Semisynthesis and in vitro cytotoxic evaluation of new analogues of 1-O-acetylbritannilactone, a sesquiterpene from Inula britannica

Article abstract of DOI:10.1016/j.ejmech.2014.04.028

Semisynthetic analogues of the natural product 1-O-acetylbritannilactone (ABL), a sesquiterpene isolated from the medicinal plant Inula britannica, have been prepared and exhibited significant in vitro cytotoxic activities against four cell lines includin

Full text of DOI:10.1016/j.ejmech.2014.04.028

European Journal of Medicinal Chemistry 80 (2014) 71e82
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Semisynthesis and in vitro cytotoxic evaluation of new analogues
of 1-O-acetylbritannilactone, a sesquiterpene from Inula britannica
*
Shuai Dong, Jiang-Jiang Tang , Cheng-Chen Zhang, Jun-Mian Tian, Jun-Tao Guo,
*
Qiang Zhang, He Li, Jin-Ming Gao
Shaanxi Engineering Center of Bioresource Chemistry & Sustainable Utilization, College of Science, Northwest A&F University, Yangling 712100, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Semisynthetic analogues of the natural product 1-O-acetylbritannilactone (ABL), a sesquiterpene isolated
from the medicinal plant Inula britannica, have been prepared and exhibited significant in vitro cytotoxic
activities against four cell lines including three human cancer cell lines (HCT116, HEp-2 and HeLa) and
one normal hamster cell line (CHO). Structureeactivity relationships indicate that esterification of 6-OH
Received 19 December 2013
Received in revised form
6 April 2014
Accepted 7 April 2014
Available online 13 April 2014
(enhanced lipophilicity) and
cytotoxicity. Among the tested compounds, 14 bearing a lauroyl group (12C) at the 6-OH position dis-
played most potent in vitro cytotoxic activity, with IC50 values between 2.91 and 6.78 M, comparable to
the positive control etoposide (VP-16, IC50 values between 2.13 and 4.79 M). Moreover, the compound
a-methylene-g-lactone functionalities play important roles in conferring
m
Keywords:
m
1-O-Acetylbritannilactone sesquiterpene
Cytotoxic activity
Structureeactivity relationship
14 triggered remarkable apoptosis at a low concentration, and induced cell cycle arrest in G2/M phase in
HCT116 cells. The biological assays conducted with normal cells (CHO) revealed that all the synthetic
compounds are no selective against cancer cell lines tested.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
An analogue of ABL, 1,6-O,O-diacetylbritannilactone (OABL,
Fig. 1) isolated from I. britannica, was ten times more potent than
Inula britannica L. (Xuan fu hua in Chinese) from Compositae
family is one of the most popular and multi-purpose traditional
Chinese medicinal herbs, and has high sesquiterpenes content [1,2].
1-O-acetylbritannilactone (ABL, Fig. 1), a 1,10-seco-eudesmanolide
sesquiterpene extracted from I. Britannica, has several biological
effects including anti-inflammatory, antibacterial, antihepatitic,
antidiabetes, and antitumour activities [3e8]. It has been shown to
possess anticancer effects in various cancer cells [4,5,9e12],
including anti-proliferation, cell cycle arrest, induction of apoptosis
and increased sensitivity to apoptosis. ABL induced cell cycle arrest
in G0/G1 phase of human colon cancer HT-29 cells accompanied by
a strong decrease of cyclin E and CDK4 protein levels, and an in-
crease in p21 protein expression [4]. ABL-induced growth inhibi-
tion is also associated with the upregulation of KLF4 expression [4]
and ABL induced phosphorylation of Bcl-2 in breast, ovary and
prostate cancer cell lines [9]. Moreover, ABL and celecoxib (Fig. 1)
could interact synergistically to suppress breast cancer cell growth
via COX-2-dependent and -independent mechanisms [13].
ABL in HL-60 and MCF-7 cells [3,5,14]. OABL also showed strong
induced-apoptosis associated with activation of caspase-8, -9, and
-3, phosphorylation of Bcl-2 and Bid, and increased release of cy-
tochrome c from mitochondria in promyelocytic leukemia HL-60
[11]. The more potent activity of OABL has been considered due
to the acetyl group at 6-hydroxy (6-OH) position enhancing the
molecular lipophilicity [15]. Another important structural factor
related to their bioactivity may be the presence of an electrophilic
a-methylene-g-lactone motif, which can bind thiols of proteins or
residues as alkylating reagent to induce the DNA-fragmentation
and apoptosis mediated by glutathione depletion of the cells [16e
18]. This was also confirmed in many studies of other sesquiter-
pene lactones [18], such as parthenolide [19,20], helenalin [21]
(Fig. 1). Besides, potent bioactivity of OABL may correlate other
factors, such as molecular geometry and chemical environment.
The high content (0.2%e0.5% weight of dry flowers and stems)
[22] of ABL and potent anticancer activity associated with ABL and
OABL in I. britannica has prompted interest in designing novel ABL
analogues with anticancer activity superior to that of the parent
compound [14,15,23,24]. So far, structural modifications of ABL
focused mainly on alterations at 6-OH moiety, and some of these
analogues have turned out to be more cytotoxic against various
human cancer cells than ABL and OABL [14,15,23,24]. For example,
* Corresponding authors.
E-mail addresses: tangjiang11@nwsuaf.edu.cn (J.-J. Tang), jinminggao@nwsuaf.
edu.cn (J.-M. Gao).
http://dx.doi.org/10.1016/j.ejmech.2014.04.028
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

72
S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
and optical rotation data. So to determine which the relative
configuration of ABL was correctly depicted in the literatures, we
obtained X-ray diffraction data of ABL from a SuperNova, Dual, Eos
diffractometer with Cu-K
Supplementary data). The X-ray diffraction analysis of ABL unam-
biguously verified the out-of-plane of 4-methyl and the 6 -orien-
a radiation [32] (Fig. 1) (see Table S1 in
a
tation of the secondary hydroxyl group. As a result, the relative
configuration of (þ)-ABL should be revised as 4S*, 6S*, which is in
accord with that of (þ)-britannilactone ([ ] ¼ þ86.0 (c ¼ 0.57 in
a
CHCl3)) obtained by total synthesis [33]. It can be concluded that in
the crystal structure (Fig. 1) a six-membered ring adopted a slightly
twisted boat conformation and was fused by a planar five-
membered ring; and 6-OH of ABL located in the backside of the two
rings with small steric hindrance could be a modifiable site.
2.1.2. Synthesis of ABL 6-OH modified analogues (1e19)
The synthesis of ABL 6-OH modified analogues 1e19
commenced from starting material ABL. In a conventional proce-
dure, ABL was converted to the corresponding ester analogues with
aliphatic side chains and aromatic moieties using different acid
anhydrides (Scheme 1) or long chain acids (Scheme 2). This upon
treatment with various acid anhydrides in the presence of trie-
thylamine (Et3N) and catalytic dimethylaminopyridine (DMAP) in
dried dichloromethane (DCM) afforded ester analogues 1e11 in
excellent yields (85%e98%) and in short time (10 mine4 h).
Treatment of ABL with various long chain fatty acids in anhydrous
DCM solution using dicyclohexylcarbodiimide (DCC) as activating
reagent of carboxylic acid gave the desired compounds 12e16 in
70%e75% yields. Furthermore, reaction of ABL with p-Br- or p-CF3-
substituted cinnamic acids provided the expected compounds 17
and 18 in 65% and 83% yields, respectively. Additionally, mild
oxidation of ABL with DesseMartin periodinane (DMP) afforded
ketone 19 in 43% yield.
Fig. 1. The structures of 1-O-acetylbritannilactone (ABL), 1,6-O,O-diacetyl-
britannilactone (OABL), X-ray of ABL, Celecoxib and two sesquiterpene lactones.
Ho and co-workers have synthesized some 1-O-acetyl-
britannilactone oxime esters, of which four esters show better
cytotoxic activity on HL-60 and human hepatoma Bel-7402 cell
lines [14,15,23].
Our ongoing efforts focused on the introduction of chemical
diversity in the molecular framework in order to prepare plant-
derived pharmacologically interesting compounds [25e27]. This
prompted us to synthesize a series of new ABL analogues and
evaluate their structureeactivity relationships. We firstly per-
formed the isolation of ABL from I. britannica, and the structure of
ABL was elucidated by NMR, ESI-MS spectroscopy and X-ray crys-
tallographic data (Fig. 1). Then, using ABL as the starting material,
we semisynthesized a series of 6-OH modified and C13-modified
arylation analogues. For 6-OH modified analogues, we tried out
several standard esterification methods to convert ABL into a series
of new esters of various aliphatic and cinnamic carboxylic acids
(Schemes 1 and 2). Furthermore, to examine effect of the oxidation
of 6-OH on cytotoxic properties, we oxidized 6-OH into 6-ketone
using DesseMartin oxidation (Scheme 3). For C13-modified aryla-
tion analogues, we conducted a series of reactions with readily
available aryl iodides using the previously reported Heck reaction
conditions [28,29] (Scheme 4). Herein, we report the semisyn-
thesis, the structureeactivity relationship of ABL analogues based
on in vitro cytotoxic activities and preliminary cytotoxic mechanism
of the most active compound.
2.1.3. Synthesis of OABL C13-modified arylation analogues (20e24)
The synthetic route of target compounds was obtained in
Scheme 4. Five OABL arylation analogues 20e24 were synthesized
by Heck coupling reaction of more active OABL with readily avail-
able aryl iodides. Indeed, when 5 mol% of Pd(OAc)2 with Et3N in
anhydrous DMF at 80 ꢁC was used, a main product with an exocyclic
olefin was isolated in each case after purification in moderate to
good yields (41%e82%). The substitution pattern on the aromatic
ring and the presence of electron-donating or electron-
withdrawing substituents did not affect the yields or the prefer-
ence for a main olefin geometry in the isolated product. The
assignment of the C11eC13 olefin geometry of 20 was determined
using NOESY NMR experiments to be the E-olefin. Specifically,
NOESY signals were readily apparent between the two protons of
phenyl ring and the protons attached to C6 and C7 on the six-
membered ring (See Fig. S1 in Supplementary data). Also, the C13
benzylic proton for 20 had a chemical shift of 7.60 ppm that sup-
ports the assignment as an E-olefin. The assignment of the E-ge-
ometry for the compound 23 was further verified following
determination of an X-ray crystal structure (Fig. 2) [32]. For 21, 22
and 24, the assignment of the E-olefin geometry was accomplished
by the diagnostic chemical shift of the C13 proton. These data
support that the preferential selectivity for the E-isomer (over the
Z-isomer) from the Heck reaction [34], which is in accord with prior
work results of Colby and co-workers [28], whom semisynthetized
sesquiterpene lactone derivatives by the palladium-catalyzed ary-
2. Results and discussion
2.1. Chemistry
2.1.1. Isolation and characterization of ABL
The AcOEt-soluble fraction of the ethanolic extract of the dried
flowers of I. britannica was repeatedly subjected to column chro-
matography over silica gel, followed by recrystallization from EtOH
to give the pure compound (þ)-ABL (½a D25
¼ þ103.5 in CHCl3),
ꢀ
which was found to be identical to the natural product [2,3,7] by
comparison of the spectral and optical rotation data. However,
three relative configurations of ABL present in the same plant have
been reported, i.e., (4S,6R) e [9,14,15], (4R,6S) e [7,30], and (4S,6S)-
ABL [2,31], but the three stereoisomers had similar NMR spectral
lation for parthenolide containing a a-methylene-g-lactone motif.
The structures of all 24 synthetic compounds were confirmed by
1D-NMR, 2D-NMR and HR-ESI-MS. Among them, 23 compounds
except 1 (OABL) were new. The purity of all compounds was
confirmed to be greater than 95% by HPLC with UV and evaporative

S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
73
Scheme 1. Conditions and reagents: (a) DMAP, Et3N, CH2Cl2, 0 ꢁC to rt.
cancer), HEp-2 (human larynx epidermal cancer) and HeLa (human
cervix cancer) using the SRB assay [35]. Etoposide was used as
positive control in this experiment. The IC50 values (the concen-
tration to cause 50% inhibition of cell viability) of these compounds
are summarized in Tables 1 and 2.
