Synthesis and antibacterial activities of cadiolides A, B and C and analogues

Article abstract of DOI:10.1016/j.bmc.2015.04.010

The one-pot multicomponent synthesis of natural butenolides named cadiolides A, B, C and analogues has been realized. The antibacterial structure activity relationship shows that the presence of phenolic hydroxyl groups and the number and position of bromine atoms on the different aromatic rings are important features for antibacterial activity, besides it was demonstrated the tolerance of both benzene and furan ring at position 3 of the butenolide nucleus. Furthermore, none of the most relevant antibacterial compounds showed any cytotoxicity in freshly isolated human neutrophils.

Full text of DOI:10.1016/j.bmc.2015.04.010

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antibacterial activities of cadiolides A, B and C
and analogues
Agathe Boulangé a, Javier Parraga b, Abraham Galán b, Nuria Cabedo b,c, Stéphane Leleu a,
Maria Jesus Sanz d,e, Diego Cortes b, Xavier Franck a,
⇑
a Normandie Univ, COBRA, UMR 6014 et FR 3038, CNRS, Univ Rouen, INSA Rouen, 1 rue Tesnières, 76821 Mont-Saint-Aignan, Cedex, France
b Departamento de Farmacología, Facultad de Farmacia, Universidad de Valencia, 46100 Burjassot Valencia, Spain
c Centro de Ecología Química Agrícola-Instituto Agroforestal Mediterraneo, UPV, Campus de Vera, Edificio 6C, 46022 Valencia, Spain
d Departamento de Farmacología, Facultad de Medicina, Universidad de Valencia, 46010 Valencia, Spain
e Institute of Health Research-INCLIVA, University Clinic Hospital of Valencia, 46010 Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The one-pot multicomponent synthesis of natural butenolides named cadiolides A, B, C and analogues has
been realized. The antibacterial structure activity relationship shows that the presence of phenolic hydro-
xyl groups and the number and position of bromine atoms on the different aromatic rings are important
features for antibacterial activity, besides it was demonstrated the tolerance of both benzene and furan
ring at position 3 of the butenolide nucleus. Furthermore, none of the most relevant antibacterial com-
pounds showed any cytotoxicity in freshly isolated human neutrophils.
Received 5 March 2015
Revised 3 April 2015
Accepted 4 April 2015
Available online xxxx
Keywords:
Cadiolides
Ó 2015 Elsevier Ltd. All rights reserved.
Antibacterial
Butenolides
Multicomponent
Natural products
1. Introduction
OH
R6
R5
O
O
Natural products are a rich source of chemical diversity by con-
stantly bringing new skeletons to light. This is highly demanded for
the development of new drugs and particularly for antibacterial
compounds due to worldwide emergence of multidrug resistance.
The development of resistance to classic antibiotics has emerged
from antibiotic use for both veterinary medicine and human med-
icine. This reduces the effectiveness of current treatments for
infections and becomes a major threat for public health causing a
large number of sick people, hospitalizations and deaths.1
Because of that, new antibacterial molecules are actively sought
and this encourages us to devote our research efforts to find new
active agents.2
O
R3
O
R1
HO
OH
O
R3
R1
HO
R4
OH
R2
R4
R2
cadiolides
rubrolides
R1, R2, R3, R4, R5, R6 = H or Br
Figure 1. General structures of cadiolides and rubrolides.
Marine butenolides of the cadiolide family, isolated from ascidi-
ans or tunicates, have shown wide spectrum antibacterial activi-
Some members of the cadiolide family, and especially cadiolide
E, have shown potent inhibition of Candida albicans isocitrate lyase
activity.3d However, the structure activity relationship of these
natural compounds has not yet been established and may be
function of the number and position of bromine atoms on the
phenolic rings. We recently developed a one pot multicomponent
procedure5 for the preparation of substituted acylfuranones by
condensation of a hydroxyketone 1, a functionalized dioxinone 2
and an aldehyde 3 and now report on the application of this
methodology to the synthesis of various cadiolide analogues5d,e
ties.3 They are characterized by
a tris-aromatic furanone
skeleton, where aryl groups are phenols bearing usually one or
two bromine atoms. Simpler related bis-aromatic furanones have
also been isolated from marine organisms and named rubrolides
(Fig. 1).3b,4
⇑
Corresponding author. Tel.: +33 (0)235522459; fax: +33 (0)235522959.
E-mail address: xavier.franck@insa-rouen.fr (X. Franck).
http://dx.doi.org/10.1016/j.bmc.2015.04.010
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
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2
A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Various methoxy-furanones 4 were then prepared by reaction
O
R5
of hydroxyketone 1a–c with dioxinone 2a–d and aldehyde 3a–c
at 150 °C under microwave irradiation (5 min), in the presence of
triethylamine and molecular sieves. Crude reaction mixtures were
then subsequently demethylated with BBr3 to free the phenols and
give furanones 5–10. Yields of 4 usually ranged from 40 to 70% and
debromination gave usually good yields of phenols 5–10
(Scheme 2, Table 1). Among them, three natural products were pre-
pared: cadiolide A (7i), cadiolide B (7g) and cadiolide C (6f).
O
O
R6
R3
R2
O
OH
O
O
Et3N, Toluene,
molecular sieves
O
1
R1
R4
microwave,
150°C, 5 min
4
furanone
- H2O
O
O
R2
R5
R1
O
O
2
O
O
R3
O
R6
H
via thermal
3
fragmentation of 2
1
2
+
2. Bactericidal activity
into acylketene
R4
Et3N
O
O
A total of 16 substituted furanones 5–10, including the three
natural cadiolides A, B and C, and 14 methoxy-furanones 4 were
screened in vitro for their antibacterial activity against several
human pathogenic bacteria by determination of their minimum
inhibitory concentrations (MICs, the lowest concentration of com-
pounds capable of inhibiting the growth of bacteria) and using the
2-fold microtiter method (Table 1, Fig. 3). 7 Compounds were
evaluated against four Gram-positive bacterial strains (Bacillus cer-
eus, Bacillus subtilis, Staphylococcus aureus and Enterococcus faecalis)
and four Gram-negative (Salmonella typhi, Escherichia coli 100,
E. coli 405 and Erwinia carotovora). Interestingly, compounds in
which the unsaturated butenolide ring is bearing phenolic hydro-
xyl groups (furanones 5–10) displayed moderate to significant
antibacterial activity against both Gram-positive and Gram-nega-
tive strains tested, whereas their methoxy-furanone analogues 4
were completely inactive at the highest concentration tested
O
R5
R6
R5
O
O
R6
O
O
Et3N
O
O
- H2O
R2
R2
R1
O
O
R1
R1, R2, R3, R4, R5, R6 = H or Br
Scheme 1. Approach to cadiolide analogues.
and their antibacterial activities. The versatility of this method
allowed us to easily vary the number and position of bromine
atoms on the different aromatic rings by preparing different
brominated substrates 1, 2 and 3 (Scheme 1).
Different aromatic substrates (Fig. 2) have then been prepared
by mono bromination of the pOMe- or dibromination of pOH-sub-
(125 lg/mL, results not shown). Therefore, the presence of pheno-
lic hydroxyl groups is crucial for antibacterial activity, probably
due to their ability to interact with proteins and bacterial cell walls
to form complexes.8 The importance of ionizable phenolic hydroxyl
groups to enhance bactericidal activity has been earlier reported.9
It seems that the phenolic protons can be replaced by another ion
(potassium ion or other cation) causing an efflux of cations and
influx of protons to the cytoplasm.
Among phenolic analogues, the most potent compounds were
the synthetic brominated tris-aromatic furanones 6c, 7c and 9,
with a MIC value of 1.95 lg/mL against the following strains
tested: B. cereus, S. aureus, E. faecalis, S. typhi and E. coli 405
(Fig. 3, Table 1). The structure-activity relationship (SAR) studies
highlighted the importance of both number and location of bro-
mine atoms. Therefore, our initial studies focused on cadiolide ana-
logues bearing a benzoyl substituent attached to position 3 of
furanone core showed that 5a analogue devoid of any bromine
atom displayed moderate to low activity (MIC from 62.5 to
stituted
aromatic
ring
using
conventional
methods.6a
Hydroxyketones 1a–1c have been prepared by bromination of
the corresponding acetophenone followed by treatment with
sodium formate.6b Dioxinones 2a–d have been synthesized from
the corresponding b-ketoesters using our described procedure.5
Aldehyde 3a–b are commercially available and 3c has been pre-
pared by bromination of pOH-benzaldehyde. Heteroaromatic
dioxinone 2d has also been used in order to determine whether
non phenolic substituents were tolerated for biological activity.
1
2
3
aldehydes
hydroxyketones
dioxinones
O
O
O
O
125 lg/mL). In addition, antibacterial activity dramatically
OH
CHO
decreased when a single bromine was added on the benzylidene
substituent attached to position 5 (compound 5b). Low activity
was also displayed for compound 5c, with only two bromine atoms
located on benzylidene and benzoyl substituents. However, the
antibacterial activity was increased when the two bromine atoms
were located one on the benzylidene and one on the phenyl ring,
O
O
1a
2a
3a
MeO
MeO
MeO
MeO
O
O
O
CHO
OH
OH
1b
1c
2b
3b
MeO
MeO
as compound 6b with MICs ranging from 15.62 to 31.25 lg/mL
Br
Br
Br
Br
Br
Br
for B. cereus, S. aureus, E. faecalis, S. typhi and E. coli 405.
