Synthesis and antifungal activity against strains of Candida albicans of 6-fluoro-4(5 or 7)-chloro-2-(difluorobenzoyl)aminobenzothiazoles

Article abstract of DOI:10.1002/jhet.5570410520

A series of 6-fluoro-4-(5 or 7)-chloro-2-(difluorobenzoyl) aminobenzothiazoles 3a-r were prepared to investigate their potential biological activity. In this work, the results of their in vitro antifungal activity against some strains of Candida albicans

Full text of DOI:10.1002/jhet.5570410520

Sep-Oct 2004
771
Synthesis and Antifungal Activity Against Strains of Candida albicans
of 6-Fluoro-4(5 or 7)-Chloro-2-(Difluorobenzoyl)aminobenzothiazoles
Domenico Armenise, Nicolino De Laurentis, Antonia Reho, Antonio Rosato and
Flaviano Morlacchi*
Dipartimento Farmaco-chimico, Facoltà di Farmacia, Università degli Studi di Bari, Via E. Orabona 4,
70125 Bari (Italy)
Received March 2, 2004
A series of 6-fluoro-4-(5 or 7)-chloro-2-(difluorobenzoyl)aminobenzothiazoles 3a-r were prepared to
investigate their potential biological activity. In this work, the results of their in vitro antifungal activity
against some strains of Candida albicans are reported. It was found that some derivatives displayed antifun-
gal activity higher than that for 3k [1a] compound already described in literature.
J. Heterocyclic Chem., 41, 771 (2004).
Introduction.
2-benzoylaminobenzothiazole compounds 3 (Figure 1),
as a SAR expansion in an attempt to enhance the anti-
fungal activity of the derivative already known 3k , as
well as to investigate their potential biological activity.
The benzothiazole heterocyclic nucleus occurs in a
number of compounds endowed with interesting biolog-
ical activities [1a], including antibacterial and antifungal
[1b], antiparasitic, antiviral and antitubercular.
M o r e o v e r, some N-benzothiazol-2-yl-amides or
thioamides 1 (X = O, S) (Figure 1) are useful for the
treatment of diseases related to the adenosine receptor
[2] and treatment or prevention of diseases and/or disor-
ders associated with the nuclear hormone receptor fami-
lies [3]. In particular, some dihalogenated benzothia-
Results and Discussion.
Chemistry.
As shown in Scheme 1, the desired compounds 3a-r
were obtained following a two step procedure involving at
first the synthesis of the appropriate 2-aminobenzothia-
zoles 5a-c by reaction of 4-fluoro-, 2- or 3–chloroaniline 4
with bromine and potassium thiocyanate [4,5].
Next, all the possible 18 isomeric compounds 3a-r were
achieved by reaction of compounds 5a-c with the suitable
difluorobenzoyl chloride in triethylamine and dry 1,4 diox-
ane. In this Scheme are also specified the structures of the
starting 2-aminobenzothiazoles 5a-c, the fluoro substituted
benzoyl chlorides employed and the structures of the cor-
responding 2-benzoylaminobenzothiazole compounds 3a-
r obtained.
The structures of the new compounds were fully sup-
1
ported by microanalytical and spectrographic (ir, H nmr,
and mass) data. Compound 3k showed spectral data con-
sistent with those reported in literature [1a]. Tables 1 - 4
show the main physical properties and spectral data for
compounds 5a-c and 3a-r.
Figure 1
As for the yields obtained it should be pointed out that
the 5-chloro–substituted compounds 3 were obtained in
yield higher than those of 4-substituted analogues. The
highest yields were observed for compounds 3n and 3b
(i.e., 87.4% and 68.1%, respectively). In some cases (i.e.,
3c, 3f, 3o, 3r) the low yields obtained are due to the for-
mation of co-products that have been removed by column
chromatography and were considered outside the scope of
the present work. In particular, from a synthetic point of
view, the results indicate that low yields were obtained for
compounds where a chloro atom at C-7 position of the
benzothiazole nucleus occurs.
