Synthesis of new lipoic acid conjugates and evaluation of their free radical scavenging and neuroprotective activities

Article abstract of DOI:10.1111/cbdd.12282

A series of new lipoic acid derivatives were designed and synthesized as multitarget ligands against Alzheimer's disease. In particular, analogues combining both lipoic acid and cysteine core structures were synthesized. The antioxidant properties of these compounds were evaluated by 1,1-diphenyl-2- picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS^?+) radical cation scavenging assays and ferrous ion chelation. The antioxidant potential of the synthesized compounds was also evaluated in a cellular context and compared to α-lipoic acid and its reduced form, dihydrolipoic acid. The antioxidant effects observed for these compounds in vitro confirmed the importance of free thiol functions for effective antioxidant capacities. However, these promising in vitro results were not mirrored by the antioxidant activity in T67 cell line. This suggests that multiple factors are at stake and warrant further investigations. A series of new lipoic acid derivatives have been synthesized and evaluated for their potential antioxidant and neuroprotective activities. Structure-activity relationship studies comparing lipoic acid derivatives and their corresponding reduced analogues revealed the importance of free thiol functions.

Full text of DOI:10.1111/cbdd.12282

Chem Biol Drug Des 2014; 83: 688–696
Research Article
Synthesis of New Lipoic Acid Conjugates and
Evaluation of Their Free Radical Scavenging and
Neuroprotective Activities
1
,
1
Maria Laura Bolognesi *, Christian Bergamini ,
contrast oxidatively induced injuries (2,3). While DHLA is
considered the active species, the antioxidant activity of
LA and DHLA is attributed to their ability to regenerate
other antioxidants, such as vitamins C and E, to induce a
variety of antioxidant enzymes, but also to serve as a
proglutathione agent (4–6). These properties prompted the
medicinal chemistry community to investigate the
therapeutic potential of LA and DHLA derivatives in various
oxidative stress-related disorders (7,8). In most of these
studies, the lipoyl fragment has been linked to an amine-
type partner through an amide bond, giving raise to hybrid
molecules with an improved biological profile with respect
to LA itself (9–18). Focusing on neurodegenerative
disorders, LA has demonstrated a variety of beneficial
properties that can positively modulate disease pathogen-
esis and progression. On this basis, some of us have
proposed LA as a privileged structure in the search for
novel ligands against neurodegeneration and have vali-
dated this concept through the development of several
series of LA-based neuroprotective agents (19–22). It was
argued that the incorporation into a single molecular entity
of a lipoyl fragment and a second one with an alternative
mechanism of action could potentially lead to compounds
retaining the pharmacological properties of the two starting
fragments and consequently showing a synergistic and
more effective mechanism of action. In other words, the
resulting hybrid molecules act as multitarget-directed
ligands to combat the neurotoxic network underlying
Alzheimer’s disease (AD) (23).
1
2
Romana Fato , Jo e€ l Oiry , Jean-Jacques
Vasseur and Michael Smietana *
2
2,
1
Department of Pharmacy and Biotechnology, University of
Bologna, Via Belmeloro 6 and Via Irnerio 4, 40126,
Bologna, Italy
Institut des Biomolecules Max Mousseron, IBMM UMR
247 CNRS, Universit eꢀ Montpellier 1, Universit eꢀ
Montpellier 2, Place Eug eꢁ ne Bataillon, 34095 Montpellier,
France
*Corresponding authors: Maria Laura Bolognesi,
marialaura.bolognesi@unibo.it;
Michael Smietana, msmietana@um2.fr
2
5
A series of new lipoic acid derivatives were designed and
synthesized as multitarget ligands against Alzheimer’s
disease. In particular, analogues combining both lipoic
acid and cysteine core structures were synthesized. The
antioxidant properties of these compounds were evalu-
ated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2´-azi-
•
+
no-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS
)
radical cation scavenging assays and ferrous ion chela-
tion. The antioxidant potential of the synthesized com-
pounds was also evaluated in a cellular context and
compared to a-lipoic acid and its reduced form, dihydr-
olipoic acid. The antioxidant effects observed for these
compounds in vitro confirmed the importance of free
thiol functions for effective antioxidant capacities. How-
ever, these promising in vitro results were not mirrored
by the antioxidant activity in T67 cell line. This suggests
that multiple factors are at stake and warrant further
investigations.
