New flurbiprofen derivatives: Synthesis, membrane affinity and evaluation of in vitro effect on β-amyloid levels

Article abstract of DOI:10.3390/molecules180910747

Alzheimer′s disease (AD) is characterized by irreversible and progressive loss of memory and cognition and profound neuronal loss. Current therapeutic strategies for the treatment of AD have been directed to a variety of targets with the aim of reversing or preventing the disease but, unfortunately, the available treatments often produce no significant clinical benefits. During the last decades compounds that inhibit or modulate γ-secretase, reducing β amyloid (Aβ) levels, have been considered as potential therapeutics for AD. Among these the (R)-enantiomer of flurbiprofen (FLU) seems to be very promising, but it shows low brain penetration. In this study, in order to improve the properties of FLU against Alzheimer′s pathogenesis we synthesized some novel FLU lipophilic analogues. Lipophilicity of the new molecules has been characterized in terms of clogP, log KC18/W and log K IAM/W values. Permeability has been determined in both gastrointestinal PAMPA (PAMPA-GI) at different pH values and in brain blood barrier PAMPA (PAMPA-BBB) models. They were also tested for their ability to inhibit in vitro γ-secretase activity using rat CTXTNA2 astrocytes. Interestingly, the investigated molecules demonstrated to reduce Aβ 42 levels without affecting the amyloid precursor protein APP level in a clear concentrations-dependent manner.

Full text of DOI:10.3390/molecules180910747

Molecules 2013, 18, 10747-10767; doi:10.3390/molecules180910747
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
New Flurbiprofen Derivatives: Synthesis, Membrane Affinity
and Evaluation of in Vitro Effect on β-Amyloid Levels
Piera Sozio 1,*, Lisa Marinelli 1, Ivana Cacciatore 1, Antonella Fontana 1, Hasan Türkez 2,
Gianfabio Giorgioni 3, Dario Ambrosini 4, Francesco Barbato 5, Lucia Grumetto 5,
Stephanie Pacella 6, Amelia Cataldi 1 and Antonio Di Stefano 1
1
Department of Pharmacy, “G. D’Annunzio” University, Via dei Vestini 31, Chieti 66100, Italy
2
Department of Molecular Biology and Genetics, Erzurum Technical University,
Erzurum 25240, Turkey
3
Medicinal Chemistry Unit, School of Pharmacy, University of Camerino, via S. Agostino 1,
Camerino 62032, Italy
4
Center for Drug Discovery 116 MU, Northeastern University, 360 Huntington Avenue,
Boston 02115-5000, MA, USA
5
Department of Pharmacy, Università di Napoli Federico II, Via D. Montesano 49, Naples 80131, Italy
6
Department of Medicine and Ageing Sciences, “G. D’Annunzio” University, Via dei Vestini 31,
Chieti 66100, Italy
* Author to whom correspondence should be addressed; E-Mail: p.sozio@unich.it;
Tel.: +39-871-3554-709; Fax: +39-871-3554-706.
Received: 28 June 2013; in revised form: 14 August 2013 / Accepted: 27 August 2013 /
Published: 3 September 2013
Abstract: Alzheimer’s disease (AD) is characterized by irreversible and progressive loss
of memory and cognition and profound neuronal loss. Current therapeutic strategies for the
treatment of AD have been directed to a variety of targets with the aim of reversing or
preventing the disease but, unfortunately, the available treatments often produce no
significant clinical benefits. During the last decades compounds that inhibit or modulate
γ-secretase, reducing β amyloid (Aβ) levels, have been considered as potential therapeutics
for AD. Among these the (R)-enantiomer of flurbiprofen (FLU) seems to be very
promising, but it shows low brain penetration. In this study, in order to improve the
properties of FLU against Alzheimer’s pathogenesis we synthesized some novel FLU
lipophilic analogues. Lipophilicity of the new molecules has been characterized in terms of
clogP, log KC18/W and log K
values. Permeability has been determined in both
IAM/W
gastrointestinal PAMPA (PAMPA-GI) at different pH values and in brain blood barrier

Molecules 2013, 18
10748
PAMPA (PAMPA-BBB) models. They were also tested for their ability to inhibit in vitro
γ-secretase activity using rat CTXTNA2 astrocytes. Interestingly, the investigated
molecules demonstrated to reduce Aβ 42 levels without affecting the amyloid precursor
protein APP level in a clear concentrations-dependent manner.
Keywords: Alzheimer’s disease; beta amyloid peptide; flurbiprofen; γ-secretase
Abbreviations: Aβ: β amyloid; AD: Alzheimer’s disease; APP: amyloid precursor protein;
BBB: blood brain barrier; EDCI: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;
FASSIF: fasted state simulated intestinal fluid; FLU: flurbiprofen; GI: gastrointestinal; GSI:
γ-secretase inhibitors; GSM: γ-secretase modulators; IAM: immobilized artificial membrane;
KHMDS: potassium hexamethyldisilazide; NSAIDs: non-steroidal anti-inflammatory drugs; PAMPA:
parallel artificial membrane permeability; SP: senile plaque.
1. Introduction
Alzheimer’s disease (AD) is one of the most common types of dementia, characterized by
irreversible and progressive loss of memory and cognition and profound neuronal loss [1]. Current
therapeutic strategies for the treatment of AD have been directed to a variety of targets with the aim of
reversing or preventing the disease but, unfortunately, the available treatments produce limited clinical
benefits [2]. During the last decades many investigations have been directed to the modulation of
amyloid precursor protein (APP) transcription, including α-, β-, and γ-secretase. In particular,
compounds that inhibit or modulate γ-secretase can be considered as a potential therapeutics for AD
because their use appears to be a rational strategy to prevent senile plaque (SP) formations [3].
The first γ-secretase inhibitor (GSI) with in vivo efficacy was DAPT (Elan and Eli Lilly), followed
by LY-450139 (also known as semagecestat) and LY-411575 (Eli Lilly) (Figure 1). When
administered in mice they inhibit APP cleavage, thus dose dependently reducing β amyloid (Aβ) levels
and avoiding plaque formation [4].The major problem associated with these inhibitors is their non
selective targeting towards APP. As a matter of fact they also inhibit the cleavage of the Notch receptor,
causing embryological defects, interferences in B and T lymphocytes proliferation, and abnormalities in
the gastrointestinal (GI) tract [5,6].
These toxic effects can be avoided by substituing GSI with γ-secretase modulators (GSMs). Indeed,
γ-secretase is a multi-subunit protease complex constituted by at least four components (presenilin,
nicastrin, aph-1 and pen-2). Two aspartates moieties lying at the interface of presenilin constitute the
catalytic site [7].

Molecules 2013, 18
10749
Figure 1. Structure of GSIs, GSMs and compounds 1–9.
F
O
H
N
O
H
N
N
H
F
O
F
N
H
OH
O
O
O
O
N
F
DAPT
LY-411575
N
OH
O
O
OH
H
N
O
N
H
F
R-Flurbiprofen
O
LY-450139
O
O
H
N
H
N
O
O
N
H
N
H
O
O
O
O
F
F
1
2
O
H
N
O
N
H
O
O
F
3
O
O
H
N
H
N
N
N
O
O
F
F
4
5
O
O
H
N
H
N
N
N
O
O
F
F
6
7
O
O
H
N
H
N
N
N
O
O
F
F
8
9