zoles 2 (Figure 1) substituted at the 2-position with a
benzoylamino moiety showed antibacterial, antifungal
and antitubercular activities [1a]. Among these last com-
pounds, 5-chloro-6-fluoro-2',6'-difluoro-2-benzoy-
laminobenzothiazole 3k was found to possess antifungal
activity against Candida albicans, Trichophyton rubrum
and Trichophyton mentagrophytes [1a]. Taking into
account this finding and as part of our in progress
research program aimed at discovering new aza-sulfu-
rated antimicrobial agents [1b], we decided to synthesize
all the regioisomers of monochloro-trifluoro-substituted

772
D. Armenise, N. De Laurentis, A. Reho, A. Rosato and F. Morlacchi
Vol. 41
Scheme 1
4
5a-c
5a
5a = 4-Cl
2-Cl
3-Cl
5b+5c 5b = 5-Cl
5c = 7-Cl
4
5
6
7
2'
3'
F
4'
F
5'
6'
F
3a
3d
3g
3j
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
F
F
F
F
F
3m
3p
F
F
F
3b
3e
3h
3k
3n
3q
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
3c
3f
3i
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
F
F
F
F
F
3l
F
3o
3r
F
F
Microbiology.
The in vitro antifungal assays of the prepared dihalo-
genated benzothiazole 3a-r compounds were carried out
(see Table 5a) against some strains of the fungus Candida
albicans. Furthermore, taking into account that sorbic acid
is widely used in food manufactoring due to its fungistatic
activity, we chosen that compound as reference. The fun-
gal strains were grown in Sabouraud dextrose agar. In this
context, it should be remembered that an interesting
antimicrobial activity against Candida albicans has previ-
ously been claimed for compound 3k [1a], although the
exact strains used were not specified.
Table 1
Yield and Physical Properties of Compounds 5
Compd
Yield (%)
m.p.
Reference
(°C)
5a
5b
75.2
30.0
205-210
229-231
217-220
198-201
189-192
[4]
[5]
[5]
5c
21.7
Table 5a shows the results obtained in our microbiologi-

Sep-Oct 2004
Synthesis and Antifungal Activity Against Strains of Candida albicans
773
Table 3
Table 2
Yield, Elemental Analyses and Physical Properties of Compounds 3a-r
1
H nmr Data of Compounds 5 [a]
Compd Yield (*)
(%)
Anal. Calcd. % for m.p.
H ClF N OS (°C)
i.r.(nujol)
R
f
5a (DMSO-d ) δ 7.25 (d, 1 H, J = 9.0 Hz, H -aromatic), 7.59 (d,
6
7
-1
C
ν ( c m )
14
6
3 2
1 H, J = 7.0 Hz, H -aromatic), 7.8 (s, broad, 2 H, NH ).
5
2
C
H
N
5b (CDCl ) δ 5.18 (s, broad, 2 H, NH ), 7.29 (d, 1 H, J = 8.0 Hz,
3
2
49.06 1.76 8.17
Found
H -aromatic), 7.46 (d, 1 H, J = 6.0 Hz, H -aromatic).
4
7
(DMSO-d ) δ 7.55 (d, 1 H, J
= 6.0 Hz, H ), 8.00 (d, 1 H,
4
6
H-F
J
= 9.0 Hz, H ), 9.75 (s, br, 2 H, NH ) [5].
7 2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
58.0
68.1
5.9
42.6
14.6
3.0
66.2
36.2
22.2
21.7
63.0
29.1
65.2
87.4
9.5
W
W
W
Y
W
W
Y
W
W
W
W
W
Y
W
W
Y
49.19 2.12 8.00 225-228 3412, 1676 0.47
48.94 2.10 7.78 260-262 3403, 1677 0.52
49.43 2.14 7.82 261-264 3403, 1678 0.53
49.25 2.16 8.40 274-277 3415, 1670 0.78
48.70 2.15 8.50 264-267 3401, 1673 0.82
48.71 2.12 8.53 235-242 3423. 1678 0.61
48.68 2.07 8.01 261-265 3414, 1677 0.73
48.69 1.87 7.80 362-365 3401, 1669 0.79
49.28 2.11 7.90 188-190 3412, 1679 0.54
48.93 2.14 7.86 243-247 3412, 1697 0.29
48.69 2.06 8.20 267-271 3400, 1684 0.32
49.43 2.09 7.77 260-266 3200, 1693 0.43
48.67 2.10 8.00 222-225 3239, 1675 0.63
49.29 1.94 7.90 245-250 3448, 1680 0.72
48.80 2.00 8.00 275-278 3200, 1671 0.54
48.70 2.10 8.13 260-262 3416, 1682 0.78
49.10 1.98 8.02 228-230 3190, 1695 0.67
48.76 2.13 7.79 198-208 3190. 1695 0.84
H-F
5c (CDCl ) δ 5.3 (s, broad, 2 H, NH ), 7.12 (t, 1 H, J = 10.0 Hz,
3
2
H -aromatic), 7.34 and 7.38 (dd, 1 H, J = 5.0 Hz, H -aromatic).
