Synthesis of naphtho[2,3-a]phenoxazinium chlorides: Structure-activity relationships of these heterocycles and benzo[a]phenoxazinium chlorides as new antimicrobials

Article abstract of DOI:10.1016/j.bmc.2007.12.013

Synthesised functionalised naphtho[2,3-a]phenoxazinium chlorides revealed great fluorescence with maximum emission wavelengths between 630 and 676 nm, in ethanol and water at physiological pH. Naphtho[2,3-a]phenoxazines, as well as a series of benzo[a]phe

Full text of DOI:10.1016/j.bmc.2007.12.013

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3274–3282
Synthesis of naphtho[2,3-a]phenoxazinium chlorides:
Structure–activity relationships of these heterocycles
and benzo[a]phenoxazinium chlorides as new antimicrobials
Vaˆnia H. J. Frade,a Maria J. Sousa,b Joao C. V. P. Mouraa and
˜
M. Sameiro T. Gonc¸alvesa,*
aCentro de Quı´mica, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
bCentro de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
Received 3 September 2007; revised 30 November 2007; accepted 7 December 2007
Available online 14 December 2007
Abstract—Synthesised functionalised naphtho[2,3-a]phenoxazinium chlorides revealed great fluorescence with maximum emission
wavelengths between 630 and 676 nm, in ethanol and water at physiological pH. Naphtho[2,3-a]phenoxazines, as well as a series
of benzo[a]phenoxazines, were evaluated against Saccharomyces cerevisiae, in a broth microdilution assay. This family of com-
pounds exhibited antifungal activity depending both on the substituents of the heterocycle nucleus as well as on its size. The best
activities were obtained for four-ring systems, and particularly for 5,9-diaminobenzo[a]phenoxazines with R = Me, R1 = H and
R2 = Et. As for R3 substitution, the greatest efficiency was obtained for R3 = (CH2)3Cl, with a MIC of 3.75 lM. The linkage of dif-
ferent amino acids to the functional group of the 5-amino position of diaminobenzo[a]phenoxazinium moiety resulted in compounds
with diverse antimicrobial efficiencies, depending on the polar character of the amino acid, on its linkage position and on the size of
the alkyl chain linker.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
says employing intact, live cells.9 Furthermore, different
modifications of the phenoxazine moiety can yield com-
pounds that directly bind to proteins, mainly through
hydrophobic interaction, and which have been devel-
oped either for the use in monitoring protein conforma-
tion alterations or for therapeutic purposes.10–13
Oxazine dyes have been a subject of much spectroscopic
research due to their great use as a tunable laser dye in
the range 600–900 nm. Alternatively, they are also stan-
dards for fluorescence studies and are of significant va-
lue as biological probes.1,2 Applications of these long-
wavelength fluorophores, namely Nile Blue and its
derivatives, include the covalent labelling of model car-
boxylic acids,3 amino acids,4 proteins,5,6 peptides and
DNA.7
Although a large number of studies have focused on the
photophysical properties, molecules possessing the
phenoxazine counterparts have also assumed increasing
importance in life sciences, due to their antiproliferative
properties with potential applications both as antitu-
mour and as antimicrobial agents.14–20 These com-
pounds can exert their antiproliferative activity
through the DNA intercalative binding of their large
planar polycycles; the stabilization of this binding arises
both from the hydrogen bonds and the p–p stacking
interactions.17,21 In addition, the phenoxazine ring sys-
tem can produce free radical intermediates leading to
oxidative stress.16 Some phenoxazine derivatives have
also been shown to display apoptotic activity against
different cell lines, both in a caspase-dependent and
independent manner.14,22
On the other hand, the use of benzo[a]phenoxazines in
non-covalent labelling is more common.8 These hetero-
cycles are valuable for staining nucleic acids in a variety
of contexts, including in solutions, in electrophoretic
gels or other matrices, in blotting experiments and in as-
Keywords: Naphtho[2,3-a]phenoxazinium salts; Benzo[a]phenoxazines;
Antifungal activity; Long-wavelength fluorescent dyes; Functionalised
probes.
*
Corresponding author. Tel.: +351 253604372; fax: +351
253604382; e-mail: msameiro@quimica.uminho.pt
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.013

V. H. J. Frade et al. / Bioorg. Med. Chem. 16 (2008) 3274–3282
3275
In spite of the interest of the abovementioned phenoxa-
zine dyes, the synthesis and application of compounds
starting with 1-aminoanthracene or its derivatives is
scarce in the literature.9
vant signals were assigned to the N-alkyl substituents in
the regions of 1.15–3.99 ppm and 11.70–69.06 ppm,
respectively.
Preparation of chloropropylamino derivative 1f was
achieved by reaction of compound 1d with thionyl chlo-
ride, at room temperature. After purification by dry
chromatography or successive washes with solvent
(1e), cationic dyes 1a–f were isolated as solid materials
in moderate to excellent yields (Table 1) and were fully
characterised by the usual analytical techniques.
Considering these facts, in addition to our interest in the
synthesis and application of heterocycle fluorescent re-
agents,4,23,24 we have synthesised new naphtho[2,3-
a]phenoxazine derivatives which possess an extended
aromatic system. Photophysical studies were carried
out in ethanol and water at physiological pH and the re-
sults were compared with those of benzo[a]phenoxazine
analogues.
The IR spectra bands at 1724, 3406 and 3197–3446 cmÀ1
were assigned to the ester (1c), the hydroxyl (1d) and the
amino (1a and 1e) groups, respectively. The 13C NMR
of compound 1c showed a signal at d 174.77 ppm which
also confirmed the presence of the carbonyl group of the
ester function.
Using Saccharomyces cerevisiae as a model organism, we
analysed the antifungal activity of the naphtho[2,3-a]phe-
noxazinium chlorides obtained in the present work. The
MIC values were then compared with those of a series
of benzo[a]phenoxazinium chloride analogues, exploring
the effect of alterations in the extent of the aromatic sys-
tem, the substitution of both the amino groups and the
10-position of the benzo[a]phenoxazinium nucleus, as
well as the linkage to amino acids.
Electronic absorption spectra of 10À6 M solutions of
naphtho[2,3-a]phenoxazinium chlorides 1a–f in de-
gassed absolute ethanol were measured (Table 1). The
longest wavelength of maximum absorption (kmax) of
all compounds was located between 615 and 647 nm.
When compared to 1a (R = H), compounds 1b–f ab-
sorbed at longer wavelengths; the bathochromic shift
was 25–32 nm. This fact showed the importance of a
substituent at the amine group in the 7-position of the
polycyclic system in the absorbance properties of these
compounds.
