Berbamine derivatives: A novel class of compounds for anti-leukemia activity

Article abstract of DOI:10.1016/j.ejmech.2009.02.018

Our previous studies showed that the natural compound berbamine, from Chinese herb Berberis amurensis, selectively induces apoptosis of imatinib (IM)-resistant-Bcr/Abl-expressing leukemia cells from the K562 cell line and CML patients. Here, a series of n

Full text of DOI:10.1016/j.ejmech.2009.02.018

European Journal of Medicinal Chemistry 44 (2009) 3293–3298
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Berbamine derivatives: A novel class of compounds for anti-leukemia activity
,
,
,
,c
Jinwen Xie a, Ting Ma a, Ying Gu b c, Xuzhao Zhang b c, Xi Qiu b c, Lei Zhang b
,
,
c
,
a,
*
Rongzhen Xu b
, Yongping Yu
**
a College of Pharmaceutical Sciences, Zhejiang University, Zijin Campus, Hangzhou 310058, China
b Department of Hematology, Second Affiliated Hospital, Zhejiang University, Hangzhou 310009, China
c Cancer Institute, School of Medicine, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Our previous studies showed that the natural compound berbamine, from Chinese herb Berberis
amurensis, selectively induces apoptosis of imatinib (IM)-resistant-Bcr/Abl-expressing leukemia cells
from the K562 cell line and CML patients. Here, a series of new berbamine derivatives were obtained by
synthesis. In this series, high to very high activity in vitro has been found. Compounds 2e, 2g, 3f, 3k, 3q
and 3u exhibited consistent high anti-tumor activity for imatinib-resistant K562 leukemia cells. Their
Received 17 October 2008
Received in revised form
13 January 2009
Accepted 12 February 2009
Available online 23 February 2009
IC50 values at 48 h were 0.36–0.55
results showed that compound 3h could reduce G0/G1 cells. In particular, these compounds displayed
potent inhibition of the cytoplasm-to-nucleus translocation of NF- B p65 which plays a critical role in
mM, whereas berbamine IC50 value was 8.9 mM. Cell cycle analysis
Keywords:
k
Berbamine derivatives
Anti-leukemia activity
Bisbenzylisoquinoline alkaloid
the survival of leukemia stem cells. These results suggest that berbamine could be a good starting point
for the development of novel lead compounds in the fight against leukemia.
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
berbamine derivatives for anti-tumor activity have been reported,
such as O-(4-ethoxybutyl)-berbamine (EBB). EBB displays various
Berbamine is a well-known bisbenzylisoquinoline alkaloid iso-
lated from traditional Chinese herbal medicines such as Berberis
amurensis (Fig. 1). Berbamine has been demonstrated to possess
a number of interesting and potent biological activities [1–8].
Gleevec (also called Imatinib or STI571), which is an inhibitor of the
bcr/abl tyrosine kinase [9], has been a remarkable success for the
treatment of chronic myelogenous leukemia (CML). However,
a significant proportion of patients chronically treated with Gleevec
develop resistance [10]. Thus, it is necessary to identify novel
inhibitors that are active against Gleevec-resistant mutants of bcr/
abl oncoprotein.
In our previous report, we found that berbamine can selectively
induce cell death of both Gleevec sensitive- and resistant-Phþ
chronic myeloid leukemia (CML) cells [11]. Moreover, we found that
berbamine can selectively induce caspase-3-dependent apoptosis
of leukemia NB4 cells via the survivin-mediated pathway, sug-
gesting that berbanine may be a novel agent for the treatment of
leukemia [12]. However, its IC50 value for various leukemia cells
activities such as inhibition of hepatoma [13], inhibition of the
trypsin-activated Ca2þ þ Mg2þ-ATPase [14]. E6, another berbamine
derivative, has also shown potent biological activity [15,16]. These
previous studies prompted us to develop novel berbamine deriva-
tives designed to treat leukemia. Interestingly, although berbamine
derivatives display various anticancer activities, there are currently
no publications that systematically demonstrated the structure–
activity relationship of this class of compounds for anticancer
activity.
In order to discover novel anti-leukemia berbamine derivatives
in this study we focus specifically on compounds that are active
against Gleevec-resistant leukemia cells. We describe in this report
the results of berbamine derivatives synthesis as well as their anti-
leukemia activity for imatinib-resistant K562 leukemia cells.
2. Results and discussions
2.1. Chemistry
was only modest (from 4 to 8.9 mM) [11,12]. Some synthetic
The phenolic hydroxyl group of berbamine could be etherified,
esterified or sulfonylated facilely. The synthesis of a series of
berbamine derivatives are illustrated in Scheme 1. Berbamine
derivatives 2a–g were synthesized according to Williamson
* Corresponding author. Department of Hematology, Second Affiliated Hospital,
Zhejiang University, Hangzhou 310009, China.
