Quinoxalin-2-ones: Part 5. Synthesis and antimicrobial evaluation of 3-alkyl-, 3-halomethyl- and 3-carboxyethylquinoxaline-2-ones variously substituted on the benzo-moiety

Article abstract of DOI:10.1016/S0014-827X(03)00198-8

A new series of 3-alkyl-, 3-trifluoromethyl-, 3-carboxyethyl- and 3-bromomethylquinoxaline-2-ones and 2,3-bis(bromomethyl)quinoxalines bearing Cl, CF₃, morpholine on the benzo-moiety, were synthesised and submitted to a preliminary in vitro eva

Full text of DOI:10.1016/S0014-827X(03)00198-8

Il Farmaco 58 (2003) 1251ꢁ1255
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Quinoxalin-2-ones
Part 5. Synthesis and antimicrobial evaluation of 3-alkyl-, 3-
halomethyl- and 3-carboxyethylquinoxaline-2-ones variously
substituted on the benzo-moiety
Antonio Carta a,*, Mario Loriga a, Stefania Zanetti b, Leonardo A. Sechi b
a Dipartimento Farmaco Chimico Tossicologico, via Muroni 23/a, 07100 Sassari, Italy
b Dipartimento di Scienze Biomediche, viale S. Pietro, 07100 Sassari, Italy
Received 4 April 2003; accepted 8 July 2003
Dedicated to the memory of Professor Paolo Sanna, died on the 28th March 2002
Abstract
A new series of 3-alkyl-, 3-trifluoromethyl-, 3-carboxyethyl- and 3-bromomethylquinoxaline-2-ones and 2,3-bis(bromo-
methyl)quinoxalines bearing Cl, CF3, morpholine on the benzo-moiety, were synthesised and submitted to a preliminary in vitro
evaluation for antibacterial and anticandida activities. Results of the screening showed that compounds 9b, 14b and 19b (MICꢀ
/
62.5 mg/ml) and 10b (MICꢀ15.6 mg/ml) were the most active against Vibrio alginolyticus.
/
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 2-Quinoxalinones; Quinoxalines; Antimicrobial activity
1. Introduction
showed to maintain this activity against C. albicans
and C. parapsilosis spp. [6], while a CF3 group at C-3
and Cl atoms in the benzo-moiety exhibited activity
against S. aureus [7]. Among the pyrido[2,3-g]quino-
xalin-2-one series, we have observed that compounds
bearing substituents such as a alkyl, CF3 or CH2Br
group at C-3 or C-2 and Cl atom at C-5 exhibited both
antibacterial and anticandida activities [8].
These results prompted us to continue our investiga-
tion on quinoxalin-2-ones in order to achieve additional
data for a structure-activity relationship study. In this
context, in order to evaluate if the concomitant presence
of two favourable substituents in the benzo-moiety
might improve antimicrobial activity, we have prepared
a new series of quinoxalin-2-ones bearing either a
chlorine atom or a CF3 group in the benzo-moiety, or
alternatively a chlorine atom and a morpholine ring in
5,6 (or 7,8) positions of quinoxaline nucleus.
Interest in the medicinal properties of quinoxalin-2-
ones has stimulated our research in this field [1ꢁ
Recently we reported the synthesis and biological
activities of over 120 quinoxalin-2-ones [4ꢁ7] and 12
/3].
/
pyrido[2,3-g]quinoxalin-2-ones [8]. Among the quinox-
alin-2-one series, we have observed that compounds
bearing a carboxyethyl at C-3 and a CF3 group or a
morpholine ring in the benzo-moiety were moderately
active against Escherichia coli and Pseudomonas aerugi-
nosa, respectively [4], while those with an ethyl at C-3
and a CF3 group in the benzo-moiety exhibited activity
against P. aeruginosa [5]. Introduction of a CH2Br
group at C-3 and a CF3, or a NO2 group in the benzo-
moiety exhibited activity against Staphylococcus aureus
and Candida spp., respectively [6]. The CF3 group at
both C-3 and the benzo-moiety of quinoxalinones
Furthermore, we have synthesised the 2,3-bis(bromo-
methyl)quinoxalines bearing the same substituents as
above in the benzo-moiety (Cl, CF3, morpholine), in
* Corrersponding author.
