4-Nerolidylcatechol and its synthetic analogues: Antioxidant activity and toxicity evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.12.028

4-Nerolidylcatechol (1) is a secondary metabolite of plants and is described as a promising anti-inflammatory, antimalarial, antiulcerogenic, analgesic and cytotoxic compound possibly due to its antioxidant profile. In this study, we evaluated the pharmacologic activity and the antioxidant and toxicological profiles of compound (1) and its synthetic analogues (2-6). The synthetic analogues were designed from the lead compound, (1), using a molecular-simplification strategy. Compound 5 showed, by 1,1-diphenyl-2- picrylhydrazyl (DPPH) and β-carotene systems, similar antioxidant activity when compared to compound (1). The oxidative stress in erythrocyte membrane demonstrated the highly protective effect of compounds (4), (5) and (6) and high antioxidant/pro-oxidant activity in relation to the concentrations of compounds (1) and (3). Compounds (2), (4), (5) and (6) were haemobiocompatible. All compounds (1-6) showed cytotoxic effects in 3T3 cells, but compounds (2) and (6) were highly cytotoxic in this lineage when compared to compound (1). Compound (5) had a lower myelosuppressive effect in haematopoietic progenitor cells compared to (1). Both compounds, (1) and (5), showed low genotoxic effects in vitro, on human lymphocyte cells. In addition, these compounds also showed low-toxicity in vivo as defined a LD₅₀ > 2000 mg/kg. In this assay, we did not observe death in the animals exposed to treatment with (1) and (5) compound. In conclusion, the structural design of the analogues as validated once compound (5) was found to have an antioxidant profile that was as potent as the lead compound (1). In addition, considering the safety profile, these compounds are promising as preventive and/or therapeutic agents against oxidative damage.

Full text of DOI:10.1016/j.ejmech.2012.12.028

European Journal of Medicinal Chemistry 62 (2013) 371e378
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
4-Nerolidylcatechol and its synthetic analogues: Antioxidant activity
and toxicity evaluation
Carla Rosane Mendanha da Cunha a, Sebastião Antônio Mendanha Neto b,
Claudio Carlos da Silva c, Alane Pereira Cortez a, Marcelo do Nascimento Gomes e,
Fabiula Ines Martins e, Antônio Alonso b, Kênnia Rocha Rezende d, Ricardo Menegatti e,
,
Mariana Torquato Quezado de Magalhães f, Marize Campos Valadares a
*
a Laboratory of Cellular Pharmacology and Toxicology, Faculty of Pharmacy, Federal University of Goiás, Goiás, Brazil
b Laboratory of Biophysics, Physics Institute, Federal University of Goiás, Goiás, Brazil
c Laboratory of Cytogenetic and Molecular Genetics, Replicon, PUC-Go, Goiás, Brazil
d BioPK Laboratory, Faculty of Pharmacy, Federal University of Goiás, Goiás, Brazil
e Laboratory of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, Federal University of Goiás, Goiás, Brazil
f Laboratory of Enzymology, Institute of Biological Sciences, Federal University of Goiás, Goiás, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 June 2012
Received in revised form
2 November 2012
Accepted 12 December 2012
Available online 20 December 2012
4-Nerolidylcatechol (1) is a secondary metabolite of plants and is described as a promising anti-
inflammatory, antimalarial, antiulcerogenic, analgesic and cytotoxic compound possibly due to its
antioxidant profile. In this study, we evaluated the pharmacologic activity and the antioxidant and
toxicological profiles of compound (1) and its synthetic analogues (2e6). The synthetic analogues were
designed from the lead compound, (1), using a molecular-simplification strategy. Compound 5 showed,
by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and
b-carotene systems, similar antioxidant activity when
compared to compound (1). The oxidative stress in erythrocyte membrane demonstrated the highly
protective effect of compounds (4), (5) and (6) and high antioxidant/pro-oxidant activity in relation to
the concentrations of compounds (1) and (3). Compounds (2), (4), (5) and (6) were haemobiocompatible.
All compounds (1e6) showed cytotoxic effects in 3T3 cells, but compounds (2) and (6) were highly
cytotoxic in this lineage when compared to compound (1). Compound (5) had a lower myelosuppressive
effect in haematopoietic progenitor cells compared to (1). Both compounds, (1) and (5), showed low
genotoxic effects in vitro, on human lymphocyte cells. In addition, these compounds also showed low-
toxicity in vivo as defined a LD50 > 2000 mg/kg. In this assay, we did not observe death in the
animals exposed to treatment with (1) and (5) compound. In conclusion, the structural design of the
analogues as validated once compound (5) was found to have an antioxidant profile that was as potent as
the lead compound (1). In addition, considering the safety profile, these compounds are promising as
preventive and/or therapeutic agents against oxidative damage.
