β-Nitrostyrene derivatives as potential antibacterial agents: A structure-property-activity relationship study

Article abstract of DOI:10.1016/j.bmc.2006.02.006

A multidisciplinary project was developed, combining the synthesis of a series of β-nitrostyrene derivatives and the determination of their physicochemical parameters (redox potentials, partition coefficients), to the evaluation of the corresponding antibacterial activity. A complete conformational analysis was also performed, in order to get relevant structural information. Subsequently, a structure-property-activity (SPAR) approach was applied, through linear regression analysis, aiming at obtaining a putative correlation between the physicochemical parameters of the compounds investigated and their antibacterial activity (both against standard strains and clinical isolates). The β-nitrostyrene compounds displayed a lower activity towards all the tested bacteria relative to the β-methyl-β-nitrostyrene analogues. This was observed particularly for the 3-hydroxy-4-methoxy-β-methyl-β-nitrostyrene (IVb) against the Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium). The SPAR results revealed the existence of a clear correlation between the redox potentials and the antibacterial activity of the series of β-nitrostyrene derivatives under study.

Full text of DOI:10.1016/j.bmc.2006.02.006

Bioorganic & Medicinal Chemistry 14 (2006) 4078–4088
b-Nitrostyrene derivatives as potential antibacterial agents:
A structure–property–activity relationship study
Nuno Milhazes,a,b,c Rita Calheiros,d M. Paula M. Marques,d,e Jorge Garrido,f
g,h
g,i
g,i
g,i
´
´
M. Natalia D. S. Cordeiro, Catia Rodrigues, Sandra Quinteira, Carla Novais,
g,i
b,d,
*
´
Luısa Peixe and Fernanda Borges
aCEQOFFUP
b
ˆ
´
Departamento de Quı´mica Organica, Faculdade de Farmacia, Universidade do Porto, 4050-047 Porto, Portugal
c
ˆ
´
Instituto Superior Ciencias da Saude, Norte, 4585-116 Gandra, PRD, Portugal
dUnidade I&D ‘Quı´mica-Fı´sica Molecular’
e
ˆ
Departamento de Bioquı´mica, Faculdade de Ciencias e Tecnologia, Universidade de Coimbra, 3001-401 Coimbra, Portugal
fDepartamento de Engenharia Quı´mica, Instituto Superior de Engenharia do Porto, Rua S. Tome´, 4200-485 Porto, Portugal
gREQUIMTE
h
ˆ
Departamento de Quı´mica, Faculdade de Ciencias, Universidade do Porto, Portugal
iDepartamento de Microbiologia, Faculdade de Farma´cia, Universidade do Porto, 4050-047 Porto, Portugal
Received 22 November 2005; revised 26 January 2006; accepted 3 February 2006
Available online 23 February 2006
Abstract—A multidisciplinary project was developed, combining the synthesis of a series of b-nitrostyrene derivatives and the deter-
mination of their physicochemical parameters (redox potentials, partition coefficients), to the evaluation of the corresponding anti-
bacterial activity. A complete conformational analysis was also performed, in order to get relevant structural information.
Subsequently, a structure–property–activity (SPAR) approach was applied, through linear regression analysis, aiming at obtaining
a putative correlation between the physicochemical parameters of the compounds investigated and their antibacterial activity (both
against standard strains and clinical isolates). The b-nitrostyrene compounds displayed a lower activity towards all the tested bac-
teria relative to the b-methyl-b-nitrostyrene analogues. This was observed particularly for the 3-hydroxy-4-methoxy-b-methyl-b-
nitrostyrene (IVb) against the Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium).
The SPAR results revealed the existence of a clear correlation between the redox potentials and the antibacterial activity of the series
of b-nitrostyrene derivatives under study.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
tors2 as well as cytotoxic for human cancer cell lines.3
Moreover, it was shown that they are able to downregu-
late the production of some interleukins, thus affecting
the immune response in humans.3 The nitrovinyl side
chain attached to the aromatic ring was recognized to
be an essential chemical feature in this type of com-
pounds and a critical conformational pattern for their
biological activity.1,4
Over the last few years, compounds comprising a
b-nitrostyrene moiety have been recognized to have a
series of relevant biological activities. In fact, some
b-nitrostyrene derivatives have recently been tested as
pro-apoptotic anticancer agents and the b-nitrostyrene
moiety was identified as the pharmacophore for this
activity.1 These compounds have also been described
as highly potent and selective human telomerase inhibi-
Although the antifungic and antibacterial properties of
this type of compounds have been studied since the
1940s, the work in this area during the last decade is
rather scarce. In general, the nitrostyrene derivatives
were found to be more effective against Gram-positive
than Gram-negative bacteria.5–7
Keywords: b-Nitrostyrene derivatives; Antibacterial activity; Redox
potentials; Structure–property–activity relationship.
*
Corresponding author. Tel.: +351 22 2078900; fax: +351
222003977; e-mail: fborges@ff.up.pt
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.006

N. Milhazes et al. / Bioorg. Med. Chem. 14 (2006) 4078–4088
4079
Drug discovery studies have been significantly intensi-
fied in recent years, aiming at the search for more effec-
tive antibacterial agents displaying activity against
Gram-positive and Gram-negative bacteria resistant to
the antimicrobials currently used.8,9 The search for nov-
el chemotherapeutic agents, as well as the attempt to
modify and improve the currently available ones, has
been a priority mainly as a consequence of the rapid
spread of multidrug-resistant bacterial pathogens. The
development of new antibacterial drugs is presently
based on structure–activity relationship (SAR), struc-
ture–property–activity relationship (SPAR) and quanti-
tative structure–activity relationship (QSAR) studies.9,10
yl-b-nitrostyrene (Ib), 3,4-dihydroxy-b-methyl-b-nitro-
styrene (IIb), 3,4-dimethoxy-b-methyl-b-nitrostyrene
(IIIb),
(IVb),
3-hydroxy-4-methoxy-b-methyl-b-nitrostyrene
4-hydroxy-3-methoxy-b-methyl-b-nitrostyrene
(Vb) and 3,4-methylenedioxy-b-methyl-b-nitrostyrene
(VIb) (Table 1).
