Total synthesis and neuroprotective effect of O-methylmurrayamine A and 7-methoxymurrayacine

Article abstract of DOI:10.1080/10286020.2017.1327952

O-Methylmurrayamine A (7) and 7-methoxymurrayacine (8) are natural products isolated from Murraya koenigii and Murraya siamensis, respectively. In this paper, we report the synthesis of 7 and 8 which are featured in the key step of cyclization to form carbazole intermediate 5 with mild conditions. The structures were confirmed by ¹H NMR, ¹³C NMR, and HR-ESI-MS. In addition, compounds 7 and 8 were tested for their neuroprotective effects against H₂O₂-induced PC12 cell damage. The results showed that compounds 7 and 8 have neuroprotective effect.

Full text of DOI:10.1080/10286020.2017.1327952

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Total synthesis and neuroprotective effect of O-
methylmurrayamine A and 7-methoxymurrayacine
Ying-Da Zang, Chuang-Jun Li, Xiu-Yun Song, Jie Ma, Jing-Zhi Yang, Nai-Hong
Chen & Dong-Ming Zhang
To cite this article: Ying-Da Zang, Chuang-Jun Li, Xiu-Yun Song, Jie Ma, Jing-Zhi Yang,
Nai-Hong Chen & Dong-Ming Zhang (2017): Total synthesis and neuroprotective effect of O-
methylmurrayamine A and 7-methoxymurrayacine, Journal of Asian Natural Products Research,
DOI: 10.1080/10286020.2017.1327952
To link to this article: http://dx.doi.org/10.1080/10286020.2017.1327952
Published online: 16 May 2017.
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Date: 18 May 2017, At: 03:29

Journal of asian natural Products research, 2017
ꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2017.1327952
ꢀ
Total synthesis and neuroprotective effect of
O-methylmurrayamine A and 7-methoxymurrayacine
Ying-Da Zang, Chuang-Jun Li, Xiu-Yun Song, Jie Ma, Jing-Zhi Yang, Nai-Hong Chen
and Dong-Ming Zhang
sꢁꢇꢁꢈ Kꢈy lꢇbꢄꢆꢇꢁꢄꢆy ꢄꢉ Bꢅꢄꢇꢊꢁꢅvꢈ sꢋbꢂꢁꢇꢌꢊꢈ ꢇꢌꢃ fꢋꢌꢊꢁꢅꢄꢌ ꢄꢉ nꢇꢁꢋꢆꢇꢍ Mꢈꢃꢅꢊꢅꢌꢈꢂ, iꢌꢂꢁꢅꢁꢋꢁꢈ ꢄꢉ Mꢇꢁꢈꢆꢅꢇ Mꢈꢃꢅꢊꢇ,
cꢀꢅꢌꢈꢂꢈ aꢊꢇꢃꢈmy ꢄꢉ Mꢈꢃꢅꢊꢇꢍ sꢊꢅꢈꢌꢊꢈꢂ ꢇꢌꢃ Pꢈkꢅꢌg uꢌꢅꢄꢌ Mꢈꢃꢅꢊꢇꢍ cꢄꢍꢍꢈgꢈ, Bꢈꢅjꢅꢌg 100050, cꢀꢅꢌꢇ
ABSTRACT
ARTICLE HISTORY
rꢈꢊꢈꢅvꢈꢃ 23 Mꢇꢆꢊꢀ 2017
aꢊꢊꢈpꢁꢈꢃ 4 Mꢇy 2017
O-Methylmurrayamine A (7) and 7-methoxymurrayacine (8) are
natural products isolated from Murraya koenigii and Murraya siamensis,
respectively. In this paper, we report the synthesis of 7 and 8 which are
featured in the key step of cyclization to form carbazole intermediate
KEYWORDS
tꢄꢁꢇꢍ ꢂyꢌꢁꢀꢈꢂꢅꢂ;
o-mꢈꢁꢀyꢍmꢋꢆꢆꢇyꢇmꢅꢌꢈ a;
7-mꢈꢁꢀꢄxymꢋꢆꢆꢇyꢇꢊꢅꢌꢈ;
ꢌꢈꢋꢆꢄpꢆꢄꢁꢈꢊꢁꢅvꢈ ꢈffꢈꢊꢁꢂ
1
13
5
with mild conditions. The structures were confirmed by H NMR, C
NMR, and HR-ESI-MS. In addition, compounds 7 and 8 were tested for
their neuroprotective effects against H O -induced PC12 cell damage.
2
2
The results showed that compounds 7 and 8 have neuroprotective
effect.
1
. Introduction
Carbazole alkaloids exhibit a broad variety of structures and biological activities.
O-Methylmurrayamine A (7), a pyrano[3,2-a]carbazole alkaloid, was mentioned first in
1
991 by Wu as a synthetic derivative of murrayamine A, which had been isolated from the
leaves of Murraya euchrestifolia collected in Taiwan [1]. In 2003, Nakatani and co-workers
described the isolation of O-methylmurrayamine A (7) from the leaves of Murraya koenigii
collected in Malaysia [2]. In 2016, compound 7 was reported to have a potential antidiabetic
activity by Patel et al. [3]. 7-Methoxymurrayacine (8) was obtained by Lange et al. in 1990
from the roots of Murraya siamensis [4]. e total synthesis of 7 and 8 has been reported,
but the key intermediate 5 was obtained in a harsh condition [5,6]. Herein, we report a
more temperate condition to prepare intermediate 5 and then the synthesis of 7 and 8. In
addition, compounds 7 and 8 were tested for their neuroprotective effects against H O -
2
2
induced PC12 cell damage by MTT assay (Figure 1).
CONTACT dꢄꢌg-Mꢅꢌg Zꢀꢇꢌg
zꢀꢇꢌgꢃm@ꢅmm.ꢇꢊ.ꢊꢌ
©
2017 iꢌꢉꢄꢆmꢇ uK lꢅmꢅꢁꢈꢃ, ꢁꢆꢇꢃꢅꢌg ꢇꢂ tꢇyꢍꢄꢆ & fꢆꢇꢌꢊꢅꢂ Gꢆꢄꢋp

