Biphenyl-derivatives possessing tertiary amino groups as β-secretase (BACE1) inhibitors

Article abstract of DOI:10.2174/157018010791526304

β-Secretase (BACE1, β-site APP cleaving enzyme) is one of the most challenging therapeutic targets in the field of Alzheimer's disease (AD) research. This enzyme catalyses the formation of neuronal amyloid β (A β) plaques, whose increased production is a key event in the initial pathogenesis of AD. As a consequence, many BACE1 inhibitors have been developed by several research groups. In the present work, after an analysis of tetraline derivatives reported in a Takeda patent, we designed and synthesized some analogues, making appropriate structural modifications, in order to try to improve the bioavailability features and the activities of Takeda compounds. All the new derivatives were tested on BACE1 with the TR-FRET (Time Resolved-Fluorescence Resonance Energy Transfer) technology and one of them showed a promising inhibitory activity value.

Full text of DOI:10.2174/157018010791526304

Letters in Drug Design & Discovery, 2010, 7, 507-515
507
Biphenyl-Derivatives Possessing Tertiary Amino Groups as ꢀ-Secretase
(BACE1) Inhibitors
Simone Bertini, Elisa Ghilardi, Valentina Asso, Carlotta Granchi, Filippo Minutolo and
Marco Macchia*
Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6, I- 56126 Pisa, Italy
Received February 03, 2010: Revised May 07 2010: Accepted May 10, 2010
Abstract: ꢀ-Secretase (BACE1, ꢀ-site APP cleaving enzyme) is one of the most challenging therapeutic targets in the
field of Alzheimer’s disease (AD) research. This enzyme catalyses the formation of neuronal amyloid ꢀ (Aꢀ) plaques,
whose increased production is a key event in the initial pathogenesis of AD. As a consequence, many BACE1 inhibitors
have been developed by several research groups. In the present work, after an analysis of tetraline derivatives reported in a
Takeda patent, we designed and synthesized some analogues, making appropriate structural modifications, in order to try
to improve the bioavailability features and the activities of Takeda compounds. All the new derivatives were tested on
BACE1 with the TR-FRET (Time Resolved-Fluorescence Resonance Energy Transfer) technology and one of them
showed a promising inhibitory activity value.
Keywords: Alzheimer’s disease, BACE1, Bicyclic core, Biphenyl substituent, Tetraline derivative, TR-FRET technology.
INTRODUCTION
The research of new non-peptidomimetic BACE1 inhibi-
tors that possess appropriate properties for drug develop-
ment, such as low molecular weight, good potency and high
selectivity, has been started from a detailed study of a Ta-
keda patent, in which tetraline derivatives as BACE inhibi-
tors are described (Fig. 1) [6].
Alzheimer’s disease is an age-related disorder that is re-
sponsible for a slow and progressive neurodegeneration, es-
pecially in the hippocampal and neocortical regions. From a
neuropathological point of view, this disease is characterized
by the formation of extracellular senile plaques, mainly
composed of amyloid ꢀ peptides (Aꢀ) aggregates [1], and
intracellular neurofibrillary tangles, made of hyperphos-
phorylated tau proteins [2]. Both of these brain lesions may
have a possible key role in the progression of AD and have
been recognized as diagnostic hallmarks of this disease [3].
R'
A
B
Ar
X
Y
N
R''
Aꢀ peptides are originated from a larger transmembrane
protein called amyloid precursor protein (APP), that is sub-
jected to the action of specific enzymes, such as ꢀ-secretase
and ꢀ-secretase. In particular, ꢀ-secretase (BACE1), a prote-
olytic enzyme belonging to the family of aspartyl proteases,
catalyzes the first and rate-limiting step of the sequential
cleavage of APP. Besides, it is well established that BACE1
-/- mice show no adverse phenotype and they have no de-
tectable levels of Aꢀ peptides, indicating that BACE1 inhibi-
tion poses no serious consequences for the health of the mice
[4]. As a result, BACE1 is considered one of the preferred
therapeutic targets for the treatment of AD and there is a
great interest in developing BACE1 inhibitors [5]. However,
some of the more potent BACE1 inhibitors so far discovered
are peptidomimetic or possess relatively high molecular
weights, in spite of the fact that BACE1 inhibitors have to be
able to cross blood-brain barrier (BBB) in order to reach
their target in the CNS. Reduction of molecular size and
generally of the peptidic nature and overall polarity of the
candidate inhibitors are strategies usually applied for this
purpose.
N
R
Fig. (1). General structures of compounds reported in the Takeda
patent [6].
In particular, the aminoethyl-substituted tetraline deriva-
tive 1 (Fig. 2) has been used as starting point for further
structural modifications as it was described as a potent
BACE1 inhibitor. However, its development as a drug may
present some difficulties because of its high lipophilicity,
that can compromise oral bioavailability, having a CLogP
value of 8.3 [7].
