Synthesis of berbamine acetyl glycosides and evaluation of antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2012.04.042

A series of berbamine glycosides was designed, synthesized and evaluated as a new class of antitumor agents. An efficient glycosylation route was developed for berbamide derivatives. The newly synthesized glycosides were evaluated for their cytotoxic activity in vitro against a human leukemia cell line K562, a human lung adenocarcinoma cell line A549 and mouse lymphocytic leukemia cells L1210. In contrast to berbamine most of its glycosides manifested potent cytotoxic activities. The acetyl glycosyl berbamine 5a, 5d caused distinct improvement against K562, A549 and L1210. It is suggested that the acetyl d-glucose residue has affinity to these cancer cells.

Full text of DOI:10.1016/j.ejmech.2012.04.042

European Journal of Medicinal Chemistry 54 (2012) 867e872
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of berbamine acetyl glycosides and evaluation of antitumor activity
,
a
b
,
,
Yanli Cui a , Minghan Xu , Jianwei Mao c, Jingfeng Ouyang d, Rongzhen Xu e f, Yongping Yu d
*
a Department of Chemistry, Zhejiang University, Zheda Road 38, Hangzhou 310027, PR China
b Department of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, PR China
c Zhejiang Provincial Key lab for Chem. & Bio. Processing Technology of Farm Produces, Hangzhou 310023, PR China
d College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
e Department of Hematology, Second Affiliated Hospital, Zhejiang University, Hangzhou 310009, PR China
f Cancer Institute, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< A series of berbamine (natural
alkaloid)
glycosides
was
synthesized.
< The new glycosides were evaluated
for their antitumor activity in vitro.
< Most of the berbamine glycosides
manifested potent cytotoxic activity.
< The most potent compound showed
an IC50 of 0.30
mM against L1210
leukemia cell line.
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of berbamine glycosides was designed, synthesized and evaluated as a new class of antitumor
agents. An efficient glycosylation route was developed for berbamide derivatives. The newly synthesized
glycosides were evaluated for their cytotoxic activity in vitro against a human leukemia cell line K562,
a human lung adenocarcinoma cell line A549 and mouse lymphocytic leukemia cells L1210. In contrast to
berbamine most of its glycosides manifested potent cytotoxic activities. The acetyl glycosyl berbamine
Received 28 January 2012
Received in revised form
24 April 2012
Accepted 27 April 2012
Available online 7 May 2012
5a, 5d caused distinct improvement against K562, A549 and L1210. It is suggested that the acetyl
glucose residue has affinity to these cancer cells.
D-
Keywords:
Berbamine
Ó 2012 Elsevier Masson SAS. All rights reserved.
Acetyl glycoside
Antitumor
Glycosylation
Glycosyl bromide
1. Introduction
advantageous strategy in drug design [1]. Berbamine is a natural
product derived from the plant Berberis amurensis (xiaoboan), which
has been used extensively in Asia and Europe for the treatment of
various ailments. Due to its bis-benzylisoquinoline alkaloid structure,
berbamine has been demonstrated to possess a number of interesting
and potent biological activities [2e6], such as an agent against human
breast cancer cells, inducing apoptosis in human myeloma cells and
suppression of human lung cancer cell growth. It was found by our
groupthatberbaminecanselectivelyinducecelldeathofbothGleevec
sensitive and resistant-Phþ chronic myeloid leukemia (CML) cells [7]
and selectively induce caspase-3-dependent apoptosis of leukemia
From ancient to modern times medicine has been closely linked to
the use of traditional medicines and natural products (NPs). Currently
roughly half (49%) of the New Chemical Entities (NCEs) introduced are
natural products, semi-synthetic natural product analogs or synthetic
compounds based on natural products. Utilizing scaffolds of natural
products combined with synthetic modifications is clearly an
* Corresponding author. Tel.:þ86 13858036095.
E-mail address: hnzzcyl@hotmail.com (Y. Cui).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.042

868
Y. Cui et al. / European Journal of Medicinal Chemistry 54 (2012) 867e872
NB4 cells via the survivin-mediated pathway [8]. In order to explore
the effects and mechanism of berbamine on antitumor activity, we
endeavored to make synthetic analogs of berbamine. A handful of the
synthetic berbamine derivatives have been reported [9e12]. However,
the glycosylation derivatives of berbamine for antitumor activity to
the best of our knowledge have not been reported.
With their high density of defined spatial orientations and their
relative rigidity, carbohydrates provide excellent platforms upon
which to explore unique features for the drug-discovery process
[13]. Substituted carbohydrate derivatives are generally quite stable
and usually display reasonable stability to gastric acids and liver
metabolism, which is in contrast to unsubstituted saccharides that
usually undergo rapid metabolism in a biological environment [14].
There is a number of acetyl glycosides lead compounds that
demonstrate more active than their unsubstituted carbohydrate
analogs [15,16]. We aimed to synthesize the acetyl glycosides of
berbamine for potential therapeutic treatment.
dihydrochloride 1 was neutralized and alkalified to afford the solid
berbamine salt 3 using sodium bicarbonate and solid KOH.
