Synthesis and fungicidal activity of 3,5-dichloropyrazin-2(1H)-one derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.06.024

We synthesized a family of 3,5-dichloropyrazin-2(1H)-one derivatives and assessed their in vitro fungicidal activity against Candida albicans. Compounds 11 and 20 were most active against C. albicans and induced accumulation of reactive oxygen species in

Full text of DOI:10.1016/j.bmcl.2009.06.024

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4064–4066
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and fungicidal activity of 3,5-dichloropyrazin-2(1H)-one derivatives
a
b
b
Isabelle E. J. A. François a, Bruno P. A. Cammue a, , Sara Bresseleers , Hein Fleuren , Georges Hoornaert ,
*
Vaibhav P. Mehta c, Sachin G. Modha c, Erik V. Van der Eycken c, Karin Thevissen a
a Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium
b Laboratory for Organic Synthesis, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee, Belgium
c Laboratory for Organic & Microwave-Assisted Chemistry (LOMAC), Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized a family of 3,5-dichloropyrazin-2(1H)-one derivatives and assessed their in vitro fungi-
cidal activity against Candida albicans. Compounds 11 and 20 were most active against C. albicans and
induced accumulation of reactive oxygen species in this pathogen. Using a genome-wide approach in
the yeast Saccharomyces cerevisiae, we demonstrated that genes involved in vacuolar functionality and
DNA-related functions play an important role in cellular mechanisms underlying the fungicidal activity
of these compounds.
Received 28 April 2009
Revised 2 June 2009
Accepted 3 June 2009
Available online 13 June 2009
Keywords:
Antifungal
Ó 2009 Elsevier Ltd. All rights reserved.
Candida
Reactive oxygen species
Since the early 1980s, fungi have emerged as major causes of
human disease, especially among immunocompromised patients.1
Several factors are associated with this increasing incidence of fun-
gal infections, including the increasing use of invasive devices and
implants that can be colonized by fungal biofilms and the larger
population of immunocompromised patients. In the latter, fungi
behave as opportunistic pathogens that can cause life-threatening
infections, which are mainly caused by Candida spp. or Aspergillus
spp. Some of the currently used antimycotics suffer from resistance
occurrence and other pharmacological limitations, such as harmful
drug–drug interactions, limited activity spectrum and/or high gen-
eral cytotoxicity. Therefore, the search for new antifungal com-
pounds with a novel mode of action is imperative.
In this context, we screened an in-house library, comprising of
approx. 50 heterocyclic compounds including pyrazin-2(1H)-ones,
oxazinones, pyridines and imidazopyrimidines, for fungicidal
activity against the human fungal pathogen Candida albicans. To
this end, we determined the minimal fungicidal activity of each
compound against C. albicans strain CAI4.2 The minimal fungicidal
concentration (MFC) for each compound was determined in the
saline buffer PBS and was calculated as the minimal concentration
of the compound resulting in less than 0.1% survival of a C. albicans
culture relative to the DMSO control.3 It should be noted that
determination of MFC values is preferred over MIC values, since
the former reflects fungicidal activity whereas the latter may ac-
count for both fungistatic as well as fungicidal activity. As such,
we identified two compounds, 1 and 2, both 3,5-dichloropyrazin-
2(1H)-one derivatives, with an MFC of 50 and 10 g/mL, respec-
l
tively. Until now, only few reports describing the antifungal
activity of pyrazinone derivatives are available.4 Hence, since these
compounds could represent a novel class of antifungal compounds,
we synthesized5 several substituted 3,5-dichloropyrazin-2(1H)-
one derivatives (Scheme 1) and analyzed their fungicidal activity
against C. albicans (Table 1).
Table 1 demonstrates that compounds 11 and 206 are charac-
terized by the lowest MFC (i.e., 5
lg/mL); other compounds with
R1
HN
R2
NaHSO3
R1 NH2 + R2 CHO
NaCN
+
H2O, MeOH,60 ºC
Overnight
CN
Dry HCl(g)
Ether, 20 min
R1
R1
R2
R2
N
O
(COCl)2, Et3NH+Cl-
.HCl
HN
Toluene, DMF (cat),
r.t., 2 days
CN
Cl
N
Cl
R1 = alkyl, aryl, benzyl
R2 = H, alkyl, aryl, benzyl
* Corresponding author. Tel.: +32 16 32 96 82; fax: +32 16 32 19 66.
E-mail address: bruno.cammue@biw.kuleuven.be (B.P.A. Cammue).
Scheme 1. Synthesis of 3,5-dichloropyrazin-2(1H)-ones.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.024

I. E. J. A. François et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4064–4066
4065
Table 1
shown). These data point to the causal link between ROS produc-
tion induced by compound 11 and its fungicidal activity.
