Synthesis and biological activity of 23-hydroxybetulinic acid c-28 ester derivatives as antitumor agent candidates

Article abstract of DOI:10.3390/molecules17088832

23-Hydroxybetulinic acid (1) served as the precursor for the synthesis of C-28 ester derivatives. The target compounds were evaluated in vitro for their antitumor activities against five cell lines (A549, BEL-7402, SF-763, B16 and HL-60). Among the obtained compounds, 6i had the most potent antitumor activity, with the IC₅₀ values of 8.35 μM in HL-60 cells and showed similar antitumor activity as cyclophosphamide in H22 liver tumor and as 5-fluorouracil in B16 melanoma in vivo.

Full text of DOI:10.3390/molecules17088832

Molecules 2012, 17, 8832-8841; doi:10.3390/molecules17088832
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Activity of 23-Hydroxybetulinic Acid
C-28 Ester Derivatives as Antitumor Agent Candidates
Yi Bi 1, Jinyi Xu 2,3,*, Fei Sun 2, Xiaoming Wu 3,*, Wencai Ye 4, Yijun Sun 5 and Wenwen Huang 1
1
School of Pharmacy, Yantai University, Yantai 264005, Shandong, China
2
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009,
Jiangsu, China
3
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009,
Jiangsu, China
4
Department of Phytochemistry, China Pharmaceutical University, Nanjing 210009, Jiangsu, China
5
Drug Screening Center, Nanjing KeyGen Biotech. Co. Ltd., Nanjing 210012, Jiangsu, China
* Authors to whom correspondence should be addressed; E-Mails: jinyixu@china.com (J.X.);
xmwu@cpu.edu.cn (X.W.); Tel.: +86-258-327-1445 (J.X.); Fax: +86-258-330-2827 (X.W.).
Received: 29 May 2012; in revised form: 17 July 2012 / Accepted: 18 July 2012 /
Published: 25 July 2012
Abstract: 23-Hydroxybetulinic acid (1) served as the precursor for the synthesis of C-28
ester derivatives. The target compounds were evaluated in vitro for their antitumor
activities against five cell lines (A549, BEL-7402, SF-763, B16 and HL-60). Among the
obtained compounds, 6i had the most potent antitumor activity, with the IC50 values of
8.35 µM in HL-60 cells and showed similar antitumor activity as cyclophosphamide in
H22 liver tumor and as 5-fluorouracil in B16 melanoma in vivo.
Keywords: 23-hydroxybetulinic acid; C-28 ester derivatives; antitumor activity;
structure-activity relationships
1. Introduction
23-Hydroxybetulinic acid (1, Figure 1) and betulinic acid (2, Figure1) are well-known members of
the class of pentacyclic triterpenes [1,2]. Many biological activities of betulinic acid have been
reported, such as antitumor, antiviral, antioxidant and so on [3–9]. As a good lead compound, betulinic

Molecules 2012, 17
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acid showed potent antitumor activity in a series of cell lines and the mechanism of this action might
be related to its effects on the proliferation, migration, cell cycle and apoptosis of tumor cells [3–9].
The chemical structure of 23-hydroxybetulinic acid is similar to that of betulinic acid, and they also
have the similar pharmaceutical activities [10]. In our previous study, many derivatives of 23-hydroxy-
betulinic acid showed antitumor activity in different cell lines in vitro and compound 3 (Figure 1)
displayed stronger antitumor activity in mice H22 and B16 than betulinic acid or 23-hydroxybetulinic
acid [11]. It was shown that the polarity and length of the chain in C-28 had an important impact on the
antitumor activity. These results motivated us to undertake further modifications of the 28-carboxylic
acid moiety of lead compound 1. In this paper, we wish to report a series of 28-carboxylic acid
modified 23-hydroxybetulinic acid ester derivatives and their antitumor activity. The preliminary
structure-activity relationships are also discussed.
Figure 1. Structure of 23-hydroxybetulinic acid (1), betulinic acid (2) and a derivative of
23-hydroxybetulinic acid (3).
COOCH2CH=CH2
COOH
COOH
HO
HO
HO
OH
OH
1
2
3
2. Results and Discussion
2.1. Chemistry
For the synthetic experiments, the starting material 23-hydroxybetulinic acid (1) was isolated from
the root of Pulsatilla chinensis. As described in Scheme 1, several derivatives of 23-hydroxybetulinic
acid were synthesized. Compounds 5a–d were readily prepared in two steps starting from compound 1.
