Synthesis, anti-mycobacterial, anti-trichomonas and anti-candida in vitro activities of 2-substituted-6,7-difluoro-3-methylquinoxaline 1,4-dioxides

Article abstract of DOI:10.1016/j.ejmech.2003.11.008

A new series of 23 6,7-difluoro-3-methyl-2-phenylthio/phenylsulfonyl/ phenylsulfinyl/benzylamino/phenylamino-quinoxaline 1,4-dioxides variously substituted in the phenyl moiety, was synthesized and submitted to in vitro evaluation for anti-mycobacterial,

Full text of DOI:10.1016/j.ejmech.2003.11.008

European Journal of Medicinal Chemistry 39 (2004) 195–203
www.elsevier.com/locate/ejmech
Short communication
Synthesis, anti-mycobacterial, anti-trichomonas and anti-candida in vitro
activities of 2-substituted-6,7-difluoro-3-methylquinoxaline 1,4-dioxides
Antonio Carta a,*, Mario Loriga a, Giuseppe Paglietti a, Antonella Mattana b, Pier Luigi Fiori c,
Paola Mollicotti c, Leonardo Sechi c, Stefania Zanetti c
a Dipartimento Farmaco Chimico Tossicologico, via Muroni 23/a, 07100 Sassari, Italy
b Dipartimento di Scienze del Farmaco, via Muroni 23/a, 07100 Sassari, Italy
c Dipartimento di Scienze Biomediche, viale S. Pietro, 07100 Sassari, Italy
Received 17 September 2003; received in revised form 17 November 2003; accepted 19 November 2003
Abstract
A new series of 23 6,7-difluoro-3-methyl-2-phenylthio/phenylsulfonyl/phenylsulfinyl/benzylamino/phenylamino-quinoxaline 1,4-
dioxides variously substituted in the phenyl moiety, was synthesized and submitted to in vitro evaluation for anti-mycobacterial, anti-
trichomonas, anti-candida, anti-mycoplasma and antibacterial activities. In anti-mycobacterial assays, several compounds resulted active
(MIC90 = 2.0–4.0 µg/ml) against Mycobacterium tuberculosis H37Rv. Anti-trichomonas screening showed a generally good activity of all
compounds (MBC = 0.39–25.0 µg/ml) versus Trichomonas vaginalis, in particular the derivatives 5a,d, 7a, 9 and 11c ranged 0.39–0.78 µg/ml
(metronidazole MBC = 12.5 µg/ml). Results of anti-candida assays showed that derivatives 7a, 8a,d and 9 were active against several species
of Candida (C. albicans, C. krusei, C. parapsilosis and C. glabrata), having MIC50 between 3.9 and 31.25 µg/ml. The latter compounds were
also submitted to anti-mycoplasma assay against Mycoplasma hominis, the results obtained showed that 7a, 8a,d and 9 inhibited the growth
of the mycoplasma at the concentration of 0.1 mg/ml. In antibacterial tests only a few compounds showed an MIC50 lower than 62.5 µg/ml
against representative strains of Gram-positive and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Staphylococcus
aureus, Vibrio alginolyticus and Pseudomonas aeruginosa).
© 2003 Elsevier SAS. All rights reserved.
Keywords: Quinoxaline 1,4-dioxides; Anti-mycobacterial; Anti-trichomonas; Anti-candida activity; SAR
1. Introduction
good anti-mycobacterial activity (MIC90 between 0.39 and
0.78 µg/ml) [11] within the TAACF screening program [12],
while a general good anti-candida activity (Candida albi-
cans, Candida glabrata, Candida krusei and Candida
parapsilosis) was recorded for all terms of the series [11].
Analogous anti-mycobacterial activities were obtained re-
cently by Zarranz et al. [13] for a new series of
3-methylquinoxaline 1,4-dioxides.
Our encouraging results as well as the reports on the
literature prompted us to extend the previous series of 6,7-
difluoroquinoxaline 1,4-dioxides in order to study structure–
activity relationships. Thus, we have designed the com-
pounds of Schemes 1 and 2 where at position 3 of the
heterocycle is always present a methyl group for synthetic
reasons, while at position 2 we placed variously mono-, di- or
tri-substituted phenyl ring linked by thio, sulfonyl or sulfinyl
bridge. In addition we have examined the substitution with
anilino or benzylamino group.
In the last two decades, an increasing interest on the
biological properties of the quinoxaline 1,4-dioxides has
been reported. Antibacterial activity [1–5], animal growth
promoting in feed additives [6–8], hypoxia-selective activity
[9], genotoxicity against Escherichia coli and S. typhimu-
rium [10] are well documented. In this context we recently
reported the anti-mycobacterial and anti-candida activities of
the compounds of Fig. 1 which confirm the potentiality of
this nucleus.
In particular, we could observe that the 3-methyl-2-
phenylthioquinoxaline 1,4-dioxides bearing alternatively a
chlorine atom or a trifluoromethyl group at C-6 or C-7 as well
as two fluorine atoms in C-6 and C-7 were endowed with
* Corresponding author.
E-mail address: acarta@ssmain.uniss.it (A. Carta).
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.11.008

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A. Carta et al. / European Journal of Medicinal Chemistry 39 (2004) 195–203
Fig. 1. Quinoxaline 1,4-dioxides previously described.
2. Chemistry
line 1,4-dioxide (7a). For the preparation of the compounds
11a–g and 13a–g, we used the well-experimented Scheme 2
first converting the sulfide 8a into the 2-chloro-3-methyl-6,7-
difluoroquinoxaline 1,4-dioxide (9) [11] by treatment with
concentrated hydrochloric acid solution. Compound 9 under-
went nucleophilic displacement of chlorine by both benzy-
lamino (10a–g) and anilino (12a–g) intermediates to give the
desired compounds 11a–g and 13a–g, respectively, in good
yields.
