Synthesis and biological evaluation of new berberine derivatives as cancer immunotherapy agents through targeting IDO1

Article abstract of DOI:10.1016/j.ejmech.2017.10.078

To discover small-molecule cancer immunotherapy candidates through targeting Indoleamine 2,3-dioxygenase 1 (IDO1), twenty–five new berberine (BBR) derivatives defined with substituents on position 3 or 9 were synthesized and examined for repression of IFN-γ-induced IDO1 promoter activities. Structure–activity relationship (SAR) indicated that large volume groups at the 9-position might be beneficial for potency. Among them, compounds 2f, 2i, 2n, 2o and 8b exhibited increased activities, with inhibition rate of 71–90% compared with BBR. Their effects on IDO1 expression were further confirmed by protein level as well. Furthermore, compounds 2i and 2n exhibited anticancer activity by enhancing the specific lysis of NK cells to A549 through IDO1, but not cytotoxicity. Preliminary mechanism revealed that both of them inhibited IFN-γ-induced IDO1 expression through activating AMPK and subsequent inhibition of STAT1 phosphorylation. Therefore, compounds 2i and 2n have been selected as IDO1 modulators for small-molecule cancer immunotherapy for next investigation.

Full text of DOI:10.1016/j.ejmech.2017.10.078

European Journal of Medicinal Chemistry xxx (2017) 1e11
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of new berberine derivatives as
cancer immunotherapy agents through targeting IDO1
,
,
YaneXiang Wang a 1, WeieQiang Pang a 1, QingeXuan Zeng a, ZheeSong Deng b,
TianeYun Fan a, JianeDong Jiang a, HongeBin Deng a *, DaneQing Song a
,
, **
a Institute of Medicinal Biotechnology, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, China
b Medical College, Nanchang University, Nanchang, Jiangxi 330006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
To discover small-molecule cancer immunotherapy candidates through targeting Indoleamine 2,3-
dioxygenase 1 (IDO1), twentyefive new berberine (BBR) derivatives defined with substituents on po-
sition 3 or 9 were synthesized and examined for repression of IFN-g-induced IDO1 promoter activities.
Structureeactivity relationship (SAR) indicated that large volume groups at the 9-position might be
beneficial for potency. Among them, compounds 2f, 2i, 2n, 2o and 8b exhibited increased activities, with
inhibition rate of 71e90% compared with BBR. Their effects on IDO1 expression were further confirmed
by protein level as well. Furthermore, compounds 2i and 2n exhibited anticancer activity by enhancing
the specific lysis of NK cells to A549 through IDO1, but not cytotoxicity. Preliminary mechanism revealed
Received 6 October 2017
Received in revised form
28 October 2017
Accepted 30 October 2017
Available online xxx
Keywords:
Berberine
IDO1
that both of them inhibited IFN-g-induced IDO1 expression through activating AMPK and subsequent
inhibition of STAT1 phosphorylation. Therefore, compounds 2i and 2n have been selected as IDO1
modulators for small-molecule cancer immunotherapy for next investigation.
© 2017 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
Structure-activity relationship
Cancer immunotherapy
Small-molecule
AMPK
1. Introduction
specific tumor treatment [4]. Phase I/II results showed that Epa-
cadostat and Keytruda combination demonstrated an ORR of 53%
Over the past few years, the cancer immunotherapy has made
great progress, which showns significant efficacy in human solid
tumors, with several drugs approved by the FDA, such as pro-
grammed cell death protein 1 (PD-1) antibody Keytruda® and
Opdivo® [1,2]. Clinical benefit of these antibodies as single agent,
however, has been limited to some factors such as a subset of pa-
tients, high cost, difficulty to generate a decent amount and efficacy
not for all tumor types. These limitations call for the development
of rational combination strategies or small-molecule therapeutics
aiming to extend therapeutic benefit to a broader range of patients
[3]. Meanwhile, as promising immunotherapy candidates, Indole-
amine 2,3-dioxygenase 1 (IDO1) inhibitors including Epacadostat
and Indoximod have already entered clinic trial. Especially, com-
bination of small-molecule IDO inhibitors with PD-1 antibodies
could significantly improve the objective response rate (ORR) in
(10 out of 19 patients) and a disease control rate of 74% (15 out of 19
patients) across multiple malignancies [5]. Since it is a single-chain
catalytic enzyme with a well-defined biochemistry [6], IDO1 is
considered to be an attractive target for small-molecule immuno-
therapeutics development. Therefore, it is a promising therapeutic
strategy to develop small-molecule IDO1 inhibitors or modulators
and provide new components for cancer immunotherapy
combination.
A number of pathways are related to IDO1 expression [7], and
IFN-g is the major inducer of IDO1 expression. In an effort to
discover and explore novel small-molecule immunotherapeutics
through targeting IDO1, the high-throughput screening model on
inhibition of IFN-g-induced IDO1 promoter activity was then
established in our laboratory. Given its inherent sensitivity, large
signal dynamics, and simple set up, the reporter assay platform has
been used as a high-throughput homogenous assay for screening
IDO1 modulators in this study. Then, a library of natural products
constructed in our lab was screened for their IDO1 regulating
ability. Luckily, berberine (BBR, Fig. 1), as a Traditional Chinese
Medicine used in China for decades against diarrhea, exhibited a
* Corresponding author.
** Corresponding author.
E-mail addresses: hdeng@imb.pumc.edu.cn (H. Deng), songdanqingsdq@
hotmail.com (D. Song).
moderate potency with inhibition rate of 17% at 10 mM [8e12]. The
1
These authors made equal contribution to this work.
https://doi.org/10.1016/j.ejmech.2017.10.078
0223-5234/© 2017 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article in press as: YaneXiang-g. Wang, et al., Synthesis and biological evaluation of new berberine derivatives as cancer
immunotherapy agents through targeting IDO1, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.078

2
Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
unique isoquinoline skeleton of BBR evokes our great interest to
carry out structure-activity relationship (SAR) so as to discover
small-molecule cancer-immunotherapy candidates targeting IDO1.
Therefore, a series of novel BBR derivatives, including esters, am-
ides and sulfonates on positions 3 and 9 as depicted in Fig. 1, were
prepared and evaluated for their IDO1 effects, as well as primary
mode of mechanism of the representative compounds.
substituted analogue 2i showed satisfactory inhibitory activity on
IDO1 activity. It seems that tertiary carbon or quaternary carbon
substituent with large volume might be beneficial for the inhibitory
ability. Based on these results, the replacement of bridged-ring
derivatives (2leo) at position 9 were created and tested. Surpris-
ingly, compounds 2n and 2o gave ideal activity with inhibitory rate
of 71% and 89% respectively, which indicated that large-volume
rigid structure might be favourable for the ability. The combina-
tion of aromatic ring and alkyl chain (compounds 2p and 2q) on
position 9 resulted in an obvious drop of inhibitory activity.
Moreover, the inhibitory activity of compounds 3a and 6aec
reduced significantly or lost completely, while the ester linker
fragment was switched to sulfonate or amide.
2. Results and discussion
2.1. Synthesis
A total of twenty-five new BBR derivatives were designed and
semi-synthesized as displayed in Schemes 1e3, taking commercial
available BBR, palmatine (PMT) or jatrorrhizine (JTH) as the starting
material respectively. As shown in Scheme 1, BBR esters 2aeq and
sulfonate 3a were obtained by esterification and sulfonation of
compound 1 using previously reported methods [13,14]. The key
intermediate 4 was prepared with 2,4-dimethoxyaniline as the
nucleophilic reagent as well as the solvent. Another key interme-
diate 5 with a free amine group was then acquired, and HCl/CH3OH
was selected to remove 2,4-dimethoxybenzyl in 80% yield. The
desired products 6aec were obtained by amidation with corre-
sponding acyl chloride using pyridine as the base with the yields of
33e37%.
As described in Scheme 2, compounds 8a and 8b were prepared
through de-methylation and esterification from PMT using the
procedures reported previously in overall yields of 25e33%. Simi-
larly, as depicted in Scheme 3, the desired products 9a and 9b were
obtained via esterification of JTH by 33e34% yields. All the final
products were purified via flash column chromatography using
CH3OH/CH2Cl2 as the gradient eluent (see Table 1).
Further SAR study was conducted for the substituents on posi-
tion 3 of ring A according to the above SAR analysis. The methyl-
enedioxy was opened while two analogues 8aeb were created, and
compound 8b gave an satisfactory activity with inhibitory rate of
72% which indicated that ring A might not be essential for the ac-
tivity maintaining. Moving the adamantate group from ring D to
position 3 of ring A, compound 9b gave acceptable potencies with
inhibition rate of 55%.
Based on the screening results, compounds 2f, 2i, 2n, 2o and 8b
exhibited the potent activities for modulating IFN-g-induced IDO1
promoter. In addition, as compounds 2c, 2e, 2q, 6a and 8a possess
different types of structure, all of them were selected as repre-
sentative compounds to investigate their inhibitory effects on
protein expression level of IDO1.