Keywords:
4-Nerolidylcatechol
Drug design
Antioxidant activity
Toxicity
Safety profile
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
of the oxidant/antioxidant balance [3e5]. In this context, ROS can
result in mutagenesis and can contribute to the initiation, promo-
Plants have a vast diversity of defence chemicals, known as
secondary metabolites. In general, these substances have specific
functions including protection against oxidative or UV irradiation.
Secondary metabolites such as catechol, flavanoids and lignins are
potent antioxidants [1,2]. Supplementation with antioxidants in
order to boost the production of endogenous antioxidants or
scavenge excessive reactive oxygen species (ROS) production could
be utilised to prevent some diseases by promoting the restoration
tion and progression of cancer and other degenerative diseases
such as heart attack, stroke, arthritis and cataracts [5,8]. Many
reports have suggested that dietary phenolic compounds also
exhibit pro-oxidant properties, depending on their concentration at
the site of action [6e8]. In human cells, pro-oxidant activity can
induce DNA damage and other pathophysiological processes [1,2].
These processes are associated with elevated levels of ROS, which
may readily react with the surrounding biological tissues and
damage lipids, nucleic acids and proteins [1,7,9].
4-Nerolidylcatechol (4-NRC) (1) is the main secondary metab-
olite found in Brazilian plants of the genus Pothomorphe, in special
* Corresponding author.
E-mail address: marizecv@farmacia.ufg.br (M.C. Valadares).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.12.028

372
C.R. Mendanha da Cunha et al. / European Journal of Medicinal Chemistry 62 (2013) 371e378
Table 1
Pothomorphe umbellata. This species grows in the states of São
Paulo, Minas Gerais, Espírito Santo and Bahia [10]. Several biolog-
ical activities have been described for 4-NRC (1), including anti-
inflammatory, antimalarial, antiulcerogenic, analgesic, and cyto-
toxic effects against MCF-7, B16 melanoma, HCT-8, CEM and HL-60
cells and, in special, antioxidant properties [10e16]. The antioxi-
dant profile of the P. umbellate ethanolic extract was superior that
Balb/c 3T3-A31 (1 ꢁ105 cells) NRU cytotoxicity assay after 48 h exposure with (1e6)
compounds using in vitro data to estimate the in vivo starting doses for acute toxicity.
Compound
test
Molecular
weight
IC50 (mM)
Estimated oral
mouse LD50
(mmol/kg)
Estimated
oral mouse
LD50 (mg/kg)
4-NRC (1)
318
137
247.2
229
245
279
0.507
0.0991
0.200
1.676
0.308
0.158
2.1170
0.9264
1.3222
3.872
1.7239
1.173
673.220
126.91
326.84
886.688
422.373
327.43
LQFM001 (2)
LQFM002 (3)
LQFM014 (4)
LQFM015 (5)
LQFM016 (6)
of a-tocopherol, which was attributed to the presence of 4-NRC (1)
in the extract [14,17,18]. Pohlit et al. (2004) showed that acetylated
4-NRC (1) loses its antioxidant properties, which may be related to
the presence of a free catechol subunit [18].
With the goal of developing drugs for the treatment of cancer, we
describe in the current study the synthesis, pharmacological evalu-
ation, and the toxicological profiles of newanalogues (2e6) of 4-NRC
(1) (Fig.1). These compounds (2e6) were designed usinga molecular
simplification strategy. Considering the structure of compound (1)
we retained the aromatic ring or substituted with heteroaromatic
ring and kept the lateral chain but without the chiral centre.
amine (Acros Organics, Belgium), NaCNBH3 (Acros Organics,
Belgium), ZnCl2 (Acros Organics, Belgium), MsCl (Acros Organics,
Belgium), Et3N (Acros Organics, Belgium), silica 60e200 mm and
70e230 mesh (Silicycle, Canada) and solvents as CH2Cl2 (Acros
Organics, Belgium), MeOH (Vetec, Brazil), CH3CN (Vetec, Brazil),
n-hexane (Vetec, Brazil), AcOEt (Vetec, Brazil) were purchased from
commercial suppliers.
2. Materials and methods
2.1.1. 4-NRC (1)
2.1. Compound test
The 4-NRC was extracted from P. umbellate according to Rezende
et al. (2004) [19].
The 1H and 13C NMR measurements were acquired using
a Bruker Avance III 500 instrument (operating at 500.13 MHz for
1H) equipped with a 5 mm tuneable multinuclear triple (TBI)
resonance probe head equipped with a z gradient. To perform the
1H and 13C experiments, the samples containing 20 mg of
substances typically (Table 1) in CDCl3 and 1% tetramethylsilane as
the internal standard were used. The 1D and 2D pulse sequences
from the Bruker user library were used for the NMR experiments.
