Phenolic compounds from the insect Blaps japanensis with inhibitory activities towards cancer cells, COX-2, ROCK1 and JAK3

Article abstract of DOI:10.1016/j.tet.2018.12.015

The insect Blaps japanensis have been used as an ethnomedicine in China for the treatment of several disorders such as cancer and inflammation. Our investigation with this insect led to the isolation of eight new and two known phenolic compounds. The structures of the new compounds, blapsins C?J (1–8), were identified by spectroscopic data. The inhibitory activities of all the compounds except of 9 against human cancer cells (A549, K562 and Huh-7), COX-2, ROCK1, and JAK3 were evaluated. Several compounds were found to be biologically active in these assays.

Full text of DOI:10.1016/j.tet.2018.12.015

Accepted Manuscript
Phenolic compounds from the insect Blaps japanensis with inhibitory activities
towards cancer cells, COX-2, ROCK1 and JAK3
Yong-Ming Yan, Hong-Jie Zhu, Feng-Jiao Zhou, Zheng-Chao Tu, Yong-Xian Cheng
PII:
S0040-4020(18)31480-7
https://doi.org/10.1016/j.tet.2018.12.015
TET 30001
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 28 September 2018
Revised Date: 1 December 2018
Accepted Date: 11 December 2018
Please cite this article as: Yan Y-M, Zhu H-J, Zhou F-J, Tu Z-C, Cheng Y-X, Phenolic compounds from
the insect Blaps japanensis with inhibitory activities towards cancer cells, COX-2, ROCK1 and JAK3,
Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2018.12.015.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Phenolic compounds from the insect Blaps
japanensis with inhibitory activities towards
cancer cells, COX-2, ROCK1 and JAK3
Leave this area blank for abstract info.
Yong-Ming Yan, Hong-Jie Zhu, Feng-Jiao Zhou, Zheng-Chao Tu, and Yong-Xian Cheng
Fonts or abstract dimensions should not be changed or altered.

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Phenolic compounds from the insect Blaps japanensis with inhibitory activities
towards cancer cells, COX-2, ROCK1 and JAK3
a,b
a
a
,c,d
,b
Yong-Ming Yan , Hong-Jie Zhu , Feng-Jiao Zhou , Zheng-Chao Tu* , and Yong-Xian Cheng*
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204,
People’s Republic of China
School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen 518060, People’s Republic of China
b
c
School of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China
d
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, People’s Republic of China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The insect Blaps japanensis have been used as an ethnomedicine in China for the treatment of
several disorders such as cancer and inflammation. Our investigation with this insect led to the
isolation of eight new and two known phenolic compounds. The structures of the new
compounds, blapsins C−J (1−8), were identified by spectroscopic data. The inhibitory activities
of all the compounds except of 9 against human cancer cells (A549, K562 and Huh-7), COX-2,
ROCK1, and JAK3 were evaluated. Several compounds were found to be biologically active in
these assays.
Available online
Keywords:
Insect
Blaps japanensis
phenolic compounds
Cytotoxicity
COX-2
2
018 Elsevier Ltd. All rights reserved.
ROCK1
JAK3
∗
Corresponding authors. e-mails: yxcheng@szu.edu.cn (Y.-X.C.); tu_zhengchao@jnu.edu.cn (Z.-C.T.).

2
Tetrahedron
1
. Introduction
part B (Fig. 2). Additional HMBC correlations of H-6′/C-4 and
ACCEPTED MANUSCRIPT
H-3/C-1′ allow parts A and B connecting via C-1′−C-4. The
observed ROESY correlations of H-3/H-6′, H-9/H-7, and H-7′/H-
Insect medicine is well known for its pronounced effects in
clinical practice. However, chemical profiling in the insects
remains largely unknown. In recent years, we have been
interested in searching non-peptide small molecules from
medicinal insects and several biologically active and structurally
interesting substances have been characterized by us from several
insects such as black ants,
Periplaneta americana,
japanensis, as a commonly used ethnomedicine, has been used
for the treatment of some refractory diseases including cancer,
infection, rheumatoid arthritis, cardiovascular and inflammatory
3
′, in consideration of the chemical shift of C-2 and the
requirement of the total degrees of unsaturation, further
confirmed the presence of part B, which is actually a coumarin
derivative. Thus far, the structure of 1 was identified and named
as blapsin C.