2.2.2. BBR analogues inhibited IFN-
protein level
g-induced IDO1 expression by
In order to further confirm the regulating effect of BBR ana-
logues, we examined whether they could block protein expression
level of IDO1 or not. A549 cells were pre-treated with 10
indicated compounds for 2 h and then treated with IFN-
IDO1 expression level in total cell lysate was detected by western
m
g
M of the
for 24 h.
2.2. Pharmacological evaluation
blot. As shown in Fig. 3, treatment with compounds 2f, 2i, 2n, 2o
and 8b significantly reduced IFN-g-induced IDO1 expression,
which was consistent with the results of dual-luciferase reporter
screening model.
2.2.1. SAR for down-regulated IFN-
activity
g-induced IDO1 promoter
Thus, we screened all the newly-synthesized BBR analogues for
their IDO1 regulating abilities by IDO1 promoter reporter assay.
A549 cells were transfected with a pGL4-IDO1-luc vector and the
inhibition rates of the BBR analogues on IFN-
moter activities are shown in Fig. 2.
First, SAR analysis was focused on the influence of substitutions
on position 9 of ring D, and 17 new ester derivatives (2aeq) were
prepared and tested. Compounds 2aed with different substituted
benzene and heterocycle were examined for their abilities to inhibit
IDO1 promoter activity, and their activities almost vanished
compared with BBR. Then, three analogues 2eeg with alkyl chain
attached were synthesized and screened, and compound 2f bearing
2-ethyl butyrate gave the most potent activity with the inhibitory
rate of 90%. Compounds 2hek with cyclic groups attached were
2.2.3. Effects of BBR analogues on A549 cell viability
g-induced IDO1 pro-
To exclude the BBR analogues-induced IDO1 down-regulation
resulted from the potential cytotoxicity, the cytotoxic effects of
the ten representative compounds on A549 cells were evaluated by
MTT assay. As displayed in Fig. 4, the results revealed that com-
pounds 2i, 2n, 6a and 8a showed no cytotoxic activities in
A549 cells, much lower than that of other BBR analogues.
2.2.4. Effects of BBR analogues on killing activity of human NK cells
toward A549 cells
To determine whether BBR analogues could enhances the
specific lysis of NK cells to A549 cells, A549 cells were pre-
tested,
and
2,2,3,3-tetramethylcyclopropane-1-carboxylate
Fig. 1. Chemical structure of BBR, and structure modification strategy.
Please cite this article in press as: YaneXiang-g. Wang, et al., Synthesis and biological evaluation of new berberine derivatives as cancer
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Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
3
Scheme 1. Synthesis of compounds 2aeq, 3a, 6aec. Reagents and conditions: (a) 195 ꢀC, 30e40 mmHg, 60 min; (b) RCOX/RSO2Cl, triethylamine, CH3CN, reflux. (c) 2,4-
Dimethoxybenzylamine, 120 ꢀC; (d) HCl, CH3OH; (e) R1COX, pyridine, CH2Cl2, reflux.
Scheme 2. Synthesis of compounds 8aeb. Reagents and conditions: (a) 195 ꢀC, 30e40 mmHg, 60 min; (b) RCOCl, triethylamine, CH3CN, reflux.
where it binds to and activates IFN-
g-responsive specific promoters
of IDO1 [15,16]. Considering that STAT1 plays a critical role in IFN-
g
-
induced IDO1 expression, preliminary mechanism study was car-
ried out to verify if the ten selected compounds work through
STAT1 pathway. STAT1 phosphorylation at residue Tyr701 is
required for acting as an active transcription factor. Therefore, we
asked whether BBR analogues interfered with STAT1 phosphory-
lation. The expression level of STAT1 and phosphorylation of STAT1
Tyr701 were detected by western blot. As shown in Fig. 6, stimu-
Scheme 3. Synthesis of compounds 9aeb. Reagents and conditions: (a) RCOX, trie-
thylamine, CH3CN, reflux.
lation of cells with IFN-
g alone resulted in a rapid increase in
Tyr701 phosphorylation of STAT1, and this increase was remarkably
inhibited by the treatment of compounds 2e, 2f, 2i, 2n, 2o and 8b
respectively.
treated with the indicated compounds with or without IFN-g for
Recent studies have shown that adenosine 50-monophosphate
(AMP)eactivated protein kinase (AMPK) activation suppresses
STAT signaling [17,18]. AMPK kinase (AMPKK), an upstream kinase
16 h. Cells were washed and then NK cells were co-cultured with
A549 cells at 10:1, NK killing activity was assessed by LDH assay
(Fig. 5A) and cell impedance assay (Fig. 5B), respectively. As
shown in Fig. 5A, compounds 2i, 2n and 8b significantly
enhanced the specific lysis of NK cells to A549 cells. In consid-
eration of the cytotoxic effects of the three compounds, we
choose compounds 2i and 2n to assess its effects on killing ac-
tivity of human NK cells toward A549 cells by cell impedance
of AMPK, phosphorylates threonine 172 of the AMPK subunit
a.
Activated AMPK phosphorylates and inactivates acetyl-CoA
carboxylase (ACC) [19,20]. To assess whether compounds 2e, 2f,
2i, 2n, 2o and 8b-induced STAT1 inactivation through AMPK
pathway, AMPK activation was determined by assessing ACC
phosphorylation on Ser79 and AMPK phosphorylation on Thr172
[21,22]. Western blot results (Fig. 7) demonstrated that compounds
2i, 2n and 8b significantly increased IFN-g-induced ACC and AMPK
phosphorylation. Compounds 2i, 2n and 8b induced AMPK acti-
vation and STAT1 dephosphorylation. Considering compounds 2i,
assay. 1-Methyl-D-tryptophan (1-MT) was used as a positive IDO1
inhibitor. As shown in Fig. 5B, significant reduction in the cell
index of A549 cells was observed following compounds 2i and 2n
treatment compared with BBR, and compound 2i exhibited
stronger activity than 1-MT.
2n and 8b markedly suppressed IFN-g-induced IDO1 promoter
activities, which demonstrated that they inhibited IFN-
g
-induced
2.2.5. BBR analogues inhibited IFN-
activity by inhibiting STAT1 phosphorylation and activating AMPK
IFN- -induced IDO1 expression involves the activation of signal
transducer and activator or transcription 1 (STAT1), which can be
activated via tyrosine phosphorylation by Janus kinases 1 (JAK1).
Activated STAT1 homodimerizes and translocates into the nucleus,
g-induced STAT1 transcription
IDO1 promoter activity by activating AMPK and subsequent inhi-
bition of STAT1 phosphorylation, indicating these compounds had
great potential as immunotherapy agents targeting inhibition of
IDO1 at the transcriptional level.
g
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4
Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
Table 1
Structures of all the newly synthesized BBR derivatives.
No
R
No
R
No.
R
2a
2j
6a
2b
2k
6b
2c
2l
6c
8a
2d
2m
2e
2f
2n
2o
8b
9a
2g
2p
9b
2h
2i
2q
3a
BBR
-
Fig. 2. Inhibition effect of BBR analogues on IFN-
control, followed by pretreatment with 10 mol/L of the indicated compounds for 2 h and stimulation with or without IFN-
and normalized to Renilla luciferase activity. #p < 0.001 compared with untreated control group; *p < 0.05, **p < 0.01, ***p < 0.001 compared with the IFN-
g
-induced IDO1 promoter activity. A549 cells were transfected with a pGL4-IDO1-luc vector and the pRL-CMV vector as an internal
for 24 h. The level of luciferase activity was measured
group.
m
g
g
Please cite this article in press as: YaneXiang-g. Wang, et al., Synthesis and biological evaluation of new berberine derivatives as cancer
immunotherapy agents through targeting IDO1, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.078

Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
5
Fig. 3. BBR analogues inhibited IFN-
g
-induced IDO1 expression. A549 cells were pre-treated with 10
mmol/L of the indicated compounds for 2 h, followed by IFN-
g
(5 ng/mL)
treatment for 24 h. The expression levels of IDO1 were measured by western blot. D: DMSO. GAPDH served as the loading control. **p < 0.01, ***p < 0.001 compared with the IFN-
group.
g
a Varian Inova 500 or 600 MHz spectrometer (Varian, San Francisco,
CA) and 13C NMR on a Bruker Avance III 500 or 600 spectrometer
with Me4Si as the internal standard, all the samples were dissolved
in DMSO-d6 before testing. ESI high-resolution mass spectra
(HRMS) was recorded on an Autospec UItima-TOF mass spec-
trometer (Micromass UK Ltd, Manchester, UK). Flash chromatog-
raphy was performed on CombiflashRf 200 (Teledyne, Nebraska,
USA), particle size 0.038 mm. Antibodies against IDO1, STAT1,
phospho-STAT1 (Thr701), AMPK
phospho-ACC (Ser79) were purchased from Cell Signaling Tech-
nology (Danvers, MA, USA). MTT, -Tubulin, GAPDH and -actin
antibody were obtained from Sigma (St. Louis, MO, USA). 1-MT (1-
Methyl- -tryptophan, IDO1 inhibitor) was purchased from Sell-
a, phospho-AMPK (Thr172) and
a
b
D
eckchem (Shanghai, China).