Molecules 2013, 18
10750
γ-Secretase can cut APP at different positions along its chain (referred to as γ, ε, and ζ), but Aβ is
released only after a γ-cut generating fragments whose length ranges from 37 to 43 amino acid residues.
The prevalence of one Aβ type rather than another influences its pathogenicity, because only the Aβ 42
is prone to aggregate and thus to form plaques. The GSIs, by targeting the catalytic pocket, inhibit the
production of all Aβ peptides without differentiating the processing of APP or Notch. On the contrary,
the GSMs can act either by blocking the Aβ production (Notch-sparing GSIs) or by modulating the
ratio between long and short Aβ peptides (GSMs, properly named), without changing the total amount
of Aβ produced. In both case GSMs do not affect Notch signaling [6]. Among the GSMs, some
non-steroidal anti-inflammatory drugs (NSAIDs), like flurbiprofen, ibuprofen, and indomethacin, seem
very promising. They are capable of reducing oligomer formation by allosteric modulation of
presenilin and their activity as GSMs is not correlated with their anti-cyclooxygenase activity [7,8].
The most studied NSAIDs in this context is the (R)-enantiomer of flurbiprofen (FLU) (Figure 1),
but unfortunately, results from the Myriad Flurizan trials have attributed to FLU poor potency due to
very poor brain penetration [9]. Therefore, new derivatives with better pharmacokinetic and
pharmacodynamic properties are required [10]. In order to improve the properties of FLU, we
synthesized some novel FLU lipophilic analogues and tested their potency on Aβ level reduction.
Starting from a previous work of Peretto and coworkers on the structure-activity relationships (SAR)
for FLU analogues we designed new peptide derivatives of FLU (compounds 1–9, Figure 1), in which
the carboxylic group was replaced by an amide moiety and linked to L-alanine-L-phenylglycine-t-butylester
or L-alanine-benzodiazepine (N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-
alanine) residues, in order to mimic the structure of the effective inhibitors DAPT and LY-411575,
respectively [8]. The substitution in the α position was also considered, due to the different activity of
the (R)- and (S)- enantiomers of FLU on both COX-1 and Aβ42 [11].
The pharmaceutical profile of the new agents was studied in order to determine the potential of both
their intestinal and Blood Brain Barrier (BBB) permeability. Therefore, the present study included
evaluation of solubility in Fasted State Simulated Intestinal Fluid (FASSIF), n-octanol partition
coefficient, membrane phospholipid affinity by immobilized artificial membrane (IAM)
chromatography and permeability coefficient by the Parallel Artificial Membrane Permeability Assay
(PAMPA) and in vitro activity [12].
2. Results and Discussion
Compounds 1–3 were synthesized as outlined in Scheme 1 by using stepwise elongation of the
peptide chain in the C-to-N direction via standard solution phase procedures. Amides 14–16 were
prepared by conventional DCC/HOBt condensation of FLU or its derivative 10 with
L-alanine-t-butylester hydrochloride and subsequent acidolysis with TFA; coupling with
L-phenylglycine-t-butylester hydrochloride gave the final products 1–3 [13,14]. FLU derivative 10 was
prepared as previously reported with a few slight modifications and in higher yield (Scheme 2) [15].

Molecules 2013, 18
10751
Scheme 1. Synthesis of compounds 1–3.
R1
R1
R2
O
R2
O
O
NH
OH
b)
a)
OtBu
F
F
R-flurbiprofen: R1=H, R2=Me
S-flurbiprofen: R1=Me, R2=H
10: R1=R2=H
11: R1=H, R2=Me
12: R1=Me, R2=H
13: R1=R2=H
R1
H
N
R2
O
O
R1
R
2 H
O
c)
OH
N
OtBu
N
H
O
O
F
F
14: R1=H, R2=Me
15: R1=Me, R2=H
16: R1=R2=H
1: R1=H, R2=Me
2: R1=Me, R2=H
3: R1=R2=H
Reagents: a) L-alanine-t-butylester hydrochloride, HOBt, Et3N, DCC, DCM; b) TFA; c) L-phenylglycine-t-
butylester hydrochloride, HOBt, Et3N, DCC, DCM.
Scheme 2. Synthesis of FLU derivative 10.
O
OH
Br
a)
b)
H
F
F
F
2-fluorobiphenyl-
4-carboxaldehyde
17
18
CN
COOH
c)
d)
F
F
19
10
Reagents: a) dry MeOH, NaBH4, THF; b) 48% hydrogen bromide; c) KCN, EtOH, H2O; d) NaOH, EtOH, H2O.
Alcohol 17, obtained by reduction of 2-fluorobiphenyl-4-carboxaldehyde with NaBH4, was treated
with 48% hydrogen bromide and then with potassium cyanide [16,17]. Hydrolysis of the cyano group
afforded the biphenylacetic acid 10 [8].
The synthesis of the target compounds 4–9 was carried out as outlined in Scheme 3: the proper
carboxylic acid (R-FLU, S-FLU or the unsubstituted FLU derivative 10, respectively) was coupled
with pure (S)- and (R)-5-amino-7-methyl-5,7-dihydro-6H-dibenz-[b,d]azepin-6-one (20a and 20b) in
the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDCI) and HOBt [18].

Molecules 2013, 18
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Scheme 3. Synthesis of compounds 4–9.
O
R
R
O
H
N
NH2
N
a)
COOH
N
+
O
F
F
20a,b
4-9
Reagents: a) EDCI, HOBt, THF.
To obtain the pure enantiomers 20a and 20b, racemic aminolactam 20 was conjugated with
(1S)-(+)-menthyl chloroformate (Scheme 4), separated by flash column chromatography and finally
hydrolyzed in the presence of triflic acid and trifluoroacetic acid [19].
Scheme 4. Separation of enentiomers 20a and 20b.
O
O
O
NH2
a)
O
O
N
+
NH
NH
N
N
20a,b
21a
21b
b)
b)
O
O
NH2
NH2
N
N
20a
20b
Reagents: a) TEA, (1S)-(+)-menthyl chloroformate, Et2O; b) CF3COOH, CF3SO3H, CH2Cl2.
The racemic aminolactam 20 was prepared as previously reported, with slight modifications
(Scheme 5) [20]. Starting from biphenyl-2-amine, an amination/reduction reaction with formaldehyde
and palladium on activated charcoal gave the methylated product 22 [21]. Compound 23 was obtained
by acetylation of 22 with chloroacetyl chloride. The cyclization, perfomed by a Friedel-Craft’s
intramolecular alkylation, allowed us to obtain 24 [22]. The oxime 25 was obtained by treating 26 with
potassium bis(trimethylsilyl)amide (KHMDS) and isopentyl nitrite. Reduction in a Parr autoclave with
palladium on activated charcoal afforded 20 [23].

Molecules 2013, 18
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Scheme 5. Synthesis of racemic aminolactam 20.
Cl
NH2
NH
O
a)
b)
N
biphenyl-2-amine
22
23
O
O
O
N
NH2
OH
c)
d)
e)
N
N
N
24
25
20
Reagents and Conditions: a) HCOH, NH4OCOH, Pd/C, iPrOH, H2O; b) ClCOCH2Cl, CH2Cl2, DIPEA; c) AlCl3,
100 °C–170 °C; d) isopentyl nitrite, KHMDS, Ph-CH3; e) H2, Pd/C, HCl, EtOH.
The pharmaceutical profile of the new agents was studied in order to determine the potential of both
their intestinal and BBB permeability. Besides, waters solubility of the investigated compounds was
determined for establishing the GI dissolution rate. Before partitioning into GI wall, a solid dosage form
should solubilize in the gastric fluids. For a good absorption profile from the GI lumen, a drug should
have solubility higher than 100 μg/mL [24]. On the other hand, the studied compounds demonstrated to
have low water and FaSSIF solubility (lower than 100 ng/mL and 10 μg/mL, respectively).
Nevertheless, this drawback could be overcome by their high n-octanol/water partition coefficients.
Indeed the synthesized molecules, designed by replacing the FLU ionizable carboxylic function with
lipophilic unionizable substituents, are characterized by high n-octanol/water lipophilicity values, as
confirmed by their clogP values, that are higher than those of (R)-flurbirpofen and other γ-secretase
inhibitors (Table 1).
Table 1. clogP of compounds 1–9.
1-2
3
4-7 8-9 R-FLU DAPT LY-411575 LY-450139 Nisoldipine Lacidipine
3.81 3.45 3.57 1.02
4.53 a 5.56 a
a Experimentally determined values.
5.71 5.40 6.08 5.77
Similarly, due to their high lipophilicity, experimental n-octanol/water lipophilicity values of
enantiomers 8/9 were determined by RP-HPLC because their characterization through the “classical”
shake-flask method was expected to be difficult and scarcely reproducible [25]. The retention factor,
determined in acetonitrile/water mixtures and extrapolated to 100% water (KC18/W) for enantiomers 8/9
was 3.94 (data not shown).
By comparing this value with KC18/W values of two analogously non-ionizable compounds such as
nisoldipine (i.e. KC18/W 2.99) and lacidipine (i.e. KC18/W 4.01) and considering their corresponding logP
values (i.e. 4.53 and 5.56, respectively), a logP value of 5.49 for enantiomers 8/9 can be calculated.
This latter value is strictly comparable with the clogP reported in Table 1 [26].

