Radical-scavenging activity of the reaction products of isoeugenol with thiol, thiophenol, mercaptothiazoline or mercaptomethylimidazole using the induction period method

Article abstract of DOI:10.3390/12081836

The reaction products in the presence of Lewis acid of isoeugenol (1) with ethanethiol, thiophenol, 2-mercaptothiazoline or 2-mercapto-1-methylimidazole (ISO-S1 - ISO-S-4) were obtained. The radical-scavenging activity of these compounds was investigated

Full text of DOI:10.3390/12081836

Molecules 2007, 12, 1836-1844
molecules
ISSN 1420-3049
© 2007 by MDPI
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Full Paper
Radical-scavenging Activity of the Reaction Products of
Isoeugenol with Thiol, Thiophenol, Mercaptothiazoline or
Mercaptomethylimidazole Using the Induction Period Method
Yoshinori Kadoma 1, Wakako Kumada 2,Yuhta Asai 2, Yoshiaki Sugita 2, Ichiro Yokoe 2 and
Seiichiro Fujisawa 2,*
1
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Kanda-
surugadai, Chiyoda-ku, Tokyo 101-0062, Japan; E-mail: y-kadoma.fm@tmd.ac.jp
2
Faculty of Pharmaceutical Sciences, Josai University, Saitama 3500295; Meikai Unversity School of
Dentistry, Sakado, Saitama 3500283 Japan; E-mails: wakako-216@ezweb.ne.jp (Kumada);
fqqrk256@yahoo.co.jp (Asai); yokoe@josai.ac.jp (Yokoe)
* Author to whom correspondence should be addressed; E-mail: fujisawa 33@nifty.com
Received: 13 July 2007; in revised form: 9 August 2007 / Accepted: 9 August 2007 / Published: 13
August 2007
Abstract: The reaction products in the presence of Lewis acid of isoeugenol (1) with
ethanethiol, thiophenol, 2-mercaptothiazoline or 2-mercapto-1-methylimidazole (ISO-S1 –
ISO-S-4) were obtained. The radical-scavenging activity of these compounds was
investigated using the induction period method for polymerization of methyl methacrylate
(MMA) initiated by thermal decomposition of 2,2'-azobisisobutyronitrile (AIBN) and
benzoyl peroxide (BPO) and monitored by differential scanning calorimetry (DSC). For
BPO, the stoichiometric factor (number of free radicals trapped by one mole of antioxidant
moiety, n) declined in the order isoeugenol (1.8) > ISO-S-1 (1.6) > ISO-S-2 (1.2 ) > ISO-
S-3 (0.9) > ISO-S-4 (0.3), whereas for AIBN, their n values were about 1, except for ISO-
S-3 (0.6). The ratio of the rate constant of inhibition to that of propagation (kinh/kp) for
BPO declined in the order ISO-S-4 (56) > ISO-S-3 (15) > ISO-S-2 (11) >ISO-S-1 (9) >
isoeugenol (8). Similarly, for AIBN the kinh/kp of the reaction products (33-57) was greater
than that of isoeugenol (31). The reaction products of isoeugenol with a SH group showed
greater inhibition rate constants (kinh) than the parent compound isoeugenol.

