Synthesis and antioxidant activity of novel Mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan

Article abstract of DOI:10.1016/j.bmc.2013.08.002

A new series of Mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan (6a-6ae) were synthesized and characterized by ¹H NMR, ESI-MS and elemental analysis. The structure of 6b was further confirmed by single crystal X-ray diffraction. All these novel compounds were screened for their in vitro antioxidant activity employing 2,2′-diphenyl-1- picrylhydrazyl radical (DPPH), 2,2′-azinobis (3-ethylbenzothiazoline-6- sulfonate) cationic radical (ABTS⁺) and ferric reducing antioxidant power (FRAP) scavenging assays. Due to the combination of 1,4-benzodioxan, 1,3,4-oxadiazoles and substituted phenyl ring, most of them exhibited nice antioxidant activities. In all of these three assays mentioned above, compounds 6f and 6e showed significant radical scavenging ability comparable to the commonly used antioxidants, BHT and Trolox. Seven compounds with representative substituents or activities were selected for further assays in chemical simulation biological systems - inhibition of microsomal lipid peroxidation (LPO) and protection against 2,2′-azobis (2-amidinopropane hydrochloride) (AAPH) induced DNA strand breakage, in which, 6f and 6e were demonstrated to be of the most potent antioxidant activities.

Full text of DOI:10.1016/j.bmc.2013.08.002

Accepted Manuscript
Synthesis and antioxidant activity of novel Mannich base of 1,3,4-oxadiazole
derivatives possessing 1,4-benzodioxan
Liang Ma, Yu Xiao, Cong Li, Zheng-Lu Xie, Dong-Dong Li, Yan-Ting Wang,
Hai-Tian Ma, Hai-Liang Zhu, Ming-Hua Wang, Yong-Hao Ye
PII:
S0968-0896(13)00684-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.08.002
BMC 11018
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
11 June 2013
31 July 2013
1 August 2013
Please cite this article as: Ma, L., Xiao, Y., Li, C., Xie, Z-L., Li, D-D., Wang, Y-T., Ma, H-T., Zhu, H-L., Wang,
M-H., Ye, Y-H., Synthesis and antioxidant activity of novel Mannich base of 1,3,4-oxadiazole derivatives possessing
,4-benzodioxan, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.08.002
1
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Synthesis and antioxidant activity of novel Mannich base of 1,3,4-oxadiazole
derivatives possessing 1,4-benzodioxan
a
a
a
c
b
b
c
Liang Ma , Yu Xiao , Cong Li , Zheng-Lu Xie , Dong-Dong Li , Yan-Ting Wang , Hai-Tian Ma ,
b,
a
a,
Hai-Liang Zhu *, Ming-Hua Wang , Yong-Hao Ye *
a. College of Plant Protection, Jiangsu Key Laboratory of Pesticide Science, Nanjing Agricultural
University, Nanjing 210095, P. R. China
b. State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
PR China
c. Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine,
Nanjing Agricultural University, Nanjing 210095, China
*
Corresponding authors. Tel.: +86-25-84395479. fax: +86-25-84395479. E-mail address:
yeyh@njau.edu.cn(Y.-H. Ye); zhuhl@nju.edu.cn (H.-L. Zhu).
1

Abstract
A new series of Mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan (6a –
1
6
ae) were synthesized and characterized by H-NMR, ESI-MS and elemental analysis. The
structure of 6b was further confirmed by single crystal X-ray diffraction. All these novel
compounds were screened for their in vitro antioxidant activity employing
2
,2’-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonate)
+
cationic radical (ABTS •) and ferric reducing antioxidant power (FRAP) scavenging assays. Due
to the combination of 1,4-benzodioxan, 1,3,4-oxadiazoles and substituted phenyl ring, most of
them exhibited nice antioxidant activities. In all of these three assays mentioned above,
compounds 6f and 6e showed significant radical scavenging ability comparable to the commonly
used antioxidants, BHT and Trolox. Seven compounds with representative substituents or
activities were selected for further assays in chemical simulation biological systems – inhibition of
microsomal lipid peroxidation (LPO) and protection against 2,2’-azobis (2-amidinopropane
hydrochloride) (AAPH) induced DNA strand breakage, in which, 6f and 6e were demonstrated to
be of the most potent antioxidant activities.
Keywords
Mannich base; 1,3,4-oxadiazole; 1,4-benzodioxan; antioxidant activity
2

1
. Introduction
Reactive oxygen species (ROS), known as mediators of intracellular signaling cascades, are
chemically reactive molecules containing oxygen. Most living organisms can produce ROS and
metabolize excessive ones by normal physiological processes. However, sometimes these efficient
protecting systems could be disrupted by external factors (smoke, alcohol, diet and some drugs) or
1
aging. Accumulation of excessive ROS can damage lipids, proteins, carbohydrates, and DNA in
cells, leading to oxidative stress, loss of cell function, and ultimate apoptosis or necrosis. And all
of the above biochemical processes are the common problems in various diseases such as cancer,
2
inflammation, atherosclerosis and cardiovascular disease. Therefore, exploring antioxidant
chemicals that scavenge ROS may be of great value in preventing the onset and propagation of
oxidative stress.
Mannich bases derived from various heterocycles exhibit unique biological activities, such as
3
4
5
6
antitubercular, antimalarial, anticancer and analgesic properties. Moreover, they were
7
recognized as a useful pharmacophore group against thrombosis caused by antioxidants. As an
important class of heterocyclic compounds, 1,3,4-oxadiazoles are associated with a broad
8
,9
8
10-13
spectrum of biological activities including anti-inflammatory, analgesic, antimicrobial,
14
10,15,16
anticancer and antioxidant
activities. And those heterocyclic derivatives bearing
1
7-19
1
,4-benzodioxan moiety have been proved to be of pharmacological value on antitumor
and
20
21
22
immunosuppressive, anti-inflammatory and antifungal activities.
The combination of different pharmacophores in the same unit is an attracting approach to
23
discover novel potent drugs, due to the possible synergistic effect. In this study, a series of
1
,3,4-oxadiazole derivatives possessing a 1,4-benzodioxan moiety were synthesized through
3

