Antioxydant activity of β-carboline derivatives in the LDL oxidation model

Article abstract of DOI:10.1016/j.ejmech.2011.03.048

A series of β-carboline compounds were synthesized, starting from compound GWC22, their antioxidant activity was determined by inhibition of lipid peroxidation. The oxidation of LDL was induced in the presence of CuSO ₄ or 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The protective actions of these compounds against the cytotoxicity were evaluated with lactate dehydrogenase (LDH) activity in bovine aortic endothelial cells (BAECs) and cellular vitality by measuring mitochondrial activity in the presence of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Most of compounds showed an higher antioxidant activity than GWC22 derivative (R = 1.6 for 5 μM CuSO₄). The best antioxidant activities are phenolic and benzyloxy derivatives with ratio R = 1.9 to 2.8 for 1 μM CuSO₄. These substances have protective actions and increase significantly the cell viability.

Full text of DOI:10.1016/j.ejmech.2011.03.048

European Journal of Medicinal Chemistry 46 (2011) 2575e2585
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antioxydant activity of b-carboline derivatives in the LDL oxidation model
Fariza Hadjaz a, Soizic Besret a, Françoise Martin-Nizard b, Saïd Yous a, Sébastien Dilly a, Nicolas Lebegue a,
,
Philippe Chavatte a, Patrick Duriez c, Pascal Berthelot a, Pascal Carato a
*
a Université Lille Nord de France, Faculté de Pharmacie, Laboratoire de Chimie Thérapeutique (EA 4481) 3, rue du Professeur Laguesse, BP 83, 59006 Lille, France
b Université Lille Nord de France, Inserm, U1011, UDSL, Institut Pasteur de Lille, Faculté de Pharmacie BP 83, 59006 Lille, France
c Université Lille Nord de France, Faculté de Pharmacie, EA 1046, Département de Pharmacologie Médicale, 59000 Lille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of b-carboline compounds were synthesized, starting from compound GWC22, their antioxidant
Received 15 September 2010
Received in revised form
16 March 2011
Accepted 22 March 2011
Available online 30 March 2011
activity was determined by inhibition of lipid peroxidation. The oxidation of LDL was induced in the
presence of CuSO4 or 2,20-azobis(2-amidinopropane) dihydrochloride (AAPH). The protective actions of
these compounds against the cytotoxicity were evaluated with lactate dehydrogenase (LDH) activity in
bovine aortic endothelial cells (BAECs) and cellular vitality by measuring mitochondrial activity in the
presence of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Most of compounds
showed an higher antioxidant activity than GWC22 derivative (R ¼ 1.6 for 5
m
M CuSO4). The best anti-
M CuSO4. These
Keywords:
oxidant activities are phenolic and benzyloxy derivatives with ratio R ¼ 1.9 to 2.8 for 1
m
b
-Carboline
substances have protective actions and increase significantly the cell viability.
Antioxidant
Inhibition LDL peroxidation
Melatonin
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
It is well known that lipoprotein oxidation is a key early stage in
the development of atherosclerosis. Modification of Low-Density
Lipoprotein (LDL) in the arterial wall, is crucial to the cellular
uptake of LDL in the first stages of atherosclerotic plaque devel-
opment [11].
Various reactive oxygen species (ROS), such as superoxide
anion, hydrogen peroxide and oxygen-derived free radicals, are
very important mediators of cell injury; they act on biological
macromolecules [1e3]. Under normal conditions, cells and tissues
are protected against ROS by an array of nonenzymatic and
enzymatic defense systems, such as superoxide dismutase, cata-
lase, and glutathione peroxidase or free radical scavengers [4].
Oxidative stress, however, occurring when antioxidant systems
are inadequate and/or active oxygen species are overproduced,
can damage the tissues and DNA, thus resulting in a progression
of a number of human diseases. It has been proposed that ROS are,
directly or indirectly, involved in a variety of pathology events
such as myocardial and cerebral ischemia, arteriosclerosis, dia-
betes, rheumatoid arthritis, inflammation, cancer-initiation, Alz-
heimer’s and other neurodegenerative diseases as well as in the
ageing process [5e9]. Hence, free radical scavengers are expected
to be promising drugs for treatment of these diseases by scav-
enging free radicals and therefore removing oxidative stress.
The role of antioxidants in human health and disease has been
reviewed [10].
Once initiated, oxidation of LDL is a free-radical-driven lipid
peroxidation chain reaction. Lipid peroxidation is initiated by free
radical attack on a double bond associated with a polyunsaturated
fatty acid (PUFA). These results in the removal of a hydrogen atom
from a methylene (CH2) group, the rate of which determines the
rate of initiation, a key step. Molecular rearrangement of the
resulting unstable carbon radical results in a more stable configu-
ration, a conjugated diene. The conjugated diene reacts very quickly
with molecular oxygen, and the peroxyl radical thus formed is
a crucial intermediate [12]. A PUFA peroxyl radical in LDL may
abstract a hydrogen atom from an adjacent PUFA to form a hydro-
peroxide and another lipid radical, a reaction which results in chain
propagation. Removal of hydrogen atoms by the peroxyl radical
from other lipids, including cholesterol, eventually yields oxy-
sterols. Lipid hydroperoxides fragment to shorter-chain aldehydes,
including malondialdehyde and 4-hydroxynonenal. These reactive
aldehydes in turn may bind to e-amino groups of apo B-100, giving
the protein an increased net negative charge. The classical LDL
receptor recognizes a specific domain of positive charges from
lysine, arginine and histidine residues on apo B. Alteration of this
domain results in failure of binding by the apo B/E receptor, and an
* Corresponding author. Tel.: þ33 20964966; fax. þ33 20 964913.
E-mail address: pascal.carato@univ-lille2.fr (P. Carato).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.048

2576
F. Hadjaz et al. / European Journal of Medicinal Chemistry 46 (2011) 2575e2585
nonyl, which could influence both the lipophilic and steric
parameters. We have also introduced propionyl and benzyl carba-
mate groups to study the importance of the basic nitrogen.
Therefore, we synthesized various novel compounds and per-
formed a wide range of screening tests for the purpose of devel-
oping an antioxidant that inhibits lipid peroxidation.
2. Results and discussion
Fig. 1. Design of b-carboline derivatives starting from GWC22.
2.1. Chemistry
increase in negative surface charge on apo B-100 results in
increased recognition by the scavenger receptor (mainly expressed
in the macrophage). It’s the first step of the atheroma formation.
A variety of compounds have been found to inhibit LDL oxida-
Final compounds 6aes were synthesized starting from key
intermediate products (4aeb) [33,37] (Scheme 1). The first step
was a diazotation reaction [38] in concentrated hydrochloric acid
with 4-ethylaniline (1), as commercial material, and sodium
nitrite, followed by a reduction of the diazonium intermediate salt
with tin chloride to afford 4-ethylphenyl hydrazine 2. Compound 2
was cyclised, by a fischer reaction [39], in a diluted aqueous
solution of sulfuric acid (4%) with 4,4-diethyloxy butyronitrile to
give (5-ethyl-1H-indol-3-yl)acetonitrile (3a). Reduction of
compounds 3a and 3b, as commercial product, in ethyl ether with
lithium aluminium hydride furnished the intermediate derivatives
4a,b.
tion, including vitamin E, b-carotene [13,14], the drugs probucol
[15], indapamide [16], lovastatin [17], propranolol [18], pindolol
[19], carvedilol [20], red wine polyphenols [21], and the hormone
melatonin [22e27].
Investigations revealed that pineal indoles can have similar or
more potent antioxydant effects than those observed with mela-
tonin [28,29]. Indeed, pinoline (6-methoxy-1,2,3,4-tetrahydro-b-
carboline) a minor cyclic metabolite of melatonin [30] has been
shown to inhibit lipid peroxidation in different model systems
[31,32]. In our laboratory, previous research reported that a series of
Compound 4a or 4b was engaged in a PicteteSpengler reaction
[40] in CH2Cl2, with TFA and the desired benzaldehyde to afford
1-aryl-1,2,3,4-tetrahydro-b-carboline display antioxidant proper-
ties [33]. In this study, the substitution of the methoxy group at the
6 position of Pinoline, by an ethyl group reinforced the antioxidant
activity of the series. In particular, compound GWC22 has been
shown to display better antioxidant properties than melatonin
towards peroxyl-induced peroxidation of a linoleate model system
[34,35]. Compound GWC22, used as reference, allowed to described
a series of benzazolone derivatives with antioxidant activity [36].
In this paper, we report the synthesis of novel antioxidants,
which have been rationally designed to present high reactivity and
efficacy against LDL oxidation. Starting from compound GWC22, we
developed and synthesized a series with three modulations a) at
b
-carboline derivatives 5aej,m. Debenzylation reaction of deriva-
tives 5i,j in methanol with Pd/C under hydrogen atmospheric
pressure, afforded derivatives 5k,l. Compounds 5aej,m were then
alkylated in CH3CN with the desired halogeno reagent and potas-
sium carbonate to furnish alkyl products (Scheme 2). Other tested
derivatives 6e,n,o were transformed in amide or carbamate, start-
ing from compounds 5a,i,j in ethyl acetate with the corresponding
chloride reagents. Protected benzyl derivatives 6l,m,r were placed
in methanol with Pd/C, under hydrogen atmospheric pressure to
give the corresponding phenols 6p,q,s. Compound 6s was the
6-hydroxy analogue of GWC22 derivative.
the 6 position of the
groups (R1) b) various substituents (R2) on the phenyl ring at the 1
position of the -carboline c) at the nitrogen atom (R3) of the
-carboline (Fig. 1). Most of the synthetised and tested compounds
have an ethyl group (R1) at the 6 position of the -carboline, we
have also introduced a benzyloxy and hydroxy groups to explore
the antioxidant activities of these compounds. On the phenyl ring,
with R2 group, we have modulated the position and the number of
the methoxy group and introduced other groups such as 3,5-
ditertBu-4eOH, benzoxy and hydroxy. It is well known that
phenolic compounds exert their antioxidant activity by acting
primarily as hydrogen atom donators, thereby inhibiting the
propagation of radical chain reactions. The antioxidant potential of
the phenolic derivatives depends on the number and the arrange-
ment of hydroxyl groups, as well as the nature of the other
substituents on the aromatic rings. For the third modulation with
R3 we have studied the length chain with methyl, propyl, pentyl,
b-carboline with ethyl, hydroxy or benzoxy
2.2. Antioxidant activity
b
b
Products can exert their antioxidant activity through various
mechanisms, such as i) scavenging radicals which initiate lipid
peroxidation and lipid peroxide radicals; ii) binding metal ions; iii)
inhibiting enzymatic systems responsible for free radical genera-
tion. To eliminate the possible role of Cu-chelation, oxidation was
induced by water- soluble 2,20-azobis(2-amidinopropane) dihy-
drochloride (AAPH), which generates free radical, by its sponta-
neous thermal decomposition. For some selected samples we have
compared the capacity of compounds to inhibit LDL oxidation when
Cu2þ or AAPH are used as pro-oxidants. The oxidation of LDL was
followed for 480min by measuring conjugated diene formation at
optical density (OD) 234 nm. LDL samples were subjected to
oxidation immediately after isolation and each experiment con-
sisted of 3 samples, which included 1 controls and 1 test.
b
CN
NH2
R 1
R 1
b
c
a
NH2
N
H
N
H
NH2
N
H
4a : R1
= C2H5
3a : R1
= C2H5
1
2
4b : R1 = OBn
3b : R1 = OBn
Reagents and conditions: (a) : i: NaNO2, HCl 6N ii: SnCl2, HCl 6N (b): 4,4-diethyloxy butyronitrile, H2SO4 4%
(c): LiAlH4, ethyl ether.