Molecules 2007, 12
Keywords: radical-scavenging activity; thioisoeugenols, BPO, AIBN; induction period
1837
method.
Introduction
Isoeugenol (4-propenyl-2-methoxyphenol, 1) is used in perfumes, soaps, detergents, air fresheners
and cosmetics. However, isoeugenol is also an important allergen [1]. Furthermore, isoeugenol has a
moderate acute toxicity by dermal and oral routes. Although isoeugenol exhibited cytotoxic and the
mentioned allergenic activity, it is a potent antioxidant [1]. It has been generally assumed that the
antioxidant effects of phenolic antioxidants are responsible for their anti-inflammatory and
chemopreventive effects. We previously synthesized a dimer of isoeugenol, dehydrodiisoeugenol,
demonstrating that this compound, but not isoeugenol, displays potent cyclooxygenase-2 (COX-2)
inhibition, an anti-inflammatory activity [2]. Also, we previously reported that the cytotoxicity of
isoeugenol may be attributed to its induction of reactive oxygen species (ROS) and the reduction of
gluthathione (GSH) levels, possibly due to its prooxidative activity [3]. Curcumin, an isoeugenol-
related compound, was previously reported to form conjugates with GSH by separating mono- and
diglutathionyl adducts of curcumin. This suggested that formation of curcumin-GSH adducts lead to
inactivation of the parent curcumin’s prooxidative activity [4]. These results led us a hypothesis:
thioisoeugenols may reduce the cytotoxic and allergenic activity without decreasing their antioxidant
activity. Thus, we synthesized ISO-S-1, a reaction product of isoeugenol (1) with ethanethiol; ISO-S-
2, a reaction product of 1 with thiophenol; ISO-S-3, its reaction product with 2-mercaptothiazoline
and ISO-S-4, a reaction product of 1 with 2-mercapto-1-methylimidazole (see Scheme 1).
Scheme 1. Preparation of products ISO-S-1 – ISO-S-4.
OH
OH
OMe
OMe
RSH
CF3SO3Si(Me)3
5'
3'
2'
1'
1
2
3
SR
S
Is o e ug e n o l ( 1 )
ISO-S-1: R=Et
ISO-S-2: R=Ph
ISO-S-3: R=
N
ISO-S-4: R=
NMe
N

Molecules 2007, 12
1838
We have previously used DSC and the induction period method to investigate the radical
scavenging activity of phenolic antioxidants under nearly anaerobic conditions, and this method has
been proven to be reliable for evaluating the activity of these compounds [5,6].
In an extension of this work, we investigated here the radical-scavenging activity of ISO-S-1, 2, 3
and 4, and their parent compound, isoeugenol, by determining the induction period for polymerization
of methyl methacrylate (MMA) initiated by thermal decomposition of 2,2'-azobisisobutyronitrile
(AIBN, a R. radical) and benzoyl peroxide (BPO, a PhCOO. radical).
Results and Discussion
Radical scavenging activities determined by the induction period method
The time-exotherm and time-conversion curves for ISO-S-1, 2, 3, 4 and isoeugenol are shown in
Figures 1 and 2, respectively.
Figure 1. Exothermic curves for the polymerization of MMA initiated by thermal
decomposition of BPO in the presence of 0.01 mol% additives.
4
BPO
A
E
D
C
B
3
2
1
0
F
A : Control
B : ISO-S-1
C : ISO-S-2
D : ISO-S-3
E : ISO-S-4
F : ISOEUGENOL
0
60
120
180
Time (min)
Figure 2. Time-conversion curves for the polymerization of MMA initiated by thermal
decomposition of BPO in the presence of 0.01 mol% additives. Curves were obtained
from findings shown in Figure 1.
100
BPO
80
AE D C BF
A : Control
60
40
B : ISO-S-1
C : ISO-S-2
D : ISO-S-3
E : ISO-S-4
F : ISOEUGENOL
20
0
0
60
120
180
Time (min)

Molecules 2007, 12
1839
The n value was determined from each slope (Figure 3, see Equation 2). Results are shown in
Table 1. For BPO, the n value declined in the order isoeugenol (1.8 )> ISO-S-1 (1.6) > ISO-S-2 (1.2)
> ISO-S-3 (0.9) > ISO-S-4 (0.3), whereas for AIBN, the n value of ISO-1, 2, and 4 and isoeugenol
were all about 1, while the value for ISO-3 was 0.6. In general, the n value of phenolic compounds is
close to 2 [7]. The n value for 2-methoxyphenol is less than 2, due to the strong internal hydrogen
bond on the OH and the methoxy group [5c]. It is well known that the one-electron oxidation of
phenolic compounds; the n of about 1 leads to dimmers and to polymers (lignin) [8]. In the present
study, compounds with the n of about 1 may occur dimerization during induction period.
Figure 3. A typical plot of the induction period vs the concentration for ISO-S-1, 2, 3,
and 4 in the polymerization of MMA initiated by thermal decomposition of AIBN (left
panel) and BPO (right panel).
2000
1000
0
10000
8000
6000
4000
2000
BPO
AIBN
ISO-S-1
ISO-S-2
ISO-S-3
ISO-S-4
ISO-S-1
IS0-S-2
ISO-S-3
IS0-S-4
0
0.00
0.10
0.20
0.00
0.10
Inhibitors (mol%)
0.20
Inhibitors (mol%)
Table 1. Radical-scavenging activity of the reaction products of isoeugenol (1) with
ethanethiol, thiophenol, 2-mercaptothiazoline or 2-mercapto-1-methylimidazole (ISO-S-1
– ISO-S-4) in the polymerization of methyl methacrylate initiated by AIBN and BPO.
AIBN
BPO
Compound
ISO-S-1
Relative n
0.92 ± 0.05
0.96 ± 0.04
0.59 ± 0.02
1.01 ± 0.05
1.11 ± 0.06
kinb/kp
Relative n
1.61 ± 0.08
1.19 ± 0.05
0.91 ± 0.04
0.25 ± 0.01
1.75 ± 0.09
kinb/kp
36.43 ± 1.78
34.81 ± 1.71
56.63 ± 2.27
32.86 ± 1.48
30.52 ± 1.36
8.88 ± 0.44
11.75 ± 0.58
15.46 ± 0.76
55.95 ± 2.79
8.16 ± 0.42
ISO-S-2
ISO-S-3
ISO-S-4
Isoeugenol
n values calculated using Eqn. 2. kinb and kp are the rate constants of inhibition and propagation,
respectively. The kinb/kp was calculated using Eqn. 5. MMA, 9.4 mol/L; compounds, 1mM;
T=70°C; conversion: 90.3-95.3%; values, the mean ± SD for three different experiments.

