Simple Analogues of Quaternary Benzo[ c]phenanthridine Alkaloids: Discovery of a Novel Antifungal 2-Phenylphthalazin-2-ium Scaffold with Excellent Potency against Phytopathogenic Fungi

Article abstract of DOI:10.1021/acs.jafc.0c06507

Inspired by sanguinarine and chelerythrine, a novel antifungal 2-phenylphthalazin-2-ium scaffold as a simple analogue was designed. Most of the 30 compounds showed excellent inhibition activity against almost all eight phytopathogenic fungi, far superior to sanguinarine and chelerythrine. A third of the compounds were more active than azoxystrobin in most cases. Compounds 26 and 27 showed the highest total activity against all the fungi with EC50 means of ca. 4.6 μg/mL. Fusarium solani showed the highest susceptibility with an EC50 mean of 3.62 μg/mL to 19 compounds. A concentration of 25.0 μg/mL 27 can fully control the Colletotrichum gloeosporioides infection in apples over 9 days. Electron microscopic observations showed that 27 was able to damage the structures of the hypha and cell membrane. The structure-activity relationship showed that the presence of electron-withdrawing groups on the C-ring increases the activity against most of the fungi. Thus, 2-phenylphthalazin-2-ium compounds represent promising leads for the development of novel fungicides.

Full text of DOI:10.1021/acs.jafc.0c06507

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Article
Simple Analogues of Quaternary Benzo[c]phenanthridine Alkaloids:
Discovery of a Novel Antifungal 2‑Phenylphthalazin-2-ium Scaffold
with Excellent Potency against Phytopathogenic Fungi
Zhi-Ming Cui, Bo-Hang Zhou, Chao Fu, Liu Chen, Juan Fu, Fang-Jun Cao, Xin-Juan Yang,*
and Le Zhou*
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ABSTRACT: Inspired by sanguinarine and chelerythrine, a novel antifungal 2-phenylphthalazin-2-ium scaffold as a simple analogue
was designed. Most of the 30 compounds showed excellent inhibition activity against almost all eight phytopathogenic fungi, far
superior to sanguinarine and chelerythrine. A third of the compounds were more active than azoxystrobin in most cases. Compounds
26 and 27 showed the highest total activity against all the fungi with EC50 means of ca. 4.6 μg/mL. Fusarium solani showed the
highest susceptibility with an EC50 mean of 3.62 μg/mL to 19 compounds. A concentration of 25.0 μg/mL 27 can fully control the
Colletotrichum gloeosporioides infection in apples over 9 days. Electron microscopic observations showed that 27 was able to damage
the structures of the hypha and cell membrane. The structure−activity relationship showed that the presence of electron-
withdrawing groups on the C-ring increases the activity against most of the fungi. Thus, 2-phenylphthalazin-2-ium compounds
represent promising leads for the development of novel fungicides.
KEYWORDS: phthalazine, 2-phenylphthalazin-2-ium, antifungal activity, plant pathogenic fungi, structure−activity relationships
INTRODUCTION
■
Plant mycoses are an important problem of agricultural
production worldwide,1 which reduce the global crop yield
by about 20% annually and further cause about 10% loss after
harvest. Additionally, some phytopathogenic fungi can also
harm animal and human health because of their mycotoxins.2
Therefore, fungicides have played a key role in improving the
crop yields and quality and in ensuring stability of production.
However, the prolonged usage of fungicides has led to several
drawbacks such as drug resistance, environmental problems,
and residue toxicity.3 Therefore, it is very necessary to develop
novel and environmentally friendly fungicides for plant
protection. In this respect, natural product-based or -derived
plant protectants have increasingly attracted the attention of
researchers because of the lower environmental and mamma-
lian toxicity of natural products.4
Quaternary benzo[c]phenanthridine alkaloids (QBAs)
(Figure 1) are a relatively small but important class of natural
Figure 1. Structures of quaternary benzo[c]phenanthridine alkaloids
and the analogues A, B, and C.
isoquinoline alkaloids. As the two most common QBAs,
sanguinarine (SA) and chelerythrine (CH) have been
considered as promising lead compounds for the development
deigned two classes of structurally simple analogues of
QBAs, that is, isoquinoline analogues (A) and β-carboline
analogues (B) (Figure 1) by structural simulation. These
analogues generally showed similar or even more potent
of novel antimicrobial agents because of their excellent
bioactivity,5−10 distinctive structure, and low mammalian
toxicity.11,12 The most striking structural feature of QBAs is
the presence of a highly active iminium moiety (CN+),
Received: October 12, 2020
Revised: November 30, 2020
Accepted: December 3, 2020
which has been proved to be the determinant for the
antibacterial and antifungal activity of SA and CH.7,8
Based on the understanding of the structure−activity
relationships (SARs) of QBAs and with the aim of seeking
more potent QBA-like antifungal agents, we previously
https://dx.doi.org/10.1021/acs.jafc.0c06507
© XXXX American Chemical Society
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A

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Article
antifungal activity than SA and CH.8,13−18 Besides, the
analogues also showed high safety for plant growth.19
Encouraged by the findings above, herein we further designed
a class of novel phthalazine analogues (C) (Figure 1). It was
expected that the structural similarity between C and A or
QBAs can lead to the discovery of a novel and more potent
antifungal scaffold for the development of new QBA-like
fungicides.
In this study, a novel 2-phenylphthalazin-2-ium scaffold and
a series of its derivatives were designed and synthesized, and
their inhibition activities were explored in vitro on eight
important plant pathogenic fungi. The most active compound
was evaluated for the activity in vivo, and the action mechanism
was investigated by scanning electron microscopy (SEM) and
transmission electron microscopy (TEM) observations. Addi-
tionally, the SARs were deduced.
2-Phenylphenylphthalazin-2-ium Bromide (1). Light yellow
powder; yield: 82% (1.18 g, 4.1 mmol); mp 211.1−211.8 °C; H
1
NMR (500 MHz, DMSO-d6): δ 11.27 (s, 1H), 10.31 (s, 1H), 8.81 (d,
J = 8.1 Hz, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.60 (t, J = 7.6 Hz, 1H),
8.50 (t, J = 7.6 Hz, 1H), 8.17 (d, J = 8.1 Hz, 2H), 7.84−7.75 (m, 3H);
13C NMR (126 MHz, DMSO-d6): δ 154.6, 151.1, 143.3, 139.7, 136.4,
131.5, 131.3, 130.1, 128.4, 128.2, 127.5, 123.9; HRMS (ESI, positive)
+
m/z: calcd for C14H11N2 [M − Br]+, 207.0917; found, 207.0903;
(negative) m/z: calcd for [Br]−, 78.9189 and 80.9168; found, 78.9185
and 80.9163. The NMR data above were consistent with those in the
literature.21
2-(2-Fluorophenyl)phthalazin-2-ium Bromide (2). Light yellow
1
powder; yield: 85% (1.30 g, 4.25 mmol); mp 211.0−212.2 °C; H
NMR (500 MHz, DMSO-d6): δ 11.17 (s, 1H), 10.31 (s, 1H), 8.80 (d,
J = 8.1 Hz, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.66 (t, J = 7.6 Hz, 1H),
8.53 (t, J = 7.6 Hz, 1H), 8.11 (t, J = 7.7 Hz, 1H), 7.86 (q, J = 7.0 Hz,
1H), 7.78−7.73 (m, 1H), 7.64 (t, J = 7.7 Hz, 1H); 13C NMR (126
MHz, DMSO-d6): δ 155.5, 154.72, 154.70, 154.6, 153.5, 140.4, 136.6,
134.0, 133.9, 131.5, 131.23, 131.15, 128.6, 127.82, 127.75, 127.5,
125.81, 125.78, 117.6, 117.4; HRMS (ESI, positive) m/z: calcd for
MATERIALS AND METHODS
■
+
C14H10FN2 [M − Br]+, 225.0823; found, 225.0824.
General Procedures. All starting materials and solvents were
commercially available and used as purchased without further
purification. All starting materials were analytically pure. The progress
of all reactions was monitored on precoated GF-254 thin-layer plates
(Qingdao Haiyang Chemical, China) using CHCl3/MeOH as the
solvent system. Spots were visualized under ultraviolet light (254 and
365 nm). The purity of each compound was analyzed by high-
performance liquid chromatography (HPLC, Agilent Technologies
1260 Infinity) using MeOH/H2O as the mobile phase at a flow rate of
0.6 mL/min on a C18 column (Agilent ZORBAX SB-C18, 5 μm, 4.6
mm × 150 mm). All final compounds exhibited purities greater than
95%. 1H NMR and 13C NMR spectra were recorded on an Avance III
500 MHz instrument (Bruker, Karlsruhe, Germany). Chemical shifts
were measured relative to the residual solvent peaks of dimethyl
sulfoxide-d6 (DMSO-d6) (1H, δ 2.50 ppm; 13C, δ 39.52 ppm).
Chemical shifts (δ values) and coupling constants (J values) are given
in parts per million and hertz, respectively. High-resolution electro-
spray ionization mass spectra (HRMS) were obtained with an LC-
30A + TripleTOF5600+ (AB SCIEX, USA).
2-(3-Fluorophenyl)phthalazin-2-ium Bromide (3). Light yellow
1
powder; yield: 85% (1.30 g, 4.25 mmol); mp 200.0−200.7 °C; H
NMR (500 MHz, DMSO-d6): δ 11.29 (s, 1H), 10.29 (s, 1H), 8.80 (d,
J = 8.0 Hz, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.62 (t, J = 7.5 Hz, 1H),
8.52 (t, J = 7.6 Hz, 1H), 8.13 (dt, J = 9.6, 2.2 Hz, 1H), 8.06 (dd, J =
8.2, 1.6 Hz, 1H), 7.88 (td, J = 8.3, 6.3 Hz, 1H), 7.68 (td, J = 8.4, 2.2
Hz, 1H); 13C NMR (126 MHz, DMSO-d6): δ 162.9, 161.0, 154.5,
151.6, 144.3, 144.2, 140.0, 136.5, 132.05, 131.98, 131.4, 128.4, 127.9,
127.6, 120.22, 120.20, 118.6, 118.4, 111.8, 111.6; HRMS (ESI,
+
positive) m/z: calcd for C14H10FN2 [M − Br]+, 225.0823; found,
225.0824.