On the basis of these data, we envisaged the possibility of
developing a new series of BACE1 inhibitor compounds
related to these tetralinic structures, but with reduced lipo-
philicity. The strategy was based on the replacement of the
central tetralinic core, reported in the patent, with less hy-
drophobic moieties or heterocycles, in order to enhance the
polarity of the resulting compounds. In the design of new
compounds, we have kept the extremities of Takeda struc-
*Address correspondence to this author at the Dipartimento di Scienze
Farmaceutiche, Università di Pisa, Via Bonanno 6, I- 56126 Pisa, Italy; Tel:
+39 050 2219553; Fax: +39 050 2219605; E-mail: mmacchia@farm.unipi.it
1570-1808/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

508 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7
Bertini et al.
mercially available 4-benzyloxy-2-hydroxy-benzaldehyde 6
was treated with epichlorohydrin to give aldehyde 7 [8]. A
Baeyer-Villiger oxidation converts product 7 to an unstable
formate intermediate, that spontaneously produced phenol 8
N
O
[9]. Phenol
8 so obtained was cyclized into 1,4-
benzodioxane 9 in the presence of KOH 2N [8]. A catalytic
reduction with Pd/C 10% yielded compound 10, bearing a
free phenol group. The introduction of a biphenyl substituent
(compound 11) was conducted by reaction with 4-
phenylbenzyl chloride in CH3CN. The subsequent transfor-
mation of the primary alcohol into iodide was achieved by
reaction with imidazole, iodine and triphenylphospine. This
modification allowed us to introduce subsequently a dimeth-
ylamino group in order to obtain the desired product 2 [6].
Finally, treatment of compound 2 with Et2O.HCl yielded its
hydrochloride salt form 2.HCl.
1
Fig. (2). Reference Takeda BACE1 inhibitor 1.
tures constant, that are, the biphenyl scaffold and the lateral
amino side chain present in the best compound (1, Fig. 2).
The selection of the central core replacements has been done
considering synthetic accessibility of the proposed struc-
tures, leading to compounds 2-5 (Fig. 3) as the best candi-
dates for a further exploration. In particular, the introduction
of oxygen atoms inside the central core increases the hydro-
philicity, as in the 1,4-benzodioxane scaffold of compound
2; in this case, the presence of a dimethylamine group in-
stead of piperidine one on the lateral chain, contributes to the
reduced hydrophobicity of the overall molecule. It should be
noted that there is a shortening of the lateral chain of com-
pound 2 when compared to patented compound 1. This
modification should further help in increasing polarity of the
new structures, and besides, in Takeda patent, molecules
with shorter lateral chain were also reported to be active [6].
Furthermore, we have observed that a shorter lateral chain,
with only a methylene, was synthetically more accessible
than the ethylene linker.
Compound 3 was prepared following the procedure de-
scribed for the patented compound 1, making some synthetic
modifications only where necessary (Scheme 2). Commer-
cially available 5-methoxy-1-indanone 13 was esterified with
BrCH2COOEt in the presence of LHMDS, to obtain the de-
sired ester 14 in good yield [10]. Product 14 was hydrolyzed
with NaOH in EtOH to give carboxylic acid 15. Then, amide
16 was obtained by reaction of 15 with N,N-dimethylamine.
In the following reduction-elimination reaction, compound
16 was treated with NaBH4 and p-TsOH, yielding indene
derivative 17. The double bond was hydrogenated in the
presence of Pd/C (compound 18) and the amide group was
reduced with LiAlH4 to obtain the desired amino derivative
19. Treatment with BBr3 caused the removal of the O-methyl
group and subsequent etherification with 4-phenylbenzyl
chloride in the presence of NaH produced the final aminoal-
kyl-substituted indane 3 in good yield.
Another modification concerned the shrinkage of the cen-
tral bicyclic core, to an indane cycle, leading to compound 3
(Fig. 3), which lacks one methylene unit when compared to
its patented counterparts and, consequently, is less lipophilic.
Finally, a non-cyclic scaffold as an isoprenoid moiety, as in
compounds 4 and 5 (Fig. 3), was considered suitable for our
goals.
The synthetic approach exploited for compounds 4 and 5
was based on procedures already developed in our research
laboratory. Both isoprenoid molecules were synthesized in
six steps, starting from commercially available 3-methyl-2-
buten-1-ol 20, as shown in Scheme 3. The protection of the
hydroxyl group of alcohol 20 was achieved by reaction with
3,4-dihydropyran in the presence of pyridinium p-
toluensulfonate [11-13], producing a tetrahydropyranyl ether
in high yield. Compound 21 was oxidized with t-butylhydro-
RESULTS AND DISCUSSION
The synthesis of compound 3 has been carried out ac-
cording to literature and Takeda patent (Scheme 1). Com-
O
N
N
O
O
O
3
2
R
O
O
4 R= -N(CH3)2
N
5 R=
Fig. (3). New structures (2-5) inspired to Takeda compounds.

