Synthesis of new 2,5-di-substituted 1,3,4-oxadiazoles bearing 2,6-di-tert-butylphenol moieties and evaluation of their antioxidant activity

Article abstract of DOI:10.3390/molecules19033436

Eleven new 2,6-di-tert-butyl-4-(5-Aryl-1,3,4-oxadiazol-2-yl)phenols 5a-k were synthesized by reacting aryl hydrazides with 3,5-di-tert butyl 4-hydroxybenzoic acid in the presence of phosphorus oxychloride. The resulting compounds were characterized based on their IR, 1H-NMR, 13C-NMR, and HRMS data. 2,2-Diphenyl-1-picrylhydrazide (DPPH) and ferric reducing antioxidant power (FRAP) assays were used to test the antioxidant properties of the compounds. Compounds 5f and 5j exhibited significant free-radical scavenging ability in both assays.

Full text of DOI:10.3390/molecules19033436

Molecules 2014, 19, 3436-3449; doi:10.3390/molecules19033436
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of New 2,5-Di-substituted 1,3,4-Oxadiazoles Bearing
2,6-Di-tert-butylphenol Moieties and Evaluation of Their
Antioxidant Activity
Raied M. Shakir 1,2, Azhar Ariffin 1,* and Mahmood Ameen Abdulla 3
1
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603,
Malaysia; E-Mail: raiedalsayab@yahoo.co.uk
2
Department of Chemistry, Ibn Al-haitham, University of Baghdad, Baghdad 61023, Iraq
3
Department of Molecular Medicine, Faculty of Medicine, University of Malaya,
Kuala Lumpur 50603, Malaysia; E-Mail: ammeen@um.edu.my
* Author to whom correspondence should be addressed; E-Mail: azhar70@um.edu.my;
Tel.: +60-3-7967-4080; Fax: +60-3-7967-4193.
Received: 7 February 2014; in revised form: 11 March 2014 / Accepted: 12 March 2014 /
Published: 20 March 2014
Abstract: Eleven new 2,6-di-tert-butyl-4-(5-aryl-1,3,4-oxadiazol-2-yl)phenols 5a–k were
synthesized by reacting aryl hydrazides with 3,5-di-tert butyl 4-hydroxybenzoic acid in the
presence of phosphorus oxychloride. The resulting compounds were characterized based
on their IR, 1H-NMR, 13C-NMR, and HRMS data. 2,2-Diphenyl-1-picrylhydrazide (DPPH)
and ferric reducing antioxidant power (FRAP) assays were used to test the antioxidant
properties of the compounds. Compounds 5f and 5j exhibited significant free-radical
scavenging ability in both assays.
Keywords: 2,6-di-tert-butylphenol; hindered phenol; antioxidant; 1,3,4-oxadiazole;
FRAP; DPPH
1. Introduction
Phenolic antioxidants inhibit or prevent oxidative stress in biological systems. Free radicals are one
of the main causes of many pathological conditions such as those that cause several degenerative [1]
and chronic diseases [2]. Furthermore, numerous heterocyclic compounds containing di-tert-butyl
phenol exhibit various types of biological activity in addition to their antioxidant ability [3,4].

Molecules 2014, 19
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Cyclo-oxygenase and 5-lipoxygenase [5,6] exhibit anti-inflammatory [3,7] and anticancer [8,9]
activities. Many synthesized compounds contain long-chain resonance and exhibit high antioxidant
activity, such as 1, 2, and 3 [10–12] (Figure 1).
Figure 1. Di-tert-butyl-phenols with long-chain resonance as antioxidants.
O
N
N
H
N
R
X
NH
N
Het
S
HO
HO
HO
O
3
2
1
Het =
X = O, S
benzothiazol-2-yl,
pyrazin-2-yl,
pyridin-2-yl,
R = SH, SMe, SEt
SCH2COOH,
NH2, NHNH2
pyrimidin-2-yl
Antioxidants donate protons to become a stable free radicals. This stability increases with the extent
of delocalisation [13] and enhances antioxidant ability [14]. A number of 1,3,4-oxadiazole derivatives
have exhibited various types of biological activity [15–17] and antioxidant ability [18,19].
This paper describes the synthesis of eleven 2,6-di-tert-butyl-4-(5-aryl-1,3,4-oxadiazole-2-yl)phenols
5a–k (Scheme 1) and evaluates their antioxidant activity. These new oxadiazoles are designed to be
effective antioxidants owing to their long-chain resonance. This study also investigates the effects of
different substituents on the phenyl ring C at position 6 of the 1,3,4-oxadiazole. This structure is likely
to possess superior antioxidant activity compared to the 2,6-di-tert-butyl phenol given the enhanced
stability of the free radical of the 1,3,4-oxadiazole as a result of resonance. The inductive and
resonance effects could have a major role in enhancing the scavenging ability of the compound. The
antioxidant ability of these compounds were measured by means of FRAP and DPPH assays.
Scheme 1. Synthesis of 2,6-di-tert-butyl-4-(5-aryl-1,3,4-oxadiazol-2-yl)phenols.
O
O
i) SOCl2
NH2
OH
N
ii) NH2NH2.H2O
H
R
R
4 a-k
POCl3
OH
COOH
N
N
8
3
9
2
1
B
6
R
4
7C
5
O
10
A
HO
12
11
5 a-k