Mannich reaction. The radical scavenging properties of these novel compounds were screened by
three quick assays first: 1) trapping 2,2’-diphenyl-1-picrylhydrazyl radical (DPPH), 2)
+
2
,2’-azinobis (3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS •), 3) and ferric
reducing antioxidant power (FRAP) methods. Then seven compounds with representative
substituents or activities were selected to evaluate their antioxidant effects against lipid
peroxidation (LPO) of mice liver microsomes and 2,2’-azobis (2-amidinopropane hydrochloride)
(AAPH) induced supercoiled DNA breakage, respectively.
2
. Results and discussion
2
.1. Chemistry
A total of 30 novel Mannich base of 1,3,4-oxadiazole derivatives possessing 1,4-benzodioxan
(
6a – 6ae) were synthesized from 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazole-2-
thiol (4) and different kinds of amine derivatives (5) by Mannich condensation reaction as
depicted in Scheme 1. The key intermediate 4 was prepared in three steps according to a
24
previously reported method. Esterification of the 2,3-dihydrobenzo[b]-[1,4]dioxine-6-carboxylic
acid (1) with methanol and concentrated sulfuric acid produced the corresponding ester 2. The
aroyl hydrazide 3 was obtained by reacting 2 with 85% hydrazine monohydrate in ethanol.
Treatment of the hydrazide 3 with carbon disulfide in the presence of KOH and 95% ethanol under
refluxing gave the key intermediate 4. After dissolved in ethanol, compound 4 was reacted with
formalin and appropriate amines (substituted aniline, cyclohexylamine or hexadecylamine) to
afford the target compounds 6a – 6ac, 6ad and 6ae, respectively. All of the synthetic compounds
gave satisfactory analytical and spectroscopic data, which were in full accordance with their
4

depicted structures. Additionally, the structure of compound 6b was further confirmed by X-ray
diffraction. Its perspective view with the atomic labeling system was shown in Figure 1, and the
2
5
crystallographic data was presented in Table S1 as supplementary material.
In order to set off the antioxidant property
of
1,4-benzodioxan,
5
-phenyl-1,3,4-oxadiazole-2-thione (7) was synthesized by the same synthetic route as compound
1
4
(Scheme 2). And its H-NMR and ESI-MS data were consistent with the ones in previous
26
report.
2
2
.2. In vitro antioxidant activity profiling
.2.1 DPPH radical scavenging activity
The DPPH radical scavenging activity assay is a simple method for measuring the antioxidant
ability to trap free radicals. The scavenging effects of compounds 6a-6ae were shown in Table 1
along with butylated hydroxytoluene (BHT) and
-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) as two positive controls. More
than half of the tested compounds showed higher DPPH radical scavenging capacities than BHT
IC50 = 43.84 μM), including seven of them exhibited stronger activity than Trolox (IC50 = 30.20
6
(
μM). Compounds 6f and 6e exhibited the highest activity with the IC50 values of 19.62 and 21.80
μM, while 6ae and 6ad presented lowest activity with IC50 values of 107.5 and 94.40 μM,
respectively.
2
.2.2. ABTS radical cation scavenging activity
27
The ABTS assay is another widely used method for measuring the in vitro antioxidant ability.
The ABTS radical cation scavenging capacities of the synthesized compounds 6a-6ae were also
shown in Table 1. A total of seven compounds displayed higher activity than Trolox (IC50 = 6.19
5

μM), in which, 6f and 6e exhibited the highest activities with the IC50 values of 2.92 and 3.32 μM,
respectively. Meanwhile, 6ae (IC50 = 22.90 μM) and 6ad (IC50 = 18.41 μM) presented significant
weak ABTS radical cation scavenging capacities compared to those compounds with substituted
aniline (6a – 6ac).
2
.2.3. Ferric reducing antioxidant power (FRAP)
In the ferric to ferrous reduction assay, the electron donation capacity (reflecting the electron
28
transfer ability) of the compounds was assessed. FRAP values of compounds 6a - 6ae were
presented in Table 1. Compounds 6f (C0.5FRAP = 239.7 μM), 6e (C0.5FRAP = 268.3 μM) and 6x
(C0.5FRAP = 291.7 μM) displayed better activities than Trolox (C0.5FRAP = 293.7 μM). And
another seven compounds were also more potent than BHT (C0.5FRAP = 546.2 μM).
In summary of the in vitro antioxidant activities of the synthesized 6a – 6ae, more than half of
the compounds exhibited higher antioxidant activity than BHT and few of them even exhibited
higher activity than Trolox, which is a better antioxidant than BHT. Introducing arylamine (6a –
6
ac) enhanced antioxidant activity of Mannich bases, but alkyl amines had no significant effect
(
6ad, 6ae).
In order to demonstrate the combination of 1,4-benzodioxan and 1,3,4-oxadiazole could
enhance overall activity as an antioxidant, 2 with only 1,4-benzodioxan, 7 with only
,3,4-oxadiazole and 4 with both moieties were also evaluated their antioxidant capacities by the
1
above-mentioned assays. As shown in Table 1, compound 4 exhibited better activity than both 2
and 7 in all three methods, but less than most of the 6a – 6ac. The results suggested
1
,4-benzodioxan, 1,3,4-oxadiazoles and Mannich base with aromatic ring all contribute to the
6

antioxidant activity.
Compounds 6e and 6f presented the best radical scavenging activity in all of the above three
assays, indicating that multi-fluoro substituent on the benzene improved the antioxidant activity.
Compounds 6x – 6ac with methyl-, methoxy- or ethyoxyl- substituent on the benzene exhibited
moderate antioxidant activities, while compounds 6t, 6u, 6v and 6w with nitro substituent
demonstrated weak radical scavenging activity. The results are partially consistent with Sergio and
29
30
1
Mónica, Babasaheb and Kotaiah's work that electron donating groups contribute more radical
scavenging activity than electron withdrawing groups attached to the phenyl ring.
In order to verify whether those Mannich base derivatives were applicable in various oxidation
environments, seven compounds with representative structure or activity were selected to be
further assayed their inhibition of mice liver microsomal LPO and protective effect for DNA
damaged induced by AAPH.
2
.3. Inhibition of microsomal LPO
As shown in Figure 2, compounds 6a, 6e, 6f, and 6x were more potent than Trolox (183.2 μM),
in inhibiting the LPO of mice liver microsomes, especially 6f showed the most potent biological
activity (IC50 = 110.3 μM). As expected, compounds 6t, 6u and 6w were less active than the
compounds mentioned above. The rank of these compounds matches the results of in vitro
antioxidant assays.
2
.4. DNA protective effect
31-33
DNA can be damaged by free radical-mediated oxidant as reported by many investigations.
The antioxidant activities of seven selected compounds (6a, 6e, 6f, 6x, 6w, 6t, 6u) were evaluated
against AAPH-induced plasmid pBR322 DNA strand breakage using agarose gel electrophoresis
7

