Synthesis, antitumour and antimicrobial activities of new peptidyl derivatives containing the 1,3-benzodioxole system

Article abstract of DOI:10.1016/j.ejmech.2004.09.007

Two series of 5 and 6-substituted 1,3-benzodioxole peptidyl derivatives were synthesized and evaluated as antitumour and antimicrobial agents. The compounds that could be conveniently prepared in a few steps processes from natural safrole have been charac

Full text of DOI:10.1016/j.ejmech.2004.09.007

European Journal of Medicinal Chemistry 39 (2004) 1059–1065
www.elsevier.com/locate/ejmech
Original article
Synthesis, antitumour and antimicrobial activities of new peptidyl
derivatives containing the 1,3-benzodioxole system
a,
a
b
Ana Cristina Lima Leite *, Kezia Peixoto da Silva , Ivone A. de Souza ,
b
a
Janete Magali de Araújo , Dalci José Brondani
a
Labsinfa, Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco – Rua Prof. Artur Sá S/N, CDU, 50740-520, Recife,
Pernambuco, Brazil
Departamento de Antibióticos, Universidade Federal de Pernambuco – Rua Prof. Artur Sá S/N, CDU, 50740-520, Recife, Pernambuco, Brazil
b
Received 5 January 2004; received in revised form 12 August 2004; accepted 8 September 2004
Abstract
Two series of 5 and 6-substituted 1,3-benzodioxole peptidyl derivatives were synthesized and evaluated as antitumour and antimicrobial
agents. The compounds that could be conveniently prepared in a few steps processes from natural safrole have been characterised by IR and
1
H-NMR spectroscopy. In vivo antitumor activity tests showed that some of the compounds were able to inhibit carcinoma S-180 tumour
growth in mice. The in vitro antimicrobial activity of all compounds revealed that they are able to promote the growth of some organisms,
including Bacillus subtilis.
©
2004 Elsevier SAS. All rights reserved.
Keywords: 1,3-Benzodioxole; Safrole; Peptidyl derivatives; Antitumour agents
1
. Introduction
them to small peptides or amino acid residues [13–17]. The
role of the conjugated peptide in these prodrugs is not to
physically locate the prodrug on tumour cells, but to serve as
a substrate for designated enzymes that are produced and
secreted preferentially by tumour cells [18]. In fact some
peptides that selectively target tumour blood vessels have
been identified. These remarks indicates that it may be pos-
sible to develop highly selective chemotherapy strategies that
are based on the selective expression of receptors in tumour
vasculature [19].
In view of the available information on structure–activity
relationships, we decided to synthesize derivatives, consider-
ing the potential of ring system from safrole and amino acid
residues. We undertook the synthesis of two series of com-
pounds exploring five and six positions in aromatic ring
Safrole, from sassafras oil (Ocotea pretiosa Mer., Lau-
raceae), is an abundant Brazilian natural product that dem-
onstrates interesting functionality and chemical reactivity
suggesting its use as an efficient and versatile natural synton
[1–6]. In 1983 the FDA reported that, safrole or sassafras, the
extract or the oil, was an ingredient in 113 over-the counter
drug formulations generally for topical application, but occa-
sionally for oral administration [7]. However, carcinogenic
and other toxicological effects to this product have been
pointed out [8–10].
The methylenedioxy unit, present in safrole, can be iden-
tified in the clinical antitumour agents etoposide and tenipo-
side [11] and lignan lactone podophylotoxin [12].
A major problem in chemotherapy today is the severe host
toxicity exhibited by most of the anticancer drugs due to their
poor selectivity toward cancer cells. Recently, antitumour
drugs with reduced toxicity have been developed by linking
1
,3-benzodioxole nucleus [20].
Safrole (1) was initially converted to 5-allyl-6-nitro-benzo
[
1,3]dioxole. This compound was then used to obtain the
intermediate product 6-allyl-benzo [1,3]dioxole-5-yl-amine
3) and (6-nitro-benzo [1,3]dioxole-5-yl) acetic acid (5). The
(
condensation reactions with amino acids were accomplished
by employing the classical method of peptide synthesis.
DCC and HOBt were used to prepare the two new series of
*
Corresponding author.
E-mail address: aclb@ufpe.br (A.C.L. Leite).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.09.007

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A.C.L. Leite et al. / European Journal of Medicinal Chemistry 39 (2004) 1059–1065
peptidyl derivatives of safrole. The structures of the com-
pounds were determined by spectroscopic methods (IR and
H-NMR).
Antimicrobial screening, acute toxicity and antitumour
activities were performed. Peptidyl derivatives and the inter-
mediate compound, 6-allyl-benzo [1,3]dioxole-5-yl-amine
an acetic acid medium suppresses (but does not completely
inhibit) the over-oxidation product. The acetic acid’s func-
tion (soluble in water, benzene, and pentane) may merely
reflect its solubility in the organic phase and rapid destruction
of the OH- formed during the disproportionation of manga-
nese (VI) intermediates [26]. An additional function is the
destruction of any excess of permanganate and prevention of
further oxidation of the initially formed carboxylic acid [27].
