Discovery, synthesis and biological evaluation of cycloprotoberberine derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2013.07.026

A series of new 1,13-cycloprotoberberine derivatives defined through variations at the 9-position were designed, synthesized and evaluated for their cytotoxicities in human HepG2 (hepatoma), HT1080 (fibrosarcoma) and HCT116 (colon cancer) cells. The preliminary structure-activity relationship (SAR) revealed that the replacement of 9-methoxyl with an ester moiety might significantly enhance the antiproliferative activity in vitro. Notably, compound 7f demonstrated equipotent cytotoxicity activity against breast cancer MCF-7 (parent) and doxorubicin (DOX)-resistant MCF-7 (MCF-7/ADrR) cells, indicating a mode of action different from that of DOX. Further mechanism study showed that 7f significantly inhibited activity of DNA topoisomerase I (Top I) and Top II. G2/M phase arrest and tumor cell growth reduction was observed thereafter. Thus, we consider cycloprotoberberine analogues to be a new family of promising antitumor agents with an advantage of inhibiting drug-resistant cancer cells.

Full text of DOI:10.1016/j.ejmech.2013.07.026

European Journal of Medicinal Chemistry 68 (2013) 463e472
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery, synthesis and biological evaluation of cycloprotoberberine
derivatives as potential antitumor agents
Yang-Biao Li 1, Wu-Li Zhao 1, Yan-Xiang Wang, Cai-Xia Zhang, Jian-Dong Jiang,
Chong-Wen Bi, Sheng Tang, Ru-Xian Chen, Rong-Guang Shao , Dan-Qing Song
**
*
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new 1,13-cycloprotoberberine derivatives defined through variations at the 9-position were
designed, synthesized and evaluated for their cytotoxicities in human HepG2 (hepatoma), HT1080
(fibrosarcoma) and HCT116 (colon cancer) cells. The preliminary structureꢀactivity relationship (SAR)
revealed that the replacement of 9-methoxyl with an ester moiety might significantly enhance the
antiproliferative activity in vitro. Notably, compound 7f demonstrated equipotent cytotoxicity activity
against breast cancer MCF-7 (parent) and doxorubicin (DOX)-resistant MCF-7 (MCF-7/ADrR) cells,
indicating a mode of action different from that of DOX. Further mechanism study showed that 7f
significantly inhibited activity of DNA topoisomerase I (Top I) and Top II. G2/M phase arrest and tumor
cell growth reduction was observed thereafter. Thus, we consider cycloprotoberberine analogues to be
a new family of promising antitumor agents with an advantage of inhibiting drug-resistant cancer
cells.
Received 22 January 2013
Received in revised form
1 July 2013
Accepted 6 July 2013
Available online 11 August 2013
Keywords:
Cycloprotoberberine
Topoisomerase
Structureꢀactivity relationship
Antiproliferative
Drug resistance
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
and identified structurally. The modification in compound 2
completely abolished the up-regulatory effect on LDLR mRNA
Berberine (BBR, 1, Fig. 1), which is an isoquinoline natural
product extracted from Chinese herbs Coptis chinensis, has been
extensively used as a nonprescription drug to treat diarrhea in
China for decades [1e4]. BBR has been extensively investigated
since last century and was found to have a variety of pharmaco-
logical and biological activities, such as antifungal [5], anti-
inflammatory [6], anti-oxidative [7], antineoplastic [8e11] and
glucose-lowering [12] effects. Recently, our work has identified BBR
as a novel cholesterol-lowering agent that up-regulates low-den-
sity-lipoprotein receptor (LDLR) gene expression through stabili-
zation of LDLR mRNA; the molecular mechanism is different from
that of the marketed statins [13].
expression (data not shown). Screened in the models constructed
in our laboratory, we found that compound 2 exerted a moderate
antiproliferative activity (Tables 1 and 2) against three human
tumor cell lines of HepG2 (hepatoma), HT1080 (fibrosarcoma) and
HCT116 (colon cancer) with inhibition rate between 33% and 53%,
greater than that of 1 in less than 5% inhibition (data not shown).
The mechanism of 1 for anticancer activity is to bind to and sta-
bilize the topoisomerase I (Top I)-mediated DNA cleavable com-
plex by forming a drugeTop IeDNA ternary complex, just as
hydroxycamptothecin (HCPT) or camptothecin does [15]. We
deduced that the factor that contributes to the increased anti-
proliferative activity in 2 is presumably the minor change of planar
structure (Fig. 1), which might enhance the potency of the com-
pound to intercalate into free DNA, and subsequently prevent DNA
cleavage by Top I.
In continuation of our ongoing efforts to search for derivatives
of 1 as a novel class of LDLR up-regulators [14], compound 1,13-
cycloprotoberberine chloride (2, Fig. 1) was obtained accidently
The unique chemical scaffold and promising antiproliferative
activity of 2 provoked our strong interest to further explore the
structureꢀactivity relationship (SAR) of this group of compounds.
Then, the SAR investigation for the cytotoxicity was carried out
with 2 as the lead in attempt to discover new chemical entity (NCE)
with anticancer activity. As the methylenedioxy group plays a key
role in keeping good anticancer activity [16], the present SAR
* Corresponding author. Tel./fax: þ86 10 63165268.
** Corresponding author. Tel.: þ86 10 63028003; fax: þ86 10 63017302.
E-mail addresses: shaorg@yahoo.com (R.-G. Shao), songdanqingsdq@
hotmail.com (D.-Q. Song).
1
These authors made equal contribution to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.026

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Y.-B. Li et al. / European Journal of Medicinal Chemistry 68 (2013) 463e472
Fig. 1. Modeled skeleton structures of compounds 1 and 2. Both steric structures were constructed with ACDLAB software 12.0 version (3D Viewer).
study was mainly focused on the influence of the substituents at
the 9-position. On the basis of this strategy, a series of novel
9-substituted cycloprotoberberine derivatives was designed, syn-
thesized and evaluated. Meanwhile, with the hit 2 selected,
we developed the concise synthetic routes for new cyclo-
protoberberine analogues (Scheme 1), which allows an extensive
SAR analysis. Herein, we report the synthesis of cyclo-
protoberberine derivatives and antiproliferative activity in HepG2
(hepatoma), HT1080 (fibrosarcoma) and HCT116 (colon cancer)
cell lines. Among these newly synthesized analogues, compound
7f was selected as a representative agent for further anti-
proliferative test in multidrug resistance (MDR) cells, as well as for
the primary mechanism of action.
column chromatography on silica gel using CH2Cl2 and MeOH as
eluent.
3. Results and discussion
3.1. SAR analysis for the antiproliferative activity in vitro
All of the newly synthesized compounds were initially exam-
ined for their antiproliferative activity against human HepG2 hep-
atoma cells using the sulforhodamine B (SRB) assay with HCPT as a
reference drug. Structures of the 20 cycloberberine analogues and
their cytotoxicity at the concentration of 0.6
rized in Table 1.
mg/mL were summa-
We started our SAR investigation with replacing the methoxyl at
the 9-position of 2 by an ether or ester moiety. Firstly, with an
introduction of a hydroxyl, ethoxyl, butyoxy, benzyloxy or 2,4-
difluorobenzyloxy at position 9, respectively, five new analogues
(6, 7aꢀd) were generated and followed by biological test. All of the
5 compounds exhibited a moderated cytotoxicity against HepG2
with inhibitory rate of 42ꢀ63%, similar to that of the lead 2 (53%). It
seemed that an ether moiety at the 9-position could retain the
antiproliferative activity on HepG2, regardless of their size of the
side-chains.