Scheme 1. Preparation of the intermediate derivatives 4a,b.

F. Hadjaz et al. / European Journal of Medicinal Chemistry 46 (2011) 2575e2585
2577
Scheme 2. Preparation of b-carboline derivatives 5aem and 6aes.
In the presence of CuSO4 or AAPH, the OD234 curve vs. time
corresponds to the typical one described by Esterbauer [41],
including the lag, propagation, and decomposition phases. With
our experimental conditions, the lag phase for the initiation of LDL
oxidation in the presence of copper or AAPH was about
100 min ꢀ 7min. Without Cu2þ or AAPH, the kinetics of conjugated
diene formation was slower, and the final concentration was
there was a strong correlation in the lag phase duration between
these two oxidative procedures of LDL (R ¼ 0.9 p < 0.05). Therefore,
the free radical scavenging mechanism contributes towards the
inhibitory action of these compounds.
Excepted for compounds 5h, 5k and 5l, the other derivatives
5aem showed less potent antioxidant activities than reference
GWC22 (R ¼ 1.6 for 5
mM CuSO4). The ditertbutylphenol group of
dramatically lower after 24
concentrations of derivatives from 0.1 to 10
h
of oxidation. However, when
M were added to the
compound 5h, well described in the literature for its antioxidant
activities [42], and the presence of hydroxy groups on the phenyl
ring of compound 5k and 5l induced excellent antioxidant activities
m
incubations, there was an increase in lag time, suggesting an
apparent antioxidant effect. The capacity of derivatives to reduce or
inhibit copper or AAPH-initiated LDL oxidation was tested and the
kinetics of LDL oxidation led us to calculate the ED50 or the ratio R
(Tables 1 and 2).
There is no significant decrease in the maximal rate of propa-
gation phase or in maximal amount of conjugated diene formation.
The most antioxidant products in a copper system were tested in
AAPH oxidation system. In the presence of the tested molecules
(R ¼ 2.7 to 2.8 for 1
of compound 5l to dihydrobenzodioxine derivative 5g induced
M CuSO4
mM CuSO4). The cyclisation of the catechol ring
a downfall of the antioxidant activities to R ¼ 1.1 for 5
m
compared with GWC22. These results show the importance of
phenolic groups for antioxidant activity. The variation of the posi-
tion of the methoxy groups (5aec) or the introduction of other
methoxy groups (5def) was followed by a reduction of the anti-
oxidant activity with R ¼ 1.1 to 1.4 for 5
mM CuSO4.
Table 1
Antioxidant activity and LDH release of derivatives 5aem.
5a-m
NH
R 1
R1 = C2H5, OBn
R 2
N
H
R2 = 3,5-ditertBu-4-OH, OH, OCH3, OBn
.
Cpd
R1
R2
ED50
(m
M)
5
m
M (Cu2þ
)
Activity (R) 1
m
M (Cu2þ
)
1
m
M (AAPH)
LDH release (%)
GWC22
5a
5b
5c
5d
e
e
4
9
7
9
10
9
10
1.6
1.2
1.4
1.2
1.1
1.2
1.1
1.2
1.1
1.2
1
1
1
15.32 1.56
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
3eOCH3
2eOCH3
4eOCH3
3,4eOCH3
2,3,4eOCH3
e
e
e
e
e
e
e
e
e
e
e
e
5e
5f
3,4,5eOCH3
1
O
3,4-
5g
C2H5
10
1.1
1
e
e
O
5h
5i
5j
5k
5l
5m
C2H5
C2H5
C2H5
C2H5
C2H5
OBn
3,5-tBu-4eOH
3-OBn
<0.1
7
7
<0.1
<0.1
5
>5
1.4
1.4
>5
>5
1.5
2.8
1
1
2.7
2.8
1.1
2.7
1
1
2.6
2.6
e
13.26 ꢀ 1.32
13.29 ꢀ 1.35
e
3,4-OBn
3eOH
3,4eOH
3eOCH3
13.31 ꢀ 1.29
13.30 ꢀ 1.30
e
ED50: Concentration in mM of compounds that increased the control lag phase by 1.5 times. The control lag phase is obtained in an oxidative procedure without tested
compounds.
R: Ratio between the length of the lag phase of the oxidative procedure of LDL with different compounds and the length of the one without compound (control).
LDH activity was expressed as the percentage of total LDH cellular release following the addition of Triton X-100 (final concentration of 0.1% v/v in ethanol). Cells were
pretreated for 24 h with 10
mM tested compounds, then for 16h with the same concentration of compounds and 100 mg/mL of air oxidized-LDL. The LDH release in presence of
air oxidized-LDL alone is: 22.76 ꢀ 1.53%.

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F. Hadjaz et al. / European Journal of Medicinal Chemistry 46 (2011) 2575e2585
Table 2
Antioxidant activity and LDH release of derivatives 6aes.
6a-s
R 3
N
R 1
R1 = C2H5, OBn, OH
R2 = 3,5-ditertBu-4-OH, OH, OCH3, OBn
R 2
N
H
R3 = CH3, C3H7, C5H11, C9H19, COC2H5, CO2Bn
.
Cpd
R1
R2
R3
ED50
(
m
M)
5
m
M (Cu2þ
)
Activity (R) 1
m
M (Cu2þ
)
1
m
M (AAPH)
LDH release (%)
GWC22 (6a)
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
3-OCH3
C3H7
CH3
C5H11
C9H19
COC2H5
C3H7
C3H7
4
1
9
9
9
4
3
2
2
1.6
1.9
1.2
1.2
1.2
1.6
1.7
1.8
1.8
1.2
1
1
1
1
1
1
1.1
1.2
e
e
e
e
e
e
e
e
e
15.32 1.56
6b
6c
6d
6e
6f
6g
6h
6i
3eOCH3
3eOCH3
3eOCH3
3eOCH3
2eOCH3
4eOCH3
3,4eOCH3
3,4,5eOCH3
e
e
e
e
e
e
e
e
C3H7
C3H7
O
3,4-
6j
C2H5
C3H7
9
1.2
1
e
e
O
6k
6l
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
OBn
OH
3,5-tBu-4eOH
3-OBn
3,4-OBn
3-OBn
3,4-OBn
3eOH
C3H7
C3H7
C3H7
CO2Bn
CO2Bn
C3H7
C3H7
C3H7
C3H7
<0.1
0.1
3
10
8
<0.1
<0.1
0.1
0.2
>5
4.2
1.8
1
1.3
>5
>5
4.4
3.2
2.7
2.2
1.3
1
1.1
2.8
2.7
2.4
1.9
2.6
2.3
e
13.21 ꢀ 1.22
13.42 ꢀ 1.29
e
6m
6n
6o
6p
6q
6r
e
e
e
e
2.7
2.7
2.4
2.1
13.20 ꢀ 1.25
13.19 ꢀ 1.52
13.22 ꢀ 1.26
14.02 ꢀ 1.56
3,4eOH
3eOCH3
3eOCH3
6s
ED50: Concentration in mM of compounds that increased the control lag phase by 1.5 times. The control lag phase is obtained in an oxidative procedure without tested
compounds.
R: Ratio between the length of the lag phase of the oxidative procedure of LDL with different compounds and the length of the one without compound (control).
LDH activity was expressed as the percentage of total LDH cellular release following the addition of Triton X-100 (final concentration of 0.1% v/v in ethanol). Cells were
pretreated for 24 h with 10 mM tested compounds, then for 16h with the same concentration of compounds and 100 mg/mL of air oxidized-LDL. The LDH release in presence of
air oxidized-LDL alone is: 22.76 ꢀ 1.53%.
Starting from compound GWC22 (6a), we have modulated the
propyl chain by various length chain (R2) with CH3 (6b), which
induced higher antioxidant activitycompared with (6a). If R2 is C5H11
(6c) or C9H19 (6d) we observed a diminution of the antioxidant
with derivatives 6l,m indicated that benzyl group allowed to
enhance the antioxidant activity compared to dihydrobenzodioxine
derivative 6j. The best antioxidant activities of the series were
observed with phenolic compounds 6k,p,q and 5h,k,l with R ¼ 2.7
activity (R ¼ 1.2 for 5
N-propylgroupofderivativeGWC22bypropionylgroupofcompound
6e lead to a diminution of the antioxidant activity (R ¼ 1.2 for 5
CuSO4), which emphazized that the nitrogen atom at the position 2 of
the -carboline was important for the antioxidant activity.
In a second time, we have modulated the position and the
mM CuSO4). Identically, the substitution of the
to 2.8 for 1 mM CuSO4.
Considering the importance of phenolic group, we replaced the
ethyl group of derivative GWC22 by benzoxy and hydroxy group to
afford the analogs 6r,s. The N-propyl derivative 6r, with a benzoxy
group at the 6 position, gave the best antioxidant activity with
mM
b
R ¼ 2.4 for 1
6r lead to the phenolic derivative 6s, which induced a diminution of
M CuSO4, contrary to the
mM CuSO4. The debenzylation reaction with compound
number of methoxy groups (6f,g,h,i) to enhance the antioxidant
activity, however we observed similar antioxidant activity (R ¼ 1.6
the antioxidant activity with R ¼ 1.9 for 1
m
to 1.8 for 5
m
M CuSO4) compared to compound GWC22 (R ¼ 1.6 for
other series. Substitution with hydroxy or benzoxy groups induced
interesting antioxidant activities in the series with compounds 5h,
5k, 5l, 6k, 6l, 6pes.
