Synthesis of aryl aldimines and their activity against fungi of clinical interest

Article abstract of DOI:10.1111/j.1747-0285.2011.01185.x

Aldimines are aldehyde-derived compounds that contain a C=N group. Besides its broad industrial applications, this class of non-naturally occurring compounds are found to possess antibacterial, antifungal, antimalarial, antiproliferative, anti-inflammatory, antiviral, and antipyretic properties. Based on this, six aryl aldimines were synthesized from the condensation of aromatic amines with benzaldehydes. The antifungal activities of synthesized compounds were evaluated against nineteen fungal strains that included Candida and Aspergillus species, Cryptococcus neoformans. The aryl aldimines 2-(benzylideneamino)phenol (3) and 4-(benzylideneamino)phenol (8) were the most active compounds against the fungi studied. Compounds 3 and 8 efficiently inhibited the metabolism of C. neoformans mature biofilm.

Full text of DOI:10.1111/j.1747-0285.2011.01185.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01185.x
Chem Biol Drug Des 2011; 78: 810–815
Research Article
Synthesis of Aryl Aldimines and Their Activity
against Fungi of Clinical Interest
1
,†
Cleiton M. da Silva , Danielle L. da
plantation among others are more susceptible to invasive fungal
infections (4).
1
,2,†
2,3
Silva
, Cleide V.B. Martins
Maria A. de Resende , Esther S. Dias ,
Thais F.F. Magalh a˜ es , Let ı´ cia P.
Rodrigues , Ad a˜ o A. Sabino , Rosemeire B.
,
2
1
2
Candida species infections may range from non-life-threatening muco-
cutaneous illnesses to invasive processes (5). Both mucosal and deep
tissue infections are mainly caused by C. albicans (5). Candida spe-
cies other than albicans have been assumed to be the causal agents
of nosocomial infections in immunocompromised patients (6,7). Asper-
gillosis also offers a risk to human health, being the major cause of
mortality in bone marrow-transplanted patients (8). Pulmonary asper-
gillosis is also a serious complication of patients undergoing lung and
heart–lung transplantation (9). The most common Aspergillus species
recovered from such patients is A. fumigatus (10,11). Cryptococcus
species are also among the life-threatening fungi. Approximately
1
1
1
1,2,
ˆ
Alves and Angelo de F a´ tima
*
1
Grupo de Estudos em Quꢀmica Orgꢁnica e Biolꢂgica (GEQOB),
Departamento de Quꢀmica, ICEx, Universidade Federal de Minas
Gerais, Belo Horizonte, MG, 31270-901, Brazil
Departamento de Microbiologia, ICB, Universidade Federal de
Minas Gerais, Belo Horizonte, MG, 31270-901, Brazil
2
3
Centro de Engenharias e CiÞncias Exatas, UNIOESTE, PR, 85903-
0
*
00, Brazil
3–30% of immunocompromised patients because of HIV ⁄AIDS,
Corresponding author: ffngelo de Fµtima, adefatima@qui.ufmg.br
hematological, or metabolic diseases develop cryptococcosis (12).
Cryptococcus neoformans is the most common cause of central
nervous system's mycosis in HIV-infected patients (12). Recent studies
have shown that the species C. neoformans is able to form biofilm, a
condition that reduces the efficacy of antifungal drugs (13).
These authors contributed equally to this work.
Aldimines are aldehyde-derived compounds that
contain a C=N group. Besides its broad industrial
applications, this class of non-naturally occurring
compounds are found to possess antibacterial,
antifungal, antimalarial, antiproliferative, anti-
inflammatory, antiviral, and antipyretic properties.
Based on this, six aryl aldimines were synthesized
from the condensation of aromatic amines with
benzaldehydes. The antifungal activities of synthe-
sized compounds were evaluated against nineteen
fungal strains that included Candida and Aspergil-
lus species, Cryptococcus neoformans. The aryl
aldimines 2-(benzylideneamino)phenol (3) and 4-
The frequent use of antifungal drugs may lead to fungal resistance,
affecting the infection treatment's efficiency. The current antifungal
drugs available in the market exhibit other drawbacks, such as tox-
icity, limited efficacy, and high cost (1). This scenario clearly indi-
cates that there is an ongoing requirement for the search of new
antifungal agents.
