Novel fungicidal benzylsulfanyl-phenylguanidines

Article abstract of DOI:10.1016/j.bmcl.2011.04.075

A series of substituted benzylsulfanyl-phenylamines was synthesized, of which four substituted benzylsulfanyl-phenylguanidines (665, 666, 667 and 684) showed potent fungicidal activity (minimal fungicidal concentration, MFC ≤ 10 μM for Candida albicans and Candida glabrata). A benzylsulfanyl-phenyl scaffold with an unsubstituted guanidine resulted in less active compounds (MFC = 50-100 μM), whereas substitution with an unsubstituted amine group resulted in compounds without fungicidal activity. Compounds 665, 666, 667 and 684 also showed activity against single C. albicans biofilms and biofilms consisting of C. albicans and Staphylococcus epidermidis (minimal concentration resulting in 50% eradication of the biofilm, BEC50 ≤ 121 μM for both biofilm setups). Compounds 665 and 666 combined potent fungicidal (MFC = 5 μM) and bactericidal activity (minimal bactericidal concentration, MBC for S. epidermidis ≤ 4 μM). In an in vivo Caenorhabditis elegans model, compounds 665 and 667 exhibited less toxicity than 666 and 684. Moreover, addition of those compounds to Candida-infected C. elegans cultures resulted in increased survival of Candida-infected worms, demonstrating their in vivo efficacy in a mini-host model.

Full text of DOI:10.1016/j.bmcl.2011.04.075

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3686–3692
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel fungicidal benzylsulfanyl-phenylguanidines
Karin Thevissen a, , Klaartje Pellens a, Katrijn De Brucker a, Isabelle E. J. A. François a, Kwok K. Chow a,
⇑
Els M. K. Meert a, Wim Meert a, Geert Van Minnebruggen b, Marcel Borgers b,c, Valérie Vroome b,
Jeremy Levin d, Dirk De Vos d, Louis Maes e, Paul Cos e, Bruno P. A. Cammue a
a Centre of Microbial and Plant Genetics (CMPG), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgium
b Barrier Therapeutics nv, a wholly owned subsidiary of Stiefel Laboratories, Wiertzstraat 50, 1047 Brussel, Belgium
c Department Molecular Cell Biology, CARIM, Maastricht University, Maastricht, The Netherlands
d Centre for Surface Chemistry and Catalysis, Katholieke Universiteit Leuven, Kasteelpark Arenberg 23, 3001 Heverlee, Belgium
e Laboratory of Microbiology, Parasitology and Hygiene, Antwerp University, Groenenborgerlaan 171, 2020 Antwerp-Wilrijk, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of substituted benzylsulfanyl-phenylamines was synthesized, of which four substituted ben-
Received 14 March 2011
Revised 13 April 2011
Accepted 19 April 2011
Available online 24 April 2011
zylsulfanyl-phenylguanidines (665, 666, 667 and 684) showed potent fungicidal activity (minimal fungi-
cidal concentration, MFC 6 10
l
M for Candida albicans and Candida glabrata). A benzylsulfanyl-phenyl
scaffold with an unsubstituted guanidine resulted in less active compounds (MFC = 50–100
lM), whereas
substitution with an unsubstituted amine group resulted in compounds without fungicidal activity. Com-
pounds 665, 666, 667 and 684 also showed activity against single C. albicans biofilms and biofilms con-
sisting of C. albicans and Staphylococcus epidermidis (minimal concentration resulting in 50% eradication
Keywords:
Fungicidal
Benzylsulfanyl-phenylguanidines
Biofilm
Candida albicans
of the biofilm, BEC50 6 121
gicidal (MFC = 5 M) and bactericidal activity (minimal bactericidal concentration, MBC for S. epidermi-
dis 6 4 M). In an in vivo Caenorhabditis elegans model, compounds 665 and 667 exhibited less toxicity
lM for both biofilm setups). Compounds 665 and 666 combined potent fun-
l
l
than 666 and 684. Moreover, addition of those compounds to Candida-infected C. elegans cultures
resulted in increased survival of Candida-infected worms, demonstrating their in vivo efficacy in a
mini-host model.
Ó 2011 Elsevier Ltd. All rights reserved.
