A partial synthesis of 23-hydroxybetulonic acid and 23-hydroxybetulinic acid starting from betulinic acid

Article abstract of DOI:10.3184/174751912X13332875186150

A partial syntheses of the biologically active 23-hydroxybetulonic acid and 23-hydroxybetulinic acid starting from the naturally abundant betulinic acid has been developed in which the key step was the Baldwin's cyclopalladation of the 23-methyl group from a 3-one oxime of betulinic acid, an important drug in Chinese medicine.

Full text of DOI:10.3184/174751912X13332875186150

254 RESEARCH PAPER
MAY, 254–257
JOURNAL OF CHEMICAL RESEARCH 2012
A partial synthesis of 23-hydroxybetulonic acid and 23-hydroxybetulinic
acid starting from betulinic acid
Fei Sun, Peiqing Zhu, Hequan Yao, Xiaoming Wu and Jinyi Xu*
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, P. R. China
A partial syntheses of the biologically active 23-hydroxybetulonic acid and 23-hydroxybetulinic acid starting from the
naturally abundant betulinic acid has been developed in which the key step was the Baldwin’s cyclopalladation of the
23-methyl group from a 3-one oxime of betulinic acid, an important drug in Chinese medicine.
Keywords: betulinic acid, 23-hydroxybetulonic acid, 23-hydroxybetulinic acid, cyclopalladation, Baldwin’s method
Betulinic acid (1), 23-hydroxybetulonic acid (Pulsatillic acid,
2) and 23-hydroxybetulinic acid (3) (Fig. 1) are all naturally
occurring pentacyclic triterpenes of the lupane family. Com-
pound 1 is easily obtained from the bark of various trees (up to
2.5% of dry weight),1 while 2 and 3 were both isolated from
the root of Pulsatilla chinensis Regel.2,3 Betulinic acid is
one of the most important crude drugs in traditional Chinese
medicine and is used for the treatment of amoebic dysentery
and malaria.4 Some evidence shown that 1 possesses a variety
of biological activities such as anti-tumour, antibacterial,
anti-malarial, anti-inflammatory, anthelmintic antioxidant and
anti-HIVactivities,5–7 whilst 2 and 3 have been proven to have
anti-tumour and anti-HIV activities.8–10 Thus, 2 and 3 showed
strong cytotoxic activity on murine melanoma B16 cells with
IC50 of 22.5 and 32 µg mL−1, respectively.
with NaOH in THF and CH3OH yielded oxime 8, which was
hydrolysed with TiCl3 to give ketone 9. Selective hydrogenoly-
sis of the benzyl group of 9 with Pd/C in THF produced
23-hydroxybetulonic acid 2 in 75% yield. This step must be
carefully monitored by TLC to ensure that the C–C double
bond of 9 was not hydrogenated. Reduction of 2 with NaBH4
in CH3OH and THF smoothly provided 23-hydroxybetulinic
acid 3 in good yield. The analytical data of synthetic 2 and
3 are in good agreement with those reported for the natural
samples (Tables 1 and 2).
Conclusions
We have developed a semisynthetic approach to 23-hydroxy-
betulonic acid (2) and 23-hydroxybetulinic acid (3) starting
from naturally abundant betulinic acid (1) in an overall yield of
34% and 29%, respectively. The present contribution should
facilitate the structural modification of the natural products
2 and 3 for further pharmacological research, which is
proceeding in our laboratory.
Compound 3 could also inhibited HMEC proliferation
significantly with IC50 of 40.44 µg mL−1 and arrested growth
and induced apoptotic cell death in human leukemia HL-60
cells. This was associated with concurrent down-regulation of
Bcl-2 and the telomerase activity.
Recently, we have carried out the structure modification of
3 for new drug screening and excellent results have been
obtained.11–15 However, further pharmacological investigation
was hampered by the limited amount of 3 which was available
from plant extraction.16,17 Though 2 and 3 have been identified
