Synthesis and in vitro antitumor evaluation of betulin acid ester derivatives as novel apoptosis inducers

Article abstract of DOI:10.1016/j.ejmech.2015.08.004

Nineteen betulin derivatives modified at the C-3 and C-28 positions were synthesized and assessed for antitumor activities against the MGC-803, PC3, Bcap-37, A375, and MCF-7 human cancer cell lines in vitro by MTT assay. Some derivatives (compounds 3a-3d

Full text of DOI:10.1016/j.ejmech.2015.08.004

Accepted Manuscript
Synthesis and in vitro antitumor evaluation of betulin acid ester derivatives as novel
apoptosis inducers
Sheng-Jie Yang, Ming-Chuan Liu, Hong-Mei Xiang, Qi Zhao, Wei Xue, Song Yang
PII:
S0223-5234(15)30188-4
10.1016/j.ejmech.2015.08.004
EJMECH 8040
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 May 2015
Revised Date: 8 July 2015
Accepted Date: 2 August 2015
Please cite this article as: S.-J. Yang, M.-C. Liu, H.-M. Xiang, Q. Zhao, W. Xue, S. Yang, Synthesis and
in vitro antitumor evaluation of betulin acid ester derivatives as novel apoptosis inducers, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.08.004.
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Graphical abstract
Novel betulin acid derivatives were synthesized and investigated for their antitumor activity.