Treatment of 5a–d with anhydrides R2(CO)2O, such as succinic anhydride, glutaric anhydride, maleic
anhydride and phthalic anhydride, in the presence of DMAP in CH2Cl2 gave compounds 6a–n in the
yields of 53%–76%.
2.2. Biological Studies
The MTT (3-(4,5)dimethylthiahiazo (-z-y1) -3,5- diphenyltetrazoliumromide) assay results (Table 1)
showed that most of the 23-hydroxybetulinic acid derivatives displayed better cytotoxic activities
against the tested cells than betulinic acid and 23-hydroxybetulinic acid, especially compounds 6f, 6h,
and 6i with IC50 values ranging from 8.35 to 28.78 μM. As the assay results showed, C-28 ester
derivatives with carboxylic acid substituents at the end of the C-28 side chain and whose C-28 side
chain had appropriate length and flexibility exhibited stronger cytotoxicity. The activities of
compounds 6k–n were weaker compared with 6f, 6h and 6i. It was possible due to the alkyne group in
the C-28 side chain blocking the bending and rotation of the compounds.

Molecules 2012, 17
Scheme 1. The synthesis of 23-hydroxybetulinic acid C-28 ester derivatives 6a–n.
8834
COOH
COOR1OH
COOH
i
ii
AcO
HO
AcO
OH
OAc
OAc
R1=(CH2)2
R1=(CH2)4
4
R1=(CH2)6
R1=CH2C CCH2
5a
5b
1
5c
5d
R2=(CH2)2
R2=(CH2)3
R2=CH=CH
R2=(CH2)2
R1=(CH2)2
R1=(CH2)6
6a
6b R1=(CH2)2
6h
R2=(CH2)3
R2=
6i R1=(CH2)6
iii
R1=(CH2)2
R1=(CH2)2
R1=(CH2)6
6c
6d
6j
COOR1OCOR2COOH
R2=
R1=CH2C CCH2
R2=(CH2)2
R2=(CH2)3
R2=CH=CH
R2=
6k
6l
6e R1=(CH2)4
R1=CH2C CCH2
R1=CH2C CCH2
R1=CH2C CCH2
R2=(CH2)2
R2=(CH2)3
AcO
R1=(CH2)4
OAc
6f
6m
6n
6g R1=(CH2)4
R2=
Reagents and conditions: (i) Ac2O, pyridine, rt, overnight; (ii) (COCl)2, CH2Cl2, rt, 4 h, then
HOR1OH, rt, 8 h; (iii) R2(CO)2O, DMAP(4-dimethylaminopyridine), CH2Cl2, rt, 10 h.
Table 1. The cytotoxicity data of 23-hydroxybetulinic acid and its derivatives [IC50 (μmol/L) ± SD].
Cell line
Compound
A549
BEL-7402
89.81 ± 7.32
52.51 ± 2.55
43.06 ± 9.71
48.90 ± 11.56
SF-763
B16
HL-60
HBA
BA
6a
6b
6c
81.36 ± 3.54
89.62 ± 11.23
34.12 ± 11.10
35.18 ± 15.42
27.12 ± 9.84
68.48 ± 13.10
31.36 ± 19.04
12.35 ± 1.80
69.82 ± 8.14
15.80 ± 6.51
10.02 ± 5.04
62.72 ± 5.03
57.09 ± 16.13
40.86 ± 8.44
51.71 ± 10.05
60.16 ± 9.58
90.09 ± 8.31 75.64 ± 8.55
78.89 ± 9.24 50.09 ± 7.32
42.45 ± 12.11 31.04 ± 9.48
51.05 ± 9.75 46.06 ± 1.56
80.54 ± 9.13
76.77 ± 10.58
22.85 ± 9.04
35.37 ± 14.35
28.63 ± 9.31
49.18 ± 10.48
14.80 ± 7.91
12.94 ± 6.55
52.73 ± 10.24
10.03 ± 1.76
8.35 ± 4.28
36.81 ± 12.05 43.67 ± 15.70 32.15 ± 4.02
6d
6e
59.37 ± 9.57
22.06 ± 11.75
17.08 ± 5.09
63.51 ± 8.92 51.57 ± 8.10
34.04 ± 9.11 29.12 ± 7.27
19.52 ± 9.01 15.22 ± 8.67
6f
6g
6h
6i
57.81 ± 15.85 73.10 ± 15.02 72.13 ± 2.52
28.78 ± 9.12
11.15 ± 9.34
21.84 ± 3.41 19.09 ± 6.42
12.32 ± 6.10 14.05 ± 2.97
6j
59.25 ± 10.84 49.61 ± 12.46 40.43 ± 10.06
39.66 ± 9.75
37.09 ± 14.63
30.53 ± 8.34
47.61 ± 2.45
6k
6l
69.09 ± 7.55
55.62 ± 9.02
68.13 ± 5.89
53.74 ± 9.82 43.48 ± 9.77
42.88 ± 10.39 35.70 ± 15.12
55.43 ± 13.74 46.56 ± 11.84
6m
6n
76.92 ± 12.70 66.75 ± 16.83 50.40 ± 15.22 56.23 ± 14.91
BA: betulinic acid, HBA: 23-hydroxybetulic acid; data is mean of three experiments.