The critical step in our analog production was optimizing the
formation of the berbamine phenolate. Carbohydrates carrying an
aromatic aglycon are important natural products and thus key
synthetic targets. The glycosylation of the phenols in an alkaloid is
a challenge. First the phenolichydroxyl phenols are ambident
nucleophiles in the glycosylation. A second problem is steric
hindrance from substituents on the alkaloid compound (Fig. 1)
[17,18]. Compared to acetate and trichloroacetimidate glycosyl
donors, a glycosyl halide is better in an alkaline acceptor system.
Initially we used the Michael procedure [19] which is predominantly
used for the glycosylation of the phenols (i.e., the use of a glycosyl
halide in combinationwith a phenolate dissolved in aprotic solvents
under phase-transfer catalyst conditions). After many trials it was
proved that the procedure was not suitable for berbamine. For our
glycosylation route a modified KoenigseKnorr procedure was
finally chosen. The glycopyranosyl bromides 4aef were prepared
according to the methods described elsewhere [20] and then the
abovementioned glycosyl bromide reacted with berbamide salt 3
via the modified KoenigseKnorr procedure to afford the corre-
sponding berbamide glycosides 5aef. After the screening of cata-
lysts and solvents, silver oxide and acetonitrile were chosen and
proved to be effective for the berbamine glycosylation.
2. Results and discussions
2.1. Chemistry
The synthesis of the berbamine glycosides is illustrated and
outlined in Schemes 1 and 2. Pharmaceutical grade berbamine
Scheme 1. Synthesis of berbamine glycosides 5aef. Reagents and conditions: (a) sat NaHCO3 aq, CH2Cl2, neutralization. (b) KOH, water, pH ¼ 13, 5 h, 40 ꢀC. (c) 4aef, Ag2O, dry
CH3CN, 4 Å ms, 40 ꢀC, overnight.

Y. Cui et al. / European Journal of Medicinal Chemistry 54 (2012) 867e872
869
Scheme 2. Reaction of berbamine glycoside 5d. Reagents and conditions: (a) 0.05 M NaOMe (CH2Cl2:MeOH ¼ 1:1 volume), rt, 24 h, 90%. (b) 5d: HCl (3%, water) ¼ 1:2 (mol ratio),
quantitatively.
A class of berbamine glycosides 5aef containing
-galactose, -xylose, -cellobiose, -lactose and -glucose residues
respectively was made. Glycosyl bromides are usually isolated as
the thermodynamically favored -anomer and due to participating
groups (e.g., esters) at sugar C2 glycosylation generally results in
-glycosides. The yields of 5aef glycosides were 62%, 50%, 50%, 65%,
37% and 21% respectively under our conditions. The preliminary
experiments show that acetyl -cellobiose derivative 5d improved
D
-glucose,
2.2. Biological activities
D
D
D
D
L
Three kinds of cancer cells were evaluated, namely, a human
leukemia cell line K562, a human lung adenocarcinoma cell line
A549 and mouse lymphocytic leukemia cells L1210. The results are
summarized in Table 1.
As shown in Table 1, most compounds exhibited antitumor
activities. From the IC50 data of these compounds, we notice that
a
b
D
their cytotoxicity against K562, L1210 and A549 distinctly. To
compare the different 5d derivatives’ influence to biological activity
compound 6d (deacetylation of 5d) and compound 5d1 (chloride
salt form of 5d) were produced. The procedures were shown in
Sections 4.1.3, 4.1.4 and Scheme 2.
The chemical structures of the compounds were confirmed by
1H NMR, 13C NMR spectroscopy and HRMS. The results are
presented in Section 4.3. The 1H NMR chemical shifts of the
berbamine glycosides 5aef showed the formation of the
acetyl
improved their cytotoxicity against K562, L1210 and A549 distinctly.
Interestingly, the cellobiose derivative 5d showed an IC50 of 0.30
against L1210 leukemia cell-line compared with native berbamine
IC50 of 2.73 M. Considering the acetyl -glucose derivative’s similar
increase in activity suggests that the acetyl -glucose residue has
affinity to these cancer cells. The acetyl -galactose and acetyl
-lactose derivatives’ cytotoxicity against A549 and L1210 corre-
sponding cancercellswas similarto thenative ordecreasedprobably
due to the acetyl -galactose residues. The addition of acetyl glycosyl
D
-glucose derivative 5a and acetyl
D
-cellobiose derivative 5d
mM
m
D
D
D
D
b
-glycosides.
D
groups affected the biology of berbamine analogs greatly. The degree
of influence achieved from smallest to largest was A549, K562 and
L1210 cells respectively. The best activity against K562, A549 and
L1210 respectively belonged toacetyl
cellobiose derivative 5d, and acetyl
In contrast with acetyl -glucose derivative 5a and acetyl
glucose derivative 5f, -glucose derivative showed more potent
cytotoxicity than -glucose residue. It was reported that a good
D
-xylosederivative 5c, acetyl
-cellobiose derivative 5d.
