Natural terpenoid glycosides with: In vitro / vivo antithrombotic profiles from the leaves of Crataegus pinnatifida

Article abstract of DOI:10.1039/c7ra10768d

Two norditerpenoids (1-2) with unique carbon skeletons, four sesquiterpenoids (3-6) and nine nor-sesquiterpenoids (7-15) were isolated from the leaves of Crataegus pinnatifida and evaluated as possessing antithrombotic activities in vitro/vivo. Their structures with absolute configurations were determined via a combination of spectroscopic data, chemical methods, and quantum-chemical calculations (ECD, NMR, and OR data). Compound 3 showed an inhibitory effect on ADP induced platelet aggregation in vitro, which is mediated through the response to the specific receptor of P2Y₁₂ by docking results. Compound 3 also clearly prolonged the time to form thrombocytes induced by FeCl₃, in the caudal vessels of zebrafish.

Full text of DOI:10.1039/c7ra10768d

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Natural terpenoid glycosides with in vitro/vivo
antithrombotic profiles from the leaves of
Cite this: RSC Adv., 2017, 7, 48466
Crataegus pinnatifida†
a
Pin-Yi Gao,ab Ling-Zhi Li, Ke-Chun Liu,c Chen Sun,c Xue Sun,a Ya-Nan Wua
*
*
a
and Shao-Jiang Song
Two norditerpenoids (1–2) with unique carbon skeletons, four sesquiterpenoids (3–6) and nine nor-
sesquiterpenoids (7–15) were isolated from the leaves of Crataegus pinnatifida and evaluated as
possessing antithrombotic activities in vitro/vivo. Their structures with absolute configurations were
determined via a combination of spectroscopic data, chemical methods, and quantum-chemical
calculations (ECD, NMR, and OR data). Compound 3 showed an inhibitory effect on ADP induced
platelet aggregation in vitro, which is mediated through the response to the specific receptor of P2Y12 by
docking results. Compound 3 also clearly prolonged the time to form thrombocytes induced by FeCl3, in
the caudal vessels of zebrafish.
Received 29th September 2017
Accepted 9th October 2017
DOI: 10.1039/c7ra10768d
rsc.li/rsc-advances
decades in Europe, China and the United States.6,7 An extract of
hawthorn leaves, Yixintong medicinal preparations, is one of
Introduction
the most effective drugs available for blood-activating and
stasis-dissolving.8,9 The avonoids have been considered as the
only effective component until terpenoids (monoterpenoid and
sesquiterpenoid) with antithrombotic activity were obtained
recently from the leaves of C. pinnatida.10–13 As a new effective
ingredient, the terpenoids exhibit potential antithrombotic
activity both in vitro and in vivo.14
In continuing the search for antithrombotic agents, two
novel norditerpenoids with unique carbon skeletons were iso-
lated as the rst diterpenoids from the leaves of the title plant,
together with two new sesquiterpenoids (3, 4) and 11 known
ones (5–15). The absolute congurations of 1–4 were deter-
mined by producing of Rh2(OCOCF3)4 complexes or Mosher
ester, and by comparison of ECD, NMR, and optical rotation
(OR) data with the calculated values. In this report, the isola-
tion, structure elucidation, and in vitro and in vivo antith-
rombotic evaluation of the isolates (Fig. 1) are described.
Abnormal thrombosis is a key factor in serious cardiovascular
diseases (CVDs) that are among the leading killers around the
world.1 The majority of cardiovascular diseases, including acute
coronary syndrome, ischemic stroke and peripheral vascular
diseases, are associated with thrombotic disorders.2 Platelet
activation in atherosclerotic arteries is central to the develop-
ment of arterial thrombosis. Therefore, the inhibition of
platelet aggregation must occur to prevent a thrombotic event.3
Current antithrombotic discovery efforts target compounds that
have improved therapeutic indices compared to the standard of
care.4 This fact has stimulated great interest in the search for
natural bioactive compounds (NBC) from herbal drugs or herbal
medicinal preparations that are highly efficacious in thrombin
reduction while having minimal or reduced bleeding liabili-
ties.2,5 Research on novel bioactives and drugs with different
mechanisms of action and increased efficacy, and low toxicity is
highly desired. The leaves of Crataegus pinnatida (hawthorn
leaves) have been used to treat cardiovascular disease for several
Results and discussion
aKey Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, People's Republic of China. E-mail: songsj99@163.com;
lilingzhijessie@163.com
The ethanol extract from the leaves of C. pinnatida was sepa-
rated and puried by macroporous resin, polyamide, silica gel,
C18 reversed silica gel and semipreparative HPLC to afford two
bCollege of Pharmaceutical and Biotechnology Engineering, Institute of Functional norditerpenoids (1–2), 13 sesquiterpenoids (3–15). The known
compounds (5–15) were identied as shnyegenin B (5),12
Molecules, Shenyang University of Chemical Technology, Shenyang 110142, People's
Republic of China
shnyeside B (6),12 (3S,5R,6R,7E,9S)-megastiman-7-ene-3,5,6,9-
cBiology Institute of Shandong Academy of Sciences, Jinan, People's Republic of China
tetrol (7),12 euodionosides D (8),13 (6R,9R)-3-oxo-a-ionol-9-O-b-
† Electronic supplementary information (ESI) available: NMR, HRESIMS, ECD
D-glucopyranoside (9),13 (6S,7E,9R)-6,9-dihydroxy-4,7-mega-
and Rh2(OCOCF3)4-ICD spectra of compounds; details of calculated and
experimental ECD, 13C NMR and ORs, MTPA esters, acid hydrolysis and in vitro
and in vivo antithrombotic assays. See DOI: 10.1039/c7ra10768d
stiymadien-3-one-9-O-[b-D-xylopyranosy-b-D-glucopyranoside]
(10),12 linarionoside (11),13 linarionoside (12),12
A
B
48466 | RSC Adv., 2017, 7, 48466–48474
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
Table 1 1H and 13C NMR date of compounds 1 and 2 (DMSO-d6)
1a
2a
dH, mult.
dH, mult.
