Antifungal 3,5-disubstituted furanones: From 5-acyloxymethyl to 5-alkylidene derivatives

Article abstract of DOI:10.1016/j.bmc.2010.01.030

5-Acetoxymethyl-3-(4-bromophenyl)-2,5-dihydrofuran-2-one previously described as highly antifungally active was found to provide the corresponding 5-methylene derivative via an unusual DMSO-promoted elimination of the ester group at C5 under antifungal assay conditions. Since the latter possessed nearly the same antifungal effect as that originally reported for the former, the 5-acetoxymethyl furanone just served as a precursor of the actual antifungally active species. A few series of compounds with alkyloxy, aryloxy and alkylidene substituents at C5 of the parent furanone structure were therefore prepared and evaluated. In line with the ease of elimination of the substituent from C5, low activities of the 5-alkoxy compounds were observed. On the other hand, their 5-aryloxymethyl congeners were found to be capable of liberating the antifungally active 5-methylene furanone into the testing medium. The antifungal effect of the 5-alkylidene derivatives was highly sensitive to substitution of the alkylidene moiety; a substituent in the allylic position was necessary for a compound to retain high activity. Parallel evaluation of cytostatic activity showed moderate activities of the antifungally active derivatives against HeLa S3 and CCRF-CEM lines. Cell cycle analysis of CCRF-CEM cells following the treatment with 5-methylene-3-(4-bromophenyl)-2,5-dihydrofuran-2-one revealed that this compound is a necrotic agent.

Full text of DOI:10.1016/j.bmc.2010.01.030

Bioorganic & Medicinal Chemistry 18 (2010) 1988–2000
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antifungal 3,5-disubstituted furanones: From 5-acyloxymethyl
to 5-alkylidene derivatives
a
a
Petr Šenel a, Lucie Tichotová a, Ivan Votruba b, Vladimír Buchta a,c, Marcel Špulák , Jirí Kuneš ,
ˇ
a
a
a,
*
ˇ
Milan Nobilis , Ondrej Krenk , Milan Pour
a Centre for New Antivirals and Antineoplastics, Department of Inorganic and Organic Chemistry, Charles University, Faculty of Pharmacy, Heyrovského 1203,
CZ-500 03 Hradec Králové, Czech Republic
b Centre for New Antivirals and Antineoplastics, Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo n. 2, CZ-166 10 Prague 6, Czech Republic
c Department of Clinical Microbiology, University Hospital and Charles University, Faculty of Medicine, Sokolská 581, CZ-500 12 Hradec Králové, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Acetoxymethyl-3-(4-bromophenyl)-2,5-dihydrofuran-2-one previously described as highly antifungal-
ly active was found to provide the corresponding 5-methylene derivative via an unusual DMSO-promoted
elimination of the ester group at C5 under antifungal assay conditions. Since the latter possessed nearly
the same antifungal effect as that originally reported for the former, the 5-acetoxymethyl furanone just
served as a precursor of the actual antifungally active species. A few series of compounds with alkyloxy,
aryloxy and alkylidene substituents at C5 of the parent furanone structure were therefore prepared and
evaluated. In line with the ease of elimination of the substituent from C5, low activities of the 5-alkoxy
compounds were observed. On the other hand, their 5-aryloxymethyl congeners were found to be capa-
ble of liberating the antifungally active 5-methylene furanone into the testing medium. The antifungal
effect of the 5-alkylidene derivatives was highly sensitive to substitution of the alkylidene moiety; a sub-
stituent in the allylic position was necessary for a compound to retain high activity. Parallel evaluation of
cytostatic activity showed moderate activities of the antifungally active derivatives against HeLa S3 and
CCRF-CEM lines. Cell cycle analysis of CCRF-CEM cells following the treatment with 5-methylene-3-(4-
bromophenyl)-2,5-dihydrofuran-2-one revealed that this compound is a necrotic agent.
Received 24 August 2009
Revised 11 January 2010
Accepted 13 January 2010
Available online 25 January 2010
Keywords:
Antifungal activity
Cytostatic activity
Furanones
Butenolides
Synthesis
Pd-catalysis
Flow cytometry
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the former suffers from a poor pharmacokinetics, potential toxicity
(conventional amphotericin B) or cost (lipid forms of amphotericin
The past three decades have witnessed a significant increase of
incidence of invasive fungal infections, especially among hospital-
izedpatients. Anincreasedmorbidityandmortalityofmycoticinfec-
tions is associated with a growing number of vulnerable patients to
opportunistic fungi as a consequence of progress in medicine, im-
proved diagnostics and awareness of fungi as etiology of these infec-
tions.1 Patients treated with immunosuppressive agents, broad-
spectrum antibiotics, antineoplastics and anti-HIV agents as well
as those undergoing extensive surgery or other invasive procedures
are at higher risk of contracting a systemic mycosis.2–4 The majority
of invasive mycotic diseases are caused by Candida and Aspergillus
species, even though other emerging fungi can play an important
role in etiology5 (Mucorales, Scedosporium, Fusarium). While there
are several classes of antifungal drugs on the market, not all of them
can be considered for a systemic use. In fact, amphotericin B of the
polyene antibiotics and antifungal azoles remain the most often
employed drugs for the treatment of systemic infections. While
B), classical azole antifungals are continuously being developed6,7
since a number of fungi can easily acquire resistance to them. More-
over, they usually have a limited effect on some new, not very com-
mon fungal species. In this regard, the shift of the spectrum in
etiology resulting from the change in epidemiology of nosocomial
and opportunistic mycoses is important to mention. For example,
the proportion of non-albicans Candida yeasts with variable or no
susceptibility to fluconazole such asCandida krusei andCandida glab-
rata, respectively, is on the rise.8 For this reason, the development of
structurally novel antifungal drugs is badly needed, with echinocan-
dins being a notable example of an emerging group of novel antifun-
gals based on a cyclic peptide structure.9
Some time ago, we reported the identification of 3,5-disubsti-
tuted furanones derived from the structure of a natural butenolide,
(ꢀ)incrustoporin (Fig. 1), as potential antifungal agents.10 Biologi-
cal screening of several small libraries of (ꢀ)incrustoporin ana-
logues has revealed10,11 that the endocyclic double bond is
necessary for antifungal effect of the compounds and that the
effect may be further boosted by the substitution of the C3-aryl
ring with halogens. Further elaboration of C5 substitution led to
* Corresponding author. Tel.: +420 495 067 277; fax: +420 495 067 166.
E-mail address: pour@faf.cuni.cz (M. Pour).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.030

P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
Br
1989
O
O
O
Z
O
3
O
O
OCOR
5
OCOR
5
1
Z = 3,4-diCl, 4-Br, 3-Cl
R = alkyl
(-)incrustoporin
Br
Br
O
O
comparable to
amphotericin B
O
O
OR(Ar)
R
O
O
Z
O
Figure 2. Proposed elaboration of substitution at C5.
Z
CH2OCOR
pectedly, the 5-methylene and 5-methyl derivatives 4 and 5 were
detected as well. In the process of further development of lactone
esters of type 1 following our first report,12 we also observed the
formation of impurities, which were tentatively assigned as the
corresponding 5-methylene derivatives based on the NMR spectra,
upon long term storage of the compounds at room temperature.
Thus, we assumed that in the biotransformation study, compound
1a afforded the 5-methylene furanone 4 first, the enzymatic reduc-
tion of which subsequently furnished the 5-methyl metabolite 5.
Given the results of the biotransformation study and the stabil-
ity of compounds 1 having arisen as an issue, the possibility that
furanones of type 4 were the actual active species attacking the
fungal cell membrane and esters of type 1 just served as their pre-
cursors was apparent. In addition, if these 5-methylene lactones
were indeed the antifungally active species, our as yet unexplained
observation12 that the antifungal effect of lactones 1 did not de-
pend on the sense of chirality at C5 would thus be logically
clarified.
2
3
no antifungal effect
no antifungal effect
non-specific Michael addition
unlikely mechanism of action
Figure 1. Overview of structure–antifungal activity relationships on (ꢀ)incrusto-
porin analogues 1–3.
5-acyloxymethyl-3-(halogenated phenyl)-2,5-dihydrofuran-2-ones
(1).12 The activities of these compounds did not depend either on
the nature of the acyl group or the sense of chirality at C5, and were
comparable to amphotericin B in antifungal potency in vitro13 (in
terms of lg/mL). Even though the structure of the 3,5-disubstituted
furanones seemed to indicate that the compounds could be non-
specific inhibitors of fungal growth due to an easy Michael addition,
ketone11 (2) and pentenolide14 (3) analogues of these butenolides
were later found to possess no antifungal activity at all. Since ke-
tones 2 in particular should be far better Michael acceptors than lac-
tones 1, a simple Michael addition to the b-carbon of the double
bond is an unlikely mechanism of action. Unfortunately though,
an in vivo evaluation in a mouse model of experimental candidiasis
or aspergillosis has so far been hampered by the relatively high lipo-
philicity of the compounds, which did not enable intravenous
administration.
Importantly, these results meant a significant shift from our ori-
ginal assumption12 that compounds 1 are just more lipophilic
transport forms of their parent 5-hydroxymethyl analogues, which
easily penetrate fungal cell walls and are hydrolyzed inside the
cells. In order to shed more light on these issues, we focused on
the issue of stability of 1a under the conditions of in vitro studies
first.
A recent flow cytometric study15 carried out with one of the
most effective compounds 1 (1a: Z = p-Br, R = CH3, Scheme 1)
revealed that this substance interacted with an as yet unknown
target in the fungal cell membrane. A preliminary evaluation16 of
metabolic conversions of 1a after intraperitoneal administration
in mice revealed that most of the compound underwent fast ester
hydrolysis and conjugation to glucuronic acid. Somewhat unex-
Second, replacement of the C5 acyloxy group with substituted
aryloxy and alkyloxy functions was studied (Fig. 2). The replace-
ment of the ester function with an ether group would prevent
in vivo enzymatic hydrolysis with subsequent conjugation to glu-
curonic acid observed16 for 1a, and the attachment of a hydrophilic
group (COOH) to the aryl or alkyl moiety would also help to im-
prove the lipophilicity profile of the compounds. Moreover, since
these groups eliminate less easily than the acyloxy function, deriv-
atives with higher stability would be obtained. Third, the influence
of the replacement of the C5 acyloxymethyl group with an alkyl-
idene side chain was evaluated. In order to obtain more diverse
SARs, cytostatic activity on a panel of cancer cell lines was also
evaluated in addition to antifungal.
Br
Br
O
O
O
O
1a
AcO
O
2. Chemistry
glc
HOOC
major
-AcOH
2.1. Stability of compound 1a
Synthetic standard of the elimination product 4 was prepared
by the reaction of the previously described alcohol 612 with
PPh3/I2/imidazole. The initially formed 5-iodomethyl derivative
underwent in situ elimination to produce the 5-methylene fura-
none 4 (Scheme 2).
Br
Br
O
O
O
O
Subsequently, stability of 1a under antifungal assay conditions
was explored. First, furanone 1a was dissolved in DMSO-d6. 1H
NMR monitoring of the solution showed the presence of a mixture
4
5
Scheme 1. Proposed metabolic conversion of 1a to 5.

