Synthesis of the hydrazones of 2-((3,5-di-tert-butyl-4-hydroxybenzyl) thio) acetohydrazide and the study of their radical scavenging activity by the DPPH assay and the computational method

Article abstract of DOI:10.1002/bkcs.10553

The synthesis of antioxidant compounds bearing pharmacologically active groups has become a research interest in the last decade.Aseries of hydrazones containing a butylated hydroxyl group were synthesized from 2-((3,5-di-tert-butyl-4-hydroxybenzyl)thio)acetohydrazide and various benzaldehyde derivatives, and their radical scavenging activity was explored by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and computational calculations. Based on the results, the effects of different substituents on the DPPH IC₅₀ values were discussed. The bond dissociation enthalpies (BDE) of O H and N Hbonds and the spin density (SD) values of the compounds were calculated using the density functional theory (DFT) to determine the theoretical capability of the hydrazones as radical scavengers. Based on the results, the effects of the thioether group and the substituent groups next to the N H bond on the BDE values were also discussed.

Full text of DOI:10.1002/bkcs.10553

Article
DOI: 10.1002/bkcs.10553
BULLETIN OF THE
M. K. Abdulwahab et al.
KOREAN CHEMICAL SOCIETY
Synthesis of the Hydrazones of 2-((3,5-Di-tert-butyl-4-hydroxybenzyl)
thio) acetohydrazide andtheStudy oftheir Radical ScavengingActivity by
the DPPH Assay and the Computational Method
‡
§
Muhammad K. Abdulwahab,† Azhar Ariffin,†, Wageeh A. Yehye, Azlina Abdul-Aziz,
*
Huda S. Kareem,† and Nurdiana Nordin†
†Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603, Malaysia.
*E-mail: azhar70@um.edu.my
‡Nanotechnology & Catalysis Research Centre (NANOCAT), University of Malaya, Kuala Lumpur 50603,
Malaysia
§Department of Molecular Medicine, Faculty of Medicine, University of Malaya,
Kuala Lumpur 50603, Malaysia
Received June 4, 2015, Accepted July 28, 2015, Published online October 7, 2015
The synthesis of antioxidant compounds bearing pharmacologically active groups has become a research
interest in the last decade. A series of hydrazones containing a butylated hydroxyl group were synthesized from
2-((3,5-di-tert-butyl-4-hydroxybenzyl)thio)acetohydrazide and various benzaldehyde derivatives, and their
radical scavenging activity was explored by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and computational
calculations. Based on the results, the effects of different substituents on the DPPH IC50 values were discussed.
The bond dissociation enthalpies (BDE) of O H and N H bonds and the spin density (SD) values of the com-
pounds were calculated using the density functional theory (DFT) to determine the theoretical capability of
the hydrazones as radical scavengers. Based on the results, the effects of the thioether group and the substituent
groups next to the N H bond on the BDE values were also discussed.
Keywords: Antioxidant, Butylated hydroxytoluene, Thioether, Hydrazones,
Density functional theory calculations
Introduction
It was reported that the addition of electron donating substi-
tuents, such as tert-butyl at the ortho- and para-positions
increased the antioxidant activity of the phenol.7 The presence
of tert-butyl groups at those positions reduces the BDE value
of the phenolic O H bond and stabilizes the phenoxyl radical
through inductive and hyperconjugative effects. In the radical
form of BHT, the bulkiness of the tert-butyl at the ortho-
positions also provides steric protection, which prevents unde-
sired reactions by blocking the access of other compounds to
the oxygen radical on the phenoxyl compound (Figure 2).
Meanwhile, preventive antioxidants, such as thioethers, are
peroxide decomposers. They can be used to improve the per-
formance of the chain breaking antioxidants.8 ROOH from the
radical scavenging reaction can be broken into two radical
compounds, alkoxyl (ROꢀ) and hydroxyl (OHꢀ). The radical
molecules can initiate another radical chain reaction.
Thioether prevents this circumstance by decomposing the per-
oxide. It undergoes reaction with hydroperoxide (ROOH) to
produce the corresponding alcohol and sulfinyl compounds,
which are nonradical and stable.9
Reactive oxygen species (ROS) are defined as highly reactive
oxygen-containing molecules that are capable of degrading
the biological system through their ability to react with and
damage the cellular contents, including membrane lipids, pro-
teins, and nucleic acids. ROS are generally considered to be
responsible for many cell disorders and the development of
a large number of human diseases, including cancer and
inflammation.1,2 One of the defenses against ROS is antioxi-
dants.3 Antioxidants are defined as substances that signifi-
cantly prevent the oxidation of substrates by pro-oxidant
species.4 Antioxidants can be categorized into two groups:
chain breaking and preventive antioxidants.5 Chain
breaking antioxidants are radical scavengers, hydrogen
donors, or chain reaction breakers. An example of this type
of antioxidants is phenolic antioxidants. Butylated hydroxyto-
luene (BHT) is a known sterically hindered phenolic antioxi-
dant. It has been used as an antioxidant in the food industry
since 1954, where it is frequently added in dried cereals and
cooking oils.6 At low concentrations, BHT is known to give
some protection against carcinogenicity and toxicity of a num-
ber of chemicals.6 A distinctive feature of BHT is the presence
of two tert-butyl groups on the ortho-positions of the phenol
ring (Figure 1).
Hydrazones are characterized as organic compounds with
the basic structure of R1R2C NNH2. It was reported that
hydrazones have a wide range of biological activities and
pharmacological applications. In previous research articles,
hydrazones have been described to have antioxidant,10,11
Bull. Korean Chem. Soc. 2015, Vol. 36, 2716–2724
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antimicrobial,12 anti-inflammation,13,14 and analgesic activ-
ities.14 In addition, hydrazones can act as the starting material
in the synthesis of various bioactive compounds, including
heterocyclic rings. We designed a series of compounds that
feature BHT, thioether and hydrazone groups with the aim that
they would have the multiple pharmacological effects men-
tioned above (Figure 3).
unless stated otherwise. Starting material 3 (0.2 g, 0.616
mmol) was dissolved in absolute ethanol along with a molar
equivalent of the respective aldehyde and was stirred at room
temperature for 2 h. At the end of the reaction, the product was
precipitatedbyaddinghexanetothereactionmixture. Thepre-
cipitate was collected through filtration and recrystallized in
hexane.