For the ABL 6-OH modified analogues 1e19, most compounds
generally showed good cytotoxicity against the three cancer cell
lines tested. Notably, acetylation of 6-OH of ABL gave OABL 1, which
showed high cytotoxicity toward HCT116, HEp-2 and HeLa cells,
with IC50 values of 10.1, 7.60 and 11.2 mM, respectively, with being
approximately 3-fold more efficacious than the parent compound
ABL. In addition, Ho et al. claimed that 1 has more anticancer effects
than ABL on four human cancer cell lines [36], promyelocytic leu-
kemia HL-60, colon adenocarcinoma HT-29, colon adenocarcinoma
COLO 205 and gastric carcinoma AGS. These results suggest that 1
would exhibit different sensitivity to various cancer cell lines.
Scheme 2. Conditions and reagents: (a) DCC, DMAP, Et3N, CH2Cl2, rt.
Compounds 2e10 (except 7 with IC50 25.0 mM to HEp-2) and 12e15,
possessing different aliphatic chains at 6-OH position, exhibited
higher cytotoxic activity than ABL against HCT116, HEp-2 and HeLa
light scattering detection (ELSD) (Supplementary data) prior to
their use in the evaluation of their biological efficacies. It should be
noted that the synthetic approach for this family of 6-OH modified
compounds used in the current study is a simple, effective method,
especially reaction with cheap acid anhydrides, short time and high
yields. For C13-modified compounds, the arylation of OABL via
cross-coupling reaction catalyzed by palladium is achievable. Be-
sides, all these compounds showed ester stability at the 10 mM of
concentration stayed for at least 3 days in PBS (pH ¼ 7.4) or culture
medium with HPLC analysis (data not shown).
cells (IC50 data ranging from 3 to 28
bearing a succinyl chain (eCOCH2CH2COOH) did not show any ac-
tivity with IC50 values all more than 70 M. These data indicate that
mM). However, in contrast, 11
m
introduction of the appropriate lipophilic aliphatic chains
improved potency in the activity.
2.2. Cytotoxicity
ABL and its analogues 1e24 were evaluated for in vitro cytotoxic
activities against three cancer cell lines HCT116 (human colorectal
Scheme 3. Conditions and reagents: (a) DMP, CH2Cl2, rt.
Fig. 2. The structure and X-ray of compound 23.
Scheme 4. Conditions and reagents: (a) Pd(OAc)2 (5 mol%), Et3N, DMF, 80 ꢁC.

74
S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
Table 1
cells (IC50 > 100 mM). This suggests that the aliphatic chain length
Cytotoxic activities (IC50) of ABL and its 6-OH modified analogues (1e19).
at 6-OH is decisive for the activity, and 12C aliphatic side chain may
be optimal length for the cytotoxic activity. The improvement of
bioactivity is a comprehensive effect of many factors, but in the case
perhaps the most important reason is the introduction of 12 car-
bons aliphatic side chain ester which perhaps fit for good binding at
the molecular target’s pocket. While the succinate 11 with terminal
COOH moiety loss the activity may not fit for good binding at the
molecular target.
a
No.
R
IC50
(
m
M)
HCT116
HEp-2
HeLa
CHO
On the other hand, it can also be seen from Table 1 that intro-
duction of substituted cinnamic acids to ABL could improve cyto-
toxicity towards HCT116, HEp-2 and HeLa. For example, 17 and 18,
ABL
1 (OABL) Ac
H
36.1 ꢂ 3.1
10.1 ꢂ 1.1
19.3 ꢂ 1.5
32.6 ꢂ 2.5
41.3 ꢂ 4.5
7.60 ꢂ 0.51 11.2 ꢂ 0.8
8.57 ꢂ 1.25
2
7.78 ꢂ 1.51 9.19 ꢂ 1.78 7.78 ꢂ 0.34 4.97 ꢂ 0.43
with IC50 data of 7e18
mM, had similar activity but had both
stronger activity than ABL. This indicates that the presence of a p-
bromo or p-CF3 (trifluoromethyl) group in the benzene ring
appeared to have no significant on the activity.
3
4
5
6
7
8
9
8.39 ꢂ 0.81 9.40 ꢂ 1.20 7.98 ꢂ 1.13 5.20 ꢂ 0.50
12.1 ꢂ 0.6
12.7 ꢂ 1.5
10.2 ꢂ 1.3
12.4 ꢂ 1.6
18.8 ꢂ 2.3
15.2 ꢂ 1.6
18.2 ꢂ 2.1
14.9 ꢂ 1.6
25.0 ꢂ 3.5
18.6 ꢂ 4.2
18.2 ꢂ 2.1
19.2 ꢂ 2.3
17.5 ꢂ 0.5
28.2 ꢂ 1.5
28.5 ꢂ 3.1
14.1 ꢂ 1.6
6.35 ꢂ 0.51
12.2 ꢂ 1.1
16.5 ꢂ 2.1
19.4 ꢂ 2.5
10.5 ꢂ 1.2
8.44 ꢂ 1.56
When 6-OH of ABL was oxidated to a ketone group providing
analogue 19 (IC50 ¼ 32.6
that of the parent (IC50 ¼ 36.1
had no effects on other two cancer cell lines tested (IC50 > 90
m
M), which had a similar cytotoxic effect to
M) against HCT116 cells, whereas it
M),
m
m
implying that conversion of 6-OH of ABL to a ketone group resulted
in loss of activity. In addition, it seems to be concluded from Table 1
that HCT116 cells, compared with other two cells, were higher
sensitivity to most ABL analogues.
In order to enrich the chemical diversity of ABL molecular
framework, aryl groups were introduced into the
lactone motif of 1 (OABL) to decrease -methylene nucleophilic
activity giving 20e24. Although the electrophilic -methylene-g-
a-methylene-g-
6.54 ꢂ 1.55 10.5 ꢂ 1.9
a
a
10
10.3 ꢂ 0.55 10.8 ꢂ 0.59 18.2 ꢂ 1.9
16.7 ꢂ 2.4
lactone is known to trap nucleophilic intracellular thiols, such as
cysteine residues [16e18], the biological effect of arylation of this
functional group for OABL is not known. From Table 2, it could be
seen that arylation analogues 20e24 resulted in decreased potency
11
12
13
14
15
16
17
>100
71.3 ꢂ 8.9
>100
91.8 ꢂ 10.5
15.2 ꢂ 3.5
11.9 ꢂ 2.5
8.97 ꢂ 2.64 16.4 ꢂ 1.8
21.3 ꢂ 2.5
(IC50 > 50
compared with that of the parent ABL and OABL, which hinted at
the importance of the unscreened -methylene functionality.
mM and >40 mM on HCT116 and HeLa cells, respectively)
8.40 ꢂ 1.54 15.0 ꢂ 1.64 16.9 ꢂ 0.7
a
Moreover, we assessed whether ABL and analogues had any
differential sensitivity to normal versus cancer cells. Recently, the
enone-containing natural product piperlongumine has been found
to possess remarkable selectivity for promoting the death of cancer
cells versus normal cells [37]. Similarly, to determine if ABL and its
analogues are selective, we measured their cytotoxicity against
normal cell lines CHO (Chinese hamster ovary). As shown in
Tables 1 and 2, the sensitivity of all compounds was low with
approximate IC50 data, suggesting that ABL analogues may have no
selectivity toward cancer cells.
2.91 ꢂ 0.61 5.85 ꢂ 0.45 6.78 ꢂ 0.23 5.97 ꢂ 0.12
5.54 ꢂ 2.51 14.0 ꢂ 1.5
21.9 ꢂ 3.5 >100
7.69 ꢂ 0.51 13.1 ꢂ 0.5
15.6 ꢂ 1.2
>100
14.9 ꢂ 2.5
>100
18.2 ꢂ 2.1
24.1 ꢂ 2.5
18
10.2 ꢂ 0.8
32.6 ꢂ 4.5
9.80 ꢂ 0.51 18.3 ꢂ 0.8
91.2 ꢂ 11.5 >100
9.54 ꢂ 1.57
91.0 ꢂ 4.5
19
Etoposide
O
e
2.13 ꢂ 0.23 4.79 ꢂ 0.54 2.97 ꢂ 0.25 2.60 ꢂ 0.15
Table 2
Cytotoxic activities (IC50) of OABL arylation analogues (20e24).
a
The IC50 values represent the concentration to cause 50% inhibition of cell
viability. Cancer cell lines HCT116 (human colorectal cancer), HEp-2 (human larynx
epidermal cancer) and HeLa (human cervix cancer) and normal cell line CHO (Chi-
nese hamster ovary) were treated with ABL and its 6-OH modified analogues for
72 h. All data (mean ꢂ SD) are the average of three or four determinations.
Interestingly, of the test synthesized esters, compound 14,
bearing a lauroyl group (12C) at the 6-OH position, displayed the
a
No.
R
IC50
(
mM)
highest potency with IC50 values of 2.91, 5.85 and 6.78
mM toward
HCT116
HEp-2
HeLa
CHO
HCT116, HEp-2 and HeLa cells, respectively. This compound was
about 10, 4 and 5 times, respectively, more active than ABL, which is
20
21
22
23
24
H
4-F
4-Br
4-OMe
3,4,5-triOMe
59.1 ꢂ 2.3
>100
e
e
e
e
e
92.1 ꢂ 2.5
73.5 ꢂ 6.1
45.5 ꢂ 4.9
77.1 ꢂ 7.0
90.9 ꢂ 1.6
42.4 ꢂ 5.5
23.6 ꢂ 3.8
10.5 ꢂ 1.4
e
70.2 ꢂ 5.3
comparable to etoposide (IC50 data of 2.13, 4.79 and 2.97
mM,
>100
respectively), the commonly used chemotherapeutic agent. In
contrast to 14, compounds 15 with a myristoyl group (14C) and 16
with a stearoyl group (18C) had low activity, especially 16 led to a
marked reduction in the cytotoxicity against HCT116 cells
>100
e
a
The IC50 values represent the concentration to cause 50% inhibition of cell
viability. Cancer cell lines HCT116, HEp-2 and HeLa and normal cell line CHO were
treated with OABL arylation analogues for 72 h. All data (mean ꢂ SD) are the average
of three or four determinations.
(IC50 ¼ 21.9
mM) and to a loss in activity against HEp-2 and HeLa

S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
75
2.3. Apoptosis-inducing effects of ABL, 1 (OABL) and 14 in
HCT116 cells
from that of ABL and similar with that of OABL in cell cycle
progression.
It is well recognized that apoptosis is a very important mecha-
nism involved in the anti-cancer effect and can be characterized by
changes in nuclear morphology [38]. In order to determine the
mechanism involved in ABL and its active analogues-induced
cytotoxicity, nuclear morphology in HCT116 cells was evaluated
by fluorescent microscopy. Staining with Hoechst 33258 showed
fragmentation and condensation of chromatin in HCT116 cells
3. Conclusion
In summary, we have described determination of the absolute
configuration of ABL, the semisynthesis of a series of new ABL an-
alogues by modifying the 6-OH and C13 methylene groups, and
evaluated their in vitro cytotoxicity to three human cancer cell lines
(HCT116, HEp-2 and HeLa) and one normal hamster cell line (CHO)
in vitro. Compounds 2e6, 8e10, 12e15, 17 and 18 were found to
have significant cytotoxic effects on these cancer cell lines, while
exhibited no selectivity toward normal cells. The present study
indicated that introduction of appropriate enhanced lipophilic
aliphatic chains at 6-OH of ABL leads to an increase in the activity,
treated with 50 mM ABL and 20 mM 1 (OABL) for 48 h, compared
with the untreated control (Fig. 3), demonstrating a proapoptotic
activity of ABL and OABL, which is similar with reported results in
HL-60, COLO 205, HT-29 and AGS cells [4,11,36]. This apoptotic
tendency was more apparent in 14 (lauroyl) at relatively low con-
centration (5 mM).
while arylation (compounds 20e24) of
a-methylene-g-lactone
resulted in decreased potency. Impressively, compound 14, bearing
a lauroyl group (12C), demonstrated most potent in vitro cytotoxic
effects, comparable to the positive drug etoposide, and cytotoxicity
induced by the compound against HCT116 cells was mediated by
apoptosis and cell cycle arrest at G2/M phase. These results indicate
that 14 could act as a potential hit for the development of newer
anticancer agents and would be further worth clarifying molecular
protein target of the compound.
