Novel benzylidenephenylpyrrolizinones with pleiotropic activities potentially useful in Alzheimer's disease treatment

Article abstract of DOI:10.1016/j.ejmech.2016.03.023

This work describes the synthesis and the biological evaluation of novel benzylidenephenylpyrrolizinones as potential antioxidant, metal chelating or amyloid β (βA) aggregation inhibitors. Some derivatives exhibited interesting results in regard to several of the performed evaluations and appear as valuable Multi-Target Directed Ligands with potential therapeutic interest in Alzheimer's disease. Among them, compound 29 particularly appears as a valuable radical and NO scavenger, a Cu(II) and Fe(II) chelating agent and exhibits moderate βA aggregation inhibition properties. These activities, associated to a good predictive bioavailability and a lack of cytotoxicity, design it as a promising hit for further in vivo investigation.

Full text of DOI:10.1016/j.ejmech.2016.03.023

Accepted Manuscript
Novel benzylidenephenylpyrrolizinones with pleiotropic activities potentially useful in
Alzheimer's disease treatment
Jean-Pierre Jourdan, Marc Since, Laïla El Kihel, Cédric Lecoutey, Sophie Corvaisier,
Rémi Legay, Jana Sopkova-de Oliveira Santos, Thierry Cresteil, Aurélie Malzert-
Fréon, Christophe Rochais, Patrick Dallemagne
PII:
S0223-5234(16)30197-0
10.1016/j.ejmech.2016.03.023
EJMECH 8448
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 February 2016
Revised Date: 9 March 2016
Accepted Date: 10 March 2016
Please cite this article as: J.-P. Jourdan, M. Since, L. El Kihel, C. Lecoutey, S. Corvaisier, R.
Legay, J.S.-d. Oliveira Santos, T. Cresteil, A. Malzert-Fréon, C. Rochais, P. Dallemagne, Novel
benzylidenephenylpyrrolizinones with pleiotropic activities potentially useful in Alzheimer's disease
treatment, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.023.
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ACCEPTED MANUSCRIPT
Novel benzylidenephenylpyrrolizinones with pleiotropic
activities potentially useful in Alzheimer's disease
treatment.
1
1
1
1
1
Jean-Pierre Jourdan, Marc Since, Laïla El Kihel, Cédric Lecoutey, Sophie Corvaisier,
1
1
2
1
Rémi Legay, Jana Sopkova-de Oliveira Santos, Thierry Cresteil, Aurélie Malzert-Fréon,
1
1
Christophe Rochais, * Patrick Dallemagne *
1
Normandie Université, UNICAEN, CERMN (Centre d’Etudes et de Recherche sur le
Médicament de Normandie), F-14032 Caen, France
2
CIBLOT, IPSIT - IFR14, 5 rue Jean Baptiste Clément, 92290 Chatenay-Malabry (France)

ACCEPTED MANUSCRIPT
Novel benzylidenephenylpyrrolizinones with pleiotropic activities
potentially useful in Alzheimer's disease treatment.
1
1
1
1
1
1
Jean-Pierre Jourdan, Marc Since, Laïla El Kihel, Cédric Lecoutey, Sophie Corvaisier, Rémi Legay,
1
2
1
1
Jana Sopkova-de Oliveira Santos, Thierry Cresteil, Aurélie Malzert-Fréon, Christophe Rochais, *
1
Patrick Dallemagne *
1
Normandie Université, UNICAEN, CERMN (Centre d’Etudes et de Recherche sur le Médicament de
Normandie), F-14032 Caen, France
2
CIBLOT, IPSIT - IFR14, 5 rue Jean Baptiste Clément, 92290 Chatenay-Malabry (France)
*
To whom correspondence should be addressed. Phone: +33 -2-3156-6813. Fax: +33 -2-3156-6803. E-
mail: patrick.dallemagne@unicaen.fr; christophe.rochais@unicaen.fr
Highlights :
•
•
•
•
Novel benzylidenephenylpyrrolizinones were synthesized.
Some of them acted as antioxidant, metal chelating and amyloid aggregation inhibitors.
Benzylidenephenylpyrrolizinones appeared devoid of cytotoxicity.
They further possess good predictive druggability parameters.
Abstract : This work describes the synthesis and the biological evaluation of novel
benzylidenephenylpyrrolizinones as potential antioxidant, metal chelating or amyloid β (βA)
aggregation inhibitors. Some derivatives exhibited interesting results in regard to several of the
performed evaluations and appear as valuable Multi-Target Directed Ligands with potential therapeutic
interest in Alzheimer's disease. Among them, compound 29 particularly appears as a valuable radical
and NO scavenger, a Cu(II) and Fe(II) chelating agent and exhibits moderate βA aggregation inhibition

ACCEPTED MANUSCRIPT
properties. These activities, associated to a good predictive bioavailability and a lack of cytotoxicity,
design it as a promising hit for further in vivo investigation.
Key words : Alzheimer; pleiotrope; MTDL; pyrrolizinone; antioxidant; antichelating; anti βA
aggregation
1
. Introduction
Due to the relative ineffectiveness of the current treatments against Alzheimer's Disease (AD),
which mostly all target the cholinergic deficiency,[1] numerous clinical trials during the past decade
focused on the inhibition of amyloid or TAU aggregation. Many of these trials however failed and some
authors attributed these failures to the extreme selectivity of the tested drugs for one target and their
clinical inefficiency towards neurodegenerative syndromes involving multiple pathogenic factors. [2,3]
In order to circumvent these problems, drug cocktails have been proposed and today donepezil, one of
the marketed acetylcholinesterase (AChE) inhibitors, is frequently added to numerous new clinical
candidates.[4] Another paradigm, however, is emerging. It concerns drugs, called Multi-Target Directed
Ligands (MTDL), which appear effective in treating complex diseases because of their capacity to
interact with several targets implied in the pathogenesis of the disease.[5-18] Besides this synergic
effectiveness, these MTDL should furthermore avoid the compliance or drug-drug interaction problems
met with drug cocktails.[19,20] Recently some MTDL with a dual mode of action against AD have been
reported.[21-32] Most of them are AChE inhibitors (AChEI) with additional biological activities useful
for treating AD, like M2 receptor antagonism,[33] direct inhibition of amyloid beta peptide (Aβ)
aggregation,[34-39] antioxidant properties,[40-43] in particular linked to MAO-B inhibition,[44-47]
calcium channel blockade,[48,49] control oxidative damage properties,[50] or SAPPα secretion
improvement.[51,52] Another class of MTDL, potentially useful against AD, lies in polyphenolic
compounds. Some of them are obtained from nature or synthetic works such as flavonoids (e.g. EGCG

ACCEPTED MANUSCRIPT
1
),[53] aurones (e.g. Maritimetin 2),[54] chalcones (e.g. compound 3),[55] or miscellaneous compounds
such as curcumin 4 (Figure 1).[56-58] They share common biological properties such as antioxidant,
metal chelation [59,60] and anti Aβ aggregation properties[61-63] which all theoretically account for a
therapeutic benefit in AD.
OH
O
OH
OH
O
Br
HO
O
HO
OH
OH
OH
OH
O
OH
O
OH
OH
OH
O
Chalcone 3
Maritimetin 2
OH
N
O
EGGC 1
R1
R2
O
OH
R3
O
O
R6
R4
R5
HO
OH
Curcumin 4
Title benzylidenephenylpyrrolizinones
Figure 1. Structure of various naturally occurring polyphenolic derivatives (1-4) and title
benzylidenephenylpyrrolizinones.
Some common features, important for these activities, have been established. The anti Aβ
aggregation potential appears especially linked to the presence of two polyphenolic rings, otherwise
needed for the antioxidant and metal chelation activities, but specifically linked by a rigid 8-16 Å long
carbon chain.[64] We postulate that the aldolisation of the phenylpyrrolizinone structural system with
aromatic benzaldehydes [65, 66] could constitute an appropriate scaffold able to match with these
structural requirements. It is the reason why we undertook the synthesis, according to an optimized
method, of such benzylidenephenylpyrrolizinones, as well as the in vitro evaluation of their potential
antioxidant, metal chelating and anti Aβ aggregation activities. Finally, in view of in vivo

ACCEPTED MANUSCRIPT
investigations, their cytotoxicity, capacity to cross the gastrointestinal and blood brain barriers and
chemical stability were also assessed.
2
. Chemistry
The phenylpyrrolizinone scaffold 5 can be prepared according to a multi-step sequence we
previously reported. The latter involved 3-amino-3-phenylpropionic acid 6 in a Clauson-Kaas reaction,
then proceeded through the activation of the resulting pyrrolyl derivative 7 under a carboxamide form 8
and then through a ring closure reaction using a Vilsmeier-Haack procedure (Scheme 1). The latter
afforded, after alkaline hydrolysis of the iminium salt 9, the expected phenylpyrrolizinone 5. An
improvement had been already brought to the sequence through the use of BBr for the direct
3
intramolecular cyclization of 7, but the methodology can't be applied to alkoxyphenyl derivatives
without partial O-dealkylation. We pursued the study of the sequence and we investigated the use of
triphosgen as ring closure reagent for the cyclization of 7 according to a methodology recently
reported.[67] It allowed the one-step synthesis of 5 in 52% yield.
NH2
O
OH
6
a
N
O
N
O
b, c
OH
N
7
8
d, e, f
h
g
N
+
N
-
-
N
Cl and PO Cl
2 2
O
5
9