Inthis paper, wediscuss thesynthesis ofthecompoundsand
the effects of the addition of the thioether and the hydrazone
groups to the radical scavenging activity of the BHT moiety.
The discussion focuses on the performance of the compounds
in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay,15–17 and
we use a computational method to support our findings from
the DPPH assay.
4-Phenylbenzaldehyde 4-hydroxy-3,5-di-tert-butylben-
zylthioacetohydrazone (4): White crystals were produced
from 4-phenylbenzaldehyde; yield 86%; mp 178 ꢁC; IR (υ,
cm−1): 3631.59 (OH), 2871.11 (CH, aliphatic), 1666.13
(CONH), 1606.93 (C N); 1H NMR (CDCl3, 400 MHz),
ppm: 1.45 [s, 18H, 2 × t-butyl], 3.38 [s, 2H, H-8Z], 3.72 [s,
2H, H-8E], 3.78[s, 2H, H-7Z], 3.91 [s, 2H, H-7E], 5.18 [s,
1H, OH-E], 5.22 [s, 1H, OHZ], 7.13 [s, 2H, H-3Z, 5Z], 7.20
[s, 2H, H-3E, 5E], 7.38 [dd, J = 10.8, 5.6, 3H, H-19Z, 20Z,
21Z], 7.46 [dd, J = 12.0, 6.0, 3H, H-19E, 20E, 21E], 7.61
[d, J = 8.4, 2H, H-13, 15, 18, 22], 7.70 [d, J = 8.4, 2H, H-
12E, 16E], 7.72 [d, J = 8.4, 2H, H-12Z, 16Z], 7.87 [s, 1H,
H-10E], 7.93 [s, 1H, H-10Z], 9.47 [s, 1H, NH-Z], 10.15 [s,
1H, NH-E]; 13C NMR (CDCl3, 100 MHz), ppm: 30.26 [6C,
CH3 on t-butyl], 31.75 [1C, C-8E], 34.28 [2C, C(CH3) on t-
butyl], 35.17 [1C, C-8Z], 37.09 [1C, C-7E], 38.25 [1C, C-
7Z], 125.55 [2C, C-3Z,5Z], 125.85 [2C, C-3E, 5E], 127.01
[4C, C-13E, 15E, 18E, 22E], 127.24 [2C, C-12Z, 16Z],
127.38 [4C, C-13Z, 15Z, 18Z, 22Z], 127.66 [2C, C-12E,
16E], 127.77 [1C, C-20Z], 127.86 [1C, C-4E], 127.91 [1C,
C-4Z], 128.18 [1C, C-20E], 128.83 [2C, C-19E, 21E],
128.86 [2C, C-19Z, 21Z], 132.19 [1C, C-11E], 132.56 [1C,
C-11Z], 135.93 [2C, C-2E, 6E], 136.45 [2C, C-2Z, 6Z],
140.11 [1C, C-17Z], 140.21 [1C, C-17E], 142.92 [1C, C-
14E], 143.19 [1C, C-14Z], 143.89 [1C, C-10E], 147.62 [1C,
C-10Z], 152.93 [1C, C-1E], 153.30 [1C, C-1Z], 164.73 [1C,
C-9Z], 171.65 [1C, C-9E]; high resolution electrospray
ionization mass spectroscopy (HREIMS) m/z 511.2248 [M
+Na+] (calc. for C30H36N2O2S 488.2497).
4-Methylbenzaldehyde 4-hydroxy-3,5-di-tert-butylben-
zylthioacetohydrazone (5): White crystals were produced
from 4-methylbenzaldehyde; yield 86%; mp 143 ꢁC; IR (υ,
cm−1): 3618.52 (OH), 2868.31 (CH, aliphatic), 1664.32
(CONH), 1614.78 (C N); 1H NMR (CDCl3, 400 MHz),
ppm: 1.43 [s, 18H, 2 × t-butyl], 2.38 [s, 3H, H−17], 3.35 [s,
2H, H-8Z], 3.66 [s, 2H, H-8E], 3.75 [s, 2H, H-7Z], 3.87
[s, 2H, H-7E], 5.15 [s, 1H, OH-E], 5.19 [s, 1H, OH-Z], 7.10
[s, 2H, H-3Z, H-5Z], 7.17 [s, 2H, H-3E, H-5E], 7.19 [d, J =
7.6, 2H, H-13, H-15], 7.51 [d, J = 7.6, 2H, H-12E, H-16E],
7.60 [d, J = 7.6, 2H, H-12Z, H-16Z], 7.75 [s, 1H, H-10E],
7.77 [s, 1H, H-10Z], 9.16 [s, 1H, NH-E], 9.32 [s, 1H, NH-
Z]; 13C NMR (CDCl3, 100 MHz), ppm: 21.48 [1C,C-17],
30.23 [6C, CH3 on t-butyl], 31.76 [1C, C-8E], 34.29 [2C,
C(CH3) on t-butyl], 35.21 [1C, C-8Z], 37.04 [1C, C-7E],
38.38 [1C, C-7Z], 125.53 [2C, C-3Z, C-5Z], 125.87 [2C, C-
3E, C-5E], 127.18 [2C, C-12E, C-16E], 127.74 [2C, C-12Z,
C-16Z], 127.86 [1C, C-4Z], 127.95 [1C, C-4E], 129.37 [2C,
C-13Z, C-15Z], 129.48 [2C, C-13E, C-15E], 130.49 [1C, C-
11Z], 130.75 [1C, C-11E], 135.91 [2C, C-2Z, C-6Z], 136.45
Experimental
General Procedure for the Synthesis of Compounds 4–11.
The general procedure for the synthesis of the compounds
from hydrazide and the respective aldehydes was as follows,
OH
Figure 1. The structure of BHT.
H
C
3
H
C
3
H
C
3
H
3C
O
H
H
C
3
Unreactive radical
because t-butyl groups
block the access
H
3C
C
3
Figure 2. The steric protection in BHT.
thioether
H
O
O
N
R
S
N
H
BHT
hydrazone
Figure 3. The design of a multi-functional antioxidant.