Next, with an introduction of an ester moiety into the C-9 of 2,
eleven new analogues (7eꢀo) were prepared and tested. 9-
Acetyloxy compound 7e exerted an increased cytotoxicity on
HepG2 with inhibition rate by 88%, in comparison with the lead 2.
9-Substituted benzoyloxy compounds (7fꢀm) including electron-
withdrawing or electron-donating groups on the phenyl ring
afforded a potential cytotoxicity inhibition rate ranging from 62% to
94%, much greater than that of the lead 2. In addition, as the
2. Chemistry
The synthetic route used for the preparation of the hit 2 and its
analogues is described in Scheme 1 with 1 as the starting material.
Compound 1 was selectively reduced to the dihydroberberine (3)
with NaBH4 as a reductive agent in the presence of 5% NaOH/K2CO3
in 62% yield [17]. Compound 3 was treated with 40% glyoxal in
refluxing HOAc/CH3CN to provide 13-acetaldehyde berberine (4)
[17,18], which was easily converted into its isomer 5 in the acid
condition. Cyclization of the intermediate 5 in 2% HCl at room
temperature afforded 2 in 38% yield. Then, compound 2 was heated
at 195ꢀ210 ꢁC under vacuum (20ꢀ30 mmHg) and acidified in
concentrated HCl/ethanol (5/95) to get the key intermediate 6 in
92% yield with the previous procedures [19]. Compound 6 was
etherified or acylated to provide the final products 7aꢀs with yields
of 15e67% [19,20]. The desired products were purified with flash

Y.-B. Li et al. / European Journal of Medicinal Chemistry 68 (2013) 463e472
Table 1 (continued )
465
Table 1
Cytotoxicity of cycloprotoberberines against human HepG2 cells with R variations
Compd
R
X
HepG2
(%).a
O
X-
SO3
N+
7s
Cl
8.9 ꢂ 5.1
O
CH3COHN
HCPT
85.4 ꢂ 3.8
R
a
% of inhibition. Cells were untreated (control) or treated with the target com-
pounds (0.6
respectively.
m
g/mL, 1.04
m
Mꢀ1.57
mM) or HCPT (0.6 mg/mL, 1.65 mM) for 48 h,
O
CH3
trimethoxyphenyl and methylenedioxyphenyl might play a key role
for the antiproliferative activity of anticancer drugs [16,21], they
were used to replace the 9-OCH3, from which two analogues
(7pꢀq) were then created. The results showed that compounds 7p
and 7q exhibited an increased activity in 87% and 89% inhibition,
respectively. In another variation, benzenesulfonyl derivative 7r
retained a cancericidal activity similar to that of lead 2, while
benzenesulfonyl derivative 7s lost the cytotoxicity completely. The
results suggested that introduction of an ester moiety at C-9 of 2
might significantly enhance the antiproliferative activity on HepG2.
Compd
R
X
HepG2
2
6
7a
7b
7c
OCH3
OH
OCH2CH3
OCH2CH2CH2CH3
OCH2Ph
Cl
Cl
Br
I
53.4 ꢂ 3.2
50.0 ꢂ 3.1
41.9 ꢂ 3.3
62.6 ꢂ 1.8
53.3 ꢂ 11.5
Br
CH2O
F
7d
Br
50.7 ꢂ 11.7
3.2. Antiproliferative activities of the part compounds in human
tumor cell lines
F
7e
7f
7g
7h
OCOCH3
OCOPh
OCOPhF-p
OCOPhF-o
Cl
Cl
Cl
Cl
88.7 ꢂ 1.1
86.6 ꢂ 2.4
70.3 ꢂ 2.6
93.7 ꢂ 16.1
Out of the 20 newly synthesized compounds, seven analogues
(7eꢀf, 7hꢀi, 7l, 7pꢀq) demonstrated a potential cancericidal effect
against HepG2 cells with above 85% growth inhibition. All of these
active derivatives were chosen to measure their IC50 values on
HepG2 and HCT116 cells for 24 and 48 h, respectively. As indicated
in Table 2, most of them showed significant antiproliferative ac-
tivities on HepG2 and HCT116 cells. Compound 7f with no sub-
stituents on the phenyl ring showed the best activity on HepG2
cells, and then was selected for the further investigation.
The growth inhibition test in drug-resistant cells of compound
7f was performed on human MCF-7 breast cancer cells, which have
developed resistance to doxorubicin (DOX), a known Top II inhibi-
tor used widely in clinic. In this experiment, we tested compound
7f for its activity against parent MCF-7 cell line and DOX-resistant
cells (MCF-7/ADrR), with DOX as a reference drug. As shown in
Fig. 2, DOX afforded a potent activity against parent MCF-7 with an
COO
F
7i
Cl
92.5 ꢂ 11.4
F
7j
7k
7l
OCOPhCl-o
OCOPhCH3-p
OCOPhOCH3-p
Cl
Cl
Cl
62.3 ꢂ 2.0
63.7 ꢂ 2.0
84.5 ꢂ 1.9
COO
7m
7n
7o
Cl
Cl
Cl
69.6 ꢂ 2.5
50.9 ꢂ 4.3
53.6 ꢂ 2.2
F
IC50 of 0.18
with an IC50 of 24.4
antiproliferative effect in MCF-7 cells with an IC50 of 2.5
almost equipotent to that in the MCF-7/ADrR cells (IC50, 3.66
m
g/mL, much greater than that in the MCF-7/ADrR cells
g/mL. Notably, compound 7f demonstrated
g/mL,
g/
NO2
m
COO
m
m
mL). It suggests that cross drug resistance between cyclo-
protoberberines and the known Top II inhibitor might not exist.
H3C
NO2
COO
Table 2
a
Antiproliferative activities of the active compounds in human tumor cell lines IC50
(m
M).
H3CO
Compd
HepG2
24 h
HCT116
24 h
NO2
48 h
48 h
COO
H3CO
H3CO
2
1.20 ꢂ 0.08
1.01 ꢂ 0.16
0.47 ꢂ 0.10
0.93 ꢂ 0.53
0.62 ꢂ 0.20
0.44 ꢂ 0.18
0.38 ꢂ 0.07
0.42 ꢂ 0.32
0.71 ꢂ 0.07
0.19 ꢂ 0.02
0.14 ꢂ 0.02
0.15 ꢂ 0.02
0.13 ꢂ 0.01
0.41 ꢂ 0.08
0.32 ꢂ 0.01
0.22 ꢂ 0.02
0.56 ꢂ 0.02
0.09 ꢂ 0.01
5.61 ꢂ 1.54
0.42 ꢂ 0.03
3.11 ꢂ 0.15
0.39 ꢂ 0.17
0.92 ꢂ 0.08
3.06 ꢂ 0.73
1.71 ꢂ 0.12
0.84 ꢂ 0.17
0.41 ꢂ 0.02
0.28 ꢂ 0.07
0.39 ꢂ 0.01
0.43 ꢂ 0.01
0.37 ꢂ 0.03
0.45 ꢂ 0.02
7e
7f
7h
7i
7l
7p
7q
7p
Cl
86.5 ꢂ 10.6
OCH3
7q
7r
piperonyloyloxy
OSO2Ph
Cl
Cl
88.6 ꢂ 10.8
56.2 ꢂ 2.0
a
IC50: a drug concentration required to inhibit human tumor cells proliferation by
50% after 24 and 48 h treatment, respectively.

