An eco-friendly synthesis and antimicrobial activities of dihydro-2H-benzo- and naphtho-1,3-oxazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.12.058

A series of 3,4-dihydro-2H-benzo[e]-, 2,3-dihydro-1H-naphtho[1,2-e]-, 3,4-dihydro-2H-naphtho[2,1-e][1,3]oxazine and 1,2-bis(3,4-dihydrobenzo[e][1,3]oxazin-3(4H)-yl)ethane derivatives was obtained through an eco-friendly Mannich type condensation-cyclization reaction of phenols or naphthols with formaldehyde and primary amines in water at ambient temperature. Preliminary in vitro antimicrobial activity of the synthesized compounds was assessed against six pathogenic fungi, two Gram-negative and two Gram-positive bacteria. Some of the screened compounds have shown significant in vitro antimicrobial effect. Cytotoxic activities of the lead compounds (2m, 2n, 3c and 3d) against mouse fibroblast cell line (L929) were determined by MTT method. The assay results revealed that these molecules offered remarkable viability (>90%) of L929 cells at concentration of 25?μg/mL.

Full text of DOI:10.1016/j.ejmech.2009.12.058

European Journal of Medicinal Chemistry 45 (2010) 1502–1507
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
An eco-friendly synthesis and antimicrobial activities of
dihydro-2H-benzo- and naphtho-1,3-oxazine derivatives
,
Bijoy P. Mathew a, Awanit Kumar b, Satyasheel Sharma a, P.K. Shukla b, Mahendra Nath a
*
a Department of Chemistry, University of Delhi, Delhi 110 007, India
b Fermentation Technology Division, Central Drug Research Institute, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,4-dihydro-2H-benzo[e]-, 2,3-dihydro-1H-naphtho[1,2-e]-, 3,4-dihydro-2H-naphtho[2,1-
e][1,3]oxazine and 1,2-bis(3,4-dihydrobenzo[e][1,3]oxazin-3(4H)-yl)ethane derivatives was obtained
through an eco-friendly Mannich type condensation–cyclization reaction of phenols or naphthols with
formaldehyde and primary amines in water at ambient temperature. Preliminary in vitro antimicrobial
activity of the synthesized compounds was assessed against six pathogenic fungi, two Gram-negative
and two Gram-positive bacteria. Some of the screened compounds have shown significant in vitro
antimicrobial effect. Cytotoxic activities of the lead compounds (2m, 2n, 3c and 3d) against mouse
fibroblast cell line (L929) were determined by MTT method. The assay results revealed that these
Received 29 September 2009
Received in revised form
18 December 2009
Accepted 21 December 2009
Available online 13 January 2010
Keywords:
Antimicrobial activity
Benzoxazine
molecules offered remarkable viability (>90%) of L929 cells at concentration of 25 mg/mL.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Naphthoxazine
Eco-friendly synthesis
1. Introduction
4a–j and 5) by using environmentally benign process in good to
excellent yields. The in vitro antimicrobial activities of these
compounds were assessed and the results are presented in this
paper.
Invasive microbial infections with multi-drug resistant bacteria
and opportunistic mycoses have become increasingly common
worldwide and are collectively a major cause of morbidity and
mortality, especially in immunocompromised patients [1].
Although, several new antimicrobial agents have been licensed in
the past decades, but some immunosuppressed persons are diffi-
cult to treat. The main reason for this include rapid transmission of
the systemic fungal pathogens, drug toxicity, drug bioavailability,
development of drug resistance and lack of safe oral or intravenous
preparations. However, the problem of toxicity is greatly addressed
by the development of several broad-spectrum imidazole [2], tri-
azole [3] and echinocandin [4] derivatives for specific use but the
problem of drug resistance is still an issue with newly discovered
antimicrobial drugs. Therefore, a search for new, safe and highly
effective chemotherapeutic agents become inevitable. Thorough
literature survey revealed that the compounds containing dihydro-
1,3-oxazine ring system exhibited a wide spectrum of pharmaco-
logical activities such as anti-tumor [5], anti-bacterial [6], anti-HIV
[7] and antimalarial [8] agents. In addition, 6-arylbenzoxazines are
reported as potent non-steroidal progesterone receptor agonists
[9]. In the course of our search for potent antifungal compounds, we
synthesized various dihydro-1,3-oxazine derivatives (2a–n, 3a–d,
2. Chemistry
The synthesis of various 3,4-dihydro-2H-benzo[e]-, 2,3-dihy-
dro-1H-naphtho[1,2-e]-, 3,4-dihydro-2H-naphtho[2,1-e][1,3]oxa-
zines and 1,2-bis[3,4-dihydrobenzo[e][1,3]oxazin-3(4H)-yl]ethanes
involves one-pot condensation–cyclization reaction of phenols or
naphthols with formaldehyde and primary amines. Various
methods have been reported [10–12] for the synthesis of dihydro-
1,3-oxazines including the reaction under neat conditions [13].
