Synthesis, antimicrobial activity and QSARs of new benzoxazine-3-ones

Article abstract of DOI:10.1016/j.ejmech.2006.06.011

New ethyl 3,4-dihydro-3-oxo-4,6,7-trisubstituted-2H-1,4-benzoxazine-2-acetate derivatives were synthesized and their structures were elucidated by IR, ¹H NMR and mass spectral data. Antimicrobial activity of the compounds was investigated by us

Full text of DOI:10.1016/j.ejmech.2006.06.011

European Journal of Medicinal Chemistry 41 (2006) 1398e1404
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antimicrobial activity and QSARs of new benzoxazine-3-ones
a
a
a
S. Alper-Hayta a, E. Akı-Sener a, , B. Tekiner-Gulbas , I. Yıldız , O. Temiz-Arpacı ,
*
I. Yalcın a, N. Altanlar b
a Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Degol Street, TR-06100 Tandogan, Ankara, Turkey
b Ankara University, Faculty of Pharmacy, Department of Microbiology, TR-06100 Tandogan, Ankara, Turkey
Received 20 February 2006; received in revised form 13 June 2006; accepted 15 June 2006
Available online 22 September 2006
Abstract
New ethyl 3,4-dihydro-3-oxo-4,6,7-trisubstituted-2H-1,4-benzoxazine-2-acetate derivatives were synthesized and their structures were eluci-
dated by IR, 1H NMR and mass spectral data. Antimicrobial activity of the compounds was investigated by using the method of twofold serial
dilution technique against different Gram-positive, Gram-negative bacteria and some Candida species in comparison to standard drugs. Micro-
biological results indicated that the synthesized compounds possessed a broad spectrum of activity having MIC values of 6.25e100 mg/ml
against the tested microorganisms. The QSAR analysis of a set of these compounds tested for growth inhibitory activity against Candida krusei
was performed by using the computer-assisted multiple regression procedure. The activity contributions for substituent effects of these com-
pounds were determined from the correlation equation for predictions of the lead optimization.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: 1,4-Benzoxazine-3-one; Antibacterial activity; Antifungal activity; QSAR
1. Introduction
glucosides from which the aglycones are released rapidly by
enzymatic hydrolysis after physical and biological injury of
the plants and they exhibit antifungal, antibacterial and insec-
ticide properties [15,16]. Only a limited number of compounds
containing 1,4-benzoxazine ring system have been studied for
their chemotherapeutic activity. Ofloxacin (Fig. 1) is one of the
antimicrobial agents possessing the 1,4-benzoxazine ring
system in its structure. All these observations prompted us to
investigate this heterocyclic system to ascertain if it would
offer any advantage over the other known clinically used anti-
microbial drugs [17,18].
The common method for preparing 1,4-benzoxazine ring
having ethyl acetate group at position 2 is by heating amino-
phenols with ethyl 2,3-dibromopropanoate and potassium car-
bonate in acetone [19].
Michael addition method is used for preparing 3,4-dihydro-
3-oxo-2H-1,4-benzoxazine-2-acetates. Reaction of various
o-aminophenols with maleic anhydride refluxed in ethanol in
the presence of triethylamino or ether or benzene gives 1,4-
benzoxazine-3-ones with ethyl acetate group at position 2 in
The usage of most antimicrobial agents is limited, not only
by the rapidly developing drug resistance, but also by the un-
satisfactory status of present treatments of bacterial and fungal
infections and drug side effects [1e4]. Therefore, the develop-
ment of new and different antimicrobial drugs is a very impor-
tant objective and much of the research program efforts are
directed towards the design of new agents.
A group of 1,4-benzoxazine-3-ones was isolated from
maize, wheat and rye several years ago [5e9]. 2,4-Dihy-
droxy-1,4-benzoxazine-3-one, DIBOA (Fig. 1) and its methoxy
derivative DIMBOA (Fig. 1) have been shown to inhibit germi-
nation of spores of the phytopathogenic fungi [10]. They play
an important role in the chemical defence of cereals against
deleterious pests such as insects, pathogenic fungi and bacteria
[11e14]. These molecules are present naturally in the plants as
* Corresponding author. Tel.: þ90 312 2239253; fax: þ90 312 2236940.
E-mail address: sener@pharmacy.ankara.edu.tr (E. Akı-Sener).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.06.011

S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 41 (2006) 1398e1404
1399
O
OR
O
F
COOH
O
N
N
N
OH
N
O
H3C
CH3
3 OFLOXACIN
1 DIBOA, R=H
2 DIMBOA, R=OCH3
Fig. 1. Biologically active 1,4-benzoxazines.
%30 yield [20e22]. Another similar method is heating o-ami-
nophenol with fumaric acid chloride monoethylester. This re-
action gives the fumaramides which upon treatment with
potassium carbonate in ethanol produced 3-oxo-2H-1,4-ben-
zoxazine-2-acetates in %26e90 yield [22,23].
to predict the lead optimization for antifungal activity against
Candida krusei.
2. Chemistry
Alkylation of 2H-1,4-benzoxazine-3-one derivatives occurs
at the ring nitrogen. Using alkyl halides and sodium hydride as
bases is the most common method for alkylation [24,25].
