2-(substituted phenyl)-3,4-dihydroisoquinolin-2-iums as novel antifungal lead compounds: Biological evaluation and structure-activity relationships

Article abstract of DOI:10.3390/molecules180910413

The title compounds are a class of structurally simple analogues of quaternary benzo[c]phenanthridine alkaloids (QBAs). In order to develop novel QBA-like antifungal drugs, in this study, 24 of the title compounds with various substituents on the N-phenylring were evaluated for bioactivity against seven phytopathogenic fungi using the mycelial growth rate method and their SAR discussed. Almost all the compounds showed definite activities in vitro against each of the test fungi at 50 μg/mL and a broad antifungal spectrum. In most cases, the mono-halogenated compounds 2-12 exhibited excellentactivities superior to the QBAs sanguinarine and chelerythrine. Compound 8 possessed the strongest activities on each of the fungi with EC₅₀ values of 8.88-19.88 μg/mL and a significant concentration-dependent relationship. The SAR is as follows: the N-phenyl group is a high sensitive structural moiety for the activity and the characteristics and position of substituents intensively influence the activity. Generally, electron-withdrawing substituents remarkably enhance the activity while electron-donating substituents cause a decrease of the activity. In most cases, ortha- and para-halogenated isomers were more active than the corresponding m-halogenated isomers. Thus, the title compounds emerged as promising lead compounds for the development of novel biomimetic antifungal agrochemicals. Compounds 8 and 2 should have great potential as new broad spectrum antifungal agents for plant protection.

Full text of DOI:10.3390/molecules180910413

Molecules 2013, 18, 10413-10424; doi:10.3390/molecules180910413
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
2-(Substituted phenyl)-3,4-dihydroisoquinolin-2-iums as
Novel Antifungal Lead Compounds: Biological Evaluation and
Structure-Activity Relationships
Zhe Hou 1,†, Rui Yang 1,†, Cen Zhang 1, Li-Fei Zhu 1, Fang Miao 2, Xin-Juan Yang 1,* and Le Zhou 1,*
1
College of Science, Northwest A&F University, Yangling 712100, Shaanxi, China;
E-Mails: houzhe561@sina.com (Z.H.); yrer01018@126.com (R.Y.); z84934067@126.com (C.Z.);
zhao_fx0309@163.com (L.-F.Z.)
2
College of Life Science, Northwest A&F University, Yangling 712100, Shaanxi, China;
E-Mail: miaofangmf@163.com
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: yxjsn2@163.com (X.-J.Y.);
zhoulechem@nwsuaf.edu.cn (L.Z.); Tel./Fax: +86-29-8709-2226 (L.Z. & X.-J.Y.).
Received: 5 July 2013; in revised form: 18 July 2013 / Accepted: 22 August 2013 /
Published: 29 August 2013
Abstract: The title compounds are a class of structurally simple analogues of quaternary
benzo[c]phenanthridine alkaloids (QBAs). In order to develop novel QBA-like antifungal
drugs, in this study, 24 of the title compounds with various substituents on the N-phenyl
ring were evaluated for bioactivity against seven phytopathogenic fungi using the mycelial
growth rate method and their SAR discussed. Almost all the compounds showed definite
activities in vitro against each of the test fungi at 50 μg/mL and a broad antifungal
spectrum. In most cases, the mono-halogenated compounds 2–12 exhibited excellent
activities superior to the QBAs sanguinarine and chelerythrine. Compound 8 possessed the
strongest activities on each of the fungi with EC50 values of 8.88–19.88 µg/mL and a
significant concentration-dependent relationship. The SAR is as follows: the N-phenyl
group is a high sensitive structural moiety for the activity and the characteristics and
position of substituents intensively influence the activity. Generally, electron-withdrawing
substituents remarkably enhance the activity while electron-donating substituents cause a
decrease of the activity. In most cases, ortha- and para-halogenated isomers were more
active than the corresponding m-halogenated isomers. Thus, the title compounds emerged
as promising lead compounds for the development of novel biomimetic antifungal

Molecules 2013, 18
10414
agrochemicals. Compounds 8 and 2 should have great potential as new broad spectrum
antifungal agents for plant protection.
