Synthesis of phthalide derivatives and evaluation on their antiplatelet aggregation and antioxidant activities

Article abstract of DOI:10.1080/10286020.2019.1681982

As part of our continuing efforts to discover structurally interesting bioactive phthalide derivatives, 23 of them with a structure incorporating thiophen or halogens were designed and synthesized, 17 of which are previously unreported. In vitro antiplatelet aggregation activity screening showed that 14b could significantly inhibit platelet aggregation induced by arachidonic acid, compared with edaravone (p < 0.01). Meanwhile, oxidative damage models using SH-SY5Y and PC12 cells induced by H₂O₂ were built to evaluate the antioxidant activity of the phthalide derivatives. In SH-SY5Y cells, compared with aspirin, 1a significantly increased the relative cell survival rate (p < 0.05). Compared with edaravone, 1a (p < 0.01) and 15b (p < 0.05) significantly increased the relative cell survival rate. In PC12 cells, 1a (p < 0.01), 15b (p < 0.01), and 12a (p < 0.05) remarkably increased the cell survival rate compared with edaravone. The present study identified lead structures to develop potential anti-ischemic stroke agents.

Full text of DOI:10.1080/10286020.2019.1681982

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
Synthesis of phthalide derivatives and evaluation
on their antiplatelet aggregation and antioxidant
activities
Xin Fang, Qiang Ma, Kai-Xia Zhang, Song-Yun Yao, Yi Feng, Yong-Sheng Jin &
Shuang Liang
To cite this article: Xin Fang, Qiang Ma, Kai-Xia Zhang, Song-Yun Yao, Yi Feng, Yong-Sheng
Jin & Shuang Liang (2019): Synthesis of phthalide derivatives and evaluation on their antiplatelet
aggregation and antioxidant activities, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2019.1681982
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2019.1681982
Synthesis of phthalide derivatives and evaluation on their
antiplatelet aggregation and antioxidant activities
Xin Fanga, Qiang Maa, Kai-Xia Zhangb, Song-Yun Yaob, Yi Fenga,
Yong-Sheng Jinb and Shuang Lianga
aInnovation Research Institute of Traditional Chinese Medicine, Shanghai University of Traditional
b
Chinese Medicine, Shanghai 201203, China; School of Pharmacy, Second Military Medical University,
Shanghai 200433, China
ABSTRACT
ARTICLE HISTORY
Received 14 June 2019
Accepted 14 October 2019
As part of our continuing efforts to discover structurally inter-
esting bioactive phthalide derivatives, 23 of them with a struc-
ture incorporating thiophen or halogens were designed and
synthesized, 17 of which are previously unreported. In vitro
antiplatelet aggregation activity screening showed that 14b
could significantly inhibit platelet aggregation induced by ara-
chidonic acid, compared with edaravone (p < 0.01). Meanwhile,
oxidative damage models using SH-SY5Y and PC12 cells
induced by H2O2 were built to evaluate the antioxidant activity
of the phthalide derivatives. In SH-SY5Y cells, compared with
aspirin, 1a significantly increased the relative cell survival rate
(p < 0.05). Compared with edaravone, 1a (p < 0.01) and 15b
(p < 0.05) significantly increased the relative cell survival rate. In
PC12 cells, 1a (p < 0.01), 15b (p < 0.01), and 12a (p < 0.05)
remarkably increased the cell survival rate compared with edar-
avone. The present study identified lead structures to develop
potential anti-ischemic stroke agents.
KEYWORDS
Phthalide derivative;
antiplatelet aggregation;
antioxidation
1. Introduction
Phthalides are widely distributed in Umbelliferae plants, such as Ligusticum chuan-
xiong and Angelica sinensis, and are usually modified by C-3 alkyl substitutions,
CONTACT Shuang Liang
ls7312@163.com
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
X. FANG ET AL.
unsaturated benzene ring substitutions, and dimers. Phthalides are considered as a
large class of characteristic compounds in these plants [1, 2]. In addition, a large
number of phthalides are also found in the metabolites of fungi and molds, such as
Phomopsis archeri [3] and Anthocleista djalonensis [4]. Phthalides possess various
pharmacological activities, such as anticoagulation, antiplatelet aggregation, antioxi-
dant, anti-inflammation, antimicrobial, and anti-tumor effects [5–11]. As a represen-
tative phthalide, n-butylphthalide (NBP) is used as a listed drug to treat ischemic
stroke in China, and can reduce ischemic brain injury by improving blood fluidity,
and through anti-thrombotic and anti-inflammatory effects [12]. In recent years, our
research group has studied the effectiveness and mechanism of a phthalide derivative,
senkynolide I. The results suggested that senkynolide I possesses an analgesic effect,
could decrease plasma and intracerebral nitric oxide (NO) levels, and improved cere-
bral edema and cerebral infarction volume in rats with focal cerebral ischemia reper-
fusion injury. Unfortunately, none of these effects are strong enough for clinical use
[13–15]. Meanwhile, 3-substituted benzo[c]thiophen-1(3H)-ones and phthalides with
halogens had been reported previously which showed superior effects to NBP [16].
Therefore, as part of our continuing efforts to discover structurally interesting bio-
active phthalide derivatives, 23 phthalide derivatives with structures incorporating thi-
ophen or halogens were designed and synthesized (Figure 1).
Figure 1. The structures of phthalide derivatives.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Scheme 1. Rationale design of the title compounds. Reagents and conditions: (a) Na2Sꢀ9H2O, THF,
room temp, 4 h; (b) R2MgX, THF, ꢁ5 ꢂC, 0.5 h, and then room temp, 5 h; (c) AcOH, aqueous HI
(57%), reflux, 0.5 h. THF, tetrahydrofuran.