N-Acetylcysteine (NAC) is another sulfur-containing natural
antioxidant, which has been addressed as patient
supplementation in several diseases. In vivo, NAC is
readily hydrolyzed to cysteine. Indeed, NAC has been
reported to be beneficial in a number of oxidative stress
models, such as systemic sclerosis, HIV infection, hypertension,
radiation injury, and others. Recently, we reported that various
NAC analogues and notably N-(N-acetyl-L-cysteinyl)-S-acety-
lcysteamine increased intracellular GSH levels in various cell lines
and displayed interesting antiviral and immunomodulatory activi-
ties (24–28). These compounds were designed to liberate after
metabolization, NAC and 2-mercaptoethylamine (MEA), two
potential pro-GSH compounds, which act as part of the glutathi-
one (GSH/GSSG) redox system (29,30). Notably, this glutathi-
one enhancing effect, which is peculiar to the mechanism of
Key words: Alzheimer’s disease, antioxidant, ferrous chela-
tion, lipoic acid, multitarget ligands
Received 25 October 2013, revised 6 December 2013 and
accepted for publication 6 January 2014
Alpha-lipoic acid (LA) is a clinically safe supplement
exhibiting significant antioxidant and cell regulatory
properties (1). Exogenously supplied LA is transported to
tissues, taken up by cells and reduced by lipoamide
dehydrogenase to the corresponding dihydrolipoic acid
(DHLA; Figure 1). Both LA and DHLA induce a variety of
major effects, including direct radical scavenging and
redox modulation of cell metabolism, and potentially
6
88
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12282

LA Conjugates as Potential Antioxidants
Figure 1: Dihydrolipoyl
dehydrogenase reduces lipoic acid
(LA) to dihydrolipoic acid (DHLA) in
the presence of NADH.
antioxidant action of NAC, has been recently proposed as par-
ticularly effective in contrasting oxidative stress in AD (31).
N,N’-(2,2′-Disulfanediylbis(ethane-2,1-diyl))bis(5-
(1,2-dithiolane-3-yl)pentanamide) (2a)
1
Yield: 62%, white solid, mp: 85–87 °C. H-NMR d 6.3 (sb,
Even more interestingly, it has been reported that a cocktail
containing both LA and NAC improves cognitive perfor-
mance while decreasing AD neuropathology in a transgenic
mouse model, and may thus represent a safe, natural
treatment for AD (32). Inspired by these findings and building
on the hybrid molecules approach, we envisioned that
merging the structural features of LA and DHLA with merca-
ptoamine-derived units (mimicking the NAC) could provide
ligands with improved antioxidant and neuroprotective
properties. Herein, the design, synthesis, and preliminary
examination of the in vitro antioxidant and neuroprotective
activities of the novel LA-NAC conjugates will be discussed.
2H), 3.42–3.55 (m, 6H), 2.96–3.18 (m, 4H), 2.76 (t,
J = 6.4 Hz, 4H), 2.41 (m, 2H), 2.17 (t, J = 7.4 Hz, 4H), 1.85
(m, 2H), 1.52–1.63 (m, 8H), 1.32–1.48 (m, 4H); C-NMR d
13
173.1, 56.5, 40.3, 38.5, 37.8, 36.2, 34.6, 30.9, 28.9, 25.4;
+
+
+
MS (ESI ) m/z: 529.12 ([M + H] 100%); HRMS-ESI m/z
+
calcd for C20
1176.
37 2 2 6
H N O S [M + H] 529.1179 Found 529.
Diethyl-3,3′-disulfanediylbis(2-(5-(1,2-dithiolane-3-
yl)pentanamido))propanoate (2b)
Yield: 60%, yellow solid, mp: 63 °C; H-NMR d 6.38 (d,
1
J = 7.31 Hz, 2H), 4.85 (m, 2H), 4.25 (q, J = 8.2 Hz, 4H),
3
.53–3.62 (m, 2H), 3.06–3.24 (m, 8H), 2.42–2.55 (m, 2H),
Methods and Materials
2.28 (t, J = 7.35 Hz, 4H,), 1.88–1.96 (m, 2H), 1.62–1.77
13
(m, 8H), 1.43–1.56 (m, 4H), 1.32 (t, J = 8.2 Hz, 6H); C-
Synthesis
NMR d 172.6, 170.4, 62.1, 56.4, 51.7, 40.8, 40.2, 38.5,
+
Reactions were conducted in oven-dried glasswares. All
reagents were purchased from Aldrich or local suppliers and
used without purification. All reactions were monitored by
TLC, and visualization was effected by UV and/or by devel-
oping in vanillin. Chromatography refers to open column
chromatography on silica gel (Merck, 40–63 lm). Melting
points were determined on a SMP3 Stuart Scientific appara-
tus and are uncorrected; NMR spectra were recorded on a
36.1, 34.6, 28.8, 25.2, 14.1; MS (ESI ) m/z: 673.16
+
+
([M + H] 100%); HRMS-ESI m/z calcd for C H N O S
6 45 2 6 6
2
+
[M + H] 673.1602 Found 673.1599.
