Curcuminoid-derived 3,5-bis(styryl)isoxazoles - Mechanochemical synthesis and antioxidant activity

Article abstract of DOI:10.1007/s12039-016-1119-8

Mechanochemical synthesis of curcuminoid-derived 3,5-bis(styryl)isoxazoles and their antioxidant activities are reported. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s12039-016-1119-8

c
J. Chem. Sci. ꢀ Indian Academy of Sciences.
DOI 10.1007/s12039-016-1119-8
Curcuminoid-derived 3,5-bis(styryl)isoxazoles - Mechanochemical
synthesis and antioxidant activity
DAISY R SHERIN∗ and KALLIKAT N RAJASEKHARAN
Department of Chemistry, University of Kerala, Kariavattom, Thiruvananthapuram 695 581,
Kerala, India
e-mail: sherindr84@gmail.com
MS received 25 April 2016; revised 26 May 2016; accepted 28 May 2016
Abstract. Mechanochemical synthesis of curcuminoid-derived 3,5-bis(styryl)isoxazoles and their antioxi-
dant activities are reported.
Keywords. Curcuminoids; 3,5-bis(styryl)isoxazoles; antioxidant
1. Introduction
2. Experimental
Curcumin, the active ingredient of turmeric, is a diaryl- 2.1 General
heptanoid natural product that is endowed with much
bioactivity;1–3 yet it has limited applications as a
drug due to its low bioavailability.4 Structural modifi-
cation of curcumin has been explored much as a strategy
to circumvent this inadequacy. Among such structurally
modified curcumin derivatives, the curcumin-derived
pyrazoles and isoxazoles have been investigated exten-
sively.5–13 With our long standing interest in the syn-
thesis and bioactivity studies on curcumin, its analogs
and derivatives,14–18 we have also investigated the struc-
tural modification approach.12,19 The natural curcumin
is a mixture of three compounds, namely 1,7-bis(4-hy-
droxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione (1)
(curcumin I), its demethoxy (curcumin II) and bisdeme-
thoxy (curcumin III) derivatives. Recently, we have re-
ported the rapid, mechanochemical synthesis and anti-
oxidant assay of 3,5-bis(4-hydroxy-3-methoxystyryl)
pyrazole (2a), its 1-aryl derivatives (2b) and 3,5-bis(4-
hydroxy-3-methoxystyryl)isoxazole (2c) prepared from
curcumin I (1).19 In continuation, we have now extended
the above method for the synthesis of 3,5-bis(styryl)
isoxazoles (4a-g) from other, differently substituted
curcuminoids (3a-g), with a view to understand how the
substituents on the aryl ring would affect the antiox-
idant activity, in the light of the continued interest
on the antioxidant activity of curcumin, its derivatives
and analogs.20–22 The thermal methods that have been
reported earlier for the synthesis of curcumin-derived
isoxazoles take, in general, 6-40 h for completion.6–9,13
Reagents and solvents used were of analytical grade.
Thin Layer Chromatography (TLC) and Preparative
Thin Layer Chromatography (PTLC) were performed
on glass plates (20 × 75 mm and 20 × 20 cm, respec-
tively) coated with TLC grade silica gel-G (E. Merck,
India). The spots or bands were visualized directly or
under UV light. The C, H, N analyses were carried out
on Vario EL Elementar analyzer. 1H and 13C NMR were
recorded on Bruker Topspin and Bruker AV, 500 MHz
1
(500 MHz for H and 125 MHz for 13C NMR), Bruker
1
Avance III, 400 MHz (400 MHz for H and 100 MHz
for 13C NMR) at room temperature. Chemical shifts are
given in ppm relative to TMS as an internal standard.
HRMS were recorded on JEOL JMS 600H mass spec-
trometer. The absorbance values were recorded on a
Jasco V-550 spectrophotometer. The curcuminoids (3a-
g) were obtained from suitably substituted benzalde-
hydes by adopting the method reported earlier by us
and their identities were established by spectral charac-
terization and comparison with samples available with
us.12,14,23
2.2 General procedure for the synthesis of 3,5-bis
(styryl)isoxazoles (4a-g)
A mixture of curcuminoid 3a-g (0.1 mmol), hydroxy-
lamine hydrochloride (0.1 mmol) and glacial acetic acid
(0.1 mL) were vigorously ground in an agate mortar
for 1–2 mins. The crude product, obtained in a powder
form, was then purified by PTLC (CHCl3: EtOAc, 3:2)
to afford the 3,5-bis(styryl)isoxazoles (4a-g).
