Synthesis and antitumour evaluation of peptidyl-like derivatives containing the 1,3-benzodioxole system

Article abstract of DOI:10.1016/j.ejmech.2006.10.007

In the scope of a research program aiming at the synthesis and pharmacological evaluation of novel possible antitumour prototype compounds, we described in this paper the synthesis of peptidyl-like derivatives containing the 1,3-benzodioxole system. The proliferation inhibitors tested against tumour cell lines identified the derivatives tyrosine (4f) and lysine (4g) as the most active among them, presenting IC₅₀ values in micromolar range and are more active than Safrole. For the study on the embryonic development, Safrole did not show any selectivity in this latter assay, which indicates that Safrole acts as a 'cell cycle-nonspecific' inhibitory agent. However, compound 4f presented a fair antimitotic effect, mainly on third cleavage and blastulae stages (38% and 1.7% of normal development, at 10 μg/mL), suggesting a time-dependent activity and a 'cell cycle-specific' agent action. Neither derivatives revealed hemolytic action in assay with mouse erythrocytes.

Full text of DOI:10.1016/j.ejmech.2006.10.007

European Journal of Medicinal Chemistry 42 (2007) 351e357
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumour evaluation of peptidyl-like derivatives
containing the 1,3-benzodioxole system
a
Diogo Rodrigo de Magalhaes Moreira , Ana Cristina Lima Leite
a,
*
~
Paulo Michel Pinheiro Ferreira , Patrıcia Marc¸al da Costa ,
,
b
b
´
Letıcia Veras Costa Lotufo , Manoel Odorico de Moraes ,
b
b
´
a
Dalci Jose Brondani , Claudia do O Pessoa
b
´
´
a LabSINFA e Laboratory of Planning, Synthesis and Evaluation of Pharmaco, Department of Pharmaceutical Science, Health Sciences Center,
Federal University of Pernambuco, 50740-520 Recife, PE, Brazil
b LOE e Laboratory of Oncology Experimental, Department of Physiology and Pharmacology, Federal University of Ceara´,
60430-270 Fortaleza, CE, Brazil
Received 7 August 2006; accepted 19 October 2006
Available online 18 December 2006
Abstract
In the scope of a research program aiming at the synthesis and pharmacological evaluation of novel possible antitumour prototype com-
pounds, we described in this paper the synthesis of peptidyl-like derivatives containing the 1,3-benzodioxole system. The proliferation inhibitors
tested against tumour cell lines identified the derivatives tyrosine (4f) and lysine (4g) as the most active among them, presenting IC50 values in
micromolar range and are more active than Safrole. For the study on the embryonic development, Safrole did not show any selectivity in this
latter assay, which indicates that Safrole acts as a ‘cell cycle-nonspecific’ inhibitory agent. However, compound 4f presented a fair antimitotic
effect, mainly on third cleavage and blastulae stages (38% and 1.7% of normal development, at 10 mg/mL), suggesting a time-dependent activity
and a ‘cell cycle-specific’ agent action. Neither derivatives revealed hemolytic action in assay with mouse erythrocytes.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: 1,3-Benzodioxole; Peptidyl-like derivatives; Cytotoxicity; Sea urchin embryos; Erythrocyte hemolysis
1. Introduction
product that demonstrates interesting functionality and chem-
ical reactivity suggesting its use as an efficient and versatile
natural synthon [4e6]. The methylenedioxy core can be iden-
tified in several bioactive natural anticancer agents, as etopo-
side, tenoposide and podophyllotoxin, among others [7,8].
Jurd et al. [9] have reported a series of simple 6-benzyl-1,3-
benzodioxoles, structurally related to podophyllotoxin, as
more potent antitumour in vivo than the prototype. In addition,
the SAR informations for podophyllotoxin and analogues have
showed that converting the methylenedioxy unit to the corre-
sponding methoxy/hydroxy group dramatically reduced the
antitumour activity [10]. Two recent publications cite refer-
ences describing the importance of 1,3-benzodioxole ring
with antitumour properties. Xia et al. described that introduction
of the methylenedioxy moiety for 2-amino-chalcone led to
According to the WHO, cancer is an important health prob-
lem that claims the lives of more than seven million people
worldwide on an annual basis [1]. The P-glycoprotein (P-gp)
has been held responsible for the effluxes of hydrophobic an-
ticancer agents including paclitaxel and docetaxel, discussing
about the occurrence of Multiple Drug Resistance (MDR)
[2,3]. Development and optimisation of new cancer treating
drugs is a must.
Safrole (1,3-benzodioxole-5-yl), from sassafras oil (Ocotea
pretiosa Mer., Lauraceae), is an abundant Brazilian natural
* Corresponding author. Tel./fax: þ55 81 2126 8511.
E-mail address: acllb2003@yahoo.com.br (A.C. Lima Leite).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.10.007

352
D.R.deM. Moreira et al. / European Journal of Medicinal Chemistry 42 (2007) 351e357
enhanced cytotoxic activity against multidrug-resistant KB car-
cinoma cells, compared with the unsubstituted analogue [11].