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2. Results and Discussion
Chemistry
Eleven aromatic acids were converted to their corresponding aryl hydrazides 4a–k by reacting the
corresponding aromatic acids with thionyl chloride and then with hydrazine hydrate in dry benzene at
0 °C. The aryl hydrazides were then reacted with 3,5-di-tert butyl 4-hydroxybenzoic acid in the presence
of POCl3 as dehydrating agent to obtain new 2,6-di-tert-butyl-4-(5-aryl-1,3,4-oxadiazol-2-yl)phenols,
as demonstrated in Scheme 1. Table 1 shows the aryl groups with the corresponding yields and
HREIM data.
Table 1. Synthesized compounds, yields, molecular formulas (MFs) and HRMS data.
No.
5a
Compounds
Yield %
MF
HREIMS found
HREIMS calc.
N
N
CH3
O
76.1
C23H28N2O2
364.2147
364.2151
HO
HO
N
N
N
OMe
OEt
O
5b
5c
5d
76
C23H28N2O3
C24H30N2O3
380.2095
394.2249
428.1093
380.2100
394.2256
428.1099
N
O
84.2
73.6
HO
HO
N
N
Br
O
C22H25BrN2O2
N
N
Cl
OH
Cl
O
5e
5f
83.1
70
C22H25ClN2O2
C22H26N2O3
384.1597
366.1938
418.1219
384.1605
366.1943
418.1215
HO
HO
N
N
O
N
N
O
5g
80.3
C22H24Cl2N2O2
Cl
HO
Cl
N
N
O
5h
5i
74.5
59.9
68.4
C22H24Cl2N2O2
C24H30N2O2
C23H28N2O2
418.1210
378.2301
364.2144
418.1215
378.2304
364.2151
Cl
HO
HO
N
N
CH3
O
H3
C
N
N
O
H3C
5j
HO
HO
N
N
O
Cl
5k
81
C22H25ClN2O2
384.1600
384.1605

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The structures of the compounds were established on the basis of their spectral data. The IR
exhibited all the expected peaks, with the OH of the hindered phenol showing medium to strong peaks
due to nonhydrogen bonding [20] at 3658–3525, CHaliphatic ones at 2963–2947, and the C=N of the
oxadiazole ring at 1624 cm−1 to 1608 cm−1. The 1H-NMR spectra displayed the di-tert butyl group with
integration equal to 18H, whose chemical shift ranged between 1.44 ppm and 1.52 ppm. The OH of the
hindered phenol appeared at δ5.63 to δ5.69. All aryl protons and their substituents appeared in the
1
expected regions. The 13C-NMR spectra were consistent with the IR and H-NMR spectra and
our expectations. The carbons of the oxadiazole ring appeared at 161 ppm to 166 ppm, which
represented 2(C=N). HMBC was employed to distinguish between C5 and C6 through the long-range
coupling J3. The weak coupling J2 was also determined (Figure 2), and it exhibited the most significant
correlations for the aromatic area.
Figure 2. HMBC expansion region of 5d.
H3 exhibited correlations with C5 and C1 and weak correlations with C2 and C4. H8 exhibited a
correlation with C6 and C10 owing to J3, and with C7 and C9 owing to J2. This spectrum confirmed that
the C5 of the oxadiazole appeared at a lower field than C6. The HREIM values for all synthesized
compounds were consistent with the calculated mass and the molecular formula. For more details, see
the Experimental section.
The EIMs show the molecular ion M•+ for all compounds and the base peak (100%) were either
the same value of the molecular ion or the molecular ion minus methyl radical [M•+−•CH3].

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The fragmentations in EIMs confirm the proposed structures and the HREIMs confirmed the accurate
mass and the molecular formula. The interesting fragmentation observed in the mass spectrum was the
loss of isocyanic acid (HNCO). This fragmentation which strated from [M•++H] was reported in
literature [21–23]. The oxadiazoles loose HNCO but not phenol. However, in our case, losing HNCO
started from molecular ion (M•+) which was subsequently protonated. The loss of HNCO can be
explained through the rearrangement of the molecular ion and migration. Scheme 2 describes the
proposed mechanism of the elimination of isocyanic acid.
Scheme 2. Proposed pathways of HNCO loss.
H
N
N
HN
N
N
N
Ar
O
Ar
HO
Ar
O
O
O
O
O
N
HN
Ar
NH Ar
N
N
N
Ar
-HNCO
Ar
B
O
O
O
O
O
A
O
C
N
NH Ar
N
N
Ar
Ar
O
O
O
Two pathways were suggested for the radical migration, path (B) and (C). Both pathways, (B) and (C)
have intermediate (A) as their starting point which is formed by intra fragmentation of the molecular
ion and migration of 2,6-di-tert-butylphenol. Path B is similar to the mass fragmentation pathway
proposed by Frański et al. [22]. Table 2 summarizes the value of the molecular ion, the base peak and
value after losing HNCO.
Table 2. Molecular ion found, calculated, base peak and the m/z after losing HNCO.
5
M•+ Found
364.2
380.2
394.3
428.2
384.2
366.2
418.2
418.2
378.3
364.3
384.2
M•+ calculated
364.21
m/z of base peak 100%
M•+-HNCO
321.1
337.1
351.1
385.1
341.1
323.1
375.1
375.0
335.1
321.1
341.1
5a
5b
5c
5d
5e
5f
5g
5h
5i
349.2
380.2
394.3
413.1
369.1
366.2
403.1
403.1
378.3
349.2
369.2
380.21
394.22
428.10
384.16
366.19
418.12
418.12
378.23
5j
5k
364.21
384.16