1,2
3-5
Aspongopus chinensis,
6,7
8
and Catharsius molossus. Blaps
OH
OH
A
HO
HO
HO
OH
O
HO
9
HO
diseases by Yi-nationality of Yunnan province of China. In
O
B
contrast to its wide medical use, chemical investigations in this
insect is scarce. We have initiated an investigation on B.
japanensis by using 5 kg of dried material, and identified two
compounds as potent 14-3-3 protein-protein interaction
O
O
O
O
2
3
1
Fig. 2. Key HMBC correlations of 1-3.
10
inhibitors.
In addition, blapsols as derivatives of N-
Compound 2 has the same molecular formula as that of 1 by
acetyldopamine and selective COX-2 inhibitors were also
13
1
11
analysis of its HRESIMS, C NMR and DEPT spectra. The H
characterized from it. These findings prompted us to conduct an
in-depth study in B. japanensis. Our follow-up investigation led
to the isolation of ten phenolic compounds (Fig. 1), eight of
which are new ones. Inspired by the traditional use of B.
japanensis, all the isolates were biologically evaluated for their
potential inhibition on human cancer cells, ROCK1, JAK3, and
COX-2 with exception of compound 9.
13
and C NMR spectra of 2 (Table 1) are very similar to those of 1.
The only difference is that a methyl group at C-4′ in 1 was
positioned at C-3′ of 2. This alternation was in accordance with
the HMBC correlations of H -7′/C-2′, C-3′, C-4′, H-4′/C-6′ (Fig.
2), and an observed meta relationship between H-4′ (δ 6.80, 1H,
H
d, J = 2.5 Hz) and H-6′. Compound 2 was therefore identified
3
and named as blapsin D.
Table 1
1
13
H (600 MHz) and C NMR (150 MHz) data of 1 and 2 in acetone-
d6 (δ, ppm)
No.
1
2
δ
C
, mult
H
δ , (mult, J in Hz)
δ
C
, mult
H
δ , (mult, J in Hz)
2
3
4
161.1 s
116.7 d
154.5 s
120.8 s
110.6 d
153.8 s
121.6 d
127.6 s
146.8 s
15.6 q
121.2 s
147.5 s
119.3 d
127.9 s
149.3 s
116.2 d
160.9 s
117.3 d
154.2 s
120.8 s
110.4 d
153.8 s
121.7 d
127.6 s
146.9 s
15.6 q
124.4 s
145.4 s
128.1 s
119.5 d
151.3 s
114.0 d
16.8 q
6.24 (s)
6.26 (s)
4
5
6
7
8
8
9
1
2
3
4
a
a
6.61 (d, 2.7)
6.96 (d, 2.7)
6.53 (overlap)
6.97 (d, 2.1)
2.37 (s)
6.80 (s)
2.38 (s)
′
′
′
′
Fig. 1. The chemical structures of compounds 1−10.
2
2
. Results and discussion
6.80 (d, 2.5)
.1. Structure elucidation
5′
6′
7′
Compound 1 was found to have the molecular formula
6.69 (s)
2.22 (s)
6.53 (overlap)
2.25 (s)
1
6.3 q
C H O (11 degrees of unsaturation) derived from its
1
7
14
5
13
1
HRESIMS, C NMR and DEPT spectra. The H NMR spectrum
Table 1) of 1 contains two meta protons (δ 6.61, 1H, d, J = 2.7
(
Compound 3 has the molecular formula C H O (7 degrees of
11 10 4
13
H
Hz, H-5; 6.96, 1H, d, J = 2.7 Hz, H-7), suggesting a 1,2,3,5-
unsaturation), derived from its HRESIMS, C NMR and DEPT
1
tetrasubstituted benzene ring, two para protons (δ 6.80, 1H, s,
spectra. The H NMR spectrum of 3 (Table 2) shows two meta
H
H-3′; 6.69, 1H, s, H-6′), suggesting a 1,2,4,5-tetrasubstituted
protons in the olefinic region (δ 6.74, 1H, d, J =2.4 Hz, H-5;
H
benzene rings, along with a resonance for olefinic proton (δ 6.24,
6.73, 1H, d, J = 2.4 Hz, H-6), indicating the presence of a
H
13
13
1
H, s). The C NMR and DEPT spectra contain 17 carbons
1,2,3,5-tetrasubstituted benzene ring. The C NMR and DEPT
2
2
ascribe to two methyl, five sp methine, and ten quaternary
carbons (one carbonyl, 9 olefinic including four oxygenated).
The structure of 1 was constructed mainly on the basis of an
HMBC experiment. HMBC correlations of H -7′/C-3′ (δ 119.3),
spectra of 3 displays 11 signals attributed to two methyl, two sp
methine, and seven quaternary carbons (one carbonyl, six olefinic
1
13
including three oxygenated). The H and C NMR data of 3 are
similar to those of part B in 1 differing in that a hydroxyl and a
methyl was respectively positioned at C-3 and C-4 of structure 3.
