Synthesis of some benzoxazinyl pyrazolone arylidenes as potent antimicrobials and antioxidants

Article abstract of DOI:10.1007/s00044-013-0719-9

2-[2-(3-Methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl]-2H-1, 4-benzoxazin-3(4H)-one (3) was obtained starting from methyl-(3,4-dihydro-3-oxo- 2H-1,4-benzoxazin-2-yl) acetate (1) through the corresponding hydrazide (2). Condensation of (3) with diff

Full text of DOI:10.1007/s00044-013-0719-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1320–1326
DOI 10.1007/s00044-013-0719-9
ORIGINAL RESEARCH
Synthesis of some benzoxazinyl pyrazolone arylidenes as potent
antimicrobials and antioxidants
•
•
•
George Sonia Knolin K. Thachil Manoj Kumar Parameswaran
Ravi Thengungal Kochupappy
Received: 8 May 2013 / Accepted: 13 August 2013 / Published online: 1 September 2013
Ó Springer Science+Business Media New York 2013
Abstract 2-[2-(3-Methyl-5-oxo-4,5-dihydro-1H-pyrazol-
1-yl)-2-oxoethyl]-2H-1,4-benzoxazin-3(4H)-one (3) was
obtained starting from methyl-(3,4-dihydro-3-oxo-2H-1,4-
benzoxazin-2-yl) acetate (1) through the corresponding
hydrazide (2). Condensation of (3) with different aromatic
aldehydes under Knoevengel condensation afforded 2-{2-
[3-methyl-4-(2-methylbenzylidine)-5-oxo-4,5-dihydro-1H-
pyrazol-1-yl]-2-oxoethyl}-2H-1,4-benzoxazin-3(4H)-ones
(4a–k). The structures of the compounds were determined
their research on these classes of compounds. Benzoxa-
zines are an important class of nitrogen and oxygen con-
taining heterocycles which have been reported to possess
antimicrobial (Dabholkar and Gavande, 2012), antioxidant
(Largeron et al., 1999), anticancer (Korolyov et al., 2010),
and antitubercular activities (Xiaokai et al., 2010). Various
alkyl benzoxazine derivatives are reported to possess free
radical scavenging property owing to its capacity to inhibit
oxidative stress (Blattes et al., 2005). Reactive oxygen
species like superoxide, hydrogen peroxide, etc., can be
neurotoxic thus damaging critical cellular components
necessary for cell viability. This is considered as one
underlying cause for cerebral palsy. Benzoxazine deriva-
tives were identified as potent neuroprotective antioxidants
possessing the antioxidant effect comparable to the stan-
dard a-tocopherol (Largeron et al., 2001). Pyrazole deriv-
atives have also gained the attraction of researchers as it is
reported to possess excellent potential as antimicrobial
(Urmila et al., 2005; Mohammad et al., 2010), antioxidant
(Manojkumar et al., 2009), and anticancer activities (Put-
hiyapurayil et al., 2012). Pyrazoles and pyrazolones are
reported for their effect as antioxidants in inhibiting the
oxidation of LDL, which otherwise responsible for causing
atherosclerosis (Jeong et al., 2004). The development of
new antimicrobial agents is the urgent need in drug dis-
covery perspective, as the development of resistance of
pathogenic bacteria to the available antibiotics is rapidly
becoming a major world-wide problem. Keeping in mind
about the potential of new drug entities obtained by the
modification of structures of the reported compounds or the
incorporation of one or more hetero moieties possessing
potential activity and also about the reports on benzoxa-
zines and pyrazoles, an attempt was made here to incor-
porate benzoxazines and pyrazoles and evaluate their
antimicrobial and antioxidant potency.
1
by FT-IR, H NMR, 13C NMR, mass spectral data, and
elemental analysis. All the synthesized compounds were
screened for their in vitro antimicrobial and antioxidant
studies.
Keywords Benzoxazine ꢀ Pyrazole ꢀ
Knoevengel condensation ꢀ Antimicrobial activity ꢀ
Antioxidant activity
Introduction
The pharmaceutical applications of nitrogen and oxygen
containing heterocycles have owed the researchers to focus
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0719-9) contains supplementary
material, which is available to authorized users.
