Synthesis and antimicrobial activity of some 2(1H)-quinoxalinone-6-sulfonyl derivatives

Article abstract of DOI:10.1080/104265090889396

The synthesis of some quinoxalinesulfonyl derivatives is described. Two of the synthesized derivatives, 2-oxo-1,2-dihydroquinoxaline-6-sulfonyl azide (3a) and 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl azide (3b), were screened in vitro for their growth inhibitory activity against nine strains of Gram-positive and seven strains of Gram-negative bacteria, along with two fungal isolates. The two compounds showed broad spectrum (in vitro) activity against the bacterial strains. Copyright Taylor & Francis Inc.

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Phosphorus, Sulfur, and Silicon
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Synthesis and Antimicrobial
Activity of Some 2(1H)-
quinoxalinone-6-sulfonyl
Derivatives
a
b
Craig A. Obafemi & D. A. Akinpelu
a
Department of Chemistry, Obafemi Awolowo
University , Ile-Ife, Nigeria
b
Department of Microbiology, Obafemi Awolowo
University , Ile-Ife, Nigeria
Published online: 18 Aug 2006.
To cite this article: Craig A. Obafemi & D. A. Akinpelu (2005) Synthesis and
Antimicrobial Activity of Some 2(1H)-quinoxalinone-6-sulfonyl Derivatives,
Phosphorus, Sulfur, and Silicon and the Related Elements, 180:8, 1795-1807, DOI:
10.1080/104265090889396
To link to this article: http://dx.doi.org/10.1080/104265090889396
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Phosphorus, Sulfur, and Silicon, 180:1795–1807, 2005
Copyright © Taylor & Francis Inc.
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/104265090889396
Synthesis and Antimicrobial Activity of Some
2(1H)-quinoxalinone-6-sulfonyl Derivatives
Craig A. Obafemi
Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria
D. A. Akinpelu
Department of Microbiology, Obafemi Awolowo University,
Ile-Ife, Nigeria
The synthesis of some quinoxalinesulfonyl derivatives is described. Two of the syn-
thesized derivatives, 2-oxo-1,2-dihydroquinoxaline-6-sulfonyl azide (3a) and 2,3-
dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl azide (3b), were screened in vitro
for their growth inhibitory activity against nine strains of Gram-positive and seven
strains of Gram-negative bacteria, along with two fungal isolates. The two com-
pounds showed broad spectrum (in vitro) activity against the bacterial strains.
Keywords 2-Oxo-1,2-dihydroquinoxaline-6-sulfonyl azide; 2,3-dioxo-1,2,3,4-tetra-
hydroquinoxaline-6-sulfonyl azide; antimicrobial activity; synthesis
INTRODUCTION
Various organosulfonyl derivatives, ArSO2X, (sulfonamides, sulfono-
hydrazides, sulfonates, thiosulfonyl compounds, etc.) have been
1
,2
shown to possess pharmacological activities, including antibacterial,
1
3
4−5
6
7
antifungal, nematicidal, diuretic,
hypoglycemic, antitumor, and
protease inhibitory8
−10
activity.
Quinoxaline derivatives constitute an important class of pharmaceu-
1
1,22
tical and agricultural chemicals:
They are used to impart a microbi-
11
12−14
15
cidal finish on cotton fabrics, and act as herbicides,
fungicides,
amongst others. In addition,
certain quinoxaline-2,3-diones and quinoxaline-2-ones have been re-
1
6
17
18,19
antagonists, antibiotics, and dyes,
2
0,21
ported to be highly potent NMDA receptor antagonists
and HIV-1
TAR-Tat inhibitors.22
Received July 26, 2004; accepted September 7, 2004.
Address correspondence to Craig A. Obafemi, Department of Chemistry, Obafemi
Awolowo University, Ile-Ife, Nigeria. E-mail: adeyemi01@yahoo.com
1795

