Synthesis, anticancer activities, antimicrobial activities and bioavailability of berberine-bile acid analogues

Article abstract of DOI:10.2174/157018012800673010

Fifteen berberine-bile acid analogues were synthesized. Anticancer activities of these analogues compared with berberine (BBR) were evaluated in vitro; among the analogues, A4, B4, and B5 had higher cytotoxicity than that of BBR. Most of the analogues showed higher antimicrobial activity against Staphylococcus aureus ATCC 25923 and Staphylococcus albus ATCC 8799 than that of BBR, but Bacillus subtilis AS 1.398 and Escherichia coli ATCC 31343 were not sensitive to all of the analogues. A4 and B4 were stable in the serum stability assay. B4 showed promising oral bioavailability in mice.

Full text of DOI:10.2174/157018012800673010

Letters in Drug Design & Discovery, 2012, 9, 573-580
573
Synthesis, Anticancer Activities, Antimicrobial Activities and
Bioavailability of Berberine–Bile Acid Analogues
Qingyong Li*, Wuna He, Li Zhang, Yuangang Zu*, Qiaochu Zhu, Xiaoqiu Deng, Tengfei Zhao,
Wenqing Gao and Baoyou Zhang
Key Laboratory of Forest Plant Ecology (Northeast Forestry University), Ministry of Education, 332# No. 26 Hexing
Road, Harbin city, Heilongjiang Province, 150040, China
Received February 08, 2012: Revised March 12, 2012: Accepted March 14, 2012
Abstract: Fifteen berberine–bile acid analogues were synthesized. Anticancer activities of these analogues compared with
berberine (BBR) were evaluated in vitro; among the analogues, A4, B4, and B5 had higher cytotoxicity than that of BBR.
Most of the analogues showed higher antimicrobial activity against Staphylococcus aureus ATCC 25923 and
Staphylococcus albus ATCC 8799 than that of BBR, but Bacillus subtilis AS 1.398 and Escherichia coli ATCC 31343
were not sensitive to all of the analogues. A4 and B4 were stable in the serum stability assay. B4 showed promising oral
bioavailability in mice.
Keywords: Berberine, Bile acids, Synthesis, Anticancer, Antimicrobial, Oral bioavailability.
1. INTRODUCTION
activities against Staphylococcus aureus ATCC 25923,
Staphylococcus albus ATCC 8799, Bacillus subtilis AS
1.398, and Escherichia coli ATCC 31343. Among these
analogues, the serum stability of A4 and B4 was tested in
vitro, and the oral bioavailability of A4 and B4 was tested in
vivo.
Berberine (BBR), a plant isoquinoline alkaloid extracted
from Coptis chinensis, has been widely used in China to treat
diarrhea safely for decades [1,2]. Berberine also possesses
other biological activities such as anticancer [3], antibacterial
[4], anti-inflammatory [5], anti-HIV [6], and cholesterol-
lowering [7] effects. Berberine is a water-soluble quaternary
ammonium salt, but the absorption of berberine in the
intestine is poor by oral administration [8]. Low oral
bioavailability leads to poor control of plasma concentrations
and therapeutic effects. The bioavailability of drugs is often
severely limited due to the presence of biological barriers in
the form of epithelial tight junctions, efflux proteins, and
enzymatic degradation. Recently, prodrug design has shifted
towards targeting specific enzymes or membrane
transporters [9,10].
O
R3
OH
R1
R2
4 Bile acid: R1=R2=R3= !- OH;
5 Dehydrocholic acid: R1=R2=R3= carbonyl;
6 Deoxycholic acid: R1=R3= !- OH;
Bile acids (BAs, Fig. 1), synthesized from cholesterol
exclusively in hepatocytes, are facially amphipathic, having
a curved profile with the angular methyl groups on the
convex hydrophobic β side and the hydroxyl groups on the
concave hydrophilic α side, and play an important role in
enterohepatic circulation and metabolic regulation [11-13].