G. Sonia (&) ꢀ K. K. Thachil ꢀ M. K. Parameswaran ꢀ
R. T. Kochupappy
Department of Pharmaceutical Chemistry, College of Pharmacy,
Sri Ramakrishna Institute of Paramedical Sciences,
Coimbatore 641 044, India
e-mail: soniashenjeev@gmail.com
123

Med Chem Res (2014) 23:1320–1326
Results and discussion
Chemistry
1321
to be active against Pseudomonas aeruginosa at MIC
15.6 lg/ml. Other compounds in the series also showed
moderate antibacterial activity. In the antifungal studies, the
compounds substituted by fluro, methoxy, and bromo group,
i.e., 4c, 4b, and 4k showed good activity with MIC at 15.6 and
31.25 lg/ml against the fungal strains Aspergillus niger and
Candida albicans. The excellent antimicrobial potential
shown by the fluro derivative may be due to its optimum
lipophilic nature and the electron-rich methoxy and trimeth-
oxy derivatives has attributed their antibacterial effect due to
its electron donating nature and the solubilizing effect.
The scheme of the present work shows that methyl-(3,4-
dihydro-3-oxo-2H-1,4-benzoxazin -2-yl) acetate 1 was
formed by the reaction of 2-amino phenol with maleic
anhydride and this with hydrazine hydrate has given 3,4-
dihydro-3-oxo-2H-1,4-benzoxazin-2-yl acetic acid hydra-
zide 2. 2-[2-(3-Methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
yl)-2-oxoethyl]-2H-1,4-benzoxazin-3-(4H)-one
3
was
obtained by treating 2 with ethyl acetoacetate in the pres-
ence of methanol. The formation of pyrazole ring was
confirmed by the IR absorption band obtained at 1721.22,
1610.80, 1501.02 corresponding to C=O, C=N, N–N,
respectively. The signal recorded at d 2.61 in the 1H NMR
spectra and at d 38.88 in the 13C NMR spectra corre-
sponded to the CH3 of the pyrazolone. The CH2 of the
pyrazole was obtained as signals at d 4.90 and d 66.24 in
the 1H NMR and 13C NMR spectra, respectively. The
pyrazole derivative 3 on Knoevengel condensation with
different aromatic aldehydes gave 2-{2-[4-(substituted)
benzylidene-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-
yl]-2-oxoethyl}-2H-1,4-benzoxazin-3(4H)-one (4a–k). The
formation of (4a–k) was confirmed by the IR absorption
band obtained for 4e at 1654.31 corresponding to the C=CH
linkage. The absorption bands at 1627.55 and 1535.78
corresponded for the pyrazole ring. The formation of the
Antioxidant activity
The antioxidant potential of all the newly synthesized
compounds was done by evaluating their scavenging effect
on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. The
antioxidant studies by DPPH assay revealed that com-
pounds 4b, 4e, and 4k with fluro, chloro, and bromo
moieties exhibited good scavenging activity. The free
radical scavenging effect of the halogen derivatives may be
due to its electron attracting and lipophilic nature.
Conclusion
In the present work, we have synthesized eleven benzoxazinyl
pyrazolone arylidenes aiming as antimicrobial and antioxi-
dant agents. The antimicrobial studies revealed that all the
titled compounds possessed significant antimicrobial activity.
Intheantibacterialstudyperformed,thederivativeswithfluro,
methoxy, dimethylamino, and trimethoxy substituent were
found to be potential. Also, compounds possessing fluro and
methoxy substituent showed excellent antifungal activity. In
the antioxidant studies, the excellent free radical scavenging
activity is attributed to the presence of alkyl group attached to
the benzoxazine and the derivatives with fluro, chloro, and
bromo substitution showed good free radical scavenging
activity. To conclude with, the novel series of benzoxazinyl
pyrazolo arylidenes synthesized by the incorporation of pyr-
azole moiety in the benzoxazinyl structure through an alkyl
linkmaybeconsideredasapromising leadfor futuredesignof
effective antimicrobial and antioxidant agents.