2. Chemistry
Naphtho[2,3-a]phenoxazinium chlorides 1a–e were pre-
pared by condensation of 5-ethylamino-4-methyl-2-
nitrosophenol hydrochloride 2 with 1-aminoanthracene
or its N-substituted-derivatives 3b–e in the presence of
hydrochloric acid, refluxed in ethanol (Scheme 1). The
required nitrosophenol 2 was synthesised using the usual
procedure involving treatment of the corresponding 3-
ethylamino-4-methylphenol with sodium nitrite in an
acid solution.25 Compounds 3b–e were prepared by
alkylation of 1-aminoanthracene with the appropriate
bromo-reagent. After dry chromatography purification
(3b, 3d and 3e; 3c was not purified), these compounds
were obtained as oils (21%, 3b; 53%, 3d; 50%, 3e) and
were characterised by high resolution mass spectrome-
try, IR and NMR (1H and 13C) spectroscopy.
Table 1. Synthesis and visible data of naphtho[2,3-a]phenoxazinium
chlorides 1a–f, in ethanol and water (pH 7.4)
Compound
Yield
(%)
kmax [nm] (e)
(ethanol)
kmax [nm] (e)
(water, pH 7.4)
1a
1b
1c
1d
1e
1f
98
43
16a
61
98
50
615 (62,112)
642 (26,100)
647 (19,595)
640 (35,000)
645 (14,781)
645 (43,300)
615 (46,296)
601 (22,000)
b
—
640 (35,000)
b
—
b
—
The IR spectra showed strong stretching vibration
bands for the hydroxyl and the amino groups at 3434
a Overall yield of the two steps.
b Compound not soluble in water (pH 7.4).
1
(3d) and 3398 cmÀ1 (3e). In the H and 13C NMR, rele-
H3C
N
O
NO
OH
H3C
H
a)
+
H
R
H
+
H
R
N
N
N
N
H3CH2C Cl -
H3CH2C
2
3a-e
1a-f
a R = H
b R = (CH2)2CH3
c R = (CH2)3CO2CH2CH3
d R = (CH2)3OH
e R = (CH2)3NH2
f R = (CH2)3Cl
b)
Scheme 1. Synthesis of naphtho[2,3-a]phenoxazinium chlorides 1a–f. Reagents and conditions: (a) H+/ethanol, reflux; (b) SOCl2, dichloromethane/
chloroform 2:1, rt.

3276
V. H. J. Frade et al. / Bioorg. Med. Chem. 16 (2008) 3274–3282
Comparison of kmax of the naphtho[2,3-a]phenoxazini-
um chlorides 1a and 1c–f synthesised with that of the
benzo[a]phenoxazinium chloride analogues in etha-
nol4,26 showed that fluorophores 1c–f presented the
highest values (kmax of compound 1a was slightly lower
than that of the benzo[a]phenoxazinium analogue, 615/
620 nm), the red shift being 25–32 nm. These results
seem to be related to the increase of the electronic con-
jugation resulting from the addition of another aromatic
ring to the polycyclic benzo[a]phenoxazine nucleus.
compound 1d, in water), the bathochromic shift was
37–44 nm in both solvents, which might be related to
the presence of the N-substituents, which is the case of
absorbance.
Comparison of kem of synthesised naphtho[2,3-a]phe-
noxazinium chlorides 1a, 1c–f with that of the
benzo[a]phenoxazinium chloride analogues in etha-
nol4,26 showed that the naphtho[2,3-a]phenoxazines pre-
sented the highest values (except in the case of
compound 1a, which was slightly lower than the
benzo[a]phenoxazinium analogue, 634/637 nm), the red
shift being from 26 to 30 nm. These results seem to be
related to an increase in the electronic conjugation
resulting from the addition of another aromatic ring in
the polycyclic benzo[a]phenoxazine nucleus, as in the
case of absorbance variation.
The absorption properties of heterocycles 1a, 1b and 1d
were also measured in water at physiological pH (pH
7.4, adjusted with HCl and NaOH) (1c, 1e and 1f were
not soluble). Comparison of kmax values in ethanol
and water showed both no variation (1a and 1d) or a
blue shift of 41 nm (1b). Figure 1 shows the normalised
absorption spectra of compounds 1a, 1c and 1d, in
ethanol.
All compounds exhibit high levels of fluorescence in eth-
anol and water (pH 7.4), with quantum yields between
0.10 (1b and 1d, in water) and 0.47 (1a, in water) and
showed low Stokes’s shifts (the highest 32 nm for 1d,
in ethanol). Figure 2 shows the normalised fluorescence
spectra of compounds 1a, 1c and 1d, in ethanol.
Studies of the fluorescent properties of compounds 1a–f
were also carried out in ethanol and water (pH 7.4) (1a,
1b and 1d). Summarised data are presented in Table 2.
The fluorescence quantum yields (UF) were calculated
using Oxazine 1 as a standard (UF = 0.11 in ethanol).27
For the determination of the relative quantum yields,
Oxazine 1 was excited at the wavelengths of excitation
of each one of the compounds tested. Emission maxima
(kem) for all compounds in ethanol and water varied
from 630 to 676 nm. When compared to 1a (R = H),
compounds 1b–f emitted at longer wavelengths (except
3. Biological activity
Antimicrobial activity of the synthesised fluorophores
was asseded using a broth microdilution method for
the antifungal susceptibility testing of yeasts (NCCLS
M27-A). The minimum inhibitory concentrations of
growth (MIC) obtained for S. cerevisiae W303-1B, used
as a reference organism, are shown in Scheme 2. The
activity of compounds 1a–f was influenced by R3 substi-
tution, although a broad variation in MIC values was
not found. In general, the substitution of the amino
group increased activity, the only exception being com-
pound 1e (entry 5) where the presence of the amine func-
tion in R3 was related to the lowest antimicrobial
activity. Compounds 1b–d (entries 2–4) and 1f (entry
6) all shared a MIC of 15 lM.
1
1c
0.8
0.6
1a
0.4
1d
0.2
0
In order to extend structure–activity relationship (SAR)
analysis, we also evaluated the antimicrobial activity
500
600
700
of
a
large series of compounds containing the
Wavelength (nm)
Figure 1. Normalised absorbance spectra of compounds 1a, 1c and 1d,
in ethanol.
