Synthesis and bioactivity of tripolinolate A from Tripolium vulgare and its analogs

Article abstract of DOI:10.1016/j.bmcl.2015.04.091

A new coniferol derivative, named as tripolinolate A (1), and 11 known compounds (2-12) were isolated from whole plants of Tripolium vulgare Nees. The structure of this new compound was determined as 4-(2S-methylbutyryl)-9-acetyl-coniferol based on its NMR and HRESIMS spectral analyses. A simple and efficient method was designed to prepare tripolinolate A and its 19 analogs including nine new chemical entities for bioactive assay. Tripolinolate A and its analog 4,9-diacetyl-coniferol were found to be the two most active compounds that significantly inhibited the proliferation of different cancer cell lines with IC₅₀ values ranging from 0.36 to 12.9 μM and induced apoptosis in tumor cells. Structure-activity relationship analysis suggested that the molecular size of acyl moieties at C-4 and C-9 position might have an effect on the activity of this type of coniferol derivatives.

Full text of DOI:10.1016/j.bmcl.2015.04.091

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2629–2633
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and bioactivity of tripolinolate A from Tripolium vulgare
and its analogs
Lu Chen a, Ying Liang a, Tengfei Song a, Komal Anjum a, Wenling Wang a, Siran Yu a, Haocai Huang a,
Xiao-Yuan Lian b, , Zhizhen Zhang a,
⇑
⇑
a Ocean College, Zhejiang University, Hangzhou 310058, China
b College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new coniferol derivative, named as tripolinolate A (1), and 11 known compounds (2–12) were isolated
from whole plants of Tripolium vulgare Nees. The structure of this new compound was determined as 4-
(2S-methylbutyryl)-9-acetyl-coniferol based on its NMR and HRESIMS spectral analyses. A simple and
efficient method was designed to prepare tripolinolate A and its 19 analogs including nine new chemical
entities for bioactive assay. Tripolinolate A and its analog 4,9-diacetyl-coniferol were found to be the two
most active compounds that significantly inhibited the proliferation of different cancer cell lines with IC50
values ranging from 0.36 to 12.9 lM and induced apoptosis in tumor cells. Structure–activity relationship
analysis suggested that the molecular size of acyl moieties at C-4 and C-9 position might have an effect on
the activity of this type of coniferol derivatives.
Received 2 March 2015
Revised 25 April 2015
Accepted 27 April 2015
Available online 6 May 2015
Keywords:
Tripolium vulgare
Compositae
Tripolinolate A
Synthesis of tripolinolate A and analogs
Inhibition of cancer cell proliferation
Apoptosis
Ó 2015 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Glioblastomas remain the most malignant brain cancers,
despite recent advances in standard therapy including surgical
resection followed by radiation and chemotherapy.1 While
chemotherapy has played an important role in treating gliomas,
temozolomide (TMZ) is the only drug that has been alone used
for the treatment of gliomas, and the efficacy of TMZ, as well as
other current drugs remains limited.1,2 There is a need to discover
lead compounds for the development of novel anti-glioma drugs.
Natural products are significant sources of new drug leads.3
The genus Tripolium (Family Compositae) consists of only one
species, Tripolium vulgare Nees, which is distributed mainly in
Europe, Asia, North America, and Northern African.4 This plant is
an annual herb that usually resides in the saline alkaline wetland
and is an indicative plant of the alkaline earths. This halophyte
was considered an invasive species and has affected the original
reed community and changed its ecosystem in the wetlands.5
Two studies previously reported the existence of inorganic ions6
and trace elements in this plant,7 so far there have been no reports
on the medicinal uses or phytochemical investigation of this spe-
cies. As a part of our continuing research on the discovery of novel
anticancer agents from natural sources,8–12 a MeOH extract pre-
pared from the whole plants of T. vulgare was found to have activ-
ity in inhibiting the proliferation of glioma cells. Separation of this
extract produced 12 compounds (1–12, Figs. 1 and S1). Compound
1 was identified as a new compound named as tripolinolate A and
found to significantly inhibit the proliferation of cancer cells and to
induce apoptosis in glioma cells. A synthetic method was designed
to generate tripolinolate A and its 19 analogs (13–31, Figs. 1 and
S1) including nine new chemical entities for bioactive assay. In this
Letter, we describe the isolation and structural determination of
tripolinolate A (1) from T. vulgare, the synthesis of tripolinolate A
and its analogs (13–31), and the activity of compounds (1–31) in
suppressing the proliferation of glioma and colorectal cancer cells.