However, adding a third bromine atom led to one of the most
potent compounds 6c when a bromine atom was located on each
aryl ring. Cadiolide analogues with four bromine atoms resulted
in two possible groups of compounds depending on the location
of bromine atoms on the aryl rings. The first group had two bro-
mine atoms in one of the aryl rings and a single bromine on each
of the other two aryl rings as it occurs for 6e, 6f (cadiolide C)
and 7c. Results showed that when the two bromine atoms were
on phenyl or benzylidene substituent (6e and 7), higher potency
was obtained than when two bromines were on the benzoyl
O
O
O
Br
Br
Br
CHO
O
MeO
2c
3c
MeO
MeO
O
O
O
2d
Figure 2. Set of hydroxyketones, dioxinones and aldehydes.
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A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
O
O
R3
R1
O
OH
R3
Ar
O
O
O
R3
O
Ar
Et3N, Toluene,
OH
R4
O
O
molecular sieves
H
R4
BBr3
1a-c
O
R2
O
microwave, 150°C, 5 min
O
R1
3a-c
R4
R2
R1
O
O
furanone 5-10
4
R2
methoxy furanones
HO
13-72% (2 steps)
Ar
O
2a-d
O
O
O
R3
R3
R3
O
O
O
R3
R3
R3
Ar
Ar
Ar
O
O
O
O
OH
R4
O
OH
R4
O
OH
R4
O
OH
R4
O
OH
R4
O
OH
R4
O
O
O
O
O
O
H
Br
H
Br
Br
Br
Br
Br
HO
HO
HO
HO
R3= R4=H
HO
HO
10
R3=H, R4=Br
5a-d
R3, R4=H or Br
6b-f
R3, R4=H or Br
7c-i
R3, R4=H or Br
8
9
R3=H, R4=Br
Scheme 2. Multicomponent reaction.
Table 1
Antibacterial activities
MIC (l
g mLꢀ1) 24 ha
Furanone 5–10
B. cereus
S. aureus
E. faecalis
S. typhi
E. coli 405
E. carotovora
B. subtilis
E. coli 100
5a
5b
5c
5d
62.5
125.0
3.90
15.62
15.62
1.95
1.95
3.90
1.95
3.90
7.81
3.90
7.81
15.62
1.95
3.90
3.90
62.5
>125
>125.0
15.62
31.25
1.95
3.90
3.90
1.95
3.90
7.81
3.90
7.81
15.62
1.95
1.95
0.24
125.0
>125
>125.0
15.62
31.25
1.95
1.95
3.90
1.95
3.90
7.81
3.90
7.81
31.25
1.95
125.0
>125
125
62.5
>125
125
125.0
>125
>125
125.0
125
125.0
125.0
125.0
125.0
125.0
>125
125.0
125.0
>125
125.0
125.0
3.90
>125.0
>125
>125
125.0
125
125.0
125.0
125.0
125.0
125.0
>125
125.0
125.0
>125
125.0
125.0
0.97
62.5
>125
>125
>125
15.62
>125
>125
>125
>125
>125
>125
>125
>125
31.25
15.62
15.62
3.90
15.62
31.25
1.95
3.90
3.90
1.95
3.90
15.62
3.90
7.81
15.62
1.95
1.95
7.81
15.62
15.62
1.95
3.90
3.90
1.95
1.95
7.81
7.81
7.81
31.25
1.95
3.90
7.81
6b
6cb
6e
6f (cadiolide C)
7cb
7e
7g (cadiolide B)
7h
7i (cadiolide A)
8
9b
10b
1.95
0.48
Tetracycline
a
Doses tested were 125, 62.5, 31.25, 15.62, 7.81, 3.9, 1.95, 0.97, 0.48 and 0.24 lg/mL. Gram-positive: Bacillus cereus (CECT 148), Staphylococcus aureus (CECT 86),
Enterococcus faecalis (CECT 481) and Bacillus subtilis (CECT 35); and four Gram-negative: Salmonella typhi (CECT 409), Escherichia coli 100 (CECT 100), E. coli 405 (CECT 405),
and Erwinia carotovora (CECT 225).
b
For 6c, IC50 = 1.95
M; IC50 = 15.62
M; IC50 = 125 g/mL = 204.57 l
l
g/mL = 3.06
l
M; IC50 = 125
M; IC50 = 125
M.
l
g/mL = 196.21
l
M; for 7c, IC50 = 1.95
l
g/mL = 2.72
l
M; IC50 = 125
l
g/mL = 174.59
l
M; for 9, IC50 = 1.95
l
l
g/
g/
mL = 3.66
mL = 25.56
l
l
lg/mL = 29.35
l
l
g/mL = 234.90
lM; for 10, IC50 = 1.95
l
g/mL = 3.19
lM; IC50 = 3.90
lg/mL = 6.38 l
M; IC50 = 15.62
l
substituent (6f, cadiolide C). The second group had two bromine
atoms on two aryl rings but the third aryl ring did not have any
bromine, like in compounds 5d, 7i (cadiolide A) and 7h. Although
the activity of this second group of compounds slightly decreased,
they followed the same trend, since 7h and 7i (cadiolide A) with
the two bromine atoms on phenyl or benzylidene substituent dis-
played higher potency than on benzoyl substituent (5d). A high
antibacterial activity was also obtained when a fifth bromine atom
was added to yield compound 7e, with MICs ranging from 1.95 to
one on the benzylidene and one on the phenyl ring (compare 9
vs 6b). In addition, the presence of a third bromine did not seem
to affect the activity since compound 10 displayed the same MIC
values than compound 9, which were one of the most potent com-
pounds of this series. Therefore, the replacement of benzoyl by fur-
oyl moiety proved to be advantageous for antibacterial activity in
cadiolide analogues.
In comparison with tetracycline, it was noteworthy that eight of
the butenolides tested including cadiolide C were able to inhibit
the bacterial growth against B. cereus, S. typhi and E. coli 405 with
MIC values lower or in the same order than the reference antibio-
3.90
when a sixth bromine was added to give natural product 7g (cadio-
lide B) (MICs ranging from 7.81 to 15.62 g/mL), even though two
lg/mL. However, the antibacterial activity slightly decreased
l
tic. For instance: 6c, 7c and 9 with MIC of 1.95
with MICs ranging from 1.95 to 3.90 g/mL, 6f (cadiolide C) with
MIC of 3.90 g/mL, and 7h with MICs ranging from 3.90 to
7.81 g/mL. Compound 7i (cadiolide A) required the same concen-
tration than tetracycline to inhibit the bacterial growth of S. typhi
and E. coli 405 with MIC of 7.81 g/mL. Full dibrominated 7g
(cadiolide B) showed the same potency than tetracycline only
against E. coli 405 (MIC of 7.81 g/mL), and 5d, 6b and 8 exhibited
lg/mL, 6e, 7e and 10
bromine atoms were located on both benzylidene and phenyl
substituents.
l
l
In order to improve the antibacterial activity we replaced the
benzoyl with furoyl substituent to obtain compounds 8–10. SAR
studies showed that surprisingly compound 8 without any bro-
mine, displayed higher activity than its benzoyl analogue 5a. The
activity increased again when two bromine atoms were added as
l
l
l
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A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
OH
OH
OH
OH
Br
Br
Br
O
O
O
O
O
O
O
O
O
O
O
O
Br
Br
OH
Br
OH
HO
HO
HO
HO
OH
OH
5b
39%
5a
5c
38%
5d
Br
72%
38%
OH
OH
OH
Br
OH
Br
Br
Br
O
O
O
O
O
O
O
O
O
O
O
O
Br
Br
OH
Br
OH
Br
OH
HO
HO
HO
OH
HO
Br
Br
Br
Br
6b
37%
6c
Br
6e
44%
Br
6f
61%
(cadiolide C)
47%
OH
OH
OH
Br
Br
OH
Br
O
O
O
O
O
O
O
O
O
O
O
Br
Br
HO
Br
O
Br
OH
Br
OH
Br
OH
Br
Br
OH
HO
HO
HO
Br
Br
Br
7c
7e
7g
Br
Br
Br
39%
21%
7h
(cadiolide B)
Br
19%
48%
OH
Br
Br
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Br
Br
Br
OH
Br
OH
HO
HO
HO
HO
OH
OH
Br
Br
Br
8
9
10
7i
37%
30%
13%
(cadiolide A)
18%
Figure 3. Synthesis of various butenolides 5–10.
a moderate antibacterial activity with MIC against B. cereus, S. aur-
eus, E. faecalis, S. typhi and E. coli 405 ranging from 15.62 to
3. Cytotoxicity studies
31.25 lg/mL. Curiously, among all the tested compounds, only
5a, 6b and the furan analogues 8, 9 and 10 displayed significant
activity against E. coli 100 with MIC values ranging from 15.62 to
62.5 lg/mL. In general, the presence of free phenolic groups as
When testing an antimicrobial activity, it is important to
evaluate the lack of toxicity for the host of the synthesized
compounds. Therefore, since the compounds 6c, 7c, 9 and 10
exerted the most relevant effects against human pathogens, they
unique substituents more than bromine atoms on the benzene
rings attached to positions 4 and 5 seems to favor the activity
against this bacterium.