N,N’-(Butane-1,4-diyl)bis(5-(1,2-dithiolane-3-yl)
pentanamide) (4)
Compound 4 is obtained directly by filtration of the reac-
1
Bruker DRX-300 MHz spectrometer at 300 ( H) and 75
tion mixture.
13
(
C) MHz at 298 K. Chemical shifts are reported in ppm rela-
1
tive to the solvent residual peak CDCl as internal standards
Yield: 86%, white solid, mp: 164 °C. H-NMR d 7.8 (sb,
3
1
13
1
(
H and C). Data for H are reported as follows: chemical
2H), 3.56 (m, 2H), 2.99–3.19 (m, 8H), 2.42 (m, 4H),
2.08 (t, J = 6.90 Hz, 4H), 1.83–1.90 (m, 2H), 1.22-1.50
shift, multiplicity (s = singlet, d = doublet, t = triplet, m = mul-
tiplet), coupling constants (J) in Hz, and integration. All com-
pounds were further characterized by mass spectrometry
13
(m, 14H). C-NMR d 171.7, 55.9, 39.6, 38.07, 37.9,
+
34.5, 33.9, 28.1, 26.4, 24.8.MS (ESI ) m/z: 465.17
+
+
(Q-TOF; Waters, Milford, MA, USA) and high-resolution mass
([M + H] 80%), 487.16 ([M + Na+], 100%; HRMS-ESI
+
spectrometry using an ESI/Q-TOF Micromass spectrometer.
m/z calcd for C20
65.1734.
H
37
N
2
O
2
S
4
[M + H] 465.1738 Found
4
General procedure for the coupling of LA activated
N-hydroxysuccinimide
N,N’-(((Disulfanediylbis(ethane-2,1-diyl))bis
To a solution of the appropriate amine (1 eq, 0.5 mmol) in
dioxane (cystamine for 2a, L-cystine diethyl ester for 2b, and
butanediamine for 4) were added N-ethyldiisopropylamine
(azanediyl))bis(2-oxoethane-2,1-diyl))bis(5-(1,2-
dithiolane-3-yl)pentanamide) (7)
1
Yield: 45%, white solid, mp: 224 °C; H-NMR d 7.95 (s,
2H), 6.95 (s, 2H), 3.96 (d, J = 5.33 Hz, 4H), 3.55–3.63 (m,
2H), 3.06–3.23 (m, 8H), 2.75 (t, J = 6.29 Hz, 4H), 2.42–
2.53 m (2H), 2.21 (t, J = 7.40 Hz, 4H), 1.84–1.96 (m, 2H),
(
(
5 eq, 2.5 mmol) and LA activated N-hydroxysuccinimide
33) (2.5 eq, 1.25 mmol), and the mixture was stirred at
room temperature overnight. The reaction was quenched
with saturated aqueous NaHCO and extracted with
CH Cl . The organic layer was washed with saturated aque-
13
3
1.62–1.76 (m, 8H), 1.421.53 (m, 4H); C-NMR d 174.2,
2
2
170.8, 56.4, 43.3, 40.2, 38.9, 38.6, 38.5, 35.9, 34.6, 28.9,
+
+
+
ous NaCl, dried over sodium sulfate, and the solvent was
evaporated in vacuo. The crude product was purified by
flash column chromatography.
25.3; MS (ESI ) m/z: 643.16 ([M + H] 100%); HRMS-ESI
+
m/z calcd for C24
H
43
N
4
O
4
S
6
[M + H] 643.1609 Found
643.1612.
Chem Biol Drug Des 2014; 83: 688–696
689

Bolognesi et al.
General procedure for the coupling of LA
6,8-Dimercapto-N-(2-mercaptoethyl)octanamide
3a)
(
1
Methyl 2-(5-(1,2-dithiolane-3-yl)pentanamido)
acetate (5)
To a solution of LA (1.5 eq, 3.6 mmol) in 10 mL of dry
DMF under N2 at 0 °C were added successively 0.5 mL
Yield 81%: colorless viscous oil. H-NMR d 5,81 (bs,
1H), 3.31–3.43 (m, 2H), 2.80–2.91 (m, 1H), 2.53–7.72
(m, 4H), 2.15 (t, J = 7.33 Hz, 2H), 1.75–1.80 (m, 1H),
13
1.33–1.60 (m, 7H), 1.22–1.33 (m, 3H);
C-NMR d
of Et
3
N, 1 eq of glycine methyl ester hydrochloride
172.8, 42.8, 42.2, 39.3, 38.7, 36.5, 26.6, 25.2, 24.7,
+
+
(2.40 mmol), and 1.5 eq (3.6 mmol) of 1-ethyl-3-[3-(dim-
22.3; MS (ESI ) m/z : 268.09 ([M + H] 100%); HRMS-
+
+
ethylamino)propyl]carbodiimide hydrochloride (EDCI•HCl).