2-(4-Fluorophenyl)phthalazin-2-ium Bromide (4). Light yellow
1
powder; yield: 86% (1.31 g, 4.30 mmol); mp 251.8−252.3 °C; H
NMR (500 MHz, DMSO-d6): δ 11.21 (s, 1H), 10.27 (s, 1H), 8.78 (d,
J = 8.1 Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.60 (t, J = 7.6 Hz, 1H),
8.50 (t, J = 7.6 Hz, 1H), 8.25−8.20 (m, 2H), 7.69 (t, J = 8.7 Hz, 2H);
13C NMR (126 MHz, DMSO-d6): δ 164.3, 162.4, 154.5, 151.3, 139.7,
136.4, 131.2, 128.4, 128.0, 127.4, 126.6, 126.5, 117.1, 116.9; HRMS
+
(ESI, positive) m/z: calcd for C14H10FN2 [M − Br]+, 225.0823;
Materials. Azoxystrobin was purchased from Jiangsu Frey
Agrochemicals Co., Ltd. (Jiangsu, China). o-Phthalaldehyde (98%)
and hydrobromic acid (40%) were purchased from Aladdin Industrial
Corporation (Shanghai, China). Substituted phenylhydrazine hydro-
chlorides (≥97%) were purchased from various commercial sources.
Both SA (>98%) and CH iodide (>98%) were obtained by isolation
from the plant of Macleaya microcarpa (Maxim.) Fedde in our
laboratory.7 Other reagents and solvents were obtained locally and
were of analytical grade. The plant pathogenic fungi Fusarium
oxysporum f. sp. niveum, Fusarium solani, Alternaria alternata,
Colletotrichum gloeosporioides, Fusarium bulbigenum, Alternaria solani,
Alternaria brassicae, and Pyricularia oryzae were provided by the
Center of Pesticide Research, Northwest A&F University, China.
These fungi were grown on potato dextrose agar (PDA) plates at 28
°C and maintained at 4 °C with periodic subculturing.
Synthesis of Compounds 1−30. The general procedure was
adapted from a previous paper.20 To a solution of o-phthalaldehyde
(0.67 g, 5 mmol) in 1,4-dioxane, substituted or unsubstituted
phenylhydrazine hydrochloride (5 mmol) was added at room
temperature (rt). The mixture was stirred for about 2 days until the
reaction was completed. The formed precipitate was filtered off and
washed with ethyl acetate (150 mL). The obtained solid was dissolved
in 100 mL of water and adjusted with saturated sodium carbonate
aqueous solution to pH 9−10. The resulting solution was extracted
with CH2Cl2 (3 × 50 mL). The combined organic phase was dried
over anhydrous sodium sulfate and removed under reduced pressure.
The resulting residue was dissolved in 1,4-dioxane (60 mL) and then
treated with 40% hydrobromic acid aqueous solution (5 mmol). The
formed solid was filtered off and completely dried in a vacuum drying
oven at 50 °C to afford the desired compounds 1−30. Yield and
characterization data for compounds 1−30 are as follows.
found, 225.0827.
2-(2-Chlorophenyl)phthalazin-2-ium Bromide (5). Light yellow
1
powder; yield: 86% (1.38 g, 4.30 mmol); mp 239.1−239.7 °C; H
NMR (500 MHz, DMSO-d6): δ 11.21 (s, 1H), 10.35 (s, 1H), 8.80 (d,
J = 8.1 Hz, 1H), 8.75 (d, J = 7.9 Hz, 1H), 8.69 (t, J = 7.6 Hz, 1H),
8.55 (t, J = 7.6 Hz, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 8.0 Hz,
1H), 7.84 (t, J = 7.2 Hz, 1H), 7.79 (t, J = 7.5 Hz, 1H); 13C NMR
(126 MHz, DMSO-d6): δ 154.9, 154.7, 140.7, 140.6, 136.8, 133.3,
131.5, 130.8, 128.9, 128.7, 128.4, 128.0, 127.53, 127.47; HRMS (ESI,
+
positive) m/z: calcd for C14H10ClN2 [M − Br]+, 241.0527; found,
241.0501.
2-(3-Chlorophenyl)phthalazin-2-ium Bromide (6). Light yellow
1
powder; yield: 85% (1.37 g, 4.25 mmol); mp 223.2−223.8 °C; H
NMR (500 MHz, DMSO-d6): δ 11.27 (s, 1H), 10.29 (s, 1H), 8.78 (d,
J = 8.1 Hz, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.62 (t, J = 7.6 Hz, 1H),
8.51 (t, J = 7.6 Hz, 1H), 8.31 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.91−
7.82 (m, 2H); 13C NMR (126 MHz, DMSO-d6): δ 154.5, 151.7,
144.1, 140.0, 136.5, 134.1, 131.7, 131.42, 131.39, 128.4, 127.9, 127.5,
+
123.9, 122.9; HRMS (ESI, positive) m/z: calcd for C14H10ClN2 [M
− Br]+, 241.0527; found, 241.0538.
2-(4-Chlorophenyl)phthalazin-2-ium Bromide (7). Light yellow
1
powder; yield: 87% (1.40 g, 4.35 mmol); mp 276.9−277.6 °C; H
NMR (500 MHz, DMSO-d6): δ 11.22 (s, 1H), 10.27 (s, 1H), 8.77 (d,
J = 8.0 Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.64−8.57 (m, 1H), 8.54−
8.47 (m, 1H), 8.22−8.15 (m, 2H), 7.95−7.88 (m, 2H); 13C NMR
(126 MHz, DMSO-d6): δ 154.5, 151.3, 142.0, 139.8, 136.4, 136.3,
131.3, 130.0, 128.4, 128.0, 127.4, 125.7; HRMS (ESI, positive) m/z:
+
calcd for C14H10ClN2 [M − Br]+, 241.0527; found, 241.0521.
2-(2-Bromophenyl)phthalazin-2-ium Bromide (8). Light yellow
1
powder; yield: 88% (1.61 g, 4.40 mmol); mp 254.3−255.0 °C; H
B
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NMR (500 MHz, DMSO-d6): δ 11.19 (s, 1H), 10.35 (s, 1H), 8.79 (d,
J = 8.1 Hz, 1H), 8.76 (d, J = 8.0 Hz, 1H), 8.73−8.67 (m, 1H), 8.59−
8.54 (m, 1H), 8.11−8.08 (m, 1H), 8.08−8.06 (m, 1H), 7.82 (td, J =
7.7, 1.2 Hz, 1H), 7.75 (td, J = 7.8, 1.6 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6): δ 154.8, 154.7, 142.2, 140.8, 136.9, 133.9, 133.5, 131.5,
129.4, 128.7, 128.4, 127.5, 127.4, 117.8; HRMS (ESI, positive) m/z:
°C; 1H NMR (500 MHz, DMSO-d6): δ 11.23 (s, 1H), 10.27 (s, 1H),
8.80 (d, J = 8.0 Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.62−8.58 (m,
1H), 8.52−8.48 (m, 1H), 7.76 (d, J = 2.1 Hz, 1H), 7.73−7.69 (m,
2H), 7.35 (dt, J = 6.5, 2.4 Hz, 1H), 3.94 (s, 3H); 13C NMR (126
MHz, DMSO-d6): δ 160.0, 154.4, 151.1, 144.2, 139.7, 136.3, 131.3,
131.0, 128.3, 128.0, 127.5, 117.2, 115.9, 109.6, 56.0; HRMS (ESI,
positive) m/z: calcd for C15H13N2O+ [M − Br]+, 237.1022; found,
237.1026.
+
calcd for C14H10BrN2 [M − Br]+, 285.0022; found, 285.0011.
2-(3-Bromophenyl)phthalazin-2-ium Bromide (9). Yellow pow-
1
der; yield: 85% (1.56 g, 4.25 mmol); mp 208.5−209.0 °C; H NMR
2-(4-Methoxyphenyl)phthalazin-2-ium Bromide (17). Yellow
1
(500 MHz, DMSO-d6): δ 11.26 (s, 1H), 10.28 (s, 1H), 8.78 (d, J =
8.0 Hz, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.64−8.60 (m, 1H), 8.54−8.49
(m, 1H), 8.43 (t, J = 1.9 Hz, 1H), 8.18 (dd, J = 8.2, 1.5 Hz, 1H),
8.03−7.98 (m, 1H), 7.78 (t, J = 8.1 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6): δ 154.5, 151.7, 144.2, 140.0, 136.5, 134.3, 131.9, 131.4,
128.4, 127.9, 127.5, 126.7, 123.2, 122.2; HRMS (ESI, positive) m/z:
powder; yield: 88% (1.40 g, 4.40 mmol); mp 214.8−215.5 °C; H
NMR (500 MHz, DMSO-d6): δ 11.16 (s, 1H), 10.26 (s, 1H), 8.78 (d,
J = 8.0 Hz, 1H), 8.67 (d, J = 8.0 Hz, 1H), 8.57−8.53 (m, 1H), 8.49−
8.45 (m, 1H), 8.14−8.10 (m, 2H), 7.34−7.30 (m, 2H), 3.91 (s, 3H);
13C NMR (126 MHz, DMSO-d6): δ 161.4, 154.4, 149.6, 139.2, 136.3,
136.2, 131.0, 128.3, 128.2, 127.1, 125.3, 115.1, 56.0; HRMS (ESI,
positive) m/z: calcd for C15H13N2O+ [M − Br]+, 237.1022; found,
237.1025. The NMR data above were consistent with those in the
literature.20
+
calcd for C14H10BrN2 [M − Br]+, 285.0022; found, 285.0011.