1990
P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
Br
OH
OH
Br
O
O
O
PPh3, I2
MOMCl
iPr2EtN
imidazole
O
OH
COOH
COOMOM
6
4
11a-b
Scheme 2. Synthesis of elimination product 4.
a: 4-OH, b: 3-OH
Scheme 4. Preparation of MOM-protected acids 11a–b.
of 1a and 4 in an approximate 1:1 ratio formed within minutes,
with the ratio of both compounds having remained the same even
after 24 h. Noteworthy, a solution of 1a in THF-d8 was stable after
one day at rt. Second, following the procedure for antifungal activ-
ity assays, the cultivation medium was added to a freshly prepared
stock solution of 1a in DMSO. HPLC analysis revealed that the med-
ium further accelerated the elimination, since only furanone 4 was
detectable in the mixture in less than 3 h.
O
O
Br
Br
FBSCl
Pyridine
O
O
O
12
13
HO
O2S
K2CO3
Z
2.2. Synthesis and stability of 5-alkoxy- and phenoxy
derivatives
F
O
OH
Br
11a-g
O
O
Ester 710 was subjected to epoxidation, and the resultant epox-
ide 8 was cleaved with sodium methanolate or ethanolate. Upon
acidic workup, saturated lactones 9a and 9b were obtained. The
double bond was then introduced via phenylselenenylation/selen-
oxide elimination10 to yield the 5-alkoxymethyl compounds 10a,b
(Scheme 3).
A series of 5-(subst. phenoxy)methyl derivatives was prepared
next, including two derivatives with a carboxyl on the phenyl ring,
which were deliberately prepared in order to have more hydrophilic
compounds. Hence, methoxymethylesters of 3- and 4-hydroxyben-
zoic acid 11a,b were prepared first by partial protection of the acids
with methoxymethyl chloride (Scheme 4). Even though the protec-
tion seems trivial, the compounds have only been mentioned in pat-
ent literature.17
Since the softer phenolate anions were unable to cleave the
epoxide ring in 8, OH group in lactone 1212 was esterified with
p-fluorobenzenesulfonyl chloride (FBSCl) to afford the sulfonyl
ester 13. Upon nucleophilic substitution with substituted pheno-
lates 11a–g, compound 13 furnished a series of 5-(subst. phen-
oxy)methyl derivatives 14a–g, which were converted into the
desired 2,5-dihydrofuranones 15a–g in the same fashion as de-
scribed above for 9a–b (Scheme 5).
14a-g
Z
1. LDA
Z: a: 4-COOMOM
b: 3-COOMOM
c: 4-OCH3
2. PhSeBr
3. H2O2/NaHCO3
d
e
: H
: 4-I
O
O
f: 3-Br
Br
g: 4-COOCH3
15a-g
O
Z
Scheme 5. Synthesis of phenoxymethyl derivatives 15a–g.
O
a: 4-COOMOM
Z:
Br
b
: 3-COOMOM
: 4-OCH3
O
O
c
Compounds 15a and 15b were deprotected on Dowex 50 in
MeOH to afford free acids 16a and 16b and compound 15c was
demethylated16 to afford 4-hydroxy derivative 16c (Scheme 6).
15a-c
Z
Dowex
BBr3
O
O
Br
Br
Br
mCPBA
O
O
O
O
Br
O
Br
O
O
O
8
7
-
R
1.
O
2. H+
O
16a
: 4-COOH
16c
COOH
16b: 3-COOH
O
O
Br
1. LDA
OH
O
O
O
O
2. PhSeBr
3. H2O2/NaHCO3
Scheme 6. Synthesis of phenoxymethyl derivatives 16a–c.
10a-b
9a-b
R
R
The stability of the target compounds was assessed using com-
pounds 10a and 16b as the representatives of both groups as de-
scribed above for 1a. While the 5-methoxymethyl furanone 10a
R = a: -CH3, b: -CH2CH3
Scheme 3. Synthesis of 5-alkoxymethyl derivatives products 10a–b.

P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
1991
was found to be perfectly stable in both DMSO solution and the
cultivation medium, elimination of the 5-(3-carboxyphen-
oxy)methyl derivative 16b cleanly afforded the methylene lactone
4, albeit more slowly than in the case of the 5-acetyloxymethyl
furanone 1a. As evidenced by HPLC, the elimination process under
antifungal assay conditions was over in 5 h, and produced
exclusively compound 4 and antifungally inactive (vide infra)
m-hydroxybenzoic acid.
O
O
O
O
PHT
Br
Br
O
R1
O
PdCl2(TFP)2
CuI, Et3N
Br
R1
21
22a-d
1. LiOH/H2O/H+
2. AgNO3
O
2.3. Syntheses of 5-alkylidene furanones and 5-unsubstituted
furanone
Br
O
R1
Pd-catalyzed carbonylative lactonization18 was used as the key
step in the preparation of the 5-ethylidene compound 20a. Propar-
gylic alcohol 17a was converted into iodo allylic alcohol 18a. Sub-
sequent oxidation19 of 18a afforded b-iodo enone 19a, suitable for
a cyclocarbonylation process. Even though the yield of the key step
was low, no optimization attempts were made due to zero biolog-
ical activity of furanone 20a. Similarly, alcohol 17b20 was
converted into furanone 20b21 (Scheme 7).
Most of the other 5-alkylidene furanones 24 were obtained via
sequential functionalization of methyl (E)-dibromoacrylate 21.22
Thus, Sonogashira coupling into b-position gave a series of b-alky-
nyl esters 22a–d, whose carboxyls were liberated by hydrolysis
and subjected to cyclization onto the triple bond to yield the corre-
23a-d
1. 4-BrPhZnI,
PdCl2(TFP)2
or Pd(TFP)2
2. DOWEX
4-BrPhZnI,
PdCl2(TFP)2
O
Br
O
Br
O
O
R2
24a-c
24d
R1
a
b
=
: -CH2OTHP, : -CH2CH2OTHP,
c: -CH2CH2CH2OTHP, d: -C6H5
R2 = a: -CH2OH, b: -CH2CH2OH,
c
: -CH2CH2CH2OH
sponding
a-bromolactones 23a–d. The 4-bromophenyl moiety
Scheme 8. Synthesis of alkylidene butenolides 24a–d.
Br
Br
Br
Red-Al
I2
BuLi
O
O
Br
O
I
(CH2)nOH
24a-b
OH
OH
18a
17a
R3COCl,
Pyridine
IBX
Br
O
Br
O
O
Br
PdCl2(PPh3)2
CO, NaHCO3
(CH2)nOCOR3
O
25a-c
I
25a: n=1, R3=CH3
25b: n=1, R3=C6H5
20a
O
25c
R
: n=2, 3=CH3
19a
Scheme 9. Preparation of esters 25a–c.
Br
Br
OTHP
Br-
Ph
1.
PdCl2(TFP)2,
CuI, Et3N
PPh3
BrCH2COOCH3
Ph P+ CH2 COOCH3
I
Ph
2. DOWEX
1. LDA
OH
17b
Red-Al
Br
O
Br
I2
2.
O
O
O
Br
Pd(OAc)2,PPh3
O
CO, Et3N
O
Br
I
O
OH
H3COOC
18b
20b
26
Scheme 7. Synthesis of lactones 20a,b.
Scheme 10. Synthesis of lactone 26.

1992
P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
bond at C5, and further investigation was carried out in this
direction.
Much surprisingly then, the 5-ethylidene derivative 20a differ-
Br
Br
O
O
DMSO then
testing medium
O
O
rt
ing from the 5-methylene furanone 4 by an additional methyl
group was completely inactive. Hence, another methyl group
added to the exocyclic double bond in 20a disabled the binding
of the compound to its cell membrane target. However, the addi-
tion of a hydroxyl into an allylic position restored the antifungal ef-
fect to a significant degree suggesting the need for the presence of
a binding group. The 5-(2-hydroxyethylidene) furanone 24a pos-
sessed antifungal activity against C. albicans ATCC44859 compara-
ble to furanone 4, even though its efficiency against all other
strains tested was somewhat reduced. Insertion of more carbons
between the double bond and the OH group led to practically inac-
tive compounds 24b–c. Similarly, esterification of the OH group in
24a–c had a detrimental effect, since ester 25a had higher MICs
than its precursor 24a, and compounds 25b–c were inactive.
The antifungal activity of furanone 4 and phenoxy derivatives
15c–16c is superior to that of azole drug standard (fluconazole),
especially against C. krusei, C. glabrata and filamentous strains. In
addition, antifungal activity of amphotericin B is also presented.
Cytostatic activity of selected compounds expressed as IC50 was
also evaluated (Table 2). Apparently, all antifungally active deriva-
tives 4 (and hence its precursors 15d–16b) as well as 24a also pos-
sessed cytostatic activities in the micromolar range against HeLa
S3 and CCRF-CEM cell lines. It is, however, interesting to note that
cytostatic and antifungal action do not parallel each other. First,
the 5-methylene furanone 4 itself has one of the highest antifungal
activities shown in Table 1, but its cytostatic activity is moderate to
low, and limited to CCRF-CEM and HeLa S3 cells. Similar data were
obtained when another efficient precursor of 4, compound 16a,
was subjected to cytostatic assays, with an even lower activity
against CCRF-CEM cells (8.70 0.43) than that of 4 (2.52 0.11).
Second, when the 5-phenoxy furanones 15d–16b were subjected
to screening, the results were somewhat different, even though
all furanones 15d–16b are prone to the elimination process leading
to the same 5-methylene furanone 4 in DMSO. Unlike 16a and 16b,
activities in the micromolar range against HL-60 cells were re-
corded upon screening of compounds 15d–g. Also, the screening
of furanone 16b eliminating a molecule of m-hydroxybenzoic acid
showed a higher effect on CCRF-CEM cells compared to the closely
related 16a releasing p-hydroxybenzoic acid on elimination. Thus,
it appears that even though all compounds furnish the 5-methy-
lene furanone 4, cytostatic activities could be modulated by the
phenolic side product. Again, a series of substituted phenols
(p-COOH, 4-I, H) was evaluated under the same conditions, and
OFG
4
1a FG = Ac
3 hrs
16b
FG = 3-HO2C-C6H4 5 hrs
Scheme 11. Production of active species 4.
was then attached via another Pd-coupling process with
4-bromophenylzinc iodide. Noteworthy, PdCl2(TFP)2 was used23,24
as the catalyst in this, somewhat difficult, coupling. Optimization
of the conditions was done in case of bromolactone 23a; replace-
ment of THF with DMF and reduction of the catalyst to a more ac-
tive Pd(TFP)2 prior to the coupling increased the yield to 65%
(Scheme 8).
Following the lead structure of active butenolides 1, we also
prepared esters of the 5-hydroxyalkylidene furanones 24a and
24b, resulting in 3 derivatives 25a–c (Scheme 9).
Finally, the derivative bearing an ester group 26 was obtained
by the Wittig condensation25 of (4-bromophenyl)maleic anhy-
dride26 with (methoxycarbonylmethyl)triphenyl phosphonium
ylide (Scheme 10).
3. Results and discussion
Evaluation of stability of the previously described furanone es-
ter 1a and furanone ether 16b as a representative of the 5-aryl-
oxymethyl derivatives has shown that both compounds smoothly
produced the 5-methylene lactone 4 via elimination under anti-
fungal assay conditions (Scheme 11). Notably, a couple of recent
examples27,28 of eliminations promoted by neat DMSO involve
heating to higher temperatures and halogen as a leaving group.
Elimination depicted in Scheme 11 is virtually unprecedented gi-
ven the mild conditions and leaving groups as poor as an ester
and even an ether (!). Hence, as the first MIC value reading is done
in 24 h, the antifungal activity previously reported for 1a can be as-
cribed to the elimination product 4 rather than lactone 1a.
Antifungal activity (IC80) evaluation (Table 1) of derivative 4 as
well as the antifungal profile of the compounds then provided con-
clusive evidence for the 5-methylene furanone 4 being the antifun-
gally active species. The synthetically prepared furanone
4
possessed excellent activity against Candida albicans ATCC44859
and C. glabrata 20/I strains, identical to that originally reported12
for its 5-acetoxymethyl precursor 1a. Of the filamentous strains,
the effect on Aspergillus fumigatus is also noteworthy. As further
shown in Table 1, the activities of the 5-aryloxymethyl and 5-alk-
oxymethyl compounds are apparently dependent on the ease of
eliminating the substituent at C5. Clearly, the 5-phenoxy deriva-
tives 15c–16c with a surprisingly good ability of the C5-phenoxy
group to leave under the assay conditions also served as precursors
of furanone 4. The screening of the compounds shows broad spec-
trum of activity, corresponding in all cases to that of 4, with the
exception of 15e against Candida parapsilosis, C. krusei 1 and 2,
and Absidia corymbifera. For the sake of excluding the contribution
of the phenols liberated into the medium, a series of substituted
phenols (p-COOH, 4-I, H) was evaluated under the same conditions,
and the compounds showed no activity whatsoever, even at
the compounds displayed no activity at all at 10
lmol/L. In
addition, antifungally inactive compounds 10a and 10b showed
cytostatic activities against HeLa S3 and CCRF-CEM cells. The 5-
methoxymethyl furanone 10a exhibited micromolar activity
against CCRF-CEM cells, and its 5-ethoxymethyl congener 10b
showed reasonable activity against HeLa S3 cells, while its effect
on CCRF-CEM cells was reduced as compared to 10a.
Since furanone 4 was the apparent active substance signifi-
cantly reducing the growth of several fungal strains and displaying
moderate cytostatic activity on HeLa S3 and CCRF-CEM lines, the
compound was subjected to flow cytometric studies with a view
to shedding more light on the nature of the cytostatic effect. The
cell cycle analysis of CCRF-CEM cells (Fig. 3) following treatment
with compound 4 at IC50 (2.5 l
mol Lꢀ1) showed G1 phase block
500
l
mol/L. On the other hand, 5-alkoxy compounds 10a and
accompanied by an accumulation of cells in S phase and depletion
of G2/M phase. Such pronounced effect resulted in a high amount
of cell debris (Fig. 3, blue area) and prompted that substance 4 in-
duces cell death.
Annexin V binding method (combination of annexin V-FITC and
propidium iodide) has shown that 72 h treatment of CCRF-CEM
10b displayed just a low effect on the growth of C. albicans
ATCC44859, and a marginal effect of 10a against C. albicans
ATCC90028 and A. fumigatus was also observed. Furanone 20b
unsubstituted at C5 was also prepared, and found to be inactive.
These results demonstrated the importance of the exocyclic double