2.4. Effects of ABL, 1 (OABL) and 14 on cell cycle of HCT116 cells
To further explore the mechanisms by which ABL, 1 (OABL) and
14 exert their cytotoxic potencies, their effects on the cell cycle
distribution of HCT116 cells were evaluated by flow cytometry after
cells were stained with propidium iodide [39]. As shown in Fig. 4,
treatment with 50 mM ABL resulted in accumulation of 46.4% cells
at G0/G1 phase, as compared to 39.1% in untreated cells at 24 h.
Similar accumulation of cells in G0/G1 phase has been reported
previously in another human colon cancer HT-29 cells exposed to
4. Materials and methods
4.1. Chemistry
ABL [4]. However, treatment of the compounds 1 (OABL, 20 mM)
resulted in a clear block of cells in G2/M phase, accumulation of
25.4% compared with 17.9% in untreated cells, which is similar to
previous reports at colorectal cancer cells and breast cancer cells
4.1.1. General
Optical rotations were measured on a Rudolph Autopol III
automatic polarimeter (Rudolph Research Analytical). All NMR
spectra were recorded on a 400 MHz AMX Bruker NMR spec-
trometer in CDCl3 with TMS as internal standard for protons and
solvent signals as internal standard for carbon spectra. Chemical
[5]. When HCT116 cells were in treatment of 14 (5 mM) for 24 h, a
clear block of cells in G2/M phase was also observed with 26.9%
cells accumulation, revealing that the superior cytotoxicity of 14
over ABL and 1 (OABL) was associated with a mechanism different
shift values are mentioned in d (ppm) and coupling constants (J) are
Fig. 3. Induction of apoptosis by ABL, 1 (OABL) and 14 at the indicated concentrations in HCT116 cells. 48 h after the treatment of these compounds at the indicated concentrations,
cells were fixed, washed with PBS, stained with Hoechst 33258, and analyzed for morphological characteristics associated with apoptosis by fluorescence microscopic analysis
(40ꢃ).

76
S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
Fig. 4. Effects of ABL, 1 (OABL) and 14 at the indicated concentrations on the cell cycle of HCT116 cells. The cultured cells were treated with these compounds at the indicated
concentrations for 24 h, then harvested, and analyzed by flow cytometry. Each experiment was performed in triplicate.
given in Hz. Mass spectra were recorded on an API2000 mass
spectrometer (AB Sciex) or ESI-Thermo Fisher LTQ Fleet instrument
spectrometer (Thermo Scientific). HR-ESI-MS spectra were ob-
tained on a Bruker Daltonics APEx II FT-ICR mass spectrometer.
Analytical HPLC was performed on a Waters 1525 series with UV
detection at 215 or 254 nm along with evaporative light scattering
4.1.2.1. 1-O-Acetylbritannilactone
a 2D5
¼ þ103.5 (c ¼ 0.52 in CHCl3; see Ref. [3] [
in CHCl3)); 1H NMR (400 MHz, CDCl3):
0.95e1.04 (m, 1H, H-3b),
(ABL). Cubic
crystals,
½
ꢀ
a]
D
¼ þ101.6 (c ¼ 0.25
d
1.07 (d, J ¼ 6.9 Hz, 3H, H-15), 1.19e1.31 (m, 2H, H-2b, H-3a), 1.34e
1.47 (m, 1H, H-2a), 1.76 (s, 3H, H-14), 2.04 (s, 3H, AcO-1), 2.46 (dd,
J ¼ 16.2, 2.0 Hz, 1H, H-9b), 2.69 (ddd, J ¼ 9.5, 6.9, 4.4 Hz, 1H, H-4),
2.77e2.90 (m, 1H, H-9a), 3.50e3.59 (m, 1H, H-7), 3.86e4.02 (m, 2H,
H-1), 4.18 (s,1H, H-6), 4.97e5.05 (m,1H, H-8), 5.72 (d, J ¼ 2.3 Hz,1H,
H-13b), 6.31 (d, J ¼ 2.7 Hz, 1H, H-13a); 13C NMR (100 MHz, CDCl3):
detection (ELSD), Method ¼ Agilent TC-C18, 5 m, 4.6 ꢃ 250 mm,
m
10 min gradient, 80%MeOH:20%H2O to 100% MeOH. Column
chromatography (CC) was performed over silica gel (200e
300 mesh, Qingdao Marine Chemical Ltd.). The progress of all re-
actions was monitored by TLC on 2 cm ꢃ 5 cm precoated silica gel
60 F254 plates of thickness of 0.25 mm (Qingdao Marine Chemical
Group, Co.). Spots were visualized UV light (254, 365 nm) and/or by
staining with 5% phosphomolybdic acid followed by heating. All
commercially available solvents and reagents were freshly purified
and dried by standard techniques prior to use.
d
19.3 (C-15), 20.6 (C-14), 20.9 (CH3CO-1), 26.5 (C-2), 31.0 (C-3), 32.9
(C-4), 34.4 (C-9), 45.0 (C-7), 64.2 (C-1), 68.3 (C-6), 75.9 (C-8), 123.8
(C-13), 131.1 (C-10), 136.5 (C-5), 136.9 (C-11), 169.9 (C-12), 171.2
(CH3CO-1); ESI-MS: 331.4 [MþNa]þ; HPLC: tR
purity > 99% at ELSD.
¼
3.184 min,
4.1.3. X-ray experimental
Single crystals of ABL and compound 23 were obtained by
recrystallization in ethanol. A suitable crystal was selected and
analysed on a SuperNova, Dual, Cu at zero, Eos diffractometer. The
crystal was kept at 293(2) K during data collection. Using Olex2
[40], the structure was solved with the Superflip [41] structure
solution program using Charge Flipping and refined with the
ShelXL [42] refinement package using Least Squares minimisation.
The X-ray data is available in the reference [32].
4.1.2. Extraction and isolation
ABL was obtained from the flowers of I. britannica according to a
reported procedure [3]. The dried flowers (50 kg) were extracted
with 95% EtOH (3 ꢃ 200 L) for 12 h under refluxing. Evaporation of
the EtOH solution under reduced pressure gave a crude extract, to
which water (20 L) was added. The aqueous extract was succes-
sively partitioned with petroleum ether (PE, 3 ꢃ 20 L), EtOAc
(3 ꢃ 20 L), and n-BuOH (3 ꢃ 20 L). The EtOAc-soluble part (2 kg) was
subjected to silica gel column chromatography eluted with a
gradient solvent system of PE/EtOAc (100:0e1:1) and then CHCl3e
MeOH (1:1) afforded 15 fractions. PE/EtOAc (1:1) fraction gave
crude ABL as crystals, and the pure ABL (ca. 25 g) was obtained by
recrystallization from anhydrous EtOH.
4.1.4. Chemical synthesis
4.1.4.1. Procedure for the synthesis of ABL analogues (1e11). To a
suspension of acid anhydride (0.15 mmol) and DMAP in anhydrous
CH2Cl2 (1 mL) in an ice-bath was added ABL (0.1 mmol) in anhy-
drous CH2Cl2 (1 mL) solution. After completion of the reaction from

S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
77
10 min to 4 h at room temperature, ice water (2 mL) was added to
the solvent and stirred for 20 min, then extracted with CH2Cl2,
dried and filtered. After removal of the solvent, the crude product
was purified by silica gel chromatography (EtOAc/PE) to afford
compounds 1e11 in 85%e98% yields.
(100 MHz, CDCl3):
14), 21.0 (CH3COO-1), 22.2 (CH3CH2CH2CH2COO-6), 26.5 (C-2), 27.0
(CH3CH2(CH2)2COO-6), 31.0 (C-3), 33.0 (C-4), 34.3
d 13.7 (CH3(CH2)3COO-6), 18.5 (C-15), 20.5 (C-
(CH3(CH2)2CH2COO-6), 34.5 (C-9), 42.9 (C-7), 64.2 (C-1), 69.0 (C-6),
74.9 (C-8), 124.9 (C-13), 132.1 (C-5), 133.7 (C-10), 136.3 (C-11), 169.5
(C-12), 171.2 (CH3COO-1), 173.7 (CH3(CH2)3COO-6); HR-ESI-MS:
4.1.4.1.1. 1,6-O,O-Diacetylbritannilactone (1). White solid. Yield:
87%; ½a 2D5
ꢀ
¼ ꢄ39.5 (c ¼ 0.57 in CHCl3; see Ref. [33] [
a
]
¼ ꢄ38.4
calcd for
C
22H32NaO6 [MþNa]þ: 415.2091; found: 415.2100,
D
(c ¼ 0.5 in CHCl3)); 1H NMR (400 MHz, CDCl3):
d
0.88 (d, J ¼ 6.9 Hz,
error ¼ 2.2 ppm; HPLC: tR ¼ 5.997 min, purity ¼ 99% at ELSD, 98% at
3H, H-15), 0.97e1.09 (m, 1H, H-3b), 1.21e1.32 (m, 2H, H-2b, H-3a),
1.35e1.46 (m, 1H, H-2a), 1.81 (s, 3H, H-14), 2.05 (s, 3H, AcO-6), 2.06
(s, 3H, AcO-1), 2.50 (dd, J ¼ 16.2, 2.1 Hz, 1H, H-9b), 2.63e2.76 (m,
2H, H-4, H-9a), 3.46e3.51 (m, 1H, H-7), 3.86e4.01 (m, 2H, H-1),
4.90e5.00 (m, 1H, H-8), 5.22 (d, J ¼ 1.70 Hz, 1H, H-6), 5.95 (d,
J ¼ 2.3 Hz, 1H, H-13b), 6.38 (d, J ¼ 2.7 Hz, 1H, H-13a); 13C NMR
215 nm.
4.1.4.1.5. 1-O-Acetyl-6-O-hexanoylbritannilactone (5). White oil.
Yield: 86%; ½a 2D5
ꢀ
¼ ꢄ45.5 (c ¼ 0.24 in CHCl3); 1H NMR (400 MHz,
CDCl3):
d 0.83e0.92 (m, 6H, H-15, CH3(CH2)4COO-6), 0.97e1.08 (m,
1H, H-3b), 1.21e1.35 (m, 6H, H-2b, H-3a, CH3CH2CH2(CH2)2COO-6),
1.36e1.45 (m, 1H, H-2a), 1.60 (q, 7.1 Hz, 2H,
J
¼
(100 MHz, CDCl3):
d
18.4 (C-15), 20.5 (C-14), 21.0 (CH3CO-6), 21.3
CH3CH2CH2CH2CH2COO-6), 1.79 (s, 3H, H-14), 2.03 (s, 3H, AcO-1),
2.27 (t, J ¼ 7.6 Hz, 2H, CH3(CH2)3CH2COO-6), 2.48 (dd, J ¼ 16.1,
1.7 Hz, 1H, H-9b), 2.61e2.77 (m, 2H, H-4, H-9a), 3.43e3.50 (m, 1H,
H-7), 3.85e4.00 (m, 2H, H-1), 4.93 (dt, J ¼ 4.0, 1.9 Hz, 1H, H-8), 5.20
(d, J ¼ 1.2 Hz, 1H, H-6), 5.93 (d, J ¼ 2.2 Hz, 1H, H-13b), 6.36 (d,
(CH3CO-1), 26.5 (C-2), 31.1 (C-3), 33.0 (C-4), 34.5 (C-9), 42.9 (C-7),
64.2 (C-1), 69.2 (C-6), 74.9 (C-8), 125.0 (C-13), 132.0 (C-5), 133.8 (C-
10), 136.2 (C-11), 169.5 (C-12), 170.9 (CH3CO-6), 171.2 (CH3CO-1);
ESI-MS: 389.0 [MþK]þ; HPLC: tR ¼ 4.164 min, purity ¼ 98% at ELSD,
97% at 215 nm.