4
5
(DMSO-d ) δ 6.93-7.28 (m, 2 H, aromatics), 7.60 (s, br, 2 H, NH ) [5]
6
2
cal assays against four strains of Candida albicans. By
comparing these results with those described for 3k [1a], it
is possible to conclude that 9 out of 17 compounds exhibit
antifungal activities higher than that of 3k, taking into
account that in our hands 3k showed a MIC > 500 µg/ml.
In Table 5b, an overall valuation of the antifungal activ-
ity of the regioisomers 3a-r as a function of the halogen
substitutions is specified. It is apparent from the results in
this Table that the best antifungal activity occurs when the
3',5' positions of the benzoyl moiety are substituted by
difluoro atoms as well as the 4 or 5 position of the ben-
19.1
36.2
6.0
W
W
(*) W = white solide; Y = yellow solide; R : Tlc (hexane-ethyl acetate
7:3 v/v).
f
Table 4
H nmr (CDCl , δ) Data of Compounds 3 [a]
1
3
3a
3b
1.6 (s, br, 1H, NH), 7.25-7.38 (m, 2 H, H + H ), 7.41-7.52 (m, 2 H, H + H ); 7.95 (t, 1 H, H , J = 7.0 Hz )
5' 6' 7 4' 5
1.6 (s, br, 1H, NH), 7.30-7.40 (m, 1 H, H ), 7.48 (q, 1 H, H , J = 8.0 Hz); 7.62 (d, 1 H, H , J = 8.0 Hz); 7.84 (d, 1 H, H , J = 6.0 Hz );
7
5'
6'
4
7.97 (t, 1 H, H , J = 6.0 Hz)
4'
3c
7.2-7.4 (m, 2 H, H + H ); 7.4-7.5 (m, 1 H, H ); 7.6-7.7 (m, 1 H, H ); 7.9-8.1 (m, 1 H, H ); 10.0 (s, br, 1H, NH)
5' 6' 4 5 4'
3d
7.02 (t, 1 H, H , J = 10.0 Hz); 7.11 (t, 1 H, H , J = 10.0 Hz); 7.28 (d, 1 H, H , J = 8.0 Hz); 7.47 (d, 1 H, H , J = 8.0 Hz); 8.28 (q, 1 H, H ,
3'
6'
5'
7
5
J = 6.0 Hz); 10.0 (s, br, 1H, NH)
3e
3f
1.6 (s, br, 1H, NH), 6.98-7.07 (m, 1 H, H ), 7.12 (t, 1 H, H , J=9.0 Hz); 7.61 (d, 1 H, H , J = 10.0 Hz); 7.84 (d, 1 H, H , J = 8.0 Hz);
6' 5' 4 7
8.28 (q, 1 H, H , J = 9.0 Hz );
3'
1.8 (s, br, 1H, NH), 6.95-7.06 (m, 1 H, H ), 7.08-7.18 (m, 1 H, H ); 7.25-7.36 (m, 1 H, H ); 7.60-7.70 (m, 1 H, H ); 8.30 (q, 1 H, H ,
3'
6'
5'
4
5
J = 7.0 Hz );
7.20-7.40 (m, 3 H, H + H + H ); 7.48 (d, 1 H, H , J = 8.0 Hz); 7.87-7.97 (m, 1 H, H ); 10.0 (s, br, 1H, NH)
3g
3h
3i
7
3'
4'
5
6'
7.2-7.4 (m, 2 H, H + H ); 7.63 (d, 1 H, H , J = 8.0 Hz); 7.85 (d, 1 H, H , J = 6.0 Hz); 7.9-8.0 (m, 1 H, H ); 10.0 (s, br, 1H, NH)
3'
4'
4
7
6'
7.20-7.40 (m, 3 H, H + H + H ); 7.6-7.7 (m, 1 H, H ); 7.88-7.98 (m, 1 H, H ); 10.0 (s, br, 1H, NH)
4
3'
4'
5
6'
3j
1.6 (s, br, 1H, NH); 6.92 (t, 2 H, H + H , J=8.0 Hz); 7.19 (d, 1 H, H , J = 8.0 Hz); 7.42 (t, 1 H, H , J = 8.0 Hz); 7.47 (d, 1 H, H ,
3' 5' 7 4' 5
J = 8.0 Hz)
3k
1.7 (s, br, 1H, NH); 7.02 (t, 1 H, H , J=7.5 Hz); 7.43 (d, 1 H, H , J = 7.5 Hz); 7.47-7.55 (m, 2 H, H + H ); 7.62 (d, 1 H, H , J = 7.5 Hz)
4'
4
5'
3'
7
7.00-7.70 (m, 5H, H-aromatics), 12.8 (s, 1H, NH) [1a].
