Synthesis of new series of 5,6-dihydro-4H-1,2-oxazines via hetero Diels-Alder reaction and evaluation of antimicrobial activity

Article abstract of DOI:10.1016/j.ejmech.2008.02.027

A new series of 5,6-dihydro-4H-1,2-oxazines were synthesized via hetero Diels-Alder reaction of α-nitrosoolefins with alkenes. α-Nitrosoolefins were generated from ketoximes by the action of chloramine-T. The newly synthesized compounds were characterized

Full text of DOI:10.1016/j.ejmech.2008.02.027

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European Journal of Medicinal Chemistry 44 (2009) 280e288
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of new series of 5,6-dihydro-4H-1,2-oxazines via hetero
DielseAlder reaction and evaluation of antimicrobial activity
a
a
b
b
M.K. Manjula a, , K.M.L. Rai , S.L. Gaonkar , K.A. Raveesha , S. Satish
*
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore, Karnataka 570006, India
b Department of Studies in Botany and Microbiology, University of Mysore, Manasagangotri, Mysore, Karnataka 570006, India
Received 23 October 2007; received in revised form 11 February 2008; accepted 14 February 2008
Available online 7 March 2008
Abstract
A new series of 5,6-dihydro-4H-1,2-oxazines were synthesized via hetero DielseAlder reaction of a-nitrosoolefins with alkenes. a-Nitro-
soolefins were generated from ketoximes by the action of chloramine-T. The newly synthesized compounds were characterized with IR,
NMR, elemental analysis and screened for their antimicrobial activity; they exhibited excellent antimicrobial activity. The minimal inhibitory
concentration of the compounds was in the range of 10e35 mg mlꢀ1 for bacteria and 10e40 mg mlꢀ1 for fungi.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Synthesis; 1,2-Oxazines; Antimicrobial activity; Chloramine-T
1. Introduction
There are few methods [22,23] described in the literature
for the preparation of 5,6-dihydro-4H-1,2-oxazine derivatives.
Many syntheses of 1,2-oxazines rely on hetero DielseAlder
reaction of alkenes with a-nitrosoalkenes, derived from
a-halo oximes [3,24,25] or on hetero DielseAlder reaction
of dienes with nitroso compounds [11,26,27]. Other methods
rely on cyclizations of alkenyl-substituted oximes [28e31].
They are also obtained by the cyclization of oxime dianions
with epibromohydrin [32]. Rai et al. obtained 5,6-dihydro-
4H-1,2-oxazine derivatives in good yield by the mediation of
chloramine-T [33].
In continuation to our previous work, we report here the
synthesis and antimicrobial activity of a new series of 5,6-
dihydro-4H-1,2-oxazine derivatives via hetero DielseAlder
reaction of nitrosoolefins derived from ketoximes with
alkenes.
a-Nitrosoalkenes which are normally generated in situ by
1,4-elimination from a-monohaloketoximes [1e4] are of
importance owing to their use as dienes for a (4 þ 2) cycload-
dition reaction. Of late, it has gained more importance since
the cycloaddition leads to the formation of 5,6-dihydro-4H-
1,2-oxazines which are of biological importance, viz: used
as intermediates during the synthesis of glycosidase inhibitor
analogues [5e7] and of functionalised pyrroles [8]. They
have got considerable synthetic potential [9e11] and many
bifunctional compounds are synthesized with them as interme-
diates [3,11e14]. Tetrahydro-4H-1,2-oxazines are the impor-
tant structural constituents of many fungicides, herbicides
[15] and broad spectrum bactericides [16]. Oxazines are
used as key building blocks for natural products [17]. For in-
stance, using oxazine as synthon one can synthesize pyrroles
[18,19], pyrrolidine [20], pyridines [2] and g-lactones [21]
via the reductive cleavage of the CeO and NeO bonds and
so on.
2. Chemistry
The required ketones were obtained from the corresponding
aromatic hydrocarbons by FriedeleCrafts reaction. Later the
ketones are converted to the corresponding ketoximes by rou-
tine method [34]. Chloramine-T was used as an effective
* Corresponding author. Tel.: þ91 821 2340890.
E-mail address: mkmraichur@yahoo.co.in (M.K. Manjula).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.027

M.K. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 280e288
281
Table 1
Structural formulae of the synthesized compounds, 3(a–y)
reagent for the generation of a-nitrosoolefins from the corre-
sponding ketoximes 1(aee) which subsequently undergo
hetero DielseAlder reaction with an olefin 2(aee), to give
5,6-dihydro-4H-1,2-oxazine derivatives 3(aey) in absolute
purity and good yield (Scheme 1) [33]; their structures are
highlighted in Table 1.
3
a
5,6-dihydro-4H-1,2-oxazines
O
N
CN
O
N
b
c
CH2Cl
3. Results and discussion
N O
N O
3.1. Chemistry
CH2OH
Formation of 5,6-dihydro-4H-1,2-oxazine derivatives (3)
was confirmed on the basis of elemental analysis, IR and
NMR.
O
O
d
The IR spectra of 1,2-oxazine derivative (3a) did not show
absorption at 3420e3430 cmꢀ1 and at 1620e1630 cmꢀ1 for
OH and C]N groups, respectively, but instead showed new
peak at 1643 cmꢀ1 owing to the ring nitrogen (C]N), at
2258 cmꢀ1 due to (C^N) and at 1598 cmꢀ1 due to aromatic
ring.
O
N
e
f
CO2Et
CN
N O
The 1H NMR spectrum of 3a showed a singlet at d 2.38 due
to the CH3 protons. It showed two multiplets resonating at
d (1.86e2.07) and d (2.57e2.79) due to the CH2 protons of
the oxazine ring.
The four protons of the phenyl moiety resonated as two
doublets at d 7.16 and 7.59 with the coupling constants of
J ¼ 6 Hz and J ¼ 2 Hz and CH proton resonated at d 4.17 to
give a triplet. In case of disubstituted benzene, instead of
two doublets, three singlets are found.
Further evidence for the formation of 3a was given with 13C
NMR and elemental analysis.
N
N
O
g
h
CH2Cl
O
N
CH2OH
O
N
O
O
i
O
j
CO2Et
N
O
3.2. Antimicrobial activity
CN
O
k
l
3.2.1. Antibacterial activity
N
O
The newly synthesized oxazine derivatives excluding ‘3e’
were screened for their antibacterial activity against four bac-
terial species, viz: Escherichia coli, Klebsiella pneumoniae,
CH2Cl
N
O
CH2OH
m
O
O
R'
R'
NOH
chloramine-T
O
TEA
N
N
N
O
O
N
ethanol
R
CH3
R
CH3
R
R
Cl
n
o
1(a-e)
2(a-e)
3(a-y)
O
N
CO2Et
CN
1
R
2
R’
a
b
c
p-CH3 . C6H4
3,4-(CH3)2. C6H3
Indan-5-yl
a
b
c
CN
N
N
O
CH2Cl
p
q
r
MeO
MeO
MeO
CH2OH
O
O
O
H2C
CH2Cl
d
e
p-(OCH3) . C6H4
d
e
O
N
3,4-(OCH3)2. C6H3
CO2Et
CH2OH
Scheme 1. Schematic representation of synthesis of 1,2-oxazine derivatives.
