A small chemical library of 2-aminoimidazole derivatives as BACE-1 inhibitors: Structure-based design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2011.12.016

In this work, we report a rational structure-based approach aimed at the discovery of new 2-aminoimidazoles as β-secretase inhibitors. Taking advantage of a microwave-assisted synthetic protocol, a small library of derivatives was obtained and biologicall

Full text of DOI:10.1016/j.ejmech.2011.12.016

European Journal of Medicinal Chemistry 48 (2012) 206e213
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A small chemical library of 2-aminoimidazole derivatives as BACE-1 inhibitors:
Structure-based design, synthesis, and biological evaluation
,
,e
Gianpaolo Chiriano a b, Angela De Simone c, Francesca Mancini c, Daniel I. Perez d, Andrea Cavalli c
,
Maria Laura Bolognesi c, Giuseppe Legname f, Ana Martinez d, Vincenza Andrisano c, Paolo Carloni g,
Marinella Roberti c
,
*
a Statistical and Biological Physics Sector, SISSA-ISAS, Via Bonomea 265, 34136 Trieste, Italy
b Italian Institute of Technology, SISSA-ISAS Unit, Italy
c Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
d Instituto de Quimica Medica-CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
e Department of Drug Discovery and Development, Italian Institute of Technology, Via Morego 30, 16163 Genova, Italy
f Neurobiology Sector, SISSA-ISAS, Via Bonomea 265, 34136 Trieste, Italy
g German Research School for Simulation Sciences GmbH, 52425 Jülich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, we report a rational structure-based approach aimed at the discovery of new 2-
Received 23 June 2011
Received in revised form
3 November 2011
Accepted 9 December 2011
Available online 16 December 2011
aminoimidazoles as
b-secretase inhibitors. Taking advantage of a microwave-assisted synthetic
protocol, a small library of derivatives was obtained and biologically evaluated. Two compounds showed
promising activities in both enzymatic and cellular assays. Moreover, one of them exhibited the capa-
bility to cross the bloodebrain barrier as assessed by the parallel artificial membrane permeability assay.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Alzheimer’s disease
Amyloid-beta peptide
Drug design
Hit identification
2-Aminoimidazole
1. Introduction
A
b
forms toxic extra-cellular (proto)-fibrils, which initiate the
pathogenic cascade [2]. Thus, the discovery of compounds able to
modulate the production and clearance of A represents a key
strategy in the field of AD [3e5]. A is generated by sequential
proteolytic cleavage of large trans-membrane protein, the
amyloid precursor protein (APP), by two proteases, - and -sec-
retase. Therefore, - and -secretase enzymes have been studied in
depth in the search for inhibitors as potential anti-AD drugs. In this
scenario, while a -secretase inhibitor, CTS-21666 from CoMentis,
advanced up to Phase II clinical trials [6], a -secretase inhibitor
Alzheimer’s disease (AD) is a progressive and fatal brain
disorder, for which there is no cure. AD causes memory loss, steady
deterioration of cognition, and dementia afflicting currently over
30 million people worldwide. By 2050, estimates range as high as
more than 100 million Alzheimer’s patients worldwide (World
Alzheimer Report 2010) (http://www.alz.org/). One of the major
characteristic and pathological hallmarks of AD is represented by
the senile plaques, whose main component is the amyloid-
b
b
a
b
g
b
g
b
g
b
peptide (Ab) [1].
failed in Phase III clinical trials because of lack of efficacy and
increased risk of skin cancer [7]. Furthermore, the presence of
several structural information related to
suitable target for structure-based drug design purposes [8].
The first -secretase inhibitors were peptide and peptidomi-
metic compounds successfully designed as transition state analogs
showing a nanomolar affinity for -secretase [9,10]. The crystal
b-secretase makes it a very
Abbreviations: AD, Alzheimer’s Disease; A
precursor protein; BBB, bloodebrain barrier; BACE-1,
enzyme; cLogP, calculated decimal logarithm of octanol/water partition coefficient;
CNS, central nervous system; ESP, electrostatic potential; HTS, high-throughput
screening; PAMPA, parallel artificial membrane permeability assay; TPSA, topo-
logical polar surface area.
b
, amyloid-
b
peptide; APP, amyloid
b
-secretase APP cleaving
b
b
structures of these inhibitors in complex with the enzyme have
been utilized for structure-based projects that have led to the
* Corresponding author. Fax: þ39 051 2099734.
E-mail address: marinella.roberti@unibo.it (M. Roberti).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.016

G. Chiriano et al. / European Journal of Medicinal Chemistry 48 (2012) 206e213
207
discovery of several classes of compounds with improved phar-
macokinetics properties [11]. In particular, there was a boom in the
and a
p
e
p
stacking with Tyr71; iii) the second phenyl ring estab-
interaction with the side chain of Arg235.
lished a cation-
p
development of non-peptidic
b
-secretase inhibitors that have been
In light of this computational result, 1 was tested against BACE-1
discovered by means of different approaches, such as high-
throughput screening (HTS), fragment-based, and structure-based
strategies [12e14]. Compared with traditional HTS, a significantly
higher hit rate can be obtained by using a structure-based
approach, which can fully exploit the large amount of structural
using an enzymatic assay [20]. It exhibited a moderate-to-low
inhibitor potency at 100
mM concentration (BACE-1 inhibition
% ¼ 19.64 ꢀ 0.69). On these bases, decorating fragment 1, we
designed and synthesized a small library of 2-aminoimidazoles.
information related to
In this paper, we report on the structure-based design and
microwave-assisted synthesis of novel small library of 2-
-secretase inhibitors.
b-secretase.