Some compounds were active against both bacterial strains B.
subtilis (Gram-positive) and E. carotovora (Gram-negative) at
were primarily tested at 30 and 100 lM for potential cytotoxic
effects in freshly isolated human neutrophils10 by the use of two
different approaches: the MTT (3[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) colorimetric assay11 and by flow
cytometry analysis12,13 of cell apoptosis and survival.14 Human
neutrophils cell types have already been used for assaying the
cytotoxic effects of different semi-synthetic alkaloids with
antibacterial activity.12 All the compounds displayed significant
125 lg/mL, for instance the partially bromineated derivatives such
as 6c, 6f (cadiolide C), 5d, 7i (cadiolide A), 7h, 7c, 6e, 7e, 6b and the
furan analogues 9 and 10. Among all tested compounds, the less
active was 5b which was able to inhibit the bacterial growth only
cytotoxicity at 100
lM. However, none of the compounds tested
against B. cereus at 125 lg/mL.
exerted any cytotoxicity at 30
l
M on neutrophils (Fig. 4). In fact,
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A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
this dose was 5 to 11-fold higher than the IC50 required to kill
S. aureus, E. faecalis, S. typhi and E. coli 405 (IC50 values ranged
between 2.72 and 6.38 lM, Table 1).
These results were further confirmed when apoptosis, necrosis
and survival in this human cell type was determined employing
the cytofluorimetric assay. In human neutrophils, most of the com-
pounds tested barely exerted any significant effect on neutrophil
apoptosis at the both doses assayed (below the vehicle, Fig. 5A).
When the survival of human cells was determined, none of the
compounds affected this parameter at 30
(Fig. 5C). In contrast, incubation of the cells with 6c and 9 at
100 M resulted in 98% and 95% of human neutrophil necrosis
lM concentration
l
respectively (Fig. 5B). Compounds 7c and 10 caused a 36% and
57% human neutrophil necrosis when they were tested at
100
lM. Nevertheless, and as found previously for the MTT assay,
it is important to note that 30
lM concentration is 5–11 fold higher
than the IC50 required to kill S. aureus, E. faecalis, S. typhi and E. coli
405 (Table 1), the most resistant pathogens.
Figure 4. Effect of 6c, 7c, 9 and 10 on viability of human neutrophils by MTT assay.
Data are presented as mean SEM of n = 6–9 independent experiments. ⁄⁄p <0.01
relative to the vehicle group.
Figure 5. Effect of 6c, 7c, 9 and 10 on neutrophil viability by cytofluorometric analysis. Percentage of apoptotic (A), necrotic (B) and survival (C) neutrophils after 24 h
incubation with 6c, 7c, 9 and 10. Apoptotic cells were quantified as the percentage of total population of annexin V+, PIꢀ cells, late apoptotic, and/or necrotic cells as annexin
V+ and PI+, and viable nonapoptotic cells as annexin Vꢀ and PIꢀ. The columns are the mean SEM of n = 3 independent experiments. Representative flow cytometry panels
showing the effects of control, vehicle, 6c, 7c, 9 and 10 on neutrophil apoptosis, necrosis and survival have been included. ⁄p <0.05 or ⁄⁄p <0.01 relative to the vehicle group.
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6
A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
UPLC-Q-TOF analysis: High resolution (HRMS) data were
4. Conclusion
recorded on a Waters Xevo quadrupole time-of-flight (Q-TOF)
spectrometer (Waters Corp., Milford, MA, USA) coupled to an
Acquity UPLC system (Waters Corp., Milford, MA, USA) via an elec-
trospray ionization (ESI) interface. Separation was performed on a
In summary, a series of 16 tris-aromatic butenolides including
natural cadiolides A, B, C and analogues has been prepared by using
a straightforward one-pot multicomponent synthesis (4 and 5–10).
The antibacterial activity has been evaluated on a panel of patho-
genic Gram+ and Gram- bacteria, showing MICs as low as
Waters Acquity BEH C18 column (50 ꢁ 2.1 mm i.d., 1.7
lm). The
solvent system consisted of 0.1% formic acid in acetonitrile (phase
A) and 0.1% formic acid in ultrapure water (phase B). Gradient con-
ditions were as follows: 20% B at 0 min to 100% A in 3 min, held for
2 min, returned to 20% B in 1 min, and equilibrated for 2 min
before the next injection; the flow rate was 0.2 mL/min; the col-
umn and sample temperatures were kept at 37 °C and 4 °C, respec-
1.95 l
g mLꢀ1. The SAR studies showed that phenolic hydroxyl
group is necessary but that the biological activity can be dramati-
cally increased by adding bromine atoms on the different aromatic
rings with optimal number of 3–4 bromine atoms (6c, 7c). We also
have shown that a phenolic substituent can be advantageously
substituted by a furan ring at position 3 of the butenolide nucleus
(9, 10). In addition, none of the most relevant antibacterial com-
pounds showed any cytotoxicity in freshly isolated human neu-
trophils. The straightforward access to the cadiolide skeleton will
easily allow other structural modifications; results will be reported
in due course.
tively; the sample injection volume was 1 lL. The ESI source was
operated in positive ionization mode using leucine-encephaline
as reference mass ([M+H]+ ion m/z 556.2771). The capillary and
cone voltages were set at 3.0 kV and 45 kV, respectively. The tem-
perature of the source and desolvation was set at 120 °C and
300 °C, respectively. The cone and desolvation gas (nitrogen) flows
were 500 L hꢀ1 and 50 L hꢀ1. The collision energy was set at 5 eV.
ESI data acquisition was collected in Centroid mode in a full scan
range from m/z 50–1500 at 0.2 s per scan. All data were acquired
using MasslynkTM NT4.1 software (Waters Corp., Milford, MA,
USA).
5. Materials and methods
5.1. General
Purity for all final compounds was checked by HPLC and was
determined P95%, each showed a parent mass ion consistent with
the desired structure (HRMS).
All reactions were carried out under a nitrogen or argon atmo-
sphere with dry solvents under anhydrous conditions, unless
otherwise noted. Dry tetrahydrofuran (THF), methylene chloride
(CH2Cl2), and toluene were obtained by distillation (from CaH2
for CH2Cl2, sodium/benzophenone for THF and from sodium for
toluene). Methanol (MeOH), N,N0-dimethylformamide (DMF) and
dimethylsulfoxide (DMSO) were purchased in anhydrous form
and used without further purification. Ethyl acetate (EtOAc),
diethyl ether (Et2O), methylene chloride (CH2Cl2), and cyclohexane
were purchased at ACS grade quality and used without further pur-
ification. Reagents were purchased at the highest commercial qual-
ity and used without further purification. Yields refer to
chromatographically and spectroscopically (1H NMR and 13C
NMR) homogeneous materials. Reactions were monitored by
thin-layer chromatography (TLC) carried out on Merck silica gel
plates with QF-254 indicator and an ethanolic solution of ammo-
nium molybdate or potassium permanganate and heat as develop-
ing agents. E. Merck silica gel (60, particle size 0.040–0.063 mm)
was used for flash column chromatography. NMR spectra were
recorded on Bruker Avance DMX-300 instrument and calibrated
using residual undeuterated solvent as an internal reference
(7.26 ppm and 77.16 ppm for 1H and 13C NMR in CDCl3;
2.50 ppm and 39.52 ppm for 1H and 13C NMR in [D6]DMSO;
2.05 ppm and 29.84 ppm for 1H and 13C NMR in [D6]Acetone).
The following abbreviations were used to describe the multiplici-
ties: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet,
m = multiplet, pent = pentet, hex = hextet, br = broad. IR spectra
were recorded on a Perkin–Elmer Spectrum One FTIR spectrometer
with diamond ATR accessory.
5.2. General procedure for the synthesis of dioxinones 2a–d
(procedure A)5a
To a stirred solution of acid (1 equiv) in CH2Cl2 (for a solution of
0.1 mol Lꢀ1) were added oxalyl chloride [(COCl)2; 1.25 equiv] and
DMF (0.004 equiv) at room temperature. After 1.5 h, the solvent
and the excess oxalyl chloride were removed by evaporation under
vacuum. CH2Cl2 (for
a
solution of 0.1 mol Lꢀ1), N,O-
dimethylhydroxylamine (1.4 equiv), and Et3N (3 equiv) were then
successively added at room temperature. After 2 h, the reaction
was quenched at room temperature with a saturated solution of
NaHCO3 and the mixture extracted twice with CH2Cl2. The com-
bined organic layers were then washed with a saturated solution
of NH4Cl and brine. The organic layer was then dried with anhy-
drous MgSO4, filtered, and concentrated under vacuum to afford
the crude Weinreb amine.
To a stirred solution of diisopropylamine (DIPA; 3 equiv) in THF
(for a solution of 0.1 mol Lꢀ1) was added n-Butyl lithium (nBuLi,
1.2 M in hexane, 3.1 equiv) at ꢀ78 °C. After 30 min at 0 °C, the
medium was recooled to ꢀ78 °C and freshly distilled tBu acetate
(3 equiv) was added. After 30 min at ꢀ78 °C, crude Weinreb amide
(1 equiv) was added at this temperature. After 1 h, the reaction was
quenched at room temperature with a saturated solution of
NaHCO3 and the mixture extracted twice with EtOAc. The com-
bined organic layers were then washed with a saturated solution
of NH4Cl. The organic layer was then dried with anhydrous
MgSO4, filtered, and concentrated under vacuum to afford the
crude tert-butyl ester.
To a stirred solution of tert-butyl ester (1 equiv) in acetone
(10 equiv) were added acetic anhydride (15 equiv) and sulfuric
acid (1 equiv) at 0 °C. The medium was then warmed slowly to
room temperature over 10 min. After 45 min, the reaction was
quenched at room temperature with an aqueous solution contain-
ing sodium carbonate (30 equiv) and EtOAc (100 mL) was added.