3l
1.7 (s, br, 1H, NH), 7.05 (t, 1 H, H , J = 8.0 Hz ), 7.2-7.3 (m, 3 H, H + H + H ), 7.4-7.6 (m, 1 H, H )
4
5
3'
5'
3m 7.28 (d, 1 H, H , J=8.0 Hz); 7.30-7.40 (m, 1 H, H ); 7.48 (d, 1 H, H , J=7.0 Hz); 7.68-7.78 (m, 1 H, H ); 7.88 (t, 1H, H , J= 8.0 Hz);
4'
7
6'
5
5'
2'
10.0 (s, br, 1H, NH)
3n
1.8 (s, br, 1H, NH); 7.35 (q, 1 H, H , J=8.0 Hz); 7.64 (d, 1 H, H , J= 8.0 Hz); 7.76 (d, 1 H, H , J=6.0 Hz); 7.8-7.9 (m, 1 H, H ); 7.93
6' 4 7 5'
(t, 1H, H , J= 8.0 Hz)
2'
3o
3p
3q
3r
1.8 (s, br, 1H, NH); 7.3-7.4 (m, 2 H, H + H ); 7.6-7.7 (m, 1 H, H ); 7.8-7.9 (m, 1 H, H ); 7.9-8.1 (m, 1 H, H )
6' 4 5 5' 2'
1.6 (s, br, 1H, NH); 7.10 (t, 1 H, H , J=7.5 Hz); 7.30 (d, 1 H, H , J= 9.0 Hz); 7.48 (d, 1 H, H , J=9.0 Hz); 7.5-7.6 (m, 2 H, H + H )
6'
4'
7
5
2'
1.7 (s, br, 1H, NH); 7.08-7.15 (m, 1 H, H ); 7.60 (d, 2 H, H + H , J= 4.0 Hz); 7.62 (d, 1 H, H , J=8.0 Hz); 7.78 (d, 1H, H , J= 6.0 Hz)
7
4'
2'
6'
4
1.7 (s, br, 1H, NH); 7.05-7.15 (m, 1 H, H ); 7.20-7.40 (m, 1 H, H ); 7.50-7.70 (m, 3 H, H + H + H )
4'
4
5
6'
2'
[a] All NH signals disappear with D O.
2

774
D. Armenise, N. De Laurentis, A. Reho, A. Rosato and F. Morlacchi
Vol. 41
Table 5b
zothiazole nucleus is substituted by a chloro atom (3p, 3q).
A lower antimicrobial activity (comparable however to
that of the reference, sorbic acid) was observed when the
2',5' positions of the benzoyl moiety are substituted by
difluoro atoms and the 5 position of the benzothiazole
nucleus is substituted by a chloro atom (3h).
Overall valuation of the antifungal activity of regioisomers 3a-r as a
function of the substituent [halogen(s)] position [a]
3 (a-r)
In conclusion, as can be seen from Tables 5a and 5b,
compounds 3 show antifungal activity comparable (3h) or
in some cases better (3p, 3q ) than that of the reference
compound sorbic acid.
R
R: 4-Cl
R: 5-Cl
R: 7-Cl
1
Further studies are currently in progress to elucidate the
biological activities of compounds 3.