ACCEPTED MANUSCRIPT
2 3 3
Reagents and conditions: a) 2,5-diOMeTHF, AcOH, 60°C, 1h30, 80%; b) TEA, ClCO Et, CH COCH ,
0
°C, 30 min; (c) Me NH, Et O, room temperature, 15 min, 71%; (d) POCl , reflux, 1 h; (e) NaHCO ,
2 2 3 3
H O; (f) HClO , H O, 0°C, 75%; (g) NaOH, H O, room temperature, 10 min, 95%; (h) triphosgen,
2
4
2
2
toluene, 110°C, 5 days (sealed tube), 52%.
Scheme 1. Synthesis of phenylpyrrolizinone 5.
This procedure was used for the cyclization of the alkoxyphenylpyrrolylpropionic acids 14 and
5, obtained in two steps from O-benzyloxyisovanillin 10 and vanillin 11 respectively, and leading to
1
the synthesis of the phenylpyrrolizinones 16, 17 (Scheme 2). The latter, as well as the unsubstituted
compound 5 were then involved in an aldolisation step with various benzaldehydes yielding compounds
1
8-28. Their benzyloxy groups (except 18) were cleaved using HBr in acetic acid to give the hydroxy
derivatives 29-38. Compound 39 was obtained from the O-demethylation of 38 which subsequently took
place after the O-debenzylation of 28, the reaction leading to a mixture of the two compounds which
had to be separated. Finally the tetrahydroxy derivative 40 was obtained from the O-demethylation of 34
3
under treatment with BBr in refluxing dichloromethane.
O
NH2
O
N
O
R1
R1
R2
H
a
OH
b
R1
R2
OH
R2
6
: R = R = H
7 : R = R = H
8 : R = R = H
1 2
1
2
1
2
1
0 : R = OBn, R = OMe
12 : R = OBn, R = OMe
14 : R = OBn, R = OMe
1 2
1
2
1
2
1
1 : R = OMe, R = OBn
13 : R = OMe, R = OBn
15 : R = OMe, R = OBn
1 2
1
2
1
2
c
N
N
N
O
O
R1
R
1
O
R1
R2
e
d
R2
R2
R3
R3
R6
R
6
R4
R
4
5 : R = R = H
1 2
R5
9-39
R
5
1
6 : R = OBn, R = OMe
1 2
f
2
29
40
18-28
17 : R = OMe, R = OBn
1 2

ACCEPTED MANUSCRIPT
Starting
material
Yield
(%)
Compd
R1
R2
R3
R4
R5
R6
8
8
9
7
6
9
9
9
8
8
5
5
8
8
5
7
5
6
5
4
4
1
7
0
4
2
0
4
4
1
0
4
7
0
3
0
0
5
6
6
0
3
7
7
5
1
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
5
H
H
H
H
H
H
H
H
5
H
H
OBn
OBn
OBn
OMe
OBn
OBn
OMe
OBn
OMe
OMe
OH
OBn
OMe
OBn
OBn
OMe
OBn
OBn
OMe
H
5
H
H
H
H
16
16
16
17
17
17
17
17
19
20
21
22
23
24
25
26
27
28
28
34
OBn
OBn
OBn
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OBn
OBn
OBn
OBn
OBn
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
OMe
H
OMe
OH
H
H
H
H
OH
OMe
OH
H
OH
OMe
OMe
OMe
OH
OH
OH
OH
OH
OH
OH
H
OH
H
OH
H
OMe
OH
OH
H
OH
H
OMe
OH
H
OMe
OMe
OMe
OMe
OMe
OMe
OH
H
OH
H
H
OMe
OH
OH
H
H
OMe
H
H
H
OMe
OMe
OH
OMe
H
OMe
OMe
H
OMe
OMe
OH
H
OH
H
Reagents and conditions: a) (CH
AcOH, 60°C, 1h30, 56-68%; c) triphosgen, toluene, 110°C, 5 days (sealed tube), 40-60%; d)
Benzaldehyde, NaOH, H O, MeOH, room temperature, 1 h, 75-95%; e) HBr, AcOH, room temperature,
h, 70-80%; f) BBr , DCM, reflux, 30 mn, 71%.
2 2 2 4
) CO H, AcONH , EtOH, reflux 8-24 h, 23-70%; b) 2,5-diOMeTHF,
2
1
3
Scheme 2. Synthesis of phenylpyrrolizinones 5,16,17 and benzylidenephenylpyrrolizinones 18-40.
All the benzylidene derivatives were selectively obtained as E-isomers. The latter was attested
by NOE experiments and confirmed by X-ray diffraction analyses (Figure 2).

ACCEPTED MANUSCRIPT
Figure 2. ORTEP diagram of compound 31.
3. Biology
All the O-deprotected benzylidenephenylpyrrolizinones 29-40 as well as the unsubstituted
derivative 18 and the tribenzyloxy compound 24 were tested in vitro in order to evaluate their potential
antioxidant, metal chelator and anti β-amyloid aggregation properties.
3.1. Antioxidant activities
The antioxidant properties of the tested compounds were evaluated by their capacity to exert
high radical scavenging activities and to inhibit FeCl -induced lipoperoxidation (Table 1).
2
The radical scavenging activity of the tested compounds was first established using 2,2-
diphenyl-1-picrylhydrazyl (DPPH) with curcumin, trolox and ferulic acid as references.[68] Some of the
tested derivatives interacted with DPPH with EC50 values ranging from 9.6 to 101.3 µM. The best
results were obtained for compounds 29, 31, 34 and 40 whose EC are close to 10 µM and therefore are
5
0
more active than the reference compounds. They all possess a catechol-type benzylidene pattern
which seems to play an important role in the activity. Conversely, the sustituents on the phenyl rings do
not appear as crucial for the activity.

ACCEPTED MANUSCRIPT
The NO scavenging activity of the tested compounds was then evaluated through the inhibition
of the production of NO generated by the interaction of sodium nitroprusside in aqueous solution at
physiological pH with oxygen and measured by the Greiss reagent.[69] Curcumin, ferulic acid and
quercetin were used as references. The most active compound was 29 with a NO scavenging activity
(65%) close to quercetin (70%).
The lipoperoxidation phenomena exert important influences on the pathogenesis of AD. A
β
causes lipoperoxidation of membranes and induces lipid peroxidation products. Lipids are modified by
reactive oxygen species (ROS) and the correlations among lipid peroxides, antioxidant enzymes, senile
plaques and neurofibrillary tangles (NFT) in AD brains are very strong. It was furthermore shown that
lipid peroxidation is a major cause of depletion of membrane phospholipids in AD.[70] On the other
hand, excess concentration of radicals, such as nitric oxide (NO), shows neurotoxicity and acts as a
pathological mediator in pathophysiological process in AD. Indeed, oxygen reacts with the excess NO
to generate nitrite and peroxynitrite anions, which act as free radicals.[71]
Lipid peroxidation assay was carried out by using the linoleic system according to the ferric
thiocyanate method.[72] The best result was obtained with compound 40 which exerted a lipid
peroxidation protection similar to those of ferulic acid used as a reference (47%). This result appeared
correlated with the radical scavenging activity of this catechol-type compound.
Radical scavenging
activity (DPPH test)
EC [µM]
NO scavenging Lipid peroxidation
activity (%)
[40µM]
n = 3
(%) after 2 h
[40µM]
Compd
5
0
n = 3
n = 3
Curcumin
Trolox
19.2 ± 3.3
60.4 ± 4.2
-
-
18.2 ± 1.6
43.2 ± 3.3
-
-
Ferulic acid
Quercetin
46.4 ± 3.9
70.7 ± 4.5
34.4 ± 3.2
30.7 ± 1.5
65.6 ± 2.2
51.7 ± 5.8
57.5 ± 3.0
53.7 ± 1.8
47.0 ± 1.7
-
18
24
29
30
31
32
nd
55.1 ± 5.2
92.8 ± 4.5
57.0 ± 2.5
67.4 ± 7.2
51.3 ± 4.1
71.0 ± 6.5
> 250
12.6 ± 1.6
> 250
12.8 ± 2.8
> 250