The biphasic medium was then stirred for 40 min (hydrolysis of
the remaining acetic anhydride) and the aqueous layer was
extracted twice with EtOAc. The combined organic layers were
UPLC-PDA analysis: Samples dissolved in MeOH were injected
onto a Waters Acquity UPLC system (Milford, MA, USA) equipped
with a binary solvent manager, simple manager, column com-
partment and 2996 PDA detector, connected to Waters
Masslynx 4.1 software. The separation was carried out using a
Waters BEH C18 column (2.1 ꢁ 100 mm, 1.7
lm) at 37 °C. The
optimal chromatographic conditions were established: solvent
system, phase A, 0.1% trifluoroacetic acid in water and phase B,
0.1% trifluoroacetic acid in acetonitrile; gradient separation sys-
tem: 80% A at 0 min for 1 min to 100% B at 5 min for 2 min, then
80% A at 8 min for 2 min; flow rate 0.4 mL/min and injection
volume of 2–5 lL.
Please cite this article in press as: Boulangé, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.010

A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
then washed with a saturated solution of NH4Cl. The organic layer
was dried with anhydrous MgSO4, filtered and concentrated under
vacuum. The crude residue was finally purified by flash chro-
matography silica gel using an appropriate gradient of a cyclohex-
ane/EtOAc mixture as eluent to give the desired dioxinone.
(300 MHz, CDCl3): d = 8.17 (s, 1H), 7.91 (d, J = 8.7 Hz, 1H), 6.93 (d,
J = 8.7 Hz, 1H), 3.97 (s, 3H), 2.56 (s, 3H) ppm. 13C NMR (75 MHz,
CDCl3): d = 195.8, 159.7, 134.0, 131.4, 129.6, 112.0, 111.2, 56.6,
26.5 ppm. IR (neat): 1670, 1590, 1260, 1230, 1043, 1012, 817,
590 cmꢀ1. MS (APCI): m/z (%) = 228 (100), 229 (10) [MꢀH+].
5.2.1. 6-(4-Methoxyphenyl)-2,2-dimethyl-4H-1,3-dioxin-4-one
(2a) [87769-44-6]
5.2.6. 3,5-Dibromo-4-methoxyacetophenone (11c) [79324-79-1]
Bromine (1.63 mL, 31.5 mmol) in acetic acid (5 mL) was added
Following procedure A, 4-methoxybenzoic acid (3.41 g,
20 mmol) was used to afford 2a as yellow solid with 77% yield
(3.57 g, 15.3 mmol) after purification by flash chromatography on
silica gel (cyclohexane/EtOAc 80:20). mp: 68 °C; 1H NMR
(300 MHz, CDCl3): d 1.77 (s, 6H), 3.84 (s, 3H), 5.77 (s, 1H), 6.92
(d, J = 9.0 Hz, 2H), 7.71 (d, J = 9.0 Hz, 2H); 13C NMR (75 MHz,
CDCl3): d 25.2, 55.7, 89.5, 106.5, 114.4, 123.5, 128.4, 162.4, 163.0,
165.1; IR (neat): 2999, 2940, 2842, 1721, 1607, 1513, 1363, 1252,
1179, 1027, 804 cmꢀ1; MS (ESI): m/z 235 (M+H+); HRMS (ESI):
calcd for [C13H14O4+H]+ 235.0970; Found 235.0976.
dropwise to a mixture of 4-hydroxyacetophenone (2.08 g,
15 mmol) and sodium acetate (3.85 g, 46.5 mmol) in acid acetic
(35 mL) at room temperature over 20 min. The reaction mixture
was stirred for 1 h at room temperature. H2O (75 mL) was added
and the solid was filtered, washed with H2O and dried under high
vacuum to afford 3,5-dibromo-4-hydroxyacetophenone 12 as a
white solid. To a stirred solution of 12 in DMF (50 mL) was added
K2CO3 (4.15 g, 30 mmol) in portions, followed by the addition of
MeI (3.9 mL, 60 mmol), and the mixture was stirred at room tem-
perature for 24 h. The reaction mixture was taken in EtOAc
(100 mL) and washed successively with H2O (50 mL), 1 N HCl
(50 mL), saturated solution of NaHCO3 (50 mL), and brine
(2 ꢁ 50 mL). The organic layer was dried with Na2SO4, filtered
and concentrated under vacuum to afford 3,5-bromo-4-methoxy-
acetophenone 11c as a white solid in 96% yield (4.45 g, 14.4 mmol).
Mp 83 °C. 1H NMR (300 MHz, CDCl3): d = 8.08 (s, 2H), 3.93 (s, 3H),
2.56 (s, 3H) ppm. 13C NMR (75 MHz, CDCl3): d = 194.7, 158.2, 135.2,
133.0, 118.8, 60.9, 26.6 ppm. IR (neat): 1684, 1380, 1349, 1263,
979, 736, 592 cmꢀ1. MS (APCI): m/z (%) = 308 (55), 228 (100)
[MꢀH+].
5.2.2. 6-(3-Bromo-4-methoxyphenyl)-2,2-dimethyl-4H-1,3-
dioxin-4-one (2b)
Following procedure A, 3-bromo-4-methoxybenzoic acid
(4.44 g, 19.2 mmol) was used to afford 2b as a orange solid in
23% yield (1.37 g, 4.42 mmol) after purification by flash chro-
matography on silica gel (cyclohexane/EtOAc, 80:20), mp 120 °C.
1H NMR (300 MHz, CDCl3): d = 7.86 (d, J = 2.1 Hz, 1H), 7.62 (dd,
J = 8.7, 2.1 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H), 5.78 (s, 1H), 3.94 (s,
3H), 1.78 (s, 6H) ppm. 13C NMR (75 MHz, CDCl3): d = 163.6,
161.8, 158.9, 131.5, 127.2, 124.7, 112.3, 111.7, 106.8, 90.4, 56.6,
25.1 ppm. IR (neat): 1713, 1597, 1498, 1357, 1251, 1203, 1037,
1013, 994, 899, 806 cmꢀ1. MS (ESI): m/z (%) = 313 (80), 315 (100)
[M+H+].
5.3. General procedure for the synthesis of
13b–c (procedure B)
a-bromo-ketones
To a stirred solution of acetophenones 11b–c (1 equiv) in THF
(30 mL) was added trimethylphenylammonium tribromide
(1.05 equiv) at 0 °C. The reaction mixture was stirred at room tem-
perature for 12 h. The solid was filtered, and to the filtrate was
added EtOAc (20 mL). The organic layer was washed successively
with H2O (20 mL) and brine (20 mL). The organic layer was then
dried over Na2SO4, filtered and concentrated under vacuum. The
crude residue was then purified by flash chromatography on silica
gel using an appropriate gradient of cyclohexane/EtOAc as eluent
5.2.3. 6-(3,5-Dibromo-4-methoxyphenyl)-2,2-dimethyl-4H-1,3-
dioxin-4-one (2c)
Following procedure A, 3,5-dibromo-4-methoxybenzoic acid
(4.90 g, 15.8 mmol) was used to afford 2c as a orange solid in
74% yield (4.57 g, 11.7 mmol) after purification by flash chro-
matography on silica gel (cyclohexane/EtOAc, 90:10), mp 127 °C.
1H NMR (300 MHz, CDCl3): d = 7.80 (s, 2H), 5.81 (s, 1H), 3.91 (s,
3H), 1.78 (s, 6H) ppm. 13C NMR (75 MHz, CDCl3): d = 161.8, 161.0,
157.2, 130.6, 129.6, 118.9, 107.2, 92.4, 60.9, 25.1 ppm. IR (neat):
1716, 1616, 1381, 1340, 1260, 1199, 984, 904, 830, 743 cmꢀ1. MS
(ESI): m/z (%) = 391 (65), 393 (100), 395 (50) [M+H+].
to give the desired a-bromo-ketone 13b–c.
5.3.1. 2-Bromo-1-(3-bromo-4-methoxyphenyl)ethanone (13b)
[6096-83-9]
5.2.4. 6-(Furan-2-yl)-2,2-dimethyl-4H-1,3-dioxin-4-one (2d)
[1293991-93-1]
Following procedure B, 3-bromo-4-methoxyacetophenone 11b
(1.01 g, 4.4 mmol) was used to afford 13b as a white solid in 89%
yield (1.20 g, 3.9 mmol) after purification by flash chromatography
on silica gel (cyclohexane/EtOAc, 90:10), mp 113 °C. 1H NMR
(300 MHz, CDCl3): d = 8.19 (d, J = 2.1 Hz, 1H), 7.94 (dd, J = 8.7,
2.1 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 4.37 (s, 2H), 3.98 (s, 3H) ppm.
13C NMR (75 MHz, CDCl3): d = 189.1, 160.4, 134.5, 130.5, 128.0,
112.4, 111.4, 56.7, 30.4 ppm. IR (neat): 1688, 1586, 1493, 1272,
1247, 1180, 1045, 1002, 813, 660, 606, 586 cmꢀ1. MS (APCI): m/z
(%) = 307 (40), 228 (100) [MꢀH+].
Following procedure A, 2-furoic acid (2.24 g, 20 mmol) was
used to afford 2d as red oil with 57% yield (2.20 g, 11.3 mmol) after
purification by flash chromatography on silica gel (cyclohexane/
EtOAc 80:20). 1H NMR (300 MHz, CDCl3): d 1.71 (s, 6H), 5.74 (s,
1H), 6.49 (dd, J = 1.7, 3.6 Hz, 1H), 6.86 (d, J = 3.6 Hz, 1H), 7.53 (br
s, 1H); 13C NMR (75 MHz, CDCl3): d 25.0, 89.6, 107.0, 112.5,
114.2, 146.2, 146.3, 156.4, 161.6; IR (neat): 3134, 3000, 2945,
1727, 1679, 1641, 1470, 1361, 1278, 1205, 1017 cmꢀ1; MS (ESI):
m/z 195 (M+H).