for many years, no practical semisynthetic approach to these
substances has been established to date. We envisioned that 2
and 3 could be achieved through a simple introduction of
hydroxyl group on the 23-methyl group of naturally abundant
betulinic acid 1. We noticed a report by Baldwin that an
unactivated methyl groups could be functionalised by a cyclo-
palladation reaction (Baldwin’s method).18,19 This method has
been extensively used in the synthesis of natural triterpene
compounds.20,21 These results led us to propose the key step
by cyclopalladation of the 23-methyl group from a 3-one
oxime functionality of 1 to obtain 2 and 3. We now report a
semisynthetic approach to 2 and 3 starting from 1 by using this
method.
Experimental
Melting points were taken on XT-4 micro melting point apparatus and
are uncorrected. H NMR and 13C NMR spectra were recorded with
1
Bruker ACF 300 spectrometer in the indicated solvents (TMS as inter-
nal standard). Mass spectra were obtained using Agilent 1100-LC-
MSD-Trap/SL and HRMS were obtained using Agilent QTOF 6520.
Column chromatography was carried out using silica gel (200–
300 mesh).
Benzyl 3β-hydroxy-lup-20(29)-en-28-oate (4): BnBr (0.30 mL,
2.5 mmol) was added to a solution of 1 (1.00 g, 2.2 mmol) and K2CO3
(0.5 g, 3.6 mmol) in DMF (20 mL). The reaction mixture was stirred
for 12 h at room temperature, poured into water (30 mL) and extracted
with ethyl acetate (3×20 mL). The combined extract was washed with
water and brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The crude product was purified by column chromatography
(petroleum ether/ethyl acetate, 6:1) to give 4 as a white solid (1.10 g,
92%). m.p. 190–193 °C; ESI-MS m/z: 547.4 [M + H]+, 564.4
[M + NH4]+; HR-MS (ESI, M+H) m/z: Calcd for C37H55O3: 547.4151,
found 547.4154.
Benzyl 3-oxo-lup-20(29)-en-28-oate (5): PCC (0.50 g, 2.33 mmol)
was added to a solution of 4 (1.05 g, 1.92 mmol) in CH2Cl2 (20 mL).
The reaction mixture was stirred for 5 h at room temperature. The
reaction mixture was washed with water and brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The crude product was purified by
column chromatography (petroleum ether/ethyl acetate, 40:1) to give
5 as a white solid (0.96 g, 92%). m.p. 127–129 °C; ESI-MS m/z: 545.4
[M + H]+, 562.5 [M + NH4]+; HR-MS (ESI, M+H) m/z: Calcd for
C37H53O3: 545.3995, found 545.3993.
Benzyl 3-hydroxyimino-lup-20(29)-en-28-oate (6): Hydroxylamine
hydrochloride (0.2 g, 2.9 mmol) was added to a solution of 5 (0.95 g,
1.7 mmol) in pyridine (20 mL. The reaction mixture was stirred at
80 °C for 4 h, cooled to r.t., ethyl acetate (40 mL) was added.
The mixture was washed with 10% HCl, water and brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The crude product was
purified by column chromatography (petroleum ether/acetone, 16:1)
to give 6 as a white solid (0.91 g, 93%). m.p. 232–235 °C; 1H NMR
Results and discussion
The synthesis of 2 and 3 from betulinic acid 1 is shown in
Scheme 1. Treatment of 1 with BnBr in DMF in the presence
of K2CO3 afforded benzyl betulinate 4, which was then
converted to ketone 5 by oxidation with PCC in CH2Cl2. The
ketone 5 was converted to the oximated with hydroxylamine
hydrochloride in pyridine to give benzyl betulinate 3-oxime 6.
Balwin’s method was then applied to introduce a hydroxyl
group on the 23-methyl group of 6. Oxime 6 was first palladated
with Na2PdCl4 to afford a dimeric organopalladium complex.
Subsequently, acetylation with Ac2O, oxidation with
Pb(OAc)4, and reduction with NaBH4 smoothly gave diacetate
7 in up to 81% yield (for three steps). Finally, hydrolysis of 7
* Correspondent. E-mail: jinyixu@china.com

