The synthesis and antistaphylococcal activity of 9, 13-disubstituted berberine derivatives

Article abstract of DOI:10.1016/j.ejmech.2017.01.012

A series of novel 9, 13-disubstituted berberine derivatives have been synthesized and evaluated for the antibacterial activities against Staphylococcus aureus, including Newman strain and multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108, and NRS-271). Compound 20 shows the most potent activity against the growth of Newman strain, with a MIC value of 0.78?μg/mL, which is comparable with the positive control vancomycin. In addition, compound 20, 21, and 33 are highly antistaphylococcal active against five strains of multidrug-resistant S.?aureus, with MIC values of 0.78–1.56?μg/mL. Of note, theses antibacterial active compounds have no obvious toxicity to the viability of human fibroblast (HAF) cells at the MIC concentration.

Full text of DOI:10.1016/j.ejmech.2017.01.012

European Journal of Medicinal Chemistry 127 (2017) 424e433
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
The synthesis and antistaphylococcal activity of 9, 13-disubstituted
berberine derivatives
Jing Wang 1, Teng Yang b , Huang Chen , Yun-Nan Xu , Li-Fang Yu , Ting Liu ,
a
,
,
c
,
1
d
a
a
a
a,
e
d
c
b,
**
, Fan Yang a
, *
Jie Tang , Zhengfang Yi , Cai-Guang Yang , Wei Xue
a
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, SCME, East China Normal University, Shanghai 200062,
China
b
Center for Research and Development of Fine Chemicals of Guizhou University, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry
of Education, Guiyang 550225, China
c
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai,
d
2
00241, China
e
Shanghai Key Laboratory of Green Chemistry and Chemical Processes, SCME, East China Normal University, Shanghai 200062, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 December 2016
Received in revised form
A series of novel 9, 13-disubstituted berberine derivatives have been synthesized and evaluated for the
antibacterial activities against Staphylococcus aureus, including Newman strain and multidrug-resistant
strains (NRS-1, NRS-70, NRS-100, NRS-108, and NRS-271). Compound 20 shows the most potent activ-
6
January 2017
ity against the growth of Newman strain, with a MIC value of 0.78
positive control vancomycin. In addition, compound 20, 21, and 33 are highly antistaphylococcal active
against five strains of multidrug-resistant S. aureus, with MIC values of 0.78e1.56 g/mL. Of note, theses
mg/mL, which is comparable with the
Accepted 8 January 2017
Available online 9 January 2017
m
antibacterial active compounds have no obvious toxicity to the viability of human fibroblast (HAF) cells at
the MIC concentration.
Keywords:
Berberine derivatives
Antistaphylococcal activity
Multidrug-resistant
Cytotoxicity
©
2017 Elsevier Masson SAS. All rights reserved.
1
. Introduction
[9]. Berberine (Scheme 1) is an isoquinoline alkaloid extracted from
Coptis chinensis (Huang-Lian, a common herb in traditional Chinese
medicine), and has been traditionally used as a nonprescription
drug with a confirmed safety to treat diarrhea and gastrointestinal
disorders for decades in China [10,11]. Recent studies show that
berberine has various biological activities such as antimalarial [12],
antileishmanial [13], anticancer [14e17], anti-alzheimer's disease
[18,19], antiviral [20,21], cholesterol lowering effect [22], hypogly-
cemic effect [23], G-quadruplex binding ligands [24], and particu-
larly antibacterial activity [25e28]. The lipophilic substituents at C-
9 position of berberine could enhance the antimicrobial activities
and broaden antimicrobial spectrum significantly. Zhou et al.
showed the synthesis of 9-O-substituted berberine derivatives with
antimicrobial activity by introducing various heterocycles such as
triazole, metronidazole, and benzimidazole in C-9 position of
berberine. Berberine-triazole A, berberine-metronidazole B, and
berberine-benzimidazole C exhibited excellent activity against
Gram-negative bacteria and fungus (Fig. 1) [29e31]. Su et al.
revealed the antibacterial activity of 9-phenoxyalkyl berberine
derivatives, for example, compound D (Fig. 1) strongly inhibited the
proliferation of Gram-positive bacteria [32]. In addition, 9-N-
Staphylococcus aureus is a Gram-positive bacterium that causes
many infections of ecological niches within the human body,
leading to a wide range of diseases [1]. Methicillin-resistant
S. aureus (MRSA) is a nosocomial and communal menace that is
resistant to most antibacterial drugs and antiseptics [2]. MRSA ac-
counts for 60e70% of S. aureus infections in hospitals and causes the
highest number of invasive infections among all antibiotic-resistant
bacteria [3e6]. It is estimated that by 2050, worldwide drug-
resistant infections will cause an additional 10 million deaths
annually [2]. Therefore the demand for the development of new
antimicrobial agents with novel chemical scaffolds and biological
mechanisms is necessary [7,8].
Natural products have been a rich resource for drug discovery
*
Corresponding author.
*
* Corresponding author.
E-mail addresses: wxue@gzu.edu.cn (W. Xue), fyang@chem.ecnu.edu.cn
(
F. Yang).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2017.01.012
223-5234/© 2017 Elsevier Masson SAS. All rights reserved.
0

J. Wang et al. / European Journal of Medicinal Chemistry 127 (2017) 424e433
425
ꢀ
Scheme 1. Reagents and conditions (a) n-pentyl amine, EtOH, reflux, 8 h, 72%; (b) 5 N NaOH, acetone, rt, 1 h; (c) various bromides, NaI, CH
3
CN, 80 C, 4 h, 65%e77% (over two steps);
ꢀ
(
d) various amine, EtOH, reflux, 8e15 h, 60%e85%; (e) NaOH, MeOH, reflux, 1 h, 71%; (f) 1,3-diiodopropane, CH
h; (h) n-pentyl amine, EtOH, reflux, 8 h, 33e42% (over two steps).
3
CN, 80 C, 6 h, 50% (over two steps); (g) various amines, EtOH, reflux,
4
substituted berberine derivatives could serve as antioxidant, G-
quadruplex binding ligands, inhibitors of acetylcholinesterase, and
butyrylcholinesterase [33,34]. Several derivatives of 9-N-
substituted berberine exhibited better antioxidant activity than 9-
O-substituted berberine derivatives [33]. However, antibacterial
activity of 9-N-substituted berberine has been poorly explored.
Moreover, 13-substituted berberine derivatives have been shown a
variety of bioactivities [35e38]. Kim et al. investigated the anti-
fungal activities of 13-(substituted benzyl) berberine and showed
that 13-(4-isopropyl benzyl) berberine E (Fig. 1) exerted the most
potent antifungal activities against Candida species [35]. Berberine
derivative F (Fig. 1) reported by Ball et al. showed even better
antibacterial activity than berberine against S. aureus strain that
overexpresses NorA protein [36]. To our knowledge, berberine de-
rivatives bearing structural modification on both C-9 and C-13
positions have been rarely reported, however. In order to search for
new chemical classes of potential antibacterial agents, we designed
and synthesized a series of 9, 13-disubstituted berberine de-
rivatives. The in vitro antibacterial activities against S. aureus
Newman and five multidrug-resistant strains were evaluated.
Scheme 1. Compound 2 was obtained by treatment of berberine
with n-pentylamine in refluxing EtOH. Compounds 4e15 were
synthesized from a key intermediate 3 that was prepared by
condensation of berberine with acetone. Treatment of 3 with
3
various bromides and sodium iodide (NaI) in CH CN yielded 13-
substituted berberine derivatives 4e15 respectively. The
berberine derivatives 16e37 were made by reaction of compounds
4e15 with primary amines under refluxing EtOH. Compound 38
was synthesized by hydrolysis of 37 in the presence of 30% aqueous
ꢀ
NaOH in MeOH at 80 C. The intermediates 40e42 were obtained
according to the published procedure [38]. Refluxing of compounds
40e42 with n-pentylamine in EtOH afforded berberine derivatives
43e45 respectively.
3. Result and discussion
3.1. Antibacterial activity
These novel berberine derivatives were first evaluated for the
in vitro antibacterial activity (MIC; the minimum concentration of
the compound that produced completely bacterial growth inhibi-
tion) against S. aureus Newman strain. The antibacterial activities of
2
. Chemistry
16, 17, 20, 21, 23, 26, and 30e34 were shown in Table 1 and
All berberine derivatives were synthesized from commercially
available berberine (1) as the starting material as described in
berberine, totarol, vancomycin were used as positive controls. The
antibacterial activities of all synthesized compounds were shown in

426
J. Wang et al. / European Journal of Medicinal Chemistry 127 (2017) 424e433
Fig. 1. Structures and antimicrobial activity of some reported compounds.
Supporting Information.
For S. aureus Newman strain, the MICs of most derivatives were
between 0.78 and 25 g/mL, except 43e45. The 9-N- and 13-
phenyl at 13-position exhibited better activity than 17 which had
no substituent on phenyl ring. Compound 34 with hydrophilic
group (OH) substituted in phenyl ring slightly decreased the anti-
bacterial potency compared to compound 17. Compound 20
m
disubstituted berberine derivatives 16e18 and 19e21 exhibited
remarkably improved antibacterial activity than 9-O- and 13-
disubstituted berberine analogues 4 and 5. For berberine ana-
logues 16e18, 19e21, 22e24 and 25e27, structure-activity rela-
tionship analysis showed that the antibacterial activity was relative
to the length of 9-N-substituted alkyl chain, and the best alkyl
length is n ¼ 4. For instance, compounds 17, 20, 23, and 26 (n ¼ 4)
exhibited more potent activity than 16, 19, 22, and 25 (n ¼ 3) and
showed the highest antibacterial activity (0.78
mg/mL), which is
better than totarol (1.56 g/mL) and comparable to vancomycin.
m
Replacement of phenyl ring at 13-position in compound 17 with
heterocycles, such as thienyl (35) and pyridyl (36), decreased the
antistaphylococcal activity. Similar, compounds with alkyl carbox-
ylic acid (38) and its corresponding ethyl formate (37) at 13-postion
displayed weakened inhibitory activities. In particular, compounds
with (1-piperidino) propyl (43), (4-morpholino) propyl (44), and
(1-pyrryl) propyl (45) substituents at 13-position lose inhibition on
1
8, 21, 24, and 27 (n ¼ 5). Replacement the methyl group (R
2
) at 9-
position of 19 with phenyl (28) and hydroxyl (29) dramatically
decreased the antibacterial activities.