The preparation of 6,7-difluoro-3-methyl-2-penylthio
(5a–e), -2-phenylsulfinyl (7a) and -2-phenylsulfonyl (8a–e)
quinoxalines is reported in Scheme 1. The intermediate ac-
etonylphenyl sulfides (3a–e) were obtained by reaction of
thiophenol derivatives (1a–e) with chloroacetone (2) in the
presence of pyridine at 100 °C, adapting a general procedure
reported by Banfield et al. [14] for the preparation of aceto-
nylaryl sulfides. Successive nucleophilic attack by the known
5,6-difluorobenzofuroxane (4) [11] in methanol, made alka-
line with saturated dry ammonia methanol solution, afforded
the compounds (5a–e) in moderate yields. Oxidation of the
sulfides with a slight excess (1:2.5 molar ratio) of
m-chloroperbenzoic acid (MCPBA) in chloroform gave the
1,4-dioxides (8a–e) in high yields. Compound 8a was previ-
ously described by us [11]. However, in this occasion, we
have discovered that when 5a underwent oxidation using
1:1.2 molar ratio with MCPBA it gave the sulfinylquinoxa-
3. Microbiology
About the biological screening, all new compounds and
some previous synthesized derivatives were submitted to
anti-mycobacterial (Mycobacterium tuberculosis H37Rv),
anti-candida (C. albicans, C. glabrata, C. krusei and
C. parapsilosis clinical isolates) and antibacterial (Gram-
positive: Staphylococcus aureus ATCC 2913 and Gram-
Scheme 1. Method of preparation of quinoxalines 6,7-difluoro-3-methyl-2-phenylthio/phenylsulfinyl/phenylsulfonyl 1,4-dioxides (5a–e, 7a, and 8a–e).
Conditions: (i) 5a and MCPBA 1:1.2 molar ratio; (ii) 5a–e and MCPBA 1:2.5 molar ratio.

A. Carta et al. / European Journal of Medicinal Chemistry 39 (2004) 195–203
197
Scheme 2. Method of preparation of quinoxalines 6,7-difluoro-3-methyl-2-benzylamino/phenylamino-quinoxaline 1,4-dioxides (11a–g, and 13a–g).
negative: E. coliATCC 25922, Klebsiella pneumoniaeATCC
700603, Pseudomonas aeruginosa ATCC 27853 and the
environmental isolate Vibrio alginolyticus) assays. Further-
more, in order to investigate this type of quinoxalin 1,4-
dioxides against sexual transmitting diseases, the same com-
pounds were also submitted to anti-trichomonas screening
against Trichomonas vaginalis, and compounds which ex-
hibited the best activity against several species of Candida,
were also submitted to screening against Mycoplasma homi-
nis.
significant activity versus the mentioned bacteria. However,
the best activity was exhibited when a more or less oxidized
sulfur atom connects the C-2 position of quinoxaline scaffold
with the phenyl moiety as phenylthio (5e), phenylsulfinyl
(7a) and phenylsulfonyl (8a,d) derivatives, or a chlorine
atom is present in C-2 (9). About the phenyl moiety, the most
favorable resulted none for 7a and 8a, a para methoxy group
for 8d, or both meta and para methoxy group for 5e.
The in vitro anti-candida activity was first tested against
clinically isolated strains of C. albicans, using miconazole as
reference drug (MIC = 3.9 µg/ml), the MICs ranged between
7.8 and 250 µg/ml. Compounds that exhibited MCs <
15.6 µg/ml (7a, 8a,d and 9) were submitted to screening
against further strains of different Candida species (C. gla-
brata, C. krusei and C. parapsilosis). From this screening,
the results reported in Table 1 show that although none of the
tested compounds exhibited a better activity than micona-
zole, MIC and minimum fungicidal concentration (MFC)
values ranged between 3.9 and 31.25 µg/ml. We must point
out that also in this screening the best derivatives were those
having phenylsulfinyl (7a), phenylsulfonyl (8a,d), or a chlo-
rine atom (9) on C-2 position, and no substituent (7a, 8a) or
a para methoxy group (8d) on the phenyl moiety. Further-
more, the same compounds (7a, 8a,d and 9) were evaluated
against M. hominis. In this case the results showed that the
growth of this bacterium was inhibited at the concentration of
0.1 mg/ml.
4. Results and discussion
Quinoxaline 1,4-dioxides (5a–e, 7a, 8a–e, 9, 11a–g and
13a–g) were evaluated in vitro for antibacterial (S. aureus, E.
coli, V. alginolyticus, K. pneumoniae and P. aeruginosa),
antifungal (C. albicans, C. glabrata, C. krusei and C. parap-
silosis), anti-mycobacterial (M. tuberculosis), anti-
trichomonas (T. vaginalis), and anti-mycoplasma (M. homi-
nis) activities. The results obtained show that various
compounds possess an interesting activity against several
tested strains.
With regard to antibacterial activity, the obtained results
mainly indicate that most of tested compounds exhibited low
activity against Gram-positive and Gram-negative bacteria
showing MIC values ranging from 62.5 to 500 µg/ml with a
few exceptions. Compounds 5e, 7a and 8a,d
(MIC = 31.25 µg/ml) and 9 (MIC = 15.6 µg/ml) were the
most active against E. coli, while only compound 5e shows
MIC = 31.25 µg/ml against S. aureus and K. pneumoniae.