JOURNAL OF CHEMICAL RESEARCH 2012 255
Fig. 1 Structures of pentacyclic triterpenes 1, 2 and 3.
Table 1 13C NMR spectral data of synthetic 2 and 3 compared
with literature (pyridine-d5, δ)
[M + H]+; HR-MS (ESI, M+H) m/z: Calcd for C37H54NO3: 560.4104,
found 560.4109.
Benzyl3-acetoxyimino-23-acetoxy-lup-20(29)-en-28-oate(7):NaOAc
(0.17 g, 2.0 mmol) and Na2PdCl4 (0.61 g, 2.1 mmol) were added to a
solution of 6 (0.9 g, 1.61 mmol) in AcOH (100 mL),. The solution was
stirred for 72 h at room temperature, and then ice water (120 mL) was
added to give a yellow precipitate. The precipitate was filtered and
dried in vacuo at 60 °C for 24 h to give a palladium complex. DMAP
(10 mg), Et3N (0.3 mL), and Ac2O (0.2 mL) were added to a solution
of this palladium complex in anhydrous CH2Cl2 (30 mL). The reaction
mixture was stirred at r.t. for 1 h, washed with water, dried over
Na2SO4, filtered, and evaporated in vacuo. The crude product was
dissolved in anhydrous THF (80 mL), and pyridine (0.2 mL) was
added. The reaction mixture was stirred for 15 min at room tempera-
ture and cooled at –80 °C, and a solution of Pb(OAc)4 (1.2 g,
2.7 mmol) in HOAc (40 mL) was added and again stirred at room
temperature for 20 h. To remove the remaining Pd salts, a solution
of NaBH4 (0.1 g) in 1 N NaOH solution (10 mL) was added to the
reaction mixture. The mixture was stirred for 15 min and filtered.
The filtrate was diluted with ethyl acetate (70 mL), and washed with
saturated aqueous NaHCO3 solution, dried over Na2SO4, filtered, and
evaporated in vacuo to give a solid. The crude product was purified by
column chromatography (petroleum ether/ethyl acetate, 10:1) to give
2
3
Ref.
2
Synth.
Ref.
3
Synth.
C1
38.4
38.53
36.60
217.09
46.96
49.76
20.03
33.60
40.94
52.67
36.77
21.82
26.16
38.72
42.88
31.21
32.80
56.62
47.73
49.68
151.28
30.22
37.56
68.2
39.2
38.99
27.76
73.31
42.72
48.67
18.44
34.39
40.98
50.86
37.22
21.10
25.98
38.48
42.85
31.06
32.71
56.48
47.63
49.62
151.17
30.14
37.42
67.8
C2
36.2
217.2
47.0
49.6
19.9
33.5
40.8
52.4
36.6
21.7
26.0
38.6
42.7
31.1
32.6
56.4
47.5
49.6
151.1
30.0
37.3
68.0
17.2
16.0
15.9
14.6
178.6
109.6
19.3
27.9
73.5
42.9
48.8
18.6
34.5
41.4
51.0
37.4
21.3
26.1
38.6
42.9
31.2
32.9
56.6
47.7
49.7
151.2
30.3
37.6
68.0
12.9
16.8
16.5
14.9
178.9
109.9
19.5
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
1
7 as a white solid (0.86 g, 81%). m.p. 64–67 °C; H NMR (CDCl3,
300 MHz): δ 0.78, 0.86, 0.94, 1.15, 1.68 (each 3H, s, 24, 25, 26, 27
and 30-CH3), 2.05, 2.16 (each 3H, s, Ac×2), 2.22–2.30 (2H, m),
2.58–2.66 (2H, m), 3.01 (1H, m, H-19), 4.13 (2H, dd, J = 27.5,
11.1 Hz, H-23), 4.60, 4.72 (each 1H, s, H-29), 5.12, 5.17 (each 1H, d,
J = 12.3 Hz, CH2-Ar), 7.31–7.37 (5H, m, H-Ar); ESI-MS m/z: 660.4
[M + H]+, 680.5 [M + NH4]+; HR-MS (ESI, M+H) m/z: Calcd for
C41H58NO6: 660.4264; found 660.4268.
Benzyl 3-hydroxyimino-23-hydroxy-lup-20(29)-en-28-oate (8): 4 N
NaOH solution (3 mL) was added to a solution of 7 (0.85 g, 1.3 mmol)
in CH3OH (15 mL) and THF (9 mL). The reaction mixture was stirred
at room temperature for 1 h, diluted with ethyl acetate (30 mL),
washed with water and brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. The crude product was purified by column chroma-
tography (petroleum ether/ethyl acetate, 16:1) to give 8 as a white
solid (0.71 g, 95%). m.p. 224–226 °C; 1H NMR (CDCl3, 300 MHz):
δ 0.79, 0.93, 0.94, 1.01, 1.67 (each 3H, s, 24, 25, 26, 27 and 30-CH3),
17.52
16.29
16.08
14.75
178.83
109.97
19.48
12.77
16.66
16.31
14.75
178.7
109.79
19.31
(CDC3, 300 MHz): δ 0.79, 0.89, 0.93, 1.03, 1.12, 1.67 (each 3H, s, 23,
24, 25, 26, 27 and 30-CH3), 2.15–2.29 (3H, m), 2.91–3.02 (2H, m,
H-19 and H-2b), 4.60, 4.72 (each 1H, s, H-29), 5.12,5.18 ( each 1H, d,
J = 12.2 Hz, CH2-Ar), 7.31–7.52 (5H, m, H-Ar); ESI-MS m/z: 560.5