3. Chemistry
a
The 2-aminoimidazoles, 1e11, were obtained taking advantage
of a microwave-assisted, one-pot, two-step protocol [18] based on
the cyclocondensation of 2-benzylaminopyrimidines 12aed and
aminoimidazoles as
b
appropriate 3-substituted-a-bromopropyl aldehydes 13aef, fol-
2. Design
lowed by the cleavage of the corresponding not isolated interme-
diate imidazo[1,2-a]pyrimidin-1-ium salts with an excess of
hydrazine (Scheme 1). The 2-benzylaminopyrimidines 12aed were
synthesized in parallel by reaction of commercially available ben-
zylbromides 14aed with excess of 2-aminopyrimidine 15 and
The b-secretase APP cleaving enzyme (BACE-1) is a member of
the pepsin-like family of aspartyl proteases. It is a class I trans-
membrane protein characterized by an NH2-terminal protease
domain structurally well-defined, a connecting strand, a trans-
membrane region, and a cytosolic domain [15].
sodium hydride (Scheme 2). The a-bromo aldehydes 13aef were
obtained in a parallel fashion using the following synthetic
pathway. A cross-coupling Suzuki reaction between the 3-(bro-
mophenyl)-propionic methyl esters 16,17, which can be easily
accessed from the corresponding 3-(bromophenyl)-propanoic
acids, and the appropriate boronic acids 18e21 in the presence of
a catalytic amount of tetrakis (triphenylphosphine) palladium
Pd(PPh3)4 gave the 3-biphenyl propanoic methyl esters 22aee,
respectively. These were reduced to the corresponding 3-biphenyl
propyl alcohols 23aee. Oxidation of 23aee and commercially
available 3-phenylpropanol 23f gave the corresponding 3-
substituted propyl aldehydes 24aef, which were brominated in
mild conditions using 0.5 equivalent of 5,5-dibromobarbituric acid
Initially, we aimed at identifying a moiety potentially interacting
with the catalytic aspartic dyad of the enzyme. In particular, among
the possible scaffolds, the 2-aminoimidazole appeared to be a very
attractive moiety for the following reasons: i) it contains the gua-
nidinium function, which can provide optimal interactions with the
catalytic aspartic dyad (see Supporting Information (SI)), as also
demonstrated by the crystal structures of several guanidinium-
carrying inhibitors in complex with BACE-1 [12]; ii) it is a privi-
leged structure [16]; iii) it allows the parallel synthesis of differ-
ently polysubstituted derivatives [17,18]. Therefore, the 2-
aminoimidazole was docked to validate its capability to interact
with the catalytic dyad of BACE-1. As expected, the 2-
aminoimidazole turned out to be oriented in the center of the
rather large BACE-1 binding pocket by interacting with both cata-
lytic aspartic acids, Asp32 and Asp228, via electrostatic and H-bond
interactions (see SI). Then, among the 2-aminoimidazoles reported
in the literature [16,18,19], the fragment 1 [18] shown in Fig. 1
turned out to be particularly well-suited for drug discovery
purposes for the following reasons: 1 has a low molecular weight
(MW ¼ 263.34) and displays a rather good chemical accessibility,
which could allow for generating library of compounds. This frag-
ment was preliminary investigated by means of docking simula-
tions. The binding mode of 1 at BACE-1 binding pocket is reported
in Fig. 1. The following interactions were identified: i) the guani-
dinium moiety of 1 interacted with both aspartic acids (Asp32 and
Asp228) side chains and with Thr232; ii) one of the two phenyl
rings formed hydrophobic interactions (with Val69, Trp76, Phe108)
(DBBA) to provide the required 3-substituted-a-bromopropyl
aldehydes 13aef (Scheme 3).
4. Biology
2e11 were first tested in biochemical assays performed using the
fluorescence resonance energy transfer (FRET) methodology [20].
The BACE-1 inhibition studies were based on the cleavage of peptide
substrate mimicking the human APP sequence with the Swedish
mutation (Methoxycoumarin-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-
Phe-Lys-dinitrophenyl, M-2420, Bachem, Germany) [20] (see SI).
2e11 were tested at a concentration of 5
mM and their BACE-1
inhibition percentages are reported in Table 1. The IC50 values of
most active compounds (7e9 and 11) were determined by using the
linear regression parameters. Subsequently, the capability of 7e9
Fig. 1. Low-energy docking model of the BACE-1/1 complex and the main interactions are highlighted by orange points. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

208
G. Chiriano et al. / European Journal of Medicinal Chemistry 48 (2012) 206e213
Scheme 1. Reagents and conditions: a) MeCN, 150 ꢁC, 150 W; b) 60% hydrazine (5 equiv), MeCN, 100 ꢁC, 100 W.
and 11 to modulate APP processing was examined by performing
a cell-based ELISA assay. This study was carried out in primary
chicken telencephalon neurons to assess the effect of the most active
inhibitors on secretion of Ab38, Ab40 and Ab42 [21] (see SI).
To increase the chemical diversity, we then synthesized a second
series of derivatives, 5e11, maintaining a halogenated benzyl group
in R1 and bearing differently substituted aromatic rings in R2 and R3
(meta and para positions, see Scheme 1). All compounds showed
a BACE-1 inhibitory profile. In particular, 7e9 and 11 showed IC50
values in the low micromolar range (see Table 1). To characterize the
binding mode of one of the most active inhibitors, docking and
molecular dynamics (MD) simulations were carried out using BACE-
1 (PDB ID: 1SGZ) [23] and 7 (see Fig. 2 and SI). The following
interactions were observed for the best-ranked pose as obtained
using the Goldscore scoring function (see SI): i) the amino group
(NH2) of 7 interacts via H-bond with the catalytic dyad; ii) the N3
nitrogen of the imidazole ring establishes electrostatic and H-bond
interactions with the side chains of Asp228 and Thr232, respec-
tively; iii) the fluorine atom interacts with the NH of Trp76 side
5. Results and discussion
As previously described, our strategy was based on 1 as the
starting fragment for generating a new series of BACE-1 inhibitors.