2
Y.ꢀD. ZANG ET AL.
2
. Results and discussion

e construction of multi-substituted carbazole through intramolecular C-C oxidative cycli-
zation mediated by palladium is the key step. Reaction condition previously reported in the
literature included 0.2 equiv. Pd(OAc) , 2.5 equiv. Cu(OAc) , PivOH, microwave (300 W),
2
2
1
10 °C [5]. However, this condition is too harsh and not suitable for the synthesis in large
quantities (Figure 2). We achieved 50-g scale reaction at 60 °C in THF by increasing the
amount of Pd(OAc) to 1.1 equiv which gave 85% yield. e black palladium powder which
2
was transformed from Pd(OAc) could be obtained through filtration af er the reaction and
2
palladium powder can react with AcOH to reproduce Pd(OAc) under the catalytic amount
2
of HNO in a 95% yield (Figure 3). is method realizes Pd(OAc) recycling use and the
3
2
reaction condition is mild and suitable for preparation in large quantities.
e synthetic routes for the compounds 7 and 8 are shown in Scheme 1. In general,
3-(tert-butyldiphenylsilyloxy)-4-methylaniline (3) was easily prepared from commercially
available 2-methyl-5-nitrophenol (1) in two steps. Compound 3 reacted with m-Bro-
moanisole by means of a Buchwald–Hartwig C–N coupling to give 4. e subsequent
palladium-catalyzed cyclization of 4 afforded the key intermediate 5 in the less-hindered
conformation. Compound 6 was obtained by the removal of the tert-butyldiphenylsilyl
(
TBDPS) protecting group with tetra-n-butylammonium fluoride (TBAF) in tetrahydro-
furan (THF). e reaction of 6 with 3-methyl-2-butene in the presence of titanium (IV)
tetraisopropoxide provided target compound 7. Oxidation of the methyl group of 7 with
2
,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) further gave target compound 8.
7
O-Methylmurrayamine A (R = CH
3
)
8
7-Methoxymurrayacine (R = CHO)
Figure 1. sꢁꢆꢋꢊꢁꢋꢆꢈꢂ ꢄꢉ o-mꢈꢁꢀyꢍmꢋꢆꢆꢇyꢇmꢅꢌꢈ a (7) ꢇꢌꢃ 7-mꢈꢁꢀꢄxymꢋꢆꢆꢇyꢇꢊꢅꢌꢈ (8).
Figure 2. hꢇꢂꢀ ꢆꢈꢇꢊꢁꢅꢄꢌ ꢊꢄꢌꢃꢅꢁꢅꢄꢌ pꢆꢈvꢅꢄꢋꢂꢍy ꢆꢈpꢄꢆꢁꢈꢃ ꢅꢌ ꢁꢀꢈ ꢍꢅꢁꢈꢆꢇꢁꢋꢆꢈ.
Figure 3. Mꢅꢍꢃ ꢆꢈꢇꢊꢁꢅꢄꢌ ꢊꢄꢌꢃꢅꢁꢅꢄꢌ ꢇꢌꢃ Pꢃ(oaꢊ) ꢆꢈꢊyꢊꢍꢅꢌg.
2

