Synthesis of pure stereoisomers of benzo[b]thienyl dehydrophenylalanines by Suzuki cross-coupling. Preliminary studies of antimicrobial activity

Article abstract of DOI:10.1016/j.tet.2004.09.107

Several benzo[b]thienyldehydrophenylalanines were synthesized from pure stereoisomers of the methyl ester of N-(tert-butoxycarbonyl)-β- bromodehydrophenylalanine as an extension of our previously reported method for the synthesis of dehydrotryptophan analogues to dehydrophenylalanine derivatives. The latter were obtained in high yields by N-deprotection and bromination of N,N-bis-(tert-butoxycarbonyl)-(Z)-dehydrophenylalanine using TFA and NBS. This was carried out in two steps or in a one pot procedure resulting in different E/Z ratios. These compounds were coupled under Suzuki cross-coupling conditions [Pd(PPh₃)₄, Na ₂CO₃, DME/H₂O] with several boronic benzo[b]thienyl acids in good to high yields maintaining the stereochemistry of the starting materials. The best yields were obtained when the boronic acid was in position 7 of the benzo[b]thiophene and with the E isomer of the brominated dehydrophenylalanine. In some cases it was possible to increase the lower yields by changing the Pd source to PdCl₂(PPh₃)₂. A model dipeptide was prepared coupling a benzo[b]thienyldehydrophenylalanine with the methyl ester of alanine. Preliminary antimicrobial studies were performed with both isomers of one of the β, β-diaryldehydroalanines obtained. The results show that the compounds are selective and very active (very low MICs) against Gram positive bacteria (B. cereus and B. subtilis) the Z-isomer being more active. The compounds are also active against Candida albicans presenting similar MICs. Graphical Abstract

Full text of DOI:10.1016/j.tet.2004.09.107

Tetrahedron 60 (2004) 11821–11828
Synthesis of pure stereoisomers of benzo[b]thienyl
dehydrophenylalanines by Suzuki cross-coupling.
Preliminary studies of antimicrobial activity
a
a
Ana S. Abreu,a Paula M. T. Ferreira,a, Luıs S. Monteiro, Maria-Joa˜o R. P. Queiroz,
*
´
b
Isabel C. F. R. Ferreira, Ricardo C. Calhelha and Letıcia M. Estevinho
b
b
´
a
´
Departamento de Quımica, Universidade do Minho, 4710-057 Braga, Portugal
b
´
´
´
Escola Superior Agraria, Instituto Politecnico de Braganc¸a, Campus de Sta Apolonia, 5300 Braganc¸a, Portugal
Received 29 March 2004; revised 23 September 2004; accepted 29 September 2004
Available online 18 October 2004
Abstract—Several benzo[b]thienyldehydrophenylalanines were synthesized from pure stereoisomers of the methyl ester of N-(tert-
butoxycarbonyl)-b-bromodehydrophenylalanine as an extension of our previously reported method for the synthesis of dehydrotryptophan
analogues to dehydrophenylalanine derivatives. The latter were obtained in high yields by N-deprotection and bromination of N,N-bis-(tert-
butoxycarbonyl)-(Z)-dehydrophenylalanine using TFA and NBS. This was carried out in two steps or in a one pot procedure resulting in
different E/Z ratios. These compounds were coupled under Suzuki cross-coupling conditions [Pd(PPh3)4, Na2CO3, DME/H2O] with several
boronic benzo[b]thienyl acids in good to high yields maintaining the stereochemistry of the starting materials. The best yields were obtained
when the boronic acid was in position 7 of the benzo[b]thiophene and with the E isomer of the brominated dehydrophenylalanine. In some
cases it was possible to increase the lower yields by changing the Pd source to PdCl2(PPh3)2. A model dipeptide was prepared coupling a
benzo[b]thienyldehydrophenylalanine with the methyl ester of alanine. Preliminary antimicrobial studies were performed with both isomers
of one of the b, b-diaryldehydroalanines obtained. The results show that the compounds are selective and very active (very low MICs) against
Gram positive bacteria (B. cereus and B. subtilis) the Z-isomer being more active. The compounds are also active against Candida albicans
presenting similar MICs.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
