3,5-Disubstituted pyranone analogues of highly antifungally active furanones: Conversion of biological effect from antifungal to cytostatic

Article abstract of DOI:10.1016/j.bmcl.2010.10.052

A series of 3-aryl-5-acyloxymethyl-5,6-dihydro-2H-pyran-2-ones, related to highly antifungally active butenolides, was synthesized via cyclization of substituted δ-hydroxy acids as the key step, and evaluated for their in vitro antifungal activity and cytostatic activity. While the extension of the furanone ring to pyranone led to a complete loss of the antifungal effect, some of the compounds displayed promising effect against several cell lines, including the resistant colorectal carcinoma cells.

Full text of DOI:10.1016/j.bmcl.2010.10.052

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7358–7360
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3,5-Disubstituted pyranone analogues of highly antifungally active
furanones: Conversion of biological effect from antifungal to cytostatic
Radan Schiller a, Lucie Tichotová a, Jan Pavlík a, Vladimír Buchta b, Bohuslav Melichar c, Ivan Votruba d,
a
a
a,
⇑
ˇ
Jirí Kuneš , Marcel Špulák , Milan Pour
a Centre For New Antivirals and Antineoplastics, Department of Inorganic and Organic Chemistry, Charles University in Prague, Faculty of Pharmacy in Hradec Králové,
Heyrovského 1203, CZ-500 03 Hradec Králové, Czech Republic
b Department of Clinical Microbiology, University Hospital and Charles University, Faculty of Medicine, Sokolská 581, CZ-500 03 Hradec Králové, Czech Republic
c Institute of Experimental Neurosurgery, University Hospital and Charles University, Sokolská 581, CZ-500 03 Hradec Králové, Czech Republic
d Institute of Organic Chemistry and Biochemistry, Flemingovo n. 2, CZ-166 10 Prague 6, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-aryl-5-acyloxymethyl-5,6-dihydro-2H-pyran-2-ones, related to highly antifungally active
butenolides, was synthesized via cyclization of substituted d-hydroxy acids as the key step, and evaluated
for their in vitro antifungal activity and cytostatic activity. While the extension of the furanone ring to
pyranone led to a complete loss of the antifungal effect, some of the compounds displayed promising
effect against several cell lines, including the resistant colorectal carcinoma cells.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 August 2010
Revised 12 October 2010
Accepted 13 October 2010
Available online 21 October 2010
Keywords:
Pyranone
Synthesis
Screening
Antifungal
Cytostatic
Several years ago, we disclosed antifungal analogues of natural
3,5-disubstituted butenolides,1–4 the structure–activity relation-
ships of which clearly indicated that the necessary pharmaco-
phoric features include: (1) double bond in conjugation with the
lactone moiety and (2) halogenated phenyl ring at C(3). The
in vitro antifungal effect of the most successful series, that is,
Further development of the furanone pharmacophore led to the
ring expansion into pyranone, with the acyloxymethyl moiety
being located at C6 (3). Surprisingly, the resultant pentenolides
showed total lack of both antifungal activity and cytostatic effect.7
Nonetheless, another possible location of the acyloxymethyl group
would logically be C5, leading onwards to 3,5-disubstituted-5,
6-dihydro-2H-pyran-2-ones (4). In this communication, we report
the synthesis and biological evaluation of these compounds, which
3-(halogenated
phenyl)-5-acyloxymethyl-2,5-dihydrofuran-2-
ones (1, Fig. 1), was comparable to that of amphotericin B, espe-
cially against the dangerous filamentous human pathogen Aspergil-
lus fumigatus.
A
flow cytometric study5 revealed that the
compounds of this series damage the fungal cell membrane of
Candida albicans. On the molecular level, the SARs of the furanon-
es1,3,6 together with zero activity of related 3,6-disubstituted pyra-
nones7 have clearly indicated that a non-specific Michael addition
of a nucleophile to the b-carbon of the butenolide ring is an unli-
kely mechanism of action. Very recently, we disclosed the finding6
that the nature of antifungal action of compounds 1 lies in the
ability of some of the derivatives (Z = 4-Br, 3,4-diCl) to produce
the corresponding, highly antifungally active 5-methylene fura-
O
O
O
O
O
Z
Z
Z
OCOR
OCOR
1
3
antifungal
assay
O
O
O
Z
nones
2 upon dissolution in DMSO under antifungal assay
conditions (Fig. 1).
