Structural modification of sanguinarine and chelerythrine and their antibacterial activity

Article abstract of DOI:10.1080/14786419.2010.482055

In this study, five derivatives of sanguinarine (1) and chelerythrine (2) were prepared, with 1 and 2 as starting materials, by reduction, oxidation and nucleophilic addition to the iminium bond C=N+. The structures of all compounds were elucidated on account of their MS, 1H-NMR and 13C-NMR data. The antibacterial activities of all compounds were screened, using Staphylococcus aureus, Escherichia coli, Aeromonas hydrophila and Pasteurella multocida as test bacteria. The minimum bacteriostatic concentration and minimum bactericidal concentration of the active compounds were determined by the turbidity method. The structure-activity relationships of 1 and 2 were discussed. The results showed that 1, 2 and their pseudoalcoholates were found to be potent inhibitors to S. aureus, E. coli and A. hydrophila, while the other derivatives were found to be inactive. The pseudoalcoholates might be the prodrugs of 1 and 2. The iminium bond in the molecules of 1 or 2 was the determinant for antibacterial activity, and the substituents at the 7 and 8 positions influenced the antibacterial activities of 1 and 2 against different bacteria.

Full text of DOI:10.1080/14786419.2010.482055

Natural Product Research
Vol. 25, No. 9, May 2011, 863–875
Structural modification of sanguinarine and chelerythrine and their
antibacterial activity
b
b
Fang Miaoa, Xin-Juan Yangab, Le Zhouab , Hai-Jun Hu , Feng Zheng ,
*
Xu-Dong Dingb, Dong-Mei Sunb, Chun-Dong Zhoub and Wei Sunc
aCollege of Life Science, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China;
bCollege of Science, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China;
cDepartment of Chemistry, Northwest University, Xi’an, Shaanxi 710068, P.R. China
(Received 12 January 2010; final version received 28 March 2010)
In this study, five derivatives of sanguinarine (1) and chelerythrine (2) were
prepared, with 1 and 2 as starting materials, by reduction, oxidation
and nucleophilic addition to the iminium bond C¼Nþ. The structures of
all compounds were elucidated on account of their MS, 1H-NMR and
13C-NMR data. The antibacterial activities of all compounds were
screened, using Staphylococcus aureus, Escherichia coli, Aeromonas hydro-
phila and Pasteurella multocida as test bacteria. The minimum bacterio-
static concentration and minimum bactericidal concentration of the active
compounds were determined by the turbidity method. The structure–activ-
ity relationships of 1 and 2 were discussed. The results showed that 1, 2 and
their pseudoalcoholates were found to be potent inhibitors to S. aureus, E.
coli and A. hydrophila, while the other derivatives were found to be inactive.
The pseudoalcoholates might be the prodrugs of 1 and 2. The iminium
bond in the molecules of 1 or 2 was the determinant for antibacterial
activity, and the substituents at the 7 and 8 positions influenced the
antibacterial activities of 1 and 2 against different bacteria.
Keywords: sanguinarine;
structure–activity relationship
chelerythrine;
antibacterial
activity;
1. Introduction
Sanguinarine (1) and chelerythrine (2) (Figure 1) belong to the quaternary
benzo[c]phenanthridine alkaloids (QBAs), which are widely distributed in the high
plant families, Papaveraceae, Fumariaceae and Rutaceae (Krane, Fagbule, Shamma,
& Gozler, 1984). The main botanical sources of QBAs are Chelidonium majus L.,
¨
Sanguinaria canadensis L., Dicranostigma lactucoides Hook. f. et Thoms., Macleaya
and Bocconia species from the Papaveraceae family, and some members of
Zanthoxylum (Rutaceae). Although the QBAs are a relatively small class
of plant products, they have attracted much attention from researchers because of
their bioactivities: anti-inflammatory (Lenfeld et al., 1981), antimicrobial (Mitscher
et al., 1978; Navarro, Villarreal, Rojas, & Lozoya, 1996; Odebiyi & Sofowora, 1979),
*Corresponding author. Emails: zhoulechem@yahoo.com.cn; betterzl@163.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2011 Taylor & Francis
DOI: 10.1080/14786419.2010.482055
http://www.informaworld.com

864
F. Miao et al.
O
O
1
12
2
3
O
O
11
12a
13
10
10b
N
4a
4b
9
8
Me
10a
R3O
1a R1=R2=H, R3 + R4 = CH2
4
R2
OR4
N
R1
6a
Me
7
R1O
6
2a R1=R2=H, R3=R4= Me
OR2
2b R1=H, R2=OCH3, R3=R4= Me
1b R1=H, R2=OCH3, R3 + R4 = CH2
1c R1=H, R2=OEt, R3 + R4 = CH2
2c R1=H, R2=OEt, R3=R4= Me
2d R1=H, R2= CH2COCH3, R3=R4= Me
2e R1 + R2 =O, R3=R4= Me
1 R1 + R2 = CH2
2 R1 = R2 = Me
1d R1=H, R= CH2COCH3, R3 + R4 = CH2
1e R1 + R2 =O, R3 + R4 = CH2
Figure 1. Compounds 1, 2 and their derivates 1a–2e.
antitumour (Nakanishi, Suzuki, Mashiba, Ishikawa, & Yokotsuka, 1998; Stermitz,
Larson, & Kim, 1973; Stermitz et al., 1975; Tin-Wa, Bell, Bevelle, Fong, &
Farnsworth, 1974; Zee-Cheng & Cheng, 1975), antiviral (Sethi, 1979), cytotoxic
(Cordell & Farnsworth, 1976) and anti-liver mitochondrial monoamine oxidase
(Iagodina, Nikol’skaia, & Faddeeva, 2003). With respect to the antimicrobial
activities of QBAs, previous research has mainly focused on the total alkaloids
containing QBAs from the plant, while only a few studies on 1 and 2 have been
reported.
