Inhibition of Enterococcus faecalis biofilm formation by highly active lactones and lactams analogues of rubrolides

Article abstract of DOI:10.1016/j.ejmech.2014.05.035

Seven β-aryl substituted γ-alkylidene-γ-lactones analogues of rubrolides were synthesized from mucobromic acid and converted through a lactamization with isobutylamine into their corresponding γ-hydroxy- γ-lactams (76-85%). These lactams were converted in

Full text of DOI:10.1016/j.ejmech.2014.05.035

European Journal of Medicinal Chemistry 82 (2014) 127e138
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibition of Enterococcus faecalis biofilm formation by highly active
lactones and lactams analogues of rubrolides
,
b
,
a
a
Ulisses A. Pereira a, Luiz C.A. Barbosa a , Célia R.A. Maltha , Antônio J. Demuner ,
*
Mohammed A. Masood c, Andréa L. Pimenta d
,
e
a Department of Chemistry, Federal University of Viçosa, Av. PH Rolfs, Viçosa, MG 36570-000, Brazil
b Department of Chemistry, Universidade Federal de Minas Gerais, Av. Pres. Antônio Carlos 6627, Campus Pampulha, CEP 31270-901, Belo Horizonte, MG,
Brazil
c Gene Solutions, LLC, One Boars Head Pointe, Suite 131, Charlottesville, VA 22903, USA
d Department of Odontology, Federal University of Santa Catarina, Florianópolis, SC 88040-900, Brazil
e Department of Biologie, University of Cergy Pontoise, 2 Av. Adolphe Chauvin, 95302 Cergy Pontoise, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven
b-aryl substituted g-alkylidene-g-lactones analogues of rubrolides were synthesized from
Received 19 September 2013
Received in revised form
6 May 2014
Accepted 11 May 2014
Available online 16 May 2014
mucobromic acid and converted through a lactamization with isobutylamine into their corresponding
g
-
hydroxy-g-lactams (76e85%). These lactams were converted into (Z)- and (E)-g-alkylidene-g-lactams (23
e45%). All compounds were fully characterized by IR, NMR (1H and 13C), COSY and HETCOR bidimen-
sional experiments, and NOE difference spectroscopy experiments when necessary. Evaluation of these
three different classes of compounds against Enterococcus faecalis biofilm formation showed that all
classes are active and the highest biofilm inhibition activity was caused by lactam 13f (IC50 ¼ 0.76
mL). Moreover, in almost all cases at least one of the lactams is more active than its correspondent
alkylidene- -lactone. The use of rubrolides as a lead structure has proven successful for the identification
of new compounds displaying novel antibacterial activities, namely biofilm inhibition, which have the
potential for the development of antimicrobial drugs targeted to inhibition of the initial stages of bac-
terial infections, rather than bacterial viability. Such drugs are less prompt to induce bacterial resistance,
being therefore a more cost-effective investment for pharmaceutical research.
mg/
Keywords:
g
-
g-Alkylidene-
g-Hydroxy- -lactams
g-Alkylidene- -lactams
g-lactones
g
g
g
Quorum sensing antagonist
Rubrolides
Anti-biofilm activity
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Biofilms are highly organized surface-associated communities
of bacteria encased within an extracellular matrix [4]. Some reports
Resistance to antibiotics has become a major clinical and public
health problem affecting the lives of a large percentage of the world
population. Confronted by increasing amounts of antibiotics over
the past 60 years, bacteria have responded by the propagation of
progeny no longer susceptible to them. While it is clear that anti-
biotics are pivotal in the selection of bacterial resistance, the spread
of resistance genes and of resistant bacteria has also contributed to
the problem. We currently face multi-resistant infectious disease
organisms that are difficult and, sometimes, impossible to treat
successfully. Misuse of antibiotics has almost certainly abrogated
the situation [1e3].
on bacterial resistance have demonstrated that, when organized in
biofilms, bacteria are able to survive to antibiotics treatments at
concentrations up to a thousand times higher than those used to
kill their planktonic counterparts [4,5]. It has been estimated that
80% of the world’s microbial biomass exists in the biofilm state and
that 65e80% of the microbial infections occurring in the human
body are biofilm-mediated [3]. Moreover, bacterial biofilm impacts
countless environments, from water pipes to indwelling devices
installed within or attached in hospitalized patients. Therefore,
identifying substances that are able to inhibit bacterial adherence
and biofilm formation constitutes a promising approach for the
development of a new generation of antimicrobial drugs targeted to
bacterial virulence rather than bacterial viability, which would
impose a low selection pressure on the bacterial populations, thus
avoiding development of resistance [6].
* Corresponding author. Department of Chemistry, Universidade Federal de
Minas Gerais, Av. Pres. Antônio Carlos 6627, Campus Pampulha, CEP 31270-901,
Belo Horizonte, MG, Brazil.
From a drug discovery perspective, little research has been done
in this arena for the past 25 years and there is a now resurgence in
E-mail addresses: lcab@ufmg.br, lcab@ufv.br (L.C.A. Barbosa).
http://dx.doi.org/10.1016/j.ejmech.2014.05.035
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

128
U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
research activities directed to finding novel mechanisms that
would circumvent bacterial resistance. Therefore, due to rapid in-
crease of bacterial resistance to currently available antimicrobial
drugs, the discovery of new molecules active against bacterial
pathogens is an important area of research in academy and in the
pharmaceutical industry [7]. In this context and in line with our
continuous effort in the search of bioactive molecules [8e11], we
embarked on a program to prepare and explore the potential
bioactivity of new natural product analogues having the rubrolides
(1) (Fig. 1) as a model compound [12e18]. This work was motivated
by recent reports on the biofilm inhibitor properties of some nat-
ural halogenated butenolides (2) (Fig. 1), isolated from the marine
red algae Delisea pulchra [19]. These compounds have been shown
to prevent biofilm formation in Escherichia coli and Bacillus subtilis
[20,21]. A similar activity was reported for the synthetic furanone 3
investigation in this area, demonstrating for the first time that
rubrolide analogues and their corresponding lactams are amongst
the most actives micromolecules in inhibiting biofilm formation by
Enterococcus faecalis, an important bacteria that resides in the hu-
man gastrointestinal tract. This Gram-positive bacteria, frequently
isolated from patients with endocarditis, bacteremia, urinary-tract
infections, and septicemia in general, is a multi-antibiotic resistant
pathogen [28]. Although there is no recent compilation data, it is
known that several types of hospitalized patients suffer a great
negative impact due to bacterial resistance to antibiotics and as a
consequence new therapeutic agents are constantly required. We
believe that the results reported here can open a new perspective
for the development of a new quorum sensing antagonists that
could help in the control of this pathogen.
which is able to inhibit E. coli biofilm formation by 80% at 141
mM
2. Results and discussion
[22]. Examination of structures depicted in Fig. 1, indicates that
natural and synthetic compounds 2 and 3 shares some structural
2.1. Synthesis
features with rubrolides and they all correspond to g-alkylidene-g-
lactones (4). We then hypothesized that the rubrolide analogues
could also have some inhibitory effect on bacterial biofilm forma-
tion. In addition, we wanted to gain understanding of the biological
mechanism by which these bacterial biofilms were prevented from
forming.
The rubrolide analogues (10aeg) were prepared from
commercially available mucobromic acid (7) as shown in Scheme 1.
In this procedure compound
7 was converted into 3,4-
dibromofuranone 8 in 83% [10]. A subsequent regioselective
SuzukieMiyaura cross-coupling between 8 and arylboronic acids in
the presence of Ag2O, AsPh3 and catalytic amount of PdCl2(MeCN)2
[16] resulted in the formation of 4-aryl-3-bromofuran-2(5H)-ones
(9ae9d) in low yields (26e37%). The reactions with boronic acids
with Br or Cl at the 50 position resulted in the required product in
slightly lower yield than the corresponding analogues without
electron withdrawing groups. Usually electron deficient arylbor-
onic acids tend to be difficult coupling partners, as they are less
nucleophilic and, hence transmetalate more slowly than electron-
neutral and -rich analogues [29]. In addition, the low conversions
of these Suzuki cross-couplings reactions can be explained in part
by the formation of the biphenyls 2,20-dimethoxybiphenyl (9a0),
5,50-dibromo-2,20-dimethoxybiphenyl (9b0), 5,50-dimethyl-2,20-
dimethoxybiphenyl (9c0), and 5,50-dichloro-2,20-dimethox-
ybiphenyl (9d0) produced in 14e23% as a result of the homocou-
pling of the boronic acids. Structural characterization of biphenyls
compounds 9a0e9d0 has already been described [30e32]. Although
the required products were obtained in low yields (26e37%), no
effort to optimize the reaction conditions was made since at this
stage we focused our attention in obtaining the final products for
biological evaluation, as no data on bacterial biofilm inhibition for
this class of compound has been reported yet.
Further insight into this work proposal came from the knowl-
edge that natural products bearing a
g-hydroxy-g-lactam (5) and g-
alkylidene- -lactam units (6) (Fig. 1), mainly produced by fungi
g
[23], also display a large array of biological activities such as anti-
fungal activity [24], cytotoxicity [25], angiogenesis inhibition
properties [26], etc.
Considering that lactones could be easily transformed into the
corresponding lactams [27], we envisaged that the rubrolide ana-
logues could be converted into the corresponding
g-hydroxy-g-
lactams and -alkylidene- -lactams. To the best of our knowledge,
g
g
there is no report on the bacterial biofilm inhibition caused by these
classes of compounds.
In light of the needs for new substances able to inhibit the
bacterial biofilm formation, we report here the results of our
On the next step 4-aryl-3-bromofuran-2(5H)-ones (9ae9d)
were treated with aromatic aldehyde, tert-butyldimethylsilyl tri-
fluoromethanesulfonate
(TBDMSOTf),
diisopropylethylamine
(DIPEA) followed by treatment of the silyl ether generated in situ
with DBU. Initially some experiments were carried out in order to
get the best reaction conditions and the
b-aryl substituted g-alky-
lidene- -lactones (10ae10g) analogues to rubrolides were pre-
g
pared stereoselectively in yields ranging from 44% to 68%. All
compounds were fully characterized by detailed IR, NMR, and MS
analyses. The configuration of the C5eC6 double bound was clari-
fied by NOE difference spectroscopy experiments. Irradiation of
hydrogen H-6 resulted in an enhancement of H-60 aromatic
hydrogen signal. In addition enhancement of H-200 and H-600 aro-
matic hydrogen signals were also observed. These results prove the
Z geometry for exocyclic double bound for compounds 10ae10g.
The stereoselectivity bias of these reactions can, in part, be related
to the steric hindrance of the aromatic group on the
b position.
While this is a possible explanation other factors can also be
involved since preferential Z geometry products have also been
reported for similar structures without b substituent [33].
Fig. 1. General structure for naturally occurring bioactive rubrolides (1), furanonas
displaying anti-biofilm activity (2 and 3),
lactams (5) and -alkylidene- -lactams (6).
g-alkylidene-g-lactones (4), g-hydroxy-g-
g
g

U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
129
Scheme 1. Preparation of g-alkylidene-g-lactones (10ae10g) and their corresponding g-hydroxy-g-lactams (11ae11g) and g-alkylidene-g-lactams (12ae12g and 13ae13g).
A subsequent treatment of compounds 10ae10g with an excess
of isobutylamine resulted in the formation of the corresponding
hydroxy- -lactams (11ae11g) in good yields (76e85%) [27]. The
hydroxylactams were then dehydrated with PTSA under reflux
affording a mixture of Z- and E- -alkylidene- -lactams (12ae12g
specifically due to an inhibition of bacterial adherence or could be
associated to an indirect effect of the compounds on bacterial
viability. For this, bacterial growth was quantified by absorbance
at 630 nm readings using a microtiter plate reader device in the
presence of each compound at concentrations of 87.5, 43.8, 21.9,
g-
g
g
g
and 13ae13g) that were isolated from all reaction after purification
using column chromatography, a separation that failed for similar
lactams as reported by Gupton and co-workers [34].
10.9, 5.5, 2.73, 1.37, 0.68, 0.34 and 0.17
m
g/mL. The results obtained
g/mL)
(Table S1) showed that -hydroxy- -lactam 11a (at 87.5 m
g
g
inhibited the planktonic growth of E. faecalis above 20%, while all
other compounds did not show any significant inhibitory effect on
E. faecalis growth to a maximum concentration of 87.5
Consequently the biofilm inhibition assays were carried out with
all compounds, except for 11a, in a maximum concentration of
87.5 mg/mL.
Detailed spectroscopic analyses (IR, 1H NMR, 13C NMR, and MS)
analyses confirmed the structures of all compounds and stereo-
chemistry characterization of the exocyclic double bond was ach-
ieved by NOE experiments. For example irradiation at H-6 signal
resulted in NOE enhancements at H-7/H-8/H-9/H-10 absorptions,
consistent with E configuration in the case of compounds 13ae13g,
once H-6 is in close proximity to isobutyl group. As shown on
Scheme 1, the E-compounds were obtained in better yields (31e
45%) than the Z-form (23e41%). At this point we have no final
explanation for such results, but it seems to be related to a balanced
steric interaction between the arylidene moiety and the N-isobutyl
and aryl group at C-4.
mg/mL.
2.3. Inhibition of E. faecalis biofilm formation
The antagonist effect of compounds 10ae10g, 11ae11g, 12ae
12g and 13ae13g on biofilm formation of E. faecalis was tested
using crystal violet staining assay and the results are shown in
Table 1. For the lactone series, 10d was the most active compound
Even though some reactions were low yielding, at this stage we
managed to prepare a series of 28 compounds closely related to
natural rubrolides, ready for the first time evaluation of their effects
on biofilm formation by E. faecalis, an important microorganism
responsible for endodontic infections [35].
(IC50 ¼ 1.51
m
m
g/mL), followed by 10b (IC50 ¼ 6.91
g/mL). A comparison between the effect of lactones
-lactams, 11a,
mg/mL) and 10c
(IC50 ¼ 18.7
10aeg and their corresponding nitrogen-analogues
g
11b, 11d, and 11g did not show good inhibition of biofilm formation
of E. faecalis (IC50 > 87.5 g/mL). Compound 11c caused some effect
(IC50 > 33
g/mL), while 11e and 11f were highly active (IC50 ¼ 1.07
and 1.29 g/mL, respectively). Among these two series of lactones
m
2.2. Effects on E. faecalis planktonic growth
m
m
In order to select the maximum concentration for the biofilm
inhibition assays all compound series (10e13) had their effect on
the planktonic growth of E. faecalis evaluated. This is important in
order to establish if eventual effects on biofilm formation were
(10aeg, 11aeg) the structural modifications were restricted to the
benzylidene ring and due to the limited number of analogues no
meaningful insight on the structureeactivity relationship can be
obtained.

