Antifungal activity of synthetic di(hetero)arylamines based on the benzo[b]thiophene moiety

Article abstract of DOI:10.1016/j.bmc.2008.07.042

The antifungal activity of several di(hetero)arylamine derivatives of the benzo[b]thiophene system was evaluated against clinically relevant Candida, Aspergillus, and dermatophyte species by a broth macrodilution test based on CLSI (formerly NCCLS) guidelines. The most active compound showed a broad spectrum of activity (against all tested fungal strains, including fluconazole-resistant fungi), with particularly low MICs for dermatophytes. Results from the inhibition of the dimorphic transition in Candida albicans and flow cytometry studies further confirmed their biological activity. With this study it was possible to establish some structure-activity relationships (SARs). The hydroxy groups proved to be essential for the activity in the aryl derivatives. Furthermore, the spectrum of activity in the pyridine derivatives was broadened by the absence of the ester group on position 2 of the benzo[b]thiophene system.

Full text of DOI:10.1016/j.bmc.2008.07.042

Bioorganic & Medicinal Chemistry 16 (2008) 8172–8177
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antifungal activity of synthetic di(hetero)arylamines based on the
benzo[b]thiophene moiety
b
a
a
Eugénia Pinto a, , Maria-João R. P. Queiroz , Luís A. Vale-Silva , João F. Oliveira ,
*
Agathe Begouin b,c, Jeanne-Marie Begouin b,c, Gilbert Kirsch c
a Centro de Química Medicinal, Faculdade de Farmácia, Rua Aníbal Cunha, 164, 4050-047 Porto, Portugal
b Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
c Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, Université Paul-Verlaine de Metz, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The antifungal activity of several di(hetero)arylamine derivatives of the benzo[b]thiophene system was
evaluated against clinically relevant Candida, Aspergillus, and dermatophyte species by a broth macrodi-
lution test based on CLSI (formerly NCCLS) guidelines. The most active compound showed a broad spec-
trum of activity (against all tested fungal strains, including fluconazole-resistant fungi), with particularly
low MICs for dermatophytes. Results from the inhibition of the dimorphic transition in Candida albicans
and flow cytometry studies further confirmed their biological activity. With this study it was possible to
establish some structure–activity relationships (SARs). The hydroxy groups proved to be essential for the
activity in the aryl derivatives. Furthermore, the spectrum of activity in the pyridine derivatives was
broadened by the absence of the ester group on position 2 of the benzo[b]thiophene system.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 16 June 2008
Accepted 16 July 2008
Available online 20 July 2008
Keywords:
Di(hetero)arylamine
Benzo[b]thiophene
Antifungal activity
Flow cytometry
Germ tubes
SARs
1. Introduction
can be inhibitors of herpes simplex virus type I (HSV-1) replication,
antimitotics, inhibitors of cysteine and serine proteases (impor-
The incidence and severity of fungal diseases has increased re-
cently, particularly in patients with impaired immunity.1 The
growing number of cases of fungi implicated in sepsis has been
shown to be a consistent trend2 and Candida is the third or fourth
most common isolate in nosocomial bloodstream infections in the
United States.3 Furthermore, invasive candidosis is a problem of
growing importance in critically ill non-immunosuppressed pa-
tients as well.4 Besides that the mortality rate due to invasive
aspergillosis has increased by 357% between 1980 and 1997 in
the United States5 and superficial mycoses, dermatophytosis (tine-
as), and candidosis, often recurrent, are an important cause of mor-
bidity and are more severe in immunocompromised patients.
Together with this increase in the clinical importance of fungal
infections, the small number of available antifungals, often show-
ing only fungistatic activity, and the increase in antifungal resis-
tance have been stimulating the search for new drugs, which are
more effective and less toxic than those already in use.6
tantly, thrombin), opioid receptor analgesics, and 5-HT6 antago-
nists, making this very attractive structure for medicinal
a
chemists.8 Its potential has, actually, already been materialized
through the development of marketed drugs, like the anti-asthma
drug zileuton9 and raloxifene, a non-hormonal drug showing estro-
gen agonist effects on the bone and the cardiovascular system and
estrogen antagonist effects on endometrial and breast tissues.10
Benzo[b]thiophene is also a structural part of the commercial imid-
azole antifungal agent sertaconazole, an antimycotic with applica-
tions in dermatology and gynecology.11
For some years now we have been interested in the functional-
ization of either the benzene or the thiophene ring of the
benzo[b]thiophene system, using palladium-catalyzed C–N cross-
couplings to obtain the corresponding diarylamines,12,13 and in
the study of their potential antimicrobial activity.13,14
In this work, we present the synthesis of two new compounds
and the screening of several di(hetero)arylamine derivatives of
benzo[b]thiophenes as potential antifungal agents against derma-
tophytes, yeasts, and Aspergillus species with clinical relevance,
using the macrodilution broth method. The antifungal activity
was also evaluated against fluconazole-resistant Candida albicans
strains, Candida krusei, Candida glabrata, and Aspergillus fumigatus,
which are intrinsically resistant to fluconazole or whose resistance
is easily inducible. For the compounds showing antifungal activity
The diarylamine skeleton and the benzo[b]thiophene system7
are often present in biologically active compounds and many
examples of biological activities found for small molecules based
on the benzo[b]thiophene moiety can be referred. Namely, they
* Corresponding author. Tel.: +351 22 2078995; fax: +351 22 2003977.