1
1c
Table 2. Fluorescence data of naphtho[2,3-a]phenoxazinium chlorides
1a–f, in ethanol and water (pH 7.4)
0.8
0.6
0.4
0.2
0
1d
Compound
Ethanol
kexc kem UF Stokes’s kexc kem UF Stokes’s
shift shift
590 634 0.34 19 590 632 0.47 17
590 676 0.10 34
Water (pH 7.4)
1a
1a
1b
1c
1d
1e
1f
590 671 0.29 29
585 674 0.20 27
600 672 0.37 32
575 674 0.24 29
590 671 0.26 26
a
—
—
—
—
600 630 0.10 10
570
670
770
a
Wavelength (nm)
—
—
—
—
—
—
—
—
a
Figure 2. Normalised fluorescence spectra of compounds 1a, 1c and
1d, in ethanol.
a Compounds not soluble in water (pH 7.4).

V. H. J. Frade et al. / Bioorg. Med. Chem. 16 (2008) 3274–3282
3277
R
N
R
N
R1
R2
+
Cl -
R1
R2
H
H
+
N
O
1
N
N
O
4
N
Cl -
R3
R3
Entry
1
Compound
1a
1b
1c
R
R1
R2
Et
R3
MICa
30
log P
2.67
3.93
3.52
2.69
2.12
3.92
1.51
2.36
1.53
0.96
2.77
1.68
2.09
1.71
1.26
0.69
1.41
4.02
0.70
0.71
0.89
0.59
-0.06
0.52
0.14
-0.04
-0.17
2.45
5.72
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
2
H
Et
(CH2)2Me
15
3
H
Et
(CH2)3CO2Et 15
4
1d
1e
H
Et
(CH2)3OH
(CH2)3NH2
(CH2)3Cl
H
15
60
15
15
5
H
Et
6
1f
H
Et
7
4a
4b
4c
H
Et
8
H
Et
(CH2)3CO2Et 7.5
9
H
Et
(CH2)3OH
(CH2)3NH2
(CH2)3Cl
60
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
4d
4e
H
Et
3026
3.75
120
H
Et
4f
H
Et
(CH2)3CO2H
4g
4h
4i
H
Et
(CH2)2CO2Et 15
(CH2)2CO2Me 15
H
Et
H
Et
(CH2)2OH
(CH2)2NH2
(CH2)2CO2H
Ph
30
4j
H
Et
120
15
4l
H
Et
4m
4n
4o
4p
4q
4r
H
Et
>120
60
Et
Et
Et
Et
Me
Me
Me
Me
Me
Me
H
Et
H
H
Et
(CH2)3OH
120
H
Et
(CH2)2CO2Me 15
H
Et
(CH2)2CO2H
H
120
>120
H
Me
Me
Me
Me
Me
Me
4s
H
(CH2)2CO2Et 15
4t
H
(CH2)2CO2Me >120
4u
4v
4x
4z
H
(CH2)3OH
(CH2)2CO2H
Ph
120
H
>120
>120
>120
H
H
(CH2)5Me
Ph
a Minimal Inhibitory Concentration of growth (μM).
Scheme 2. Activity against Saccharomyces cerevisiae W303-1B and logP values of naphtho[2,3-a]phenoxazinium chlorides 1a–f and benzo[a]phe-
noxazinium chlorides 4a–z.
5,9-diaminobenzo[a]phenoxazinium moiety, which had
been previously obtained.4,24,28
1c/4b, entries 3/8; 1e/4d, entries 5/10 and 1f/4e, entries
6/11). On the other hand, decreasing the alkyl chain size
in the R3 position either increased or decreased the effi-
ciency of the compounds depending on the functional
group of R3 (for the increase, compare 4c/4i, entries 9/
15 and 4f/4l, entries 12/17; for the decrease, compare
4b/4g, entries 8/13 and 4d/4j, entries 10/16). Alterations
The decrease in the aromatic ring system, by replacing
fused naphthalene with a benzene ring in the phenoxa-
zine skeleton, generally increased the activity of the
compounds (compare compounds 1a/4a, entries 1/7;

3278
V. H. J. Frade et al. / Bioorg. Med. Chem. 16 (2008) 3274–3282
in R, R1 and R2 also influenced antifungal activity, in
general the combination R = Me, R1 = H and R2 = Et
was associated with the best activity, which decreased
for R = H, R1 = R2 = Et and for R = H, R1 = R2 = Me
(compare, for example, compounds 4a/4n/4r, entries 7/
19/23 or 4c/4o/4u, entries 9/20/26). A phenyl substituent
at R3 (4m, 4x and 4z, entries 18, 28 and 29) completely
abolished antifungal activity and was independent from
the different combinations of R, R1 and R2.
acid was associated with a better activity. Consequently,
we used one of the apolar amino acids, valine, bonded
at its C-terminus or at the amino function to benzo[a]-
phenoxazine through an ester or amide linkage, respec-
tively, and varied the size of the alkyl chain linker
(compounds 5g–5l, entries 7–11). Size increase of the
alkyl chain link as well as its removal decreased the
activity of the compounds (compare compounds 5a/5i,
entries 1/9; 5g/5j/5l, entries 7/10/11). On the other hand,
binding of the amino acid with its carboxylic group,
rather than with its amino group, increased the activity
of the compounds with –NH(CH2)2 (compare 5a/5g/5h,
entries 1/7/8) and decreased its value with –NH(CH2)3
(compare 5i/5j, entries 9/10).
The effect of coupling 5,9-diaminobenzo[a]phenoxazini-
um chlorides to a-amino acids was also tested (Scheme
3). The linkage of the amino group of six different amino
acids to the carboxylic acid of R3 from 4l produced com-
pounds whose MIC varied from 15 to 120 lM. These
can be ordered as follows, according to their activity:
phenylalanine (5c) > alanine (5b) = valine (5a) > glycine
(5d) = lysine (5e) = glutamate (5f). These results seemed
to indicate that the non-polar character of the amino
For the two compounds showing the best antifungal
activities, 4b and 4e (Scheme 2, entries 8 and 11), MIC
values were also determined varying the initial cell
densities in the assays (Table 3). Four different initial
H3C
N
H +
N
H
R
O
5
N
Cl -
H3CH2C
Entry
1
Compound
R
MICa
30
log P
O
C
CH(CH3)2
5a
2.24
(CH2)2
N
H
C
CO2CH3
H
O
C
CH3
2
3
5b
5c
30
15
1.46
2.71
(CH2)2
N
H
C
CO2CH3
H
CH2Ph
C
H
O
C
(CH2)2
N
H
CO2CH3
H
C
H
O
C
4
5
6
7
8
5d
5e
5f
120
120
120
15
1.13
1.31
1.21
5.48
4.72
(CH2)2
N
H
CO2CH3
H
O
(CH2)2 C
N
H
(CH2)4C CO2CH3
NHCOCH3
CH2CH2CO2CH3
C
H
O
C
(CH2)2
(H2C)2
N
H
CO2CH3
O
CH(CH3)2
5g
5h
O
C
C
H
N
H
Boc
O
CH(CH2)3
(H2C)2 N C C N Boc
H H
15
H
O
C
CH(CH3)2
9
5i
5j
5l
60
2.51
5.75
4.94
(CH2)3
(H2C)3
N
H
C
CO2CH3
H
O
CH(CH3)2
10
11
>120
60
O
C
C
H
N
H
Boc
O
CH(CH3)2
N C
H
C
H
N
H
Boc
a Minimal Inhibitory Concentration of growth (μM). Boc = N-tert-butyloxycarbonyl.
