Antioxidant activity of unexplored indole derivatives: Synthesis and screening

Article abstract of DOI:10.1016/j.ejmech.2010.07.059

The present study envisaged the development of novel antioxidant candidates using the indole scaffold. Several tryptophan and tryptamine derivatives were synthesized, in particular prenylated indole compounds, and their scavenging activity for reactive oxygen species (ROS) and reactive nitrogen species (RNS) was investigated. The library substitution pattern included several alkyl chains at positions N-1, C-2 of the indole nucleus, including prenyl and isopentyl chain, as well as different groups at the side chain (C-3) that allowed the investigation of a possible radical stabilization. The results obtained showed that tryptophan (8), tryptamine (9), N-phthaloyl tryptamine (5) and N-prenyl tryptophan (13) were the most active against peroxyl radical (ROO ^?) with activities higher than Trolox, which was used as control. The scavenging of hypochlorous acid (HOCl) was also evaluated and tryptophan (8) and tryptamine (9) showed IC₅₀ of 3.50 ± 0.4 and 6.00 ± 0.60 μM, respectively. Significant activity was also found for the N-prenyl tryptophan (13) with an IC₅₀ of 4.13 ± 0.17 μM and C-2 prenylated derivative (14), with 4.56 ± 0.48 μM. The studies were extended to RNS and best results were obtained against peroxynitrite anion (ONOO^-) in the presence of NaHCO₃. N-alkylated tryptophan (18) showed a high activity with an IC₅₀ of 14.0 ± 6.8 μM. The results show that the tested compounds are effective scavengers of ROS and RNS, and suggest that the radical stabilization is strongly dependent on the type of substituents on the indolic moiety and on their relative positions. In addition, the radical dissipation inside the indolic system is mandatory for the observed antioxidant activity.

Full text of DOI:10.1016/j.ejmech.2010.07.059

European Journal of Medicinal Chemistry 45 (2010) 4869e4878
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antioxidant activity of unexplored indole derivatives: Synthesis and screening
Mónica S. Estevão a, Luísa C. Carvalho a, Daniela Ribeiro b, Diana Couto b, Marisa Freitas b, Ana Gomes b,
,
L.M. Ferreira a, Eduarda Fernandes b, M. Manuel B. Marques a
*
a REQUIMTE-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Monte de Caparica, 2829-516 Caparica, Portugal
b REQUIMTE, Departamento de Química-Física, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4099-030 Porto, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study envisaged the development of novel antioxidant candidates using the indole scaffold.
Several tryptophan and tryptamine derivatives were synthesized, in particular prenylated indole
compounds, and their scavenging activity for reactive oxygen species (ROS) and reactive nitrogen species
(RNS) was investigated. The library substitution pattern included several alkyl chains at positions N-1,
C-2 of the indole nucleus, including prenyl and isopentyl chain, as well as different groups at the side
chain (C-3) that allowed the investigation of a possible radical stabilization. The results obtained showed
that tryptophan (8), tryptamine (9), N-phthaloyl tryptamine (5) and N-prenyl tryptophan (13) were the
most active against peroxyl radical (ROOꢀ) with activities higher than Trolox, which was used as control.
The scavenging of hypochlorous acid (HOCl) was also evaluated and tryptophan (8) and tryptamine (9)
Received 24 April 2010
Received in revised form
20 July 2010
Accepted 29 July 2010
Available online 6 August 2010
Keywords:
Antioxidant activity
Tryptophan and tryptamine derivatives
Scavenging activity
Reactive oxygen species
Reactive nitrogen species
Prenylated indoles
showed IC50 of 3.50 ꢁ 0.4 and 6.00 ꢁ 0.60
N-prenyl tryptophan (13) with an IC50 of 4.13 ꢁ 0.17
4.56 ꢁ 0.48 M. The studies were extended to RNS and best results were obtained against peroxynitrite
anion (ONOOL) in the presence of NaHCO3. N-alkylated tryptophan (18) showed a high activity with an
M. The results show that the tested compounds are effective scavengers of ROS and
m
M, respectively. Significant activity was also found for the
m
M and C-2 prenylated derivative (14), with
m
IC50 of 14.0 ꢁ 6.8
m
RNS, and suggest that the radical stabilization is strongly dependent on the type of substituents on the
indolic moiety and on their relative positions. In addition, the radical dissipation inside the indolic
system is mandatory for the observed antioxidant activity.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
On the other hand, ROS are also produced during inflammatory
response as a consequence of cyclooxygenase activity. Additionally,
The search and evaluation of chemical compounds and their
derivatives with a specific pharmacological activity is a demanding
task in the drug discovery process. In this context, the study of
structureeactivity relationship (SAR) is a valuable tool for the
planning of potential novel drugs and, in a later stage, for the
development of novel treatments.
During the inflammatory processes many reactive species are
produced, among them reactive oxygen species (ROS) such as,
superoxide radical (O2ꢀL), hydrogen peroxide (H2O2), hypochlorous
acid (HOCl), singlet oxygen (1O2) and peroxyl radical (ROOꢀ), as well
as reactive nitrogen species (RNS), like nitric oxide (ꢀNO) and per-
oxynitrite anion (ONOOL). Indeed, ROS and RNS are produced by
the endothelial cells, Kupffer cells, neutrophils and macrophages as
mechanism of defense against foreign infectious pathogens [1,2].
mitochondria are also a major cellular source of ROS [3]. ROS and
RNS are associated with a wide range of pathologies and diseases
such as cancer, rheumatoid arthritis, atherosclerosis, Alzheimer and
Parkinson disease, among others [4]. To date, the prevention of
oxidative stress related diseases has been tentatively achieved by
the development of antioxidant compounds that are able to scav-
enge ROS and RNS, and thus avoid radical-induced oxidation
damage. When a validated scaffold has been identified, the study of
SAR may provide useful information for the understanding of its
mode of action and to the improvement of the antioxidant activity
of future generated compounds.
The indole framework is a medicinally relevant scaffold and has
become widely identified as a privileged structure or pharmaco-
phore [5]. The indole scaffold is present in thousands of isolated
natural products and also medicinal (synthetic) compounds with
diverse therapeutic activity. For instance, the non-steroidal anti-
inflammatory drugs (NSAIDs) constitute the most important class
of therapeutic agents for the treatment of inflammatory related
diseases. Indomethacin, acemetacin and etodolac are NSAIDs
* Corresponding author.