Molecules 2007, 12
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The inhibition rate constant, kinh plays a more important role for evaluating the activity, so we
investigated this parameter. The relationships between propagation rate (Rp), the initial rate of
polymerization and concentration of the ISO-S compounds are shown in Figure 4. Plots of the
Rpinh/Rpcon vs concentrations for both initiators decreased linearly as the concentration increased. In
particular, ISO-S-2 was strongly reduced in both initiators, followed by the great retardation of the
growing MMA radicals. This was possibly due to the strong interaction between the oxidized products
of ISO-S-2 with a benzene ring and the growing MMA radicals. For AIBN and BPO, the kinh/kp was
calculated using Equation 5.
Figure 4. A typical plot of the Rpinh/Rpcon vs the concentration for ISO-S-1, 2, 3, and 4.
1.1
1.0
0.9
0.8
1.2
1.0
0.8
0.6
0.4
0.2
AIBN
BPO
S-1
S-2
S-3
S-4
S-1
S-2
S-3
S-4
0.00
0.10
0.20
0.00
0.10
0.20
Inhibitors (mol%)
Inhibitors (mol%)
Results are shown also in Table 1. For AIBN, the kinh/kp value for isoeugenol (31) was the smallest.
The kinh/kp value of ISO-S-3 (57) was the greatest, but was about only twice as large as that of other
reaction products. For BPO, the kinh/kp declined in the order ISO-S-4 (56) > ISO-S-3 (15) > ISO-S-2
(12) >ISO-S-1 (9) > isoeugenol (8). The kinh of ISO-S-4 was estimated as 4.4 x 104 mol-1s-1, assuming
that the kp value for MMA is 797 mol-1s-1 [5c]. Antioxidants having a n value of about 1 or less than 1
may undergo dimerization during the induction period. In contrast, the n of isoeugenol for BPO was
about 2, suggesting the formation of vinyl quinone methide derived from a two-electron oxidation of
this compound [9]. The radical-scavenging activity of ISO-S-1, 2, 3, and 4 against PhCOO., an
oxygen-centered radical derived from BPO, was clearly different from that against R., a carbon-
centered radical derived from AIBN. These compounds preferred an oxygen-centered radical rather
than a carbon-centered radical.
Conclusions
ISO-S-1, 2, 3, and 4 were obtained by reaction of isoeugenol with compounds having a SH group
Their radical-scavenging activity using two methods under nearly anaerobic conditions (n and kinh)