2-(4-Bromophenyl)phthalazin-2-ium Bromide (10). White pow-
1
der; yield: 87% (1.59 g, 4.35 mmol); mp 271.8−272.4 °C; H NMR
(500 MHz, DMSO-d6): δ 11.23 (s, 1H), 10.27 (s, 1H), 8.78 (d, J =
8.0 Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.61 (t, J = 7.2 Hz, 1H), 8.50
(t, J = 7.2 Hz, 1H), 8.11 (d, J = 8.9 Hz, 2H), 8.05 (d, J = 8.9 Hz, 2H);
13C NMR (126 MHz, DMSO-d6): δ 154.5, 151.2, 142.4, 139.8, 136.5,
133.0, 131.3, 128.4, 128.0, 127.5, 125.9, 125.0; HRMS (ESI, positive)
2-(2-Trifluoromethylphenyl)phthalazin-2-ium Bromide (18).
Light yellow powder; yield: 89% (1.58 g, 4.45 mmol); mp 230.2−
231.0 °C; 1H NMR (500 MHz, DMSO-d6): δ 11.27 (s, 1H), 10.33 (s,
1H), 8.79 (d, J = 8.1 Hz, 1H), 8.76 (d, J = 8.0 Hz, 1H), 8.71 (t, J = 7.6
Hz, 1H), 8.59−8.55 (m, 1H), 8.22 (dd, J = 11.8, 8.0 Hz, 2H), 8.15 (t,
J = 7.6 Hz, 1H), 8.06 (t, J = 7.7 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6): δ 154.8, 154.3, 141.0, 140.1, 137.0, 134.6, 132.8, 131.6,
128.8, 128.7, 128.2, 128.1, 127.5, 127.2, 124.1, 123.8, 123.6, 121.4;
+
m/z: calcd for C14H10BrN2 [M − Br]+, 285.0022; found, 285.0029.
2-(2-Iodophenyl)phthalazin-2-ium Bromide (11). Light yellow
1
powder; yield: 75% (1.55 g, 3.75 mmol); mp 283.2−283.8 °C; H
+
NMR (500 MHz, DMSO-d6): δ 11.21 (s, 1H), 10.26 (s, 1H), 8.77 (d,
J = 8.1 Hz, 1H), 8.68 (d, J = 8.0 Hz, 1H), 8.62−8.58 (m, 1H), 8.52−
8.47 (m, 1H), 8.20 (d, J = 8.8 Hz, 2H), 7.94 (d, J = 8.8 Hz, 2H); 13C
NMR (126 MHz, DMSO-d6): δ 154.5, 151.0, 142.9, 139.8, 138.8,
136.4, 131.3, 128.4, 128.0, 127.5, 125.7, 99.0; HRMS (ESI, positive)
HRMS (ESI, positive) m/z: calcd for C15H10F3N2 [M − Br]+,
275.0791; found, 275.0790.
2-(3-Trifluoromethylphenyl)phthalazin-2-ium Bromide (19). Yel-
low powder; yield: 85% (1.51 g, 4.25 mmol); mp 209.5−210.3 °C; 1H
NMR (500 MHz, DMSO-d6): δ 11.35 (s, 1H), 10.31 (s, 1H), 8.80 (d,
J = 8.0 Hz, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.64 (t, J = 7.6 Hz, 1H),
8.57 (s, 1H), 8.55−8.51 (m, 1H), 8.49 (d, J = 8.1 Hz, 1H), 8.19 (d, J
= 7.9 Hz, 1H), 8.08 (t, J = 8.0 Hz, 1H); 13C NMR (126 MHz,
DMSO-d6): δ 154.5, 152.1, 143.7, 140.1, 136.6, 131.6, 131.5, 130.6,
130.3, 128.5, 128.3, 128.2, 127.9, 127.5, 124.5, 122.3, 121.2, 121.1;
+
m/z: calcd for C14H10IN2 [M − Br]+, 332.9883; found, 332.9883.
2-(2-Methylphenyl)phthalazin-2-ium Bromide (12). Yellow pow-
1
der; yield: 88% (1.33 g, 4.40 mmol); mp 183.5−183.8 °C; H NMR
(500 MHz, DMSO-d6): δ 11.20 (s, 1H), 10.27 (s, 1H), 8.79 (d, J =
8.0 Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.61−8.57 (m, 1H), 8.51−8.47
(m, 1H), 8.00 (s, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.68 (t, J = 7.9 Hz,
1H), 7.59 (d, J = 7.6 Hz, 1H), 2.51 (s, 3H); 13C NMR (126 MHz,
DMSO-d6): δ 154.5, 150.8, 143.2, 139.9, 139.6, 136.3, 132.0, 131.2,
129.8, 128.3, 128.1, 127.4, 124.1, 121.0, 20.8; HRMS (ESI, positive)
+
HRMS (ESI, positive) m/z: calcd for C15H10F3N2 [M − Br]+,
275.0791; found, 275.0805.
2-(4-Trifluoromethylphenyl)phthalazin-2-ium Bromide (20).
White powder; yield: 85% (1.51 g, 4.25 mmol); mp 263.0−264.3
°C; 1H NMR (500 MHz, DMSO-d6): δ 11.38 (s, 1H), 10.33 (s, 1H),
8.83 (d, J = 8.0 Hz, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.64 (t, J = 7.5 Hz,
1H), 8.53 (t, J = 7.5 Hz, 1H), 8.41 (d, J = 8.5 Hz, 2H), 8.24 (d, J = 8.6
Hz, 2H); 13C NMR (126 MHz, DMSO-d6): δ 154.6, 152.0, 145.9,
140.1, 136.5, 131.53, 131.46, 131.2, 128.4, 127.9, 127.6, 127.34,
127.31, 127.28, 127.25, 125.1, 124.6, 122.4; HRMS (ESI, positive) m/
+
m/z: calcd for C15H13N2 [M − Br]+, 221.1073; found, 221.1075.
2-(3-Methylphenyl)phthalazin-2-ium Bromide (13). Light yellow
1
powder; yield: 88% (1.33 g, 4.40 mmol); mp 205.6−206.2 °C; H
NMR (500 MHz, DMSO-d6): δ 11.01 (s, 1H), 10.27 (s, 1H), 8.77 (d,
J = 8.1 Hz, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.66−8.61 (m, 1H), 8.54−
8.49 (m, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.68 (td, J = 7.6, 1.0 Hz, 1H),
7.63−7.56 (m, 2H), 2.31 (s, 3H); 13C NMR (126 MHz, DMSO-d6):
δ 154.4, 153.8, 142.9, 139.8, 136.3, 133.0, 131.8, 131.4, 131.2, 128.4,
128.0, 127.5, 127.3, 126.3, 17.0; HRMS (ESI, positive) m/z: calcd for
+
z: calcd for C15H10F3N2 [M − Br]+, 275.0791; found, 275.0796.
2-(3-Cyanophenyl)phthalazin-2-ium Bromide (21). Light yellow
1
powder; yield: 88% (1.37 g, 4.40 mmol); mp 268.2−268.4 °C; H
+
C15H13N2 [M − Br]+, 221.1073; found, 221.1067.
NMR (500 MHz, DMSO-d6): δ 11.34 (s, 1H), 10.31 (s, 1H), 8.78 (d,
J = 8.0 Hz, 1H), 8.73−8.69 (m, 2H), 8.64 (t, J = 7.4 Hz, 1H), 8.55−
8.48 (m, 2H), 8.28 (d, J = 7.8 Hz, 1H), 8.04 (t, J = 8.1 Hz, 1H); 13C
NMR (126 MHz, DMSO-d6): δ 154.6, 152.1, 143.5, 140.2, 136.7,
135.1, 131.5, 131.5, 128.9, 128.5, 127.8, 127.7, 127.6, 117.4, 112.7;
2-(4-Methylphenyl)phthalazin-2-ium Bromide (14). Light yellow
1
powder; yield: 89% (1.34 g, 4.45 mmol); mp 217.8−218.9 °C; H
NMR (500 MHz, DMSO-d6): δ 11.21 (s, 1H), 10.28 (s, 1H), 8.80 (d,
J = 8.0 Hz, 1H), 8.68 (d, J = 8.0 Hz, 1H), 8.60−8.56 (m, 1H), 8.50−
8.46 (m, 1H), 8.06 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.3 Hz, 2H), 2.48
(s, 3H); 13C NMR (126 MHz, DMSO-d6): δ 154.5, 150.3, 141.7,
141.0, 139.4, 136.3, 131.1, 130.4, 128.3, 128.2, 127.4, 123.6, 20.7;
+
HRMS (ESI, positive) m/z: calcd for C15H10N3 [M − Br]+,
232.0869; found, 232.0838.
2-(2-Nitrophenyl)phthalazin-2-ium Bromide (22). Light yellow
+
1
HRMS (ESI, positive) m/z: calcd for C15H13N2 [M − Br]+,
powder; yield: 80% (1.33 g, 4.00 mmol); mp 243.4−244.3 °C; H
221.1073; found, 221.1068.
NMR (500 MHz, DMSO-d6): δ 11.38 (s, 1H), 10.30 (s, 1H), 8.82 (d,
J = 7.8 Hz, 1H), 8.72 (dt, J = 14.3, 7.4 Hz, 2H), 8.58 (t, J = 7.0 Hz,
1H), 8.51 (d, J = 8.0 Hz, 1H), 8.35 (d, J = 7.7 Hz, 1H), 8.23 (t, J = 7.2
Hz, 1H), 8.10 (t, J = 7.4 Hz, 1H). 13C NMR (126 MHz, DMSO): δ
154.7, 154.3, 142.8, 141.0, 137.0, 135.9, 135.7, 133.5, 131.8, 128.9,
128.8, 127.5, 127.4, 126.6; HRMS (ESI, positive) m/z: calcd for
2-(4-Ethylphenyl)phthalazin-2-ium Bromide (15). Light yellow
1
powder; yield: 86% (1.36 g, 4.30 mmol); mp 196.5−197.5 °C; H
NMR (500 MHz, DMSO-d6): δ 11.2 (s, 1H), 10.3 (s, 1H), 8.8 (d, J =
8.0 Hz, 1H), 8.7 (d, J = 8.1 Hz, 1H), 8.6−8.5 (m, 1H), 8.5−8.4 (m,
1H), 8.1 (d, J = 8.6 Hz, 2H), 7.6 (d, J = 8.5 Hz, 2H), 2.8 (q, J = 7.6
Hz, 2H), 1.3 (t, J = 7.6 Hz, 3H); 13C NMR (126 MHz, DMSO-d6): δ
154.5, 150.4, 147.8, 141.2, 139.5, 136.3, 131.1, 129.3, 128.3, 128.2,
127.4, 123.7, 27.8, 15.4; HRMS (ESI, positive) m/z: calcd for
+
C14H10N3O2 [M − Br]+, 252.0768; found, 252.0765.
