Synthesis and evaluation for biological activity of 3-alkyl and 3-halogenoalkyl-quinoxalin-2-ones variously substituted. Part 4

Article abstract of DOI:10.1016/S0014-827X(01)01153-3

A new series of 3-isopropyl-, 3-trifluoromethyl- and 3-bromomethylquinoxaline-2-ones variously substituted on the benzo-moiety were synthesized and submitted to a preliminary in vitro evaluation for antibacterial, antifungal and anti-HIV activities. Furth

Full text of DOI:10.1016/S0014-827X(01)01153-3

Il Farmaco 57 (2002) 19–25
www.elsevier.com/locate/farmac
Synthesis and evaluation for biological activity of 3-alkyl and
3-halogenoalkyl-quinoxalin-2-ones variously substituted. Part 4
Antonio Carta a, Paolo Sanna a,*, Mario Loriga a, Maria Giovanna Setzu b,
Paolo La Colla b, Roberta Loddo b
a Dipartimento Farmaco Chimico Tossicologico, Uni6ersita´ di Sassari, 6ia Muroni 23/a, 07100 Sassari, Italy
b Dipartimento di Biologia Sperimentale-Sezione di Microbiologia, Cittadella Uni6ersitaria, Monserrato, Italy
Received 5 June 2001; accepted 30 July 2001
Abstract
A new series of 3-isopropyl-, 3-trifluoromethyl- and 3-bromomethylquinoxaline-2-ones variously substituted on the benzo-moi-
ety were synthesized and submitted to a preliminary in vitro evaluation for antibacterial, antifungal and anti-HIV activities.
Furthermore, all compounds were also tested for cytotoxicity. Results of the screening showed that compound 10 exhibits
moderate antimicrobial activity against Staphylococcus aureus (MIC=33 mM), and that 25 and 26 showed interesting cytotoxicity
versus mock-infected MT-4 cells. All the other compounds were inactive. © 2002 Elsevier Science S.A. All rights reserved.
Keywords: 2-Quinoxalinones; Antimicrobial activity; Cytotoxic activity
1. Introduction
These results prompted us to prepare a new series of
quinoxalin-2-one derivatives in the hope of increasing
the activity and obtaining additional data for struc-
ture–activity relationships. In this paper we describe
the synthesis and microbiological and cytotoxic activi-
ties of 24 derivatives, the structure of which is summa-
rized in Formula II.
In previous reports we described the synthesis and
antimicrobial and anticancer activities of various
quinoxalin-2-one derivatives [1–3]. Among these, com-
pounds with a CF3 or NO2 in C-6 or C-7 and a CH2Br
or CF3 at C-3 showed good activity against various
strains of Candida (C. albicans, C. krusei, C. parapsilosis,
C. tropicalis, C. glabrata and various clinical isolated of
Candida spp.), whereas derivatives bearing Cl or CF3 at
C-6 or C-7 and a carboxyethyl, ethyl or CH2Br group at
C-3 exhibited moderate antibacterial activity. Moreover,
3-bromomethyl-7-trifluoromethyl-2(1H)-quinoxalinone
(Formula I) proved fairly active in vitro as an anticancer
agent against about 60 human tumor cell lines and it is
actually tested in vivo at the National Cancer Institute
(NCI) of Bethesda, MD, USA.
In these new compounds we introduced in the benzo-
moiety alternatively one or two atoms of chlorine, one
electron-releasing group (CH3O), or one electron-with-
drawing (Cl or CF3) group together with an electron-re-
leasing group (CH3O or NH2), whereas we introduced
at the C-3 position an isopropyl, bromomethyl or trifl-
uoromethyl group in order to evaluate the effect of
electron-releasing and/or electron-withdrawing sub-
stituents on biological activities.
* Corresponding author.
E-mail address: psanna@ssmain.uniss.it (P. Sanna).
0014-827X/02/$ - see front matter © 2002 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01153-3

20
A. Carta et al. / Il Farmaco 57 (2002) 19–25
2. Chemistry
It is important to note that when 1,2-diamino-4-
methoxybenzene (1b) and 1,2-diamino-3-chloro-4-
methoxybenzene (1d) were condensed with 2a and 2c
under acidic conditions, we obtained prevalently (9\4,
11\6) or exclusively (26) the 6-methoxy derivatives.
