Synthesis and antimycobacterial activity of new quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives

Article abstract of DOI:10.1016/S0968-0896(03)00119-6

As a continuation of our research and with the aim of obtaining new antituberculosis agents which can improve the current chemotherapeutic antituberculosis treatments, new series of quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives were synthesized and

Full text of DOI:10.1016/S0968-0896(03)00119-6

Bioorganic & Medicinal Chemistry 11 (2003) 2149–2156
Synthesis and Antimycobacterial Activity of New Quinoxaline-2-
carboxamide 1,4-di-N-Oxide Derivatives
Belen Zarranz, Andres Jaso, Ignacio Aldana* and Antonio Monge
´
Unidad en Investigacion y Desarrollo de Medicamentos, Centro de Investigacion en Farmacobiologıa Aplicada (CIFA),
´
´
Universidad de Navarra, Pamplona, Spain
Received 20 December 2002; accepted 18 February 2003
Abstract—As a continuation of our research and with the aim of obtaining new antituberculosis agents which can improve the current
chemotherapeutic antituberculosis treatments, new series of quinoxaline-2-carboxamide 1,4-di-N-oxide derivatives were synthesized and
evaluated for in vitro antituberculosis activity against Mycobacterium tuberculosis strain H37Rv, using the radiometric BACTEC 460-TB
methodology. Active compounds were also screened by serial dilution to assess toxicity to a VERO cell line. The results indicate that
some compounds exhibited a good antituberculosis activity and the arylcarboxamide analogues 3, 8, and 9 were the most active com-
pounds (EC90/MIC1). Also, the cytotoxic effects indicate that these compounds have a good Selectivity Index (SI).
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
As a result of the antituberculosis research project, our
group has had several papers published in which the
synthesis and biological assessment of a large amount of
quinoxaline and quinoxaline 1,4-di-N-oxide derivatives
have been described.5,6 Therefore, for example, different
7-chloro-3-(p-substituted)phenylaminoquinoxaline-2-
carbonitrile 1,4-di-N-oxides (I, Fig. 1) have been shown
to possess M. tuberculosis growth inhibition values of
99%7 and 6,7-dichloro-2-ethoxycarbonyl-3-methylqui-
noxaline 1,4-di-N-oxide (II, Fig. 1) and 3-acetamide-6,7-
dichloroquinoxaline-2-carbonitrile 1,4-di-N-oxide deri-
vatives produced growth inhibition values of 100%.8,9
On the other hand, we observed that the lack of the two
N-oxide groups generally led to the loss of the anti-
mycobacterial activity.9,10
Tuberculosis (TB), an infection of Mycobacterium
tuberculosis, still remains the leading cause of worldwide
death among infectious diseases. One-third of the
population is infected with M. tuberculosis and the
World Health Organization (WHO) estimates that
within the next 20 years about 30 million people will be
infected with the bacillus.1
The mycobacterial cell wall is the site of action of many
of the first line antimycobacterial agents and contains
numerous components that are presumed to be required
for cell viability or survival in the host and therefore,
are attractive drug targets.2
Current frontline therapy consists of administering one
of three drugs (isoniazid, rifampin and pyrazinamide)
for 2 months followed by 4 months of follow up therapy
with isoniazid and rifampin.3 But the ineffectiveness of
the present therapy is found to be responsible for a very
long duration of the therapy as well as the emergence of
resistance to these drugs. Thus, the problem arising due
to MDR-TB requires the development of new ther-
apeutic agents that have unique mechanism of action
from presently used antitubercular drugs in order to
treat drug resistant forms of the disease.4
As a consequence of this research and with the aim of
obtaining new and more potent antituberculosis com-
pounds which can improve the current chemothera-
peutic antituberculosis treatments, we have synthesized
and evaluated 31new carbonylquinoxaline ,14-di- N-
oxide derivatives possessing different amine groups in
the 2-position and methyl group in the 3-position (III,
Fig. 1).
Chemistry
The aforementioned compounds were prepared accord-
ing to the synthetic sequences illustrated in Scheme 1.
The starting compounds, 5-substituted benzofuroxane
*Corresponding author. Tel.: +34-948-425653; fax: +34-948-425652;
e-mail: ialdana@unav.es
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00119-6

2150
B. Zarranz et al. / Bioorg. Med. Chem. 11 (2003) 2149–2156
chloromethane. The organic solvent was eliminated
under pressure and a solid or oil was obtained.
Formation of isomeric quinoxaline 1,4-di-N-oxides was
observed in the case of monosubstituted benzofuroxanes.
According to previous reports,12 we have observed that
7-substituted quinoxaline 1,4-di-N-oxides were prevailing
over the 6-isomer, or the only 7-isomer formed in the case
of methoxy substituent. In practice, the workup and pur-
ification allows isolation of the 7 isomer.13
Figure 1.
All of the compounds were chemically characterized by
thin-layer chromatography (TLC), melting point, infra-
red (IR) and nuclear magnetic resonance (1H NMR)
spectra as well as elemental microanalysis.
or 5,6-disubstituted benzofuroxane were obtained by
previously described methods.6
The synthesis of compounds 1–14 was carried out by
reaction of the appropriate benzofuroxane with the
corresponding 3-oxobutyramide, using triethylamine.
The 2-carboxamide derivatives with general structure 2-
carbonylpiperazine, namely 15–31, were obtained by the
reaction of benzofuroxane with the corresponding N-(3-
oxobutyryl)-piperazine, using morpholine as catalyst.
Pharmacology
In vitro evaluation of the antituberculosis activity was
carried out at the GWL Hansen’s Disease Center within
the Tuberculosis Antimicrobial Acquisition & Coordi-
nating Facility (TAACF) screening program for the
discovery of novel drugs for the treatment of tubercu-
losis. Under the direction of the US National Institute
of Allergy and Infectious Disease (NIAID), Southern
Research Institute coordinates the overall program.
The intermediates N-(3-oxobutyryl)-piperazine were
prepared according to the synthetic procedure for simi-
lar compounds.11 The methyl acetoacetate was heated
with the corresponding piperazine in the presence of
2-hydroxypyridine as catalyst in a bath at 169 ꢀC for 5
h, under nitrogen atmosphere as illustrated in Scheme 2.
