Facile synthesis of tetrahydroprotoberberine and protoberberine alkaloids from protopines and study on their antibacterial activities

Article abstract of DOI:10.1080/14786419.2013.867344

A series of isoquinoline alkaloids including tetrahydroprotoberberines, N-methyl tetrahydroprotoberberines and protoberberines were facile synthesised with protopines as the starting material. All compounds were evaluated for their antibacterial activities against four pathogenic bacteria Escherichia coli, Staphyloccocus aureus, Staphylococcus gallinarum and Salmonella 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 aromatisation level (e.g. coptisine 5 and berberine 6) or a positive charge of the molecules (e.g. N-methyl tetrahydroprotoberberines 11 and 12) had a positive influence on the antibacterial effects.

Full text of DOI:10.1080/14786419.2013.867344

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Facile synthesis of
tetrahydroprotoberberine and
protoberberine alkaloids from
protopines and study on their
antibacterial activities
Pi Chengab, Bin Wangc, Xiubin Liua, Wei Liua, Weisong Kangc, Jie
Zhouc & Jianguo Zengabc
a Pre-State Key Laboratory for Germplasm Innovation and
Utilization of Crop, Hunan Agricultural University, Changsha,
Hunan 410128, P.R. China
b National Research Center of Engineering Technology for
Utilization of Functional Ingredients from Botanicals, Hunan
Agricultural University, Changsha, Hunan 410128, P.R. China
c School of Pharmaceutical, Hunan University of Chinese Medicine,
Changsha, Hunan 410208, P.R. China
Published online: 14 Jan 2014.
To cite this article: Pi Cheng, Bin Wang, Xiubin Liu, Wei Liu, Weisong Kang, Jie Zhou & Jianguo
Zeng (2014) Facile synthesis of tetrahydroprotoberberine and protoberberine alkaloids from
protopines and study on their antibacterial activities, Natural Product Research: Formerly Natural
Product Letters, 28:7, 413-419, DOI: 10.1080/14786419.2013.867344
To link to this article: http://dx.doi.org/10.1080/14786419.2013.867344
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Natural Product Research, 2014
Vol. 28, No. 7, 413–419, http://dx.doi.org/10.1080/14786419.2013.867344
Facile synthesis of tetrahydroprotoberberine and protoberberine alkaloids
from protopines and study on their antibacterial activities
Pi Chengab*, Bin Wangc, Xiubin Liua, Wei Liua, Weisong Kangc, Jie Zhouc and
Jianguo Zengabc
*
aPre-State Key Laboratory for Germplasm Innovation and Utilization of Crop, Hunan Agricultural
University, Changsha, Hunan 410128, P.R. China; bNational Research Center of Engineering Technology
for Utilization of Functional Ingredients from Botanicals, Hunan Agricultural University, Changsha,
Hunan 410128, P.R. China; cSchool of Pharmaceutical, Hunan University of Chinese Medicine, Changsha,
Hunan 410208, P.R. China
(Received 7 August 2013; final version received 14 November 2013)
A series of isoquinoline alkaloids including tetrahydroprotoberberines, N-methyl
tetrahydroprotoberberines and protoberberines were facile synthesised with protopines
as the starting material. All compounds were evaluated for their antibacterial activities
against four pathogenic bacteria Escherichia coli, Staphyloccocus aureus, Staphylococcus
gallinarum and Salmonella choleraesuis. Experimental results indicated that protoberber-
ines were the most active compounds to the target bacteria among the tested alkaloids. It
was suggested that planar molecule with high aromatisation level (e.g. coptisine 5 and
berberine 6) or a positive charge of the molecules (e.g. N-methyl tetrahydroprotoberberines
11 and 12) had a positive influence on the antibacterial effects.
