Antioxidant and antimicrobial studies on fused-ring pyrazolones and isoxazolones

Article abstract of DOI:10.1016/j.bmc.2014.10.015

A series of 3-nitrochalcones have been synthesized enroute towards fused ring pyrazolones and isoxazolones. Base catalyzed condensation of the chalcones with ethylacetoacetate yielded cyclohexenones in good yields (74-76%). The treatment of cyclohexenones with hydrazine hydrate or hydroxylamine chloride in the presence of a base afforded the corresponding fused-ring pyrazolinones (70-78% yield) and isoxazolinones (58-66% yield). The newly synthesized compounds were characterized by IR, 1D and 2D NMR and HRMS spectral analysis. The compounds were screened for their antioxidant and antimicrobial activities. Pyrazolinones showed good DPPH radical scavenging and iron metal chelating properties. The para-hydroxy group was important for a compound to have enhanced antioxidant activity. Pyrazolinones and isoxazolinone exhibited a wider range of antimicrobial activities compared to cyclohexenones. Pyrazolinones and isoxazolinone bearing a thiophene ring were the most potent type of compounds against Bacillus subtilis and Candida albicans with MIC values of 0.313-1.25 μg/mL. Some of the synthesized compounds were found to have promising antioxidant, metal chelation and antimicrobial activities.

Full text of DOI:10.1016/j.bmc.2014.10.015

Bioorganic & Medicinal Chemistry 22 (2014) 6564–6569
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antioxidant and antimicrobial studies on fused-ring pyrazolones
and isoxazolones
b
b
b
Ofentse Mazimba a, , Kabo Wale , Daniel Loeto , Tebogo Kwape
⇑
a Botswana Institute for Technology Research and Innovation, Private Bag 0082, Gaborone, Botswana
b Department of Biological Sciences, University of Botswana, Private Bag 00704, Gaborone, Botswana
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-nitrochalcones have been synthesized enroute towards fused ring pyrazolones and isoxazol-
ones. Base catalyzed condensation of the chalcones with ethylacetoacetate yielded cyclohexenones in
good yields (74–76%). The treatment of cyclohexenones with hydrazine hydrate or hydroxylamine chlo-
ride in the presence of a base afforded the corresponding fused-ring pyrazolinones (70–78% yield) and
isoxazolinones (58–66% yield). The newly synthesized compounds were characterized by IR, 1D and
2D NMR and HRMS spectral analysis. The compounds were screened for their antioxidant and antimicro-
bial activities. Pyrazolinones showed good DPPH radical scavenging and iron metal chelating properties.
The para-hydroxy group was important for a compound to have enhanced antioxidant activity. Pyrazoli-
nones and isoxazolinone exhibited a wider range of antimicrobial activities compared to cyclohexenones.
Pyrazolinones and isoxazolinone bearing a thiophene ring were the most potent type of compounds
Received 21 August 2014
Revised 3 October 2014
Accepted 13 October 2014
Available online 22 October 2014
Keywords:
Chalcones
Cyclohexenones
Pyrazolinones
Isoxazolinones
Antioxidant
Antimicrobial
against Bacillus subtilis and Candida albicans with MIC values of 0.313–1.25 lg/mL. Some of the synthe-
sized compounds were found to have promising antioxidant, metal chelation and antimicrobial activities.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
indazol-3(3aH)-one) and isoxazol-5(4H)-one (or 4,5-dihydro-
benzo[c]isoxazol-3(3aH)-one). The preparation of the target com-
Pyrazolone and oxazolones are nitrogen containing five-mem-
bered heterocycles that forms a key pharmacophore in numerous
compounds of biological importance. A large number of these com-
pounds and their derivatives have been described in the chemistry
and pharmacological literature. Pyrazolone and oxazolones possess
pounds was achieved through the reaction of cyclohexenones
with hydrazine hydrate or hydroxyl amine. The cyclohexenones
were derived from 3-nitrochalcones. The chalcones were obtained
from the reactions of substituted acetophenones and 3-nitrobenz-
aldehyde. The nitro group was incorporated due to the biological
activities of nitro aromatic compounds, some of which have anti-
bacterial, antifungal and cytotoxic activities.10,11 The antimicrobial
and antioxidant properties of the synthesized compounds are also
presented.
similar general properties in
a wide spectrum of biological
activities such as anti-tubercular, antiviral, anti-hypertensive,
antioxidant, anti-depressant, neuroprotective, anti-diabetic, anti-
bacterial, anti-fungal, anti-inflammatory and anticancer.1–6 Anti-
pyrine (2,3-dimethyl-1-phenyl-3-pyrazolin-5-one) is a pyrazolone
used as an anti-fever and in management of pain and inflamma-
tion,7 while linezolid, Zyvox™ is an oxazolidinone used against
infections caused by multi-drug resistant Gram-positive bacteria.8
Several synthetic methods have been employed in literature for the
synthesis of pyrazolone and oxazolones. The classical synthetic
strategies involves the reaction of 1,3-diketones or b-keto esters
and hydrazine derivatives7 that yields cyclic hydrazines.9 Based
on the biological properties of pyrazolone and oxazolones and their
simplistic synthetic approach it was envisaged to be worthwhile to
prepare novel fused 1H-pyrazol-5(4H)-one (or 4,5-dihydro-2H-
2. Results and discussion
2.1. Pyrazolone and isoxazolones
The preparation of the various fused 1H-pyrazol-5(4H)-one and
isoxazol-5(4H)-one is as follows. First the required intermediary
chalcone synthons were prepared by the NaOH catalysed Aldol con-
densation of 3-nitrobenzaldehydes 1 and acetophenones 2. The
Michael addition of ethylacetoacetate to the resulting 3-nitrochal-
cones 3–8 in presence of NaOH yielded cyclohexenones 9–14
(Scheme 1).12 The basic conditions were necessary in this reaction
to turn the intermediate Michael addition product into cyclohexe-
nones through the intramolecular cyclocondensation of the
⇑
Corresponding author. Tel.: +267 3607580; fax: +267 3974677.