1
compound was further confirmed by the H NMR signal
obtained as singlet at around d 6.23 and 13C NMR at around
d 112.33 corresponding to C=CH group. The aromatic
protons were shown as multiplets at d 7.24–7.76 and aro-
matic carbons in the range of d 118.70–132.15. The for-
mation of the compound was further confirmed by the mass
spectral data where molecular ion peak was obtained cor-
responding to the molecular weight. The successful for-
mation of the compound was further confirmed by the
elemental analysis data. All the derivatives were obtained in
quite good yields.
Antimicrobial screening
All the newly synthesized compounds (4a–k) were screened
for their in vitro antibacterial activity against Staphylococcus
aureus NCIM 2079, Bacillus subtilis NCIM 2063, Esche-
richia coli NCIM 2118, and Pseudomonas aeruginosa NCIM
2036 and antifungal screening against Aspergillus niger
NCIM 545 and Candida albicans NCIM 3100. The antibac-
terial studies revealed that compounds 4c, 4f, and 4j con-
taining the methoxy, dimethyl amino, and trimethoxy
moieties, respectively, showed the minimum inhibitory con-
centration as low as 15.6 lg/ml against all the bacterial
strains. Also compound 4b with fluro substitution was found
Experimental
Chemistry
General informations for chemicals and instruments
Melting points were determined by using melting point
apparatus MR-VIS (MR08190508). IR spectra were
123

1322
Med Chem Res (2014) 23:1320–1326
recorded on JASCO FT/IR-410 spectrometer on KBr pel-
1
acetate 1 was formed by the reaction of 2-amino phenol
with maleic anhydride and the reaction of 1 with hydrazine
hydrate gave 3,4-dihydro-3-oxo-2H-1,4-benzoxazin-2-yl
acetic acid hydrazide 2 (Jayamma et al., 1989).
lets. H NMR and 13C NMR spectra were recorded on a
Bruker 300 MHz NMR spectrometer. Mass spectra were
recorded on Shimadzu, LCMS 2010 EV. The elemental
analysis was done on Vario EL III CHNS and the analysis
data (C, H, and N) were within the range of 0.4 % of the
calculated values. Purity of all the compounds were
checked by thin layer chromatography using silica gel—G
as adsorbent and iodine vapor as detecting agent.
Synthesis of 2-[2-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-
1-yl)-2-oxoethyl]-2H-1,4 benzoxazin-3(4H)-one (3)
The aryl hydrazide 2 and ethyl acetoacetate both in equi-
molar amounts (0.002 mol) were refluxed in methanol
(20 ml) for 5–6 h. After cooling the reaction mixture, a
The synthetic pathway is enumerated in Scheme 1.
Methyl-(3,4-dihydro-3-oxo-2H-1,4-benzoxazin-2-yl)
Scheme 1 Formation of
benzoxazinyl pyrazolo
methylidenes 4a–k
O
NH
NH2
O
N
H
O
(2)
CH COCH COOC
H
3
2
2
5
Reflux 5-6 hr
CH3
N
N
O
O
O
N
H
O
(3)
RCHO / CH COONa/ CH COOH
3
3
Reflux 2 hr
CH3
N
O
N
R
O
O
N
H
O
(4a - k)
R = H, F, OCH3, OH (OCH3), Cl, N(CH3)2, CH3, OH, NO2, (OCH3)3, Br
123

Med Chem Res (2014) 23:1320–1326
1323
solid which was separated out was filtered and dried after
washing with pet ether (60–80 °C). Recrystallization was
done from methanol. The solvent system used was meth-
anol:benzene (3:7).
(CH3 of pyrazolone), 65.68 (CH2–CO), 112.33 (C=CH),
120.19–131.15 (aromatic carbons), 165.62 ([NCO),
170.39 (C=O of pyrazolone), 172.78 (C=O of benzoxazi-
none); (M)? ion peak at 393; Anal. Calcd. (%) for
C21H16N3O4F: C, 64.12; H, 4.07; N, 10.61. Found: C,
64.22; H, 4.12; N, 10.58.