Keywords: protopines; isoquinoline alkaloids; synthesis; antibacterial activities
1. Introduction
The protopine alkaloids, for instance, protopine and allocryptopine, are a type of isoquinoline
alkaloids mainly found in the plants of the Papaveraceae and Fumariaceae families and have
been the focus of a number of biological studies such as anti-parasitic activity (Satou et al. 2002),
anti-arrhythmic (Song et al. 2000), anti-thrombotic and hepatoprotective effects in animal
models (Rathi et al. 2008).
Protopine alkaloids contain the unique structural feature of a nitrogen-containing 10-
membered cyclic ketone. In biological systems, protopine alkaloids are considered to exist in
equilibrium between their tricyclic bases and tetracyclic quaternary salts N-methyl-16-hydroxyl-
tetrahydroprotoberberines (Vacek et al. 2010). In the metabolism pathway of protopines,
(S)-reticuline that is derived from L-tyrosine underwent cyclisation, N-methylation and
14-hydroxylation to afford tetrahydroprotoberberine, N-methyl tetrahydroprotoberberine and
N-methyl-16-hydroxyl-tetrahydroprotoberberine successively. N-methyl-16-hydroxyl-tetrahy-
droprotoberberines have been demonstrated to be the direct biosynthetic precursors of
protopines (Zeng et al. 2013).
As the intermediates in the metabolism pathway of protopines, berbines (tetrahydropro-
toberberine and N-methyl tetrahydroprotoberberine) were generally distributed in protopine-
containing plants such as Macleaya cordata, a Papaveraceae berb medicine, with very low
accumulation levels (Chen et al. 2009). In this study, the berbines and protoberberines were
*Corresponding authors. Email: picheng55@126.com; ginkgo@world-way.net
q 2014 Taylor & Francis

414
P. Cheng et al.
facile synthesised with protopine alkaloids as the starting material. To gain further insight into
the pharmacological effects of alkaloids involved in the biosynthesis of protopines, all alkaloids
were evaluated for their antibacterial activities against several animal pathogens.
2. Results and discussion
2.1. Chemistry
In protopines, the presence of nucleophilic amino group and electrophilic carbonyl group in the
10-membered ring generates a strong intramolecular interaction. As shown in Scheme 1, the
reaction of protopine (1) or allocryptopine (2) with oxalyl chloride provided the N-methy-
13,14-dehydroprotoberberine quaternary salts 3 and 4, which contain four 6-membered ring
system. A dimethyl sulfoxide (DMSO) solution of 3 or 4 was heated at 1208C to yield
protoberberine salts coptisine (5) and berberine (6) as reported by Valpuesta et al. (2002).
Coptisine and berberine were reduced by NaBH4 to yield tetrahydroprotoberberine alkaloids
stylopine (7) and canadine (8).
Dihydroprotopines (9, 10) were obtained through an NaBH4-mediated reduction of
protopines, and then a reaction of compound 9 or 10 with CF3COOH followed by treatment with
HCl afforded N-methyl stylopine (11) and N-methyl canadine salts (12) successfully. With
natural protopine-type alkaloids as starting materials, tetrahydroprotoberberine alkaloids (7, 8)
and their methylated salt (11, 12) which were involved in the biosynthesis pathway of protopines
were synthesised. Compounds 3 and 4 and hydroxyl derivatives 9 and 10 were unnatural and
never existed in plants.