Scheme 3. Activity against Saccharomyces cerevisiae W303-1B and logP values of benzo[a]phenoxazinium chlorides 5a–l.

V. H. J. Frade et al. / Bioorg. Med. Chem. 16 (2008) 3274–3282
3279
Table 3. Activity against Saccharomyces cerevisiae W303-1B of
compounds 4b and 4e, measured with different initial cell densities
best results being obtained for the four-ring compounds.
As for substitution of the 5,9-diaminobenzo[a]phenoxa-
zine nucleus, the most efficient compounds were ob-
tained when R = Me, R1 = H and R2 = Et, and
particularly for R3 = (CH2)3Cl, with an MIC of
3.75 lM. The high efficiency of the halogenated com-
pound could be related to its capacity to increase ROS
production and subsequently oxidative damage as has
been reported in previous work where Nile Blue was
used as a lead compound.29 The linkage of different a-
amino acids to N-functionalised substituents at the 5-
position of 5,9-diaminobenzo[a]phenoxazinium moiety
resulted in compounds with diverse antimicrobial effi-
ciencies, which depended on the amino acid, on its link-
age position and on the size of the alkyl linker.
Phenylalanine and valine residues yielded the most ac-
tive compounds (5c, 5g and 5h) with MIC values of
15 lM. A specific correlation between partition coeffi-
cients, expressed as logP, and the antifungal activities
(MIC values) was not found for the compounds under
study.
Cell concentration (cell/mL)
MICa
4b
4e
0.5 · 103
0.5 · 104
0.5 · 105
0.5 · 106
7.5
7.5
7.5
30
3.75
7.5
7.5
15
a Minimal inhibitory concentration of growth (lM).
concentrations with 10-fold increases, in a range from
0.5 · 103 to 0.5 · 106 cells/mL, were used. Although
higher MIC values were found for the higher cell con-
centrations, there was not a direct correlation between
these parameters, and effective drug/cell ratios decreased
with increasing cell concentrations. For both com-
pounds, a 1000-fold increase in cell concentration re-
sulted only in a 4-fold increase in the MIC value.
However, the variation pattern was different for the
two compounds, showing for compound 4e an almost
log correlation. The results suggest that the antifungal
activity of these compounds may be exerted through dif-
ferent mechanisms.
As a whole, the results are compatible with a DNA
binding mechanism, as proposed for other compounds
of this class, since we observed that antimicrobial activ-
ity depended both on the size of the aromatic system and
on the size and character of the functional groups
bounded, suggesting steric interference and group inter-
actions. However, further studies are required in order
to clarify this aspect.
Considering that the octanol–water partition coefficient,
expressed as logP, is one physical parameter often
linked with biological effects, it was predicted for all
compounds under study and correlated with MIC values
(Schemes 2 and 3).30,31 The results showed that there
was not a specific correlation between the lipophilicity
and the antimicrobial activity for the naphtho[2,3-
a]phenoxazine (1a–f) and benzo[a]phenoxazine deriva-
tives tested (4a–z and 5a–l), the same MIC values being
observed for compounds exhibiting a wide variation in
logP. For example, with a MIC value of 15 lM, com-
pounds 1f, 4s, 5c and 5g have logP of 3.92, 0.52, 2.71
and 5.48, respectively.
5. Experimental
5.1. General
All melting points were measured on a Gallenkamp
melting point apparatus and are uncorrected. TLC anal-
yses were carried out on precoated silica plates, 0.25 mm
thick (Merck Fertigplatten Kieselgel 60F254) and spots
were visualised under UV light. Dry chromatography
on silica gel was carried out on Merck Kieselgel (230–
240 mesh). IR spectra were determined on a Perkin-El-
mer FTIR-1600 using KBr discs, Nujol or neat samples.
UV/visible spectra were run on a Hitachi U-2000 spec-
4. Conclusion
In this work, several naphtho[2,3-a]phenoxazinium
chlorides containing different N-substituents were syn-
thesised in low to excellent yields. These compounds
with absorption in the range of 601–647 nm were highly
fluorescent, revealing maximum emission wavelengths
from 630 to 676 nm in ethanol and water at physiologi-
cal pH. Their photophysical properties, as well as the
cationic structure and the presence of functional groups
(except in compound 1b; the ester group of 1c could be
hydrolysed to the corresponding carboxylic acid),
strongly suggested that these polycyclic heterocycles
are promising fluorescent probes for biological applica-
tions, either by non-covalent or covalent bonds.
1
trophotometer. H NMR spectra were recorded on a
Varian 300 spectrometer in CDCl3 or CD3OD solution
at 300 MHz at 25 ꢁC. All chemical shifts are given in
ppm using dHMe4Si = 0 ppm as reference and J values
are given in Hz. 13C NMR spectra were run on the same
instrument at 75.4 MHz using the solvent peak as the
internal reference. Assignments were made by compari-
son of chemical shifts, peak multiplicities and J values
and were supported by spin decoupling-double reso-
nance and bidimensional heteronuclear correlation
HMBC and HMQC techniques. Mass spectrometry
analyses were performed at the ‘C.A.C.T.I.—Unidad
de Espectrometria de Masas’ of the University of Vigo,
Spain, on a Hewlett Packard 5989 A spectrometer for
low resolution spectra and an Autospec M spectrometer
for high resolution mass spectra. Fluorescence spectra
Growth inhibition assays showed that the naphtho[2,3-
a]phenoxazinium and 5,9-diaminobenzo[a]phenoxazini-
um chlorides studied in this work present significant
antifungal activity, emerging as good candidates for fur-
ther studies. Regarding SAR analysis, we concluded that
the extent of the aromatic system influenced activity, the
were collected using
Spectrometer.
a
Spex Fluorolog 1680

3280
V. H. J. Frade et al. / Bioorg. Med. Chem. 16 (2008) 3274–3282
5.2. General method for the synthesis of compounds
(1a–e)
160.03 (Ar-C). IR (Nujol, cmÀ1): mmax 3420, 2954, 2924,
2854, 1653, 1647, 1636, 1558, 1457, 1541, 1521, 1507,
1489, 1376, 1289, 1124, 1016. HRMS (FAB) Calcd for
C26H26N3O [M+]: 396.2076. Found: 396.2076.
