Synthesis and antiproliferative evaluation of 23-hydroxybetulinic acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.03.038

Based on structural modifications of the natural 23-hydroxybetulinic acid, a series of novel its derivatives had been synthesized. The new compounds were screened for in vitro antiproliferative activity against cancer cell lines HeLa, MCF-7, HepG2, B16 and A375 using doxorubicin as a reference. The vast majority of derivatives had exhibited potent tumor growth inhibitory activity than original compound. The derivatives 4, 5, 7, 20, 23, 26, 43 and 44 with IC ₅₀ values lower than 10 μM on all tested cell lines were regarded as the most promising compounds. The structure-activity relationships of 23-hydroxybetulinic acid derivatives were also discussed in the present investigations.

Full text of DOI:10.1016/j.ejmech.2011.03.038

European Journal of Medicinal Chemistry 46 (2011) 2490e2502
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative evaluation of 23-hydroxybetulinic acid derivatives
,
,
Ping Lan a 1, Jiao Wang a 1, Dong-Mei Zhang a, Chang Shu a, Hui-Hui Cao a, Ping-Hua Sun a, Xiao-Ming Wu b,
**
Wen-Cai Ye a , Wei-Min Chen
,
a,
*
a Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, PR China
b College of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on structural modifications of the natural 23-hydroxybetulinic acid, a series of novel its derivatives
had been synthesized. The new compounds were screened for in vitro antiproliferative activity against
cancer cell lines HeLa, MCF-7, HepG2, B16 and A375 using doxorubicin as a reference. The vast majority of
derivatives had exhibited potent tumor growth inhibitory activity than original compound. The deriv-
atives 4, 5, 7, 20, 23, 26, 43 and 44 with IC50 values lower than 10 mM on all tested cell lines were
regarded as the most promising compounds. The structureeactivity relationships of 23-hydroxybetulinic
acid derivatives were also discussed in the present investigations.
Received 12 February 2011
Received in revised form
10 March 2011
Accepted 16 March 2011
Available online 25 March 2011
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
23-Hydroxybetulinic acid
Structural modification
Antiproliferative activity
Structureeactivity relationship
1. Introduction
investigated. A series of 23-hydroxybetulinic acid derivatives
exhibiting improved GP inhibitory potencies were reported by
Lupane triterpenoids such as betulinic acid are prevalent in
natural sources and have various biological activities. Previous
experimental and epidemiological studies have indicated that
betulinic acid as well as its analogues-betulin and 23-hydrox-
ybetulinic acid (Fig. 1) may be developed as potent anti-HIV and
anti-tumor drugs [1e6]. A great deal of investigations on the
structural modifications of betulinic acid and betulin were carried
out, and many derivatives with excellent anti-HIV and anti-tumor
activities have been obtained [3,7e20]. The 3-O-(30,30-dime-
thylsuccinyl)-betulinic acid (DSB) [19,20], one of the most famous
derivative of betulinic acid, is currently under Phase-IIb clinical
evaluation [10]. Nevertheless, there is rare report about the struc-
tural modifications of 23-hydroxybetulinic acid.
The 23-hydroxybetulinic acid, isolated from the root of Pulsa-
tilla chinensis, displayed similar anti-HIV and anti-tumor activities
as betulinic acid [21e24]. Besides the structural similarity and
pharmacological comparability of 23-hydroxybetulinic acid with
betulinic acid and betulin, the glycogen phosphorylase (GP)
inhibitory activity of 23-hydroxybetulinic acid has also been
literature [21]. Furthermore, 23-hydroxybetulinic acid exerted
a synergistic effect on the cytotoxicity of doxorubicin (DOX) in
vitro and in vivo in the recent study [22]. The results indicated that
23-hydroxybetulinic acid had the prospect to be developed as
a novel chemosensitizer. Meanwhile, many attentions have also
been paid to the antiproliferative mechanism of 23-hydrox-
ybetulinic acid. Previous pharmacological studies suggested that
the antiproliferative effect of 23-hydroxybetulinic acid was
mediated by apoptotic induction and down-regulation of pro-
apoptotic gene bcl-2. The telomerase may play a critical role in
23-hydroxybetulinic acid induced apoptosis [23]. This apoptosis
inducing ability along with the apparent lack of toxicity in normal
cells has made 23-hydroxybetulinic acid as a promising and
potential anti-tumor agent [22,23].
Modification of potential plant-derived natural products is
continuing goals of our laboratory. Suitable modification at right
positions of these natural products may yield analogues with
significantly improved potency. The structure of 23-hydrox-
ybetulinic acid consists of a 30-carbon skeleton which has four
available sites for chemical modifications at C-3, C-23, C-17 and
C-19. A set of 17-carboxylic acid modified 23-hydroxybetulinic
acid ester derivatives which exhibited slightly improved anti-
proliferative potencies were reported previously [24]. By utilizing
the structureeactivity relationships obtained by the previous
betulinic acid and betulin modification researches, we conducted
* Corresponding author. Tel.: þ86 20 85224497; fax: þ86 20 85224766.
** Corresponding author. Tel.: þ86 20 85220936; fax: þ86 20 85224766.
E-mail addresses: chywc@yahoo.com.cn (W.-C. Ye), twmchen@jnu.edu.cn
(W.-M. Chen).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.038

P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
2491
2. Results and discussion
19
17
30
2.1. Chemistry
R1
28
Firstly, a series of 23-hydroxybetulinic derivatives bearing the C-17
modified carboxylate or amide have been designed and synthesized.
Furthermore, based onthestructuralavailabilitiesof thesederivatives,
a set of C-30 modified derivatives were also obtained. The general
procedure for the synthesis of derivatives 4e42 was described in
Scheme 1. The 23-hydroxybetulinic acid (1) was isolated by the
laboratory of Prof. Wen-Cai Ye. It was treated with acetic anhydride in
pyridine to yield 3,23-O-diacetyl betulinic acid (2). Compound 2
upon reaction with oxalyl chloride using DMF as a catalyst in CH2Cl2
afforded 3,23-O-diacetyl-17-acyl chloride (3). Upon reaction with
3
HO
Betulinic acid: R1= COOH R2= H
Betulin: R1= CH2OH R2= H
23-Hydroxy betulinic acid: R1= COOH R2= OH
R2
23
Fig. 1. The chemical structures of betulinic acid, betulin and 23-hydroxybetulinic acid.
the synthesis and antiproliferative activity evaluation of novel
23-hydroxybetulinic acid derivatives modified at C-3, C-23,
C-17 and C-19 sites. Based on the observed antiproliferative
data, preliminary structureeactivity relationships have been
established.
1,40-dipiperidine,
4-(5-pyridine-2-tetrazolium-1-)-n-butylamine,
diallylamine, ethylendiamine, morpholine, piperidine, piperazidine,
4-(2-hydroxyethyl) morpholine, cis-2-butene-1,4-diol, isopropyl
alcohol, tert-butyl alcohol, tetrahydrofurfuryl alcohol, isopentyl
OH
OH
Cl
i
ii
O
O
O
HO
AcO
AcO
OH
OAc
OAc
3
1
2
iii
R
4,20
N
N
R
R
N
N
iv
O
O
N
N
5,21
NH
N
AcO
HO
OAc
20-31
4-19
OH
6,22
7,23
N
NH
NH2
v
8,24,36
9,25,37
N
N
O
HO
OH
HO
R
R
10,26
11,27
N
O
NH
N
vi
O
O
O
HO
AcO
N
39-42
OAc
32-38
12,28
O
OH
17,38
N
29
OMe
(Pro-OMe)
OH
13,32,39
14,33,40
O
O
(Pro)
O
O
O
O
H
N
H
N
18
30
31
OMe
(Gly-OMe)
OH
H
O
H
(Gly)
H
O
15,34,41
16,35,42
O
O
H
O
19 NH
NH
OMe
OH
(Met-OMe)
S
(Met)
S
Scheme 1. Synthesis of derivatives 4e42. Reagents and conditions: (i) Ac2O/Pyr, rt, 8 h; (ii) (COCl)2/CH2Cl2/cat.DMF, rt, 4 h; (iii) RNH, RNH2 or ROH/CH2Cl2, rt or reflux, 8e72 h; (iv)
NaOH/THF/CH3OH, reflux, 4 h; (v) m-CPBA/CHCl3, reflux, 6 h; (vi) NaOH/THF/CH3OH, reflux, 4 h.

2492
P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
O
O
R2
H
H
N
N
OMe
OH
N
H
i
O
R1
O
R1
O
HO
HO
OH
OH
43-47
29-31
R1
R2
ii
-H (Gly)
43,48
44,49
-CH3 (Ala)
-(CH2)3- (Pro)
-H (Gly)
O
R2
45,50
(Met)
S
(Met)
H
N
S
OH
N
H
46,51 -(CH2)3- (Pro)
(Met)
(Trp)
S
O
R1
O
HO
-H (Gly)
47,52
OH
48-52
N
H
Scheme 2. Synthesis of derivatives 43e52. Reagents and conditions: (i) EDC$HCl/HoBt/CH2Cl2, reflux, 8e12 h; (ii) NaOH/THF/CH3OH, reflux, 4 h.
acohol,
hydrochloride and
L
-proline methyl ester hydrochloride, glycine methyl ester
inferred that the C-17 region was close to the C-23 position in space,
therefore, once the C-17 was occupied, the 1,40-dipiperidine-1-
carbonyl chloride was unable to attack the C-23 hydroxyl group due
to the steric hindrance. Meanwhile, the excess triphosgene (used to
prepare the 1,40-dipiperidine-1-carbonyl chloride) in the reaction
mixture was activated by nucleophile (pyridine or triethylamine)
and then upon reaction with the C-23 hydroxyl group to afford
3-hydroxy-23-O-carbonochloridate-17-1,40-bipiperidinyl betulinic
amide and finally formed the compound 54. The procedure of this
kind of reaction was illustrated in Fig. 2.
To investigate whether the carboxyl group at the terminal of
the C-17 side chain is essential for the antiproliferative activities,
compound 7 was selected for further modification. We intro-
duced a 3-carboxypropanoyl in the terminal of the C-28 side
chain of compound 7 by treating it with succinic anhydride.
(Scheme 5)
L-methionine methyl ester hydrochloride, yielded
corresponding 3,23-O-diacetyl-17-carboxylate or amide derivatives
4e19. Compounds 4e12 as well as 17e19 were then hydrolyzed to
give corresponding 3,23-dihydroxy-17-carboxylate or amide deriva-
tives 20e28 and 29e31. Meanwhile, compounds 13e17 and 8e9 were
treated with metachloroperbenzoic acid (m-CPBA) to yield subse-
quent 3,23-O-diacetyl-30-hydroxy-17-carboxylate or amide deriva-
tives 32e38. Finally, compounds 32e35 were hydrolyzed to afford
3,23-dihydroxy-30-hydroxy-17-carboxylate derivatives 39e42.
The previous researches have revealed that amino acid conju-
gates of betulinic acid have improved water solubility and were
selectively antiproliferative toward cancer cells [12]. Thus, we have
designed and synthesized a series of 23-hydroxybetulinic acid
derivatives with conjugation of amino acids and dipeptides. The
general procedures for the synthesis of the amino acid conjugates
derivatives 29e31 and the dipeptide conjugates compounds 43e52
were described in Scheme 1 and Scheme 2. The amino acid
2.2. Antiproliferative activity in vitro
conjugates derivatives 29e31 upon reaction with
ester hydrochloride, -proline methyl ester hydrochloride,
-methionin methyl ester hydrochloride and -tryptophane methyl
ester hydrochloride in CH2Cl2 yielded corresponding 3,23-dihy-
droxy-17-dipeptide methyl ester derivatives 43e47. Finally,
compounds 43e47 were hydrolyzed to give corresponding 3,23-
dihydroxy-17-dipiptide derivatives 48e52.
L-alanine methyl
L
To evaluate the anticancer potencies of these newly synthesized
23-hydroxybetulinic acid derivatives, the antiproliferative activities
of compounds 1e55 were tested against five cancer cell lines,
L
L
O
The 23-hydroxybetulinic acid possessed a peculiar hydroxyl
group at C-23 position, while the betulinic acid and betulin have
not such a moiety. It is the first time to report a chemical modifi-
cation at this hydroxyl group. We have introduced a 1,40-dipiper-
idine-1-carbonyl moiety into this C-23 position by following the
successful preparation of irinotecan. 1,40-dipiperidine upon reac-
tion with triposgene in dry CH2Cl2 afforded 1,40-dipiperidine-1-
carbonyl chloride, then it was reacted with 23-hydroxybetulinic
acid to give 3-hydroxy-23-O-(1,40-bipiperidine-1-carbonyl)betu-
linic acid (53) in a high yield (Scheme 3). Meanwhile, since
compound 53 displayed excellent potency, we have also designed
a set of 3-hydroxy-23-O-(1,40-bipiperidine-1-carbonyl)-17-carbox-
ylate or amide derivatives, unfortunately, none of the designed
molecules was obtained. For instance, the compound 20 upon
reaction with 1,40-dipiperidine-1-carbonyl chloride in pyridine
yielded derivative 54, not the designed one (Scheme 4). It can be
i
N
N
NH
Cl
N
OH
OH
ii
O
O
HO
HO
OH
O
N
1
N
O
53
Scheme 3. Synthesis of compound 53. Reagents and conditions: (i) Triphosgene/Et3N/
Pyr, rt, 8 h; (ii) 1,40-dipiperidine-1-carbonyl chloride/Pyr, rt, 16 h.