5
m
M CuSO4). The benzoxy group on the 3 position of the phenyl
ring of compound 6l gave excellent antioxidant activity with R ¼ 4.2
for 5 M, however the disubstitution with 3,4-
M and R ¼ 2.2 for 1
benzoxy groups of derivative 6m induced a reduction of the anti-
M CuSO4. Some authors [43],
m
m
It has been proposed that ROS are, directly or indirectly, involved
in a variety of pathology events such as myocardial and cerebral
ischemia, arteriosclerosis, Alzheimer’s and other neurodegenera-
tive diseases etc. Hence, free radical scavengers are expected to be
promising drugs for treatment of these diseases by scavenging free
radicals and therefore removing oxidative stress. In a previous
paper we discussed about the relationship between melatonin
derivatives and atherosclerosis [36]. The biological properties look
important in term of prevention of cardiovascular diseases but the
capacity of molecules to inhibit atherogenic lipoprotein oxidation
in vitro offers no prediction of their capacity to inhibit in vivo
atherosclerosis development. In addition to its protective effects as
free radical scavenger and antioxidant, abundant studies showed
that melatonin or derivatives has multifaceted properties acting
oxidant activity with R ¼ 1.8 for 5
m
showed that benzyl carbamate group enhances the antioxidant
activity for described derivatives, so we have synthesized
compounds 6n,o which showed disappointing results with a lower
antioxidant activity (R ¼ 1 and 1.3 for 5
m
M CuSO4) compared to
their propyl analogs 6l,m (R ¼ 4.2 and 1.8 for 5
mM CuSO4).
The debenzylation reaction of compounds 6l,m gave phenolic
derivatives 6p,q with higher antioxidant activity, the ratio R was
superior to 5 for 5
mM and respectively 2.8 and 2.7 for 1 mM.
However, the cyclisation of the phenolic groups of derivatives 6q to
compounds 6j indiced a downfall of the antioxidant activity with
R ¼ 1.2 for 5
mM. These protections of phenolic compounds 6p,q

F. Hadjaz et al. / European Journal of Medicinal Chemistry 46 (2011) 2575e2585
2579
against cerebral hypoxia/ischemia and its protection could be long-
3. Conclusion
lasting over months. Indeed melatonin crosses the bloodebrain
barrier. Contrary to experiments made with mice to study regres-
sion of atherosclerotic plaque [44,45] administering melatonin
significantly reduces infract volume, oedema and oxidative
damage, in animal stroke models [46].
All synthetized compounds displayed antioxidant activities
superior to those of the widly studied melatonin. Some of them (5h,
5k, 5l, 6k, 6per) presented very strong antioxidant activities
compared to GWC22 derivatives. In this paper we showed the
importance of phenolic groups. The benzyloxy group (6l,r) induced
also a good antioxidant activity. For all compounds, the presence of
Hence,
b-carboline derivatives show free radical scavengers
properties. They are expected to be promising drugs for treatment
of these diseases by scavenging free radicals and therefore
removing oxidative stress.
the propyl group intheN-2 positionof the b-carbolinesisessential for
antioxidant activities, particularly for compounds 5i versus 6l. The
series, with benzyloxy and hydroxy groups at the 6 position of the
2.3. Cytotoxicity assays
b-carboline (6r,s), will be interesting to modulate on the phenyl ring.
The oxidative modification of LDL has been alleged to play an
In vivo low density protein (LDL) oxidation is a progressive
phenomenon leading to the presence of minimally and highly
oxidized LDLs in the subendothelial arterial space. Oxidized LDLs
have been reported to be cytotoxic against endothelial cells. Fur-
man [47] showed the differential toxicities of air (minimally)
or copper-oxidized-LDL toward endothelial cells. Both the
morphological aspect of the cells themselves and LDH test revealed
that air oxidized-LDLs had cytotoxicity with up to 8 h of oxidation.
The protective effect of derivatives against cytotoxics oxidized-
LDL was studied in cultured bovine aortic endothelial cells (BAECs).
This model of primary culture cells is commonly used in our
laboratory.
When the medium is cytotoxic, the initial visual change con-
sisted in cell contraction with the appearance of cytoplasmic
ramifications, then some cells rounded up and detached from the
bottom of the wells. The attached cells showed marked alterations,
such as the disappearance of the characteristic double refraction of
intact membranes and nuclear condensation. When nonoxidized
important role in the development of human pathologies Thus,
protecting LDL from oxidation by these compounds can delay or
prevent the progression of these diseases. LDL resistance to
oxidation is one of the models used by many researchers to
investigate the efficacy of new drugs as antioxidants against radi-
cals generated in the lipophilic phase.
Our results show that most antioxidant products are also the
less cytotoxic while they increase the vital capacity of the cell. They
exert their antioxidant effect on lipid peroxidation primarily by
scavenging free radicals.
However these very encouraging results do not prove a benefi-
cial effect in Vivo. Specific experiments in animals and humans are
essential.
4. Experimental
Compounds were purified on a glass column using Merck silica
gel 60 (230e400 mesh). Melting points were determined by a Büchi
510 capillary apparatus and are uncorrected. 1H and 13C NMR
proton spectra were recorded on a Bruker AVANCE 300 spectrom-
eter and chemical shifts are in ppm with TMS as internal standard.
Mass spectra were recorded on a quadripolar Finnigan Mat SSQ 710
instrument. All compounds were found to be >96% pure by HPLC
analysis.
LDL (100
shape changes are induced. After 16 h of incubation most cells
remained attached to the bottom of culture wells. If 100
mg protein/ml) is added in cellular medium, minor cellular
mg
proteins/ml air oxidized-LDL are added in cellular medium for 16 h
there are major morphological cellular changes:
In spite of oxygen is essential to life, it damages the cells and
organelles, this phenomenon is named “the oxygen paradox”. We
have pointed compounds that have both antioxidant activities but
also cytoprotective effects.
4.1. 2-(5-Benzoxy-1H-indol-3-yl) ethylamine (4b)
Cytotoxicity was quantified using LDH release. LDH release
increased when cells were treated with air oxidized-LDL in
In diethyl ether (100 mL), (5-benzoxy-1H-indol-3-yl)acetoni-
trile (3b) (4 g, 15.3 mmol), as commercial product, and lithium
aluminium hydride (2.9g, 76.5 mmol) were added. The reaction
mixture was refluxed for 1 day and hydrolysed in cold water
(50 mL). The solution was extracted with ethyl ether. The organic
layer was dried with magnesium sulfate, filtered and concentrated
under reduced pressure. The residue was purified by column
chromatography using dichloromethane/methanol (97/3, v/v) as
eluent to afford compound 4b, as an oily product. Yield 87%; 1H
comparison with non oxidized- LDL (22.76
ꢀ
1.53% versus
11.07 ꢀ 2.8 respectively). In presence of non oxidized- LDL the
b-
carboline derivatives didn’t involve any significant effect on cellular
LDH release. In presence of air oxidized-LDL, the addition of GWC
22 or other
b-carboline derivatives significantly reduced LDH
activity in the extra cellular medium. Only compounds with the
best antioxidant activities were tested.
Our results show that the most antioxidant products are also the
less cytotoxics. For exemple, for compounds GWC22 (R ¼ 1.6 for
NMR (300 MHz, CDCl3),
d
¼ 1.35 (br s, 2H, NH2), 2.90 (t, 2H, CH2,
J ¼ 6.4 Hz), 3.10 (t, 2H, CH2, J ¼ 6.4 Hz), 5.15 (s, 2H, OCH2), 6.96 (dd,
1H, H6, J ¼ 8.8 Hz, J ¼ 2.1 Hz), 7.02 (s, 1H, H2), 7.15 (d, 1H, H4,
J ¼ 2.1 Hz), 7.27 (d, 1H, H7, J ¼ 8.8 Hz), 7.40 (m, 3H, HAr), 7.40 (m, 3H,
HAr), 7.52 (d, 2H, HAr, J ¼ 7.3 Hz), 8.35 (br s, 1H, NH); 13C NMR
5
m
M Cuþþ), LDH release reaches 15.32 ꢀ 1.56%. Compounds 6p and
6p, with hydroxy group, with the best antioxidant activities (R ¼ 2.8
and 2.7 respectively for 1
13.20 ꢀ 1.25% and 13.19 ꢀ 1.52% respectively. Derivative 6r (R ¼ 2.4
for 1
M Cuþþ), with benzyloxy group, also shows good LDH release
m
M Cuþþ) shows a lower LDH release
(300 MHz, CDCl3),
d
¼ 20.1, 26.4, 69.4, 109.5, 111.1, 113.6, 114.2, 116.7,
m
122.1, 125.4, 133.5, 133.9, 134.6, 135.1. SM (APCIþ) m/z 267.5
13.22 ꢀ 1.26%.
[M þ H]þ.
In parallel, the MTT assay was performed in order to test cellular
vitality by measuring mitochondrial activity in the presence of MTT:
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).
After 16 h of incubationwith air oxidized-LDL alone MTTreactivity is
4.2. General procedure for the synthesis of b-carboline derivatives
(5aej,m)
about 60%. In presence of anti oxidant
b
-carboline derivatives the
In dichloromethane (50 mL), 2-(5-ethyl-1H-indol-3-yl) ethyl-
amine (4a) or 2-(5-benzoxy-1H-indol-3-yl) ethylamine (4b) (2.5 g,
13.3 mmol), trifluoroacetic acid (1.35 mL, 20.0 mmol) and the
desired benzaldehyde (20.0 mmol) were added. The reaction
activities reached nearly 85 ꢀ 4% without significant differences
between them Both of the tested derivatives increased (p < 0.05) the
cell viability as indicated by MTT assay.

2580
F. Hadjaz et al. / European Journal of Medicinal Chemistry 46 (2011) 2575e2585
mixture was stirred for 1 day at room temperature and then
evaporated. Ethyle acetate (80 mL) and 10% K2CO3 aqueous solution
(50 mL) were added. The organic layer was dried with magnesium
sulfate, filtered and concentrated under reduced pressure. The
residue was purified by column chromatography using dichloro-
methane/methanol (94/6, v/v) as eluent to afford compounds
(5aej). Solid products were recrystallized with the appropriate
solvent, while the oily products were solubilized in dry ethyl
acetate and treated with gazeous hydrochloric acid in diethyl ether
(20 mL). The obtained precipitate was filtered and washed with
ethyl acetate and the residue was recrystallized with the appro-
priate solvent.