bond of dehydroamino acids does not in itself cause reversal
in the peptide backbone and the preferred conformation of a
dehydropeptide may be decided by the nature of the
b-substituents. Thus, different dehydroamino acids can be
used to introduce different kinds of constraints in peptides.4
Dehydroamino acids have been found in several natural
peptides from microbial or marine sources. The incorpor-
ation of a,b-dehydroamino acids constitutes a valuable tool
in structure-activity relationship (SAR) studies, due to the
conformational constraints they impose. These restrained
analogues of amino acids mainly dehydrophenylalanine and
dehydrotyrosine have been introduced in peptide sequences
to probe the preferred orientations of these residues once
bound to the receptors.1 These molecules can also be very
useful as pharmaceutical probes towards various proteases,
namely HIV-proteases.2
Recently, we have been interested in the synthesis of new
b-substituted dehydroamino acids either by Michael
additions5 or by palladium catalyzed cross-couplings.6a–c
The fluorescence studies performed on b-benzo[b]thienyl-
dehydroamino acids already prepared by us showed that
some of them can also be used as fluorescent probes.6c
Here we describe the synthesis of benzo[b]thienyldehydro-
phenylalanines using Suzuki cross-coupling7 of several
boronic benzo[b]thienyl acids with pure stereoisomers of a
b-bromodehydrophenylalanine derivative. Two of the b,
b-diaryldehydroalanines obtained were tested for anti-
microbial activity and showed to be active with low
minimal inhibitory concentration (MIC). The insertion of
this type of compounds in peptides was demonstrated
preparing a model dipeptide using DCC/HOBt.
The dehydrophenylalanine residue as a constrained phenyl-
alanine mimic has gained much importance, in particular,
because of its turn inducing as well as helix-forming
propensity.3 Studies have indicated that the a,b-double
Keywords: Amino acids; Dehydrophenylalanines; Benzo[b]thiophenes;
Suzuki coupling; Palladium; Antimicrobial.
* Corresponding author. Tel.: C351 253604372; fax: C351 253678983;
e-mail: pmf@quimica.uminho.pt
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.09.107

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A. S. Abreu et al. / Tetrahedron 60 (2004) 11821–11828
Scheme 1. Synthesis of compound (E)-1 and (Z)-1 in two steps or in a one pot procedure.
2. Results and discussion
monodeprotected5 with TFA and b-brominated with NBS
followed by treatment with NEt3 to give the methyl ester of
N-(tert-butoxycarbonyl)-b-bromodehydrophenylalanine
[Boc-DPhe(b-Br)-OMe)] as a 1:2 E/Z mixture in an overall
The methyl ester of N,N-bis-(tert-butoxycarbonyl)-(Z)-
dehydrophenylalanine5 (Boc2-DPhe-OMe) was N-
Figure 1. 1H NMR spectrum (CDCl3) of (E)-1 and NOE difference experiment irradiating the a-NH and observing the effect on the signal of the phenyl
protons.

A. S. Abreu et al. / Tetrahedron 60 (2004) 11821–11828
11823
Figure 2. 1H NMR spectrum (CDCl3) of (Z)-1 and NOE difference experiment irradiating the a-NH.
yield of 89%. The same reactions performed in a one pot
procedure gave the products in a similar yield but resulted in
a higher selectivity towards the Z isomer (1:6 E/Z)
(Scheme 1). These results are similar to those obtained by
us in the bromination of dehydroaminobutyric acid.6a
In order to increase the yields of the Z isomers, another
palladium catalyst [PdCl2(PPh3)2], that had already given
good results in the synthesis of b,b-bis-(benzo[b]thienyl)-
dehydroalanines from a b,b-dibromodehydroalanine
derivative, was used.6c In these conditions (ii), when the
boronic acids are in the thiophene ring the yields increased
from 61 to 74% in the synthesis of (Z)-2a and from 50 to
66% in the synthesis of (Z)-2b. This increase was not
observed in the synthesis of compound (Z)-2c which was
obtained in similar yields using both catalytic systems
(Scheme 2).