This alternation was supported by HMBC correlations of Me-
3
C
C-4′, C-5′ (δC 149.3), H-6′/C-1′, C-4, in consideration of the
chemical shifts of C-2′ and C-5′ and a para relationship between
H-3′ and H-6′, indicates the presence of substructure A (Fig. 2).
HMBC correlations of H -9/C-7 (δ 121.6), C-8, C-8a (δ 146.8),
4
/C-3, C-4, C-5 and OH-3/C-2, C-3, C-4 (Fig. 2). As a result, the
structure of 3 was assigned as shown and named as blapsin E.
Compound 4 has a molecular formula C H O (8 degrees of
3
C
C
1
6
18
4
H-3/C-2, C-4, C-4a, and H-5/C-4, in conjunction with a meta
relationship of H-5 and H-7, suggest a substructure like that of
1
3
unsaturation), by analysis of its HREIMS, C NMR and DEPT

3
1
3
spectra. The C NMR and DEPT spectra of 4 show 16 carbons
= 7.3 Hz). Consequently, the structure of 6 was identified and
named as blapsin H.
ACCEPTED MANUSCRIPT
2
ascribe to two methyl, two aliphatic methylene, four sp methine,
and eight olefinic quaternary carbons (four oxygenated). Careful
inspection of C NMR spectrum of 3 found that signals appear in
pairs, suggesting that 4 might be a dimer. Two para protons (H-
Table 3
13
1
13
H (600 MHz) and C NMR (150 MHz) data of 4 in acetone-d (δ,
6
ppm)
No.
3
H
′ and H-6′) and two meta protons (H-4 and H-6) observed in the
δ
C
δ
H
, (m, J in Hz)
δ
C
H
δ , (m, J in Hz)
1
NMR spectrum reveal the presence of
a
1,2,3,5-
1
2
3
4
5
6
7
8
128.6 s
145.6 s
134.3 s
116.1 d
151.7 s
115.4 d
24.5 t
1′
2′
3′
4′
5′
6′
7′
8′
125.3 s
146.6 s
117.6 d
131.9 s
149.7 s
118.3 d
23.7 t
tetrasubstituted benzene ring and a 1,2,4,5-tetrasubstituted
1
1
6.80 (s)
benzene ring, respectively. The H- H COSY cross peak of H-
/H-8 and HMBC correlations of H-7/C-2, C-3, C-4 (Fig. 3),
6.65 (d, 2.9)
7
ROESY correlations of H-6/H-6′, H-3′/H-7′, and H-7/H-4
suggest that the monomer of 4 is a 2-ethylbenzene-1,4-diol. The
two monomers were connected via C-1′−C-1 evidenced from H-
6.57 (d, 2.9)
2.64 (q, 7.5)
1.19 (t, 7.5)
6.73 (s)
2.61 (q, 7.5)
1.17 (t, 7.5)
14.9 q
14.5 q
6
/C-1′ and H-6′/C-1 correlations. As a result, the structure of 4
Compound 7 has the same molecular formula as that of 6. Its
NMR data are also similar to those 6, indicating that they might
be isomers. Careful comparison of NMR data of 6 and 7 found
that they differ only in the configuration of glycosidic bond. The
coupling constant of H-1′ is 3.6 Hz in 7 suggests an α-form
configuration. In the same manner as that of 6, acid hydrolysis
followed by TLC comparison with an authentic sample and
optical rotation measurement confirmed the above conclusion.
The structure of 7 was thus identified and named blapsin I.
Compound 8 has the same molecular formula and similar
NMR data as those of 6. Inspection of NMR data of compounds
6 and 8 found that they differ only in the position of the glucose
moiety, which could be evidently confirmed by the observed
HMBC correlation of H-1′/C-4. Evidence for the presence of a
D-glucose moiety in 8 comes from analysis of the acid hydrolysis
product in the manner described for 6. Therefore, the structure of
was deduced as shown and named as blapsin F.
Table 2
1
13
H (600 MHz) and C NMR (150 MHz) data of 3 in DMSO-d (δ,
6
ppm)
No.