The MeOH extract was successively partitioned with cyclohex-
ane (CHX) and n-butanol (BuOH). Both CHX and BuOH parts were
repeatedly separated by column chromatography, following by
HPLC purification to give the individual 12 compounds
(Supplementary data). On the basis of the NMR analyses, HRESIMS
data, and HPLC comparison with reference compounds, isolates 2–
12 were identified as a
-tocoquinone (2),13 trans-phytol (3),14 E-fer-
ulic acid eicosyl ester (4),15 E-p-coumaric acid eicosyl ester (5),16
(22E,24S)-ergosta-4,6,8(14),22-tetraen-3-one (6),17 (22E,24R)-er-
gosta-4,6,8(14),22-tetraen-3-one (7),17 3,4-dicaffeoylquinic acid
(8), 3,5-dicaffeoylquinic acid (9), 4,5-dicaffeoylquinic acid (10),
⇑
Corresponding authors. Tel./fax: +86 57188208432 (X.-Y.L.); tel.: +86
57188206577; fax: +86 57188208891 (Z.Z.).
E-mail addresses: xylian@zju.edu.cn (X.-Y. Lian), zzhang88@zju.edu.cn
(Z. Zhang).
http://dx.doi.org/10.1016/j.bmcl.2015.04.091
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

2630
L. Chen et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2629–2633
O
O
O
O
O
O
7
9
1''
O
1
O
O
O
O
O
O
O
O
O
2''
4
2'
O
O
OCH3
O
OCH3
1'
O
3
4'
1
13
19
14
5'
OCH3
15
OCH3
O
O
O
O
O
O
O
O
O
O
O
OCH3
O
OCH3
O
OCH3
O
OCH3
20
21
17
O
O
O
O
O
O
O
O
O
OCH3
O
24
25
26
OCH3
OCH3
Figure 1. Structures of tripolinolate A (1) and its new synthetic analogs (14, 15, 17, 19–21, 24–26) and the bioactive compound (13).
astragalin (11),18 and 1-[(butanoyl)phloroglucinyl]-b-
noside (12).19
D
-glucopyra-
their structure–activity relationships. The methodology is outlined
in Figure 3 and detailed in the Supplementary data. The prepara-
tion started from vanillin (1a, 36.51 g), which was reacted with
malonic acid (1b, 31.2 g) at 90–95°C in the presence of pyridine
and aniline to produce (E)-ferulic acid (1c, 41.96 g, 90.0% yield).
The (E)-ferulic acid (1c, 40.66 g) was treated with SOCl2
(20.1 mL) in anhydrous methanol to furnish (E)-ferulic acid methyl
ester (1d, 40.59 g, 92.8% yield). The coniferol (1e, 21.68 g, 80.2%
yield) was obtained by the reduction of (E)-ferulic acid methyl
ester (1d, 31.24 g) with LiAlH4 (11.38 g) in anhydrous THF at room
temperature. Finally, coniferol (1e) was acylated by different anhy-
drides (1f) in pyridine to give the desired compounds 1 and 13–31.