With versatile derivatization possibilities, a rigid steroidal
backbone, enantiomeric purity, availability, and low cost
combined with unique physiological properties, bile acids
have been used to improve intestinal absorption and increase
the metabolic stability of pharmaceuticals in organs involved
in enterohepatic circulation [14,15].
7 Chenodeoxycholic acid: R1= R2= !- OH;
8 Ursodeoxycholic acid:R1= !- OH, R2= "- OH.
Fig. (1). Structures of bile acids (4–8).
2. MATERIALS AND METHODS
2.1. Synthesis
Structural modifications at C-9 of BBR (1) can be
generated after converting the methoxy group to hydroxyl to
obtain berberrubine (2). In our study, 1 was converted to 2
with 74% yields by using vacuum pyrolysis [16]. Different
berberine–bile acid analogues (A1–A5, B1–B5, and C1–C5)
were prepared using different bile acids as shown in Fig. (1).
In this report, we synthesized 15 berberine–bile acid
analogues, assessed their cytotoxicity against SGC-7901,
HCT 116, SMMC-7721, BEL-7402 cancer cell lines, and
normal liver cells HL-7702 and evaluated the antimicrobial
Scheme 1a shows the synthesis of intermediates of bile
acids. Scheme 1b illustrates three synthetic methods. Route
A used 2 as the starting material. Alkylation of 2 with 1, 3-
dibromopropane in N,N-dimethylformamide (DMF) was
stirred at 60°C for 6 h to afford 3 [17], and 3 was aminated
by NH3 in dimethylsulfoxide (DMSO) at room temperature
for 30 minutes, then bile-acid active esters (9–13) and
triethylamine (TEA) were added in the reacting solution. The
*Address correspondence to these authors at the Key Laboratory of Forest
Plant Ecology (Northeast Forestry University), Ministry of Education, 332#
No. 26 Hexing Road, Harbin city, Heilongjiang Province, 150040, China;
Tel: +86-451-82192336; Fax: +86-451-82102082;
E-mails: li_qingyong@126.com, zygorl@vip.hl.cn
1875-628X/12 $58.00+.00
© 2012 Bentham Science Publishers

574 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6
Li et al.
mixture was stirred for 4 h and then suitable absolute ethyl
ether was added. Filter residues were purified to obtain
products A1–A5, whose linkers were amide bonds between
BBR and bile acids.
was obtained in KBr pellets using a Nicolet Magna-560 IR
Spectrometer. Electrospray ionization (ESI) mass
spectrometric analysis was performed on an ABI-3000 LC-
MS-MS Spectrometer. All compounds tested displayed more
than 98% purity. HPLC was recorded on a Waters Alliance
2690 apparatus using a reverse-phase HiQ sil C18 (Ø 4.6
mm × 150 mm) and UV photoiode array detection at 347
nm. All reactions were monitored by thin layer
chromatography (TLC) using 0.2 mm silica gel plates G
(Sijiashenghua, Taizhou, China). Column chromatography
was carried out using silica gel zcx.II (300–400 Mesh,
Qingdao Haiyang Chemical Co. Ltd, China). Chemicals and
solvents used were commercially available. Reaction
components were visualized initially under UV (365 nm) or
color reactions were visualized after the silica gel plates with
vanillin-concentrated sulfuric acid-ethanol (10%) were
heated.
In route B, bile acids were used as the start materials. The
substitution of bile acids with 1,3-dibromopropane in DMF
was catalyzed by K2CO3 to obtain 14–18 [18]. Solutions
containing 2 and 14–18 in DMF were stirred at 60°C for 6 h
and then suitable absolute ethyl ether was added. After
filtration, the crude products were purified to give products
B1–B5.
In route C, amidation of bile-acid active esters (9–13)
with glycine in DMSO was performed using TEA as the
catalyst to obtain glycocholic acids (19–23), then 19–23 and
1,3-dibromopropane were stirred in DMF with K2CO3 as the
catalyst for 4 h to produce 24–28 [18]. Subsequently, 24–28
and 2 were stirred in DMF at 60°C for 6 h, and the resultant
mixture and suitable absolute ethyl ether were filtered. The
crude products were purified to obtain products C1–C5, with
linkers of amide and ester bonds between BBR and bile
acids.