** Corresponding author. Tel./fax: þ86 571 88208452.
E-mail addresses: zrxyk10@zju.edu.cn (R. Xu), yyu@zju.edu.cn (Y. Yu).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.02.018

3294
J. Xie et al. / European Journal of Medicinal Chemistry 44 (2009) 3293–3298
2.2. Biological activities
All compounds were evaluated for their cytotoxic activity in
vitro against human imatinib-resistant K562 leukemia cell line. The
results are summarized in Table 1.
As shown in Table 1, most compounds exhibited inhibitions on
the growth of imatinib-resistant K562 leukemia cells. Preliminary
structure–activity relationship of this series of compounds for
cytotoxicity against imatinib-resistant K562 leukemia cell line was
investigated. From the IC50 data of these compounds, we find that
etherification, esterification or sulfonylation of berbamine could
improve its cytotoxicity against imatinib-resistant K562 leukemia
cell line significantly. In general, when the R1, R2 or R3 group con-
tained an aromatic ring, the activity was better than when these
substituents were aliphatic chain. For example, 2a (R1 ¼ CH3CH2,
Fig. 1. The structure of berbamine.
reaction. Etherified derivatives (route a) were synthesized starting
from berbamine dihydrochloride 1, which was dissolved in DMF,
the solution was cooled to 0 ꢀC. NaH was added to the solution and
the solution was stirred for 1 h at 0 ꢀC. Then an alkyl halide such as
benzyl bromide was added dropwise and the solution was stirred
for 1 h at 0 ꢀC. The solution was allowed to warm to room
temperature and was stirred for 1–8 h to afford products 2a–g.
Esterfied derivatives (route b) were synthesized starting with the
treatment of berbamine dihydrochloride 1 with Et3N at 0 ꢀC in
CH2Cl2 for 1 h. Then an acyl chloride was added dropwise and the
solution was stirred for 1 h at 0 ꢀC. The solution was allowed to
warm to room temperature to afford products 3a–t. An additional
esterfied derivative, compound 3u, was synthesized (route c) via
reaction of berbamine with 1H-indole-2-carboxylic acid in the
presence of N,N0-dicyclohexylcarbodiimide (DCC) in CH2Cl2 at room
temperature. Finally sulfonylated derivatives were formed (route d)
by first neutralizing berbamine dihydrochloride 1 with Et3N. A
sulfonyl chloride was then added dropwise to the solution. The
mixture was stirred at 0 ꢀC for 1 h to afford products 4a and 4b.
The yields of berbamine derivatives 2–4 are listed in Table 1. The
products were characterized by ESI-MS and NMR spectra.
IC50 ¼ 3.14
m
M) compared to 2c (R1 ¼ PhCH2, IC50 ¼ 0.80
mM), 3a
(R2 ¼ CH3, IC50 ¼ 6.60
m
M) compared to 3e (R2 ¼ Ph, IC50 ¼ 2.16
mM),
and 4a (R3 ¼ CH3, IC50 ¼ 4.74
m
M) compared to 4b (R3 ¼ Ph,
IC50 ¼ 1.99
mM). Comparing the IC50 data for 2c–f allows us to
evaluate the SAR, when an electron withdrawing substituent is
present on the phenyl ring of the R1 group with the etherified
berbamine derivatives. The data suggest that cytotoxicity against
imatinib-resistant K562 leukemia cell line is better in most cases
than when the electron donating group is present, such as with 2e
(R1 ¼4-NO2PhCH2,
IC50 ¼ 0.36
m
M)
and
2f
(R1 ¼3,4,5-
(CH3O)3PhCH2, IC50 ¼ 1.53
mM). The data of 3e–s likewise suggests
that an electron donating substituent is present on the phenyl ring
could increase the activity of esterified berbamine derivative’s
cytotoxicity against imatinib-resistant K562 leukemia cell line such
as 3f (R2 ¼ 4-CH3OPh, IC50 ¼ 0.55
m
M), 3h (R2 ¼ 2-CH3Ph,
IC50 ¼ 0.69
m
M), 3n (R2 ¼ 4-NO2Ph, IC50 ¼ 2.13
m
M) and 3o (R2 ¼ 3,
5-diNO2Ph, IC50 ¼ 3.75
mM). Finally the activity of the compounds
in this series is better when R1 or R2 is a heterocycle containing
nitrogen
IC50 ¼ 0.40
such
m
as
2g
(R2 ¼ (6-chloropyridin-3-yl)methyl,
M) and 3u (R2 ¼1H-indol-2-yl, IC50 ¼ 0.46
mM) rather
Scheme 1. Synthesis of berbamine derivatives 2–4. Reagents and conditions: (a) NaH (4 equiv), R1X (1.1 equiv or 5 equiv), DMF, 0 ꢀC to r.t. (b) Et3N (4 equiv), R2COCl (1.1 equiv), 1 h,
ꢀC, CH2Cl2. (c) 1H-Indole-2-carboxylic acid, DCC (2 equiv), CH2Cl2, overnight. (d) Et3N (4 equiv), R3SO2Cl (1.1 equiv), 1 h, 0 ꢀC, CH2Cl2.