ACCEPTED MANUSCRIPT
33
34
35
36
37
38
39
40
99.5 ± 13.1
55.1 ± 5.1
58.0 ± 0.9
57.7 ± 3.2
57.0 ± 0.6
40.3 ± 2.7
32.6 ± 4.6
35.3 ± 2.5
47.1 ± 2.4
56.4 ± 3.8
50.6 ± 6.6
58.2 ± 6.8
48.5 ± 6.9
67.5 ± 7.5
67.1 ± 4.0
70.4 ± 3.3
47.0 ± 6.7
12.0 ± 0.9
> 250
101.3 ± 23.2
> 250
> 250
246 ± 9.7
9.6 ± 1.0
Table 1. Antioxidant activities of curcumin, trolox, ferulic acid, quercetin and compounds 18, 24, 29-40
nd : not determined).
(
3
.2. Metal chelation
The increased level of oxidative stress in AD brain is reflected by the increased brain content of
iron (Fe) and copper (Cu) both able to stimulate free radical formation (e.g. hydroxyl radicals via Fenton
reaction), to enhance protein and DNA oxidation and lipid peroxidation.[73] Moreover, Cu(II) was
detected in vivo at elevated concentrations in amyloid plaques, suggesting that it was bound to A
β
peptides.[74] From that, development of new chemical entities able to chelate in the brain Cu(II) or
other metal dications as well as Fe(II) or Zn(II) appears to be a valuable therapeutic target against
AD.[75] Previous studies showed the possibility to investigate the complexation reactivity of drugs
using UV-visible spectral changes of an organic compound incubated with a metal dication. Some of
these studies allowed to obtain only qualitative results, [76] whereas some others delivered quantitative
data through the determination of dissociation constant.[77,78] Thus, the chelating activity of our novel
compounds was explored using these two approaches. First, a qualitative assay was performed on all
compounds in order to select active chelator compounds among all newly synthesized derivatives. On
the basis of the first results, four derivatives were selected and their reactivity with Cu(II) were deeply
explored to determine the stoichiometry and dissociation constant of the reaction.
Thus, the UV-Visible spectral changes of compounds 29-40 at 50 µM in presence of Cu(II) or
Fe(II) (50 µM in methanol) were established (Table 2).

ACCEPTED MANUSCRIPT
Compd
Cu (II)
Fe (II)
curcumine
+
+
-
+
+
nd
+
+
+
+
-
2
3
3
3
3
3
3
3
3
3
3
4
9
0
1
2
3
4
5
6
7
8
9
0
+
+
+
+
-
+
-
+
-
-
-
-
-
+
+
Table 2 : Chelating ability screening of curcumine and compounds 29-40 using UV-Visible
spectrophometry (nd : not determined).
Significant spectral changes were measured for curcumin as already reported by Baum [79] and also for
compounds 29, 31- 34, 36 and 40. As an illustration of positive and negative results, figure 3 shows
spectral curves obtained for 29 and 38 respectively.
A
B
Figure 3 : UV-visible spectra of compounds 29 (A) and 38 (B) alone and in presence of Cu and Fe
dications.
Considering the antioxidant activity expressed by the catechol-type derivatives 31, 34, 40 and
29, they were deeply investigated to determine their stoichiometric coefficient and their dissociation

ACCEPTED MANUSCRIPT
constant with Cu(II) in Methanol-Tris pH 7.4 buffer. A first assay was conducted at a fixed
concentration of compound (50 µM) with increasing concentration (0 to 200 µM) of Cu(II) (see for
example 29 in Figure 4). For all the considered compounds, an hypochromic effect at 384 nm and a
new absorbance band at around 448 nm appeared and grew up when Cu(II) concentration increased.
Such a band appeared to be very specific of Cu(II)-compound complex formation. This was illustrated
for compound 29 in Figure 4A. This test allowed us to determine a specific wavelength for each
complex formation (29: 448 nm; 31: 476 nm; 34: 500 nm; 40: 476 nm). Considering that at these
specific wavelengths it was possible to study the influence of Cu(II) concentration on the complex
formation, these compounds were considered for Job plots assay and for dissociation constant
determination.
A
B
Figure 4 : A) UV-visible spectra of 29 at 50 µM with rising concentration (0 to 200 µM) of Cu(II); B)
Variation of absorbance at 384 and 448 nm versus Cu(II) concentration.
For all the tested compounds, as illustrated for 29 in Figure 4B, above 100 µM of Cu(II) added,
the signal became stable and remains also stable at higher concentrations (500, 1000, 5000 and 10 000
µM of Cu (II), results not shown). It appears from this result that, above 100 µM Cu(II), 100 % of
compound 29 was implied in complex formation, and from the presence of an isobestic point at 404 nm,
only one type of complex product is present. Similar results were obtained with 31, 34 and 40.

ACCEPTED MANUSCRIPT
Using method of continuous variation (MCV, Job plots) it was then possible to determine the
stoichiometry of the reaction (Figure 5, Table 2).
Figure 5 : Job plots of compound 29.
Contrarily to saturation curve, in MCV method, the addition of ligand (i.e., studied compound)
and receptor (i.e., Cu(II)) concentration was fixed (100 µM) and the relative mole fraction of the two
reagents varied).[80] From results, 31 revealed a stoichiometry of 2:1 with Cu(II) whereas for
compounds 29, 34 and 40, reactions appeared equimolar as illustrated for 29 in Figure 5.
A fixed concentration (50 µM) of Cu(II), considered as receptor, was then incubated with
growing concentrations of the tested compounds (0 to 200 µM). Difference of absorbance monitored in
absence versus presence of Cu(II) was determined at the previously fixed wavelength to extract specific
signal of the Cu(II)-compound complex. Scatchard analysis of these data provided same results for
stoichiometry than Job plots (data not shown). The dissociation constant (Kd) and the logarithm of the
association constant (log K ) were established using non-linear regression (Table 3). From the results, it
f
appears that in comparison with other compounds, the Cu(II)-29 bond was significantly stronger.
Compd
Job’s plots
Kd
Log Kf
stoichiometry
(µM)
2
+
X : Cu
29
31
34
40
1 : 1
2.2
5.65
4.15
4.65
4.70
2 : 1
1 : 1
1 : 1
70.5
22.1
19.9

ACCEPTED MANUSCRIPT
Table 3 : Stoichiometry, dissociation constant and logarithm of association constant of complexation
reaction between compounds 29, 31, 34, 40 and Cu(II).
3
.3. Self-mediated Aβ1-42 aggregation inhibition
The ability of the pyrrolizinones to inhibit the self-mediated Aβ1-42 aggregation was assessed
using the thioflavin (ThT) fluorescence assay with curcumin as standard (Table 4).[39] The results
showed that some tested compounds exhibited moderate potencies compared to that of curcumin
(68.9%). Concerning the SAR, the analysis of these results allows some assertions. The nature of the
substituents on the benzylidene ring appears more critical for the activities than those of the phenyl ring.
Indeed, compound 29, devoid of substituents on the phenyl ring, remains active. Conversely the absence
of substituents on the benzylidene ring (18) or the only presence of O-benzyl protecting groups (24),
without hydroxyl one, dramatically decreases the activity. Curiously, the presence of either a vanillyl-
(
36) or isovanillyl-type (30, 32, 35) catechol benzylidene pattern such as in curcumine structure, did not
afford the best results, the most active compounds remaining the catechol- (29, 34, 40), syringyl- (31,
9) or trimethoxyphenyl-type (38) catechol benzylidene derivatives whatever the substituents on their
pending phenyl ring. The best results were obtained with 39, 29 and 40 (% inhibition = 30.1, 28.7 and
7.0 respectively).
3
2
inhibition of A 1
β
aggregation
-42
Compd
(%) [10 µM]
n = 1 triplicate
Curcumin
68.9 ± 4.9
1.4 ± 1.4
18
24
29
30
31
32
33
34
35
4.2 ± 3.7
28.7 ± 3.3
14.5 ± 1.7
24.9 ± 4.2
8.4 ± 3.1
12.8 ± 1.2
26.1 ± 7.1
15.9 ± 1.3

ACCEPTED MANUSCRIPT
3
3
3
3
4
6
10.4 ± 2.1
6.0 ± 10.3
23.6 ± 0.6
30.1 ± 0.1
27.0 ± 3.1
7
8
9
0
Table 4. Aggregation inhibition activities.
4
. Drugability
In view of performing additional in vivo tests upon the synthesized compounds, we undertook
the study of their drugability and especially the evaluation of their cytoxicity, their capacity to cross the
gastrointestinal and blood-brain barriers and their chemical stability.
4
.1. Cytotoxicity
The cytotoxicity of the synthesized compounds was evaluated against KB cells, using curcumin
as a reference. None of the tested derivatives exhibited a significant cytotoxicity in this model, the more
-5
cytotoxic derivatives (18, 37) remaining two time less active (near 40% inhibition at 10 M) in regard to
curcumin (83%) (Table 5).
4
.2. PAMPA assays
Parallel artificial membrane permeability assay (PAMPA) is a high-throughput technique
commonly used in drug discovery to predict passive permeability through biological membranes.[82,83]
Thus, the ability of the new compounds to cross the gastrointestinal tract (GIT), and to penetrate the
Blood-Brain-Barrier (BBB) was evaluated by PAMPA-GIT and PAMPA-BBB assays, respectively.
From results (Table 5), it appears that, except 24 and to a lesser degree 40, all compounds could be well
absorbed after oral administration, and could penetrate into the CNS and reach their biological targets
located in the CNS.
Cytotoxicity -
−1
−1
5
-log Pe (cm s ) for -log Pe (cm s ) for
PAMPA-BBB assay PAMPA-GIT assay
Compd
Inhibition % at 10 M
KB cells) n = 3
(