Infrared (IR) spectra were obtained with a Nicolet-55a Magna
spectrophotometer using potassium bromide plates. Mass spectra
were obtained with an electrospray mass spectrometry (ESI)
[M þ H]þ. The progress of all reactions was monitored by thin layer
chromatography (TLC), which was performed on 2.0e6.0 cm
aluminium sheets precoated with silica gel 60 (Merck) to a thick-
ness of 0.25 mm. The developed chromatograms were viewed
under ultraviolet light (254e265 nm) and treated with
iodine vapour. For column chromatography, we used Merck silica
gel (70e230 mesh). The reagents aniline (Acros Organics, Belgium),
4-aminophenol (Acros Organics, Belgium), 3,4-(methylenedioxy)
aniline (Acros Organics, Belgium), 1,3-dimethyl-1H-pyrazol-5-
2.1.2. Procedure general to reductive amination (Luo et al., 2004)
[20]
Toa mixtureof thearomaticamines(1.05equiv),citral(1.0equiv),
NaBH3CN (0.5 equiv) in 5 mL of MeOH was added ZnCl2 (0.5 equiv) in
one portion. The mixture was stirred at room temperature for
20 min. The residue was partitioned between saturated NaHCO3 and
CH2Cl2. The combined organic layers were dried (Na2SO4), concen-
trated in vacuo, and the residue was purified through column chro-
matography using n-hexane:AcOEt (6:4) as mobile phase and silica
(60e200 mm, 70e230 mesh) as stationary phase.
2.1.2.1. (E)-N-(3,7-Dimethylocta-2,6-dienyl)benzo[d][1,3]dioxol-5-
amine. Compound (2) was obtained as a brown oil, with a yield of
72%. 1H NMR (CDCl3)
d: 1.61 (3H, s, CH3-10), 1.69 (3H, s, CH3-8), 1.74
(3H, s, CH3-9), 2.11e2.32 (4H, m, H-4 and 5), 3.63 (2H, s, H-1), 5.08
(1H, m, H-6), 5.32 (1H, m, H-2), 5.86 (2H, s, CH2), 6.13 (1H, dd,
J ¼ 2.2 and 8.2 Hz, H-60), 6.29 (1H, d, J ¼ 2.2 Hz, H-20) and 6.62 (1H,
d, J ¼ 8.2 Hz, H-50). 13C NMR (CDCl3)
d: 17.2 (1CeC9), 19.6 (1CeC10),
25.6 (1CeC8), 26.4 (1CeC6), 39.0 (1CeC4), 39.5 (1CeC1), 98.0 (1Ce
C20), 101.5 (1CeC10), 106.8 (1CeC60), 108.5 (1CeC50), 120.0 (1CeC2),
123.5 (1CeC6), 132.1 (1CeC7), 135.6 (1CeC3), 141.1 (1CeC30) and
148.1 (1CeC40). IR (KBr) cmꢀ1: 3301 (
n
NeH), 3100 (n CeH), 2936 (n
CH3), 1684 (
n C]C) and 1373 (n CeN); ESI-MS calculated for
C17H24NO2 [M þ H]þ: 274.18, found: 274.12.
2.1.2.2. (E)-N-(3,7-Dimethylocta-2,6-dienyl)-1,3-dimethyl-1H-pyr-
azol-5-amine. Compound (3) was obtained as a yellow oil, with
a yield of 76%. 1H NMR (CDCl3) : 1.61 (3H, s, CH3-100), 1.67 (3H, s,
d
CH3-8), 1.69 (3H, s, CH3-9), 2.07e2.14 (4H, m, H-4 and 5), 2.16 (3H, s,
CH3-30), 3.53 (3H, s, CH3-10), 3.59e3.66 (2H, m, H-1), 5.06e5.12 (1H,
m, H-6), 5.27 (1H, H-40) and 5.29e5.36 (1H, m, H-6). 13C NMR
(CDCl3) d
: 13.8 (1CeC30),16.4 (1CeC3),17.8 (1CeC10), 25.8 (1CeC8),
26.4 (1CeC5), 39.7 (1CeC4), 43.9 (1CeC1), 88.1 (1CeC40),
120.8 (1CeC2), 124.0 (1CeC6), 131.7 (1CeC7), 139.4 (1CeC3), 147.1
(1Ce3), and 148.5 (1CeC50). IR (KBr) cmꢀ1: 3218 (
n
NeH), 1398
(n CeN), 1268 (n CeN) and 835 (n NeH); ESI-MS calculated for
C15H26N3 [M þ H]þ: 248.21, found: 248.18.