Compound Activity Compound Activity Compound Activity
2',3'-F
2',4'-F
2',5'-F
2',6'-F
3',4'-F
3',5'-F
3a
3d
3g
3j
3m
3p
*
*
3b
3e
3h
3k
3n
3q
3c
3f
3i
3l
3o
3r
*
*
Table 5a
*
**
Antifungal Activities of Compounds 3a-r against Candida albicans
(MIC, µg/ml)
*
***
***
**
Compounds C. albicans
C. albicans
C. albicans
C. albicans
Sorbic Acid **
**
ATCC 10231 ATCC 14053 NRRL Y12983 NRRL Y896
[a] For sake of clarity about the structure-antifungal activity relationship,
in this Table the regioisomers 3a-r are listed as a function of the sub-
stituent [halogen(s)] position. R indicates the position of chloro-atom on
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
500
-----
500
-----
500
-----
500
250
-----
-----
-----
-----
500
-----
-----
250
250
-----
250
500
-----
500
-----
500
-----
500
500
500
-----
-----
-----
500
-----
-----
250
250
-----
500
500
-----
250
-----
500
-----
500
250
-----
-----
-----
-----
500
-----
-----
250
250
-----
250
500
-----
500
-----
500
-----
500
500
500
-----
-----
-----
500
-----
-----
500
500
-----
500
the benzothiazole nucleus, R . indicates the position of the fluoro-atoms
1
on the benzoyl nucleus. The MIC values reported in Table 5a for each of
the 4 strains of Candida albicans , in this Table are indicated as follows: *
for MIC values < sorbic acid standard, ** for MIC values = standard, ***
for MIC values > standard.
70°)-ethyl acetate (7:3 v/v) or ethyl acetate-hexane (7:3 v/v) as
eluent, in each case specified, and tlc was carried out by using
hexane-ethyl acetate (7:3 v/v) as eluent.
General Procedure for the Preparation of Compounds 5 [5].
3q
3r
A mixture of the 2- or 3-chloro-4-fluoroaniline (30.0 g, 206
mmol) and potassium thiocyanate (40.0 g, 412 mmol) in glacial
acetic acid (500 ml) was stirred for 5 min. Bromine (48.0 g, 300
mmol) in glacial acetic acid (500 ml) was added dropwise to this
mixture, the temperature being kept below 30-35 °C throughout
the addition. Stirring was continued for an additional 1 hour after
the bromine addition. After cooling, the residue was removed by
filtration. The filtered solution was made alkaline with 28%
ammonium hydroxide and the solid precipitate was collected
washed with water (3 x 50 ml). The combined water layers were
made alkaline with 28% ammonium hydroxide and the resulting
precipitate was combined with that previously collected.
Sorbic Acid
--- = MIC > 500 µg/ml.
EXPERIMENTAL
Chemistry.
Melting points were determined in open capillary tubes with a
Büchi apparatus and are uncorrected. Ir spectra were obtained on
a Perkin-Elmer 283 spectrophotometer (nujol mull or potassium
In the following cases, alternative procedures were also used.
Thus, for 5a , the combined precipitates were dissolved in
toluene, and the mixture was distilled in a Dean Stark apparatus
using toluene/water azeotropic phase. The toluene layer was sep-
ared, dried (sodium sulfate) and evaporated in vacuo.
For 5b and 5c, the combined precipitates were extracted with
ethyl acetate (3 x 200 ml). The organic layers were separated,
dried (sodium sulfate) and evaporated in vacuo. The residue on
1
bromide pellets, in each case specified). H nmr spectra were
recorded on Varian-Mercury instrument operating at 300 MHz.
Chemical shifts are given in δ values (ppm) and the coupling
constants are express in J values (Hz) downfield from tetram-
ethylsilane as internal standard. Mass spectra were recorded on a
Hewlett-Packard 5995c GC-MS low resolution spectrometer. All
1
compounds showed appropriate ir, H nmr and mass spectra.
Elemental analyses were carried out with Eurovector EuroEA
3000 analyzer and the results were within ± 0.40% of the theoret-
ical values. Column chromatography on silica gel 60 (Merck 70-
230 mesh) was carried out by using light petroleum ether (bp 40-
tlc showed R 0.73 and 0.65. Column chromatography (eluent
f
hexane-ethyl acetate 3:7) of the residue gave compounds 5b as a
pink solid, R 0.73 (Yield 30.0%) and compound 5c as a white
f
solid, R 0.65 (Yield 21.7%).
f

Sep-Oct 2004
Synthesis and Antifungal Activity Against Strains of Candida albicans
775
According to one of the two procedures, starting from 4-fluo-
roaniline (2 or 3 respectively)-Cl-substituted, as reported in
Scheme 1 the three isomers 5a-c were prepared, showing M.W.
µg/ml. A series of twofold dilutions of each agent were prepared
in yeast malt broth in a 96 wells microdilution. Each well was
3
inoculated with a final concentration of approximately 1 x 10 to
3
3x10 cfu/ml as confirmed by viable counts. Microdilution trays
202, Anal. Calcd. for C H ClFN S: C, 41.58; H, 1.98; N, 13.86.