Hence, Micale et al. [12] reported that 1,3-benzodioxole deriv-
atives exhibited in vitro tumour growth inhibition activity,
which led to 6-(4-aminobenzoyl)-1,3-benzodioxole-5-acetic
acid methyl ester as the most active compound of this series.
More recently, novel analogues of podophyllotoxin and thu-
riferic acid containing different heterocyclic moieties, such as
furan, thiophene and carbazole ring systems, as well as the
less polar naphthalene moiety substituting the benzodioxole
system of the prototype have been evaluated which revealed
that benzodioxole system is an important requirement for anti-
tumour activity for these natural lignan, being the bioisoster ring
derivatives less active than the prototype [13].
some compounds were substituted in 6-position by a nitro
group. It is well known that nitro group could act to form ox-
ygen species reactive or as internal catalyst and to promote
chemical breakdown in bioactive compounds [22]. To exploit
the activities of the benzodioxole system, we also describe
here, the in vitro evaluation of proliferation inhibitors against
tumour cell lines, embryonic development with sea urchin (Ly-
techinus variegatus) embryos and hemolytic action for pep-
tidyl-like derivatives containing the 1,3-benzodioxole system.
2. Results and discussion
2.1. Chemistry and spectroscopy mean
According to carcinogenesis and metabolism studies, Saf-
role is metabolically activated with the electrophilic interme-
diates that bind with cellular DNA, to form adduct as
guanine derivatives [14]. In addition, recently it was reported
that Safrole induced an increase in Ca2þ levels in PC3 prostate
cancer cell, with decrease in cell viability in a concentration-
dependent manner [15].
Antitumour drugs that present reduced toxicity have been
developed by linking them to small peptides or amino acids
residues [16]. It is supposed that, the conjugated peptide, in
these drugs serves as a substrate for designated enzymes that
are produced and secreted preferentially by tumour tissue
[17]. The rules for the peptidyl-prodrugs on antitumour activ-
ity have not yet fully established. Actually, some approaches
have shown the use of specific sequence of complementary
amino acids for tumour-associated enzymes, with the objective
for rational selection of the peptidyl residues to be conjugated
as tumour recognition moiety, such as a plasmin system (an in-
active pro-enzyme plasminogen form) [18,19]. However, this
strategy does not demonstrate to be absolute and new predic-
tion for selection of these tumour recognition moieties is
necessary.
Among several obvious synthetic route to obtain the pep-
tidyl-like derivatives 4aeh, we decided to explore the 5-
position on aromatic ring of Safrole. Besides, the compounds
4deg were substituted in 6-position, in order to investigate the
importance of nitro group for the cytotoxic activity. Safrole (1)
and 5-allyl-6-nitro-benzo[1,3]dioxole (2) were used to obtain
the intermediate product (6-nitro-benzo[1,3]dioxole-5-yl) ace-
tic acid (3a) or (6-H-benzo[1,3]dioxole-5-yl) acetic acid (3b).
The condensation reactions with L-a-amino acids (S enantio-
mer) were accomplished by employing the classical method
of peptide synthesis (Scheme 1) [23]. DCC (dicyclohexylcar-
bodimide) and HOBt (hydroxybenzotriazole) were used to
prepare the series of peptidyl derivatives of Safrole [20].
Side chains of lysine were protected by a tert-butoxycarbonyl
and tyrosine by 2,6-di-chlorobenzyl ether.
Structures of these compounds were characterized using IR,
1H NMR and elemental analyses. Physical and spectral data
are given in Tables 1 and 2. The common characteristic signals
of all compounds showed a characteristic NeH stretching
vibration around 3193 and 3387 cmꢀ1, overlaps C ¼ O
stretching, amide first band around 1627 and 1671 cmꢀ1 and
CeOeC symmetrical stretching appears near 1020e1088
1
In view of the available information on StructureeActivity
Relationships (SAR) considering the potential of ring system
from Safrole, in previous works we undertook the synthesis
of compounds exploring five and six positions in aromatic
ring from 1,3-benzodioxole core. In vivo assays showed that,
some 1,3-benzodioxole peptidyl derivatives were able to in-
hibit sarcoma S-180 tumour at 35 mg/kg with low toxicity
when compared to intermediary 6-allyl-benzo[1,3]dioxole-5-
yl-amine that was toxic and lethal at the same dose [20].
Our results demonstrate that the linking of 1,3-benzodioxole
core to an amino acid moiety reduced the intrinsic toxicity.
