Synthesis and evaluation of cytotoxic effects of novel α- methylenelactone tetracyclic diterpenoids

Article abstract of DOI:10.1016/j.bmcl.2012.01.051

A series of tetracyclic diterpenoids bearing the α-methylenelactone group have been synthesized and screened for their in vitro anti-tumor activities against six human cancer cell lines. The results showed that compounds 1c, 2a and 2b exhibited significan

Full text of DOI:10.1016/j.bmcl.2012.01.051

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1922–1925
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of cytotoxic effects of novel
tetracyclic diterpenoids
a-methylenelactone
Yao-fu Zeng a, Jia-qiang Wu a, Lian-yong Shi a, Ke Wang a, Bin Zhou a, Yong Tang a, Da-yong Zhang a,
,
⇑
Yang-chang Wu b, Wei-yi Hua a, Xiao-ming Wu a
a Center of Drug Discovery, College of Pharmacy, China Pharmaceutical University, 24 Road Tongjia Xiang, Nanjing 210009, China
b Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tetracyclic diterpenoids bearing the
a-methylenelactone group have been synthesized and
Received 8 November 2011
Revised 22 December 2011
Accepted 16 January 2012
Available online 26 January 2012
screened for their in vitro anti-tumor activities against six human cancer cell lines. The results showed
that compounds 1c, 2a and 2b exhibited significant cytotoxicity superior to the positive control
doxorubicin hydrochloride against MDA-MB-231, K562 and HepG2 cell lines. In particular, compound
2b was identified as the most promising anticancer agent against HepG2 cells with IC50 value of 0.09 lM.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Tetracyclic diterpenoids
Steviol
Isosteviol
a-Methylenelactone
Anti-tumor activity
Cancer is one of the leading causes of death worldwide and
causes serious problems in human life. Therefore, various catego-
ries of anti-tumor agents have been developed. However, some
side effects could happen simultaneously and the resistance to
available chemo-therapeutic agents was rising. Hence, it is urgent
to develop novel compounds as anticancer agents with higher
bioactivities and lower toxicities.1,2
Natural products have always been interesting sources for
developing novel leading compounds. Stevioside (Fig. 1) is the pri-
mary sweet component in the leaves of Stevia rebaudiana Bertoni
which is a plant native to South America.3,4 Stevioside consists of
three molecules of glucose and steviol as its aglycone. A large num-
ber of researches have suggested that stevioside tastes 300 times
sweeter than sucrose and can be used as a non-caloric sweetener
in South America, Japan, and China. Moreover, stevioside along
with its metabolic components steviol5 and isosteviol6 possesses
multiple pharmacological activities including anti-hyperglycemic,
anti-inflammatory, anti-tumor and anti-diarrheal. It has been
shown that these three compounds strongly inhibited the cancer
formation induced by TPA (12-o-tetra-decanoylphorbol-13-ace-
chemopreventive agents against chemical carcinogenesis.7,8 In or-
der to develop potential anticancer agents of higher cytotoxicity,
some structural modifications have been done. In our previous
work, we built up a crucial fragment of exo-methylene cyclopenta-
none in the ring D of steviol and isosteviol and obtained some com-
pounds with significantly improved cytotoxicity.9 Tao and co-
workers. synthesized various 15- and 16-substituted isosteviol
derivatives by means of functional interconversions, then obtained
some compounds with promising activities against B16-F10 mela-
noma cells.10
Plenty of investigations have reported that a-methylenelactone
is a crucial building block of many natural products and exhibits
wide-ranging biological activities such as anti-tumor, anti-inflam-
matory, antimicrobial and so on.11 Therefore, the synthesis of this
structural moiety has received much attention,12,13 and the rela-
tionship between its activities and structure has also been studied.
It appears that
agents by virtue of Michael addition with biological nucleophiles
such as
-cysteine or thiol-containing enzymes (Enz-SH).14 Many
a-methylenelactone may be regarded as alkylating
L
sesquiterpene lactones isolated from different kinds of natural
plants have been reported to display interesting biological activi-
ties. For instance, costunolide (Fig. 1) isolated from the root of Sau-
ssurea lappa exhibited potent cytotoxicity against HepG2, OVCAR-3
tate) and DMBA (7,12-dimethylbenz[a]anthracene) in a two-stage
carcinogenesis test in mouse. In addition, isosteviol inhibited both
mammalian DNA polymerases and human DNA topoisomerase II.