2
OCOR
active species
4
Z = 4-Br, 3,4-diCl
⇑
Corresponding author. Tel.: +420 49 5067277; fax: +420 49 5067166.
E-mail address: milan.pour@faf.cuni.cz (M. Pour).
Figure 1. Design of the title pyranones 4.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.052

R. Schiller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7358–7360
7359
may serve as novel natural product-derived leads in anticancer
drug development.
MeOOC
COOMe
While a number of inventive approaches towards 6-substituted
pentenolides have appeared in the literature over the past 10 years,8
3,5-disubstituted compounds received practically no attention.
Thus, because of the simplicity of the methodology as well as com-
mercial availability of substituted phenylacetic acids, we employed
a proven cyclization strategy which had been successfully applied to
the synthesis of 3,5-disubstituted-2,5-dihydrofuran-2-ones,1–3 and
recently in the construction of the 3,6-disubstituted pentenolide
ring.7 Hence, given the structure of the title compounds 4, the se-
quence is based on a-alkylation of protected phenylacetic acids with
a suitably substituted alkylating agent followed by cyclization and
introduction of the double bond into the ring.
As regards the alkylating agent, 5-iodomethyl-2,2-dimethyl-1,3-
dioxan (7), obtainable from the commercially available diethyl-
bis(hydroxymethyl)malonate 5 (Scheme 1) over four steps9 was a
convenient choice.
The preparation of the key intermediates, 3-aryl-5-hydroxy-
methyl-5,6-dihydro-2H-pyran-2-ones (12) is outlined in Scheme 2.
Thus, methyl esters of substituted phenylacetic acids (8a–f) were
deprotonated with LDA, and the enolates quenched with iodide 7
to afford the methyl esters of 2-aryl-3-(2,2-dimethyl-1,3-dioxan-
5-yl)propanoic acids (9a–f). Even though a spontaneous cyclization
to six-membered lactones following the deprotection of esters 9a–
f with Dowex 50 in MeOH/H2O could be expected, we obtained a
mixture of the desired products together with acyclic material in
approximately 1:1 ratio. Fortunately, the cyclization could be dri-
ven to completion7 by replacing the solvent with MeCN. In order
to introduce the double bond, the hydroxy group was protected
as a TBS ether, the product enolized with LDA and the enolate
quenched with PhSeBr. The intermediate 3-phenylselanyl deriva-
tives were, due to their limited stability, immediately oxidized to
afford the corresponding selenoxides, which underwent a sponta-
neous syn-elimination. Subsequent acidic removal of the silyl
group then furnished the target pyranone alcohols 12a–f.
Finally, alcohols 12a–f were treated with selected acyl chlorides
under mild conditions2 in the presence of pyridine (Scheme 3).
All target esters 4ax–fz were evaluated for their in vitro anti-
fungal activity against a set of human pathogenic fungi including
the representatives of both yeast (C. albicans ATCC 44859, C. albi-
cans ATCC 90028, Candida krusei ATCC 6258, C. krusei E28, Candida
parapsilosis ATCC 22019, Candida glabrata 20/I, Candida tropicalis
156, Candida lusitaniae 2446/I, and Trichosporon beigelii 1188) and
filamentous strains ( A. fumigatus 231, Absidia corymbifera 272,
Microsporum gypseum, and Trichophyton mentagrophytes 445) using
the microdilution format of the NCCLS M27-A guidelines.10 In gen-
eral, the antifungal effect of pyranones 4 was low to negligible
LDA, 7
Z
8a-f
Z
O
O
9a-f
Dowex 50
MeOH/H2O
O
COOMe
O
Z
+
Z
OH OH
OH
11a-f
10a-f
Dowex 50, MeCN
1.TBSCl
2.LDA
3.PhSeBr
4.mCPBA
5.H+, H2O
O
O
Z
OH
12a-f
a
b
c
Z = H; Z = 4-Br; Z = 3,4-Cl2;
d Z = 3-Cl; e Z = 4-Cl; f Z = 4-OMe
Scheme 2. Synthesis of pyranone alcohols 12.
RCOCl
py, CH2Cl2
0 ºC
O
O
O
O
O
Z
Z
O
OH
R
4ax-fz
12a-f
a
b
c
Z = H; Z = 4-Br; Z = 3,4-Cl2;
d Z = 3-Cl; e Z = 4-Cl; f Z = 4-OMe
x:
y
R = CH3; : R = (CH3)3C;
z: R = phenyl
Scheme 3. Preparation of pyranone esters 4.