Staphylococcal growth (>25
mg/mL). Taken together, these 9, 13-
Compounds 17, 20, 23, 26, and 30e34 with various benzyl
groups at 13-position showed better antibacterial activity than
compound 2 without substituent at 13-position. Compounds 20,
disubstituted berberine derivatives (16e35) exhibited better anti-
bacterial activity than berberine. Especially compounds 17, 20, 21,
23, 24, 26 and 30e33 showed potent activity against S. aureus strain
2
3, 26, and 30e33 with various hydrophobic groups substituted in
Newman with low MIC values of 0.78e1.56 mg/mL.
Table 1
MICs of berberine derivatives 16, 17, 20, 21, 23, 26, and 30e34 against S. aureus strain Newman (Newman), S. aureus NRS-1, NRS-70, NRS-100, NRS-108, NRS-271 and their IC50
values against the growth of HAF.
Compound
MIC (
m
g/mL)
IC50 (mg/mL)
Newman
NRS-1
NRS-70
NRS-100
NRS-108
NRS-271
1
1
2
2
2
2
3
3
3
3
3
6
7
0
1
3
6
0
1
2
3
4
3.13
1.56
0.78
1.56
1.25
1.25
1.25
1.25
1.25
1.25
2.5
n.t.a
3.13
1.56
1.56
3.13
1.56
3.13
3.13
3.13
1.56
6.25
n.t.
n.t.
n.t.
n.t.
n.t.
>20
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
n.t.
1.56
0.78
1.56
3.13
3.13
3.13
3.13
1.56
1.56
3.13
n.t.
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
1.56
3.13
n.t.
1.56
1.56
1.56
1.56
1.56
3.13
1.56
1.56
1.56
3.13
n.t.
15.8 ± 3.48
12.512 ± 0.373
12.784 ± 0.452
12.702 ± 1.325
13.79 ± 1.602
13.013 ± 0.483
12.678 ± 1.039
12.498 ± 0.539
11.446 ± 1.291
>20
Berberine
Vancomycin
Totarolb
>25
0.78
1.56
n.t.
n.t.
8.46 ± 0.87
1.56
3.13
0.39
1.56
0.78
1.56
0.39
3.13
0.39
3.13
a
n.t. ¼ not tested.
b
These data were reported previously in reference [8].

J. Wang et al. / European Journal of Medicinal Chemistry 127 (2017) 424e433
427
Since compounds 17e27 and 30e34 possessed excellent activity
against S. aureus Newman strain with MIC values below 2.5 g/mL,
For Newman, compound 20 showed the highest activity (MIC,
0.78 g/mL), which is comparable to the positive control vanco-
mycin. For multidrug-resistant bacteria, compounds 20, 21, and 33
exhibited the most potent activity (MIC, 0.78e1.56 g/mL) which
are comparable or even superior to totarol. All the active antimi-
crobial compounds did not show obvious toxicity to human fibro-
blast (HAF) cells at MIC dose. In summary, we showed a series of 9,
13-disubstituted berberine derivatives for discovery of new
chemical entities against multidrug-resistant S. aureus. The mech-
anism of these berberine derivatives for antistaphylococcal activity
will be further explored.
m
m
we further evaluated the antibacterial activities of these com-
pounds against multidrug-resistant S. aureus strain NRS-1 (resis-
tant to aminoglycosides and tetracycline), NRS-70 (resistant to
erythromycin), NRS-100 (resistant to oxacillin and tetracycline),
NRS-108 (resistant to gentamicin) and NRS-271 (linezolid-resistant,
containing phage type E-MRSA 15). Compounds 20, 21 and 33
displayed the most potent activity against drug-resistant strains
with MIC values of 0.78e1.56 mg/mL, which was 1e4 folds more
potent than totarol. For S. aureus strain NRS-1, compounds 18e21
and 26e33 showed equivalent antibacterial activity to vancomycin.
m
5. Experimental section
3.2. In vitro toxicity study
5.1. General methods
Berberine derivatives (16, 17, 20, 21, 23, 26, and 30e34) with low
MIC values against Newman or multidrug-resistant strains were
tested the cytotoxic activity on a human fibroblast (HAF) cell line
using the MTS assay. The cytotoxicity results of these compounds
on normal mammalian cells were summarized in Table 1. The
Starting materials, reagents and chemicals were purchased from
commercial suppliers and used without further purification unless
otherwise stated. Flash column chromatography was performed
using Qingdao Haiyang silica gel (200e300) with the indicated
1
tested compounds showed cytotoxicity with IC50 11e20
which is lower than totarol. In addition, compounds 20, 21, 33, and
4 were selected to investigate the cellular morphology of HAF
under the concentration of MIC (1.56 g/mL), and IC50 (12.5 g/mL)
respectively. As shown in Fig. 2, the compounds in a dose of 1.56 g/
mg/mL,
eluents. H NMR spectra were recorded at a spectrometer fre-
13
quency of 400 MHz, and C NMR spectra at 100 MHz. Chemical
shifts are reported in (ppm) using the 0 signal of tetrame-
3
d
d
m
m
thylsilane (TMS) as internal standard. High resolution mass spectra
were performed using a Bruker ESI-TOF high-resolution mass
spectrometer. Melting points were uncorrected and were recorded
on a Buchi B-54 melting point apparatus.
m
mL exhibited minimal toxicity to HAF cells that showed normal
morphology, however, the cells lost normal morphology when
exposed to the tested compounds at 12.5 mg/mL. The above cyto-
toxicity assay demonstrated that the novel 9, 13-disubstituted
berberine derivatives bear good selectivity for bacterial over
mammalian cells.
5
.2. Synthesis of 9-N-n-pentyl-berberine 2
Berberine chloride (0.37 g, 10 mmol) was dissolved in n-pentyl
amine (40 mmol, 4.6 mL), the mixture was stirred and heated for
ꢀ
4
. Conclusion
8 h at 100 C. After the reaction completed (monitored by TLC), the
mixture was concentrated in vacuo and the residual oil was purified
A series of novel 9, 13-disubstituted berberine derivatives were
3 3
on silica gel chromatography (CHCl /CH OH, 50/1e20/1 v/v) to give
synthesized and investigated for the antibacterial activity in vitro
against S. aureus strain Newman and five multidrug-resistant S.
aureus (NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271). For these
Gram-positive bacteria species, most synthetic derivatives showed
the proposed compound 2 as a red solid; yield: 72%; mp:
121.4e123.2 C. H NMR (400 MHz, CDCl ) d 11.57 (s, 1H), 7.86 (s,
3
1H), 7.62 (br s, 1H), 7.51 (d, J ¼ 8.5 Hz, 1H), 7.28 (s, 1H), 7.09 (d,
J ¼ 8.5 Hz, 1H), 6.78 (s, 1H), 6.06 (s, 2H), 5.09e5.06 (m, 2H), 3.91 (s,
3H), 3.82e3.76 (m, 2H), 3.16e3.13 (m, 2H), 1.82e1.75 (m, 2H),
ꢀ
1
satisfactory inhibitory activity with MIC values of 0.78e2.5 mg/mL.
Fig. 2. Normal growth (control) of human fibroblast (HAF) cells and morphological changes in the cells exposure to compound 20, 21, 33, and 34 at concentrations of 1.56
2.5 g/mL, respectively.
mg/mL and
1
m

428
J. Wang et al. / European Journal of Medicinal Chemistry 127 (2017) 424e433
1
CDCl
.38e1.35 (m, 4H), 0.88 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz,
7.62 (d, J ¼ 9.3 Hz, 1H), 7.16 (d, J ¼ 7.9 Hz, 2H), 7.00 (s, 1H), 6.98 (s,
3
)
d
150.28, 148.45, 148.03, 146.17, 140.36, 135.51, 133.40,
2H), 6.86 (s, 1H), 5.99 (s, 2H), 5.21e5.17 (m, 2H), 4.62 (s, 2H), 4.41 (s,
1
29.91, 125.47, 120.73, 118.47, 117.16, 113.13, 108.78, 104.80, 102.18,
3H), 4.01 (s, 3H), 3.31e3.27 (m, 2H), 2.34 (s, 3H); HRMS (ESI): calcd
þ
57.25, 54.17, 46.96, 31.23, 29.35, 28.14, 22.66, 14.28; HRMS (ESI):
for C28
H
26NO
4
[MeBr] 440.1856; found 440.1862.
þ
27 2 3
calcd for C24H N O [MeCl] 391.2016; found 391.2020.