The mixture was stirred at 0 °C for further 15 min and at
rt until completion (TLC monitoring). The mixture was
then diluted with water and extracted with ethyl acetate.
The organic layer was washed with brine, dried over
ESI m/z calcd for
C
10
H
22NOS
3
[M + H] 268.0864
Found 268.0862.
Ethyl 2-(6,8-dimercaptooctanamido)-3-
mercaptopropanoate (3b)
2 4
Na SO , and evaporated under vacuum. The crude
1
product was purified by flash column chromatography.
Yield 63%, colorless oil; H-NMR d 6.02 (bs, 1H), 4.05
Yield: 90%, colorless viscous oil; H-NMR d 6.28 (bs, 1H),
1
4.82-4.92 (m, 1H), 4.23-4.32 (m, 2H), 3.05 (dd,
J = 8.0 Hz, 2H), 2.85–2.96 (m, 1H), 2.63–2.74 (m, 3H),
2.28 (t, J = 7.7 Hz, 2H), 1.82–1.92 (m, 1H), 1.43–1.78 (m,
(
3
d, J = 6.0 Hz, 2H), 3.76 (s, 3H), 3.57 (m, 1H),
.07–3.19 (m, 2H), 2.41–2.49 (m, 1H), 2.26 (t,
J = 7.5 Hz, 2H), 1.74–1.97 (m, 1H), 1.64–1.72 (m, 4H),
13
6H), 1.26–1.47 (m, 6H); C-NMR d 172.5, 170.1, 62.1,
13
1
4
2
.44–1.53 (m, 2H); C-NMR d 172.8, 170.5, 56.4, 52.4,
1.2, 40.2, 38.5, 36.0, 34.6, 28.8, 25.2; MS (ESI ) m/z:
56.3, 53.4, 42.7, 39.3, 38.7, 36.2, 34.6, 26.9, 25.1, 14.2;
+
+
+
+
MS (ESI ) m/z: 340.11 ([M + H] 100%); HRMS-ESI m/z
+
+
+
78.09 ([M + H] 50%), 300.07 ([M + Na] 50%); HRMS-
calcd for
340.1079.
C
13
H
26NO
3
S
3
[M + H]
340.1075 Found
+
+
ESI m/z calcd for C H20NO3S2 [M + H] 278.0885
11
Found 278.0885.
6
,8-Dimercapto-N-(2-((2-mercaptoethyl)amino)-2-
2
-(5-(1,2-Dithiolane-3-yl)pentanamido)acetic acid (6)
oxoethyl)octanamide (8)
1
To a solution of 1.77 g. (6.4 mmol) of 5 in 24 mL of meth-
anol/THF (1/1) was added dropwise 12 mL of a 1.0 N
sodium hydroxide solution. The resulting mixture was stir-
red at rt overnight and then neutralized with a 1.0 N
hydrochloric acid solution. The mixture was then diluted
with water and extracted with ethyl acetate. The organic
Yield 81%, white solid, mp: 108 °C; H-NMR d 6.36 (s,
1H), 6.12 (s, 1H), 3.95 (d, J = 5.26 Hz, 2H), 3.42–3.51 (m,
2H), 3.88–3.95 (m, 1H), 2.62–2.78 (m, 4H), 2.28 (t,
J = 7.3 Hz, 2H), 1.83–1.98 (m, 1H), 1.62–1.81 (m, 4H),
13
1.42–1.53 (m, 3H), 1.25–1.42 (m, 3H); C-NMR d 173.5,
169.0, 43.4, 42.8, 42.4, 39.3, 38.7, 36.1, 26.6, 25.1,
+
+
layer was washed with brine, dried over Na
2
SO
4
, and
24.5, 22.3; MS (ESI ) m/z: 325.11 ([M + H] 100%);
+
+
evaporated under vacuum. The crude product was purified
HRMS-ESI
m/z calcd for
C
12
H
25
N
2
O
2
S
3
[M + H]
