Novel 3-(2-Oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoates from dimethyl-2-oxoglutarate and test of their biological activity

Article abstract of DOI:10.1002/jhet.1520

Five novel 3-(2-oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoates were synthesized under mild conditions from 2-aminophenols and dimethyl-2- oxoglutarate. Biological assays of these 1,4-benzoxazinones were conducted with three bacterial strains and one yeast. All compounds were active against a Candida albicans ATCC 10231, whereas only methyl 3-(6-methyl-2-oxo-2H-benzo[b] [1,4]oxazin-3-yl)propanoate showed a general moderate activity against the bacterial strains tested.

Full text of DOI:10.1002/jhet.1520

March 2013
Novel 3-(2-Oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoates from
413
Dimethyl-2-oxoglutarate and Test of Their Biological Activity
a
a
b
a
c
c
*
Kamel Hachama, Mohamed Khodja, Saad Moulay, Hocine Boutoumi, Lothar Hennig, and Dieter Sicker
aLaboratoire de Recherche Plantes Médicinales et Aromatiques, Département de Chimie Industrielle, Université Saâd
Dahlab de Blida, 09000 Blida, B. P. 270, Algeria
bLaboratoire Chimie-Physique Moléculaire et Macromoléculaire, Département de Chimie Industrielle, Université Saâd
Dahlab de Blida, 09000 Blida, B. P. 270, Algeria
cInstitut für Organische Chemie der Universität Leipzig, Johannisallee 29, D-04103 Leipzig, Germany
*
E-mail: khodjamed@yahoo.fr
Received June 29, 2011
DOI 10.1002/jhet.1520
Published online 20 March 2013 in Wiley Online Library (wileyonlinelibrary.com).
R3
R2
O
N
O
R3
R2
OH
O
O
MeOH, r.t., 1 h
40 - 60 %
+
O
O
O
NH2
O
R1
R1
O
Five novel 3-(2-oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoates were synthesized under mild conditions
from 2-aminophenols and dimethyl-2-oxoglutarate. Biological assays of these 1,4-benzoxazinones were con-
ducted with three bacterial strains and one yeast. All compounds were active against a Candida albicans
ATCC 10231, whereas only methyl 3-(6-methyl-2-oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoate showed a
general moderate activity against the bacterial strains tested.
J. Heterocyclic Chem., 50, 413 (2013).
INTRODUCTION
RESULTS AND DISCUSSION
Synthesis of 3-(2-oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoates
3a–e. Dimethyl-2-oxoglutarate 2 (Scheme 1) was prepared
This article was inspired by two conceptually different
areas of our research. On the one hand, 2-oxoglutaric acid,
well known as a part of the citric acid cycle, is—in accor-
dance to the White biotechnology for green chemistry
concept—a 2-oxocarboxylic acid [1] now accessible in large
quantities by fermentation of paraffines, glycerol, ethanol,
glucose, or sunflower oil. Hence, 2-oxoglutaric acid is on its
way from a laboratory chemical to a biotechnologically man-
ufactured industrial product [2–4]. Earlier, we reported on the
synthesis of heterocycles based on this building block [5]. On
the other hand, naturally occurring 2H-1,4-benzoxazin-3
(4H)-ones from cereal plants are well known for their high
biological activity as intrinsic resistance factors of these plants
[6]. From this perspective, 1,4-benzoxazines are of general
interest for biological testing. The versatility of related 1,
4-benzoxazin-2-ones is similarly linked to their potential
pharmacological uses [7–9] and their photochemical activity
[10–13]. For example, 1,4-benzoxazin-2-ones prepared from
o-aminophenols and methyl acylpyruvates show significant
antimicrobial activities against Staphylococcus aureus and
Escherichia coli. Common strategies that have been
employed for their synthesis are the reaction of substituted
2-aminophenols with 2-oxoesters [14–17], alkyl propiolates
[18], or b-nitroacrylates [19].