2.1.2.3. (E)-N-(3,7-Dimethylocta-2,6-dienyl)
benzenamine. Comp-
Fig. 1. Design of compounds (2e6) from the 4-NRC (1) lead compound.
ound (4) was obtained as a brown oil, with a yield of 77%. 1H NMR

C.R. Mendanha da Cunha et al. / European Journal of Medicinal Chemistry 62 (2013) 371e378
373
(CDCl3)
d: 1.60 (3H, s, H-10), 1.68 (3H, s, H-8), 1.70 (3H, s, H-9), 2.01e
acclimatisation period of five days. This study was approved by the
2.06 (2H, m, H-5), 2.07e2.13(2H, m, H-4), 3.67 (1H, d, J ¼ 6.8 Hz, H-1),
3.79 (1H, d, J ¼ 6.8 Hz, H-1), 5.07e5.13 (1H, m, H-6), 5.31e5.36(1H, m,
H-2), 6.59e6.63 (2H, m, H-20 and 60), 6.68e7.72 (1H, m, H-40) and
Ethical Committee of this University (protocol number 37/2009).
2.4. Antioxidant activity
7.15e7.19 (2H, m, H-30 and 50). 13C NMR (CDCl3)
d: 16.4 (1CeC9), 17.5
(1CeC10), 25.5 (1CeC8), 26.4 (1CeC5), 39.4 (1CeC4), 43.2 (1CeC1),
113.7 (2CeC 20 and 60), 117.7 (1CeC40), 121.2 (1CeC2), 123.0 (1Ce
C6), 130.4 (1CeC30 and 50), 131.0 (1CeC7), 139.0 (1CeC3), 148.3
2.4.1.
The antioxidant activities of the compounds (1e6) at concen-
trations of 200e12.5 g/mL were evaluated using the -carotene/
linoleic acid model system according to Miller (1971) [22]. Briefly,
5.0 mL of the solution of the -carotene/linoleic acid solution
(20 mg/mL) was mixed with 0.4 mL of the test compound. Trolox
(400 g/mL) was used as the positive control. The absorbance was
b-Carotene and linoleic acid system
m
b
(1CeC10). IR (KBr) cmꢀ1: 3558 (
n NeH), 3100 (n CeH), 2906 (n CH3),
and 1375 (
n
CeN); ESI-MS calculated for C16H24N [M þ H]þ: 230.19,
b
found: 231.06.
m
read at 470 nm at 37 ꢂC, 2 min after exposure and then every 15 min
for 2 h total. The assays were carried out in duplicate. Oxidation
inhibition was measured using the equation: Absorbance reduction
(ABS reduction) ¼ ABSstart ꢀ ABSfinish. The percentage of the
oxidation and the protective effect of the compounds were
measured using the equations:
2.1.2.4. (E ) -N -4- (3, 7 -Dim e thyl oc ta-2 , 6- die nyla min o)
phenol. Compound (5) was obtained as a black solid, with a yield
of 60%. 1H NMR (CDCl3)
d: 1.60 (3H, s, H-10), 1.66 (3H, s, H-8), 1.68
(3H, s, H-9), 1.96e2.04 (4H, m, H-5), 2.04e2.11(2H, m, H-4), 3.62
(1H, d, J ¼ 6.8 Hz), 3.65 (1H, d, J ¼ 6.8 Hz), 5.02e5.13 (1H, m, H-6),
5.28e5.36 (1H, m, H-2), 6.56 (2H, d, J ¼ 8.6 Hz, H-20 and 60), 6.70
(2H, d, J ¼ 8.6 Hz, H-30 and 50). 13C NMR (CDCl3)
d: 16.5 (1CeC9),
Â
Ã
ðABS reductionÞtest ꢁ 100
ðABS reductionÞsystem
17.5 (1CeC10), 26.0 (1CeC8), 26.6 (1CeC5), 38.7 (1CeC5), 39.7
(1CeC1), 115.6 (1CeC20 and 60), 116.6 (1CeC30 and 50), 121.9 (1Ce
C2), 124.2 (1CeC6), 131.7 (1CeC7), 139.0 (1CeC3), 141.8 (1CeC10),
% Oxidation ¼
149.0 (1CeC40). IR (KBr) cmꢀ1: 3427 (
n
OeH), 3300 (
n NeH), 2946
% Protective effect ¼ 100 ꢀ (% Oxidation).
(n CH3), 1686 (n C]C) and 1606 (n NeH); ESI-MS calculated for
C16H24NO [M þ H]þ: 246.19, found: 246.19.
2.4.2. DPPH system
The capability of these compounds (1e6) to scavenge the DPPH
free radical was assayed according to the method of Duarte-Almeida
et al. (2006) [23]. Each test compound (500e0.48 mg/mL), 2.5 mL,
2.1.3. Proceduretoproduce(E)-N-(3,7-dimethylocta-2,6-dienyl)-N-(4-
hydroxyphenyl)methanesulfonamide compound (6)(Vogel, 1989) [21]
To a mixture of compound (5) (1.0 mmol, 111 mg), MsCl
was mixed with 1 mL of DPPH radical ethanolic solution (0.3 mM).