QBAs are highly reactive to reductants such as NaBH4 and nicotinamide adenine
dinucleotide (NADH), a biological reducing agent, and nucleophiles such as OHꢀ,
CNꢀ, NH3, ROH and acetone, and are easily converted to their corresponding
derivatives, such as dihydrosanguinarine, 6-hydroxydihydrosanguinarine (pseudo-
base or alkanolamine), aminal, bimolecular aminoacetals and 6-alkoxysanguinarine
(pseudoalcoholate) (Eldin & Jencks, 1995; Nakanishi et al., 2000; Parenty, Smith,
Pickering, Long, & Cronin, 2004; Shimizu, Itou, & Miura, 2005; Yoshida et al.,
1999). On the other hand, the pseudobase, aminal and pseudoalcoholate of QBAs
may be converted to their corresponding iminium ion form in an acidic condition.
Therefore, QBAs have different forms depending on the different solvent conditions,
and these different existing forms will have different effects on the biological activity
of QBAs. It is this reason that led to the conclusion that the results of the previous
research on the biological activities of 1 and 2 were not consistent with each other,
but contradictory. Lenfeld et al. (1981) reported that 2 exhibited significant
antimicrobial effects upon Gram-positive bacteria and Candida albicans, but was
ineffective upon the Gram-negative strains Escherichia coli, Pseudomonas aeruginosa
and Klebsiella pneumoniae (Navarro et al., 1996). But Odebiyi and Sofowora (1979)
proved that 2 was highly active to the Gram-negative strains E. coli, K. pneumoniae,
P. aeruginosa and Proteus sp. Zhao, Yu, Zhou, Meng and Wu (2005) reported that 1
exhibited antibacterial activities against Staphyloccus aureus, E. coli, Tetracoccus,
Bacillus cereus and Bacillus subtilis. Navarro & Delgado (1999) reported that
dihydrosanguinarine (1a) and dihydrochelerythrine (2a) (see Figure 1) displayed
significant antimicrobial activities against S. aureus, Streptococcus faecalis, E. coli,
P. aeruginosa, Proteus mirabilis and C. albicans. However, more recently Zuo et al.
(2008) found that 1a and 2a were almost inactive to S. aureus and that
8-hydroxydihydrosanguinarine and 8-hydroxydihydrochelerythrine were more
active against clinical strains of methicillin-resistant Staphylococcus aureus
(MRSA), evidencing bacteriostatic and bactericidal effects. In addition, Mitscher

Natural Product Research
865
O
O
O
O
O
O
a
b
N+
N
N
Me
R3O
R3O
Me
R3O
Me
OR4
OR4
1a, 2a
OR4
OMe
1b, 2b
1 or 2
c
d
e
O
O
O
O
O
O
N
N
N
Me
R3O
R3O
Me
R3O
Me
OR4
OR4
O
OEt
1c, 2c
OR4
CH2COCH3
1e, 2e
1d, 2d
Scheme 1. (a) NaBH4, MeOH, room temperature; (b) MeONa, reflux; (c) EtONa, EtOH,
reflux; (d) acetone, Na2CO3, reflux; (e) K3Fe (CN)6, 1% HCL/H2O, 90ꢁC.
et al. (1978) reported that the pseudoalcoholates of 1 and 2 also exhibited
antimicrobial activities. In order to clearly understand which forms of 1 and 2 act as
the principal active compounds as well as the structural requirement for antibacterial
activity, in this research, we prepared a series of derivatives of 1 and 2 and examined
their antibacterial activities. As far as we are aware, this is the first report of the
structure–antibacterial activity relationship between 1 and 2.
2. Results and discussion
2.1. Chemistry
The compounds 1, 2 and their derivatives (compounds 1a–1e and 2a–2e) prepared in
this study for bioactivity investigations are shown in Figure 1. Structural modifi-
cations included introduction of alkoxyl and acetonyl at C-6 in 1 and 2 by
nucleophilic addition, and the reduction and oxidation of the C¼N double bond at
C-6 in 1 and 2. The synthesis routes of the derivatives are shown in Scheme 1. The
structures of all of the compounds were elucidated on the basis of spectroscopic
1
evidence, including MS, H-NMR and 13C-NMR (Table 1 and Section 2).
2.2. Bioactivity and discussion
Our previous research showed that the total alkaloids of M. microcarpa exhibited
antibacterial activities against S. aureus, Streptococcus agalactiae, E. coli,
P. multocida, B. cereus, A. hydrophila, Aeromonas punctata f. intestinalis and
Vibrio anguillarum (Hu et al., 2009). Therefore, in this research, S. aureus, E. coli,
A. hydrophila and P. multocida were first selected as the indicator organisms with
which to determine the in vitro antibacterial activities of 1, 2 and 1a–2e. The bioassay
results are summarised in Table 1.