P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
2493
N
N
N
N
O
O
N
i
HO
HO
OH
20
O
N
ii
O
N
N
O
O
54
O
O
Scheme 4. Synthesis of derivative 54. Reagents and conditions: (i) 1,40-dipiperidine-1-carbonyl chloride/Pyr, rt; (ii) Triphosgene/Et3N/Pyr, rt.
including HepG2 human hepatocellular carcinoma, HeLa human
cervical adenocarcinoma, MCF-7 breast adenocarcinoma, B16 mice
melanoma and A375 human melanoma cells by performing MTT
assay. Doxorubicin was selected as positive control. These results
were summarized in Table 1 and presented as the concentration of
drug inhibiting 50% cell growth (IC50).
tested cancer cell lines. This observation indicated that the acetyl at
C-3 and C-23 positions markedly influenced the antiproliferative
potential, which was in agreement with the previous betulinic acid
researches [7e12]. Compared compound 9 with 10, as well as 25
with 26, it can be easily found that an electron-donating and
hydrophilic substituent such as groups bearing an N atom at the
terminal of C-17 side chain would benefit the potency. This also
contributed to explain why compounds 4e5, 7, 23 as well as 26
bearing one or several N atoms at C-17 side chain, were the most
potential derivatives. The C-17 ester derivatives (11, 12) displayed
improved activities than 23-hydroxybetulinic acid and were found
to be as potent as C-17 amide compounds (4e8). The compounds
43e47 with C-17 dipeptide methyl ester exhibited extremely better
potencies compared to their corresponding C-17 dipeptides deriv-
atives 48e52, suggesting that the carboxyl group at the terminal of
the C-17 side chain was not essential for antiproliferative activity.
To validate this observation, one of the most potential derivative 7
was selected for further investigation, compared compound 7 with
55, it was also found that the 30-N-succinic acid-40-carbonyl
derivative 55 displayed significantly decreased potency. This
observation varied from some previous betulinic acid researches
2.3. Structureeactivity relationship
The present results demonstrate that nearly all synthesized 23-
hydroxybetulinic acid derivatives can markedly inhibit the prolif-
eration of cancer cells. Among them, compounds 4, 5, 7, 20, 23, 26,
43 and 44 with an IC50 around 10 mM on all tested cell lines were
the most promising derivatives. Comparing the activities of all
derivatives with that of 23-hydroxybetulinic acid in each panel, it
can be found that the vast majority of derivatives have shown
a better antiproliferative profile than 23-hydroxybetulinic acid
except 9, 29, 41, 49e52 and 55.
The results also revealed that, 3,23-O-diacetyl derivatives (e.g.
4e8, 11e12, 17 and 32e35) were more potent than corresponding
3,23-dihydroxy compounds (e.g. 20e24, 27e29 and 39e42) in all
Cl
C
Nu
O
Cl
N
N
N
N
O
O
Nu= pyridine
HO
Cl
HO
OH
20
O
O
N
N
O
O
O
54
O
Fig. 2. The reaction mechanism for the synthesis of compound 54.

2494
P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
O
H
H
N
N
OH
NH2
N
i
H
O
O
O
AcO
AcO
OAc
OAc
55
7
Scheme 5. Synthesis of derivative 55. Reagent and condition: (i) Succinic anhydride/DMAP/CH2Cl2, reflux, 10 h.
[12,25] suggesting a carboxyl group at this site would be favorable.
The introduction of the 10,40-dipiperidine-10-carbonyl at C-23
position yielded potent derivative 53 revealing that bulky, hydro-
philic groups at C-23 position would be favored. Since the betulinic
acid and betulin have not such a hydroxyl group at C-23 position,
the hydroxyl group at C-23 site of 23-hydroxybetulinic acid is
worthy of further modification and research. Compared 3,23-
dihydroxy derivative 20 with corresponding 3,23-dioxylcarbonyl
compound 54, it was observed that protection of the C-3 and C-23
with dioxylcarbonyl group may result in decreased activities. The
introduction of the hydroxyl at C-30 position yielded derivatives
with slightly decreased antiproliferative activities (36 and 38)
compared to C-30 unsubstituted compounds (8 and 17). Mean-
while, compared to those 3,23-diacetic-30-hydroxy derivatives,
the 3,23-dihydroxy-30-hydroxy compounds (39e42) exhibited
decreased antiproliferative potencies, indicating that a hydrophilic
group at C-30 position may be unfavorable. In fact, most of the
efforts on introducing various moieties into the C-19 site failed in
yielding potential betulinic acid and betulin derivatives [5,26].
were employed when necessary. For thin layer chromatography
(TLC) analysis Qingdao haiyang GF254 silica gel plates were used.
All NMR spectra were recorded on a Mercury-400 spectrometer
in DMSO-d6, CDCl3, CD3OD or pyridine-d5. The chemical shift
was reported in ppm using TMS as the internal standard. The
coupling constant (J) was presented in hertz (Hz). Electrospray
ionization mass spectra (ESI-MS) were obtained on a Finnigan
LCQ Advantage MAX mass spectrometer (Applied Biosystems,
4000 Q TRAP). High-resolution mass spectra (HR-MS) were
obtained on an Aglient 6210 series LC/MSD TOF mass
spectrometer.
4.1.2. 3,23-O-Diacetyl betulinic acid (2)
To a solution of compound 1 (0.30 g, 0.63 mmol) in dry pyridine
(12 mL) was added acetic anhydride (1 mL, 9.8 mmol), the solution
was stirred at room temperature for 8 h. After reaction completion,
25 mL of ethyl acetate was added. And then the pH of solution was
adjusted to 4e5 by 10% HCl. Then the organic layer was washed by
brine (50 mLꢀ3), dried with anhydrous Na2SO4 for 6 h. Filtered, the
filtrate was concentrated, the residue was dissolved in dichloro-
methane and purified on a silica gel column (200 g, petroleum
ethereethyl acetate 10:1) to give 2 (0.32 g, 91.4%) as white foam. MS
(ESI): m/z 579.5 [M þ Na]þ; HR-ESI-MS: m/z 579.3652 [M þ Na]þ
3. Conclusion
The chemically synthesized anticancer drugs in clinic usually
displayed unacceptable adverse effects and toxicity. Nevertheless,
the naturally occurring 23-hydroxybetulinic acid selectively killed
cancer cells and was non-toxic to normal tissue in animal models.
A large number of 23-hydroxybetulinic acid derivatives as possible
anti-tumor agents were reported. The present study demonstrate
that introduction with proper substituents at C-3, C-23 and C-17
positions of 23-hydroxybetulinic acid can produce various
potentially important derivatives with improved antiproliferative
activity. Derivatives 4, 5, 7, 20, 23, 26, 43 and 44 are potential lead
compounds for the development of novel anti-tumor agent. Based
on the observed antiproliferative evaluation, the structur-
eeactivity relationship of these derivatives has been established.
In detail, the acetyl groups at both C-3 and C-23 positions would
be favorable; a bulky, electron-donating and hydrophilic moiety at
C-23 site may benefit the potency; a carboxyl group at the
terminal of the C-17 side chain was not essential for the anti-
proliferative activity; the C-19 position was not eligible for
a successful modification; modification at the C-17 site with
suitable carboxylate or amide can produce potent derivatives. The
results taken from present study can be served as a valuable
guideline for further modification of 23-hydroxybetulinic acid
with improved biological response.
(calcd: 579.3656); 1H NMR (CDCl3, 400 MHz)
d: 0.80, 0.88, 0.93,
0.97 (each 3H, s, Me-24, -25, -26, -27), 1.69 (3H, s, Me-30), 1.79 (1H,
m, H-18), 1.95 (1H, m, H-19), 2.01 (3H, s, 3-OAc), 2.06 (3H, s, 23-
OAc), 3.02 (1H, m, H-3), 3.68 (1H, d, J ¼ 11.6 Hz, H-23
J ¼ 11.6 Hz, H-23 ), 4.61 (1H, s, H-29 ), 4.77 (1H, s, H-29
(CDCl3, 100 MHz) : 12.8, 14.5, 16.0, 16.5, 17.9, 19.3, 20.9, 21.1, 23.1,
a
), 3.83 (1H, d,
b
b
a
); 13C NMR
d
29.6, 36.9, 38.0, 38.3, 40.6, 40.6, 42.3, 46.9, 48.0, 49.2, 50.5, 56.3,
65.4, 74.5, 109.7, 150.3, 170.6, 171.0, 181.5.
4.1.3. 3,23-O-Diacetyl-17-acyl chloride (3)
To a solution of compound 2 (0.5 g, 0.87 mmol) in dry
dichloromethane (30 mL) was added oxalyl chloride (0.5 mL,
2.65 mmol) and dry DMF (0.1 mL). The solution was stirred at room
temperature for 4 h. After quenching the reaction, the solution was
concentrated to give compound 3 (about 0.5 g) as yellow foam.
4.1.4. 3,23-O-Diacetyl-17-1,40-bipiperidinyl betulinic amide (4)
To a solution of compound 3 (0.38 g, 0.66 mmol) in dry
dichloromethane (40 mL) was added 1,40-dipiperidine (0.66 g,
4.0 mmol). The solution was stirred and refluxed for 10 h. At which
time the reaction was completed, the solution was concentrated,
the residue was dissolved in ethyl acetate (40 mL) and 10% HCl
(40 mL), the organic layer was separated and washed by brine
(50 mL), dried with anhydrous Na2SO4. Filtered, the filtrate was
concentrated, the residue was dissolved in methanol and purified
on a silica gel column (200 g, ethyl acetate-methanol 3:1) to give 4
(0.40 g, 85.0%) as white foam. MS (ESI): m/z 708.6 [M þ H]þ; 1H
4. Experimental
4.1. Chemistry
4.1.1. General
NMR (CD3OD, 400 MHz) d: 0.84, 0.94, 0.97, 1.02 (each 3H, s, Me-24,
All reagents and solvents were purchased from commercial
sources. Further purification and drying by standard methods
-25, -26, and -27), 1.70 (3H, s, Me-30), 2.02 (1H, m, H-18), 1.95
(1H, m, H-19), 2.01 (3H, s, 3-OAc), 2.04 (3H, s, 23-OAc), 2.18

P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
2495
Table 1
Antiproliferative activities of compounds 1e55 against HeLa, MCF-7, HepG2, B16 and A375 cells.
a
Compounds
IC50
(m
M ꢁ SD)
HeLa
MCF-7
HepG2
B16
A375
1
4
5
6
7
8
9
46.22 ꢁ 1.34
10.80 ꢁ 1.06
17.87 ꢁ 0.28
9.84 ꢁ 0.32
7.39 ꢁ 0.56
7.47 ꢁ 0.65
69.20 ꢁ 6.47
18.84 ꢁ 1.61
9.18 ꢁ 0.28
16.35 ꢁ 1.67
ntb
46.61 ꢁ 1.69
6.86 ꢁ 0.73
16.10 ꢁ 1.10
47.10 ꢁ 1.07
3.93 ꢁ 0.60
26.44 ꢁ 0.88
>100
4.89 ꢁ 0.63
97.57 ꢁ 8.75
15.91 ꢁ 0.17
nt
41.41 ꢁ 2.94
11.14 ꢁ 0.68
3.91 ꢁ 0.57
8.41 ꢁ 0.71
7.99 ꢁ 0.77
9.74 ꢁ 0.79
66.34 ꢁ 6.04
17.65 ꢁ 0.72
12.28 ꢁ 0.42
7.43 ꢁ 0.80
nt
18.68 ꢁ 1.24
9.57 ꢁ 0.85
4.75 ꢁ 0.29
>100
4.61 ꢁ 0.32
18.48 ꢁ 1.64
>100
13.07 ꢁ 1.23
24.21 ꢁ 2.69
>100
41.99 ꢁ 4.86
8.42 ꢁ 0.97
1.81 ꢁ 0.43
19.04 ꢁ 0.89
9.99 ꢁ 0.27
4.33 ꢁ 0.35
>100
18.79 ꢁ 1.69
8.71 ꢁ 0.93
5.72 ꢁ 0.76
nt
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
DOX
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
9.78 ꢁ 0.18
27.22 ꢁ 2.15
9.78 ꢁ 0.97
93.67 ꢁ 6.70
5.83 ꢁ 0.53
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
8.42 ꢁ 0.45
46.26 ꢁ 3.65
59.01 ꢁ 5.57
8.92 ꢁ 0.74
34.29 ꢁ 1.16
66.45 ꢁ 2.97
7.12 ꢁ 1.27
>100
6.78 ꢁ 0.85
17.68 ꢁ 0.97
17.70 ꢁ 0.54
2.93 ꢁ 0.32
19.84 ꢁ 1.51
16.63 ꢁ 1.16
2.27 ꢁ 0.35
34.30 ꢁ 0.67
36.03 ꢁ 0.80
>100
12.64 ꢁ 1.50
48.27 ꢁ 4.83
59.77 ꢁ 5.44
9.57 ꢁ 0.12
41.97 ꢁ 1.45
68.60 ꢁ 3.82
8.85 ꢁ 0.42
59.21 ꢁ 5.29
38.00 ꢁ 2.98
72.21 ꢁ 5.64
nt
5.71 ꢁ 0.26
66.91 ꢁ 3.46
60.40 ꢁ 6.44
8.38 ꢁ 0.81
42.70 ꢁ 3.03
43.66 ꢁ 2.02
4.39 ꢁ 0.46
45.85 ꢁ 3.34
19.08 ꢁ 1.64
87.34 ꢁ 3.44
nt
9.67 ꢁ 0.67
30.58 ꢁ 1.02
17.18 ꢁ 0.52
4.83 ꢁ 0.26
25.06 ꢁ 2.64
17.88 ꢁ 0.80
2.30 ꢁ 0.19
34.24 ꢁ 3.93
55.69 ꢁ 4.74
>100
44.44 ꢁ 4.12
95.61 ꢁ 3.79
nt
nt
nt
nt
nt
nt
nt
nt
19.46 ꢁ 1.39
23.28 ꢁ 1.84
17.79 ꢁ 1.72
20.84 ꢁ 1.21
18.31 ꢁ 1.23
11.02 ꢁ 1.27
13.08 ꢁ 1.01
22.27 ꢁ 1.46
30.65 ꢁ 0.82
79.35 ꢁ 5.95
20.85 ꢁ 1.40
8.27 ꢁ 0.91
4.84 ꢁ 0.51
44.39 ꢁ 3.95
41.26 ꢁ 3.19
66.33 ꢁ 6.64
22.10 ꢁ 1.74
>100
17.21 ꢁ 0.96
14.12 ꢁ 1.92
33.65 ꢁ 0.87
15.10 ꢁ 1.49
17.32 ꢁ 0.55
22.20 ꢁ 2.10
31.98 ꢁ 1.37
16.57 ꢁ 1.22
16.40 ꢁ 0.62
76.30 ꢁ 8.44
12.53 ꢁ 1.76
8.95 ꢁ 0.20
7.58 ꢁ 0.62
66.09 ꢁ 4.30
19.77 ꢁ 1.56
>100
10.71 ꢁ 1.34
15.75 ꢁ 1.62
18.65 ꢁ 2.54
10.72 ꢁ 0.66
20.68 ꢁ 1.63
11.27 ꢁ 1.11
10.00 ꢁ 1.18
20.98 ꢁ 1.80
22.68 ꢁ 1.64
81.23 ꢁ 6.97
34.62 ꢁ 1.87
12.09 ꢁ 1.01
9.33 ꢁ 0.69
23.05 ꢁ 1.73
40.46 ꢁ 2.27
46.79 ꢁ 4.34
32.36 ꢁ 2.20
>100
23.65 ꢁ 1.63
49.45 ꢁ 1.29
41.80 ꢁ 3.89
35.88 ꢁ 1.27
39.27 ꢁ 3.94
64.26 ꢁ 5.52
68.97 ꢁ 2.73
38.57 ꢁ 0.99
84.08 ꢁ 8.31
64.35 ꢁ 0.35
51.20 ꢁ 4.30
11.31 ꢁ 1.14
8.23 ꢁ 0.87
20.39 ꢁ 1.89
20.73 ꢁ 0.44
44.35 ꢁ 2.75
22.25 ꢁ 0.82
>100
65.09 ꢁ 3.47
76.46 ꢁ 0.31
>100
7.25 ꢁ 1.25
8.69 ꢁ 1.53
82.57 ꢁ 3.37
0.14 ꢁ 0.02
21.54 ꢁ 1.54
34.72 ꢁ 2.75
12.47 ꢁ 1.54
7.80 ꢁ 0.82
31.84 ꢁ 1.17
53.74 ꢁ 4.57
11.75 ꢁ 1.56
21.20 ꢁ 2.16
42.71 ꢁ 3.58
70.37 ꢁ 5.13
17.94 ꢁ 1.29
5.01 ꢁ 0.60
3.34 ꢁ 0.37
26.80 ꢁ 0.71
30.14 ꢁ 2.60
>100
17.25 ꢁ 1.31
18.38 ꢁ 0.41
>100
>100
>100
84.72 ꢁ 6.72
41.30 ꢁ 1.51
>100
9.95 ꢁ 0.98
17.56 ꢁ 0.52
51.00 ꢁ 2.14
0.19 ꢁ 0.02
>100
89.30 ꢁ 0.98
66.50 ꢁ 5.28
>100
9.23 ꢁ 0.67
20.41 ꢁ 1.06
59.53 ꢁ 4.83
0.13 ꢁ 0.01
81.13 ꢁ 8.31
91.70 ꢁ 6.82
>100
>100
12.52 ꢁ 1.85
20.55 ꢁ 2.06
72.90 ꢁ 6.68
0.13 ꢁ 0.02
18.54 ꢁ 1.36
10.28 ꢁ 0.96
76.09 ꢁ 3.57
0.19 ꢁ 0.02
Antiproliferative activity was determined by MTT assay as shown in Experimental 4.2 part. All data are presented as means ꢁ standard deviation of at least three independent
experiments.
DOX: Doxorubicin.
a
IC50: concentration of the tested compound that inhibits 50% of cell growth.
nt: not test.
b
(4H, m, H-200, 600), 2.90 (1H, m, H-3), 2.95 (4H, m, H-20, 60), 2.91
(1H, d, J ¼ 11.8 Hz, H-23 ), 3.81 (1H, d, J ¼ 11.8 Hz, H-23 ), 4.59
(1H, s, H-29 ), 4.72 (1H, s, H-29
); 13C NMR (CD3OD, 100 MHz)
18.0, 18.6, 20.3, 21.1, 22.1, 22.5, 23.7, 24.2, 25.4, 25.7, 28.2, 29.3,
29.6, 32.4, 33.7, 43.7, 36.5, 38.3, 39.5, 39.7, 40.6, 43.2, 43.3, 44.4,
48.6, 49.7, 50.8, 51.0, 52.6, 53.6, 55.2, 57.4, 66.1, 67.5, 77.2, 80.9,
111.3, 153.8, 173.9, 173.9, 176.9.
4.1.5. 3,23-O-Diacetyl-17-[4-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)-n-
butyl]betulinic amide (5)
a
b
a
b
d
:
White foam, yield: 87.0%. MS (ESI): m/z 758.3 [M þ H]þ, 780.1
[M þ Na]þ; 1H NMR (CDCl3, 400 MHz)
d: 0.62, 0.84, 0.88, 1.27 (each
3H, s, Me-24, -25, -26, and-27),1.43 (3H, s, Me-30),1.78 (1H, m, H-18),
2.14 (1H, m, H-19), 2.26 (6H, s, 3,23-OAc), 3.00 (1H, m, H-3), 3.62 (1H,
m, H-20
J ¼ 8.0 Hz, H-23
5.29 (1H, s, H-29
a
), 3.64 (1H, m, H-20
b
), 4.07 (1H, m, H-50
a
), 4.54 (1H, d,
), 5.02 (1H, m, H-50
),
b
), 7.42 (1H, m, H-140), 7.54 (1H,
The compounds 5e10 were obtained by using the similar
synthetic procedure of compound 4.
a
a
), 4.59 (1H, d, J ¼ 8.0 Hz, H-23
b
b
), 5.50 (1H, s, H-29