The increasing number of immunocompromised patients, com-
bined with advances in medical technology, has led to an increase
in fungal infections, with Candida albicans as the major fungal
pathogen. These infections are, especially in immunocompromised
patients, an important cause of morbidity and mortality, despite
aggressive treatment with new or more established licensed anti-
fungal agents.1 Apart from their existence under free-living or
planktonic form, fungi and bacteria are known to form biofilms
upon contact with various surfaces. Fungal biofilms, especially
those of C. albicans, can cause infections associated with medical
devices like indwelling intravascular catheters. Such infections
are particularly serious because biofilm-associated Candida cells
are relatively resistant to a wide spectrum of antifungal drugs.2
Due to this resistance, removal of the catheter is often required
to cure the infection and this can be a serious risk for the patient.3
Biofilms can also consist of mixed species, like C. albicans and
Staphylococcus epidermidis.4 Such mixed fungal-bacterial biofilms
are more resistant to antimycotics, like fluconazole, compared to
single species C. albicans biofilms.5 Due to toxicity, drug–drug
interactions and the increasing occurrence of resistance of current
antimycotics, there is an urgent need to identify novel fungicidal
compounds, preferentially with activity against fungal and mixed
species biofilms.6
In the present study, we identified novel fungicidal compounds
with activity against single C. albicans and mixed biofilms, consist-
ing of C. albicans and S. epidermidis, two organisms commonly
found in catheter-associated infections.5 We focused on the class
of piperazine-1-carboxamidine compounds, which were recently
shown to exhibit fungicidal activity against C. albicans.7 Their mode
of action comprises the induction of endogenous reactive oxygen
species, resulting in apoptosis in susceptible yeast.7,8 These piper-
azine-1-carboxamidines share overall 3D structural similarity with
abafungin, a compound belonging to a new class of microbiocidal
arylguanidines. Abafungin is characterized by broad fungicidal
activity against various species of pathogenic fungi and dermato-
phytes.9 Based on this overall 3D structural similarity between
piperazine-1-carboxamidines and arylguanidines, we hypothe-
sized that benzylsulfanyl-phenylamines, uniting structural fea-
tures of piperazine-1-carboxamidines and arylguanidines (Fig. 1),
are characterized by increased fungicidal activity compared to
Abbreviations: MFC, minimal fungicidal concentration; MBC, minimal bacteri-
cidal concentration; BEC, biofilm eradicating concentration.
⇑
Corresponding author. Tel.: +32 16329688; fax: +32 16321966.
E-mail address: karin.thevissen@biw.kuleuven.be (K. Thevissen).
piperazine-1-carboxamidines against C. albicans (MFC >100 l
M).7
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.075

K. Thevissen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3686–3692
3687
(20 mL mmolÀ1) was added and washed sequentially with brine
(20 mL mmolÀ1), H2O (20 mL mmolÀ1), brine (20 mL mmolÀ1),
H2O (20 mL mmolÀ1) and NH4Cl (20 mL mmolÀ1). The organic layer
was dried (MgSO4), filtered and concentrated. The residue was puri-
fied by flash-chromatography on SiO2 (Hex/AcOEt; 14:1) yielding
pure o-, m- or p-1,2-dicyclohexylguanidyl derivatives 665, 666,
667, 677, 679 and 680, respectively (Scheme 1).
To synthesize m- and p-1-formimidamideguanidyl derivatives
684, 687, 688 and 692 cyanoguanidine and HCl (1:1) were added
to respective solutions of the m- and p-amino intermediates (com-
pounds 657, 669, 655 and 651) dissolved in MeCN (4 mL mmolÀ1).
The reaction mixture was heated under microwaves at 150 °C for
15 min. After cooling to rt, the solid precipitate was filtered and
washed with MeCN yielding pure m- and p-1-formimidamideguan-
idyl derivatives 684, 687, 688 and 692, respectively (Scheme 1).13
The fungicidal activity of these newly synthesized compounds
was tested against the human fungal pathogens C. albicans (strain
SC5314)14 and C. glabrata (strain BG2).15 The minimal fungicidal
concentration (MFC) of the benzylsulfanyl-phenylamines is shown
in Table 1.16–19 Fluconazole (Sigma, St. Louis, MO) was used as ref-
erence compound. We identified four potent fungicidal com-
Figure 1. Overall 3D structural similarity of abafungin (A), piperazine carboxami-
dine (B) and benzylsulfanyl-phenylguanidine derivatives (C).