The stereochemistry of (E) and (Z)-1 was determined by
NOE difference experiments irradiating the a-NH and
observing a NOE effect on the signal of the phenyl protons
of (E)-1 (Fig. 1) while for compound (Z)-1 this was not
observed (Fig. 2).
The pure stereoisomers (E) and (Z)-1 were coupled with
several boronic benzo[b]thienyl acids under Suzuki cross-
coupling conditions6a to give b-benzo[b]thienyldehydro-
phenylalanines in good to high yields (Scheme 2). NOE
difference experiments irradiating the a-NH confirmed that
the coupled products maintained the stereochemistry of the
starting materials.
A dipeptide was prepared from (E)-2b by C-deprotection
and coupling with the methyl ester of alanine using DCC/
HOBt (Scheme 3). This result in the synthesis of a model
dipeptide shows that our b,b-diaryldehydroamino acids can
be inserted into peptides.
A screening of antibacterial activities using two Gram
negative (Escherichia coli and Pseudomonas aeruginosa)
and two Gram positive bacteria (Bacillus subtilis and
Bacillus cereus) and antifungal activity using Candida
albicans as a representative of fungi was assessed for
compounds (Z)-2c and (E)-2c. The MIC (in mg/mL) was
determined (Table 1) using an adaptation of agar streak
The best yields were obtained when the boronic acid was in
position 7 of the benzo[b]thiophene moiety. In all cases
using the same catalytic conditions (i), the higher yields
were obtained from compound (E)-1 which can be due to the
lower steric hindrance of this derivative.

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A. S. Abreu et al. / Tetrahedron 60 (2004) 11821–11828
Scheme 2. Synthesis of compounds (E)-2a–c and (Z)-2a–c under Suzuki cross-coupling conditions. (i) Pd(PPh3)4 (10 mol%), Na2CO3 (2 equiv), boronic acid
(1.3 equiv), DME/H2O (4:1), 90 8C, 3–5 h. (ii) Same conditions but using PdCl2(PPh3)2 (10 mol%).
dilution method based on radial diffusion.8 In the same
conditions, different concentrated solutions of Ampicillin
(antibacterial) and Cyclohexamide (antifungal) were used as
standards. The MIC was considered to be the lowest
concentration of the tested compound which inhibits
growth of bacteria or fungi on the plate. The compounds
tested were inactive against the Gram- bacteria
(Escherichia coli and Pseudomonas aeruginosa). The
diameters of the inhibition zones corresponding to the
MICs for the GramC bacteria and for C. albicans are
presented in Table 1.
conclude that the compounds tested are active
against B.cereus, B.subtilis and C. albicans presenting
MICs very much lower than those obtained with
Ampicillin
(antifungal).
(antibacterial)
and
Cyclohexamide
Compound (Z)-2c shows lower MICs than (E)-2c for
GramC bacteria but the results against C. albicans are
similar for both isomers. These results indicate that the
compounds are selective and very active (very low MICs)
against the GramC bacteria tested and against a represen-
tative of fungi, thus showing very interesting antimicrobial
properties.
From the inspection of Table 1 it is possible to

A. S. Abreu et al. / Tetrahedron 60 (2004) 11821–11828
11825
Scheme 3. Synthesis of dipeptide 4 from (E)-2b and the methylester of alanine.
Table 1. Antimicrobial activity of compounds (Z)-2c and (E)-2c
Compounds
MIC (mg/mL) (zone of inhibition in mm)
Bacillus subtilis CECT498
Bacillus cereus CECT148
Candida albicans CECT 1394
(E)-2c
(Z)-2c
Ampicillin
Cyclohexamide
0.125 (13)
1.25!10K3 (15)
3.13 (13)
—
0.125 (15)
1.25!10K3 (11)
12.5 (10)
—
0.125 (6)
0.125 (5)
—
12.5 (5)
CECT—Spanish type culture collection of Valencia University.
3. Conclusion
was used, the increase in polarity was made from neat
petroleum ether to mixtures of ether/petroleum ether,
increasing 10% of ether each time until the isolation of
the product.