ACCEPTED MANUSCRIPT
Curcumin
83 ± 1
4.6 ± 0.0
4.5 ± 0.1
10.0 ± 0.0
4.1 ± 0.0
4.2 ± 0.1
4.4 ± 0.1
4.3 ± 0.1
4.3 ± 0.1
4.8 ± 0.1
4.7 ± 0.0
4.3 ± 0.0
4.6 ± 0.1
4.7 ± 0.0
4.8 ± 0.0
5.4 ± 0.1
4.3 ± 0.2
1
2
2
3
3
3
3
3
3
3
3
3
3
4
8
4
9
0
1
2
3
4
5
6
7
8
9
0
41 ± 9
0 ± 18
0 ± 3
4.0 ± 0.0
10.0 ± 0.0
3.9 ± 0.0
3.9 ± 0.0
4.4 ± 0.0
3.9 ± 0.0
3.9 ± 0.0
4.9 ± 0.1
4.4 ± 0.3
3.9 ± 0.0
3.9 ± 0.1
3.9 ± 0.0
3.9 ± 0.0
5.7 ± 0.1
24 ± 6
0 ± 3
24 ± 3
29 ± 4
0 ± 24
0 ± 9
0 ± 4
38 ± 14
nd
0 ± 7
0 ± 14
Table 5. Cytotoxicity towards KB cells and PAMPA BBB and GIT assays results for curcumin and
compounds 18, 24, 29-40.
4
.3. Chemical stability
Considering chemical stability in physiological condition could be a discriminant parameter for
our compounds. Then the chemical stability of the four catechol-type benzylidene derivatives (29, 31,
3
4, 40) was investigated. A 20 µM solution was incubated at 25°C in a 10 mM phosphate buffer at pH
7
.4 with ionic strength fixed at 150 mM. Solutions was analysed using UHPLC with PDA detector.
Product disappearance was plotted versus time during 5 - 8 hours, and half-life was calculated from
exponential regression (Table 6). Only derivative 29 was stable after a 5 hours incubation.
Compd
Calculated
half-life (hour)
11.9
R²
3
3
4
2
1
4
0
9
> 0.99
> 0.99
> 0.99
-
6.8
6.4
no degradation

ACCEPTED MANUSCRIPT
Table 6: Chemical stability of compounds 31, 34, 40, 29 in pH 7.4 buffer.
5
. Discussion and conclusion
We have performed, during this work, the synthesis of fourteen novel benzylidene-
phenylpyrrolizinones whose antioxidant, metal chelating and anti Aβ aggregation activities were
evaluated in various tests to potentially attest their therapeutic interest in AD treatment. Some of these
compounds exerted interesting antioxidant and chelating activities and promising self-mediated Aβ1-42
aggregation inhibitory activity.
If we consider the compounds exhibiting the best results in each test, it seems possible to
establish some global structure-activity relationships on the basis of the overall results. Indeed some
compounds were found exhibiting the best results in several tests, accounting for common
pharmacophoric features for the various considered activities. The analysis of these results allows some
assertions. The nature of the substituents on the benzylidene ring appears more critical for the activities
than those of the phenyl ring. Indeed, compound 29, without substituents on the phenyl ring, remains
active as radical and NO scavenger and Aβ aggregation inhibitor. Conversely, the absence of
substituents on the benzylidene ring (18) or the only presence of benzyloxy groups (24) or methoxy
groups (37, 38), without hydroxyl one, dramatically decreases all the activities, except for the Fe
chelating activity of 24 and 38 which remains high. A vanillyl (36), isovanillyl (35) or syringyl-type
benzylidene group (39), consequently, appears important for the activities, on the condition that a
vanillyl-type phenyl group was associated to the structure. Indeed, compounds bearing such a
benzylidene group but with an additional unsubstituted (30) or isovanillyl-type phenyl group (32, 33)
are almost devoid of activities. Finally, the best results were globally obtained with the compounds
bearing a catechol-type benzylidene group (29, 31, 34, 40) whose activities are preserved whatever the
nature of the pending phenyl group.

ACCEPTED MANUSCRIPT
Among them, compound 29 further exhibits good predictive drugability parameters in term of
permeability, lack of cytotoxicity and chemical stability which design it as a promising hit for in vivo
investigations.
7
7
. Experimental section
.1. General
Melting points were determined on a Köfler melting point apparatus and are uncorrected. IR
1
spectra were recorded on a Perkin Elmer BX FT-IR apparatus using KBr pellets. The H (400 MHz) and
1
3
C (100 MHz) NMR spectra were obtained on a Jeol Lambda 400 spectrometer using DMSO-d6 or
CDCl as solvent and TMS as internal standard. The chemical shifts (δ) are reported in ppm, and the
3
coupling constants are in Hertz. Reactions were monitored by LC/MS and stopped when all starting
material had disappeared. LC/MS (ESI) analyses were realized with a Waters alliance 2695 as
separating module using the following gradient: A (95%)/B (5%) to A (5%)/B (95%) in 10 min. This
ratio was hold for 3 min before return to initial conditions in 1 min. Initial conditions were then
maintained for 5 min (A: H O, B: CH CN; each containing HCOOH: 0.1%; Column: C18 Xterra
2
3
MSC118/2.1_50 mm). MS detection was performed with a SQ Detector by positive ESI. High-
resolution mass spectra (EIMS) were obtained using a Jeol JMS GCMate spectrometer. High-resolution
mass spectra were performed at 70 eV by electronic impact. Reactions were monitored by thin-layer
chromatography (TLC) using 0.2 mm Polygram Sil silica gel G/UV 254 precoated plates with
visualisation by irradiation with a short-wavelength UV light. Silica gel flash chromatography was
performed using 63-200 mM Kieselgel Merck 60 silica gel.
PAMPA, DPPH and chelating activity assays were performed from a single DMSO (Fisher
Scientific, Illkirch, France) stock solution at 50 mM of newly synthesized compounds in order to
consume the least amount of product.

ACCEPTED MANUSCRIPT
All tested compounds for their antioxidant activity were solubilized in ethanol at 100 µM. The
analyses were performed in triplicate and the results were averaged.
7
7
.2. Chemistry
.2.1. General procedure (A) to obtain benzylated products
Unprotected starting material (1 Eq), benzyl bromide (1.2 Eq) and dipotassic carbonate (1,2 Eq)
-1
in MeOH (5 mL.mmol ) were stirred at reflux for 2 hours. The reaction mixture was filtered and
-1
-1
evaporated to vacuo, taken up with DCM (15 mL.mmol ) and washed by water (3 × 1 mL.mmol ).
Organic layer was dried over MgSO and evaporated in vacuo to give the expected protected product.
4
7
.2.2. General procedure (B) to obtain beta-aminopropionic acids
Protected aldehyde (1 Eq) was suspended in hot EtOH (2.5 mL.mmol ). After return to room
temperature, malonic acid (1 Eq) and ammonium acetate (2 Eq) were added and the mixture was stirred
-1
-1
at reflux for 24 hours before being immediately filtered and washed by hot EtOH (5 mL.mmol ).[84]
7
.2.3. General procedure (C) to obtain pyrrolyl propanoic acids
Appropriate
β-amino propionic acid (1 Eq) and 2,5-dimethoxytetrahydrofuran (1 Eq) were
-1
suspended in acetic acid (8 mL.mmol ) and heated at 60 °C for 1.5 hour. After return to room
-1
temperature, the mixture was evaporated in vacuo, taken up with DCM (8 mL.mmol ) and washed by
-1
HCl 2M (3 × 1 mL.mmol ). Organic layer was dried under MgSO and evaporated in vacuo. The crude
4
was purified on silica gel column (Cyclohexane/EtOAc; 9:1).
7
.2.4. General procedure (D) to obtain phenylpyrrolizinones

ACCEPTED MANUSCRIPT
-1
Pyrrolyl propanoic acid (1 Eq.) was dissolved in toluene (25 mL.mmol ) and stirred at 0 °C
under N flux then we added triphosgen (1/3 Eq.) and the tube was sealed and stirred at 110 °C for 5
2
days. After return to room temperature, the mixture was evaporated to dryness, taken up with DCM (50
-1
-1
mL.mmol ) and washed by a solution of saturated K CO (3 × 20 mL.mmol ). Organic layer was dried
2
3
under MgSO then evaporated in vacuo and was purified on silica gel column (cyclohexane/EtOAc;
4
8
:2).
7
.2.5. General procedure (E) for aldolisation
Phenylpyrrolizinone (1 Eq) and appropriated aldehyde (1 Eq) were suspended in a mixture of
-1
MeOH (8 mL.mmol ) and 5N aqueous NaOH (2Eq) and heated at 60 °C for 1.5 hour. After return to
-1
room temperature, the mixture was evaporated in vacuo, taken up with DCM (8 mL.mmol ) and
-1
washed by HCl 2M (3×1 mL.mmol ). Organic layer was dried under MgSO and evaporated in vacuo.
4
The crude was purified on silica gel column (cyclohexane/EtOAc; 9:1).
7
.2.6. General procedure (F) to obtain unprotected benzylidenephenylpyrrolizinones
Benzylated product (1 Eq) was stirred in bromhydric acid 33 % diluted in acetic acid (8
-1
mL.mmol ) at room temperature for 1.5 hour before ice was introduced in the flask. The mixture was
-1
extracted by EtOAc (8 mL.mmol ). Organic layer was dried under MgSO and evaporated in vacuo.
4
The crude was purified on silica gel column (cyclohexane/EtOAc; 1:1).
7
.2.7. 3-Phenyl-3-pyrrol-1-yl-propanoic acid (7) [65]
Using general procedure (C), starting from commercial 3-amino-3-phenylpropanoic acid 6 (1.0
1
g) to afford 7 (1.1 g, 80 %) as a white solid. Rf = 0.60 (1:1/cyclohexane:EtOAc); mp = 120 °C; H NMR
(400 MHz, CDCl3): δ = 7.32 (m, 3 H), 7.16 (m, 2 H), 6.75 (m, 2 H), 6.19 (t, Ј = 2 Hz, 2 H), 5.65 (m, 1