analysis (Figure 3A). The native DNA was mostly in its supercoiled form, which would be
transformed to open-circular form completely in the presence of AAPH. Most of the newly
synthetic compounds showed better activity than Trolox regarding protection against
AAPH-induced DNA strand breakage. On the basis of the relative amount of intact supercoiled
DNA (Figure 3B), the activity of the selected compounds followed the order: 6a ≈ 6e ≈ 6f ≈ 6x >
6
w > 6t ≈ 6u, which was the same order as our previous results. Compounds 6u, 6v and 6t
appeared less active than the others, suggesting the introduction of electron-withdrawing
substituents such as nitro decrease the activity significantly.
3
. Conclusion
In conclusion, a new class of Mannich base of 1,3,4-oxadiazole derivatives possessing
,4-benzodioxan were prepared from simple starting materials and investigated for their
1
antioxidant activities in vitro. In the DPPH, ABTS and FRAP chemical models, most of the
compounds with substituted phenyl exhibited better antioxidant activity than the standard
antioxidant agents – Trolox and BHT, in which, 6f and 6e with multi-fluoro substituent on the
benzene displayed the most potent activity. Four high-active compounds, 6a, 6e, 6f and 6x, and
three less-active compounds, 6t, 6u and 6w, with distinct representative substituents were selected
to be assessed using inhibition of microsomal LPO and AAPH-induced oxidation of DNA models.
Highly consistent results were obtained and the SAR of these synthetic compounds was further
defined. This conclusion may lead to the development of novel drugs for treating the diseases
caused by oxidative stress.
8

4
. Experimental protocols
4
.1. Chemistry general
All chemicals and reagents used in current study were of analytical grade. The reactions were
monitored by thin layer chromatography (TLC) using pre-coated silica gel GF254 plates from
Qingdao Haiyang Chemical, China. Melting points (uncorrected) were determined on a SPSIC
WRS-1B digital melting-point apparatus (Shanghai, China). IR spectra were recorded as KBr
disks on a Bruker Tensor 27 IR spectrophotometer. ESI-MS spectra were obtained on a Mariner
1
Mass 5304 instrument. H-NMR spectra were collected on a Bruker Avance III 400 NMR
3
spectrometer with CDCl as solvent at room temperature, The chemical shifts(δ) were reported in
ppm with reference to internal TMS and coupling constants (J) were given in Hz. Elemental
analyzes were performed on a CHN-O-Rapid instrument and were within ± 0.4 % of the
theoretical values.
4
.2. General procedure for synthesis of the target compounds (6a ~ 6ae)
Formalin (150 μL, 2 mmol) was added to a stirred solution of 5-(2,3-dihydrobenzo[b][1,4]
dioxin-6-yl)-1,3,4-oxadiazole-2(3H)-thione (4) (0.236g, 1 mmol) in ethanol (10 mL ). An
ethanolic solution (5 mL) of the appropriate amine (1 mmol) was added portionwise to the
reaction mixture, stirred for 3 h at room temperature and left overnight in a 4 °C refrigerator. The
precipitate was filtered, washed with cold ethanol, dried, and crystallized from the suitable
solvent.
4
.2.1. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-((phenylamino)methyl)-1,3,4-oxadiazole-2(3H)
-
thione (6a)
1
Light white crystal, yield: 63 %, mp: 147.3-147.9 ºC. H-NMR (400 MHz, CDCl
3
) δ 7.41 – 7.44
9

(
(
m, 2H, ), 7.27 (t, J = 8.0 Hz, 2H), 6.94 – 7.02 (m, 3H), 6.87 (t, 1H, J = 7.3 Hz), 5.59 (s, 2H), 5.18
+
br s, 1H), 4.33 – 4.36 (m, 4H). MS (ESI): 342.08 (C17
15
H N
3
O
3
S, [M+H] ). Anal. Calcd. For
17 15 3 3
C H N O S: C, 59.81; H, 4.43; N, 12.31; Found: C, 59.65; H, 4.15; N, 12.38.
4
.2.2.
oxadiazole-2(3H)-thione (6b)
Colorless crystal, yield: 77 %, mp: 171.4-171.6 ºC. H-NMR (400 MHz, CDCl
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((2-fluorophenyl)amino)methyl)-1,3,4-
1
3
) δ 7.37 – 7.42
(
(
(
m, 2H), 7.22 (td, 1H, J = 8.0, 1.3 Hz), 7.02 – 7.04 (d, 1H, J = 8.6 Hz), 6.97 – 7.00 (m, 1H), 6.93
d, 1H, J = 8.4 Hz), 6.73 – 6.78 (m, 1H), 5.55 (s, 2H), 5.32 (br s, 1H), 4.28 – 4.32 (m, 4H). MS
+
ESI): 359.07 (C17
H
3
14FN O
3
S, [M+H] ). Anal. Calcd. For C17
H
14FN
3
O
3
S: C, 56.82; H, 3.93; N,
11.69; Found: C, 56.83; H, 3.54; N, 11.85.
4
.2.3.
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((3-fluorophenyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (6c)
Colorless podwer, yield: 54 %, mp: 149.5-150.4 ºC. H NMR (400 MHz, CDCl
m, 2H), 7.15 (dd, 1H, J = 15.0 7.8 Hz), 6.93 (d, J = 8.5 Hz, 1H), 6.67 (dd, 1H, J = 9.7 1.32 Hz),
1
3
) δ 7.38 – 7.44
(
6
3 3
.51 (m, 1H), 5.48 (s, 2H), 5.22 (br s, 1H), 4.28 – 4.33 (m, 4H). MS (ESI): 359.07 (C17H14FN O S,
+
[
M+H] ). Anal. Calcd. For C17
H
14FN
3
O
3
S: C, 56.82; H, 3.93; N, 11.69; Found: C, 56.43; H, 3.99;
N, 11.88.
4
.2.4.
oxadiazole-2(3H)-thione (6d)
Colorless powder, yield: 54 %, mp: 189.8-190.6 ºC. H NMR (400 MHz, CDCl
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((4-fluorophenyl)amino)methyl)-1,3,4-
1
3
) δ 7.36 – 7.40
(
m, 3H), 6.89 – 6.95 (m, 2H), 6.84 – 6.87 (m, 2H), 6.01 (s, 1H), 5.47 (s, 2H), 4.23 – 4.37 (m, 4H).
+
MS (ESI): 359.07 (C17H14FN
3
O
3
S, [M+H] ). Anal. Calcd. For C17
H
3 3
14FN O S: C, 56.82; H, 3.93;
1
0