Both 1 and 2 exhibited antibacterial activities against S. aureus, E. coli and
A. hydrophila to different extents at 1.0 mg mLꢀ1, whereas, with respect to

866
F. Miao et al.
Table 1. Antibacterial activity of 1, 2 and their derivates at 1.0 mg mLꢀ1
.
Inhibition zone (mm)
Compound
S. aureus
E. coli
A. hydrophila
P. multocida
1
1a
1b
1c
1d
1e
17.7
11.7
19.3
18.0
–
15.3
–
14.3
14.7
–
9.7
–
8.7
10.3
–
8.7
–
–
–
–
–
–
–
–
2
2a
20.7
–
11.7
–
6.7
–
–
–
2b
2c
2d
2e
17.7
20.0
–
10.7
12.3
–
7.3
7.3
–
–
–
–
–
–
–
–
Penicillin sodium
Ceftriaxone sodium
Control
36.7
13.3
–
14.0
17.3
–
–
18.7
–
–
21.7
–
Note: –, no activity.
P. multocida, only 1 showed a moderate activity among the compounds tested.
Therefore, 1 exhibited a broader antibacterial spectrum than 2.
By comparing the activities of 1 with those of its derivates (1a, 1d and 1e), it can
clearly be seen that the activity reduced or disappeared when the double bond of
C¼Nþ in the molecule of 1 was reduced to a tertiary amine (1a) or oxidised to an
amide (1e), or had acetone added to form a 6-acetonyl-substituted derivate (1d).
Similar results were also obtained in the comparison of the activities of 2 with those
of its derivates (2a, 2d and 2e). The above results strongly suggest that the double
bond of C¼Nþ in the molecules of 1 or 2 is the determinant for their antibacterial
activity. On the other hand, it is worthwhile noting that the 6-methyoxyl or
6-ethyloxyl-substituted derivates (1b, 1c, 2b, 2c) showed good activities, comparable
to those of their corresponding parent compound (1 or 2), despite there being no
occurrence of a C¼Nþ bond in the molecules.
Based on the above results, the minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) values of 1, 1b, 1c, 2, 2b and 2c were
further determined for S. aureus, E. coli and A. hydrophila. The results are listed in
Table 2.
All the tested compounds showed antibacterial activities against S. aureus, E. coli
and A. hydrophila with 12.5–100 mg mLꢀ1 of MIC and bactericidal activities against
S. aureus and E. coli with 25–100 mg mLꢀ1 of MBC. Among all the tested
compounds, only 1 exhibited bactericidal activity against A. hydrophila at
100 mg mLꢀ1. For S. aureus, 2 (MIC 12.5 mg mLꢀ1, MBC 25 mg mLꢀ1) displayed
higher bacteriostatic activity than 1 (MIC 25 mg mLꢀ1) and the same level of
bactericidal activity as 1. For E. coli, 1 showed the same level of bacteriostatic
activity as 2, with 25 mg mLꢀ1 of MIC value, and higher bactericidal activity (MBC
25 mg mLꢀ1) than 2 (MBC 50 mg mLꢀ1). The above results demonstrated that 1 and 2
are effective upon both Gram-positive bacteria and Gram-negative bacteria, which is

Natural Product Research
Table 2. MIC and MBC values (mg mLꢀ1) of 1, 2 and their derivates.
867
S. aureus
E. coli
A. hydrophila
Compound
MIC
MBC
MIC
MBC
MIC
MBC
1
1b
1c
2
2b
25
12.5
25
12.5
25
50
25
25
25
25
25
50
25
50
25
25
50
25
25
100
100
50
100
100
4200
25
25
25
50
50
100
4200
4200
4200
4200
4200
4200
12.5
100
100
4200
2c
Penicillin sodium
Ceftriaxone sodium
3.2
100
6.3
4200
4200
12.5
6.3
a different result from that reported by Lenfeld et al. (1981). On the other hand, the
activity difference between 1 and 2 suggested that the substituents at the 7 and 8
positions influenced the antibacterial susceptibility of 1 and 2 to some extent.
Furthermore, it is worth noting that the 6-alkoxy-substituted derivatives (1b, 1c, 2b
and 2c) showed similar MIC and MBC values to those of their corresponding parent
compounds (1 or 2), in agreement with the results seen in Table 1.
QBAs, unlike other alkaloids, undergo a dynamic equilibrium between the
pseudobase (base form) and iminium ion form in any protic solvent due to the
sensitivity of the polar bond (C¼Nþ) to the attack of nucleophiles (Harkrader &
Jones, 1991; Jones, Harkrader, & Southard, 1986). The pseudobase has higher
lipophilicity than its corresponding iminium ion form. Walterova et al. (1995)
reported that sanguinarine and chelerythrine were able to penetrate mammalian cell
membranes at pH 7.2–7.4 as hydrophobic pseudobases, where they were then able to
accumulate to high levels in the acidic environment of the lysosome and convert back
to the iminium ion. Similarly, the pseudoalcoholates of QBAs could also be easily
converted back to the corresponding iminium form in acidic medium (Dosta
Slavık, 2002). Therefore, we suggest that the pseudoalcoholates (1b, 1c, 2b and 2c), like
´
l &
´
the pseudobases, might be prodrugs of 1 and 2, and that the real active compounds
should be 1 and 2, not the pseudoalcoholates. The viewpoint given above is further
confirmed by the comparison of the activities of the pseudoalcoholates and those of
1a, 2a, 1d, 2d, 1e and 2e. Although 1a, 2a, 1d, 2d, 1e and 2e had similar molecular
structures to the pseudoalcoholates (1b, 1c, 2b and 2c), the former were inactive or less
active because they cannot be converted back to 1 and 2 under physiological
conditions. Zuo et al. (2008) also reported that 1a and 2a did not possess antibacterial
activity. Obviously, the double bond of C¼Nþ in the molecules of 1 and 2 was the
determinant not only for their antitumour properties (Nakanishi, Suzuki, Saimoto, &
Kabasawa, 1999), but also for their antibacterial activities.