E-mail address: mazimbaof@yahoo.com (O. Mazimba).
http://dx.doi.org/10.1016/j.bmc.2014.10.015
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

O. Mazimba et al. / Bioorg. Med. Chem. 22 (2014) 6564–6569
6565
O
2.2.1. Antioxidant activity
O
The in vitro antioxidant activity was screened using two models
viz DPPH (1,1-diphenyl-2-picryl hydrazyl) free radical scavenging
and chelating effect on ferrous ions. The DPPH radical scavenging
activities are shown in Table 1, ascorbic acid was used as a
standard.
+
NO2
1
R
2
i
The result shows that isoxazolinones exhibited poor radical
scavenging capability while chalcones and cyclohexenones show
moderate activities. The data shows the importance of the para
hydroxy group in radical scavenging activities.14 The chalcone 7
and pyrazolinones 19 which bears a para-hydroxy group and the
standard are the only compounds that show greater than 40%
O
O
O
OEt
ii
R
R
NO2
NO2
DPPH inhibition activity at concentration 10 lg/mL after 3 h.
Figure 1 shows the DPPH activity profile for compounds bearing
a hydroxy group.
The higher activity of pyrazolinones was attributed to the pres-
ence of amino group which like hydroxy groups transfers a hydro-
gen atom to DPPH radical to form DPPH-H. Electron withdrawing
groups reduce the efficiency of DPPH radical inhibition by pyrazoli-
3
9 R= H
4
5
6
7
8
10 R= p-OMe
11 R= p-Br
12 R= o,p-diMeO
13 R= p-OH
14 R-Ar = Thiophene
Scheme 1. Reagents and conditions: (i) 20% NaOH, EtOH, rt, 24 h. (ii) Ethylace-
toacetate, 10% NaOH, EtOH, reflux, 2 h. Yields (%): 3 (76), 4 (74), 5 (75), 6 (76), 7
(70), 8 (75), 9 (79), 10 (81), 11 (82), 12 (81), 13 (66), 14 (74).
nones. The para-substituted halogen (Br) 17 (IC50 = 77.89 lg/mL
after 3 h) reduce the activity of pyrazolinone, while in the Litera-
ture para-fluoro substituted pyrazolinone exhibited poor DPPH
radical inhibition activity.13
acetoacetate methyl group and the chalcones keto-group. The
cyclocondensation of ethylacetoacetate with chalcones generate
two chiral carbon atoms. The diastereomeric cyclohexenones were
characterized12,13 and utilized as such in the preparation of target
compounds.
Treatment of cyclohexenones with hydrazine hydrate or
hydroxylamine chloride in ethanol in the presence of 10% NaOH
afforded the required fused pyrazolinones and isoxazolinones in
good yields (Scheme 2).
High electron releasing methoxy groups facilitates free radical
inhibition after long reaction times. Pyrazolinone 16 activity, IC50
increased from 86.74 to 38.38 lg/mL between 0.5 and 3 h of reac-
tion time intervals. Previous studies on isoxazole and pyrazolines
pointed out that electron withdrawing groups degrades the antiox-
idant potentials.15,16 These studies also revealed that pyrazoline (%
inhibition >50%) show higher DPPH radical scavenging activity
compared to isoxazoles (% inhibition <50%) when tested at
100 lg/mL for 20 min. The replacement of the aromatic ring in pyr-
The synthesized compounds have been characterized by IR, 1D
and 2D-NMR data and HRMS spectral analysis. The key structural
differences between the target compounds are the presence of
the NH group in pyrazolinones 15–20 and oxygen atom in isoxaz-
olinones 21–26. On the IR spectra pyrazolinones have NH band at
3666 cmꢀ1, while isoxazolinones exhibited prominent peaks at
1730 and 1700–1690 cmꢀ1 corresponding to C@O bond stretching
azolinones 15 with a thiophene ring in analogue 20 does not have a
significant influence of the DPPH activity.
The metal chelation profile of the compounds was in the
order pyrazolinones 15–20 > chalcones 3–8 > isoxazolinones
21–26 > cyclohexenones 9–14 (Table 2). Electron donating groups
enhanced metal chelation properties of pyrazolines as previously
reported by other investigators.17,18 The presence of the thiophene
ring slightly increased the metal chelation of pyrazolinone 20
frequencies. The pyrazolinones carbonyl bands (1661–1666 cmꢀ1
)
on the IR spectra rule out the enol tautomer forms.13 Generally
pyrazolinones were obtained in higher yields (70–78%) than isox-
azolinones (58–66%), while it was also observed that the hydroxy
group retarded the isoxalizolinones yields (Scheme 2).
(IC50 = 86.96 lg/mL) compared to IC50 = 93.04 lg/mL for 15. Metal
ions are associated with the production of reactive oxygen species
via the Fenton type reactions.19 The metal ion chelation activities
shown by pyrazolinones and chalcones would reduce radical gen-
eration by the metal ions and play a role in reducing metal over-
loading diseases.
2.2. Biological activity
The synthesized compounds were screened for antioxidant and
antimicrobial activities.
Table 1
DPPH assay antioxidant activity of synthesized compounds
Entry
IC50
lg/mL
O
O
N
X
30 min
165.21 0.23 112.50 0.11 15
135.27 0.10 92.46 0.12 16
170.00 0.10 122.80 0.11 17
3 h
Entry 30 min
3 h
59.50 0.10
38.38 0.12
77.89 0.09
39.67 0.09
30.23 0.12
63.78 0.08
3a
OEt
3
4
5
6
7
98.53 0.12
86.74 0.10
103.60 0.09
88.75 0.40
55.88 0.12
100.61 0.08
312.28 0.11 215.71 0.11
258.75 0.07 118.69 0.23
305.01 0.11 218.75 0.08
275.00 0.09 140.02 0.09
202.15 0.08 118.69 0.10
309.29 0.11 197.78 0.13
O
4
1
8
6″
7
1′
133.57 0.06
92.75 0.12
112.29 0.10
96.22 0.12 18
40.67 0.07 19
90.73 0.20 20
R
3″
R
5′
NO2
X = NH 15 R= H
NO2
8
9
9
X =O 21 R= H
165.21 0.19 143.60 0.11 21
196.50 0.06 98.53 0.30 22
409.10 0.14 409.10 0.12 23
10
11
12
13
14
16 R= p-OMe
22 R= p-OMe
23 R= p-Br
10
11
12
13
14
17 R= p-Br
18 R= o,p-diMeO
19 R= p-OH
24 R= o,p-diMeO
25 R= p-OH
195.65 0.07
175.00 0.04
95.43 0.09 24
86.67 0.04 25
20 R-Ar = Thiophene
26 R-Ar = Thiophene
208.95 0.25 133.66 0.12 26
Asc
Scheme 2. Reagents and conditions: NH2NH2ꢁH2O or NH2OHꢁHCl, 10% NaOH, EtOH,
reflux, 2 h. Yield (%), 15 (76), 16 (78), 17 (71), 18 (78), 19 (70), 20 (75), 21 (63), 22
(65), 23 (61), 24 (66), 25 (58), 26 (62).