(MIC values P62.5 l
mol LÀ1), and there appeared to be no signifi-
cant differences in the activity of the phenyl derivatives (4ax–4az),
p-methoxyphenyl derivatives (4fx–4fz), and the halophenyl com-
pounds (4bx–4bz, 4cx–4cz, 4dx–4dz, and 4ex–4ez). Importantly,
unlike the analogous furanone esters6 (1), pyranone esters 4
showed no sign of an easy elimination to furnish the corresponding
5-methylene derivatives analogous to 2 (see Fig. 1). For example,
the solution of pentenolide 4ax has been stable in DMSO-d6 solu-
tion for an indefinitely long time, as evidenced by NMR. Also, in or-
der to prepare the corresponding 5-methylene compound from
derivative 12a, alcohol 12a had to be tosylated and the tosylate
treated with DBU.
1. cat. TsOH
OMe
O
O
O
O
O
OMe
2. NaCl, DMSO
O
O
O
OH OH
5
6
However, initial screening for cytostatic activity11,12 on mouse
lymphocytic leukemia L1210 cells (ATCC CCL 219), CCRF-CEM T
lymphoblastoid cells (human acute lymphoblastic leukemia, ATCC
CCL 119), human promyelocytic leukemia HL-60 cells (ATCC CCL
240), and human cervix carcinoma HeLa S3 cells (ATCC CCL 2.2) re-
vealed potentially interesting activity of compounds 4cx, 4cz, and
1. LiAlH4
2. I2, PPh3
I
O
O
4ez at the concentration of 10 l
mol LÀ1. While all other derivatives
displayed marginal or no inhibition of cell growth at this concen-
7
tration, these compounds substituted with chlorine on the phenyl
Scheme 1. Synthesis of alkylating agent 7.

7360
R. Schiller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7358–7360
Table 1
might play a role in the cytostatic effect, it should be noted that the
Growth inhibitory activity (IC50
,
l
mol LÀ1) of compounds 12c, 4cx, 4cz, and 4ez
compounds do not easily undergo the addition of a nucleophile as
strong as a thiolate. Especially remarkable was the activity against
the resistant colorectal carcinoma cells, which renders the sub-
stances possible leads for further investigation and development
as potential anticancer agents. Despite recent progress of combina-
tion chemotherapy and introduction of biological agents, such as
bevacizumab and cetuximab, more active agents are urgently
needed for the therapy of colorectal carcinoma.
Cell line
Compound
12c
4cx
4cz
4ez
L1210
HL-60
HeLa S3
CCRF-CEM
NAa
NA
NA
NA
7.3 0.42
7.3 0.44
6.8 0.41
3.6 0.26
5.9 0.35
6.1 0.36
6.7 0.40
4.5 0.27
NA
NA
NA
7.1 0.50
a
Not active at relevant concentrations.
Acknowledgments
Table 2
Comparison of growth inhibitory activity (IC50, l
mol LÀ1) of compounds 12c, 4cx, and
This work was supported by Charles University (project No.
SVV/2010/261/001) and the Ministry of Education, Youth and
Sports of the Czech Republic (projects Nos. 1M0508 and
MSM0021620822).
4ez and two drug standards (OxPt—oxaliplatin, Irt—irinotecan)
Cell line
HT-29
Compound
4ez
12c
4cx
OxPt
Irt
NA
2.45 0.15
2.93 0.18
NA
4.73 0.28
References and notes
1. Pour, M.; Špulák, M.; Balšánek, V.; Kuneš, J.; Buchta, V.; Waisser, K. Bioorg. Med.
Chem. Lett. 2000, 10, 1893.
2. 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.
3. Pour, M.; Špulák, M.; Balšánek, V.; Kuneš, J.; Kubanová, P.; Buchta, V. Bioorg.
Med. Chem. 2003, 11, 2843.
ring and easy-to-hydrolyze acyl group showed a promising inhib-
itory effect. The determination of their IC50 values (Table 1) re-
vealed that they were in the range of 3–7 l .
mol LÀ1
Selected compounds (12c, 4cx, and 4ez) were subsequently sub-
jected to screening against colorectal carcinoma cell line HT-29
(ATCC HTB 38), resistant to most cytostatic agents. Among the three
drugs currently used in therapy of colorectal carcinoma (5-fluoro-
uracil, irinotecan, and oxaliplatin), HT-29 cells are moderately
sensitive only to irinotecan. Thus, the low IC50 values observed in
HT-29 cells (Table 2) are of particular interest. It is also worthy to
note that the 4-chlorophenyl derivative 4ez showed interesting
activity only against the CCRF-CEM and HT-29 cells, which might
be a sign of possible selectivity.