Biphenyl-Derivatives Possessing Tertiary Amino Groups
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7 509
CHO
CHO
OH
OH
OH
OH
O
Cl
O
Cl
a
c
b
OBn
OBn
OBn
6
7
8
O
O
OH
O
O
e
d
OH
OH
O
BnO
O
HO
O
9
10
11
O
O
I
N
g
f
h
O
O
O
O
12
2
O
O
. HCl
N
O
2.HCl
Scheme 1.a Synthesis of benzodioxanic compound 2.
aKey: (a) epichlorohydrin, piperidine, 100 °C; (b) H2O2 35%, KHSO4, MeOH; (c) KOH 2N; (d) H2, Pd/C 10%, EtOH-EtOAc; (e) 4-
phenylbenzyl-chloride, K2CO3, KI, CH3CN, 60 °C to 80 °C; (f) I2, Ph3P, imidazole/THF; (g) dimethylamine, K2CO3, I2, THF; (h) Et2O.HCl.
a
b
OEt
MeO
MeO
MeO
MeO
O
O
O
O
13
15
14
c
d
N
OH
MeO
O
O
O
O
16
e
f
N
N
MeO
O
17
18
g
N
O
N
MeO
3
19
Scheme 2.a Synthesis of indane compound 3.
aKey: (a) LHMDS, HMPA, BrCH2COOEt, THF, -60 °C; (b) NaOH 2N, EtOH; (c) [3-(dimethylamino)propyl]-3-ethyl-carbodiimide hydro-
chloride, hydroxybenzotriazole, N,N-dimethylamine, dry DMF, 0 °C; (d) 1) NaBH4, MeOH, 0 °C; 2) dry PhCH3, p-TsOH, 120 °C; (e) H2,
Pd/C, EtOH; (f) LiAlH4, dry THF, 70 °C; (g) 1) BBr3, CH2Cl2, -78 °C to 0 °C; 2) NaH, 4-phenylbenzyl chloride, dry DMF, 0 °C to 50 °C,
then 0 °C to r. t..

510 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7
Bertini et al.
O
O
O
O
a
OH
b
HO
20
21
22
O
O
OH
d
c
O
O
23
24
e
O
O
R
4 R= -N(CH3)2
N
5 R=
Scheme 3.a Synthesis of isoprenoid compounds 4 and 5.
aKey: (a) 3,4-DHP, PPTS, CH2Cl2; (b) t-BuOOH, H2SeO3, salicylic acid; CH2Cl2; (c) NaH, 4-phenylbenzyl chloride, dry DMF, 0 °C to 50 °C,
then 0 °C to r. t.; (d) PPTS, EtOH, 55 °C; (e) NaH, chloroethyl-N,N-dimethylamine or N-chloroethylpiperidine, dry DMF, 0 °C to 50 °C, then
0 °C to r. t..
peroxide and catalytic H2SeO3 in the presence of salicylic
acid to give compound 22, which was then submitted to de-
protonation by NaH and, subsequently, to a reaction with 4-
phenylbenzyl chloride in DMF, to obtain compound 23. Af-
ter deprotection of 23 with pyridinium p-toluensulfonate in
EtOH, the resulting alcohol 24 was treated with chloroethyl-
N,N-dimethylamine or N-chloroethylpiperidine, affording the
two desired final products 4 and 5, respectively.
EXPERIMENTAL
Biological Methods
The time resolved fluorescence quenching assay devel-
oped for BACE1 used as substrate a peptide made of 10
amino acids. The sequence contains the KM - NL APP
(Amyloid Precursor Protein) Swedish and has a fluorescent
Eu3+-chelate coupled to the N-terminus as donor (Ex max
330 nm- Em max 615 nm) and a quenching organic fluoro-
phore at the C-terminus as acceptor. The proteolytic cleavage
of the substrate by BACE1 interrupts the energy transfer
between donor and acceptor, leading to a linear increase of
fluorescence emission at 615 nm of the donor, that is directly
related to the enzyme activity and can be followed by kinetic
TRF measurements. Problems with compound interference,
frequently occurring with fluorescent FRET substrates, are
not significant in this assay. A high signal-to-noise ratio of
13 and a Z' factor of 0.76 make this assay well suitable for
HTS. The assay was validated by reproducing the IC50 val-
ues of known and patented inhibitors and by determining the
Ki value and binding mechanism of a known Stat-Val pep-
tide inhibitor. All measured IC50 values correlated with pub-
lished data. Ki for the Stat-Val peptide inhibitor was deter-
mined with various substrate concentrations (0.1-7 mM) and
four different peptide concentrations (6, 15, 30 and 60 nM).
Data were plotted with the double reciprocal Lineweaver-
Burk plot and Ki was graphically determined through a Cor-
nish-Bowden plot. Reversibility and time dependency were
established to better characterize the Stat-Val peptide inhibi-
tion. The reversibility was assessed through 10-times dilu-
tion of a mixture of 100 ng enzyme and compounds at 10-
times the IC50 concentration after a pre-incubation of 60 min
at 25 °C. The obtained activity was normalized to the control
and compared to the activity of the concentrated mixture.