in a high yield (85%) by esterification of 2-oxoglutaric
acid with methanol according to the procedure formerly
reported [20]. As shown in Scheme 1, the reaction of
dimethyl-2-oxoglutarate with an equimolar amount of
2-aminophenol 1a, 2-amino-5-methylphenol 1b, 2-amino-4-
methylphenol 1c, 2-amino-3-methylphenol 1d, and
2-amino-4-chlorophenol 1e afforded the corresponding
3-(2-oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoates 3a–e
in good yields under very mild conditions. All novel
compounds 3a–e obtained have been characterized by
spectroscopic data and elemental analysis as shown in
the Experimental section. The commercial availability of
2-aminophenols 1a–e was the reason for the pattern of
substituents described here. Heterocycles 3a–e have not
been reported yet.
Antimicrobial activity of compounds 3a–e.
Biological
activity of the products (3a–e) in form of DMSO solutions
was assessed against fungus and yeast by the method of
discs, also known as Bauer-Kirby method as described in
the Biological Assay section. The biological strains studied
were S. aureus ATCC 6538 (gram-positive bacteria), E.
coli ATCC 10536 and Proteus vulgaris ATCC 6896
(gram-negative bacteria), and Candida albicans ATCC
10231 (yeast). A blank test was performed with DMSO,
and the result was negative, that is, no aureola was
formed indicating no inhibition of microbial growth. The
Here, we report a facile synthesis of novel 3-(2-oxo-2H-
benzo[b][1,4]oxazin-3-yl)propanoates from 2-aminophenols
and dimethyl-2-oxoglutarate and results of their antimicrobial
and antifungal testing.
© 2013 HeteroCorporation

414
K. Hachama, M. Khodja, S. Moulay, H. Boutoumi, L. Hennig, and D. Sicker
Vol 50
Scheme 1. Synthetic route to substituted 3-(2-oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoates (3a–e).
R3
OH
R3
O
O
O
O
MeOH, r.t., 1 h
40 - 60 %
+
O
O
O
R2
N
R2
NH2
O
R1
R1
3 a - e
O
1 a -e
2
R2
1,3
R1
R3
H
a
b
c
d
e
H
H
H
H
CH3
H
H
CH3
H
H
CH3
H
H
Cl
screening results shown in Table 1 clearly suggest that all
compounds exhibited antifungal activity in the order
3a > 3c > 3d > 3e > 3b. Conspicuously, methyl 3-(6-
methyl-2-oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoate (3c)
was active towards all the biological strains, and its
antifungal activity was 2.5 times higher than its
antibacterial one. Compounds 3a and 3e were active
against P. vulgaris ATCC 6896, and compound 3c was
active against all bacteria strains tested. No other
suppression of bacterial growth was observed. On the other
hand, the minimum inhibiting concentrations (MIC),
defined as the lowest concentration of an antimicrobial that
will inhibit the visible growth of a microorganism after
incubation, of 1,4-benzoxazin-2-ones were also determined
[21] (Table 1). As shown, all MICs were found to be in the
spectrum of 1 250 to 2 500 mg/mL for 3c towards S. aureus
ATCC 6538, E. coli ATCC 10536 and P. vulgaris ATCC
6896. It is worth recalling that the reported MICs for
(7)-substitued-3-acylmethylene-1,4-benzoxazin-2-ones and
ethacridine lactate towards the first two bacteria were in the
range of 500–2000 mg/mL [22]. The antimicrobial activity
of the present compounds strongly depends on the type of
strain tested and the nature and position of the substituent
at the benzene ring. Clearly, the 6-methyl derivative 3c
showed the highest bioactivity.
In summary, five novel 3-(2-oxo-2H-benzo[b][1,4]
oxazin-3-yl)propanoates (3a–e) have been prepared from
dimethyl-2-oxoglutarate, and their biological activity has
been tested toward three bacterial strains and one yeast.
All compounds were active against a C. albicans strain,
whereas only 3c clearly showed the highest activity against
the bacteria strains tested.