0

J. Xie et al. / European Journal of Medicinal Chemistry 44 (2009) 3293–3298
3295
Table 1
Berbamine derivatives and IC50 values of them for anti-leukemia activity (K562-R).
Compounds
R1/R2/R3
Yields (%)
IC50
(
m
M) a
Compounds
R1/R2/R3
Yields (%)
IC50 (m
M) a
2a
2b
2c
2d
2e
2f
CH3CH2
BrCH2CH2CH2
PhCH2
4-BrPhCH2
4-NO2PhCH2
3,4,5-(CH3O)3PhCH2
43
37
79
75
77
70
3.14
1.66
0.80
0.71
0.36
1.53
3j
3k
3l
3m
3n
3o
2-ClPh
4-BrPh
2-BrPh
3-FPh
4-NO2Ph
3,5-(NO2)2Ph
70
72
71
74
75
78
0.60
0.40
0.78
1.97
2.13
3.75
Cl
N
2g
78
0.40
3p
3-CF3Ph
76
0.89
3a
3b
3c
CH3
(CH3)3C
CH3C]CH2
65
70
65
6.60
1.01
3.81
3q
3r
3s
3-ClCH2Ph
PhCH2
3,4,6-(F)3PhCH2
72
75
75
0.50
2.23
3.02
O
3d
ClCH2
60
2.70
3t
72
2.78
3e
Ph
70
2.16
3u
75
0.46
N
H
3f
4-CH3OPh
3,4,5-(CH3O)3Ph
2-CH3Ph
70
72
76
71
0.55
0.69
0.69
0.98
4a
4b
CH3
Ph
H
77
78
–
4.74
1.99
8.93
4.08
3g
3h
3i
Berbamine
EBB
4-ClPh
C2H5O(CH2)4
–
a
Mean values of three independent determinations.
than a heterocycle containing oxygen such as 3t (R3 ¼ furan-2-yl,
the percentage of G0/G1 phase cells (non-proliferating cells) before
and after treatment of the compound 3h at 1 g/ml were 49.05%
IC50 ¼ 2.78
mM).
m
NF-kB, which is thought to be an important signal molecule
and 32.37%, respectively whereas, the percentage of sub-G1 phase
cells (apoptotic cells) were 8.0%, and 31.71%, respectively.
Compound 3h also reduced proliferating cells (S phase cells). The
percentage of S phase cells was 34.99% in untreated K562-R cells,
but deceased to 22.89% after treatment with the derivative 3h at
downstream p210Bcr/Abl oncoprotein [17–19], is constitutively
active in AML [18], MDS [20] and T-cell acute lymphoblastic
leukemia (T-ALL) [21] but not in normal hematopoietic cells [22],
and its inhibition is correlated with leukemia-specific cell death
[20,21,23]. Therefore, we sought to investigate whether berbamine
2 mg/ml for 48 h. These observations suggest that the compound 3h
derivatives can affect NF-
and nuclear levels of NF-
exposure to berbamine derivatives for 24 h. The treatment of K562
cells with berbamine derivatives led to the disappearance of NF-
from the nuclei (Fig. 2). However, total level of NF- B subunit p65
k
B signaling pathway. We analyzed total
can induce apoptosis of K562-R cells and may have potential to kill
leukemia stem cells that are quiescent.
k
B p65/relA subunit in leukemia cells after
k
B
3. Conclusion
k
were not obviously affected (data not shown). These data indicate
that berbamine derivatives can effectively inhibit the cytoplasm-to-
nucleus translocation of NF-kB p65/relA subunit of leukemia cells.
Flow cytometry was used to evaluate the effects of compound
3h on the cell cycles and apoptosis of K562-R cells. As summarized
in Fig. 3, and Table 2, treatment of K562-R cells with compound 3h
resulted in a significant decrease of G0/G1 phase cells accompanied
with a parallel increase of sub-G1 phase cells. As indicated in Fig. 3,
A series of berbamine derivatives were synthesized and the
anti-leukemia activity of them against imatinib-resistant K562
leukemia cell line was evaluated. Etherified, esterified or sulfony-
lated of the phenolic hydroxyl group of berbamine increased the
cytotoxic activity of berbamine against leukemia cell lines signifi-
cantly. Most compounds displayed potent cytotoxic activity in the
micromolar range. Compounds 2e, 2g, 3f, 3k, 3q and 3u, whose IC50
values were 0.36
0.46 M showed the best activity. In particular, these compounds
also displayed potent inhibition of the cytoplasm-to-nucleus
translocation of NF- B p65 which is critical for survival of leukemia
mM, 0.40 mM, 0.55 mM, 0.40 mM, 0.50 mM and
m
k
stem cells. The cell cycle analysis on K562-R cell line indicated that
these compounds can reduce quiescent (G0/G1) phase cells, sug-
gesting that berbamine derivatives may have potential to kill
leukemia stem cells.