4.7. 6-Ethyl-1-(2,3,4-trimethoxyphenyl)-2,3,4,9-tetrahydro-1H-b-
carboline (5e)
Yield 78% (ethanol 95ꢁ); mp 100e102 ꢁC; 1H NMR (300 MHz,
CDCl3),
d
¼ 1.35 (t, 3H, CH3, J ¼ 7.6 Hz), 1.80 (br s, 1H, NH), 2.75 (m,
3H, CH2), 2.90 (m, 1H, CH2), 3.15 (m, 1H, CH2), 3.40 (m, 1H, CH2),
3.70 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 5.60
(s, 1H, CH), 6.60 (s, 1H, HAr), 6.75 (s, 1H, HAr), 7.00 (d, 1H, H7,
J ¼ 8.5 Hz), 7.18 (d, 1H, H8, J ¼ 8.5 Hz), 7.38 (s, 1H, H5), 7.90 (br s, 1H,
NH); 13C NMR (300 MHz, CDCl3),
d
¼ 16.1, 22.1, 29.7, 41.5, 55.1, 55.3,
55.6, 57.8, 109.3, 110.4, 112.7, 114.8, 117.3, 122.8, 129.2, 130.7, 133.5,
134.8, 135.8, 158.6, 158.9, 159.4; SM (APCIþ) m/z 367.5 [M þ H]þ.
4.3. 6-Ethyl-1-(3-methoxyphenyl)-2,3,4,9-tetrahydro-1H-
b
-
4.8. 6-Ethyl-1-(3,4,5-trimethoxyphenyl)-2,3,4,9-tetrahydro-1H-b-
carboline (5a)
carboline hydrochloride (5f)
Yield 85% (ethanol 95ꢁ); mp 152e153 ꢁC; 1H NMR (300 MHz,
DMSO-d6),
J ¼ 7.6 Hz), 3.00 (m, 1H, CH2), 3.10 (m, 1H, CH2), 3.35 (m, 2H, CH2),
3.80 (s, 3H, OCH3), 5.90 (s, 1H, CH), 6.92 (d, 1H, HAr, J ¼ 7.6 Hz), 6.95
(dd,1H, H7, J ¼ 8.2 Hz, J ¼ 1.7 Hz), 7.08 (m, 2H, H5,et HAr), 7.20 (d, 1H,
H8, J ¼ 8.2 Hz), 7.33 (s, 1H, HAr), 7.40 (t, 1H, H5’, J ¼ 7.6 Hz, J ¼ 7.2 Hz),
9.50 (br s, 1H, NH), 10.80 (br s, 1H, NH); 13C NMR (300 MHz, CDCl3),
Yield 90% (methanol); mp 142e144 ꢁC; 1H NMR (300 MHz,
DMSO-d6),
d
¼ 1.20 (t, 3H, CH3, J ¼ 7.6 Hz), 2.70 (q, 2H, CH2,
d
¼ 1.20 (t, 3H, CH3, J ¼ 7.6 Hz), 2.70 (q, 2H, CH2,
J ¼ 7.6 Hz), 3.00 (m, 1H, CH2), 3.10 (m, 1H, CH2), 3.40 (m, 1H, CH2),
3.60 (m, 1H, CH2), 3.75 (s, 3H, OCH3), 3.80 (s, 6H, OCH3), 5.95 (s, 1H,
CH), 6.70 (s, 2H, HAr), 7.00 (dd,1H, H7, J ¼ 8.2 Hz, J ¼ 1.5 Hz), 7.20 (d,
1H, H8, J ¼ 8.2 Hz), 7.40 (s, 1H, H5), 9.70 (br s, 2H, NH2þ), 10.80 (br s,
1H, NH); 13C NMR (300 MHz, DMSO-d6),
d
¼ 17.1, 18.6, 28.9, 56.1,
d
¼ 16.8, 22.7, 29.3, 43.1, 50.8, 55.3, 109.5, 110.2, 110.8, 115.3, 119.8,
56.4, 56.7, 60.4, 107.3, 107.5, 111.8, 117.1, 122.9, 126.3, 128.9, 130.5,
134.9, 135.5, 138.9, 153.5, 158.9, 160.2; SM (APCIþ) m/z 367.3
[M þ H]þ.
121.5, 127.5, 129, 129.7, 130.1, 133.9, 134.3, 135.3, 158.1; SM (APCIþ)
m/z 307.3 [M þ H]þ.
4.4. 6-Ethyl-1-(2-methoxyphenyl)-2,3,4,9-tetrahydro-1H-
b
-
4.9. 1-(2,3-Dihydrobenzo [1,4]dioxin-6-yl)-6-ethyl-2,3,4,9-
carboline (5b)
tetrahydro-1H-
b-carboline hydrochloride (5g)
Yield 80% (absolute ethanol); mp 120e121 ꢁC; 1H NMR
(300 MHz, CDCl3),
Yield 80% (methanol); mp 240e241 ꢁC; 1H NMR (300 MHz,
d
¼ 1.20 (t, 3H, CH3, J ¼ 7.6 Hz), 2.70 (m, 4H,
DMSO-d6),
d
¼ 1.20 (t, 3H, CH3, J ¼ 7.6 Hz), 2.70 (q, 2H, CH2,
CH2), 2.90 (m, 2H, CH2 and NH), 3.60 (m, 1H, CH2), 3.90 (s, 3H,
OCH3), 5.50 (s, 1H, CH), 6.79 (m, 2H, HAr), 6.85 (dd, 1H, H7,
J ¼ 8.2 Hz, J ¼ 1.5 Hz), 7.05 (d, 1H, HAr, J ¼ 8.1 Hz), 7.12 (d, 1H, HAr,
J ¼ 8.1 Hz), 7.20 (s, 1H, H5), 7.26 (m, 1H, HAr), 10.30 (br s, 1H, NH);
J ¼ 7.6 Hz), 2.95 (m, 1H, CH2), 3.15 (m, 1H, CH2), 3.40 (m, 2H, CH2),
4.25 (s, 4H, OCH2CH2O), 5.80 (s, 1H, CH), 6.90 (m, 4H, H7, HAr), 7.20
(d,1H, H8, J ¼ 8.2 Hz), 7.30 (s,1H, H5), 9.40 (br s,1H, NH),10.30 (br s,
1H, NHþ), 10.70 (br s, 1H, NH); 13CNMR (300 MHz, DMSO-d6),
13C NMR (300 MHz, CDCl3),
d
¼ 16.7, 22.5, 29.1, 41.7, 50.8, 55.5,
d
¼ 17.1, 21.7, 28.9, 55.4, 64.6, 64.8, 107.4, 111.7, 114.4, 117, 117.7, 119.1,
109.9, 110.5, 110.6, 116.6, 120.6, 121.9, 127.6, 129, 129.3, 129.9, 134.3,
126.3, 128, 129.1, 134.3, 134.8, 135.4, 143.7, 144.9; SM (APCIþ) m/z
134.4, 135.1, 157.2; SM (APCIþ) m/z 307.2 [M þ H]þ.
335.1 [M þ H]þ
4.5. 6-Ethyl-1-(4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-
b
-
4.10. 6-Ethyl-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2,3,4,9-
carboline (5c)
tetrahydro-1H-
b-carboline (5h)
Yield 87% (absolute ethanol); mp 78e79 ꢁC; 1H NMR (300 MHz,
CDCl3),
Yield 60% (ethanol 95ꢁ); mp 180e181 ꢁC; 1H NMR (300 MHz,
d
¼ 1.30 (t, 3H, CH3, J ¼ 7.6 Hz), 2.70 (m, 4H, CH2), 2.90 (m,
CDCl3),
d
¼ 1.30 (t, 3H, CH3, J ¼ 7.6 Hz), 1.40 (s, 18H, CH3), 2.00 (br s,
1H, CH2), 3.20 (m,1H, CH2), 3.80 (s, 3H, OCH3), 5.60 (s,1H, CH), 6.97
(d, 2H, HAr, J ¼ 8.8 Hz), 7.01 (dd, 1H, H7, J ¼ 8.3 Hz, J ¼ 1.7 Hz), 7.17
(d, 1H, H8, J ¼ 8.3 Hz), 7.27 (m, 3H, HAr, NH), 7.36 (s, 1H, H5), 10.60
1H, NH), 2.80 (q, 2H, CH2, J ¼ 7.6 Hz), 2.80 (m, 1H, CH2), 2.95 (m, 1H,
CH2), 3.15 (m, 1H, CH2), 3.50 (m, 1H, CH2), 5.10 (br s, 1H, OH), 5.30
(s, 1H, CH), 7.10 (dd, 1H, H7, J ¼ 8.2 Hz, J ¼ 1.5 Hz), 7.12 (s, 2H, HAr),
7.15 (d, 1H, H8, J ¼ 8.2 Hz), 7.40 (s, 1H, H5), 7.50 (br s, 1H, NH);
(br s, 1H, NH); 13C NMR (300 MHz, CDCl3),
d
¼ 16.7, 22.5, 29.1, 42.7,
55.3, 57.3, 109.7, 110.7, 113.3, 114.1, 116.7, 122.1, 127.6, 129.7, 130.1,
13CNMR (300 MHz, CDCl3),
d
¼ 16.6, 22.7, 29.1, 30.3, 34.3, 43.8, 58.9,
133.9, 134.4, 134.9, 135.3, 159.4; SM (APCIþ) m/z 307.1 [M þ H]þ.
109.6, 110.6, 116.6, 121.9, 125.1, 127.5, 127.8, 132.4, 134.3, 135.2, 135.5,
135.9, 136.2, 153.7; SM (APCIþ) m/z 405.3 [M þ H]þ.
4.6. 6-Ethyl-1-(3,4-dimethoxyphenyl)-2,3,4,9-tetrahydro-1H-b-
carboline (5d)
4.11. 6-Ethyl-1-(3-benzoxyphenyl)-2,3,4,9-tetrahydro-1H-b-
carboline hydrochloride (5i)
Yield 82% (acetonitrile); mp 172e173 ꢁC; 1H NMR (300 MHz,
CDCl3),
d
¼ 1.40 (t, 3H, CH3, J ¼ 7.6 Hz), 2.00 (br s, 1H, NH), 2.80 (m,
Yield 88% (acetonitrile); mp 130e131 ꢁC; 1H NMR (300 MHz,
DMSO-d6),
3H, CH2), 2.95 (m, 1H, CH2), 3.25 (m, 1H, CH2), 3.41 (m, 1H, CH2),
3.80 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 5.10 (s, 1H, CH), 6.80 (m, 3H,
HAr), 7.10 (d, 1H, H7, J ¼ 8.2 Hz), 7.18 (d, 1H, H8, J ¼ 8.2 Hz), 7.40 (s,
d
¼ 1.30 (t, 3H, CH3, J ¼ 7.6 Hz), 2.80 (q, 2H, CH2,
J ¼ 7.6 Hz), 2.90 (m, 2H, CH2), 3.15 (m, 1H, CH2), 3.40 (m, 1H, CH2),
5.00 (s, 2H, OCH2), 5.10(s, 1H, CH), 6.90 (m, 3H, HAr), 7.05 (dd, 1H,
H7, J ¼ 8.2 Hz, J ¼ 1.8 Hz), 7.12 (d, 1H, H8, J ¼ 8.2 Hz), 7.30 (m, 7H,
H5, HAr), 9.80 (br s, 1H, NH), 10.20 (br s, 1H, NHþ), 10.50 (br s, 1H,
1H, H5), 7.60 (br s, 1H, NH); 13C NMR (300 MHz, CDCl3),
d
¼ 16.6,
22.5, 29.1, 43.3, 55.7, 55.9, 58.2, 109.7, 110.9, 111.3, 113.1, 114.9, 116.7,
120.7, 122.1, 127.6, 134.3, 134.9, 135.4, 149.3, 152.6; SM (APCIþ) m/z
337.3 [M þ H]þ.