E-mail addresses: egracas@ff.up.pt (E. Fernandes), mmbmarques@dq.fct.unl.pt
(M.M.B. Marques).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.059

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M.S. Estevão et al. / European Journal of Medicinal Chemistry 45 (2010) 4869e4878
possessing an indole ring, and their ROS and RNS scavenging
activity has already been reported [6,7]. In animals, the most
significant indolic compounds are the tryptophan derivatives
serotonin (5-hydroxytryptamine; 5-HT) and melatonin (N-acetyl-
5-methoxytryptamine), and related compounds while, in medicinal
plants, indole alkaloids, especially the plant hormone, indole-3-
acetic acid (IAA) and its catabolic products, seem to display relevant
biological activities [8]. One of the most promising endowments
displayed by indole derivatives is their antioxidant potential,
conferred by their ability to scavenge ROS and RNS [9]. With
reference to ROS, melatonin has a well-known potent scavenging
effect against 1O2, O2ꢀL, H2O2, hydroxyl radical (HOꢀ), and ROOꢀ
[10,11]. The high reactivity of melatonin with reactive species is
probably due to the presence of an electron-rich aromatic ring
system, allowing the indoleamine to easily function as an electron
donor, to form the melatoninyl cation radical, or through an elec-
trophilic radical addition at the C-3 position [12]. This type of
reactivity with ROS is extended to other indole derivatives, namely
tryptophan, serotonin, 5-methoxytryptamine, 6-chloromelatonin
and related indoles [10a,10b,10d], IAA and other plant-derived
indoles [13] indole-3-propionic acid [13], indomethacin and indo-
methacin derivatives [6,14], indole derivatives containing an
amine-triazole moiety [15], indolin-2-one and indoline-2-thione
derivatives [16], 2-phenylindole derivatives [17], and stobadine
[18]. Some of these representative examples of indole derivatives
showing antioxidant activity are displayed in Fig. 1.
reactive species that react via an ionic mechanism. Scheme 1 shows
the proposed plan for substitution pattern of the indole library. The
prenyl chain at N-1 or C-2 was chosen on the basis of a possible H-
abstraction from the
a position (Scheme 1), allowing a radical
stabilization by the allylic double bond as well as by dissipation into
the indole ring. Some reactive species are known to react with
double bonds by a radical whether ionic mechanism, such as the
1O2 and the ꢀNO. Thus the presence of an exocyclic double bond
would enhance the scavenging activity with these species by both
mechanisms. The influence of the position of the prenyl chain was
also evaluated, and consequently the importance of a free indolic
nitrogen.
Moreover, the substitution of the alkyl chain at C-3 was further
explored. Different electron withdrawing groups were used at the
nitrogen atom from the side chain, such as the acetyl group and the
phthaloyl group. These groups were selected due to their easy
preparation, stability and most important the radical stabilization
whether H-abstraction occurs at the
b position. The different
reactivity of tryptamine and tryptophan derivatives provided
evidences of the relevance of a carbomethoxy group at the indolic
side chain for the scavenging of the studied ROS and RNS.
2. Results and discussion
2.1. Chemistry
The development of novel indole derivatives with ROS and RNS
scavenging activities constitutes a required step forward in this
area in view of the myriad of different biological activities displayed
by compounds belonging to this chemical family. The discovery of
new derivatives with a high antioxidant potential, but without
other biological effects may be important to avert adverse reactions
that may arise within a clinical setting. Moreover, HOCl scavenging
activity has been investigated for the marketed indole derived
NSAIDs, but no activity was found against this ROS [6].
Thus, we were encouraged to explore the radical scavenging
potency of a small library of novel synthetic tryptophan and
tryptamine derivatives, by evaluating their in vitro scavenging
activity for ROS (O2ꢀL, H2O2, HOCl, ROOꢀ, 1O2) and RNS (ꢀNO and
ONOOL). These ROS and RNS were chosen due to their hitherto
relatively unexplored reactivity with indole derivatives, in partic-
ular the HOCl.
Our strategy consisted on the synthesis of an indole based
library of compounds, bearing different substituents on positions 1,
2 and 3 of the indole nucleus (Fig. 2), and its evaluation as anti-
oxidants. The obtained results revealed a promising antioxidant
activity against the studied species for the novel synthesized
compounds.
As described above, free radical scavenging and antioxidant
properties have been already reported for some indole derivatives
[6,10a,b,d,], [13e18] thus we were encouraged to explore the radical
scavenging potency of a small library of tryptophan and tryptamine
derivatives [19]. Prenylated indole derivatives belong to a large
class of alkaloids isolated from fungi, possessing a tryptophan
moiety substituted with isoprenic units and exhibiting very
important biological activities such as inhibitors of the cell cycle. To
our knowledge antioxidant property has not been investigated for
any prenylated indole derivative. Moreover, the choice of preny-
lated indole derivatives relies on the assumption that if the radical
Our strategy envisaged the installation of several groups at the
indole moiety that facilitate either a radical scavenging mechanism
via electron donation/H-abstraction or would serve as traps for
is formed in
a position of the prenyl chain (at C-2 or N-1 position of
the indole) there will be conjugation with the indolic ring.
Thus, in this work, several compounds were synthesized start-
ing from L-tryptophan methyl ester and tryptamine according to
the synthetic plan depicted on Scheme 2, and their scavenging
activity against ROS and RNS was evaluated.
Compounds 1 [20a,21a], 5 [20b], 6 [21], 12[20c] and 13[20d]
were synthesized as previously described in the literature. The
synthesis started with nitrogen-protection of 9 and 10, with
phthalimide and acetyl groups being chosen as N-protecting
groups. Compounds 5, 11[20e] and 12 were prepared in moderate
to high yields. Subsequent N-prenylation with NaH in DMF at 0 ꢂC
using either 3,3-dimethylallyl bromide or 3-bromo 1-phenyl
1-propene afforded compounds 3, 4, 6 and 7. Removal of the
phthalimide group from 4 was achieved by treatment with excess
of hydrazine hydrate solution under reflux conditions [22],
provided 2 in 63% yield. Treatment of 6 with BF3.OEt2 at low
temperature afforded 1, according to the Lobo-Prabhakar sigma-
tropic rearrangement conditions of N-prenylindole derivatives [21].
Indoles 13 and 14 were synthesized from the corresponding pre-
nylated precursors 6 and 1 by treatment with hydrazine followed
by alkaline hydrolysis.
Fig. 1. Indole derivatives with antioxidant activity.

M.S. Estevão et al. / European Journal of Medicinal Chemistry 45 (2010) 4869e4878
4871
Scheme 1. Structural requirements for the indole library.
In order to study the influence of the exocyclic double bond on
the reactivity, some hydrogenated compounds were prepared.
Thus, compounds 15, 16, 17 and 18 were obtained by hydrogenation
of the corresponding prenylated precursors (4, 3, 7 and 1, respec-
tively) under Pd/C at room temperature.
chain (C-3) seems to be detrimental for activity (compounds 4 and
15).
In order to investigate the relevance of the unsaturated chain at
the C-2 and N-1 position of indole, several hydrogenated
compounds were prepared (compounds 15 to 18, Fig. 1) and tested.
The total order of reactivity found was 8 > 10 > 9 > 5 > 11 z 12z
13 z 18z2 > 14z1 z 17z7 > 6 > 16 z 3. The hydrogenated
compounds were shown to be effective scavengers of the ROOꢀ, and
some with higher activities than the corresponding unsaturated
compounds (compounds 18 and 1). These results indicate that the
external double bond is not crucial for measured activity, and
suggest that for the scavenging of ROOꢀ the indole nitrogen is the
active redox center of indoles. The indole nucleus is already
reported as the reactive center towards radical species, and this
reactivity has been attributed to its high resonance stability and
very low energy barrier towards radical reactions [23]. Neverthe-
less, the evaluated prenylated compounds possessing a free amine
at the side chain (e.g.13 and 2) have a potency close to that observed
for Trolox (used as control), and most of the tested compounds are
even more active than indomethacin (ORAC reported 0.17) [6].