2. Results and discussion
2.1. Chemistry
b-Nitrostyrene and b-methyl-b-nitrostyrene derivatives
were prepared by a nitroaldol condensation reaction
(Henry reaction).11 The compounds were synthesized
by the reaction of the corresponding benzaldehydes—
with the same aromatic substitution pattern of the de-
sired product—and nitromethane or nitroethane,
respectively, for b-nitrostyrenes and b-methyl-b-nitro-
styrenes, using ammonium acetate as the catalyst
(Scheme 1). These experimental conditions led to mod-
erate/ high yields. The final products were identified by
NMR (1H NMR and 13C NMR), FT-IR and MS-EI
spectroscopic techniques. The purity of the target com-
pounds was determined by two different HPLC systems
(see Section 4). The structures of the synthesized com-
pounds are depicted in Table 1.
The aim of the present work is to be aware of the signif-
icance of b-nitrostyrene as a putative pharmacophore
for antibacterial activity. The antibacterial activity of a
series of compounds was evaluated against several type
strains and clinical isolates of different bacterial species
pathogenic to humans. Simultaneously, relevant physi-
cochemical parameters, such as redox potentials (Ep)
and partition coefficients (logP), were determined and/
or calculated. A complete conformational analysis of
the b-nitrostyrene derivatives under study was also per-
formed through theoretical methods—ab initio molecu-
lar orbital calculations—gathering information of the
utmost relevance for correlating structural features with
the physicochemical properties and antimicrobial activ-
ity of this class of compounds.
Several b-nitrostyrene derivatives were synthesized in
order to study the influence of different aromatic substi-
tution patterns, which confer diverse electronic environ-
ments, in the antibacterial activity. In this kind of
systems, the nitro group is relatively distant from the
aromatic ring and conjugated with an ethylenic double
bond, thus allowing long-distance transmission of elec-
tronic effects.12 One methyl group was also added to
the b-carbon of the double bond, to verify the influence
of conformational and steric parameters on the redox
and antibacterial profiles of this chemical family.
The compounds studied are chemically related to the
parent compound with slight modifications in their
aromatic substitution pattern: b-nitrostyrene (Ia), 3,4-
dihydroxy-b-nitrostyrene (IIa), 3,4-dimethoxy-b-nitro-
styrene (IIIa), 3-hydroxy-4-methoxy-b-nitrostyrene
(IVa), 4-hydroxy-3-methoxy-b-nitrostyrene (Va) and
3,4-methylenedioxy-b-nitrostyrene (VIa) (Table 1). In
addition, another series of compounds was synthesized,
displaying a methyl group in the b-carbon of the vinyl
side chain, the aromatic part being kept unchanged.
The following compounds were thus obtained: b-meth-
2.2. Conformational analysis
In order to establish the effect of the structural prefer-
ences of b-nitrostyrene derivatives on their biological
activity, a complete conformational analysis was carried
out, through theoretical (ab initio) methods. The effects
of three structural parameters on the overall stability of
these molecules were investigated: (i) relative orientation
of the hydroxy and/or methoxy group(s) in the aromatic
ring; (ii) orientation of the benzene and the nitro group
relative to the double bond, Ca@Cb, defining either a Z
or an E configuration; (iii) position of the NO2 and
Table 1. b-Nitrostyrene and b-methyl-b-nitrostyrene derivatives syn-
thesized in the present work
α
1
R1
R2
3
NO2
β
R3
4
Compound
R1
R2
R3
Ia
H
H
H
Ib
H
OH
H
OH
CH3
H
IIa
IIb
IIIa
IIIb
IVa
IVb
Va
NO2
R1
NH4OAc, MeNO2
OH
OH
CH3
H
OCH3
OCH3
OH
OCH3
OCH3
OCH3
OCH3
OH
reflux
R2
CH3
H
CHO
R1
R2
OH
OCH3
OCH3
CH3
H
NO2
NH4OAc, EtNO2
reflux
R1
R2
Vb
OH
CH3
H
R3
VIa
VIb
–OCH2O–
–OCH2O–
CH3
Scheme 1.

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N. Milhazes et al. / Bioorg. Med. Chem. 14 (2006) 4078–4088
methyl group (in b-methyl-b-nitrostyrenes) relative to
the ring. For all the geometries obtained calculation of
the harmonic vibrational frequencies was also per-
formed, allowing to determine which ones were real
energy minima in the potential energy surface.
angle around C1–Ca changing from 0ꢁ to 28ꢁ. Concern-
ing the relative orientation of the ring substituent
groups, it was found that it is identical whenever two
OHs or one OH and one OMe are present, in order to
allow the formation of intramolecular hydrogen bonds.
When there are two OMe substituents, an opposite ori-
entation is favoured (Fig. 1), in view of the minimization
of OCH3ꢀ ꢀ ꢀH3CO steric repulsions.
Since the more substituted b-nitrostyrene derivatives
display a higher degree of freedom, several low energy
conformers were calculated for them. Two stable geom-
etries were obtained for compounds Ia and Ib (only one
of them being populated at room temperature), 4 con-
formers were found for VIa and VIb, 8 were determined
for Va, 10 for IIb, 11 for IVb and 12 for IIa, IIIa, IIIb,
IVa and Vb (two of these having significant populations
at room temperature). Figure 1 comprises the most sta-
ble structures calculated for each of the compounds
presently studied.
