Butylated hydroxytoluene analogs: Synthesis and evaluation of their multipotent antioxidant activities

Article abstract of DOI:10.3390/molecules17077645

A computer-aided predictions of antioxidant activities were performed with the Prediction Activity Spectra of Substances (PASS) program. Antioxidant activity of compounds 1, 3, 4 and 5 were studied using 1,1-diphenyl-2- picrylhydrazyl (DPPH) and lipid peroxidation assays to verify the predictions obtained by the PASS program. Compounds 3 and 5 showed more inhibition of DPPH stable free radical at 10^?4 M than the well-known standard antioxidant, butylated hydroxytoluene (BHT). Compound 5 exhibited promising in vitro inhibition of Fe²⁺-induced lipid peroxidation of the essential egg yolk as a lipid-rich medium (83.99%, IC₅₀ 16.07 ± 3.51 μM/mL) compared to α-tocopherol (α-TOH, 84.6%, IC50 5.6 ± 1.09 μM/mL). The parameters for drug-likeness of these BHT analogues were also evaluated according to the Lipinski's rule-of-five (RO5). All the BHT analogues were found to violate one of the Lipinski's parameters (LogP > 5), even though they have been found to be soluble in protic solvents. The predictive polar surface area (PSA) and absorption percent (% ABS) data allow us to conclude that they could have a good capacity for penetrating cell membranes. Therefore, one can propose these new multipotent antioxidants (MPAOs) as potential antioxidants for tackling oxidative stress and lipid peroxidation processes.

Full text of DOI:10.3390/molecules17077645

Molecules 2012, 17, 7645-7665; doi:10.3390/molecules17077645
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Butylated Hydroxytoluene Analogs: Synthesis and Evaluation of
Their Multipotent Antioxidant Activities
Wageeh A. Yehye 1, Noorsaadah Abdul Rahman 1, Abeer A. Alhadi 1, Hamid Khaledi 1,
Seik Weng Ng 1,2 and Azhar Ariffin 1,*
1
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia
2
Chemistry Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203,
Jeddah 21589, Saudi Arabia
* Author to whom correspondence should be addressed; E-Mail: azhar70@um.edu.my;
Tel.: +603-7967-4080.
Received: 9 April 2012; in revised form: 1 June 2012 / Accepted: 12 June 2012 /
Published: 25 June 2012
Abstract: A computer-aided predictions of antioxidant activities were performed with the
Prediction Activity Spectra of Substances (PASS) program. Antioxidant activity of
compounds 1, 3, 4 and 5 were studied using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and
lipid peroxidation assays to verify the predictions obtained by the PASS program.
Compounds 3 and 5 showed more inhibition of DPPH stable free radical at 10−4 M than the
well-known standard antioxidant, butylated hydroxytoluene (BHT). Compound 5 exhibited
promising in vitro inhibition of Fe2+-induced lipid peroxidation of the essential egg yolk as
a lipid-rich medium (83.99%, IC50 16.07 ± 3.51 µM/mL) compared to -tocopherol
(-TOH, 84.6%, IC50 5.6 ± 1.09 µM/mL). The parameters for drug-likeness of these BHT
analogues were also evaluated according to the Lipinski’s “rule-of-five” (RO5). All the
BHT analogues were found to violate one of the Lipinski’s parameters (LogP > 5), even
though they have been found to be soluble in protic solvents. The predictive polar surface
area (PSA) and absorption percent (% ABS) data allow us to conclude that they could have
a good capacity for penetrating cell membranes. Therefore, one can propose these new
multipotent antioxidants (MPAOs) as potential antioxidants for tackling oxidative stress
and lipid peroxidation processes.

Molecules 2012, 17
Keywords: butylated hydroxyltolouene; drug-likeness properties; DPPH; lipid peroxidation;
7646
multipotent antioxidant; rule-of-five; PASS and activity prediction; thiosemicarbazide;
1,2,4-triazole; 1,3,4-thiadiazole
1. Introduction
Reactive oxygen species (ROS) are generally considered responsible for many cell disorders and the
development of many undesired processes, including aging [1], inflammatory [2] and many others [3–7].
Phenolic primary antioxidants are the most active dietary antioxidants [8]. The commonly used
synthetic antioxidants in foods are butylated hydroxyanisole (BHA) [9], butylated hydroxytoluene
(BHT) [10] (Figure 1).
Figure 1. Chemical structures of compounds BHA, BHT, 1 and 2.
O
O
OH
OH
OH
NH
NH2
S
S
HO
HO
OMe
BHA
BHT
1
2
BHT (CAS 128-37-0; NCI C03598) was patented in 1947 [11]. It was found that two tert-butyl
groups flanking the OH group are required to retain in vivo anti-inflammatory potency [12]. Following
the same route, researchers of Parke-Davis have disclosed a new class of potent, selective and orally
active COX-2 inhibitors incorporating the 2,6-di-tert-butyl phenol moiety, such as PD 164387 and PD
138387 (Figure 2) [13,14].
Figure 2. Parke-Davis COX-2 inhibitors.
S
OCH3
N
H
O
N
S
N
S
N
OH
OH
PD 164387
PD 138387
Based on the Parke-Davis COX-2 inhibitors, researchers have investigated their SAR to improve
COX-2 selective inhibitorscontaining a 2,6-di-tert-butylphenol substituent as an antioxidant moiety [15].
Their selectivity might be related to their free-radical scavenging potency [15].

Molecules 2012, 17
7647
On the other hand, sterically hindered phenols linked with heterocyclic rings have also been
extensively studied as dual COX/5-LOX inhibitors [16–18]. A combination of radical scavenging
properties and anti-inflammatory activity has already been proven for a number of non-steroidal
anti-inflammatory drugs (NSAIDs) [19,20]. However, BHT derivatives have become attractive antioxidant
or co-antioxidant groups [21]. Therefore, it is no surprise that BHT has been modified to prepare a
series of new antioxidants having new medicinal properties in the pharmaceutical industry [12,22,23].
Recently, qualitative SARS and rational-design strategies for antioxidants have been used to combine
multiple functions; including various multiple antioxidant properties such as radical-scavenging ability
and diversified pharmacological activities to offer hybrid compounds, which can exert multiple
pharmacological functions through one molecular framework [24–26].
Thiosemicarbazides have been reported to show antibacterial [27], antimicrobial [28,29],
anti-toxoplasmagondii [29] and antioxidant [30] activities. To date acyl derivatives of thiosemicarbazide
bearing BHT moiety have been rarely synthesized.
Compounds containing the 1,3,4-thiadiazole nucleus have a wide range of pharmacological activities
that include antimicrobial [31], antitubercular [32], anticancer [33,34] and antioxidant [35] properties.
1,2,4-Triazoles are an important class of five membered heterocyclic compounds. 1,2,4-Triazole-5-
thiones are known for their anti-inflammatory [36], selective COX-2 inhibitor [37] and antimycotic [38]
and antioxidant [35] activity.
Regarding the effect of m-substituents, Tetsuto et al. [39] have evaluated the antioxidant activity of
different donating substituents on a m-substituted phenol, and found that the m-substituent does not
influence the antioxidant activity of a phenol at all. This is probably because a m-substituent shows
only a small resonance effect. In contrast to this fact, previous reports found that heterocyclic systems
with a halogen substituted at the m-position show greater antioxidant properties than those with other
substituents [40,41].
Since we could not find any reports on the use of electron withdrawing groups to enhance the
antioxidant activity of BHT, therefore, we used MPAO as an effective strategy to enhance the
antioxidant activity of BHT even if a strong electron withdrawing group (m-fluoro substituent) is a
basic part of the hybrid molecule.
In the present study four BHT derivatives have been synthesized. The acid-(base-)catalyzed
intramolecular dehydrative cyclization reaction of acylthiosemicarbazide 3 to the corresponding
1,3,4-thiadiazole 4 and 1,2,4-triazole 5 are described. Synthesized compounds have been characterized
by IR, NMR and mass spectral analysis. X-ray structures of 3 and 4 will be further discussed in this
paper. Potential biological effects of new compounds were predicted based on structure-activity
relationships with the PASS software. Antioxidant activities predicted by the PASS program were
experimentally verified by DPPH and TBARS (thiobarbituric acid reactive substance) assays.
Furthermore, a computational study for prediction of absorption, distribution (ADMET) [42] properties
of the synthesized compounds was performed by determination of polar surface area (PSA), absorption
(ABS) and Lipinski parameters.