Yield: 63 %; m.p. 160 °C. Spectroscopic analysis:
IR(KBr) mmax/cm-1: 3218.06, 3103.27, 1721.22, 1610.80,
1501.02,1219.63, 753.52; H NMR (DMSO-d6, 300 MHz,
1
d ppm): 2.61 (s, 3H, CH3), 3.55 (s, 2H,CH2 of pyrazole),
4.90 (s, 2H, CH2), 5.13 (s, 1H, CH), 7.26–7.48 (m, 4H, Ar–
H), 10.92 (bs, 1H, NH of benzoxazine); 13C NMR
(DMSOd6, 300 MHz, d ppm): 38.88 (CH3 of pyrazolone),
66.24 (CH2–CO), 113.90 (C4 of pyrazolone), 118.40–132.61
(aromatic carbons), 166.14 ([NCO), 171.80 (C=O of pyr-
azolone), 172.14 (C=O of benzoxazinone); (M ? 1)? ion
peak at 288; Anal. Calcd. (%) for C14H13N3O4: C, 58.53; H,
4.53; N, 14.63. Found: C, 58.50; H, 4.57; N, 14.68.
2-(2-{4-[(4-Methoxyphenyl)methylidene]-3-methyl-5-oxo-
4, 5-dihydro-1H-pyrazol-1-yl} -2-oxoethyl)-2H-1,4-benz-
oxazin-3(4H)-one (4c) Yield: 57 %; m.p. 226 °C. Spec-
troscopic analysis: IR(KBr) mmax/cm-1: 3107.35, 2981.23,
1
1667.75, 1628.63, 1565.71, 1221.19, 1064.33, 771.53; H
NMR (DMSO-d6, 300 MHz, d ppm): 2.53 (s, 3H, CH3),
3.76 (s, 3H, OCH3), 4.66 (s, 2H, CH2), 5.16 (s, 1H, CH),
6.18 (s, 1H, C=CH), 7.24–7.83 (m, 8H, Ar–H), 10.42 (bs,
1H, NH of benzoxazine); 13C NMR (DMSOd6, 300 MHz,
d ppm): 38.36 (CH3 of pyrazolone), 65.72 (CH2–CO),
112.23 (C=CH), 118.20–130.15 (aromatic carbons), 165.16
([NCO), 170.89 (C=O of pyrazolone), 172.67 (C=O of
benzoxazinone); (M ? 1)? ion peak at 406; Anal. Calcd.
(%) for C22H19N3O5: C, 65.18; H, 4.69; N, 10.37. Found:
C, 65.23; H, 4.71; N, 10.43.
Synthesis of 2-{2-[4-(substituted) benzylidene-3-methyl-5-
oxo-4,5-dihydro-1H-pyrazol-1-yl]-2-oxoethyl}-2H-1,4-
benzoxazin-3(4H)-one (4a–k)
To compound 3 (0.01 mol) dissolved in glacial acetic acid
(10 ml) was added fused sodium acetate (0.015 mol) and
various aldehydes (0.01 mol) and the mixture was refluxed for
2 h. It was then cooled and poured into crushed ice. The
resulting solid was washed and recrystallized from aqueous
ethanol. The solvent system used was methanol:benzene (4:6).
2-(2-{4-[(3-Hydroxy-4-methoxyphenyl) methylidene]-3-methyl-
5-oxo-4,5-dihydro-1H-pyrazol-1-yl}-2-oxoethyl)-2H-1,4-benz-
oxazin-3(4H)-one (4d) Yield: 62 %; m.p. 203 °C. Spec-
troscopic analysis: IR(KBr) mmax/cm-1: 3110.42, 3098.45,
2978.32, 1670.67, 1629.25, 1560.43, 1219.23, 1053.64,
754.64; 1H NMR (DMSO-d6, 300 MHz, d ppm): 2.54 (s, 3H,
CH3), 3.76 (s, 3H, OCH3), 4.56 (s, 2H, CH2), 5.23 (s, 1H,
CH), 6.20 (s, 1H, C=CH), 7.16–7.63 (m, 7H, Ar–H), 9.87 (bs,
1H, NH of benzoxazine), 11.02 (s, 1H, OH); 13C NMR
(DMSOd6, 300 MHz, d ppm): 38.38 (CH3 of pyrazolone),
65.76 (CH2–CO), 112.23 (C=CH), 118.19–129.20 (aromatic
carbons), 165.66 ([NCO), 171.03 (C=O of pyrazolone),
172.78 (C=O of benzoxazinone); (M ? 1)? ion peak at 422;
Anal. Calcd. (%) for C22H19N3O6: C, 62.70; H, 4.51; N, 9.97.
