Berberine derivatives with a long alkyl chain branched by hydroxyl group and methoxycarbonyl group at 9-position show improved anti-proliferation activity and membrane permeability in A549 cells

Article abstract of DOI:10.1038/s41401-019-0346-1

Berberine (BBR) exhibits diverse bioactivities, including anticancer activity; but its poor druggability limits its applications. In this study, we designed and synthesized a series of 9-O position modified BBR derivatives aiming to improve its cell permeability and anticancer activity, utilizing a long alkyl chain branched by hydroxyl group and methoxycarbonyl group. Among these compounds, B10 showed 3.6-fold higher intracellular concentration than BBR, as well as 60-fold increased anti-proliferation activity against human lung cancer A549 cells compared with BBR. Treatment with B10 (1, 2 μM) induced apoptosis of A549 cells. Further investigations showed that B10 treatment dose-dependently affected mitochondrial functions, including oxygen consumption rate (OCR), mitochondrial membrane potential (MMP) and the morphology of mitochondria in A549 cells. Therefore, this work offers a new way for BBR structural modification through improving cell membrane permeability to affect mitochondrial functions and potential anti-tumor therapy in the future.

Full text of DOI:10.1038/s41401-019-0346-1

www.nature.com/aps
ARTICLE
Berberine derivatives with a long alkyl chain branched by
hydroxyl group and methoxycarbonyl group at 9-position
show improved anti-proliferation activity and membrane
permeability in A549 cells
Yi Liu1, Ke-xin Zhu2,3, Lei Cao2, Zhi-fu Xie2, Min Gu2, Wei Lü1, Jing-ya Li2 and Fa-jun Nan2
Berberine (BBR) exhibits diverse bioactivities, including anticancer activity; but its poor druggability limits its applications. In this
study, we designed and synthesized a series of 9-O position modified BBR derivatives aiming to improve its cell permeability and
anticancer activity, utilizing a long alkyl chain branched by hydroxyl group and methoxycarbonyl group. Among these compounds,
B10 showed 3.6-fold higher intracellular concentration than BBR, as well as 60-fold increased anti-proliferation activity against
human lung cancer A549 cells compared with BBR. Treatment with B10 (1, 2 μM) induced apoptosis of A549 cells. Further
investigations showed that B10 treatment dose-dependently affected mitochondrial functions, including oxygen consumption rate
(OCR), mitochondrial membrane potential (MMP) and the morphology of mitochondria in A549 cells. Therefore, this work offers a
new way for BBR structural modification through improving cell membrane permeability to affect mitochondrial functions and
potential anti-tumor therapy in the future.
Keywords: berberine; anticancer; membrane permeability; apoptosis; mitochondria
Acta Pharmacologica Sinica (2020) 0:1–12; https://doi.org/10.1038/s41401-019-0346-1
INTRODUCTION
[19, 20]. Vascular endothelial growth factor (VEGF) plays a vital role
in tumor cell neovascularization. BBR shows an inhibitory effect on
VEGF expression in the melanoma cell line B16F-10 [21, 22]. In colon
cancer cells, BBR can significantly inhibit the synthesis of COX-2 and
block the activation of the STAT3 signaling pathway induced by
COX-2, thereby inhibiting the invasion and metastasis of tumor cells
[23]. Moreover, BBR affects apoptosis-related proteins and pathways,
which lead to the inhibition of tumor cell proliferation and the
induction of tumor cell apoptosis. Caspase can be activated by BBR
on different tumor cells [24–26]. In addition, BBR inhibits the
proliferation of various tumor cells by inducing cell cycle arrest in
in vitro cell experiments [27–29].
Mitochondria play a key role in the antitumor effects of BBR.
Since most cancer cells have a higher mitochondrial membrane
potential (MMP) than nontransformed cells [30], the positively
charged alkaloid can selectively accumulate in mitochondria
driven by the negatively charged inner mitochondrial membranes
(IMMs). BBR treatment results in decreased MMP, inhibition of
respiratory chain complex I, increased mitochondrial permeability
transition (MPT), damage to the mitochondrial structure, and
decreased mitochondrial oxidative stress and mitochondrial DNA
copy number, which together finally causes mitochondrial
dysfunction and leads to cell death [31–33].
Berberine (BBR) is an isoquinoline quaternary alkaloid isolated
from Rhizoma coptidis (Fig. 1a), which has been widely used for
decades as a nonprescription drug to treat diarrhea in China with
good safety [1]. It has been reported in many studies that BBR
possesses diverse bioactivities, including antidiarrheal [2], anti-
bacterial [3, 4], anti-inflammatory [5], antidiabetic [6, 7] and
antihyperlipidemic [2, 8] effects. Particularly, some recent studies
have shown that BBR exhibits inhibitory effects on a variety of
tumors, such as lymphoma [9], breast [10], colorectal [11],
hepatocellular [12], lung [13], esophageal [14] and pancreatic
[15] cancers. The antitumor activity of BBR has been confirmed by
many studies that revealed that BBR could inhibit the growth of
cancer cells, induce cell apoptosis and arrest the cell cycle [16, 17].
Some studies have indicated that BBR can inhibit the migration
and invasion of tumor cells by affecting the related proteins. For
example, the expression of E-cadherin, which is a significant
mediator that regulates cell–cell adhesion and is an important
molecule for maintaining the morphological and structural integrity
of epithelial cells, can be enhanced by BBR in A549 lung cancer cells
[18]. BBR also inhibits the expression of matrix metalloproteinases,
an important class of proteins involved in the degradation of the
extracellular matrix barrier, as the first step in tumor cell metastasis
1Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University,
Shanghai 200062, China; 2State Key Laboratory of Drug Research, the National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China and 3University of Chinese Academy of Sciences, Beijing 100049, China
Correspondence: Wei Lü (wlu@chem.ecnu.edu.cn) or Jing-ya Li (jyli@simm.ac.cn) or Fa-jun Nan (fjnan@simm.ac.cn)
These authors contributed equally: Yi Liu, Ke-xin Zhu
Received: 29 July 2019 Accepted: 11 November 2019
© CPS and SIMM 2020

High membrane permeable berberine derivatives
Y Liu et al.
