Synthesis and Antitumor Activity of C-3(R) Hydroxy Modified Betulinic Acid Derivatives

Article abstract of DOI:10.1007/s10600-019-02899-x

Four novel betulinic acid (BA) derivatives modified at the C-3 position were prepared by the Mitsunobu reaction and subsequently evaluated for antitumor activity against four common cancer cell lines in vitro. The results from the MTT assay revealed that

Full text of DOI:10.1007/s10600-019-02899-x

DOI 10.1007/s10600-019-02899-x
Chemistry of Natural Compounds, Vol. 55, No. 6, November, 2019
SYNTHESIS AND ANTITUMOR ACTIVITY
OF C-3(R) HYDROXY MODIFIED
BETULINIC ACID DERIVATIVES
1,2
1,2
2
Pan Yu, Dong-dong Li, Jun-jun Ni,
1,2
3
Chao-jie Xia, Zhen-zhong Wang,
3*
3
1,2*
Wei Xiao, Gang Ding, and Lin-guo Zhao
Four novel betulinic acid (BA) derivatives modified at the C-3 position were prepared by the Mitsunobu
reaction and subsequently evaluated for antitumor activity against four common cancer cell lines in vitro.
The results from the MTT assay revealed that the four tested compounds showed remarkable cytotoxic activity
against four cancer cell lines, much better than BA. Western blotting results indicated that the four compounds,
especially compound 2c, caused a significant up-regulation expression of the caspase-12 in CT26 cell.
Our results showed that configuration inversions of chiral C-3 of BA could also produce BA derivatives with
potent antitumor activity.
Keywords: betulinic acid, antitumor activity, cinnamic acids, MTT assay.
Betulinic acid (1, BA) is a pentacyclic triterpene of the lupane group. Due to its potential biological activities, especially
antitumor activity, hundreds of BA derivatives were synthesized for clinical trial [1–7]. Modifications of BA have focused
mainly on the C-3 hydroxyl, C-20 allylic, C-28 carboxylic acid, A-ring, D-ring, and E-ring positions [8, 9]. Among these target
sites, the 3β-hydroxy moiety is a primary location for chemical modifications, including esterification, amination, etherification,
oxidation, and so on [10]. Recently, a series of C-3 modified BA derivatives was synthesized and exhibited higher antitumor
ability [11–15]. Several structure activity relationship studies [16–18] have indicated that a moderately bulky and
electron-withdrawing group at or near the C-3 site would improve the antitumor activity regardless of the configuration of
3-OH (whether the configuration of the hydroxy group at C-3 is R or S). However, it was noted that most of the C-3 modified
BA derivatives had (S)-configuration, and there are very few reports on the antitumor activities of compounds with
(R)-configuration at this site. Thus, the synthesis of C-3(R) BA derivatives is of great interest and, based on it, we developed
novel BA derivatives by introducing key substituting groups into the 3β-hydroxy group of BA. Cinnamic acid possesses
potential antitumor activity [19, 20] and could be widely employed for the construction of new molecules as basic blocks in the
field of medicinal chemistry. We selected four frequently used cinnamic acids, including three cinnamic acids with the
electron-withdrawing groups as substituents for synthesizing novel BA derivatives with the (R)-configuration of the C-3 OH
site by the classical Mitsunobu reaction. Biological evaluation of these BA analogues against four cancer cell lines was carried
out, and the cytotoxicity of C-3 modified BA derivatives against 4T1 cell was studied for the first time.
Four new BA derivatives 1a–d were synthesized according to the procedure described in Scheme 1. The BA esters
2a–d were synthesized by interaction with the corresponding cinnamic acids in the presence of DEAD, PPh , and dry THF at
3
0°C under an inert (N ) atmosphere according to the Mitsunobu reaction [21].
2
1) Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, 159 Long Pan
Road, 210037, Nanjing, P. R. China; 2) College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road,
210037, Nanjing, P. R. China, e-mail: lg.zhao@163.com; 3) Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Haichang South
Road, Lianyungang 222001, Jiangsu Province, P. R. China, e-mail: xw@kanion.com. Published in Khimiya Prirodnykh
Soedinenii, No. 6, November–December, 2019, pp. 930–933. Original article submitted August 7, 2018.