Aldimines, compounds containing the C=N group, are some of the
most widely used organic compounds. Besides industrial applica-
tions, they show a broad range of biological activities, such as anti-
(
benzylideneamino)phenol (8) were the most active
compounds against the fungi studied. Compounds
and 8 efficiently inhibited the metabolism of
3
fungal,
antibacterial,
antimalarial,
antiproliferative,
anti-
C. neoformans mature biofilm.
inflammatory, antiviral, and antipyretic properties (14,15). A number
of studies report the antifungal activity of several aldimines. Some
aryl aldimines have been checked for antifungal activity (Figure 1).
The compound 4-(4-(3-methoxy-4-hydroxybenzylidene-imino)-phenyl)-
morpholine (1) was shown to inhibit the growth of A. niger and
C. albicans at minimal inhibitory concentration (MIC) of 23 and
Key words: Antifungal activity, aryl aldimines, Aspergillus spp., bio-
film, Candida spp., Cryptococcus neoformans
Received 1 May 2010, revised 3 July 2011 and accepted for publication
3
July 2011
30 lg ⁄ mL, respectively (16). The growth of C. albicans also was
inhibited by 2-((2-hydroxynaphthalen-1-yl)methyleneamino)acetic acid
(2) that was more active than its corresponding manganese(III) com-
plex (17). The aryl aldimine 4-(benzylideneamino)phenol (3) inhibited
the budded-to-hyphal form transition in the C. albicans (18). Such
transition is essential for the establishment of systemic fungal
infections. Thus, the ability of compound 3 to inhibit the transition
of budded-to-hyphal form brings new perspectives for the develop-
ment of more effective antifungal therapeutics in the future.
Fungi have remained as some of the neglected pathogens in terms
of new drug discovery (1). Consequently, the incidence of invasive
fungal infections has steadily increased in the last two decades.
Fungal infections are potentially life threatening to patients of
compromised immunological system (2,3). Indeed, patients under
advanced age, major surgery, immunosuppressive therapy, AIDS and
cancer treatment, solid-organ and hematopoietic stem cell trans-
8
10

Antifungal Activity of Aryl Aldimines
)
1
Overall, aldimines are interesting prototypes for the design of new
antifungal agents (15). Here, we report the synthesis and the
antifungal activity of six aryl aldimines (compounds 3–8) against
nineteen fungal strains. The effect of the aryl aldimines 4-(benzyl-
ideneamino)phenol (3) and 2-(benzylideneamino)phenol (8) on the
metabolism of C. neoformans biofilm is also discussed.
Mp: 50–51 ꢀC [(21): 53–54 ꢀC)]. IR (neat, cm ) m : 3061, 3029,
2891, 1625, 1590, 1577, 1483, 1450, 1366, 1192, 1170, 1073, 976,
1
906, 867, 754, 690. H-NMR (CDCl
3
, 200 MHz): d 7.10–7.61 (m, 8H,
, H ), 8.44
H
3 4
, H , H
5
, H10, H11, H12, H13, H14), 7.81–8.00 (m, 2H, H
2
6
13
(s, 1H, H7). C-NMR (CDCl , 50 MHz): 121.1 (C , C ), 126.1 (C ),
3
10
14
12
129.0, 129.3 (C
2
, C , C , C
3 5 6
, C11, C13), 131.6 (C
4
), 136.4 (C
1
), 152.3
(
C ), 160.6 (C ). Elemental Analysis for (C H N): calculated, C:
9 7 13 11
86.15, H: 6.12, N: 7.73; found: C: 86.07, H: 6.08, N: 7.74.
Experimental
General experimental procedures
2-[(phenylimino)methyl]phenol (5)
All reagents used in the experiments were obtained from commer-
cial sources and used without purification. The determination of
compounds melting point (uncorrected) was carried out in an
MQAPF-302 apparatus. Infrared spectra were recorded on Perkin–
Elmer Spectrum One spectrophotometer. NMR spectra were
recorded in a Bruker Avance DRX-200 spectrometer. Chemical shifts
are reported in d units downfield from TMS, and J values are given
in Hertz. Elemental analyses were performed by using a CHN Per-
kin–Elmer 2400 analyzer (Boston, MA, USA).