E-mail address: epinto@ff.up.pt (E. Pinto).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.042

E. Pinto et al. / Bioorg. Med. Chem. 16 (2008) 8172–8177
8173
against C. albicans, we have conducted a study of the effect upon
germ tube formation, an important virulence factor in this yeast.15
Further investigations by flow cytometry, using the fluorescent
dyes PI (propidium iodide) and FUN-1, were also conducted. With
the battery of compounds available, it was tried to establish some
structure–activity relationships (SARs).
the spectrum of activity to include yeasts, but with higher MICs
(25–50 g/mL) than those for dermatophytes (6.25–12.5 g/mL).
l
l
The hydroxylated compounds 2 and 5 are actually fungicidal for
dermatophytes, presenting the same or similar MICs and MFCs.
Comparing compounds 6 and 7 (the pyridine derivatives), it is
possible to conclude that the absence of the ester group in the lat-
ter widens the spectrum of activity. Nevertheless, the MICs for der-
matophytes remain lower (3.13–12.5
l
g/mL) than those for
g/mL).
2. Results and discussion
2.1. Chemistry
Candida (50–100 g/mL) and Aspergillus (25–50
l
l
If compound 5 adds a moderate activity against the tested
yeasts to its referred high activity against dermatophytes, com-
pound 7 achieves the broadest spectrum of activity, including all
tested pathogenic yeasts and moulds. It is, thus, active against fun-
gi with decreased susceptibility to fluconazole, such as C. krusei, C.
glabrata, and Aspergillus spp. The activity of these compounds was
not affected by the fluconazole susceptibility profile of the tested
strains.
The di(hetero)arylamines 1, 3, 4, and 6 had previously been pre-
pared by palladium-catalyzed C–N cross-coupling, as had the
diarylamine 2, in this case by demethylation of compound 1 using
BBr3 (Scheme 1).13 Here, we present the synthesis of compound 5
by demethylation of compound 4 and of the diheteroarylamine 7
by palladium-catalyzed C–N cross-coupling of 3-bromobenzo[b]-
thiophene and 3-aminopyridine under the conditions required
for the coupling of heteroarylamines13 (Scheme 1). The synthesis
of diarylamines 5 and 7 allowed the enlargement of the SARs study.
2.3. Germ tube inhibition
Sub-inhibitory concentrations of compounds 5 and 7 strongly
prevented germ tube formation, widely regarded as an important
mechanism of pathogenicity in this yeast.15 The highest concentra-
tion, half of the MIC, caused an almost complete inhibition in all
tested strains (Fig. 1). C. albicans D5, the strain showing the lowest
germ tube production in the controls, appears to be especially sus-
ceptible to this effect. In this strain, both compounds caused a vir-
tually complete inhibition of the dimorphic transition even at
concentrations four times lower than the respective MICs.
2.2. Antifungal activity
Compounds 1–7 were screened for antifungal activity against
clinical isolates and reference strains of Candida species (C. albi-
cans, C. glabrata, C. krusei, and C. tropicalis), Aspergillus species
(A. fumigatus, A. niger, and A. flavus), and dermatophytes (Microspo-
r u m c a n i s , Microsporum gypseum, Thichophyton mentagrophytes,
Thichophyton rubrum, and Epidermophyton floccosum). The MICs
and MFCs of the tested and reference (amphotericin B and fluco-
nazole) compounds are presented in Table 1.
2.4. Flow cytometry studies
No activity was detected against any of the tested strains for
compounds 1, 3, and 4 at concentrations up to 200 lg/mL. Derma-
Regarding the flow cytometric results for C. albicans cells
stained with FUN-1, the three highest test concentrations yielded
over 90% of metabolically inactive cells, similarly to what hap-
pened to amphotericin B-treated cells (Fig. 2A). FUN-1 is a mem-
brane-permeant fluorescent probe which is converted into
orange/red cylindrical intra-vacuolar structures (CIVS) by metabol-
ically active fungal cells, while remaining in the cytoplasm as a
bright diffuse green/yellow stain in cells with impaired metabo-
lism,16 thus indicating the cells’ metabolic vitality state. With the
tophytes were found to be sensitive to compounds 2 and 6,
whereas these compounds exhibited no antifungal activity against
Aspergillus and Candida species. Compounds 5 and 7 exhibited a
larger spectrum of activity than compounds 2 and 6, particularly
compound 7, additionally showing moderate activity against both
Candida and Aspergillus species.