The partly cooled mixture was then stirred into hot
water. The resulting suspension was dissolved in di-
The purpose of the screening program is to provide a
resource whereby new experimental compounds can be
tested for their capacity to inhibit the growth of virulent
M. tuberculosis. Biological tests have been performed
according to the previously described method.14,15
Scheme 1. Synthesis of compounds 1–31.
Scheme 2. Synthesis of the intermediates N-(3-oxobutyryl)-piperazine derivatives.

B. Zarranz et al. / Bioorg. Med. Chem. 11 (2003) 2149–2156
2151
Results and Discussion
active in the first level screening were then tested to
determine the actual MIC. MIC is defined as the lowest
concentration effecting a reduction in fluorescence of
90% relative to controls. Therefore, compounds 3, 4, 5,
and 14 have proven to be the most efficient ones, with
MIC values ranging from 0.78 to 3.13 mM. Concurrent
with the determination of MICs, compounds were tes-
ted for cytotoxicity (IC50) in VERO cells, and the
Selectivity Index (SI=IC50/MIC) was calculated.
The results of the in vitro evaluation of antituberculosis
activity are reported in Tables 1–3. As described in pre-
vious papers, the presence of 1,4-di-N-oxides is essential
for the activity of the compounds.6 In general, most com-
pounds with a N-arylcarboxamide group in position 2, 1–
10, have been shown to possess good antimycobacterial
activity in the run of the first level screening, with growth
inhibition values ranging from 98 to 100%. Only com-
pounds 1, 2, 6 and 7 showed low or no activity (0%,
62%, 22% and 70% growth inhibition, respectively).
On the contrary, 2-piperazinylcarbonyl quinoxaline
derivatives 15–31, showed no activity (Table 1).
Three compounds, 3, 8 and 9, with a high SI value
(>40.06, >10 and >10, respectively), were then tested
for efficacy in vitro in a TB-infected macrophage model,
showing good values of EC90 and EC99. Macrophage
assay results are reported in Table 3.
Compounds with a chloro in the 7- or 6,7-positions (4,
5, 9 and 14) and corresponding unsubstituted deriva-
tives (3 and 8) showed the best activity. An electron-
releasing group (CH3) on the benzene moiety reduces
the activity.
On the basis of the obtained data we can remark the
interesting antitubercular properties showed by some N-
phenylquinoxaline-2-carboxamide 1,4-di-N-oxide deri-
vatives, namely 3, 8 and 9. These compounds have
shown very good activity in macrophage assay (EC90/
MIC=0.89, 0.42 and 0.14, respectively). SDR-MIC and
MBC studies are scheduled. The activity is significantly
Second level assays and cytotoxicity results are sum-
marized in Table 2. All of the compounds that were
Table 1. Results of the first antituberculosis screening
Compd
R
Ra
Rb
MIC (mg/mL)a
GI (%)b
1
2
3
4
5
6
7
8
NH–C6H5
NH–C6H5
NH–C6H5
CH3
CH3
H
Cl
Cl
CH3
CH3
H
Cl
Cl
CH3
H
Cl
Cl
CH3
CH3
Cl
CH3
H
CH3
H
H
H
Cl
CH3
H
H
H
Cl
CH3
H
H
Cl
CH3
H
Cl
CH3
H
CH3
H
H
Cl
H
CH3
Cl
H
Cl
>6.25
>6.25
<6.25
<6.25
<6.25
>6.25
>6.25
<6.25
<6.25
>6.25
>6.25
>6.25
>6.25
<6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
0
62
100
99
100
22
70
98
98
100
0
10
51
100
0
0
0
0
0
0
0
0
0
0
0
NH–C6H5
NH–C6H5
20-CH3–C6H4–NH
20-CH3–C6H4–NH
20-CH3–C6H4–NH
9
20-CH3–C6H4–NH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
20-CH3–C6H4–NH
NH–C(CH3)3
NH–C(CH3)3
NH–C(CH3)3
NH–C(CH3)3
4-C6H5-Piperazin-1-yl
4-C6H5-Piperazin-1-yl
4-C6H5-Piperazin-1-yl
4-(40-Chlorophenyl)piperazin-1-yl
4-(40-Chlorophenyl)piperazin-1-yl
4-(40-Nitrophenyl)piperazin-1-yl
4-(40-Nitrophenyl)piperazin-1-yl
4-(40-Nitrophenyl)piperazin-1-yl
4-(40-Nitrophenyl)piperazin-1-yl
4-(40-Nitrophenyl)piperazin-1-yl
4-(20-Methoxyphenyl)piperazin-1-yl
4-(20-Methoxyphenyl)piperazin-1-yl
4-(40-Methoxyphenyl)piperazin-1-yl
4-(40-Methoxyphenyl)piperazin-1-yl
4-Benzylpiperazin-1-yl
4-Benzylpiperazin-1-yl
4-Benzylpiperazin-1-yl
CH3
CH3
H
Cl
OCH3
CH3
Cl
H
Cl
CH3
H
Cl
0
0
0
0
0
0
CH3
H
Cl
aMIC of Rifampin: 0.125 mg/mL versus M. tuberculosis H37Rv (97% inhibition).
bGrowth Inhibition of virulent H37Rv strain of M. tuberculosis. According to the TAACF program, compounds effecting less than 90% inhibition
are considered to be inactive.

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B. Zarranz et al. / Bioorg. Med. Chem. 11 (2003) 2149–2156
Table 2. Results of second level and cytotoxicity antituberculosis
assays
A 4 mm (3.9ꢁ150 mm); mobile phase: acetonitrile/iso-
propanol 50/50; flux: 1mL/min.
Compd
MIC (mM)a
IC50 (mM)b
SI (IC50/MIC)c
Chemicals were purchased from E. Merck (Darmstadt,
Germany), Scharlau (F.E.R.O.S.A., Barcelona, Spain),
Panreac Quımica S.A. (Montcada i Reixac, Barcelona,
Spain), Sigma-Aldrich Quımica, S.A., (Alcobendas,
Madrid), Acros Organics (Janssen Pharmaceuticalaan
3a, 2440 Geel, Belgie) and Lancaster (Bischheim-Stras-
bourg, France).
3
4
5
8
9
14
3.13
1.56
0.78
6.25
6.25
1.56
1
>10
Insoluble
>62.5
>62.5
>10
>40.06
>6.41
Insoluble
>10
>10
>6.41
aActual minimun inhibitory concentration (MABA assay).
bMeasurement of cytotoxicity in VERO cells.
cSelectivity Index.