11
OH
7
1
9
13
N
5
3
)
1
Mp: 50–51 ꢀC [(21): 51–52 ꢀC)]. IR (neat, cm ) m : 3058, 2991,
614, 1589, 1567, 1484, 1458, 1403, 1361, 1280, 1182, 1171, 1150,
08, 834, 757, 687. H-NMR (CDCl , 200 MHz): d 6.85–7.08 (m, 2H,
3
1
9
1
H , H ), 7.17–7.54 (m, 7H, H , H , H , H , H , H , H ), 8.59 (s,
4
6
3
1
5
10
11
12
13
14
3
Synthesis of aldimines
1
1
1
7 3 6
H, H ), 13.28 (s, 1H, OH). C-NMR (CDCl , 50 MHz): 117.4 (C ),
The aldimines 3–8 were prepared from the condensation of appropri-
ate amines with benzaldehydes in toluene using a Dean Stark appara-
tus according to methods previously described (19,20). Solvent was
evaporated, and the compounds were purified by crystallization.
19.3 (C ), 119.4 (C ), 121.4 (C , C ), 127.1 (C ), 129.6 (C , C ),
4
2
10 14
12
11 13
32.5 (C
3
), 133.3 (C
5
), 148.7 (C
9
), 161.3 (C
1
), 162.9 (C
7
). Elemental
Analysis for (C13
H
11NO): calculated, C: 79.16, H: 5.62, N: 7.10, O:
8.11; found: C: 79.43, H: 5.52, N: 7.15, O: 7.90.
4
-(benzylideneamino)phenol (3)
3
-[(phenylimino)methyl]phenol (6)
11
1
OH
3
7
9
7
N
HO
1
9
3
11
13
N
5
13
5
)
1
)1
Mp: 184–186 ꢀC [(21): 184–185 ꢀC)]. IR (neat, cm ) m : 3062, 3008,
Mp: 90–91 ꢀC [(22): 90 ꢀC)]. IR (neat, cm ) m : 3051, 2915, 2691
2
918, 2789, 2720, 2666, 2578, 2466, 1618, 1604, 1587, 1506, 1450,
2557, 2444, 1621, 1596, 1581, 1447, 1389, 1274, 1221, 1207, 1166,
1
1
1372, 1274, 1235, 1182, 1172, 1106, 978, 835, 823, 753, 717, 685. H-
1156, 1080, 857, 782, 756, 691, 681. H-NMR [(CD
3
)
2
SO, 200 MHz]:
, H
), 9.73 (s, 1H, OH). C-NMR [(CD
), 118.8 (C ), 120.4 (C ), 121.0 (C10, C14), 125.9
), 137.4 (C ), 151.5 (C ), 157.7 (C ),
). Elemental Analysis for (C13 11NO): calculated, C: 79.16,
H: 5.62, N: 7.10, O: 8.11; found: C: 79.50, H: 5.14, N: 7.10, O: 8.27.
NMR [(CD
7
3
)
2
SO, 200 MHz]: d 6.84 (d, 2H, J2,3 = J6,5 = 8.7, H
2 6
, H ),
d 6.89–7.01 (m, 1H, H
6
), 7.19–7.47 (m, 8H, H
2
, H
4
5
, H10, H11,
1
3
.23 (d, 2H, J3,2 = J5,6 = 8.7, H
3
, H ), 7.45–7.59 (m, 3H, H11, H12
5
,
H
12, H13, H14), 8.52 (s, 1H, H
7
3 2
) SO,
H ), 7.85–7.99 (m, 2H, H , H ), 8.62 (s, 1H, H ), 9.57 (s, 1H, OH).
50 MHz]: 114.3 (C
2
6
4
1
3
10
14
8
1
3
C-NMR [(CD
3
)
2
SO, 50 MHz]: 115.8 (C
2
, C
6
), 122.5 (C
3
, C
5
), 128.3,
(C12), 129.2 (C11, C13), 129.9 (C
160.7 (C
5
3
9
1
1
1
5
28.8 (C , C , C , C ), 130.9 (C ), 136.5 (C ), 142.6 (C ), 156.3 (C ),
7
H
1
0
11 13 14
12
9
4
1
57.2 (C
8
). Elemental Analysis for (C13H11NO): calculated, C: 79.16, H:
.62, N: 7.10, O: 8.11; found: C: 79.24, H: 5.46, N: 7.09, O: 8.21.