1
796
C. A. Obafemi and D. A. Akinpelu
There is the need for continuous search for simple new compounds
with more potent antimicrobial activities because of constant emer-
gence of drug-resistant microorganisms.
In this article, we report the synthesis of some quinoxalinonesul-
fonyl derivatives and the antimicrobial evaluation of two sulfonyl azide
derivatives.
RESULTS AND DISCUSSION
Chemistry
The quinoxalinesulfonyl derivatives 2 were prepared following the gen-
eral route of chlorosulfonation of aromatic compounds, followed by
the reaction of the formed organosulfonyl chlorides with the desired
nucleophile.
1
,2,3,4-Tetrahydroquinoxaline-2,3-dione (1b) was prepared via two
2
3
routes: (i) by oxidation of quinoxalin-2(1H)-one (1a) with KMnO and
4
(
ii) by the reaction of 1,2-diaminobenzene with oxalic acid under mi-
crowave irradiation.
a was reacted with excess chlorosulfonic acid at about 110 C to
◦
1
give 2-oxo-1,2-dihydroquinoxaline-6-sulfonyl chloride (2a) as the ma-
jor isomer. 2,3-Dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride
(
2b) was similarly prepared from 1b.
Chlorination of 2a and 2b with thionyl chloride-DMF under re-
flux afforded 2-chloroquinoxaline-6-sulfonyl chloride (4a) and 2,3-
dichloroquinoxaline-6-sulfonyl chloride (4b), respectively. Compound
4
a was also obtained from the reaction of 1a with a mixture of chloro-
sulfonic acid and thionyl chloride, together with the formation of 2-
chloroquinoxaline (8).
In addition, the reaction of 2a and 2b with sodium azide gave the
corresponding sulfonyl azides: 2-oxo-1,2-dihydroquinoxaline-6-sulfonyl
azide (3a) and 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl azide
(
3b).
Chlorination of 3a and 3b with thionyl chloride-DMF gave 2-
chloroquinoxaline-6-sulfonyl azide (5a) and 2,3-dichloroquinoxaline-6-
sulfonyl azide (5b), respectively.
Treatment of 2a with hydrazine hydrate gave 2-oxo-1,2-dihydro-
quinoxaline-6-sulfonohydrazide (6), which was further reacted
with 3-methylthiophene-2-carbaldehyde to give 3-methylthiophene-2-
carbaldehyde[2-oxo-1,2-dihydroquinoxalin-6-yl)sulfonyl]hydrazone (7).
These reactions are summarized in Scheme 1.
The yields, physical properties, and analytical data of the synthesized
compounds are given in Table I. The structures of the compounds were

Quinoxalinone Sulfonyl Derivatives
1797
SCHEME 1 Synthetic route to quinoxaline derivatives 1–8. (a) CISO
3
H/heat,
(
3 2
b) NaN /DMF, and (c) SOCl -DMF.
1
assigned by their chemical analysis and infrared, mass, and H-nmr
spectra.
1
The H-nmr data are listed in Table II. The infrared spectra of the
compounds show absorptions due to the stretching vibrations of N3,
C O, and SO2 groups in the range that agree with literature data and
are given in the experimental section.