The presence of a free OH group (compound 2) instead of an
OMe group (compound 1) is responsible for the antidermatophyte
activity. The presence of two OH groups (compound 5) broadens
three test concentrations above 50 lg/mL resulting in over 90%
R1
R1
R2
Pd(OAc)2 (3 mol%)
H
H2N
Br
+
N
BINAP (4 mol%)
R2
CO2Et
CO2Et
Cs2CO3 (1.4 equiv.)
S
S
toluene, 100 ºC, Argon
1 R1= H, R2 = OMe
2 R1= H, R2 = OH
3 R1= H, R2 = F
4 R1= R2 = OMe
5 R1= R2 = OH
i
i
BINAP = rac-2,2'-bis(diphenylphosphane)-1,1'binaphtyl
i ) BBr3, CH2Cl2, 0 ºC
H
N
N
H2N
Pd(OAc)2 (8 mol%)
Br
R
+
R
Xantphos (10 mol%)
Cs2CO3 (2 equiv.)
dioxane, 110 ºC, Argon
N
S
6 R = CO2Et
S
1.2 equiv.
7 R = H
R = CO2Et or H
Scheme 1. Structures of the tested compounds 1–7. Diarylamines 5 and 7 were synthesized and characterized in this work.

8174
E. Pinto et al. / Bioorg. Med. Chem. 16 (2008) 8172–8177
Table 1
Antifungal activity—MIC (MFC)—of seven tested compounds against yeasts and filamentous fungi (defined MIC values are shown in bold font)
Strains
Compounds/MIC (MFC)
l
g/mL (W/V)
Fluconazole
Amphotericin B
1
2
3
4
5
6
7
Yeasts
ATCC
Candida albicans
ATCC 10231
>200
(>200)
>200
(>200)
>200
(>200)
>200
(>200)
25–50
(>200)
>200
(>200)
50
(100)
1
N.T.
N.T.
N.T.
(>128)
C. krusei
ATCC 6258
>200
(>200)
>200
(>200)
>200
(>200)
>200
(>200)
25–50
(>50)
>200
(>200)
100
(>100)
64
(64–128)
C. tropicalis
>200
>200
>200
>200
50
>200
100
4
ATCC 13803
(>200)
(>200)
(>200)
(>200)
(>50)
(>200)
(>100)
(>128)
Clinical
C. albicans
D5
>200
(>200)
>200
(>200)
>200
(>200)
>200
(>200)
25
(>50)
>200
(>200)
50
(>50)
>128
(>128)
N.T.
N.T.
C. glabrata
>200
>200
>200
>200
25–50
>200
50
32
D 10R
(>200)
(>200)
(>200)
(>200)
(>50)
(>200)
(>50)
(32)
Filamentous fungi
Dermatophytes
Epidermophyton floccosum
FF9
>200
(>200)
3.13
(12.5)
>200
(>200)
>200
(>200)
6.25–12.5
(12.5)
6.25
(>50)
12.5
(>12.5)
16
(16)
N.T.
N.T.
N.T.
N.T.
N.T.
Trichophyton rubrum
FF5
>200
(>200)
6.25
(6.25)
>200
(>200)
>200
(>200)
12.5–25
(25)
3.13
(>50)
3.13
(12.5)
16–32
(32)
T. mentagrophytes
FF7
>200
(>200)
6.25
(6.25)
>200
(>200)
>200
(>200)
12.5
(25)
3.13
(>50)
12.5
(>12.5)
16–32
(32–64)
Microsporum canis
FF1
>200
(>200)
1.56
(1.56)
>200
(>200)
>200
(>200)
12.5
(12.5)
3.13
(>50)
12.5
(>12.5)
128
(128)
M. gypseum
>200
6.25
>200
>200
12.5
6.25
12.5
>128
FF3
(>200)
(12.5)
(>200)
(>200)
(25)
(>50)
(>12.5)
(>128)
Aspergillus
Aspergillus flavus
F44
>200
(>200)
>200
(>200)
>200
(>200)
>200
(>200)
>100
(>100)
>200
(>200)
50
(100)
N.T.
N.T.
N.T.