To a cold solution (ice bath) of 5-ethylamino-4-methyl-
2-nitrosophenol hydrochloride 2 in ethanol (2 mL),
compounds 3a–e and concentrated hydrochloric acid
(5.0 · 10À2 mL) were added. The mixture was refluxed
during the time given below and monitored by TLC
(chloroform or dichloromethane/methanol). After evap-
oration of the solvent and dry chromatography on silica
gel or successive washes with a solvent (1e), compounds
1a–e were obtained as blue solids.
5.2.3.
N-[7-(4-Ethoxy-4-oxobutylamino)-2-methyl-3H-
naphtho[2,3-a]phenoxazin-3-ylidene] ethanaminium chlo-
ride (1c). The product of the reaction of 2 (0.279 g,
1.55 · 10À3 mol) with 3c (0.436 g, 1.55 · 10À3 mol) (re-
flux time, 16 h) was purified by chromatography
(CHCl3/MeOH, 5.6:0.4) to give compound 1c (0.118 g,
16%). Mp above 300 ꢁC. TLC Rf 0.46 (CHCl3/MeOH,
6:1). 1H NMR (CD3OD, 300 MHz): d 1.20–1.40 (m,
6H, NHCH2CH3 and OCH2CH3), 2.10 (broad s, 5H,
NHCH2CH2CH2 and CH3), 2.56 (t, J = 6.6 Hz, 2H,
NHCH2CH2CH2), 3.20–3.40 (m, 2H, NHCH2CH3),
3.35–3.45 (m, 2H, NHCH2CH2CH2), 4.19 and 4.23 (q,
J = 6.9 Hz, 2H, OCH2CH3), 6.02 (s, 1H, 4-H), 6.30 (s,
1H, 6-H), 6.92 (s, 1H, 1-H), 7.40–7.60 (m, 2H, 6-H
naphthyl and 7-H naphthyl), 7.72 (d, J = 7.2 Hz, 2H,
5-H naphthyl and 8-H naphthyl), 8.31 (s, 1H, 1-H naph-
thyl), 8.46 (s, 1H, 4-H naphthyl). 13C NMR (CD3OD,
75.4 MHz) d 14.15 (NHCH2CH3), 14.67 (OCH2CH3),
17.83 (CH3), 24.71 (NHCH2CH2CH2), 31.91 (NHCH2
CH2CH2), 39.57 (NHCH2CH3), 44.86 (NHCH2CH2
CH2), 61.93 (OCH2CH3), 93.67 (6-C), 94.12 (4-C),
121.36 (Ar-C), 124.69 (4-C naphthyl), 124.88 (1-C naph-
thyl), 127.26 (Ar-C), 127.57 (Ar-C), 128.90 (7-C naph-
thyl or 6-C naphthyl), 129.53 (Ar-C), 130.03 (5-C
naphthyl and 8-C naphthyl), 130.14 (6-C naphthyl or
7-C naphthyl), 131.66 (1-C), 133.32 (Ar-C), 134.19
(Ar-C), 134.51 (Ar-C), 147.55 (Ar-C), 153.35 (3-C),
155.06 (Ar-C), 158.75 (Ar-C), 174.77 (CO). IR (KBr
1%, cmÀ1): mmax 3422, 2956, 2925, 2854, 1724, 1638,
1585, 1549, 1512, 1467, 1450, 1376, 1354, 1297, 1262,
1210, 1165, 1141, 1085, 1016. HRMS (FAB) Calcd for
C29H30N3O3 [M+]: 468.2287. Found: 468.2292.
5.2.1. N-(7-Amino-2-methyl-3H-naphtho[2,3-a]phenoxa-
zin-3-ylidene)ethanaminium chloride (1a). The product
of the reaction of 2 (0.209 g, 1.16 · 10À3 mol) with 3a
(0.165 g, 8.54 · 10À4 mol) (reflux time, 1 h) was purified
by chromatography (CH2Cl2/MeOH, 5.6:0.4) to give
compound 1a (0.302 g, 98%). Mp above 300 ꢁC. TLC
Rf 0.62 (CHCl3/MeOH, 6:1). 1H NMR (CD3OD,
300 MHz): d 1.20 (broad s, 3H, NHCH2CH3), 2.0
(broad s, 3H, CH3), 3.20 (broad s, 2H, NHCH2CH3),
6.30 (broad s, 1H, 4-H), 7.0 (broad s, 1H, 6-H), 7.53
(broad s, 3H, 1-H, 6-H naphthyl and 7-H naphthyl),
7.75 (broad s, 3H, 5-H naphthyl, 8-H naphthyl and 1-
H naphthyl), 8.57 (broad s, 1H, 4-H naphthyl). 13C
NMR (CD3OD, 75.4 MHz): d 14.20 (NHCH2CH3),
17.73 (CH3), 39.50 (NHCH2CH3), 94.22 (4-C), 97.91
(6-C), 122.81 (Ar-C), 124.68 (Ar-C), 125.85 (Ar-C),
126.92 (4-C naphthyl), 128.07 (1-C naphthyl), 128.92
(7-C or 6-C naphthyl), 129.77 (5-C and 8-C naphthyl),
131.84 (6-C or 7-C naphthyl), 131.93 (1-C), 134.86
(Ar-C), 136.45 (Ar-C), 138.74 (Ar-C), 140.14 (Ar-C),
147.54 (Ar-C), 153.61 (3-C), 154.78 (7-C), 163.0 (Ar-
C). IR (KBr 1%, cmÀ1): mmax 3446, 2923, 2853, 1653,
1636, 1558, 1541, 1522, 1507, 1489, 1473, 1457, 1398,
1301, 1261, 1154, 1014. HRMS (FAB) Calcd for
C23H20N3O [M+]: 354.1606. Found: 354.1605.