5.3.2. 2-Bromo-1-(3,5-dibromo-4-methoxyphenyl)ethanone
[1563037-34-2] (13c)
5.2.5. 3-Bromo-4-methoxyacetophenone (11b) [35310-75-9]6a
Bromine (0.54 mL, 10.5 mmol) in acetic acid (1.7 mL) was added
Following procedure B, 3,5-dibromo-4-methoxyacetophenone
11c (4.36 g, 14.2 mmol) was used to afford 13c as a white solid
in 99% yield (5.42 g, 14.0 mmol) after purification by flash chro-
matography on silica gel (cyclohexane/EtOAc, 90:10), mp 101 °C.
1H NMR (300 MHz, CDCl3): d = 8.12 (s, 2H), 4.36 (s, 2H), 3.96 (s,
3H) ppm. 13C NMR (75 MHz, CDCl3): d = 188.3, 158.9, 133.6,
132.0, 119.0, 61.0, 30.0 ppm. IR (neat): 1698, 1469, 1377, 1273,
dropwise to
a mixture of 4-methoxyacetophenone (758 mg,
5 mmol) and sodium acetate (1.28 g, 15.5 mmol) in acid acetic
(10 mL) at room temperature over 10 min. The reaction mixture
was stirred for 1 h at room temperature. H2O (30 mL) was added
and the solid was filtered, washed with H2O and dried under high
vacuum to afford 3-bromo-4-methoxyacetophenone 11b as a
white solid in 88% yield (1.01 g, 4.4 mmol). Mp 86 °C. 1H NMR
1178, 985, 851, 735, 608 cmꢀ1
.
Please cite this article in press as: Boulangé, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.010

8
A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5.4. General procedure for the synthesis of
1a–c (procedure C)
a
-hydroxy-ketones
microwave reactor. Over 1.5 min, the temperature was raised to
150 °C, held for 5 min, and then cooled for 10 min. The reaction
was quenched at room temperature with 1 M HCl and the mixture
extracted with EtOAc. The combined organic layers were then
dried with Na2SO4, filtered and concentrated under vacuum.
Filtration through a plug of silica and elution with (cyclohexane/
EtOAc, 80:20) afforded a mixture of bis-methoxylated and mono-
methoxylated acylfuranones 4, which was used directly without
further purification (partial de-methoxylation resulted during the
microwave-assisted multi-component recation).
Sodium formate (3 equiv) was stirred in ethanol (0.1 mol Lꢀ1
for 15 min and -bromo ketone 13 (1 equiv) was then added. The
)
a
mixture was stirred at 70 °C overnight. The solution was filtered
hot and concentrated under vacuum. The crude residue was then
purified by flash chromatography on silica gel using an appropriate
gradient of cyclohexane/EtOAc as eluent to give the desired a-hy-
droxy-ketone 1a–c.
To a stirred solution of bis-methoxylated and mono-methoxy-
lated acylfuranones 4 (obtained above) in CH2Cl2 (5 mL) was added
BBr3 (10 equiv) at ꢀ78 °C. The mixture was then slowly warmed to
room temperature over 20 h. The reaction was quenched at ꢀ78 °C
with MeOH then at room temperature with H2O and the mixture
extracted twice with EtOAc. The combined organic layers were
then dried with Na2SO4, filtered and concentrated under vacuum.
The crude product was purified by flash chromatography on silica
gel using a gradient of EtOAc/MeOH (100:0 to 95:5) to give cadio-
lide analogues 5–10.
5.4.1. 2-Hydroxy-1-(3-bromo-4-methoxyphenyl)-ethanone (1b)
[927802-90-2]
Following procedure C, 2-bromo-1-(3-bromo-4-methoxyphe-
nyl)ethanone 13b (1.16 g, 3.8 mmol) was used to afford 1b as a
white solid in 58% yield (533 mg, 2.2 mmol) after purification by
flash chromatography on silica gel (cyclohexane/EtOAc, 80:20),
mp 115 °C. 1H NMR (300 MHz, CDCl3): d = 8.12 (d, J = 2.1 Hz, 1H),
7.87 (dd, J = 8.7, 2.1 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 4.80 (d,
J = 3.6 Hz, 2H), 3.98 (s, 3H), 3.47 (t, J = 3.6 Hz, 1H) ppm. 13C NMR
(75 MHz, CDCl3): d = 196.1, 160.6, 133.3, 129.0, 127.5, 112.5,
111.5, 65.2, 56.7 ppm. IR (neat): 3389, 1671, 1590, 1414, 1256,
5.5.1. (Z)-3-(4-Hydroxybenzoyl)-5-(4-hydroxybenzylidene)-4-
(4-hydroxyphenyl)furan-2(5H)-one (5a)
1209, 1108, 1049, 1002, 916, 676, 622, 597 cmꢀ1
.
Following procedure D,
a-hydroxy-ketone 1a (83 mg,
5.4.2. 2-Hydroxy-1-(3,5-dibromo-4-methoxyphenyl)-ethanone
(1c)
0.5 mmol), dioxinone 2a (234 mg, 1.0 mmol) and 4-methoxyben-
zaldehyde 3a (0.06 mL, 0.5 mmol) were used to afford 5a as yellow
solid in 72% yield (142 mg, 0.36 mmol). Analyses are similar to
those described in the literature.5d Mp 262 °C. 1H NMR (300 MHz,
[D6]DMSO): d = 10.69 (br s, 1H), 10.28 (br s, 1H), 10.15 (br s, 1H),
7.74 (d, J = 8.7 Hz, 2H), 7.68 (d, J = 8.7 Hz, 2H), 7.22 (d, J = 8.7 Hz,
2H), 6.89 (d, J = 8.7 Hz, 2H), 6.82 (d, J = 8.7 Hz, 2H), 6.78 (d,
J = 8.7 Hz, 2H), 6.33 (s, 1H) ppm. 13C NMR (75 MHz, [D6]DMSO):
d = 188.0, 166.1, 163.2, 159.6, 159.5, 155.9, 144.6, 133.0, 132.1,
130.8, 127.4, 124.0, 121.4, 119.3, 116.4, 116.1, 115.8, 115.5 ppm.
HRMS (ESI): calculated for C24H17O6 401.1025 found 401.1016.
Following procedure C, 2-bromo-1-(3,5-di bromo-4-methoxy-
phenyl)ethanone 13c (5.41 g, 14 mmol) was used to afford 1c as
a white solid in 60% yield (2.71 g, 8.4 mmol) after purification by
flash chromatography on silica gel (cyclohexane/EtOAc, 80:20),
mp 124 °C. 1H NMR (300 MHz, CDCl3): d = 8.06 (s, 2H), 4.81 (s,
2H), 3.96 (s, 3H), 3.34 (br s, 1H) ppm. 13C NMR (75 MHz, CDCl3):
d = 195.6, 159.2, 132.4, 131.5, 119.3, 65.6, 61.0 ppm. IR (neat):
3248, 1695, 1262, 1234, 981, 736 cmꢀ1
.
5.4.3. 3,5-Dibromo-4-methoxybenzaldehyde (3c) [108940-96-1]
Bromine (1.63 mL, 31.5 mmol) in acetic acid (5 mL) was added
5.5.2. (Z)-5-(3-Bromo-4-hydroxybenzylidene)-3-(4-hydroxy-
benzoyl)-4-(4-hydroxyphenyl)furan-2(5H)-one (5b)
dropwise to
a mixture of 4-hydroxybenzaldehyde (1.85 g,
15 mmol) and sodium acetate (3.85 g, 46.5 mmol) in acid acetic
(35 mL) at room temperature over 20 min. The reaction mixture
was stirred for 1 h at room temperature. H2O (75 mL) was added
and the solid was filtered, washed with H2O and dried under high
vacuum to afford 3,5-dibromo-4-hydroxybenzaldehyde 14 as a
white solid.
Following procedure D,
a-hydroxy-ketone 1a (41 mg,
0.25 mmol), dioxinone 2a (117 mg, 0.5 mmol) and 3-bromo-4-
methoxybenzaldehyde 3b (54 mg, 0.25 mmol) were used to afford
5b as yellow solid in 39% yield (43 mg, 0.09 mmol). Mp 159 °C. 1H
NMR (300 MHz, [D6]DMSO): d = 11.03 (br s, 1H), 10.61 (br s, 1H),
10.09 (br s, 1H), 8.08 (d, J = 2.1 Hz, 1H), 7.76 (dd, J = 8.7, 2.1 Hz,
1H), 7.71 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 8.6 Hz, 2H), 7.05 (d,
J = 8.7 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 8.6 Hz, 2H),
6.36 (s, 1H) ppm. 13C NMR (75 MHz, [D6]DMSO): d = 188.3, 166.3,
163.5, 160.0, 155.9, 145.7, 135.1, 132.6, 132.2, 131.2, 127.7,
126.1, 122.4, 119.6, 117.1, 116.1, 115.8, 114.9, 110.3 ppm. IR
To a stirred solution of 14 in DMF (50 mL) was added K2CO3
(4.15 g, 30 mmol) in portions, followed by the addition of MeI
(3.9 mL, 60 mmol), and the mixture was stirred at room tempera-
ture for 24 h. The reaction mixture was taken in EtOAc (100 mL)
and washed successively with H2O (50 mL), 1 M HCl (50 mL), satu-
rated solution of NaHCO3 (50 mL), and brine (2 ꢁ 50 mL). The
organic layer was dried with Na2SO4, filtered and concentrated
under vacuum. The crude product was purified by chromatography
on silica gel by using cyclohexane/EtOAc (90:10) as eluent to give
3,5-dibromo-4-methoxybenzaldehyde 3c as a white solid in 82%
yield (3.62 g, 12.3 mmol). Mp 97 °C. 1H NMR (300 MHz, CDCl3):
d = 9.86 (s, 1H), 8.02 (s, 2H), 3.96 (s, 3H) ppm. 13C NMR (75 MHz,
CDCl3): d = 188.6, 159.3, 134.4, 134.1, 119.5, 61.0 ppm. IR (neat):
(neat):
1221, 1152, 1041, 863, 841 cmꢀ1. HRMS (ESI): calculated for
24H16BrO6 479.0130 found 479.0116.