This hypothesis was further substantiated by a report on the anti-
fungal activity of structurally related alkylsulfanyl-pyridinylguani-
dines.10 In this study, we synthesized a series of benzylsulfanyl-
phenylamines and assessed their fungicidal activity against C. albi-
cans and Candida glabrata. Next, we assessed the bactericidal activ-
ity of the four most potent fungicidal benzylsulfanyl-phenylamines
against S. epidermidis, as well as their potential to eradicate single
and mixed species biofilms. Furthermore, we assessed toxicity and
efficacy of these molecules in the mini-host Caenorhabditis elegans
model.11 The important advantage of such mini-host models is the
possibility to test toxicity and efficacy of compounds in vivo on a
small scale (in microtiter plates). Inclusion of the C. elegans model
system in early stages of antifungal drug development allows the
determination of in vivo nontoxic doses, the selection of the
in vivo most promising molecules and the assessment of the effec-
tive concentration in vivo in a single assay, hence reducing animal
testing considerably.
pounds, namely 665, 666, 667 and 684, with MFC 6 10
both pathogens. Compounds with moderate fungicidal activity
(MFC = 10–50 M) were compounds 640, 642, 643, 649, 679, 687
and 688 (Table 1). The MFC of fluconazole for both species was
>100 M. Hence, compounds 665, 666, 667, 684, 640, 642, 643,
lM for
l
l
649, 679, 687 and 688 are all characterized by an increased fungi-
cidal activity compared to the most potent piperazine-1-carbox-
amidine derivatives.7 In general, benzylsulfanyl-phenylguanidines
displaying the highest fungicidal activity were characterized by
either R1 benzyl substituted with bromine on positions 3 and 5
in combination with R2 thiophenyl substituted with 1,2-dic-
yclohexylguanidine in meta (667) or para position (665) or imid-
odicarbonimidic diamide in meta position (684), or R1 benzyl
substituted with chlorine on position 4 and R2 thiophenyl substi-
tuted with 1,2-dicyclohexylguanidine in meta position (666). This
structure–activity relationship (SAR) study revealed that to detect
The synthetic route of the different compounds is outlined in
Scheme 1. Compound 1 (1 equiv) was added to a solution of Et3N
(1 equiv) dissolved in DMF (dimethyl formamide, 3 mL mmolÀ1),
followed by compound 2 (1 equiv). The mixture was stirred at rt
for 5 h. AcOEt (20 mL mmolÀ1) and brine (20 mL mmolÀ1) were
added and the layers were separated. The organic layer was dried
(MgSO4), filtered and concentrated. The residue was purified by
flash-chromatography on SiO2 (Hex/AcOEt; 10:1) yielding pure o-
, m- or p-amino derivatives 650 (Scheme 1, panel A), 651 (Scheme 1,
panel B), 655 (Scheme 1, panel C), 657 (Scheme 1, panel D), 669
(Scheme 1, panel E) and 670 (Scheme 1, panel F). To synthesize
compounds 640, 641, 642, 643, 647 and 649, Di-Boc thiourea, DI-
PEA (diisopropyl ethyl amine) and EDCI (1:1.2:1.2) were added to
respective solutions of the o-, m- and p-amino derivatives (com-
pounds 651, 670, 669, 650, 655 and 657) dissolved in DMF
(3 mL mmolÀ1). The mixture was stirred at rt for 48 h. Afterwards,
additional Di-Boc thiourea, DIPEA and EDCI (1.2:1.2:1.2) were
high fungicidal activity against Candida species (MFC 6 10 lM), the
benzylsulfanyl-phenyl scaffold should bear a R1 benzyl substituted
with bromine on positions 3 and 5 and a substituted guanidine at
the meta or para position of the R2 thiophenyl moiety. A ben-
zylsulfanyl-phenyl scaffold with an unsubstituted guanidine re-
sulted in less active compounds (MFC = 50–100 lM), whereas
substitution with an (unsubstituted) amine group resulted in com-
pounds without fungicidal activity (Table 1). Whether the in-
creased fungicidal activity of such substituted benzylsulfanyl-
phenylguanidines as compared to unsubstituted benzylsulfanyl-
phenylguanidines or benzylsulfanyl-phenylamines results from
an increased interaction with their fungal target, or alternatively,
results from increased hydrophobicity of the molecules and, conse-
quently, increased intracellular uptake, needs to be determined.