5
.4.3. 13-(4-Fluorobenzyl) berberine 6
Yellow solid; yield: 65% (over two steps); mp: 238.4e240.2 C;
H NMR (400 MHz, CDCl
ꢀ
5.3. Synthesis of 4-(bromomethyl) phenyl acetate
1
3
)
d
10.39 (s, 1H), 7.71 (d, J ¼ 9.4 Hz, 1H),
In a round bottomed flask were placed of 4-hydroxy benzalde-
7.58 (d, J ¼ 9.3 Hz,1H), 7.12e7.08 (m, 2H), 7.07e7.03 (m, 2H), 6.90 (s,
hyde (2.00 g, 16.4 mmol), acetic anhydride (4.20 g, 41 mmol),
pyridine (19 mL) and DMAP (0.10 g, 0.82 mmol). The reaction
mixture was maintained under room temperature and the reaction
was checked by TLC. After disappearance of the starting material,
1H), 6.87 (s, 1H), 6.00 (s, 2H), 5.18e5.14 (m, 2H), 4.64 (s, 2H), 4.39 (s,
3H), 4.02 (s, 3H), 3.31e3.27 (m, 2H); HRMS (ESI): calcd for
þ
C
27
H
23FNO
4
[MeBr] 444.1606; found 444.1629.
the mixture was hydrolyzed with a saturated solution NaHCO
5 mL) and the medium was poured in a separating funnel con-
taining 50 mL of diethyl ether. The aqueous layer was removed and
the organic phase was washed with an aqueous solution of CuSO
20%) until the aqueous layer remained light blue. The mixture was
3
5.4.4. 13-(4-Methoxybenzyl) berberine 7
Yellow solid; yield: 68% (over two steps); mp: 187.7e189.7 C;
H NMR (400 MHz, CDCl
ꢀ
(
1
3
)
d
10.42 (s, 1H), 7.69 (d, J ¼ 9.4 Hz, 1H),
4
7.63 (d, J ¼ 9.4 Hz, 1H), 7.02 (d, J ¼ 8.3 Hz, 2H), 6.99 (s, 1H), 6.90 (d,
J ¼ 8.6 Hz, 2H), 6.87 (s, 1H), 6.00 (s, 2H), 5.21e5.17 (m, 2H), 4.60 (s,
(
then washed several times with water (5 ꢁ 5 mL). The organic layer
was dried and the solvents evaporated under vacuum. The crude
obtained was purified over silica (petroleum ether/AcOEt, 5/1 v/v)
2H), 4.41 (s, 3H), 4.01 (s, 3H), 3.80 (s, 3H), 3.31e3.27 (m, 2H); HRMS
þ
(ESI): calcd for C28
H26NO
5
[MeBr] 456.1805; found 456.1824.
1
to give the 4-formylphenyl acetate as clear oil, yield 85%. H NMR
5.4.5. 13-(2-Methylbenzyl) berberine 8
Yellow solid; yield: 73% (over two steps); mp: 198.6e200.4 C;
H NMR (400 MHz, CDCl
ꢀ
(
400 MHz, CDCl
3
)
d
9.97 (s, 1H), 7.95 (d, J ¼ 8.6 Hz, 2H), 7.23 (d,
1
J ¼ 8.6 Hz, 2H), 2.32 (s, 3H).
3
)
d
10.42 (s, 1H), 7.69 (d, J ¼ 9.3 Hz, 1H),
4
-formylphenyl acetate (2.20 g, 13.4 mmol) which was dissolved
7.52 (d, J ¼ 9.3 Hz, 1H), 7.35 (d, J ¼ 7.5 Hz, 1H), 7.25e7.21 (m, 1H),
7.08e7.04 (m, 1H), 6.86 (s, 1H), 6.82 (s, 1H), 6.64 (d, J ¼ 7.6 Hz, 1H),
5.98 (s, 2H), 5.21e5.17 (m, 2H), 4.46 (s, 2H), 4.41 (s, 3H), 4.01 (s, 3H),
ꢀ
in THF (40 mL) and the solution was cooled to 0 C, then NaBH
(
4
ꢀ
0.56 g, 14.7 mmol) was added. After the solution was stirred at 0 C
for 1.5 h, the reaction was complete according to TLC analysis. The
solution was washed with NH
Cl (2 ꢁ 50 mL) and the organic layer
was dried with Na SO . The solvent evaporated under vacuum and
3.31e3.29 (s, 2H), 2.45 (s, 3H); HRMS (ESI): calcd for C28
[MeBr] 440.1856; found 440.1833.
H26NO
4
þ
4
2
4
the crude product 4-(hydroxymethyl) phenyl acetate was used for
the next reaction without further purification. To the solution of 4-
5.4.6. 13-(3-Methylbenzyl) berberine 9
ꢀ
Yellow solid; yield: 76% (over two steps); mp: 229.2e230.9 C;
1
(
(
1
hydroxymethyl) phenyl acetate (2.20 g, 13.2 mmol) and CBr
5.80 g, 17.5 mmol) in CH Cl (40 mL), then added PPh (4.14 g,
5.8 mmol), the mixture was left at room under stirring and the
4
H NMR (400 MHz, CDCl
3
)
d
10.43 (s, 1H), 7.69 (d, J ¼ 9.4 Hz, 1H),
2
2
3
7.61 (d, J ¼ 9.3 Hz, 1H), 7.23 (d, J ¼ 7.6 Hz, 1H), 7.10 (d, J ¼ 7.6 Hz, 1H),
6.98 (s, 1H), 6.95e6.82 (m, 3H), 5.99 (s, 2H), 5.21e5.17 (m, 2H), 4.63
reaction was checked by TLC. After disappearance of the starting
material, the mixture was the residue was purified by silica gel
chromatography (petroleum ether/AcOEt, 10/1 v/v) to give 4-(bro-
momethyl) phenyl acetate (2.40 g, 60% (over two steps)) as a white
(s, 2H), 4.41 (s, 3H), 4.01 (s, 3H), 3.30e3.28 (m, 2H), 2.31 (s, 3H);
þ
HRMS (ESI): calcd for C28
440.1842.
H26NO
4
[MeBr] 440.1856; found
ꢀ
1
solid; mp: 61.7e63.2 C; H NMR (400 MHz, CDCl
3
)
d
7.40 (d,
5.4.7. 13-(3, 4-Dimethylbenzyl) berberine 10
Yellow solid; yield: 71% (over two steps); mp: 227.4e229.2 C;
H NMR (400 MHz, CDCl
10.43 (s, 1H), 7.65 (d, J ¼ 9.3 Hz, 1H),
7.61 (d, J ¼ 9.3 Hz, 1H), 7.16 (s, 1H), 7.00 (s, 1H), 6.98 (s, 2H), 6.86 (s,
H), 6.00 (s, 2H), 5.21e5.17 (m, 2H), 4.62 (s, 2H), 4.41 (s, 3H), 4.01 (s,
ꢀ
J ¼ 8.0 Hz, 2H), 7.06 (d, J ¼ 8.0 Hz, 2H), 4.47 (s, 2H), 2.29 (s, 3H).
1
3
) d
5.4. General procedure for the synthesis of compounds 4e15
1
Berberine chloride (5.0 g, 13.5 mmol) was dissolved in 5 N NaOH
23 mL). While stirring, acetone (5 mL) was added dropwise. After
3H), 3.31e3.27 (m, 2H), 2.21 (s, 3H), 2.16 (s, 3H); HRMS (ESI): calcd
þ
(
for C29
H
28NO
4
[MeBr] 454.2013; found 454.2041.
stirring for 1 h at room temperature, the reaction mixture was
filtered and washed with 80% MeOH to give compound 3 without
purified by silica gel chromatography. Compound 3 (4.0 g,
5.4.8. 13-(4-Trifluoromethylbenzyl) berberine 11
Yellow solid; yield: 66% (over two steps); mp: 221.6e223.5 C;
H NMR (400 MHz, CDCl
ꢀ
1
1
0.2 mmol) dissolved in acetonitrile was reacted with NaI (1.87 g,
3
)
d
10.85 (s, 1H), 7.61 (d, J ¼ 8.1 Hz, 2H),
ꢀ
12.5 mmol) and various bromide (15.3 mmol) at 80 C for 4 h. The
reaction mixture was concentrated and chromatographed on silica
7.40 (d, J ¼ 8.9 Hz, 2H), 7.25 (s, 1H), 6.92 (d, J ¼ 8.8 Hz, 1H), 6.85 (s,
1H), 6.79 (s, 1H), 6.00 (s, 2H), 5.18e5.14 (m, 2H), 4.64 (s, 2H), 4.39 (s,
gel (CHCl
3
/CH
3
OH, 100/1e50/1 v/v) to give compounds 4e15.
3H), 4.02 (s, 3H), 3.31e3.27 (m, 2H); HRMS (ESI): calcd for
þ
C
28
H F
23 3
NO
4
[MeBr] 494.1574; found 494.1561.
5
.4.1. 13-Benzyl berberine 4
Yellow solid; yield: 77% (over two steps); mp: 186.2e188.1 C;
H NMR (400 MHz, CDCl
10.44 (s, 1H), 7.69 (d, J ¼ 9.4 Hz, 1H),
.62 (d, J ¼ 9.3 Hz,1H), 7.39e7.35 (m, 2H), 7.34e7.28 (m, 1H), 7.12 (d,
ꢀ
5.4.9. 13-(4-Acetoxybenzyl) berberine 12
Yellow solid; yield 65% (over two steps); mp: 202.8e204.2 C;
H NMR (400 MHz, CDCl
1
ꢀ
3
) d
1
7
3
)
d
10.39 (s, 1H), 7.71 (d, J ¼ 9.4 Hz, 1H),
J ¼ 7.3 Hz, 2H), 6.96 (s, 1H), 6.88 (s, 1H), 6.00 (s, 2H), 5.22e5.18 (m,
7.58 (d, J ¼ 9.3 Hz,1H), 7.12e7.08 (m, 2H), 7.07e7.03 (m, 2H), 6.90 (s,
2
H), 4.68 (s, 2H), 4.43 (s, 3H), 4.02 (s, 3H), 3.33e3.10 (m, 2H); HRMS
1H), 6.87 (s, 1H), 6.00 (s, 2H), 5.18e5.14 (m, 2H), 4.64 (s, 2H), 4.39 (s,
þ
(
ESI): calcd for C27
H24NO
4
[MeBr] 426.1700; found 426.1714.