Position dC
Type (J in Hz)
dC
Type (J in Hz)
1
2
36.5
C
75.3
37.1
CH2 3.65 m
41.0
21.0
31.0
CH2 a 1.30 m
b 1.40 m
CH2 1.54 m
CH
1.52 m
3
4
24.0
CH2 a 1.55 m
b 2.19 m
CH2 a 2.04 m
134.4 CH
5.54 m
b 2.30 m
5
6
7
132.1
140.8
45.2
C
C
133.6
141.7 CH
118.8 CH
C
6.28 d (15.3)
5.54 m
CH2 2.89 m
8
9
10
11
67.0
138.6
124.2 CH
84.9 CH
CH
C
3.80 m
58.2
86.0
38.6
37.1
CH
C
CH
2.48 m
5.49 d (9.6)
5.23 d (9.6)
1.96 m
CH2 a 1.45 m
b 1.57 m
12
13
14
15
16
17
151.7 CH
7.26 s
46.7
179.5
22.5
12.7
23.8
C
C
128.6
174.5
28.2
C
C
CH3 1.16 d (6.0)
CH3 1.75 s
CH3 1.25 s
CH2 3.51 m
CH3 0.82 s
CH3 0.88 s
28.1
70.7
CH2 a 3.90 d (10.8) 64.6
b 4.48 d (10.8)
18
19
10
14.7
10.9
103.6 CH
74.2
77.6
70.8
77.6
61.9
CH3 1.57 s
CH3 1.78 s
19.5
CH3 0.94 s
4.17 d (7.5)
2.90 m
3.11 m
3.08 m
3.10 m
103.4 CH
4.16 d (7.8)
2.92 m
3.12 m
3.09 m
3.10 m
20
CH
CH
CH
CH
74.3
77.5
70.8
77.5
61.9
CH
CH
CH
CH
30
40
50
Fig. 1 The structures of compounds 1–15.
60
CH2 a 3.33 m
b 3.60 m
CH2 a 3.33 m
b 3.60 m
Measured at 150 MHz for 13C in DMSO-d6, and measured in 600 MHz
for 1H in DMSO-d6, (s) singlet, (d) doublet, (m) multiplet.
a
linarionoside C (13),12 3,9-dihydroxy-5-megastigmen-3-O-[b-D-
xylopyranosy-b-D-glucopyranoside] (14),12 pinnatidanoside C
(15)14 by comparison of their spectroscopic data with literature
data (ESI-E.2.†).
obtained from an HMBC experiment. The key correlations of 1
(Fig. 2), dH 1.78 (19-Me)/dC 151.7 (C-12), 128.6 (C-13), 174.5 (C-
14), dH 5.23 (H-11)/dC 124.2 (C-10), 151.7 (C-12), 128.6 (C-13)
and dH 7.26 (H-12)/dC 84.9 (C-11), 128.6 (C-13), 174.5 (C-14),
10.9 (C-19), suggested the presence of an a, b unsaturated
lactone. The other key correlations of 1 around dH 0.82 (15-Me),
0.88 (16-Me), 3.90 and 4.48 (17-CH2OH), 1.57 (18-Me) coupling
with above part suggested the presence of the C19-norditerpe-
noid skeleton. In addition, the glycosidic site was established by
a HMBC correlation from H-10 (dH 4.17) and C-17 (dC 70.7). The
geometry of the double bond at C-9 was assigned as E on the
Compound 1, a colorless oil, was assigned the molecular
formula of C25H38O9 as determined by the HRESIMS ion at m/z
1
483.2582 [M + H]+ (calc. 483.2589). The H NMR data (Table 1)
showed the presence of four tertiary methyls at dH 0.82 (3H, s),
0.88 (3H, s), 1.57 (3H, s) and 1.78 (3H, s), a pair of oxygenated
methylene protons at dH 3.90 (1H, d, J ¼ 10.8 Hz) and 4.48 (1H,
d, J ¼ 10.8 Hz), two oxygenated methine protons at dH 3.80 (1H,
m) and 5.23 (1H, d, J ¼ 9.6 Hz), and there were two olenic H-
atoms which appeared to be at two double bonds separately
5.49 (1H, d, J ¼ 9.6 Hz) and 7.26 (1H, s). In addition, an
anomeric proton signal appeared to be at 4.17 (1H, d, J ¼
7.5 Hz), while six oxygenated H-atoms at dH 2.90–3.60 indicated
the presence of a sugar moiety. The 13C NMR spectrum (Table 1)
exhibited 25 carbon resonances, from which, four methyls (dC
10.9, 14.7, 28.1, 28.2), four methylenes (dC 21.0, 31.0, 41.0, 45.2),
a quaternary carbon (dC 36.5), three carbons bearing oxygen (dC
67.0, 70.7, 84.9), three double bond groups (dC 124.2, 128.6,
132.1, 138.6, 140.8, 151.7), one ester carbonyl group (dC 174.5),
and a sugar group (dC 61.9, 70.8, 74.2, 77.6, 77.6 and 103.6) were
identied, which suggested that compound 1 is a norditerpene
glycoside. Further information about the 2D structure of 1 was
basis of the upeld shi of the methyl carbon signal in the 13
C
NMR, dC 14.7 for C-18,15 which was conrmed by NOE correla-
tions with H-8 and H-10, as well as H-11 and H3-18 (Fig. 3).
The absolute conguration of the norditerpenoid genin (10)
obtained from acid hydrolysis of 1, was established by calcula-
tions of its ECD data,16,17 and the Bulkiness Rule for Rh2(-
OCOCF3)4 complexes of
a
secondary alcohol.18,19
A
conformational search (MMFFs force eld) for 10a and its
enantiomer 10b led to 12 conformers, respectively, followed by
geometric optimization of each one.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 48466–48474 | 48467

View Article Online
RSC Advances
Paper
Fig. 2 HMBC correlations (arrows) of 1–3, and 1H–1H COSY (bold
lines) of 4.
Fig. 4 Experimental and calculated ECD for 1 and 2.
Fig. 3 Key NOESY correlations (H–H) of 1 and 3.
methyls (dC 12.7, 19.5, 22.5, 23.8), two methylenes (dC 24.0,
37.1), three methines (dC 37.1, 38.6, 58.2), a quaternary carbon
(dC 46.7), four carbons bearing oxygen (dC 64.6, 75.3, 86.0), two
double bond groups (dC 118.8, 133.6, 134.4, 141.7), one ester
carbonyl group (dC 179.5), and a sugar group (dC 61.9, 70.8, 74.3,
77.5, 77.5 and 103.4) were identied, which suggested that
compound 2 has a norditerpenoid skeleton. Analysis of the 1H-,
13C-NMR and HSQC data helped us to assign H- to their bonded
C-atoms (Table 1), and further information about the 2D
structure of 2 (Fig. 2) was obtained from the HMBC experiment.