This moderate activity confirms our previous report [11] that
3-methylquinoxaline 1,4-dioxides in general do not display
In Table 2 we have reported the results of an in vitro
anti-tubercular screening of 16 derivatives that showed a
general good activity against M. tuberculosis
(MCs < 4 µg/ml). In particular, the compounds 5b,d,e and 9,
exhibited MIC = 2 µg/ml (in comparison with rifampin
MIC = 1 µg/ml). Antitubercular assays seem to confirm our

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A. Carta et al. / European Journal of Medicinal Chemistry 39 (2004) 195–203
Table 1
In vitro evaluation of anti-candida activity against clinical isolates C. glabrata, C. krusei and C. parapsilosis (MIC100 µg/ml) of compounds having
MICs = 15.6 µg/ml against C. albicans
7a
8a
8d
9
Miconazole
MIC
MFC
7.8
MIC
7.8
MFC
15.6
15.6
7.8
MIC
15.6
15.6
3.9
MFC
15.6
31.25
7.8
MIC
7.8
3.9
3.9
15.6
MFC
15.6
7.8
MIC
3.9
C. albicans 7.8
C. glabrata 7.8
C. krusei 3.9
7.8
15.6
7.8
0.4
7.8
3.9
0.9
C. parapsilosis15.6
31.25
31.25
31.25
31.25
31.25
31.25
0.4
Table 2
In vitro evaluation of compounds having MICs = 4 µg/ml against M. tuberculosis H37Rv
Compound
MIC (µg/ml)
Compound
MIC (µg/ml)
Compound
13a
13b
13c
13d
MIC (µg/ml)
5b
5c
5d
5e
8a
8b
2
4
2
2
4
4
8c
8d
9
11a
11b
11d
4
4
2
4
4
4
4
4
4
4
Rifampin
1
Table 3
In vitro evaluation of anti-trichomonas activity against T. vaginalis (MIC100 µg/ml)
Compound
MIC
After 24 h
3.12
>50
Compound
MIC
After 24 h
6.25
>50
25
After 48 h
0.78
50
After 72 h
0.39
50
After 96 h
0.39
50
After 48 h
After 72 h
0.78
25
After 96 h
0.78
25
5a
5b
5c
5d
5e
7a
8a
8b
8c
8d
8e
9
11c
11d
11e
11f
11g
13a
13b
13c
13d
13e
13f
13g
0.78
25
12.5
6.25
>50
3.12
0.78
25
3.12
0.78
25
3.12
0.78
12.5
0.78
1.56
3.12
3.12
1.56
1.56
0.39
12.5
25
12.5
12.5
12.5
25
6.25
6.25
6.25
25
6.25
6.25
6.25
25
25
25
1.56
1.56
6.25
6.25
6.25
3.12
0.78
>50
1.56
1.56
3.12
3.12
3.12
3.12
0.39
12.5
25
0.78
1.56
3.12
3.12
1.56
1.56
0.39
12.5
25
>50
>50
>50
>50
>50
50
12.5
25
3.12
25
3.12
25
25
12.5
12.5
50
12.5
12.5
50
25
50
>50
25
12.5
6.25
11a
11b
>50
Metronidazole 50
12.5
12.5
12.5
previous observations on structure–activity relationships
[11]. In fact this activity resulted significant when a phe-
nylthio (5b,d,e) group or a chlorine atom (9) in C-2 of the
quinoxaline 1,4-dioxide system was present. Furthermore, a
para fluorine atom (5b), a para methoxy group (5d) or both
meta and para methoxy group (5e) on the phenylthio moiety
were more favorable than chlorine atoms or methoxy groups
in different positions. Oxidation of sulfur bridge to yield
phenylsulfinyl and phenylsulfonyl derivatives or its replace-
ment with benzylamino or phenylamino group slightly re-
duces this activity.
About anti-trichomonas assay, the results obtained are
reported in Table 3. The quinoxaline 1,4-dioxides tested
exhibited a general good activity against T. vaginalis. Com-
pounds 5a,c,d, 7a, 8a–e, 9, 11c, and 13b showed
MCs < 3.12 µg/ml. In particular compounds 5a,d, 7a, 9 and
11c exhibited the best activity (MIC = 0.39–0.78 µg/ml).
Also in this case the data seem to confirm that the sulfur atom
in C-2 position is favorable for anti-trichomonas activity.
Mono-oxidation of phenylthio moiety to phenylsulfinyl de-
rivative or substitution with 3,4-dichlorobenzylamino group
retains the biological activity, whereas di-oxidation to phe-
nylsulfonyl derivative or substitution with phenylamino
group reduces this activity. About the substituents on the
phenylthio moiety, no substituent or a para methoxy group
resulted the most favorable.
5. Conclusions
In the light of the results obtained from the screening of
the phenyl substituted 6,7-difluoro-3-methyl-2-phenyl-
thio/phenylsulfinyl/phenylsulfonyl/benzylamino/phenylami-
no-quinoxaline 1,4-dioxides synthesized, we can confirm
that this scaffold is certainly endowed with anti-tubercular
and anti-candida activities, associating for some quinoxaline
1,4-dioxides a moderate anti-mycoplasma activity against
M. hominis. In addition, a very interesting activity emerged
against T. vaginalis for compounds 5a,d, 7a, 9 and 11c
(MIC = 0.39–0.78 µg/ml) which were much more potent than

A. Carta et al. / European Journal of Medicinal Chemistry 39 (2004) 195–203
199
reference drug (Metronidazole). This observation may pro-
mote a further development of our research in this field.
(1H, d, J = 2.2, H-2), 7.36 (1H, d, J = 8.2, H-5), 7.15 (1H, dd,
J = 8.2 and 2.2, H-6), 3.69 (2H, s, CH2), 2.29 (3H, s, CH3).
MS: m/z 238, 236, 234 [M]+. Anal. C9H8Cl2OS (C, H, N).
6. Experimental
6.1.2.3. Acetonyl-4-methoxyphenyl sulfide (3d). This com-
pound was obtained in 95% yield; 1H NMR (CDCl3): d 7.35
(2H, d, J = 8.6, H-2 + H-6), 6.84 (2H, d, J = 8.6, H-3 + H-5),
3.79 (3H, s, OCH3), 3.55 (2H, s, CH2), 2.26 (3H, s, CH3).
MS: m/z 196 [M]+. Anal. C10H12O2S (C, H, N).