Molecules 2012, 17
7648
2. Results and Discussion
2.1. Chemistry
Carboxylic acid 1 (Figure 1) was synthesized according to an established solvent-free procedure [43]
through the reaction between 2,6-di-tert-butyl-phenol with formaldehyde and thioglycolic acid in the
presence of di-n-butylamine. Hydrazide 2 (Figure 1) was synthesized using the indirect hydazination
method through the reaction of 3,5-di-t-butyl-4-hydroxybenzyl chloride with the sodium salt of
thioglycolic acid hydrazide [44].
The synthesis of thiosemicarbazide 3, 1,3,4-thiadiazole 4 and 1,2,4-triazole 5, described in this
study are outlined in Scheme 1. Acid hydrazide 2 was treated with 3-fluorophenylisothiocyanate to
give the corresponding acylthiosemicarbazide 3 in good yield. Compound 3 under acidic and basic
conditions gave thiadiazole 4 and triazole 5, respectively. The structure of the synthesized compounds
was confirmed based on their physical and spectral data. The structures of compounds 3 and 4 were
further confirmed by X-ray crystallography.
Scheme 1. Synthesis of compounds 3–5.
F
15
13
11
N
2
NH
N
10
9
S
Toluene
RT 6 h
8
Ar-NCS
O
S
3
7
50% H2SO4
HO
1
5
1
60%
4
NH
HN
S
10
9
8
H
N
S
3
5
7
15
2
S
11
13
HO
1
HN
10
N
3
9
N
8
F
S
3
7
11
3
92%
1) 25% K2CO3
2) AcOH
HO
1
15
5
13
F
70%
5
The IR spectra of all synthesized compounds showed strong absorptions at 3,623–3,653 cm−1,
attributed to free ν(Ar-O-H). Acylthiosemicarbazide 3 showed NH stretching bands between
3,285–3,340 cm−1, a C=O stretching band at 1,713 cm−1, and did not show any (S-H) band at 2,570 cm−1,
while the presence of a C=S stretching band at 1,248 cm−1 indicated that 3 exists in the thione form in
the solid-state [45,46]. Compound 4 showed two C=N stretching bands at 1,616 and 1,610 cm−1
attributed to the presence of the thiadiazole ring C=N. Compound 5 did not show any (S-H) band at
2,570 cm−1, while the presence of C=S stretching band at 1,252 cm−1 indicated that 5 exists in the
thione form in the solid state [42,43].
The 1H-NMR spectra of compound 3, recorded in CDCl3, showed a singlet peak at 8.32 ppm due to
NH attached to a phenyl group while the other two singlets at 9.26 ppm and 9.84 were attributed to the
hydrazido group NHs. Both appear as a broad band which supports the formation of intramolecular

Molecules 2012, 17
7649
hydrogen bonding [45,47] (Figure 3). The disappearance of the NH-1 and NH-2 groups of 3, combined
with a singlet at 11.75 ppm due to the presence of the NH-triazole of 5 suggest that a 1,2,4-triazole-5-
thione, and not a 1,2,4-triazole-5-thiol was formed.
Figure 3. Intramolecular hydrogen bonding of thioxo form of 1-acylthiosemicarbazide 3.
H
F
S
HO
N
N
S
O
N
H
H
In the 13C-NMR spectra, the greatest differences between the acylthiosemicarbazide 3 and its cyclic
derivatives 4 and 5 were found at the carbons designated C-8, C-9 and C-10. For C-8 in 3, this carbon
was bonded to C-9 (C=O). The electron-withdrawing effects of the oxygen of the C=O greatly
deshielded the C-8 and caused it to appear at a high chemical shift of 33.76 ppm. After acid
(base)-catalysed intramolecular dehydrative cyclizations of 3, the carbon at the same position (C-8) in
4 and 5 were bonded to the new C-9 due to the formation of thiadiazole and thiadiazole rings, respectively,
so it had a new electronic environment. This greatly lowered the chemical shift of the methylene (C-8)
13
to values of 29.96 and 24.68 ppm in 4 and 5, respectively. Interestingly, the heteronuclear C-19F
coupling constants of compounds 3, 4 and 5 were 972, 984 and 1,004 Hz, respectively.
2.2. Single Crystal X-ray Crystallography of Compounds 3 and 4
The crystal structure of molecule 3 is depicted in Figure 4 and the selected bond lengths and angles
are given in Table 1. In the crystal the molecule exists in its thione form. The two methylene carbon
atoms, C15 and C16, subtend an angle of 100.77 (7) at S1 atom. Several atom pairs of the molecule are
connected via N-H…O hydrogen bonding (Table 2) in a bifurcated system to form centro-symmetric
dimers. The hydroxyl group is shielded by the two di-tert-butyl residues and therefore is not involved
in any hydrogen bonding.
Figure 4. The molecular structures and labeling schemes of 3 (50% probability ellipsoids).

Molecules 2012, 17
Table 1. Selected bond lengths [Å] and bond angles [°] for 3 and 4.
7650
3
4
Bond lengths
1.6714(15)
S(2)-C(18)
S(2)-C(17)
S(2)-C(18)
O(1)-C(1)
N(1)-C(17)
N(1)-N(2)
N(2)-C(18)
N(3)-C(18)
1.728(6)
1.749(6)
1.376(6)
1.293(7)
1.395(6)
1.296(6)
1.365(7)
O(2)-C(17)
N(1)-C(17)
N(1)-N(2)
N(2)-C(18)
N(3)-C(18)
N(3)-C(19)
1.2417(18)
1.3201(18)
1.3757(17)
1.3490(18)
1.3563(19)
1.4108(18)
Bond angles
C(16)-S(1)-C(15) 100.77(7)
C(16)-S(1)-C(15) 100.4(3)
C(18)-N(3)-C(19) 130.8(5)
C(18)-N(2)-N(1)
N(2)-C(18)-S(2)
N(3)-C(18)-S(2)
120.45(13)
121.18(11)
128.05(11)
C(4)-C(15)-S(1)
113.1(4)
C(17)-C(16)-S(1) 112.1(4)
Table 2. Hydrogen-bond geometry for 3 and 4.
D-H···A
H···A [Å]
D···A [Å]
D-H···A [°]
3
N(3)-H(3N)...O(2) #1
N(2)-H(2N)...O(2) #1
2.040 (15)
2.019 (16)
2.8570 (16)
2.7927 (17)
158.1 (17)
150.4 (17)
4
N(3)-H(3N)...N(2) #2
2.05 (2)
2.910 (7)
169 (5)
Symmetry transformations used to generate equivalent atoms: #1 -x+1,-y+1,-z+1; #2 x+1/2,-y+1/2,-z+1.
Figure 5 presents the molecular structure of compound 4 and Table 1 compiles the selected bond
lengths and angles. The thiadiazole ring makes dihedral angles of 14.6 (3)° and 29.6(2)° with the C10-C24
and C1-C6 aromatic rings, respectively. Similar to what was observed in the structure of the
semicarbazide 3, the hydroxyl group is not involved in any hydrogen bonding as it is shielded by the
two sterically hindered tert-butyl groups. In the crystal, pairs of N3-H…N2 hydrogen bonds (Table 2)
link the molecules into centro-symmetric dimers.
Figure 5. The molecular structure and labeling scheme of 4 (50% probability ellipsoids).
The crystal data and structure refinement for compounds 3 and 4 is summarized in Table 3.

Molecules 2012, 17
7651
Table 3. Crystal data and refinement parameters for 3 and 4.
3
4
Empirical formula
Formula weight
C24 H32 F N3 O2 S2
477.65
C24 H30 F N3 O S2
459.63
Crystal system
Space group
Triclinic
P-1
Monoclinic
C 2/c
Unit cell dimensions
a [Å]
b [Å]
c [Å]
 [°]
9.3441 (5)
11.1548 (6)
11.7683 (6)
91.189 (2)
93.388 (2)
93.224 (2)
1,222.19 (11)
2
28.911 (11)
5.731 (2)
28.430 (11)
β [°]
99.330 (5)
γ [°]
Volume [Å3]
4,648 (3)
8
Z
Independent reflections
Observed reflections [I > 2σ(I)]
Final R indices [I > 2σ(I)]
R indices (all data)
5,587 [Rint = 0.0428]
4,988
R1 = 0.0405, wR2 = 0.1135 R1 = 0.0690, wR2 = 0.1340
R1 = 0.0448, wR2 =0.1160 R1 = 0.1792, wR2 = 0.1820
4,202 [Rint = 0.2119]
1,848
2.3. Computational Evaluation of Biological Activity
The biological activity spectra of the target compounds were obtained using the Prediction of
Activity Spectra for Substances (PASS) software [48]. PASS prediction tools are constructed using
20,000 principal compounds from the MDDR database (produced by Accelrys and Prous Science) [49].
The database contains over 180,000 biologically relevant compounds and is constantly updated [49].
PASS web tool has the ability to predict about 4,000 kinds of biological activity on the basis of
structural formula with mean accuracy of about 90% [50,51], therefore, it is reasonable to use PASS
for finding and optimizing new lead compounds. The PASS training set which has been compiled from
various sources including publications, patents, chemical databases, and “gray” literatures consists of
over 26,000 biological compounds and includes drugs, lead compounds, drug-candidates, and toxic
substances. Algorithm of activity spectrum estimation is based on Bayesian approach. The result of
prediction is presented as the list of activities with appropriate Pa and Pi ratio. Pa and Pi are the
estimates of probability for the compound to be active and inactive respectively for each type of
activity from the biological. It is reasonable that only those types of activities may be revealed by the
compound, which Pa > Pi. If Pa > 0.3 the compound is likely to reveal this activity in experiments, but
in this case the chance of being the analogue of the known pharmaceutical agents for this compound is
also high. A portion of the predicted biological activity spectra (lipid peroxidase inhibitor, antioxidant,
free radical scavenger and antiinflammatory) for the synthesized compounds and BHT are given in
Table 4.