δ
C
H
δ , (m, J in Hz)
δ
C
H
δ , (m, J in Hz)
2
3
4
4
5
6
7
4
158.2 s
137.9 s
123.7 s
121.9 s
106.3 d
153.5 s
117.1 d
123.7 s
8
8a
9
125.7 s
140.2 s
15.3 q
10.6 q
2.29 (s)
2.17 (s)
9.71 (s)
9.52 (s)
a
4-CH
3
6.74 (d, 2.4)
6.73 (d, 2.4)
3-OH
6-OH
8
125.7 s
Compound 5 was determined to be C H O (10 degrees of
17
16
7
1
3
unsaturation) by analysis of its HRESIMS, C NMR and DEPT
spectra. The H NMR spectrum of this substance contains a
typical ABX spin system (δ 7.32, 1H, d, J = 1.8 Hz, H-2′; 6.90,
1
8
was identified and named blapsin J.
H
1
H, d, J = 8.2 Hz, H-5′; 7.19, 1H, dd, J = 8.2, 1.8 Hz, H-6′) and
13
two para protons (δ 6.83, 1H, s, H-3; 6.87, 1H, s, H-6). The
C
H
NMR and DEPT spectra of 5 give 17 carbons classified into one
methyl, two aliphatic methylene (one oxygenated), five olefinic
methine, and nine quaternary carbons (one ketone, one carbonyl,
1
1
seven olefinic including four oxygenated). The H- H COSY
correlation of H-7/H-8 and HMBC correlations of H-8, H-10/C-9
allow the assignment of the side chain (Fig. 3), whose position at
the ring A was established by HMBC correlations of H-8/C-1, H-
Fig. 3. Key HMBC correlations of 4-6 and 8.
7
/C-1, C-2, C-6. The positions of two hydroxyl groups in ring A
was deduced as shown from HMBC correlations of H-7/ C-1, C-
, C-6 and H-3/C-7′, C-2 and a para relationship between H-3
Two known compounds were isolated and identified as
13
12
2
transtorine (9) and 2,5-dihydroxy-4-methyl acetophenone (10)
and H-6. The presence of an ABX system along with HMBC
correlations of H-2′, H-6′/C-1′, C-7′ suggests the substituted
pattern in ring B which was connected to ring A via C-7′. The
structure of 5 was therefore identified and named as blapsin G.
by comparing their spectroscopic properties with those
previously reported for these substances. Of note, all the
compounds with exception of 5 are hydroquinone derivatives.
Compound 4 is a dimer of 2-ethylbenzene-1,4-diol, and 1-3
might be hybrids of hydroquinone derivatives with coumarin.
Compound 6 was found to have a molecular formula C H O
on the basis of its HRESIMS, C NMR and DEPT spectra. The
H NMR spectrum (Table 4) of 6 exhibits a typical ABX spin
14
20
7
1
3
1
2
.2. Biological evaluation
system (δ 6.59, 1H, d, J = 2.9 Hz, H-3; 7.00, 1H, d, J = 8.7 Hz,
H
13
H-6; 6.54, 1H, dd, J = 8.7, 2.9 Hz, H-5). The C NMR and
DEPT spectra of this substance give 14 resonances attributable to
one methyl, two methylene, seven methine (three olefinic and
four oxygenated), and three olefinic quaternary carbons (two
Biological activities of all the isolates except of 9 against
cancer cells (A549, Huh-7, K562), COX-2, ROCK1 and JAK3
were evaluated. For cytotoxic activities by using CCK8 assay, we
found that compounds 1, 2, and 4 are active towards the above
three cancer cell lines (Table 5), among them, 1 appears to be the
most active with IC50 values less than 10 ꢀM. We also noted that
compound 3 is not active towards these cell lines, indicating the
importance of part A in the structure of 1 for keeping the activity.
Activated COX-2 is known to promote inflammation, selective
inhibition of COX-2 will be beneficial for anti-inflammatory
therapy. In this study, we found that compounds 2-4 show COX-
13
oxygenated). Inspection of C NMR of 6 found that compound 6
is a glycoside. Acid hydrolysis of 6 produced D-glucose, which
was identified by TLC comparison with an authentic sample and
from its positive sign of optical rotation. In addition to the signals
of a glucose residue, the remaining resonances are in accordance
with the monomer of 4. This conclusion could be confirmed by
detailed interpretation of its 2D NMR data (Fig. 3). The glucose
moiety is connected to C-1 supported by the observed HMBC
correlation of H-1′/C-1. The configuration of glycosidic bond
was assigned as β-form based on the coupling constant of H-1′ (J
2
inhibitory activity with respective

4
Tetrahedron
ACCEPTED MANUSCRIPT
Table 4
1
13
H (600 MHz) and C NMR (150 MHz) data of 5–8 (δ, ppm)
a
b
b
b
No.