6.1. Chemistry
Melting points (m.p.) were uncorrected and were taken in
open capillaries in a Digital Electrothermal IA9100 melting
point apparatus. 1H NMR spectra were recorded on a Varian
XL-200 (200 MHz) instrument, using TMS as internal stan-
dard. The chemical shift values are reported in ppm (d) and
coupling constants (J) in Hertz (Hz). Signal multiplicities are
represented by: s (singlet), d (doublet), dd (double doublet),
m (multiplet), and br s (broad singlet). MS spectra of aceto-
nylphenyl sulfides (3a–e) were performed on a combined HP
5790 (GC)–HP 5970 (MS) apparatus, while the products
(5b–e, 7a, 8b–e, 11a–g and 13a–g) were performed with a
combined Liquid Chromatograph-Agilent 1100 series Mass
Selective Detector (MSD). Column chromatography was
performed using 70–230 mesh (Merck silica gel 60). Light
petroleum refers to the fraction with b.p. 40–60 °C. The
progress of the reactions, the Rf and the purity of the final
compounds were monitored by TLC using Merck F-254
commercial plates. Analyses indicated by the symbols of the
elements were within 0.4% of the theoretical values.
6.1.2.4. Acetonyl-3,4-dimethoxyphenyl sulfide (3e). This
compound was obtained in 73% yield; 1H NMR (CDCl3): d
7.00–6.94 (2H, m, H-2 + H-6), 6.80 (1H, d, J = 8.2, H-5),
3.87 (6H, s, 2OCH3), 3.59 (2H, s, CH2), 2.27 (3H, s, CH3).
MS: m/z 226 [M]+. Anal. C11H14O3S (C, H, N).
6.1.3. General procedure for preparation
of 6,7-difluoro-3-methyl-2-phenylthioquinoxaline
1,4-dioxides (5a–e)
The title compounds were prepared modifying the known
Beirut reaction. Equimolar amounts (3.0 mmol) of the appro-
priate acetonylphenyl sulfides (3a–e) and 5,6-difluoro-
benzofuroxan (4) [11] were added to methanol (20 ml) and
methanol saturated with dry ammonia gas (5 ml), then stirred
at r.t. for 2 h. The resulting precipitate was filtered off,
washed with little portions of cold methanol and dried, to
afford the desired derivatives 5a–e.
6.1.1. Starting materials and intermediates
Thiophenol derivatives (1a–e), chloroacetone (2), benzy-
lamine derivatives (10a–g) and aniline derivatives (12a–g)
were commercially available. Acetonylphenyl sulfide (3a),
5,6-difluorobenzofuroxan (4), 6,7-difluoro-3-methyl-2-
phenylthioquinoxaline 1,4-dioxide (5a), 6,7-difluoro-3-
methyl-2-fenylsulfonylquinoxaline 1,4-dioxide (8a) and
2-chloro-6,7-difluoro-3-methylquinoxaline 1,4-dioxide (9)
were known and prepared as previously described [11].
6.1.3.1. 6,7-Difluoro-3-methyl-2-(4-fluorophenylthio)quino-
xaline 1,4-dioxide (5b). This compound was obtained in
39% yield; m.p. 161–162 °C (ethanol); TLC (light petroleu-
m–ethyl acetate 70:30): Rf 0.15; 1H NMR (CDCl3): d 8.47–
8.28 (2H, m, H-5 + H-8), 7.50–7.43 (2H, m, H-3′ + H-5′),
7.09–7.00 (2H, m, H-2′ + H-6′), 2.91 (3H, s, CH3). LC/MS:
339 [M + H], 361 [M + Na]. Anal. C15H9F3N2O2S (C, H, N).
6.1.3.2. 6,7-Difluoro-3-methyl-2-(3,4-dichlorophenylthio)-
quinoxaline 1,4-dioxide (5c). This compound was obtained
in 31% yield; m.p. 191–192 °C (ethanol); TLC (light petro-
6.1.2. General procedure for preparation
of acetonylphenyl sulfides (3a–e)
A solution of the appropriate thiophenol derivatives
(1a–e) (6.0 mmol) and chloroacetone (2) (7.8 mmol) in
pyridine (5 ml) was heated to 100 °C for 1 h. On cooling to
room temperature (r.t.), the reaction mixture was added to
6 N hydrochloric acid solution (20 ml), then extracted with
dichloromethane and dried on anhydrous sodium sulfate. On
evaporation of the liquor mother, a yellow oil corresponding
to acetonylphenyl sulfides (3a–e) was collected and the ana-
lytical properties are reported below.
1
leum–ethyl acetate 70:30): Rf 0.15; H NMR (CDCl3): d
8.50–8.27 (2H, m, H-5 + H-8), 7.45 (1H, s, H-2′) 7.41 (1H, d,
J = 8.8, H-5′), 7.24 (1H, d, J = 8.8, H-6′), 2.93 (3H, s, CH3).
LC/MS: 411 [M + Na]. Anal. C15H8Cl2F2N2O2S (C, H, N).
6.1.3.3. 6,7-Difluoro-3-methyl-2-(4-methoxyphenylthio)qui-
noxaline 1,4-dioxide (5d). This compound was obtained in
39% yield; m.p. 166–167 °C (ethanol); TLC (light petroleu-
m–ethyl acetate 1:1): Rf 0.48; 1H NMR (CDCl3): d 8.46–8.30
(2H, m, H-5 + H-8), 7.45 (2H, d, J = 8.8, H-2′ + H-6′), 6.87
(2H, d, J = 8.8, H-3′ + H-5′), 3.80 (3H, s, OCH3), 2.86 (3H, s,
CH3). LC/MS: 351 [M + H], 373 [M + Na], 389 [M + K].
Anal. C16H12F2N2O3S (C, H, N).
6.1.2.1. Acetonyl-4-fluorophenyl sulfide (3b). This com-
1
pound was obtained in 89% yield; H NMR (CDCl3): d
7.40–7.33 (2H, m, H-3 + H-5), 7.05–6.96 (2H, m, H-2 +
H-6), 3.61 (2H, s, CH2), 2.27 (3H, s, CH3). MS: m/z 184
[M]+. Anal. C9H9FOS (C, H, N).