In this research, some novel ethyl 3,4-dihydro-3-oxo-6 and/
or 7-disubstituted-2H-1,4-benzoxazine-2-acetate compounds
and their N-alkylated derivatives were synthesized in order
to interpret their antifungal activity against different Candida
species and antibacterial activity against different Gram-posi-
tive and Gram-negative bacteria. The target of this research
was to observe differences between the derivatives with free
NH and N-alkylated ones towards antimicrobial activity. The
alkylated derivatives were synthesized to avoid the behaviour
of free NH as a hydrogen bond donor. 2D-QSAR analysis was
also aimed to explain clearly the relation of physicochemical
parameters with the activity. QSAR analysis of these com-
pounds was performed by multiple regression analysis in order
Monoethyl fumaryl chloride was added to a mixture of ap-
propriate o-aminophenol and sodium bicarbonate in dioxane.
The prepared phenylcarbamoyl acrylate derivatives 4 were
then stirred with potassium carbonate in ethanol at room tem-
perature to form compound 5. Alkylated compounds 6 were
prepared by stirring compound 5 in acetone with methyliodide
and/or ethyliodide and potassium carbonate (Scheme 1).
3. Results and discussion
A series of ethyl 3,4-dihydro-3-oxo-4,6,7-trisubstituted-2H-1,
4-benzoxazine-2-acetate derivatives (7e23) have been synthe-
sized by using a three-step procedure as shown in Scheme 1.
All of the derivatives were supported by spectral data. The
1
IR and H NMR spectra are in agreement with the proposed
R2
R2
OH
OH
COOC2H5
NaHCO3
Dioxane
CHCOOC2H5
+
COCl
NH
NH2
R1
R1
O
4
R2
R2
CH2COOC2H5
CH2COOC2H5
O
O
K2CO3
EtOH
K2CO3
RI
N
H
N
R
R1
R1
O
O
5
6
Scheme 1. Synthesis of 1,4-benzoxazine derivatives.

1400
S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 41 (2006) 1398e1404
structures. Physical and spectral data of the compounds are re-
ported in Section 4. In order to determine the antimicrobial ac-
tivity of the synthesized compounds (7e23), three Gram-
positive, two Gram-negative bacteria and three Candida
species were screened using the twofold serial dilution tech-
nique [26]. All biological results of the compounds are given
in Table 1.
Although compounds 13, 14, 15, 17 and 19 showed activity
having an MIC value of 12.5 mg/ml against C. albicans, they
exhibited lower potencies than the compared control drugs.
Compounds 8 and 12 were found to be the most active deriv-
atives against C. krusei and C. glabrata at an MIC value of
6.25 mg/ml among the other compounds whereas compound
10 showed the same activity against C. krusei.
Compounds showed antimicrobial activity against Staphy-
lococcus aureus and Streptococcus faecalis at MIC values of
25e50 mg/ml. Compounds 11 and 13 were found to be
more active than the others at an MIC value of 12.5 mg/ml
against Bacillus subtilis. The synthesized compounds
showed antimicrobial activity with MIC values between 25
and 100 mg/ml against Escherichia coli which exhibited
lower potencies than the compared control drugs. Com-
pound 7 was found to be the most active derivative against
Pseudomonas aeruginosa at an MIC value of 12.5 mg/ml
among the tested compounds, and showed the same potency
with Gentamycin.
According to structureeactivity relationships (SAR), it can
be concluded that compounds with a free NH (7e13) are gen-
erally more active than the N-alkylated ones (14e23) against
selected microorganisms and an electron-withdrawing substit-
uent at position 6 of benzoxazine moderately favours the
inhibition of the growth. Additionally, a nitro group at position
7 of the fused ring slightly helped to inhibit the growth of the
microorganisms.
Furthermore, the QSARs of these molecules were analysed
by multiple regression analysis (MRA) in order to predict the
lead optimization in this set of compounds. From the data as
seen in Table 2, the following QSAR was developed:
Compounds 7e23 were also tested against Candida albi-
cans, Candida krusei, and Candida glabrata for their antimy-
cotic activity, and they indicated significant antimycotic
activity displaying MIC values between 6.25 and 50 mg/ml.
log 1=C ¼ þ0:0499ð ꢀ 0:039ÞB1R þ 0:169ð ꢀ 0:053ÞsR
1
þ 0:0569ð ꢀ 0:020ÞIxR þ 3:950
ð1Þ
2
Table 1
The in vitro antimicrobial activity of the synthesized compounds and the control drugs
O
R2
CH2COOC2H5
N
R
R1
O
Comp. no.