Antifungal activity of pyrazin-2(1H)-one derivatives against C. albicans
R1
In order to further unravel the mode of antifungal action of
compound 11, a genome-wide genetic approach in Saccharomyces
cerevisiae was followed. This approach consisted of screening the
complete haploid collection of 4853 S. cerevisiae deletion mutants
(BY4741-Mat A collection, Invitrogen), individually deleted for
non-essential genes, for mutants that were either resistant or
hypersensitive towards compound 11 in agar. Theoretically, yeast
mutants that are resistant to an antifungal compound are affected
in a sensitivity gene, whereas yeast mutants that are hypersensi-
tive to an antifungal compound are affected in a tolerance gene.
The nature of sensitivity and tolerance genes provides complemen-
tary information regarding the mode of action of the antifungal
compound. First, we determined the minimal inhibitory concentra-
tion (MIC) of compound 11 for the parental strain BY4741 in YPD
R2
N
N
O
Cl
R3
Compds R1
R2
R3
MFC
ROS
(
lg/mL)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Benzyl
Phenyl
p-Methoxybenzyl
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
50
10
50
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
p-Methoxybenzyl p-Methoxyphenyl
p-Methoxybenzyl Methyl
>100
>100
>100
>100
>100
>100
>100
5
>100
10
50
>100
100
>100
>100
>100
5
Benzyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
p-Methoxyphenyl
Phenethyl
Cyclohexyl
Cyclohexylmethyl
Methyl
p-Methoxyphenyl
o-Methoxyphenyl
o-Tolyl
p-Carbomethoxyphenyl Cl
Chloromethyl
agar plates as 2.5 lg/mL. Screening for compound 11 hypersensi-
tive or resistant mutants was performed on YPD agar plates con-
taining a compound 11 concentration which was fivefold lower
(0.5 lg/mL) or higher (12.5 l
g/mL), respectively, than the MIC.10
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Methyl
Using this genome-wide approach, we could only identify yeast
mutants that were hypersensitive to compound 11. The fact that
no compound 11 resistant S. cerevisiae deletion mutants could be
identified could imply that the target for compound 11 is encoded
by an essential gene and as such, could point to a low frequency of
resistance occurrence in vivo. However, note that resistance
against an antifungal compound can also occur via other mecha-
nisms than mutation/deletion of the target gene, namely via in-
creased efflux of the compound, modulation of its target, or via a
compensatory mutation in a related biochemical pathway, etc.
We identified 11 deletion mutants with at least fivefold in-
creased sensitivity to compound 11 (listed in Table 2). Most genes
fall into a limited set of functional classes, including (i) vacuolar
functionality and assembly of vacuolar proton-translocating ATP-
ase, (ii) DNA synthesis and repair, transcription and chromatin
structure and (iii) meiotic cell division. We could further demon-
strate that these compound 11 hypersensitive yeast deletion
mutants are not hypersensitive to the structurally very similar
non-ROS inducing compound 2 (results not shown), pointing to
the specificity of the identified tolerance genes towards compound
11. we will briefly discuss the two major functional classes of
compound 11 tolerance genes in the next paragraphs.
H
H
H
H
o-Methoxybenzyl Benzyl
Methyl
2-Methoxyethyl
Phenyl
Phenyl
Phenyl
m-Methoxyphenyl
Phenyl
Bromomethyl
Chloromethyl
Chloromethyl
Methoxy >100
Phenyl >100
ꢀ
ꢀ
+ indicates compounds with a corrected fluorescence value (CFV)6 >500 units when
assayed at 100
g/mL; ꢀ indicates compounds with a CFV<100 units when assayed
at 100 g/mL.
l
l
fungicidal activity were compounds 2 and 13 (MFC = 10
compounds 1, 3 and 14 (MFC = 50 g/mL); and compound 16
(MFC = 100 g/mL). Replacing chlorine at 3-position of compound
11 by methoxy or phenyl resulted in a complete loss of fungicidal
activity (compounds 21 and 22,6 respectively). This structure–
activity relationship (SAR) study revealed that to detect high fungi-
cidal activity, the pyrazin-2(1H)-one scaffold should bear both, a
chlorine at the 3-position and a chloro- or bromomethyl at the 6-
position. Note that MFC of fluconazole, a frequently used azole
lg/mL);
l
l
(i) The largest proportion of compound 11 tolerance genes
fall in the functional class of vacuolar functionality and more
specifically in assembly and functionality of vacuolar H+ ATPase
(V-ATPase). V-ATPase has a role in the acidification of vacuoles,
antimycotic, is greater than 200 lg/mL (data not shown).