During our continuous effort aiming the synthesis and phar-
macological evaluation of novel possible antitumour com-
pounds, we began to optimize the 1,3-benzodioxole peptidyl
derivatives by chemical modification at 5-position on aromatic
ring to obtain new SAR data. It is well documented that the
toxicophore unit from Safrole devils in C-5 moiety, with for-
mation of diepoxide species (butadiene dimers) [21]. Then,
we have decided introducing some lipophilic or basic pro-
tected amino acids at 5-position. Besides, in order to investi-
gate the importance of nitro group for the cytotoxic activity,
cmꢀ1. In the H NMR spectra, methylene protons displayed
as one singlet at 4.21e3.35 ppm and at 6.02e6.27 ppm
attributed to the methylenedioxy group. The methin protons
of the chiral centre showed resonances at 3.35e4.68 ppm,
and for compounds 4c and 4d, the other methin protons
gave a multiplet at 1.59e2.22 ppm in each case. The NH2
signals appear as two singlets or only one singlet at 6.95e
7.42 ppm and NH protons resonated as a singlet or a doublet
non-equivalents at 7.98e8.60. The tert-butoxycarbonyl peak
appeared as a large and broad singlet at 1.10 ppm and the
2,6-di-chlorobenzyl ether displayed a multiplet at 7.45e7.56
ppm.
2.2. Cancer cell assays
The peptidyl-like derivatives were evaluated against four
tumour cell lines (MDA/MB-435, HCT-8, HL-60, and SF-
295), using a previously described MTT assay. Table 3 sum-
marizes the IC50 data (mM) for antitumour activity. The results
indicated that compounds 4a (glycine), 4f (tyrosine) and 4g
(lysine) have comparable and significant activities and are

D.R.deM. Moreira et al. / European Journal of Medicinal Chemistry 42 (2007) 351e357
353
KMnO4(aq), C6H6, AcOH/
Phase Transfer Catalyst
NaHSO4/H2SO4
O
O
O
COOH
O
R1
R1
1 - R1 = H
2- R1 = NO2
1
3a-b
DMF, DCC, NH2CH(R2)COP
HOBt, 0ºC
R2
H
Comp.
Entry
O
H
N
Gly
Ala
Val
4a
4b
O
CH3
P
CH(CH3)2
4c
4d
4e
O
R2
CH CH(CH3)2
2
O
Ile
R1
CH2C6H5
Phe
Tyr
4
CH2-C6H4-O-(2,6-di-Cl)-C6H3
4f
(CH2)4-NH-Boc
CH(CH3)C2H5
4g
Lys
Leu
4h
P = OEt: 4a; P = NH2: 4b-h. R1 = H: 3a, 4a-c,h. R1 = NO2:3b, 4d-g.
Scheme 1.
the most potent proliferation inhibitors in this series, with best
IC50 values of 4.3 (HCT-8), 8.5 (HCT-8) and 5.4 mM (HL-60),
respectively. The growth of cancer cell lines was also inhibited
by Safrole, with IC50 values of 32.6 mM for SF-295 and
36.1 mM for HCT-8 cells. The peptidyl-like derivatives con-
taining the nitro group were the most potent than non-nitrated
analogues. The other compounds studied in this series were
not able to inhibit cell growth in this assay (IC50 > 50 mM).
The differences in potency among the derivatives and Safrole
shown here may indicate that their antiproliferative effects are
not solely due to the reactive benzodioxole system, since their
activity was influenced by the pattern of nitro group in aro-
matic ring. Regarding the use of different amino acids, the
bulk and basic groups in the side chain (like lysine and tyro-
sine) can be a recognition pattern to answer the increase of
the cytotoxic activity.
Despite Safrole being classified as a weak hepatocarcinogen
in rodents and possibly in human [24], it has epoxy structures
which are important in many compounds with physiological
activity [25]. The carcinogenicity of Safrole is usually thought
to be caused by SafroleeDNA adducts formation [26]. After
Safrole exposure, SafroleeDNA adducts have been found in
many tissues in animal models and human [27]. In addition,
potent effects of Safrole oxide were observed on vascular
endothelial cells (VECs), suggesting that Safrole oxide might
be promising for angiogenesis inhibition [28]. Recently,
studies suggest that Fas/FasL pathway might be activated by
Safrole oxide and then mediated the apoptosis of tumour cell
lines, and this may constitute a potential mechanism in the
mediation of anticancer drug-induced apoptosis [29].
and 100 mg/mL). In contrast, the peptidyl-like derivatives (4a,
4c and 4g) presented a fair antimitotic effect, mainly on third
cleavage and blastulae stages and at the highest tested concen-
trations. Compound 4f presented inhibitory activity at both
tested concentrations but it was not able to inhibit the first
cleavage, while compound 4e completely inhibited the devel-
opment of sea urchin eggs at the highest tested concentration
(100 mg/mL) since the first cleavage. The remaining tested
compounds (4b, 4d and 4h) were not able to inhibit mitosis
on the embryos in this assay. Jacobs et al. [30] have considered
very active a substance that promotes 100% of inhibition in
this assay at a concentration of 16 mg/mL or less. Based on
this consideration, 4f was the most active compound tested,
whereas it was able to inhibit almost any division at the blas-
tulae phase.