2.3. Antibacterial activity
The antibacterial properties of the b-nitrostyrene deriv-
atives investigated were measured against selected type
strains of Gram-positive (Staphylococcus aureus ATCC
29213 and Enterococcus faecalis ATCC 29212) and
Gram-negative (Escherichia coli ATCC 25922 and Pseu-
domonas aeruginosa ATCC 27853) bacteria. Salmonella
typhimurium FFUP1 and Acinetobacter baumannii
FFUP10 are clinical isolates susceptible to all antibiot-
ics, according to CLSI (formerly NCCLS)
recommendations.14
The conformational behaviour of these phenolic com-
pounds is mainly determined by electronic effects, as
well as by the possibility of occurrence of (C)Hꢀ ꢀ ꢀO
intramolecular hydrogen bonds, which are medium
strength, directional interactions (with a preference for
linearity) known to be relevant for the overall stability
of this kind of systems.13 The most stable calculated
structures were found to display a planar or quasi-pla-
nar geometry and an E conformation, which corre-
sponds to a more effective p-electron delocalization, as
well as to a minimization of repulsive effects, relative
to the Z conformers. In fact, even for those compounds
where two conformers exist at room temperature, they
differ only in the value of the C1–Ca torsion angle: either
0ꢁ or 180ꢁ. By comparing the b-nitrostyrenes with the b-
methyl-b-nitrostyrenes, it was verified that the almost
perfect planarity detected for the former is lost upon
methyl substitution in the double bond Cb, the aromatic
ring being tilted relative to the carbon side chain, the
Table 2 comprises the in vitro results of antibacterial
activity for the b-nitrostyrenes under study, expressed
as minimum inhibitory concentrations (MICs). The low-
est antibacterial activity was observed for the b-nitrosty-
rene series, with MICs ranging from 128 to P512 mg/L.
In general, from the activity measured for the b-methyl-
b-nitrostyrenes it is possible to conclude that the inclu-
sion of a methyl group in the molecule’s side chain leads
to an increase of the inhibitory effect—MICs are be-
tween 16 and 256 mg/L. This activity enhancement due
to the methyl substitution was found to be more pro-
nounced for Gram-positive bacteria (S. aureus and
Enterococcus spp.)—MICs ranging from 16 to 32 mg/
L. Among the b-methyl-b-nitrostyrenes, compound
IVb displayed the highest activity against Gram-positive
Ia
Ib
IIa
IIb
IIIa
IIIb
IVa
IVb
Va
Vb
VIa
VIb
Figure 1. Schematic representation of the most stable geometries calculated (B3LYP/6-31G ) for the b-nitrostyrene derivatives under study.
**

N. Milhazes et al. / Bioorg. Med. Chem. 14 (2006) 4078–4088
4081
Table 2. In vitro antibacterial activity of the b-nitrostyrene derivatives against several bacterial strains
Bacterial strain
Minimum inhibitory concentration, MIC (mg/L)
Ia
Ib
IIa
IIb
IIIa
IIIb
IVa
IVb
Va
Vb
VIa
VIb
Escherichia coli ATCC 25922
256
256
256
256
256
128
128
128
256
128
64
256
256
256
256
256
256
64
64
P512
P512
512
256
256
256
256
32
512
512
256
256
256
256
128
128
256
128
16
512
512
512
256
256
256
256
256
256
256
32
128
128
128
128
128
128
256
256
256
256
32
Salmonella typhimurium FFUP1
Pseudomonas aeruginosa ATCC 27853
Acinetobacter baumannii FFUP10
Enterococcus faecalis ATCC 29212
Staphylococcus aureus ATCC 29213
256
64
512
512
64
64
64
512
32
16
32
32
bacteria. Interestingly, compound IIb showed the high-
est activity against all the tested Gram-negative bacteria
(MIC of 64 mg/L), except for P. aeruginosa.
2.4. Electrochemical and partition coefficient
measurements
As the antibacterial activity and the mechanism of action
of the nitro compounds, commonly used in therapy, are
largely a function of their redox potential, it was found
important to study the physicochemical properties of
the synthesized b-nitrostyrene derivatives (Table 1).15,16
The antibacterial activity of the compounds under study
was also evaluated against clinical isolates with different
resistance profiles to several antimicrobial agents,
including sulfonamide-resistant Salmonella and ampicil-
lin, aminoglycoside, tetracycline, macrolide and glyco-
peptide-resistant Enterococcus (Table 3). The results
obtained corroborate previously reported patterns ob-
served for these type strains. It may then be concluded
that the activity of b-nitrostyrene derivatives against
antibiotic-resistant Salmonella and Enterococcus is not
affected by their resistance mechanisms, which are capa-
ble of inactivating several antimicrobial agents.
Thus, the standard redox potentials were determined at
physiological pH 7.3, using differential pulse polarogra-
phy, as well as cyclic and square wave voltammetry,
with a glassy carbon working electrode.
A well-defined cathodic peak can be observed at physi-
ological pH for all the compounds studied using differ-
ential pulse polarography (Fig. 2). This wave is due to
a four-electron, four-proton reduction of the nitro
group yielding the corresponding hydroxylamine deriva-
tive, according to the following equation:12,17
Based on the presently obtained results, a series of other
compounds based on the same structural templates were
designed, aiming at the screening of their antibacterial
activity. These derivatives display different aromatic ring
substituents (changing their number and polarity de-
gree), thus promoting changes in their electronic charac-
teristics and lipophilicity. Neither of these chemical
modifications led to an increase of the activity (data
not shown). In order to check the relevance of the nitro
group in the antimicrobial activity of this kind of sys-
tems, the amine analogues of some of the b-nitrostyrene
and b-methyl-b-nitrostyrenes presently studied were
synthesized and tested. In addition, various saturated
analogues were also obtained, in order to check the rel-
evance of the ethylenic double bond on the antibacterial
properties. In both cases, no significant activity was ob-
served (data not shown). These results reinforce the
mechanistic hypothesis proposed in the present work.