General procedure for compounds 1–14
The corresponding 3-oxobutyramide (4,8 mmol) was
added to a solution of the appropriate benzofuroxane
(2,4 mmol) in dry chloroform (35 mL). The mixture was
allowed to stand at 0 ꢀC. Next, triethylamine was added
drop by drop (0.1mL). The solution was stirred at room
temperature for 24 h. The solvent was eliminated under
pressure. The resulting solid was washed with ethyl
ether (or n-hexane). The obtained yellow precipitate was
purified by recrystallization using methanol. Yields:
5–64%.
Table 3. Results of macrophage assay
a
a
Compd
EC90
EC99
EC90/MICb
3
8
9
2.79
2.60
0.86
10.07
11.08
6.75
0.89
0.42
0.14
aThe EC90 and EC99 are defined as the concentrations effecting 90 and
99% reduction in residual mycobacterial growth after 7 days, as com-
pared to untreated controls.
bCompounds with EC90>16ꢁMIC are considered inactive.
3,6,7-Trimethyl-N-phenylquinoxaline-2-carboxamide 1,4-
dioxide (1). Mp 229–230 ꢀC. IR (KBr) n 3250, 1682,
affected by substituents, both on position 7 of quinox-
aline nucleus and position 20 of the phenyl ring. Com-
pounds with a chloro in 7 position and corresponding
unsubstituted derivatives showed the best activity. With
regard to the benzene moiety, greater effectiveness
resulted for ortho-methyl substituents.
1
1334 cmꢂ1; H NMR (DMSO-d6) d 2.50 (s, 3H, C3–
0
CH3), 2.54 (s, 6H, 2CH3–Ar), 7.21(t, 1H, H 4 ), 7.45 (t,
0
0
0
0
0
0
2H, H3 +H5 , J3 ꢂ4 =7.7 Hz), 7.71(d, 2H, H 2 +H6 ,
0
0
J2 ꢂ3 =7.9 Hz), 8.29 (s, 1H, H5), 8.33 (s, 1H, H8), 11.03
(s, 1H, NH) ppm. Anal. (C18H17N3O3) C, H, N; C (%):
calcd: 66.87; found: 66.51. H (%): calcd: 5.26; found:
5.15. N (%): calcd: 13.00; found: 13.05 (33% yield).
Conclusion
3,7-Dimethyl-N-phenylquinoxaline-2-carboxamide 1,4-di-
oxide (2). Mp 206–207 ꢀC. IR (KBr) n 3249, 1687,
In conclusion, we can assert that the promising results
obtained for the new N-phenylquinoxaline-2-carbox-
amide 1,4-di-N-oxide derivatives presented in this paper
make them possible candidates for the treatment of
tuberculosis and encourage us to advance in the synth-
esis and evaluation of new derivatives.
1
1554, 1333 cmꢂ1; H NMR (DMSO-d6) d 2.47 (s, 3H,
0
C3–CH3), 2.59 (s, 3H, CH3–Ar), 7.20 (t, 1H, H4 ), 7.41
0
0
0
0
0
0
(t, 2H, H3 +H5 , J3 ꢂ4 =7.7 Hz); 7.67 (d, 2H, H2 +H6 ,
0
0
J2 ꢂ3 =8.0 Hz), 7.85 (d, 1H, H6, J6ꢂ5=8.0 Hz), 8.29 (s,
1H, H8), 8.41(d, 1H, H 5), 10.98 (s, 1H, NH) ppm. Anal.
(C17H15N3O3) C, H, N; C (%): calcd: 66.02; found:
66.05. H (%): calcd: 4.85; found: 4.75. N (%): calcd:
13.59; found: 13.35 (35% yield).
Experimental
Chemistry
3-Methyl-N-phenylquinoxaline-2-carboxamide 1,4-diox-
ide (3). Mp 225–226 ꢀC. IR (KBr) n 3251, 1686, 1309,
Melting points were determined with
FP82+FP80 apparatus (Greifense, Switzerland) and
a
Mettler
757 cmꢂ1 1H NMR (DMSO-d6) d 2.51(s, 3H, C
. –
3
0
0
0
CH3), 7.20 (t, 1H, H4 , J4 ꢂ3 =6.9 Hz), 7.43 (t, 2H,
1
0
0
0
0
0
0
have not been corrected. The H NMR spectra were
H3 +H5 ), 7.70 (d, 2H, H2 +H6 , J2 ꢂ3 =8.2 Hz), 8.04–
8.08 (m, 2H, H6+H7), 8.49–8.57 (m, 2H, H5+H8), 11.00
(s, 1H, NH) ppm. Anal. (C16H13N3O3) C, H, N; C (%):
calcd: 65.08; found: 64.60. H (%): calcd: 4.36; found:
4.38. N (%): calcd: 14.24; found: 14.20 (33% yield).
recorded on a Bruker AC-200E (Rheinstetten, Ger-
many) using TMS as the internal standard. The IR
spectra were performed on a Perkin Elmer 1600 FTIR
(Norwalk, CT, USA) in KBr pellets. Elemental micro-
analyses were obtained on a Elemental Analyzer (Carlo
Erba 1106, Milan, Italy) from vacuum-dried samples;
for each compound, the calculated and found values are
being reported.
7-Chloro-3-methyl-N-phenylquinoxaline-2-carboxamide
1,4-dioxide (4). Mp 189–190 ꢀC. IR (KBr) n 3248, 1677,
1
1333, 743 cmꢂ1. H NMR (DMSO-d6) d 2.49 (s, 3H,
0
0
0
C3–CH3), 7.20 (t, 1H, H4 , J4 ꢂ3 =7.2 Hz), 7.42 (t, 2H,
Alugram1 SIL G/UV254 (Layer: 0.2 mm) (Macherey-
Nagel GmbH & Co. KG. Postfach 101352. D-52313
Duren, Germany) was used for Thin Layer Chromato-
graphy. HPLC conditions: Column Nova Pack C18 60
H3 +H5 ), 7.66 (d, 2H, H2 +H6 , J2 ꢂ 3 =8.0 Hz),
8.05(d, 1H, H6, J6ꢂ5=7.3 Hz); 8.49 (s, 1H, H8), 8.54 (d,
1H, H5), 11.01(s, 1H, NH) ppm. Anal. (C16H12ClN3O3)
C, H, N; C (%): calcd: 58.27; found: 58.21. H (%):
0
0
0
0
0
0

B. Zarranz et al. / Bioorg. Med. Chem. 11 (2003) 2149–2156
2153
0
0
calcd: 3.64; found: 3.90. N (%): calcd: 12.75; found:
12.61. HPLC: Rt=3.06 min (27% yield).
J6 ꢂ5 =6.4 Hz), 8.73 (bs, 2H, H5+H8), 10.37 (s, 1H,
NH) ppm. Anal. (C17H13Cl2N3O3) C, H, N; C (%):
calcd: 53.97; found: 53.66. H (%): calcd: 3.44; found:
3.34. N (%): calcd: 11.11; found: 10.91 (14% yield).
6,7-Dichloro-3-methyl-N-phenylquinoxaline-2-carboxa-
mide 1,4-dioxide (5). Mp 213–214 ꢀC. IR (KBr) n 3255,
1
1677, 1312, 743 cmꢂ1. H NMR (DMSO-d6) d 2.49 (s,
N-(tert-Butyl)-3,6,7-trimethylquinoxaline-2-carboxamide
1,4-dioxide (11). Mp 261–262 ꢀC. IR (KBr) n 3256,
0
0
0
3H, C3–CH3), 7.19 (t, 1H, H4 , J4 ꢂ3 =7.3 Hz), 7.41(t,
1
0
0
0
0
0
0
2H, H3 +H5 ), 7.66 (d, 2H, H2 +H6 , J2 ꢂ3 =7.9 Hz),
8.68 (s, 1H, H8), 8.70 (s, 1H, H5), 10.97 (s, 1H, NH)
ppm. Anal. (C16H11Cl2N3O3) C, H, N; C (%): calcd:
52.75; found: 53.26. H (%): calcd: 3.02; found: 3.13. N
(%): calcd: 11.54; found: 11.24 (15% yield).
1686, 1544, 1331 cmꢂ1; H NMR (DMSO-d6) d 1.36 (s,
9H, C(CH3)3), 2.39 (s, 3H, C3–CH3), 2.47 (s, 6H, 2CH3–
Ar), 8.18 (s, 1H, H5), 8.24 (s, 1H, H8), 8.51(s, 1H, NH)
.
ppm. Anal. (C16H21N3O3 1=4H2O) C, H, N; C (%):
calcd: 62.43; found: 62.39. H (%): calcd: 6.99; found:
7.18. N (%): calcd: 13.66; found: 13.80. HPLC:
Rt=2.61min (34% yield).
3,6,7-Trimethyl-N-(2-methylphenyl)quinoxaline-2-carbox-
amide 1,4-dioxide (6). Mp 228–229 ꢀC. IR (KBr) n
1
3262, 1664, 1590, 1333 cmꢂ1; H NMR (DMSO-d6) d
N-(tert-Butyl)-3-methylquinoxaline-2-carboxamide 1,4-
dioxide (12). Mp 213–214 ꢀC. IR (KBr) n 3260, 1685,
0
2.31(s, 3H, C 2 –CH3), 2.52 (s, 3H, C3–CH3), 2.54 (s,
1333 cmꢂ1 1H NMR (DMSO-d6) d 1.39 (s, 9H,
.
C(CH3)3), 2.44 (s, 3H, C3–CH3), 7.96–8.00 (m, 2H,
H6+H7), 8.48–8.53 (m, 2H, H5+H8) 8.58 (s, 1H, NH)
0
6H, 2CH3–Ar), 7.16–7.32 (m, 3H, H3 +H4 +H5 ), 7.64
0
0
0
0
0
(d, 1H, H6 , J6 ꢂ5 =6.4 Hz); 8.30 (s, 1H, H5), 8.31(s, 1H,
H8), 10.35 (s, 1H, NH) ppm. Anal. (C19H19N3O3) C, H,
N; C (%): calcd: 67.65; found: 67.58. H (%): calcd: 5.64;
found: 5.43. N (%): calcd: 12.46; found: 12.15. HPLC:
Rt=3.09 min (38% yield).
.
ppm. Anal. (C14H17N3O3 1=4H2O) C, H, N; C (%):
calcd: 60.11; found: 59.70. H (%): calcd: 6.26; found:
6.51. N (%): calcd: 15.02; found: 14.62. HPLC:
Rt=3.07 min (5% yield).
3,7-Dimethyl-N-(2-methylphenyl)quinoxaline-2-carbox-
amide 1,4-dioxide (7). Mp 196–197 ꢀC. IR (KBr) n
7-Chloro-N-(tert-butyl)-3-methylquinoxaline-2-carbox-
amide 1,4-dioxide (13). Mp 209–210 ꢀC. IR (KBr) n
1
3260, 1659, 1544, 1331 cmꢂ1; H NMR (DMSO-d6) d
1
2.33 (s, 3H, C ,–CH3), 2.57 (s, 3H, C3–CH3), 2.64 (s, 3H,
3248, 1673, 1327, 802 cmꢂ1. H NMR (DMSO-d6) d
1.38 (s, 9H, C(CH3)3), 2.42 (s, 3H, C3–CH3), 8.01(d,
0
0
Ar–CH3), 7.22–7.35 (m, 3H, H3 +H4 +H5 ), 7.69 (d,
0
0
0
0
0
1H, H6 , J6 ꢂ5 =7.4 Hz); 7.89 (d, 1H, H6, J6ꢂ5=8.0 Hz),
8.36 (s, 1H, H8), 8.46 (d, 1H, H5, J5ꢂ6=8.0 Hz), 10.35
(s, 1H, NH) ppm. Anal. (C18H17N3O3) C, H, N; C (%):
calcd: 66.87; found: 66.77. H (%): calcd: 5.26; found:
5.41. N (%): calcd: 13.00; found: 12.61. HPLC:
Rt=3.14 min (46% yield).
1H, H6, J6ꢂ5=7.0 Hz), 8.44 (s, 1H, H8), 8.52 (d, 1H, H5)
8.46 (s, 1H, NH). Anal. (C14H16ClN3O3 1=4H2O) C, H,
N; C (%): calcd: 53.51; found: 53.73. H (%): calcd: 5.25;
found: 5.10. N (%): calcd: 13.37; found: 13.22. HPLC:
Rt=3.14 min (6% yield).