Molecules 2012, 17
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Compound 6i was chosen to evaluated its antitumor activities in mice in vivo based on its
significant efficiency in vitro. As illustrated in Tables 2 and 3, in H22 mice, compound 6i exhibited
stronger activity than 23-hydroxybetulinic acid and comparable activity to cyclophosphamide, which
was used as a positive control. In the B16 group, compound 6i also showed more potent activity than
23-hydroxybetulinic acid but somewhat weaker than 5-fluorouracil.
Table 2. The cytoxicity data of 23-hydroxybetulinic acid and its derivative 6i in mice H22 in vivo.
Number of
mice
Weight of
Ratio of
Weight of mice (g)
Start End
P
Drugs
Dose
Injection
tumor X ± SD inhibition
value
(g)
(%)
Start End
Normal
saline
CP
0.4 mL/mouse
iv
10
10
18.8 ± 1.1 25.3 ± 1.2
1.70 ± 0.45
30 mg/kg
25 mg/kg
25 mg/kg
iv
ip
ip
10
10
10
10
10
10
18.4 ± 1.2 25.2 ± 1.6
18.5 ± 1.6 26.1 ± 1.5
18.3 ± 0.9 25.7 ± 1.4
0.71 ± 0.27
1.31 ± 0.75
0.75 ± 0.63
58.2
22.9
55.9
<0.01
>0.05
<0.01
HBA
6i
CP – cyclophosphamide; HBA – 23-hydroxybetulinic acid.
Table 3. The cytoxicity data of 23-hydroxybetulinic acid and its derivative 6i in mice B16 in vivo.
Number of
mice
Weight of
Ratio of
Weight of mice (g)
Start End
P
Drugs
Dose
Injection
tumor X ± SD inhibition
value
(g)
(%)
Start End
Normal
Saline
5-FU
HBA
6i
0.4 mL/mouse
iv
10
10
18.9 ± 2.1 23.8 ± 1.6
1.96 ± 0.52
25 mg/kg
25 mg/kg
25 mg/kg
iv
ip
ip
10
10
10
10
10
10
19.4 ± 1.8 23.2 ± 1.8
19.1 ± 1.7 24.2 ± 1.7
18.9 ± 1.4 24.7 ± 1.1
0.59 ± 0.31
1.59 ± 0.44
0.81 ± 0.21
69.9
18.9
58.7
<0.01
>0.05
<0.01
5-FU – 5-fluorouracil; HBA – 23-hydroxybetulinic acid.
3. Experimental
3.1. Synthesis
3.1.1. General
Melting points were obtained on a MEL-TEMP II melting-point apparatus and are uncorrected. IR were
1
recorded on Nicolet Impact 410 or Bruker FT-IR TENSOR27 instruments. H-NMR spectra were
recorded on a Bruker-ACF-300 (chemical shifts are expressed as values relative to TMS as internal
standard). HR-MS were obtained using a Agilent QTOF 6520 instrument.
3.1.2. General Procedure for the Synthesis of 6a–n
Ac2O (0.15 mL, 1.5 mmol) was added to a solution of 23-hydroxybetulinic acid (1, 120.0 mg,
0.25 mmol) in dry pyridine (5 mL). The mixture was stirred overnight at room temperature. After
adding acetic ether (10 mL), the mixture was washed with 9% HCl (20 mL × 3) and brine (20 mL × 3),
dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The residue was purified by

Molecules 2012, 17
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crystallization from ethyl acetate to afford the desired compound 4 as yellow powder (136.0 mg, 98%).
(COCl)2 (0.1 mL) was added to a solution of 4 (100.0 mg, 0.18 mmol) in dry CH2Cl2 (10 mL). The
mixture was stirred for 4 h at room temperature and evaporated to dryness. Immediately, the
corresponding alcohol (5 equiv.) and CH2Cl2 (10 mL) were added and the mixture allowed to stir for
8vh at room temperature, evaporated to dryness and purified by column chromatography on silica gel,
eluted with an mixture of petroleum ether/ethyl acetate to give compounds 5a–d. The corresponding
R2(CO)2O (4 equiv.) was added to a solution of compounds 5a–d in CH2Cl2 (10 mL) in the presence of
DMAP (1.5 equiv.). The mixture was stirred for 10 h at room temperature and washed with 9% HCl
(20 mL × 2), H2O (20 mL × 2), brine (20 mL × 2), dried over anhydrous Na2SO4, filtered, and
concentrated to dryness. The residue was purified by column chromatography on silica gel to afford
compounds 6a–n.