The sea urchin egg development possesses some peculiari-
ties, making possible to suggest how the tested substances
acted. The inhibition at the first cleavage is related to DNA
and/or protein synthesis or microtubule assembly, once RNA
synthesis is very slow or absent after fertilization. At this
time, the rapid increase in the rate of protein synthesis is
largely due to the recruitment of maternal mRNA into poly-
somes [31]. However, when a compound blocks microtubule
assembly, clear spots corresponding to nucleus duplication
can be observed in the cytoplasm. Since cells treated with Saf-
role (1) and compound 4e presented a homogeneous cyto-
plasm, this process appears not to have been affected [32].
Hence, these compounds might affect DNA and/or protein
synthesis. On the other hand, based on the present data it is
not possible to affirm whether the delayed activity of com-
pounds 4a, 4c, 4f and 4g is only a result of the chemical prop-
erties of these compounds or a different target though the
antimitotic activity shown by these peptidyl derivatives was
in agreement with Batra et al. [33] who showed that com-
pounds containing methylenedioxy unit are capable to inhibit
the assembly of microtubules. Moreover, the benzodioxole
system also prevented microtubule formation in the ovaries
of Drosophila melanogaster [34].
2.3. Antimitotic activity
The antimitotic activity was performed on the embryonic
development of fertilized sea urchin eggs assay and is shown
in Table 4. Safrole inhibited the cleavages of sea urchin eggs
in a concentration-dependent manner. This inhibition is signif-
icant since the first cleavage for both tested concentrations (10

354
D.R.deM. Moreira et al. / European Journal of Medicinal Chemistry 42 (2007) 351e357
Table 1
1H NMR spectral data of newly synthesized derivatives
2.4. Hemolytic activity
Compound
1H NMR (DMSO-d6) d
In order to verify whether the observed cytotoxic and anti-
mitotic activities are related to membrane disruption, com-
pounds were tested for their ability to induce lysis of mouse
erythrocytes. The erythrocyte membrane is a dynamic struc-
ture that can dictate significant changes in its interaction
with drugs [36]. The results revealed that only Safrole showed
hemolytic activity at the highest tested concentration (200 mg/
mL), suggesting that both cytotoxic and antimitotic activities
were not related to the lytic properties or membrane instability
induced by the Safrole derivatives, which may be probably
caused by a more specific pathway.
4a
1.19 (t, J ¼ 7.19 Hz, 3H, CH3b), 3.82 (d, J ¼ 5,7 Hz, 2H,
CH2a), 3.85 (s, 2H, CH2a0), 4.08 (q, J ¼ 7.19 Hz,
2H, CH2a00), 6.21 (s, 2H, CH22), 7.06 (d, J ¼ 1.50 Hz,
1H, Ar-4), 7.49 (d, J ¼ 8.00 Hz, 1H, Ar-6), 7.61
(s, 1H, Ar-7), 8.33 (broad, 1H, NH-10).
4b
4c
1.19 (t, J ¼ 11.09 Hz, 3H, CH3a), 3.35e3.55 (m, 1H, CHa),
4.16 (s, 2H, CH2g0), 6.06 (s, 2H, CH22), 6.88 (d, J ¼ 8.39 Hz,
1H, Ar-4), 7.15 (s, 1H, NH-13), 7.49 (d, J ¼ 8.39 Hz,
1H, Ar-6), 7.75 (s, 1H, Ar-7), 8.35 (s, 1H, NH-11).
0.91 (d, J ¼ 7.19 Hz, 3H, CH3D), 2.05e2.22 (m, 1H,
CHg), 3.82 (d, J ¼ 6.50 Hz, 1H, CHb), 3.83 (s, 2H,
CH2a), 5.32 (s, 1H, NH2-12), 6.07 (s, 2H, CH22), 6.88
(d, J ¼ 8.09 Hz, Ar-4), 7.49 (d, J ¼ 7.49 Hz, Ar-6), 7.75
(s, 1H, Ar-7), 8.47 (s, 1H, NH-10).
3. Conclusion
4d
0.79 (dd, J ¼ 7.49 Hz, J ¼ 6.8 Hz, 3H, CH3D), 0.83
(d, J ¼ 5.89 Hz, 3H, CH2g), 1.02e1.09 (m, 1H, CH2g),
1.18e1.3 (m, 1H, CH2g), 1.59e1.73 (m, 1H, CHb), 3.80
(d, J ¼ 15.89 Hz, 1H, CH2g2), 3.91 (d, J ¼ 16.19 Hz, 1H,
CH2a), 4.11 (dd, J ¼ 6.59 Hz and 9.00 Hz, 1H, CHa0),
6.21 (s, 2H, CH22), 7.03 (s, 1H, NH2-13), 7.05 (s, 1H,
Ar-7), 7.35 (s, 1H, NH2-13), 7.62 (s, 1H, Ar-4), 7.98
(d, J ¼ 9.29 Hz, 1H, NH-10).