by flash column chromatography. Yield 65%, yellow solid,
325.1078 Found 325.1081.
1
mp: 206 °C; H-NMR
d
8.09 (bs, 1H), 3.73 (d,
J = 5.85 Hz, 2H), 3.63 (m, 1H), 3.12–3.26 (m, 2H), 3.39–
3
1
1
.49 (m, 1H), 2.16 (t, J = 7.15 Hz, 2H), 1.87–1.92 (m, 1H),
Methyl 2-(6,8-dimercaptooctanamido)acetate (9)
13
1
.52–1.72 (m, 4H), 1.41 (m, 2H);
C-NMR d 172.3,
Yield 74%, colorless oil; H-NMR d 5.88 (bs, 1H), 4.03 (d,
J = 5.1 Hz, 2H,), 3.77 (s, 3H), 2.83–2.96 (m, 1H), 2.62–
2.78 (m, 2H), 2.27 (t, J = 7.3 Hz, 2H), 1.86–1.96 (m, 1H),
1.63–1.82 (m, 4H), 1.42–1.53 (m, 3H), 1.28–1.38 (m, 2H);
71.3, 66.3, 56.1, 41.4, 38.0, 34.8, 34.0, 28.2, 24.9; MS
+
+
+
(ESI ) m/z: 286.05 ([M + Na] 100%); HRMS-ESI m/z
+
calcd for C10
86.0549.
3 2
H17NO S Na [M + Na] 286.0548 Found
1
3
2
C-NMR d 173.2, 170.2, 52.4, 42.8, 41.2, 39.3, 38.7,
+
+
3
1
6.1, 26.6, 25.1, 22.3; MS (ESI ) m/z: 280.10 ([M + H]
00%); HRMS-ESI m/z calcd for C11H NO S [M + H]
+
+
22
3 2
General procedure for the reduction in lipoic
derivatives
280.1041 Found 280.1045.
To a solution of the corresponding lipoic derivatives
(0.54 mmol) in 15 mL abs, EtOH was added at 0 °C
Antioxidant activity
NaBH4 (0.08 g, 2.2 mmol), and the mixture was stirred
at room temperature overnight. The reaction was
UV spectra were recorded on a Varian Cary 300 Bio UV/
Vis spectrometer.
quenched with H O, and EtOH was evaporated in
2
2 2 2
vacuo. The residue was extracted with CH Cl and H O.
The organic layer was washed with saturated aqueous
NaCl, dried over sodium sulfate, and the solvent was
evaporated in vacuo. The residue was purified by flash
column chromatography.
Scavenging effect on 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical
The scavenging effect of the synthesized compounds on
the DPPH radical was evaluated according to previously
6
90
Chem Biol Drug Des 2014; 83: 688–696

LA Conjugates as Potential Antioxidants
published methods (34,35). Various concentrations of the
test compounds were incubated in 1 mL of a 60% ethanolic
solution containing the DDPH radical (60 lM). The mixture
was shaken vigorously and allowed to stand for 30 min;
absorbance at 517 nm was determined, and the percentage
of activity was calculated by the following equation:
gemistocytic astrocytoma. T67 cells were cultured in Dul-
becco’s modified Eagle’s medium (DMEM; BioWhittaker,
Cambrex BioScience, Belgium), supplemented with 10%
fetal bovine serum (FBS; BioWhittaker), 100 UI/mL penicil-
lin, 100 lg/mL streptomycin, and 40 lg/mL gentamicin, in
a 5% CO atmosphere at 37 °C, with saturating humidity.
2
Cell stocks were cryopreserved by standard methods and
stored in liquid nitrogen. Cell viability was measured by try-
pan blue exclusion (21).
ꢀ
ꢁ
ðAcontrol ꢀ AtestÞ
Acontrol
%
inhibition ¼
ꢁ 100:
To evaluate the antioxidant activity of the compounds, T67
cells were seeded in 24-well plates at 1 9 105 cells/well.
Experiments were performed after 24 h of incubation at
All tests and analyses were undertaken in triplicate and
the results averaged.
3
2
7 °C in 5% CO . After this time cells were washed and
•
+
treated for 24 h with the compounds, the antioxidant
activity was evaluated after 30 min of incubation with
ABTS radical cation scavenging assay
The scavenging effect of the synthesized compounds on
•
+
1
0 lM fluorescent probe (2′,7′-dichlorofluorescein diace-
the ABTS radical cation (ABTS ) was evaluated according
to previously published methods (36). The radical cation
was prepared by mixing ABTS stock solution (7 mM in
water) with 2.45 mM potassium persulfate (final concentra-
tion). This mixture was allowed to stand in the dark at room
temperature for 12–16 h before use. The ABTS solution
was diluted with ethanol to an absorbance of 0.70 at
34 nm. Diluted ABTS solution (0.9 mL) and 100 lL of
tate, DCFH-DA) in DMEM, by measuring the intracellular
ROS formation evoked by 30-min exposure of T67 cells to
1
00 lM tert-butyl hydroperoxide (t-BuOOH) in PBS. The
fluorescence increase in the cells from each well was mea-
sured (kexc = 485 nm; kem = 535 nm) with a spectrofluo-
rometer (Wallac Victor multilabel S9 counter; Perkin-Elmer
Inc., Boston, MA, USA). Data are reported as the
mean ꢂ standard deviation of at least three independent
experiments (21).