.
6,7-Dichloro-N-(tert-butyl)-3-methylquinoxaline-2-carbox-
amide 1,4-dioxide (14). Mp 220–221 ꢀC. IR (KBr) n
3254, 2968, 1681, 1329, 757 cmꢂ1. 1H NMR (DMSO-d6)
d 1.38 (s, 9H, C(CH3)3), 2.42 (s, 3H, C3–CH3), 8.54 (s,
1H, NH), 8.63 (s, 1H, H5), 8.66 (s, 1H, H8) ppm. Anal.
(C14H15Cl2N3O3) C, H, N; C (%): calcd: 48.84; found:
49.03. H (%): calcd: 4.36; found: 4.39. N (%): calcd:
12.21; found: 11.88 (34% yield).
3-Methyl-N-(2-methylphenyl)quinoxaline-2-carboxamide
1,4-dioxide (8). Mp 195–196 ꢀC. IR (KBr) n 3254, 1663,
1
1339, 751 cmꢂ1. H NMR (DMSO-d6) d 2.28 (s, 3H,
0
C2 –CH3), 2.48 (s, 3H, C3–CH3), 7.12–7.29 (m, 3H,
0
0
0
0
0
0
H3 +H4 +H5 ), 7.63 (d, 1H, H6 , J6 ꢂ5 =8.0 Hz), 7.96–
8.04 (m, 2H, H6+H7), 8.48–8.54 (m, 2H, H5+H8) ppm.
Anal. (C17H15N3O3) C, H, N; C (%): calcd: 66.88;
found: 66.55. H (%):calcd: 4.92; found: 4.77. N
(%):calcd: 13.77; found: 13.47. HPLC: Rt=1.38 min
(64% yield).
General procedure for compounds 15–31
The corresponding N-(3-oxobutyryl)piperazine (4.8
mmol) was added to a solution of the appropriate ben-
zofuroxane (2.4 mmol) in dry chloroform (35 mL). The
mixture was allowed to stand at 0 ꢀC. Next, morpholine
was added drop by drop (0.1mL). The solution was
stirred at room temperature for 48 h. The solvent was
eliminated under pressure. The resulting solid was
washed with ethyl ether (or methanol). The compounds
were purified by recrystallization using methanol.
Yields: 4–44%.
7-Chloro-3-methyl-N-(2-methylphenyl)quinoxaline-2-car-
boxamide 1,4-dioxide (9). Mp 202–203 ꢀC. IR (KBr) n
1
3249, 1658, 1326, 757 cmꢂ1. H NMR (DMSO-d6) d
0
2.29 (s, 3H, C2 –CH3), 2.54 (s, 3H, C3–CH3), 7.07–7.39
0
0
0
0
(m, 4H, H3 +H4 +H5 +H6 ), 7.64 (d, 1H, H6, J6ꢂ5=7.1
Hz), 8.04 (s, 1H, H8), 8.53 (d, 1H, H5), 10.32 (s, 1H,
.
NH) ppm. Anal. (C17H14ClN3O3 1=4H2O) C, H, N; C
(%): calcd: 58.62; found: 58.93. H (%):calcd: 4.17;
found: 4.11. N (%):calcd: 12.07; found: 11.67. HPLC:
Rt=3.11 min (7% yield).
3,6,7-Trimethyl-2-(4-phenylpiperazin-1-yl)carbonylquinox-
aline 1,4-dioxide (15). Mp 221–222 ꢀC. IR (KBr) n 1652,
6,7-Dichloro-3-methyl-N-(2-methylphenyl)quinoxaline-2-
carboxamide 1,4-dioxide (10). Mp 224–225 ꢀC. IR
(KBr) n 3261, 1672, 1327, 758 cmꢂ1. 1H NMR (DMSO-
1
1598, 1474, 1327, 1012 cmꢂ1; H NMR (DMSO-d6) d
2.46 (s, 3H, C3–CH3), 2.54 (s, 6H, 2CH3–Ar), 3.03–4.04
0
0
(m, 8H, 4CH2 piperazine), 6.86 (t, 1H, H4 ),7.00 (d, 2H,
d6) d 2.30 (s, 3H, C2 –CH3), 2.52 (s, 3H, C3–CH3), 7.20–
0
7.33 (m, 3H, H3 +H4 +H5 ), 7.64 (d, 1H, H6 ,
0
0
0
0
0
0
0
0
0
H2 +H6 , J2 ꢂ3 =8.2 Hz), 7.27 (t, 2H, H3 +H5 ,

2154
B. Zarranz et al. / Bioorg. Med. Chem. 11 (2003) 2149–2156
0
0
J3 ꢂ4 =7.6 Hz), 8.25 (s, 1H, H5), 8.31(s, 1H, H 8) ppm.
Anal. (C22H24N4O3) C, H, N; C (%): calcd: 67.34;
found: 67.41. H (%): calcd: 6.12; found: 6.06. N (%):
calcd: 14.28; found: 14.01. HPLC: Rt=3.01min (44%
yield).
3,7-Dimethyl-2-4-(4-nitrophenyl)piperazin-1-ylcarbonyl-
quinoxaline 1,4-dioxide (21). Mp 274–275 ꢀC. IR (KBr)
n 1642, 1598, 1492, 1332, 1243 cmꢂ1; 1H NMR (DMSO-
d6) d 2.43 (s, 3H, C3–CH3), 2.60 (s, 3H, CH3–Ar), 3.46-
0
0
3.92 (m, 8H, 4CH2 piperazine), 7.07 (d, 2H, H2 +H6 ,
0
0
J2 ꢂ3 =8.9 Hz), 7.83 (d, 1H, H6, J6ꢂ5=8.0 Hz), 8.09 (d,
0
0
0
0
3,7-Dimethyl-2-(4-phenylpiperazin-1-yl)carbonylquinoxa-
line 1,4-dioxide (16). Mp 232–233 ꢀC. IR (KBr) n 1646,
2H, H3 +H5 , J3 ꢂ4 =8.8 Hz), 8.26 (s, 1H, H8) ppm.
Anal. (C21H21N5O5 1=2H2O) C, H, N; C (%): calcd:
.