Molecules 2007, 12
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was determined using the induction period method. The reaction products showed the greater kinh /kp
than that of isoeugenol.
Experimental
General
The chemicals were purchased from Tokyo Kasei Kogyo Ltd. (Tokyo Japan) . NMR spectra were
measured in CDCl3 solutions at ambient temperature on a JEOL, JNM-A500 spectrometer (JEOL,
Tokyo, Japan). The chemical shifts δ are given in ppm related to tetramethylsilane, TMS as internal
standard. Mass spectra were measured on aJMS/700 (JEOL).
General procedure for the synthesis of ISO-S-1, 2, 3, and 4: reaction of isoeugenol (1) with thiols in
the presence of Lewis acid
To a solution of 1 (0.5 g, 3 mmol) and ethanethiol (0.76 g, 12 mmol) in dry chloroform (20 mL)
trimethylsilyl trifloromethanesulfonate (0.5 mL) was added and the reaction mixture was stirred at
room temperature for 0.5 h. After washing with 5% sodium carbonate solution the mixture was
evaporated and purified by silicagel chromatography to give an oily product (ISO-S-1) in 66.0% yield.
MS; m/z 165 (100%), 226 (M+, C12H18O2S); 1H-NMR (CDCl3) δ (ppm): 6.86 (1H, d, J=1.8 Hz, C2’-H),
6.83 (1H, d, J=8.0 Hz, C5’-H), 6.75 (1H, dd, J=8.0, 1.8 Hz, C6’-H), 3.90 (3H, s, C3’-OCH3), 3.65 (1H,
dd, J=8.7, 6.1 Hz, C1-H), 2.3-2.4 (2H, m, SCH2-CH3), 1.78-1.92 (2H, m, C1-CH2CH3), 1.15 (3H, t,
J=7.3 Hz, S-CH2CH3), 0.90 (3H, t, J=7.3 Hz, C1-CH2CH3).
Reaction of 1 with thiophenol
According to the general procedure, the reaction of isoeugenol with thiophenol for 1.5 h gave an
oily product (ISO-S-2) in 75.0% yield. MS; m/z 165 (100%), 274 (M+, C16H18O2S); 1H-NMR (CDCl3)
δ: 7.18-7.26 (5H, m, S-Ph), 6.80 (1H, d, J=8.0 Hz, C5’’-H), 6.74 (1H, d, J=2.1 Hz, C2’-H), 6.72 (1H, dd,
J=8.0, 2.1 Hz, C6’-H), 3.97 (1H, dd, J=8.9, 6.1 Hz, C1-CH-), 3.83 (3H, s, C3’-OCH3), 1.80-1.98 (2H,
m, C1-CH2CH3), 0.90 (3H, t, J=7.3Hz, C1-CH2CH3).
Reaction of 1 with 2-mercaptothiazoline
According to the general procedure, the reaction of isoeugenol with 2-mercaptothiazoline for 24 h
1
gave an oily product (ISO-S-3) in 36.5% yield. MS; m/z 165 (100%), 283 (M+, C13H17O2NS2); H-
NMR (CDCl3) δ: 6.96 (1H, d, J=1.7 Hz,C2’-H), 6.89 (1H, d, J=8.2 Hz, C5’-H), 6.86 (1H, dd, J=8.2,
1.7 Hz, C6’-H), 6.10 (1H, m, C1-CH-), 3.88 (3H, s, C3’-OCH3), 3.6-3.9 (2H, m, -S-CH2CH2-N), 3.05-
3.24 (2H, m, -S-CH2CH2-N=), 1.88-2.12 (2H, m, C1-CH2CH3), 1.01(3H, t, J=7.3Hz, C1-CH2CH3).