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Synthesis and in vitro antitumor evaluation of betulin acid ester
derivatives as novel apoptosis inducers
Sheng-Jie Yanga,b¶, Ming-Chuan Liua,b¶, Hong-Mei Xianga, Qi Zhaoa, Wei Xuea, and Song Yanga*
a State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou
University, Guiyang 550025, P.R. China
b Sinphar Tian-Li Pharmaceutical Co., LTD, Hangzhou, 311100, P.R. China
¶All authors contributed equally to this work.
*Corresponding author. Ctr for R&D of Fine Chemicals, Guizhou University, Huaxi St., Guiyang,
China 550025. Tel.: +86 851 829 2171; Fax: +86 851 829 2170.
E-mail address: jhzx.msm@gmail.com
Conflicts of interest: None.
Author’s contributions:
Sheng-Jie Yang and Ming-Chuan Liu synthesized the compounds and carried out most of the
bioassay experiments. Hong-Mei Xiang took part in the compound structural elucidation and
bioassay experiments. Qi Zhao carried out some structure elucidation experiments. Wei Xue
assisted in structural elucidation experiments. Prof. Song Yang is the co-corresponding author for
this work.
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Abbreviation list:
ADM: adriamycin
AO/EB: acridine orange/ethidium bromide
BE: betulin
13C NMR: 13C Nuclear Magnetic Resonance
DMF: N, N-dimethylformamide
DMSO: dimethyl sulfoxide
HCPT: 10-hydroxyl camptothecine
1H NMR: Proton Nuclear Magnetic Resonance
IR: Infra-red
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
TUNEL: terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling
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Abstract
Nineteen betulin derivatives modified at the C-3 and C-28 positions were synthesized and
assessed for antitumor activities against the MGC-803, PC3, Bcap-37, A375, and MCF-7 human
cancer cell lines in vitro by MTT assay. Some derivatives (compounds 3a–3d and 5) displayed
strong antitumor properties, with IC50 values between 4 and 18 ꢀM. Compound 3c, containing
piperidine group at C-28 positon, had IC50 values of 4.3, 4.5, 5.2, 7.5, and 5.2 ꢀM on the five
cancer cell lines, respectively. Subsequent fluorescence staining and flow cytometric analysis
indicated that compound 3c induced apoptosis in MGC-803 cell line, with an apoptosis ratio of
31.11% after 36 h of treatment at 10 ꢀM 3c.
Keywords: Betulin derivatives; Synthesis; Antitumor; Cancer cells; Apoptosis
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1. Introduction
Betulin (BE; 1; lup-20(29)-ene-3β, 28-diol), is an abundant and naturally occurring pentacyclic
triterpenoid [1,2]. This compound is a major effective component of many traditional Chinese
medicines that is widely distributed in plants [3-5], and forms up to 30% of the dry weight of the
extractive (e.g., the bark of birch trees) [6-8]. BE possesses various biological and
pharmacological activities [9], such as anti-HIV [10], anti-inflammatory [11,12], , antiretroviral
[13], antibacterial properties [14], and anticancer properties [15,16]. Recently, some studies have
shown that BE had marked antitumor activities against various types of cancer cell lines [17], such
as colorectal (DLD-1), prostate (PC3), breast (MCF-7), and lung (A549) cancer cell lines, and
could induce A549 cell-line apoptosis [18-21]. Furthermore, a recent report found BE to be more
active against other melanoma lines (G361, SK-MEL-28), neuroblastoma cell lines (GOTO,
NB-1), and leukemia lines (HL-60, U937, K562) [22]. However, compared to other pentacyclic
triterpenoids, BE is inactive against melanoma cell line (MEL-2) [23]. Researchers had
demonstrated that keeping a polar substituent at the C-3 position was essential for the
pharmacological activities of pentacyclic triterpenes [24]. According to the structure-activity
relationship, a hydrogen donor group at either C-3 or C-28 position can improve cell proliferation
inhibition significantly [25]. In recent years, the drug, with introduced amino alkyl groups has
attracted considerable attention. Recent studies have shown that the introduction of amino alky
groups had unexpected improvements in the anti-HIV or antitumor activities of compounds [25].
In 2010, Kommera et al. found that the antitumor activities of BE derivatives with amino acyl
groups introduced at C-28 position were significantly improved, and had the therapeutic potential
in the treatment of gastric carcinoma [26]. Furthermore, previous work already indicated that the
introduction of aromatic carboxyl groups to pentacyclic triterpenoids could result in good
cytotoxicity [27,28].
Thus, in view of the previous rationale and in continuation of an ongoing program aiming at
developing more potential anticancer drugs, in the present study nineteen BE derivatives modified
at C-3 and C-28 positions were designed and synthesized. The antitumor activities of these
derivatives were screened in vitro by MTT assay using five human cancer cell lines MGC-803
(human gastric carcinoma cell line), PC3 (human prostate carcinoma cell line), Bcap-37 (human
breast carcinoma cell line), A375 (human malignant melanoma cell line), and MCF-7 (human
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breast carcinoma cell line). Additionally, the antitumor mechanism of the BE derivatives was
investigated through acridine orange/ethidium bromide staining (AO/EB), Hoechst 33258 staining,
terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and flow
cytometric analysis.
2. Results and discussion
2.1. Chemistry
The syntheses of BE derivatives are summarized in Scheme 1. The structure of BE was modified
at C-3 and C-28 positions. Succinic anhydride and maleic anhydride were introduced at the C-28
position in dry dichloromethane (DCM) in the presence of pyridine to obtain compounds 2 and 4.
Compound 2 was treated with corresponding amines in dry DCM, in the presence of PyBOP to
afford compounds 3a–3d. The 28-OH and 3-OH of BE were acetylated with aromatic carboxyl
group in the presence of DCC and DMAP in dry DCM at room temperature to gain the
compounds 6a–6d. Finally, only the 28-OH of BE was acetylated using EDCI instead of DCC to
obtain the compounds 7a–7g in high yields. All the compounds were fully characterized by
various spectroscopic methods.
Scheme 1
2.2. Evaluation of antitumor activities
In vitro inhibitory activities of the synthesized BE derivatives were evaluated against MGC-803,
PC3, Bcap-37, A375, and MCF-7 cell lines by MTT assay. Hydroxycamptothecine (HCPT) and
Adriamycin (ADM) were used as positive controls. A culture medium containing 0.1% DMSO
served as negative control. All BE derivatives including ADM and HCPT were dissolved in
DMSO. Each experiment was repeated thrice. The IC50 values are summarized in Table 1.
Table 1
Table 1 presents the modified compounds at the C-28 position with selected amino groups
displaying potent antitumor activities against the five cancer cell lines. These compounds had IC50
values between 4 and 18 ꢀM. Furthermore, the most active compound among the synthesized BE
derivatives was compound 3c. This compound had approximately four times more activity than
that of BE, and the IC50 values were 4.3, 4.5, 5.2, 7.5, and 5.2 ꢀM on the five cancer cell lines,
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respectively. Further experiments indicated that proliferation of the five cancer cells were
significantly inhibited by compound 3c in a concentration-dependent manner (Figure 1).
Figure 1
Compounds 2 and 4, with succinic anhydride and maleic anhydride introduced at the C-28
position, had more activity than that of the parent BE. This result may be attributed to the
introduced carboxyl group, which was more hydrophilic than the hydroxyl group. This phenomena
was similar to Drag-Zalesinska observations, wherein the most active compounds were those with
the highest solubility in water [29].
Recently, some researchers studied the structure-activity relationships of other pentacyclic
triterpenes including Betulinic acid, Ursolic acid, and Oleanolic acid. These pentacyclic
triterpenes had similar structure-activity relationships despite the different structures.30 Previous
studies showed that the introduction of amino alkyl groups at the C-28 position further enhanced
the antitumor activities. In this study, the compounds modified at the C-28 position with selected
amino groups displayed potent antitumor activities (IC50 values, between 4 and 18 ꢀM), identical
with the previous reports [30].
Remarkably, when 28-OH was esterified with aromatic acid (7a–7g), the modification led to a
decline in activity. In addition, when 28-OH and 3-OH were esterified at the same time (6a–6d),
the activity was reduced or even disappeared. The low activity can be explained by the
observation of Kommera et al. [26]: “One reason for low activity can be due to the straight and
rigid phenyl groups and the possible interactions with the π-system of the phenyl ring lead to a
much bulky molecule, thereby with a lower ability to penetrate the cell membrane.”
In addition, the active compounds 2, 3a–3d, 4, and 5 were also tested on normal cell line NIH3T3.
As shown in Table 2, compounds 2 and 3a–3d were less toxic on normal cell lines than that on
cancer cell lines, and more selective to cancer cell lines than compounds 4 and 5. Compounds 4
and 5 were selective to cancer cells, and were, to some extent, cytotoxic on normal cells.
Table 2
2.3. Preliminary investigation of the apoptosis-inducing effect of title compound 3c
BE and its derivatives have been reported to induce apoptosis in certain cancer cell lines [31]. In
the present study, compound 3c was selected to analyze the mechanism of growth inhibition of
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MGC-803 cells. To determine whether or not the growth inhibitory activity of compound 3c were
related to the induction of apoptosis, the morphological changes of MGC-803 cells were
investigated by AO/EB staining, Hoechst 33258 staining, and TUNEL assay.
The cell morphological changes brought by compound 3c were assessed using fluorescence
microscopy after AO/EB staining. AO permeates all the cells and stains the nuclei green, whereas
EB only penetrates the cells when cytoplasmic membrane integrity is lost, and stains the nucleus
red, dominating over AO. The stained cells revealed four different types under a fluorescence
microscope: the chromatin of living cells was green with normal structure; the chromatin of
non-apoptotic dead cells was red with normal structure; yellow coloration of early apoptotic cells
with pyknotic morphology; and orange coloration of late apoptotic cells with pyknotic
morphology. With HCPT as positive control, compound 3c at 5 ꢀM against MGC-803 cells from
12 h to 48 h was detected via AO/EB staining. The HCPT concentration was 10 ꢀM against
MGC-803 cells for 48 h. The results are shown in Figure 2A. Cells treated with compound 3c
from 12 to 48 h had morphological changes. The nuclei stained as yellow green or orange, and the
morphology showed pyknosis, membrane blebbing, and cell budding. These phenomena were
associated with cell apoptosis.
The morphological changes during cell apoptosis were also assayed using Hoechst 33258 staining.
Live cells with uniformly light blue nuclei were treated with Hoechst 33258 and observed under a
fluorescence microscope. Four types of stained cells were observed, characterized by cytoplasmic
and nuclear shrinkage, chromatin condensation, and apoptotic body. With HCPT (at 10 ꢀM for 48
h) as positive control, compound 3c at 5 ꢀM was tested against MGC-803 cells from 12 h to 48 h
via Hoechst 33258 staining. Results are shown in Figure 2B, which shows that the cells of the
negative group were stained blue, indicating normal condition. However, the cells of HCPT group
appeared compact and condensed, with strong blue fluorescence, signifying typical apoptosis
characteristics. The cells treated with compound 3c from 12 to 48 h had morphological changes.
The blue emission light in apoptotic cells was brighter than the negative group. Condensed
chromatin could also be found in many treated cells and some cells formed apoptotic bodies,
which is a characteristic of apoptotic cells. These results were similar with the previous AO/EB
dual staining results.
TUNEL, one of the popular methods to investigate the apoptosis induction, relies on the presence
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of nicks in the DNA. DNA nicks can be identified by terminal deoxynucleotidyl transferase, an
enzyme that will catalyze the addition of dUTPs, which are secondarily labeled with a marker.
Cells with brown precipitate are positive to apoptosis. With HCPT used as positive control at 10
ꢀM for 48 h, compound 3c at 5 ꢀM was tested against MGC-803 cells from 12 to 48 h and
apoptosis was detected using TUNEL assay. Results are shown in Figure 2C. Cells of the negative
group did not appear as brown precipitates, whereas HCPT group appeared as brown precipitates.
After 48 h, most of the cells treated with compound 3c also appeared as brown precipitates.
Therefore, compound 3c induced apoptosis against MGC-803 cells. The results were identical
with the previous experiments.
Figure 2
The apoptosis ratios induced by compound 3c in cancer cells were quantitatively assessed by FCM.
In early apoptotic cells, phosphatidylserine (PS), which is distributed inside the lipid bilayer of
normal cells, was transferred from the inside of the cell membrane to the outside. Annexin V, a
Ca2+ dependent phospholipid-binding protein with a high affinity for PS, was used to detect early
apoptosis. Propidine Iodide (PI) is a red fluorescent dye, which stains cells that have lost
membrane integrity. When the cells are dual stained with annexin V-FITC and PI, four different
periods of apoptotic cells can be observed. Cells that are not stained with either annexin V or PI