37.78 0.04
25.26 0.07
Data is a mean of triplicate analysis SD n = 3. Asc = ascorbic acid.

6566
O. Mazimba et al. / Bioorg. Med. Chem. 22 (2014) 6564–6569
Pyrazolinones and isoxazolinones exhibited varied activities
compared to cyclohexenones 9–14 which indicated resistance to
antimicrobial activity. Compounds 13 and 14 are the only active
cyclohexenones against B. subtilis and C. albicans. E. coli and P. aeru-
ginosa were resistant to most compounds with activity shown by
compounds 20, 21, 25 and 26. The thiophene ring moiety was
important in enhancing the activities of compounds 20 and 26
which were activity against all tested microbes. Compounds 20
and 26 showed significant activities against B. subtilis and C. albi-
cans, MIC = 0.313–1.25 lg/mL, where the activity of pyrazolinones
20 was better than that of isoxazolinone 26. Antifungal activities
were also shown by compounds 13, 14, 17, 19, 21, 23, and 25, with
8–11 mm inhibition zones.
3. Conclusion
Figure 1. DPPH activity profiles for compounds bearing a hydroxy group. C7:
chalcone 7; ch: cyclohexenone 13; co7: isoxazolinones 25; cn7: pyrazolinones 19.
Data is a average for triplicate determinations.
We have reported the synthesis and evaluation of the antioxidant
and antimicrobial activities of chalcones, cyclohexenone and fused
isoxazolones and pyrazolones. The compounds were synthesized
in moderate to good yields, 58–78%. The fused pyrazolone 19 antiox-
idant potentials were comparable to standard ascorbic acid. The
findings are significant and show that electron donating groups
would improve pyrazoline analogs antioxidant potential. The good
chelating effects and DPPH radical scavenging activities would allow
pyrazolinones to act as both chelator and radical quencher in the
Fenton reaction assays. The present study is purely a preliminary
investigation such it is difficult to suggest any possible mechanism
for the antioxidant effects of the synthesized compounds. But still
the results of this study will provide useful information for the
design of novel molecules as antioxidant agents. Antimicrobial
screening indicated that pyrazolinones and isoxazolinones exhib-
ited a wide range of activities when compared to cyclohexenones.
Thiophene substituted pyrazolinones 20 and isoxazolinone 26 were
the most potent type of compounds against B. subtilis and C. albicans.
Table 2
Metal chelation of synthesized compounds
Entry
IC50
l
g/mL
Entry
IC50 lg/mL
3
4
5
6
7
104.76 0.24
97.32 0.30
202.56 0.18
95.13 0.22
15
16
17
18
19
20
21
22
23
24
25
26
93.04 0.30
85.58 0.41
106.36 0.30
84.39 0.36
80.91 0.70
86.96 0.33
750.45 1.07
704.31 0.35
816.48 0.17
746.04 0.22
360.98 0.35
675.67 0.31
84.09 0.35
8
9
1029.41 0.17
1028.57 0.86
1028.00 0.41
1148.57 0.35
1006.67 0.51
1014 0.33
10
11
12
13
14
EDTA
1030.56 0.45
50.98 0.17
2.3. Antimicrobial screening
4. Experimental section
The newly synthesized compounds were screened for antimi-
crobial activity against Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans
(Table 3).
4.1. Materials and methods
Melting points were determined on a Stuart melting point
apparatus SMP1 (UK) and are uncorrected. Infrared spectra were
recorded neat on a Perkin Elmer FT-IR spectrophotometer 1000.
Electron impact (EI) High resolution mass spectra (HR-MS) were
carried on GCT Premier Mass Spectrometer (Waters) ionization
energy 70 eV, at the Chemistry Department, University of Bots-
wana. 1H, 13C and 2D-NMR spectra were recorded on a Bruker
Avance DPX 300 MHz NMR spectrometer in CDCl3 (or acetone-d6)
with TMS as an internal standard at room temperature. All reac-
tions were monitored by TLC, which was carried out on 0.25 mm
layer of Merck silica gel 60 F254 pre-coated on aluminium sheets.
Laboratory grade chemicals and solvents available commercially in
high purity were used. All the prepared compounds were identified
by physical properties, IR, HRMS and NMR data. NMR data were
recorded as follow: chemical shift measured in parts per million
(ppm), multiplicity, observed coupling constant (J) in Hertz (Hz),
proton count. Multiplicities are reported as singlet (s), broad sin-
glet (br s), doublet (d), triplet (t), quartet (q), quintet (quin) and
multiplet (m). 13C NMR chemical shifts are also reported in ppm
downfield from TMS and identifiable carbons are given. Yields
reported are isolated yields unless indicated otherwise.
Table 3
Antimicrobial activity of synthesized compounds
Entry
ZI (mm) [MIC (
l
g/mL)]
B. subtilis
S aureus
E. Coli
Pa
Ca
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
G
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
12 [2.5]
12 [2.5]
13 [2.5]
11 [2.5]
13 [2.5]
9 [2.5]
—
22 [0.313]
—
12 [2.5]
—
13 [2.5]
8 [2.5]
—
12 [2.5]
—
13 [2.5]
—
11 [2.5]
9 [2.5]
—
—
—
—
—
—
—
—
—
—
—
10 [2.5]
—
11 [2.5]
23 [0.313]
10 [2.5]
—
9 [2.5]
—
10 [2.5]
15 [1.25]
11 [2.5]
—
—
—
10 [2.5]
11 [2.5]
—
—
—
—
11 [2.5]
[<0.02]
nt
—
—
—
10 [2.5]
12 [2.5]
[<0.02]
nt
10 [2.5]
13 [2.5]
[<0.02]
nt
14 [1.25]
[<0.02]
nt
4.2. General procedure for the synthesis of pyrazolone and
isoxazolones
N
[<0.02]
G: Gentamycin; N: Nystatin—: no activity; nt: not tested; Ca: C. albicans; Pa: P.
aeruginosa, ZI: zone of inhibition tested at 100 g/mL. Activity: 5–10 mm: poor; 10–
15 mm: moderate; 15–20 mm: good; 20–25 mm: very good.