2-(3-Nitrophenyl)phthalazin-2-ium Bromide (23). Light yellow
1
powder; yield: 88% (1.46 g, 4.40 mmol); mp 258.8−259.6 °C; H
+
C16H15N2 [M − Br]+, 235.1230; found, 235.1222.
NMR (500 MHz, DMSO-d6): δ 11.42 (s, 1H), 10.34 (s, 1H), 9.03 (s,
1H), 8.82 (d, J = 7.9 Hz, 1H), 8.73 (d, J = 7.9 Hz, 1H), 8.68−8.58
(m, 3H), 8.54 (t, J = 7.2 Hz, 1H), 8.12 (t, J = 8.2 Hz, 1H); 13C NMR
2-(3-Methoxyphenyl)phthalazin-2-ium Bromide (16). Light
yellow powder; yield: 81% (1.28 g, 4.05 mmol); mp 188.4−189.3
C
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(126 MHz, DMSO-d6): δ 154.6, 152.4, 148.2, 143.6, 140.3, 136.7,
Petri dish (ca. 15 mL for each plate) in a laminar flow chamber. The
concentration of the compound in PDA was 50 μg/mL. The
commercial fungicide standard azoxystrobin at 50 μg/mL in culture
medium and 0.5% DMSO in culture medium without the test
compounds were used as a positive control and a blank control,
respectively. SA and CH were used as the reference controls. When
the medium became semisolidified, a mycelial dish (d = 5 mm) cut
from the subcultured medium was inoculated in the center of the
medium under sterile conditions. The dishes were incubated at 28 °C
for ca. 72 h. Each treatment was repeated three times. Each colony
diameter (in mm) was measured three times by the cross-bracketing
method. The growth inhibition rates were calculated according to the
method reported6 and expressed as means standard deviation.
Based on the initial screening results, the more active compounds
were further measured for median effective concentrations (EC50) by
using the same method as described above. A set of stock solutions of
compounds in 5% DMSO aqueous solution was prepared by the
double-fold dilution method. Each stock solution (10 mL) was mixed
with autoclaved liquid PDA medium (190 mL) to provide a series of
culture media with various concentrations (50, 30, 25, 20, 12.5, 6.25,
3.13, 1.56, and 0.78 μg/mL) of the compound. Toxicity regression
equations were established according to a method previously
reported.22 The EC50 value and 95% confidence interval were
calculated from the equations using Prism 7 software (GraphPad
Software Inc., San Diego, CA, USA).
Antifungal Activity Assay in Vivo. As a representative
compound, the most active compound 27 was further tested for the
inhibition activity in vivo against C. gloeosporioides on apples. The
same kind of ripened apples with similar shapes were used as test
materials. The apples were disinfected in 1% sodium hypochlorite
solution for 2 min, and then rinsed with running water for 5 min.
After the surface of the apples dried naturally, a hole with a diameter
of 5 mm and a depth of 3 mm was punched on the surface of the
apple using a sterile hole puncher. After the hole dried naturally, 40
μL of a solution of 27 in acetone (four concentrations: 50, 25, 12.5,
and 6.25 μg/mL) was added to each hole. After the acetone in the
hole evaporated naturally, a mycelial dish (d = 5 mm) cut from the
subcultured medium was inoculated in the hole under sterile
conditions. The apples were placed in an incubator with 70%
humidity at 28 °C for 9−10 days. Acetone and sterile distilled water
were used as a blank control and a negative control, respectively. Each
treatment was repeated three times.
131.8, 131.6, 130.4, 128.5, 127.8, 127.6, 126.1, 119.3; HRMS (ESI,
+
positive) m/z: calcd for C14H10N3O2 [M − Br]+, 252.0768; found,
252.0776.
2-(4-Nitrophenyl)phthalazin-2-ium Bromide (24). Light yellow
1
powder; yield: 88% (1.46 g, 4.40 mmol); mp 273.6−274.3 °C; H
NMR (500 MHz, DMSO-d6): δ 11.4 (s, 1H), 10.3 (s, 1H), 8.8 (d, J =
8.0 Hz, 1H), 8.7 (d, J = 8.0 Hz, 1H), 8.6 (t, J = 7.9 Hz, 3H), 8.5 (t, J =
7.6 Hz, 1H), 8.4 (d, J = 9.0 Hz, 2H); 13C NMR (126 MHz, DMSO-
d6): δ 154.5, 152.4, 148.8, 147.0, 140.3, 136.6, 131.6, 128.5, 127.9,
127.6, 125.5, 125.4; HRMS (ESI, positive) m/z: calcd for
+
C14H10N3O2 [M − Br]+, 252.0768; found, 252.0751.
2-(2,3-Dichlorophenyl)phthalazin-2-ium Bromide (25). Light
yellow powder; yield: 84% (1.50 g, 4.20 mmol); mp 224.7−225.6
°C; 1H NMR (500 MHz, DMSO-d6): δ 11.20 (s, 1H), 10.35 (s, 1H),
8.79 (d, J = 8.1 Hz, 1H), 8.75 (d, J = 8.1 Hz, 1H), 8.70 (t, J = 7.5 Hz,
1H), 8.12 (t, J = 9.0 Hz, 2H), 7.83 (t, J = 8.1 Hz, 1H); 13C NMR
(126 MHz, DMSO-d6): δ 155.2, 154.8, 142.0, 140.9, 136.9, 133.7,
133.2, 131.7, 129.5, 128.8, 127.6, 127.4, 127.4, 127.3; HRMS (ESI,
+
positive) m/z: calcd for C14H9Cl2N2 [M − Br]+, 275.0137; found,
275.0139.
2-(2,4-Dichlorophenyl)phthalazin-2-ium Bromide (26). Light
yellow powder; yield: 88% (1.57 g, 4.40 mmol); mp 245.6−246.4
°C; 1H NMR (500 MHz, DMSO-d6): δ 11.20 (s, 1H), 10.35 (s, 1H),
8.79 (d, J = 7.8 Hz, 1H), 8.75 (d, J = 7.8 Hz, 1H), 8.69 (t, J = 7.1 Hz,
1H), 8.55 (t, J = 7.0 Hz, 1H), 8.21 (s, 1H), 8.14 (d, J = 8.6 Hz, 1H),
7.93 (d, J = 7.2 Hz, 1H); 13C NMR (126 MHz, DMSO-d6): δ 155.2,
154.8, 140.9, 139.6, 137.2, 136.8, 131.6, 130.5, 129.7, 129.5, 129.1,
128.7, 127.6, 127.4; HRMS (ESI, positive) m/z: calcd for
+
C14H9Cl2N2 [M − Br]+, 275.0137; found, 275.0136.
2-(2,5-Dichlorophenyl)phthalazin-2-ium Bromide (27). Light
yellow powder; yield: 79% (1.41 g, 3.95 mmol); mp 240.8−241.8
°C; 1H NMR (500 MHz, DMSO-d6): δ 11.23 (s, 1H), 10.35 (s, 1H),
8.78 (d, J = 7.9 Hz, 1H), 8.75 (d, J = 8.1 Hz, 1H), 8.70 (t, J = 7.1 Hz,
1H), 8.56 (t, J = 7.0 Hz, 1H), 8.34 (s, 1H), 8.00 (d, J = 8.7 Hz, 1H),
7.96 (d, J = 7.0 Hz, 1H); 13C NMR (126 MHz, DMSO-d6): δ 155.4,
154.7, 141.1, 141.0, 136.9, 133.2, 132.6, 132.4, 131.7, 128.8, 128.5,
127.6, 127.4, 127.3; HRMS (ESI, positive) m/z: calcd for
+
C14H9Cl2N2 [M − Br]+, 275.0137; found, 275.0135.
2-(2,6-Dichlorophenyl)phthalazin-2-ium Bromide (28). Light
yellow powder; yield: 85% (1.51 g, 4.25 mmol); mp 226.6−227.5
°C; 1H NMR (500 MHz, DMSO-d6): δ 11.40 (s, 1H), 10.50 (s, 1H),
8.85 (d, J = 7.7 Hz, 1H), 8.80 (t, J = 9.5 Hz, 2H), 8.61 (d, J = 6.7 Hz,
1H), 8.00 (d, J = 8.0 Hz, 2H), 7.90 (t, J = 8.0 Hz, 1H); 13C NMR
(126 MHz, DMSO-d6): δ 156.4, 156.2, 142.1, 137.9, 137.4, 134.4,
132.3, 130.9, 129.9, 129.3, 127.8, 127.1; HRMS (ESI, positive) m/z:
Analysis of Mycelial Morphology and Cell Ultrastructure. C.
gloeosporioides was chosen as the test fungus; 27 as the most active
compound was selected as the test compound. The mycelium was
grown on PDA plates with or without 4.03 μg/mL compound 27 for
72 h at 28 °C.
+
calcd for C14H9Cl2N2 [M − Br]+, 275.0137; found, 275.0134.
2-(3,4-Dichlorophenyl)phthalazin-2-ium Bromide (29). Light
yellow powder; yield: 88% (1.57 g, 4.40 mmol); mp 242.8−243.3
°C; 1H NMR (500 MHz, DMSO-d6): δ 11.25 (s, 1H), 10.27 (s, 1H),
8.75 (d, J = 8.1 Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.63 (t, J = 7.6 Hz,
1H), 8.55−8.49 (m, 2H), 8.19−8.11 (m, 2H); 13C NMR (126 MHz,
DMSO-d6): δ 154.5, 151.8, 142.4, 140.2, 136.6, 134.5, 132.4, 132.0,
131.5, 128.5, 127.8, 127.5, 125.9, 124.3; HRMS (ESI, positive) m/z:
SEM of hyphal morphological and ultrastructural changes of C.
gloeosporioides was conducted according to the method described by
Wang et al.23 Mycelium disks (5 mm diameter) were taken from the
periphery of the colony grown on PDA medium and fixed in 2.5%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 24 h at 4 °C.