are viable and reside in the lower lift quadrant. Cells that are only stained with annexin V are in
the stage of early apoptosis and reside in the lower right quadrant. Cells that are stained by both
chemicals are nonviable apoptotic/necrotic cells and scatter in the upper right quadrant, while the
upper left quadrant represents the nuclear fragments/necrotic cells stained only with PI. As shown
in Figure 3A, with HCPT as positive control, compound 3c (10 ꢀM) could induce apoptosis of
MGC-803 cells. The highest apoptosis ratio, 31.11% was obtained after 36 h of treatment at a
concentration of 10 ꢀM. Furthermore, as shown in Figure 3B, the apoptosis of MGC-803 cells
increased gradually in a time-dependent manner.
Figure 3
3. Conclusions
In summary, nineteen BE derivatives were designed and synthesized. The growth inhibitory
activities were evaluated against MGC-803, PC3, Bcap-37, A375, and MCF-7 human cell lines in
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vitro for all derivatives. Among the BE derivatives synthesized, compound 2, 3a–3c, and 5
displayed strong antitumor properties showing IC50 values between 4 and 18 ꢀM. Compound 3c
possessed the most potent activities, with IC50 values of 4.3, 4.5, 5.2, 7.5, and 5.2 ꢀM for the five
cancer cell lines, respectively. However, after introduction of aromatic acid in BE, the activity was
reduced or even disappeared. Structure-activity relationship was also investigated in this study.
Moreover, compound 3c was selected to analyze the mechanism of growth inhibition of MGC-803
cell lines by AO/EB staining, Hoechst 33258 staining, TUNEL assay, and flow cytometric analysis.
These mechanistic studies demonstrated that compound 3c inhibited cell growths by inducing cell
apoptosis. Apoptosis ratio reached 31.11% after 36 h of treatment at 10 ꢀM, which is higher than
the ratio observed for the positive control HCPT. These findings provide a powerful incentive for
further research on the chemical modification and structure-activity relationships of BE and other
triterpenoid acids.
4. Experimental section
4.1. General
Betulin with more than 98% purity was purchased from Zhejiang Tiancao Biotech Co., Ltd.
Reagents of analytical grade were obtained from Yuda Chemistry Co., Ltd., and used without
further purification unless otherwise noted. Silica gel (200–300 mesh) used in column
chromatography was provided by Tsingtao Marine Chemistry Co., Ltd. IR spectra were recorded
on a Bruker VECTOR22 spectrophotometer in KBr disks. 1H NMR and 13C NMR were performed
on a JEOL-ECX500 spectrometer at 22 °C, with TMS as the internal standard.
4.2. Synthesis
4.2.1. General procedure for compounds 2, 3a-3d, and 4-5.
Betulin reacted with succinic anhydride or maleic anhydride (0.50 g, 5.00 mmol) in dry DCM (35
mL) containing DMAP (0.06 g, 0.50 mmol) and the reaction mixture was reflux for 5 h, then the
mixture was poured into the 100 mL distilled water which containing 1N HCl (10 mL) and
partitioned with ethyl acetate that containing 20% butanol (3×30 mL), The organic layer was dried
over Na2SO4, and purified via silica gel column chromatography (Flash column) with
chloroform/methanol (20:1, v/v) to obtain the compounds 2 and 4. And then, compounds 2 or 4 (1
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mmol), amines (1.2 mmol), and PyBOP (1.2 mmol) were added to DCM (10 mL) containing Et3N
(0.5 mmol); the mixture was then stirred at room temperature for 6–12 h. After the reaction was
completed, the mixture was poured onto 100 mL of distilled water and partitioned with ethyl
acetate (3×50 mL). The target compounds were purified on a flash column with
chloroform/methanol (20:1, v/v) to yield compounds 3a and 5.
o
Compound (2): Yield: 73.3%; white power, mp: 223-225 C; IR (KBr, cm-1): νmax 3445, 3071,
1
2930, 2873, 1730, 1640, 882; MS (EI): 542 (M+); H-NMR (CDCl3, 500 MHz) δ: 4.81 (1H, s,
Hb-29), 4.67 (1H, s, Ha-29), 4.50 (1H, d, J = 8.5 Hz, Hb-28), 4.09 (1H, d, J = 10.2 Hz, Ha-28),
3.15~3.17 (1H, m, H-3), 2.85~2.89 (4H, m, CH2), 1.66 (3H, s, H-30), 1.16 (3H, s, CH3), 0.96 (3H, s,
CH3), 0.92 (3H, s, CH3), 0.87 (3H, s, CH3), 0.78 (3H, s, CH3); 13C-NMR (CDCl3, 125 MHz) δ: 174.8
(C=O), 173.2 (C=O), 150.4 (C-20), 110.1 (C-29), 77.9 (C-3), 62.8 (C-28), 55.6 (C-5), 50.5 (C-9), 48.9
(C-19), 47.7 (C-17), 46.7 (C-18), 42.7 (C-14), 40.9 (C-8), 39.3 (C-4), 39.0 (C-1), 37.6 (C-13), 37.2
(C-10), 34.7 (C-7), 34.3 (C-22), 29.9 (CH2), 29.9 (C-21), 29.8 (CH2), 29.8 (C-16), 28.4 (C-23), 28.1
(C-2), 27.2 (C-15), 25.4 (C-12), 20.7 (C-11), 19.1 (C-30), 18.6 (C-6), 16.2 (C-25), 16.1 (C-26), 15.9
(C-24), 14.7 (C-27).
4.2.2. General procedure for compounds 6a-6d.
Betulin (1 mmol), aromatic acids (2 mmol), DCC (2 mmol), and DMAP (1 mmol) were added to
DCM (25 mL); the mixture was then stirred at room temperature for 6–12 h. After the reaction
was completed, the mixture was poured onto 100 mL of distilled water and partitioned with ethyl
acetate (3×50 mL). The target compounds were purified on a flash column with petroleum
ether/EtOAc (20:1, v/v) to yield compounds 6a-6d.
Compound (6a): Yield: 73.9%; colorless oil; IR (KBr, cm-1): νmax 3438, 1715, 2945, 1640, 1377,
1030; MS (EI): 650 (M+); 1H-NMR (CDCl3, 500 MHz) δ: 8.04~8.06 (4H, m, PhH), 7.53~7.55 (2H, m,
PhH), 7.41~7.45 (4H, m, PhH), 4.72~4.74 (1H, m, H-3), 4.70 (1H, s, Hb-29), 4.69 (1H, s, Ha-29), 4.54
(1H, d, J = 10 Hz, Hb-28), 4.10 (1H, d, J = 10.8 Hz, Ha-28), 1.74 (3H, s, H-30), 1.07 (3H, s, CH3), 1.01
(3H, s, CH3), 0.99 (3H, s, CH3), 0.92 (3H, s, CH3), 0.86(3H, s, CH3); 13C-NMR (CDCl3, 125 MHz) δ:
167.1 (C=O), 166.4 (C=O), 150.2 (C-20), 132.9 (C), 132.8 (C), 131.0 (CH), 130.6 (CH), 129.7 (CH),
129.6 (CH), 128.5 (CH), 128.4 (CH), 110.0 (C-29), 81.7 (C-3), 63.4 (C-28), 55.4 (C-5), 50.4 (C-9),
48.9 (C-19), 47.8 (C-17), 46.8 (C-18), 42.9 (C-14), 41.0 (C-8), 38.5 (C-4), 38.3 (C-1), 37.8 (C-13),
37.2 (C-10), 34.8 (C-7), 34.2 (C-22), 30.0 (C-21), 29.8 (C-16), 28.2 (C-23), 27.2 (C-2), 25.3 (C-15),
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23.9 (C-12), 20.9 (C-11), 19.2 (C-30), 18.3 (C-6), 16.9 (C-25), 16.3 (C-26), 16.2 (C-24), 14.9 (C-27).
4.2.3. General procedure for compounds 7a-7g.
Betulin (0.45 g, 1.00 mmol), aromatic acids (1.20 mmol), EDCI (0.23 g, 1.20 mmol), and DMAP
(0.06 g, 0.50 mmol) were added to DCM (10 mL) containing Et3N (0.05 g, 0.50 mmol); the
mixture was then stirred at room temperature for 6-12 h. After the reaction was completed, the
mixture was poured onto 100 mL of distilled water and partitioned with ethyl acetate (3×50 mL).
The target compounds were purified on a flash column with petroleum ether/EtOAc (10:1, v/v) to
yield compounds 7a-7g.
Compound (7a): Yield: 72.2%; colorless oil; IR (KBr, cm-1): νmax 2944, 2873, 1711, 1640, 1012,
1
880, 754, 703; MS (EI): 546 (M+); H-NMR (CDCl3, 500 MHz) δ: 8.05 (2H, d, J = 7.3 Hz, PhH),
7.56 (2H, t, J = 11.4 Hz, PhH), 7.45 (2H, t, J = 14.4 Hz, PhH), 4.71 (1H, s, Hb-29), 4.60 (1H, s, Ha-29),
4.50 (1H, d, J = 9.5 Hz, Hb-28), 4.09 (1H, d, J = 10.3 Hz, Ha-28), 3.14~3.17 (1H, m, H-3), 1.69 (3H, s,
H-30), 1.05 (3H, s, CH3), 1.01 (3H, s, CH3), 0.95 (3H, s, CH3), 0.83 (3H, s, CH3), 0.75 (3H, s, CH3);
13C-NMR (CDCl3, 125 MHz) δ: 167.0 (C=O), 150.2 (C-20), 132.9 (C), 130.6 (CH), 129.6 (CH), 128.4
(CH), 109.9 (C-29), 77.1 (C-3), 63.4 (C-28), 55.4 (C-5), 50.5 (C-9), 48.9 (C-19), 47.8 (C-17), 46.8
(C-18), 42.8 (C-14), 40.9 (C-8), 38.9 (C-4), 38.8 (C-1), 37.7 (C-13), 37.2 (C-10), 34.8 (C-7), 34.3
(C-22), 30.1 (C-21), 29.7 (C-16), 28.1 (C-23), 27.5 (C-2), 27.2 (C-15), 25.3 (C-12), 20.9 (C-11), 19.3
(C-30), 18.4 (C-6), 16.2 (C-25), 16.1 (C-26), 15.4 (C-24), 14.9 (C-27).
4.3. Cell lines and culture
MGC-803, PC3, Bcap-37, A375, MCF-7, and NIH3T3 cell lines were obtained from the Institute
of Biochemistry and Cell Biology, China Academy of Science. MGC-803 is gastric cancer cell
line, PC3 is prostate cancer cell line, A375 is malignant melanoma cell line, Bcap-37 and MCF-7
are breast cancer cell lines. The entire cancer cell lines were maintained in the RPMI 1640
medium, whereas the NIH3T3 cell line was maintained in DMEM medium. They were
supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere of
5% CO2 at 37 °C. All cell lines were maintained at 37 °C in a humidified 5% carbon dioxide and
95% air incubator.
4.4. MTT Assays
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All tested compounds were dissolved in DMSO and subsequently diluted in the culture medium
before treatment of the cultured cells. When the cells were 80-90% confluent, they were harvested
by treatment with a solution containing 0.25% trypsin, thoroughly washed and resuspended in
supplemented growth medium. Cells (2×103/well) were plated in 100 ꢀL of medium/well in
96-well plate. After incubations overnight, the cells were treated with different concentrations of
extracts in RPMI 1640 with 10% FBS for 72 h. In parallel, the cells treated with 0.1% DMSO
served as negative control and ADM (Adriamycin) as positive control. Finally, 100 ꢀL of MTT
was added, and the cells were incubated for 4 h. The MTT-formazan formed by metabolically
viable cells was dissolved in 100 ꢀL of SDS for 12 h. The absorbance was then measured at 595
nm with a microplate reader, which is directly proportional to the number of living cells in culture.
4.5. AO/EB Staining
The cells were seeded at a concentration of 5×104 cell/mL in a volume of 0.6 mL on a sterile cover
slip in 6-well tissue culture plates. Following incubation, the medium was removed and replaced
with fresh medium plus 10% FBS and then supplemented with compounds. After the treatment
period, the cover slip with monolayer cells was inverted on the glass slide with 20 ꢀL of AO/EB
stain (100 ꢀg/mL). The fluorescence was read using an IX71SIF-3 fluorescence microscope.
4.6. Hoechst 33258 Staining
The cells grown on the sterile cover slip in 6-well tissue culture plates were treated with
compounds for a certain range of treatment time. The culture medium containing compounds was
removed, and the cells were fixed in 4% paraformaldehyde for 10 min. The cells were washed
twice with PBS, and were consequently stained with 0.5 mL of Hoechst 33258 staining for 5 min.
The stained nuclei were washed twice with PBS, and were consequently observed under an
IX71SIF-3 fluorescence microscope at 350 nm excitation and 460 nm emissions.
4.7. TUNEL Assay
TUNEL assays were performed using a colorimetric TUNEL apoptosis assay kit according to the
manufacturer’s instructions. The cells grown in 6-well culture clusters were treated as mentioned
in mitochondrial depolarization assay. The MGC-803 grown in 6-well tissue culture plates were
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washed with PBS and fixed in 4% paraformaldehyde for 40 min. The cells were washed once with
PBS, and were consequently permeabilized with immunol staining wash buffer for 2 min on ice.
The cells were rewashed once with PBS, and were consequently incubated in 0.3% H2O2 in
methanol at room temperature for 20 min to inactivate the endogenous peroxidases, after which
the cells were washed thrice with PBS. Thereafter, the cells were incubated with 2 ꢀL of
TdT-enzyme and 48 ꢀL of Biotin-dUTP per specimen for 60 min at 37 °C. The cells were
terminated for 10 min, and were consequently incubated with streptavidin-HRP (50 ꢀL per
specimen) conjugate diluted at 1:50 in sample diluent for 30 min. The cells were washed three
times with PBS, and were consequently incubated with diaminobenzidine solution (200 ꢀL per
specimen) for 10 min. Thereafter, the cells were rewashed twice with PBS, and were consequently
imaged under an XDS-1B inverted biological microscope.
4.8. Flow Cytometry Analysis
Prepared MGC-803 cells (1×106/mL) were washed twice with cold PBS and then re-suspended
gently in 500 ꢀL binding buffer. Thereafter, cells were stained in 5 ꢀL Annexin V-FITC and
shaked well. Finally, 5 ꢀL PI was added to these cells and incubated for 20 min in a dark place,
analyzed by FACS Calibur, Becton Dickinson.
4.9. Statistical Analysis
All statistical analyses were performed using SPSS 10.0, and the data were analyzed using
one-way ANOVA. The mean separations were performed using the least significant difference
method. Each experiment was performed in triplicate, and all experiments were run thrice and
yielded similar results. Measurements from all the replicates were combined, and the treatment
effects were analyzed.
Acknowledgments
This work was financially supported by the National Nature Science Foundation of China (Grant
Nos. 21172048, 21372052), Key Technologies R&D Program (Nos. 2011BAE06B02), the
Research Project of Chinese Ministry of Education (No. 213033A), and Guizhou Key S&T
Project (Nos. [2012]6012, [2011]3016, [2008]70011) for the financial support.
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Captions for Figures and Tables
Figure 1. Effect of compound 3c on cancer cell proliferation.
Figure 2. Apoptosis induction studies of compound 3c. (A) AO\EB staining. (B) Hoechst 33258
staining. (C) TUNEL assay.
Figure 3. Annexin V/PI dual staining of MGC-803 cell lines. (A) Apoptosis ratios. (B) A: negative
control; B–D: cells treated with HCPT (10 ꢀM) as positive control; E–G: cells treated with
compound 3c (10 ꢀM).
Table 1 IC50 (ꢀM) for 72 h of antitumor activities of BE derivatives on human cancer cell lines in
vitro
Table 2 Selectivity index a of 2, 3a–3d, 4, and 5 toward cancer cells
a
selectivity index (IC50 on mouse embryo fibroblast cell line/IC50 on corresponding cancer cell
line)
Scheme 1. Reagents and conditions: (a) Succinic anhydride, DMAP, pyridine, DCM, r.t., 8 h; (b)
R1H, PyBOP, DCM, r.t., 12 h; (c) Maleic anhydride, DMAP, pyridine, DCM, r.t., 24 h; (d)
Piperidine, PyBOP, DCM, r.t., 12 h; (e) R2COOH, DCC, DMAP, DCM, r.t., 24 h; (f) R3COOH,
EDCI, DMAP, DCM, r.t., 24 h.
16

