Structure-activity relationships of memoquin: Influence of the chain chirality in the multi-target mechanism of action

Article abstract of DOI:10.1016/j.bmcl.2009.05.087

The present article expands on the study of structure-activity relationships of the novel class of quinone-bearing polyamines, as multi-target-directed ligands against Alzheimer's disease. Namely, the effect of inserting a methyl substituent at the α posi

Full text of DOI:10.1016/j.bmcl.2009.05.087

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4312–4315
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationships of memoquin: Influence of the chain chirality
in the multi-target mechanism of action
*
Maria Laura Bolognesi , Manuela Bartolini, Michela Rosini, Vincenza Andrisano, Carlo Melchiorre
Department of Pharmaceutical Sciences, Alma Mater Studiorum-Bologna University, Via Belmeloro 6, I-40126 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The present article expands on the study of structure–activity relationships of the novel class of quinone-
bearing polyamines, as multi-target-directed ligands against Alzheimer’s disease. Namely, the effect of
Received 22 April 2009
Revised 19 May 2009
Accepted 20 May 2009
Available online 28 May 2009
inserting a methyl substituent at the
a position of the terminal benzyl amine moieties of lead candidate
1 (memoquin) was evaluated at the multiple targets involved in the multifunctional mechanism of
action. The RR stereoisomer 2 resulted more effective than 1 in reverting two important effects mediated
by acetylcholinesterase (AChE), that is, acetylcholine hydrolysis and AChE-induced amyloid-b
aggregation.
Keywords:
Alzheimer’s disease
Acetylcholinesterase
Butyrylcholinesterase
Amyloid-beta
Ó 2009 Elsevier Ltd. All rights reserved.
Small molecule protein-protein interaction
inhibitors
Despite the huge research effort and the considerable strides
already made, Alzheimer’s disease (AD) still is an incurable pathol-
ogy which provokes considerable concern inside the pharmaceuti-
cal community.1,2 Over the past decade, trying to address the
pitfalls that have long hindered the identification of effective ther-
apeutic tools, we developed an innovative concept, which embod-
ies in the design of Multi-Target Directed Ligands (MTDLs) as
response to the multifactorial nature of AD.3 This approach has
proven particularly fruitful and allowed the identification of
several MTDLs, some of which have emerged as interesting phar-
macological tools for the investigation of neurodegenerative disor-
ders, or as innovative drug candidates for combating AD.3–5
Among them, memoquin (1) is a promising new chemical entity
that has been rationally designed with the deliberate aim of creat-
ing a multifunctional small molecule acting at different levels of
the neurodegenerative cascade underlying AD pathology.6
By inserting the antioxidant benzoquinone moiety of the prom-
ising drug candidate ubiquinone (2,3-dimethoxy-5-methyl-6-
decaprenyl-1,4-benzoquinone) into a polyamine scaffold endowed
with acetylcholinesterase (AChE) inhibition activity 1 was gener-
ated (Fig. 1). An in-depth in vitro and in vivo characterization has
confirmed its multifunctional mechanism of action and verified
the interaction of 1 with different crucial molecular targets in AD
neurotoxic pathways, such as AChE and b-secretase (BACE-1) en-
zymes, b-amyloid (Ab) and oxidative processes.7 The ability of 1
to exert a positive pharmacological effect on AD neurodegenera-
tion in vivo was verified by extending the studies to different ani-
mal models for AD.7 Administration of 1 to anti NGF AD11 mice
showed that it is able to ameliorate neuropathological signs and
cognitive deficits, confirming its capability to impinge on different
points of the cascade/s leading to neurodegeneration.6,7
In a search of memoquin derivatives with an improved MTDL
profile, we previously synthesized a small unbiased library of
1-derivatives, and analyzed the contributions of the different
O
O
H
N
N
N
N
H
O
O
1
O
O
H
*
N
N
*
N
N
H
O
O
2: RR
3: SS
4: RS
* Corresponding author. Tel.: +39 051 2099718; fax: +39 051 2099734.
E-mail address: marialaura.bolognesi@unibo.it (M.L. Bolognesi).
Figure 1. Chemical structures of 1 and its chiral derivatives 2–4.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.087