However, many of these processes have several disadvantages such
as the need for a prolonged reaction time, high temperature, use of
volatile and toxic organic solvents and occurrence of side products.
Thus, the development of simple, efficient and green procedure for
the synthesis of these molecules is highly desirable. In continuation
of our efforts to develop a simple and economical methodology
for the synthesis of target compounds of biological interests, we
have successfully developed an efficient and eco-friendly
synthetic strategy [14] for the construction of a series of 3,4-dihy-
dro-2H-benzo[e][1,3]oxazines (2a–n), 1,2-bis[3,4-dihydrobenzo-
[e][1,3]oxazin-3(4H)-yl]ethanes (3a–d), 2,3-dihydro-1H-naphtho-
[1,2-e][1,3]oxazines (4a–j) and 3,4-dihydro-3-phenyl-2H-naph-
tho[2,1-e][1,3]oxazine (5) in good to excellent yields via Mannich
* Corresponding author. Tel.: þ91 11 27667405; fax: þ91 11 27666605.
E-mail address: mnath@chemistry.du.ac.in (M. Nath).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.058

B.P. Mathew et al. / European Journal of Medicinal Chemistry 45 (2010) 1502–1507
1503
type condensation–cyclization reaction of phenols or naphthols
with HCHO and primary amines in water at ambient temperature
(Scheme 1). The structures of all the synthesized molecules were
confirmed by elemental analyses and spectral (IR, 1H NMR, 13C
NMR, and mass) data. The IR spectrum of compound 2i showed the
characteristic absorption peaks of benzoxazine ring structure at
1226 cmꢀ1 (asymmetric stretching of C–O–C), and at 1035 cmꢀ1
(symmetric stretching of C–O–C). In 1H NMR, characteristic peaks of
Candida parapsilosis (Cp) by broth micro-dilution technique as per
guidelines of National Committee for Clinical Laboratory Standards
[15] using RPMI Medium 1640 buffered with MOPS [3-(N-morpho-
lino)propanesulphonic acid] in microtitre plates. The starting inocu-
lums of test culture were 1–5 ꢂ 103 cfu/mL. Microtitre plates were
incubated at 35 ꢁC. Minimal inhibitory concentrations (MICs) were
determined by spectrophotometric method at 492 nm after 24–48 h
(yeasts) and 72–96 h (mycelial fungi) incubation. Ketoconazole and
fluconazole were used as reference fungicides.
two methylene groups of 1,3-oxazine ring are observed at
d 4.53
corresponding to Ar–CH2–N and 5.26 corresponding to O–CH2–N.
d
Further evidence for the formation of 3-(4-fluorophenyl)-6-
methyl-3,4-dihydro-2H-benzo-[e][1,3]oxazine (2i) was obtained by
recording its mass spectrum. The mass spectrum of compound (2i)
showed Mþ ion peak at m/z 243 for the molecular formula
C15H14FNO. In addition, differential scanning calorimetry (DSC)
thermogram of 2i showed an endothermic peak at 82.2 ꢁC which
was attributed to its melting point. The characteristic exothermic
peak was observed due to ring-opening polymerization [10j] with
onset at 141.2 ꢁC and maximum at 165.7 ꢁC. The amount of heat of
polymerization was found to be 66.4 J/g. The typical synthetic
procedure and characterization data of compounds (2i–j, 2n, 3b,
4b, and 4d–e) are presented in the Experimental Section. The
physical data of all the known compounds (2a–h, 2k–m, 3a, 3c–d,
4a, 4c, 4f–j and 5) are in agreement with those of reported data
[10–14].
3.2. Anti-bacterial studies
The anti-bacterial activity of all the prepared compounds was
evaluated against two Gram-positive bacteria viz. Klebsiella
pneumonia (Kp) and Staphylococcus aureus (Sa), and two Gram-
negative bacteria viz. Escherichia coli (Ec) and Pseudomonas aeru-
ginosa (Pa) by the NCCLS Method using Mueller Hinton broth in
96-well tissue culture plates. The bacterial strains were grown on
nutrient agar at 37 ꢁC. After 24 h of incubation, bacterial cells were
suspended in normal saline containing Tween 20 at 0.05%
concentration of approximately 1.0–2.0
ꢂ
107 cells/mL by
matching with McFarland standards. Proper growth control, drug
control and the negative control were adjusted on to the plate.