3H), 4.02 (s, 3H), 3.31e3.27 (m, 2H), 2.29 (s, 3H); HRMS (ESI): calcd
þ
for C29
H
26NO
6
[MeBr] 484.1755; found 484.1742.
5
.4.2. 13-(4-Methylbenzyl) berberine 5
Yellow solid; yield: 72% (over two steps); mp: 235.5e237.2 C;
H NMR (400 MHz, CDCl
10.42 (s, 1H), 7.68 (d, J ¼ 9.4 Hz, 1H),
ꢀ
5.4.10. 13-(2-Thienylmethyl) berberine 13
Yellow solid; yield: 65% (over two steps); mp: 217.4e219.0 C;
1
ꢀ
3
) d

J. Wang et al. / European Journal of Medicinal Chemistry 127 (2017) 424e433
429
1H NMR (400 MHz, CDCl
)
d
10.41 (s, 1H), 7.79 (s, 2H), 7.30 (d,
1.35e1.28 (m, 4H), 0.87 (t, J ¼ 6.7 Hz, 3H); 13C NMR (100 MHz,
3
J ¼ 8.2 Hz,1H), 7.11 (s, 1H), 6.96 (s, 1H), 6.88 (s, 1H), 6.67 (s, 1H), 6.03
3
CD OD) d 151.20, 149.29, 148.49, 147.06, 140.27, 140.19, 137.33,
(
s, 2H), 5.17e5.13 (m, 2H), 4.73 (s, 2H), 4.39 (s, 3H), 4.03 (s, 3H),
134.75, 134.36, 131.48, 130.26 (2C), 129.23 (2C), 127.94, 123.48,
121.86, 118.49, 116.73, 109.78, 109.31, 103.51, 58.46, 57.31, 51.11,
þ
3
.29e3.25 (m, 2H); HRMS (ESI): calcd for C25
H22NO
4
S [MeBr]
4
32.1264; found 432.1249.
37.16, 32.79, 32.31, 29.39, 27.65, 23.71, 14.42; HRMS (ESI): calcd for
þ
C
32
35
H N
2
O
3
[MeBr] 495.2642; found 495.2640.
5
.4.11. 13-(2-Pyridylmethyl) berberine 14
Yellow solid; yield: 69% (over two steps); mp: 221.3e222.9 C;
ꢀ
5.5.4. 9-N-n-butyl-13-(4-methylbenzyl) berberine 19
1
ꢀ
1
H NMR (400 MHz, CDCl
3
)
d
10.25 (s, 1H), 8.45 (d, J ¼ 4.2 Hz, 1H),
Red solid, yield: 70%; mp: 179.1e181.0 C. H NMR (400 MHz,
CDCl
7
.78e7.74 (m, 1H), 7.68 (d, J ¼ 9.4 Hz, 1H), 7.56 (d, J ¼ 9.3 Hz, 1H),
3
)
d
10.89 (s, 1H), 7.40 (d, J ¼ 8.8 Hz, 1H), 7.15 (d, J ¼ 7.8 Hz, 2H),
7.46 (d, J ¼ 7.8 Hz, 1H), 7.33 (s, 1H), 7.21 (d, J ¼ 7.0 Hz, 1H), 6.85 (s,
7.06 (d, J ¼ 8.8 Hz, 1H), 6.99 (d, J ¼ 7.7 Hz, 2H), 6.95 (s, 1H), 6.84 (s,
1H), 6.58 (br s, 1H), 5.98 (s, 2H), 5.10 (br s, 2H), 4.52 (s, 2H), 3.90 (s,
3H), 3.88e3.83 (m, 2H), 3.13e3.10 (m, 2H), 2.34 (s, 3H), 1.81e1.73
1
3
4
H), 5.97 (s, 2H), 5.14e5.10 (m, 2H), 4.82 (s, 2H), 4.29 (s, 3H), 3.96 (s,
H), 3.28e3.24 (m, 2H); HRMS (ESI): calcd for C26
27.1652; found 427.1649.
þ
H
23
N
2
O
4
[MeBr]
13
(m, 2H), 1.50e1.41 (m, 2H), 0.95 (t, J ¼ 7.4 Hz, 3H); C NMR
(
100 MHz, CDCl 149.64, 147.20, 146.59, 145.76, 139.97, 136.80,
3
) d
5
.4.12. 13-(Ethyl acetate) berberine 15
Yellow solid; yield: 65% (over two steps); mp: 216.2e218.1 C;
135.44, 135.23, 133.52, 132.98, 130.13 (2C), 129.42, 127.86 (2C),
123.54, 120.60, 116.36, 112.86, 109.05, 108.52, 102.00, 77.16, 56.83,
ꢀ
1
H NMR (400 MHz, CDCl
3
)
d
10.81 (s, 1H), 7.56 (d, J ¼ 8.7 Hz, 1H),
47.53, 36.21, 33.73, 28.99, 21.18, 20.34, 14.16; HRMS (ESI): calcd for
þ
7.28 (s, 1H), 7.15 (d, J ¼ 8.7 Hz, 1H), 6.88 (s, 1H), 6.09 (s, 2H),
5
3
C H N
31 33 2
O
3
[MeBr] 481.2483; found 481.2483.
.07e5.03 (m, 2H), 4.33 (q, J ¼ 7.0 Hz, 2H), 4.17 (s, 2H), 3.97 (s, 3H),
.80 (s, 3H), 3.33e3.29 (m, 2H), 1.22 (t, J ¼ 7.0 Hz, 3H); HRMS (ESI):
5.5.5. 9-N-n-pentyl-13-(4-methylbenzyl) berberine 20
þ
ꢀ
1
calcd for C24
H24NO
6
[MeBr] 422.1598; found 422.1563.
Red solid, yield: 83%; mp: 170.2e171.9 C. H NMR (400 MHz,
OD)
9.77 (s, 1H), 7.71 (d, J ¼ 9.0 Hz, 1H), 7.41 (d, J ¼ 9.0 Hz, 1H),
7.16 (d, J ¼ 7.8 Hz, 2H), 7.07e7.01 (m, 3H), 7.00 (s, 1H), 5.99 (s, 2H),
.82e4.79 (m, 2H), 4.64 (s, 2H), 4.00 (s, 3H), 3.67e3.63 (m, 2H),
3.17e3.14 (m, 2H), 2.32 (s, 3H),1.76e1.69 (m, 2H) 1.46e1.33 (m, 4H),
CD
3
d
5.5. General procedure for the synthesis of compounds 16e37
4
To a solution of intermediates 4e15 (1 mmol) in anhydrous
1
3
ethanol (2 mL), the substituted amine (40 mmol) were added. The
mixture was stirred and heated for 8e15 h at 78 C. After the re-
action completed (monitored by TLC), the mixture was concen-
0.93 (t, J ¼ 7.0 Hz, 3H); C NMR (100 MHz, CD
3
OD) d 151.23, 149.38,
ꢀ
148.52, 146.96, 140.12, 137.76, 137.30, 137.14, 134.72, 134.45, 131.81,
130.88 (2C), 129.11 (2C), 123.53, 121.89, 118.61, 116.92, 109.84,
trated in vacuo and the residual oil was purified on silica gel
109.29, 103.52, 58.47, 57.32, 51.17, 36.79, 32.04, 30.22, 29.39, 23.59,
þ
chromatography (CHCl
posed compound.
3
/CH
3
OH, 100/1e50/1, v/v) to give the pro-
21.08, 14.45; HRMS (ESI): calcd for C32
found 495.2674.
35
H N
2
O
3
[MeBr] 495.2642;
5
.5.1. 9-N-n-butyl-13-benzyl berberine 16
Red solid, yield: 82%; mp: 169.5e171.2 C. H NMR (400 MHz,
CDCl
10.92 (s,1H), 7.41 (d, J ¼ 8.9 Hz,1H), 7.38e7.34 (m, 2H), 7.30
5.5.6. 9-N-n-hexyl-13-(4-methylbenzyl) berberine 21
ꢀ
1
ꢀ
1
Red solid, yield: 75%; mp: 190.6e192.3 C. H NMR (400 MHz,
CDCl
3
)
d
3
)
d
10.88 (s, 1H), 7.40 (d, J ¼ 8.9 Hz, 1H), 7.15 (d, J ¼ 7.7 Hz, 2H),
(
(
(
(
(
d, J ¼ 7.2 Hz,1H), 7.12 (d, J ¼ 7.5 Hz, 2H), 7.06 (d, J ¼ 8.8 Hz,1H), 6.93
7.06 (d, J ¼ 8.9 Hz, 1H), 6.99 (d, J ¼ 7.7 Hz, 2H), 6.95 (s, 1H), 6.83 (s,
1H), 6.57 (br s, 1H), 5.98 (s, 2H), 5.10 (br s, 2H), 4.52 (s, 2H), 3.90 (s,
3H), 3.87e3.82 (m, 2H), 3.13e3.10 (m, 2H), 2.34 (s, 3H), 1.82e1.74
(m, 2H), 1.45e1.38 (m, 2H), 1.35e1.28 (m, 4H), 0.87 (t, J ¼ 6.8 Hz,
s, 1H), 6.85 (s, 1H), 6.63 (br s, 1H), 5.99 (s, 2H), 5.11 (br s, 2H), 4.58
s, 2H), 3.91 (s, 3H), 3.89e3.83 (m, 2H), 3.14e3.11 (m, 2H), 1.82e1.75
13
m, 2H), 1.52e1.42 (m, 2H), 0.96 (t, J ¼ 7.4 Hz, 3H); C NMR
100 MHz, CD OD) 151.26, 149.39, 148.54, 147.03, 140.28, 140.20,
37.38, 134.78, 134.42, 131.56, 130.28 (2C), 129.22 (2C), 127.97,
23.58, 121.88, 118.60, 116.82, 109.82, 109.32, 103.53, 58.48, 57.34,
13
3
d
3
3H); C NMR (100 MHz, CD OD) d 151.23, 149.38, 148.53, 146.97,
1
1
140.15, 137.76, 137.29, 137.13, 134.72, 134.43, 131.81, 130.88 (2C),
129.10 (2C), 123.47, 121.89, 118.59, 116.92, 109.83, 109.28, 103.52,
5
C
0.89, 37.16, 34.47, 29.41, 21.14, 14.30; HRMS (ESI): calcd for
58.45, 57.30, 51.23, 36.78, 32.80, 32.31, 29.38, 27.66, 23.72, 21.08,
þ
þ
30 31
H N
2
O
3
[MeBr] 467.2329; found 467.2342.