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Scheme 1. tꢀꢈ ꢂyꢌꢁꢀꢈꢁꢅꢊ ꢆꢄꢋꢁꢈ ꢄꢉ o-mꢈꢁꢀyꢍmꢋꢆꢆꢇyꢇmꢅꢌꢈ a (7) ꢇꢌꢃ 7-mꢈꢁꢀꢄxymꢋꢆꢆꢇyꢇꢊꢅꢌꢈ (8). rꢈꢇgꢈꢌꢁꢂ
ꢇꢌꢃ ꢊꢄꢌꢃꢅꢁꢅꢄꢌꢂ: (ꢇ). tBdPscꢍ, ꢅmꢅꢃꢇzꢄꢍꢈ, ch cꢍ , rt; (b). h , Pꢃ/c, thf; (ꢊ). m-bꢆꢄmꢄꢇꢌꢅꢂꢄꢍꢈꢂ, Pꢃ(oaꢊ) ,
2
2
2
2
BinaP, cꢂ co , ꢁꢄꢍꢋꢈꢌꢈ, ꢆꢈflꢋx, 24 ꢀ; (ꢃ). Pꢃ(oaꢊ) , thf, 60 °c, 24 ꢀ; (ꢈ). tBaf, thf, 0 °c; (ꢉ). 3-mꢈꢁꢀyꢍ-2-
2
3
2
bꢋꢁꢈꢌꢈ, ꢁꢅꢁꢇꢌꢅꢋm (iV) ꢅꢂꢄpꢆꢄpꢄxꢅꢃꢈ, −78 °c-rt, g). ddQ, thf-Mꢈoh-h o, rt.
2
Table 1. nꢈꢋꢆꢄpꢆꢄꢁꢈꢊꢁꢅvꢈ ꢇꢊꢁꢅvꢅꢁy ꢂꢊꢆꢈꢈꢌꢅꢌg ꢄꢉ ꢊꢄmpꢄꢋꢌꢃꢂ 7 ꢇꢌꢃ 8 ꢇꢁ 10 μM ꢅꢌ Pc12 ꢊꢈꢍꢍꢂ ꢅꢌꢃꢋꢊꢈꢃ by
h o .
2
2
Group
Concentration (μM)
The survival rate (%)
cꢄꢌꢁꢆꢄꢍ
Mꢄꢃꢈꢍ
7
8
–
–
10
10
100.0 ± 5.0
#
##
70.1 ± 3.7
82.8 ± 7.5
**
84.7 ± 5.5**
nꢄꢁꢈ: eꢆꢆꢄꢆ bꢇꢆꢂ ꢆꢈpꢆꢈꢂꢈꢌꢁ ꢁꢀꢈ ꢂꢁꢇꢌꢃꢇꢆꢃ ꢃꢈvꢅꢇꢁꢅꢄꢌ.
#
##
p < 0.001 vꢂ ꢊꢄꢌꢁꢆꢄꢍ.
*
*
p < 0.01 vꢂ mꢄꢃꢈꢍ (mꢈꢇꢌꢂ ± ꢂꢁꢇꢌꢃꢇꢆꢃ ꢃꢈvꢅꢇꢁꢅꢄꢌ, n = 6).