This behavior is reversed in the case of the condensa-
tion of the same diamines 1b,d with 2b. In the former
case only the 7-methoxy derivative 14 is obtained
whereas in the latter case 16 is prevalent over 20. These
results show that protonation of the amino group on
the para position of the methoxy group precedes the
condensation, and that the steric hindrance of the iso-
propyl group in the case of 2b promotes an inversion of
reactivity in comparison with other a-ketoesters.
Quinoxalinones 3 and 8 were already described by
The quinoxalinones 3–26, listed in Table 1, were
prepared following the procedure depicted in Scheme 1.
According to our previous report [3], the diamine 1a–d
was reacted with the suitable a-ketoester 2a–c in 10%
aqueous solution of sulfuric acid to give compounds
3–6, 8–11, 13–16, 18–20, 23, 25 and 26 in good yields,
while the condensation of 1a with 2c, to obtain com-
pounds 22 and 24, failed. The latter compounds were
obtained by condensation in ethanol at room tempera-
ture for 1 h, while the isomeric pairs 7/12 and 17/21
were obtained by condensation in ethanol under reflux
for 3 h of the diamine 1e with the a-ketoesters 2a,b,
respectively.
Table 1
Compounds of Scheme 1
Comp.
R
R1
R2
Comp.
R
R1
R2
Comp.
R
R1
R2
1a
1b
1c
1d
1e
3
4
5
6
7
H
H
Cl
Cl
NH2
H
H
Cl
Cl
Cl
OCH3
Cl
OCH3
H
Cl
OCH3
Cl
OCH3
H
H
H
H
H
CF3
H
H
H
H
8
9
H
H
Cl
Cl
NH2
H
H
Cl
Cl
Cl
OCH3
Cl
OCH3
H
Cl
OCH3
Cl
OCH3
H
H
H
H
H
CF3
H
H
H
H
18
19
20
21
22
23
24
25
26
H
Cl
Cl
OCH3
H
Cl
Cl
Cl
Cl
OCH3
H
H
H
CF3
H
H
H
H
H
Cl
Cl
NH2
H
Cl
H
10
11
12
13
14
15
16
17
Cl
Cl
NH2
CF3
NH2
CF3
Scheme 1. Preparation of substituted quinoxalin-2-ones (3–26). Reagents: (2a) CF3COCO2Et; (2b) (CH3)2CHCOCO2Et; (2c) CH2BrCOCO2Et.
Conditions: (i) EtOH, under reflux for 3 h; (ii) H2SO4 10% aqueous solution at 60–100 °C for 1–3 h; (iii) EtOH r.t. for 1 h; (iv) MeONa/MeOH,
under reflux for 8 h; (v) H2 on 10% Pd/C at 3 atm.

A. Carta et al. / Il Farmaco 57 (2002) 19–25
21
Mustafa et al. [4], but we reprepared and concluded on
3.1.2. General procedure for preparation of quinoxa-
linones 3–6, 8–11, 13–16, 18–20, 23, 25 and 26
the basis of 1H NMR data that the previous assignment
of the structure to two isomers must be reversed. The
structures of all quinoxalinones 3–26 have been sup-
ported by us by analytical and spectroscopic data (MS
A mixture of equimolar amounts (3.5 mmol) of the
appropriate diamine 1a–d and the suitable a-ketoester
2a,c in 10% aqueous solution of sulfuric acid was stirred
at 100 °C for 1 h in the case of 3, 4, 8, 9, 13, 14, 18 and
24 or at 60 °C for 3 h in the case of 5, 6, 10, 11, 15, 16,
19, 20, 23, 25 and 26. After cooling of the reaction
mixture to room temperature (r.t.), the crude precipitate
formed was collected by filtration and chromatographed
on a silica gel column, eluting as reported below, in order
to separate the mixture of isomers where present.
1
spectra, IR, UV, H NMR) and are in agreement with
those previously reported for similar compounds [1–3].