The detailed data about compounds can be found in the
Supplementary Materials.
2.2. MTT Assays
Melting points (mp) were determined using a WRS-1B
digital melting point apparatus (Shanghai, China) and
uncorrected. H NMR spectra was recorded on a Bruker
Avance 500 spectrometer (500 MHz). Chemical shifts are
expressed as δ values (ppm) relative to tetramethylsilane
(TMS) as an internal standard (s = singlet, d = doublet, t =
triplet, q = quadruplet, m = multiplet and b = broad).
Coupling constants (J) are given in Hertz (Hz). IR spectra
MTT assays were used to assess cytotoxicity of the
analogues [19]. Human hepatoma cells SMMC-7721, BEL-
7402, colon cancer cells HCT 116, gastric cancer cells SGC-
7901 and normal liver cell HL-7702 provided by the Chinese
Academy of Sciences cell bank (Shanghai, China) were
plated in 96-well plates by a density of 105 cells/mL for 24 h
(37°C, 5% CO2). Then cells were exposed continuously for
72 h to various concentrations of tested drugs. MTT
1
O
O
R3
R3
OH
c
O
R1
R2
R1
R2
Br
O
N
14-18
4-8
O
O
R3
a
R3
O
NH
O
O
b
HO
R1
R2
R1
R2
19-23
9-13
O
c
R3
NH
O
O
4, 9, 14, 19,24: R1=R2=R3= !-OH;
5, 10, 15, 20, 25: R1=R2=R3= carbonyl;
6, 11, 16, 21, 26: R1=R3= !-OH;
7, 12, 17, 22, 27: R1=R2= !-OH;
8, 13, 18, 23, 28: R1= !-OH, R2= "-OH.
Br
R1
R2
24-28
a

Synthesis, Anticancer Activities, Antimicrobial Activities
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6 575
O
O
N+Cl-
H
A1, B1, C1: R1=R2=R3= !- OH;
A2, B2, C2: R1=R2=R3= carbonyl;
A3, B3, C3: R1=R3= !- OH;
O
O
N
O
A4, B4, C4: R1=R2= !- OH;
A5, B5, C5: R1= !- OH, R2= "- OH.
d
3
1
r
o
R2
2
1
,
O
O
1
1
R3
,
0
1
,
c
9
N+Cl-
+
O
O
Br
R1
3
A1-A5
O
O
N+Cl-
b
O
O
O
O
O
O
N+Cl-
N+Cl-
O
a
O
e
OH
O
O
+ 14, 15,16, 17 or 18
R2
R3
O
1
2
+
24,
25,
26,
27
or
e
R1
B1-B5
O
O
28
N+Cl-
R2
O
O
O
O
O
R1
N
H
R3
C1-C5
b
Scheme 1. a. Synthesis of intermediates of bile acids. Reagents and conditions: (a) N-hydroxysuccinimide, 1-ethyl-3-(3-
dimethyllaminopropyl) carbodiimide hydrochloride, CH2Cl2, 4 h; (b) glycin, DMSO, triethylamine, rt, 4 h; (c) 1,3-dibromopropane, DMF,
K2CO3, 4 h.
b. Synthesis of berberine–bile acid analogues. Reagents and conditions: (a) 20–30 mmHg, 195–210°C, 30 min; (b) DMF, 1,3-
dibromopropane, 60°C, 6 h; (c) NH3, DMSO, triethylamine, 30 min; (d) DMSO, triethylamine, 4 h; (e) DMF, 60°C, 6 h.
solution (5 mg/mL in PBS) was added (20 µL/well), and the
plates were incubated for another 4 h at 37°C. All media was
removed and formazan crystals were dissolved with DMSO
100 µL per well. After mixing equally, the absorbance was
read at 570 nm (630 nm used as a reference) on a microplate
reader (Awareness Technology, Palm City, USA). Wells
containing no drugs were used as blanks, and BBR was a
reference. Assays were performed in three independent
experiments. The IC50 was defined as the concentration of
compound that produced a 50% reduction of surviving cells
and calculated using the logit method.