1
1523, 1327, 1011 cmꢂ1; H NMR (DMSO-d6) d 2.42 (s,
3H, C3–CH3), 2.58 (s, 3H, CH3–Ar), 2.97-3.94 (m, 8H,
58.33; found: 58.31. H (%): calcd: 5.09; found: 4.95. N
(%): calcd: 16.20; found: 15.94 (29% yield).
0
4CH2 piperazine), 6.84 (t, 1H, H4 ), 6.96 (d, 2H,
0
0
0
0
0
0
H2 +H6 , J2 ꢂ3 =8.0 Hz), 7.23 (t, 2H, H3 +H5 ,
3-Methyl-2-4-(4-nitrophenyl)piperazin-1-ylcarbonylqui-
noxaline 1,4-dioxide (22). Mp 220–221 ꢀC. IR (KBr) n
0
0
J3 ꢂ4 =7.2 Hz), 7.81(d, 1H, H 6, J6ꢂ5=8.4 Hz), 8.24 (s,
1H, H8), 8.38 (d, 1H, H5) ppm. Anal. (C21H22N4O3) C,
H, N; C (%): calcd: 66.66; found: 66.31. H (%): calcd:
5.82; found: 5.72. N (%): calcd: 14.81; found: 14.49.
HPLC: Rt=3.02 min (30% yield).
1
1643, 1597, 1334, 1241 cmꢂ1; H NMR (DMSO-d6) d
2.42 (s, 3H, CH3), 3.49–3.89 (m, 8H, 4CH2 piperazine),
0
0
0
0
7.04 (d, 2H, H2 +H6 , J2 ꢂ3 =9.2 Hz), 7.94–7.99 (m, 2H,
H6+H7), 8.07 (d, 2H, H3 +H5 ); 8.41–8.51 (m, 2H,
0
0
.
H5+H8) ppm. Anal. (C20H19N5O5 1=2H2O) C, H, N; C
6,7-Dichloro-3-methyl-2-(4-phenylpiperazin-1-yl)carbonyl-
quinoxaline 1,4-dioxide (17). Mp 221–222 ꢀC. IR (KBr)
n 3067, 1655, 1324, 882. 1H NMR (DMSO-d6) d 2.43 (s,
3H, C3-CH3), 2.95–3.92 (m, 8H, 4CH2 piperazine), 6.83
(%): calcd: 57.41; found: 57.26. H (%): calcd: 4.78;
found: 4.62. N (%): calcd: 16.74; found: 16.64 (5%
yield).
0
0
0
0
0
(t, 1H, H4 , J4 ꢂ3 =7.1Hz), 6.98 (d, 2H, H 2 +H6 ,
6,7-Dichloro-3-methyl-2-4-(4-nitrophenyl)piperazin-1-yl-
carbonylquinoxaline 1,4-dioxide (23). Mp 223–224 ꢀC.
0
0
0
0
J2 ꢂ3 =8.0 Hz), 7.24 (t, 2H, H3 +H5 ), 8.63 (s, 1H, H5),
8.67 (s, 1H, H8) ppm. Anal. (C20H18Cl2N4O3) C, H, N;
C (%): calcd: 55.43; found: 55.71. H (%): calcd: 4.16;
found: 4.23. N (%): calcd: 12.93; found: 12.75. HPLC:
Rt=3.14 min (12% yield).
1
IR (KBr) n 1656, 1325 cmꢂ1. H NMR (DMSO-d6) d
2.43 (s, 3H, C3–CH3), 3.45–3.89 (m, 8H, 4CH2 piper-
0
0
0
0
azine), 7.07 (d, 2H, H3 +H5 , J3 ꢂ2 =9.4 Hz), 8.08 (d,
0
0
2H, H2 +H6 ), 8.62 (s,1H, H5), 8.67 (s;1H, H8) ppm.
Anal. (C20H17Cl2N5O5) C, H, N; C (%): calcd: 50.21;
found: 50.87. H (%): calcd: 3.55; found: 3.62. N (%):
calcd: 14.64; found: 14.48. HPLC: Rt=3.01min (16%
yield).
3,6,7-Trimethyl-2-4-(4-chlorophenyl)piperazin-1-ylcarbo-
nylquinoxaline 1,4-dioxide (18). Mp 214–215 ꢀC. IR
(KBr) n 1646, 1496, 1329, 1000 cmꢂ1
;
1H NMR
(DMSO-d6) d 2.41(s, 3H, C –CH3), 2.50 (s, 6H, 2CH3–
3
Ar), 3.01–4.00 (m, 8H, 4CH2 piperazine), 6.98 (d, 2H,
0
3-Methyl-7-methoxy-2-4-(4-nitrophenyl)piperazin-1-yl-
carbonylquinoxaline 1,4-dioxide (24). Mp 264–265 ꢀC.
0
0
0
0
0
H2 +H6 , J2 ꢂ3 =7.4 Hz), 7.26 (d, 2H, H3 +H5 ,
J3 ꢂ4 =7.0 Hz), 8.21(s, 1H, H 5); 8.27 (s, 1H, H8) ppm.
Anal. (C22H23ClN4O3 1=4H2O) C, H, N; C (%): calcd:
IR (KBr) n 1643, 1598, 1492, 1330, 1241 cmꢂ1 1H
;
0
0
.
NMR (DMSO-d6) d 2.42 (s, 3H, CH3), 3.57–3.78 (m,
8H, 4CH2 piperazine), 4.00 (s, 3H, OCH3), 7.07 (d, 2H,
61.26; found: 61.39. H (%): calcd: 5.45; found: 5.47. N
(%): calcd: 12.99; found: 12.46. HPLC: Rt=3.05 min
(5% yield).
0
0
0
0
H2 +H6 , J2 ꢂ3 =7.9 Hz), 7.60 (d, 1H, H6, J6ꢂ5=8.9
0
0
0
0
Hz), 7.77 (s, 1H, H8); 8.09 (d, 2H, H3 +H5 , J3 ꢂ4 =8.3
Hz), 8.42 (d, 1H, H5) ppm. Anal. (C21H21N5O6.H2O) C,
H, N; C (%): calcd: 55.14; found: 55.34. H (%): calcd:
5.03; found: 4.81. N (%): calcd: 15.31; found: 14.98
(33% yield).