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Synthesis of the Hydrazones of 2-((3,5-Di-tert-butyl-4-hydroxybenzyl)thio)
acetohydrazide
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[2C, C-2E, C-6E], 140.61 [1C, C-14E], 140.97 [1C, C-14Z],
143.90 [1C, C-10E], 148.09 [1C, C-10Z], 152.92 [1C, C-1E],
153.30 [1C, C-1Z], 164.48 [1C, C-9Z], 171.99 [1C, C-9E];
HREIMS m/z 449.2069 [M+Na+] (calc. for C25H34N2O2S
426.2341).
cm−1): 3624.44 (OH), 2940.00 (CH, aliphatic), 2227.33
(C N), 1666.13 (CONH), 1606.93 (C N); 1H NMR
(CDCl3, 400 MHz), ppm: 1.41 [s, 18H, 2 × t-butyl], 3.37 [s,
2H, H-8Z], 3.64 [s, 2H, H-8E], 3.74 [s, 2H, H-7Z], 3.85 [s,
2H, H-7E], 5.17 [s, 1H, OH-E], 5.19 [s, 1H, OH-Z], 7.08 [s,
2H, H-3Z, H-5Z], 7.16 [s, 2H, H-3E, H-5E], 7.66 [d, J =
8.0, 2H, H-13, H-15], 7.71 [d, J = 8.4, 2H, H-12E, H-16E],
7.77 [d, J = 8.4, 2H, H-12Z, H-16Z], 7.88 [s, 1H, H-10],
9.44 [s, 1H, NH-Z], 10.23 [s, 1H, NH-E]; 13C NMR
(CDCl3, 100 MHz), ppm: 30.3 [6C, CH3 on t-butyl], 31.62
[1C, C-8E], 34.34 [2C, C(CH3) on t-butyl], 35.35 [1C, C-
8Z], 37.11 [1C, C-7E], 38.45 [1C, C-7Z], 113.36 [1C, C-
14E], 113.60 [1C, C-14Z], 118.43 [1C,C-17], 125.56 [2C,
C-3Z, C-5Z], 125.85 [2C, C-3E, C-5E], 127.54 [2C, C-12E,
C-16E], 127.67 [2C, C-12Z, C-16Z], 127.93 [1C,C-4],
132.43 [2C, C-13Z, C-15Z], 132.56 [2C, C-13E, C-15E],
136.03 [2C, C-2E, C-6E], 136.56 [2C, C-2Z, C-6Z], 137.76
[1C, C-11Z], 137.83 [1C, C-11E], 141.80 [1C, C-10E],
145.50 [1C, C-10Z], 153.05 [1C, C-1E], 153.38 [1C, C-1Z],
165.08 [1C, C-9Z], 172.92 [1C, C-9E]; HREIMS m/z
460.1838 [M+Na+] (calc. for C25H31N3O2S 437.2137).
4-(Trifluoromethyl)benzaldehyde 4-hydroxy-3,5-di-tert-
butylbenzylthioacetohydrazone (9): White crystals were pro-
duced from 4-(trifluoromethyl)benzaldehyde; yield 74%; mp
156 ꢁC; IR (υ, cm−1): 3620.65 (OH), 2918.11 (CH, aliphatic),
4-Bromobenzaldehyde 4-hydroxy-3,5-di-tert-butylben-
zylthioacetohydrazone (6): White crystals were produced
from 4-bromobenzaldehyde; yield 83%; mp 156 ꢁC; IR (υ,
cm−1): 3620.05 (OH), 2948.00 (CH, aliphatic), 1661.79
(CONH), 1609.17 (C N); 1H NMR (CDCl3, 400 MHz),
ppm: 1.43 [s, 18H, 2 × t-butyl], 3.87 [s, 2H, H-8Z], 3.65 [s,
2H, H-8E], 3.76 [s, 2H, H-7Z], 3.87 [s, 2H, H-7E], 5.18 [s,
1H, OH-E], 5.21 [s, 1H, OH-Z], 7.10 [s, 2H, H-3Z, H-5Z],
7.18 [s, 2H, H-3E, H-5E], 7.49 [d, J = 8.4, 2H, H-12E, H-
16E], 7.53 [d, J = 6.0, 2H, H-13, H-15], 7.58 [d, J = 8.8, 2H,
H-12Z, H-16Z], 7.76 [s, 1H, H-10E], 7.77 [s, 1H, H-10Z],
9.36 [s, 1H, NH-E], 9.38 [s, 1H, NH-Z]; 13C NMR (CDCl3,
100 MHz), ppm: 30.22 [6C, CH3 on t-butyl], 31.67 [1C, C-
8E], 34.28 [2C, C(CH3) on t-butyl], 35.23 [1C, C-8Z],
37.05 [1C, C-7E], 38.34 [1C, C-7Z], 124.45 [1C, C-4E],
124.86 [1C, C-4Z], 125.51 [2C, C-3Z, C-5Z], 125.82 [2C,
C-3E, C-5E], 127.79 [1C, C-4E], 127.91 [1C, C-4Z],
128.55 [2C, C-12E, C-16E], 129.02 [2C, C-12Z, C-16Z],
131.89 [2C, C-13Z, C-15Z], 131.98 [2C, C-13E, C-15E],
132.25 [1C, C-11Z], 132.52 [1C, C-11E], 135.94 [2C, C-
2E, C-6E], 136.47 [2C, C-2Z, C-6Z], 142.94 [1C, C-10E],
146.67 [1C, C-10Z], 152.95 [1C, C-1E], 153.30 [1C, C-1Z],
164.82 [1C, C-9Z], 172.61 [1C, C-9E]; HREIMS m/z
513.1036 [M+Na+] (calc. for C24H31BrN2O2S 490.1290).