5
6
7
8
δ
C
H
δ , (m, J in Hz)
δ
C
H
δ , (m, J in Hz)
δ
C
H
δ , (m, J in Hz)
δ
C
H
δ , (m, J in Hz)
1
2
3
4
5
6
7
8
9
1
1
2
3
4
5
6
6
131.9 s
131.7 s
117.6 d
143.3 s
147.8 s
118.6 d
32.5 t
65.9 t
170.9 s
20.7 q
130.6 s
117.5 d
145.6 s
150.9 s
115.5 d
124.8 d
150.2 s
136.5 s
116.5 d
153.6 s
113.8 d
118.4 d
24.1 t
149.7 s
136.4 s
116.7 d
153.4 s
113.8 d
118.1 d
24.3 t
151.4 s
132.6 s
119.4 d
152.4 s
116.2 d
116.1 d
24.3 t
6.83 (s)
6.59 (d, 2.9)
6.60 (d, 3.0)
6.88 (d, 2.9)
6.54 (dd, 8.7, 2.9)
7.00 (d, 8.7)
2.65 (q, 7.5)
6.53 (dd, 8.7, 3.0)
7.04 (d, 8.7)
2.68 (q, 7.5)
6.79 (dd, 8.7, 2.9)
6.65 (d, 8.7)
2.57 (q, 7.5)
6.87 (s)
2.87 (t, 7.0)
4.14 (t, 7.0)
15.0 q
1.17 (t, 7.5)
15.1 q
1.19 (t, 7.5)
14.6 q
1.17 (t, 7.5)
0
′
′
′
′
1.86 (s)
104.0 d
75.1 d
78.3 d
71.5 d
78.0 d
62.6 t
4.72 (d, 7.3)
100.3 d
73.6 d
75.0 d
71.7 d
74.4 d
62.5 t
5.31 (d, 3.6)
3.56 (dd, 9.8,3.6)
3.86 (t, 9.3)
103.7 d
75.1 d
78.3 d
78.0 d
71.5 d
62.6 t
4.73 (d, 7.2)
7.32 (d, 1.8)
3.42 (overlap)
3.42 (overlap)
3.36 (overlap)
3.36 (overlap)
3.88 (dd, 12.0, 2.0)
3.70 (dd, 12.0, 5.1)
3.42 (overlap)
3.42 (overlap)
3.36 (overlap)
3.36 (overlap)
3.88 (dd, 12.0, 2.0)
3.69 (dd, 12.0, 4.9)
3.43 (t, 9.2)
′
6.90 (d, 8.2)
7.19 (dd, 8.2, 1.8)
3.71 (overlap)
3.77 (dd, 13.5, 4.0)
3.71 (overlap)
′a
′b
′
7
a
196.1 s
b
in acetone-d6, in methanol-d4
IC50 value of 6.12 ꢀM, 10.70 ꢀM, and 83.20 ꢀM. It was noted
spectra were recorded on a Shimadzu UV-2401PC spectrometer.
CD spectra were measured on a Chirascan instrument. Semi-
preparative HPLC was carried out using an Agilent 1200 liquid
chromatograph, the column used was a 250 mm × 9.4 mm, i.d., 5
ꢀm, Zorbax SB-C and a 250 mm × 4.6 mm, i.d., 5 ꢀm, Daicel
Chiralpak IC. NMR spectra were recorded on a Bruker AV-500
or an AV-600 spectrometer, with TMS as an internal standard.
EIMS and HREIMS were collected by AutoSpec Premier P776
spectrometer. ESIMS and HRESIMS were collected by API
QSTAR Pulsar 1 spectrometer.
that compound 1 is not active towards COX-2 in spite of its slight
difference in the structure from 2. ROCK1 regulates a wide range
of fundamental cell functions, inhibition of ROCK1 has been
proved to be beneficial for the treatment of oncological,
cardiovascular and neurological disorders and cardiorenal
18
14
syndrome. Our study showed that compounds 1, 2 and 4
possess ROCK1 inhibitory activity with respective IC50 value of
.77 ꢀM, 9.31 ꢀM, and 4.61 ꢀM. JAK3 is a member of the JAK
9
family of non-receptor protein tyrosine kinases, selective JAK3
inhibitors might be useful as therapeutic agents in the areas of
15,16
3.2. Insect material
oncology, organ transplantation and autoimmune diseases.