14.42; HRMS (ESI): calcd for C33
H
37
N
2
O
3
[MeBr] 509.2799; found
509.2862.
5
.5.2. 9-N-n-pentyl-13-benzyl berberine 17
ꢀ
1
Red solid, yield: 76%; mp: 208.5e210.4 C. H NMR (400 MHz,
CDCl
10.89 (s, 1H), 7.41e7.29 (m, 4H), 7.11 (d, J ¼ 7.4 Hz, 2H), 7.05
5.5.7. 9-N-n-butyl-13-(4-fluorobenzyl) berberine 22
ꢀ
1
3
)
d
Red solid, yield: 70%; mp: 188.3e190.1 C. H NMR (400 MHz,
OD)
(
2
d, J ¼ 8.8 Hz, 1H), 6.92 (s, 1H), 6.84 (s, 1H), 6.60 (br s, 1H), 5.98 (s,
CD
3
d
9.79 (s, 1H), 7.73 (d, J ¼ 9.0 Hz, 1H), 7.40 (d, J ¼ 9.0 Hz, 1H),
H), 5.11 (br s, 2H), 4.57 (s, 2H), 3.90 (s, 3H), 3.88e3.83 (m, 2H),
.13e3.10 (m, 2H), 1.83e1.76 (m, 2H), 1.43e1.33 (m, 4H), 0.90 (t,
7.24e7.16 (m, 2H), 7.09e7.05 (m, 2H), 7.00 (s, 1H), 6.98 (s, 1H), 6.00
(s, 2H), 4.80 (br s, 2H), 4.69 (s, 2H), 4.00 (s, 3H), 3.70e3.66 (m, 2H),
3.18e3.15 (m, 2H), 1.75e1.68 (m, 2H), 1.52e1.43 (m, 2H), 0.97 (t,
3
13
J ¼ 6.9 Hz, 3H); C NMR (100 MHz, CD
47.05, 140.27, 140.17, 137.33, 134.76, 134.37, 131.49, 130.26 (2C),
29.23 (2C), 127.95, 123.51, 121.87, 118.51, 116.74, 109.79, 109.31,
03.51, 58.46, 57.32, 51.10, 37.16, 32.04, 30.21, 29.39, 23.59, 14.46;
3
OD) d 151.20, 149.30, 148.50,
1
3
1
1
1
J ¼ 7.4 Hz, 3H); C NMR (100 MHz, CD
3
OD) d 163.10 (d, JC-
F
J
¼ 245.4 Hz), 151.21, 149.28,148.49, 147.10,140.24, 137.35,136.14 (d,
C-F ¼ 3.2 Hz), 134.78, 134.21, 131.27, 131.06, 130.98, 123.55, 121.78,
þ
HRMS (ESI): calcd for C31
33
H N
2
O
3
[MeBr] 481.2486; found
118.49, 116.97, 116.75, 116.55, 109.68, 109.32, 103.52, 58.44, 57.32,
4
81.2500.
50.78, 36.36, 34.43, 29.36, 21.09, 14.28; HRMS (ESI): calcd for
þ
C
30
H30FN
2
O
3
[MeBr] 485.2235; found 485.2243.
5
.5.3. 9-N-n-hexyl-13-benzyl berberine 18
ꢀ
1
Red solid, yield: 72%; mp: 175.2e177.1 C. H NMR (400 MHz,
CDCl
10.87 (s, 1H), 7.44e7.26 (m, 4H), 7.11 (d, J ¼ 7.5 Hz, 2H), 7.05
5.5.8. 9-N-n-pentyl-13-(4-fluorobenzyl) berberine 23
ꢀ
1
3
)
d
Red solid, yield: 76%; mp: 204.2e206.1 C. H NMR (400 MHz,
OD)
7.24e7.16 (m, 2H), 7.10e7.06 (m, 2H), 7.01 (s, 1H), 6.98 (s, 1H), 6.01
(s, 2H), 4.82e4.79 (m, 2H), 4.70 (s, 2H), 4.01 (s, 3H), 3.68e3.65 (m,
(
2
d, J ¼ 8.8 Hz, 1H), 6.92 (s, 1H), 6.84 (s, 1H), 6.57 (br s, 1H), 5.98 (s,
CD
3
d
9.77 (s, 1H), 7.74 (d, J ¼ 9.0 Hz, 1H), 7.41 (d, J ¼ 9.0 Hz, 1H),
H), 5.10 (br s, 2H), 4.57 (s, 2H), 3.90 (s, 3H), 3.87e3.82 (m, 2H),
.13e3.10 (t, J ¼ 5.8 Hz, 2H), 1.82e1.73 (m, 2H), 1.45e1.38 (m, 2H),
3

430
J. Wang et al. / European Journal of Medicinal Chemistry 127 (2017) 424e433
2
H), 3.18e3.15 (m, 2H), 1.77e1.70 (m, 2H), 1.46e1.36 (m, 4H), 0.93
3
) d 160.72 (d, JC-
¼ 247.4 Hz), 148.63, 146.13, 145.51, 144.86, 139.01, 134.40, 132.87
4.72e4.69 (m, 2H), 4.65 (s, 2H), 3.98 (s, 3H), 3.66e3.63 (m, 2H),
3.15e3.12 (m, 2H), 2.75e2.72 (m, 2H), 2.33 (s, 3H), 2.06e1.99 (m,
2H); C NMR (100 MHz, CDCl ) d 149.52, 147.10, 146.45, 145.75,
3
142.21, 139.56, 136.67, 135.28, 135.11, 133.43, 132.80, 130.00 (2C),
129.26, 128.58 (2C), 128.17 (2C), 127.73 (2C), 125.51, 123.76, 120.45,
116.50, 112.90, 108.92, 108.39, 101.90, 56.56, 46.87, 41.02, 36.05,
13
(
t, J ¼ 6.7 Hz, 3H); C NMR (100 MHz, CDCl
13
F
(
1
d, JC-F ¼ 4.0 Hz), 132.08, 131.93, 128.45, 128.37, 127.64, 122.30,
19.30, 115.37, 115.16, 115.08, 111.44, 107.64, 107.48, 100.95, 55.72,
4
6.72, 34.72, 30.21, 29.93, 28.13, 27.83, 21.50, 13.11; HRMS (ESI):
þ
calcd for C31
H32FN
2
O
3
[MeBr] 499.2391; found 499.2364.
33.38, 32.94, 28.88, 21.05; HRMS (ESI): calcd for C36
35
H N
2
O
3
þ
[MeBr] 543.2642; found 543.2660.
5
.5.9. 9-N-n-hexyl-13-(4-fluorobenzyl) berberine 24
ꢀ
1
Red solid, yield: 66%; mp: 188.6e190.5 C. H NMR (400 MHz,
CDCl
10.82 (s,1H), 7.44 (d, J ¼ 8.9 Hz,1H), 7.14e7.01 (m, 5H), 6.88
s, 1H), 6.86 (s, 1H), 6.54 (br s, 1H), 6.01 (s, 2H), 5.09 (br s, 2H), 4.56
s, 2H), 3.92 (s, 3H), 3.88e3.83 (m, 2H), 3.15e3.12 (t, J ¼ 5.9 Hz, 2H),
.82e1.75 (m, 2H), 1.44e1.39 (m, 2H), 1.34e1.29 (m, 4H), 0.88 (t,
5.5.14. 9-N-(3-hydroxypropyl)-13-(4-methylbenzyl) berberine 29
ꢀ
1
3
)
d
Red solid, yield: 66%; mp: 223.1e225.0 C; H NMR (400 MHz,
OD)
(
(
1
CD
3
d
9.89 (s, 1H), 7.71 (d, J ¼ 9.0 Hz, 1H), 7.44 (d, J ¼ 8.9 Hz, 1H),
7.16 (d, J ¼ 7.7 Hz, 2H), 7.08e6.95 (m, 4H), 6.00 (s, 2H), 4.81e4.79
(m, 2H), 4.65 (s, 2H), 4.01 (s, 3H), 3.82e3.79 (m, 2H), 3.77e3.73 (m,
13
13
J ¼ 6.6 Hz, 3H); C NMR (100 MHz, CDCl
3
)
d
161.77 (d, JC-
¼ 247.4 Hz), 149.69, 147.19, 146.63, 145.88, 139.99, 135.49, 133.94
d, JC-F ¼ 3.0 Hz), 133.12, 132.98, 129.53, 129.45, 128.80, 123.32,
20.36, 116.42, 116.21, 116.19, 112.68, 108.70, 108.54, 102.03, 56.82,
2H), 3.18e3.15 (m, 2H), 2.33 (s, 3H), 1.99e1.92 (m, 2H); C NMR
F
(
1
3
(100 MHz, CDCl ) d 149.77, 147.25, 147.08, 145.84, 139.65, 136.87,
135.80, 135.20, 133.54, 133.11, 130.17 (2C), 129.91, 127.88 (2C),
123.19, 120.50, 116.93, 114.03, 109.06, 108.57, 102.03, 59.23, 57.39,
5
6.79, 47.93, 35.77, 31.67, 31.53, 28.88, 26.69, 22.67, 14.12; HRMS
56.79, 45.66, 36.24, 33.26, 29.03, 21.18; HRMS (ESI): calcd for
þ
þ
(
ESI): calcd for C32
H34FN
2
O
3
[MeBr] 513.2548; found 513.2563.