2
Cellular proliferation assays (dose-dependent)
A549 cells were plated into 96-well plates at a density of 3 × 104
cells/mL in triplicate and incubated with escalating concentrations
of different compounds for 72 h. CellTiter 96 AQueous One
Solution reagent (G5430, Promega) (20 μL) was added per well.
The absorbance at 490 nm was measured using a 340PC384
SpectraMAX microplate reader (Molecular Devices). IC50 values are
reported as the mean of three independent experiments
performed in triplicate. The inhibitor concentration range for
IC50 determinations was 0–200 μM.
Proliferation (time-dependent)
A549 cells were cultured in 96-well plates at a density of 3 × 104
cells/mL and incubated overnight for adherence before blank
treatment or treatment with different compounds. CellTiter 96
AQueous One Solution reagent (G5430, Promega) (20 μL) was
added per well. The absorbance at 490 nm was measured using a
340PC384 SpectraMAX microplate reader (Molecular Devices) at 0,
24, 48 and 72 h to determine cell proliferation.
Cell energy metabolic analysis
Fig. 1 Structures of berberine, its derivatives and ETC-1002.
A549 cells were cultured in Seahorse XF96 plate at a density of 3 ×
105 cells/mL and incubated 24 h for adherence. Then, the medium
was replaced by preheated oxygen consumption rate (OCR) assay
buffer after the cells were gently rinsed with phosphate-buffered
saline (PBS). Each measurement consisted of four injections, and
the working compound and final concentration were as follows:
injection A, test compounds of various concentrations; injection B,
1 μM oligomycin as an ATP synthase inhibitor; injection C, 0.6 μM
FCCP (carbonyl cyanide-p-tri-fluoromethoxyphenyl-hydrazone) as
a mitochondrial uncoupler; and injection D, 10 μM rotenone and
10 μM antimycin as electron transport chain (ETC) of oxidative
phosphorylation inhibitors. After incubation at 37 °C without CO2
for ~1 h, the OCR of the cells was measured with a Seahorse XF
extracellular flux analyzer.
However, the poor druggability of BBR leads to extremely low
plasma exposure and oral bioavailability (<5%) or acute toxicity after
intravenous injection (LD50 < 10 mg/kg), which limits its further
development as a potential drug candidate [34, 35]. To solve these
problems, some studies have been carried out that mainly focused
on modifications to C-8, C-9, C-10, C-12 and C-13 (Fig. 1b) [36–38].
Most of these studies focused on lipophilic modifications by
introducing long chain alkyl groups. For example, 8-cetylberberine
is a long chain alkylate derivative of BBR, which can inhibit the
growth of lung cancer in vitro and in vivo [39]. A palmitate ester at
the 9-position of BBR showed enhanced lipid-lowering efficacy [40].
13-N-Octylberberine derivatives had more effective antimycobacter-
ial activity [4]. Similarly, the replacement of other nonalkylate
moieties also improved BBR activity. Kang et al. synthesized 9-O-
benzoyl-substituted analogs that exerted triglyceride-lowering
effects [41]. 9-N-Substituted BBR derivatives were synthesized and
evaluated as a new class of G-quadruplex binding ligands or as
cancer immunotherapy agents [42]. Although the majority of the
modifications were centralized at the 9-position, there was a limited
report on applying this strategy to improve anticancer activity.
To explore whether the modification of the 9-position could
improve the anticancer activity of BBR, we creatively designed and
synthesized several compounds by introducing a long alkyl chain
branched by hydroxyl group and a methoxycarbonyl group at the
9-position. The substituted group shared a similar structure with
the known antihyperlipidemic drug ETC-1002 (Fig. 1c). We
evaluated the effects on proliferation and mitochondrial functions
in A549 lung cancer cells. Some compounds exhibited better
anticancer effects than BBR. Further, we found that improved cell
membrane permeability was crucial in the enhancement of anti-
proliferative activity. Therefore, we offer a new approach to
develop BBR derivatives as potential anticancer drug candidates.
Compounds cell permeability
A549 cells at a density of 2.5 × 105 cells/mL were seeded into 12-
well plates in triplicate and incubated overnight at 37 °C. After 8 h
of exposure to 5 μM different compounds, the medium was
removed by aspiration from the wells. Then, the cells were washed
three times with ice-cold PBS and lysed on ice for 20 min in 1 mL
of lysis buffer (acetonitrile:PBS = 1:2). After sonication, the super-
natants were collected and centrifuged for 10 min at 12,000 rpm
(4 °C) to remove cell debris. The supernatants were dried in a
vacuum, and then 100 μL of acetonitrile was added to the residue.
The mixture was vortexed for 3 min, and after centrifugation at
9600 × g for 5 min, the supernatant was collected. The concentra-
tion of compounds was determined by LC-MS.
Imaging and quantification of TMRE mitochondrial fluorescence
staining
A549 cells were seeded in 96-well μCLEAR black F-bottom plates
at a density of 3 × 105 cells/mL and incubated overnight for
adherence. Then, the cells were treated with different concen-
trations of compounds for 24 h. Next, cells were incubated with
100 nM tetramethylrhodamine ethyl ester perchlorate (TMRE) for
30 min and 1 μg/mL Hoechst 33342 (the final concentration) for
the next 10 min in the dark before analysis. Then, the medium
was replaced with PBS. Cells were analyzed with an Operetta
high content screening system. Images of bright field, TMRE and
Hoechst were captured with a 40× microscope, and the
fluorescence intensity of TMRE was measured.