2.3. Antimicrobial Testing
The microorganisms used in this study were S. aureus
ATCC 25923, S. albus ATCC 8799, and E. coli ATCC
31343 provided by the American Type Culture Collection
(Maryland, America) and B. subtilis AS 1.398 provided by

576 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6
Li et al.
the China General Microbiological Culture Collection Center
(Beijing, China). MIC was determined by the meams of a
serial 2-fold dilution following the recommendations of the
Clinical and Laboratory Standards Institute (CLSI). Bacterial
cells (105 CFU/mL) were inoculated into a nutrient broth at
0.1 mL per well of 96-well microtiter plates, in the presence
of derivates and BBR dissolved in sterilized physiological
saline solution (0.9%) at a final concentration (0, 3.9, 7.8,
15.7, 31.3, 62.5, 125, 250, 500 µg/mL). The plate was
incubated under anaerobic conditions at 37°C for 24 h. After
incubation, the wells were examined for growth of
microorganisms and the MIC was defined as the minimal
concentration of derivates that yielded no visible growth.
[20] MIC values were determined by three independent
assays. Subculturing (10 µL) from each well from the MIC
assays was reinoculated on nutrient agar plates. The plates
were incubated at 37°C for 24 h. The MBC was defined as
the minimal concentration of samples at which all inoculated
microorganisms were killed. [21] All determinations were
performed in duplicate.
of 3 by phosphoric acid. The separation of A4 and B4 was
carried out with a mobile phase of a (25:75) water/methanol
mixture, and the separation of BBR was carried out with a
mobile phase of a (55:45) water/methanol mixture. The flow
rate was 1.0 mL/min and the elution profile was monitored
by recording the UV absorbance at 347 nm. Assays were
performed in three independent experiments.
2.5. Calibration Curve
The stock solutions (3 000 ng/mL) of A4, B4, and BBR
were dissolved in methanol and diluted to give
concentrations of 3–1500 ng/mL. The control plasma
samples (200 µL) with a range of drug concentrations (20
µL) and the internal standard (20 µL) were diluted with 2-
fold methanol and then centrifuged at 12 000 r/min for 10
min. A 10 µL volume of each sample supernatant was
injected into LC-MS-MS for analysis.
2.6. Oral Bioavailability
Twelve female Wistar mice (180–220 g) were from the
Institute of Laboratory Animal Science (Shanghai, China),
and housed with 3 mice per cage to evaluate the oral
bioavailability of A4, B4, and BBR. [23] The drugs were
dissolved and diluted in physiological saline including
DMSO (3%) and ethanol (10%). The drug was administered
by oral gavage at a dose of 62.2mmol/kg. Blood was
collected from the orbital sinus at 0, 15, 30, 60, 120, 180,
240, 360, 480, and 720 min and centrifuged at 4000 r/min for
10 min. 10-Hydroxycamptothecin was used as the internal
standard of BBR, then A4 and B4 were used as the internal
2.4. Serum Stability Studies
Serum stability assays were carried out on mouse serum
to measure the stability of A4, B4, and BBR. [22] The
compounds were dissolved in 100 µL DMSO solution (4
mmol/L), then incubated in 4 mL rat blood serum at 37°C.
Aliquots were taken at different time points (0, 1, 2, 4, 6, 8,
10, 12, and 24 h). Samples (200 µL) with chromatographic-
grade methanol (600 µL) were centrifuged at 12 000 r/min
for 10 min. Liquid supernatant was analyzed by HPLC.