∗For correspondence

Daisy R Sherin and Kallikat N Rajasekharan
2.2a 3,5-Bis(4-hydroxystyryl)isoxazole (4a): Yield 87%; 2.2e 3,5-Bis(3,4-dimethoxystyryl)isoxazole (4e): Yield
1
1
M.p. 198◦C; H NMR (500 MHz, Acetone-d6): δ 5.62 82%; M.p. 16◦C; H NMR (500 MHz, Acetone-d6): δ
(s, 1H, 4-H), 5.94 (s, 2H, ArOH), 6.64 (d, 2H, 2,6-H, 3.84 (s, 12H, OCH3), 5.64 (s, 1H, 4-H), 6.64 (d, 2H,
J = 16.0 Hz), 6.86 (d, 2H, ArH, J = 8.0 Hz), 7.14– 2,6-H, J = 16.0 Hz), 6.86 (d, 2H, ArH, J = 8.0 Hz),
7.15 (dd, 4H, ArH), 7.37 (s, 2H, ArH), 7.52 (d, 2H, 7.14-7.15 (dd, 2H, ArH), 7.36 (s, 2H, ArH), 7.52 (d, 2H,
1,7-H, J = 16.0 Hz); 13C NMR (100 MHz, DMSO- 1,7-H, J = 16.0 Hz); 13C NMR (100 MHz, DMSO-d6):
d6): δ 168.6, 162.1, 148.1, 147.9, 147.8, 136.3, 134.7, δ 168.9, 162.1, 148.3, 147.9, 147.1, 136.7, 134.3, 127.3,
127.3, 126.9, 121.4, 121.2, 115.6, 115.5, 112.6, 110.4, 126.8, 121.5, 121.2, 115.6, 115.4, 112.6, 110.6, 110.3,
110.3, 110.1, 97.8; HRMS (ESI): Calcd for C19H15NO3, 110.1, 55.7, 55.6HRMS (ESI): Calcd for C23H23NO5,
(M+H): 306.11302; Found: 306.11308; Anal. Calcd. (M+Na): 416.14739; Found: 416.14736; Anal. Calcd.
(%) For C19H15NO3: C, 74.74; H, 4.95; N, 4.59; Found (%) For C23H23NO5: C, 70.21; H, 5.89; N, 3.56; Found
(%): C, 74.53; H, 5.17; N, 4.69.
(%): C, 70.06; H, 6.13; N, 3.83.
2.2b 3,5-Bis(3-hydroxy-4-methoxystyryl)isoxazole (4b):
Yield 85%; M.p. 168◦C; 1H NMR (500 MHz, Acetone-
d6): δ 3.82 (s, 6H, OCH3), 5.60 (s, 1H, 4-H), 5.92 (s, 2H,
ArOH), 6.64 (d, 2H, 2,6-H, J = 16.0 Hz), 6.86 (d, 2H,
ArH, J = 8.0 Hz), 7.14-7.16 (dd, 2H, ArH), 7.36 (s,
2H, ArH), 7.52 (d, 2H, 1,7-H, J = 16.0 Hz); 13C NMR
(100 MHz, DMSO-d6): δ 168.3, 162.1, 148.1, 147.9,
147.9, 147.8, 136.3, 134.7, 127.3, 126.9, 121.6, 121.2,
115.6, 115.5, 112.6, 110.4, 110.3, 110.1, 97.8, 55.7,
55.6 HRMS (ESI): Calcd. for C21H19NO5, (M+Na):
388.11609; Found: 388.11608; Anal. Calcd. (%) For
C21H19NO5: C, 69.03; H, 5.24; N, 3.83; Found (%): C,
68.98; H, 5.28; N, 3.96.
2.2f 3,5-Bis(3,4-methylenedioxystyryl)isoxazole (4f):
Yield 82%; M.p. 152◦C; 1H NMR (500 MHz, Acetone-
d6): δ 5.55 (s, 1H, 4-H), 5.99 (s, 4H, CH2), 6.59 (d,
2H, 2,6-H, J = 16.0 Hz), 6.90 (s, 2H, ArH), 7.11
(m, 4H, ArH), 7.52 (d, 2H, 1,7-H, J = 15.5 Hz);
13C NMR (125 MHz, Acetone-d6): δ 150.1, 148.6,
141.8, 128.1, 123.6, 122.3, 116.2, 111.1, 106.5, 92.6,
91.5; HRMS (ESI): Calcd for C21H15NO5, (M+H):
362.10285; Found: 362.10286; Anal. Calcd. (%) For
C21H15NO5: C, 69.80; H, 4.18; N, 3.88; Found (%): C,
69.98; H, 4.39; N, 3.52.