Taken together, these compounds could be served as promising
and HeLa cell lines with CD50 values of 1.6, 2.0, 2.0
tively.15 Kupchan et al. discovered that vernolepin (Fig. 1) bearing
two -methylenelactone groups showed significant cytotoxicity
activity against Walker intramuscular carcinosarcoma in vitro
lg/mL, respec-
a
⇑
Corresponding author. Tel./fax: +86 25 83271307.
E-mail address: zhangdayong@cpu.edu.cn (D. Zhang).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.051

Y. Zeng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1922–1925
1923
then reaction of 4 with selenium oxide and tert-butyl hydroperox-
ide led to 5 (Scheme 1).18 Oxidation of 5 with PDC provided com-
pound 6.19 We next tried a phenylthio group as a stable protecting
O-β−glu-β−glu(2→1)
OH
O
H
H
group of the a-methylene unit. Conjugate addition of p-thiocresol
O
to enone 6 produced b-thioketone 720 which was successfully
transformed into sulfone lactone 8 by Baeyer–Villiger oxidation
with excessive mCPBA.21,22 Finally, desulfonylation of 8 with DBU
in THF under mild condition gave the desired compound 1a.23
Compound 1b was prepared from 1a by deprotection of methoxy-
methyl group with 10% HCl in THF.24 Esterification of 1b with dif-
ferent kinds of halohyrocarbons afforded 1c–f. Compound 1g could
be obtained by acylation of 1f with acetic anhydride in the pres-
ence of DMAP.25
H
O
O
COO-β−glu
stevioside
O
O
vernolepin
costunolide
Figure 1. Chemical structures of stevioside, costunolide and vernolepin.
and in vivo in rats.16 However, ent-kaurane diterpenoids possess-
ing -methylenelactone group are rare in the natural products dis-
covered recently. Therefore, we tried to introduce this critical
moiety into steviol and isosteviol and obtained three scaffolds of
ent-kaurene diterpenoids. Some derivatives were also synthesized
and screened for their anticancer activities against six cancer cell
lines in vitro by MTT method.
The synthetic route towards the target compounds was de-
scribed as follows: first, treatment of steviol with chloromethyl
methyl ether and N,N-diisopropylethylamine afforded 4 in 1 h,17
a
The synthetic approach employed to prepare 2a was outlined in
Scheme 2. First, we attempted to reduce 4 with LiAlH4 in anhy-
drous THF under refluxing condition. Although the reaction could
proceed smoothly, the yield was very low due to the poor liposol-
ubility of the product. Therefore, we had to protect the 13-hydroxy
of steviol with excessive MOM ether as well. By doing this, com-
pound 10 could be obtained in a good yield (86%). Acylation of
OH
OH
OH
OH
a
b
d
c
H
H
H
H
H
H
H
H
OH
O
COOMOM
COOMOM
COOH
COOMOM
4
5
steviol
6
OH
OH
OH
S
SO2
H
H
e
g
f
H
H
O
O
H
H
O
O
O
COOMOM
COOMOM
1a
COOMOM
7
8
OR2
OH
1c R1=CH3, R2=H
1d R1=CH2CH2CH3, R2=H
h
H
H
O
O
H
H
O
O
1
2
1e
1f
R =CH2CH=CH2, R =H
R1=Bn, R2=H
COOR1
COOH
i
1
2
1g
R =Bn, R =Ac
1b
Scheme 1. Reagents and conditions: (a) MOMCl, DIPEA, DMF (90.0%); (b) SeO2, t-BuOOH, THF (85.0%); (c) PDC, DMF (75.0%); (d) p-thiocresol, Et3N, THF (65.6%); (e) 85%
mCPBA, NaHCO3, CH2Cl2 (53.8%); (f) DBU, THF (69.9%); (g) 10% HCl, THF, H2O (84.0%); (h) R1R (for 1c, R = I; for 1d and 1f, R = Br; for 1e, R = Cl), K2CO3, DMF, KI (64.0–81.4%); (i)
Ac2O, Et3N, THF, DMAP (65.2%).