4.2. Chemistry
4.2.1. General synthesis procedure for synthesis of compounds
2aeq and 3a
Fig. 4. Effects of BBR analogues on A549 cell viability. A549 cells were treated with
10 mol/L of the indicated compounds for 24 h, cell toxicity was measured by MTT
assay. Control cells were treated with 0.5% (v/v) DMSO. The results are presented as
means standard error and represent three individual experiments. *p 0.05,
m
BBR (3.71 g, 10 mmol) was heated at 195e210 ꢀC for 10e15 min
under vacuum (30e40 mmHg) to afford the black oil, which was
acidified with ethanol/concentrated HCl (95:5). The solvent was
removed by evaporation, the residue was collected and then puri-
fied by flash chromatography over silica gel using CH2Cl2/CH3OH as
the gradient eluent, affording the title compound 1 (2.85 g, 80%) as
a yellow solid.
<
**p < 0.01 compared with the untreated control group.
3. Conclusions
Taking IDO1 as the target, a series of novel BBR derivatives
including esters, amides and sulfonates on different positions were
designed, prepared and examined for their activity for suppression
To a stirred solution of 1 (100 mg, 0.28 mmol) in anhydrous
CH3CN, triethylamine (175 mL, 1.26 mmol) was added and heated to
70 ꢀC. Then the RCOCl/RSO2Cl (1.1e1.2 eq) was added and stirred for
5e6 h. The mixture was cooled to precipitate completely, filtrated
and washed with CH2Cl2 to afford compounds 2aeq and 3a.
Compounds 2aeg, 2i, 2neq and 3a were gained following the same
procedure using purchased acyl chloride or sulfuryl chloride as
material. Compounds 2h, 2jem were gained by the same procedure
using purchased acid which was reflux in SOCl2 to afford acyl
chloride.
of IFN-g-induced IDO1 promoter expression. SAR analysis indicated
that large volume substituent at the 9-position might be beneficial
for enhancing the potency. Among them, compounds 2f, 2i, 2n, 2o
and 8b exhibited increased potency with inhibitory rate of 71e90%
compared with BBR. Their activities were further confirmed by
protein level. Furthermore, compounds 2i and 2n exhibited their
anticancer activity by enhancing the specific lysis of NK cells to
A549 cells, as through targeting IDO1, but not cytotoxicity. Pre-
liminary mechanism revealed that compounds 2i and 2n inhibited
IFN-g-induced IDO1 expression through activating AMPK and
4.2.1.1. 2,3-Methylenedioxy-9-((2,5-difluorobenzoyl)oxy)-10-
subsequent inhibition of STAT1 phosphorylation. Thus, the results
provided powerful information on further strategic optimization,
and compounds 2i and 2n have been selected as promising IDO1
modulators for cancer immunotherapy for next investigation.
methoxyprotoberberine chloride (2a). Compound
0.28 mmol) was treated with 2,5-difluorobenzoyl chloride (39
1
(100 mg,
L,
m
0.31 mmol) according to the general procedure to give the desired
product 2a as a yellow solid, yield: 43%; Mp: 184e186 ꢀC (dec.); 1H
NMR (500 MHz)
d
10.07 (s, 1H), 9.14 (s, 1H), 8.37 (d, J ¼ 9.3 Hz, 1H),
4. Experimental section
8.30 (d, J ¼ 9.3 Hz, 1H), 8.10 (s, 1H), 7.85 (s, 1H), 7.84e7.77 (m, 1H),
7.67e7.61 (m, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 4.92 (t, J ¼ 6.4 Hz, 2H),
4.1. General
4.06 (s, 3H), 3.22 (t, J ¼ 6.4 Hz, 2H); 13C NMR (126 MHz)
d 160.1,
159.5, 157.6, 150.9, 150.7, 148.4, 145.2, 138.9, 133.6, 133.4, 131.5,
127.9, 126.5, 124.3, 121.6, 121.3, 121.0, 120.2, 119.4, 118.2, 109.1, 106.2,
102.8, 58.03, 56.0, 26.8; HRMS: calcd for C26H18F2NO5Cl [M ꢁ Cl]þ
Melting point (mp) was obtained with CXM-300 melting point
apparatus and uncorrected. The 1H NMR spectra was performed on
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6
Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
Fig. 5. BBR analogues induce killing activity of human NK cells toward A549 cells. A549 cells were pre-treated with the indicated compounds with or without IFN-g (10 ng/mL) for
16 h. Cells were washed and then NK cells were co-cultured with A549 cells at 10:1 in RPMI 1640 plus 10% FBS. (A): After 4 h, the specific lysis of NK cells to A549 cells was
determined by LDH releasing assay. All of the experiments were performed in triplicated and the results were calculated as means standard error. #p < 0.05 compared with
untreated control group; *p < 0.05 compared with the IFN-g group. (B): A549 cells were freshly plated in E16 plates and the killing activity of human NK cells toward A549 cells was
measured by a cell impedance assay. Data was analyzed using the RTCA Software 1.2 program (Roche Diagnostics). All data is presented as the mean (n ¼ 2) normalized cellular
index SEM over time.
Fig. 6. BBR analogues suppressed IFN-
g-induced STAT1 phosphorylation. A549 cells were pre-treated with 10 mmol/L of the indicated compounds for 2 h, followed by IFN-g (5 ng/
mL) treatment for 24 h. The expression level of STAT1 and phosphorylation of STAT1 (Tyr701) were measured by western blot using corresponding antibodies. D: DMSO. GAPDH
served as the loading control. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the IFN-
g
group.
462.1148, found 462.1155.
1H), 7.11 (s, 1H), 6.19 (s, 2H), 4.91 (t, J ¼ 6.3 Hz, 2H), 4.06 (s, 3H), 3.22
(t, J ¼ 6.3 Hz, 2H); 13C NMR (126 MHz)
d 160.0, 154.7, 152.1, 150.9,
150.7, 148.4, 145.1, 140.5, 139.0, 133.6, 133.3, 131.5, 128.6, 128.0,
126.5, 121.6, 121.3, 121.0, 115.2, 114.2, 109.1, 106.2, 102.8, 58.0, 56.0,
26.8; HRMS: calcd for C26H17F3NO5Cl [M ꢁ Cl]þ 480.1053, found
480.1058.
4.2.1.2. 2,3-Methylenedioxy-9-((2,3,4-trifluorobenzoyl)oxy)-10-
methoxyprotoberberine chloride (2b). Compound
0.28 mmol) was treated with 2,3,4-trifluorobenzoyl chloride (40
1
(100 mg,
L,
m
0.31 mmol) according to the general procedure to give the desired
product 2b as a yellow solid, yield: 36%; Mp: 186e188 ꢀC (dec.); 1H
NMR (500 MHz)
d
10.06 (s, 1H), 9.13 (s, 1H), 8.37 (d, J ¼ 9.2 Hz, 1H),
4.2.1.3. 2,3-Methylenedioxy-9-((6-fluoronicotinoyl)oxy)-10-
8.30 (d, J ¼ 9.2 Hz,1H), 8.22e8.12 (m,1H), 7.85 (s,1H), 7.75e7.63 (m,
methoxyprotoberberine chloride (2c). Compound
1 (100 mg,
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Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
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Fig. 7. BBR analogues induces AMPK activation. A549 cells were pre-treated with 10
mmol/L of the indicated compounds for 2 h, followed by IFN-
g
(5 ng/mL) treatment for 24 h. The
expression level of phosphor-ACC (Ser79), AMPK and phosphor-AMPK (Thr172) were measured by western blot using corresponding antibodies. D: DMSO. GAPDH served as the
loading control. **p < 0.01, ***p < 0.001 compared with the IFN- group.
g
0.28 mmol) was treated with 6-fluoropyridine-3-carbonyl chloride
(54 mg, 0.34 mmol) according to the general procedure to give the
desired product 2c as a yellow solid, 39%; Mp: 187e189 ꢀC (dec.);
150.8, 150.6, 148.4, 144.4, 138.7, 134.4, 133.6, 131.6, 127.3, 126.5,
121.6, 121.4, 121.0, 109.1, 106.2,102.8, 57.8, 56.4, 43.6, 33.5, 26.8, 24.9
(2), 9.6; HRMS: calcd for C25H26NO5Cl [M ꢁ Cl]þ 420.1806, found
420.1813.