130
U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
Table 1
Antagonistic effect of compounds 10ae10g, 11ae11g, 12ae12g and 13ae13g against E. faecalis biofilm formation.
Compound
IC50
(m
g/mL)
Compound
IC50
(m
g/mL)
Compound
IC50
(
m
g/mL)
Compound
IC50 (mg/mL)
a
a
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
12.01 ꢀ 4.62
3.31 ꢀ 1.34
6.62 ꢀ 0.35
62.64 ꢀ 9.68
1.09 ꢀ 0.30
1.48 ꢀ 0.10
1.46 ꢀ 0.33
13a
13b
13c
13d
13e
13f
2.96 ꢀ 0.67
3.46 ꢀ 0.24
3.35 ꢀ 0.43
59.34 ꢀ 11.13
1.04 ꢀ 0.23
0.76 ꢀ 0.19
3.26 ꢀ 1.52
6.91 ꢀ 1.73
18.7 ꢀ 5.1
>87.5
33.41 ꢀ 16.96
1.51 ꢀ 0.11
>87.5
a
1.07 ꢀ 0.06
1.29 ꢀ 0.19
>87.5
>87.5
53.11 ꢀ 16.72
10g
11g
12g
13g
IC50: concentration of compound needed to inhibit biofilm formation by 50%.
a
The IC50 was not possible to be calculated due to irregular response of inhibition of E. faecalis biofilm in function of concentration of the solution of the compound tested.
Considering the lactams (series 12 and 13) obtained by dehy-
dration of the series 11, it can be observed that 10 out of 14 com-
of all the compounds synthesized and tested was below 75 and
there was only 1 hydrogen bond donor group or less and these
features have been shown to give drugs the ability to penetrate the
blood brain barrier potentially leading to harmful side effects [38].
To the best of our knowledge, none of these synthetic com-
pounds have been tested previously as antagonists of quorum
sensing regulated biofilm formation in E. faecalis. In the case of the
Gram-positive bacteria E. faecalis, the quorum sensing autoinducers
are constituted by cyclic peptides. One of the autoinducers named
gelatinase biosynthesis-activating pheromone (GBAP) is consti-
tuted by 11 aminoacids residues forming a macrolactone ring
pounds were highly active (IC50 < 3.5
3 out of 14 compounds from the previously discussed series 10aeg
and 11aeg had IC50 < 3.5 g/mL.
A comparison between the bioactivity of isomers (Z)- and (E)-
alkylidene- -lactams indicates that both series of compounds have,
in general, the same effect on E. faecalis biofilm formation.
mg/mL). For comparison, only
m
g-
g
Considering all lactams, those bearing chlorine or bromine at
the ortho position on the benzylidene ring were the most active.
As the physicochemical properties of the molecules are an
important aspect to be considered during a drug discovery process,
we have calculated some properties for all compounds using
MedChem Designer software (Table 2) [36]. The compounds were
designed to determine if any structureeactivity relationship could
be found at this stage. Considering all 28 compounds, the “rule of
five” was violated twice by 10 of them (10c, 11b, 11c, 11f, 12b, 12c,
12f, 13b, 13c and 13f). Among such compounds are the most active
ones as 11f, 12f, 13f. These data suggest that the compounds might
have poor oral bioavailability [37], and further work has to be done
in order to better evaluate such hypothesis, especially considering
that compounds like 10a that was inactive did not violate any
parameter of the Lipinsky’s rule of five. The low polar surface area
involving the a-carboxyl group at the C-terminal methionine res-
idue and the hydroxyl group of a serine at the third position on the
other side of the peptide chain. The lactone unit was shown to be
essential for the activity [39]. Based on the structure of GBAP,
Nakayama, et al. (2013) [40] have used a unique drug design
approach to produce a series of polypeptides capable of inhibiting
the GBAP signals. The complex peptide named ZBzl-YAA5911
showed the strongest quorum sensing of E. faecalis antagonist ac-
tivity (IC50 ¼ 262 nM). Although a direct quantitative comparison of
the levels of activities of small molecules can be misleading due to a
lack of standardization between the assays [41], the most active
lactam 13f (IC50 ¼ 760 nM) was only three time less active then the
Table 2
Predicted physicochemical properties of compounds 10e13.
Compound
S þ logP
S þ logD
MlogP
MWt
HBDH
M-NO
T-PSA
Rule of 5
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
11e
11f
11g
12a
12b
12c
12d
12e
12f
12g
13a
13b
13c
13d
13e
13f
5.705
6.253
6.353
5.884
5.852
6.133
5.715
5.184
5.307
5.439
4.964
4.909
5.223
4.824
6.281
6.657
6.773
6.321
6.277
6.555
6.153
6.281
6.657
6.773
6.321
6.277
6.555
6.153
5.705
6.253
6.353
5.884
5.852
6.133
5.715
5.184
5.307
5.439
4.964
4.909
5.223
4.824
6.281
6.657
6.773
6.321
6.277
6.555
6.153
6.281
6.657
6.773
6.321
6.277
6.555
6.153
3.883
4.151
4.259
3.774
3.774
4.151
3.234
4.395
4.663
4.764
4.293
4.293
4.663
3.774
4.721
4.989
5.090
4.619
4.619
4.989
4.079
4.721
4.989
5.090
4.619
4.806
4.989
4.079
425.209
470.557
515.013
405.683
405.683
470.557
421.682
498.348
543.695
588.151
478.821
478.821
543.695
494.821
480.332
525.680
570.136
460.806
460.806
525.680
476.806
480.332
525.680
570.136
460.806
460.806
525.680
476.806
0
0
0
0
0
0
0
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
3
3
3
3
3
4
4
4
4
4
4
4
5
3
3
3
3
3
3
4
3
3
3
3
3
3
4
35.530
35.530
35.530
35.530
35.530
35.530
44.760
49.770
49.770
49.770
49.770
49.770
49.770
59.000
29.540
29.540
29.540
29.540
29.540
29.540
38.770
29.540
29.540
29.540
29.540
29.540
29.540
38.770
0
1
2
0
0
1
0
1
2
2
1
1
2
0
1
2
2
1
1
2
0
1
2
2
1
1
2
0
13g

U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
131
complex peptide ZBzl-YAA5911. Considering the simplicity of the
molecule compared with polypeptides, and the possibility of easily
increasing the molecular diversity in these rubrolide derived lac-
tams, we believe that such compounds can be used as a new
scaffold for the discovery of even more active antagonist to GBAP.
Among the quorum sensing antagonists reported, several
brominated furanones share some structural features with the
rubrolide derivatives reported here [22]. The authors suggested
that a conjugated exocyclic vinylbromide on the furanone ring of
several analogues of 3 is the most important structural feature for
the biofilm inhibition formation by E. coli. In this work the most
active compounds reported here, 11e and 11f, lack such function-
ality. We should also note that none of the active lactones or lac-
tams has a bromine atom attached to the exocyclic C]C double
bond. Han et al. (2008) [22] suggested that one bromine atom
directly attached to the furanone ring seems not to be a require-
ment for the activity. Although all active compounds tested by us
have one bromine atom on the furan ring, we cannot draw any
conclusion about the influence of such group on the activity since
no derivative deprived of this group was assayed.
resolution mass spectra were recorded on a Bruker MicroTof
(resolution ¼ 10,000 FWHM) under electrospray ionization (ESI)
and are given to four decimal places. Analytical thin layer chro-
matography analysis was conducted on aluminum packed pre-
coated silica gel plates. Column chromatography was performed
over silica gel (60e230 mesh).
4.2. Synthesis
4.2.1. General procedure for the synthesis of 9ae9d
4.2.1.1. 3-Bromo-4-(2-methoxyphenyl)furan-2(5H)-one (9a). To
two neck round-bottomed flask (100 mL) were added 3,4-
dibromofuran-2(5H)-ona (1.00 g; 4.13 mmol), 2-
a
8
methoxyphenylboronic acid (0.69 g; 4.54 mmol), bis(acetonitrile)
dichloropalladium(II) (PdCl2(CH3CN)2) (0.054 g; 0.21 mmol), tri-
phenylarsine (0.254 g; 0.83 mmol) and silver oxide (2.871 g;
12.39 mmol). Under nitrogen was added anhydrous THF (18 mL).
This system was maintained under magnetic stirring at 65 ꢂC for
24 h. The reaction mixture was cooled to room temperature, diluted
with ethyl acetate and filtered through a Celite pad. The filtrate was
concentrated in a rotary evaporator to afford the resulting material
of the reaction, which was purified by column chromatography on
silica gel employing elutions with mixtures of dichloromethanee
hexane 3:1, 2:1, 1: 1, and 1:2 v/v. This procedure afforded com-
pound 9a as a white solid in 35% yield (0.436 g; 1.62 mmol), m.p.
169.2e171.5 ꢂC. Rf ¼ 0.28 (hexane:dichloromethane, 1:1.5, v/v). IR
(ATR) nmax 3072, 2958, 1769, 1614, 1598, 1486, 1264, 1248, 1178,
3. Conclusion
In the present study we synthesized
g-hydroxy-g-lactams and
(Z)- and (E)- -alkylidene- -lactams from the correspondent
g
g
rubrolides analogues. The potential of these compounds as in-
hibitors of bacterial biofilm formation was tested against E. faecalis,
and results showed that these classes of compounds are very active,
1012, 978, 744, 721 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 3.87 (s, 3H, e
with twelve of them exhibiting IC50 less than 3.5
mg/mL.
OCH3), 5.26 (s, 2H, H-5), 6.99 (dd, 1H, J ¼ 8.4, 0.9 Hz, H-30), 7.09
(ddd, 1H, J ¼ 7.5, 7.5, 0.9 Hz, H-50), 7.48 (ddd, 1H, J ¼ 8.4, 7.5, 1.5 Hz,
H-40), 7.80 (dd, 1H, J ¼ 7.5, 1.5 Hz, H-60). 13C NMR (75 MHz, CDCl3):
Moreover, by comparing the activity of these classes of com-
pounds against E. faecalis biofilm formation, we showed that the
majority of the
g-alkylidene-
g-lactams were more active than the
d
55.8 (eOCH3), 73.8 (C-5), 108.2 (C-3), 111.6 (C-30), 118.9 (C-10),
precursor lactones. Although the data obtained did not allow us to
draw a clear structureeactivity relationship, it has successfully
allowed us to show that the nature of the substituents on both
aromatic rings has a dramatic effect on the bioactivity, such effects
being different for each class of compound tested. Considering that
many of the compounds prepared are amongst the most active
biofilm inhibitors described in the literature, further studies toward
the identification of the structural elements important for the
toxicity of these substances against E. faecalis, may result in the
development of new anti-infective agents to attenuate virulence
expression in this opportunistic important pathogen.
121.1 (C-50), 130.3 (C-60), 132.8 (C-40), 157.4 (C-20)*, 157.7 (C-4)*,
170,0 (C-2). *These assignments could be reversed; MS, m/z (%): 270
ꢃ
([Mþ2]þ , 35); 268 ([Mꢃ]þ, C11H9BrO3, 36), 241 (32), 239 (33), 189
(100), 161 (19), 145 (27), 133 (38), 132 (18), 131 (52), 118 (17), 115
(24), 105 (87),103 (21), 102 (39), 89 (39), 88 (28), 71 (49), 76 (19), 75
(28), 74 (25), 63 (37), 62 (30), 51 (39), 50 (28), 39 (43), 38 (18).
HRMS (ESI TOF-MS) calcd. for [C11H9BrNaO3]þ: 290.9627, found
290.9628.
4.2.1.2. 3-Bromo-4-(5-bromo-2-methoxyphenyl)furan-2(5H)-one
(9b). Compound 9b was synthesized using a method similar to that
of 9a and was isolated as a white solid in 29% yield; purified by
column chromatography, eluent hexane/dichloromethane (1:1.5 v/
v), m.p. 169.2e171.5 ꢂC. Rf ¼ 0.28 (hexane:dichloromethane, 1:1.5,
v/v). IR (ATR) nmax 2997, 2964, 1769, 1618, 1591, 1482, 1470, 1401,
1266, 1245, 1181, 1139, 1062, 1012, 983, 880, 805, 749, 621 cmꢁ1. 1H
4. Material and methods
4.1. Synthesis
Reagents and solvents were purified, when necessary, according
to procedures described by Perrin and Armarego [42]. The 3,4-
dibromofuran-2(5H)-ona 8 was obtained by sodium borohydride
reduction of mucobromic acid (7) commercially available (Aldrich
e Milwaukee, USA) according to a procedure described in the
literature [43]. It is important to mention that compound 7 can
alternatively be prepared from furfural as previously described
[44]. All reactions were carried out under a protective atmosphere
of dry nitrogen. The 1H and 13C NMR spectra were recorded on a
Varian Mercury 300 instrument (300 MHz and 75 MHz, respec-
tively), using deuterated chloroform as a solvent and tetrame-
NMR (300 MHz, CDCl3): d 3.86 (s, 3H, eOCH3), 5.21 (s, 2H, H-5), 6.88
(d, 1H, J ¼ 8.8 Hz, H-30), 7.56 (dd, 1H, J ¼ 8.8, 2.4 Hz, H-40), 7.88 (d,
1H, J ¼ 2.4 Hz, H-60). 13C NMR (75 MHz, CDCl3):
d 56.1 (eOCH3), 73.5
(C-5), 109.5 (C-3), 113.3 (C-50), 113.4 (C-30), 120.7 (C-10), 132.6 (C-60),
135.2 (C-40), 156.0 (C-20)*, 156.5 (C-4)*, 169.5 (C-2). *These assign-
ꢃ
ments could be reversed; MS, m/z (%): 350 ([Mþ4]þ , 3), 348
ꢃ
ꢃ
([Mþ2]þ , 9), 346 ([M]þ , C11H8Br2O3, 2), 269 (21), 267 (19), 132
(100), 131 (32), 102 (27), 101 (15), 99 (11), 89 (14), 88 (14), 87 (33),
77 (11), 75 (26), 74 (20), 63 (24), 62 (14), 51 (33), 50 (34), 44 (22), 38
(14). HRMS (ESI TOF-MS) calcd. for [C11H8Br2NaO3]þ: 368.8732,
found 368.8731.
thylsilane (TMS) as internal standard (
d
¼ 0). Infrared spectra were
recorded on a Varian 660-IR, equipped with GladiATR scanning
from 4000 to 500 cmꢁ1. Mass spectra were recorded on a Shimadzu
GCMS-QP5050A instrument under electron impact (70 eV) condi-
tions. Melting points are uncorrected and were obtained from
MQAPF-301 melting point apparatus (Microquimica, Brazil). High
4.2.1.3. 3-Bromo-4-(5-methyl-2-methoxyphenyl)furan-2(5H)-one
(9c). Compound 9c was synthesized using a method similar to that
of 9a and was isolated as a white solid in 37% yield; purified by
column chromatography, eluent hexane/dichloromethane (1:1.5 v/
v); m.p. 97.9e99.5 ꢂC; Rf ¼ 0.20 (hexane:dichlorometane, 1:1 v/v);