OMOM
OH
OMOM
OMOM
OH
e
b
d
a
g
H
H
H
H
H
H
H
H
CH2OAc
COOMOM
CH2OR
COOH
steviol
9
12
10
11
R=H
R=Ac
c
OH
OH
OH
OR
O
SO2
S
h
i
H
H
O
O
H
H
H
H
O
O
H
H
O
CH2OAc
CH2OAc
CH2OR
CH2OAc
15
16
13
14
R=MOM
R=H
2a
2b
R=Ac
R=H
f
j
Scheme 2. Reagents and conditions: (a) MOMCl, DIPEA, DMF (85.3%); (b) LiAlH4, THF, reflux (86.0%); (c) Ac2O, Et3N, THF, DMAP (77.6%); (d) SeO2, t-BuOOH, THF (75.6%); (e)
PDC, DMF (72.0%); (f) 10% HCl, THF, H2O (82.3%); (g) p-thiocresol, Et3N, THF (87.6%); (h) 85% mCPBA, NaHCO3, CH2Cl2 (55.2%); (i) DBU, THF (70.6%); (j) 10% KHCO3, CH3OH,
reflux (71.0%).

1924
Y. Zeng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1922–1925
Table 1
10 with acetic anhydride afforded 11. The following procedures for
preparing 2a were exactly the same as for preparing 1a. Finally, 2a
was converted to 2b with 10% KHCO3 in refluxing CH3OH. It was
noteworthy that conjugate addition of p-thiocresol to 13 could
not get the b-thioketone, so we had to deprotect the MOM ether
to reduce the steric hindrance.
Scheme 3 illustrated the synthesis of compounds 3a–d starting
from isosteviol. First of all, we attempted to construct a hydroxy-
methyl in the a-position of 16-ketone with aqueous formaldehyde
under base condition. Surprisingly, the 16-ketone was reduced at
the same time. The mechanism of the reaction had been reported
and was proposed as a one-spot ‘Aldol–Cannizzaro reaction’ pro-
cess.26 After the diol 18 had been successfully achieved, we chose
acetyl, a cleanly and conveniently deprotected group, to protect
the primary alcohol selectively. Oxidation of 19 with PDC provided
20. However, the key intermediate lactone 22 couldn’t be prepared
from 20 with excessive mCPBA until the deprotection of acetyl was
completed. Treatment of 22 with p-toluenesulfonyl chloride in pyr-
idine produced tosylate 23 which was heated under reflux in pyr-
idine for 6 h to give the target compound 3a.27 Compound 3b was
obtained via elimination of 24 which was accessed by removing
the benzyl of 23 with 10% Pd-C. Eventually, two analogues (3c,
d) of 3b have been synthesized.
Cytotoxicities of compounds 1a–3e in vitroa
Compound
Anti-tumor activity in 48 hb (IC50
,
l
M)
PC-3
HCT-
116
MDA-MB-
231
K562
HepG2 MGC803
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
3a
3b
3c
3d
3e
Doxc
0.12
8.32
1.56
2.56
2.31
0.22
0.78
21.10
0.56
24.21
18.4
0.13
24.43
22.12
6.23
0.34
102.10
0.89
0.38
5.68
0.16
67.21
67.21
78.15
25.23
48.34
0.12
2.23
3.23
1.21
35.14
34.18
2.12
1.56
29.97
2.56
88.67
56.32
15.52
10.11
56.24
5.71
67.56
24.12
45.24
35.23
45.46
0.34
36.43
37.32
28.23
18.10
38.69
1.10
2.10
3.83
0.22
0.16
0.09
1.26
88.76
56.21
34.21
65.35
189.92 222.62
1.18 3.03
78.90
35.23
56.45
23.45
102.50
78.43
22.87
56.76
127.35
1.45
19.53
56.24
25.45
35.23
28.65
46.54
35.24
12.39
19.92
98.78
11.23
19.76
87.81 231.26 123.54
2.35 1.09 2.56
a
Inhibition of cell growth by the listed compounds was determined using MTT
assay.
b
Data represent the mean value of three independent determinations.
Dox = Doxorubicin hydrochloride.
c
In order to investigate whether the
a-methylenelactone group
is essential for the bioactivity of the compound; we changed the
colotectal carcinoma (HCT-116), breast carcinoma (MDA-MB-
231), human erythroleukemic cell line (K562), hepatocellular car-
cinoma (HepG2) and gastric carcinoma (MGC-803). Doxorubicin
hydrochloride was selected as a positive control. The IC50 values
were used to determine the growth inhibition in the presence of
tetracyclic diterpenoids 1a–3e against PC-3, HCT-116, MDA-MB-
231, K562, HepG2 and MGC-803 cancer cell lines. From the IC50
values summarized in Table 1, the compounds 1c, 2a and 2b have
a
-methylene group into the epoxy. Oxidation of 1f and 3a with
excessive mCPBA afforded compounds 1h28 and 3e, respectively
(Scheme 4).