2.2. Antioxidant activity
The antioxidant activity of the synthesized library was evaluated
by in vitro studies with different methodologies. Table 1 shows the
results obtained for the screening tests against ROS, in particular
HOCl, ROOꢀ and 1O2.
All compounds except 4 and 15 were shown to be effective
scavengers of the ROOꢀ. Indeed some were more active than trolox
(used as control), and also more active than some NSAIDs pos-
sessing the indole ring, like indomethacin, etodolac and acemetacin
[6,7]. The scavenging activity order found for compounds 1 to 14
was: 8 > 10 > 9 > 5 > 11 z 12z13 z 2 > 14 z 1z7 > 6 > 3.
According to the results obtained, the compounds’ library can be
divided in two sets: the non-alkylated compounds (without the
prenyl chain) and the alkylated (prenylated) ones. The presence of
a free amine in the side chain, as well as free indolic nitrogen is
important for scavenging activity. Under these conditions (physi-
ological pH), compounds 14 and 13 exist in solution in the zwit-
terionic form. The non-alkylated compounds revealed to be the
best scavengers of ROOꢀ. Indeed the protection of both nitrogen
atoms (from the side chain C-3 and the indolic nitrogen N-1),
combined with the lack of the carbomethoxy group at the side
Furthermore, the presence of
a prenyl chain makes these
compounds more lipophilic. Lipophilicity is a major determinant
for the disposition and biological action of drugs, making them
suitable for membranes protection against peroxidation.
The same library was screened as scavenger of HOCl (Table 1).
Scavengers of HOCl are of reassured relevance since most NSAIDs
(indole derived) are not able to scavenge this ROS. All the tested
compounds were found to be active against HOCl, and all

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M.S. Estevão et al. / European Journal of Medicinal Chemistry 45 (2010) 4869e4878
Fig. 2. Synthesized indole based library of compounds.
hydrogenated compounds (except 15) were more active than the
corresponding unsaturated compounds. The order of potencies
found was 8 z 18 > 13z14 > 9 z 4 > 1 > 16z10 z 15z5 > 17 >
2 z 12 > 3 z 6> > 11 > 7. Considering that the reaction of amines
with HOCl is well known [24], it was anticipated that compounds
possessing a free amine on the side chain would show higher
activity. Indeed compounds 8, 13, 9, 10 and 2 revealed to be potent
scavengers of HOCl. The carboxylic acid at the side chain (C-3) (8
and 13), revealed to be important for activity since when the
carboxylic acid is methylated (8 vs 10) or is absent (13 vs 2) its
potency decreases.
and 5-methoxytryptamine. On the other hand, the N-acetyl group
may be physiologically important due to its preventing effect on the
degradation of these compounds by monoamine oxidase. In that
aspect, the replacement of the secondary amine by the N-phthaloyl
group may achieve the same protective effect, while maintaining an
effective scavenging activity, as shown for compounds 1, 4, 5, 6, 12,
15 and specially 18. It was not possible to conclude the effect of the
loss of saturation at the exocyclic double bond in the scavenging
activity of this family of compounds. Compound 18 showed a high
potency when compared to the corresponding unsaturated
compound 1. The same was observed for compounds 16 and 3, and
for compounds 17 and 7. However, compounds 4 and 15 showed
inverted reactivity. The results obtained suggest that the observed
reactivity with HOCl results from a conjugation of different func-
tions within the tested substitution pattern and not from an iso-
lated functionalization.
The mechanism involved in the reaction of HOCl with indole
derivatives has not yet been investigated. Nevertheless, while
a recent study in vitro and in vivo concludes that the activity of HOCl
occurs by radical processes [25], other published reports, which face
the problem from the chemical side, always consider ionic mecha-
nisms [26e30]. In addition, in a recent theoretical study, the possible
transition states were calculated for several ionic reaction pathways
[31]. Nevertheless, radical dissociation of HOCl is only discussed
under photolytic catalysis [32]. On the other hand, if peroxides are
The influence of the side chains of the indole nucleus on the HOꢀ
scavenging ability by comparing melatonin with several analogs
was previously evaluated. It was found that the 5-methoxy group as
well the N-acetyl group at the side chain (C-3) were essential for
melatonin to display potent HOꢀ scavenging activity [10c].
However, in the present study, the N-acetyl group, by itself, was
detrimental for HOCl scavenging activity, as may be ascertained by
comparing compound 10 with N-acetyl-methyltryptophan
(compound 11), and also for the lower activity found for
compounds 3 and 7, suggesting that a different reaction mechanism
is involved.
Nevertheless, our results are in accordance to those of Poeggeler
et al. [10a], who demonstrated that tryptamine is a better HOꢀ
scavenger in the ABTS competition assay than N-acetyltryptamine

M.S. Estevão et al. / European Journal of Medicinal Chemistry 45 (2010) 4869e4878
4873
Scheme 2. i) Phthalic anhydride, TEA, toluene, reflux; ii) Ac2O, TEA, AcOEt, DMAP, RT; iii) NaH, DMF, 0 ꢂC, alkyl bromide; iv) NH2NH2.H2O, EtOH, reflux; v) BF3.OEt2, CH2Cl2, ꢃ 14 ꢂC;
vi) 1) NH2NH2.H2O, CH2Cl2/MeOH, RT; 2) 1 M NaOH, MeOH, RT; vii) 10% Pd/C, MeOH, H2, RT.
used instead of HOCl, it seems to be no doubt on the radical character
of the reaction mechanisms involved, as recently discussed by Wang
et al. [33]. Thus, mechanistic studies of the reaction between indole
derivatives and HOCl are being undertaken.
8, 9, 13 and 15 were able to scavenge ꢀNO achieving the IC50 in the
tested range, and the order of reactivity found was
13 z1 >9 > 3 > 8 > 2 >15 (Table 2). The presence of a prenyl chain is
important for the activity against ꢀNO, on position N-1 as well as at
position C-2, compounds 13 and 1 are the most actives. The N-
alkylated compounds are more active against ꢀNO than those pos-
sessing an indolic free nitrogen, with exception of compound 9,
which has a free amine at the side chain. In addition, considering the
IC50, the hydrogenated compound 15 is the less active, supporting
that the loss of unsaturation is detrimental for the activity with this
species. Nitric oxide is known as a nitrating agent in many processes
and it has been used in the nitration of olefins as an economic and
straightforwardly available source of nitrogen [34,35]. This may
explain the scavenging activity found for the prenylated compounds
of this library.
The same library was also evaluated as scavenger of 1O2. All the
assayed compounds, except 4 and 5, were shown to be effective
scavengers of 1O2, in a concentration dependent manner, and the
sequence of activity found was 17 z 2z9 z 16z18 z 3 > 13 z
12z7 > 8z15 z10 > 11 >1 >6. Since reactions with 1O2 could take
place at the exocyclic double bond, we investigated the reactivity of
hydrogenated compounds. Compounds 17, 16 and 18 were more
active than the corresponding unsaturated compounds. From the
activity results obtained, it is not possible to understand which
structural motifs control reactivity of the tested compounds with 1O2.