N, 11.69; Found: C, 56.17; H, 3.98; N, 11.55.
.2.5. 3-(((2,6-difluorophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
oxadiazole-2(3H)-thione (6e)
White powder, yield: 50 %, mp: 148.5-148.5 ºC. H NMR (400 MHz, CDCl
4
1
3
) δ 7.37 – 7.39 (m,
2
H), 6.94 – 6.98 (m, 2H), 6.86 – 6.89 (m, 2H), 5.62 (s, 2H), 5.09 (br s, 1H), 4.32 – 4.37 (m, 4H).
+
MS (ESI): 378.07 (C17
H
13
F
2 3
N O
3
S, [M+H] ). Anal. Calcd. For C17
13
H F
2
N
3
O
3
S: C, 54.11; H, 3.47;
N, 11.14; Found: C, 54.01; H, 3.39; N, 11.23.
.2.6. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((3,4,5-trifluorophenyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (6f)
White powder, yield: 67 %, mp: 203.1-204.2 ºC. H NMR (400 MHz, CDCl
4
1
3
) δ 7.41 – 7.49 (m,
3
H, H-), 6.99(d, 1H, J = 8.8), 5.71 (t, 1H, J = 7.5), 5.57 (d, 2H, J = 7.6), 4.33 – 4.38 (m, 4H). MS
+
(
ESI): 396.05 (C17
H
13
F
2 3
N O
3
S, [M+H] ). Anal. Calcd. For C17
H
12
F
3
N
3 3
O S: C, 51.65; H, 3.06; N,
1
4
1
0.63; Found: C, 51.78; H, 3.01; N, 10.02.
.2.7. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((2-(trifluoromethyl)phenyl)amino)methyl)-
,3,4-oxadiazole-2(3H)-thione (6g)
White crystal, yield: 80 %, mp: 185.4-186.2 ºC. H NMR (400 MHz, CDCl
1
3
) δ 7.33 – 7.40 (m,
3
H), 6.90 – 6.94 (m, 2H), 6.81 – 6.85 (m, 3H), 5.58 (s, 2H), 5.05 (br s, 1H), 4.33 – 4.37 (m, 4H).
+
MS (ESI): 410.07 (C18
H
14
F
3 3
N O
3
S, [M+H] ). Anal. Calcd. For C18
H
14
F
3 3 3
N O S: C, 52.81; H, 3.45;
N, 10.26; Found: C, 52.86; H, 3.21; N, 10.11.
4
1
.2.8.
,3,4-oxadiazole-2(3H)-thione (6h)
White crystal, yield: 85 %, mp: 200.4-201.0 ºC. H NMR (400 MHz, CDCl
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((3-(trifluoromethyl)phenyl)amino)methyl)-
1
3
) δ 7.37 – 7.40 (m,
11

3
H), 7.32 (t, 1H, J = 7.90 Hz), 7.20(s, 1H), 7.08 (t, 1H, J = 8.6 Hz), 6.93 (dd, 1H, J = 7.90, 0.90
14 3 3 3
Hz), 5.53 (s, 2H), 5.31 (br s, 1H), 4.28 - 4.32(m, 4H). MS (ESI): 410.07 (C18H F N O S,
+
[
M+H] ). Anal. Calcd. For C18
14
H F
3
N
3
O
3
S: C, 52.81; H, 3.45; N, 10.26; Found: C,52.59; H, 3.21;
N, 10.20.
.2.9. 3-(((2,5-bis(trifluoromethyl)phenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)
1,3,4-oxadiazole-2(3H)-thione (6i)
White powder, yield: 77 %, mp: 177.3-178.3 ºC. H NMR (400 MHz, CDCl
4
-
1
3
) δ 7.38 – 7.40 (m,
1
H), 7.33 – 7.35 (m, 2H), 6.91 (dd, 1H, J = 8.6, 0.8 Hz), 6.81 – 6.85 (m, 2H) 5.58 (s, 2H), 5.05 (br
+
s, 1H), 4.27 – 4.33(m,4H). MS (ESI): 478.08 (C19
H
13
F
6 3
N O
3
S, [M+H] ). Anal. Calcd. For
C
19 13
H F
6
N
3
O
3
S: C, 47.80; H, 2.74; N, 8.80; Found: C, 47.38; H, 2.61; N, 8.54.
4
.2.10.
3-(((2-chlorophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
oxadiazole-2(3H)-thione (6j)
White powder, yield: 61 %, mp: 179.6-180.4 ºC. H NMR (400 MHz, CDCl
H), 7.24 (dd, 1H, J = 7.9, 1.6 Hz), 7.19 – 7.20 (m, 1H), 6.93 (d, 1H, J = 8.6 Hz), 6.76 (td, 1H, J =
1
3
) δ 7.38 – 7.41 (m,
2
7
3 3
.9, 1,7 Hz) 5.70 (br s, 1H), 5.58 (s, 2H), 4.27 – 4.33(m,4H). MS (ESI): 375.04 (C17H14ClN O S,
+
[
M+H] ). Anal. Calcd. For C17
H
14ClN
3
O
3
S: C, 54.33; H, 3.75; N, 11.18; Found: C, 54.12; H, 3.71;
N, 11.22.
4
.2.11.
oxadiazole-2(3H)-thione (6k)
White powder, yield: 61 %, mp: 139.9-140.0 ºC. H NMR (400 MHz, CDCl
H), 7.34 (d, 1H, J = 8.2 Hz), 6.98 (d, 1H, J = 8.0 Hz), 6.93 (t, 1H, J = 7.6 Hz), 5.74 (t, 1H, J =
.2 Hz), 5.61 (d, 2H, J = 7.6 Hz), 4.33 – 4.37(m, 4H). MS (ESI): 375.04 (C17 S,
3-(((3-chlorophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
1
3
) δ 7.43 – 7.54 (m,
4
7
H
3 3
14ClN O
1
2