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Table 1 IC50 (ꢀM) for 72 h of antitumor activities of BE derivatives on human cancer cell lines in
vitro
Compound
MGC-803
30.2 ± 0.8
11.3 ± 0.2
8.5 ± 0.2
8.9 ± 0.3
4.3 ± 0.4
9.2 ± 0.6
21.3 ± 0.5
5.3 ± 0.2
>100
PC3
Bcap-37
25.5 ± 0.5
15.7 ± 0.9
6.7 ± 0.3
7.9 ± 0.1
5.2 ± 0.4
6.4 ± 0.3
18.3 ± 0.1
7.4 ± 0.5
>100
A375
MCF-7
22.4 ± 0.9
12.5 ± 0.3
9.6 ± 0.4
8.3 ± 0.2
4.5 ± 0.2
11.2 ± 0.7
25.8 ± 0.4
9.6 ± 0.2
>100
28.0 ± 0.3
17.9 ± 0.5
10.3 ± 0.8
12.6 ± 0.6
7.5 ± 0.6
18.5 ± 0.9
23.2 ± 0.2
14.4 ± 0.4
>100
19.3 ± 0.5
14.5 ± 0.6
9.2 ± 0.6
10.1 ± 0.3
5.2 ± 0.7
11.5 ± 0.4
17.1 ± 0.1
6.3 ± 0.4
>100
1
2
3a
3b
3c
3d
4
5
6a-6d
7a-7g
HCPT
ADM
>100
>100
>100
>100
>100
29.1 ± 2.6
0.7 ± 0.2
34.5 ± 1.5
0.6 ± 0.1
28.1 ± 1.0
1.2 ± 0.2
27.8 ± 1.2
1.0 ± 0.6
48.2 ± 0.4
1.35 ± 0.1
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Table 2 Selectivity index a of 2, 3a-3d, 4, and 5 toward cancer cells
Compound
MGC-803
6.45
PC3
5.83
6.45
5.33
7.86
5.12
< 3
Bcap-37
4.64
A375
4.07
6.01
3.52
4.72
3.10
< 3
MCF-7
5.02
6.73
4.38
6.80
4.99
< 3
2
7.29
9.25
5.60
6.81
8.97
< 3
3a
3b
3c
3d
4
4.97
8.23
6.23
< 3
< 3
< 3
< 3
< 3
< 3
5
a
selectivity index (IC50 on mouse embryo fibroblast cell line/IC50 on corresponding cancer cell
line)
18