Molecules 2013, 18
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With the aim to evaluate a parameter that closely ensures the affinity of our neutral compounds for
membrane phospholipids, we chose to measure their retention factors on phospholipid-based stationary
phase by performing IAM-HPLC measurements (Table 2). Indeed, it is well known that phospholipid
layers, being an anisotropic partition phase, have superior biomimetic properties than n-octanol for
neutral compounds [27].
Table 2. LogKIAM/W of compounds 1–9.
1
2
3
4/7
5/6
8/9 R-FLU DAPT LY-411575 LY-450139 Nisoldipine Lacidipine
1.82 1.61 1.85 0.11 2.63 3.52
3.67 3.80 3.64 3.21 3.93 3.58
It is worth noting that, although the experimental measurements were performed on all the
compounds under investigation, being the IAM phase not enantioselective, a single logKIAM/W value for
each of the three enantiomer pairs, i.e., compounds 4 and 7, 5 and 6, 8 and 9, is reported in Table 2.
On the other hand, whereas clogP values do not discriminate between diastereomers, IAM-HPLC
retention values do.
Our results show that there is a broadly agreement between clogP and logKIAM/W data. The only
exceptions being compounds 3 and the 4/7 enantiomer pair. In particular, compound 3 is the only
compound that has a clogP value lower than lacidipine and enantiomers 4/7 are the only compounds
that have a logKIAM/W value lower than lacidipine. All other compounds have a lipohilicity higher than
lacidipine and the other reference compounds and the enantiomers 5/6 are the most prone to partition
in the immobilized membrane.
In order to better predict the ability of drugs to diffuse through the biological membranes, PAMPA
has been used as a non cell based assay for measuring passive permeability of the investigated
compounds [28]. Depending on the phospholipid type, PAMPA can mimic different
adsorption/permeation environments. Lecithin in dodecane has been used for GI permeation studies
(PAMPA-GI), whereas porcine polar brain lipid is used for BBB permeation assays (PAMPA-BBB) [29].
In brief our results indicate that all the synthesized compounds showed good permeability through GI
membranes, being their permeability coefficient (Pe) higher than 2 × 10−6 cm/s. Moreover, they have
also good brain permeability: in particular, enantiomers 4/7 seem very promising in BBB penetration,
respect to the drug parent flurbiprofen (Pe ≥ 4×10−6 cm/s instead of Pe ≥ 2×10−6 cm/s) (Table 3) [30,31].
Table 3. PAMPA-GI and PAMPA-BBB permeability. Pe (10−6 cm/s) of compounds 1–9
after 2 h of incubation for PAMPA-GI and after 18 h of incubation for PAMPA-BBB.
Compounds
Time (h) pH
1
2
3
4–7 8–9 FLU DAPT LY-411575 LY-450139
7.4
6.5
7.4
4.8 4.1 2.0 4.6 2.6 2.1
3.7 3.6 2.1 4.5 2.5 2.4
2.4 2.1 2.0 4.5 2.3 1.6
2.6
2.5
2.2
2.5
2.3
2.0
1.8
1.6
1.4
2
18
Compounds 1–9 were profiled for their in vitro toxicity and inhibitory activity on γ-secretase. For
this purpose CTXTNA2 rat astrocytes have been treated with compounds 1–9 at concentrations ranging
from 1 µM to 100 µM for 24 h, using LY-450139 and LY-411575 as the reference compounds [32].
With the exception of 1, 3 and 4, Aβ-42 production was reduced by increasing the concentration of

Molecules 2013, 18
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investigated compounds without affecting the APP level (Table 4) and all compounds did not cause
reduction in cell viability (data not shown) [33]. This behavior was consistent with that of LY-411575
and LY-450139 and was not related to compound toxicity because no reduction in cellular viability
was observed (data not shown) [26].
Table 4. Expression of APP and Aβ-42 in CTXNA2 astrocytes exposed to different
concentrations of the investigated 1–9 derivatives for 24 h (the most representative out of
three different consistent experiments). Samples have been normalized by incubating
membranes in the presence of β tubulin monoclonal antibody. Western blotting is the most
representative out of three different consistent experiments.
C
1
1 µM
10 µM
100 µM
APP
(63.1 ± 3.2) × 10−2
(23.2 ± 0.8)×10−2
(93.1 ± 3.8) × 10−2
(29.2 ± 1.2) × 10−2
(89.4 ± 3.2) × 10−2
(43.5 ± 1.6) × 10−2
3
(120 ± 3.2) × 10−2
(45.8. ± 1.3) × 10−2
Aβ-42
2
1 µM
10 µM
100 µM
1 µM
10 µM
100 µM
(93.1 ± 2.6) × 10−2 (93.3 ± 3.5) × 10−2
(20.0 ± 1.1) × 10−2 (12.1 ± 0.6) × 10−2
(63.6 ± 2.8) × 10−2
(8.4 ± 0.2) × 10−2
(64.1 ± 2.3) × 10−2
(8.8. ± 0.4) × 10−2
(72.2 ± 2.1) × 10−2
(10.5 ± 0.4) × 10−2
5
(105 ± 3.2) × 10−2
(45.8. ± 1.3) × 10−2
4
1 µM
10 µM
100 µM
1 µM
10 µM
100 µM
(51.3 ± 2.6) × 10−2 (39.9 ± 1.6) × 10−2
(11.0 ± 0.4) × 10−2 (12.1 ± 0.4) × 10−2
(60.1 ± 2.7) × 10−2
(16.2 ± 0.7) × 10−2
(41.1 ± 1.7) × 10−2
(17.4. ± 0.3) × 10−2
(44.2 ± 2.1) × 10−2
(17.1 ± 0.6) × 10−2
7
(49.2 ± 3.2) × 10−2
(17.4. ± 0.5) × 10−2
6
1 µM
10 µM
100 µM
1 µM
10 µM
100 µM
(11.3 ± 0.4) × 10−2 (10.4 ± 0.2) × 10−2
(19.0 ± 0.6) × 10−2 (16.2 ± 0.5) × 10−2
(13.5 ± 0.5) × 10−2
(13.4 ± 0.7) × 10−2
(9.0 ± 0.3) × 10−2
(12.4. ± 0.1) × 10−2
(8.0 ± 0.3) × 10−2
(11.1 ± 0.3) × 10−2
9
(10.1 ± 3.2) × 10−2
(9.2 ± 0.3) × 10−2
8
1 µM
10 µM
100 µM
1 µM
10 µM
100 µM
(82.3 ± 3.2) × 10−2 (68.1 ± 3.2) × 10−2
(31.1 ± 0.9) × 10−2 (25.0 ± 1.0) × 10−2
LY411575
(94.4 ± 4.2) × 10−2
(35.2 ± 1.1) × 10−2
(68.7 ± 2.2) × 10−2
(43.2 ± 2.1) × 10−2
(69.2 ± 1.4) × 10−2
(38.4 ± 1.0) × 10−2
LY450139
(94.3 ± 3.2) × 10−2
(14.0 ± 0.5) × 10−2
1 µM
10 µM
100 µM
1 µM
10 µM
100 µM
(64.0 ± 2.4) × 10−2 (54.7 ± 2.0) × 10−2
(57.2 ± 1.2) × 10−2
(4.1 ± 0.2) × 10−2
(65.9 ± 2.3) × 10−2
(9.4. ± 0.3) × 10−2
(60.0 ± 1.6) × 10−2
(5.1 ± 0.1) × 10−2
S-Flurbiprofen
10 µM
(58.8 ± 2.5) × 10−2
(5.4 ± 0.2) × 10−2
(8.2 ± 0.1) × 10−2
(6.0 ± 0.1) × 10−2
R-Flurbiprofen
10 µM
1 µM
100 µM
1 µM
100 µM
(60.0 ± 1.3) × 10−2 (54.5 ± 2.3) × 10−2
(20.5 ± 0.7) × 10−2 (19.1 ± 0.5) × 10−2
DAPT
(52.1 ± 2.1) × 10−2
(14.1 ± 0.7) × 10−2
(60.6 ± 2.5) × 10−2
(18.8. ± 0.6) × 10−2
(65.2 ± 1.8) × 10−2
(19.0 ± 0.7) × 10−2
(53.2 ± 1.9) × 10−2
(15.4 ± 0.3) × 10−2
1 µM
10 µM
100 µM
(65.4 ± 2.8) × 10−2 (62.1 ± 2.8) × 10−2 (58.3 ± 1.9) × 10−2
(8.3 ± 0.2) × 10−2 (6.4 ± 0.1) × 10−2 (5.5 ± 0.3) × 10−2
It is worth noting that enantiomers display different activity in agreement with the previously
highlighted different activity of the (R)- and (S)-enantiomers of FLU on both COX-1 and Aβ42 [11].