2. Results and discussion
2.1. Chemistry
The synthesis of the a and b series of target compounds is outlined in Scheme 1.
Starting from phthalic anhydride, 4-chloro phthalic anhydride, and 3-chloro phthalic
anhydride, three intermediates of benzo[c]thiophene-1,3-dione 2 were prepared at
50–80% yield by reacting them with Na2Sꢀ9H2O. Then, a series of target products
were synthesized by treatment with corresponding Grignard reagents to introduce dif-
ferent alkyls and hydroxyls to the C-3 lactone ring. Finally, the b series of target
products were obtained using a one-pot reaction comprising dehydration and reduc-
tion (Scheme 1).
2.2. Antiplatelet aggregation activity
Thromboembolism is associated with the pathophysiological process of cerebral ische-
mia, and previous studies have shown that platelets are an important factor affecting
thrombosis [16–18]. Platelet status is an important parameter when evaluating the
severity of cerebral ischemia [19]. Thus, all the compounds obtained were evaluated
for their activity against platelet aggregation induced by arachidonic acid (AA) and
adenosine diphosphate (ADP). The screening results showed that the phthalide deriv-
atives selectively inhibited the platelet aggregation induced by ADP and AA
(Supporting Information Table 3). The structure-activity analysis of the two inducers
showed that the electronegativity, length, and space steric resistance of the C-3 sub-
stituents greatly influenced their activity; but no substitution of OH and smaller alkyl
hindrance in C-3 benefited the antiplatelet aggregation activity. The results of ADP
experiment showed that chloro-substituted phthalides had no marked influence on
their activity, regardless of the substitution position. The results of AA experiment
showed that, compared with the positive control drug Eda, 14b could significantly
inhibit platelet aggregation (Table 1). The electron-withdrawing group chlorine on
the benzene ring might be beneficial to the antiplatelet aggregation activity; however,
it was not influenced by the substitution site. Compared with the results in the litera-
ture [16], the bioactivities measured were not consistent well with each other, and the
reason may be related to different measurement methods.

4
X. FANG ET AL.
Table 1. Effect of phthalide derivatives on platelet aggregation and oxidative damage of SH-
ꢀ
SY5Y/PC 12 cells induced by H2O2. (x6s, n ¼ 3).
Aggregation inhibition rate (%)
Cell relative survival rate (%)
NO.
ADP
AA
SH-SY5Y
PC 12
Asp
Eda
NBP
1a
72.8 1.1
80.0 4.1
48.9 7.8
33.4 4.7
14.6 7.8
ꢁ8.0 7.3
54.5 0.9
56.0 4.0
50.2 3.2
23.3 10.9
0.2 3.9
85.9 4.9
74.7 6.7
57.6 4.5
97.1 8.8
69.1 7.3
56.0 7.5
88.2 0.3
56.8 2.3
46.5 5.1
74.5 4.1##
67.4 5.9#
47.8 2.8
93.8 3.7##
,##
ꢃ
12a
14b
15b
ꢃꢃ
15.6 9.8
ꢃꢃ
65.1 5.5
44.5 7.1
92.7 5.8#
p < 0.01 vs. Edaravone (Eda);
p < 0.05 vs. Asp,
#p < 0.05,
ꢃ
##p < 0.01 vs. Eda.
SPSS 21.0 software was used for data processing and one-way ANOVA was used for statistical analysis.
2.3. Antioxidation activity
As one of the important factors involved in neuronal injury, oxidative stress is associ-
ated with the pathophysiological processes of various neurological diseases, and is one
of the main research strategies for anti-stroke treatment [20]. H2O2, a toxic byproduct
of aerobic metabolism, can cross the cell membrane and participate in the Fenton
reaction to generate the most active free radical, OHꢁ, result a series of chain reac-
tions [21]. Oxidative damage models comprising SH-SY5Y and PC12 cells induced by
H2O2 were used to evaluate the antioxidant activity of the compounds. The relative
cell survival rate was determined using the cell counting kit-8 (CCK-8) method, with
edaravone (Eda) and aspirin (Asp) as positive controls. The compounds exhibited
some selective protection in both models (Supporting Information Table 3). The
results of the SH-SY5Y cell experiment showed that compared with Asp, 1a signifi-
cantly increased the relative cell survival rate (p < 0.05). Compared with Eda, 1a very
significantly increased the relative cell survival rate (p < 0.01) and 15b significantly
increased the relative cell survival rate (p < 0.05) (Table 1). The length and steric hin-
drance of the C-3 side chain greatly influenced the oxidation activity, with ethyl and
propyl groups showing better activity. In addition, the activity decreased with increas-
ing alkyl side chain length. The results of the PC12 cell experiment showed that com-
pared with Eda, 1a and 15b very significantly increased the relative cell survival rate
(p < 0.01) and 12a significantly increased the relative cell survival rate (p < 0.05)
(Table 1). No explicit structure-activity relationships were observed.
In conclusion, 23 phthalide derivatives were designed and synthesized, among
which seventeen were unreported. In vitro antiplatelet aggregation and antioxidant
models were successfully constructed to evaluate the activity of the phthalide
derivatives. The screening results indicated that the phthalide derivatives displayed
selective inhibition of platelet aggregation induced by ADP and AA, and selective
protection from oxidative damage induced by H2O2 in SH-SY5Y and PC12 cells.
The two sets of experiments revealed the following structure-activity rules:
Substituting ethyl and propyl for C-3 alkyl side chains was beneficial to the com-
pound’s activity; the substitution of C-3 hydroxyl reduced the original activity and

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
increased the structural instability. The introduction of electron-absorbing chlorine
atoms on the benzene ring had no clear guiding significance for the change in
activity, and was independent of the replacement position. The substitution of
2-sulfur atoms tended to increase the antioxidant activity, and most of the deriva-
tives were superior to butylphthalide, which indicated a direction for further struc-
tural modification.