Because all pyranones 4 can act as Michael acceptors, derivative
4ez was subjected to a reaction with PrSH in THF–H2O and the
presence of K2CO3. While no conjugate addition was observed at
0 °C, less than 5% of the addition product was detected in the 1H
NMR spectrum of the crude reaction mixture after 5 h at rt.13
Apparently, the 3-arylated pyranones do not easily undergo conju-
gate addition of a thiolate.
In summary, we have prepared a series of 3-aryl-5-acyloxy-
methyl-5,6-dihydro-2H-pyran-2-ones,14 derived from the struc-
ture of highly antifungally active furanones. Similar to the
recently described7 6-acyloxymethyl analogues, this change led
to a complete loss of antifungal activity, which is thus specifically
linked to furanone (butenolide) ring. Most noteworthy, some of the
3,5-disubstituted pentenolides showed promising cytostatic activ-
ity. While it could be argued that a non-specific conjugate addition
4. Buchta, V.; Pour, M.; Kubanová, P.; Silva, L.; Votruba, I.; Vopršálová, M.; Schiller,
R.; Fáková, H.; Špulák, M. Antimicrob. Agents Chemother. 2004, 48, 873.
5. Vale-Silva, L. A.; Buchta, V.; Vokurková, D.; Pour, M. Bioorg. Med. Chem. Lett.
2006, 16, 2492.
6. Šenel, P.; Tichotová, L.; Votruba, I.; Buchta, V.; Špulák, M.; Kuneš, J.; Nobilis, M.;
Krenk, O.; Pour, M. Bioorg. Med. Chem. 2010, 18, 1988.
7. Šnajdr, I.; Pavlík, J.; Schiller, R.; Kuneš, J.; Pour, M. Collect. Czech. Chem. Commun.
2007, 72, 1472.
8. For an up-to-date review on novel strategies, see: Boucard, V.; Broustal, G.;
Campagne, J. M. Eur. J. Org. Chem. 2007, 225.
9. Iwata, C.; Fujita, M.; Moritani, Y.; Sugiyama, K.; Hattori, K.; Imanishi, T.
Tetrahedron Lett. 1987, 28, 3131.
10. 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.
ˇ
11. Hocek, M.; Holy´, A.; Votruba, I.; Dvoráková, H. J. Med. Chem. 2000, 43, 1817.
12. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer
Res. 1987, 47, 936.
13. The rest was unchanged starting material.
14. Analytical data for 5-acetyloxymethyl-3-(3,4-dichlorophenyl)-5,6-dihydro-2H-
pyran-2-one (4cx): white crystals, mp 75–78 °C; 1H NMR: (300 MHz, CDCl3) d
7.58 (1H, d, J = 2.2 Hz, ArH2), 7.45 (1H, d, J = 8.5 Hz, ArH5), 7.33 (1H, dd,
J1 = 2.2 Hz, J2 = 8.5 Hz, ArH6), 6.91 (1H, d, J = 4.4 Hz, H4), 4.58–4.51 (1H, m, H6),
4.45–4.37 (1H, m, H6), 4.28 (1H, dd, J1 = 5.5 Hz, J2 = 11.3 Hz, CH2O), 4.19 (1H,
dd, J1 = 7.4 Hz, J2 = 11.3 Hz, CH2O), 3.10–2.98 (1H, m, H5), 2.10 (3H, s, CH3COO);
13C NMR: (75 MHz, CDCl3) d 170.6, 162.5, 141.8, 134.7, 133.0, 132.5, 132.3,
130.6, 130.2, 127.7, 67.9, 62.2, 34.6, 20.7; IR: (CDCl3) mmax 2956 (w), 2899 (w),
1731 (s), 1471 (m), 1366 (m) cmÀ1; LRMS (ESI): m/z (relative intensity) 315
[M+H]+ (2), 281 (8), 265 (7), 247 (5), 235 (49), 221 (12), 207 (8), 191 (4), 173
(100), 158 (15), 147 (87), 134 (20), 121 (25), 105 (21), 91 (30), 73 (75), 59 (20);
C,H,N: calcd for C14H12Cl2O4: C, 53.36; H, 3.84; found: C, 53.35; H, 4.12.