Compounds were dissolved in dimethyl sulfoxide (DMSO) at
a concentration of 1 mg/mL and 20
each well of 96-well tissue culture plate having 180
m
L of this solution was added to
L Mueller
m
Hinton broth. The solution was then serially diluted to afford
twofold serial dilutions of the test compounds in the subsequent
3. Pharmacology
wells. Then McFarland matched bacterial suspension (100
diluted with 10 mL of media. The diluted bacterial suspension
(100 L) was added to each well and then kept for incubation. The
maximum concentration of compounds tested was 50 g/mL.
Microtitre plates were incubated at 35 ꢁC in a moist, dark chamber.
MICs were recorded spectrophotometrically after 24 h incubation.
Gentamycin and ampicillin were used as reference anti-bacterial
agents.
mL) was
3.1. Antifungal studies
m
The in vitro antifungal activity of 3,4-dihydro-2H-ben-
zo[e][1,3]oxazines (2a–n), 1,2-bis[3,4-dihydrobenzo[e][1,3]oxazin-
3(4H)-yl]ethanes (3a–d), 2,3-dihydro-1H-naphtho[1,2-e][1,3]oxa-
zines (4a–j) and 3,4-dihydro-3-phenyl-2H-naphtho[2,1-e][1,3]oxa-
zine (5) was investigated against six pathogenic fungi viz. – Candida
albicans (Ca), Cryptococcus neoformans (Cn), Sporothrix schenckii (Ss),
Trichophyton mentagrophytes (Tm), Aspergillus fumigatus (Af) and
m
3.3. Cytotoxicity evaluation
The cytotoxic effect of the lead compounds (2m, 2n, 3c and 3d)
against mammalian cells, mouse fibroblast cell line L929 was deter-
mined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) method [16]. The stock solutions (1.0 mg/mL) of
the test compounds were prepared in DMSO. The cells (L929) were
grown at 37 ꢁC in RPMI Medium 1640 supplemented with 10% (v/v)
foetal bovine serum (FBS) and antimycotic and anti-bacterial solu-
O
N
5
tions (Sigma, USA) in humidified atmosphere having 5% CO2.100 mL of
the confluent fibroblast stock suspension (1.0 ꢂ 105 cells/mL) was
X = OH,
Y = H
R
N
R
N
b
dispensed in 96-well tissue culture plates. The original medium was
X
Y
replaced from the wells with 100 mL serum-free RPMI Medium 1640
R3
R2
O
when the cells reached 80% confluence after incubation at 37 ꢁC in
humidified incubator with 5% CO2. L929 cells were exposed to
various concentrations (25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19 and
R3
R2
O
a
a
X = H,
X = H,
R1
R1
Y = OH
Y = OH
4a-j
2a-n
1
0.09
incubation for 24 h, cells were washed twice in phosphate-buffered
saline (PBS) and 200 L of MTT solution (0.5 mg/mL of MTT in RPMI
Medium 1640) was added to each well and then incubated for 4 h at
37 ꢁC to allow MTT metabolism. An aliquot of 100
L DMSO solvent
mg/mL) of test compounds along with control experiment. After
X = H,
c
m
Y = OH
R1
R3
m
R2
O
N
was added to each well and the plate was incubated for 30 min at
room temperature. Response of L929 cells to the test compounds was
determined spectrophotometrically at 570 and 630 nm (reference
wavelength). The ratio of differences in absorbance values at these
two wavelengths (A570 and A630) for both treated and untreated
cultured cells were used to calculate % viability using the equation
N
R2
O
R3
R1
3a-d
Scheme 1. Reagents and conditions: (a) RNH2, HCHO, H2O, 25 ꢁC, 0.5–1 h; (b)
C6H5NH2, HCHO, H2O, 25 ꢁC, 30 min; (c) NH2CH2CH2NH2, HCHO, H2O, 25 ꢁC, 1 h.
[(A570–A630)sample/(A570-A630)control]
ꢂ
100%. Further, the

1504
B.P. Mathew et al. / European Journal of Medicinal Chemistry 45 (2010) 1502–1507
morphology of cells was observed by using Giemsa stain under phase
contrast microscope. After fixation of the cells in the wells of 96-well
tissue culture plates, Giemsa stain was added to each well and incu-
bated for 30 min at 37 ꢁC. The excess stain was removed by thorough
washing with PBS. The culture plates were dried in air and observed
under phase contrast microscope.
depends mainly on the presence of substituent in the fused aryl ring.