R1R2–Ph–CH@CR3–NO2 + 4eꢁ + 4Hþ
! R1R2–Ph–CH@CR3–NHOH þ H2O
Cyclic voltammograms were recorded at different sweep
rates. All compounds were found to produce a well-de-
fined and irreversible peak at physiological pH (Fig. 3).
This peak corresponds to the above-described wave ob-
served by differential pulse polarography. From the
dependence of the peak current on the sweep rate, it
was possible to conclude that the process follows a
mixed diffusion–adsorption control, since the experi-
mentally obtained slopes (dlogIp/dlogm) display values
between 0.5 and 1.18
Table 3. In vitro antibacterial activity of the b-nitrostyrene derivatives against several clinical isolates
Clinical isolates
Minimum inhibitory concentration, MIC (mg/L)
Ia
Ib
IIa
IIb
IIIa
IIIb
IVa
IVb
Va
Vb
VIa
VIb
Salmonella typhimurium FFUP40
Salmonella enteritidis 09 FF-D
Enterococcus faecalis H 318
Enterococcus faecalis H 318
Enterococcus faecalis H 181A
Enterococcus faecalis 536540
Enterococcus faecium Med150
Enterococcus faecium Med100 F3
Enterococcus faecium KE77F2
256
256
256
256
256
256
256
128
128
128
128
64
256
256
256
256
256
256
256
256
256
64
64
64
64
64
64
64
64
64
P512
P512
512
256
256
32
512
512
256
256
256
256
256
256
256
128
128
16
512
512
256
256
256
256
256
256
256
256
256
32
128
128
128
128
128
128
128
128
128
256
256
32
64
64
512
512
32
32
16
16
32
32
32
32
64
64
512
512
32
32
16
16
32
32
32
32
64
64
512
512
32
32
16
32
32
32
32
32

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N. Milhazes et al. / Bioorg. Med. Chem. 14 (2006) 4078–4088
sion angle,12 also predicted by the ab initio calculations.
Although the increase in the electron-donor properties
of the meta substituent influences the reduction potential
of the compounds, this effect is less pronounced than the
one resulting from a para-substitution. Moreover, it is
more significant for b-nitrostyrenes than for the b-meth-
yl-b-nitrostyrenes, as expected.
200 nA
In view of better understanding of the overall properties
of the compounds, the lipophilicity, expressed as the oct-
anol–water partition coefficient and herein called logP,
was calculated according to Parham et al.19 LogP be-
tween 1.5 and 2.5 were obtained (Table 4).
0.0
0.2
0.4
0.6
0.8
1.0
2.5. Structure–property–activity relationship (SPAR)
- E / V vs. Ag/AgCl
In this work, two types of bioisosteric replacements (mod-
ifications in the substitution pattern of both the unsatu-
rated side chain and the aromatic ring) were performed
in the parent compound—b-nitrostyrene—and the phys-
icochemical properties, as well as antibacterial activity of
the series, were examined in order to perform structure–
property–activity relationship studies. The chemical
structure of each compound was analyzed through three
types of parameters: steric interference, electronic distri-
bution and hydrophobicity, since these appear to signifi-
cantly influence other biological activities.20,21
Figure 2. Differential pulse voltammogram for reduction of VIb, in pH
7.3 supporting electrolyte, in the absence (ꢀ ꢀ ꢀꢀ ꢀ ꢀ) and presence (—) of
100 lM VIb. Scan rate 5 mV sꢁ1
.
The results obtained are in agreement with the data
reported for other b-nitrostyrene derivatives.12,17 The
shifts detected in the standard redox potential values
(Table 4) for the different b-nitrostyrene derivatives are
probably due to two distinct substituent effects: (i) sub-
stitution of Hb by a methyl group; (ii) different meta and
para substitutions on the aromatic ring.
In these SPAR studies, the antimicrobial activities ob-
tained for the nitrostyrene compounds against the S.
aureus ATCC 29213 and E. faecalis ATCC 29212 strains
were represented as log 1/MIC. These values were then
plotted against Ep and logP (Figs. 4 and 5, respectively).
These results strongly suggest a correlation between
antibacterial activity and redox potential. In turn, no
correlation was found between antibacterial activity
and lipophilicity. A statistical treatment of the redox po-
tential and partition coefficient results was performed, in
view of achieving a better understanding of these find-
ings. An analysis of variance test was carried out, in or-
der to compare the different Ep and logP values for the
b-nitrostyrenes and the b-methyl-b-nitrostyrenes. Since
the experimental values of F (between 1.1 and 2.6) were
always lower than the theoretical value (F5,5 = 7.1;
p = 0.05), it was concluded that there were no significant
differences between the variances obtained for those two
series of compounds, for neither of the two parameters
studied. A comparison of the experimental mean values
for the two series of nitrostyrene derivatives was also
performed, using the t test. Comparison of the redox
potentials of b-nitrostyrenes and b-methyl-b-nitrosty-
renes yielded t test values (t = 4.2) higher than the theo-
retical one (t = 2.6; p = 0.05). There was no statistical
evidence linking the presence of a b-methyl group to a
significant effect on the partition coefficient value.
Therefore, it may be concluded that the redox potential
might be an important parameter ruling the antibacteri-
al activity of b-nitrostyrene derivatives. In fact, it was
verified that the more negative the Ep the stronger the
antibacterial promoting effect.