In fact, the desired (6-nitro-benzo [1,3]dioxole-5-yl) acetic
acid (5) was obtained, but it was contaminated with
6-nitrobenzo [1,3]dioxole-5-carboxylic acid (5%). This
made it necessary to purify it (5) before amino acid conden-
sation.
1
(3), were able to induce the growth of some bacteria which
are important in phytopathogenic control in agriculture.
The acute toxicity of the N-(6-allyl-1,3-benzodioxol-5-
yl)-N-(tert-butoxy carbonyl)phenylalaninamide (4e) deriva-
tive was evaluated and revealed a LD50 at 250 mg/kg in
mice. The antitumour activity of our compounds was
checked against sarcoma 180. Peptidyl derivatives of leucine,
valine, glycine and glutamic acid significantly reduced the
growth of the tumour at 35 mg/kg. All of the compounds that
were tested revealed low toxicity when compared to interme-
diary 6-allyl-benzo [1,3]dioxole-5-yl-amine (3), that was
toxic/lethal at the same dose.
For this series, the synthetic routes were applied on gly-
cine methyl ester, phenylalaninamide, leucinamide and tyro-
sinamide. The side chain of tyrosinamide residue was pro-
tected by a 2,6-dichloro-benzyl group. The expected amino-
acyl derivatives 6a–d were synthesized by condensation with
(6-nitro-benzo [1,3]dioxole-5-yl) acetic acid (5) to a-amino
acids using the same procedure used to the series 4a–h. The
safrole derivatives were obtained at 18 in 99% yield. The
obvious synthetic route planned to achieve the new safrole
derivative compounds 4a–h and 6a–d is shown in Fig. 1.
The formation of 4a–h and 6a–d, was supported by IR and
2
. Chemistry
The general synthetic route that was chosen for the prepa-
ration of peptidyl derivatives of safrole included two stages.
Initially, our synthetic strategy was based on the selective and
classical nitration with nitric acid in acetic acid, on the five
position of the 1,3-benzodioxole moiety [20].
The derivatives of the series 4a–h, were prepared, from
the functionalised nitro safrole, by reduction in mild condi-
1
H-NMR spectral data. The common characteristic signals to
all compounds are described here. The IR spectra showed a
characteristic N–H stretching vibration around 3193 and
–
1
3387 cm , overlap C=O stretching, amide I band around
–
1
1627 and 1671 cm . Asymmetrical C–O–C stretching band
–
1
tions using iron in powder and NH Cl in ethanol/water to
around 1200–1275 cm and symmetrical stretching near
4
–
1
1
afford the amino derivative [21]. The synthetic routes were
applied on appropriately protected lipophilic (glycine, phe-
nylalanine, alanine, valine, leucine), acid (aspartic and
glutamic acid) and basic (lysine) amino acids. The amino
group in the side chain of lysine was protected by a fluore-
nylmethyloxycarbonil (Fmoc) group. The expected peptidyl
derivatives 4a–f have been synthesized by condensation with
1020–1075 cm . The H-NMR spectra of compounds
showed the same characteristic at (D) 5.75–5.91 ppm attrib-
uted to ethylenic proton, at 4.97–4.99 and 5.03–5.05 ppm,
corresponding to methylenic protons. Another representative
signal occurs at 5.98–6.21 ppm was attributed to the methyl-
enedioxy group. A characteristic doublet around 6.91–
8.60 ppm for the NH indicating a coupling constant with CH
chiral. In the 4a–h series, two signals occurring between
6.70 and 6.74 ppm appeared as two singlets indicating a
typical para-aromatic hydrogen pattern. The NH, linked to
aromatic ring showed a singlet around 9.07 and 9.25 ppm. In
the 9a–d series, an aromatic hydrogen pattern near to nitro
group appeared as two signals occurring between 7.53 and
7.72 ppm. Another aromatic proton occurring between
6-allyl-benzo [1,3]dioxole-5-ylamine (3) to a-amino acids
using diciclohexylcarbodiimide and 1-hydroxybenzotriazole
to activate the carboxylic acid function of the amino acid to
generate an active ester in situ [22–24]. The peptidyl deriva-
tives were obtained at 25–95% yield.
The next step was the synthesis of compounds 6a–d.
Among all possible methods for oxidation of 5-allyl-6-nitro-
benzo [1,3]dioxole (2), a heterogeneous two-phase water–
benzene system in permanganate and a phase-transfer cata-
lyst is one of the simplest and most convenient routes [25].
We have tested several phase-transfer agents. Treatment of
6.77 and 7.06 ppm. The NH signal appears as two singlets at
2
about 7.35–7.42 and 7.00–7.09 ppm.
(2) with permanganate in benzene water and acetic acid
3. In vitro and in vivo results and discussions
medium with tetrabutylammonium tetrafluorborate pro-
duced the intermediate (5) with 25% yield. In the same
conditions, benzyldimethyltetradecylammonium chloride
dihydrate was used as phase-transfer agent. The desired car-
boxylic acid was obtained in 60%. Our attention was next
turned to the over-oxidation product resulting from a loss of
two carbons in permanganate oxidation of olefins. The use of
3.1. Antimicrobial activity
The antimicrobial activity of compounds were determined
in vitro by the disc diffusion method [28] against some
strains of Staphylococcus aureus (DAUFPE 02), Bacillus
subtilis (DAUFPE 16), Escherichia coli (DAUFPE 224),

A.C.L. Leite et al. / European Journal of Medicinal Chemistry 39 (2004) 1059–1065
1061
Fig. 1
.