t = 3233, 1729, 1576, 1557, 1501, 1440, 1410, 1371, 1274,
C
5.5.3. (Z)-3-(3-Bromo-4-hydroxybenzoyl)-5-(3-bromo-4-
hydroxybenzylidene)-4-(4-hydroxyphenyl)furan-2(5H)-one (5c)
Following procedure D,
a-hydroxy-ketone 1a (41 mg,
0.25 mmol), dioxinone 2b (156 mg, 0.5 mmol) and 3-bromo-4-
methoxybenzaldehyde 3b (54 mg, 0.25 mmol) were used to afford
5c as yellow solid in 38% yield (52 mg, 0.09 mmol). Mp 219 °C. 1H
NMR (300 MHz, [D6]DMSO): d = 11.10 (br s, 1H), 10.62 (br s, 1H),
10.11 (br s, 1H), 8.09 (d, J = 2.1 Hz, 1H), 7.95 (d, J = 2.2 Hz, 1H),
7.76 (dd, J = 8.7, 2.1 Hz, 1H), 7.69 (dd, J = 8.5, 2.2 Hz, 1H), 7.25 (d,
J = 8.8 Hz, 2H), 7.05 (d, J = 8.5 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H),
6.79 (d, J = 8.8 Hz, 2H), 6.37 (s, 1H) ppm. 13C NMR (75 MHz,
[D6]DMSO): 187.4, 166.2, 160.1, 159.8, 157.1, 156.1, 145.8, 135.8,
133.4, 132.5, 132.3, 131.4, 129.0, 126.1, 121.5, 119.5, 117.1,
1685, 1545, 1468, 1365, 1258, 1186, 979, 877, 730, 653, 531 cmꢀ1
.
5.5. General procedure for the synthesis of cadiolide analogues
5–10
Toluene (for a solution of 1.0 mol Lꢀ1), Et3N (2.0 equiv) and the
appropriate
a-hydroxy-ketone 1a–c (1 equiv), dioxinone 2a–d
(2 equiv) and aldehyde 3a–c (1 equiv) were added to a 15 mL tube
flushed with argon. The tube was sealed and then heated in a
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A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
9
116.1, 115.5, 110.3, 110.0 ppm. IR (neat):
t
= 3336, 1747, 1714,
182 °C. 1H NMR (300 MHz, [D6]DMSO): d = 11.48 (br s, 1H), 10.94
(br s, 1H), 10.65 (br s, 1H), 8.10 (s, 2H), 7.97 (d, J = 2.0 Hz, 1H),
7.70 (dd, J = 8.5, 2.0 Hz, 1H), 7.49 (d, J = 2.0 Hz, 1H), 7.22 (dd,
J = 8.4, 2.0 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H),
6.39 (s, 1H) ppm. 13C NMR (75 MHz, [D6]DMSO): d = 186.6, 165.6,
159.7, 156.4, 155.6, 152.1, 146.5, 135.2, 134.8, 133.8, 131.1,
130.3, 128.6, 127.6, 122.8, 120.6, 116.6, 116.2, 113.6, 112.1 (2C),
109.8 ppm. IR (neat): 3188, 1737, 1476, 1403, 1367, 1296, 1191,
978, 708, 635, 545 cmꢀ1. MS (ESI): m/z (%) = 719 (26), 718 (21),
717 (100), 716 (55), 715 (96), 714 (30), 713 (80), 711 (27). HRMS
(ESI): calculated for C24H13Br4O6 716.7407 found 716.7411.
1584, 1556, 1504, 1409, 1383, 1303, 1218, 1179, 1150, 1081,
848, 825 cmꢀ1. HRMS (ESI): calculated for C24H15Br2O6 558.9217
found 558.9201.
5.5.4. (Z)-3-(3,5-Dibromo-4-hydroxybenzoyl)-5-(3,5-dibromo-4-
hydroxybenzylidene)-4-(4-hydroxyphenyl)furan-2(5H)-one
(5d)
Following procedure D,
a-hydroxy-ketone 1a (44 mg,
0.26 mmol), dioxinone 2c (204 mg, 0.52 mmol) and 3,5-dibromo-
4-methoxybenzaldehyde 3c (76 mg, 0.26 mmol) were used to
afford 5d as a yellow solid in 38% yield (70 mg, 0.01 mmol).
Decomp. 150 °C. 1H NMR (300 MHz, [D6]DMSO): d = 10.15 (br s,
1H), 8.12 (s, 2H), 7.97 (s, 2H), 7.21 (d, J = 8.7 Hz, 2H), 6.79 (d,
J = 8.7 Hz, 2H), 6.42 (s, 1H) ppm. 13C NMR (75 MHz, [D6]DMSO):
d = 186.0, 165.6, 159.9, 157.7, 156.0, 152.1, 146.6, 134.7, 133.9,
131.3, 129.5, 127.6, 121.0, 119.0, 115.9, 113.7, 112.1, 111.5 ppm.
IR (neat): 3208, 1748, 1578, 1476, 1376, 1297, 1152, 981,
679 cmꢀ1; MS (ESI): m/z (%) = 719 (15), 718 (19), 717 (70), 716
(30), 715 (100), 714 (22), 713 (60), 711 (15). HRMS (ESI): calcu-
lated for C24H13Br4O6 716.7407 found 716.7396.
5.5.8. Cadiolide C: (Z)-5-(3-bromo-4-hydroxybenzylidene)-
4-(3-bromo-4-hydroxyphenyl)-3-(3,5-dibromo-4-hydroxy-
benzoyl)furan-2(5H)-one [1414518-14-1] (6f)
Following procedure D,
a-hydroxy-ketone 1b (123 mg,
0.5 mmol), dioxinone 2c (392 mg, 1.0 mmol) and 3-bromo-4-
methoxybenzaldehyde 3b (110 mg, 0.5 mmol) were used to afford
6f as an orange solid in 47% yield (168 mg, 0.24 mmol). Decomp.
116 °C. 1H NMR (300 MHz, [D6]DMSO): d = 11.11 (br s, 1H), 10.95
(br s, 1H), 8.09 (s, 1H), 7.96 (s, 2H), 7.77 (d, J = 8.4 Hz, 1H), 7.50
(s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.97 (d,
J = 8.4 Hz, 1H), 6.38 (s, 1H) ppm. 13C NMR (75 MHz, [D6]DMSO):
d = 185.7, 165.6, 156.9, 156.2, 156.0, 155.7, 145.5, 135.8, 133.8,
132.2, 130.3, 129.8, 125.8, 121.0, 120.7, 116.8, 116.4, 116.0, 111.4
(2C), 110.0, 109.7 ppm. IR (neat): 3073, 1739, 1547, 1400, 1295,
979, 685 cmꢀ1; MS (ESI): m/z (%) = 719 (25), 718 (24), 717 (70),
716 (30), 715 (100), 714 (22), 713 (72), 711 (17). HRMS (ESI): cal-
culated for C24H13Br4O6 716.7407 found 716.7406.
5.5.5. (Z)-5-(3-Bromo-4-hydroxybenzylidene)-4-(3-bromo-4-
hydroxyphenyl)-3-(4-hydroxybenzoyl)furan-2(5H)-one (6b)
Following procedure D,
a-hydroxy-ketone 1b (61 mg,
0.25 mmol), dioxinone 2a (117 mg, 0.5 mmol) and 3-bromo-4-
methoxybenzaldehyde 3b (54 mg, 0.25 mmol) were used to afford
6b as yellow solid in 37% yield (51 mg, 0.09 mmol). Mp 148 °C. 1
H
NMR (300 MHz, [D6]DMSO): d = 10.92 (br s, 1H), 10.71 (br s, 1H),
8.30 (br s, 1H), 8.09 (d, J = 1.9 Hz, 1H), 7.76 (dd, J = 8.6, 1.9 Hz,
1H), 7.72 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 2.1 Hz, 1H), 7.25 (dd,
J = 8.4, 2.1 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H),
6.79 (d, J = 8.8 Hz, 2H), 6.35 (s, 1H) ppm. 13C NMR (75 MHz,
[D6]DMSO): 187.9, 166.1, 163.6, 156.5, 156.1, 154.9, 145.8, 135.9,
133.8, 132.6, 132.3, 130.3, 127.7, 126.0, 121.0, 117.1, 116.8,
5.5.9. (Z)-3-(3-Bromo-4-hydroxybenzoyl)-5-(3-bromo-4-
hydroxybenzylidene)-4-(3,5-dibromo-4-hydroxyphenyl)-
furan-2(5H)-one (7c)
Following procedure D,
a-hydroxy-ketone 1c (162 mg,
0.5 mmol), dioxinone 2b (313 mg, 1.0 mmol) and 3-bromo-4-
methoxybenzaldehyde 3b (110 mg, 0.5 mmol) were used to afford
7c as an orange solid in 39% yield (140 mg, 0.2 mmol). Decomp.