The redox potential values presented in Table 4 evidence
that in all cases the b-methyl-b-nitrostyrene derivatives
are reduced at more negative potentials than their corre-
sponding b-nitrostyrenes. Hence, the reduction potential
seems to be highly sensitive to a methyl substitution in
the b-position. A shift of several tens of millivolts to-
wards more negative potentials is observed when going
from b-nitrostyrene to b-methyl-b-nitrostyrene deriva-
tives, which can be attributed to a lack of planarity
due to b-methyl substitution.12 In fact, this distortion
of the coplanar arrangement in b-nitrostyrenes leads to
a decrease of the resonance interaction between the
electroactive nitro group and the aromatic ring, thus
causing the observed shift towards negative potentials.12
This conformational change upon b-methyl substitution
is corroborated, in the present study, by the theoretical
results that yielded a deviation of about 28ꢁ between
the planar b-nitrostyrenes and the corresponding
b-methyl-b-nitrostyrenes. Also, when such kind of
distortion takes place an accumulation of charge on
the Ca atom was verified—for example, from +0.050
to ꢁ0.008 for compounds Ia and Ib, respectively—which
is probably due to the less effective electronic delocaliza-
tion to the nitro group in the latter.
The substitution on the aromatic ring in meta and espe-
cially in para position also has a marked effect on the re-
dox potentials. In fact, an increase in the electron-donor
properties of the para substituent significantly increases
the magnitude of the reduction potential, that is, reduc-
tion is unlikely to occur (Table 4). As expected, this ef-
fect is more pronounced in the b-nitrostyrenes than in
the b-methyl-b-nitrostyrenes, due to the decrease in the
conjugation coupled with the increase in the C1–Ca tor-
In the light of the results presently obtained, it may be
suggested that the mode of action of this kind of b-nitro-

N. Milhazes et al. / Bioorg. Med. Chem. 14 (2006) 4078–4088
4083
a
b
500 nA
500 nA
c
d
500 nA
500 nA
e
f
500 nA
500 nA
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
- E / V vs. Ag/AgCl
- E / V vs. Ag/AgCl
Figure 3. Cyclic voltammograms for several b-nitrostyrene (ꢀ ꢀ ꢀꢀ ꢀ ꢀ) and b-methyl-b-nitrostyrene derivatives (—), in 100 lM solution, at physiological
pH. (a) Ia, Ib; (b) IIa, IIb; (c) IIIa, IIIb; (d) IVa, IVb; (e) Va, Vb; (f) VIa, VIb. Scan rate 75 mV sꢁ1
.
styrene derivatives is closely related to their electrophi-
licity, thus being a consequence of their ability to accept
electrons, which enables the reduction of the nitro
group. The antimicrobial action of nitro compounds is
generally believed to result from a 4-electron reduction
of the nitro group, carried on by bacterial nitroreducta-
ses, to short-lived redox active intermediates, like
hydroxylamine adducts, known to be biologically
active.15,16,22,23
(<650 Da) through porine channels located on their out-
er membranes.24 As shown in Figure 1 and Table 4, the
conformational and chemical characteristics of com-
pound IIb are in complete accordance with these requi-
sites. Nevertheless, further work is needed in order to
prove this hypothesis.
3. Conclusions
A possible explanation for the higher antimicrobial
activity verified for compound IIb (one of the b-meth-
yl-b-nitrostyrene derivatives) against some of the
Gram-negative bacteria, including members of the
Enterobacteriaceae family, may be its rather high hydro-
philicity (calculated logP value of 1.51). In fact, these
bacterial strains are known to uptake hydrophilic drugs
The increasing resistance to available antibiotics against
clinically important bacteria represents a serious prob-
lem in public health which requires the development of
new therapeutic alternatives.
The results on the antibacterial activity of b-nitrostyrene
derivatives, namely b-methyl-b-nitrostyrenes, towards

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N. Milhazes et al. / Bioorg. Med. Chem. 14 (2006) 4078–4088
Table 4. Redox potential and partition coefficient values for the b-
nitrostyrene and b-methyl-b-nitrostyrene derivatives studied in this
work
Even though further research must be conducted in
order to study other physicochemical parameters, the
redox potential value seems to be a crucial parameter
for the displayed antimicrobial activity. It was verified
that the conformational behaviour of these molecules
is mainly determined by the stabilizing effect of p-elec-
tron delocalization, which is reflected in the most
stable geometries calculated for the b-nitrostyrene
derivatives, and is in accordance with the electrochem-
ical data.
a
Compound
Ep (V)
DEp (mV)
LogP
Ia
Ib
ꢁ0.455
ꢁ0.547
92
2.28
2.40
IIa
IIb
ꢁ0.497
77
87
87
77
92
1.46
1.51
ꢁ0.574
IIIa
IIIb
ꢁ0.471
ꢁ0.558
2.34
2.50
The results gathered along this work provide a ra-
tional approach that will hopefully allow the design
of selective and effective antibacterial agents. The
b-nitrostyrene family seems to represent a novel class
of antibacterial agents possessing a surprisingly small
pharmacophore. Changes in the ethylenic side chain
of the b-nitrostyrenes must be performed, namely in
the length and volume of its substituents, in order
to check its influence in the activity versus electro-
chemical properties.
IVa
IVb
ꢁ0.477
1.78
2.11
ꢁ0.564
Va
Vb
ꢁ0.492
ꢁ0.569
1.79
2.15
VIa
VIb
ꢁ0.473
1.85
2.07
ꢁ0.565
a Ep difference between the b-nitrostyrenes and the corresponding
b-methyl-b-nitrostyrenes.
standard strains suggest that these could constitute a
group of promising agents for the most clinically rele-
vant Gram-positive bacteria, S. aureus and Enterococcus
spp. This propensity is in accordance with previously
obtained results with similar compounds.5–7 The high
selectivity exhibited by these systems is unequivocally
related to their structural features.