Found: C, 62.66; H, 4.46; N, 9.92.
2-{2-[3-Methyl-5-oxo-4-(phenylmethylidene)-4,5-dihydro-
1H-pyrazol-1-yl]-2-oxoethyl}-2H-1,4-benzoxazin-3(4H)-
one (4a) Yield: 58 %; m.p. 184 °C. Spectroscopic anal-
ysis: IR(KBr) mmax/cm-1: 3027.09, 2965.22, 1675.14,
1629.21, 1560.01,1220.65,1035.65, 752.73; 1H NMR
(DMSO-d6, 300 MHz, d ppm): 2.58 (s, 3H, CH3), 4.85 (s,
2H, CH2), 5.12 (s, 1H, CH), 6.12 (s, 1H, C=CH), 7.18–7.73
(m, 9H, Ar–H), 9.83 (bs, 1H, NH of benzoxazine); 13C
NMR (DMSOd6, 300 MHz, d ppm): 38.68 (CH3 of pyr-
azolone), 65.56 (CH2–CO), 112.43 (C=CH), 119.43–132.05
(aromatic carbons), 166.29 ([NCO), 170.54 (C=O of pyr-
azolone), 172.34 (C=O of benzoxazinone); (M)? ion peak
at 375; Anal. Calcd. (%) for C21H17N3O4: C, 67.20; H,
4.53; N, 11.12. Found: C, 67.24; H, 4.49; N, 11.09.
2-(2-{4-[(4-Chlorophenyl)methylidene]-3-methyl-5-oxo-4,5-
dihydro-1H-pyrazol-1-yl}-2-oxoethyl)-2H-1,4-benzoxazin-
3(4H)-one (4e) Yield: 59 %; m.p. 240 °C. Spectro-
scopic analysis: IR(KBr) mmax/cm-1: 3106.70, 2934.45,
1654.31,1627.55, 1535.78, 1229.19, 1064.76, 763.21; 1H
NMR (DMSO-d6, 300 MHz, d ppm): 2.46 (s, 3H, CH3),
4.44 (s, 2H, CH2), 5.36 (s, 1H, CH), 6.13 (s, 1H, C=CH),
7.24–7.76 (m, 8H, Ar–H), 10.34 (bs, 1H, NH of benzox-
azine); 13C NMR (DMSOd6, 300 MHz, d ppm): 38.24 (CH3
of pyrazolone), 65.56 (CH2–CO), 112.56 (C=CH), 118.
20–132.15 (aromatic carbons), 165.56 ([NCO), 170.43 (C=
O of pyrazolone), 172.67 (C=O of benzoxazinone); (M)?
ion peak at 409; Anal. Calcd. (%) for C21H16N3O4Cl: C,
61.61; H, 3.91; N, 10.26. Found: C, 61.67; H, 3.96; N, 10.30.