4-Chlorobenzaldehyde 4-hydroxy-3,5-di-tert-butylben-
zylthioacetohydrazone (7): White crystals were produced
from 4-chlorobenzaldehyde; yield 80%; mp 151 ꢁC; IR (υ,
cm−1): 3618.99 (OH), 2916.24 (CH, aliphatic), 1663.59
(CONH), 1607.47 (C N); 1H NMR (CDCl3, 400 MHz),
ppm: 1.44 [s, 18H, 2 × t-butyl], 3.38 [s, 2H, H-8Z], 3.66 [s,
2H, H-8E], 3.76 [s, 2H, H-7Z], 3.88 [s, 2H, H-7E], 5.18 [s,
1H, OH-E], 5.21 [s, 1H, OH-Z], 7.11 [s, 2H, H-3Z, H-5Z],
7.18 [s, 2H, H-3E, H-5E], 7.37 [d, J = 8.4, 2H, H-13, H-15],
7.56 [d, J = 8.8, 2H, H-12E, H-16E], 7.64 [d, J = 8.4, 2H,
H-12Z, H-16Z], 7.79 [s, 1H, H-10Z], 7.80 [s, 1H, H-10E],
9.37 [s, 1H, NH-E], 9.62 [s, 1H, NH-Z]; 13C NMR (CDCl3,
100 MHz), ppm: 30.22 [6C, CH3 on t-butyl], 31.66 [1C, C-
8E], 34.29 [2C, C(CH3) on t-butyl], 35.26 [1C, C-8Z],
37.06 [1C, C-7E], 38.36 [1C, C-7Z], 125.52 [2C, C-3Z, C-
5Z], 125.83 [2C, C-3E, C-5E], 127.81 [1C, C-4Z], 127.92
[1C, C-4E], 128.34 [2C, C-12E, C-16E], 128.81 [2C, C-
12Z, C-16Z], 128.94 [2C, C-13Z, C-15Z], 129.03 [2C, C-
13E, C-15E], 131.85 [1C, C-11Z], 132.09 [1C, C-11E],
135.95 [2C, C-2E, C-6E], 136.11 [2C, C-2Z, C-6Z], 136.47
[1C,C-14], 142.79 [1C, C-10Z], 146.58[1C, C-10E], 152.96
[1C, C-1E], 153.31 [1C, C-1Z], 164.72 [1C, C-9Z], 172.57
[1C, C-9E]; HREIMS m/z 469.1529 [M+Na+] (calc. for
C24H31ClN2O2S 446.1795).
1
1666.77 (CONH), 1607.35 (C N); H NMR (CDCl3, 400
MHz), ppm: 1.44 [s, 18H, 2 × t-butyl], 3.39 [s, 2H, H-8Z],
3.67 [s, 2H, H-8E], 3.77[s, 2H, H-7Z], 3.88 [s, 2H, H-7E],
5.18 [s, 1H, OH-E], 5.22 [s, 1H, OH-Z], 7.11 [s, 2H, H-3Z,
H-5Z], 7.18 [s, 2H, H-3E, H-5E], 7.65 [d, J = 8.0, 2H, H-13,
H-15], 7.74 [d, J = 8.4, 2H, H-12E, H-16E], 7.82 [d, J = 8.0,
2H, H-12Z, H-16Z], 7.86 [s, 1H, H-10E], 7.89 [s, 1H, H-
10Z], 9.42 [s, 1H, NH-E], 9.60 [s, 1H, NH-Z]; 13C NMR
(CDCl3, 100 MHz), ppm: 30.29 [6C, CH3 on t-butyl], 31.73
[1C, C-8E], 34.34 [2C, C(CH3) on t-butyl], 35.25 [1C, C-
8Z], 37.17 [1C, C-7E], 38.38 [1C, C-7Z], 122.53 [1C,C-
17E], 125.24 [1C,C-17Z], 125.60 [2C, C-13E, C-15E],
125.72 [2C, C-13Z, C-15Z], 127.75 [2C, C-3Z, C-5Z],
125.86 [2C, C-3E, C-5E], 127.39 [2C, C-12E, C-16E],
127.77 [2C, C-12Z, C-16Z], 127.81 [1C, C-4E], 127.99
[1C, C-4Z], 131.57 [1C, C-14E], 131.89 [1C, C-14Z],
136.05 [2C, C-2E, C-6E], 136.55 [2C, C-2Z, C-6Z], 136.79
[1C, C-11Z], 137.08 [1C, C-11E], 142.92 [1C, C-10E],
146.22 [1C, C-10Z], 153.05 [1C, C-1E], 153.37 [1C, C-1Z],
165.22 [1C, C-9Z], 173.39 [1C, C-9E]; HREIMS m/z
503.1809 [M+Na+] (calc. for C25H31F3N2O2S 480.2058).
4-Methoxybenzaldehyde 4-hydroxy-3,5-di-tert-butylben-
zylthioacetohydrazone (10): White crystals were produced
from 4-methoxybenzaldehyde; yield 78%; mp 157 ꢁC; IR
(υ, cm−1): 3617.90 (OH), 3179.58 (N H), 2868.38 (CH, ali-
phatic), 1661.30 (CONH), 1611.98 (C N); 1H NMR (CDCl3,
400 MHz), ppm: 1.42 [s, 18H, 2 × t-butyl], 3.35 [s, 2H, H-8Z],
3.65 [s, 2H, H-8E], 3.74[s, 2H, H-7Z], 3.85 [s, 3H, H-17], 3.87
[s, 2H, H-7E], 5.15 [s, 1H, OH-E], 5.20 [s, 1H, OH-Z], 6.90 [d,
J = 8.8, 2H, H-13, H-15], 7.10 [s, 2H, H-3Z, H-5Z], 7.17 [s,
2H, H-3E, H-5E], 7.55 [d, J = 8.4, 2H, H-12E, H-16E], 7.65
4-Cyanobenzaldehyde 4-hydroxy-3,5-di-tert-butylben-
zylthioacetohydrazone (8): White crystals were produced
from 4-cyanobenzaldehyde; yield 90%; mp 170 ꢁC; IR (υ,
Bull. Korean Chem. Soc. 2015, Vol. 36, 2716–2724
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Synthesis of the Hydrazones of 2-((3,5-Di-tert-butyl-4-hydroxybenzyl)thio)
acetohydrazide
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[d, J = 8.4, 2H, H-12Z, H-16Z], 7.74 [s, 1H, H-10], 9.18 [s, 1H,
NH-E], 9.30 [s, 1H, NH-Z]; 13C NMR (CDCl3, 100 MHz),
ppm: 30.30 [6C, CH3 on t-butyl], 31.81 [1C, C-8E], 34.33
[2C, C(CH3) on t-butyl], 35.20 [1C, C-8Z], 37.11 [1C, C-
7E], 38.28 [1C, C-7Z], 55.39 [1C,C-17], 114.12 [2C, C-
13Z, C-15Z], 114.23 [2C, C-13E, C-15E], 125.59 [2C, C-
3Z, C-5Z], 125.89 [2C, C-3E, C-5E], 125.97 [1C, C-11Z],
126.41 [1C, C-11E], 127.93 [1C, C-14Z], 128.04 [1C, C-
14E], 128.80 [2C, C-12E, C-16E], 129.41 [2C, C-12Z, C-
16Z], 135.95 [2C, C-2E, C-6E], 136.46 [2C, C-2Z, C-6Z],
144.06 [1C, C-10Z], 147.88 [1C, C-10E], 152.95 [1C, C-
4E], 153.32 [1C, C-4Z], 161.31 [1C, C-1E], 161.61 [1C, C-
1Z], 164.54 [1C, C-9Z], 172.45 [1C, C-9E]; HREIMS m/z
465.2029 [M+Na+] (calc. for C25H34N2O3S 442.2290).