J ¼ 2.6 Hz, 1H, H-13a); 13C NMR (100 MHz, CDCl3):
d 13.8
4.1.4.1.2. 1-O-Acetyl-6-O-propionylbritannilactone
(2).
(CH3(CH2)4COO-6), 18.5 (C-15), 20.4 (C-14), 20.9 (CH3COO-1), 22.2
(CH3CH2CH2(CH2)2COO-6), 24.6 (CH3CH2CH2(CH2)2COO-6), 26.4 (C-
2), 31.0 (C-3), 31.2 (CH3CH2CH2CH2CH2COO-6), 33.0 (C-4), 34.5 (C-
9), 34.5 (CH3(CH2)3CH2COO-6), 42.8 (C-7), 64.2 (C-1), 69.0 (C-6),
74.9 (C-8), 124.9 (C-13), 132.0 (C-5), 133.7 (C-10), 136.2 (C-11), 169.5
(C-12), 171.2 (CH3COO-1), 173.7 (CH3(CH2)4COO-6); ESI-MS: 429.2
[MþNa]þ; HR-ESI-MS: calcd for C23H34NaO6 [MþNa]þ: 429.2248;
found: 429.2244, error ¼ 0.9 ppm; HPLC: tR ¼ 7.352 min,
purity > 99% at ELSD, 98% at 215 nm.
a 25
Yellow solid. Yield: 98%; ½ ꢀD ¼ ꢄ32.3 (c ¼ 0.16 in CHCl3); 1H NMR
(400 MHz, CDCl3):
d
0.87 (d, J ¼ 6.9 Hz, 3H, H-15), 0.98e1.07 (m, 1H,
H-3b), 1.10e1.21 (m, 3H, CH3CH2COO-6), 1.23e1.30 (m, 2H, H-2b, H-
3a), 1.34e1.46 (m, 1H, H-2a), 1.80 (s, 3H, H-14), 2.04 (s, 3H, AcO-1),
2.26e2.34 (m, 2H, CH3CH2COO-6), 2.45e2.52 (m, 1H, H-9b), 2.62e
2.75 (m, 2H, H-4, H-9a), 3.42e3.52 (m, 1H, H-7), 3.86e4.00 (m, 2H,
H-1), 4.90e4.98 (m, 1H, H-8), 5.21 (d, J ¼ 1.70 Hz, 1H, H-6), 5.94 (d,
J ¼ 2.31 Hz, 1H, H-13b), 6.37 (d, J ¼ 2.68 Hz, 1H, H-13a); 13C NMR
(100 MHz, CDCl3):
d
9.1 (CH3CH2COO-6), 18.5 (C-15), 20.5 (C-14),
4.1.4.1.6. 1-O-Acetyl-6-O-heptanoylbritannilactone
(6).
21.0 (CH3COO-1), 26.5 (C-2), 27.8 (CH3CH2COO-6), 31.0 (C-3), 33.0
(C-4), 34.5 (C-9), 42.9 (C-7), 64.2 (C-1), 69.1 (C-6), 74.9 (C-8), 124.9
(C-13), 132.0 (C-5), 133.7 (C-10), 136.3 (C-11), 169.5 (C-12), 171.2
(CH3COO-1),174.3 (CH3CH2COO-6); ESI-MS: 403.0 [MþK]þ; HR-ESI-
MS: calcd for C20H28NaO6 [MþNa]þ: 387.1778; found: 387.1782,
error ¼ 1 ppm; HPLC: tR ¼ 4.758 min, purity ¼ 99% at ELSD, 98% at
215 nm.
Colourless oil. Yield: 85%; ½a D25
ꢀ
¼ ꢄ58.6 (c ¼ 0.11 in CHCl3); 1H NMR
(400 MHz, CDCl3):
d 0.83e0.92 (m, 6H, H-15, CH3(CH2)5COO-6),
0.97e1.08 (m, 1H, H-3b), 1.22e1.34 (m, 8H, H-2b, H-3a,
CH3(CH2)3(CH2)2COO-6), 1.35e1.46 (m, 1H, H-2a), 1.54e1.68 (m, 2H,
CH3(CH2)3CH2CH2COO-6), 1.80 (s, 3H, H-14), 2.04 (s, 3H, AcO-1),
2.28 (t, J ¼ 7.5 Hz, 2H, CH3(CH2)4CH2COO-6), 2.49 (dd, J ¼ 16.1,
2.0 Hz, 1H, H-9b), 2.59e2.79 (m, 2H, H-4, H-9a), 3.43e3.52 (m, 1H,
H-7), 3.83e4.03 (m, 2H, H-1), 4.86e4.99 (m, 1H, H-8), 5.21 (d,
J ¼ 1.7 Hz,1H, H-6), 5.94 (d, J ¼ 2.3 Hz,1H, H-13b), 6.37 (d, J ¼ 2.7 Hz,
4.1.4.1.3. 1-O-Acetyl-6-O-butyrylbritannilactone (3). White oil.
Yield: 89%; ½a 2D5
ꢀ
¼ ꢄ47.5 (c ¼ 0.14 in CHCl3); 1H NMR (400 MHz,
CDCl3):
d
0.87 (d, J ¼ 6.9 Hz, 3H, H-15), 0.94 (t, J ¼ 7.4 Hz, 3H,
1H, H-13a); 13C NMR (100 MHz, CDCl3):
d
14.0 (CH3(CH2)5COO-6),
(CH3COO-1), 22.4
(CH3CH2CH2(CH2)3COO-6), 24.9 (CH3CH2CH2(CH2)3COO-6), 26.5 (C-
2), 28.8 (CH3(CH2)2CH2(CH2)2COO-6), 31.0 (C-3), 31.4
CH3(CH2)2COO-6), 0.98e1.08 (m, 1H, H-3b), 1.20e1.32 (m, 2H, H-2b,
H-3a), 1.33e1.50 (m, 1H, H-2a), 1.55e1.71 (m, 2H, CH3CH2CH2COO-
6), 1.80 (s, 3H, H-14), 2.04 (s, 3H, AcO-1), 2.22e2.32 (m, 2H,
CH3CH2CH2COO-6), 2.49 (dd, J ¼ 16.2, 2.1 Hz, 1H, H-9b), 2.61e2.79
(m, 2H, H-4, H-9a), 3.43e3.54 (m, 1H, H-7), 3.82e4.02 (m, 2H, 1-H),
4.88e5.00 (m, 1H, H-8), 5.22 (d, J ¼ 1.8 Hz, 1H, H-6), 5.94 (d,
J ¼ 2.4 Hz, 1H, H-13b), 6.37 (d, J ¼ 2.7 Hz, 1H, H-13a); 13C NMR
18.5
(C-15),
20.5
(C-14),
21.0
(CH3(CH2)3CH2CH2COO-6), 33.0 (C-4), 34.5 (C-9), 34.5
(CH3(CH2)4CH2COO-6), 42.9 (C-7), 64.2 (C-1), 69.0 (C-6), 75.1 (C-8),
124.9 (C-13), 132.1 (C-5), 133.7 (C-10), 136.3 (C-11), 169.5 (C-12),
171.2 (CH3COO-1),173.7 (CH3(CH2)5COO-6); ESI-MS: 459.0 [MþK]þ;
HR-ESI-MS: calcd for C24H36NaO6 [MþNa]þ: 443.2404; found:
443.2409, error ¼ 1.1 ppm; HPLC: tR ¼ 9.587 min, purity > 99% at
ELSD.
(100 MHz, CDCl3): d 13.6 (CH3(CH2)2COO-6), 18.4 (CH3CH2CH2COO-
6), 18.5 (C-15), 20.5 (C-14), 21.0 (CH3COO-1), 26.5 (C-2), 31.0 (C-3),
33.0 (C-4), 34.5 (C-9), 36.4 (CH3CH2CH2COO-6), 42.9 (C-7), 63.9 (C-
1), 69.0 (C-6), 74.9 (C-8), 124.9 (C-13), 132.1 (C-5), 133.7 (C-10),
136.3 (C-11), 169.5 (C-12), 171.2 (CH3COO-1), 173.5 (CH3(CH2)2COO-
6); ESI-MS: 401.0 [MþNa]þ; HR-ESI-MS: calcd for C21H30NaO6
[MþNa]þ: 401.1935; found: 401.1943, error ¼ 2 ppm; HPLC:
tR ¼ 5.217 min, purity > 99% at ELSD, >99% at 215 nm.
4.1.4.1.7. 1-O-Acetyl-6-O-decoylbritannilactone
(7).
Colourless oil. Yield: 85%; ½a D25
ꢀ
¼ ꢄ38.8 (c ¼ 0.07 in CHCl3); 1H NMR
(400 MHz, CDCl3): d 0.83e0.91 (m, 6H, H-15, CH3(CH2)6COO-6), 1.01
(d,
J
¼
9.5 Hz, 1H, H-3b), 1.19e1.34 (m, 10H, H-2b, H-3a,
CH3(CH2)4CH2CH2COO-6), 1.35e1.43 (m, 1H, H-2a), 1.54e1.66 (m,
2H, CH3(CH2)4CH2CH2COO-6), 1.79 (s, 3H, H-14), 2.03 (s, 3H, AcO-1),
2.27 (t, J ¼ 7.4 Hz, 2H, CH3(CH2)5 CH2COO-6), 2.48 (dd, J ¼ 16.1,
2.0 Hz, 1H, H-9b), 2.62e2.74 (m, 2H, H-4, H-9a), 3.42e3.50 (m, 1H,
H-7), 3.85e3.99 (m, 2H, H-1), 4.90e4.95 (m, 1H, H-8), 5.20 (d,
J ¼ 1.8 Hz,1H, H-6), 5.93 (d, J ¼ 2.3 Hz,1H, H-13b), 6.36 (d, J ¼ 2.9 Hz,
4.1.4.1.4. 1-O-Acetyl-6-O-valerylbritannilactone (4). White solid.