Molecules 2013, 18
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It is particularly intriguing that the highly effective 2 and 7 compounds derive from the less studied
(S)-enantiomer of FLU.
3. Experimental
3.1. General
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). LY-411575 and
LY-450139 were bought from Haoyuan Chemexpress (Shanghai, China), whereas compounds 1–9
were synthesized in our labs. Microanalyses were performed on a 1106 Carlo Erba CHN analyzer, and
13
the results were within (0.4%) of the calculated values. 1H- and C-NMR spectra were recorded on a
Varian VXR 300-MHz spectrometer. Chemical shifts are reported in parts per million (δ) downfield
from the internal standard tetramethylsilane (Me4Si). Optical rotations were taken at 20 °C with a
Perkin/Elmer 241 polarimeter. Melting points were determined on a Büchi B-450 apparatus and are
uncorrected. The LC-MS/MS system used consisted of an LCQ ion trap mass spectrometer (Thermo
Finnigan, San Jose, CA, USA) equipped with an electrospray ionization (ESI) source. The capillary
temperature was set at 300 °C and the spray voltage at 4.25 kV. The fluid was nebulized using
nitrogen (N2) as both the sheath gas and the auxiliary gas. The identity of all new compounds was
confirmed by elemental analysis, NMR data and LC-MS/MS system; homogeneity was confirmed by
TLC on silica gel Merck 60 F254. Solutions were routinely dried over anhydrous sodium sulphate
prior to evaporation. Chromatographic purifications were performed by Merck 60 70-230 mesh ASTM
silica gel column. The HPLC system for solubility determination was a Waters Pump 600 Controller,
equipped with a Waters 2996 photodiode array detector, a 20-μl Rheodyne injector and a Waters
Symmetry RP-C18 column (4.6 mm × 150 mm, 5 µm); the mobile phase was a mixture of
water/methanol 10/90. The flow rate was 1 mL/min and the wavelength for compound detection was
246 nm.
(2-Fluoro-1,1'-biphenyl-4-yl)methanol (17): NaBH4 (0.15 g, 4.0 mmol) was added to 2-fluoro-1,
1'-biphenyl-4-carbaldehyde (0.20 g, 1 mmol) in MeOH/THF (1:1, v/v, 4 mL). After 12 h at r.t., HCl
2N (1 mL) was carefully added and the product was extracted with Et2O. The organic layer was
washed with brine, dried and evaporated. Crystallization from hexane afforded 17; Rf: 0.51
(CHCl3/acetone 7:3); yield 85%; 1H-NMR (CDCl3): δ 7.59–7.18 (m, 8H, ArH), 4.75 (s, 2H, CH2), 1.84
(bs, 1H, OH).
4-(Bromomethyl)-2-fluoro-1,1'-biphenyl (18): The alcohol 17 (0.17 g, 0.84 mmol) was refluxed with
48% hydrogen bromide (1 mL) for 4 h at 120 °C. After cooling, water was added and the product was
extracted with ether. The organic phase was purified by column chromatography with cyclohexane/Et2O
1
95:5 as eluent; Rf: 0.46 (cyclohexane/Et2O 95:5); yield 86%; H-NMR (CDCl3): δ 7.56–7.19 (m, 8H,
ArH), 4.50 (s, 2H, CH2).
(2-Fluoro-1,1′-biphenyl-4-yl)acetonitrile (19): To a solution of KCN (0.11 g, 1.72 mmol) in water (2.5 mL)
was slowly added a solution of 18 (0.46 g, 1.72 mmol) in ethanol (5 mL). After 2 h at 120 °C, the
ethanol was evaporated and the product was extracted with ether and purified by column

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chromatography with cyclohexane/EtOAc 6:4; Rf: 0.56 (cyclohexane/EtOAc 6:4); yield 50%;
1H-NMR (CDCl3): δ 7.56–7.14 (m, 8H, ArH), 3.79 (s, 2H, CH2).
(2-Fluoro-1,1'-biphenyl-4-yl)acetic acid (10): To a solution of 19 (0.17 g, 0.83 mmol) in methanol
(1.2 mL), 35% NaOH (4.8 mL) was added. The mixture was stirred for 8 h at 100 °C and then was
cooled at r.t.. After acidification with HCl 2N, the precipitate formed was filtered, washed with water
and dried under vacuum; Rf: 0.31 (EtOAc/Cyclohexane 6:4); yield 71%; 1H-NMR (CDCl3): δ 7.55–7.10
(m, 8H, ArH), 3.70 (s, 2H, CH2).
N-Methylbiphenyl-2-amine (22): To a stirred solution of biphenyl-2-amine (0.029 mol) in
2-propanol (125 mL) a solution 37% of formaldehyde in water (0.059 mol) was added and the solution
was stirred for 1 h. In another flask, to a suspension of palladium on activated charcoal (0.003 mol) in
2-propanol (125 mL), a solution of ammonium formate (0.29 mol) in water (50 mL) was added. The
two solutions were combined and the new suspension was stirred overnight at r.t. and then refluxed for
1 h. After cooling, the suspension was filtered over a Celite pad and the solvent was evaporated. The
residue was dissolved in dichloromethane and washed with water. The organic layer was dried over
Na2SO4 and evaporated. The crude product was purified by flash column chromatography
1
n-hexane/EtOAc 98:2; Rf: 0.41 (n-hexane/EtOAc 98:2); yield: 43%; H-NMR (CDCl3): δ 7.50–7.42
(m, 4H, ArH); 7.38–7.35 (m, 1H, ArH); 7.24–7.22 (m, 1H, ArH); 7.14–7.10 (m, 1H, ArH); 6.84–6.80
(m, 1H, ArH); 6.73–6.70 (m, 1H, ArH), 4.05 (brs, 1H, NH); 2.78 (s, 3H, CH3).
N-(Biphenyl-2-yl)-2-chloro-N-methylacetamide (23): In a perfectly dried two-necked flask, to a cooled
(ice bath) solution of 22 in dry DCM (10 mL), N,N-diisopropylethylamine (1.5 eq.) and 2-chloroacetyl
chloride (1.5 eq.) were added. The solution was allowed to raise r.t. and stirred under a nitrogen stream
for 2 h and then HCl 0.5 N was added. The two phases were separated and the organic layer was dried
over Na2SO4 and evaporated. The crude product was recrystallized from EtOH; Rf: 0.60
1
(n-hexane/EtOAc 98:2); yield: 83%. H-NMR (CDCl3): δ 7.46–7.22 (m, 9H, ArH); 3.90 (d, 1H, CH2,
J = 12 Hz); 3.75 (d, 1H, CH2, J=12 Hz); 3.10 (s, 3H, CH3).
N-Methyl-5H-dibenzo[b,d]azepin-6(7H)-one (24): In a perfectly dried three-necked flask, aluminum
chloride (4 eq.) was added portionwise to 23 under a nitrogen stream. The mixture was vigorously
stirred and heated at 100 °C for 1 h and then the temperature was raised to 170 °C until gas evolution
ceased. After removing the oil bath, ice was added and the mixture was vigorously stirred for
additional 30 min, then HCl 2N was added. The aqueous layer was extracted exhaustively with
chloroform. The combined organic phases were washed with brine, dried over Na2SO4 and evaporated.
The crude product was recrystallized from EtOH; Rf: 0.55 (n-hexane/EtOAc 98:2); yield: 59%.
1H-NMR (CDCl3): δ 7.65–7.60 (m, 2H, ArH); 7.42–7.25 (m, 6 H, ArH); 3.59-3.55 (m, 1H, CH2);
3.45–3.42 (m, 1H, CH2); 3.35 (s, 3H, CH3).
(E)-7-(Hydroxyimmino)-5-metil-5H-dibenzo[b,d]azepin-6(7H)-one (25): In a perfectly dried two-necked
flask, to a cooled (ice bath) solution of 24 in dry toluene potassium-bis(trimethylsilyl)-amide (0.5 M
solution in toluene, 1.1 eq.) was added dropwise, under a nitrogen stream. The solution was stirred for
20 min, and isopentyl nitrite (1.1 eq.) was added dropwise. The solution was allowed to raise to r.t. and