3,5-Di-tert-butyl-4-hydroxybenzaldehyde 4-hydroxy-3,5-
di-tert-butylbenzylthioacetohydrazone (11): White crystals
were produced from 3,5-di-t-butyl-4-hydroxybenzaldehyde;
yield 70%; mp 170 ꢁC; IR (υ, cm−1): 3635.97 (OH),
2872.19 (CH, aliphatic), 1654.88 (CONH), 1600.04 (C N);
1H NMR (CDCl3, 400 MHz), ppm: 1.42 [s, 18H, 2 × t-butyl],
1.45 [s, 18H, 2 × t-butyl], 3.35 [s, 2H, H-8Z], 3.65 [s, 2H, H-
8E], 3.74 [s, 2H, H-7Z], 3.89 [s, 2H, H-7E], 5.14 [s, 1H, OH-
1E], 5.18 [s, 1H, OH-1Z], 5.48 [s, 1H, OH-2E], 5.51 [s, 1H,
OH-2Z], 7.10 [s, 2H, H-3E, H-5E], 7.16 [s, 2H, H-3Z, H-
5Z], 7.46 [s, 2H, H-12E, H-16E], 7.50 [s, 2H, H-12Z, H-
16Z], 7.70 [s, 1H, H-10E], 7.80 [s, 1H, H-10Z], 8.84 [s, 1H,
NH-E], 9.24 [s, 1H, NH-Z]; 13C NMR (CDCl3, 100 MHz),
ppm: 30.18 [6C, CH3 on t-butyl], 30.30 [6C, CH3 on t-butyl],
31.91 [1C, C-8E], 34.34 [2C, C(CH3) on t-butyl], 34.36 [2C,
C(CH3)ont-butyl], 35.31[1C, C-8Z], 37.30[1C, C-7E], 38.31
[1C, C-7Z], 124.48 [2C, C-12E, C-16E], 124.57 [1C, C-11Z],
124.95 [1C, C-11E], 125.19 [2C, C-12Z, C-16Z], 125.55 [2C,
C-3Z, C-5Z], 125.90 [2C, C-3E, C-5E], 127.97 [1C, C-4Z],
128.80 [1C, C-4E], 135.91 [2C, C-2E, C-6E], 136.22 [2C,
C-2Z, C-6Z], 136.36 [2C, C-13E, C-15E], 136.46 [2C, C-
13Z, C-15Z], 145.10 [1C, C-10E], 149.75 [1C, C-10Z],
152.93 [1C, C-1E], 153.32 [1C, C-1Z], 156.01 [1C, C-14E],
156.35 [1C, C-14Z], 164.32 [1C, C-9Z], 172.32 [1C, C-9E];
HREIMS m/z 563.3162 [M+Na+] (calc. for C32H48N2O3S
540.3386).
percentage (%) of the DPPH radical scavenging activity in
each extract was calculated using the following equation:
ÀÀ
Á
Á
Scavenging activity ð%Þ =
A
control – Asample =Acontrol × 100
where Acontrol is the absorbance of DPPH radicals without
sample or standard and Asample is theabsorbanceof DPPH with
sample or standard. The IC50 value was calculated as the effi-
cient concentration of the samples that inhibits 50% of the
DPPH activity and was expressed in μM.
Computational Calculation. All of the molecular modeling
calculations were performed using NWChem software.20 The
structures of the compounds were optimized using the density
functional theory (DFT) quantum mechanical method at the
B3LYP/6-311G (d,p) energy level. The radical forms of the
compounds were treated as open shell systems. The value
for BDE was calculated using the following formulas.
BDE = EðPhOÞ + EðHÞ – EðPhOHÞ
Results and Discussion
Chemistry. The starting material, 2-((3,5-di-tert-butyl-4-
hydroxybenzyl)thio)acetohydrazide, 3, was obtained from a
series of reactions involving thioglycollic acid and 2,6-di-
tert-butyl-4-(hydroxymethyl)phenol (Scheme 1).21
The hydrazones were synthesized using the procedure
reported in the literature,22 with some modification. The gen-
eral procedure is depicted in Scheme 2. Starting material 3 was
dissolved in absolute ethanol along with the respective alde-
hydes and was stirred at room temperature for 2 h. At the
end of the reaction, the product was precipitated by adding
hexane to the reaction mixture. The precipitate was collected
by filtration and recrystallized in hexane. The yields of com-
pounds 4–11 were good and ranged within 70–90%. The
values are reported in Table 1.
The IR spectra for compounds 4–11 showed the disappear-
ance of the NHNH2 peak from the starting material and the
emergence of the C N peak at approximately 1600–1610
cm−1. The mass spectrometry analysis of the products also
confirmed the HREIMS values of compounds 4–11.
Determination of 1,1-Diphenyl-2-picryl hydrazyl Radical
Scavenging Activity. The procedure for determining the free
radical scavenging activity of the compounds in the DPPH
assay was adopted from Kong et al., with slight modifica-
tions.18 Instead of methanol, dimethyl sulfoxide (DMSO)
was used as the solvent. Amorati and Valgimigli argued that
in methanol, the acidic phenols react with DPPH much faster
than expected, which would overturn the order of reactivity
with respect to their actual antioxidant activity.19 Solutions
of the hydrazones (50 μL) at different concentrations
(0–1000 μg/mL) were mixed with 195 μL of a 100 μM DPPH
solution in the solvent. After 30 min, the absorbance of the
reaction mixture was read at 515 nm. Different concentrations
(0–1000 μg/mL) of known antioxidant standards, namely,
BHT and ascorbic acid (AA), were used as positive controls
and were run in parallel. Using the absorbance data, the
The 1H and 13C NMR spectra of the compounds in CDCl3
indicated that two configurations, E and Z, were present in the
products. Palla et al. reported that in solution, N-
acylhydrazone existed as four different isomers, E/Z geomet-
rical isomers and cis/trans conformational isomers.23 The E/Z
geometrical isomers existed due to the N C bond, while
the cis/trans conformational isomers were due to the hindered
rotation around the C N amide bond (Isomer I, Scheme 3).