The synthetic strategy towards the target compounds have pre-
viously been reported.12,13,20 Briefly, sodium hydroxide (50%,
l

O. Mazimba et al. / Bioorg. Med. Chem. 22 (2014) 6564–6569
6567
20 mL) was added drop wise to a mixture of acetophenone
(10 mmol) and 3-nitrobenzaldehydes (10 mmol) in ethanol
(30 mL) with continuous stirring. The mixture was stirred over-
night at room temperature and then it was poured into crushed
ice and acidified with dilute hydrochloric acid. The solid was
collected and recrystallized from ethanol to yield solid chalcones.
3-nitrochalcones (2 mmol) and ethyl acetoacetate (4 mmol) in
20 mL of ethanol were refluxed for 2 h in the presence of 0.5 mL
10% NaOH. The reaction mixture poured into cold water and kept
overnight at room temperature. The precipitate was filtered off
and recrystallized from ethanol to yield the required cyclohexe-
none, as an isomeric mixture. The satisfactory purity of the chal-
cones and cyclohexenones was affirmed by TLC analysis and 1H
NMR data before use in further reactions.
A mixture of the appropriate cyclohexenone (6 mmol), hydrox-
ylamine hydrochloride/hydrazine hydrate (6 mmol), 10% NaOH
solution (1 mL) and ethanol (20 mL) was refluxed for 2 h. The mix-
ture poured into cold water and solid mass was filtered, washed
with ethanol (60%). The solid was recrystallized from ethanol to
afford the corresponding pyrazolone or isoxazolones. Purity of
the synthesized compound was checked by TLC analysis.
(dd, J = 1.9, 8.1 Hz, H-500), 7.22 (dd, J = 2.1, 7.8 Hz, H-600), 7.55 (dd,
J = 1.5, 9.3 Hz, H-400), 7.59 (dd, J = 0.9, 2.1 Hz, H-200), 9.20 (s, NH);
13C NMR (75.6 MHz, acetone-d6): 34.1 (C-7), 35.7 (C-6), 40.3
(C-8), 97.6 (C-4), 120.6 (C-400), 121.1 (C-200), 121.5 (C-40), 127.2
(C-20 & 60), 131.4 (C-30 & 50), 129.6 (C-500), 133.8 (C-600), 134.9
(C-10), 139.2 (C-5), 147.8 (C-100), 147.4 (C-300), 152.5 (C-3a), 167.8
(C-1); HRMS (EI) m/z Calcd for C19H14BrN3O3 (M)+ 411.0220,
[M+2]+ 413.0198. Found 411.0219.
4.3.4. 6-(3,4-Dimethoxyphenyl)-4-(3-nitrophenyl)-4,5-dihydro-
2H-indazol-3(3aH)-one (18)
Yield = 78%; pale yellow solid; mp = 208–210 °C; FT-IR (ATR)
m
max/cmꢀ1 3269 (N–H), 1661 (C@O), 1602 (C@N); 1H NMR
:
(300 MHz, acetone-d6): 2.91 (dd, J = 5.1, 17.4 Hz, H-6a), 2.98 (dd,
J = 2.1, 16.9 Hz, H-6b), 3.21 (dd, J = 2.1, 8.4 Hz, H-8), 4.35 (dd,
J = 5.4, 8.4 Hz, H-7), 6.52 (d, J = 2.1 Hz, H-4), 6.63 (dd, J = 1.9,
8.1 Hz, H-50), 7.23 (dd, J = 1.5, 7.8 Hz, H-60), 7.55 (d, J = 1.5 Hz, H-
20), 7.76 (t, J = 7.2 Hz, H-500), 8.06 (dd, J = 2.1, 8.1 Hz, H-600), 8.16
(dd, J = 1.9, 7.5 Hz, H-400), 8.26 (d, J = 2.1 Hz, H-200), 9.61 (s, NH);
13C NMR (75.6 MHz, acetone-d6): 34.1 (C-7), 35.7 (C-6), 40.3
(C-8), 54.8 (MeO-40), 55.2 (MeO-30), 98.6 (C-4), 104.8 (C-20), 120.7
(C-50), 121.0 (C-400), 121.4 (C-60), 122.0 (C-200), 129.4 (C-500), 133.8
(C-600), 138.2 (C-10), 147.2 (C-5), 147.6 (C-300), 147.8 (C-100), 148.3
(C-40), 148.6 (C-30), 153.4 (C-3a), 161.2 (C-1); HRMS (EI) m/z Calcd
for C21H19N3O5 (M)+ 393.1325, Found 393.1358.