4
-[(phenylimino)methyl]phenol (7)
11
N-benzylideneaniline (4)
11
3
7
9
13
N
7
1
9
3
1
13
5
HO
N
)
1
Mp: 194–195 ꢀC [(21): 195–196 ꢀC)]. IR (neat, cm ) m : 3047,
5
2867, 2795, 2733, 2673, 2568, 2478, 1602, 1574, 1515, 1485,
Chem Biol Drug Des 2011; 78: 810–815
811

da Silva et al.
443, 1387, 1284, 1241, 1189, 1164, 978, 936, 840, 758, 723, 689.
1
All compounds (3–8) were primarily dissolved in dimethylsulfox-
1
H-NMR [(CD ) SO, 200 MHz]: d 6.90 (d, 2H, J = J6,5 = 8.6, H2,
ide (DMSO) and then diluted in NaHCO -free RPMI-1640 medium
3
2
2,3
3
H
6
), 7.13–7.45 (m, 5H, H10, H11, H12, H13, H14), 7.78 (d, 2H, J3,2
=
(INLAB, Diadema, SP, Brazil) buffered with 165 mM morpholine
propanesulfonic acid (MOPS, pH 7.0) and supplemented with
4 mM L-glutamine. The final concentration of DMSO was 5%
and did not affect fungal growth as attested for the control
treatment. Each compound was tested at concentrations ranging
from 0.5 to 256 lg ⁄ mL, and fluconazole was used as a positive
control.
13
J
3 5 7
5,6 = 8.6, H , H ), 8.45 (s, 1H, H ), 10.15 (s, 1H, OH). C-NMR
[
(CD ) SO, 50 MHz]: 115.7 (C , C ), 120.9 (C , C ), 125.3 (C ),
3
2
2
6
10
14
12
1
1
27.5 (C
4
), 129.2, 130.7 (C
3 5 9 7
, C , C11, C13), 152.0 (C ), 160.0 (C ),
60.7 (C ). Elemental Analysis for (C H NO): calculated, C: 79.16,
1
13 11
H: 5.62, N: 7.10, O: 8.11; found: C: 78.45, H: 5.29, N: 6.66, O:
.45.
7
Yeast species were cultured on SDA for 24 or 48 h, while the fila-
mentous ones were cultured on PDA for 7 days. Fungal inoculum
2
-(benzylideneamino)phenol (8)
3
concentrations were adjusted to 1–5 · 10 cells ⁄ mL for yeasts and
4
HO
1
5
0
.4–5 · 10 cells ⁄ mL for filamentous fungi and diluted two-fold in
the incubation medium. Three independent experiments were per-
formed each done in duplicate. The MIC end-point for test com-
pounds was defined as the lowest concentration of tested
compound necessary to abolish the micro-organism growth. Mini-
mal inhibitory concentration value for fluconazole was defined as
the lowest concentration that prevented the fungal growth by
50%.
7
N
9
11
3
13
)
1
Mp: 89–90 ꢀC [(21): 89–90 ꢀC)]. IR (neat, cm ) m : 3323, 3020,
2
902, 1625, 1584, 1574, 1481, 1450, 1380, 1358, 1285, 1249,
1
1194, 1167, 1147, 1026, 967, 874, 850, 790, 763, 733, 688. H-
NMR [(CD
)
3 2
SO, 200 MHz]: d 6.85 (t, 1H, J4,3
=
J
4,5 = 7.7, H
4
),
Biofilm assay
6
.90 (d, 1H, J6,5 = 7.7, H ), 7.11 (t, 1H, J = J5,6 = 7.7, H ), 7.22
Biofilm assay was performed as described by Ramage and co-work-
ers (2001) (25) with some modifications. Briefly, C. neoformans
strain was inoculated in yeast peptone dextrose medium (YPD; Dif-
co) and then incubated at 37 ꢀC under stirring at 100 rpm for
24 h. Cells were centrifuged, washed three times in sterile phos-
phate-buffered saline (PBS) solution, and re-suspended in RPMI-
6
5,4
5
(
d, 1H, J3,4 = 7.7, H
3
), 7.46–7.59 (m, 3H, H11, H12, H13), 7.98–8.13
13
(
m, 2H, H10, H14), 8.72 (s, 1H, H ), 9.04 (s, 1H, OH). C-NMR
8
[
(CD ) SO, 50 MHz]: 116.1 (C ), 119.2, 119.5 (C , C ), 127.4 (C ),
3
2
3
4
6
5
1
1
28.7, 128.9 (C10, C11, C13, C14), 131.3 (C12), 136.4 (C
51.2 (C ), 159.3 (C ). Elemental Analysis for (C H NO): calculated,
9
), 138.0 (C
2
),
1
8
13 11
6
C: 79.16, H: 5.62, N: 7.10, O: 8.11; found: C: 79.19, H: 5.46, N:
.12, O: 8.25.