The presence of bicyclic [2.2.1] rigid rings of 2 was supported by
the long-range HMBC interactions dH 1.25 (16-Me)/dC 58.2 (C-8),
86.0 (C-9), 38.6 (C-10); dH 3.51 (17-CH2OH)/dC 86.0 (C-9), 38.6 (C-
10), 37.1 (C-11); dH 0.94 (18-Me)/dC 58.2 (C-8), 37.1 (C-11), 46.7
(C-12), 179.5 (C-13). The presence of the C9 side chain was
established by other key correlations around dH 1.16 (14-Me),
1.75 (15-Me), 5.54 and 6.28 (olenic H – 4, 6, 7). In addition, C-8
was assigned to the linkage site of above two moieties due to the
key correlations between dH 2.48 (H-8) and dC 141.7 (C-6), 118.8
(C-7), 86.0 (C-9), 37.1 (C-11), 46.7 (C-12), 23.8 (C-16), 19.5 (C-18).
The location of the sugar moiety was determined by the HMBC
correlations between H-10 (dH 4.16) and C-1 (dC 70.3) (Fig. 2).
Furthermore, In the 1H NMR data, the vicinal coupling constant
J(6,7) of 15.3 Hz suggested a trans relationship between the
protons H-6 and H-7. The geometry of the double bond at C-4
was assigned as E on the basis of the upeld shi of the
The optimized conformers subjected to ECD calculations in
MeOH (CPCM) using the B3LYP functional and the 6-311+G(d)
basis set for TDDFT. The nal calculated ECD was obtained as
the result of the Boltzmann-weighted average. Comparison of
the experimental ECD curve 10 and calculated ECD curves
(Fig. 4) permitted the assignment of the absolute conguration
of 10 as 8R, 11R (ESI-Table 1†). In addition, the Rh2(OCOCF3)4-
induced ECD experiment on the 8-OH of 10 also conrmed the
above result (negative E band at 340 nm in spectra Fig. S24†).
The D-glucose was identied by acid hydrolysis of 1 then
comparison with an authentic sample by an HPLC analysis
using OR detector (ESI-Fig. 3†). Consequently, the structure of
compound 1 was established and named norhawthornoid A.
Compound 2 was obtained as a colorless oil, and its HRE-
SIMS exhibited a sodium adduct ion peak at m/z 471.2588 [M +
H]+ (calc. 471.2589), corresponding to the molecular formula
1
C
24H38O9. In the H NMR spectrum (Table 1) of 2 there were
resonances attributable to four methyls at dH 1.75 (3H, s), 1.25
(3H, s), 0.94 (3H, s) and 1.16 (3H, d, J ¼ 6.0 Hz), two pair of
oxygenated methylene protons at dH 3.51 (2H, m) and 3.65 (2H,
m), three olenic H-atoms at dH 6.28 (1H, d, J ¼ 15.3 Hz), 5.54
(2H, m), an anomeric proton at dH 4.16 (1H, d, J ¼ 7.8 Hz), and
six oxygenated H-atoms at dH 2.92–3.60. The 13C NMR spectrum
(Table 1) exhibited 24 carbon resonances, from which, four
48468 | RSC Adv., 2017, 7, 48466–48474
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
methyl carbon signal in the 13C NMR spectrum (dC 12.7 for which, four methyls (dC 22.8, 24.0, 24.0, 24.0), ve methylenes
C-15).15
(dC 24.0, 27.1, 27.3, 27.3, 31.1), two methines (dC 43.7, 85.4), two
The absolute conguration of the genin 20 obtained from quaternary carbons (dC 78.4, 85.2), a double bond group (dC
acid hydrolysis of 2, was established by comparison of the 121.5, 133.9), and a sugar group (dC 61.9, 71.1, 74.4, 77.2, 77.8
experimental and calculated ECD and NMR data.16,17 The and 98.0) were identied, which suggested that compound 3 is
experimental ECD curves of 20 (Fig. 4) matched well with the a sesquiterpenoid glycoside. In the HMBC spectrum (Fig. 2), the
theoretically calculated ECD data of 20b using the TDDFT key correlations of 3 (Fig. 2), dH 1.07 (12-Me)/dC 78.4 (C-1), 85.4
method at the B3LYP/6-311+G (d) level (ESI-Table 2†). Thus, the (C-2), 24.0 (C-13); dH 1.16 (13-Me)/dC 78.4 (C-1), 85.4 (C-2), 22.8
absolute conguration of 20 was determined to 8R/9S/10R/12S. (C-12); dH 1.02 (14-Me)/dC 27.3 (C-4), 85.2 (C-5), 43.7 (C-6), and
Furthermore, the conguration of C-2 was indicated by calcu- dH 1.59 (15-Me)/dC 121.5 (C-8), 133.9 (C-9), 31.1 (C-10), suggested
lated NMR data. For this, our high-accuracy 13C-NMR calcula- the presence of the bisabolane sesquiterpene skeleton. A long-
tion of the two possible stereoisomers (2R/8R/9S/10R/12S or 2S/ range correlation between H-2 (dH 3.82) and C-5 (dC 85.2) sug-
8R/9S/10R/12S) indicated that the potential one is 2S/8R/9S/10R/ gested the presence of 2,5-ether moieties. In addition, the
12S (R2 ¼ 0.9875, AveDev ¼ 3.5) (ESI-Fig. 5†). Finally, the glycosidic site (C-1) was established unambiguously by an
D conguration of the sugar moiety was determined by acid HMBC experiment, in which a long-range correlation between
hydrolysis followed by an HPLC analysis with an authentic H-10 (dH 4.37) and C-17 (dC 78.4) was observed (Fig. 2). The
sample using an OR detector (ESI-Fig. 3†). On the basis of the relative conguration of 3 was determined by the key NOE
above evidence, the structure of 2 was determined and named correlations between H-2/H-6, H-2/H2-7, which suggested that
norhawthornoid B.
they were located on the same side of the tetrahydrofuran (THF)
Compound 3, a colorless oil, showed the molecular formula ring (C2–C5) (Fig. 3).