2496
P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
m, H-150), 8.01 (2H, m, H-160, 130); 13C NMR (CDCl3, 100 MHz)
13.7, 14.7, 15.7, 19.8, 20.7, 21.0, 22.5, 32.4, 33.1, 34.1, 35.1, 40.7, 45.8,
49.9, 51.1, 52.0, 63.5, 69.3, 70.4, 71.9, 73.2, 75.5, 79.7, 82.5,101.4,128.3,
129.6, 129.7, 130.4, 132.3, 132.8, 138.4, 163.2, 165.1, 169.5.
d:10.2,
(0.1 mL, 5.22mmol). The solutionwasstirredatroom temperaturefor
8 h. At which time the reaction was completed, the solution was
washed by water (40 mL) and brine (40 mL), dried with anhydrous
Na2SO4. Filtered, the filtrate was concentrated, the residue was dis-
solved in dichloromethane and purified on a silica gel column (200 g,
petroleum ethereethyl acetate 1:1) to give 11 (0.18 g, 51.4%) as white
foam. MS(ESI): m/z 670.8 [M þ H]þ;692.9 [Mþ Na]þ; 1H NMR (CDCl3,
4.1.6. 3, 23-O-Diacetyl-17-diallyl betulinic amide (6)
White foam, yield: 91.0%. MS (ESI): m/z 536.4 [M þ H]þ, 658.5
[M þ Na]þ; 1H NMR (CDCl3, 400 MHz)
d
: 0.80, 0.88, 0.94, 0.95 (each
400 MHz) d: 0.80, 0.87, 0.92, 0.96 (each 3H, s, Me-24, -25, -26, and
3H, s, Me-24, -25, -26, and -27), 1.68 (3H, s, Me-30), 1.83 (1H, m,
H-18), 1.96 (1H, m, H-19), 2.01 (3H, s, 3-OAc), 2.06 (3H, s, 23-OAc),
-27), 1.68 (3H, s, Me-30), 1.90 (1H, m, H-18), 1.98 (1H, m, H-19), 2.01
(3H, s, 3-OAc), 2.06 (3H, s, 23-OAc), 2.51 (4H, m, H-40, 80), 2.67 (2H, t,
H-20), 3.01 (1H, m, H-3), 3.70 (4H, m, H-50, 70), 3.72 (1H, d, J ¼ 11.6 Hz,
3.04 (1H, m, H-3), 3.67 (1H, d, J ¼ 11.6 Hz, H-23
J ¼ 11.6 Hz, H-23
), 3.87 (2H, m, H-20), 4.09 (2H, m, H-50), 4.58 (1H, s,
H-29 ), 4.76 (1H, s, H-29
), 5.15 (4H, m, H-40, 70), 5.74 (2H, m, H-30,
60); 13C NMR (CDCl3,100 MHz)
: 12.8,14.6,16.0,16.6,17.9, 20.9, 21.2,
a), 3.86 (1H, d,
b
H-23
a
), 3.83 (1H, d, J ¼ 11.6 Hz, H-23
b
), 4.21 (2H, t, H-10), 4.60 (1H, s,
a
b
H-29a
), 4.74 (1H, s, H-29b d: 12.8, 14.5,
); 13C NMR (CDCl3, 100 MHz)
d
16.0, 16.5, 17.8, 19.2, 20.9, 21.2, 29.5, 36.9, 36.9, 38.1, 40.5, 40.6, 42.3,
46.9, 47.9, 49.2, 50.5, 53.7, 56.4, 57.2, 60.7, 65.3, 66.9, 74.4,109.6,150.4,
170.6, 171.0, 175.8.
23.1, 25.6, 29.8, 31.4, 32.2, 34.0, 36.8, 37.0, 40.6, 40.7, 41.9, 45.6, 48.1,
50.9, 52.9, 54.8, 65.4, 74.5, 109.0, 133.6, 151.4, 170.6, 171.0, 174.4.
Compounds 12e16 were obtained by using the similar synthetic
procedure of compound 11.
4.1.7. 3,23-O-Diacetyl-17-(2-aminoethyl)betulinic amide (7)
White foam, yield: 76.9%. MS (ESI): m/z 599.8 [M þ H]þ; 1H NMR
(CDCl3, 400 MHz)
d
: 0.74, 0.83, 0.87, 0.97 (each 3H, s, Me-24, -25,
4.1.12. 3,23-O-Diacetyl-17-(E-4-hydroxybut-2-enyl)betulinic ester
(12)
-26, and -27), 1.68 (3H, s, Me-30), 1.97 (1H, m, H-18), 2.01 (3H, s,
3-OAc), 2.06 (3H, s, 23-OAc), 2.39 (1H, m, H-19), 2.44 (2H, m, H-30),
White foam, yield: 71.9%. MS (ESI): m/z 649.6 [M þ Na]þ; 1H
3.06 (1H, m, H-3), 3.24 (2H, t, H-20), 3.68 (1H, d, J ¼ 11.5 Hz, H-23
a
),
NMR (CDCl3, 400 MHz) d: 0.81, 0.88, 0.91, 0.96 (each 3H, s, Me-24,
3.84 (1H, d, J ¼ 11.5 Hz, H-23
b
), 4.60 (1H, s, H-29
a
), 4.75 (1H, s,
-25, -26, and -27),1.68 (3H, s, Me-30),1.88 (1H, m, H-18), 2.01 (3H, s,
3-OAc), 2.06 (3H, s, 23-OAc), 2.22 (1H, m, H-19), 2.98 (1H, m, H-3),
H-29b d: 12.8, 13.0,
), 5.39 (1H, s, H-10); 13C NMR (CDCl3, 100 MHz)
14.5, 15.8, 16.1, 16.5, 17.9, 19.3, 20.8, 21.1, 23.1, 23.5, 30.6, 36.9, 38.0,
39.3, 40.5, 40.5, 40.7, 42.4, 46.8, 48.0, 50.6, 55.6, 65.4, 74.4, 109.3,
150.6, 170.6, 170.9, 171.0.
3.69 (1H, d, J ¼ 11.6 Hz, H-23
(2H, m, H-40), 4.60 (1H, s, H-29
H-10), 5.63 (1H, m, H-20), 5.88 (1H, m, H-30); 13C NMR (CDCl3,
100 MHz) : 12.8, 14.5, 15.9, 16.5, 19.3, 20.8, 21.1, 23.0, 29.5, 30.5,
a
), 3.83 (1H, d, J ¼ 11.6 Hz, H-23
b), 4.28
a
), 4.73 (1H, s, H-29 ), 4.77 (2H, m,
b
d
4.1.8. 3,23-O-Diacetyl-17-morpholinyl betulinic amide (8)
33.9, 36.9, 38.1, 40.5, 40.7, 42.3, 46.8, 48.0, 49.4, 50.5, 56.5, 58.4,
59.3, 65.4, 74.4, 109.6, 125.8, 133.3, 150.3, 170.5, 170.9, 176.1.
White foam, yield: 83.3%. MS (ESI): m/z 627.0 [M þ H]þ; 649.0
[M þ Na]þ; 1H NMR (CDCl3, 400 MHz)
d: 0.82, 0.89, 0.95, 0.97 (each
3H, s, Me-24, -25, -26, and -27), 1.69 (3H, s, Me-30), 1.95 (1H, m,
H-18), 2.02 (3H, s, 3-OAc), 2.07 (3H, s, 23-OAc), 2.10 (1H, m, H-19),
2.99 (1H, m, H-3), 3.66 (8H, m, H-20, 30, 50, 60), 3.70 (1H, d, J ¼ 11.6 Hz,
4.1.13. 3,23-O-Diacetyl-17-isopropyl betulinic ester (13)
White foam, yield: 77.4%. MS (ESI): m/z 599.9 [M þ H]þ, 621.9
[M þ Na]þ; 1H NMR (CDCl3, 400 MHz)
d: 0.81, 0.88, 0.93, 0.97 (each
H-23
a
), 3.85 (1H, d, J ¼ 11.6 Hz, H-23
b
), 4.60 (1H, s, H-29
a
), 4.74 (1H,
3H, s, Me-24, -25, -26, and -27), 1.23 (3H, d, J ¼ 6.2 Hz, Me-20), 1.24
(3H, d, J ¼ 6.2 Hz, Me-30), 1.68 (3H, s, Me-30), 1.81 (1H, m, H-18),
2.02 (3H, s, 3-OAc), 2.07 (3H, s, 23-OAc), 2.22 (1H, m, H-19), 3.03
s, H-29
b
); 13C NMR (CDCl3, 100 MHz)
d: 12.7, 14.4, 15.9, 16.5, 17.8,
20.7, 21.0, 29.6, 31.1, 33.9, 36.7, 36.9, 37.9, 40.4, 40.5, 41.7, 45.5, 48.0,
50.7, 52.5, 54.3, 66.8, 74.4, 109.1, 151.0, 170.5, 170.8, 173.5.
(1H, m, H-3), 3.70 (1H, d, J ¼ 11.6 Hz, H-23
H-23 ), 4.60 (1H, s, H-29 ), 4.75 (1H, s, H-29
13C NMR (CDCl3, 100 MHz)
: 12.7, 14.4, 15.8, 16.4, 17.7, 19.2, 20.7,
a
), 3.83 (1H, d, J ¼ 11.6 Hz,
b
a
b
), 5.01 (1H, m, H-10);
4.1.9. 3,23-O-Diacetyl-28-piperidinyl betulinic amide (9)
d
White foam, yield: 74.0%. MS (ESI): m/z 646.6 [M þ Na]þ; 1H
21.0, 21.6, 23.0, 30.5, 31.9, 36.8, 38.1, 40.4, 40.6, 42.2, 46.9, 47.9, 49.2,
50.4, 56.1, 65.3, 66.6, 74.4, 109.3, 150.5, 170.5, 170.9, 175.4.
NMR (CDCl3, 400 MHz) d: 0.85, 0.94, 0.95, 0.99 (each 3H, s, Me-24,
-25, -26, and -27),1.70 (3H, s, Me-30),1.95 (1H, m, H-18), 2.01 (3H, s,
3-OAc), 2.06 (3H, s, 23-OAc), 2.10 (1H, m, H-19), 3.04 (1H, m, H-3),
4.1.14. 3,23-O-Diacetyl-28-tert-butyl betulinic ester (14)
3.48 (4H, m, H-20, 60), 3.67 (1H, d, J ¼ 11.5 Hz, H-23
a
), 3.83 (1H, d,
b
White foam, yield: 92.7%. MS (ESI): m/z 613.9 [M þ H]þ, 635.9
J ¼ 11.5 Hz, H-23
b
), 4.57 (1H, s, H-29
a
), 4.76 (1H, s, H-29
); 13C NMR
[M þ Na]þ; 1H NMR (CDCl3, 400 MHz)
d: 0.80, 0.87, 0.94, 0.95 (each
(CDCl3, 100 MHz)
d: 12.8, 14.5, 16.0, 16.6, 20.9, 21.1, 21.2, 23.1, 25.5,
3H, s, Me-24, -25, -26, and -27), 1.45 (9H, s, Me-20, -30, -40), 1.68 (3H,
s, Me-30), 1.81 (1H, m, H-18), 2.01 (3H, s, 3-OAc), 2.06 (3H, s, 23-
OAc), 2.22 (1H, m, H-19), 3.03 (1H, m, H-3), 3.68 (1H, d, J ¼ 11.6 Hz,
26.1, 29.8, 31.3, 32.4, 36.8, 36.9, 37.9, 40.5, 40.6, 41.8, 45.7, 48.0, 50.8,
52.7, 54.6, 65.4, 74.5, 109.0, 151.5, 170.6, 171.0, 173.1.
H-23
a
), 3.83 (1H, d, J ¼ 11.6 Hz, H-23
b), 4.59 (1H, s, H-29
a), 4.78
4.1.10. 3,23-O-Diacetyl-28-piperizinyl betulinic amide (10)
(1H, s, H-29
b
); 13C NMR (CDCl3, 100 MHz)
d
: 12.8, 14.5, 15.9, 16.5,
White foam, yield: 74.1%. MS (ESI): m/z 625.8 [M þ H]þ; 1H NMR
19.4, 20.9, 21.1, 23.1, 28.0, 30.7, 32.3, 36.9, 38.0, 38.2, 40.5, 40.7, 42.4,
47.0, 48.0, 49.2, 50.6, 56.8, 65.4, 74.5, 109.3, 150.8, 170.6,170.9,175.4.
(CDCl3, 400 MHz) d: 0.81, 0.88, 0.94, 0.95 (each 3H, s, Me-24, -25, -26,
and -27), 1.68 (3H, s, Me-30), 1.95 (1H, m, H-18), 2.01 (3H, s, 3-OAc),
2.06 (3H, s, 23-OAc), 2.12 (1H, m, H-19), 2.48 (4H, m, H-30, 50), 2.89
4.1.15. 3,23-O-Diacetyl-17-(tetrahydrofufuran-2-yl)methyl
betulinic ester (15)
(1H, m, H-3), 3.60 (4H, m, H-20, 60), 3.68 (1H, d, J ¼ 11.6 Hz, H-23
a),
3.84 (1H, d, J ¼ 11.6 Hz, H-23
b
), 4.58 (1H, s, H-29
a
), 4.72 (1H, s,
White foam, yield: 80.6%. MS (ESI): m/z 641.9 [M þ H]þ; 663.9
H-29
b
); 13C NMR (CDCl3, 100 MHz)
d: 12.8, 14.5, 16.1, 16.6, 17.9, 19.6,
[M þ Na]þ; 1H NMR (CDCl3, 400 MHz)
d: 0.80, 0.87, 0.92, 0.96 (each
3H, s, Me-24, -25, –26, and -27), 1.68 (3H, s, Me-30), 1.81 (1H, m,
20.9, 21.2, 23.1, 29.7, 31.3, 34.0, 36.9, 37.0, 38.0, 40.6, 40.7, 41.8, 45.7,
48.1, 50.9, 52.7, 54.5, 65.5, 74.5, 109.1, 151.3, 170.6, 170.9, 173.5.
H-18),1.90 (4H, m, H-30, 40), 2.01 (3H, s, 3-OAc), 2.06 (3H, s, 23-OAc),
2.22 (1H, m, H-19), 3.03 (1H, m, H-3), 3.68 (1H, d, J ¼ 11.6 Hz, H-23
a
b
),
),
4.1.11. 3,23-O-Diacetyl-17-(2-morpholinylethyl)betulinic ester (11)
To a solution of compound 3 (0.30 g, 0.52 mmol) in dry
dichloromethane (30 mL) wasadded4-(2-hydroxyethyl) morpholine
3.88 (2H, m, H-50), 4.04 (1H, m, H-20), 4.12 (1H, d, J ¼ 11.6 Hz, H-23
4.16 (2H, m, H-10), 4.60 (1H, s, H-29
a), 4.73 (1H, s, H-29
b
); 13C NMR
(CDCl3, 100 MHz) d: 12.8, 14.5, 15.9, 16.5, 17.9, 19.3, 20.9, 20.9, 21.1,