R
R1
R2
Fungi
Gram-positive
Gram-negative
C. albicans
C. krusei
C. glabrata
S. aureus
St. faecalis
B. subtilis
E. coli
P. aeruginosa
7
H
H
CH3
H
25
25
12.5
6.25
12.5
6.25
12.5
6.25
12.5
12.5
12.5
50
12.5
6.25
12.5
12.5
12.5
6.25
12.5
12.5
12.5
25
50
50
50
50
25
25
25
50
50
50
50
50
50
25
25
50
25
e
50
50
50
50
25
50
25
50
50
50
50
50
50
e
25
25
50
25
50
50
25
50
25
50
25
50
50
25
50
100
50
50
100
e
12.5
25
50
25
50
25
25
25
25
25
25
25
25
e
8
H
H
9
10
H
Cl
COOC2H5
H
25
25
25
25
H
H
11
12
H
H
NO2
NH2
NO2
H
25
50
12.5
25
H
H
Cl
13
14
H
12.5
12.5
12.5
25
12.5
25
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
C2H5
C2H5
e
H
CH3
15
16
H
25
50
Cl
COOC2H5
H
17
18
H
12.5
25
12.5
12.5
12.5
25
12.5
12.5
12.5
25
25
50
Cl
H
NO2
NO2
H
19
20
12.5
25
50
25
H
21
22
Cl
H
H
25
25
25
50
25
25
e
50
50
e
NO2
NO2
e
e
e
e
e
23
Cl
e
e
e
e
e
25
25
e
25
e
100
e
Oxiconazole
Haloprogin
Miconazole
Ciprofloxacin
Gentamycin
6.25
3.12
3.12
e
e
e
e
e
e
e
e
1.56
e
e
3.12
e
e
e
e
e
e
e
e
e
e
e
e
e
3.125
3.125
e
12.5
1.56
1.56
3.125
12.5
0.78
12.5
e
e
e
e
e

S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 41 (2006) 1398e1404
1401
Table 2
The parameters used in Eq. (1)
position R is important for the antifungal activity against
C. krusei. These observations could guide us to design further
new lead antifungal compounds.
Comp. no. IxR
sR
B1R
Obs. log 1/C Calc. log 1/C Residuals
2
1
7
0
0
0
0
1
e
1
0
0
0
0
1
1
0
0
1
1
0.00 1.00 3.973
ꢂ0.17 1.00 3.998
0.23 1.00 4.032
0.45 1.00 4.089
0.00 1.00 4.049
4.000
3.972
4.039
4.076
4.057
e
ꢂ0.027
0.026
8
4. Experimental procedures
9
ꢂ0.007
0.013
10
11
12a
13
14
15
16
17
18
19
20
21
22
23
a
4.1. Chemistry
ꢂ0.008
e
0.003
e
e
e
Silicagel HF254 chromatoplates (0.3 mm) were used for
TLC. The solvent systems were chloroform:methanol
(15:0.5) and ethyl acetate:n-hexane (20:10) for compounds
7e23. Melting points were taken on a Buchi SMP 20 capillary
apparatus and are uncorrected. IR spectra were recorded by
FT/IR-420 in KBr discs. 1H NMR spectra were obtained
with a Bruker GmbH D PX-400 MHz spectrometer in chloro-
form; tetramethylsilane (TMS) was used as an internal stan-
dard. Elemental analyses were carried out with a Perkin
Elmer model 240-C apparatus. The results (C, H, N) were
within ꢀ0.4% of the calculated values.
0.23 1.00 4.099
0.00 1.52 3.998
4.096
4.026
3.997
4.065
4.102
4.122
4.083
4.026
4.065
4.083
4.122
ꢂ0.028
0.025
ꢂ0.17 1.52 4.022
0.23 1.52 4.054
0.45 1.52 4.106
0.23 1.52 4.118
0.00 1.52 4.070
0.00 1.52 4.022
0.23 1.52 4.075
0.00 1.52 4.091
0.23 1.52 4.136
ꢂ0.011
0.004
ꢂ0.004
ꢂ0.013
ꢂ0.004
0.010
0.008
0.014
This compound is outlier in QSAR analysis.
n ¼ 16, R2 ¼ 0.942, s ¼ 0.018, F ¼ 31.471 > Ftab(3, 12, 0.05)
3.49, Q2 ¼ 0.790, SPRESS ¼ 0.024.
¼
4.2. General procedure for compounds 7e23
Monoethyl fumaric acid (10 mmol) and thionyl chloride
(5 mmol) were stirred for 3 h at 80 ꢁC in benzene (15 ml).
At the end of the reaction, excess thionyl chloride was evapo-
rated. Obtained monoethyl fumaryl chloride was added
dropwise to a suspension of appropriate o-aminophenol
(10 mmol) and NaHCO3 (10 mmol) in dioxane. The reaction
mixture was stirred for 24 h at room temperature, then poured
into water and extracted with ethyl acetate (15 ml). The extract
was washed with water, dried and evaporated, respectively.
The residue was recrystallised from EtOH to give 4 ethyl 3-
[(4- and/or 5-substituted-2-hydroxyphenyl)carbamoyl]acrylate
derivatives. A suspension of these compounds (10 mmol) and
K2CO3 (5 mmol) in EtOH (70 ml) was stirred for 3 h at room
temperature. The reaction mixture was poured into water and
extracted with ethyl acetate (15 ml). The extracts were washed
with water, dried and evaporated, respectively [22,23]. The
residue was recrystallised from EtOH.
The general synthetic procedure as seen above was
employed to prepare compounds 14e23 involving the reaction
of methyliodide or ethyliodide (15 mmol) and ethyl 3,4-dihy-
dro-6-and/or 7-substituted-3-oxo-2H-1,4-benzoxazine-2-ace-
tate derivatives (10 mmol) 5, by heating in the presence of
potassium carbonate (15 mmol) in acetone (100 ml) for 4 h
at 40 ꢁC. After removal of the solvent, the residue was ex-
tracted with ethyl acetate (15 ml). The extracts were washed
with water, then dried and evaporated, respectively [24,25].