Klebsiella pneumoniae (DAUFPE 396), Salmonella enteriti-
dis (DAUFPE 414), Pseudomonas aeruginosa (DAUFPE
that are inoculated with B. subtilis [30] and 33% on the pea
production that are inoculated with Pseudomonas fluore-
scens [31].
39), Shigella sonei (DAUFPE 413), Sarcinea lutea
(
DAUFPE 100). All the strains came from the collection of
The present work, suggests that the peptidyl derivatives of
safrole could offer an exciting new method to promote
B. subtilis growth for agricultural use.
the Department of Antibiotics (UFPE-BR). One strain, Can-
dida spp., that was isolated from immune suppressed patients
was also used.
All compounds of the series 4a–f, promoted an increase in
the growth of the micro-organisms P. aeruginosa, Shigella
sonei, B. subtilis, Staphylococcus aureus and K. pneumo-
niae. All compounds of series 6a–d promoted an increase in
the growth of the micro-organism E. coli.
In this way, it was verified that our products did not reveal
antimicrobial activity. However, the literature tells us that
some micro-organisms like B. subtilis, Pseudomonas spp,
are being studied as rhyzobacteria, acting in the promotion of
plant growth. The interest in this kind of alternative method
for the control of phytopathogens has been increasing be-
cause they do not require the use of many chemical products
that elevate the manufacturing costs of conventional plant
protection products and cause environmental damage. As
examples, the innoculation of carrots with B. subtilis has
resulted in a tax increase of 48% [29], 37% on the peanuts
3.2. Behavioral effects and in vivo acute toxicity
The acute toxicity test of N-(6-allyl-1,3-benzodioxol-5-
yl)-N-(tert-butoxy carbonyl)phenylalaninamide (4e), was
realised through intraperitoneal way, on the concentrations:
5, 50, 100, 250 and 500 mg/kg of the animal weight. All
animals were monitored continuously for 72 h after dosing
for signs of toxicosis.
In the present study, we verified that the effects were not
noticed until the dose of 250 mg/kg, presenting acute toxicity
with cumulative action, irreversible and lethal hydric reten-
tion of two animals. This group of animals presented aug-
mented hearty reactions. Two animals presented cardio-
respiratory failure and then death. There was one death
50 min after the administration and other 20 h later. Higher
doses present toxicity, but in lower level. Clinical signals

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A.C.L. Leite et al. / European Journal of Medicinal Chemistry 39 (2004) 1059–1065
included motor uncoordination, hyperactivity, tremors, de-
crease of muscle tone, prostração and labored brating. There
was only one death in the group that received the dose of
raphy was carried out on silica gel plates having fluorescence
indicator F254 (0.2 mm, E. Merck); the spots were visualised
with UV light, and by spraying with a 2% ethanol solution of
ninhydrin or charging reagent. Column chromatography was
performed on silica using Kiesegel 60 (230–400 mesh, E.
Merck). All reagents used in the present work were of ana-
lytical grade.
500 mg/kg.
These results lead us to conclude that the dose of 25 mg/kg
(10% of the acute toxicity) will be the ideal for evaluation of
the antitumour activity. However, in an attempt to find a
better therapeutic response, we began with a dose of
35 mg/kg.
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]di-
oxole, 0.062 mol of acetic acid under 5 °C, 0.062 mol of
concentrated 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
(Na SO ). The residue was chromatographed on silica gel
3
.3. In vivo antitumour activity
The antitumour activity of compounds 4a–h and interme-
diary compound 6-allyl-benzo(1,3)dioxol-5-ylamine (3) was
tested against sarcoma S-180 in six Swiss albino mice (25–
28 g) for each tested product. The pre-clinical trial began 48 h
2
4
after the implantation of the tumour and longed for 8 days of
treatment, with the dose of 35 mg/kg/day, using the intraperi-
toneal way.
with 2% ethyl acetate in hexane to give 8.9 g (70%), of a
1
yellow oil. H-NMR (D ppm): 3.8 (d, 2H, CH 8); 4.98 (dd,
2
1H, CH 10a), 5.05 (dd, 1H, CH 10b), 5.97 (m, 1H, CH9);
2
2
All the products of the series 4a–h were able to inhibit the
growth of the tumour mass on the assays. The products 4b
6.00 (s, 2H, CH 2), 6.70 (s, 1H, Ar-7), 6.74 (s, 1H, Ar-4).
2
Anal. CHN C H O N.