MATERIALS AND METHODS
Synthesis
The experimental procedures and characterization of all com-
pounds are provided in the Supplementary Information.
Biological assay
A549 cells were purchased from the Cell Bank of the Chinese
Academy of Sciences (TCHu150, Shanghai, China). The A549 cell
lines were maintained in F12K medium (Gibco, CA, USA)
supplemented with 10% fetal bovine serum (FBS) (Gibco, CA,
USA) and cultured at 37 °C in 5% CO2.
Mitochondrial morphological observation by Mito Tracker Green
fluorescence staining
A549 cells were seeded in 96-well μCLEAR black F-bottom plates
at a density of 1.5 × 105 cells/mL and incubated overnight for
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
3
adherence. Cells were then incubated with different concentra-
tions of compounds for 24 h. Twenty minutes before the end of
incubation, a final concentration of 100 nM Mito Tracker Green
was added to each well and incubation continued in the dark.
After the medium was replaced with PBS, the cells were analyzed
with an Opera Phenix high content screening system. Confocal
images of Mito Tracker Green were captured under a 63×
microscope.
The synthetic route for compound B1 is illustrated in Scheme 2.
Starting from the commercially available ETC-1002, intermediate 5
was synthesized using Dess-Martin oxidation (DMP), followed by
condensation with intermediate 1. The reaction was quenched
with methanol to give the methyl esterification intermediate 6.
We chose sodium borohydride (NaBH4) to achieve the reduction
of 6, followed by oxidation with iodine to afford the final
compound B1.
Derivative B2, with a free carboxyl group, was synthesized as
shown in Scheme 3. Starting from intermediate 5, one of the
carboxyl groups of intermediate 5 was selectively protected with a
benzyl group. The resulting intermediate 8 was then condensed
with intermediate 2 to give compound 9. After reduction,
deprotection and oxidation reactions, derivative B2 was obtained.
To further explore the structure activity relationship (SAR), we
introduced different side chain substitutions with the same length
of 15 carbons and synthesized compounds B3–B7 (Scheme 4).
Different substituted pentadecanoic acids 12–15 were reacted
with oxalyl chloride and condensed with intermediate 1 to obtain
the derivatives B3–B6. Reagent 16 was condensed with inter-
mediate 2 to give compound 17. After reduction and oxidation
reactions, derivative B7 was obtained.
Western blots
Proteins of cell lysates or the cytoplasm (without mitochondria)
were separated using SDS-PAGE (10%) and transferred to NC
membranes. Nonspecific binding was blocked with 5% milk (in
TBST) for 1 h, and membranes were probed with antibodies
against β-actin (ABGENT, #AM1021B), cytochrome c (11940, Cell
Signaling Technologies), Bcl-2 (2764s, Cell Signaling Technologies),
cleaved-caspase-3 (9542, Cell Signaling Technologies) and Mcl-1
(5453s, Cell Signaling Technologies). Membranes were incubated
with secondary anti IgG-HRP, and chemiluminescence was
detected using ECL substrate (GE Healthcare, #RPN2232) and
captured with a ChemiDoc MP imaging system.
Apoptosis analysis by flow cytometry
To further enrich the diversity of structures, we used similar
methods to those used for the synthesis of B1 and B2 to
synthesize amide derivatives B8 and B9 (Scheme 5). To simplify
the synthesis, we removed the two α-methyl groups and obtained
ether derivatives B10 and B11. Alkyl bromide 26 was obtained by
starting with the reported intermediate 24 according to a previous
procedure [43]. Finally, derivative B10 was obtained by condensa-
tion of intermediate 1 with intermediate 26. Derivative B11 was
obtained by the heating and hydrolysis of B10.
A549 cells (1.5 × 105) were cultured for 24 h in 12-well plates with
or without BBR or B10 (1 μM or 2 μM) treatment. Twenty-four
hours after incubation, the cells were harvested, washed with PBS
and centrifuged at 400 × g for 2 min three times. The cells were
resuspended in 100 μL of 1× binding buffer and then incubated
with 2 μL of Annexin-V-APC and 2 μL of PI (Meilunbio, MA0220) for
10 min and then diluted with 100 μL of 1× binding buffer. Flow
cytometric data were acquired from 1 × 104 cells with a FACS
Calibur cytometer (NovoCyte, D2060R), and data were analyzed by
Flowjo software.
Activities of the BBR derivatives
We tested the activity of compound B1 on the A549 cell line
(Table 1), and the results showed a considerable increase in
potency (8.6 μM) compared with BBR (54.5 μM). Compound B1
showed a dramatic increase in cLog P (cLog P = 3.175) compared
with BBR (cLog P = −0.771), indicating that B1 might have better
lipophilicity and cell membrane permeability. BBR can inhibit
cell proliferation in a dose-dependent manner and achieves
complete suppression at the concentration of 60 μM [33]. To
investigate whether B1 could inhibit cell proliferation as well,
A549 cells were incubated in the absence and presence of
increasing concentrations of drug (0.5, 1 μM) for 24, 48 and 72 h.
BBR and ETC-1002 had no obvious effect on A549 cells (Fig. 2a, b).
However, at the same concentration, B1 had a dose- and time-
dependent effect on cell proliferation (Fig. 2c). We speculated that
one of the reasons might be the improved permeability. To verify
Statistical analysis
Data were analyzed and are expressed as the mean standard
error of the mean (S.E.M.). Difference between the two groups
were analyzed using Student's t-tests. The differences among
multiple groups were compared by one-way ANOVA. Differences
were considered significant when P < 0.05.
RESULTS
Synthesis
Eleven new BBR derivatives were designed and synthesized as
displayed in Schemes 1–5. As shown in Scheme 1, taking the
commercially available BBR as the starting material, intermediates
1–4 were obtained according to a previous report [42].