E-mail address: acarta@uniss.it (A. Carta).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00198-8

1252
A. Carta et al. / Il Farmaco 58 (2003) 1251ꢁ1255
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order to verify if this type of biological activity was
maintained or not on the quinoxaline scaffold.
3. Experimental
3.1. Chemistry
2. Chemistry
Melting points were determined by a Kofler hot stage
or Digital Electrothermal apparatus, and are uncor-
rected. 1H NMR spectra were recorded on a Varian XL-
200 (200 MHz) instrument, using TMS as internal
standard. The chemical shift values are reported in parts
per million (d) and coupling constants (J) in Hertz (Hz).
Signal multiplicities are represented by: s (singlet), d
(doublet), t (triplet), q (quadruplet) and m (multiplet).
The starting diamines 4b,c were commercially avai-
lable, while 4a was prepared according to Scheme 1. The
quinoxalinones (6a, 7a, 8aꢁ
13aꢁb, 14aꢁb, 15aꢁb, 16aꢁb and 17a) listed in Table 1,
were prepared following the procedure depicted in
Scheme 2 according to our previous reports [4ꢁ7]. The
reaction of the diamines 4a,b with the suitable a-
ketoesters 5cꢁh was performed in various conditions:
/
b, 9b, 10aꢁ
/
b, 11a, 12aꢁ
/
b,
/
/
/
/
/
Light petroleum refers to the fraction with b.p. 40ꢁ
/
/
60 8C. Elemental analyses were performed by the
Dipartimento di Chimica, Universita` di Sassari (Italy).
in ethanol under reflux (method A) or in 10% sulfuric
acid aqueous solution at 50 8C (method B). In the last
case we could observe that when the 1,2-diamino-3-
chloro-4-morpholinobenzene (4a) was condensed with
the a-ketoesters 5e, 5f and 5h under acidic conditions,
The analytical results for C, H, N, were within 90.4% of
/
the theoretical values.
3.1.1. Intermediates
we were able to obtain prevalently (11aꢀ
/
10a, 13aꢀ12a
/
The diaminobenzene derivatives 4b,c were commer-
cially available, compound 4a is a new derivative and its
preparation is described below.
in 3:1 and 4:1 ratio, respectively) or exclusively (17a) the
7-morpholino derivatives than the 6-morpholino sub-
stituted-ones. These results seem to indicate that proto-
nation of the amino group on the meta position to the
morpholino group takes place thus forcing condensation
of the para amino group with the ketocarbonyl group of
the ester prior to ring closure. This behaviour was in
part reversed in the case of the condensation of 4a with
3.1.1.1. Preparation
of
2-chloro-3-morpholino-6-
nitroaniline (3). A mixture of 2 g (9.6 mmol) of 2,3-
dichloro-6-nitroaniline (1), prepared as described [10],
and 14.9 g (172 mmol) of morpholine was reacted under
reflux for 2 h. On cooling, the reaction mixture was
diluted with 100 ml of water. The precipitate formed was
5g to give 14aꢀ15a in a ratio of 3:2, whereas the steric
/
hindrance of isopropyl group possibly promotes an
inversion of reactivity of the ketocarbonyl group in
comparison with that of the other a-ketoesters.
The different behaviour of reactivity of the examined
diamine (4a) with the a-ketoesters in comparison with
that previously observed in the case of 1,2-diamino-3-
chloro-4-metoxybenzene [7] is probably due to the
concomitant protonation of the morpholine nitrogen
atom which makes less basic the amino group on the
para position with respect to that in meta position.
collected by filtration to give 3 in 90% yield as crystals,
1
m.p. 142ꢁ
8.00 (1H, d, Jꢀ
(1H, d, Jꢀ9.6 Hz, H-4), 3.88 (4H, t, Jꢀ
3?ꢂCH2-5?), 3.19 (4H, t, Jꢀ4.6 Hz, CH2-2?ꢂ
Anal. C10H12ClN3O3 (C, H, N).
/
143 8C (diethyl ether), H NMR (CDCl3): d
/
9.6 Hz, H-5), 6.72 (2H, s, NH2), 6.24
4.6 Hz, CH2-
CH2-6?).