P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
1993
Table 1
Antifungal activity of prepared compounds and drug standards (MIC [
l
mol Lꢀ1ꢀ1])
No.
Time (h)
CA1a
CA2b
CPc
CK1d
CK2e
CTf
CGg
CLh
TBi
AFj
ACk
TMl
1a
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
24
48
0.49
1.95
0.49
1.95
15.62
31.25
31.25
62.5
0.97
1.95
0.97
1.95
0.48
1.95
0.97
1.95
0.48
1.95
0.48
1.95
0.97
1.95
0.97
1.95
250
>500
250
250
1.95
7.81
62.5
125
62.5
125
>125
>125
7.81
31.25
>500
>500
15.62
500
>125
>125
0.03
0.06
1
3.90
3.90
3.90
7.81
31.25
31.25
125
3.90
15.62
7.81
15.62
125
250
125
250
7.81
15.62
7.81
31.25
31.25
>500
7.81
31.25
15.62
15.62
7.81
15.62
15.62
15.62
7.81
15.62
>500
>500
250
3.90
7.81
7.81
7.81
250
250
250
250
7.81
15.62
7.81
31.25
31.25
>500
7.81
15.62
15.62
15.62
7.81
15.62
15.62
15.62
7.81
15.62
>500
>500
250
7.81
7.81
7.81
7.81
250
500
250
250
7.81
15.62
15.62
31.25
31.25
>500
7.81
15.62
15.62
15.62
7.81
15.62
15.62
15.62
7.81
15.62
>500
>500
250
7.81
7.81
7.81
15.62
62.5
62.5
250
1.95
1.95
1.95
1.95
125
3.90
7.81
7.81
15.62
62.5
125
7.81
7.81
3.90
15.62
62.5
125
0.98
3.91
1.95
7.81
31.25
62.5
62.5
125
3.90
7.81
1.95
15.62
3.90
7.81
3.90
15.62
3.90
7.81
1.95
3.90
1.95
7.81
1.95
7.81
>500
>500
250
7.81
15.62
31.25
125
125
250
125
125
31.25
62.5
125
1.95
3.90
1.95
3.90
31.25
31.25
15.62
15.62
1.95
1.95
1.95
3.90
1.95
3.90
1.95
1.95
3.90
3.90
0.97
0.97
0.97
1.95
1.95
1.95
31.25
31.25
250
4
10a
10b
15c
15d
15e
15f
125
62.5
62.5
0.97
1.95
1.95
1.95
1.95
3.90
1.95
1.95
3.90
3.90
0.97
1.95
1.95
1.95
1.95
1.95
>500
>500
500
125
250
125
250
125
250
1.95
7.81
1.95
7.81
3.90
7.81
3.90
7.81
3.90
7.81
0.97
3.90
3.90
15.62
1.95
3.90
>500
>500
500
7.81
7.81
7.81
15.62
15.62
31.25
7.81
15.62
15.62
15.62
3.90
7.81
7.81
7.81
7.81
7.81
>500
>500
250
3.90
7.81
3.90
7.81
7.81
31.25
7.81
15.62
7.81
15.62
3.90
7.81
7.81
7.81
3.90
7.81
>500
>500
500
7.81
15.62
7.81
31.25
7.81
500
7.81
62.5
7.81
15.62
7.81
15.62
15.62
15.62
7.81
15.62
>500
>500
250
500
>500
>500
31.25
62.5
62.5
125
15.62
62.5
62.5
62.5
15.62
62.5
>500
>500
500
15g
16a
16b
16c
20a
20b
24a
24b
24c
24d
25a
25b
25c
26
500
500
500
500
500
500
500
500
500
500
250
7.81
15.62
125
31.25
62.5
125
15.62
31.25
125
31.25
62.5
250
15.62
31.25
125
15.62
62.5
62.5
250
>500
>500
>125
>125
31.25
62.5
>500
>500
250
>500
>125
>125
0.03
0.09
22
7.81
15.62
125
31.25
62.5
125
7.81
15.62
125
15.62
15.62
62.5
62.5
62.5
62.5
>125
>125
31.25
31.25
>500
>500
250
7.81
7.81
31.25
31.25
62.5
62.5
>125
>125
7.81
15.62
>500
>500
15.62
15.62
>125
>125
1.08
1.08
17
250
125
250
250
250
250
125
250
>500
>500
>125
>125
31.25
62.5
>500
>500
>500
>500
>125
>125
0.07
0.14
0.8
>500
>500
>125
>125
31.25
62.5
>500
>500
250
>500
>125
>125
0.03
0.14
6.5
>500
>500
>125
>125
7.81
62.5
>500
>500
500
>500
>125
>125
0.14
0.14
>50
>500
>500
>125
>125
62.5
62.5
>500
>500
500
>500
>125
>125
0.14
0.18
>50
>500
>500
>125
>125
31.25
125
>500
>500
>500
>500
>125
>125
0.09
0.11
3
>500
>500
>125
>125
62.5
125
>500
>500
>500
>500
>125
>125
0.14
0.27
3.3
>500
>500
>125
>125
31.25
31.25
>500
>500
>500
>500
>125
>125
1.08
1.80
4
>500
>500
>125
>125
15.62
31.25
>500
>500
>500
>500
>125
>125
0.18
0.23
>50
250
>125
>125
1.08
2.16
>50
AmBm
FLUn
2
3.3
13
>50
>50
5
>50
3.3
9
>50
>50
26
MIC were defined as 80% inhibition of the growth of control.
a
Candida albicans ATCC44859.
C. albicans ATCC90028.
C. parapsilosis ATCC22019.
C. krusei ATCC 6258.
C. krusei E28.
C. tropicalis 156.
C. glabrata 20/I.
C. lusitaniae 2446/I.
b
c
d
e
f
g
h
i
Trichosporon beigelii 1188.
Aspergillus fumigatus 231.
Absidia corymbifera 272.
j
k
l
Trichophyton mentagrophytes 445 (MIC values were determined after 72 and 120 h).
Amphotericin B (MIC were defined as 95% inhibition of the growth of control).
Fluconazole.
m
n
cells with furanone 4 (2.5 l
mol Lꢀ1ꢀ1) mostly induces necrosis
4. Conclusion
(Fig. 4B, Q2) while the early apoptosis was not detected (Fig. 4B,
Q1). At IC50 and higher concentrations, more than 80% of cells
are in necrotic stage. These data indicate that the tested compound
is a necrotic agent, which causes a concentration-dependent loss of
plasma and nuclear membrane integrity. This result clearly falls in
line with our previous finding that derivative 1a destroys the fun-
gal cell membrane in C. albicans and, given the above results, it
does so via liberating the actual active compound 4.
We have shown that 5-acyloxymethyl- and 5-aryloxymethyl-3-
aryl-2,5-dihydrofuran-2-ones produce 5-methylene furanones
such as 4 under standard antifungal assay conditions. The latter
compounds then act as the active species that destroy fungal cell
membranes. Notably, while the limited stability and fast biotrans-
formation of the 5-acyloxymethyl furanones into the correspond-
ing glucuronides precludes their further utilization in models

1994
P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
Table 2
Cytostatic activity (IC50
in vivo, their 5-aryloxy counterparts could be interesting for in vivo
[
l
mol Lꢀ1])
HL60
experiments, since they (1) are stable upon storage, (2) can release
the active 5-methylene furanones, and (3) the ether bond is not
degradable by plasmatic esterases. The importance of the methy-
lene group at C5 was further demonstrated by the screening of
the 5-alkyloxymethyl derivatives, which were antifungally inac-
tive. As regards further substitution of the 5-methylene moiety,
its replacement of the 5-methylene group with more complex
alkylidene substituents was unproductive, with a notable excep-
tion of 2-hydroxyethylidene (compound 24a), which restored the
antifungal activity close to the level of 4. It is therefore likely that
a substituent with a binding ability in the allylic position on the
alkylidene side chain is necessary.
The antifungally active substances 4 (and hence its precursors
15d–16b), and 24a as well as inactive derivatives 10a and 10b also
displayed cytostatic activities against HeLa S3 a CCRF-CEM cell
lines, even though they can be generally referred to as moderate
to low. Interestingly, the differences observed upon cytostatic
activity screening of the 5-phenoxymethyl furanones 15d–16b
which are all subject to the elimination affording 4 seem to
No.
L1210
HeLa S3
CCRF-CEM
4
21.00 0.75
18.00 1.66
16.00 0.63
NA
4.37 0.42
7.22 0.19
4.05 0.60
19.35 1.02
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
2.09 0.16
NAb
2.52 0.11
2.21 0.09
9.82 0.68
1.09 0.09
1.19 0.19
1.25 0.05
8.70 0.43
1.64 0.20
NA
NA
2.07 0.07
NA
NA
NA
10a
10b
15d
15e
15g
16a
16b
20a
20b
24a
24b
24c
24d
25a
25b
26
NTa
NA
NT
NT
1.94 0.12
4.67 0.30
4.00 0.26
4.50 0.30
2.69 0.22
1.44 0.08
NA
NA
3.06 0.26
NA
NA
NA
4.18 0.89
NA
NA
NT
12.31 2.00
7.42 0.22
NA
NA
7.71 0.15
NA
NA
NA
NA
NA
4.32 0.52
NA
NA
NA
a
Not tested.
Not active at relevant concentrations.
b
0
30
60
90
120
150
0
30
60
90
120
150
Channels (PI-A)
Channels (PI-A)
Figure 3. Cell cycle profile in CCRF-CEM cells treated with compound 4. Control cells (left) and cells grown in the presence of tested compound 4 (2.5
l
mol Lꢀ1ꢀ1, right).
Figure 4. Annexin V binding—detection of apoptotic/necrotic cells. Control cells (A) and cells grown in the presence of tested compound 4 (2.5 l
mol Lꢀ1ꢀ1, B).