•
+
•
+
7
various concentrations of the test compounds were mixed
and measured immediately after 6 min at 734 nm. The anti-
oxidant activity was calculated using the previous equation.
Results and discussion
2
+
Chelation activity on Fe
Chemistry
The synthesized compounds were evaluated for their ability
to compete with ferrozine for iron (II) ions in solution accord-
ing to previously published methods (37). Various concen-
trations of the test compounds (1 mL in water) were added
to a solution of 2 mM FeCl .4H O (0.4 mL). The reaction
was initiated by the addition of 2.5 mM ferrozine (0.1 mL);
the mixture was shaken vigorously and left standing at room
temperature for 10 min. Absorbance of the solution was
then measured at 562 nm against the blank performed in
The syntheses of the target compounds are shown in
Scheme 1. In details, the carboxylic acid functionality of LA
was coupled to different sulfur-containing amines. Hybrid
structures 2a, 2b, and 4 were prepared from N-hydrosuc-
cinimide-activated LA (39) and cystamine, L-cystine diethyl
ester, and butanediamine, respectively. Reduction in the
2
2
4
disulfide bonds in 2a and 2b using NaBH yielded the corre-
sponding trithiols 3a and 3b. Compound 3b thus features
an unprecedented LA-cysteine core structure. To obtain a
better understanding of the structure–activity relationship
and to probe the influence of the added disulfide unit, LA
was also coupled with glycine methyl ester upon activation
of LA in the presence of 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDCI). The corresponding ester
the same way using FeCl and water. The percentage of
2
+
2
inhibition of ferrozine–Fe complex formation was calcu-
lated using the following equation:
ꢀ
ꢁ
1
ꢀ Atest
%
Chelating activity ¼
ꢁ 100:
Acontrol
5
was then hydrolyzed in the presence of sodium hydroxide
and further coupled with cystamine leading to the original
Biological evaluation
bis-lipoyl derivative 7. Compounds 5 and 7 were then
4
reduced using NaBH affording the desired free thiols 9 and
Reagents
8, respectively (Scheme 1).
All chemicals used throughout this study were of the high-
est analytical grade, purchased from Sigma Chemical
Company, Milan, Italy, unless otherwise specified.
Free radical scavenging activities
First, we evaluated the antioxidant activity of these com-
•
pounds using the 2,2-diphenyl-1-picrylhydrazyl (DPPH )
Cell culture
(34) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic
acid (ABTS ) (36) antioxidant assays. For that purpose,
various concentrations of the test compounds were
•
+
The T67 human glioma cell line was derived by Lauro
et al.(38) from a World Health Organization Grade III
Chem Biol Drug Des 2014; 83: 688–696
691

Bolognesi et al.
Scheme 1: Synthesis of the target compounds
incubated for 30 min in a solution containing the stable
free radical. The antioxidant activities shown in Table 1 are
ols are necessary for maximal free radical scavenging effi-
cacy, a third sulphydryl group does not seem to be
crucial. Indeed, IC50 values of compounds 3a, 3b, and 8
are comparable with those obtained with DHLA. However,
while almost no difference was observed depending on
the nature of the N-amido group inside this series, we
found that coupling the carboxylic acid moiety present in
DHLA with glycine methyl ester (9) induces a slight
decrease in activity. In general, the results indicate that
free sulphydryl groups confer to these molecules
interesting free radical scavenging properties similar to
those of DHLA, Trolox, and ascorbic acid, thus validating
a multi-antioxidant approach with compounds covering
both lipophilic and hydrophilic areas of the cell.
