Multitarget drug discovery for Alzheimer's disease: Triazinones as BACE-1 and GSK-3β inhibitors

Article abstract of DOI:10.1002/anie.201410456

Cumulative evidence strongly supports that the amyloid and tau hypotheses are not mutually exclusive, but concomitantly contribute to neurodegeneration in Alzheimer's disease (AD). Thus, the development of multitarget drugs which are involved in both pathways might represent a promising therapeutic strategy. Accordingly, reported here in is the discovery of 6-amino-4-phenyl-3, 4-dihydro-1, 3, 5-triazin-2(1H)-ones as the first class of molecules able to simultaneously modulate BACE-1 and GSK-3β. Notably, one triazinone showed well-balanced in vitro potencies against the two enzymes (IC₅₀ of (18.03 ±0.01) μM and (14.67±0.78)μUM for BACE-1 and GSK-3β, respectively). In cell-based assays, it displayed effective neuroprotective and neurogenic activities and no neurotoxicity. It also showed good brain permeability in a preliminary pharmacokinetic assessment in mice. Overall, triazinones might represent a promising starting point towards high quality lead compounds with an AD-modifying potential.

Full text of DOI:10.1002/anie.201410456

Angewandte
Chemie
DOI: 10.1002/anie.201410456
Drug Discovery
Multitarget Drug Discovery for Alzheimerꢀs Disease: Triazinones as
BACE-1 and GSK-3b Inhibitors**
Federica Prati, Angela De Simone, Paola Bisignano, Andrea Armirotti, Maria Summa,
Daniela Pizzirani, Rita Scarpelli, Daniel I. Perez, Vincenza Andrisano, Ana Perez-Castillo,
Barbara Monti, Francesca Massenzio, Letizia Polito, Marco Racchi, Angelo D. Favia,
Giovanni Bottegoni, Ana Martinez, Maria Laura Bolognesi,* and Andrea Cavalli*
Abstract: Cumulative evidence strongly supports that the
amyloid and tau hypotheses are not mutually exclusive, but
concomitantly contribute to neurodegeneration in Alzheimer’s
disease (AD). Thus, the development of multitarget drugs
which are involved in both pathways might represent a promis-
ing therapeutic strategy. Accordingly, reported here in is the
discovery of 6-amino-4-phenyl-3,4-dihydro-1,3,5-triazin-
2(1H)-ones as the first class of molecules able to simultane-
ously modulate BACE-1 and GSK-3b. Notably, one triazinone
showed well-balanced in vitro potencies against the two
enzymes (IC50 of (18.03 Æ 0.01) mm and (14.67 Æ 0.78) mm for
BACE-1 and GSK-3b, respectively). In cell-based assays, it
displayed effective neuroprotective and neurogenic activities
and no neurotoxicity. It also showed good brain permeability
in a preliminary pharmacokinetic assessment in mice. Overall,
triazinones might represent a promising starting point towards
high quality lead compounds with an AD-modifying potential.
drug discovery efforts.[2] Ab is generated by the cleavage of
the membrane-associated amyloid precursor protein (APP).
A key enzyme has been identified as being responsible for Ab
formation, namely, the aspartyl protease b-secretase (BACE-
1).[3] BACE-1 inhibitors are in clinical development, with the
most advanced ones in phase III of clinical trials.[4] As for the
tau hypothesis, glycogen synthase kinase-3b (GSK-3b) has
been recognized as one of the key players. GSK-3b is
responsible for tau hyperphosphorylation, which causes tau
to be detached from the microtubules and precipitate as
tangle aggregates.[5] Therefore, the development of selective
GSK-3b inhibitors has been recently pursued.[6] Notably,
these molecules also enhance neurogenesis and could be
promising from a regenerative medicine standpoint.[7] Fur-
thermore, since GSK-3b is involved in neuroinflammatory
cascades, its inhibitors might hamper the production of
inflammatory mediators and neurotoxic factors.[8] Thus far,
neither approach has led to effective drugs to combat AD.