Several b,b-diaryldehydroamino acids in the benzo[b]thio-
phene series were synthesized in good to high yields from
pure stereoisomers of a b-bromodehydrophenylalanine
derivative and benzo[b]thienylboronic acids using C–C
palladium-catalyzed cross-couplings. From the results
obtained we can conclude that the E isomer of the b-
brominated dehydrophenylalanine (E)-1 is more reactive
under Suzuki cross coupling conditions then the Z isomer. It
is also possible to conclude that the 7-benzo[b]thienyl-
boronic acid is the most reactive, and its reactivity does not
depend on the Pd source. The insertion of this type of
compounds in peptides was tested preparing a model
dipeptide in high yield. Preliminary antimicrobial studies
were performed using both isomers of one of the b,b-
diaryldehydroalanines obtained. The results show that the
compounds are selective and very active (very low MICs)
against GramCbacteria (B. cereus and B. subtilis), the
Z-isomer being more active. The compounds are also active
against Candida albicans presenting similar MICs.
4.2. Synthesis of Boc-(E)-DPhe(b-Br)-OMe (E)-1 and
Boc-(Z)-DPhe(b-Br)-OMe (Z)-1
Boc-DPhe-OMe5 (1.39 g, 5.00 mmol) was dissolved in
dichloromethane (0.1 M) and 1.2 equiv of N-bromo-
succinimide (0.980 g, 5.50 mmol) were added with vigorous
stirring. After reacting for 16 h, triethylamine (1.5 equiv)
was added and stirring continued for an additional hour.
Dicloromethane (50 mL) was added and the organic phase
was washed with water and brine (3!30 mL each). After
drying over MgSO4 the extract was taken to dryness at
reduced pressure to give (E)-1 and (Z)-1 (1.73 g, 97%) as a
1:2 mixture. The diastereomers were separated by column
chromatography using solvent gradient from neat petroleum
ether to 20% diethyl ether/petroleum ether to give (Z)-1 mp
101–103 8C (from diethyl ether/n-hexane). 1H NMR
(CDCl3): 1.49 (9H, s, CH3 Boc), 3.53 (3H, s, OCH3), 6.55
(1H, s, aNH), 7.34 (5H, br s, ArH). 13C NMR (CDCl3):
28.06 (C(CH3)3), 52.43 (OCH3), 82.10 (OC(CH3)3), 128.21
(CH), 128.41 (C), 128.94 (CH), 129.11 (CH), 129.31 (C),
137.39 (C), 151.89 (C]O), 163.47 (C]O) ppm. Anal.
Calcd for C15H18NO4Br (356.22): C, 50.58; H, 5.09; N,
3.93. Found: C, 50.61; H, 5.12; N, 4.01. (E)-1 mp 80–81 8C
(from petroleum ether). 1H NMR (CDCl3): 1.43 (9H, s, CH3
Boc), 3.93 (3H, s, OCH3), 6.08 (1H, s, aNH), 7.43 (5H, br s,
ArH). 13C NMR (CDCl3): 28.01 (C(CH3)3), 52.58 (OCH3),
82.00 (OC(CH3)3), 128.58 (C), 128.94 (CH), 129.04 (CH),
129.47 (CH), 136.50 (C), 151.56 (C]O), 164.55
(C]O) ppm. Anal. Calcd for C15H18NO4Br (356.22): C,
50.58; H, 5.09; N, 3.93. Found: C, 50.74; H, 5.21; N, 4.09.
4. Experimental
4.1. General
Melting points (8C) were determined in a Gallenkamp
1
apparatus and are uncorrected. H and 13C NMR spectra
were recorded on a Varian Unity Plus at 300 and 75.4 MHz,
respectively. H–1H spin–spin decoupling and DEPT q 458
1
were used. Chemical shifts are given in ppm and coupling
constants in Hz. MS and HRMS data were recorded by the
mass spectrometry service of the University of Vigo, Spain.
Elemental analysis was performed on a LECO CHNS 932
elemental analyser.