P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
1995
indicate an influence of the liberated phenolic derivative on the
5.3. Antifungal activity
cytostatic effect of 4. Furanone 4 itself was found to be a necrotic
agent.
Finally, future work will be directed towards the introduction of
a hydrophilic group onto the phenyl ring at C3 of the furanone core
in compounds similar to 4, and towards further elaboration of the
allylic position in compounds similar to 24a.
In vitro antifungal activities of the compounds were evaluated
on a panel of four ATCC (C. albicans ATCC 44859, C. albicans ATCC
90028, C. parapsilosis ATCC 22019, C. krusei ATCC 6258) and eight
clinical isolates of yeasts (C. krusei E28, C. tropicalis 156, C. glabrata
20/I, Candida lusitaniae 2446/I, Trichosporon beigelii 1188) and fila-
mentous fungi (A. fumigatus 231, A. corymbifera 272, Trichophyton
mentagrophytes 445) from the collection of fungal strains deposited
at the Department of Biological and Medical Sciences, Faculty of
Pharmacy, Charles University, Hradec Králové, Czech Republic.
Three of the above ATCC strains (C. albicans ATCC 90028, C. parapsi-
losis ATCC 22019, C. krusei ATCC 6258) also served as the quality
control strains. All the isolates were maintained on Sabouraud dex-
trose agar prior to being tested.
Minimum inhibitory concentrations (MICs) were determined
by the microdilution format of the NCCLS M27-A guidelines.29 Di-
methyl sulfoxide (100%) served as a diluent for all compounds;
the final concentration did not exceed 2%. RPMI 1640 (Sevaphar-
ma, Prague) medium supplemented with L-glutamine and buf-
fered with 0.165 M morpholinepropanesulfonic acid (Serva) to
pH 7.0 by 10 N NaOH was used as the test medium. The wells
5. Experimental
5.1. General experimental procedures
All substances were purchased from Sigma–Aldrich and used as
received. THF was freshly distilled from sodium benzophenone ke-
tyl. DMF was dried over 3 Å molecular sieves. All anhydrous reac-
tions were performed in flame-dried Schlenk tubes under Ar
atmosphere. Analytical thin-layer chromatography (TLC) was con-
ducted on E. Merck TLC plates (Silica Gel 60 F254, aluminum back).
Silica Gel 60 (230–400 mesh) for column chromatography was pur-
chased from E. Merck. Melting points were determined on Kofler
block or Büchi B-545 Melting point apparatus and are uncorrected.
1H and 13C NMR spectra were recorded for CDCl3, DMSO-d6, CD3OD
and THF-d8 solutions at ambient temperature on a Varian Mer-
cury-Vx BB 300 spectrometer operating at 300 MHz for 1H and
75 MHz for 13C. Chemical shifts were recorded as d values in parts
per million (ppm), and were indirectly referenced to tetramethyl-
silane (TMS) via the solvent signal. Coupling constants (J) are
given in hertz. All assignments were made on the basis of
DPFG-NOE, gHSQC and gHMBC experiments. In certain cases of
mixtures of isomers, these were referred to as A and B. Infrared
spectra were recorded on a Nicolet 6700 FT-IR spectrophotometer.
Low resolution mass spectra were measured on Finnigan LTQ XL
apparatus. The purity of all target compounds was established
by elemental analysis carried out on a CHNS-OCE FISONS EA
1110 instrument.
of the microdilution tray contained 100
medium with twofold serial dilutions of the compounds (500–
0.48 mol/L for the new compounds) and 100 L of inoculum sus-
lL of the RPMI 1640
l
l
pension. Fungal inoculum in RPMI 1640 was prepared to give a
final concentration of 5 ꢁ 103 0.2 cfu mLꢀ1. The trays were incu-
bated at 35 °C and MICs were read visually for filamentous fungi
and photometrically for yeasts as an absorbance at 540 nm after
24 and 48 h. The MIC values for the dermatophytic strain
(T. mentagrophytes) were determined after 72 h and 120 h. The
MICs were defined as 80% inhibition of the growth of control.
MICs were determined twice and in duplicate. The deviations
from the usually obtained values given in Tables 1 and 2 were
no higher than the nearest concentration value up and down
the dilution scale.
5.2. Stability evaluation (HPLC)
5.4. Cytostatic activity assays
Routine chromatographic analyses were performed using a
Thermo Electron chromatograph. The chromatographic system
was composed of an SCM1000 solvent degasser, P4000 quaternary
Inhibition of the cell growth was estimated in mouse lympho-
cytic leukemia L1210 cells (ATCC CCL 219), CCRF-CEM T lymphoblas-
toid cells (human acute lymphoblastic leukemia, ATCC CCL 119),
human promyelocytic leukemia HL-60 cells (ATCC CCL 240) and hu-
man cervix carcinoma HeLa S3 cells (ATCC CCL 2.2).30 L1210 cells,
CCRF-CEM cells and HL-60 cells were cultivated in RPMI 1640 med-
ium supplemented with calf fetal serum using 24-well tissue cul-
ture plates. The endpoint of the cell growth was 72 h following
the drug addition. HeLa S3 cells were seeded to 24-well dishes
in RPMI 1640 HEPES modification with fetal calf serum. 48 h fol-
lowing the drug addition the cultivation was stopped and the cell
growth was evaluated. In parallel, the cell viability was quanti-
fied using XTT standard spectrophotometric assay.31 The inhibi-
tory potency of the compound tested was expressed as IC50
values.
gradient pump, AS 3000 autosampler with a 100 lL sample loop,
UV6000LP photodiode-array detector with Light Pipe Technology,
SN4000 system controller and data station with the ChromQuest
4 analytical software (Thermo Electron, San Jose, CA, USA). The
chromatographic conditions described in our previous paper16
had to be modified in order to accelerate the acquisition of chro-
matographic data as a fast decomposition of the compounds 1a
and 16b was expected.
A chromatographic column 250–4.6 mm containing LiChrospher
60 RP-selectB 5
lm with a 4 mm ꢁ 4 mm guard column (the same
stationary phase, all from Merck) and an isocratic mobile phase con-
taining only acetonitrile and UHQ water (6:4, v/v) with a flow rate of
1 mL minꢀ1 were used for the separation of compounds 1a (tR =
1.5 min)and 16b(tR = 1.2 min)and theirdegradation productsolefin
4 (tR = 5.6 min) and 3-hydroxybenzoic acid (HBA, tR = 0.86 min).
A single wavelength mode at 263 nm was used to obtain chromato-
gramsanda photodiode-arraymodeintherange195–380 nmwitha
1 nm scanning step was applied to record UV spectra of individual
compounds.
5.5. Flow cytometry
The flow cytometry cell cycle analysis (BD FACSAria) was per-
formed by using ethanol-fixed cells stained with propidium iodide
in buffer containing RNase A at the endpoint of the cell growth
(72 h). Tested compound was added to a culture medium at IC50
concentration. The cell cycle events were evaluated with the aid
of BD FACSDiva™ Software v5.3. Apoptosis was evaluated by flow
cytometry using Annexin V-FITC apoptosis kit (Clontech Laborato-
ries, Inc.) according manufacturer’s instructions.
Compounds 1a (4.7 mg) and 16b (5.8 mg) were dissolved in
DMSO (280 lL) and 30 lL of the resultant mixture was added to
2.97 mL of RPMI 1640 medium (vide infra). The solution was kept
at rt and samples were analyzed after period of 0.5, 1, 2, 3, 5, 10
and 24 h.