4. Experimental
4.1. Chemicals
b-Nitrostyrene (Ia), ammonium acetate, nitroethane
and nitromethane were purchased from Sigma–Aldrich
Log (1/MIC) = f (Log P)
Log P
A
B
Log (1/MIC) = f (E p)
E p
-0,7
-1,0
-0,65
-0,6
-0,55 -0,5
-0,45 -0,4 -0,35
-0,3
1
1,2
1,4 1,6
1,8
2
2,2
2,4 2,6
2,8
3
-1,0
-1,5
-2,0
-2,5
-3,0
-3,5
-4,0
-1,5
-2,0
-2,5
-3,0
-3,5
-4,0
Figure 4. Regression plots of log(1/MIC) towards Staphylococcus aureus ATCC 29213, for the b-nitrostyrene derivatives tested: (A) standard redox
potential values at physiological pH; (B) logP values.
Log (1/MIC) = f (E p)
A
Log (1/MIC) = f (Log P)
Log P
B
E p
1
1,2 1,4
1,6
1,8
2
2,2 2,4 2,6
2,8
3
-0,7
-1,0
-0,65
-0,6
-0,55
-0,5 -0,45
-0,4
-0,35 -0,3
-1,0
-1,5
-2,0
-2,5
-3,0
-3,5
-4,0
-1,5
-2,0
-2,5
-3,0
-3,5
-4,0
Figure 5. Regression plots of log(1/MIC) towards Enterococcus faecalis ATCC 29212, for the b-nitrostyrene derivatives tested: (A) standard redox
potential values at physiological pH; (B) logP values.

N. Milhazes et al. / Bioorg. Med. Chem. 14 (2006) 4078–4088
4085
´
Quımica SA. (Sintra, Portugal). All other reagents and
solvents (pro analysis grade) were acquired from Merck
(Lisbon, Portugal). Deionized water (conductivity
<0.1 lS cmꢁ1) was used in all experiments. Trypticase
soy broth was obtained from Oxoid (Basingstone, UK)
4.3. General synthetic procedure and structural
elucidation
The method of Parker et al.,25 slightly modified, was
used for the synthesis of the compounds. The benzalde-
hyde with the same aromatic substitution pattern of the
desired final product (25.0 mmol) and ammonium
acetate (6.5 mmol) were dissolved in nitromethane
(50 mL)—for the b-nitrostyrene derivatives (IIIa, IVa,
Va and VIa)—or in nitroethane (50 mL)—for the
b-methyl-b-nitrostyrene derivatives (compounds IIIb,
IVb and Vb), and refluxed for 6 h. The reactions were
followed by thin-layer chromatography (TLC). After
cooling the reaction mixtures to room temperature, the
solvents were partially evaporated, diluted with diethyl
ether and washed twice with water. The organic layer
was dried over anhydrous magnesium sulfate, filtered
and concentrated. The remaining residues were recrys-
tallized from diethyl ether/petroleum ether (40–60 ꢁC)
or from methanol/water.
´
and Mueller–Hinton II from Biomerieux (Marcy L’Eto-
ile, France). All reagents were used without further
purification.
4.2. Analysis
1H and 13C NMR data were acquired, at room tem-
perature, on a Bruker AMX 300 spectrometer operat-
¨
ing at 300.13 and 75.47 MHz, respectively.
Dimethylsulfoxide-d6 was used as a solvent; chemical
shifts are expressed in d (ppm) values, relative to tet-
ramethylsilane (TMS) (as internal reference); coupling
constants (J) are given in Hz. Assignments were also
made from Distortionless Enhancement by Polariza-
tion Transfer (DEPT) experiments (underlined values).
Electron impact mass spectra (EI-MS) were obtained
on a VG AutoSpec instrument; data are reported as
m/z (% of relative intensity for the most important
fragments). Melting points were measured on a Ko¨fler
The synthesized compounds were identified by both
NMR and EI-MS. In the present study, the nitro com-
pounds were systematically characterized, although
some of them had been previously reported.26–30 The
syntheses of the remaining compounds used in this study
(Ib, IIa, IIb and VIb) are described elsewhere.31,32
microscope
uncorrected.
(Reichert
Thermovar)
and
are
The purity of the final products (higher than 98%)
was verified using two different high-performance li-
quid chromatography (HPLC) systems, one equipped
with UV and the other with an electrochemical detec-
tor. HPLC-UV chromatograms were obtained in a
Jasco instrument (pumps model 880-PU and solvent
mixing model 880-30, Tokyo, Japan), equipped with
a Nucleosil RP-18 column (250 mm · 4.6 mm, 5 lm,
4.3.1. 3,4-Dimethoxy-b-nitrostyrene (IIIa). Yield 77%;
1H NMR d: 3.82 (3H, s, 3-OCH3), 3.83 (3H, s, 4-
OCH3), 7.06 (1H, d, J = 8.4, H(5)), 7.43 (1H, d,
J = 8.4, H(6)), 7.49 (1H, s, H(2)), 8.07 (1H, d,
J = 13.4, H(a)), 8.22 (1H, d, J = 13.5, H(b)); 13C NMR
d: 55.8 (4-OCH3), 55.9 (3-OCH3), 111.2, 111.8 C(2)
and C(5), 123.0 C(1), 125.9 C(6), 136.0 C(a), 140.1
C(b), 149.2 C(4), 152.7 C(3); EI-MS m/z (%): 209
(M+Å, 100), 162 (53), 147 (19), 119 (15), 91 (11), 77
(11), 63 (7); mp 142–144 ꢁC.