The four substituted benzylsulfanyl-phenylguanidines with
added to the mixture and stirred for 24 h. AcOEt (20 mL mmolÀ1
)
was added À1and the solution was washed with H2O
(20 mL mmol ), NaHCO3 (3Â) (20 mL mmolÀ1
)
and brine
(20 mL mmolÀ1). The organic layer was dried (MgSO4), filtered
and concentrated to give an intermediate compound, which was
used in the next step without purification. This compound was dis-
solved in CH2Cl2 (1.5 mL mmolÀ1) and cooled on an ice-bath. TFA
(1.5 mL mmolÀ1) was added and the reaction mixture was stirred
for 6 h at 0 °C. The reaction mixture was concentrated in vacuo
and the residue was purified by RP-HPLC to yield pure o-, m- or
p-guanidyl derivatives 640, 641, 642, 643, 647 and 649, respec-
tively (Scheme 1).12
MFC 6 10 lM for both pathogens were selected for subsequent
biological evaluation (Table 2). Their bactericidal activity against
S. epidermidis was determined as well as their potential to eradi-
cate single and mixed species biofilms. Compounds 665 and 666
were characterized by high bactericidal activity against S. epidermi-
dis (MBC 6 4
l
M), while compounds 667 and 684showed moder-
ate bactericidal activity (35
lM 6 MBC 6 75
l
M).20 Activity of
To synthesize o-, m- and p-1,2-dicyclohexylguanidyl derivatives
665, 666, 667, 677, 679 and 680 dicyclohexyl carbodiimide (DCC;
2 equiv) were added to respective solutions of the o-, m- and p-ami-
no derivatives (compounds 651, 655, 657, 670, 650 and 669) dis-
solved in dioxane (3 mL mmolÀ1). The reaction mixture was
heated at 130 °C and stirred for 12 h. Additional DCC (0.5 equiv)
was added and the mixture was stirred at 130 °C for 4 h. At rt, AcOEt
these compounds was further tested against single C. albicans bio-
films and mixed biofilms using the crystal violet quantification
method.19,21 The biofilm-eradicating concentration (BEC50), that
is, the concentration of a compound resulting in 50% eradication
of the biofilm, was determined (Table 2).22 The BEC50 for fluconaz-
ole against single and mixed biofilms was above 240
lM. Com-
pounds 666, 667, 684 and 665 were all active against single as

3688
K. Thevissen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3686–3692
Scheme 1. Synthesis of benzylsulfanyl-phenylamines. (A) Synthesis of 650, 643 and 679. (B) Synthesis of 651, 640, 692 and 665. (C) Synthesis of 655, 647, 666 and 688. (D)
Synthesis of 657, 649, 667 and 684. (E) Synthesis of 669, 687, 680 and 642. (F) Synthesis of 670, 641 and 677. Reagents and conditions: (a) Et3N/DMF/rt; (b) dioxane/
dicyclohexyl carbodiimide; (c) DMF/Di-Boc thiourea/DIPEA/EDCI; (d) CH2Cl2/TFA; (e) MeCN, cyanoguanidine, HCL (c), 150 °C, w. See text for more details.
D
,
l

K. Thevissen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3686–3692
3689
Scheme 1 (continued)
well as mixed biofilms (BEC50 6 121
l
M). Compounds 666, 667
in vivo toxicity and efficacy of the compounds was further assessed
in C. elegans model system as described by Breger and
and 684 were most active (BEC50 6 55
l
M) in this respect. The
a

3690
K. Thevissen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3686–3692
Table 1
Antifungal activity of substituted benzylsulfanyl-phenylamines (lM)
R1
R2
S
Compound
R1
R2
o-Guanidyl
o-1,2-Dicyclohexylguanidyl
o-Amino
m-Guanidyl
m-1,2-Dicyclohexylguanidyl
m-1-Formimidamideguanidyl
p-Guanidyl
p-1,2-Dicyclohexylguanidyl
p-1-Formimidamideguanidyl
p-Amino
MFC (Ca)a
MFC (Cg)a
641
677
670
647
666
688
642
680
687
669
4-Chlorine
4-Chlorine
4-Chlorine
4-Chlorine
4-Chlorine
4-Chlorine
4-Chlorine
4-Chlorine
4-Chlorine
4-Chlorine
>100
>100
>100
100
5
50
50
>100
50
>100
>100
50
>100
100
5
50
50
>100
50
>100
643
679
650
649
667
684
640
665
3,5-Dibromine
3,5-Dibromine
3,5-Dibromine
3,5-Dibromine
3,5-Dibromine
3,5-Dibromine
3,5-Dibromine
3,5-Dibromine
3,5-Dibromine
3,5-Dibromine
o-Guanidyl
o-1,2-Dicyclohexylguanidyl
o-Amino
50
5
>100
50
5
5
50
5
50
50
>100
50
5
10
50
5
m-Guanidyl
m-1,2-Dicyclohexylguanidyl
m-1-Formimidamideguanidyl
p-Guanidyl
p-1,2-Dicyclohexylguanidyl
p-1-Formimidamideguanidyl
p-Amino
692
651
Fluconazole
>100
>100
>100
>100
>100
>100
a
Minimal fungicidal concentration (lM) for Candida albicans (Ca) and Candida glabrata (Cg).