1640 medium. Cells (100 lL of 10 units ⁄ mL) were transferred to a
7
microplate and incubated at 37 ꢀC for 24 h to allow biofilm forma-
tion. Non-adherent cells were carefully removed and the remainder
ones washed with sterile PBS solution. An equal volume of com-
pounds 3, 8, or fluconazole solutions (at MIC values) was added
to yeast cells and the system incubated at 37 ꢀC for 48 h. Formed
biofilms were washed three times with sterile PBS solution follow-
ing the metabolism analysis.
Micro-organisms
Candida albicans ATCC 18804, Candida tropicalis ATCC 750, Can-
dida krusei ATCC 20298, Candida parapsilosis ATCC 22019, Can-
dida glabrata ATCC 2001, Aspergillus fumigatus ATCC 16913,
A. nomius ATCC 15546, and C. neoformans ATCC 24067 were
obtained from the American Type Culture Collection (Manassas,
VA, USA), while A. flavus IMI 190443 was acquired from the Inter-
national Mycological Institute (IMI, England). Clinical isolates of
Candida dubliniensis (Cd22, Cd23, Cd25, Cd27, Cd28, and Cd29),
A. clavatus, A. tamarii, A. fumigatus, and A. terreus were also
used.
2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-
tetrazolium-5-carboxanilide (XTT) reduction
assay
Cell viability of cryptococcal biofilms was determined semi-quantita-
tively by the XTT reduction assay as reported elsewhere (13).
Briefly, newly formed biofilms were incubated with 50 lL XTT solu-
tion (1 mg ⁄ mL in PBS) and 4 lM menadione (1 mM in acetone) in
the dark at 37 ꢀC for 5 h. The supernatant was then transferred to
a new microplate and analyzed spectrophotometrically at 492 nm to
determine cell viability.
All strains are maintained in the Laboratory of Mycology of the
Institute of Biological Sciences at the Federal University of Minas
Gerais (Brazil) on slopes of Sabouraud dextrose agar (SDA, Difco,
Detroit, MI, USA) or potato dextrose agar (PDA; Difco) and sub-
cultured every 2 months.
Statistical analysis
Susceptibility assay
Statistical analysis was carried out by using the SIGMA STAT Soft-
ware. Experimental data were submitted to one-way analysis of
variance (ANOVA). Pairwise comparisons between control and drug-
exposed fungi were carried out with Tukey's test. p-value of less
than 0.05 was considered statistically significant.
The minimum inhibitory concentration (MIC) for each synthesized
compound was determined for yeasts and filamentous fungi accord-
ing to the protocols M27-A2 (NCCLS, 2002) (23) and M38-A (NCCLS,
2002) (24), respectively.
8
12
Chem Biol Drug Des 2011; 78: 810–815

Antifungal Activity of Aryl Aldimines
Results and Discussion
Among the Candida species, C. dubliniensis Cd22 (clinical isolate)
was the most sensitive to compound 3 whose MIC value was
64.0 lg ⁄ mL. The growth of C. glabrata ATCC 2001 and C. tropicallis
ATCC 750 was also affected by compound 3 that exhibited MIC val-
ues around 99.0 lg ⁄ mL.
All aryl aldimines (compounds 3–8) were obtained in good yields
(
Table 1). The synthesized compounds were fully characterized by
1
13
elemental analysis, IR, H, and C NMR spectroscopy, and the data
were consistent with those reported in the literature (21,22).
Eight Candida species of eleven strains were inhibited by the aryl
aldimine 8 (Table 2). The MIC values for this compound ranged
from 124.5 to 176.0 lg ⁄ mL. At the studied experimental conditions,
no detectable growth-inhibiting activity was observed for aryl aldi-
mines 4–7 against Candida species.
