In vitro antifungal activity of sanguinarine and chelerythrine derivatives against phytopathogenic fungi

Article abstract of DOI:10.3390/molecules171113026

In order to understand the antifungal activity of some derivatives of sanguinarine (S) and chelerythrine (C) and their structure-activity relationships, sixteen derivatives of S and C were prepared and evaluated for in vitro antifungal activity against seven phytopathogenic fungi by the mycelial growth rate method. The results showed that S, C and their 6-alkoxy dihydro derivatives S₁-S₄, C₁-C₄ and 6-cyanodihydro derivatives S₅, C₅ showed significant antifungal activity at 100 μg/mL against all the tested fungi. For most tested fungi, the median effective concentrations of S, S₁, C and C₁ were in a range of 14-50 μg/mL. The structure-activity relationship showed that the C=N⁺ moiety was the determinant for the antifungal activity of S and C. S₁-S₅ and C ₁-C₅ could be considered as the precursors of S and C, respectively. Thus, the present results strongly suggested that S and C or their derivatives S₁-S₅ and C₁-C₅ should be considered as good lead compounds or model molecules to develop new anti-phytopathogenic fungal agents.

Full text of DOI:10.3390/molecules171113026

Molecules 2012, 17, 13026-13035; doi:10.3390/molecules171113026
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
In Vitro Antifungal Activity of Sanguinarine and Chelerythrine
Derivatives against Phytopathogenic Fungi
Xin-Juan Yang 1,2,†, Fang Miao 2,†, Yao Yao 1, Fang-Jun Cao 1, Rui Yang 1, Yan-Ni Ma 1,
Bao-Fu Qin 2 and Le Zhou 1,*
1
College of Science, Northwest A&F University, Yangling 712100, Shaanxi, China;
E-Mails: yxjsn2@163.com (X.-J.Y.); mumu2006yo@126.com (Y.Y.);
caofangjun@yahoo.com.cn (F.-J.C.); yrer@126.com (R.Y.); ni-2003@163.com (Y.-N.M.)
2
College of Life Science, Northwest A&F University, Yangling 712100, Shaanxi, China;
E-Mails: miaofangmf@163.com (F.M.); baofu_qin@yahoo.com.cn (B.-F.Q.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: zhoulechem@yahoo.com.cn;
Tel.: +86-029-8709-2048; Fax: +86-029-8709-2226.
Received: 8 October 2012; in revised form: 29 October 2012 / Accepted: 30 October 2012 /
Published: 2 November 2012
Abstract: In order to understand the antifungal activity of some derivatives of
sanguinarine (S) and chelerythrine (C) and their structure-activity relationships, sixteen
derivatives of S and C were prepared and evaluated for in vitro antifungal activity against
seven phytopathogenic fungi by the mycelial growth rate method. The results showed that
S, C and their 6-alkoxy dihydro derivatives S1–S4, C1–C4 and 6-cyanodihydro derivatives
S5, C5 showed significant antifungal activity at 100 µg/mL against all the tested fungi. For
most tested fungi, the median effective concentrations of S, S1, C and C1 were in a range of
14–50 µg/mL. The structure-activity relationship showed that the C=N+ moiety was the
determinant for the antifungal activity of S and C. S1–S5 and C1–C5 could be considered as
the precursors of S and C, respectively. Thus, the present results strongly suggested that S
and C or their derivatives S1–S5 and C1–C5 should be considered as good lead compounds
or model molecules to develop new anti-phytopathogenic fungal agents.
Keywords: sanguinarine; chelerythrine; quaternary benzo[c]phenanthridine alkaloids;
antifungal activity; phytopathogenic fungi

Molecules 2012, 17
13027
1. Introduction
The continuing development of fungicidal resistance in plant and human pathogens necessitates the
discovery and development of new fungicides. In the past decades, natural product-based plant
protectants have attracted a lot of attention from researchers owing to the fact they are perceived to
have lower environmental and mammalian toxicity [1].
Natural quaternary benzo[c]phenanthridine alkaloids (QBAs) constuitute a relative small class of
isoquinoline alkaloids that nevertheless are widely distributed in the higher plant families Fumariaceae,
Papaveraceae and Rutaceae [2,3]. Among QBAs, sanguinarine (S) and chelerythrine (C) (Figure 1) are
the most common and their richest natural sources are the plants Sanguinaria Canadensis L.,
Dicranostigma lacucoides Hook.f. & T. Thoms., Chelidonium majus L., Macleaya, Bocconia species
from the Papaveraceae family and some members of Zanthoxylum (Rutaceae). In the past decades,
QBAs had attracted much attention from investigators because of their extensive and important
bioactivities, which include antitumour [4,5], antimicrobial [6–9], anti-inflammatory [10], antiviral [11],
anti-HIV [12], antiparasitic action against Trichodina sp. [13], Dactylogyrus intermedius [14] and
malaria [15], anti-platelet aggregation [16], anti-angiogenesis [17] and anti-acetylcholinesterase
properties [18]. We recently also found that 1-alkoxydihydro derivatives of S had significant acaricidal
activity against Psoroptes cuniculi [19].