The presence of chloro substituent at position 6 and H at 5, 8 posi-
tions in 2m and 3c displayed significant activity against Ca (12.5
mL), Cn(12.5 g/mL), Ss (6.25 g/mL), Tm (3.12 g/mL)and Cp(25
m
m
g/
g/
m
m
m
mL). Introduction of chloro substituent at position 8 in 3d and 5,8-
positions in 2n did not improve the antifungal activity. As it is
evident from the activity data that a mere change of 6-chloro
substituent in 2m or 3c by electron-donating methyl group as in 2g
or 3b displayed low order of inhibitory effect against all the tested
mycoses.
4. Results and discussion
Most target compounds showing minimum inhibitory
Structure–activity relationship revealed that the substituent at
position 3 in structure 2 is also responsible for antimycotic activity.
An exchange of 2-chlorophenyl substituent at position 3 in 2m by
4-methylphenyl, phenyl or butyl substituent as in 2l, 2k and 2j
respectively led to reduce the antifungal activity. Additionally,
2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazines (4a–j) and 3,4-dihydro-
3-phenyl-2H-naphtho[2,1-e][1,3]oxazine (5) have demonstrated
poor antifungal activity compared to 3,4-dihydro-2H-benzo[e][1,3]-
oxazines.
concentration (MIC) values between 0.39 and 50 mg/mL were able
to have a significant in vitro inhibitory effect against the screened
microbes. The activity data are presented in Tables 1 and 2. Among
all the synthesized compounds, 2m was found to be the most
active against Ca, Cn, Ss, Tm, Af and Cp with MIC values 12.5, 12.5,
6.25, 3.12, 25 and 25
was found equipotent to 2m except it showed higher MIC value
(50 g/mL) against Af. However, this trend does not translate
mg/mL respectively. The other compound 3c
m
exactly to anti-bacterial activities. As shown in Table 2,
compounds 2m, 3c and 3d were found active against Sa but latter
was most promising against Sa and Kp with MIC values 0.39 and
Besides significant antifungal effect, some of the synthesized
compounds indicated potent inhibitory effect against tested Gram-
positive and Gram-negative bacteria (Table 2). Among all, the
1.56 mg/mL respectively.
compound 3d having MIC value 0.39 mg/mL was found to be most
As seen in Table 1; although none of the compounds showed
more efficacy than fluconazole against tested fungi, but some of the
title compounds were found to be significantly potent with low MIC
active and equipotent to standard drug gentamycin, used against
Gram-positive bacterium, S. aureus. Surprisingly, the 3,4-dihydro-
2H-benzo[e][1,3]oxazines (2d–f, 2k–l) were found to be more
values in the range of 3.12–25
2m and 3c have displayed better fungicidal activity (3.12
than ketoconazole (4 g/mL) against Tm. The antifungal
activity profile of 3,4-dihydro-2H-benzo[e][1,3]oxazines (2a–n) and
mg/mL. The most active compounds
active against Ec than that of standard drug, ampicillin (MIC 50
mL). In contrast, compounds 2f, 2h, 2k–n, 3c–d have shown
significant activity against Sa (MICs ranged from 0.39 to 25 g/mL).
Similarly, derivatives 2a, 2c, 2g, 2i, 4a, and 4f were found
mg/
mg/mL)
m
m
1,2-bis[3,4-dihydrobenzo[e][1,3]oxazin-3(4H)-yl]ethanes
(3a–d)
Table 1
Antifungal activity of 3,4-dihydro-2H-benzo[e][1,3]oxazines (2a–n), 1,2-bis(3,4-dihydrobenzo[e][1,3]oxazin-3(4H)-yl)ethanes (3a–d), 2,3-dihydro-1H-naphtho[1,2-e][1,3]oxa-
zines (4a–j) and 3,4-dihydro-3-phenyl-2H-naphtho[2,1-e][1,3]oxazine (5).