We could recently demonstrate a link between fungicidal activ-
ity against C. albicans and induction of reactive oxygen species
(ROS) in C. albicans.7 As a first step to unravel the mode of anti-
fungal action of the fungicidal 3,5-dichloropyrazin-2(1H)-ones,
we determined their ROS induction capacity using 20,70-dichloro-
fluorescin diacetate staining (Table 1).7 Compounds 11 and 20
induce ROS in C. albicans. We next focused on unraveling the cellu-
lar mechanisms underlying the fungicidal activity of compound 11.
In a first instance, we analyzed whether the induction of ROS by
compound 11 is causally linked with its fungicidal activity. To this
end, we determined the percentage survival of an overnight C. albi-
cans culture after treatment with compound 11 in the presence
and absence of the antioxidant ascorbic acid (AA). The low molec-
ular weight antioxidant AA is one of the most studied and powerful
antioxidants in plant cells.8 AA can directly scavenge superoxide,
ÅOH and singlet oxygen and can reduce H2O2 to water via an ascor-
bate peroxidase reaction.9 Percentage survival of C. albicans upon
Table 2
Compound 11 tolerance genes
Gene
Function
Vacuolar functionality
VPS15
CUP5
Serine/threonine protein kinase involved in vacuolar protein sorting
Proteolipid subunit of the vacuolar H+ proton-translocating ATPase (V-
ATPase) V0 sector
VPH2
VMA7
Membrane protein required for V-ATPase function
Subunit of the V1 sector of V-ATPase
Transcription
ADA2
HTL1
Transcriptional co-activator, component of the ADA/SAGA complexes
Component of the RSC chromatin remodeling complex
Subunit of the SAGA transcriptional regulatory complex
SPT7
Meiotic cell division
GET2
Protein required for meiotic nuclear division
YKL118W Hypothetical protein, involved in meiotic cell division
incubation with 5
percentage survival of C. albicans upon coincubation with 5
mL compound 11 and 80 mM AA was 64%. Control application of
80 mM AA alone did not influence the percentage (results not
l
g/mL compound 11 was 0.009%, whereas the
Varia
REG1
AMD1
Regulatory subunit of type 1 protein phosphatase Glc7p
AMP deaminase, possibly involved in regulation of intracellular
adenine nucleotide pools
lg/

4066
I. E. J. A. François et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4064–4066
was determined as the ratio of the number of CFUs after compound treatment
to the number of CFUs after 2% DMSO (control) treatment.
4. (a) Jiang, B.; Yang, C. G.; Wang, J. J. Org. Chem. 2001, 66, 4865; (b) Bereznak, J.F.;
Sharpe, P.L.; Sheth, R.; Stevenson, T.M.; Taggi, A.E.; Tseng, C.P.; Zhang, W. World
Patent Application WO 06/089060.
5. General procedure for the synthesis of substituted 3, 5-dichloropyrazin-2(1H)-ones.
In a 500 mL round-bottomed flask NaHSO3 (0.05 mol) was dissolved in water
and aldehyde (0.05 mol) was added dropwise under an argon atmosphere.
controlling several antiport systems for ions and organic molecules
and playing a role in maintaining cellular electrochemical equilib-
rium. A link between oxidative stress and V-ATPase was demon-
strated in Caenorhabditis elegans in which a gene encoding a V-
ATPase subunit was upregulated upon application of oxidative
stress through incubation under high oxygen levels.11 Additionally,
V-ATPase was reported to play a role in resistance to cellular tox-
ins,12 the drug trifluoroperazine13 and the antimicrobial peptide
MiAMP1 isolated from Macadamia integrifolia.14
(ii) Yeast mutants affected in genes involved in DNA synthesis
and repair, transcription and chromatin structure (including
ADA/SAGA histone acetyltransferase complexes or SWI/SNF nucle-
osome remodeling complex) were previously identified as hyper-
After stirring for 45 min,
a solution of substituted amine (0.05 mol) in
methanol was added dropwise to the reaction mixture and stirring was
continued for 2 h at 60 °C. Subsequently, NaCN (0.05 mol) was added and the
reaction mixture was left stirring overnight at 60 °C (under hood). The reaction
mixture was cooled to room temperature and extracted with dichloromethane
(3 ꢂ 50 mL). The organic phases were combined, washed with brine (50 mL),
then dried for 2 h over Na2SO4 and concentrated. The residue was dissolved in
dry ether (200 mL) and the resulting solution was cooled to 0 °C. Dry HCl gas
was bubbled through the solution upon vigorous stirring for 20 min and the
precipitate was filtered off and dried. Subsequently, the precipitate was
suspended in 500 mL flask in dry toluene and oxalyl chloride (0.2 mol,
4.0 equiv) was added dropwise under an argon atmosphere. After stirring for
45 min, triethylamine hydrochloride (0.075 mol, 1.5 equiv) was added by small
portions and DMF (0.1 equiv) as catalyst and the reaction mixture was kept
stirring for 2 days. It was then concentrated under reduced pressure and the
residue was purified by silica gel column chromatography (from 10% to 30%
EtOAc in petroleum ether) to obtain substituted 3,5-dichloropyrazin-2(1H)-
ones 1–10, 12–19.
sensitive to
a variety of stresses, including oxidative and
chemical stress.12,15 This finding highlights the requirement for
de novo transcription in response to environmental stress.