None of the tested compounds was able to scavenge O2ꢀL and in
what concerns to H2O2 only a residual activity was found for some
of the compounds in the highest tested concentration (2 mM) (data
not shown).
Despite the important role of ꢀNO as vasodilator and platelet
aggregation inhibitor [36], its overproduction has been involved in
pathological conditions such as inflammation and several neuro-
degenerative disorders (Alzheimer and Parkinson’s diseases)
[36,37]. Therefore the ꢀNO scavenging activity found in this library
might be of therapeutical significance.
Table 2 shows the results obtained for the scavenging activity of
the synthesized library against RNS, in particular the scavenging
effect of ꢀNO and ONOOL. From the studied compounds, only 1, 2, 3,

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M.S. Estevão et al. / European Journal of Medicinal Chemistry 45 (2010) 4869e4878
Table 1
Screening test against ROS.
Compound
R1
R2
R3
R4
R5
ROO.
(ORAC)a
HOCl
IC50
1O2
(mM)
1
2
3
4
5
6
7
H
CH2CHC(Me)2
H
H
H
H
H
H
H
H
H
H
H
CO2Me
H
CO2Me
H
Phth
H
H
Phth
Phth
Phth
H
H
H
H
H
Phth
H
H
Phth
H
H
Phth
H
Ac
Phth
Phth
Phth
Ac
H
H
Ac
Ac
Phth
H
H
Phth
Ac
Ac
0.45 ꢁ 0.05
0.96 ꢁ 0.07
0.21 ꢁ 0.03
e
7.18 ꢁ 0.41
26.2 ꢁ 9.55
32.8 ꢁ 6.82
6.34 ꢁ 1.54
11.1 ꢁ 0.78
34.3 ꢁ 6.26
67.7 ꢁ 9.24
3.50 ꢁ 0.40
6.0 ꢁ 0.60
9.60 ꢁ 0.76
z50
27.3 ꢁ 4.84
4.13 ꢁ 0.17
4.56 ꢁ 0.48
10.6 ꢁ 4.80
8.35 ꢁ 1.08
14.2 ꢁ 2.95
3.75 ꢁ 0.52
2307 ꢁ 447
618 ꢁ 94
777 ꢁ 155
e
CH2CHC(Me)2
CH2CHC(Me)2
CH2CHC(Me)2
H
CH2CHC(Me)2
CH2CHCHPh
H
H
H
1.46 ꢁ 0.08
0.36 ꢁ 0.01
0.44 ꢁ 0.01
2.74 ꢁ 0.07
1.86 ꢁ 0.03
2.60 ꢁ 0.16
1.21 ꢁ 0.06
1.09 ꢁ 0.13
1.05 ꢁ 0.05
0.48 ꢁ 0.04
e
e
CO2Me
CO2Me
CO2H
H
2859 ꢁ 499
971 ꢁ 69
992 ꢁ 220
682 ꢁ 102
1043 ꢁ 320
1559 ꢁ 271
943 ꢁ 168
841 ꢁ 59
*
8b
9c
10
11
12
13d
14d
15
16
17
18
H
H
H
CO2Me
CO2Me
CO2Me
COꢃ2
CH2CHC(Me)2
H
CH2CH2CH(Me)2
CH2CH2CH(Me)2
CH2CH2CH2Ph
H
H
CH2CHC(Me)2
H
H
H
COꢃ2
H
1029 ꢁ 216
718 ꢁ 128
506 ꢁ 44
750 ꢁ 128
CO2Me
CO2Me
CO2Me
0.28 ꢁ 0.02
0.44 ꢁ 0.07
0.97 ꢁ 0.09
CH2CH2CH(Me)2
Phth
Phth
* Compound not tested; — not active.
a
Trolox was used as control (ORAC ¼ 1).
b
Tryptophan.
c
Tryptamine.
d
Under physiological conditions 13 and 14 exist in the zwitterionic form.
Concerning the ONOOL, scavenging activity was observed for all
the studied compounds when the study was carried in the presence
of NaHCO3, under these conditions the activity increased. The aim
of performing scavenging studies of ONOOL, in the presence of
NaHCO3 is to simulate the physiological concentrations of CO2 due
to its fast reaction in vivo with ONOOL with formation of CO3ꢀL and
ꢀNO2 leading to oxidation and nitration, respectively [38]. The fact
that a scavenging of ꢀNO2 and CO3ꢀL can enhance the compounds’
Table 2
Screening test against RNS.
IC50 (mM)
Compound
R1
R2
R3
R4
R5
ꢀNO
ONOOꢃ without NaHCO3
ONOOꢃ with NaHCO3
1
2
3
4
5
6
7
H
CH2CHC(Me)2
H
H
H
H
H
H
H
H
H
H
H
CO2Me
H
CO2Me
H
Phth
H
H
Phth
Phth
Phth
H
H
H
H
H
Phth
H
H
Phth
H
H
Phth
H
Ac
Phth
Phth
Phth
Ac
H
H
Ac
Ac
Phth
H
H
Phth
Ac
Ac
239 ꢁ 7
e
28.2 ꢁ 10.2
234 ꢁ 46
162 ꢁ 76
192 ꢁ 31
40.9 ꢁ 9.5
241 ꢁ 92.9
36.3 ꢁ 2.8
19.2 ꢁ 2.1
10.4 ꢁ 3.2
13.1 ꢁ 5.9
22.9 ꢁ 8.9
29.5 ꢁ 3.6
42.4 ꢁ 22.8
*
CH2CHC(Me)2
CH2CHC(Me)2
CH2CHC(Me)2
H
CH2CHC(Me)2
CH2CHCHPh
H
H
1430 ꢁ 108
905 ꢁ 66
545 ꢁ 118
e
980 ꢁ 142
e
H
e
93.0 ꢁ 36.3
e
CO2Me
CO2Me
CO2H
H
CO2Me
CO2Me
CO2Me
CO2H
CO2H
H
e
e
218 ꢁ 26
31.9 ꢁ 9.6
31.8 ꢁ 9.9
47.8 ꢁ 7.4
223 ꢁ 128
208 ꢁ 49
94.7 ꢁ 40.0
*
8a
9b
10
11
12
13c
14
15
16
17
18
1151 ꢁ 203
672 ꢁ 105
H
H
H
e
e
e
CH2CHC(Me)2
H
CH2CH2CH(Me)2
CH2CH2CH(Me)2
CH2CH2CH2Ph
H
H
208 ꢁ 26
CH2CHC(Me)2
H
H
H
*
1526 ꢁ 62
e
237 ꢁ 55
315 ꢁ 83
142 ꢁ 24
14.0 ꢁ 6.8
CO2Me
CO2Me
CO2Me
e
e
e
548 ꢁ 154
e
CH2CH2CH(Me)2
Phth
Phth
e
* Compound not tested;; — not active.
a
Tryptophan.
Tryptamine.
b
c
Under physiological conditions 13 exits in the zwitterionic form.