2.2g 3,5-Bis(styryl)isoxazole (4g): Yield 83%; M.p.
1
128◦C; H NMR (500 MHz, Acetone-d6): δ 5.54 (s,
2.2c 3,5-Bis(2-hydroxystyryl)isoxazole (4c): Yield 84%;
1H, 4-H), 6.59 (d, 2H, 2,6-H, J = 16.0 Hz), 6.90
(s, 2H, ArH), 7.11 (m, 8H, ArH), 7.52 (d, 2H, 1,7-
H, J = 15.5 Hz); 13C NMR (125 MHz, Acetone-
d6): δ 141.1, 133.0, 128.0, 123.4, 122.3, 116.1, 111.6,
108.1; HRMS (ESI): Calcd for C19H15NO, (M+Na):
296.10513; Found: 296.10511; Anal. Calcd. (%) For
C19H15NO: C, 83.49; H, 5.53; N, 5.12; Found (%): C,
83.67; H, 5.75; N, 5.23.
1
M.p. 152◦C; H NMR (500 MHz, Acetone-d6): δ 5.64
(s, 1H, 4-H), 5.97 (s, 2H, ArOH), 6.64 (d, 2H, 2,6-H,
J = 16.0 Hz), 6.85 (d, 2H, ArH, J = 8.0 Hz), 7.14-
7.15 (dd, 4H, ArH), 7.37 (s, 2H, ArH), 7.52 (d, 2H, 1,7-
H, J = 16.0 Hz); 13C NMR (100 MHz, DMSO-d6): δ
168.9, 162.1, 148.3, 147.9, 147.8, 147.6, 136.1, 134.7,
127.6, 126.9, 121.4, 121.2, 115.5, 115.3, 112.8, 110.4,
110.3, 110.1, 97.6; HRMS (ESI): Calcd for C19H15NO3,
(M+H): 306.11302; Found: 306.11306; Anal. Calcd.
(%) For C19H15NO3: C, 74.74; H, 4.95; N, 4.59; Found
(%): C, 74.51; H, 5.12; N, 4.66.
2.3 Antioxidant capacity assays
2.3a DPPH assay: Methanolic solutions of 3,5-bis
(styryl)isoxazoles (4a-g), at six different concentrations
2.2d 3,5-Bis(4-methoxystyryl)isoxazole (4d): Yield 85%; (0.05, 0.1, 0.25, 0.5, 0.75 and 1 mM), were prepa-
1
M.p. 143◦C; H NMR (500 MHz, Acetone-d6): δ 3.84 red. These test solutions (0.04 mL) containing bis(styr-
(s, 6H, OCH3), 5.64 (s, 1H, 4-H), 6.64 (d, 2H, 2,6-H, yl)isoxazoles (4a-g) were added to DPPH solution in
J = 16.0 Hz), 6.86 (d, 2H, ArH, J = 8.0 Hz), 7.14- methanol (10−5 M; 2.8 mL). The absorbance was moni-
7.15 (dd, 4H, ArH), 7.36 (s, 2H, ArH), 7.52 (d, 2H, tored at 517 nm, after the reaction mixture was allowed
1,7-H, J = 16.0 Hz); 13C NMR (100 MHz, DMSO- to stand for 30 min. From the absorbance values moni-
d6): δ 168.7, 162. 9, 148.1, 147.9, 147.8, 136.3, 134.3, tored, the percentage inhibition was calculated and the
127.3, 126.1, 121.6, 121.2, 115.6, 115.4, 112.6, 110.5, result is expressed in terms of EC50. The percentage of
110.3, 110.1, 55.71, 55.67; HRMS (ESI): Calcd for inhibition of DPPH by the antioxidant at a particular
C21H19NO3, (M+Na): 356.12626; Found: 356.12628; time was calculated as % inhibition of DPPH = [(Abc−
Anal. Calcd. (%) For C21H19NO3: C, 75.66; H, 5.74; N, Abs)/Abc] × 100, where Abc is the absorbance of the
4.20; Found (%): C, 75.52; H, 5.79; N, 4.06.
control and Abs is the absorbance of the sample.