Scheme 1 a 195 °C, 30–40 mmHg, 15 min; b NaBH4, CH3OH, 0 °C, 30 min; c 2,4-Dimethoxybenzylamine, 120 °C; d HCl, CH3OH.
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
4
Scheme 2 a DMP, DCM, rt, 1 h; b (1) oxalyl chloride, dry DCM, DMF, rt, 2 h, (2) 1, CH3CN, reflux, overnight, CH3OH; c NaBH4, CH3OH, 0 °C, 1 h; d I2,
KOAc, EtOH, 2 h.
Scheme 3 a Benzyl chloride, K2CO3, DMF, 50 °C, overnight; b (1) oxalyl chloride, dry DCM, DMF, room temperature (rt), 2 h, (2) 2, Et3N, DMAP, dry
DCM overnight; c NaBH4, CH3OH, 0 °C, 1 h; d Pd/C, H2, MeOH, rt, 3 h; e I2, KOAc, EtOH, 2 h.
Scheme 4 a (1) Oxalyl chloride, dry DCM, DMF, rt, 2 h, (2) 1, pyridine, CH3CN, overnight, reflux; b 2, EDCI, DMAP, dry DCM, rt, overnight; c NaBH4,
CH3OH, 0 °C, 1 h; d I2, KOAc, EtOH, 2 h.
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
5
Scheme 5 a (1) Oxalyl chloride, dry DCM, DMF, rt, 2 h, (2) 4, pyridine, CH3CN, overnight, CH3OH; b NaBH4, CH3OH, 0 °C, 1 h; c I2, KOAc, EtOH, 2 h;
d (1) oxalyl chloride, dry DCM, DMF, rt, 2 h, (2) 4, pyridine, CH3CN, overnight; e Pd/C, H2, CH3OH, rt, 3 h; f (1) 1, 7-Dibromoheptane, potassium
tert-butoxide, dry DMF, rt, 2 h, (2) HCl (12 N), DCM, 2 h; g 1, CH3CN, overnight, reflux; h sodium hydroxide, CH3OH : H2O = 2:1, reflux, overnight.
our speculation, we conducted a cell membrane permeability
experiment in which A549 cells were incubated with BBR and B1
(5 μM) for 8 h. The results showed that the concentration of
intracellular compound B1 was higher than that of intracellular
BBR, which was consistent with the IC50 value (Fig. 2e). Therefore,
we found a compound (B1) with better permeability and
antitumor activity than BBR.
Encouraged by the results above, we next introduced
different side chain substitutions with the same length of 15
carbons to further investigate the SAR. After deprotecting the
methyl group, the acid from compound B2 completely lost its
activity, revealing that the ester moiety was important to
maintain the anticancer activity in A549 cells. To simplify the
structure, the four α-methyl groups were removed, and
compounds B3–B7 were designed and synthesized. Unfortu-
nately, all of them showed dramatic declines in their IC50 values
toward A549 cells compared with B1. Several of the compounds
were completely inactive. The above results indicated that the α-
methyl groups were beneficial for maintaining the activity.
Amide derivatives B8 and B9 both displayed weaker activity
compared to B1, indicating the importance of the oxygen at the
9-position. We then kept the 9-position oxygen to synthesize
ester derivatives B10 and B11. To our surprise, compound B10
exhibited a 60-fold increase in activity compared to BBR (IC50
0.9 μM). In addition, the acid form compound B11 had no
=
activity in accordance with the above SAR.
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
6
Table 1. Structures, cLog P valuesa and effects on the survival rate (IC50) of A549 cells by B1–B11.
O
Cl
N
O
R
O
R
Compd
BBR
cLog P
IC50 (μM)
O
-0.771
54.5 13.9
OH
O
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
3.175
2.799
5.536
3.590
4.495
3.966
3.329
3.010
2.634
4.095
3.190
8.6 2.3
135.4 2.5
20.4 0.9
158.2 57.0
37.6 13.2
95.5 31.6
46.7 6.3
27.6 9.5
>200
COOCH3
COOH
5
5
O
OH
O
5
5
O
O
12
O
O
COOH
12
O
O
O
OAc
12
O
COOCH3
12
O
OH
O
5
5
O
OH
H
N
COOCH3
5
5
O
OH
H
N
COOH
5
5
O
OH
O
O
0.9 0.6
COOEt
COOH
5
5
OH
121.9 12.9
5
5
acLog P means the logarithm of the n-octanol/water coefficients by calculation
We selected compound B10 with good potency to test its
effects on cell proliferation and cell membrane permeability. As
shown in Fig. 2d, B10 significantly inhibited cell proliferation in a
dose-dependent manner. Moreover, the inhibitory effect of B10
was stronger than that of B1, which matched well with their IC50
values in A549 cells. We also evaluated the activity of compounds
B2 and B11 in the MTS assay (Supplementary Fig. S1a, b), and
compounds B2 and B11 showed no inhibitory effect. There was
no doubt that the permeability experiment showed that the
concentration of intracellular compound B10 was higher than that
of BBR (Fig. 2f). The membrane permeabilities of compounds B2
and B11 were worse than that of BBR, which demonstrated that
the increase in activity was correlated with the increase in
permeability (Supplementary Fig. S1c).
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
7
Fig. 2 Anti-proliferative activities and permeability of compounds B1 and B10 on A549 cells. a–d Relative optical density (OD) value
measured at 490 nm on A549 cells after treatment of BBR, ETC-1002, B1 and B10 at 0, 24, 48 and 72 h. All data were shown as mean S.E.M of
three independent replications. Asterisks indicate P-values (*P < 0.05, **P < 0.01, ***P < 0.001) of control versus treated groups. e, f Permeability
experiment performed in A549 cells with 5 μM compounds treatment for 8 h to measure intracellular concentration.