1080
0009-3130/19/5506-1080 ©2019 Springer Science+Business Media, LLC

TABLE 1. Cytotoxicity Profile of 1, 2a–d against Selected Cancer Cell Lines (IC , μM SD)
50
Compound
7721
A549
4T1
CT26
1
30.33 1.52
17.48 0.87
19.98 0.10
17.53 0.88
21.42 1.07
45.31 2.26
23.11 1.15
23.81 1.19
20.14 1.01
26.90 1.35
41.06 2.05
38.99 1.95
33.50 1.65
22.34 1.12
27.61 1.38
50.11 2.51
26.92 1.35
28.84 1.44
16.98 0.85
33.88 1.69
2a
2b
2c
2d
29
20
19
30
O
H
H
E
22
13
OH
H
O
R
O
25
17
11
C
D
1
28
OH
O
O
OH
10
A
4
B
DEAD, PPh
7
3
27
26
HO
R
H
23 24
1
2a-d
R = H (a), 4-Br (b), 4-NO (c), 4-Cl (d)
2
Scheme 1. Synthesis of the BA derivatives 2a–d. Reagents and conditions: diethyl azodicarboxylate (DEAD),
triphenylphosphine (PPh ), dry tetrahydrofuran (THF), –30°C, 15 min, 0°C, 2–4h.
3
The cytotoxic activity of BA and its derivatives 2a–d in vitro was determined by using the MTT assay, and the result
of testing against four cancer cell lines, human hepatocellular carcinoma cell (7721), human lung adenocarcinoma cell (A549),
mouse breast cancer cell (4T1), and human colon cancer cell (CT26), is summarized in Table 1. The up-regulation effects of
compounds 2a–d was studied by Western blot analysis. As shown in Fig. 1, pretreatment with 25 μM of the four compounds
led to the increase in the caspase-12 protein level. Among them, compound 2c was the most potent derivative, showing the
strongest cytotoxic effect on caspase-12 protein expression. Different concentrations (10, 30, and 100 μM) of compound 2c
were subsequently selected to study its effect through the caspase signaling pathway to up-regulate the proteinase with only
cell sap added and BA (50 μM) as the reference. These results indicated that compounds 2a–d can accelerate cell apoptosis in
CT26 cell through up-regulation of caspase-12 expression. The antitumor activity of compounds 2a–d might be attributed to
the caspase signaling pathway. Thus, the studied BA derivatives displayed good antitumor activity, and compound 2c is a lead
compound.
In human hepatocellular carcinoma cell (7721), compounds 2a–d had better cytotoxicity than BA. Compounds 2a
and 2c were almost 1.8-fold more potent than BA. In human lung carcinoma cell (A549), compounds 2a–d also showed higher
potency than BA. Compound 2c was the most active compound, which displayed 2.3-fold better cytotoxicity than BA.
In mouse breast cancer cell (4T1), although compounds 2a and 2b showed comparable activity with BA, compounds 2c and
2d exhibited higher cytotoxicity than BA. In human colon cancer cell (CT26), all the compounds tested showed stronger
cytotoxic activities than BA. Compound 2c was the most potent, possessing an almost threefold higher inhibitory activity than
BA. It is a noteworthy that all four BA derivatives studied had the C-3(R) OH configuration. Thus, compared to the C-3(S) BA
derivatives reported [11–15], the antitumor activity of the four C-3(R) BAderivatives are as follows: (1) compound 2c exhibited
similar potency to compound 3 against human colon cancer cell lines CT26 and DLD-1; (2) the cytotoxicity of C-3(R) BA
derivatives was close to C-3(S) BA derivatives against human lung carcinoma cell, such as A549 cell; (3) the four C-3(R) BA
derivatives 2a–d showed stronger cytotoxic activity than BA against human hepatocellular carcinoma cell (HepG2 and 7721).
As discussed above, modifications at the C-3 position in BA seemed to be critical to antitumor activity, particularly
for the C-3(R) structure. However, the synthesis and antitumor activities of C-3(R) BAderivatives have rarely been reported so
far. In this study, we show that the introduction of electron-withdrawing groups in the C-3(R) site of BAring also significantly
elevated cytotoxicity, and the obtained assay results showed that the introduction of an aromatic lipophilic side chain and
electron-withdrawing groups such as the nitro group (NO ) could obviously increase cytotoxic activity.
2
1081

0.3
0.2
250
200
150
100
50
100
80
60
40
20
0
0.55
0.50
0.45
0.40
0.35
0.1
0
0.30
0.25
0
0.20
Control
2b
2d
Control
BA
2a
2c
2c,10
μ
M
2c, 30
μ
M
BA, 50 μM
M
2c, 100
μ
25 μM
Relative band density of caspase-12
Survival rate, % of control
Fig. 1. Effects of compounds 2a–d on cytotoxic activity in CT26 cell. Image J software was used to analyze the levels of
caspase-12 with GAPDH as the reference. Data represent mean SD of three independent experiments.