Keywords: 2-aryl-3,4-dihydroisoquinolin-2-ium; antifungal activity; phytopathogenic fungi;
structure-activity relationship; sanguinarine; chelerythrine
1. Introduction
Fungal plant diseases are one of the important concerns to agricultural production and food safety
worldwide [1]. Phytopathogenic fungi are able to infect any tissue at any stage of plant growth, and
result in severe yield losses and the quality decrease of agricultural products. In addition, many of the
phytofungal pathogens may produce mycotoxins harmful to animal and human health [2]. Therefore,
various plant fungicides are extensively used in current agriculture. However, many of the currently
used antifungal agents have several shortcomings, such as affecting human health, environmental
pollution and development of pathogen resistance [3]. Thus, the worsening problems of phytofungal
disease control urgently necessitate the discovery and development of new antifungal agents for
plant protection.
In the past decades, natural product-based antimicrobial agents had attracted a lot of attention from
researchers owing to the fact they are perceived to have less environmental toxicity and lower
mammalian toxicity [4]. Although most natural antifungal compounds are not suitable for direct or
extensive application as agricultural fungicides because of their lower activity than that of artificial
antimicrobial agents, limited sources or higher cost, they have been considered as ideal leads or model
compounds for the development of new environmentally acceptable antimicrobial agents.
Quaternary benzo[c]phenanthridine alkaloids (QBAs) are a relatively small but important class of
natural isoquinolines [5,6]. Among them, sanguinarine (SA) and chelerythrine (CH) (Figure 1) are the
most common. QBAs had been proven to have low toxicity to mammals [7–9] and extensive
pharmacological activities such as antitumor [10], antimicrobial [11,12], anti-inflammatory [7],
anti-HIV [13], anti-angiogenesis [14], anti-acetylcholinesterase [15] and antiparasitic actions against
Trichodina sp. [16], Dactylogyrus intermedius [17], malaria [18] and Psoroptes cuniculi [6]. QBAs
extract from plants have been used in toothpastes and mouthwashes as antiplaque agents, a veterinary
preparation for mastoiditis in cows and an additive to animal feeds [8]. Our previous studies also
demonstrated that sanguinarine and chelerythrine possessed excellent bioactivities against
phytopathogenic fungi [19]. Particularly, it was worth mentioning that the iminium moiety (C=N+) in
SA and CH had been proven to be the key structural moiety for their antimicrobial activity [12,19] and
acaricidal activity [6]. The similar case was also found for their anti-tumor activity [20].
In order to develop more potent isoquinoline drugs, we designed a class of structurally simple
QBAs-like compounds, i.e., 2-aryl-3,4-dihydroisoquinolin-2-iums (1–24, Figure 1), based on a
structural biomimetic strategy. Like QBAs, the compounds 1–24 possess an isoquinoline framework
containing an iminium moiety (C=N+). Besides, the compounds have approximate equal molecular
length to QBAs. We expected that the structural similarity between the designed compounds and their
model compounds can lead to discovery of new compounds with higher bioactivity than the model

Molecules 2013, 18
10415
compounds. It should be noted that unlike the complete planarity of QBAs, the compounds 1–24 have
different stereo structures from QBAs due to the existence of a dihedral angle between two phenyl
rings. The changeable dihedral angle endows the molecules with flexibility and was expected to make the
molecules better match the environment of the binding site of their biotarget and display higher bioactivity.
The title compounds may be easily prepared from commercially available isochroman according to
our previously reported method [21]. The reaction of isochroman with 57% hydroiodic acid under
reflux yielded 1-(2-iodoethyl)-2-(iodomethyl)benzene in 96% yield. A solution of the resultant
diiodide and aniline or substituted aniline in water was refluxed in the presence of sodium dodecyl
sulfonate (SDS) as phase-transfer catalyst to afford the corresponding 2-aryl-1,2,3,4-tetrahydroisoquinoline
in 86%–94% yield. Finally, the 2-aryl-1,2,3,4-tetrahydroisoquinolines were selectively oxidized with
DDQ (2,3-dichloro-5,6-dicyano-p-benzoquinone) or CuCl2·2H2O followed by treatment of
hydrobromic acid to provide the title compounds as solids in 40%–99% yield.
Figure 1. Structures of the target compounds and their model compounds.
O
R
O
Imitation design
N
Br-
N+
R'O
Me
OR"
Target compounds (1-24)
Sanguinarine (SA): R' + R" = CH2
Chelerythrine (CH): R' = R" = Me
(Natural model compounds)
As a part of our ongoing program for the development of QBAs-like isoquinoline drugs, we report
herein the evaluation of the antifungal activity of compounds 1–24 against various plant pathogenic
fungi and the establishment of their structure-activity relationships. To our knowledge, there has been
no information on the antifungal activity of the title compounds in the literature until now.