4.3. Characterization of the pyrazolone and isoxazolones
4.3.1. 4-(3-Nitrophenyl)-6-phenyl-4,5-dihydro-2H-indazol-
3(3aH)-one (15)
Yield = 76%; pale yellow solid; mp = 208–210 °C; FT-IR (ATR)
4.3.5. 6-(4-Hydroxyphenyl)-4-(3-nitrophenyl)-4,5-dihydro-2H-
indazol-3(3aH)-one (19)
m
max/cmꢀ1 3269 (N-H), 1658 (C@O), 1603 (C@N); 1H NMR
:
(300 MHz, acetone-d6): 3.06 (dd, J = 5.1, 17.1 Hz, H-6a), 3.36 (dd,
J = 2.1, 17.1 Hz, H-6b), 3.42 (dd, J = 2.1, 8.4 Hz, H-8), 4.50 (dd,
J = 5.1, 8.4 Hz, H-7), 6.91 (d, J = 1.8 Hz, H-4), 7.32 (t, J = 7.2 Hz,
H-40), 7.37 (dd, J = 1.5, 7.6 Hz, H-30 & 50), 7.55 (dd, J = 1.5, 8.7 Hz,
H-20 & 60), 7.58 (t, J = 8.1 Hz, H-500), 7.75 (dd, J = 2.1, 7.8 Hz, H-600),
8.09 (dd, J = 1.5, 7.2 Hz, H-400), 8.19 (dd, J = 2.1 Hz, H-200), 9.20 (s,
NH); 13C NMR (75.6 MHz, acetone-d6): 35.1 (C-7), 36.6 (C-6), 59.6
(C-8), 97.7 (C-4), 112.9 (C-400), 121.2 (C-200), 122.0 (C-40), 125.4
(C-20 & 60), 127.8 (C-30 & 50), 128.6 (C-500), 129.5 (C-600), 133.8
(C-10), 138.2 (C-5), 140.3 (C-100), 147.6 (C-300), 148.4 (C-3a), 167.7
(C-1); HRMS (EI) m/z Calcd for C19H15N3O3 (M)+ 333.1113. Found
3333.1111.
Yield = 70%; pale yellow solid; mp = 192–194 °C; FT-IR (ATR)
m
max/cmꢀ1 3266 (N–H), 1666 (C@O), 1605 (C@N); 1H NMR
:
(300 MHz, acetone-d6): 3.00 (dd, J = 3.9, 15.6 Hz, H-6a), 3.23 (dd,
J = 5.1, 15.6 Hz, H-6b), 3.29 (dd, J = 5.1, 9.3 Hz, H-8), 4.43 (dd,
J = 3.9, 9.3 Hz, H-7), 6.74 (d, J = 2.1 Hz, H-4), 6.91 (d, J = 8.7 Hz,
H-30 & 50), 7.68 (d, J = 8.7 Hz, H-20 & 60), 7.55 (t, J = 7.2 Hz, H-500),
7.71 (dd, J = 1.8, 9.0 Hz, H-600), 8.05 (dd, J = 1.2, 8.1 Hz, H-400), 8.14
(d, J = 2.1 Hz, H-200), 9.28 (s, NH); 13C NMR (75.6 MHz, acetone-
d6): 19.7 (C-7), 34.8 (C-6), 59.2 (C-8), 97.4 (C-4), 115.4 (C-30 & 50),
120.7 (C-400), 122.1 (C-200), 126.7 (C-20 & 60), 129.3 (C-500), 132.3
(C-600), 138.2 (C-10), 144.9 (C-300), 147.7 (C-5), 148.5 (C-100), 154.3
(C-3a), 159.8 (C-40), 168.0 (C-1); HRMS (EI) m/z Calcd for
C19H15N3O4 (M)+ 349.1063. Found 349.1083.
4.3.2. 6-(4-Methoxyphenyl)-4-(3-nitrophenyl)-4,5-dihydro-2H-
indazol-3(3aH)-one (16)
4.3.6. 4-(3-Nitrophenyl)-6-(thiophen-2-yl)-4,5-dihydro-2H-
indazol-3(3aH)-one (20)
Yield = 78%; pale yellow solid; mp = 142–144 °C; FT-IR (ATR)
m
max/cmꢀ1
:
3263 (N–H), 1663 (C@O), 1602 (C@N); 1H NMR
Yield = 75%; pale yellow solid; mp = 96–98 °C; FT-IR (ATR) mmax/
(300 MHz, acetone-d6): 3.07 (dd, J = 5.4, 17.1 Hz, H-6a), 3.28 (dd,
J = 2.1, 17.1 Hz, H-6b), 3.32 (dd, J = 2.4, 8.4 Hz, H-8), 4.47 (dd,
J = 5.1, 8.4 Hz, H-7), 6.82 (d, J = 1.8 Hz, H-4), 3.80 (MeO-40), 6.93
(dd, J = 1.8, 8.7 Hz, H-30 & 50), 7.51 (dd, J = 2.1, 8.7 Hz, H-20 & 60),
7.56 (t, J = 8.1 Hz, H-500), 7.76 (dd, J = 1.8, 7.2 Hz, H-600), 8.08 (dd,
J = 1.2, 9.3 Hz, H-400), 8.17 (t, J = 2.1 Hz, H-200), 9.15 (s, NH); 13C
NMR (75.6 MHz, acetone-d6): 19.7 (C-7), 36.4 (C-6), 54.7 (MeO-
40), 59.2 (C-8), 97.6 (C-4), 111.0 (C-400), 121.9 (C-200), 113.9 (C-30 &
50), 126.7 (C-20 & 60), 132.5 (C-500), 133.8 (C-600), 138.0 (C-10),
141.6 (C-5), 148.4 (C-100), 147.7 (C-300), 158.2 (C-3a), 167.9 (C-1);
cmꢀ1: 3272 (N–H), 1654 (C@O), 1606 (C@N); 1H NMR (300 MHz,
acetone-d6): 3.06 (dd, J = 3.6, 16.1 Hz, H-6a), 3.18 (dd, J = 3.6,
16.1 Hz, H-6b), 3.28 (dd, J = 2.4, 8.4 Hz, H-8), 4.48 (dd, J = 3.6,
8.4 Hz, H-7), 6.89 (d, J = 2.1 Hz, H-4), 7.03 (dd, J = 1.5, 2.4 Hz,
H-50), 7.26 (dd, J = 2.4, 3.6 Hz, H-40), 7.38 (dd, J = 1.5, 3.6 Hz, H-30),
7.57 (t, J = 8.1 Hz, H-500), 7.72 (dd, J = 2.1, 7.8 Hz, H-600), 8.05 (dd,
J = 1.9, 8.1 Hz, H-400), 8.13 (dd, J = 2.1 Hz, H-200), 9.18 (s, NH); 13C
NMR (75.6 MHz, acetone-d6): 19.7 (C-7), 34.9 (C-6), 59.4 (C-8),
98.0 (C-4), 121.1 (C-400), 122.2 (C-200), 129.5 (C-500), 129.7 (C-40 &
50), 127.9 (C-30), 133.8 (C-600), 135.6 (C-10), 144.4 (C-300), 147.5
(C-100), 148.2 (C-5), 150.9 (C-3a), 167.8 (C-1); HRMS (EI) m/z Calcd
for C17H13N3O3S (M)+ 339.0678. Found 339.0618.