Yield: 94%; ½a 2D5
ꢀ
¼ ꢄ49.0 (c ¼ 0.12 in CHCl3); 1H NMR (400 MHz,
CDCl3):
d
0.87 (d, J ¼ 6.9 Hz, 3H, H-15), 0.92 (t, J ¼ 7.4 Hz, 3H,
CH3(CH2)3COO-6), 0.95e1.09 (m, 1H, H-3b), 1.19e1.48 (m, 5H, H-2,
H-3a, CH3CH2(CH2)2COO-6),1.50e1.66 (m, 2H, CH3CH2CH2CH2COO-
6), 1.80 (s, 3H, H-14), 2.04 (s, 3H, AcO-1), 2.28 (t, J ¼ 7.6 Hz, 2H,
CH3(CH2)2CH2COO-6), 2.49 (dd, J ¼ 16.1, 2.0 Hz, 1H, H-9b), 2.60e
2.78 (m, 2H, H-4, H-9a), 3.33e3.54 (m, 1H, H-7), 3.83e4.04 (m, 2H,
H-1), 4.81e5.01 (m, 1H, H-8), 5.21 (d, J ¼ 1.6 Hz, 1H, H-6), 5.94 (d,
J ¼ 2.3 Hz, 1H, H-13b), 6.37 (d, J ¼ 2.7 Hz, 1H, H-13a); 13C NMR
1H, H-13a); 13C NMR (100 MHz, CDCl3):
d
14.0 (CH3(CH2)6COO-6),
18.5 (C-15), 20.4 (C-14), 20.9 (CH3COO-1), 22.5 (CH3CH2(CH2)5COO-
6), 24.9 (CH3CH2CH2(CH2)4COO-6), 26.4 (C-2), 28.8
(CH3(CH2)2CH2(CH2)3COO-6), 29.0 (CH3(CH2)3CH2(CH2)2COO-6),
31.0 (C-3), 31.6 (CH3(CH2)4CH2CH2COO-6), 33.0 (C-4), 34.5 (C-9),

78
S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
42.8 (C-7), 53.4 (CH3(CH2)5CH2COO-6), 64.2 (C-1), 69.0 (C-6), 74.9
(C-8), 124.9 (C-13), 132.0 (C-5), 133.7 (C-10), 136.2 (C-11), 169.5 (C-
12), 171.2 (CH3COO-1), 173.7 (CH3(CH2)6COO-6); ESI-MS: 457.3
[MþNa]þ; HR-ESI-MS: calcd for C25H37NaO6 [MþNa]þ: 457.2561;
(C-7), 64.2 (C-1), 69.6 (C-6), 74.9 (C-8), 125.1 (C-13), 131.7 (C-5),
134.0 (C-10), 136.1 (C-11), 169.6 (C-12), 171.3 (CH3COO-1), 172.0
(HOOC(CH2)2COO-6), 177.7 (HOOC(CH2)2COO-6); ESI-MS: 431.1
[MþNa]þ; HR-ESI-MS: calcd for C21H29O8 [MþH]þ: 409.1857;
found: 409.1865, error ¼ 2 ppm; HPLC: tR ¼ 0.410 min, purity > 99%
at ELSD, >99% at 215 nm.
found: 457.2554, error ¼ 1.5 ppm; HPLC: tR
¼
9.502 min,
purity > 99% at ELSD, >99% at 215 nm.
4.1.4.1.8. 1-O-Acetyl-6-O-chloracetylbritannilactone
(8).
White solid. Yield: 99%; ½a D25
ꢀ
¼ ꢄ54.0 (c ¼ 0.13 in CHCl3); 1H NMR
4.1.4.2. Procedure for the synthesis of ABL analogues (12e18).
To a suspension of a corresponding acid (pelargonic acid for 12,
capric acid for 13, lauric acid for 14, myristic acid for 15, stearic acid
for 16, p-bromocinnamic acid for 17, p-trifluoromethylcinnamic
acid for 18) (0.2 mmol), DMAP and DCC in anhydrous CH2Cl2 (1 mL)
was added ABL (0.1 mmol) in anhydrous CH2Cl2 (1 mL) solution.
After completion of the reaction from 5 to 20 h at room tempera-
ture, water (2 mL) was added to the mixture and stirred for 20 min,
then extracted with CH2Cl2, dried and filtered. After removal of the
solvent, the crude product was purified by silica gel chromatog-
raphy (EtOAc/PE) to afford analogues 12e18 in the yield from 65% to
83%.
(400 MHz, CDCl3):
d
0.87 (d, J ¼ 6.8 Hz, 3H, H-15), 0.93e1.06 (m, 1H,
H-3b), 1.17e1.30 (m, 2H, H-2b, H-3a), 1.32e1.43 (m, 1H, H-2a), 1.80
(s, 3H, H-14), 2.02 (s, 3H, AcO-1), 2.50 (d, J ¼ 16.2 Hz, 1H, H-9b),
2.61e2.75 (m, 2H, H-4, H-9a), 3.45e3.55 (m, 1H, H-7), 3.85e3.98
(m, 2H, H-1), 3.99e4.05 (m, 2H, ClCH2COO-6), 4.91e4.99 (m, 1H, H-
8), 5.25 (s, 1H, H-6), 5.93 (s, 1H, H-13b), 6.38 (s, 1H, H-13a); 13C NMR
(100 MHz, CDCl3): d 18.5 (C-15), 20.5 (C-14), 20.9 (CH3COO-1), 26.4
(C-2), 31.0 (C-3), 32.9 (C-4), 34.5 (C-9), 40.9 (ClCH2COO-6), 42.7 (C-
7), 64.1 (C-1), 70.9 (C-6), 74.5 (C-8), 125.2 (C-13), 131.2 (C-5), 134.8
(C-10), 135.8 (C-11), 167.1 (CH2ClCOO-6), 169.2 (C-12), 171.1
(CH3COO-1); ESI-MS: 407.2 [MþNa]þ; HR-ESI-MS: calcd for
C
19H26ClO6 [MþH]þ: 385.1412; found: 385.1428, error ¼ 4.2 ppm;
4.1.4.2.1. 1-O-Acetyl-6-O-nonanoylbritannilactone
(12).
HPLC: tR ¼ 4.338 min, purity > 99% at ELSD, 99% at 215 nm.
Colourless oil. Yield: 71%; ½a D25
ꢀ
¼ ꢄ50.7 (c ¼ 0.17 in CHCl3); 1H NMR
4.1.4.1.9. 1-O-Acetyl-6-O-isobutyrylbritannilactone
(9).
(400 MHz, CDCl3): d 0.77e0.89 (m, 6H, H-15, CH3(CH2)7COO-6),
White solid. Yield: 99%; ½a D25
ꢀ
¼ ꢄ49.0 (c ¼ 0.09 in CHCl3); 1H NMR
0.94e1.08 (m, 1H, H-3b), 1.19e1.31 (m, 13H, H-2, H-3a,
CH3(CH2)5CH2CH2COO-6), 1.37 (ddd, J ¼ 18.08, 7.12, 3.53 Hz, 1H),
1.51e1.67 (m, 2H, CH3(CH2)5CH2CH2COO-6), 1.78 (s, 3H, H-14), 2.02
(s, 3H, AcO-1), 2.21e2.32 (m, 2H, H-4, H-9a), 2.47 (dd, J ¼ 16.1,
2.0 Hz, 1H, H-9b), 2.59e2.74 (m, 2H, CH3(CH2)5CH2CH2COO-6),
3.40e3.50 (m, 1H, H-7), 3.83e4.02 (m, 2H, H-1), 4.86e4.96 (m, 1H,
H-8), 5.19 (d, J ¼ 1.7 Hz,1H, H-6), 5.93 (d, J ¼ 2.2 Hz, 1H, H-13b), 6.35
(400 MHz, CDCl3):
d
0.86 (d, J ¼ 6.9 Hz, 3H, H-15), 0.97e1.08 (m, 1H,
H-3b), 1.15 (d, J ¼ 6.9 Hz, 6H, (CH3)2CHCOO-6), 1.21e1.31 (m, 2H, H-
2b, H-3a), 1.34e1.45 (m, 1H, H-2a), 1.80 (s, 3H, H-14), 2.04 (s, 3H,
AcO-1), 2.45e2.56 (m, 2H, H-9b, (CH3)2CHCOO-6), 2.60e2.76 (m,
2H, H-4, H-9a), 3.41e3.49 (m, 1H, H-7), 3.85e4.00 (m, 2H, H-1),
4.90e4.98 (m, 1H, H-8), 5.19 (d, J ¼ 1.7 Hz, 1H, H-6), 5.94 (d,
J ¼ 2.3 Hz, 1H, H-13b), 6.37 (d, J ¼ 2.9 Hz, 1H, H-13a); 13C NMR
(d, J ¼ 2.6 Hz, 1H, H-13a); 13C NMR (100 MHz, CDCl3):
d 14.3
(100 MHz, CDCl3)
d
18.6 (C-15), 18.7 (CH3CH3CHCOO-6), 18.8
(CH3(CH2)7COO-6), 18.8 (C-15), 20.7 (C-14), 21.2 (CH3COO-1), 22.9
(CH3CH2(CH2)6COO-6), 25.2 (CH3(CH2)5CH2CH2COO-6), 26.7 (C-2),
(CH3CH3CHCOO-6), 20.5 (C-14), 21.0 (CH3COO-1), 26.5 (C-2), 31.0
(C-3), 33.0 (C-4), 34.1, 34.6 (C-9), 42.9 (C-7), 64.2 (C-1), 69.0 (C-6),
74.9 (C-8), 124.9 (C-13), 132.1 (C-5), 133.6 (C-10), 136.3 (C-11), 169.5
(C-12), 171.2 (CH3COO-1), 176.9 ((CH3)2CHCOO-6); ESI-MS: 401.2
[MþNa]þ; HR-ESI-MS: calcd for C21H30NaO6 [MþNa]þ: 401.1935;
29.4
(CH3(CH2)2(CH2)3(CH2)2COO-6),
31.3
(C-3),
32.0
(CH3CH2CH2(CH2)5COO-6), 33.3 (C-4), 34.8 (C-9), 34.8
(CH3(CH2)6CH2COO-6), 43.1 (C-7), 64.5 (C-1), 69.3 (C-6), 75.2 (C-8),
125.2 (C-13), 132.3 (C-5), 134.0 (C-10), 136.5 (C-11), 169.8 (C-12),
171.5 (CH3COO-1), 174.0 (CH3(CH2)7COO-6); ESI-MS: 487.2 [MþK]þ;
HR-ESI-MS: calcd for C26H40NaO6 [MþNa]þ: 471.2717; found:
471.2710, error ¼ 1.5 ppm; HPLC: tR ¼ 11.531 min, purity > 99% at
ELSD, >99% at 215 nm.
found: 401.1946, error
purity > 99% at ELSD, >99% at 215 nm.
¼
2.7 ppm; HPLC: tR
¼
5.394 min,
4.1.4.1.10. 1-O-Acetyl-6-O-pivaloylbritannilactone (10). Pale yel-
low oil. Yield: 86%; ½a D25
ꢀ
¼ ꢄ31.0 (c ¼ 0.10 in CHCl3); 1H NMR
(100 MHz, CDCl3):
d
0.83e0.90 (m, 3H, H-15), 1.00e1.09 (m, 1H, H-
4.1.4.2.2. 1-O-Acetyl-6-O-decanoylbritannilactone
(13).
3b), 1.15e1.19 (m, 9H, (CH3)3CCOO-6), 1.21e1.31 (m, 3H, H-2, H-3a),
1.81 (d, J ¼ 0.9 Hz, 3H, H-14), 2.04 (s, 3H, AcO-1), 2.49 (dd, J ¼ 16.1,
2.1 Hz, 1H, H-9b), 2.67 (dd, J ¼ 3.6, 1.0 Hz, 2H, H-4, H-9a), 3.42e3.46
(m, 1H, H-7), 3.93 (m, 2H, H-1), 4.90e4.98 (m, 1H, H-8), 5.17 (d,
J ¼ 1.8 Hz,1H, H-6), 5.94 (d, J ¼ 2.4 Hz,1H, H-13b), 6.37 (d, J ¼ 2.8 Hz,
Colourless oil. Yield: 74%; ½a D25
ꢀ
¼ ꢄ40.4 (c ¼ 0.20 in CHCl3); 1H NMR
(400 MHz, CDCl3):
d 0.84e0.91 (m, 6H, H-15, CH3(CH2)8COO-6),
0.98e1.08 (m, 1H, H-3b), 1.20e1.34 (m, 14H, H-2b, H-3a,
CH3(CH2)6CH2CH2COO-6), 1.35e1.45 (m, 1H, m, 1H, H-2a),1.54e1.70
(m, 2H, CH3(CH2)6CH2CH2COO-6), 1.80 (s, 3H, H-14), 2.03 (s, 3H,
AcO-1), 2.27 (t, J ¼ 7.5 Hz, 2H, CH3(CH2)6CH2CH2COO-6), 2.48 (dd,
J ¼ 16.1, 2.0 Hz, 1H, H-9b), 2.63e2.75 (m, 2H, H-4, H-9a), 3.43e3.52
(m, 1H, H-7), 3.86e4.01 (m, 2H, H-1), 4.88e4.98 (m, 1H, H-8), 5.21
(d, J ¼ 1.8 Hz, 1H, H-6), 5.94 (d, J ¼ 2.3 Hz, 1H, H-13b), 6.36 (d,
1H, H-13a); 13C NMR (100 MHz, CDCl3):
d 18.7 (C-15), 20.5 (C-14),
20.9 (CH3COO-1), 26.5 (C-2), 26.9 (3C, (CH3)3CCOO-6), 31.0 (C-3),
33.0 (C-4), 34.6 (C-9), 42.8 (C-7), 64.2 (C-1), 69.1 (C-6), 74.9 (C-8),
124.9 (C-13), 132.3 (C-5), 133.6 (C-10), 136.2 (C-11), 169.5 (C-12),
171.2 (CH3COO-1), 178.2 ((CH3)3CCOO-6); ESI-MS: 415.1 [MþNa]þ;
HR-ESI-MS: calcd for C22H32NaO6 [MþNa]þ: 415.2091; found:
415.2099, error ¼ 1.9 ppm; HPLC: tR ¼ 11.152 min, purity > 99% at
ELSD, 98% at 215 nm.