Molecules 2012, 17
Table 4. Part of the predicted biological activity spectra for the compounds 1, 3–5 and BHT.
7652
Mode of
1
3
4
5
BHT
biological activity
Pa
Pi
Pa
Pi
Pa
Pi
Pa
Pi
pa
Pi
Lipid peroxidase 0.652 0.006 0.436 0.027 0.485 0.019 0.639 0.007 0.843 0.003
inhibitor
Antioxidant
Free radical
scavenger
0.712 0.004 0.385 0.035 0.420 0.028 0.529 0.015 0.845 0.003
0.807 0.004 0.585 0.025 0.554 0.031 0.506 0.042 0.797 0.004
Antiinflammatory 0.659 0.017 0.375 0.136 0.649 0.018 0.479 0.065 0.804 0.005
Pa—probability “to be active”; Pi—probability “to be inactive”.
2.4. Molecular Properties and Drug-Likeness
2.4.1. Calculation of Drug-Likeness Properties
Drug-likeness can be defined as a delicate balance between various structural features, which
determine whether a particular molecule is similar to recognized drugs. It generally means molecules,
which contain functional groups and/or have physical properties consistent with most of the known
drugs. These properties are; absorption, distribution, metabolism, and excretion in the human body like
a drug. Lipinski [52] used these molecular properties in formulating his Rule of Five. The rule states
that the most molecules with good membrane permeability have logP < 5, molecular weight <500,
number of hydrogen bond acceptors <10, number of hydrogen bond donors <5. Number of rotatable
bonds is important for conformational changes of molecules under study and ultimately for the binding
with receptors or channels [53]. It is revealed that for passing oral bioavailability criteria, number of
rotatable bond should be (≤10). The synthesized compounds 1 and 3–5 in general possessed high
number of rotatable bonds (6–10) and therefore, obviously exhibit large conformational flexibility.
2.4.2. Lipophilicity
Generally, lipophilic antioxidants demonstrated more potent scavenging properties than hydrophilic
antioxidants. Vitamin E is a fat-soluble antioxidant [54]. Distribution of this vitamin within the
membrane has been shown to alter its antioxidant potency [55]. The Partition Coefficient is a measure
of how well a substance partitions between a lipid and water. It is therefore important to determine
these physico-chemical properties associated with an antioxidant activity. LogP measurements (P is
partition coefficient of the molecule in the water-octanol system) are a useful parameter for use in
understanding the behaviour of antioxidant molecules. For calculation of logP, we used computed
logP values by using ADMET predictive software as shown in Table 5. Compounds having logP ≥ 5
should be considered as a higher lipophilicity have higher permeation across biological membranes
(but lower aqueous solubility) [56]. The logP values showed that the order of lipophilicity was
1-acyl-semicarbazide 3 < triazole 5 (thione form) < thiadiazole 4 and 5 (thiol form). Compounds 3–5
showed lipophilic properties with logP values between 6.439–6.967 while natural lipophilic
antioxidant, -tocopherol (-TOH) (10.44) and, hydrophilic antioxidant, ascorbic acid (−1.70).

Molecules 2012, 17
7653
Table 5. Predicted ADMET and Lipinski’s parameters.
Violation of
Rule of 5 (≤1) (≤10) (≤5)
HBA HBD LogP
(≤5)
MW
(≤500)
NROTB
(≤10)
PSA A2
Compound
%ABS
≤90
58.93
76.54
56.14
48.30
48.68
20.81
29.74
109.49
1
3
4
0
1
1
1
4
4
5
4
5
1
-
2
4
2
2
2
1
-
4.41 310.452
6.43 477.658
6.94 459.643
6.84 459.643
6.96 459.643
4.87 220.350
10.44 430.71
−1.70 176.12
6
10
8
7
7
2
-
-
88.66
82.59
89.63
92.33
92.21
101.81
98.73
71.23
5 (thione)
5 (thiol)
BHT
Vitamin E *
Vitamin C *
0
-
-
-
-
* Vitamin E and C are outside the “rule of 5” [52], BHT—butylated hydroxytoluene,
HBA—hydrogen bond acceptor, HBD—hydrogen bond donor, NROTB—number of rotated bonds,
PSA—polar surface area.
2.4.3. Violations of Lipinski’s Rule of Five
It is very important to note that there are many violations of this rule among currently existing drugs
and vice versa, but in general, an orally active drug has no more than one violation. Compounds
violating more than one of the Lipinski Rules are reasonably assumed to have bioavailability problems
and are therefore, presumably unsuitable as drugs [57]. However, qualifying the RO5 does not absolute
guarantee that a molecule is “drug-like” [58]. Moreover, the RO5 says nothing about specific
chemistry structural features found in drugs or non-drugs [59]. Polar surface area (PSA) is basically
recognized as a good indicator of drug absorbance in the intestines, Caco-2 monolayers penetration,
and blood-brain barrier crossing [58].
The earlier mentioned parameters were calculated for the BHT derivatives in analysis, and the
results are depicted in Table 4. From the data obtained, one can notice that the synthesized compounds
possess an adequate number of proton acceptor and proton donor groups to ensure efficient interaction
with the hydrogen-bonding groups of the receptors. Hydrogen-bonding capacity has been also
identified as an important parameter for describing drug permeability [60]. All the BHT derivatives
were found to violate one of the Lipinski’s parameters (logP ≤ 5), although they have been found to
have solubility in protic solvents. Magnitude of absorption is expressed by the percentage of
absorption; Absorption percent was calculated [61] using the equation: % ABS = 109 – (0.345 × PSA).
Accordingly, to their predictive low PSA (PSA values are considerably less than 90 A2) and high %
ABS data it seems that this type of antioxidants could have a good capacity for penetrating cell
membranes [53].
2.5. In Vitro Antioxidant Activities
In present study, antioxidant activities of five BHT derivatives were carried out by two well known
in vitro antioxidant assays, DPPH and TBARS. The effects of antioxidants in the DPPH-radical-
scavenging test reflects the hydrogen-donating capacity of a compound. In its radical form, DPPH•
absorbs at 570 nm. The radical form of DPPH is scavenged by an antioxidant through the donation of a

Molecules 2012, 17
7654
hydrogen atom to form a stable DPPH molecule. This leads to a colour change from purple to yellow,
and a corresponding decrease in absorbance [62,63].
The thiobarbituric acid reactive substances (TBARS) assay is used for screening and monitoring
lipid peroxidation. The basis of TBARS assay is the absorbance of a pink coloured complex that is
monitored spectrophotometrically at 532–535 nm which is formed between thiobarbituric acid and the
oxidation products of unsaturated lipids [64,65].
2.5.1. In Vitro DPPH Free Radical Scavenging Activity
Compound 3 showed significant levels of % inhibition of DPPH radical compared to the standard
antioxidant (BHT) used in the study (Table 6). As shown in this Table, thiosemicarbazide 3 exhibited
strong scavengings effect on DPPH stable radical, with an IC50 value of 68.03 ± 1.27 µM/mL.
Table 6. IC50 value and % inhibition of the DPPH radical scavenging and lipid peroxidation
inhibition assays.
IC50 a Values (µM/mL) ± S.E.M b and Max. inhibition % ± S.E.M
Compounds
DPPH Radical Scavenging
96.73 ± 1.87 (51.25 ± 0.82)
68.03 ± 1.27 (65.21 ± 0.55)
> 100 c (26.09 ± 0.33)
85.30 ± 1.16 (65.26 ± 0.38)
>100 c (25.23 ± 0.17)
67.77 ± 0.17 (71.93 ± 1.61)
-
Lipid Peroxidation Inhibition
38.84 ± 1.54 (73.99 ± 1.30)
56.00 ± 5.05 (74.64 ± 1.68)
33.20 ± 2.91 (84.99 ± 1.37)
16.07 ± 3.51 (83.99 ± 1.65)
36.67 ± 1.78 (79.45 ± 1.27)
-
1
3
4
5
BHT
Ascorbic acid
-TOH
5.63 ± 1.09 (84.69 ± 1.23)
a IC50: 50% effective concentration; b S.E.M: standard error of the mean; c did not reach 50% inhibition.
This value was lower than that of the thiadiazole 4, triazole 5, BHT and quite close to that of
ascorbic acid (67.77 ± 0.17 µM/mL), which was used as a positive control in the study, clearly
indicating that thiosemicarbazide 3 has good radical scavenging activity. Presumably, significantly
greater inhibition of lipid peroxidation could be attributed to the phenylthiourea system which is the
active center of antioxidation, acts as the active center of containing thione and secondary aromatic
amine antioxidants (Figure 6).
Figure 6. Phenylthiourea system.
thione
S
active redox center
aromatic amine
primary antioxidant
N
NH
H
This result is in agreement with previous reports whereby thiourea compounds exhibited
antioxidant activity, while S-alkylation virtually eliminated the antioxidant activity, which indicated