3. Experimental
3.1. Chemistry
Melting points were determined in a Kofler hot stage
or Digital Electrothermal apparatus, and are uncor-
rected. Infrared spectra were recorded in Nujol mulls
and were recorded using a Perkin–Elmer 781 spec-
trophotometer. Qualitative UV spectra (ethanol) were
recorded in a Perkin–Elmer Lambda 5 spectrophotome-
3.1.2.1. 7-Chloro-3-trifluoromethyl-2(1H)-quinoxalinone
(3). This compound was obtained in 38% yield by eluting
with a 8:2 mixture of light petroleum–ethyl acetate; m.p.
244–246 °C; IR: w 1670, 1610 cm−1; UV: u 345, 276,
1
219, 194 nm; H NMR (CDCl3+DMSO-d6): l 13.08
(1H, br s, NH), 7.84 (1H, d, J=8.8 Hz, H-5), 7.43 (1H,
d, J=2.4 Hz, H-8), 7.31 (1H, dd, J=8.8 and 2.4 Hz,
H-6); MS: m/z 248 (M+).
1
ter and are reported in nanometers. H NMR spectra
were recorded in a Varian XL-200 (200 MHz) instru-
ment, using TMS as internal standard. The chemical
shift values are reported in ppm (l) 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 were recorded in a combined HP 5790-HP 5970
GC/MS apparatus. Column chromatography was per-
formed using 230–400 mesh silica gel (Merck silica gel
60). Light petroleum refers to the fraction with b.p.
40–60 °C. Elemental analyses were performed by the
Laboratorio di Microanalisi, Dipartimento di Scienze
Farmaceutiche, Universita` di Padova (Padua). The ana-
lytical results for C, H, N and halogen, when present,
were within 90.4% of the theoretical values.
3.1.2.2. 6-Chloro-3-trifluoromethyl-2(1H)-quinoxalinone
(8). This compound (42% yield) was eluted as described
for 3; m.p. 208–210 °C (EtOH); IR: w 1570 cm−1; UV:
1
u 352, 269, 218 nm; H NMR (CDCl3+DMSO-d6): l
13.12 (1H, br s, NH), 7.69 (1H, d, J=2.0 Hz, H-5), 7.57
(1H, dd, J=8.8 and 2.0 Hz, H-7), 7.38 (1H, d, J=8.8
Hz, H-8); MS: m/z 248 (M+).
3.1.2.3. 7-Methoxy-3-trifluoromethyl-2(1H)-quinoxali-
none (4). This compound was obtained in 10% yield by
eluting with a 1:1 mixture of light petroleum–ethyl
acetate; m.p. 260–262 °C; IR: w 1660, 1620 cm−1; UV:
u 358, 264, 232, 214 nm; 1H NMR (CDCl3+DMSO-d6):
l 12.75 (1H, br s, NH), 7.71 (1H, d, J=8.8 Hz,
H-5), 6.86 (1H, dd, J=8.8 and 2.6 Hz, H-6), 6.72 (1H,
d, J=2.6 Hz, H-8), 3.83 (3H, s, CH3); MS: m/z 244
(M+).
3.1.1. Intermediates
The diaminobenzene derivatives 1a,b were commer-
cially available, 1e was prepared following the literature
procedure [5]; 1c [6] was reprepared by us following a
new procedure by hydrogenation of the parent 2,3-
dichloro-6-nitroaniline [7] using 10% Pd/C catalyst at 3
atm. Diamine 1d is a new compound and was prepared
in 80% yield by hydrogenation using 10% Pd/C catalyst
at 3 atm, starting from the parent 2-chloro-3-methoxy-6-
nitroaniline. The latter compound [8] was reprepared by
us in 96% yield following a new procedure by treatment
of the 2,3-dichloro-6-nitroaniline with MeONa/MeOH,
under reflux for 8 h, followed by evaporation in vacuo
of the solvent and treatment with water.
3.1.2.4. 6-Methoxy-3-trifluoromethyl-2(1H)-quinoxali-
none (9). This compound (57% yield) was eluted as
described for 4; m.p. 230–232 °C; IR: w 1670 cm−1; UV:
1
u 288, 237, 206 nm; H NMR (CDCl3+DMSO-d6): l
12.93 (1H, br s, NH), 7.37 (1H, d, J=9.0 Hz, H-8), 7.34
(1H, d, J=1.2 Hz, H-5), 7.24 (1H, dd, J=9.0 and 1.2
Hz, H-7), 3.88 (3H, s, CH3); MS: m/z 244 (M+).