2
7
4
+,
Each of the isomers showed m/z 202 (M base), 175, 140. The
yields and the chemical and physical properties are reported in
Tables 1 and 3.
were incubated for 48 h at 30 °C and than the growth was moni-
tored visually. The minimum inhibitory concentration was
defined as the lowest concentration required to arrest the growth
of the fungi at the end of 48 h of incubation at 30 °C. Each isolate
was tested at least twice in duplicate [8,9]. The results are
reported in Tables 5a and b.
General Procedure for the Preparation of Compounds 3.
A mixture of 5 (0.406 g, 2.00 mmol) and triethylamine (0.202
g, d = 0.726, 2.00 mmol) in dry 1,4-dioxane (20 ml) was stirred
for 30 min at 50-60 °C. A solution of appropriate difluorine-ben-
zoyl chloride (0.354 g, 2.00 mmol) in dry 1,4-dioxane (20 ml)
was added dropwise, at the same temperature. The mixture was
stirred for 2 hours at 50-60 °C and then poured into crushed ice.
The resulting solid, so separated, was collected by filtration and
washed with 1% potassium bicarbonate and water.
In the following cases, alternative procedures were also used.
For 3 a, d, h, i, n, q, r, the crude precipitate was treated three
times with boiling ethyl alcohol 95% and filtered; the combined
filtrates, after cooling, gave crystals which were separated by
filtration.
Acknowledgements.
This work was supported by a grant of Ministero
dell'Istruzione, dell'Università e della Ricerca (M.I.U.R.) -
Rome.
REFERENCES AND NOTES
*
Correspondence to: Prof. Flaviano Morlacchi - Dipartimento
Farmaco-chimico, Facoltà di Farmacia, Università degli Studi di Bari,
Via E. Orabona 4, 70125 Bari, Italy.
[1a] Anup R. Barde, H. Barsu, Khadse and Anil S. Bobade,
Indian Drugs, 35, 554, (1998); [b] G. Trapani, A. Latrofa, M. Franco, D.
Armenise, F. Morlacchi, G. Liso, Arzneim. –Forsch./Drug Res., 44 (II),
8, 969, (1994).
For 3b, c, e, f, g, j, k, l, m, o, p, the crude precipitate was puri-
fied by column chromatography, eluent light petroleum ether (bp
40-70°)-ethyl acetate (7:3, v/v).
All the prepared 18 isomeric compounds 3(a-r) show appro-
+
priate mass spectra. Each of the isomers showed m/z 342 (M ),
141 (base), 113. The yields, the elemental analyses and the chem-
ical and physical properties are reported in Tables 2 and 4.
[2] A. Alanine, A. Flohr, A. K. Miller, R. D. Norcross and C.
R i e m e r. (F. Hoffman – La Roche A. – G. Switz.) PCT Int. Appl.,
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(1998).
Microbiology - Determination of Fungistatic Activity.
[4] C. Zagar, E. Heistracher, R. Reinhard, G. Hamprecht, M.
Menges, O. Menke, P. Schafer and K. O. Westphalen, PCT Int. Appl.,
WO9833796, 40 pp, (1998); Chem. Abstr., 129, 148990 (1998).
[5] V. Cecchetti, A. Fravolini, R. Fringuelli, G. Mascellani, P.
Pagella, M. Palmioli, G. Segre and P. Terni, J. Med. Chem., 30, 3, 465,
(1987).
[6] American Type Colture Collection.
[7] United States Department of Agriculture.
[8] Manual of Clinical Microbiology. Murray et al. 7th ed.,
ASM (USA) ed. (1999).
Standard strains of Candida albicans ATCC 10231, Candida
albicans ATCC [6] 14053, Candida albicans NRRL y 869 and
Candida albicans NRRL 12983 [7] was grown and maintained
on sabouraud agar slants. The fungal strains were grown in
Sabouraud dextrose agar. Five colonies were picked and suspen-
6
sions were adjusted to approximately 1x10 cfu/ml. They were
diluted appropriately in yeast malt broth medium (National
Committee Clinical Laboratory Standard USA -NCCLS 1997,
3
m27-a) to give an inoculum of 1x10 cfu/ml [8,9]. Stock solu-
tions of the compounds were adjusted in DMSO [8]. Final con-
centrations ranges were for each compound from 500 to 62.5
[9] NCCLS M27-A Reference Method for Broth Dilution
Antifungal Susceptibility Testing of Yeasts; Approved Standard.