1H NMR (500 MHz)
d
10.07 (s, 1H), 9.17 (d, J ¼ 2.4 Hz, 1H), 9.10 (s,
1H), 8.82e8.75 (m, 1H), 8.37 (d, J ¼ 9.3 Hz, 1H), 8.29 (d, J ¼ 9.3 Hz,
1H), 7.84 (s, 1H), 7.59e7.55 (m, 1H), 7.11 (s, 1H), 6.20 (s, 2H), 4.90 (t,
J ¼ 6.3 Hz, 2H), 4.05 (s, 3H), 3.21 (t, J ¼ 6.4 Hz, 2H);13C NMR
4.2.1.6. 2,3-Methylenedioxy-9-((2-ethylbutanoyl)oxy)-10-
methoxyprotoberberine chloride (2f). Compound
0.28 mmol) was treated with 2-ethylbutanoyl chloride (42
1
(100 mg,
L,
(151 MHz)
d 166.2, 161.7, 151.3, 150.8, 150.5, 148.2, 144.9, 144.8,
m
138.8, 133.4, 133.2, 131.3, 127.7, 126.4, 123.5, 121.4, 121.1, 120.8, 111.0,
108.9, 106.0, 102.6, 57.8, 55.8, 26.6; HRMS: calcd for C25H18FN2O5Cl
[M ꢁ Cl]þ 445.1194, found 445.1201.
0.31 mmol) according to the general procedure to give the desired
product 2f as a yellow solid, yield: 27%; Mp: 200e201 ꢀC (dec.); 1H
NMR (500 MHz)
d
9.72 (s, 1H), 9.09 (s, 1H), 8.30 (d, J ¼ 9.2 Hz, 1H),
8.23 (d, J ¼ 9.2 Hz, 1H), 7.83 (s, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 4.96 (t,
J ¼ 6.3 Hz, 2H), 4.03 (s, 3H), 3.22 (t, J ¼ 6.3 Hz, 2H), 2.85 (p,
J ¼ 6.6 Hz, 1H), 1.93e1.70 (m, 4H), 1.06 (t, J ¼ 7.4 Hz, 6H); 13C NMR
4. 2 .1. 4. 2, 3-Methylen ed ioxy-9-((3 -methylsu lfonyl- 2-
oxoimidazolidine-1-carbonyl)oxy) -10-methoxyprotoberberine chlo-
ride (2d). Compound 1 (100 mg, 0.28 mmol) was treated with 1-
chlorocarbonyl-3-methanesulfonyl-2-imidazolidinone (70 mg,
0.31 mmol) according to the general procedure to give the desired
product 2d as a yellow solid, yield: 34%; Mp: 147e149 ꢀC (dec.); 1H
(126 MHz)
d 173.2, 150.9, 150.6, 148.3, 144.7, 138.7, 134.1, 133.6,
131.5, 127.3, 126.5, 121.7, 121.3, 121.0, 109.1, 106.2, 102.8, 57.7, 56.2,
47.6, 26.8, 24.0 (2), 11.8 (2); HRMS: calcd for C25H26NO5Cl [M ꢁ Cl]þ
420.1806, found 420.1816.
NMR (600 MHz)
d
9.96 (s, 1H), 9.03 (s, 1H), 8.30 (d, J ¼ 9.4 Hz, 1H),
8.23 (d, J ¼ 8.8 Hz, 1H), 7.79 (s, 1H), 7.09 (s, 1H), 6.16 (s, 2H), 4.91 (t,
J ¼ 6.3 Hz, 2H), 4.13 (s, 2H), 3.98e3.94 (m, 2H), 3.41 (s, 3H), 3.22 (t,
4.2.1.7. 2,3-Methylenedioxy-9-((3,5,5-trimethylhexanoyl)oxy)-10-
J ¼ 6.3 Hz, 3H); 13C NMR (151 MHz)
d 151.1, 150.5, 150.1, 148.2, 147.9,
methoxyprotoberbe rine chloride (2g). Compound
0.28 mmol) was treated with 3,5,5-trimethylhexanoyl chloride
(65 L, 0.34 mmol) according to the general procedure to give the
desired product 2g as a yellow solid, 29%; Mp: 206e207 ꢀC (dec.);
1H NMR (500 MHz)
1 (100 mg,
144.7,138.8,133.4, 132.6,131.3,127.8,126.4,121.5,121.1, 120.8,108.9,
106.0, 102.6, 57.8, 56.0, 41.6, 41.0, 29.3, 26.6; HRMS: calcd for
m
C
24H22N3O8SCl [M ꢁ Cl]þ 512.1122, found 512.1126.
d
9.97 (s, 1H), 9.07 (s, 1H), 8.30 (d, J ¼ 9.2 Hz,
4.2.1.5. 2,3-Methylenedioxy-9-((3,3-dimethylbutanoyl)oxy)-10-
methoxyprotoberberine chloride (2e). Compound (100 mg,
0.28 mmol) was treated with 2,2-dimethylbutyryl chloride (43 L,
1H), 8.22 (d, J ¼ 9.2 Hz, 1H), 7.83 (s, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 4.97
(t, J ¼ 6.3 Hz, 2H), 4.03 (s, 3H), 3.23 (t, J ¼ 6.3 Hz, 2H), 2.90 (dd,
J ¼ 15.6, 5.8 Hz, 1H), 2.69 (dd, J ¼ 15.6, 8.1 Hz, 1H), 2.22e2.12 (m,
J ¼ 6.8 Hz, 1H), 1.45 (dd, J ¼ 14.0, 4.1 Hz, 1H), 1.25 (dd, J ¼ 14.0,
6.6 Hz, 1H), 1.14 (d, J ¼ 6.6 Hz, 3H), 0.97 (s, 9H); 13C NMR (126 MHz)
1
m
0.31 mmol) according to the general procedure to give the desired
product 2e as a yellow solid, yield: 37%; Mp: 205e207 ꢀC (dec.); 1H
NMR (500 MHz)
d
9.47 (s, 1H), 9.10 (s, 1H), 8.30 (d, J ¼ 9.2 Hz, 1H),
d 170.5, 151.0, 150.6, 148.3, 145.1, 138.7, 134.2133.6, 131.5, 127.3,
8.23 (d, J ¼ 9.2 Hz, 1H), 7.83 (s, 1H), 7.12 (s, 1H), 6.19 (s, 2H), 4.98 (t,
J ¼ 6.3 Hz, 2H), 4.03 (s, 3H), 3.22 (t, J ¼ 6.3 Hz, 2H),1.83 (q, J ¼ 7.4 Hz,
126.5, 121.8, 121.2, 121.0, 109.1, 106.2, 102.8, 57.8, 55.9, 50.6, 43.5,
31.45, 30.6 (3), 27.3, 26.8, 22.8; HRMS: calcd for C28H32NO5Cl
[M ꢁ Cl]þ 462.2275, found 462.2285.
2H), 1.44 (s, 6H), 1.03 (t, J ¼ 7.4 Hz, 3H); 13C NMR (126 MHz)
d 175.3,
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8
Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
4.2.1.8. 2,3-Methylenedioxy-9-((1-methylcyclopropane-1-carbonyl)
oxy)-10-methoxy protoberberine chloride (2h). Compound
3.54e3.47 (m, 1H), 3.22 (t, J ¼ 6.5 Hz, 2H), 2.83e2.77 (m, 1H),
2.36e2.32 (m, 1H), 1.90e1.81 (m, 1H), 1.73e1.53 (m, 5H), 1.48e1.41
1
(100 mg, 0.28 mmol) was treated with 1-methylcyclopro pan-
ecarboxylic acid (40 mg, 0.34 mmol) according to the general
procedure to give the desired product 2h as a brown solid, yield:
(m, 1H), 1.33e1.19 (m, 1H); 13C NMR (151 MHz)
d 172.3, 151.0, 150.9,
148.5, 143.8, 138.8, 134.4, 133.7, 130.6, 126.4, 125.5, 121.9, 120.6,
120.3, 107.9, 105.2, 102.3, 56.1, 55.9, 45.7, 40.7, 39.8, 37.0, 31.9, 28.8,
26.6, 24.2; HRMS: calcd for C27H26NO5Cl [M ꢁ Cl]þ 444.1806, found
444.1809.