C
30
H
31
N
2
O
4
[MeBr] 483.2278; found 483.2280.
5
.5.10. 9-N-n-butyl-13-(4-methoxybenzyl) berberine 25
5.5.15. 9-N-n-pentyl-13-(2-methylbenzyl) berberine 30
ꢀ
1
ꢀ
1
Red solid, yield: 66%; mp: 212.4e214.3 C. H NMR (400 MHz,
OD)
9.76 (s, 1H), 7.90 (s, 1H), 7.73 (d, J ¼ 9.1 Hz, 1H), 7.44 (d,
Red solid, yield: 82%; mp: 216.3e218.2 C. H NMR (400 MHz,
CDCl
CD
3
d
3
)
d
10.91 (s, 1H), 7.40 (d, J ¼ 8.9 Hz, 1H), 7.34 (d, J ¼ 7.5 Hz, 1H),
J ¼ 9.1 Hz, 1H), 7.12e7.02 (m, 3H), 7.00 (s, 1H), 6.91 (s, 1H), 6.89 (s,
H), 6.00 (s, 2H), 4.82e4.79 (m, 2H), 4.63 (s, 2H), 4.01 (s, 3H), 3.78
s, 3H), 3.68e3.64 (m, 2H), 3.17e3.14 (m, 2H), 1.75e1.68 (m, 2H),
7.24e7.21 (m,1H), 7.09e7.05 (m, 1H), 6.95 (d, J ¼ 8.8 Hz,1H), 6.84 (s,
1H), 6.80 (s, 1H), 6.68 (d, J ¼ 7.6 Hz, 1H), 6.61 (br s, 1H), 5.97 (s, 2H),
5.12 (br s, 2H), 4.36 (s, 2H), 3.90 (s, 3H), 3.89e3.84 (m, 2H),
3.14e3.11 (m, 2H), 2.43 (s, 3H),1.84e1.77 (m, 2H),1.47e1.31 (m, 4H),
1
(
13
1
CDCl
.52e1.43 (m, 2H), 0.98 (t, J ¼ 7.4 Hz, 3H); C NMR (100 MHz,
1
3
3
)
d
158.51,149.54,147.10,146.50,145.62,139.81,135.32,133.36,
0.91 (t, J ¼ 6.9 Hz, 3H); C NMR (100 MHz, CD
3
OD) d 151.21, 149.44,
132.84, 130.06, 129.42, 128.91 (2C), 123.38, 120.46, 116.26, 114.72
148.56, 147.00, 140.15, 138.18, 137.55, 137.33, 134.78, 134.61, 131.92,
131.75, 128.76, 128.22, 127.66, 123.70, 121.91, 118.56, 116.77, 109.40,
(
2C), 112.82, 108.90, 108.40, 101.90, 56.74, 56.72, 55.31, 47.47, 35.64,
3
3.61, 28.85, 20.22, 14.04; HRMS (ESI): calcd for C31
H N
33 2
O
4
109.31, 103.53, 58.49, 57.35, 51.17, 35.57, 32.05, 30.23, 29.39, 23.59,
þ
þ
[MeBr] 497.2435; found 497.2400.
19.90, 14.45; HRMS (ESI): calcd for C32
found 495.2658.
35
H N
2
O
3
[MeBr] 495.2642;
5
.5.11. 9-N-n-pentyl-13-(4-methoxybenzyl) berberine 26
ꢀ
1
Red solid, yield: 72%; mp: 205.6e207.3 C. H NMR (400 MHz,
CDCl
10.81 (s, 1H), 7.44 (d, J ¼ 8.9 Hz, 1H), 7.11 (d, J ¼ 8.9 Hz, 1H),
5.5.16. 9-N-n-pentyl-13-(3-methylbenzyl) berberine 31
ꢀ
1
3
)
d
Red solid, yield: 85%; mp: 225.4e227.3 C. H NMR (400 MHz,
CDCl
7.03 (d, J ¼ 8.2 Hz, 2H), 6.98 (s, 1H), 6.88 (d, J ¼ 8.6 Hz, 2H), 6.85 (s,
1
3
3
)
d
10.88 (s,1H), 7.40 (d, J ¼ 8.9 Hz,1H), 7.25e7.21 (m, 1H), 7.09
H), 6.49 (br s, 1H), 6.00 (s, 2H), 5.10 (br s, 2H), 4.52 (s, 2H), 3.92 (s,
H), 3.87e3.81 (m, 2H), 3.80 (s, 3H), 3.14e3.11 (m, 2H), 1.83e1.75
(d, J ¼ 7.6 Hz, 1H), 7.05 (d, J ¼ 8.9 Hz, 1H), 6.95 (s, 1H), 6.91e6.88 (m,
2H), 6.83 (s, 1H), 6.59 (br s, 1H), 5.98 (s, 2H), 5.10 (br s, 2H), 4.52 (s,
2H), 3.90 (s, 3H), 3.87e3.82 (m, 2H), 3.11e3.10 (m, 2H), 2.31 (s, 3H),
13
(
(
1
1
m, 2H), 1.45e1.32 (m, 4H), 0.90 (t, J ¼ 6.9 Hz, 3H); C NMR
100 MHz, CDCl 158.53, 149.57, 147.14, 146.58, 145.61, 139.74,
35.37, 133.39, 132.84, 130.10, 129.56, 128.94 (2C), 123.41, 120.48,
16.34, 114.75 (2C), 113.04, 108.93, 108.41, 101.95, 56.84, 56.80,
13
3
)
d
1.83e1.76 (m, 2H), 1.45e1.30 (m, 4H), 0.90 (t, J ¼ 6.9 Hz, 3H);
C
NMR (100 MHz, CDCl 149.53, 147.10, 146.46, 145.66, 139.83,
3
) d
139.09, 138.20, 135.34, 133.36, 132.86, 129.19, 129.16, 128.51, 127.83,
124.94, 123.40, 120.47, 116.22, 112.77, 108.93, 108.40, 101.89, 56.75,
5
5.35, 47.86, 35.67, 31.22, 29.17, 28.88, 22.53, 14.15; HRMS (ESI):
þ
calcd for C32
H N
35 2
O
4
[MeBr] 511.2591; found 511.2583.
56.71, 47.70, 36.41, 31.24, 29.16, 28.88, 22.52, 21.52, 14.14; HRMS
þ
(ESI): calcd for C32
35
H N
2
O
3
[MeBr] 495.2642; found 495.2660.
5
.5.12. 9-N-n-hexyl-13-(4-methoxybenzyl) berberine 27
ꢀ
1
Red solid, yield: 69%; mp: 202.3e204.3 C; H NMR (400 MHz,
CDCl
10.86 (s, 1H), 7.41 (d, J ¼ 8.9 Hz, 1H), 7.07 (d, J ¼ 8.9 Hz, 1H),
5.5.17. 9-N-n-pentyl-13-(3,4-dimethylbenzyl) berberine 32
ꢀ
1
3
)
d
Red solid, yield: 81%; mp: 199.3e201.3 C; H NMR (400 MHz,
OD)
7.01 (d, J ¼ 8.2 Hz, 2H), 6.96 (s, 1H), 6.87 (d, J ¼ 8.3 Hz, 2H), 6.83 (s,
1
3
CD
3
d
9.87 (s, 1H), 7.66 (d, J ¼ 9.0 Hz, 1H), 7.39 (d, J ¼ 9.0 Hz, 1H),
H), 6.56 (br s, 1H), 5.99 (s, 2H), 5.09 (br s, 2H), 4.49 (s, 2H), 3.90 (s,
H), 3.87e3.81 (m, 2H), 3.79 (s, 3H), 3.12e3.09 (m, 2H), 1.81e1.74
7.09 (d, J ¼ 7.7 Hz, 1H), 7.05 (s, 1H), 6.96e6.94 (m, 2H), 6.84 (d,
J ¼ 7.9 Hz, 1H), 6.00 (s, 2H), 4.88e4.85 (m, 2H), 4.61 (s, 2H), 4.00 (s,
(
3
m, 2H), 1.45e1.38 (m, 2H), 1.33e1.28 (m, 4H), 0.87 (t, J ¼ 6.8 Hz,
3H), 3.70e3.67 (m, 2H), 3.19e3.16 (m, 2H), 2.25 (s, 3H), 2.22 (s, 3H),
13
13
H); C NMR (100 MHz, CD
3
OD)
d
160.00, 151.14, 149.24, 148.45,
1.79e1.72 (m, 2H), 1.47e1.36 (m, 4H), 0.93 (t, J ¼ 6.9 Hz, 3H);
C
146.89, 140.05, 137.20, 134.65, 134.41, 131.93, 131.88, 130.22 (2C),
NMR (100 MHz, CD OD/CDCl
3
3
)
d
150.76, 148.67, 148.13, 146.21,
1
5
23.52, 121.85, 118.55, 116.82, 115.59 (2C), 109.81, 109.25, 103.47,
139.61, 138.25, 136.75, 136.72, 135.97, 134.07, 133.90, 131.54, 131.09,
129.86, 126.13, 123.25, 121.30, 117.98, 116.38, 109.62, 108.99, 103.00,
8.46, 57.32, 55.77, 49.43, 36.33, 32.76, 32.28, 29.36, 27.62, 23.67,
þ
14.39; HRMS (ESI): calcd for C33
H
37
N
2
O
4
[MeBr] 525.2748; found
58.17, 57.12, 50.61, 36.64, 31.70, 29.80, 29.22, 23.18, 19.98, 19.43,
þ
5
25.2769.