The structures of the target compounds were elucidated using
spectroscopic techniques (IR, 1H and 13C NMR, ESI/MS, HRMS).
The cytotoxic activities29 of compounds 1a-3e30 were deter-
mined in vitro against six cell lines: prostatic carcinoma (PC-3),
O
OH
OH
OH
O
a
c
d
H
H
H
H
H
H
OAc
H
H
OR
COOH
COOR
COOBn
19
COOBn
17
isosteviol
R=H
20
R=Ac
b
e
18 R=Bn
21 R=H
O
O
O
f
g
O
i
O
H
H
H
H
H
H
O
OH
OTs
COOBn
22
COOR
COOR
3a
23
R=Bn
h
24 R=H
R=Bn
3b R=H
j
j
3c
3d
R=CH3
R=CH2CH2CH3
Scheme 3. Reagents and conditions: (a) HCHO (aq), NaOH, C2H5OH–H2O, 75 °C, 3 h (56.1%); (b) BnBr, K2CO3, DMF, KI (70.4%); (c) Ac2O, Et3N, THF, DMAP, 45 min (85.2%); (d)
PDC, DMF (83.1%); (e) 10% KOH, CH3OH, rt (89.4%); (f) 85% mCPBA, NaHCO3, CH2Cl2 (56.1%); (g) TsCl, pyridine, DMAP (53.7%); (h) 10% Pd-C, H2, C2H5OH (92.3%); (i) pyridine,
DMAP, reflux (66.2%); (j) RR1(for 3c, R1 = I; for 3d, R1 = Br), K2CO3, DMF, KI (75.2%, 80.1%).
OH
OH
O
O
O
a
a
H
H
O
H
H
O
H
H
O
O
H
H
O
O
O
COOBn
3a
COOBn
COOBn
COOBn
3e
1h
1f
Scheme 4. Reagents and conditions: (a) 85% mCPBA, NaHCO3, CH2Cl2 (51.3%, 55.7%).

Y. Zeng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1922–1925
1925
22. Whitesell, J. K.; Matthews, R. S.; Minton, M. A.; Helbling, A. M. J. Am. Chem. Soc.
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2011, 13, 787.
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27. Kretchmer, R. A.; Thompson, W. J. J. Am. Chem. Soc. 1976, 98, 3379.
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Lin, S. J. Phytochemistry 2007, 68, 562.
29. Cytotoxicity assay in vitro: The human cancer cell lines were provided by Mr.
Yangchang Wu research group (China Medical University) and maintained in a
humidified atmosphere at 37 °C in 5% CO2. The cells were cultured in RPMI-
shown significant cytotoxities against all the six cell lines with the
IC50 values ranging from 0.09 to 5.71 M. Compounds 1a and 1c
l
were found to be more effective than doxorubicin hydrochloride
in HCT-116, MDA-MB-231, HepG2 and MGC803 cell lines. Esterifi-
cation of 19-acid with MOM ether or methyl improved the cytotox-
icity (1a vs 1b, 1b vs 1c), while esterification of 19-acid with
propyl, allyl and benzyl group decreased the activity (1b vs 1d,
1b vs 1e, 1b vs 1f). Compound 1f showed selective inhibition
against K562 (IC50 = 6.23 lM) and MGC803 (IC50 = 2.12 lM) cell
lines. Compound 1g with the 13-hydrogen acylated exhibited
slightly higher activity compared to compound 1f. On the contrary,
removing the 19-acetyl of 2a afforded 2b with better cytotoxity
against all the cell lines except HCT-116. Compared with 1h, com-
1640 media containing 10% FBS, 100 IU/mL penicillin and 100 lg/mL
streptomycin. Cell cytotoxity was determined by MTT assay. Briefly, cells
were seeded in 96-well-plate (1 Â 104 cells/well) and incubated for 48 h. Then,
the tested compounds with various concentrations were added to the wells
and 48 h later the MTT solution (0.5 mg/mL) was added and incubated for 4 h.