(continued on next page)

282
M.K. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 280e288
Table 1 (continued)
flavus, Aspergillus niger, Fusarium moniliforme and Fusarium
oxysporum by cup diffusion method [35]. All compounds ex-
hibited in vitro antifungal activity against all the tested fungal
strains. The results are summarized in Table 4. Nystatin was
used as positive reference to determine the sensitivity of
each fungal species tested. The results were subjected to statis-
tical analysis and are summarized in Table 5.
Table 4 clearly reveals that, most of the compounds are
even more effective than the standard Nystatin. With all the
four tested fungal strains, the maximum activity is exhibited
by 3l and the least by 3a; 3o and 3t exhibited excellent activity
almost on par with 3l. The activity may be due to, the aromatic
ring being common, the chloro (3l), ester (3o), methoxy and
ester groups (3t).
3
5,6-dihydro-4H-1,2-oxazines
O
N
O
O
MeO
s
t
N
O
CO2Et
MeO
MeO
MeO
N O
u
CN
MeO
MeO
O
N
v
CH2Cl
O
4. Conclusion
MeO
MeO
N
w
CH2OH
In conclusion a new series of 5,6-dihydro-4H-1,2-oxazine
derivatives were synthesized and their antimicrobial activities
have been evaluated. The antibacterial and antifungal activi-
ties were observed in all the tested compounds. Majority of
the compounds proved even better than the standard Gentami-
cin against the Gram-negative bacteria; with Gram-positive
bacteria the activity is less than the control. The compounds
exhibited excellent antifungal activity. Majority of the com-
pounds exhibited better activity against the tested fungal
strains than the standard Nystatin. Hence, it is concluded
that there is ample scope for further developing this field.
MeO
MeO
O
N
O
O
x
y
MeO
MeO
N O
CO2Et
Pseudomonas aeruginosa, Staphylococcus aureus by cup dif-
fusion method [35]. Penicillin G and Gentamicin were used
as positive reference to determine the sensitivity of each
bacterial species tested. All compounds exhibited excellent
in vitro antibacterial activity against Gram-positive and
Gram-negative organisms. The results are summarized in
Table 2. The results were subjected to statistical analysis and
are summarized in Table 3.
5. Experimental protocols
Melting points were determined on Thomas Hoover melt-
ing point apparatus and are uncorrected. The IR spectra (in
KBr pellets) were recorded on JASCO FT/IR e 460/113257
spectrometer (Japan) in the wave number range of 4000e
1
400 cmꢀ1. H NMR spectra were recorded on a Bruker AM
The investigation of antibacterial screening data revealed
that all the tested compounds showed considerable and varied
activity against the four human pathogenic bacteria. Com-
pound 3t showed the maximum activity against the E. coli,
which may be attributed to the aromatic ring, methoxy and
carboxylate groups in para positions and in case of 3s, the
activity may be due to the methoxy and benzene dioxo group
in para positions, the less activity may be due to the interven-
ing methylene group. With K. pneumoniae 3l showed the max-
imum activity, while 3k and 3y exhibited almost same level of
activity with the said bacterium; with P. aeruginosa 3l showed
the maximum activity while 3k and 3s showed almost the
same level of activity as 3l. Against S. aureus, 3a showed
the maximum activity. The antibacterial activity may be due
to the highly active chloro, nitrile, ester and methylene dioxyl
groups. Most of the compounds exhibited better activity while
some are even better than the standards.
300 MHz spectrometer using CDCl3 as solvent and tetrame-
thylsilane as an internal standard. 13C NMR spectra were
measured on Jeol 400 (75 MHz) instrument. The chemical
shifts are expressed in d and the following abbreviations are
used: s ¼ singlet, d ¼ doublet, t ¼ triplet and m ¼ multiplet.
Elemental analyses were obtained on a Vario-EL instrument.
The purity of the compounds was checked by thin layer
chromatography (TLC) on silica gel plates using chloro-
formeacetone (8:2) as eluent.
5.1. General procedure for the synthesis of oxazine
derivatives 3(aey)
5.1.1. Synthesis of 3-p-tolyl-5,6-dihydro-4H-1,2-oxazine-6-
carbonitrile 3a
Typical procedure. A mixture of 1a (0.5 g, 3.35 mmol) and
chloramine-T trihydrate (0.99 g, 3.52 mmol) in ethanol was
taken in a 100 ml r.b. flask and refluxed for 2 h. The mixture
was cooled to room temperature; triethylamine (1 ml) was
added and stirred at room temperature for 15 min. To this, a so-
lution of 2a (1.8 g, 3.33 mmol) in ethanol (5 ml) was added
3.2.2. Antifungal activity
Newly prepared compounds were screened for their anti-
fungal activity against four fungal species namely Aspergillus

M.K. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 280e288
283
Table 2
Bacterial activity of the newly synthesized compounds and antibiotics against human pathogenic bacteria
Compound
Escherichia coli MTCC443
Klebsiella pneumoniae TCC109
Pseudomonas aeruginosa MTCC1688
Staphylococcus aureus MTCC 737
3a
3b
11.50 ꢂ 0.13
15.66 ꢂ 0.15
08.83 ꢂ 0.14
10.66 ꢂ 0.11
15.50 ꢂ 0.27
14.83 ꢂ 0.20
13.66 ꢂ 0.25
11.50 ꢂ 0.12
13.50 ꢂ 0.27
15.50 ꢂ 0.25
11.83 ꢂ 0.20
10.50 ꢂ 0.12
11.50 ꢂ 0.25
10.66 ꢂ 0.06
10.16 ꢂ 0.12
09.50 ꢂ 0.12
10.66 ꢂ 0.25
21.83 ꢂ 0.14
24.66 ꢂ 0.06
10.50 ꢂ 0.25
13.83 ꢂ 0.14
08.50 ꢂ 0.13
08.12 ꢂ 0.19
14.66 ꢂ 0.11
0.00 ꢂ 0.00
16.66 ꢂ 0.06
16.50 ꢂ 0.27
19.83 ꢂ 0.20
19.66 ꢂ 0.25
19.75 ꢂ 0.16
15.83 ꢂ 0.14
17.66 ꢂ 0.15
15.50 ꢂ 0.25
16.66 ꢂ 0.66
23.83 ꢂ 0.20
25.83 ꢂ 0.20
17.66 ꢂ 0.25
16.50 ꢂ 0.17
13.50 ꢂ 0.19
06.66 ꢂ 0.12
17.66 ꢂ 0.11
15.50 ꢂ 0.13
19.66 ꢂ 0.15
14.83 ꢂ 0.14
07.75 ꢂ 0.15
17.00 ꢂ 0.18
14.50 ꢂ 0.27
17.50 ꢂ 0.17
20.66 ꢂ 0.15
0.00 ꢂ 0.00
12.66 ꢂ 0.15
11.50 ꢂ 0.25
10.75 ꢂ 0.16
11.25 ꢂ 0.25
15.25 ꢂ 0.18
17.63 ꢂ 0.25
12.75 ꢂ 0.15
11.00 ꢂ 0.18
12.83 ꢂ 0.26
31.50 ꢂ 0.27
34.50 ꢂ 0.17
13.66 ꢂ 0.15
21.50 ꢂ 0.27
13.38 ꢂ 0.18
12.16 ꢂ 0.12
29.50 ꢂ 0.12
16.66 ꢂ 0.25
31.83 ꢂ 0.14
23.66 ꢂ 0.06
24.50 ꢂ 0.25
09.83 ꢂ 0.14
10.50 ꢂ 0.13
14.12 ꢂ 0.19
25.66 ꢂ 0.11
0.00 ꢂ 0.00
23.50 ꢂ 0.25
16.66 ꢂ 0.66
07.50 ꢂ 0.19
12.83 ꢂ 0.14
21.50 ꢂ 0.25
20.66 ꢂ 0.66
14.83 ꢂ 0.20
21.33 ꢂ 0.17
17.83 ꢂ 0.20
17.66 ꢂ 0.25
13.50 ꢂ 0.17
10.66 ꢂ 0.12
15.66 ꢂ 0.11
18.66 ꢂ 0.15
09.50 ꢂ 0.13
14.66 ꢂ 0.15
10.83 ꢂ 0.14
21.66 ꢂ 0.11
16.66 ꢂ 0.06
15.50 ꢂ 0.27
17.83 ꢂ 0.20
07.66 ꢂ 0.25
16.50 ꢂ 0.12
16.50 ꢂ 0.12
9.38 ꢂ 0.18
3c
3d
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
3y
Penicillin G
Gentamicin
10.25 ꢂ 0.25
17.50 ꢂ 0.25
12.63 ꢂ 0.18
24.63 ꢂ 0.18
Zone of inhibition in mm (mean of six replicas ꢂ standard error), p < 0.05.