The minimum inhibitory concentration (MIC) of aryl aldimines 3–8
necessary to completely suppress the growth of fungal strains is
shown in Table 2. The aryl aldimines 3 and 8 were the most effec-
tive compounds inhibiting the growth of all fungi studied, except
A. nomius (ATCC 15546), A. flavus (IMI 190443), and A. terreus
(clinical isolate).
Aspergillus fumigatus ATCC 16913 and clinical isolate, A. clavatus
(
clinical isolate), and A. tamarii (clinical isolate) were the most sen-
Table 1: Synthesis of aryl aldimines 3–8
sitive to aryl aldimines 3 and 8, among the Aspergillus species.
Notably, aryl aldimine 8 (MIC = 10.0 lg ⁄ mL) was 6-fold more
potent than the reference drug fluconazole in inhibiting the growth
of A. clavatus (clinical isolate). Also, the MIC value for compound 8
against A. fumigatus ATCC 16913 was three-fold lower than that of
fluconazole. The aryl aldimine 8 was also more potent than fluco-
nazole in the growth inhibition of the clinical isolates of A. tamari
and A. fumigatus (Table 2). Even though the most expressive results
against Aspergillus species were obtained from the cell treatment
with compound 8, promising results were achieved from aryl aldi-
mine 3 treatment of A. fumigatus ATCC 16913 and clinical isolate,
and A. clavatus and A. tamarii clinical isolates. The aryl aldimines
CHO
NH2
toluene
reflux
N
R2
+
R1
R2
R1
1
2
a
Compound
R
R
Time (h)
Yields (%)
3
4
5
6
7
8
H
H
2-OH
3-OH
4-OH
H
4-OH
H
H
H
H
2-OH
16
36
22
20
16
24
83
65
83
84
81
63
4
–7 were also not active against Aspergillus species (Table 2). The
stability of such compounds was checked by incubating each com-
pound in RPMI medium (pH 7.0) containing DMSO 5% for 3 h at
3
7 ꢀC in the absence of fungus. The UV–vis spectrum for each
a
Isolated yields.
compound revealed that only 5 underwent decomposition (data not
a
Table 2: Minimal inhibitory concentration (MIC ; lg ⁄ mL) of the aryl aldimines 3–8 against fungal strains
Aryl aldimines
b
Micro-organism
3
4
5
6
7
8
Flu
Candida albicans ATCC 18804
Candida tropicalis ATCC 750
Candida krusei ATCC 20298
Candida parapsilosis ATCC 22019
Candida glabrata ATCC 2001
157.5
99.5
157.5
125.0
99.0
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
88.0
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
256.0
128.0
256.0
124.5
256.0
176.0
128.0
128.0
128.0
256.0
128.0
128.0
20.0
256.0
>256
10.0
64.0
64.0
2.0
2.0
32.0
1.0
1.0
*
*
*
*
*
*
Candida dubliniensis Cd 22
C. dubliniensis Cd 23
C. dubliniensis Cd 25
C. dubliniensis Cd 27
C. dubliniensis Cd 28
C. dubliniensis Cd 29
64.0
0.12
0.18
0.18
0.25
0.12
0.25
64.0
>64
>64
64.0
>64
>64
>64
16.0
128.0
128.0
128.0
128.0
128.0
128.0
>256
>256
128.0
128.0
128.0
>256
30.0
Aspergillus fumigatus ATCC 16913
Aspergillus nomius ATCC 15546
Aspergillus flavus IMI 190443
*
*
*
*
Aspergillus clavatus
Aspergillus tamarii
Aspergillus fumigatus
Aspergillus terreus
>256
8.0
Cryptococcus neoformans ATCC 24067
a
Data are geometric means of three independent experiments each carried out in duplicate.
Flu stands for fluconazole (positive control).
b
Fungal strains marked with an asterisk (*) are clinical isolate.
Chem Biol Drug Des 2011; 78: 810–815
813

da Silva et al.
shown). Indeed, imines derived from salicylidene aldehydes, like 5,
are known to be easily hydrolyzed in aqueous medium (26). A six-
membered conformer is established between the o-hydroxyl group
and the nitrogen atom from C=N bond, which activates the addition
and 8 prompted us to further investigate their effect on the metab-
olism of C. neoformans ATCC 24067 mature biofilm. Compounds 3
and 8 at their MIC values (30.0 and 8.0 lg ⁄ mL, respectively)
impaired the viability of C. neoformans ATCC 24067 mature biofilm
(Figure 2). Compounds 3 and 8 inhibited the metabolic activity of
C. neoformans biofilm by 43% and 49%, respectively. Fluconazole
at 16.0 lg ⁄ mL inhibited the biofilm metabolic activity by 85%.