Figure 1. Structures of sanguinarine (S) and chelerythrine (C).
1
4
12
12a
2
3
O
O
O
O
11
13
10
10b
4a
9
8
10a
4b
N
N
6a
H3CO
Me
Me
O
7
6
OCH3
O
14
S
C
Previous research had proven that S and C had significant activities against plant microbial
pathogens. As early as in the period of 1939 to 1973, S had been found to be fungistatic on several plant
fungal pathogens including Phytomatotrichum omnivorum, Sclerotium rolfisii, Gaeumannomyces grminis,
Rhizoctonia solani, Amillaria mellea, Fusarium oxysyporum and Verticillium alboatrum [20–22]. In
1999, Matos et al. demonstrated that S and C were the active antifungal components of the plant
extracts from Chelidonium majus L. or Macleaye cordata (Willd) R. Br. against 13 strains of
phytopanthogenic fungi from the genus Fusarium [23]. The results were further confirmed by Liu et al.
using bioassay-guided fractionation of the extract of Macleaya cordata R. Br. [24]. In addition, the
extract from Macleaye cordata (Willd) R. Br. was able to effectively control powdery mildew
(Sphaerotheca pannosa var. rosae) of greenhouse roses in vivo [25]. However, until now the activity of
the derivatives of S and C against phytopathogenic fungi and their structure-activity relationships were
not reported. The objective of the present study was to systematically evaluate the anti-
phytopanthogenic fungal activity of a series of the derivatives of S and C and understand their
structure-activity relationships.

Molecules 2012, 17
13028
2. Results and Discussion
2.1. Chemistry
Compounds S and C were obtained by isolation from the entire plant of Macleaya microcarpa
(Maxim) Fedde according to the method reported by us [26] and used as the starting materials to
synthesize S1–S8 and C1–C8 (Figure 2), respectively, using the synthetic route outlined in Scheme 1.
Figure 2. Derivatives of sanguinarine S1–S8 and chelerythrine C1–C8.
O
O
O
O
N
N
MeO
Me
Me
O
R'
R'
MeO
O
R
R
S1
S
C1 C
-
-
8
8
S1 R = H, R' = MeO
S2 R = H, R' = EtO
C1 R = H, R' = MeO
C2 R = H, R' = EtO
C3
R = H, R' = PrO
S3
R = H, R' = PrO
S4 R = H, R' = iso-PrO
S5 R = H, R' = CN
S6
R = H, R' = acetonyl
R = R' = H
S7
S8 R + R' = O
C8 R + R' = O
Scheme 1. Synthetic pathway for compounds S1–S8 and C1–C8.
Reagents and conditions: (a) NaBH4, MeOH, r.t.; (b) R'OH, CuCl2·2H2O, 80 °C, 10 h; (c) NaCN,
EtOH, r.t.; (d) Acetone, Na2CO3, reflux; (e) K3Fe(CN)6, 1% HCl/H2O, 90 °C.
Structural modifications of S and C included introduction of alkoxyl, cyano and acetonyl at C-6 by
nucleophilic addition, and the reduction and oxidation of the C=N double bond at the C-6 position.
Compounds S1–S5/C1–C5 and S6–S8/C6–C8 were prepared according to the methods recently reported
by us [19,26].
1
The structures of all compounds were elucidated by spectroscopic analyses including ESI-MS, H-
13
and C-NMR spectra. The spectral data of all the compounds were in agreement with that previously
reported by us [19,26]. It should be noted that S1–S6 and C1–C6 were racemates and used as such for

Molecules 2012, 17
13029
the bioactivity assays. We tried to resolve the racemates to their enantiomers but were unsuccessful.
The main reason is that these compounds very easily convert back to their corresponding parent
compounds S or C under acidic or even weakly acidic conditions.
2.2. Antifungal Activity
2.2.1. Screening of Antifungal Activity in Vitro
The in vitro antifungal activities of S, C and their derivatives at a concentration of 100 μg/mL were
assayed by the linear growth rate method. Thiabendazole (TBZ), a commercial fungicide, was used a
control. The results are shown in Table 1.