Molecules 2012, 17
7655
that an unalkylated thiourea group is critical to antioxidant activity. In addition, according to the
published report, thiosemicarbazide obviously showed greater antioxidant property than triazole and
1
thiadiazole [66]. The X-ray structures (Figure 4) and also the H-NMR data of 3 revealed that the
thione form is formed. Thus, the DPPH inhibition result of compound 3 suggested that the thione form
is largely responsible for the antioxidant activity due to its favourable electron-donating characteristics.
In addition to that, the active redox center of compound 3 is the Ar-NH- (Figure 6) which increases
the rate of H-atom transfer to neutralize the DPPH• by delocalization of the nitrogen electron pair over
the aromatic system and the thione group, forming a stable aminyl radical, thus yielding a DPPH•
inhibition of 65.21% for 3, while ascorbic acid and BHT gave values of 71.93% and 25.23%, respectively.
Compound 4 did not show any DPPH radical-scavenging activity compared to BHT and 1. This
could be attributed to the fact that the electron-withdrawing groups produce a strengthening of the N-H
bond. This behavior can be attributed to the conjugative effect of the substituents [67,68].
Table 6 clearly shown the % inhibition of compound 5 to be higher than BHT and close to that of
ascorbic acid. Presumably, like 4, the 1,2,4-triazole is present in the thione form as a planar molecule.
The increased stability of the thione form (I) over thiol form (II) (Scheme 2) has been shown
theoretically, and the sulfur atom is found to be the atom with largest electron density in the molecule [69].
Thus, the chemical behaviour of the system with respect to free radicals would largely depend on the
tautomeric form present in the reaction, reasonably assuming that the sulfur atom is involved in the
reactive site.
Scheme 2. Thione I and thiol II tautomers of 5.
H
N N
N N
H
S
S
N
N
Ar
I
Ar
II
Although we don’t have an exact explanation for the greater activity of 5, we have some logical
arguments. Heterocyclic systems with halogen substituents have shown greater antioxidant properties
than other substituents [40,41]. Takanori et al. [70] studied the antioxidant activity of o-benzyl-substituted
phenols and found that electron-withdrawing groups on benzyl substituent decreases the electron
density of methylene, making hydrogen transfer to a phenoxy radical easier and regenerated the
antioxidant activity of phenols. Thus, the fluorine group in compound 5 seemed to enhance free radical
scavenging by making the NH of triazole ring easy to regenerate reversibly recycling antioxidant
activity [71] or this might be due to its electron-donating mesomeric effect [39].
2.5.2. In Vitro Lipid Peroxidation Activity
Contrary to the DPPH assay results, compound 3 exhibited the lowest lipid peroxidation activity
yielding the highest IC50 value of 56 ± 5.05 µM/mL. Compound 4 was higher than BHT, with an IC50
value of 33.20 ± 2.91 µM/mL. Interestingly, thiadiazole 5 exhibited the strongest inhibitory activity
with IC50 16.07 ± 3.51 µM/mL (inhibition = 83.99%) which is two times better than the IC50 of
the standard antioxidant BHT with IC50 = 36.67 ± 1.1.78 µM/mL and close to that of vitamin E

Molecules 2012, 17
7656
(IC50 = 5.63 ± 1.09 µM/mL, % inhibition = 84.69). It is known that -TOH and BHT are strongly
lipophilic antioxidants due to their long hydrocarbon chain and di-tert-butyl groups similar to the tail
of a fatty acid and therefore, reach a higher level of bioavailability [72]. Presumably, the significantly
greater differences between the lipid peroxidation values of 3, 4 and 5 could be attributed to the
balance between the hydrophilicity of the polar moieties and the lipophilicity of the hydrocarbon
moieties. Thus, the stronger inhibitory activity of compound 5 could be attributed to hydrophilic and also
lipophilic effects on emulsified oils. In emulsions, which is the medium of the TBARS assay, non-polar
free radical scavengers accumulate in the lipid phase and at the oil-water interface where interactions
between hydroperoxides at the droplet surface and prooxidants in the aqueous phase occur [73–75].
Structurally, in 2010 Imtiaz et al. [76] found triazole derivatives to be relatively more active than
the derivatives of thiadiazole. These may be regarded as substrate-like inhibitors on the basis of their
structural similarity with the natural substrate of urease (i.e., urea). Similarly, hydroxyphenylurea
derivatives exhibit 10 times higher antioxidant activity than -TOH [24]. Thus, results of lipid
peroxidation inhibition of compound 5 suggested that a 1,2,4-triazole bearing a BHT moiety and
m-flourophenyl can donate electron/hydrogen easily. However, the m-flouro substituent could also
enhance the antioxidant activity of BHT similar to donating groups, which could be attributed to its
electron-donating mesomeric effect [41] but not to the conjugative effect [67,68], and maybe to
intermolecular lipophilicity effects [77].
LogP calculations of 4 and 5 (Table 5) showed the thione form of 5 (logP = 6.84) to have only
slight more polarity than 4 (logP = 6.94) due to thiol-thione tautomerism effect, which suggested
compound 4 and 5 to have hydrophilic and lipophilic groups, which may act as amphiphilic
antioxidants in one molecule rather than having to use two antioxidants separately. Thus, results of
lipid peroxidation inhibition of compound 5 suggested that it to be possible candidate with promising
anti-oxidant activities.
3. Experimental
3.1. General
All materials and solvents were obtained from Sigma-Aldrich. Melting points were determined on a
MEL-TEMP II melting point instrument. IR spectra were recorded on a Perkin-Elmer RX1 FT-IR
1
13
spectrometer. The H- (400 MHz) and C-NMR (100 MHz) spectra were recorded on JEOL ECA
400 MHz FT-NMR using CDCl3 or DMSO-d6 as solvent and tetramethylsilane as an internal standard.
HR-mass spectra (ESI) were obtained with a MAT 95 xl-T mass spectrometer operating at 70 ev.
UV-visible spectra were recorded on a UV-1650PC model UV-visible spectrophotometer.
2-(2-(3,5-di-tert-Butyl-4-hydroxybenzylthio)acetyl)-N-(3-luorophenyl)hydrazinecarbothioamide
(3).
To a solution of S-(3,5-di-t-butyl-4-hydroxybenzyl)thioglycolic acid hydrazide (2, 0.45 g, 1.39 mmol)
in dry toluene or benzene (5 mL), was added 3-fluorophenylisothiocyanate (0.21 g, 1.39 mmol) and the
reaction mixture was stirred for 2 h at room temperature. The resulting precipitate was collected by
filtration, washed with boiled hexane, dried at room temperature, and recrystallized from toluene to
give white solid, yield 92% (0.60 g), mp 180–182 °C. IR (KBr pellet), cm−1: ν = 3,633 (free OH,
BHP), 3,340, 3,314 and 3,285 (NH), 2,873–2,961 (C-H of t-Bu), 1,713 (C=O), 1,248 ν (C=S). 1H-NMR