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Y.-B. Li et al. / European Journal of Medicinal Chemistry 68 (2013) 463e472
O
O
O
O
O
O
CH3COO-
Cl-
N+
N+
b
a
62%
N
O
O
O
O
O
OHC
CH3
CH3
CH3
O
CH3
CH3
CH3
4
1
3
O
O
O
CH3COO-
O
Cl-
N+
N+
O
c
38%
d
92%
HOHC
O
O
CH3
CH3
CH3
CH3
5
2
O
O
O
Cl-
X-
N+
N+
O
f or g
OH
O
15-67%
e
R
O
O
RCOOH
CH3
CH3
7a-s
6
Scheme 1. Reagents and conditions: (a) NaBH4, 5% NaOH/K2CO3, CH3OH, rt, 2 h; (b) 40% glyoxal, HOAc/CH3CN, reflux, 5 h; (c) 2% HCl, rt, 5 d; (d) 20ꢀ30 mmHg, 195ꢀ210 ꢁC; (e)
SOCl2, reflux, 2 h; (f) RCOCl, pyridine, CH3CN, reflux, 6 h; (g) RX, KOH, DMF, 60 ꢁC, 12 h.
3.3. Primary mechanism
After the activity evaluation, we extended our work to the pri-
mary mechanism investigation of compound 7f. Flow cytometric
analysis of the DNA profile in the HCT116 cells (Fig. 3) showed that
7f treatment (0.5 mg/mL for 24 h) caused a major shift of the cell
population from G0/G1 to G2/M phase, revealing a significant
accumulation of cells in the G2/M phase. Next, we accessed the
inhibitory activity of 7f on DNA Top I, with HCPT as a reference drug.
As shown in Fig. 4 (top), compound 7f significantly inhibited the
activity of Top I at the concentration of 15
mg/mL, which was
consistent with that of HCPT. Meanwhile, the inhibitory activity of
7f on the DNA Top II was also carried out with Etoposide (VP16) as a
reference, a known Top II inhibitor. As shown in Fig. 4 (bottom),
compound 7f at 15
mg/mL also significantly inhibited Top II activity,
demonstrating potency greater than that of VP16 (100
mg/mL). The
results indicated that compound 7f had a potent inhibitory activity
on either DNA Top I or DNA Top II.
In order to further understand the binding situation between 7f
and its targets, the docking analysis of 7f with Top I was carried out
with eHiTS software. As illustrated in Fig. 5 (top), the results indi-
cated an intercalation of 7f into DNA molecules. As shown in Fig. 5
(bottom), the described binding model of the 7feTop IeDNA
showed an extended region of pep stacking interactions between
the core of 7f and the DNA base pairs G 11 and A 113, as well as a
number of hydrogen bonds formed between the oxygen atoms in 7f
and Top IeDNA residues. Besides, the benzoyloxy group at the 9-
positions of 7f might insert into the hydrophobic pocket formed
by Ala 351, Met 428, Pro 431 and Lys 436.
Fig. 2. IC50 values of 7f and DOX in MCF-7 (parent) and DOX-resistant (MCF-7/ADrR)
cell lines.

Y.-B. Li et al. / European Journal of Medicinal Chemistry 68 (2013) 463e472
467
Fig. 3. Cell cycle analysis of 7f. HCT116 cells were incubated without (control) or with 7f (0.5
distribution using flow cytometry.
mg/mL) for 4, 12, or 24 h, respectively. Cells were then analyzed for their cell cycle
Fig. 4. DNA Top I (top) and Top II (bottom) inhibitory activity of 7f at different concentration using HCPT and VP16 as a positive control respectively.

468
Y.-B. Li et al. / European Journal of Medicinal Chemistry 68 (2013) 463e472
an Autospec Uitima-TOF mass spectrometer. Flash column chro-
matography was performed on Combiflash Rf 200, particle size
0.038 mm. Melting points (mp) were obtained with CXM-300
melting point apparatus. HPLC analyses were performed on an
Agilent 1200 using a 5.0 mm C18 reversed phase column. Purities of
biologically important compounds were ꢃ95%. For purities esti-
mated by HPLC, the major peak accounted for ꢃ95% of the com-
bined total peak area when monitored by an MWD detector at
278 nm.
5.1.1. 2,3-Methylenedioxy-9,10-dimethoxy-1,13-
cycloprotoberberine chloride (2)
To a stirred solution of 1 (7.4 g, 20 mmol) and K2CO3 (8.3 g,
60 mmol) in methanol (250 mL), 5% NaOH (10 mL) solution con-
taining NaBH4 (1.2 g, 32 mmol) was added dropwise. The reaction
mixture was stirred at room temperature for 2 h and the precipi-
tated solid was filtered, washed with distilled water (100 mL) and
80% ethanol (100 mL) to give kelly solid 3 (4.2 g, 62%). Intermediate
3 (4.0 g, 12 mmol) was then reacted with 40% glyoxal (2 mL) in the
stirred solvent mixture of CH3CN (160 mL) and HOAc (40 mL),
which was heated to 85ꢀ95 ꢁC for 5 h to prepare the desired
compound 4 (5). The solvent was evaporated under vacuum, and 2%
HCl (200 mL) was added into the residue. The reaction mixture was
stirred at room temperature for 1 h and filtered to remove undis-
solved material. The filtrate was stirred at room temperature for 5
d, then evaporated under vacuum and recrystallized from 95%
ethanol to obtain orange solid 2 (1.8 g, 38%). Mp 185ꢀ187 ꢁC; 1H
NMR (CD3SOCD3, 400 MHz):
d
3.62 (t, J ¼ 6.8 Hz, 2H), 4.12 (s, 3H),
4.16 (s, 3H), 5.24 (t, J ¼ 6.8 Hz, 2H), 6.39 (s, 2H), 7.59 (s, 1H), 8.21 (d,
J ¼ 9.2 Hz, 1H), 8.31 (d, J ¼ 9.6 Hz, 1H), 8.85 (d, J ¼ 9.6 Hz, 1H), 8.89
(d, J ¼ 9.2 Hz, 1H), 10.14 (s, 1H); 13C NMR (CD3SOCD3, 400 MHz)
d:
26.07, 55.72, 57.01, 62.18, 102.89, 110.32, 115.97, 117.00, 119.46,
120.02, 120.98, 122.15, 122.86, 126.03, 126.50, 128.06, 129.35,
140.90, 145.60, 146.61, 146.96, 150.58; HRMS: calcd for C22H18NO4Cl
[M ꢀ Cl]þ, 360.1226; found, 360.1216.
Fig. 5. Binding modes of 7f within Top I/DNA covalent complex (top) and Top IeDNA
domain (bottom). Molecular figures were generated by PyMOL.
4. Conclusions
5.1.2. 2,3-Methylenedioxy-9-hydroxy-10-methoxy-1,13-
cycloprotoberberine chloride (6)
A series of novel cycloprotoberberines defined through modifi-
cations at the 9-posotion were prepared and evaluated for their
antiproliferative activities against human tumor cell lines with 2 as
the lead. The SAR analysis indicated that replacement of the
9-methoxyl in 2 by an ester moiety might significantly enhance the
antiproliferative activity. Notably, compound 7f showed promising
potency in vitro in either naive breast cancer MCF-7 or MCF-7/ADrR
cells, suggesting an antiproliferative mechanism different from that
of DOX. Further study showed that compound 7f inhibited the ac-
tivity of DNA Top I and Top II, and arrested the cell cycle at G2/M
phase. The results suggest that these cycloprotoberberine de-
rivatives constitute a novel family of promising antitumor agents
with an advantage of inhibiting drug-resistant cancer cells and
merit further investigation.