The structures of these synthetic analogs (13–31, Figs. 1 and S1)
were confirmed by their NMR and HRESIMS spectral data. They
are 4,9-diacetyl-coniferol (13),21 4-acetyl-9-propionyl-coniferol
(14), 4-propionyl-9-acetyl-coniferol (15), 4,9-dipropionyl-coniferol
(16),21 4-acetyl-9-isobutyryl-coniferol (17), 4-isobutyryl-9-acetyl-
coniferol (18),22 4-acetyl-9-butyryl-coniferol (19), 4-butyryl-9-
acetyl-coniferol (20), 4-acetyl-9-(2S-methylbutyryl)-coniferol
(21), 4,9-diisobutyryl-coniferol (22),22 4,9-dibutyryl-coniferol
(23),21 4-butyryl-9-isovaleryl-coniferol (24), 4-isovaleryl-9-bu-
tyryl-coniferol (25), 4,9-di(2S-methylbutyryl)-coniferol (26), 4,9-
diisovaleryl-coniferol (27),23 4,9-divaleryl-coniferol (28),21 4,9-di-
hexanoyl-coniferol (29),21 4,9-diheptanoyl-coniferol (30),21 and
4,9-dioctanoyl-coniferol (31).21 Compounds 14, 15, 17, 19–21,
and 24–26 are nine new compounds. The 1H, 13C NMR, and
HRESIMS data of these new compounds were summarized in
Tables 1 and S1. Compounds 1 and 21, 14 and 15, 17 and 18, 19
and 20, 24 and 25 are five pairs of isomers with two different acyl
groups at C-4 and C-9. It was found that the two isomers of each
pair can be differentiated by their HPLC retention times (tR), where
compound with shorter chain acyl group at C-4 had a shorter tR
value, while compound with shorter chain acyl group at C-9 had
a longer tR value. It is also worthwhile to mention that each of
the 1H signals of the acyl group at C-4 usually resonated at down-
field, when compared to its counterparts of the same acyl group at
C-9.
Compound 1 was obtained as a colorless oil and had a molecular
formula of C17H22O5, which was deduced from its HRESIMS at m/z
[M+Na]+ 329.1357 (calcd for C17H22NaO5, 329.1365). Its 13C NMR
spectrum displayed 17 signals for two carbonyls (d 174.9 and
170.9), eight olefinic and aromatic carbons, one oxymethylene (d
65.3), one methoxyl (d 56.2), one methine (d 41.6), one methylene
(d 27.5), and three methyls (d 21.1, 17.2, and 12.0). A pair of exo-
cyclic double bond displayed characteristic NMR signals for CH-7
(dC 133.7; dH 6.71, 1H, d, J = 16.0 Hz) and CH-8 (dC 124.9, dH 6.37,
1H, m). The larger coupling constant (J = 16.0 Hz) between H-7
and H-8 implied an E configuration for this double bond. The six
aromatic carbons (d 152.4, 140.9, 136.2, 124.2, 120.1, and 111.4)
were assigned to the benzene ring. Three aromatic proton signals
at
d 7.23 (1H, H-5), d 7.22 (1H, H-2), and 7.10 (1H, dd,
J = 8.0/1.8 Hz, H-6) suggested that this aromatic ring was trisubsti-
tuted. The NMR spectra of 1 displayed resonances due to a meth-
oxyl (d 56.2; dH 3.76) and an acetyl (dC 170.9, 21.1; dH 2.05).
C
These two substitutes were attached to C-3 and C-9, respectively,
as established by HMBC correlations (Fig. 2) of dH 3.76 (OCH3-3)
with dC 152.4 (C-3) and dH 4.80 (2H, dd, J = 6.3/1.2 Hz, H-9) with
dC 170.9 (C-100). In addition, the presence of a 2-methylbutyryl moi-
ety at the C-4 position was indicated by its carbon signals at d
174.9 (C-10), 41.6 (C-20), 27.5 (C-30), 12.0 (C-40), 17.2 (C-50) and pro-
ton signals at dH 2.70 (H-20), 1.86 (H-30a), 1.61 (H-30b), 1.00 (H-40),
and 1.29 (H-50). Long-range HMBC correlations as depicted in
Figure 2 further supported the structure of this methylbutyryl
group. The stereochemistry of C-20 in 1 could be assigned based
on its aD value,20 that is, a negative value for 2R and a positive
value for 2S. The configuration of C-20 in 1 was assigned to be S
25
by a positive aD value ([
a]
+10.6). The full proton and carbon
D
assignments were made by a combination of 1H, 13C, HSQC, and
HMBC spectral analyses. The forementioned evidence led to the
structural determination of 1 as 4-(2S-methylbutyryl)-9-acetyl-
coniferol, a new coniferol derivative, named as tripolinolate A.