Molecules 2012, 17
7657
(CDCl3), δ, ppm: 1.40 (s, 18H, 2 × t-Bu), 3.22 (s, 2H, H-8), 3.78 (s, 2H, H-7), 5.20 (s, 1H, OH, BHP),
6.62–6.66 (dt, 1H, H-14, 3JF-H = 8.2, 3J = 8, 4J = 2.3 Hz), 6.85–6.87 (d, 1H, H-16, 3J = 8 Hz), 7.04–7.10
(m, 1H, H-15), 7.10 (2H, H-3, H-5), 7.20–7.25 (m, 1H, H-12), 7.97 (b, 2H, NH-2, NH-3), 8.90 (1H,
NH-1). 13C-NMR (CDCl3), δ, ppm: 30.30 (6C, 2 × -C(CH3)3, 33.51 (1C, C-8), 34.41 (2C, 2 × -C(CH3)3),
2
2
37.48 (1C, C-7), 106.65 (d, 1C, C-12, JC-F = 104 Hz ), 110.01 (d, 1C, C-14, JC-F = 88 Hz ), 114.52
(1C, C-16), 125.89 (2C, C-3, C-5), 126.93 (1C, C-4), 129.94 (d, 1C, C-15, 3JC-F = 36 Hz), 136.41 (2C,
C-2, C-6), 139.60 (d, 1C, C-11, 3JC-F = 44 Hz), 153.35 (1C, C-1), 154.55 (1C, C-10), 163.02 (d, 1C, C-13,
1JC-F = 972 Hz), 170.13(1C, C-9). HREIMS m/z 477.1918 [M]+ (calcd for C24H32O2N3 FS 477.1920).
139.60
3J
(d, C-F = 44)
106.65
(d, 2JC-F = 104)
5.20
(m, 7.20-7.25)
34.41
136.41
129.93
154.55
(b, 7.97)
163.02
8.9
S
H
N
2
H
N
(1JC-F = 972)
153.35
H
N
H
N
OH
F
OH
1
NH
F
3
NH 10
3
11
13
3
11
13
1
1
9
5
15
5
15
S
S
8
S
7
7
8
110.01
O
O
6.62-6.66
(d, 2JC-F = 88)
(dt, JF-H =8.2, 3J=8, 4J=2.3)
114.52
6.85-6.87
37.48
129.94 (d, 3JC-F = 36)
(d, 3J=8)
170.13
3.78 3.22
30.30
125.89
7.10
1.40
(m, 7.04-7.10)
33.51
2,6-di-tert-Butyl-4-(((5-(3-fluorophenylamino)-1,3,4-thiadiazol-2-yl)methylthio)methyl)phenol
(4).
Thiosemicarbazide 3 (0.23 g, 0.48 mmol) was added gradually under stirring to a cooled sulphuric acid
solution (50%, 5 mL) for 10 min. The reaction mixture was heated for 20 min at 100 °C. It was then
poured over crushed ice under stirring. The solid precipitated was filtered, washed with water, dried
and recrystallized from MeOH 4:1 H2O, white solid, Yield 60% (0.13 g). mp 175–177 °C. IR (KBr
pellet), cm−1: ν ꢀ 3,623, 3,631, 3,653 (free OH, BHP), 2,873–2,958 (C-H of t-Bu), 1,616, 1,610
(2 C=N). 1H-NMR (CDCl3), δ, ppm: 1.40 (s,18H, 2 × t-Bu), 3.69 (s, 2H, H-7), 3.89 (s, 2H, H-8), 5.12
3
3
4
(s, 1H, OH, BHP), 6.76–6.81 (ddt, 1H, H-14, JF-H = 8.5, J = 8.4, J = 0.8 Hz), 7.07 (2H, H-3, H-5),
3
4
3
4
7.09–7.12 (dd, 1H, H-16, J = 8.2, J = 0.92 Hz), 7.14–7.18 (dt, 1H, H-12, JF-H = 10.4, J = 2.7,
4J = 2.4 Hz), 7.27–7.33 (dt, 1H, H-15, J = 8.5, J = 8 Hz), 8–9 (b, NH). C-NMR (CDCl3), δ, ppm:
3
3
13
29.96 (1C, C-8), 30.33 (6C, 2 × -C(CH3)3), 34.40 (2C, 2 × -C(CH3)3), 36.62 (1C, C-7), 112.20 (d, 1C,
2
2
4
C-12, JC-F = 103 Hz), 110.3 (d, 1C, C-14, JC-F = 88 Hz), 113.62 (1C, C-16, JC-F = 11.2 Hz), 125.97
(2C, C-3, C-5), 127.50 (1C, C-4), 130.96 (d, 1C, C-15, 3JC-F = 38.4 Hz), 136.18(2C, C-2, C-6), 141.73
3
(d, 1C, C-11, JC-F = 42 Hz), 153.18 (1C, C-1), 160.17 (1C, C-9), 163.54 (d, 1C, C-13,
1JC-F = 984 Hz), 166.59 (1C, C-10). HREIMS m/z 459.1759 [M]+ (calcd for C24H30O1N3FS2 459.1814).
7.14-7.18
112.20 (d, 2 C-F = 103)
J
3
(dt, JF-H = 10.4, 4J = 2.7, 4J = 2.4)
29.96
3.89
163.54 (d, 1JC-F = 984)
36.62
3.69
F
F
6.76-6.81
127.50
(ddt, 3 F-H = 8.5,
J
N
N
N
N
3
13
= 8.4, 4 =0.8)
110.3 (d, 2 C-F = 88)
136.18
13
15
J
J
J
9
9
10
10
8
8
11
11
130.96 (d, 3JC-F = 38.4)
15
S
S
S
S
N
H
N
H
7
7
7.27-7.33
(dt, 3J= 8.5, 3J=8)
3
3
166.59
125.97
5
5
1
113.62 (4
= 11.2)
C-F
1
7.07
J
(b, 8-9).
7.09-7.12
HO
HO
(dd, 3J = 8.2, 4J = 0.92)
141.73 (d, 3JC-F = 42)
34.40
30.33
1.40
153.18
5.12

Molecules 2012, 17
7658
3-((3,5-di-tert-Butyl-4-hydroxybenzylthio)methyl)-4-(3-fluorophenyl)-1H-1,2,4-triazole-5(4H)-thione (5).
A mixture of thiosemicarbazide 3 (0.23 g, 0.48 mmol) and potassium carbonate (25%, 5 L) was stirred
for 18 h. Water (250 mL) was added and the solution was stirred for 1 h, the solution was adjusted to
pH 5–6 with dilute hydrochloric acid and was kept aside for 1 h. The resulting white precipitate was
filtered, washed with water, dried and and recrystallized from MeOH 4:1 H2O to give white sold, yield
70%, (0.16 g), mp 183–185 °C. IR (KBr pellet), cm−1: ν = 3,633 (free OH, BHP), 3,088, 3,014 (NH),
1
2,874, 2,930, 2,956 (C-H of t-Bu), 1,608 (C=N). 1,252.9 (C=S). H-NMR (CDCl3), δ, ppm: 1.41
(s, 18H, 2 × t-Bu), 3.36 (s, 2H, H-8), 3.64 (s, 2H, H-7), 5.18 (s, 1H, OH, BHP), 7.08 (2H, H-3, H-5),
3
3
4
7.14–7.19 (m, 2H, H-12, H-16), 7.21–7.26 (dt, 1H, H-14, JF-H = 10.4, J = 8, J = 4 Hz), 7.49–7.54
(dt, 1H, H-15, 3J = 8, 3J = 8 Hz), 11.75 (b, NH). 13C-NMR (CDCl3), δ, ppm: 24.67 (1C, C-8), 30.35 (6C,
2 × -C(CH3)3), 34.43 (2C, 2 × -C(CH3)3), 36.26 (1C, C-7), 116.29 (d,1C, C-12, 2JC-F = 96 Hz), 117.65 (d,
4
1C, C-14, 2JC-F = 84 Hz), 124.25 (d, 1C, C-16, JC-F = 16 Hz), 126.06 (2C, C-3, C-5), 126.93 (1C, C-4),
3
3
131.16 (d, 1C, C-15, JC-F = 36 Hz), 134.13 (d, 1C, C-11, JC-F = 40 Hz), 136.19 (2C, C-2, C-6),
150.12 (1C, C-9), 153.25 (1C, C-1), 162.88 (1C, C-13, 1JC-F = 1004 Hz), 169.14 (1C, C-10). HREIMS
m/z 459.1793 [M]+ (calcd for C24H30O1N3 F1 32S2 459.1814).
169.14
(b, 11.75)
150.12
30.35
1.41
7.08
H
H
134.13 (d, 3JC-F = 40)
124.25 (d, 4JC-F = 16)
34.43
N
N
S
S
10
10
N
126.93
N
9
(m, 7.14-7.19)
9
N
N
8
8
S
S
11
11
3
3
7.49-7.54
7
7
15
15
(dt, 3J= 8, 3J=8)
HO
1
HO
1
13
13
131.16 (d, 3JC-F = 36)
117.65 (d, 2JC-F = 84)
5
5
24.67
36.26
3.36
F
F
3.64
7.21-7.26
(dt, 3JF-H=10.4, 3J=8, 4J=4)
5.18
162.88 (d, 1JC-F = 1004)
153.25
136.19
116.29 (d, 2 C-F = 96)
126.06
J
3.2. X-ray Crystallography
Diffraction data were measured using a Bruker SMART Apex II CCD area-detector diffractometer
(graphite-monochromated Mo K radiation, =0.71073 Å). The orientation matrix, unit cell refinement
and data reduction were all handled by the Apex2 software (SAINT integration, SADABS absorption
correction) [78]. The structures were solved using direct method in the program SHELXS-97 [79] and
were refined by the full matrix least-squares method on F2 with SHELXL-97. Drawings of the
molecules were produced with XSEED [80]. Crystal data and refinement are summarized in Table 3.
CCDC 874287 and CCDC 874288 contain the supplementary crystallographic data for 3 and 4,
respectively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2
1EZ, UK; fax: (+44)-1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
3.3. Antioxidant Assays
3.3.1. DPPH Free Radical Scavenging Assay
DPPH radical scavenging assay was carried out according to the literature [81] with some
modifications. To a range of various concentrations 100, 10, 1, 0.1 and 0.01 µM/mL of samples DPPH