3. Experimental
3.1. General experimental procedures
Bruker Spectrospin AC-300P/AC 300 MHz spectrometers and Bruker AVANCE III
400 MHz spectrometers (Bruker, Reinstetten, Germany) recorded the corresponding
nuclear magnetic resonance (NMR) spectra. Chemical shifts are indicated in d-values
(ppm) in relation to tetramethylsilane (TMS) used as an internal standard. The electro-
spray ionization mass spectrometry (ESIMS) was measured on an Aglilent LC/MS-6210
mass spectrometer (Aglilent Technologies Inc., California, USA). Semi-preparative high
performance liquid chromatography (HPLC) was carried out on a Shimadzu LC-6AD
HPLC instrument (Shimadzu, Kyoto, Japan) with a Shim-pack Prep-octadecyl silica
(ODS) column (Shimadzu, Kyoto, Japan, NO. 2025B41). Thin-layer chromatography
(TLC) was performed on precoated silica gel plate (SGF254, 0.2 mm, Yantai Chemical
Industry Research Institute, Yantai, China). Flash column chromatography was per-
formed using silica gel (200–300 mesh, Yantai Jiangyou Silica Gel Development Co.,
Ltd., Yantai, China). All solvents and reagents were of analytical pure grade, and no fur-
ther purification was needed. All starting materials were commercially available.
3.2. Synthesis
3.2.1. Preparation of intermediates
I. Phthalic anhydride (2.96 g, 20 mmol) was dissolved in 4 ml tetrahydrofuran (THF);
4 ml Na2Sꢀ9H2O (2.40 g, 10 mmol) water solution was added one drop at a time.
During the process, the reaction solution turned light yellow, and a small amount of
white solids were precipitated. At room temperature, stirring was continued for
4 hours. 20 ml H2O was added to the reaction solution to quench, and a large amount
of white solids were precipitated out. 5 ml 2 mol/L HCl was adjusted to pH 6, and
1.27 g white crystals were extracted, with a yield of 85%.
II. Using 4-chloro-phthalic anhydride (3.65 g, 20 mmol) as raw material, 1.60 g of
white powder was obtained with the yield of 87.6%, following the above method.
III. Using 3-chloro-phthalic anhydride (3.65 g, 20 mmol) as raw material, 1.40 g
grey powder was obtained with a yield of 76.7%, following the above method.
3.2.2. Preparation of a-series derivatives
Because the synthesis steps were very similar, 2a was taken as an example. Dry
phthalic anhydride (4.50 g, 27.44 mmol) was dissolved in 40 ml THF, slowly drop
with isopropyl magnesium chloride (anhydrous THF solution, 28 ml, 60.36 mmol)
below ꢁ5 ꢂC, until the reaction fluid changed from yellow to orange. The reaction

6
X. FANG ET AL.
was continued at low temperature for 0.5 h, and moved to room temperature for 5 h.
The reaction was quenched with a saturated ammonium chloride solution below 0 ꢂC.
The reaction mixture was acidified to pH 2 with 10% HCl, and extracted with
EtOAc, then dried with anhydrous Na2SO4. Yellow oily products were obtained by
chromatographic separation with a yield of 30%. The yield of this reaction step is
generally 20–30%.
3.2.2.1. 3-Butyl-3-hydroxybenzo[c]thiophen-1(3H)-one (compound 1a). C12H14O2S,
1
yellow oil. H-NMR (300 MHz, CDCl3) d (ppm): 7.50–7.62 (3H, m, ArH), 7.39 (1H, t,
J ¼ 7.2 Hz, ArH), 4.62 (1H, s, OH), 2.30 (1H, td, J ¼ 4.5, 13.8Hz, CH2), 2.03 (1H, td,
J ¼ 4.2, 12.3 Hz, CH2), 1.54–1.58 (1H, m, CH2), 1.22–1.32 (2H, m, CH2), 1.04–1.15 (1H,
m, CH2), 0.83 (3H, t, J ¼ 7.2 Hz, CH3); 13C-NMR (75MHz, CDCl3) d (ppm): 195.8, 153.0,
134.8, 134.3, 129.8, 123.8, 123.3, 95.9, 42.5, 27.8, 22.6, 13.9. ESI-MS m/z: 221.8 [M-H]ꢁ.
The spectroscopic data of 1a were in agreement with those of known compound reported
in literatures [16].
3.2.2.2.
3-Hydroxy-3-isopropylbenzo[c]thiophen-1(3H)-one
(compound
2a).
C11H12O2S, light yellow oil. 1H-NMR (300 MHz, CDCl3) d (ppm): 7.60 (3H, m,
ArH), 7.43 (1H, t, H-4, ArH), 3.98 (1H, s, OH), 2.47–2.56 (1H, m, CH), 1.23 (3H, d,
J ¼ 6.3 Hz, CH3), 0.63 (3H, d, J ¼ 6.6 Hz, CH3); 13C-NMR (75 MHz, CDCl3) d (ppm):
196.4 (C-1), 152.9 (C-3a), 135.2 (C-1), 134.4 (C-5), 129.9 (C-4), 123.6 (C-7a), 123.2
(C-6), 100.7 (C-3), 77.6 (C-1), 77.2 (C-1), 76.7 (C-1), 38.8 (C-8), 19.2 (C-10), 17.8
(C-9). ESI-MS m/z: 209 [M þ H]þ, 231 [M þ Na]þ.
3.2.2.3. 3-Butyl-5-chloro-3-hydroxybenzo[c]thiophen-1(3H)-one (compound 3a).