2-(2-{4-[(4-Fluorophenyl) methylidene]-3-methyl-5-oxo-
4,5-dihydro-1H-pyrazol-1-yl}-2-oxoethyl)-2H-1,4-benzox-
azin-3(4H)-one (4b) Yield: 65 %; m.p. 222 °C. Spectro-
scopic analysis: IR(KBr) mmax/cm-1: 3003.31, 2950.32,
1680.41, 1628.42, 1556.12,1227.20,1056.35, 762.52; 1H
NMR (DMSO-d6, 300 MHz, d ppm): 2.56 (s, 3H, CH3),
4.96 (s, 2H, CH2), 5.22 (s, 1H, CH), 6.22 (s, 1H, C=CH),
7.23–7.65 (m, 8H, Ar–H), 10.23 (bs, 1H, NH of benzox-
azine); 13C NMR (DMSOd6, 300 MHz, d ppm): 38.45
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Med Chem Res (2014) 23:1320–1326
2-(2-{4-[(4-Dimethylaminophenyl)methylidene]-3-methyl-5-
oxo-4,5-dihydro-1H-pyrazol-1-yl}-2-oxoethyl)-2H-1,4-benz-
oxazin-3(4H)-one (4f) Yield: 67 %; m.p. 190 °C. Spec-
troscopic analysis: IR(KBr) mmax/cm-1: 3006.65, 2945.34,
(DMSO-d6, 300 MHz, d ppm): 2.40 (s, 3H, CH3), 4.40 (s,
2H, CH2), 5.38 (s, 1H, CH), 6.25 (s, 1H, C=CH), 7.18–7.67
(m, 8H, Ar–H), 9.42 (bs, 1H, NH of benzoxazine); 13C
NMR (DMSOd6, 300 MHz, d ppm): 38.59 (CH3 of pyr-
azolone), 65.50 (CH2–CO), 112.28 (C=CH), 119.30–130.23
(aromatic carbons), 165.68 ([NCO), 170.51 (C=O of pyr-
azolone), 172.76 (C=O of benzoxazinone); (M ? 1)? ion
peak at 421; Anal. Calcd. (%) for C21H16N4O6: C, 60.32; H,
3.81; N, 13.33. Found: C, 60.49; H, 3.88; N, 13.36.
1
1652.43, 1626.14, 1532.34, 1230.34, 1072.32, 756.90; H
NMR (DMSO-d6, 300 MHz, d ppm): 2.39 (s, 3H, CH3),
3.07 (s, 6H, N(CH3)2), 4.52 (s, 2H, CH2), 5.37 (s, 1H, CH),
6.22 (s, 1H, C=CH), 7.28–7.82 (m, 8H, Ar–H), 10.44 (bs,
1H, NH of benzoxazine); 13C NMR (DMSOd6, 300 MHz,
d ppm): 38.16 (CH3 of pyrazolone), 65.28 (CH2–CO),
112.09 (C=CH), 118.43–130.11 (aromatic carbons), 165.60
([NCO), 170.56 (C=O of pyrazolone), 172.56 (C=O of
benzoxazinone); (M ? 1)? ion peak at 419; Anal. Calcd.
(%) for C23H22N4O4: C, 66.02; H, 5.26; N, 13.39. Found:
C, 66.09; H, 5.22; N, 13.43.
2-(2-{4-[(3,4,5-Trimethoxyphenyl)methylidene]-3-methyl-
5-oxo-4,5-dihydro-1H-pyrazol-1-yl}-2-oxoethyl)-2H-1,4-
benzoxazin-3(4H)-one (4j) Yield: 60 %; m.p. 156 °C.
Spectroscopic analysis: IR(KBr) mmax/cm-1: 3036.82,
2963.56, 1656.32, 1628.72, 1542.21, 1231.09, 10782.33,
1
761.52; H NMR (DMSO-d6, 300 MHz, d ppm): 2.56 (s,
2-(2-{3-Methyl-4-[(4-methylphenyl)methylidene]-5-oxo-4,5-
dihydro-1H-pyrazol-1-yl}-2-oxoethyl)-2H-1,4-benzoxazin-3
(4H)-one (4g) Yield: 62 %; m.p. 214 °C. Spectroscopic
analysis: IR(KBr) mmax/cm-1: 3023.78, 2938.45, 1645.43,
1627.41, 1536.32, 1228.34, 1068.65, 754.56; 1H NMR
(DMSO-d6, 300 MHz, d ppm): 2.38 (s, 3H, CH3), 4.43 (s,
2H, CH2), 5.36 (s, 1H, CH), 6.18 (s, 1H, C=CH), 7.26–7.83
(m, 8H, Ar–H), 9.35 (bs, 1H, NH of benzoxazine); 13C
NMR (DMSOd6, 300 MHz, d ppm): 38.55 (CH3 of pyr-
azolone), 39.68 (CH3 of phenyl) 65.72 (CH2–CO), 112.44
(C=CH), 121.13–132.17 (aromatic carbons), 165.26
([NCO), 170.45 (C=O of pyrazolone), 172.54 (C=O of
benzoxazinone); (M)? ion peak at 389; Anal. Calcd. (%)
for C22H19N3O4: C, 67.86; H, 4.88; N, 10.79. Found: C,
67.82; H, 4.93; N, 10.74.