-acetyl
D-
D
D
L
D
L
number of active natural products owned unsubstituted carbohy-
drate moiety, then the deacetylation structure 6d demonstrated less
active than its acetyl carbohydrate analog 5d. Additionally the
hydrochloride salt 5d1 of
D-cellobiose derivative 5d and berbamine
hydrochloride salt have similar solubility, but protonation
1
decreased the cytotoxicity. Work to uncover the molecular mecha-
Fig. 1. The structure of berbamine.
nism of berbamine’s cytotoxicity is ongoing.

870
Y. Cui et al. / European Journal of Medicinal Chemistry 54 (2012) 867e872
Table 1
Berbamine glycosides and their IC50 values for antitumor.
Compounds 5
Compounds 5 structures
Compounds 5 sugar residues
A549 IC50
29.51
(m
M)
L1210 IC50
0.57
(m
M)
K562 IC50
1.96
(mM)
5a
Acetyl-
Acetyl-
D
-Glucose
5b
D
-Galactose
42.99
2.17
2.03
5c
Acetyl-
Acetyl-
D
D
-Xylose
NA
1.27
0.30
1.55
2.23
5d
-cellobiose
27.75
5e
Acetyl-
Acetyl-
D
-Lactose
NA
2.03
3.56
5f
L-glucose
NA
NA
2.74
NA
NA
6d
D
-cellobiose
10.62
5d1
1
Acetyl-
/
D
-cellobiose
NA
1.85
2.73
NA
Bera
39.72
4.00
Ber ¼ berbamine; Bera ¼ berbamine dihydrochloride; NA: IC50 ꢁ 50 (
mM).
3. Conclusions
The other commercial reagents were used directly without further
purification.
An efficient glycosylation route of berbamine (a natural alkaloid)
was found and a series of berbamine glycosides was achieved. The
novel glycosyl derivatives of berbamine were evaluated for their
cytotoxic activity in vitro against three cancer cell lines (K562, A549
and L1210). Most of the glycosyl derivatives of berbamine exhibited
potent cytotoxicities, several showed more significant cytotoxicity
than native berbamine. The acetyl glycosides of berbamine 5a, 5d
caused distinct improvement against K562, A549 and L1210. It is
4.1. General procedure for berbamine derivatives
4.1.1. Procedure for berbamine salt 3
Pharmaceutical grade berbamine dihydrochloride (1, 1.4944 g,
2.19 mmol) was washed with saturated sodium bicarbonate solu-
tion and then washed with CH2Cl2. The organic phase was
concentrated in vacuum to afford neutral berbamine (2, 1.2842 g,
2.11 mmol). Neutral berbamine dissolved in 21.1 mL water was
adjusted to pH ¼ 13 with solid KOH. After 5 h stirring at 40 ꢀC, the
solution was evaporated in vacuum with ethanol to afford the solid
berbamine salt 3. It could be directly used for the next reaction step.
suggested that the acetyl
cancer cells.
D-glucose residue has affinity to these
4. Experimental protocols
Electrospray ionization (ESI) mass spectra were recorded on an
Esquire 3000 plus mass spectrometer. NMR spectra (1H, 13C) were
recorded on a Bruker DRX-400 instrument (Karlsruhe, Germany) at
400 MHz for 1H NMR and at 100 MHz for 13C NMR and tetrame-
thylsilane was used as an internal standard; coupling constants are
represented in hertz. Reactions were monitored by thin-layer
chromatography (TLC) on an aluminum sheet precoated with
Silica gel 60 (Merck, Germany) and detected by charring with 10%
sulfuric acid in ethanol solution. Column chromatography was
performed on silica gel (300e400 mesh; Qingdao Marine Chemical
Ltd., Qingdao, PRC). CH3CN and CH2Cl2 were distilled from P2O5.
4.1.2. Procedure for 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl
berbamine 5a
2,3,4,6-Tetra-O-acetyl-a-D-glucopyranosyl bromide (4a, 100 mg,
0.24 mmol), the berbamine salt (3, 52.4 mg, 0.08 mmol) and the
activated 4 Å molecular sieve (50 mg) were put into 4 mL anhy-
drous acetonitrile. The solution was stirred at room temperature for
15 min in inert atmosphere, then silver oxide (56.4 mg, 0.24 mmol)
was slowly added into the system with the reaction temperature to
40 ꢀC overnight (avoiding light). TLC (Rf ¼ 0.6, CH2Cl2:MeOH ¼ 10:1,
containing 0.01% Et3N) was used to detect the reaction completion.
The solution was filtered with kieselguhr, concentrated in vacuum,

Y. Cui et al. / European Journal of Medicinal Chemistry 54 (2012) 867e872
871
the residue was purified by silica gel chromatography
(CH2Cl2:MeOH ¼ 20:1, containing 0.01% Et3N) to afford the desired
product 5a (yield 62%).