6.1.3.4. 6,7-Difluoro-3-methyl-2-(3,4-dimethoxyphenylthio-
)quinoxaline 1,4-dioxide (5e). This compound was obtained
in 46% yield; m.p. 182–184 °C (ethanol); TLC (light petro-
6.1.2.2. Acetonyl-3,4-dichlorophenyl sulfide (3c). This com-
pound was obtained in 95% yield; 1H NMR (CDCl3): d 7.42

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1
leum–ethyl acetate 70:30): Rf 0.14; H NMR (CDCl3): d
8.47–8.31 (2H, m, H-5 + H-8), 7.06 (1H, s, H-2′), 7.04 (1H,
d, J = 8.0, H-6′), 6.81 (1H, d, J = 8.0, H-5′), 3.87 (6H, s,
2OCH3), 2.86 (3H, s, CH3). LC/MS: 381 [M + H], 403 [M +
Na]. Anal. C17H14F2N2O4S (C, H, N).
(ethanol); TLC (light petroleum–ethyl acetate 1:1): Rf 0.48;
1H NMR (CDCl3): d 8.40 (1H, m, H-8), 8.23 (1H, m, H-5),
8.11 (2H, d, J = 8.8, H-2′ + H-6′), 7.03 (2H, d, J = 8.8, H-3′ +
H-5′), 3.89 (3H, s, OCH3), 3.19 (3H, s, CH3). LC/MS: 405
[M + Na]. Anal. C16H12F2N2O5S (C, H, N).
6.1.4. General procedure for preparation of 6,7-difluoro-3-
methyl-2-phenylsulfinylquinoxaline 1,4-dioxide (7a) and
6,7-difluoro-3-methyl-2-phenylsulfonylquinoxaline
6.1.4.5. 6,7-Difluoro-3-methyl-2-(3,4-dimethoxyphenylsul-
fonyl)quinoxaline 1,4-dioxide (8e). This compound was ob-
tained in 85% yield after stirring for 5 h; m.p. 169–170 °C
(ethanol); TLC (light petroleum–ethyl acetate 70:30): Rf
0.14; 1H NMR (CDCl3): d 8.43 (1H, m, H-8), 8.24 (1H, m,
H-5), 7.75 (1H, dd, J = 8.4 and 2.2, H-6′), 7.66 (1H, d, J = 2.2,
H-2′), 6.98 (1H, d, J = 8.4, H-5′), 4.00 (3H, s, OCH3), 3.96
(3H, s, OCH3), 3.20 (3H, s, CH3). LC/MS: 413 [M + H], 435
[M + Na], 451 [M + K]. Anal. C17H14F2N2O6S (C, H, N).
1,4-dioxides (8a–e)
The title compounds were prepared following the proce-
dure previously described by Abushanab [15]. To a solution
of the appropriate 6,7-difluoro-3-methyl-2-phenylthioqui-
noxaline 1,4-dioxide (5a–e) (3.0 mmol) in chloroform
(40 ml) a solution of 3-chloroperoxybenzoic acid (MCPBA)
in 1:2.5 molar ratio (7.5 mmol) in chloroform (15 ml) was
slowly added. After the addition was complete, the reaction
mixture was stirred at r.t. for an additional time as indicated
below, then the chloroform solution was washed with 5%
sodium bicarbonate aqueous solution, dried over anhydrous
sodium sulfate, filtered off and evaporated. A solid corre-
sponding to derivatives 8a–e was obtained and purified as
indicated below. Compound 7a was in turn obtained in simi-
lar conditions but using 6,7-difluoro-3-methyl-2-
phenylthioquinoxaline 1,4-dioxide (5a) (3.0 mmol) and
MCPBA (3.6 mmol) in 1:1.2 molar ratio.
6.1.5. General procedure for preparation of 6,7-difluoro-3-
methyl-2-benzylaminoquinoxaline 1,4-dioxides (11a–g)
and 6,7-difluoro-3-methyl-2-phenylaminoquinoxaline
1,4-dioxides (13a–g)
Equimolar amounts (1.0 mmol) of 2-chloro-6,7-difluoro-
3-methylquinoxaline 1,4-dioxide (9) [11], and the appropri-
ate benzylamine (10a–g) or phenylamine (12a–g) in dry
DMF (6 ml) were heated to 70 °C under stirring for 14 h. On
cooling to r.t., the reaction mixture was diluted with 50 ml of
water. A crude precipitate was formed and collected by filtra-
tion.
6.1.4.1. 6,7-Difluoro-3-methyl-2-phenylsulfinylquinoxaline
1,4-dioxide (7a). This compound was obtained in 50% yield
after stirring for 5 h and crystallization from ethanol; m.p.
187–188 °C; TLC (light petroleum–ethyl acetate 80:20): Rf
Purification of all compounds was carried out by crystal-
lization from aqueous ethanol.
1
0.17; H NMR (CDCl3): d 8.44–8.25 (2H, m, H-5 + H-8),
6.1.5.1. 6,7-Difluoro-3-methyl-2-benzylaminoquinoxaline
1,4-dioxide (11a). This compound was obtained in 62%
yield; m.p. 102–104 °C (aqueous ethanol); TLC (light petro-
8.09–8.04 (2H, m, H-2′ + H-6′), 7.56–7.53 (3H, m, H-3′ +
H-4′ + H-5′), 2.95 (3H, s, CH3). LC/MS: 359 [M + Na], 375
[M + K]. Anal. C15H10F2N2O3S (C, H, N).
1
leum–ethyl acetate 1:1): Rf 0.22; H NMR (CDCl3): d 8.18
(1H, m, H-8), 7.62 (1H, m, H-5), 7.50–7.30 (5H, m, 5 phenyl
H), 5.18 (1H, br s, NH), 4.53 (2H, d, J = 4.8, CH2), 2.79 (3H,
s, CH3). LC/MS: 356 [M + K]. Anal. C16H13F2N3O2 (C, H,
N).