of C21H36O7 from the HRESIMS ion at m/z 401.2536 [M + H]+
The absolute conguration of the sesquiterpene skeleton (30)
1
(calc. 401.2534). The H and 13C NMR data (Table 2), assigned obtained by acid hydrolysis was established by comparison of
by HSQC and HMBC spectra, showed signals of four tertiary the experimental and calculated OR.20 Due to the above NMR
methyls at dH 1.59 (3H, s), 1.16 (3H, s), 1.07 (3H, s), 1.02 (3H, s), data, the conguration of 30 was identied as one of the two
an oxygenated methine proton at dH 3.82 (1H, m), an olenic isomers (2R, 5R, 6S or 2S, 5S, 6R). A conformational search using
proton at dH 5.35 (1H, br s). Furthermore, an anomeric proton the MMFFs force eld for the (2R, 5R, 6S)-stereoisomer and its
signal at 4.37 (1H, d, J ¼ 7.8 Hz), and six oxygenated H-atoms at enantiomer led to the identication of 11 conformers, followed
dH 2.86–3.62 indicated the presence of a sugar moiety. The 13C by geometric optimization of each one. The optimized
NMR spectrum (Table 2) exhibited 21 carbon resonances, from conformers were subjected to OR calculations in MeOH (CPCM)
Table 2 1H and 13C NMR date of compounds 3 and 4 (DMSO-d6)
3a
4a
Position
dC
Type
dH, mult. (J in Hz)
dC
Type
dH, mult. (J in Hz)
1
2
3
4
5
6
7
8
78.4
85.4
24.0
27.3
85.2
43.7
27.1
121.5
133.9
31.1
27.3
22.8
24.0
24.0
24.0
98.0
74.4
77.8
71.1
77.2
61.9
C
77.4
80.2
21.2
38.2
72.5
42.3
24.5
121.5
133.6
31.1
26.9
23.7
23.8
23.8
23.5
97.5
74.0
77.2
70.6
77.0
61.5
C
CH
CH2
CH2
C
CH
CH2
CH
C
CH2
CH2
CH3
CH3
CH3
CH3
CH
CH
CH
CH
CH
CH2
3.82 m
1.71 m
1.52 m
CH
CH2
CH2
C
CH
CH2
CH
C
CH2
CH2
CH3
CH3
CH3
CH3
CH
CH
CH
CH
CH
CH2
3.91 m
1.71 m
1.53 m
1.96 m
1.92 m
5.35 br s
1.88 m
1.88 m
5.34 br s
9
10
11
12
13
14
15
10
20
30
40
50
60
1.95 m
1.65 m
1.07 s
1.16 s
1.02 s
1.59 s
4.37 d (7.8)
2.86 m
3.12 m
3.04 m
3.06 m
a 3.30 br d (11.4)
b 3.62 br d (11.4)
1.88 (2H, m)
1.67 (2H, m)
1.10 s
1.10 s
0.83 s
1.59 s
4.34 d (7.5)
2.89 m
3.11 m
3.02 m
3.04 m
a 3.35 br d (10.8)
b 3.62 br d (10.8)
a
1
Measured at 150 MHz for 13C in DMSO-d6, and measured in 600 MHz for H in DMSO-d6, (s) singlet, (d) doublet, (m) multiplet.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 48466–48474 | 48469

View Article Online
RSC Advances
Paper
using the B3LYP functional and the 6-311+G(d) basis set for
DFT. Final calculated ORs were obtained as the result of the
Boltzmann-weighted average (ESI-Table 6†). From these results,
the negative calculated ORs ([a]D20 ꢀ8.7) of the (2R, 5R, 6S)-
stereoisomer was a better t to the experimental OR value
([a]D20 ꢀ2.8, c 0.4, MeOH) than the positive one ([a]2D0 +8.7). In
addition, the D-glucosyl moiety was conrmed by acid hydro-
lysis of 3 and then comparison with an authentic sample.
Consequently, the structure of compound 3 was given the trivial
name pinnatidanoside F.
Compound 4 was obtained as colorless oil and its molecular
1
formula was determined as C21H38O8 by HRESIMS. In the H
NMR spectrum (Table 2), the presence of four tertiary methyls at
dH 1.59 (3H, s), 1.10 (6H, s), 0.83 (3H, s), a oxygenated methine
proton at dH 3.91 (1H, m), a olenic proton at dH 5.34 (1H, br s),
were revealed, in addition, the anomeric proton signal at 4.34
(1H, d, J ¼ 7.5 Hz) and six oxygenated H-atoms at dH 2.89–3.62
demonstrated the presence of a saccharide group. The 13C NMR
spectrum (Table 2) exhibited 21 carbon resonances, from
which, four methyls (dC 23.5, 23.7, 23.8, 23.8), ve methylenes
(dC 21.2, 24.5, 26.9, 31.1, 38.2), two methines (dC 42.3, 80.2), two
quaternary carbons (dC 72.5, 77.4), a double bond group (dC
121.5, 133.6), and a sugar group (dC 61.5, 70.6, 74.0, 77.0, 77.2
and 97.5) were identied, which suggested that compound 4
has a sesquiterpenoid skeleton closed to the structure of 3. A
careful comparison of the NMR spectroscopic data of 4 with that
of 3 (Table 2) suggested that they have a closed sesquiterpenoid
skeleton. Following analysis of their NMR data, the C2 and C5
atoms in 4 exhibiting ꢀ5.2 and ꢀ12.7 ppm deviations respec-
tively indicated that the THF ring was replaced by a 2,5-dihy-
droxy moiety, which was conrmed by HRESIMS data (Fig. 1).
The multi-method combination of Mosher's method and
comparison of the experimental and calculated NMR and OR
values21–23 was presented here for support in the study of the
absolute conguration of the sesquiterpene genin (40). Firstly,
according to the MTPA shielding/deshielding effects of the two
esters, the DdH (S–R) values (ESI-Fig. 4†) revealed a 2R congu-
ration for 40. Then, the congurations of C-5 and C-6 were
indicated by calculated NMR and OR data. For this, our high-
accuracy 13C-NMR calculation of the four possible stereoiso-
mers (2R/5S/6S, 2R/5S/6R, 2R/5R/6S or 2R/5R/6R) indicated that
the potential one is 2R/5S/6S (R2 ¼ 0.9980 larger than others)
(Fig. 5, ESI-Fig. 6 and 7†). Meanwhile, the OR value of the (2R/
5S/6S)-stereoisomer predicted to be ꢀ21.8 at the DFT level was
in agreement with the experimental OR value ([a]D20 ꢀ7.5, c 0.2,
MeOH). Furthermore, HPLC analysis of the acidic hydrolysate
of 4 showed that the sugar moiety was a D-glucose. Thus, the
structure of compound 4 was named pinnatidanoside G.