Therefore JAK3 has become a drug target which attracted great
interest in academia and pharmaceutical industry. Since the title
insect has been used for treating rheumatoid arthritis that is
closely associated with dysimmunity, all the isolates were thus
submitted to test their potential against JAK3. It was found that
compounds 1-5 show potent inhibitory activity with respective
IC50 value of 2.88 ꢀM, 5.27 ꢀM, 35.46 ꢀM, 1.87 ꢀM, and 6.72
B. japanensis was collected from YuanJiang County, Yunnan
Province, People’s Republic of China in September 2012, and
authenticated by Professor Dazhi Dong at Kunming Institute of
Zoology, Chinese Academy of Sciences, People’s Republic of
China. A voucher specimen (CHYX0469) was deposited at the
State Key Laboratory of Phytochemistry and Plant Resources in
West China of our institute.
ꢀ
M, with 4 is the most active.
3
.3. Extraction and isolation
Table 5
Compounds against cancer cells (A549, Huh-7, K562), COX-2,
The air-dried powder of B. japanensis bodies (50 kg) was
ROCK1, and JAK3
soaked with 70% aqueous EtOH (3 × 300 L) at room
temperature. The combined extracts were concentrated to obtain
a crude extract (4 Kg), which was suspended in water followed
by extracted with EtOAc to afford a EtOAc soluble extract (1.0
kg). The EtOAc extract was divided into 7 parts (Fr.1–Fr.7) by
using a MCI gel CHP 20P column eluting with gradient aqueous
MeOH (5:95–100:0). Fr. 3 (65 g) was separated by using MCI
gel CHP 20P eluted with a gradient of aqueous MeOH
IC50 (ꢀM)
Compd
1
2
3
4
5
A549
9.96
Huh-7
2.35
K562
4.06
5.45
JAK3
2.88
5.27
35.46
1.87
ROCK1 COX-2
9.77
9.31
NA
NA
6.12
13.10
3.86
a
a
a
a
NAa
NA
NA
10.70
83.20
NAa
14.20
a
8.04
a
4.61
a
a
NA
NA
NA
6.72
NA
NT
NA
NT
b
b
b
Toxol
0.0009
0.0052
0.0025
NT
b
b
b
b
b
Celecoxib
Staurosporine
NT
NT
NT
NT
NT
0.0032
NT
b
b
b
NT
NT
NT
0.0001 0.0074
a
b
(MeOH/H
.6). Fr. 3.2 (4.5 g) was subjected to gel filtration on Sephadex
LH-20 (MeOH) to give five portions (Fr. 3.2.1−Fr. 3.2.5). Fr.
2
O, 10:90–50:50) to yield six fractions (Fr. 3.1−Fr.
No activity, No test.
3
3
3
. Experimental section
.1. General procedure
3
5
.2.2 (400 mg) was separated by preparative TLC (MeOH/H O,
:1) to produce Fr. 3.2.2.1−Fr. 3.2.2.3. Fr. 3.2.2.2 (80 mg) was
2
purified vis semi-preparative HPLC (MeOH/H O, 10:90) to
2
Column chromatography was performed on silica gel (200-300
mesh; Qingdao Marine Chemical Inc., P. R. China), on C-18
silica gel (40−60 ꢀm; Daiso Co., Japan), MCI gel CHP 20P (75-
obtain 6 (R
t
= 24.1 min, 3.0 mg), 7 (R = 26.5 min, 8.0 mg) and 8
t
(R = 28.8 min, 5.0 mg). Fr. 5 (55 g) was separated by using a
t
MCI gel CHP 20P column eluting with gradient aqueous MeOH
(40:60–100:0) to provide eight portions (Fr. 5.1−Fr. 5.8). Fr. 5.4
(6.2 g) was separated by using Sephadex LH-20 (MeOH)
1
50 ꢀm, Mitsubishi Chemical Industries, Tokyo, Japan) and on
Sephadex LH-20 (Amersham Pharmacia, Sweden). Optical
rotations were recorded on a Horiba SEPA-300 polarimeter. UV
followed by a RP-18 column (MeOH/H O, 20:80–70:30) to
2

5
produce Fr. 5.4.1−Fr. 5.4.6. Fr. 5.4.2 (2.5 g)
using Sephadex LH-20 (MeOH) to give five portions (Fr.
.4.2.1−Fr. 5.4.2.5). Fr. 5.4.2.4 (200 mg) was purified by semi-
preparative HPLC (MeOH/H O, 42:58) to obtain 3 (R = 15.3
A
wa
C
s
C
se
E
pa
P
ra
T
te
E
d
D
by MAN
A
U
ll t
S
he
C
is
R
ol
I
at
P
es except of 9 were evaluated for their inhibitory
T
17,18
2
effects against cancer cells (A549, Huh-7, K562),
ROCK1, and JAK3 as previously described methods.