Butanedioic acid, 1-[2-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6a). From
1,2-ethanediol (56.0 mg, 0.9 mmol) and succinic anhydride (55.0 mg, 0.55 mmol); column
chromatography with petroleum ether/ethyl acetate/acetic acid = 50:50:1 (v:v:v). Yield: 73.0 mg (76%)
as a yellow solid, mp 68 °C–70 °C; IR (KBr, cm−1) ν 3429, 2985, 2947, 2872, 1738, 1731, 1649, 1453,
1
1370, 1246, 1151, 1039, 881; H-NMR (CDCl3, 300 MHz) δ 0.80, 0.88, 0.91, 0.96, 1.68 (3H each, s,
24, 25, 26, 27, 30-CH3), 2.02, 2.06 (3H each, s, 3 and 23-OCOCH3), 2.67 (4H, m, 28-COOCH2CH2O
COCH2CH2), 2.99 (1H, m, 19-CH), 3.68 (1H, d, J = 11.9 Hz, 23-CH2), 3.84 (1H, d, J = 11.9 Hz,
23-CH2), 4.29 (4H, m, 17-COOCH2CH2), 4.61 (1H, s, 29=CH2), 4.72 (1H, s, 29=CH2), 4.74 (1H, m,
3-CH); HR-MS (ESI, M+H) m/z: calcd for C40H61O10 701.4108, found 701.4104.
Pentanedioic acid, 1-[2-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6b). From
1,2-ethanediol (56.0 mg, 0.9 mmol) and glutaric anhydride (63.0 mg, 0.55 mmol); column
chromatography with petroleum ether/ethyl acetate/acetic acid = 50:50:1 (v:v:v). Yield: 71.0 mg (73%)
as a yellow oil; IR (KBr, cm−1) ν 3457, 2948, 2873, 1738, 1711, 1642, 1451, 1375, 1245, 1153, 1039,
1
885, 756; H-NMR (CDCl3, 300 MHz) δ 0.80, 0.87, 0.91, 0.96, 1.68 (3H each, s, 24, 25, 26, 27,
30-CH3), 2.01, 2.06 (3H each, s, 3 and 23-OCOCH3), 2.43 (4H, m, 28-COOCH2CH2OCOCHCH2),
2.98 (1H, m, 19-CH), 3.68 (1H, d, J = 11.6 Hz, 23-CH2), 3.85 (1H, d, J = 11.6 Hz, 23-CH2), 4.29 (4H,
m, 17-COOCH2CH2), 4.60 (1H, s, 29=CH2), 4.72 (1H, s, 29=CH2), 4.75 (1H, m, 3-CH); HR-MS (ESI,
M+H) m/z: calcd for C41H63O10 715.4265, found 715.4262.
Butylenedioic acid, 1-[2-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6c). From
1,2-ethanediol (56.0 mg, 0.9 mmol) and maleic anhydride (54.0 mg, 0.55 mmol); column
chromatography with petroleum ether/ethyl acetate = 1:1 (v:v). Yield: 59.0 mg (62%) as a white solid;
mp 68 -70 °C; IR (KBr, cm−1) ν 3458, 3418, 2945, 2869, 1732, 1640, 1452, 1375, 1245, 1155, 1038,
1
758; H-NMR (CDCl3, 300 MHz) δ 0.80, 0.87, 0.91, 0.96, 1.68 (3H each, s, 24, 25, 26, 27, 30-CH3),
2.01, 2.06 (3H each, s, 3 and 23-OCOCH3), 2.95 (1H, m, 19-CH), 3.69(1H, d, J = 11.3 Hz, 23-CH2),
3.83 (1H, d, J = 11.3 Hz, 23-CH2), 4.36 (2H, m, 17-COOCH2CH2), 4.44 (2H, m, 17-COOCH2CH2),
4.61 (1H, s, 29=CH2), 4.72 (1H, s, 29=CH2), 4.76 (1H, m, 3-CH), 6.36(1H, d, J = 12.8 Hz,
28-COOCH2CH2OCOCH=CH), 6.50 (1H, d, J = 12.8 Hz, 28-COOCH2CH2OCOCH=CH). HR-MS
(ESI, M+H) m/z: calcd for C40H59O10 699.3952, found 699.3959.