Molecules 2013, 18
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was stirred overnight. The solution was quenched with HCl 1N; the two phases were separated and the
organic layer was washed with brine, dried over Na2SO4 and evaporated. The crude product was
purified by flash column chromatography (n-hexane/EtOAc 75:25); Rf: 0.40 (n-hexane/EtOAc 75:25);
yield: 36%. 1H-NMR (CDCl3): δ 7.71–7.28 (m, 8H, ArH); 3.42 (s, 3H, CH3).
7-Amino-5-methyl-5H-dibenzo[b,d]azepin-6(7H)-one (20): A solution of 25 (200 mg) in EtOH (10 mL)
was placed in a Parr autoclave. Palladium on activated charcoal (0.1 eq.) and HCl 2N were added. The
suspension was hydrogenated at exactly 5 Bars for 24 h. The solution was filtered through a Celite pad
and the solvent evaporated. The residue was added of HCl 4N and washed with EtOAc. The aqueous
layer was basified with NaOH 2N and extracted exhaustively with EtOAc. The combined organic
layers were dried over Na2SO4 and evaporated. No further purification was required; Rf: 0.20
1
(n-hexane/EtOAc 98:2); yield: 41%. H-NMR (CDCl3): δ 7.60–7.23 (m, 8H, ArH); 4.35 (s, 1H, CH);
3.36 (s, 3H, CH3); 2.38 (brs, 2H, NH2).
Racemic resolution of the amine 20: The hydrochloride salt of 20 was suspended in dry diethyl ether
and triethylamine (3 eq.) was added. To the mixture a solution of (1S)-(+)-menthylchloroformate
(1.5 eq.) in dry diethyl ether was added dropwise. The suspension was stirred at r.t. for 2 h.
The reaction was quenched with NaOH 2N, the phases were separated and the organic layer was dried
over Na2SO4 and evaporated. The diastoisomers were separated by flash column chromatography
(dichloromethane/cyclohexane 9:1) to afford compounds 21a and 21b. Overall yield: 89.7%. 1H-NMR
(CDCl3) of compound 21a: δ 7.58 (dd, 2H, ArH); 7.30-7.49 (m, 6H, ArH); 6.37 (d, 1H); 5.16 (d, 1H);
4.51 (m, 1H, CH); 3.37 (s, 3H, CH3); 2.09 (m, 1H); 1.93 (dd, 1H); 1.63 (m, 3H); 1.41 (m, 3H); 1.03
1
(m, 1H); 0.98 (d, 3H, CH3); 0.86 (d, 3H, CH3); 0.79 (d, 3H, CH3). H-NMR (CDCl3) of compound
21b: δ 7.58 (dd, 2H, ArH); 7.30–7.49 (m, 6H, ArH); 6.37 (d, 1H); 5.16 (d, 1H); 4.51 (m, 1H, CH);
3.37 (s, 3H, CH3); 2.09 (m, 1H); 1.93 (dd, 1H); 1.63 (m, 3H); 1.41 (m, 3H); 1.03 (m, 1H); 0.88 (dd,
6H, 2CH3); 0.71 (d, 3H, CH3).
Compounds 21a or 21b (1 eq.) were dissolved in dry dichloromethane (10 mL) and the solution was
cooled down (ice bath). Then trifluoroacetic acid (10 eq.) and triflic acid (3 eq.) were added dropwise.
The reaction was stirred at 0 °C for 2 h and then was quenched with a saturated solution of NaHCO3.
The two layers were separated and the aqueous phase was extracted with dichloromethane. The
combined organic layers were dried over Na2SO4 and evaporated to afford one of the enantiomers of
1
amine 20 as shown by TLC and its H-NMR spectrum. No further purification was required; Rf: 0.35
(n-hexane/EtOAc 98:2); Yield: 93%.
3.2. General Procedure for Synthesis of Compunds 11–13
To a solution of flurbiprofen (0.82 mmol) in DCM (2.7 mL) was added a solution of DCC (0.17 g,
0.82 mmol) in DCM (2.7 mL). After 15 min, L-alanine-t-butylester hydrochloride (0.15 g, 0.82 mmol)
in DCM (2.7 mL) and Et3N (107 µL, 0.82 mmol) was added. The mixture was stirred at room
temperature for 3 h. The precipitated dicyclohexylurea was filtered and the solvent was evaporated.
The product was purified by column chromatography with DCM/EtOAc 9:1 as eluent.