Notably, although in the past the two hypotheses have been
considered to be at odds, recent evidence suggests that they
concomitantly contribute to neuronal death in AD.[9] So,
although drug development has been focused on targeting Ab
and tau pathology independently, a disease-modifying treat-
ment for AD should address both amyloid and tau cas-
cades.[10] In light of these considerations, multitarget (MT)
Alzheimer’s disease (AD) is the major unmet medical need
in neurology,[1] and despite massive investments, there are no
effective treatments. Amyloid beta (Ab) aggregates and
hyperphosphorylated neurofibrillary tangles (NFT) of tau
represent the two main pathological hallmarks of AD. These
distinctive features have fostered the proposal of two
etiological hypotheses which have inspired most of the AD
[*] F. Prati, Dr. A. De Simone, Dr. P. Bisignano, Dr. A. Armirotti,
Dr. M. Summa, Dr. D. Pizzirani, Dr. R. Scarpelli, Dr. A. D. Favia,
Dr. G. Bottegoni, Prof. Dr. A. Cavalli
and
Centro Investigaciꢁn Biomꢀdica en Red Enfermedades Neuro-
degenerativas (CIBERNED) (Spain)
Istituto Italiano di Tecnologia, D3
via Morego 30, 16163 Genova (Italy)
E-mail: andrea.cavalli@iit.it
Dr. L. Polito
Fondazione Golgi Cenci
Corso San Martino 10, 20081 Abbiategrasso (Milan) (Italy)
F. Prati, Prof. Dr. B. Monti, F. Massenzio, Prof. Dr. M. L. Bolognesi,
Prof. Dr. A. Cavalli
Department of Pharmacy and Biotechonology
University of Bologna
via Belmeloro 6/Selmi 3, 40126 Bologna (Italy)
E-mail: marialaura.bolognesi@unibo.it
Prof. Dr. M. Racchi
Department of Drug Sciences—Pharmacology, University of Pavia
viale Taramelli 12, 27100 Pavia (Italy)
[**] We thank S. Mandrup Bertozzi, G. Ottonello, and L. Goldoni for
analytical analysis and S. Venzano for compound handling support
(IIT) as well as G. Forloni and D. Albani for providing H4-APPsw
cells (Mario Negri Institute). This work was supported by IIT,
UNIBO, PRIN (20103W4779), MINECO (SAF2012-37979-C03-01),
Ministerio de Ciencia y Tecnologia (SAF2010-16365), Instituto de
Salud Carlos III, UniRimini, and CIRI (PORFESR project).
Dr. A. De Simone, Prof. Dr. V. Andrisano
Department for Life Quality Studies, University of Bologna
Corso D’Augusto 237, 47921 Rimini (Italy)
Dr. D. I. Perez, Prof. Dr. A. Martinez
Centro de Investigaciones Biologica, CSIC
Ramiro de Maetzu 9, 28040 Madrid (Spain)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201410456.
Dr. A. Perez-Castillo
Instituto de Investigaciones Biomꢀdicas, CSIC-UAM
Arturo Duperier 4, 28029 Madrid (Spain)
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drugs, namely small molecules which are able to hit multiple
targets responsible for the disease, emerge as promising
therapeutic tools.[11]
Herein we report on the development of the first class of
MT molecules which are able to modulate BACE-1 and GSK-
3b activity simultaneously. We envisaged that such dual-target
inhibitors, which intervene at two crucial points in the
neurotoxic pathways, might represent a breakthrough in the
quest for clinical efficacy.
In MT drug discovery, fragment-based strategies play
a pivotal role.[12] On these premises,[13] we utilized a fragment-
based approach to design dual inhibitors against BACE-1 and
GSK-3b, two enzymes ancestrally quite divergent and sharing
a sequence identity of only 19%. We exploited a ligand-based
approach by combining the pharmacophoric features respon-
sible for binding to BACE-1 and GSK-3b, such as a guanidino
motif and a cyclic amide group, respectively, into a single
scaffold (Figure 1).[14] As a result, we identified the 6-amino-
Figure 2. (S)-1 docked into the catalytic region of BACE-1 (a) and GSK-
3b (b). a) (S)-1 participated in a H-bond with D32, D228, and T231 in
the BACE-1 catalytic pocket. The binding affinity associated to this
pose is À7.291 kcalmolÀ1. b) (S)-1 participated in two H-bonds with
V135 and stacking interactions with R141 (À6.372 kcalmolÀ1). The
best orientation for (R)-1 presented similar binding affinities on both
targets.
Scheme 1. Synthesis of 1–7.
GSK-3b dual-inhibition profile, and are potentially useful as
AD modifying agents.
Figure 1. Design strategy to dual BACE-1/GSK-3b inhibitors.