3.1.2.5. 7,8-Dichloro-3-trifluoromethyl-2(1H)-quinoxali-
none (5). This compound was obtained in 46% yield by
eluting with diethyl ether; m.p. 242–244 °C; IR: w 1680,
3.1.1.1. 1,2-Diamino-3-chloro-4-methoxybenzene (1d).
M.p. 100–101 °C; IR: w 3400, 3360, 3280, 3240, 1660,
1640, 1610 cm−1; UV: u 301, 221 nm; 1H NMR (CDCl3):
l 6.59 (1H, d, J=8.2 Hz, H-6), 6.28 (1H, d, J=8.2 Hz,
H-5), 3.82 (1H, s, CH3), 3.70–3.10 (4H, m, 2NH2).
1
1595 cm−1; UV: u 379, 296, 253, 216 nm; H NMR
(CDCl3+DMSO-d6): l 7.82 (1H, d, J=8.8 Hz, H-6),
7.49 (1H, d, J=8.8 Hz, H-5); MS: m/z 281 (M+), 282
(M+).

22
A. Carta et al. / Il Farmaco 57 (2002) 19–25
3.1.2.6. 5,6-Dichloro-3-trifluoromethyl-2(1H)-quinoxali-
none (10). This compound (28% yield) was eluted as
described for 5; m.p. 252–254 °C; IR: w 1685, 1595,
ether; m.p. 236–237 °C; IR: w 1670, 1600 cm−1; UV: u
350 infl, 340, 330 infl, 280 sh, 236, 211 nm; H NMR
(CDCl3+DMSO-d6): l 11.75 (1H, br s, NH), 7.67 (1H,
d, J=8.6 Hz, H-6), 7.40 (1H, d, J=8.6 Hz, H-5), 3.54
(1H, m, CH), 1.26 (6H, d, J=6.8 Hz, 2 CH3); MS: m/z
256 (M+).
1
1
1580 cm−1; UV: u 381, 300 sh, 256, 215 nm; H NMR
(CDCl3+DMSO-d6): l 13.32 (1H, br s, NH), 7.66 (1H,
d, J=8.8 Hz, H-7), 7.33 (1H, d, J=8.8 Hz, H-8); MS:
m/z 282 (M+).
3.1.2.13. 5,6-Dichloro-3-isopropyl-2(1H)-quinoxalinone
(19). This compound (25% yield) was eluted as de-
scribed for 15; m.p. 247–248 °C; IR: w 1665, 1565,
1580 cm−1; UV: u 360 infl, 348, 338 infl, 285, 241, 213
3.1.2.7. 8-Chloro-7-methoxy-3-trifluoromethyl-2(1H)-
quinoxalinone (6). This compound was obtained in 25%
yield by eluting with diethyl ether; m.p. 272–274 °C;
IR: w 1690, 1620, 1600 cm−1; UV: u 362, 253, 220 nm;
1H NMR (CDCl3+DMSO-d6): l 12.40 (1H, br s, NH),
7.88 (1H, d, J=9.2 Hz, H-5), 7.26 (1H, d, J=9.2 Hz,
H-6), 4.04 (3H, s, CH3); MS: m/z 278 (M+).
1
nm; H NMR (CDCl3+DMSO-d6): l 12.58 (1H, br s,
NH), 7.62 (1H, d, J=8.8 Hz, H-7), 7.24 (1H, d, J=8.8
Hz, H-8); 3.51 (1H, m, CH), 1.26 (6H, d, J=6.8 Hz,
2CH3); MS: m/z 256 (M+).
3.1.2.8. 5-Chloro-6-methoxy-3-trifluoromethyl-2(1H)-
quinoxalinone (11). This compound (55% yield) was
eluted as described for 6; m.p. 288–290 °C; IR: w 1680,
3.1.2.14.
8-Chloro-7-methoxy-3-isopropyl-2(1H)-
quinoxalinone (16). This compound was obtained in
47% yield by eluting with diethyl ether; m.p. 244–
245 °C; IR: w 1665, 1620 cm−1; UV: u 348 infl, 338,
1
1610, 1600 cm−1; UV: u 396, 299, 240, 212 nm; H
1
NMR (CDCl3+DMSO-d6): l 13.45 (1H, br s, NH),
7.56 (1H, d, J=9.2 Hz, H-8), 7.34 (1H, d, J=9.2 Hz,
H-7), 3.95 (3H, s, CH3); MS: m/z 278 (M+).