A statement such as this is a typical feature of experi-
ments with substituents of different kind and at different
positions, for example, at aromatic rings. The reasons
are very difficult to unravel in a preliminary investiga-
tion as described here. However, it is known from
many examples that already minor changes may alter
the biological performance of small molecules dramati-
cally. The difference in the behavior of the allelopathic
natural products benzoxazolin-2(3H)-one and 6-methoxy-
benzoxazolin-2(3H)-one known from rye and maize
exudates, respectively, is one of the most convincing
examples [23].
Table 1
Results of the antimicrobial tests of 3a–e; concentration 160 mg/mL in DMSO.
Antibacterial activitya
Gram-positive (+)
Gram-negative (À)
Antifungal activitya
Compound
tested
Staphylococcus aureus
Escherichia coli
ATCC 10536
Proteus vulgaris
ATCC 6896
Candida albicans
ATCC 10231
ATCC 6538
3a
3b
3c
3d
3e
00
00
00
00
09 (s = 0.47)(20 000)
34 (s = 0.94)(10 000)
16 (s = 0.47)(5000)
30 (s = 0.94)(40 000)
25 (s = 0.47)(20 000)
21 (s = 1.41)(10 000)
00
13 (s = 2.49)(2500)
13 (s = 2.16)(2500)
12 (s = 0.47)(1250)
00
13 (s = 0.47)(40 000)
00
00
00
00
aDiameter of aureola formed in millimeter (mm); the mean of three experimental values, and s is the standard deviation; the numbers below and in paren-
theses are the minimum inhibiting concentration values in mg/mL. The minimum inhibitory concentration was assessed as described in the literature using the
method of dilution [21].
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

March 2013
Novel 3-(2-Oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoates
415
from Dimethyl-2-oxoglutarate and Test of Their Biological Activity
CH2, J = 7.1 Hz, CH2); 3.02 (t, 2H, CH2, J = 7.1 Hz); 3.59
(3H, s, OCH3); 7.25 (d, 1H, Harom, J = 8.7 Hz); 7.34 (dd, 1H,
Harom, J = 8.7 Hz, J = 1.5 Hz); 7.46 (d, 1H, Harom, J = 1.5 Hz).
13C NMR: d 20.16 (CH3); 28.08 (CH2); 29.06 (CH2); 51.45
(OCH3); 115.81 (C-8); 128.04 (C-6); 130.24 (C-5); 131.15
(C-7); 134.79 (C-4a); 143.97 (C-8a); 152.69 (C═O); 156.37
(N═C); 172.63 (COO); ms: m/z 247 (M+, 81%), 216
(M+ À OCH3, 43%), 188 (M+ À CO2CH3, 19%), 160
EXPERIMENTAL
Melting points were determined on a Boetius micro hot stage
and are corrected. IR spectra were measured in KBr on spectro-
photometer Specord M80, Carl Zeiss, Jena, Germany.. 1H and
13C NMR spectra were recorded in DMSO-d6 with Varian
Mercury plus 300 or 400 MHz spectrometers. Chemical shifts
for NMR signals are reported in parts per million from tetra-
methylsilane. 70 eV Electron impact mass spectra were recorded
with a MAT 8230 Thermo Finnigan mass spectrometer. Elemen-
tal analyses were measured with a Vario El elemental analyzer
from Elementar Analysensysteme GmbH Hanau, Germany.
Dimethyl-2-oxoglutarate (2). A 73.05 g (0.5 mole) of 2-
oxoglutaric acid was dissolved in dry methanol (300 mL)
(M+ À CH2-CH2-CO2CH3,
100%).
Anal.
Calcd
for
C13H13NO4: C, 63.15; H, 5.30; N, 5.67. Found: C, 63.03; H,
5.04; N, 5.59.
Methyl 3-(5-methyl-2-oxo-2H-benzo[b][1,4]oxazin-3-yl)pro-
panoate (3d). This compound was obtained as colorless
crystals, 1.23 g (50%), mp 132–134ꢀC; ir n C═O 1740,
N═C, 1612 cmÀ1;- 1H NMR: d 2.48 (s, 3H, CH3); 2.74 (t,
2H, CH2, J = 6.6 Hz); 3.04 (t, 2H, CH2, J = 6.6 Hz); 3.60 (s,
and allowed to stand for 5 days at 20ꢀC.