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3. Antioxidant Assays
3.1. FRAP Assay
The FRAP assay was performed according to the Benzie and Strain [24] method. The FRAP
reagent was prepared by combining 300 mM acetate buffer and 10 mM 2,4,6-tripyridyl-s-triazine
(TPTZ) solution in 40 mM HCl and 20 mM FeCl3·6H2O, in a ratio of 10:1:1. The FRAP reagent was
incubated at 37 °C prior to use. Ten microliters of the sample was reconstituted in the carrier (solvent
or ultrapure water) and mixed with 300 μL of FRAP reagent. The mixture was incubated at 37 °C for
4 min in a microplate reader. The absorbance of the complex was 593 nm. The FRAP value can be
calculated using the following equation [25]:
FRAP = [(0–4 min ∆A593 nm of test sample)/(0–4 min ∆A593 nm of standard)]
× [standard] (µM) × Y × 1000
where Y is absorbance of the spectrophotometer.
3.2. DPPH Assay
The assay was performed as reported by Gerhauser et al. [26]. Five microliters of the sample
(dissolved in ethanol) was added into 195 μL of 100 μM DPPH reagent in ethanol (96%) and mixed in
a 96-well plate. The intensity of the color was measured for 3 h at an interval of 20 min at 515 nm.
Ascorbic acid and BHT were used as reference.
3.3. Antioxidant Activity
Differences occurred between the structures of the synthesized compound in ring C owing to
different substituent and positions, whereas rings A and B were the same. Various antioxidant abilities
were exhibited in both assays based on the type of substituent and their position, which have important
roles in enhancing or negating antioxidant ability. The inductive effects of the electron-donating group +I
and electron-withdrawing group (EWG) −I, the mesomeric effect (electron-releasing group +M or
electron-withdrawing group −M), and the resonance effect directly affected antioxidant ability.
Compound 5f exhibited higher antioxidant capacity, with a FRAP value of 2207.25 (see Table 3).
This result is consistent with the concept that the hydroxyl group enhances antioxidant ability [27–30].
The +M of the hydroxyl at the para position is more important than the −I. Compound 5j exhibited
excellent antioxidant ability (1538.9).
The FRAP value and the substituent followed the following sequence: 4-OH > 2-Me > 2,4-di-Me >
4-Me > 4-OMe ≈ 4-OEt > 4-Cl> 2-Cl > 4-Br ≈ 3,4-di Cl ≈ 3,5-di Cl. This sequence demonstrates that
the electron-releasing group, which exerts mesomeric and inductive effects, enhances antioxidant
ability, whereas the inductive-withdrawing group decreases antioxidant ability. The results show that
the position of substituent also affects antioxidant ability, as illustrated in Figure 3.

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Table 3. Antioxidant activity of the synthesized oxadiazoles.
Compound
FRAP a
648.3
629.4
613.3
302.2
640.0
2207.2
322.8
212.8
987.2
1538.9
500.6
488.3
2421.1
848.9
445.0
2090.6
779.4
DPPH Inhibition % ±SD
76.02 ± 0.059
62.03 ± 0.327
56.04 ± 0.187
30.85 ± 0.166
50.44 ± 0.045
89.05 ± 0.024
30.35 ± 0.038
29.26 ± 0.041
79.22 ± 0.037
87.21 ± 0.084
42.14 ± 0.078
66.03 ± 0.051
-
IC50 ± SEM b (100 µg/mL)
5a
41.76 ± 0.042
50.69 ± 0.181
54.60 ± 0.469
>100
5b
5c
5d
5e
99.2 ± 0.032
15.79 ± 0.017
>100
5f
5g
5h
>100
5i
41.27 ± 0.027
15.9 ± 0.054
>100
5j
5k
BHT
79.84 ± 0.036
-
Gallic acid
Ascorbic acid
Rutin
Quercetin
Trolox
90.65 ± 0.122
-
22.71 ± 0.086
-
-
-
-
-
a Standard deviation (SD) value in FRAP was between 0.01–0.16; b SED standard mean error and IC50: 50%
effective concentration.
Figure 3. FRAP assay for the synthesized 2,6-di-tert-butyl-4-(5-aryl-1,3,4-oxadiazol-2-yl)phenols.
The methyl group at position 2 enhances the antioxidant ability more than that at position 4, and
exhibits higher antioxidant ability than 4-methoxy. However, the differences between 4-methoxy and
4-ethoxy were too small. The analogue with EWG in positions 2, 3, and 4, i.e., 4-Br, 4-Cl, 3,4-diCl,

Molecules 2014, 19
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3,5-diCl reduced or negated antioxidant activity. The results may be explained by the fact that
previously described analogues with EWG’s, increase the bond dissociation energy. Another
possibility is that they exhibit decreased antioxidant ability [31,32]. The DPPH results (Table 3) were
compatible with, and possessed the same sequence, as the FRAP assay. However, all values for DPPH
are on the whole lower than the values for the FRAP assay in comparison to ascorbic acid. For
instance, in FRAP assay, 5f showed about twice the antioxidant ability of ascorbic acid, whereas in the
DPPH assay, 5f exhibited similar antioxidant ability to ascorbic acid. This difference could be
attributed to the different mechanisms for FRAP and DPPH. FRAP involves the single electron
transfer mechanism, whereas DPPH assay depends on the H-atom transfer mechanism [33]. The steric
hindrance between the synthesized compound and DPPH may account for the difference [34].
Compounds 5f and 5j exhibited lower IC50 values of 15.79 and 15.9 µg/mL, respectively, compared
to ascorbic acid. The IC50 of para substituted analogues with methyl, methoxy, and ethoxy were lower
than that of BHT. By contrast, for electron withdrawing group at para, meta, and ortho, the analogues
showed reduced antioxidant activity. All compounds were screened in terms of their free radical
scavenging properties using five concentrations: 12.5, 25, 50, 75, and 100 µg/mL. Both 5f and 5j
exhibited significant antioxidant capability at low concentrations, as depicted in Figure 4.
Figure 4. DPPH inhibitions of 5f and 5j at different concentration
.
4. Experimental
General Information
The chemicals used for the synthesis were supplied by Sigma-Aldrich (Petaling Jaya, Selangor,
Malaysia), Fisher (Shah Alam, Selangor, Malaysia), and Merck (Petaling Jaya, Selangor, Malaysia).
The melting point was determined by open capillary tube method using an MEL-TEMP II apparatus
and was uncorrected. The purity of the compounds was checked through thin layer chromatography
(silica gel TLC) using Merck plates. The plates were visualized by mean of iodine vapors and UV