NH); 13CNMR (300 MHz, DMSO-d6),
d
¼ 17.1, 21.1, 28.9, 55.8, 69.5,
107.4, 111.8, 116.1, 117.1, 122.9, 126.2, 128.2, 128.4, 128.8, 128.9,

F. Hadjaz et al. / European Journal of Medicinal Chemistry 46 (2011) 2575e2585
2581
129.2, 129.5, 130.4, 130.7, 134.9, 135.5, 136.7, 137.2, 158.9; SM
117.7, 119.1, 126.3, 128, 129.1, 134.3, 134.8, 135.5, 143.7, 144.9; SM
(APCIþ) m/z 383.2 [M þ H]þ.
(APCIþ) m/z 309.3 [M þ H]þ.
4.12. 6-Ethyl-1-[3,4-bis(benzoxy)phenyl]-2,3,4,9-tetrahydro-1H-
b
-
4.17. General procedure for the synthesis of derivatives 6aed,fem,r
carboline hydrochloride (5j)
In CH3CN (80 mL), compounds 5aej,m (10 mmol), potassium
carbonate (4.2 g, 30 mmol) and the desired alkyle iodide (15 mmol)
were added. The reaction mixture was refluxed for 1 day and then
evaporated. Ethyle acetate (80 mL) and water (50 mL) were added.
The organic layer was dried with magnesium sulfate, filtered and
concentrated under reduced pressure. The residue was purified by
column chromatography using dichloromethane/methanol (96/4,
v/v) as eluent to afford compounds 6aed,fem,r. Solid products
were recrystallized with the appropriate solvant, while the oily
products were solubilized in dry ethyl acetate and treated with
gazeous hydrochloric acid in diethyl ether (20 mL). The obtained
precipitates were filtered and washed with ethyl acetate and the
residues were recrystallized with the appropriate solvant.
Yield 85% (methanol); mp > 260 ꢁC; 1H NMR (300 MHz, DMSO-
d6),
d
¼ 1.35 (t, 3H, CH3, J ¼ 7.6 Hz), 2.70 (q, 2H, CH2, J ¼ 7.6 Hz),
3.00 (m, 1H, CH2), 3.15 (m, 1H, CH2), 3.40 (m, 2H, CH2), 4.90 (s, 2H,
OCH2), 5.20 (s, 2H, OCH2), 5.80 (s, 1H, CH), 6.90(dd, 1H, HAr,
J ¼ 8.2 Hz, J ¼ 1.8 Hz), 7.00 (dd, 1H, H7, J ¼ 8.2 Hz, J ¼ 1.2 Hz), 7.15 (d,
1H, H8, J ¼ 8.2 Hz), 7.20 (d, 1H, HAr, J ¼ 8.3 Hz), 7.40 (m, 12H, H5,
HAr), 9.70 (br s, 1H, NH), 10.30 (br s, 1H, NHþ), 10.70 (br s, 1H, NH);
13CNMR (300 MHz, DMSO-d6),
d
¼ 17.1, 21.1, 28.9, 55.8, 68.3, 70.3,
107.4, 109.2, 111.8, 114.2, 116.2, 116.4, 117, 122.8, 123.4, 126.4, 127.7,
127.9, 128.1, 128.3, 128.7, 128.9, 129.2, 134.8, 135.5, 137.4, 137.5, 137.7,
148.6, 149.6; SM (APCIþ) m/z 489.5 [M þ H]þ.
4.13. 6-Benzyloxy-1-(3-methoxyphenyl)-2,3,4,9-tetrahydro-1H-b-
carboline hydrochloride (5m)
4.18. 6-Ethyl-1-(3-methoxyphenyl)-2-propyl-2,3,4,9-tetrahydro-
1H-
b-carboline (6a)
Yield 73% (absolute ethanol), mp 191e192 ꢁC; 1H NMR
(300 MHz, DMSO-d6),
(m, 2H, CH2), 3.75 (s, 3H, OCH3), 5.10 (s, 2H, OCH2), 5.83 (s, 1H, CH),
6.84 (d, 1H, H7, J ¼ 8.7 Hz), 6.95 (d, 1H, HAr, J ¼ 7.4 Hz), 7.05 (d, 1H,
HAr, J ¼ 8.0 Hz), 7.13 (m, 2H, H), 7.22 (d, 1H, H8, J ¼ 8.7 Hz), 7.38 (m,
4H, H5 HAr), 7.50 (d, 2H, HAr, J ¼ 8.5 Hz), 9.75 (br s, 1H, NH), 10.60 (br
s, 1H, NHþ), 10.70 (br s, 1H, NH); 13CNMR (300 MHz, DMSO-d6),
d
¼ 2.95 (m, 1H, CH2), 3.13 (m, 1H, CH2), 3.35
Yield 60% (absolute ethanol); mp 115e116 ꢁC; 1H NMR
(300 MHz, DMSO-d6),
d
¼ 0.80 (t, 3H, CH3, J ¼ 7.5 Hz), 1.20 (t, 3H,
CH3, J ¼ 7.6 Hz), 1.50 (m, 2H, CH2), 2.40 (m, 1H, CH2), 2.60 (m, 3H,
CH2), 2.80 (m, 2H, CH2), 3.10 (m, 1H, CH2), 3.40 (m, 1H, CH2), 3.70 (s,
3H, OCH3), 4.50 (s, 1H, CH), 6.85 (m, 4H, H7, HAr), 7.10 (d, 1H, H8,
J ¼ 8.2 Hz), 7.20 (s, 1H, H5), 7.21 (m, 1H, HAr), 10.00 (br s, 1H, NH);
d
¼ 18.6, 55.7, 56.1, 70.3, 102.2, 107.5, 112.7, 112.9, 115.6, 116, 122.4,
13CNMR (300 MHz, CDCl3),
d
¼ 11.5, 16.8, 17.4, 29.4, 46.4, 54.8, 55.7,
126.5, 128, 128.2, 128.3, 128.8, 129.7, 130.3, 132.2, 136.6, 138.2, 152.9,
56.5, 62.9, 106.9, 110.7, 117.5, 118.3, 122.6, 126, 126.3, 127.5, 131,
159.8; SM (APCIþ) m/z 385.1 [M þ H]þ.
132.4, 134.9, 135.6, 135.9, 158.6; SM (APCIþ) m/z 349.2 [M þ H]þ.
4.14. General procedure for the synthesis of phenolic derivatives
4.19. 6-Ethyl-1-(3-methoxyphenyl)-2-methyl-2,3,4,9-tetrahydro-
(5k,l)
1H-b-carboline (6b)
In 20 mL of methanol, compound 5i or 5j (2.5 mmol), and
Palladium/C were added. The reaction mixture was placed under
hydrogen atmosphere and stirred for 1 day. Palladium/C was filtered
and the solvent was removed in vacuo. The oily product was dis-
solved in EtOAc and ether saturated with gaseous HCl was added.
The corresponding hydrochloride product (5k or 5l) was filtered and
recrystallized from acetonitrile.
Yield 20% (absolute ethanol); mp 100e102 ꢁC; 1H NMR
(300 MHz, DMSO-d6),
d
¼ 1.30 (t, 3H, CH3, J ¼ 7.6 Hz), 2.40 (s, 3H,
NCH3), 2.60 (m, 3H, CH2), 2.75 (m, 1H, CH2), 3.20 (m, 1H, CH2), 3.40
(m, 1H, CH2), 3.75 (s, 3H, OCH3), 4.35 (s, 1H, CH), 6.90 (m, 4H, H8
HAr), 7.10 (d, 1H, H8, J ¼ 8.2 Hz), 7.20 (s, 1H, H5), 7.40 (m, 1H, HAr),
10.00 (br s, 1H, NH); 13CNMR (300 MHz, CDCl3),
d
¼ 11.7, 16.8, 29.8,
48.7, 56.1, 57.2, 62.9, 106.9, 112.5, 117.1, 117.5, 122.1, 126, 126.4, 127.4,
131.4, 132, 135, 135.3, 135.9, 158.2; SM (APCIþ) m/z 322.2 [M þ H]þ.
4.15. 6-Ethyl-1-(3-hydroxyphenyl)-2,3,4,9-tetrahydro-1H-b-
carboline hydrochloride (5k)
4.20. 6-Ethyl-1-(3-methoxyphenyl)-2-pentyl-2,3,4,9-tetrahydro-
1H-
b-carboline (6c)
Yield 85%; mp > 260 ꢁC; 1H NMR (300 MHz, DMSO-d6),
d
¼ 1.30
(t, 3H, CH3, J ¼ 7.6 Hz), 2.70 (q, 2H, CH2, J ¼ 7.6 Hz), 3.10 (m, 2H, CH2),
3.40 (m, 2H, CH2), 5.70 (s, 1H, CH), 6.70 (s, 1H, HAr), 6.90 (m, 2H,
HAr), 7.10 (dd, 1H, H7, J ¼ 8.5 Hz, J ¼ 1.5 Hz), 7.30 (m, 3H, H5, H8, HAr),
9.40 (br s, 1H, NH), 9.80 (br s, 1H, OH), 10.40 (br s, 1H, NHþ), 10.70
Yield 65% (ethanol 95ꢁ); mp 78e80 ꢁC; 1H NMR (300 MHz,
CDCl3),
d
¼ 0.80 (t, 3H, CH3, J ¼ 7.5 Hz), 1.34 (m, 7H, CH3, CH2), 1.50
(m, 1H, CH2), 2.40 (m, 1H, CH2), 2.60 (m, 3H, CH2), 2.90 (m, 2H,
CH2), 3.10 (m, 1H, CH2), 3.40 (m, 1H, CH2), 3.70 (s, 3H, OCH3), 4.50
(s, 1H, CH), 6.90 (dd, 1H, HAr, J ¼ 8.2 Hz, J ¼ 2.6 Hz), 7.00 (m, 3H, H7,
HAr), 7.10 (d, 1H, H8, J ¼ 8.2 Hz), 7.20 (br s, 1H, NH), 7.30 (m, 1H,
(br s, 1H, NH); 13CNMR (300 MHz, DMSO-d6),
d
¼ 17.8, 19.9, 28.9,
64.7, 107.4, 111.5, 118.2, 120.4, 122.2, 126.3, 127.3, 128.1, 129.1, 134.8,
135.4, 143.7, 144.9, 146.3; SM (APCIþ) m/z 293.4 [M þ H]þ.