(1.0 mole) of 2,2-dimethoxypropane was added, and the
solution was allowed to stand for an additional day.
Methanol and acetone were removed in vacuo. The
A 104.5 g
3H, OCH3); 7.17 (d, 1H, Harom
,
J = 8.4 Hz); 7.22
(d, 1H, Harom., J = 6.9 Hz) 7.39 (dd, 1H, Harom, J = 8.4 Hz,
J = 6.9 Hz). 13C NMR: d 16.11 (CH3); 28.17 (CH2); 29.05
(CH2); 51.35 (OCH3); 113.81 (C-8); 126.24 (C-6); 128.87
(C-5); 130.11 (C-7); 136.92 (C-4a); 146.28 (C-8a); 152.53
(C═O); 154.73 (N═C); 172.73 (COO); ms: m/z 247
(M+, 80%), 215 (M+ À CH3OH, 100%), 188 (M+ À CO2CH3,
10%), 160 (M+ À CH2-CH2-CO2CH3, 80%). Anal. Calcd for
C13H13NO4: C, 63.15; H, 5.30; N, 5.67. Found: C, 63.10;
H, 5.14; N, 5.58.
Methyl 3-(6-chloro-2-oxo-2H-benzo[b][1,4]oxazin-3-yl)pro-
panoate (3e). This compound was obtained as colorless crystaÀls1,
1.23 g (50%), mp 114–116ꢀC; ir n C═O 1740, N═C 1615 cm
(N═C); 1H NMR: d 2.77 (t, 2H, CH2, J = 7.2 Hz, CH2); 3.06
(t, 2H, CH2, J = 7.2 Hz); 3.62 (s, 3H, OCH3); 7.45 (d, 1H,
Harom, J = 6.5 Hz); 7.59 (dd, 1H, Harom, J = 6.5 Hz, J = 2.1 Hz);
7.74 (d, 1H, Harom, J = 2.1 Hz). 13C NMR: d 28.19 (CH2); 28.96
(CH2); 51.46 (OCH3); 117.94 (C-8); 127.27 (C-5); 128.73
(C-7); 129.97 (C-6); 131.32 (C-4a); 145.02 (C-8a); 152.12
(C═O); 158.19 (N═C); 172.49 (COO); ms: m/z 267 (M+,
16%), 236 (M+ À OCH3, 15%), 208 (M+ À CO2CH3, 5%),
180 (M+ À CH2-CH2-CO2CH3, 100%). Anal. Calcd for
C12H10ClNO4: C, 53.85; H, 3.77; N, 5.23. Found: C, 53.83;
H, 3.73; N, 5.16.
remaining oily crude product was then subjected to
a
fractional distillation in vacuo. Dimethyl-2-oxoglutarate was
isolated as colorless oil, 74.0 g (85%), bp 102–104ꢀC/
2.66 mbar, n2D0 1.4414.
General procedure for the synthesis of substituted 3-(2-
oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoates 3a–e. A solution
of 1.74 g (0.01mole) of dimethyl-2-oxoglutarate (2) in 10mL of
dry methanol was added dropwise under stirring to a solution of
the corresponding substituted 2-aminophenol (1a–e) (0.01mole)
in 20mL of dry methanol at 20ꢀC. After stirring for 1 h at 20ꢀC,
the crystals formed were filtered off and recrystallized from
methanol.
Methyl 3-(2-oxo-2H-benzo[b][1,4]oxazin-3-yl)propanoate (3a).