HRMS (EI) m/z Calcd for
363.1253.
C
20H17N3O4 (M)+ 363.1219. Found
4.3.3. 6-(4-Bromophenyl)-4-(3-nitrophenyl)-4,5-dihydro-2H-
indazol-3(3aH)-one (17)
4.3.7. 4,5-Dihydro-4-(3-nitrophenyl)-6-phenylbenzo[c]isoxazol-
3(3aH)-one (21)
Yield = 71%; pale yellow solid; mp = 192–194 °C; FT-IR (ATR)
Yield = 63%; pale yellow solid; mp = 76–78 °C; FT-IR (ATR) mmax/
m
max/cmꢀ1
:
3256 (N–H), 1663 (C@O), 1599 (C@N); 1H NMR
cmꢀ1: 1665 (C@O), 1602 (C@N); 1H NMR (300 MHz, MeOD): 2.43
(m, H-6a), 2.70 (m, H-6b), 3.24 (m, H-8), 3.27 (m, H-7), 6.59 (s,
H-4), 7.31–7.32 (m, H-20, 30,40,50 & 60), 7.54 (t, J = 8.4 Hz, H-500),
7.71 (dd, J = 2.1, 8.4 Hz, H-600), 8.06 (br d, J = 1.5 Hz, H-400), 8.15
(br s, H-200); 13C NMR (75.6 MHz, MeOD): 28.2 (C-7), 34.5 (C-6),
(300 MHz, acetone-d6): 2.49 (dd, J = 4.2, 16.9 Hz, H-6a), 2.76
(dd, J = 2.4, 16.9 Hz, H-6b), 2.82 (dd, J = 2.4, 8.7 Hz, H-8), 3.95
(dd, J = 4.2, 8.4 Hz, H-7), 6.41 (d, J = 2.1 Hz, H-4), 7.06
(d, J = 7.5 Hz, H-20 & 60), 7.15 (d, J = 7.2 Hz, H-30 & 50), 7.10

6568
O. Mazimba et al. / Bioorg. Med. Chem. 22 (2014) 6564–6569
39.9 (C-8), 97.2 (C-4), 112.3 (C-400), 121.5 (C-200), 125.6 (C-20 & 60),
127.9 (C-40), 128.4 (C-30 & 50), 129.5 (C-500), 133.3 (C-600), 139.7
(C-10), 143.7 (C-300), 146.9 (C-5), 148.4 (C-100), 156.8 (C-3a), 166.4
(C-1); HRMS (EI) m/z Calcd for C19H14N2O4 (M)+ 334.0954. Found
334.0987.
HRMS (EI) m/z Calcd for C19H14N2O5 (M)+ 350.0903. Found
350.0904.
4.3.12. 4,5-Dihydro-4-(3-nitrophenyl)-6-(thiophen-2-yl)benzo-
[c]isoxazol-3(3aH)-one (26)
Yield = 62%; brown solid; mp = 102–104 °C; FT-IR (ATR) mmax
/
4.3.8. 4,5-Dihydro-6-(4-methoxyphenyl)-4-(3-nitrophenyl)-
benzo[c]isoxazol-3(3aH)-one (22)
cmꢀ1: 1664 (C@O), 1603 (C@N); 1H NMR (300 MHz, acetone-d6):
2.41 (m, H-6a), 2.86 (m, H-6b), 3.21 (m, H-8), 3.83 (m, H-7), 6.47
(s, H-4), 6.92 (dd, J = 1.5, 5.1 Hz, H-50), 7.19 (dd, J = 2.7, 6.9 Hz, H-
40), 7.28 (dd, J = 1.8, 5.4 Hz, H-30), 7.55 (t, J = 7.2 Hz, H-500), 7.77
(dd, J = 3.9, 7.8 Hz, H-600), 8.03 (dd, J = 1.8, 9.3 Hz, H-400), 8.18 (dd,
J = 1.8 Hz, H-200); 13C NMR (75.6 MHz, acetone-d6): 20.7 (C-7),
34.5 (C-6), 43.3 (C-8), 119.3 (C-4), 121.6 (C-400), 122.6 (C-200),
125.4 (C-50), 127.8 (C-40), 128.1 (C-30), 129.8 (C-500), 133.7 (C-600),
136.7 (C-10), 144.2 (C-300), 148.6 (C-100), 152.4 (C-5), 154.7 (C-3a),
171.1 (C-1); HRMS (EI) m/z Calcd for C17H12N2O4S (M)+ 340.0518.
Found 340.0518.
Yield = 65%; Brown solid; mp = 70–72 °C; FT-IR (ATR) mmax
/
cmꢀ1: 1664 (C@O), 1600 (C@N); 1H NMR (300 MHz, acetone-d6):
2.55 (dd, J = 4.2, 16.8 Hz, H-6a), 2,78 (dd, J = 4.2, 16.8 Hz, H-6b),
2.95 (dd, J = 3.9, 8.4 Hz, H-8), 3.48 (dd, J = 3.6, 8.4 Hz, H-7), 6.63
(d, J = 1.5 Hz, H-4), 3.88 (s, MeO-40), 6.93 (d, J = 8.7 Hz, H-30 & 50),
7.47 (d, J = 8.9 Hz, H-20 & 60), 7.56 (dd, J = 2.1, 8.9 Hz, H-500), 7.68
(dd, J = 2.1, 7.8 Hz, H-600), 8.17 (dd, J = 1.2, 8.9 Hz, H-400), 8.25 (d,
J = 2.1 Hz, H-200); 13C NMR (75.6 MHz, acetone-d6): 34.4 (C-7),
38.9 (C-6), 40.0 (C-8), 54.7 (MeO-40), 111.3 (C-4), 113.9 (C-30 &
50), 119.4 (C-400), 121.5 (C-200), 126.6 (C-20 & 60), 129.8 (C-500),
132.3 (C-10), 133.8 (C-600), 145.0 (C-5), 147.3 (C-300), 148.6 (C-100),
155.2 (C-3a), 160.4 (C-1); HRMS (EI) m/z Calcd for C20H16N2O5
(M)+ 364.1059. Found 364.1059.