132
U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
IR (ATR) nmax 3026, 2989, 2955, 2910, 2854, 1749, 1610, 1575, 1494,
102 (25),101 (17), 91 (27), 89 (53), 77 (100), 63 (24), 62 (24), 39 (35).
HRMS (ESI TOF-MS) calcd. for [C19H12BrF3NaO3]þ: 446.9814, found
446.9816.
1444, 1346, 1260, 1190, 989, 822, 752, 726, 660, 560 cmꢁ1. 1H NMR
(300 MHz, CDCl3): d
2.34 (s, 3H, 50-CH3), 3.83 (s, 3H, 20-OCH3), 5.24
(s, 2H, H-5), 6.90 (d, 1H, J ¼ 8.4 Hz, H-30), 7.26 (dd,1H, J ¼ 8.4, 2.0 Hz,
H-40), 7.60 (d, 1H, J ¼ 2.0 Hz, H-60). 13C NMR (75 MHz, CDCl3):
d
20.6
4.2.2.2. (Z)-3-bromo-4-(5-bromo-2-methoxyphenyl)-5-(3-
chlorobenzylidene)furan-2(5H)-one (10b). Compound 10b was
synthesized using a method similar to that of 10a and was isolated
as pale green solid in 44% yield; purified by column chromatog-
raphy, eluent hexane/dichloromethane (3:2 v/v). m.p. 149.5e
150.6 ꢂC. Rf ¼ 0.31 (hexane:dichloromethane, 3:2, v/v). IR (ATR)
nmax 3057, 2963, 2937,1764,1600,1488,1459,1260,1226,1119,1019,
(50-CH3), 55.8 (20-OCH3), 73.8 (C-5), 107.9 (C-3), 111.6 (C-30), 118.5
(C-10), 130.4 (C-50), 130.5 (C-60), 133.3 (C-40), 155.4 (C-20)*, 157.8 (C-
4)*, 170.1 (C-2). *These assignments could be reversed; MS, m/z (%)
ꢃ
ꢃ
284 ([Mþ2]þ , 46), 282 ([M]þ , C12H11BrO3, 45), 255 (27), 253 (29),
238 (17), 203 (87), 175 (25), 174 (15), 159 (42), 147 (84), 146 (23),145
(60),132 (26),131 (33),131 (33),129 (30),119 (90),117 (28),116 (42),
115 (100), 103 (33), 102 (57), 91 (77), 89 (32), 87 (16), 78 (22), 76
(29), 75 (34), 74 (31), 63 (56), 62 (24), 52 (32), 51 (91), 50 (48);
HRMS (ESI TOF-MS) calcd. for [C12H12BrO3]þ: 282.9964, found
282.9882.
984, 960, 883, 780, 679 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 3.84 (s,
3H, eOCH3), 5.84 (s, 1H, H-6), 6.95 (d, 1H, J ¼ 9.0 Hz, H-30), 7.30e
7.32 (m, 2H, H-400 and H-500), 7.37 (d, 1H, J ¼ 2.4 Hz, H-60), 7.63 (dd,
1H, J ¼ 9.0, 2.4 Hz, H-40); 7.66e7.67 (m, 1H, H-600), 7.71e7.72 (m, 1H,
H-200). 13C NMR (75 MHz, CDCl3):
d 56.2 (eOCH3), 112.2 (C-6), 112.3
4.2.1.4. 3-Bromo-4-(5-chloro-2-methoxyphenyl)furan-2(5H)-one
(9d). Compound 9d was synthesized using a method similar to that
of 9a and was isolated as white solid in 26% yield; purified by
column chromatography, eluent hexane/dichloromethane (1:1 v/
v). m.p. 159.4e160.6 ꢂC. Rf ¼ 0.23 (hexane:dichloromethane, 1:1, v/
v). IR (ATR) nmax 2997, 2961, 2855, 1769, 1615, 1596, 1485, 1471, 1441,
1407, 1266, 1245, 1182, 1012, 982, 880, 808, 749, 679, 640 cmꢁ1. 1H
(C-3), 113.0 (C-50), 113.6 (C-30), 119.6 (C-10), 129.0 (C-600), 129.7 (C-
500)*, 130.3 (C-400)*, 130.6 (C-200), 132.8 (C-60), 134.4 (C-100)**, 134.9
(C-40), 135.0 (C-300)**, 148.2 (C-5), 151.0 (C-4), 156.0 (C-20), 164.8 (C-
2). *, **These assignments could be reversed; MS, m/z (%) 474
ꢃ
ꢃ
ꢃ
ꢃ
([Mþ6]þ , 1), 472 ([Mþ4]þ , 7), 470 ([Mþ2]þ , 11), 468 ([M]þ
,
C18H11Br2ClO3, 4), 310 (16), 211 (11), 209 (14), 176 (12), 152 (18), 130
(25), 124 (21), 118 (28), 102 (61), 89 (100), 88 (26), 87 (47), 75 (21),
63 (35), 39 (28), 36 (15). HRMS (ESI TOF-MS) calcd. for
[C18H12Br2ClO3]þ: 468.8836, found 468.8886.
NMR (300 MHz, CDCl3): d 3.87 (s, 3H, eOCH3), 5.23 (s, 2H, H-5), 6.95
(d, 1H, J ¼ 9.0 Hz, H-30), 7.43 (dd, 1H, J ¼ 9.0, 2.7 Hz, H-40), 7.77 (d,
1H, J ¼ 2.7 Hz, H-60); 13C NMR (75 MHz, CDCl3):
d 56.2 (eOCH3),
73.5 (C-5), 109.4 (C-3), 113.0 (C-30), 120.2 (C-10), 126.2 (C-50), 129.8
(C-60), 132.3 (C-40), 156.0 (C-20)*, 156.1 (C-4)*, 169.6 (C-2). *These
4.2.2.3. (Z)-3-bromo-4-(5-bromo-2-methoxyphenyl)-5-(4-
bromobenzylidene)furan-2(5H)-one (10c). Compound 10c was syn-
thesized using a method similar to that of 10a and was isolated as
white solid in 45% yield; purified by column chromatography,
eluent hexane/dichloromethane (3:2 v/v). m.p. 157.5e158.6 ꢂC.
Rf ¼ 0.32 (hexane:dichloromethane, 3:2, v/v). IR (ATR) nmax 3022,
2941, 2843, 1773, 1644, 1575, 1481, 1403, 1266, 1250, 1178, 1121,
1008, 978, 874, 836, 800, 693, 750, 622 cmꢁ1. 1H NMR (300 MHz,
ꢃ
assignments could be reversed; MS, m/z (%): 306 ([Mþ4]þ , 21), 304
ꢃ
ꢃ
([Mþ2]þ , 85), 302 ([M]þ , C11H8BrClO3, 64), 275 (71), 273 (54), 260
(30), 225 (35), 223 (100), 179 (42), 167 (57), 165 (62), 139 (48), 136
(35), 132 (49), 101 (54), 87 (46), 75 (53). HRMS (ESI TOF-MS) calcd.
for [C11H9BrClO3]þ: 302.9418, found 302.9367.
4.2.2. General procedure for the synthesis of 10ae10g
CDCl3): d 3.83 (s, 3H, eOCH3), 5.85 (s, 1H, H-6), 6.96 (d, 1H,
4 . 2 . 2 . 1. ( Z ) - 3 - b r o m o - 4 - ( 2 - m e t h o x y p h e n y l ) - 5 - ( 4 -
trifluoromethylbenzylidene)furan-2(5H)-one (10a). To a two-neck
round-bottom flask under nitrogen atmosphere were added 3-
J ¼ 9.0 Hz, H-30), 7.37 (d, 1H, J ¼ 2.4 Hz, H-60), 7.50 (d, 2H, J ¼ 8.4 Hz,
H-300 and H-500), 7.62 (d, 2H, J ¼ 8.4 Hz, H-200 and H-600), 7.63 (dd, 1H,
J ¼ 9.0, 2.4 Hz, H-40). 13C NMR (75 MHz, CDCl3):
d 56.2 (eOCH3),
bromo-4-(2-methoxyphenyl)furan-2(5H)-one
9a
(900
mg,
111.9 (C-3), 112.6 (C-6), 112.9 (C-50), 113.6 (C-30), 119.7 (C-10)*, 124.2
(C-400)*, 131.7 (C-100), 132.3 (C-200, C-300, C-500 and C-600), 132.8 (C-60),
134.9 (C-40), 147.9 (C-5), 150.9 (C-4), 156.0 (C-20), 164.9 (C-2). *These
3.34 mmol), dichloromethane (20 mL), TBDMSOTf (1.688 mL,
7.35 mmol), DIPEA (1.745 mL, 10.02 mmol), and p-tri-
fluoromethylbenzaldehyde (0.651 mL, 3.74 mmol). The resulting
mixture was stirred at room temperature over 1 h. After DBU
(1.099 mL, 7.35 mmol) was added, the reaction mixture was
refluxed for an additional 3 h before the addition of dichloro-
methane (70 mL). The resulting organic layer was washed with
aqueous HCl 3 mol Lꢁ1 solution (2 ꢄ 40 mL) and brine (3 ꢄ 40 mL).
After separation, the organic layer was dried over MgSO4, filtered,
and concentrated under reduced pressure. The resulting material
was purified by column chromatography on silica gel eluted with
hexane/dichloromethane (3:2 v/v) to afford compound 10a in 63%
yield (896 mg, 2.11 mmol). m.p. 147.6e149.3 ꢂC. IR (ATR) nmax 3006,
1779, 1608, 1492, 1322, 1249, 1164, 1066, 1020, 975, 872, 840,
ꢃ
assignments could be reversed; MS, m/z (%) 518 (Mþ6]þ , 4), 516
ꢃ
ꢃ
ꢃ
([Mþ4]þ , 11), 514 ([Mþ2]þ , 12), 512 ([M]þ , C18H11Br3O3, 4), 197
(11), 196 (15), 168 (12), 130 (13), 118 (13), 102 (30), 89 (100), 87 (26),
63 (32), 39 (22). HRMS (ESI TOF-MS) calcd. for [C18H12Br3Oþ3 :
512.8331, found 512.8310.
4.2.2.4. (Z)-3-bromo-5-(3-chlorobenzylidene)-4-(5-methyl-2-
methoxyphenyl)furan-2(5H)-one (10d). Compound 10d was syn-
thesized using a method similar to that of 10a and was isolated as
pale yellow solid in 63% yield; purified by column chromatography,
eluent hexane/dichloromethane (3:2 v/v). m.p. 138.2e139.6 ꢂC.
Rf ¼ 0.25 (hexane:dichloromethane, 3:2, v/v). IR (ATR) nmax 3008,
2929, 1769, 1644, 1497, 1457, 1248, 1230, 986, 928, 882, 810, 780,
597 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 3.86 (s, 3H, eOCH3), 5.94 (s,
1H, H-6), 7.09 (dd, 1H, J ¼ 8.4, 0.9 Hz, H-30), 7.12 (ddd, 1H, J ¼ 7.5, 7.5,
0.9 Hz, H-50), 7.27 (dd, 1H, J ¼ 7.5, 1.8 Hz, H-60), 7.54 (ddd, 1H, J ¼ 8.4,
7.5, 1.8 Hz, H-40), 7.61 (d, 2H, J ¼ 8.4 Hz, H-300 and H-500), 7.85 (d, 2H,
697, 679 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 2.37 (s, 3H, AreCH3),
3.81 (s, 3H, eOCH3), 5.87 (s, 1H, H-6), 6.95 (d, 1H, J ¼ 8.4 Hz, H-30);
7.04 (d, 1H, J ¼ 1.8 Hz, H-60), 7.28e7.31 (m, 2H, H-400 and H-500),
7.30e7.33 (m, 1H, H-40), 7.63e7.67 (m, 1H, H-600), 7.71 (m, 1H, H-200).
J ¼ 8.4 Hz, H-200 and H-600). 13C NMR (75 MHz, CDCl3):
d 55.9 (e
OCH3); 111.6 (C-6), 111.9 (C-30), 112.2 (C-3), 117.7 (C-10), 121.0 (C-50),
124.1 (q, J ¼ 270.6 Hz, eCF3), 125.9 (q, J ¼ 3.8 Hz, C-300 and C-500),
130.5 (C-60), 130.5 (q, J ¼ 32.2 Hz, C-400), 130.9 (C-200 and C-600), 132.4
13C NMR (75 MHz, CDCl3):
d 20.7 (AreCH3), 55.9 (eOCH3), 111.5 (C-
3), 111.8 (C-30), 112.1 (C-6), 117.4 (C-10), 128.9 (C-600), 129.4 (C-400)*,
130.2 (C-500)*, 130.3 (C-50), 130.5 (C-200), 130.7 (C-60), 132.7 (C-40),
134.7 (C-100), 135.0 (C-300),148.7 (C-5), 152.7 (C-4), 154.6 (C-20), 165.3
(C-2). *These assignments could be reversed; MS, m/z (%) 408
(C-40), 136.3 (C-100), 149.3 (C-5), 152.5 (C-4)*, 156.8 (C-20)*, 165 (C-2).
ꢃ
*These assignments could be reversed. MS, m/z (%) 426 ([Mþ2]þ
,
ꢃ
ꢃ
ꢃ
ꢃ
68), 424 ([M]þ , C19H12BrF3O3, 66), 345 (41), 317 (33), 167 (22), 163
(37),159 (31),158 (43),135 (18),131 (81),119 (54),115 (45),103 (30),
([Mþ4]þ , 20), 406 ([Mþ2]þ , 76), 404 ([M]þ , C19H14BrClO3, 58), 325
(26), 297 (29), 279 (17), 219 (21), 177 (67), 165 (17), 149 (42), 145