Fig. 3 B10 reduced mitochondrial membrane potential in A549. TMRE assay in A549 cells following 24 h treatment with BBR, B1 or B10. a
Representative merged fluorescent images of A549 cells under Operetta 40× objective. Blue: Hoechst for nuclei; orange: TMRE for
mitochondria. Images were presented under same parameters. b Quantification of TMRE intensity in a. All data were shown as mean S.E.M
of three independent replications. Asterisks indicate P-values (*P < 0.05, **P < 0.01) of control versus treated groups.
Due to the positive charge on BBR and its derivatives, they could
accumulate into the mitochondria, driven by the strong negative
charge on the mitochondrial inner membrane. Moreover, mito-
chondria play a vital role in the physiological activity of BBR. As a
membrane fluorescent potential-dependent mitochondrial indica-
tor dye, TMRE fluorescence would increase with the enlarged MMP
difference and vice versa, which could reflect the morphology and
membrane potential of the mitochondria. To confirm the effect of
the compounds on the mitochondria, we investigated the effects
of B1, B10 and BBR on mitochondria via TMRE MMP experiments
and fluorescence microscopy experiments.
The results revealed that B10 dramatically decreased the MMP
in a dose-dependent manner, while BBR did not affect the MMP
even at the highest concentration (10 μM). B10 was obviously
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
8
Fig. 4 Mitochondrial morphological observation after compounds treatment for 24 h. The fluorescent signal of Mito Tracker Green was
observed at the concentrations of 1.25, 2.5, 5.0 and 10 μM B1, B10 and BBR under 63× microscope. Scale bar = 10 μm.
much stronger than B1 in the TMRE-based MMP assay, which was
consistent with their IC50 values (Fig. 3b). The MMP was almost
fully lost after treatment with high concentrations of B10 (10 μM).
In the fluorescence microscopy experiment, the results confirmed
that B10 could lower the MMP in a dose-dependent manner, and
the fluorescent signal of TMRE fully disappeared at the 10 μM
concentration of B10, which was much stronger than that of B1. In
addition, the fluorescence intensity had no obvious change with
BBR treatment, indicating that BBR had no effect on MMP at this
concentration (Fig. 3a). In addition, we noticed that treatment with
B1 and B10 could lead to changes in mitochondrial morphology.
BBR can induce mitochondrial structure collapse and fragmenta-
tion at 60 μM in HepG2 cells [33]. Our results showed that 10 μM
B1 could slightly change the mitochondrial morphology, while 2.5
μM B10 initiated significant mitochondrial fragmentation. On the
other hand, 10 μM BBR treatment had no obvious effects on
mitochondrial morphology, as indicated in Fig. 4. These results
revealed that B1 and B10 could influence mitochondrial functions
and change the normal mitochondrial structure, which could
finally lead to the death of cancer cells.
not affect the OCR. The OCR dramatically decreased in a dose-
dependent manner after treatment with compound B1. In
summary, the anticancer activity of compound B1 was better
than BBR. Compound B10 significantly caused a decline in the
OCR at the lowest concentration (5 μM) and lead to a sharp and
rapid loss of OCR, reaching 30% after 10 min of incubation with
high concentration treatment (>20 μM). In addition, the mito-
chondrial uncoupler FCCP did not elicit the full percent of
oligomycin-blocked ETC respiration, indicating that the ETC
respiration capacity of treated A549 cells was suppressed by
compound B10 in a dose-dependent manner. However, com-
pounds B2 and B11 had no obvious effect on the OCR
(Supplementary Fig. S1d, e).
In addition to proliferation inhibition, we regarded apoptosis
promotion as another marker of anticancer efficacy. BBR has
been reported to induce apoptosis in SiHa and HeLa cells at
doses of 100 μg/mL (297.6 μM) and 250 μg/mL (744.0 μM). In our
case, we conducted flow cytometry to evaluate whether B10
could promote A549 apoptosis with a higher capability than
BBR. We chose 1 μM and 2 μM to maintain consistency with the
proliferation inhibitory assay, in which 24 h was the intermediate
duration of treatment. As shown in Fig. 6a, the percent of
apoptotic cells was significantly increased after B10 treatment
for 24 h in a dose-dependent manner, and B10 showed
To investigate the effects of the compounds on mitochondrial
ETC function, we then investigated the OCR after B1 and B10
treatment (Fig. 5). The results showed that BBR could only slightly
inhibit OCR at high concentrations (>20 μM), while ETC-1002 did
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
9
Fig. 5 Inhibition of OCR by BBR (a), ETC-1002 (b), B1 (c) and B10 (d) in A549 cells. Mean S.E.M. Asterisks indicate P-values (**P < 0.01,
***P < 0.001) of control versus treated groups.
increased potency compared to BBR (Fig. 6a). To further confirm
the induced apoptotic mechanism, A549 cells were treated with
BBR and B10 to examine the effect on caspase-3 cleavage by
Western blot. As shown in Fig. 6b, significant caspase-3 cleavage
was observed after B10 treatment, which indicated that B10
induced A549 cell apoptosis. Mitochondrial membrane depolar-
ization is an early event in apoptosis. Cytosolic cytochrome c
was necessary for the initiation of the apoptotic program. B10
treatment induced A549 cell apoptosis, so we next detected the
levels of mitochondrial proteins related to apoptosis. After B10
treatment for 24 h, the expressions of Bcl-2 and MCL-1, the key
proteins that prevent apoptosis, were much lower than the
levels in the control group as well as the levels in the BBR-
treated group (Fig. 6b). More importantly, the release of
cytochrome c from the mitochondria into the cytoplasm was
much greater than in the control group after B10 treatment.