256 JOURNAL OF CHEMICAL RESEARCH 2012
Scheme 1 Reagents and conditions: (i) BnBr, K2CO3, DMF, r.t., 12 h (92%); (ii) PCC, CH2Cl2, r.t., 2 h, (92%); (iii) HONH2.HCl,
pyridine, 80 °C, 4h, (93%); (iv) (a) Na2PdCl4, NaOAc, HOAc, r.t., 72 h; (b) Ac2O, DMAP, Et3N, CH2Cl2, r.t., 1h; (c) Pb(OAc)4, pyridine,
HOAc, THF, −78 °C to r.t., 20 h (81% for 3 steps ); (v) NaOH, THF, CH3OH, r.t., 1 h (95%); (vi) TiCl3, HCl, NH4OAc, H2O, THF, r.t., 6 h,
(75%); (vii) H2, Pd/C, THF, r.t., (75%); (viii) NaBH4, THF, CH3OH, r.t., 2 h (87%).

JOURNAL OF CHEMICAL RESEARCH 2012 257
Table 2 1H NMR spectral data of synthetic 2 and 3 compared to the literature
2
2
2
3
3
3
Ref. 2 CDCl3
(synth.) CDCl3
(synth.) pyridine-d5
Ref. 3 CDCl3
(synth.) CDCl3
(synth.) pyridine-d5
H-24
H-25
H-26
H-27
H-30
H-19
H-23
H-3
0.99
0.99
0.99
1.00
0.81
0.96
0.86
0.86
0.86
0.86
0.88
1.00
1.01
1.00
1.01
0.94
0.94
1.02
1.04
1.04
1.04
0.98
0.97
1.06
1.69
1.70
1.77
1.70
1.69
1.76
3.04
3.01
3.51
3.02
3.00
3.52
3.41, 3.64
—
3.41, 3.64
—
3.66, 3.95
—
3.44, 3.74
3.64
3.42, 3.72
3.62
3.69, 4.16
4.19
H-29
4.62, 4.74
4.62, 4.75
4.75, 4.92
4.63, 4.74
4.61, 4.74
4.74, 4.91
Table 3 Melting point of synthetic 2 and 3 compared with
This work was supported by National Natural Science Fund
Committee (No. 30472083) and Jiangsu Natural Science
Foundation (Grant BK2005101) and Simcere PhD Program
Foundation (No. 02704003)
literature
Compound
2
3
Ref.
2
Synth.
Ref.
3
Synth.
Received 12 January 2012; accepted 29 February 2012
Paper 1201102 doi: 10.3184/174751912X13332875186150
Published online: 10 May 2012
M.p.
214–217 °
C
219–221 °
C
300–302 °
C
303–305 °C
2.06–2.30 (3H, m), 3.01–3.10 (2H, m, H-19 and H-2b), 3.48, 3.63
(each 1H, d, J = 11.5 Hz, H-23), 4.59, 4.72 (each 1H, s, H-29), 5.12,
5.17 (each 1H, d, J = 12.3 Hz, CH2-Ar), 7.31–7.36 (5H, m, H-Ar);
ESI-MS m/z: 576.4 [M + H]+; HR-MS (ESI, M+H) m/z: Calcd for
C37H54NO4:576.4053, found 576.4049.
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M.p. 187–189 °C; H NMR (CDCl3, 300 MHz): δ 0.81, 0.95, 0.99,
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+57.7 (c, 0.84, pyridine); ESI-MS: m/z = 469 [M - H]–.
8
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