Between the conformational isomers, the cis-isomer was more
stable due to the intramolecular hydrogen bond between C O
and N H (Isomer III, Scheme 3). For the geometrical isomers,
the E isomer was reported to be more stable due to less steric
hindrance.24 The conversion between the conformational
Bull. Korean Chem. Soc. 2015, Vol. 36, 2716–2724
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Synthesis of the Hydrazones of 2-((3,5-Di-tert-butyl-4-hydroxybenzyl)thio)
acetohydrazide
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H
O
O
H
O
PTSA, reflux
toluene
O
+
H
S
H
O
S
H
O
H
O
1
thioglycollic
acid
2,6-di-tert-butyl-4-
Esterification
CH3OH
(hydroxymethyl)phenol
reflux
H
O
H
O
O
O
RT, hexane, 24 hrs
NH2NH2.H2O
NH2
S
S
N
H
H
OC
3
3
2
Scheme 1. The pathway of the synthesis of the starting material.
cis/trans
HO
HO
E Z
/
O
O
O
NH2
N
R
S
EtOH, 2hr, RT
S
O
O
N
+
N
R
H
slow
H
H
N
H
R'
N
H
R
N
H
R
N
H
4-11
11
3
R'
R
4
5
6
7
8
9
10
I-trans,E
II-trans,Z
fast
fast
H C 3C
C C
H
( )
3 3
(
)
3
OH
Br
CH3
Cl
CN
CF3
OCH3
R
R
slow
N
H
N
R'
Scheme 2. The synthesis of a series of hydrazones.
N
H
O
N
H
O
H
R'
Table 1. The structures, yields, and ratios of the E to Z isomers by the
1H NMR spectra and DPPH Ic50 values for compounds 4–11.
IV-cis,Z
III-cis,E
Scheme 3. The isomers of N-acylhydrazone.
Compound
Yield (%)
E to Z ratio
DPPH (IC50 μM)
4
5
6
7
8
9
10
11
86
86
83
80
90
74
78
70
—
—
1.95:1
1.40:1
1.31:1
1.44:1
1.85:1
2.80:1
1.26:1
1.26:1
—
176.27 ꢂ 2.92
162.12 ꢂ 16.99
138.60 ꢂ 7.46
150.65 ꢂ 4.50
225.98 ꢂ 1.96
202.21 ꢂ 0.79
195.05 ꢂ 16.58
26.00 ꢂ 0.20
H
R
H
R
R
H
C
N
R
H
C
N
C
N
C
N
H
N
H
H+
N
N
N
R'
R'
R'
R'
Z
E
Scheme 4. The mechanism of the conversion of the E/Z geometric
isomers of the hydrazones.
Ascorbic acid
BHT
80.55 ꢂ 1.03
—
270.95 ꢂ 18.97
the N C (Scheme 4). When the azo tautomerized back
to the hydrazone, two geometrical isomers were formed.
In the NMR spectra of the compounds, only two sets of
peaks were observable; thus, we believed that they belonged
tothe cis, Eand cis, and Z isomers. We assigned the moredom-
inant peaks in the NMR spectra to the E isomer. The X-ray
structural analysis on compound 4 showed that in solid form,
the compound only existed in the cis, E-configuration
(Figure 4).26
isomers in chloroform was reported to be fast, while the con-
version between the geometrical isomers was slow.23
The mechanism of the E/Z geometrical isomerisation
involved hydrazone-azo tautomerization.25 Tautomerization
into the azo- caused the double bond in N C to become
a single bond, N C . In this case, the C atom attached to
the N atom could be rotated to either side to be in cis- or
trans-position with respect to the N R0 group attached to
Bull. Korean Chem. Soc. 2015, Vol. 36, 2716–2724
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Synthesis of the Hydrazones of 2-((3,5-Di-tert-butyl-4-hydroxybenzyl)thio)
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Radical Scavenging Activity in the DPPH Assay. The
DPPH assay is one of the most frequently employed methods
to evaluate the radical scavenging activity of antioxidant com-
pounds.27 DPPH is described as a very stable and long-lived
organic nitrogen radical. Its stability is attributed to two fac-
tors. According to Kedare and Singh, the stability of DPPH
is due to its ability to delocalize the radical electron over the
molecule as a whole.28 On the other hand, Foti and Daquino
suggested that the stability is essentially due to the steric hin-
drance around the divalent N atom.29
The delocalization of the electron gives a deep violet color,
with an absorption at 515 nm in solution.28 When the DPPH is
mixed with an H-atom donor, the divalent N is reduced to
N H and the color of the solution changes from violet to yel-
low because the absorption band at 515 nm disappears. The
radical scavenging activity is expressed as the amount of the
sample needed to inhibit the DPPH activity by 50%, which
is also known as the IC50 value. The IC50 values for the com-
pounds, along with the references BHT and AA are reported in
Table 1.
The IC50 values of compounds 4–10 indicated that eachhad
better radical scavenging activity than BHT. This observation
could be explained by the number of sites that could donate an
H atom for the compounds. The NH group of hydrazone was
described to display radical scavenging activity (Scheme 5).
Several researchers synthesized hydrazones without a pheno-
lic group, and they reported that the synthesized hydrazones
showed radical scavenging activity in the DPPH assay.11,30
Therefore, compounds 4–10, which possessed two sites,
had better radical scavenging activity than BHT, which had
only one site. This explanation was strengthened by another
observation: compound 11, which had three sites, had the
lowest IC50 value (26 μM). The radical scavenging activity
shown by compound 11 was even better than AA. The IC50
value of compound 11 proved that by adding more sites that
could donate an H atom, the radical scavenging activity would
be increased.
From the DPPH result, it seemed that the different substitu-
ents on the hydrazones played a minor role in stabilizing the
radicals. Thisobservation mightbe becausethegroupsexerted
their electron contribution effects only on the NH and not on
the OH. In terms of H-atom transfer, the OH group plays a
far more dominant role compared to the NH group. This will
be explained using our computational method in the next
section.