In summary, new peptidyl-like derivatives containing the
1,3-benzodioxole system were synthesized using accessible
methodologies. Some derivatives exhibit significant in vitro
activity against cancer cell lines, particularly compounds 4f
and 4g. The cytotoxic effect of the peptidyl-like derivatives
can be associated with the DNA-binding in tumour cells, as
it is told for Safrole, however, with potentialization of antipro-
liferative action and selectivity for the phase specifies cellular
division.
4e
2.80 (dd, J ¼ 13.80 Hz and J ¼ 4.50 Hz, 1H, CH2b),
3.01 (dd, J ¼ 13.80 Hz and 4.80 Hz, 1H, CH2b0), 3.73
(d, J ¼ 16.20 Hz, 1H, CH2a), 3.82 (d, J ¼ 16.20 Hz,
1H, CH2a), 4.43e4.41 (m, 1H, CH2a0), 6.20 (s, 2H,
CH22), 6.88 (s, 1H, Ar-7), 7.0 (s, 1H, NH2-13),
7.21e7.29 (1H of NH2-13, 5H of Ar-m, p and o),
8.07 (d, J ¼ 7.80 Hz, 1H, NH-10).
4. Materials and methods
4f
2.85 (dd, J ¼ 9.10 Hz and 14.10 Hz, 1H, CH2b),
3.06 (dd, J ¼ 5.10 Hz and 14.10 Hz, 1H, CH2b0),
3.35e3.34 (m, 2H, CH2a), 4.61e4.54 (m, 1H, CHa0),
6.77 (s, 1H, Ar-4), 6.97 (d, J ¼ 8.70 Hz, 2H, Ar, H-110
and H-130), 7.09 (s, 1H, NH2-13), 7.22 (d, J ¼ 8.70 Hz,
2H, Ar-, H-120 and H-140), 7.42 (s, 1H, NH2-13),
7.53 (s, 1H, Ar-7), 7.45e7.56 (m, 3H, Ar), 8.60
(s, 1H, NH-16).
4.1. Chemistry
All melting points were determined using a Thomas Hoo-
ver apparatus and are uncorrected. FTIR spectra were obtained
on Brukker spectrophotometer, model IFS66 using KBr pel-
1
lets. H NMR spectra were measured using a Varian UNITY-
4g
0.76e0.91 (m, 2H, CHg), 1.10 (s, 9H, (CH3)3),
1.15e1.28 (m, 2H, CH2g0), 1.68e1.98 (m, 2H,
CH2eNH-11), 4.12 (t, J ¼ 8.30 Hz, 1H, NH-11),
4.21 (s, 2H, CH2b), 4.63e4.68 (m, 1H, CHa), 6.27
(s, 2H, CH22), 7.18 (s, 1H, NH2-14), 7.34 (s, 1H,
NH2-14), 7.44e7.48 (m, 1H, Ar-4), 7.61 (s, 1H, Ar-7),
8.50 (d, J ¼ 8.39 Hz, 1H, NH-3).
plus-300 MHz NMR spectrophotometer using DMSO-d6 as
solvent and tetramethylsilane as an internal standard. Elemen-
tal analyses were performed on a PE-2400 instrument and the
results were in acceptable range. Thin layer chromatography
(TLC) was carried out on silica gel plates with a fluorescence
indicator of F254 (0.2 mm, E. Merck); the spots were visual-
ized in UV light, and by spraying a 2% ethanol solution of nin-
hydrin or charing reagent. Column chromatography was
performed on silica gel using Kiesegel 60 (230e400 Mesh,
E. Merck). All reagents used in the present study were of an-
alytical grade.
4h
1.12 (d, J ¼ 6.8 Hz, 3H, CH3D), 1.31e1.60 (m, 1H,
CHD), 1.76e1.93 (m, 2H, CH2g), 4.12 (s, 1H, CH2g0),
4.24 (dd, J ¼ 3.9 Hz and 7.79 Hz, 1H, CHg0), 6.27
(s, 2H, CH22), 7.03 (d, 1H, J ¼ 7.5 Hz, Ar-6), 7.15
(s, 1H, NH2-13), 7.21 (d, J ¼ 7.50 Hz, 1H, Ar-6),
7.30 (d, 1H, NH2-13), 7.41 (d, J ¼ 1.50 Hz, 1H, Ar-4),
7.57 (d, J ¼ 7.50 Hz, 1H, Ar-7), 8.47 (d, J ¼ 8.3 Hz,
1H, NH-10).
4.1.1. 5-Allyl-6-nitro-benzo[1,3]dioxole (2)
To a stirred mixture of 0.062 mol of 5-allyl-benzo[1,3]diox-
ole and 0.062 mol of acetic acid under 5 ꢁC, 0.062 mol of con-
centrated nitric acid in 1.5 mL of acetic acid was slowly
added. After 2 h, the mixture was taken up in 100 mL of water
and then extracted with three 50 mL portions of ethyl acetate.
The organic phase was washed with water, filtered and dried
(Na2SO4). The residue was chromatographed on silica gel
with 2% ethyl acetate in n-hexane to give 8.9 g (70%) as a
yellow oil.