Both Takeda compounds and new derivatives were
evaluated at a concentration of 20 μM on BACE1 with a TR-
FRET (Time Resolved-Fluorescence Resonance Energy
Transfer) assay [14]. IC50 values were calculated only for
compounds that showed an enzymatic inhibition greater than
50% at 10 ꢀM and are reported in Table 1. Reference com-
pound 1 was synthesized by following the procedure re-
ported in the Takeda patent [6]. As regards newly synthe-
sized compounds, only the indane analogue 3 showed a no-
table inhibitory activity in the μM range, possessing an IC50
of 12 μM, which is comparable to that of reference com-
pound 1 [15], whereas the other, more polar, derivatives
showed an inhibition level lower than 50% at a concentration
of 20 μM. These results show that the contraction of the cen-
tral saturated ring of compound 1 from six to five members
and the replacement of the piperidine substituent with a di-
methylamino group as in the derivative 3, led to maintenance
of a good level of inhibitory activity on BACE1. On the con-
trary, the replacement of the bicyclic core with an acyclic
isoprenoid moiety, as in compounds 4 and 5, was detrimental
for the activity. A similar decrement was found for com-
pound 2, suggesting that the introduction of oxygen atoms
into the central core and the presence of a methylenic side
chain are not suitable strategies to obtain good BACE1 in-
hibitors.

Biphenyl-Derivatives Possessing Tertiary Amino Groups
Table 1. BACE1-Inhibitory Activitiesa of Takeda Compound 1 and Derivatives 2-5
Compound Structure
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7 511
IC50 (μM)
N
1
11
O
O
O
N
O
2
> 20
N
O
3
12
O
O
N
CH3
4
5
> 20
> 20
O
O
N
CH3
aDetermined by a TR-FRET assay (Time-Resolved Fluorescence Resonance Energy Transfer).
Chemistry
dimethylformamide (DMF) was purchased from Aldrich in
SureSeal containers. Where necessary, reaction requiring
anhydrous condition were performed in a flame or oven-
dried apparatus under nitrogen atmosphere.
NMR spectra were obtained with a Varian Gemini 200
MHz spectrometer. Chemical shifts (ꢀ) are reported in parts
per million downfield from tetramethylsilane and referenced
from solvent references. Chromatographic separations were
performed on silica gel columns by flash (Kieselgel 40,
0.040–0.063 mm; Merck) or gravity column (Kieselgel 60,
0.063–0.200 mm; Merck) chromatography, or on Isolute pre-
packed silica columns and Isolute ionic exchanged pre-
packed silica columns as the stationary phase. Reactions
were followed by thin-layer chromatography (TLC) on
Merck aluminum silica gel (60 F254) sheets that were visual-
ized under a UV lamp and/or with phosphomolybdic acid
solution. Evaporation was performed in vacuo (rotating
evaporator). Sodium sulfate was always used as the drying
agent. The organic solvents were dried by distillation from
calcium hydride, magnesium methoxide or sodium metal and
stored under nitrogen and/or over sodium wire. N,N-
4-(Benzyloxy)-2-(3-chloro-2-hydroxypropoxy)benzaldehyde
(7)
Commercially available 4-benzyloxy-2-hydroxy-benzal-
dehyde 6 (2.00 g, 8.70 mmol) was treated with epichlorohy-
drin (2.1 mL, 26.1 mmol) and 7-8 drops of piperidine were
added. The reaction mixture was stirred for 4 hours at 100 °C
in a closed vial. After complete reaction, the reaction mixture
was diluted with CHCl3 and concentrated HCl was added to
open the unreacted epoxide. The organic phase was sepa-
rated and washed with water, then evaporated in vacuo. The
desired product 7 (1.45 g, 52% yield) was purified by flash
chromatography (90 to 80 Hexane/EtOAc); 1H-NMR
(CDCl3) ꢀ (ppm): 3.77 (dd, 2H, J = 5.2, 1.3 Hz), 4.40-4.00
(m, 3H), 5.13 (s, 2H, benzylic -CH2-), 6.56 (d, 1H, J = 2.1

512 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7
Bertini et al.
Hz), 6.68 (dd, 1H, J = 8.6, 2.0 Hz), 7.26-7.50 (m, 5H), 7.77
(d, 1H, J = 8.7 Hz), 10.19 (s, 1H, -CHO).
organic phase was dried over Na2SO4 and evaporated in
vacuo. The crude residue almost entirely constituted by
compound 12 was used for the next reaction without further
4-(Benzyloxy)-2-(3-chloro-2-hydroxypropoxy)phenol (8)
1
purification. H-NMR (CDCl3) ꢀ (ppm): 3.31-3.34 (m, 2H),
Aldehyde 7 (1.45 g, 4.54 mmol) was dissolved in MeOH,
then H2O2 sol. 35% (0.5 ml, 5.90 mmol) and KHSO4 (96 mg,
cat) were added. After 1 night at room temperature the mix-
ture was diluted with H2O and extracted with CHCl3. The
organic phase was dried over Na2SO4 and evaporated in
vacuo. The crude residue almost entirely constituted by
compound 8 was used for the next reaction without further
4.09-4.39 (m, 3H), 5.02 (s, 2H, benzylic -CH2-), 6.50-6.56
(m, 2H), 6.79-6.84 (m, 1H), 7.34-7.73 (m, 9H).