Table 1. Linear Growth inhibitory rates (means ± S.D.%) of 18 compounds against seven
phytopathogenic fungi (100 µg/mL).
Seven Tested Phytopathogenic Fungi *
Compd.
C.L.
V.M.
F.S.
F.O.N.
F.O.V.
P. O.
A.A.
S
S1
78.6 ± 3.4DE **
80.8 ± 2.0BCDE
87.5 ± 2.8A
84.8 ± 0.8BC 81.4 ± 1.0BC
88.1 ± 0.4AB 83.8 ± 2.6BC
72.1 ± 3.1E
86.6 ± 1.5C
69.7 ± 1.7E
87.6 ± 2.3C
79.6 ± 5.4D
63.2 ± 0.0F
−0.5 ± 0.9K
2.0 ± 3.4JK
0.5 ± 0.9JK
85.6 ± 0.9C
94.0 ± 0.0B
82.0 ± 2.2D
84.0 ± 0.0CD
85.6 ± 1.6CD
87.2 ± 1.6C
84.5 ± 1.0CD
71.6 ± 0.7E
−4.4 ± 0.9J
−0.1 ± 1.8HI
2.6 ± 3.9H
60.4 ± 2.1D
70.8 ± 2.1C
70.1 ± 3.2C
69.4 ± 1.2C
61.1 ± 1.2D
47.0 ± 0.6E
9.7 ± 1.2HI
20.8 ± 0.0G
5.8 ± 1.2IJ
74.3 ± 1.2BC
71.2 ± 0.0C
74.6 ± 0.9C
70.0 ± 2.3C
73.6 ± 0.9C
64.9 ± 1.5D
44.9 ± 1.6E
16.2 ± 1.5H
21.2 ± 0.9G
11.0 ± 1.5I
83.7 ± 1.8B
S2
85.8 ± 5.9BC
93.2 ± 0.6A
85.4 ± 2.6B
S3
86.1 ± 0.8AB
78.1 ± 1.5DE
79.7 ± 0.8CDE
19.1 ± 2.0HI
33.0 ± 1.3F
86.9 ± 4.6AB
S4
84.6 ± 1.8BC 82.0 ± 2.1BC
S5
80.1 ± 0.4C
−4.0 ± 3.5FG
−8.3 ± 1.7H
3.4 ± 0.7DE
73.3 ± 0.0D
−0.1 ± 3.2F
7.5 ± 1.9E
6.3 ± 0.7E
S6
S7
S8
18.0 ± 1.3I
C
81.5 ± 2.2BCDE
79.9 ± 3.5CDE
76.3 ± 0.8E
88.5 ± 2.0AB 87.3 ± 0.2AB
93.0 ± 1.7A 92.4 ± 0.4A
94.2 ± 0.0B
C1
100.0 ± 0.0A 77.1 ± 2.1AB 85.8 ± 0.9AB
C2
86.9 ± 0.4AB 83.4 ± 0.2BC 100.0 ± 0.0A 95.7 ± 3.7AB 79.9 ± 1.2AB 86.8 ± 0.9AB
C3
85.7 ± 5.1ABC
89.8 ± 2.1AB 86.4 ± 6.4AB
94.0 ± 0.0B
100.0 ± 0.0A
92.0 ± 0.0B
69.1 ± 1.1E
−3.3 ± 3.3IJ
12.7 ± 2.4F
6.8 ± 0.9G
81.9 ± 2.4A
83.0 ± 0.6A
48.4 ± 1.1E
1.4 ± 2.4J
87.3 ± 0.9AB
88.8 ± 0.9A
62.5 ± 0.8D
5.5 ± 1.5J
C4
82.6 ± 4.0ABCD 83.5 ± 0.8BC 82.2 ± 0.9BC 90.5 ± 0.9BC
C5
80.8 ± 0.3BCDE
5.7 ± 3.4J
84.4 ± 0.4BC 78.7 ± 0.4CD
40.2 ± 3.4G
5.0 ± 0.9IJ
13.4 ± 0.0H
9.5 ± 4.8HI
C6
−1.4 ± 2.8EF
5.1 ± 6.9D
8.6 ± 3.9E
10.5 ± 3.2E
10.5 ± 1.3E
C7
18.0 ± 1.3I
28.9 ± 5.9F
11.9 ± 1.3H
47.9 ± 5.9E
24.7 ± 1.8G
30.6 ± 4.0F
C8
24.0 ± 2.0GH
27.1 ± 1.5G
−5.2 ± 2.4FG
TBZ ***
88.4 ± 0.8AB 83.9 ± 3.5BC 100.0 ± 0.0A 100.0 ± 0.0A 15.3 ± 6.7GH
* C.L.: Curvularia lunata; V.M.: Valsa mali; F.S.: Fusarium solani; F.O.V.: Fusarium oxysporum f. sp.
vasinfectum; A.A.: Alternaria alternate; P.O.: Pyricularia oryza; F.O.N.: Fusarium oxysporum sp. niveum. ** The
differences between data with different capital letters within a column are significant for the same tested fungus
(p < 0.01) with respect to S and its pseudoalcoholates or C and its pseudoalcoholates. *** TBZ: thiabendazole.