e rat PC12 cell line is a well-established model for studying many aspects of the cellular
biology of neurons. Hydrogen peroxide (H O ) can react with metals (i.e. copper ions) in
2
2
biological systems via the Fenton reaction to produce hydroxyl radicals (·OH), and H O
2
2
and ·OH can break DNA and cause strong cytotoxicity. To investigate the possible protective
effects of compounds 7 and 8 against apoptosis induced by H O , PC12 cells were treated
2
2
with 200 μM H O , and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
2
2
(
MTT) assays were performed to test cell viability. As shown in Table 1, PC12 cells treated
only with H O exhibited significantly decreased cell viability (~70%). e administration
2
2
of compounds 7 and 8 promoted cell viability to ~82% and ~84%, respectively.
3
. Experimental
3
.1. General experimental procedures
1
13
H NMR and C NMR spectral data were obtained using Bruker Avance 500 MHz and
4
00 MHz spectrometers at 300 K using tetramethylsilane (TMS) as an internal standard
Bruker, Billerica, Massachusetts, USA). HRESIMS was measured with an Agilent 6520
(
Accurate-Mass Q-TOF LC/MS (Agilent, Waldbronn, Germany). Silica gel thin-layer

4
Y.ꢀD. ZANG ET AL.
chromatography (TLC) plates (Qing Dao Marine Chemical Factory, Qingdao, China) were
used to monitor the reaction progress. Column chromatography was performed using
silica gel (100–200 mesh size, Qing Dao Marine Chemical Factory, Qingdao, China). All
commercial chemicals were used as supplied unless otherwise indicated. All reactions were
performed in oven-dried glassware. All yields reported refer to the yields of the isolated
compounds.
3
.2. 3-tert-Butyldiphenylsilyloxy-4-methylnitrobenzene (2)
Compound 1 (153 g, 1 mol) and imidazole (75 g, 1.1 mol) were dissolved in CH Cl (750 ml),
2
2
and a solution of tert-butyldiphenylchlorosilane (302.5 g, 1.1 mol) in CH Cl (250 ml) was
2
2
added dropwise at room temperature (RT). Af er the reaction mixture was stirred for 5
hours at RT, the mixture was washed with water, saturated NaHCO solution, and brine. e
3
organic layers were dried over sodium sulfate, and the solvent was evaporated. e solid was
1
washed with a small quantity of CH OH to obtain light yellow crystal 2 (391 g, 88%). H
3
NMR (500 MHz, CD COCD ) δ 7.80-7.78 (m, 4H), 7.70 (dd, J = 1.5, 8.4 Hz, 1H), 7.53-7.50