(dd, J ¼ 8.0 Hz, 1H), 7.02 (d, J ¼ 8.0 Hz, 1H), 6.97 (dd, J1 ¼ 1.6 Hz,
J2 ¼ 8.0 Hz, 1H), 6.90 (dd, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, 1H), 6.74 (dd,
J ¼ 8.0 Hz, 1H), 6.64e6.60 (m, 1H), 6.59 (s, 1H), 6.40e6.35 (m, 1H),
6.30 (dd, J ¼ 8.4 Hz, 1H), 6.28 (s, 1H), 5.58e5.41 (m, 2H), 5.09 (dd,
J1 ¼ 3.2 Hz, J2 ¼ 10.4 Hz, 1H), 4.88 (d, J ¼ 8.0 Hz, 1H, anomeric
proton), 4.24 (dd, J1 ¼ 6.8 Hz, J2 ¼ 11.2 Hz, 1H), 4.20e4.14 (m, 2H),
3.97e3.92 (m, 1H), 3.78 (s, 3H), 3.63 (s, 3H), 3.40e3.29 (m, 2H), 3.17
(s, 3H), 3.10e2.61 (m, 10H), 2.56 (s, 3H), 2.53 (br, 1H), 2.40e2.25 (m,
3H), 2.18 (s, 3H), 2.04 (s, 3H), 1.98 (s, 3H), 1.92 (s, 3H); 13C NMR
4.1.3. Procedure for b-D-cellobiopyranosyl berbamine 6d
Compound 5d (243.5 mg, 0.198 mmol) was added into 10 mL of
0.05 M NaOMe (MeOH:CH2Cl2 ¼ 1:1 volume) solution. The mixture
was stirred at room temperature under argon for 24 h. The solution
was neutralized by 732 cation exchange resins, then was filtered
and concentrated. The residue was purified by silica gel chroma-
tography (CH2Cl2:MeOH ¼ 1:1, containing 0.01% Et3N) to afford 6d
(yield 90%).
(100 MHz, CDCl3):
d 170.21, 170.15, 170.00, 169.57, 153.91, 151.74,
151.02, 149.63, 148.16, 144.20, 143.45, 139.31, 136.96, 135.35, 132.25,
130.06, 128.80, 127.56, 125.15, 122.85, 121.84, 121.07, 120.43, 120.22,
119.58, 116.40, 111.16, 105.49, 102.34 (anomeric carbon), 70.76,
70.64, 68.57, 66.88, 63.62, 61.72, 61.21, 60.30, 55.60, 55.39, 46.00,
43.95, 42.68, 42.40, 38.72, 37.57, 29.54, 25.60, 20.64e20.44.
4.1.4. Procedure for 2,3,6,20,30,40,60-hepta-O-acetyl-
b-D-
cellobiopyranosyl berbamine dihydrochloride 5d1
Compound 5d (60 mg, 0.049 mmol) was mixed with 3 mL water.
The 3% HCl (0.098 mmol HCl) was added dropwise to the mixture.
The resulting mixture was stirred for 30 min and filted. The filtrate
was concentrated. The resulting yellow solid was washed by 3 mL
CH2Cl2 and dried under reduced pressure to afford 3d1 (yield 60%).
4.3.3. 2,3,4-Tri-O-acetyl-
Light yellow powder. HRMS (TOF-EI) Calcd for C48H54N2O13
[M]þ: 866.3625, Found: 866.3632. 1H NMR (400 MHz, CDCl3):
7.29
b-D-xylopyranosyl berbamine (5c)
d
(dd, J1 ¼ 1.6 Hz, J2 ¼ 8.0 Hz, 1H), 7.09e7.03 (m, 1H), 7.03 (d,
J ¼ 8.0 Hz, 1H), 6.75 (dd, J ¼ 8.0 Hz, 1H), 6.58 (dd, J ¼ 6.8 Hz, 1H),
6.54 (s, 1H), 6.40 (s, br, 2H), 6.28 (s, 1H), 5.95 (s, br, 1H), 5.26e5.20
(m, 2H), 5.15e5.10 (m, 1H), 5.01 (d, J ¼ 7.6 Hz, 1H, anomeric proton),
4.26 (dd, J1 ¼ 4.8 Hz, J2 ¼ 12.0 Hz, 1H), 3.91e3.80 (m, 3H), 3.75 (s,
3H), 3.60 (s, 3H), 3.53e3.40 (m, 2H), 3.32e3.22 (m, 2H), 3.13 (s, 3H),
3.07e2.75 (m, 6H), 2.67e2.58 (m, 1H), 2.56 (s, 3H), 2.47e2.31 (m,
1H), 2.26 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H); 13C NMR
4.2. Biology
To evaluate the cytotoxicity of compounds used in this experi-
ment, a MTT assay was performed. Cells were seeded into 96-well
micro-culture plates at a concentration of 5 ꢂ 103 cells per well,
and then cells were exposed to compounds at concentrations of 50,
25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.39
controls were set. After incubation (37 ꢀC, 5% CO2) for 2 days, 20
of MTT solution (5 mg/mL) was transferred to each well to yield
a final assay volume of 220 L/well. Plates were incubated for 4 h at
37 ꢀC and 5% CO2. After incubation, supernatants were removed,
and 150 L of dimethyl sulfoxide (DMSO) was added. The solution
m
M, while positive and blank
(100 MHz, CDCl3): d 169.90, 169.72, 169.43, 154.08, 151.75, 150.96,
mL
149.74, 148.02, 143.73, 143.47, 138.34, 136.97, 134.36, 132.18, 130.06,
128.51, 127.11, 124.94, 122.97, 121.59, 121.12, 120.96, 120.26, 119.62,
116.52, 111.13, 105.48, 100.73 (anomeric carbon), 71.47, 70.04, 69.43,
62.32, 61.83, 61.71, 60.29, 55.60, 55.39, 45.59, 45.35, 42.49, 42.43,
38.51, 37.69, 25.17, 25.12, 20.60, 20.57, 20.54.