J ¼ 2.6 Hz, 1H, H-13a); 13C NMR (100 MHz, CDCl3):
d 14.0
(CH3(CH2)8COO-6), 18.5 (C-15), 20.5 (C-14), 20.9 (CH3COO-1), 22.6
(CH3CH2(CH2)7COO-6), 24.9 (CH3(CH2)6CH2CH2COO-6), 26.5 (C-2),
29.1e29.4 (4C, CH3(CH2)2(CH2)4(CH2)2COO-6), 31.1 (C-3), 31.8
(CH3CH2CH2(CH2)6COO-6), 33.1 (C-4), 34.5 (C-9), 34.6
(CH3(CH2)7CH2COO-6), 42.9 (C-7), 64.2 (C-1), 69.0 (C-6), 74.9 (C-8),
124.9 (C-13), 132.1 (C-5), 133.7 (C-10), 136.3 (C-11), 169.5 (C-12),
171.1 (CH3COO-1), 173.7 (CH3(CH2)8COO-6); ESI-MS: 485.1
[MþNa]þ; HR-ESI-MS: calcd for C27H42NaO6 [MþNa]þ: 485.2874;
found: 485.2886, error ¼ 2.5 ppm; HPLC: tR ¼ 12.862 min,
purity ¼ 95% at ELSD.
4.1.4.1.11. 1-O-Acetyl-6-O-carboxypropionylbritannilactone (11).
White oil. Yield: 88%; ½a D25
ꢀ
¼ ꢄ112.0 (c ¼ 0.13 in CHCl3); 1H NMR
(400 MHz, CDCl3):
d
0.86 (d, J ¼ 6.8 Hz, 3H, H-15), 0.95e1.06 (m, 1H,
H-3b), 1.19e1.31 (m, 2H, H-2b, H-3a), 1.39 (m, 1H, H-2a), 1.79 (s, 3H,
H-14), 2.02 (s, 3H, AcO-1), 2.47 (dd, J ¼ 16.2, 2.0 Hz,1H, H-9b), 2.54e
2.60 (m, 2H, H-4, H-9a), 2.61e2.73 (m, 4H, HOOC(CH2)2COO-6),
3.45e3.51 (m, 1H, H-7), 3.85e3.99 (m, 2H, H-1), 4.87e4.97 (m, 1H,
H-8), 5.22 (d, J ¼ 1.7 Hz,1H, H-6), 5.92 (d, J ¼ 2.3 Hz,1H, H-13b), 6.36
4.1.4.2.3. 1-O-Acetyl-6-O-lauroylbritannilactone (14). White oil.
(d, J ¼ 2.6 Hz, 1H, H-13a); 13C NMR (100 MHz, CDCl3):
d
18.4 (C-15),
Yield: 73%; ½a 2D5
ꢀ
¼ ꢄ37.7 (c ¼ 0.23 in CHCl3); 1H NMR (400 MHz,
20.4 (C-14), 20.9 (CH3COO-1), 26.4 (C-2), 28.8 (HOOCCH2CH2COO-
6), 29.1 (HOOCCH2CH2COO-6), 31.0 (C-3), 33.0 (C-4), 34.5 (C-9), 42.6
CDCl3): d 0.83e0.94 (m, 6H, H-15, CH3(CH2)10COO-6), 0.97e1.11 (m,
1H, H-3b), 1.19e1.47 (m, 19H, H-2, H-3a, CH3(CH2)8CH2CH2COO-6),

S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
79
1.54e1.69 (m, 2H, CH3(CH2)8CH2CH2COO-6), 1.80 (s, 3H, H-14),
2.02e2.08 (m, 3H, AcO-1), 2.27 (t, 7.5 Hz, 2H,
6.39 (d, J ¼ 2.7 Hz, 1H, H-13a), 6.40 (d, J ¼ 16.0 Hz, 1H, p-
BrPhCH ¼ CHCOO-6), 7.38 (d, J ¼ 8.5 Hz, 2H, p-BrPhCH]CHCOO-6),
7.52 (d, J ¼ 8.5 Hz, 2H, p-BrPhCH]CHCOO-6), 7.59 (d, J ¼ 16.0 Hz,
J
¼
CH3(CH2)8CH2CH2COO-6), 2.44e2.53 (m, 1H, H-9b), 2.61e2.78 (m,
2H, H-4, H-9a), 3.43e3.52 (m, 1H, H-7), 3.85e4.03 (m, 2H, H-1),
4.93 (ddd, J ¼ 5.8, 3.8, 1.8 Hz, H-8), 5.21 (d, J ¼ 1.6 Hz, 1H, H-6), 5.94
(d, J ¼ 2.3 Hz, 1H, H-13b), 6.37 (d, J ¼ 2.8 Hz, 1H, H-13a); 13C NMR
1H, p-BrPhCH]CHCOO-6); 13C NMR (100 MHz, CDCl3):
d 18.5 (C-
15), 20.5 (C-14), 20.9 (CH3COO-1), 26.4 (C-2), 31.1 (C-3), 33.1 (C-4),
34.6 (C-9), 42.5 (C-7), 64.2 (C-1), 69.4 (C-6), 74.9 (C-8), 118.3 (p-
BrPhCH]CHCOO-6), 124.8 (C-13), 125.0 (p-BrPhCH]CHCOO-6),
129.5 (2C, p-BrPhCH]CHCOO-6), 132.0 (p-BrPhCH]CHCOO-6),
132.1 (2C, p-BrPhCH]CHCOO-6), 132.9 (C-5), 133.9 (C-10), 136.2 (C-
11), 144.0 (p-BrPhCH]CHCOO-6), 166.4 (p-BrPhCH]CHCOO-6),
169.5 (C-12), 171.1 (CH3COO-1); ESI-MS: 539.0 [MþNa]þ; HR-ESI-
MS: calcd for C26H29BrNaO6 [MþNa]þ: 539.1040; found: 539.1022,
error ¼ 3.3 ppm; HPLC: tR ¼ 9.184 min, purity ¼ 98% at ELSD, 96% at
215 nm.
(100 MHz, CDCl3):
d 14.3 (CH3(CH2)10COO-6), 18.7 (C-15), 20.6 (C-
14), 21.1 (CH3COO-1), 22.8 (CH3CH2(CH2)9COO-6), 25.1
(CH3(CH2)8CH2CH2COO-6), 26.7 (C-2), 29.2e29.7 (6C, CH3(CH2)2
(CH2)6(CH2)2COO-6), 31.2 (C-3), 32.0 (CH3CH2CH2(CH2)8COO-6),
33.2 (C-4), 34.7 (C-9), 34.74 (CH3(CH2)9CH2COO-6), 43.1 (C-7), 64.4
(C-1), 69.2 (C-6), 75.1 (C-8), 125.1 (C-13), 132.3 (C-5), 133.8 (C-10),
136.5
(C-11), 169.7
(C-12), 171.3
(CH3COO-1), 178.9
(CH3(CH2)10COO-6); ESI-MS: 513.0 [MþNa]þ; HR-ESI-MS: calcd for
C
29H46NaO6 [MþNa]þ: 513.3187; found: 513.3180, error ¼ 1.4 ppm;
4.1.4.2.7. 1-O-Acetyl-6-O-(p-trifluoromethylcinnamoyl)britanni-
HPLC: tR ¼ 12.873 min, purity ¼ 99% at ELSD, 98% at 215 nm.
lactone (18). Yellow oil. Yield: 83%; ½a D25
¼ ꢄ111.8 (c ¼ 0.07 in
ꢀ
4.1.4.2.4. 1-O-Acetyl-6-O-myristoylbritannilactone
(15).
CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.91 (d, J ¼ 6.9 Hz, 3H, H-15),
White solid. Yield: 70%; ½a D25
ꢀ
¼ ꢄ37.4 (c ¼ 0.10 in CHCl3); 1H NMR
1.02e1.13 (m, 1H, H-3b), 1.23e1.35 (m, 2H, H-2b, H-3a), 1.38e1.47
(m, 1H, H-2a), 1.85 (s, 3H, H-14), 2.05 (s, 3H, AcO-1), 2.55 (dd,
J ¼ 16.2, 2.0 Hz, 1H, H-9b), 2.72 (ddd, J ¼ 9.4, 6.9, 4.6 Hz, 1H, H-4),
2.80 (dd, J ¼ 16.2, 2.6 Hz, 1H, H-9a), 3.55e3.61 (m, 1H, H-7), 3.88e
4.03 (m, 2H, H-1), 4.95e5.01 (m, 1H, H-8), 5.37 (d, J ¼ 1.8 Hz, 1H, H-
6), 6.00 (d, J ¼ 2.3 Hz,1H, H-13b), 6.41 (d, J ¼ 2.6 Hz,1H, H-13a), 6.48
(d, J ¼ 16.1 Hz, 1H, p-F3CPhCH]CHCOO-6), 7.61e7.71 (m, 5H, p-
(400 MHz, CDCl3):
d 0.83e0.92 (m, 6H, H-15, CH3(CH2)12COO-6),
0.97e1.09 (m, 1H, H-3b), 1.19e1.45 (m, 23H, H-2, H-3a,
CH3(CH2)10CH2CH2COO-6), 1.54e1.71 (m, 2H, CH3(CH2)10CH2CH2
COO-6), 1.80 (s, 3H, H-14), 2.04 (d, J ¼ 1.3 Hz, 3H, AcO-1), 2.24e2.32
(m, 2H, CH3(CH2)10CH2CH2COO-6), 2.49 (d, J ¼ 16.2 Hz, 1H, H-9b),
2.62e2.76 (m, 2H, H-4, H-9a), 3.47 (d, J ¼ 7.8 Hz, 1H, H-7), 3.85e
4.01 (m, 2H, H-1), 4.89e4.97 (m, 1H, H-8), 5.21 (s, 1H, H-6), 5.94 (s,
1H, H-13b), 6.37 (d, J ¼ 2.3 Hz, 1H, H-13a); 13C NMR (100 MHz,
F3CPhCH]CHCOO-6); 13C NMR (100 MHz, CDCl3):
d 18.5 (C-15),
20.6 (C-14), 20.9 (CH3COO-1), 26.5 (C-2), 31.1 (C-3), 33.1 (C-4), 34.7
(C-9), 42.9 (C-7), 64.2 (C-1), 69.7 (C-6), 74.9 (C-8), 120.3 (p-
F3CPhCH]CHCOO-6), 122.4 (p-F3CPhCH]CHCOO-6), 125.1 (C-13),
125.9 (2C, p-F3CPhCH]CHCOO-6), 128.3 (3C, p-F3CPhCH]CHCOO-
6), 132.1 (C-5), 134.1 (C-10), 136.2 (C-11), 137.4 (p-F3CPhCH]
CHCOO-6), 143.6 (p-F3CPhCH]CHCOO-6), 166.1 (p-F3CPhCH]
CHCOO-6), 169.4 (C-12), 171.1 (CH3COO-1); ESI-MS: 529.0
[MþNa]þ; HR-ESI-MS: calcd for C27H29F3NaO6 [MþNa]þ: 529.1808;
CDCl3):
d 14.1 (CH3(CH2)12COO-6), 18.5 (C-15), 20.5 (C-14), 20.9
(CH3COO-1), 22.6 (CH3CH2(CH2)11COO-6), 24.9 (CH3(CH2)10CH2CH2
COO-6), 26.5 (C-2), 29.1e29.7 (8C, CH3(CH2)2(CH2)8(CH2)2COO-6),
31.1 (C-3), 31.9 (CH3CH2CH2(CH2)10COO-6), 33.1 (C-4), 34.5
(CH3(CH2)11CH2COO-6), 34.6 (C-9), 42.9 (C-7), 64.2 (C-1), 69.0 (C-6),
74.9 (C-8), 124.9 (C-13), 132.1 (C-5), 133.7 (C-10), 136.3 (C-11), 169.5
(C-12), 171.1 (CH3COO-1), 173.7 (CH3(CH2)12COO-6); ESI-MS: 541.2
[MþNa]þ; HR-ESI-MS: calcd for C31H50NaO6 [MþNa]þ: 541.3500;
found: 541.3506, error ¼ 1.1 ppm; HPLC: tR ¼ 14.696 min,
purity ¼ 99% at ELSD, 96% at 215 nm.
found: 529.1800, error
purity > 99% at ELSD, 98% at 215 nm.