14.31; HRMS (ESI): calcd for C33
37
H N
2
O
3
[MeBr] 509.2799; found
509.2780.
5
.5.13. 9-N-(3-phenylpropyl)-13-(4-methylbenzyl) berberine 28
ꢀ
1
Red solid, yield: 66%; mp: 185.9e187.8 C; H NMR (400 MHz,
OD)
9.63 (s, 1H), 7.90 (s, 1H), 7.72 (d, J ¼ 9.1 Hz, 1H), 7.43 (d,
5.5.18. 9-N-n-pentyl-13-(4-trifluoromethylbenzyl) berberine 33
ꢀ
1
CD
3
d
Red solid, yield: 79%; mp: 204.2e206.1 C; H NMR (400 MHz,
CDCl
10.85 (s, 1H), 7.62 (s, 1H), 7.60 (s, 1H), 7.40 (d, J ¼ 8.9 Hz,
J ¼ 9.1 Hz, 1H), 7.25e7.07 (m, 6H), 7.04e7.00 (m, 4H), 6.00 (s, 2H),
3
) d

J. Wang et al. / European Journal of Medicinal Chemistry 127 (2017) 424e433
431
1
6
3
4
CDCl
1
H), 7.27 (s, 1H), 7.25 (s, 1H), 6.92 (d, J ¼ 8.8 Hz, 1H), 6.85 (s, 1H),
.79 (s, 1H), 6.61 (br s, 1H), 5.99 (s, 2H), 5.08 (br s, 2H), 4.63 (s, 2H),
.90 (s, 3H), 3.88e3.83 (m, 2H), 3.14e3.11 (m, 2H), 1.82e1.75 (m,
(12 mL) and 30% aqueous NaOH (7.5 mL) and the mixture was
heated to reflux for 1 h. The reaction mixture was concentrated
under reduced pressure and treated with 20 mL of water. The so-
13
H), 1.42e1.33 (m, 2H), 0.89 (t, J ¼ 7.0 Hz, 3H); C NMR (100 MHz,
149.74, 147.20, 146.52, 146.33, 142.53, 140.31, 135.55,
33.09, 132.85, 129.43 (q, JC-F ¼ 32.4 Hz), 128.35 (2C), 127.73, 126.29
q, JC-F ¼ 3.7 Hz, 2C), 124.00 (q, JC-F ¼ 270.2 Hz), 123.42, 120.23,
16.07, 112.18, 108.62, 108.50, 102.03, 56.74, 56.59, 47.68, 36.39,
1.22, 29.15, 28.91, 22.51, 14.12; HRMS (ESI): calcd for C32
lution was extracted three times with CH
2 2
Cl , and the combined
3
)
d
extracts were dried over Na SO . The organic phase was evaporated
2
4
under reduced pressure and crude product was recrystallized from
(
1
EtOAc to obtain compound 38 (0.185 g, 71%) as a red solid; mp:
ꢀ
1
138.6e139.9 C; H NMR (400 MHz, CD OD) d 9.62 (s, 1H), 7.82 (d,
3
3
[
32
H N
2
O
3
F
3
J ¼ 9.0 Hz, 1H), 7.68e7.65 (m, 2H), 6.96 (s, 1H), 6.04 (s, 2H),
4.71e4.68 (m, 2H), 4.09 (s, 2H), 4.02 (s, 3H), 3.55e3.51 (m, 2H),
þ
MeBr] 529.2360; found 529.2365.
3
.09e3.07 (m, 2H), 1.69e1.62 (m, 2H), 1.39e1.33 (m, 4H), 0.91 (t,
13
5
.5.19. 9-N-n-pentyl-13-(4-hydroxylbenzyl) berberine 34
J ¼ 6.9 Hz, 3H); C NMR (100 MHz, CD
3
OD) d 177.26, 151.01, 149.31,
ꢀ
1
Red solid, yield: 68%; mp: 218.7e220.6 C; H NMR (400 MHz,
OD)
9.81 (s, 1H), 7.70 (d, J ¼ 9.0 Hz, 1H), 7.42 (d, J ¼ 9.0 Hz, 1H),
148.58, 145.78, 139.60, 136.73, 134.99, 134.58, 131.03, 123.62, 122.18,
118.75, 116.11, 111.00, 109.06, 103.42, 58.17, 57.30, 50.92, 41.21, 31.87,
CD
3
d
7.06 (s, 1H), 7.00e6.91 (m, 3H), 6.75 (s, 1H), 6.73 (s, 1H), 5.98 (s, 2H),
30.18, 29.31, 23.52, 14.39; HRMS (ESI): calcd for C26
[MeBr] 449.2071; found 449.2096.
H
29
N
2
O
5
þ
4
3
.80 (br s, 2H), 4.56 (s, 2H), 3.98 (s, 3H), 3.67e3.63 (m, 2H),
.16e3.13 (m, 2H), 1.76e1.69 (m, 2H), 1.43e1.34 (m, 4H), 0.92 (t,
13
J ¼ 6.9 Hz, 3H); C NMR (100 MHz, CD
48.51, 146.81, 140.02, 137.21, 134.66, 134.53, 132.15, 130.68, 130.16
2C), 123.65, 121.89, 118.69, 117.02, 116.97 (2C), 109.88, 109.23,
03.48, 58.42, 57.30, 51.08, 36.31, 31.98, 30.19, 29.37, 23.54, 14.39;
3
OD)
d
157.43, 151.19, 149.38,
5.7. General procedure for the synthesis of compounds 43e45
Compound 3 (2.65 g, 6.7 mmol) dissolved in CH CN (75 mL) and
1
(
1
3
1, 3-diiodopropane (18.92 g, 64 mmol) was added, then stirred at
þ
ꢀ
HRMS (ESI): calcd for C31
H N
33 2
O
4
[MeBr] 497.2435; found
80 C for 6 h. After cooling, the reaction mixture was concentrated
4
97.2460.
under reduced pressure and crude product was purified by column
3 3
chromatography on silica gel (CHCl /CH OH, 100/1e50/1 v/v) to
5
.5.20. 9-N-n-pentyl-13-(2-thienylmethyl) berberine 35
give 13-(3-iodopropyl) berberine 39 2.11 g (yield 50%, over two
steps); mp: 207.3e209.3 C; H NMR (400 MHz, CDCl ) d 10.79 (s,
3
Red solid, yield: 80%; mp: 176.1e178.4 C; 1H NMR (400 MHz,
10.85 (s, 1H), 7.49 (d, J ¼ 8.9 Hz, 1H), 7.29 (s, 1H), 7.24 (d,
J ¼ 8.9 Hz,1H), 7.08 (s,1H), 6.96 (s,1H), 6.86 (s,1H), 6.67 (s,1H), 6.55
br s, 1H), 6.04 (s, 2H), 5.10 (br s, 2H), 4.64 (s, 2H), 3.94 (s, 3H),
.89e3.84 (m, 2H), 3.14e3.11 (m, 2H), 1.83e1.76 (m, 2H), 1.43e1.34
ꢀ
ꢀ
1
CDCl
3
)
d
1H), 7.88 (d, J ¼ 9.1 Hz, 1H), 7.76 (d, J ¼ 9.0 Hz, 1H), 7.23 (s, 1H), 7.03
(s, 1H), 6.01 (s, 2H), 5.10 (br s, 2H), 4.38 (s, 3H), 4.08 (s, 3H),
3.63e3.58 (m, 2H), 3.13e3.11 (m, 2H), 2.93e2.88 (m, 2H), 2.30e2.14
(m, 2H). Compound 39 (370 mg, 0.59 mmol) dissolved in EtOH
(36 mL) and various amines (8.8 mmol) was added, then stirred at
(
3
13
(
1
1
1
m, 4H), 0.90 (t, J ¼ 6.9 Hz, 3H); C NMR (100 MHz, CDCl
47.24, 146.47, 146.20, 141.16, 140.03, 134.99, 133.07, 132.97, 128.75,
27.45, 126.03, 124.98, 123.59, 120.25, 116.36, 112.33, 108.91, 108.43,
02.01, 56.78, 56.50, 47.61, 31.68, 31.17, 29.13, 28.88, 22.49, 14.12;
3
) d 149.65,
ꢀ
78 C for 4 h. After cooling, the reaction mixture was dried under
vacuum and the crude product 40e42 was used for the next re-
action without further purification. To a solution of compound 40
(0.5 mmol) in anhydrous ethanol (1 mL), n-pentylamine (2.3 mL,
þ
HRMS (ESI): calcd for C29H N O S [MeBr] 487.2050; found
31 2 3
4
87.2033.