3,5-Bis(styryl)isoxazoles
2.3b Ferric ion reducing antioxidant power (FRAP) and control, respectively. At and Aot are the absorbance
assay: Freshly prepared FRAP reagent was used values of the sample and control after incubation time.
for the analysis. It was obtained by mixing acetate
buffer (300 mM each of acetic acid and sodium
acetate), tripyridyltriazine (10 mM in 40 mM aqueous
3. Results and Discussion
hydrochloric acid solution) and FeCl3.6H2O (20 mM)
aqueous solutions in the ratio 10:1:1. Methanolic solu-
tion of FeSO4.6H2O was prepared at different con-
centrations (0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 1.5 and
2.0 mM). The solutions of bis(styryl)isoxazoles (4a-
g) were also prepared in methanol (0.5 mM) and after
adding these (0.04 mL) to the FRAP reagent (3 mL), the
reaction mixtures were incubated at 37◦C for 30 min.
The absorbance was measured at 596 nm and the results
were expressed as micro molar Fe2+ equivalents.
3.1 Synthesis
The precursor curcuminoids (3a-g) were obtained by
microwave assisted synthetic protocol23 by the modi-
fication of a conventional thermal method developed
earlier by our group.14 Sui et al., seemingly in the first
report on the synthesis of an isoxazole derivative (2c) of
a curcuminoid, had reacted curcumin I (1) with hydrox-
ylamine hydrochloride in ethanol under reflux for
16 h.13 The methods subsequently reported for this reac-
2.3c β-Carotene bleaching assay: β-Carotene (0.2 mg), tion could be seen to generally take 6–40 h for the com-
linoleic acid (0.02 mL) and Tween 20 (0.18 mL) were pletion under conventional heating.5–9 In continuation
mixed with chloroform (0.5 mL) and the solvent was to our work on mechanochemical synthesis of curcumin
removed by evaporation. The residue obtained was derivatives,19 we have now reacted curcuminoids (3a-
mixed with distilled water (50 mL) and the result- g) with hydroxylamine hydrochloride in presence of
ing emulsion (4 mL aliquots each) was added to catalytic amount of acetic acid. The room temperature
3,5-bis(styryl)isoxazoles (4a-g) in methanol (0.2 mL, mechanical grinding in an agate mortar for about 1–
1 mmol). Absorbance was measured at 470 nm, using a 2 minutes rapidly afforded the 3,5-bis(styryl)isoxazoles
mixture of the emulsion (4 mL) and methanol (0.2 mL) (4a-g) in powder form. We observed that the com-
as the control. The absorbance was again recorded after pounds obtained in better purity and yield than reported
incubation for 1 h at 50◦C in the dark. The antioxidant earlier, as judged by TLC. The formation and identity of
activity (AA) was evaluated in terms of bleaching of the these isoxazoles were confirmed by physical and spec-
β-carotene using the formula AA = [1 − (Ao– At)/(Aoo– tral methods. It appears that the initially formed oxime
Aot )] ×100, where Ao and Aoo are the absorbance values derivatives proceeded to cyclize affording the isoxazole
measured at zero time of the incubation for test sample ring (scheme 1). Among the 3,5-bis(styryl)isoxazoles
O
OH
H3CO
HO
OCH3
OH
1
N
X
H3CO
HO
OCH3
2a-c X= a NH; b N-Aryl; c O
OH
O
N
O
OH
NH2OH.HCl, Gl. HOAc;
Grind, 1-2 mins
Ar
Ar
Ar
Ar
4a-g
3a-g
3, 4 Ar = a 4-HOC6H4; b 3-HO-4-MeOC6H3; c 2-HOC6H4; d 4-MeOC6H4;
e 3,4-(MeO)2C6H3;f 3,4-O-CH2-O-C6H3; g C6H5
Scheme 1. Structures of curcumin I (1) and its 3,5-bis(styryl)azole derivatives.

Daisy R Sherin and Kallikat N Rajasekharan
Table 2. The FeSO4 equivalent of 3,5-bis(styryl)isoxazo-
les (4a-g) in the presence of FRAP reagent and the Antioxi-
dant Activity (AA) % of 4a-g in β–carotene assay.
(4a-g) now synthesized, five examples (4b, 4c, 4d, 4f
and 4g) appear to be hitherto unreported.