1
0 9 4
(leucine), 4c (valine), 4h (glutamic acid) and 4e (glycine)
derivatives, presented an antitumour profile with reduction of
the tumour mass in 66.44%; 83.48%; 71.49%; 77.49%, re-
spectively. The other tested products 4a (phenylalanine), 4d
4.1.2. 6-Allyl-benzo [1,3]dioxole-5-ilamine (3)
A mixture of 2 (0.017 mol), iron powder (0.095 mol) and
NH Cl (0.01 mol) in 150 ml of EtOH:H O (2:1) was refluxed
4
2
(
lysine), 4f (alanine) and 4g (aspartic acid) derivatives were
for 1 h. The hot mixture was filtered through Celite and
concentrated under reduced pressure. The residue was di-
less active, with inhibition rates of 37.30%; 37.56%; 52.17%
and 51.78%, respectively. Concerning compound 6-allyl-
benzo(1,3)dioxol-5-ylamine (3), this product was very toxic,
with cumulative and lethal action. Six animals were tested
with this product, just one did not die.
luted with H O and extracted with EtOAc. The EtOAc ex-
2
tract was dried over anhydrous MgSO and concentrated to
4
1
give 3 (80%) as a light brown solid. H-NMR (D ppm): 3.10
(d, 2H, CH 8); 4.52 (s, 2H, NH ); 4.98 (dd, 1H, CH 10a),
2
2
2
This data clearly demonstrates that the linking of the
5.03 (dd, 1H, CH 10b), 5.88 (m, 1H, CH9); 5.96 (s, 2H,
2
1,3-benzodioxole nucleus from safrole to an amino acid
CH 2), 6.31 (s, 1H, Ar-4), 6.49 (s, 1H, Ar-7). Anal. CHN
2
moiety reduced the intrinsic toxicity. Six tested products of
this series were able to reduce the tumour in 50% or more.
Our results are according to the literature shown in all
cases, were that the drugs were linked to amino acids or
peptide fragments as a delivery system the resulting prodrug
was less toxic [13–17]. The reduced toxicity of the peptidyl
derivatives of safrole, in comparison to its prototype (inter-
mediary 3), could indicate an effective enzymatic cleavage
process selective to the tumour cell.
C H O N.
1
0 11 2
4.1.3. General procedure for the synthesis of peptidyl
derivatives from safrole (4a–h)
To a stirred solution of 2.5 mmol of the N-protected amino
acid in dimethylformamide under 0 °C, 6.8 mmol of the
diciclohexylcarbodiimide, 5.1 mmol of hydroxybenzotriaz-
ole and 1.7 mmoles of 6-allyl-benzo [1,3]dioxole-5-ylamine
(3) was added and was allowed to warm to room temperature.
After that, the reaction mixture was filtered, and the filtrate
was treated with ethyl acetate. The organic phase washed
with NaHCO , water and aqueous NaCl and dried (Na SO4)
4. Experimental
3
2
and concentrated. The residue was treated with hexane and
filtered.
4.1. Chemistry
1
H-NMR spectra were recorded on a Varian UNITY plus-
4.1.3.1. N-(6-Allyl-1,3-benzodioxol-5-yl)-N-(benzyloxy car-
3
00 MHz NMR spectrophotometer using DMSO-d as sol-
bonyl)glycinamide (4a). Yield 95.45%; m.p. 118–120 °C;
6
–
1
vent as tetramethylsilane as an internal standard. IR spectra
were obtained on using KBr pellets. Microanalyses for C, H,
N were performed in a EA1110 apparatus and were within
TLC R = 0.46 (3:2 hexane/ethyl acetate); I.R. (cm ): 1040
f
NC–O–C sym, 1245 NC–O–C assym, 1167 NC–C(=O)–O,
3338 NN–H, 1672 NC=O amide, 1685 NC=O carbamate,
1
±
0.4% of the required theoretical values. The melting points
1736 NC=O ester; H-NMR (D ppm): 1.37 (s, 9H, (CH ) ),
3
3
were determined using a Thomas Hoover apparatus and the
results are given without correction. Thin-layer chromatog-
2.49 (dd, J = 2.1 Hz, J = 6.3 Hz, 1H, CH b), 2.51 (dd, J
2
= 2.1 Hz, J = 6.0 Hz, 1H, CH b), 3.20 (d, J = 6.60 Hz, 2H,
2

A.C.L. Leite et al. / European Journal of Medicinal Chemistry 39 (2004) 1059–1065
1063
–
1
CH 8), 4.43 (ddd, J = 8.69 Hz, J = 6.30 Hz, J = 6.00 Hz, 1H,
(cm ): 1044 NC–O–C sym, 1219 NC–O–C assym, 1169
2
CHa), 4.98 (dd, J = 2.69 Hz, J = = 17.09 Hz, 1H, CH 10a),
NC–C(=O)–O, 3323 NN–H, 1658 NC=O amide, 1690 NC=O
2
1
5
.05 (dd, J = 2.69 Hz, J = 9.89 Hz, 1H, CH 10b), 5.12 (s, 2H,
carbamate; H-NMR (D ppm): 1.33 (s, 9H, (CH ) ), 2.84 (dd,
2
3 3
CH a″) 5.75-5.86 (m, 1H, CH9), 5.98 (s, 2H, CH 2), 6.70 (s,
J = 13.2 Hz, J = 9.6 Hz, 1H, CH b), 3.01 (dd, J = 13.49 Hz, J
2
2
2
1H, Ar-4), 6.79 (s, 1H, Ar-7), 7.27 (d, J = 8.69 Hz, 1H,
= 5.69 Hz, 1H, CH b), 3.15 (d, J = 6.89 Hz, 2H, CH 8),
2
2
NH-14), 7.35 (s, 5H, Ar-m, p and o), 9.25 (s, 1H, NH-11).