P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
2497
23.1, 25.5, 25.6, 28.1, 30.6, 36.9, 38.0, 40.6, 40.7, 42.3, 48.0, 50.6, 56.5,
65.4, 65.7, 68.3, 74.5, 109.5, 150.5, 170.6, 170.9, 175.9.
(CDCl3, 100 MHz) d: 12.6, 15.1, 15.3, 16.1, 20.4, 20.4, 20.7, 23.1, 25.1,
29.5, 30.2, 32.5, 36.2, 38.9, 40.0, 41.4, 41.5, 46.0, 47.2, 51.3, 51.9, 55.3,
64.9, 74.0, 122.7, 143.2, 170.1, 170.4, 172.0, 177.3.
4.1.16. 3,23-O-Diacetyl-17-isopentyl betulinic ester (16)
White foam, yield: 63.6%. MS (ESI): m/z 543.8 [M þ H]þ, 565.8
4.1.20. 3,23-Dihydroxy-17-1,40-bipiperidinyl betulinic amide (20)
To a solution of compound 4 (0.08 g, 0.11 mmol) in meth-
anol:THF (2:5 mL) was added a NaOH solution (4 mol/L, 2 mL). The
solution was stirred and refluxed for 4 h. At which time the reaction
was completed, the pH of the solution was adjusted to 4e5 by 10%
HCl, filtered, the filter cake was collected and purified on a silica gel
column (200 g, 1:1 chloroform-methanol) to give 20 (0.058 g,
[M þ Na]þ; 1H NMR (CDCl3, 400 MHz)
d: 0.80, 0.87, 0.91, 0.92, 0.94,
0.96 (each 3H, s, Me-24, -25, -26, -27, -40, -50), 1.68 (2H, m, H-20),
1.70 (3H, s, Me-30), 1.86 (1H, m, H-18), 2.01 (3H, s, 3-OAc), 2.06 (3H,
s, 23-OAc), 2.24 (1H, m, H-19), 3.03 (1H, m, H-3), 3.68 (1H, d,
J ¼ 11.6 Hz, H-23
H-10), 4.60 (1H, s, H-29
100 MHz) : 12.8, 14.5, 15.9, 16.5, 19.3, 20.9, 21.2, 22.3, 22.4, 23.0,
a
), 3.83 (1H, d, J ¼ 11.6 Hz, H-23
b), 4.07 (2H, m,
a
), 4.75 (1H, s, H-29
b
); 13C NMR (CDCl3,
d
82.0%) as white foam. MS (ESI): m/z 624.4 [M þ H]þ; HR-ESI-MS:
þ
25.1, 36.9, 37.4, 38.2, 40.5, 40.6, 42.3, 46.9, 47.9, 49.3, 50.5, 56.4, 62.4,
65.3, 74.4, 109.5, 150.5, 170.6, 171.0, 176.1.
m/z 623.5160 [M þ H]
(calcd: 623.5146); 1H NMR (CD3OD,
400 MHz) d: 0.66, 0.88, 0.94, 1.00 (each 3H, s, Me-24, -25, -26, and
-27), 1.68 (3H, s, Me-30), 2.02 (1H, m, H-18), 2.15 (1H, m, H-19), 2.18
4.1.17. N-(3,23-O-Diacetyl-17-betulinic acyl)-L-proline methyl ester
(4H, m, H-200, 600), 2.83 (4H, m, H-20, 60), 2.99 (1H, m, H-3), 3.26(1H,
(17)
d, J ¼ 10.9 Hz, H-23
a
), 3.50 (1H, d, J ¼ 10.9 Hz, H-23
b
), 4.57 (1H, s,
: 13.7,
To a solution of compound 3 (0.30 g, 0.52 mmol) in dry
dichloromethane (20 mL) was added -proline methyl ester
H-29 ), 4.64 (1H, s, H-29
a
b
); 13C NMR (CD3OD, 100 MHz)
d
L
16.3, 17.9, 18.3, 25.6, 39.3, 43.1, 44.3, 44.6, 49.5, 49.8, 50.0, 50.4, 50.5,
50.6, 50.7, 50.8, 52.5, 53.4, 57.3, 66.1, 68.7, 75.1, 111.1, 153.6, 177.0.
Compounds 21e28 were obtained by using the similar synthetic
procedure of compound 20.
hydrochloride (0.26 g, 1.56 mmol), DMAP (0.14 g, 1.0 mmol) and dry
triethylamine (0.1 mL). The solution was stirred and refluxed for
24 h. At which time the reaction was completed, the solution was
concentrated, the residue was dissolved in ethyl acetate (30 mL)
and washed by 10% HCl (20 mL) and brine (30 mL), dried with
anhydrous Na2SO4. Filtered, the filtrate was concentrated, the
residue was dissolved in dichloromethane and purified on a silica
gel column (200 g, petroleum ethereethyl acetate 10:1) to give 17
(0.26 g, 74.3.0%) as white foam. MS (ESI): m/z 690.6 [M þ Na]þ; HR-
ESI-MS: m/z 690.4336 [M þ Na]þ (calcd: 690.4340); 1H NMR (CDCl3,
4.1.21. 3,23-Dihydroxy-17-[4-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)-
n-butyl]betulinic amide (21)
White foam, yield: 80.0%. MS (ESI): m/z 673.6 [M þ H]þ; HR-ESI-
þ
MS: 643.7814 [M þ H]
(calcd: 643.7800); 1H NMR (CD3OD,
400 MHz)
-27), 2.00 (3H, s, Me-30), 2.11 (1H, m, H-18), 2.17 (1H, m, H-19), 3.21
(1H, m, H-3), 3.49 (1H, d, J ¼ 10.8 Hz, H-23 ),
), 3.58 (1H, m, H-20
3.64 (1H, m, H-20 ), 3.82 (1H, m, H-50 ), 4.81 (1H, m, H-50
), 4.92
), 5.47 (1H, s, H-29 ),
d: 0.63, 0.78, 0.79, 1.09 (each 3H, s, Me-24, -25, -26, and
400 MHz)
d
: 0.78, 0.84, 0.90, 0.94 (each 3H, s, Me-24, -25, -26, and
a
a
-27), 1.44 (4H, m, H-30, 40), 1.66 (3H, s, Me-30), 1.91 (1H, m, H-18),
1.99 (3H, s, 3-OAc), 2.04 (3H, s, 23-OAc), 2.31 (1H, m, H-19), 3.00
b
a
b
(1H, d, J ¼ 10.8 Hz, H-23
b), 4.83 (1H, s, H-29
a
b
(1H, m, H-3), 3.53 (1H, m, H-20
a
), 3.65 (1H, d, J ¼ 11.6 Hz, H-23
3.68 (3H, s, OMe-60), 3.75 (1H, m, H-20
), 3.82 (1H, d, J ¼ 11.6 Hz, H-
23 ), 4.69 (1H, s, H-29
), 4.44 (1H, t, H-50), 4.55 (1H, s, H-29 ); 13
NMR (CDCl3, 100 MHz) : 14.9, 16.6, 18.2, 18.6, 20.0, 21.8, 22.9, 23.0,
a
),
8.18 (1H, m, H-140), 8.96 (1H, m, H-150), 9.12 (1H, d, J ¼ 8.1 Hz, H-
b
160), 9.51 (1H, s, H-130); 13C NMR (CD3OD, 100 MHz)
d: 5.0, 6.6, 8.5,
b
a
b
C
9.9, 16.4, 20.0, 26.7, 30.5, 33.6, 34.1, 35.8, 40.8, 41.0, 41.1, 41.2, 41.4,
41.7, 41.9, 42.1, 45.0, 59.6, 66.0, 92.5, 121.2, 121.5, 134.2, 134.5, 136.0,
136.3, 137.7, 154.0, 177.0.
d
23.2, 25.1, 27.7, 36.1, 39.0, 40.0, 42.6, 42.7, 44.1, 47.9, 50.1, 52.8, 54.0,
57.3, 62.2, 67.5, 76.6, 110.9, 153.5, 172.6, 173.0, 175.5, 176.1.
Compounds 18 and 19 were obtained by using the similar
synthetic procedure of compound 17.
4.1.22. 3,23-Dihydroxy-17-diallyl betulinic amide (22)
White foam, yield: 85.7%. MS (ESI): m/z 552.4 [M þ H]þ; HR-ESI-
MS: m/z 574.4219 [M þ Na]þ (cacld: 574.4231); 1H NMR (pyridine-
4.1.18. N-(3,23-O-Diacetyl-17-betulinic acyl)-
L
-glycine methyl ester
d5, 400 MHz) d: 0.91, 0.98, 1.04,1.10 (each 3H, s, Me-24, -25, -26, and
(18)
-27), 1.74 (3H, s, Me-30), 2.05 (1H, m, H-18), 2.16 (1H, m, H-19), 3.24
White foam, yield: 91.0%. MS (ESI): m/z 626.4 [M ꢂ H]ꢂ, 650.5
(1H, m, H-3), 3.70 (1H, d, J ¼ 11.1 Hz, H-23
a
), 3.95 (2H, m, H-20), 4.15
[M þ Na]þ; HR-ESI-MS: m/z 626.4040 [M ꢂ H]ꢂ (calcd: 626.4062);
(2H, m, H-50), 4.19 (1H, d, J ¼ 11.1 Hz, H-23
b), 4.72 (1H, s, H-29a),
1H NMR (CDCl3, 400 MHz)
d
: 0.79, 0.86, 0.92, 0.95 (each 3H, s, Me-
4.87 (1H, s, H-29
b
), 5.16 (2H, d, J ¼ 8.5 Hz, H-40), 5.21 (2H, d,
24, -25, -26, and -27), 1.67 (3H, s, Me-30), 1.96 (1H, m, H-18), 1.99
(3H, s, 3-OAc), 2.04 (3H, s, 23-OAc), 2.41 (1H, m, H-19), 3.07 (1H, m,
J ¼ 12.2 Hz, H-70), 5.83 (2H, br s, H-30, 60); 13C NMR (pyridine-d5,
100 MHz) d: 14.3, 16.4, 17.9, 18.3, 20.0, 21.2, 31.7, 33.3, 33.8, 36.0,
H-3), 3.66 (1H, d, J ¼ 11.6 Hz, H-23
(1H, d, J ¼ 11.6 Hz, H-23
), 3.99 (2H, s, H-20), 4.58 (1H, s, H-29
4.71 (1H, s, H-29
), 6.07 (1H, t, H-10); 13C NMR (CDCl3, 100 MHz)
: 12.8, 14.5, 16.0, 16.5, 17.9, 19.4, 20.8, 21.1, 29.3, 36.9, 37.6, 38.0,
a
), 3.73 (3H, s, H-30eOCH3), 3.84
37.8, 38.7, 38.8, 40.6, 42.6, 43.7, 44.4, 47.9, 50.4, 52.7, 54.7, 56.5, 69.4,
74.9, 111.0, 118.2, 124.4, 125.4, 136.0, 153.3, 175.9.
b
a),
b
d
4.1.23. 3,23-Dihydroxy-17-(2-aminoethyl)betulinic amide (23)
White foam, yield: 84.6%. MS (ESI): m/z 515.9 [M þ H]þ; HR-ESI-
MS: m/z 515.4211 [M þ H]þ (cacld: 515.4207); 1H NMR (pyridine-d5,
40.5, 40.7, 41.0, 42.4, 46.6, 48.0, 50.0, 50.6, 52.1, 55.7, 65.4, 74.5,
109.3, 150.7, 170.6, 170.8, 170.9, 176.5.
400 MHz) d: 0.94, 1.01, 1.06, 1.15 (each 3H, s, Me-24, -25, -26, and
4.1.19. N-(3,23-O-Diacetyl-17-betulinic acyl)-
L
-methionine methyl
-27), 1.78 (3H, s, Me-30), 2.10 (1H, m, H-18), 2.18 (1H, m, H-19), 3.04
ester (19)
(1H, m, H-3), 3.62 (2H, t, H-20), 3.72 (1H, d, J ¼ 10.2 Hz, H-23
a
), 4.18
),
d: 13.7, 15.6, 17.4,
White foam, yield: 56.0%. MS (ESI): m/z 724.6 [M þ Na]þ; HR-ESI-
(1H, d, J ¼ 10.2 Hz, H-23
b), 4.76 (1H, s, H-29a), 4.94 (1H, s, H-29b
MS: m/z 700.4249 [M ꢂ H]ꢂ (calcd: 700.4252); 1H NMR (CDCl3,
5.21 (1H, s, H-10); 13C NMR (pyridine-d5, 100 MHz)
400 MHz)
d
: 0.70, 0.83, 0.90, 1.15 (each 3H, s, Me-24, -25, -26, and
17.6, 19.4, 20.4, 27.0, 28.7, 32.2, 35.4, 38.2, 38.6, 39.9, 41.3, 42.0, 43.6,
43.8, 48.1, 49.7, 51.5, 51.9, 56.7, 68.7, 74.3, 110.5, 152.4, 178.3.
-27), 1.70 (3H, s, Me-30), 1.93 (2H, m, H-100), 1.94 (1H, m, H-18), 2.02
(3H, s, Me-400), 2.06 (3H, s, 3-OAc), 2.09 (3H, s, 23-OAc), 2.11 (2H, m, H-
200), 2.56 (1H, m, H-19), 3.09 (1H, m, H-3), 3.69 (1H, d, J ¼ 11.6 Hz,
4.1.24. 3,23-Dihydroxy-17-morpholinyl betulinic amide (24)
White foam, yield: 93.8%. MS (ESI): m/z 564.7 [M þ Na]þ; HR-
ESI-MS: m/z 542.4207 [M þ H]þ (cacld: 542.4204); 1H NMR
H-23a
), 3.74 (3H, s, OMe-30), 3.87 (1H, d, J ¼ 11.6 Hz, H-23
b), 4.68 (1H,
s, H-29a), 4.77 (1H, s, H-29
b
), 6.61 (1H, d, J ¼ 6.2 Hz, H-10); 13C NMR