1
C12H13ClO2S, light yellow oil. H-NMR (300 MHz, CDCl3) d (ppm): 7.58 (2H, d,
J ¼ 8.1 Hz, ArH), 7.45 (1H, d, J ¼ 8.1 Hz, ArH), 3.76 (1H, s, OH), 2.30–2.39 (1H, m,
CH2), 2.02–2.11 (1H, m, CH2), 1.10–1.44 (4H, m, 2 ꢄ CH2), 0.89 (3H, t, CH3,
J ¼ 6.9 Hz, CH3); 13C-NMR (75 MHz, CDCl3) d (ppm): 194.2, 154.6, 141.3, 133.2,
130.4, 124.4, 124.3, 95.1, 42.5, 27.9, 22.7, 14.0. ESI-MS m/z: [M þ H]þ 257:259 ¼ 3:1.
3.2.2.4. 3-Butyl-7-chloro-3-hydroxybenzo[c]thiophen-1(3H)-one (compound 4a).
1
C12H13ClO2S, light yellow oil. H-NMR (300 MHz, CDCl3) d (ppm): 7.67 (1H, d,
J ¼ 7.5 Hz, ArH), 7.61 (1H, d, J ¼ 7.8 Hz, ArH), 7.44 (1H, t, J ¼ 7.5 Hz, ArH), 3.39
(1H, s, OH), 2.50–2.64 (2H, m, CH2), 1.55–1.67 (2H, m, CH2), 1.25–1.39 (2H, m,
CH2), 0.82 (3H, t, J ¼ 7.2 Hz, CH3); 13C-NMR (75 MHz, CDCl3) d (ppm): 193.4,
146.8, 138.1, 136.1, 131.4, 131.2, 122.3, 96.6, 39.3, 27.8, 22.6, 14.0. ESI-MS m/z:
[M þ H]þ 257:259 ¼ 3:1.
3.2.2.5. 3-Hydroxy-3-isopropylbenzo[c]thiophen-1(3H)-one (compound 5a). C10H10O2S,
light yellow oil. ESI-MS m/z: 195 [M þ H]þ, 217 [M þ Na]þ.
3.2.2.6. 3-Hydroxy-3-pentylbenzo[c]thiophen-1(3H)-one (compound 6a). C13H16O2S,
1
light yellow oil. H-NMR (300 MHz, CDCl3) d (ppm): 7.61–7.71 (m, 3H, ArH), 7.49
(1H, t, J ¼ 7.5 Hz, ArH), 2.33–2.43 (1H, m, CH2), 2.02–2.15 (1H, m, CH2), 1.17–1.30

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
(6H, m, 3 ꢄ CH2), 0.86 (3H, t, J ¼ 6.3 Hz, CH3); 13C-NMR (75 MHz, CDCl3) d (ppm):
195.0, 153.0, 134.9, 134.3, 130.0, 123.8, 123.5, 95.8, 42.8, 31.7, 25.5, 22.5, 14.1. ESI-MS
m/z: 237 [M þ H]þ, 261 [M þ Na]þ. The spectroscopic data of 6a were in agreement
with those of known compound reported in literatures [16].
3.2.2.7. 5-Chloro-3-hydroxy-3-pentylbenzo[c]thiophen-1(3H)-one (compound 7a).
C13H15ClO2S, light yellow oil. ESI-MS m/z: [M þ H]þ 271:273 ¼ 3:1, [M þ Na]þ
293:295 ¼ 3:1.
3.2.2.8. 5-Chloro-3-hydroxy-3-isopropylbenzo[c]thiophen-1(3H)-one (compound 8a).
1
C11H11ClO2S, light yellow oil. H-NMR (400 MHz, CDCl3) d (ppm): 7.56–7.59 (2H,
m, ArH), 7.45 (1H, dd, J ¼ 1.6, 8.4 Hz, ArH), 2.48–2.55 (1H, m, CH), 1.26 (3H, d,
J ¼ 6.4 Hz, CH3), 0.70 (3H, d, J ¼ 6.4 Hz, CH3); 13C-NMR (100 MHz, CDCl3) d
(ppm): 194.3, 154.5, 141.4, 133.7, 130.5, 124.3, 124.1, 99.8, 38.8, 19.1, 17.9. ESI-MS m/
z: [M þ H]þ 243:245 ¼ 3:1.
3.2.2.9. 4-Chloro-3-hydroxy-3-pentylbenzo[c]thiophen-1(3H)-one (compound 9a).
C13H15ClO2S, light yellow oil. ESI-MS m/z: [M þ H]þ 271:273 ¼ 3:1.
3.2.2.10. 7-Chloro-3-hydroxy-3-isopropylbenzo[c]thiophen-1(3H)-one (compound
10a). C11H11ClO2S, light yellow oil. ESI-MS m/z: [M þ H]þ 243:245 ¼ 3:1.
3.2.2.11. 3-Hydroxy-3-propylbenzo[c]thiophen-1(3H)-one (compound 11a). C11H12O2S,
light yellow oil. ESI-MS m/z: 209 [M þ H]þ.
3.2.2.12. 5-Chloro-3-hydroxy-3-propylbenzo[c]thiophen-1(3H)-one (compound 12a).
1
C11H11ClO2S, light yellow oil. H-NMR (400 MHz, CDCl3) d (ppm): 7.57–7.59 (2H, m,
ArH), 7.45 (1H, dd, J ¼ 1.2, 8.4 Hz, ArH), 3.63 (1H, s, OH), 2.29–2.37 (1H, m, CH2),
2.05 (1H, td, J¼ 4.4, 12.0 Hz, CH2), 1.66–1.74 (1H, m, CH2), 1.23–1.26 (1H, m, CH2),
0.95 (3H, t, J ¼ 3.2 Hz, CH3); 13C-NMR (100 MHz, CDCl3) d (ppm): 194.0, 154.6, 141.3,
133.2, 130.5, 124.4, 124.3, 95.0, 44.9, 19.2, 14.0. ESI-MS m/z: [M þ H]þ 243:245 ¼ 3:1.