+
[
M+H] ). Anal. Calcd. For C17
N, 11.05.
.2.12.
oxadiazole-2(3H)-thione (6l)
White powder, yield: 71 %, mp: 216.1-216.1 ºC. H NMR (400 MHz, CDCl
3 3
H14ClN O S: C, 54.33; H, 3.75; N, 11.18; Found: C, 54.09; H, 3.63;
4
3-(((4-chlorophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
1
3
) δ 7.40 – 7.43 (m,
2
1
H), 7.15 (t, 1H, J = 8.0 Hz), 6.95 – 6.97 (m, 2H), 6.80 – 6.83 (m, 2H), 5.50 (s, 2H), 5.22 (br s,
+
H), 4.31 – 4.35 (m, 4H). MS (ESI): 375.04 (C17
H
14ClN
3
O
3
S, [M+H] ). Anal. Calcd. For
17 3 3
C H14ClN O S: C, 54.33; H, 3.75; N, 11.18; Found: C, 54.25; H, 3.70; N, 10.99.
4
.2.13.
oxadiazole-2(3H)-thione (6m)
White powder, yield: 89 %, mp: 155.9-156.1 ºC. H NMR (400 MHz, CDCl
3-(((2,4-dichlorophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
1
3
) δ 7.38 – 7.40 (m,
2
H), 7.28 (d, 1H, J = 1.6 Hz), 7.14 – 7.20 (m, 2H), 6.93 (d, 1H, J = 8.1 Hz), 5.65 (t, 1H, J = 7.7
+
Hz), 5.54 (d, 2H, J = 7.6 Hz), 4.28 – 4.32 (m, 4H). MS (ESI): 410.01 (C17
H13Cl
N
2 3
O
3
S, [M+H] ).
2 3 3
Anal. Calcd. For C17H13Cl N O S: C, 49.77; H, 3.19; N, 10.24; Found: C, 49.61; H, 3.10; N,
1
0.19.
4
.2.14.
3-(((2,5-dichlorophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
oxadiazole-2(3H)-thione (6n)
White powder, yield: 61 %, mp: 166.3-167.7 ºC. H NMR (400 MHz, CDCl
1
3
) δ 7.40 – 7.42 (m,
2
8
4
1
H), 7.32 (d, 1H, J = 2.5 Hz), 7.18 (d, 1H, J = 8.4 Hz), 6.95 (d, 1H, J = 8.5 Hz), 6.74 (dd, 1H, J =
.3 2.2 Hz), 5.72 (t, 1H, J = 7.6, Hz), 5.54 (d, 2H, J = 7.7 Hz), 4.29 – 4.33 (m, 4H). MS (ESI):
+
10.01 (C17
H
13Cl
2
N
3
O
3
S, [M+H] ). Anal. Calcd. For C17
H13Cl
2
N
3
O
3
S: C, 49.77; H, 3.19; N,
0.24; Found: C, 49.53; H, 3.09; N, 10.17.
1
3

4
.2.15.
oxadiazole-2(3H)-thione (6o)
White powder, yield: 51 %, mp: 168.3-168.9 ºC. H NMR (400 MHz, CDCl
3-(((3,4-dichlorophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
1
3
) δ 7.38 – 7.40 (m,
2
5
H), 7.24 (s, 1H), 7.06 (d, 1H, J = 2.7 Hz), 6.94 (d, 1H, J = 4.6 Hz), 6.78 (dd, 1H, J = 4.7 2.8 Hz),
+
.46 (s, 2H), 5.18 (br s, 1H), 4.29 – 4.33 (m, 4H). MS (ESI): 410.01 (C17
H
13Cl
2
N
3
O
3
S, [M+H] ).
2 3 3
Anal. Calcd. For C17H13Cl N O S: C, 49.77; H, 3.19; N, 10.24; Found: C, 49.37; H, 3.01; N,
1
0.02.
4
.2.16.
3-(((2-bromophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
oxadiazole-2(3H)-thione (6p)
Light yellow powder, yield: 81 %, mp: 158.8-159.6 ºC. H NMR (400 MHz, CDCl
1
3
) δ 7.38 –
7
5
.45 (m, 3H), 7.22 – 7.24 (m, 2H), 6.93 (d, 1H, J = 8.4 Hz), 6.68 – 6.72 (m, 1H), 5.69 (br s, 1H),
+
.57 (s, 2H), 4.28 – 4.32 (m, 4H). MS (ESI): 420.00 (C17
3
H14BrN O
3
S, [M+H] ). Anal. Calcd. For
17 3 3
C H14BrN O S: C, 48.58; H, 3.36; N, 10.00; Found: C, 48.11; H, 3.40; N, 9.98.
4
.2.17.
oxadiazole-2(3H)-thione (6q)
White powder, yield: 78 %, mp: 150.0-150.7 ºC. H NMR (400 MHz, CDCl
3-(((3-bromophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
1
3
) δ 7.38 – 7.41 (m,
2
H), 7.05 – 7.11 (m, 2H), 6.93 – 6.95 (m, 2H), 6.83 (dd, 1H, J = 4.0 1.6 Hz), 5.47 (d, 2H, J = 4.0
+
Hz), 5.17 (br s, 1H), 4.29 – 4.33 (m, 4H). MS (ESI): 420.00 (C17
H
14BrN
3
O
3
S, [M+H] ). Anal.
Calcd. For C17
.2.18.
oxadiazole-2(3H)-thione (6r)
White crystal, yield: 59 %, mp: 205.3-206.2 ºC. H NMR (400 MHz, CDCl
H
14BrN
3
O
3
S: C, 48.58; H, 3.36; N, 10.00; Found: C, 48.62; H, 3.09; N, 9.92.
4
3-(((4-bromophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
1
3
) δ 7.38 – 7.40 (m,
1
4

2
H), 7.05 – 7.11 (m, 2H), 6.93 – 6.94 (m, 2H), 6.84 (dd, 1H, J = 4.1 1.7 Hz), 5.47 (d, 2H, J = 5.2
+
Hz), 5.18 (br s, 1H), 4.28 – 4.32 (m, 4H). MS (ESI): 420.00 (C17
H
14BrN
3
O
3
S, [M+H] ). Anal.
Calcd. For C17
.2.19. 3-(((2,4-dibromophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-
oxadiazole-2(3H)-thione (6s)
White crystal, yield: 76 %, mp: 187.6-188.1 ºC. H NMR (400 MHz, CDCl
.2 Hz), 7.42 – 7.45 (m, 2H), 7.38 (dd, 1H, J = 8.7 2.1 Hz), 7.16 (d, 1H, J = 8.8 Hz), 6.98 (dd, 1H,
3 3
H14BrN O S: C, 48.58; H, 3.36; N, 10.00; Found: C, 48.71; H, 3.02; N, 9.85.
4
1
3
) δ 7.62 (d, 1H, J =
2
J = 8.2 0.4 Hz), 5.72 (t, 1H, J = 7.7 Hz), 5.58 (d, 2H, J = 7.8 Hz), 4.33 – 4.37 (m, 4H). MS (ESI):
+
4
97.90 (C17
H
13Br N
2 3
O
3
S, [M+H] ). Anal. Calcd. For C17
2
H13Br N
O
3 3
S: C, 40.90; H, 2.62; N, 8.42;
Found: C, 40.53; H, 2.54; N, 8.52.
.2.20. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((2-nitrophenyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (6t)
Yellow powder, yield: 84 %, mp: 191.9-193.2 ºC. H NMR (400 MHz, CDCl
4
1
3
) δ 8.80 (t, 1H, J =
6
2
4
.6 Hz), 8.20 (d, 1H, J = 8.4 Hz), 7.55 (t, 1H, J = 7.1 Hz), 7.48 (d, 1H, J = 8.5 Hz), 7.39 – 7.42 (m,
H), 6.94 (d, 1H, J = 8.4 Hz), 6.85 (t, 1H, J = 8.1 Hz), 5.66 (d, 2H, J = 7.8 Hz), 4.33 – 4.37 (m,
+
H). MS (ESI): 387.07 (C17
H
14
N
4
O
5
S, [M+H] ). Anal. Calcd. For C17
14
H N
4 5
O S: C, 52.84; H, 3.65;
N, 14.50; 8.42; Found: C, 51.99; H, 3.55; N, 14.12.
.2.21. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((3-nitrophenyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (6u)
Yellow powder, yield: 85.0%, mp: 181.4-181.4 ºC. H NMR (400 MHz, CDCl
2.1 Hz), 7.65 (dd, 1H, J = 10.5 0.6 Hz), 7.39 – 7.41 (m, 2H), 7.35 (d, 1H, J = 8.0 Hz), 7.22 (dd,
H, J = 7.6 0.8 Hz), 5.56 (d, 2H, J = 7.9 Hz), 5.44 (t, 1H, J = 4.2 Hz), 4.28 – 4.32 (m, 4H). MS
4
1
3
) δ 7.84 (t, 1H, J
=
1
1
5