Compound 2 (3.6 g, 9.1 mmol) was heated at 195ꢀ210 ꢁC in a dry
oven under vacuum (20ꢀ30 mmHg) for 30 min and the crude
material was acidified with concentrated HCl/ethanol (5/95) to
obtain red solid 6 (3.2 g, 92%). Mp 269ꢀ271 ꢁC; 1H NMR (CD3SOCD3,
400 MHz):
d
3.56 (t, J ¼ 6.8 Hz, 2H), 4.03 (s, 3H), 5.10 (t, J ¼ 6.8 Hz,
2H), 6.36 (s, 2H), 7.52 (s, 1H), 8.02 (d, J ¼ 8.8 Hz, 1H), 8.10 (d,
J ¼ 9.2 Hz, 1H), 8.26 (d, J ¼ 8.4 Hz, 1H), 8.75 (d, J ¼ 9.2 Hz, 1H), 9.96
(s, 1H); 13C NMR (CD3SOCD3, 400 MHz)
d: 26.17, 55.30, 56.93,
102.79, 110.21, 113.86, 115.85, 116.13, 117.00, 121.10, 121.98, 122.45,
124.44, 126.37, 127.48, 128.89, 140.80, 145.79, 145.99, 146.32, 147.15;
HRMS: calcd for C21H16NO4Cl [M ꢀ Cl]þ, 346.1073; found, 346.1069.
5.1.3. General procedures for the synthesis of compounds (7aꢀd)
A mixture of 6 (198 mg, 0.52 mmol), alkyl halide (2.08 mmol)
and KOH (60 mg, 1.04 mmol) in DMF (10 mL) was heated for 12 h at
60 ꢁC. The solvent was evaporated under vacuum, and dilute HCl
was added into the residue to get a suspension of pH 3. After
filtration, the resulting residue was purified with flash column
chromatography on silica gel using CH2Cl2 and MeOH as eluent to
obtain the desired compound.
5. Experimental section
5.1. Chemistry
Unless otherwise noted, all commercial reagents and solvents
were obtained from the commercial provider and used without
further purification. The 1H NMR spectra was performed on a
Varian Inova 400 MHz spectrometer (Varian, San Francisco, CA) and
13C NMR on a Bruker Avance III 400 spectrometer in CD3OD or
DMSO-d6, with Me4Si as the internal standard. Chemical shifts are
given in ppm scale and coupling constants (J values) are expressed
in Hz. ESI high-resolution mass spectra (HRMS) were recorded on
5.1.3.1. 2,3-Methylenedioxy-9-ethoxy-10-methoxy-1,13-
cycloprotoberberine bromide (7a). The title compound was pre-
pared from 6 and ethyl bromide (2 mL) in the same manner as
described above. Yield: 22%; Red solid; mp 216ꢀ218 ꢁC; 1H NMR
(CD3SOCD3, 400 MHz):
2H), 4.10 (s, 3H), 4.44 (q, J ¼ 7.2 Hz, 2H), 5.26 (t, J ¼ 6.8 Hz, 2H), 6.39
d
1.49 (t, J ¼ 7.2 Hz, 3H), 3.62 (t, J ¼ 6.8 Hz,

Y.-B. Li et al. / European Journal of Medicinal Chemistry 68 (2013) 463e472
469
(s, 2H), 7.58 (s, 1H), 8.20 (d, J ¼ 9.2 Hz, 1H), 8.29 (d, J ¼ 9.2 Hz, 1H),
8.83 (d, J ¼ 9.2 Hz, 1H), 8.88 (d, J ¼ 9.2 Hz, 1H), 10.05 (s, 1H); 13C
J ¼ 9.2 Hz, 1H), 8.40 (d, J ¼ 9.2 Hz, 1H), 8.95 (d, J ¼ 9.2 Hz, 1H),
9.09 (d, J ¼ 9.2 Hz, 1H), 10.15 (s, 1H); 13C NMR (CD3SOCD3,
NMR (CD3SOCD3, 400 MHz)
d
: 15.36, 26.06, 55.76, 56.96, 70.22,
400 MHz) d: 20.68, 25.91, 55.92, 57.22, 102.96, 110.37, 115.88,
102.88, 110.29, 115.95, 116.96, 119.25, 120.42, 120.93, 122.11, 122.82,
125.83,126.49,128.04,129.30,140.87,144.53,146.57,146.87,150.67;
HRMS: calcd for C23H20NO4Br [M ꢀ Br]þ, 374.1391; found, 374.1397.
117.01, 119.81, 120.87, 122.25, 122.70, 123.28, 125.30, 126.62,
128.03, 129.74, 135.44, 140.97, 145.79, 146.81, 150.43, 168.02;
HRMS: calcd for C23H18NO5Cl [M
388.1188.
ꢀ
Cl]þ, 388.1181; found,
5.1.3.2. 2,3-Methylenedioxy-9-butoxy-10-methoxy-1,13-
cycloprotoberberine iodide (7b). The title compound was prepared
from 6 and 1-butyl iodide in the same manner as described
above. Yield: 27%; Red solid; mp 202ꢀ204 ꢁC; 1H NMR
5.1.4.2. 2,3-Methylenedioxy-9-benzoyloxy-10-methoxy-1,13-
cycloprotoberberine chloride (7f). The title compound was prepared
from 6 and benzoyl chloride in the same manner as described
above. Yield: 67%; Orange solid; mp 228ꢀ230 ꢁC; 1H NMR
(CD3SOCD3, 400 MHz):
d
1.00 (t, J ¼ 7.2 Hz, 3H), 1.51e1.57 (m,
2H), 1.87e1.94 (m, 2H), 3.62 (t, J ¼ 6.8 Hz, 2H), 4.10 (s, 3H), 4.37
(t, J ¼ 6.8 Hz, 2H), 5.25 (t, J ¼ 6.8 Hz, 2H), 6.40 (s, 2H), 7.59 (s, 1H),
8.22 (d, J ¼ 9.2 Hz, 1H), 8.30 (d, J ¼ 9.2 Hz, 1H), 8.84 (d, J ¼ 9.2 Hz,
1H), 8.89 (d, J ¼ 9.2 Hz, 1H), 9.98 (s, 1H); 13C NMR (CD3SOCD3,
(CD3SOCD3, 400 MHz):
d
3.61 (t, J ¼ 6.8 Hz, 2H), 4.07 (s, 3H), 5.22 (t,
J ¼ 6.8 Hz, 2H), 6.41 (s, 2H), 7.60 (s, 1H), 7.71 (t, J ¼ 7.6 Hz, 2H), 7.86
(t, J ¼ 7.6 Hz, 1H), 8.26e8.31 (m, 3H), 8.46 (d, J ¼ 9.6 Hz, 1H), 8.98 (d,
J ¼ 9.6 Hz, 1H), 9.15 (d, J ¼ 9.2 Hz, 1H), 10.24 (s, 1H); 13C NMR
400 MHz)
d
: 13.76, 18.55, 26.07, 31.51, 55.85, 56.99, 74.20, 102.88,
(CD3SOCD3, 400 MHz) d: 25.89, 55.86, 57.30, 102.96, 110.36, 115.87,
110.29, 115.97, 117.00, 119.19, 120.23, 120.96, 122.17, 122.87,
125.94, 126.49, 128.11, 129.34, 140.90, 144.81, 146.60, 146.70,
150.56; HRMS: calcd for C25H24NO4I [M ꢀ I]þ, 402.1705; found,
402.1702.