3.2.2.13. 4-Chloro-3-hydroxy-3-propylbenzo[c]thiophen-1(3H)-one (compound 13a).
1
C11H11ClO2S, light yellow oil. H-NMR (400 MHz, CDCl3) d (ppm): 7.67 (1H, d,
J ¼ 7.6 Hz, ArH), 7.61 (1H, d, J ¼ 8.0 Hz, ArH), 7.45 (1H, d, J ¼ 8.0 Hz, ArH), 3.17
(1H, br s, OH), 2.60–2.68 (1H, m, CH2), 2.43–2.51 (1H, m, CH2), 1.62–1.65 (1H, m,
CH2), 1.18–1.21 (1H, m, CH2), 0.96 (3H, t, J ¼ 7.2 Hz, CH3); 13C-NMR (100 MHz,
CDCl3) d (ppm): 193.2, 146.8, 138.1, 136.1, 131.4, 131.2, 122.3, 96.5, 41.7, 19.2, 14.0.
ESI-MS m/z: [M þ H]þ 243:245 ¼ 3:1.
3.2.2.14. 5-Chloro-3-ethyl-3-hydroxybenzo[c]thiophen-1(3H)-one (compound 14a).
C10H9ClOS, light yellow oil. ESI-MS m/z: [M þ H]þ 213:215 ¼ 3:1.
3.2.2.15. 4-Chloro-3-ethyl-3-hydroxybenzo[c]thiophen-1(3H)-one (compound 15a).
C10H9ClO2S, light yellow oil. ESI-MS m/z: [M þ H]þ 229:231 ¼ 3:1.

8
X. FANG ET AL.
3.2.3. Preparation of b-series derivatives
Because of the very similar synthesis steps, 2b was taken as an example. 2a was dis-
solved in 2 ml glacial acetic acid, added 57% HI after reflux, and continued reflux
reaction for 0.5 h. Then the mixture was washed by a saturated solution of NaHSO3
until a negative potassium iodine-starch test was given. The solution was evaporated
in a vacuum to remove acetic acid, then ethyl acetate was added to the residues.
Anhydrous Na2SO4 was added to the resulted solution to remove the water. After
removing Na2SO4 by filtration, the filtrate was subject to column chromatography to
give the title compound. The yield is generally 70–80%.
3.2.3.1. 3-Butylbenzo[c]thiophen-1(3H)-one (compound 1b). C12H14OS, yellow oil.
1H-NMR (300 MHz, CDCl3) d (ppm): 7.78 (1H, d, J ¼ 7.5 Hz, ArH), 7.62 (1H, t,
J ¼ 7.5 Hz, ArH), 7.53 (1H, d, J ¼ 7.5 Hz, ArH), 7.45 (1H, t, J ¼ 7.5 Hz, ArH), 4.83
(1H, d, J ¼ 9.6 Hz, CH), 2.30 (1H, dd, J ¼ 9.9, 18.9 Hz, CH2), 1.82 (1H, dd, J ¼ 9.9,
20.4 Hz, CH2), 1.38–1.64 (4H, m, 2 ꢄ CH2), 0.91 (3H, t, J ¼ 6.0 Hz, CH3); 13C-
NMR (100 MHz, CDCl3) d (ppm): 197.6, 151.6, 136.1, 133.4, 128.3, 125.2, 123.8,
51.7, 36.3, 30.3, 22.6, 14.0. ESI-MS m/z: 207 [M þ H]þ, 229 [M þ Na]þ. The spec-
troscopic data of 1b were in agreement with those of known compound reported
in literatures [16].
3.2.3.2. 3-Isopropylbenzo[c]thiophen-1(3H)-one (compound 2b). C11H12O2S, light yel-
1
low oil. H-NMR (300 MHz, CDCl3) d (ppm): 7.70 (1H, d, J ¼ 7.8 Hz, ArH), 7.61
(1H, t, J ¼ 7.5 Hz, ArH), 7.41–7.51 (2H, m, ArH), 4.94 (1H, d, J ¼ 2.7 Hz, CH),
2.58–2.68 (1H, m, CH), 1.18 (3H, d, J ¼ 6.6 Hz, CH3), 0.62 (3H, d, J ¼ 6.6 Hz, CH3);
13C-NMR (75 MHz, CDCl3) d (ppm): 197.8, 150.7, 136.7, 133.4, 128.2, 125.1, 123.6,
58.7, 32.6, 22.9, 15.0. ESI-MS m/z: 193 [M þ H]þ.
3.2.3.3. 3-Butyl-5-chlorobenzo[c]thiophen-1(3H)-one (compound 3b). C12H13ClOS,
1
light yellow oil. H-NMR (300 MHz, CDCl3) d (ppm): 7.71 (1H, d, J ¼ 8.4 Hz, ArH),
7.51 (1H, br s, ArH), 7.43 (1H, d, J ¼ 8.1 Hz, ArH), 4.78–4.81 (1H, m, CH), 2.23–2.27
(1H, m, CH2), 1.73–1.81 (1H, m, CH2), 1.32–1.45 (4H, m, 2 ꢄ CH2), 0.93 (3H, t,
J ¼ 6.0 Hz, CH3); 13C-NMR (75 MHz, CDCl3) d (ppm): 196.0, 153.0, 140.3, 133.3,
129.0, 125.5, 124.7, 51.1, 36.2, 30.2, 22.6, 14.0. ESI-MS m/z: [M þ H]þ 241:243 ¼ 3:1.