+
(
ESI): 387.07 (C17
14
H N
4
O
5
S, [M+H] ). Anal. Calcd. For C17
4.50; 8.42; Found: C, 52.37; H, 3.60; N, 14.31.
.2.22. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((4-nitrophenyl)amino)methyl)-1,3,4-
14 4 5
H N O S: C, 52.84; H, 3.65; N,
1
4
oxadiazole-2(3H)-thione (6v)
Light yellow powder, yield: 83 %, mp: 233.7-234.5 ºC. H NMR (400 MHz, CDCl
1
3
) δ 7.40 –
7
1
.42 (m, 2H), 7.32 (d, 1H, J = 2.2 Hz), 7.18 (d, 1H, J = 8.4 Hz), 6.96 (d, 1H, J = 8.8 Hz), 6.73 (dd,
H, J = 8.4 2.2 Hz), 5.71 (t, 1H, J = 7.6 Hz), 5.54 (d, 2H, J = 7.7 Hz), 4.29 – 4.33 (m, 4H). MS
+
(
14
ESI): 387.07 (C17H N
4
O
5
S, [M+H] ). Anal. Calcd. For C17
H
14
N
4
5
O S: C, 52.84; H, 3.65; N,
1
4
1
4.50; Found: C, 52.29; H, 3.42; N, 14.34.
.2.23. 3-(((2-chloro-4-nitrophenyl)amino)methyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
,3,4-oxadiazole-2(3H)-thione (6w)
Light yellow powder, yield: 59 %, mp: 167.2-168.1 ºC. H NMR (400 MHz, CDCl
H, J = 2.5 Hz), 8.14 (dd, 1H, J = 8.3 2.5 Hz), 7.39 – 7.41 (m, 2H), 7.35 (d, 1H, J = 9.4 Hz), 6.95
1
3
) δ 8.24 (d,
1
(
(
d, 1H, J = 8.0 Hz), 6.21 (t, 1H, J = 7.8 Hz), 5.64 (d, 2H, J = 7.5 Hz), 4.29 – 4.33 (m, 4H). MS
+
ESI): 421.03 (C17
H
4
13ClN O
5
S, [M+H] ). Anal. Calcd. For C17
H
4
13ClN O
5
S:C, 48.52; H, 3.11; N,
1
4
2
3.31; Found: C, 48.43; H, 3.02; N, 13.07.
.2.24. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-((o-tolylamino)methyl)-1,3,4-oxadiazole-
(3H)-thione (6x)
Colorless crystal, yield: 69 %, mp: 147.2-147.5 ºC. H NMR (400 MHz, CDCl
m, 2H), 7.19 (d, 1H, J = 7.4 Hz), 7.11 – 7.15 (m, 2H), 6.97 (d, 1H, J = 8.2 Hz), 6.81 (t, 1H, J =
1
3
) δ 7.42 – 7.44
(
5
3
.1 Hz), 5.62 (d, 2H, J = 5.2 Hz), 5.15 (br s, 1H), 4.23 – 4.37 (m, 4H), 2.27 (s, 3H). MS (ESI):
+
56.10 (C18
H
17
N
3
O
3
S, [M+H] ). Anal. Calcd. For C18
H
17
3
N O
3
S: C, 60.83; H, 4.82; N, 11.82;
1
6

Found: C, 60.41; H, 4.32; N, 11.56.
4
2
.2.25.
(3H) –thione (6y)
Colorless crystal, yield: 51 %, mp: 161.3-161.3 ºC. H NMR (400 MHz, DMSO-d
H, J = 8.5 2.00 Hz), 7.27 (d, 1H, J = 2.0 Hz), 7.05 (d, 1H, J = 8.5 Hz), 6.93 – 6.95 (m, 2H), 6.78
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-((p-tolylamino)methyl)-1,3,4-oxadiazole-
1
6
) δ 7.34 (dd,
1
(
(
d, 2H, J = 8.4 Hz), 5.43 (d, 2H, J = 7.4 Hz), 4.28 – 4.34 (m, 4H), 2.14 (s, 3H). MS (ESI): 356.10
+
C
18
H
17
N
3
O
3
S, [M+H] ). Anal. Calcd. For C18
H
17
N
3
O
3
S: C, 60.83; H, 4.82; N, 11.82; Found: C,
6
0.73; H, 4.55; N, 11.88.
.2.26. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((2-methoxyphenyl)amino)methyl)-1,3,4-
4
oxadiazole-2(3H)-thione (6z)
Light yellow powder, yield: 44 %, mp: 156.3-157.5 ºC. H NMR (400 MHz, DMSO-d
dd, 1H, J = 8.3 2.0 Hz), 7.27 (d, 1H, J = 2.0 Hz), 6.96 (dd, 1H, J = 7.9 1.1 Hz), 6.87 (d, 1H, J =
1
6
) δ 7.34
(
7
.9 Hz), 6.79 (t, 1H, J = 7.6 Hz), 6.68 (td, 1H, J = 7.7 1.2 Hz), 6.37 (t, 1H, J = 7.4 Hz), 5.50 (d, 2H,
+
J = 7.3 Hz), 4.30 – 4.40 (m, 4H), 3.80 (s, 3H). MS (ESI): 372.09 (C18
17
H N
3
O
3
S, [M+H] ). Anal.
Calcd. For C18
.2.27. 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((4-methoxyphenyl)amino)methyl)-1,3,4-
oxadiazole-2(3H)-thione (6ab)
White powder, yield: 80 %, mp: 191.0-191.1 ºC. H NMR (400 MHz, DMSO-d
17 3 3
H N O S: C, 58.21; H, 4.61; N, 11.31; Found: C, 58.29; H, 4.72; N, 11.11.
4
1
6
) δ 7.28 – 7.37
(
m, 2H), 7.06 (d, 1H, J = 8.5 Hz), 6.96 – 6.97 (m, 1H), 6.74 – 6.84 (m, 3H), 5.42 (d, 2H, J = 7.4
+
Hz), 4.32 – 4.35 (m, 4H), 3.64 (s, 3H). MS (ESI): 372.09 (C18
17
H N
3
O
3
S, [M+H] ). Anal. Calcd.
17 3 3
For C18H N O S: C, 58.21; H, 4.61; N, 11.31; Found: C, 58.03; H, 4.69; N, 11.27.
4
.2.28.
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-(((2-ethoxyphenyl)amino)methyl)-1,3,4-
1
7