Deionized water with 2% triethylamine was adjusted to a pH
Table 1. Anticancer Activities of A1–A5, B1–B5, and C1–C5
Cell lines/IC50 (µM)a
Compd
SGC-7901b
HCT 116
SMMC-7721
BEL-7402
BBR
A1
A2
A3
A4
A5
B1
B2
B3
B4
B5
C1
C2
C3
C4
C5
3.33±0.26
16.58±0.39
52.45±0.35
7.64±0.38
3.13±0.29
7.17±0.53
4.52±0.59
7.10±0.38
3.78±0.54
1.13±0.08
2.08±0.13
23.07±0.34
48.36±0.35
35.79±0.26
8.84±0.24
28.32±0.43
4.16±0.14
20.19±0.40
NAc
4.55±0.15
12.40±0.25
43.33±0.57
14.38±0.65
1.44±0.08
3.60±0.09
2.97±0.09
6.26±0.33
2.74±0.07
0.50±0.02
1.62±0.10
6.05±0.28
27.86±0.50
13.13±0.26
25.24±0.38
34.92±0.33
28.06±0.34
29.78±0.40
47.63±0.80
28.10±0.16
10.38±0.45
14.00±0.10
11.18±0.32
5.66±0.32
19.33±0.44
3.33±0.51
6.52±0.39
6.73±0.61
7.74±0.49
6.27±0.35
1.52±0.43
4.14±0.35
19.60±0.41
42.48±0.39
31.39±0.12
17.47±0.22
40.25±0.21
25.91±0.64
24.28±0.35
5.82±0.13
31.20±0.52
47.42±0.54
12.11±0.23
17.62±0.41
46.32±0.93
aEffects on cancer cell line replication were determined using a standard method. Anticancer activities are expressed as IC50 values for each cell line, the concentration of compound
that caused 50% inhibition.
bSGC-7901, human gastric cancer cells; HCT 116, human colon cancer cells; SMMC-7721, human hepatocellular cells; Bel-7402, human hepatocellular cancer cells.
cNA, not active.

Synthesis, Anticancer Activities, Antimicrobial Activities
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6 577
Table 2. Cytotoxicity of A4, B4, C4 and BBR Against a Normal Liver Cell Line HL-7702 with the Concentration of 2.000, 0.400,
0.080 and 0.016(µM)
cell viability(% of control)
c(µM)
BBR
A4
B4
C4
2.000
0.400
0.080
0.016
69.44±0.44
70.38±0.35
85.79±0.18
92.76±0.67
101.82±0.23
102.47±0.97
104.24±0.35
105.55±0.49
72.28±0.62
76.71±0.68
85.47±0.41
90.12±0.70
74.50±0.36
84.38±0.21
96.42±0.77
101.60±0.50
standard for each other. A liquid supernatant/methanol (1:2)
mixture was centrifuged at 12 000 r/min for 10 min after
blending. The sample (10 µL) was injected into LC-MS-MS.
The pharmacokinetic parameters were analyzed by
noncompartmental analysis using PKSolver 2.0.
3.3. Cytotoxicity Assays on Liver Cells
The cytotoxicity against the liver cells HL-7702 of A4,
B4, C4, and BBR was mesured. The results in Table 2
indicated that A4, B4, and C4 showed lower cytotoxicity
than BBR at the same concentration. The specific carrier
proteins responsible for liver bile acid uptake might carry
compouds through the cell membrane [29].
3. RESULTS AND CONCLUSIONS
3.1. Synthesis
3.4. Antimicrobial Activities
The yields of B1–B5, whose linkers were ester bonds,
were higher than those of A1–A5 and C1–C5. It was
presumed that the lipophilicity of B1–B5 was higher than
that of the others. A1–A5 and C1–C5 were not only
quaternary ammonium salts but also linked by amide
bonding and, therefore, might be more easily adsorbed by
silica gel and more difficult to elute.
The antimicrobial activities of these new compounds
were evaluated by MIC [20] and the MBC [21] against the
gram-positive bacteria S. aureus ATCC 25923, S. albus
ATCC 8799, and B. subtilis AS 1.398 and the gram-negative
bacterium E. coli ATCC 31343 in vitro. BBR was used as a
positive control. MIC and MBC values are shown in Table 3.