In particular, we attempted to improve the low potency of 1 (see
Fig. 1) by initially modifying the electronic and hydrophobic
properties of the benzyl group in position R1 (see Scheme 1)
through the introduction of fluorine and chlorine atoms in different
positions (see compounds 2e4 in Table 1). These substitutions
allowed 2e4 to consolidate the hydrophobic interactions observed
for 1 (Fig. 1) and to potentially establish H-bonds (for the fluorine
derivatives) with the OH of Tyr71 and NH of Trp76 side chains. As
chain; iv) the benzyl ring establishes favorable
pep stacking with
the side chain of Tyr71 and hydrophobic interactions with Val69,
Trp76, and Phe108; v) the phenyl group mounted on the C4 of the
expected, 2e4 showed
a BACE-1 inhibitory potency notably
imidazole ring interacts via cation-p with the Arg235; vi) the pol-
increased when compared to that of 1 (see Table 1). To inspect for
possible correlations between electronic properties and BACE-1
inhibition, the electrostatic potential (ESP) surfaces were calcu-
lated for 2e4. A qualitative relationship was observed between an
increased inhibition percentage and a decreased negative character
of the aromatic ring in R1 (see Fig. S4 in SI). In particular, when
compared to 1, 2e4 appeared to have an electron-poorer benzyl
group (R1) that could allow this moiety to establish more favorable
ymethoxylated substituent in R3 might establish H-bond interac-
tions with the side chains of Asn233 and Lys321, both residues
located in a solvent-exposed region of the active site. Notably, once
this complex was already computationally generated, the X-ray
structure of a 2-aminoimidazole derivative in complex with BACE-1
was published by researchers from Merck [24,25]. Interestingly
enough, our predicted binding mode was remarkably similar to that
reported [24,25] showing as pivotal interactions the salt-bridge
between the guanidinium moiety and the aspartic dyad. To
further investigate the role of these electrostatic interactions, we
monitored the stability of these salt bridges throughout 100 ns of
MD simulations (see SI for further details). Both interactions were
remarkably stable showing that the guanidinium was the anchoring
point of our inhibitors at BACE-1 active site.
pep stacking with electron-rich aromatic residues located in the
binding pocket (i.e. Tyr71 and Trp76). In addition, from a pharma-
cokinetic perspective, the presence of fluorine atoms on an
aromatic ring could improve the metabolic stability, by avoiding
a probable aromatic hydroxylation mechanism [22].
In light of these results, 7e9 and 11 were tested using cellular
assays based on the secretion of Ab38, Ab40 and Ab42 and on cell
viability in primary chicken telencephalon neurons [21]. The IC50
values reported in Table 2 have been corrected with mean neurons
viability obtained in the MTT reduction assay (see SI). 7 was the
most active compound inhibiting Ab38, Ab40, and Ab42 secretion
with IC50 values of 15, 23 and 19
moderately toxic at 25 M. Otherwise, 8 started to display toxic
effects at 50 M, and reduced Ab38, Ab40, and Ab42 secretion with
higher IC50 values than 7 (33, 35 and 27 M respectively). In
mM respectively, and starting to be
m
m
m
contrast, 9 and 11 resulted inactive. To explain the different activity
of derivatives 7e9 and 11 in cellular assays, we explored some of
Scheme 2. Reagents and conditions: a) NaH, THF, 24 h, room temperature.

G. Chiriano et al. / European Journal of Medicinal Chemistry 48 (2012) 206e213
209
Scheme 3. Reagents and conditions: a) Pd(PPh3)4, Na2CO3 (aq), toluene:EtOH (2:1), 5 h, reflux; b) LiAlH4, Et2O, 0 ꢁC, 5 h; c) PCC (1.4 equiv), CH2Cl2, 0 ꢁC, 3 h; d) DBBA (0.5 equiv),
Et2O, HCl (cat).
their molecular descriptors such as calculated decimal logarithm of
octanol/water partition coefficient (cLogP) and topological polar
surface area (TPSA). 9 and 11 showed higher cLogP and lower TPSA
values when compared to 7 and 8 (see Table 2). Finally, since an
anti-AD drug candidate must work at central nervous system (CNS)
level, we studied the capability of 7e9 and 11 to cross the
bloodebrain barrier (BBB) by using the parallel artificial membrane
permeability assay (PAMPA), as described by Di et al. [26] (see SI).
As shown by the in vitro permeability (Pe) values (Table 2), 8 BBB
permeation was predicted to be low, 9 was not examined because
of its insolubility in the experimental conditions here employed,
whereas 7 and 11 were predicted to be able to cross the BBB by
passive permeation. Moreover, the calculated LogP and TPSA values
of 7 and 8 in their protonated form are in agreement with the
optimal physicalechemical parameters for targeting CNS drugs
[27,28]. Differently, the cLogP values are to high both for 9 and 11,
whereas the TPSA value is low for 9 and proper for 11 (see Table 2).
In light of this series of experiments, it turned out that 7 was
a promising hit to undergo to a subsequent hit-to-lead campaign.
Interestingly, structurally similar compounds recently reported by
Hills et al. [25] have shown relatively low Pgp efflux, pointing to this
class of molecules as promising BACE-1 lead candidates. Indeed, our
result could be particularly hopeful in the context of the 2-
aminoimidazole-based BACE-1 inhibitors, where a major issue is
the BBB penetration that may hamper the further development of 2-
aminoimidazoles endowed with in vitro low nanomolar profiles [29].
chemical accessibility that allows to carry out extensive SAR studies;
ii) a low micromolar inhibitory profile against BACE-1, as assessed by
enzymatic and cellular assays; iii) the capability to cross in vitro the
BBB. In conclusions, 7 can represent the starting point for an extensive
campaign of hit-to-lead and eventually lead optimization.