ACCEPTED MANUSCRIPT
-1
H), 3.24 (m, 2 H) ppm; IR (KBr):
;
ν = 2959, 2927, 1712, 1518, 1490, 1190, 1083, 848, 724 cm Anal.
calc. for C H N O : C, 72.54; H, 6.09; N, 6.51. Found: C, 72.67; H, 6.04; N, 6.47.
1
2
12
2
4
7
.2.8. 3-Phenyl-2,3-dihydropyrrolizin-1-one (5)
Using general procedure (D), starting from 7 (100 mg) to afford 5 (47.6 mg, 52 %) as a white
1
solid. [65] mp = 104 °C; H NMR (400 MHz, CDCl
3
):
δ
= 7.35 (m, 3 H), 7.12 (m, 2 H), 6.85 (d, J = 2
= 8; 4 Hz, 1 H), 3.54 (dd, J
= 2924, 1698, 1527, 1370, 1309, 1065,
47 cm ; Anal. Calc. for C H NO: C, 79.17; H, 5.62; N, 7.10. Found : C, 79.11; H, 5.70; N, 7.13.
Hz, 1 H), 6.80 (dd, J = 4; 1 Hz, 1 H), 6.55 (dd, J = 4; 2 Hz, 1 H), 5.54 (dd,
18; 8 Hz, 1 H), 2.96 (dd, J = 18; 4 Hz, 1 H) ppm; IR (KBr):
Ј
=
ν
-1
7
13
11
7
.2.9. 3-Benzyloxy-4-methoxy-benzaldehyde (10)
Using general procedure (A), starting from commercial iso-vanilline to afford 10 in quantitative
1
yield as a pale yellow powder. Rf = 0.57 (1:1/cyclohexane:EtOAc); mp = 67 °C; H NMR (400 MHz,
1
3
CDCl3):
δ
= 9.81 (s, 1 H), 7.39 (m, 7H), 6.98 (dd, J= 8; 1 Hz, 1 H), 5.18 (s, 2 H), 3.95 (s, 3 H) ppm; C
= 190.8, 154.9, 148.6, 136.2, 129.9, 128.5 (2 C), 128.0, 127.4 (2 C), 126.8,
11.2, 110.7, 70.7, 56.1 ppm; IR (KBr): = 3342, 2933, 2837, 2748, 1677, 1583, 1506, 1431, 1235,
009, 735 cm ; HREIMS [M+H ] m/z 243.101444 (calcd for C H O 243.101571).
NMR (100 MHz, CDCl3):
δ
1
1
ν
−
1
+
1
5
14
3
7
.2.10. 4-Benzyloxy-3-methoxy-benzaldehyde (11)
Using general procedure (A), starting from commercial vanillin to afford 11 in quantitative yield
1
as pale yellow powder. Rf = 0.57 (1:1/cyclohexane:EtOAc); mp = 63 °C; H NMR (400 MHz, CDCl ) :
3
1
3
δ
= 9.84 (s, 1 H), 7.40 (m, 7H), 6.99 (d, J = 8 Hz, 1 H), 5.25 (s, 2 H), 3.95 (s, 3 H) ppm; C NMR (100
MHz, CDCl3): = 190.9, 153.6, 150.1, 136.0, 130.3, 128.7 (2 C), 128.2, 127.3 (2 C), 126.6, 112.4,
09.4, 70.9, 56.0 ppm; IR (KBr):
δ
−
1
1
ν = 3412, 2839, 1676, 1538, 1260, 1133, 989, 812, 747, 561 cm .

ACCEPTED MANUSCRIPT
7
.2.11. 3-Amino-3-(3-benzyloxy-4-methoxy-phenyl)propanoic acid (12)
Using general procedure (B), starting from 10 (33.0 g) to afford 12 (23.8 g, yield 58 %) as a
1
white solid. mp = 266 °C; H NMR (400 MHz, CD OD):
δ
= 7.39 (m, 5 H), 7.14 (s, 1 H), 7.04 (m, 2 H),
3
1
3
5
.12 (s, 2 H), 4.60 (dd, J = 8; 6 Hz, 1 H), 3.86 (s, 3 H), 2.96 (m, 2 H) ppm; C NMR (100 MHz,
CD OD): = 173.2, 152.1, 150.0, 138.3, 129.7, 129.5 (2 C), 129.1, 128.8 (2 C), 121.6, 114.6, 113.5,
2.3, 57.6, 52.9, 39.1 ppm; IR (KBr): = 3436, 3031, 2930, 2834, 1571, 1522, 1395, 1271, 1144, 1021,
94 cm ; HREIMS [M+H ] m/z 302.138747 (calc. for C17 302.138685).
δ
3
7
6
ν
−
1
+
H20NO
4
7
.2.12 3-amino-3-(4-benzyloxy-3-methoxy-phenyl)propanoic acid (13)
Using general procedure (B), starting from 11 (9.90 g) to afford 13 (9.30 g, 75 %) as a white
1
solid. mp = 258 °C; H NMR (400 MHz, DMSO-d6, 383 K):
δ = 7.37 (m, 5 H), 7.06 (d, J = 2 Hz, 1 H),
6
2
1
1
.97 (d, J = 8 Hz, 1 H), 6.87 (dd, J = 8; 2 Hz, 1 H), 5.07 (s, 2 H), 4.17 (t, J = 8 Hz, 1 H), 3.81 (s, 3 H),
1
3
.45 (d, 2 H) ppm; C NMR (100 MHz, DMSO-d6, 383 K):
δ
= 172.9, 150.2, 147.9, 138.0, 137.9,
= 2937,
630, 1580, 1539, 1520, 1272, 1147, 1026, 693 cm ; HREIMS [M+H ] m/z 302.138159 (calc. for
28.7 (2 C), 128.1, 127.9 (2 C), 119.2, 115.5, 112.5, 79.6, 71.4, 56.7, 52.2 ppm; IR (KBr):
ν
−
1
+
C H NO 302.138685).
17
20
4
7
.2.13 3-(3-Benzyloxy-4-methoxy-phenyl)-3-pyrrol-1-yl-propanoic acid (14)
Using general procedure (C), starting from 12 (3.47 g), to afford 14 (2.27 g, 56 %) as a white
1
solid. Rf = 0.57 (1:1/cyclohexane:EtOAc); mp = 105 °C; H NMR (400 MHz, CDCl ):
δ
7.34 (m, 5 H),
3
6
.82 (d,
t,
CDCl3):
Ј
= 8 Hz, 1 H), 6.74 (dd,
Ј
= 8; 2 Hz, 1 H), 6.65 (d,
Ј
= 2 Hz, 1H), 6.64 (t, Ј = 2 Hz, 2 H), 6.13
1
3
(
Ј
= 2 Hz, 2 H), 5.51 (m, 1 H), 5.06 (s, 2 H), 3.85 (s, 3 H), 3.11 (m, 2 H) ppm; C NMR (100 MHz,
δ
= 175.5, 149.4, 148.0, 136.6, 132.5, 128.5 (2 C), 127.9, 127.5 (2 C), 119.4, 118.9, 112.5,
11.6, 108.5, 71.0, 70.9, 58.5, 56.0, 55.9, 40.7 ppm; IR (KBr): = 3468, 2046, 1638, 1516, 1427, 1260,
139, 728, 697 cm ; HREIMS [M+H ] m/z 352.154187 (calc. for C21 352.154335).
1
1
ν
-1
+
H22NO
4