M.S. Estevão et al. / European Journal of Medicinal Chemistry 45 (2010) 4869e4878
4875
efficiency [39] may explain why the library compounds were more
active in the presence of NaHCO3.
internal reference (dH 0.00) for 1H-NMR spectra and to CDCl3
(dC
77.00) for 13C-NMR spectra. High resolution mass spectra were
recorded on an AutoSpecQ spectrometer. IR spectra were run on an
FT PerkineElmer 683 instrument, with absorption frequencies
expressed in reciprocal centimetres. Thin-layer chromatography
was performed on Merck silica gel 60 F254 plates and PTLC on
0.5 mm thick plates. Column chromatography was carried out on
Merck silica gel 60 (70e230 mesh). Usual work-up implies drying
the water- or brine-washed organic extracts over anhydrous sodium
sulfate or magnesium sulfate, followed by filtration and evaporation
of the solvent from the filtrate under reduced pressure. Anhydrous
solvents were dried as described [37] and freshly distilled.
The results obtained for this library show that the presence of
a free amine at the side chain, as well as a free indolic nitrogen atom
is important for activity. For the scavenging of ONOOL, alkylation at
N-1 is detrimental. However when alkylation occurs at C-2
(compounds 18 and 1), leaving the N-1 position free, the reactivity
of the indolic moiety is increased, since compound 12 is less active.
The fact that the prenylated and hydrogenated compounds’ activ-
ities were slightly different (4/15, 18/1 and 16/3) indicated that the
reaction with this radical has a different mechanism than with ꢀNO,
and that the indolic moiety is the crucial reaction center. NSAIDs
derived from indole, like indomethacin and etodolac, also
demonstrated to be good scavengers of this radical [6].
4.1.1. Na-(3,3-Dimethylallyl)-tryptamine (2)
The involvement of ONOOL in diseases like atherosclerosis has
already been reported, thus the scavenging effect of ONOOL
demonstrated by this library may be therapeutically useful for the
design of improved agents [40].
A solution of Na-(3,3-Dimethylallyl)-Nb-phthaloyl-tryptamine
(4) (300 mg, 0.84 mmol) in ethanol (29 mL) was heated to reflux till
complete solubilisation. The solution was cooled and hydrazine
hydrate (153 mL, 4.21 mmol) was added. The mixture was refluxed
These results are now being used for the rationalisation of the
reaction mechanism of these compounds with the ROS and RNS.
overnight and the reaction progress was monitored by TLC (CH2Cl2/
MeOH/Et3N, 10:1:0.5). The solvent was removed under reduced
pressure and the crude dissolved in an aqueous solution of NaOH
2 M (40 mL) and extracted three times with CHCl3/i-PrOH (4:1)
(40 mL). The organic layer was dried over anhydrous MgSO4,
filtered and the solvent removed. After column chromatography
(CH2Cl2/MeOH/Et3N, 10:1:0.5), compound 2 was obtained in 63%
3. Conclusion
A
library of tryptamine and tryptophan derivatives was
prepared and evaluated for the scavenging activity against ROS and
RNS. The tested indole derivatives share a heteroaromatic ring
system, differing among them by the presence of diverse side
chains with different functionalization. The results obtained in the
present study reveal a strong scavenging effect of tryptophan and
tryptamine for the tested ROS and RNS, which activity varies in
different extensions, depending on the substituents. Best results of
HOCl scavenging were obtained for the tryptophan (8) and trypt-
yield (119 mg) as yellow oil [38]. 1H-NMR ([D1]CHCl3):
d
¼ 7.57 (d,
3J ¼ 7.8 Hz, 1H; Ar-H), 7.28 (d, 3J ¼ 8.0 Hz, 1H; Ar-H), 7.18
(t, 3J ¼ 7.3 Hz, 1H; Ar-H), 7.07 (t, 3J ¼ 7.3 Hz, 1H; Ar-H), 6.90 (s, 1H;
NCH ¼ C), 5.34 (t, 3J ¼ 6.5 Hz, 1H; CH]C(CH3)2), 4.61 (d, 3J ¼ 6.8 Hz,
2H; CH2CH]C(CH3)2), 2.97 (t, 3J ¼ 6.5 Hz, 2H; NCH2CH2), 2.86
(t, 3J ¼ 6.5 Hz, 2H; NCH2CH2), 1.87 (sl, 2H; NH2 exchangeable with
D2O), 1.79 (s, 3H; CH]C(CH3)2), 1.74 (s, 3H; CH]C(CH3)2) ppm. 13
C
amine (9) with IC50s of 3.50 ꢁ 0.4 and 6.00 ꢁ 0.60
Furthermore, the prenylated compounds 13 and 14 also demon-
strated to be potent scavengers with IC50s of 4.13 ꢁ 0.17 M and
4.56 ꢁ 0.48 M, respectively. Although the position of the prenyl
m
M, respectively.
NMR ([D1]CHCl3):
d
¼ 136.4, 135.9, 128.1, 125.4, 121.4, 120.1, 118.9,
118.6, 112.1, 109.4, 43.7, 42.2, 29.1, 25.4, 17.7 ppm; IR (NaCl):
m
y
¼ 3300, 3050, 2960, 2920 cmꢃ1; HRMS (ESI-TOF) [M þ 1]þ m/z
m
C15H20N20O2: 228.16265 found 229.1699.
chain is not relevant for this ROS, these are relevant results since
the presence of a prenyl chain turns these compounds more lipo-
philic. For the ROOꢀ some compounds were more potent than
trolox (control). The library was also evaluated against RNS and the
best result was obtained for the scavenging of ONOOL, in the
presence of NaHCO3, by the N-alkylated tryptophan (18), with an
4.1.2. Na-(3,3-dimethylallyl)-Nb-acetyl-tryptophan methyl ester (3)
To a solution of N-acetyl- -tryptophan methyl ester (11) (0.5 g,
L
1.9 mmol) in dry DMF (5 mL) was added NaH (93 mg, 2.30 mmol,
60% oil dispersion) and the resulting mixture was stirred for 10 min
in an ice bath. 3,3-Dimethylallyl bromide (0.41 mL, 3.5 mmol) was
added and the resulting mixture stirred for 30 min at 0 ꢂC. The
mixture was diluted with AcOEt (25 mL), washed five times with
distilled water (25 mL). The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent removed. The crude residue was
IC50 of 14 ꢁ 6.8
mM, that demonstrated that the double bond is not
crucial for the reactivity with this species.