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tert-Butyl N-[(2R)-2-(2-fluoro-1,1′-biphenyl-4-yl)propanoyl]-L-alaninate (11): Rf: 0.49 (DCM/EtOAc
9:1); yield 50%; 1H-NMR (CDCl3): δ 7.53-7.14 (m, 8H, ArH), 6.04 (d, 1H, J = 7.2 Hz, NH), 4.48–4.41
(m, 1H, αCH), 3.62–3.55 (m, 1H, CH), 1.53 (d, 3H, J = 7.2 Hz, CH3), 1.45 (s, 9H, CH3 t-Bu), 1.31 (d,
3H, J = 7.2 Hz, CH3).
tert-Butyl N-[(2S)-2-(2-fluoro-1,1′-biphenyl-4-yl)propanoyl]-L-alaninate (12): Rf: 0.49 (DCM/EtOAc
9:1); yield 50%; 1H-NMR (CDCl3): δ 7.58–7.19 (m, 8H, ArH), 6.07 (d, 1H, J = 7.2 Hz, NH), 4.46–4.41
(m, 1H, αCH), 3.62-3.55 (m, 1H, CH), 1.54 (d, 3H, J = 7.2 Hz, CH3), 1.41 (s, 9H, CH3 t-Bu), 1.36
(d, 3H, J = 7.2 Hz, CH3); 13C-NMR (DMSO-d6): δ 172.93 (s, 1C, CONH), 135.68–115.21 (10 s, 12 C,
Ar), 82.33 (s, 1C, C t-Bu), 49.93 (s, 1C, αCH), 46.88 (s, 1C, CH), 28.41 (s, 3C, CH3 t-Bu), 19.41 (s,
1C, CH3), 18.10 (s, 1C, CH3).
tert-Butyl N-[(2-fluoro-1,1′-biphenyl-4-yl)acetyl]-L-alaninate (13): Rf: 0.38 (DCM/EtOAc 9:1); yield
1
74%; H-NMR (CDCl3): δ 7.56-7.09 (m, 8H, ArH), 6.14 (d, 1H, J = 6.6 Hz, NH), 4.50-4.46 (m, 1H,
αCH), 3.60 (s, 2H, CH2), 1.44 (s, 9H, CH3 t-Bu), 1.36 (d, 3H, J = 7.2 Hz, CH3).
3.3. General Procedure for Synthesis of Compunds 14–16
Compounds 11–13 (0.14 g, 0.38 mmol) were dissolved in TFA (7 mL). After 30 min at r.t., the
solvent was evaporated the residue was repeatedly washed with ether, to give compounds 14–16
respectively in quantitative yield.
N-[(2R)-2-(2-Fluoro-1,1′-biphenyl-4-yl)propanoyl]-L-alanine (14): Rf: 0.28 (DCM/EtOAc 9:1);
1H-NMR (CDCl3): δ 8.58 (bs, 1H, COOH), 7.54–7.10 (m, 8H, ArH), 6.08 (d, 1H, J = 6.9 Hz, NH),
4.62–4.53 (m, 1H, αCH), 3.67–3.60 (m, 1H, CH), 1.54 (d, 3H, J = 7.2 Hz, CH3), 1.40 (d, 3H,
J = 7.2 Hz, CH3).
N-[(2S)-2-(2-Fluoro-1,1′-biphenyl-4-yl)propanoyl]-L-alanine (15): Rf: 0.28 (DCM/EtOAc 9:1);
1H-NMR (CDCl3): δ 9.88 (bs, 1H, COOH), 7.54–7.09 (m, 8H, ArH), 6.10 (d, 1H, J = 6.9 Hz, NH),
4.58–4.53 (m, 1H, αCH), 3.64-3.61 (m, 1H, CH), 1.53 (d, 3H, J = 7.2 Hz, CH3), 1.42 (d, 3H,
J = 7.2 Hz, CH3).
N-[(2-Fluoro-1,1′-biphenyl-4-yl)acetyl]-L-alanine (16): Rf: 0.18 (DCM/EtOAc 9:1); 1H-NMR (DMSO):
δ 12.51 (bs, 1H, COOH), 8.48 (d, 1H, J = 7.5 Hz, NH), 7.53–7.15 (m, 8H, ArH), 4.20–4.16 (m, 1H,
αCH), 3.51 (s, 2H, CH2), 1.27 (d, 3H, J = 7.5 Hz, CH3).
3.4. General Procedure for Synthesis of Final Products 1–3
To a solution of compounds 14–16 (0.63 mmol) in DCM (2.1 mL) was added a solution of DCC
(0.13 g, 0.63 mmol) in DCM (2.1 mL). After 15 min, L-phenylglycine-t-butylester hydrochloride (0.15 g,
0.63 mmol) in DCM (2.1 mL) and Et3N (82 µL, 0.63 mmol) was added. The mixture was stirred at
room temperature for 2 h. The precipitated dicyclohexylurea was filtered and the solvent was
evaporated. The final products were purified by column chromatography with DCM/EtOAc as eluent.

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tert-Butyl (2S)-({N-[(2R)-2-(2-fluoro-1,1'-biphenyl-4-yl)propanoyl]-L-alanyl}amino)(phenyl)acetate
(1): Rf: 0.59 (DCM/EtOAc 7:3); yield 70%; M.p: 105–107 °C; ꢀꢁꢂꢄꢃꢅ = + 127.2 (c = 1, EtOH);
1H-NMR (CDCl3): δ 7.54–7.08 (m, 14H, ArH and NH), 6.18 (d, 1H, J = 7.2 Hz, NH), 5.38 (d, 1H,
J = 7.2 Hz, αCH), 4.61–4.52 (m, 1H, αCH), 3.55-3.48 (m, 1H, CH), 1.46 (d, 3H, J = 7.2 Hz, CH3),
13
1.38 (s, 9H, CH3 t-Bu), 1.34 (d, 3H, J = 6.9 Hz, CH3); C-NMR (CDCl3): δ 173.5 (s, 1C, CONH),
171.4 (s, 1C, CONH), 169.6 (s, 1C, COO), 160.0 (d, 1C, J = 987.6 Hz, CF), 142.6 (d, 1C, J = 29.4 Hz,
Ar), 137.0 (s, 1C, Ar), 135.6 (s, 1C, Ar), 131.3 (d, 1C, J = 14.7 Hz, Ar), 129.2–127.2 (7s, 11C, Ar),
123.7 (d, 1C, J = 13.5 Hz, Ar), 115.4 (d, 1C, J = 94.5 Hz, Ar), 83.0 (s, 1C, C t-Bu), 57.3 (s, 1C, αCH),
48.9 (s, 1C, αCH), 46.6 (s, 1C, CH), 28.0 (s, 3C, CH3 t-Bu), 18.8 (s, 1C, CH3), 18.4 (s, 1C, CH3); MS
(ESI) m/z 505 (M-H)+.
tert-Butyl(2S)-({N-[(2S)-2-(2-fluoro-1,1'-biphenyl-4-yl)propanoyl]-L-alanyl}amino)(phenyl)acetate
(2): Rf: 0.59 (DCM/EtOAc 7:3); yield 40%; M.p: 106–108 °C; ꢀꢁꢂꢄꢃꢅ = + 213.4 (c = 1, EtOH);
1H-NMR (CDCl3): δ 7.54-7.10 (m, 14H, ArH and NH), 6.41 (d, 1H, J = 7.8 Hz, NH), 5.41 (d, 1H,
J = 7.2 Hz, αCH), 4.64-4.59 (m, 1H, αCH), 3.65-3.58 (m, 1H, CH), 1.54 (d, 3H, J = 7.2 Hz, CH3), 1.38
13
(s, 9H, CH3 t-Bu), 1.26 (d, 3H, J = 6.6 Hz, CH3); C-NMR (CDCl3): δ 173.7 (s, 1C, CONH), 171.7
(s, 1C, CONH), 169.7 (s, 1C, COO), 160.0 (d, 1C, J = 987.6 Hz, CF), 142.7 (d, 1C, J = 30.9 Hz, Ar),
137.1 (s, 1C, Ar), 135.7 (s, 1C, Ar), 131.2 (d, 1C, J = 15.9 Hz, Ar), 129.3-127.3 (7s, 11C, Ar), 123.8
(d, 1C, J = 13.5 Hz, Ar), 115.4 (d, 1C, J = 94.2 Hz, Ar), 82.9 (s, 1C, C t-Bu), 57.2 (s, 1C, αCH), 48.9
(s, 1C, αCH), 46.6 (s, 1C, CH), 28.0 (s, 3C, CH3 t-Bu), 18.8 (s, 1C, CH3), 18.4 (s, 1C, CH3); MS (ESI)
m/z 505 (M-H)+.
tert-butyl (2S)-({N-[(2-fluoro-1,1'-biphenyl-4-yl)acetyl]-L-alanyl}amino)(phenyl)acetate (3): Rf: 0.62
(DCM/EtOAc 9:1); yield 66%; M.p: 104–106 °C; ꢀꢁꢂꢄꢃꢅ = + 170.3 (c = 1, EtOH); 1H-NMR (CDCl3): δ
7.53–7.09 (m, 14H, ArH and NH), 6.39 (d, 1H, J = 7.2 Hz, NH), 5.37 (d, 1H, J = 7.2 Hz, αCH),
4.62–4.58 (m, 1H, αCH), 3.60 (s, 2H, CH2), 1.36 (s, 9H, CH3 t-Bu), 1.30 (d, 3H, J = 7.2 Hz, CH3);
13C-NMR (CDCl3): δ 171.5 (s, 1C, CONH), 170.5 (s, 1C, CONH), 169.7 (s, 1C, COO), 160.0 (d, 1C,
J = 990.0 Hz, CF), 137.0 (s, 1C, Ar), 136.0 (d, 1C, J = 33.0 Hz, Ar), 135.6 (s, 1C, Ar), 131.3 (d, 1C,
J = 15.9 Hz, Ar), 129.2-127.1 (7s, 11C, Ar), 125.6 (d, 1C, J = 13.5 Hz, Ar), 117.8 (d, 1C, J = 93.3 Hz,
Ar), 83.0 (s, 1C, C t-Bu), 57.3 (s, 1C, αCH), 48.9 (s, 1C, αCH), 43.0 (s, 1C, CH2), 27.9 (s, 3C, CH3
t-Bu), 18.3 (s, 1C, CH3); MS (ESI) m/z 491 (M-H)+.
3.5. General Procedure for Synthesis of Final Products 4–9
The corresponding carboxylic acid (1 eq.) was dissolved in dry THF. 20a or 20b (1 eq.),
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (1 eq.), HOBt (1 eq.) and
diisopropylethylamine (1 eq.) were added and the mixture was stirred at r.t. for 16 h. The reaction was
quenched with HCl 0.5 N and the aqueous layer was extracted with DCM. The combined organic
phases were washed twice with NaHCO3 2.5% and once with brine. The organic layer was dried over
Na2SO4 and evaporated.
(2R)-2-(2-Fluoro-1,1'-biphenyl-4-yl)-N-[(7R)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-
yl]propanamide (4): Rf: 0.63 (DCM/EtOAc 7:3); Yield: 44%; ꢀꢁꢂꢄꢃꢅ = +43.1 (c = 1, EtOH); M.p:

Molecules 2013, 18
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1
100–103 °C; H-NMR (CDCl3): δ 7.65–7.20 (m, 16H, ArH); 6.98–6.95 (m, 1H, NH); 5.41-5.37 (m,
1H, CH); 3.88–3.83 (m, 1H, CH); 3,38 (s, 3H, CH3); 1,59 (s, 3H, CH3); 13C-NMR (CDCl3): δ 171.4 (s,
1C, CONH), 169.2 (s, 1C, CONH), 162.7-111.4 (22s, 24C, Ar), 60.3 (s, 1C, αCH), 47.9 (s, 1C, αCH),
32.1 (s, 1C, CH3), 17.8 (s, 1C, CH3); MS (ESI) m/z 465 (M−H)+.
(2R)-2-(2-Fluoro-1,1'-biphenyl-4-yl)-N-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-
yl]propanamide (5): Rf: 0.63 (DCM/EtOAc 7:3); Yield: 44%; M.p: 102–103 °C; ꢀꢁꢂꢄꢃꢅ = +42.8
1
(c = 1, EtOH); H-NMR (CDCl3): δ 7.62-7.20 (m, 16H, ArH and 1H, NH); 5.35–5.32 (m, 1H, CH);
3.89–3.85 (m, 1H, CH); 3.35 (s, 3H, CH3); 1.62 (s, 3H, CH3); 13C-NMR (CDCl3): δ 172.2 (s, 1C,
CONH), 170.1 (s, 1C, CONH), 163.2-111.2 (22s, 24C, Ar), 59.9 (s, 1C, αCH), 47.00 (s, 1C, αCH),
32.8 (s, 1C, CH3), 17.9 (s, 1C, CH3); MS (ESI) m/z 465 (M−H)+.
(2S)-2-(2-Fluoro-1,1'-biphenyl-4-yl)-N-[(7R)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-
yl]propanamide (6): Rf: 0.63 (DCM/EtOAc 7:3); Yield: 47%; M.p: 104–105 °C; ꢀꢁꢂꢄꢃꢅ = −42.8 (c = 1,
EtOH); 1H-NMR (CDCl3): δ 7.65–7.20 (m, 16H, ArH); 6.99-6.95 (m,1H, NH); 5.40-5.37 (m, 1H, CH);
3.88–3.83 (m, 1H, CH); 3.38 (s, 3H, CH3); 1.59 (d, 3H, CH3); 13C-NMR (CDCl3): δ 172.2 (s, 1C,
CONH), 171.0 (s, 1C, CONH), 164.0-112.0 (22s, 24C, Ar), 61.0 (s, 1C, αCH), 48.2 (s, 1C, αCH), 32.4
(s, 1C, CH3), 18.0 (s, 1C, CH3); MS (ESI) m/z 465 (M-H)+.
(2S)-2-(2-Fluoro-1,1'-biphenyl-4-yl)-N-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-
yl]propanamide (7): Rf: 0.63 (DCM/EtOAc 7:3); Yield: 47%; M.p: 102–104 °C; ꢀꢁꢂꢄꢃꢅ = −43.1
1
(c = 1, EtOH); H-NMR (CDCl3): δ 7.62–7.20 (m, 16H, ArH and 1H, NH); 5.36–5.32 (m, 1H, CH);
3.90–3.85 (m, 1H, CH); 3.35 (s, 3H, CH3); 1.62 (s, 3H, CH3); 13C-NMR (CDCl3): δ 172.1 (s, 1C,
CONH), 170.2 (s, 1C, CONH), 164.8–112.3 (22s, 24C, Ar), 60.4 (s, 1C, αCH), 48.5 (s, 1C, αCH), 32.6
(s, 1C, CH3), 17.6 (s, 1C, CH3); MS (ESI) m/z 465 (M-H)+.
2-(2-Fluoro-1,1'-biphenyl-4-yl)-N-[(7R)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-
acetamide (8): Rf: 0.56 (DCM/EtOAc 7:3); Yield: 55%; M.p: 96–100 °C; ꢀꢁꢂꢄꢃꢅ = +0.2 (c = 1, EtOH);
1H-NMR (CDCl3): δ 7.62-7.16 (m, 17H, ArH and NH); 5.36-5.31 (m, 1H, CH); 3.77 (s, 2H, CH2);
3.36 (s, 3H, CH3); 13C-NMR (CDCl3): δ 172.2 (s, 1C, CONH), 171.0 (s, 1C, CONH),
164.0–112.0 (22s, 24C, Ar), 61.0 (s, 1C, αCH), 45.2 (s, 1C, CH2), 32.4 (s, 1C, CH3); MS (ESI) m/z
451 (M−H)+.
2-(2-Fluoro-1,1'-biphenyl-4-yl)-N-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-
acetamide (9): Rf: 0.56 (DCM/EtOAc 7:3); Yield: 79%; M.p: 96–100 °C; ꢀꢁꢂꢄꢃꢅ = −0.2 (c = 1, EtOH);
1H-NMR (CDCl3): δ 7.62-7.16 (m, 17H, ArH and NH); 5.35–5.31 (m, 1H, CH); 3.77 (s, 2H, CH2); 3.36 (s,
3H, CH3). 13C-NMR (CDCl3): δ 171.4 (s, 1C, CONH), 169.2 (s, 1C, CONH), 162.7–111.4 (22s, 24C, Ar),
60.3 (s, 1C, αCH), 46.2 (s, 1C, CH2), 32.1 (s, 1C, CH3); MS (ESI) m/z 451 (M−H)+.
3.6. Water Solubility
Compounds 1–9 (50 mg) were placed in deionized water (1 mL), shaken at 25 °C for 1 h to ensure
the solubility equilibrium and then centrifuged. The supernatant (20 µL) was analyzed by HPLC [18].