U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
133
(76),133 (64),129 (29),116 (35),115 (75),105 (31),102 (86),111 (49),
91 (48), 89 (100), 63 (43), 51 (23). HRMS (ESI TOF-MS) calcd. for
[C19H15BrClO3]þ: 404.9888, found 404.9779.
C19H14BrClO4, 75), 341 (24), 278 (38), 250 (14), 225 (22), 223 (23),
189 (16), 165 (22), 161 (29), 149 (24),148 (67), 121 (26), 102 (26), 101
(24), 91 (58), 89 (25), 77 (34), 51 (41). HRMS (ESI TOF-MS) calcd. for
[C19H14BrClNaO4]þ: 442.9656, found 446.9661.
4.2.2.5. (Z)-3-bromo-5-(2-chlorobenzylidene)-4-(5-methyl-2-
methoxyphenyl)furan-2(5H)-one (10e). Compound 10e was syn-
thesized using a method similar to that of 10a and was isolated as
pale yellow solid in 68% yield; purified by column chromatography,
eluent hexane/dichloromethane (3:2 v/v). m.p. 121.3e122.6 ꢂC.
Rf ¼ 0.27 (hexane:dichloromethane, 3:2, v/v). IR (ATR) nmax 3063,
2998, 2964, 2832, 1766, 1613, 1584, 1499, 1436, 1250, 1109, 996, 979,
4.2.3. General procedure for the synthesis of 11ae11g
4.2.3.1. 3-Bromo-5-(4-trifluoromethylbenzyl)-5-hydroxy-1-isobutyl-
4-(2-methoxyphenyl)pyrrol-2(5H)-one (11a). To a two-neck round-
bottom flask were added (Z)-3-bromo-4-(2-methoxyphenyl)-5-(4-
trifluoromethylbenzylidene)furan-2(5H)-one (10a) (650 mg,
1.53 mmol) dissolved in dichloromethane (8 mL) and the solution
was cooled to 0 ꢂC. Then the isobutylamine (560 mg, 7.65 mmol)
dissolved in dichloromethane was added in a dropwise fashion and
the resulting mixture was stirred at 0 ꢂC for 3 h before the addition
of dichloromethane (70 mL). After this time, the mixture was
quenched with HCl aqueous solution (2 mol Lꢁ1, 2 ꢄ 55 mL), and
washed with saturated NaHCO3 solution (2 ꢄ 55 mL) and brine
(2 ꢄ 55 mL). After separation, the organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure. The
resulting material was purified by column chromatography on sil-
ica gel eluted with hexane/dichloromethane (1:7 v/v). The proce-
dure described afforded compound 11a in 85% yield (646 mg,
1.30 mmol). m.p. 160.4e161.2 ꢂC. IR (ATR) nmax 3450e3180, 2963,
2873, 1695, 1682, 1492, 1418, 1324, 1250, 1158, 1115, 1067, 1019, 927,
814, 737 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 2.37 (s, 3H, AreCH3),
3.83 (s, 3H, eOCH3), 6.49 (s, 1H, H-6), 6.97 (d, 1H, J ¼ 8.4 Hz, H-30),
7.11 (d, 1H, J ¼ 2.4 Hz, H-60), 7.25 (ddd, 1H, J ¼ 7.8, 7.5, 1.8 Hz, H-500),
7.32 (ddd, 1H, J ¼ 7.8, 7.5, 1.8 Hz, H-400), 7.32 (dd, 1H, J ¼ 8.4, 2.4 Hz,
H-40), 7.37 (dd, 1H, J ¼ 7.8, 1.8 Hz, H-300), 8.24 (dd, 1H, J ¼ 7.8, 1.8 Hz,
H-600). 13C NMR (75 MHz, CDCl3):
d 20.7 (AreCH3), 55.9 (eOCH3),
109.2 (C-6), 111.3 (C-3), 111.8 (C-30), 117.4 (C-10), 127.5 (C-400), 129.9
(C-300), 130.3 (C-50), 130.4 (C-500), 130.9 (C-100), 131.0 (C-60), 132.1 (C-
600), 132.8 (C-40), 134.9 (C-200), 148.7 (C-5), 153.0 (C-4), 154.7 (C-20),
165.5 (C-2). *These assignments could be reversed; MS, m/z (%) 408
ꢃ
ꢃ
ꢃ
([Mþ4]þ , 26), 406 ([Mþ2]þ , 100), 404 ([M]þ , C19H14BrClO3, 77),
325 (31), 297 (30), 279 (15), 253 (20), 251 (19), 189 (17), 177 (48),
173 (16), 149 (35), 145 (61), 133 (39), 129 (23), 116 (26), 115 (54), 102
(65), 95 (40), 89 (73), 63 (27). HRMS (ESI TOF-MS) calcd. for
[C19H15BrClO3]þ: 404.9888, found 404.9878.
862, 753, 642, 618, 500 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 0.96 (d,
3H, J ¼ 6.6 Hz, H-9), 0.98 (d, 3H, J ¼ 6.6 Hz, H-10), 2.25e2.39 (m, 1H,
H-8), 3.12 (d, 1H, J ¼ 14.4 Hz, H-6a), 3.13 (dd, 1H, J ¼ 13.8, 7.8 Hz, H-
7a), 3.20 (d, 1H, J ¼ 14.4 Hz, H-6b), 3.53 (dd, 1H, J ¼ 13.8, 8.1 Hz, H-
7b), 3.76 (s, 3H, eOCH3), 5.13 (s, 1H, eOH), 6.83 (dd, 1H, J ¼ 8.4,
0.9 Hz, H-30), 6.98 (ddd, 1H, J ¼ 7.5, 7.5, 0.9 Hz, H-50), 7.05 (dd, 1H,
J ¼ 7.5, 1.8 Hz, H-60), 7.07 (d, 2H, J ¼ 8.1 Hz, H-200 and H-600), 7.34 (d,
2H, J ¼ 8.1 Hz, H-300 and H-500), 7.36e7.31 (m, 1H, H-40). 13C NMR
4.2.2.6. (Z)-3-bromo-5-(2-bromobenzylidene)-4-(5-chloro-2-
methoxyphenyl)furan-2(5H)-one (10f). Compound 10f was synthe-
sized using a method similar to that of 10a and was isolated as pale
yellow solid in 58% yield, purified by column chromatography,
eluent hexane/dichloromethane (3:2 v/v). m.p. 154.3e155.7 ꢂC.
Rf ¼ 0.25 (hexane:dichloromethane, 3:2, v/v). IR (ATR) nmax 3097,
3061, 3010, 2968, 2845, 1768, 1645, 1487, 1462, 1249, 1184, 1025,
(75 MHz, CDCl3):
d 20.7 (C-9), 20.9 (C-10), 27.9 (C-8), 42.9 (C-6),
48.3 (C-7), 56.2 (eOCH3), 93.3 (C-5), 111.7 (C-30), 120.7 (C-3)**, 121.7
(C-50), 122.1 (C-10)**, 124.3 (q, J ¼ 270.5 Hz, eCF3), 124.9 (q,
J ¼ 3.7 Hz, C-300 and C-500),129.2 (q, J ¼ 32.3 Hz, C400),130.4 (C-200 and
C-600), 131.4 (C-60), 131.5 (C-40), 138.5 (C-100), 153.3 (C-20)*, 155.8 (C-
4)*,164 (C-2). *, **These assignments could be reversed; MS, m/z (%)
481 (7), 479 (6), 356 (9), 341 (17), 340 (100), 339 (19), 338 (99), 284
(25), 282 (25), 241 (11), 239 (11), 159 (43), 132 (13), 131 (26), 115
(14), 109 (12), 89 (11), 77 (19), 57 (23), 55 (12), 43 (21), 41 (61), 39
(18), 30 (17). HRMS (ESI TOF-MS) calcd. for [C23H23BrF3NNaO3]þ:
520.0706, found 520.0705.
981, 825, 751, 671 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 3.87 (s, 3H, e
OCH3), 6.44 (s, 1H, H-6), 7.02 (d, 1H, J ¼ 9.0 Hz, H-30), 7.18 (ddd, 1H,
J ¼ 7.8, 7.8,1.8 Hz, H-400), 7.32 (dd,1H, J ¼ 2.7 Hz, H-60), 7.37 (ddd,1H,
J ¼ 7.8, 7.8, 1.2 Hz, H-500), 7.49 (dd, 1H, J ¼ 9.0, 2.7 Hz, H-40), 7.58 (dd,
1H, J ¼ 7.8, 1.2 Hz, H-300), 8.20 (dd, 1H, J ¼ 7.8, 1.8 Hz, H-600). 13C NMR
(75 MHz, CDCl3):
d 56.2 (eOCH3), 112.0 (C-3), 112.1 (C-6), 113.1 (C-
30), 119.1 (C-10), 125.8 (C-200), 126.0 (C-50), 128.1 (C-500), 130.3 (C-60),
130.8 (C-400), 132.0 (C-40), 132.3 (C-600), 132.3 (C-100), 133.3 (C-300),
148.1 (C-5), 151.3 (C-4), 155.4 (C-20), 165.1 (C-2); MS, m/z (%) 474
ꢃ
ꢃ
ꢃ
ꢃ
([Mþ6]þ , 8), 472 ([Mþ4]þ , 38), 470 ([Mþ2]þ , 48), 468 ([M]þ
,
C18H11Br2ClO3, 23), 391 (20), 273 (19), 198 (16), 197 (18), 196 (21),
176 (17), 169 (17), 165 (27), 102 (32), 101(29), 94 (18), 89 (100), 88
(24), 87 (25), 63 (28). HRMS (ESI TOF-MS) calcd. for
[C18H12Br2ClO3]þ: 468.8836, found 468.8822.
4.2.3.2. 3-Bromo-4-(5-bromo-2-methoxyphenyl)-5-(3-
chlorobenzyl)-5-hydroxy-1-isobutylpyrrol-2(5H)-one
(11b).
Compound 11b was synthesized using a method similar to that of
11a and was isolated as white solid in 78% yield, purified by column
chromatography, eluent hexane/dichloromethane (1:9 v/v). m.p.
171.4e173.0 ꢂC. Rf ¼ 0.17 (hexane:dichloromethane, 1:9, v/v). IR
(ATR) nmax 3385e3205, 3007, 2960, 2928, 2868, 2843, 1693, 1680,
1485, 1415, 1386, 1245, 1183, 1083, 1025, 780, 701, 682, 650,
4.2.2.7. (Z)-3-bromo-4-(5-chloro-2-methoxyphenyl)-5-(3-
methoxybenzylidene)furan-2(5H)-one (10g). Compound 10g was
synthesized using a method similar to that of 10a and was isolated
as pale green solid in 65% yield, purified by column chromatog-
raphy, eluent hexane/dichloromethane (1:2 v/v). m.p. 137.8e
139.4 ꢂC. Rf ¼ 0.44 (hexane:dichloromethane, 1:2, v/v). IR (ATR)
nmax 3049, 3009, 2831,1767,1756,1640,1598,1487,1459,1250,1161,
1123, 989, 869, 822, 789, 686, 641 cmꢁ1. 1H NMR (300 MHz, CDCl3):
621 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d
0.95 (d, 3H, J ¼ 6.9 Hz, H-9),
0.97 (d, 3H, J ¼ 6.9 Hz, H-10), 2.22e2.36 (m, 1H, H-8), 2.97 (d, 1H,
J ¼ 14.4 Hz, H-6a), 3.15 (dd, 1H, J ¼ 14.1, 7.5 Hz, H-7a), 3.16 (d, 1H,
J ¼ 14.4 Hz, H-6b), 3.52 (dd, 1H, J ¼ 14.1, 7.8 Hz, H-7b), 3.77 (s, 3H, e
OCH3), 6.73 (d, 1H, J ¼ 9.0 Hz, H-30), 6.86 (ddd, 1H, J ¼ 7.8, 1.6, 1.6 Hz,
H-600), 6.96e6.98 (m, 1H, H-200), 7.05 (dd, 1H, J ¼ 7.8, 7.8 Hz, H-500),
7.09 (ddd, 1H, J ¼ 7.8, 1.6, 1.6 Hz, H-400), 7.18 (d, 1H, J ¼ 2.4 Hz, H-60),
d
3.84 (s, 6H, 2 ꢄ eOCH3), 5.90 (s, 1H, H-6), 6.89e6.93 (m, 1H, H-400),
7.01 (d, 1H, J ¼ 9.0 Hz, H-30), 7.25 (d, 1H, J ¼ 2.4 Hz, H-60), 7.26e7.35
(m, 3H, H-200, H-500 and H-600), 7.48 (dd, J ¼ 9.0, 2.7 Hz, H-40). 13C
7.40 (dd, 1H, J ¼ 9.0, 2.4 Hz, H-40). 13C NMR (75 MHz, CDCl3):
d 20.8
NMR (75 MHz, CDCl3):
d
55.6 (eOCH3), 56.6 (eOCH3), 111.6 (C-3),
(C-9), 20.9 (C-10), 28.0 (C-8), 42.7 (C-6), 48.2 (C-7), 56.4 (eOCH3),
93.4 (C-5), 113.1 (C-30), 113.6 (C-50), 122.6 (C-3 and C-10), 127.4 (C-
400), 128.1 (C-600), 129.3 (C-500), 130.4 (C-200), 133.4 (C-60), 133.9 (C-40
and C-300), 136.1 (C-100), 151.6 (C-20), 155.0 (C-4), 164.5 (C-2); MS, m/z
(%) 420 (48), 418 (100), 416 (50), 362 (25), 360 (13), 283 (15), 281
113.2 (C-30), 113.9 (C-6), 115.6 (C-200), 116.1 (C-400), 119.4 (C-10), 123.8
(C-600), 125.9 (C-50), 130.0 (C-500), 130.1 (C-60), 131.8 (C-40), 133.9 (C-
100), 147.6 (C-5)*, 151.1 (C-4), 155.5 (C-20), 160.0 (C-300), 165.1 (C-2);
ꢃ
ꢃ
ꢃ
MS, m/z (%) 424 ([Mþ4]þ , 26), 422 ([Mþ2]þ , 100), 420 ([M]þ
,