m
m
was at room temperature for 20 min. The optical density (OD) was
then recorded at 570 nm using a spectrophotometer. The mean OD
570 of the control cells exposed to test compound-free culture
medium was set to represent 100% of viability and the results were
expressed as a percentage of these controls. The average 50%
inhibitory concentration (IC50) was evaluated by MTT tetrazolium
dye assay. Each experiment was performed three times.
4.3.4. 2,3,6,20,30,40,60-Hepta-O-acetyl-
b-D-cellobiopyranosyl
berbamine (5d)
Light yellow powder. HRMS (ESI) Calcd for C63H74N2O23
[M þ H]þ: 1227.4760, Found: 1227.4718. 1H NMR (400 MHz, CDCl3):
d
7.28 (dd, J ¼ 8.0 Hz, 1H), 7.04 (d, J ¼ 8.0 Hz, 1H), 7.04e6.98 (m, 1H),
6.73 (dd, J ¼ 8.0 Hz,1H), 6.58 (dd, J ¼ 7.2 Hz,1H), 6.54 (s,1H), 6.43 (s,
br, 2H), 6.27 (s, 1H), 5.95 (s, br, 1H), 5.29 (dd, J1 ¼ 4.4 Hz, J2 ¼ 9.2 Hz,
1H), 5.21 (d, J ¼ 8.0 Hz, 1H, anomeric proton), 5.16 (t, J ¼ 8.8 Hz, 1H),
5.07 (t, J ¼ 9.6 Hz, 1H), 4.98e4.92 (m, 2H), 4.56 (d, J ¼ 9.6 Hz, 1H,
anomeric hydrogen) 4.57e4.53 (m, 1H), 4.38 (dd, J1 ¼ 4.4 Hz,
J2 ¼ 12.4 Hz, 1H), 4.16 (dd, J1 ¼ 5.2 Hz, J2 ¼ 12.0 Hz, 1H), 4.05 (d,
J ¼ 10.8 Hz, 1H), 3.92e3.78 (m, 3H), 3.75 (s, 3H), 3.72e3.65 (m, 2H),
3.61 (s, 3H), 3.45e3.33 (m, 1H), 3.34e3.19 (m, 2H), 3.13 (s, 3H),
3.05e2.75 (m, 7H), 2.61 (s, 1H), 2.57 (s, 3H), 2.41e2.30 (m, 1H), 2.27
(s, 3H), 2.11 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.01 (s,
4.3. Analytical data for compounds 5a, 5b, 5c, 5d, 5e, 5f, 6d and 5d1
4.3.1. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl berbamine (5a)
Light yellow powder. HRMS (TOF-EI) Calcd for C51H58N2O15
[M]þ: 938.3837, Found: 938.3828. 1H NMR (400 MHz, CDCl3):
d
7.31e7.25 (m, 1H), 7.09 (d, J ¼ 8.0 Hz, 1H), 7.07e6.99 (m, 1H),
6.79e6.70 (m, 1H), 6.61e6.52 (m, 1H), 6.55 (s, 1H), 6.45e6.36 (m,
2H), 6.28 (s, 1H), 5.96 (s, br, 1H), 5.34e5.26 (m, 1H), 5.30 (d,
J ¼ 8.0 Hz, 1H, anomeric proton), 5.17 (t, J ¼ 7.6 Hz, 1H), 4.98 (d,
J ¼ 5.6 Hz, 1H), 4.30 (dd, J1 ¼ 4.0 Hz, J2 ¼ 9.6 Hz, 1H), 4.19 (dd,
J1 ¼ 1.6 Hz, J2 ¼ 9.6 Hz, 1H), 3.94e3.81 (m, 2H), 3.79 (s, 3H), 3.61 (s,
3H), 3.53e3.24 (m, 4H), 3.14 (s, 3H), 3.05e3.77 (m, 7H), 2.60 (s, 4H),
2.38e2.23 (m, 4H), 2.09 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.97 (s,
3H), 1.98 (s, 3H), 1.96 (s, 3H); 13C NMR (100 MHz, CDCl3):
d 170.12,
170.09, 170.05, 169.98, 169.89, 169.72, 169.42, 154.13, 151.78, 150.97,
149.78, 148.02, 143.77, 143.51, 138.82, 136.93, 134.92, 132.19, 130.07,
129.08, 128.45, 127.13, 123.02, 121.60, 121.12, 120.22, 120.08, 119.63,
116.66, 111.16, 105.51, 100.73 (anomeric carbon), 95.59 (anomeric
carbon), 71.47, 70.53, 70.06, 69.44, 69.06, 68.62, 67.33, 66.11, 65.85,
63.53, 62.33, 61.66, 60.29, 55.62, 55.40, 45.59, 44.33, 42.49, 42.38,
38.50, 37.62, 29.49, 25.12, 20.87, 20.59e20.48.