2.2. Bioactivity and discussion
Compounds 1–12 were evaluated for their antibacterial activities against four Gram-negative
pathogenic bacteria: Escherichia coli, Staphyloccocus aureus, Staphylococcus gallinarum and
4
1
5
O
O
O
O
O
O
6
Me
Me
N
N
N
O
a
b
OR1
OR2
OR1
OR2
OR1
OR2
R1 + R2= –CH2–
R1 + R2= –CH2–
5:
R1 + R2= –CH2–
1 Protopine (PR):
3:
2 Allocryptopine (ALL): R1 = R2 = CH3
4: R1 = R2 = CH3
6: R1 = R2 = CH3
c
d
O
O
O
O
Me
N
Me
N
N
O
O
e
HO
OR1
OR2
OR1
OR1
OR2
OR2
R1 + R2= –CH2–
11:
R1 + R2= –CH2–
R1 + R2= –CH2–
9:
10: R1 = R2 = CH3
7:
12: R1 = R2 = CH3
8: R1 = R2 = CH3
Scheme 1. Reagents and conditions: (a) (COCl)2, CHCl3, reflux, 1 h; (b) DMSO, 1208C, 90 min; (c) NaBH4
(10 equiv.), MeOH, room temperature, 24 h; (d) NaBH4 (10 equiv.), MeOH, reflux, 48 h; (e) CF3COOH,
CHCl2, room temperature 1.5 h, then HCl, MeOH.

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415
Salmonella choleraesuis. Firstly, the Kirby-Bauer test (Kong & Jang 2008) was conducted to
determine the sensitivity of the four selected bacteria to various alkaloids. The results are
summarised in Table 1.
E. coli was resistant to the protopine-type alkaloids (1, 2) and their cyclisation salts (3, 4)
(inhibition zone , 10 mm). However, when the protopines were converted to the
protoberberine-type alkaloids, berberine (6) exhibited increased inhibitory activity to E. coli
(inhibition zone ¼ 15.8 mm). For comparison, the tetrahydroprotoberberine-type (7, 8),
hydroxyl derivatives (9, 10) and N-methyl tetrahydroprotoberberine salts (11, 12) lost
antibacterial activities. S. aureus, S. gallinarum and S. choleraesuis were moderately sensitive or
resistant to naturally existed alkaloids 1, 2 and their cyclisation salts 3, 4. S. aureus,
S. gallinarum and S. choleraesuis showed higher sensitivity (inhibition zone ¼ 30.6, 28.6 and
24.0 mm, respectively) to berberine 6 than that of E. coli and this is in accord with previous study
(Cernakova & Kostalova 2002). Tetrahydroprotoberberine alkaloids 7 and 8 exhibited
significantly decreased inhibitory activities to the bacteria compared with the aromatised
protoberberine-type alkaloids 5 and 6. But it was interesting to note that the N-methyl
tetrahydroprotoberberine quaternary salts 11 and 12 showed moderate inhibitory activities to
S. aureus and S. gallinarum compared to tetrahydroprotoberberines 7 and 8. With respect to the
positive control, all the four bacteria are moderately sensitive to Mequindox.
Minimum bactericidal concentration (MBC) values of all compounds were further
determined for E. coli, S. aureus, S. gallinarum and S. choleraesuis. The results are listed
in Table 2. For E. coli, all alkaloids showed a minimum inhibitory concentration (MIC) value
above 100 mg mL21. The protoberberine-type salt berberine (6) displayed low MIC and MBC
values against S. aureus, S. gallinarum and S. choleraesuis. This result is compatible with the
data in Table 1. Coptisine (5) displayed better inhibitory activity against S. choleraesuis (MIC
¼ 52.5 mg mL21) compared to protopine derivative 1. For S. aureus and S. gallinarum, the
Table 1. Kirby-Bauer antibacterial activities of 1 and 2 and their derivatives.
Inhibition zone (mm)a
E. coli
(CMCC44717)
S. aureus
(CMCC56002)
S. gallinarum
(NO2-2)
S. choleraesuis
(CVCC504)
Compounds
1
2
3
4
5
6
7
8
9
10
11
12
9.0
8.4
7.6
8.0
8.0
15.8
7.5
8.0
7.5
7.4
8.0
7.9
10.5
–
7.0
7.1
7.0
10.2
7.0
16.5
10.3
28.6
–
9.8
11.9
13.9
7.0
20.5
24.0
7.0
7.0
7.0
7.0
6.4
13.1
12.1
12.5
30.6
d
–
7.0
–
–
–
7.1
15.0
18.9
13.1
–
7.0
15.3
19.3
13.5
–
7.0
13.6
–
Mequindoxb
Controlc
Note: All data are mean values of two separate experiments.
a Inhibition zone . 20 mm: sensitive; 20–10 mm: moderate sensitive; ,10 mm: resistant.
b Mequindox: an antibacterial agent used as positive control.
c Negative control: 20% DMSO.
d No inhibitory activity at the experimental conditions.