The results in Table 1 show that among all the derivatives only the 6-alkoxydihydro derivatives S1–S4,
C1–C4 and 6-cyanodihydro derivatives S5, C5 displayed significant activities (40.2%–100% inhibitory
rate) against all seven tested fungi at 100 µg/mL. On the contrary, the other derivatives S6–S8, C6–C8
gave lower or no activity at the same concentration. With respect to C. lunata, P. oryzae and
A. alternate, S, C and all of their pseudoalcoholates (compounds S1–S4, C1–C4) were much more

Molecules 2012, 17
13030
active than TBZ, a commercial fungicide (p < 0.01). For V. mali and F. solani, S, S1–S4, C and C1–C4
showed the same activities as TBZ (p > 0.01). For F. oxysporum sp. niveum, F. oxysporum f. sp.
vasinfectum, P. oryza, and A. alternate, the activities of C and its pseudoalcoholates C1–C4 were
significantly stronger than that of S and its corresponding pseudoalcoholates S1–S4 (p < 0.01). However,
for the other three fungi, no significant differences between S or S1–S4 and C or C1–C4 was observed
(p > 0.01). In addition, in most cases, there was also no significant difference between the activities of
pseudoalcoholates S1–S4 or C1–C4 and their corresponding parent compound S or C and between the
different pseudoalcoholates S1–S4 or C1–C4 from the same parent compound S or C (p < 0.01).
2.2.2. Antifungal Toxicity
Based on the results above, S1 and C1 were used as representative pseudoalcoholates to further
determine their regression equations and median effective concentration (EC50 values) towards the
seven tested fungi. S and C as parent compounds were used as control. The results are listed
in Table 2. The inhibition rates of the four compounds increased as the concentration increased. All the
compounds showed a significant linear correlation between the inhibition rate and log[concentration]
value (R2 values = 0.9193–0.9925, p < 0.01). C and C1 gave the highest activity against C. lunata with
EC50 values of 15.43 µg/mL (32.5 µM) and 14.23 µg/mL (37.5 µM), respectively, and S and S1
showed the lowest activity against P. oryza with EC50 values of 101.6 µg/mL (2291.2 µM) and
96.63 µg/mL (265.9 µM). The other EC50 values were in a range of 24.28 to 53.8 µg/mL. Comparison
of the EC50 values of the four compounds for the same fungus showed that C and C1 were more active
than S and S1 for C. lunata, F. solani, F. oxysporum sp. niveum, P. oryzae and A. alternate. However,
for F. oxysporum f. sp. vasinfectum and V. mali, S and S1 were more active than C and C1. These
results were not exactly the same as those obtained from Table 1. This is because the various
compounds had different slope values. On the other hand, compared the EC50 value (µM) of S (or C)
with that of S1 (or C1) for the same fungi, it was found that in most cases S (or C) was slightly more
active than its correspongding pseudoalcoholate S1 (or C1).
Besides EC50 value, the slope value (k) in a toxicity regression equation is also an important factor
for evaluation of bioactivity of a compound. A slope value reflects the concentration effect (CE) of a
compound on its bioactivity. The k values in Table 2 showed that the CEs of S, S1, C and C1 on the
activities against the seven fungi were different. In order to comprehensively compare the activities of
the different compounds, the value of k/EC50 (nM) of each the compound, here named as
comprehensive activity (CA), was calculated and shown in Table 2. Comparing the CA values of the
compounds for each fungus, it may be obviously seen that for most of the fungi both C and C1 were
more active than S or S1. However, the CAs of S or C were close to that of its pseudoalcoholate S1 or
C1. This conclusion is basically consistent with that obtained from Table 1.

Molecules 2012, 17
13031
Table 2. Toxicity regression equations and EC50 values of compounds S, C, S1 and C1 against seven fungi.
EC50 value
(µg/mL)
Toxicity regression
equation *
CI 95% **
(µg/mL)
Fungus Compd.