ACCEPTED MANUSCRIPT
7
.2.14. 3-(4-Benzyloxy-3-methoxy-phenyl)-3-pyrrol-1-yl-propanoic acid (15)
Using general procedure (C), starting from 14 (2.64 g) to afford 15 (2.59 g, 85 %) as a white
1
solid. Rf = 0.60 (1:1/cyclohexane:EtOAc); mp = 100 °C; H NMR (400 MHz, CDCl ):
δ
= 7.35 (m, 5
= 2 Hz, 2 H), 5.58 (m, 1 H), 5.13 (s, 2
H), 3.82 (s, 3 H), 3.20 (m, 2 H) ppm; C NMR (100 MHz, CDCl ): = 175.9, 149.8, 148.0, 136.9,
33.2, 128.6 (2 C), 127.9, 127.2 (2 C), 119.6 (2 C), 118.3, 113.8, 110.4, 108.7 (2 C), 71.0, 58.7, 56.0,
3
H), 6.82 (m, 1 H), 6.73 (t, Ј = 2 Hz, 1 H), 6.68 (m, 2 H), 6.17 (t, Ј
1
3
δ
3
1
4
-1
+
0.9 ppm; IR (KBr):
ν = 3037, 2924, 1701, 1518, 1421, 1230, 1143, 1005, 728 cm ; HREIMS [M+H ]
m/z 352.154171 (calc. for C H NO 352.154335).
2
1
22
4
7
.2.15. 3-(3-Benzyloxy-4-methoxy-phenyl)-2,3-dihydropyrrolizin-1-one (16)
Using general procedure (D), starting from 14 (500 mg) to afford 16 (220 mg, 46 %) as a white
1
solid. mp = 127 °C; H NMR (400 MHz, CDCl
3
):
= 2 Hz, 1 H), 6.50 (m, 1 H), 5.40 (dd,
H), 3.88 (s, 3 H), 3.46 (dd, J = 18; 8 Hz, 1 H), 2.85 (dd, J = 18; 4 Hz, 1 H) ppm; C NMR (100 MHz,
CDCl3): = 188.3, 149.9, 148.5, 136.4, 133.0, 132.7, 128.5 (2 C), 127.9, 127.4 (2 C), 122.5, 119.1,
17.3, 111.8, 111.5, 107.5, 71.0, 57.8, 55.9, 49.6 ppm; IR (KBr): = 3479, 1682, 1519, 1368, 1258,
230, 1067, 741 cm ; HREIMS [M+H ] m/z 334.143544 (calc. for C H NO 334.143770).
δ
= 7.30 (m, 5 H), 6.85 (d,
Ј
= 8 Hz, 1 H), 6.78 (d,
Ј =
4
Hz, 1 H), 6.72 (m, 2 H), 6.51 (d,
Ј
Ј = 8; 4 Hz, 1 H), 5.06 (m, 2
1
3
δ
1
1
ν
-1
+
2
1
20
3
7
.2.16. 3-(4-Benzyloxy-3-methoxy-phenyl)-2,3-dihydropyrrolizin-1-one (17)
Using general procedure (D), starting 15 (0.50 g) to afford 17 (240 mg, 50 %) as a white solid.
1
mp = 121 °C; H NMR (400 MHz, CDCl ):
δ
= 7.37 (m, 5 H), 6.85 (d,
= 8; 2 Hz, 1 H), 6.48 (d, J = 2 Hz, 1 H), 6.46 (dd, J = 4; 2 Hz, 1
= 8; 4 Hz, 1 H), 5.08 (s, 2 H), 3.75 (s, 3 H), 3.46 (dd, J = 18; 8 Hz, 1 H), 2.85 (dd, J =
Ј = 8 Hz, 1 H), 6.83 (m, 1 H),
3
6
.72 (dd, J = 4; 1 Hz, 1 H), 6.56 (dd,
H), 5.40 (dd,
8; 4 Hz, 1 H) ppm; C NMR (100 MHz, CDCl ):
Ј
Ј
1
3
1
δ
= 188.4, 150.3, 148.4, 136.8, 133.4, 133.2, 128.6
3
(2 C), 127.9, 127.2 (2 C), 122.6, 118.6, 117.4, 114.0, 109.1, 107.6, 71.0, 58.5, 56.1, 49.7 ppm; IR

ACCEPTED MANUSCRIPT
-1
+
(
KBr):
ν 3345, 3111, 1695, 1517, 1370, 1258, 1267, 1070, 747 cm ; HREIMS [M+H ] m/z 334.143504
(
calc. for C H NO 334.143770).
2
1
20
3
7
.2.17. (2E)-2-Benzylidene-3-phenyl-3H-pyrrolizin-1-one (18)
Using general procedure (E), starting from 5 (135 mg) and benzaldehyde (73 mg) to afford 18 as
1
a yellow solid (170 mg, 80 %). mp = 178 °C; H NMR (400 MHz, CDCl
3
):
δ
= 7.64 (d, J= 2 Hz, 1 H),
1
3
7
.22 (m, 10 H), 6.85 (m, 2 H), 6.40 (m, 1 H), 6.28 (d, J = 2 Hz, 1 H) ppm; C NMR (100 MHz,
CDCl3):
δ
= 179.3, 138.8, 137.8, 133.4, 133.3, 133.2, 130.6, 129.5 (2 C), 129.1, 128.9 (2 C), 128.4 (2
= 3434, 2924, 1688, 1630,1524, 1366, 1306,
119, 744, 697 cm ; HREIMS [M+H ] m/z 286.122461 (calc. for C H NO 286.122641).
C), 127.5 (2 C), 121.2, 117.2, 108.9, 62.1 ppm; IR (KBr):
ν
-
1
+
1
2
0
16
7
.2.18. (2E)-2-[(3,4-Dibenzyloxyphenyl)methylene]-3-phenyl-3H-pyrrolizin-1-one (19)
Using general procedure (E), starting from 5 (100 mg) and 3,4-dibenzyloxybenzaldehyde
synthesized from commercially available 3,4-dihydroxybenzaldehyde[85] (161 mg) to afford 19 as a
1
yellow solid (210 mg, 84 %). mp = 115 °C; H NMR (400 MHz, CDCl ):
δ = 7.62 (d, J= 2 Hz, 1 H),
3
7
1
.34 (m, 15 H), 6.99 (dd, J = 8; 2 Hz, 1 H), 6.91 (dd, J = 4; 1 Hz, 1 H), 6.86 (m, 2 H), 6.82 (d, J = 8 Hz,
H), 6.45 (dd, = 4; 2 Hz, 1 H), 6.23 (d, J = 2 Hz, 1 H), 5.12 (m, 2 H), 4.88 (d, J = 12 Hz, 1 H), 4.71
Ј
1
3
(
d, J = 12 Hz, 1 H), ppm; C NMR (100 MHz, CDCl ):
δ
= 179.6, 150.5, 148.6, 138.1, 136.7, 136.6,
35.5, 133.6, 133.2, 129.5 (2 C), 129.1, 128.6 (4 C), 128.0, 127.9, 127.6 (2 C), 127.2 (2 C), 127.1 (2 C),
26.8, 126.3, 120.8, 116.9, 116.4, 113.9, 108.6, 71.2, 70.8, 62.2 ppm; IR (KBr): = 3435, 1678, 1620,
513, 1453, 1367, 1268, 1132, 734, 695 cm ; HREIMS [M+H ] m/z 498.206378 (calc. for
3
1
1
1
ν
-1
+
C H NO 498.206370).
34
28
3
7
.2.19. (2E)-2-[(4-Benzyloxy-3-methoxy-phenyl)methylene]-3-phenyl-3H-pyrrolizin-1-one (20)