2
(8)
A. fumigatus
ATCC 46645
>200
(>200)
>200
(>200)
>200
(>200)
>200
(>200)
>100
(>100)
>200
(>200)
25
(>50)
2
(4)
A. niger
ATCC 16404
>200
(>200)
>200
(>200)
>200
(>200)
>200
(>200)
>100
(>100)
>200
(>200)
50–100
(>100)
1–2
(4)
100
ATCC 10231
90
80
70
60
50
40
30
20
10
0
D5
M1
5-MIC/2
5-MIC/4
5-MIC/8
7-MIC/2
7-MIC/4
7-MIC/8
C+DMSO
Figure 1. Percentage of positive germ tube bearing cells after treatment of C. albicans strains ATCC 10231, D5, and M1 with sub-inhibitory concentrations of compounds 5 and
7.
of cells with impaired metabolism, manifested by a reduction in
fluorescence in the FL-2 channel, these results show a strict corre-
lation to the determined MIC. The concentration immediately be-
low still yielded over 60% of cells with decreased CIVS formation
in comparison to the control sample (Fig. 2A). Similar results were
obtained for conidia of A. fumigatus, in which case a greater influ-
ence of lower tested concentrations (below the MIC) was found
(Fig. 2A).
Concerning PI, after short incubation times of both C. albicans
and A. fumigatus (a maximum of four hours), neither the test com-
pound nor amphotericin B, in fungicidal concentrations, produced
a shift of more than 40% of the cells to higher values of red fluores-
cence in comparison to the stained controls (data not shown). After
24 h, however, the two highest test concentrations of compound 7,
together with amphotericin B, led to the penetration of PI in over
80% of the C. albicans cells (Fig. 2B). Regarding A. fumigatus conidia,

E. Pinto et al. / Bioorg. Med. Chem. 16 (2008) 8172–8177
8175
100
80
60
40
20
0
C. albicans
A. fumigatus
Controlo AMB 2/4
C7 2
C7 4
C7 8
C7 16
C7 32
C7 64
C7 128
C7 256
100
80
60
40
20
0
C. albicans
A. fumigatus
Controlo
AMB 2/4 Heat-killed
C7 16
C7 32
C7 64
C7 128
C7 256
Figure 2. Flow cytometry results after treatment of C. albicans cells and A. fumigatus conidia with compound 7 and staining with the fluorescent probes FUN-1 (A) and PI (B).
similar results were obtained with the highest concentration of
compound 7 and with the reference compound (Fig. 2B). A longer
incubation time with both the test compound and amphotericin
B was, this way, required to produce positivity. PI is a fluorescent
marker that only penetrates cells with severe damage of the mem-
brane, cells that are already dead, where it binds to nucleic acids
and leads to increased red fluorescence.17 Permeation to PI means
that the compound was responsible for a fungicidal effect, result-
ing in extensive lesion to the plasmatic membrane, either from a
direct effect or as a secondary result of metabolic impairment.
Under our experimental conditions compound 7 showed similar
effects to the reference fungicidal compound amphotericin B.
4. Experimental
4.1. Synthesis
General procedures: Melting points were determined on a Stuart
SMP3 apparatus and are uncorrected. The 1H NMR spectra were
measured on a Varian Unity Plus at 300 MHz. Spin–spin decoupling
was used to assign the signals. The 13C NMR spectra were mea-
sured in the same instrument at 75.4 MHz (using DEPT h 45°).
Elemental analyses were determined on a LECO CHNS 932 ele-
mental analyzer. Mass spectra (EI) and HRMS were performed by
the mass spectrometry service of University of Vigo-Spain.
Column chromatography was performed on Macherey–Nagel
silica gel 230–400 mesh. Preparative layer chromatography (PLC)
was performed on Macherey–Nagel 20 ꢀ 20 cm2 silica plates, layer
2 mm SIL G-200 UV254. Petroleum ether refers to the boiling range
40–60 °C. Ether refers to diethyl ether.
3. Conclusion
With the battery of di(hetero)arylamine derivatives of
benzo[b]thiophenes tested it was possible to establish some struc-
ture–activity relationships. The presence of hydroxy groups in the
aryl derivatives was found to be essential for the antifungal activ-
ity. The spectrum of activity in the pyridine derivatives was broad-
ened by the absence of the ester group on position 2 of the
benzo[b]thiophene system, with compound 7 inhibiting all tested
fungal species.
Xantphos corresponds to 4,5-bis(diphenylphosphino)-9,9-dim-
ethylxanthene and was purchased from Strem.
Compounds 1–4 and 6, as mentioned before, had been prepared
and characterized by us in a previous work.13
4.1.1. Ethyl 3-(2,4-dihydroxyphenylamino)benzo[b]thiophene-
2-carboxylate (5)
The dermatophytes were found to be more sensitive to the ac-
tive tested compounds than Candida and Aspergillus species, with
four compounds (2 and 5–7) showing a considerably high activity.