After a wash with the same buffer, the disk was dehydrated in a
gradient series of ethanol (30, 50, 70, 80, and 90%, v/v, each one
time, and finally, 100%, two times) for 15 min per time. The resulted
disk was critical point-dried and gold-coated. SEM observations were
performed with a Hitachi S-4800 scanning electron microscope at an
accelerating voltage of 10 kV.
TEM observation was conducted according to the method
described by Shang et al.24 A small piece of mycelia (2 mm diameter)
grown on PDA medium was sampled, prefixed in 2.5% glutaraldehyde
in 0.1 M phosphate buffer (pH 7.2) for 24 h at 4 °C, washed with the
same buffer, and postfixed in 1% osmium tetroxide. Next, the mycelia
were rinsed in phosphate buffer, dehydrated in a gradient series of
ethanol (30, 50, 70, 80, and 90%, v/v) for 10 min each time, followed
by three consecutive 20 min periods in 100% ethanol, then infiltrated
with increasing concentrations of White’s resin (London Resin
Company Ltd.), and finally embedded in dry capsules filled with pure
White’s resin and polymerized at 60 °C for 2 days. Sections were cut
to 70 nm thickness using an ultramicrotome (Leica EM UC7)
+
calcd for C14H9Cl2N2 [M − Br]+, 275.0137; found, 275.0137.
2-(3,5-Dichlorophenyl)phthalazin-2-ium Bromide (30). Yellow
1
powder; yield: 84% (1.50 g, 4.20 mmol); mp 259.8−260.3 °C; H
NMR (500 MHz, DMSO-d6): δ 11.37 (s, 1H), 10.31 (s, 1H), 8.78 (d,
J = 7.7 Hz, 1H), 8.72 (d, J = 7.7 Hz, 1H), 8.65 (d, J = 6.9 Hz, 1H),
8.53 (d, J = 7.2 Hz, 1H), 8.32 (s, 2H), 8.13 (s, 1H); 13C NMR (126
MHz, DMSO-d6): δ 154.5, 152.3, 144.6, 140.4, 136.7, 135.2, 131.7,
131.1, 128.6, 127.8, 127.6, 123.2; HRMS (ESI, positive) m/z: calcd
+
for C14H9Cl2N2 [M − Br]+, 275.0137; found, 275.0138.
Antifungal Activity Assay in Vitro. Compounds 1−30 were
screened for inhibition activity on eight strains of plant pathogenic
fungi by the mycelium linear growth rate method reported.6 Briefly,
PDA was prepared and sterilized with an autoclave. The compound
(10 mg) was dissolved in 0.5 mL of DMSO, and diluted with 9.5 mL
of sterile water. The resulting solution was fully mixed with molten
PDA (190 mL) at ca. 50 °C and then was poured into a sterilized
D
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a
Scheme 1. Synthesis of 2-Substituted Phenylphthalazin-2-ium Bromides
a
Reagents and conditions: (a) phenylhydrazine-HCl, 1,4-dioxane, 3−4 days at rt; (b) treated with Na2CO3/H2O; and (c) treated with 40% HBr.
equipped with a diamond knife and then stained using uranyl acetate
Synthesis of Compounds. Target compounds 1−30 were
synthesized according to a method reported in the literature.20
and lead citrate and observed with a transmission electron microscope
(Tecnai G2 Spirit BioTWIN, FEI Corporation) at 80 kV. The
experiment was performed in triplicate.
The synthetic route is outlined in Scheme 1. Commercially
available o-phthalaldehyde reacted with substituted or
unsubstituted phenylhydrazine hydrochlorides in 1,4-dioxane
RESULTS AND DISCUSSION
at rt to afford 2-arylphthalazin-2-ium chlorides in >80% yield.
The chlorides were transferred to the corresponding pseudo-
bases by treatment with sodium carbonate aqueous solution.
The pseudo-bases without purification were treated with
aqueous hydrobromic acid to give the corresponding bromide
salts C. The synthetic route mentioned above has some
obvious advantages such as high yields, no harsh reaction
conditions, and a simple workup operation.
■
Design of Target Compounds. Our previous studies
showed that the analogue A possesses a variety of excellent
pharmacological activities of QBAs such as antimicro-
bial,8,13−16 anticancer,25−27 acaricidal,28,29 and anti-acetylcho-
linesterase activities.30 Therefore, the analogue A can be used
as the secondary lead of QBAs to develop novel QBA-like
antifungal agents.13−16
The structures of the target compounds were confirmed by
The phthalazine skeleton has been found to be a remarkable
scaffold in drug discovery.31 A lot of phthalazine-containing
compounds showed potent antimicrobial activity. Therefore,
the phthalazine skeleton can be considered as a promising
antimicrobial pharmacophore.32,33 Based on the principle of
analogue design and the hybridized combination strategy of
double pharmacophores, we designed one novel 2-phenyl-
phthalazin-2-ium skeleton (C) (Figure 1) by replacing the 3,4-
dihydropyridine moiety of the analogue A (Figure 1) with a
pyridazine unit. The skeletons C and A possess some obvious
similarities such as the same molecular length and width, the
presence of one polar iminium moiety (CN+), and planar or
approximately planar molecules, suggesting that the two
compounds should have a similar pharmacological activity. It
was expected that the structural similarity and the presence of
double pharmacophores could endow the target compounds
with more potent antifungal activity.
Our previous studies on the antifungal activity of compound
A found that the substitution pattern of the C-ring is able to
significantly affect the activity. Theoretically, therefore, the
structural similarity of analogues C should show similar
structure−activity effects. Thus, we synthesized analogues C
with a large variety of substituents on the C-ring. The
substituents include a variety of electron-withdrawing groups
(EWGs) and electron-donating groups (EDGs), such as
halogens, trifluoromethyl, cyano, methyl, ethyl, and methoxy.
The substitution sites encompass the ortho-, meta-, and para-
positions. These substituents (especially o-substituents) can
not only affect the electron density distribution of the target
compounds but also affect the conformation of the bond
between the B- and C-rings.
1
1H NMR, 13C NMR, and HR-ESI-MS analyses. In their H
NMR and 13C NMR spectra, all compounds showed similar
spectroscopic characteristics, as expected. Each compound
revealed two singlets (1H each) in the range of δH 11.4−10.3
due to H-1 and H-4 and two carbon signals in the range of δC
155−150 due to C-1 and C-4. For the positive ESI-MS spectra,
all the compounds showed a characteristic ion peak of [M −
Br]+. For the negative ESI-MS spectra, each compound also
showed two ion peaks of [79Br−] and [81Br−].
Antifungal Activity in Vitro. According to the mycelium
linear growth rate method,6 compounds 1−30 were screened
for inhibition activity in vitro at 50 μg/mL on eight strains of
plant pathogenic fungi, F. oxysporum f. sp. niveum, F. solani, A.
alternata, C. gloeosporioides, F. bulbigenum, A. solani, A.
brassicae, and P. oryzae.
The activity screening found that most of the compounds
displayed definite inhibition activity against all the fungi at 50
μg/mL (see the Supporting Information). As for all the
compounds, 74.6% of the trials gave good to excellent activity
with inhibition rates of 50 to 100%. Among them, 19
compounds (2, 3, 5−9, 18, 19, and 21−30) were more active
against all or most of the fungi than azoxystrobin, a fungicide
standard. In terms of the test fungi, F. oxysporum f. sp. niveum,
F. solani, A. alternata, C. gloeosporioides, and F. bulbigenum
showed higher susceptibility to the compounds.
In order to understand in more detail the antifungal
potential of the compounds and the SAR, the compounds
with inhibition rates of >50% at 50 μg/mL were further
measured for median effective concentrations (EC50). The
commercial fungicide standard azoxystrobin was used as the
E
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a
Table 1. EC50 Vales of Target Compounds against Eight Strains of Fungi
EC50 vales (95% confidence interval) (μg/mL)
CG FB
b
no.