Table 2
Biological evaluation of substituted benzylsulfanyl-phenylguanidines
Compounds
MBC (Se)a
BEC50 (Ca)b
BEC50 (Ca/Se)b
665
666
667
684
3.0 0.5
4.0 1.1
75.0 5.0
35.0 8.8
ndc
121.0 17.2
18.0 5.5
19.0 12.1
31.0 6.3
>240
84.0 17.2
22.0 6.8
55.0 23.3
55.0 15.1
>240
Fluconazole
a
Minimal bactericidal concentration (
lM) of the compounds for Staphylococcus
epidermidis (Se).
b
Biofilm eradicating concentration (lM) of the compounds for C. albicans bio-
films (Ca) or mixed species (C. albicans/S. epidermidis) biofilms (Ca/Se).
c
Not determined.
co-workers.11,23 Administration of DMSO (0.5%), the compound’s
solvent, resulted in 59 1.5% nematode survival after 11 days.
Figure 2. In vivo performance of benzylsulfanyl-phenylguanidines in a C. elegans
model for C. albicans infection. Nematodes were infected with C. albicans for 4 h and
then moved to pathogen-free liquid media in the presence of 60 lM of compounds
665 (black triangles), 666 (black circles), 667 (black diamonds), 684 (black squares)
or DMSO (open diamonds) in PBS. Living worms were counted daily and percentage
survival was calculated relative to the survival at day 0. Data are means of SEM of
duplicate measurements and experiments were performed at least twice.
Compounds (50
lM) 665 and 667 exhibited minor toxicity
(51 0.0% and 33 0.3% survival, respectively) whereas50
lM of
compounds 666 and 684 showed higher toxicity in the C. elegans
model (19 5.0% and 5 2.4% survival, respectively) upon the
same incubation period. To test the efficacy of the compounds in
the C. elegans infection model, survival of the Candida infected
worms was monitored in the absence (DMSO control) or presence
effect on survival of the Candida-infected worms as compared to
DMSO treatment (18.5 5.3% and 15.4 0.3%, respectively, after
5 days of incubation). These data indicate that, using the miniatur-
ized host model C. elegans infected with C. albicans, compounds
667 and 665 show in vivo activity against C. albicans.
In summary, based on all above in vitro and in vivo data, com-
pounds 665 and 666 combine potent fungicidal and bactericidal
activity, whereas compound 666 additionally exerts high activity
against single C. albicans and mixed C. albicans/S. epidermidis species
biofilms. Moreover, addition of 665 (Fig. 3A) and 667 to Candida-in-
of 60 l
M of compounds 665, 666, 667 and 684.24 When a chemical
compound has no antifungal activity in the C. elegans in vivo infec-
tion model, only an average of 10–20% of the worms are still alive
on day 5.25 As shown in Figure 2, survival of the worms in the pres-
ence of DMSO (0.6%) was 20.0 2.8% after 5 days of incubation.
Addition of compounds 665 or 667 increased survival of the Can-
dida-infected worms (49.0 6.8% and 55.5 4.0%, respectively)
after 5 days of incubation and these survival percentages leveled
off over time. Addition of compounds 666 or 684 had no significant

K. Thevissen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3686–3692
3691
ing medicinal chemistry and Professor Tom Coenye (University
Ghent, Belgium) for technical assistance with Candida biofilm set-
up. This work was supported by a grant from IWT-Vlaanderen (No.
060013). K.T. acknowledges the receipt of a research manager fel-
lowship from K.U. Leuven (IOF, Industrial Research Fellow).
References and notes
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2. Ramage, G.; Mowat, E.; Jones, B.; Williams, C.; Lopez-Ribot, J. Crit. Rev.