1
798

Quinoxalinone Sulfonyl Derivatives
1799
TABLE II 1H NMR Data of Quinoxaline Derivatives
Product
1H NMR (DMSO-d6 or CDCl3, ppm)
∗
1
a
12.40 (br s, 1H, NH, D2O exchangeable), 8.20 (s, 1H, H-3), 7.81–7.30
(
m, 4H, Ar).
11.91 (s, 2H, 2NH, D2O exchangeable), 7.13–7.04 (m, 4H, Ar).
12.76 (br s, 1H, NH, D2O exchangeable), 8.32 (s, 1H, H-3), 8.30 (d, 1H, J =
∗
∗
1
2
b
a
2
.00 Hz, H-5), 8.12 (dd, 1H, H-7, J = 8.69 Hz, J = 2.04 Hz), 7.60 (d, 1H,
H–8, J = 8.70 Hz).
∗
∗
2
b
12.30 (s, 1H, NH, D2O exchangeable), 12.01 (s, 1H, NH, D2O
exchangeable), 7.84–7.81 (m, 2H, Ar), 7.15 (d, 1H, Ar).
12.94 (br s, 1H, NH, D2O exchangeable), 8.34 (s, 1H, H-3), 8.32 (d, 1H, H-5
J = 1.98 Hz), 8.13 (dd, 1H, H-7, J = 8.67 Hz, J = 2.06 Hz), 7.56 (d, 1H,
H-8, J = 8.71 Hz).
3
a
∗
∗
∗
∗
∗
∗
4
a
8.98 (s, 1H, H-3), 8.87 (d, 1H, H-5, J = 2.12 Hz), 8.36 (dd, 1H, H-7, J =
8
.98 Hz, J = 2.15 Hz), 8.28 (d, 1H, H-8, J = 9.10).
4
b
8.85 (d, 1H, H-5, J = 2.30 Hz), 8.40 (dd, 1H, H-7, J = 8.90 Hz, J = 2.10 Hz),
8
.30 (d, 1H, H-8, J = 9.15).
5
6
a
8.96 (s, 1H, H-3), 8.80 (t, 1H, J = 1.11 Hz), 8.26 (s, 1H), 8.25 (s, 1H).
12.74 (br s, 1H, NH, D2O exchangeable), 8.46 (br s, 1H, NH, D2O
exchangeable), 8.27 (s, 1H, H-3), 8.13 (d, 1H, H-5, J = 1.95), 7.90 (dd,
∗
1
H, H-7, J = 8.62 Hz, J = 2.00 Hz), 7.43 (d, 1H, H-8, J = 8.64 Hz), 4.17
(
br s, 2H, NH2, D2O exchangeable).
∗
7
12.75 (s, 1H, NH, D2O exchangeable), 11.35 (s, 1H, NH, D2O
exchangeable), 8.25 (s, 1H, H-3), 8.14 (s, 1H), 8.09 (s, 1H), 7.94 (d, 1H,
J = 8.52 Hz), 7.48 −7.41 (m, 2H), 6.86 (d, 1H, J = 4.88), 2.19 (s, 3H, CH3).
8.97 (s, 1H, H-3), 8.14–8.00 (m, 2H, Ar), 7.98–7.84 (m, 2H, Ar).
∗
∗
8
∗
in DMSO-d6 solvent.
in CDCl3 solvent.
∗∗
Antimicrobial Activity
The two quinoxaline-6-sulfonyl azides 3a and 3b were screened in vitro
for possible antibacterial activity. The sensitivity testings (inhibition
−
1
zones (mm)) of 3a and 3b (at 2 mg mL ), streptomycin (a reference
−1
clinical antibiotic, at 1 mg mL ), and DMSO (solvent) against nine
species of Gram-positive and seven Gram-negative bacteria, along with
two fungal isolates, are reported in Table III.
The results of the minimum inhibitory concentration (MIC) (defined
as the lowest concentration of drug that completely inhibited the growth
of the organism) of the two compounds are shown in Table IV.
The results in Table III indicated that the two compounds showed
broad spectrum activity against bacteria strains (a variety of Gram pos-
itive and Gram negative), comparable to the standard reference strep-
tomycin. However, 3b showed larger zones of inhibition than 3a for
most strains.

1
800
C. A. Obafemi and D. A. Akinpelu
TABLE III Antimicrobial Activity of 3a, 3b, and Streptomycin (stm.)
as Determined by Dilution Techniques
Inhibition Zone (mm)
3
a
3b
Stm
2
mL
mg
2 mg
mL
1 mg
mL
−
1
−1
−1
Microorganism
Gram
DMSO
Clostridium sporogenes (NCIB 532)
Escherichia coli (NCIB 86)
Klebsiella pneumoniae (NCIB 418)
Proteus vulgaris (NCIB 67)
−
−
−
−
−
22
0
32
30
24
0
25
0
0
28
30
0
0
0
0
0
17
15
17
18
Pseudomonas fluorescens
(
NCIB 3756)
Serratia marcescens (NCIB 1377)
Shigella dysenteriae (LIO)
Bacillus anthracis (LIO)
Bacillus cereus (NCIB 6349)
Bacillus polymyxa (LIO)
−
−
+
+
+
+
16
25
0
20
0
25
18
24
16
17
31
19
22
18
28
15
23
0
0
0
0
0
0
Bacillus stearothermophilus
18
(
NCIB 8222)
Bacillus subtilis (NCIB 3610)
Corynebacterium pycogenes (LIO)
Micrococcus luteus (NCIB 196)
Staphylococcus aureus (NCIB 8588)
Streptococcus faecalis (NCIB 755)
Aspergillus flavus
+
+
+
+
+
12
11
0
27
14
18
26
32
15
0
21
13
15
25
20
20
25
21
23
0
0
0
0
0
0
0
ND
ND
Candida albicaus
NCIB = National Collection of Industrial Bacteria.
LIO = Locally Isolated Organism.
ND = Not done.
−
1
−1
The MICS of 3a varied between 31.3 µg mL and 1000 µg mL for
−1
−1
all strains, and between 15.6 µg mL and 500 µg mL for compound
3
b. These results indicated that 3b has a greater activity than 3a.
The two quinoxalinesulfonyl azides were further characterized in the
time-kill studies, using Escherichia coli as a Gram-negative represen-
tative and Staphylococcus aureus (NCIB 8588) as a representative of
Gram-positive organism, and the results are shown in Figures 1 and 2,
respectively.
−
1
As shown in Figure 1, at their MIC values (250 µg mL for 3a and
−1
6
2.5 µg mL for 3b), compound 3a exhibited a higher degree of killing,
reaching 96% killing at 90 min compared to 3b which reached only 52%
killing at 90 min. At four times the MICs, the two compounds showed
a similar trend, with 3a reaching a bactericidal activity (100% killing)
at 60 min, while 3b only reached 100% killing at 90 min.