ACCEPTED MANUSCRIPT
Using general procedure (E), starting from 5 (133 mg) and 11 (163 mg) to afford 20 (261 mg,
1
9
2 %) as a yellow powder. mp = 140 °C; H NMR (400 MHz, CDCl ):
δ
= 7.65 (d, J = 1 Hz, 1 H), 7.34
3
(
m, 10 H), 6.96 (dd, J = 8; 2 Hz, 1 H), 6.93 (d, J = 2 Hz, 1 H), 6.86 (m, 1 H), 6.77 (d, J = 8 Hz, 1 H),
.75 (d, J = 1 Hz, 1 H), 6.45 (dd, J = 4; 2 Hz, 1 H), 6.29 (d, = 1 Hz, 1 H), 5.12 (s, 2 H), 3.60 (s, 3 H)
= 179.6, 149.7, 149.1, 138.0, 136.4, 135.6, 133.8, 133.1, 129.4 (2
C), 129.1, 128.6 (2 C), 128.0, 127.5 (2 C), 127.1 (2 C), 126.6, 125.8, 120.8, 116.9, 113.2, 112.9, 108.6,
6
Ј
1
3
ppm; C NMR (100 MHz, CDCl ):
δ
3
-1
7
0.6, 62.2, 55.9 ppm; IR (KBr):
ν
= 3005, 2946, 2849, 1681, 1521, 1266, 1115, 999, 738, 698 cm ;
+
HREIMS [M+H ] m/z 422.174942 (calc. for C28
H
24NO
3
422.175070).
7
.2.20.
(2E)-3-(3-Benzyloxy-4-methoxy-phenyl)-2-[(3,4-dibenzyloxyphenyl)
methylene]-3H-
pyrrolizin-1-one (21)
Using general procedure (E), starting from 16 (94 mg) and 3,4-dibenzyloxybenzaldehyde
synthesized from commercially available 3,4-dihydroxybenzaldehyde (90 mg) to afford 21 yellow solid
1
(
126 mg, 70 %). mp = 167 °C; H NMR (400 MHz, CDCl
3
):
δ
= 7.56 (d, J= 2 Hz, 1 H), 7.20 (m, 15 H),
6
.97 (dd, J = 8; 2 Hz, 1 H), 6.94 (dd, J = 8; 2 Hz, 1 H), 6.87 (d, J = 2 Hz, 1 H), 6.83 (dd, J = 4; 1 Hz, 1
H), 6.79 (m, 2 H), 6.76 (d, J = 2 Hz, 1 H), 6.70 (m, 1 H), 6.40 (dd, = 4; 2 Hz, 1 H), 6.10 (d, J = 2 Hz, 1
H), 5.13 (m, 2 H), 5.03 (d, J = 12 Hz, 1 H), 4.95 (d, J = 12 Hz, 1 H), 4.86 (d, J = 12 Hz, 1 H), 4.67 (d, J
Ј
13
=
12 Hz, 1 H), 3.80 (s, 3 H) ppm; C NMR (100 MHz, CDCl ): δ = 179.6, 150.5, 150.3, 148.6, 148.5,
3
1
1
1
1
36.8, 136.7, 136.4, 135.8, 133.6, 133.0, 130.3, 128.6 (2 C), 128.6 (2 C), 128.5 (2 C), 128.0 (2 C),
27.9, 127.3 (2 C), 127.2 (2 C), 127.1 (2 C), 126.8, 126.3, 120.7, 120.6, 116.8, 116.6, 113.9, 113.1,
11.9, 108.5, 71.2, 70.9, 70.8, 61.8, 56.0 ppm; IR (KBr):
ν
= 3435, 2926, 1691, 1511, 1360, 1278, 1140,
-1
+
014, 729, 696 cm ; HREIMS [M+H ] m/z 634.258839 (calc. for C42 634.258800).
H
36NO
5
7
3
.2.21.
(2E)-3-(3-Benzyloxy-4-methoxy-phenyl)-2-[(3-benzyloxy-4-methoxy-phenyl)methylene]-
H-pyrrolizin-1-one (22)

ACCEPTED MANUSCRIPT
Using general procedure (E), starting from 16 (100 mg) and 10 (72 mg) to afford 22 as a yellow
1
solid (106 mg, 64 %). mp = 162 °C; H NMR (400 MHz, CDCl ):
δ = 7.58 (d, J= 2 Hz, 1 H), 7.29 (m,
3
1
0 H), 6.98 (m, 2 H), 6.83 (dd, J = 4; 1 Hz, 1 H), 6.79 (m, 4 H), 6.71 (dd, J = 2; 1 Hz, 1 H), 6.40 (dd, Ј
=
4; 2 Hz, 1 H), 6.10 (d, J = 2 Hz, 1 H), 5.02 (d, J = 12 Hz, 1 H), 4.94 (d, J = 12 Hz, 1 H), 4.84 (d, J =
1
3
1
2 Hz, 1 H), 4.64 (d, J = 12 Hz, 1 H), 3.85 (s, 3 H), 3.80 (s, 3 H) ppm; C NMR (100 MHz, CDCl ): δ
3
=
179.8, 151.3, 150.4, 148.6, 148.1, 136.6, 136.5, 135.6, 133.9, 133.1, 130.4, 128.8 (2 C), 128.7 (2 C),
28.2, 128.0, 127.6 (2 C), 127.5 (2 C), 126.5, 126.4, 120.9, 120.8, 116.9, 115.6, 113.2, 112.0, 111.4,
08.6, 71.1, 71.0, 61.9, 56.1, 56.0 ppm; IR (KBr): = 3431, 2915, 1688, 1522, 1332, 1266, 1117, 1014,
33 cm ; HREIMS [M+H ] m/z 558.228392 (calc. forC36 558.227500).
1
1
7
ν
-1
+
H32NO
5
7
3
.2.22.
(2E)-3-(3-Benzyloxy-4-methoxy-phenyl)-2-[(4-benzyloxy-3-methoxy-phenyl)methylene]-
H-pyrrolizin-1-one (23)
Using general procedure (E), starting from 16 (114 mg) and 11 (83 mg) to afford 23 as a yellow
1
solid (180 mg, 94 %). mp = 146 °C; H NMR (400 MHz, CDCl
3
):
δ
= 7.58 (d, J = 2 Hz, 1 H), 7.30 (m,
1
6
0 H), 6.98 (dd, J = 8; 2 Hz, 1 H), 6.91 (dd, J = 8; 2 Hz, 1 H), 6.83 (m, 2 H), 6.79 (d, J = 2 Hz, 1 H),
.71 (m, 2 H), 6.69 (m, 1 H), 6.40 (dd, = 4; 2 Hz, 1 H), 6.15 (d, J = 2 Hz, 1 H), 5.12 (m, 2 H), 5.06 (d,
Ј
1
3
J = 12 Hz, 1 H), 4.97 (d, J = 12 Hz, 1 H), 3.83 (s, 3 H), 3.60 (s, 3 H) ppm; C NMR (100 MHz,
CDCl3): = 179.8, 151.3, 150.4, 148.6, 148.1, 136.6, 136.5, 135.6, 133.9, 133.1, 130.4, 128.8 (2 C),
28.7 (2 C), 128.2, 128.0, 127.6 (2 C), 127.5 (2 C), 126.5, 126.4, 120.9, 120.8, 116.9, 115.6, 113.2,
12.0, 111.4, 108.6, 71.1, 71.0, 61.9, 56.1, 56.0 ppm; IR (KBr): = 3431, 2932, 1680, 1513, 1306,
260, 1139, 1005, 742, 693 cm ; HREIMS [M+H ] m/z 558.227078 (calc. for C36 558.227500).
δ
1
1
1
ν
-1
+
H32NO
5
7
.2.23.
(2E)-3-(4-Benzyloxy-3-methoxy-phenyl)-2-[(3,4-dibenzyloxyphenyl)
methylene]-3H-
pyrrolizin-1-one (24)

ACCEPTED MANUSCRIPT
Using general procedure (E), starting from 17 (200 mg) and 3,4-dibenzyloxybenzaldehyde
synthesized from commercially available 3,4-dihydroxybenzaldehyde (210 mg) to afford 24 as a yellow
1
solid (345 mg, 91 %). mp = 64 °C; H NMR (400 MHz, CDCl ):
δ
= 7.58 (d, J = 2 Hz, 1 H), 7.29 (m, 15
3
H), 6.97 (dd, J = 8; 2 Hz, 1 H), 6.92 (dd, J = 8; 2 Hz, 1 H), 6.88 (dd, J = 2; 1 Hz, 1 H), 6.87 (d, J = 2 Hz,
H), 6.83 (dd, = 4; 1 Hz, 1 H), 6.79 (m, 2 H), 6.68 (d, J = 2 Hz, 1 H), 6.43 (dd, J = 4; 2 Hz, 1 H), 6.13
d, J = 2 Hz, 1 H), 5.11 (m, 2 H), 5.08 (s, 2 H), 4.84 (d, J = 12 Hz, 1 H), 4.71 (d, J = 12 Hz, 1 H), 3.67
1
Ј
(
(
1
3
s, 3 H); C NMR (100 MHz, CDCl
3
):
35.9, 133.7, 133.1, 130.9, 128.6 (2 C), 128.6 (2 C), 128.5 (2 C), 128.0 (2 C), 127.3 (2 C), 127.2 (2 C),
27.1 (2 C), 126.8, 126.5, 120.9, 120.4, 116.9, 116.6, 113.8 (2 C), 110.1, 108.5, 71.3, 70.9, 70.8, 61.9,
δ = 179.6, 150.6, 150.4, 148.7, 148.6, 136.9, 136.8, 136.7, 136.6,
1
1
5
-1
6.1 ppm; IR (KBr):
ν = 3434, 2923, 1685, 1624, 1511,1453, 1269, 1142, 1024, 735 cm ; HREIMS
+
[
M+H ] m/z 634.258067 (calc. for C H NO 634.258800).
4
2
36
5
7
3
.2.24.
(2E)-3-(4-Benzyloxy-3-methoxy-phenyl)-2-[(3-benzyloxy-4-methoxy-phenyl)methylene]-
H-pyrrolizin-1-one (25)
Using general procedure (E), starting from 17 (100 mg) and 10 (80 mg) to afford 25 (153 mg, 90
1
%
) as a yellow powder. mp = 87 °C; H NMR (400 MHz, CDCl ):
δ
= 7.62 (d, J = 2 Hz, 1 H), 7.29 (m,
3
1
6
0 H), 7.03 (dd, J = 8; 2 Hz, 1 H), 6.93 (dd, J = 8; 2 Hz, 1 H), 6.90 (dd, J = 2; 1 Hz, 1 H), 6.86 (m, 1 H),
.84 (dd, J = 4; 1 Hz, 1 H), 6.82 (d, J = 8 Hz, 1 H), 6.79 (d, J = 9 Hz, 1 H), 6.70 (d, J = 2 Hz, 1 H), 6.45
(dd, J = 4; 2 Hz, 1 H), 6.15 (d,
Ј = 2 Hz, 1 H), 5.05 (s, 2 H), 4.86 (d, J = 12 Hz, 1 H), 4.71 (d, J = 12 Hz,
1
3
1
1
1
5
H), 3.86 (s, 3 H), 3.68 (s, 3 H) ppm; C NMR (100 MHz, CDCl ):
δ = 179.8, 151.3, 150.4, 148.6,
3
48.1, 136.6, 136.5, 135.6, 133.9, 133.1, 130.4, 128.8 (2 C), 128.7 (2 C), 128.2, 128.0, 127.6 (2 C),
27.5 (2 C), 126.5, 126.4, 120.9, 120.8, 116.9, 115.6, 113.2, 112.0, 111.4, 108.6, 71.1, 71.0, 61.9, 56.1,
-1;
+
6.0 ppm; IR (KBr): ν = 2925, 2849, 1686, 1513, 1366, 1272, 1236, 1025, 735 cm HREIMS [M+H ]
m/z 558.227446 (calc. for C H NO 558.227500).
3
6
32
5