2. Results and Discussion
2.1. Screening of Antifungal Activity in Vitro
Compounds 1–24 were obtained by chemical synthesis according to our previous method [21] and
their structures and substituent patterns are shown in Figure 1 and Table 1. According to the mycelial
linear growth rate method [19], 1–24 were screened for antifungal activities in vitro against seven
phytopathogenic fungi (Alternaria alternate, Curvularia lunata, Pyricularia oryza, Fusarium solani,
Valsa mali, Fusarium oxysporum sp. niveum and Fusarium oxysporum f. sp. vasinfectum.) at 50 μg/mL.
SA iodide and CH iodide as model compounds, and triabendazole (TBZ, ≥99%), a commercial
fungicide standard, were used as positive controls. Mean inhibition rates of all the test compounds against
the same fungus were pairwise compared by Duncan’s multiple test. The results are listed in Table 1.

Molecules 2013, 18
Table 1. Substituent patterns and antifungal activities of compounds at 50 μg/mL (72 h). *
10416
Compound
Linear growth inhibitory rates (means, %) **
No.
1
R
H
A.A.
56.8 h
78.4 b
66.4 def
78.4 b
84.1 a
68.3 d
78.4 b
78.1 b
72.1 c
65.9 def
64.2 f
C.L.
76.7 ef
95.1 a
P.O.
F.S.
V.M.
32.4 l
F.O.N.
50.8 h
80.1 c
44.1 i
71.1 de
74.0 f
36.6 j
74.0 f
91.7 b
65.9 f
67.3 ef
28.5 k
44.1 i
48.4 hi
35.1 j
36.1 j
5.3 m
56.9 g
36.1 j
13.8 l
8.1 m
33.2 j
25.2 k
35.1 j
36.6 j
50.5 h
63.2 f
100.0 a
F.O.V.
50.6 hi
23.5 k
25.6 m
71.3 c
43.1 h
59.5 e
55.8 f
34.9 ij
65.1 d
74.6 b
61.5 e
52.8 f
25.6 m
43.6 h
48.2g
2
o-F
69.9 b
85.4 b
80.8 bc
54.2 i
3
m-F
86.9 bcd
73.4 fg
84.1 cd
84.5 cd
89.0 bc
92.0 ab
69.4 g
62.2 cdef
62.2 cdef
67.9 bc
60.9 def
48.5 ghij
59.7 def
59.2 def
62.1 de
4
p-F
66.4 g
72.9 ef
72.1 ef
61.2 g
71.7 ef
55.5 i
5
o-Cl
m-Cl
p-Cl
o-Br
p-Br
o-I
6
7
64.1 bcde 55.8 defgh
8
82.3 a
62.8 cde
60.3 def
59.0 defg
53.8 gh
65.4 bcd
63.5 cde
58.3 efg
12.8 n
93.9 a
49.0 ghij
55.3 defghi
54.3 efghi
47.0 ij
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
SA
CH
TBZ
81.6 de
84.9 cd
73.4 fg
73.9 fg
86.1 bcd
87.7 bcd
4.0 l
70.8 f
53.3 i
m-I
p-I
60.6 g
64.0 f
77.3 cd
55.9 i
47.2 j
o-CF3
m-CF3
p-CF3
o-NO2
m-NO2
p-NO2
o-Me
m-Me
p-Me
o-OH
p-OH
p-OMe
—
53.8 efghi
47.5 hij
44.6 j
64.9 ef
67.8 de
11.1 m
73.1 c
63.5 f
31.3 kl
33.8 jk
8.2 o
38.5 k
2.7 op
47.2 j
82.3 ab
2.3 p
−12.2 n
56.81 defg
58.7 def
−0.6 m
73.9 fg
60.8 h
70.4 b
52.8 f
37.9 i
10.8 o
11.3 o
21.0 n
20.0 n
30.8 kl
29.7 l
59.7 e
47.6 g
100.0 a
62.2 cdef
32.7 l
51.0 i
58.7 ghi
41.6 k
11.6 m
60.1 g
47.2 k
58.2 gh
55.8 h
47.7 jk
50.3 ij
16.4 l
19.2 m
35.3 kl
44.9 ij
2.6 m
11.6 n
6.6 o
57.1 ghi
53.8 ij
87.7b cd
57.5 hi
58.7 hi
48.7 j
13.5 l
53.4 fghi
73.3 c
11.0 n
76.0 de
25.0 m
69.1 fg
57.1 i
86.0 a
55.8 fgh
46.8 i
22.7 k
40.7 jk
50.1 hi
6.4 o
56.4 defg
63.5 d
—
—
9.3 l
81.5 b
* A.A., Alternaria alternate; C.L., Curvularia lunata; P.O., Pyricularia oryza; F.S., Fusarium solani; V.M.,
Valsa mali; F.O.N., Fusarium oxysporum sp. niveum; F.O.V., Fusarium oxysporum f. sp. vasinfectum.