These experiments showed that B10 induces apoptosis in A549
cells and that this induction may be associated with the
modulation of mitochondria.
accumulates in the negatively charged inner membrane of the
mitochondria. Thus, we assumed that the derivatives could also
accumulate in and affect the mitochondria. In addition to the
generation of reactive oxygen species, oxidation and reduction of
materials and regulation of related signaling, mitochondria, as the
energy plant of the whole cell, generates ATP to maintain
physiological activities through aerobic respiration. Furthermore,
for cancer cells, the specific role varies according to both genetic
and environmental factors. Generally, mitochondrial metabolism,
bioenergetics, oxidative stress regulation, fission and fusion
dynamics, cell death susceptibility, mitochondria biogenesis,
turnover, and retrograde signaling can all contribute as mediators
of tumorigenesis. The roles of mitochondria differ with different
stages of cancer proliferation [44]. During the initial stages of
cancer, genesis can be induced by oxidative stress and signaling
pathway alterations in mitochondria. Then, in the growth stage, in
addition to the factors mentioned above, mitochondria biogenesis
and structural dynamics can be involved. During the survival
stage, mitochondria may experience metabolic reprogramming
and morphological changes. In the metastasis stage, reprogram-
ming, biogenesis and dynamics are considered to be relative.
Recent experimental results indicate that the mitochondrial
ultrastructure is involved in the relationship between the
bioenergetics and apoptotic (cell life/death decision) roles of
mitochondria [45]. The drop in the MMP and ETC respiration rate is
the first stage of the apoptosis process by mediation of
DISCUSSION
Although BBR may be used as a lead compound for the
prevention of human cancer, the poor druggability of BBR has
limited its applications. Inspired by previous studies of BBR
derivatives, we introduced long chains at the 9-position to design
and synthesize derivatives (B1–B11) that possessed different
substituent groups, such as hydroxyl and methoxycarbonyl.
Among these derivatives, compound B10 showed dramatically
increased anti-proliferative activity compared with BBR. Various
factors can lead to this phenotype. First, the physical properties
and accessibility to cells were considered. We detected the
intracellular concentration after treatment for 8 h. B1 and B10
both showed significantly higher concentrations compared to BBR
due to the introduction of the lipophilic side chains at the 9-
position. Thus, higher activities and druggability became possible
and were deserving of further research.
cytochrome
c release. In these processes, the imbalanced
distributions of protons and other various ions contribute to the
MMP between the two sides of the inner membrane to store
energy. Stable MMP is necessary for oxidative phosphorylation
and ATP generation. Thus, the MMP assay was regarded as a
classical evaluation method for mitochondrial function, in which
we chose the potential-related fluorescent dye TMRE to indicate.
The intracellular intensity of TMRE is positively correlated to the
MMP. Not surprisingly, B10 represented a significant ability to
reduce the MMP compared to BBR and B1, indicating more severe
mitochondrial dysfunction.
Second, the positive charges as another physical property of the
compounds were taken into consideration. Positively charged BBR
Third, under normal circumstances, mitochondria in the cell
are thought to connect with each other dynamically, forming a
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
10
Fig. 6 B10 induced apoptosis in A549 cells. a Flow cytometry plots of Annexin V/PI assay in A549 cells, following 24 h treatment with BBR or
B10. b Immunoblot analysis of A549 cells lysate following 24 h treatment with blank, BBR or B10 for detection of apoptosis-related proteins.
All data were shown as mean S.E.M. of three independent replications. Asterisks indicate P-values ( **P < 0.01, ***P < 0.001) of control versus
treated groups. ###P < 0.001 of BBR versus the same dose of B10.
clear web structure. In the MMP assay, fluorescent images
indicated that not only did the MMP decrease, but the structures
of some mitochondria were also altered after treatment with
B10. To further confirm this morphological change with
relatively stable intensity, we turned to Mito Tracker Green as
an MMP-independent mitochondria labeling tool. In represen-
tative images, mitochondria lost their clear and web-like
structures in the blank control group, tending to be obscure
and dot-like after treatment with B10, especially in the relatively
high dose groups. Thus, these two aspects—MMP and morphol-
ogy—reflected the negative effects on mitochondria caused by
B10, which could lead to obstacles in energy generation, mutual
cooperation or dynamic balance among the mitochondria. OCR
detection representing respiration capability also consisted of
the assumptions mentioned above. On one hand, damages to
the energy-generation ability may fail to meet the requirements
of cell activities; thus, apoptosis may be induced; on the other
hand, the initial anti-proliferative effects observed can also
result from enhanced apoptosis. Thus, the apoptosis rate was
compared with the control group after treatment with
compounds by flow cytometry. The results showed that the
rates of apoptosis were significantly improved after treatment
with B10 compared to both the blank control group and
BBR group.
Taken together, with the novel modification strategy of
introducing a long chain alkyl chain branched by a hydroxyl
group and a methoxycarbonyl group at the 9-position, we found
one BBR derivative, B10, with a higher anti-proliferative effect in
A549 cells and had initial trials to investigate the molecular
mechanism, in which we thought that the mitochondria and
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
11
caspase-3-related apoptosis were at least partly involved. Further
pharmacological activity evaluations, such as specific targets and
druggability, still deserve systematic investigation.
15. Park SH, Sung JH, Kim EJ, Chung N. Berberine induces apoptosis via ROS generation
in PANC-1 and MIA-PaCa2 pancreatic cell lines. Braz J Med Biol Res. 2015;48:111–9.
16. Yan K, Zhang C, Feng J, Hou L, Yan L, Zhou Z, et al. Induction of G1 cell cycle
arrest and apoptosis by berberine in bladder cancer cells. Eur J Pharmacol.
2011;661:1–7.
17. Chai YS, Hu J, Lei F, Wang YG, Yuan ZY, Lu X, et al. Effect of berberine on cell cycle
arrest and cell survival during cerebral ischemia and reperfusion and correlations
with p53/cyclin D1 and PI3K/Akt. Eur J Pharmacol. 2013;708:44–55.