Unquestionably, thepotentfreeradicalscavengingcapacityfound
for the tested compounds relies mainly on their electron-rich indole
moieties, which hold high resonance stability. On the other hand, the
substituents at the indole backbone substantially change the reac-
tivity, potency and efficiency of radical scavenging. The effect of the
prenyl chain was sometimes favourable and others unfavourable,
depending on the reactive species. The indole derivatives synthe-
sized and tested in the present study were shown to be of reassuring
importance for the development of new antioxidant drugs.
purified by column chromatography (silica, Et2O) and compound 3
20
was isolated as an yellow oil in 57% yield (0.36 g): [
a]
¼ þ 27.8 (c
D
3.8, Et2O); 1H-NMR ([D1]CHCl3):
d
¼ 7.50 (d, 3J ¼ 7.8 Hz, 1H; Ar-H),
7.29 (d, 3J ¼ 8.2 Hz, 1H; Ar-H), 7.19 (t, 3J ¼ 7.4 Hz, 1H; Ar-H), 7.09 (t,
3J ¼ 7.3 Hz, 1H; Ar-H), 6.85 (s, 1H; Ar-H), 5.99 (d, 3J ¼ 7.2 Hz, 1H; NH,
exchangeable with D2O), 5.35e5.32 (m, 1H; CH]C(CH3)2),
4.94e4.90 (m, 1H; CHCO2CH3), 4.63 (d, 3J ¼ 6.8 Hz, 2H; CH2CH]C
(CH3)2), 3.69 (s, 3H; OCH3), 3.35e3.25 (m, 2H; CH2CHCO2CH3), 1.96
(s, 3H; NCOCH3), 1.81 (s, 3H; CH]C(CH3)2), 1.76 (s, 3H; CH]C
4. Experimental
(CH3)2) ppm; 13C-NMR ([D1]CHCl3):
128.5, 126.0, 121.7, 119.8, 119.3, 118.7, 109.7, 108.5, 53.3, 52.3, 44.0,
d
¼ 172.4, 169.7, 136.5, 136.2,
4.1. General
27.6, 25.6, 23.3, 18.0 ppm; IR (NaCl):
y
¼ 1743,1657 cmꢃ1; HRMS (EI)
Melting points were determined on a Reichert Thermovar
apparatus and are uncorrected. Optical rotations were determined
[M þ 1]þ m/z: C19H24N2O3: 329.17047 found 328.177956.
in a PerkineElmer 241 MC polarimeter at RT, and [
a
]
values are
4.1.3. Na-(3,3-Dimethylallyl)-Nb-phthaloyl-tryptamine (4)
To a solution of N-phthaloyl-tryptamine (5) (50 mg, 0.17 mmol)
in dry DMF (0.5 ml) was added NaH (10 mg, 0.24 mmol, 60% oil
dispersion) and the resulting mixture was stirred for 45 min in an
D
given in 10ꢃ1 deg cm2 gꢃ1. Ordinary mass spectra were recorded on
a Fisons TRIO 2000 or AEI MS-9 spectrometers. 1H- and 13C-NMR
spectra were recorded in CDCl3 on a Bruker ARX 400 spectrometer.
Chemical shifts are reported relative to tetramethylsilane as the
ice bath. 3,3-Dimethylallyl bromide (19 ml, 0.17 mmol) was added

4876
M.S. Estevão et al. / European Journal of Medicinal Chemistry 45 (2010) 4869e4878
and the resulting mixture stirred for 2.5 h at room temperature. The
mixture was diluted with AcOEt (25 mL), washed five times with
distilled water (25 mL). The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent removed. The crude residue was
purified by crystallization (CH2Cl2, petroleum ether) and
compound 4 was obtained as yellow crystals in 53% yield (33 mg)
25.5, 24.7, 17.8 ppm; IR (NaCl):
m/z C16H20N2O2: 272.1525 found: 272.1528.
y
¼ 1748 cmꢃ1; HRMS (ESI-TOF) [M]þ
4.1.6. General procedure for hydrogenation
To a solution of tryptophan or tryptamine derivatives in MeOH
(55 mM) was added 10% Pd/C (ca. 61 mg per mmol). The mixture
was stirred at room temperature under H2 atmosphere. After
starting material consumption (TLC), the reaction mixture was
filtered and concentrated under reduced pressure.
[38]: M.p. ¼ 130e132 ꢂC; 1H-NMR ([D1]CHCl3):
d
¼ 7.80e7.83 (m,
2H; Ar-H), 7.73 (d, 3J ¼ 7.6 Hz, 1H; Ar-H), 7.69e7.66 (m, 2H; Ar-H),
7.28 (d, 3J ¼ 8.0 Hz, 1H; Ar-H), 7.19 (t, 3J ¼ 7.4 Hz, 1H; Ar-H), 7.11 (t,
3J ¼ 7.2 Hz, 1H; Ar-H), 6.99 (s, 1H; NCH ¼ C), 5.34 (t, 3J ¼ 6.2 Hz, 1H;
CH]C(CH3)2), 4.62 (d, 3J ¼ 6.7 Hz, 2H; CH2CH]C(CH3)2), 3.97
(t, 3J ¼ 7.9 Hz 2H; NCH2CH2), 3.12 (t, 3J ¼ 7.9 Hz, 2H; NCH2CH2), 1.80
(s, 3H; CH]C(CH3)2),1.75 (s, 3H; CH]C(CH3)2) ppm; 13C-NMR ([D1]
4.1.6.1. Na-(Isopentyl)-Nb-phthaloyl-tryptamine (15). Purified by
column chromatography (silica, hexane/Et2O, 3:2) affording
compound 15 with 88% yield as an yellow oil. 1H-NMR ([D1]CHCl3):
CHCl3):
d
¼ 168.3, 136.3, 136.2, 133.8, 132.3, 128.1, 125.4, 123.1, 121.5,
d
¼ 7.85e7.83 (m, 2H; Ar-H), 7.73 (d, 3J ¼ 8.1 Hz, 1H; Ar-H),
120.0, 119.0, 119.0, 110.8, 109.5, 43.9, 38.7, 25.7, 24.5, 18.0 ppm; IR
(NaCl):
C23H22N2O3: 359.17595 found 359.1754.
7.71e7.69 (m, 2H; Ar-H), 7.30 (d, 3J ¼ 8.1 Hz, 1H; Ar-H), 7.20 (t,
3J ¼ 7.5 Hz, 1H; Ar-H), 7.11 (t, 3J ¼ 7.3 Hz, 1H; Ar-H), 6.70 (s, 1H;
NCH ¼ C), 4.07 (t, 3J ¼ 7.3 Hz, 2H; NCH2CH2CH(CH3)2), 3.99 (t,
3J ¼ 7.8 Hz, 2H; NCH2CH2C(CH3)2), 3.14 (t, 3J ¼ 7.7 Hz, 2H;
NCH2CH2C), 1.68 (dd, 3J ¼ 7.0, 14.3 Hz, 2H, NCH2CH2CH(CH3)2),
1.57e1.52 (m, 1H; NCH2CH2CH(CH3)2), 0.94 (d, 3J ¼ 6.4 Hz, 6H,
y
¼ 1764, 1708 cmꢃ1; HRMS (ESI-TOF) [M þ 1]þ m/z
4.1.4. Na-(E-cinnamyl)-Nb-acetyl-tryptophan methyl ester (7)
To a solution of N-acetyl- -tryptophan methyl ester (11) (50 mg,
L
0.2 mmol) in dry DMF (0.5 mL) was added NaH (10 mg, 0.24 mmol,
60% oil dispersion) and the resulting mixture was stirred for 10 min
in an ice bath. 3-bromo-1-phenyl-1-propene (45 mg, 0.24 mmol)
was added and the resulting mixture stirred for 2 h at 0 ꢂC. The
mixture was diluted with AcOEt (25 mL), washed five times with
distilled water (25 mL). The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent removed. The crude residue was
purified by column chromatography (silica gel, ethyl ether) and
NCH2CH2CH(CH3)2) ppm; 13C-NMR ([D1]CHCl3):
d
¼ 168.3, 136.2,
133.8, 132.2, 127.8, 125.6, 123.1, 121.5, 119.1, 118.8, 110.7, 109.3, 44.3,
39.0, 38.6, 25.6, 24.4, 22.4 ppm; IR (NaCl):
y
¼ 2955, 1770, 1714,
1396 cmꢃ1; HRMS (EI) [M]þ m/z C23H24N2O2: 360.18378 found
360.1836.