Among the presented set of compounds, compound 7 shows the
highest potential. Although somewhat expressing its counterparts
selectivity to dermatophytes, it inhibited all tested fungal strains,
including fluconazole-resistant yeasts and A. fumigatus, especially
important organisms from the clinical point of view. In fact, its
activity was not affected by the fluconazole susceptibility profile
of the tested strains. It clearly evidences a broad spectrum of activ-
ity that has been further confirmed by the prevention of germ tube
production in C. albicans and the flow cytometry studies.
Given the results described, and as a future perspective, the
structure–activity oriented preparation of a wider set of deriva-
tives could, probably, allow the tuning of the antifungal activity
of the compounds, particularly regarding the reduction of MICs
for yeasts and Aspergillus species. Overall, these compounds show
an interesting potential for development as antifungal agents and
certainly deserve further investigation.
To a solution of diarylamine 413 (100 mg, 0.280 mmol) in dry
dichloromethane (10 mL), a solution of BBr3 in dichloromethane
1 M (5 equiv) was added at 0 °C under argon and the mixture
was stirred for 3 h. A saturated solution of NaHCO3 (15 mL) was
then slowly added and the extractions were done with dichloro-
methane (2ꢀ 20mL). The organic phases were collected, dried
(MgSO4), filtered, and removal of the solvent gave a yellow oil
which was submitted to PLC (ether/petroleum ether 1:1) to give
compound 5 as a yellow green solid (65 mg, 70%) mp 178–
180 °C. 1H NMR (CDCl3): d 1.44 (3H, t, J = 7.5 Hz, CH2CH3), 4.41
(2H, q, J = 7.5 Hz, CH2CH3), 4.87 (1H, s, OH), 6.28 (1H, s, OH), 6.32
(1H, dd, J = 8.4 and 2.7 Hz, 50-H), 6.61 (1H, d, J = 2.7 Hz, 30-H),
6.92 (1H, d, J = 8.4 Hz, 60-H), 6.99–7.12 (2H, m, ArH), 7.34–7.42
(1H, m, ArH), 7.70–7.73 (1H, br d, J = 8.1 Hz, ArH), 8.45 (1H, br s,
NH) ppm. 13C NMR (CDCl3): d 14.42 (CH2CH3), 61.00 (CH2CH3),
102.45 (CH), 104.62 (C), 107.62 (CH), 121.31 (C), 123.14 (CH),
123.91 (CH), 124.68 (CH), 127.72 (CH), 128.50 (CH), 131.37 (C),
140.25 (C), 149.01 (C), 153.63 (C), 155.77 (C), 165.99 (C@O) ppm.

8176
E. Pinto et al. / Bioorg. Med. Chem. 16 (2008) 8172–8177
MS (EI) m/z (%): 331 (M++2, 5), 330 (M++1, 17), 329 (M+, 81), 283
dida, and at 30 °C for filamentous fungi. The strains were then sub-
cultured under the same conditions. Yeasts and conidial
suspensions of these recent cultures were prepared in sterile
0.85% saline solution. Each suspension was diluted in RPMI 1640
(100). HRMS M+ calcd for C17H15NO4S: 329.0722, found 329.0727.
4.1.2. N-(Benzo[b]thien-3-yl)pyridine-3-amine (7)
To
a
dried Schlenk tube dry dioxane (3 mL), 3-bro-
medium, with L-glutamine and without sodium bicarbonate (Bio-
mobenzo[b]thiophene (150 mg, 0.704 mmol), Pd(OAc)2 (8 mol%),
xantphos (10 mol%), Cs2CO3 (2 equiv), and 3-aminopyridine
(80.0 mg, 0.845 mmol) were added under argon and the mixture
was heated at 110 °C for 3 h:30 min. After cooling, the mixture
was filtered and the filtrate was evaporated to give an oil. This
was submitted to column chromatography using a gradient from
10% ethyl acetate/petroleum ether to 80% ethyl acetate/petroleum
ether to give diarylamine 7 as an orange oil (128 mg, 80%). Crystal-
lization from ethyl acetate/petroleum ether gave orange crystals
mp114–116 °C. 1H NMR (CDCl3): d 5.80 (1H, s, NH), 7.09 (1H, s,
20-H), 7.16 (1H, dd, J = 8.3 and 4.7 Hz, 5-H), 7.24–7.28 (1H, m, 4-
H), 7.37–7.44 (2H, m, ArH), 7.63–7.67 (1H, m, ArH), 7.85–7.89
(1H, m, ArH), 8.15 (1H, dd, J = 4.7 and 1.3 Hz, 6-H), 8.37 (1H, br
d, J = 2.6 Hz, 2-H) ppm. 13C NMR (CDCl3): d 111.33 (CH), 120.65
(CH), 121.82 (CH), 123.27 (CH), 123.78 (CH), 124.12 (CH), 125.05
(CH), 133.77 (C), 134.38 (C), 138.36 (CH), 138.90 (C), 140.97 (CH),
141.41 (C) ppm. MS (EI) m/z (%) 228 (M++2, 5.75), 227 (M++1,
17.30), 226 (M+, 100) 225 (M+-1, 50), 224 (M+-2, 15). Calcd for
chrom AG), buffered to pH 7.0 0.2 with 0.165 M morpholinepro-
panesulfonic acid (MOPS; Sigma), and adjusted to 1–2 ꢀ 103 CFU/
mL for yeasts and 1–2 ꢀ 104 CFU/mL for filamentous fungi. The
concentration of each inoculum was confirmed by viable count
on Sabouraud agar plates by plating 100 lL of serial dilutions onto
the surface and incubating until visible growth.