One pot procedure. Boc2-DPhe-OMe5 (1.86 g, 5.00 mmol)
was dissolved in dichloromethane (0.1 M) and 2% of TFA
was slowly added with vigorous stirring. The reaction was
monitored by TLC and when no starting material was
detected (z1 h) 1.2 equiv of N-bromosuccinimide (1.34 g,
The reactions were monitored by thin layer chromatography
(TLC). Column chromatography was performed on
Macherey-Nagel silica gel 230–400 mesh. Petroleum ether
refers to the boiling range 40–60 8C. When solvent gradient

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A. S. Abreu et al. / Tetrahedron 60 (2004) 11821–11828
7.50 mmol) were added. After reacting for 16 h triethyl-
amine (15.0 mmol) was added and stirring continued for an
additional hour. Dichoromethane was added (50 mL) and
the organic phase was then washed with water and brine
(2!30 mL each). After drying over MgSO4, the extract was
taken to dryness at reduce pressure to afford a 1:6 mixture of
(E)-1 and (Z)-1 (1.48 g, 83%).
4.3.3. Boc-(E)-DPhe(b-benzo[b]thien-3-yl)-OMe ((E)-
2b). The procedure described above was applied using
compound (E)-1 (178 mg, 0.500 mmol) and the 3-benzo-
[b]thienylboronic acid (98.0 mg, 0.550 mmol) and heating
for 3 h. Column chromatography using a solvent gradient
from pure petroleum ether to 30% diethyl ether in petroleum
ether, gave compound (E)-2b (112 mg, 60%) as an oil.
Recrystallization from diethyl ether/petroleum ether
afforded colourless crystals, mp 171–172 8C. 1H NMR
(CDCl3): 1.48 (9H, s, CH3 Boc), 3.46 (3H, s, OCH3), 6.33
(1H, br s, NH), 7.19 (1H, d, JZ7.8 Hz, ArH), 7.26–7.40
(8H, m, ArH), 7.81 (1H, d, JZ7.5 Hz, ArH) ppm 13C NMR
(CDCl3): 28.09 (C(CH3)3), 52.09 (OCH3), 81.46
(OC(CH3)3), 122.49 (CH), 123.21 (CH), 124.08 (CH),
124.30 (CH), 125.63 (CH), 127.57 (C), 128.45 (CH), 128.89
(CH), 129.20 (CH), 131.94 (C), 134.59 (C), 136.75 (C),
138.09 (C), 139.81 (C), 152.54 (C]O), 166.34
(C]O) ppm. Anal. Calcd for C23H23NO4S (409.50): C,
67.46; H, 5.66; N, 3.42; S, 7.83. Found: C, 67.30; H, 5.94;
N, 3.51; S, 7.64.
4.3. General procedure for Suzuki cross couplings
To a solution of compound (E)-1 or (Z)-1 in DME/water
(4:1), benzo[b]thienylboronic acids (1.1 equiv), Na2CO3
(2 equiv) and Pd(PPh3)4 (10 mol%) were added and the
mixture was heated at 90 8C while the reaction was
monitored by TLC. The DME was removed under reduced
pressure and the residue was dissolved in ethyl acetate
(15 mL). The organic layer was then washed with water and
brine (3!5 mL) dried with MgSO4 and the solvent
evaporated at reduce pressure to give an oil which was
submitted to column chromatography.
4.3.4. Boc-(Z)-DPhe(b-benzo[b]thien-3-yl)-OMe ((Z)-
2b). The procedure described above was applied using
compound (Z)-1 (107 mg, 0.300 mmol) and the 3-benzo-
[b]thienylboronic acid (59.0 g, 0.330 mmol) and heating for
3 h. Column chromatography using a solvent gradient from
pure petroleum ether to 30% diethyl ether in petroleum
ether, gave compound (Z)-2b (62.0 mg, 50%) as an oil.