3H); 13C NMR (100 MHz, CDCl3):
d 170.65, 170.25, 169.59, 169.40,
154.10, 151.89, 151.17, 150.02, 149.83, 149.66, 144.26, 143.58, 137.07,
135.35, 135.21, 132.36, 130.15, 128.77, 127.50, 123.96, 123.02, 122.22,
121.19, 120.61, 119.72, 116.50, 111.27, 105.61, 101.92 (anomeric
carbon), 74.58, 72.63, 71.93, 71.20, 68.73, 63.57, 61.96, 60.44, 55.73,
55.52, 45.97, 45.93, 42.67, 42.48, 38.81, 37.48, 29.64, 25.53, 20.69,
20.65, 20.59, 20.56.
4.3.5. 2,3,6,20,30,40,60-Hepta-O-acetyl-
b-D-lactopyranosyl berbamine
(5e)
Light yellow powder. HRMS (ESI) Calcd for C63H74N2O23
[M þ H]þ: 1227.4760, Found: 1227.4745. 1H NMR (400 MHz, CDCl3):
4.3.2. 2,3,4,6-Tetra-O-acetyl-
Light yellow powder. HRMS (TOF-EI) Calcd for C51H58N2O15
[M]þ: 938.3837, Found: 938.3827. 1H NMR (400 MHz, CDCl3):
7.42
b
-D
-galactopyranosyl berbamine (5b)
d
7.28 (dd, J ¼ 8.0 Hz, 1H), 7.08e6.98 (m, 2H), 6.73 (dd, J ¼ 7.6 Hz,
1H), 6.58 (dd, J ¼ 6.8 Hz, 1H), 6.54 (s, 1H), 6.43 (s, br, 2H), 6.27 (s,
1H), 5.95 (s, br, 1H), 5.35 (d, J ¼ 3.6 Hz, 1H), 5.30 (t, J ¼ 9.2 Hz, 1H),
d

872
Y. Cui et al. / European Journal of Medicinal Chemistry 54 (2012) 867e872
5.20 (dd, J1 ¼ 8.0 Hz, J2 ¼ 9.6 Hz, 1H), 5.13 (dd, J1 ¼ 7.6 Hz,
J2 ¼ 10.0 Hz, 1H), 4.97 (d, J ¼ 7.2 Hz, 1H, anomeric proton), 4.97 (dd,
J1 ¼ 3.6 Hz, J2 ¼ 10.8 Hz, 1H), 4.52 (d, J ¼ 8.0 Hz, 1H, anomeric
proton), 4.55e4.50 (m, 1H), 4.19e4.06 (m, 3H), 3.93e3.86 (m, 2H),
3.86e3.78 (m, 2H), 3.75 (s, 3H), 3.72e3.64 (m, 1H), 3.61 (s, 3H),
3.44e3.33 (m, 1H), 3.33e3.18 (m, 2H), 3.13 (s, 3H), 3.06e2.73 (m,
7H), 2.61 (br,1H), 2.57 (s, 3H), 2.37e2.19 (m, 4H), 2.16 (s, 3H), 2.10 (s,
4.3.8. 2,3,6,20,30,40,60-Hepta-O-acetyl-
b-D-cellobiopyranosyl
berbamine dihydrochloride (5d1)
Light yellow powder. HRMS (ESI) Calcd for C63H74N2O23
[M þ H]þ: 1227.4760, Found: 1227.4715. 1H NMR (400 MHz, DMSO-
d6):
d
7.55e7.39 (m,1H), 7.27e7.07 (m, 3H), 6.82 (dd, J ¼ 11.0 Hz,1H),
6.64 (d, J ¼ 12.0 Hz, 1H), 6.59e6.49 (m, 1H), 6.43 (s, 1H), 6.40e6.29
(m, 1H), 6.12 (dd, J ¼ 12.0 Hz, 1H), 5.34 (d, J ¼ 8.0 Hz, 1H), 5.28 (d,
J ¼ 8.0 Hz, 1H, anomeric proton), 5.28e5.22 (m, 1H), 5.99 (t,
J ¼ 8.2 Hz, 1H), 4.92e4.81 (m, 2H), 4.65 (t, J ¼ 8.2 Hz, 1H), 4.38 (d,
J ¼ 8.0 Hz, 1H, anomeric hydrogen), 4.28e4.20 (m, 1H), 4.15e3.99
(m, 3H), 3.94 (d, J ¼ 12.0 Hz, 1H), 3.86 (dd, J1 ¼ 8.0 Hz, J2 ¼ 12.0 Hz,
1H), 3.72 (s, 3H), 3.65e3.36 (m, 6H), 3.32e3.08 (m, 6H), 3.04 (s, 3H),
2.96 (s, 3H), 2.75 (s, 1H), 2.50 (s, 6H), 2.36 (s, 1H), 2.41e2.30 (m, 1H),
2.05 (s, 3H), 2.00 (s, 3H), 1.99 (s, 3H), 1.96 (s, 6H), 1.91 (s, 3H), 1.90 (s,
3H), 2.06 (s, 9H), 1.97 (s, 6H); 13C NMR (100 MHz, CDCl3):
d 170.32,
170.26, 170.08, 169.97, 169.79, 169.68, 169.00, 153.99, 151.83, 151.08,
149.74, 148.26, 144.15, 143.53, 139.40, 137.03, 135.54, 132.28, 130.15,
129.38, 128.89, 127.76, 122.95, 121.88, 121.17, 120.37, 120.23, 119.68,
116.51, 111.24, 105.56, 101.56 (anomeric carbon), 100.99 (anomeric
carbon), 76.23, 72.71, 72.57, 71.56, 70.95, 70.67, 69.08, 68.65, 66.61,
63.73, 61.95, 60.80, 60.40, 55.69, 55.49, 46.13, 46.00, 42.77, 42.51,
38.75, 37.70, 28.47, 25.69, 20.74e20.42.