and refluxed for an hour. It was then concentrated under re-
duced pressure and the residue was extracted with ether. The
extract then washed with water (15 ml), with 1 N aq. NaOH
and then with water and dried over anhydrous Na2SO4. Yellow
solid, recrystallised from alcoholen-hexane (0.36 g, 72%),
5.1.3. Synthesis of 6-hydroxymethyl-3-p-tolyl-5,6-
dihydro-4H-1,2-oxazine 3c
Obtained from 1a (0.5 g, 3.35 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2c (1.6 g, 2.56 mmol) as a colourless
solid, recrystallised from alcoholen-hexane (0.29 g, 58%),
1
1
m.p. 91 ꢁC. H NMR (CDCl3, 300 MHz): d 1.86e2.07 (m,
m.p. 80 ꢁC. H NMR (CDCl3, 300 MHz): d 1.87e2.08 (m,
2H, CH2), 2.38 (s, 3H, CH3), 2.57e2.79 (m, 2H, CH2), 4.17
(t, H, CH), 7.16 (dd, J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH), 7.59 (dd,
J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH). 13C NMR (75 MHz, CDCl3):
d 25.2 (q, CH3), 16.8 (t, CH2), 22.3 (t, CH2), 72.1 (d, CH),
130.3 (d, CH), 117.2 (s, C), 133.0 (s, C), 141.2 (s, C), 150.3
(s, C). IR (KBr pellets cmꢀ1) n 1598, 1643, 2258. Anal.
CHN: calcd C 71.98, H 6.04, N 13.99, O 7.99, found C
71.95, H 6.04, N 13.98, O 7.99.
2H, CH2), 2.13 (br, 1H, OH), 2.37 (s, 3H, CH3), 2.56e2.78
(m, 2H, CH2), 3.74e3.93 (m, 3H, CH2eCH), 7.18 (dd,
J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH), 7.59 (dd, J ¼ 6 Hz, J ¼ 2 Hz,
2H, CH). 13C NMR (75 MHz, CDCl3): d 25.2 (q, CH3), 10.5
(t, CH2), 23.3 (t, CH2), 66.7 (t, CH2), 82.4 (d, CH), 130.2
(d, CH), 132.5 (s, C), 141.9 (s, C), 151.9 (s, C). IR (KBr pel-
lets cmꢀ1) n 1600, 1647, 3340. Anal. CHN: calcd C 70.22, H
7.37, N 6.82, O 15.59, found C 70.22, H 7.37, N 6.80, O 15.60.
5.1.4. Synthesis of 6-(benzo[1,3]dioxol-5-yl)methyl-3-
5.1.2. Synthesis of 6-chloromethyl-3-p-tolyl-5,6-
dihydro-4H-1,2-oxazine 3b
p-tolyl-5,6-dihydro-4H-1,2-oxazine 3d
Obtained from 1a (0.5 g, 3.35 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2d (1.37 g, 0.85 mmol) as a red liquid
(0.325 g, 65%). 1H NMR (CDCl3, 300 MHz): d 1.87e2.09 (m,
2H, CH2), 2.36 (s, 3H, CH3), 2.55e2.77 (m, 2H, CH2), 3.51e
3.72 (m, 3H, CH2eCH), 5.93 (s, 2H, OeCH2eO), 6.51e6.62
(m, 3H, CH), 7.17 (dd, J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH), 7.59 (dd,
J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH). 13C NMR (75 MHz, CDCl3):
d 25.2 (q, CH3), 14.6 (t, CH2), 23.2 (t, CH2), 41.9 (t, CH2),
102.5 (t, CH2), 81.9 (d, CH), 114.5 (d, CH), 116.3 (d, CH),
122.6 (d, CH), 130.1 (d, CH), 131.9 (s, C), 133.2 (s, C),
141.5 (s, C), 146.7 (s, C), 149.2 (s, C), 152.3 (s, C). IR
(KBr pellets cmꢀ1) n 1600, 1642. Anal. CHN: calcd C
Obtained from 1a (0.5 g, 3.35 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2b (1.6 g, 2.09 mmol) as a red liquid
1
(0.35 g, 70%). H NMR (CDCl3, 300 MHz): d 1.88e2.09 (m,
2H, CH2), 2.36 (s, 3H, CH3), 2.55e2.75 (m, 2H, CH2), 3.55e
3.79 (m, 3H, CH2eCH), 7.17 (dd, J ¼ 6 Hz, J ¼ 2 Hz, 2H,
CH), 7.59 (dd, J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH). 13C NMR
(75 MHz, CDCl3): d 25.2 (q, CH3), 11.5 (t, CH2), 22.5 (t,
CH2), 47.9 (t, CH2), 82.2 (d, CH), 130.1 (d, CH); 133.2 (s,
C), 142.3 (s, C), 151.4 (s, C). IR (KBr pellets cmꢀ1) n 782,
1595, 1637. Anal. CHN: calcd C 64.43, H 6.31, Cl 15.85, N
6.26, O 7.15, found C 64.42, H 6.31, Cl 15.83, N 6.25, O 7.15.