In general, the antimitotic compounds assayed in the sea ur-
chin eggs presented a higher IC50 than that observed for tu-
mour cell lines [35]. However, Safrole was the most active
compound in the sea urchin egg’s model, while it was only
weakly active against tumour cell lines. Probably, the struc-
tural requirements for cytotoxic action will depend upon the
model used.

D.R.deM. Moreira et al. / European Journal of Medicinal Chemistry 42 (2007) 351e357
355
Table 2
Physical and spectral data for 4aeg
Compound
Molecular formulaa
M.p. (ꢁC)b
Rfc
Yield (%)
IR (KBr) n (cmꢀ1
)
4a
4b
C
C
13H15NO5
12H14N2O4
130e133
135e136
0.83
0.85
85
80
3324 NNeH, 1628 NC]O amide, 1739 NC]O ester, 1020 NCeOeC.
3381 NNeH amide 2nd, 1729 NC]O assym,
1626 NC]O sym, 1503 NC]O amide 1st, 1073 NCeOeC.
3315 NNeH amide 2nd, 3405 NNH2 assym, 3347 NNH2 sym, 1689
NC]O assym,
4c
C14H18N2O4
125e126
0.35
75
1626 NC]O sym, 1503 NC]O amide 1st, 1073 NCeOeC.
3326 NNeH amide 2nd, 1629 NC]O amide, 1574 NAreNO2 assym,
1244 NAreNO2 sym, 1050 NCeOeC.
4d
4e
4f
4g
4h
a
C
C
15H19N3O6
18H17N3O6
175e176
158e160
150e153
160e162
153e154
0.30
0.40
0.40
0.60
0.70
70
76
46
60
80
3320 NNeH amide 2nd, 1627 NC]O amide 1st, 1527 NAreNO2 assym,
1330 NAreNO2 sym, 1058 NCeOeC.
C24H19N3O7Cl2
20H28N4O8
C15H20N2O4
3326 NNeH amide 2nd, 3326 NNeH assym, 3193 NNeH sym,
1628 NC]O amide 2nd, 1532 NAreNO2 assym, 1243 NAreNO2 sym.
3382 NNeH of amide 2nd, 1740 NC]O amide 1st, 1691 NC]O assym,
1658 NC]O sym, 1368 NAreNO2, 1041 NCeOeC.
3325 NNH amide 2nd, 3380 NNH2 assym, 3323 NNH2 sym,
1672 NC]O assym, 1626 NC]O sym, 1088 NCeOeC.
C
Microanalysis for the synthesized compounds; Anal. Calc./Found: Compound 4a: C, 58.86; H, 5.70; N, 5.28/C, 58.01; H, 5.15; N, 4.95. Compound 4b: C,
57.59; H, 5.64; N, 11.19/C, 57.021; H, 5.11; N, 10.86. Compound 4c: C, 60.42; H, 6.52; N, 10.07/C, 60.01; H, 6.15; N, 9.86. Compound 4d: C, 53.41; H,
5.68; N, 12.46/C, 53.00; H, 5.15; N, 12.05. Compound 4e: C, 58.22; H, 4.61; N, 11.31/C, 58.25; H, 4.65; N, 11.50. Compound 4f: C, 54.15; H, 3.60; N, 7.98/
C, 54.54; H, 3.85; N, 7.82. Compound 4g: C, 53.09; H, 6.24; N, 12.38/C, 52.89; H, 6.59; N, 12.00. Compound 4h: C, 61.63; H, 6.90; N, 9.59/C, 61.30; H,
7.10; N, 10.02.
b
Crystallized from ethyl acetate/n-hexane (1:2).
Solvent system ethyl acetate/n-hexane (8:2).
c
4.1.2. (6-Nitro-benzo[1,3]dioxole-5-yl)acetic acid or
(6-H-benzo[1,3]dioxole-5-yl) acetic acid (3aeb)
H2SO4 in 100 mL of water). Two clear layers resulted. The
layers were separated and the organic layer was washed
once with a 100 mL portion of water. The organic layer was
dried over anhydrous sodium sulfate, the drying agent was
The aqueous KMnO4 (0.135 mol) in about 150 mL of water
was stirred and cooled in an ice bath. A solution of Safrole or
compound 2 (0.024 mol), benzyldimethyltetradecylammo-
nium chloride dihydrate (0.0012 mol), 30 mL of acetone,
150 mL of benzene, and 30 mL of acetic acid was added in
one portion. After 2 h, stirring continued without any further
addition of ice to the bath for 70 h. A total of 40 g of NaHSO3
was added to the cooled reaction mixture followed by the slow
addition of 70 mL of aqueous H2SO4 (25 g of concentrated
Table 4
Inhibition of the cell cycle by the peptidyl-like derivatives 4aeh on the em-
bryos of the sea urchin Lytechinus variegatus on the first and third cleavage
and blastulae stages
Compound
Concentration Sea urchin egg developmenta (%)
(mg/mL)
(mean ꢂ S.E.M.)