The residue was recrystallised from petroleum ether.
Eq. (1) was found to be the best fit for the predictions
according to the examined validation test results. As can be
deduced from Table 2, the goodness of fit of equation is signif-
icant, possessing a high R2 (0.942) and a small s (0.018) with
an overall F test value of 31.471 at a significant level of
p < 0.05. The correlation coefficients which are given in
Table 3 revealed that there was no colinearity between the
independent variables used in Eq. (1). The parameters used
in this QSAR analysis together with the observed, calculated
and residual values are given in Table 2.
QSAR analysis revealed that the substitution of positions R
and R1 was more significant than the position R2 for the tested
antifungal activity against C. krusei. The QSAR was devel-
oped based on the s values of the substituents at position 6,
where C is the minimum concentration of the compound
that inhibited the growth of C. krusei. From this relationship,
we see that an electron-withdrawing substituent at position 6
of benzoxazine favours inhibition of growth. The positive
coefficients of B1 parameter indicated that the width of the
substituent at position 4 was conducive for the activity. There-
fore, the compounds having an NeH instead of N-alkylated of
1,4-benzoxazine moiety were found to be more active than the
others.
In conclusion, it was found that the 2D-QSAR study
confirmed the SAR study for antimycotic activity against
C. krusei. We could conclude that the width substituent on
For the preparation of compounds 10 and 17, first of all,
mixture of 3-amino-4-hydroxy benzoic acid (10 mmol), sul-
phuric acid (0.5 ml) and absolute ethanol (30 ml) was cooled
in an ice bath and dry HCl gas was passed through it. When
the mixture was saturated with HCl (g), it was stirred at
room temperature for 5 h. At the end of the reaction, the pre-
cipitates were collected by filtration, washed with water and
dried in vacuo to give ethyl 3-amino-4-hydroxy benzoate
Table 3
Correlation matrix of variables used in Eq. (1)
X-1
X-2
X-3
X-1
X-2
X-3
1.000
0.001
1.000
0.004
0.000
1.000
X ꢂ 1 ¼ IxR ; X ꢂ 2 ¼ sR ; X ꢂ 3 ¼ B1R
:
2
1

1402
S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 41 (2006) 1398e1404
crystals [27]. Then the general synthetic procedure as seen
above was employed. Quantitative reduction of nitro group
in compound 11 was achieved by catalytic hydrogenation to
make compound 12. Compound 11 (3.5 mmol) in EtOH
(75 ml) was subjected to hydrogenation using 40 psi of H2
and 10% PdeC (40 mg) until uptake of H2 ceased. The cata-
lyst was filtered on a bed of celite, washed with EtOH, and the
filtrate was concentrated in vacuo to give compound 12 [28].
8.30 (s, 1H, NH ); IR (cmꢂ1): 3200e2900, 1740e1690,
1613, 1550e1510, 1440e1320, 1260e1030, 990e730; MS
(70 eV) m/z: 280 (Mþ), 42 (100%).
4.2.6. 3,4-Dihydro-3-oxo-7-amino-2H-1,4-benzoxazine-2-
acetate (12)
C12H14N2O4 e m.p.: 150 ꢁC; yield (%): 35; 1H NMR
(ppm): 1.25 (t, 3H, CH2COOCH2CH3, J ¼ 7.2), 3.00 (dd,
2H, CH2COOCH2CH3), 3.50 (s, 2H, NH2), 4.20 (q, 2H,
CH2COOCH2CH3), 5.00 (q, 1H, CH ), 6.20e6.50 (m, 3H, ar-
omatic H, Jm ¼ 8.3), 7.60 (s, 1H, NH ); IR (cmꢂ1): 3400e
2800, 1733e1682, 1523, 1375, 1278e1050, 855e439; MS
(70 eV) m/z: 250 (Mþ), 83 (100%).
4.2.1. 3,4-Dihydro-3-oxo-2H-1,4-benzoxazine-2-acetate (7)
C12H13NO4 e m.p.: 106e107 ꢁC [22]; yield (%): 80;
1H NMR (ppm): 1.25 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 3.00
(dd, 2H, CH2COOCH2CH3), 4.20 (q, 2H, CH2COOCH2CH3,
J ¼ 7.2), 5.00 (q, 1H, CH ), 6.80e7.10 (m, 4H, aromatic H ),
8.50 (s, 1H, NH ); IR (cmꢂ1): 3230e2860, 1740, 1695,
1625, 1520, 1480e1280, 1240e1030, 950e585; MS (70 eV)
m/z: 235 (Mþ), 161 (100%).
4.2.7. 3,4-Dihydro-3-oxo-6-chloro-7-nitro-2H-1,4-
benzoxazine-2-acetate (13)
C12H11N2O6Cl e m.p.: 162e165 ꢁC [23]; yield (%): 50; 1H
NMR (ppm): 1.25 (t, 3H, CH2COOCH2CH3, J ¼ 7.2), 3.00
(dd, 2H, CH2COOCH2CH3), 4.10 (q, 2H, CH2COOCH2CH3,
J ¼ 7.1), 5.00 (q, 1H, CH ), 6.90 (s, 1H, 5-H ), 7.60 (s, 1H,
8-H ), 8.40 (s, 1H, NH ); IR (cmꢂ1): 3200e2900, 1740e
1690, 1615, 1550e1515, 1440e1319, 1250e1000, 950e
685; MS (70 eV) m/z: 314 (Mþ), 43 (100%).