/
/
/
/
/
3.1.1.2. Preparation
diaminobenzene (4a). Compound 3 (1.75 g, 6.8 mmol)
of
3-chloro-4-morpholino-1,2-
Quinoxalines (19aꢁ
procedure reported by Huffman for the known (19c) [9],
by condensation of the diamines 4aꢁc with 1,4-
/
c) were prepared, according to the
in ethanol (100 ml) and in the presence of 10%
palladiumꢁcharcoal (0.2 g) was hydrogenated under 3
/
/
atm until the uptake of the theoretical amount of
hydrogen was reached. After filtration of the catalyst,
the mother liquors were evaporated to dryness to give 4a
dibromo-2,3-butanedione (18) in ethanol (Scheme 3).
Structures of all compounds synthesised have been
supported by analytical and spectroscopical data (Ele-
in 98% yield as a solid, m.p. 147ꢁ
1H NMR (CDCl3): d 6.64 (2H, s, NH2), 6.60 (1H, d,
Jꢀ8.2, H-6), 6.41 (2H, s, NH2), 6.37 (1H, d, Jꢀ8.2, H-
5), 3.85 (4H, t, Jꢀ4.6, CH2-3?ꢂCH2-5?), 2.93 (4H, t,
/
148 8C (diethyl ether),
1
mental analyses and H NMR) and were in agreement
with those previously reported for similar compounds
/
/
[4ꢁ
/
7].
/
/
Scheme 1. Preparation of the intermediate (4a).

A. Carta et al. / Il Farmaco 58 (2003) 1251ꢁ
/
1255
1253
Table 1
Analytical and spectroscopic data of compounds (6ꢁ
/
17 and 19) obtained according to Schemes 2 and 3
Comp.
R
R1 R2 M.p.
(8C)
Yield
(%)
1H NMR (solvent), d (ppm), J (Hz)
6a
mor
mor
mor
H
H
H
H
239ꢁ
/
16 a
25 a
44 a
84 a
13 a
(CDCl3) 10.22 (1H, s, NH), 7.28 (1H, d, Jꢀ
CH2-3?ꢂCH2-5?), 3.10 (4H, t, Jꢀ4.6, CH2-2?ꢂ
(CDCl3) 9.18 (1H, s, NH), 7.62 (1H, d, Jꢀ8.8, H-5), 6.96 (1H, d, Jꢀ
CH2-3?ꢂCH2-5?), 3.09 (4H, t, Jꢀ4.6, CH2-2?ꢂCH2-6?), 2.50 (3H, s, CH3)
(DMSO-d6) 12.69 (1H, s, NH), 7.49 (1H, d, Jꢀ8.4, H-8), 7.25 (1H, d, Jꢀ
CH2Br), 3.76 (4H, t, Jꢀ4.6, CH2-3?ꢂCH2-5?), 2.98 (4H, t, Jꢀ4.6, CH2-2?ꢂ
(CDCl3ꢂDMSO-d6) 13.07 (1H, s, NH), 7.60 (1H, s, H-8), 7.57 (1H, s, H-6), 4.65 (2H, s, CH2Br)
/
9.2, H-8), 7.20 (1H, d, Jꢀ
CH2-6?), 2.69 (3H, s, CH3)
8.8, H-6), 3.84 (4H, t, Jꢀ
/
9.2, H-7), 3.93 (4H, t, Jꢀ
/
4.6,
240a
/
/
/
7a
234ꢁ
/
/
/
/4.6,
235
/
/
/
8a
ꢀ/
300
/
/8.8, H-7), 4.62 (2H, s,
/
/
/
/CH2-6?)
8b
CF3 219ꢁ
/
/
220
9b
H
CF3 167ꢁ
/
(CDCl3) 9.81 (1H, br s, NH), 8.09 (1H, s, H-5), 7.84 (1H, s, H-7), 4.64 (2H, s, CH2Br)
168
10a
10b
11a
12a
12b
13a
mor
H
H
277ꢁ
278
CF3 209ꢁ
211
/
15 a, 14 b (CDCl3ꢂ
t, Jꢀ4.6, CH2-3?ꢂ
10 a
(CDCl3) 12.87 (1H, s, NH), 7.63 (1H, s, H-8), 7.56 (1H, s, H-6)
/
DMSO-d6) 13.13 (1H, s, NH), 7.45 (1H, d, Jꢀ
/
9.0, H-8), 7.35 (1H, d, Jꢀ
/
9.0, H-7), 3.88 (4H,
/
/
CH2-5?), 3.08 (4H, t, Jꢀ4.6, CH2-2?ꢂ
/
/CH2-6?)