4.27–4.32 (m, 1H, Ha), 4.97 (dd, J = 1.8 Hz, J = 16.8 Hz, 1H,
CH 10a), 5.05 (dd, J = 1.8 Hz, J = 9.90 Hz, 1H, CH 10b),
Anal. CHN C H O N .
2
0
20
5
2
2
2
5
.75–5.88 (m, 1H, CH9), 5.98 (s, 2H, CH 2), 6.72 (s, 1H,
2
4
.1.3.2.
N-(6-Allyl-1,3-benzodioxol-5-yl)-N-(tert-butoxy
Ar-4), 6.73 (s, 1H, Ar-7), 7.13 (d, J = 8.40 Hz, 1H, NH-14),
7.29–7.30 (m, 5H, Ar-m, p and o prótons), 9.23 (s, 1H,
NH-11). Anal. CHN C H O N .
carbonyl)alanynamide (4b). Yield 42.85%; m.p. 93–95 °C;
TLC R = 0.44 (7:3 hexane/ethyl acetate); I.R. (cm ): 1037
–1
f
24 28 5 2
NC–O–C sym, 1250 NC–O–C assym, 1172 NC–C(=O)–O,
3
1
1
341 NN–H, 1659 NC=O amide, 1684 NC=O carbamate,
6
4
9
.1.3.6. N-(6-Allyl-1,3-benzodioxol-5-yl)- N -(9H-fluoren-
-ylmetoxicarbonylamino) N-(tert-butoxy carbonyl)lysin-
1
730 NC=O ester; H-NMR (D ppm): 1.38 (s, 9H, (CH ) ),
3
3
.82–1.92 (m, 1H, CH b), 1.98–2.04 (m, 1H, CH b), 2.45
2
2
amide (4f). Yield 92.60%; m.p. 115–117 °C; TLC R = 0.51
3:2 hexane/ethyl acetate); I.R. (cm ): 1027 NC–O–C sym,
262 NC–O–C assym, 1165 NC–C(=O)–O, 3317 NN–H,
654 NC=O amide, 1689 NC=O carbamate; H-NMR (D
ppm): 1.14–1.23 (m, 2H, CH c), 1.36–1.37 (m, 2H, Hb),
.38 (s, 9H, (CH ) ), 1.44–1.47 (m, 2H, HD), 2.96–3.00 (m,
H, He), 3.20 (d, J = 6.30 Hz, 2H, CH 8), 3.27 (s, 1H, CHb′),
.99–4.02 (m, 1H, CHa), 4.98 (dd, J = 1.79 Hz, J = 17.09 Hz,
H, CH 10a), 5.05 (dd, J = 1.79 Hz, J = 10.19 Hz, 1H,
CH 10b), 5.08 (s, 2H, CH a′) 5.79–5.88 (m, 1H, CH9), 5.97
s, 2H, CH 2), 6.64 (broad s, 1H, NH-f), 6.73 (s, 1H, Ar-4),
.85 (s, 1H, Ar-7), 6.99 (d, J = 8.39 Hz, 1H, NH-14), 7.39–
.34 (m, 4H, Ar-c′), 7.44–7.49 (m, 4H, Ar-c′), 9.12 (s, 1H,
f
(broad, 2H, CH c), 3.20 (d, J = 6.89 Hz, 2H, CH 8), 4.07–
–1
2
2
(
1
1
4.09 (m, 1H, CHa), 4.96 (dd, J = 1.79 Hz, J = 17.09 Hz, 1H,
CH 10a), 5.04 (dd, J = 1.79 Hz, J = 10.19 Hz, 1H, CH 10b),
1
2
2
5.09 (s, 2H, CH a″), 5.76–5.89 (m, 1H, CH9), 5.98 (s, 2H,
2
2
CH 2), 6.73 (s, 1H, Ar-4), 6.86 (s, 1H, Ar-7), 7.11 (d, J
=
2
1
2
3
1
3 3
7.49 Hz, 1H, NH-14), 7.32-7.37 (m, 5H, Ar-m, p and o),
2
9.14 (s, 1H, NH-11). Anal. CHN C H O N .
18 24 5 2
2
4.1.3.3.
N-(6-Allyl-1,3-benzodioxol-5-yl)-N-(tert-butoxy
2
2
carbonyl)valynamide (4c). Yield 24.27%; m.p. 203–205 °C;
TLC R = 0.74 (3:2 hexane/ethyl acetate); I.R. (cm ): 1043
NC–O–C sym, 1248 NC–O–C assym, 1171 NC–C(=O)–O,
(
6
7
–1
2
f
3
322 NN–H, 1653 NC=O amide, 1690 NC=O carbamate;
NH-11). Anal. CHN C H O N Cl .