4.4. Biological activity assays
4.4.1. DPPH radical scavenging assay
DPPH free radical scavenging activity was determined by the
method described by Mazimba et al. (2011).21 Briefly, DPPH solu-
tion (1.5 mL, 0.51 mM in methanol) was incubated with 1.5 mL
of varying concentration of the compounds (100, 50, 25
4.3.9. 6-(4-Bromophenyl)-4,5-dihydro-4-(3-nitrophenyl)benzo-
[c]isoxazol-3(3aH)-one (23)
Yield = 61%; white solid; mp = 100–102 °C; FT-IR (ATR) mmax
/
cmꢀ1: 1662 (C@O), 1609 (C@N); 1H NMR (300 MHz, acetone-d6):
2.40 (dd, J = 3.9, 14.4 Hz, H-6a), 2.57 (dd, J = 2.4, 14.4 Hz, H-6b),
2.90 (dd, J = 5.7, 9.6 Hz, H-8), 3.24 (dd, J = 6.6, 9.6 Hz, H-7), 6.54
(d, J = 2.1 Hz, H-4), 7.45 (dd, J = 0.9, 7.9 Hz, H-20 & 60), 7.54 (d,
J = 7.8 Hz, H-30 & 50), 7.78 (t, J = 7.5 Hz, H-500), 8.02 (dd, J = 2.4,
7.2 Hz, H-600), 8.17 (dd, J = 1.9, 7.8 Hz, H-400), 8.19 (dd, J = 1.9,
2.1 Hz, H-200); 13C NMR (75.6 MHz, acetone-d6): 34.2 (C-7), 38.8
(C-6), 48.8 (C-8), 110.0 (C-4), 121.6 (C-400), 122.0 (C-200), 127.3 (C-
20 & 60), 127,8 (C-40), 131.5 (C-30 & 50), 129.8 (C-500), 133.7 (C-600),
139.3 (C-10), 141.3 (C-5), 144.4 (C-300), 147.3 (C-100), 154.9 (C-3a),
and10 lg/mL). The absorbance was read at 517 nm against a meth-
anol blank after 0.5 and 3 h incubation time. Ascorbic acid was
used as a reference compound. The percentage of DPPH radical dis-
colouration was calculated using the equation: % DPPH inhibition
[Ao ꢀ As)/Ao] ꢂ 100. Where Ao is the absorbance of the control
and As is the absorbance of the test compound. Measurements
were carried out in triplicates.
4.4.2. Metal chelating assay
The chelating effect on ferrous ions of the prepared extracts was
estimated using reported methods18,22 with slight modifications.
The principle of this assay is based on the O-phenanthroline–Fe2+
complex formation and its disruption in the presence of chelating
agents. The reaction mixture containing O-phenanthroline (1 mL,
0.05% in methanol), FeCl2 (2 mL, 0.1%) and 2 mL sample of various
166.0 (C-1); HRMS (EI) m/z Calcd for
C
19H13BrN2O4 (M)+
412.0059, [M+2]+ 414.0039. Found 412.0087.
4.3.10. 4,5-Dihydro-6-(3,4-dimethoxyphenyl)-4-(3-nitrophenyl)-
benzo[c]isoxazol-3(3aH)-one (24)
Yield = 66%; brown paste; FT-IR (ATR)
m
max/cmꢀ1: 1664 (C@O),
concentrations (100, 200, 500 and 1000 lg/mL) was incubated at
1604 (C@N); 1H NMR (300 MHz, acetone-d6): 2.58 (dd, J = 4.4,
16.2 Hz, H-6a), 2.82 (dd, J = 3.6, 16.2 Hz, H-6b), 2.89 (dd, J = 2.1,
8.4 Hz, H-8), 3.29 (dd, J = 2.1, 8.4 Hz, H-7), 6.34 (d, J = 2.1 Hz, H-
4), 6.52 (d, J = 8.4 Hz, H-50), 7.67 (td, J = 2.4, 8.2 Hz, H-60), 7.18 (d,
J = 2.1 Hz, H-20), 7.87 (dd, J = 1.5, 8.2 Hz, H-500), 8.14 (dd, J = 1.5,
8.4 Hz, H-600), 8.27 (dd, J = 2.1, 8.4 Hz, H-400), 8.29 (d, J = 2.1 Hz,
H-200); 13C NMR (75.6 MHz, acetone-d6): 19.7 (C-7), 36.6 (C-6),
40.1 (C-8), 54.8 (MeO-40), 55.1 (MeO-30), 98.7 (C-4), 104.9 (C-20),
111.0 (C-50), 121.4 (C-400), 121.9 (C-60), 123.4 (C-200), 129.4 (C-10),
129.7 (C-500), 133.8 (C-600), 142.9 (C-300), 146.4 (C-5), 147.5 (C-30),
148.5 (C-40), 152.5 (C-100), 154.9 (C-3a), 161.0 (C-1); HRMS (EI)
m/z Calcd for C21H18N2O6 (M)+ 394.1165, Found 394.1198.
room temperature for 20 min and the absorbance was measured
at 510 nm. EDTA was used as a standard metal chelator. The exper-
iment was performed in triplicates. The ratio of inhibition of com-
plex formation was calculated using the equation: Chelation
activity (%) = [Acontrol ꢀ Asample/Acontrol] ꢂ 100. Plots of % Chelation
activity vs concentration were made to determine IC50 using
Microsoft office 2010 Excel™.