/
mor
mor
H
H
243ꢁ
244
/
10 a, 40 b (CDCl3ꢂ
t, Jꢀ4.6, CH2-3?ꢂ
18 a, 7 b (CDCl3) 12.05 (1H, s, NH), 7.21ꢁ
(4H, t, Jꢀ4.6, CH2-2?ꢂCH2-6?), 2.99 (2H, q, Jꢀ
(CDCl3) 12.10 (1H, s, NH), 7.66 (1H, s, H-8), 7.50 (1H, s, H-6), 3.10 (2H, q, Jꢀ
Jꢀ7.4, CH3)
20 a30 b (CDCl3) 9.71 (1H, s, NH), 7.71 (1H, d, Jꢀ
CH2-3?ꢂCH2-5?), 3.16 (4H, t, Jꢀ4.6, CH2-2?ꢂ
CH3)
(CDCl3) 9.76 (1H, s, NH), 8.06 (1H, s, H-5), 7.77 (1H, s, H-7), 3.01 (2H, q, Jꢀ
Jꢀ7.4, CH3)
12 a22 b (DMSO-d6) 12.45 (1H, s, NH), 7.48 (1H, d, Jꢀ
4.6, CH2-3?ꢂCH2-5?), 3.60 (1H, m, CH), 3.05 (4H, t, Jꢀ
CH3)
(CDCl3ꢂ
(6H, d, Jꢀ
60 a, 13 b (DMSO-d6) 11.84 (1H, s, NH), 7.87 (1H, d, Jꢀ
4.6, CH2-3?ꢂCH2-5?), 3.66 (1H, m, CH), 3.25 (4H, t, Jꢀ
CH3)
/
DMSO-d6) 11.75 (1H, s, NH), 7.81 (1H, d, Jꢀ
CH2-5?), 3.25 (4H, t, Jꢀ4.6, CH2-2?ꢂ
719 (2H, m, H-7ꢂH-8), 3.91 (4H, t, Jꢀ
7.4, CH2), 1.38 (3H, t, Jꢀ
/
9.0, H-5), 7.11 (1H, d, Jꢀ
/9.0, H-6), 3.87 (4H,
/
/
/
/CH2-6?)
H
252ꢁ
/
/
/
/
4.6, CH2-3?ꢂ
7.4, CH3)
7.4, CH2), 1.43 (3H, t,
/CH2-5?), 3.07
254
/
/
/
/
CF3 229ꢁ
/
13 a
/
231
/
mor
H
204ꢁ
/
/
8.8, H-5), 7.03 (1H, d, Jꢀ
/
8.8, H-6), 3.90 (4H, t, Jꢀ
7.4, CH2), 1.34 (3H, t, Jꢀ
/
4.6,
7.4,
206
/
/
/
CH2-6?), 2.95 (2H, q, Jꢀ
/
/
13b
14a
H
CF3 173ꢁ
/
23 a
/
7.4, CH2), 1.36 (3H, t,
174
/
mor
H
233ꢁ
/
/
8.8, H-8), 7.29 (1H, d, Jꢀ
/
8.8, H-7), 3.84 (4H, t, Jꢀ
/
234
/
/
4.6, CH2-2?ꢂCH2-6?), 1.31 (6H, d, Jꢀ
/
/
7.0, 2
14b
15a
H
CF3 242ꢁ
/
23 a
/
DMSO-d6) 12.61 (1H, s, NH), 7.62 (1H, s, H-8), 7.52 (1H, s, H-6), 3.53 (1H, m, CH), 1.34
244
/6.8, 2 CH3)
mor
H
204ꢁ
/
/
8.8, H-5), 7.29 (1H, d, Jꢀ
/
8.8, H-6), 3.85 (4H, t, Jꢀ
/
205
/
/
4.6, CH2-2?ꢂCH2-6?), 1.37 (6H, d, Jꢀ
/
/
7.0, 2
15b
16a
H
CF3 160ꢁ
/
29 a
(CDCl3) 9.32 (1H, s, NH), 8.07 (1H, s, H-5), 7.56 (1H, s, H-7), 3.62 (1H, m, CH), 1.33 (6H, d, Jꢀ6.8, 2
/
162
CH3)
34 a, 0 b (CDCl3) 12.85 (1H, s, NH), 7.40 (1H, d, Jꢀ
mor
H
171ꢁ
/
/
9.0, H-8), 7.30 (1H, d, Jꢀ
/
9.0, H-7), 4.55 (2H, q, Jꢀ
/7.2,
172
O-CH2), 3.90 (4H, t, Jꢀ
7.2, CH3)
(CDCl3) 12.73 (1H, s, NH), 7.72 (1H, s, H-8), 7.63 (1H, s, H-6), 4.57 (2H, q, Jꢀ
t, Jꢀ7.2, CH3)
22 a, 58 b (CDCl3) 9.54 (1H, s, NH), 7.85 (1H, d, Jꢀ
CH2), 3.92 (4H, t, Jꢀ4.6, CH2-3?ꢂCH2-5?), 3.25 (4H, t, Jꢀ
CH3)
(CDCl3ꢂ