284
M.K. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 280e288
Table 3
The minimal inhibitory concentration (MIC) in mg/mL of synthesized com-
pounds against tested strains
Table 5
The minimal inhibitory concentration (MIC) in mg/mL of synthesized com-
pounds against fungal strains
Compound
Escherichia Klebsiella
coli
MTCC443
Pseudomonas Staphylococcus
aureus
Compound
Aspergillus
flavus
Aspergillus
niger
Fusarium
moniliforme
Fusarium
oxysporum
pneumoniae aeruginosa
TCC109
MTCC1688
MTCC 737
3a
3b
3c
35
15
30
15
20
15
35
10
15
40
10
30
15
10
35
15
35
40
10
35
15
40
40
35
15
40
15
35
20
20
15
30
15
20
35
15
30
20
15
40
20
40
35
15
35
20
40
40
35
10
30
10
30
20
15
20
35
20
25
40
20
35
20
20
35
15
35
35
20
30
15
35
35
30
15
25
15
25
10
15
10
25
10
15
35
10
25
15
10
30
10
30
35
10
30
10
30
30
35
20
3a
3b
25
20
35
25
25
20
25
30
20
25
20
35
25
30
25
20
35
25
35
25
20
35
25
30
e
20
15
30
20
15
15
20
20
15
20
15
25
15
25
15
10
25
15
25
15
10
25
15
20
e
20
15
25
20
15
15
25
20
20
15
15
20
15
20
15
15
20
20
20
10
15
20
20
15
e
15
10
25
15
10
10
15
15
10
15
10
20
10
20
10
10
20
15
20
15
10
20
10
15
e
3c
3d
3d
3f
3f
3g
3g
3h
3i
3h
3i
3j
3j
3k
3k
3l
3l
3m
3m
3n
3o
3p
3q
3r
3n
3o
3p
3q
3r
3s
3s
3t
3t
3u
3u
3v
3w
3x
3y
Nystatin
3v
3w
3x
3y
Penicillin G
Gentamicin
20
15
15
15
73.77, H 6.19, N 4.53, O 15.52, found C 73.75, H 6.19, N 4.53,
O 15.50.
Table 4
Antifungal activity of the newly synthesized compounds and antifungal agent
(Nystatin) against pathogenic fungi
5.1.5. Synthesis of 3-p-tolyl-5,6-dihydro-4H-1,2-oxazine-6-
carboxylic acid ethylester 3e
Obtained from 1a (0.5 g, 3.35 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2e (1.63 g, 1.63 mmol) as a pale yel-
low solid recrystallised from ethanolen-hexane (0.275 g,
55%), m.p.99 ꢁC. 1H NMR (CDCl3, 300 MHz): d 1.27 (t,
3H, CH3), 2.28e2.52 (m, 2H, CH2), 2.37 (s, 3H, CH3),
2.56e2.78 (m, 2H, CH2), 3.99e4.02 (m, 3H, CH2eCH),
7.15 (dd, J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH), 7.55 (dd, J ¼ 6 Hz,
J ¼ 2 Hz, 2H, CH). 13C NMR (75 MHz, CDCl3): d 15.2 (q,
CH3), 25.1 (q, CH3), 20.9 (t, CH2), 22.0 (t, CH2), 62.2 (t,
CH2), 86.3 (d, CH), 129.9 (d, CH), 132.0 (s, C), 141.5 (s,
C), 152.2 (s, C), 171.5 (s, C). IR (KBr pellets cmꢀ1) n 1600,
1648, 1778. Anal. CHN: calcd C 68.00, H 6.93, N 5.66, O
19.41, found C 68.00, H 6.93, N 5.66, O 19.40.
Compound
Aspergillus
flavus
Aspergillus
niger
Fusarium
moniliforme
Fusarium
oxysporum
3a
3b
3c
07.50 ꢂ 0.25
14.50 ꢂ 0.27
12.50 ꢂ 0.12
17.83 ꢂ 0.20
12.16 ꢂ 0.12
09.50 ꢂ 0.12
08.66 ꢂ 0.25
18.83 ꢂ 0.14
12.66 ꢂ 0.06
09.50 ꢂ 0.19
32.75 ꢂ 0.16
09.83 ꢂ 0.14
20.66 ꢂ 0.15
28.50 ꢂ 0.25
08.50 ꢂ 0.25
21.66 ꢂ 0.66
07.83 ꢂ 0.20
09.66 ꢂ 0.25
25.50 ꢂ 0.12
10.50 ꢂ 0.25
20.83 ꢂ 0.14
08.50 ꢂ 0.13
08.12 ꢂ 0.19
09.66 ꢂ 0.11
13.25 ꢂ 0.25
06.83 ꢂ 0.20
13.83 ꢂ 0.20
11.50 ꢂ 0.17
15.66 ꢂ 0.25
11.66 ꢂ 0.11
08.50 ꢂ 0.13
10.66 ꢂ 0.12
15.66 ꢂ 0.11
11.50 ꢂ 0.13
10.66 ꢂ 0.15
28.83 ꢂ 0.14
07.66 ꢂ 0.11
18.66 ꢂ 0.06
26.66 ꢂ 0.12
09.50 ꢂ 0.27
19.50 ꢂ 0.17
08.66 ꢂ 0.15
10.66 ꢂ 0.15
24.16 ꢂ 0.12
11.50 ꢂ 0.12
18.66 ꢂ 0.25
07.83 ꢂ 0.14
07.75 ꢂ 0.15
08.00 ꢂ 0.18
11.50 ꢂ 0.25
10.50 ꢂ 0.12
17.66 ꢂ 0.15
15.83 ꢂ 0.14
19.50 ꢂ 0.25
15.66 ꢂ 0.15
11.50 ꢂ 0.25
14.66 ꢂ 0.66
22.83 ꢂ 0.20
16.66 ꢂ 0.25
13.50 ꢂ 0.27
34.50 ꢂ 0.17
13.66 ꢂ 0.15
21.50 ꢂ 0.27
32.83 ꢂ 0.14
13.38 ꢂ 0.18
25.25 ꢂ 0.18
17.63 ꢂ 0.25
12.75 ꢂ 0.15
28.00 ꢂ 0.18
12.83 ꢂ 0.26
26.50 ꢂ 0.25
10.75 ꢂ 0.16
11.25 ꢂ 0.25
11.66 ꢂ 0.11
15.63 ꢂ 0.18
13.66 ꢂ 0.06
20.83 ꢂ 0.14
19.83 ꢂ 0.14
24.50 ꢂ 0.25
21.50 ꢂ 0.25
20.66 ꢂ 0.66
19.83 ꢂ 0.20
25.33 ꢂ 0.17
19.83 ꢂ 0.20
17.66 ꢂ 0.25
38.50 ꢂ 0.17
18.83 ꢂ 0.14
26.66 ꢂ 0.06
36.50 ꢂ 0.13
18.50 ꢂ 0.25
29.66 ꢂ 0.66
19.50 ꢂ 0.19
16.83 ꢂ 0.14
30.66 ꢂ 0.15
15.50 ꢂ 0.27
28.66 ꢂ 0.06
16.50 ꢂ 0.27
15.83 ꢂ 0.20
16.66 ꢂ 0.25
16.63 ꢂ 0.18
3d
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
5.1.6. Synthesis of 3-(3,4-dimethylphenyl)-5,6-dihydro-4H-
1,2-oxazine-6-carbonitrile 3f
3s
Obtained from 1b (0.5 g, 3 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2a (1.8 g, 3.33 mmol) as a pale brown
solid, recrystallised from ethanolen-hexane (0.365 g, 73%),
3t
3u
3v
3w
3x
3y
Nystatin
1
m.p. 121 ꢁC. H NMR (CDCl3, 300 MHz): d 1.85e2.08 (m,
2H, CH2), 2.38 (s, 6H, CH3), 2.56e2.78 (m, 2H, CH2), 4.18
(t, H, CH), 7.05e7.34 (m, 3H, CH). 13C NMR (75 MHz,
CDCl3): d 18.5 (q, CH3), 16.5 (t, CH2), 21.9 (t, CH2), 71.1
(d, CH), 126.9 (d, CH), 130.1 (d, CH), 119.3 (s, C), 131.5
Zone of inhibition in mm (mean of six replicas ꢂ standard error), p < 0.05.