416
P. Cheng et al.
Table 2. MIC and MBC values (mg mL21) of 1 and 2 and synthetic compounds.
E. coli
(CMCC44717)
S. aureus
(CMCC56002
S. gallinarum
(NO2-2)
S. choleraesuis
(CVCC504)
Compounds
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
1
2
3
4
5
6
7
8
9
10
11
12
1000
500
1000
500
250
.1000
1000
1000
1000
1000
500
.1000
1000
1000
1000
500
500
500
.1000
31.25
250
15.63
.1000
.1000
.1000
250
125
31.25
31.25
1000
1000
.1000
62.5
500
31.25
.1000
.1000
.1000
500
250
62.5
62.5
500
250
.1000
31.25
500
15.63
.1000
.1000
500
250
62.5
1000
1000
.1000
31.25
500
15.63
.1000
.1000
1000
500
500
500
1000
250
62.5
62.5
500
250
250
250
500
250
7.8
500
1000
1000
500
62.5
125
1000
500
500
500
500
250
.1000
500
500
500
250
500
62.5
125
62.5
62.5
1000
125
31.25
31.25
250
15.63
Mequindox
Note: All data are mean values of two separated experiments.
antibacterial data of N-methyl tetrahydroprotoberberine quaternary salts 11 and 12 also
exhibited good consonance with the Kirby-Bauer tests showed in Table 1, and this strongly
suggests that N-methylation of tetrahydroprotoberberine will increase the antibacterial activities.
3. Experimental
3.1. Chemistry
Column chromatography silica gel (200–300 mesh) was used for compounds’ purification; thin
layer chromatography plate (Qingdao Meijin Chemical, Inc., Qingdao, China) was used for
monitoring the chemical reactions; 1H and 13C NMR spectra were recorded on Bruker 400 MHz
spectrometers (Bruker Corporation, Germany) and chemical shifts were given in d with
tetramethylsilane as an internal reference. HRMS data were obtained on an Agilent UPLC-QTOF
(6530) instrument (Agilent Technologies, USA). All the reagents are commercially available.
Protopine and allocryptopine were isolated from M. cordata. All compounds were synthesised as
reported in previous literatures with slight modifications (Valpuesta et al. 2002; Grycova et al.
2007), and the detailed procedure can be seen in the Supplementary material (available online).
Compound (3): obtained as pale yellow amorphous powder, isolated yield: 49.7%, 1H NMR
(DMSO-d6, 400 MHz) d 7.74 (s, 1H, H-1), 7.61 (s, 1H, H-13), 7.07 (d, 1H, J ¼ 8.6 Hz, H-11),
7.05 (d, 1H, J ¼ 8.6 Hz, H-12), 6.21, 6.11 (two brs, each 2H, 2 £ OCH2O), 5.08–5.00 (m, 2H,
H-8), 4.23–4.18 (m, 1H, H-6), 4.02–3.89 (m, 1H, H-60), 3.38–3.66 (m, 1H, overlapped, H-5),
3.13–3.09(m, 1H, overlapped, H-50), 3.09 (s, 3H, NMe); 13C NMR (DMSO-d6, 100 MHz) d
149.5 (C-2), 149.2 (C-3), 148.3 (C-9), 144.9 (C-10), 135.2 (C-14), 126.6 (C-4a), 123.3 (C-12),
122.4 (C-12a), 118.9 (C-14a), 116.1 (C-13), 109.5 (C-1), 108.9 (C-11), 106.5 (C-8a), 103.7
(OCH2O), 103.1 (C-4), 102.3 (OCH2O), 60.7 (C-6), 59.6 (C-8), 45.9 (NMe), 23.9 (C-5); ESI-
HRMS [M]þ C20H18NO4 336.1230, found 336.1176.