7. Experimental section
7.1. General chemical methods
Reaction progress was monitored by TLC on precoated silica gel
plates (Kieselgel60 F254, Merck) andvisualized by UV254light. Flash
column chromatography was performed on silica gel (particle size
40e63 mM, Merck). Tetrahydrofuran (THF) and Et2O were freshly
distilled over sodium/benzoketal. Unless otherwise stated, all
reagents were obtained from commercial sources and used without
further purification. Compounds 16, 17 were obtained following
a standard procedure as described in SI. Compounds were named
relying on the naming algorithm developed by CambridgeSoft
Corporation and used in Chem-BioDraw Ultra 11.0. 1H NMR and 13
C
NMR spectra were recorded at 200e400 and 50e100 MHz, respec-
tively. All the NMR experiments were performed by using CDCl3 as
solvent. Chemical shifts (d) are reported in parts per millions (ppm)
relative to TMS as internal standard. Coupling constants (J), when
given, are reported in Hertz (Hz). For microwave-assisted organic
synthesis a CEM Discover BenchMate reactor was used in the stan-
dard configuration as delivered, including proprietary software. All
microwave-assisted reactions were carried out in sealed quartz
process vials (15 mL). IR-FT spectra were performed in Nujol and
obtained on a Nicolet Avatar 320 E.S.P. instrument; nmax is expressed
in cmꢂ1. Mass spectrawere recordedona V.G. 7070E spectrometeror
on a Waters ZQ 4000 apparatus operating in electrospray (ES) mode.
Purity of compounds was determined by elemental analyses; purity
for all the tested compounds was ꢃ95%.
6. Conclusion
In this paper, we have described a rational structure-based
approach, integrated with a synthetic protocol amenable to parallel
synthesis, aimed at the discovery of new 2-aminoimidazole deriva-
tives as BACE-1 inhibitors. Among 10 novel derivatives, 7 hasemerged
as a promising anti-BACE-1 hit compound thanks to: i) a rather good

210
G. Chiriano et al. / European Journal of Medicinal Chemistry 48 (2012) 206e213
150 W at the temperature of 150 ꢁC for 75 min. After the reaction
Table 1
BACE-1 inhibition profile of compounds 2e11.
mixture was cooled with an air flow for 15 min, a hydrazine hydrate
60% solution (5 equiv) was added, and the mixture was irradiated at
100 W to heat at the temperature of 100 ꢁC for 15 min. The reaction
mixture was diluted by CH2Cl2 (20 mL), washed with a saturated
NH4Cl solution (10 mL), brine (10 mL) and H2O (2 ꢄ 10 mL). The
organic layer was dried over Na2SO4, then filtered and concen-
trated. The resulting residue was purified by flash chromatography
on silica gel (CH2Cl2/MeOH ¼ 9.5/0.5) (Scheme 1).
Cpds Chemical structure
BACE-1 inhibition (%)a,b BACE-1 IC50 M)a
(m
2
26.40 ꢀ 0.02
20.27 ꢀ 0.01
n.d.c
3
n.d.
7.2.1. 4-Benzyl-1-(4-fluorobenzyl)-1H-imidazol-2-amine 2
Reaction of N-(4-fluorobenzyl)pyrimidin-2-amine 12b (0.25 g,
1.25 mmol) and 2-bromo-3-phenylpropanal 13f (0.36 g, 1.68 mmol)
gave the crude final product 2 that was purified by flash chroma-
tography (CH2Cl2/MeOH ¼ 9.5/0.5). Yield 29%; brown semisolid;
4
5
23.31 ꢀ 0.01
32.51 ꢀ 0.01
n.d.
n.d.
ESI-MS (m/z): 282 (M þ Hþ); 1H NMR (200 MHz):
d 7.28e7.12 (m,
7H), 7.05e6.96 (m, 2H), 6.04 (s, 1H), 5.94 (br-s, 2H, NH2), 4.87 (s,
2H), 3.73 (s, 2H) ppm; 13C NMR (50 MHz):
d 164.7, 159.8, 147.4,
138.3, 131.0, 130.9, 129.2, 129.1, 128.7, 128.3, 126.3, 116.0, 115.5, 111.5,
47.8, 33.0 ppm. IR:
n
¼ 3422 cmꢂ1
.
7.2.2. 4-Benzyl-1-(3,5-difluorobenzyl)-1H-imidazol-2-amine 3
Reaction of N-(3,5-difluorobenzyl)pyrimidin-2-amine 12c
(0.27 g, 1.25 mmol) and 2-bromo-3-phenylpropanal 13f (0.36 g,
1.68 mmol) gave the crude final product 3 that was purified by flash
chromatography (CH2Cl2/MeOH ¼ 9.5/0.5). Yield 21%; brown
semisolid; ESI-MS (m/z): 300 (M þ Hþ); 1H NMR (200 MHz):
6
32.48 ꢀ 0.01
n.d.
d
7.29e7.20 (m, 5H), 6.71e6.67 (m, 3H), 6.10 (s, 1H), 5.10 (br-s, 2H,
NH2), 4.89 (s, 2H), 3.77 (s, 2H) ppm; 13C NMR (50 MHz):
d
165.8 (d,
7
8
40.25 ꢀ 0.01
38.17 ꢀ 0.05
7.40 ꢀ 1.20
7.32 ꢀ 0.54
J ¼ 12.5), 160.8 (d, J ¼ 12.5), 148.2, 139.0, 138.7, 137.7, 132.2, 128.8,
128.5, 126.6, 111.2, 110.0, 103.8 (t, J ¼ 25), 47.6, 32.6 ppm. IR:
n
¼ 3421 cmꢂ1
.