Although compounds 3 and 8 were less effective than fluconazole,
the results suggest that these aryl aldimines 3 and 8 are promising
prototypes for the design of new C. neoformans anti-biofilm agents.
of a nucleophile (e.g., H O) to the carbon atom at C=N bond (26).
2
Thus, the lack of activity for compounds 4, 6, and 7 cannot be
attributed to a possible decomposition in the medium.
The growth of C. neoformans ATCC 24067 was inhibited by treat-
ment with the aryl aldimines 3, 5, and 8 (Table 2). The moderate-
to-low activity of 5 is probably derived from its decomposition prod-
uct(s). Interestingly, compound 8 was twice as potent as fluconaz-
ole against C. neoformans ATCC 24067. The aryl aldimines 3 and 5
exhibited promising results against C. neoformans ATCC 24067
reaching MIC values as low as 30.0 and 88.0 lg ⁄ mL, respectively.
Conclusions
The aryl aldimines 3 and ⁄ or 8 showed to have promising antifungal
activities against C. albicans ATCC 18804, C. glabrata ATCC 2001,
C. dubliniensis Cd 22 CI, A. fumigatus ATCC 16913, A. clavatus CI,
A. tamarii CI, A. fumigatus CI, and C. neoformans ATCC 24067.
These compounds were also able to reduce by 40–50% the meta-
bolic activity of C. neoformans ATCC 24067 mature biofilm. In con-
junction, these results point out aryl aldimine 3 and 8 as lead
Cryptococcus neoformans is opportunistic and accounts for a large
number of deaths in Brazil because of systemic mycosis with under-
lying cause of AIDS (27). The efficacy of antifungal drugs used to
treat cryptococcal infections may be compromised by fungal biofilm
formation. The promising results obtained with the aryl aldimines 3
Figure 1: Examples of aryl
aldimines of recognized antifungal
activities.
OH
HO
N
N
N
N
N
F
OH
N
N
N
F
Figure 2: Effect of the aryl aldimines 3 and 8 on the metabolic activity of Cryptococcus neoformans biofilms. Biofilms were exposed to
the aryl aldimines 3 and 8 at their minimal inhibitory concentration values (30.0 and 8.0 lg ⁄ mL, respectively) for 24 h. Metabolic activity
was assessed by XTT reduction assay. NC, biofilms incubated with RPMI-1640 medium; Flu, biofilms incubated with fluconazole at MIC value
(
16.0 lg ⁄ mL). Bars represent the means € SE of three independent experiments carried out in duplicate. Different letters indicate significant
difference (p £ 0.05) in relation to results obtained for the negative control.
8
14
Chem Biol Drug Des 2011; 78: 810–815

Antifungal Activity of Aryl Aldimines
compounds for the development of new antifungal agents. To the
best of our knowledge, this is the first report on the effect of aryl
aldimines on the viability of C. neoformans ATCC 24067 biofilms.
13. Martinez L.R., Casadevall A. (2006) Susceptibility of Cryptococ-
cus neoformans biofilms to antifungal agents in vitro. Antimicrob
Agents Chemother;50:1021–1033.
1
4. Przybylski P., Huczynski A., Pyta K., Brzezinski B., Bartl F. (2009)
Biological properties of Schiff bases and azo derivatives of phe-
nols. Curr Org Chem;13:124–148.
Acknowledgments
1
5. da Silva C.M., da Silva D.L., Modolo L.V., Alves R.B., Martins
C.V.B., de Resende M.A., de Fꢀtima A. (2011) Schiff bases: a
short review of their biological activities. J Adv Res;2:1–8.
The authors are grateful to Dr Luzia V. Modolo for critical reading
of this manuscript. This work was funded by FAPEMIG, CAPES, and
CNPq. ADF and MAR are supported by research fellowships from
CNPq.
16. Panneerselvam P., Nair R.R., Vijayalakshmi G., Subramanian E.H.,
Sridhar S.K. (2005) Synthesis of Schiff bases of 4-(4-aminophe-
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