Molecules 2014, 19
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light. The IR spectra were obtained using a PerkinElmer 400 Fourier transform infrared spectrometer.
All NMR spectra were recorded on either a JEOL-ECA 400 MHz or JEOL-Lambda 400 MHz
spectrometer. CDCl3 and DMSO-d6 were used as solvents with TMS as the internal standard. Mass
spectra were recorded using a TSQ7000 for HREI/MS (NUS Singapore). For UV spectroscopy, a
Power Wave X340 (BIO-TEK Instruments, Inc., Winooski, VT, USA) was used to record the FRAP
and DPPH assays.
General Synthesis of 2,6-Di-tert-butyl-4-(5-aryl-1,3,4-oxadiazol-2-yl)phenols
To a mixture of (0.31 g, 1.24 mmol) of 3,5-di-tert-butyl-4-hydroxybenzoic acid and 1.24 mmole
aryl acid hydrazide in a 50 mL round bottom flask, 5 mL of phosphorus oxychloride was added in a few
portions at room temperature. The mixture was refluxed for 3 h with stirring on water bath 80–90 °C.
After cooling, the mixture poured onto 100 mL crushed ice and stirred for 15 min. Sodium bicarbonate
was added in a few portions until the pH was around to 7–8. The precipitate was filtered, washed with
water and dried then purified either by column chromatography or by crystallization from suitable solvent.
2,6-Di-tert-butyl-4-[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]phenol (5a). The product was recrystallized
from chloroform-ethanol (1-1) to obtain white crystals. Yield 0.343 g (76.0%), m.p. 196–197 °C,
IR (KBr, νmax/cm−1): 3658 (OH), 3011 (CHaromatic), 2962–2947 (CHaliphatic), 1610 (C=N), 1585, 1498 (C=C),
1
1219 (C-O), H-NMR (CDCl3, 400 MHz, ppm): 1.51 (s, 18H, H14, 2 × t-Bu), 2.42 (s, 3H, p-CH3-ph),
5.67 (s, 1H, OH), 7.31 (d, 2H, J = 8.28 Hz, H9, H11), 7.94 (s, 2H, H3), 8.01 (d, 2H, J = 8.28 Hz, H8, H12),
13C-NMR (CDCl3, 100 MHz, ppm): 21.73 (p-CH3Ph), 30.25 (6C, C14, 2 × C(CH3)3), 34.56 (2C, C13,
2 × C(CH3)3), 115.43 (C4), 121.52 (C7), 124.35 (C3), 126.87 (2C, C8 & C12), 129.77 (2C, C9 & C11),
136.81 (C2), 141.99 (C10), 157.12 (C1), 164.22 & 165.21 (C5 & C6). HREIMs, m/z = 364.2147 [M•+]
(calc. for C23H28O2N2, 364.2151).
2,6-Di-tert-butyl-4-[5-(4-methox yphenyl)-1,3,4-oxadiazol-2-yl]phenol (5b). The solid product was
recrystallized from ethyl acetate to yield white crystals. Yield 0.346 g (73%), m.p. 179–181 °C,
IR (KBr, νmax/cm−1): 3625 (OHphenol), 3006 (CHaromatic), 2955 (CHaliphatic), 1611 (C=N), 1585, 1495 (C=C),
1
1219 (C-O), 1020 (O-CH3), H-NMR (CDCl3, 400 MHz, ppm): 1.51 (s, 18H, H14, 2 × t-Bu),
3.89 (s, 3H, OCH3), 5.63 (s, 1H, OH), 7.03 (d, 2H, J = 9.04 Hz, H9, H11), 7.92 (s, 2H, H3), 8.06 (d, 2H,
13
J = 8.56 Hz, H8, H12), C-NMR (CDCl3, 100 MHz, ppm): 30.13 (6C, C14, 2 × C(CH3)3), 34.45 (2C,
C13, 2 × C(CH3)3), 55.44 (OCH3), 114.40 (2C, C8 & C12), 115.37 (C4), 116.72 (C7), 124.19 (C3),
128.58 (2C, C9 & C11), 136.68 (C2), 156.95 (C1), 162.11 (C10), 163.89 & 164.75 (C6 & C5). HREIMs,
m/z = 380.2095 [M•+] (calc. for C23H28O3N2, 380.2100).
2,6-Di-tert-butyl-4-[5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenol (5c). The crude solid was
recrystallized from ethyl acetate-methanol (1:1) to give a white amorphous solid. Yield 0.371 g (76%),
m.p. 176–178 °C, IR (KBr, νmax/cm−1), 3628 (OHphenol), 3009 (CHaromatic), 2957 (CHaliphatic), 1611 (C=N),
1
1543, 1495 (C=C), 1221 (C-O), 1111 (O-CH2), H-NMR (CDCl3 ,400 MHz, ppm): 1.46 (t, 3H,
J = 7.32 Hz, OCH2CH3), 1.51 (s, 18H, H14, 2 × t-Bu), 4.11 (q, 2H, J = 8 Hz, OCH2), 5.64 (s, 1H, OH),
13
7.1 (d, 2H, J = 8.8 Hz, H9 & H11), 7.92 (s, 1H, H3), 8.05 (d, 2H, J = 8.04 Hz, H8 & H12), C-NMR
(CDCl3, 100 MHz, ppm): 14.71 (OCH2CH3), 30.17 (6C, C14, 2 × C(CH3)3), 34.48 (2C, C13, 2 × C(CH3)3),