1996
P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
5.6. Chemistry
5.6.2.2. 3-(4-Bromophenyl)-5-ethoxymethyl-2,5-dihydrofuran-
2-one (10b). Yield: 58%. Colorless crystals, mp 43–45 °C; 1H
NMR: (300 MHz, CDCl3) d 7.79–7.72 (2H, m, AA0, BB0, H20, H60),
7.63 (1H, d, J = 1.9 Hz, H4), 7.59–7.51 (2H, m, AA0, BB0, H30, H50),
5.15 (1H, td, J1 = 5.5 Hz, J2 = 1.9 Hz, H5), 3.77 (1H, dd, J1 = 10.4 Hz,
J2 = 5.5 Hz, OCH2), 3.66 (1H, dd, J1 = 10.4 Hz, J2 = 5.5 Hz, OCH2),
3.63–3.50 (2H, m, OCH2), 1.20 (3H, t, J = 7.0 Hz, CH3); 13C NMR:
(75 MHz, CDCl3) d 171.0, 145.9, 131.9, 131.7, 128.6, 128.3, 123.8,
79.5, 70.4, 67.5, 15.0; IR: (ATR) mmax 1110, 1294, 1333, 1487,
1588, 1748, 2866, 2928, 2975, 3097 cmꢀ1; LRMS: m/z (relative
intensity) 297.0 [M+H]+ (100), 291.5 (28), 265.1 (34), 251.2 (92),
225.2 (8), 207.2 (5). Anal. Calcd for C13H13BrO3: C, 52.55; H, 4.41.
Found: C, 52.73; H, 4.61.
5.6.1. 3-(4-Bromophenyl)-5-methylene-2,5-dihydrofuran-2-
one (4)
Lactone 612 (0.075 g, 0.28 mmol), triphenylphosphine (0.088 g,
0.34 mmol) and imidazole (0.023 g, 0.34 mmol) were dissolved in
dry dichloromethane (3 mL), and the solution was stirred at room
temperature for 30 min. The mixture was then cooled down to
ꢀ10 °C, and a solution of iodine (0.085 mg, 0.34 mmol) in dry
dichloromethane (3 mL) was added dropwise. The resultant
mixture was allowed to warm up to room temperature, diluted
with ethyl acetate (20 mL) and washed with saturated aqueous so-
dium thiosulphate solution (30 mL). The organic layer was dried
(Na2SO4), solvents were removed under reduced pressure and the
product was purified by column chromatography (gradient elution,
hexane/hexane–ethyl acetate 9:1) to afford lactone 4.16 Yield: 64%.
White crystals, mp 85–87 °C; 1H NMR: (300 MHz, CDCl3) d 7.83–
7.76 (2H, m, AA0, BB0, H20, H60), 7.60–7.54 (2H, m, AA0, BB0, H30,
H50), 7.53 (1H, s, H4), 5.29 (1H, d, J = 2.6 Hz, CH2), 4.99 (1H, d,
J = 2.6 Hz, CH2); 13C NMR: (75 MHz, CDCl3) d 168.2, 153.3, 134.5,
132.1, 131.0, 128.8, 127.8, 124.4, 98.1; IR: (ATR) mmax 1293, 1404,
1486, 1584, 1642, 1765, 2852, 2922, 3098 cmꢀ1; LRMS: m/z (rela-
tive intensity) 251.1 [M+H]+ (2), 233.2 (2), 217.2 (1), 201.1 (1),
183.1 (1), 102.2 (1). Anal. Calcd for C11H7BrO2: C, 52.62; H, 2.81.
Found: C, 52.42; H, 2.93.
5.6.3. General procedure for preparation of compounds 15a–
15g
A
mixture of LDA (1.5 M THF complex in cyclohexane,
2.25 mmol) in dry THF (10 mL) under argon atmosphere was
cooled down to ꢀ70 °C, then corresponding lactone 14a–g
(1.88 mmol) in 3 mL of dry THF was added dropwise. The resultant
mixture was stirred at ꢀ70 °C for 1 h and a solution of phenylse-
lenenyl bromide (2.63 mmol) in 3 mL of dry THF was added. Reac-
tion mixture was allowed to warm to rt in period of 2 h and
solvents were removed under reduced pressure. The crude product
was dissolved in ethyl acetate (20 mL), washed with saturated
aqueous ammonium chloride solution (50 mL), organic layer was
dried (Na2SO4) and solvents were removed under reduced pres-
sure. Product was immediately purified by column chromatogra-
phy (gradient elution, hexane/hexane–ethyl acetate 9:1) to afford
phenylselenenyl lactones, which were dissolved in mixture of ethyl
acetate–THF (0.4 mL:0.25 mL). Solution was cooled down to 0 °C
and sodium hydrogen carbonate (2.12 mmol) and 30% aqueous
hydrogen peroxide solution (0.16 mL) were added. Reaction mix-
ture was stirred at rt for 1 h, then it was diluted with ethyl acetate
(20 mL), washed with brine (50 mL), organic layer was dried
(Na2SO4) and solvents were removed under reduced pressure.
Product was recrystallized in ethyl acetate–hexane mixture to af-
ford lactones 15a–g.
5.6.2. General procedure for preparation of compounds 10a–
10b
A
mixture of LDA (1.5 M THF complex in cyclohexane,
8.42 mmol) in dry THF (40 mL) under argon atmosphere was
cooled down to ꢀ70 °C, then corresponding lactone 9a–b
(7.17 mmol) in 20 mL of dry THF was added dropwise. The resul-
tant mixture was stirred at ꢀ70 °C for 1 h and a solution of phe-
nylselenenyl bromide (9.32 mmol) in 10 mL of dry THF was
added. Reaction mixture was allowed to warm to rt in period of
2 h and solvents were removed under reduced pressure. The crude
product was dissolved in ethyl acetate (200 mL), washed with sat-
urated aqueous ammonium chloride solution (200 mL), organic
layer was dried (Na2SO4) and solvents were removed under re-
duced pressure. Product was immediately purified by column
chromatography (gradient elution, hexane/hexane–ethyl acetate
9:1) to afford phenylselenenyl lactones, which were dissolved in
mixture of ethyl acetate–THF (2 mL:1.25 mL). Solution was cooled
down to 0 °C and sodium hydrogen carbonate (8.06 mmol) and 30%
aqueous hydrogen peroxide solution (0.61 mL) were added. Reac-
tion mixture was stirred at rt for 1 h, then it was diluted with ethyl
acetate (100 mL), washed with brine (150 mL), organic layer was
dried (Na2SO4) and solvents were removed under reduced pres-
sure. The product was purified by column chromatography (gradi-
ent elution, hexane/hexane–ethyl acetate 7:3) to afford lactones
10a–b.
5.6.3.1. Methoxymethyl 4-{[4-(4-bromophenyl)-5-oxo-2,5-dihy-
drofuran-2-yl]methoxy}benzoate (15a). Yield: 41%. White crys-
tals, mp 90–92 °C; 1H NMR: (300 MHz, CDCl3) d 8.07–8.01 (2H, m,
AA0, BB0, H2, H6), 7.80–7.74 (2H, m, AA0, BB0, H200, H600), 7.70 (1H, d,
J = 1.9 Hz, H40) 7.60–7.52 (2H, m, AA0, BB0, H300, H500), 6.97–6.90
(2H, m, AA0, BB0, H3, H5), 5.46 (2H, s, OCH2), 5.39 (1H, td,
J1 = 5.2 Hz, J2 = 1.9 Hz, H50), 4.37 (1H, dd, J1 = 10.0 Hz, J2 = 5.2 Hz,
OCH2), 4.28 (1H, dd, J1 = 10.0 Hz, J2 = 5.2 Hz, OCH2), 3.54 (3H, s,
OCH3); 13C NMR: (75 MHz, CDCl3) d 170.6, 165.5, 161.7, 144.4,
132.2, 132.1, 132.0, 128.6, 127.9, 124.2, 123.3, 114.2, 90.8, 78.0,
67.5, 57.7; IR: (KBr) mmax 1161, 1252, 1288, 1490, 1510, 1606,
1720, 1757, 2946, 3073 cmꢀ1; LRMS: m/z (relative intensity) 433.1
[M+H]+ (100), 422.4 (18), 402.5 (12), 343.3 (6), 205.0 (4), 177.0 (4),
144.4 (8), 130.1 (42).
5.6.2.1. 3-(4-Bromophenyl)-5-methoxymethyl-2,5-dihydrofu-
ran-2-one (10a). Yield: 65%. White crystals, mp 56–58 °C; 1H
NMR: (300 MHz, CDCl3) d 7.78–7.70 (2H, m, AA0, BB0, H20, H60),
7.60 (1H, d, J = 1.7 Hz, H4), 7.56–7.49 (2H, m, AA0, BB0, H30, H50),
5.14 (1H, td, J1 = 5.1 Hz, J2 = 1.9 Hz, H5), 3.70–3.63 (2H, t,
J = 4.8 Hz, OCH2), 3.40 (3H, s, CH3); 13C NMR: (75 MHz, CDCl3) d
171.0, 145.5, 131.9, 131.6, 128.6, 128.2, 123.8, 79.3, 72.2, 59.7;
IR: (KBr) mmax 1123, 1198, 1292, 1332, 1475, 1490, 1588, 1738,
2832, 2866, 2897, 2930, 2992, 3076 cmꢀ1; LRMS: m/z (relative
intensity) 283.0 [M+H]+ (21), 251.0 (9), 225.1 (28), 212.1 (11),
196.0 (8), 184.1 (28), 129.9 (20), 117.1 (40), 105.0 (52), 76.1 (30).
Anal. Calcd for C12H11BrO3: C, 50.91; H, 3.92. Found: C, 51.01; H,
3.88.
5.6.3.2. Methoxymethyl 3-{[4-(4-bromophenyl)-5-oxo-2,5-dihy-
drofuran-2-yl]methoxy}benzoate (15b). Yield: 40%. White crys-
tals, mp 89–92 °C; 1H NMR: (300 MHz, DMSO-d6) d 8.22 (1H, d,
J = 1.9 Hz, H40), 7.91–7.84 (2H, m, AA0, BB0, H200, H600), 7.71–7.64
(2H, m, AA0, BB0, H300, H500), 7.64–7.59 (1H, m, H4), 7.50–7.43 (2H,
m, H2, H6), 7.35–7.24 (1H, m, H5), 5.63–5.57 (1H, m, H50), 5.42
(2H, s, OCH2), 4.53 (1H, dd, J1 = 11.1 Hz, J2 = 4.4 Hz, OCH2), 4.34
(1H, dd, J1 = 11.1 Hz, J2 = 4.4 Hz, OCH2), 3.44 (3H, s, OCH3); 13C
NMR: (75 MHz, DMSO-d6) d 171.2, 165.1, 158.3, 148.3, 131.9,
131.1, 130.4, 129.9, 129.0, 128.9, 122.9, 122.5, 120.6, 115.1, 91.0,
79.4, 67.6, 57.3; IR: (ATR)
mmax 1157, 1229, 1276, 1295, 1340, 1403,
1440, 1485, 1584, 1719, 1756, 2950, 3071 cmꢀ1; LRMS: m/z (relative