6.1.4.2. 6,7-Difluoro-3-methyl-2-(4-florophenylsulfonyl)qui-
noxaline 1,4-dioxide (8b). This compound was obtained in
91% yield after stirring for 10 h; m.p. 117–119 °C (ethanol);
TLC (light petroleum–ethyl acetate 70:30): Rf 0.15; 1H NMR
(CDCl3): d 8.43 (1H, m, H-8), 7.89 (1H, m, H-5), 7.68–7.54
(4H, m, 4 phenyl H), 3.21 (3H, s, CH3). LC/MS: 371 [M +
H]. Anal. C15H9F3N2O4S (C, H, N).
6.1.5.2. 6,7-Difluoro-3-methyl-2-(4-fluorobenzylamino)qui-
noxaline 1,4-dioxide (11b). This compound was obtained in
59% yield; m.p. 180–181 °C (aqueous ethanol); TLC (light
petroleum–ethyl acetate 40:60): Rf 0.24; 1H NMR (CDCl3): d
7.60 (1H, m, H-8), 7.41–734 (3H, m, H-5 + H-3′ + H-5′),
7.13–7.04 (2H, m, H-2′ + H-6′), 5.16 (1H, br s, NH), 4.51
(2H, d, J = 5.4, CH2), 2.78 (3H, s, CH3). LC/MS: 336 [M +
H], 358 [M + Na]. Anal. C16H12F3N3O2 (C, H, N).
6.1.4.3. 6,7-Difluoro3-methyl-2-(3,4-dichlorophenylsulfo-
nyl)quinoxaline 1,4-dioxide (8c). This compound was ob-
tained in 80% yield after stirring for 15 h; m.p. 123–126 °C
(ethanol); TLC (light petroleum–ethyl acetate 70:30): Rf
0.15; 1H NMR (CDCl3): d 8.42 (1H, m, H-8), 8.20 (1H, m,
H-5), 8.18 (1H, d, J = 2.2, H-2′), 7.68–7.46 (2H, m, H-3′ +
H-5′), 3.18 (3H, s, CH3). LC/MS: 421 [M + H], 459 [M + K].
Anal. C15H8Cl2F2N2O4S (C, H, N).
6.1.5.3. 6,7-Difluoro-3-methyl-2-(3,4-dichlorobenzylamino)
quinoxaline 1,4-dioxide (11c). This compound was obtained
in 73% yield; m.p. 194–196 °C (aqueous ethanol); TLC
1
(light petroleum–ethyl acetate 30:70): Rf 0.27; H NMR
6.1.4.4.
6,7-Difluoro-3-methyl-2-(4-methoxyphenylsulfo-
(CDCl3): d 8.20 (1H, m, H-8), 7.56–7.22 (4H, m, H-5 +
3 phenyl H), 5.26 (1H, br s, NH), 4.53 (2H, d, J = 4.8, CH2),
nyl)quinoxaline 1,4-dioxide (8d). This compound was ob-
tained in 82% yield after stirring for 5 h; m.p. 142–144 °C

A. Carta et al. / European Journal of Medicinal Chemistry 39 (2004) 195–203
201
2.78 (3H, s, CH3). LC/MS: 386 [M + H]. Anal.
C16H11Cl2F2N3O2 (C, H, N).
6.1.5.10. 6,7-Difluoro-3-methyl-2-(3,4-dichlorophenylami-
no)quinoxaline 1,4-dioxide (13c). This compound was ob-
tained in 33% yield; m.p. 224–226 °C (aqueous ethanol);
1
6.1.5.4. 6,7-Difluoro-3-methyl-2-(4-methoxybenzylamino)
quinoxaline 1,4-dioxide (11d). This compound was obtained
in 78% yield; m.p. 83–85 °C (aqueous ethanol); TLC (light
TLC (light petroleum–ethyl acetate 1:1): Rf 0.46; H NMR
(CDCl3): d 8.55 (1H, br s, NH), 8.47–8.32 (2H, m, H-5 +
H-8), 7.45 (1H, d, J = 8.6, H-5′), 7.14 (1H, d, J = 2.4, H-2′),
6.89 (1H, dd, J = 8.6 and 2.4, H-6′), 2.35 (3H, s, CH3).
LC/MS: 410 [M + K]. Anal. C15H9Cl2F2N3O2 (C, H, N).
1
petroleum–ethyl acetate 1:1): Rf 0.16; H NMR (CDCl3): d
8.18 (1H, m, H-8), 7.62 (1H, m, H-5), 7.32 (2H, d, J = 7.8,
H-2′ + H-6′), 6.91 (2H, d, J = 7.8, H-3′ + H-5′), 5.12 (1H, br
s, NH), 4.45 (2H, d, J = 4.8, CH2), 3.81 (3H, s, OCH3), 2.78
(3H, s, CH3). LC/MS: 386 [M + K]. Anal. C17H15F2N3O3 (C,
H, N).
6.1.5.11. 6,7-Difluoro-3-methyl-2-(4-methoxyphenylamino-
)quinoxaline 1,4-dioxide (13d). This compound was ob-
tained in 74% yield; m.p. 203–204 °C (aqueous ethanol);
1
TLC (light petroleum–ethyl acetate 1:1): Rf 0.44; H NMR
6.1.5.5. 6,7-Difluoro-3-methyl-2-(3,4-dimethoxybenzylami-
no)quinoxaline 1,4-dioxide (11e). This compound was ob-
tained in 53% yield; m.p. 103–105 °C (aqueous ethanol);
TLC (light petroleum–ethyl acetate 70:30): Rf 0.18; 1H NMR
(CDCl3): d 8.18 (1H, m, H-8), 7.63 (1H, m, H-5), 6.98–6.84
(3H, m, 3 phenyl H), 5.17 (1H, br s, NH), 4.45 (2H, d, J = 4.8,
CH2), 3.89 (6H, s, 2OCH3), 2.78 (3H, s, CH3). LC/MS: 416
[M + K]. Anal. C18H17F2N3O4 (C, H, N).
(CDCl3): d 8.71 (1H, br s, NH), 8.44–8.30 (2H, m, H-5 +
H-8), 7.06 (2H, d, J = 8.8, H-2′ + H-6′), 6.91 (2H, d, J = 8.8,
H-3′ + H-5′), 3.84 (3H, s, OCH3), 2.25 (3H, s, CH3). LC/MS:
334 [M + H], 356 [M + Na], 372 [M + K]. Anal.