Molecules 2012, 17
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1,2-Xylenedioic acid, 1-[2-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6d). From
1,2-ethanediol (56.0 mg, 0.9 mmol) and phthalic anhydride (81.0 mg, 0.55 mmol); column
chromatography with petroleum ether/ethyl acetate/acetic acid = 50:50:1 (v:v:v). Yield: 75.0 mg (73%)
as a white solid, mp 70 °C–72 °C; IR (KBr, cm−1) ν 3450, 2947, 2871, 1731, 1635, 1448, 1380, 1248,
1
1128, 1038, 883, 743; H-NMR (CDCl3, 300 MHz) δ 0.79, 0.82, 0.89, 0.92, 1.65 (3H each, s, 24, 25,
26, 27, 30-CH3), 2.01, 2.06 (3H each, s, 3 and 23-OCOCH3), 2.97 (1H, m, 19-CH), 3.68 (1H, d,
J = 11.6 Hz, 23-CH2), 3.83 (1H, d, J = 11.6 Hz, 23-CH2), 4.58 (7H, m, 29 = CH2, 3-CH and
17-COOCH2CH2), 7.70 (4H, m, 17-COOCH2CH2OCOC6H4COOH); HR-MS (ESI, M+H) m/z: calcd
for C44H61O10 749.4108, found 749.4111.
Butanedioic acid, 1-[4-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6e). From 1,4-butanediol
(81.0 mg, 0.9 mmol) and succinic anhydride (44.0 mg, 0.44 mmol); column chromatography with
petroleum ether/ethyl acetate/acetic acid = 50:50:1 (v:v:v). Yield: 56.0 mg (70%) as a yellow oil; IR
(KBr, cm−1) ν 3488, 3422, 2923, 2854, 1738, 1641, 1461, 1377, 1245, 1157, 1037, 974, 883; 1H-NMR
(CDCl3, 300 MHz) δ 0.80, 0.88, 0.91, 0.96, 1.68 (3H each, s, 24, 25, 26, 27, 30-CH3), 2.01, 2.06
(3H each, s, 3 and 23-OCOCH3), 2.65 (4H, m, 28-COOCH2(CH2)2CH2OCOCH2CH2), 2.99 (1H, m,
19-CH), 3.68 (1H, d, J = 11.7 Hz, 23-CH2), 3.84 (1H, d, J = 11.7 Hz, 23-CH2), 4.13 (4H, m,
17-COOCH2(CH2)2CH2), 4.60 (1H, s, 29=CH2), 4.72 (1H, s, 29=CH2), 4.74 (1H, m, 3-CH); HR-MS
(ESI, M+H) m/z: calcd for C42H65O10 729.4421, found 729.4428.
Pentanedioic acid, 1-[4-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6f). From 1,4-butanediol
(81.0 mg, 0.9 mmol) and glutaric anhydride (50.0 mg, 0.44 mmol); column chromatography with
petroleum ether/ethyl acetate/acetic acid = 50:50:1 (v:v:v). Yield: 55.0 mg (67%) as a yellow oil; IR
1
(KBr, cm−1) ν 3425, 2947, 2871, 1735, 1641, 1451, 1384, 1244, 1155, 1036, 975; H-NMR (CDCl3,
300 MHz) δ 0.80, 0.87, 0.91, 0.96, 1.70 (3H each, s, 24, 25, 26, 27, 30-CH3), 2.01, 2.06 (3H each, s,
3 and 23-OCOCH3), 2.43 (4H, m, 28-COOCH2(CH2)2CH2OCOCH2CH2CH2), 2.99 (1H, m,
19-CH), 3.68 (1H, d, J = 12.3 Hz, 23-CH2), 3.85 (1H, d, J = 12.3 Hz, 23-CH2), 4.10 (4H, m,
28-COOCH2(CH2)2CH2), 4.60 (1H, s, 29=CH2), 4.73 (1H, s, 29=CH2), 4.76 (1H, m, 3-CH); HR-MS
(ESI, M+H) m/z: calcd for C43H67O10 743.4578, found 743.4573.