4.1.6.2. Na-(Isopentyl)-Nb-acetyl-tryptophan
methyl
ester
(16). Purified by column chromatography (silica, Et2O) affording
compound 7 was isolated as white crystals in 72% yield (52 mg):
compound 16 with 85% yield as colorless oil. 1H-NMR ([D1]CHCl3):
20
M.p. 76e77 ꢂC; [
d
a
]
¼ þ 15.4 (c 4.8, Et2O); 1H-NMR ([D1]CHCl3):
d
¼ 7.51 (d, 3J ¼ 7.9 Hz; 1H, Ar-H), 7.31 (d, 3J ¼ 8.2 Hz, 1H; Ar-H), 7.20
D
¼ 7.56 (d, 3J ¼ 7.8 Hz, 1H; Ar-H), 7.38e7.01 (m, 8H; Ar-H), 6.94 (s,
(t, 3J ¼ 7.4 Hz, 1H; Ar-H), 7.10 (t, 3J ¼ 7.3 Hz, 1H; Ar-H), 6.86 (s, 1H;
Ar-H), 5.99 (d, 3J ¼ 7.2 Hz, 1H; NH), 4.96e4.91 (m, 1H; CHCO2CH3),
4.08 (t, 3J ¼ 7.3 Hz, 2H; NCH2CH2CH(CH3)2), 3.70 (s, 3H; OCH3),
3.35e3.25 (m, 2H; CH2CHCO2CH3), 1.96 (s, 3H; NCOCH3), 1.69 (dd,
3J ¼ 14.5, 7.2 Hz, 2H; NCH2CH2CH(CH3)2), 1.61e1.51 (m, 1H;
NCH2CH2CH(CH3)2), 0.96 (d, 3J ¼ 6.4 Hz, 6H, NCH2CH2CH(CH3)2)
3
1H; Ar-H), 6.43 (d, J ¼ 16 Hz, 1H; CH ¼ CHCH2), 6.32 (dt, J ¼ 15.8,
6.0 Hz, 1H; CH ¼ CHCH2), 6.14 (d, 3J ¼ 7.3 Hz, 1H; NH, exchangeable
with D2O), 4.99e4.95 (m, 1H; CHCO2CH3), 4.86 (d, 3J ¼ 5.3 Hz, 2H;
NCH2), 3.68 (s, 3H; OCH3), 3.40e3.29 (m, 2H; CH2CHCO2CH3), 1.97
(s, 3H; NCOCH3) ppm; 13C-NMR ([D1]CHCl3):
d
¼ 172.4, 169.7, 136.3,
136.1, 132.3, 128.6, 128.5, 127.9, 126.4, 126.3, 124.7, 121.9, 119.4, 118.7,
ppm. 13C-NMR ([D1]CHCl3):
d
¼ 172.4, 169,6, 136.1, 128.3, 126.2,
109.7, 109.2, 52.2, 52.3, 48.2, 27.6, 23.2 ppm; IR (NaCl):
y
¼ 3399,
121.6, 119.1, 118.7, 109.5, 108.4, 53.1, 52.2, 44.4, 38.9, 27.5, 25.6, 23.2,
2853, 1741, 1651 cmꢃ1; HRMS (ESI-TOF) [M þ 1]þ m/z C23H24N2O3:
22.4 ppm; IR (NaCl):
y
¼ 3286, 2955, 1745, 1656, 1469 cmꢃ1; HRMS
377.18652 found 377.1860.
(EI) [M]þ m/z C19H26N2O3: 330.19434 found 330.1941.
4.1.5. 2-(3,3-Dimethylallyl)-tryptophan (14)
4.1.6.3. Na-(3-Phenylpropyl)-Nb-acetyl-tryptophan methyl ester
(17). Purified by column chromatography (silica, Et2O) affording
compound 17 as pale yellow oil with 88% yield. 1H-NMR ([D1]
To a solution of 2-(3,3-dimethylallyl)-Nb-phthaloyl-tryptophan
methyl ester (1) (636 mg, 1.53 mmol) in 3:1 MeOH/CH2Cl2
(11.5:3.8 mL) was added hydrazine hydrate (0.26 mL, 5.36 mmol).
The flask was capped and the solution stirred at ambient temper-
ature for 24 h, during which time a white precipitate formed. The
solution was poured into water (73 mL) and extracted with CH2Cl2
(4 ꢄ 15 mL). The combined organic extracts were dried (Na2SO4),
filtered, concentrated. The resulting crude was purified by column
chromatography (CH2Cl2/MeOH 10:1). The product was isolated as
pale yellow oil in 70% yield (307 mg). The product previously
prepared (30 mg, 0.105 mmol) was dissolved in 1 M NaOH (0.1 mL)
and MeOH (0.16 mL). The resulting mixture was stirred at room
temperature overnight. The mixture was concentrated and acidi-
fied with 1 M HCl to pH 7e8 at 0 ꢂC. The precipitate which formed
was filtrated, washed with cold water and dried. Compound 14 was
isolated as light beige solid in 87% yield (24.8 mg): M.p. > 300 ꢂC
CHCl3):
d
¼ 7.52 (d, 3J ¼ 7.8 Hz, 1H; Ar-H), 7.32e7.09 (m, 8H; Ar-H),
6.87 (s, 1H; Ar-H), 5.99 (d, 3J ¼ 7.3 Hz, 1H; NH), 4.98e4.93 (m, 1H;
CHCO2CH3), 4.10 (t, 3J ¼ 6.9 Hz, 2H; NCH2CH2CH2), 3.70 (s, 3H;
OCH3), 3.37e3.26 (m, 2H; CH2CHCO2CH3), 2.61 (t, 3J ¼ 7.5 Hz, 2H,
NCH2CH2CH2), 2.19e2.12 (m, 2H; NCH2CH2CH2), 1.96 (s, 3H;
NCOCH3) ppm; 13C-NMR ([D1]CHCl3):
128.5, 128.3, 126.3, 126.1, 121.7, 119.2, 118.8, 109.5, 108.6, 53.2, 52.3,
d
¼ 172.4, 169.6, 140.8, 136.2,
45.5, 32.9, 31.5, 27.6, 23.2 ppm; IR (NaCl):
y
¼ 3392, 2926, 1744,
1656, 1469 cmꢃ1; HRMS (EI) [M]þ m/z C23H26N2O3: 378.19434
found 378.1948.
4.1.6.4. 2-(Isopentyl)-Nb-phthaloyl-tryptophan
methyl
ester
(18). Purified by column chromatography (silica, hexane/Et2O, 1:1)
affording compound 18 with 83% yield as yellow solid.