134
U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
(15), 202 (13), 127 (15), 125 (41), 89 (14), 57 (55), 56 (17), 55 (18).
HRMS (ESI TOF-MS) calcd. for [C22H23Br2ClNO3]þ: 541.9728, found
541.9572.
7.04 (m, 2H, H-400 and H-500), 7.08 (dd, 1H, J ¼ 8.7, 2.1 Hz, H-40), 7.17
(dd, 1H, J ¼ 6.6, 2.7 Hz, H-300), 7.25 (dd, 1H, J ¼ 6.9, 2.7 Hz, H-600). 13C
NMR (75 MHz, CDCl3): d 20.5 (AreCH3), 20.7 (C-9), 20.8 (C-10), 27.8
(C-8), 38.7 (C-6), 48.2 (C-7), 56.5 (eOCH3), 93.4 (C-5), 111.5 (C-30),
120.3 (C-3)**, 121.7 (C-10)**, 126.6 (C-500), 128.3 (C-400), 129.3 (C-300),
130.4 (C-600), 131.5 (C-60), 131.5 (C-40), 131.2 (C-50), 132.8 (C-200),
135.2 (C-100), 153.6 (C-20)*, 154.1 (C-4)*, 164.7 (C-2). *, **These as-
signments could be reversed; MS, m/z (%) 354 (98), 352 (100), 298
(19), 296 (19), 145 (17), 127 (11), 125 (27), 115 (12), 57 (19). HRMS
(ESI TOF-MS) calcd. for [C23H26BrClNO3]þ: 478.0779, found
478.0804.
4.2.3.3. 3-Bromo-4-(5-bromo-2-methoxyphenyl)-5-(4-
bromobenzyl)-5-hydroxy-1-isobutylpyrrol-2(5H)-one
(11c).
Compound 11c was synthesized using a method similar to that of
11a and was isolated as white solid in 85% yield, purified by column
chromatography, eluent hexane/dichloromethane (1:7 v/v). m.p.
172.5e173.5 ꢂC. Rf ¼ 0.22 (hexane:dichloromethane, 1:7, v/v). IR
(ATR) nmax 3399e3154, 2957, 2869, 2844, 1676, 1645, 1486, 1385,
.
1247, 1050, 1026, 809, 653, 620, 506 cmꢁ1 1H NMR (300 MHz,
CDCl3):
d
0.96 (d, 3H, J ¼ 6.6 Hz, H-9), 0.98 (d, 3H, J ¼ 6.6 Hz, H-10),
4 . 2 . 3 . 6 . 3 - B ro m o - 5 - ( 2 - b ro m o b e n z yl ) - 4 - ( 5 - ch l o ro - 2 -
2.23e2.37 (m, 1H, H-8), 3.02 (d, 1H, J ¼ 14.7 Hz, H-6a), 3.11 (d, 1H,
J ¼ 14.7 Hz, H-6b), 3.13 (dd, 1H, J ¼ 14.1, 7.2 Hz, H-7a), 3.53 (dd, 1H,
J ¼ 14.1, 8.1 Hz, H-7b), 3.73 (s, 3H, eOCH3), 6.74 (d, 1H, J ¼ 8.7 Hz, H-
30), 6.83 (d, 2H, J ¼ 8.4 Hz, H-200 and H-600), 7.10 (d, 1H, J ¼ 2.7 Hz, H-
60), 7.25 (d, 2H, J ¼ 8.4 Hz, H-300 and H-500), 7.45 (dd, 1H, J ¼ 8.7,
methoxyphenyl)-5-hydroxy-1-isobutylpyrrol-2(5H)-one
(11f).
Compound 11f was synthesized using a method similar to that of
11a and was isolated as white solid in 76% yield, purified by col-
umn chromatography, eluent hexane/dichloromethane (1:9 v/v).
m.p. 159.3e160.5 ꢂC. Rf ¼ 0.31 (hexane:dichloromethane, 1:9, v/v).
IR (ATR) nmax 3221e3397, 3068, 2936, 2864, 2842, 1688, 1673,
2.7 Hz, H-40). 13C NMR (75 MHz, CDCl3):
d 20.7 (C-9), 21.0 (C-10),
28.0 (C-8), 42.3 (C-6), 48.3 (C-7), 56.5 (eOCH3), 93.5 (C-5), 113.2 (C-
30), 113.6 (C-50), 121.4 (C-400), 122.6 (C-10 and C-3), 131.2 (C-300 and C-
500), 131.6 (C-200 and C-600), 133.0 (C-100), 133.6 (C-60), 133.7 (C-40),
151.7 (C-20), 155.0 (C-4), 164.4 (C-2); MS, m/z (%) 420 (47), 418 (100),
416 (50), 364 (10), 362 (20), 360 (10), 283 (13), 281 (13), 171 (12),
169 (16), 90 (15), 89 (12), 57 (26). HRMS (ESI TOF-MS) calcd. for
[C22H22Br3NNaO3]þ: 607.9042, found 607.9052.
1486, 1411, 1390, 1239, 1137, 1046, 890, 806, 751 cmꢁ1
.
1H NMR
0.94 (d, 3H, J ¼ 6.6 Hz, H-9), 0.95 (d, 3H,
(300 MHz, CDCl3):
d
J ¼ 6.6 Hz, H-10), 2.19e2.36 (m, 1H, H-8), 3.07 (dd, 1H, J ¼ 13.8,
7.8 Hz, H-7a), 3.30 (d, 1H, J ¼ 15 Hz, H-6a), 3.41 (d, 1H, J ¼ 15 Hz, H-
6b), 3.50 (dd, 1H, J ¼ 13.8, 7.8 Hz, H-7b), 3.76 (s, 3H, eOCH3), 4.86
(s, 1H, eOH), 6.75 (d, 1H, J ¼ 9.0 Hz, H-30), 6.96 (ddd, 1H, J ¼ 7.8, 7.8,
1.8 Hz, H-400), 6.99 (d, 1H, J ¼ 2.4 Hz, H-60), 7.06 (ddd, 1H, J ¼ 7.8,
7.8, 1.5 Hz, H-500), 7.23 (dd, 1H, J ¼ 9.0, 2.4 Hz, H-40), 7.25 (dd, 1H,
J ¼ 7.8, 1.8 Hz, H-600), 7.41 (dd, 1H, J ¼ 7.8, 1.5 Hz, H-300). 13C NMR
4.2.3.4. 3-Bromo-5-(3-chlorobenzyl)-5-hydroxy-1-isobutyl-4-(5-
methyl-2-methoxyphenyl)pyrrol-2(5H)-one (11d). Compound 11d
was synthesized using a method similar to that of 11a and was
isolated as white solid in 84% yield, purified by column chroma-
tography, eluent hexane/dichloromethane (1:8 v/v). m.p. 140.0e
140.5 ꢂC. Rf ¼ 0.30 (hexane:dichloromethane, 1:8, v/v). IR (ATR)
nmax 3376e3226, 2957, 2928, 2869, 1691, 1678, 1638, 1500, 1434,
(75 MHz, CDCl3):
d 20.7 (C-9), 20.9 (C-10), 27.8 (C-8), 41.5 (C-6),
48.2 (C-7), 56.6 (eOCH3), 93.6 (C-5), 112.6 (C-30), 122.0 (C-3)*,
122.6 (C-10)*, 126.4 (C-200): 126.5 (C-50), 127.3 (C-500), 128.8 (C-400),
130.3 (C-600), 130.6 (C-60), 130.7 (C-40), 132.8 (C-300), 134.1 (C-100),
152.2 (C-20), 154.4 (C-4), 164.6 (C-2). *These assignments could be
ꢃ
ꢃ
reversed; MS, m/z (%) 529 ([Mþ6]þ e H2O, 3), 527 ([Mþ4]þ
e
1414, 1248, 1064, 1017, 882, 801, 785, 686, 656 cmꢁ1
(300 MHz, CDCl3):
.
1H NMR
0.96 (d, 3H, J ¼ 6.9 Hz, H-10), 0.99 (d, 3H,
H2O, 16), 525 ([Mþ2]þ e H2O, 24), 523 ([M]þꢃ e H2O, 10), 376 (17),
375 (16), 374 (100), 372 (73), 317 (22), 316 (16), 171 (10), 169 (14),
165 (12), 57 (18), 41 (41). HRMS (ESI TOF-MS) calcd. for
[C22H23Br2ClNO3]þ: 541.9728, found 541.9700.
ꢃ
d
J ¼ 6.9 Hz, H-9), 2.30 (s, 3H, AreCH3), 2.26e2.38 (m, 1H, H-8), 3.05
(s, 2H, H-6), 3.14 (dd, 1H, J ¼ 13.8, 7.2 Hz, H-7a), 3.55 (dd, 1H,
J ¼ 13.8, 8.1 Hz, H-7b), 3.78 (s, 3H, eOCH3), 5.37 (s, 1H, eOH), 6.75e
6.77 (m, 1H, H-200), 6.78 (d, 1H, J ¼ 9.0 Hz, H-30), 6.85 (ddd, 1H,
J ¼ 8.1, 1.5, 1.5 Hz, H-600), 6.91 (d, 1H, J ¼ 2.1 Hz, H-60), 7.05 (dd, 1H,
J ¼ 8.1, 8.1 Hz, H-500), 7.10 (ddd, 1H, J ¼ 8.1, 1.5, 1.5 Hz, H-400), 7.14 (dd,
4.2.3.7. 3-Bromo-4-(5-chloro-2-methoxyphenyl)-5-hydroxy-1-
isobutyl-5-(3-methoxybenzyl)pyrrol-2(5H)-one
(11g).
J ¼ 9.0, 2.1 Hz, H-40) 13C NMR (75 MHz, CDCl3):
d
20.6 (C-8), 20.7 (C-
Compound 11g was synthesized using a method similar to that of
11a and was isolated as white solid in 76% yield, purified by column
chromatography, eluent dichloromethane. m.p. 152.0e153.3 ꢂC.
Rf ¼ 0.20 (dichloromethane). IR (ATR) nmax 3187e3416, 3069, 3003,
2960, 2931, 2868, 2837, 1685, 1671, 1592, 1490, 1434, 1412, 1253,
9), 20.9 (C-10), 28.0 (AreCH3), 42.9 (C-6), 48.3 (C-7), 56.5 (eOCH3),
93.4 (C-5), 111.7 (C-30), 120.4 (C-3)**, 121.7 (C-10)**, 127.3 (C-400),
127.8 (C-600), 129.3 (C-500), 130.8 (C-60), 131.3 (C-50), 131.9 (C-40),
132.0 (C-200), 133.7 (C-300), 136.3 (C-100), 153.4 (C-20)*, 153.7 (C-4)*,
164.6 (C-2). *, **These assignments could be reversed. MS, m/z (%)
354 (99), 352 (100), 298 (18), 296 (19), 146 (11), 145 (17), 125 (21),
115 (13), 57 (27). HRMS (ESI TOF-MS) calcd. for [C23H26BrClNO3]þ:
478.0779, found 478.0798.
1104, 701, 655, 640 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 0.97 (d, 3H,
J ¼ 6.6 Hz, H-9), 0.98 (d, 3H, J ¼ 6.6 Hz, H-10), 2.25e2.39 (m, 1H, H-
8), 3.07 (d, 1H, J ¼ 14.7 Hz, H-6a), 3.14 (d, 1H, J ¼ 14.7 Hz, H-6b), 3.14
(dd, 1H, J ¼ 13.8, 7.2 Hz, H-7a), 3.56 (dd, 1H, J ¼ 13.8, 8.1 Hz, H-7b),
3.73 (s, 3H, eOCH3), 3.80 (s, 3H, eOCH3), 4.63 (s, 1H, eOH), 6.50 (s,
1H, H-200), 6.56 (d, 1H, J ¼ 7.8 Hz, H-400), 6.71 (m, 1H, H-600), 6.84 (d,
1H, J ¼ 8.7 Hz, H-30), 6.94 (d, 1H, J ¼ 2.4 Hz, H-60), 7.09 (dd, 1H,
J ¼ 7.8, 7.8 Hz, H-500), 7.30 (dd, 1H, J ¼ 8.7, 2.4 Hz, H-40). 13C NMR
4.2.3.5. 3-Bromo-5-(2-chlorobenzyl)-5-hydroxy-1-isobutyl-4-(5-
methyl-2-methoxyphenyl)pyrrol-2(5H)-one (11e). Compound 11e
was synthesized using a method similar to that of 11a and was
isolated as white solid in 84% yield, purified by column chroma-
tography, eluent hexane/dichloromethane (1:8 v/v). m.p. 128.3e
129.8 ꢂC. Rf ¼ 0.28 (hexane:dichloromethane, 1:8, v/v). IR (ATR)
nmax 3401e3174, 2959, 2928, 2668, 1689, 1675, 1411, 1385, 1244,
(75 MHz, CDCl3):
d 20.7 (C-9), 20.9 (C-10), 27.9 (C-8), 42.5 (C-6),
48.4 (C-7), 55.4 (eOCH3), 56.7 (eOCH3), 93.8 (C-5), 112.9 (C-30),
113.3 (C-600), 115.1 (C-200), 122.2 (C-400), 122.3 (C-3)*, 122.7 (C-10)*,
126.5 (C-50), 129.3 (C-500), 130.8 (C-40), 131.0 (C-60), 135.5 (C-100),
151.9 (C-20), 154.6 (C-300), 159.5 (C-4), 164.3 (C-2). *These assign-
ments could be reversed; MS, m/z (%) 479 ([Mþ4]þꢃ e H2O, 10), 477
1056, 806, 730, 686, 656, 620 cmꢁ1 1H NMR (300 MHz, CDCl3):
.
d
0.93 (d, 3H, J ¼ 6.9 Hz, H-9), 0.95 (d, 3H, J ¼ 6.9 Hz, H-10), 2.21 (s,
ꢃ
3H, AreCH3), 2.25e2.35 (m, 1H, H-8), 3.05 (dd, 1H, J ¼ 14.1, 7.2 Hz,
H-7a), 3.26 (d, 1H, J ¼ 15.0 Hz, H-6a), 3.35 (d, 1H, J ¼ 15.0 Hz, H-6b),
3.50 (dd, 1H, J ¼ 14.1, 7.8 Hz, H-7b), 3.77 (s, 3H, eOCH3), 5.22 (s, 1H,
eOH), 6.71e6.72 (m, 1H, H-60), 6.73 (d, 1H, J ¼ 8.7 Hz, H-30), 7.01e
([Mþ2]þ e H2O, 37), 475 ([M]þꢃ e H2O, 29), 396 (15), 375 (17), 374
(100), 373 (11), 372 (76), 340 (29), 318 (25), 316 (18), 122 (25), 121
(25), 91 (15), 57 (26), 41 (44). HRMS (ESI TOF-MS) calcd. for
[C23H26BrClNO4]þ: 494.0728, found 494.0837.