3-Methyl-2-4-(4-chlorophenyl)piperazin-1-ylcarbonylqui-
noxaline 1,4-dioxide (19). Mp 216–217 ꢀC. IR (KBr) n
1
1648, 1496, 1330, 1232 cmꢂ1; H NMR (DMSO-d6) d
2.44 (s, 3H, CH3), 3.03–4.00 (m, 8H, 4CH2 piperazine),
0
0
0
0
6.99 (d, 2H, H2 +H6 , J2 ꢂ3 =7.3 Hz), 7.26 (d, 2H,
3,6,7-Trimethyl-2-4-(2-methoxyphenyl)piperazin-1-ylcar-
bonylquinoxaline 1,4-dioxide (25). Mp 229–230 ꢀC. IR
0
0
0
0
H3 +H5 , J3 ꢂ4 =7.4 Hz), 7.95–8.02 (m, 2H, H6+H7);
8.43–8.52 (m, 2H, H5+H8) ppm. Anal.
(C20H19ClN4O3 1=2H2O) C, H, N; C (%): calcd: 58.90;
found: 59.01. H (%): calcd: 4.90; found: 4.82. N (%):
calcd: 13.74; found: 13.25. HPLC: Rt=3.02 min (4%
yield).
(KBr) n 1650, 1443, 1328, 1241 cmꢂ1
;
1H NMR
.
(DMSO-d6) d 2.42 (s, 3H, C3–CH3), 2.49 (s, 6H, 2CH3–
Ar), 2.67–4.05 (m, 8H, 4CH2 piperazine), 3.77 (s, 3H,
0
0
0
0
OCH3), 6.88–6.96 (m, 4H, H3 +H4 +H5 +H6 ), 8.21(s,
1H, H5); 8.27 (s, 1H, H8) ppm. Anal.
.
(C23H26N4O4 1=2H2O) C, H, N; C (%): calcd: 64.03;
3,6,7-Trimethyl-2-4-(4-nitrophenyl)piperazin-1-ylcarbonyl-
quinoxaline 1,4-dioxide (20). Mp 235–236 ꢀC. IR (KBr)
found: 64.02. H (%): calcd: 6.26; found: 6.02. N (%):
calcd: 12.99; found: 12.80 (4% yield).
1
n 1645, 1595, 1493, 1330 cmꢂ1; H NMR (DMSO-d6) d
2.42 (s, 3H, C3–CH3), 2.49 (s, 3H, C6 or C7-CH3), 2.50
6,7-Dichloro-3-methyl-2-4-(2-methoxyphenyl)piperazin-
1-ylcarbonylquinoxaline 1,4-dioxide (26). Mp 212–
(s, 3H, C7 or C6-CH3), 3.38–3.89 (m, 8H, 4CH2 piper-
0
ꢀ
1
0
0
0
azine), 7.04 (d, 2H, H2 +H6 , J2 ꢂ3 =9.4 Hz), 8.07 (d,
2H, H3 +H5 ), 8.21(s, 1H, H 5), 8.27 (s, 1H, H8) ppm.
213 C. IR (KBr) n 3065, 1649, 1462, 1323. H NMR
(DMSO-d6) d 2.44 (s, 3H, C3–CH3), 3.78 (s, 3H, OCH3),
2.84–3.92 (m, 8H, 4CH2 piperazine), 6.90–6.96 (m, 4H,
0
0
.
Anal. (C22H23N5O5 1=2H2O) C, H, N; C (%): calcd:
0
0
0
0
59.19; found: 59.10. H (%): calcd: 5.38; found: 5.40. N
(%): calcd: 15.69; found: 15.59 (15% yield).
H3 +H4 +H5 +H6 ), 8.63 (s, 1H, H5), 8.65 (s, 1H, H8)
ppm. Anal. (C21H20Cl2N4O4) C, H, N; C (%): calcd:

B. Zarranz et al. / Bioorg. Med. Chem. 11 (2003) 2149–2156
2155
54.42; found: 53.91. H (%): calcd: 4.32; found: 4.43. N
(%): calcd: 12.10; found: 11.80. HPLC: Rt=1.58 min
(19% yield).
medium using a broth microdilution assay, the Micro-
plate Alamar Blue Assay (MABA).14 Compounds exhi-
biting fluorescence were tested in the BACTEC 460
radiometric system.16 Compounds showing ꢃ90%
inhibition in the primary screening were considered
active and then re-tested at lower concentration against
M. tuberculosis H37Rv in order to determine the actual
minimum inhibitory concentration (MIC), using
MABA. The MIC is defined as the lowest concentration
effecting a reduction in fluorescence of 90% with respect
to the controls.
3-Methyl-2-4-(4-methoxyphenyl)piperazin-1-ylcarbonyl-
quinoxaline 1,4-dioxide (27). Mp 216–217 ꢀC. IR (KBr)
1
n 3065, 1649, 1322, 1240, 831 cmꢂ1. H NMR (DMSO-
d6) d 2.48 (s, 3H, C3–CH3), 2.88–3.99 (m, 8H, 4CH2
0
0
piperazine), 3.72 (s, 3H, OCH3), 6.87 (d, 2H, H2 +H6 ,
0
0
0
0
J2 ꢂ3 =9.0 Hz), 6.97 (d, 2H, H3 +H5 ), 7.98–8.04 (m,
2H, H6+H7), 8.46–8.56 (m, 2H, H5+H8) ppm. Anal.
(C21H22N4O4) C, H, N; C (%): calcd: 63.96; found:
63.80. H (%): calcd: 5.58; found: 5.30. N (%): calcd:
14.21; found: 13.81. HPLC: Rt=2.36 min (14% yield).
Compounds were also tested for cytotoxicity (IC50) in
VERO cell line at a concentration equal to and greater
than the MIC for M. tuberculosis H37Rv. After 72 h
exposure, viability was assessed on the basis of cellular
conversion of MTT into a formazan product using the
Promega Cell Titer 96 Non-radio-active Cell Prolifera-
tion assay. The Selectivity Index was also determined; it
is considered significant when >10. Rifampin (RMP)
was used as reference compound.
6,7-Dichloro-3-methyl-2-4-(4-methoxyphenyl)piperazin-
1-ylcarbonylquinoxaline 1,4-dioxide (28). Mp 219–
220 ꢀC. IR (KBr) n 3065, 1657, 1326, 1240, 831 cmꢂ1
.