Molecules 2012, 17
7659
solution (1.0 mL, 200 µM in DMSO) was added. 22.03–47.76 mg (1 × 10−4 M) of test compound was
dissolved in DMSO (1.0 mL, 100%) as a stock solution. This stock solution was then diluted to a range
of final extraction concentrations 100, 10, 1, 0.1 and 0.01 µM. A negative control with the same DPPH
concentration in DMSO without sample was used. Each assay was carried out in triplicates. The
mixture was then incubated in dark for 60 min at room temperature. Absorbance at 570 nm for each
sample was then measured. Ascorbic acid was used as positive control. The free radical scavenging
activity of the compounds was calculated as a percentage of radical inhibition by using the formula:
Percentage of Inhibition (%) = [(Ac − As)/Ac] × 100
in which As = Absorbance of the compounds/ positive control and Ac = Absorbance of control (DPPH
solution and DMSO). To determine the concentration required to achieve 50% inhibition (IC50) of DPPH
radical, the percentage of DPPH inhibition for each compound was plotted against extract concentration.
3.3.2. Lipid Peroxidation Inhibition Assay
The lipid peroxidation inhibition assay was carried out according to the reported method with some
modifications [82]. Fowl egg yolk, comprising mainly of phospholipids, proteins and triacyglycerol,
was used as an alternative to rat liver microsomes and linoleic acid. The reactive mixture for the
induction of lipid peroxidation included egg yolk (1.0 mL) emulsified with phosphate buffer saline
(0.1 M, pH7.4), to a final concentration of 12.5 g/L and 3,000 µM FeSO4 (200 µL). Test compound
(22.03–47.76 mg, 1 × 10−4 M) was dissolved in DMSO (1.0 mL, 100%) as a stock solution. This stock
solution was then diluted to a range of final extraction concentrations 100, 10, 1, 0.1, 0.01 and 0.001 µM.
Each assay was carried out in triplicates. The mixture was incubated at 37 °C for 1 h, after which it
was treated with freshly prepared trichloracetic acid (TCA, 15%, 0.5 mL) and thiobarbituric acid
(TBA, 1%, 1.0 mL). The reaction mixtures were then incubated in boiling water for 10 min. Upon
cooling, the mixtures were centrifuged at 3,500 rpm for 10 min. The formation of TBARS was
measured by removing 100 µL of supernatant and measuring the absorbance at 532 nm. α-TOH was
used as positive control. The percentage of inhibition was calculated from the following equation:
% inhibition = [Ac − As/Ac] × 100
where As = Absorbance of the compound and Ac = Absorbance of control.
To determine the concentration required to achieve 50% inhibition (IC50) of phospholipid oxidation
in egg yolk, the percentage of lipid peroxidation inhibition was plotted against extract concentration.
4. Conclusions
Our design strategy involved combining the good features of two or more antioxidants into one
structure. This strategy was applied in an attempt to significantly improve the antioxidant activities of
the well-known antioxidant BHT to create novel MPAOs bearing meta electron withdrawing groups.
We have demonstrated the improvement in the free-radical scavenging capacity of BHT as determined
by inhibition (25.23%) of DPPH free radical to be more than two-fold. Compound 5 is the most potent
compound in the in vitro lipid peroxidation and obviously exhibits promising in vitro inhibition of
Fe2+-induced lipid peroxidation of essential oils. The synthesized compounds 3–5 satisfied Lipinski’s

Molecules 2012, 17
7660
RO5 and ADMET properties. RO5 and ADMET predictions can be important initial steps toward the
development of novel pharmaceuticals in the fight against free radicals. Compounds 4 and 5 have
exactly same molecular weight but different polarity and antioxidant activity, and therefore, PASS and
MPAO design strategies can be effectively used for finding of compounds with required properties and
without undesirable side effects. m-Flouro substituents could significantly enhance the antioxidant
activity of BHT derivatives like other electron donating groups.
Acknowledgments
The authors wish to acknowledge the grants (PS190/2008A, PS381/2009B and RG149-11AFR)
provided by the University of Malaya to conduct this study.
References and Notes
1. Schöneich, C. Reactive oxygen species and biological aging: A mechanistic approach.
Exp. Gerontol. 1999, 34, 19–34.
2. Sahinoglu, T.; Stevens, C.R.; Bhatt, B.; Blake, D.R. The role of reactive oxygen species in
inflammatory disease: Evaluation of methodology. Methods 1996, 9, 628–634.
3. Schöneich, C. Methionine oxidation by reactive oxygen species: Reaction mechanisms and
relevance to alzheimer’s disease. Biochim. Biophys. Acta 2005, 1703, 111–119.
4. Oka, M.; Tachibana, M.; Noda, K.; Inoue, N.; Tanaka, M.; Kuwabara, K. Relevance of
anti-reactive oxygen species activity to anti-inflammatory activity of components of eviprostat, a
phytotherapeutic agent for benign prostatic hyperplasia. Phytomedicine 2007, 14, 465–472.
5. Kirkinezos, I.G.; Moraes, C.T. Reactive oxygen species and mitochondrial diseases. Semin. Cell
Dev. Biol. 2001, 12, 449–457.
6. de Maria, N.; Colantonl, A.; Fagiuoli, S.; Liu, G.-J.; Rogers, B.K.; Farinati, F.; van Thiel, D.H.;
Floyd, R.A. Association between reactive oxygen species and disease activity in chronic hepatitis C.
Free Radic. Biol. Med. 1996, 21, 291–295.
7. Smith, C.; Zhang, Y.; Koboldt, C.; Muhammad, J.; Zweifel, B.; Shaffer, A.; Talley, J.; Masferrer, J.;
Seibert, K.; Isakson, P. Pharmacological analysis of cyclooxygenase-1 in inflammation.
Proc. Natl. Acad. Sci. USA 1998, 95, 13313–13318.
8. Shahidi, F.; Naczk, M.; Griffiths, W. Food Phenolics: Sources, Chemistry, Effects, Applications;
Technomic Publishing: Lancaster, PA, USA, 1995.
9. Bandyopadhyay, M.; Chakraborty, R.; Raychaudhuri, U. A process for preparing a natural
antioxidant enriched dairy product (sandesh). LWT-Food Sci. Technol. 2007, 40, 842–851.
10. Dacre, J. The metabolism of 3: 5-di-tert-butyl-4-hydroxytoluene and 3: 5-di-tert-butyl-4-
hydroxybenzoic acid in the rabbit. Biochem. J. 1961, 78, 758–766.
11. Stecher, P. Butylated hydroxytoluene. In The Merck Index, 8th ed.; O’Neil, M.J., Ed.;
Merck Research Laboratories: Rahway, NJ, USA, 1968; p. 179.
12. Lazer, E.S.; Wong, H.C.; Possanza, G.J.; Graham, A.G.; Farina, P.R. Antiinflammatory
2,6-di-tert-butyl-4-(2-arylethenyl)phenols. J. Med. Chem. 1989, 32, 100–104.