2498
P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
(pyridine-d5, 400 MHz)
d
: 0.92, 0.99, 1.04, 1.11 (each 3H, s, Me-24,
and purified on a silica gel column (200 g, chloroformemethanol
10:1) to give 29 (0.26 g, 89.7%) as white foam. MS (ESI): m/z 568.6
[M ꢂ H]ꢂ, 592.4 [M þ Na]þ; HR-ESI-MS: m/z 568.4010 [M ꢂ H]ꢂ
-25, -26, and -27), 1.75 (3H, s, Me-30), 1.92 (1H, m, H-18), 1.99 (1H,
m, H-19), 3.35 (1H, m, H-3), 3.62 (8H, m, H-20, 30, 50, 60), 3.70 (1H, d,
J ¼ 10.5 Hz, H-23
a
), 4.17 (1H, d, J ¼ 10.5 Hz, H-23
b
), 4.73 (1H, s,
: 12.2,
(calcd: 568.4007); 1H NMR (pyridine-d5, 400 MHz)
d: 0.99, 1.03,
H-29 ), 4.89 (1H, s, H-29
a
b
); 13C NMR (pyridine-d5,100 MHz)
d
1.06, 1.09 (each 3H, s, Me-24, -25, -26, and -27), 1.77 (3H, s, Me-30),
14.2, 15.8, 16.1, 17.9, 19.0, 20.7, 27.2, 29.5, 31.8, 35.3, 36.5, 36.7, 38.4,
40.4, 41.5, 42.3, 45.7, 48.3, 50.6, 52.3, 54.0, 66.4, 67.3, 72.8, 108.9,
151.0, 172.8.
2.03 (1H, m, H-18), 2.08 (4H, m, H-30, 40), 2.34 (1H, m, H-19), 3.22
(1H, m, H-3), 3.52 (1H, d, J ¼ 10.5 Hz, H-23
J ¼ 10.5 Hz, H-23
), 3.91 (1H, t, H-50), 4.64 (1H, s, H-29
s, H-29 : 12.8, 13.1, 14.8, 16.5,
); 13C NMR (pyridine-d5, 100 MHz)
a
), 3.73 (1H, d,
b
a
), 4.70 (1H,
b
d
4.1.25. 3,23-Dihydroxy-17-piperidinyl betulinic amide (25)
White foam, yield: 83.9%. MS (ESI): m/z 562.16 [M þ Na]þ; HR-
ESI-MS: m/z 538.4266 [M ꢂ H]ꢂ (calcd: 538.4266); 1H NMR (pyri-
16.8, 18.5, 19.7, 26.1, 27.8, 30.5, 37.2, 37.2, 37.3, 41.0, 42.4, 42.9, 42.9,
48.7, 51.1, 55.3, 55.4, 61.1, 61.3, 67.7, 73.3, 109.2, 151.9, 173.9, 175.6.
Compounds 30 and 31 were obtained by using the similar
synthetic procedure of compound 29.
dine-d5, 400 MHz) d: 0.92, 1.00, 1.02, 1.10 (each 3H, s, Me-24, -25,
-26, and -27), 1.75 (3H, s, Me-30), 1.93 (1H, m, H-18), 2.15 (1H, m,
H-19), 3.28 (1H, m, H-3), 3.50 (2H, t, H-20), 3.56 (2H, t, H-60), 3.68
4.1.30. N-(3,23-Dihydroxy-17-acyl betulinic acyl)-L-glycine acid
(1H, d, J ¼ 10.2 Hz, H-23
a
), 4.13 (1H, d, J ¼ 10.2 Hz, H-23
b
), 4.87 (1H,
(30)
s, H-29 ), 5.06 (1H, s, H-29
a
b
); 13C NMR (pyridine-d5, 100 MHz)
d:
White foam, yield: 75.0%. MS (ESI): m/z 528.7 [M ꢂ H]ꢂ; HR-ESI-
13.3, 15.3, 16.9, 17.3, 19.0, 20.2, 21.9, 26.9, 28.2, 30.7, 32.2, 35.0, 37.6,
37.8, 39.6, 41.5, 42.7, 43.3, 46.9, 49.4, 51.7, 53.6, 55.3, 68.5, 74.0,
109.9, 152.3, 173.5.
MS: m/z 528.3674 [M ꢂ H]ꢂ (calcd: 528.3694); 1H NMR (CD3OD,
400 MHz) d: 0.66, 0.86, 0.94, 0.98 (each 3H, s, Me-24, -25, -26, and
-27), 1.66 (3H, s, Me-30), 1.97 (1H, m, H-18), 2.10 (1H, m, H-19), 2.59
(1H, m, H-3), 3.49 (1H, d, J ¼ 12.7 Hz, H-23 ),
), 3.03 (1H, s, H-20
3.68 (1H, s, H-20
), 3.90 (1H, d, J ¼ 12.7 Hz, H-23 ), 4.55 (1H, s,
H-29 ), 4.67 (1H, s, H-29 : 12.4,
); 13C NMR (CD3OD, 100 MHz)
15.0, 17.0, 19.1, 19.6, 22.0, 26.9, 38.0, 38.8, 41.9, 43.5, 47.9, 48.3, 49.2,
49.4, 49.6, 51.9, 56.9, 74.0, 109.8, 152.2, 172.0, 179.7.
a
a
4.1.26. 3,23-Dihydroxy-17-piperizinyl betulinic amide (26)
White foam, yield: 76.9%. MS (ESI): m/z 541.5 [M þ H]þ; HR-ESI-
MS: m/z 541.4372 [M þ H]þ (calcd: 541.4364); 1H NMR (pyridine-d5,
b
b
a
b
d
400 MHz) d: 0.90, 0.98, 1.02, 1.10 (each 3H, s, Me-24, -25, -26, and
-27), 1.74 (3H, s, Me-30), 2.02 (1H, m, H-18), 2.12 (1H, m, H-19), 2.84
(4H, m, H-30, 50), 3.28 (1H, m, H-3), 3.68 (4H, m, H-20, 60), 3.73 (1H, d,
4.1.31. N-(3,23-Dihydroxy-17-betulinic acyl)-L-methionine acid
J ¼ 10.3 Hz, H-23
a
), 4.16 (1H, d, J ¼ 10.3 Hz, H-23
b
), 4.72 (1H, s, H-
(31)
29 ), 4.88 (1H, s, H-29
a
b
); 13C NMR (pyridine-d5, 100 MHz)
d: 12.3,
White foam, yield: 82.3%. MS (ESI): m/z 602.8 [M ꢂ H]ꢂ; HR-ESI-
14.2, 15.8, 16.2, 17.9, 19.0, 25.6, 27.2, 29.5, 31.9, 33.9, 36.5, 36.7, 38.5,
40.5, 41.6, 42.3, 45.8, 45.9, 48.2, 50.6, 52.4, 54.1, 67.1, 72.8, 108.9,
151.2, 172.7.
MS: m/z 602.3882 [M ꢂ H]ꢂ (calcd: 602.3885); 1H NMR (pyridine-
d5, 400 MHz) d: 0.88, 0.98,1.03,1.15 (each 3H, s, Me-24, -25, -26, and
-27), 1.73 (3H, s, Me-30), 1.90 (2H, m, H-100), 2.02 (1H, m, H-18), 2.08
(3H, s, Me-400), 2.10 (2H, m, H-200), 2.39 (1H, m, H-19), 2.91 (1H, m,
4.1.27. 3,23-Dihydroxy-17-(2-morpholinylethyl)betulinic ester (27)
White foam, yield: 85.7%. MS (ESI): m/z 586.6 [M þ H]þ; HR-ESI-
MS: m/z 586.4467 [M þ H]þ (cacld: 586.4466); 1H NMR (pyridine-
H-3), 3.57 (1H, t, H-20), 3.70 (1H, d, J ¼ 10.2 Hz, H-23
a
), 4.17 (1H, d,
J ¼ 10.2 Hz, H-23
b), 4.72 (1H, s, H-29a), 4.89 (1H, s, H-29b
); 13C
NMR (pyridine-d5, 100 MHz) d: 12.8, 14.8, 15.2, 16.7, 16.8, 19.5, 31.1,
d5, 400 MHz)
d
: 0.90, 0.95, 1.03, 1.05 (each 3H, s, Me-24, -25, -26,
31.3, 34.6, 37.3, 37.7, 39.0, 41.1, 42.7, 42.8, 42.9, 47.1, 48.7, 50.6, 51.0,
55.9, 67.7, 73.3, 110.5, 151.7, 177.6.
and -27), 1.74 (3H, s, Me-30), 2.04 (1H, m, H-18), 2.09 (1H, m, H-19),
2.46 (4H, m, H-40, 80), 2.60 (2H, t, H-20), 3.35 (1H, m, H-3), 3.73 (4H,
m, H-50, 70), 3.70 (1H, d, J ¼ 11.5 Hz, H-23
a
), 4.18 (1H, d, J ¼ 11.5 Hz,
), 4.89 (1H, s, H-29 );
d: 12.4, 14.3, 15.8, 16.2, 18.8, 25.4,
4.1.32. 3,23-O-Diacetyl-30-hydroxy-17-isopropyl betulinic ester (32)
To a solution of compound 13 (0.34 g, 0.57 mmol) in chloroform
(30 mL) was added m-CPBA (0.088 g, 0.51 mmol). The solution was
stirred and refluxed for 8 h. At which time the reaction was
completed, the solution was washed by saturated NaHCO3 solution
(40 mL), water (40 mL) and brine (40 mL), dried with anhydrous
Na2SO4. Filtered, the filtrate was concentrated, the residue was
dissolved in dichloromethane and purified on a silica gel column
(200 g, petroleum ethereethyl acetate 5:1) to give 32 (0.16 g, 45.7%)
as white foam. MS (ESI): m/z 615.9 [M þ H]þ, 637.9 [M þ Na]þ; 1H
H-23
b
), 4.39 (2H, t, H-10), 4.74 (1H, s, H-29
a
b
13C NMR (pyridine-d5, 100 MHz)
27.3, 29.5, 31.8, 36.7, 37.9, 38.5, 40.5, 42.1, 42.4, 47.0, 48.1, 49.1, 50.3,
53.5, 56.2, 57.0, 60.4, 66.6, 72.7, 86.0, 109.5, 150.4, 175.4.
4.1.28. 3,23-Dihydroxy-17-(E-4-hydroxybut-2-enyl)betulinic ester
(28)
White foam, yield: 77.8%. MS (ESI): m/z 565.6 [M þ Na]þ; HR-
ESI-MS: m/z 565.3865 [M þ Na]þ (cacld: 565.3863); 1H NMR
(pyridine-d5, 400 MHz)
d
: 0.90, 0.95, 1.03, 1.07 (each 3H, s, Me-24,
NMR (CDCl3, 400 MHz) d: 0.79, 0.85, 0.90, 0.95 (each 3H, s, Me-24,
-25, -26, and -27), 1.77 (3H, s, Me-30), 2.22 (1H, m, H-18), 2.47 (1H,
m, H-19), 3.40 (1H, m, H-3), 3.70 (1H, d, J ¼ 10.5 Hz, H-23 ), 4.16
(1H, d, J ¼ 10.5 Hz, H-23 ),
), 4.19 (2H, m, H-40), 4.60 (1H, s, H-29
4.73 (1H, s, H-29
), 5.13 (2H, m, H-10), 5.81 (1H, m, H-20), 6.17 (1H,
m, H-30); 13C NMR (pyridine-d5, 100 MHz)
: 14.1, 16.0, 17.5, 18.0,
-25, -26, and -27), 1.20 (3H, d, J ¼ 6.2 Hz, Me-20), 1.22 (3H, d,
J ¼ 6.2 Hz, Me-30),1.81 (1H, m, H-18), 2.00 (3H, s, 3-OAc), 2.05 (3H, s,
23-OAc), 2.22 (1H, m, H-19), 2.89 (1H, m, H-3), 3.68 (1H, d,
a
b
a
b
J ¼ 11.6 Hz, H-23
H-30), 4.75 (1H, m, H-10), 4.90 (1H, s, H-29
13C NMR (CDCl3, 100 MHz)
: 13.0, 14.7, 16.0, 16.7, 18.0, 21.1, 21.1,
21.3, 21.8, 21.9, 23.2, 29.6, 32.0, 34.0, 38.1, 38.3, 40.7, 48.1, 50.1, 50.6,
65.2, 65.5, 67.0, 74.6, 76.8, 106.7, 155.0, 170.8, 171.2, 175.6.
Compounds 33e38 were obtained by using the similar synthetic
procedure of compound 32.
a
), 3.82 (1H, d, J ¼ 11.6 Hz, H-23
b
), 4.10 (2H, s,
d
a
), 4.97 (1H, s, H-29
b);
19.7, 20.6, 22.3, 27.2, 29.0, 31.2, 35.6, 38.5, 39.7, 40.3, 42.3, 43.9, 44.2,
48.7, 50.0, 51.0, 52.1, 57.9, 59.6, 61.4, 69.2, 74.7, 111.2, 126.0, 137.1,
152.1, 177.0.
d
4.1.29. N-(3,23-Dihydroxy-17-betulinic acyl)- -proline acid (29)
L
To a solution of compound 17 (0.34 g, 0.51 mmol) in meth-
anol:THF (4:10 mL) was added a NaOH solution (4 mol/L, 8 mL). The
solution was stirred and refluxed for 4 h. At which time the reaction
was completed, the pH of the solution was adjusted to 1e2 by 10%
HCl, filtered, the filter cake was collected and dissolved in methanol
4.1.33. 3,23-O-Diacetyl-30-hydroxy-17-tert-butyl betulinic ester
(33)
White foam, yield: 55.0%. MS (ESI): m/z 651.6 [M þ Na]þ; 1H
NMR (CDCl3, 400 MHz) d: 0.79, 0.86, 0.92, 0.95 (each 3H, s, Me-24,