3H, CH3), 3.68 (s, 9H, (OCH3)3), 4.50 (s, 2H, CH2), 5.46
(s, 1H, CH), 6.32 (s, 1H, C=CH), 7.10–7.82 (m, 6H, Ar–H),
9.50(bs, 1H, NH of benzoxazine); 13C NMR (DMSOd6,
300 MHz, d ppm): 38.23 (CH3 of pyrazolone), 64.65
(CH2–CO), 114.23(C=CH), 118.34–132.01(aromatic car-
bons), 163.57 ([NCO), 171.06 (C=O of pyrazolone),
171.29 (C=O of benzoxazinone); (M)? ion peak at 465;
Anal. Calcd. (%) for C24H23N3O7: C, 61.93; H, 4.94; N,
9.03. Found: C, 61.78; H, 4.88; N, 9.10.
2-(2-{4-[(4-Bromophenyl)methylidene]-3-methyl-5-oxo-4,5-
dihydro-1H-pyrazol-1-yl}-2-oxoethyl)-2H-1,4-benzoxazin-
3(4H)-one (4k) Yield: 54 %; m.p. 203 °C. Spectroscopic
analysis: IR(KBr) mmax/cm-1: 3063.87, 2966.56, 1643.25,
1628.53, 1542.65, 1228.14, 1070.22, 756.65; 1H NMR
(DMSO-d6, 300 MHz, d ppm): 2.47 (s, 3H, CH3), 4.48 (s,
2H, CH2), 5.36 (s, 1H, CH), 6.27 (s, 1H, C=CH), 7.20–7.80
(m, 8H, Ar–H), 9.38 (bs, 1H, NH of benzoxazine); 13C
NMR (DMSOd6, 300 MHz, d ppm): 37.23(CH3 of pyraz-
olone), 65.56 (CH2–CO), 111.56(C=CH), 121.03–131.87
(aromatic carbons), 164.54 ([NCO), 168.71 (C=O of pyr-
azolone), 170.95(C=O of benzoxazinone); (M ? 1)? ion
peak at 455; Anal. Calcd. (%) for C21H16BrN3O4: C, 55.51;
H, 3.52; N, 9.25. Found: C, 55.64; H, 3.48; N, 9.32.
2-(2-{4-[(4-Hydroxyphenyl)methylidene]-3-methyl-5-oxo-4,
5-dihydro-1H-pyrazol-1-yl}-2-oxoethyl)-2H-1,4-benzoxa-
zin-3(4H)-one (4h) Yield: 58 %; m.p. 174 °C. Spectro-
scopic analysis: IR(KBr) mmax/cm-1:3112.34, 3044.84,
2956.45, 1645.43, 1629.85, 1540.87, 1230.97, 1072.33,
1
765.33; H NMR (DMSO-d6, 300 MHz, d ppm): 2.43 (s,
3H, CH3), 4.44 (s, 2H, CH2), 5.40 (s, 1H, CH), 6.23 (s, 1H,
C=CH), 7.22–7.65 (m, 8H, Ar–H), 9.44 (bs, 1H, NH of
benzoxazine), 11.03 (s, 1H, OH); 13C NMR (DMSOd6,
300 MHz, d ppm): 38.64 (CH3 of pyrazolone), 65.56
(CH2–CO), 112.33 (C=CH), 119.32–130.19 (aromatic
carbons), 165.76 ([NCO), 170.56 (C=O of pyrazolone),
172.96 (C=O of benzoxazinone); (M ? 1)? ion peak at
392; Anal. Calcd. (%) for C21H17N3O5: C, 64.45; H, 4.34;
N, 10.74. Found: C, 64.49; H, 4.38; N, 10.79.
Biological activity
Antimicrobial activity
The synthesized compounds were screened for their
in vitro antibacterial activity against Staphylococcus aur-
eus NCIM 2079, Bacillus subtilis NCIM 2063, Escherichia
coli NCIM 2118, and Pseudomonas aeruginosa NCIM
2036 and antifungal activity against Aspergillus niger
NCIM 545 and Candida albicans NCIM 3100 by twofold
serial dilution method (Linnette et al., 1985).