Microbiol. 2009, 35, 340.
3. Kojic, E. M.; Darouiche, R. O. Clin. Microbiol. Rev. 2004, 17, 255.
4. Costerton, J. W.; Marrie, T. J.; Cheng, K. J. In Phenomena of Bacterial Adhesion;
Savage, D. C., Fletcher, M., Eds.; Plenum Press: New York, 1985; pp 3–43.
5. Adam, B.; Baillie, G. S.; Douglas, L. J. J. Med. Microbiol. 2002, 51, 344.
6. Mathew, B. P.; Nath, M. ChemMedChem 2009, 4, 310.
7. François, I. E.; Thevissen, K.; Pellens, K.; Meert, E. M.; Heeres, J.; Freyne, E.;
Coesemans, E.; Viellevoye, M.; Deroose, F.; Martinez Gonzalez, S.; Pastor, J.;
Corens, D.; Meerpoel, L.; Borgers, M.; Ausma, J.; Dispersyn, G. D.; Cammue, B. P.
ChemMedChem 2009, 4, 1714.
8. Bink, A.; Govaert, G.; François, I. E.; Pellens, K.; Meerpoel, L.; Borgers, M.; Van
Minnebruggen, G.; Vroome, V.; Cammue, B. P.; Thevissen, K. FEMS Yeast Res.
2010, 10, 812.
9. Borelli, C.; Schaller, M.; Niewerth, M.; Nocker, K.; Baasner, B.; Berg, D.;
Tiemann, R.; Tietjen, K.; Fugmann, B.; Lang-Fugmann, S.; Korting, H. C.
Chemotherapy 2008, 54, 245.
10. Pálat, K.; Braunerová, G.; Miletın, M.; Buchta, V. Chem. Pap. 2007, 61, 507.
¯
11. Breger, J.; Fuchs, B. B.; Aperis, G.; Moy, T. I.; Ausubel, F. M.; Mylonakis, E. PLoS
Pathog. 2007, 3, e18.
12. HPLC was carried out on a YMC-Pack ODS-AQ column (3
with a column temperature set at 35 °C, a flow rate of 2.6 mL minÀ1 and an
injection volume of 10 L; or on Zorbax SB-C18 column (1.8 m,
4.6 Â 30 mm) with column temperature set at 65 °C, flow rate of
4 mL minÀ1 and an injection volume of
L. Solvent: acetonitrile/H2O
lm, 4.6 Â 50 mm)
l
a
l
a
a
1
l
containing 0.1% of/HCOOH. The used detection method is UV detection at
254 nm.
Figure 3. Chemical structure of benzylsulfanyl-phenylguanidine derivative 665 (A)
and related alkylsulfanyl-pyridinylguanidines (B, C).
13. Purity of all compounds was checked by LC–MS and was >95%. LC–MS data
were obtained on a LC-MS agilent 1100 series instrument. Mass spectra were
obtained in API-ES (Atmospheric Pressure Ionization, Electro Spray) mode and
were acquired by scanning from 50 to 1500 mass units. The capillary needle
voltage was 4 kV and the gas temperature was maintained at 140 °C. Nitrogen
was used as the nebulizer gas.
fected C. elegans cultures increased survival of the Candida-infected
worms and proved not toxic in an in vivo C. elegans model system.
Although use of this C. elegans model allows the evaluation of tox-
icity and antifungal activity of the compounds, it is unlikely that
this will completely eliminate all toxic compounds,11 so further
toxicity test will be performed to determine the clinical potential
of these compounds. In this respect, the most toxic compound,
684, was used in a preliminary toxicity experiment in mice. The
data indicated that a single intraperitoneal dose of 10 mg/kg, a dose
level that is frequently used to assess in vivo efficacy of the antifun-
gals in a murine candidiasis model,26,27 was well tolerated. This
indicates that this dose may be suitable to test the in vivo efficacy
of the different compounds in Candida-infected mice in the future.