The adenosine diphosphate (ADP), a granular content, act as
agonists to activate more platelets and recruit them onto the
subendothelial matrix, which contains cells, collagen, and von
Willebrand factor, is exposed and tether to the site of injury.
Our current study identied a key mechanism underlying
antithrombotic effect of terpenoids from hawthorn leaves,
showing that they blocked platelet activation by inhibiting the
ADP pathway.14,24 In this study, all the isolates were tested in
vitro for their antithrombotic effects by ADP induced platelet
13
Fig. 5 Experimental and calculated C chemical shifts of 40a.
aggregation experiment.25 The inhibitory activity of individual
compounds is summarized in Table 3. Compounds 1–4, 8, 11
and 15 showed potent anti-platelet activities. Compound 3
a sesquiterpenoid displayed the best inhibitory activity in
particular, inhibiting platelet aggregation by 84.00 ꢁ 4.00% at
the nal concentration of 0.25 mg mLꢀ1 during the test interval.
These results indicated that sesquiterpenoid inhibit platelet
aggregations induced by ADP agonists, and thus it may have
benecial potential for the prevention of platelet-involved
cardiovascular disease.
The P2 family of receptors mediates the platelet response to
ADP, and mammalian platelets express three ADP subtypes
receptors, namely, P2Y1, P2Y12, and P2X1.24,26 Among them, the
P2Y12 receptor (P2Y12R), a member of the P2Y purinergic GPCR
family stimulated by ADP, is one of the most important clinical
drug targets for inhibition of platelet aggregation.26 Clopidogrel
inhibits platelet activation through irreversible binding to the
P2Y12 ADP receptor on platelet membrane. Therefore,
compound 3, the most potent inhibitor of the ADP-induced
platelet aggregation, was investigated as an antagonist of the
direct-acting P2Y12 receptor by molecular modeling studies
(Fig. 6). Also AZD1283 (the co-crystal ligand from crystal protein
PDB code 4PXZ) was used as a positive control.26 These inves-
tigations indicate that (a) the 30-OH and 1-O- of 3 were in close
ꢀ
Table 3 Antiplatelet aggregation activity of compounds 1–15 (x ꢁ SD,
n ¼ 3)
Samples
Inhibition rate (%)
Samples
Inhibition rate (%)
1
2
3
4
5
6
7
8
69.20 ꢁ 6.35
74.00 ꢁ 4.50
84.00 ꢁ 4.00
76.25 ꢁ 7.25
<50
9
<50
<50
68.25 ꢁ 1.35
64.55 ꢁ 1.75
<50
10
11
12
13
14
15
<50
64.25 ꢁ 2.35
54.05 ꢁ 5.25
76.95 ꢁ 2.45
82.65 ꢁ 4.65
74.95 ꢁ 10.45
Aspirin
48470 | RSC Adv., 2017, 7, 48466–48474
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
150 mg mLꢀ1. Next, different doses of each compound were
investigated using this thrombosis model. Compounds 1–3
clearly prolonged the time to form a thrombus in the tested
zebrash (Fig. 7). Among them, the thrombosis time of 3 was
around or over 2.2-fold that of the model control and was the
most potential.
Experimental
General experimental procedures
Optical rotations were obtained using an Autopol IV automatic
polarimeter (Rudolph Research Analytical). UV spectra were
recorded on a Shimadzu double-beam 210A spectrometer. The
ECD spectra were measured with a BioLogic MOS-450 spec-
tropolarimeter. NMR spectra were recorded on a Bruker ARX-
400 and 600 spectrometer with TMS as internal standard in
dimethyl sulfoxide-d6 (DMSO-d6). Mass spectra were deter-
mined on a HRESI-MS: MicroTOF spectrometer (Bruker Dal-
tonics, CA). TLC silica gel (GF254), silica gel (200–300 mesh) and
polyamide (200–400 mesh) were purchased from Qingdao
Marine Chemical Co., China. Reversed-phase C18 silica gel (ODS
70–80 mm Merck, Germany). Solvents were of industrial purity
and distilled prior to use.
Fig. 6 Amino acids interacting with compound 3 and AZD1283.
Hydrogen bonds are shown as green dots.
˚
proximity to the catalytic residues Tyr 109 (2.97 A) and Arg 256
˚
(5.01 A) and underwent hydrogen bonding interactions. Also,
the same hydrogen bonding interactions were oriented between
˚
AZD1283 and Tyr 109 and Arg 256 (distance 3.29 and 3.18 A)
(Fig. 7) (b) both AZD1283 and compound 3 were oriented in an
aromatic pocket consisting of Tyr 109, Phe 252, Arg 256, and Lys
280. In addition, Tyr 105, Asn 191, Gln 195, Tyr 259, and Leu 276
are also considered as key active residues interacting with the
natural ligand. In addition, the results of the ADP-induced
platelet aggregation experiment indicated that these isolates
may block platelet activation by inhibiting P2Y12R.
Plant material and animals
The leaves of Crataegus pinnatida (Rosaceae) were gathered
from Liaoning in November 2015, and authenticated by phar-
macognosy Prof. Lu Jin-Cai. A voucher specimen (HY151108) is
kept in the Nature Products Laboratory of Shenyang Pharma-
ceutical University, Shenyang, China.
More recently, the genetic screening of natural products has
been developed for an FeCl3-induced thrombosis model in
zebrash in vivo, which overcomes the limitations of the mouse
model.27,28 Prior to investigating the antithrombotic effect, we
determined the viability of zebrash embryos pretreated with
increasing doses of isolates (50 and 150 mg mLꢀ1) for 48 h. The
results obtained showed that compounds 1–4, 8, 11 and 15 did
not inuence the embryo viability in cultured zebrashes up to
Sprague–Dawley (SD) rats (8 weeks, 220–240 g) were obtained
from Liao Ning Chang Sheng Biotechnology Co., Ltd. They were
housed in a conventional animal facility with free access to food
and water in a temperature and relative humidity controlled
environment under a 12 h light/dark schedule. Adult zebrash
(AB) were obtained from Biology Institute of Shandong Academy
of Sciences (Jinan, China), and sh were maintained with a 14/
10 h light/dark cycle. Zebrash were fed with Tetramin granu-
lated food and live brine shrimps (Artemia nauplii). Embryos
were obtained from natural spawning, which was induced in
the next morning aer mating, and were placed in the embryos
medium (5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, 0.16 mM
ꢂ
MgSO4) under 28 C, to feed them for three days post fertiliza-
tion (dpf). All animal procedures were carried out according to
the Regulations of Experimental Animal Administration issued
by State Committee of Science and Technology of China, and
approved by the institutional ethical committee (IEC) of She-
nyang Pharmaceutical University.