2-(2-{4-[(4-Nitrophenyl)methylidene]-3-methyl-5-oxo-4,5-
dihydro-1H-pyrazol-1-yl}-2-oxoethyl)-2H-1,4-benzoxazin-
3(4H)-one (4i) Yield: 62 %; m.p. 121 °C. Spectroscopic
analysis: IR(KBr) mmax/cm-1: 3056.73, 2943.34, 1644.23,
1628.34, 1535.34, 1224.72, 1087.45, 766.32; 1H NMR
123

Med Chem Res (2014) 23:1320–1326
1325
The in vitro antimicrobial activities of the compounds
were tested in Muller Hinton broth for bacteria and in
Sabourauds dextrose broth for fungal strains by twofold
serial dilution method. The test compounds were dissolved
in dimethylsulfoxide (DMSO) to obtain 1 mg/ml stock
solutions.
The final inoculum size was 105 cfu/ml for antibac-
terial assay and 102 cfu/ml for antifungal assay and
testing was performed at pH 7.4 0.2. The tubes were
incubated at 37 1 °C for bacteria and 28 1 °C for
fungi. The minimum inhibitory concentrations (MICs)
were recorded by visual observations after 24 h for
bacteria and 48 h of incubation for fungi. Ciprofloxacin
and fluconazole were used as standards, respectively, for
bacterial and fungal studies. The antimicrobial results are
given in Table 1.
scavenging activity was calculated using the formula
shown below. Ascorbic acid was used as the reference
compound. All tests and analyses were undertaken on
three replicates and the results were averaged. The
results are given in Table 2.
Scavenging activity ð%Þ ¼ f½ðAb þ AsÞ ꢁ Amꢂ=Abg
ꢃ 100 %
Ab: absorbance of 0.1 mM methanolic solution of DPPH at
517 nm, As: absorbance of 0.1 mM methanolic solution of
test compound at 517 nm, Am: absorbance of methanolic
mixture of the drug and DPPH at 517 nm.
Table 2 Antioxidant activity data of synthesized compounds (4a–k)
Compound
Scavenging activity (%)
Antioxidant activity
4a
53
82
65
69
86
35
76
78
64
65
86
98
The antioxidant potential of all the newly synthesized
compounds were done by evaluating their scavenging
effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical by
using ascorbic acid as the reference compound (Demetrios
et al., 1998).
4b
4c
4d
4e
4f
1.5 ml methanolic solution of the synthesized com-
pounds (0.2 mM) was added to 1.5 ml (0.2 mM) solu-
tion of DPPH radical in methanol to make the final
concentration of DPPH and synthesized compounds to
0.1 mM. The mixture was shaken vigorously and
allowed to stand for 30 min. After this, the absorbance
at 517 nm was determined and the percentage of
4g
4h
4i
4j
4k
Ascorbic acid
Table 1 Antimicrobial activity data of synthesized compounds (4a–k)
Compound
MIC (lg/ml)
Antibacterial activity
B. subtilis
Antifungal activity
S. aureus
E. coli
P. aeruginosa
A. niger
C. albicans
4a
62.5
31.25
31.25
31.25
125
125
15.625
15.625
31.25
250
125
62.5
31.25
15.625
62.5
31.25
62.5
31.25
62.5
62.5
31.25
31.25
–
4b
15.625
15.625
31.25
125
31.25
15.625
62.5
31.25
15.625
125
31.25
15.625
31.25
250
4c
4d
4e
31.25
15.625
250
250
4f
31.25
62.5
31.25
62.5
125
125
4g
62.5
4h
125
62.5
31.25
125
31.25
62.5
4i
62.5
31.25
15.625
62.5
125
4j
31.25
7.812
31.25
–
31.25
62.5
3.91
–
15.625
125
15.625
31.25
4k
Ciprofloxacin
Fluconazole
3.91
1.953
–
–
3.91
1.9
123

1326
Med Chem Res (2014) 23:1320–1326
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