** The differences between data with the different lowercase letters within a column are significant for the
same tested fungus (p < 0.05).
The results in Table 1 show that almost all the target compounds 1–24 displayed activities in
varying degrees against each of the test fungi at 50 μg/mL, indicating that the compounds had a wide
antifungal spectrum similar to that of SA and CH. For every one of the fungi, all or some of the
compounds were found to be more active than SA, CH and/or TBZ (p < 0.05). For the fungi
A. alternate, C. lunata and P. oryza, all or most of the compounds were more active than SA, CH
and/or TBZ (p < 0.05). Among them, 2, 4, 5, 7 and 8 showed the greatest or greater inhibition rates
(78.4%, 78.4%, 84.1%, 78.4% and 78.1%) on A. alternate, 2, 3, 7, 8, 14, 15 and 23 displayed the
strongest or stronger activities with inhibition rates of 95.1%, 86.9%, 89.0%, 92.0%, 86.1%, 87.7%
and 87.7% on C. lunata, and 8 exhibited the best activity (82.3%) on P. oryza, followed by 2 (69.9%),
5 (67.9%), 7 (64.1%), 13 (65.4%) and 17 (70.4%).

Molecules 2013, 18
10417
For each of the fungi F., V. mali, solani, F. oxysporum sp. niveum and F. oxysporum f. sp.
vasinfectum, some of the compounds showed higher or equal activity to that of SA, CH or TBZ. For
F. solani, 15 of the compounds showed the same or higher activities (48.5–93.9%) than that (56.4%) of
SA while 10 compounds gave the same or higher activities (55.3–93.9%) than that (63.5%) of CH
(p > 0.05). Among them, 8 and 2 gave the strongest activity (93.9%, 85.4%) over or equal to that
(81.5%) of TBZ (p > 0.05). For V. mali, 10 of the compounds were equally or more active
(53.3~82.3%) than CH (57.1%) and 15 of the compounds were equally or more active (61.2–82.3%)
than SA (69.1%) (p > 0.05). 18 (R = p-NO2) revealed the highest activity (82.3%), equal to that of
TBZ (86.0%) (p > 0.05).
As to F. oxysporum sp. niveum, 10 of the 24 compounds were equally or more active (48.4–91.7%)
than SA (50.5%) while seven of the compounds were equally or more active than CH (63.2%)
(p > 0.05). For F. oxysporum f. sp. vasinfectum, five of the compounds were equally or more active
than SA (59.7%) while nine of the compounds were equally or more active (48.2–74.6%) than CH
(47.6%). In both cases mentioned above, 8 revealed the highest activities (91.7%, 74.6%), but the
activities of all the compounds were weaker than that of TBZ (100.0%).
2.2. Structure-Activity Relationship
Qualitative structure-activity relationship was established by comparison of both the activities and
structures of various test compounds 1–24. By comparison of the activity of 1 (R = H) with that of the
other compounds (2–24) on each the fungus (Table 1), it was clearly seen that the introduction of
various substituents to N-aromatic ring had significant effects on the activity, indicating that the
N-phenyl group was a high sensitive structural moiety for the activity. The general trend was that
electron-withdrawing substituents like halogen atoms (2–12), trifluoromethyl groups (-CF3) (13–15)
and nitro groups (m- and p-NO2) (17, 18), especially halogen atoms, remarkably enhanced the activity.
On the contrary, the presence of electron-donating groups like -CH3 (19–21), o-OH (22), and p-OCH3
(24) led to a decrease of the activity in most cases (Figure 2).
Figure 2. Structure-activity relationship of the compounds 1–24.