Recrystallization from diethyl ether/petroleum ether
afforded colourless crystals, mp 139–140 8C. 1H NMR
(CDCl3): 1.40 (9H, s, CH3 Boc), 3.61 (3H, s, OCH3), 5.92
(1H, br s, NH), 7.16–7.20 (2H, m, ArH), 7.27–7.44 (6H, m,
ArH), 7.58 (1H, d, JZ8.1 Hz, ArH), 7.90 (1H, d, JZ8.1 Hz,
ArH) ppm. 13C NMR (CDCl3): 28.07 (C(CH3)3), 52.13
(OCH3), 81.38 (OC(CH3)3), 122.79 (CH), 123.25 (C),
124.67 (CH), 124.82 (CH), 127.22 (C), 128.05 (CH),
128.17 (CH), 128.41 (CH), 128.70 (CH), 134.16 (C),
136.84 (C), 139.39 (C), 140.12 (C), 152.73 (C]O),
166.18 (C]O) ppm. Anal. Calcd for C23H23NO4S
(409.50): C, 67.46; H, 5.66; N, 3.42; S, 7.83. Found: C,
67.53; H, 5.79; N, 3.50; S, 7.72. The procedure described
above using PdCl2 (PPh3)2 gave compound (Z)-2b (81.0 mg,
66%).
4.3.1. Boc-(E)-DPhe(b-benzo[b]thien-2-yl)-OMe ((E)-
2a). The procedure described above was applied using
compound (E)-1 (107 mg, 0.300 mmol) and the 2-benzo-
[b]thienylboronic acid (0.330 mmol, 59.0 mg) and heating
for 3 h 30 min. Column chromatography using a solvent
gradient from pure petroleum ether to 30% diethyl ether in
petroleum ether, gave compound (E)-2a (86.0 mg, 70%) as
an oil. Recrystallization from diethyl ether/petroleum ether
afforded light yellow crystals, mp 97–99 8C. 1H NMR
(CDCl3): 1.46 (9H, s, CH3 Boc), 3.69 (3H, s, OCH3), 6.17
(1H, br s, NH), 7.16 (1H, s, ArH), 7.29–7.43 (7H, m, ArH),
7.69–7.74 (2H, m, ArH) ppm. 13C NMR (CDCl3): 28.09
(C(CH3)3), 52.58 (OCH3), 81.77 (OC(CH3)3), 122.05 (CH),
123.60 (CH), 124.33 (CH), 124.43 (CH), 124.55 (CH),
127.44 (C), 128.83 (CH), 129.02 (CH), 129.62 (CH), 137.33
(C), 139.31 (C), 140.72 (C), 141.97 (C), 152.12 (C]O),
166.01 (C]O) ppm. Anal. Calcd for C23H23NO4S (409.50):
C, 67.46; H, 5.66; N, 3.42; S, 7.83. Found: C, 67.25; H, 5.93;
N, 3.39; S, 7.36.
4.3.2. Boc-(Z)-DPhe(b-benzo[b]thien-2-yl)-OMe ((Z)-
2a). The procedure described above was applied using
compound (Z)-1 (107 mg, 0.300 mmol) and the 2-benzo-
[b]thienylboronic acid (59.0 mg, 0.330 mmol) and heating
for 5 h. Column chromatography using a solvent gradient
from pure petroleum ether to 30% diethyl ether in petroleum
ether, gave compound (Z)-2a (75.0 mg, 61%) as an oil.
Recrystallization from diethyl ether/petroleum ether
afforded colourless crystals, mp 123–124 8C. 1H NMR
(CDCl3): 1.52 (9H, s, CH3 Boc), 3.49 (3H, s, OCH3), 6.55
(1H, br s, NH), 7.08 (1H, s, ArH), 7.28–7.41 (7H, m, ArH),
7.67–7.76 (1H, m, ArH), 7.80–7.83 (1H, m, ArH) ppm. 13C
NMR (CDCl3): 28.15 (C(CH3)3), 51.96 (OCH3), 81.59
(OC(CH3)3), 122.01 (CH), 123.57 (C), 124.00 (CH), 124.59
(CH), 125.31 (CH), 125.87 (C), 127.80 (CH), 128.07 (CH),
128.24 (CH), 129.38 (CH), 138.79 (C), 138.85 (C), 140.77
(C), 152.84 (C]O), 165.89 (C]O) ppm. Anal. Calcd for
C23H23NO4S (409.50): C, 67.46; H, 5.66; N, 3.42; S, 7.83.