Molecules 2013, 18
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3.7. FaSSIF Solubility
The Fasted State Simulated Intestinal Fluid (FaSSIF) medium was prepared as shown in Table 5 [34].
Table 5. FaSSIF composition.
pH
6.8
29
KH2PO4 (mM)
Bile salts (mM)
Phospholipids (mM)
2.5
0.5
An excess of each compound (about 2 mg) was added to a screw cap vial containing 1 mL medium
heated to 37 °C. The sample was gently stirred for 24 h and then it was centrifuged at 4,500 rpm for
15 min: all these procedures were conducted at 37 °C. The supernatant was filtered, diluted with a
same volume of methanol and analyzed by HPLC.
3.8. Lipophilicity
n-Octanol/water partition coefficients were theoretically calculated by the program clogP for
windows version 2.0 (Biobyte Corp., Claremont, CA, USA). Affinity for membrane phospholipids, so
called “phospholipophilicity”, was measured by HPLC on a phospholipid stationary phase.
A 600E liquid chromatograph (Waters-Millipore, Milford, MA, USA), equipped with a 7125
Rheodyne injection valve and a 486 UV detector (Waters) set at 246 nm, was used. The column
was an IAM.PC.MG (Immobilized Artificial Membrane-PhosphatidylCholine-MethylGlycolate)
(4.6 mm × 150 mm) (Regis Chemical Company, Morton Grove, IL, USA). The mobile phase was
0.01 M phosphate buffer at pH 7.0 and acetonitrile at percentages ranging from 10 to 35 percent (v/v)
(from 1.0 to 4.0 mL/min). The chromatography was carried out at room temperature. All samples were
dissolved in methanol (ca. 10−3 M) and 20 μL samples were injected in the chromatograph.
Chromatographic retention data are expressed as the logarithm of the capacity factor, log k, defined as:
log k = log [(tr − t0)/t0]
(1)
where tr and t0 are the retention times of the drug and a non-retained compound (citric acid),
respectively. The log k values relative to 100% aqueous eluent (logKIAM/W) were calculated by
performing a polycratic method of extrapolation [35,36]. Each compound was eluted with at least three
different mobile phases containing acetonitrile in different percentages (ϕ). Linear relationships
between log k and ϕ values were found for all the compounds in the range of eluent composition
examined (r2 ≥ 0.994). All values of log k reported are the average of at least three measurements; the
95% confidence interval never exceeded 0.04 for each log k value. Possible occurrence of retention
changes due to column aging was monitored by checking the retention times of five test compounds
(amlodipine, p-nitroaniline, toluene, isradipine, and ketoprofen). During the study no retention value of
test compounds changed more than 4% and no correction was done to the retention values
experimentally determined for the analytes.

Molecules 2013, 18
10763
3.9. PAMPA Method
The following protocol was applied to measure the Pe through the artificial membrane to predict
oral absorption and BBB permeation. In order to study the g.i. permeability, a 2% solution (w/v) of
egg lecithin (phosphatidyl choline > 98%, PC) in dodecane was employed as an artificial membrane
solution. The effective permeability of BBB was measured using phospholipid mixture from porcine
polar brain lipid extract, composed by PC 12.6%, phosphatidylethanolamine (PE) 33.1%,
phosphatidylserine (PS) 18.5%, phosphatidylinositol (PI) 4.1%, phospatidic acid (PA) 0.8% and 30.9%
of other compounds (purchased from Avantis Polar Lipids, Alabaster, AL, USA). Each donor filtration
plate well was then impregnated with 5 µL of this solution carefully, and immediately after 150 µL of
phosphate buffer (pH 7.4/pH 6.5), containing co-drug 1 500 µM and iPrOH 20% as co-solvent was
added. Then the drug-filled donor plate was placed into the acceptor plate that had been prefilled with
the same buffer (300 µL) as acceptor solution. After plate lid was replaced, the resulting assembled
donor-acceptor plates were incubated at r.t. for 2 h for PAMPA-GI and 18 h for PAMPA-BBB,
following which drugs concentration in the acceptor and donor solutions were determined by HPLC [37].
Log Pe can be calculated from the equation below:
ꢀ
ꢂ
ꢜꢝꢝꢞꢟꢠꢡꢢ
ꢙꢚꢛꢈ
ꢎꢃ ꢏ ꢎ
ꢐ
ꢆꢇꢈꢉꢊ ꢋ ꢌꢍ
ꢖ ꢗ ln ꢘ1 ꢗ
ꢩꢪ
(2)
ꢑ
ꢓ
ꢎꢃ ꢒ ꢎ ꢏ ꢔ ꢏ ꢕ
ꢀꢙꢚꢛꢈꢂꢞꢣꢤꢥꢦꢥꢧꢢꢥꢤꢨ
ꢐ
where Pe is the effective permeability coefficient (cm x s−1), VD is volume of donor compartment
(0.15 cm3) and VA is volume of acceptor compartment (0.30 cm3), A is effective filter area (0.28 cm2),
t is incubation time for the assay (s), Cacceptor is the concentration of the compound in the acceptor
compartment at the completion of the assay, and Cequilibrium is the concentration of compound at
theoretical equilibrium.
3.10. Cell Culture and Drug Treatment
CTXTNA2 cell line, established from primary cultures of type 1 astrocytes from brain frontal
cortex tissue of 1 day old rats, was obtained by ECACC (Salisbury, UK). Cells have been maintained
in DMEM, 10% FBS at 37 °C, 5% CO2 until sub-confluence and incubated for 24 h with a DMSO
solution of drug (concentrations ranging from 1 µM to 100 µM). For detection of cell viability, a
Trypan blue dye exclusion test has been performed [33].
3.11. Western Blotting
Cell lysates (20 µg) have been electrophoresed and transferred to nitrocellulose membrane.
Nitrocellulose membranes, blocked in 5% non-fat milk, 10 mmol/L Tris pH 7.5, 100 mmol/L NaCl,
0.1% Tween-20, have been probed with mouse APP (Millipore, Billerica, MA, USA) and Aβ (Alpha
Diagnostic Intl, San Antonio, TX, USA) monoclonal antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA, USA), then incubated in the presence of specific enzyme conjugated IgG horseradish
peroxidase. Immunoreactive bands have been detected by ECL detection system (Amersham Intl.,
Buckinghamshire, UK) and analysed by densitometry. Densitometric values, expressed as Integrated
Optical Intensity (I.O.I.), have been estimated in a CHEMIDOC XRS system by the QuantiOne 1-D
analysis software (BioRad, Richmond, CA, USA). Values obtained have been normalized basing on

Molecules 2013, 18
10764
densitometric values of internal β tubulin. Statistical analysis has been performed using the analysis of
variance (ANOVA). Results have been expressed as mean ± SD. Values of p < 0.05 have been
considered statistically significant.
3.12. MTT Assay
Viability of cells was assessed by measuring the formation of a formazan from MTT
spectrophotometrically via commercial kits (Cayman Chemical®, Ann Arbor, Michigan, USA). At the
end of the experiment, the cells were incubated with 0.7 mg/mL MTT for 30 min at 37 °C. After
washing the blue formazan was extracted from cells with isopropanol/formic acid (95:5) and was
photometrically determined at 560 nm. The density of formazan formed in untreated cells was taken as
100% viability.
4. Conclusions
In conclusion, the data obtained highlight that both a high clogP and a high logKIAM/W are necessary
for the derivatives to be active. It appears also that, contrary to data on properties of FLU, the more
in vitro active novel compounds are characterized by a permeability in PAMPA-GI much higher
(about 20 times) than that in PAMPA-BBB. These results allow us to confirm that the newly
synthesized compounds are not toxic and are able to reduce Aβ levels, especially compounds 2 and 7.
However, the site of action of these compounds has not been identified yet. Nevertheless the similar
stereochemistry of the latter effective compounds and the evidenced important role of lipophilicity and
PAMPA permeability could help in elucidating this point. The improvement of potency and the
investigation of in vivo activity are subject to further investigations. In particular studies on Aβ-40 and
42 quantification and Notch processing are under way to understand if compound 2 and 7 behave as
GSIs or GSMs.
Acknoledgments
This work was supported by MIUR 2012 60% Grant.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–9 are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).