EXPERIMENTAL
1
General. H NMR spectra were recorded on Bruker AM 600 MHz spectrometers with tetramethylsilane (TMS) as
internal standard. Melting points (mp) were recorded on an SRS OptiMelt-100 fully automatic micro melting point instrument.
Electrospray ionization mass spectra (ESI-MS) were recorded on anAgilent 6520B Q-TOF system. FT-IR spectra were recorded
–1
on a Nicolet 380 fourier transform infrared spectrometer using KBr pellets in the 400–4000 cm range. Column chromatography
(CC) was performed on silica gel (200–300 mesh; Qingdao Makall Group Co., Ltd.; Qingdao, China). All reactions were
monitored using thin-layer chromatography (TLC) on silica gel plates. Reagents were analytical reagent grade and purchased
from Aladdin.
General Procedure for the Preparation of Compounds 2a–d. A mixture of betulinic acid (0.31 g, 0.67 mmol),
PPh (0.21 g, 0.8 mmol), and cinnamic acid (0.8 mmol) in dry THF (10 mL) was stirred at –30°C under an inert (N ) atmosphere;
3
2
then a solution of DEAD (130 μL, 0.8 mmol) in dry THF (10 mL) was added dropwise over 15 min while the temperature was
maintained at –30°C. When the addition was complete, the mixture was allowed to warm gradually to 0°C and was vigorously
stirred for 2–4 h. After completion of the reaction as indicated by TLC, saturated aqueous NaHCO (20 mL) was added.
3
The aqueous phase was separated and extracted with ether (3 × 10 mL). The organic extracts were combined, dried, and
concentrated. To the residue was added ether (3 × 10 mL) and hexane (3 × 10 mL) whereupon the bulk of the triphenylphosphine
oxide was filtered off. Concentration of the residue gave a viscous oil, which was purified by CC using ethyl acetate–petroleum
ether (3:1) to give 2a–d as white solids.
(1R,3aS,5aR,5bR,7aR,9R,11aR,11bR,13aR,13bR)-9-(Cinnamoyloxy)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-
2-yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylic Acid (2a). White solid, yield 65%, mp 241–243°C. ESI-MS
–1
m/z 586.7. IR (KBr, ν, cm ): 3473, 3238, 2940, 2863 (C–H), 1747 (COOH), 1682 (C=O), 1640, 1533, 1482, 1435 (C=C).
PMR (600 MHz, DMSO-d , δ, ppm, J/Hz): 7.62 (3H, dd, J = 12.4, 7.5), 7.55 (2H, dd, J = 7.3, 2.7), 4.68 (1H, d, J = 1.8), 4.56
6
(1H, s), 4.03 (1H, q, J = 7.1), 2.98–2.91 (2H, m), 2.22 (1H, td, J = 12.7, 3.4), 2.14–2.09 (1H, m), 1.83–1.77 (2H, m), 1.64 (3H,
s), 1.61–1.52 (2H, m), 1.50 (1H, d, J = 11.3), 1.43 (4H, dd, J = 8.7, 4.4), 1.40 (1H, d, J = 4.3), 1.39–1.33 (3H, m), 1.33–1.27
(4H, m), 1.24 (1H, dd, J = 12.7, 2.2), 1.17 (2H, t, J = 7.1), 1.09 (1H, d, J = 12.4), 0.97 (1H, dd, J = 13.0, 4.5), 0.92 (3H, s), 0.86
(6H, d, J = 2.9), 0.85–0.80 (1H, m), 0.76 (3H, s), 0.65 (3H, s).
(1R,3aS,5aR,5bR,7aR,9R,11aR,11bR,13aR,13bR)-9-(((E)-3-(4-Bromophenyl)acryloyl)oxy)-5a,5b,8,8,11a-
pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylic Acid (2b). White solid, yield 63%.
–1
mp 240–242°C. ESI-MS m/z 666.6. IR (KBr, ν, cm ): 3423, 3244, 2993, 2861 (C–H), 1747 (COOH), 1692 (C=O), 1626,
1583, 1530, 1482 (C=C), 1070 (C–Br). PMR (600 MHz, DMSO-d , δ, ppm, J/Hz): 12.36 (1H, s), 7.61 (3H, d, J = 8.6), 7.58
6
(1H, s), 7.55 (1H, s), 6.57 (1H, d, J = 16.0), 4.69 (1H, d, J = 1.8), 4.56 (1H, s), 4.27 (1H, d, J = 5.1), 4.03 (1H, dd, J = 14.1, 7.0),
2.96 (2H, ddd, J = 16.0, 10.6, 5.4), 2.22 (1H, td, J = 12.8, 3.4), 2.11 (1H, d, J = 9.7), 1.83–1.76 (2H, m), 1.64 (3H, s), 1.56 (2H,
dd, J = 28.5, 15.4), 1.50 (1H, d, J = 11.3), 1.45–1.41 (3H, m), 1.35 (3H, dd, J = 12.3, 10.0), 1.33–1.29 (3H, m), 1.25 (2H, d,
1082

J = 13.8), 1.17 (2H, t, J = 7.1), 1.09 (1H, d, J = 10.3), 0.97 (1H, dd, J = 13.0, 4.5), 0.93 (3H, s), 0.86 (6H, d, J = 2.6), 0.76
(3H, s), 0.65 (3H, s).