1,2-xylenedioic acid, 1-[4-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6g). From
1,4-butanediol (81.0 mg, 0.9 mmol) and phthalic anhydride (65.0 mg, 0.44 mmol); column
chromatography with petroleum ether/ethyl acetate/acetic acid = 50:50:1 (v:v:v). Yield: 53.0 mg (62%)
as a white solid, mp 163 °C–165 °C; IR (KBr, cm−1) ν 3457, 2973, 2866, 1643, 1455, 1381, 1055,
1025, 1012, 771; 1H-NMR (CDCl3, 300 MHz) δ 0.80, 0.88, 0.91, 0.95, 1.68 (3H each, s, 24, 25, 26, 27,
30-CH3), 2.02, 2.06 (3H each, s, 3 and 23-OCOCH3), 2.99 (1H, m, 19-CH), 3.69 (1H, d, J = 11.6 Hz,
23-CH2), 3.84 (1H, d, J = 11.6 Hz, 23-CH2), 4.12 (4H, m, 28-COOCH2(CH2)2CH2), 4.60 (1H, s,
29=CH2), 4.72 (1H, s, 29 = CH2), 4.76 (1H, m, 3-CH), 7.52 (4H, m, 17-COO(CH2)4OCOC6H4COOH);
HR-MS (ESI, M+H) m/z: calcd for C46H65O10 777.4421, found 777.4427.
Butanedioic acid, 1-[6-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6h). From
1,6-hexanediol (106.0 mg, 0.9 mmol) and succinic anhydride (42.0 mg, 0.42 mmol); column
chromatography with petroleum ether/ethyl acetate = 2:1 (v:v). Yield: 58.0 mg (73%) as a yellow oil;

Molecules 2012, 17
8838
IR (KBr, cm−1) ν 3480, 3417, 2945, 2867, 1738, 1641, 1454, 1372, 1244, 1160, 1038, 885, 756;
1H-NMR (CDCl3, 300 MHz) δ 0.80, 0.88, 0.91, 0.96, 1.68 (3H each, s, 24, 25, 26, 27, 30-CH3), 2.01,
2.06 (3H each, s, 3 and 23-OCOCH3), 2.65 (4H, m, 17-COOCH2(CH2)4CH2OCOCH2CH2), 2.99 (1H,
m, 19-CH), 3.68 (1H, d, J = 11.5 Hz, 23-CH2), 3.86 (1H, d, J = 11.5 Hz, 23-CH2), 4.05 (4H, m,
17-COOCH2(CH2)4CH2), 4.60 (1H, s, 29=CH2), 4.72 (1H, s, 29=CH2), 4.74 (1H, m, 3-CH); HR-MS
(ESI, M+H) m/z: calcd for C44H69O10 757.4734, found 757.4738.
Pentanedioic acid, 1-[6-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6i). From 1,6-hexanediol
(106 mg, 0.9 mmol) and glutaric anhydride (48.0 mg, 0.42 mmol); column chromatography with
petroleum ether/ethyl acetate = 1:2 (v:v). Yield: 55.0 mg (68%) as a yellow oil; IR (KBr, cm−1) ν 3454,
2943, 2866, 1731, 1642, 1454, 1378, 1247, 1157, 1054, 771; 1H-NMR (CDCl3, 300 MHz) δ 0.80, 0.87,
0.91, 0.96, 1.68 (3H each, s, 24, 25, 26, 27, 30-CH3), 1.98, 2.06 (3H each, s, 3 and 23-OCOCH3), 2.41
(4H, m, 28-COOCH2(CH2)4CH2OCOCH2CH2CH2), 2.99 (1H, m, 19-CH), 3.69 (1H, d, J = 10.2 Hz,
23-CH2), 3.83 (1H, d, J = 10.2 Hz, 23-CH2), 4.06 (4H, m, 28-COOCH2(CH2)4 CH2), 4.60 (1H, s,
29=CH2), 4.72 (1H, s, 29=CH2), 4.74 (1H, m, 3-CH); HR-MS (ESI, M+H) m/z: calcd for C45H71O10
771.4891, found 771.4896.
1,2-Xylenedioic acid, 1-[6-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]ethyl] ester (6j). From
1,6-hexanediol (106.0 mg, 0.9 mmol) and phthalic anhydride (62.0 mg, 0.42 mmol); column
chromatography with petroleum ether/ethyl acetate/acetic acid = 50:50:1 (v:v:v). Yield: 50.0 mg (59%)
as a yellow oil; IR (KBr, cm−1) ν 3479, 2946, 2866, 1735, 1704, 1591, 1384, 1369, 1246, 1130, 1040,
1
652; H-NMR (CDCl3, 300 MHz) δ 0.79, 0.87, 0.91, 0.95, 1.68 (3H each, s, 24, 25, 26, 27, 30-CH3),
2.01, 2.05 (3H each, s, 3 and 23-OCOCH3), 2.99 (1H, m, 19-CH), 3.68(1H, d, J = 11.6 Hz, 23-CH2),
3.84 (1H, d, J = 11.6 Hz, 23-CH2), 4.07 (4H, m, 28-COOCH2(CH2)4CH2), 4.60 (1H, s, 29=CH2), 4.73
(1H, s, 29=CH2), 4.76 (1H, m, 3-CH), 7.48 (4H, m, 17-COO(CH2)6OCOC6H4COOH); HR-MS (ESI,
M+H) m/z: calcd for C48H69O10 805.4734, found 805.4739.