110 °C. 1H NMR (300 MHz, [D6]DMSO): d = 11.48 (br s, 1H), 11.11
(br s, 1H), 8.09 (d, J = 2.1 Hz, 1H), 7.97 (d, J = 2.1 Hz, 1H), 7.78
(dd, J = 2.1, 8.7 Hz, 1H), 7.70 (dd, J = 2.1, 8.7 Hz, 1H), 7.55 (s, 2H),
7.06 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.38 (s, 1H) ppm.
13C NMR (75 MHz, [D6]DMSO): d = 186.2, 165.6, 159.5, 156.0,
154.8, 152.8, 145.4, 135.9, 135.2, 132.9, 132.2, 130.8, 128.6,
125.7, 122.8, 122.5, 116.8, 116.0, 115.8, 111.8, 110.0, 109.6 ppm.
IR (neat): 2919, 1746, 1592, 1296, 1164, 1022, 978, 824, 744,
634 cmꢀ1. MS (ESI): m/z (%) = 719 (20), 718 (18), 717 (65), 716
(27), 715 (100), 714 (16), 713 (61), 711 (16). HRMS (ESI): calcu-
lated for C24H13Br4O6 716.7407 found 716.7400.
115.9, 115.2, 110.3, 109.9 ppm. IR (neat):
t = 3160, 1732, 1561,
1548, 1504, 1406, 1370, 1287, 1225, 1161, 1042, 866, 848 cmꢀ1
.
HRMS (ESI): calculated for C24H15Br2O6 558.9217 found 558.9219.
5.5.6. (Z)-3-(3-Bromo-4-hydroxybenzoyl)-5-(3-bromo-4-
hydroxybenzylidene)-4-(3-bromo-4-hydroxyphenyl)furan-
2(5H)-one (6c)
Following procedure D,
a-hydroxy-ketone 1b (123 mg,
0.5 mmol), dioxinone 2b (313 mg, 1.0 mmol) and aldehyde 3b
(110 mg, 0.5 mmol) were used to afford 6c as an orange solid in
44% yield (158 mg, 0.22 mmol). Decomp. 98 °C. 1H NMR
(300 MHz, [D6]DMSO): d = 11.47 (br s, 1H), 11.06 (br s, 1H), 10.93
(br s, 1H), 8.08 (d, J = 2.1 Hz, 1H), 7.97 (d, J = 2.1 Hz, 1H), 7.75
(dd, J = 8.7, 2.1 Hz, 1H), 7.70 (dd, J = 8.7, 2.1 Hz, 1H), 7.50 (d,
J = 2.1 Hz, 1H), 7.23 (dd, J = 8.7, 2.1 Hz, 1H), 7.05 (d, J = 8.7 Hz,
1H), 6.98 (d, J = 8.7 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.36 (s, 1H)
ppm. 13C NMR (75 MHz, [D6]DMSO): d = 186.7, 165.7, 159.5,
156.2, 155.9, 155.7, 145.4, 135.7, 135.0, 133.7, 132.1, 131.0,
130.2, 128.7, 125.8, 122.0, 120.7, 116.8, 116.5, 116.0, 115.5,
110.0, 109.7 (2C) ppm. IR (neat): 2925, 1745, 1555, 1495, 1367,
1291, 974, 820 cmꢀ1; MS (ESI): m/z (%) = 641 (39), 640 (22), 639
(100), 638 (32), 637 (58), 636 (30). HRMS (ESI): calculated for
5.5.10. (Z)-3-(3-Bromo-4-hydroxybenzoyl)-5-(3,5-dibromo-4-
hydroxybenzylidene)-4-(3,5-dibromo-4-hydroxyphenyl)furan-
2(5H)-one (7e)
Following procedure D,
a-hydroxy-ketone 1c (162 mg,
0.5 mmol), dioxinone 2b (313 mg, 1.0 mmol) and 3,5-dibromo-4-
methoxybenzaldehyde 3c (147 mg, 0.5 mmol) were used to afford
7e as a brown solid in 21% yield (85 mg, 0.11 mmol). Decomp.
110 °C. 1H NMR (300 MHz, [D6]DMSO): d = 11.51 (br s, 1H), 10.61
(br s, 1H), 8.13 (s, 2H), 8.0 (s, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.55 (s,
2H), 6.96 (d, J = 8.6 Hz, 1H), 6.42 (s, 1H) ppm. 13C NMR (75 MHz,
[D6]DMSO): d = 186.1, 165.3, 159.7, 154.6, 152.6, 152.2, 146.4,
135.3, 134.7, 133.4, 133.0, 130.8, 128.5, 127.5, 123.6, 122.3,
116.1, 113.7, 112.1, 111.9, 111.4, 109.6 ppm. IR (neat): 3201,
1754, 1589, 1476, 1406, 1296, 1194, 1152, 1021, 977, 816, 740,
634 cmꢀ1. MS (ESI): m/z (%) = 799 (15), 798 (20), 797 (53), 796
(26), 795 (96), 794 (32), 793 (100), 791 (51), 789 (12). HRMS
(ESI): calculated for C24H12Br5O6 794.6512 found 794.6512.
C24H14Br3O6 636.8322 found 636.8324.
5.5.7. (Z)-3-(3-Bromo-4-hydroxybenzoyl)-4-(3-bromo-4-
hydroxyphenyl)-5-(3,5-dibromo-4-hydroxybenzylidene)furan-
2(5H)-one (6e)
Following procedure D,
a-hydroxy-ketone 1b (123 mg,
0.5 mmol), dioxinone 2b (313 mg, 1.0 mmol) and 3,5-dibromo-4-
methoxybenzaldehyde 3c (147 mg, 0.5 mmol) were used to afford
6e as a brown solid in 61% yield (220 mg, 0.31 mmol). Decomp.
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10
A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5.5.11. Cadiolide B: (Z)-3-(3,5-dibromo-4-hydroxybenzoyl)-5-
(3,5-dibromo-4-hydroxybenzylidene)-4-(3,5-dibromo-4-
hydroxyphenyl)furan-2(5H)-one [206763-42-0]: (7g)
157.5, 151.3, 149.3, 144.5, 133.3, 131.0, 124.1, 122.6, 119.9,
119.3, 117.6, 116.2, 115.9, 113.0 ppm. MS (ESI): m/z = 373
[MꢀH]+. HRMS (ESI): calculated for C22H15O6 375.0869 found
375.0857.
To a solution of acylfuranone 5a (26 mg, 0.065 mmol) in diox-
ane-H2O (2 mL, 1:1) was added dropwise a brominating agent
(0.69 mL) prepared from Br2 (0.03 mL) and KBr (150 mg) in H2O
(1 mL). The mixture was stirred for 24 h, treated with brine, and
extracted with Et2O. The organic layer was washed with brine then
with Na2S2O3. The combined organic layer was then dried over
Na2SO4, filtered and concentrated under vacuum. The crude pro-
duct was chromatographied on silica gel using a gradient of
EtOAc/MeOH mixture (100:0 to 90:10) to give Cadiolide B as a
black solid with 67% yield (38 mg, 0.044 mmol). Analyses are simi-
lar to those described in the literature.3a,b 1H NMR (300 MHz,
[D6]DMSO): 8.12 (s, 2H), 7.91 (s, 2H), 7.55 (s, 2H), 6.40 (s, 1H)
ppm13C NMR (75 MHz, [D6]DMSO): d = 184.3, 165.3, 156.2, 154.8,
152.6, 146.2, 134.8, 133.7, 133.0, 130.2, 127.1, 122.8, 122.3,
114.0, 112.2, 112.0, 111.8, 109.7 ppm. MS (ESI): m/z 879 (8), 878
(14), 877 (30), 874 (78), 873 (100), 871 (84), 868 (30), 867 (6).
HRMS (ESI): calculated for C24H11Br6O6 874.5590 found 874.5598.
5.5.15. (Z)-5-(3-Bromo-4-hydroxybenzylidene)-4-(3-bromo-4-
hydroxyphenyl)-3-(furan-2-carbonyl)furan-2(5H)-one (9)
Following procedure D,
a-hydroxy-ketone 1b (61 mg,
0.25 mmol), dioxinone 2d (97 mg, 0.5 mmol) and 3-bromo-4-
methoxybenzaldehyde 3b (54 mg, 0.25 mmol) were used to afford
9 as a brown solid in 30% yield (40 mg, 0.075 mmol). Mp = 246 °C.
1H NMR (300 MHz, [D6]DMSO): d = 11.05 (br s, 1H), 10.98 (br s,
1H), 8.10 (d, J = 2.1 Hz, 1H), 8.02 (d, J = 0.6 Hz, 1H), 7.78 (dd,
J = 8.7, 2.1 Hz, 1H), 7.55 (d, J = 2.1 Hz, 1H), 7.46 (dd, J = 3.9,
0.9 Hz, 1H), 7.29 (dd, J = 8.4, 2.1 Hz, 1H), 7.04 (t, J = 7.8 Hz, 2H),
6.69 (dd, J = 3.9, 1.8 Hz, 1H), 6.39 (s, 1H) ppm. 13C NMR (75 MHz,
[D6]Acetone): d = 176.3, 166.1, 156.84, 156.81, 152.8, 149.5,
147.0, 136.8, 134.8, 132.9, 131.1, 127.6, 123.5, 122.6, 117.6,
117.3, 116.2, 113.6, 110.8, 110.6 ppm. IR (neat): 2289, 1743,
1600, 1543, 1496, 1453, 1398, 1299, 1182, 986, 822, 772, 671,
587, 529 cmꢀ1. MS (ESI): m/z = 531 [MꢀH]+. HRMS (ESI): calculated
for C22H12Br2O6Na 552.8898 found 552.8903.