3
3
1
3
(
m, 2H), 7.48-7.45 (m, 5H), 7.24 (d, J = 2.3 Hz, 1H), 2.52 (s, 3H), 1.15 (s, 9H). C NMR
(
2
125 MHz, CD COCD ) δ 154.8, 147.3, 138.0, 136.2, 132.3, 132.2, 131.4, 129.1, 116.9, 113.5,
3
3
+
6.8, 20.0, 17.5. HRESIMS: m/z 414.1491 [M + Na] (calcd for C H NO SiNa, 414.1496).
23 25 3
3
.3. 3-tert-Butyldiphenylsilyloxy-4-methylaniline (3)
A mixture of 2 (150 g, 0.38 mol) and 10% palladium on activated charcoal (Pd-C) catalyst
(
15 g) in THF (400 ml) was stirred at ambient temperature under a hydrogen atmosphere
for three hours. e catalyst was removed by filtration, and the solvent was removed by
1
evaporation to obtain white powder 3 (138.5 g, 94%). H NMR (400 MHz, DMSO-d ) δ
6
7
.71-7.69 (m, 4H), 7.48-7.45 (m, 6H), 6.78 (d, J = 8.0 Hz, 1H), 6.04 (d, J = 7.6 Hz, 1H), 5.76
1
3
(
s, 1H), 4.57 (s, 2H), 2.15 (s, 3H), 1.02 (s, 9H). C NMR (100 MHz, DMSO-d ) δ 153.6,
6
1
3
47.3, 134.9, 132.5, 130.7, 130.0, 127.9, 114.2, 107.4, 104.6, 26.4, 19.1, 16.0. HRESIMS: m/z
+
62.1940 [M + H] (calcd for C H NOSi, 362.1935).
2
3
28
3
.4. 3-(tert-Butyldiphenylsilyloxy)-N-(3-methoxyphenyl)-4-methylaniline (4)
In a dry round-bottom flask, 3 (50 g, 1 equiv), m-bromoanisoles (25.6 g, 1equiv), and
,2’-bis(diphenylphosphino)-1,1’-binaphyl (BINAP, 5.18 g, 0.06 equiv) were dissolved in
dry toluene (1000 ml). Pd(OAc) (1.86 g, 0.06 equiv) and CsCO (54.2 g, 1.2 equiv) were
2
2
3
added, and the resulting mixture was gently refluxed under nitrogen until consumption of
the starting materials as indicated by TLC. Af er cooling, the mixture was filtrated and the
solvent was evaporated to give a residue, which was purified on silica gel (petroleum ether/
1
EtOAc = 20:1) to afford 5 (53 g, 82%) as a white solid. H NMR (500 MHz, CD COCD )
3
3
δ 7.76 (d, J = 7.4 Hz, 4H), 7.48-7.46 (m, 2H), 7.44-7.41 (m, 4H), 7.08 (br s, 1H), 7.04 (d,
J = 8.1 Hz, 1H), 6.80-6.77 (m, 1H), 6.54 (d, J = 8.1 Hz, 1H), 6.40 (s, 1H), 6.30 (s, 1H), 6.24
1
3
(
d, J = 8.1 Hz, 1H), 6.12 (d, J = 8.1 Hz, 1H), 3.59 (s, 3H), 2.33 (s, 3H), 1.09 (s, 9H). C
NMR (125 MHz, CD COCD ) δ 161.4, 154.9, 146.0, 142.7, 136.2, 133.4, 132.0, 130.9, 130.5,
3
3
1
28.8, 120.7, 112.2, 109.4, 108.7, 105.7, 103.0, 55.1, 26.9, 20.0, 16.7. HRESIMS: m/z 468.2353
+
[
M + H] (calcd for C H NO Si, 468.2353).
3
0
34
2