FON
FS
AA
AS
AB
PO
TAI
1
24.3
(19.4−32.2)
9.52
(8.49−10.8)
9.36
(7.80−11.5)
14.8
(13.0−17.0)
7.50
(6.38−8.91)
18.5
(12.7−33.7)
15.7
(11.1−24.5)
6.86
(5.65−8.38)
>50
>50
>50
>50
>50
>50
18.1
(16.3−20.2)
20.5
(19.4−21.7)
>50
>50
18.0
(16.5−19.7)
15.9
(15.0−16.9)
≈50
2
3
4
5
6
7
8
9
1.74
(1.49−2.00)
2.27
(1.92−2.63)
>50
7.84
(6.68−9.16)
8.47
(7.45−9.67)
48.7
(42.0−57.9)
6.28
(5.74−6.86)
6.66
(6.05−7.30)
10.3
(8.95−12.0)
7.38
(6.01−8.98)
24.5
13.2
15.1
2.64
2.82
(17.9−38.4)
7.07
(6.26−8.03)
>50
(12.5−13.8)
23.9
(21.5−27.0)
≈50
(14.0−16.4)
5.32
(4.59−6.04)
25−50
1.07
9.67
(7.69−13.1)
6.83
(5.73−8.18)
7.99
(6.63−9.81)
9.90
(7.40−15.7)
10.3
(9.57−11.0)
9.58
(8.92−10.2)
10.9
(10.3−11.5)
11.3
(10.7−12.0)
10.6
(9.72−11.4)
11.1
(9.91−12.3)
8.15
(6.95−9.34)
6.08
(5.18−6.96)
9.51
(8.68−10.3)
15.1
(14.3−15.9)
14.0
(13.2−14.9)
10.9
(10.1−11.6)
10.8
(10.2−11.6)
14.1
(13.4−14.8)
18.9
(18.3−19.4)
11.8
(10.9−12.6)
3.30
2.80
2.51
3.11
3.09
(0.97−1.18)
2.38
(1.43−3.32)
5.03
(3.97−6.10)
1.79
(1.39−2.18)
15.2
(14.1−16.5)
1.66
(1.10−2.26)
7.18
(6.88−7.49)
5.29
(4.48−6.20)
18.3
(16.9−19.8)
5.68
(4.77−6.57)
11.5
(10.8−12.2)
10.9
(10.2−11.7)
10
11
>50
>50
≈17.3
13.7
(11.3−17.2)
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
12
13
14
15
16
≈36.3
25−50
>50
>50
>50
≈16.52
>50
>50
≈50
19.6
≈41.91
≈50
>50
≈39.1
≈44.6
>50
>50
>50
≈50
>50
≈50
>50
>50
≈50
>50
25−50
>50
>50
>50
25−50
>50
>50
>50
>50
≈50
25−50
>50
>50
25−50
25−50
(15.6−25.1)
17
18
>50
10.1
(8.71−11.8)
16.2
(15.7−16.8)
>50
3.22
(2.87−3.59)
4.59
(3.95−5.30)
>50
24.2
(20.2−30.1)
7.29
(6.76−7.88)
>50
16.6
(15.1−18.1)
6.95
(6.61−7.33)
>50
14.9
(12.4−18.2)
14.8
(13.2−16.3)
>50
24.9
(20.1−32.9)
10.6
(9.37−11.8)
>50
35.4
(27.8−48.6)
14.3
(13.6−15.0)
>50
24.3
(21.6−27.8)
9.36
(8.29−10.4)
2.15
2.62
19
20
21
>50
28.9
(28.1−29.7)
≈39.8
≈20.4
5.08
(4.03−6.28)
7.16
(6.28−8.12)
2.82
(2.50−3.15)
8.02
(7.14−8.99)
2.58
(1.99−3.29)
2.10
(1.68−2.55)
2.03
(1.74−2.34)
2.89
(2.64−3.15)
6.50
(5.51−7.68)
5.80
(5.09−6.61)
41.7
(31.6−54.9)
26.5
(15.9−44.2)
5.56
(5.22−5.89)
11.2
>50
9.81
(8.75−10.9)
15.2
(12.7−18.5)
6.46
(6.08−6.87)
7.84
(7.33−8.38)
6.28
(5.58−7.03)
3.83
(3.65−4.02)
4.23
(3.72−4.78)
8.16
(7.48−8.86)
8.40
(7.17−9.82)
9.15
(8.03−10.3)
≈50
6.41
(5.73−7.11)
17.7
(14.8−21.7)
4.19
(3.49−5.16)
7.39
(5.25−9.85)
5.89
(4.30−9.02)
4.52
(3.90−5.33)
4.03
(2.86−5.52)
10.8
(9.81−12.0)
5.81
(5.37−6.28)
12.6
(11.6−13.6)
≈50
27.6
(25.7−29.9)
42.5
(38.9−47.4)
26.9
(24.8−29.6)
46.7
(42.1−53.2)
5.91
(5.30−6.51)
6.50
(6.18−6.82)
9.30
(8.35−10.2)
9.02
(7.93−10.1)
14.0
(12.7−15.4)
16.6
(15.4−17.9)
>100
>100
3.13
(2.89−3.36)
5.13
≈50
8.24
(6.88−9.51)
25.3
(23.4−27.5)
5.70
(4.87−6.53)
6.23
(5.21−7.22)
3.97
(3.08−4.86)
1.79
(1.27−2.33)
2.75
(2.09−3.42)
8.90
(7.69−10.1)
3.29
(2.61−3.97)
7.12
(6.16−8.07)
>50
15.2
>50
12.7
2.42
1.79
2.95
2.54
3.42
4.12
3.99
2.91
2.90
2.54
(14.3−16.1)
20.9
(19.4−22.6)
12.7
(11.5−13.9)
7.31
(6.35−8.25)
9.58
(9.05−10.1)
3.72
(2.99−4.45)
3.75
(3.42−4.07)
11.1
(9.99−12.3)
9.83
(9.00−10.7)
8.22
(7.17−9.26)
(12.4−13.0)
26.8
(25.5−28.4)
8.77
(7.95−9.59)
11.3
(10.3−12.3)
9.12
(8.75−9.50)
5.23
(4.96−5.50)
4.41
(3.86−4.95)
8.93
(8.19−9.68)
6.20
(5.42−6.96)
7.28
(6.71−7.84)
101
(72.6−141)
46.7
(39.0−55.9)
22
23
24
25
26
27
28
29
30
SA
17.2
(14.4−21.5)
16.4
(13.8−19.8)
5.68
(4.80−6.91)
8.12
(6.46−11.2)
6.94
(6.29−7.68)
7.45
(6.86−8.12)
24.0
(19.6−30.8)
33.3
(27.1−44.2)
50.2 (43−58)
c
53.0 (28−100) >100
≈100
≈100
≈100
>50
d
CH
28.9
(23.8−35.1)
3.86
(3.42−4.31)
49.5 (21−114) 50−100
50−100
e
AZ
29.3
(26.0−33.5)
6.86
>50
9.89
(8.63−11.2)
7.46
>50
f
TSI
5.43
6.88
5.79
5.87
a
FON, F. oxysporum f. sp. niveum; FS, F. solani; AA, A. alternata; CG, C. gloeosporioides; FB, F. bulbigenum; AS, A. solani; AB, A. brassicae; and PO, P.
oryzae. TAI: total activity index of the specific compound on all the fungi, equal to n (1/ EC50
)
n. Sanguinarine. Chelerythrine. Azoxystrobin.
b
c
d
e
∑
1
TSI: total susceptibility index of the specific fungus to compounds 2, 3, 5−9, 18, 19, and 21−30, equal to n (1/ EC50
)
.
f
∑
n
1
positive control. SA and CH were used as the reference
controls. The results are listed in Table 1.
EC50 values in Table 1 clearly show that 19 (2, 3, 5−9, 18,
19, and 21−30) out of the 30 compounds showed excellent
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Journal of Agricultural and Food Chemistry
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antifungal activity, not only far superior to model compounds
SA and CH on almost all the fungi but also superior to the
positive drug azoxystrobin (EC50 = 5.56 to >50 μg/mL) on
most of the fungi (F. solani, A. alternata, C. gloeosporioides, A.
solani, A. brassicae, and P. oryzae).
In order to compare the total activity of the different
compounds against all the test fungi, herein we introduce a
concept of the activity index (AI), which is defined as
( EC50 )−1. For the test fungi, AI may also be called
susceptibility index (SI). The AI or SI is positively correlated
with the activity intensity of the compounds or the
susceptibility of the test fungi to the test compound.
Furthermore, AI or SI may also represent the level of the
importance of the activity strength of the compounds. The
total activity of one compound on multiple fungi may be
represented by using the sum of the AIs of the compound on
each of the fungi, that is, the total activity index (TAI, equal to
∑ni=1AIi). Similarly, the total susceptibility of the specific
fungus to a group of compounds may also be represented by
using the sum of SIs (TSI) of the fungus on each of the
compounds. Compared with the average EC50 values, TAI can
better reflect the total activity of the compounds and the total
susceptibility of the fungi.
treatments with 27 at 6.25, 12.5, 25.0, and 50.0 μg/mL had
obvious inhibition efficacy on the infection of C. gloeosporioides
(see the Supporting Information). This inhibition phenomen-
on became more obvious over time. The treatment with 25.0
μg/mL 27 almost completely inhibited the growth of the fungi
over the post-treatment 9th day. On the other hand, the
inhibition efficacy of 27 showed concentration dependency.
The results mentioned above, in combination with those in
Table 1, suggest that 2-phenylphthalazin-2-iums are very
promising candidate compounds for the development of novel
antifungal agents.
Structure−Activity Relationship. Comparison of the
EC50 values in Table 1 and/or the inhibition rates at 50 μg/mL
(see the Supporting Information) showed that the substitution
pattern of the C-ring significantly affects the activity (Figure
2). The general trend is that the presence of EWGs such as
Among all the compounds, 26, 27, 25, and 5 possessed the
top four highest TAIs (=4.12, 3.99, 3.42, and 3.30) for all the
eight fungi (see Figure 1 in the Supporting Information),
where 26 showed the most potent activity on A. alternata, A.
solani, and A. brassicae (EC50 = 3.83, 1.79, 3.72 μg/mL) (Table
1); 27 was most active against C. gloeosporioides, A. brassicae,
and P. oryzae (EC50 = 4.03, 3.75, 4.41 μg/mL); 25 had the
highest activity against F. oxysporum f. sp. niveum, and F.
bulbigenum (EC50 = 5.68, 5.91 μg/mL); and 5 was most active
against F. solani (EC50 = 1.07 μg/mL).
Among the eight fungi, F. solani showed the highest
sensitivity (TSI = 11.2) to 19 compounds (2, 3, 5−9, 18,
19, and 21−30), A. solani, C. gloeosporioides, and A. alternata
showed moderate sensitivity (TSIs = 7.46 to 6.86), and P.
oryzae, A. brassicae, F. oxysporum f. sp. niveum, and F.
bulbigenum had lower sensitivity (TSIs = 5.38 to 4.65) (see
the Supporting Information). The sensitivity order of the eight
fungi is F. solani (TSI = 11.2) ≫ A. solani (TSI = 7.46) > C.
gloeosporioides (TSI = 6.88) ≈ A. alternata (TSI = 6.86) > P.
oryzae (TSI = 5.87) ≈ A. brassicae (TSI = 5.79) > F. oxysporum
f. sp. niveum (TSI = 5.43) ≈ F. bulbigenum (TSI = 5.13). For
the most sensitive fungus, F. solani, 15 (2, 3, 5−9, 18, 19, 21,
23, and 25−28) out of the 19 compounds (EC50 = 1.07 to 5.08
μg/mL) were more active than azoxystrobin (EC50 = 5.56 μg/
mL) (Table 1). For the moderately sensitive fungi, A. solani, C.
gloeosporioides, and A. alternata, 13 (3, 8, 9, 21, and 23−29)
out of the 19 compounds gave higher activity (EC50 = 1.79 to
9.90 μg/mL) than azoxystrobin on each of the fungi. For each
of the low-sensitive fungi, P. oryzae, A. brassicae, F. oxysporum f.
sp. niveum, and F. bulbigenum, there were still five to eight
compounds that gave higher activity (EC50 < 10 μg/mL) than
azoxystrobin on each of the fungi.