Another explanation as to why the substituent groups could
only exert a small effect was the extent of the delocalization of
the radical electron on the N atom over the aromatic ring.
According to Harej and Dolenc, the substituents on the hydra-
zine moiety (labeled as 1 in Figure 5) of the hydrazone exerted
a greater effect on the strength of the NH bond compared
with the substituents on the aldehyde/ketone moiety (labeled
as 2 in Figure 5).31 Therefore, the radical on the N atom would
mostly be delocalized over the C(O)R instead of the aro-
matic ring and its substituents.
Having said how little the effect the substituents would
exert, there were still patterns among the substituents that
could be discussed. Compounds 8 (225.98 μM) and
9 (202.21 μM), which had inductive electron withdrawing
groups, CN and CF3, respectively, had the highest IC50
values. On the other hand, compounds that contained induc-
tive electron donating groups,
4
(176.27 μM) and
5 (162.12 μM), and an electron donating group by resonance,
10 (195.05 μM), had lower IC50 values. This finding was in
agreement with many previous research articles that indicated
that electron donating groups would stabilize the radical elec-
tron, while the electron withdrawing group would destabi-
lize it.32
However, the observation that perplexed us was the IC50
values of compounds 6 (with Br substituent, 138.60 μM)
and 7 (with Cl substituent, 150.65 μM), which were quite
low and comparable to the IC50 values of the compounds with
electron donating groups.
Br and Cl were known to exert inductive electron with-
drawing effects. However, in our case, these halogen groups
had lone pair electrons. When they are directly connected to
a radical center, they helped stabilize the center by donating
Figure 4. Oak ridge thermal ellipsoid plot (ORTEP) diagram of
compound 4 at the 50% probability level.
1
R1
R
R
R
R
O
N
O
O
N
N
N
N
O
O
N
N
N
N
N
R
H
2
R1
R1
R1
R1
Figure 5. The hydrazine and aldehyde/ketone moieties of the
Scheme 5. The stabilization of the radical formed on the N atom.
hydrazones.
Bull. Korean Chem. Soc. 2015, Vol. 36, 2716–2724
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Synthesis of the Hydrazones of 2-((3,5-Di-tert-butyl-4-hydroxybenzyl)thio)
acetohydrazide
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one of the electrons (Scheme 6). A similar result was reported
by Nazarbahjat, et al. and Lone et al., in which the compounds
with halogen substituents on the para-position had good
DPPH IC50 values.33,34
delocalized, which causes the radical compound to become
more stable.38
Calculation of the Parameters. All of the values were calcu-
lated using the DFT quantum mechanical method at the
B3LYP/6-311G (d,p) level.33 The BDE values of the hydra-
zones were calculated using the following formula.
Predictionof Radical Scavenging Activity Using a Compu-
tational Method. One of the pathways in which antioxidants
can play a radical scavenger role is H-atom transfer.35 The
pathway was described for the important case of lipid perox-
idation (Scheme 7). Once a free radical Rꢀ has been initiated in
Step (1), a chain of reactions is formed in Steps (2) and (3),
yielding many lipid molecules (R H) that are converted into
lipid hydroperoxide (ROOH). Equation (2) is very fast,
ca. 109/M/s, whereas Eq. (3) is slower at 101/M/s. The role
of the antioxidant ArOH is to interrupt the chain reaction
according to Step (4). To be effective, the ArOꢀ must be a rel-
atively stable radical so that it reacts slowly with the substrate,
RH, but rapidly with ROOꢀ.
The rate of Eq. (3) depends on the energy barrier of the H-
atom transfer from RH to RO2ꢀ . The same case applies for the
chain breaking Eq. (4). To effectively prevent the reaction of
ROꢀ2 with the substrate RH, the energy barrier of the reaction of
antioxidant ArOH with ROꢀ2 should be lower than that of
Eq. (3).36 The parameter that can be used to determine the
energy barrier is bond dissociation enthalpy (BDE).36 BDE
of the O H bond of the antioxidant calculates the energy
needed to overcome the energy barrier of the H-atom transfer.
Better antioxidants will have lower BDE O H values.
Another important parameter in evaluating the radical
scavenging activity of antioxidants is spin density (SD).37
SD characterizes the stability of the free radical. SD is
described as the population difference between the spin-up
(α) electron and spin-down (β) electron. In a fully occupied
molecular orbital (FOMO), the SD is zero because there is
one α-spin electron and one β-spin electron. On the other hand,
a singly occupied molecular orbital (SOMO) has a SD value of
one or negative one. The SD distribution calculates the delo-
calization of the unpaired electron on the radical atom. Low
SD on a radical atom in the radical form of antioxidants indi-
cates that the unpaired electron on the atom is highly
ArOH ! ArOꢀ + H
BDE = EðArOÞ + EðHÞ – EðArOHÞ
ð5Þ
ð6Þ
BDE was calculated as the sum of the total energy for the
formed radical and the hydrogen atom minus the total energy
of the antioxidant before the dissociation.37 Jursic et al. sug-
gested that the DFT calculation overestimates the total energy
of the H atom.39 To counter this problem, an alternative
method was applied to obtain the value. Hoelz et al. used phe-
nol as the reference in calculating the total energy of the H
atom, as shown in the following equation.40
BDE = EðPhOÞ + EðHÞ – EðPhOHÞ
EðHÞ = BDE – EðPhOÞ + EðPhOHÞ
ð7Þ
ð8Þ
BDE is the experimental value of the BDE O H of phenol,
which is 86.5 kcal/mol, while E(PhOH) and E(PhOꢀ) are the
total energy of phenol and the radical form of phenol, respec-
tively.40 Both values were calculated using DFT. The SD
values of the radical compounds were obtained by calculating
the Mulliken population analysis using DFT.33
There were two sites for compounds 4–10, OH-1 and NH,
and three for compound 11, OH-1, NH, and OH-2, that might
havethecapabilityof donating an Hatom (Figure 6). The BDE
and SD values were calculated for each site. The result of the
calculations is summarized in Table 2.