R2
CA ***
(µM)
76.6
79.4
32.5
37.5
52.9
67.5
82.9
77.4
88.3
96.4
55.8
71.8
106.2
90.2
61.7
74.6
72.3
95.5
109.1
79.0
221.2
265.9
101.9
115.3
115.6
119.6
S
y = 0.5758x − 0.3905
y = 0.5452x − 0.2960
y = 0.3413x + 0.0944
y = 0.3635x + 0.0808
y = 0.6317x − 0.3751
y = 0.5275x − 0.2329
y = 0.6918x − 0.6038
y = 0.6420x − 0.4423
y = 0.7415x − 0.6922
y = 0.5949x − 0.4189
y = 0.5005x − 0.2123
y = 0.5949x − 0.3540
y = 0.5139x − 0.3676
y = 0.5487x − 0.3317
y = 0.6186x − 0.4076
y = 0.6629x − 0.4624
y = 0.6007x − 0.4137
y = 0.6320x − 0.4735
y = 1.0309x − 1.2677
y = 0.6303x − 0.4308
y = 0.3182x − 0.1386
y = 0.4238x − 0.3413
y = 0.7447x − 0.7550
y = 0.7178x − 0.6779
y = 0.7921x − 0.8663
y = 0.5176x − 0.3453
0.9628
0.9848
0.9621
0.9914
0.9901
0.9860
0.9505
0.9904
0.9717
0.957
35.20
28.84
15.43
14.23
24.28
24.51
39.40
29.36
40.53
35.04
26.50
27.26
48.79
32.79
29.32
28.30
33.20
34.70
51.84
29.98
101.6
96.63
48.44
43.75
53.08
43.47
35.13–35.27
28.81–28.85
15.21–15.65
14.18–14.28
24.24–24.32
23.97–25.05
39.30–39.50
29.34–29.38
40.46–40.60
34.95–35.13
26.41–26.59
27.24–27.28
48.73–48.85
32.73–32.85
29.30–29.34
28.26–28.34
33.14–33.26
34.66–34.74
51.80–51.88
29.91–30.06
101.2–102.00
96.61–96.63
48.41–48.47
43.72–43.78
53.03–53.13
43.10–43.84
16.4
18.9
22.1
25.5
26.0
21.5
17.6
21.9
18.3
17.0
18.9
21.8
10.9
16.7
21.1
23.4
18.1
18.2
19.9
21.0
3.1
S1
C.L.
C
C1
S
S1
V.M.
C
C1
S
S1
F.S.
C
0.9600
0.9925
0.9768
0.9705
0.9891
0.9836
0.9735
0.9826
0.9600
0.9684
0.9687
0.9914
0.9727
0.9843
0.9608
0.9193
C1
S
S1
F.O.N.
C
C1
S
S1
F.O.V.
C
C1
S
S1
4.4
P.O.
C
15.4
16.4
14.9
11.9
C1
S
S1
A.A.
C
y = 0.9270x − 1.0756
y = 0.6218x − 0.4104
0.9572
0.9659
50.08
29.11
105.4
76.7
50.03–50.13
29.04–29.18
18.5
21.4
C1
* y: Inhibitory rate. x: Log10[concentration(mg/L)]; ** CI 95%: Confidence interval at 95% probability
(µg/mL). *** Slope value/EC50 (μg/mL) × 1,000.
2.3. Structure-Activity Relationship
Unlike the parent compounds S or C, all the derivatives of S or C lack the C=N+ moiety, and their
only structural difference lies in their different substituents at the 6 position. S7 and C7 were the
reduction products of S and C, respectively. The significant difference of the activities between S and
S7 or C and C7 showed that the iminium moiety in the molecule S or C is the determinant for their
antifungal activity.
S1–S4 or C1–C4 have N,O-acetal structures and both S5 and C5 are structurally α-hydroxynitrile
derivatives. All these compounds are able to easily convert back to the corresponding parent
compound S or C under acidic conditions [27,28], including the slightly acidic environment of the
lysosome of cells [29]. Unlike S1–S5 and C1–C5, compounds S6–S8 and C6–C8 could not undergo a
similar transformation under the same conditions. The significant difference in activities between S1–S5
and S6–S8 or between C1–C5 and C6–C8 as well as the lack of a significant difference between the of

Molecules 2012, 17
13032
S1–S5 or C1–C5 strongly suggest that the activities of S1–S5 and C1–C5 might well come from their
corresponding hydrolytic products, i.e., S or C. In other words, S and C might be the real active
compounds of S1–S5 and C1–C5, respectively, while S1–S5 and C1–C5 might be only the precursors of
S and C. In addition, comparing the activities of C with that of S or C1–C5 with S1–S5, we could find
that for most of the fungi, a 7,8-methoxy group, relative to a 7,8-methylenedioxy group, could enhance
the activity to a certain degree. It was worth noting that the structure and antifungal activity
relationships were very similar to the case of their reported antibacterial activities [26].