Quinoxalinone Sulfonyl Derivatives
1801
TABLE IV Minimum Inhibitory Concentration (MIC) for 3a and 3b
3a (µg mL−1)
−1
3b (µg mL )
Microorganism
Clostridium sporogenes (NCIB 532)
Escherichia coli (NCIB 86)
Klebsiella pneumoniae (NCIB 418)
Proteus vulgaris (NCIB 67)
Pseudomonas fluorescens (NCIB 3756)
Serratia marcescens (NCIB 1377)
Shigella dysenteriae (LIO)
Bacillus anthracis (LIO)
Bacillus cereus (NCIB 6349)
Bacillus polymyxa (LIO)
Bacillus stearothermophilus (NCIB 8222)
Bacillus subtilis (NCIB 3610)
Corynebacterium pyogenes (LIO)
Micrococcus luteus (NCIB 196)
Staphylococcus aureus (NCIB 8588)
Streptococcus faecalis
250
250
31.3
31.3
250
250
250
—
125
—
250
125
500
—
—
62.5
31.3
250
—
15.6
15.6
500
15.6
500
62.5
15.6
62.5
—
1000
500
15.6
250
NCIB = National Collection of Industrial Bacteria.
LIO = Locally Isolated Organism.
In the case of S. aureus as shown in Figure 2, at their MIC values
1000 µg mL for 3a and 15.6 µg mL for 3b), 3a again showed a
−
1
−1
(
higher rate of killing and reaching 100% killing within 90 min, and 3b
reached only 90% killing. There was no significant effective change at
two times the MIC of 3b. Even at four times the MIC, compound 3b
Figure 1 Rate of killing of E. coli (representing Gram-Organism) by 3a and
3b.

1
802
C. A. Obafemi and D. A. Akinpelu
aF ni gd u3 rb e. 2 Rate of killing of S. aureus (representing Gram + Organism) by 3a
still exhibited a lower rate of killing compared to the killing rate by 3a
at its MIC.
EXPERIMENTAL
Chemistry
Melting points were determined on a Gallenkamp melting point appara-
tus and no corrections. For TLC, precoated silica gel thin-layer sheets F
1
13
2
54 from MERCK was used. H- and C-NMR were recorded as CDCl3
or DMSO-d6 solutions on a Brucker-AC-250 and JOEL-JNMGX 400-
MHZ spectrometer (δ in ppm relative to Me4Si and H3PO4). Mass spec-
tra (E1–MS) were recorded on a finnigan MAT 312 machine (in m/z (rel.
%
)) (all at the Fakultat fur Chemie, Universitat Konstanz, Konstanz,
Germany). Infrared spectra were recorded on a BUCK Scientific
spectrometer.
1
,2,3,4-Tetrahydroquinoxaline-2,3-dione (1b)
Method a. A solution of KMnO4 (27.0 g, 171 mmol) in water (300
mL) was added dropwise to a boiling solution of quinoxalin-2(1H)-one
1a) (5.0 g, 34 mmol) and NaOH (1.4 g, 35 mmol) in water (300 mL)
(
for a period of 2 h with vigorous stirring. The resulting mixture was
refluxed for another 1 h and then cooled to about 60 . The MnO2 was
◦
filtered off and washed with hot water twice (2 × 50 mL). The combined
filterate was concentrated to 50 mL and then acidified with acetic acid.
The resulting solid product was purified by column chromatography