¼
1.5 ppm; HPLC: tR
¼
7.019 min,
4.1.4.2.5. 1-O-Acetyl-6-O-stearoylbritannilactone
(16).
4.1.4.3. Procedure for the synthesis of ABL analogue (19). ABL
(0.1 mmol) in CH2Cl2 (1 mL) was added to a suspension of DMP
(42.4 mg, 0.1 mmol) in CH2Cl2 (1 mL). After stirring at 20 ꢁC for 3 h,
the complete solution was subjected to silica gel chromatography
(PE:EtOAc ¼ 1:1) to give compound 19.
White waxy solid. Yield: 75%; ½a D25
ꢀ
¼ ꢄ30.1 (c ¼ 0.112 in CHCl3); 1H
NMR (400 MHz, CDCl3): d 0.84e0.92 (m, 6H, H-15, CH3(CH2)16COO-
6), 0.99e1.08 (m, 1H, H-3b), 1.20e1.35 (m, 30H, H-2b, H-3a,
CH3(CH2)14CH2CH2COO-6), 1.35e1.45 (m, 1H, H-2a), 1.55e1.65 (m,
2H, CH3(CH2)14CH2CH2COO-6), 1.81 (s, 3H, H-14), 2.03e2.07 (m, 3H,
AcO-1), 2.28 (t, J ¼ 7.5 Hz, 2H, CH3(CH2)14CH2CH2COO-6), 2.49 (dd,
J ¼ 16.1, 2.0 Hz, 1H, H-9b), 2.62e2.76 (m, 2H, H-4, H-9a), 3.44e3.52
(m, 1H, H-7), 3.85e4.01 (m, 2H, H-1), 4.89e4.98 (m, 1H, H-8), 5.21
(d, J ¼ 1.8 Hz, 1H, H-6), 5.95 (d, J ¼ 2.3 Hz, 1H, H-13b), 6.38 (d,
4.1.4.3.1. 1-O-Acetyl-6-oxobritannilactone (19). Yellow oil. Yield:
43%; ½a 2D5
ꢀ
¼ þ54.6 (c ¼ 0.17 in CHCl3); 1H NMR (400 MHz, CDCl3):
d
1.19 (d, 3H, H-15), 1.23e1.35 (m, 1H, H-3b), 1.41e1.53 (m, 3H, H-2,
H-3a), 2.00 (s, 3H, H-14), 2.02 (s, 3H, AcO-1), 2.64e2.84 (m, 3H, H-9,
H-4), 3.74 (dt, J ¼ 7.1, 2.3 Hz, 1H, H-7), 3.94 (t, J ¼ 6.5 Hz, 2H, H-1),
5.00 (ddd, J ¼ 7.2, 4.7, 3.4 Hz, 1H, H-8), 5.98 (d, J ¼ 2.3 Hz, 1H, H-
13b), 6.32 (d, J ¼ 2.4 Hz, 1H, H-13a); 13C NMR (100 MHz, CDCl3):
J ¼ 2.8 Hz, 1H, H-13a); 13C NMR (100 MHz, CDCl3):
d 14.1
(CH3(CH2)16COO-6), 18.5 (C-15), 20.5 (C-14), 21.0 (CH3COO-1), 22.7
(CH3CH2(CH2)15COO-6), 25.0 (CH3(CH2)14CH2CH2COO-6), 26.5 (C-
2), 29.1e29.7 (12C, CH3(CH2)2(CH2)12(CH2)2COO-6), 31.0 (C-3), 31.9
(CH3CH2CH2(CH2)14COO-6), 33.1 (C-4), 34.6 (C-9), 34.6
(CH3(CH2)15CH2COO-6), 42.9 (C-7), 64.2 (C-1), 69.0 (C-6), 75.0 (C-8),
125.0 (C-13), 132.1 (C-5), 133.7 (C-10), 136.3 (C-11), 169.5 (C-12),
171.2 (CH3COO-1), 173.7 (CH3(CH2)16COO-6); ESI-MS: 597.2
[MþNa]þ; HR-ESI-MS: calcd for C35H58NaO6 [MþNa]þ: 597.4126;
d
19.3 (C-15), 20.9 (C-14), 21.6 (CH3CO-1), 27.3 (C-2), 29.9 (C-3), 33.3
(C-4), 34.6 (C-9), 50.0 (C-7), 64.3 (C-1), 73.2 (C-8), 125.5 (C-13),
133.3 (C-11), 139.0 (C-5), 149.6 (C-10), 168.7 (C-12), 171.1 (CH3CO-1),
191.8 (C-12); ESI-MS: 329.0 [MþNa]þ; HR-ESI-MS: calcd for
C
17H23O5 [MþH]þ: 307.1540; found: 307.1554, error ¼ 4.6 ppm;
HPLC: tR ¼ 3.694 min, purity > 99% at 254 nm.
found: 597.4109, error
¼
2.2 ppm; HPLC: tR
¼
6.846 min,
4.1.4.4. Procedure for the synthesis of OABL C13-modified arylation
analogues (20e24). A mixture of OABL (35 mg, 0.1 mmol), trie-
thylamine (30 mg, 0.3 mmol), and Iodobenzene (40.8 mg,
purity ¼ 97% at ELSD, 95% at 215 nm.
4.1.4.2.6. 1-O-Acetyl-6-O-(p-bromocinnamoyl)britannilactone
(17). Colourless oil. Yield: 65%; ½a D25
ꢀ
¼ ꢄ133.8 (c ¼ 0.50 in CHCl3);
0.2 mmol) in DMF (750 mL) was treated with palladium(II) acetate
1H NMR (400 MHz, CDCl3):
d
0.89 (d, J ¼ 6.9 Hz, 3H, H-15),1.00e1.11
(1.12 mg, 0.005 mmol) and then heated at 80 ꢁC under Ar atmo-
sphere. After stirring for 24e40 h, the reaction mixture was allowed
to cool to rt, water (5 mL) was added, and the resultant mixture was
extracted with dichloromethane (5 mL ꢃ 3). The organic extract
was evaporated to give an oily residue which was further purified
by silica gel chromatography (PE: EtOAc ¼ 5:1 to 1:1).
(m, 1H, H-3b), 1.21e1.34 (m, 2H, H-2b, H-3a), 1.35e1.46 (m, 1H, H-
2a), 1.83 (s, 3H, H-14), 2.04 (s, 3H, AcO-1), 2.53 (dd, J ¼ 16.2, 2.1 Hz,
1H, H-9b), 2.66e2.74 (m, 1H, H-4), 2.78 (dd, J ¼ 16.2, 2.7 Hz, 1H, H-
9a), 3.53e3.59 (m, 1H, H-7), 3.87e4.01 (m, 2H, H-1), 4.94e5.00 (m,
1H, H-8), 5.34 (d, J ¼ 1.7 Hz, 1H, H-6), 5.99 (d, J ¼ 2.2 Hz, 1H, H-13b),

80
S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
4.1.4.4.1. 1,6-O,O-Diacetyl-(E)-13-phenylbritannilactone
(20).
(CH3CO-1), 26.6 (C-2), 30.6 (C-3), 33.2 (C-4), 35.3 (C-9), 42.8 (C-7),
Yellow oil. Yield: 41%; ½a D25
ꢀ
¼ þ10.3 (c ¼ 0.35 in CHCl3); 1H NMR
55.4 (CH3O-Ar-13), 64.3 (C-1), 64.7 (C-6), 74.5 (C-8), 114.5 (2C, Ar-
13), 120.7 (C-11), 125.4 (Ar-13), 133.0 (2C, Ar-13), 133.2 (C-5),
133.8 (C-10), 139.6 (C-13), 161.5 (Ar-13), 170.4 (C-12), 171.2 (CH3CO-
6), 172.0 (CH3CO-1); ESI-MS: 456.9 [MþH]þ; HR-ESI-MS: calcd for
(400 MHz, CDCl3):
d 0.67e0.79 (m, 4H, H-15, H-3b), 0.88e1.01 (m,
1H, H-3a), 1.22e1.44 (m, 2H, H-2), 1.80 (s, 3H, H-14), 2.03 (s, 3H,
AcO-6), 2.06 (s, 3H, AcO-1), 2.52 (dd, J ¼ 15.8, 2.7 Hz, 1H, H-9b),
2.55e2.64 (m, 1H, H-4), 2.86 (dd, J ¼ 15.8, 2.7 Hz, 1H, H-9a), 3.80e
3.88 (m, 1H, H-1a), 3.89e3.98 (m, 1H, H-1b), 4.08 (dt, J ¼ 7.8, 2.3 Hz,
1H, H-7), 5.02e5.11 (m, 1H, H-8), 5.57 (d, J ¼ 2.1 Hz, 1H, H-6), 7.40e
7.55 (m, 3H, Ar-13), 7.60 (d, J ¼ 2.6 Hz, 1H, H-13), 7.78 (d, J ¼ 7.2 Hz,
C
26H32NaO7
[MþNa]þ:
479.2040;
found:
479.2054,
error ¼ 2.9 ppm; HPLC: tR ¼ 6.66 min, purity > 99% at ELSD.
4.1.4.4.5. 1,6-O,O-Diacetyl-(E)-13-(3,4,5-trimethoxyphenyl)bri-
tannilactone (24). White solid. Yield: 82%; ½a D25
¼ þ30.9 (c ¼ 0.24 in
ꢀ
2H, Ar-13); 13C NMR (100 MHz, CDCl3):
d
18.3 (C-15), 20.2 (C-14),
CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.75 (d, J ¼ 6.9 Hz, 3H, H-15),
21.0 (CH3CO-6), 21.3 (CH3CO-1), 26.6 (C-2), 30.6 (C-3), 33.2 (C-4),
35.2 (C-9), 42.7 (C-7), 64.2 (C-1), 64.7 (C-6), 74.7 (C-8), 124.0 (C-11),
129.0 (2C, Ar-13), 130.6 (Ar-13), 130.9 (2C, Ar-13), 132.7 (C-5), 133.2
(C-10), 133.9 (Ar-13), 139.9 (C-13), 170.2 (C-12), 171.2 (CH3CO-6),
171.5 (CH3CO-1); ESI-MS: 426.8 [MþH]þ; HR-ESI-MS: calcd for
0.70e0.90 (m, 1H, H-3b), 0.95e1.04 (m, 1H, H-3a), 1.23e1.34 (m, 1H,
H-2b), 1.35e1.45 (m, 1H, H-2a), 1.79 (s, 3H, C-14), 2.01 (s, 3H, AcO-
6), 2.03 (s, 3H, AcO-1), 2.51 (dd, J ¼ 15.9, 2.0 Hz, 1H, H-9b), 2.54e
2.62 (m,1H, H-4), 2.83 (dd, J ¼ 15.6, 2.4 Hz,1H, H-9a), 3.78e3.87 (m,
1H, H-1b), 3.93 (s, 9H, CH3O-Ar-13), 3.88e3.96 (m, 1H, H-1a), 4.03e
4.10 (m, 1H, H-7), 4.99e5.07 (m, 1H, H-8), 5.50 (d, J ¼ 2.2 Hz, 1H, H-
6), 6.92 (s, 2H, Ar-13), 7.49 (d, J ¼ 2.3 Hz, 1H, H-13); 13C NMR
C
25H30NaO6 [MþNa]þ: 449.1935; found: 449.1942, error ¼ 1.6 ppm;
HPLC: tR ¼ 6.480 min, purity ¼ 98% at ELSD.