Withtheexception ofcompound4, theBDEvaluesofOH-1
for the hydrazones did not differ too much from each other; all
of them were within the range of 78.3–78.8 kcal/mol. The dif-
ferent substituents on the compounds did not exert any elec-
tronic effect on the BDE value of OH-1 because the
delocalization of the radical electron stopped at the 4th carbon
and the substituents were too far away from the radical oxygen
to apply any inductive effect. The SD values of OH-1 for the
compounds were also close to each other, indicating that the
stability and the delocalization of the radicals were about
the same (Scheme 8).
R1
R1
R1
R1
R1
O
O
O
N
N
N
N
N
N
O
O
N
N
N
N
Cl
Cl
Cl
Cl
Cl
OH-1
OH-1
Scheme 6. The stabilization of the radical center by Cl group for
compound 7.
OH-2
R
H
O
H
O
H
O
O
O
N
N
S
S
N
N
H
H
+
O2
O2
RH
O2
R
R
H
(initiation)
(1)
(2)
(3)
(4)
+
R
R
(addition of O2)
(H-atomexchange)
(chainbreaking)
NH
NH
+
+
R
R
R
R
H
O2
+
4-10
11
r
+
A
O
R
ArOH
H
O2
O2
Figure 6. Active sites with transferable H atoms for com-
pounds 4–11.
Scheme 7. H-atom transfer mechanism of antioxidants.
Bull. Korean Chem. Soc. 2015, Vol. 36, 2716–2724
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Synthesis of the Hydrazones of 2-((3,5-Di-tert-butyl-4-hydroxybenzyl)thio)
acetohydrazide
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Table 2. Computational calculation results for compounds 4–11 and BHT.
BDE OH-1
(kcal/mol)
BDE OH-2
(kcal/mol)
BDE NH
(kcal/mol)
Spin density
OH-1
Spin density
OH-2
Spin
density NH
Compound
4
5
6
7
8
9
10
11
BHT
80.47
78.41
78.58
78.58
78.75
78.68
78.35
78.31
76.58
—
—
94.11
91.25
93.72
91.75
92.23
92.04
93.54
96.04
—
0.3462
0.3459
0.3469
0.3466
0.3473
0.3469
0.3457
0.3457
0.3459
—
—
0.5156
0.4841
0.4935
0.4932
0.4975
0.4981
0.4980
0.4887
—
—
—
—
—
—
—
—
—
—
—
71.28
—
0.2766
—
0.3641
R1
0.2347
0.3888
O
O
O
O
HN
O
0.3012
0.3550
R
R
R
R
N
0.3012
Scheme 8. The delocalization of radical form OH-1 and the spin den-
sity values of the radicals for compound 5.
O
0.2213
0.2766
Compound 11
The BDE OH-1 values of the compounds were higher com-
pared to those of BHT(76.55 kcal/mol). Theelectron donating
substituent at the ortho- and para-positions of the phenolic
antioxidant would lower its BDE OH value.7 The absence
of an electronegative atom next to it allowed the methyl group
in BHT to give better electron donating effect as compared
with the hydrazone compounds. Hence, the BHT radical
formed were more stabilized, which generate a lesser BDE
OH-1 value. The calculation of the energies was carried out
with the assumption that the molecules were in the gas phase.
The experimental value of the BDE OH of BHT in benzene
was reported to be 79.9 kcal/mol.41
The BDE OH-2 was 71.28 kcal/mol, which was the lowest
among the compounds. This meant that the radical formed on
the OH-2 site on compound 11 was the most stable. The sta-
bility was attributed by the number of atoms that the radical
electron could be delocalized on (Figure 7).
The BDE NH values of the compounds (91–96 kcal/mol)
seemed quite high. We were hoping for lower values due
to the intended radical delocalization over many atoms, as
suggested by Belkheiri et al. (Scheme 5).10 In general, good
antioxidants are characterized by BDE values that are lower
than the BDE value of the O H bond of hydroperoxide
(88 kcal/mol).41 The high values for the BDE of the NH com-
pounds might be due to the presence of an electron withdraw-
ing group next to the N atom ( C(O)R), which caused the
electron delocalization over the aromatic ring to become very
minimal. This assumption was in agreement with what we dis-
cussed earlier regarding the little effect that the substituents
exerted onthe DPPH assay and with the literature that wemen-
tioned earlier that the substituents on the hydrazine moiety of
the hydrazone exerted a greater effect on the strength of the
NH bond compared with the substituents on the aldehyde/
ketone moiety.31
Figure 7. The delocalization of radical form OH-2 and the spin den-
sity values of the radicals for compound 11.
Conclusion
The combination of BHT and a thioether in a single structure
was believed to increase the overall antioxidant activity due to
thesynergy between thechain breaking andpreventive antiox-
idants. However, based on the calculated BDE OH-1 values of
the compounds, we discovered that the location of thioether at
the 4th carbon of the BHT decreased the radical scavenging
activity of the chain breaking antioxidant. Relocating the
thioether group farther away from the BHT ring might be able
to prevent the decrease in the BDE value of the BHT.
The presence of a hydrazone group in the structure
increased the radical scavenging activity of the compound,
as shown by the comparison of the IC50 values of compound
4–10 and BHT. However, the calculations of the BDE NH
values of those compounds gave quite large figures. Based
on the previous literature, it was suggested that the BDE
NH value could be lowered by replacing the C O group next
to the NH with an electron donating group. By doing so, the
radical electron could be efficiently delocalized over the aro-
matic ring; thus, the BDE NH values would be lowered and the
overall radical scavenging activity would be increased.31
Electron donating group substituents on the para-position
of the aromatic ring would increase the radical scavenging
activity, while the electron withdrawing group would have
the opposite effect. However, there was an exception in the
halogen group substituents, where the lone pair electrons on
the atoms could stabilize the radical on the neighboring carbon
to increase the radical scavenging activity.
Bull. Korean Chem. Soc. 2015, Vol. 36, 2716–2724
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Synthesis of the Hydrazones of 2-((3,5-Di-tert-butyl-4-hydroxybenzyl)thio)
acetohydrazide
BULLETIN OF THE
Article
KOREAN CHEMICAL SOCIETY
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Acknowledgments. We would like to thank the University of
Malaya for PPP grants (PG028-2013B and PG030-2013B) for
supporting this study. Muhammad Abdulwahab wishes to
thank the Ministry of Education (MOE) of Malaysia for the
scholarship (SLAB Scheme).
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Bull. Korean Chem. Soc. 2015, Vol. 36, 2716–2724
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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