Macherey-Nagel, Duren, Germany) and UV detection
¨
at 214 nm (Jasco model 875-UV). The mobile phase
was acetonitrile/water, at a flow rate of 1 mL/min.
A linear gradient of acetonitrile from 10% to 100%
during a period of 35 min was used. The chromato-
graphic data were processed in a Compaq computer,
fitted with CSW 1.7 software (DataApex, Czech
Republic).
4.3.2. 3,4-Dimethoxy-b-methyl-b-nitrostyrene (IIIb).
1
Yield 68%; H NMR d: 2.44 (3H, s, CH3), 3.80 (3H, s,
3-OCH3), 3.82 (3H, s, 4-OCH3), 7.08 (1H, d, J = 8.5,
H(5)), 7.22 (1H, s, H(2)), 7.23 (1H, d, J = 8.5, H(6)),
8.08 (1H, s, H(a)); 13C NMR d: 14.0 CH3, 55.6 (4-
OCH3), 55.7 (3-OCH3), 111.8, 113.9 C(2) and C(5),
124.3 C(6), 124.4 C(1), 133.8 C(a), 145.6 C(b), 148.7
C(4), 150.7 C(3); EI-MS m/z (%): 223 (M+Å, 100), 176
(78), 165 (22), 161 (37), 146 (47), 131 (46), 119 (36),
103 (37), 91 (47), 77 (38), 65 (34); mp 71–72 ꢁC.
A Waters 2690 Alliance system equipped with a
CONCORDE Electrochemical Detector (Waters Cor-
poration, Milford, USA). The electrochemical cell
was a VT-03 flow cell (Antec Leyden, Zoeterwoude,
Netherlands) with a confined wall-jet design, in a
three-electrode configuration: a 2 mm diameter glassy
carbon working electrode, an in situ Ag/AgCl refer-
ence electrode and a stainless steel auxiliary elec-
trode. The electrochemical detector was operated in
reductive amperometric mode with the working
potentials between ꢁ450 and ꢁ600 mV. The HPLC
separation was carried out in a reverse-phase LC-
18-S Supelcosil analytical column (150 mm · 4.6 mm,
5 lm, Supelco, Bellefonte, USA). A 50 mM phos-
phate buffer of pH 6.8 with 6% MeOH was used
as mobile phase, in isocratic mode at 1 mL/min flow
rate. Chromatograms were acquired in a Millenium
32 Chromatography Manager (Waters Corporation,
Milford, USA).
4.3.3. 3-Hydroxy-4-methoxy-b-nitrostyrene (IVa). Yield
92%; 1H NMR d: 3.84 (3H, s, OCH3), 7.02 (1H, d,
J = 8.4, H(5)), 7.24 (1H, d, J = 2.0, H(2)), 7.32 (1H,
dd, J = 8.4; 2.1, H(6)), 8.01 (2H, s, H(a), H(b)), 9.35
(1H, br s, OH); 13C NMR d: 55.8 (OCH3), 112.0,
115.6 C(2) and C(5), 122.9 C(1), 123.6 C(6), 135.6
C(a), 139.9 C(b), 146.8 C(3), 151.6 C(4); EI-MS m/z
(%): 195 (M+Å, 100), 148 (53), 133 (44), 105 (25), 89
(31), 77 (25), 63 (14); mp 158–161 ꢁC.
4.3.4.
3-Hydroxy-4-methoxy-b-methyl-b-nitrostyrene
1
(IVb). Yield 97%; H NMR d: 2.40 (3H, s, CH3), 3.83
(3H, s, OCH3), 7.04 (1H, d, J = 8.2, H(5)), 7.06–7.10

4086
N. Milhazes et al. / Bioorg. Med. Chem. 14 (2006) 4078–4088
(2H, m, H(2), H(6)), 7.98 (1H, s, H(a)), 9.39 (1H, br s,
OH); 13C NMR d: 14.0 CH3, 55.6 (OCH3), 112.1,
116.9 C(2) and C(5), 123.5 C(6), 124.5 C(1), 133.7
C(a), 145.2 C(b), 146.6 C(3), 149.8 C (4); EI-MS m/z
(%): 209 (M+Å, 100), 162 (56), 147 (53), 131 (23), 119
(29), 103 (75), 91 (50), 83 (62), 77 (31), 65 (29); mp
79–81 ꢁC.
4.5. Antibacterial activity
4.5.1. Test strains and medium. The antibacterial activi-
ty, expressed as MIC values (minimum inhibitory con-
centration), was determined on both clinical isolates
and bacterial strains obtained from the American Type
Culture Collection (ATCC) and from the Microbiology
Laboratory of Faculty of Pharmacy of University of
Porto (FFUP). All bacterial strains were maintained as
frozen stocks at ꢁ80 ꢁC in protected vials. The stock
cultures were propagated overnight in trypticase soy
broth at 35 ꢁC, before experimental use.
4.3.5. 4-Hydroxy-3-methoxy-b-nitrostyrene (Va). Yield
91%; 1H NMR d: 3.82 (3H, s, OCH3), 6.84 (1H, d,
J = 8.2, H(5)), 7.30 (1H, dd, J = 8.2; 1.9, H(6)), 7.47
(1H, d, J = 1.8, H(2)), 8.02 (1H, d, J = 13.4, H(a)),
8.16 (1H, d, J = 13.4, H(b)), 10.15 (1H, br s, OH); 13C
NMR d: 55.8 (OCH3), 112.2, 115.8 C(2) and C(5),
121.5 C(1), 126.0 C(6), 134.9 C(a), 140.3 C(b), 148.2
C(4), 151.5 C(3); EI-MS m/z (%): 195 (M+Å, 100), 148
(77), 133 (46), 105 (28), 89 (38), 78 (28), 63 (19); mp
168–171 ꢁC.