COX-2,
1
2
5
2
t
Acknowledgments
min, 4.5 mg) and 2 (R = 24.5 min, 2.5 mg). Fr. 5.4.2.5 (150 mg)
t
was purified by semi-preparative HPLC (MeOH/H O, 38:62) to
This work was financially supported by National Science Fund
for Distinguished Young Scholars (81525026), Shenzhen
2
obtain 9 (R = 12.2 min, 8.5 mg) and 10 (R = 28.5 min, 25 mg).
t
t
Fr. 5.4.3 (120 mg) was separated vis semi-preparative HPLC
Government’s
Plan
of
Science
and
Technology
(
2
MeOH/H O, 40:60) to yield 1 (R = 20.5 min, 10 mg), 5 (R =
(JCYJ20170412110504956), and National Natural Science
Foundation of China (21172223).
2
t
t
8.3 min, 22mg), and 4 (R = 35.2 min, 15 mg).
t
Supplementary material
3
.4. Spectral data of the new compounds
3
.4.1. Blapsin C (1). Yellow solid; UV (MeOH) λmax (logε) 350
HRMS, and 1D and 2D NMR spectra of compounds 1−8. This
(
[
2
3.78), 286 (4.13), 221 (4.44), 205 (4,68) nm; ESIMS m/z 297
material is available free of charge via the Internet at http://
–
–
M–H] ; HRESIMS m/z 297.0777 [M–H] (calcd for C H O ,
17
14
5
1
13
97.0768). H and C NMR data, see Table 1.
References and notes
3
(
[
2
.4.2. Blapsin D (2). Yellow solid; UV (MeOH) λmax (logε) 350
(
1) Tang, J. J.; Zhang, L.; Jiang, L. P.; Di, L.; Yan, Y. M.; Tu, Z. C.; Yang,
C. P.; Zuo, Z. L.; Hou, B.; Xia, H. L.; Chen, Y. B.; Cheng, Y. X.
Tetrahedron. 2014, 70, 8852–8857.
3.68), 286 (4.08), 219 (4.42), 206 (4.62) nm; ESIMS m/z 297
–
–
M–H] ; HRESIMS m/z 297.0773 [M–H] (calcd for C H O ,
17
14
5
1
13
97.0768). H and C NMR data, see Table 1.
(2) Tang, J. J.; Fang, P.; Xia, H. L; Tu, Z. C.; Hou, B. Y.; Yan, Y. M.; Di,
L.; Zhang, L.; Cheng, Y. X. Food Res. Int. 2015, 67, 163–168.
3) Yan, Y. M.; Ai, J.; Shi, Y. N.; Zuo, Z. L.; Hou, B.; Luo, J.; Cheng, Y.
X. Org. Lett. 2014, 16, 532–535.
(4) Shi, Y. N.; Tu, Z. C.; Wang, X. L.; Yan, Y. M.; Fang, P.; Zuo, Z. L.;
3
3
.4.3. Blapsin E (3). Yellowish solid; UV (MeOH) λmax (logε)
(
–
41 (4.16), 248 (3.97), 213 (4.53) nm; ESIMS m/z 205 [M–H] ;
–
HRESIMS m/z 205.0507 [M–H] (calcd for C H O , 205.0506).
11
10
4
1
13
Hou, B.; Yang, T. H.; Cheng, Y. X. Bioorg. Med. Chem. Lett. 2014, 24,
H and C NMR data, see Table 2.
5
164–5169.
.4.4. Blapsin F (4). Yellow gum; UV (MeOH) λmax (logε) 304
(
5) Di, L.; Shi, Y. N.; Yan, Y. M.; Jiang, L. P.; Hou, B.; Wang, X. L.; Zuo,
Z. L.; Chen, Y. B.; Yang, C. P.; Cheng, Y. X. RSC Adv. 2015, 5,
70985–70991.
3
(
2
–
4.06), 207 (4.64) nm; ESIMS m/z 273 [M–H] ; HRESIMS m/z
–
1
13
73.1132 [M–H] (calcd for C H O , 273.1132). H and
C
17
14
5
(6) Yan, Y. M.; Zhu, H. J.; Xiang, B.; Qi, J. J.; Cheng, Y. X. Nat. Prod.
Comm. 2017, 12, 1769–1772.
NMR data, see Table 3.
(
7) Yang, Y. X.; Luo, Q.; Hou, B.; Yan, Y. M.; Wang, Y. H.; Tang, J. J.;
Dong, X. P.; Ma, X. Y.; Yang, T. H.; Zuo, Z. L.; Cheng, Y. X. J. Asian
Nat. Prod. Res. 2015, 17, 988–995.