Extraction and isolation
Exhaustive extraction of C. pinnatida leaves (15 kg) with 50%
ethanol (total 270 L) followed by reux two times, which was
prepared according to Pharmacopoeia of People's Republic of
China (Yixintong medicinal preparations in page 396–397). The
concentrated crude extract (2.0 kg) was separated by macro-
porous resin (20 kg) with EtOH/H2O (v/v ¼ 0 : 100, 10 : 90,
Fig. 7 Antithrombotic activity of compounds 1–4, 8, 11 and 15 on
zebrafish.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 48466–48474 | 48471

View Article Online
RSC Advances
Paper
60 : 40, 95 : 5). The 60% ethanol fraction (280 g) was further time evolution was monitored until stationary (10–15 min aer
separated by polyamide (2 kg) column chromatography (EtOH/ mixing). The inherent ECD was subtracted. The observed sign of
H2O 20 : 70, 40 : 60, 60 : 40, 95 : 5) to provide 4 fractions the E band at about 350 nm in the induced ECD spectrum was
(Frs. a–d). Fr. a (26 g) was subjected to silica gel (200 g) chro- correlated to the absolute conguration of the C-8 of 10
matography eluting with a CH2Cl2–EtOH (v/v ¼ 30 : 1, 15 : 1, (secondary alcohol moiety), as well as the C-3 of 5 (tertiary
10 : 1, 8 : 1) and further puried by ODS (50 g) column chro- alcohol moiety).29
matography with EtOH : H2O (3 : 4) to yield compounds 8
(12 mg), 10 (23 mg), 13 (23 mg), 14 (9 mg) and 15 (15 mg) which
were further puried by preparative HPLC. And the same
Computational methods
Quantum chemical ECD calculation. Initial structures were
constructed based on information of NOESY correlations, and
conformational analysis was performed by using the MMFF
molecular mechanics force eld. Further optimization of the
structures in methanol was performed using the Gaussian 09
program.30 The optimized structures were shown in ESI-Tables 1
and 2.† Optimization was conrmed by computation of
frequency. Conformational distribution of the optimized
structures was investigated at B3LYP/6-31G (d) and suggested
the major conformers (>98%). Energies of the geometric
conformations in MeOH were calculated at the B3LYP/6-311+G
(d) level. Solvent effects were considered by using the
conductor-like polarizable continuum model (CPCM). The
hybrid B3LYP functionals were choosed to run the TDDFT
calculations, solving for 30 states for per molecule of 10 and 20.
The ECD spectra were generated using the program SpecDis
with a half-bandwidth of 0.30 eV (for 10 and 20). Aer
hypsochromic-shiing 8 nm (for 10) and ꢀ20 nm (for 20) by UV
correction, in the 190–400 nm region, the theoretically calcu-
lated ECD spectra of 10a and 20b were in good agreement with
the experimental ECD spectra of 10 and 20 (Fig. 3).
method was employed to obtain compounds 3 (27 mg), 4
(11 mg) and 6 (22 mg), 9 (16 mg), 11 (53 mg) and 12 (45 mg) from
the next fraction (Frs. b, 35 g). Compounds 1 (9 mg), 2 (12 mg), 5
(28 mg) and 7 (19 mg) from Frs. c, (23 g).
Norhawthornoid A (1). Colorless oil; [a]D20 ꢀ3.5, (c 0.15,
MeOH); UV (MeOH) lmax (log 3) 208 (2.21) nm; ECD (MeOH) l
(D3) 209 (ꢀ39.50) nm; see Table 1 for 1H NMR, 13C NMR;
HRESIMS m/z: 483.2582 [M + H]+ (calcd C25H39O9 483.2589).
Norhawthornoid B (2). Colorless oil; [a]D20 ꢀ8.5, (c 0.12,
MeOH); UV (MeOH) lmax (log 3) 228 (2.21) nm; ECD (MeOH) l
1
(D3) 230 (ꢀ9.80) nm; see Table 1 for H NMR, 13C NMR; HRE-
SIMS m/z: 471.2588 [M + H]+ (calcd C24H38O9 471.2589).
Pinnatidanoside F (3). Colorless oil; [a]D20 ꢀ2.8, (c 0.4,
MeOH); see Table 2 for 1H NMR, 13C NMR; HRESIMS m/z:
401.2536 [M + H]+ (calcd C21H37O7 401.2534).
Pinnatidanoside G (4). Colorless oil; [a]D20 ꢀ7.5, (c 0.2,
MeOH); see Table 2 for 1H NMR, 13C NMR; HRESIMS m/z:
419.2636 [M + H]+ (calcd C21H39O8 419.2639).
Characteristic data of compounds 5–15 see ESI-E.2.†
Acid hydrolysis of 1–4
13C NMR calculation section (ESI-Tables 3–5†). Aer opti-
mization of the major conformers (>98%) was performed using
the Gaussian 09 program at B3LYP/6-31G(d) level. Computed
chemical shis reported in this study were determined using
the GIAO method in Gaussian 09 at the B3LYP/6-311+G(d) level
of theory.30 The scaled calculated 13C NMR chemical shis were
obtained from the following: dscal.calc. ¼ (dcalc. ꢀ intercept)/
slope.31 The results were evaluated in terms of R2, MaxDev and
AveDev. Among them, R2 is its coefficient of determination.