7.2.3. 4-Benzyl-1-(2-chlorobenzyl)-1H-imidazol-2-amine 4
Reaction of N-(2-chlorobenzyl)pyrimidin-2-amine 12d (0.27 g,
1.25 mmol) and 2-bromo-3-phenylpropanal 13f (0.36 g, 1.68 mmol)
gave the crude final product 4 that was purified by flash chroma-
tography (CH2Cl2/MeOH ¼ 9.5/0.5). Yield 11%; brown semisolid;
9
41.34 ꢀ 0.01
24.25 ꢀ 0.02
5.59 ꢀ 0.06
ESI-MS (m/z): 299 (M þ Hþ); 1H NMR (200 MHz):
d 7.40e7.23 (m,
8H), 7.07e7.02 (m, 1H), 5.96 (s, 1H), 4.95 (s, 2H), 4.80 (br-s, 2H, NH2)
3.80 (s, 2H) ppm; 13C NMR (50 MHz):
d 148.1, 137.8, 133.0, 132.3,
10
n.d.
131.5, 129.9, 129.8, 128.9, 128.7, 128.5, 127.5, 126.6, 111.3, 46.2,
32.5 ppm. IR:
n
¼ 3420 cmꢂ1
.
7.2.4. 1-(4-Fluorobenzyl)-4-((30,50-dimethoxybiphenyl-3-yl)
methyl)-1H-imidazol-2-amine 5
11
37.78 ꢀ 0.01
5.95 ꢀ 0.17
0.01 ꢀ 0.00
Reaction of N-(4-fluorobenzyl)pyrimidin-2-amine 12b (0.25 g,
1.25 mmol) and 2-bromo-3-(30,50-dimethoxybiphenyl-3-yl)propa-
nal 13a (0.58 g, 1.68 mmol) gave the crude final product 5 that was
purified by flash chromatography (CH2Cl2/MeOH ¼ 9.5/0.5). Yield
21%; orange solid; m.p. 76.5e80.0 ꢁC decomposed; ESI-MS (m/z):
IVd
>80
418 (M þ Hþ); 1H NMR (200 MHz):
d 7.48e7.25 (m, 4H), 7.15e6.99
(m, 4H), 6.74e6.73 (m, 2H), 6.48e6.46 (m, 1H), 6.21 (s, 1H), 4.79 (s,
a
Values are mean ꢀ S.D. of two independent experiments for BACE-1 inhibition
2H), 3.99 (br-s, 2H, NH2), 3.85 (s, 6H), 3.82 (s, 2H) ppm; 13C NMR
[20].
b
% inhibition of BACE-1 activity at the concentration of 5
mM of the tested
(50 MHz): d 164.8, 161.0, 159.9, 147.5, 143.5, 141.1, 140.4, 136.7, 131.8,
compounds 2e11.
c
131.7, 128.7, 128.5, 128.1, 127.7, 124.9, 116.1, 115.7, 112.4, 105.5, 99.1,
n.d. ¼ not determined.
¼ 3414 cmꢂ1
.
d
55.4, 47.8, 34.8 ppm. IR:
n
Reference BACE-1 inhibitor, b-secretase inhibitor IV, Calbiochem, UK.
7.2. General procedure for the microwave-assisted synthesis of
2-aminoimidazoles, 1e11
7.2.5. 1-(3,5-Difluorobenzyl)-4-((30,50-dimethoxybiphenyl-3-yl)
methyl)-1H-imidazol-2-amine 6
Reaction of N-(3,5-difluorobenzyl)pyrimidin-2-amine 12c
(0.27 g, 1.25 mmol) and 2-bromo-3-(30,50-dimethoxybiphenyl-3-yl)
propanal 13a (0.58 g, 1.68 mmol) gave the crude final product 6 that
was purified by flash chromatography (CH2Cl2/MeOH ¼ 9.5/0.5).
Yield 22%; brown semisolid; ESI-MS (m/z): 436 (M þ Hþ); 1H NMR
In a 10 mL microwave vial, 2-benzylaminopyrimidines 12aed
(1.0 equiv) and 3-substituted-a-bromopropyl aldehydes 13aef
(1.35 equiv) were successively dissolved in dry CH3CN (2e3 mL).
The microwave reactor was irradiated by maximum power of

G. Chiriano et al. / European Journal of Medicinal Chemistry 48 (2012) 206e213
211
Fig. 2. The binding mode of 7 at the proteasic domain of BACE-1 (PDB ID: 1SGZ) [23] with the key interactions shown by orange points. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
(400 MHz):
d
7.46e7.44 (m, 1H), 7.42e7.40 (m, 1H), 7.35e7.31 (m,
7.2.7. 1-(3,5-Difluorobenzyl)-4-((30,50-dimethoxybiphenyl-4-yl)
methyl)-1H-imidazol-2-amine 8
1H), 7.24e7.21 (m, 2H), 6.75e6.70 (m, 2H), 6.69e6.61 (m, 2H),
6.44e6.43 (m,1H), 6.04 (s,1H), 4.79 (s, 2H), 4.82 (s, 6H), 3.80 (s, 2H),
Reaction of N-(3,5-difluorobenzyl)pyrimidin-2-amine 12c
(0.28 g, 1.25 mmol) and 2-bromo-3-(30,50-dimethoxybiphenyl-4-yl)
propanal 13b (0.59 g,1.69 mmol) gave the crude final product 8 that
was purified by flash chromatography (CH2Cl2/MeOH ¼ 9.5/0.5).
Yield 31%; yellow solid; m.p. 142.5e145.5 ꢁC decomposed; ESI-MS
2.78 (br-s, 2H, NH2) ppm; 13C NMR (50 MHz):
d
165.7 (d, J ¼ 12.5),
161.7 (d, J ¼ 12.5), 148.0,143.3, 141.0, 140.3, 140.2, 136.9, 128.6, 128.1,
127.7, 124.9, 112.0, 109.8, 109.6, 109.3, 105.4, 103.3 (t, J ¼ 25), 99.2,
55.3, 47.4, 34.7 ppm. IR:
n
¼ 3415 cmꢂ1
.