3.2.3.4. 3-Butyl-7-chlorobenzo[c]thiophen-1(3H)-one (compound 4b). C12H13ClOS,
1
light yellow oil. H-NMR (300 MHz, CDCl3) d (ppm): 7.70 (1H, d, J ¼ 7.8 Hz, ArH),
7.60 (1H, d, J ¼ 7.8 Hz, ArH), 7.42 (1H, t, J ¼ 7.5 Hz, ArH), 4.88 (1H, dd, J ¼ 2.1,
9.6 Hz, CH2), 2.56–2.60 (1H, m, CH2), 1.70–1.79 (2H, m, CH2), 1.25–1.52 (4H, m,
2 ꢄ CH2), 0.84 (3H, t, J ¼ 6.9 Hz, CH3); 13C-NMR (75 MHz, CDCl3) d (ppm): 196.3,
147.9, 138.3, 134.2, 132.0, 129.8, 122.4, 51.0, 34.1, 30.1, 22.4, 14.0. ESI-MS m/z:
[M þ H]þ 241:243 ¼ 3:1.
3.2.3.5. 3-Ethylbenzo[c]thiophen-1(3H)-one (compound 5b). C10H10OS, light yellow
1
oil. H-NMR (300 MHz, CDCl3) d (ppm): 7.79 (1H, d, J¼ 7.5 Hz, ArH), 7.62 (1H, t,
J ¼ 7.2 Hz, ArH), 7.54 (1H, d, J¼ 7.8 Hz, ArH), 7.45 (1H, t, J ¼ 7.2 Hz, ArH), 4.83 (1H,

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
dd, J¼ 3.3, 8.7 Hz, CH), 2.32–2.38 (1H, m, CH2), 1.84–1.94 (1H, m, CH2), 1.04 (3H, t,
J ¼ 7.5 Hz, CH3); 13C-NMR (75 MHz, CDCl3) d (ppm): 197.6, 151.2, 136.3, 133.4, 128.3,
125.2, 123.8, 53.0, 29.3, 11.8. ESI-MS m/z: 179 [M þ H]þ. The spectroscopic data of 5b
were in agreement with those of known compound reported in literatures [16].
3.2.3.6. 3-Pentylbenzo[c]thiophen-1(3H)-one (compound 6b). C13H16OS, light yellow
1
oil. H NMR (300 MHz, CDCl3) d (ppm): 7.79 (1H, d, J ¼ 7.8 Hz, ArH), 7.62 (1H, t,
J ¼ 7.5 Hz, ArH), 7.53 (1H, d, J ¼ 7.8 Hz, ArH), 7.45 (1H, t, J ¼ 7.2 Hz, ArH), 4.83
(1H, dd, J ¼ 3.6, 9.6 Hz, CH), 2.24–2.32 (1H, m, CH2), 1.77–1.85 (1H, m, CH2),
1.50–1.59 (1H, m, CH2), 1.25–1.45 (3H, m, 2 ꢄ CH2), 0.89 (3H, t, J ¼ 6.6 Hz, CH3);
13C-NMR (75 MHz, CDCl3) d (ppm): 197.5, 151.6, 136.1, 133.3, 128.3, 125.2, 123.8,
51.7, 36.6, 31.6, 27.8, 22.5, 14.1. ESI-MS m/z: 221 [M þ H]þ. The spectroscopic data
of 6b were in agreement with those of known compound reported in literatures [16].
3.2.3.7. 5-Chloro-3-pentylbenzo[c]thiophen-1(3H)-one (compound 7b). C13H15ClOS,
1
light yellow oil. H-NMR (300 MHz, CDCl3) d (ppm): 7.70 (1H, d, J ¼ 8.4 Hz, ArH),
7.51 (1H, br s, ArH), 7.44 (1H, d, J ¼ 8.4 Hz, ArH), 4.78–4.82 (1H, m, CH), 2.24–2.27
(1H, m, CH2), 1.77–1.80 (1H, m, CH2), 1.25–1.44 (6H, m, 3 ꢄ CH2), 0.88–0.90 (3H,
m, CH3); 13C-NMR (75 MHz, CDCl3) d (ppm): 195.9, 153.0, 140.3, 133.3, 128.9,
125.5, 124.7, 51.1, 36.5, 31.6, 27.8, 22.5, 14.1. ESI-MS m/z: [M þ H]þ 255:256 ¼ 3:1.
3.2.3.8.
5-Chloro-3-isopropylbenzo[c]thiophen-1(3H)-one
(compound
8b).
C11H11ClOS, light yellow oil. 1H-NMR (400 MHz, CDCl3) d (ppm): 7.70 (1H, d,
J ¼ 8.0 Hz, ArH), 7.49 (1H, d, J¼ 1.2 Hz, ArH), 7.44 (1H, dd, J ¼ 1.2, 8.4 Hz, ArH), 4.90
(1H, d, J ¼ 3.2 Hz, CH), 2.57–2.64 (1H, m, CH), 1.19 (3H, d, J ¼ 6.8 Hz, CH3), 0.68 (3H,
d, J¼ 6.8 Hz, CH3); 13C-NMR (100 MHz, CDCl3) d (ppm): 196.2, 152.2, 140.4, 135.3,
129.0, 125.4, 124.7, 58.1, 32.7, 22.8, 15.2. ESI-MS m/z: [M þ H]þ 227:229 ¼ 3:1.