P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
1997
intensity) 433.1 [M+H]+ (100), 402.9 (14), 388.9 (12), 339.2 (19),
294.3 (10), 251.0 (3), 219.3 (3), 184.3 (2), 139.1 (3), 102.2 (7).
BB0, H2, H6), 7.80–7.74 (2H, m, AA0, BB0, H200, H600), 7.70 (1H, d,
J = 1.9 Hz, H40), 7.60–7.52 (2H, m, AA0, BB0, H300, H500), 6.96–6.87
(2H, m, AA0, BB0, H3, H5), 5.39 (1H, td, J1 = 5.3 Hz, J2 = 1.9 Hz, H50),
4.37 (1H, dd, J1 = 9.9 Hz, J2 = 5.3 Hz, OCH2), 4.26 (1H, dd,
J1 = 9.9 Hz, J2 = 5.3 Hz, OCH2), 3.89 (3H, s, OCH3); 13C NMR:
(75 MHz, CDCl3) d 170.6, 166.6, 161.4, 144.5, 132.2, 132.0, 131.8,
128.6, 127.9, 124.2, 123.7, 114.2, 78.0, 67.5, 52.0; IR: (KBr) mmax
1170, 1254, 1283, 1317, 1436, 1490, 1511, 1589, 1606, 1717,
1759, 2952, 3076 cmꢀ1; LRMS: m/z (relative intensity) 403.0
[M+H]+ (2), 372.0 (1), 283.5 (1), 252.1 (1), 177.9 (14), 131.1 (2),
122.2 (3), 90.3 (100) 76.8 (14). Anal. Calcd for C19H15BrO5: C,
56.59; H, 3.75. Found: C, 56.91; H, 4.02.
5.6.3.3. 3-(4-Bromophenyl)-5-[(4-methoxyphenoxy)methyl]-
2,5-dihydrofuran-2-one (15c). Yield: 42%. Brownish crystals, mp
1
89–91 °C; H NMR: (300 MHz, CDCl3) d 7.78–7.75 (2H, m, AA0, BB0,
H20, H60), 7.71 (1H, d, J = 1.9 Hz, H4) 7.57–7.54 (2H, m, AA0, BB0, H30,
H50), 6.86–6.82 (4H, m, H200, H300, H500, H600), 5.33 (1H, td,
J1 = 5.2 Hz, J2 = 1.9 Hz, H5), 4.28 (1H, dd, J1 = 10.0 Hz, J2 = 5.4 Hz,
OCH2), 4.14 (1H, dd, J1 = 10.0 Hz, J2 = 5.4 Hz, OCH2), 3.77 (3H, s,
OCH3); 13C NMR: (75 MHz, CDCl3) d 170.9, 154.5, 152.0, 145.2,
131.9, 131.8, 128.6, 128.1, 124.0, 115.9, 114.7, 78.4, 68.5, 55.7;
IR: (KBr) mmax 1235, 1490, 1508, 1756, 2926, 3076 cmꢀ1; LRMS:
m/z (relative intensity) 375.1 [M+H]+ (100), 356.7 (53), 304.5
(38), 251.4 (28), 235.9 (31), 217.3 (14), 149.3 (22), 102.3 (29). Anal.
Calcd for C18H15BrO4: C, 57.62; H, 4.03. Found: C, 57.96; H, 4.33.
5.6.4. General procedure for preparation of compounds 16a–
16b
A mixture of corresponding methoxymethyl derivative 15a–b
(0.27 mmol) and DOWEXÒ 50 W (0.034 g) in 6 mL of methanol
was stirred at 40 °C for 1 h. The precipitated pure product and resin
were filtered off, product was dissolved in ethyl acetate (50 mL)
and solvent was removed under reduced pressure.
5.6.3.4. 3-(4-Bromophenyl)-5-phenoxymethyl-2,5-dihydrofu-
ran-2-one (15d). Yield: 43%. White crystals, mp 113 °C; 1H NMR:
(300 MHz, CDCl3) d7.80–7.74 (2H, m, AA0, BB0, H20, H60), 7.72 (1H, d,
J = 1.9 Hz, H4), 7.59–7.53 (2H, m, AA0, BB0, H30, H50), 7.34–7.27 (2H,
m, H300, H500), 7.04–6.97 (1H, m, H400), 6.93–6.86 (2H, m, H200, H600),
5.37 (1H, td, J1 = 5.5 Hz, J2 = 1.9 Hz, H5), 4.35 (1H, dd, J1 = 9.9 Hz,
J2 = 5.5 Hz, OCH2), 4.18 (1H, dd, J1 = 9.9 Hz, J2 = 5.5 Hz, OCH2). 13C
NMR: (75 MHz, CDCl3) d 170.8, 157.9, 145.2, 132.0, 131.9, 129.7,
128.6, 128.1, 124.0, 121.8, 114.7, 78.3, 67.6; IR: (KBr) mmax 1127,
5.6.4.1. 4-{[4-(4-Bromophenyl)-5-oxo-2,5-dihydrofuran-2-yl]-
methoxy}benzoic acid (16a). Yield: 92%. White crystals, mp
224 °C; 1H NMR: (300 MHz, DMSO-d6) d 12.68 (1H, bs, OH), 8.22
(1H, d, J = 1.9 Hz, H40), 7.91–7.84 (4H, m, H200, H600, H2, H6),
7.70–7.63 (2H, m, H300, H500), 7.06–6.99 (2H, m, H3, H5), 5.64–
5.58 (1H, m, H50), 4.53 (1H, dd, J1 = 11.2 Hz, J2 = 4.4 Hz, OCH2),
4.33 (1H, dd, J1 = 11.2 Hz, J2 = 4.4 Hz, OCH2); 13C NMR: (75 MHz,
DMSO-d6) d 171.2, 167.1, 161.7, 148.2, 132.1, 131.9, 131.6, 129.9,
129.3, 123.8, 122.9, 114.7, 79.3, 67.4; IR: (KBr) mmax 1170, 1256,
1244, 1491, 1497, 1589, 1599, 1745, 2926, 2953, 3065 cmꢀ1
;
LRMS: m/z (relative intensity) 345.0 [M+H]+ (1), 282.0 (3), 250.1
(52), 194.0 (8), 180.2 (42), 171.1 (66), 115.0 (100), 101.1 (54),
75.2 (43). Anal. Calcd for C17H13BrO4: C, 59.15; H, 3.80. Found: C,
59.49; H, 3.95.
1425, 1490, 1513, 1607, 1685, 1717, 1751, 2954, 3079 cmꢀ1
;
LRMS: m/z (relative intensity) 389.9 [M+H]+ (56), 371.2 (12),
282.3 (100), 277.2 (39), 266.2 (58), 251.0 (6), 194.9 (7), 171.0
(16), 163.0 (6), 149.1 (6), 133.0 (7), 102.2 (7). Anal. Calcd for
C18H13BrO5: C, 55.55; H, 3.37. Found: C, 55.72; H, 3.14.
5.6.3.5. 3-(4-Bromophenyl)-5-[(4-iodophenoxy)methyl]-2,5-
dihydrofuran-2-one (15e). Yield: 43%. White crystals, mp 149–
1
151 °C; H NMR: (300 MHz, CDCl3) d 7.79–7.73 (2H, m, AA0, BB0,
H20, H60), 7.68 (1H, d, J = 1.9 Hz, H4), 7.60–7.53 (4H, m, H30, H50,
H300, H500), 6.71–6.64 (2H, m, AA0, BB0, H200, H600), 5.35 (1H, td,
J1 = 5.2 Hz, J2 = 1.9 Hz, H5), 4.28 (1H, dd, J1 = 10.1 Hz, J2 = 5.2 Hz,
OCH2), 4.17 (1H, dd, J1 = 10.1 Hz, J2 = 5.2 Hz, OCH2); 13C NMR:
(75 MHz, CDCl3) d 170.7, 157.8, 144.6, 138.4, 132.1, 131.9, 128.6,
127.9, 124.1, 117.0, 84.1, 78.1, 67.6; IR: (KBr) mmax 1126, 1245,
1284, 1301, 1485, 1584, 1749, 2920, 2949, 3088 cmꢀ1; LRMS: m/
z (relative intensity) 470.9 [M+H]+ (1), 407.0 (1), 329.1 (1), 251.0
(49), 180.9 (40), 172.0 (65), 128.1 (24), 115.9 (100), 101.9 (53),
76.0 (40). Anal. Calcd for C17H12BrIO3: C, 43.34; H, 2.57. Found: C,
43.69; H, 2.86.
5.6.4.2. 3-{[4-(4-Bromophenyl)-5-oxo-2,5-dihydrofuran-2-yl]-
methoxy}benzoic acid (16b). Yield: 95%. White crystals, mp
206 °C; 1H NMR: (300 MHz, DMSO-d6) d 13.01 (1H, bbs, OH), 8.22
(1H, d, J = 1.9 Hz, H40), 7.93–7.83 (2H, m, AA0, BB0, H200, H600),
7.72–7.63 (2H, m, AA0, BB0, H300, H500), 7.58–7.52 (1H, m, H2),
7.45–7.37 (2H, m, H4, H6), 7.24–7.16 (1H, m, H5), 5.64–5.55 (1H,
m, H50), 4.51 (1H, dd, J1 = 11.1 Hz, J2 = 4.4 Hz, OCH2), 4.31 (1H, dd,
J1 = 11.1 Hz, J2 = 4.4 Hz, OCH2); 13C NMR: (75 MHz, DMSO-d6) d
171.3, 167.2, 158.1, 148.3, 132.5, 131.9, 130.1, 129.9, 129.0,
128.9, 122.9, 122.4, 119.8, 115.1, 79.4, 67.5; IR: (ATR) mmax 1243,
1257, 1308, 1385, 1421, 1458, 1489, 1587, 1677, 1707, 1757,
2951, 3069 cmꢀ1; LRMS: m/z (relative intensity) 388.9 [M+H]+
(100), 369. (18), 329.1 (7), 301.1 (9), 291.4 (17), 269.3 (8), 251.1
(30), 227.3 (5), 213.5 (6). Anal. Calcd for C18H13BrO5: C, 55.55; H,
3.37. Found: C, 55.88; H, 3.28.
5.6.3.6. 3-(4-Bromophenyl)-5-[(3-bromophenoxy)methyl]-2,5-
dihydrofuran-2-one (15f). Yield: 38%. Brownish crystals, mp
1
115–117 °C; H NMR: (300 MHz, CDCl3) d 7.80–7.74 (2H, m, AA0,
BB0, H20, H60), 7.68 (1H, d, J = 1.9 Hz, H4), 7.59–7.53 (2H, m, AA0,
BB0, H30, H50), 7.17–7.12 (2H, m, H400, H500), 7.07–7.04 (1H, m,
H200), 6.86–6.80 (1H, m, H600), 5.36 (1H, td, J1 = 5.2 Hz, J2 = 1.9 Hz,
H5), 4.30 (1H, dd, J1 = 10.0 Hz, J2 = 5.2 Hz, OCH2), 4.19 (1H, dd,
5.6.4.3. 3-(4-Bromophenyl)-5-[(4-hydroxyphenoxy)methyl]-
2,5-dihydrofuran-2-one (16c). Methoxy derivative 15c (0.111 g,
0.29 mmol) was dissolved in dry dichloromethane (2 mL), solu-
tion was cooled down to ꢀ50 °C, then solution of BBr3 (1.0 M
solution in dichloromethane, 1.20 mL) was added dropwise. Reac-
tion mixture was allowed to warm to 5 °C in period of 1 h and
water (2 mL) was added. After stirring at rt for further 30 min,
reaction mixture was diluted with ethyl acetate (20 mL), washed
with saturated aqueous sodium chloride solution (50 mL), organic
layer was dried (Na2SO4) and solvents were removed under re-
duced pressure. Product was recrystallized in ethyl acetate–hex-
ane mixture to afford lactone 16c. Yield: 75%. Brownish crystals,
mp 118–120 °C; 1H NMR: (300 MHz, CD3OD) d 7.95 (1H, d,
J = 1.9, H4), 7.84–7.81 (2H, m, AA0, BB0, H30, H50), 7.60–7.57 (2H,
J1 = 10.0 Hz, J2 = 5.2 Hz, OCH2); 13C NMR: (75 MHz, CDCl3)
d
170.6, 158.6, 144.6, 132.1, 132.0, 130.7, 128.6, 127.9, 125.0,
124.1, 122.9, 118.1, 113.5, 78.0, 67.7; IR: (KBr) mmax 1126, 1230,
1287, 1474, 1490, 1575, 1587, 1750, 2926, 3088 cmꢀ1; LRMS: m/
z (relative intensity) 422.9 [M+H]+ (1), 403.9 (1), 329.0 (1), 281.1
(1), 251.0 (53), 180.9 (41), 172.1 (67), 116.0 (100), 101.9 (55),
75.9 (45). Anal. Calcd for C17H12Br2O3: C, 48.15; H, 2.85. Found:
C, 47.92; H, 3.03.
5.6.3.7. Methyl 4-{[4-(4-bromophenyl)-5-oxo-2,5-dihydrofuran-
2-yl]methoxy}benzoate (15g). Yield: 41%. White crystals, mp
1
148–151 °C; H NMR: (300 MHz, CDCl3) d 8.03–7.96 (2H, m, AA0,