4.2.4. Antifungal susceptibility testing
Broth macrodilution methods based on the CLSI (formerly
NCCLS) reference documents M27-A218 and M38-A,19 for yeasts
and filamentous fungi, respectively, with minor modifications,
were used to determine minimum inhibitory concentrations
(MICs) and minimum fungicidal concentrations (MFCs). Since no
reference method is available for dermatophytes, protocol M38-
A19 and previously published data on the subject20 were taken into
account. Briefly, serial twofold dilutions of each di(hetero)aryl-
amine were prepared over the range of 200–0.8 lg/mL in RPMI
1640 medium immediately before testing. Each test tube contain-
ing a given concentration of the drug being tested was inoculated
with the inoculum suspension and then incubated aerobically at
37 °C for 48 h (yeasts) or at 30 °C for 3/5 days (filamentous fungi/
dermatophytes). The fungal growth was indicated by the turbidity
and MICs were defined as the lowest drug concentration that re-
duced growth by 80% in comparison to the drug-free controls. To
C13H10N2S: C 69.00, H 4.45, N 12.38, S 14.17%, found C 68.88, H
4.62, N 12.06, S 14.23%.
4.2. Antifungal evaluation
4.2.1. Antifungal drugs
Amphotericin B (Sigma) and fluconazole (Pfizer) were used as
standard antifungal drugs. Amphotericin B was dissolved in 100%
dimethyl sulfoxide (DMSO; Sigma) at a starting concentration of
evaluate the MFCs, 20 lL aliquots were subcultured from each neg-
ative tube (optically clear tube) and the last positive tube, after MIC
reading, onto Sabouraud dextrose agar plates. The plates were then
incubated at 37 °C (yeasts) or 30 °C (Aspergillus species/dermato-
phytes) until growth was seen in the last positive tube subculture.
Minimum fungicidal concentration was the lowest concentration
of antifungal yielding subcultures without any visible fungal
growth. In addition, a reference antifungal compound, fluconazole
or amphotericin B, was used as the standard antifungal drug. Two-
1600
at 12,800
l
g/mL and fluconazole was dissolved in sterile distilled water
g/mL.
l
The test compounds 1–4 and 6 were prepared as previously re-
ported13 and compounds 5 and 7 (see chemical structures in
Scheme 1) were prepared as described above. A 10 mg/mL stock
solution of each of the test derivatives was prepared in 100% DMSO
and then diluted in the test medium to achieve the required test
concentrations. The final concentration of DMSO did not exceed
2%. According to previously performed tests (data not shown),
DMSO at that concentration did not affect fungal growth.
fold serial dilutions, ranging from 128 to 0.25
lg/mL for fluconaz-
ole and 8 to 0.25 g/mL for amphotericin B, were used. Quality
l
control determinations of the MIC of fluconazole were ensured
by testing C. parapsilosis ATCC 90018. The results obtained were
within the recommended limits (data not shown). All results are
from three independent and concordant experiments, performed
in duplicate. A range of values are presented when different results
were obtained. Two growth controls, using test medium alone and
with 2.0% (v/v) DMSO, and a sterility control (drug-free medium
only) were included in all assays.
4.2.2. Fungal organisms
The antifungal activity of di(hetero)arylamines 1–7 was evalu-
ated against Candida, Aspergillus, and dermatophyte strains: two
clinical strains of Candida isolated from recurrent cases of oral can-
didosis (C. albicans D5 and C. glabrata D 10R); three Candida refer-
ence strains (C. albicans ATCC 10231, C. tropicalis ATCC 13803, and
C. krusei ATCC 6258); three Aspergillus strains (A. niger ATCC 16404,
A. fumigatus ATCC 46645, and A. flavus F44); and five clinical strains
of dermatophytes isolated from nails and skin (M. canis FF1, M. gyp-
seum FF3, T. rubrum FF5, T. mentagrophytes FF7, and E. floccosum
FF9). Candida parapsilosis ATCC 90018 was used for quality control.