20%; Mp: 184e186 ꢀC (dec.); 1H NMR (500 MHz)
d 9.78 (s, 1H), 9.07
(s, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.20 (d, J ¼ 9.2 Hz, 1H), 7.82 (s, 1H),
7.11 (s, 1H), 6.19 (s, 2H), 4.98 (t, J ¼ 6.2 Hz, 2H), 4.04 (s, 3H), 3.22 (t,
J ¼ 6.2 Hz, 2H), 1.56 (s, 2H), 1.49 (s, 3H), 1.09 (d, J ¼ 3.6 Hz, 2H); 13
C
4.2.1.13. 2,3-Methylenedioxy-9-((noradamantane-1-carbonyl)oxy)-
10-methoxyproto berberine chloride (2m). Compound 1 (100 mg,
0.28 mmol) was treated with 3-noradamantanecarboxylic acid
(56 mg, 0.34 mmol) according to the general procedure to give the
desired product 2m as a yellow solid, yield: 36%; Mp: 201e203 ꢀC
NMR (126 MHz)
d 173.6, 150.9, 150.6, 148.3, 144.9, 138.7, 134.5,
133.5, 131.5, 127.2, 126.5, 121.8, 121.3, 121.0, 109.1, 106.2, 102.8, 58.0,
56.0, 26.8, 19.6, 19.3, 18.1 (2); HRMS: calcd for C24H22NO5Cl
[M ꢁ Cl]þ 404.1493, found 404.1500.
(dec.); 1H NMR (500 MHz)
d 9.62 (s, 1H), 9.08 (s, 1H), 8.29 (d,
4.2.1.9. 2,3-Methylenedioxy-9-((2,2,3,3-tetramethylcyclopropane-1-
J ¼ 9.2 Hz, 1H), 8.22 (d, J ¼ 9.2 Hz, 1H), 7.84 (s, 1H), 7.12 (s, 1H), 6.19
(s, 2H), 4.98 (t, J ¼ 6.3 Hz, 2H), 4.03 (s, 3H), 3.22 (t, J ¼ 6.3 Hz, 2H),
3.08 (t, J ¼ 6.9 Hz, 1H), 2.42e2.26 (m, 4H), 2.10e2.02 (m, 2H),
carbonyl)oxy)-10-
Compound 1 (100 mg, 0.28 mmol) was treated with 2,2,3,3-
tetramethylcyclo propane-1-carbonyl chloride (48 L, 0.31 mmol)
methoxyprotoberberine
chloride
(2i).
m
1.95e1.88 (m, 2H), 1.80e1.63 (m, 4H); 13C NMR (126 MHz)
d 174.8,
according to the general procedure to give the desired product 2i as
150.8, 150.6, 148.4, 144.8, 138.6, 134.8, 133.6, 131.6, 127.2, 126.5,
121.7, 121.3, 121.0, 109.1, 106.2, 102.8, 58.0, 56.2, 54.4, 47.2 (2), 44.4,
43.8 (2), 37.6 (2), 34.7, 26.8; HRMS: calcd for C29H28NO5Cl [M ꢁ Cl]þ
470.1962, found 470.1966.
a yellow solid, yield: 43%; Mp: 198e200 ꢀC (dec.); 1H NMR
(500 MHz)
d
9.89 (s, 1H), 9.06 (s, 1H), 8.28 (d, J ¼ 9.2 Hz, 1H), 8.19 (d,
J ¼ 9.2 Hz, 1H), 7.83 (s, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 4.98 (t,
J ¼ 6.3 Hz, 2H), 4.02 (s, 3H), 3.23 (t, J ¼ 6.3 Hz, 2H), 1.81 (s, 1H), 1.34
(s, 6H), 1.28 (s, 6H); 13C NMR (126 MHz)
d
169.1, 150.9, 150.6, 148.3,
4.2.1.14. 2,3-Methylenedioxy-9-(2-(adamantan-1-yl)acetoxy)-10-
methoxyberberine chloride (2n). Compound 1 (100 mg, 0.28 mmol)
was treated with 2-(1-adamantyl)acetyl chloride (66 mg,
0.31 mmol) according to the general procedure to give the desired
product 2n as a yellow solid, yield: 43%; Mp: 188e190 ꢀC (dec.); 1H
145.0, 138.6, 134.8, 133.5, 131.5, 126.9, 126.5, 121.9, 121.2, 121.1, 109.1,
106.2, 102.8, 57.7, 56.0, 35.2, 31.8 (2), 26.8, 23.8 (2), 17.2 (2); HRMS:
calcd for C27H28NO5Cl [M ꢁ Cl]þ 446.1962, found 446.1973.
4.2.1.10. 2,3-Methylenedioxy-9-((cyclobutanecarbonyl)oxy)-10-
NMR (500 MHz)
d
9.96 (s, 1H), 9.07 (s, 1H), 8.30 (d, J ¼ 9.2 Hz, 1H),
methoxyprotoberbe rine chloride (2j). Compound
0.28 mmol) was treated with cyclobutanecarboxylic acid chloride
(35 L, 0.31 mmol) according to the general procedure to give the
desired product 2j as a brown solid, yield: 32%; Mp: 197e199 ꢀC
(dec.); 1H NMR (500 MHz)
9.92 (s, 1H), 9.08 (s, 1H), 8.29 (d,
1
(100 mg,
8.21 (d, J ¼ 9.2 Hz, 1H), 7.82 (s, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 4.96 (t,
J ¼ 6.3 Hz, 2H), 4.05 (s, 3H), 3.23 (t, J ¼ 6.3 Hz, 2H), 2.59 (s, 2H),
2.07e1.98 (m, 3H), 1.84e1.77 (m, 6H), 1.78e1.63 (m, 6H); 13C NMR
m
(126 MHz)
d 168.8, 150.9, 150.6, 148.3, 145.1, 138.7, 134.2, 133.6,
d
131.5, 127.3, 126.5, 121.8, 121.2, 121.0, 109.1, 106.2, 102.8, 57.7, 55.9,
48.7, 42.2 (3), 37.0 (3), 33.3, 28.7 (3), 26.8; HRMS: calcd for
J ¼ 9.2 Hz, 1H), 8.21 (d, J ¼ 9.2 Hz, 1H), 7.82 (s, 1H), 7.11 (s, 1H), 6.19
(s, 2H), 4.96 (t, J ¼ 6.3 Hz, 2H), 4.05 (s, 3H), 3.82e3.73 (m, 1H), 3.22
(t, J ¼ 6.3 Hz, 2H), 2.50e2.35 (m, 4H), 2.16e2.04 (m, 1H), 2.02e1.90
C
31H32NO5Cl [M ꢁ Cl]þ 498.2275, found 498.2278.
(m, 1H); 13C NMR (126 MHz)
d
172.9, 151.0, 150.6, 148.3, 145.1, 138.7,
4.2.1.15. 2,3-Methylenedioxy-9-((3-bromoadamantane-1-carbonyl)
oxy)-10-methoxy protoberberine chloride (2o). Compound
134.3, 133.5, 131.5, 127.2, 126.4, 121.8, 121.2, 121.0, 109.1, 106.2, 102.8,
57.9, 56.0, 37.7, 26.8, 25.4 (2), 18.6; HRMS: calcd for C24H22NO5Cl
[M ꢁ Cl]þ 404.1492, found 404.1496.
1
(100 mg, 0.28 mmol) was treated with 3-bromoadaman tane-1-
carbonyl chloride (94 mg, 0.34 mmol) according to the general
procedure to give the desired product 2o as a yellow solid, yield:
4.2.1.11. 2,3-Methylenedioxy-9-((cyclohexanecarbonyl)oxy)-10-
39%; Mp: 191e193 ꢀC (dec.); 1H NMR (500 MHz)
d 9.63 (s, 1H), 9.08
methoxyprotoberbe rine chloride (2k). Compound
0.28 mmol) was treated with cyclohexanecarboxylic acid chloride
(41 L, 0.31 mmol) according to the general procedure to give the
desired product 2k as a yellow solid, yield: 32%; Mp: 178e180 ꢀC
(dec.); 1H NMR (500 MHz)
9.90 (s, 1H), 9.08 (s, 1H), 8.28 (d,
1
(100 mg,
(s, 1H), 8.29 (d, J ¼ 9.2 Hz, 1H), 8.22 (d, J ¼ 9.2 Hz, 1H), 7.83 (s, 1H),
7.12 (s, 1H), 6.19 (s, 2H), 4.99 (t, J ¼ 6.3 Hz, 2H), 4.03 (s, 3H), 3.23 (t,
J ¼ 6.3 Hz, 2H), 2.78 (s, 2H), 2.45e2.35 (m, 4H), 2.34e2.26 (m, 2H),
m
2.26e2.13 (m, 4H), 1.84e1.74 (m, 2H); 13C NMR (126 MHz)
d 173.0,
d
150.6 (2), 148.4, 144.6, 138.7, 134.3, 133.6, 131.6, 127.4, 126.5, 121.5,
121.4, 121.0, 109.1, 106.2, 102.8, 66.2, 58.0, 56.2, 49.4, 48.2 (2), 45.8,
37.1 (2), 34.3, 32.0 (2), 26.8; HRMS: calcd for C30H29BrNO5Cl
[M ꢁ Cl]þ 562.1224, found 562.1240.