Quinoxalinone Sulfonyl Derivatives
1803
(
1
(
alumina-neutral). Elution with CHCl3/CH3OH (9/1) gave unreacted
◦
a (2.2 g), while acetic acid gave 1b (2.9 g, 52%). mp > 320 C. MS: 162
+
+
+
17, M ), 134 (46, [M-CO] ), 106 (100, [M-2CO] ).
Method b: Microwave-assisted synthesis. A mixture of oxalic
acid dihydrate (10.0 g, 79.4 mmol) and benzene-1,2-diamine (8.6 g,
9.6 mmol) was ground in a mortar and placed in an open beaker. Water
1 mL) was added and mixed thoroughly and then irradiated in a do-
7
(
mestic MW oven at an emitted power of 400 W for 3 min. Water (100 mL)
was added and irradiated for 1 min to give a clear solution and then
left to stand at room temperature to give colorless needles of 1b (12.8 g,
9
9%). The physical and spectroscopic data of 1b were identical to those
of authentic 1,2,3,4-tetrahydroquinoxaline-2,3-dione.
2
-Oxo-1,2-dihydroquinoxaline-6-sulfonyl chloride (2a)
Quinoxalin-2(1H)-one (1a) (10.0 g, 68.5 mmol) was added in portions
to chlorosulfonic acid (46 mL, 10 mol. Equiv.), after which the mixture
◦
was heated (under reflux) at 110 for 8 h. The reaction mixture was
cooled in ice water before it was poured into crushed ice. The resulting
solid was filtered and washed with cold water and dried. Recrystal-
−
1
lization from toluene-acetone gave 2a (13.5 g). Ir (cm ) 3300 (NH),
+
+
1
680 (C O), 1380, 1165 (SO2). MS: 246 (7.8, [M + 2] ), 244 (20.0 [M ],
+
+
2
09 (55.3, [M Cl] ), 145 (43.2, [M Cl SO2] ), 117 (100), 90 (25.4), 64
(
18.7).
Compound 2b was similarly obtained from 1b. Ir (cm ): 3380 (NH),
−
1
+
+
1
2
680 (C O), 1355, 1140 (SO2). MS: 262 (5.3, [M + 2] ), 260 (14.0, [M ],
+
+
25 (48.1, [M Cl] ), 161 (100, [M Cl SO2] ), 133 (45.4), 105 (70.5), 78
(
45.2), 51 (82.3).
2
-Oxo-1,2-dihydroquinoxaline-6-sulfonyl azide (3a)
Sodium azide (7.0 g, 108 mmol) was added to a stirring solution
of 2a (13.2 g, 54 mmol in acetone (250 mL)), followed by addition
of water (5.0 mL) and the mixture stirred at room temperature for
8
h. Removal of acetone followed by addition of water gave 2-oxo-1,2-
dihydroquinoxaline-6-sulfonyl azide as light pink crystals. Recrystal-
lization from ethanol gave pure 3a (13.0 g). Ir (cm ): 3290 (w, NH),
−
1
+
2
131 (N3), 1664 (C O), 1350, 1173 (SO2). MS: 251 (28.0, M ), 209 (59.3,
+
[
M N3] ), 145 (77.2), 117 (100), 90 (12.5).
Compound 3b was prepared in a similar manner from 2b.
−
1
3
b: Ir (cm ): 2159 (N3), 1690 (C O), 1365, 1160 (SO2). MS: 267 ( 100,
+
+
+
M ), 239 (17.0, [M CO] ), 225 (18.3, [M N3] ), 211 (6.0), 161 (10.0),
33 (5.0), 105 (22.1).
1