3. Experimental
3.1. General experimental procedures
Melting points were taken on a XT-4 microscopic melting point apparatus. NMR
spectra were recorded on a Bruker AM-500 FT-NMR spectrometer using TMS as

868
F. Miao et al.
the internal standard at 500 MHz for 1H-NMR and 125 MHz for 13C-NMR,
respectively. ESI–MS data were obtained with a Trace DSQ spectrometer. TLC was
performed on silica gel GF254 (0–40 mm, Qingdao Haiyang Chemical Group Co.,
Qingdao, China) activated at 110ꢁC for 1 h. The compounds were visualised with UV
light.
3.2. Plant material
Plants of Macleaya microcarpa (Maxim) Fedde were collected from Qingling
Mountain, Shaanxi Province, China, in August 2006, and were identiEed by Vice
Professor Fang Miao, one of the authors. A voucher specimen is deposited at the
Botanic Specimen Centre of Northwest A&F University, Yangling, China.
3.3. Isolation of 1 and 2
The entire plant of M. microcarpa (Maxim) Fedde (500 g, dry weight) was extracted
with 0.1 mol Lꢀ1 aqueous hydrochloric acid at 35ꢁC under an ultrasound-assisted
flow through a column packed with D101 macroporous resin. The column adsorbing
total alkaloids was eluted with water, 50% ethanol in water and finally acetone.
After the solvent was removed at reduced pressure, acetone eluent provided 9.0 g of
fraction B. Fraction B was repeatedly chromatographed on silica gel with petroleum
ether–ethyl acetate (5 : 1) to yield crude 1 and 2, which were subjected to
recrystallisation in 0.1 mol Lꢀ1 aqueous hydrochloric acid to provide 1 (520 mg)
and 2 (610 mg), respectively.
3.4. Syntheses of 1a–2e
Compounds 1 and 2 were used as starting materials to synthesise 1a–1e and 2a–2e,
respectively. Compounds 1a and 2a were synthesised by the literatural method
(Slavik & Slavikova, 1977). Compounds 1b, 1c, 2b and 2c were prepared according to
the method of Stermitz et al. (1973). Compounds 1d and 2d were prepared by
previously described methods (Maclean, Gracey, & Saunders, 1969). Compounds 1e
and 2e were obtained by the oxidation of 1 and 2 with K3Fe (CN)6 (Masayuki,
Kaoru, Kyoko, Naofumi, & Teruo, 1970). The structures of all of the above
compounds were elucidated on the basis of spectroscopic evidence, including MS,
1H-NMR and 13C-NMR.
3.4.1. Syntheses of 1a and 2a
To the solution of 1 or 2 (0.33 mmol) in ca 50 mL of MeOH, 0.5 mmol (20 mg) of
NaBH4 was added. The reaction solution was stirred for 30 min at room
temperature. The solvent was evaporated to dryness under reduced pressure.
The residue was subjected to column chromatography over silica gel using petroleum
ether–ethyl acetate (20 : 1) as eluent, and recrystallised from petroleum ether–ethyl
acetate (20 : 1) to yield 1a or 2a.

Natural Product Research
869
3.4.2. Syntheses of 1b, 1c, 2b and 2c
To the solution of 1 or 2 (0.33 mmol) in ca 40 mL of MeOH, ca 4 mL of the solution
of CH3ONa in MeOH was added for 1b or 2b (or EtONa in EtOH for 1c or 2c) to
pH 10, and refluxed for 40 min. Fifteen millilitres of H2O were added to the reaction
solution, and extracted with CHCl3. The solvent was evaporated to dryness under
reduced pressure, and the residue was recrystallised in MeOH to provide 1b or 2b
(or 1c or 2c).
3.4.3. Syntheses of 1d and 2d
To the solution of 1 or 2 (0.33 mmol) in ca 40 mL of acetone, ca 2 mL of the solution
of 20% Na2CO3 in water was added. The resulting solution was refluxed for 7 h, and
the solvent was then evaporated to dryness under reduced pressure. The residue was
subjected to column chromatography over silica gel using petroleum ether–ethyl
acetate (8 : 1) as eluent, and then recrystallised in a solution of petroleum ether–ethyl
acetate (8 : 1) to yield 1d or 2d.
3.4.4. Syntheses of 1e and 2e
To a hot solution (90ꢁC) of sanguinarine (1) or chelerythrine (2) (0.33 mmol) in
50 mL of 0.2% HCl in water was added a hot solution (80ꢁC) of K3Fe (CN)6 (0.8 g)
in H2O (10 mL) with stirring. Stirring was continued for 3 h at 90ꢁC. During the
reaction, 5 mL of 3% KOH in water was added every 30 min. After cooling, the
precipitate was collected. The solid was dissolved in CHCl3, washed with water
and dried over Na2SO4. The residue was recrystallised from CHCl3–acetone to yield
1e or 2e.