M.K. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 280e288
285
(s, C), 137.9 (s, C), 140.8 (s, C), 151.6 (s, C). IR (KBr pellets
cmꢀ1) n 1598, 1645, 2260. Anal. CHN: calcd C 72.87, H 6.59,
N 13.07, O 7.47, found C 72.85, H 6.58, N 13.07, O 7.47.
CH3), 2.55e2.78 (m, 2H, CH2), 3.99e4.03 (m, 3H, CH2e
CH), 7.03e7.32 (m, 3H, CH). 13C NMR (75 MHz, CDCl3):
d 15.2 (q, CH3), 18.9 (q, CH3), 21.5 (t, CH2), 22.5 (t, CH2),
62.5 (t, CH2), 86.6 (d, CH), 127.5 (d, CH), 130.3 (d, CH),
131.8 (s, C), 138.2 (s, C), 140.3 (s, C), 152.6 (s, C), 171.5
(s, C). IR (KBr pellets cmꢀ1) n 1605, 1645, 1778. Anal.
CHN: calcd C 68.94, H 7.33, N 5.36, O 18.37, found C
68.94, H 7.30, N 5.36, O 18.35.
5.1.7. Synthesis of 6-chloromethyl-3-(3,4-dimethylphenyl)-
5,6-dihydro-4H-1,2-oxazine 3g
Obtained from 1b (0.5 g, 3 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2b (1.6 g, 2.09 mmol) as a pale yel-
low solid, recrystallised from ethanolen-hexane (0.375 g,
75%), m.p. 77 ꢁC. 1H NMR (CDCl3, 300 MHz): d 1.87e
2.09 (m, 2H, CH2), 2.37 (s, 6H, CH3), 2.54e2.75 (m, 2H,
CH2), 3.54e3.79 (m, 3H, CH2eCH), 7.06e7.35 (m, 3H,
CH). 13C NMR (75 MHz, CDCl3): d 18.6 (q, CH3), 11.5 (t,
CH2), 22.4 (t, CH2), 47.8 (t, CH2), 82.3 (d, CH), 127.6 (d,
CH), 130.5 (d, CH), 131.8 (s, C), 137.8 (s, C), 139.9 (s, C),
152.3 (s, C). IR (KBr pellets cmꢀ1) n 779, 1600, 1640.
Anal. CHN: calcd C 65.68, H 6.78, Cl 14.91, N 5.89, O
6.73, found C 65.66, H 6.75, Cl 14.91, N 5.89, O 6.73.
5.1.11. Synthesis of 3-indan-5-yl-5,6-dihydro-4H-
1,2-oxazine-6-carbonitrile 3k
Obtained from 1c (0.5 g, 2.85 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2a (1.8 g, 3.33 mmol) as a reddish
brown oil (0.375 g, 75%). 1H NMR (CDCl3, 300 MHz):
d 1.86e2.07 (m, 4H, CH2), 2.57e2.78 (m, 2H, CH2), 2.86
(t, 4H, CH2), 4.19 (t, H, CH), 7.48e7.82 (m, 3H, CH). 13C
NMR (75 MHz, CDCl3): d 16.6 (t, CH2), 22.1 (t, CH2), 26.5
(t, CH2), 34.2 (t, CH2), 71.4 (d, CH), 127.2 (d, CH), 129.2
(d, CH), 119.2 (s, C), 132.5 (s, C), 145.1 (s, C), 147.3 (s,
C), 152.0 (s, C). IR (KBr pellets cmꢀ1) n 1600, 1642, 2255.
Anal. CHN: calcd C 74.31, H 6.24, N 12.38, O 7.07, found
C 74.30, H 6.25, N 12.35, O 7.07.
5.1.8. Synthesis of [3-(3,4-dimethylphenyl)-5,6-dihydro-4H-
1,2-oxazin-6-yl]-methanol 3h
Obtained from 1b (0.5 g, 3 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2c (1.6 g, 2.56 mmol) as a colourless
solid, recrystallised from ethanolen-hexane (0.36 g, 72%),
5.1.12. Synthesis of 6-chloromethyl-3-indan-5-yl-5,6-
dihydro-4H-1,2-oxazine 3l
1
m.p. 91 ꢁC. H NMR (CDCl3, 300 MHz): d 1.87e2.08 (m,
2H, CH2), 2.13 (br, 1H, OH), 2.36 (s, 6H, CH3), 2.56e2.79
(m, 2H, CH2), 3.73e3.92 (m, 3H, CH2eCH), 7.03e7.32 (m,
3H, CH). 13C NMR (75 MHz, CDCl3): d 18.7 (q, CH3), 10.8
(t, CH2), 23.2 (t, CH2), 66.3 (t, CH2), 82.6 (d, CH), 127.2
(d, CH), 130.5 (d, CH), 132.0 (s, C), 138.1 (s, C), 140.2 (s,
C), 152.7 (s, C). IR (KBr pellets cmꢀ1) n 1598, 1646, 3300.
Anal. CHN: calcd C 71.21, H 7.81, N 6.39, O 14.59, found
C 71.19, H 7.80, N 6.39, O 14.58.
Obtained from 1c (0.5 g, 2.85 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2b (1.6 g, 2.09 mmol) as a red oil
1
(0.3 g, 60%). H NMR (CDCl3, 300 MHz): d 1.88e2.08 (m,
4H, CH2), 2.55e2.89 (m, 6H, CH2), 3.55e3.78 (m, 3H,
CH2eCH), 7.45e7.82 (m, 3H, CH). 13C NMR (75 MHz,
CDCl3): d 11.6 (t, CH2), 22.4 (t, CH2), 26.4 (t, CH2), 34.3
(t, CH2), 47.1 (t, CH2), 82.3 (d, CH), 127.3 (d, CH), 129.4
(d, CH), 132.3 (s, C), 145.1 (s, C), 147.3 (s, C), 152.3 (s,
C). IR (KBr pellets cmꢀ1) n 780, 1599, 1640. Anal. CHN:
calcd C 67.33, H 6.46, Cl 14.20, N 5.61, O 6.41. found C
67.33, H 6.44, Cl 14.20, N 5.60, O 6.40.
5.1.9. Synthesis of 6-(benzo[1,3]dioxol-5-yl)methyl-3-(3,4-
dimethylphenyl)-5,6-dihydro-4H-1,2-oxazine 3i
Obtained from 1b (0.5 g, 3 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2d (1.37 g, 0.85 mmol) as a red oil
(0.35 g, 70%). 1H NMR (CDCl3, 300 MHz): d 1.88e2.09
(m, 2H, CH2), 2.37 (s, 6H, CH3), 2.55e2.76 (m, 2H, CH2),
3.51e3.73 (m, 3H, CH2eCH), 5.92 (s, 2H, OeCH2eO),
6.52e6.63 (m, 3H, CH), 7.06e7.33 (m, 3H, CH). 13C NMR
(75 MHz, CDCl3): d 18.8 (q, CH3), 14.5 (t, CH2), 23.4 (t,
CH2), 41.8 (t, CH2), 102.5 (t, CH2), 82.1 (d, CH), 114.3 (d,
CH), 116.3 (d, CH), 122.7 (d, CH), 127.2 (d, CH), 130.5 (d,
CH), 131.8 (s, C), 138.1 (s, C), 140.5 (s, C), 147.5 (s, C),
149.8 (s, C), 152.3 (s, C). IR (KBr pellets cmꢀ1) n 1599,
1643. Anal. CHN: calcd C 74.28, H 6.55, N 4.33, O 14.84,
found C 74.25, H 6.55, N 4.30, O 14.84.