The mixture was then incubated for 30 min at 25 ꢂC in the dark. The
reduction of the DPPH was determined by measuring the absor-
bance at 517 nm. The antioxidant activity (AA%) was calculated as
demonstrated in the equation: AA% ¼ 100 ꢀ {[(ABST ꢀ ABSB) ꢁ 100]/
ABSC}, where AA% ¼ Antioxidant activity; ABST ¼ Test absorbance
(1.0 mmol, 77
amine (1.0 mmol, 138
m
L mg) in 5 mL of acetonitrile was added triethyl-
L). The mixture was stirred at room
m
temperature for 1 h. The residue was then partitioned between
saturated NaHCO3 and CH2Cl2. The combined organic layers were
dried (Na2SO4), concentrated in vacuo and the residue was purified
by through column chromatography using n-hexane:AcOEt (5:5)
(compound
þ
DPPH);
ABSB
¼
Blank
absorbance
(compound þ ethanol); and ABSC ¼ Negative control Absorbance
as mobile phase and silica (60e200
mm, 70e230 mesh) as
(DPPH þ ethanol).
stationary phase to generate (E)-N-(3,7-dimethylocta-2,6-dienyl)-
N-(4-hydroxyphenyl)methanesulfonamide, which was obtained as
a black oil with a yield of 50%.
2.4.3. Oxidative stress in erythrocyte membranes
The antioxidant activities of compounds (1e6) at the 100, 50, 25
and 12.5 mM concentrations were evaluated using the erythrocyte
membrane model of oxidative stress [23]. The blood samples were
obtained from different blood banks and diluted in saline phosphate
containing 1 mM NaN3 at 4 ꢂC; the plasma and white cells were
removed. The isolated erythrocytes were then incubated for 30 min
at 37 ꢂC with compounds test. Next, PBS was added until the hae-
matocrit was stable at 2%. Oxidative stress was induced by adding
1H NMR (CDCl3)
d: 1.60 (3H, s, H-10),1.68 (3H, s, H-8),1.70 (3H, s, H-
9), 1.94e2.13 (4H, m, H-4 and 5), 3.07 (3H, s, SO2CH3), 3.65 (1H, d,
J ¼ 6.6 Hz, H-1), 3.68 (1H, d, J ¼ 6.6 Hz, H-1), 5.02e5.13 (1H, m, H-6),
5.27e5.34(1H, m, H-2), 6.57 (2H, d, J ¼ 9.3 Hz, H-30 and 50), 7.08 (2H, d,
J¼9.3Hz, H-20 and 60).13CNMR(CDCl3)
d:16.3(1CeC9),17.6(1CeC10),
25.6 (1CeC8), 26.1 (1CeC5), 36.7 (1CeSO2CH3), 39.6 (1CeC4), 41.9
(1CeC1), 113.0 (2CeC20 and 60),120.9 (1CeC2),122.6 (2CeC30 and 50),
123.7 (1CeC6), 131.0 (1CeC7), 141.0 (1CeC3), 140.2 (1CeC10), 147.4
(1CeC40). IR (KBr) cmꢀ1: 3322 (
n OeH), 3200 (n CeH) and 1307 (n Ne
H2O2 (300 mM). The test compounds in the erythrocyte solutionwere
incubatedat37 ꢂC for3 h. Theabsorbances weremeasuredat540nm.
H); ESI-MS calculated for C17H26NO3S [Mþ H]þ: 324.16, found:324.13.
2.5. Biocompatibility
2.2. Cell lines and culture conditions
The biocompatibility of compounds (1e6) (50e0.419
mg/mL)
The mouse Balb/c 3T3 fibroblast cell line was donated by Dr. Mari
Cleide Sogayar, Biochemistry Department of the Chemistry Institute
of University of São Paulo, São Paulo, Brazil. The cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% bovine serum and were routinely grown as a monolayer in
75-cm2 tissue culture flasks at 37 ꢂC, 90% humidity, 5.0% CO2/air.
was studied using the haemolytic assay according to Laranjeira
et al. (2010) [24]. The blood samples were obtained from different
blood banks. After separation, the erythrocytes were prepared as
a suspension in a 2% saline solution. The erythrocytes were incu-
bated with the test sample for 1 h. Triton X (5 mg/mL) was used as
the positive control. The absorbance was then read at 450 nm.
2.3. Animals
2.6. Toxicity profile
The mice used in this study were supplied by IQUEGO (Industria
Química do Estado de Goiás) and raised under specific pathogen-
free conditions. The female Swiss mice (eight weeks old) were
maintained at 22 ꢂC and given food and water ad libitum for an
2.6.1. Cytotoxicity in 3T3 cells using the neutral red uptake assay
The cytotoxic effects of compounds (1e6) at concentrations
of 4000e20 mM were evaluated according to ECVAM (2003) [25].