Nuclear NF- κ Bp65
Lamin
4. Experimental protocols
BBM 8μg/ml; BBDs 0.5μg/ml
Melting points were recorded on a Bu¨chi B-540 apparatus
and were uncorrected. NMR spectra were recorded at 500 MHz
Fig. 2. Berbamine and its derivatives inhibit the cytoplasm-to-nucleus translocation of
NF-kappaB in leukemia cells.

3296
J. Xie et al. / European Journal of Medicinal Chemistry 44 (2009) 3293–3298
Fig. 3. Induction of apoptosis by 3h in K562-R cells.
(1H NMR) or 125 MHz (13C NMR) with CDCl3 as solvent and TMS
as the internal standard. J values are in Hertz and chemical shifts
are expressed in ppm downfield from internal TMS. DMF and
CH2Cl2 were distilled from CaH2. The other commercial reagents
were used directly without further purification. Column
chromatography was performed on silica gel. MS (ESI) spectra
chromatography on silica gel (CH2Cl2:CH3OH ¼ 8:1) to afford the
desired product.
4.2. General procedure for the preparation of berbamine
derivatives 3a–t
were recorded on
Spectrophotometer.
a
Thermo Finnigan LCQ Advantage mass
Berbamine dihydrochloride 1 (681 mg, 1 mmol) was suspended
in 10 mL of CH2Cl2. The mixture was cooled to 0 ꢀC under N2. Et3N
(404 mg, 4 mmol) was added to the solution. After stirring for 1 h at
0 ꢀC, an acyl halide (1.1 mmol, in 3 mL of CH2Cl2) was added
dropwise to the solution over 15 min. The resulting solution was
stirred for 1 h at 0 ꢀC and was stirred another hour at room
temperature. The solution was washed with water and brine, dried
over anhydrous Na2SO4 and filtered. After being concentrated in
vacuo, the residue was purified by flash chromatography on silica
gel (CH2Cl2:CH3OH ¼ 8:1) to afford the desired product.
4.1. General procedure for the preparation of berbamine
derivatives 2a–g
Berbamine dihydrochloride 1 (681 mg, 1 mmol) was dissolved
in 10 mL of DMF, the solution was cooled to 0 ꢀC under N2. NaH
(96 mg, 4 mmol) was added to the solution. The mixture was stirred
for 1 h and an alkyl halide (1.1 mmol, for 2a and 2b, 5 mmol, in 3 mL
of DMF) was added dropwise over 15 min. After stirring for 1 h at
0 ꢀC, the solution was allowed to warm to room temperature and
was stirred for 1–8 h (tested by TLC). The reaction mixture was
evaporated in vacuo, diluted with water and extracted with CH2Cl2
(3 ꢁ10 mL). The combined organic phase was washed with water
and brine, dried over anhydrous Na2SO4 and filtered. After being
concentrated in vacuo, the residue was purified by flash
4.3. Procedure for the preparation of berbamine derivative 3u
1H-indole-2-carboxylic acid (161 mg, 1 mmol) was dissolved in
15 mL of CH2Cl2. The solution was cooled to 0 ꢀC under N2. DCC
(2 mmol) was added to the solution. The solution was stirred for 1 h
at 0 ꢀC. Berbamine (1 mmol) (berbamine dihydrochloride 1 was
neutralized with Et3N and dried in vacuo) was added to the solu-
tion, and then DMAP (0.5 mmol) was added. The reaction mixture
was allowed to warm to room temperature and was stirred over-
night, during which time a white precipitate was formed. The
reaction mixture was filtered and washed with CH2Cl2, the filtrate
was washed with water and brine, dried over anhydrous Na2SO4
and filtered. After being concentrated in vacuo, the residue
was purified by flash chromatography on silica gel
(CH2Cl2:CH3OH ¼ 8:1) to afford the desired product.
Table 2
Cell cycle analysis results of K562-R treated with compound 3h.