18. Qi HW, Xin LY, Xu X, Ji XX, Fan LH. Epithelial-to-mesenchymal transition markers
to predict response of Berberine in suppressing lung cancer invasion and
metastasis. J Transl Med. 2014;12:22.
19. Sengupta D, Chowdhury KD, Chatterjee S, Sarkar A, Paul S, Sur PK, et al. Mod-
ulation of adenylate cyclase signaling in association with MKK3/6 stabilization
under combination of SAC and berberine to reduce HepG2 cell survivability.
Apoptosis. 2017;22:1362–79.
ACKNOWLEDGEMENTS
This work was supported by a grant from the Shanghai Commission of Science and
Technology (16JC1405000); the "Personalized Medicines–Molecular Signature-based
Drug Discovery and Development" Strategic Priority Research Program of the Chinese
Academy of Sciences (XDA12040328); and the China Postdoctoral Science Founda-
tion (2018M632186).
AUTHOR CONTRIBUTIONS
YL and KXZ conducted the key experiments and contributed equally to this work; YL
conducted the chemical experiments, analyzed the results and wrote the paper; KXZ
conducted the biological experiments and analyzed the results; LC and ZFX
performed part of the cell studies and analyzed the results; MG performed the LC/MS
analysis; FJN, JYL, and WL conceived the idea for the project, analyzed the results and
wrote the paper. All authors reviewed the results and approved the final version of
the manuscript.
20. Hamsa TP, Kuttan G. Berberine inhibits pulmonary metastasis through down-
regulation of MMP in metastatic B16F-10 melanoma cells. Phytother Res.
2012;26:568–78.
21. Hamsa TP, Kuttan G. Antiangiogenic activity of berberine is mediated through
the downregulation of hypoxia-inducible factor-1, VEGF, and proinflammatory
mediators. Drug Chem Toxicol. 2012;35:57–70.
22. Liu H, Ren X, Ma C. Effect of berberine on angiogenesis and HIF-1α / VEGF signal
transduction pathway in rats with cerebral ischemia—reperfusion injury. J Coll
Physicians Surg Pak. 2018;28:753–7.
ADDITIONAL INFORMATION
The online version of this article (https://doi.org/10.1038/s41401-019-0346-1)
contains supplementary material, which is available to authorized users.
23. Thirupurasundari CJ, Padmini R, Devaraj SN. Effect of berberine on the
antioxidant status, ultrastructural modifications and protein bound carbohy-
drates in azoxymethane-induced colon cancer in rats. Chem Biol Interact.
2009;177:190–5.
24. Zhao Y, Jing Z, Lv J, Zhang Z, Lin J, Cao X, et al. Berberine activates caspase-9/
cytochrome c-mediated apoptosis to suppress triple-negative breast cancer cells
in vitro and in vivo. Biomed Pharmacother. 2017;95:18–24.
Competing interests: The authors declare no competing interests.
25. Okubo S, Uto T, Goto A, Tanaka H, Nishioku T, Yamada K, et al. Berberine induces
apoptotic cell death via activation of caspase-3 and -8 in HL-60 human leukemia
cells: nuclear localization and structure-activity relationships. Am J Chin Med.
2017;45:1497–511.
26. Tong L, Xie C, Wei Y, Qu Y, Liang H, Zhang Y, et al. Antitumor effects of berberine
on gliomas via inactivation of caspase-1-mediated IL-1beta and IL-18 release.
Front Oncol. 2019;9:364.
27. Lu W, Du S, Wang J. Berberine inhibits the proliferation of prostate cancer cells
and induces G(0)/G(1) or G(2)/M phase arrest at different concentrations. Mol
Med Rep. 2015;11:3920–4.
28. Yip NK, Ho WS. Berberine induces apoptosis via the mitochondrial pathway in
liver cancer cells. Oncol Rep. 2013;30:1107–12.
29. Li L, Wang X, Sharvan R, Gao J, Qu S. Berberine could inhibit thyroid carcinoma
cells by inducing mitochondrial apoptosis, G0/G1 cell cycle arrest and suppres-
sing migration via PI3K-AKT and MAPK signaling pathways. Biomed Pharmac-
other. 2017;95:1225–31.
30. Modica-Napolitano JS, Aprille JR. Delocalized lipophilic cations selectively target
the mitochondria of carcinoma cells. Adv Drug Deliv Rev. 2001;49:63–70.
31. Pereira GC, Branco AF, Matos JA, Pereira SL, Parke D, Perkins EL, et al. Mito-
chondrially targeted effects of berberine [natural yellow 18, 5,6-dihydro-9,10-
dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a) quinolizinium] on K1735-M2 mouse
melanoma cells: comparison with direct effects on isolated mitochondrial frac-
tions. J Pharmacol Exp Ther. 2007;323:636–49.
32. Diogo CV, Machado NG, Barbosa IA, Serafim TL, Burgeiro A, Oliveira PJ. Berberine
as a promising safe anti-cancer agent—is there a role for mitochondria? Curr
Drug Targets. 2011;12:850–9.
33. Yan XJ, Yu X, Wang XP, Jiang JF, Yuan ZY, Lu X, et al. Mitochondria play an
important role in the cell proliferation suppressing activity of berberine. Sci Rep.
2017;7:41712.
REFERENCES
1. Tillhon M, Guaman Ortiz LM, Lombardi P, Scovassi AI. Berberine: new perspec-
tives for old remedies. Biochem Pharmacol. 2012;84:1260–7.
2. Kong W, Wei J, Abidi P, Lin M, Inaba S, Li C, et al. Berberine is a novel cholesterol-
lowering drug working through a unique mechanism distinct from statins. Nat
Med. 2004;10:1344–51.