(m/z): 436 (M þ Hþ); 1H NMR (400 MHz):
d
7.49 (d, J ¼ 8.4, 2H),
7.2.6. 1-(4-Fluorobenzyl)-4-((30,50-dimethoxybiphenyl-4-yl)
methyl)-1H-imidazol-2-amine 7
7.32 (d, J ¼ 8.4, 2H), 6.73e6.70 (m, 1H), 6.69e6.68 (m, 2H),
6.65e6.62 (m, 2H), 6.43e6.42 (m, 1H), 6.21 (s, 1H), 4.81 (s, 2H), 3.83
(s, 2H), 3.82 (s, 6H), 1.64 (br-s, 2H, NH2) ppm; 13C NMR (50 MHz)
Reaction of N-(4-fluorobenzyl)pyrimidin-2-amine 12b (0.25 g,
1.25 mmol) and 2-bromo-3-(30,50-dimethoxybiphenyl-4-yl)propa-
nal 13b (0.58 g, 1.68 mmol) gave the crude final product 7 that was
purified by flash chromatography (CH2Cl2/MeOH ¼ 9.5/0.5). Yield
30%; yellow solid; m.p. 156.5e161.2 ꢁC decomposed; ESI-MS (m/z):
d
165.8 (d, J ¼ 12.5), 161.0 (d, J ¼ 12.5), 147.7, 143.2, 140.4, 140.2,
140.1, 139.4, 138.9, 137.2, 129.2, 127.1, 112.2, 109.8, 109.3, 105.2, 103.4
(t, J ¼ 25), 99.0, 55.3, 47.6, 34.3 ppm. IR:
n
¼ 3415 cmꢂ1
.
418 (M þ Hþ); 1H NMR (400 MHz):
d
7.47 (d, J ¼ 8.4, 2H), 7.31 (d,
7.2.8. 1-(3,5-Difluorobenzyl)-4-((40-(trifluoromethyl)biphenyl-4-yl)
methyl)-1H-imidazol-2-amine 9
Reaction of N-(3,5-difluorobenzyl)pyrimidin-2-amine 12c
(0.15 g, 0.70 mmol) and 2-bromo-3-(40-(trifluoromethyl)biphenyl-
4-yl)propanal 13c (0.34 g, 0.95 mmol) gave the crude final product
9 that was purified by flash chromatography. Yield 26%; yellow
solid; m.p. 164.0e169.0 ꢁC decomposed; ESI-MS (m/z): 444
J ¼ 8.4, 2H), 7.10e7.08 (m, 2H), 7.04e6.99 (m, 2H), 6.70e6.69 (m,
2H), 6.44e6.43 (m,1H), 6.02 (s,1H), 4.79 (s, 2H), 3.82 (s, 6H), 3.79 (s,
2H), 1.96 (br-s, 2H, NH2) ppm; 13C NMR (50 MHz)
d 164.0, 161.0,
159.0,147.4,143.3, 139.3, 139.0, 136.6,131.7,129.2, 128.7, 128.5, 127.1,
116.2, 115.8, 112.4, 105.3, 99.1, 55.4, 47.9, 34.3 ppm. IR:
n
¼ 3414 cmꢂ1
.
Table 2
Inhibition of Ab38, Ab40 and Ab42 secretion,a in vitro permeability (Pe) valuesb with related predictive penetration into the CNSc and molecular descriptorsd of compounds 7e9
and 11.
b
Cpds
A
b38 IC50
(
m
M)a
A
b40 IC50
(
m
M)a
A
b42 IC50
(
m
M)a Pe (10ꢂ6 cm sꢂ1
)
Predictionc cLogP (prot.)d cLogP (not prot.)d TPSA (prot.)d TPSA (not prot.)d
7
8
9
11
15
23
35
n.a.
n.a.
19
27
n.a.
n.a.
4.0 ꢀ 1.0
3.2 ꢀ 0.2
n.d.f
4.0 ꢀ 0.7
CNSþ
CNSþ/ꢂ
n.d.
CNSþ
2.572
2.663
3.517
4.581
5.593
5.685
6.539
7.602
63.562
63.562
45.094
58.234
62.317
62.317
43.849
56.989
33
n.a.e
n.a.
a
Values are mean of three independent experiments for reduction of Ab secretion. All data were corrected with mean neurons viability obtained in the MTT reduction assay,
performed after 24 h of treatment with these BACE-1 inhibitors to evaluate their potential cell toxicity.
b
Values are mean ꢀ S.D. of two independent experiments (PBS/EtOH ¼ 70/30 was used as solvent).
c
The compounds were classified [26] as CNSþ when they present a Pe value >3.55 ꢄ 10ꢂ6 cm sꢂ1, and as CNSþ/ꢂ when the Pe value is between 3.55 ꢄ 10ꢂ6 and
2.00 ꢄ 10ꢂ6 cm sꢂ1
.
d
cLogP and TPSA in both protonated and not protonated were calculated by Molinspiration, a free-online cheminformatics tool (http://www.molinspiration.com).
e
f
n.a. ¼ not active.
n.d. ¼ not determined.