Control (%)
1
m
g/ml (%)
2 mg/ml (%)
Apoptosis
G0/G1
S
8.0
31.71
32.37
29.28
7.30
51.91
20.04
22.89
6.01
49.05
34.99
8.90
G2/M

J. Xie et al. / European Journal of Medicinal Chemistry 44 (2009) 3293–3298
3297
4.4. General procedure for the preparation of berbamine
derivatives 4a–b
(d, 2H, J ¼ 8.4 Hz), 7.45–7.42 (d, 1H, J ¼ 8.4 Hz), 6.99–6.95 (m, 3H),
6.78–6.64 (m, 3H), 6.40–6.32 (m, 3H), 5.53–5.52 (d, 1H, J ¼ 1.6 Hz),
5.25 (s, 2H), 4.26–4.18 (dd, 1H, J ¼ 4.4 Hz, 5.6 Hz), 3.78 (s, 3H), 3.64
(s, 3H), 3.38–3.31 (m, 2H), 3.19 (s, 3H), 3.15–3.07 (m, 2H), 3.04–3.00
(m, 2H), 2.95–2.88 (m, 4H), 2.85–2.72 (m, 3H), 2.66 (s, 3H), 2.52 (s,
Berbamine dihydrochloride 1 (681 mg, 1 mmol) was suspended
in 10 mL of CH2Cl2. The mixture was cooled to 0 ꢀC under N2. Et3N
(404 mg, 4 mmol) was added to the solution. The mixture was
stirred for 1 h and a sulfonyl chloride (1.1 mmol, in 3 mL of CH2Cl2)
was added dropwise over 15 min. The solution was stirred for 1 h at
0 ꢀC and was stirred another hour at room temperature. The solu-
tion was washed with water and brine, dried over anhydrous
Na2SO4 and filtered. After being concentrated in vacuo, the residue
was purified by flash chromatography on silica gel
(CH2Cl2:CH3OH ¼ 8:1) to afford the desired product.
2H). 13C NMR (125 MHz, CDCl3)
d 25.3, 28.8, 37.5, 39.7, 42.1, 43.8,
45.3, 51.2, 54.9, 55.9, 60.4, 61.5, 64.0, 70.5, 105.8, 110.9, 114.5, 116.6,
117.4, 120.9, 122.1, 123.2, 123.6 (2C), 123.8, 127.5, 127.6, 127.7 (2C),
127.9, 130.9,131.5,132.6,137.0,139.3,143.8,145.0,145.2,147.4,147.5,
148.2, 149.9, 151.5, 152.2.
4.6.3. O-((6-Chloropyridin-3-yl)methyl)-berbamine (2g)
White powder, m.p.: 112–115 ꢀC. MS (ESI) m/z 735.2 (M þ Hþ).1H
NMR (500 MHz, CDCl3)
d
8.43 (s, 1H), 7.82–7.79 (d, 1H, J ¼ 8.0 Hz),
4.5. Biology
7.43–7.41 (d, 1H, J ¼ 7.2 Hz), 7.32–7.27 (m, 2H), 7.00–6.91 (m, 2H),
6.80–6.73 (dd, 2H, J ¼ 8.4 Hz, 8.0 Hz), 6.65–6.6.63 (s, 1H), 6.39–6.27
(m, 3H), 5.50–5.49 (d, 1H, J ¼ 1.5 Hz), 5.13 (s, 2H), 4.22–4.18 (dd, 1H,
J ¼ 4.0 Hz, 5.6 Hz), 3.78 (s, 3H), 3.63 (s, 3H), 3.37–3.31 (m, 2H), 3.19 (s,
3H), 3.14–3.09 (m, 2H), 3.03–3.02 (m, 2H), 2.92–2.72 (m, 4H), 2.65 (s,
4.5.1. Cell line, culture conditions, and growth assays
Human chronic myeloid leukemia cell line imatinib-resistant-
K562 (K562-R), which constitutively express endogenous p210Bcr/
Abl oncoprotein, were grown in RPMI-1640 medium supplemented
with 10% fetal calf serum (FCS), 100 units/ml penicillin G, and
3H), 2.55 (s, 3H), 2.37(m, 2H).13C NMR (125 MHz, CDCl3)
d 25.3, 28.6,
37.5, 39.6, 42.1, 43.7, 45.2, 51.1, 54.8, 55.9, 60.4, 61.4, 63.9, 68.7, 105.7,
110.8, 115.1, 116.6, 117.3, 120.8, 122.0, 123.1, 123.7, 124.1, 127.4, 127.7,
130.9, 131.5, 132.0, 132.8, 137.0, 137.6, 138.4, 139.1, 143.7, 144.8, 147.4,
148.2, 148.8, 150.0, 150.8, 151.5, 152.1.
100 m
g/ml streptomycin at 37 ꢀC in a 95% air, 5% CO2 humidified
incubator. K562-R cells expresse multipledrug resistance-1(MDR1)
and are highly resistant to IM and conventional chemotherapeutic
agents. Cell number and viabilities were monitored by hemocy-
tomer and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay, respectively.
4.6.4. O-(2-Methylbenzoyl)-berbamine (3h)
White powder, m.p.: 122–125 ꢀC. MS (ESI) m/z 727.6 (M þ Hþ).