The Mueller–Hinton II culture medium, containing beef
infusion 300 g/L, bio-Case 17.5 g/L, starch 1.5 g/L and
agar 17 g/L, was used in the antibacterial assays.
4.5.2. Antibacterial assays. The antibacterial activity of
the compounds was evaluated through the agar dilution
procedure outlined by the National Committee for Clin-
ical Laboratory Standards (NCCLS).14 Stock solutions
of the b-nitrostyrene derivatives were prepared either
in ethanol or water depending on the compounds solu-
bility, and dilutions were carried out with sterilized dis-
tilled water. Different concentrations were obtained by
2-fold plate-dilution of a stock containing 1280 mg/L
of test substance, ranging from 16 to 512 mg/L, pre-
pared immediately before use and sterilized by filtration
through a 0.2 lm membrane filter.
4.3.6.
4-Hydroxy-3-methoxy-b-methyl-b-nitrostyrene
1
(Vb). Yield 95%; H NMR d: 2.44 (3H, s, CH3), 3.81
(3H, s, OCH3), 6.89 (1H, d, J = 8.2, H(5)), 7.12 (1H,
dd, J = 8.2; 1.9, H(6)), 7.20 (1H, d, J = 1.9, H(2)), 8.05
(1H, s, H(a)), 9.85 (1H, br s, OH); 13C NMR d: 14.0
CH3, 55.7 (OCH3), 114.8, 115.9 C(2) and C(5), 123.0
C(1), 124.8 C(6), 134.2 C(a), 144.7 C(b), 147.7 C(4),
149.2 C(3); EI-MS m/z (%): 209 (M+Å, 100), 162 (81),
147 (53), 131 (33), 119 (30), 103 (84), 91 (56), 77 (36),
65 (32); mp 97–99 ꢁC.
4.3.7. 3,4-Methylenedioxy-b-nitrostyrene (VIa). Yield
91%; 1H NMR d: 6.09 (2H, s, CH2), 7.00 (1H, d,
J = 8.0, H(5)), 7.34 (1H, dd, J = 8.0; 1.7, H(6)), 7.46
(1H, d, J = 1.6, H(2)), 8.01 (1H, d, J = 13.5, H(a)),
8.07 (1H, d,J = 13.5, H(b)); 13C NMR d: 102.3 CH2,
107.8, 109.2 C(2) and C(5), 124.6 C(1), 128.0 C(6),
136.4 C(a), 139.9 C(b), 148.5 C(4), 151.2 C(3); EI-MS
m/z (%): 193 (M+Å, 100), 146 (92), 117 (17), 89 (70), 71
(24), 63 (46); mp 164–165 ꢁC.
The inoculated plates were incubated overnight at 35 ꢁC.
All the experiments were conducted in triplicate. The
lowest concentration of the compounds that prevented
visible growth was considered to be the MIC value.
The solvent was found to have no effect on the microor-
ganisms, for all the concentrations tested (data not
shown).
4.6. Electrochemical determinations
4.4. Ab initio MO calculations
4.6.1. Preparation of solutions. Ten millimole stock solu-
tions of the b-nitrostyrene derivatives were prepared by
dissolving an appropriate amount in ethanol. The polar-
ographic working solutions were prepared, in the elec-
trochemical cell, by diluting 100 lL of the stock
solution in 10 mL of supporting electrolyte, in order to
get a final concentration of 0.1 mM.
The ab initio calculations—full geometry optimization
and calculation of the harmonic vibrational frequen-
cies—were performed using the GAUSSIAN 98W pro-
gram,33 within the Density Functional Theory (DFT)
approach, in order to properly account for the electron
correlation effects. The widely employed hybrid method
denoted by B3LYP,34–39 which includes a mixture of
HF and DFT exchange terms and the gradient-corrected
correlation functional of Lee, Yang and Parr,40,41 as pro-
The pH 7.3 supporting electrolyte was prepared by
diluting 6.2 mL of 0.2 M dipotassium hydrogen phos-
phate and 43.8 mL of 0.2 M potassium dihydrogen
phosphate to 100 mL.
posed and parameterized by Becke,42,43 was used, along
with the double-zeta split valence basis set 6-31G
44,45
.
**
4.6.2. Voltammetric measurements. Differential pulse
polarograms, cyclic and square wave voltammograms
were obtained in a PalmSens potentiostat/galvanostat
(Palm Instruments, The Netherlands) and a one-com-
partment glass electrochemical cell. A 2 mm diameter
glassy carbon working electrode (GCE), a platinum wire
counterelectrode and an Ag/AgCl saturated KCl refer-
ence electrode were used. pH measurements were carried
out in a Metrohm E520 pH-meter with a glass electrode
(Metrohm, Switzerland).
Molecular geometries were fully optimized by the Berny
algorithm, using redundant internal coordinates:46 The
bond lengths to within ca. 0.1 pm and the bond angles
to within ca. 0.1ꢁ. The final root-mean-square (rms)
gradients were always less than 3 · 10ꢁ4 hartree bohrꢁ1
or hartree radianꢁ1. No geometrical constraints were
imposed on the molecules under study. The ZPE scaling
was employed for all minima in the potential energy
surface.

N. Milhazes et al. / Bioorg. Med. Chem. 14 (2006) 4078–4088
4087
tests for bacteria that grow aerobically. Approved stan-
dard M7-A5. National Committee for Clinical Laboratory
Standards, Wayne PA, 2000.
All measurements were performed at room temperature
and purified nitrogen was used for oxygen displacement.
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Miller, A. F.; Cenas, N. Arch. Biochem. Biophys. 2001,
385, 170.
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pounds. Property descriptors encompass Electrotopolog-
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4.8. Statistical analysis
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