P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
2499
-25, -26, and -27), 1.44 (9H, s, Me-20, -30, -40), 1.85 (1H, m, H-18),
2.00 (3H, s, 3-OAc), 2.05 (3H, s, 23-OAc), 2.20 (1H, m, H-19), 2.88
(1H, m, H-3), 3.67 (1H, d, J ¼ 11.6 Hz, H-23 ), 3.82 (1H, d, J ¼ 11.6 Hz,
H-23 ), 4.10 (2H, m, H-30), 4.90 (1H, s, H-29 ), 4.95 (1H, s, H-29 );
13C NMR (CDCl3, 100 MHz)
: 13.9, 15.5, 16.9, 17.5, 18.9, 21.9, 22.0,
m, H-20), 4.10 (1H, d, J ¼ 11.5 Hz, H-23
a
), 3.72 (3H, s, H-60eOCH3),
3.84 (2H, s, H-30), 4.44 (1H, d, J ¼ 11.5 Hz, H-23
b
), 4.78 (1H, s,
a
H-29a), 4.92 (1H, s, H-29b d: 12.9, 14.4,
); 13C NMR (CDCl3, 100 MHz)
b
a
b
14.6, 16.2, 16.6, 16.6, 17.9, 20.9, 21.2, 23.1, 26.8, 29.7, 36.9, 36.9, 38.0,
40.6, 40.7, 42.0, 48.1, 48.9, 50.5, 50.8, 52.0, 60.1, 65.3, 65.4, 74.5,
105.8, 155.8, 170.7, 171.1, 173.5, 174.1.
d
22.2, 24.1, 29.0, 30.5, 37.9, 37.9, 39.0, 39.1, 41.6, 41.8, 43.3, 49.0, 50.9,
51.6, 57.7, 66.2, 66.4, 75.4, 80.6, 107.5, 156.0, 171.6, 172.0, 176.3.
4.1.39. 3,23-Dihydroxy-30-hydroxy-17-isopropyl betulinic ester
(39)
4.1.34. 3,23-O-Diacetyl-30-hydroxy-17-(tetrahydrofufuran-2-yl)
methyl betulinic ester (34)
To a solution of compound 32 (0.24 g, 0.39 mmol) in THF (20 mL)
were added methanol (8 mL) and NaOH solution (4 mol/L, 8 mL).
The solution was stirred and refluxed for 4 h. At which time the
reaction was completed, the pH of the solution was adjusted to 5e6
by 10% HCl, filtered, the filter cake was collected and purified on
a silica gel column (200 g, 1:1 petroleum ethereacetone) to give 39
(0.18 g, 85.7%) as white foam. MS (ESI): m/z 531.9 [M þ H]þ, 553.8
[M þ Na]þ; HR-ESI-MS: m/z 553.3865 [M þ Na]þ (calcd: 553.3863);
White foam, yield: 59.7%. MS (ESI): m/z 679.8 [M þ Na]þ; 1H
NMR (CDCl3, 400 MHz) d: 0.78, 0.85, 0.89, 0.94 (each 3H, s, Me-24,
-25, -26, and -27),1.66 (4H, m, H-30, 40), 1.86 (1H, m, H-18), 2.00 (3H,
s, 3-OAc), 2.06 (3H, s, 23-OAc), 2.16 (1H, m, H-19), 3.03 (1H, m, H-3),
3.66 (1H, d, J ¼ 11.6 Hz, H-23
(6H, m, H-10, 50, 30), 4.75 (1H, m, H-20), 4.89 (1H, s, H-29
s, H-29 : 13.0, 14.7, 16.0, 16.7, 18.0,
); 13C NMR (CDCl3, 100 MHz)
a
), 3.81 (1H, d, J ¼ 11.6 Hz, H-23
b), 4.12
), 4.95 (1H,
a
b
d
21.0, 21.3, 23.2, 25.8, 32.0, 32.4, 36.8, 37.0, 38.1, 40.6, 40.8, 48.1, 50.6,
65.2, 65.5, 68.4, 74.6, 77.4, 106.7, 154.8, 170.9, 171.2, 175.9.
1H NMR (pyridine-d5, 400 MHz)
d: 0.90, 0.95, 1.02, 1.04 (each 3H, s,
Me-24, -25, -26, and -27), 1.20 (3H, d, J ¼ 6.2 Hz, Me-20), 1.24 (3H, d,
J ¼ 6.2 Hz, H-30-Me),1.91 (1H, m, H-18), 2.19 (1H, m, H-19), 3.32 (1H,
4.1.35. 3,23-O-Diacetyl-30-hydroxy-17-isopentyl betulinic ester (35)
White foam, yield: 31.9%. MS (ESI): m/z 665.9 [M þ Na]þ; 1H
m, H-3), 3.70 (1H, d, J ¼ 10.6 Hz, H-23 ), 4.17 (1H, d, J ¼ 10.6 Hz, H-
a
23
b
), 4.52 (2H, s, H-30), 5.12 (1H, s, H-29
a
), 5.16 (1H, m, H-10), 5.49
: 13.0, 14.9, 16.4,
NMR (CDCl3, 400 MHz)
d
: 0.80, 0.87, 0.91, 0.94, 0.95, 0.96 (each 3H,
(1H, s, H-29
b
); 13C NMR (pyridine-d5, 100 MHz)
d
s, Me-24, -25, -26, -27, -40, -50),1.70 (2H, m, H-20),1.85 (1H, m, H-18),
16.9, 18.5, 21.3, 21.8, 21.8, 27.9, 30.1, 34.5, 37.1, 37.3, 38.6, 39.2, 41.1,
42.7, 43.0, 48.6, 50.2, 50.9, 56.2, 56.6, 64.5, 67.0, 67.6, 73.2, 106.2,
156.8, 175.5.
Compounds 40e42 were obtained by using the similar synthetic
procedure of compound 39.
1.87 (1H, m, H-30), 2.01 (3H, s, 3-OAc), 2.06 (3H, s, 23-OAc), 2.65 (1H,
m, H-19), 2.89 (1H, m, H-3), 3.68 (1H, d, J ¼ 11.6 Hz, H-23
(1H, d, J ¼ 11.6 Hz, H-23
), 4.14 (2H, m, H-10), 4.75 (2H, m, H-30),
4.91 (1H, s, H-29 ), 4.97 (1H, s, H-29
); 13C NMR (CDCl3, 100 MHz)
: 12.8, 14.5, 15.9, 16.5, 17.8, 20.9, 21.0, 21.2, 22.3, 22.4, 23.0, 25.1,
a), 3.83
b
a
b
d
36.9, 37.4, 37.9, 38.1, 40.5, 40.6, 42.2, 47.9, 50.0, 50.5, 56.4, 62.4, 65.3,
74.4, 106.5, 154.8, 170.6, 171.0, 176.0.
4.1.40. 3,23-Dihydroxy-30-hydroxy-17-tert-butyl betulinic ester
(40)
White foam, yield: 82.4%. MS (ESI): m/z 567.4 [M þ Na]þ; HR-
4.1.36. 3,23-O-Diacetyl-30-hydroxy-17-morpholinyl betulinic
amide (36)
ESI-MS: m/z 567.4023 [M þ Na]þ (cacld: 567.4020); 1H NMR
(pyridine-d5, 400 MHz) d: 0.94, 0.98, 1.06, 1.06 (each 3H, s, Me-24,
White foam, yield: 24.5%. MS (ESI): m/z 664.7 [M þ Na]þ; HR-
-25, -26, and -27), 1.53 (9H, s, Me-20, 30, 40), 2.11 (1H, m, H-18), 2.48
(1H, m, H-19), 3.30 (1H, m, H-3), 3.72 (1H, d, J ¼ 10.3 Hz, H-23 ),
4.18 (1H, d, J ¼ 10.3 Hz, H-23 ), 4.49 (2H, m, H-30), 5.13 (1H, s,
H-29 ), 5.50 (1H, s, H-29 : 13.1,
); 13C NMR (pyridine-d5, 100 MHz)
ESI-MS: m/z 642.4268 [M þ H]þ (calcd: 642.4364); 1H NMR (CDCl3,
a
400 MHz)
d
: 0.81, 0.87, 0.92, 0.96 (each 3H, s, Me-24, -25, -26, and
b
-27), 1.95 (1H, m, H-18), 2.02 (3H, s, 3-OAc), 2.07 (3H, s, 23-OAc),
a
b
d
2.20 (1H, m, H-19), 3.56 (1H, m, H-3), 3.70 (4H, m, H-30, 50), 3.84
15.1, 16.6, 17.1, 21.5, 27.4, 28.1, 28.3, 30.2, 30.3, 32.8, 34.7, 37.6, 38.8,
39.4, 41.4, 42.9, 43.2, 43.6, 49.0, 50.4, 51.2, 57.3, 64.8, 68.1, 73.7, 79.6,
106.3, 157.2, 175.6.
(2H, s, H-30), 4.12 (1H, d, J ¼ 11.6 Hz, H-23
H-23 ), 4.76 (1H, s, H-29 ), 4.95 (1H, s, H-29
100 MHz) : 12.8,14.4,14.6,16.2,16.5,16.6,17.9, 20.9, 21.2, 23.1, 26.8,
a
), 4.49 (1H, d, J ¼ 11.6 Hz,
b
a
b
); 13C NMR (CDCl3,
d
29.7, 36.9, 36.9, 38.0, 40.6, 40.7, 42.0, 48.1, 48.1, 48.9, 50.5, 50.8, 52.3,
60.6, 65.3, 65.4, 74.5, 105.8, 155.8, 170.7, 171.1, 174.1.
4.1.41. 3,23-Dihydroxy-30-hydroxy-17-(tetrahydrofufuran-2-yl)
methyl betulinic ester (41)
White foam, yield: 78.3%. MS (ESI): m/z 573.9 [M þ H]þ, 595.8
4.1.37. 3,23-O-Diacetyl-30-hydroxy-17-piperidinyl betulinic amide
(37)
[M þ Na]þ; HR-ESI-MS: m/z 595.3968 [M þ Na]þ (cacld: 595.3969);
1H NMR (pyridine-d5, 400 MHz)
d: 0.61, 0.67, 0.75, 0.77 (each 3H, s,
White foam, yield: 53.9%. MS (ESI): m/z 640.6 [M þ H]þ; HR-ESI-
Me-24, -25, -26, and -27), 1.67 (4H, m, H-30, 40), 1.86 (1H, m, H-18),
2.16 (1H, m, H-19), 3.06 (1H, m, H-3), 3.43 (2H, m, H-50), 3.56 (1H, d,
MS: m/z 640.4580 [M þ H]þ (calcd: 640.4572); 1H NMR (CDCl3,
400 MHz)
d
: 0.80, 0.88, 0.94, 0.96 (each 3H, s, Me-24, -25, -26, and
J ¼ 14.4 Hz, H-23
(1H, d, J ¼ 14.4 Hz, H-23
13C NMR (pyridine-d5, 100 MHz)
a
), 3.92 (4H, m, H-10, 30), 4.07 (1H, m, H-20), 4.25
-27), 1.95 (1H, m, H-18), 2.01 (3H, s, 3-OAc), 2.06 (3H, s, 23-OAc),
b), 4.83 (1H, s, H-29
a), 5.21 (1H, s, H-29b);
2.10 (1H, m, H-19), 3.03 (1H, m, H-3), 3.49 (4H, m, H-20, 60), 3.69 (1H,
d
: 13.0, 14.9, 16.3, 16.8, 18.5, 21.2,
d, J ¼ 11.6 Hz, H-23
H-30), 4.90 (1H, s, H-29
100 MHz) : 12.4, 14.1, 15.6, 16.2, 17.4, 20.5, 20.8, 22.6, 24.3, 25.8,
a
), 3.84 (1H, d, J ¼ 11.6 Hz, H-23
b
), 4.09 (2H, s,
26.0, 27.8, 28.3, 32.3, 32.8, 34.5, 37.1, 37.3, 39.1, 41.1, 42.7, 43.0, 48.7,
50.2, 50.9, 56.9, 64.5, 67.7, 68.3, 73.3, 76.9, 77.7, 99.0, 106.2, 156.7,
176.0.
a
), 4.94 (1H, s, H-29
b
); 13C NMR (CDCl3,
d
33.6, 35.3, 36.4, 36.6, 37.6, 40.2, 40.2, 41.4, 47.7, 50.5, 53.2, 54.2, 54.6,
64.8, 65.0, 74.1, 105.3, 155.4, 170.2, 170.6, 172.7.
4.1.42. 3,23-Dihydroxy-30-hydroxy-17-isopentyl betulinic ester
(42)
4.1.38. N-(3,23-O-Diacetyl-30-hydroxy-17-betulinic acyl)-
L
-proline
White foam, yield: 81.2%. MS (ESI): m/z 581.6 [M þ Na]þ; HR-
methyl ester (38)
ESI-MS: m/z 581.4168 [M þ Na]þ (cacld: 581.4176); 1H NMR (pyri-
White foam, yield: 69.4%. MS (ESI): m/z 684.7 [M þ H]þ; HR-ESI-
dine-d5, 400 MHz) d: 0.88, 0.90, 0.96, 0.99, 1.06, 1.07 (each 3H, s,
MS: m/z 684.4472 [M þ H]þ (calcd: 684.4470); 1H NMR (CDCl3,
Me-24, -25, -26, -27, -40, -50), 1.74 (2H, m, H-20), 1.94 (1H, m, H-18),
1.95 (1H, m, H-19), 2.01 (1H, m, H-30), 3.31 (1H, m, H-3), 3.71 (1H, d,
400 MHz) d: 0.81, 0.87, 0.92, 0.97 (each 3H, s, Me-24, -25, -26, and
-27), 1.37 (4H, m, H-30, 40), 1.91 (1H, m, H-18), 2.01 (3H, s, 3-OAc),
2.07 (3H, s, 23-OAc), 2.74 (1H, m, H-19), 2.92 (1H, m, H-3), 3.56 (2H,
J ¼ 10.3 Hz, H-23
a
), 4.17 (1H, d, J ¼ 10.3 Hz, H-23
b
), 4.31 (2H, m,
);
H-10), 4.53 (2H, m, H-30), 5.14 (1H, s, H-29
a
), 5.50 (1H, s, H-29
b