Entry
FeSO4 equivalents (μmol)
AA %
3.2 Antioxidant Studies
4a
4b
4c
4d
4e
4f
1182
1195
997
883
592
1036
475
87.59
90.91
76.99
69.52
65.31
80.23
61.12
The antioxidant (AO) activities of the 3,5-bis(styryl)
isoxazoles (4a-g) were evaluated by 2,2-diphenylpicryl-
hydrazyl (DPPH), ferric ion reducing antioxidant power
(FRAP) and β-carotene assays. The AO activity by the
DPPH assay is expressed in terms of EC50 values, cal-
culated by plotting the percentage inhibition of DPPH
against various test concentrations (figure 1, table 1).
We have earlier shown that the isoxazole derivative of
curcumin I (2c) exhibits better AO capacity than the
parent curcumin I (1) in DPPH assay under similar
experimental conditions;19 the corresponding EC50 val-
ues for the percentage inhibition of DPPH being 8
0.011 μmol and 40 0.06 μmol, respectively for (2c)
and (1). A comparison of this data with those obtained
now for the 3,5-bis(styryl)isoxazoles (4a-g) shows that
the isoxazoles (4a-g) are less active than the isoxa-
zole derivative of curcumin I (2c). However, two among
these, 4a and 4b, are more active than curcumin I (1).
Thus, the presence of a hydroxy group juxtaposed with
4g
a methoxy group on the terminal aryl rings enhances the
AO activity of curcuminoid derived isoxazoles.
The results of FRAP assay are expressed as micro-
molar Fe2+ equivalents (table 2); a higher value of fer-
rous sulphate equivalent being indicative of a better AO
capacity. Our earlier sttudies19 have shown that under
identical assay conditions, the curcumin I derived isox-
azole (2c) and curcumin I (1) showed Fe2+ equiva-
lent of 1304 μmol and 1164 μmol, respectively. The
present results based on FRAP assay is now seen to
follow a similar pattern as seen in the DPPH assay,
wherein the presence of a hydroxy group along with a
methoxy group on the terminal aryl rings is found to
be beneficial. A similar conclusion could now be drawn
based on the β-carotene bleaching assay of the 3,5-
bis(styryl)isoxazoles (4a-g) (table 2) and in the light of
our earlier study.19
100
4a
4b
80
4c
60
4d
4e
4. Conclusions
40
4f
We report the synthesis of curcuminoid-derived 3,5-
bis(styryl)isoxazoles (4a-g) by mechanochemical grind-
ing of the respective curcuminoid and hydroxylamine
hydrochloride catalyzed by gl. acetic acid. The anti-
oxidant studies of 4a-g revealed that hydroxy and
methoxy groups present in the terminal aryl moieties
of 3,5-bis(styryl)isoxazoles improve their anti-oxidant
activity.
4g
20
0
0
0.02
0.04 0.06
0.08
0.1
0.12
Conentration (mmol)
Figure 1. Percentage inhibition of DPPH (10−2 mM) at
various concentrations of 3,5-bis(styryl)isoxazoles (4a-g).
Table 1. Percentage inhibition of DPPH (10−2 mmol) by 3,5-bis(styryl)isoxazoles (4a-g).
Percentage inhibition by 3,5-bis(styryl)pyrazoles (4a-g) at various concentrations (mM)
Entry
0.005
0.01
0.025
0.05
0.075
0.1
EC50 (μmol)
4a
4b
4c
4d
4e
4f
23.91 0.21 37.15 0.06 49.98 0.31 60.29 0.18 79.08 0.12
27.12 0.18 40.36 0.11 54.25 0.22 64.29 0.37 82.13 0.19
18.28 0.23 26.32 0.09 33.74 0.16 39.62 0.22 47.64 0.07
16.27 0.16 19.11 0.23 27.18 0.17 31.13 0.25 40.77 0.06
15.03 0.29 18.20 0.18 24.09 0.08 28.78 0.24 35.43 0.17
19.26 0.33 29.88 0.17 38.56 0.24 47.23 0.29 55.64 0.07
13.26 0.11 16.27 0.21 21.78 0.09 25.98 0.12 33.06 0.19
87.42 0.16
91.05 0.16
63.29 0.34
53.02 0.14
48.78 0.09
74.28 0.13
46.52 0.17
28 0.19
20 0.11
80 0.48
96 0.52
102 0.49
60 0.31
4g
108 0.22

3,5-Bis(styryl)isoxazoles
9. Das J, Pany S, Panchal S, Majhi A and Rahman G M
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Maulana Azad National Fellowship. KNR thanks UGC
for UGC-BSR Faculty and Emeritus Fellowships. The
authors also thank SAIF-STIC at CUSAT, Kochi and
NIIST Trivandrum, for elemental analysis, NMR and
MS spectra.
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