M. L. Bolognesi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4312–4315
4313
structural elements to the different activities.8 The data obtained
from this SAR study confirmed that 1 was indeed the best ligand
targeting the different biological targets relevant in the AD pathol-
ogy. However, the resulting information, in addition to the molec-
ular modeling studies on 1, allowed us to hypothesize some further
structural manipulation potentially leading to lead optimization.
Therefore, starting from the 1-AChE complex obtained by docking
simulations,7 and being aware that AChE peripheral and catalytic
sites are rich in hydrophobic aminoacids, we explored the intro-
stitution reaction conditions on 13 by reacting an excess of it with
diamine 11, and isolating the monosubstituted derivative 14 in
acceptable yield. Then, the remaining methoxy group of 14 was
displaced by the primary amino group of 12, affording the final
meso compound 4 (Scheme 2).16
The influence of chirality of memoquin and other MTDLs at the
different molecular targets has never been explored before. There-
fore, this investigation was directed toward evaluating changes in
the polypharmacological profile resulting from introducing chiral-
ity into the terminal benzyl groups of 1. We thought to preliminary
disclose the effect of this modification on chiral protein targets
such as AChE, BChE and Ab. We assumed that this effect would
be negligible on the antioxidant profile, as trolox equivalent anti-
oxidant capacity (TEAC) is mediated by the quinone moiety.7,8
To determine the biological profile of 2–4 as MTDLs for the
treatment of AD, they were screened at the four selected targets,
that is, (a) AChE and BChE, (b) Ab aggregation induced by human
recombinant AChE, (c) self-induced Ab aggregation, using experi-
mental procedures reported before.8,10,17 To allow comparison of
the results, 1 was used as reference compound. From the data re-
ported in Table 1 some interesting conclusions can be drawn; AChE
inhibition can be affected by the introduction of a methyl group on
the benzyl moiety of 1 and chirality of this stereocenter modulates
AChE activity. Specifically, the inhibitory potency of the RR enan-
tiomer 2 and the meso 4 resulted slightly improved with respect
to that of 1, reaching values in the sub-nanomolar range. Notably,
being endowed with an IC50 value of 0.36 nM, 4 emerges as the
most active AChE inhibitor among 1-derivatives developed so far.
Conversely, the SS enantiomer 3 was 2.6-fold less potent than 1.
Regarding the inhibitory profile against human BChE, all the
stereoisomers maintained, although at a different extent, the
remarkable selectivity displayed by the prototype 1. Despite no
significant differences in the IC50 values against BChE for the differ-
ent enantiomers might be found, the overall BChE/AChE selectivity
ratio for 2 and 3 differs of one order of magnitude (2960 vs 238).
This is due to the different trend in activities for 2 and 3 towards
the two target proteins.
duction of a methyl substituent at the
a position of the terminal
benzyl amine moieties. Due to the symmetrical nature of 1, this
modification generates two equivalent stereogenic centers, and
three possible stereoisomers: the pair of enantiomers 2 and 3
and the meso stereoisomer 4, as shown in Figure 1.
Chiral discrimination has tremendous importance in medicinal
chemistry as a means to gain insight into the mechanisms of inter-
action and to better understand structural features of ligand-target
recognitions.
On the basis of this rational design, 2–4 should possess a better
inhibition profile against AChE and more importantly, could bind
more tightly to the allosteric peripheral anionic site (PAS) of AChE,
with a concomitant higher prevention of AChE-promoted Ab aggre-
gation. The theory that AChE acts as a pathologic chaperone, accel-
erating the formation of amyloid fibrils in the brain and forming
stable complexes with Ab at the PAS region, has found general
acceptance.9,10
Consequently, AChE has been recently revaluated as a key target
for AD drug discovery.11–14
The RR, SS and RS-meso isomers 2–4 have been separately
synthesized, by exploiting a route different from the standard syn-
thetic protocol used for memoquin.8 Commercial availability of
both (1R)- and (1S)-1-(2-methoxyphenyl)ethanamines (5 and 6)
prompted us to use them as chiral building blocks. They were N-
ethylated by a reductive amination procedure (7 and 8) and then
alkylated by reaction with (6-chloro-hexyl)-carbamic acid tert-bu-
tyl ester.15 The obtained intermediates 9 and 10 were deprotected
at the terminal amino function to get the required diamines 11 and
12 (Scheme 1). Afterward, in the case of 2 and 3 the reaction
scheme proceeds as reported for 1,8 exploiting the efficient substi-
tution reaction on 2,5-dimethoxybenzoquinone (13). Conversely,
for the synthesis of the meso derivative 4, we optimized monosub-
In parallel to the improved activity profile against human AChE,
the new derivatives showed a better efficacy in inhibiting the AChE
pro-aggregating action. As shown in Table 2, compounds 2–4
blocked the amyloid aggregation induced by AChE with IC50 values
O
O
O
*
c
*
NHBOC
a, b
*
N
H
N
NH2
9: R
10: S
7: R
8: S
5: R
6: S
d
O
e
*
NH2
N
11: R
12: S
O
O
H
N
*
N
*
N
N
H
O
O
2, 3
Scheme 1. Reagents and conditions: (a) 1.1 equiv CH3CHO, EtOH, 3 h, rt; (b) NaBH4, 12 h, rt; (c) 2 equiv (6-chlorohexyl)carbamic acid tert-butyl ester, 8 equiv KI, 4 equiv
EtN(iPr)2, EtOH, reflux, 48 h; (d) TFA, 2H, rt; (e) 0.5 equiv 13, EtOH, 50 °C for 3 h, then rt, overnight.