212
G. Chiriano et al. / European Journal of Medicinal Chemistry 48 (2012) 206e213
(M þ Hþ); 1H NMR (400 MHz):
d
7.66e7.64 (m, 4H), 7.53 (d, J ¼ 8.4,
7.4. General parallel procedure for the synthesis of biphenyl
propanoic acid methyl esters 22aee
2H), 7.36 (d, J ¼ 8.4, 2H), 6.76e6.75 (m, 1H), 6.66e6.65 (m, 2H), 6.08
(s, 1H), 4.81 (s, 2H), 3.83 (s, 2H), 3.06 (br-s, 2H, NH2) ppm; 13C NMR
(100 MHz):
d
164.6 (d, J ¼ 12.3), 162.1 (d, J ¼ 12.3), 148.3, 144.3,
In distinct reactors, 3-(bromophenyl)-propionic methyl esters 16,
17 (1.0 equiv) were dissolved in toluene (7 mL). Phenyl boronic acids
18e21 (2 equiv) in EtOH (3.5 mL) and Na2CO3 2 M (2 M, aq, 3.0 equiv)
were then added to the corresponding reactors, and the resulting
mixtures were deoxygenated with a stream of N2. After 10 min,
Pd(PPh3)4 (0.005 equiv) was added and each mixture was stirred at
reflux temperature for 5 h under N2, then cooled to room tempera-
ture and treated as follows. Each solution was poured into a mixture
of H2O (5 mL) and Et2O (5 mL), and the two phases were separated.
The aqueous layer was washed with Et2O (5 mL), and the organic
phases were combined and washed with 1 M NaOH (5 mL), followed
by brine (5 mL), then dried over Na2SO4 and evaporated. Purification
of each crude product performed by flash chromatography on silica
gel (petroleum ether/EtOAc ¼ 8/2) yielded the corresponding
biphenyl propanoic acid methyl esters 22aee (Scheme 3).
138.5, 138.1, 137.9, 132.1, 129.5, 129.1, 127.5, 127.2, 125.7, 125.6, 122.1,
111.2, 110.1, 109.9, 104.0 (t,
J
¼
24.6), 47.6, 32.2 ppm. IR:
n
¼ 3418 cmꢂ1
.
7.2.9. 1-(3,5-Difluorobenzyl)-4-((30-(benzyloxy)biphenyl-4-yl)
methyl)-1H-imidazol-2-amine 10
Reaction of N-(3,5-difluorobenzyl)pyrimidin-2-amine 12c
(0.18 g, 0.82 mmol) and 2-bromo-3-(30-(benzyloxy)biphenyl-4-yl)
propanal 13d (0.44 g, 1.11 mmol) gave the crude final product 10
that was purified by flash chromatography (CH2Cl2/MeOH ¼ 9.5/
0.5). Yield 23%; yellow solid; m.p. 166.0e171.0 ꢁC decomposed; ESI-
MS (m/z): 482 (M þ Hþ); 1H NMR (200 MHz):
d 7.56e7.29 (m, 10H),
7.21e7.18 (m, 2H), 6.99e6.96 (m, 1H), 6.82e6.98 (m, 3H), 6.01 (s,
1H), 5.14 (s, 2H), 4.88 (s, 2H), 3.84 (s, 2H), 2.02 (br-s, 2H, NH2) ppm;
13C NMR (50 MHz):
d
160.8 (d, J ¼ 12.5), 159.1 (d, J ¼ 12.5), 147.3,
142.6, 139.2, 138.8, 137.6, 137.0, 130.9, 129.7, 129.3, 128.6, 128.0,
7.4.1. Methyl 3-(30,50-dimethoxybiphenyl-4-yl)propanoate 22b
Reaction of 3-(bromophenyl)-propionic methyl ester 17 (0.3 g,
1.23 mmol) and boronic acid 18 (0.45 g, 2.46 mmol) afforded
compound 22b that was purified by flash chromatography on silica
gel (petroleum ether/EtOAc ¼ 8/2). Yield 94%; white powder; m.p.
127.5, 127.2, 119.8; 113.7, 113.3, 112.6, 109.9, 109.4, 103.6 (t, J ¼ 25),
70.1, 47.7, 34.5 ppm. IR:
n
¼ 3415 cmꢂ1
.
7.2.10. 1-(2-Chlorobenzyl)-4-(4-(dibenzo[b,d]furan-1-yl)benzyl)-
1H-imidazol-2-amine 11
50.0e52.0 ꢁC; 1H NMR (200 MHz):
J ¼ 8.0, 2H), 6.75e6.74 (m, 2H), 6.49e6.48 (m, 1H), 3.87 (s, 6H), 3.71
(s, 3H), 3.02 (t, J ¼ 7.6, 2H), 2.69 (t, J ¼ 7.6, 2H) ppm.
d
7.52 (d, J ¼ 7.8, 2H), 7.29 (d,
Reaction of N-(2-chlorobenzyl)pyrimidin-2-amine 12d (0.15 g,
0.68 mmol) and 2-bromo-3-(4-(dibenzo[b,d]furan-1-yl)phenyl)
propanal 13e (0.35 g, 0.92 mmol) gave the crude final product 11
that was purified by flash chromatography (CH2Cl2/MeOH ¼ 9.5/
0.5). Yield 13%; yellow solid; m.p. 76.2e81.2 ꢁC decomposed; ESI-
7.5. General procedure for the synthesis of 3-biphenyl propyl
alcohols 23aee
MS (m/z): 465 (M þ Hþ); 1H NMR (400 MHz):
d 7.97e7.95 (m,
1H), 7.91e7.88 (m, 1H), 7.83e7.81 (m, 2H), 7.58e7.55 (m, 2H),
7.46e7.31 (m, 6H), 7.25e7.22 (m, 2H), 6.94e6.92 (m, 1H), 6.25 (s,
1H), 4.92 (s, 2H), 3.87 (s, 2H), 2.63 (br-s, 2H, NH2) ppm; 13C NMR
LiAlH4 (1.2 equiv) was dissolved in dry Et2O (2 mL) and to the
resulting suspension cooled at 0 ꢁC a solution of the appropriate
biphenyl propanoic methyl esters 22aee (1.0 equiv) in dry Et2O was
added dropwise. The reaction mixture was stirred at room
temperature for 5 h under N2 atmosphere. The mixture was cooled
to 0 ꢁC and quenched with sequential additions of H2O (1 mL),
NaOH 10% (0.8 mL) and H2O (1 mL). The aqueous suspension was
extracted with Et2O (3 ꢄ 10 mL). The combined organic layers were
dried over Na2SO4 and evaporated to dryness. The crude products
were used in a further reaction without purification (Scheme 3).