Figure 2. SAR of compounds 1−24.
halogen atoms, trifluoromethyl, nitro, and cyano significantly
increases the activity for all or most of the fungi, whereas
EDGs such as Me, Et, and MeO cause a relatively small change
in activity in most cases.
For halogen atoms as weak EWGs, o- and m-substitution
can dramatically increase the activity against all the fungi (2, 3,
5, 6, 8, and 9 vs 1). By contrast, p-halogen atom (F, Br, and I)
substitution only makes little effect on the activity in most
cases (4, 10, and 11 vs 1). The activity order of the three
fluorinated (2−4) or brominated (8−9) isomers is the m-
isomer ≈ the o-isomer ≫ the p-isomer. The exception is that
the presence of chlorine atom at the o-, m-, or p-site causes
remarkable improvement of the activity (5−7 vs 1).
Strong EWGs, trifluoromethyl and nitro at the o-, m-, or p-
site, can improve the activity, but the two substituents have
different position effect trends. The three trifluoromethyl-
substituted isomers displayed the activity order of the m-
isomer (19, TAI = 2.62) > the o-isomer (18, TAI = 2.15) ≫
the p-isomer (20, TAI < 1.2), whereas the activity order of the
nitro-substituted isomers is the m-isomer (23, TAI = 2.95) >
the p-isomer (24, TAI = 2.54) > the o-isomer (22, TAI =
1.79). Additionally, m-cyano (21), as a strong EWG, can also
greatly increase the activity against almost all the fungi.
The enhanced activity of single chlorine atom analogues (5−
6), compared with compound 1 (R = H, TAI < 1.2), is also
seen dramatically with the presence of two chlorine atom
analogues (25−30, TAIs = 2.54 to 4.12) for almost all the
fungi. However, compared with the corresponding mono-
chlorides (5−6, TAIs < 3.31), the effect of the two chlorine
atoms in the dichlorides (25−30) on the activity varies with
the relative positions of the two chlorine atoms and the type of
It was worth mentioning that compared with the analogues
A6,13−16 and B,17,18 compound C had more potent activity and
a broader antifungal spectrum.
Antifungal Activity in Vivo. Compound 27, with the
highest in vitro activity (EC50 = 4.03 μg/mL) against C.
gloeosporioides, was further tested in vivo to assay the infectivity
of C. gloeosporioides on apples. The results showed that all the
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Article
Figure 3. Electron microscopic observation of hyphal morphological and ultrastructural changes of C. gloeosporioides treated with 4.03 μg/mL 27
(EC50) for 72 h at 28 °C (C−E,H−J). (A,B,F,G) Control group untreated with 27. (A−E) SEM. (F−J) TEM.
the fungi. Compared with the corresponding monochlorinated
compounds, the 2′,3′-dichloro (25), 2′,4′-dichloro (26), or
2′,5′-dichloro (27) combination (TAIs = 3.42 to 4.12) can
enhance the inhibition activity on most of the fungi, whereas
the 2′,6′-dichloro (28), 3′,4′-dichloro (29), or 3′,5′-dichloro
(30) combination (TAIs < 2.92) has only a slight effect on the
activity on most of the fungi. It is worth noting that all or most
of the dichloro combinations (25−30) can significantly
improve the inhibition activity on C. gloeosporioides, F.
bulbigenum, A. solani, A. brassicae, and P. oryzae, especially A.
solani and P. oryzae. In general, 2′,3′-dichloride (25), 2′,4′-
dichloride (26), and 2′,5′-dichloride (27) showed the highest
total activity (see the Supporting Information).
Unlike EWGs, the presence of EDGs such as Me, Et, and
MeO only causes a slight change (increase or decrease) of the
activity for most of the fungi, whether it is at the o-, m-, or p-
position. Compared with EWGs, the effect of EDGs on the
activity is more complex. As for methyl, the o-substitution
slightly increases the activity in most cases, while m- and p-
substitution decreases the activity. Like p-methyl, p-methoxy
also causes decrease in the activity in most cases, but m-
methoxy can increase the activity. Unlike p-methyl and p-
methoxy, the presence of p-ethyl can increase the activity in
most cases.
Most organelles were indistinct. Large cytoplasmic blebs
appeared in the cell (Figure 3H−J).
The results above indicated that 27 was able to damage the
integrity of the mycelial structure by affecting the structure of
the cell membrane of mycelium. In the treated cells, the lack of
organelles may be related to the change of the permeability of
the cell membrane, the separation of the cell membrane and
wall, and the leakage of the cell contents. Thus, the damage
effect on the cell membrane may be considered as the main
reason for the inhibition action of 27 on the fungi.
In summary, a novel and simple antifungal 2-phenyl-
phthalazin-2-ium scaffold was identified. Systematic structural
optimization and SAR studies led to the discovery of 19 2-
phenylphthalazin-2-ium derivatives with potent inhibition
activity in vitro against eight strains of phytopathogenic fungi.
26 and 27 showed the higher activity against each of the fungi.
F. solani showed the highest susceptibility to the compounds
among all the fungi. Tests in vivo revealed that 27 can
completely control C. gloeosporioides infections in apples at
25.0 μg/mL. Electron microscopic observation showed that 27
can damage the integrity of the mycelial structure and affect
the structure of the cell membrane and the tight junction
between the cell membrane and wall. SAR showed that the
presence of EWGs, especially chlorine atoms, on the C-ring
can dramatically increase the activity against all the fungi.
Compared with the natural model compounds, SA and CH,
the present compounds have some obvious advantages, such as
more potent activity, wider antifungal spectrum, simpler
structure, easier synthesis and modification, and lower cost.
Thus, the 2-phenylphthalazin-2-ium scaffold can be considered
a promising lead to develop novel QBA-like fungicides for
plant protection. 26 and 27 represent promising candidates for
the development of novel antifungal agents. Safety evaluations
of the compounds on plants and animals are currently
underway in our laboratories.
In general view, the effect of EWGs on the activity is much
stronger than EDGs, and the former is much more beneficial
for improvement of the activity than the latter.
Effects of Compound 27 on Hyphal Morphology and
Cell Ultrastructure. In order to understand preliminarily the
action mechanism of the compounds, we carried out analysis of
the hyphal morphology and cell ultrastructure by using SEM
and TEM. C. gloeosporioides was chosen as the test fungi. The
most active compound 27 against C. gloeosporioides was
selected as the test compound. The test concentration was
set to the EC50 of 27. The results are shown in Figure 3.
The SEM images showed the untreated mycelia were
regular, intact, and plump and had a smooth surface (Figure
3A,B). By contrast, the treatment with 27 for 72 h led to
shrinkage, collapse, and twisting of the mycelia (Figure 3C−E).
The surface was rough and had some fragments. The hyphal
apexes had an obvious opening.
In the TEM images, the untreated fungi had a typical cell
ultrastructure, where the cell membrane and cell wall were
complete and normal; the normal organelles, especially
mitochondria, were clearly visible (Figure 3F,G). After
treatment with 27, the cell membrane became wrinkled,
contracted inward, and partially detached from the cell wall.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c06507.
■
sı
1H NMR, 13C NMR, and HRMS spectra of compounds
1−30, HPLC of compounds 1−30 for purity analysis,
antifungal activity in vitro of compounds 1−30 at 50 μg/
mL, TAIs of individual compounds against all the eight
fungi, TAIs of 19 compounds against the same fungi,
and control effect of compound 27 on C. gloeosporioides
infections in apples (PDF)
H
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Article
̈
(2) Brase, S.; Encinas, A.; Keck, J.; Nising, C. F. Chemistry and
biology of mycotoxins and related fungal metabolites. Chem. Rev.
2009, 109, 3903−3990.
AUTHOR INFORMATION
Corresponding Authors
■
Xin-Juan Yang − College of Chemistry & Pharmacy,
Northwest A&F University, Yangling 712100, Shaanxi,
China; Phone: +86-029-87092662; Email: yxjsn2@
163.com
Le Zhou − College of Chemistry & Pharmacy, Northwest A&F
University, Yangling 712100, Shaanxi, China; orcid.org/
0000-0001-9043-6657; Phone: +86-029-87092662;
Email: zhoulechem@nwsuaf.edu.cn
(3) Delp, C. J. Coping with resistance to plant disease. Plant Dis.
1980, 64, 652−657.
(4) 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.
(5) Beuria, T. K.; Santra, M. K.; Panda, D. Sanguinarine blocks
cytokinesis in bacteria by inhibiting FtsZ assembly and bundling.
Biochemistry 2005, 44, 16584−16593.
(6) Yang, R.; Gao, Z.-F.; Zhao, J.-Y.; Li, W.-B.; Zhou, L.; Miao, F.
New class of 2-aryl-6-chloro-3,4-dihydroisoquinolinium salts as
potential antifungal agents for plant protection: synthesis, bioactivity
and structure-activity relationships. J. Agric. Food Chem. 2015, 63,
1906−1914.
Authors
Zhi-Ming Cui − College of Chemistry & Pharmacy, Northwest
A&F University, Yangling 712100, Shaanxi, China
Bo-Hang Zhou − College of Chemistry & Pharmacy,
Northwest A&F University, Yangling 712100, Shaanxi,
China
Chao Fu − College of Chemistry & Pharmacy, Northwest A&F
University, Yangling 712100, Shaanxi, China
Liu Chen − College of Chemistry & Pharmacy, Northwest
A&F University, Yangling 712100, Shaanxi, China
Juan Fu − College of Chemistry & Pharmacy, Northwest A&F
University, Yangling 712100, Shaanxi, China
Fang-Jun Cao − Shaanxi Institute of Zoology, Xi’an 710032,
China
(7) 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.
(8) Yang, X.-J.; Miao, F.; Yao, Y.; Cao, F.-J.; Yang, R.; Ma, Y.-N.;
Qin, B.-F.; Zhou, L. In vitro antifungal activity of sanguinarine and
chelerythrine derivatives against phytopathogenic fungi. Molecules
2012, 17, 13026−13035.