C16H13F2N3O3 (C, H, N).
6.1.5.12.
6,7-Difluoro-3-methyl-2-(3,4-dimethoxyphenyl-
amino)quinoxaline 1,4-dioxide (13e). This compound was
obtained in 64% yield; m.p. 214–216 °C (aqueous ethanol);
TLC (light petroleum–ethyl acetate 70:30): Rf 0.33; 1H NMR
(CDCl3): d 8.70 (1H, br s, NH), 8.44–8.30 (2H, m, H-5 +
H-8), 6.86–6.67 (3H, m, 3 phenyl H), 3.91 (3H, s, OCH3),
3.87 (3H, s, OCH3), 2.28 (3H, s, CH3). LC/MS: 364 [M + H],
386 [M + Na]. Anal. C17H15F2N3O4 (C, H, N).
6.1.5.6. 6,7-Difluoro-3-methyl-2-(3,5-dimethoxybenzylami-
no)quinoxaline 1,4-dioxide (11f). This compound was ob-
tained in 62% yield; m.p. 196–197 °C (aqueous ethanol);
TLC (light petroleum–ethyl acetate 70:30): Rf 0.25; 1H NMR
(CDCl3): d 8.18 (1H, m, H-8), 7.60 (1H, m, H-5), 6.51 (2H, s,
H-2′ + H-6′), 6.40 (1H, s, H-4′), 5.17 (1H, br s, NH), 4.46
(2H, d, J = 4.8, CH2), 3.79 (6H, s, 2OCH3), 2.78 (3H, s, CH3).
LC/MS: 416 [M + K]. Anal. C18H17F2N3O4 (C, H, N).
6.1.5.13.
6,7-Difluoro-3-methyl-2-(3,5-dimethoxyphenyl-
amino)quinoxaline 1,4-dioxide (13f). This compound was
obtained in 59% yield; m.p. 217–218 °C (aqueous ethanol);
TLC (light petroleum–ethyl acetate 1:1): Rf 0.39; H NMR
(CDCl3): d 8.55 (1H, br s, NH), 8.45–8.33 (2H, m, H-5 +
H-8), 6.34 (1H, m, H-4′), 6.19 (2H, s, H-2′ + H-6′), 3.78 (6H,
s, 2OCH3), 2.37 (3H, s, CH3). LC/MS: 364 [M + H], 386 [M
+ Na]. Anal. C17H15F2N3O4 (C, H, N).
6.1.5.7. 6,7-Difluoro-3-methyl-2-(3,4,5-trimethoxybenzyl-
amino)quinoxaline 1,4-dioxide (11g). This compound was
obtained in 61% yield; m.p. 193–194 °C (aqueous ethanol);
TLC (chloroform–methanol 95:5): Rf 0.45; 1H NMR
(CDCl3): d 8.20 (1H, m, H-8), 7.64 (1H, m, H-5), 6.62 (2H, s,
H-2′ + H-6′), 5.15 (1H, br s, NH), 4.45 (2H, d, J = 5.0, CH2),
3.88 (6H, s, C-3′ OCH3 + C-5′ OCH3), 3.85 (3H, s, C-4′
OCH3), 2.79 (3H, s, CH3). LC/MS: 408 [M + H], 430 [M +
Na]. Anal. C19H19F2N3O5 (C, H, N).
1
6.1.5.14. 6,7-Difluoro-3-methyl-2-(3,4,5-trimethoxyphenyl-
amino)quinoxaline 1,4-dioxide (13g). This compound was
obtained in 85% yield; m.p. 198–199 °C (aqueous ethanol);
TLC (light petroleum–ethyl acetate 40:60): Rf 0.22; 1H NMR
(CDCl3): d 8.62 (1H, br s, NH), 8.45–8.32 (2H, m, H-5 +
H-8), 6.31 (2H, s, H-2′ + H-6′), 3.87 (3H, s, C4′–OCH3), 3.82
(6H, s, C3′–OCH3 + C5′–OCH3), 2.35 (3H, s, CH3). LC/MS:
3.94 [M + H], 416 [M + Na]. Anal. C18H17F2N3O5 (C, H, N).
6.1.5.8. 6,7-Difluoro-3-methyl-2-phenylaminoquinoxaline
1,4-dioxide (13a). This compound was obtained in 81%
yield; m.p. 230–231 °C (aqueous ethanol); TLC (light petro-
1
leum–ethyl acetate 1:1): Rf 0.40; H NMR (CDCl3): d 8.67
(1H, br s, NH), 8.45–8.32 (2H, m, H-5 + H-8), 7.41–7.30
(3H, m, H-3′ + H-4′ + H-5′), 7.08–7.05 (2H, m, H-2′ + H-6′),
2.28 (3H, s, CH3). LC/MS: 304 [M + H], 326 [M + Na].Anal.
C15H11F2N3O2 (C, H, N).
6.2. Microbiological assays
6.2.1. Antibacterial assays
The strains used in these tests were from American Type
Culture Collection (ATCC): S. aureus ATCC 2913, E. coli
ATCC 25922, K. pneumoniae ATCC 700603, and P. aerugi-
nosa ATCC 27853, or were environmental isolate (V. algi-
nolyticus). A logarithmic phase culture 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
6.1.5.9. 6,7-Difluoro-3-methyl-2-(4-fluorophenylamino)qui-
noxaline 1,4-dioxide (13b). This compound was obtained in
81% yield; m.p. 218–219 °C (aqueous ethanol); TLC (light
1
petroleum–ethyl acetate 1:1): Rf 0.30; H NMR (CDCl3): d
8.63 (1H, br s, NH), 8.45–8.32 (2H, m, H-5 + H-8), 7.11–
7.07 (4H, m, 4 phenyl H), 2.27 (3H, s, CH3). LC/MS: 322 [M
+ H], 344 [M + Na]. Anal. C15H10F3N3O2 (C, H, N).