The 1H and 13C NMR data of tripolinolate A (1) were listed in
Table 1.
Sulforhodamine B (SRB) assay (Supplementary data) was used
to evaluate the activity of compounds 1–31 and 1c–1e in inhibiting
the proliferation of glioma cells (U251 and U87-MG) and colorectal
cancer cells (HCT-15 and SW620). Doxorubicin (DOX, one of the
most potent of the chemotherapeutic drugs)24 was used as the pos-
itive control. The results (Table 2 and Fig. 4) indicated that tripoli-
nolate A (1) and 4,9-diacetyl-coniferol (13) are the two most active
compounds. Both compounds showed dose-dependent activity in
inhibiting the proliferation of all tested cancer cells with IC50 val-
A simple and efficient method was applied to prepare tripolino-
late A (1) and its analogs (13–31) for bioactive assay to investigate
O
O
O
O
ues in a range of 0.36–12.9
eicosyl ester (4), astragalin (11), and 1-[(butanoyl)phloroglu-
cinyl]-b- -glucopyranoside (12) also had activity against these
tested cancer cell lines and these four compounds were more
lM. Trans-phytol (3), E-ferulic acid
OCH3
Figure 2. Key HMBC correlations of tripolinolate A (1).
D

L. Chen et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2629–2633
2631
Table 1
13C, 1H NMR, and HRESIMS data of compounds 1 and 20 (in MeOD-d4)
No.
1a
1
20
dC
dH (J = Hz)
dC
dH (J = Hz)
dC
dH (J = Hz)
1
2
3
4
5
6
7
8
136.2
111.4
152.4
140.9
124.2
120.1
133.7
124.9
65.3
56.2
174.9
41.6
27.5
12.0
137.1
111.6
152.9
141.2
123.9
120.4
134.5
125.0
66.2
56.4
176.6
42.4
28.1
12.0
137.1
111.5
152.9
141.2
124.0
120.4
134.5
125.0
66.2
56.5
173.5
36.7
19.7
14.0
7.22b, 1H
7.14, 1H, d (1.8)
7.14, 1H, d (1.7)
7.23b, 1H
7.10, 1H, dd (8.0, 1.8)
6.71, 1H, d (16.0)
6.37, 1H, m
4.80, 2H, dd (6.3, 1.2)
3.76, 3H, s
6.94, 1H, d (8.2)
7.01, 1H, dd (8.2, 1.8)
6.66, 1H, d (15.9)
6.31, 1H, m
4.72, 2H, dd (6.3, 1.3)
3.82, 3H, s
6.96, 1H, d (8.1)
7.00, 1H, dd (8.1, 1.7)
6.65, 1H, d (15.9)
6.31, 1H, m
4.72, 2H, dd (6.3, 1.3)
3.83, 3H, s
9
3-OCH3
10
20
2.70, 1H, m
2.62, 1H, sext (7.0)
1.60, 1H, m; 1.77, 1H, m
1.02, 3H, t (7.4)
2.53, 2H, t (7.2)
1.73, 2H, sext (7.2)
1.03, 3H, t (7.2)
30
1.61, 1H, m; 1.86, 1H, m
1.00, 3H, t (7.3)
1.29. 3H, d (7.0)
40
50
17.2
17.3
1.27. 3H, d (7.0)
100
170.9
21.1
172.8
21.0
172.8
21.0
200
2.05, 3H, s
2.08, 3H, s
2.08, 3H, s
HRESIMS
[M+Na]+ 329.1357
[M+Na]+ 329.1357
[M+Na]+ 315.1201
a
Data were recorded in pyridine-d5.