117.04, 119.92, 120.91, 122.27, 122.94, 123.32, 125.30, 126.65, 127.96,
128.09, 129.10 (2), 129.77, 130.45 (2), 134.63, 135.61, 140.98, 145.86,
146.82, 150.39, 163.44; HRMS: calcd for C28H20NO5Cl [M ꢀ Cl]þ,
450.1346; found, 450.1336.
5.1.3.3. 2,3-Methylenedioxy-9-benzyloxy-10-methoxy-1,13-
cycloprotoberberine bromide (7c). The title compound was pre-
pared from 6 and benzyl bromide in the same manner as described
above. Yield: 60%; Orange solid; mp 131ꢀ133 ꢁC; 1H NMR
5.1.4.3. 2,3-Methylenedioxy-9-(p-fluorobenzoyloxy)-10-methoxy-
1,13-cycloprotoberberine chloride (7g). The title compound was
prepared from 6 and 4-fluorobenzoyl chloride in the same
manner as described above. Yield: 31%; Orange solid; mp
(CD3SOCD3, 400 MHz):
d
3.61 (t, J ¼ 6.8 Hz, 2H), 4.14 (s, 3H), 5.22 (t,
218ꢀ220 ꢁC; 1H NMR (CD3SOCD3, 400 MHz):
d
3.61 (t, J ¼ 6.4 Hz,
J ¼ 6.8 Hz, 2H), 5.43 (s, 2H), 6.39 (s, 2H), 7.35e7.43 (m, 3H), 7.59 (s,
1H), 7.62 (d, J ¼ 6.8 Hz, 2H), 8.21 (d, J ¼ 9.2 Hz, 1H), 8.32 (d,
J ¼ 9.6 Hz, 1H), 8.86 (t, J ¼ 9.2 Hz, 2H), 9.98 (s, 1H); 13C NMR
2H), 4.08 (s, 3H), 5.21 (t, J ¼ 6.4 Hz, 2H), 6.41 (s, 2H), 7.56 (t,
J ¼ 8.8 Hz, 2H), 7.60 (s, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.35e8.39 (m,
2H), 8.46 (d, J ¼ 9.6 Hz, 1H), 8.99 (d, J ¼ 9.2 Hz, 1H), 9.16 (d,
(CD3SOCD3, 400 MHz)
d: 26.09, 55.88, 57.01, 75.60, 102.90, 110.34,
J ¼ 9.6 Hz, 1H), 10.24 (s, 1H); 13C NMR (CD3SOCD3, 400 MHz)
d:
115.95, 117.01, 119.72, 120.45, 120.97, 122.14, 122.98, 125.88, 126.48,
128.03, 128.38 (2), 128.46, 128.82 (2), 129.29, 136.33, 140.93, 143.94,
146.65, 146.77, 150.87; HRMS: calcd for C28H22NO4Br [M ꢀ Br]þ,
436.1547; found, 436.1562.
25.89, 55.87, 57.29, 102.97, 110.35, 115.80, 116.23, 116.45, 116.98,
119.81, 120.81, 122.18, 122.96, 123.26, 124.61, 125.22, 126.60,
128.02, 129.69, 133.49, 133.58, 135.41, 140.94, 145.81, 146.81,
150.34, 162.50, 164.64; HRMS: calcd for C28H19NO5FCl [M ꢀ Cl]þ,
468.1248; found, 468.1256.
5.1.3.4. 2,3-Methylenedioxy-9-(2,4-difluorobenzyloxy)-10-methoxy-
1,13-cycloprotoberberine bromide (7d). The title compound was
prepared from 6 and 2,4-difluorobenzyl bromide in the same
manner as described above. Yield: 55%; Red solid; mp 138ꢀ140 ꢁC;
5.1.4.4. 2,3-Methylenedioxy-9-(o-fluorobenzoyloxy)-10-methoxy-
1,13-cycloprotoberberine chloride (7h). The title compound was
prepared from 6 and 2-fluorobenzoyl chloride in the same manner
as described above. Yield: 27%; Red solid; mp 181ꢀ183 ꢁC; 1H NMR
1H NMR (CD3SOCD3, 400 MHz):
d
3.61 (t, J ¼ 6.8 Hz, 2H), 4.11 (s, 3H),
5.22 (t, J ¼ 6.8 Hz, 2H), 5.47 (s, 2H), 6.40 (s, 2H), 7.14e7.19 (m, 1H),
7.28e7.33 (m, 1H), 7.60 (s, 1H), 7.81 (q, J ¼ 8.4 Hz, 1H), 8.22 (d,
J ¼ 9.2 Hz, 1H), 8.33 (d, J ¼ 9.6 Hz, 1H), 8.88 (d, J ¼ 4.4 Hz, 1H), 8.90
(CD3SOCD3, 400 MHz):
d
3.62 (t, J ¼ 6.8 Hz, 2H), 4.10 (s, 3H), 5.22
(t, J ¼ 6.8 Hz, 2H), 6.41 (s, 2H), 7.51e7.57 (m, 2H), 7.60 (s, 1H),
7.88e7.92 (m, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.30e8.32 (m, 1H), 8.46
(d, J ¼ 9.2 Hz, 1H), 8.98 (d, J ¼ 9.2 Hz, 1H), 9.16 (d, J ¼ 9.2 Hz, 1H),
(d, J ¼ 4.0 Hz, 1H), 9.93 (s, 1H); 13C NMR (CD3SOCD3, 600 MHz)
d:
26.46, 56.34, 57.39, 69.16, 103.30, 104.23, 104.41, 110.75, 111.95,
112.09, 116.36, 117.43, 120.43, 120.71, 121.39, 122.61, 123.45, 126.27,
126.90, 128.51, 129.80, 133.37, 133.44, 141.35, 143.94, 146.90, 147.08,
151.27; HRMS: calcd for C28H20NO4F2Br [M ꢀ Br]þ, 472.1360; found,
472.1347.
10.25 (s, 1H); 13C NMR (CD3SOCD3, 400 MHz)
d: 25.88, 55.93,
57.35, 102.97, 110.36, 115.82, 116.41, 116.99, 117.60, 119.78, 120.86,
122.22, 123.07, 123.28, 125.01, 125.28, 126.63, 128.07, 129.75,
132.95, 135.15, 136.95, 140.95, 145.80, 146.82, 150.28, 160.54,
163.22; HRMS: calcd for C28H19NO5FCl [M ꢀ Cl]þ, 468.1247; found,
468.1247.
5.1.4. General procedures for the synthesis of compounds (7eꢀs)
A mixture of 6 (99 mg, 0.26 mmol), acyl chloride (1.04 mmol)
and anhydrous pyridine (84 mL, 1.04 mmol) in anhydrous CH3CN
(5 mL) was refluxed for 6 h. After cooling, the mixture was filtered
and the resulting residue was purified with flash column chroma-
tography on silica gel using CH2Cl2 and MeOH as gradient eluent to
obtain desired compounds.