R = F or Cl increases activity against all the fungi
R = Br, I or CF3 increases activity against 6 of 7 the fungi
R = NO2 increases activity against 5 of 7 the fungi
R = OH increases activity against 4 of 7 the fungi
R = Me or MeO dereases activity against 6 of 7 the fungi
R = F, Cl, CF3 or NO2 increases activity against 6 of 7 the fungi
R =I increases activity against 5 of 7 the fungi
p
R
R = Me dereases activity against all the fungi
m
N
o
R = F, Cl, Br, I or CF3 increases activity against all the fungi
R = CF3 increases activity against 6 of 7 the fungi
R = OH decreases activity against 6 of 7 the fungi
R = NO2 or Me dereases activity against all the fungi

Molecules 2013, 18
10418
On the other hand, by comparison of the activity of various isomers on each the fungus, a
significant effect of the position of the substituents on the activity was observed. For halogenated
isomers (2–12), the order of position effect of the substituents (-F, -Br, -Cl, -I) was o-substituted
isomer > p-substituted isomer > m-substituted isomer in most cases. For example, for the three
fluorinated compounds 2–4, the o-F isomer 2 showed the strongest activity against each the test fungi,
the p-F isomer 4 was the second, except for C. lunata, and the m-F isomer 3 gave the weakest activity
except for C. lunata. A similar trend was also observed for chlorinated, brominated and iodinated
isomers 5–12. The case for trifluoromethyl-substituted isomers was similar, but not identical to that of
halogen atoms. Like the halogenated isomers 2–12, the o-CF3 isomer 13 also gave the stronger activity
on most of the fungi (6/7) than the m-CF3 isomer 14 and p-CF3 isomer 15, but the activities of 14 and
15 showed no significant difference for all tested fungi except for V. mali (p > 0.05). The results above
showed that for halogen atoms and trifluoromethyl group, the 2-site of the N-phenyl ring was
beneficial for the improvement of the activity. Unlike the cases of halogen atoms and -CF3 group, an
o-nitro group (compound 16) caused an obvious decrease of the activity for all the fungi while a
m-nitro group (compound 17) and p-nitro group (compound 18) led to an enhancement of the activity.
Among them, the m-nitro-substituted isomer 17 showed the best activity on 6 of 7 the fungi.
The results above indicate that the activity of the target compounds greatly depended on the
characteristic and position of substituents on the N-aromatic ring. Generally, the introduction of F, Cl
or Br to the I site in the N-phenyl ring resulted in a very great improvement of the activity.
2.3. Antifungal Toxicity
In order to more fully understand the antifungal activities of the compounds, the most active
compounds 2, 4, 5, 7 and 8 were further evaluated as representative compounds for antifungal toxicity
on seven phytophathogenic fungi.
Table 2. Toxicity regression equations and EC50 values of 2, 4, 5, 7 and 8 against 7 fungi.
EC50
Toxicity regression
equation *
2
Fungus
Compd.
R
95%CI ** of EC50
(μg/mL)
22.12
53.12
39.67
39.39
12.39
33.71
52.58
30.80
26.42
19.86
30.97
10.49
50.20
28.89
(μmol/L)
72.2
F.O.V.
2 (o-F)
4 (p-F)
5 (o-Cl)
7 (p-Cl)
8 (o-Br)
SA
y = 1.4300x + 3.0769
y = 2.6168x + 0.4853
y = 1.2450x + 3.0099
y = 2.3044x + 1.3235
y = 1.0996x + 3.7981
y = 1.6623x + 2.4605
y = 3.0902x − 0.3177
y = 2.4275x + 1.3864
y = 2.4139x + 1.5676
y = 1.3796x + 3.2094
y = 2.7227x + 0.9408
y = 1.8107x + 3.1516
y = 1.4175x + 2.5893
y = 1.7052x + 2.5090
0.9780
0.9381
0.9433
0.9971
0.9958
0.9730
0.9737
0.9769
0.9669
0.9373
0.9988
0.9414
0.9853
0.9864
20.74–23.60
42.10–67.02
36.31–72.17
39.29–40.05
12.09–12.70
23.26–48.85
29.43–93.95
29.98–31.64
25.36–27.52
18.05–37.91
30.88–31.06
7.48–14.71
173.5
123.0
122.1
33.8
73.4
CH
110.6
100.6
86.3
F.O.N.
2 (o-F)
4 (p-F)
5 (o-Cl)
7 (p-Cl)
8 (o-Br)
SA
61.6
96.0
28.6
109.3
60.8
43.44–58.01
23.77–35.12
CH

Molecules 2013, 18
10419
Table 2. Cont.