374
C.R. Mendanha da Cunha et al. / European Journal of Medicinal Chemistry 62 (2013) 371e378
Balb/c 3T3-A31 fibroblasts were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% bovine serum
at 37 ꢂC, 90% humidity, 5.0% CO2/air. 3T3 cells were incubated
with the synthetic analogues, and the control wells (blanks)
received culture medium supplemented with 10% bovine serum.
2.7. Statistical analysis
The data were analysed using the GraphPad Prism program
(version 5.00 for Windows XP, GraphPad Software, San Diego,
California, USA). All compounds were compared to the control
using analyses of variance (ANOVA). In cases of significant differ-
ences, the Tukey test was used. Statistical significance was set at
p < 0.05.
After 48 h, the solutions were removed and 250
mL of neutral
red (NR) solution was added to all wells and incubated for 3 h
(37 ꢂC, 90% humidity, 5.0% CO2/air). After 3 h, the NR medium
(250
removed and the cells were carefully rinsed with 250
pre-warmed PBS. The PBS was decanted from the plate and
100 L of NR solvent (50 EtOH: 1 acetic acid: 49 water) solution
m
g/mL of VN diluted in DMEM culture medium) was
m
L/well of
3. Results and discussion
m
3.1. Chemistry
were added to all wells, including the blanks. The plates were
rapidly shaken on a microplate shaker for 20 min to extract NR
from the cells and form a homogeneous solution. The absorption
was measured at 550 nm in a microtiter plate reader (spectro-
photometer). The optical density (OD) was calculated as the
difference between the absorbance at the test wavelength and
that at the reference wavelength. For each concentration tested,
wells containing all the reagents used but no cells served as
reference blanks.
The compounds (2e5) were produced through a reductive
amination reaction, which was carried out using aromatic amines
(7, 9e11), citral (8), zinc chloride, and sodium cyanoborohydride in
a methanol medium. Compounds (2e5) were formed, yielding
among 60% and 77%, after 20 min (Fig. 2) [20]. Compound (5) was
mesylated by using methanesulphonyl chloride, triethylamine and
acetonitrile as the solvent. This procedure resulted in the formation
of compound (6) at a yield of 50% after 1 h at room temperature
(Fig. 2) (Vogel, 1989) [21]. We mesylated the compound (5) since
some sulphonamides like nimesulide have anti-inflammatory
profile as demonstrated by 4-nerolidilcathecol (1).
2.6.2. Clonal culture haematopoietic progenitor cell assay
The potential myelosuppressive effect of the ethanolic solutions
of compounds (1) and (5) (534e4.17
mM) was evaluated using
1 ꢁ105 bone marrow cells obtained from Swiss mouse according to
Parchment et al. (1998) [26]. The incubation medium was prepared
with agar (bacto agar), 2ꢁ DMEM, mouse GM-CSF and bovine
serum. Next, the cells and compounds (1) and (5) were added to the
incubation medium. The negative control contained only the
incubation medium and cells. The cells were incubated with the
test compounds for seven days. A criterion for scoring this test was
read in glass with a 32ꢁ increase.
3.2. Antioxidant activity
3.2.1. DPPH and the
The effective concentration for the antioxidant activity of
compounds (1e6) was evaluated using the -carotene/linoleic acid
system (200e12.5 g/mL) and the DPPH system (500e0.48 g/mL)
b-carotene and linoleic acid system
b
m
m
as shown in Fig. 3. Using the DPPH assay, as reported previously,
compound (1) showed approximately 75% antioxidant activity. A
similar result was only observed with compound (5). Compounds
(2), (3) and (4) showed antioxidant activities that were lower than
2.6.3. Comet assay for measuring DNA damage
The DNA damage caused by 100, 50 and 25 mM of compounds (1)
4-NRC (1). In the b-carotene/linoleic acid assay, better antioxidant
and (5) dissolved in DMSO was assessed using the comet assay.
The gel to electrophoresis (agarose solution to 1.5%), was in the
glass laminate and briefly melt agarose and added the blood with
heparin maintained the suspension at 37 ꢂC. Next, the test
compound was added and incubated for 1 h. Electrophoresis was
performed at 25 V and 300 mA for 25 min. Then, the gel was fixed
activity results were observed with compounds (1), (4), (5) and (6).
As expected, these behaviours can be explained by the absence
of phenolic subunit in compound structures [28]. In this regard, Ujo
et al. (2001) showed that the antioxidant activity of compound (1)
was high but was limited by the formation of semiquinone [28]. On
the other hand, the high antioxidant activities of compounds (5)
and (6) were, at least in part, due to the presence of the phenolic
subunits. The introduction of the methylsulphonamide subunit in
(6), such as nimesulide, which is an anti-inflammatory drug with
antitumour properties, did not produce significant changes in the
compounds’ antioxidant profiles [29,30].
with ethanol, and the DNA was visualised with 25
mL ethidium
bromide (0.5 g/mL). The DNA damage was observed using a fluo-
m
rescence microscope set to 515e560 nm (400e600ꢁ). After ana-
lysing 50 cells, these cells were classified as either without genomic
damage (WGD) or with genomic damage (GD).