4314
M. L. Bolognesi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4312–4315
O
O
O
O
H
N
*
*
13
NH2
12
N
4
N
H
b
a
O
11: R
14: R
Scheme 2. Reagents and conditions: (a) 7 equiv 13, CHCl3/EtOH, 50 °C for 100, then rt overnight; (b) 1 equiv 13, CH2Cl2, rt, overnight.
Table 1
Inhibition of AChE and BChE activities, by 2ꢀ4 and reference compound 1
Compd
IC50 (nM)a
AChE
BChE
BChE/AChEb
1c
2
3
—
1.55 ( 0.11)
0.50 ( 0.02)
4.03 ( 0.20)
0.36 ( 0.02)
1440 ( 100)
1480 ( 40)
958 ( 18)
982 ( 37)
929
2960
238
R,R
S,S
R,S
4
2728
a
Values are mean of three experiments, standard deviation is given in
parentheses.
b
The AChE/BChE selectivity ratio is the ratio of IC50 values at BChE and AChE,
respectively.
c
From Ref. 7.
lower than that displayed by the drug candidate 1. Remarkably, 3
was the most potent in the series and resulted 3.6-fold more effi-
cient than 1. Nevertheless, 2 resulted only slightly less potent than
3. The most potent published compounds acting as AChE-induced
Ab aggregation inhibitors display a potency in the same range as
3 and 2.18–21 As verified in other cases, these profiles do not per-
fectly parallel the relative AChE inhibitory activities. In fact, 3 dis-
played a higher anti-aggregating activity with respect to 1, 2 and 4,
despite its lower AChE inhibitory potency. Figure 2 shows the inhi-
bition profile obtained for compound 2 in the thioflavin T flores-
cence-based assay for the anti-aggregating potency determination.
Concerning amyloid self-induced aggregation, compounds were
screened at the same concentration used for characterizing 1
Figure 2. Inhibition of AChE-induced Ab aggregation by 2. (A) Bar plot showing
experimental florescence values obtained for AChE-induced Ab aggregation in the
absence and in the presence of increasing concentrations of inhibitor. AChE and Ab
florescence values are used as blanks. (B) Inhibition plot. When measuring the
AChE-mediated aggregation, the concentration of Ab(1–40) was 230
the Ab(1–40)/AChE ratio was equal to 100:1.
lM, whereas
(10 lM). The almost identical inhibition percentage provided by
2–4 with respect to 1 points to the conclusion that the presence
of additional substituents on the benzyl moiety is irrelevant
against the self-induced Ab aggregation. Notably, also 2–4 emerge
as small molecule capable to inhibit protein–protein interaction in
AD. It is also highly conceivable that they share with 1 the same
anti-amyloidogenic mechanism of action, by binding to Ab in the
prevailing b-sheet conformation and by preventing the induction
of the conformational shift on vicinal peptides.17
effective than 1 in altering two important effects in AD neurotoxic
cascade mediated by AChE (prevention of Ab fibrils formation in
addition to simple inhibition of acetylcholine hydrolysis). From
these results, 2 emerges as a small-molecule-based MTDL that
can serve as powerful tool both in understanding the role and func-
tion of the multiple biological targets and in further validating the
design rationale.
In conclusion, owing to the improved inhibitory potency against
AChE and AChE-induced aggregation, 2 is the first memoquin
derivative showing a better MTDL profile; in particular 2 is more
Acknowledgement
This work was supported by the University of Bologna. Federica
Lizzi, Fabio Magnani and Ioannis Matziris are acknowledged for
excellent technical assistance.
Table 2
Inhibition of AChE-mediated and self-induced Ab aggregation by 2ꢀ4 and reference
compound 1
References and notes
Compd
Inhibition of Ab aggregationa
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2. Gura, T. Nat. Med. 2008, 14, 894.
AChE-induced IC50
(lM)
Self-inducedc (%)
b
1d
2
3
—
28.3 ( 0.3)
9.34 ( 1.42)
7.91 ( 0.69)
11.0 ( 1.4)
66.8 ( 4.4)
63.9 ( 1.4)
62.3 ( 0.3)
63.5 ( 2.0)
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R,R
S,S
R,S
4
a
Values are mean of three experiments, standard deviation is given in
parentheses.
b
Inhibition of AChE-induced Ab(1–40). The Ab(1–40)/AChE ratio was equal to
100:1.
c
Inhibition of self-induced Ab(1–42) aggregation (50 lM) produced by the tested
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Minarini, A.; Rosini, M.; Tumiatti, V.; Bergamini, C.; Fato, R.; Lenaz, G.; Hrelia,
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d
From Ref. 7.