EC50
Toxicity regression
2
Fungus
Compd.
R
95%CI ** of EC50
equation *
(μg/mL)
22.27
36.33
24.65
26.45
19.88
25.59
39.68
22.25
15.74
24.02
33.84
8.88
(μmol/L)
72.7
118.7
76.4
82.0
54.2
55.7
83.5
72.7
51.4
74.5
104.9
24.2
90.8
55.8
58.6
76.4
56.7
46.6
25.9
115.5
104.2
33.1
44.7
28.2
40.2
24.4
77.4
33.1
53.3
108.0
67.0
89.9
34.46
220.1
98.3
V.M.
2 (o-F)
4 (p-F)
5 (o-Cl)
7 (p-Cl)
8 (o-Br)
SA
y = 2.3031x + 1.8962
y = 2.3521x + 1.3300
y = 2.0946x + 2.0847
y = 1.4730x + 2.9048
y = 1.1731x + 3.4768
y = 1.9432x + 2.2639
y = 1.9658x + 1.8575
y = 2.1583x + 2.0918
y = 1.2769x + 3.4716
y = 1.0950x + 3.4884
y = 0.9805x + 3.5004
y = 2.1420x + 2.9683
y = 2.1928x + 1.4479
y = 1.3614x + 3.0620
y = 1.3559x + 3.2997
y = 2.6327x + 1.3957
y = 1.1348x + 3.5676
y = 1.3719x + 3.3852
y = 1.1204x + 3.9040
y = 2.2140x + 1.1814
y = 2.6508x + 0.5078
y = 1.3293x + 3.6629
y = 1.0827x + 3.7694
y = 1.3088x + 3.7443
y = 1.4152x + 3.4244
y = 1.7125x + 3.3707
y = 1.5699x + 2.5652
y = 0.9154x + 3.9048
y = 1.0351x + 3.7446
y = 1.4434x + 2.8066
y = 0.8119x + 3.9161
y = 1.6338x + 2.6110
y = 1.4194x + 3.4358
y = 0.8326x + 3.3309
y = 1.6792x + 2.1968
0.9836
0.9162
0.9651
0.9749
0.9575
0.9880
0.9541
0.9938
0.9846
0.9583
0.9534
0.9833
0.9893
0.9466
0.9835
0.9876
0.9738
0.9813
0.9953
0.9550
0.9586
0.9725
0.9198
0.9169
0.9675
0.9373
0.9621
0.9621
0.9531
0.9892
0.9818
0.9893
0.9806
0.9754
0.9629
21.71–26.42
33.79–59.37
23.21–26.18
25.87–30.72
16.03–24.66
17.19–38.09
16.17–97.36
22.05–23.64
15.52–17.30
22.95–32.70
29.63–38.65
8.04–9.81
CH
F.S.
A.A.
C.L.
P.O.
2 (o-F)
4 (p-F)
5 (o-Cl)
7 (p-Cl)
8 (o-Br)
SA
41.68
26.52
17.95
23.39
18.29
15.03
9.51
31.65–54.89
15.92–44.19
17.50–21.32
22.96–26.56
17.57–24.00
14.60–18.29
9.25–9.78
CH
2 (o-F)
4 (p-F)
5 (o-Cl)
7 (p-Cl)
8 (o-Br)
SA
53.03
49.51
10.14
13.70
9.11
28.22–99.67
21.52–113.92
7.28–14.11
12.10–31.73
7.10–11.69
12.33–18.34
6.38–12.53
23.37–54.07
6.08–40.66
15.63–21.97
32.78–35.15
20.66–22.82
28.73–30.81
11.15–14.35
72.61–140.73
39.01–55.93
CH
2 (o-F)
4 (p-F)
5 (o-Cl)
7 (p-Cl)
8 (o-Br)
SA
12.98
8.94
35.55
15.72
16.33
33.08
21.63
28.99
12.65
101.09
46.71
CH
2 (o-F)
4 (p-F)
5 (o-Cl)
7 (p-Cl)
8 (o-Br)
SA
CH
* y: The probability of the inhibition rate; x: log[concentration (μg/mL)]. ** 95% CI: Lower and upper values
of the confidence interval of EC50 (μg/mL) at 95% probability.