(2H, s, CH2Br), 3.96 (4H, t, Jꢀ
(CDCl3) 8.31 (1H, s, H-8), 8.06 (1H, s, H-6), 5.00 (2H, s, CH2Br), 4.96 (2H, s, CH2Br)
/
4.6, CH2-3?ꢂ
/
CH2-5?), 3.11 (4H, t, Jꢀ4.6, CH2-2?ꢂ
/
/
CH2-6?), 1.48 (3H, t, Jꢀ
/
16b
17a
H
CF3 168ꢁ
/
23 a
/7.2, O-CH2), 1.48 (3H,
170
/
mor
H
H
180ꢁ
181
/
/9.0, H-5), 7.07 (1H, d, Jꢀ/9.0, H-6), 4.51 (2H, q, Jꢀ
/
7.2, O-
7.2,
/
/
/
4.6, CH2-2?ꢂ
/
CH2-6?), 1.45 (3H, t, Jꢀ
/
19a
19b
Cl mor
Cl
110ꢁ
112
/
73
92
/
DMSO-d6) 7.96 (1H, d, Jꢀ
/9.2, H-8), 7.61 (1H, d, Jꢀ
/
9.2, H-7), 4.97 (2H, s, CH2Br), 4.93
4.6, CH2-2?ꢂCH2-6?)
/4.6, CH2-3?ꢂ
/
CH2-5?), 3.30 (4H, t, Jꢀ
/
/
H
CF3 78ꢁ80
/
Mor, Morpholine.
a
Yield of method A.
Yield of method B.
b
Jꢀ
N).
/
4.6, CH2-2?ꢂ
/
CH2-6?). Anal. C10H14ClN3O (C, H,
the suitable a-ketoesters 5dꢁ
ethanol (20 ml) was stirred and refluxed for 2 h in the
case of 6a, 7a, 8aꢁb, 9b, 10a, 11a, 12aꢁb, 13aꢁb, 14a,
15a, 16aꢁb, 17a, for 48 and 10 h for 10b and for the
/
h, and 4a with 5c, in
/
/
/
3.1.2. General procedure for preparation of
quinoxalinones 6a, 7a, 8aꢁb, 9b, 10aꢁb, 11a, 12aꢁ
13aꢁb, 14aꢁb, 15aꢁb, 16aꢁb and 17a
/
/
/
/
b,
mixture 14b/15b, respectively.
/
/
/
/
After evaporation of the solvent, a residue was
chromatographed on silica gel column, eluting with a
8:2 mixture of diethyl ether/light petroleum, in order to
separate the mixture of isomers when present. In any
3.1.2.1. Method A. A solution of equimolar amounts
(3.0ꢁ4.0 mmol) of the appropriate diamines 4aꢁb and
/
/

1254
A. Carta et al. / Il Farmaco 58 (2003) 1251ꢁ1255
/
Scheme 2. Preparation of quinoxalin-2-ones 6a, 7a, 8aꢁ
/
b, 9b, 10aꢁ
/
b, 11a, 12aꢁ
/
b, 13aꢁ
/
b, 14aꢁ
/
b, 15aꢁ
/
b, 16aꢁb and 17a. Conditions: (i) EtOH, under
/
reflux for 2ꢁ48 h; (ii) H2SO4 10% aqueous solution at room temperature or 50 8C for 1 h.
/
case we observed that the 8-chloro derivatives move
faster in the eluate than the 5 substituted-ones. In Table
1 we report the structures, melting points, yields, and 1H
NMR data of all compounds synthesised.
precipitate formed was purified by chromatography on
silica gel column, eluting with a 7:3 mixture of diethyl
ether/light petroleum. Melting points, yields, and 1H
NMR data are reported in Table 1.