In conclusion, we demonstrated high fungicidal activity against
C. albicans of 3,5-dichloropyrazin-2(1H)-ones with chlorine at 3-
position and chloromethyl (or bromomethyl) at 6-position (com-
pounds 11 and 20). Both compounds induced accumulation of
ROS in C. albicans. Moreover, this ROS induction was causally
linked with the antifungal activity of compound 11. Additionally,
in order to identify general cellular mechanisms underlying the
fungicidal activity of compound 11, we demonstrated that genes
involved in vacuolar functionality (including V-ATPases) and
DNA-related functions play an important role.
6. See Supplementary data for the experimental procedure.
7. François, I. E. J. A.; Cammue, B. P. A.; Borgers, M.; Ausma, J.; Dispersyn, G. D.;
Thevissen, K. Anti-Infect. Ag. Med. Chem. 2006, 5, 3. Briefly, 5 mL of an overnight
C. albicans culture in YPD (grown at 37 °C) was resuspended in 8 mL phosphate-
buffered saline (PBS). Forty-microliter aliquots of this cell suspension were
mixed with 20
and incubated in white microtiterplates (Perkin–Elmer, Norwalk, Connecticut).
After 1 h of incubation at 37 °C, 40-
aliquots of 20,70-dichlorofluorescin
diacetate (DCFHDA, Molecular Probers, Inc. Eugene, Oregon) stock solution
(25 M) were added to the cell suspensions. Fluorescence emitted by the cells
lL of 100 lg/mL of each compound in 10% DMSO or 10% DMSO
lL
l
Acknowledgments
was measured with a Perkin–Elmer LS50B fluorescence spectrometer at an
excitation wavelength of 485 nm (slit, 5 nm) and an emission wavelength of
525 nm (slit, 5 nm). Fluorescence was measured after 3 h of incubation.
Fluorescence values of the samples were corrected by subtracting the
fluorescence value of the antifungal compound without cells but with
DCFHDA. These values were denominated the corrected fluorescence values
(CFVs).
A postdoctoral fellowship to K.T. from K.U. Leuven (Industrial
Research Fellow) is gratefully acknowledged. Support was pro-
vided to E.V.d.E. by the research fund of the University of Leuven
and the FWO (Fund for Scientific Research—Flanders (Belgium)).
V.P.M. is grateful to the IRO (Interfacultaire Raad voor Ontwikkel-
ings-samenwerking) for obtaining a doctoral scholarship.
8. (a) Smirnoff, N. Ann. Bot. 1996, 78, 661; (b) Arrigoni, O.; De Tullio, M. C. J. Plant
Physiol. 2000, 157, 481; (c) Horemans, N.; Foyer, C. H.; Potters, G.; Asard, H.
Plant Physiol. Biochem. 2000, 38, 531; (d) Smirnoff, N. Curr. Opin. Plant Biol. 2000,
3, 229.
9. Noctor, G.; Foyer, C. H. Ann. Rev. Plant Phys. Plant Mol. Biol. 1998, 49, 249.
10. The individual yeast deletion mutants were grown in 96 well microtiterplates
Supplementary data
containing 100
mutants were spotted on YPD agar plates containing 0.5
compound 11 using a 96-pin replicator. The wild type strain BY4741 failed to
grow on plates containing 2.5 g/mL compound 11. After 48–72 h incubation
l
L YPD. After 48 h incubation at 30 °C, the individual deletion
l
g/mL or 12.5 g/mL
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.024.
l
at 30 °C, plates were scored and hypersensitive mutants were identified. The
identified compound 11 hypersensitive mutants were retested as follows. Five-
microliter samples of fivefold serial dilutions of each yeast cell culture
including the wild type strain BY4741 (grown to stationary phase in YPD in
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glucose) was diluted in PBS (ꢁ2 ꢂ 106 cells/mL) and treated with various
concentrations of the compounds of interest in 2% DMSO. After 5 h incubation
at 37 °C, colony forming units (CFUs) were determined. Percentage survival
microtiterplates) were spotted on YPD plates containing
0 or 2.5 lg/mL
compound 11. Growth was assessed after 48 h of incubation at 30 °C. Only
mutants displaying less growth than the wild type strain BY4741 in this assay
were considered compound 11 hypersensitive mutants.
11. Yanase, S.; Ishi, N. J. Radiat. Res. 1999, 40, 39.
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