oxadiazole-2(3H)-thione (6ac)
White powder, yield: 79 %, mp: 178.2-179.0 ºC. H NMR (400 MHz, DMSO-d
J = 8.4 2.0 Hz), 7.27 (d, 1H, J = 2.0 Hz), 6.96 (d, 1H, J = 8.5 1.1 Hz), 6.96 (d, 1H, J = 7.8 Hz),
.85 (d, 1H, J = 7.5 Hz), 6.78 (t, 1H, J = 7.7 1.24 Hz), 6.30 (t, 1H, J = 7.4 Hz), 5.51 (d, 2H, J = 7.3
Hz), 4.30 – 4.34 (m, 4H), 4.03 (dd, 2H, J = 13.7 7.0 Hz), 1.38 (t, 3H, J = 6.9 Hz). MS (ESI):
1
6
) δ 7.34 (dd, 1H,
6
+
+
3
17 3 3 17 3 3
86.11 (C18H N O S, [M+H] ). MS (ESI): 386.11 (C18H N O S, [M+H] ). Anal. Calcd. For
18 17 3 3
C H N O S: C, 59.21; H, 4.97; N, 10.90; Found: C, 59.11; H, 4.33; N, 10.34
4
2
.2.29.
(3H)-thione (6ad)
White powder, yield: 90 %, mp: 129.9-130.1 ºC. H NMR (400 MHz, CDCl
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-((hexadecylamino)methyl)-1,3,4-oxadiazole-
1
3
) δ 7.39 – 7.41 (m,
2
3
H), 6.89 – 6.92 (m, 1H), 5.47 (s, 2H), 4.29 – 4.33 (m, 6H), 1.19 – 1.79 (m, 8H). MS (ESI):
+
48.13 (C18
H
17
N
3
O
3
S, [M+H] ). Anal. Calcd. For C18
H
17
N
3
O
3
S: C, 58.77; H, 6.09; N, 12.09;
Found: C, 58.43; H, 6.02; N, 12.20.
4
2
.2.30.
(3H)-thione (6ae)
White powder, yield: 89 %, mp: 131.0-131.0 ºC. H NMR (400 MHz, CDCl
5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-((hexadecylamino)methyl)-1,3,4-oxadiazole-
1
3
) δ 7.30 – 7.39 (m,
2
2
4
H), 6.92 (d, 1H, J = 8.3 Hz), 5.41 (s, 2H), 4.33 – 4.37 (m, 6H), 3.09 (t, 1H, J = 7.2 Hz), 1.66 (s,
+
H), 1.34(s, 26H), 0.88(t, 3H, J = 2.1 Hz). MS (ESI): 490.30 (C18
17
H N
3
O
3
S, [M+H] ). MS (ESI):
+
90.30 (C18H N
17 3
O
3
S, [M+H] ). Anal. Calcd. For C18
17
H N
3
O
3
S: C, 66.22; H, 8.85; N, 8.58;
Found: C, 66.21; H, 8.80; N, 8.64
.3. Procedure for synthesis of the compounds 7
Be similar with synthesis of compound 4, benzoic acid was used as starting reactant, followed
4
1
8

by esterification, hydrazintion and cyclization reaction to get compound 7.
4
.4 Crystal structure determination
X-ray single-crystal diffraction data for compound 6b were collected on a Bruker SMART
APEX CCD equipped with graphitemonochromated MoKα (λ = 0.71073 Å) radiation. The
2
structure was solved by direct methods and refined on F by full-matrix least-squares methods
34
using SHELX-97. All the non-hydrogen atoms were refined anisotropically. All the hydrogen
atoms were placed in calculated positions and were assigned fixed isotropic thermal parameters at
1
.2 times the equivalent isotropic U of the atoms to which they are attached and allowed to ride on
their respective parent atoms. The contributions of these hydrogen atoms were included in the
structure-factors calculations. The crystal data and refinement parameter for compound 6b are
listed in Table S1 in supplementary material.
4
4
.5. In vitro antioxidant activity assay
.5.1 DPPH scavenging capacity assay
The scavenging rate of DPPH radical was carried out by the improved protocol of previously
35
reported method by Skrede et al. In brief, in 96-well microliter plates, appropriately serial
dilution (100, 50, 25, 12.5, 6.25, 3.125 μg/mL, respectively) of each test compound (100 μL) was
mixed with DPPH reagent (100 μL, 0.2 mmol/L DPPH in ethanol). Ethanol was used as a blank
experiment while BHT was assayed as controls. The absorbance of the mixture was read at 515
nm after 0.5 h at 25 °C using microplate reader infinite M200 (Tecan, Switzerland). Antioxidant
capacity was evaluated as IC50 (sample concentration that produced 50% scavenging of the DPPH
radical).
4
.5.2 ABTS scavenging capacity assay
1
9