The new compounds displayed superior activity against S.
aureus ATCC 25923 and S. albus ATCC 8799, and most of
the compounds displayed lower MIC, MBC values and
better antimicrobial activity than that of BBR. A3, B3, and
C3 connected with deoxycholic acid or A4, B4, and C4
connected with chenodeoxycholic acid showed better
inhibitory activity than that of A1, B1, and C1 connected
with bile acid. A2, B2, and C2 with poor activity might be
due to the connection with dehydrocholic acid.
3.2. Anticancer Activities
All new analogues were evaluated against 4 human
tumor cell lines (SGC-7901, HCT 116, SMMC-7721, and
BEL-7402) by using an MTT assay [19]. Table 1 lists the
values of IC50 of A1–A5, B1–B5, and C1–C5 with BBR as
the positive control. Table 1 indicated that most of B1–B5
compounds with ester linkers showed lower values of IC50
and better anticancer activities than that of BBR. However,
A1, A2, A3, A5 and C1–C5 displayed inferior anticancer
activities compared with BBR. A4, B4 and B5 showed
higher cytotoxicities against the tested cancer cell lines than
BBR.
The structure of the quaternary ammonium salt in
berberine analogues potentially destroys the cell membrane
of gram-positive bacteria due to disturbing the charge
balances of the cell membrane. [30] BBR modified by bile
acids acquires facial amphiphiles, which may improve
uptake by bacterial cell membranes. The substituents of the
bile acid steroid nucleus were also important to the affinity
and uptake rates of the bile acid conjugates. [31] Two
hydroxyls at C3′, C7′ or C12′ on the steroid might be optimal
for antibacterial activity, but three hydroxyl groups could
decrease the activity. The α-hydroxyl groups at C7′ or C12′
acted as hydrogen bond donors, and the hydroxylation
degree of the steroid skeleton had a significant effect on the
The complexes of bile acid could interreact with DNA to
inhibit DNA synthesis and to reduce cell proliferation [24].
But the anticancer effect of these analogues were diferent. It
might be accounted for by the structure/activity relationship.
It was presumed that the bile acid lipophilicity influenced
cytotoxicity and that bile acids linked to BBR might produce
appropriate lipophilicity and polarity for absorption [25]. The
hydroxyl functionality at the C7′ position of the steroid
nucleus may be crucial for the cytotoxic activity. Moreover,
the hydroxyl functionality at C12′ may suppress the activity
even in the presence of the hydroxyl group at C7′ [26,27]. B4
modified by chenodeoxycholic acid exhibited higher
cytotoxicty than B5 modified by ursodeoxycholic acid, that
is consistent with the result the hydroxyl at the C7′ position
in the stereochemistry alpha was suggested to enhance the
anticancer activity [26]. It suggests that bile acids play an
important role in entry into cancer cells [28].
transport
activity.[15,32,33] Chenodeoxycholic
acid
conjugates exhibited greater inhibitory potency than
ursodeoxycholic acid conjugates. This suggests that the α-
OH is favored over the β-OH at C7′ [15,34].
3.5. Serum Stability Assays
The ideal prodrug should withstand the rigors of the
gastrointestinal environment with physiochemical and

578 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6
Li et al.
Table 3. Antimicrobial Activities of A1–A5, B1–B5, and C1–C5 (µg/mL)
Compd
S. aereus ATCC 25923
S. albus ATCC 8799
B. subtilis AS 1.398
E. coli ATCC 31343
MICa
MBCb
MIC
MBC
MIC
MBC
MIC
MBC
BBR
A1
A2
A3
A4
A5
B1
B2
B3
B4
B5
C1
C2
C3
C4
C5
125
125
>500
7.8
>500
125
125
125
>500
7.8
>500
125
250
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
31.3
31.3
125
>500
31.3
31.3
125
7.8
7.8
125
7.8
125
7.8
15.7
>500
15.7
15.7
15.7
62.5
>500
62.5
62.5
500
31.3
>500
15.7
250
>500
7.8
>500
7.8
7.8
62.5
3.9
7.8
7.8
62.5
>500
15.7
15.7
250
62.5
>500
15.7
15.7
250
62.5
>500
250
31.3
500
aThe MIC (µg/mL) was defined as the concentration of the agent that completely inhibited cell growth during a 24 h incubation at 37 °C.