C. albicans strains ATCC 10231 and D5, plus an additional clinical
isolate, C. albicans M1, were used for the germ tube inhibition as-
say, while C. albicans ATCC 10231 and A. fumigatus ATCC 46645
were included in the flow cytometry studies. The clinical isolates
were identified using standard microbiological methods. All strains
were stored in Sabouraud dextrose broth (Becton–Dickinson) with
20% glycerol at ꢁ80 °C and passaged on Sabouraud dextrose agar
(SDA; Becton–Dickinson) before use to ensure purity and viability.
4.2.5. Germ tube inhibition assay
Cell suspensions from overnight SDA cultures of C. albicans
strains ATCC 10231, D5, and M1, at 37 °C, were prepared in NYP
medium (N-acetylglucosamine [Sigma; 10ꢁ3 mol/L], Yeast Nitro-
gen Base [Difco; 3.35 g/L], proline [Fluka; 10ꢁ3 mol/L]) with NaCl
(4.5 g/L, pH 6.7 0.1)21 and adjusted to obtain a density of
(1.0 0.2) ꢀ 106 CFU/mL. The compounds were dissolved and di-
luted in DMSO and added in a volume of 10 lL to 990 lL of the
yeast suspensions (final DMSO concentration of 1%) to obtain
appropriate sub-inhibitory concentrations (1/2, 1/4, and 1/8 of
the MIC). After a 3-h incubation at 37 °C, 100 cells from each sam-
ple were counted, using a hemocytometer, and the percentage of
germ tubes was determined. Germ tubes were considered positive
when they were at least as long as the diameter of the blastospore.
Protuberances showing a constriction at the point of connection to
the mother cell, typical for pseudohyphae, were excluded. The re-
4.2.3. Inoculum preparation
The isolates of yeasts, Aspergillus spp., and dermatophytes were
grown for 1, 3, and 5 days, respectively, on SDA at 37 °C for Can-

E. Pinto et al. / Bioorg. Med. Chem. 16 (2008) 8172–8177
8177
laser emitting at 488-nm wavelength and the results were ana-
lyzed using CellQuest Pro Software (Becton–Dickinson). Intrinsic
parameters (forward and side scatter, for cell size and complexity
analysis, respectively) and fluorescence in the FL2 channel (log yel-
low/orange fluorescence, around 575 nm) for FUN-1 and the FL3
channel (log red fluorescence, above 605 nm) for PI were acquired
and recorded, using logarithmic scales, for a minimum of 7500
events/sample. Markers (M1) were adjusted in monoparametric
histograms of sample fluorescence intensity in order to include a
maximum of 5% of the cells and then used in the analysis of the
remaining samples to quantify the percentages of positive cells:
cells showing altered fluorescence in comparison to the drug-free
controls (see Fig. 3 for examples of such histograms). The results
are presented as averages SD of at least three replicate
experiments.
Figure 3. Monoparametric flow cytometry histograms showing overlaid peaks of
different cell samples and the marker (M1), set to include a maximum of 5% of
control cells and used to analyze drug-treated samples. (A) Samples of A. fumigatus
conidia stained with FUN-1; (B) Samples of C. albicans cells stained with PI. (Af,
auto-fluorescence of unstained cells; control, stained drug-free samples; AmB, cells
treated with amphotericin B.)
Acknowledgements
The authors thank the Foundation for the Science and Technol-
ogy (Portugal) and FEDER for financial support through the re-
search centres, project POCI/QUI/59407/2004 and for the post-
doc grant attributed to Luís Vale-Silva (SFRH/BPD/29112/2006).
The authors also thank the Socrates/Erasmus programme for finan-
cial support in Portugal to A.B. and J.-M.B. (Univ. Metz ? Univ.
Minho).
sults are presented as averages standard deviations (SD) of three
separate experiments.
4.2.6. Cell treatment for flow cytometry
Yeast and conidia suspensions in RPMI 1640 medium and ster-
ile water with 0.01% Tween 80, respectively, were prepared from
overnight SDA cultures of C. albicans ATCC 10231 and 4–7 days
old SDA cultures of A. fumigatus ATCC 46645 at 35 °C and adjusted
using a hemocytometer to a final density of (2.0 0.2) ꢀ 106 CFU/
mL. A suspension of heat-killed conidia (95 °C, 30 min) at the same
density was also prepared. Serial twofold dilutions of compound 7
References and notes
1. Odds, F. C. Trends Microbiol. 2000, 8, 200.
2. Martin, G. S.; Mannino, D. M.; Eaton, S.; Moss, M. N. Engl. J. Med. 2003, 348,
1546.