Found: C, 67.58; H, 5.74; N, 3.48; S, 7.70. The procedure
described above using 0.5 mmol of (Z)-1 and using PdCl2
(PPh3)2 gave compound (Z)-2a (150 mg, 74%).
4.3.5. Boc-(E)-DPhe(b-2,3-dimethylbenzo[b]thien-7-yl)-
OMe ((E)-2c). The procedure described above was applied
using compound (E)-1 (178 mg, 0.500 mmol) and the 2,3-
dimethyl-7-benzo[b]thienylboronic
acid
(113 mg,
0.550 mmol) and heating for 4 h 30 min. Column chroma-
tography using a solvent gradient from pure petroleum ether
to 30% diethyl ether in petroleum ether, gave compound
(E)-2c (209 mg, 96%) as an oil. Recrystallization from
petroleum ether afforded colourless crystals, mp 152–
1
154 8C. H NMR (CDCl3): 1.47 (9H, s, CH3 Boc), 2.26
(3H, s, ArCH3), 2.36 (3H, s, ArCH3), 3.41 (3H, s, OCH3),
6.24 (1H, br s, NH), 7.15 (1H, br d, JZ6.9 Hz, ArH), 7.31–
7.36 (6H, m, ArH), 7.53 (1H, dd, JZ8.1, 0.9 Hz, ArH) ppm.
13C NMR (CDCl3): 11.41 (CH3), 13.63 (CH3), 28.13
(C(CH3)3), 51.99 (OCH3), 81.27 (OC(CH3)3), 120.89
(CH), 123.71 (CH), 124.47 (CH), 126.70 (C), 127.06 (C),
128.06 (C), 128.45 (CH), 128.76 (CH), 129.67 (CH), 133.50
(C), 134.48 (C), 136.60 (C), 138.07 (C), 141.48 (C), 152.70
(C]O), 166.03 (C]O) ppm. Anal. Calcd for C25H27NO4S

A. S. Abreu et al. / Tetrahedron 60 (2004) 11821–11828
11827
(437.55): C, 68.62; H, 6.22; N, 3.20; S, 7.33. Found: C,
68.64; H, 6.44; N, 3.29; S, 7.02.
4 was isolated as a white solid (74 mg, 80%), mp 168–
169 8C (from diethyl ether/n-hexane). H NMR (CDCl3):
1
1.10 (3H, d, JZ7.2 Hz, bCH3 Ala), 1.44 (9H, s, CH3 Boc),
3.43 (3H, s, OMe), 4.54–4.63 (1H, m, aCH Ala), 6.06 (1H,
s, NH), 6.42 (1H, d, JZ7.5 Hz, NH), 7.27–7.41 (8H, m,
ArH), 7.73–7.78 (2H, m, ArH) ppm. 13C NMR (CDCl3):
17.82 (CH3), 28.13 (C(CH3)3), 48.33 (CH), 52.17 (OMe),
81.52 (OC(CH3)3), 122.07 (CH), 123.57 (C), 123.67 (CH),
124.45 (CH), 124.56 (CH), 125.18 (CH), 128.73 (CH),
128.96 (CH), 129.54 (CH), 131.11 (C), 137.50 (C), 139.44
(C), 140.82 (C), 141.44 (C), 152.16 (C]O), 164.29 (C]O),
172.60 (C]O) ppm. Anal. Calcd for C26H28N2O5S
(480.58): C, 64.98; H, 5.87; N, 5.83; S, 6.67. Found: C,
64.94; H, 5.95; N, 5.82; S, 6.66.