1-(6-(Biphenyl-4-ylmethoxy)-2,3-dihydrobenzo[b][1,4]dioxin-
2-yl)-N,N-dimethylmethanamine (2)
To a solution of compound 12 (261 mg, 0.569 mmol) and
dimethylamine (3 mL, 4.55 mmol) in THF (1 mL), K2CO3
(197 mg, 1.42 mmol) and a solution of I2 (250 mg, 0.985
mmol) in THF (2.5 mL) were added. The mixture was stirred
at room temperature for 3 days. The mixture was washed
with H2O and extracted with CH2Cl2. Purification by flash
chromatography (80 to 50% Hexane/EtOAc) afforded the
final product 2 as a white solid, which has been treated with
HCl to form the hydrochloride salt 2.HCl as a white solid
1
purification. H-NMR (CDCl3) ꢀ (ppm): 3.71-3.76 (m, 2H),
4.03-4.31 (m, 3H), 4.99 (s, 2H, benzylic -CH2-), 6.53 (dd,
1H, J = 8.6, 2.7 Hz), 6.60 (d, 1H, J = 2.7 Hz), 6.85 (d, 1H, J
= 8.6 Hz), 7.32-7.44 (m, 5H).
(6-(Benzyloxy)-2,3-dihydrobenzo[b][1,4]dioxin-2-yl)meth-
anol (9)
1
Phenol 8 (1.30 g, 4.23 mmol) was treated with an excess
of KOH 2N solution. The mixture was allowed to react 1
hour at room temperature and an oil appeared in the mixture.
The product was extracted with CH2Cl2 and purified by flash
chromatography (8:2 Hexane/EtOAc). The purification af-
forded the desired product 9 as an yellow oil (933 mg, 81%
(39.0 mg, 17% yield); H-NMR (CDCl3) ꢀ (ppm): 2.33 (s,
6H, -N(CH3)2), 2.40-2.70 (m, 2H), 3.86-4.04 (m, 1H), 4.14-
4.36 (m, 2H), 5.02 (s, 2H, benzylic -CH2-), 6.46-6.60 (m,
2H), 6.83 (d, 1H, J = 8.4 Hz), 7.80-7.26 (m, 9H).
Ethyl 2-(6-methoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)ace-
tate (14)
1
yield); H-NMR (CDCl3) ꢀ (ppm): 3.70-3.88 (m, 2H), 3.88-
4.40 (m, 3H), 4.98 (s, 2H, benzylic -CH2-), 6.40-6.60 (m,
2H), 6.80 (d, 1H, J = 8.4 Hz), 7.25-7.50 (m, 5H).
A solution of the commercial available 6-methoxy-2,3-
dihydro-1H-inden-1-one 13 (1.0 eq) in a minimal amount of
THF was added dropwise to a stirred solution of LHMDS
(1.2 eq) at -60 °C. After 30 minutes, HMPA (1.2 eq) and
BrCH2COOEt (2.0 eq) were added and the reaction was left
under stirring for 1 h at -60 °C. After TLC analysis, the mix-
ture was stopped: NH4Cl solution was added and the organic
layer extracted with AcOEt, dried over Na2SO4 and concen-
trated. The desired product was purified with silica and ionic
exchange pre-packed cartridges (yield 70%); 1H-NMR
(CDCl3) ꢀ (ppm): 1.22 (t, 3H, J = 7.1 Hz, -C(=O)OCH2CH3),
2.66 (dd, 1H, J = 16.8, 8.8 Hz), 2.87 (dd, 1H, J = 15.2, 3.9
Hz), 2.71-3.09 (m, 2H), 3.38 (dd, 1H, J = 16.6, 7.6 Hz), 3.83
(s, 3H, -OCH3), 4.13 (q, 2H, J = 7.1 Hz, -C(=O)OCH2CH3),
716-7.37 (m, 3H).
2-(Hydroxymethyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-ol
(10)
1,4-benzodioxane 9 (887 mg, 3.26 mmol) was dissolved
in EtOH (15 mL) and EtOAc (6-7 drops). Pd/C 10% was
added and the mixture was allowed to react under H2 for 5
hours. The mixture was filtered on Celite and the solution
was evaporated in vacuo, affording pure compound 10 (563
1
mg, 94% yield); H-NMR (CDCl3) ꢀ (ppm): 3.60-4.35 (m,
5H), 6.28-6.44 (m, 2H), 6.75 (d, 1H, J = 8.6 Hz).