3. Experimental
3.1. General
Sanguinarine iodide (S) and chelerythrine iodide (C) were obtained in our laboratory by isolation
from the whole plant of M. microcarpa (Maxim) Fedde [24]. Melting points (m.p.) were determined on
1
XT-4 micro-melting point apparatus and are uncorrected. H-NMR and 13C-NMR spectra were
recorded with a Bruker AVANCE Ⅲ instrument operating at 500 and 125 MHz, respectively, using
TMS as an internal standard. ESI-MS was measured on a Trace mass spectrometer. Thiabendazole
(TBZ, ≥99.1%), a commercial fungicide standard, was purchased from Sigma-Aldrich Trading Co. Ltd.
(Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from J&K China Chemical Ltd.
(Beijing, China). Other reagents were obtained locally and of analytical grade. The water used was
redistilled and ion-free. Curvularia lunata, Alternaria alternate, Fusarium solani, Fusarium
oxysporum sp. vasinfectum, Valsa mali, Fusarium oxysporum sp. niveum and Pyricularia oryzae were
isolated, identified and provided by the Center of Pesticide Research, Northwest A&F University,
(Shaanxi, China). These fungi were grown on PDA plates at 28 °C and maintained by periodic
subculturing at 4 °C.
3.2. Synthesis of S1–S8 and C1–C8
Compounds S1–S5 and C1–C5 were prepared according to the methods and the procedure recently
reported by us [19]. Compounds S6–S8 and C6–C8 were prepared the procedure according to the
methods previously reported by us [26]. The spectral data of S1–S8 and C1–C8 are consistent with our
previous papers [19,26].
3.3. Screening of Antifungal Activity in Vitro
The antifungal activity in vitro was assayed by the growth rate method [30] with slight
modifications. The tested fungi maintained on potato-dextrose-agar (PDA) medium slants were
subcultured for 48 h in Petri dishes prior to testing and used for inoculation of fungal strains on PDA
plates. The tested compounds were completely dissolved in DMSO, and then diluted by water to
provide the stock solution (1.0 µg/mL) in 5% DMSO aqueous solution. The stock solution was
completely mixed with the autoclaved PDA medium to provide a medium containing 100 µg/mL of
sample and then poured into the Petri dishes in a laminar flow chamber. Thiabendazole solution
(1.0 µg/mL) in 5% DMSO aqueous solution and 5% DMSO aqueous solution were used as the treated
control and the untreated control, respectively. When the medium in the plates was partially solidified,

Molecules 2012, 17
13033
a 5-mm thick and 4-mm diameter disc of fungus cut from earlier subcultured Petri dishes was placed at
the centre of the semi-solid medium. The dishes were kept in an incubator at 28 °C for 72 h. Each
experiment was carried out in triplicates. The diameters (in mm) of inhibition zones were measured in
three different directions and the growth inhibition rates were calculated according to the following
formula and expressed as means ± S.D.:
Growth inhibition rate (%) = [(dc − d0) − (ds − d0)]/(dc − d0) × 100
where d0: Diameter of the fungus cut, dc: Diameter of the untreated control fungus, ds: Diameter of the
sample-treated fungus.
3.4. Toxicity Assays
Based on in vitro antifungal activity screening results, S, S1, C and C1 were selected to determine
their toxicity. According to the method described above, a stock solution containing 800 μg/mL
samples was prepared in 5% DMSO aqueous solution. Then the stock solution was completely mixed
with the autoclaved PDA medium to prepare a set of media containing 120, 90, 80, 40, 20, 10, 5 and
2.5 μg/mL samples, respectively. 5% DMSO aqueous solution was used as the untreated control.
According to the method described above, the antifungal activity for each concentration was
determined. Each experiment was carried out in triplicate. The average inhibition rate for each test was
calculated. The concentration of the compound was transformed to the corresponding logarithm value
(log10C). A linear regression, namely toxicity regression equation, was established by using the least
square method. EC50 values were calculated from the toxicity regression equation.
3.5. Statistic Analysis
SPSS statistics V17.0 software was used to analyze the data and establish toxicity regression
equationa. Duncan multiple comparison test was performed on the data to determine significant
differences between the inhibition rates of different compounds at the same concentration.