4.3.6. Boc-(Z)-DPhe(b-2,3-dimethylbenzo[b]thien-7-yl)-
OMe ((Z)-2c). The procedure described above was applied
using compound (Z)-1 (178 mg, 0.500 mmol) and the 2,3-
dimethyl-7-benzo[b]thienylboronic
acid
(113 mg,
0.550 mmol) and heating for 4 h 30 min. Column chroma-
tography using a solvent gradient from pure petroleum ether
to 30% diethyl ether in petroleum ether, gave compound
(E)-2c (162 mg, 74%) as an oil. Recrystallization from
petroleum ether afforded colourless crystals, mp 140–
1
142 8C. H NMR (CDCl3): 1.42 (9H, s, CH3 Boc), 2.31
(3H, s, ArCH3), 2.42 (3H, s, ArCH3), 3.60 (3H, s, OCH3),
5.89 (1H, br s, NH), 7.08–7.21 (3H, m, ArH), 7.23–7.30
(3H, m, ArH), 7.38 (1H, t, JZ8.1 Hz, ArH), 7.59 (1H, d, JZ
8.1 Hz, ArH) ppm. 13C NMR (CDCl3): 11.46 (CH3), 13.67
(CH3), 28.08 (C(CH3)3), 52.19 (OCH3), 81.14 (OC(CH3)3),
121.26 (CH), 124.38 (CH), 125.33 (CH), 126.99 (C), 127.93
(CH), 128.08 (CH), 128.78 (CH), 129.69 (C), 132.00 (C),
134.87 (C), 137.87 (C), 138.59 (C), 141.78 (C), 152.67
(C]O), 166.30 (C]O) ppm. Anal. Calcd for C25H27NO4S
(437.55): C, 68.62; H, 6.22; N, 3.20; S, 7.33. Found: C,
68.54; H, 6.35; N, 3.24; S, 7.05. The procedure described
above using PdCl2 (PPh3)2 gave compound (Z)-2c (160 mg,
73%).
4.5. In vitro antimicrobial activity
Suspensions of the microorganism were prepared to contain
approximately 108 cfu/mL and the plates were inoculated. A
stock solution of the synthesized compound (1000 mg/mL)
in DMSO was prepared and graded dilutions of the tested
compounds were incorporated in a cavity (depth 3 mm,
diameter 4 mm) made in the center of the Petri dish (nutrient
agar for antibacterial activity and sabouraud dextrose agar
medium for antifungal activity). The plates were incubated
at 37 8C (for bacteria) and at 30 8C (for fungi) for 24 h in
duplicate. Positive control using only inoculation and
negative control using only DMSO in the cavity were
carried out.
4.4. Synthesis of the model dipeptide Boc-(E)-DPhe-(b-
benzo[b]thien-2-yl)-Ala-OMe
4.4.1. Synthesis of Boc-(E)-DPhe-(b-benzo[b]thien-2-yl)-
OH ((E)-3). To a solution of Boc-E-DPhe(b-benzo[b]thien-
2-yl)-OMe (0.34 mmol, 137 mg) in dioxane (3 mL), 1 equiv
of NaOH 1 M was added. The solution was left stirring for
18 h at rt (the reaction was followed by TLC until no
starting material was detected). The reaction mixture was
acidified to pH 2–3 with KHSO4 1 M and the solid formed
filtered. Crystallization from ethyl acetate/n-hexane
afforded compound (E)-3 (117 mg, 87%) as a light yellow
solid, mp 189–191 8C (from ethyl acetate/n-hexane). 1H
NMR (CDCl3): 1.43 (9H, s, CH3 Boc), 6.18 (1H, s, NH),
7.29–7.40 (8H, m, ArH), 7.70–7.73 (2H, m, ArH) ppm. 13C
NMR (CDCl3): 28.08 (C(CH3)3), 82.01 (OC(CH3)3), 114.48
(C), 122.09 (CH), 123.87 (CH), 124.38 (CH), 124.72 (CH),
125.23 (CH), 126.32 (C), 128.95 (CH), 129.02 (CH), 129.58
(CH), 137.46 (C), 139.36 (C), 141.05 (C), 141.34 (C),
151.32 (C]O), 152.56 (C]O) ppm.
Acknowledgements
We thank the Foundation for Science and Technology
(Portugal) for financial support through IBQF-Univ. Minho,
POCTI/1999/QUI/32689
SFRH/BD/4709/2001, Ph.D. financial support of A. S.
Abreu.
project
and
through
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