5.1.4.5. 2,3-Methylenedioxy-9-(2,4-difluorobenzoyloxy)-10-
methoxy-1,13-cycloprotoberberine chloride (7i). The title compound
was prepared from 6 and 2,4-difluorobenzoyl chloride in the same
manner as described above. Yield: 17%; Red solid; mp 191ꢀ193 ꢁC;
1H NMR (CD3SOCD3, 400 MHz):
d
3.62 (t, J ¼ 6.4 Hz, 2H), 4.10 (s, 3H),
5.21 (t, J ¼ 6.4 Hz, 2H), 6.41 (s, 2H), 7.41e7.46 (m, 1H), 7.61 (s, 1H),
7.62e7.68 (m, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.39 (q, J ¼ 8.4 Hz, 1H),
8.46 (d, J ¼ 9.6 Hz, 1H), 8.98 (d, J ¼ 9.6 Hz, 1H), 9.16 (d, J ¼ 9.6 Hz,
5.1.4.1. 2,3-Methylenedioxy-9-acetyloxy-10-methoxy-1,13-
cycloprotoberberine chloride (7e). The title compound was pre-
pared from 6 and acetyl chloride in the same manner as described
above. Yield: 64%; Red solid; mp 183ꢀ185 ꢁC; 1H NMR
1H), 10.24 (s, 1H); 13C NMR (CD3SOCD3, 400 MHz)
d: 25.89, 55.96,
57.37, 102.98, 106.07, 110.39, 112.60, 112.81, 113.32, 115.87, 117.05,
119.79, 120.91, 122.28, 123.17, 123.36, 125.33, 126.66, 128.14, 129.84,
135.01, 135.10, 135.21, 141.00, 145.79, 146.86, 150.31, 159.79; HRMS:
calcd for C28H18NO5F2Cl [M ꢀ Cl]þ, 486.1153; found, 486.1151.
(CD3SOCD3, 400 MHz):
(s, 3H), 5.24 (t, J ¼ 6.8 Hz, 2H), 6.41 (s, 2H), 7.61 (s, 1H), 8.26 (d,
d
2.56 (s, 3H), 3.64 (t, J ¼ 6.8 Hz, 2H), 4.09

470
Y.-B. Li et al. / European Journal of Medicinal Chemistry 68 (2013) 463e472
5.1.4.6. 2,3-Methylenedioxy-9-(o-chlorobenzoyloxy)-10-methoxy-
1,13-cycloprotoberberine chloride (7j). The title compound was
prepared from 6 and 2-chlorobenzoyl chloride in the same manner
as described above. Yield: 23%; Red solid; mp 164ꢀ166 ꢁC; 1H NMR
prepared from 6 and 4- methyl-3-nitrobenzoyl chloride in the same
manner as described above. Yield: 25%; Dark red solid; mp
165ꢀ167 ꢁC; 1H NMR (CD3SOCD3, 400 MHz):
d 2.70 (s, 3H), 3.62 (t,
J ¼ 6.8 Hz, 2H), 4.08 (s, 3H), 5.20 (t, J ¼ 6.8 Hz, 2H), 6.41 (s, 2H), 7.61
(s, 1H), 7.87 (d, J ¼ 8.0 Hz, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.47 (d,
J ¼ 9.2 Hz, 2H), 8.79 (d, J ¼ 1.2 Hz, 1H), 8.99 (d, J ¼ 9.6 Hz, 1H), 9.17
(CD3SOCD3, 400 MHz):
d
3.63 (t, J ¼ 6.8 Hz, 2H), 4.11 (s, 3H), 5.24 (t,
J ¼ 6.8 Hz, 2H), 6.41 (s, 2H), 7.61 (s, 1H), 7.66e7.69 (m, 1H), 7.79e
7.81 (m, 2H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.45e8.48 (m, 2H), 8.99 (d,
J ¼ 9.6 Hz, 1H), 9.16 (d, J ¼ 9.2 Hz, 1H), 10.24 (s, 1H); 13C NMR
(d, J ¼ 9.2 Hz, 1H), 10.27 (s, 1H); 13C NMR (CD3SOCD3, 400 MHz)
d:
19.81, 25.89, 55.94, 57.35, 102.98, 110.38, 115.85, 117.05, 119.71,
120.88, 122.24, 123.35, 125.29, 126.02, 126.63, 127.09, 128.11, 129.81,
133.39, 133.91, 134.33, 135.06, 139.59, 140.99, 145.85, 146.86, 149.12,
150.28, 161.77; HRMS: calcd for C29H21N2O7Cl [M ꢀ Cl]þ, 509.1348;
found, 509.1343.
(CD3SOCD3, 400 MHz) d: 25.90, 55.96, 57.39, 102.97, 110.38, 115.86,
117.05, 119.73, 120.91, 122.27, 123.09, 123.33, 125.36, 126.66, 126.86,
127.64, 128.14, 129.81, 131.64, 133.18, 133.99, 134.99, 135.29, 140.99,
145.77, 146.85, 150.23, 161.41; HRMS: calcd for C28H19NO5Cl2
[M ꢀ Cl]þ, 484.0952; found, 484.0952.
5.1.4.11. 2,3-Methylenedioxy-9-(m-nitro-p-methoxybenzoyloxy)-10-
methoxy-1,13-cycloprotoberberine chloride (7o). A mixture of 4-
methoxy-3-nitrobenzoic acid (205 mg, 1.04 mmol) and SOCl2
(5 mL) was refluxed for 2 h, then the surplus SOCl2 was evaporated
under vacuum to prepare acyl chloride. The title compound was
prepared from 6 and 4-methoxy-3-nitrobenzoyl chloride in the
same manner as described above. Yield: 15%; Dark red solid; mp
5.1.4.7. 2,3-Methylenedioxy-9-(p-methylbenzoyloxy)-10-methoxy-
1,13-cycloprotoberberine chloride (7k). The title compound was
prepared from 6 and 4-methylbenzoyl chloride in the same manner
as described above. Yield: 41%; Red solid; mp 221ꢀ223 ꢁC; 1H NMR
(CD3OD, 400 MHz):
d
2.47 (s, 3H), 3.60 (t, J ¼ 6.8 Hz, 2H), 4.06 (s,
3H), 5.17 (t, J ¼ 6.8 Hz, 2H), 6.29 (s, 2H), 7.40 (s, 1H), 7.43 (d,
J ¼ 8.0 Hz, 2H), 8.19 (d, J ¼ 8.0 Hz, 2H), 8.27 (d, J ¼ 9.6 Hz, 1H), 8.32
(d, J ¼ 9.6 Hz, 1H), 8.76 (d, J ¼ 9.2 Hz, 1H), 8.96 (d, J ¼ 9.2 Hz, 1H),
158ꢀ160 ꢁC; 1H NMR (CD3SOCD3, 400 MHz):
d
3.62 (t, J ¼ 6.4 Hz,
2H), 4.08 (s, 3H), 4.11 (s, 3H), 5.20 (t, J ¼ 6.4 Hz, 2H), 6.41 (s, 2H),
7.60 (s, 1H), 7.68 (d, J ¼ 8.8 Hz, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.46 (d,
J ¼ 9.6 Hz, 1H), 8.51 (dd, J ¼ 2.4, 8.8 Hz, 1H), 8.74 (d, J ¼ 2.4 Hz, 1H),
8.98 (d, J ¼ 9.6 Hz, 1H), 9.16 (d, J ¼ 9.2 Hz, 1H), 10.25 (s, 1H); 13C
9.89 (s, 1H); 13C NMR (CD3SOCD3, 400 MHz)
d: 21.35, 25.90, 55.87,
57.29, 102.96, 110.37, 115.91, 117.07, 120.00, 120.94, 122.30, 122.86,
123.34, 125.21, 125.32, 126.67, 128.11, 129.62 (2), 129.80, 130.53 (2),
135.75, 141.00, 145.29, 145.87, 146.83, 150.45, 163.43; HRMS: calcd
for C29H22NO5Cl [M ꢀ Cl]þ, 464.1499; found, 464.1489.