U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
135
4.2.4. General procedure for the synthesis of 12ae12g and 13ae
13g
synthesized using a method similar to that of 12a and 13a were
isolated in 28% and 31% yield, respectively.
Data for 12b: pale yellow solid, purified by column chroma-
tography, eluent hexane/dichloromethane (1:3 v/v). m.p. 120.2e
122.0 ꢂC. Rf ¼ 0.41 (hexane:dichloromethane,1:3, v/v). IR (ATR) nmax
3060, 2957, 2927, 2870, 1702, 1632, 1484, 1250, 1080, 1049, 1024,
4.2.4.1. Synthesis of (Z)-3-bromo-5-(4-trifluoromethylbenzylidene)-
1-isobutyl-4-(2-methoxyphenyl)pyrrol-2(5H)-one (12a) and (E)-3-
bromo-5-(4-trifluoromethylbenzylidene)-1-isobutyl-4-(2-
methoxyphenyl)pyrrol-2(5H)-one (13a). To a round-bottom flask
under nitrogen atmosphere were added 3-bromo-5-(4-
trifluoromethylbenzyl)-5-hydroxy-1-isobutyl-4-(2-
914, 810, 736, 688, 621 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 0.54 (d,
3H, J ¼ 6.9 Hz, H-9), 0.56 (d, 3H, J ¼ 6.9 Hz, H-10), 1.36e1.49 (m, 1H,
H-8), 3.47 (d, 2H, J ¼ 7.0 Hz, H-7), 3.82 (s, 3H, eOCH3), 5.99 (s,1H, H-
6), 6.90 (d, 1H, J ¼ 9.0 Hz, H-30), 7.13e7.16 (m, 1H, H-600), 7.26e7.27
(m, 1H, H-200), 7.29e7.31 (m, 2H, H-400 and H-500), 7.35 (d, 1H,
J ¼ 2.7 Hz, H-60), 7.55 (dd, 1H, J ¼ 9.0, 2.7 Hz, H-40). 13C NMR
methoxyphenyl)pyrrol-2(5H)-one 11a (250 mg, 0.502 mmol) dis-
solved in dry CHCl3 (20 mL) and p-toluenesulfonic acid (14 mg,
0.075 mmol). After reflux for 2 h, the reaction was diluted with
additional chloroform (20 mL) and quenched with saturated
aqueous NaHCO3 solution (3 ꢄ 20 mL) and washed with brine
(3 ꢄ 10 mL). The organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure. The resulting material was
purified by column chromatography on silica gel eluted with hex-
ane/dichloromethane (1:2.5 v/v). The procedure described afforded
compound 12a in 36% yield (87 mg, 0.18 mmol) and 13a in 39% yield
(95 mg, 0.20 mmol).
(75 MHz, CDCl3): d 19.6 (C-9),19.7 (C-10), 28.0 (C-8), 49.6 (C-7), 56.2
(eOCH3), 112.7 (C-50), 113.4 (C-6), 113.5 (C-30), 117.0 (C-3), 121.8 (C-
10), 127.7 (C-600), 128.3 (C-200)*, 129.5 (C-400)*, 129.6 (C-500)*, 133.5 (C-
60), 134.0 (C-40), 134.3 (C-300), 136.5 (C-100), 139.4 (C-5), 145.3 (C-4),
ꢃ
156.4 (C-20), 166.8 (C-2); MS, m/z (%) 529 ([Mþ6]þ , 6), 527
ꢃ
ꢃ
ꢃ
([Mþ4]þ , 32), 525 ([Mþ2]þ , 42), 523 ([M]þ , C22H20Br2ClNO2, 19),
446 (29), 444 (16), 403 (14), 402 (27), 401 (13), 400 (30), 390 (31),
388 (29), 125 (20), 114 (20), 102 (33), 101 (29), 100 (15), 94 (17), 57
(33). HRMS (ESI TOF-MS) calcd. for [C22H21Br2ClNO2]þ: 523.9622,
found 523.9589.
Data for 12a: pale yellow solid. m.p. 100.3e101.3 ꢂC. Rf ¼ 0.38
(hexane:dichloromethane, 1:3, v/v). IR (ATR) nmax 3050, 2958, 2921,
2850, 1698, 1615, 1491, 1455, 1407, 1322, 1254, 1161, 1120, 1065, 908,
Data for 13b: amorphous green solid, purified by column chro-
matography, eluent hexane/dichloromethane (1:3 v/v). Rf ¼ 0.31
(hexane:dichloromethane, 1:3, v/v). IR (ATR) nmax 2958, 2929, 2871,
1702, 1484, 1462, 1370, 1254, 1136, 1074, 1048, 1025, 915, 809, 740,
843, 751, 655 cmꢁ1 1H NMR (300 MHz, CDCl3):
. d 0.53 (d, 6H,
J ¼ 6.9 Hz, H-9 and H-10), 1.33e1.45 (m, 1H, H-8), 3.42 (dd, 1H,
J ¼ 14.1, 7.5 Hz, H-7a), 3.47 (dd, 1H, J ¼ 14.1, 7.5 Hz, H-7b), 3.84 (s,
3H, eOCH3), 6.07 (s,1H, H-6), 7.04 (dd, 1H, J ¼ 8.4, 0.9 Hz, H-30), 7.07
(ddd, 1H, J ¼ 7.5, 7.5, 0.9 Hz, H-50), 7.25 (dd, 1H, J ¼ 7.5, 1.8 Hz, H-60),
7.37 (d, 2H, J ¼ 8.1 Hz, H-200 and H-600), 7.47 (ddd, 1H, J ¼ 8.4, 7.5,
1.8 Hz, H-40), 7.61 (d, 2H, J ¼ 8.1 Hz, H-300 and H-500). 13C NMR
688, 621 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d
1.01 (d, 3H, J ¼ 6.6 Hz,
H-9), 1.02 (d, 3H, J ¼ 6.6 Hz, H-10), 2.07e2.21 (m, 1H, H-8), 3.59 (dd,
1H, J ¼ 14.4, 7.2 Hz, H-7a), 3.64 (s, 3H, eOCH3), 3.67 (dd,1H, J ¼ 14.4,
8.1 Hz, H-7b), 6.48 (s, 1H, H-6), 6.49 (d, 1H, J ¼ 8.7 Hz, H-30), 6.81e
6.84 (m, 2H, H-200 and H-600), 6.95e6.98 (m, 3H, H-60, H-400 and H-
500), 7.20 (dd, 1H, J ¼ 8.7, 2.4 Hz, H-40); 13C NMR (75 MHz, CDCl3):
(75 MHz, CDCl3): d 19.5 (C-9), 19.6 (C-10), 27.9 (C-8), 49.6 (C-7), 55.9
(eOCH3), 111.8 (C-30), 113.0 (C-6), 116.6 (C-3), 119.7 (C-10), 120.7 (C-
50), 124.1 (q, J ¼ 270 Hz, eCF3), 125.2 (q, J ¼ 3.7 Hz, C-300 and C-500),
129.9 (C-200 and C-600), 130.1 (q, J ¼ 32.5 Hz, C-400), 131.2 (C-60), 131.3
(C-40), 138.8 (C-100), 140.1 (C-5), 146.9 (C-4), 157.2 (C-20), 167.1 (C-2);
d
20.4 (C-9), 20.5 (C-10), 28.4 (C-8), 47.9 (C-7), 55.6 (eOCH3), 112.2
(C-30), 112.4 (C-50), 113.6 (C-6), 121.6 (C-3), 123.4 (C-10), 127.5 (C-600)
**, 127.6 (C-400)*, 128.6 (C-500)*, 129.6 (C-200)**, 132.6 (C-60)*, 133.1 (C-
40), 133.4 (C-300), 135.5 (C-100), 139.3 (C-5), 141.0 (C-4), 155.4 (C-20),
164.4 (C-2). *, **These assignments could be reversed; MS, m/z (%)
ꢃ
ꢃ
MS, m/z (%) 481 ([Mþ2]þ , 74), 479 ([M]þ , C23H21BrF3NO2, 74), 401
(14), 400 (51), 358 (15), 357 (57), 356 (100), 354 (24), 345 (17), 344
(80), 342 (15), 322 (23), 320 (23), 314 (19), 313 (24), 286 (14), 272
(15), 241 (15), 207 (42), 189 (16), 159 (51), 131 (28), 115 (31), 77 (38),
57 (20): 55 (21), 41 (97), 39 (40). HRMS (ESI TOF-MS) calcd. for
[C23H22BrF3NO2]þ: 480.0781, found 480.0834.
ꢃ
ꢃ
ꢃ
ꢃ
529 ([Mþ6]þ , 6), 527 ([Mþ4]þ , 29), 525 ([Mþ2]þ , 40), 523 ([M]þ
,
C
22H20Br2ClNO2, 17), 446 (27), 444 (13), 434 (15), 403 (14), 402 (27),
401 (12), 400 (28), 390 (29), 388 (26), 202 (13), 189 (15), 187 (14),
125 (18), 115 (14), 114 (18), 102 (34), 101 (26), 100 (15), 57 (36), 41
(100). HRMS (ESI TOF-MS) calcd. for [C22H21Br2ClNO2]þ: 523.9622,
found 523.9589.
Data for 13a: amorphous green solid. Rf ¼ 0.27 (hexane:di-
chloromethane, 1:3, v/v). IR (ATR) nmax 2960, 2929, 2873, 1704,
1614, 1490, 1463, 1322, 1163, 1122, 1089, 1066, 1018, 753 cmꢁ1. 1H
NMR (300 MHz, CDCl3):
d
1.02 (d, 3H, J ¼ 6.9 Hz, H-9); 1.03 (d, 3H,
4.2.4.3. (Z)-3-bromo-4-(5-bromo-2-methoxyphenyl)-5-(4-
bromobenzylidene)-1-isobutylpyrrol-2(5H)-one (12c) and (E)-3-
bromo-4-(5-bromo-2-methoxyphenyl)-5-(4-bromobenzylidene)-1-
isobutylpyrrol-2(5H)-one (13c). Compounds 12c and 13c were
synthesized using a method similar to that of 12a and 13a were
isolated in 23% and 33% yield, respectively.
J ¼ 6.9 Hz, H-10), 2.10e2.23 (m, 1H, H-8), 3.50 (s, 3H, eOCH3), 3.63
(dd, 1H, J ¼ 14.4, 7.5 Hz, H-7a), 3.69 (dd, 1H, J ¼ 14.4, 7.5 Hz, H-7b),
6.41 (dd, 1H, J ¼ 8.4, 0.9 Hz, H-30), 6.51 (s, 1H, H-6), 6.74 (ddd, 1H,
J ¼ 7.5, 7.5, 0.9 Hz, H-50), 6.93e6.96 (m, 3H, H-200, H-60 and H-600),
7.07e7.13 (m, 3H, H-300, H-40 and H-500). 13C NMR (75 MHz, CDCl3):
d
20.2 (C-9 and C-10), 28.2 (C-8), 47.6 (C-7), 54.9 (eOCH3), 110.1 (C-
Data for 12c: pale yellow solid, purified by column chromatog-
raphy, eluent hexane/dichloromethane (1:3 v/v). m.p. 191.6e
192.4 ꢂC. Rf ¼ 0.37 (hexane:dichloromethane,1:3, v/v). IR (ATR) nmax
3059, 2958, 2932, 2871, 1701, 1483, 1386, 1278, 1250, 1070, 1010,
30), 113.1 (C-6), 120.1 (C-50), 120.6 (C-3)*, 121.3 (C-10)*, 123.5 (q,
J ¼ 3.7 Hz, C-300 and C-500), 123.9 (q, J ¼ 270.6 Hz, eCF3), 129.0 (q,
J ¼ 32.3 Hz, C-400), 129.4 (C-200 and C-600), 130.4 (C-60), 130.5 (C-40),
137.3 (C-100), 139.5 (C-5), 142.3 (C-4), 155.8 (C-20), 164.5 (C-2).
913, 808, 736, 664, 620 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 0.54 (d,
ꢃ
*These assignments could be reversed; MS, m/z (%) 481 ([Mþ2]þ
,
3H, J ¼ 6.6 Hz, H-9), 0.55 (d, 3H, J ¼ 6.6 Hz, H-10), 1.36e1.50 (m, 1H,
H-8), 3.47 (d, 2H, J ¼ 7.5 Hz, H-7), 3.81 (s, 3H, eOCH3), 5.96 (s,1H, H-
6), 6.91 (d, 1H, J ¼ 8.7 Hz, H-30), 7.13 (d, 2H, J ¼ 8.7 Hz, H-200 and H-
600), 7.33 (d, 1H, J ¼ 2.4 Hz, H-60), 7.50 (d, 2H, J ¼ 8.7 Hz, H-300 and H-
500), 7.54 (dd, 1H, J ¼ 8.7, 2.4 Hz, H-40). 13C NMR (75 MHz, CDCl3):
ꢃ
43), 479 ([M]þ , C23H21BrF3NO2, 44), 400 (23), 357 (26), 356 (50),
344 (38), 159 (28), 77 (22), 57 (31), 55 (29), 41 (33), 41 (100). HRMS
(ESI TOF-MS) calcd. for [C23H22BrF3NO2]þ: 480.0781, found
480.0833.
d
19.6 (C-9), 19.7 (C-10), 27.9 (C-8), 49.7 (C-7), 56.2 (eOCH3), 112.7
4.2.4.2. (Z)-3-bromo-4-(5-bromo-2-methoxyphenyl)-5-(3-
chlorobenzylidene)-1-isobutylpyrrol-2(5H)-one (12b) and (E)-3-
bromo-4-(5-bromo-2-methoxyphenyl)-5-(3-chlorobenzylidene)-1-
isobutylpyrrol-2(5H)-one (13b). Compounds 12b and 13b were
(C-50), 113.5 (C-30), 113.8 (C-6), 116.9 (C-3), 121.9 (C-10), 122.4 (C-400),
131.2 (C-200 and C-600), 131.5 (C-300 and C-500), 133.5 (C-60), 133.6 (C-
100), 133.9 (C-40), 139.1 (C-5), 145.3 (C-4), 156.4 (C-20), 166.9 (C-2);
ꢃ
ꢃ
ꢃ
MS, m/z (%) 573 ([Mþ6]þ , 10), 571 ([Mþ4]þ , 29), 569 ([Mþ2]þ , 28),

