SUPPLEMENTARY MATERIAL
Facile synthesis of tetrahydroprotoberberine and protoberberine
alkaloids from protopines and study on their antibacterial activities
Pi Cheng ab*, Bin Wang c, Xiubin Liua, Wei Liua, Weisong Kang c, Jie
Zhouc and Jianguo Zeng abc*
a
Pre-State Key Laboratory for Germplasm Innovation and Utilization of Crop,
Hunan Agricultural University, Changsha, Hunan 410128, China
b
National Research Center of Engineering Technology For Utilization of Functional
Ingredients From Botanicals, Hunan Agricultural University, Changsha, Hunan
410128, China
c
School of Pharmaceutical, Hunan University of Chinese Medicine, Changsha,
Hunan 410128, China
*Corresponding authors: E-Mail: picheng55@126.com (Pi Cheng); ginkgo@worldway.net (Jianguo Zeng); Tel.: +86-731-8468-6560; Fax: +86-731-8468-6560.
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Facile synthesis of tetrahydroprotoberberine and protoberberine
alkaloids from protopines and study on their antibacterial activities
A series of isoquinoline alkaloids including tetrahydroprotoberberines, Nmethyl tetrahydroprotoberberines and protoberberines were facile synthesized
with protopines as starting material. All compounds were evaluated for their
antibacterial activities against four pathogenic bacteria E. coli, S. aureus, Avian
Staphylocosis and S. choleraesuis. Experimental results indicated that
protoberberines were the most active compounds to the target bacteria among
the tested alkaloids. It was suggested that planar molecule with high
aromatization level (eg. coptisine 5 and berberine 6) or a positive charge of the
molecules (eg. N-methyl tetrahrydroprotoberberines 11 and 12) had a positive
influence on the antibacterial effects.
Keywords: protopines; isoquinoline alkaloids; synthesis; antibacterial activities
1 Experimental
1.1 Chemistry
Column chromatography silica gel (200-300 mesh) and TLC plate (Qingdao Meijin
Chemical Inc.; Qingdao; China); 1H NMR and
13
C NMR spectra were recorded on
Bruker 400M spectrometers and chemical shifts were given in  with TMS as an
internal reference. HRMS data were obtained on an Agilent UPLC-QTOF(6530)
instrument. All the reagents are commercially available. Protopine and allycryptopine
isolated from M. cordata.
1.1.1. Synthesis of N-Methyl-13,14-dehydrostylopinium (3) and N-Methyl-7,8dihydroberberine (4)
To a solution of protopine (1) or allycryptopine (2) (2 mmol) in chloroform (30mL),
oxalyl chloride (1.6 mL) was added and the mixture refluxed for 1h. After this period,
the solvent was removed in vacuum. The reaction crude was purified on silica column
eluted by dichloromethane/methanol/acetone（7.5:1:0.1）to provide compounds 3
and 4 respectively.
1.1.2. Synthesis of Coptisine (5) and Berberine (6)
A solution of compound 3 or 4 (0.5 mmol) in DMSO (10 mL) was heated at 115°C
for 1.5 h. The solvent was removed in vacuum at 80 °C for 24 h to give a residue. The
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residue
was
purified
on
silica
column
chromatography
eluted
by
dichloromethane/methanol/acetone （ 7.5:1:0.1 ） to provide compounds 5 and 6
respectively.
1.1.3. Synthesis of Stylopine (7) and Canadine(8)
To a solution of compounds 5 or 6 (0.5 mmol) in methanol (15 mL), NaBH4 (380mg,
10mmol) was added in 15min and the reaction mixture was stirred for 24h. Then the
reaction mixture was poured to ice-cooled water and extracted with chloroform. The
organic solvent was removed in vacuum to give a residue which was further purified
on silica column with chloroform/acetone (10:0.2) as eluant to afford the desired
compounds.
1.1.4. Synthesis of Dihydroprotopine (9) and Dihydroallycryptopine (10)
To a solution of protopine or allycryptopine (1 or 2, 10 mmol) in methanol (100 mL),
NaBH4 (3.8g, 100mmol) was added and the mixture was refluxed for 48h. The solvent
was removed in vacuum. Water 100 mL was added to the residue and the mixture was
extracted with chloroform. The organic solvent was dried over Na2SO4, concentrated
and purified by column chromatography (hexane/acetone/triethylamine, 3:2:0.1) to
give the target compound.