M. L. Bolognesi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4312–4315
4315
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(2H, t, J = 7.8 Hz, ArH), 7.20 (2H, t, J = 8 Hz, ArH), 7.45 (2H, d, J = 8 Hz, ArH); ESI-
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a-methoxy-
a
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16. Spectral data for target compounds. Compounds 2 and 3: red solids, mp 63–
65 °C; 1H NMR (300 MHz,CDCl3) d 1.02 (6H, t, J = 7.0 Hz, CH2CH3), 1.29 (6H, d,
J = 6.9 Hz, CHCH3), 1.27–1.64 (16H, m complex, aliphatic chain), 2.38–2.76 (8H,
m complex, CH2N) 3.13 (4H, q, J = 6.4 Hz, NHCH2), 3.83 (6H, s, OCH3), 4.32 (2H,
q, J = 6.9 Hz, CHCH3), 5.31 (2H, s, quinone), 6.61 (2H, br t exch with D2O, NH),
6.88 (2H, d, J = 8 Hz, ArH), 6.95 (2H, t, ArH), 7.20 (2H, t, J = 9 Hz, ArH), 7.45 (2H,
d, J = 9 Hz, ArH); ESI-MS (m/z): 662 (M+H+). Compound 4: red waxy solid; 1H
NMR (300 MHz,CDCl3) d 1.01 (6H, t, J = 7.0 Hz, CH2CH3), 1.17–1.84 (22H, m
complex, aliphatic chain + CHCH3), 2.33–2.96 (8H, m complex, CH2N), 3.13 (4H,
21. Xie, Q.; Wang, H.; Xia, Z.; Lu, M.; Zhang, W.; Wang, X.; Fu, W.; Tang, Y.;
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