1
31 32
6
2
2
H-NMR (D ppm): 0.89 (d, J = 6.89 Hz, 3H, CH c ), 0.92 (d,
3
1
J = 6.89 Hz, 3H, CH c ),1.39 (s, 9H, (CH ) ), 1.99–2.02 (m,
3
2
3 3
4
4
.1.3.7. N-(6-Allyl-1,3-benzodioxol-5-yl)-O -benzyl ester-
N-(tert-butoxy carbonyl)aspartic 1-amide (4g). Yield
0.65%; m.p. 110–112 °C; TLC R = 0.46 (3:2 hexane/ethyl
1
H, CHb), 3.22 (d, J = 6.60 Hz, 2H, CH 8), 3.77 (dd, J
2
=
=
=
5.70 Hz, J = 7.79 Hz, 1H, CHa), 4.97 (dd, J = 1.20 Hz, J
3
f
16.79 Hz, 1H, CH 10a), 5.05 (dd, J = 1.20 Hz, J
2
–
1
acetate) I.R. (cm ): 1040 NC–O–C sym, 1219 NC–O–C
assym, 1147 NC–C(=O)–O, 3263 NN–H, 1666 NC=O
amide, 1744 NC=O ester; H-NMR (D ppm): 3.21 (d, J
10.19 Hz, 1H, CH 10b), 5.77–5.91 (m, 1H, CH9), 5.98 (s,
2
2
H, CH 2), 6.73 (s, 1H, Ar-4), 6.81 (s, 1H, Ar-7), 6.91 (d, J
2
1
=
7.79 Hz, 1H, NH-14), 9.18 (s, 1H, NH-11). Anal. CHN
=
(
6,30 Hz, 2H, CH 8), 3.78 (d, J = 6.30 Hz, 2H, CH a), 4.99
C H O N .
2
2
20 28 5 2
dd, J = 1.80 Hz, J = 16.79 Hz, 1H, CH 10a), 5.03 (dd, J
2
=
1.80 Hz, J = 10.19 Hz, 1H, CH 10b), 5.05 (s, 2H, CH a′),
2 2
4
.1.3.4.
N-(6-Allyl-1,3-benzodioxol-5-yl)-N-(tert-butoxy
5
.77–5.86 (m, 1H, CH9), 5.98 (s, 2H, CH 2), 6.74 (s, 1H,
carbonyl)leucynamide (4d). Yield 46.4%, m.p. 211–212 °C;
TLC R = 0.87 (3:2 hexane/ethyl acetate I.R. (cm ): 1042
NC–O–C sym, 1250 NC–O–C assym, 1172 NC–C(=O)–O,
2
–1
Ar-4), 6.89 (s, 1H, Ar-7), 7.31–7.37 (m, 5H, Ar-b′), 7.57 (t, J
6.30 Hz, 1H, NH-14), 9.15 (s, 1H, NH-11). Anal. CHN
C H O N .
f
=
3
363 NN–H, 1649 NC=O amide, 1691 NC=O carbamate;
26 30 7 2
4
1
N-(6-Allyl-1,3-benzodioxol-5-yl)-O -benzyl
tert-butoxy carbonyl)glutamic (4h)
ester-N-
H-NMR (D ppm): 0.82 (d, J = 6.90 Hz, 3H, CH D), 0.85 (d,
3
(
(
J = 6.60 Hz, 3H, CH D), 0.90 (t, J = 6.60 Hz, 2H, CH b),
3
2
Yield 31.35%; m.p. 75–78 °C; TLC R = 0.70
1
=
=
.37 (s, 9H, (CH ) ), 1.43–1.53 (m, 1H, CHc), 3.20 (d, J
f
3
3
–
1
7:3 hexane/ethyl acetate); I.R. (cm ): 1038 NC–O–C sym,
215 NC–O–C assym, 1167 NC–C(=O)–O, 3324 NN–H,
5.09 Hz, 2H, CH 8), 4.05–4.13 (m, 1H, CHa), 4.99 (dd, J
2.02 Hz, J = 17.09 Hz, 1H, CH 10a), 5.06 (dd, J = 2.02 Hz,
2
1
2
1
J = 9.90 Hz, 1H, CH 10b), 5.79–5.88 (m, 1H, CH9), 5.98 (s,
2
=
1659 NC=O amide, 1690 NC=O carbamate; H-NMR (D
ppm): 1.25 (d, J = 7.49 Hz, 3H, CH b), 1.38 (s, 9H, (CH ),
3.20 (d, J = 6.89 Hz, 2H, CH 8), 4.07–4.12 (m, 1H, CHa),
5.03 (dd, J = 2.20 Hz, J = 17.09 Hz, 1H, CH 10a), 5.05 (dd,
2
H, CH 2), 6.73 (s, 1H, Ar-4), 6.84 (s, 1H, Ar-7), 7.04 (d, J
8.09 Hz, 1H, NH-14), 9.11 (s, 1H, NH-11). Anal. CHN
3
)
3 3
2
2
C H O N .