202
A. Carta et al. / European Journal of Medicinal Chemistry 39 (2004) 195–203
microtiter plate in a final volume of 100 µl. Test compounds
were dissolved in dimethyl sulfoxide at an initial concentra-
tion of 1000 µg/ml and serially diluted in the plate (500–
7.8 µg/ml) using Luria broth. Each well was then inoculated
with the standardized bacterial suspension and incubated at
37 °C for 18–24 h. One well containing bacteria without
sample (growth control, GC), and one well containing broth
only (sterility control) were also used. After the incubation
the growth (or its lack) of the bacteria was determined visu-
ally in both containing compound well and control well. The
lowest concentration at which there was no visible growth
(turbidity) was taken as the MIC. In addition 5 µl of suspen-
sion from each well were inoculated in a Mueller Hinton agar
plate to control bacterial viability.
lum was prepared by pipetting 0.1 ml of the test inoculum
with 10 ml of sterile saline to make a 1:100 dilution and
0.5 ml added to the MIGT tube. All the inoculated tubes were
placed into the Bactec MGIT 960 instrument the same day of
inoculation.
The relative growth ratio between the compound-
containing tube and the compound-free GC tube was deter-
mined by the system’s software algorithm. If the relative
growth in the compound-containing tube was equal to or
exceeded that of the GC tube, the isolate was considered
compound resistant; if the relative growth was less than that
of the GC tube, the isolate was considered compound suscep-
tible.
6.2.4. Anti-mycoplasma assay
6.2.2. Anti-candida assay
M. hominis used for this assay was a clinical isolate strain
and was grown on BEa broth [18]. The test was performed in
a 96-well microtiter plate in a concentration of bacteria in
BEa broth of 106 bacteria per well. Test compounds were
dissolved in dimethyl sulfoxide at an initial concentration of
10 mg/ml and serially diluted to 0.1 µg/ml, then added at the
plate. Each well was then incubated at 37 °C in humid
atmosphere containing 5% CO2. The MBC determination
was defined as the lowest concentration of the compound,
which did not produce color change of the broth, after re-
moval of test compound, the bacteria showed no growth.
Wells containing bacteria on BEa broth and the same amount
of dimethyl sulfoxide, without sample (GC), were also used.
In these cases each well produced color change indicating the
bacterial growth. Gentamicine used as control drug showed
MBC = 5–25 µg/ml in different experiments.
Antifungal activity was determined by the tube dilution
method on four clinical isolates of Candida species (C.
albicans, C. glabrata, C. krusei and C. parapsilosis) coming
from patients withAIDS and candidemia.Yeast inocula were
obtained by properly diluting cultures incubated at 35 °C for
48 h in Sabouraud Dextran agar to obtain a density of 106
CFU/ml. Test compounds were dissolved in dimethyl sulfox-
ide at an initial concentration of 1000 µg/ml and then were
serially diluted in Brain Hearth fusion broth to 0.1 µg/ml.
Then, 0.5 ml of the above serial dilutions of test compounds
were added, in sterile polystyrene tubes, with an equal vol-
ume of fungal suspension and incubated at 35 °C for 48 h.
Preliminary tests with dimethyl sulfoxide were performed to
assure that no Candida species inhibition occurred at used
concentrations.
The MIC100 determination was performed in duplicate,
and defined as the lowest concentration of the compound
which produced no visible growth. MFC determinations
were done for all compounds by plating 20 µl of each well on
Sabouraud dextrose agar (SDA) plates after 48 h of incuba-
tion at 35 °C. The MFC was the lowest drug concentration
giving no growth on the (SDA) plates (>98% killing). A
sample of compound-free GC and a set of tubes with sample
alone for monitoring contamination of the medium were
used.
6.2.5. Anti-trichomonas assay
Our study was performed by using T. vaginalis (SS-22),
Mycoplasma-free, isolated in Sassari (Italy) from a case of
acute vaginal trichomoniasis, axenically grown at 37 °C in
Diamond medium [19] supplemented with 10% heat-
inactivated fetal calf serum (FCS). Trichomonas were used
immediately after isolation, or frozen in liquid nitrogen.
Stock solutions (2 mg/ml) of metronidazole (SIGMA) and
other compounds tested were prepared in dimethyl sulfoxide,
and stored in small aliquots at 4 °C.
6.2.3. Anti-mycobacterial assay
The susceptibility of T. vaginalis was determined by the
assessment of growth and motility of flagellates exposed to
different concentrations of drugs. Tests were performed in
sterile tissue-culture microtiter (Corning). Serial twofold di-
lution of the compounds were made in Diamond medium
containing 10% FCS. Control wells received 100 µl of com-
plete Diamond medium. Protozoa, washed twice in
phosphate-buffered saline (PBS, pH 7.2), were suspended in
the same medium at the density of 2.5 × 105 cells per ml and
100 µl of the calibrate suspension was added to each well.
Final concentrations obtained ranged from 50 to
0.006 µg/ml. Tests were performed in duplicate and repeated
at least three times. Microtiter plates were then incubated
aerobically at 37 °C, in a humid atmosphere containing 5%
Anti-mycobacterial activity was determined by the new
method Bactec MGIT 960 [16,17] against M. tuberculosis
H37Rv (ATCC 27294). A suspension of micro-organisms
was prepared in 7H9 medium at a density of 0.5 Mc Farland
and was diluted (1:5) with sterile saline solution. Test com-
pounds were dissolved in dimethyl sulfoxide at an initial
concentration of 2240 mg/ml and diluted to obtain a final
concentration of 8, 4, 2 and 1 µg/ml (rifampin was used as
reference drug). To each 7 ml MGIT tube, 0.8 ml of MGIT
960 growth supplement and 0.1 ml of the test compound
were aseptically added, and finally 0.5 ml of the test inocu-
lum was added.
For each isolate a GC tube with growth supplement and
without test compound was included. For this GC, the inocu-

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