4.2.2. 3,4-Dihydro-3-oxo-6-methyl-2H-1,4-benzoxazine-2-
acetate (8)
1
C13H15NO4 e m.p.: 115e116 ꢁC [22]; yield (%): 36; H
NMR (ppm): 1.25 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 2.25
(s, 3H, CH3), 2.80e3.00 (dd, 2H, CH2COOCH2CH3), 4.20
(q, 2H, CH2COOCH2CH3, J ¼ 7.1), 5.00 (q, 1H, CH ), 6.50
(d, 1H, 5-H, J5,7 ¼ 1.1), 6.75e6.85 (m, 2H, 7-H/8-H ), 8.10
(s, 1H, NH ); IR (cmꢂ1): 3220e2890, 1750, 1690, 1618,
1430e1500, 1460e1330, 1300e1035, 990e590; MS
(70 eV) m/z: 249 (Mþ), 42 (100%).
4.2.8. 3,4-Dihydro-3-oxo-4-methyl-2H-1,4-benzoxazine-
2-acetate (14)
1
C13H15NO4 e m.p.: 35 ꢁC; yield (%): 40; H NMR (ppm):
1.25 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 3.00 (dd, 2H,
CH2COOCH2CH3), 3.40 (s, 3H, CH3), 4.20 (q, 2H,
CH2COOCH2CH3, J ¼ 7.2), 5.00 (q, 1H, CH ), 7.10 (m, 4H,
5-H, 6-H, 7-H, 8-H ); IR (cmꢂ1): 1744e1683; MS (70 eV)
m/z: 249 (Mþ), 249 (100%).
4.2.3. 3,4-Dihydro-3-oxo-6-chloro-2H-1,4-benzoxazine-2-
acetate (9)
C12H12NO4Cl e m.p.: 143e145 ꢁC [22]; yield (%): 50; 1H
NMR (ppm): 1.25 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 3.00
(dd, 2H, CH2COOCH2CH3), 4.20 (q, 2H, CH2COOCH2CH3,
J ¼ 7.1), 4.95 (q, 1H, CH ), 6.75e6.95 (m, 3H, aromatic H ),
8.30 (s, 1H, NH ); IR (cmꢂ1): 3200e2895, 1740e1700,
1615, 1510, 1430e1330, 1300e1030, 990e590; MS (70 eV)
m/z: 269 (Mþ), 195 (100%).
4.2.9. 3,4-Dihydro-3-oxo-4,6-dimethyl-2H-1,4-benzoxazine-
2-acetate (15)
1
C14H17NO4 e m.p.: 58 ꢁC; yield (%): 45; H NMR (ppm):
1.30 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 2.30 (s, 3H, CH3),
3.00 (dd, 2H, CH2COOCH2CH3), 3.45 (s, 3H, CH3), 4.25 (q,
2H, CH2COOCH2CH3, J ¼ 7.1), 5.00 (q, 1H, CH ), 6.80 (m,
3H, 5-H, 7-H, 8-H ); IR (cmꢂ1): 1743e1691; MS (70 eV)
m/z: 263 (Mþ), 189 (100%).
4.2.4. 3,4-Dihydro-3-oxo-6-ethoxycarbonyl-2H-1,4-
benzoxazine-2-acetate (10)
1
C15H17NO6 e m.p.: 125e130 ꢁC; yield (%): 60; H NMR
(ppm): 1.15 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 1.30 (t, 3H,
COOCH2CH3, J ¼ 7.1), 3.00 (dd, 2H, CH2COOCH2CH3),
4.30 (q, 2H, CH2COOCH2CH3, J ¼ 7.1), 4.90 (q, 2H,
COOCH2CH3, J ¼ 7.1), 5.10 (q, 1H, CH ), 6.70e7.70 (q,
3H, aromatic H, J8,5 ¼ 8.5, J0 ¼ 2.0, J7,5 ¼ 8.5), 8.70 (s, 1H,
NH ); IR (cmꢂ1): 3200e2850, 1687, 1615, 1493, 1376,
1298e1132, 989e420; MS (70 eV) m/z: 308 (Mþ), 45
(100%).
4.2.10. 3,4-Dihydro-3-oxo-4-methyl-6-chloro-2H-1,4-
benzoxazine-2-acetate (16)
C13H14NO4Cl e m.p.: 56 ꢁC; yield (%): 41; 1H NMR
(ppm): 1.25 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 3.00 (dd,
2H, CH2COOCH2CH3), 3.40 (s, 3H, CH3), 4.20 (q, 2H,
CH2COOCH2CH3, J ¼ 7.1), 5.00 (q, 1H, CH ), 6.90 (m, 3H,
5-H, 7-H, 8-H ); IR (cmꢂ1): 1741e1692; MS (70 eV) m/z:
283 (Mþ), 82 (100%).
