduction assay. Conversely, because of
their toxicity, a clear concentration-dependent decrease in Aβ
In light of the remarkable antiaggregating properties of 134
and several other bivalent ligands,22,35 the ability of 2−9 to
reduce Aβ42 spontaneous aggregation was then investigated.
Data in Table 1 show that 2−9 at 10 μM inhibited Aβ self-
aggregation in a range from 14% to 46%. At the same
concentration, 1 displayed 68%, which is less than 2 times
higher than that of the most potent compound 5. Interestingly,
two protonable diamino chains of 1 do not appear to be
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secretion could not be observed for 1, 5, and 9 (see Supporting
Information).
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 0512099718. E-mail: marialaura.bolognesi@
unibo.it.
■
There is a growing body of evidence that multitarget-directed
ligands (MTDLs)4 provide a viable area for AD drug discovery.
One limitation to this approach is that most of the hits
discovered so far tend to have high MW, resulting in new NCEs
that are eventually associated with poor oral bioavailability.41
The low ligand efficiency of MTDLs is a critical issue. This is
because affinity at the different targets usually parallels the
increase in MW, whereas pharmacokinetic properties are
improved by reducing the MW.41 However, small molecule
MTDLs (MW ≈ 500) have been reported.42 Interestingly, in
the present series of 1 derivatives, the reduction of MW did not
necessarily correspond to a reduction in the multiple activities.
In fact, for the most active compound 2, the MTDL activity
profile remains almost unchanged with respect to 1, with the
toxicity profile actually being improved.
ACKNOWLEDGMENTS
This research was supported by grants from MIUR (PRIN
2009_ESXPT2_001) and the University of Bologna, Italy.
■
́
Crude 5 was a gift of Martine Fayolle (Universite de Grenoble,
France). Federica Lizzi, Federica Prati, and Birgit Hutter-Paier
(JSW Lifesciences) are acknowledged for their excellent
assistance. We thank Grace Fox for editing and proofreading
the manuscript.
ABBREVIATIONS USED
■
Aβ, β-amyloid; AChE, acetylcholinesterase; AD, Alzheimer’s
disease; APP, amyloid precursor protein; BACE-1, β-secretase;
BuChE, butyrylcholinesterase; MTDL, multitarget-directed
ligand; MW, molecular weight; NCE, new chemical entity;
PAS, peripheral anionic site
In conclusion, if the decrease in MW translates into the
expected superior bioavailability, 2 could be a promising
starting point in the search for new MTDLs against AD.
EXPERIMENTAL SECTION
■
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* Supporting Information
Experimental details of the synthesis of 2−9 and computational
and biological methods. This material is available free of charge
via the Internet at http://pubs.acs.org.
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