136
U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
ꢃ
ꢃ
ꢃ
567 ([M]þ , C22H20Br3NO2, 9), 448 (19), 447 (19), 446 (32), 444 (15),
436 (18), 434 (38), 432 (22), 355 (25), 353 (31), 352 (27), 350 (23),
189 (17), 187 (15), 102 (37), 101 (26), 94 (21), 57 (32). HRMS (ESI
TOF-MS) calcd. for [C22H21Br3NO2]þ: 567.9117, found 567.9079.
Data for 13c: amorphous green solid, purified by column chro-
matography, eluent hexane/dichloromethane (1:3 v/v). Rf ¼ 0.26
(hexane:dichloromethane, 1:3, v/v). IR (ATR) nmax 2958, 2928, 2871,
1700, 1484, 1461, 1389, 1262, 1070, 1010, 912, 848, 801, 743,
could be reversed; MS, m/z (%) 463 ([Mþ4]þ , 27), 461 ([Mþ2]þ
,
ꢃ
100), 459 ([M]þ , C23H23BrClNO2, 74), 430 (18), 383 (18), 382 (39),
381 (27), 380 (72), 370 (22), 368 (23), 338 (25), 337 (39), 336 (71),
334 (29), 326 (28), 324 (87), 286 (31), 202 (20), 173 (34), 145 (25),
115 (42), 57 (31), 41 (99). HRMS (ESI TOF-MS) calcd. for
[C23H24BrClNO2]þ: 460.0673, found 460.0745.
4.2.4.5. (Z)-3-bromo-5-(2-chlorobenzylidene)-1-isobutyl-4-(5-
methyl-2-methoxyphenyl)pyrrol-2(5H)-one (12e) and (E)-3-bromo-
5-(2-chlorobenzylidene)-1-isobutyl-4-(5-methyl-2-methoxyphenyl)
pyrrol-2(5H)-one (13e). Compounds 12e and 13e were synthesized
using a method similar to that of 12a and 13a were isolated in 29%
and 39% yield, respectively.
Data for 12e: amorphous yellow solid, purified by column
chromatography, eluent hexane/dichloromethane (1:3.5 v/v).
Rf ¼ 0.34 (hexane:dichloromethane, 1:3.5, v/v). IR (ATR) nmax 3056,
2957, 2927, 2870, 1702, 1498, 1249, 1087, 1048, 1030, 938, 902, 809,
663 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d
0.99 (d, 3H, J ¼ 6.6 Hz, H-9),
1.01 (d, 3H, J ¼ 6.6 Hz, H-10), 2.07e2.21 (m, 1H, H-8), 3.56 (s, 3H, e
OCH3), 3.60 (dd, 1H, J ¼ 14.1, 7.8 Hz, H-7a), 3.67 (dd, 1H, J ¼ 14.1,
7.8 Hz, H-7b), 6.42 (d, 1H, J ¼ 9.0 Hz, H-30), 6.47 (s, 1H, H-6), 6.73 (d,
2H, J ¼ 8.1 Hz, H-200 and H-600), 6.98 (d, 1H, J ¼ 2.7 Hz, H-60), 7.05 (d,
2H, J ¼ 8.1 Hz, H-300 and H-500), 7.26 (dd, 1H, J ¼ 9.0, 2.7 Hz, H-40). 13C
NMR (75 MHz, CDCl3):
d 20.4 (C-9), 20.5 (C-10), 28.4 (C-8), 47.9 (C-
7), 55.7 (eOCH3), 112.3 (C-30), 112.4 (C-50), 114.1 (C-6), 121.3 (C-3),
121.9 (C-10), 123.5 (C-400), 130.4 (C-300 and C-500), 130.8 (C-200 and C-
600), 132.6 (C-100), 132.8 (C-40), 133.2 (C-60), 138.8 (C-5), 140.8 (C-4),
753, 735 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d
0.52 (d, 3H, J ¼ 6.9 Hz,
ꢃ
155.4 (C-20), 164.4 (C-2); MS, m/z (%) 573 ([Mþ6]þ , 9), 571
H-9), 0.54 (d, 3H, J ¼ 6.9 Hz, H-10), 1.37e1.51 (m, 1H, H-8), 2.34 (s,
3H, AreCH3), 3.36 (dd, 1H, J ¼ 14.1, 7.5 Hz, H-7a), 3.43 (dd, 1H,
J ¼ 14.1, 7.5 Hz, H-7b), 6.04 (s, 1H, H-6), 6.91 (d, 1H, J ¼ 8.4 Hz, H-30),
7.08 (d, 1H, J ¼ 2.4 Hz, H-60), 7.22 (dd, J ¼ 8.4, 2.4 Hz, H-40), 7.26e
7.28 (m, 3H, H-400, H-500 and H-600), 7.39 (m, 1H, H-300). 13C NMR
ꢃ
ꢃ
ꢃ
([Mþ4]þ , 28), 569 ([Mþ2]þ , 28), 567 ([M]þ , C22H20Br3NO2, 9), 447
(20), 446 (32), 444 (15), 436 (17), 434 (37), 432 (21), 400 (15), 355
(24), 353 (29), 352 (27), 350 (22), 202 (15), 189 (16), 115 (18), 114
(19), 102 (36), 101 (25), 100 (15), 89 (20), 57 (35), 41 (100). HRMS
(ESI TOF-MS) calcd. for [C22H21Br3NO2]þ: 567.9117, found 567.9085.
(75 MHz, CDCl3): d 19.7 (C-9), 19.8 (C-10), 20.7 (AreCH3), 27.9 (C-8),
49.4 (C-7), 56.0 (eOCH3), 111.7 (C-30), 112.2 (C-6), 116.0 (C-3), 119.5
(C-10), 126.3 (C-600)*, 129.5 (C-300)*, 129.6 (C-500)*, 130.0 (C-100), 131.7
(C-60)*, 131.8 (C-40)*, 131.8 (C-400)*, 133.7 (C-50), 134.2 (C-200), 139.5
(C-5), 146.9 (C-4), 155.2 (C-20), 167.1 (C-2). *These assignments
4.2.4.4. (Z)-3-bromo-5-(3-chlorobenzylidene)-1-isobutyl-4-(5-
methyl-2-methoxyphenyl)pyrrol-2(5H)-one (12d) and (E)-3-bromo-
5-(3-chlorobenzylidene)-1-isobutyl-4-(5-methyl-2-methoxyphenyl)
pyrrol-2(5H)-one (13d). Compounds 12d and 13d were synthesized
using a method similar to that of 12a and 13a were isolated in 35%
and 45% yield, respectively.
ꢃ
ꢃ
could be reversed; MS, m/z (%) 463 ([Mþ4]þ , 26), 461 ([Mþ2]þ
,
ꢃ
100), 459 ([M]þ , C23H23BrClNO2, 83), 383 (31), 382 (63), 381 (45),
380 (93), 370 (61), 368 (58), 337 (37), 336 (56), 324 (34), 289 (44),
287 (16), 286 (53), 173 (27), 116 (16), 115 (37), 102 (21), 101 (19), 57
(31). HRMS (ESI TOF-MS) calcd. for [C23H24BrClNO2]þ: 460.0673,
found 460.0604.
Data for 12d: yellow solid, purified by column chromatography,
eluent hexane/dichloromethane (1:4 v/v). m.p. 150.7e151.7 ꢂC.
Rf ¼ 0.35 (hexane:dichloromethane, 1:4, v/v). IR (ATR) nmax 3056,
2957, 2926, 2870, 2836, 1701, 1632, 1498, 1462, 1249, 1084, 1050,
Data for 13e: green solid, purified by column chromatography,
eluent hexane/dichloromethane (1:3.5 v/v). m.p. 123.0e124.2 ꢂC.
Rf ¼ 0.26 (hexane:dichloromethane, 1:3.5, v/v). IR (ATR) nmax
3054, 2959, 2927, 2871, 2835, 1702, 1499, 1402, 1264, 1246, 1090,
1029, 809, 736, 689 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 0.56 (d, 6H,
J ¼ 6.9 Hz, H-9 and H-10), 1.37e1.50 (m, 1H, H-8), 2.35 (s, 3H, Are
CH3), 3.45 (dd, 1H, J ¼ 14.1, 7.5 Hz, H-7a), 3.50 (dd, 1H, J ¼ 14.1,
7.5 Hz, H-7b), 3.80 (s, 3H, eOCH3), 6.02 (s, 1H, H-6), 6.92 (d, 1H,
J ¼ 8.4 Hz, H-30), 7.01 (d, 1H, J ¼ 1.8 Hz, H-60), 7.12e7.16 (m, 1H, H-
600), 7.22e7.30 (m, 4H, H-40, H-200, H-400 and H-500). 13C NMR
1048, 1013, 806, 747, 737, 689 cmꢁ1 1H NMR (300 MHz, CDCl3):
.
d
1.03 (d, 6H, J ¼ 6.6 Hz, H-9 and H-10), 2.11 (s, 3H, AreCH3), 2.14e
2.27 (m, 1H, H-8), 3.56 (s, 3H, eOCH3), 3.63 (dd, 1H, J ¼ 14.4, 7.5 Hz,
H-7a), 3.69 (dd, 1H, J ¼ 14.1, 7.5 Hz, H-7b), 6.35 (d,1H, J ¼ 8.4 Hz, H-
30), 6.48 (s, 1H, H-6), 6.52 (ddd, 1H, J ¼ 7.8, 7.8, 1.2 Hz, H-500), 6.66
(dd, 1H, J ¼ 7.8, 1.5 Hz, H-600), 6.74 (d, 1H, J ¼ 2.1 Hz, H-60), 6.84 (dd,
1H, J ¼ 8.4, 2.1 Hz, H-40), 6.89 (ddd, 1H, J ¼ 7.8, 7.8, 1.5 Hz, H-400),
(75 MHz, CDCl3): d 19.6 (C-9), 19.7 (C-10), 20.7 (AreCH3), 28.0 (C-8),
49.6 (C-7), 56.1 (eOCH3), 111.8 (C-30), 113.3 (C-6), 116.2 (C-3), 119.6
(C-10), 127.7 (C-600), 128.1 (C-400)*, 129.5 (C-200)*, 129.5 (C-40)*, 130.0
(C-50), 131.5 (C-60), 131.7 (C-500)*, 134.3 (C-300), 136.8 (C-100), 139.7 (C-
5), 147.1 (C-4), 155.1 (C-20), 167.2 (C-2). *These assignments could be
7.19 (dd, 1H, J ¼ 7.8, 1.2 Hz, H-300). 13C NMR (75 MHz, CDCl3):
d 20.3
ꢃ
ꢃ
reversed; MS, m/z (%) 463 ([Mþ4]þ , 27), 461 ([Mþ2]þ , 100), 459
(AreCH3), 20.5 (C-9 and C-10), 28.5 (C-8), 47.8 (C-7), 55.3 (e
OCH3), 110.1 (C-30), 112.8 (C-6), 120.4 (C-3), 121.0 (C-10), 125.2 (C-
500), 128.1 (C-300), 128.8 (C-400), 129.3 (C-100), 130.8 (C-40), 130.9 (C-
60), 131.9 (C-600), 132.7 (C-50), 133.8 (C-200), 139.2 (C-5), 142.7 (C-4),
ꢃ
([M]þ , C23H23BrClNO2, 74), 382 (39), 381 (27), 380 (72), 337 (39),
336 (71), 334 (29), 326 (28), 324 (87), 293 (21), 286 (31), 173 (34),
145 (25), 116 (20), 115 (42), 102 (24), 101 (24), 57 (31). HRMS (ESI
TOF-MS) calcd. for [C23H24BrClNO2]þ: 460.0673, found 460.0740.
Data for 13d: amorphous green solid, purified by column chro-
matography, eluent hexane/dichloromethane (1:4 v/v). Rf ¼ 0.26
(hexane:dichloromethane, 1:4, v/v). IR (ATR) nmax 2958, 2926, 2872,
ꢃ
154.2 (C-20), 164.7 (C-2); MS, m/z (%) 463 ([Mþ4]þ , 16), 461
ꢃ
ꢃ
([Mþ2]þ , 72), 459 ([M]þ , C23H23BrClNO2, 56), 383 (27), 382 (52),
381 (40), 380 (71), 370 (50), 368 (48), 338 (18), 336 (43), 324 (29),
287 (15), 286 (51), 202 (30), 115 (53), 102 (29), 101 (29), 57 (36), 41
(100). HRMS (ESI TOF-MS) calcd. for [C23H24BrClNO2]þ: 460.0673,
found 460.0629.
1706, 1631, 1500, 1464, 1251, 1087, 1051, 1030, 810, 690 cmꢁ1
NMR (300 MHz, CDCl3):
J ¼ 6.6 Hz, H-10), 2.11 (s, 3H, AreCH3), 2.14 (m, 1H, H-8), 3.59 (s, 3H,
eOCH3), 3.59 (dd, 1H, J ¼ 14.1, 7.2 Hz, H-7a), 3.67 (dd, 1H, J ¼ 14.1,
7.8 Hz, H-7b), 6.45 (s, 1H, H-6), 6.47 (d, 1H, J ¼ 8.4 Hz, H-30), 6.69e
6.94 (m, 6H, H-40, H-60, H-200, H-400, H-500 and H-600). 13C NMR
.
1H
1.00 (d, 3H, J ¼ 6.6 Hz, H-9), 1.02 (d, 3H,
d
4.2.4.6. (Z)-3-bromo-5-(2-bromobenzylidene)-4-(5-chloro-2-
methoxyphenyl)-1-isobutylpyrrol-2(5H)-one (12f) and (E)-3-bromo-
5-(2-bromobenzylidene)-4-(5-chloro-2-methoxyphenyl)-1-
isobutylpyrrol-2(5H)-one (13f). Compounds 12f and 13f were syn-
thesized using a method similar to that of 12a and 13a were iso-
lated in 29% and 43% yield, respectively.
(75 MHz, CDCl3):
d 20.4 (AreCH3), 20.5 (C-9 and C-10), 28.5 (C-8),
47.8 (C-7), 55.4 (eOCH3), 110.4 (C-30), 113.6 (C-6), 120.6 (C-3), 121.0
(C-10), 127.2 (C-60)*, 127.4 (C-600)*, 128.3 (C-400)*, 129.6 (C-50), 129.7
(C-200)*, 130.6 (C-40)*, 131.0 (C-500)*, 133.1 (C-300), 135.7 (C-100), 139.5
(C-5), 142.7 (C-4), 154.1 (C-20), 164.7 (C-2). *These assignments
Data for 12f: pale yellow solid, purified by column chromatog-
raphy, eluent hexane/dichloromethane (1:2 v/v). m.p. 162.7e