2
21 30 5 2
J = 2.20 Hz, J = 10,19 Hz, 1H, CH 10b), 5.77–5.91 (m, 1H,
2
4
.1.3.5.
carbonyl)phenylalaninamide (4e). Yield 36.6%, m.p. 110–
13 °C; TLC R = 0.53 (7:3 hexane/ethyl acetate); I.R.
N-(6-Allyl-1,3-benzodioxol-5-yl)-N-(tert-butoxy
CH9), 5.98 (s, 2H, CH 2), 6.73 (s, 1H, Ar-4), 6.86 (s, 1H,
Ar-7), 7.06 (d, J = 6.59 Hz, 1H, NH-14), 9.07 (s, 1H, NH-11).
Anal. CHN C H O N .
27 32 7 2
2
1
f

1064
A.C.L. Leite et al. / European Journal of Medicinal Chemistry 39 (2004) 1059–1065
4.1.4. (6-Nitro-benzo [1,3]dioxole-5-yl) acetic acid (5)
CH 2), 7.03 (s, 1H, NH -13), 7.05 (s, 1H, Ar-7), 7.35 (s, 1H,
2 2
The aqueous KMnO (0.135 mol) in about 150 ml of water
NH -13), 7.62 (s, 1H, Ar-4), 7.98 (d, J = 9.29 Hz, 1H,
4
2
was stirred and cooled out in an ice bath. A solution of
compound 2 (0.024 mol), benzyldimethyltetradecylammo-
nium chloride dihydrate (0.0012 mol), and 30 ml of acetone,
NH-10). Anal. CHN C H O N .
1
5 19 6 3
4.1.5.3. N-[(6-nitro-1,3-benzodioxol-5-yl) acetyl]phenyl-
1
50 ml of benzene, and 30 ml of acetic acid was added in one
alaninamide (6c). Yield 76.40%; m.p. 158–160 °C; TLC R
f
–
1
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 solution of aqueous H SO (25 g of
= 0.40 (1:1 hexane/ethyl acetate); I.R. (cm ): 3320 NN–H
a
amide 2 , 1627 NC=O amide, 1527 NAr–NO assym, 1330
2
1
NAr–NO sym, 3320 NN–H assym, 3196 NN–H sym; H-
2
NMR (D ppm): 2.80 (dd, J = 13.80 Hz, J = 4.50 Hz, 1H,
CH b), 3.01 (dd, J = 13.80 Hz, J = 4.80, 1H, CH b); 3.73 (d,
2
4
concentrated H SO in 100 ml of water). Two clear layers
2
4
2
2
resulted. The layers were separated and the organic layer was
washed once with a 100 ml of portion of water. The organic
layer was dried over anhydrous sodium sulfate, the drying
agent was removed by filtration, and the solvent was removed
on a rotary evaporator. The crude product was chromato-
J = 16.20 Hz, 1H, CH a), 3.82 (d, J = 16.20 Hz, 1H, CH a);
2
2
4.43 – 4.41 (m, 1H, CH a′); 6.20 (s, 2H, CH 2), 6.88 (s, 1H,
2
2
Ar-7), 7.0 (s, 1H, NH -13); 7.29 – 7.21 (1H do NH -13, 5H
2
2
do Ar-m, p and o); 7.57 (s, 1H, Ar-4), 8.07 (d, J = 7.80 Hz,
1H, NH-10). Anal. CHN C H O N .
1
8 17 6 3
graphed on silica gel with 5% ethyl acetate in hexane to give
1
4
3.25 g (60%), of a yellow solid. m.p. 180–182 °C. H-NMR
4.1.5.4.
N-[(6-nitro-1,3-benzodioxol-5-yl)
acetyl]-O -
(
D ppm): 3.9 (s, 2H, CH 8); 6.23 (s, 2H, CH 2); 7.12 (s, 1H,
(2,6-dichlorophenyl)tyrosinamide (6d). Yield 46.15%; m.p.
2 2
–
1
Ar-7); 7.69 (s, 1H, Ar-4); 12.48 (s, 1H, COOH). Anal. CHN
C H O N.
150–153 °C; TLC R = 0.80 (ethyl acetate); I.R. (cm ): 3326
f
a
NN–H amide 2 , 3326 NN–H assym, 3193 NN–H sym, 1628
9
7 6
NC=O amide, 1532 NAr–NO assym, 1243 NAr–NO sym;
2
2
1
4
.1.5. General procedure for the synthesis of peptidyl
H-NMR (D ppm): 2.85 (dd, J = 9.10 Hz, J = 14.10 Hz, 1H,
CH b), 3.06 (dd, J = 5.10 Hz, J = 14.10, 1H, CH b); 3.35–
3.34 (m, 2H, CH a), 4.61–4.54 (m, 1H, CHa′); 5.21 (s, 2H,
CH a″); 6.25 (s, 2H, CH 2), 6.77 (s, 1H, Ar-7), 6.97 (d, J
8.70 Hz, 2H, Ar-c); 7.09 (s, 1H, NH -13); 7.22 (d, J
derivatives of safrole (6a–d)
2
2
To a stirred solution of 2.5 mmol of the N-protected amino
acid in dimethylformamide under 0 °C, 6.8 mmol of the
diciclohexylcarbodiimide, 5.1 mmol of hydroxybenzotriaz-
ole and 1.7 mmoles of 6-alyl-benzo [1,3]dioxole-5-ilamine
2
2
2
=
=
2
8.70 Hz, 2H, Ar-c); 7.42 (s, 1H, NH -13); 7.53 (s, 1H,
2
Ar-4), 7.56–7.45 (3H, Ar - b′), 8.60 (s, 1H, NH-10). Anal.