3.4.5. Sanguinarine (1)
Orange–red needle crystal; m.p. 244–245ꢁC (H2O–MeOH); 13C-NMR (125 MHz,
CD3OD) ꢀ ¼ 107.2(C-1), 150.6(C-2), 150.6(C-3), 105.2(C-4), 121.8(C-4a),
133.0(C-4b), 150.7(C-6), 111.1(C-6a), 148.1(C-7), 149.3(C-8), 121.4(C-9),
118.4(C-10), 128.9(C-10a), 127.4(C-10b), 119.8(C-11), 133.0(C-12), 133.9(C-12a),
1
104.3(2,3-OCH2O), 106.5(7,8-OCH2O) and 53.2(N–Me). H-NMR (CDOD3, TMS)
ꢀ ¼ 9.95(1H, s, H-6), 8.57(1H, d, J ¼ 8.9 Hz, H-11), 8.48(1H, d, J ¼ 8.8 Hz, H-10),
8.19(1H, d, J ¼ 8.9 Hz, H-12), 8.13(1H, s, H-4), 7.95(1H, d, J ¼ 8.8 Hz, H-9),
7.55(1H, s, H-1), 6.54(2H, s, 1,2-OCH2O), 6.30(2H, s, 7,8-OCH2O) and 4.49(3H, s,
NCH3); ESI–MS (positive mode) m/z ¼ 332[M]þ.
3.4.6. Chelerythrine (2)
Yellow needle crystal; m.p. 199–200ꢁC (H2O–MeOH); 13C-NMR (125 MHz,
CD3OD) ꢀ ¼ 107.3(C-1), 151.0(C-2), 150.9(C-3), 105.3(C-4), 121.8(C-4a),
133.5(C-4b), 152.1(C-6), 120.1(C-6a), 147.6(C-7), 151.8(C-8), 127.6(C-9),
120.9(C-10), 130.1(C-10a), 127.2(C-10b), 119.7(C-11), 63.2(7-OMe), 57.8(8-OMe),
132.8(C-12), 134.3(C-12a), 104.5(2,3-OCH2O) and 53.2(N–Me). 1H-NMR (CDOD3,
TMS) ꢀ ¼ 9.92(1H, s, H-6), 8.60(1H, d, J ¼ 9.0 Hz, H-10), 8.56(1H, d, J ¼ 9.0 Hz,
H-11), 8.10(1H, d, J ¼ 9.0 Hz, H-12), 8.08(1H, s, H-4), 8.10(1H, d, J ¼ 9.0 Hz, H-9),

870
F. Miao et al.
7.49(1H, s, H-1), 6.26(2H, s, OCH2O), 4.97 (3H, s, NCH3), 4.27(3H, s, 7-OCH3) and
4.12(3H, s, 8-OCH3); ESI–MS (positive mode) m/z ¼ 348[M]þ.
3.4.7. Dihydrosanguinarine (1a)
Yield: 93 mg (92.5%), red–white granule crystal; m.p. 186–187ꢁC (petroleum
ether–ethyl acetate); 13C-NMR (125 MHz, CD3OD) ꢀ ¼ 104.3(C-1), 148.1(C-2),
147.5(C-3), 100.7(C-4), 126.5(C-4a), 142.5(C-4b), 48.4(C-6), 113.6(C-6a), 144.6(C-7),
147.0(C-8), 107.1(C-9), 116.1(C-10), 127.2(C-10a), 124.3(C-10b), 120.3(C-11),
123.9(C-12), 130.8(C-12a), 101.0(2,3-OCH2O), 101.3(7,8-OCH2O), 41.5(N–Me).
1H-NMR (CDOD3, TMS) ꢀ ¼ 7.67(1H, d, J ¼ 8.6 Hz, H-11), 7.66(1H, s, H-4),
7.45(1H, d, J ¼ 8.6 Hz, H-12), 7.28(1H, d, J ¼ 8.1 Hz, H-10), 7.09(1H, s, H-1),
6.83(1H, d, J ¼ 8.1 Hz, H-9), 6.02(2H, s, OCH2O), 6.01(2H, s, OCH2O), 4.17(2H, s,
H-6) and 2.62(3H, s, NCH3); ESI–MS (positive mode) m/z ¼ 333[M]þ.
3.4.8. 6-Methoxysanguinarine (1b)
Yield: 61 mg (50.9%), white crystal; m.p. 194–194.5ꢁC (MeOH); 13C-NMR
(125 MHz, CD3OD) ꢀ ¼ 104.6(C-1), 148.1(C-2), 147.4(C-3), 100.6(C-4), 126.9(C-
4a), 138.2(C-4b), 85.9(C-6), 108.8(C-6a), 145.3(C-7), 147.2(C-8), 113.2(C-9),
116.4(C-10), 125.8(C-10a), 122.8(C-10b), 120.1(C-11), 123.7(C-12), 131.1(C-12a),
101.1(2,3-OCH2O), 101.7(7,8-OCH2O), 40.9(N–Me) and 54.1(6-OMe). 1H-NMR
(CDOD3, TMS) ꢀ ¼ 7.76(1H, d, J ¼ 8.6 Hz, H-11), 7.69(1H, s, H-4), 7.48(1H, d,
J ¼ 8.6 Hz, H-12), 7.20(1H, d, J ¼ 8.2 Hz, H-10), 7.12(1H, s, H-1), 6.93(1H, d,
J ¼ 8.2 Hz, H-9), 6.11(2H, s, OCH2O), 6.04(2H, s, OCH2O), 5.37(1H, s, H-6),
3.46(3H, s, OCH3) and 2.79(3H, s, NCH3); ESI–MS (positive mode) m/z ¼ 363[M]þ.