1998
P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
m, AA0, BB0, H20, H60), 6.80–6.76 (2H, m, AA0, BB0, H300, H500), 6.71–
6.68 (2H, m, AA0, BB0, H200, H600), 5.42–5.39 (1H, m, H5), 4.31 (1H,
dd, J1 = 10.9 Hz, J2 = 4.4 Hz, OCH2), 4.19 (1H, dd, J1 = 10.9 Hz,
J2 = 4.4 Hz, OCH2); 13C NMR: (75 MHz, CD3OD) d 172.0, 153.1,
153.1, 148.4, 132.9, 132.2, 130.2, 129.9, 124.5, 117.2, 116.9,
81.2, 69.5; IR: (KBr) mmax 1215, 1234, 1489, 1511, 1758, 2872,
2923, 3077 cmꢀ1; LRMS: m/z (relative intensity) 369.1 [M+H]+
(22), 360.9 (100), 304.7 (18), 271.9 (11), 251.2 (48), 238.1 (21),
225.1 (9), 194.9 (12), 164.9 (16), 149.0 (9), 133.0 (6), 102.1 (6).
Anal. Calcd for C17H13BrO4: C, 56.53; H, 3.63. Found: C, 56.83;
H, 3.38.
Solution C: The solvent from 4-bromophenylzinciodide (0.5 M
solution in THF, 3.7 mmol) was almost completely removed under
reduced pressure, then dry DMF (3 mL) was added under argon
atmosphere.
Solution B was added via cannula to a stirred solution A at
ꢀ30 °C under argon atmosphere. The reaction mixture was allowed
to warm up to rt in period of 1 h, then solution C was added via
cannula. The resultant mixture was stirred at rt for further 3 h, it
was then diluted with ethyl acetate (25 mL) and washed with sat-
urated aqueous ammonium chloride solution (50 mL). The organic
layer was dried (Na2SO4), solvents were removed under reduced
pressure and the crude product was purified by column chroma-
tography (gradient elution, hexane/hexane–ethyl acetate 94:6).
The resultant THP-protected alcohol was dissolved in methanol
(22 mL), 220 mg of DOWEXÒ 50 W was added and the reaction
mixture was stirred at rt for 1 h. The resin was filtered off, solvent
was removed under reduced pressure, the resultant solid was di-
luted with ethyl acetate (50 mL), washed with saturated aqueous
sodium chloride solution (50 mL), the organic layer was dried
(Na2SO4) and solvents were removed under reduced pressure.
The crude product was purified by column chromatography (gradi-
ent elution, hexane/hexane–ethyl acetate 6:4) and further recrys-
tallized in mixture of hexane–ethyl acetate to afford lactone 24a
as (Z) isomer. Yield: 65%. White crystals, mp 131 °C; 1H NMR:
(300 MHz, CDCl3) d 7.82–7.76 (2H, m, AA0, BB0, H20, H60), 7.59–
7.54 (2H, m, AA0, BB0, H30, H50), 7.51 (1H, s, H4), 5.55 (1H, t,
J = 6.9 Hz, CH), 4.56 (2H, d, J = 6.9 Hz, OCH2); 13C NMR: (75 MHz,
CDCl3) d 167.6, 134.8, 132.1, 131.8, 128.8, 128.6, 127.9, 124.4,
114.1, 57.4; IR: (KBr) mmax 1176, 1234, 1296, 1311, 1404, 1490,
5.6.4.4. (Z)-3-(4-Bromophenyl)-5-ethylidene-2,5-dihydrofuran-
2-one (20a). Iodoenone 19a (0.610 g, 1.67 mmol) was dissolved
in dry DMF (10 mL), and PdCl2(PPh3)2 (0.061 g, 0.0835 mmol) and
NaHCO3 (0.280 g, 3.34 mmol) were added. The reaction flask was
evacuated and vacuum was replaced by carbon monooxide atmo-
sphere. The reaction mixture was stirred at 60 °C for 24 h, then it
was diluted with ethyl acetate (50 mL), washed with 5% aqueous
sodium chloride solution (50 mL). The organic layer was dried
(Na2SO4) and solvents were removed under reduced pressure.
The crude product was purified by column chromatography (gradi-
ent elution, hexane/hexane–ethyl acetate 8:2) and further recrys-
tallized in mixture of hexane–ethyl acetate to afford lactone 20a
as (Z) isomer. Yield: 5%. White crystals, mp 122–124 °C; 1H NMR:
(300 MHz, CDCl3) d 7.82–7.75 (2H, m, AA0, BB0, H20, H60), 7.59–
7.52 (2H, m, AA0, BB0, H30, H50), 7.47 (1H, s, H4), 5.43 (1H, q,
J1 = 7.5 Hz, CH), 2.02 (3H, d, J = 7.5 Hz, CH3); 13C NMR: (75 MHz,
CDCl3) d 168.4, 148.8, 135.0, 132.0, 128.7, 128.5, 128.4, 123.7,
112.6, 12.2; IR: (KBr) mmax 1295, 1323, 1405, 1489, 1582, 1667,
1744, 2854, 2939, 3072, 3086 cmꢀ1; LRMS: m/z (relative intensity)
265.0 [M+H]+ (58), 225.0 (16), 186.1 (42), 181.0 (30), 129.9 (100),
102.1 (55), 75.4 (35). Anal. Calcd for C12H9BrO2: C, 54.37; H, 3.42.
Found: C, 54.09; H, 3.22.
1584, 1601, 1672, 1762, 2862, 2923, 2951, 3090, 3281 cmꢀ1
;
LRMS: m/z (relative intensity) 280.9 [M+H]+ (50), 263.1 (16),
237.1 (10), 203.2 (5), 187.2 (6), 158.0 (10), 130.0 (100), 113.3
(12). Anal. Calcd for C12H9BrO3: C, 51.27; H, 3.23. Found: C,
51.55; H, 3.50.
5.6.4.5. 3-(4-Bromophenyl)-2,5-dihydrofuran-2-one (20b).
Iodoalcohol 18b32 (1.140 g, 3.36 mmol) was dissolved in dry
DMF (20 mL) and Pd(OAc)2 (0.039 g, 0.168 mmol), triphenylphos-
phine (0.093 g, 0.355 mmol) and triethylamine (1.20 mL, 8.61
mmol) were added. The reaction flask was evacuated and vacuum
was replaced by carbon monooxide atmosphere. The reaction mix-
ture was stirred at 60 °C for 24 h, then it was diluted with ethyl
acetate (50 mL), washed with 5% aqueous sodium chloride solution
(50 mL). The organic layer was dried (Na2SO4) and solvents were
removed under reduced pressure. The crude product was purified
by column chromatography (gradient elution, hexane/hexane–
ethyl acetate 9:1) and further recrystallized in mixture of hex-
ane–ethyl acetate to afford lactone 20b.21 Yield: 41%. Yellowish
crystals, mp 117 °C; 1H NMR: (300 MHz, CDCl3) d 7.78–7.72 (2H,
m, AA0, BB0, H20, H60), 7.67 (1H, t, J = 2.1 Hz, H4), 7.58–7.52 (2H,
m, AA0, BB0, H30, H50), 4.93 (2H, d, J = 2.1 Hz, H5); 13C NMR:
(75 MHz, CDCl3) d 171.8, 144.5, 131.9, 130.7, 128.5, 128.3, 123.7,
69.5; IR: (ATR) mmax 1116, 1296, 1315, 1343, 1403, 1442, 1485,
1586, 1737, 2852, 2943, 3085 cmꢀ1; LRMS: m/z (relative intensity)
239.1 [M+H]+ (100), 221.1 (5), 211.1 (6), 193.1 (4), 180.9 (3), 160.1
(13), 149.1 (3), 116.2 (4). Anal. Calcd for C10H7BrO2: C, 50.24; H,
2.95. Found: C, 50.74; H, 3.24.
5.6.4.7. (Z)-3-(4-Bromophenyl)-5-(3-hydroxypropylidene)-2,5-
dihydrofuran-2-one (24b). PdCl2(TFP)2 (25.0 mg, 0.039 mmol)
and 4-bromophenylzinciodide (0.5 M solution in THF, 1.0 mmol)
were added to a stirred solution 23b (0.235 g, 0.78 mmol) in dry
DMF (1 mL) at rt under argon atmosphere. The reaction mixture
was stirred at rt for 2 h, it was then diluted with ethyl acetate
(15 mL) and washed with saturated aqueous ammonium chloride
solution (20 mL). The organic layer was dried (Na2SO4), solvents
were removed under reduced pressure and the crude product
was purified by column chromatography (gradient elution, hex-
ane/hexane–ethyl acetate 95:5). The resultant THP-protected alco-
hol was dissolved in methanol (6 mL), 75 mg of DOWEXÒ 50 W
was added and the reaction mixture was stirred at rt for 1 h. The
resin was filtered off, solvent was removed under reduced pres-
sure, the resultant solid was diluted with ethyl acetate (20 mL),
washed with saturated aqueous sodium chloride solution
(20 mL), the organic layer was dried (Na2SO4) and solvents were
removed under reduced pressure. The crude product was purified
by column chromatography (gradient elution, hexane/hexane–
ethyl acetate 6:4) and further recrystallized in mixture of hex-
ane–ethyl acetate to afford lactone 24b as (Z) isomer. Yield: 59%.
White crystals, mp 98–100 °C; 1H NMR: (300 MHz, CD3OD) d
7.89 (1H, s, H4), 7.88–7.84 (2H, m, AA0, BB0, H20, H60), 7.60–7.54
(2H, m, AA0, BB0, H30, H50), 5.58 (1H, t, J = 8.0 Hz, CH), 3.70 (2H, t,
J = 6.3 Hz, OCH2), 2.68–2.59 (2H, m, CH2); 13C NMR: (75 MHz,
CD3OD) d 169.9, 150.7, 137.6, 133.0, 130.1, 129.8, 129.4, 124.5,
115.5, 61.7, 31.1; IR: (KBr) mmax 1296, 1384, 1404, 1489, 1583,
1758, 2884, 2954, 3089, 3423 cmꢀ1; LRMS: m/z (relative intensity)
295.1 [M+H]+ (11), 277.1 (18), 263.1 (12), 251.2 (7), 237.1 (6),
214.3 (9), 204.2 (15), 177.1 (100). Anal. Calcd for C13H11BrO3: C,
52.91; H, 3.76. Found: C, 53.21; H, 4.01.
5.6.4.6.
(Z)-3-(4-Bromophenyl)-5-(2-hydroxyethylidene)-2,5-
dihydrofuran-2-one (24a). Solution A: Dry THF (2 mL) was added
to PdCl2(TFP)2 (84.0 mg, 0.13 mmol) at ꢀ80 °C under argon atmo-
sphere; to the resultant stirred suspension butyllithium (1.6 M
solution in hexanes, 0.26 mmol) was added dropwise. The reaction
mixture was allowed to warm up to ꢀ30 °C in period of 1 h.
Solution B: Solution of 23a (0.753 g, 2.60 mmol) in dry DMF
(2 mL).