First cleavage Third cleavage Blastulae
Negative control e
Doxorubicin
89.3 ꢂ 2.8
36.0 ꢂ 1.7*
16.3 ꢂ 2.4*
59.5 ꢂ 6.4*
0.7 ꢂ 1.2*
83.0 ꢂ 10.4
79.7 ꢂ 6.8
76.3 ꢂ 7.5
76.5 ꢂ 9.2
88.0 ꢂ 1.0
70.7 ꢂ 2.3*
88.3 ꢂ 6.0
94.3 ꢂ 1.2
86.3 ꢂ 4.5
1.7 ꢂ 2.1*
92.3 ꢂ 4.5
85.3 ꢂ 1.5
88.7 ꢂ 3.5
88.7 ꢂ 4.0
89.3 ꢂ 3.5
87.7 ꢂ 6.1
76.3 ꢂ 1.1
0.0 ꢂ 0.0*
1.5 ꢂ 1.5*
33.7 ꢂ 3.5*
0.0 ꢂ 0.0*
71.3 ꢂ 11.6
15.7 ꢂ 3.2*
68.3 ꢂ 2.9
78.7 ꢂ 2.3
67.0 ꢂ 3.6
61.7 ꢂ 2.9
77.0 ꢂ 5.3
72.7 ꢂ 4.7
73.3 ꢂ 15.5
5.3 ꢂ 4.6*
38.0 ꢂ 4.0*
46.0 ꢂ 2.6*
72.3 ꢂ 11.6
66.3 ꢂ 12.9
62.0 ꢂ 3.5
70.3 ꢂ 9.3
81.0 ꢂ 3.5
0.3 ꢂ 0.3*
0.0 ꢂ 0.0*
31.7 ꢂ 9.3*
0.3 ꢂ 0.6*
83.7 ꢂ 3.2
0.3 ꢂ 0.6*
92.0 ꢂ 2.0
87.7 ꢂ 4.9
96.7 ꢂ 2.3
1.0 ꢂ 1.7*
80.3 ꢂ 4.2
89.0 ꢂ 1.7
92.0 ꢂ 1.0
0.0 ꢂ 0.0*
1.7 ꢂ 1.5*
15.0 ꢂ 2.8*
79.7 ꢂ 4.5
3.7 ꢂ 3.2*
94.7 ꢂ 2.3
84.7 ꢂ 7.5
Table 3
Cytotoxic activity of the peptidyl-like derivatives on human tumour cell linesa
10
100
10
Compound Entryb HL-60
HCT-8
MDA-MB SF-295
435
1
100
10
Doxorubicin
1
e
e
0.03
104.5
0.03
36.1
0.83
104.7
0.42
32.6
4a
4b
4c
4d
4e
4f
100
10
(84.0e128.4) (31.5e41.3) (72.8e150) (4.1e6.6)
13.5
4a
Gly
18.7
4.3
>50
100
10
(14.4e24)
>50
>50
(2.5e7.9)
>50
>50
(9.6e18.5)
>50
>50
4b
4c
4d
Ala
Val
Ileu
>50
>50
>50
100
10
>50
>50
19.1
100
10
(10.4e34.2)
>50
>50
4e
4f
Phe
Tyr
>50
>50
>50
8.5
>50
9.9
100
10
(4.3e9.5)
(8.6e11.4)
9.2
100
10
4g
Lys
Leu
5.4 (3.0e8.5) >50
31.6
4g
4h
(8.5e11.3) (27.2e36.4)
>50 >50
100
10
4h
>50 >50
100
a
Data are presented as IC50 (mM) values and 95% confidence interval ob-
tained from at least three independent experiments and standard deviation is
given in parentheses.
*p < 0.05, ANOVA followed by Dunnett’s Multiple Comparison test
compared to negative control.
b
a
Entry represents the amino acid residue of the compounds.
The compounds were added 2 min after fertilization of the eggs.

356
D.R.deM. Moreira et al. / European Journal of Medicinal Chemistry 42 (2007) 351e357
removed by filtration, and the solvent was removed on a rotary
evaporator. The crude product was chromatographed on silica
gel with 5% ethyl acetate in n-hexane to give 3.25 g (60%), as
a yellow solid with m.p. 180e182 ꢁC (3a) and 3.15 g (58%) as
well crystal with m.p. of 192 ꢁC (3b).
a Pasteur pipette and maintained under low temperature until
the moment of fertilization. For fertilization, 1 mL of a sperm
suspension (0.05 mL of concentrated sperm in 2.45 mL of fil-
tered seawater) was added to every 50 mL of egg solution.
Each well received 1 mL of fertilized egg suspension. The com-
pounds were added immediately after fertilization (within
2 min) to get concentrations of 10 and 100 mg/mL in a final vol-
ume of 2 mL. Doxorubicin was used as positive control. The
plates were then shaken in a constant temperature water bath
at 26 ꢂ 2 ꢁC. At appropriate intervals, aliquots of 200 mL were
fixed with the same volume of 10% formaldehyde to obtain first
and third cleavages, and blastulae. One hundred eggsor embryos
were counted for each concentration of test substance to obtain
the percentage of normal cells.