1
Compound (4): obtained as yellow amorphous powder, isolated yield: 60.0%, H NMR
(DMSO-d6, 400 MHz) d 7.74 (s, 1H, H-1), 7.60 (s, 1H, H-13),7.26 (d, 1H, J ¼ 8.4 Hz, H-11),
7.21 (d, 1H, J ¼ 8.4 Hz, H-12), 6.94 (s, 1H, H-4), 6.12, 6.10 (two brs, each 1H, ZOCH2OZ),
5.10 (d, 1H, J ¼ 14.8 Hz, H-8), 4.94 (d, 1H, J ¼ 14.8 Hz, H-80), 4.12–4.02 (m, 1H, H-6), 3.99–
3.89 (m, 1H, overlapped, H-60), 3.89 (s, 3H, O-Me), 3.82 (s, 3H, OMe), 3.42–3.55 (m, 1H,
overlapped, H-5), 3.11–3.06 (m, 1H, overlapped, H-50), 3.06 (s, 3H, N-Me); 13C NMR (DMSO-

Natural Product Research
417
d6, 100 MHz) d 154.2 (C-2), 149.2 (C-3), 148.2 (C-9), 145.8(C-10), 135.2 (C-14), 126.6 (C-4a),
124.3 (C-12), 122.1 (C-12a), 119.5 (C-14a), 118.8 (C-13), 115.9 (C-1), 113.9 (C-11), 108.9
(C-8a), 103.7 (C-4), 102.3 (OCH2O), 61.5 (C-6), 60.7 (C-8), 60.4 (9-OMe), 56.5 (10-OMe), 45.6
(NMe), 23.9 (C-5); ESI-HRMS [M]þ calcd for C21H22NO4 352.1543, found 352.1483.
1
Compound (5): obtained as yellow amorphous powder, isolated yield: 20.9%, H NMR
(DMSO-d6, 400 MHz) d 9.96 (s, 1H, H-8), 8.97 (s, 1H, H-13), 8.04 (d, 1H, J ¼ 8.8 Hz, H-11),
7.82 (d, 1H, J ¼ 8.8 Hz, H-12), 7.79 (s, 1H, H-1), 7.08 (s, 1H, H-4), 6.53, 6.17 (two s, each 2H, 2
£ OCH2O), 4.88 (t, 2H, J ¼ 6.0 Hz, H-6), 3.19 (t, 2H, J ¼ 6.0 Hz, H-5); 13C NMR (DMSO-d6,
100 MHz) d 150.3 (C-2), 148.2 (C-3), 147.6 (C-8), 145.0 (C-9), 144.3 (C-10), 137.3 (C-14),
132.8 (C-4a), 131.0 (C-12a), 122.2 (C-12), 121.5 (C-13), 121.4 (C-11), 121.0 (C-14a), 112.1
(C-8a), 108.9 (C-1), 105.8 (C-4), 105.0, 102.6 (2 £ OCH2O), 55.7 (C-6), 26.8 (C-5); ESI-HRMS
[M]þ C19H14NO4 320.0917, found 320.0888.