Molecules 2014, 19
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63.73 (OCH2), 114.91 (2C, C8 & C12), 115.50 (C4), 116.60 (C7), 124.18 (C3), 128.57 (2C, C9 & C11),
136.69 (C2), 156.93 (C1), 161.52 (C10), 164.00 & 164.90 (C6 & C5). HREIMs, m/z = 394.2249 [M•+]
(calc. for C24H30O3N2, 394.2256).
4-(5-(4-Bromophenyl)-1,3,4-oxadiazol-2-yl)-2,6-di-tert-butylphenol (5d). The crude product was
recrystallized from ethyl acetate-methanol (1-1) to give white crystals. Yield 0.447 g (84%),
m.p. 168–170 °C, IR (KBr, νmax/cm−1): 3525 (OHphenol), 3005 (CHaromatic), 2953 (CHaliphatic), 1602 (C=N),
1
1543, 1495 (C=C), 1244 (C-O), H-NMR (CDCl3, 400 MHz, ppm): 1.51 (s, 18H, H14, 2 × t-Bu),
5.66 (s, 1H, OH), 7.67 (d, 2H, J = 8.42 Hz, H9 & H11), 7.92 (s, 2H, H3), 7.9 (d, 2H, J = 8.54 Hz, H8 & H12),
13C-NMR (CDCl3, 100 MHz, ppm): 30.23 (6C, C14, 2 × C(CH3)3), 34.56 (2C, C13, 2 × C(CH3)3),
115.12 (C4), 123.22 (C7), 124.43 (C3), 126.1 (2C, C8 & C12), 128.31 (2C, C9 & C11), 132.41 (C10),
136.87 (C2), 157.33 (C1), 163.37 & 165.66 (C5 & C6) ppm. HREIMs m/z = 428.1093 [M•+] (calc. for
C22H25O2N2Br, 428.1099).
4-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-2,6-di-tert-butylphenol (5e). The crude material was
recrystallized from ethyl acetate-methanol (1:1) to obtain a white solid. Yield 0.396 g (83%),
m.p. 162–164 °C, IR (KBr, νmax/cm−1): 3583 (OHphenol), 3004 (CHaromatic), 2959 (CHaliphatic), 1607 (C=N),
1
1571, 1540 (C=C), 1239 (C-O). H-NMR (CDCl3-400 MHz, ppm): 1.51 (s, 18H, H14, 2 × t-Bu),
5.67 (s, 1H, OH), 7.51 (d, 2H, J = 8.52 Hz, H9 & H11), 7.93 (s, 2H, H3), 8.07 (d, 2H, J = 8.52 Hz, H8 & H12),
13C-NMR (CDCl3,100 MHz, ppm): 30.23 (6C, C14, 2 × C(CH3)3), 34.56 (2C, C13, 2 × C(CH3)3),
115 (C4), 122.78 (C7), 124.27 (C3), 129.46 (2C, C9 & C11) 128.18 (2C, C8 & C12), 136.87 (C2),
137.71 (C10), 157.32 (C1), 163.29 & 165.64 (C5 & C6). HREIMs m/z = 384.1597 [M•+] (calc. for
C22H25O2N2Cl, 384.1605).
2,6-Di-tert-butyl-4-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)phenol (5f). The crude mixture was purified
by column chromatography using (6:1) hexane ethyl acetate as eluent to give a white amorphous solid.
Yield 0.318 g (70%), m.p. 144–146 °C, IR (KBr, νmax/cm−1): 3617 (OHphenol), 2958 (CHaliphatic),
1
1609 (C=N), 1546, 1506 (C=C), 1250 (C-O), H-NMR (CDCl3, 400 MHz, ppm): 1.51 (s, 18H, H14,
2 × t-Bu), 5.65 (s, 1H, OH), 6.70 (bs, 1H, OH), 7.01 (d, 2H, J = 8.76 Hz, H9 & H11), 7.92 (s, 1H, H3),
8.01 (d, 2H, J = 8.8 Hz, H8 & H12), 13C-NMR (CDCl3, 100 MHz, ppm): 31.06 (6C, C14, 2 × C(CH3)3),
34.56 (2C, C13, 2 × C(CH3)3), 115.27 (C4), 115.58 (C7), 116.40 (2C, C8 & C12), 124.37 (C3),
128.97 (2C, C9 & C11), 136.85 (C2), 157.24 (C1) 159.83 (C10), 164.33 & 165.05 (C5 & C6). HREIMs
m/z = 366.1938 [M•+] (calc. for C22H26O3N2, 366.1943).
2,6-Di-tert-butyl-4-(5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)phenol (5g). The crude product was
recrystallized from benzene to give a white solid. Yield 0.416 g (80%), m.p. 222–224 °C, IR (KBr,
νmax/cm−1): 3580 (OHphenol), 3003(CHaromatic), 2952 (CHaliphatic), 1606 (C=N), 1546, 1462 (C=C),
1
1239 (C-O). H-NMR (CDCl3, 400 MHz, ppm): 1.44 (s, 18H, H14, 2 × t-Bu), 5.67 (s, 1H, OH),
13
7.59 (d, 2H, J = 8.52 Hz, H11), 7.91–7.95 (m, 3H, H12 & H3'), 8.19 (d, J = 2.2 Hz, 1H, H8), C-NMR
(CDCl3, 100 MHz, ppm): 30.23 (6C, C14, 2 × C(CH3)3), 34.57 (2C, C13, 2 × C(CH3)3), 114.89 (C4),
124.11 (C7), 124.51 (C3), 125.95 (C12), 128.53 (C11), 131.28 (C8), 133.68 (C9), 135.89 (C10),
136.94 (C2), 157.50 (C1), 162.3 & 165.94 (C5 & C6). HREIMs m/z = 418.1219[M•+] (calc. for
C22H24O2N2Cl2, 418.1215).