J ¼ 9.2 Hz, 1H), 8.21 (d, J ¼ 9.2 Hz, 1H), 7.82 (s, 1H), 7.11 (s, 1H), 6.19
(s, 2H), 4.97 (t, J ¼ 6.3 Hz, 2H), 4.03 (s, 3H), 3.23 (t, J ¼ 6.3 Hz, 2H),
3.02e2.92 (m, 1H), 2.18e2.10 (m, 2H), 1.85e1.77 (m, 2H), 1.71e1.59
(m, 3H), 1.48e1.38 (m, 2H), 1.34 (td, J ¼ 11.8, 3.8 Hz, 1H); 13C NMR
(126 MHz)
d
173.2, 150.9, 150.6, 148.3, 145.0, 138.7, 134.4, 133.5,
4.2.1.16. 2,3-Methylenedioxy-9-((1R,2R)-2-phenylcyclopropane-1-
131.5, 127.2, 126.5, 121.8, 121.2, 121.0, 109.1, 106.2, 102.8, 57.9, 56.0,
42.5, 29.1 (2), 26.8, 26.0, 25.4 (2); HRMS: calcd for C26H26NO5Cl
[M ꢁ Cl]þ 432.1806, found 432.1809.
carbonyloxy)-10-
Compound 1 (100 mg, 0.28 mmol) was treated with (1R,2R)-2-
phenylcyclo propane-1-carbonyl chloride (45 L, 0.31 mmol) ac-
methoxyprotoberberine
chloride
(2p).
m
cording to the general procedure to give the desired product 2p as a
4.2.1.12. 2,3-Methylenedioxy-9-((bicyclo[2.2.1]heptane-2-carbonyl)
brown solid, yield: 34%; Mp: 172e174 ꢀC (dec.); 1H NMR (500 MHz)
oxy)-10-methoxy protoberberine chloride (2l). Compound
1
d
9.95 (s, 1H), 9.09 (s, 1H), 8.30 (d, J ¼ 9.2 Hz, 1H), 8.23 (d, J ¼ 9.2 Hz,
(100 mg, 0.28 mmol) was treated with bicyclo[2.2.1]heptane-2-
carboxylic acid (48 mg, 0.34 mmol) according to the general pro-
cedure to give the desired product 2l as a brown solid, yield: 31%;
1H), 7.83 (s, 1H), 7.41e7.32 (m, 4H), 7.32e7.25 (m, 1H), 7.11 (s, 1H),
6.19 (s, 2H), 4.96 (t, J ¼ 6.3 Hz, 2H), 4.06 (s, 3H), 3.22 (t, J ¼ 6.3 Hz,
2H), 2.85e2.79 (m, 1H), 2.45e2.39 (m, 1H), 1.84e1.77 (m, 1H),
Mp: 202e204 ꢀC (dec.); 1H NMR (500 MHz)
d
9.90 (d, J ¼ 5.3 Hz,
1.77e1.71 (m, 1H); 13C NMR (126 MHz)
d 170.8, 150.9, 150.6, 148.3,
1H), 9.06 (s, 1H), 8.29 (d, J ¼ 9.2 Hz, 1H), 8.21 (d, J ¼ 9.2 Hz, 1H), 7.82
145.1, 140.1, 138.7, 134.2, 133.5, 131.5, 129.2 (2), 127.4, 127.4, 126.9
(2), 126.5, 121.7, 121.3, 121.0, 109.1, 106.2, 102.8, 58.0, 56.0, 27.6, 26.8,
(s, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 5.03e4.86 (m, 2H), 4.03 (s, 3H),
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Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
9
24.6, 18.8; HRMS: calcd for C29H24NO5Cl [M ꢁ Cl]þ 466.1649, found
4.2.2.1. 2,3-Methylenedioxy-9-(adamantane-1-carboxamido)-10-
methoxyprotoberbe rine chloride (6a). Compound (100 mg,
466.1651.
5
0.28 mmol) was treated with adamantane-1-carbonyl chloride
(167 mg, 0.84 mmol) according to the general procedure to give the
desired product 6a as a yellow solid, yield: 35%; Mp: 242e243 ꢀC
4.2.1.17. 2,3-Methylenedioxy-9-((2-methyl-2-phenylpropanoyl)oxy)-
10-methoxyproto berberine chloride (2q). Compound 1 (100 mg,
0.28 mmol) was treated with 2-methyl-2-phenyl propanoyl chlo-
(dec.); 1H NMR (500 MHz)
d
9.40 (s,1H), 9.29 (d, J ¼ 4.6 Hz,1H), 8.21
ride (57
the desired product 2q as a yellow solid, yield: 35%; Mp:
198e200 ꢀC (dec.); 1H NMR (500 MHz)
9.23 (s, 1H), 9.08 (s, 1H),
mL, 0.31 mmol) according to the general procedure to give
(s, 2H), 7.82 (s, 1H), 7.11 (s, 1H), 6.18 (s, 2H), 5.00 (t, J ¼ 6.3 Hz, 2H),
4.07e3.96 (m, 3H), 3.21 (t, J ¼ 6.3 Hz, 2H), 2.07 (s, 9H), 1.76 (s, 6H);
d
13C NMR (126 MHz)
d 177.9, 155.2, 150.3, 148.1, 146.0, 137.8, 133.6,
8.25 (d, J ¼ 9.2 Hz,1H), 8.20 (d, J ¼ 9.2 Hz,1H), 7.82 (s,1H), 7.66e7.61
(m, 2H), 7.52e7.47 (m, 2H), 7.41e7.35 (m, 1H), 7.12 (s, 1H), 6.19 (s,
2H), 4.90 (t, J ¼ 6.3 Hz, 2H), 3.95 (s, 3H), 3.22 (t, J ¼ 6.3 Hz, 2H), 1.84
131.2, 127.9, 125.7, 124.9, 122.9, 121.3, 120.9, 108.9, 106.0, 102.5, 57.5,
56.0, 41.3, 38.9, 36.6 (3), 28.2 (3), 26.8 (3); HRMS: calcd for
C
30H31N2Oþ4 Cl [M ꢁ Cl]þ 483.2278, found 483.2280.
(s, 6H); 13C NMR (126 MHz)
d 174.1, 151.0, 150.7, 148.4, 144.2, 144.2,
138.7, 134.0, 133.6, 131.5, 129.2 (2), 127.8, 127.5, 126.8 (2), 126.6,
121.4, 121.4, 120.9, 109.09, 106.2, 102.8, 57.6, 56.5, 47.6 (2), 26.9,
26.8; HRMS: calcd for C29H26NO5Cl [M ꢁ Cl]þ 468.1806, found
468.1809.
4.2.2.2. 2,3-Methylenedioxy-9-(3,3-dimethylbutanamido)-10-
methoxyprotoberberine chloride (6b). Compound (100 mg,
0.28 mmol) was treated with 3,3-dimethylbutyryl chloride (116 L,
5
m
0.84 mmol) according to the general procedure to give the desired
product 6b as a yellow solid, yield: 37%; Mp: 222e224 ꢀC (dec.); 1H
4.2.1.18. 2,3-Methylenedioxy-9-((1-methyl-1H-imidazole-4-yl)sulfo-
NMR (500 MHz)
d
10.00 (s, 1H), 9.62 (s, 1H), 8.23 (d, J ¼ 9.2 Hz, 1H),
nyloxy)-10-methoxy protoberberine chloride (3a). Compound
1
8.20 (d, J ¼ 9.2 Hz, 1H), 7.82 (s, 1H), 7.11 (s, 1H), 6.19 (s, 2H), 4.94 (t,
(100 mg, 0.28 mmol) was treated with 1-methyl-1H-imidazole-4-
sulfonyl chloride (45 mg, 0.31 mmol) according to the general
procedure to give the desired product 3a as a yellow solid, yield:
J ¼ 6.4 Hz, 2H), 4.03 (s, 3H), 3.22 (t, J ¼ 6.4 Hz, 2H), 2.42 (s, 2H), 1.11
(s, 9H); 13C NMR (126 MHz)
d 171.6, 154.9, 150.5, 148.3, 146.9, 138.0,
133.8, 131.3, 127.8, 125.9, 124.4, 122.7, 121.3, 121.1, 109.1, 106.1, 102.8,
57.5, 56.0, 49.6, 31.4 (3), 30.4, 26.97.; HRMS: calcd for C25H27N2O4Cl
[M ꢁ Cl]þ 419.1965, found 419.1966.