4.2.5. 3,4-Dihydro-3-oxo-7-nitro-2H-1,4-benzoxazine-2-
acetate (11)
4.2.11. 3,4-Dihydro-3-oxo-4-methyl-6-ethoxycarbonyl-
2H-1,4-benzoxazine-2-acetate (17)
1
1
C12H12N2O6 e m.p.: 172 ꢁC [23]; yield (%): 39; H NMR
C16H19NO6 e m.p.: 40 ꢁC; yield (%): 35; H NMR (ppm):
(ppm): 1.25 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 3.00 (dd, 2H,
CH2COOCH2CH3), 4.20 (q, 2H, CH2COOCH2CH3), 5.00 (q,
1H, CH ), 6.80 (d, 1H, 5-H, Jm ¼ 8.7), 7.80 (m, 2H, 6-H/8-H ),
1.25 (t, 3H, CH2COOCH2CH3, J ¼ 7.2), 1.30 (t, 3H, J ¼ 7.1),
3.00 (dd, 2H, CH2COOCH2CH3), 3.45 (s, 3H, CH3), 4.25 (q,
2H, CH2COOCH2CH3, J ¼ 7.1), 4.35 (q, 2H, J ¼ 7.1), 5.00

S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 41 (2006) 1398e1404
1403
(q, 1H, CH ), 7e7.75 (m, 3H, 5-H, 7-H, 8-H ); IR (cmꢂ1):
4.3. Microbiology
1711; MS (70 eV) m/z: 321 (Mþ), 247 (100%).
For the antibacterial and antimycotic assays, the com-
pounds were dissolved in absolute ethanol (0.8 mg/ml). Fur-
ther dilutions of the compounds and standard drugs in the
test medium were prepared at the required quantities of 400,
200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 mg/ml concentra-
tions with MuellereHinton broth and Sabouraud dextrose
broth. The minimum inhibitory concentrations (MICs) were
determined using the twofold serial dilution technique [26].
A control test was also performed containing inoculated broth
supplemented with only ethanol at the same dilutions used in
our experiments and found inactive in the culture medium. All
the compounds were tested for their in vitro growth inhibitory
activity against different bacteria and the yeasts C. albicans
ATCC 10145, C. krusei ATCC 6258, C. glabrata (isolated).
Origin of bacterial strains are S. aureus ATCC 25923, B. subtilis
ATCC 6633 and St. faecalis ATCC 10541 as Gram-positive
and E. coli ATCC 23556 and P. aeruginosa ATCC 10145 as
Gram-negative bacteria. ATCC strains of the microorganisms
used in this study were obtained from the culture collection
of Refik Saydam Health Institution of Health Ministry, Ankara,
and maintained at the Microbiology Department of Faculty of
Pharmacy of Ankara University.
4.2.12. 3,4-Dihydro-3-oxo-4-methyl-6-chloro-7-nitro-2H-
1,4-benzoxazine-2-acetate (18)
C13H13N2O6Cl e m.p.: not detected; yield (%): 19; 1H
NMR (ppm): 1.30 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 3.00
(dd, 2H, CH2COOCH2CH3), 3.45 (s, 3H, CH3), 4.20 (q, 2H,
CH2COOCH2CH3), 5.00 (q, 1H, CH ), 6.80 (s, 1H, 5-H ),
7.30 (s, 1H, 8-H ); IR (cmꢂ1): 1699; MS (70 eV) m/z: 328
(Mþ), 55 (100%).
4.2.13. 3,4-Dihydro-3-oxo-4-methyl-7-nitro-2H-1,4-
benzoxazine-2-acetate (19)
1
C13H14N2O6 e m.p.: 95e100 ꢁC; yield (%): 50; H NMR
(ppm): 1.35 (t, 3H, CH2COOCH2CH3, J ¼ 7.1), 3.10 (m, 2H,
CH2COOCH2CH3), 3.45 (s, 3H, CH3), 4.30 (q, 2H,
CH2COOCH2CH3, J ¼ 7.1), 5.10 (q, 1H, CH ), 7.10 (d, 1H,
5-H, J ¼ 9.0), 7.90 (d, 1H, 8-H, J0 ¼ 2.5), 8.10 (dd, 1H, 6-H,
J ¼ 2.5); IR (cmꢂ1): 1716e1598; MS (70 eV) m/z: 294
(Mþ), 220 (100%).
4.2.14. 3,4-Dihydro-3-oxo-4-ethyl-2H-1,4-benzoxazine-
2-acetate (20)
1
C14H17NO4 e m.p.: 40e45 ꢁC; yield (%): 34; H NMR
Oxiconazole, Haloprogin, Ciprofloxacin, Gentamycin and
Miconazole were used as control drugs. The data on the anti-
fungal and antibacterial activities of the compounds and the
control drugs as MIC, mg/ml, values are given in Table 1.
(ppm): 1.10e1.40 (overlapped, 6H, CH3), 3.00 (dd, 2H,
CH2COOCH2CH3), 3.90 (q, 2H, CH2CH3), 4.10 (q, 2H,
COOCH2CH3), 4.90 (q, 1H, CH ), 7.00 (m, 4H, 5-H, 6-H,
7-H, 8-H ); IR (cmꢂ1): 1744e1683; MS (70 eV) m/z: 263
(Mþ), 263 (100%).