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3446
2,6-Di-tert-butyl-4-(5-(3,5-dichlorophenyl)-1,3,4-oxadiazol-2-yl)phenol (5h). The crude product was
purified by recrystallized from acetonitrile to afford a white amorphous solid. Yield 0.386 g (74%),
m.p. 195–197 °C, IR (KBr, νmax/cm−1): 3600 (OHphenol), 3007 (CHaromatic), 2961 (CHaliphatic), 1606 (C=N),
1
1574, 1550 (C=C), 1240 (C-O), H-NMR (CDCl3, 400 MHz, ppm): 1.52 (s, 18H, H14, 2 × t-Bu),
5.69 (s, 1H, OH), 7.51 (t, 1H, J = 1.24 Hz, H10), 7.93 (s, 2H, H3), 8.01 (t, 2H, J = 1.72 Hz, H8 & H12),
13C-NMR (CDCl3, 100 MHz, ppm): 30.39 (6C, C14, 2 × C(CH3)3), 34.50 (2C, C13, 2 × C(CH3)3),
114.73 (C4), 124.48 (C3), 125.02 (2C, C8 & C12), 126.87 (C7), 131.24 (C10), 135.92 (C9 & C11),
136.93 (C2), 157.50 (C1), 161.877 (C5), 166.05 (C6). HREIMs m/z = 418.1210 [M•+] (calc. for
C22H24O2N2Cl2, 418.1215).
2,6-Di-tert-butyl-4-(5-(2,4-dimethylphenyl)-1,3,4-oxadiazol-2-yl)phenol (5i). The crude product was purified
by recrystallized from toluene to give white crystalline needles. Yield 0.28 g (60%), m.p. 170–172 °C.
IR (KBr, νmax/cm−1): 3587 (OHphenol), 2957 (CHaliphatic), 1614 (C=N), 1550, 1536 (C=C), 1238 (C-O),
1H-NMR (CDCl3, 400 MHz, ppm): 1.50 (s, 18H, H14, 2 × t-Bu), 2.39 (s, 3H, H15), 2.70 (s, 3H, H16),
5.64 (s, 1H, OH), 7.17–7.14 (m, 2H, H9 & H11), 7.95–7.90 (m, 3H, H12, H3'), 13C-NMR (CDCl3, 100 MHz,
ppm): 21.50 (C16, o-CH3), 22.04 (C15, p-CH3), 30.22 (6C, C14, 2 × C(CH3)3), 34.55 (2C, C13, 2 × C(CH3)3),
115.40 (C4), 12.60 (C7), 124.38 (C3), 126.98 (C11), 128.96 (C12), 132.59 (C9), 136.79 (C2), 138.19 (C8),
141.43 (C10), 157.11 (C1), 164.49 & 164.81 (C5 & C6). HREIMs m/z =378.2301 [M•+] (calc. for
C24H30O2N2, 378.2307).
2,6-Di-tert-butyl-4-[5-(2-methylphenyl)-1,3,4-oxadiazol-2-yl] phenol (5j). The product was recrystallized
from ethyl acetate to afford white crystals, 0.308 g (68.5%), m.p. 132–134 °C, IR (KBr, νmax/cm−1):
3588 (OHphenol), 3008 (CHaromatic), 2963 (CHaliphatic), 1607 (C=N), 1592, 1537 (C=C), 1238 (C-O),
1H-NMR (CDCl3,400 MHz, ppm): 1.52 (s, 18H, H14, 2 × t-Bu), 5.66 (s, 1H, OH), 7.44–7.34 (m, 3H,
H9, H10, H11), 7.96 (s, 2H, H3, H3'), 8.02 (d, 1H, J = 7.32 Hz, H12), 13C-NMR (CDCl3, 100 MHz, ppm):
2 2.11 (o-CH3), 30.22 (6C, C14, 2 × C(CH3)3), 34.52 (2C, C13, 2 × C(CH3)3), 115.53 (C4), 123.40 (C7),
124.37 (C3), 126.21 (C11), 128.96 (C12), 131.03 (C10), 131.80 (C9), 136.80 (C2), 138.35 (C8), 157.14 (C1),
164.32 & 165.04 (C5 & C6), HREIMs m/z = 364.2144 [M•+] (calc. for C23H28O2N2, 364.2151).
2,6-Di-tert-butyl-4-(5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl)phenol (5k). The crude product was
recrystallized from ethyl acetate to obtain white crystals. Yield 0.386 g (81%), m.p. 113–115 °C,
IR (KBr, νmax/cm−1): 3584 (OHphenol), 3004 (CHaromatic), 2959 (CHaliphatic), 1607 (C=N), 1570, 1539 (C=C),
1
1239 (C-O), H-NMR (CDCl3, 400 MHz, ppm): 1.48(s, 18H, H14, 2 × t-Bu), 5.66 (s, 1H, OH),
7.38–7.46 (m, 2H, H10, H11), 7.53 (d, 1H, J = 8.04 Hz, H12), 7.95 (s, 2H, H3, H3'), 8.05 (d, 1H, J = 6.32 Hz,
H9), 13C-NMR (CDCl3, 100 MHz, ppm): 30.27 (6C, C14, 2 × C(CH3)3), 34.61 (2C, C13, 2 × C(CH3)3),
115.18 (C4), 123.65 (C7), 124.61 (C3), 127.21 (C11), 131.33 (C12), 132.32 (C9), 133.14 (C8),
136.79 (C9), 136.94 (C2), 157.28 (C1), 162.55 & 166.04 (C5 & C6). HREIMs m/z = 384.1600 [M•+]
(calc. for C22H25O2N2Cl, 384.1605).
5. Conclusions
A series of new 1,3,4-oxadiazole compound incorporating hindered phenol moities were successfully
synthesized and characterized. All of the new compounds were screened for antioxidant activity using