4.4.3. Antimicrobial screening
Initial screening of the test samples were done to check for anti-
microbial activity. Extract stock-solution were prepared by placing
10 mg of plant extract into 1 mL of EtOH and diluted with 1:1 (v/v)
Muller-Hinton medium. Agar well diffusion method was used for
4.3.11. 4,5-Dihydro-6-(4-hydroxyphenyl)-4-(3-nitrophenyl)-
benzo[c]isoxazol-3(3aH)-one (25)
initial screening at concentrations of 100 l
g/mL.23,24 Minimum
inhibitory concentrations (MIC) were determined for samples
which showed activity.23,24 Gram-positive organisms (Staphylococ-
cus aureus ATCC 25923, Bacillus subtilis NCIB 3610), Gram-negative
organisms (Pseudomonas aeruginosa NCIMB 8295, Escherichia coli
ATCC 8739) and a fungal yeast Candida albicans were obtained
and confirmed at the Microbiology Laboratory, Department of Bio-
logical Sciences, University of Botswana. The microbial cultures
were maintained on Mueller-Hinton medium (Oxoid, UK) and
eighteen hour old cultures were used in the bioassay. Nystatin
(Sigma–Aldrich, Czech Republic) and Gentamycin (Sigma–Aldrich,
Czech Republic) were used as standards.
Yield = 58%; pale yellow solid; mp = 108–110 °C; FT-IR (ATR)
m
max/cmꢀ1: 1662 (C@O), 1607 (C@N); 1H NMR (300 MHz, ace-
tone-d6): 2.16 (m, H-6a), 2.27 (m, H-6b), 3.24 (dd, J = 6.6, 9.4 Hz,
H-8), 3.96 (dd, J = 6.9, 9.4 Hz, H-7), 7.03 (d, J = 8.7 Hz, H-30 & 50),
7.41 (s, H-4), 7.83 (d, J = 8.7 Hz, H-20 & 60), 7.88 (t, J = 8.1 Hz, H-
500), 8.40 (dd, J = 2.4, 7.2 Hz, H-400 & 600), 8.75 (t, J = 1.8 Hz, H-200);
13C NMR (75.6 MHz, acetone-d6): 19.6 (C-7), 34.6 (C-6), 38.9 (C-
8), 96.1 (C-4), 115.3 (C-30 & 50), 118.9 (C-400), 121.2 (C-200), 127.5
(C-20 & 60), 130.5 (C-500), 131.2 (C-10), 132.5 (C-600), 144.3 (C-300),
151.1 (C-5), 153.0 (C-100), 153.7 (C-3a), 159.7 (C-40), 161.1 (C-1);

O. Mazimba et al. / Bioorg. Med. Chem. 22 (2014) 6564–6569
10. Ju, K.-S.; Parales, R. E. Microbiol. Mol. Biol. R. 2010, 74, 250.
6569
Acknowledgements
11. Al-Zereini, W.; Schuhmann, I.; Laatsch, H.; Helmke, E.; Anke, H. J. Antibiot. 2007,
60, 301.
O.M. would like to thank the Chemistry Department, University
of Botswana for availing their Laboratories used for the synthetic
work and Spectrometers used in structural determination and con-
firmations, and during antioxidant assays.
12. Sreevidya, T. V.; Narayana, B.; Yathirajan, H. S. Cent. Eur. J. Chem. 2010, 8, 174.
13. Shakil, N. A.; Singh, K. M.; Sathiyendiran, M.; Kumar, J.; Padaria, C. J. Eur. Med.
Chem. 2013, 59, 120.
14. Murti, Y.; Goswami, A.; Mishra, P. Int. J. PharmTech Res. 2013, 5, 811.
15. Madhavi, K.; Bharathi, K.; Prasad, K. V. S. R. G. Res. J. Pharm. Biol. Chem. Sci. 2010,
1, 1073.
16. Kumar, A.; Varadaraj, B. G.; Singla, R. K. Bull. Fac. Pharm. Cairo Univ. 2013, 51,
167.
References and notes
17. Perjési, P.; Rozmer, Z. Open Med. Chem. J. 2011, 5, 61.
18. Rajendran, M.; Devi, M. T. J. Chem. Biol. Phys. Sci. Sec. A 2014, 4, 038.
19. Pan, Y. M.; Wang, K.; Huang, S. Q.; Wang, H.; Mu, X.; He, C.; Ji, X.; Zhang, J.;
Huang, F. J. Agric. Food Chem. 2008, 106, 1264.
20. Padmavathi, V.; Sharmila, K.; Balaiah, A.; Reddy, A. S.; Reddy, D. B. Synth.
Commun. 2001, 31, 2119.
21. Mazimba, O.; Majinda, R. R. T.; Modibedi, C.; Masesane, I. B.; Cencic, A.;
Chingwaru, W. Bioorg. Med. Chem. 2011, 19, 5225.
22. Kumar, D. S.; Muthu, A. K.; Smith, A. A.; Manavalan, R. Int. J. PharmTech Res.
2010, 2, 2063.
1. Kumar, S.; Bawa, S.; Drabu, S.; Rajiv Kumar, R.; Gupta, H. Recent Pat. AntiInfect.
Drug Discovery 2009, 4, 154.
2. Padmaja, A.; Rajasekhar, C.; Muralikrishna, A.; Padmavathi, V. Eur. J. Med. Chem.
2011, 46, 5034.
3. Pattan, S. R.; Rabara, P. A.; Pattan, J. S.; Bukitagar, A. A.; Wakale, V. S.; Musmade,
D. S. Indian J. Chem. 2009, 48B, 1453.
4. Shankarananth, V.; Ranganayakulu, D.; Sridhar, C.; Rajasekhar, K. K. IJPA 2012,
1, 47.
5. Shriram, P.; Kumar, D.; Sudhir, I.; Asif, K. UJPB 2013, 01, 31.
6. Kumar, H.; Jain, S. IJPSR 2013, 4, 453.
7. Subramanyam, S.; Raja, S.; Jayaveera, K. N.; Kumar, S. K. Int. J. Innov. Pharm. Res.
2012, 3, 187.
8. Leach, K. L.; Brickner, S. J.; Noe, M. C.; Miller, P. F. Acad. Sci. 2011, 1222, 49.
9. Kelekci, N. G.; Koyunoglu, S.; Yabanoglu, S.; Yelekci, K.; Ozgen, O.; Ucar, G.; Erol,
K.; Kendi, E.; Yesilada, A. Bioorg. Med. Chem. 2008, 22, 23.
23. Anyanwu, C. U.; Nwosu, G. C. J. Med. Plants Res. 2014, 8, 436.
24. Prabuseenivasan, S.; Jayakumar, M.; Ignacimuthu, S. BMC Complement. Altern.
Med. 2006, 6, 39.