4. Conclusions
In this study, sixteen derivatives of S and C were obtained by modification of their C=N+ groups
and evaluated for in vitro antifungal activity against seven phytopathogenic fungi by the mycelial
growth rate method. The structure-activity relationships were also discussed. S, C, S1–S5 and C1–C5
showed significant antifungal activities against all the tested fungi at 100 μg/mL. For most tested fungi,
the median effective concentrations of S, S1, C and C1 were in a range of 14–50 μg/mL. The iminium bond
was proven to be the determinant for the antifungal activity of S and C. S1–S5 and C1–C5 were considered
as the precursors of S and C, respectively. Based on the present results, S and C should be considered
as good lead compounds or model molecules to develop new anti-phytopathogenic fungal agents.
Acknowledgments
This project was supported by the National Natural Science Foundation of China (NNSF; Nos.
31172365, 31000865).

Molecules 2012, 17
13034
References
1. Wedge, D.E.; Camper, N.D. Biologically Active Natural Products. In Agrochemicals and
Pharmaceuticals; Cutler, H.G., Cutler, S.J., Eds.; CRC Press: Boca Raton, FL, USA, 2000; pp. 1–15.
2. Krane, B.D.; Fagbule, M.O.; Shamma, M.; Gözler, B. The benzophenanthridine alkaloids.
J. Nat. Prod. 1984, 47, 1–43.
3. Šimánek, V. Benzophenanthridine Alkaloids. In The Alkaloids; Brossi, A., Ed.; Academic Press:
New York, NY, USA, 1985; Volume 26, pp. 185–240.
4. Ahsan, H.; Reagan-Shaw, S.; Breur, J.; Ahmad, N. Sanguinarine induces apoptosis of human
pancreatic carcinoma AsPC-1 and BxPC-3 cells via modulations in Bcl-2 family proteins. Cancer Lett.
2007, 249, 198–208.
5. Jang, B.C.; Park, J.G.; Song, D.K.; Baek, W.K.; Yoo, S.K.; Jung, K.H.; Park, G.Y.; Lee, T.Y.;
Suh, S.I. Sanguinarine induces apoptosis in A549 human lung cancer cells primarily via cellular
glutathione depletion. Toxicol. In Vitro 2009, 23, 281–287.
6. Godowski, K.C. Antimicrobial action of sanguinarine. J. Clin. Dent. 1989, 1, 96–101.
7. Navarro, V.; Villarreal, M.L.; Rojas, G.; Lozoya, X. Antimicrobial evaluation of some plants used
in Mexican traditional medicine for the treatment of infectious diseases. J. Ethnopharmacol.
1996, 53, 143–147.
8. Zuo, G.Y.; Meng, F.Y.; Hao, X.Y.; Zhang, Y.L.; Wang, G.C.; Xu, G.L. Antibacterial alkaloids
from Chelidonium majus Linn (Papaveraceae) against clinical isolates of methicillin-resistant
Staphylococcus aureus. J. Pharm. Pharmaceut. Sci. 2008, 11, 90–94.
9. Meng, F.Y.; Zuo, G.Y.; Hao, X.Y.; Wang, G.C.; Xiao, H.T.; Zhang, J.Q.; Xu, G.L. Antifungal
activity of the benzo[c]phenanthridine alkaloids from Chelidonium majus Linn against resistant
clinical yeast isolates. J. Ethnopharmacol. 2009, 125, 494–496.
10. Lenfeld, J.; Kroutil, M.; Maršálek, E.; Slavík, J.; Preininger, V.; Šimánek, V. Antiinflammatory
activity of quaternary benzophenanthridine alkaloids from Chelidonoum majus. Planta Med.
1981, 43, 161–165.
11. Colombo, M.L.; Bosisio, E. Pharmacological activities of Chelidonium majus L. (Papaveraceae).
Pharmacol. Res. 1996, 33, 127–134.
12. Tan, G.T.; Pezzuto, J.M.; Kinghorn, A.D.; Hughes, S.H. Evaluation of natural products as
inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. J. Nat. Prod.
1991, 54, 143–154.
13. Yao, J.Y.; Li, X.L.; Shen, J.Y.; Pan, X.Y.; Hao, G.J.; Xu, Y.; Ying, W.L.; Ru, H.S.; Liu, X.L.
Isolation of bioactive components from Chelidonium majus L. with activity against Trichodina sp.
Aquaculture 2011, 318, 235–238.
14. Wang, G.X.; Zhou, Z.; Jiang, D.X.; Han, J.; Wang, J.F.; Zhao, L.W.; Li, J. In vivo anthelmintic
activity of five alkaloids from Macleaya microcarpa (Maxim) Fedde against Dactylogyrus
intermedius in Carassius auratus. Vet. Parasitol. 2010, 171, 305–313.