1H NMR (500 MHz, CDCl3)
d
8.15–8.13 (d, 1H, J ¼ 7.6 Hz), 7.60–7.58
4.5.2. Detection of NF-
Leukemia cells were treated with berbamine (8
amine derivatives (0.5 g/ml), and total and nuclear cellular proteins
were extracted using the Mammalian Protein Extraction Reagent
(Pierce). NF- B p65 protein was detected using Western blot.
kB p65 proteins
(d, 1H, J ¼ 8.0 Hz), 7.43–7.40 (m, 2H), 7.28–7.25 (m, 3H), 7.16 (s, 1H),
7.04–6.97 (dd, 2H, J ¼ 8.0 Hz, 8.4 Hz), 6.92–6.90 (d, 1H, J ¼ 8.0 Hz),
6.68 (s, 1H), 6.43–6.35 (m, 2H,), 5.57–5.56 (d, 1H, J ¼ 1.6 Hz), 4.39–
4.37 (dd, 1H, J ¼ 4 Hz, 5.5 Hz), 3.80 (s, 3H), 3.64 (s, 3H), 3.41–3.35
(m, 2H), 3.21 (s, 3H), 3.17–3.06 (m, 4H), 2.96–2.81 (m, 4H), 2.73 (s,
3H), 2.64 (brs, 6H), 2.59–2.48 (m, 2H). 13C NMR (125 MHz, CDCl3)
m
g/ml) or berb-
m
k
4.5.3. Flow cytometry
d 21.7, 24.2, 28.2, 37.7, 41.3, 41.8, 43.5, 44.7, 50.6, 54.8, 55.7, 60.4,
K562-R cells at a density of 2 ꢁ105 cells/mL were treated with
61.6, 63.8, 105.7, 111.0, 116.6, 117.7, 120.8, 121.8, 122.1, 124.1, 125.7,
125.8, 126.8 128.0, 129.0, 130.7, 131.1, 131.2, 131.3, 131.6, 132.3, 136.5,
137.2, 137.7, 138.3, 140.7, 143.7, 147.6, 148.2, 151.0, 151.8, 152.4, 165.6.
various concentrations of compound 3h (1.0 mg/ml, 2.0 mg/ml) for
48 h. The cells were harvested, washed with PBS and centrifuged.
The fixation of cells was relized through the addition of 4 ml
ethanol (70% ice-cold) and then keeping cells at 4 ꢀC overnight until
DNA staining. The fixed cells were treated with 100
in PBS for 1 h, followed by staining with 50 g/ml propidium iodide
4.6.5. O-(4-Chlorobenzoyl)-berbamine (3i)
mg/ml Rnase A
White powder, m.p.: 148–151 ꢀC. MS (ESI) m/z 748.2 (M þ Hþ).
m
1H NMR (500 MHz, CDCl3)
d
8.15–8.13 (d, 2H, J ¼ 8.5 Hz), 7.45–7.43
in PBS in the dark. The DNA content of eukaryotic cells was then
measured with flow cytometery.
(d, 2H, J ¼ 9.0 Hz), 7.41–7.39 (d, 1H, J ¼ 8.0 Hz), 7.24–7.20 (m, 1H),
7.01–6.99 (d, 1H, J ¼ 8.0 Hz), 6.96–6.91 (m, 2H), 6.88–6.86 (d, 1H,
J ¼ 8.5 Hz), 6.64 (s, 1H), 6.41–6.31 (m, 3H), 5.62–5.61 (d, 1H,
J ¼ 1.5 Hz), 4.29–4.18 (dd, 1H, J ¼ 3.5 Hz, 5.5 Hz), 3.78 (s, 3H), 3.63 (s,
3H), 3.39–3.29 (m, 2H), 3.20 (s, 3H), 3.07–2.99 (m, 2H), 2.93–2.87
(m, 2H), 2.82–2.69 (m, 4H), 2.65 (s, 3H), 2.58 (s, 3H), 2.44–2.37(m,
4.6. Analytical data for compounds 2d, 2e, 2g, 3h, 3i, 3u and 4a
4.6.1. O-(4-Bromobenzyl)-berbamine (2d)
White powder, m.p.: 108–111 ꢀC. MS (ESI) m/z 778.2 (M þ Hþ).
2H). 13C NMR (125 MHz, CDCl3)
d 24.2, 28.1, 37.8, 40.2, 41.2, 43.4,
1H NMR (500 MHz, CDCl3)
d
7.47–7.46 (d, 2H, J ¼ 7.0 Hz), 7.42–7.40
44.5, 50.6, 54.8, 55.9, 60.4, 61.4, 63.7, 105.7, 111.9, 116.6, 117.8, 120.7,
121.5, 122.0, 124.0, 126.9, 127.9, 128.7 (2C), 128.8, 130.8, 130.9, 131.3,
131.6 (2C), 131.7, 136.9, 137.1, 137.4, 138.5, 139.7, 143.7, 147.6, 148.2,
150.9, 151.8, 152.2, 163.9.