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Figure 1. Effect of compound 3c on cancer cell proliferation.
19

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Figure 2. Apoptosis induction studies of compound 3c. (A) AO\EB staining. (B) Hoechst 33258
staining. (C) TUNEL assay.
20

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Figure 3. Annexin V/PI dual staining of MGC-803 cell lines. (A) Apoptosis ratios. (B) A: negative
control; B–D: cells treated with HCPT (10 ꢀM) as positive control; E–G: cells treated with
compound 3c (10 ꢀM).
21

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Scheme 1. Reagents and conditions: (a) Succinic anhydride, DMAP, pyridine, DCM, r.t., 8 h; (b)
R1H, PyBOP, DCM, r.t., 12 h; (c) Maleic anhydride, DMAP, pyridine, DCM, r.t., 24 h; (d)
Piperidine, PyBOP, DCM, r.t., 12 h; (e) R2COOH, DCC, DMAP, DCM, r.t., 24 h; (f) R3COOH,
EDCI, DMAP, DCM, r.t., 24 h.
22

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Highlights
1. Nineteen betulin derivatives modified at the C-3 and C-28 positions were synthesized
2. The antitumor activities were investigated against various cancer cell lines.
3. Compound 3c displayed the most potent antitumor activity.
4. Further mechanism study indicated compound 3c could induce apoptosis of cancer cells.

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Supporting Information
Synthesis and in vitro antitumor evaluation of betulin acid ester
derivatives as novel apoptosis inducers
Sheng-Jie Yanga,b¶, Ming-Chuan Liua,b¶, Hong-Mei Xianga, Qi Zhaoa, Wei Xuea, and Song Yanga*
a State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou
University, Guiyang 550025, P.R. China
b Sinphar Tian-Li Pharmaceutical Co., LTD, Hangzhou, 311100, P.R. China
¶All authors contributed equally to this work.
*Corresponding author. Ctr for R&D of Fine Chemicals, Guizhou University, Huaxi St., Guiyang,
China 550025. Tel.: +86 851 829 2171; Fax: +86 851 829 2170.
E-mail address: jhzx.msm@gmail.com
Data of other compounds
Compound (3a): Yield: 61.1%; pale yellow oil; IR (KBr, cm-1): νmax 3445, 2947, 2870, 1728, 1033,
1
882; MS (EI): 597 (M+); H-NMR (CDCl3, 500 MHz) δ: 4.63 (1H, s, Hb-29), 4.53 (1H, s, Ha-29),
4.23 (1H, d, J = 9.2 Hz, Hb-28), 3.84 (1H, d, J = 10.8 Hz, Ha-28), 3.30~3.33 (4H, m, CH2), 3.15~3.17
(1H, m, H-3), 2.62~2.64 (4H, m, CH2), 1.63 (3H, s, H-30), 1.15 (3H, s, CH3), 1.05 (3H, s, CH3), 0.97
(3H, s, CH3), 0.92 (3H, s, CH3), 0.87 (3H, s, CH3), 0.78 (3H, s, CH3); 13C-NMR (CDCl3, 125 MHz) δ:
173.7 (C=O), 170.3 (C=O), 150.3 (C-20), 109.8 (C-29), 78.9 (C-3), 62.8 (C-28), 55.3 (C-5), 50.4 (C-9),
48.9 (C-19), 47.7 (C-17), 46.7 (C-18), 42.7 (C-14), 41.9 (CH2), 40.9 (C-8), 40.4 (C-4), 38.9 (C-1), 38.7
(C-13), 37.6 (CH2), 37.1 (C-10), 34.6 (C-7), 34.2 (C-22), 29.8 (CH2), 29.6 (C-21), 28.0 (CH2), 27.9
(C-16), 27.8 (C-23), 27.4 (C-2), 27.1 (C-15), 25.2 (C-12), 20.8 (C-11), 19.2 (C-30), 18.6 (C-6), 16.2
(C-25), 16.1 (C-26), 15.5 (C-24), 14.8 (C-27), 14.2 (CH3), 13.1 (CH3).
Compound (3b): Yield: 62.8%; pale yellow oil; IR (KBr, cm-1): νmax 3444. 2942, 2860, 1728,
1641459, 882; MS (EI): 595 (M+); 1H-NMR (CDCl3, 500 MHz) δ: 4.63 (1H, s, Hb-29), 4.53 (1H, s,
1