3H); 13C NMR (100 MHz, DMSO-d6):
d 170.54,170.38,169.96,169.77,
169.57, 169.40, 169.31, 154.13, 151.78, 150.97, 149.78, 148.02, 143.77,
143.51, 138.82, 136.93, 134.92, 132.19, 130.07, 129.08, 128.45, 127.13,
123.02, 121.60, 121.12, 120.22, 120.08, 119.63, 116.45, 111.95, 106.66,
99.88 (anomeric carbon), 99.41 (anomeric carbon), 76.79, 72.56,
72.33, 72.16, 71.54, 71.26, 70.81, 68.04, 65.10, 61.83, 61.37, 60.59,
60.30, 56.90, 56.01, 45.02, 44.29, 43.04, 42.20, 38.07, 37.59, 24.61,
23.95, 21.42, 20.97, 20.29, 20.77, 20.69, 20.60, 20.56.
4.3.6. 2,3,4,6-Tetra-O-acetyl-b-L-glucopyranosyl berbamine (5f)
Light yellow powder. MS (ESI) Calcd for C51H58N2O15 [M þ H]þ:
939.3915, Found: 939.3876. 1H NMR (400 MHz, CDCl3):
d 7.31e7.25
(m, 1H), 7.08 (dd, J ¼ 8.1 Hz, 1H), 6.85 (d, J ¼ 7.9 Hz, 1H), 6.77 (d,
J ¼ 7.8 Hz,1H), 6.63 (d, J ¼ 7.8 Hz,1H), 6.53 (s,1H), 6.52 (d, J ¼ 4.1 Hz,
1H), 6.45e6.37 (m, 1H), 6.29 (s, 1H), 5.99 (s, br, 1H), 5.31e5.26 (m,
2H), 5.17 (t, J ¼ 9.3 Hz, 1H), 4.98 (d, J ¼ 7.2 Hz, 1H, anomeric proton),
4.29 (dd, J1 ¼ 5.0 Hz, J2 ¼ 12.2 Hz, 1H), 4.15 (d, J ¼ 11.3 Hz, 1H),
3.92e3.78 (m, 3H), 3.76 (s, 3H), 3.60 (s, 3H), 3.49e3.19 (m, 4H), 3.12
(s, 3H), 3.08e3.72 (m, 7H), 2.58 (s, 3H), 2.45e2.33 (m, 1H), 2.33 (s,
3H), 2.09 (s, 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.87 (s, 3H); 13C NMR
Acknowledgements
The financial support from the National Natural Science Foun-
dation of China under Grant No. 30870553 and the Key Interna-
tional S&T Cooperation Projects under Grant No. 2010DFA34370 for
this work is greatly appreciated.
(100 MHz, CDCl3):
d 170.57, 170.21, 169.46, 169.30, 153.87, 151.80,
151.06, 149.74, 148.20, 143.91, 143.27, 137.79, 136.95, 135.40, 132.10,
130.25, 128.72, 128.06, 127.53, 123.21, 121.87, 121.23, 120.57, 119.75,
116.87, 116.44, 111.06, 105.45, 101.70 (anomeric carbon), 72.56,
71.83, 71.18, 68.41, 63.56, 62.03, 61.87, 60.40, 55.66, 55.38, 46.00,
44.50, 42.79, 42.66, 38.85, 37.57, 30.22, 25.62, 20.63e20.49.
References
[1] K. Lee, J. Nat. Prod. 67 (2004) 273e283.
[2] S. Wang, Q. Liu, Y. Zhang, K. Liu, P. Yu, J. Luan, H. Duan, Z. Lu, F. Wang, E. Wu,
K. Yagasaki, G. Zhang, Mol. Canc. 8 (81) (2009) 1e18.
4.3.7.
b-D-Cellobiopyranosyl berbamine (6d)
[3] Y. Liang, R. Xu, L. Zhang, X. Zhao, Acta Pharmacol. Sin. 30 (2009) 1659e1665.