(d, 1H, J ¼ 8.5 Hz), 7.36–7.33 (t, 2H, J ¼ 7.5 Hz, 7.5 Hz), 7.29–7.28 (d,
1H, J ¼ 10.0 Hz), 6.97–6.95 (d, 2H, J ¼ 8.5 Hz), 6.81–6.79 (d, 1H,
J ¼ 8.5 Hz), 6.72–6.68 (m, 1H), 6.63 (s, 1H), 6.39–6.32 (m, 3H,), 5.50–
5.49 (d, 1H, J ¼ 1.5 Hz), 5.17 (s, 2H), 4.24–4.18 (dd, 1H, J ¼ 4 Hz,
5.5 Hz), 3.78 (s, 3H), 3.63 (s, 3H), 3.37–3.31 (m, 2H), 3.19 (s, 3H),
3.15–3.09 (m, 2H), 3.07–3.00 (m, 2H), 2.91–2.71 (m, 4H), 2.67 (s,
4.6.6. O-(1H-Indole-2-carbonyl)-berbamine (3u)
White powder, m.p.: 178–181 ꢀC. MS (ESI) m/z 752.8 (M þ Hþ).1H
3H), 2.55 (s, 3H), 2.37(s, 2H). 13C NMR (125 MHz, CDCl3)
d
25.5, 28.8,
NMR (500 MHz, CDCl3)
d
9.30 (s, 1H), 7.73–7.70 (d, 1H, J ¼ 8.4 Hz),
37.5, 39.6, 42.2, 43.8, 45.4, 51.1, 54.9, 55.9, 60.5, 61.5, 64.0, 71.5,
105.7, 110.9, 114.3, 116.7, 117.1, 121.1, 122.2, 123.4, 123.5, 127.3, 127.5
(2C), 127.7, 127.9, 128.4 (2C), 128.6, 130.9, 131.4, 131.8, 137.0, 137.6,
139.1, 143.7, 145.7, 147.5, 148.2, 149.9, 151.5, 152.4.
7.47–7.40 (m, 3H), 7.35–7.30 (m,1H), 7.22–7.14 (m, 2H), 7.08–7.06 (d,
1H, J ¼ 8.0 Hz), 6.98–6.87 (m, 3H), 6.67 (s, 1H), 6.43–6.33 (m, 3H,),
5.64–5.63 (d, 1H, J ¼ 1.6 Hz), 4.33–4.21 (dd, 1H, J ¼ 3.2 Hz, 5.2 Hz),
3.80(s, 3H), 3.64 (s, 3H), 3.40–3.24 (m, 2H), 3.21 (s, 3H), 3.17–3.00 (m,
2H), 2.96–2.91 (m, 2H), 2.84–2.71 (m, 4H), 2.67 (s, 3H), 2.60 (s, 3H),
4.6.2. O-(4-Nitrobenzyl)-berbamine (2e)
2.47–2.39 (m, 2H). 13C NMR (125 MHz, CDCl3)
d 25.0, 28.7, 37.7, 39.8,
Yellow powder, m.p.: 131–134 ꢀC. MS (ESI) m/z 744.4 (M þ Hþ).
41.9, 43.8, 45.1, 51.2, 54.8, 55.8, 60.4, 61.4, 64.0, 105.7, 110.2, 110.9,
112.1, 116.5, 117.9, 120.7, 120.8, 121.6, 121.9, 122.6, 123.0, 124.1, 124.8,
1H NMR (500 MHz, CDCl3)
d
8.22–8.20 (d, 2H, J ¼ 8.8 Hz), 7.65–7.63

3298
J. Xie et al. / European Journal of Medicinal Chemistry 44 (2009) 3293–3298
[2] C. Luo, X. Lin, W. Li, X. Yang, L. Wang, S. Xie, P. Xiao, Phytother. Res. 11 (1997)
585–587.
[3] N. Shiraishi, S. Akiyama, M. Nakagawa, M. Kobayashi, M. Kuwano, Cancer Res.
47 (1987) 2413–2416.
125.4, 126.5, 127.3, 127.4, 127.7, 131.0, 131.3, 136.9, 140.0, 137.2, 137.3,
139.2, 143.8, 147.5, 148.2, 151.0, 151.5, 152.1, 160.0.
[4] T. Matsuno, K. Orita, K. Edashige, H. Kobuchi, E.F. Sato, B. Inouye, M. Inoue,
K. Utsumi, Biochem. Pharmacol. 39 (1990) 1255–1259.
[5] H. Ju, X. Li, B. Zhao, Z. Han, W. Xin, Biochem. Pharmacol. 39 (1990) 1673–1678.
[6] S. Akiba, R. Nagatomo, T. Ishimoto, T. Sato, Eur. J. Pharmacol 291 (1995) 343–
350.
[7] S. Kanako, N. Fumiko, O. Minoru, A. Naoto, Biochem. Pharmacol. 66 (2003)
379–385.
[8] M. Karin, S. Frank, S. Dietmar, W. Michael, Planta Med. 67 (2001) 623–627.
[9] B.J. Druker, M. Talpaz, D.J. Resta, B. Peng, E. Buchdunger, J.M. Ford, N.B. Lydon,
H. Kantarjian, R. Capdeville, S. Ohno-Jones, C.L. Sawyers, N. Engl. J. Med. 344
(2001) 1031–1037.