5.5.12. (Z)-5-(3,5-Dibromo-4-hydroxybenzylidene)-4-(3,5-
dibromo-4-hydroxyphenyl)-3-(4-hydroxybenzoyl)furan-2(5H)-
one (7h)
5.5.16. (Z)-5-(3-Bromo-4-hydroxybenzylidene)-4-(3,5-dibromo-
4-hydroxyphenyl)-3-(furan-2-carbonyl)furan-2(5H)-one (10)
Following procedure D,
a-hydroxy-ketone 1c (162 mg,
0.5 mmol), dioxinone 2a (234 mg, 1.0 mmol) and 3,5-dibromo-4-
methoxybenzaldehyde 3c (147 mg, 0.5 mmol) were used to afford
7h as a brown solid in 19% yield (69 mg, 0.96 mmol). Decomp.
128 °C. 1H NMR (300 MHz, [D6]DMSO): d = 10.67 (br s, 1H), 8.13
(s, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.55 (s, 2H), 6.79 (d, J = 8.7 Hz,
2H), 6.41 (s, 1H) ppm. 13C NMR (75 MHz, [D6]DMSO): d = 187.0,
165.4, 163.4, 153.4, 152.6, 152.2, 146.2, 134.6, 132.8, 132.4, 127.3
(2C), 124.4, 122.3, 115.5, 113.2, 112.1, 111.8 ppm. IR (neat):
3073, 1753, 1575, 1476, 1291, 1246, 1161, 974, 874, 741,
620 cmꢀ1. MS (ESI): m/z (%) = 719 (20), 718 (23), 717 (85), 716
(28), 715 (100), 714 (24), 713 (74), 711 (30). HRMS (ESI): calcu-
lated for C24H13Br4O6 716.7407 found 716.7394.
Following procedure D,
a-hydroxy-ketone 1c (81 mg,
0.25 mmol), dioxinone 2d (97 mg, 0.5 mmol) and 3-bromo-4-
methoxybenzaldehyde 3b (54 mg, 0.25 mmol) were used to afford
10 as a brown solid in 13% yield (20 mg, 0.03 mmol). Mp >266 °C.
1H NMR (300 MHz, [D6]Acetone): d = 8.13 (d, J = 0.7 Hz, 1H), 7.86
(d, J = 0.7 Hz, 1H), 7.82 (dd, J = 8.7, 2.1 Hz, 1H), 7.73 (s, 2H), 7.46
(dd, J = 3.7, 0.7 Hz, 1H), 7.14 (d, J = 8.5 Hz, 1H), 6.67 (dd, J = 3.7,
1.7 Hz, 1H), 6.47 (s, 1H), 5.63 (s, residual CH2Cl2) ppm. 13C NMR
(75 MHz, [D6]Acetone): d = 175.9, 166.0, 156.7, 155.8, 153.6,
152.8, 149.7, 146.9, 136.9, 134.0, 133.0, 127.5, 124.1, 123.8,
122.8, 117.7, 116.6, 113.7, 111.7, 111.0 ppm. IR (neat): 3077,
1736, 1643, 1591, 1561, 1461, 1416, 1331, 1277, 1166, 830, 807,
755, 655, 561 cmꢀ1. MS (ESI): m/z = 610 [MꢀH]+. HRMS (ESI): cal-
culated for C22H12Br3O6 608.8184 found 608.8180.
5.5.13. Cadiolide A: (Z)-3-(3,5-dibromo-4-hydroxybenzoyl)-4-
(3,5-dibromo-4-hydroxyphenyl)-5-(4-hydroxybenzylidene)-
furan-2(5H)-one [206763-40-8]: (7i)
5.6. Antibacterial activity
Following procedure D,
a-hydroxy-ketone 1c (162 mg,
0.5 mmol), dioxinone 2c (392 mg, 1.0 mmol) and p-methoxyben-
zaldehyde 3a (0.06 mL, 0.5 mmol) were used to afford 7i as an
orange solid in 18% yield (64 mg, 0.09 mmol). Decomp. 168 °C. 1H
NMR (300 MHz, [D6]DMSO): d = 10.56 (br s, 1H), 10.37 (br s, 1H),
7.93 (s, 2H), 7.79 (d, J = 8.7 Hz, 2H), 7.56 (s, 2H), 6.88 (d,
J = 8.7 Hz, 2H), 6.37 (s, 1H) ppm. 13C NMR (75 MHz, [D6]DMSO):
d = 185.3, 165.5, 159.9, 156.0, 155.6, 152.4, 144.6, 133.7, 133.6,
133.0, 129.8, 124.1, 122.7, 121.1, 118.3, 116.2, 111.7, 111.3 ppm.
IR (neat): 3073, 1749, 1597, 1538, 1293, 1170, 987, 740 cmꢀ1. MS
(ESI): m/z (%) = 719 (20), 718 (20), 717 (62), 716 (26), 715 (100),
714 (22), 713 (60), 711 (13). HRMS (ESI): calculated for
The following eight bacteria were provided by the Spanish
Type Culture Collection (CECT): Bacillus cereus (CECT 148),
Staphylococcus aureus (CECT 86), Enterococcus faecalis (CECT
481), Bacillus subtilis (CECT 35), Salmonella typhi (CECT 409),
Escherichia coli 405 (CECT 405), Escherichia coli 100 (CECT 100)
and Erwinia carotovora (CECT 225). The minimum inhibitory con-
centration (MIC) was determined as the lowest compound con-
centration required to completely inhibit growth of bacteria, by
the 2-fold microtiter broth dilution method and according to
CLSI guidelines.7 Each compound was dissolved in dimethyl sul-
foxide (DMSO), and ten 2-fold serial dilutions of the stock were
prepared. Stock suspensions of bacteria were prepared to
OD620 = 0.001 in Nutrient Broth (Difco). Then, this suspension
C24H13Br4O6 716.7407 found 716.7402.
5.5.14. (Z)-3-(Furan-2-carbonyl)-5-(4-hydroxybenzylidene)-4-
(4-hydroxyphenyl)furan-2(5H)-one (8)
of bacteria (100
lL) was mixed with serial dilutions of each com-
pound (100 L) in each well of a 96-well of polypropylene
l
Following procedure D,
a
-hydroxy-ketone 1a (166 mg,
microplates and incubated at 28 °C or 37 °C, depending on each
strain growth conditions overnight (18 h). After, bacteria growth
was determined turbidimetrically at OD620 in each well using a
microplate reader (Thermo Scientific Multiskan FC multimode).
Each experiment was performed by triplicate. The antibacterial
activity was expressed as the MIC range and defined as the low-
est concentration of test compounds that inhibited bacterial
1.0 mmol), dioxinone 2d (388 mg, 2.0 mmol) and 4-methoxyben-
zaldehyde 3a (0.11 mL, 1.0 mmol) were used to afford 8 as a yellow
solid in 37% yield (143 mg, 0.37 mmol). 1H NMR (300 MHz,
[D6]DMSO): d = 10.24 (br s, 1H), 10.11 (br s, 1H), 7.97 (d,
J = 1.8 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.35 (d, J = 3.6 Hz, 1H),
7.28 (dd, J = 6.9, 1.8 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 6.85 (d,
J = 6.9, 9.0 Hz, 2H), 6.64 (dd, J = 3.6, 1.8 Hz, 1H), 6.37 (s, 1H) ppm.
13C NMR (75 MHz, [D6]DMSO): d = 176.0, 165.8, 159.8, 159.6,
growth. Tetracycline hydrochloride was used as
compound.
a reference
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A. Boulangé et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
11
5.7. Cytotoxicity studies
Competiveness, the European Regional Development Fund
(FEDER) and research grants from Generalitat Valenciana
(GVACOMP2014-006 and PROMETEO II/2013/014). A.G. is thankful
to the Spanish Ministry of Education, Culture and Sport for a fel-
lowship from FPU program.
The cytotoxicity of the most active compounds, 6c, 7c, 9 and 10
was determined at 30 and 100 lM on freshly isolated human neu-
trophils by the use of two different approaches: the MTT colori-
metric assay and by flow cytometry analysis of cell apoptosis and
survival.14 Human neutrophils were obtained from buffy coats of
healthy donors by Ficoll-Hypaque density gradient centrifugation
as described.10
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.04.010.
5.8. MTT assay
References and notes
The viability of neutrophils was determined using the
previously described MTT (3[4,5-dimethylthiazol-2-yl]-2,5-
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lL of
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Cells were incubated in the absence or presence of compounds at
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100
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200 L of DMSO was added to each well to dissolve the formazan.
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l
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Mannedorf, Switzerland).
5.9. Cytofluorometric analysis of apoptosis, necrosis and
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Freshly isolated neutrophils were resuspended in supple-
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lL were cultured
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and/or necrotic (annexin V+ and PI+), and viable nonapoptotic
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Acknowledgments
We gratefully acknowledge the Région Haute Normandie for
financial support to A.B. This study was supported by grants
14. Smith, L. E.; Rimmer, S.; MacNeil, S. Biomaterials 2006, 27, 2806.
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Please cite this article in press as: Boulangé, A.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.04.010