3.2.3.9. 4-Chloro-3-pentylbenzo[c]thiophen-1(3H)-one (compound 9b). C13H15ClOS,
1
light yellow oil. H-NMR (400 MHz, CDCl3) d (ppm): 7.50 (1H, t, J ¼ 7.6 Hz, ArH),
7.42 (1H, d, J ¼ 7.6 Hz, ArH), 7.40 (1H, t, J ¼ 8.0 Hz, ArH), 4.74 (1H, dd, J ¼ 3.6,
10.0 Hz, CH), 2.18–2.28 (1H, m, CH2), 1.76–1.79 (1H, m, CH2), 1.33–1.37 (1H, m,
CH2), 1.25–1.32 (5H, m, CH2), 0.88 (3H, t, J ¼ 6.8 Hz, CH3); 13C-NMR (100 MHz,
CDCl3) d (ppm): 194.1, 154.7, 133.4, 131.7, 131.4, 130.5, 123.9, 49.5, 36.8, 31.6, 27.6,
22.5, 14.1. ESI-MS m/z: [M þ H]þ 255:257 ¼ 3:1.
3.2.3.10.
7-Chloro-3-isopropylbenzo[c]thiophen-1(3H)-one
(compound
10b).
C11H11ClOS, light yellow oil. 1H-NMR (400 MHz, CDCl3) d (ppm): 7.50 (1H, t,
J ¼ 8.0 Hz, ArH), 7.42 (1H, d, J ¼ 8.0 Hz, ArH), 7.40 (1H, d, J ¼ 7.6 Hz, ArH), 4.85
(1H, d, J ¼ 3.6 Hz, CH), 2.58–2.63 (1H, m, CH), 1.18 (3H, d, J ¼ 6.8 Hz, CH3), 0.64
(3H, d, J ¼ 6.8 Hz, CH3); 13C-NMR (100 MHz, CDCl3) d (ppm): 194.3, 154.0, 133.5,
132.0, 131.7, 130.6, 123.8, 56.4, 32.9, 22.9, 14.9. ESI-MS m/z: [M þ H]þ 227:229 ¼ 3:1.
3.2.3.11. 3-Propylbenzo[c]thiophen-1(3H)-one (compound 11b). C11H12OS, light yel-
low oil. 1H-NMR (400 MHz, CDCl3) d (ppm): 7.71 (1H, d, J ¼ 7.6 Hz, ArH),

10
X. FANG ET AL.
7.55–7.59 (1H, m, ArH), 7.49 (1H, t, J ¼ 7.6 Hz, ArH), 7.39 (1H, t, J ¼ 7.2 Hz, ArH),
4.79 (1H, dd, J ¼ 3.6, 8.4 Hz, CH), 2.19–2.23 (1H, m, CH2), 1.70–1.74 (1H, m, CH2),
1.50–1.53 (1H, m, CH2), 1.39–1.41 (1H, m, CH2), 0.93 (1H, d, J ¼ 7.6 Hz, CH3); 13C-
NMR (100 MHz, CDCl3) d (ppm): 197.2, 151.4, 135.7, 133.2, 128.0, 125.1, 123.5, 51.3,
38.5, 21.3, 13.7. ESI-MS m/z: 193 [M þ H]þ. The spectroscopic data of 11b were in
agreement with those of known compound reported in literatures [16].
3.2.3.12.
7-Chloro-3-isopropylbenzo[c]thiophen-1(3H)-one
(compound
12b).
C11H11ClOS, light yellow oil. 1H-NMR (400 MHz, CDCl3) d (ppm): 7.71 (1H, d,
J ¼ 8.4 Hz, ArH), 7.51 (1H, br s, ArH), 7.43 (1H, dd, J ¼ 1.2, 8.4 Hz, ArH), 4.80 (1H,
dd, J ¼ 3.6, 9.6 Hz, CH), 2.22–2.25 (1H, m, CH2), 1.77–1.82 (1H, m, CH2), 1.54–1.58
(1H, m, CH2), 1.41–1.49 (1H, m, CH2), 1.00 (3H, t, J ¼ 7.2 Hz, CH3); 13C-NMR
(100 MHz, CDCl3) d (ppm): 195.9, 153.0, 140.3, 134.6, 129.0, 125.5, 124.8, 50.9, 38.6,
21.5, 13.9. ESI-MS m/z: [M þ H]þ 227:229 ¼ 3:1.
3.2.3.13.
4-Chloro-3-propylbenzo[c]thiophen-1(3H)-one
(compound
13b).
C11H11ClOS, light yellow oil. 1H-NMR (400 MHz, CDCl3) d (ppm): 7.70 (1H, d,
J ¼ 7.6 Hz, ArH), 7.59 (1H, d, J ¼ 7.2 Hz, ArH), 7.41 (1H, t, J ¼ 7.6 Hz, ArH), 4.88
(1H, dd, J ¼ 2.8, 9.6 Hz, OH), 2.52–2.54 (1H, m, CH2), 1.71–1.76 (1H, m, CH2),
1.51–1.53 (1H, m, CH2), 1.39–1.42 (1H, m, CH2), 0.97 (3H, t, J ¼ 7.2 Hz, CH3); 13C-
NMR (100 MHz, CDCl3) d (ppm): 196.2, 147.9, 138.2, 134.2, 131.9, 129.8, 122.4, 50.8,
36.5, 21.4, 13.7. ESI-MS m/z: [M þ H]þ 227:229 ¼ 3:1.