NMR (CD3SOCD3, 400 MHz) d: 25.91, 55.95, 57.35, 57.63, 102.98,
110.39, 115.07, 115.91, 117.09, 119.83, 119.90, 120.94, 122.30, 123.15,
123.38, 125.34, 126.66, 127.23, 128.15, 129.85, 135.22, 136.36,
139.19, 141.02, 145.87, 146.87, 150.38, 156.40, 161.63; HRMS: calcd
for C29H21N2O8Cl [M ꢀ Cl]þ, 525.1299; found, 525.1295.
5.1.4.8. 2,3-Methylenedioxy-9-(p-methoxybenzoyloxy)-10-methoxy-
1,13-cycloprotoberberine chloride (7l). The title compound was
prepared from 6 and 4-methoxybenzoyl chloride in the same
manner as described above. Yield: 51%; Orange solid; mp
5.1.4.12. 2,3-Methylenedioxy-9-(3,4,5-trimethoxybenzoyloxy)-10-
methoxy-1,13-cycloprotoberberine chloride (7p). A mixture of 3,4,5-
trimethoxybenzoic acid (221 mg, 1.04 mmol) and SOCl2 (5 mL) was
refluxed for 2 h, then the surplus SOCl2 was evaporated under
vacuum to prepare acyl chloride. The title compound was prepared
from 6 and 3,4,5-trimethoxybenzoyl chloride in the same manner
as described above. Yield: 21%; Orange solid; mp 210ꢀ212 ꢁC; 1H
202ꢀ204 ꢁC; 1H NMR (CD3SOCD3, 400 MHz):
d
3.61 (t, J ¼ 6.8 Hz,
2H), 3.92 (s, 3H), 4.06 (s, 3H), 5.22 (t, J ¼ 6.8 Hz, 2H), 6.41 (s, 2H),
7.22 (d, J ¼ 8.8 Hz, 2H), 7.59 (s, 1H), 8.24 (d, J ¼ 8.8 Hz, 2H), 8.27 (d,
J ¼ 9.6 Hz, 1H), 8.44 (d, J ¼ 9.2 Hz, 1H), 8.98 (d, J ¼ 9.6 Hz, 1H), 9.13
(d, J ¼ 9.6 Hz, 1H), 10.20 (s, 1H); 13C NMR (CD3SOCD3, 500 MHz)
d:
26.02, 55.93 (2), 57.37, 103.07, 110.46, 114.52 (2), 116.03, 117.17,
120.08, 120.19, 121.06, 122.43, 122.87, 123.44, 125.42, 126.77, 128.22,
129.89, 132.86 (2), 135.98, 141.10, 145.98, 146.93, 150.62, 163.19,
164.35; HRMS: calcd for C29H22NO6Cl [M ꢀ Cl]þ, 480.1449; found,
480.1442.
NMR (CD3SOCD3, 400 MHz):
d
3.62 (t, J ¼ 6.4 Hz, 2H), 3.83 (s, 3H),
3.92 (s, 6H), 4.08 (s, 3H), 5.24 (t, J ¼ 6.4 Hz, 2H), 6.41 (s, 2H), 7.56 (s,
2H), 7.60 (s, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.46 (d, J ¼ 9.6 Hz, 1H), 8.99
(d, J ¼ 9.2 Hz, 1H), 9.16 (d, J ¼ 9.6 Hz, 1H), 10.20 (s, 1H); HRMS: calcd
for C31H26NO8Cl [M ꢀ Cl]þ, 540.1658; found, 540.1685.
5.1.4.9. 2,3-Methylenedioxy-9-(m-nitro-p-fluorobenzoyloxy)-10-
methoxy-1,13-cycloprotoberberine chloride (7m). A mixture of 4-
fluoro-3-nitrobenzoic acid (193 mg, 1.04 mmol) and SOCl2 (5 mL)
was refluxed for 2 h, then the surplus SOCl2 was evaporated under
vacuum to prepare acyl chloride. The title compound was prepared
from 6 and 4-fluoro-3-nitrobenzoyl chloride in the same manner as
described above. Yield: 25%; Red solid; mp 187ꢀ189 ꢁC; 1H NMR
5.1.4.13. 2,3-Methylenedioxy-9-piperonyloyloxy-10-methoxy-1,13-
cycloprotoberberine chloride (7q). A mixture of piperonylic acid
(173 mg, 1.04 mmol) and SOCl2 (5 mL) was refluxed for 2 h, then the
surplus SOCl2 was evaporated under vacuum to prepare acyl
chloride. The title compound was prepared from 6 and piperonyloyl
chloride in the same manner as described above. Yield: 21%; Orange
(CD3SOCD3, 400 MHz):
d
3.62 (t, J ¼ 6.4 Hz, 2H), 4.09 (s, 3H), 5.20 (t,
solid; mp 192ꢀ194 ꢁC; 1H NMR (CD3SOCD3, 400 MHz):
d 3.61 (t,
J ¼ 6.4 Hz, 2H), 6.41 (s, 2H), 7.61 (s, 1H), 7.93e7.98 (m, 1H), 8.29 (d,
J ¼ 9.2 Hz, 1H), 8.48 (d, J ¼ 9.2 Hz, 1H), 8.67e8.68 (m, 1H), 8.94 (dd,
J ¼ 2.0, 7.2 Hz, 1H), 8.99 (d, J ¼ 9.2 Hz, 1H), 9.18 (d, J ¼ 9.6 Hz, 1H),
J ¼ 6.4 Hz, 2H), 4.07 (s, 3H), 5.23 (t, J ¼ 6.4 Hz, 2H), 6.25 (s, 2H), 6.41
(s, 2H), 7.22 (d, J ¼ 8.0 Hz, 1H), 7.60 (s, 1H), 7.73 (d, J ¼ 1.6 Hz, 1H),
7.91 (dd, J ¼ 1.6, 8.4 Hz, 1H), 8.27 (d, J ¼ 9.2 Hz, 1H), 8.44 (d,
J ¼ 9.6 Hz, 1H), 8.97 (d, J ¼ 9.6 Hz, 1H), 9.13 (d, J ¼ 9.6 Hz, 1H), 10.19
10.29 (s, 1H); 13C NMR (CD3SOCD3, 400 MHz)
d: 25.90, 55.97, 57.39,
102.99, 110.41, 115.87, 117.07, 119.66, 119.79, 120.01, 120.92, 122.26,
123.39 (2), 125.11, 125.34, 126.65, 128.16, 128.40, 129.86, 134.88,
137.31, 138.11, 141.01, 145.87, 146.89, 150.26, 161.04; HRMS: calcd for
C28H18N2O7FCl [M ꢀ Cl]þ, 513.1096; found, 513.1083.
(s, 1H); 13C NMR (CD3SOCD3, 500 MHz)
d: 26.02, 55.99, 57.38,
102.63, 103.07, 108.74, 109.79, 110.46, 115.99, 117.14, 120.03, 121.00,
121.59, 122.36, 122.91, 123.41, 125.39, 126.75, 127.02, 128.17, 129.85,
135.80, 141.09, 145.95, 146.92, 147.99, 150.56, 152.76, 162.78; HRMS:
calcd for C29H20NO7Cl [M ꢀ Cl]þ, 494.1240; found, 494.1239.