3.1.2.2. Method B. A mixture of equimolar amounts
3.2. Microbiology
(3.5 mmol) of 4a and the suitable a-ketoester (5eꢁ
/
h) in
10% sulfuric acid aqueous solution (20 ml) was stirred at
50 8C for 1 h in the case of 10a, 11a, 12a, 13a, 14a and
15a, or at room temperature for 16a and 17a. On
cooling to room temperature from the reaction mixture
a crude precipitate was formed and collected by filtra-
tion. Purification by chromatography on silica gel
column was carried out, eluting with a mixture of
solvents as reported above.
3.2.1. Antibacterial assay
Antibacterial activity was investigated in vitro on
Gram positive and Gram negative bacteria. The strains
used in these tests were from American Type Culture
Collection (ATCC): S. aureus ATCC 25923, E. coli
ATCC 3853, Klebsiella pneumoniae ATCC 700603, and
P. aeruginosa ATCC 27853, or were environmental
isolate (Vibrio alginolyticus). A logarithmic phase cul-
ture of each bacterial strain was diluted with Luria broth
in order to obtain a density of 106 CFU/ml. The test was
performed in a 96 well microtiter plate in a final volume
of 100 ml. Test compounds were dissolved in dimethyl
sulfoxide at an initial concentration of 1000 mg/ml and
3.1.3. General procedure for preparation of 2,3-
bis(bromomethyl)quinoxalines 19aꢁ
A solution of equimolar amounts (2.5 mmol) of the
appropriate diamine (4aꢁc) and 1,4-dibromo-2,3-buta-
/
c
/
nedione (18) in ethanol (15 ml) was refluxed for 10 min
and then diluted with 100 ml of water. The crude
serially diluted in the plate (500ꢁ
broth. Each well was then inoculated with the standar-
/
7.8 mg/ml) using Luria
Scheme 3. Preparation of quinoxalines (19aꢁc).
/

A. Carta et al. / Il Farmaco 58 (2003) 1251ꢁ
/
1255
1255
dised bacterial suspension and incubated at 37 8C for
18ꢁ24 h. One well containing bacteria without sample
quinoxaline ring [4ꢁ
/
7], the data obtained from the
/
actual screening allow us to make the following ob-
servations. The concomitant presence of a CF3 group
with a chlorine atom on the benzo-moiety associated
with the substituent at C-3 position as CH2Br, CF3, or
C3H7 groups in quinoxalinone derivatives, in general
seem to induce a lower activity against bacteria and
Candida spp. whilst promoting a better activity against
V. alginolyticus. Analogous behaviour was observed in
the series of quinoxalines having identical substituted
ring counterpart and two CH2Br groups in both C-2 and
C-3. The presence of a chlorine atom associated with
morpholine in the benzene moiety makes the quinox-
alinone derivatives completely inactive.
(growth control), and one well containing broth only
(sterility control) were also used. After the incubation,
the growth (or its lack) of the bacteria was determined
visually in both containing compound well sand control
well. The lowest concentration at which there was no
visible growth (turbidity) was taken as the MIC. In
addition 5 ml of suspension from each well were
inoculated in a Muller Hinton agar plate to control
bacterial viability.
3.2.2. Antimycotic assay
Antifungal activity was determined by the tube
dilution method on clinical isolates of Candida spp.
Yeast inocula were obtained by properly diluting
cultures, previously incubated at 35 8C for 48 h in
Sabouraud Dextran agar, to obtain a density of 106
CFU/ml. Test compounds were dissolved in dimethyl
sulfoxide at an initial concentration of 1000 mg/ml and
then were serially diluted in culture medium to 15.6 mg/
ml. Then, 0.5 ml of the above serial dilutions of test
compounds were added, in sterile polystyrene tubes,
with an equal volume of fungal suspension and incu-
bated at 35 8C for 48 h. The MIC determination was
performed in duplicate, and defined as the lowest
concentration of the compound which produced no
visible growth. A sample of compound free growth
control and a set of tubes with sample alone for
monitoring contamination of the medium were used.
Acknowledgements
The authors thank Prof. Giuseppe Paglietti for
fruitful discussion and the review of the manuscript.
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