P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
1999
1
5.6.4.8.
(Z)-3-(4-Bromophenyl)-5-(4-hydroxybutylidene)-2,5-
99.6 °C; H NMR: (300 MHz, CDCl3) d 7.80–7.75 (2H, m, AA0, BB0,
H20, H60), 7.59–7.54 (2H, m, AA0, BB0, H30, H50), 7.49 (1H, s, H4),
5.48 (1H, t, J = 7.2 Hz, CH), 4.93 (2H, d, J = 7.2 Hz, OCH2), 2.10
(3H, s, OCH3); 13C NMR: (75 MHz, CDCl3) d 170.7, 167.3, 149.4,
134.4, 132.1, 130.7, 128.7, 127.7, 124.5, 108.7, 58.5, 20.8; IR:
(KBr) mmax 1110, 1225, 1308, 1365, 1382, 1406, 1450, 1490, 1582,
1676, 1744, 1761, 2872, 2924, 3088 cmꢀ1; LRMS: m/z (relative
intensity) 309.0 [M+H]+ (1), 295.1 (42), 263.3 (100), 251.4 (2),
237.3 (7), 184.4 (10). Anal. Calcd for C14H11BrO4: C, 52.04; H,
3.43. Found: C, 51.88; H, 3.09.
dihydrofuran-2-one (24c). PdCl2(TFP)2 (12.0 mg, 0.019 mmol)
and 4-bromophenylzinciodide (0.5 M solution in THF, 0.5 mmol)
were added to a stirred solution 23c (0.110 g, 0.35 mmol) in dry
DMF (1 mL) at rt under argon atmosphere. The reaction was stirred
at rt for 2 h, it was then diluted with ethyl acetate (10 mL) and
washed with saturated aqueous ammonium chloride solution
(15 mL). The organic layer was dried (Na2SO4), solvents were re-
moved under reduced pressure and the crude product was purified
by column chromatography (gradient elution, hexane/hexane–
ethyl acetate 95:5). The resultant THP-protected alcohol was dis-
solved in methanol (3 mL), 30 mg of DOWEXÒ 50 W was added
and the reaction mixture was stirred at rt for 1 h. The resin was fil-
tered off, solvent was removed under reduced pressure, the resul-
tant solid was diluted with ethyl acetate (10 mL), washed with
saturated aqueous sodium chloride solution (10 mL), the organic
layer was dried (Na2SO4) and solvents were removed under re-
duced pressure. The crude product was purified by column chro-
matography (gradient elution, hexane/hexane–ethyl acetate 6:4)
and further recrystallized in mixture of hexane–ethyl acetate to af-
ford lactone 24c as (Z) isomer. Yield: 56%. White crystals, mp 99–
5.6.5.2. (Z)-4-[(4-Bromophenyl)-5-oxo-2,5-dihydrofuran-2-yli-
dene]methyl benzoate (25b). Yield: 70%. White crystals, mp
106 °C; 1H NMR: (300 MHz, CDCl3) d 8.10–8.02 (2H, m, H2, H6),
7.86–7.74 (2H, m, H200, H600), 7.64–7.39 (5H, m, H300, H500, H3, H4,
H5), 7.52 (1H, s, H40), 5.62 (1H, t, J = 7.2 Hz, CH), 5.20 (2H, d,
J = 7.2 Hz, OCH2); 13C NMR: (75 MHz, CDCl3) d 167.4, 166.3,
149.6, 134.4, 133.3, 132.1, 130.7, 129.7, 128.9, 128.7, 128.4,
127.8, 124.6, 108.8, 58.9; IR: (KBr) mmax 1117, 1176, 1268, 1319,
1378, 1407, 1451, 1491, 1585, 1602, 1678, 1731, 1750, 3071,
3106 cmꢀ1; LRMS: m/z (relative intensity) 385.0 [M+H]+ (100),
329.1 (1), 295.0 (11), 282.3 (57), 263.1 (99), 254.3 (2), 239.1 (4),
184.1 (6), 149.2 (3). Anal. Calcd for C19H13BrO4: C, 59.24; H, 3.40.
Found: C, 59.51; H, 3.66.
1
102 °C; H NMR: (300 MHz, CD3OD) d 7.89–7.80 (3H, m, H4, H20,
H60), 7.61–7.52 (2H, m, AA0, BB0, H30, H50), 5.56 (1H, t, J = 8.0 Hz,
CH), 3.61 (2H, t, J = 6.3 Hz, OCH2), 2.56–2.42 (2H, m, CH2), 1.80–
1.66 (2H, m, CH2); 13C NMR: (75 MHz, CD3OD) d 169.9, 149.9,
137.6, 132.9, 129.8, 129.1, 128.0, 124.4, 118.5, 62.3, 32.9, 24.2;
IR: (KBr) mmax 1295, 1405, 1489, 1582, 1668, 1755, 2876, 2939,
3093, 3421 cmꢀ1; LRMS: m/z (relative intensity) 309.5 [M+H]+
(97), 291.8 (23), 263.7 (6), 251.1 (2), 231.9 (2), 213.7 (2), 177.7
(100). Anal. Calcd for C14H13BrO3: C, 54.39; H, 4.24. Found: C,
54.68; H, 4.04.
5.6.5.3. (Z)-4-[(4-Bromophenyl)-5-oxo-2,5-dihydrofuran-2-yli-
dene]ethyl acetate (25c). Yield: 80%. White crystals, mp 89–
1
92 °C; H NMR: (300 MHz, CDCl3) d 7.82–7.75 (2H, m, AA0, BB0,
H20, H60), 7.59–7.52 (2H, m, AA0, BB0, H30, H50), 7.48 (1H, s, H4),
5.38 (1H, t, J = 7.8 Hz, CH), 4.21 (2H, t, J = 6.3 Hz, OCH2), 2.78 (2H,
m, CH2), 2.07 (3H, s, CH3); 13C NMR: (75 MHz, CDCl3) d 171.0,
168.0, 149.2, 134.7, 132.0, 129.3, 128.6, 128.1, 124.1, 112.1, 62.7,
26.2, 20.9; IR: (KBr) mmax 1101, 1251, 1312, 1368, 1393, 1404,
1473, 1489, 1585, 1673, 1739, 1750, 2854, 2880, 2923, 2966,
3089 cmꢀ1; LRMS: m/z (relative intensity) 323.0 [M+H]+ (1),
295.1 (16), 277.1 (100), 268.7 (6), 258.7 (4), 185.0 (1). Anal. Calcd
for C15H13BrO4: C, 53.43; H, 3.89. Found: C, 53.29; H, 4.29.
5.6.4.9.
(Z)-3-(4-Bromophenyl)-5-benzylidene-2,5-dihydrofu-
ran-2-one (24d). PdCl2(TFP)2 (15.0 mg, 0.023 mmol) and 4-brom-
ophenylzinciodide (0.5 M solution in THF, 0.6 mmol) were added
to a stirred solution 23d (0.115 g, 0.46 mmol) in dry DMF (1 mL)
at rt under argon atmosphere. The reaction was stirred at rt for
3 h, it was then diluted with ethyl acetate (10 mL) and washed
with saturated aqueous sodium chloride solution (15 mL). The or-
ganic layer was dried (Na2SO4), solvents were removed under re-
duced pressure and the crude product was purified by column
chromatography (gradient elution, hexane/hexane–ethyl acetate
95:5) to afford lactone 24d as (Z) isomer. Yield: 30%. Yellow crys-
tals, mp 206–207 °C; 1H NMR: (300 MHz, CDCl3) d 7.86–7.79 (4H,
m, H20, H60, H200, H600), 7.62 (1H, s, H4), 7.60–7.54 (2H, m, H30,
H50), 7.46–7.31 (3H, m, H300, H400, H500), 6.11 (1H, s, CH); 13C
NMR: (75 MHz, CDCl3) d 168.5, 147.0, 136.6, 133.1, 132.1, 130.7,
129.3, 128.9, 128.5, 128.3, 127.6, 123.9, 111.5; IR: (KBr) mmax
1103, 1179, 1293, 1359, 1449, 1485, 1580, 1643, 1746, 2926,
3024, 3068, 3103 cmꢀ1; LRMS: m/z (relative intensity) 327.0
[M+H]+ (54), 299.1 (5), 282.0 (7), 247.9 (13), 219.9 (38), 208.0
(18), 192.1 (100), 181.1 (20), 119.0 (62). Anal. Calcd for
C17H11BrO2: C, 62.41; H, 3.39. Found: C, 62.79; H, 3.26.
5.6.5.4. (E)-Methyl 2-[4-(4-bromophenyl)-5-oxo-2,5-dihydrofu-
ran-2-ylidene]acetate (26). Methyl bromoacetate (1.523 g,
10.00 mmol) and triphenylphosphine (3.150 g, 12.00 mmol) were
dissolved in toluene (100 mL) and the resultant mixture was
heated under reflux for 5 h. Precipitated product was filtered and
washed with toluene (50 mL). Dry THF (20 mL) was added to the
prepared phosphonium salt (1.530 g, 3.68 mmol), the suspension
was cooled down to ꢀ80 °C and LDA (1.5 M THF complex in
cyclohexane, 4.05 mmol) was added dropwise at argon atmo-
sphere. The reaction mixture was heated up to ꢀ40 °C in period
of 1 h and
a
solution of 4-bromophenylmaleic anhydride26
(0.911 g, 3.60 mmol) in dry THF (20 mL) was slowly added. The
resultant mixture was allowed to heat up to rt in period of 2 h, it
was then diluted with ethyl acetate (50 mL) and washed with sat-
urated aqueous ammonium chloride solution (50 mL). The organic
layer was dried (Na2SO4), solvents were removed under reduced
pressure and the crude product was purified by column chroma-
tography (gradient elution, hexane/hexane–ethyl acetate 95:5) to
afford lactone 26 as (E) isomer. Yield: 15%. White crystals, mp
146 °C; 1H NMR: (300 MHz, CDCl3) d 8.50 (1H, s, H4), 7.92–7.83
(2H, m, AA0, BB0, H20, H60), 7.64–7.56 (2H, m, AA0, BB0, H30, H50)
5.96 (1H, s, CH), 3.83 (3H, s, OCH3); 13C NMR: (75 MHz, CDCl3) d
166.6, 165.7, 159.1, 133.7, 132.4, 132.3, 129.2, 127.3, 125.7,
101.8, 52.1; IR: (KBr) mmax 1142, 1215, 1254, 1316, 1379, 1405,
5.6.5. General procedure for preparation of compounds 25a–c
A solution of corresponding alcohol 24 (1.00 mmol), dry pyri-
dine (1.10 mmol) and acyl chloride (1.10 mmol) in dry dichloro-
methane (15 mL) was stirred at rt for 1.5 h. Mixture was diluted
with ethyl acetate (50 mL), washed with saturated aqueous so-
dium chloride solution (50 mL), the organic layer was dried
(Na2SO4) and solvent was removed under reduced pressure. The
crude product was purified by column chromatography (gradient
elution, hexane/hexane–ethyl acetate 8:2) to afford esters 25a–c.
1434, 1487, 1581, 1594, 1648, 1721, 1776, 2951, 3087 cmꢀ1
;
LRMS: m/z (relative intensity) 309.0 [M+H]+ (62), 278.1 (27),
251.4 (44), 221.0 (5), 193.9 (8), 181.1 (27), 101.0 (63). Anal. Calcd
for C13H9BrO2: C, 50.51; H, 2.93. Found: C, 50.27; H, 3.22.
5.6.5.1. (Z)-4-[(4-Bromophenyl)-5-oxo-2,5-dihydrofuran-2-yli-
dene]methyl acetate (25a). Yield: 87%. White crystals, mp

2000
P. Šenel et al. / Bioorg. Med. Chem. 18 (2010) 1988–2000
13. Buchta, V.; Pour, M.; Kubanová, P.; Silva, L.; Votruba, I.; Vopršalová, M.; Schiller,
Acknowledgments
R.; Fáková, H.; Špulák, M. Antimicrob. Agents Chemother. 2004, 48, 873.
14. Šnajdr, I.; Pavlík, J.; Schiller, R.; Kuneš, J.; Pour, M. Collect. Czech. Chem. Commun.
2007, 72, 1472.
15. Vale-Silva, L. A.; Buchta, V.; Vokurková, D.; Pour, M. Bioorg. Med. Chem. Lett.
2006, 16, 2492.
This work was supported by the Czech Science Foundation (Pro-
ject No. 203/07/1302) and the Ministry of Education, Youth and
Sports of the Czech Republic (Project Nos. 1M0508 and
MSM0021620822).
ˇ
16. Nobilis, M.; Pour, M.; Šenel, P.; Pavlík, J.; Kuneš, J.; Vopršalová, M.; Kolárová, L.;
10.
ˇ
Holcapek, M. J. Chromatogr., B 2007, 853,
17. Horimoto, A. Jpn. Kokai Tokkyo Koho 2007277137, 2007.
18. Fáková, H.; Pour, M.; Kuneš, J.; Šenel, P. Tetrahedron Lett. 2005, 46, 8137.
19. More, J. D.; Finney, N. S. Org. Lett. 2002, 4, 3001.
Supplementary data
20. Tretyakov, E. V.; Tkachev, A. V.; Rybalova, T. V.; Gatilov, Y. V.; Knight, D. W.;
Vasilevsky, S. F. Tetrahedron 2000, 56, 10075.
21. Gabriele, B.; Salerno, G.; Costa, M.; Chiusoli, G. P. Tetrahedron Lett. 1999, 40,
989.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.01.030.
22. (a) Rossi, R.; Bellina, F.; Mannina, L. Tetrahedron Lett. 1998, 39, 3017; (b) Rossi,
R.; Bellina, F.; Biagetti, M.; Mannina, L. Tetrahedron Lett. 1998, 39, 7799; (c)
Biagetti, M.; Bellina, F.; Carpita, A.; Viel, S.; Mannina, L.; Rossi, R. Eur. J. Org.
Chem. 2002, 1063.
23. Pavlík, J.; Šnajdr, I.; Kuneš, J.; Špulák, M.; Pour, M. J. Org. Chem. 2009, 74, 703.
24. Pour, M.; Negishi, E.-I. Tetrahedron Lett. 1997, 38, 525.
25. Li, X.-C.; Ferreira, D.; Jacob, M. R.; Zhang, Q.; Khan, S. I.; ElSohly, H. N.; Nagle, D.
G.; Smillie, T. J.; Khan, I. A.; Walker, L. A.; Clark, A. M. J. Am. Chem. Soc. 2004,
126, 6872.
26. Dean, W. D.; Blum, D. M. J. Org. Chem. 1993, 58, 7916.
27. Li, W.; Li, J.; Lin, M.; Wacharasindhu, S.; Tabei, K.; Mansour, T. S. J. Org. Chem.
2007, 72, 6016.
28. Li, W.; Li, J.; Wan, Z.-K.; Wu, J.; Massefski, W. Org. Lett. 2007, 9, 4607.
29. National Committee for Clinical Laboratory Standards. Reference method for
broth dilution antifungal susceptibility testing of yeasts: Approved Standard,
NCCLS document M27-A, 771 E. Lancaster Avenue, Villanova, PA, 19085, 1997.
References and notes
1. Richardson, M. D. J. Antimicrob. Chemother. 2005, 56, i5.
2. Kale, P.; Johnson, L. B. Drugs Today 2005, 41, 91.
3. Spellberg, B. J.; Filler, S. G.; Edwards, J. E., Jr. Clin. Pract. 2006, 42, 244.
4. Oppenheim, B. A.; Herbrecht, R. Clin. Infect. Dis. 1995, 21, 1145.
5. Bhatti, Z.; Shaukat, A.; Almyroudis, N. G.; Segal, B. H. Mycopathologia 2006,
162, 1.
6. De Sarro, A.; La Camera, E.; Fera, M. T. J. Chemother. 2008, 20, 661.
7. La Regina, G.; D’Auria, F. D.; Tafi, A.; Piscitelli, F.; Olla, S.; Caporuscio, F.;
Nencioni, L.; Cirilli, R.; La Torre, F.; De Melo, N. R.; Kelly, S. L.; Lamb, D. C.;
Artico, M.; Botta, M.; Palamara, A. T.; Silvestri, R. J. Med. Chem. 2008, 51, 3841.
8. Pfaller, M. A.; Diekema, D. J. Clin. Microbiol. Rev. 2007, 20, 133.
9. Playford, E. G.; Eggimann, P.; Calandra, T. Curr. Opin. Infect. Dis. 2008, 21, 610.
10. Pour, M.; Špulák, M.; Balšánek, V.; Kuneš, J.; Buchta, V.; Waisser, K. Bioorg. Med.
Chem. Lett. 2000, 10, 1893.
ˇ
30. Hocek, M.; Holy´, A.; Votruba, I.; Dvoráková, H. J. Med. Chem. 2000, 43, 1817.
31. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer
Res. 1987, 47, 936.
32. Havranek, M.; Sauerberg, P.; Mogensen, J. P.; Kratina, P.; Jeppesen, C. B.;
Pettersson, I.; Pihera, P. Bioorg. Med. Chem. Lett. 2007, 17, 4144.
11. Pour, M.; Špulák, M.; Balšánek, V.; Kuneš, J.; Buchta, V. Bioorg. Med. Chem. 2003,
11, 2843.
12. Pour, M.; Špulák, M.; Buchta, V.; Kubanová, P.; Vopršalová, M.; Wsól, V.;
Fáková, H.; Koudelka, P.; Pourová, H.; Schiller, R. J. Med. Chem. 2001, 44, 2701.