Based on the results that will be obtained using this model, it can be
decided if further optimization of these compounds is required. Un-
til now, only one report describing the antifungal activity of struc-
1H NMR spectra were recorded in CDCl3with TMS as internal reference at room
temperature on
a
300 MHz Bruker spectrometer. 13C NMR spectra were
recorded in CDCl3 with TMS as internal reference at room temperature on a
600 MHz Bruker spectrometer.
Compound 665: 1H NMR (300 MHz, DMSO): d 9.29 (1H, s), 7.70 (1H, s), 7.57
(2H, s), 7.33–7.04 (4H, dd), 4.22 (2H, s), 3.47 (2H, s), 1.80 (4H, s), 1.69 (4H, s),
1.56 (2H, d), 1.32–1.03 (10H, m). 13C NMR (300 MHz, DMSO): d 143.40, 132.42,
131.19, 130.66, 129.79, 127.81, 124.29, 122.65, 99.99, 40.93, 32.65, 25.28,
24.98, 22.61.
Compound 666: 1H NMR (300 MHz, DMSO): d 9.51 (1H, s), 7.38 (4H, dd), 7.27
(1H, m), 7.09 (1H, d), 7.01 (1H, s), 6.88 (1H, d), 4.24 (2H, s), 3.50 (2H, s), 1.80
(4H, s), 1.69 (4H, s), 1.55 (2H, d), 1.30–0.96 (10H, m). 13C NMR (300 MHz,
DMSO):
d 152.22, 137.3, 137.07, 132.15, 131.19, 131.02, 130.26, 128.83,
124.06, 122.69, 120.80, 51.48, 40.95, 36.14, 32.70, 25.37, 24.99.
Compound 667: 1H NMR (300 MHz, DMSO): d 70 (1H, s), 7.71 (1H, s), 7.57 (2H,
s), 7.42 (1H, s), 7.34 (4H, s), 7.24–6.99 (4H, m), 4.23 (2H, s). 13C NMR (300 MHz,
DMSO): d 161.17, 154.92, 142.61, 139.21, 135.29, 132.25, 132.07, 130.82,
129.22, 122.25, 120.29, 118.50, 35.14.
turally
related
compounds,
namely
alkylsulfanyl-
Compound 684: 1H NMR (300 MHz, DMSO): d 9.37 (1H, s), 7.71 (1H, s), 7.59
(2H, s), 7.32 (1H, m), 7.15 (1H, d), 7.02 (1H, s), 6.94 (1H, d), 4.26 (2H, s), 3.50
(2H, s), 1.80 (4H, s), 1.71 (4H, s), 1.57 (2H, d), 1.31–1.01 (10H, m). 13C NMR
(300 MHz, DMSO): d 150.30, 141.31, 134.80, 130.48, 129.09, 128.42, 127.76,
126.89, 120.97, 119.11, 49.76, 49.67, 33.50, 30.1, 23.30, 23.07.
pyridinylguanidines, is available.10 Apparently, the antifungal
activity of substituted alkylsulfanyl-pyridinylguanidines improved
by increasing the length of the aliphatic chain: derivatives with C7
and longer aliphatic chains showed antifungal activity, with 6-
(undecylsulfanyl)pyridin-3-ylguanidinium dinitrate being the most
potent compound (Fig. 3B). However, substitution of the pyridinyl-
guanidine scaffold with a benzylsulfanyl moiety, as in 6-(ben-
zylsulfanyl)pyridin-3-ylguanidinium dinitrate (Fig. 3C), resulted
in loss of the antifungal activity.10 Hence, as substituted guanidines
with an alkylsulfanyl moiety are characterized by increased anti-
fungal activity as compared to guanidines with a benzylsulfanyl
moiety,10 future research will be directed at assessing the fungicidal
activity of alkylsulfanyl-phenylguanidines.
14. Fonzi, W. A.; Irwin, M. Y. Genetics 1993, 134, 717.
15. Kaur, R.; Ma, B.; Cormack, B. P. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 7628.
16. An overnight culture was diluted in PBS (5 Â 105 CFU/ml) and incubated for 2 h
with different concentrations of the compounds. Cells were washed, plated on
YPD (1% yeast extract, 2% peptone, 2% glucose) and after incubation, CFUs
(Colony forming units) were determined. MFC, defined as the minimal
concentration of the compound resulting in less than 0.1% survival of the
yeast culture, was determined for both yeast species, relative to the DMSO
control.
17. Graybill, J. R.; Burgess, D. S.; Hardin, T. C. Eur. J. Clin. Microbiol. Infect. Dis. 1997,
16, 42.
18. Thevissen, K.; Hillaert, U.; Meert, E. M.; Chow, K. K.; Cammue, B. P.; Van
Calenbergh, S.; François, I. E. Bioorg. Med. Chem. Lett. 2008, 18, 3728.