Butanedioic acid, 1-[4-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]butynyl] ester (6k). Butyne-1,4-diol
(77.0 mg, 0.9 mmol) and succinic anhydride (37.0 mg, 0.37 mmol); column chromatography with
petroleum ether/ethyl acetate/acetic acid = 50:50:1 (v:v:v). Yield: 50.0 mg (64%) as a yellow solid, mp
99 °C–101 °C; IR (KBr, cm−1) ν 3450, 2929, 2870, 1736, 1649, 1427, 1370, 1246, 1148, 1041, 971,
1
803, 641; H-NMR (CDCl3, 300 MHz) δ 0.80, 0.87, 0.90, 0.96, 1.68(3H each, s, 24, 25, 26, 27,
30-CH3), 2.01, 2.06 (3H each, s, 3 and 23-OCOCH3), 2.69 (4H, t, 17- 17-COOCH2C≡CCH2O
COCH2CH2COOH), 2.99 (1H, m, 19-CH), 3.68 (1H, d, J = 11.7 Hz, 23-CH2), 3.85 (1H, d, J = 11.7
Hz, 23-CH2), 4.69 (7H, m, 29=CH2, 3-CH and 17-COOCH2C≡CCH2); HR-MS (ESI, M+H) m/z: calcd
for C42H61O10 725.4108, found 725.4102.
Pentanedioic acid, 1-[4-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]butynyl] ester (6l). From
butyne-1,4-diol (77.0 mg, 0.9 mmol) and glutaric anhydride (42.0 mg, 0.37 mmo); column
chromatography with petroleum ether/ethyl acetate/acetic acid = 50:50:1 (v:v:v). Yield: 50.0 mg (67%)
as a yellow solid, mp 90 °C–92 °C; IR (KBr, cm−1) ν 3438, 2946, 2871, 1737, 1643, 1582, 1451, 1369,
1245, 1146, 1123, 1041, 973; 1H-NMR (DMSO, 300 MHz) δ 0.76, 0.83, 0.87, 0.94, 1.65 (3H each, s,
24, 25, 26, 27, 30-CH3), 1.96, 1.99 (3H each, s, 3 and 23-OCOCH3), 2.19 (4H, m, 17-COOCH2

Molecules 2012, 17
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C≡CCH2OCOCH2CH2CH2COOH), 2.91 (1H, m, 19-CH), 3.63 (1H, d, J = 11.9 Hz, 23-CH2), 3.78 (1H,
d, J = 11.9 Hz, 23-CH2), 4.70 (7H, m, 29=CH2, 3-CH and 17-COOCH2≡CCH2); HR-MS (ESI, M+H)
m/z: calcd for C43H63O10 739.4265, found 739.4261.
Butylenedioic acid, 1-[4-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]butynyl] ester (6m). From
butyne-1,4-diol (77.0 mg, 0.9 mmol) and maleic anhydride (36.0 mg, 0.37 mmol); column
chromatography with petroleum ether/ethyl acetate = 1:2 (v:v). Yield: 36.0 mg (53%) as a yellow oil;
IR (KBr, cm−1) ν 3405, 2967, 2922, 2872, 1731, 1650, 1584, 1437, 1407, 1334, 1126, 1044, 953;
1H-NMR (DMSO, 300 MHz) δ 0.76, 0.82, 0.87, 0.94, 1.65 (3H each, s, 24, 25, 26, 27, 30-CH3), 1.96,
1.99 (3H each, s, 3 and 23-OCOCH3), 3.39 (1H, m, 19-CH), 3.63 (1H, d, J = 11.4 Hz, 23-CH2),
3.78 (1H, d, J = 11.4 Hz, 23-CH2), 4.69 (7H, m, 29=CH2, 3-CH and 17-COOCH2C≡CCH2),
6.22 (1H, d, J = 15.8 Hz, 17-COOCH2C≡CCH2OCOCH=CHCOOH), 6.77 (1H, d, J = 15.8 Hz,
17-COOCH2C≡CCH2OCO CH=CHCOOH); HR-MS (ESI, M+H) m/z: calcd for C42H59O10 723.3952,
found 723.3948.