2.6.4. Acute oral toxicity evaluation
3.2.2. Oxidative stress in erythrocyte membranes
The starting dose for the acute oral systemic toxicity assays in
Swiss mice (LD50) was estimated by using the prediction model
described in the ICCVAM (2001) validation study, and these results
were compared to the results obtained by in vivo testing performed
according to OECD Test Guideline 423 (2001) [27]. After the cyto-
toxic tests, the estimated LD50 values were calculated based on the
equation:
In the erythrocyte membrane model of oxidative stress, all
compounds (1e6) showed protective antioxidant effects. Our
results showed that compounds (2), (4), (5) and (6) were highly
protective at all concentrations tested than to (1) compound.
Compound (1) demonstrated a protective effect at approximately
35
mM. However, when higher concentrations of (1) and (3)
compounds were tested, they did not show any protective effect on
erythrocyte membranes and also were able to increase the hae-
molysis as shown in Fig. 4. Sakihama et al. (2002) reported that
when phenolic compounds accumulate inside cells, they can
increase the formation of ROS and trigger cellular damage [8]. It has
been reported that high concentrations of some commercial anti-
oxidants can demonstrate a pro-oxidant profile, e.g., gallic acid
(3,4,5-trihydroxybenzoic acid) [31]. Yamamoto (2001) reported
Log (LD50) ¼ 0.506 ꢁ log (IC50) þ 0.475.
For the in vivo tests, 2000 mg/kg solutions of compounds (1) and
(5) were incorporated in sunflower oil under agitation, according to
OECD Test guideline 423. The test compounds (1) and (5) were
administered in Swiss mice (n ¼ 3) as a single dose by oral gavage.
These animals were observed individually after dosing at least once
during the first 30 min and periodically during the first 24 h, with
special attention given during the first 4 h. Daily observations were
continued for 14 days.
that
a-tocopherol is associated with the first defence system to
protect the cellular membrane from oxidative stress [32]. Usually,

C.R. Mendanha da Cunha et al. / European Journal of Medicinal Chemistry 62 (2013) 371e378
375
Fig. 2. General route of synthesis for compounds (2e6) production.
this compound inhibits the formation of lipid hydroperoxide, in
early stage of oxidative process, producing the radical -tocopherol.
In general, this radical can be reverted to -tocopherol by endog-
enous and exogenous antioxidants. However, when this process is
blocked, lipoperoxidation occurs if the concentration of -tocoph-
erol is higher or similar to hydroperoxide. Considering that
compound (1) has a chemical structure that is related to
more toxic to human erythrocytes, with haemolytic potentials of
approximately 75%. Our results showed that compounds (1) and (3)
both induced haemolytic rates higher than 5% at almost all
concentrations studied. Compounds (2), (4), (5) and (6) demon-
strated low toxicity in erythrocyte cells in comparison to the lead
compound (1), resulting in less than 5% haemolysis. These results
indicated haemo/biocompatibility.
a
a
a
a
-
tocopherol, it is possible that antioxidant or pro-oxidant effects
occur in a concentration-dependent manner. The same profile was
observed for compound (3).
3.4. Toxicology profile
3.4.1. Cytotoxicity in 3T3 cells using the neutral red assay
The cytotoxic effects of compounds (1e6) (1068e16.6 mM) using
3.3. Biocompatibility
3T3 cells are shown in Fig. 6. In this assay, all compounds (1e6)
showed cytotoxic effects; however (2), (3), (5) and (6) were more
cytotoxic when compared to (1). Compound (4) demonstrated the
lowest cytotoxicity. The estimated LD50 values for compounds (1)
and (2e6) were found to be higher than 300 mg/kg (Table 1).
In general, phenolic compounds can demonstrate a toxic profile
due to the possible formation of reactive intermediates, such as
The haemolytic test is frequently used to evaluate the haemo-
compatibility of compounds by detecting the disruption of the
membrane of the human erythrocytes. The haemolytic potentials of
compounds (1e6) (50e0.419
Compounds (1) and (3) showed IC50 values of approximately
3.76 g/mL and 1.76 g/mL, respectively. These compounds were
mg/mL) are illustrated in Fig. 5.
m
m
Fig. 4. Protective effect of compounds (1e6) (100e6.25
membrane. Erythrocytes were incubated with compound test for 30 min and the
oxidative stress was induced by H2O2 (300 M). After for 3 h, the absorbances were
mM) on the erythrocyte
Fig. 3. Antioxidant activities of compounds (1e6) using DPPH (500e0.48
-carotene/linoleic acid (B/A) (200e12.5 g/mL) assays. The figure shows the EC50
values in relation to the test compounds for the two assays.
m
g/mL) and
m
b
m
measured at 540 nm. The data are presented as the mean ꢃ S.D. of three experiments
run in duplicate.