2500
P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
13C NMR (pyridine-d5, 100 MHz)
23.7, 26.6, 29.1, 31.4, 35.8, 38.6, 39.0, 39.9, 40.4, 42.4, 44.0, 44.2, 50.1,
51.5, 52.2, 58.1, 63.8, 65.8, 69.2, 69.8, 74.7, 107.5, 158.0, 177.2.
d
: 14.1, 16.1, 17.6, 18.0, 19.8, 22.5,
H-23
a
), 3.62 (2H, t, H-20), 3.70 (1H, d, J ¼ 10.2 Hz, H-23
b
), 3.74 (3H,
s, Me-90), 4.55 (1H, s, H-29 ), 4.61 (1H, m, H-50), 4.71 (1H, s, H-29
a b);
13C NMR (CDCl3, 100 MHz)
d: 11.2, 14.7, 15.4, 16.1, 16.5, 18.4, 19.6,
21.0, 25.6, 29.9, 36.9, 37.1, 38.4, 40.6, 41.8, 42.1, 46.0, 50.0, 50.8, 51.7,
52.3, 52.4, 55.5, 72.0, 76.6, 76.8, 109.2, 151.2, 172.2, 172.5, 175.1.
4.1.43. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-L-glycine acyl]-L-
alanine methyl ester (43)
To a solution of compound 30 (0.40 g, 0.75 mmol) in dry
dichloromethane (50 mL) were added -alanine methyl ester
4.1.47. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-L-glycine acyl]-L-
L
tryptophane methyl ester (47)
hydrochloride (0.42 g, 3.0 mmol), EDC$HCl (1.15 g, 6.0 mmol) and
HoBt (0.82 g, 6.0 mmol). The solution was stirred and refluxed for
10 h. At which time the reaction was completed, the solution was
washed by water (100 mL) and brine (100 mL), dried with anhy-
drous Na2SO4. Filtered, the filtrate was concentrated, the residue
was dissolved in dichloromethane and purified on a silica gel
column (200 g, petroleum ethereacetone 3:1) to give 43 (0.33 g,
72.0%) as white foam. MS (ESI): m/z 637.6 [M þ Na]þ; HR-ESI-MS:
m/z 613.4214 [M ꢂ H]ꢂ (calcd: 613.4222); 1H NMR (CDCl3,
White foam, yield: 55.9%. MS (ESI): m/z 730.3 [M þ H]þ; HR-ESI-
MS: m/z 752.4582 [M þ Na]þ (calcd: 752.4606); 1H NMR (CDCl3,
400 MHz) d: 0.80, 0.83, 0.87, 0.91 (each 3H, s, Me-24, -25, -26, and
-27), 1.64 (3H, s, Me-30), 1.75 (1H, m, H-18), 2.34 (1H, m, H-19), 3.00
(1H, m, H-3), 3.31 (2H, m, H-100), 3.38 (1H, d, J ¼ 11.2 Hz, H-23
a),
3.70 (3H, s, Me-60), 3.77 (2H, m, H-20), 3.95 (1H, d, J ¼ 11.2 Hz,
H-23b), 4.57 (1H, s, H-29a), 4.69 (1H, s, H-29b
), 4.92 (1H, m, H-50),
6.97 (1H, d, J ¼ 2.0 Hz, H-400), 7.09 (1H, t, H-700), 7.18 (1H, t, H-800),
7.34 (1H, s, H-300), 7.42 (1H, d, J ¼ 8.0 Hz, H-600), 7.52 (1H, d,
400 MHz)
-27), 1.40 (3H, s, Me-50), 1.64 (3H, s, Me-30), 1.81 (1H, m, H-18), 2.43
(1H, m, H-19), 3.05 (1H, m, H-3), 3.38 (1H, d, J ¼ 10.3 Hz, H-23 ),
d
: 0.84, 0.84, 0.88, 0.94 (each 3H, s, Me-24, -25, -26, and
J ¼ 7.8 Hz, H-900); 13C NMR (CDCl3, 100 MHz)
d: 14.5, 15.9, 16.4, 19.3,
23.1, 27.5, 27.5, 29.6, 30.6, 37.0, 37.6, 38.3, 40.6, 41.8, 42.4, 42.8, 46.6,
49.9, 52.3, 52.4, 53.1, 55.7, 76.6, 76.8, 109.4, 111.2, 116.9, 118.3, 119.6,
122.2, 126.4, 128.4, 136.1, 150.6, 171.8, 172.3, 177.1.
a
3.69 (1H, d, J ¼ 10.3 Hz, H-23
b
), 3.72 (3H, s, OMe-60), 3.90 (2H, d,
J ¼ 5.2 Hz, H-20), 4.52 (1H, m, H-50), 4.58 (1H, s, H-29
a), 4.71 (1H, s,
H-29
b
), 6.51 (1H, t, H-10), 6.86 (1H, d, J ¼ 7.0 Hz, H-40); 13C NMR
4.1.48. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-L-glycine acyl]-L-
alanine acid (48)
(CDCl3, 100 MHz)
d
: 11.2, 14.6, 16.1, 16.4, 18.1, 18.3, 19.4, 30.7, 37.0,
37.6, 40.7, 41.8, 42.4, 43.1, 46.6, 48.1, 49.9, 50.0, 50.5, 52.4, 55.8, 71.8,
76.6, 109.4, 150.6, 169.1, 173.0, 177.2.
Compounds 44e47 were obtained by using the similar synthetic
procedure of compound 43.
To a solution of compound 43 (0.29 g, 0.47 mmol) in meth-
anol:THF (20:20 mL) was added a NaOH solution (4 mol/L, 10 mL).
The solution was stirred and refluxed for 4 h. At which time the
reaction was completed, the pH of the solution was adjusted to
1e2 by 10% HCl, filtered, the filter cake was collected and dissolved
in methanol and purified on a silica gel column (200 g, petroleum
ethereacetone 1:1) to give 48 (0.25 g, 89.0%) as white foam. MS
(ESI): m/z 599.5 [M ꢂ H]ꢂ; HR-ESI-MS: m/z 599.4059 [M ꢂ H]ꢂ
4.1.44. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-L-glycine acyl]-L-
proline methyl ester (44)
White foam, yield: 50.0%. MS (ESI): m/z 639.4 [M ꢂ H]ꢂ, 663.6
[M þ Na]þ; HR-ESI-MS: m/z 639.4371 [M ꢂ H]ꢂ (calcd: 639.4379);
(calcd: 599.4066); 1H NMR (pyridine-d5, 400 MHz)
d: 0.77, 0.89,
1H NMR (CDCl3, 400 MHz)
d: 0.79, 0.81, 0.89, 0.94 (each 3H, s, Me-
0.90, 1.06 (each 3H, s, Me-24, -25, -26, and -27), 1.41 (3H, s, Me-50),
1.66 (3H, s, Me-30), 2.20 (2H, m, H-18, 19), 2.86 (1H, m, H-3), 3.51
24, -25, -26, and -27), 1.66 (3H, s, Me-30), 1.87 (1H, m, H-18), 2.04
(4H, m, H-60, 70), 2.43 (1H, m, H-19), 3.05 (1H, m, H-3), 3.39 (1H, d,
(1H, d, J ¼ 10.1 Hz, H-23
a
), 3.59 (1H, d, J ¼ 10.1 Hz, H-23
b
), 4.18
),
J ¼ 11.0 Hz, H-23
H-23
H-20
6.60 (1H, t, H-10); 13C NMR (CDCl3, 100 MHz)
a
), 3.53 (2H, m, H-50), 3.65 (1H, d, J ¼ 11.0 Hz,
(2H, d, J ¼ 5.2 Hz, H-20), 4.38 (1H, m, H-50), 4.65 (1H, s, H-29
a
b
b
), 3.75 (3H, s, OMe-90), 3.90 (1H, m, H-20
a
), 4.15 (1H, m,
4.78 (1H, s, H-29b d: 13.3, 15.2,
); 13C NMR (pyridine-d5, 100 MHz)
), 4.52 (1H, m, H-80), 4.58 (1H, s, H-29
a
), 4.71 (1H, s, H-29
b
),
16.9, 17.2, 18.9, 19.9, 21.6, 26.6, 30.2, 33.9, 37.6, 39.5, 41.5, 42.0, 43.1,
43.3, 47.6, 48.8, 50.0, 51.4, 52.5, 56.5, 67.5, 73.5, 110.1, 152.1, 172.1,
174.4, 178.6.
Compounds 49e52 were obtained by using the similar synthetic
procedure of compound 48.
d
: 11.2, 14.5, 16.1, 16.4,
18.3, 19.3, 24.5, 29.0, 37.0, 37.5, 40.6, 41.7, 41.8, 42.4, 45.9, 46.6, 49.9,
50.0, 50.5, 52.4, 55.7, 58.8, 76.6, 109.3, 150.8, 167.5, 172.3, 176.7.
4.1.45. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-L-methionine
acyl]-
L
-methionine methyl ester (45)
4.1.49. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-L-glycine acyl]-L-
White foam, yield: 83.0%. MS (ESI): m/z 750.1 [M þ H]þ; HR-ESI-
proline acid (49)
MS: m/z [M ꢂ H]ꢂ 747.4735 (calcd: 747.4446); 1H NMR (CDCl3,
White foam, yield: 86.2%. MS (ESI): m/z 625.6 [M ꢂ H]ꢂ; HR-ESI-
400 MHz) d: 0.85, 0.86, 0.88, 0.95 (each 3H, s, Me-24, -25, -26, and
MS: m/z 625.4220 [M ꢂ H]ꢂ (calcd: 625.4222); 1H NMR (pyridine-
-27), 1.67 (3H, s, Me-30),1.98 (1H, m, H-18), 2.09 (3H, s, Me-400), 2.13
(3H, s, Me-800), 2.41 (1H, m, H-19), 2.50 (4H, m, H-100, 500), 2.64 (4H,
d5, 400 MHz) d: 0.88, 0.99, 1.03,1.11 (each 3H, s, Me-24, -25, -26, and
-27), 1.70 (3H, s, Me-30), 1.77 (1H, m, H-18), 1.86 (4H, m, H-30, 40),
2.12 (2H, m, H-60), 2.28 (1H, m, H-19), 2.41 (2H, m, H-70), 2.97 (1H,
m, H-200, 600), 3.10 (1H, m, H-3), 3.40 (1H, d, J ¼ 10.3 Hz, H-23
a
), 3.70
),
); 13C NMR (CDCl3,
: 11.2, 14.6, 15.1, 15.4, 16.1, 16.4, 19.4, 29.9, 30.0, 30.2, 31.4,
(1H, d, J ¼ 10.3 Hz, H-23
b
), 3.75 (3H, s, Me-60), 4.55 (1H, s, H-29
a
m, H-3), 3.32 (2H, m, H-20), 3.67 (1H, d, J ¼ 11.3 Hz, H-23
a
), 4.01 (1H,
), 4.78
); 13C NMR (pyridine-d5, 100 MHz)
(1H, s, H-29b d: 12.2, 13.5, 14.2,
4.64 (2H, m, H-20, 50), 4.73 (1H, s, H-29
100 MHz)
b
d, J ¼ 11.3 Hz, H-23
b a
), 4.41 (1H, m, H-80), 4.67 (1H, s, H-29
d
37.1, 40.7, 41.9, 42.5, 49.9, 50.6, 51.5, 51.6, 52.5, 55.8, 76.5, 76.6,109.5,
150.6, 171.3, 171.8, 176.5.
16.0, 16.2, 18.0, 18.9, 18.9, 25.6, 27.1, 36.7, 37.1, 38.5, 40.6, 42.2, 42.2,
42.3, 46.5, 48.2, 50.5, 55.5, 59.6, 61.4, 67.3, 72.9, 108.9, 151.1, 176.5.
4.1.46. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-
L
-proline acyl]-
L
-
4.1.50. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-L-methionine
methionine methyl ester (46)
acyl]-L-methionine acid (50)
White foam, yield: 64.0%. MS (ESI): m/z 716.0 [M þ H]þ, 737.8
White foam, yield: 92.0%. MS (ESI): m/z 733.9 [M ꢂ H]ꢂ, 757.6
[M þ Na]þ; HR-ESI-MS: m/z [M ꢂ H]ꢂ 713.4563 (calcd: 713.4569);
[M þ Na]þ; HR-ESI-MS: m/z [M ꢂ H]ꢂ 733.4280 (calcd: 733.4290);
1H NMR (CDCl3, 400 MHz)
d
: 0.84, 0.85, 0.88, 0.94 (each 3H, s, Me-
1H NMR (CD3OD, 400 MHz)
d: 0.87, 0.90, 0.95, 1.00 (each 3H, s, Me-
24, -25, -26, and -27), 1.42 (4H, m, H-30, 40), 1.67 (3H, s, Me-30), 2.05
(1H, m, H-18), 2.08 (3H, s, Me-400), 2.19 (2H, m, H-100), 2.50 (2H, m,
H-200), 2.73 (1H, m, H-19), 2.95 (1H, m, H-3), 3.40 (1H, d, J ¼ 10.2 Hz,
24, -25, -26, and -27), 1.76 (3H, s, Me-30), 1.97 (1H, m, H-18), 2.08
(3H, s, Me-400), 2.08 (3H, s, Me-800), 2.19 (1H, m, H-19), 2.54 (8H, m,
H-100, 200, 500, 600), 3.03 (1H, m, H-3), 3.50 (1H, d, J ¼ 11.2 Hz, H-23
a),