Two hundred microliters of DMSO was added to each well to dissolve the
reduced MTT crystals. The absorbance of each well was measured at 570 nm
with a microplate reader.
pound 1f bearing the
activity, which indicated that
an important role in their anticancer activities. However, the com-
pounds (3a–e) with the isosteviol scaffold exhibited weak
activities.
a
-methylenelactone group displayed better
a
-methylenelactone group played
In summary, we have successfully synthesized three scaffolds of
30. Selected spectral data for compounds 1a–2b: Compound 1a: white solid, mp
172–174 °C; IR (KBr, cmÀ1) 3415, 2952, 2928, 2879, 1731, 1717, 1696, 1629,
1466, 1384, 1370, 1311, 1170, 1136, 1073, 1035, 999, 942; 1H NMR (300 MHz,
CDCl3) d: 6.56(s, 1H), 6.07(s, 1H), 5.29(d, J=6 Hz, 1H), 5.16(d, J = 6 Hz, 1H),
3.49(s, 3H), 2.35(d, J = 13.2 Hz, 1H), 2.26(d, J = 13.41 Hz, 1H), 1.98–2.04(m, 3H),
1.86–1.94(m, 4H), 1.43–1.83(m, 6H), 1.26(s, 3H), 0.91–1.21(m, 3H), 0.88(s, 3H);
13C NMR (75 MHz, CDCl3) d: 176.6, 165.4, 143.3, 125.8, 90.5, 84.9, 70.3, 57.9,
56.1, 50.2, 43.9, 42.6, 41.7, 40.9, 39.7, 38.1, 37.4, 28.8, 20.6, 19.1, 18.8, 16.4; ESI/
MS: 393 [M+H]+; HRMS: calcd for C22H32NaO6 [M+Na]+ 415.2091, found
415.2102. Compound 1b: white solid, mp 218–220 °C; IR (KBr, cmÀ1) 3539,
3423, 3221, 2957, 2868, 1704, 1630, 1467, 1385, 1370, 1353, 1292, 1264, 1234,
1202, 1164, 1095, 999, 970, 956, 812, 499; 1H NMR (300 MHz, DMSO-d6) d:
12.1(s, 1H), 6.26(d, J = 1.77 Hz, 1H), 5.94(d, J = 1.1 Hz, 1H), 2.20(d, J = 13.38 Hz,
1H), 2.04(d, J = 12.93 Hz, 1H), 1.77–1.90(m, 5H), 1.61–1.75(m, 4H), 1.41–
1.45(m, 1H), 1.33(m, 1H), 1.18–1.27(m, 2H), 1.14(s, 3H), 0.93–1.05(m, 2H),
0.88(s, 3H), 0.74–0.85(m, 1H); 13C NMR (75 MHz, DMSO-d6) d: 178.5, 164.7,
144.1, 124.6, 84.4, 68.6, 54.9, 50.1, 42.6, 41.6, 41.2, 40.4, 39.2, 38.3, 37.1, 28.4,
20.4, 18.9, 18.5, 15.9; ESI/MS: 331 [M+1–H2O]+. Compound 1c: white solid, mp
200ꢀ202 °C; IR (KBr, cmÀ1) 3437, 2954, 2928, 2868, 1721, 1697, 1625, 1456,
1437, 1371, 1307, 1291, 1237, 1202, 1167, 1149, 1118, 1083, 1039, 995, 971;
1H NMR (300 MHz, CDCl3) d: 6.55(s, 1H), 6.07(s, 1H), 3.65(s, 3H), 2.35(d,
J = 13.2 Hz, 1H), 2.23(d, J = 13.32 Hz, 1H), 1.96–2.0(m, 2H), 1.85–1.91(m, 4H),
1.76–1.82(m, 2H), 1.62–1.74(m, 2H), 1.25–1.58(m, 4H), 1.21(s, 3H), 1.03–
1.18(m, 1H), 0.93–1.00(m, 1H), 0.85(s, 3H); 13C NMR (75 MHz, CDCl3) d: 177.6,
165.4, 143.3, 125.8, 85.0, 70.3, 56.0, 51.3, 50.2, 43.6, 42.5, 41.7, 40.9, 39.6, 38.1,
37.5, 28.7, 20.6, 19.1, 18.8, 16.2; ESI/MS: 380 [M+NH4]+; HRMS: calcd for
tetracyclic diterpenoids bearing the
and evaluated their anticancer activities against six cell lines. We
also proved that -methylenelactone group was essential for the
a-methylenelactone moiety
a
bioactivity of the compound, which was consistent with the previ-
ous literature.11 Compound 2b was found to be the most potent
compound in HepG2 with IC50 value of 0.09 lM. Further researches
on identifying their cellular targets are ongoing in our laboratory
and the results will be reported in due course.