expressed as the 50% inhibitory concentration (IC50
)
based on the amount of compound required for a 50%
decrease in the initial free radical concentration. The
results obtained were compared with LA, DHLA, and two
well-known antioxidants, Trolox (a polar analogue of vita-
min E) and ascorbic acid. Both assays are valuable for
ranking the potency of an antioxidant. In this context, we
observed that the relative IC50 values obtained agreed well
with one another. Overall, it was found that while free thi-
Table 1: Antioxidant activities of synthesized product derivatives
•
•+
2
a–9 in DPPH and ABTS radical scavenging assays
•
•+
DPPH IC50
ABTS IC50
Compounds
(lM)
(lM)
Iron chelating assay
2
2
3
3
4
5
6
7
8
9
a
b
a
b
>100
80
23
>100
>100
10
To further explore the potential neuroprotective properties
of these compounds, we investigated their ability to
chelate ferrous ions. These ions play a critical role in the
free radicals formation, as they can convert hydrogen
peroxide to hydroxyl radicals through the Fenton reaction
and many drug discovery programs are devoted to the
development of efficient iron chelators (40). Thus, the
29
9.4
>100
>100
>100
>100
26
33
>100
22
>100
>100
>100
>100
8.9
15.2
>100
11
2+
ability of molecules to chelate Fe
parameter to assess their antioxidant effects. The Fe
ions is a practical
2+
Lipoic acid
Dihydrolipoic acid
Trolox
chelating activities of the synthesized compounds were
assessed by differences in absorbance at 562 nm, with
the appearance of an absorption peak upon reaction of
20
19
6.2
8.8
Ascorbic acid
2+
Fe with ferrozine and compared with the results obtained
6
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Chem Biol Drug Des 2014; 83: 688–696

LA Conjugates as Potential Antioxidants
EDTA. However, none of the lipoyl derivatives exhibited a
chelating capacity comparable to the positive control,
EDTA-2Na (EC50 = 32 lM). Observed values ranged from
ROS levels, the DCFDA fluorescent probe was used.
Initially, all the synthesized compounds were used at a
concentration that did not affect cell viability (i.e., 500 nM),
and the antioxidant profile is reported in Figure 3A.
Surprisingly, at this concentration, nearly all compounds
only slightly counteracted the evoked oxidative stress. For
the DHLA derivatives 3b and 9, even an increase in ROS
formation was evident. This behavior and the high stan-
dard deviations made us postulating that some toxic
effects could take place and mask the antioxidant activity.
Thus, to rule out this possibility, we repeated the same
experiment, but using a lower (less toxic) concentration of
tested compounds (100 nM). However, in this case, no sig-
nificant protection against oxidative stress by the synthe-
sized conjugates was detected (Figure 3B). This outcome
was better appreciated for compound 6, which was tested
in a wider range of concentrations: at 125 and 250 nM a
dose-dependent effect could be seen, but at the higher
concentrations it is highly feasible that toxic effects may
counterbalance the beneficial ones (Figure 4A).
300 to 3000 lM without any indication of structure–activity
relationships (data not shown). These results were rather
surprising as iron(II) is a medium soft Lewis acid and
prefers ligation to soft Lewis base donors like thiols. While
complexes of iron(II) and iron(III) with LA and DHLA have
been reported, iron binding is not as effective as iron
reduction (41). Ferrozine is acknowledged as an effective
chelator of ferrous iron and has been used for the
determination of iron in biological samples and none of the
synthesized compounds was able to compete with the
2
+
ferrozine–Fe preformed complex.
Biological activities
To explore the antioxidant potential of the synthesized
compounds in
a cellular context, cell viability and
neuroprotective activity against an oxidative insult were
assayed in the human astrocytoma cell line T67, in com-
parison with LA as reference compound.
Actually, these findings are not surprising, albeit disappoint-
ing. In fact, we evaluated LA antioxidant capacities under
the same experimental conditions. As evident from the
graph of Figure 4B, LA starts to display an antioxidant effect
at a concentration of 5 lM, which is a tenfold higher concen-
tration than that used for our compounds (500 nM). A 50%
reduction in ROS formation was achieved only at a LA con-
centration of 50 lM. Of course, this was possible thanks to
the minimal cytotoxicity shown by LA in this cell line.
Cytotoxic effects on T67 cells
To assess the potential cytotoxicity, we used the MTT
assay to examine the viability of T67 cells treated with
three doses of the compounds (0.5, 1, and 5 lM).
Treatment concentrations of 0.5 lM had no effects on cell
viability, while the viability of T67 cells treated with 5 lM
was decreased by 40–70% (Figure 2). Notably, LA was
not toxic to the same cell line up to a concentration of
Thus, we can conclude that in the tested conditions, 2a–9
were found to be weak antioxidants. In this respect, it
should be noticed that, despite it has been clearly
demonstrated that LA/DHLA supplementation decreases
oxidative stress and restores reduced levels of other
antioxidants in vivo, there is also evidence indicating that
they may exert pro-oxidant properties in vitro (42). The
ability of LA and/or DHLA to act as either anti- or pro-oxi-
dants, at least in part, has been deemed dependent on
50 lM (data not shown).