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Ha-29), 4.22 (1H, d, J = 11.5 Hz, Hb-28), 3.84 (1H, d, J = 10.3 Hz, Ha-28), 3.14~3.16 (1H, m, H-3),
2.63~2.66 (4H, m, CH2), 2.52 (4H, t, J = 12.5 Hz, CH2), 1.79~1.82 (m, CH2), 1.63 (3H, s, H-30), 1.01
(3H, s, CH3), 0.96 (3H, s, CH3), 0.81 (3H, s, CH3), 0.71 (3H, s, CH3); 13C-NMR (CDCl3, 125 MHz) δ:
173.4 (C=O), 169.7 (C=O), 150.2 (C-20), 109.9 (C-29), 77.4 (C-3), 62.8 (C-28), 55.3 (C-5), 50.4 (C-9),
48.9 (C-19), 47.7 (C-17), 46.5 (CH2), 46.4 (C-18), 45.8 (CH2), 42.7 (C-14), 40.9 (C-8), 39.0 (C-4), 38.7
(C-1), 37.6 (C-13), 37.2 (C-10), 34.5 (C-7), 34.2 (C-22), 29.8 (CH2), 29.7 (C-21), 29.4 (CH2), 29.2
(C-16), 28.1 (C-23), 27.4 (C-2), 27.1 (C-15), 26.1 (CH2), 25.2 (C-12), 24.5 (CH2), 20.7 (C-11), 19.1
(C-30), 18.6 (C-6), 16.2 (C-25), 16.1 (C-26), 15.9 (C-24), 14.7 (C-27).
Compound (3c): Yield: 63.6%; pale yellow oil; IR (KBr, cm-1): νmax 3439, 2943, 2868, 1733, 1453,
1
1030, 880; MS (EI): 609 (M+); H-NMR (CDCl3, 500 MHz) δ: 4.64 (1H, s, Hb-29), 4.54 (1H, s,
Ha-29), 4.25 (1H, d, J = 6.5 Hz, Hb-28), 3.84 (1H, d, J = 11.8 Hz, Ha-28), 3.48~3.50 (4H, m, CH2),
3.14~3.16 (1H, m, H-3), 2.62~2.64 (4H, m, CH2), 1.64 (3H, s ,H-30), 1.57~1.59 (m, CH2), 1.06 (3H, s,
CH3), 0.96 (3H, s, CH3), 0.92 (3H, s, CH3), 0.78 (3H, s, CH3), 0.72 (3H, s, CH3); 13C-NMR (CDCl3,
125 MHz) δ: 173.7 (C=O), 169.4 (C=O), 150.3 (C-20), 109.9 (C-29), 78.9 (C-3), 62.8 (C-28), 55.3
(C-5), 50.4 (C-9), 48.8 (C-19), 47.7 (C-17), 46.5 (CH2), 46.4 (C-18), 42.9 (CH2), 42.7 (C-14), 40.9
(C-8), 39.0 (C-4), 38.7 (C-1), 37.6 (C-13), 37.2 (C-10), 34.6 (C-7), 34.2 (C-22), 29.8 (CH2), 29.7
(C-21), 29.4 (CH2), 29.6 (C-16), 28.1 (C-23), 27.4 (C-2), 27.1 (C-15), 26.4 (CH2), 25.5 (CH2), 25.2
(C-12), 24.6 (CH2), 20.8 (C-11), 19.2 (C-30), 18.4 (C-6), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.8
(C-27).
Compound (3d): Yield: 64.4%; pale yellow oil; IR (KBr, cm-1): νmax 3446, 2945, 2860, 1730, 1647,
1
1028, 881; MS (EI): 611 (M+); H-NMR (CDCl3, 500 MHz) δ: 4.64 (1H, s, Hb-29), 4.54 (1H, s,
Ha-29), 4.25 (1H, d, J = 13.3 Hz, Hb-28), 3.85 (1H, d, J = 8.4 Hz, Ha-28), 3.63~3.65 (4H, m, CH2),
3.47 (4H, t, J = 5.6 Hz, CH3), 3.14~3.17 (1H, m, H-3), 2.62~2.65 (4H, m, CH2), 1.66 (3H, s, H-30),
1.01 (3H, s, CH3), 0.96 (3H, s, CH3), 0.92 (3H, s, CH3), 0.77 (3H, s, CH3), 0.71 (3H, s, CH3); 13C-NMR
(CDCl3, 125 MHz) δ: 173.4 (C=O), 169.9 (C=O), 150.2 (C-20), 109.9 (C-29), 78.9 (C-3), 66.9 (CH2),
66.5 (CH2), 62.9 (C-28), 55.3 (C-5), 51.9(CH2), 50.4 (C-9), 48.8 (C-19), 47.7 (C-17), 46.5 (C-18), 45.7
(CH2), 42.2 (C-14), 40.9 (C-8), 38.9 (C-4), 38.7 (C-1), 37.6 (C-13), 37.2 (C-10), 34.6 (C-7), 34.2
(C-22), 29.8 (CH2), 29.3 (C-21), 29.0 (CH2), 28.0 (C-16), 27.8 (C-23), 27.4 (C-2), 27.1 (C-15), 25.2
(C-12), 20.8 (C-11), 19.2 (C-30), 18.4 (C-6), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.8 (C-27).
o
Compound (4): Yield: 65.8%; white power, mp: 232-234 C; IR (KBr, cm-1): νmax 3435, 2942,
2