4.3.1. Antibacterial and antifungal assays
The cultures were obtained from MuellereHinton broth
(Difco) for all the bacterial strains after 24 h of incubation at
37 ꢀ 1 ꢁC. C. albicans, C. krusei and C. glabrata were main-
tained in Sabouraud dextrose broth (Difco) after incubation for
24 h at 25 ꢀ 1 ꢁC. Testing was carried out in MuellereHinton
broth and Sabouraud dextrose broth (Difco) at pH 7.4 and the
twofold serial dilution technique was applied. The final inoc-
ulum size was 105 CFU/ml for the antibacterial assay and
104 CFU/ml for the antifungal assay. A set of tubes containing
only inoculated broth were used as controls. For the antibacte-
rial assay after incubation for 24 h at 37 ꢀ 1 ꢁC and after incu-
bation for 48 h at 25 ꢀ 1 ꢁC for the antifungal assay, the last
tube with no growth of microorganism and/or yeast was re-
corded to represent the MIC expressed in mg/ml. Every exper-
iment in the antibacterial and antifungal assays was done in
duplicate.
4.2.15. 3,4-Dihydro-3-oxo-4-ethyl-6-chloro-2H-1,4-
benzoxazine-2-acetate (21)
C14H16NO4Cl e m.p.: 65 ꢁC; yield (%): 46; 1H NMR
(ppm): 1.10e1.20 (overlapped, 6H, CH3), 3.00 (dd, 2H,
CH2COOCH2CH3), 3.90 (q, 2H, CH2CH3), 4.20 (q, 2H,
COOCH2CH3), 5.00 (q, 1H, CH ), 6.90e7.00 (m, 3H, 5-H,
7-H, 8-H ); IR (cmꢂ1): 1741e1692; MS (70 eV) m/z: 297
(Mþ), 297 (100%).
4.2.16. 3,4-Dihydro-3-oxo-4-ethyl-7-nitro-2H-1,4-
benzoxazine-2-acetate (22)
C14H16N2O6 e m.p.: 100 ꢁC; yield (%): 52; 1H NMR
(ppm): 1.10e1.30 (overlapped, 6H, CH3), 3.00 (dd, 2H,
CH2COOCH2CH3), 3.90 (q, 2H, CH2CH3), 4.10 (q, 2H,
COOCH2CH3), 4.90 (q, 1H, CH ), 7.10 (d, 1H, 5-H ), 7.90
(d, 1H, 8-H ), 8.00 (dd, 1H, 6-H ); IR (cmꢂ1): 1716e1598;
MS (70 eV) m/z: 308 (Mþ), 97 (100%).
4.4. Data processing
4.2.17. 3,4-Dihydro-3-oxo-4-ethyl-6-chloro-7-nitro-2H-1,4-
benzoxazine-2-acetate (23)
Hansch analysis method which is an extra-thermodynamic
approach in QSAR analysis was applied in order to determine
the lead optimization due to the various physicochemical
(electronic, steric and hydrophobic) parameters and structural
indicator parameters [29,30].
Regression analysis and calculations were run on a PC us-
ing the BILIN statistical program package [31]. In equations,
the figures in parentheses are the standard errors of the
C14H15N2O6Cl e m.p.: 75e80 ꢁC; yield (%): 31; 1H NMR
(ppm): 1.10e1.35 (overlapped, 6H, CH3), 3.00 (dd, 2H,
CH2COOCH2CH3), 4.00 (q, 2H, CH2CH3), 4.10 (q, 2H,
COOCH2CH3), 5.00 (q, 1H, CH ), 6.80 (s, 1H, 5-H ), 7.30
(s, 1H, 8-H ); IR (cmꢂ1): 1699; MS (70 eV) m/z: 342 (Mþ),
264 (100%).

1404
S. Alper-Hayta et al. / European Journal of Medicinal Chemistry 41 (2006) 1398e1404
[6] A.I. Virtanen, P.K. Hietala, Acta Chem. Scand. 14 (2) (1960) 499e502.
[7] P.K. Hietala, A.I. Virtanen, Acta Chem. Scand. 14 (2) (1960) 502e504.
[8] C.L. Tipton, J.A. Klun, R.R. Husted, M.D. Pierson, Biochemistry 6
(1967) 2866e2870.
[9] J.A. Klun, C.L. Tipton, J.F. Robinson, D.L. Ostreem, M. Beroza, Agric.
Food Chem. 18 (4) (1970) 663e665.
regression coefficients. For a given equation, n is the number
of compounds, R2 denotes the square of the multiple correla-
tion coefficients, F is the significance test and s represents
the residual standard deviation.
On the other hand, in order to judge the predictive power as
Q2 and/or SPRESS values of the performed QSAR model was
also calculated by cross-validation technique which is
a method to check the validity of regression models by elim-
inating each object leave-one-out technique [32].
[10] C.L. Tipton, M.C. Wang, F.H. Tasao, C.C. Lintu, R.R. Husted, Phyto-
chemistry 12 (3) (1973) 347e352.
[11] H.M. Niemeyer, Phytochemistry 27 (11) (1988) 3349e3358.
[12] G.E. Zuniga, F. Massardo, Phytochemistry 30 (10) (1991) 3281e3283.