267, 260 sh, 228 sh, 220 nm; H NMR (DMSO-d6): l
11.73 (1H, br s, NH), 7.72 (1H, d, J=9.4 Hz, H-5),
7.17 (1H, d, J=9.4 Hz, H-6), 3.97 (3H, s, OCH3), 3.90
(1H, m, CH), 1.24 (6H, d, J=6.8 Hz, 2CH3); MS: m/z
252 (M+).
3.1.2.9. 7-Chloro-3-isopropyl-2(1H)-quinoxalinone (13).
This compound was obtained in 24% yield by eluting
with a 8:2 mixture of light petroleum–ethyl acetate;
m.p. 207–209 °C; IR: w 1670, 1610 cm−1; UV: u 339,
275, 232, 208 nm; 1H NMR (CDCl3+DMSO-d6): l
12.32 (1H, br s, NH), 7.68 (1H, d, J=8.6 Hz, H-5),
7.28 (1H, d, J=2.0 Hz, H-8), 7.20 (1H, dd, J=8.6 and
2.0 Hz, H-6), 3.52 (1H, m, CH), 1.29 (6H, d, J=6.8
Hz, 2 CH3); MS: m/z 222 (M+).
3.1.2.15. 5-Chloro-6-methoxy-3-isopropyl-2(1H)-quin-
oxalinone (20). This compound (3% yield) was eluted as
described for 16; m.p. 242–243 °C; IR: w 1675, 1610,
1590 cm−1; UV: u 358, 289, 268, 240, 216 nm; 1H
NMR (DMSO-d6): l 12.36 (1H, br s, NH), 7.41 (1H, d,
J=9.2 Hz, H-8), 7.22 (1H, d, J=9.2 Hz, H-7), 3.92
(1H, m, CH), 3.90 (3H, s, OCH3), 1.24 (6H, d, J=6.6
Hz, 2CH3).
3.1.2.10.
6-Chloro-3-isopropyl-2(1H)-quinoxalinone
(18). This compound (16% yield) was eluted as de-
3.1.2.16. 3-Bromomethyl-7,8-dichloro-2(1H)-quinoxali-
none (23). This compound was obtained in 33% yield by
eluting with a 7:3 mixture of diethyl ether–light
petroleum; m.p. 215–216 °C; IR: w 1670, 1595, 1580
cm−1; UV: u 371, 300, 255, 217 nm; 1H NMR (CDCl3):
l 9.60 (1H, br s, NH), 7.73 (1H, d, J=8.4 Hz, H-6),
7.45 (1H, d, J=8.4 Hz, H-5), 4.62 (2H, s, CH2); MS:
m/z 306 (M+).
scribed for 13; m.p. 229–231 °C; IR: w 1660 cm−1
;
UV: u 337, 280, 231, 211 nm; 1H NMR (CDCl3+
DMSO-d6): l 12.06 (1H, br s, NH), 7.79 (1H, d, J=2.6
Hz, H-5), 7.35 (1H, dd, J=8.8 and 2.6 Hz, H-7), 7.22
(1H, d, J=8.8 Hz, H-8), 3.59 (1H, m, CH), 1.30 (6H,
d, J=6.8 Hz, 2CH3); MS: m/z 222 (M+), 222 (M+).
3.1.2.11. 7-Methoxy-3-isopropyl-2(1H)-quinoxalinone
(14). This compound was obtained pure in 79% yield;
m.p. 223–225 °C; IR: w 1670, 1610, 1600 cm−1; UV: u
340, 265, 211 nm; 1H NMR (CDCl3+DMSO-d6): l
12.06 (1H, br s, NH), 7.62 (1H, d, J=8.8 Hz, H-5),
6.80 (1H, dd, J=8.6 and 2.8 Hz, H-6), 6.74 (1H, d,
J=2.8 Hz, H-8), 3.85 (3H, s, CH3O), 3.49 (1H, m,
CH), 1.26 (6H, d, J=6.8 Hz, 2 CH3); MS: m/z 218
(M+).