Molecules 2012, 17
7661
13. Song, Y.; Connor, D.; Sercel, A.; Sorenson, R.; Doubleday, R.; Unangst, P.; Roth, B.; Beylin, V.;
Gilbertsen, R.; Chan, K. 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. 2. 1,3,4- and 1,2,4-Thiadiazole series. J. Med. Chem. 1999, 42,
1161–1169.
14. Marnett, L.J.; Kalgutkar, A.S. Design of selective inhibitors of cyclooxygenase-2 as
nonulcerogenic anti-inflammatory agents. Curr. Opin. Chem. Biol. 1998, 2, 482–490.
15. Inagaki, M.; Tsuri, T.; Jyoyama, H.; Ono, T.; Yamada, K.; Kobayashi, M.; Hori, Y.; Arimura, A.;
Yasui, K.; Ohno, K. Novel antiarthritic agents with 1,2-isothiazolidine-1,1-dioxide ([gamma]-sultam)
skeleton: Cytokine suppressive dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase. J. Med.
Chem. 2000, 43, 2040–2048.
16. Unangst, P.C.; Shrum, G.P.; Connor, D.T.; Dyer, R.D.; Schrier, D.J. Novel 1,2,4-oxadiazoles and
1,2,4-thiadiazoles as dual 5-lipoxygenase and cyclooxygenase inhibitors. J. Med. Chem. 1992, 35,
3691–3698.
17. Unangst, P.C.; Connor, D.T.; Cetenko, W.A.; Sorenson, R.J.; Kostlan, C.R.; Sircar, J.C.;
Wright, C.D.; Schrier, D.J.; Dyer, R.D. Synthesis and biological evaluation of 5-[[3,5-bis(1,1-
dimethylethyl)-4-hydroxyphenyl]methylene]oxazoles, -thiazoles, and -imidazoles: Novel dual
5-lipoxygenase and cyclooxygenase inhibitors with antiinflammatory activity. J. Med. Chem.
1994, 37, 322–328.
18. Mullican, M.D.; Wilson, M.W.; Conner, D.T.; Kostlan, C.R.; Schrier, D.J.; Dyer, R.D. Design of
5-(3,5-di-tert-butyl-4-hydroxyphenyl)-1,3,4-thiadiazoles, -1,3,4-oxadiazoles, and -1,2,4-triazoles
as orally active, nonulcerogenic antiinflammatory agents. J. Med. Chem. 1993, 36, 1090–1099.
19. Leventis, I.; Andreadou, I.; Papalois, A.; Sfiniadakis, I.; Gorgoulis, V.; Korkolis, D.;
Hadjipavlou-Litina, D.; Kourounakis, P.; Fotiadis, C. A novel antioxidant non-steroidal
anti-inflammatory agent protects rat liver against ischemia-reperfusion injury. In Vivo 2004, 18,
161–169.
20. Weber, V.; Rubat, C.; Duroux, E.; Lartigue, C.; Madesclaire, M.; Coudert, P. New 3- and
4-hydroxyfuranones as anti-oxidants and anti-inflammatory agents. Bioorg. Med. Chem. 2005, 13,
4552–4564.
21. Amorati, R.; Lucarini, M.; Mugnaini, V.; Pedulli, G. Antioxidant activity of o-bisphenols: The
role of intramolecular hydrogen bonding. J. Org. Chem. 2003, 68, 5198–5204.
22. Moore, G.; Swingle, K. 2,6-di-tert-butyl-4-(2-thenoyl)phenol(r-830): A novel nonsteroidal
anti-inflammatory agent with antioxidant properties. Inflamm. Res. 1982, 12, 674–683.
23. Grosso, P.; Vogl, O. Functional polymers. Polym. Bull. 1985, 14, 245–250.
24. Zhang, H. Structure-activity relationships and rational design strategies for radical-scavenging
antioxidants. Curr. Comput. Aided Drug Des. 2005, 1, 257–273.
25. Zhang, H.-Y.; Yang, D.-P.; Tang, G.-Y. Multipotent antioxidants: From screening to design.
Drug Discov. Today 2006, 11, 749–754.
26. Kato, T.; Ozaki, T.; Tamura, K.; Suzuki, Y.; Akima, M.; Ohi, N. Novel calcium antagonists with
both calcium overload inhibition and antioxidant activity. 1. 2-(3,5-di-tert-butyl-4-hydroxyphenyl)-
3-(aminopropyl)thiazolidinones. J. Med. Chem. 1998, 41, 4309–4316.

Molecules 2012, 17
7662
27. Dobek, A.; Klayman, D.; Dickson, E., Jr.; Scovill, J.; Oster, C. Thiosemicarbazones of
2-acetylpyridine, 2-acetylquinoline, 1-and 3-acetylisoquinoline and related compounds as
inhibitors of clinically significant bacteria in vitro. Arzneimittelforschung 1983, 33, 1583–1591.
28. Eid, A.I.; Ragab, F.A.; El-Ansary, S.L.; El-Gazayerly, S.M.; Mourad, F.E. Synthesis of new
7-substituted 4-methylcoumarin derivatives of antimicrobial activity. Arch. Pharm. 1994, 327,
211–213.
29. Liesen, A.P.; de Aquino, T.M.; Carvalho, C.S.; Lima, V.T.; de Araújo, J.M.; de Lima, J.G.;
de Faria, A.R.; de Melo, E.J.T.; Alves, A.J.; Alves, E.W.; et al. Synthesis and evaluation of
anti-toxoplasma gondii and antimicrobial activities of thiosemicarbazides, 4-thiazolidinones and
1,3,4-thiadiazoles. Eur. J. Med. Chem. 2010, 45, 3685–3691.
30. Kus, C.; Ayhan-Kilcigil, G.; Eke, B.; iŞcan, M. Synthesis and antioxidant properties of some
novel benzimidazole derivatives on lipid peroxidation in the rat liver. Arch. Pharm. Res. 2004, 27,
156–163.
31. Hussain, S.; Sharma, J.; Amir, M. Synthesis and antimicrobial activities of 1,2,4-triazole and
1,3,4-thiadiazole derivatives of 5-amino-2-hydroxybenzoic acid. E-J. Chem. 2008, 5, 963–968.
32. Vasoya, S.L.; Paghdar, D.J.; Chovatia, P.T.; Joshi, H.S. Synthesis of some new thiosemicarbazide
and 1,3,4-thiadiazole heterocycles bearing benzo[b]thiophene nucleus as a potent antitubercular
and antimicrobial agents. J. Sci. Islam. Repub. Iran 2005, 16, 33–36.
33. Rzeski, W.; Matysiak, J.; Kandefer-Szerszen, M. Anticancer, neuroprotective activities and
computational studies of 2-amino-1,3,4-thiadiazole based compound. Bioorg. Med. Chem. 2007,
15, 3201–3207.
34. Abdel-Rahman, T.M. Synthesis, reactions, and anticancer activity of some 1,3,4-thiadiazole/
thiadiazine derivatives of carbazole. Phosphorus Sulfur Silicon Relat. Elem. 2006, 181, 1737–1754.
35. Khan, I.; Ali, S.; Hameed, S.; Rama, N.H.; Hussain, M.T.; Wadood, A.; Uddin, R.; Ul-Haq, Z.;
Khan, A.; Ali, S.; et al. Synthesis, antioxidant activities and urease inhibition of some new
1,2,4-triazole and 1,3,4-thiadiazole derivatives. Eur. J. Med. Chem. 2010, 45, 5200–5207.
36. Palaska, E.; Sahin, G.; Kelicen, P.; Durlu, N.T.; Altinok, G. Synthesis and anti-inflammatory
activity of 1-acylthiosemicarbazides, 1,3,4-oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazole-3-
thiones. Il Farmaco 2002, 57, 101–107.
37. Navidpour, L.; Shafaroodi, H.; Abdi, K.; Amini, M.; Ghahremani, M.H.; Dehpour, A.R.; Shafiee, A.
Design, synthesis, and biological evaluation of substituted 3-alkylthio-4,5-diaryl-4h-1,2,4-
triazoles as selective COX-2 inhibitors. Bioorg. Med. Chem. 2006, 14, 2507–2517.
38. Wujec, M.; Pitucha, M.; Dobosz, M.; Kosikowska, U.; Malm, A. Synthesis and potential
antimycotic activity of 4-substituted-3-(thiophene-2-yl-methyl)-delta2-1,2,4-triazoline-5-thiones.
Acta Pharm. 2004, 54, 251–260.
39. Kajiyama, T.; Ohkatsu, Y. Effect of meta-substituents of phenolic antioxidants—Proposal of
secondary substituent effect. Polym. Degrad. Stab. 2002, 75, 535–542.
40. Hossain, M.; Shaha, S.; Aziz, F. Antioxidant potential study of some synthesized N-heterocycles.
Bangladesh Med. Res. Counc. Bull. 2009, 35, 49–52.
41. Ferreira, I.; Queiroz, M.; Vilas-Boas, M.; Estevinho, L. Evaluation of the antioxidant properties of
diarylamines in the benzo [b] thiophene series by free radical scavenging activity and reducing
power. Bioorg. Med. Chem. Lett. 2006, 16, 1384–1387.