5.2.4. N-[7-(3-Hydroxypropylamino)-2-methyl-3H-naph-
tho[2,3-a]phenoxazin-3-ylidene]ethanaminium
5.2.2. N-(2-Methyl-7-propylamino-3H-naphtho[2,3-a]phen-
oxazin-3-ylidene)ethanaminium chloride (1b). The product
of the reaction of 2 (0.008 g, 4.26 10À5 mol) with 3b
(0.010 g, 4.26 · 10À5 mol) (reflux time, 5 h) was purified
by chromatography (CHCl3/MeOH, 5.5:0.5) to give com-
pound 1b (0.072 g, 43%). Mp above 300 ꢁC. TLC Rf 0.30
chloride
(1d). The product of the reaction of 2 (0.082 g,
4.54 · 10À4 mol) with 3d (0.114 g, 4.54 · 10À4 mol) (re-
flux time, 4 h and 30 min), was purified by chromatogra-
phy (CH2Cl2/MeOH, 5.5:0.5) to give compound 1d (0.114
g, 61%). Mp above 300 ꢁC. TLC Rf 0.68 (CH2Cl2/MeOH,
6:1). 1H NMR (CD3OD, 300 MHz): d 1.20–1.40 (m, 3H,
NHCH2CH3), 2.10–2.20 (m, 2H, NHCH2CH2CH2), 2.31
(broad s, 2H, NHCH2CH2CH2), 2.36 (s, 3H, CH3), 3.50–
3.60 (m, 2H, NHCH2CH3), 3.85 (broad s, 2H,
NHCH2CH2CH2), 6.71 (broad s, 1H, 4-H), 6.91 (broad
s, 1H, 6-H), 7.63 (s, 1H, 1-H), 7.73 (broad s, 2H, 6-H
naphthyl and 7-H naphthyl), 8.16 (broad s, 2H, 5-H
naphthyl and 8-H naphthyl), 8.80 (broad s, 1H, 1-H
naphthyl), 9.28 (broad s, 1H, 4-H naphthyl). 13C NMR
(CD3OD, 75.4 MHz): d 14.66 (NHCH2CH3), 18.33
(CH3), 30.21 (NHCH2CH2CH2), 32.75 (NHCH2CH2
CH2), 40.06 (NHCH2CH3), 43.95 (NHCH2CH2CH2),
94.27 (4-C), 94.67 (6-C), 122.00 (Ar-C), 125.28 (Ar-C),
125.45 (Ar-C), 127.83 (4-C naphthyl), 127.94 (1-C naph-
thyl), 129.45 (7-C naphthyl or 6-C naphthyl), 130.08 (5-C
naphthyl and 8-C naphthyl), 130.61 (6-C naphthyl or 7-C
naphthyl), 132.19 (1-C), 133.87 (Ar-C), 134.95 (Ar-C),
135.06 (Ar-C), 135.49 (Ar-C), 148.08 (Ar-C), 154.0
1
(CHCl3/MeOH, 5.5:0.5). H NMR (CD3OD, 300 MHz):
d 1.17 (t, J = 6.6 Hz, 3H, NHCH2CH3), 1.34–1.46 (m,
2H, NHCH2CH2CH2), 1.94 (broad s, 2H, NHCH2
CH2CH2), 2.32 (s, 3H, CH3), 3.38–3.50 (m, 2H,
NHCH2CH3), 3.62–3.75 (m, 2H, NHCH2CH2CH2),
6.70 (s, 1H, 4-H), 6.85 (s, 1H, 6-H), 7.60 (s, 1H, 1-H),
7.75 (t, J = 7.2 Hz, 2H, 6-H naphthyl and 7-H naphthyl),
8.06–8.20 (m, 2H, 5-H naphthyl and 8-H naphthyl), 8.88
(s, 1H, 1-H naphthyl), 9.27 (s, 1H, 4-H naphthyl). 13C
NMR (CD3OD, 75.4 MHz): d 14.19 (NHCH2CH3),
17.71 (CH3), 30.76 (NHCH2CH2CH2 and NHCH2CH2
CH2), 39.53 (NHCH2CH3), 47.57 (NHCH2CH2CH2),
94.31 (6-C), 94.50 (4-C), 121.0 (Ar-C), 125.10 (4-C naph-
thyl), 125.58 (1-C naphthyl), 127.65 (2· Ar-C), 129.32 (7-
C naphthyl or 6-C naphthyl), 129.87 (5-C naphthyl and
8-C naphthyl), 130.35 (6-C naphthyl or 7-C naphthyl),
130.69 (Ar-C), 132.22 (C-1), 134.05 (2· Ar-C), 135.76
(Ar-C), 148.58 (Ar-C), 154.83 (Ar-C), 155.41 (Ar-C),

V. H. J. Frade et al. / Bioorg. Med. Chem. 16 (2008) 3274–3282
3281
(3-C), 155.50 (2-C), 159.42 (Ar-C). IR (Nujol, cmÀ1): mmax
3406, 2954, 2924, 2854, 1658, 1638, 1549, 1463, 1455,
1377, 1278, 1124, 1016. HRMS (FAB) Calcd for
C26H26N3O2 [M+]: 412.2025. Found: 412.2021.
5-C naphthyl or 8-C naphthyl), 130.85 (Ar-C), 132.07
(1-C), 133.71 (2· Ar-C), 134.93 (Ar-C), 148.27 (Ar-C),
154.10 (3-C), 155.54 (7-C), 159.40 (Ar-C). IR (Nujol,
cmÀ1): mmax 3412, 2954, 2925, 2854, 1638, 1583, 1547,
1463, 1377, 1281, 1208, 1163, 1139, 1014. HRMS
(FAB): Calcd for C26H25N3O35Cl [M+]: 430.1686.
Found: 430.1693. Calcd for C26H25N3O37Cl [M+]:
432.1657. Found: 432.1665.
5.2.5. N-[7-(3-Aminopropylamino)-2-methyl-3H-naphtho
[2,3-a]phenoxazin-3-ylidene]ethanaminium chloride (1e).
The product of the reaction of 2 (0.122 g, 6.80 ·
10À4 mol) with 3e (0.170 g, 6.80 · 10À4 mol) (reflux time,
9 h) was successively washed with chloroform and mix-
tures of increasing polarity of chloroform and methanol
to give compound 1e (0.273 g, 98%). Mp above 300 ꢁC.