1
804
C. A. Obafemi and D. A. Akinpelu
2
-Chloroquinoxaline-6-sulfonyl chloride (4a)
A mixture of 2a (2.0 g, 8.2 mmol), DMF (0.1 mL) and excess SOCl2
5.0 mL) was heated to reflux for 1.5 h with continuous stirring. The
(
excess SOCl2 was distilled off, with the last traces removed on a ro-
tatory evaporator. The remaining solid residue was washed with cold
water (3 × 10 mL), dried in air, and then recrystallized from petroleum
◦
−1
ether (bp 30–80 ) to give 4a (1.9 g). Ir (cm ): 1380, 1180, 1160
+
+
(
[
(
SO2). MS: 264 (16.4, [M + 2] ), 262 (22.4, M ), 229 (25.1), 227 (71.0
M Cl] ), 165 (32.2), 163 (100, [M Cl SO2] ), 127 (10.2). 101 (15.6), 75
24.1).
+
+
Compounds 4b, 5a, and 5b were prepared in a similar manner:
−
1
+
+
4
b: Ir (cm ): 1370, 1170 (SO2), MS: 300 (6.7 [M + 4] ), 298 (18.4,
+
+
[
1
M + 2] ), 296 (19.1, M ), 263 (39.5), 261 (55.5, [M Cl] ), 199 (54.4),
+
+
97 (88.1, [M Cl SO2] ), 162 (14.4, [M Cl2 SO2] ), 136 (18.5), 101
(
34.7), 75 (85).
−
1
+
5
a: Ir (cm ): 2160 (N3), 1380, 1160 (SO2). MS: 271 (5.9, [M + 2] ),
+
+
2
69 (14.2, M ), 229 (26.3), 227 (72.8, [M N3] ), 165 (35.8), 163 (100,
+
+
[
M-N3-SO2] ), 127 (8.6, [M-N3-SO2-HCl] ), 101 (13.7), 75 (20.4).
−
1
+
5
b: Ir (cm ): 2120 (N3), 1370, 1160 (SO2), MS: 305 (11.7, [M + 2] ),
+
+
3
03 (16.5, M ), 263 (49.9), 261 (69.9, [M-N3] ), 199 (66.4), 197 (100,
+
[
M-N3-SO2] ), 162 (15.8), 136 (21.0), 101 (33.8), 75 (84.1).
2
-Oxo-1,2-dihydroquinoxaline-6-sulfonohydrazide (6)
2
-Oxo-1,2-dihydroquinoxaline-6-sulfonyl chloride (2a) (2.0 g, 8.2
mmol) was added in portions to hydrazine hydrate (2.0 g, 34 mmol)
in alcohol, after which the reaction mixture was stirred at room tem-
perature overnight. The mixture was poured into cold water to give a
solid product. Recrystallization from ethanol gave the pure hydrazide 6
−
1
(
(
1
(
1.3 g). Ir (cm ): 3320, 3300, 3210, 3200 (NH), 1670 (C O), 1355, 1155
+
+
SO2). MS: 240 (2.0, M ), 225 (10.0), 210 (11.3), 209 (2.0, [M NHNH2] ),
93 (6.4), 178 (11.9), 161 (17.6), 146 (40.9), 145 (11.4), 117 (54.5), 106
100).
3
-Methylthiophene-2-carbaldehyde[2-oxo-1,2-
dihydroquinoxaline-6-yl) sulfonyl]hydrazone (7)
A mixture of 2-oxo-1,2-dihydroquinoxaline-6-sulfonohydrazide (6)
(
(
1.0 g, 4.2 mmol) in ethanol and 3-methyl-thiophene-2-carbaldehyde
0.5 mL, 4.6 mmol) was refluxed for 1 h and then allowed to cool to room
−
1
temperature to give analytically pure 7 (1.4 g). Ir (cm ): 1665 (C O),
1
+
610, 1369, 1165 (SO2). MS: 348 (11.8, M ), 248 (31.8), 221 (15.1), 210
(
11.0), 193 (12.6), 178 (74.7), 150 (17.8), 146 (45.9), 140 (10.6), 139 (100),