U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
137
164.2 ꢂC. Rf ¼ 0.22 (hexane:dichloromethane, 1:2, v/v). IR (ATR)
nmax 3058, 2959, 2871, 1706, 1643, 1486, 1463, 1281, 1251, 1026, 919,
(24), 55 (21). HRMS (ESI TOF-MS) calcd. for [C23H24BrClNO3]þ:
476.0623, found 476.0547.
811, 755, 670 cmꢁ1
.
1H NMR (300 MHz, CDCl3):
d
0.52 (d, 3H,
Data for 13g: green solid, purified by column chromatography,
eluent hexane/dichloromethane (1:6 v/v). m.p. 92.4e94.7 oC.
Rf ¼ 0.15 (hexane:dichloromethane, 1:6, v/v). IR (ATR) nmax 3002,
2958, 2933, 2872, 2835, 1698, 1576, 1486, 1462, 1399, 1325, 1268,
J ¼ 6.9 Hz, H-9), 0.54 (d, 3H, J ¼ 6.9 Hz, H-10), 1.36e1.50 (m, 1H, H-
8), 3.35 (dd, 1H, J ¼ 14.1, 7.5 Hz, H-7a), 3.42 (dd, 1H, J ¼ 14.1, 7.5 Hz,
H-7b), 3.83 (s, 3H, eOCH3), 5.98 (s, 1H, H-6), 6.96 (d, 1H, J ¼ 8.7 Hz,
H-30), 7.20 (ddd, 1H, J ¼ 8.1, 6.9, 2.4 Hz, H-500), 7.29 (d, 1H, J ¼ 2.7 Hz,
H-60), 7.27e7.36 (m, 2H, H-300 and H-400), 7.41 (dd, 1H, J ¼ 8.7, 2.7 Hz,
H-40), 7.59 (dd, 1H, J ¼ 8.1, 1.2 Hz, H-600). 13C NMR (75 MHz, CDCl3):
1158, 1088, 863, 733 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 1.00 (d, 3H,
J ¼ 6.6 Hz, H-9), 1.01 (d, 3H, J ¼ 6.6 Hz, H-10), 2.08e2.22 (m, 1H, H-
8), 3.56 (s, 3H, eOCH3), 3.60 (dd, 1H, J ¼ 14.0, 7.0 Hz, H-7a), 3.61 (s,
3H, eOCH3), 3.68 (dd, 1H, J ¼ 14.0, 7.0 Hz, H-7b), 6.38 (m, 1H, H-200),
6.44 (d,1H, J ¼ 8.7 Hz, H-30), 6.50e6.56 (m, 2H, H-400 and H-600), 6.56
(s,1H, H-6), 6.90 (d, 1H, J ¼ 2.7 Hz, H-60), 6.91 (dd,1H, J ¼ 8.0, 8.0 Hz,
H-500), 7.03 (dd, 1H, J ¼ 8.7, 2.7 Hz, H-40). 13C NMR (75 MHz, CDCl3):
d
19.7 (C-9), 19.9 (C-10), 28.0 (C-8), 49.5 (C-7), 56.3 (eOCH3), 113.0
(C-30), 114.4 (C-6), 116.8 (C-3), 121.2 (C-10), 124.5 (C-200), 125.6 (C-50),
127.0 (C-400)*, 129.9 (C-500), 131.0 (C-40), 131.0 (C-60)*, 131.7 (C-300)*,
132.7 (C-600), 135.3 (C-100), 138.8 (C-5), 145.2 (C-4), 155.9 (C-20), 166.7
(C-2). *These assignments could be reversed; MS, m/z (%) 529
d
20.4 (C-9), 20.5 (C-10), 28.5 (C-8), 47.8 (C-7), 55.2 (eOCH3), 55.7
ꢃ
ꢃ
ꢃ
ꢃ
([Mþ6]þ , 9), 527 ([Mþ4]þ , 53), 525 ([Mþ2]þ , 70), 523 ([M]þ
,
(eOCH3), 111.6 (C-30), 113.6 (C-400)*, 114.9 (C-200), 115.7 (C-6), 121.0
(C-3), 121.9 (C-600)*, 123.4 (C-50), 125.1 (C-10), 128.4 (C-60)**, 129.8
(C-40), 130.1 (C-500)**, 135.0 (C-100), 138.4 (C-5), 141.2 (C-4), 155.0 (C-
20), 158.8 (C-300), 164.4 (C-2). *, **These assignments could be
C
22H20Br2ClNO2, 31), 446 (27), 404 (23), 403 (36), 402 (59), 401
(30), 400 (42), 390 (54), 388 (42), 309 (42), 306 (47), 189 (15), 102
(21), 101 (19), 57 (24), 55 (23). HRMS (ESI TOF-MS) calcd. for
[C22H21Br2ClNO2]þ: 523.9622, found 523.9599.
ꢃ
ꢃ
reversed; MS, m/z (%) 479 ([Mþ4]þ , 26), 477 ([Mþ2]þ , 100), 475
ꢃ
Data for 13f: green solid, purified by column chromatography,
eluent hexane/dichloromethane (1:2 v/v). m.p. 140.7e142.4 ꢂC.
Rf ¼ 0.16 (hexane:dichloromethane, 1:2, v/v). IR (ATR) nmax 3061,
2959, 2929, 2872, 1699, 1628, 1485, 1462, 1399, 1261, 1245, 1088,
([M]þ , C23H23BrClNO3, 76), 434 (16), 421 (16), 398 (15), 396 (40),
354 (35), 353 (34), 352 (49), 341 (20), 340 (85), 338 (25), 309 (26),
57 (23), 43 (25), 41 (88). HRMS (ESI TOF-MS) calcd. for
[C23H24BrClNO3]þ: 476.0623, found 476.0583.
1024, 912, 742, 710, 668 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d 1.03 (d,
6H, J ¼ 6.6 Hz, H-9 and H-10), 2.22 (m,1H, H-8), 3.58 (s, 3H, eOCH3),
3.67 (d, 2H, J ¼ 7.5 Hz, H-7), 6.36 (d, 1H, J ¼ 8.7 Hz, H-30), 6.46 (s, 1H,
H-6), 6.63e6.73 (m, 2H, H-500 and H-600), 6.84e6.89 (m, 1H, H-400),
6.98 (d, 1H, J ¼ 2.7 Hz, H-60), 7.02 (dd, 1H, J ¼ 8.7, 2.7 Hz, H-40), 7.42
4.2.5. Bacterial growth biofilm inhibition assays
Biofilm production and quantifications were performed as
described previously [45]. In brief, polystyrene 96-well microtiter
plates were inoculated with bacterial suspension prepared at
5 ꢄ 108 UFC/mL in TSB supplemented with 4% sucrose (w/v) and
3.5% (v/v) DMSO, to ensure that the concentration of this solvent
remained constant as the tested molecules were sequentially
diluted for the inhibition assays. Previous results indicated that at
this concentration, DMSO is innocuous both to bacterial growth
and biofilm formation (data not shown). Compounds to be tested
for antibiofilm activity were added at an initial concentration of
(dd, 1H, J ¼ 7.5, 1.2 Hz, H-300). 13C NMR (75 MHz, CDCl3):
d 20.6 (C-9
and C-10), 28.5 (C-8), 47.9 (C-7), 55.8 (eOCH3), 111.4 (C-30), 115.1 (C-
6), 121.2 (C-3), 122.9 (C-10), 124.4 (C-200), 125.1 (C-50), 125.9 (C-600),
129.3 (C-400), 130.0 (C-40), 130.2 (C-60), 131.6 (C-300), 131.9 (C-500),
134.2 (C-100), 138.7 (C-5), 141.2 (C-4), 154.8 (C-20), 164.4 (C-2); MS,
ꢃ
ꢃ
ꢃ
m/z (%) 529 ([Mþ6]þ , 15), 527 ([Mþ4]þ , 70), 525 ([Mþ2]þ , 99), 523
ꢃ
([M]þ , C22H20Br2ClNO2, 43), 446 (36), 404 (28), 403 (52), 402 (76),
400 (54), 392 (19), 391 (16), 390 (71), 389 (16), 388 (57), 356 (25),
354 (20), 309 (58), 306 (58), 102 (23), 101 (26), 57 (25), 41 (100).
HRMS (ESI TOF-MS) calcd. for [C22H21Br2ClNO2]þ: 523.9622, found
523.9576.
87.5
tration of 0.17
m
g/mL, and serially diluted at 1:2 ratio until a final concen-
g/mL was reached. Microtiter plates were incu-
m
bated for 20 h at 37 ꢂC in a humidified chamber, and bacterial
growth was quantified by absorbance at 630 nm readings using a
microtiter plate reader device, to assess effects on bacterial
growth prior to biofilm quantifications. Bacterial suspensions
were then discarded, and after washing, remaining biofilms were
stained with a 0.1% (w/v) crystal violet solution, washed and
solubilized in 1% sodium dodecyl sulphate (SDS). Biofilm quanti-
fication was assessed by measuring the absorbance at 595 nm
using a microtiter plate reader device. Each assay was performed
in triplicate. Data were transferred into a graphics program (Ori-
ginPro8), sigmoidal doseeresponse curves determined, and IC50
values calculated.
4.2.4.7. (Z)-3-bromo-4-(5-chloro-2-methoxyphenyl)-1-isobutyl-5-
(3-methoxybenzylidene)pyrrol-2(5H)-one (12g) and (E)-3-bromo-4-
(5-chloro-2-methoxyphenyl)-1-isobutyl-5-(3-methoxybenzylidene)
pyrrol-2(5H)-one (13g). Compounds 12g and 13g were synthesized
using a method similar to that of 12a and 13a were isolated in 41%
and 35% yield, respectively.
Data for 12g: amorphous green solid, purified by column chro-
matography, eluent hexane/dichloromethane (1:6 v/v). Rf ¼ 0.22
(hexane:dichloromethane, 1:6, v/v). IR (ATR) nmax 3066, 3002, 2957,
2871, 2838, 1702, 1595, 1485, 1459, 1272, 1250, 1158, 1046, 920, 812,
700, 643 cmꢁ1. 1H NMR (300 MHz, CDCl3):
d
0.53 (d, 3H, J ¼ 6.6 Hz,
H-9), 0.55 (d, 3H, J ¼ 6.6 Hz, H-10), 1.41e1.55 (m, 1H, H-8), 3.52 (d,
2H, J ¼ 7.5 Hz, H-7), 3.81 (s, 3H, eOCH3), 3.82 (s, 3H, eOCH3), 6.01 (s,
1H, H-6), 6.77e6.78 (m, 1H, H-200), 6.83e6.88 (m, 2H, H-400 and H-
600), 6.96 (d, 1H, J ¼ 9.0 Hz, H-30), 7.23 (d, 1H, J ¼ 3.0 Hz, H-60), 7.27
(dd, 1H, J ¼ 8.0, 8.0 Hz, H-500), 7.40 (dd, 1H, J ¼ 9.0, 3.0 Hz, H-40). 13C
Acknowledgements
We are grateful to the following Brazilian agencies: Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for
research fellowships (AJD, CRAM, LCAB, ALP), Fundação de Amparo
NMR (75 MHz, CDCl3):
d 16.7 (C-9), 19.7 (C-10), 28.0 (C-8), 49.7 (C-
à
Pesquisa de Minas Gerais (FAPEMIG-APQ-00276-13), Coor-
7), 55.6 (eOCH3), 56.3 (eOCH3), 113.0 (C-30), 114.0 (C-400), 115.0 (C-
200), 115.3 (C-6), 116.6 (C-3), 121.6 (C-10), 122.1 (C-600), 125.6 (C-50),
129.4 (C-500), 130.8 (C-40), 130.8 (C-60), 135.9 (C-100), 138.7 (C-5),
145.4 (C-4), 155.9 (C-20), 159.5 (C-300), 166.9 (C-2). *These assign-
denação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
and FINEP for financial support.
ꢃ
ments could be reversed; MS, m/z (%) 479 ([Mþ4]þ , 26), 477
Appendix A. Supplementary data
ꢃ
ꢃ
([Mþ2]þ , 100), 475 ([M]þ , C23H23BrClNO3, 78), 398 (16), 396 (43),
356 (17), 355 (16), 354 (37), 353 (36), 352 (50), 342 (24), 341 (21),
340 (88), 338 (24), 309 (25), 149 (14), 148 (15), 121 (23), 102 (18), 57
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.035.

138
U.A. Pereira et al. / European Journal of Medicinal Chemistry 82 (2014) 127e138
[25] T. Yamada, E. Imai, K. Nakatuji, A. Numada, R. Tanaka, Cephalimysin A, a
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