3.4.9. 6-Ethoxysanguinarine (1c)
Yield: 53 mg (42.6%), white crystal; m.p. 210–212ꢁC (EtOH); 13C-NMR (125 MHz,
CD3OD) ꢀ ¼ 104.6(C-1), 148.0(C-2), 147.4(C-3), 100.7(C-4), 126.9(C-4a),
138.5(C-4b), 84.2(C-6), 108.7(C-6a), 145.2(C-7), 147.3(C-8), 113.4(C-9),
116.4(C-10), 125.8(C-10a), 122.9(C-10b), 120.3(C-11), 123.6(C-12), 131.0(C-12a),
101.0(2,3-OCH2O), 101.7(7,8-OCH2O), 40.9(N–Me), 61.6(OCH2CH3) and
15.0(OCH2CH3). 1H-NMR (CDOD3, TMS) ꢀ ¼ 7.76(1H, d, J ¼ 8.6 Hz, H-11),
7.66(1H, s, H-4), 7.48(1H, d, J ¼ 8.6 Hz, H-12), 7.40(1H, d, J ¼ 8.2 Hz, H-10),
7.12(1H, s, H-1), 6.92(1H, d, J ¼ 8.2 Hz, H-9), 6.11(2H, s, OCH2O), 6.08(2H, s,
OCH2O), 5.48(1H, s, H-6), 3.90–3.93(1H, m, OCH2CH3), 3.65(1H, q, J ¼ 7.0 Hz,
OCH2CH3), 2.76(3H, s, NCH3) and 1.08(3H, t, J ¼ 7.0 Hz, OCH2CH3); ESI–MS
( positive mode) m/z ¼ 377[M]þ.
3.4.10. 6-Acetonylsanguinarine (1d)
Yield: 62 mg (48.3%), yellow–white crystal; m.p. 189–190ꢁC (petroleum ether–ethyl
acetate); Rf ¼ 0.37 (petroleum ether–acetone, 5 : 1), Rf ¼ 0.70 (chloroform–acetone,
19 : 1); 13C-NMR (125 MHz, CD3OD) ꢀ ¼ 104.3(C-1), 148.2(C-2), 147.6(C-3),
100.5(C-4), 127.4(C-4a), 139.2(C-4b), 54.4(C-6), 123.4(C-6a), 144.2(C-7),
147.1(C-8), 107.5(C-9), 116.4(C-10), 125.6(C-10a), 116.0(C-10b), 120.0(C-11),
124.0(C-12), 131.0(C-12a), 101.1(2,3-OCH2O), 101.5(7,8-OCH2O), 43.0(N–Me),

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1
46.5(CH2COCH3), 207.3(CH2COCH3) and 31.7(CH2COCH3). H-NMR (CDOD3,
TMS) ꢀ ¼ 7.69(1H, d, J ¼ 8.4 Hz, H-11), 7.52(1H, s, H-4), 7.48(1H, d, J ¼ 8.4 Hz,
H-12), 7.33(1H, d, J ¼ 8.4 Hz, H-10), 7.10(1H, s, H-1), 6.86(1H, d, J ¼ 8.4 Hz, H-9),
6.04(2H, t, J ¼ 1.2 Hz, OCH2O), 6.03(2H, t, J ¼ 1.2 Hz, OCH2O), 4.87(1H, dd,
J ¼ 4.4, 9.6 Hz, H-6), 2.60–2.66(1H, m, CH2COCH3), 2.62(3H, s, NCH3), 2.30(1H,
dd, J ¼ 3.6, 15.2 Hz, CH2COCH3) and 2.06(3H, s, CH2COCH3); ESI–MS (positive
mode) m/z ¼ 389[M]þ.
3.4.11. Oxysanguinarine (1e)
Yield: 74 mg (64.6%), grey amorphous powder; m.p. 4350ꢁC (chloroform–acetone);
13C-NMR (125 MHz, CD3OD) ꢀ ¼ 104.7(C-1), 147.6(C-2), 147.1(C-3), 102.5(C-4),
121.2(C-4a), 136.7(C-4b), 162.7(C-6), 118.7(C-6a), 147.8(C-7), 147.7(C-8),
115.4(C-9), 118.7(C-10), 128.8(C-10a), 113.2(C-10b), 123.6(C-11), 117.3(C-12),
131.9(C-12a), 101.5(2,3-OCH2O), 102.9(7,8-OCH2O) and 40.8(N–Me). 1H-NMR
(CDOD3, TMS) ꢀ ¼ 7.97(1H, d, J ¼ 8.8 Hz, H-11), 7.76(1H, d, J ¼ 8.7 Hz, H-9),
7.57(1H, d, J ¼ 8.8 Hz, H-12), 7.53(1H, d, J ¼ 8.7 Hz, H-10), 7.23(1H, s, H-4),
7.16(1H, s, H-1), 6.27(2H, s, 7,8-OCH2O), 6.09(2H, s, 2,3-OCH2O) and 3.91 (3H, s,
NCH3); ESI–MS (positive mode) m/z ¼ 347[M]þ.