1H NMR (CD3OD, 300 MHz): d 1.30–1.50 (m, 3H,
NHCH2CH3), 2.12–2.20 (m, 2H, NHCH2CH2CH2),
2.35 (broad s, 2H, NHCH2CH2CH2), 2.36 (s, 3H,
CH3), 3.56–3.66 (m, 2H, NHCH2CH3), 3.90 (broad s,
2H, NHCH2CH2CH2), 6.76 (broad s, 1H, 4-H), 6.95
(broad s, 1H, 6-H), 7.67 (s, 1H, 1-H), 7.80 (broad s,
2H, 6-H naphthyl and 7-H naphthyl), 8.20 (broad s,
2H, 5-H naphthyl and 8-H naphthyl), 8.86 (broad s,
1H, 1-H naphthyl), 9.38 (broad s, 1H, 4-H naphthyl).
13C NMR (CD3OD, 75.4 MHz): d 14.70 (NHCH2CH3),
18.40 (CH3), 30.31 (NHCH2CH2CH2), 32.85 (NHCH2
CH2CH2), 40.20 (NHCH2CH3), 44.0 (NHCH2CH2CH2),
95.30 (4-C), 96.70 (6-C), 124.00 (Ar-C), 126.38 (Ar-C),
126.50 (Ar-C), 128.50 (4-C naphthyl), 128.60 (1-C naph-
thyl), 130.0 (7-C naphthyl or 6-C naphthyl), 131.08 (5-C
naphthyl and 8-C naphthyl), 132.60 (6-C naphthyl or 7-C
naphthyl), 133.20 (1-C), 134.0 (Ar-C), 135.90 (Ar-C),
136.10 (Ar-C), 137.60 (Ar-C), 149.00 (Ar-C), 155.0 (3-
C), 156.0 (2-C), 160.20 (Ar-C). IR (Nujol, cmÀ1): mmax
3396, 3197, 2927, 1633, 1604, 1581, 1548, 1514, 1465,
1451, 1403, 1289, 1262, 1211, 1165 1143, 1015. HRMS
(FAB) Calcd for C26H27N4O [M+]: 411.2185. Found:
411.2188.
5.3. Synthesis of compounds (3b–e)
5.3.1. N-Propylanthracen-1-amine (3b). To a solution of
1-aminoanthracene (0.300 g, 1.55 · 10À3 mol) in ethanol
(2 mL), 1-bromopropane (0.30 mL, 3.26 · 10À3 mol)
was added and the resulting mixture was refluxed for
14 h and monitored by TLC (CH2Cl2). The solvent
was removed under reduced pressure and purified by
dry chromatography (n-hexane/CHCl3, 8.5:1.5) to pro-
duce compound 3b as a greenish oil (0.077 g, 21%).
1
TLC Rf 0.55 (n-hexane/CHCl3, 4:3). H NMR (CDCl3,
300 MHz): d 1.15 (t, J = 7.5 Hz, 3H, CH3), 1.80–2.0
(m, 2H, NHCH2CH2CH3), 3.34 (t, J = 7.2 Hz, 2H,
NHCH2CH2CH3), 6.58 (d, J = 6.9 Hz, 1H, 4-H), 7.32–
7.50 (m, 4H, 3-H, 2-H, 7-H and 8-H), 7.94–8.60 (m,
2H, 6-H and 9-H), 8.39 (s, 1H, 5-H or 10-H), 8.39 (s,
1H, 10-H or 5-H). 13C NMR (CDCl3, 75.4 MHz): d
11.70 (NHCH2CH2CH3), 21.90 (NHCH2CH2CH2),
47.68 (NHCH2CH2CH2), 109.70 (4-C), 118.95 (2-C
and 5-C), 123.66 (4a-C), 125.25 (8-C), 125.64 (7-C),
125.71 (3-C), 126.74 (10-C), 127.72 (6-C), 128.48 (9-C),
131.07 (5a-C), 131.57 (9a-C), 132.41 (10a-C), 140.65
(1-C). IR (Nujol, cmÀ1): mmax 3425, 2954, 2924, 2854,
1569, 1552, 1462, 1377, 1308, 1154, 1042. HRMS (EI)
Calcd for C17H17N [M+]: 235.1361. Found: 235.1363.
5.3.2. Ethyl 4-(anthracen-1-ylamino)butanoate (3c). The
product of the reaction of 1-aminoanthracene (0.300 g,
1.55 · 10À3 mol) with ethyl-4-bromobutyrate (0.23 mL,
1.63 · 10À3 mol), according to the procedure described
above for the preparation of compound 3b (reflux time
25 h) was used in the following reaction without any
purification (0.476 g, 100%, crude mixture).
5.2.6.
N-[7-(3-Chloropropylamino)-2-methyl-3H-naph-
chloride
tho[2,3-a]phenoxazin-3-ylidene]ethanaminium
(1f). Thionyl chloride (3.0 · 10À2 mL, 4.12 · 10À4 mol)
was added to a solution of compound 1d (0.020 g,
4.85 · 10À5 mol) in dichloromethane/chloroform, 2:1
(3.0 mL) and the reaction mixture was stirred at room
temperature for 74 h. The solvent was removed under
reduced pressure and the crude mixture was purified
by dry chromatography (CHCl3/MeOH, 5.7:0.3). Com-
pound 1f was obtained as a blue solid (0.011 g, 50%).