bThe MBC (µg/mL) was defined as the concentration of the agent that sterilized the cells.
biological properties permitting good absorption and
efficient conversion to the active parent compound once
absorbed. A4 and B4, conjugated with chenodeoxycholic
acid via different bonds, had significant effects in anticancer
and antimicrobial activities. To obtain further information on
bioavailability, A4, B4, and BBR were incubated in mouse
serum to determine the stability by HPLC. These compounds
did not degrade over a 24-hour period. A4 and B4 showed
good solubility and chemical stability in vitro.
Therefore, B4 is more easily absorbed by the gastrointestinal
system. For enterohepatic cycling of bile acids, both the
hepatocyte and the enterocyte must efficiently transport bile
acids. The major site of bile acid absorption from the small
intestine is in the terminal ileum. [35] However, the ester-
linked berberine derivative enhanced oral bioavailability;
therefore, the linking of the prodrug may have a determinant
effect in the oral bioavailability in mice.
In summary, we have designed and synthesized a novel
series of BBR analogues conjugated with bile acids to
increase the oral bioavailability of BBR. A4, B4, and B5
showed superior cytotoxity against the tested cancer cell
lines, but showed lower cytotoxity against the normal liver
cells HL-7702. All of the analogues except for A2, B2, and
C2 displayed significantly greater antimicrobial activity than
that of BBR against S. aureus ATCC 25923 and S. albus
ATCC 8799. A4 and B4 were stable in serum. B4 showed
superior oral bioavailability in vivo. The oral bioavailability
of BBR was improved by chenodeoxycholic acid via ester
bonding. Reports from our backup program where efforts are
focused on berberine–bile acid analogues with improved
pharmacokinetic properties will be forthcoming.
3.6. Oral Bioavailability Studies
The oral bioavailability of A4 and B4 was evaluated in
female Wistar mice compared with that of BBR. The drug
was administered by oral gavage at a dose of 62.2mmol/kg.
Blood was collected at various time points and plasma
samples were analyzed by LC-MS-MS. The pharmacokinetic
parameters are shown in Table 4. The time (tmax) taken to
reach the maximum plasma concentration (cmax) of B4 was
shorter than that of BBR. Compared with BBR, the AUC of
B4 was significantly higher. Conversely, the AUC of oral A4
was less than that of BBR. The clearance of B4 presented
group was significantly less than that of the other groups.
Table 4. Oral Bioavailability of A4, B4, and BBR
Compd
cmax (nM)
tmax (min)
AUC0 t (nM/ mL*min)a
Clearance (mmol/kg)/(nmol/ml)/min

BBR
A4
0.0086
0.0017
0.0324
180
180
60
1.424
0.3549
6.755
27.15
154.17
6.99
B4
aAUC0 t was defined as the area under curve from 0 min to t min.


Synthesis, Anticancer Activities, Antimicrobial Activities
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 6 579
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CONFLICT OF INTEREST
Declared none.
ACKNOWLEDGEMENTS
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This work was supported by the Program for Innovation
for graduates of NEFU, the Young Science Foundation of
Heilongjiang Province QC08C30, the Scientific Research
Foundation for Returned Scholars, Heilongjiang Province,
and funding from the Northeast Forestry University
(DL09DAQ02) and the Talents Foundation of Harbin City
(2010RFLXS013).
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ABBREVIATIONS
BBR
BA
= berberine
= bile acid
DMF
= N,N-dimethylformamide
Lianquan,
G.;
Zhishu,
H.
Quinolino-benzo-[5,
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DMSO = dimethylsulfoxide
TEA
MIC
MBC
= triethylamine
[20]
= the minimum inhibitory concentration
= the minimum bactericidal concentration
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Supplementary material is available on the publishers
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