3.4.12. Dihydrochelerythrine (2a)
Yield: 75 mg (65.1%), yellow–white needle crystal; m.p. 163–164ꢁC (petroleum
ether–ethyl acetate); Rf ¼ 0.34 (petroleum ether–ethyl acetate, 17 : 3), Rf ¼ 0.70
(chloroform–acetone, 20 : 1); 13C-NMR(125 MHz, CD3OD) ꢀ ¼ 104.3(C-1),
148.0(C-2), 147.4(C-3), 100.6(C-4), 126.3(C-4a), 142.6(C-4b), 48.7(C-6),
126.2(C-6a), 146.0(C-7), 152.2(C-8), 110.8(C-9), 118.6(C-10), 126.2(C-10a),
124.2(C-10b), 120.1(C-11), 61.1(7-OMe), 55.7(8-OMe), 123.7(C-12), 130.7(C-12a)
and 101.0(2,3-OCH2O), 40.7(N–Me). 1H-NMR (CDOD3, TMS) ꢀ ¼ 7.70(1H, d,
J ¼ 8.4 Hz, H-11), 7.67(1H, s, H-4), 7.50(1H, d, J ¼ 8.4 Hz, H-10), 7.48(1H, d,
J ¼ 8.4 Hz, H-12), 7.11(1H, s, H-1), 6.93(1H, d, J ¼ 8.4 Hz, H-9), 6.05(2H, s,
OCH2O), 3.94(3H, s, 8-OCH3), 3.87(3H, s, 7-OCH3), 4.30(2H, s, H-6) and 2.59(3H,
s, NCH3); ESI–MS (positive mode) m/z ¼ 349[M]þ.
3.4.13. 6-Methoxychelerythrine (2b)
Yield: 57 mg (45.6%), yellow–white prism; m.p. 199–200ꢁC (MeOH); 13C-NMR
(125 MHz, CD3OD) ꢀ ¼ 104.7(C-1), 148.0(C-2), 147.4(C-3), 100.7(C-4), 126.8(C-4a),
138.4(C-4b), 86.1(C-6), 125.7(C-6a), 146.7(C-7), 152.1(C-8), 113.0(C-9), 118.9(C-10),
124.9(C-10a), 122.6(C-10b), 120.1(C-11), 61.7(7-OMe), 56.0(8-OMe), 123.5(C-12),
131.0(C-12a), 101.0(2,3-OCH2O), 40.6(N–Me) and 54.0(6-OMe). 1H-NMR
(CDOD3, TMS) ꢀ ¼ 7.77(1H, d, J ¼ 8.5 Hz, H-11), 7.69(1H, s, H-4), 7.62(1H, d,
J ¼ 8.6 Hz, H-10), 7.47(1H, d, J ¼ 8.5 Hz, H-12), 7.12(1H, s, H-1), 7.04(1H, d,
J ¼ 8.6 Hz, H-9), 6.05(2H, s, OCH2O), 5.54(1H, s, H-6), 3.96(3H, s, 8-OCH3), 3.92
(3H, s, 7-OCH3), 3.45(3H, s, 6-OCH3) and 2.60(3H, s, NCH3); ESI–MS (positive
mode) m/z ¼ 379[M]þ.

872
F. Miao et al.
3.4.14. 6-Ethoxychelerythrine (2c)
Yield: 76 mg (61.1%), yellow–white prism; m.p. 206–206.5ꢁC (EtOH); 13C-NMR
(125 MHz, CD3OD) ꢀ ¼ 104.6(C-1), 147.8(C-2), 147.3(C-3), 100.7(C-4), 126.8(C-4a),
138.7(C-4b), 84.5(C-6), 126.0(C-6a), 146.6(C-7), 152.2(C-8), 112.9(C-9), 119.0(C-10),
124.9(C-10a), 122.7(C-10b), 120.1(C-11), 61.7(7-OMe), 56.0(8-OMe), 123.3(C-12),
131.0(C-12a),
101.0(2,3-OCH2O),
40.7(N–Me),
61.6(OCH2CH3)
and
15.2(OCH2CH3). 1H-NMR (CDOD3, TMS) ꢀ ¼ 7.76(1H, d, J ¼ 8.5 Hz, H-11),
7.66(1H, s, H-4), 7.62(1H, d, J ¼ 8.6 Hz, H-10), 7.45(1H, d, J ¼ 8.5 Hz, H-12),
7.12(1H, s, H-1), 7.02(1H, d, J ¼ 8.6 Hz, H-9), 6.04 (2H, s, OCH2O), 5.67(1H, s,
H-6), 3.96(3H, s, 8-OCH3), 3.92(3H, s, 7-OCH3), 3.72(2H, q, J ¼ 7.0 Hz, OCH2CH3),
2.74(3H, s, NCH3) and 1.09(3H, t, J ¼ 7.0 Hz, OCH2CH3); ESI–MS (positive mode)
m/z ¼ 393[M]þ.