(6-(Biphenyl-4-ylmethoxy)-2,3-dihydrobenzo[b][1,4]dioxin-
2-yl)methanol (11)
Phenol 10 (563 mg, 3.09 mmol) was dissolved in CH3CN
(5 mL), then K2CO3 (768 mg, 5.55 mmol) was added and the
mixture was allowed to warm to 60 °C. A solution of 4-
phenylbenzyl-chloride (689 mg, 2.81 mmol) in CH3CN (1
mL) was added. Subsequently, catalytic KI (a spatula tip)
was added. After the mixture was stirred at 80 °C for 1 night,
CH3CN was evaporated. The crude residue was acidified
with HCl 4N solution and extracted with CHCl3. The desired
product 11 (968 mg, 90% yield) was obtained after purifica-
tion by flash chromatography (90 to 80% Hexane/EtOAc);
1H-NMR (CDCl3) ꢀ (ppm): 3.70-3.98 (m, 2H), 3.98-4.40 (m,
3H), 5.02 (s, 2H, benzylic -CH2-), 6.43-6.62 (m, 2H), 6.82
(d, 1H, J = 8.6 Hz), 7.25-7.70 (m, 9H).
2-(6-Methoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)acetic acid
(15)
A solution of NaOH 2N (10 eq) was added to a solution
of compound 14 (1.0 eq) in a minimal amount of EtOH and
the resulting mixture was allowed to react for 5 h at room
temperature. The reaction was stopped by adding HCl 10%
solution and then the organic layer was extracted with Et2O,
dried over Na2SO4 and concentrated. The crude residue was
purified with silica and ionic exchange pre-packed cartridges
1
to obtain the desired acid 15 (yield 97%); H-NMR (CDCl3)
ꢀ (ppm): 2.64 (dd, 1H, J = 17.8, 9.4 Hz), 2.81 (dd, 1H, J =
16.7, 3.9 Hz), 2.97-3.10 (m, 2H), 3.42 (dd, 1H, J = 16.6, 7.5
Hz), 3.84 (s, 3H, -OCH3), 7.15-7.38 (m, 3H).
6-(Biphenyl-4-ylmethoxy)-2-(iodomethyl)-2,3-dihydrobenzo
[b][1,4]dioxine (12)
2-(6-Methoxy-1-oxo-2,3-dihydro-1H-inden-2-yl)-N,N-dime-
thylacetamide (16)
To a solution of Ph3P (88.1 mg, 0.518 mmol) in THF (1.5
mL), imidazole (21.8 mg, 0.320 mmol) and I2 (85.2 mg,
0.336 mmol) were added. Then, a solution of alcohol 11
(200 mg, 0.574 mmol) in THF (1.5 mL) was added. After 5
hours at room temperature, the mixture was treated with
Na2S2O3 saturated solution and extracted with CH2Cl2. The
To a cooled (0 °C) and stirred solution of carboxylic acid
15 (1.0 eq) in dry DMF, 1-[3-(dimethylamino)propyl]-3-
ethyl-carbodiimide hydrochloride (1.1 eq), hydroxybenzotri-
azole (1.0 eq) and N,N-dimethylamine (1.1 eq) were added.
The solution was stirred for 2 h. After evaporation of the

Biphenyl-Derivatives Possessing Tertiary Amino Groups
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7 513
solvent, the residue was poured into water and extracted with
AcOEt. The extracts were dried over Na2SO4 and concen-
trated. The residue was purified by column chromatography
with AcOEt as eluent (yield 40%); 1H-NMR (CDCl3) ꢀ
(ppm): 2.63 (dd, 1H, J = 17.1, 9.2 Hz), 2.81 (dd, 1H, J =
16.7, 3.9 Hz), 2.94 (s, 3H), 3.02 (s, 3H), 2.97-3.09 (m, 2H),
3.41 (dd, 1H, J = 16.7, 7.4 Hz), 3.83 (s, 3H, -OCH3), 7.14-
7.21 (m, 2H), 7.33 (d, 1H, J = 8.2 Hz).
4-phenylbenzyl chloride (1.1 eq) was added and the resulting
mixture was stirred at room temperature for 2 h. After
evaporation of the solvent, the residue was diluted with wa-
ter and extracted with AcOEt. The organic layer was dried
over Na2SO4 and concentrated, affording a crude residue that
was purified by silica and ionic exchange pre-packed car-
1
tridges. Yield 41%, H-NMR (CDCl3) ꢀ (ppm): 1.46-1.84
(m, 2H), 2.36 (s, 6H, -N(CH3)2), 2.42-2.66 (m, 5H), 2.90-
3.10 (m, 2H), 5.07 (s, 2H, benzylic -CH2-), 6.74-6.88 (m,
2H), 7.08 (d, 1H, J = 8.4 Hz), 7.34-7.63 (m, 9H).