[10] P. le Coutre, E. Tassi, M. Varella-Garcia, R. Barni, L. Mologni, G. Cabrita,
E. Marchesi, R. Supino, C. Gambacorti-Passerini, Blood 95 (2000) 1758–1766.
[11] R. Xu, Q. Dong, Y. Yu, X. Zhao, X. Gan, D. Wu, Q. Lu, X. Xu, X. Yu, Leuk. Res. 30
(2006) 17–23.
[12] X. Zhao, Z. He, D. Wu, R. Xu, Chin. Med. J. 120 (2007) 802–806.
[13] J. Liu, S. Qi, H. Zhu, J. Zhang, Z. Li, T. Wang, Chin. Med. J. 115 (2002) 759–762.
[14] Y. Xu, S. Zhang, Biochem. Biophys. Res. Commun. 140 (1986) 461–467.
[15] Z. Hu, Y. Gong, W. Huang, Biochem. Pharmacol. 44 (1992) 1543–1547.
[16] H. Zhu, J. Wang, Q. Guo, Y. Jiang, G. Liu, Biol. Pharm. Bull. 28 (2005) 1974–1978.
[17] A.K. Samanta, H. Lin, T. Sun, H. Kantarjian, R.B. Arlinghaus, Cancer Res. 66
(2006) 6468–6472.
[18] A. Mukhopadhyay, S. Shishodia, J. Suttles, K. Brittingham, B. Lamothe,
R. Nimmanapalli, K.N. Bhalla, B.B. Aggarwal, J. Biol. Chem. 277 (2002) 30622–
30628.
[19] D. Kirchner, J. Duyster, O. Ottmann, R.M. Schmid, L. Bergmann, G. Munzert,
Exp. Hematol. 31 (2003) 504–511.
4.6.7. O-(Methylsulfonyl)-berbamine (4a)
White powder, m.p.: 135–138 ꢀC. MS (ESI) m/z 687.8 (M þ Hþ).
1H NMR (500 MHz, CDCl3)
d
7.48–7.46 (d, 1H, J ¼ 8.4 Hz), 7.17–7.15
(d, 1H, J ¼ 8.0 Hz), 6.99–6.94 (m, 2H), 6.86–6.84 (d, 1H, J ¼ 8.5 Hz),
6.63 (s, 1H), 6.44–6.41 (m, 1H), 6.38 (s, 1H), 6.33–6.31 (m, 2H,),
5.60–5.59 (d, 1H, J ¼ 1.5 Hz), 4.33–4.19 (dd, 1H, J ¼ 3.5 Hz, 5.6 Hz),
3.78 (s, 3H), 3.64 (s, 3H), 3.41–3.27 (m, 2H), 3.23 (s, 3H), 3.20 (s, 3H),
3.13–3.01 (m, 2H), 2.97–2.92 (m, 2H), 2.87–2.82 (m, 2H), 2.79–2.72
(m, 2H), 2.68 (s, 3H), 2.57 (s, 3H), 2.40–2.37(m, 2H). 13C NMR
(125 MHz, CDCl3)
d 25.1, 28.9, 37.8, 38.3, 40.0, 42.0, 43.9, 45.2, 51.4,
54.9, 55.9, 60.4, 61.5, 64.0, 105.8, 111.0, 116.5, 118.1, 119.1, 120.6,
121.8, 122.9, 123.2, 124.6,127.2, 127.8, 128.0, 131.2, 131.7, 136.0, 137.0,
138.7, 140.0, 143.8, 147.5, 148.2, 150.9, 151.5.
Acknowledgements
Yongping Yu thanks for the Program for New Century Excellent
Talents in University (NCET-05-0523), National Natural Science
Foundation of China (NSFC 30772652, 90813026). Rongzhen Xu
would like to thank the National Natural Science Foundation of
China (NSFC 30672381, 30873095) and Zhejiang Provincial
program for the Cultivation of High-level Innovative Health talents.
[20] T. Braun, G. Carvalho, A. Coquelle, M.C. Vozenin, P. Lepelley, F. Hirsch,
J.J. Kiladjian, V. Ribrag, P. Fenaux, G. Kroemer, Blood 107 (2006) 1156–1165.
[21] T. Vilimas, J. Mascarenhas, T. Palomero, M. Mandal, S. Buonamici, F. Meng,
B. Thompson, C. Spaulding, S. Macaroun, M.L. Alegre, B.L. Kee, A. Ferrando,
L. Miele, I. Aifantis, Nat. Med. 13 (2007) 70–77.
References
[22] M.L. Guzman, S.J. Neering, D. Upchurch, B. Grimes, D.S. Howard, D.A. Rizzieri,
S.M. Luger, C.T. Jordan, Blood 98 (2001) 2301–2307.
[23] M.S. Holtz, S.J. Forman, R. Bhatia, Leukemia 19 (2005) 1034–1041.
[1] C.K. Angerhofer, H. Guinaudeau, V. Wongpanich, J.M. Pezzuto, G.A. Cordell, J.
Nat. Prod. 62 (1999) 59–66.