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1728, 1375, 1030, 881; MS (EI): 540 (M+); 1H-NMR (CDCl3, 500 MHz) δ: 6.35 (1H, d, J = 10.4 Hz,
CH), 6.23 (1H, d, J = 14.8 Hz, CH), 4.66 (1H, s, Hb-29), 4.56 (1H, s, Ha-29), 4.37 (1H, d, J = 11.3 Hz,
Hb-28), 3.97 (1H, d, J = 7.9 Hz, Ha-28), 3.14~3.16 (1H, m, H-3), 1.64 (3H, s, H-30), 0.99 (3H, s, CH3),
0.94 (3H, s, CH3), 0.92 (3H, s, CH3), 0.79 (3H, s, CH3), 0.72 (3H, s, CH3); 13C-NMR (CDCl3, 125
MHz) δ: 167.2 (C=O), 167.2 (C=O), 149.9 (C-20), 133.9 (CH), 128.6(CH), 110.1 (C-29), 79.0 (C-3),
64.8 (C-28), 55.3 (C-5), 50.4 (C-9), 48.9 (C-19), 47.7 (C-17), 46.5 (C-18), 42.7 (C-14), 40.9 (C-8),
38.9 (C-4), 38.7 (C-1), 37.8 (C-13), 37.2 (C-10), 34.5 (C-7), 34.2 (C-22), 29.7 (C-21), 29.5 (C-16),
28.0 (C-23), 27.2 (C-2), 27.0 (C-15), 25.2 (C-12), 20.8 (C-11), 19.1 (C-30), 18.3 (C-6), 16.1 (C-25),
16.0 (C-26), 15.4 (C-24), 14.8 (C-27).
Compound (5): Yield: 60.7%; pale yellow oil; IR (KBr, cm-1): νmax 3240, 2930, 1734, 1640, 1455,
883; MS (EI): 607 (M+); 1H-NMR (CDCl3, 500 MHz) δ: 6.50 (1H, d, J = 10.2 Hz, CH), 5.95 (1H, d, J
= 11.5 Hz, CH), 4.62 (1H, s, Hb-29), 4.52 (1H, s, Ha-29), 4.23 (1H, d, J = 9.1 Hz, Hb-28), 3.87 (1H, d,
J = 12.6 Hz, Ha-28), 3.55~3.58 (4H, m, CH2), 3.12~3.14 (1H, m, H-3), 1.61 (3H, s, H-30), 1.48~1.51
(m, CH2), 0.95 (3H, s, CH3), 0.92 (3H, s, CH3), 0.89 (3H, s, CH3), 0.75 (3H, s, CH3), 0.69 (3H, s, CH3);
13C-NMR (CDCl3, 125 MHz) δ: 165.3 (C=O), 165.2 (C=O), 150.1 (C-20), 138.4 (CH), 122.8 (CH),
110.0 (C-29), 77.4 (C-3), 63.5 (C-28), 55.3 (C-5), 50.4 (C-9), 48.9 (C-19), 47.7 (C-17), 47.4 (CH2),
46.3 (C-18), 42.7 (C-14), 42.1 (CH2), 40.9 (C-8), 38.9 (C-4), 38.7 (C-1), 37.6 (C-13), 37.2 (C-10), 34.6
(C-7), 34.2 (C-22), 29.7 (C-21), 29.5 (C-16), 28.1 (C-23), 27.4 (C-2), 27.1 (C-15), 26.1 (CH2), 25.2
(C-12), 24.6 (CH2), 20.8 (C-11), 19.2 (C-30), 18.3 (C-6), 16.2 (C-25), 16.1 (C-26), 15.4 (C-24), 14.8
(C-27).
Compound (6b): Yield: 75.2%; white solid; mp: 222-225 oC;IR (KBr, cm-1): νmax 3456, 1698, 1638,
1
1377, 1032, 882, 744; H-NMR (CDCl3, 500 MHz) δ: 8.83 (1H, s, PhH), 8.81 (1H, s, PhH),
8.37~8.41 (m, PhH), 7.62~7.67 (2H, m, PhH), 4.75~4.77 (1H, m, H-3), 4.76 (1H, s, Hb-29), 4.61 (1H,
s, Ha-29), 4.58 (1H, d, J = 10.5 Hz, Hb-28), 4.15 (1H, d, J = 10.8 Hz, Ha-28), 1.72 (3H, s, H-30), 1.08
(3H, s, CH3), 1.01 (3H, s, CH3), 1.00 (3H, s, CH3), 0.90 (3H, s, CH3); 13C-NMR (CDCl3, 125 MHz) δ:
164.9 (C=O), 164.2 (C=O), 150.0 (C-20), 148.4 (C), 148.4 (C), 135.3 (C), 135.2 (C), 132.7 (CH),
132.3 (CH), 129.8 (CH), 129.7 (CH), 127.5 (CH), 127.3 (CH), 124.6 (CH), 124.5 (CH), 110.2 (C-29),
82.9 (C-3), 64.4 (C-28), 55.5 (C-5), 50.4 (C-9), 48.9 (C-19), 47.8 (C-17), 46.8 (C-18), 42.9 (C-14),
41.0 (C-8), 38.4 (C-4), 38.3 (C-1), 37.8 (C-13), 37.2 (C-10), 34.8 (C-7), 34.2 (C-22), 30.0 (C-21), 29.6
(C-16), 28.2 (C-23), 27.1 (C-2), 25.2 (C-15), 23.8 (C-12), 20.9 (C-11), 19.2 (C-30), 18.3 (C-6), 16.9
3

ACCEPTED MANUSCRIPT
(C-25), 16.3 (C-26), 16.2 (C-24), 14.9 (C-27).
o
Compound (6c): Yield: 65.4%; white solid; mp: 234-236 C; IR (KBr, cm-1): νmax 3446, 2945,
1
2870, 1711, 1642, 1033; H-NMR (CDCl3, 500 MHz) δ: 7.76~7.78 (m, PhH), 7.59~7.61 (m, PhH),
4.75~4.77 (1H, m, H-3), 4.74 (1H, s, Hb-29), 4.60 (1H, s, Ha-29), 4.55 (1H, d, J = 10.8 Hz, Hb-28),
4.12 (1H, d, J = 10.4 Hz, Ha-28), 1.66 (3H, s, H-30), 1.12 (3H, s, CH3), 1.03 (3H, s, CH3), 1.00 (3H, s,
CH3), 0.93 (3H, s, CH3), 0.88 (3H, s, CH3); 13C-NMR (CDCl3, 125 MHz) δ: 167.5 (C=O), 166.8
(C=O), 150.1 (C-20), 132.3 (C), 131.8 (C), 131.7(C), 131.6 (C), 131.1 (CH), 130.8 (CH), 130.2 (CH),
129.9 (CH), 128.7 (CH), 128.3 (CH), 126.8 (CH), 126.7 (CH), 124.6 (CF3), 122.4 (CF3), 110.0 (C-29),
83.2 (C-3), 64.7 (C-28), 55.6 (C-5), 50.3 (C-9), 49.0 (C-19), 47.8 (C-17), 46.5 (C-18), 42.8 (C-14),
40.9 (C-8), 38.5 (C-4), 38.0(C-1), 37.7 (C-13), 37.2 (C-10), 34.5 (C-7), 34.1(C-22), 29.7 (C-21), 29.6
(C-16), 28.0 (C-23), 27.0 (C-2), 25.2 (C-15), 23.3 (C-12), 20.9 (C-11), 19.2 (C-30), 18.2 (C-6), 16.6
(C-25), 16.2 (C-26), 14.8 (C-24), 14.3 (C-27).
o
Compound (6d): Yield: 72.5%; white solid; mp: 212-214 C; IR (KBr, cm-1): νmax 3433, 2942,
2899, 1718, 1640, 882, 734; 1H-NMR (CDCl3, 500 MHz) δ: 7.84 (1H, d, J = 7.9 Hz, PhH), 7.80 (1H,
d, J = 7.4 Hz, PhH), 7.40~7.42 (m, PhH), 7.26~7.28 (m, PhH), 4.73~4.76 (1H, m, H-3), 4.71 (1H, s,
Hb-29), 4.60 (1H, s, Ha-29), 4.53 (1H, d, J = 10.8 Hz, Hb-28), 4.12 (1H, d, J = 11.1 Hz, Ha-28), 1.70
(3H, s, H-30), 1.08 (3H, s, CH3), 1.06 (3H, s, CH3), 0.95 (3H, s, CH3), 0.93 (3H, s, CH3), 0.88 (3H, s,
CH3); 13C-NMR (CDCl3, 125 MHz) δ: 166.3 (C=O), 165.7(C=O), 150.1 (C-20), 133.7 (C), 133.5 (C),
132.6 (C), 132.3 (C), 131.6 (CH), 131.3 (CH), 131.2 (CH), 131.1 (CH), 131.0 (CH), 130.5 (CH), 126.7
(CH), 126.6 (CH), 110.0 (C-29), 82.7 (C-3), 64.2 (C-28), 55.6 (C-5), 50.4 (C-9), 48.9 (C-19), 47.8
(C-17), 46.7 (C-18), 42.8 (C-14), 41.0 (C-8), 38.5 (C-4), 38.1 (C-1), 37.8 (C-13), 37.2 (C-10), 34.8
(C-7), 34.2 (C-22), 29.9 (C-21), 29.7 (C-16), 29.6 (C-23), 28.2 (C-2), 27.2 (C-15), 25.3 (C-12), 20.9
(C-11), 19.2 (C-30), 18.3 (C-6), 16.9 (C-25), 16.3 (C-26), 16.1 (C-24), 14.9 (C-27).
Compound (7b): Yield: 69.4%; colorless oil; IR (KBr, cm-1): νmax 3444, 2930, 2866, 1712, 1455,
881, 739; MS (EI): 581 (M+); 1H-NMR (CDCl3, 500 MHz) δ: 7.99 (1H, s, PhH), 7.93 (1H, d, J = 7.9
Hz, PhH), 7.52 (1H, d, J = 8.2 Hz, PhH), 7.39 (1H, t, J = 15.1 Hz, PhH), 4.70 (1H, s, Hb-29), 4.59 (1H,
s, Ha-29), 4.52 (1H, d, J = 10.4 Hz, Hb-28), 4.09 (1H, d, J = 12.3 Hz, Ha-28), 3.15~3.18 (1H, m, H-3),
1.73 (3H, s, H-30), 1.08 (3H, s, CH3), 0.98 (3H, s, CH3), 0.95 (3H, s, CH3), 0.81 (3H, s, CH3), 0.74 (3H,
s, CH3); 13C-NMR (CDCl3, 125 MHz) δ: 165.8 (C=O), 150.1 (C-20), 134.6 (C), 133.0 (C), 132.3 (CH),
129.8 (CH), 129.7 (CH), 127.8 (CH), 110.0 (C-29), 79.0 (C-3), 63.8 (C-28), 55.4 (C-5), 50.5 (C-9),
4