3.4.15. 6-Acetonylchelerythrine (2d)
Yield: 89 mg (66.6%), yellow–white crystal; m.p. 196–197ꢁC (petroleum ether–ethyl
acetate); 13C-NMR (125 MHz, CD3OD) ꢀ ¼ 104.3(C-1), 148.2(C-2), 147.6(C-3),
100.6(C-4), 128.2(C-4a), 139.3(C-4b), 54.9(C-6), 127.4(C-6a), 145.5(C-7), 152.1(C-8),
111.6(C-9), 118.8(C-10), 124.8(C-10a), 123.3(C-10b), 119.8(C-11), 61.0(7-OMe),
55.8(8-OMe), 123.8(C-12), 131.1(C-12a), 101.0(2,3-OCH2O), 42.8(N–Me),
1
46.9(CH2COCH3), 207.5(CH2COCH3) and 31.1(CH2COCH3). H-NMR (CDOD3,
TMS) ꢀ ¼ 7.70(1H, d, J ¼ 8.6 Hz, H-11), 7.53(1H, d, J ¼ 8.4 Hz, H-10), 7.51(1H, s,
H-4), 7.47(1H, d, J ¼ 8.6 Hz, H-12), 7.09(1H, s, H-1), 6.94(1H, d, J ¼ 8.6 Hz, H-9),
6.02(2H, t, J ¼ 1.2 Hz, OCH2O), 5.04(1H, dd, J ¼ 3.6, 11.1 Hz, H-6), 3.96(3H, s,
8-OCH3), 3.92(3H, s, 7-OCH3), 2.64(3H, s, NCH3), 2.57(1H, t, J ¼ 3.6 Hz,
CH2COCH3), 2.25(1H, dd, J ¼ 3.6, 11.1 Hz, CH2COCH3) and 2.05(3H, s,
CH2COCH3); ESI–MS (positive mode) m/z ¼ 405[M]þ.
3.4.16. Oxychelerythrine (2e)
Yield: 37 mg (30.9%), silver grey amorphous powder; m.p. 198–199ꢁC
(chloroform–acetone); 13C-NMR (125 MHz, CD3OD) ꢀ ¼ 104.7(C-1), 152.7(C-2),
150.2(C-3), 102.5(C-4), 121.0(C-4a), 135.6(C-4b), 162.6(C-6), 119.8(C-6a),
147.5(C-7), 147.1(C-8), 117.9(C-9), 118.5(C-10), 129.0(C-10a), 117.2(C-10b),
123.2(C-11),
61.7(7-OMe),
56.6(8-OMe),
117.8(C-12),
131.7(C-12a),
101.5(2,3-OCH2O) and 40.8(N–Me). 1H-NMR (CDOD3, TMS) ꢀ ¼ 7.97(1H, d,
J ¼ 8.5 Hz, H-11), 7.97(1H, d, J ¼ 8.5 Hz, H-9), 7.51(1H, d, J ¼ 8.5 Hz, H-12),
7.37(1H, d, J ¼ 8.5 Hz, H-10), 7.15(1H, s, H-4), 7.13(1H, s, H-1), 6.08(2H, s,
OCH2O), 4.08(3H, s, 8-OCH3), 3.98(3H, s, 7-OCH3) and 3.89(3H, s, NCH3);
ESI–MS (positive mode) m/z ¼ 363[M]þ.
3.5. Bioactivity assay
3.5.1. Screening of antibacterial activity of 1, 2 and their derivatives (1a–1e, 2a–2e)
Compounds 1, 2 and their derivatives were screened for their antibacterial activity
using the paper disc diffusion method (Iwasal, Kamigauchi, Ueki, & Taniguchi, 1996),
with slight modification. S. aureus, E. coli, Aeromonas hydrophila and Pasteurella

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873
multocida were used as the test bacteria. The test compound was dissolved in MeOH at
a concentration of 1 mg mLꢀ1 and a paper disc (d ¼ 6 mm) was immersed in the sample
solution for 3 h, and then dried in an oven at 40ꢁC and placed on an agar plate seeded
with the 20–24 h cultured fresh bacteria at 28ꢁC. After incubation for 24 h at 37ꢁC, the
diameter of the inhibitory zone around the disc was measured. A paper disc saturated
with MeOH was used, after drying, as a negative control. Penicillin sodium and
ceftriaxone sodium were used as positive controls.
3.5.2. Determination of MIC and MBC
MIC and MBC values of 1, 2, 1b, 1c, 2b and 2c were determined by the turbidity
method (Urzu´ a et al., 2006) with slight modification, using S. aureus, E. coli and A.
hydrophila as the test bacteria.
A Baird–Parker agar (BPA) broth consisting of beef extract (0.3%), peptone (1%)
and NaCl (0.5%) was used for culturing the test bacteria. A 20–24 h cultured fresh
broth (E. coli and S. aureus at 37ꢁC for 20 h and A. hydrophila at 28ꢁC for 24 h) was
diluted by the same broth to 2.2 ꢂ 107 colony forming units (CFU) for S. aureus,
3.6 ꢂ 107 CFU for E. coli and 2.3 ꢂ 108 CFU for A. hydrophila to serve as inocula.
The test sample was dissolved in methanol, and 20 mL of the resulting solution was
added to a first tube containing 980 mL of BPA broth. Two-fold serial dilutions were
made by adding the same BPA broth to obtain concentrations of 400–3.12 mg mLꢀ1
.
A bacterial suspension (0.5 mL) was added to each tube and incubated for 24 h at
37ꢁC for S. aureus and E. coli, and for 24 h at 28ꢁC for A. hydrophila. The MIC was
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 of
the MIC determination. After the bacterial suspensions had fully absorbed into the
agar, the plates were further incubated at 28ꢁC for 24 h and were examined for
growth in daylight. The MBC was defined as the concentration at which no colony
was observed after incubation. All experiments were performed in triplicate.
Acknowledgement
This project was supported by the National Natural Science Foundation of China (NNSF;
nos. 30571402, 30771454).
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