1.1.5. Synthesis of N-methyl stylopine (11) and N-methyl tetrahydroberberine (12)
To a solution of dihydroprotopine (9) dihydroallycryptopine (10) (1mmol) in
chloroform (50 mL), a few drops of TFA were added and the mixture was stirred for
0.5 h. The reaction mixture was then evaporated and the residue was purified on silica
gel chromatography eluted by dichloromethane/MeOH/triethylamine (7.5:1:0.1) to
provide the target compound.
1.2. General procedure for anti-bacteria assay
1.2.1. Kirby-Bauer experiments
Compounds 1-4 and their derivatives were screened for their antibacterial activity
using the Kirby-Bauer paper disc diffusion method (Melaiye et al., 2004; Kong et al.,
2008). S. aureus, Avian Staphylocosis and S. choleraesuis were used as the test
bacteria. The test compounds were dissolved in 20% DMSO at a concentration of
1mgmL-1 and a paper disc (d = 6 mm) was evenly soaked with 100 L compound
solution, and then dried in an oven at 40°C and placed on an agar plate seeded with
the 20–24 h cultured fresh bacteria at 28°C. After incubation for 24 h at 37°C, the
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diameter of the inhibitory zone around the disc was measured. A paper disc saturated
with 20% DMSO was used, after drying, as a negative control. Mequindox was used
as positive control.
1.2.2 Determination of MIC and MBC
MIC and MBC values of all compounds were determined by the turbidity method
with slight modification, using S. aureus, Avian Staphylocosis and S. choleraesuis as
the test bacteria. A Baird-Parker agar (BPA) broth consisting of beef extract (0.3%),
peptone and NaCl (0.5%) was used for culturing the test bacteria. A 24 h cultured
broth diluted by the same broth to 2.2×107 colony forming units (CFU) for S. aureus,
Avian Staphylocosis and S. choleraesuis, 3.6×107CFU for E. coli to serve as inocula.
The test sample was dissolved in 20% DMSO, and 20 mL of the resulting solution
added to a first tube containing 980 L of BPA broth. Two-fold serial dilutions made
by adding the same BPA broth to obtain concentrations from 400 gmL-1 to 3.12
gmL-1. A bacterial suspension (0.5 mL) was added to each tube and incubated for 24
h at 37°C for bacteria. The MIC determined by visually judging the bacterial growth
in the series of test tubes, which was defined as the lowest concentration at which no
bacterial growth was observed after incubation. The MBCs were determined by
inoculating the surfaces of Mueller-Hinton agar (MHA) plates with 25 mL of the
samples taken from the clear tubes the MIC determination. After the bacterial
suspensions had fully absorbed into agar, the plates were further incubated at 28°C for
24 h and were examined growth in daylight. The MBC was defined as the
concentration at which no colony was observed after incubation. All experiments
were performed in triplicate.
1.3 Original spectra of typical synthetic compounds
1.3.1 ESI-HRMS and 1H-NMR spectra of compound 3
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1.3.2 ESI-HRMS and 1H-NMR spectra of compound 4
1.3.3 ESI-HRMS and 1H-NMR spectra of compound 5
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1.3.4 ESI-HRMS and 1H-NMR spectra of compound 6
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1.3.5 ESI-HRMS and 1H-NMR spectra of compound 7
1.3.6 ESI-HRMS and 1H-NMR spectra of compound 8
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1.3.7 ESI-HRMS and 1H-NMR spectra of compound 10
1.3.8 ESI-HRMS and 1H-NMR spectra of compound 12
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Acknowledgements
This work was financially supported by the Important Science & Technology Specific
Projects of Hunan province (No. 2012FJ1004), the Talent Introduction Project in
Hunan Agricultural University (No. 11YJ04) and TCM Scientific Research Project
(No. 201259) of state Administration of Traditional Chinese Medicine of Hunan
Province).
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