HAr), 7.40 (s, 1H, H5); 13CNMR (300 MHz, CDCl3),
d
¼ 14, 16, 21.3,
22.6, 26.7, 29.1, 29.57, 48.5, 54.1, 55.1, 65, 108.5, 111, 113.5, 114, 117.1,
122, 123.1, 127.4, 128.7, 134.7, 135.2, 135.3, 143.4, 159.9; SM (APCIþ)
m/z 377.3 [M þ H]þ.
4.16. 6-Ethyl-1-(3,4-dihydroxyphenyl)-2,3,4,9-tetrahydro-1H-b-
carboline hydrochloride (5l)
Yield 80%; mp > 260 ꢁC; 1H NMR (300 MHz, DMSO-d6),
(t, 3H, CH3, J ¼ 7.6 Hz), 2.70 (q, 2H, CH2, J ¼ 7.6 Hz), 3.10 (m, 2H, CH2),
3.40 (m, 2H, CH2), 5.70 (s, 1H, CH), 6.70 (m, 2H, HAr), 6.85 (d,1H, HAr
J ¼ 7.8 Hz), 7.10 (dd, 1H, H7, J ¼ 8.5 Hz, J ¼ 1.5 Hz), 7.20 (d, 1H, H8,
J ¼ 8.5 Hz), 7.30 (s, 1H, H5), 8.10 (br s, 1H, NH), 9.15 (br s, 1H, OH),
9.35 (br s,1H, OH), 9.70 (br s,1H, NHþ),10.70 (br s,1H, NH); 13CNMR
d
¼ 1.30
4.21. 6-Ethyl-1-(3-methoxyphenyl)-2-nonyl-2,3,4,9-tetrahydro-
1H-b-carboline (6d)
,
Yield 60% (isopropanol); mp 50e52 ꢁC; 1H NMR (300 MHz,
CDCl3),
d
¼ 0.80 (t, 3H, CH3, J ¼ 7.5 Hz),1.30 (m,15H, CH3, CH2),1.50
(m, 2H, CH2), 2.40 (m, 1H, CH2), 2.60 (m, 5H, CH2), 3.10 (m, 1H,
CH2), 3.40 (m, 1H, CH2), 3.80 (s, 3H, OCH3), 4.50 (s, 1H, CH), 6.85
(300 MHz, DMSO-d6),
d
¼ 17.1, 19.4, 28.9, 55.5, 107.4, 111.7, 114.4, 117,

2582
F. Hadjaz et al. / European Journal of Medicinal Chemistry 46 (2011) 2575e2585
(dd,1H, HAr, J ¼ 8.2 Hz, J ¼ 2.6 Hz), 7.00 (m, 3H, H7, HAr), 7.10 (d,1H,
H8, J ¼ 8.2 Hz), 7.20 (br s, 1H, NH), 7.30 (m, 1H, HAr, J ¼ 8.2 Hz), 7.40
4.26. 1-(2,3-Dihydrobenzo [1,4]dioxin-6-yl)-6-ethyl-2-propyl-
2,3,4,9-tetrahydro-1H- -carboline (6j)
b
(s, 1H, H5); 13CNMR (300 MHz, CDCl3),
d
¼ 14.1, 16.6, 21.3, 22.7, 27,
27.3, 29.1, 29.3, 29.6, 29.8, 31.9, 48.6, 54.1, 55.2, 64.9, 108.5, 110.5,
113.6, 114, 116.7, 121.3, 121.8, 127.4, 129.5, 134.7, 135.1, 135.3, 143.4,
159.9; SM (APCIþ) m/z 433.5 [M þ H]þ.
Yield 61% (ethanol 95ꢁ); mp 62e64 ꢁC; 1H NMR (300 MHz,
CDCl3),
d
¼ 0.90 (t, 3H, CH3, J ¼ 7.4 Hz), 1.20 (t, 3H, CH3, J ¼ 7.6 Hz),
1.60 (m, 2H, CH2), 2.40 (m, 1H, CH2), 2.60 (m, 1H, CH2), 2.80 (m, 4H,
CH2), 300 (m, 1H, CH2), 3.40 (m, 1H, CH2), 4.25 (s, 4H, OCH2CH2O),
4.50 (s, 1H, CH), 6.85 (d, 1H, HAr, J ¼ 1.3 Hz), 6.90 (m, 2H, HAr), 7.00
(dd, 1H, H7, J ¼ 8.5 Hz, J ¼ 1.6 Hz), 7.12 (d, 1H, H8, J ¼ 8.5 Hz), 7.30 (s,
4.22. 6-Ethyl-1-(2-methoxyphenyl)-2-propyl-2,3,4,9-tetrahydro-
1H-b-carboline hydrochloride (6f)
1H, NH), 7.40 (s, 1H, H5); 13CNMR (300 MHz, CDCl3),
d
¼ 11.9, 16.7,
20.2, 21.2, 29.1, 48.2, 55.9, 63.9, 64.3, 108.5, 110.5, 116.8, 117.2, 117.7,
121.8, 121.5, 127.4,134.7, 134.9, 135.3,135.4,143.1,143.5; SM (APCIþ)
m/z 377.2 [M þ H]þ.
Yield 40% (methanol); mp 215e216 ꢁC; 1H NMR (300 MHz,
DMSO-d6),
d
¼ 0.90 (t, 3H, CH3, J ¼ 7.5 Hz), 1.30 (t, 3H, CH3,
J ¼ 7.5 Hz), 1.90 (m, 2H, CH2), 2.70 (q, 2H, CH2, J ¼ 7.5 Hz), 3.10 (m,
4H, CH2), 3.40 (m, 1H, CH2), 3.60 (s, 1H, CH2), 4.00 (s, 3H, OCH3),
6.20 (s, 1H, CH), 6.85 (dd,1H, HAr, J ¼ 7.5 Hz, J ¼ 1.5 Hz), 6.90 (m, 1H,
HAr), 6.99 (dd, 1H, H7, J ¼ 8.4 Hz, J ¼ 1.6 Hz), 7.20 (m, 2H, H8, HAr),
7.40 (s, 1H, H5), 7.50 (m, 1H, HAr), 9.70 (br s, 1H, NHþ), 10.50 (br s,
4.27. 6-Ethyl-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-propyl-
2,3,4,9-tetrahydro-1H-b-carboline (6k)
Yield 60% (absolute ethanol); mp 55e56 ꢁC; 1H NMR (300 MHz,
1H, NH); 13CNMR (300 MHz, DMSO-d6),
d
¼ 11.3, 16.5, 17.1, 28.9,
CDCl3),
d
¼ 0.80 (t, 3H, CH3, J ¼ 7.4 Hz), 1.30 (t, 3H, CH3, J ¼ 7.6 Hz),
46.9, 54, 56, 56.3, 62.7, 106.7, 111.8, 117, 117.2, 122.3, 126, 126.4, 127,
1.40 (s, 18H, CH3), 1.60 (m, 2H, CH2), 2.30 (m, 1H, CH2), 2.50 (m, 1H,
CH2), 2.70 (m, 4H, CH2), 300 (m, 1H, CH2), 3.40 (m, 1H, CH2), 4.50
(br s, 1H, OH), 5.20 (s, 1H, CH), 7.10 (dd, 1H, H7, J ¼ 82 Hz, J ¼ 1.5 Hz),
7.13 (s, 3H, H8, HAr), 7.20 (br s, 1H, NH), 7.35 (s, 1H, H5); 13CNMR
131, 132.1, 134.9, 135.3, 135.7, 157.9; SM (APCIþ) m/z 307.2 [M þ H]þ.
4.23. 6-Ethyl-1-(4-methoxyphenyl)-2-propyl-2,3,4,9-tetrahydro-
1H-
b
-carboline (6g)
(300 MHz, CDCl3),
d
¼ 11.5, 16.6, 20.3, 22.5, 29.3, 30.1, 34.1, 43.4,
48.1, 58.7, 109.76, 110.7, 116.5, 121.7, 125.3, 127.6, 128.3, 132.4, 134.3,
Yield 48% (absolute ethanol); mp 145e146 ꢁC; 1H NMR
(300 MHz, CDCl3),
135.3, 135.7, 135.9, 136.5, 153.9; SM (APCIþ) m/z 450.1 [M þ H]þ.
d
¼ 0.80 (t, 3H, CH3, J ¼ 7.5 Hz), 1.30 (t, 3H, CH3,
J ¼ 7.5 Hz), 1.40 (m, 2H, CH2), 2.30 (m, 1H, CH2), 2.60 (m, 1H, CH2),
2.70 (m, 4H, CH2), 3.00 (m, 1H, CH2), 3.40 (m, 1H, CH2), 3.80 (s, 3H,
OCH3), 4.50 (s,1H, CH), 6.80 (d, 2H, HAr, J ¼ 8.3 Hz), 7.00 (dd,1H, H7,
J ¼ 8.2 Hz, J ¼ 1.5 Hz), 7.10 (d, 1H, H8, J ¼ 8.2 Hz), 7.10 (br s, 1H, NH),
7.20 (d, 2H, HAr, J ¼ 8.3 Hz), 7.30 (s, 1H, H5); 13CNMR (300 MHz,
4.28. 6-Ethyl-1-(3-benzoxyphenyl)-2-propyl-2,3,4,9-tetrahydro-
1H-b-carboline hydrochloride (6l)
Yield 60% (methanol); mp 241e242 ꢁC; 1H NMR (300 MHz,
DMSO-d6),
d
¼ 0.90 (t, 3H, CH3, J ¼ 7.5 Hz), 1.30 (t, 3H, CH3,
CDCl3),
d
¼ 11.6, 16.6, 20.1, 21.2, 29, 48.3, 55.3, 55.8, 63.9, 108.5,
J ¼ 7.6 Hz), 1.50 (m, 2H, CH2), 1.60 (br s, 1H, NHþ), 2.35 (m, 1H, CH2),
2.60 (m, 1H, CH2), 2.80 (m, 4H, CH2), 3.00 (m, 1H, CH2), 3.40 (m, 1H,
CH2), 4.50 (s, 1H, CH), 500 (s, 2H, OCH2), 7.00 (m, 4H, H7, HAr), 7.10
(d, 1H, H8, J ¼ 8.4 Hz), 7.30 (m, 1H, HAr), 7.40 (m, 6H, H5, HAr), 9.80
(br s, 1H, NHþ), 10.60 (br s, 1H, NH); 13CNMR (300 MHz, DMSO-d6),
113.5, 113.8, 116.8, 121.8, 127.6, 130.2, 130.5, 133, 133.5, 134.3, 134.6,
135, 159.2; SM (APCIþ) m/z 349.3 [M þ H]þ.