20 mol) were added. The mixture was stirred and heated for 8 h at
ꢀ
78 C. After the reaction completed, the mixture was concentrated
5
.5.21. 9-N-n-pentyl-13-(2-pyridylmethyl) berberine 36
in vacuo and the residual oil was purified on silica gel chroma-
tography (CHCl /CH OH, 100/1e50/1 v/v) to give compound 43.
ꢀ
1
Red solid, yield: 78%; mp: 143.6e145.7 C; H NMR (400 MHz,
CDCl 10.77 (s, 1H), 8.54 (s, 1H), 7.74e7.70 (m, 1H), 7.37 (d,
3
3
3
) d
J ¼ 8.9 Hz, 1H), 7.29 (s, 1H), 7.27 (s, 1H), 7.24e7.20 (m, 1H), 6.98 (d,
5.7.1. 9-N-n-pentyl-13-(3-(piperidin-1-yl) propyl) berberine 43
ꢀ
1
J ¼ 8.9 Hz, 1H), 6.84 (s, 1H), 6.39 (br s, 1H), 5.98 (s, 2H), 5.08 (br s,
Red solid; yield: 35% (over two steps); mp: 163.2e165.3 C; H
2
H), 4.71 (s, 2H), 3.88 (s, 3H), 3.84e3.79 (m, 2H), 3.13e3.10 (m, 2H),
NMR (400 MHz, CD
3
OD)
d
9.64 (s, 1H), 7.88 (d, J ¼ 9.1 Hz, 1H), 7.76
13
1.78e1.72 (m, 2H), 1.39e1.30 (m, 4H), 0.87 (t, J ¼ 6.8 Hz, 3H);
C
(d, J ¼ 9.0 Hz, 1H), 7.23 (s, 1H), 7.03 (s, 1H), 6.11 (s, 2H), 4.71e4.68
(m, 2H), 4.08 (s, 3H), 3.62e3.59 (m, 2H), 3.51 (d, J ¼ 12.3 Hz, 2H),
3.46e3.42 (m, 2H), 3.22e3.18 (m, 2H), 3.11e3.08 (m, 2H), 2.91 (t,
J ¼ 12.3 Hz, 2H), 2.30e2.14 (m, 2H),1.93e1.90 (m, 2H),1.85e1.65 (m,
3
NMR (100 MHz, CDCl ) d 158.80, 150.02, 149.59, 147.14, 146.37,
1
1
4
C
46.01, 140.04, 137.45, 135.59, 133.20, 132.97, 128.76, 123.43, 123.20,
22.19, 120.70, 116.50, 112.45, 109.17, 108.39, 101.90, 56.77, 56.42,
7.79, 39.23, 31.16, 29.13, 28.97, 22.50, 14.13; HRMS (ESI): calcd for
13
5H), 1.56e1.32 (m, 5H), 0.96e0.84 (m, 3H); C NMR (100 MHz,
þ
H
30 32
N
3
O
3
[MeBr] 482.2438; found 482.2439.
CDCl
31.76, 122.09, 120.13, 116.72, 115.34, 109.59, 108.43, 102.31, 57.27,
56.62, 55.75, 52.87 (2C), 49.98, 31.10, 28.99, 28.72, 26.79, 24.46,
3
) d 149.99, 147.65 (2C), 145.41, 138.88, 134.49, 132.90, 131.96,
1
5
.5.22. 9-N-n-pentyl-13-(2-ethoxy-2-oxoethyl) berberine 37
ꢀ
1
Red solid, yield: 60%; mp: 146.2e148.2 C; H NMR (400 MHz,
CDCl
10.81 (s, 1H), 7.56 (d, J ¼ 8.8 Hz, 1H), 7.28 (s, 1H), 7.15 (d,
22.72 (2C), 22.46, 21.53, 14.05; HRMS (ESI): calcd for C32
[MeI] 516.3221; found 516.3252.
H
42
N
3
O
3
þ
3
)
d
J ¼ 8.8 Hz, 1H), 6.88 (s, 1H), 6.51 (br s, 1H), 6.09 (s, 2H), 5.05 (br s,
2
3
H), 4.36e4.30 (m, 2H), 4.17 (s, 2H), 3.97 (s, 3H), 3.88e3.83 (m, 2H),
.11e3.08 (m, 2H), 1.78e1.76 (m, 2H), 1.39e1.33 (m, 7H), 0.90 (t,
5.7.2. 9-N-n-pentyl-13-(3-morpholinopropyl) berberine 44
Compound 41 was treated with n-pentylamine according to
general procedure to give the desired product 44 as a red solid,
13
J ¼ 6.9 Hz, 3H); C NMR (100 MHz, CDCl
46.67, 145.97, 140.35, 135.66, 133.30, 133.07, 124.80, 123.21, 120.22,
15.83, 110.71, 109.22, 108.58, 102.11, 62.15, 56.83, 56.39, 47.93,
7.12, 31.18, 29.09, 28.82, 22.49, 14.25, 14.10; HRMS (ESI): calcd for
3
) d 170.83, 149.84, 147.46,
ꢀ
1
1
1
3
yield 33% (over two steps), mp: 152.5e154.3 C; H NMR (400 MHz,
CD OD)
3
d
9.74 (s, 1H), 7.88 (d, J ¼ 9.0 Hz, 1H), 7.77 (d, J ¼ 9.0 Hz, 1H),
7.21 (s, 1H), 7.00 (s, 1H), 6.10 (s, 2H), 4.76e4.74 (m, 2H), 4.04 (s, 3H),
3.69e3.67 (m, 4H), 3.63e3.60 (m, 2H), 3.39e3.35 (m, 2H),
þ
28 33 2 5
C H N O [MeBr] 477.2384; found 477.2376.
3
.12e3.09 (m, 2H), 2.60e2.50 (m, 6H), 2.06e1.98 (m, 2H), 1.72e1.65
13
5.6. Synthesis of 9-N-n-pentyl-13-(carboxymethyl) berberine 38
(m, 2H), 1.40e1.32 (m, 4H), 0.90 (t, J ¼ 6.8 Hz, 3H); C NMR
100 MHz, CD OD) 150.90, 149.17, 148.48, 146.01, 139.97, 135.88,
134.83, 134.75, 133.76, 123.65, 122.03, 118.54, 116.03, 110.43, 109.30,
(
3
d
Compound 37 (0.28 g, 0.5 mmol) was dissolved in warm MeOH

432
J. Wang et al. / European Journal of Medicinal Chemistry 127 (2017) 424e433
103.57, 67.35 (2C), 58.79, 58.38, 57.43, 54.58 (2C), 50.77, 31.94,
differentiation, the cells were immersed in 500 mL 0.5% dilute
3
C
0.12, 29.42, 28.30, 27.89, 23.49, 14.43; HRMS (ESI): calcd for
ammonia water for 10 min to make the cell nucleuses turn to blue.
Then the plate was washed by water for three times and each well
þ
31
40
H N
3
O
4
[MeI] 518.3013; found 518.3028.
was added 500 mL eosin solutions for 5 min to stain the cytoplasm.
5
.7.3. 9-N-n-pentyl-13-(3-(pyrrolidin-1-yl) propyl) berberine 45
Compound 42 was treated with n-pentylamine according to
After staining, the plate was washed by water for five times until
the background was almost colorless. At last, the morphology was
observed under a contrast microscope.
general procedure to give the desired product 45 as a red solid,
ꢀ
1
yield 42% (over two steps), mp: 149.3e151.3 C; H NMR (400 MHz,
CD OD)
9.74 (s, 1H), 7.90 (d, J ¼ 9.0 Hz, 1H), 7.85 (d, J ¼ 9.0 Hz, 1H),
.21 (s, 1H), 7.01 (s, 1H), 6.10 (s, 2H), 4.76e4.72 (m, 2H), 4.04 (s, 3H),
3
d
Acknowledgments
7
3
3
.65e3.62 (m, 2H), 3.49e3.45 (m, 2H), 3.43e3.32 (m, 6H),
.16e3.13 (m, 2H), 2.26e2.20 (m, 2H), 2.10e2.01 (m, 4H), 1.73e1.66
This research was partially supported by the National Key
Technology R&D Program (2015BAK45B00), the National Natural
Science Foundation of China (81472788, 81272463, and
81661138004), Major State Basic Research Development Program of
China (2015CB910400), and Shanghai Science and Technology
Council (14DZ0511800 and 14142201200). We also thank the Lab-
oratory of Organic Functional Molecules, Sino-French Institute of
ECNU for support.
13
(
(
1
1
m, 2H), 1.40e1.32 (m, 4H), 0.91 (t, J ¼ 6.8 Hz, 3H); C NMR
100 MHz, CD OD) 150.92, 149.26, 148.47, 145.98, 140.01, 135.91,
34.99, 134.86, 133.85, 123.67, 122.14, 118.65, 116.10, 110.50, 109.31,
03.57, 58.38, 58.16, 57.44, 55.40, 53.05, 50.84, 49.00, 45.40, 31.95,
3
d
3
0.13, 29.44, 28.46, 28.36, 23.50, 14.41; HRMS (ESI): calcd for
þ
C
31
40
H N
3
O
3
[MeI] 502.3064; found 502.3087.
5
5
.8. Biological assay
Appendix A. Supplementary data
.8.1. In vitro antibacterial assay
The strains used in this study were S. aureus Newman and five
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.01.012.
multidrug-resistant S. aureus (NRS-1, NRS-70, NRS-100, NRS-108
and NRS-271). All strains were grown at 37 C overnight in TSB
without the antibiotic. Overnight cultures diluted 1000 fold were
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