19. Thevissen, K.; Marchand, A.; Chaltin, P.; Meert, E. M.; Cammue, B. P. Curr. Med.
Chem. 2009, 16, 2205.
Acknowledgements
20. To determine the bactericidal activity against S. epidermidis, an overnight
culture of S. epidermidis in TSB (5% Tryptic Soy Broth; BD Diagnostics, MD, USA)
was diluted in PBS (2 Â 105 CFU/ml) and incubated for 2 h with different
concentrations of the compounds. After the incubation period, cells were
We kindly acknowledge Arnaud Marchand (Centre for Drug De-
sign and Discovery (CD3), 3000 Leuven, Belgium) for input regard-

3692
K. Thevissen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3686–3692
10 l
g mLÀ1 cholesterol and 100 g mLÀ1 kanamycin). For toxicity testing, 40 to
washed, plated on TSB and CFUs were determined. Afterwards, MBC (Minimal
l
bactericidal concentration), defined as the minimal concentration resulting in
less than 0.1% survival of the bacterial culture, relative to the DMSO control
treatment, was determined.
50 worms were suspended in 1 mL M9 buffer in each well of 24-well microtiter
plates, in the presence or absence (DMSO control) of the fungicidal compounds
665, 666, 667 and 684 (50 lM). Survival of the worms was monitored daily.
21. Peeters, E.; Nelis, H. J.; Coenye, T. J. Microbiol. Methods 2008, 72, 157.
22. To assess the activity of the compounds against mixed biofilms, composed of C.
albicans SC5314 and S. epidermidis in a 50/50 ratio, overnight cultures of both
The percentage survival of the worms in the presence or absence of antifungal
compounds was calculated each day relative to the survival at day 0.
24. The efficacy of the compounds in the C. elegans infection model was tested
using L4 larvae which were fed for 4 h on C. albicans SC5314 agar plates (YPD
organisms were suspended in 1/20 TSB at OD590nm = 0.5 and 50 lL of both
cultures were mixed in the wells of a 96-well plate. After an adhesion phase of
24 h, planktonic cells were removed and fresh 1/20 TSB-medium was added for
the 48-h growth phase. Mature biofilms were incubated for 24 h in PBS
containing the fungicidal compounds and biofilm mass was quantified using
the crystal violet staining.18,20 To assess the number of bacterial and fungal
cells in these biofilms, biofilm cells were suspended and plated on media
promoting growth of both C. albicans and S. epidermidis (YPD) or of C. albicans
agar plates on the surface inoculated with 100 lL of an overnight culture in
YPD and incubated for 16 h at 37 °C).11,28 Worms were collected and washed
with M9 buffer. Survival of the worms in absence (DMSO control) or presence
of compounds 665, 666, 667 and 684 (60 lM) was monitored daily. The
percentage survival of the worms in presence or absence of antifungal
compounds was calculated relative to the survival at day 0.
25. Tampakakis, E.; Okoli, I.; Mylonakis, E. Nat. Protocols 2008, 3, 1925.
26. Andes, D. R.; Diekema, D. J.; Pfaller, M. A.; Marchillo, K.; Bohrmueller, J.
Antimicrob. Agents Chemother. 2008, 10, 3497.
alone (YPD + 100 l
g mLÀ1 ampicillin) after which the colony forming units
(CFUs) were determined.
23. Larvae of a double mutant (glp-4
D
sek-1
D
) of C. elegans were grown on NGM/
L of an
27. Andes, D.; Diekema, D. J.; Pfaller, M. A.; Prince, R. A.; Marchillo, K.; Ashbeck, J.;
Hou, J. Antimicrob. Agents Chemother. 2008, 52, 539.
OP50 agar plates (NGM agar plates on the surface inoculated with 100
l
overnight culture of OP50 Escherichia coli and incubated for 16 h at 37 °C) until
all larvae had reached the L4 stage. Worms were collected and washed with
M9 buffer (3 g LÀ1 KH2PO4, 6 g LÀ1 Na2HPO4, 5 g LÀ1 NaCl, 1 mM MgSO4,
28. Okoli, I.; Coleman, J. J.; Tampakakis, E.; An, W. F.; Holson, E.; Wagner, F.;
Conery, A. L.; Larkins-Ford, J.; Wu, G.; Stern, A.; Ausubel, F. M.; Mylonakis, E.
PLoS One 2009, 4, e7025.