ACCEPTED MANUSCRIPT
48.9 (C-19), 47.8 (C-17), 46.7 (C-18), 42.8 (C-14), 40.9 (C-8), 38.9 (C-4), 38.8 (C-1), 37.7 (C-13),
37.2 (C-10), 34.8 (C-7), 34.3 (C-22), 30.0 (C-21), 29.7 (C-16), 28.1 (C-23), 27.5 (C-2), 27.2 (C-15),
25.3 (C-12), 20.9 (C-11), 19.3 (C-30), 18.4 (C-6), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.9 (C-27).
Compound (7c): Yield: 67.6%; colorless oil; IR (KBr, cm-1): νmax 3433, 2951, 2931, 1701, 1022,
1
882, 735; MS (EI): 615 (M+); H-NMR (CDCl3, 500 MHz) δ: 7.76 (1H, d, J = 12.3 Hz, PhH), 7.71
(1H, d, J = 14.6 Hz, PhH), 7.58~7.60 (2H, m, PhH), 4.69 (1H, s, Hb-29), 4.58 (1H, s, Ha-29), 4.51 (1H,
d, J = 10.5 Hz, Hb-28), 4.13 (1H, d, J = 9.7 Hz, Ha-28), 3.15~3.18 (1H, m, H-3), 1.71 (3H, s, H-30),
1.07 (3H, s, CH3), 0.97 (3H, s, CH3), 0.92 (3H, s, CH3), 0.81 (3H, s, CH3), 0.75 (3H, s, CH3); 13C-NMR
(CDCl3, 125 MHz) δ: 167.5 (C=O), 150.1 (C-20), 131.8 (C), 131.1 (CH), 130.2 (CH), 128.5 (CH),
126.8 (CH), 126.7 (C), 121.7(CF3), 110.0 (C-29), 79.0 (C-3), 64.9 (C-28), 55.4 (C-5), 50.5 (C-9), 49.0
(C-19), 47.7 (C-17), 46.5 (C-18), 42.7 (C-14), 40.9 (C-8), 38.9 (C-4), 38.8 (C-1), 37.7 (C-13), 37.2
(C-10), 34.5 (C-7), 34.3 (C-22), 29.7 (C-21), 29.6 (C-16), 28.1 (C-23), 27.5 (C-2), 27.1 (C-15), 25.3
(C-12), 20.9 (C-11), 19.3 (C-30), 18.4 (C-6), 16.2 (C-25), 16.1 (C-26), 15.4 (C-24), 14.9 (C-27).
Compound (7d): Yield: 65.2%; colorless oil; IR (KBr, cm-1): νmax 3448, 2927, 1698, 1457, 880,
730; MS (EI): 560 (M+); 1H-NMR (CDCl3, 500 MHz) δ: 7.92 (1H, d, J = 8.2 Hz, PhH), 7.04 (1H, d, J
= 8.3 Hz, PhH), 7.25~7.27 (2H, m, PhH), 4.70 (1H, s, Hb-29), 4.59 (1H, s, Ha-29), 4.46 (1H, d, J =
10.8 Hz, Hb-28), 4.09 (1H, d, J = 11.5 Hz, Ha-28), 3.16~3.18 (1H, m, H-3), 2.61 (3H, s, CH3), 1.69
(3H, s, H-30), 1.05 (3H, s, CH3), 1.03 (3H, s, CH3), 0.96 (3H, s, CH3), 0.82 (3H, s, CH3), 0.75 (3H, s,
CH3); 13C-NMR (CDCl3, 125 MHz) δ: 168.1 (C=O), 150.2 (C-20), 140.2 (C), 132.0 (C), 131.8 (CH),
129.9 (CH), 129.9 (CH), 125.7 (CH), 110.0 (C-29), 79.0 (C-3), 63.2 (C-28), 55.4 (C-5), 50.5 (C-9),
49.0 (C-19), 47.8 (C-17), 46.7 (C-18), 42.8 (C-14), 40.9 (C-8), 38.9 (C-4), 38.8 (C-1), 37.7 (C-13),
37.2 (C-10), 34.9 (C-7), 34.3 (C-22), 30.0 (C-21), 29.7 (C-16), 28.0 (C-23), 27.5 (C-2), 27.2 (C-15),
25.3 (C-12), 22.1 (CH3), 20.9 (C-11), 19.3 (C-30), 18.4 (C-6), 16.1(C-25), 16.1 (C-26), 15.5 (C-24),
14.9 (C-27).
Compound (7e): Yield: 68.1%; colorless oil; IR (KBr, cm-1): νmax 2947, 2866, 1688, 1455, 882,
1
744; MS (EI): 565 (M+); H-NMR (CDCl3, 500 MHz) δ: 8.03~8.06 (2H, m, PhH), 7.11 (2H, t, J =
14.5 Hz, PhH), 4.70 (1H, s, Hb-29), 4.59 (1H, s, Ha-29), 4.50 (1H, d, J = 10.1 Hz, Hb-28), 4.07 (1H, d,
J = 8.4 Hz, Ha-28), 3.16~3.18 (1H, m, H-3), 1.68 (3H, s, H-30), 1.04 (3H, s, CH3), 0.99 (3H, s, CH3),
0.95 (3H, s, CH3), 0.81 (3H, s, CH3), 0.75 (3H, s, CH3); 13C-NMR (CDCl3, 125 MHz) δ: 166.0 (C=O),
150.1 (C-20), 132.1 (C), 126.8 (C), 115.7 (CH), 115.5 (CH), 110.0 (C-29), 79.0 (C-3), 63.5 (C-28),
5