4.1.4.4.2. 1,6-O,O-Diacetyl-(E)-13-(4-fluorophenyl)britannilactone
(100 MHz, CDCl3): d 18.4 (C-15), 20.3 (C-14), 20.9 (CH3CO-6), 21.2
(21). Yellow oil. Yield: 52%; ½a D25
ꢀ
¼ þ16.7 (c ¼ 0.21 in CHCl3); 1H
(CH3CO-1), 26.7 (C-2), 30.7 (C-3), 33.4 (C-4), 35.3 (C-9), 42.4 (C-7),
56.4 (2C, CH3O-Ar-13), 61.0 (CH3O-Ar-13), 64.3 (C-1), 65.2 (C-6),
74.6 (C-8), 108.4 (2C, Ar-13), 122.9 (C-11), 128.3 (Ar-13), 133.3 (C-5),
134.0 (C-10), 140.2 (Ar-13), 153.5 (2C, Ar-13), 169.7 (C-12), 171.2
(CH3CO-6), 171.6 (CH3CO-1); ESI-MS: 533.8 [MþNH4]þ; HPLC:
tR ¼ 15.2 min, purity ¼ 97.5% at ELSD.
NMR (400 MHz, CDCl3): d 0.63e0.76 (m, 4H, H-15, H-3b), 0.86e1.02
(m, 1H, H-3a), 1.19e1.30 (m, 1H, H-2b), 1.35 (dt, J ¼ 10.7, 6.6 Hz, 1H,
H-2a), 1.80 (s, 3H, H-14), 2.03 (s, 3H, AcO-6), 2.06 (s, 3H, AcO-1),
2.52 (dd, J ¼ 16.0, 2.0 Hz, 1H, H-9b), 2.55e2.64 (m, 1H, H-4), 2.86
(dd, J ¼ 16.0, 2.0 Hz, 1H, H-9a), 3.82 (dt, J ¼ 10.9, 7.1 Hz, 1H, H-1b),
3.93 (dt, J ¼ 10.9, 7.1 Hz, 1H, H-1a), 4.03 (d, J ¼ 7.8 Hz, 1H, H-7),
5.02e5.12 (m, 1H, H-8), 5.51 (d, J ¼ 2.0 Hz, 1H, H-6), 7.19 (t,
J ¼ 8.7 Hz, 2H, Ar-13), 7.55 (d, J ¼ 2.6 Hz, 1H, H-13), 7.81 (dd, J ¼ 8.7,
4.2. Assay for cytotoxicity
5.4 Hz, 2H, Ar-13); 13C NMR (100 MHz, CDCl3):
d
18.3 (C-15), 20.2 (C-
4.2.1. Cell culture
14), 21.0 (CH3CO-6), 21.2 (CH3CO-1), 26.6 (C-2), 30.7 (C-3), 33.2 (C-
4), 35.2 (C-9), 42.5 (C-7), 64.2 (C-1), 64.6 (C-6), 74.6 (C-8), 116.3 (2C,
JC-F ¼ 22.0 Hz, Ar-13), 123.4 (C-11), 129.0 (JC-F ¼ 2.9 Hz, Ar-13), 133.1
(2C, JC-F ¼ 8.8 Hz, Ar-13), 133.4 (C-5), 133.6 (C-10), 138.6 (C-13),
163.8 (JC-F ¼ 253.1 Hz, Ar-13), 170.4 (C-12), 171.2 (CH3CO-6), 171.4
The HCT116 cell line was originally obtained from Shanghai
Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences. The HEp-2, HeLa and CHO cells were granted by Prof. Lei
group of college of life sciences, Northwest A&F university. The
HCT116 cells were grown in RPMI-1640 (Gibco), and HEp-2, HeLa
and CHO cells were grown in high glucose-DMEM (Gibco) medium
containing 10% (v/v) thermally inactivated foetal bovine serum
(FBS), penicillin (100 KU/L) and streptomycin (100 KU/L) at 37 ꢁC in
a 5% CO2 humidified incubator. Cells were always used at <90% of
confluence. All compounds were dissolved in DMSO and diluted in
cell culture media to a final concentration of less than or equal to
0.1% (v/v), which did not interfere with the cell growth. The 6-OH
ester analogues 6e8, 12e18 and C13 arylation analogues 20e24
although soluble in DMSO, started slightly precipitating (visible
only under a microscope) in the cell culture medium, and hence
were added into 96-well plates through violently vortex and taken
forward for the estimation of IC50 values.
(CH3CO-1); ESI-MS: 467.0 [MþNa]þ; HPLC: tR
purity > 99% at ELSD.
¼
16.5 min,
4.1.4.4.3. 1,6-O,O-Diacetyl-(E)-13-(4-bromophenyl)britanni-
lactone (22). Yellow oil. Yield: 67%; ½a D25
¼ þ9.5 (c ¼ 0.38 in CHCl3);
ꢀ
1H NMR (400 MHz, CDCl3):
d
0.69 (dd, J ¼ 10.3, 4.9 Hz, 1H, H-3b),
0.73 (d, J ¼ 6.9 Hz, 3H, H-15), 0.88 (dd, J ¼ 6.8, 4.9 Hz, 1H, H-3a),
0.92e1.04 (m, 1H, H-2b), 1.12e1.28 (m, 1H, H-2a), 1.80 (s, 3H, H-14),
2.03 (s, 3H, AcO-6), 2.06 (s, 3H, AcO-1), 2.49e2.55 (m, 1H, H-9b),
2.55e2.62 (m,1H, H-4), 2.86 (dd, J ¼ 16.0, 2.9 Hz,1H, H-9a), 3.82 (dt,
J ¼ 10.9, 7.0 Hz, 1H, H-1b), 3.94 (dt, J ¼ 10.9, 6.5 Hz, 1H, H-1a), 4.02
(dt, J ¼ 7.8, 2.3 Hz, 1H, H-7), 5.00e5.10 (m, 1H, H-8), 5.49 (d,
J ¼ 2.0 Hz, 1H, H-6), 7.51 (d, J ¼ 2.6 Hz, 1H, H-13), 7.64 (d, J ¼ 8.6 Hz,
2H, Ar-13), 7.69 (d, J ¼ 8.6 Hz, 2H, Ar-13); 13C NMR (100 MHz,
CDCl3):
d
18.3 (C-15), 20.2 (C-14), 21.0 (CH3CO-6), 21.2 (CH3CO-1),
4.2.2. Cytotoxicity assays
26.6 (C-2), 30.7 (C-3), 33.2 (C-4), 35.1 (C-9), 42.6 (C-7), 64.1 (C-1),
64.6 (C-6), 74.7 (C-8),124.6 (Ar-13),125.2 (C-11),131.5 (Ar-13),132.3
(4C, Ar-13), 132.4 (C-5), 133.4 (C-10), 133.6 (C-13), 138.5 (Ar-13),
170.4 (C-12), 171.2 (CH3CO-6), 171.3 (CH3CO-1); ESI-MS: 505.0
[MþH]þ; HR-ESI-MS: calcd for C25H30BrO6 [MþH]þ: 505.1220;
found: 505.1227, error ¼ 1.4 ppm; HPLC: tR ¼ 7.99 min, purity ¼ 97%
at ELSD.
Cytotoxicity in vitro was assessed by the SRB colorimetric assay,
which estimates cell number indirectly by measuring total basic
amino acids of cultured cells [35]. Briefly 100 mL aliquots of the
exponentially growing cells containing 2.5 ꢃ 104 cells/mL were
added to each well of a 96-well flat-microtiter plate and let cells
attach for 24 h. Then the medium was replaced by fresh medium
and cells were incubated with various amounts of the test com-
pound for an additional 72 h. Four replicate wells were used in each
point in the experiments. After incubation at 37 ꢁC, culture medium
4.1.4.4.4. 1,6-O,O-Diacetyl-(E)-13-(4-methoxyphenyl)britanni-
lactone (23). White solid. Yield: 43%; ½a D25
¼ þ34.9 (c ¼ 0.19 in
ꢀ
CHCl3); 1H NMR (400 MHz, CDCl3):
d
0.67e0.78 (4H, H-15, H-3b),
was moved and cells were fixed in situ with 100
10% trichloroacetic acid (TCA), and plates were incubated for 1 h at
ꢁC. Thereafter, supernatant was discarded and plates were
mL aliquots of cold
0.90e1.03 (m, 1H, H-3a), 1.19e1.40 (2H, H-2), 1.79 (s, 3H, C-14), 2.02
(s, 3H, AcO-6), 2.07 (s, H, AcO-1), 2.51 (dd, J ¼ 15.9, 2.0 Hz, 1H, H-
9b), 2.54e2.65 (m, 1H, H-4), 2.86 (dd, J ¼ 15.6, 2.4 Hz, 1H, H-9a),
3.78e3.85 (m, 1H, H-1b), 3.87 (s, 3H, CH3O-Ar-13), 3.88e3.96 (m,
1H, H-1a), 4.01 (dt, J ¼ 7.8, 2.2 Hz, 1H, H-7), 4.99e5.07 (m, 1H, H-8),
5.61 (d, J ¼ 2.1 Hz, 1H, H-6), 7.01 (d, J ¼ 8.9 Hz, 1H, Ar-13), 7.53 (d,
J ¼ 2.6 Hz, 1H, H-13), 7.78 (d, J ¼ 8.9 Hz, 1H, Ar-13); 13C NMR
4
washed 5 times with distilled water and air dried. Sulforhodamine
B solution at 0.4% (w/v) in 1% acetic acid was added to each well and
plates were incubated for 20e30 min at room temperature. The
unbound dye is removed by washing 5 times with 1% acetic acid
and plates were air dried. Bound sulforhodamine B was subse-
quently solubilized with 10 mM Tris base, and the absorbance was
(100 MHz, CDCl3):
d 18.4 (C-15), 20.2 (C-14), 21.0 (CH3CO-6), 21.3

S. Dong et al. / European Journal of Medicinal Chemistry 80 (2014) 71e82
81
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Condensation of chromatin is usually the late event in apoptosis
and is detected by nuclear staining with Hoechst 33258 as
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treatment. To observe cells undergoing apoptosis after 48 h treat-
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were then incubated with tested compound ABL, 1 (OABL) and 14.
Untreated cells (control, 0.1% DMSO) or cells treated with the sol-
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m
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Acknowledgements
[22] W.-N. Wang, Y.-Z. Wang, D.-Q. Zhang, Study on RP-HPLC determination of 1-
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This project was supported by the National Natural Science
Foundation of China (31200254) as well as Chinese Universities
Scientific Fund (QN2012047).
Appendix A. Supplementary data
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antifeedant activity of new derivatives of a dihydro-beta-agarofuran from
Parnassia wightiana, International Journal of Molecular Sciences 14 (2013)
19484e19493.
Supplementary data related to this article can be found at http://
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