5.1.4.10. 2,3-Methylenedioxy-9-(m-nitro-p-methylbenzoyloxy)-10-
methoxy-1,13-cycloprotoberberine chloride (7n). A mixture of 4-
methyl-3-nitrobenzoic acid (188 mg, 1.04 mmol) and SOCl2
(5 mL) was refluxed for 2 h, then the surplus SOCl2 was evaporated
under vacuum to prepare acyl chloride. The title compound was
5.1.4.14. 2,3-Methylenedioxy-9-benzenesulfonyloxy-10-methoxy-
1,13-cycloprotoberberine chloride (7r). The title compound was
prepared from 6 and benzenesulfonyl chloride in the same manner
as described above. Yield: 28%; Red solid; mp 223ꢀ225 ꢁC; 1H NMR

Y.-B. Li et al. / European Journal of Medicinal Chemistry 68 (2013) 463e472
471
(CD3SOCD3, 400 MHz):
d
3.62 (t, J ¼ 6.8 Hz, 2H), 3.76 (s, 3H), 5.20 (t,
concentrations compound were added and mixed, the reaction
took place at 37 ꢁC for 20 min, then 0.5
g pBR322 DNA (Beyotime,
J ¼ 6.8 Hz, 2H), 6.42 (s, 2H), 7.63 (s, 1H), 7.73 (t, J ¼ 8.0 Hz, 2H), 7.91
(t, J ¼ 7.6 Hz,1H), 7.97 (d, J ¼ 7.2 Hz, 2H), 8.28 (d, J ¼ 9.6 Hz,1H), 8.33
(d, J ¼ 9.6 Hz, 1H), 8.95 (d, J ¼ 9.2 Hz, 1H), 9.16 (d, J ¼ 9.6 Hz, 1H),
m
Haimen, Jiangsu, China) was added and the reaction continued to
take place at 37 ꢁC for 30 min and was terminated by adding 0.5%
9.79 (s, 1H); 13C NMR (CD3SOCD3, 400 MHz)
d: 25.98, 56.47, 56.95,
SDS, 0.25 mg/mL bromophenol blue, and 15% glycerol.
103.05, 110.51, 115.73, 117.09, 120.51, 120.86, 122.42, 123.74, 124.55,
125.68, 126.70, 128.60, 128.68 (2), 129.75 (2), 130.06, 132.86, 134.40,
135.65, 141.10, 145.17, 147.08, 151.63; HRMS: calcd for C27H20NO6SCl
[M ꢀ Cl]þ, 486.1017; found, 486.1001.
5.2.5. Top II inhibition assay
Top II enzyme activity was assessed by measuring the decate-
nation of kinetoplast DNA using the Top II Assay Kit (TopoGEN,
Columbus, OH) [24]. Briefly, 0.15
bated with 4 U of human Top II (TopoGEN) in 20
buffer [50 mmol/L TriseHCl (pH 8), 120 mmol/L KCl, 10 mmol/L
m
g of kinetoplast DNA was incu-
5.1.4.15. 2,3-Methylenedioxy-9-(N-acetylsulfanilyloxy)-10-methoxy-
1,13-cycloprotoberberine chloride (7s). The title compound was
prepared from 6 and N-acetylsulfanilyl chloride in the same
manner as described above. Yield: 15%; Red solid; mp 205ꢀ207 ꢁC;
ml of the assay
MgCl2, 0.5 mmol/L ATP, 0.5 mmol/L dithiothreitol and 30 mg/mL
bovine serum albumin] at 37 ꢁC for 30 min in the presence or
1H NMR (CD3SOCD3, 400 MHz):
d
2.12 (s, 3H), 3.60 (t, J ¼ 6.4 Hz, 2H),
absence of 7f or VP16. The reaction was stopped by the addition of
5 ml of gel loading dye [5%Sarkosyl, 0.0025% bromophenol blue and
25% glycerol]. The reaction products were electrophoresed in a
horizontal 1% agarose gel in tris-acetate/EDTA buffer at 4 V/cm for
40 min at room temperature stained with 5 mg/mL ethidium bro-
3.81 (s, 3H), 5.18 (t, J ¼ 6.4 Hz, 2H), 6.42 (s, 2H), 7.63 (s, 1H), 7.86 (s,
4H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.34 (d, J ¼ 9.6 Hz, 1H), 8.95 (d,
J ¼ 9.2 Hz, 1H), 9.15 (d, J ¼ 9.6 Hz, 1H), 9.74 (s, 1H), 10.61 (s, 1H); 13C
NMR (CD3SOCD3, 400 MHz)
d
: 24.19, 25.91, 56.48, 57.01, 103.03,
110.49, 115.71, 117.05, 118.62 (2), 120.48, 120.86, 122.40, 123.67,
124.40, 125.69, 126.62, 126.94, 128.55, 129.94, 130.28 (2), 133.04,
141.06, 145.26, 145.57, 147.03, 151.76, 169.49; HRMS: calcd for
C29H23N2O7SCl [M ꢀ Cl]þ, 543.1227; found, 543.1203.
mide, distained in water, and photographed under UV light.
5.2.6. Molecular docking
The X-ray crystallographic structures of the Indolocarbazole
(SA315F)eDNAeTop I complex (PDB: 1SEU) [25] in Protein Data
Bank were selected for the docking study. The eHiTS software
package was used for flexible docking. eHiTS is an exhaustive
flexible docking algorithm with a scoring function which in-
corporates both empirical and knowledge-based features [26,27].
Active site detection of the Indolocarbazole (SA315F)eDNAeTop I
complex was carried out using the “-complex” parameter. The
program automatically detected the SA315F in the complex and
selected the part of enzyme and DNA within a 7 Å radius around the
SA315F to be the active site. The compound 7f was then docked into
the active site using the highest accuracy mode of docking (accu-
racy set to 6). Finally, the program gave 32 docked conformers
ranking the Scores. We selected the conformer with the best score
and speculated the detail binding patterns. Molecular figures were
generated by PyMOL [28].
5.2. Biological methods
5.2.1. Tumor cell lines
The human tumor cell lines HepG2, HCT116, HT1080, MCF-7
were obtained from American Type Culture Collection, Cells were
cultured in the MEM-EBSS medium (Invitrogen) supplemented
with 100 units/mL penicillin and 100 mg/mL streptomycin and 10%
heat-inactivated fetal bovine serum at 37 ꢁC in a humidified at-
mosphere containing 5% CO2. MCF-7/ADrR cells were kindly a gift
from Dr Qiyang He (Institute of Medicinal Biotechnology) and
grown in medium containing 10 mM DOX.
5.2.2. Cell growth inhibition assay
The effect on cell growth was determined using an SRB assay
[22]. Cells were seeded in 96-well plates at a density of 4 ꢄ 103e
8 ꢄ 103/mL and treated with the tested compounds (100
ml) at
Acknowledgements
different concentrations of 0.039, 0.15, 0.63 and 2.5 mg/mL respec-
tively. After 24 h and 48 h, cells were fixed with 50% trichloroacetic
acid (TCA), and then 0.4% (w/v) SRB in acetic acid (1%) was added to
stain cells. Unbound SRB was washed out by 1% acetic acid and SRB
bound cells were solubilized with 10 mM Trizma base. The absor-
bance was read at a wavelength of 492 nm. The growth inhibition
(%) was calculated at each concentration and the IC50 for reducing
the cell number to 50% of the control was obtained. Results were
obtained from triplicate determinations and are shown as mean
and SD.
This work was supported by the “National Mega-project for
Innovative Drugs” (2012ZX09301002-001-017) and the National
Natural Science Fund for Young Scientists (81102464) and the IMB
Research Foundation.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.07.026.
5.2.3. Flow cytometric analysis
Flow cytometric analysis was performed in our previous report
[22]. After treated with compound 7f (0.5 mg/mL) for 0 h, 4 h, 12 h
References
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content was analyzed with a FACScan flow cytometer (COULTER
EPICS XL, Fullerton, Cal, USA).
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