This compound was obtained asÀo1range crystals, 1.39 g, (60%), mp
134–135ꢀC; ir: n CO 1740 cm (C═O), N═C 1612cmÀ1 1H
;
NMR: d 2.76 (t, 2H, CH2, J = 7.2Hz); 3.03 (t, 2H, CH2,
J = 7.2Hz); 3.59 (s, 3H, OCH3); 7.36 (dd, 1H, Harom, J = 8.6Hz,
J = 7.5Hz); 7.38 (d, 1H, Harom, J = 8.5Hz); 7.51 (dd, 1H, Harom
,
J = 8.5Hz, 7.5 Hz); 7.67 (d, 1H, Harom, J = 8.6 Hz). 13C NMR:
d 28.14 (CH2); 29.11 (CH2); 51.48 (OCH3); 116.23 (C-8);
125.31 (C-6); 128.24 (C-5); 130.49 (C-7); 130.57 (C-4a); 146.10
(C-8a); 152.28 (C═O); 156.60 (N═C); 172.67 (COO); ms: m/z
233 (M+, 58%), 231 (M+ À OCH3, 37%), 174 (M+ À CO2CH3,
35%), 146 (M+ À CH2-CH2-CO2CH3, 100%). Anal. Calcd for
C12H11NO4: C, 61.80; H, 4.75; N, 6.01. Found: C, 61.63; H,
4.74; N, 5.91.
BIOLOGICAL ASSAYS
Methyl 3-(7-methyl-2-oxo-2H-benzo[b][1,4]oxazin-3-yl)pro-
panoate (3b). This compound was obtained as colorless
crystals, 0.98 g (40%), mp 99–100ꢀC; ir n C═O 1740, N═C
Antimicrobial activity of the substituted 3-(2-oxo-
2H-benzo[b][1,4]oxazin-3-yl)propanoates (3a–e) was
performed with the Bauer-Kirby method by the well
diffusion technique using Petri plates made by Muller
Hinton agar containing the culture [24,25]. This run
involved the preparation of sterilized Whatman filter
paper cut in form of disc of 0.6 cm of diameter, which
was impregnated with the compound to be tested
(160 mg/mL in DMSO), followed by adhering it to the
surface of Petri plate that was previously inoculated with
diluted culture. The incubation was realized at 37ꢀC for
48 h for bacteria and at 25ꢀC for 3–4 days for the yeast.
The results, expressed as the inhibition aureola diameters
in millimeter (mm) and the MIC (mg/mL), are shown in
Table 1.
1616 cmÀ1; H NMR: d 2.38 (s, 3H, CH3); 2.74 (t, 2H, CH2,
1
J = 7.1 Hz); 3.01 (t, 2H, CH2, J = 7.1 Hz); 3.59 (s, 3H, OCH3);
7.18 (d, 1H, Harom, J = 8.4 Hz); 7.20 (s, 1H, Harom); 7.54
(d, 1H, Harom, J = 8.4 Hz). 13C NMR: d 20.98 (CH3); 28.02
(CH2); 29.12 (CH2); 51.44 (OCH3); 116.18 (C-8); 126.20
(C-6); 127.85 (C-5); 128.55 (C-7); 141.17 (C-4a); 145.93
(C-8a); 152.69 (C═O); 155.21 (N═C); 172.66 (COO); ms: m/z
247 (M+, 88%), 216 (M+ À OCH3, 51%), 188 (M+ À CO2CH3,
36%), 160 (M+ À CH2-CH2-CO2CH3, 100%). Anal. Calcd for
C13H13NO4: C, 63.15; H, 5.30; N, 5.67. Found: C, 63.08; H,
5.11; N, 5.61.
Methyl 3-(6-methyl-2-oxo-2H-benzo[b][1,4]oxazin-3-yl)pro-
panoate (3c). This compound was obtained as colorless
crystals,1.23 g (50%), mp 100–101ꢀC; ir n C═O 1740, N═C
1612 cmÀ1 1H NMR: d 2.35 (s, 3H, CH3); 2.74 (t, 2H,
;
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

416
K. Hachama, M. Khodja, S. Moulay, H. Boutoumi, L. Hennig, and D. Sicker
Vol 50
[13] Azuma, K.; Suzuki, S.; Uchiyama, S.; Kajiro, T.; Santa, T.;
Imai, K. Photochem Photobiol Sci 2003, 2, 443.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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