1
Compound (6): obtained as yellow amorphous powder, isolated yield: 20.7%, H NMR
(DMSO-d6, 400 MHz) d 9.90 (s, 1H, H-8), 8.95 (s, 1H, H-13), 8.21 (d, 1H, J ¼ 9.2 Hz, H-11), 8.00
(d, 1H, J ¼ 9.2 Hz, H-12), 7.80 (s, 1H, H-1), 7.09 (s, 1H, H-4), 6.17 (s, 1H, OCH2O), 4.93 (t, 2H,
J ¼ 6.0 Hz, H-6), 4.09 (s, 3H, 9-OMe), 4.07 (s, 3H, 10-OMe), 3.20 (t, 2H, J ¼ 6.0 Hz, H-5); 13
C
NMR (DMSO-d6, 100 MHz) d 150.9 (C-2), 150.3 (C-3), 148.2 (C-9), 145.9 (C-10), 144.2(C-8),
138.0 (C-14), 133.5 (C-4a), 131.2 (C-12), 127.3 (C-12a), 124.0 (C-14a), 121.9 (C-13), 120.9
(C-1), 120.7 (C-11), 108.9 (C-8a), 105.9 (C-4), 102.6 (OCH2O), 62.4 (C-6), 57.6 (9-OMe), 55.7
(10-OMe), 26.9 (C-5); ESI-HRMS [M]þ calcd for C20H18NO4 336.1230, found 336.1185.
1
Compound (7) obtained as white amorphous powder, isolated yield: 68.0%, H NMR
(DMSO-d6, 400 MHz) d 6.92 (s, 1H, H-1), 6.75 (d, 1H, J ¼ 8.0 Hz, H-11), 6.70 (s, 1H, H-4),
6.63 (d, 1H, J ¼ 8.0 Hz, H-12), 6.00–5.94 (m, 4H, 2 £ OCH2O), 3.95 (d, 1H, J ¼ 16 Hz, H-8),
3.46–3.32(m, 2H, overlapped, H-80, H-14), 3.07 (1H, dd, J ¼ 12.0, 4.0 Hz, H-6), 2.94–2.85 (m,
1H, H-13), 2.62–2.43 (m, 3H, H-130, 80, 60); 13C NMR (DMSO-d6, 100 MHz) d 146.2 (C-2),
145.9 (C-3), 145.0 (C-9), 143.2 (C-10), 131.2 (C-4a), 129.1 (C-12a), 128.0 (C-14a), 121.4
(C-12), 117.1 (C-8a), 108.6 (C-1), 107.0 (C-11), 106.2 (C-4), 101.3, 101.0 (2 £ OCH2O), 59.6
(C-14), 52.7 (C-8), 51.0 (C-6), 36.2 (C-13), 29.4 (C-5); ESI-HRMS [M þ H]þ C19H18NO4
324.1236, found 324.1182.
1
Compound (8): obtained as white amorphous powder, isolated yield: 58.4%, H NMR
(DMSO-d6, 400 MHz) d 6.91 (s, 1H, H-1), 6.88 (d, 1H, J ¼ 8.4 Hz, H-11), 6.84 (d, 1H,
J ¼ 8.4 Hz, H-12), 6.66 (s, 1H, H-4), 5.94 (brs, 2H, OCH2O), 4.05 (d, 1H, J ¼ 16 Hz, H-8) 3.77,
3.72 (two s, 6H, 2 £ OCH3), 3.38–3.28 (m, 2H, overlapped, H-80, H-14), 3.08 (1H, dd, J ¼ 11.2,
4.0 Hz, H-6), 2.94–2.86 (m, 1H, H-13), 2.61–2.41 (m, 3H, H-130, 80, 60); 13C NMR (DMSO-d6,
100 MHz) d 150.3 (C-2), 146.2 (C-3), 145.9 (C-9), 144.9 (C-10), 131.4 (C-4), 128.7 (C-4a),
128.1 (C-12a), 128.0 (C-14a), 124.2 (C-12), 111.7 (C-1), 108.5 (C-8a), 106.2 (C-11), 101.0
(OCH2O), 60.0 (9-OMe), 59.5 (C-14), 56.2 (10-OMe), 54.0 (C-8), 51.2 (C-6), 36.2 (C-13), 29.5
(C-5); ESI-HRMS [M þ H]þ C20H22NO4 340.1549, found 340.1539.