P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
2501
3.59 (1H, d, J ¼ 11.2 Hz, H-23
b
), 4.49 (1H, s, H-29
a
), 4.57 (2H, m,
: 12.5,
4.1.55. 3,23-Dioxylcarbonyl-17-1,40-bipiperidinyl betulinic amide
(54)
H-20, 50), 4.69 (1H, s, H-29 ); 13C NMR (CD3OD, 100 MHz)
b
d
15.0, 15.2, 15.3, 17.1, 19.1, 38.1, 41.9, 43.3, 43.6, 48.3, 48.5, 48.7, 49.2,
49.4, 49.6, 52.0, 57.1, 67.6, 74.0, 109.9, 152.1, 174.3, 174.6, 179.0.
The synthesis of compound 54 has been discussed in Scheme 4
and Fig. 2. MS (ESI): m/z 650.0 [M þ H]þ; HR-ESI-MS: m/z 649.4940
[M þ H]þ (calcd: 649.4949); 1H NMR (CDCl3, 400 MHz)
d: 0.85, 0.85,
4.1.51. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-
L
-proline acyl]-
L
-
0.90, 0.92 (each 3H, s, Me-24, -25, -26, and -27), 1.48 (3H, s, Me-30),
methionine acid (51)
1.86 (8H, H-20,200,60,600), 2.02 (1H, m, H-18), 1.95 (1H, m, H-19), 2.96
White foam, yield: 80.0%. MS (ESI): m/z 699.9 [M ꢂ H]ꢂ, 701.9
(1H, m, H-3), 3.76 (1H, d, J ¼ 7.2 Hz, H-23
H-23 ), 4.52 (1H, s, H-29 ), 4.72 (1H, s, H-29
100 MHz) : 11.2,14.6,16.1,16.4,19.4,19.6, 21.1, 24.6, 25.6, 26.2, 29.0,
a
), 3.98 (1H, d, J ¼ 7.2 Hz,
[M þ H]þ; HR-ESI-MS: m/z 723.4383 [M þ Na]þ(calcd: 727.4377); 1H
b
a
b
); 13C NMR (CDCl3,
NMR (pyridine-d5, 400 MHz)
d
: 0.97,1.02,1.06,1.17 (each 3H, s, Me-24,
d
-25, -26, and -27),1.80 (3H, s, Me-30),1.97 (4H, m, Me-30, 40), 2.05 (1H,
29.9, 31.4, 35.9, 36.8, 37.1, 40.6, 41.8, 41.9, 45.6, 50.4, 52.7, 54.6, 62.7,
70.5, 73.3, 76.7, 109.0, 151.4, 173.2.
m, H-18), 2.11 (3H, s, Me-400), 2.15 (1H, m, H-19), 2.31 (4H, m, H-100, 200),
3.05 (1H, m, H-3), 3.72 (1H, d, J ¼ 11.4 Hz, H-23
J ¼ 11.4 Hz, H-23
), 4.22 (2H, t, H-20), 4.76 (1H, s, H-29
H-50), 4.99 (1H, s, H-29 ); 13C NMR (pyridine-d5, 100 MHz)
a
a
), 4.19 (1H, d,
), 4.95 (1H, m,
: 12.8,
b
4.1.56. 3,23-O-Diacetyl-17-(2-(3-carboxypropanamido)
ethylcarbamoyl)betulinic amide (55)
b
d
14.8,15.1,16.6,16.8,18.5,19.8, 21.4, 26.2, 27.8, 29.8, 30.7, 34.5, 37.2, 37.3,
39.1, 41.1, 42.4, 42.9, 48.8, 51.1, 55.4, 61.7, 67.9, 73.4, 109.4, 152.0, 174.1.
To a solution of compound 7 (0.15 g, 0.25 mmol) in dry
dichloromethane (40 mL) was added succinic anhydride (0.084 g,
0.84 mmol), DMAP (0.10 g, 0.84 mmol). The solution was stirred and
refluxed for 6 h. At which time the reaction was completed, the
solution was washed by water (40 mL) and brine (40 mL), dried
with anhydrous Na2SO4. Filtered, the filtrate was concentrated, the
residue was dissolved in dichloromethane and purified on a silica
gel column (200 g, 1:1 petroleum ethereacetone) to give 55 (0.13 g,
72.2%) as white foam. MS (ESI): m/z 700.0 [M þ H]þ, 697.9 [M ꢂ H]ꢂ,
721.9 [M þ Na]þ; HR-ESI-MS: m/z 721.4402 [M þ Na]þ (calcd:
4.1.52. N-[N-(3,23-Dihydroxy-17-betulinic acyl)-L-glycine acyl]-L-
tryptophane acid (52)
White foam, yield: 58.4%. MS (ESI): m/z 715.0 [M ꢂ H]ꢂ; HR-ESI-
MS: m/z 714.4482 [M ꢂ H]ꢂ (calcd: 714.4488); 1H NMR (CD3OD,
400 MHz) d: 0.65, 0.81, 0.83, 0.95 (each 3H, s, Me-24, -25, -26, and
-27), 1.67 (3H, s, Me-30), 2.09 (1H, m, H-18), 2.47 (1H, m, H-19), 3.19
(1H, m, H-3), 3.27 (1H, d, J ¼ 10.8 Hz, H-23 ), 3.49 (1H, d,
J ¼ 10.8 Hz, H-23
), 3.55 (2H, m, H-100), 3.84 (2H, m, H-20), 4.55 (1H,
s, H-29
a
b
721.4398); 1H NMR (CDCl3, 400 MHz)
d: 0.82, 0.88, 0.93, 0.97 (each
a
), 4.57 (1H, m, H-50), 4.67 (1H, s, H-29
b
), 6.97 (1H, t, H-700),
3H, s, Me-24, -25, -26, and -27), 1.69 (3H, s, Me-30), 1.98 (1H, m,
H-18), 2.03 (3H, s, 3-OAc), 2.07 (3H, s, 23-OAc), 2.45 (1H, m, H-19),
2.53 (2H, t, H-20), 2.69 (2H, t, H-30), 3.07 (1H, m, H-3), 3.39 (4H, m,
7.04 (1H, t, H-800), 7.09 (1H, s, H-300), 7.27 (1H, d, J ¼ 8.0 Hz, H-600),
7.54 (1H, d, J ¼ 7.8 Hz, H-900); 13C NMR (CD3OD, 100 MHz)
d: 10.6,
15.2, 21.5, 36.2, 40.0, 41.4, 46.5, 46.7, 46.9, 47.1, 47.2, 47.2, 47.4, 47.4,
47.5, 47.6, 47.7, 55.2, 65.7, 72.1, 108.1, 109.8, 110.3, 117.6, 117.8, 120.4,
122.4, 127.5, 136.1, 150.4, 169.8, 177.7.
H-60, 70), 3.69 (1H, d, J ¼ 11.7 Hz, H-23
a
), 3.85 (1H, d, J ¼ 11.7 Hz, H-
23b), 4.61 (1H, s, H-29a), 4.77 (1H, s, H-29b
); 13C NMR (CDCl3,
100 MHz) d: 12.5, 14.1, 15.7, 16.2, 17.5, 18.9, 20.5, 20.8, 29.5, 30.4,
36.6, 37.3, 40.2, 40.3, 42.0, 46.3, 47.6, 49.6, 50.2, 55.3, 65.0, 74.1,
109.1, 150.2, 170.2, 170.6, 172.9, 174.8, 177.8.
4.1.53. 1,40-Dipiperidine-1-carbonyl chloride
To a solution of triphosgene (8 g, 26.9 mmol) in dry dichloro-
methane (40 mL) at ꢂ7 ꢃC were added dry triethylamine (1 mL,
7.1 mmol) and dropwise a solution of 1,40-dipiperidine (5 g,
29.7 mmol) in dry dichloromethane (35 mL) over a period of
50 min. Then the solution was stirred at room temperature for 8 h,
at which time the reaction was completed, the solution was filtered,
the filtrate was concentrated, the residue was reacted with
23-hydroxybetulinic acid directly without further purification.
4.2. Antiproliferative activity
4.2.1. Cell culture
The human breast carcinoma cell line MCF-7 and human hepa-
tocellular carcinoma cell line HepG2 were purchased from American
Type Culture Collection; the human cervical carcinoma cell line
Hela, human melanoma cell line A375 and mice melanoma cell line
B16 were obtained from Chinese Academy of Sciences Committee
Type Culture Collection. All cell lines were cultured in RPMI 1640
(Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin
streptomycin (Gibco) at 37 ꢃC in the presence of 5% CO2.
4.1.54. 3-Hydroxy-23-O-(1,40-bipiperidine-1-carbonyl)betulinic
acid (53)
To a solution of 1,40-dipiperidine-1-carbonyl chloride mixture
obtained from previous reaction in dry pyridine (120 mL) was added
23-hydroxybetulinic acid (1.0 g, 2.1 mmol). The solution was stirred
at room temperature for 16 h. At which time the reaction was
completed, ethyl acetate (100 mL) was added, the pH of the solution
was adjusted to 4e5 by 10% HCl, the organic layer was separated and
washed by brine (100 mL), dried with anhydrous Na2SO4. Filtered,
the filtrate was concentrated, the residue was dissolved in methanol
and purified on a silica gel column (200 g, chloroformemethanol
5:1) to give 53 (1.2 g, 85.7%) as white foam. MS (ESI): m/z 667.5
4.2.2. Viability assay
MTT assay was used to evaluate the antiproliferative activity of
these compounds. Briefly, cells were seeded in 96-well plates
overnight. All of the reported 23-hydroxybetulinic acid derivatives
were dissolved in DMSO while the positive control doxorubicin was
dissolved in PBS. These tested compounds at different concentra-
tions were added into wells and cells were treated at 37 ꢃC for 72 h.
Then MTT (5 mg/mL, in PBS) was added into each well and cultured
for another 4 h. The optical density was detected in a microplate
reader at 570 nm. IC50 values were calculated according to the
dose-dependent curves.
[M þ H]þ, 665.9 [M ꢂ H]ꢂ; HR-ESI-MS: m/z 667.5056 [M þ H]þ
1
(calcd: 667.5044); H NMR (DMSO-d6, 400 MHz)
d
: 0.51, 0.78, 0.86,
0.94 (each 3H, s, Me-24, -25, -26, and -27), 1.36 (6H, H-300,400,500),1.46
(4H, H-30, 50), 1.66 (3H, s, Me-30), 1.92 (1H, m, H-18), 2.12 (1H, m, H-
19), 2.42 (2H, t, H-200), 2.50 (2H, t, H-600), 2.86 (2H, t, H-20), 3.02 (2H, t,
Acknowledgments
H-60), 3.06 (1H, m, H-3), 3.84 (1H, d, J ¼ 11.6 Hz, H-23
a
), 4.01 (1H, d,
J ¼ 11.6 Hz, H-23
b
), 4.58 (1H, s, H-29
a), 4.71 (1H, s, H-29
b
); 13C NMR
We gratefully acknowledge support for this research from the
Natural Science Foundation of Guangdong Province (No.
9151063201000053), Program for Changjiang Scholars and Inno-
vative Research Team in University (IRT0965), Science and
(DMSO-d6, 100 MHz) d: 12.3, 13.4, 14.2, 18.5, 18.8, 24.4, 26.0, 29.9,
36.3, 38.8, 39.2, 39.5, 39.7, 39.9, 40.1, 41.8, 42.0, 46.6, 49.5, 56.4, 60.7,
64.9, 70.2, 109.9, 149.6, 166.8, 171.6.

2502
P. Lan et al. / European Journal of Medicinal Chemistry 46 (2011) 2490e2502
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Appendix. Supplementary material
Supplementary material associated with this article can be
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