4.24. 6-Ethyl-1-(3,4-dimethoxyphenyl)-2-propyl-2,3,4,9-
tetrahydro-1H-
d
¼ 11.2, 17, 18.1, 28.8, 49.4, 64.7, 69.8, 106.4, 111.8, 117.2, 123, 123.1,
b-carboline (6h)
123.2, 125.9, 128.2, 128.5, 128.6, 128.9, 131.3, 134.5, 134.9, 135.2,
135.6, 136.1, 136.9, 158.9; SM (APCIþ) m/z 462.3 [M þ H]þ.
Yield 52% (absolute ethanol); mp 140e142 ꢁC; 1H NMR
(300 MHz, CDCl3),
d
¼ 0.90 (t, 3H, CH3, J ¼ 7.5 Hz), 1.40 (t, 3H, CH3,
4.29. 6-Ethyl-1-[3,4-bis(benzoxy)phenyl]-2-propyl-2,3,4,9-
J ¼ 7.6 Hz), 1.60 (m, 2H, CH2), 2.30 (m, 1H, CH2), 2.80 (m, 4H, CH2),
2.90 (m, 1H, CH2), 3.10 (m, 1H, CH2), 3.40 (m, 1H, CH2), 3.80 (s, 3H,
OCH3), 3.90 (s, 3H, OCH3), 4.50 (s, 1H, CH), 6.80 (d, 1H, HAr,
J ¼ 7.9 Hz), 6.90 (m, 2H, HAr), 7.00 (dd,1H, H7, J ¼ 8.2 Hz, J ¼ 1.4 Hz),
7.10 (d, 1H, H8, J ¼ 8.2 Hz), 7.30 (s, 1H, H5), 7.40 (br s, 1H, NH);
tetrahydro-1H-
b-carboline (6m)
Yield 65% (absolute ethanol); mp 112e114 ꢁC; 1H NMR
(300 MHz, CDCl3), ¼ 0.90 (t, 3H, CH3, J ¼ 7.5 Hz), 1.35 (t, 3H, CH3,
d
J ¼ 7.6 Hz), 1.50 (m, 2H, CH2), 2.30 (m, 1H, CH2), 2.60 (m, 1H, CH2),
2.80 (m, 4H, CH2), 3.00 (m, 1H, CH2), 3.30 (m, 1H, CH2), 4.40 (s, 1H,
CH), 5.10 (s, 2H, OCH2), 5.20 (s, 2H, OCH2), 6.90 (dd, 1H, H7,
J ¼ 8.1 Hz, J ¼ 1.5 Hz), 6.94 (d, 1H, HAr, J ¼ 8.1 Hz), 7.00 (dd, 2H,
J ¼ 8.7 Hz, J ¼ 1.5 Hz), 7.07 (br s, 1H, NH), 7.12 (d, 1H, H8, J ¼ 8.3 Hz),
7.35 (m, 9H, H8 HAr), 7.50 (d, 2H, HAr, J ¼ 7.9 Hz); 13CNMR
13CNMR (300 MHz, CDCl3),
d
¼ 11.9, 16.7, 18.7, 20.1, 21.4, 29.1, 48.8,
55.8, 55.9, 64.9, 108.3, 110.5, 110.6, 111.3, 116.7, 121.2, 121.8, 127.4,
134, 134.7, 135.3, 135.6, 148.7, 149.4; SM (APCIþ) m/z 379.5
[M þ H]þ.
4.25. 6-Ethyl-1-(3,4,5-trimethoxyphenyl)-2-propyl-2,3,4,9-
(300 MHz, CDCl3),
d
¼ 11.9, 16.7, 20.2, 21.3, 29.1, 48.5, 55.9, 64.4,
tetrahydro-1H-
b
-carboline (6i)
70.9, 71.2, 108.4, 110.6, 114.5, 115.2, 116.7, 116.8, 121.8, 121.9, 127.3,
127.4, 127.5, 127.7, 127.8, 128.4, 128.5,134.7, 134.8, 135.3,135.5,137.2,
137.4, 148.6, 148.9; SM (APCIþ) m/z 531.2 [M þ H]þ.
Yield 60% (absolute ethanol); mp 145e147 ꢁC; 1H NMR
(300 MHz, CDCl3),
d
¼ 0.90 (t, 3H, CH3, J ¼ 7.5 Hz), 1.40 (t, 3H, CH3,
J ¼ 7.6 Hz), 1.60 (m, 2H, CH2), 2.30 (m, 1H, CH2), 2.60 (m, 2H, CH2),
2.80 (m, 3H, CH2), 3.10 (m, 1H, CH2), 3.40 (m, 1H, CH2), 3.80 (s, 6H,
OCH3), 3.90 (s, 3H, OCH3), 4.50 (s, 1H, CH), 6.70 (m, 2H, HAr), 7.00
(d, 1H, H7, J ¼ 8.2 Hz), 7.20 (d, 1H, H8, J ¼ 8.2 Hz), 7.35 (s, 1H, H5),
4.30. 6-Benzoxy-1-(3-methoxyphenyl)-2-propyl-2,3,4,9-
tetrahydro-1H-
b-carboline hydrochloride (6r)
Yield 63% (absolute ethanol); mp 132e132.8 ꢁC; 1H NMR
7.40 (br s, 1H, NH); 13CNMR (300 MHz, CDCl3),
d
¼ 11.9, 16.6, 20.1,
(300 MHz, DMSO-d6),
d
¼ 0.80 (t, 3H, CH3, J ¼ 7.3 Hz), 1.84 (m, 2H,
21.5, 29.1, 49, 55, 55.9, 56.1, 60.8, 65.7, 105.4, 106, 108.2, 110.6, 116.7,
121.8, 127.4, 134.7, 135.3, 135.5, 137.2, 137.5, 153.3; SM (APCIþ) m/z
409.3 [M þ H]þ.
CH2), 3.10 (m, 3H, CH2), 3.35 (m, 2H, CH2), 3.65 (m, 1H, CH2), 3.75
(s, 3H, OCH3), 5.10 (s, 2H, OCH2), 5.85 (s, 1H, CH), 6.83 (dd, 1H, H7,
J ¼ 8.7 Hz, J ¼ 2.5 Hz), 7.08 (m, 1H, HAr), 7.17 (m, 2H, H8, HAr), 7.25 (s,

F. Hadjaz et al. / European Journal of Medicinal Chemistry 46 (2011) 2575e2585
2583
1H, HAr), 7.38 (m, 5H, H5, HAr), 7.47 (d, 2H, HAr, J ¼ 8.3 Hz),; 13CNMR
hydrogen atmosphere and stirred for 1 day. Palladium/C was
filtered and the solvent was removed in vacuo. The residue was
purified by column chromatography using dichloromethane/
methanol (96/4, v/v) as eluent to afford solid compounds 6p,q,s.
(300 MHz, DMSO-d6),
d
¼ 11.4, 16.8, 18.1, 20.3, 48.4, 54.4, 55.8, 64.1,
70.3, 102.3, 106.6, 112.8, 113, 116.3, 123.1, 126.2, 127.9, 128, 128.2,
128.5, 128.9, 129.6, 130.5, 132.5, 133.7, 135.2, 138.2, 152.9, 159.9; SM
(APCIþ) m/z 427.2 [M þ H]þ.
4.36. 6-Ethyl-1-(3-hydroxyphenyl)-2-propyl-2,3,4,9-tetrahydro-
4.31. General procedure for the synthesis of derivatives 6e,n,o
1H-b-carboline (6p)
Yield 75% (ethanol 95ꢁ); mp 80e82 ꢁC; 1H NMR (300 MHz,
To a solution of 10% K2CO3 aqueous (30 mL) and ethyl acetate
(50 mL), b-carboline derivative (5a,i,j) (10 mmol) was solubilised.
CDCl3),
d
¼ 0.80 (t, 3H, CH3, J ¼ 7.4 Hz), 1.30 (t, 3H, CH3, J ¼ 7.6 Hz),
The mixture was cooled to 0 ꢁC, and benzyl chloroformate (2.1 mL,
15 mmol) or propionyl chloride (0.9 mL, 15 mmol) was added
dropwise. The reaction mixture was stirred for 3 h at room
temperature. The organic layer was washed with HCl 1M (50 mL),
dried with magnesium sulfate, filtered, and concentrated under
reduced pressure. The residue was purified by column chroma-
tography using dichloromethane/petroleum ether (90/10, v/v) as
eluent to afford solid compounds 6e,n,o.
1.60 (m, 2H, CH2), 2.40 (m, 1H, CH2), 2.60 (m, 1H, CH2), 2.80 (m, 4H,
CH2), 300 (m, 1H, CH2), 3.40 (m, 1H, CH2), 4.50 (s, 1H, CH), 6.40 (br s,
1H, OH), 6.70 (m, 2H, HAr), 6.80 (d, 1H, HAr, J ¼ 7.3 Hz), 7.10 (dd, 1H,
H7, J ¼ 8.5 Hz, J ¼ 1.5 Hz), 7.10 (m, 2H, H8, HAr), 7.40 (m, 2H, H5, NH);
13CNMR (300 MHz, CDCl3),
d
¼ 11.9, 16.6, 19.7, 29.1, 33.9, 47.7, 55.8,
63, 108.4, 110.7, 111.4, 115.5, 116.2, 121.2, 122.1, 123.3, 127.1, 129.6,
134.7, 135.4, 136.6, 156.3; SM (APCIþ) m/z 335.2 [M þ H]þ.