1
Compound (9): obtained as white amorphous powder, isolated yield: 65.8%, H NMR
(DMSO-d6, 400 MHz) d 7.00 (s, 1H, H-1), 6.84, 6.75 (two d, 1H each, J ¼ 8.0 Hz, H-11, H-12),
6.70 (s, d, H-4), 6.00–5.95 (m, 4H, 2 £ OCH2O), 5.11–5.00 (m, 1H, H-14), 4.88–4.63 (m, 1H,
H-8), 4.01 (d, 1H, J ¼ 14.0 Hz, H-13), 3.48 (d, 1H, J ¼ 14.0 Hz, H-130), 3,40–3.35 (m, 1H,
overlapped, H-80), 2.96 (t, 1H, J ¼ 12.0 Hz, H-5), 2.66 (d, 1H, J ¼ 12.0 Hz, H-6), 2.55–2.43(m,
2H, H-50, H-60), 1.99 (s, 3H, NCH3); 13C NMR (DMSO-d6, 100 MHz) d 150.7 (C-2), 146.6
(C-3), 145.6 (C-9), 145.2 (C-10), 141.9 (C-12a), 132.1 (C-14a), 130.9 (C-4a), 130.3 (C-8a),
126.1 (C-12), 110.5 (C-1), 110.0 (C-11), 104.8 (C-4), 101.0, 100.8(2 £ OCH2O), 69.5 (C-14),
60.7 (C-6), 51.9 (C-8), 47.7 (C-13), 42.0 (N-CH3), 32.0 (C-5); ESI-HRMS [M þ H]þ calcd for
C20H22NO5 356.1498, found 356.1490.
1
Compound (10): obtained as white amorphous powder, isolated yield: 85.8%, H NMR
(DMSO-d6, 400 MHz) d 7.01 (s, 1H, H-1), 6.85, 6.80 (two d, 1H each, J ¼ 8.4 Hz, H-11, H-12),

418
P. Cheng et al.
6.72 (s, d, H-4), 5.96, 5.92 (two brs, 1H each, OCH2O), 5.07–5.04 (m, 1H, H-14), 4.86 (d, 1H,
J ¼ 4.8 Hz, H-8), 4.04 (d, 1H, J ¼ 14.8Hz, H-13), 3.76, 3,64 (two s, 3H each, 2 £ OCH3), 3.51 (d,
1H, J ¼ 14.8Hz, H-130), 3,35–3.31 (m, 1H, overlapped, H-80), 2.98 (t, 1H, J ¼ 12.8Hz, H-5), 2.71
(d, 1H, J ¼ 12.8Hz,H-6), 2.54–2.40(m,1H, H-50, H-60),1.97(s, 3H, NCH3);13CNMR(DMSO-d6,
100 MHz) d 151.2 (C-2), 147.6 (C-3), 146.1 (C-9), 145.7 (C-10), 142.0 (C-12a), 133.1 (C-14a),
131.5 (C-4a), 131.3 (C-8a), 127.1 (C-12), 110.6 (C-1), 110.4 (C-11), 105.8 (C-4), 100.8 (OCH2O),
69.2 (C-14), 60.5 (9-OCH3), 60.2 (C-6), 56.0 (10-OCH3), 52.0 (C-8), 47.5 (C-13), 42.9 (N-CH3),
32.7 (C-5); ESI-HRMS [M þ H]þ calcd for C21H26NO5 372.1811, found 372.1807.