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3447
the FRAP and DPPH assays. The substituents on ring C demonstrated a significant role in improving
or negating the antioxidant activity of the compounds. The analogues incorporating electron releasing
substituents exhibited high antioxidant activity, whereas those with electron withdrawing substituent
demonstrated reduced antioxidant activity.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/3/3436/s1.
Acknowledgments
We would like to thank the University of Malaya for a UMRG grant (RG149-11AFR) and a PPP
grant (PS345-2009B) and the Ministry of Higher Education of Malaysia for supporting this study.
We are also grateful to Amir Atto for his contributions and advice and to Noel F. Thomas for
proofreading the manuscript.
Author Contributions
All authors contributed equally for the manuscript. Raied M. Shakir (PhD candidates), Mahmood
Ameen Abdulla (DPPH and FRAP assays).
Conflicts of Interest
The authors report no conflicts of interest. The authors alone are responsible for the content and
writing of the paper.
References
1. Kar, S.; Subbaram, S.; Carrico, P.M.; Melendez, J.A. Redox-control of matrix metalloproteinase-1:
A critical link between free radicals, matrix remodeling and degenerative disease.
Respir. Physiol. Neurobiol. 2010, 174, 299–306.
2. Khalil, Z.; Khodr, B. A role for free radicals and nitric oxide in delayed recovery in aged rats with
chronic constriction nerve injury. Free Radic. Biol. Med. 2001, 31, 430–439.
3. Ziakas, G.N.; Rekka, E.A.; Gavalas, A.M.; Eleftheriou, P.T.; Kourounakis, P.N. New analogues
of butylated hydroxytoluene as anti-inflammatory and antioxidant agents. Bioorg. Med. Chem.
2006, 14, 5616–5624.
4. Yehye, W.A.; Abdul Rahman, N.; Alhadi, A.A.; Khaledi, H.; Ng, S.W.; Ariffin, A. Butylated
hydroxytoluene analogs: Synthesis and evaluation of their multipotent antioxidant activities.
Molecules 2012, 17, 7645–7665.
5. Ruiz, J.; Pẻrez, C.; Pouplana, R. QSAR study of dual cyclooxygenase and 5-lipoxygenase
inhibitors 2,6-di-tert-butylphenol derivatives. Bioorg. Med. Chem. 2003, 11, 4207–4216.
6. Mullican, M.D.; Wilson, M.W.; Connor, D.T.; Kostlan, C.R.; Schrier, D.J.; Dyer, R.D. Design of
5-(3,5-di-tert-butyl-4-hydroxyphenyl)-l,3,4-thiadiazo-l 1,3,4 -oxadiazoles and -1,2,4-triazoles as
orally-active, nonulcerogenic antiinflammatory agents. J. Med. Chem. 1993, 36, 1090–1099.

Molecules 2014, 19
3448
7. Moore, G.G.; Swingle, K.F. 2,6-Di-tert-butyl-4-(2'-thenoyl)phenol(R-830): A novel nonsteroidal
anti-inflammatory agent with antioxidant properties. Agents Actions 1982, 12, 674–683.
8. Ye, X.; Zhou, W.; Li, Y.; Sun, Y.; Zhang, Y.; Ji, H.; Lai, Y. Darbufelone, a novel anti-inflammatory
drug, induces growth inhibition of lung cancer cells both in vitro and in vivo. Cancer Chemother.
Pharmacol. 2010, 66, 277–285.
9. Sagar, B.H.; Stephen, M. Antioxidants during anticancer therapy. Focus Altern. Complement. Ther.
2004, 9, 96–106.
10. Jeong, T.-S.; Kim, K.S.; Kim, J.-R.; Cho, K.-H.; Lee, S.; Lee, W.S. Inhibitory efects of
multi-substituted benzylidenethiazolidine-2,4-diones on LDL oxidation. Bioorg. Med. Chem.
2004, 12, 4017–4023.
11. Song, Y.; Connor, D.T.; Doubleday, R.; Sorenson, R.J.; Sercel, A.D.; Unangst, P.C.; Roth, B.D.;
Gilbertsen, R.B.; Chan, K.; Schrier, D.J.; et al. Synthesis, structure-activity relationships, and
in vivo evaluations of substituted di-tert-butylphenols as a novel class of potent, selective, and
orally active cyclooxygenase-2 inhibitors. 1. Thiazolone and oxazolone series. J. Med. Chem.
1999, 42, 1151–1160.
12. Cuadro, A.M.; Valenciano, J.S.; Vaquero, J.J.; Alvarez-Builla, J.; Sunkel, C.; de Casa-Juana, M.F.;
Ortega, M.P. Synthesis and biological evaluation of 2,6-di-tert-butylphenol hydrazones as
5-lipoxygenase inhibitors. Bioorg. Med. Chem. 1998, 6, 173–180.
13. Nonhebel, D.C.; Walton, J.C. Free-Radical Chemistry, Structure and Mechanism;
Cambridge University Press: Cambridge, UK, 1974; p. 477.
14. Hung, C.-Y.; Yen, G.-C. Antioxidant activity of phenolic compounds isolated from mesona
procumbens hemsl. J. Agric. Food Chem. 2002, 50, 2993–2997.
15. De Oliveira, C.S.; Lira, B.F.; Barbosa-Filho, J.M.; Lorenzo, J.G.F.; de Athayde-Filho, P.F.
Synthetic approaches and pharmacological activity of 1,3,4-oxadiazoles: A review of the literature
from 2000–2012. Molecules 2012, 17, 10192–10231.
16. De Oliveira, C.S.; Lira, B.F.; dos Santos Falcão-Silva, V.; Siqueira-Junior, J.P.; Barbosa-Filho, J.M.;
de Athayde-Filho, P.F. Synthesis, molecular properties prediction, and anti-staphylococcal activity
of N-acylhydrazones and new 1,3,4-oxadiazole derivatives. Molecules 2012, 17, 5095–5107.
17. El Sadek, M.; Hassan, S.; Abdelwahab, H.; Yacout, G. Synthesis and bioassay of a new class of
furanyl-1,3,4-oxadiazole derivatives. Molecules 2013, 18, 8550–8562.
18. Musad, E.A.; Mohamed, R.; Ali Saeed, B.; Vishwanath, B.S.; Lokanatha Rai, K.M. Synthesis and
evaluation of antioxidant and antibacterial activities of new substituted bis(1,3,4-oxadiazoles),
3,5-bis(substituted) pyrazoles and isoxazoles. Bioorg. Med. Chem. Lett. 2011, 21, 3536–3540.
19. Maheshwari, R.; Chawla, P.; Saraf, S. Comparison between antioxidant activity of 2,5-disubstituted
1,3,4-oxadiazoles containing heteroaromatic ring and aromatic ring at 2nd position. Med. Chem. Res.
2011, 20, 1650–1655.
20. Silverstein, R.; Webster, F. Spectromeetric Identification Of Organic Compounds; John Wiley &
Sons Inc.: New York, NY, USA, 1998; pp. 90–91.
21. Frański, R.; Eitner, K.; Schroeder, G. Mass spectrometric study of some protonated and lithiated
2,5-disubstituted-1,3,4-oxadiazoles. J. Am. Soc. Mass Spectrom. 2003, 14, 289–294.