Compound
R1
R2
R3
R
Minimum inhibitory concentration (MIC) in mg/mL
Caa
Cna
Ssa
Tma
Afa
Cpa
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
H
H
H
CH3
H
H
H
H
H
H
H
H
H
Cl
H
H
H
Cl
–
–
–
–
–
–
–
–
–
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
–
–
–
–
–
–
–
–
–
C4H9
C6H11
C6H5
C6H5
C6H5
4-CH3C6H4
2-ClC6H4
4-ClC6H4
4-FC6H4
C4H9
>50
>50
>50
50
>50
50
50
50
>50
>50
50
25
12.5
>50
>50
50
12.5
50
50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
>50
>50
50
25
25
50
>50
50
50
12.5
50
50
50
12.5
25
>50
50
50
50
>50
25
25
12.5
12.5
12.5
12.5
25
25
6.25
6.25
6.25
12.5
25
12.5
6.25
>50
25
50
50
50
50
50
25
25
25
>50
>50
50
50
50
50
50
25
>50
>50
25
25
25
50
50
50
50
>50
>50
50
50
50
50
50
50
>50
>50
50
25
25
50
>50
50
25
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
CH3
CH3
CH3
CH3
CH3
Cl
Cl
Cl
Cl
H
12.5
12.5
12.5
25
50
12.5
12.5
3.12
12.5
25
25
3.12
25
>50
25
50
25
50
50
50
25
C6H5
4-CH3C6H4
2-ClC6H4
2-ClC6H4
–
–
–
H
CH3
Cl
Cl
–
–
–
–
–
–
–
–
C6H5
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
4-ClC6H4
4-BrC6H4
4-FC6H4
2,4-Cl2C6H3
2,5-Cl2C6H3
2,4,6-Cl3C6H2
C4H9
C6H11
CH2C6H5
–
50
>50
>50
>50
50
50
>50
>50
>50
>50
>50
50
50
50
–
–
–
–
25
50
>50
–
–
–
–
>50
>50
5
>50
Ketoconazole
Fluconazole
–
–
–
–
–
–
–
–
0.002
0.5
0.001
1.0
0.031
2.0
4.0
1.0
2.0
2.0
0.031
1.0
a
Ca – Candida albicans; Cn – Cryptococcus neoformans; Ss – Sporothrix schenckii; Tm – Trichophyton mentagrophytes; Af – Aspergillus fumigatus; Cp – Candida parapsilosis
(ATCC 22019).

B.P. Mathew et al. / European Journal of Medicinal Chemistry 45 (2010) 1502–1507
1505
Table 2
Anti-bacterial activity of the compounds 2a-n, 3a-d, 4a-j and 5.
a
Minimum inhibitory concentration in mg/mL
Kpa
Eca
Paa
Saa
2a
2b
2c
2d
2e
2f
2g
2h
>50
>50
>50
>50
50
50
>50
25
50
>50
50
25
25
25
50
>50
50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
50
50
25
>50
25
50
50
50
2i
50
2j
2k
2l
2m
2n
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
4g
>50
50
25
>50
6.25
>50
>50
>50
1.56
>50
50
>50
12.5
6.25
>50
>50
>50
>50
50
>50
>50
>50
>50
50
50
50
50
>50
>50
>50
>50
>50
>50
>50
>50
25
12.5
6.25
12.5
>50
>50
12.5
0.39
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
>50
4h
4i
4j
5
Gentamycin
Ampicillin
0.78
0.39
0.78
50
0.78
50
0.39
0.19
a
Kp – Klebsiella pneumoniae; Ec – Escherichia coli; Pa – Pseudomonas aeruginosa;
Sa – Staphylococcus aureus.
equipotent (MIC 50
to ampicillin.
mg/mL) against Ec, 2g–i, 2n and 4a–d against Pa,
The structure–activity relationships showed that the anti-
bacterial activity of 1,2-bis[3,4-dihydrobenzo[e][1,3]oxazin-3(4H)-
yl]ethanes (3a–d) based on the substituents present in the fused
aryl ring and their efficacy is in the order of 6,8-dichloro > 6-
chloro > 6-methyl, H. The activity of 4a–j depends on the nature of
N-substituent at position 3. In 4a–d the N-aryl substituent such as
C6H5, 4-ClC6H4, 4-BrC6H4, 4-FC6H4 have shown comparable activity
(MIC 50 mg/mL) against Pa where as compound 4a with N–C6H5 and
4f with N–C6H3Cl2 (2, 5) demonstrated similar effect against Ec. In
addition, compounds 4b with N–C6H4Cl(4) and 4d with N–C6H4F(4)
were also found equally potent (MIC 50 mg/mL) against Kp. Other
compounds with N-substituents as 2,4-Cl2C6H3, 2,4,6-Cl3C6H2,
C4H9, C6H11 and benzyl were found inactive against all the tested
bacteria. However, these results could be guide for further anti-
bacterial studies.