3.1.2.17. 3-Bromomethyl-5,6-dichloro-2(1H)-quinoxali-
none (25). This compound (20% yield) was eluted as
described for 23; m.p. \300 °C; IR: w 1675, 1600,
1
1590 cm−1; UV: u 372, 296, 250, 216 nm; H NMR
(CDCl3+DMSO-d6): l 12.92 (1H, br s, NH), 7.65 (1H,
d, J=8.6 Hz, H-7), 7.30 (1H, d, J=8.6 Hz, H-8), 4.67
(2H, s, CH2).
3.1.2.18. 3-Bromomethyl-5-chloro-methoxy-2(1H)-quin-
oxalinone (26). This compound was obtained in 20%
yield by eluting with diethyl ether; m.p. 226–228 °C;
IR: w 1665, 1610, 1590 cm−1; UV: u 386, 301, 260 sh,
3.1.2.12. 7,8-Dichloro-3-isopropyl-2(1H)-quinoxalinone
(15). This compound was obtained in 29% yield by
eluting with a 1:1 mixture of light petroleum–diethyl

A. Carta et al. / Il Farmaco 57 (2002) 19–25
23
1
242, 216 nm; H NMR (DMSO-d6): l 12.71 (1H, br
d6): l 12.62 (1H, br s, NH), 7.71 (1H, d, J=8.6 Hz,
H-5), 7.36 (1H, d, J=2.4 Hz, H-8), 7.24 (1H, dd,
J=8.6 and 2.4 Hz, H-6), 4.59 (2H, s, CH2); MS: m/z
272 (M+).
s, NH), 7.54 (1H, d, J=9.2 Hz, H-8), 7.29 (1H, d,
J=9.2 Hz, H-7), 4.65 (2H, s, CH2), 3.93 (3H, s,
CH3).
3.1.3.6. 3-Bromomethyl-6-chloro-2(1H)-quinoxalinone
(24). This compound (34% yield) was eluted as de-
3.1.3. General procedure for preparation of
quinoxalinones 7, 12, 17, 21, 22 and 24
scribed for 22; m.p. 223–225 °C; IR: w 1660 cm−1
;
A mixture of equimolar amounts (2.5 mmol) of the
appropriate diamine and the suitable a-ketoester in
ethanol (10 ml) was stirred at r.t. for 1 h (1a with 2c)
or at reflux for 3 h in the case of 1e with 2a and 2b,
respectively. After evaporation of the solvent, the
crude solid residue was purified by chromatography
on a silica gel column, eluting as reported below.
UV: u 341, 269, 222, 193 nm; 1H NMR (CDCl3+
DMSO-d6): l 12.66 (1H, br s, NH), 7.41 (1H, d,
J=2.4 Hz, H-5), 7.46 (1H, dd, J=8.6 and 2.4 Hz,
H-7), 7.34 (1H, d, J=8.6 Hz, H-8), 4.59 (2H, s,
CH2); MS: m/z 272 (M+).
3.2. Biological assays
3.1.3.1. 8-Amino-3,6-bis(trifluoromethyl)-2(1H)-quinox-
alinone (7). This compound was obtained in 37% yield
by eluting with a 8:2 mixture of diethyl ether–light
petroleum; m.p. 220–222 °C; IR: w 3450, 3390, 1680,
1610 cm−1; UV: u 282, 250, 231 nm; 1H NMR
(CDCl3+DMSO-d6): l 12.50 (1H, br s, NH), 7.41
(1H, s, H-7), 7.21 (1H, s, H-5), 5.89 (2H, br s, NH2);
MS: m/z 297 (M+).
All the compounds were evaluated in vitro for an-
timicrobial activity against yeast (C. albicans), Gram
positive (Staphylococcus aureus) and Gram negative
(Salmonella spp.) bacteria. Miconazole and strepto-
mycin were used as reference drugs in antifungal and
antibacterial assays, respectively. Test compounds
were also evaluated for antiretroviral activity in MT-4
cells infected with HIV-1. Cytotoxicity for MT-4 cells,
carried out in parallel with the anti-HIV-1 activity,
was evaluated to determine whether the test com-
pounds were endowed with selective antimicrobial/an-
tiviral activity.