2-(5-Methoxy-1H-inden-2-yl)-N,N-dimethylacetamide (17)
To a cooled (0 °C) and stirred solution of amide 16 (1 eq)
in a minimal amount of MeOH, NaBH4 (2.5 eq) was added
dropwise and the resulting solution was stirred for 1 h. After
evaporation of the solvent, the residue was neutralized with
HCl 1N, extracted with CH2Cl2, dried over Na2SO4 and con-
centrated. The residue was diluted in dry toluene and p-
TsOH (cat) was added. The reaction mixture was heated to
reflux at 120 °C and stirred for 1 h . After TLC analysis, the
reaction was stopped by adding NaHCO3 sat. solution and
brine. The organic layer was dried over Na2SO4 and concen-
trated. The crude residue was used for the next step without
2-(3-methylbut-2-enyloxy)tetrahydro-2H-pyran (21)
To a solution of the commercial available 3-methyl-2-
buten-1-ol (1.0 eq) in a minimal amount of CH2Cl2, 3,4-
dihydropyrane (1.5 eq) and PPTs (0.10 eq) were added and
the mixture was stirred for 4 h at room temperature. After
TLC analysis, the reaction mixture was partially evaporated,
diluted with Et2O and washed with NaHCO3 sat. solution.
The organic layer was dried over Na2SO4 and concentrated
affording the desired product, that was purified with silica
1
anionic exchange pre-packed cartridges (yield 87%); H-
1
further purification (yield 65%); H-NMR (CDCl3) ꢀ (ppm):
NMR (CDCl3) ꢀ (ppm): 1.40-1.85 (m, 6H), 1.64 (s, 3H), 1.70
(s, 3H), 3.40-3.51 (m, 1H), 3.80-3.86 (m, 1H), 3.92 (dd, 1H,
J = 12.1, 6.0 Hz), 4.17 (dd, 1H, J = 12.1, 6.0 Hz), 4.58 (t,
1H, J = 4.0 Hz), 5.28 (t, 1H, J = 6.0 Hz).
2.98 (s, 3H), 3.05 (s, 3H), 3.37 (s, 2H), 3.57 (s, 2H), 3.81 (s,
3H,-OCH3), 6.69 (dd, 1H, J = 8.1, 2.4 Hz), 6.85-6.99 (m,
1H); 7.14-7.28 (m, 2H).
2-(5-Methoxy-2,3-dihydro-1H-inden-2-yl)-N,N-dimethylac-
etamide (18)
(E)-2-methyl-4-(tetrahydro-2H-pyran-2-yloxy)but-2-en-1-ol
(22)
To a stirred solution of amide 17 (1.0 eq) in a minimal
amount of EtOH, Pd/C (0.1 eq) was added, under vacuum
and N2. The mixture was allowed to react under H2 for 13 h.
After TLC analysis, the desired product was collected by
filtration (yield 76%); H-NMR (CDCl3) ꢀ (ppm): 2.46-2.69
(m, 4H), 2.96 (s, 3H), 2.98 (s, 3H), 2.83-3.21 (m, 3H), 3.77
A
solution of compound 21 (1.0 eq) and t-
butylhydroperoxide (3.2 eq) in CH2Cl2 was treated with
H2SeO3 (0.11 eq) and salicylic acid (0.11 eq) and the reac-
tion mixture was stirred for 14 h at room temperature. The
solvent was removed in vacuo and t-butylhydroperoxide was
removed by washing repeatedly with toluene. The residue
was dissolved in Et2O, washed with NaHCO3 sat. solution to
remove H2SeO3, dried over Na2SO4 and concentrated. The
residue was purified by silica and ionic exchange pre-packed
cartridges (yield 40%); 1H NMR (CDCl3) ꢀ (ppm): 1.54-1.85
(m, 9H), 3.47-3.57 (m, 1H), 3.83-3.94 (m, 1H), 4.01-4.10
(m, 4H), 4.29 (dd, 1H, J = 12.1, 6.4 Hz), 5.63 (t, 1H, J = 6.1
Hz).
1
(s, 3H, -OCH3), 6.66-6.78 (m, 2H), 7.08 (d, 1H, J = 8.0 Hz).
2-(5-Methoxy-2,3-dihydro-1H-inden-2-yl)-N,N-dimethyle-
thanamine (19)
To a stirred solution of amide 18 (1.0 eq) in dry THF,
LiAlH4 (1.5 eq) was added and the mixture was heated at 70
°C for 1 h. After TLC analysis, water and NaOH 1N were
added, then the organic layer was dried over Na2SO4 and
concentrated. The residue was purified by silica and ionic
exchange pre-packed cartridges (yield 63%); 1H-NMR
(CDCl3) ꢀ (ppm): 1.65-1.76 (m, 2H), 2.29 (s, 6H, -N(CH3)2),
2.37-2.63 (m, 4H), 2.68-2.84 (m, 1H), 2.92-3.07 (m, 2H),
3.77 (s, 3H, -OCH3), 6.64-6.73 (m, 2H), 7.06 (d, 1H, J = 8.0
Hz).