(9) 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 Staph-
ylococcus aureus. J. Pharm. Pharm. Sci. 2008, 11, 90−94.
(10) Meng, F.; Zuo, G.; Hao, X.; Wang, G.; Xiao, H.; Zhang, J.; Xu,
G. Antifungal activity of the benzo[c]phenanthridine alkaloids from
Chelidonium majus Linn against resistant clinical yeast isolates. J.
Ethnopharmacol. 2009, 125, 494−496.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c06507
Author Contributions
́
(11) Psotova, J.; Vecera, R.; Zdarilova, A.; Anzenbacherova, E.;
Z.-M.C. and B.-H.Z. contributed equally to this work. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Kosina, P.; Svobodova, A.; Hrbac, J.; Jirovsky, D.; Stiborova, M.;
Lichnovsky, V.; Vicar, J.; Simanek, V.; Ulrichova, J. Safety assessment
of sanguiritrin, alkaloid fraction of Macleaya cordata, in rats. Vet. Med.
2006, 51, 145−155.
(12) Kosina, P.; Walterova, D.; Ulrichova, J.; Lichnovsky, V.;
Stiborova, M.; Rydlova, H.; Vicar, J.; Krecman, V.; Brabec, M. J.;
Funding
́
́
́
This work was financially supported by the National Natural
Science Foundation of China (no. 31872008) and the Key
Research and Development Project of Shaanxi Province
(2019NY-195).
́
́
́
̌
̌
̌
́
Simanek, V. Sanguinarine and chelerythrine: assessment of safety on
pigs in ninety days feeding experiment. Food Chem. Toxicol. 2004, 42,
85−91.
(13) Zhu, L.; Zhou, B.; Zhang, B.; Xu, M.; Geng, H.; Zhou, L. New
2-aryl-7,8-dimethoxy-3,4-dihydroisoquinolin-2-ium salts as potential
antifungal agents: synthesis, bioactivity and structure-activity relation-
ships. Sci. Rep. 2017, 7, 7537.
(14) Zhu, L.-F.; Hou, Z.; Zhou, K.; Tong, Z.-B.; Kuang, Q.; Geng,
H.-L.; Zhou, L. Synthesis, bioactivity and structure-activity relation-
ships of new 2-aryl-8-OR-3,4-dihydroisoquinolin-2-iums salts as
potential antifungal agents. Bioorg. Med. Chem. Lett. 2016, 26,
2413−2417.
(15) Hou, Z.; Yang, R.; Zhang, C.; Zhu, L.-F.; Miao, F.; Yang, X.-J.;
Zhou, L. 2-(Substituted phenyl)-3,4-dihydroisoquinolin-2-iums as
novel antifungal lead compounds: biological evaluation and structure-
activity relationships. Molecules 2013, 18, 10413−10424.
(16) Yang, X.; Yao, Y.; Qin, Y.; Hou, Z.; Yang, R.; Miao, F.; Zhou, L.
Synthesis and in vitro antifungal activities of new 2-aryl-6,7-
methylenedioxy-3,4-dihydroisoquinolin-2-ium bromides. Chem.
Pharm. Bull. 2013, 61, 731−739.
(17) Hou, Z.; Zhu, L.-F.; Yu, X.-C.; Sun, M.-Q.; Miao, F.; Zhou, L.
Design, synthesis, and structure-activity relationship of new 2-aryl-3,4-
dihydro-β-carbolin-2-ium salts as antifungal agents. J. Agric. Food
Chem. 2016, 64, 2847−2854.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Life Science Research Core Services
and the State Key Laboratory of Crop Stress Biology for Arid
Areas for providing us with the nuclear magnetic resonance
and electron microscopy analysis.
ABBREVIATIONS
■
QBAs, quaternary benzo[c]phenanthridine alkaloids; SA,
sanguinarine; CH, chelerythrine; br s, broad signal; IC50,
median inhibition concentration; mp, melting point; PDA,
potato dextrose agar; SD, standard deviation; DMSO, dimethyl
sulfoxide; SAR, structure−activity relationship; EWGs, elec-
tron-withdrawing groups; EDGs, electron-donating groups; AI,
activity index; TAI, total activity index; SI, susceptibility index;
TSI, total susceptibility index; SEM, scanning electron
microscopy; TEM, transmission electron microscopy
(18) Li, X.; Zhang, B.; Zhao, W.; Yang, S.; Yang, X.; Zhou, L. New 2-
aryl-6-methyl-3,4-dihydro-β-carbolin-2-iums as potential antifungal
agents: Synthesis, bioactivity and structure-activity relationship. Sci.
Rep. 2019, 9, 1941.
(19) Zheng, Z.-L.; Miao, F.; Yang, X.-J.; Zhou, L. Effects of 2-aryl-
3,4-dihydroisoquinolin-2-iums as antifungal agents on plant seed
REFERENCES
■
(1) Savary, S.; Teng, P. S.; Willocquet, L.; Nutter, F. W., Jr.
Quantification and modeling of crop losses: a review of purposes.
Annu. Rev. Phytopathol. 2006, 44, 89−112.
I
https://dx.doi.org/10.1021/acs.jafc.0c06507
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
germination and seedling growth. J. Northwest A&F Univ. (Nat. Sci.
Ed.) 2015, 43, 116−122.
́
́
́
(20) Magriz, A.; Gomez-Bujedo, S.; Alvarez, E.; Fernandez, R.;
Lassaletta, J. M. Phthalazin-2-ylidenes as cyclic amino aryl carbene
ligands in rhodium(I) and iridium(I) complexes. Organometallics
2010, 29, 5941−5945.
(21) Butler, R. N.; Pyne, C. S. N-Arylphthalazinium cations:
Synthons for biphthalazinylidenes via endocyclic phthalazinium ylide
intermediates. J. Chem. Soc., Perkin Trans. 1 1992, 3387−3389.
(22) Zhou, K.; Chen, D.; Li, B.; Zhang, B.; Miao, F.; Zhou, L.
Bioactivity and structure-activity relationship of cinnamic acid esters
and their derivatives as potential antifungal agents for plant
protection. PLoS One 2017, 12, No. e0176189.
(23) Wang, D.; Zhang, J.; Jia, X.; Xin, L.; Zhai, H. Antifungal effects
and potential mechanism of essential oils on Collelotrichum
gloeosporioides in vitro and in vivo. Molecules 2019, 24, 3386.
(24) Shang, S.; Liang, X.; Liu, G.; Zhang, S.; Lu, Z.; Zhang, R.;
Gleason, M. L.; Sun, G. Histological and ultrastructural character-
ization of the leaf infection events of Colletotrichum fructicola on
Malus domestica ‘Gala’. Plant Pathol. 2020, 69, 538−548.
(25) Cao, F.-J.; Xu, M.-X.; Zhou, B.-H.; Du, Y.-S.; Yao, J.-H.; Zhou,
L. Effects of 2-aryl-1-cyano-1,2,3,4-tetrohydroisoquinolines on
apoptosis induction mechanism in NB4 and MKN-45 cells. Toxicol.
in Vitro 2019, 54, 295−303.
(26) Cao, F.-J.; Yang, R.; Lv, C.; Ma, Q.; Lei, M.; Geng, H.-L.; Zhou,
L. Pseudocyanides of sanguinarine and chelerythrine and their series
of structurally simple analogues as new anticancer lead compounds:
cytotoxic activity, structure-activity relationship and apoptosis
induction. Eur. J. Pharm. Sci. 2015, 67, 45−54.
(27) Cao, F.-J.; Zhu, L.-F.; Kuang, Q.; Li, X.-Q.; Zhou, B.-H.; Yang,
X.-J.; Zhou, L. Cytotoxic activity, apoptosis induction and structure−
activity relationship of 8-OR-2-aryl-3,4-dihydroisoquinolin-2-ium salts
as promising anticancer agents. Bioorg. Med. Chem. Lett. 2017, 27,
55−60.
(28) Yang, R.; Ruan, Q.; Zhang, B.-Y.; Zheng, Z.-L.; Miao, F.; Zhou,
L.; Geng, H.-L. A class of promising acaricidal tetrahydroisoquinoline
derivatives: synthesis, biological evaluation and structure-activity
relationships. Molecules 2014, 19, 8051−8066.
(29) Ma, Y.-N.; Yang, X.-J.; Pan, L.; Hou, Z.; Geng, H.-L.; Song, X.-
P.; Zhou, L.; Miao, F. Synthesis of 2-aryl-3,4-dihydroisoquinolin-2-
ium bromides and their in vitro acaricidal activity against Psoroptes
cuniculi. Chem. Pharm. Bull. 2013, 61, 204−211.
(30) Zhou, B.; Li, H.; Cui, Z.; Li, D.; Geng, H.; Gao, J.; Zhou, L.
Simple analogues of natural product chelerythrine: discovery of a
novel anticholinesterase 2-phenylisoquinolin-2-ium scaffold with
excellent potency against acetylcholinesterase. Eur. J. Med. Chem.
2020, 200, 112415.
́
(31) Vila, N.; Besada, P.; Costas, T.; Costas-Lago, M. C.; Teran, C.
Phthalazin-1(2H)-one as a remarkable scaffold in drug discovery. Eur.
J. Med. Chem. 2015, 97, 462−482.
(32) Abbasi, M.; Nazifi, S. M. R.; Nazifi, Z. S.; Massah, A. R.
Synthesis, characterization and in vitro antibacterial activity of novel
phthalazine sulfonamide derivatives. J. Chem. Sci. 2017, 129, 1257−
1266.
(33) Onkol, T.; Dogruer, D. S.; Uzun, L.; Adak, S.; Oezkan, S.;
Sahin, M. F. Synthesis and antimicrobial activity of new 1,2,4-triazole
and 1,3,4-thiadiazole derivatives. J. Enzyme Inhib. Med. Chem. 2008,
23, 277−284.
J
https://dx.doi.org/10.1021/acs.jafc.0c06507
J. Agric. Food Chem. XXXX, XXX, XXX−XXX