The two-step synthetic route to the target compounds 1–7
is depicted in Scheme 1. First, the acid-catalyzed hydration of
the 1-cyanoguanidines 8 and 9 provided the guanylurea 10
and (N’-ethylcarbamimidoyl)urea 11, respectively, in excel-
lent yield. Subsequently, 10 and 11 were coupled to the
aromatic aldehydes of interest (12–17) through a condensation
reaction to provide 1–7.[16]
The compounds 1–7 showed high aqueous kinetic sol-
ubility (ca. 250 mm in PBS, pH 7.4), which enabled further
investigation. The ability to inhibit both enzymes was inves-
tigated in comparison to inhibitor IV and SB415286, which
were used as reference compounds for BACE-1 and GSK-3b,
respectively (Table 1).[17]
4-substituted triazinone scaffold as potential starting point
(Figure 1). We subsequently performed structure-based dock-
ing simulations[15] to study the interactions of triazinone-
based compounds with the catalytic pocket of both enzymes
(Figure 2). We found that 6-amino-4-phenyl-3,4-dihydro-
1,3,5-triazin-2(1H)-one (1; Scheme 1) should efficiently bind
to both targets.
The molecule 1 was then explored in terms of preliminary
structure–activity relationships (SARs), and a first set of
molecules (2–7) was synthesized and tested against the two
enzymes. Experiments were also carried out at cellular levels.
Finally, for one of the most promising compounds, 3, we
generated pharmacokinetics data in mice. Anticipating our
results, it turned out that triazinones are promising fragments
for developing lead compounds endowed with a BACE-1 and
The compounds 1, bearing no substituents, and 2, bearing
a dimethylamino group on the aromatic ring, did not show any
significant effect in BACE-1 inhibition assay. The methyl
derivative 5 showed modest activity. However, the halogen-
2
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Table 1: Aqueous kinetic solubility (Skinetic) and inhibitory potencies (IC50)
of 1–7 and reference compounds against BACE-1 and GSK-3b.
phase was performed. The compound 7 was selected for these
studies because of its higher solubility in the solvents used for
the normal-phase HPLC method. (À)-7 and (+)-7 turned out
to be equally potent against BACE-1 and GSK-3b, that is,
there is no enantiomer discrimination.
Compound
Skinetic
[mm]
BACE-1
GSK-3b
IC50 [mm][a] ÆS.E.M.
IC50 [mm][a] ÆS.E.M.
1
2
3
4
5
6
7
>250
249
>250
>250
247
>250
>250
–
n.a.
n.a.
30.91Æ0.50
26.44Æ0.01
14.67Æ0.78
37.78Æ4.50
27.37Æ6.13
25.75Æ0.26
7.11Æ0.37
12.69Æ0.44
16.00Æ0.84
–
From a MT drug discovery standpoint, the most promising
result of the present study is the remarkably similar enzymatic
activities of 3, despite the two-digit micromolar range. In fact,
where connections exist between two targets, as it seems to be
the case for GSK-3b and BACE-1, inhibitors with only
moderate potency are expected to produce superior in vivo
effects compared to higher-potency single-target compounds,
and fewer side effects.[18,19] Therefore, despite the moderate
activity, we propose that 3, due to its structural simplicity and
low molecular weight, might be a more suitable binder than
reference inhibitors. In fact, it exhibits a ligand efficiency
(LE = 0.32) which is higher than those of inhibitor IV (0.19)
and SB415286 (0.29). In addition, 3 showed a promising
lipophilic efficiency (LipE; see Table S1 in the Supporting
Information).
Based on these considerations, 3 was selected for addi-
tional cellular studies aimed at assessing its effect on neuro-
protection and neurogenesis, two crucial properties for AD-
modifying drugs. GSK-3b overactivity results in increased
microglia activation, thus leading to the neuronal cell death in
AD. Furthermore, GSK-3b regulates inflammatory tolerance
in astrocytes.[20] Accordingly, we explored the potential
neuroprotective activity of 3 in primary cultures of astrocytes
and microglia by evaluating nitrite production. Glial cells
were first incubated with 3 (10 mm) for 1 hour, and then
cultured for another 24 hours with lipopolysaccharide (LPS),
a potent inducer of inflammatory reaction and of a cascade of
intracellular events involved in neuronal death. We observed
an important induction of nitrite production, which was
significantly reduced by treatment with 3 (Figure 4). Similar
18.03Æ0.01
61.53Æ14.2
222.37Æ60.2
41.24Æ1.29
16.05Æ0.64
14.23Æ0.01
12.55Æ0.01
0.02Æ0.00
–
(+)-7
(À)-7
–
–
–
Inhibitor IV
SB415286
0.05Æ0.01
[a] IC50 values are reported as a mean value of three or more
determinations. n.a.=not active up to a concentration of 100 mm.
ated derivatives 3, 4, 6, and 7 resulted in moderate inhibition.