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
3
.5. 2-(tert-Butyldiphenylsilyloxy)-7-methoxy-3-methyl-9H-carbazole (5)
Pd(OAc) (26.5 g, 1.1 equiv) was added at room temperature to a solution of 4 (50 g, 1
2
equiv) in THF (500 ml), and the mixture was stirred for 24 hours at 60 °C. e mixture
was filtered, and the solvent was removed by evaporation. Purification by column chroma-
1
tography (petroleum ether/ethyl acetate = 20:1) gave 5 (37.3 g, 75%) as a white solid. H
NMR (500 MHz, CD COCD ) δ 9.70 (br s, 1H), 7.82-7.81 (m, 5H), 7.77 (s, 1H), 7.49-7.46
3
3
(
m, 2H), 7.44-7.41 (m, 4H), 6.84 (s, 1H), 6.72 (d, J = 8.5 Hz, 1H), 6.51 (s, 1H), 3.79 (s, 3H),
1
3
2
1
.54 (s, 3H), 1.13 (s, 9H). C NMR (125 MHz, CD COCD ) δ 159.1, 152.5, 142.2, 140.0,
3 3
36.2, 133.7, 130.9, 128.8, 121.4, 120.6, 118.3, 117.8, 108.2, 101.4, 95.3, 55.6, 26.9, 20.1, 17.9.
+
HRESIMS: m/z 466.2215 [M + H] (calcd for C H NO Si, 466.2197).
3
0
32
2
3
.6. Recycling method of Pd(OAc)2
Recycling palladium powder (10.3 g, 0.9 equiv) was added into AcOH (200 ml) and HNO3
(
0.5 ml), followed by refluxing for 30 min. When the reaction was stopped, the hot brown
solution was filtrated. e solution was concentrated until a little AcOH (~20 ml) was lef .
At room temperature, Pd(OAc) crystals precipitated at the bottom of beaker. rough
filtration, we obtained recycling Pd(OAc) (20 g, 92%).
2
2
3
.7. 7-Methoxy-3-methyl-9H-carbazol-2-ol (6)
A solution of tetrabutylammonium fluoride (TBAF, 33.7 g, 2 equiv) in THF (100 ml) was
added to a stirred solution of 5 (30 g, 1 equiv) in THF (200 ml) at 0 °C. Af er stirring at
room temperature for 2 h, the reaction mixture was quenched by addition of water (300 ml).

e mixture was extracted with EtOAc (3 × 300 ml) and the combined organic layer was
washed with brine, dried over anhydrous Na SO and concentrated in vacuo. e crude
2
4,
product was purified by column chromatography (petroleum ether/EtOAc = 2:1) to afford
1
6
(13.8 g, 94%) as a white solid. H NMR (500 MHz, CD COCD ) δ 9.81 (br s, 1H), 8.10 (s,
3
3
1
H), 7.78 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 6.93-6.92 (m, 2H), 6.72 (d, J = 8.4 Hz, 1H), 3.82
1
3
(
s, 3H), 2.32 (s, 3H). C NMR (125 MHz, CD COCD ) δ 158.8, 154.5, 142.0, 140.8, 121.4,
3 3
+
1
20.2, 118.3, 117.3, 117.1, 107.9, 97.1, 95.5, 55.6, 16.7. HRESIMS: m/z 228.1024 [M + H]
(
calcd for C H NO , 228.1019).
14 14 2
3
.8. O-Methylmurrayamine A (7)
Briefly, 3-methyl-2-butene (7.4 g, 2 equiv) was added at −78 °C to a solution of 6 (10 g,
1
4
equiv) in toluene (100 ml). At the same temperature, titanium (IV) isopropoxide (50 g,
equiv) was added slowly, and the reaction mixture was allowed to warm to RT over a
period of 22 h. e solvent was removed by evaporation, and the residue was diluted with
EtOAc. e solution was washed with water, dried over MgSO , filtered, and concentrated
4
in vacuo. Purification by column chromatography (petroleum ether/ethyl acetate, 5:1) and
1
recrystallization with methanol gave 7 (8.4 g, 65%). H NMR (500 MHz, CD COCD ) δ
3
3
1
0.13 (br s, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.60 (s, 1H), 6.93 (d, J = 2.2 Hz, 1H), 6.88 (d,
J = 9.8 Hz, 1H), 6.75 (dd, J = 8.6, 2.2 Hz, 1H), 5.76 (d, J = 9.8 Hz, 1H), 3.83 (s, 3H), 2.27
1
3
(s, 3H), 1.45 (s, 6H). C NMR (125 MHz, CD COCD ) δ 159.1, 149.5, 142.3, 136.2, 130.0,
3 3