MaxDev is the maximum absolute deviation with respect to the
Compounds 1–4 (each 2.0 mg) were individually hydrolyzed by
6% HCl (1.0 mL) under reux for 2 h. The reaction mixtures
were extracted with CH2Cl2, and the organic layers evaporated
under reduced pressure to yield 10 (0.2 mg), 20 (0.5 mg), 30
(0.7 mg), and 40 (0.8 mg). D-Glucose was obtained from the
aqueous layers of 1–4 and identied by HPLC conrmation
(Shodex Asahipak NH2P-50 4E, 250 ꢃ 4.6 mm no. N1490155;
ow rate 1.0 mL minꢀ1; eluent CH3CN/H2O, 3 : 1) with the
authentic sample using optical rotation detector (Jasco OR-4090).
experimental chemical shis dexp. AveDev is the average abso-
Preparation of the (R)- and (S)-MTPA esters of 40
n
X
ꢀ
ꢀ
ꢀ
ꢀ
To a solution of 40 (1.0 mg) in dry pyridine (200 mL) was added
(S)-MTPA chloride (20 mL). The mixture was allowed to stand
under N2 at room temperature for 2 h. The solution was
concentrated under vacuum to afford a residue, which was
subjected to semipreparative HPLC with an RP-C18 column
afforded the (S)-MTPA esters of 40. The (R)-MTPA ester of 40 was
prepared with (R)-MTPA chloride and was puried in the same
manner. 40S: from 1.0 mg of 40 was obtained 0.3 mg of (S)-MTPA
ester. 40R: from 1.0 mg of 40 was obtained 0.2 mg of (R)-MTPA
ester.
lute deviation, computed as ð1=nÞ
dscal:calc: ꢀ dexp:
.
i¼1
Optical rotations (ORs) calculation section (ESI-Table 6†). A
conformational search using the Molecular Merck force eld
(MMFF) led to the identication of the major conformers
(>98%), which was performed using the Gaussian 09 program at
B3LYP/6-31G(d) level.30 Geometry optimization followed by OR
calculations at D-sodium line radiation (wavelength of 589 nm)
in MeOH (CPCM) using the B3LYP functional and the 6-
311+G(d) basis set for DFT. Final calculated ORs were obtained
as the result of the Boltzmann-weighted average.
Rh2(OCOCF3)4-induced ECD experiment of 10 and 5
Evaluation of antiplatelet activity in rat PRP
Compound 10 and 5 (0.5 mg) were dissolved in a dry solution of
[Rh2(OCOCF3)4] complex (1.0 mg) in CDCl3 (700 mL). The rst Evaluation of the antiplatelet aggregation activities of pure
ECD spectrum was recorded immediately aer mixing, and its compounds 1–15, positive control (aspirin) and blank (normal
48472 | RSC Adv., 2017, 7, 48466–48474
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
saline) were performed using an antiplatelet aggregation
method.25 SD rats weighing 200–240 g were lightly anesthetized
with diethyl ether. A volume of 8–10 mL of blood was collected
from the abdominal aorta into a syringe containing 3.8%
sodium citrate. The ratio of blood to 3.8% sodium citrate was
adjusted to 1 : 9 v/v and then centrifuged at 150g for 10 min to
obtain platelet-rich plasma (PRP) used for the aggregation
Conclusions
In summary, our current study describes the discovery of two
uncommon norditerpenoids (1–2) and 13 sesquiterpenoids (3–
15). The compounds 1 and 2 represent the rst examples of
diterpenoids obtained from Crataegus genus. The new
compounds 1–3 exhibited exceptionally potent antithrombotic
activities in vitro/vivo. The most potent one, pinnatidanoside F
(3), is about 2.2-fold more potent compared with the model
control in the zebrash model for thrombosis (in vivo). It is
interesting to study the kinds of antithrombotic ingredients
present in the title plant. Particularly, these terpenoids could
play an important role on their own or in good synergy with
avonoids. This need to be examined in future research studies.
ꢂ
study. PRP (2 ꢃ 108 platelets per mL) was prewarmed at 37 C
for 2 min. The isolates 1–15 dissolved respectively in 5%
dimethylsulfoxide at a concentration of 6.25 mg mLꢀ1 (10 mL)
were added to the diluted PRP (230 mL) before the addition of
the platelet aggregation agent. PRP aggregation was induced by
10 mL ADP (62.5 mM). The nal concentration of all samples was
0.25 mg mLꢀ1. In positive and blank control experiments,
normal saline and aspirin were added instead of the isolates.
From each series of experiments, the inhibitors were tested in at
least three times, and the extent of antiplatelet aggregation was
measured by the percentage inhibition of the control value. The
results obtained are shown in Table 3, ESI-Fig. 8.†
Conflicts of interest
There are no conicts to declare.
Acknowledgements
Financial support from the Project of National Natural Science
Foundation of P. R. China (81302661), Innovation Team
(LT2015027) of Liaoning of P. R. China, and Foundation
(L2015419) from the Project of Education Department of
Liaoning province of P. R. China are gratefully acknowledged.
The authors thank Molegro ApS for kindly providing us with
a free evaluation of their soware packages. The authors also
thank Li W. and Sha Y. of Shenyang Pharmaceutical University
for recording NMR spectra.
Antithrombotic assay using a zebrash system
The arachidonic acid (FeCl3) was used to induce larval zebrash
thrombus as model group. Three dpf (days post fertilization)
AB-type zebrash embryos were placed into 24 well plates with
ten ones per well. Then the embryos were exposed to select
positive control (20 U mLꢀ1 heparin sodium) and test sample
solutions at nal concentrations (50, 150 mg mLꢀ1) in 0.2%
DMSO for 48 h until ve dpf. The negative control group was the
solvent group, which not only represented the back ground
value of the test group, but also made sure that the cardiac red
blood cells of larval zebra sh were normal. The larval zebrash
were treatment with 60 mg mLꢀ1 of FeCl3, and the time was
recorded until a thrombus was observed in the venous system.
Meanwhile, it also needed to be observed if there was the
presence of embryonic death or deformity. The thrombosis time
of the heparin sodium was normalised to 100% and the results
were expressed as a percentage of the control (Fig. 7).
Notes and references
1 N. L. Smith, L. B. Harrington, M. Blondon, K. L. Wiggins,
J. S. Floyd, C. M. Sitlani, B. McKnight, E. B. Larson,
F. R. Rosendaal, S. R. Heckbert and B. M. Psaty, J. Thromb.
Haemostasis, 2016, 7, 1384.
2 E. Fuentes and I. Palomo, J. Funct. Foods, 2014, 6, 73.
3 Y. N. Shi, Y. M. Shi, L. Yang, X. C. Li, Z. H. Zhao, Y. Qu,
H. T. Zhu, D. Wang, R. R. Cheng, C. R. Yang, M. Xu and
Y. J. Zhang, J. Ethnopharmacol., 2015, 162, 87.
4 J. X. Qiao, T. C. Wang, S. Hiebert, C. H. Hu,
W. A. Schumacher, S. A. Spronk, C. G. Clark, Y. Han,
J. Hua, L. A. Price, H. Shen, S. A. Chacko, G. Everlof,
J. S. Bostwick, T. E. Steinbacher, Y. X. Li, C. S. Huang,
D. A. Seiffert, R. Rehfuss, R. R. Wexler and P. Y. S. Lam,
ChemMedChem, 2014, 9, 2327.