3. Djebbi MA, Elabed A, Bouaziz Z, Sadiki M, Elabed S, Namour P, et al. Delivery
system for berberine chloride based on the nanocarrier ZnAl-layered double
hydroxide: physicochemical characterization, release behavior and evaluation of
anti-bacterial potential. Int J Pharm. 2016;515:422–30.
4. Liu YX, Xiao CL, Wang YX, Li YH, Yang YH, Li YB, et al. Synthesis, structure-activity
relationship and in vitro anti-mycobacterial evaluation of 13-n-octylberberine
derivatives. Eur J Med Chem. 2012;52:151–8.
5. Fan FL, Dart AM. Anti-inflammatory treatment in patients after percutaneous
coronary intervention: another potential use for berberine? Clin Exp Pharmacol
Physiol. 2012;39:404–5.
6. Zhang H, Wei J, Xue R, Wu JD, Zhao W, Wang ZZ, et al. Berberine lowers blood
glucose in type 2 diabetes mellitus patients through increasing insulin receptor
expression. Metabolism. 2010;59:285–92.
7. Yin J, Xing H, Ye J. Efficacy of berberine in patients with type 2 diabetes mellitus.
Metabolism. 2008;57:712–7.
8. Li YH, Yang P, Kong WJ, Wang YX, Hu CQ, Zuo ZY, et al. Berberine analogues as a
novel class of the low-density-lipoprotein receptor up-regulators: synthesis,
structure-activity relationships, and cholesterol-lowering efficacy. J Med Chem.
2009;52:492–501.
9. Goto H, Kariya R, Shimamoto M, Kudo E, Taura M, Katano H, et al. Antitumor effect
of berberine against primary effusion lymphoma via inhibition of NF-kappaB
pathway. Cancer Sci. 2012;103:775–81.
10. Barzegar E, Fouladdel S, Movahhed TK, Atashpour S, Ghahremani MH, Ostad SN,
et al. Effects of berberine on proliferation, cell cycle distribution and apoptosis of
34. Maeng HJ, Yoo HJ, Kim IW, Song IS, Chung SJ, Shim CK. P-glycoprotein-mediated
transport of berberine across Caco-2 cell monolayers.
J Pharm Sci.
human breast cancer T47D and MCF7 cell lines. Iran
2015;18:334–42.
J Basic Med Sci.
2002;91:2614–21.
35. Kheir MM, Wang Y, Hua L, Hu J, Li L, Lei F, et al. Acute toxicity of berberine and its
correlation with the blood concentration in mice. Food Chem Toxicol.
2010;48:1105–10.
36. Jin Y, Khadka DB, Cho WJ. Pharmacological effects of berberine and its deriva-
tives: a patent update. Expert Opin Ther Pat. 2016;26:229–43.
37. Singh IP, Mahajan S. Berberine and its derivatives: a patent review (2009 - 2012).
Expert Opin Ther Pat. 2013;23:215–31.
38. Fu S, Xie Y, Tuo J, Wang Y, Zhu W, Wu S, et al. Discovery of mitochondria-
targeting berberine derivatives as the inhibitors of proliferation, invasion and
migration against rat C6 and human U87 glioma cells. MedChemComm.
2015;6:164–73.
11. Cai Y, Xia Q, Luo R, Huang P, Sun Y, Shi Y, et al. Berberine inhibits the growth of
human colorectal adenocarcinoma in vitro and in vivo. J Nat Med. 2014;68:53–62.
12. Hou Q, Tang X, Liu H, Tang J, Yang Y, Jing X, et al. Berberine induces cell death in
human hepatoma cells in vitro by downregulating CD147. Cancer Sci.
2011;102:1287–92.
13. Katiyar SK, Meeran SM, Katiyar N, Akhtar S. p53 Cooperates berberine-induced
growth inhibition and apoptosis of non-small cell human lung cancer cells
in vitro and tumor xenograft growth in vivo. Mol Carcinog. 2009;48:24–37.
14. Iizuka N, Miyamoto K, Okita K, Tangoku A, Hayashi H, Yosino S, et al. Inhibitory
effect of coptidis rhizoma and berberine on the proliferation of human eso-
phageal cancer cell lines. Cancer Lett. 2000;148:19–25.
Acta Pharmacologica Sinica (2020) 0:1 – 12

High membrane permeable berberine derivatives
Y Liu et al.
12
39. Han B, Jiang P, Xu H, Liu W, Zhang J, Wu S, et al. 8-Cetylcoptisine, a new coptisine
derivative, induces mitochondria-dependent apoptosis and G0/G1 cell cycle arrest
in human A549 cells. Chem Biol Interact. 2019;299:27–36.
40. Li YH, Li Y, Yang P, Kong WJ, You XF, Ren G, et al. Design, synthesis, and
cholesterol-lowering efficacy for prodrugs of berberrubine. Bioorg Med Chem.
2010;18:6422–8.
42. Wang YX, Pang WQ, Zeng QX, Deng ZS, Fan TY, Jiang JD, et al. Synthesis and
biological evaluation of new berberine derivatives as cancer immunotherapy
agents through targeting IDO1. Eur J Med Chem. 2018;143:1858–68.
43. Dasseux JLH, Oniciu DC, inventors. Hydroxyl compounds and compositions for
cholesterol management and related uses. United States Patent.
WO2004067489A2. 2003 Jan 23.
41. Cao S, Yu S, Cheng L, Yan J, Zhu Y, Deng Y, et al. 9-O-benzoyl-substituted ber-
berine exerts a triglyceride-lowering effect through AMPK signaling pathway in
human hepatoma HepG2 cells. Environ Toxicol Pharmacol. 2018;64:11–7.
44. Vyas S, Zaganjor E, Haigis MC. Mitochondria and cancer. Cell. 2016;166:555–66.
45. Burke PJ. Mitochondria, bioenergetics and apoptosis in cancer. Trends Cancer.
2017;3:857–70.
Acta Pharmacologica Sinica (2020) 0:1 – 12