(E)-2-(4-(biphenyl-4-ylmethoxy)-3-methylbut-2-enyloxy)tet-
rahydro-2H-pyran (23)
Alcohol 22 (1.0 eq) was dissolved in dry DMF and
cooled to 0 °C prior to NaH (60% oil dispersion, 2.5 eq) ad-
dition, then the mixture was stirred at 50 °C for 1 h. Then,
the mixture was cooled at 0 °C and 4-phenylbenzyl chloride
(1.1 eq) was added. The resulting mixture was stirred for 24
h at room temperature, then poured into water and extracted
with AcOEt. The combined extracts were dried over Na2SO4
2-(5-(Biphenyl-4-ylmethoxy)-2,3-dihydro-1H-inden-2-yl)-
N,N-dimethylethanamine (3)
1
A solution of BBr3 1M in CH2Cl2 (3 eq) was added
dropwise at -78 °C to a stirred solution of amine 19 (1.0 eq)
in CH2Cl2. After 30 minutes, the mixture was allowed to
warm to room temperature and left stirring for 1 h. After
TLC analysis, NaHCO3 sat. solution was added and the or-
ganic layer was extracted with CH2Cl2, dried over Na2SO4
and concentrated. The residue was purified by SCX pre-
packed cartridges. To a stirred solution of alcohol resulting
from the previous step (1 eq) in dry DMF, at 0 °C, NaH
(60% oil dispersion, 2.5 eq) was added and the mixture was
stirred at 50 °C for 1 h. The mixture was then cooled to 0 °C,
and concentrated (yield 30%); H-NMR (CDCl3) ꢀ (ppm):
1.70-1.82 (m, 9H), 3.48-3.54 (m, 1H), 3.80-3.90 (m, 1H),
3.98 (s, 2H), 4.10-4.20 (m, 2H), 4.52 (s, 2H, benzylic -CH2-),
4.60 (dd, 1H, J = 14.8, 2.9 Hz), 5.66-5.80 (m, 1H), 7.33-7.48
(m, 5H), 7.55-7.62 (m, 4H).
(E)-4-(biphenyl-4-ylmethoxy)-3-methylbut-2-en-1-ol (24)
To a solution of compound 23 (1.0 eq) in a minimal
amount of EtOH, PPTs (0.10 eq) was added and the reaction
mixture stirred at 55 °C for 6 h. The solvent was partially
evaporated in vacuo and the resulting solution was diluted

514 Letters in Drug Design & Discovery, 2010, Vol. 7, No. 7
Bertini et al.
ACKNOWLEDGEMENTS
with Et2O and washed with half-saturated brine to remove
the catalyst. The organic layer was dried over Na2SO4 and
concentrated affording a crude residue that was purified with
silica and ionic exchange pre-packed cartridges (yield 62%);
1H-NMR (CDCl3) ꢀ (ppm): 1.72 (d, 3H, J = 7.3 Hz), 3.87 (d,
2H, J = 18.0 Hz), 4.18 (dd, 2H, J = 24.1, 6.5 Hz), 4.55 (s,
2H, benzylic -CH2-), 5.60-5.80 (m, 1H), 7.31-7.48 (m, 5H),
7.56-7.61 (m, 4H).
The authors are grateful to Siena Biotech SpA - Italy, for
scientific and financial support. MIUR - “grandi programmi
strategici” is gratefully acknowledged for a Ph.D. fellowship
(to C.G.).
ABBREVIATIONS
BACE1
AD
=
=
=
ꢀ-secretase or ꢀ-site APP cleaving enzyme
Alzheimer’s disease
(E)-2-(4-(Biphenyl-4-ylmethoxy)-3-methylbut-2-enyloxy)-
N,N-dimethylethanamine (4), (E)-1-(2-(4-(biphenyl-4-
ylmethoxy)-3-methylbut-2-enyloxy)ethyl)piperidine (5)
Aꢀ
amyloid ꢀ
Alcohol 24 (1.0 eq) was dissolved in dry DMF and
cooled to 0 °C prior to NaH (60% oil dispersion, 2.5 eq) ad-
dition. The mixture was stirred at 50 °C for 1 h. Then, after
cooling to 0 °C, chloroethylamine (1.1 eq) was added and the
resulting mixture was stirred for 18-24 h at room tempera-
ture. Then, the reaction mixture was poured into water and
extracted with AcOEt. The combined extracts were dried
over Na2SO4 and concentrated, then purified with silica and
ionic exchange pre-packed cartridges (95:5 CH2Cl2/MeOH);
TR-FRET = Time Resolved-Fluorescence Resonance
Energy Transfer
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There are many evidences that BACE1 inhibitors prevent
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evaluation of a series of BACE1 inhibitors, using tetraline
derivatives patented by Takeda as a starting point. One of
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tive 1, showed a good inhibitory activity, with an IC50 in the
micromolar range, but it possesses an high degree of lipo-
philicity, resulting in diminished drug-like properties. De-
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the common tetralinic core with more polar scaffolds, keep-
ing the biphenyl group and the amino side-chain constant.
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showed a good IC50 value, comparable to that of reference
compound 1, demonstrating that the contraction of the satu-
rated ring of the tetraline moiety from six to five atoms is a
good structural modification for maintaining a good BACE1
inhibitory potency and, at the same time, for enhancing the
polarity of the structure. On the contrary, other more polar
analogues (2, 4, and 5) did not reach a 50% inhibition at 20
μM. These findings may constitute a basis for the develop-
ment of more potent BACE1 inhibitors possessing better
physico-chemical properties and, consequently, a higher de-
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