[13] H.M. Niemeyer, E. Pesel, S. Franke, W. Francke, Phytochemistry 28 (9)
(1989) 2307e2310.
[14] E. Glawischnig, S. Grun, M. Frey, A. Gierl, Phytochemistry 50 (6) (1999)
925e930.
4.5. Determination of parameters
[15] T. Ishizaki, Y. Hashimoto, K. Shudo, T. Okamato, Tetrahedron Lett. 23
(39) (1982) 4055e4056.
In this study, the model is based on the in vitro activity of cer-
tain ethyl 3,4-dihydro-3-oxo-4,6,7-trisubstituted-2H-1,4-ben-
zoxazine-2-acetate derivatives against C. krusei, where C is
the MIC value expressed in molar concentration units (Table 1).
The variables used as descriptors in the analysis are elec-
tronic, steric and structural parameters. The structural indica-
tor variable Ix expresses the replacement of H by nitro group
at R2 (position 7). Ix defined as 1 for NO2, and as 0 for H.
The screened physicochemical parameters in this QSAR
study are p for the hydrophobic effects, s, F (field effect), R
(resonance effect) as electronic influences and Verloop’s
STERIMOL parameters (L and B1) for the steric interactions
of the substituents R, R1 and R2. Values for all candidate phys-
icochemical variables used in this QSAR study were taken
from the table of Hansch and Leo [33]. The values of the de-
scriptors used in the best equation are shown in Table 2.
[16] S. Ozden, A.M. Ozturk, H. Goker, N. Altanlar, Il Farmaco 55 (2000)
715e718.
[17] I. Hayakawa, S. Atarashi, S. Yokohama, M. Imamura, K. Sakano,
M. Frukawa, Antimicrob. Agents Chemother. 29 (1) (1986) 163e164.
[18] A. Abdi-Ali, M. Mohammadi-Mehr, Y. Agha Alaei, Int. J. Antimicrob.
Agents 27 (3) (2006) 196e200.
[19] A.S. Bourlot, I. Sanchez, G. Dureng, G. Guillaumet, R. Massingham,
A. Monteil, E. Winslow, M.D. Pujol, J.Y. Merour, J. Med. Chem. 41
(1998) 3142e3158.
[20] D.R. Shridhar, B. Ram, R.K. Srinivasa, M.L. Jain, Indian J. Chem. 24B
(1985) 992e994.
[21] T. Teitei, Aust. J. Chem. 39 (3) (1986) 503e510.
[22] Y. Masuoka, T. Asako, G. Goto, N. Shunsaku, Chem. Pharm. Bull. 34 (1)
(1986) 130e139.
[23] I. Yalcın, B.P. Tekiner, I. Yıldız-Oren, O. Temiz-Arpacı, E. Sener-Akı,
N. Altanlar, Indian J. Chem. 42B (2003) 905e909.
[24] P. Stefanic, M. Breznik, N. Lah, I. Leban, J. Plavec, D. Kikelj, Tetrahe-
dron Lett. 42 (2001) 5295e5297.
[25] T. Yamamoto, M. Hori, I. Watanabe, H. Tsutsui, S. Ikeda, H. Ohtaka,
Chem. Pharm. Bull. 46 (8) (1998) 1317e1320.
Acknowledgment
[26] S. Shadomy, A. Espinel, Manual of Clinical Microbiology American
Society for Microbiology, Washington DC, 1980, pp. 647.
[27] H. Goker, D.W. Boykin, S. Yıldız, Bioorg. Med. Chem. 13 (2005)
1707e1714.
We thank the Research Fund of Ankara University (Grant
No. 2001-08-03-27) for the financial support of this research.
[28] H. Goker, M. Alp, S. Yıldız, Molecules 10 (2005) 1377e1386.
[29] C. Hansch, J. Med. Chem. 19 (1) (1976) 1e6.
References
[30] C. Hansch, S.D. Rockwell, P.W.C. Jow, A. Leo, E.E. Steller, J. Med.
Chem. 20 (29) (1977) 304e306.
[1] D.F. Fidler, Emerg. Infect. Dis. 4 (1998) 169e177.
[2] I. Oren, O. Temiz, I. Yalcin, E. Sener, N. Altanlar, Eur. J. Pharm. Sci. 7
(1998) 153e160.
[31] H. Kubinyi, Quantitative models, in: R. Mannhold, P. Krogsgaard-
Larsen, H. Timmerman (Eds.), QSAR: Hansch Analysis and Related
Approaches, vol. 1, VCH Verlagsgesellschaft mbH, Weinheim, 1993,
pp. 57e89.
[3] C.Y.J. Hong, Farmaco 56 (2001) 41e44.
[4] A. Macchiarulo, G. Constantino, D. Fringuelli, A. Vecchiarelli,
F. Schiaffella, R. Fringuelli, Bioorg. Med. Chem. 10 (11) (2002)
3415e3423.
[32] S. Wold, Quant. Struct. Act. Relat. 10 (1991) 191e193.
[33] C. Hansch, A. Leo, Substituent Constant for Correlation Analysis in
Chemistry and Biology, John Wiley & Sons, New York, 1979.
[5] O. Wahlroos, A.I. Virtanen, Acta Chem. Scand. 13 (1959) 1906e1908.