(1R,3aS,5aR,5bR,7aR,9R,11aR,11bR,13aR,13bR)-5a,5b,8,8,11a-Pentamethyl-9-(((E)-3-(4-nitrophenyl)
acryloyl)oxy)-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylic Acid (2c). White solid, yield 58%,
–1
mp 254–256°C. ESI-MS m/z 632.7. IR (KBr, ν, cm ): 3441, 3116, 2934, 2863 (C–H), 1746 (COOH), 1685 (C=O), 1626,
1599, 1524, 1423 (C=C), 1343 (C-NO ). PMR (600 MHz, DMSO-d , δ, ppm, J/Hz): 12.08 (1H, s), 7.56 (2H, d, J = 3.0), 7.54
2
6
(2H, d, J = 2.7), 4.69 (1H, d, J = 1.8), 4.56 (1H, s), 4.27 (1H, d, J = 5.0), 4.03 (2H, q, J = 7.1), 3.00–2.93 (2H, m), 2.22 (1H, td,
J = 12.7, 3.4), 2.13–2.09 (1H, m), 1.80 (2H, dd, J = 8.7, 4.8), 1.64 (3H, s), 1.53 (1H, d, J = 6.4), 1.50 (1H, d, J = 11.3), 1.46–1.41
(4H, m), 1.39–1.34 (3H, m), 1.33–1.29 (3H, m), 1.26–1.22 (1H, m), 1.20 (1H, dd, J = 10.0, 4.1), 1.17 (3H, t, J = 7.1), 1.09 (1H,
d, J = 12.4), 0.97 (1H, dd, J = 13.0, 4.6), 0.92 (3H, s), 0.86 (6H, d, J = 3.2), 0.76 (3H, s), 0.65 (3H, s).
(1R,3aS,5aR,5bR,7aR,9R,11aR,11bR,13aR,13bR)-9-(((E)-3-(4-Chlorophenyl)acryloyl)oxy)-5a,5b,8,8,11a-
pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylic Acid (2d). White solid, yield 62%,
–1
mp 253–255°C. ESI-MS m/z 622.2. IR (KBr, ν, cm ): 3449, 3238, 2940, 2866 (C–H), 1746 (COOH), 1682 (C=O), 1622,
1587, 1488, 1399 (C=C), 1230 (C–Cl). PMR (600 MHz, DMSO-d , δ, ppm, J/Hz): 12.25 (1H, s), 7.73 (2H, d, J = 8.5), 7.58
6
(1H, d, J = 16.0), 7.51–7.44 (2H, m), 6.56 (1H, d, J = 16.0), 4.69 (1H, d, J = 2.0), 4.56 (1H, s), 4.27 (1H, d, J = 5.1), 4.03 (1H,
q, J = 7.1), 2.95 (2H, qd, J = 10.7, 5.4), 2.22 (1H, td, J = 12.7, 3.5), 2.15–2.08 (1H, m), 1.84–1.76 (2H, m), 1.64 (3H, s), 1.54
(1H, d, J = 6.8), 1.50 (1H, d, J = 11.3), 1.43 (4H, dd, J = 12.0, 7.3), 1.41–1.37 (2H, m), 1.36–1.33 (2H, m), 1.31 (2H, d, J = 6.1),
1.25 (2H, dd, J = 12.9, 2.3), 1.16 (3H, dt, J = 12.8, 5.7), 1.09 (1H, d, J = 10.4), 0.93 (3H, s), 0.87 (6H, d, J = 2.6), 0.76 (3H, s),
0.65 (3H, s).
Reagents and Cell Culture. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) and
96-well plates (Beyotime Biotechnology) were used for studies. All cells were purchased from the American Tissue Culture
Collection (ATTC). Cells were grown in high glucose Dulbecco′s modified Eagle′s medium (DMEM) containing 10% (v/v)
fetal calf serum and 100 mM penicillin and 100 mM streptomycin. All cells were propagated at 37°C in a humidified atmosphere
containing 5% CO in air. Compounds 2a–d were dissolved in dimethyl sulfoxide to make stock solutions and kept at –20°C.