ACCEPTED MANUSCRIPT
7
3
.2.25.
(2E)-3-(4-Benzyloxy-3-methoxy-phenyl)-2-[(4-benzyloxy-3-methoxy-phenyl)methylene]-
H-pyrrolizin-1-one (26)
Using general procedure (E), starting from 17 (90 mg) and 11 (72 mg) to afford 26 (127 mg, 84
1
%
) as a yellow powder. mp = 77 °C; H NMR (400 MHz, CDCl ):
δ
= 7.64 (d, J = 2 Hz, 1 H), 7.35 (m,
3
1
1
Ј
0 H), 7.00 (dd, J = 8; 2 Hz, 1 H), 6.96 (dd, J = 8; 2 Hz, 1 H), 6.92 (dd, J = 2; 1 Hz, 1 H), 6.86 (m, 1 H,
H), 6.84 (d, J = 8 Hz, 1 H), 6.77 (d, J = 4 Hz, 1 H), 6.76 (m, 2 H), 6.45 (dd, J = 4; 2 Hz, 1 H), 6.21 (d,
1
3
3
= 2 Hz, 1 H), 5.12 (m, 4 H), 3.71 (s, 3 H), 3.55 (s, 3 H) ppm; C NMR (100 MHz, CDCl ): δ = 179.8,
1
1
7
51.3, 150.4, 148.6, 148.1, 136.6, 136.5, 135.6, 133.9, 133.1, 130.4, 128.8 (2 C), 128.7 (2 C), 128.2,
28.0, 127.6 (2 C), 127.5 (2 C), 126.5, 126.4, 120.9, 120.8, 116.9, 115.6, 113.2, 112.0, 111.4, 108.6,
1.1, 71.0, 61.9, 56.1, 56.0 ppm; IR (KBr):
ν = 3438, 2927,1684, 1511, 1366, 1268, 1236, 1021, 732
-1
+
cm ; HREIMS [M+H ] m/z 558.228555 (calc. for C H NO 558.227500).
3
6
32
5
7
.2.26.
(2E)-3-(4-Benzyloxy-3-methoxy-phenyl)-2-[(2,4-dimethoxyphenyl)
methylene]-3H-
pyrrolizin-1-one (27)
Using general procedure (E), starting from 17 (100 mg) and commercial 2,4-
1
dimethoxybenzaldehyde (50 mg) to afford 27 (yellow solid (125 mg, 87 %). mp = 72 °C; H NMR (400
MHz, CDCl3):
J = 2 Hz, 1 H), 6.76 (d, J = 8 Hz, 1 H), 6.61 (d, J = 2 Hz, 1 H), 6.43 (dd,
Hz, 1 H), 6.26 (dd, = 8; 2 Hz, 1 H), 6.18 (d, J = 2 Hz, 1 H), 5.06 (s, 2 H), 3.79 (s, 3 H), 3.76 (s, 3 H),
δ
= 7.99 (d, J = 2 Hz, 1 H), 7.33 (m, 5 H), 7.09 (d, J = 8 Hz, 1 H), 6.85 (m, 2 H), 6.80 (d,
Ј
= 4; 2 Hz, 1 H), 6.32 (d, J = 2
Ј
1
3
3
1
1
1
.69 (s, 3 H) ppm; C NMR (100 MHz, CDCl ):
δ
= 178.9, 161.4, 158.9, 148.9, 147.3, 135.8, 135.6,
3
32.6, 130.7, 130.2, 127.6, 127.5 (2 C), 126.9, 126.2 (2 C), 119.7, 119.4, 115.5, 114.7, 112.7, 109.3,
07.1, 103.6, 97.0, 69.9, 61.2, 54.9, 54.5, 54.4 ppm; IR (KBr):
ν
= 3434, 2926, 2850, 1685, 1603, 1512,
-1
+
462, 1263, 1140, 1024, 742 cm ; HREIMS [M+H ] m/z 482.196603 (calc. for
C H NO 482.196199).
30
28
5

ACCEPTED MANUSCRIPT
7
.2.27.
(2E)-3-(4-Benzyloxy-3-methoxy-phenyl)-2-[(3,4,5-trimethoxyphenyl)
methylene]-3H-
pyrrolizin-1-one (28)
Using general procedure (E), starting from 17 (50 mg) and commercial 3,4,5-
1
trimethoxybenzaldehyde (30 mg) to afford 28 yellow solid (38 mg, 50 %). mp = 68 °C; H NMR (400
MHz, CDCl3):
Hz, 1 H), 6.87 (dd, J = 4; 1 Hz, 1 H), 6.85 (d, J = 8 Hz, 1 H), 6.74 (d, J = 2 Hz, 1 H), 6.55 (s, 2 H), 6.47
dd, J = 4; 2 Hz, 1 H), 6.21 ( d, J = 2 Hz, 1 H), 5.15 (m, 2 H), 3.83 (s, 3 H), 3.74 (s, 3 H), 3.61 (s, 6 H)
δ = 7.63 (d, J = 2 Hz, 1 H), 7.35 (m, 5 H), 7.01 (dd, J = 8; 2 Hz, 1 H), 6.93 (dd, J = 2; 1
(
1
3
ppm; C NMR (100 MHz, CDCl
32.9, 131.0, 128.9, 128.6 (2 C), 128.0, 127.1 (2 C), 121.1, 120.3, 117.1, 113.9, 109.9, 108.8, 108.5 (2
C), 70.8, 61.9, 60.9, 56.1 (3 C) ppm; IR (KBr): = 3435, 2933, 2837, 1688, 1626, 1505, 1461, 1332,
239, 1123, 1025, 744 cm ; HREIMS [M+H ] m/z 512.207468 (calc. for C H NO 512.206764).
3
): δ = 179.1, 153.0 (2 C), 150.5, 148.7, 139.6, 136.8, 136.6, 133.9,
1
ν
-1
+
1
31
30
6
7
.2.28. (2E)-2-[(3,4-Dihydroxyphenyl)methylene]-3-phenyl-3H-pyrrolizin-1-one (29)
Using general procedure (F), starting from 19 (180 mg) to afford 29 (60 mg, 53 %) as a yellow
1
powder. mp > 260 °C; H NMR (400 MHz, CD
3
OD):
δ
= 7.49 (d, J = 2 Hz, 1 H), 7.32 (m, 5 H), 7.08
= 2 Hz,
= 180.7, 147.8, 144.9, 138.2,
35.7, 134.1, 132.9, 128.7 (2 C), 128.4, 127.4 (2 C), 125.0, 124.2, 121.9, 117.8, 116.8, 114.9, 108.2,
(
dd, J = 2; 1 Hz, 1 H), 6.90 (d, J = 2 Hz, 1 H), 6.83 (m, 2 H), 6.64 (d, J = 8 Hz, 1 H), 6.55 (d, Ј
1
3
1
1
6
H), 6.49 (dd, J = 4; 2 Hz, 1 H) ppm; C NMR (100 MHz, CD OD): δ
3
-1
1.8 ppm; IR (KBr):
ν = 3467, 1671, 1592, 1521, 1461, 1362, 1283, 1108, 1032, 735 cm ; HREIMS
+
[
M+H ] m/z 318.112239 (calc. for C H NO 318.112470).
2
0
16
3
7
.2.29. (2E)-2-[(4-hydroxy-3-methoxy-phenyl)methylene]-3-phenyl-3H-pyrrolizin-1-one (30)
Using general procedure (F), starting from 20 (40 mg) to afford 30 as a pale yellow solid (25 mg,
1
8
0 %). mp = 208 °C; H NMR (400 MHz, CDCl ) :
δ
= 7.65 (d, J = 1 Hz, 1 H), 7.34 (m, 5 H), 6.96 (dd,
3
J = 8; 2 Hz, 1 H), 6.93 (d, J = 2 Hz, 1 H), 6.86 (m, 1 H), 6.80 (d, J = 8 Hz, 1 H), 6.73 (d, J = 1 Hz, 1 H),