Both signals overlapped with the signal of pyridine-d5.
b
H3COOC
OHC
HOOC
O
O
SOCl2, MeOH, 0oC
Aniline, pyridine
90−95oC, 3 h
+
HO
HO
OH
RT, 7−8 h
OH
OH
OH
1d
1a
1b
1c
OCH3
OCH3
OCH3
O
O
O
O
O
R0
O
O
R
O
R
LiAlH4, THF
Pyridine
RT, 24 h
+
OH
O
R0
RT, 9−10 h
R1
O
R1
OCH3
1e
OCH3
(R0 = R or R1)
1 13 31
1f
,
−
Figure 3. Synthetic route leading to tripolinolate A (1) and its derivatives (13–31) (R, R1 = –CH3, –CH2CH3, –CH(CH3)2, –(CH2)2CH3, –(2S)-CH(CH3)CH2CH3, –CH2CH(CH3)2, –
CH2(CH2)2CH3, –CH2(CH2)3CH3, –CH2(CH2)4CH3, –CH2(CH2)5CH3).
Table 2
Activity of compounds inhibiting the proliferation of cancer cells (IC50: lM)
Compounds
Cancer cells
Compounds
Cancer cells
U87-MG
U251
HCT-15
SW620
U87-MG
U251
HCT-15
SW620
1
3
4
11
12
13
16
0.48 0.08
8.02 0.56
5.24 0.19
3.12 0.36
3.89 0.11
1.06 0.03
28.8 0.59
12.9 0.97
27.8 4.61
26.8 4.37
37.6 5.79
19.7 0.27
9.71 0.36
52.6 3.03
4.58 0.48
22.5 0.83
15.1 0.21
23.5 2.38
1.91 0.24
3.26 0.58
29.6 3.64
0.99 0.18
5.85 0.54
5.25 0.37
4.60 0.13
0.41 003
0.36 0.19
47.4 6.18
19
20
21
22
23
DOX
18.5 0.95
10.5 1.94
15.0 1.55
29.4 1.80
17.3 0.72
0.93 0.07
33.3 0.92
45.7 2.04
28.1 0.33
>50
>50
2.20 0.11
22.8 1.16
17.1 2.68
34.1 0.68
34.6 2.97
28.8 1.79
1.13 0.08
10.23 0.42
7.59 0.74
21.9 0.49
35.5 1.36
7.85 1.50
1.33 0.06
Figure 4. Tripolinolate A (1) and 4,9-diacetyl-coniferol (13) inhibiting the growth of different glioma and colorectal cancer cells. Tumor cells were treated with different
concentrations of tested compounds 1 and 13 for 72 h, and then assayed by SRB.
potent against U87-MG and SW620 cells (IC50 0.41–8.02
against U251 and HCT-15 cells (IC50 15.1–37.6 M). The com-
pounds 16 and 19–23 showed moderate activity (IC50: 7.59–
l
M) than
45.7
activity (IC50 >50
tripolinolate A (1) and its analogs (13–31) suggested that the
l
M) and the rest of the tested compounds had weak to no
l
lM). Structure–activity relationship analysis for

2632
L. Chen et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2629–2633
molecular size of acyl groups at C-4 and C-9 had an effect on the
activity of this type of coniferol derivatives. It seems that com-
pounds with larger molecular acyls at C-4 and C-9 had less activity
than those with small molecular acyls at C-4 and C-9. For example,
4,9-dihexanoyl-coniferol (29), 4,9-diheptanoyl-coniferol (30), and
4,9-dioctanoyl-coniferol (31) showed no activity even at high
apoptosis in U87-MG cells with a 38.5% (for 1) or 50.69% (for 13)
increase in total apoptotic cells. The positive control DOX also
induced apoptosis in U87-MG cells with a 24.53% increase in total
apoptotic cells.
Compounds 1–31 and 1c–1e were also assayed for their activity
in inhibiting the growth of Staphylococcus aureus and Escherichia
coli (Supplementary data). However, none of these compounds
was found to be active.
concentration of 200 lM.
Tripolinolate A (1) and 4,9-diacetyl-coniferol (13) were evalu-
ated on their ability to induce apoptosis in glioma U87-MG cells.