4.1.3. General procedure for the synthesis of peptidyl
derivatives of the Safrole (4aeh)
To a stirred solution of 2.5 mmol of the TFAeamino acyla-
mide in N,N-dimethylformamide (20 mL) under 0 ꢁC,
6.8 mmol of the dicyclohexylcarbodiimide, 5.1 mmol of 1-hy-
droxybenzotriazole and 1.7 mmol of compound 3a or 3b were
added and was allowed to warm at room temperature. After
that, the reaction mixture was filtered, and the filtrate was
treated with ethyl acetate. The organic phase was washed
with the NaHCO3 solution (50 mL), 1 M citric acid (50 mL),
aqueous NaCl (25 mL) and dried (Na2SO4) and concentrated.
The residue was treated with n-hexane and filtered. The deriv-
atives 4aeh were obtained as yellow crystals.
4.4. Hemolytic assay
The test was performed in 96-well plates following the
method described by Costa-Lotufo et al. [35]. Each well re-
ceived 100 mL of 0.85% NaCl solution containing 10 mM
CaCl2. The first well was the negative control that contained
only the vehicle (distilled water or DMSO 10%), and, in the
second well, 100 mL of test substance that was diluted to
half was added. The compounds were tested at concentrations
ranging from 10 to 200 mg/mL. The serial dilution continued
until the eleventh well. The last well received 20 mL of
0.1% Triton X-100 (in 0.85% saline) to obtain 100% hemoly-
sis (positive control). Then, each well received 100 mL of a 2%
suspension of mouse erythrocytes in 0.85% saline containing
10 mM CaCl2. After incubation at room temperature for
30 min and centrifugation, the supernatant was removed and
the liberated hemoglobin was measured spectroscopically as
absorbance at 540 nm.
4.2. MTT assay
The cytotoxicity of the compounds was tested against four
tumour cell lines: SF-295 (human glyoblastoma), HCT-8 (hu-
man colon carcinoma), HL-60 (human myeloblastic leukemia)
and MDA/MB-435 (human breast) (National Cancer Institute,
USA). Cells were cultured in RPMI-1640 medium, supple-
mented with 10% fetal calf serum, 2 mM glutamine, 100 mg/
mL streptomycin and 100 U/mL penicillin at 37 ꢁC with 5%
CO2. For experiments, cells were plated in 96-well plates
(105 cells/well for adherent cells or 0.3 ꢃ 106 cells/well for
suspended cells in 100 mL medium). After 24 h, the com-
pounds (0.39e25 mg/mL) dissolved in DMSO (5%) were
added to each well and incubated for 3 days (72 h). Control
groups received the same amount of DMSO. Doxorubicin
was used as positive control. Growth of tumoural cells was
quantitated by the ability of living cells to reduce the yellow
dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) to a blue formazan product. At the end of
72 h incubation, the medium in each well was replaced by
fresh medium (200 mL) containing 0.5 mg/mL of MTT. Three
hours later, the formazan product of MTT reduction was dis-
solved in DMSO, and absorbance was measured using
a multi-plate reader (Spectra Count, Packard, Ont., Canada).
Drug effect was quantified as the percentage of control absor-
bance of reduced dye at 590 nm.
4.5. Statistical analysis
The IC50 values and their 95% confidence intervals (CI 95%)
were obtained by nonlinear regression using the GRAPHPAD
program (Intuitive Software for Science, San Diego, CA). For
the sea urchin egg’s assay, the differences were analyzed by AN-
OVA followed by Dunnett’s Multiple Comparison test com-
pared to negative control at a significance level of 5%.
Acknowledgements
4.3. Assay on sea urchins
We would like to thank the Brazilian National Research
Council (CNPq #471834/2006-8), the Research Foundation
of Pernambuco State (FACEPE), Northeast Bank of Brazil
(BNB) and FINEP for financial support. P.M.P.F and
D.R.M.M. are recipients of CAPES (masters) fellowships.
Our thanks are also due to the Department of Chemistry at
the Federal University of Pernambuco (UFPE), for recording
the H NMR, IR spectra and elemental analyses of all com-
´
pounds and to Ms. Celio R.M. Nascimento for the help in tech-
nical works (CNPq #500361/2004-5).
The test was performed in 24-well plates following the
method described by Costa-Lotufo et al. [35]. Adult sea urchins
(Lytechinus variegatus) were collected at Pecem beach, on the
northeastern coast of Brazil. The gamete elimination was in-
duced by injecting 3.0 mL of 0.5 M KCl into the urchin’s coelo-
mic cavity via the periostomial membrane. The eggs were
washed twice using filtered seawater to remove the jelly coat
surrounding the cells. Concentrated sperm was collected with
´
1

D.R.deM. Moreira et al. / European Journal of Medicinal Chemistry 42 (2007) 351e357
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