The ABTS radical-scavenging capacity assay of each test compound was conducted according
27
2 2 8
to the previous method reported by Re et al. Briefly, K S O (88 μL, 140 mmol/L) was mixed
with ABTS [5 mL, 7 mmol/L in PBS (pH 7.4)]. The mixture was stored in the dark for 12-16 h and
the resulting ABTS radical solution was adjusted to an absorbance of 0.700 ± 0.020 at 734 nm
with the addition of PBS. Diluted sample solutions (20 μL) were mixed with ABTS radical
solution (180 μL). The mixture was reacted at 37 °C under restricted light for 6 min. The decrease
of absorbance at 734 nm was recorded and the results were evaluated as IC50 (The amount of
samples required to decrease the ABTS radical concentration by 50%).
4
.5.3 FRAP scavenging capacity assay
The FRAP assay was performed according to the previous procedure reported by Benzie and
36
Strain. Specifically, the same dilution described in DPPH assay (20 μL) were mixed with the
freshly prepared ferric-tripyridyltriazine (TPTZ) reagent [180 μL, a mixture of 300 mmol/L
acetate buffer (pH 3.6), 10 mmol/L 2,4,6-tris(2-pyridyl)-s-triazine in 40 mmol/L HCl, and 20
3
mmol/L FeCl (v/v/v: 10/1/1)]. The absorbance of the reaction mixture was measured at 593 nm
after 4 min. The standard curve was established using the ferrous sulfate solutions (range 0.1-1.0
mmol/L with n = 6 concentrations), and the results were expressed as C0.5FRAP (the concentration
of samples with the antioxidant capacity equivalent to that of ferrous sulfate at 0.5 mmol/L).
4
.5.4. Inhibition of Microsomal LPO
Mouse liver microsomes used for the LPO studies were prepared adopting the method of Gao
37
and Wang. The adult male mice (8 - 12 weeks old) of KM strain (purchased from the
Comparative Medicine Center of Yangzhou University) were used for the preparation of liver
38
microsomes. The protein content of microsomes was measured using the Bradford method.
2
0

Microsomes (0.67 mg protein/mL) were incubated at 37 ºC for 60 min with test compounds of
varying concentrations,10 mM FeSO and 0.1 mM ascorbic acid in 1.0 mL potassium phosphate
4
buffer solution (0.2 M, pH 7.4). The reaction was stopped by 20 % (w/v) trichloroacetic acid (1.0
mL) and 0.67 % (w/v) 2-thiobarbituric acid (1.5 mL) in succession, and the solution was then
heated to 100 ºC for 15 min. After centrifugation of precipitated protein, the color reaction of
malondialdehyde (MDA)-TBA complex was detected at 535 nm.
4
.5.5. DNA nicking assay for AAPH scavenging activity
The method of AAPH induced DNA breakage in plasmid pBR322 was modified from Jie et
39
al. . Briefly, in an Eppendorf tube was conducted at total volume of 25μL containing
pBR322DNA (purchased from Shanghai Sangon Biological Company) and AAPH in phosphate
2 4 2 4
buffered solution (PBS: 8.1 mM Na HPO , 1.9 mM NaH PO , 10.0 mM EDTA) with the final
concentrations at 4 μg/mL and 4 mM, respectively. Samples and Trolox (standard) both at 100 μM
in DMSO were added to the above mixture. After incubation at 37 ºC for 1 hour, 2 μL of
electrophoresis loading buffer (0.25% bromophenol blue and 30% (w/v) glycerol) were added to
1
0 μL the reaction mixture, and an aliquot (10 μL) was then loaded onto a 1% agarose gel and
electrophoresed in Tris-acetate-EDTA buffer at 60 V for 100 min. DNA bands (supercoiled and
open circular) were stained with ethidium bromide. PBR322 DNA bands were visualized and
photographed with a Gel Imaging System (ImageQuant 400, GE) under UV illuminator. One-way
ANOVA was used for the comparison of the DNA damage control with the other treatments. A
difference was considered statistically significant when P < 0.05 (*). Values represent the mean ±
SD of triplicate samples.
2
1

Acknowledgments
This work was co-supported by National Basic Research Program of China (2010CB126100),
National Natural Science Foundation of China (30901854), Fundamental Research Funds for the
Central Universities (KYZ201107) and Special Fund for Agro-scientific Research in the Public
Interest (201303023).
References and notes
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2
4

Table 1. The in vitro antioxidant activity of 6a - 6ae in DPPH, ABTS and FRAP methods.
Figure 1. ORTEP drawing of molecular structure of compound 6b.
Figure 2. The IC50 values of microsomal lipid peroxidation activity (μM).
Figure 3. Protection against AAPH-induced pBR322 DNA strand breakage by synthetic
compounds.
(
A) Lanes1: blank, native DNA; Lanes 2-9, DNA, AAPH and test compounds (6a, 6e, 6f, 6x, 6t,
u, 6w and Trolox, respectively).
B) The relative amount of supercoiled (SC) DNA of test compounds and control.
6
(
Scheme 1. General synthesis of compounds (6a-6ae).
Scheme 2. Synthesis of compound 7.
2
5

Scheme 1. General synthesis of compounds (6a-6ae).
6
6
6
6
6
6
3 6 4
y = 4-CH -C H -
6
6
6
6
6
6
6
6
a = C
6
H
5
-
6i = 3,5-2CF
3
-C
6
H
3
-
6 4
6q = 3-Br-C H -
3 6 4
z = 2-OCH -C H -
b = 2-F-C
6
H
4
-
6j = 2-Cl-C
6
H
4
-
6 4
6r = 4-Br-C H -
3 6 4
ab = 4-OCH -C H -
c = 3-F-C
6
H
4
-
6k = 3-Cl-C
6
4
H -
6 3
6s = 2,4-2Br-C H -
2 3 6 4
ac = 2-OCH CH -C H -
d = 4-F-C
6
H
4
-
6l = 4-Cl-C
6
H
4
-
2 6 4
6t = 2-NO -C H -
ad = -cyclohexane
ae = -hexadecyl
e = 2,6-2F-C
6
H
3
-
6m = 2,4-2Cl-C
6
H
3
-
2 6 4
6u = 3-NO -C H -
f = 3,4,5-3F-C
6
H
2
-
6n = 2,5-2Cl-C
6
H
3
-
2 6 4
6v = 4-NO -C H -
g = 2-CF
3
-C
6
H
4
-
6o = 3,4-2Cl-C
6
H
3
-
6w = 2-Cl-4-NO
2
-C
-
6 3
H -
h = 3-CF
3
-C
6
H
4
-
6p = 2-Br-C
6
H
4
-
3 6
6x = 2-CH -C H
4
Reagents and conditions: (a) methanol, concentrated sulfuric acid; reflux, 8-12 h; (b)
2 2 2 2
NH NH .H O (85%), ethanol; reflux, 8–12 h; (c) (1) CS /KOH, ethanol (95%), reflux, 24 h; (2)
HCl, pH 5–6; (d) HCHO(40%), ethanol, rt, 3 h.
2
6

Scheme 2. Synthesis of compound 7.
Reagents and conditions (e): (1) methanol, concentrated sulfuric acid; reflux, 8-12 h; (2)
NH NH ∙H O (85%), ethanol; reflux, 8-12 h; (3-i) CS /KOH, ethanol (95%), reflux, 24 h; (3-ii)
2
2
2
2
HCl, pH 5-6.
2
7

Figure 1. ORTEP drawing of molecular structure of compound 6b.
2
8

Figure 2. The IC50 values of microsomal lipid peroxidation activity (μM).
2
9