6
Y.ꢀD. ZANG ET AL.
1
21.0, 120.5, 118.7, 118.3, 118.1, 117.8, 108.3, 105.5, 95.7, 76.3, 55.7, 27.9, 16.2. HRESIMS:
+
m/z 294.1491 [M + H] (calcd for C H NO , 294.1489).
1
9
20
2
3
.9. 7-Methoxymurrayacine (8)
To a solution of 7 (8 g, 1 equiv) in a mixed solvent (160 ml) of MeOH–THF–H O (3:3:1),
2
2
,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 12.4 g, 2 equiv) was added. e mixture
was allowed to stir at room temperature for 2 h before being poured into saturated NaHCO3.
e product was extracted with EtOAc and the combined organic layer was washed with

brine, dried over Na SO and concentrated in vacuo. e residue was purified by silica gel
2
4,
column chromatography (petroleum ether/EtOAc = 4:1) to give 8 (7.2 g, 86%) as a yellow
1
solid. H NMR (500 MHz, CD COCD ) δ 10.68 (br s, 1H), 10.46 (s, 1H), 8.24 (s, 1H), 7.79
3
3
(d, J = 8.6 Hz, 1H), 6.99 (d, J = 2.2 Hz, 1H), 6.93 (d, J = 9.9 Hz, 1H), 6.85 (dd, J = 8.6, 2.2 Hz,
1
3
1
1
9
H), 5.92 (d, J = 9.9 Hz, 1H), 3.86 (s, 3H), 1.55 (s, 6H). C NMR (125 MHz, CD COCD ) δ
3 3
88.5, 160.2, 154.6, 143.3, 141.7, 130.9, 121.6, 119.2, 118.5, 118.2, 117.6, 110.9, 109.5, 105.4,
6.3, 77.9, 55.8, 27.7. HRESIMS: m/z 308.1290 [M + H] (calcd for C H NO , 308.1281).
+
1
9
18
3
3
.10. Neuroprotective activity assay
PC12 cells were maintained routinely in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 5% horse serum (ES) and 5% fetal bovine serum (FBS) at 37 °C in
a humidified atmosphere of 5% CO and 95% air. e medium was changed every two
2
days, and cells were seeded on poly-L-lysine (PLL)-coated plates at an appropriate density
according to each experimental scale. Af er 24 h of subculture, the cells were switched to
fresh medium for treatment. e attached cells were exposed to 200 μM H O in the pres-
2
2
ence or absence of compounds for 24 h. Cell viability was determined by MTT assay. Cells
were incubated with 5 mg/ml MTT (10 μl) at 37 °C for 4 h. e formazan crystals were
dissolved, and absorbance was determined at 570 nm using a Microplate Reader (ermo,
Germany). e cell viability was expressed as a percentage of the optical density (OD) value
of the control cultures.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is study was financially supported by the National Natural Science Foundation of China [grant
number 21272278] and [NNSFC 81630094].
References
[
[
[
1] T.S. Wu, Phytochemistry 30, 1048 (1991).
2] Y. Tachibana, H. Kikuzaki, N.H. Lajis, and N. Nakatani, J. Agric. Food Chem. 51, 6461 (2003).
3] O.P.S. Patel, A. Mishra, R. Maurya, D. Saini, J. Pandey, I. Taneja, K.S.R. Raju, S. Kanojiya, S.K.
Shukla, M.N. Srivastava, M. Wahajuddin, A.K. Tamrakar, A.K. Srivastava, and P.P. Yadav, J.
Nat. Prod. 79, 1276 (2016).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
[
[
[
4] N. Ruangrungsi, J. Ariyaprayoon, G.L. Lange, and M.G. Organ, J. Nat. Prod. 53, 946 (1990).
5] R. Hesse, A. Jäger, A.W. Schmidt, and H.J. Knölker, Org. Biomol. Chem. 12, 3866 (2014).
6] R. Hesse, O. Kataeva, A.W. Schmidt, and H.J. Knölker, Chem. Eur. J. 20, 9504 (2014).