(100 MHz):
d 156.1, 153.3, 148.2, 136.9, 135.0, 133.1, 131.8, 130.3,
130.0, 129.9, 129.1, 129.0, 128.8, 127.6, 127.2, 126.7, 125.4, 124.9,
124.1, 123.2, 122.8, 120.6, 119.6, 111.8, 111.3, 46.4, 31.8 ppm. IR:
n
¼ 3413 cmꢂ1
.
7.3. General parallel procedure for the synthesis of 2-benzylaminopyr-
imidines 12aed
In distinct reactors, 2-aminopyrimidine 15 (4 equiv) was dis-
solved in dry THF (12 mL) and the resulting solution was cooled in
an ice bath. To these solutions NaH (4 equiv) was added resulting in
effervescence and in the formation of suspensions. The mixtures
were stirred in the ice bath for 15 min, and then the appropriate
benzyl bromide derivates 14aed (1 equiv) were added dropwise to
each reactor. The mixtures were stirred at room temperature for
24 h and each one was treated as follow. The solvent was evapo-
rated under vacuo, and H2O (25 mL) was added. The resulting
aqueous solution was extracted with CH2Cl2 (3 ꢄ 25 mL) and the
combined organic layers were washed with H2O (25 mL), saturated
NaHCO3 solution (25 mL) and brine (25 mL), dried over anhydrous
Na2SO4 and concentrated under vacuo. Each crude residue was
purified by flash chromatography on silica gel (petroleum ether/
EtOAc ¼ 1/1) (Scheme 2).
7.5.1. 3-(30,50-Dimethoxybiphenyl-4-yl)propan-1-ol 23b
Biphenyl propanoic methyl ester 22b (0.15 g, 0.50 mmol) led to
compound 23b. Yield 99%; white powder; m.p. 80.5e81.5 ꢁC; 1H
NMR (200 MHz):
d
7.40 (d, J ¼ 8.2, 2H), 7.14 (d, J ¼ 7.8, 2H),
6.63e6.62 (m, 2H), 6.36e6.34 (m, 1H), 3.73 (s, 6H), 3.58 (t, J ¼ 6.6,
2H), 2.64 (t, J ¼ 7.2, 2H), 1.88e1.74 (m, 2H), 1.82 (br-s, 1H, OH) ppm.
7.6. General procedure for the synthesis of 3-susbstituted propyl
aldehydes 24aef
A solution of the appropriate 3-biphenyl propyl alcohols 23aef
(1.0 equiv) in CH2Cl2 (5 mL) was added dropwise to the suspension
of PCC (1.4 equiv) in CH2Cl2 (15 mL) cooled to 0 ꢁC. The reaction
mixture was stirred in an ice bath for 3e4 h, then filtered on silica
gel and evaporated to dryness. All the compounds were used in the
next step without any further purification (Scheme 3).
7.3.1. N-(4-fluorobenzyl)pyrimidin-2-amine 12b
Reaction of 2-aminopyrimidine 15 (1.5 g, 15.75 mmol) and the
benzyl bromide 14b (0.49 mL, 3.96 mmol) afforded compound 12b
that was purified on silica gel (petroleum ether/EtOAc ¼ 1/1). Yield
7.6.1. 3-(30,50-Dimethoxybiphenyl-4-yl)propanal 24b
3-Biphenyl propyl alcohol 23b (0.42 g, 1.54 mmol) lead to
compound 24b. Yield 72%; yellow oil; 1H NMR (400 MHz):
d 9.82 (t,
83%; white solid; m.p. 89.5e90.5 ꢁC; 1H NMR (200 MHz):
d
8.29 (d,
J ¼ 1.2, 1H), 7.50 (d, J ¼ 6.8, 2H), 7.24 (d, J ¼ 7.6, 2H), 6.72e6.71 (m,
2H), 6.47e6.45 (m, 1H), 3.83 (s, 6H), 2.98 (t, J ¼ 7.6, 2H), 2.79 (t,
J ¼ 7.2, 2H) ppm.
J ¼ 4.6, 2H), 7.38e7.29 (m, 2H), 7.08e6.99 (m, 2H), 6.58 (t, J ¼ 4.8,
1H), 5.62 (br-s, 1H, NH), 4.63 (d, J ¼ 6.0, 2H) ppm.

G. Chiriano et al. / European Journal of Medicinal Chemistry 48 (2012) 206e213
213
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A solution of the appropriate 3-susbstituted propyl aldehydes
24aef (1.0 equiv) in dry Et2O (2 mL) was added dropwise to
a solution of DBBA (0.5 equiv) in dry Et2O (4 mL). Successively,
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d
9.55 (d, J ¼ 1.8, 1H), 7.57e7.54 (m, 1H), 7.40e7.28 (m,
3H), 6.74e6.73 (m, 1H), 6.53e6.49 (m, 2H), 4.57e4.46 (m, 1H), 3.87
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Acknowledgments
The authors acknowledge the Italian Institute of Technology
(IIT) and the University of Bologna for the financial support. G.C.
thanks Dr. M. Masetti for valuable scientific discussions, and Ms S.
Leone and Ms R. Manfroni for their technical assistance. M.L.B.
thanks Dr. S. Czvitkovich and Dr. M. Windisch from JSW for their
scientific contribution. D.I.P. acknowledges a postdoctoral fellow-
ship from the CSIC (JAE program). Thanks are due to CIRB
(University of Bologna) for the use of Fluoroskan Ascent multiwell
spectrofluorometer and to Prof. D. Simoni (University of Ferrara) for
the use of CEM Discover BenchMate reactor.
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