Quinoxalinone Sulfonyl Derivatives
1805
1
26 (37.4), 125 (52.4), 123 (35.3), 122 (37.2), 118 (18.2), 117 (19.3), 112
(
23.9), 110 (77.2), 98 (22.8), 97 (28.2), 69 (25.8).
Chlorosulfonation of Quinoxalin-2(1H)-one (1a) in the
Presence of Thionyl Chloride
Quinoxalin-2(1H)-one (1a) (10.0 g, 68.5 mmol) was added in portions
to chlorosulfonic acid (46 mL, 697 mmol), followed by addition of thionyl
chloride (25 mL, 348 mmol) and the mixture heated at 110 for 11 h.
Excess thionyl chloride was distilled off, the remaining mixture cooled
in ice-water and then poured carefully into crushed ice with continuous
stirring. The resulting solid was filtered, washed with cold water and
dried in air. Repeated recrystallizations from petroleum either (bp 30–
◦
8
2
0 ) gave pure 4a (8.0 g, 44%). From the mother liquors was obtained
1
-chloroquinoxaline (8) (8.5 g) (mp, mmp, H—and mass spectra).
Antimicrobial Screening
Media Used
Nutrient broth (Oxoid Ltd.) and nutrient agar (Oxoid Ltd.) were used
for subculturing the bacterial isolates, while diagnostic sensitivity test
agar (Oxoid Ltd.) was used for sensitivity testing.
Microorganisms Used
The following microorganisms were used Clostridium sporogenes
(
4
NCIB 532), Escherichia coli (NCIB 86), Klebsiella pneumoniae (NCIB
18), Proteus vulgaris (NCIB 67), Pseudomonas aeruginosa (NCIB 950),
Pseudomonas fluorescens (NCIB 3756), Serratia marcescens (NCIB
377), Shigella dyseuteriae (LIO), Bacillus anthracis (LIO), Bacil-
1
lus cereus (NCIB 6349), Bacillus polymyxa (LIO), Bacillus stearother-
mophilus (NCIB 8222), Bacillus subtilis (NCIB 3610), Corynebacterium
pyogenes (LIO), Micrococcus luteus (NCIB 196), Staphylococcus aureus
(
NCIB 8588), Streptococcus faecalis (NCIB 755), Aspergillus flavus, and
Candida albicans.
Sensitivity Testing27
The sensitivity testing of the two synthetic compounds were deter-
mined using agar-well diffusion method.
The bacterial isolates were first grown in nutrient broth (Ox-
oid Ltd.) for 18 h before use. The inoculum suspensions were stan-
dardized and then tested against the effect of the two compounds
at a concentration of 2 mg/mL each in diagnostic sensitivity test
agar (Oxoid Ltd.). The plates were observed for zones of inhibi-
◦
tion after 24 h incubation at 37 C. The effects were compared with

1
806
C. A. Obafemi and D. A. Akinpelu
that of the streptomycin standard antibiotic at a concentration of
mg/mL.
1
Minimum Inhibitory Concentration (MIC)28
The MIC of different concentrations of the two compounds was de-
termined using two-fold dilutions method. Different concentrations of
the synthetic compound ranging between 0.0313 mg/mL and 1 mg/mL
were prepared. Two millimetres of the concentrate from each dilution
was added to 18 mL of molten sterile nutrient agar (Oxoid Ltd.) asep-
tically, and thoroughly mixed together in a sterile Petri dish. This was
allowed to set. The surface of the nutrient agar was allowed to dry prop-
erly before streaking with the appropriate bacterial isolate. The plates
◦
were then incubated at 37 C for up to 72 h. The lowest concentration
preventing the growth was taken as the minimum inhibitory concen-
tration of the compound.
Determination of the Rate of Killing of the Compounds
(
Bactericidal Activity of the Compounds)
The bactericidal activity of different concentrations of the two com-
pounds were determined by the extent of killing at time intervals 0,
5, 30, 45, 60, 75, and 90 min. Viable counts of the bacterial cells to
1
be used were determined first. Two representatives of the bacterial iso-
lates were used; these were Staphylococcus aureus representing Gram-
positive bacteria and Escherichia coli representing Gram-negative bac-
teria. Serial dilutions of the test organism was done and about 1 mL of
−
5
10
dilution of the test organism was added to 4 mL of the test con-
centration of the compound. The suspension was thoroughly mixed and
◦
held at room temperature (28 C). Exactly 1 mL volume of each sus-
pension was withdrawn at the appropriate intervals and transferred
to 4.0 mL of nutrient broth (Oxoid Ltd.) recovery medium containing
3
% Tween 80 to neutralize the effects of any antimicrobial compound
carry-overs from the suspension. One mL of the final dilution was plated
out and incubated at 37 C for 72 h. Depressions in the viable counts
◦
indicated killing by the compound.
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