3.1.3.2. 5-Amino-3,7-bis(trifluoromethyl)-2(1H)-quinox-
alinone (12). This compound (37% yield) was eluted as
described for 7; m.p. 280–282 °C; IR: w 3500, 3400,
1
1680, 1610 cm−1; UV: u 343, 284, 222 nm; H NMR
(CDCl3+DMSO-d6): l 12.50 (1H, br s, NH), 6.80
(1H, s, H-7), 6.68 (1H, s, H-5), 6.34 (2H, br s, NH2);
MS: m/z 297 (M+).
3.2.1. Material and method
3.2.1.1. Compounds. The test compounds were dis-
solved in DMSO at an initial concentration of 200
mM and then were serially diluted in culture medium.
3.1.3.3. 8-Amino-6-trifluoromethyl-3-isopropyl-2(1H)-
quinoxalinone (17). This compound was obtained in
60% yield by eluting with a 8:2 mixture of diethyl
ether–light petroleum; m.p. 270–272 °C; IR: w 3480,
3.2.1.2. Cells. Cells lines were from the American
Type Culture Collection (ATCC). Bacterial and fungal
strains were either clinical isolates (obtained from
Clinica Dermosifilopatica, University of Cagliari) or
collection strains form ATCC. H9/IIIB, MT-4 and
C8166 cells [grown in RPMI 1640 containing 10%
fetal calf serum (FCS), 100 UI/ml penicillin G and
100 mg/ml streptomycin] were used for anti-HIV-1 as-
says. Cell cultures were checked periodically for the
absence of mycoplasma contamination with a Myco-
Tect Kit (GIBCO).
1
3400, 1660, 1630 cm−1; UV: u 277, 229, 211 nm; H
NMR (CDCl3+DMSO-d6): l 11.80 (1H, br s, NH),
7.36 (1H, s, H-7), 7.01 (1H, s, H-5), 3.56 (1H, m,
CH), 1.30 (6H, d, J=6.8 Hz, 2CH3); MS: m/z 271
(M+).
3.1.3.4. 5-Amino-7-trifluoromethyl-3-isopropyl-2(1H)-
quinoxalinone (21). This compound (21% yield) was
eluted as described for 17; m.p. 256–258 °C; IR: w
3490, 3380, 1660, 1610 cm−1; UV: u 328, 280, 228,
1
212 nm; H NMR (CDCl3+DMSO-d6): l 12.18 (1H,
3.2.1.3. Viruses. Human immunodeficiency virus type-
1 (HIV-1, IIIB strain) was obtained from supernatants
of persistently infected H9/IIIB cells. HIV-1 stock so-
lutions had a titer of 5×107 cell culture infectious
dose fifty (CCID50)/ml.
br s, NH), 6.75 (2H, s, H-6+H-8), 3.49 (1H, m,
CH), 1.28 (6H, d, J=6.8 Hz, 2CH3); MS: m/z 271
(M+).
3.1.3.5. 3-Bromomethyl-7-chloro-2(1H)-quinoxalinone
(22). This compound was obtained in 32% yield by
eluting with a 7:3 mixture of light petroleum–ethyl
acetate; m.p. 216–218 °C; IR: w 1670 cm−1; UV: u
336, 277, 219, 195 nm; 1H NMR (CDCl3+DMSO-
3.2.1.4. Anti6iral assay. Activity against the HIV-1
multiplication in acutely infected cells was based on
inhibition of virus-induced cytopathogenicity in MT-4
cells [9]. Briefly, 50 ml of RPMI 10% FCS containing

24
A. Carta et al. / Il Farmaco 57 (2002) 19–25
Table 2
Compound dose (mM) required to reduce the viability of mock-infected MT-4 cells by 50%; (reference compound I CC50=62 mM)
Comp.
CC50
Comp.
CC50
Comp.
CC50
Comp.
CC50
3
4
5
6
7
8
\200
\200
11993
\200
17392.5
16194
9
\200
15
16
17
18
19
20
\200
\200
\200
\200
\200
6492
21
22
23
24
25
26
11495
14692
11293
13993
2492
10
11
12
13
14
10895
9691.5
14391.5
\200
200
2191.8
Data represent mean values of three separate experiments. Variation among triplicate samples was less than 15%.
1×104 cells were added to each well of flat-bottomed
microtiter trays containing 50 ml of medium and serial
dilutions of test compounds. Then, 20 ml of an HIV-1
suspension containing 100 CCID50 were added. After a
4 day incubation at 37 °C, the number of viable cells
was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method [10,11].