5.1.13. Synthesis of (3-indan-5-yl-5,6-dihydro-4H-
1,2-oxazin-6-yl)-methanol 3m
Obtained from 1c (0.5 g, 2.85 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2c (1.6 g, 2.56 mmol) as a red liquid
1
(0.32 g, 64%). H NMR (CDCl3, 300 MHz): d 1.87e2.09 (m,
4H, CH2), 2.13 (br, 1H, OH), 2.57e2.90 (m, 6H, CH2), 3.74e
3.93 (m, 3H, CH2eCH), 7.46e7.83 (m, 3H, CH). 13C NMR
(75 MHz, CDCl3): d 10.6 (t, CH2), 22.9 (t, CH2), 26.2 (t,
CH2), 33.9 (t, CH2), 66.2 (t, CH2), 82.3 (d, CH), 127.7 (d,
CH), 129.3 (d, CH), 132.2 (s, C), 145.1 (s, C), 147.2 (s, C),
152.4 (s, C). IR (KBr pellets cmꢀ1) n 1599, 1642, 3330.
Anal. CHN: calcd C 72.70, H 7.41, N 6.06, O 13.83, found
C 72.70, H 7.40, N 6.06, O 13.80.
5.1.10. Synthesis of 3-(3,4-dimethylphenyl)-5,6-dihydro-4H-
1,2-oxazine-6-carboxylic acid ethylester 3j
5.1.14. Synthesis of 6-(benzo[1,3]dioxol-5-yl)methyl-3-
indan-5-yl-5,6-dihydro-4H-1,2-oxazine 3n
Obtained from 1c (0.5 g, 2.85 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2d (1.37 g, 0.85 mmol) as a reddish
1
liquid (0.325 g, 65%). H NMR (CDCl3, 300 MHz): d 1.88e
2.08 (m, 4H, CH2), 2.56e2.88 (m, 6H, CH2), 3.51e3.74 (m,
Obtained from 1b (0.5 g, 3 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2e (1.63 g, 1.63 mmol) as a yellow
crystalline solid, recrystallised from ethanolen-hexane
(0.29 g, 58%), m.p. 115 ꢁC. 1H NMR (CDCl3, 300 MHz):
d 1.28 (t, 3H, CH3), 2.28e2.53 (m, 2H, CH2), 2.37 (s, 6H,

286
M.K. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 280e288
3H, CH2eCH), 5.93 (s, 2H, OeCH2eO), 6.53e6.62 (m, 3H,
CH), 7.45e7.82 (m, 3H, CH). 13C NMR (75 MHz, CDCl3):
d 14.2 (t, CH2), 23.0 (t, CH2), 26.7 (t, CH2), 34.1 (t, CH2),
41.2 (t, CH2), 102.3 (t, CH2), 81.5 (d, CH), 114.2 (d, CH),
116.1 (d, CH), 122.2 (d, CH), 127.5 (d, CH), 129.5 (d, CH),
133.0 (s, C), 145.3 (s, C), 147.1 (s, C), 149.5 (s, C), 152.2
(s, C). IR (KBr pellets cmꢀ1) n 1602, 1645. Anal. CHN: calcd
C 75.20, H 6.31, N 4.18, O 14.31, found C 75.20, H 6.30, N
4.15, O 14.30.
5.1.18. Synthesis of [3-(4-methoxyphenyl)-5,6-dihydro-4H-
1,2-oxazin-6-yl)-methanol 3r
Obtained from 1d (0.5 g, 3.05 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2c (1.6 g, 2.56 mmol) as a yellow oil
1
(0.32 g, 64%). H NMR (CDCl3, 300 MHz): d 1.88e2.09 (m,
2H, CH2), 2.13 (br, 1H, OH), 2.55e2.78 (m, 2H, CH2), 3.72e
3.93 (m, 3H, CH2eCH), 3.76 (s, 3H, OMe), 6.87 (dd,
J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH), 7.58 (dd, J ¼ 6 Hz, J ¼ 2 Hz,
2H, CH). 13C NMR (75 MHz, CDCl3): d 56.8 (q, CH3), 10.8
(t, CH2), 23.0 (t, CH2), 66.2 (t, CH2), 82.2 (d, CH), 115.2
(d, CH), 131.3 (d, CH), 127.4 (s, C), 152.6 (s, C), 164.1 (s,
C). IR (KBr pellets cmꢀ1) n 1600, 1642, 3370. Anal. CHN:
calcd C 65.14, H 6.83, N 6.33, O 21.69, found C 65.11, H
6.83, N 6.33, O 21.66.
5.1.15. Synthesis of 3-indan-5-yl-5,6-dihydro-4H-
1,2-oxazine-6-carboxylic acid ethylester 3o
Obtained from 1c (0.5 g, 2.85 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2e (1.63 g, 1.63 mmol) as a reddish
1
brown oil (0.3 g, 60%). H NMR (CDCl3, 300 MHz): d 1.28
(t, 3H, CH3), 1.98 (q, 2H, CH2), 2.28e2.52 (m, 2H, CH2),
2.56e2.87 (m, 6H, CH2), 3.98e4.02 (m, 3H, CH2eCH),
7.46e7.83 (m, 3H, CH). 13C NMR (75 MHz, CDCl3):
d 15.6 (q, CH3), 21.4 (t, CH2), 22.6 (t, CH2), 26.4 (t, CH2),
34.2 (t, CH2), 62.8 (t, CH2), 86.3 (d, CH), 127.4 (d, CH),
129.3 (d, CH), 132.3 (s, C), 145.1 (s, C), 147.1 (s, C), 152.6
(s, C), 171.5 (s, C). IR (KBr pellets cmꢀ1) n 1598, 1643,
1774. Anal. CHN: calcd C 70.31, H 7.01, N 5.12, O 17.56,
found C 70.31, H 7.01, N 5.10, O 17.55.
5.1.19. Synthesis of 6-(benzo[1,3]dioxol-5-yl)methyl-3-(4-
methoxyphenyl)-5,6-dihydro-4H-1,2-oxazine 3s
Obtained from 1d (0.5 g, 3.05 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2d (1.37 g, 0.85 mmol) as a red oil
1
(0.375 g, 75%), H NMR (CDCl3, 300 MHz): d 1.87e2.08
(m, 2H, CH2), 2.55e2.77 (m, 2H, CH2), 3.51e3.73 (m, 3H,
CH2eCH), 3.75 (s, 3H, OMe), 5.92 (s, 2H, OeCH2eO),
6.86 (dd, J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH), 7.59 (dd, J ¼ 6 Hz,
J ¼ 2 Hz, 2H, CH). 13C NMR (75 MHz, CDCl3): d 56.8 (q,
CH3), 14.1 (t, CH2), 22.9 (t, CH2), 41.8 (t, CH2), 102.3 (t,
CH2), 81.5 (d, CH), 114.1 (d, CH), 115.1 (d, CH), 116.1 (d,
CH), 122.4 (d, CH), 131.1 (d, CH), 127.2 (s, C), 133.0 (s,
C), 147.1 (s, C), 149.5 (s, C), 152.2 (s, C), 164.0 (s, C). IR
(KBr pellets cmꢀ1) n 1600, 1640. Anal. CHN: calcd C
70.14, H 5.89, N 4.31, O 19.67, found C 70.14, H 5.89, N
4.30, O 19.65.