376
C.R. Mendanha da Cunha et al. / European Journal of Medicinal Chemistry 62 (2013) 371e378
Fig. 5. Effects of the compounds (1e6) (50e0.419 mg/mL) on human erythrocytes. The
Fig. 7. Effects of 4-NRC (1) and 5 (LQFM015) (4.17e534
mM) on the growth and
erythrocytes (2% saline solution) were incubated with the test sample for 1 h. The
absorbance was read at 450 nm. Variance was calculated by ANOVA assay and the
Tukey test, and significance was set at p < 0.05.
differentiation of bone marrow granulocyte/macrophagic progenitor cells (CFU-GM).
The cells (1 ꢁ 105 cell/mL) were incubated with these compounds for seven days, and
after that the number of CFU-GM was counted. *A (p < 0.05) in relation to the control
(ANOVA).
quinones or semiquinones, which can generate Michael acceptor
subunits. Michael acceptors are known to be reactive intermediates
that deplete endogenous nucleophiles, i.e., proteins, DNA and
others, and can cause significant damage to cells (Rodriguez et al.,
2004) [33]. Zapor (2004) evaluated the cytotoxic effects of five
phenol derivatives (phenol, catechol, resorcinol, hydroquinone and
phloroglucinol) in mouse 3T3 fibroblast cells and suggested that
the cytotoxic activity of the phenolic compound most likely
depends on the number of hydroxyl subunits as well as their
position on the aromatic ring [34].
control, non-exposed progenitor cells. Additionally, compound (1)
(IC50 293
mM) was more toxic to granulocyte/macrophagic
progenitor cells when compared to (5) (IC50 460.4
mM). However,
when these IC50 values were compared with the IC50 values of
classical antineoplastic compounds, such as 5-fluoruacil and
cyclophosphamide, (1) and (5) showed lower haematotoxicity [35].
Usually, antineoplastic drugs and some phenols, catechols and
hydroquinone compounds are able to cause myelosuppression with
a direct reduction in circulating blood cells and bone marrow
cellularity [36e38]. These toxic effects can cause leukopaenia,
anaemia and thrombocytopaenia, which are the main limiting
factors of the antitumoural treatments [39].
Considering that compounds (1) and (5) showed comparable
antioxidant profiles to one another, beside biocompatibility, both
were selected for the subsequent tests.
3.4.2. Clonal culture haematopoietic progenitor cell assay
3.4.3. Comet assay for measuring DNA damage
Considering that antitumour agents are frequently haemato-
toxic, the myelosuppressive potentials of compounds (1) and (5)
were studied and are shown in Fig. 7. Both compounds inhibited the
growth and differentiation of granulocyte/macrophagic progenitor
cells in a concentration-dependent manner when compared to
DNA damage in lymphocyte cells was evaluated after exposure
to compounds (1) or (5) at different concentrations (100, 50 and
25 mM). The results are illustrated in Table 2. DNA damage was
observed only at higher concentrations of both compounds, which
Table 2
DNA damage in T lymphocytes exposed to three different concentrations (100, 50
and 25
m
M) of compounds (1) and (5).
Group
Cells numbers
Scores
0
Scores
total
WGDa
(%)
GDb
(%)
1
5
2
3
Control
Exposed 1
Negative
4-NRC
45
43
0
0
0
0
5
7
90
86
10
14
7
25
4-NRC
50
4-NRC
100
LQFM015
25
LQFM015
50
LQFM015
100
mM
39
37
40
36
35
11
13
9
0
0
1
0
0
0
0
0
0
0
11
13
11
14
15
76
74
78
72
70
24
26
22
28
30
m
M
m
M
Exposed 2
m
M
14
15
m
M
m
M
Fig. 6. Cytotoxic activity of compounds (1e6) on 3T3 cells (1 ꢁ 105 cells) after a 48 h
incubation. The curves show the cytotoxic effects of compounds (1e6) using the
neutral red assay. Inhibition is expressed relative to control cell viability (100%), and
n ¼ 50. Score 0: without genomic damage, score 1: low genomic damage, Score 2:
moderate genomic damage and score 3: high genomic damage.
a
each point represents the mean
triplicate.
ꢃ
S.D. of two independent experiments run in
WGD: cell numbers without genomic damage.
GD: cells number with genomic damage.
b

C.R. Mendanha da Cunha et al. / European Journal of Medicinal Chemistry 62 (2013) 371e378
377
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