Neuroprotective effects on T67 cells
To mimic oxidative stress conditions, ROS production in
T67 cells pretreated with the test compound has been
stimulated by treatment with the radical initiator tert-butyl
hydroperoxide (TBHP). Then, to measure the intracellular
Figure 2: Effects of compounds
on cell viability in T67 cells. The cell
viability was determined by the MTT
assay (as described in Methods
and Materials) after 24 h of
incubation with compounds’
concentrations of 0.5, 1, and 5 lM.
The results were expressed as a
percentage of control cells. Values
are reported as the mean ꢂ SD of
three independent experiments.
Chem Biol Drug Des 2014; 83: 688–696
693

Bolognesi et al.
A
A
B
B
Figure 3: Effects of the lipoic acid conjugate on intracellular ROS
formation in T67 cells at two different concentrations: 500 nM in
(A) and 100 nM in (B). The results are expressed as percentage
increase in intracellular ROS evoked by exposure to tert-butyl
hydroperoxide (TBHP) for 30 min. Values are the mean ꢂ SD of
three independent experiments.
Figure 4: (A) Effects of different concentrations of 6 on ROS
production in T67 cells exposed to 100 lM TBHP. The results,
expressed as percentage of the control, are means ꢂ SD of three
independent experiments. (B) Dose-dependent ROS scavenging
effects of lipoic acid (LA) in T67 cells exposed to 100 lM TBHP.
The results, expressed as percentage of the control, are
means ꢂ SD of three independent experiments.
the type of oxidant stress and the physiological circum-
stances. These pro-oxidant actions suggest that LA and
DHLA act by multiple mechanisms, many of which are
only recently being explored. Thus, it has been proposed
that the antioxidant properties of LA and DHLA should be
tested experimentally in different oxidative stress condi-
tions. The same might apply to our compounds, and this
point will be taken into due consideration in the next
endeavors on these and analogous compounds.
these analogues showed remarkable free radical scaveng-
ing abilities, similar to those of well-known antioxidants such
as Trolox and ascorbic acid. The results obtained in the
•
•+
DPPH and ABTS assays highlighted the importance of
sulphydryl functional groups for maximal free radical scav-
enging efficacy. However, this promising in vitro profile was
not mirrored by the antioxidant activity in T67 cell line. In
fact, all compounds when tested at a concentration of
500 nM displayed marginal antioxidant capacities, while they
seemed to develop some cytotoxicity.
Conclusion
In conclusion, a new series of LA derivatives were
synthesized and evaluated as potential antioxidants. In vitro,
6
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Chem Biol Drug Des 2014; 83: 688–696

LA Conjugates as Potential Antioxidants
We suggest that these results do not discard the idea of
combining the LA moiety with those of other therapeuti-
cally relevant antioxidants. Conversely, they might serve as
the basis for developing new LA-based hybrids that could
exert beneficial effects against the oxidative processes
underlying AD, following the fine optimization of their toxic-
ity properties.
10. Antonello A., Tarozzi A., Morroni F., Cavalli A., Rosini
M., Hrelia P., Bolognesi M.L., Melchiorre C. (2006)
Multitarget-directed drug design strategy: a novel mol-
ecule designed to block epidermal growth factor
receptor (EGFR) and to exert proapoptotic effects. J
Med Chem;49:6642–6645.
11. Packer L., Tritschler H.J., Wessel K. (1997) Neuropro-
tection by the metabolic antioxidant alpha-lipoic acid.
Free Radic Biol Med;22:359–378.
Acknowledgment
12. Harnett J.J., Auguet M., Viossat I., Dolo C., Bigg D.,
Chabrier P.E. (2002) Novel lipoic acid analogues that
inhibit nitric oxide synthase. Bioorg Med Chem
Lett;12:1439–1442.
Mrs A. Siffre, M. Sellak, and F. Maait are acknowledged
for technical assistance.
1
3. Koufaki M., Calogeropoulou T., Detsi A., Roditis A.,
Kourounakis A.P., Papazafiri P.,Tsiakitzis K., Gaitanaki
C., Beis I., Kourounakis P.N. (2001) Novel potent
inhibitors of lipid peroxidation with protective effects
Conflict of Interest
The authors declare no conflict of interest.
against
reperfusion
arrhythmias.
J
Med
Chem;44:4300–4303.
1
4. Koufaki M., Detsi A., Theodorou E., Kiziridi C., Calog-
eropoulou T., Vassilopoulos A., Kourounakis A.P.,
Rekka E., Kourounakis P.N., Gaitanaki C., Papazafiri
P. (2004) Synthesis of chroman analogues of lipoic
acid and evaluation of their activity against reperfusion
arrhythmias. Bioorg Med Chem;12:4835–4841.
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