5.1.16. Synthesis of 3-(4-methoxyphenyl)-5,6-dihydro-4H-
1,2-oxazine-6-carbonitrile 3p
Obtained from 1d (0.5 g, 3.05 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2a (1.8 g, 3.33 mmol) as a yellow
solid, recrystallised from ethanolen-hexane (0.38 g, 76%),
m.p. 131 ꢁC. 1H NMR (CDCl3, 300 MHz): d 1.85e2.07
(m, 2H, CH2), 2.55e2.79 (m, 2H, CH2), 3.75 (s, 3H,
OMe), 4.18 (t, H, CH), 6.88 (dd, J ¼ 6 Hz, J ¼ 2 Hz, 2H,
CH), 7.57 (dd, J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH). 13C NMR
(75 MHz, CDCl3): d 56.9 (q, CH3), 16.5 (t, CH2), 22.0 (t,
CH2), 71.3 (d, CH), 115.6 (d, CH), 131.3 (d, CH), 119.2
(s, C), 127.1 (s, C), 152.0 (s, C), 164.2 (s, C). IR (KBr pel-
lets cmꢀ1) n 1600, 1642, 2258. Anal. CHN: calcd C 66.65, H
5.59, N 12.96, O 14.80, found C 66.65, H 5.55, N 12.93, O
14.80.
5.1.20. Synthesis of 3-(4-methoxyphenyl)-5,6-dihydro-4H-
1,2-oxazine-6-carboxylic acid ethylester 3t
Obtained from 1d (0.50 g, 3.05 mmol), chloramine-
T$3H2O (0.99 g, 3.52 mmol) and 2e (1.63 g, 1.63 mmol) as
1
a red oil (0.34 g, 68%). H NMR (CDCl3, 300 MHz): d 1.28
(t, 3H, CH3), 2.28e2.52 (m, 2H, CH2), 2.57e2.79 (m, 2H,
CH2), 3.76 (s, 3H, OMe), 3.98e4.01 (m, 3H, CH2eCH),
6.87 (dd, J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH), 7.58 (dd, J ¼ 6 Hz,
J ¼ 2 Hz, 2H, CH). 13C NMR (75 MHz, CDCl3): d 15.2 (q,
CH3), 56.7 (q, CH3), 21.6 (t, CH2), 22.3 (t, CH2), 62.3 (t,
CH2), 86.7 (d, CH), 115.5 (d, CH), 131.5 (d, CH), 127.5 (s,
C), 152.5 (s, C), 164.3 (s, C), 171.8 (s, C). IR (KBr pellets
cmꢀ1) n 1598, 1645, 1775. Anal. CHN: calcd C 63.87, H
6.51, N 5.32, O 24.31, found C 63.82, H 6.51, N 5.32, O 24.30.
5.1.17. Synthesis of 6-chloromethyl-3-(4-methoxyphenyl)-
5,6-dihydro-4H-1,2-oxazine 3q
Obtained from 1d (0.5 g, 3.05 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2b (1.6 g, 2.09 mmol) as a reddish
brown oil (0.275 g, 55%). 1H NMR (CDCl3, 300 MHz):
d 1.86e2.08 (m, 2H, CH2), 2.53e2.74 (m, 2H, CH2), 3.54e
3.73 (m, 3H, CH2eCH), 3.75 (s, 3H, OMe), 6.89 (dd,
J ¼ 6 Hz, J ¼ 2 Hz, 2H, CH), 7.58 (dd, J ¼ 6 Hz, J ¼ 2 Hz,
2H, CH). 13C NMR (75 MHz, CDCl3): d 56.8 (q, CH3), 11.7
(t, CH2), 21.9 (t, CH2), 47.2 (t, CH2), 82.3 (d, CH), 115.5
(d, CH), 131.4 (d, CH), 127.5 (s, C), 151.9 (s, C), 164.3 (s,
C). IR (KBr pellets cmꢀ1) n 780, 1600, 1645. Anal. CHN:
calcd C 60.13, H 5.89, Cl 14.79, N 5.84, O 13.35, found C
60.13, H 5.87, Cl 14.79, N 5.82, O 13.35.
5.1.21. Synthesis of 3-(3,4-dimethoxyphenyl)-5,6-dihydro-
4H-1,2-oxazine-6-carbonitrile 3u
Obtained from 1e (0.5 g, 2.5 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2a (1.8 g, 3.33 mmol) as a yellow
solid, recrystallised from ethanolen-hexane (0.4 g, 80%),
1
m.p. 155 ꢁC. H NMR (CDCl3, 300 MHz): d 1.86e2.08 (m,
2H, CH2), 2.56e2.78 (m, 2H, CH2), 3.76 (s, 6H, OMe), 4.19
(t, H, CH), 6.77e7.19 (m, 3H, CH). 13C NMR (75 MHz,
CDCl3): d 57.3 (q, CH3), 16.8 (t, CH2), 22.2 (t, CH2), 71.2
(d, CH), 115.5 (d, CH), 116.9 (d, CH), 123.8 (d, CH), 119.2

M.K. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 280e288
287
(s, C), 128.5 (s, C), 150.8 (s, C), 152.6 (s, C), 153.2 (s, C). IR
(KBr pellets cmꢀ1) n 1596, 1638, 2260. Anal. CHN: calcd C
63.40, H 5.73, N 11.38, O 19.49, found C 63.40, H 5.70, N
11.35, O 19.49.
3H, CH2eCH), 6.76e7.19 (m, 3H, CH). 13C NMR
(75 MHz, CDCl3): d 15.2 (q, CH3), 57.2 (q, CH3), 21.3 (t,
CH2), 22.6 (t, CH2), 62.5 (t, CH2), 86.6 (d, CH), 115.2 (d,
CH), 116.3 (d, CH), 123.4 (d, CH), 128.3 (s, C), 150.5 (s,
C), 152.6 (s, C) 153.0 (s, C), 171.8 (s, C). IR (KBr pellets
cmꢀ1) n 1605, 1641, 1776. Anal. CHN: calcd C 61.42, H
6.53, N 4.78, O 27.27, found C 61.42, H 6.50, N 4.75, O 27.27.
5.1.22. Synthesis of 6-chloromethyl-3-(3,4-
dimethoxyphenyl)-5,6-dihydro-4H-1,2-oxazine 3v
Obtained from 1e (0.5 g, 2.5 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2b (1.6 g, 2.09 mmol) as a reddish
brown oil (0.35 g, 70%). 1H NMR (CDCl3, 300 MHz):
d 1.87e2.09 (m, 2H, CH2), 2.54e2.75 (m, 2H, CH2), 3.54e
3.73 (m, 3H, CH2eCH), 3.76 (s, 6H, OMe), 6.78e7.19 (m,
3H, CH). 13C NMR (75 MHz, CDCl3): d 57.2 (q, CH3), 11.9
(t, CH2), 22.5 (t, CH2), 47.7 (t, CH2), 82.5 (d, CH), 115.9
(d, CH), 116.8 (d, CH), 123.6 (d, CH), 128.4 (s, C), 150.7
(s, C), 152.3 (s, C), 153.1 (s, C). IR (KBr pellets cmꢀ1) n
782, 1602, 1648. Anal. CHN: calcd C 57.89, H 5.98, Cl
13.14, N 5.19, O 17.80, found C 57.85, H 5.98, Cl 13.14, N
5.15, O 17.80.