5 D. J. Newman and G. M. Cragg, J. Nat. Prod., 2016, 79, 629.
6 R. Guo, M. H. Pittler and E. Ernst, Cochrane Database Syst.
Rev., 2008, 1, 1.
Molecular modeling (docking) studies
Docking of AZD1283 and compound 3 into the P2Y12 was per-
formed with Molegro virtual docker (Molegro). The X-ray crystal
structure of the P2Y receptor P2Y12 (PDB code 4PXZ) was ob-
tained from the RCSB Protein Data Bank. The co-crystal ligand
AZD1283 was extracted to dene the binding site in docking.
The structure of compound 3 was constructed and energy
minimized for 1000 iterations reaching a convergence of
0.01 kcal molꢀ1
ꢀ1. The docking experiments on P2Y12 were
˚
A
¨
carried out by superimposing the minimized ligand into the
receptor. Ten runs were performed and ve poses returned. All
the other docking parameters were set as default. The pose for
which the biotin entity of AZD1283 and 3 binds to P2Y12
receptor in a similar way as observed in the original ligand–
enzyme complex was exported and examined with Discovery
Studio Visualizer 4.0 (Accelrys).
7 P. Z. Liu, H. K. Kallio, D. G. Lu, C. S. Zhou, S. Ou and
B. J. Yang, J. Agric. Food Chem., 2010, 58, 1012.
8 M. Luo, J. Y. Hu, Z. Y. Song, J. Jiao, F. S. Mu, X. Ruan,
Q. Y. Gai, Q. Qiao, Y. G. Zu and Y. J. Fu, RSC Adv., 2015, 5,
67532.
9 H. F. Pan, X. Wang, Y. L. Du, Y. R. Li, S. N. Zhao and
W. D. Pan, Anal. Methods, 2014, 6, 7475.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 48466–48474 | 48473

View Article Online
RSC Advances
Paper
10 B. M. Fraga, Nat. Prod. Rep., 2012, 29, 1334.
11 J. E. Edwards, P. N. Brown, N. Talent, T. A. Dickinson and
P. R. Shipley, Phytochemistry, 2012, 79, 5.
12 S. J. Song, L. Z. Li, P. Y. Gao, Y. Peng, J. Y. Yang and C. F. Wu,
Food Chem., 2011, 129, 933.
13 P. Y. Gao, L. Z. Li, Y. Peng, F. F. Li, C. Niu, X. X. Huang,
M. Ming and S. J. Song, Biochem. Syst. Ecol., 2010, 38, 988.
14 L. Z. Li, P. Y. Gao, S. J. Song, Y. Q. Yuan, C. T. Liu,
X. X. Huang and Q. B. Liu, J. Funct. Foods, 2015, 12, 237.
15 J. H. Shin, Y. W. Seo and K. W. Cho, J. Org. Chem., 1999, 64,
1853.
26 J. Zhang, K. Zhang, Z. G. Gao, S. Paoletta, D. Zhang,
G. W. Han, T. Li, L. Ma, W. Zhang, C. E. Muller, H. Yang,
H. Jiang, V. Cherezov, V. Katritch, K. A. Jacobson,
R. C. Stevens, B. Wu and Q. Zhao, Nature, 2014, 509, 119.
27 K. D. Kurz, B. W. Main and G. E. Sandusky, Thromb. Res.,
1990, 60, 269.
¨
28 M. R. Lang, G. Gihr, M. P. Gawaz and I. I. Muller, J. Thromb.
Haemostasis, 2010, 8, 1159.
29 L. Xiong, C. G. Zhu, Y. R. Li, Y. Tian, S. Lin, S. P. Yuan,
J. F. Hu, Q. Hou, N. H. Chen, Y. H. Yang and J. P. Shi, J.
Nat. Prod., 2011, 74, 1188.
16 Y. Tang, Y. B. Xue, G. Du, J. P. Wang, J. J. Liu, B. Sun, X. N. Li, 30 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
G. M. Yao, Z. W. Luo and Y. H. Zhang, Angew. Chem., Int. Ed.,
2016, 55, 4069.
17 G. Q. Zhan, J. J. Liu, J. F. Zhou, B. Sun, H. J. Akber Aisa and
G. M. Yao, Eur. J. Med. Chem., 2017, 127, 771.
18 J. Frelek and W. J. Szczepek, Tetrahedron: Asymmetry, 1999,
10, 1507.
19 M. T. Liu, S. Lin, M. L. Gan, M. H. Chen, L. Li, S. J. Wang,
J. C. Zi, X. N. Fan, Y. Liu, Y. K. Si, Y. C. Yang, X. G. Chen
and J. G. Shi, Org. Lett., 2012, 14, 1004.
20 L. W. Tian, Y. J. Feng, T. D. Tran, Y. Shimizu, T. Pfeifer,
P. I. Forster and R. J. Quinn, J. Nat. Prod., 2015, 78, 1756.
21 D. P. Curran, Q. S. Zhang, H. J. Lu and V. Gudipati, J. Am.
Chem. Soc., 2006, 128, 9943.
22 M. Diaz, J. Jaballas, J. Arias, H. Lee and T. Onak, J. Am. Chem.
Soc., 1996, 118, 4405.
23 G. Barone, D. Duca, A. Silvestri, L. Gomez-Paloma, R. Riccio
and G. Bifulco, Chem.–Eur. J., 2002, 8, 3240.
24 P. Jagadeeswaran, M. Gregory, K. Day, M. Cykowski and
B. Thattaliyath, J. Thromb. Haemostasis, 2005, 3, 46.
25 G. H. Tang, G. H. Jiang, S. Z. Wang and L. F. Zheng, Chin. J.
Pharmacol. Toxicol., 2001, 15, 317.
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng,
J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota,
R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,
O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery,
J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd,
E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith,
R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell,
J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega,
J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken,
C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann,
O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli,
J. W. Ochterski, R. L. Martin, K. Morokuma,
V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg,
S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman,
J. V. Ortiz, J. Cioslowski and D. J. Fox, Gaussian 09,
Gaussian, Inc., C. T. Wallingford, 2010.
31 G. Barone, L. Gomez-Paloma, D. Duca, A. Silvestri, R. Riccio
and G. Bifulco, Chem.–Eur. J., 2002, 8, 3233.
48474 | RSC Adv., 2017, 7, 48466–48474
This journal is © The Royal Society of Chemistry 2017