3
3
3
.4.5. Blapsin G (5). Yellowish solid; UV (MeOH) λmax (logε)
20 (3.96), 291 (3.89), 240 (4.03), 207 (4.28) nm; ESIMS m/z
+
+
55 [M+Na] ; HRESIMS m/z 355.0793 [M+Na] (calcd for
(8) Lu, J.; Sun, Q.; Tu, Z. C.; Lv, Q.; Shui, P. X.; Cheng, Y. X. Molecules
015, 20, 15589–15596.
1
13
2
C H NaO , 355.0788). H and C NMR data, see Table 4.
17
16
7
(9) Zhang, L. S.; Xia, C. L.; Yang, Y. S.; Liu, G. M. Lishizhen Med. Mater.
Med. Res 2009, 20, 3113–3114.
3
2
[
(
.4.6. Blapsin H (6). Yellowish solid; UV (MeOH) λmax (logε)
86 (3.32), 215 (3.79), 202 (4.08) nm; ESIMS m/z 345
(
10) Yan, Y. M.; Dai, H. Q.; Du, Y. H.; Schneider, B.; Guo, H.; Li, D. P.;
Zhang, L. X.; Fu, H.; Dong, X. P.; Cheng, Y. X. Bioorg. Med. Chem.
Lett. 2012, 22, 4179–4181.
–
–
M+HCOOH–H] ; HRESIMS m/z 345.1199 [M+HCOOH–H]
1 13
calcd for C H O , 345.1191). H and C NMR data, see Table
14 20 7
(11) Yan, Y. M.; Li, L. J.; Qin, X. C.; Lu, Q.; Tu, Z. C.; Cheng, Y.X. Bioorg.
4
.
Med. Chem. Lett. 2015, 25, 2469–2472.
(
(
12) Hangauer, D. G. Tetrahedron Lett. 1986, 27, 5799–5802.
13) Sabui, S. K.; Venkateswaran, R. V. Tetrahedron 2003, 59, 8375–8381.
3
(
.4.7. Blapsin I (7). Yellowish solid; UV (MeOH) λmax (logε) 286
3.35), 216 (3.78), 202 (4.09) nm; ESIMS m/z 345 [M+HCOOH–
(14) Pan, P. C.; Shen, M. Y.; Yu, H. D.; Li, Y. Y.; Li, D.; Hou, T. J. Drug
Discov. Today 2013, 18, 1323–1333.
–
–
H] ; HRESIMS m/z 345.1201 [M+HCOOH–H] (calcd for
1
13
(
(
15) Worbleski, S.T.; Pitts, W. J. Annu. Rep. Med. Chem. 2009, 44, 247–248.
16) Wilson, L. J. Expert Opin. Ther. Pat. 2010, 20, 609–623.
C H O , 345.1191). H and C NMR data, see Table 4.
4
1
20
7
3
(
.4.8. Blapsin J (8). Yellowish solid; UV (MeOH) λmax (logε) 286
3.27), 215 (3.69), 200 (3.93) nm; ESIMS m/z 345 [M+HCOOH–
(17) Chen, S. T.; Wang, J. F.; Lin, X. P.; Zhao, B. X.; Wei, X. Y.; Li, G. Q.;
Kaliaperumal, K.; Liao, S. R.; Yang, B.; Zhou, X. F.; Liu, J.; Xu, S. H.;
Liu, Y. H. Org. Lett. 2016, 18, 3650–3653.
–
–
H] ; HRESIMS m/z 345.1197 [M+HCOOH–H] (calcd for
(18) Qin, F. Y.; Yan, Y. M.; Tu, Z. C.; Cheng, Y.X. Fitoterapia 2018, 129,
1
13
C H O , 345.1191). H and C NMR data, see Table 4.
14
20
7
167–172.
3
.5. Acid hydrolysis of 6–8
A solution of 6–8 (1.0 mg) in 6 N HCl was stirred at 60℃ for
1
.5 h. After cooling, the mixtures were extracted with EtOAc.
The aqueous layer was concentrated in vacuo followed by TLC
examination and optical rotation measurement. The optical
2
5
rotation of the glucose of 6–8 are as follows: [α]
+ 45.0 (c
D
25
25
0
0
[
.044, H O), [α] + 42.3 (c 0.052, H O), and [α] + 40.9 (c
2 D 2 D
.044, H O). By comparing optical rotation with D-glucose:
α]D + 42.3 (c 0.106, H O), the glucose in compounds 6–8 were
2
25
2
determined to be D-configurations.
.6. Procedure for biological evaluation
3