(decomp.); 1H-NMR ([D6]DMSO):
d
¼ 10.72 (s, 1H; NH), 7.50 (d,
M.p. ¼ 51e53 ꢂC; 1H-NMR ([d1]CHCl3):
d
¼ 7.75e7.72 (m, 3H; NH
3J ¼ 7.6 Hz, 1H; Ar-H), 7.25 (d, 3J ¼ 8.0 Hz, 1H; Ar-H), 6.98e6.88 (m,
2H; Ar-H), 5.29 (m, 1H; CH]C(CH3)2), 4.03e3.98 (m, 1H;
CHCO2CH3), 3.30 (2H; CH2CH]C(CH3)2, under water peak),
2.89e2.83 (m, 2H; CH2CHCO2H), 1.70, (s, 3H; CH]C(CH3)2), 1.66 (s,
and Ar-H), 7.65e7.63 (m, 2H; Ar-H), 7.48 (d, 3J ¼ 7.6 Hz, 1H; Ar-H),
7.16 (d, 3J ¼ 7.8 Hz, 1H; Ar-H), 7.03e6.94 (m, 2H; Ar-H), 5.21 (dd,
3J ¼ 5.5, 10.0 Hz, 1H; CHCO2CH3), 3.79 (s, 3H; OCH3), 3.68e3.64 (m,
2H; CH2CHCO2CH3), 2.74e2.57 (m, 2H, CCH2CH2CH(CH3)2),
1.56e1.44 (m, 1H; CCH2CH2CH(CH3)2), 1.38e1.30 (m, 2H;
CCH2CH2CH(CH3)2), 0.90 (d, 3J ¼ 6.4 Hz, 3H; CCH2CH2CH(CH3)2),
3H; CH]C(CH3)2) ppm; 13C-NMR ([D6]DMSO):
d
¼ 170.8, 136.8,
135.5, 131.7, 128.1, 121.7, 120.0, 118.1, 117.7, 110.6, 105.0, 55.6, 26.5,

M.S. Estevão et al. / European Journal of Medicinal Chemistry 45 (2010) 4869e4878
4877
0.87 (d, 3J ¼ 6.4 Hz, 3H; CCH2CH2CH(CH3)2) ppm; 13C-NMR ([D1]
4.2.1.7. Peroxyl radical scavenging assay. The ROOꢀ scavenging
activity was measured by monitoring the effect of the tested
compounds on the fluorescence decay resulting from ROOꢀ-
induced oxidation of fluorescein and expressed as the “Oxygen
Radical Absorbance Capacity” (ORAC), as previously described [43].
Trolox was used as the standard control in each assay. The results
were expressed as ORAC values. Each study corresponds to four
experiments, performed in triplicate.
CHCl3):
d
¼ 169.7, 167.4, 137.3, 135.1, 133.9, 131.7, 128.5, 123.3, 121.0,
119.3, 117.8, 110.2, 106.0, 52.7, 52.5, 38.6, 27.9, 24.0, 23.8, 22.3 ppm;
IR (NaCl):
C25H26N2O4: 418.18926 found 418.1894.
y
¼ 3401, 2955, 1715 cmꢃ1; HRMS (EI) [M]þ m/z
4.2. ROS and RNS scavenging assays
4.2.1. Materials and methods
4.2.1.1. Equipment. A microplate reader (Synergy HT, BIO-TEK), for
fluorescence, absorbance in UV/vis and luminescence measure-
ments, equipped with a thermostat, was used for all the assays.
4.2.1.8. Peroxynitrite scavenging assay. The ONOOꢃ scavenging
activity was measured by monitoring the effect of the tested
compoundsonONOOꢃ-induced oxidation ofnon-fluorescent DHRto
fluorescentrhodamine123, aspreviouslydescribed[41]. Ebselenwas
used as positive control. In a parallel set of experiments, the assays
were performed in the presence of 25 mM NaHCO3 in order to
simulate the physiological CO2 concentrations. This evaluation is
important because, under physiological conditions, the reaction
between ONOOꢃ and bicarbonate is predominant, with a very fast
4.2.1.2. Chemicals. All the chemicals and reagents were of
analytical grade. Dihydrorhodamine 123 (DHR), 4,5-diamino-
fluorescein (DAF-2), 30% hydrogen peroxide, sodium hypochlorite
solution, with 4% available chlorine, diethylenetriaminepenta-
acetic acid (DTPA), 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-
oxo-1-triazene (NOC-5),
b
-nicotinamide adenine dinucleotide
rate constant (k2 ¼ 3e5.8 ꢄ 104 Mꢃ1
s
ꢃ1) [42]. The inhibition (in
(NADH), phenazine methosulfate (PMS), nitroblue tetrazolium
chloride (NBT), tiron, lucigenin, ascorbic acid, lipoic acid, ebselen,
and rutin were obtained from SigmaeAldrich (St. Louis, USA).
percentage) of ONOOꢃ-induced oxidation of DHR byeach compound
was calculated. The results were expressed by the IC50 values. Each
study corresponds to four experiments, performed in triplicate.
a,a
0-Azodiisobutyramidine dihydrochloride (AAPH), histidine and
trolox were obtained from Fluka Chemie GmbH (Steinheim,
Germany). Fluorescein sodium salt was obtained from Aldrich
(Milwaukee, USA).
4.2.1.9. Nitric oxide scavenging assay. The ꢀNO scavenging activity
was measured by monitoring the effect of the tested compounds on
ꢀNO-induced oxidation of non-fluorescent DAF-2 to the fluorescent
triazolo fluorescein (DAF-2T), as previously described [41]. Rutin
was used as positive control. The inhibition (in percentage) of ꢀNO-
induced oxidation of DAF-2 by each compound was calculated. The
results were expressed by the IC50 values. Each study corresponds
to four experiments, performed in triplicate.
ꢀL
4.2.1.3. Superoxide radical scavenging assay.ꢀLThe O2
was gener-
ated by the NADH/PMS system and the O2
scavenging activity
was determined by monitoring the effect of the tested compounds
on the O2ꢀL-induced reduction of NBT at 560 nm as previously
described [41]. The antioxidant tiron was used as positive control.
The inhibition (in percentage) of the NBT reduction to diformazan
by each compound was calculated. The results were expressed by
the IC50 values. Each study corresponds to four experiments, per-
formed in triplicate.
Acknowledgments
The authors greatly acknowledge FCT financial support for the
project PTDC/QUI/65187/2006 and for the fellowships to M. S.
Estevão SFRH/BD/46234/2008, L. C. Carvalho SFRH/BD/63407/2009,
Marisa Freitas (SFRH/BD/28502/2006) and Ana Gomes (SFRH/BPD/
63179/2009).
4.2.1.4. Hydrogen peroxide scavenging assay. The H2O2 scavenging
activity was measured by monitoring the effect of the tested
compounds on the H2O2-induced oxidation of lucigenin as previ-
ously described [41]. The antioxidant ascorbic acid was used as
positive control. The inhibition (in percentage) of the H2O2-induced
oxidation of lucigenin by each compound was calculated. The
results were expressed by the IC50 values. Each study corresponds
to four experiments, performed in triplicate.
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