Meanwhile, both SA and CH were used as positive controls. Based on the mycelial growth
inhibitory rates of the compounds at a series of concentrations, toxicity regression equations of the
compounds were established between log[concentration] value and probit value of inhibition rates by
using the least square method. EC50 (median effective concentration) values of the compounds and

Molecules 2013, 18
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their confidence intervals at 95% probability were calculated from the corresponding toxicity
regression equations. The results are listed in Table 2.
All the tested compounds showed significant concentration-dependent antifungal activity toward
every one of the tested fungi (R2 values = 0.9162–0.9988) (Table 2) and their EC50 values were in a
range of 8.88 to 53.12 µg/mL. From Table 2, it was clearly seen that most of the EC50 values (27/35)
of the tested compounds were lower than 30 µg/mL. By contrast, most of EC50 values (10/14) of SA
and CH were greater than 30 µg/mL. Among the tested compounds, 8 possessed the strongest activity
on each the fungi and most of its EC50 values (6/7) were lower than 15 µg/mL. Therefore, as far as the
EC50 values were concerned, 2, 4, 5, 7 and 8 possessed stronger activity than SA and CH in most
cases. In addition, It was worth mentioning that all the test compounds showed the highest activity on
C. lunata (EC50 = 8.94–13.7 µg/mL) among the seven fungi.
A slope value (k) in a toxicity regression equation reflects concentration effect of a compound on its
bioactivity. A greater k value implies that the antifungal activity of a test compound is more
susceptible to its concentration change. The results in Table 2 showed that the k values of each the test
compound on various fungi or the k values of various compounds on the same fungus were different,
indicating that both the concentration effect of each the compound on the various fungi and the
concentration effect of the various compounds on the same fungus were different.
A compound with better bioactivity should have a greater slope value and a smaller EC50 value at
the same time. The ratio value of k/EC50 of a compound reflects its comprehensive activity (CA) to
certain degree. In order to conveniently compare the CAs of the various compounds, we calculated the
ratio of CA of each the compound to that of CH for the same fungus, i.e., relative comprehensive
activity (RCA). As shown in Figure 3, 30 of 35 the RCA values of the five test compounds were
greater than 1.0, i.e., the RCA value of CH. This result indicated that all the tested compounds
possessed stronger RCA than CH in most cases, in agreement with the data obtained from EC50 values
in Table 2. In particular, both 2 and 8 (RCA = 1.10–4.70) showed higher activity than CH against all
the tested fungi. Among the tested compounds, 2 showed the strongest activity against V. mail while 8
showed the strongest activity against the other fungi.
Figure 3. Relative comprehensive activities of the compounds against seven fungi.
6.00
F. solani
C. lunata
5.00
P. oryza
F. oxysporum sp. niveum
4.00
A. alternata
F. oxysporum f. sp. vasinfectum
3.00
V. mali
2.00
1.00
0.00
o-Br
8
o-F
2
p-F
4
o-Cl
p-Cl
7
5
SA
CH
Compound

Molecules 2013, 18
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3. Experimental
3.1. General
Compounds 1–24 were obtained by synthesis according to our previously reported methods and
their structures were determined by spectroscopic analysis [21]. Sanguinarine iodide (SA, >98%) and
chelerythrine iodide (CH, >99%) were obtained in our laboratory by isolation from the whole plant of
M. microcarpa (Maxim) Fedde [12]. 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 Chemical Ltd. (Beijing, China). Other reagents were obtained locally and of
analytical grade. The water used was redistilled and ion-free.
Test fungi Fusarium oxysporum sp. vasinfectum, Fusarium oxysporum sp. niveum, Valsa mali,
Fusarium solani, Alternaria alternate, Curvularia lunata and Pyricularia oryzae were isolated,
identified and provided by the Center of Pesticide Research, Northwest A&F University, Yangling,
China. These fungi were grown on potato-dextrose-agar (PDA) plates at 28 °C and maintained at 4 °C
by periodic subculturing.
3.2. Assay of Antifungal Activity in Vitro
The antifungal activity in vitro was assayed by the growth rate method reported by us [19]. The test
fungi maintained on 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 a stock solution (1.2 mg/mL) in 5% DMSO aqueous
solution. Thiabendazole solution (1.2 μg/mL) in 5% DMSO aqueous solution and 5% DMSO aqueous
solution were used as positive drug control and blank control, respectively. SA and CH solutions
(1.2 μg/mL) in 5% DMSO aqueous solution were used as the model compound controls at the same
time. The stock solution (10 mL) was completely mixed with the autoclaved PDA medium (230 mL)
to provide a medium containing 50 μg/mL of test compounds and then poured into the Petri dishes in a
laminar flow chamber. When the medium in the plate was partially solidified, 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 triplicate. 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.:
(1)
Growth inhibition rate (%) = [(dc − d0) − (ds − d0)]/(dc − d0) × 100
where d0: Diameter of the fungus cut, dc: Diameter of the blank control fungus, ds: Diameter of the
compound-treated fungus.