1,2-Xylenedioic acid, 1-[4-[(3β,23-diacetoxylup-20(29)-en-28-oyl)oxy]butynyl] ester (6n). From
butyne-1,4-diol (77.0 mg, 0.9 mmol) and phthalic anhydride (55.0 mg, 0.37 mmol); column
chromatography with petroleum ether/ethyl acetate/acetic acid = 50:50:0.4 (v:v:v). Yield: 44.0 mg
(61%) as a yellow oil; IR (KBr, cm−1) ν 3435, 3070, 2948, 2871, 1728, 1591, 1450, 1385, 1247, 1131,
1
1040, 747; H-NMR (CDCl3, 300 MHz) δ 0.82, 0.88, 0.91, 0.98, 1.71 (3H each, s, 24, 25, 26, 27,
30-CH3), 2.03, 2.08 (3H each, s, 3 and 23-OCOCH3), 3.06 (1H, m, 19-CH), 3.71 (1H, d, J = 11.7 Hz,
23-CH2), 3.86 (1H, d, J = 11.7 Hz, 23-CH2), 4.83 (7H, m, 29=CH2, 3-CH and 17-COOCH2C≡CCH2),
7.78 (4H, m, 17-COOCH2C≡CCH2OCOC6H4COOH); HR-MS (ESI, M+H) m/z: calcd for C46H61O10
773.4108, found 773.4101.
3.2. Pharmacology
In vitro, the cytotoxic activities of 23-hydroxybetulinic acid, betulinic acid and all derivatives were
determined by the MTT cytotoxicity assay, which was performed in 96-well plates. The tumor cell line
panel consisted of A549 (human lung carcinoma), BEL-7402 (human hepatoma), SF-763 (human
cerebroma), B16 (mice melanoma), HL-60 (human leukaemia) (final concentration in the growth
medium was 2~4 × 104 /mL). MTT solution (20 L/well) was added after cells were treated with drug
for 48 h, and cells were incubated for a further 4 h at 37 °C. The purple formazan crystals were
dissolved in 150 L DMSO. After 5 min, the plates were read on an automated microplate
spectrophotometer at 570 nm. Assays were performed in triplicate in three independent experiments.
The concentration required for 50% inhibition of cell viability (IC50) what was calculated. In all of
these experiments, three replicate wells were used to determine each point [11].
In vivo, ICR female mice with body weight of 18–22 g were transplanted with H22 and B16
subcutaneously into the right axilla according to protocols of transplant tumor research. After 24 h of
tumor transplantation, mice were weighed, and each model group was at random divided into 4 groups,
each of which had 10 mice in H22 and B16 group. The groups with 23-OH betulinic acid and 6i were
administered intraperitoneously 25 mg/kg in a vehicle of 20% DMSO/80% saline. respectively. The
positive control group was treated with cyclophosphamide (30 mg/kg) in H22 group and 5-fluorouracil

Molecules 2012, 17
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(25 mg/kg) in B16 group through intravenous injection in a vehicle of 20% DMSO/80% saline. The
negative control group received 0.9% normal saline through intravenous injection. All test compounds
were given through injections 24 h after tumor transplantation (or inoculation). Treatments were done
at a frequency of intravenous or intraperitoneal injection one dose per day for a total of four
consecutive days in H22 group and for a total of 11 consecutive days in B16 group. After the
treatments, all mice were killed and weighed simultaneously, and then segregated and weighed the
tumor [11]. Tumor inhibitory ratio was calculated by the following formula and perform T test:
Tumor inhibitory ratio (%) = (1-average tumor weight of treated group/average tumor
weight of control group) × 100%
4. Conclusions
In summary, a series of novel of 23-hydroxybetulinic acid C-28 ester derivatives were synthesized
and tested for their in vitro cytotoxic activities against five human tumor cell lines. Most of the
compounds showed moderate potent cytotoxic activities on all the tested cells. The results of
preliminary biological activity showed that three compounds (6f, 6h, 6i) possessed impressive
cytotoxicities. Compound 6i was chosen to evaluate its antitumor activities in vivo. Compound 6i
showed similar antitumor activity as cyclophosphamide in H22 and as 5-fluorouracil in B16. Further
structure modification and SAR studies of antitumor 23-hydroxybetulinic acid derivatives are in
progress in our laboratory and the results will be reported in due course [12].
Acknowledgements
This study was supported by grant from National Natural Science Fund (No. 30472083), Key fund
of Ministry of Education of China (No. 108069) and Taishan Scholar Project to Fenghua Fu for
financial assistance.
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Sample Availability: Samples of the compounds 5a–6n are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).