28%; Mp: 181e183 ꢀC (dec.); 1H NMR (500 MHz)
d 9.54 (s, 1H), 9.07
(s, 1H), 8.30 (d, J ¼ 9.2 Hz, 1H), 8.25 (d, J ¼ 9.2 Hz, 1H), 8.22 (s, 1H),
8.05 (s, 1H), 7.82 (s, 1H), 7.13 (s, 1H), 6.19 (s, 2H), 4.88 (t, J ¼ 6.3 Hz,
2H), 3.95 (s, 3H), 3.77 (s, 3H), 3.21 (t, J ¼ 6.3 Hz, 2H); 13C NMR
4.2.2.3. 2,3-Methylenedioxy-9-(2,2-dimethylbutanamido)-10-
methoxyprotoberberine chloride (6c). Compound
0.28 mmol) was treated with 2,2-dimethylbutyryl chloride (77
(126 MHz)
d
152.7, 150.6, 148.2, 144.7, 141.6, 138.8, 133.9, 133.7,
5
(100 mg,
L,
132.1, 131. 3, 129.6, 128.6, 126.8, 122.0, 121.2, 120.7, 108.9, 106.0,
102.7, 57.7, 56.1, 34.5, 26.7; HRMS: calcd for C23H20N3O6SCl
[M ꢁ Cl]þ 466.1067, found 466.1073.
m
0.56 mmol) according to the general procedure to give the desired
product 6c as a yellow solid, yield: 33%; Mp: 247e249 ꢀC (dec.); 1H
NMR (600 MHz) d 9.43 (s, 1H), 9.25 (s, 1H), 8.99 (s, 1H), 8.20 (s, 2H),
4.2.2. General synthesis procedure for synthesis of compounds
6aec
7.79 (s, 1H), 7.08 (s, 1H), 6.15 (s, 2H), 4.95 (s, 2H), 3.98 (s, 3H), 1.71 (d,
J ¼ 9.0 Hz, 2H), 1.30 (s, 6H), 0.93 (t, J ¼ 7.6 Hz, 3H); 13C NMR
The solution of BBR (7.4 g, 20 mmol) in 2,4-
dimethoxybenzylamine (15 mL, 78 mmol) was stirred at 120 ꢀC
for 6e8 h. The mixture was cooled to room temperature and
washed with acetone (3 ꢂ 50 mL) to remove the remaining amine.
The reside was purified by flash chromatography over silica gel
using CH2Cl2/CH3OH (96.5:3.5) as the gradient eluent to afford
compound 4 (3.5 g, 37%). Mp: 239e240 ꢀC (Dec.); 1H NMR
(151 MHz) d 177.8, 155.2, 150.3, 148.1, 145.9, 137.8, 133.7, 131.1, 127.9,
125.7, 124.9, 122.9, 121.3, 120.9, 108.9, 106.0, 102.5, 57.3, 56.1, 43.2,
33.6, 26.8, 25.2 (2), 9.5; HRMS: calcd for C25H27N2O4Cl [M ꢁ Cl]þ
419.1965, found 419.1966.
4.2.3. General synthesis procedure for synthesis of compounds 8a
and 8b
(500 MHz)
d
9.98 (s, 1H), 8.73 (s, 1H), 7.88 (d, J ¼ 8.8, 1H), 7.77 (s,
PMT (3.87 g, 10 mmol) was heated at 195e210 ꢀC for 10e15 min
under vacuum (30e40 mmHg) to afford the black oil, which was
acidified with ethanol/concentrated HCl (95:5). The solvent was
removed by evaporation, the residue was collected and then puri-
fied by flash chromatography over silica gel using CH2Cl2/CH3OH as
the gradient eluent, affording the title compound 7 (3.2 g, 86%) as a
yellow solid.
1H), 7.52 (d, J ¼ 8.8 Hz, 1H), 7.14 (d, J ¼ 8.3 Hz, 1H), 7.09 (s, 1H), 6.52
(d, J ¼ 2.2 Hz, 1H), 6.44 (t, J ¼ 6.5 Hz, 1H), 6.42e6.39 (m, 1H), 6.17 (s,
2H), 4.80 (t, J ¼ 6.2 Hz, 2H), 4.66 (d, J ¼ 6.3 Hz, 2H), 3.87 (s, 3H), 3.77
(s, 3H), 3.71 (s, 3H), 3.21 (t, J ¼ 6.3 Hz, 2H); 13C NMR (126 MHz)
d
160.5, 158.5, 150.1, 148.2, 148.1, 147.1, 137.5, 136.3, 133.5, 130.9,
130.3,124.8, 121.2, 120.5,120.3,118.0,117.7, 109.1,105.9,104.8,102.6,
98.9, 57.5, 56.0, 55.8, 55.6, 47.0, 27.3; HRMS: calcd for C28H27N2O5Cl
[M ꢁ Cl]þ 471.1914, found 471.1919.
To a stirred solution of compound 7 (100 mg, 0.29 mmol) in
anhydrous CH3CN, triethylamine (175 mL, 1.26 mmol) was added
Compound 4 (3 g, 6.4 mmol) was dissolved in CH3OH, and hy-
drochloric acid 3 mL was added. The mixture was stirred for 5e6 h,
filtered and washed with 80% ethanol to afford compound 5 (1.8 g,
and heated to 70 ꢀC. Then the 2-(1-adamantyl)acetyl chloride
(66 mg, 0.31 mmol) was added and stirred for 5e6 h. The mixture
was cooled to precipitate completely, filtrated and washed by
CH2Cl2 to afford compounds 8aeb.
80%). Mp: 212e214 ꢀC (Dec.); 1H NMR (500 MHz)
d 10.19 (s, 1H),
8.64 (s, 1H), 7.84 (d, J ¼ 8.6 Hz, 1H), 7.76 (s, 1H), 7.32 (d, J ¼ 8.6 Hz,
1H), 7.08 (s,1H), 6.89 (s, 2H), 6.16 (s, 2H), 4.70 (t, J ¼ 6.3 Hz, 2H), 3.98
4.2.3.1. 2,3,10-trimethoxy-9-((adamantane-1-carbonyl)oxy)proto-
berberine chloride (8a). Compound 7 (100 mg, 0.29 mmol) was
treated with adamantane-1-carbonyl chloride (61 mg, 0.31 mmol)
according to the general procedure to give the desired product 8a as
a brown solid, yield: 25%; Mp: 158e160 ꢀC (dec.); 1H NMR
(s, 3H), 3.20 (t, J ¼ 6.3 Hz, 2H); 13C NMR (126 MHz)
d 149.8, 148.0,
147.0, 143.9, 138.1, 135.4, 132.1, 130.4, 123.0, 121.2, 119.8, 113.7, 113.3,
108.9, 105.6, 102.4, 56.9, 55.1, 27.2; HRMS: calcd for C19H17N2O3Cl
[M ꢁ Cl]þ 321.12337, found 321.12352.
To a stirred solution of 5 (100 mg, 0.28 mmol) and pyridine
(500 MHz)
d
9.51 (s, 1H), 9.20 (s, 1H), 8.29 (d, J ¼ 9.2 Hz, 1H), 8.25 (d,
(100
m
L,1.24 mmol) in anhydrous CH2Cl2 (5 mL), the RCOCl (2e3 eq)
J ¼ 9.2 Hz, 1H), 7.76 (s, 1H), 7.12 (s, 1H), 5.00 (t, J ¼ 6.3 Hz, 2H), 4.03
(s, 3H), 3.96 (s, 3H), 3.88 (s, 3H), 3.24 (t, J ¼ 6.3 Hz, 2H), 2.18 (d,
J ¼ 3.0 Hz, 6H), 2.15e2.10 (m, 3H), 1.83e1.74 (m, 6H); 13C NMR
was added, and refluxed for 10e12 h. The solvent was removed by
evaporation and purified by flash chromatography over silica gel
using CH2Cl2/CH3OH (95:5) as the gradient eluent to give 6aec.
(126 MHz)
d 174.8, 152.2, 150.6, 149.4, 144.5, 138.9, 134.5, 133.7,
Please cite this article in press as: YaneXiang-g. Wang, et al., Synthesis and biological evaluation of new berberine derivatives as cancer
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10
Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
129.4, 127.1, 126.5, 121.5, 121.0, 119.5, 111.9, 109.4, 57.9, 56.8, 56.5,
56.5, 41.6, 38.9 (2), 36.5 (2), 27.9 (2), 26.5; HRMS: calcd for
C
1 mM Na3VO4, 0.5% Triton X-100, and 1 mM DTT) supplemented
with protease inhibitor cocktail (Sigma P8340). Proteins were
separated by SDS-PAGE and were electrically transferred to a PVDF
membrane. The membrane was probed with the appropriate pri-
mary antibody and with a HRP-conjugated secondary antibody.
Blots were visualized by Tanon 5200 system (Tanon, Shanghai,
China).
31H34NO5Cl [M ꢁ Cl]þ 500.2432, found 500.2432.