This inhibition could be due the F, CF3, and Br substituents
establishing polar and hydrophobic interactions with a cage
made by Leu30, Ile118, Phe108, and Trp115 at subsite P1.
Remarkably, the fluorinated 3 and 7 were the most active of
the series, with IC50 values of 18.03 and 16.05 mm, respectively.
Importantly, their enzymatic activity was mirrored by a mod-
erate reduction of cellular Ab and no cytotoxicity (Figure 3).
Figure 3. H4APPsw cells were treated with 3, 7, or reference com-
pounds. a) Cell viability was assessed through MTT assay. Data
represent mean ÆSD. **p<0.01 Student t-Test compared to cell
treated with vehicle alone. b) Ab(1–40) levels in conditioned media
were quantified through ELISA, and adjusted to take into account
differences in cell proliferation. Data represent mean ÆSD. *p<0.05,
*** p<0.001, Student t-Test compared to cell treated with vehicle
alone.
Figure 3a shows that exposing neuroglioma cells, overex-
pressing APP harboring the Swedish mutation, for 24 hours to
3 and 7 did not affect viability. Furthermore, after the same
time frame 3 and 7 slightly reduced Ab(1–40) levels,
compared to results obtained with inhibitor IV (Figure 3b).
With regards to GSK-3b, all compounds modulated the
enzyme activity with similar potency, ranging from 7.11 to
37.78 mm. As a result, 3 showed a well-balanced in vitro
BACE-1/GSK-3b inhibitory profile (IC50 values of 18.03 mm
and 14.67 mm).
Figure 4. Primary microglia and astrocyte cells were treated with LPS
in the absence and presence of 3. The production of nitrites from the
medium was measured with the Griess reaction.
results were obtained by co-treatment of primary rat glial
cells with LPS and 3 (data not shown).
GSK-3b activity promotes inducible nitric oxide synthase
(iNOS) induction,[21] thus resulting in the elevated NO
concentrations associated with the neurodegenerative path-
To explore the enantioselectivity of these compounds,
enantiomer separation by HPLC using a chiral stationary
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Figure 6. The neurospheres were cultivated in the absence (a) or
presence (b) of 3 during a week. After that, they were first incubated
with anti-b-tubulin and anti-MAP2 antibodies, and treated with the
corresponding Alexa-labeled secondary antibodies (green and red
labels to reveal b-tubulin and MAP2 respectively). DAPI staining (blue)
was used as nuclear marker.
neuroprotective one. Moreover, 3 does not display any
toxicity in glial and neuronal cells (see Figure S1).
To evaluate the potential neurogenic effect of 3, we
analyzed whether its addition to neurospheres of primary rat
neural stem cells could regulate differentiation toward
a neuronal phenotype. As shown in Figure 6, some neuro-
genic effects were evident after treatment with 3. Particularly,
when compared to the control, the number of MAP-2 positive
cells (mature neuronal marker) was increased in treated
cultures. Notably, this underlines the ability of 3 to differ-
entiate neural stem cells into mature neurons.
Finally, pharmacokinetic (PK) analyses in mice showed
that 3 has good oral bioavailability and blood-brain-barrier
(BBB) penetration. After dosing 3 intravenously and orally,
we monitored plasma concentrations for 8 hours. After
intravenous dosing (3 mgkgÀ1), we measured a maximal
plasma concentration of 713 ngmLÀ1. The half-life of 3 for
the elimination phase was 31 minutes. The compound 3
showed a volume of distribution of 3.4 LkgÀ1 and disappeared
Figure 5. Primary rat microglia and astrocyte cells were treated with
LPS in the presence or absence of 3. a–d) Immunomodulatory activity
of 3 in glial cells was evaluated through Western blot analysis of iNOS
expression, using b-actin as control. d,e) The modulatory action of 3
on M1/M2 phenotypes was assessed by studying TREM2 expression in
microglia.
from the systemic circulation with
a
clearance of
77 mLminkgÀ1. After oral administration (10 mgkgÀ1), the
maximal plasma concentration of 3 was observed 30 minutes
after dosing (665 ngmLÀ1). The oral bioavailability of 3 was
66%. Furthermore, 3 showed good BBB penetration, thus
reaching a maximum concentration in 1 mL of brain homo-
genate of 0.613 ngmgproteinÀ1, 30 minutes after oral adminis-
tration (see Table S2). Accordingly, 3 reaches a total cerebral
concentration of 0.62 mm (see the Supporting Information).