Based on the in vitro antifungal activity screening results, the more active compounds 2, 4, 5, 7 and
8 were selected to determine their antifungal toxicity according to the method described above [19]. A
stock solution of the compounds was prepared in 5% DMSO aqueous solution, and then diluted by 5%
DMSO in water using serial two-fold dilution method to obtain a series of stock solutions. Each stock
solution (10 mL) was respectively mixed with the autoclaved PDA medium to prepare a set of media

Molecules 2013, 18
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containing 120, 90, 80, 40, 20, 10, 5 and 2.5 μg/mL compounds. Meanwhile, 5% DMSO aqueous
solution was used as blank control. The antifungal activity for each concentration of the compounds
was determined. Each experiment was performed in triplicate. The average inhibition rate for each test
was calculated and then transformed to the corresponding probit value. The concentration of the
compound was transformed to the corresponding logarithm value (log10C). Log10C values for each
compound and its corresponding probit values were used to establish toxicity regression equation by
using the least square method. EC50 values and their confidence interval at 95% probability were
calculated from the toxicity regression equations. RCA of each compound was calculated according to
the following formula:
RCA = (slope value of the test compound/slope value of CH) ×
(2)
(EC50 value of CH/EC50 value of the test compound)
3.3. Statistic Analysis
SPSS V17.0 statistical software was used to analyze the data and establish toxicity regression
equations. Duncan multiple comparison test was performed on the data to determine significant
difference between the inhibition rates of various compounds at the same concentration.
4. Conclusions
2-Aryl-3,4-dihydroisoquinolin-2-ium bromides are a class of structurally simple QBA-like
compounds. In the present study, we evaluated in vitro antifungal activities of a series of synthesized
2-aryl-3,4-dihydroisoquinolin-2-ium bromides with various substituents on the N-aromatic ring against
seven pathogenic fungi and discussed their structure-activity relationships as well as compared their
activities with that of their natural model compounds SA and CH. Like SA and CH, almost all the
compounds 1–24 displayed the activities against all the fungi in varying degrees at 50 μg/mL and a
broad antifungal spectrum. The N-phenyl ring was proven to be a highly influential structural moiety
for the activity. The introduction of substituents to N-phenyl ring led to significant change of the
activity. Generally, electron-withdrawing substituents such as -X (X = F, Cl, Br or I), -CF3 and -NO2,
especially -X, remarkably enhanced the activity. On the contrary, electron-donating groups such as
-CH3, o-OH, and p-OCH3 result in the decrease of the activity in most cases. In addition, the position
of substituents was able to significantly influence the activity also. For halogenated compounds, the
order of the activity of various isomers was generally o-substituted isomer > p-substituted isomer >
m-substituted isomer. In most cases, most of the halogenated compounds 2~12 showed higher activity
than SA and CH. 2, 4, 5, 7 and 8 showed significant concentration-dependent antifungal activity
toward the tested fungi. Both 2 and 8, especially 8, showed higher RCAs than SA and CH against all
the 7 tested fungi. Thus, 2-aryl-3,4-dihydroisoquinolin-2-ium compounds might be considered as
promising lead compounds for the development of novel isoquinoline antifungal agrochemicals.
Compounds 2, 4, 5, 7 and 8, and especially 2 and 8, are of great potential as new antifungal agents for
plant protection.
Compared with the natural model compounds SA or CH, the title compounds had some obvious
advantages such as more simple structure, higher bioactivity, higher high atom economy, easy

Molecules 2013, 18
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chemical synthesis on a large scale and lower cost, etc. In addition, our recent research had
demonstrated that the title compounds were safe for plant growth (unpublished data). Therefore, the
title compounds should have broad application prospects in agriculture as novel biomimetic fungicides
and alternatives to the natural model compounds.
Acknowledgments
This project was financially supported by the National Natural Science Foundation of China
(NNSF; No. 31000865, 31172365, 31101469).
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–24 are available from the authors.
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