Finally, the lead compounds (2m, 2n, 3c and 3d) were studied
for their cytotoxic effects against mammalian L929 cells by MTT
assay. Morphological anomalies of L929 cells caused due to the test
compounds were evident under phase contrast microscope. In
control experiment, L929 cells were found fairly transparent, well
spaced and attached to the well’s surface of tissue culture plate
(Fig. 1A). After exposure of the L929 cells to test compounds, no
Fig. 1. Morphological changes of L929 cells exposed to compounds 2m, 2n, 3c and 3d
for 24 h. (A) Control; (B) treated at MIC (6.25 mg/mL); (C) treated at higher concen-
trations (12.5 and 25 mg/mL).
toxicity was observed at MIC (6.25 mg/mL) and lower concentra-
tions as evident from the normal morphology of cells (Fig. 1B).
However, when these cells exposed to higher concentrations (12.5
5. Conclusions
and 25
m
g/mL) of test compounds they lost their normal
In conclusion, various 3,4-dihydro-2H-benzo[e]-, 2,3-dihydro-
1H-naphtho[1,2-e]- and 3,4-dihydro-2H-naphtho[2,1-e][1,3]oxa-
zines have been synthesized by using green methodology and
evaluated for their antimicrobial activity. Most of the analogues
morphology (Fig. 1C) but the absorbance studies at 570 and 630 nm
during MTT assay revealed >90% viability of L929 cells even at
higher concentrations of test compounds.

1506
B.P. Mathew et al. / European Journal of Medicinal Chemistry 45 (2010) 1502–1507
have shown significant activity with MIC values in the range of
0.39–50 g/mL against the tested microbes. The MTT assay results
revealed that the compounds 2m, 2n, 3c and 3d offered remarkable
viability (>90%) of L929 cells at 25 g/mL. Henceforth, these results
(1H, d, J ¼ 8.7 Hz, ArH); 13C NMR (300 MHz, CDCl3):
d: 13.8, 20.3,
m
29.7, 49.9, 51.1, 82.6, 117.7, 121.7, 124.9, 127.0, 127.2, 152.9; MS (ESI):
m/z 225 (Mþ).
m
may be useful for the further development of new antifungal and
anti-bacterial agents.
6.2.3. 5,8-Dichloro-3-(2-chlorophenyl)-3,4-dihydro-2H-
benzo[e][1,3]oxazine (2n)
Pale yellow viscous liquid (60%); bp 162 ꢁC; ymax(film, cmꢀ1):
2924, 2854, 1588, 1566, 1484, 1454, 1422, 1375, 1240 (asymmetric
stretching, C–O–C), 1154, 1051 (symmetric stretching, C–O–C), 975,
6. Experimental protocols
6.1. Chemistry
937, 799, 758; 1H NMR (300 MHz, CDCl3):
d: 4.58 (2H, s, Ar–CH2–N),
5.40 (2H, s, O–CH2–N), 6.93 (1H, d, J ¼ 8.6 Hz, ArH), 7.04–7.10 (1H,
Thin-layer chromatography was performed on pre-coated Merck
silica 60 F254 plates and spots were detected under UV light
(254 nm). All the compounds were purified by column chromatog-
raphy using silica gel (60–120 mesh, Ranbaxy). Mass spectra were
recorded on ESI-MS mass spectrometer in positive ionization mode.
Infrared spectra were recorded on Perkin–Elmer IR spectrometer in
film; absorption maxima (ymax) are given in cmꢀ1. The 1H and 13C
NMR spectra were measured on Bruker 300 MHz spectrometer in
CDCl3. The calibration of spectra was carried out on solvent signal
m, ArH), 7.15–7.24 (2H, m, ArH), 7.27–7.30 (1H, m, ArH), 7.42 (1H,
dd, J ¼ 1.5, 6.4 Hz, ArH); 13C NMR (300 MHz, CDCl3):
d: 49.6, 81.2,
111.9, 115.9, 118.6, 119.8, 121.8, 122.7, 125.5, 127.8, 129.5, 142.3, 145.7,
150.9; MS (ESI): m/z 314 (Mþ).
6.2.4. 2-(4-Chlorophenyl)-2,3-dihydro-1H-naphtho[1,2-e]-
[1,3]oxazine (4b)
Pale yellow solid (82%); mp 109.5 ꢁC; ymax(film, cmꢀ1): 3054,
2950, 2900, 1625, 1595, 1495, 1472, 1398, 1230 (asymmetric
stretching, C–O–C),1157, 1095, 1058 (symmetric stretching, C–O–C),
(CDCl3: dH ¼ 7.26 and dC ¼ 77.36) and chemical shifts (d) are reported
in parts per million (ppm). Coupling constants J are reported in hertz
(Hz). DSC traces were recorded on Perkin–Elmer differential calo-
rimetry. Elemental analysis was performed on Elementar Analy-
sensysteme GmbH VarioEL V3.00 for compounds 2i, 3b, 4b and 4d.