Although below the in vivo target engagement levels, this
represents an attractive starting point towards a promising hit
optimization campaign.
In conclusion, we have reported on the first class of
BACE-1 and GSK-3b dual-target inhibitors in the search for
innovative AD modifiers. One of these new derivatives (3)
has shown balanced in vitro activities against the two targets,
with potency in the low micromolar range. In this respect, it is
worth noting that 3 displays fragment-like features, and has
a high LE against both targets, as well as several possibilities
in terms of chemical tractability and functionalization. In
addition, 3 has shown a promising profile in terms of
neuroprotection and neurogenesis in cells, as well as of
immunomodulation, with no sign of toxicity. It has also
displayed good brain exposure, a fundamental property for
CNS-directed drugs. Indeed, it is widely acknowledged that
ology.[22] Moreover, iNOS is considered a marker of the
inflammatory M1 microglial phenotype, and iNOS inhibitors
have been shown to exert beneficial anti-inflammatory
properties.[23]
Therefore, we evaluated the effect of 3 on iNOS
expression in parallel cultures of astrocytes and microglia.
When cells were stimulated with LPS, we observed the
expected iNOS induction, which was significantly reduced by
3, in a dose dependent manner (Figure 5a–d). In addition, the
modulatory action of 3 was assessed by studying the micro-
glial M1/M2 phenotypes through the evaluation of the
phagocytic receptor TREM2.[24] Compounds that induce
microglia switch from inflammatory M1-type to anti-inflam-
matory M2-type have been proposed to attenuate neuro-
inflammation and bolster neuronal protection and recov-
ery.[25] Remarkably, 3 was able to shift microglia phenotype
from M1 to M2, with a decrease in iNOS expression, but there
were no changes in the positive microglial phagocytic activity,
as assessed by TREM2 expression (Figure 5d,e).[26] Alto-
gether these results indicate that 3 is a highly promising anti-
inflammatory and neuroprotective agent, thereby decreasing
the neurotoxic microglial activation without affecting the
4
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347 – 372; b) E. Viayna, I. Sola, M. Bartolini, A. De Simone, C.
combining a desirable balanced biological profile with drug-
like properties is a critical aspect of MT drug discovery.[19] In
light of these features, we can point to triazinones as
a promising class of MT fragments able to tackle amyloid
and tau pathological cascades, and they deserve to be further
explored in the field of AD.
The challenge in the fragment evolution process is to
transform these low affinity binders into potent leads, while
maintaining reasonable LE and an attractive PK profile. Such
studies have been undertaken, and progress on the develop-
ment of optimized dual BACE-1/GSK-3b inhibitors derived
from 3 will be reported in due course.
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Keywords: drug design · drug discovery · enzymes ·
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heterocycles · neurochemistry
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Communications
Drug Discovery
Two for one: Alzheimer’s disease is
a complex multifactorial syndrome which
calls for the development of multitarget
drugs. Accordingly, triazinones are
reported here as the first molecule class
which is able to simultaneously modulate
BACE-1 and GSK-3b activity. Such dual-
target inhibitors, by acting against two
crucial enzymes in the neurotoxic path-
ways, might represent a breakthrough in
the quest for disease-modifying drugs.
F. Prati, A. De Simone, P. Bisignano,
A. Armirotti, M. Summa, D. Pizzirani,
R. Scarpelli, D. I. Perez, V. Andrisano,
A. Perez-Castillo, B. Monti, F. Massenzio,
L. Polito, M. Racchi, A. D. Favia,
G. Bottegoni, A. Martinez,
M. L. Bolognesi,*
A. Cavalli*
&&&& — &&&&
Multitarget Drug Discovery for
Alzheimer’s Disease: Triazinones as
BACE-1 and GSK-3b Inhibitors
6
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Angew. Chem. Int. Ed. 2014, 53, 1 – 6
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