Mp above 300 ꢁC. TLC Rf 0.32 (CH2Cl2/MeOH,
5.5:0.5). 1H NMR (CD3OD, 300 MHz): d 1.20–1.40
(m, 5H, NHCH2CH3 and NHCH2CH2CH2), 2.23 (s,
3H, CH3), 2.34 (broad s, 2H, NHCH2CH2CH2), 3.30–
3.40 (m, 2H, NHCH2CH3), 3.81–3.90 (m, 2H,
NHCH2CH2CH2), 6.45 (s, 1H, 4-H), 6.59 (s, 1H, 6-H),
7.34 (s, 1H, 1-H), 7.66 (t, J = 9.0 Hz, 2H, 6-H naphthyl
and 7-H naphthyl), 7.90–8.04 (m, 2H, 5-H naphthyl and
8-H naphthyl), 8.63 (s, 1H, 1-H naphthyl), 8.90 (s, 1H,
4-H naphthyl). 13C NMR (CD3OD, 75.4 MHz): d
14.18 (NHCH2CH3), 17.81 (CH3), 30.77 (NHCH2
CH2CH2), 32.49 (NHCH2CH2CH2), 39.63 (NHCH2
CH3), 43.23 (NHCH2CH2CH2), 93.85 (6-C), 94.40 (4-
C), 121.96 (Ar-C), 125.12 (4-C naphthyl and 1-C naph-
thyl), 127.92 (Ar-C), 128.02 (Ar-C), 129.17 (7-C naph-
thyl or 6-C naphthyl), 129.72 (5-C naphthyl or 8-C
naphthyl), 130.28 (6-C naphthyl or 7-C naphthyl and
5.3.3. 3-(Anthracen-1-ylamino)propan-1-ol (3d). The
product of the reaction of 1-aminoanthracene (0.300 g,
1.55 · 10À3 mol) with 3-bromo-1-propanol (0.147 mL,
1.63 · 10À2 mol), according to the procedure described
above for the preparation of compound 3b (reflux time,
20 h) was purified by chromatography (CH2Cl2 and
CH2Cl2/MeOH, 5.9:0.1) to produce compound 3d as a
brown oil (0.205 g, 53%). TLC Rf 0.42 (CH2Cl2/MeOH,
5.8:0.2). 1H NMR (CDCl3, 300 MHz): d 2.14 (t,
J = 5.7 Hz, 2H, NHCH2CH2CH2), 3.54 (t, J = 6.3 Hz,
2H, NHCH2CH2CH2), 3.99 (t, J = 5.7 Hz, 2H,
NHCH2CH2CH2), 6.58 (d, J = 7.5 Hz, 1H, 4-H), 7.30–
7.38 (m, 2H, 3-H and 2-H), 7.40–7.50 (m, 2H, 7-H
and 8-H), 7.94–8.04 (m, 2H, 6-H and 9-H), 8.37 (s,
2H, 5-H and 10-H). 13C NMR (CDCl3, 75.4 MHz) d
31.45 (NHCH2CH2CH2), 42.52 (NHCH2CH2CH2),
62.17 (NHCH2CH2CH2), 102.13 (4-C), 117.59 (2-C),
118.65 (5-C), 123.75 (4a-C), 124.97 (8-C), 125.44 (7-C),
126.17 (3-C), 126.53 (10-C), 127.73 (6-C), 128.40 (9-C),
130.82 (5a-C), 131.51 (9a-C), 132.49 (10a-C), 143.27

3282
V. H. J. Frade et al. / Bioorg. Med. Chem. 16 (2008) 3274–3282
(1-C). IR (neat, cmÀ1): mmax 3434, 2956, 2925, 2854,
1642, 1462, 1378, 1121, 1031. HRMS (EI): Calcd for
C17H17NO [M+]: 251.1310. Found: 251.1318.
4. Frade, V. H. J.; Coutinho, P. J. G.; Moura, J. C. V. P.;
Gonc¸alves, M. S. T. Tetrahedron 2007, 63, 1654–1663.
5. Theodoropulos, S. U.S. Patent 4,873,318, 1989.
6. Salomi, B. S. B.; Mitra, C. K.; Gorton, L. Synth. Met.
2005, 155, 426–429.
7. Simmonds, A.; Miller, J. N.; Moody, C. J.; Swann, E.;
Briggs, M. S. J.; Bruce, I. E. Eur. Patent WO97,29154,
1997.
8. Soto, C. Y.; Andreu, N.; Gibert, I.; Luquin, M. J. Clin.
Microbiol. 2002, 40, 3021–3024.
9. Yan, X.; Miragila, S.; Yuan, P.M. U.S. Patent 6,140,500,
2000.
10. Lahti, R.; Salminen, T.; Latonen, S.; Heikinheimo, P.;
Pohjanoksa, K.; Heinonen, J. Eur. J. Biochem. 1991, 198,
293–297.
11. Nakanishi, J.; Nakajima, T.; Sato, M.; Ozawa, T.; Tohda,
K.; Umezawa, Y. Anal. Chem. 2001, 73, 2920–2928.
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J. Am. Chem. Soc. 2000, 122, 2178–2192.
5.3.4. N1-(Anthracen-1-yl) propane-1,3-diamine (3e). The
product of the reaction of 1-aminoanthracene (0.300 g,
1.55 · 10À3 mol) with 3-bromopropylamine hydrobro-
mide (0.357 g, 1.63 · 10À3 mol), according to the proce-
dure described above for the preparation of compound
3b (reflux time, 26 h) was purified by chromatography
(CH2Cl2/MeOH, 5.5:0.5) to produce compound 3e as a
brown oil (0.191 g, 50%). TLC Rf 0.46 (CH2Cl2/MeOH,
5:1). 1H NMR (CD3OD, 300 MHz): d 2.14–2.28 (m,
2H, NHCH2CH2CH2), 3.20 (t, J = 7.5 Hz, 1H, NHCH2
CH2CH2), 3.53 (t, J = 6.6 Hz, 2H, NHCH2CH2CH2),
6.58 (d, J = 6.6 Hz, 1H, 4-H), 7.30–7.39 (m, 2H, 2-H
and 3-H), 7.40–7.50 (m, 2H, 7-H and 8-H), 7.98 (dd,
J = 9.3 and 3.9 Hz, 1H, 6-H), 8.07 (dd, J = 9.0 and
3.6 Hz, 1H, 9-H), 8.34 (s, 1H, 5-H or 10-H), 8.70 (s,
1H, 10-H or 5-H). 13C NMR (CD3OD, 75.4 MHz): d
31.40 (NHCH2CH2CH2), 39.39 (NHCH2CH2CH2),
69.06 (NHCH2CH2CH2), 118.16 (4-C and 2-C), 120.80
(5-C), 122.77 (4a-C), 125.80 (8-C), 126.50 (7-C), 127.10
(3-C), 127.23 (10-C), 128.64 (6-C), 129.59 (9-C), 130.52
(5a-C), 132.03 (9a-C), 132.98 (10a-C), 144.56 (1-C). IR
(Nujol, cmÀ1): mmax 3398, 2954, 2923, 2854, 1627, 1504,
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Minimum inhibitory concentrations of growth (MIC) for
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microdilution method for the antifungal susceptibility
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cerevisiae W303-1B (MATa, ade2, his3, leu2, trp1, ura3)
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medium, buffered to pH 7.0 with 0.165 M morpholene-
propanesulfonic acid (MOPS) buffer (Sigma) and supple-
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the text. MIC values were determined visually after 48 h
of incubation, as the lowest concentration of drug that re-
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compounds were prepared in DMSO and a final dilution
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ˆ
We thank the Fundac¸a˜o para a Ciencia e Tecnologia (Por-
tugal) for their financial support to the Centro de Quımica
and Centro de Biologia (Universidade do Minho).
´
29. Cincotta, L.; Foley, J. W. U.S. Patent 4,962,197, 1990.
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