[4] H. Duan, J. Luan, Q. Liu, K. Yagasaki, G. Zhang, Cytotechnology 62(2010) 341e348.
[5] S.J. Marshall, P.F. Russell, C.W. Wright, M.M. Anderson, J.D. Phillipson,
E.C. Kirby, D.C. Warhurst, P.L. Schiff, Antimicrob. Agents Chemother. 38 (1994)
96e103.
[6] X. Cheng, Y. Lui, B. Zhou, X. Xiao, Y. Liu, Spectrochim. Acta A 72 (2009) 922e928.
[7] R. Xu, Q. Dong, Y. Yu, X. Zhao, X. Gan, D. Wu, Q. Lu, X. Xu, X. Yu, Leuk. Res. 30
(2006) 17e23.
[8] X. Zhao, Z. He, D. Wu, R. Xu, Chin. Med. J. 120 (2007) 802e806.
[9] J. Xie, T. Ma, Y. Gu, X. Zhang, X. Qiu, L. Zhang, R. Xu, Y. Yu, Eur. J. Med. Chem. 44
(2009) 3293e3298.
[10] J. Liu, S. Qi, H. Zhu, J. Zhang, Z. Li, T. Wang, Chin. Med. J. 115 (2002) 759e762.
[11] D. Hornd, W. Huang, US patent. 0298369 A1 (2010).
[12] B. Fang, M. Yu, S. Yang, L. Liao, J. Liu, R. Zhao, World J. Gastroenterol. 10 (2004)
950e953.
[13] W. Meutermans, G.T. Le, B. Becker, ChemMedChem 1 (2006) 1164e1194.
[14] B. Becker, G.C. Condie, G.T. Le, W. Meutermans, Mini-Rev. Med. Chem. 6
(2006) 1299e1309.
[15] Q. Yan, R. Cao, W. Yi, L. Yu, Z. Chen, L. Ma, H. Song, Bioorg. Med. Chem. Lett. 19
(2009) 4055e4058.
[16] N.B. Drinnan, F. Vari, Mini-Rev. Med. Chem. 3 (2003) 641.
[17] M. Jacobsson, J. Malmberg, U. Ellervik, Carbohydr. Res. 341 (2006) 1266e1281.
[18] K.J. Jensen, J. Chem. Soc. Perkin Trans. 1 (2002) 2219e2233.
[19] A. Michael, Am. Chem. J. 1 (1879) 305e312.
Light yellow powder. HRMS (ESI) Calcd for C49H60N2O16
[M þ H]þ: 933.4016, Found: 933.4022. 1H NMR (400 MHz, pyridine-
d5):
d
7.48 (d, J ¼ 8.0 Hz, 1H), 7.31 (d, J ¼ 8.0 Hz, 1H), 7.14 (d,
J ¼ 8.0 Hz, 1H), 6.99 (d, J ¼ 4.0 Hz, 1H), 6.81e6.67 (m, 1H), 6.77 (s,
1H), 6.45 (s, 1H), 6.41e6.24 (s, br, 3H), 5.29 (dd, J1 ¼ 4.4 Hz,
J2 ¼ 9.2 Hz, 1H), 5.21 (d, J ¼ 8.0 Hz, 1H, anomeric proton), 5.16 (t,
J ¼ 8.8 Hz, 1H), 5.07 (t, J ¼ 9.6 Hz, 1H), 4.98e4.92 (m, 2H), 4.56 (d,
J ¼ 9.6 Hz, 1H, anomeric proton) 4.57e4.53 (m, 1H), 4.38 (dd,
J1 ¼ 4.4 Hz, J2 ¼ 12.4 Hz, 1H), 4.16 (dd, J1 ¼ 5.2 Hz, J2 ¼ 12.0 Hz, 1H),
4.05 (d, J ¼ 10.8 Hz, 1H), 3.92e3.78 (m, 3H), 3.75 (s, 3H), 3.72e3.65
(m, 2H), 3.61 (s, 3H), 3.45e3.33 (m, 1H), 3.34e3.19 (m, 2H), 3.13 (s,
3H), 3.05e2.75 (m, 7H), 2.61 (s, 1H), 2.57 (s, 3H), 2.41e2.30 (m, 1H),
2.27 (s, 3H); 13C NMR (100 MHz, pyridine-d5):
d 153.46, 151.78,
149.77, 149.03, 148.43, 144.50, 143.43, 137.23, 136.14, 135.98, 132.25,
131.19, 129.51, 128.73, 128.16, 123.45, 123.25, 121.76, 120.87, 120.43,
119.48, 115.58, 111.85, 106.71, 103.52 (anomeric carbon), 100.57
(anomeric carbon), 80.33, 77.16, 76.77, 75.53, 75.29, 73.63, 73.27,
70.36, 63.02, 62.04, 61.35, 60.45, 60.06, 55.94, 55.62, 46.53, 45.85,
42.92, 42.46, 38.66, 36.21, 26.02, 21.86.
[20] J. Leroux, S.A. Perlin, Carbohydr. Res. 67 (1978) 163e178.