Acknowledgment
We thank the National Natural Science Foundation of China
(No. 30973607 and No. 81172934) for financial support.
References and notes
1. Wang, T. T.; Liu, J.; Zhong, H. Y.; Chen, H.; Lv, Z. L.; Zhang, Y. K.; Zhang, M. F.;
Geng, D. P.; Niu, C. J.; Li, Y. M.; Li, K. Bioorg. Med. Chem. Lett. 2011, 21, 3381.
2. Sangthong, S.; Krusong, K.; Ngamrojanavanich, N.; Vilaivan, T.; Puthong, S.;
Chandchawan, S.; Muangsin, N. Bioorg. Med. Chem. Lett. 2011, 21, 4813.
3. Brusick, D. J. Food Chem. Toxicol. 2008, 46, S83.
C
21H30NaO5 [M+Na]+ 385.1985, found 385.1996. Compound 1f: colorless oil, IR
(KBr, cmÀ1) 3427, 2956, 2932, 2871, 1718, 1627, 1455, 1370, 1351, 1275, 1259,
1231, 1207, 1163, 1141, 1092, 999, 996, 806, 755, 745, 699; 1H NMR (300 MHz,
CDCl3) d: 7.34(m, 5H), 6.54(s, 1H), 6.05(s, 1H), 5.15(d, J = 12.34 Hz, 1H), 5.04(d,
J = 12.33 Hz, 1H), 2.25(d, J = 13.17 Hz, 1H), 1.68–2.00(m, 9H), 1.57–1.63(m, 2H),
1.45–1.55(m, 3H), 1.24(s, 3H), 0.92–1.22(m, 3H), 0.76(s, 3H); 13C NMR
(75 MHz, CDCl3) d: 176.8, 165.3, 143.3, 135.8, 128.5, 128.3(2), 128.2(2),
125.8, 84.9, 70.4, 66.1, 56.2, 50.1, 43.76, 42.5, 41.7, 40.9, 39.6, 38.1, 37.5,
28.8, 20.6, 19.1, 18.8, 16.3; ESI/MS: 461 [M+Na]+. Compound 2a: white solid,
mp 152–154 °C; IR (KBr, cmÀ1) 3433, 2935, 2866, 1717, 1654, 1629, 1458,
1384, 1374, 1307, 1275, 1260, 1241, 1159, 1090, 1036, 1000; 1H NMR
(300 MHz, CDCl3) d: 6.48(s, 1H), 6.00(s, 1H), 4.11(d, J = 11.1 Hz, 1H), 3.83(d,
J = 11 Hz, 1H), 2.26(d, J = 13.2 Hz, 1H), 1.98(s, 3H), 1.82–1.94(m, 5H), 1.71–
1.81(m, 2H), 1.58–1.67(m, 2H), 1.36–1.53(m, 4H), 1.26–1.32(m, 1H), 1.15–
1.23(m, 1H), 1.03–1.09(m, 1H), 0.99(s, 3H), 0.93(s, 3H), 0.78–0.90(m, 1H); ESI/
MS: 375 [MÀH]À. 2b: white solid, 92–94 °C; IR (KBr, cmÀ1) 3424, 2961, 2930,
2870, 1701, 1626, 1475, 1446, 1384, 1369, 1348, 1307, 1267, 1174, 1164, 1029,
1000; 1H NMR (300 MHz, DMSO-d6) d: 6.26(s, 1H), 5.94(s, 1H), 3.45–3.51(m,
1H), 3.16–3.22(m, 1H), 2.22(d, J = 13.4 Hz, 1H), 1.80–1.98(m, 4H), 1.67–1.76(m,
4H), 1.42–1.54(m, 3H), 1.23–1.36(m, 3H), 1.18(m, 1H), 1.03–1.15(m, 1H),
0.99(s, 3H), 0.89(s, 3H), 0.83(m, 1H); 13C NMR (75 MHz, DMSO-d6) d: 164.7,
144.3, 124.5, 84.6, 68.6, 63.0, 55.5, 51.1, 43.5, 41.8, 41.7, 40.1, 39.2, 38.3, 35.1,
27.7, 18.8, 18.4, 18.0, 17.7; ESI/MS: 333 [MÀH]À; HRMS: calcd for C40H60NaO8
[2M+Na]+ 691.4180, found 691.4207.
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