4.2.3.2. 2,3,10-trimethoxy-9-(2-(adamantan-1-yl)acetoxy)proto-
berberine chloride (8b). Compound 7 (100 mg, 0.29 mmol) was
treated with 2-(1-adamantyl)acetyl chloride (66 mg, 0.31 mmol)
according to the general procedure to give the desired product 8b
as a brown solid, yield: 33%; Mp: 142e144 ꢀC (dec.); 1H NMR
4.4. MTT assay
(600 MHz)
d
9.87 (s, 1H), 9.12 (s, 1H), 8.27 (d, J ¼ 9.2 Hz, 1H), 8.21 (d,
J ¼ 9.2 Hz, 1H), 7.71 (s, 1H), 7.07 (s, 1H), 4.92 (t, J ¼ 6.4 Hz, 2H), 4.02
(s, 3H), 3.92 (s, 3H), 3.85 (s, 3H), 3.21 (t, J ¼ 6.4 Hz, 2H), 2.55 (s, 2H),
2.03e1.97 (m, 3H), 1.79e1.75 (m, 6H), 1.72e1.62 (m, 6H); 13C NMR
The effect of the indicated compounds on the cell viability of
A549 cells was evaluated using the MTT assay [24]. Briefly, Cells
were seeded (5 ꢂ 103/well) into 96-well plate and the investigated
compounds were added at indicated concentrations for 48 h. Next,
MTT solution at a concentration of 5 mg/mL was added to each well.
After subsequent 4 h, the culture medium was removed and for-
(151 MHz)
d 168.5, 152.1, 150.6, 149.2, 144.8, 138.8, 133.9, 133.5,
129.2, 127.0, 126.4, 121.5, 120.7, 119.2, 111.7, 109.3, 57.4, 56.6, 56.3,
56.0, 48.5, 42.0 (3), 36.7 (3), 33.0, 28.5 (3), 26.2; HRMS: calcd for
C
32H36NO5Cl [M ꢁ Cl]þ 514.2588, found 514.2590.
mazan crystals were dissolved with 150
mL DMSO. Finally, the
absorbance was measured at 570 nm using a microplate reader
(Multiskan FC, Thermo, USA).
4.2.4. Synthesis of 3-((adamantane-1-carbonyl)oxy)-2,9,10-
trimethoxyprotoberberine chloride (9a)
To a stirred solution of JTH (100 mg, 0.29 mmol) in anhydrous
4.5. IDO1 promoter activity assay
CH3CN, triethylamine (175 mL, 1.26 mmol) was added and heated to
70 ꢀC. Then the adamantane-1-carbonyl chloride (61 mg,
0.31 mmol) was added and stirred for 5e6 h. The mixture was
cooled to precipitate completely, filtrated and washed by CH2Cl2 to
afford compound 9a as a yellow solid, yield: 34%; Mp: 186e188 ꢀC
A549 cells were co-transfected with pGL4-IDO1-luc with the
pRL-CMV plasmid using the Vigofect transfection reagent (Beijing,
China) as instructed by the manufacturers. After 24 h of trans-
fection, cells were pretreated with the indicated compounds
(dec.); 1H NMR (500 MHz)
d 9.98 (s, 1H), 9.21 (s, 1H), 8.27 (d,
(10
mmol/L) for 2 h and then stimulated with or without IFN-
g
J ¼ 9.1 Hz, 1H), 8.09 (d, J ¼ 9.1 Hz, 1H), 7.91 (s, 1H), 7.24 (s, 1H), 4.99
(t, J ¼ 6.3 Hz, 2H), 4.12 (s, 3H), 4.10 (s, 3H), 3.95 (s, 3H), 3.24 (t,
J ¼ 6.3 Hz, 2H), 2.08e2.04 (m, 3H), 2.04e2.00 (m, 6H),1.78e1.71 (m,
(10 ng/mL) for 24 h. Following IFN-
g
treatment, the cells were
lysed, and the luciferase activity was determined using the lucif-
erase reporter assay system (Promega, Madison, CA, USA) according
to manufacturer's protocols. The luciferase activity values were
normalized to the expression of the Renilla luciferase, and pre-
sented as the percentages of luciferase activity.
6H); 13C NMR (126 MHz)
d 175.2, 151.5, 151.4, 146.6, 144.4, 142.5,
137.5, 133.3, 128.6, 127.4, 125.9, 124.4, 123.5, 122.4, 121.9, 110.7, 62.6,
57.7, 57.3, 56.0, 41.2, 39.0 (3), 36.5 (3), 27.9 (3), 26.06; HRMS: calcd
for C31H34NO5Cl [M ꢁ Cl]þ 500.2432, found 500.2434.
4.6. LDH release assay
4.2.5. Synthesis of 3-(2-(adamantan-1-yl)acetoxy)-2,9,10-
trimethoxyprotoberberine chloride (9b)
Cytotoxicity of human NK cells against A549 cells was assessed
with LDH release assay, as previously described [25]. Briefly,
A549 cells were freshly plated at 5 ꢂ 103/well in 96-well plates and
To a stirred solution of JTH (100 mg, 0.29 mmol) in anhydrous
CH3CN, triethylamine (175 mL, 1.26 mmol) was added and heated to
70 ꢀC. Then the 2-(1-adamantyl)acetyl chloride (66 mg, 0.31 mmol)
was added and stirred for 5e6 h. The mixture was cooled to pre-
cipitate completely, filtrated and washed by CH2Cl2 to afford
compound 9b as a yellow solid, yield: 33%; Mp: 149e151 ꢀC (dec.);
pretreated with the indicated compounds (10 mmol/L) for 2 h and
then stimulated with IFN-g (10 ng/mL) for 16 h. A549 cells were
washed and then were cocultured with NK cells at 1:10 in triplicate
in RPMI 1640 plus 10% FBS. Four hours later, cytotoxicity assay was
conducted using non-radioactive lactate dehydrogenase (LDH)
release using a cytotoxicity detection kit (CytoTox 96, Promega,
Madison, WI, USA) as the manufacturer's instructions. Spontaneous
release and maximum release were determined by incubating
target cells without effector cells in medium alone or in 0.5% NP40,
respectively. The percent cytotoxicity was calculated as follows:
(experimental release-spontaneous release)/(maximum release-
spontaneous release) ꢂ 100%.
1H NMR (500 MHz)
d
9.99 (s, 1H), 9.26 (s, 1H), 8.26 (d, J ¼ 9.1 Hz,
1H), 8.11 (d, J ¼ 9.1 Hz, 1H), 7.94 (s, 1H), 7.25 (s, 1H), 5.00 (t,
J ¼ 6.3 Hz, 2H), 4.12 (s, 3H), 4.09 (s, 3H), 3.97 (s, 3H), 3.25 (t,
J ¼ 6.3 Hz, 2H), 2.34 (s, 2H), 2.04e1.96 (m, 3H), 1.77e1.64 (m, 12H);
13C NMR (126 MHz)
d 169.3, 151.5, 151.4, 146.6, 144.4, 142.1, 137.4,
133.3, 128.6, 127.3, 126.1, 124.4, 123.6, 122.4, 122.0, 110.7, 62.6, 57.7,
57.2, 56.6, 48.4, 45.9, 42.2 (3), 36.9 (3), 33.3, 28.7 (3), 26.1; HRMS:
calcd for C32H36NO5Cl [M ꢁ Cl]þ 514.2588, found 514.2587.
4.3. Cell culture and western blot analysis
4.7. Cell impedance assay
A549 human lung cancer cell lines were procured from ATCC.
The cells were cultured in DME/F-12 (Hyclone, UT, USA) supple-
mented with 10% fetal bovine serum (Hyclone, UT, USA), 100 U/ml
A549 cells were freshly plated at 5 ꢂ 103/well in E16 plates
(Roche Diagnostics, Basel, Switzerland). The plate was connected to
an xCELLigence RTCA SP instrument (Roche Diagnostics) within a
penicillin, and 100 mg/mL streptomycin sulfate, and incubated at
37 ꢀC in a humidified atmosphere with 5% CO2. Actived NK cells
were purchased from Stemcell Technologies (Vancouver, BC,
Canada).
humidified cell culture incubator. After
A549 cells were treated with the indicated compounds (10
with IFN- (10 ng/mL). After 16 h, A549 cells were washed and co-
4
h
incubation and
m
mol/L)
g
Western blot was performed as described previously [23].
Briefly, A549 cells were washed with PBS and lysed in M2 lysis
cultured with NK cells at 1:10 in triplicate in RPMI 1640 plus 10%
FBS. Data was analyzed using the RTCA Software 1.2 program
(Roche Diagnostics). All data is presented as the mean normalized
cellular index SEM over time.
buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM
b-glycer-
ophosphate, 5 mM EGTA, 1 mM sodium pyrophoshate, 5 mM NaF,
Please cite this article in press as: YaneXiang-g. Wang, et al., Synthesis and biological evaluation of new berberine derivatives as cancer
immunotherapy agents through targeting IDO1, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.078

Y. Wang et al. / European Journal of Medicinal Chemistry xxx (2017) 1e11
agents, Acta Pharm. Sin. B 2 (2012) 581e587.
11
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immunotherapy agents through targeting IDO1, European Journal of Medicinal Chemistry (2017), https://doi.org/10.1016/j.ejmech.2017.10.078