The apoptosis and necrosis induced by 1 (20 lM) and 13 (20 lM)
Summarily, a new coniferol derivative, tripolinolate A, was
isolated from a halophyte of T. vulgare that was collected from a
sea beach. The structure of this novel compound was determined
as 4-(2S-methylbutyryl)-9-acetyl-coniferol. A synthetic method
was designed to generate tripolinolate A and its 19 analogs, includ-
ing nine new chemical entities. Tripolinolate A (1) and its analog
4,9-diacetyl-coniferol (13) were the two most active compounds
and significantly inhibited the proliferation of glioma and colorec-
in U87-MG cells were determined by DAPI (4,6-diamidino-2-
phenylindole) and PI (propidium iodide) double staining
(Supplementary data). Under fluorescence microscope, the control
glioma U87-MG cells displayed blue fluorescence with consistent
nucleus intensity. By contrast, apoptotic cells induced by 1 and
13 showed bright blue nuclear pyknosis stained by DAPI and
necrotic cells displayed red fluorescence when stained by PI
(Fig. 5). Annexin V-FITC/PI double staining using flow cytometry
(Supplementary data) was used to further quantify the apoptotic
cells. As shown in Table 3 and Figure 6, compared to the control
(CON), tripolinolate A (1) and 4,9-diacetyl-coniferol (13) induced
tal cancer cells with IC50 values in range of 0.36–12.9 lM and
induced apoptosis in cancer cells. Structure–activity relationship
analysis suggested that the molecular size of acyl moieties at C-4
and C-9 might have an effect on the activity of this type of coniferol
derivatives. The in vivo anti-tumor potential of tripolinolate A and
Figure 5. Tripolinolate A (1) and 4,9-diacetyl-coniferol (13) induced apoptosis and necrosis in U87-MG cells. U87-MG cells were treated with tripolinolate A (1, 20
lM) and
4,9-diacetyl-coniferol (13, 20 M) for 72 h and then double stained with DAPI and PI. Apoptotic cells showed bright blue nuclear condensation stained by DAPI and necrotic
l
cells stained by PI displayed red fluorescence.
Table 3
Tripolinolate A (1) and 4,9-diacetyl-coniferol (13) induced apoptosis in U87-MG cells
Apoptotic cells
CON
DOX (10
lM)
1 (20
l
M)
13 (40
lM)
DOX–CON
1–CON
13–CON
Early apoptotic cells (%)
Late apoptotic cells (%)
Total apoptotic cells (%)
3.41
3.23
6.64
20.15
11.02
31.17
9.02
36.12
45.14
21.62
35.71
57.33
16.74
7.79
24.53
5.61
32.89
38.50
18.21
32.48
50.69
Figure 6. Tripolinolate A (1) and 4,9-diacetyl-coniferol (13) induced apoptosis in U87-MG cells quantified by cytometric analysis with annexin V-FITC/PI double staining.
U87-MG cells were treated with tested compound 1 (20 M) or 13 (40 M) for 72 h, stained with annexin-V FITC and PI, and then analyzed by flow cytometry. Fractions B1,
B2, B3 and B4 represent necrotic cells, late apoptotic cells, glioma cells, and early apoptotic cells, respectively. Doxorubicin (DOX, 10 M) was used as positive control.
l
l
l

L. Chen et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2629–2633
2633
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We greatly appreciate Professor Shuili Zhang at the Zhejiang
University of Traditional Chinese Medicine for the authentication
of Tripolium vulgare, and Dr. Zhongjun Ma and Dr. Pinmei Wang
at Ocean College of Zhejiang University for the gifts of
Staphylococcus aureus and Escherichia coli. Authors also thank
Mrs. Jianyang Pan at the Pharmaceutical Informatics Institute of
Zhejiang University for performing NMR spectrometry for struc-
ture elucidation. This research was supported by Grants from the
National Natural Science Foundation of China (No. 81273428)
and the Cross-Disciplinary Research for Ocean Science of
Zhejiang University (No. 2012HY018B).
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Supplementary data
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Supplementary data associated with this article can be found, in
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