Molecules 2007, 12
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Reaction of 1 with 2-mercapto-1-methylimidazole
According to the general procedure, the reaction of eugenol with 2-mercapto-1-methylimidazole
for 24 h gave a crystalline product (ISO-S-4); mp 137-138°C (hexane-AcOEt) in 48.2% yield. MS;
1
m/z 165 (100%), 246 (M+, C14H18O2N2S); H-NMR (CDCl3) δ: 6.96 (1H, d, J=1.7Hz, C2’-H), 6.87-
6.91 (2H, m, C5’, 6’-H), 6.64 (2H, s, imidazole-CH=CH-), 5.94 (1H, t, J=7.9Hz, C1-CH-), 3.87 (3H, s,
C3’-OCH3), 3.62 (3H, s, N-Me), 2.08-2.19 (2H, m, C1-CH2CH3), 0.95 (3H, t, J=7.9Hz, C1-CH2CH3).
DSC measurements
The induction period (IP) and initial rate of polymerization in the presence (Rpinh) or absence
(Rpcon) of ISO-S-1, 2, 3, 4 and isoeugenol (1) (Figure 2) were determined by the previously reported
method [6]. In brief, the experimental resin consisted of MMA and AIBN (or BPO) with or without
additives. AIBN (or BPO) were added at 1.0 mol%, and the additives were used at 0, 0.001, 0.01, 0.02
and 0.1 mol%. Approximately 10 µL of the experimental resin (MMA: 9.12-9.96 mg) was loaded into
an aluminum sample container and sealed by applying pressure. The container was placed in a
differential scanning calorimeter (model DSC 3100; MAC Science Co., Tokyo, Japan) kept at 70°C,
and the thermal changes induced by polymerization were recorded for the appropriate periods. The
heat due to polymerization of MMA was 13.0 kcal/mole in this experiment. The conversion of all
samples, as calculated from the DSC thermograms, was 91-96%. Polymerization curves were derived
from DSC thermograms using the integrated heat evoked by the polymerization of MMA.
Polymerization curves break when an inhibitor is consumed (Figure 2). These breaks are sharp and
provide a reliable measure of the IP of the inhibitor. The presence of oxygen retards polymerization
because oxygen reacts with MMA radicals activated by the initiator and then subsequently produces a
non-radical product. Thus, polymerization of the control was slightly inhibited, even though the
reaction was carried out in a sealed DSC pan, because the pan contained a small amount of oxygen;
approximately 8.128 x 10-8 mole/L since it had been sealed in air. Tangents were drawn to
polymerization curves at an early stage in the run. The IP of test compounds was determined from the
length of time between the zero point on the abscissa and the point of intersection of tangents drawn to
the early stage of polymerization. The IP was calculated from the difference between the induction
period of specimens and that of controls. The initial rates of polymerization in the absence (Rpcon) and
presence (Rpinh) of natural and synthetic antioxidants were calculated from the slope of the plots of the
first linear line of the conversion rate of MMA polymerization (tangent drawn at the early
polymerization stage).
Rate of initiation
The induction period method was used to determine the rate of initiation (Ri) due to the thermal
decomposition of AIBN or BPO according to Eqn. (1):
Ri = n [IH]0/[IP]
(1)
where [IH]0 is the concentration of the inhibitor at time zero and [IP] is the induction period. 2,6-di-
tert-butyl-4-methoxyphenol (DTBM) was used to determine Ri, since its stoichiometric factor, n, is

Molecules 2007, 12
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known to be 2.00 [7]. In the case of [MMA] = 9.4 M and [AIBN or BPO] = 0.1 M at 70°C, the
induction period method using DTBM gave the rate of initiation, Ri, at 70°C. The Ri values of AIBN
and BPO were 5.66 x 10-6 Ms-1 and 2.28 x 10-6 Ms-1, respectively.
Measurement of stoichiometric factor (n)
The relative n value in Eqn. (2) can be calculated from the induction period in the presence of
inhibitors:
n = Ri[IP]/[IH]
(2)
where [IP] is the induction period in the presence of an inhibitor. The number of moles of peroxy
radicals trapped by the antioxidant is calculated with respect to 1 mole of inhibitor moiety unit.
Measurement of the inhibition rate constant (kinh)
When Ri is constant, i.e. when new chains are started at a constant rate, a steady-state treatment can
be applied and the initial rate of polymerization of MMA is given by Eqn. (3) [6]:
Rpcon = {kp [MMA] Ri1/2}/(2kt)1/2
(3)
where MMA represents methyl methacrylate and kp and kt are the rate constants for chain propagation
and termination, respectively.
The kp/(2kt)1/2 rate of polymerization of MMA (9.4 M) by AIBN (1 mol%) and BPO (1 mol%) at
70°C was a constant value, 9.86 x 10-2 M-1/2 s-1/2. The Rpinh rates are determined by Eqn. (4):
Rpinh = {kp [MMA] Ri} /{n kinh [IH]}
(4)
in which Rpinh is the initial rate of inhibited polymerization, [MMA], n, [IH] and kp are defined above,
and kinh is the rate constant for scavenging (inhibiting) of MMA radicals by an antioxidant. From Eqn.
(2) and Eqn. (4), kinh/kp can be calculated (Eqn. 5):
kinh/kp = [MMA]/{[IP] x [Rpinh]}
(5)
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Sample Availability: Available from the authors.
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