3.2.3.14. 5-Chloro-3-ethylbenzo[c]thiophen-1(3H)-one (compound 14b). C10H9ClOS,
1
light yellow oil. H NMR (400 MHz, CDCl3) d (ppm): 7.71 (1H, d, J ¼ 8.4 Hz, ArH),
7.51 (1H, br s, ArH), 7.45 (1H, d, J ¼ 8.4 Hz, ArH), 4.80 (1H, dd, J ¼ 3.6, 8.8 Hz, CH),
2.29–2.35 (1H, m, CH2), 1.88–1.93 (1H, m, CH2), 1.05 (3H, t, J ¼ 7.2 Hz, CH3); 13C
NMR (100 MHz, CDCl3) d (ppm): 195.9, 152.6, 140.4, 137.1, 134.9, 129.0, 125.5,
124.8, 52.4, 29.2, 11.7. ESI-MS m/z: [M þ H]þ 213:215 ¼ 3:1.
3.2.3.15. 4-Chloro-3-ethylbenzo[c]thiophen-1(3H)-one (compound 15b). C10H9ClOS,
1
light yellow oil. H-NMR (400 MHz, CDCl3) d (ppm): 7.70 (1H, dd, J ¼ 1.2, 7.6 Hz,
ArH), 7.59 (1H, dd, J ¼ 1.2, 7.6 Hz, ArH), 7.41 (1H, t, J ¼ 7.6 Hz, ArH), 4.88 (1H, dd,
J ¼ 2.8, 8.8 Hz, CH), 2.52–2.58 (1H, m, CH2), 1.88–1.95 (1H, m, CH2), 0.98 (3H, t,
J ¼ 7.2 Hz, CH3); 13C-NMR (100 MHz, CDCl3) d (ppm): 196.1, 147.5, 138.5, 134.1,
132.0, 129.9, 122.4, 52.3, 27.3, 11.6. ESI-MS m/z: [M þ H]þ 213:215 ¼ 3:1,
[M þ Na]þ 235:237 ¼ 3:1.
3.3. In vitro antiplatelet aggregation assay
Blood was drawn from rabbit aorta abdominalis and mixed with 3.8% sodium citrate
(9:1, v/v). After centrifugation at 900 rpm for 5 min at room temperature to obtain plate-
let-rich plasma (PRP), the remaining blood was further centrifuged at 3000 rpm for
another 15 min to obtain platelet-poor plasma (PPP). A microtiter plate method for
detection of platelet aggregation was used according to previous literatures [22, 23].

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
Microscopically, PRP count was adjusted to 5 ꢄ 108/ml using PPP. Briefly, PRP (90 ll)
was preincubated in 96-well plates in duplicate for 10 min at 37 ꢂC with vehicle, the
individual compounds, or reference drugs (ASP and Eda) at the same concentrations
(100 lM). After the samples were shaken and mixed, the absorbance value was meas-
ured at 570 nm with microplate reader (Synergy 4 MLFPTAD microplate reader, BioTek
Instruments, Inc.). The absorbance value was measured continuously for two times until
the datum was stable. The average absorbance of two times per well was recorded as
APRP. APPP was measured in the same way as APRP, except 90ll PPP was added into
5 ll blank solvent. The samples were mixed with 5 ll inducer ADP or AA, and vibrated
for 5 min. The mean absorbance of the parallel wells was denoted as Asample. Results
ꢀ
indicated by absorbance value x6s at 570 nm, aggregation rate (AR) and aggregation
inhibition rate (AIR) of each group were calculated according to the following formula:
APRPꢁAsample
AR ¼
ꢄ 100%
APRP ꢁ APRP
ARcontrolꢁARexperiment
AIR ¼
ꢄ 100%
ARcontrol
ARcontrol represented the platelet aggregation rate of the blank control group;
ARexperiment represented the platelet aggregation rate of the drug group.
3.4. In vitro antioxidant assay
SH-SY5Y cells were cultured in F12K Dulbecco’s modified Eagle’s medium (DMEM)
culture medium containing 10% fetal bovine serum, under the condition of 37 ꢂC and
5% CO2. After 2–3 days, the mixture of 0.25% trypsin and 0.02% ethylene diamine
tetraacetic acid (EDTA) (1:1) was used to cells passage once at 1:5, and the logarith-
mic growth stage cells were taken for the next experiment. The treatment of PC 12
cell culture was the same. The toxicity of the synthetic compounds was detected by
CCK-8 method first. Based on the CCK-8 experimental results, compounds with cyto-
toxic IC50 > 30 lM were selected for further experiments. Cells (1 ꢄ 105 cells/ml,
100 ll) were inoculated in 96-well plates, to join the H2O2 solution to 300 lM, com-
pounds to 30 lM. The samples were incubated at 37 ꢂC, 5% CO2 for 24 h, and the
cell survival rate was determined by CCK-8 method. The medium was changed to the
medium containing 10% CCK-8 reagent. After incubating for 4 h, the absorbance of
450 nm was measured with an enzyme marker (H1M microplate reader, BioTek
Instruments, Inc.), that is, the OD values [24, 25]. The cell relative survival rate of
the tested individual compounds on the antioxidant activity was calculated as the fol-
lowing formula: Cell relative survival rate (CRSR) ¼ (ODexperiment – ODmodel)/
(ODcontrol – ODmodel) ꢄ 100%. Control group (control), dimethylsulfoxide (DMSO)
was added, but H2O2 and compounds were not added. Model group (model), H2O2
and DMSO were added, but no compounds treatment.
Disclosure statement
No potential conflict of interest was reported by the authors.

12
X. FANG ET AL.
Funding
This work was financially supported by the Shanghai Municipal Commission of Health and
Family Planning [grant number ZY3-CCCX-3-5001], the Science and Technology Commission
of Shanghai Municipality [grant numbers 14401901400, 15DZ2292000], the Expenditure
Budget Program of Shanghai Municipal Education Commission [grant number 2016YSN24],
and Xinglin Young Talent Program of Shanghai University of Traditional Chinese Medicine.
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