Molecules 2014, 19
3449
22. Frański, R.; Schroeder, G.; Rybachenko, V.; Szwajka, O.P. Loss of isocyanic acid from the internal
oxadiazole ring of protonated molecules of some 2,5-diaryl-1,3,4-oxadiazoles. Rapid Commun.
Mass Spectrom. 2002, 16, 390–395.
23. Frański, R.; Gierczyk, B.; Schroeder, G. Mass spectrometric fragmentation pathways of isotope
labeled 2,5-disubstituted-1,3,4-oxadiazoles and thiadiazoles. Int. J. Mass Spectrom. 2004, 231,
47–49.
24. Benzie, I.F.; Strain, J.J. The Ferric Reducing Ability of Plasma (FRAP) as a measure of
“antioxidant power”: The FRAP assay. Anal. Biochem. 1996, 239, 70–76.
25. Rustaiyan, A.; Javidnia, K.; Farjam, M.H.; Aboee-Mehrizi, F.; Ezzatzadeh, E. Antimicrobial
and antioxidant activity of the Ephedra sarcocarpa growing in Iran. J. Med. Plants Res. 2011, 5,
4251–4255.
26. Gerhäuser, C.; Klimo, K.; Heiss, E.; Neumann, I.; Gamal-Eldeen, A.; Knauft, J.; Liu, G.-Y.;
Sitthimonchai, S.; Frank, N. Mechanism-based in vitro screening of potential cancer chemopreventive
agents. Mutat. Res. Fundam. Mol. Mech. Mutagen. 2003, 523–524, 163–172.
27. Shahidi, F. Natural Antioxidants: Chemistry, Health Effects, and Applications; AOCS Press:
Champaign, IL, USA, 1997; p. 18.
28. Simić, A.; Manojlović, D.; Šegan, D.; Todorović, M. Electrochemical behavior and antioxidant
and prooxidant activity of natural phenolics. Molecules 2007, 12, 2327–2340.
29. Moalin, M.; Strijdonck, G.P.F.; Beckers, M.; Hagemen, G.J.; Borm, P.J.; Bast, A.; Haenen, G.R.
A planar conformation and the hydroxyl groups in the B and C rings play a pivotal role in the
antioxidant capacity of quercetin and quercetin derivatives. Molecules 2011, 16, 9636–9650.
30. Torres, R.; Urbina, F.; Morales, C.; Modak, B.; Monache, F.D. Antioxidant properties of
lignans and ferulic acid from the resinous exudate of larrea nitida. J. Chil. Chem. Soc. 2003,
doi:10.4067/S0717-97072003000300012.
31. Lucarini, M.; Pedrielli, P.; Pedulli, G.F.; Cabiddu, S.; Fattuoni, C. Bond dissociation energies of
O-H bonds in substituted phenols from equilibration studies. J. Org. Chem. 1996, 61, 9259–9263.
32. Inami, K.; Iizuka, Y.; Furukawa, M.; Nakanishi, I.; Ohkubo, K.; Fukuhara, K.; Fukuzumi, S.;
Mochizuki, M. Chlorine atom substitution influences radical scavenging activity of 6-chromanol.
Bioorg. Med. Chem. 2012, 20, 4049–4055.
33. Huang, D.; Ou, B.; Prior, R.L. The chemistry behind antioxidant capacity assays. J. Agric. Food
Chem. 2005, 53, 1841–1856.
34. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate
antioxidant activity. LWT Food Sci. Technol. 1995, 28, 25–30.
Sample Availability: Samples of the compounds 5a–k are available from the authors.
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