2
The final concentration of the vehicle in the solution never exceeded 0.1% and had no effects on cell viability.
Cell Proliferative Assay. Cell proliferative assays were carried out using 96-well plate cultures and MTT staining.
Briefly, test compounds were added from stock solutions at 10 mM in the incubation mixture in a small volume of dimethyl
sulfoxide (DMSO). Selected cancer cell lines (3000 cells/well) were treated with serial dilutions (100, 80, 40, 30, 20, and
10 μM) of BA and derivatives in 96-well culture plates for 72 h. Then 20 μL of MTT solution was added to each well, and the
microplates were further incubated at 37°C for 4 h. The medium was aspirated, and the insoluble formazan product was
dissolved in DMSO for at least 2 h in the dark. Then the extent of MTT reduction was quantified by measuring the absorbance
at 540 nm. IC values were calculated using the formula IC = 1 – [(C
– C
)/(C
– C
)] through
50
50
experimental group
blank
control
blank
nonlinear fitting in Origin software. C
, C
, and C
are mean the concentrations of the experimental
experimental group
control
blank
group, the only cell sap added group, and the no-additions (NA) group, respectively. The results were determined from replicates
of 96 wells from at least three independent experiments
Protein Extraction and Western Blotting Analysis. The operation was conducted as follows [22]. The CT26 cells
on 6-well plates were rinsed twice with cold PBS and lysed in 100 μL RIPA lysis buffer (10 mM HEPES, 2 mM EDTA, 0.1%
CHAPS, 5 mM DTT, and 1 mM PMSF) on ice for 30 min. Insoluble components of cell lysates were removed by centrifugation
at 12000 g for 5 min at 4°C, and protein concentrations were measured using the BCATM protein quantification kit. The
caspase-12 protein level was determined by western blotting analysis of 50 μg of cell extract using antibody against caspase-12
(2202). Briefly, the protein samples were centrifuged (4°C, 12000g, 10 min) and boiled for 10 min, then subjected to 10%
SDS-polyacrylamide gel electrophoresis at a constant 20 mA current for 1 h. The resolved proteins were transferred to a PVDF
membrane by wet rotation at 70 mV and blocked with a blocking buffer (2% fat-free milk, 10 mM Tris-Cl, 50 mM NaCl, 0.1%
Tween 20, pH 7.4) at room temperature for 1 h. The membrane was incubated with primary antibodies against caspase-12 and
GAPDH overnight on the converter at low temperature (4°C). The next day, the membrane was washed using the washing
buffer (10 mM Tris-Cl, 50 mM NaCl, 0.1% Tween 20, pH 7.4) three times, twice for 5 min and once for 10 min, to remove any
nonspecific primary antibody binding. Then the membrane was incubated with an appropriate dilution of secondary antibody
at room temperature for 2 h. After washing three times with washing buffer, the membrane was illuminated with ECL reagent
according to the manufacturer′s instructions. A photograph of the gel was taken, and the relative band density was analyzed by
optical densitometry using Image J. Survival rate values were calculated using the formula SR = (D
– D
)/
experimental group
control
(D
) through nonlinear fitting in Origin software. D
, D
, and D
are the mean band density of the
control
experimental group
control
blank
1083

experimental group, the only cell sap added group, and the no-additions (NA) group, respectively. The results were determined
from replicates of six wells from at least three independent experiments.
In summary, compounds 2a–d exhibited more potent cytotoxic activity than BA in four cancer cell lines.
Especially, compound 2c was the most active in A549, 4T1 with CT26 cell lines and may be chosen for further studies. The
electron-withdrawing groups on the aromatic ring at the C-3 position showed stronger cytotoxicity. Moreover, the stronger the
electron-withdrawing ability, the better the cytotoxicity when comparing compound 2c with 2a, 2b, and 2d. This brief SAR
study should not be overlooked for a better understanding of the structural requirements at the C-3 position of BA for antitumor
effects.
ACKNOWLEDGMENT
This work was supported by the National Key R&D Program of China (2016YFD0600805), the Jiangsu “333”
project of cultivation of high-level talents, the 11th Six Talents Peak Project of Jiangsu Province, a Project Funded by the
National First-class Disciplines (PNFD), a project funded by the priority academic program development of Jiangsu Higher
Education Institutions (PAPD), and the Doctorate Fellowship Foundation of Nanjing Forestry University (163030748).
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