5.2. Biological activity
5.2.1. Antibacterial studies (materials and methods)
Four bacterial species, viz: E. coli, K. pneumoniae, P. aer-
uginosa, S. aureus were used as the antibacterial test strains.
The in vitro antibacterial activity of the compounds was tested
by cup diffusion method [35] by using the test bacteria main-
tained on the nutrient agar (NA) medium at 37 ꢁC at 104 cells/
mL and incubating in LB medium containing the indicated
amount of the given compound. After overnight incubation
at 37 ꢁC, the optical density of the culture was determined.
All compounds exhibited excellent in vitro antibacterial activ-
ity against Gram-positive and Gram-negative organisms.
The minimal inhibitory concentration (MIC) was deter-
mined by assaying the effect of each compound at concentra-
tions of 0.1, 5, 10, 15, 20, 25, 30, 35 and 40 mg/mL. The
sterile medium (20 ml) was poured onto 9 cm Petri plates.
The medium was allowed to cool in a sterile condition and
plates were then inculcated with 1 ꢃ 105 cfu cultures of test
bacteria. The concentration of bacterial cells in the suspension
was adjusted to a minimum of 1 ꢃ 105 cfu/ml in nutrient broth
solution. Agar cups of 5 mm diameter were made in the plates.
Each test sample was dissolved in dimethylformamide (DMF)
and 50 ml of test solution containing 20 mg of the test
compound was placed in each cup. The plates were left to
stay for an hour in order to facilitate the diffusion of the
drug solution. Negative controls were prepared using the
same solvent (DMF) employed to dissolve the test compounds.
Then the plates were incubated at 37 ꢁC for 24 h. The zone of
inhibition, if any, against the test bacteria was measured in
mm. Penicillin G and Gentamicin were used as positive refer-
ence to determine the sensitivity of each bacterial species
tested.
5.1.23. Synthesis of [3-(3,4-dimethoxyphenyl)-5,6-dihydro-
4H-1,2-oxazin-6-yl]-methanol 3w
Obtained from 1e (0.5 g, 2.5 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2c (1.6 g, 2.56 mmol) as a red oil
1
(0.4 g, 80%). H NMR (CDCl3, 300 MHz): d 1.87e2.08 (m,
2H, CH2), 2.13 (br, 1H, OH), 2.54e2.78 (m, 2H, CH2),
3.72e3.93 (m, 3H, CH2eCH), 3.75 (s, 6H, OMe), 6.77e
7.18 (m, 3H, CH). 13C NMR (75 MHz, CDCl3): d 57.3 (q,
CH3), 10.5 (t, CH2), 23.0 (t, CH2), 66.2 (t, CH2), 82.6 (d,
CH), 115.8 (d, CH), 116.7 (d, CH), 123.4 (d, CH), 128.5 (s,
C), 150.8 (s, C), 152.6 (s, C), 153.1 (s, C). IR (KBr pellets
cmꢀ1) n 1600, 1640, 3360. Anal. CHN: calcd C 62.14, H
6.82, N 5.57, O 25.47, found C 62.14, H 6.82, N 5.55, O 25.45.
5.1.24. Synthesis of 6-(benzo[1,3]dioxol-5-yl)methyl-3-(3,4-
dimethoxyphenyl)-5,6-dihydro-4H-1,2- oxazine 3x
Obtained from (0.5 g, 2.5 mmol) 1e, chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2d (1.37 g, 0.85 mmol) as a reddish
brown oil (0.375 g, 75%). 1H NMR (CDCl3, 300 MHz):
d 1.86e2.07 (m, 2H, CH2), 2.56e2.77 (m, 2H, CH2), 3.51e
3.73 (m, 3H, CH2eCH), 3.75 (s, 6H, OMe), 5.93 (s, 2H,
OeCH2eO), 6.75e7.18 (m, 3H, CH). 13C NMR (75 MHz,
CDCl3): d 57.2 (q, CH3), 14.2 (t, CH2), 23.1 (t, CH2), 41.9
(t, CH2), 102.2 (t, CH2), 81.9 (d, CH), 114.3 (d, CH), 115.7
(d, CH), 116.9 (d, CH), 122.6 (d, CH), 123.5 (d, CH), 128.3
(s, C), 133.0 (s, C), 147.2 (s, C), 149.2 (s, C), 150.8 (s, C),
152.4 (s, C), 153.1 (s, C). IR (KBr pellets cmꢀ1) n 1600,
1645. Anal. CHN: calcd C 67.59, H 5.96, N 3.94, O 22.51,
found C 67.59, H 5.95, N 3.90, O 22.51.
5.2.2. Antifungal studies (materials and methods)
Four fungal species namely A. flavus, A. niger, F. monili-
forme and F. oxysporum were used as antifungal test strains
to determine the antifungal activity of the synthesized com-
pounds by cup diffusion method [35]. The filamentous fungi
were maintained on potato dextrose agar (PDA) medium at
28 ꢁC. The in vitro antifungal activity of the compounds was
tested by using the test fungus at 106 spores/mL and was incu-
bated in potato dextrose liquid medium containing the indi-
cated amount of the given compound. After incubation for
48 h at 28 ꢁC, the optical density of the culture was deter-
mined. All compounds exhibited in vitro antifungal activity
against all the tested fungal strains.
5.1.25. Synthesis of 3-(3,4-dimethoxyphenyl)-5,6-dihydro-
4H-1,2-oxazine-6-carboxylic acid ethylester 3y
Obtained from 1e (0.5 g, 2.5 mmol), chloramine-T$3H2O
(0.99 g, 3.52 mmol) and 2e (1.63 g, 1.63 mmol) as a blood
red viscous oil. (0.425 g, 85%). 1H NMR (CDCl3,
300 MHz): d 1.29 (t, 3H, CH3), 2.29e2.54 (m, 2H, CH2),
2.56e2.79 (m, 2H, CH2), 3.75 (s, 6H, OMe), 3.99e4.02 (m,
MIC was determined by assaying the effect of each com-
pound at concentrations of 0.1, 5, 10, 15, 20, 25, 30, 35 and

288
M.K. Manjula et al. / European Journal of Medicinal Chemistry 44 (2009) 280e288
[5] A. Defoin, H. Sarazin, C. Strehler, J. Streith, Tetrahedron Lett. 35 (1994)
5653e5656.
40 mg/mL. The sterile medium (20 ml) was poured onto 9 cm
Petri plates. The medium was allowed to cool in a sterile
condition and plates were then inculcated with 1 ꢃ 106 cfu
cultures of test fungi. The concentration of spores in the
suspension was adjusted to a minimum of 1 ꢃ 106 cfu/ml in
potato dextrose broth solution. Agar cup of 5 mm diameter
was made in the plates. Each test sample was dissolved in
dimethylformamide (DMF), 50 ml of test solution containing
20 mg of the test compound was placed in the cup. The plates
were left to stay for an hour in order to facilitate the diffusion
of the drug solution. The same procedure was followed for the
remaining compounds. Negative controls were prepared using
the same solvent (DMF) employed to dissolve the test com-
pounds. Then the plates were incubated at 28 ꢁC for 48 h.
The zone of inhibition, if any, against the test fungi were
measured in mm. Nystatin was used as positive reference to
determine the sensitivity of each fungal species tested. The
results were subjected to statistical analysis.
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