(3) was added and was allowed to warm at room temperature.
CHN C H O N Cl .
After that, the reaction mixture was filtered, and the filtrate
was treated with ethyl acetate. The organic phase was washed
with the NaHCO water, aqueous NaCl and dried (Na SO4)
25 21
7
3
2
4.2. Biology
3
2
and concentrated. The residue was treated with hexane and
filtered. The safrole derivatives 4a–d, were obtained as white
crystals.
4.2.1. Antimicrobial activity
The antimicrobial activity was determined in vitro by disc
diffusion method against Staphylococcus aureus, Sarcina
lutea, B. subtilis, Streptococcus faecalis, 381, Staphylococ-
cus aureus, 466 Staphylococcus aureus, E. coli, K. pneu-
moniae, Shigella sonei, Salmonella enteritides, P. aerugi-
nosa and Candida albicans from Departamento de
Antibióticos da Universidade Federal de Pernambuco collec-
tion. It is dispensed into Petri dishes to yield a uniform depth
of 6 mm. Overnight cultures were grown in adequate broth
and temperature, and diluted to obtain an opacity equivalent
to 0.5 on the Mc Farland scale. The drugs are weighed and
dissolved in acetone to give concentrations equal to 2 mg/ml.
The discs were placed in the surface of culture medium after
the complete evaporation of acetone. The plates are incu-
bated for 18 h at adequate temperature.
4.1.5.1. Ethyl N-[(6-nitro-1,3-benzodioxol-5-yl) acetyl]
glycinate (6a). Yield 99%; m.p. 130–133 °C; TLC R = 0.83
f
–1
a
(
ethyl acetate); I.R. (cm ): 3324 NN–H amide 2 , 1628
NC=O amide, 1524 NAr–NO assym, 1260 NAr–NO sym,
2
2
1
1
3
.739 NC=O ester; H-NMR (D ppm): 1.19 (t, J = 7.19 Hz,
H, CH b), 3.82 (d, J = 5,7 Hz, 2H, CH a′); 3.85 (s, 2H,
3
2
CH a), 4.08 (q, J = 7.19 Hz, 2H, CH a″); 6.21 (s, 2H, CH 2),
2
2
2
7.06 (s, 1H, Ar-7), 7.61 (s, 1H, Ar-4), 8.33 (broad, 1H,
NH-10). Anal. CHN C H O N .
13 14 7 2
4.1.5.2. N-[(6-nitro-1,3-benzodioxol-5-yl) acetyl]leucin-
amide (6b). Yield 17.80%; m.p. 175–176 °C; TLC R = 0.30
f
–1
(
2
1:1 hexane/ethyl acetate); I.R. (cm ): 3326 NN–H amide
, 1629 NC=O amide, 1574 NAr–NO assym, 1244 NAr–
NO sym, 3387 NN–H assym, 3199 NN–H sym; H-NMR (D
a
2
1
4.2.2. Toxicity tests
2
ppm): 0.79 (dd, J = 7.49 Hz, J = 6.8 Hz, 3H, CH D), 0.83 (d,
Thirty-six male Swiss adult white mice (ca. 25–28 g) were
divided into six groups and privately fed 20 h before injec-
tions. The preliminary phase dosages are 500, 250, 100, 50,
5 mg/kg to compound 4e. It was diluted in Tween 80 (two
crops) and saline (0.9%). The single intraperitoneal injec-
3
J = 5.89 Hz, 3H, CH c); 1.02–1.09 (m, 1H, CH c ), 1.18–
2
2
2
1
.30 (m, 1H, CH c ), 1.59–1.73 (m, 1H, CHb), 3.80 (d, J
2 2
=
15.89 Hz, 1H, CH a), 3.91 (d, J = 16.19 Hz, 1H, CH a),
2 2
4.11 (dd, J = 6.59 Hz, J = 9.00 Hz, 1H, CHa′), 6.21 (s, 2H,

A.C.L. Leite et al. / European Journal of Medicinal Chemistry 39 (2004) 1059–1065
1065
tions are given and the general effects were observed during
0 min and continued about 72 h after injection. The control
group, receiving solvent only.
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[
7] Available from http://ntp-server.niehs.nih.gov/htdocs/8_Roc/RAC/
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6
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4
.2.3. Antitumor activity
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for 8 days. After treatment, tumours were removed and mea-
sured. The efficacy of treatment was estimated by the ellip-
soid formula, and V of control group was taken in calcula-
tions for 100%.
(
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“
Fundação de Amparo a Ciência do Estado de Pernambuco”
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1
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