4.37. 6-Ethyl-1-(3,4-dihydroxyphenyl)-2-propyl-2,3,4,9-
4.32. 6-Ethyl-1-(3-methoxyphenyl)-2-propionyl-2,3,4,9-
tetrahydro-1H-
b-carboline (6q)
tetrahydro-1H-b-carboline (6e)
Yield 80% (ethanol 95ꢁ); mp 123e124 ꢁC; 1H NMR (300 MHz,
Yield 75% (absolute ethanol); mp 216e218 ꢁC; 1H NMR
CDCl3),
d
¼ 0.80 (m, 3H, CH3), 1.50 (m, 5H, CH3, CH2), 2.50 (m, 2H,
(300 MHz, CDCl3),
d
¼ 1.20 (t, 3H, CH3, J ¼ 7.3 Hz), 1.40 (t, 3H, CH3,
CH2), 2.80 (m, 5H, CH2), 3.20 (m, 1H, CH2), 4.50 (s, 1H, CH), 6.50 (m,
3H, HAr), 700 (m, 2H, H8, H7), 7.40 (s, 1H, H5), 8.50 (br s, 3H, OH,
J ¼ 7.6 Hz), 2.50 (q, 2H, CH2, J ¼ 7.3 Hz), 2.80 (q, 2H, CH2, J ¼ 7.6 Hz),
3.00 (m, 2H, CH2), 3.50 (m, 2H, CH2), 3.80 (s, 3H, OCH3), 4.00 (s, 1H,
CH), 6.80 (m, 2H, HAr), 7.00 (s, 1H, HAr), 7.08 (dd, 1H, H7, J ¼ 8.2 Hz,
J ¼ 1.5 Hz), 7.18 (m, 1H, HAr), 7.25 (d, 1H, H8, J ¼ 8.2 Hz), 7.35 (s, 1H,
NH); 13CNMR (300 MHz, CDCl3),
d
¼ 11.9, 16.6, 19.7, 29.1, 33.9, 47.7,
55.8, 63, 110.7, 114.7, 115.5, 116.1, 121.2, 122.1, 123.4, 127.1, 129.6,
134.7, 135.4, 136, 156, 157; SM (APCIþ) m/z 351.2 [M þ H]þ.
H5), 8.10 (br s, 1H, NH); 13CNMR (300 MHz, CDCl3),
d
¼ 9.6, 16.6,
26.8, 29.1, 39.7, 51.8, 55.2, 64.2, 110.9, 113.3, 114.3, 116.5, 118.7, 121.1,
121.9, 122.4, 126.7, 131.9, 134.8, 135.4, 141.9, 159.6, 172.41; SM
(APCIþ) m/z 363.3 [M þ H]þ.
4.38. 6-Hydroxy-1-(3-methoxyphenyl)-2-propyl-2,3,4,9-
tetrahydro-1H-
b-carboline hydrochloride (6s)
Yield 87% (absolute ethanol); mp 222e223 ꢁC; 1H NMR
4.33. 6-Ethyl-1-(3-benzoxyphenyl)-2-benzylcarbamate-2,3,4,9-
(300 MHz, DMSO-d6),
d
¼ 0.85 (t, 3H, CH3, J ¼ 7.0 Hz), 1.80 (m, 2H,
tetrahydro-1H-b-carboline (6n)
CH2), 3.00 (m, 3H, CH2), 3.35 (m, 1H, CH2), 3.70 (m, 2H, CH2), 3.80 (s,
3H, OCH3), 5.86 (s, 1H, CH), 6.65 (dd, 1H, H7, J ¼ 8.5 Hz, J ¼ 2.2 Hz),
6.83 (d, 1H, H5, J ¼ 2.2 Hz), 7.07 (m, 3H, H8, HAr), 7.23 (s, 1H, HAr),
7.38 (m, 1H, HAr), 8.80 (br s, 1H, OH), 10.30 (br s, 1H, NH), 11.10 (br s,
Yield 71% (absolute ethanol); mp 65e66 ꢁC; 1H NMR (300 MHz,
CDCl3),
d
¼ 1.45 (t, 3H, CH3, J ¼ 7.6 Hz), 2.80 (m, 4H, CH2), 3.20
(m, 1H, CH2), 4.50 (m, 1H, CH2), 5.10 (m, 2H, OCH2), 5.25 (m, 2H,
OCH2), 6.40 (s, 1H, CH), 6.80 (s, 1H, HAr), 7.05 (dd, 1H, H7, J ¼ 8.1 Hz,
J ¼ 1.7 Hz), 7.17 (d,1H, H8, J ¼ 8.1 Hz), 7.40 (m,14H, H5 HAr), 8.40 (br
1H, NHþ); 13CNMR (300 MHz, DMSO-d6),
d
¼ 11.4, 16.9, 18, 48.2,
54.3, 55.8, 64.1, 102.6, 105.8, 112.4, 112.6, 116.1, 116.3, 123, 126.6,
129.3, 130.4, 131.7, 135.2, 151.3, 159.9; SM (APCIþ) m/z 337.2
[M þ H]þ.
s,1H, NH); 13CNMR (300 MHz, CDCl3),
d
¼ 16.8, 21.6, 30.2, 37.8, 55.9,
70.5, 71.8, 108.9, 111.2, 114.6, 115.1, 115.5, 116.5, 121, 121.4, 121.8,
122.6, 127.3, 127.5, 127.8, 128.4, 128.6, 134.3, 135.5, 135.8, 137, 137.3,
137.8, 148.4; SM (APCIþ) m/z 517.8 [M þ H]þ.
4.39. Antioxydant activity
4.34. 6-Ethyl-1-[3,4-bis(benzoxy)phenyl]-2-benzylcarbamate-
4.39.1. Low Density Lipoprotein (LDL) oxidation inhibition test
Human LDLs were isolated from freshly drawn blood from
healthy, normolipidemic, fasting volunteers. LDLs were isolated by
sequential density gradient ultracentrifugation in sodium bromide
density solutions in the density range of 1.019e1.063 g/mL as
previously reported [48]. The protein concentration was deter-
mined by Peterson’s method [49] using bovine serum albumin as
the standard. Before oxidation, LDLs were extensively dialyzed
against 0.01 M phosphate-buffered-saline (PBS) pH 7.4 under N2 at
2,3,4,9-tetrahydro-1H-
b-carboline (6o)
Yield 75% (absolute ethanol); mp 54e56 ꢁC; 1H NMR (300 MHz,
CDCl3),
d
¼ 1.40 (t, 3H, CH3, J ¼ 7.6 Hz), 2.90 (m, 4H, CH2), 3.10 (m,
1H, CH2), 4.40 (m, 1H, CH2), 5.00 (m, 2H, OCH2), 5.20 (m, 4H, OCH2),
6.30 (s, 1H, CH), 6.75 (s, 2H, HAr), 7.05 (dd, 1H, H7, J ¼ 8.2 Hz,
J ¼ 1.8 Hz), 7.17 (d, 1H, H8, J ¼ 8.2 Hz), 7.40 (m, 17H, H5 HAr), 8.00 (br
s, 1H, NH); 13CNMR (300 MHz, CDCl3),
d
¼ 16.6, 21.8, 29.1, 38.2, 55.7,
70.3, 71.1, 72.9,108.6,110.8,114.3,115.3,115.7, 116.3,121,121.2,121.4,
121.6, 122.4, 122.6, 127.2, 127.5, 127.8, 128.4, 128.5, 128.6, 134.3,
134.5, 135.5, 135.7, 135.8, 137, 137.2, 137.4, 148.6, 148.8; SM (APCIþ)
m/z 623.1 [M þ H]þ.
4
ꢁC. Then oxidation was induced at 30 ꢁC by adding 20
16.6 M CuSO4 or 2 mM AAPH to 160 L of LDL (125 g of protein/
mL) in the presence or absence of 20 l derivatives from 0.1 M to
10 M or PBS for control. A mixture containing LDL and the tested
mL of
m
m
m
m
m
m
molecules without Cu2þ or AAPH was also used as control. During
copper or AAPH-induced LDL oxidation, diene-conjugated forma-
tion was followed by the measurement of optical density at 234 nm
every 10 min for 8 h at 30 ꢁC with a thermostated Spectra Max Plus
Molecular Devices spectrophotometer (96 wells, Molecular Devices
Corporation Sunnyvale, California 94089) [50].
4.35. General procedure for the synthesis of phenolic derivatives
(6p,q,s)
In 20 mL of methanol, compound 6l,m,r (2.5 mmol), and
Palladium/C were added. The reaction mixture was placed under

2584
F. Hadjaz et al. / European Journal of Medicinal Chemistry 46 (2011) 2575e2585
cultures given different treatments are assayed, then the amount of
LDH activity can be used as an indicator of relative cell viability as
well as a function of membrane integrity.10 mL drugs in methanol
solution (1.10ꢃ3 M) were added to the cell culture medium during
24h. Then cells were incubated at 37 ꢁC in the presence of LDL or
air- oxidized LDL at 100
mg/mL with an addition of 10
mL methanol
or drugs in methanol solution (final concentration 10
mM). After
16h of incubation, Lactate DesHydrogenase (LDH) activity in the
medium was measured by the spectrometric analysis of NAD
reduction using the LDH kit (sigma, Saint Quentin Fallavier, France).
LDH activity was expressed as the percentage of total LDH cellular
release following the addition of Triton X-100 (final concentration
of 0.1% v/v in ethanol). Results are the mean (þ/-SD) of 2 different
incubations each performed in triplicate.
Cellular vitality was determined by measuring mitochondrial
activity in the presence of MTT:(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide). MTT is reduced to purple formazan
Three phases could be distinguished from the Optical Density
in the mitochondria of living cells. 100 mL of a 5 mg solution of MTT
(OD) change pattern as illustrated below: (a) a lag phase, during
which OD did not increase significantly, indicating that LDL are
resistant to copper-catalysed modification; (b) a propagation phase,
during which OD increases rapidly; and (c) a degradation phase,
starting from the point at which the increase in OD reaches its
plateau.
in PBS were added to each culture well and incubated for 5 h.
Following this period, the medium was removed and the crystals
were dissolved in 4 mL of dimethylformamide. Absorbance was
read at 590 nm, and the results were expressed as the percentage of
the value of control wells [52].
The lag phase is determined by drawing tangents to the linear
segments of the curve during the lag phase and the propagation
phase. The perpendicular projection on the horizontal axis (time) of
the intersection point of the two tangents is defined as the lag time,
expressed in minutes. The propagation rate is calculated from the
slope of the tangent to the curve during the propagation phase, by
using a molecular extinction coefficient for conjugated dienes of
4.39.3. Data analysis
The results were expressed as mean ꢀ SD when a minimal
number of three independent experiments were performed in
triplicate. Differences between the experimental results were
tested using the Mann Whitney test.
Acknowledgments
3
¼ 2.95 ꢂ 104 Mꢃ1cmꢃ1
.
In vitro studies of LDL oxidation have verified the existence of
The 300 MHz NMR facilities were funded by the Région Nord-
Pas de Calais (France), the Ministère de la Jeunesse, de l’Education
Nationale et de la Recherche (MJENR), and the Fonds Européens de
Développement Régional (FEDER).
a ’lag phase’, during which significant oxidation of LDL cannot be
detected, prior to the onset of the ’propagation phase’, presumably
after the endogenous antioxidants have been consumed. There
follows a steady increase in the detectable by-products of oxida-
tion, until the substrate, i.e. the PUFAs, has been depleted, and
a plateau phase is reached. Assessment of resistance of LDL to
oxidation has generally involved measurement of the duration of
the lag phase, although other variables, such as the rate of propa-
gation, can also be noted.
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