Anhydrous sodium sulfate (Na2SO4) was used as a drying agent. All
chemicals were used without further purification. Solvents were
distilled off under reduced pressure on a rotary evaporator.
1005, 947, 812, 746; 1H NMR (300 MHz, CDCl3):
d: 4.89 (2H, s, Ar–
CH2–N), 5.41 (2H, s, O–CH2–N), 7.00–7.06 (3H, m, ArH), 7.16–7.22
(2H, m, ArH), 7.36 (1H, dd, J ¼ 7.11, 7.83 Hz, ArH), 7.50 (1H, dd,
J ¼ 7.05, 7.92 Hz, ArH), 7.64 (2H, dd, J ¼ 5.52, 6.03 Hz, ArH), 7.75 (1H,
d, J ¼ 8.04 Hz, ArH); MS (ESI): m/z 295 (Mþ); Anal. Calcd for
C18H14ClNO (295.7): C, 73.10; H, 4.77; N, 4.74. Found: C, 72.66; H,
5.01; N, 4.67.
6.2. General method for the preparation of dihydrobenzo/
naphtho[e][1,3]oxazines (2a–n, 4a–j and 5)
6.2.5. 2-(4-Fluorophenyl)-2,3-dihydro-1H-naphtho[1,2-e]-
[1,3]oxazine (4d)
Dark brown solid (77%); mp 56.9 ꢁC; ymax(film, cmꢀ1): 3061,
2921, 2851, 1625, 1598, 1509, 1471, 1401, 1230 (asymmetric
stretching, C–O–C), 1157, 1100, 1058 (symmetric stretching, C–O–C),
To
a mixture of the corresponding phenol or naphthol
(10 mmol) and primary amine (10 mmol) in water (20 mL), formalin
(37%, w/v, 20 mmol) was added. The reaction mixture was stirred at
25 ꢁC for 30 min to 1 h. After completion of the reaction, the
product was extracted with ethyl acetate (20 mL, 2 times). The
organic layers were combined and washed with 10% aqueous NaOH
solution (30 mL, 2 times) followed by water (50 mL). The organic
layer was dried over sodium sulfate and evaporated under reduced
pressure. The crude product was purified by column chromatog-
raphy on silica gel using ethyl acetate/heptane as eluent to afford
the desired product.
1005, 945, 813, 746; 1H NMR (300 MHz, CDCl3):
d: 4.87 (2H, s, Ar–
CH2–N), 5.33 (2H, s, O–CH2–N), 6.91 (2H, dd, J ¼ 8.52, 8.82 Hz, ArH),
7.01–7.10 (3H, m, ArH), 7.36 (1H, dd, J ¼ 7.08, 7.80 Hz, ArH), 7.49 (1H,
dd, J ¼ 7.05, 8.13 Hz, ArH), 7.63 (2H, d, J ¼ 8.9 Hz, ArH), 7.76 (1H, d,
J ¼ 8.1 Hz, ArH); 13C NMR (300 MHz, CDCl3):
d: 48.7, 80.3, 112.3,
115.7, 115.9, 118.8, 120.7, 120.8, 120.9, 123.8, 126.8, 129.1, 129.2,
129.4, 131.2, 145.3, 152.3, 156.7; MS (ESI): m/z 280 (MHþ); Anal.
Calcd for C18H14FNO (279.3): C, 77.40; H, 5.05; N, 5.01. Found: C,
77.60; H, 4.85; N, 4.90.