Cytotoxicity of compounds, based on the viability of
mock-infected cells, as monitored by the MTT method,
was evaluated in parallel with antiviral activity.
resulting in 50% (EC50, IC50) growth inhibition was
determined by linear regression analysis.
4. Results and discussion
All the quinoxalinones 3–26 were tested for antibac-
terial and antifungal activities in vitro by the microdilu-
tion method. Compound 10 bearing CF3 group in C-3
exhibited only a marginal activity against S. aureus
(MIC=33 mM, MBC=200 mM), while none of the
remaining compounds was found to be active at con-
centrations lower than 200 mM in antibacterial and
antifungal assays. None of the compounds tested was
capable of protecting MT-4 cells from the cytopathic
effect induced by HIV-1, at least at concentrations that
were lower than those cytotoxic.
The results of the in vitro cytotoxicity concerning all
the new quinoxalinones are reported in Table 2.
These data show a generally low cytotoxicity for all
compounds tested with the exception of 25 and 26
which exhibited a higher activity than I [3], which was
used as reference compound.
These results seem to confirm our previous observa-
tions that a CH2Br group at C-3 position promotes
cytotoxic activity, especially when a chlorine atom is
present in C-5 (compounds 25 and 26), while shifting
the chlorine atom in other positions (compounds 22–
24) were less effective.
Despite our expectations, quinoxalinones having a
CF3 group in C-3 (compounds 3–12) were quite inac-
tive in the antifungal assay, while showing only a
moderate antibacterial activity when two chlorine
atoms were present in both C-5 and C-6 positions
(compound 10). On the contrary, the presence of only
one chlorine atom at C-6 or C-7 position, or introduc-
tion of electron-releasing groups (CH3O or NH2) in the
benzo-moiety completely canceled the activity.
In conclusion, the results obtained with this series of
quinoxalinones provide evidence that both pattern and
position of substituents in the benzo-moiety is equally
as important as the substituent in C-3 for biological
activities.
3.2.1.5. Antibacterial assay. S. aureus, group D Strepto-
coccus, Salmonella and Shigella spp. were recent clinical
isolates. Assays were carried out in nutrient broth, pH
7.2, with an inoculum of 103 bacterial cells/tube. Mini-
mum inhibitory concentrations (MIC) were determined
after incubation at 37 °C for 18 h in the presence of
serial dilutions of test compounds. The minimal bacteri-
cidal concentrations (MBC) were determined by subcul-
tivating in Triptosio agar samples from cultures with no
apparent growth.
3.2.1.6. Antimycotic assay. Yeast inocula were obtained
by properly diluting cultures incubated at 37 °C for 30
h in Sabouraud dextrose broth to obtain 5×103 cells/
ml. On the contrary, dermatophyte inocula were ob-
tained from cultures grown at 37 °C for 5 days in
Sabouraud dextrose broth by finely dispersing clumps
with a glass homogenizer before diluting to 0.05
OD590/ml. Then, 20 ml of the above suspensions were
added to each well of flat-bottomed microtiter trays
containing 80 ml of medium with serial dilutions of test
compounds, and were incubated at 37 °C. Growth
controls were visually determined after 2 (yeasts) or 3
days (dermatophytes). MIC was defined as the com-
pound concentration at which no macroscopic sign of
fungal growth was detected. The minimal fungicidal
concentrations (MFC) were determined by subcultivat-
ing in Sabouraud dextrose agar samples from cultures
with no apparent growth.
3.2.1.7. Linear regression analysis. Viral and tumor cell
growth at each drug concentration was expressed as
percentage of untreated controls and the concentrations

A. Carta et al. / Il Farmaco 57 (2002) 19–25
25
uoromethylated five, six and seven membered heterocycles with
two or more heteroatoms, Bull. Soc. Chim. Fr. 6 (1986) 944–
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Acknowledgements
We are very grateful to Mrs A. Canu and Mr P. Fiori
for collaboration on the experimental part and Dr D.
Ghiani for the editing of this manuscript. The authors
thank the Italian Ministero della Sanita` —Istituto Supe-
riore di Sanita` —Progetto AIDS 2000 (grant nn.
40C.47) and Italian MURST (40% 2000) for financial
support.
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