15. Nyangulu, J.M.; Hargreaves, S.L.; Sharples, S.L.; Mackay, S.P.; Waigh, R.D.; Duval, O.;
Mberu, E.K.; Watkins, W.M. Antimalarial benzo[c]phenanthridines. Bioorg. Med. Chem. Lett.
2005, 15, 2007–2010.
16. Tsai, I.L.; Wun, M.F.; Teng, C.M.; Ishikawa, T.; Chen, I.S. Anti-platelet aggregation constituents
from formosan Toddalia asiatica. Phytochemistry 1998, 48, 1377–1382.

Molecules 2012, 17
13035
17. Eun, J.P.; Koh, G.Y. Suppression of angiogenesis by the plant alkaloid, sanguinarine. Biochem.
Biophys. Res. Commun. 2004, 317, 618–624.
18. Cho, K.M.; Yoo, I.D.; Kim, W.G. 8-Hydroxy dihydrochelerythrine and 8-hydroxy
dihydrosanguinarine with a potent acetylcholinesterase inhibitory activity from Chelidonium
majus L. Biol. Pharm. Bull. 2006, 29, 2317–2320.
19. Miao, F.; Yang, X.J.; Ma, Y.N.; Zheng, F.; Song, X.P.; Zhou, L. Structural modification of
sanguinarine and chelerythrine and their in vitro acaricidal activity against Psoroptes cuniculi.
Chem. Pharm. Bull. 2012, doi:10.1248/cpb.12−00618.
20. Greathouse, G.A. Alkaloids from Sanguinaria canadensis and their influence on growth of
Phymatotrichum omnivorum. Plant Physiol. 1939, 14, 377–380.
21. Presley, J. Growth response of Verticillium albo-atrum to sanguinarine in nutrient agar.
Phytopathology 1969, 59, 1968–1969.
22. Howell, C.R.; Bell, A.A.; Stipanovic, R.D. Virulence to cotton and tolerance to sanguinarine
among Verticillium species. Can. J. Microbiol. 1973, 19, 1367–1371.
23. Matos, O.C.; Baeta, J.; Silva, M.J.; Pinto Ricardo, C.P. Sensitivity of Fusarium strains to
Chelidonium majus L. extracts. J. Ethnopharmacol. 1999, 66, 151–158.
24. Liu, H.; Wang, J.; Zhao, J.; Lu, S.; Wang, J.; Jiang, W.; Ma, Z.; Zhou, L. Isoquinoline alkaloids
from Macleaya cordata active against plant microbial pathogens. Nat. Prod. Commun. 2009, 4,
1557–1560.
25. Newman, S.E.; Roll, M.J.; Harkrader, R.J. A naturally occurring compound for controlling
powdery mildew of greenhouse roses. HortScience 1999, 34, 686–689.
26. Miao, F.; Yang, X.J.; Zhou, L.; Hu, H.J.; Zheng, F.; Ding, X.D.; Sun, D.M.; Zhou, C.D.; Sun, W.
Structural modification of sanguinarine and chelerythrine and their antibacterial activity.
Nat. Prod. Res. 2011, 25, 863–875.
27. Dostál, J.; Slavík, J. Some Aspects of the Chemistry of Quaternary Benzo[c]phenanthridine
Alkaloids. In Studies in Natural Products Chemistry, 1st ed.; Atta-ur-Rahman, Ed.; Elsevier
Scicence B.V: Oxford, UK, 2002; Volume 27, Part H, pp. 155–184.
28. Slavík, J.; Hanuš, V.; Slavíková, L. Alkaloids from Stylophorum lasiocarpum (Oliv.) Fedde.
Collec. Czech. Chem. Commun. 1991, 56, 1116–1122.
29. Walterová, D.; Ulrichová, J.; Válka, I.; Vicar, J.; Vavrecková, C.; Táborská, E.; Harjrader, R.J.;
Meyer, D.L.; Cerná, H.; Simánek, V. Benzo[c]phenanthridine alkaloids sanguinarine and
chelerythrine: Biological activities and dental care applications. Acta Univ. Palacki. Olomuc. Fac.
Med. 1995, 139, 7–16.
30. Zhang, J.W.; Li, S.K.; Wu, W.J. The main chemical composition and in vitro antifungal activity
of the essential oils of Ocimum basilicum Linn. var. pilosum (Willd.) Benth. Molecules 2009, 14,
273–278.
Sample Availability: Samples of the compounds S1–S8 and C1–C8 are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).