1
Compound (11): obtained as white amorphous powder, isolated yield 48.0%, H NMR
(DMSO-d6, 400 MHz) d 7.21 (d, 1H, J ¼ 8.4 Hz, H-11), 7.17 (d, 1H, J ¼ 8.4 Hz, H-12), 7.09
(s, 1H, H-1), 6.88 (s, 1H, H-4), 6.10–6.02 (m, 4H, 2 £ OCH2O), 5.00 (dd, 1H, J ¼ 12.0, 6.0 Hz,
H-14), 4.78 (d, 1H, J ¼ 15.2 Hz, H-8), 4.70 (d, 1H, J ¼ 15.2 Hz, H-80), 4.10–4.00 (m, 2H, H-6),
3.33–3.15 (m, 1H, H-5), 3.10 (dd, 1H, J ¼ 18.0, 4.0 Hz, H-50), 2.90–2.81 (m, 2H, H-13), 2.88
(s, 3H, NCH3); 13C NMR (DMSO-d6, 100 MHz) d 154.3 (C-2), 147.0 (C-3), 146.3 (C-9), 142.5
(C-10), 141.1 (C-12a), 133.3 (C-14a), 131.9 (C-4a), 130.0 (C-8a), 119.1 (C-12), 110.8 (C-1),
110.0 (C-11), 106.6 (C-4), 101.9, 101.2 (2 £ OCH2O), 70.1 (C-14), 60.2 (C-6), 51.5 (C-8), 40.8
(N-CH3), 32.4 (C-13), 28.1 (C-5); ESI-HRMS [M]þ calcd for C20H20NO4 338.1392, found
338.1377.
1
Compound (12): obtained as white amorphous powder, isolated yield 38.9%, H NMR
(DMSO-d6, 400 MHz) d 7.15 (d, 1H, J ¼ 8.4 Hz, H-11), 7.09 (d, 1H, J ¼ 8.4 Hz, H-12), 7.10 (s,
1H, H-1), 6.90 (s, 1H, H-4), 6.07, 6.05 (s, 1H each, OCH2O), 5.00 (dd, 1H, J ¼ 12.4, 5.8 Hz,
H-14), 4.84 (d, 1H, J ¼ 15.6 Hz, H-8), 4.72 (d, 1H, J ¼ 15.6 Hz, H-80), 4.05–3.98 (m, 2H, H-6),
3.82 (brs, 3H, 10-OCH3), 3.78 (brs, 3H, 9-OCH3), 3.27–3.10 (m, 1H, H-5), 3.08 (dd, 1H,
J ¼ 18.0, 4.0 Hz, H-50), 2.93 (dd, 2H, J ¼ 17.6, 12.4 Hz, H-13), 2.81 (s, 3H, NCH3); 13C NMR
(DMSO-d6, 100 MHz) d 153.3 (C-2), 147.8 (C-3), 145.3 (C-9), 143.6 (C-10), 141.2 (C-12a),
133.4 (C-14a), 132.4 (C-4a), 130.9 (C-8a), 122.0 (C-12), 110.1 (C-1), 110.4 (C-11), 105.8 (C-4),
101.9 (OCH2O), 69.5 (C-14), 61.2 (C-6), 60.9 (9-OCH3), 56.8 (10-OCH3), 52.0 (C-8), 39.6
(N-CH3), 31.3 (C-13), 27.9 (C-5); ESI-HRMS [M]þ calcd for C21H24NO4 354.1700, found
354.1640.
3.2. Bioactivity assay
Kirby-Bauer experiments and MIC and MBC determination were done as reported in the
previous literature (Miao et al. 2011). The detailed procedure can be found in the Supplementary
material.
4. Conclusions
In this study, several types of isoquinoline alkaloids involved in the biosynthesis of protopines
were synthesised with protopines as starting materials and evaluated for their antibacterial
activities. High aromatisation level of the alkaloids generates positive influence on the
antibacterial activities. Planar cation molecules berberine and coptisine exhibited good
antibacterial activities and act as bacterial DNA topoisomerase inhibitors as reported previously
(Tse-Dinh 2007). N-methyl tetrahydroprotoberberines were found to display antibacterial
activities for the first time.
Supplementary material
Further experimental details are available online alongside the original spectra of compounds
3–8, 10 and 12.

Natural Product Research
419
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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