1H NMR (DMSO-d6) d 2.47 (s, 3H, C3–CH3), 2.88–3.90
(m, 8H, 4CH2 piperazine), 3.72 (s, 3H, OCH3), 6.87 (d,
0
0
0
0
0
0
2H, H2 +H6 , J2 ꢂ3 =9.1Hz), 6.98 (d, 2H, H 3 +H5 ),
8.67 (s, 1H, H5), 8.71(s, H1, H 8) ppm. Anal.
(C21H20Cl2N4O4) C, H, N; C (%): calcd: 54,42; found:
54,15. H (%): calcd: 4,32; found: 4,35. N (%): calcd:
12,10; found: 12,01. HPLC: Rt=2.40 min (16% yield).
Macrophage assay. Compounds with a MICꢄ6.25 mg/
mL and a SI>10 were then tested to evaluate efficacy
in vitro in a TB-infected macrophage model.15 The
EC90 and EC99 are defined as the concentrations
effecting 90 and 99% reduction in residual myco-
bacterial growth after 7 days, compared to untreated
controls. Compounds with EC90>16ꢁMIC are con-
sidered inactive.
3,6,7-Trimethyl-2-(4-benzylpiperazin-1-yl)carbonylqui-
noxaline 1,4-dioxide (29). Mp 210-211 ꢀC. IR (KBr) n
1
1649, 1474, 1330, 1019 cmꢂ1; H NMR (DMSO-d6) d
2.41(s, 3H, C 3–CH3 ), 2.51(s, 6H, 2CH 3–Ar), 2.40–3.80
(m, 8H, 4CH2 piperazine), 3.55 (s, 2H, NCH2Ph), 7.34
0
0
0
0
0
(bs, 5H, H2 +H3 +H4 +H5 +H6 ), 8.22 (s, 1H, H5);
.
8.27 (s, 1H, H8) ppm. Anal. (C23H26N4O3 1=2H2O) C,
Acknowledgements
H, N; C (%): calcd: 66.50; found: 66.31. H (%): calcd:
6.50; found: 6.77. N (%): calcd: 13.49; found: 13.52.
HPLC: Rt=3.26 min (11% yield).
Antimycobacterial data were provided by the Tubercu-
losis Antimicrobial Acquisition and Coordinating
Facility (TAACF) through a research and development
contract with the U.S. National Institute of Allergy and
Infectious Diseases. Thanks are addressed to Dr. Joseph
A. Maddry (TAACF) and his team for their collabora-
tion. We wish to thank the Gobierno de Navarra for
grants given to B. Zarranz and to CYTED (Red Iber-
oamericana para la Investigacion y Descubrimiento de
Medicamentos).
3-Methyl-2-(4-benzylpiperazin-1-yl)carbonylquinoxaline
1,4-dioxide (30). Mp 194–195 ꢀC. IR (KBr) n 1655, 1473,
1331, 995 cmꢂ1; 1H NMR (DMSO-d6) d 2.41(s, 3H, CH 3),
2.25–3.85 (m, 8H, 4CH2 piperazine), 3.52 (s, 2H,
0
0
0
0
0
NCH2Ph), 7.32 (bs, 5H, H2 +H3 +H4 +H5 +H6 ), 7.93–
7.98 (m, 2H, H6+H7), 8.40–8.50 (m, 2H, H5+H8) ppm.
.
Anal. (C21H22O3N4 1=2H2O) C, H, N; C (%): calcd: 65.88;
found: 65.52. H (%): calcd: 5.88; found: 6.22. N (%): calcd:
14.64; found: 14.97. HPLC: Rt=3.33 min (35% yield).
References and Notes
6,7-Dichloro-3-methyl-2-(4-benzylpiperazin-1-yl)carbonyl-
quinoxaline 1,4-dioxide (31). Mp 182–183 ꢀC. IR (KBr)
n 1649, 1442, 1321. H NMR (DMSO-d6) d 2.39 (s, 3H,
C3–CH3), 2.42–3.79 (m, 8H, 4CH2, piperazine), 3.53
1. Savini, L.; Chiasserini, L.; Gaeta, A.; Pellerano, C. Bioorg.
Med. Chem. 2002, 10, 2193.
2. Pathak, R.; Pant, C. S.; Shaw, A. K.; Bhaduri, A. P.;
Gaikwad, A. N.; Sinha, S.; Srivastava, A.; Srivastava, K. K.;
Chaturvedi, V.; Srivastava, R.; Srivastava, B. S. Bioorg. Med.
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3. Bass, J. B., Jr.; Farer, L. S.; Hopewell, P. C.; O’Brien, R.;
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1
0
0
(s, 2H, NCH2Ph), 7.31(bs, 5H, H 2 +H3 +H4 +
0
0
0
H5 +H6 ), 8.61(d, 1H, H 5), 8.65 (d, 1H, 1H8) ppm.
Anal. (C21H20Cl2N4O3) C, H, N; C (%): calcd: 56.38;
found: 56.51. H (%): calcd: 4.47; found: 4.47. N (%):
calcd: 12.53; found: 12.43. HPLC: Rt=1.8 min (29%
yield).
4. Pathak, A. K.; Pathak, V.; Seitz, L.; Maddry, J. A.;
Gurcha, S. S.; Besra, G. S.; Suling, W. J.; Reynolds, R. C.
Bioorg. Med. Chem. 2001, 9, 3129.
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Tirapu, I.; Aldana, I.; Monge, A. Die Pharmazie. 2001, 56,
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Biological evaluation
In vitro evaluation of anti-tuberculosis activity. Primary
screening was conducted at 6.25 mg/mL against M.
tuberculosis H37Rv (ATCC 27294) in BACTEC 12B

2156
B. Zarranz et al. / Bioorg. Med. Chem. 11 (2003) 2149–2156
6. Ortega, M. A.; Sainz, Y.; Montoya, M. E.; Jaso, A.;
Zarranz, B.; Aldana, I.; Monge, A. Arzneim. Forsch./Drug
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7. Ortega, M. A.; Sainz, Y.; Montoya, M. E.; Lopez de
Cerain, A.; Monge, A. Die Pharmazie. 1999, 54, 24.
8. Sainz, Y.; Montoya, M. E.; Martınez-Crespo, F. J.; Ortega,
M. A.; Lopez de Cerain, A.; Monge, A. Arzneim. -Forsch.
Drug Res. 1999, 49, 55.
12. Cheeseman, G. W. H.; Cookson, R. F. In Condensed
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