6.2.1. 3-(4-Fluoro-phenyl)-6-methyl-3,4-dihydro-2H-
benzo[e][1,3]oxazine (2i)
6.2.6. 2-(2,4-Dichlorophenyl)-2,3-dihydro-1H-naphtho[1,2-e]-
[1,3]oxazine (4e)
Dark yellow solid (75%); mp 82.2 ꢁC; ymax(film, cmꢀ1): 2920,
2854, 1618, 1508, 1375, 1226 (asymmetric stretching, C–O–C), 1160,
1035 (symmetric stretching, C–O–C), 971, 948, 915, 821; 1H NMR
Pale yellow viscous liquid (78%); bp 237 ꢁC; ymax(film, cmꢀ1):
3064, 2932, 2890, 2852, 1625, 1599, 1513, 1481, 1436, 1382, 1311,
1265, 1230 (asymmetric stretching, C–O–C), 1159, 1108, 1051
(symmetric stretching, C–O–C), 1004, 948, 813, 747, 686; 1H NMR
(300 MHz, CDCl3): d: 2.30 (3H, s, CH3), 4.53 (2H, s, Ar–CH2–N), 5.26
(2H, s, O–CH2–N), 6.70 (1H, d, J ¼ 8.3 Hz, ArH), 6.80 (1H, s, ArH),
(300 MHz, CDCl3): d: 4.86 (2H, s, Ar–CH2–N), 5.32 (2H, s, O–CH2–N),
6.90–6.97 (3H, m, ArH), 7.02–7.08 (2H, m, ArH); 13C NMR (300 MHz,
7.02–7.08 (2H, m, ArH), 7.24 (1H, d, J ¼ 8.6 Hz, ArH), 7.35–7.40 (2H,
CDCl3): d: 20.6, 51.1, 80.2, 115.8, 115.9, 116.7, 120.2, 120.4, 120.5,
m, ArH), 7.50 (1H, dd, J ¼ 7.11, 8.01 Hz, ArH), 7.61–7.69 (2H, m, ArH),
127.0, 128.6, 130.2, 145.0, 152.0, 156.6; MS (ESI): m/z 243 (Mþ); Anal.
Calcd for C15H14FNO (243.3): C, 74.06; H, 5.80; N, 5.76. Found: C,
74.36; H, 5.49; N, 5.56.
7.78 (1H, d, J ¼ 8.0 Hz, ArH); 13C NMR (300 MHz, CDCl3):
d: 48.8,
80.1, 112.2, 118.6, 120.8, 123.7, 123.8, 126.8, 127.7, 128.5, 128.7, 129.1,
129.5, 129.6, 130.1, 130.9, 145.3, 151.8; MS (ESI): m/z 330 (Mþ).
6.2.2. 3-n-Butyl-6-chloro-3,4-dihydro-2H-benzo[e][1,3]oxazine (2j)
Brown viscous liquid (84%); bp 186 ꢁC; ymax(film, cmꢀ1) 2957,
2932, 2861, 1578, 1485, 1415, 1377, 1343, 1320, 1226 (asymmetric
stretching, C–O–C), 1198, 1170, 1141, 1118, 1092, 1034 (symmetric
stretching, C–O–C), 926, 873, 815, 634; 1H NMR (300 MHz, CDCl3):
6.3. General method for preparation of 1,2-bis(3,4-
dihydrobenzo[e][1,3]oxazin-3(4H)-yl)ethanes (3a–d)
To a mixture of the corresponding phenol (20 mmol) and ethylene
diamine (10 mmol) in water (20 mL), formalin (37%, w/v, 40 mmol)
was added. The reaction mixture was stirred at 25 ꢁC for 30 min. After
completion of the reaction, the product was extracted with ethyl
acetate (20 mL, 2 times). The combined organic layer was washed
d: 0.92 (3H, t, J ¼ 7.3 Hz, CH3), 1.26–1.38 (2H, m, CH2), 1.48–1.55 (2H,
m, CH2), 2.70 (2H, t, J ¼ 7.4 Hz, CH2), 3.94 (2H, s, Ar–CH2–N), 4.84
(2H, s, O–CH2–N), 6.69 (1H, d, J ¼ 8.7 Hz, ArH), 6.92 (1H, s, ArH), 7.05

B.P. Mathew et al. / European Journal of Medicinal Chemistry 45 (2010) 1502–1507
1507
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yl)ethane (3b)
White solid (76%); mp 110 ꢁC; ymax(film, cmꢀ1): 2921, 2857,1619,
1588, 1501, 1322, 1227, (asymmetric stretching, C–O–C), 1121, 1032
(symmetric stretching, C–O–C), 937, 910, 815, 745; 1H NMR
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(300 MHz, CDCl3): d: 2.24 (6H, s, 2CH3), 2.96 (4H, s, NCH2CH2N),
4.00 (4H, s, 2Ar–CH2–N), 4.87 (4H, s, 2O–CH2–N), 6.67 (2H, d,
J ¼ 8.24 Hz, ArH), 6.76 (2H, s, ArH), 6.92 (2H, d, J ¼ 7.90 Hz, ArH); 13C
NMR (300 MHz, CDCl3): d: 20.6, 49.6, 50.4, 82.7, 116.2, 119.7, 128.6,
128.9, 129.8, 151.8; MS (ESI): m/z 323 (MHþ); Anal. Calcd for
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72.24; H, 7.61; N, 8.35.
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Acknowledgements
The authors are thankful to the University of Delhi, India for
financial support. BPM and SS are grateful to Council of Scientific
and Industrial Research, New Delhi, India, for Junior Research
Fellowships.
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