3. Edmond, M. B.; Wallace, S. E.; McClish, D. K.; Pfaller, M. A.; Jones, R. N.; Wenzel,
R. P. Clin. Infect. Dis. 1999, 29, 239.
4. Eggimann, P.; Garbino, J.; Pittet, D. Lancet Infect. Dis. 2003, 3, 685.
5. McNeil, M. M.; Nash, S. L.; Hajjeh, R. A.; Conn, L. A.; Plikaytis, B. P.; Warnock, D.
W. Clin. Infect. Dis. 2001, 33, 641.
(256 to one lg/mL) and amphotericin B (two and four lg/mL) were
then added to the cell suspensions and incubated at 35 °C in humid
atmosphere without agitation for 2, 4, and 24 h for yeasts or in a
water bath with moderate agitation for 3, 4, and 24 h for conidia,
respectively. Drug-free control tubes were included in every
experiment.
6. Rapp, R. P. Pharmacotherapy 2004, 24, 4S.
7. (a) Campaigne, E.. In Comprehensive Heterocyclic Chemistry; Katritzky, A. R., Rees,
C. W., Eds.; Pergamon Press: Oxford, 1984; Vol. 2, pp 863–934; (b) Ronald, K. R.;
Jefery, B. P.. In Comprehensive Heterocyclic Chemistry II; Katritzky, A. R., Rees, C. W.,
Scriven, E. F. V., Eds.; Pergamon Press: Oxford, 1996; Vol. 2, pp 679–729.
8. Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem. Rev. 2003, 103, 893.
9. Berger, W.; De Chandt, M. T.; Cairns, C. B. Int. J. Clin. Pract. 2007, 61, 663.
10. Vogelvang, T. E.; van der Mooren, M. J.; Mijatovic, V.; Kenemans, P. Drugs 2006,
66, 191.
4.2.7. Cell staining and flow cytometry readings
After the incubations the cells were washed and resuspended in
500 lL of phosphate-buffered saline (PBS) with 2% D-glucose for
FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-
yl)-methylidene)-1-phenylquinolinium iodide; Invitrogen, USA]
staining and 0.125% sodium deoxycholate (Sigma) for PI (propidi-
um iodide; Sigma) staining. Five microliters of the FUN-1 and PI
solutions in DMSO and PBS, respectively, at appropriate concentra-
tions (determined through preliminary tests, data not shown),
were added to the cell suspensions in order to obtain final concen-
11. Carrillo-Muñoz, A.; Guisiano, G.; Ezkurra, P. A.; Quindós, G. Expert Rev. Anti
Infect. Ther. 2005, 3, 333.
12. Ferreira, I. C. F. R.; Queiroz, M.-J. R. P.; Kirsch, G. Tetrahedron 2003, 59, 975.
13. Queiroz, M.-J. R. P.; Begouin, A.; Ferreira, I. C. F. R.; Kirsch, G.; Calhelha, R. C.;
Barbosa, S.; Estevinho, L. M. Eur. J. Org. Chem. 2004, 3679.
14. Ferreira, I. C. F. R.; Calhelha, R. C.; Estevinho, L. M.; Queiroz, M.-J. R. P. Bioorg.
Med. Chem. Lett. 2004, 14, 5831.
15. Mitchell, A. P. Curr. Opin. Microbiol. 1998, 1, 687.
16. Millard, P. J.; Roth, B. L.; Thi, H. P.; Yue, S. T.; Haugland, R. P. Appl. Environ.
Microbiol. 1993, 63, 2897.
17. Deere, D.; Shen, J.; Vesey, G.; Bell, P.; Bissinger, P.; Veal, D. Yeast 1998, 14, 147.
18. National Committee for Clinical Laboratory Standards. Approved standard
M27-A2, Wayne, PA, USA, 2002.
19. National Committee for Clinical Laboratory Standards. Approved Standard
M38-A, Wayne, PA, USA, 2002.
20. Fernández-Torres, B.; Cabañes, F. J.; Carrillo-Muñoz, A. J.; Esteban, A.; Inza, I.;
Abarca, L.; Guarro, J. J. Clin. Microbiol. 2002, 40, 3999.
21. Marichal, P.; Gorrens, J.;VanCutsem, J.; VandenBossche, H. Mykosen1986, 29, 76.
trations of 0.5 lM of FUN-1 and 1 lg/mL of PI. FUN-1 stained cells
were then incubated at 35 °C, away from incident light, for 20 min,
while PI stained samples were read after about 10 min at room
temperature for yeast cells and after 1 h incubation at 35 °C, away
from incident light, for A. fumigatus conidia. Unstained cell suspen-
sions were always included, as auto-fluorescence controls. Flow
cytometry was performed using a FACSCaliburÒ (Becton–Dickinson
Biosciences, San Jose, CA) flow cytometer with a 15 mW blue argon