Molecules 2012, 17
7663
42. Accelrys Homepage. Available online: http://accelrys.com/ (accessed on 12 January 2012).
43. Phillips, E.; Wasson, R.C. Phenols as corrosion inhibitors and antioxidants. EP330613A2,
30 August 1989.
44. Macleay, R.E.; Myers, T.N. Multipurpose polymer bound stabilizers. EP303986A2, 7 July 1993.
45. Akinchan, N.T.; West, D.X.; Yang, Y.; Salberg, M.M.; Klein, T.L. Magnetic and spectroscopic
properties of copper(II) complexes with 1-salicoyl-4-phenylthiosemicarbazide. Transit. Met. Chem.
1995, 20, 481–484.
46. Angelusiu, M.V.; Almajan, G.L.; Rosu, T.; Negoiu, M.; Almajan, E.-R.; Roy, J. Copper(II) and
uranyl(II) complexes with acylthiosemicarbazide: Synthesis, characterization, antibacterial
activity and effects on the growth of promyelocytic leukemia cells HL-60. Eur. J. Med. Chem.
2009, 44, 3323–3329.
47. Jampílek, J.; Doležal, M.; Kuneš, J.; Raich, I.; Liška, F. 4-substituted aryl bromides coupling with
4-methoxybenzene-1-thiol by means of copper catalysts. Chem. Papers 2005, 59, 178–181.
48. PharmaExpert. Available online: http://www.pharmaexpert.ru/passonline/ (accessed on 4 March
2012).
49. Parasuraman, S. Prediction of activity spectra for substances. J. Pharmacol. Pharmacother. 2011,
2, 52–53.
50. Stepanchikova, A.V.; Lagunin, A.A.; Filimonov, D.A.; Poroikov, V.V. Prediction of biological
activity spectra for substances: Evaluation on the diverse sets of drug-like structures. Curr. Med.
Chem. 2003, 10, 225–233.
51. Anzali, S.; Barnickel, G.; Cezanne, B.; Krug, M.; Filimonov, D.; Poroikov, V. Discriminating
between drugs and nondrugs by prediction of activity spectra for substances (pass). J. Med. Chem.
2001, 44, 2432–2437.
52. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational
approaches to estimate solubility and permeability in drug discovery and development settings.
Adv. Drug Deliv. Rev. 1997, 23, 3–25.
53. Veber, D.F.; Johnson, S.R.; Cheng, H.-Y.; Smith, B.R.; Ward, K.W.; Kopple, K.D. Molecular
properties that influence the oral bioavailability of drug candidates. J. Med. Chem. 2002, 45,
2615–2623.
54. Tafazoli, S.; Wright, J.S.; O’Brien, P.J. Prooxidant and antioxidant activity of vitamin e analogues
and troglitazone. Chem. Res. Toxicol. 2005, 18, 1567–1574.
55. Massaeli, H.; Sobrattee, S.; Pierce, G.N. The importance of lipid solubility in antioxidants and
free radical generating systems for determining lipoprotein peroxidation. Free Radic. Biol. Med.
1999, 26, 1524–1530.
56. Bakht, M.A.; Yar, M.S.; Abdel-Hamid, S.G.; Al Qasoumi, S.I.; Samad, A. Molecular properties
prediction, synthesis and antimicrobial activity of some newer oxadiazole derivatives. Eur. J.
Med. Chem. 2010, 45, 5862–5869.
57. Ingold, K.; Burton, G. Vitamin E: Or why we don’t go rancid. J. Chin. Chem. Soc. 1992, 39,
199–204.
58. Mishra, H.; Singh, N.; Lahiri, T.; Misra, K. A comparative study on the molecular descriptors for
predicting drug-likeness of small molecules. Bioinformation 2009, 3, 384–388.

Molecules 2012, 17
7664
59. Lipinski, C.A. Lead- and drug-like compounds: The rule-of-five revolution. Drug Discov. Today
Technol. 2004, 1, 337–341.
60. Refsgaard, H.H.F.; Jensen, B.F.; Brockhoff, P.B.; Guldbrandt, M.; Christensen, M.S. In silico
prediction of membrane permeability from calculated molecular parameters. J. Med. Chem. 2005,
48, 805–811.
61. Wang, R.; Fu, Y.; Lai, L. A new atom-additive method for calculating partition coefficients.
J. Chem. Inf. Comput. Sci. 1997, 37, 615–621.
62. Eklund, P.C.; Langvik, O.K.; Warna, J.P.; Salmi, T.O.; Willfor, S.M.; Sjoholm, R.E. Chemical
studies on antioxidant mechanisms and free radical scavenging properties of lignans. Org. Biomol.
Chem. 2005, 3, 3336–3347.
63. Sharma, O.P.; Bhat, T.K. Dpph antioxidant assay revisited. Food Chem. 2009, 113, 1202–1205.
64. Frankel, E. Lipid Oxidation, 2 ed.; Oily Press Lipid Library Bridgewater: Bridgewater, UK, 2005;
Volume 10, pp. 1–470.
65. Nawar, W. Lipids, Chapter 5. In Food Chemistry, 3rd ed.; Fennema, O.R., Ed.; Marcel Dekker,
Inc.: New York, NY, USA, 1996; pp. 254–299.
66. Kus, C.; Ayhan-KIlcIgil, G.; Özbey, S.; Kaynak, F.; Kaya, M.; Çoban, T.; Can-Eke, B. Synthesis
and antioxidant properties of novel N-methyl-1,3,4-thiadiazol-2-amine and 4-methyl-2h-1,2,4-
triazole-3(4h)-thione derivatives of benzimidazole class. Bioorg. Med. Chem. 2008, 16, 4294–4303.
667. Lucarini, M.; Pedrielli, P.; Pedulli, G.F.; Valgimigli, L.; Gigmes, D.; Tordo, P. Bond dissociation
energies of the N-H bond and rate constants for the reaction with alkyl, alkoxyl, and peroxyl
radicals of phenothiazines and related compounds. J. Am. Chem. Soc. 1999, 121, 11546–11553.
68. Scott, G. Antioxidants. Bull. Chem. Soc. Jpn. 1988, 61, 165–170.
69. Escobar-Valderrama, J.; Garcia-Tapia, J.; Ramirez-Ortiz, J.; Rosales, M.; Toscano, R.;
Valdes-Martinez, J. Crystal, molecular and electronic structure of 1-h-3-methyl-4-amine-5-thione-
1,2,4-triazol. Can. J. Chem. 1989, 67, 198–201.
70. Matsuura, T.; Ohkatsu, Y. Phenolic antioxidants: Effect of O-benzyl substituents. Polym. Degrad.
Stab. 2000, 70, 59–63.
71. Amorati, R.; Ferroni, F.; Pedulli, G.F.; Valgimigli, L. Modeling the co-antioxidant behavior of
monofunctional phenols. Applications to some relevant compounds. J. Org. Chem. 2003, 68,
9654–9658.
72. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J.; Deemer, E. Development and validation
of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly
methylated-cyclodextrin as the solubility enhancer. J. Agric. Food Chem. 2002, 50, 1815–1821.
73. McClements, D.; Decker, E. Lipid oxidation in oil in water emulsions: Impact of molecular
environment on chemical reactions in heterogeneous food systems. J. Food Sci. 2000, 65, 1270–1282.
74. Alamed, J. Impact of Chemical and Physical Properties on the Ability of Antioxidants to Inhibit
Lipid Oxidation in Foods; University of Massachusetts Amherst, Amherst, MA, USA, 2008.
75. Decker, E.A. Strategies for manipulating the prooxidative/antioxidative balance of foods to
maximize oxidative stability. Trends Food Sci. Technol. 1998, 9, 241–248.
76. Khan, I.; Ali, S.; Hameed, S.; Rama, N.; Hussain, M.; Wadood, A.; Uddin, R.; Ul-Haq, Z.; Khan, A.
Synthesis, antioxidant activities and urease inhibition of some new 1,2,4-triazole and 1,3,4-thiadiazole
derivatives. Eur. J. Med. Chem. 2010, 45, 5200–5207.

Molecules 2012, 17
7665
77. Hilton, J. Antioxidants: Function, types and necessity of inclusion in pet foods. Can. Vet. J. 1989,
30, 682–684.
78. Bruker. APEX2 and SAINT; Bruker AXS Inc.: Madison, WI, USA, 2007.
79. Sheldrick, G. A short history of shelx. Acta Crystallogr. A 2008, 64, 112–122.
80. Barbour, L.J. X-seed—A software tool for supramolecular crystallography. J. Supramol. Chem.
2001, 1, 189–191.
81. Blois, M.S. Antioxidant determination by the use of a stable free radical. Nature 1958, 181,
1199–1200.
82. Daker, M.; Abdullah, N.; Vikineswary, S.; Goh, P.C.; Kuppusamy, U.R. Antioxidant from maize
and maize fermented by Marasmiellus sp. As stabiliser of lipid-rich foods. Food Chem. 2008,
107, 1092–1098
Sample Availability: Samples of the compounds 1–3 are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).